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2003 saab 9 5 for sale on US's largest classifieds sites (160 results)
Used 2003 Saab 9-5 For Sale
We have 2003 SAAB 9.5 TURBO Auto.with 93,308 Orig. Miles with Cold A/C, Hot Heat,PWR Windows,PWR Locks,Heated PWR Mirrors,DUAL PWR Seats,Dual Heated Seats,Cruise Control, AM/FM/CD Player,Keyless Entry,Sport/Winter Mode Transmission,Rear Parking Assistance, Fog Lights with Leather Interior in excellent...
2003 Saab 9-5 Aero wagon
2003 Saab 9-5 Aero station wagon / sport combi. Four cylinders in line 2.3 TURBO, 185 Hp engine, automatic with paddle shifters and sports mode, 140k miles. Loaded with heated/vented leather seats, power everything, sunroof, reverse sensors, Xenon Hid headlights, 19 inch alloy rims with great tires. T...
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2003 *Saab* *9-5* Linear 2.3t 4dr Turbo Wagon - Multiple Locations to
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Used 2003 Saab 9-3 For Sale
This 2003 Saab 9-3 Linear with leather & heated seats features a 2.0L 4 cylinder 4cyl Gasoline engine. It is equipped with a Automatic transmission. The vehicle is Dolphin Gray with a Parchment Leather Interior. Air Conditioning, Climate Control, Cruise Control, Power Steering, Power Windows, Power Do...
2003 Saab 9-5 Aero Wagon 137xxx miles Collectible REDUCED $3900 OBO
2003 Saab 9-5 Aero Wagon 137xxx miles Rare Collectible5 Speed Auto with Winter and Sport ModePaddle shifters on the steering wheel250 HP 258 ft-lbs 2.3 Turbo engine 240 watts 8 speakers Harman/kardon premium stereo system with factory subwooferPremium sport seats Traction/Stab
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2003 Saab4 Cylinder w/ Turbo 2.3TMileage 121632Leather InteriorThis vehicle belonged to my deceased father. Approximately 3 years ago the engine was replaced with a rebuilt engine to include the turbo by our local foreign mechanical shop. The vehicle runs very well and is in good s
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2003 Saab 9-5 ARC Sedan
2003 Saab 9-5 ARC sedan 3.0L V6 turbo in excellent condition. Full power everything. Extras include xenon headlights, rain-sensing wipers, sun/moon roof, heated/cooled front seats, heated rear seats, back-up proximity sensor. Radiator replaced in 2015. Tires in good shape. The car is loaded, ever...
2003 SAAB 9-5 2.3 Turbo
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V Series Premiere: Instant Edification
The aliens are greeted with a prolonged ovation, as though the entire planet had just watched Nathan Lane on Broadway.By Adam Sternbergh
The aliens are greeted with a prolonged ovation, as though the entire planet had just watched Nathan Lane on Broadway.By Adam Sternbergh
The former teen heartthrob discusses the new show, and why he's not sick of 'Party of Five.'By Katie Goldsmith
Clearly the most ambitious, daring, and artistically successful three-and-a-half-hour Holocaust-allegory-featuring-evil-lizard-people show that's ever aired on TV.By Adam Sternbergh
Why so much of this lately?By Amy Odell
But it was such a good idea.By Adam K. Raymond
Check out the Victoria's Secret model's latest editorial.By Amy Odell
Christian groups probably won't be happy to learn that a statue of Jesus gets shattered into a thousand pieces during the show's first four minutes.By Mark Graham
Somehow it was inspired by Marc's show.By Amy Odell
They have peel-off electrostatic sunglasses on them!By Amy Odell
Production on ABC's 'V' remake has been halted for two weeks while its makers take a "creative hiatus," presumably to retool scripts.By Lane Brown
At least some of us are keeping warm this season!By Amy Odell
And we just died.By Jada Yuan
And she's the color of an Oompa Loompa.By Amy Odell
And her famous "Orbit" hat is back!By Amy Odell
The men just aren't hot enough for him. Neither was the soup, for that matter.By Amy Odell
It would appear that not even aliens have fully bought into 3-D technology.By Mark Graham
Elizabeth Mitchell will be a full-time cast member on ABC's 'V' remake, but her 'Lost' character will appear in an "unspecified number of episodes next season."By Lane Brown
Looks like the Jack-Sawyer-Kate triangle will be back in play!By Mark Graham
Mario Testino captures lady models doing everything but swimming, and much more than simply writhing on a patch of damp sand.By Amy Odell
Why, of course you do. See that and more male-model hotness from 'V' magazine's summer-swim issue.By Amy Odell
Curt Schilling Joins Breitbart
If You're Ever Dissed in a Hacked Email, Try to Respond Like Larry Lessig
New Polls Show More Trouble for Trump and the GOP
Saturday Night Live's 'A Girl's Halloween' Is Too Real
Alec Baldwin's Donald Trump and Kate McKinnon's Hillary Clinton Return to SNL for One Final Debate
Every Episode of Black Mirror, Ranked From Worst to Best
SNL Spoofs the Age-Old Question: How the Hell Is This TV Drama Considered a Comedy?
Taylor Swift Performs 'This Is What You Came For' During Only Concert in 2016, Giving the People That for Which They Came — More or Less
Why Saturday Night Live Destroys Trump, and John Oliver Doesn't
Taylor Swift's Groping-Lawsuit Deposition Released: 'I Remember Being Frantic, Distressed, Feeling Violated in a Way I Had Never Experienced Before'

subset: commoncrawl


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|Paizo Pathfinder® Paizo Games
|Pathfinder Adventure Card Game Pathfinder Roleplaying Game Pathfinder Adventures Pathfinder Campaign Setting Pathfinder Player Companion Pathfinder Accessories Pathfinder Miniatures Pathfinder Fiction Pathfinder Society Pathfinder Roleplaying Game Compatible Products Pathfinder Apparel & Gifts Pathfinder Licensed Products
Pathfinder Society Scenario #8-05: Ungrounded but Unbroken (PFRPG) PDF
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Corruption Codex (PFRPG) PDF
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Pathfinder Society Scenario #7–02: Six Seconds to Midnight (PFRPG) PDF
Pathfinder Society Scenario #4–04: King of the Storval Stairs (PFRPG) PDF
Paizo Inc.(based on 10 ratings)
A Pathfinder Society Scenario designed for levels 7–11.
The PCs are sent to map the Storval Stairs and ensure the stairs provide a safe route to the Storval Rise from Magnimar, but upon their arrival, they find the ancient site claimed and "ruled" by the self-proclaimed King of the Storval Stairs. Only through guile, diplomacy, or cold steel will the Pathfinders ensure access to iconic Thassilonian location.
Written by Dennis Baker.
This scenario is designed for play in Pathfinder Society Organized Play, but can easily be adapted for use with any world. This scenario is compliant with the Open Game License (OGL) and is suitable for use with the Pathfinder Roleplaying Game.
Product Availability
Will be added to your My Downloads Page immediately upon purchase of PDF.
Are there errors or omissions in this product information? Got corrections? Let us know at firstname.lastname@example.com.
See Also:
Last one to post wins,
Paladin Sword of Beserking,
Incubus Bladebound / Kensai,
Tracking creatures in the ocean,
Stat the Gods!!!,
On Will Saves And Narrative Agency,
Is the Samsung Galaxy Note 7 a leagal weapon for PFS?,
Deinonychus and Eldritch Heritage (Ghoul Bloodline),
The Monkey's Treefort,
[Kobold Press] Demon Cults & Secret Societies Kickstarter is Live! (Pathfinder RPG & 5E),
Big Finish and Paizo Announce the Launch of Curse of the Crimson Throne Audio Series!,
Pathfinder Adventures: Now Available on Phones and Tablets!,
Cthulhu Mythos Comes to Pathfinder's World of Golarion,
Paizo Announces Bestiary 6 for Pathfinder Roleplaying Game,
Four Paizo Employees Among Gen Con's Industry Insider Featured Presenters,


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Is Foley's Bar incorrect or missing key business information? Update your Hotfrog listing today.
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Hotfrog Ireland provides information regarding Foley's Bar in Tubbercurry Sligo. Foley's Bar is located at The Square, Tubbercurry Sligo and provides Social services. Contact them on (071) 918 5113.
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Line Number: 207
One thing you must realise is that there is no such thing as pure iron/steel these days. Iron/steel isn't nearly as strong now as it was in medieval times. However, with that said, iron in early medieval times was so soft you could hack right through a helm with a sword and leave a nice lil mark on the skull (depending on the grade of iron used on the sword and the helm, ofcaurse). After many hundreds of years of fine tuning, however, the only use the sword had was to puncture the plate. That was very difficult, however, since the grade of steel was so hard... only blunt instruments and weighted axes had any use against plate armor in later medieval times. Makes me wonder why rapiers were so popular then and why less people wore plate (Other than it's obsene costs... a nice suit of armor would cost as much as a nice lexus does now... and a kings suit would be as much as a rols royce).
Appearing a small ballista bolt, this 'bolt' is actually an arrow used by a god from a long-dead pantheon. Some sages theorize it was a weapon crafted by Loki to slay the world tree. Why it was never used escapes them however.
The spear has a divine-level anti-plant effect. When driven into the soil point first, a ever widening circle of destruction radiates out from the point, killing any plant material. Perfect for holding a kingdom's food supply for ransom.
How far it extends depends of course on the GM, of course.


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|Road Name:
|Black Horse Mews
|Road Type:
Black Horse Mews is a street in the settlement of Borough Green governed by Borough Green Civil Parish council which is within the electoral area of Borough Green and Long Mill Ward in the administration area of Tonbridge and Malling District Council within Kent County Council located in the South East Euro Region of the UK.
Black Horse Mews is located 23.24 miles South East from the centre of London, 9.21 miles West from the centre of Maidstone, 31.49 miles North from the centre of Lewes and 31.51 miles South from the centre of Chelmsford.
Black Horse Mews is approximately 6.61 miles from the coastal tidal high water mark) and is 90 metres above sea level
Postcodes for Black Horse Mews
|The Central point of Black Horse Mews is approximately 90 Metres above sea level
Hotel information updated 09/10/2016
|The Bull Hotel Maidstone/Sevenoaks - wrotham
|Star Rating:
|4 Stars
|Situated between Sevenoaks and Maidstone with easy access to M20, M26 and M25. Newly renovated in 2012, this privately owned 13th century inn is now a 4 star AA rated hotel.
|Bull Lane, Wrotham Village, TN15 7RF, Sevenoaks, Kent
|Number of Rooms:
|Lowest Price Room:
|Highest Price Room:
|Holiday Inn Maidstone-Sevenoaks - wrotham
|Star Rating:
|4 Stars
|Situated between Maidstone and Sevenoaks, this Holiday Inn offers free parking, a pool and a gym. There is easy access to the M25 and M26 motorways. Spirit Health & Fitness Club has a hot tub, a sauna and beauty treatments (booking required).
|London Road, Wrotham Heath, TN15 7RS, Sevenoaks, Kent
|Number of Rooms:
|Lowest Price Room:
|Highest Price Room:
|Premier Inn Sevenoaks/Maidstone - wrotham
|Star Rating:
|3 Stars
|Just off junction 2A of the M26 motorway and only 6.5 km from junction 4 of the M20, the hotel offers great value, with free parking and spacious, modern en suite rooms.
|London Road, TN15 7RX, Sevenoaks
|Number of Rooms:
|Lowest Price Room:
|Highest Price Room:
|Pretty Maid House B&B - wrotham
|Star Rating:
|4 Stars
|Situated in Kent, on the A20 midway between Sevenoaks and Maidstone, this luxurious guest house offers bed and breakfast accommodation with free Wi-Fi throughout and free parking on site.
|London Road, Wrotham Heath, TN15 7RU, Sevenoaks
|Number of Rooms:
|Lowest Price Room:
|Highest Price Room:
|Violets - hadlow
|Violets is a holiday home located in Hadlow, 10 km from The Hop Farm Country Park. It provides free private parking. An oven and a toaster can be found in the kitchen. Towels and bed linen are available in this cottage.
|, TN11, Shipbourne
|Number of Rooms:
|Lowest Price Room:
Local Council Details
|Within the area of Tonbridge and Malling District
|Visit the Council's Website
|Gibson Building
Gibson Drive
Kings Hill
West Malling
|Send Mail
|Telephone Number: ME19 4LZ
|County Council: Kent County
|Within the electoral area of Borough Green and Long Mill Ward -
|Within the Westminster Parliamentary Constituency of Tonbridge and Malling
|The Member of Parliament is Stanley, John
|Within the area of Borough Green Civil Parish Council
Water and Drainage Details
|Southern Water
|Visit Southern Water's Website
Recent House Sales in Black Horse Mews
|16 Black Horse Mews
|Sale Price: £245,000
|Sale Date: 30/05/2014
|7 Black Horse Mews
|Sale Price: £245,000
|Sale Date: 20/09/2012
|Source: Land Registry Price Paid Data accessed via API
Population Statistics
|ONS Area Code: E01024726
|Ons Area Name:Tonbridge and Malling 006D
|Average Age : 43.62
|Population: 1719.00
|Male Population: 830.00 (48.28%)
|Female Population: 889.00 (51.72%)
|Number Claiming Benefits: 24 (1.4%)
|Source: Office of National Statistics 2011 Census Data
Neighbourhood Policing
|Police Force:
|Find a Local Neighbourhood Watch Scheme
|Visit Police Website
|Email Police
|Telephone: 101
|Policing Links:
|Tonbridge and Malling Borough Council
Recent Crime in the Area
|Source: data.police.uk accessed via API
Map of Crimes Committed Near Black Horse Mews
Random Street
Carnegie Place
Location Details
Black Horse Mews
|OS Reference: 561158, 157092
OS Grid Reference: TQ612571
|Latitude and Longitude: 51.289618625591, 0.31146812319694
|Approximate distance to the Coast: 6.61 miles
Ordnance Survey Landranger Map Series for Black Horse Mews
|Map Number: 188
|Map Name: Maidstone & The Weald of Kent
|Borough Green
|Tonbridge and Malling
|South East
Nearest Railway Stations
|Borough Green & Wrotham Rail Station is 0.42 Kilometers away (0.26 miles)
|Kemsing Rail Station is 4.42 Kilometers away (2.75 miles)
|West Malling Rail Station is 7.59 Kilometers away (4.72 miles)
|Bat & Ball Rail Station is 8.06 Kilometers away (5.01 miles)
Nearest Airports
|Biggin Hill Airport is 19.85 Kilometers away
|London City Airport is 29.92 Kilometers away
|London Gatwick Airport is 36.20 Kilometers away
|London Southend Airport is 41.34 Kilometers away
Nearest Ferries
|Gravesend Pier is 17.77 Kilometers away
|Tilbury Ferry Terminal is 18.29 Kilometers away
|Woolwich Arsenal Pier is 28.24 Kilometers away
|Woolwich Ferry South Pier is 28.59 Kilometers away
Nearest Motorway or Major Trunk Road
|Black Horse Mews is approximately 0.72 miles from the M26
Nearest Motorway Junctions
|Junction: M26 J2A/A20 - 2.20 Km Away
|Junction: M20 J2/A20/A227 - 2.32 Km Away
|Junction: M20 J2/A20 - 3.23 Km Away
Health Clinics
|Ent Sevenoaks
|Borough Green Med Pract, Quarry Hill Road, Borough Green, Kent, TN15 8RQ
|Distance: 0.44 Kilometers
|Telephone: 01732 883161
|Borough Green Medical Practice
|Borough Green Med.pract., Quarry Hill Road, Borough Green, Kent, TN158RQ
|Distance: 0.44 Kilometers
|Telephone: 01732 883161
|Dermatology Ad Sevenoaks
|Borough Green Med Prac, Quarry Hill Road, Borough Green, Kent, TN15 8RQ
|Distance: 0.44 Kilometers
|Telephone: 01732 883161
|West Kingsdown Medical Centre
|West Kingsdown Med.ctr., London Rd, West Kingsdown, Sevenoaks, Kent, TN156EJ
|Distance: 6.55 Kilometers
|Telephone: 01474 855016
Nearest Libraries
Borough Green Library
|High Street, Borough Green, TN158BJ
|Distance: 0.21 Miles
|Visit Website
Vigo Library
|The Bay, Vigo, DA130TD
|Distance: 3.51 Miles
|Visit Website
Seal Library
|High Street, Sevenoaks, TN150AL
|Distance: 3.88 Miles
|Visit Website
Nearest Tourist Attractions
|Attraction Name: Great Comp Garden
|Distance: 2.14 (km)
Landmark or Antiquity
|Attraction Name: Old Soar Manor
|Distance: 3.04 (km)
Historic House
|Attraction Name: Old Soar Manor
|Distance: 3.41 (km)
Landmark or Antiquity
|Attraction Name: Ightham Mote
|Distance: 4.50 (km)
Nearest Schools
Borough Green Primary School
|Distance: 0.32 Kilometers
|Type: Primary
|Address: School Approach, Borough Green, Sevenoaks, Kent, TN15 8JZ
|Telephone: (01732) 883459
Grange Park School
|Distance: 1.07 Kilometers
|Type: Not applicable
|Address: Borough Green Road, Wrotham, Sevenoaks, Kent, TN15 7RD
|Telephone: (01732) 882111
Wrotham School
|Distance: 1.07 Kilometers
|Type: Secondary
|Address: Borough Green Road, Wrotham, Sevenoaks, Kent, TN15 7RD
|Telephone: (01732) 884207
Platt Church of England Voluntary Aided Primary School
|Distance: 1.21 Kilometers
|Type: Primary
|Address: St Mary's Platt, Sevenoaks, Kent, TN15 8JY
|Telephone: (01732) 882596
Broadband Speed (megabits per second)
|Speed: Less than 2 Mbps
|Average Speed: 12.9
|Median Speed: 13.9
|Maximum Speed: 21.6
|Next-generation access (NGA)?: N
|Number of Connections: 11


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Garmin's latest running watch tracks your suffering
Garmin's latest running watch can track multiple sports, measure your heart rate and even give you a "suffer score," but the price might make you gasp. The Forerunner 735XT now occupies the top of the company's run tracker lineup next to the two-year-old 920XT with the same $450 price. For that substantial sum, you do get a lot: it packs the company's Elevate heart rate monitor and is the first sports tracker with Strava's Suffer Score. That app measures your heart rate compared to a baseline, so casual or serious athletes can see how much they're pushing it.
The watch is compatible with Garmin's Vector pedal tracking and Varia radar accessories that help keep you safe in traffic. It's also waterproof to 50 meters and has custom tracking features for swimmers. Other sports tracked include hiking, cross-country skiing, strength training, cardio and multisport. The latest model is made from soft silicon, has a color display, runs up to 14 hours in training mode and goes 11 days as a regular watch.
The wearable links with Garmin's Connect IQ store, letting you get free apps, watch faces and more. Users can also track workout, sleeping and others stats on the Connect online community, or compete against friends and join larger competitions (then brag about the results, if merited). The devic also connects with your smartphone to control your music and can notify you for calls, emails and social network alerts. The Forerunner 735XT comes in black/gray or midnight blue/frost blue and is available for $450.
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If you log in, you can comment on buildings, submit new photos or update photos that you've already submitted.
We need to upgrade the server that this website runs on. Can you spare a quid to help?.
Description: Lorimar House
Grade: II
Date Listed: 13 July 1987
English Heritage Building ID: 287037
OS Grid Reference: TQ0777864175
OS Grid Coordinates: 507778, 164175
Latitude/Longitude: 51.3664, -0.4532
Location: Hanger Hill, West Molesey, Surrey KT13 9YG
Locality: Weybridge
Local Authority: Elmbridge Borough Council
County: Surrey
Country: England
Postcode: KT13 9YG
Incorrect location/postcode? Submit a correction!
(east side)
Nos. 69 and 71
(Lorimar House)
House, now 2 dwellings. 1854 for Benjamin Scott with addition of C1860 by Edmund
Woodthorpe (Handbook, p178). Yellowish brick in Flemish bond with painted ashlar
dressings; Welsh slate roof. 2 storeys with basement at rear and 4-storey tower.
Original house L-shaped, the gabled wings enfolding tower set in angle and a single-
storey room on right; 4-bay addition on left is set back slightly, the left bay
narrower and further recessed. In Italianate style with tabled plinth, rusticated
corner pilasters, ground floor entableture and console-bracketed oversailing eaves.
Single-storey entrance on right of tower has long flight of stone steps with chequered
bottom step and flat-coped side-walls with lions couchant at bottom; 4-panel double
door with fanlight under keyed archivolt on pilasters with capitals. Ground-floor
windows are round-arched sashes with archivolts on pilasters with capitals and
corbelled sills; single-storey room on right has quadrupled windows; gabled bay has
flat-roofed bay window with tripled windows. On 1st floor windows are square-headed
having wooden cross-windows with arched top lights in shouldered surrounds with consoled
segmental pediment to gabled bay; roof consoles spring from window surrounds of left-
hand windows. Tower has similar window to 2nd floor and 4th floor tripled windows as
ground floor with keyed archivolts, columns and paired side pilasters. Single-storey
room has corrugated asbestos sheet roof, hipped at right end. Various ashlar-corniced
stacks. Rear: 6 bays set forward on right plainer, having flat-arched windows with
4-pane sashes to addition, sashes with glazing bars to earlier part; a tripartite
window to ground floor left. Verandah to addition with decorative S-section iron
balustrade and slender fluted columns supporting glass roof; formerly extended further
left (Handbook, picture on P178). At left end, iron stair in same style up to door in
left return. Entrance to No. 69 on left return. Interior: No. 71 has original open-
well stair, rising under tower with open string, twisted wooden balusters; in front
left room decorative fireplace and cornice; in single-storey room, false decorative
fireplace and Adam-style ceiling. The front room of No. 69, former music room,
decorated in Pompeian style by Crace (Handbook. p178) with panelled pilasters and
heavy floral cornice and ceiling border. Top floor of tower intended to be an
[Handbook of Chertsey and its Neighbourhood]
Listing NGR: TQ0777864175
This text is a legacy record and has not been updated since the building was originally listed. Details of the building may have changed in the intervening time. You should not rely on this listing as an accurate description of the building.
Source: English Heritage
Listed building text is © Crown Copyright. Reproduced under licence.


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hiya guys new to the astra seen just need some advice really.
couple of questions:
My car currently has a poo air box. so would it be advise able to buy a 1.9 or 2.2 airbox as apposed to a standard one as i have the intensions of going ph4 eventually? will a VXR AFM fit with a standard airbox and just need a reducer? whats the bore size of a standard airbox and a 2.2 airbox?
i want a front mount aswell and will a universal one with 57mm inlet and outlet or would it be adviseable to get one with bigger inlets and outlets i.e 63mm?
can you get a hose and pipe kit for a front mount or do oyu jsut need to order all your parts yourself for the bends etc?
do you need a VXR AFM for ph4?
all help will be greatly appreciated?


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??Glass slide planned for LA towerDevelopers of the US Bank Tower in Los Angeles have unveiled plans to include a 36ft glass slide on the outside of the building, 1,000ft above the city's streets. To share with our mains we ordered the rosemary & garlic bread which was amazing and so fresh and also the heritage tomatoes which had a great flavour. ??School The ABC of Race Author: Franz Lüke 1935 Image WL14653 Indoctrination within schools covered a number of issues and paved the way for the youth&rsquo.
Desktop & web trading Mobile Trading Learn Legal To read PDF documents we recommend downloading latest version of Adobe Reader. :)I have seen many car dealerships and most of them are dumps and look really dodgy, but not these guys. Melbourne Water Download for iOS Android Melbourne Zoo The Melbourne Zoo is the official iPhone trail map for all ages to guide you on your walk through Melbourne Zoo.
a fascinating insight into the colourful characters instrumental in the design and history of these impressive old houses&rdquo. 3PB barristers have acted in heavier clinical negligence matters such as brain damage cases and are familiar with claims which include Court of Protection and receivers' fees. How can local people accept that changes may be for the better when politicians promote opposition and criticism for their own agenda.


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Using Vintage Images For Scrapbook Projects
If you would like to add in a little touch of nostalgia to your scrapbook projects, you should try to incorporate vintage images into your scrapbooking. Vintage images have the ability to give an instant artistic facelift to your scrapbook projects. It could be old photographs, early illustrations, antique media such as newspaper cuttings or advertisements, or perhaps other ephemera.
Vintage images also adds more than just artistic value. It is proof that there are important things in our lives that never change even though the test of time. It helps to capture nostalgic essence of the past, helping to convey those emotions. While you could go through the efforts of finding actual pieces of nostalgic images, it is much easier for you to find them online nowadays.
Some examples of vintage images are the likes of book illustrations, newspaper and magazine cuttings, sheet music, postcards, greeting cards, paper decorations, games and toys, clothing patterns, product boxes and labels, advertisements, personal documents, as well as personal photos. You could find these vintage images from places such as estate sales, yard sales, antique stores, book stores, flea markets, and now easily on the internet.
Once you get your hands on some vintage images, it is time to put them into play within you scrapbook projects. You could use a huge vintage image as the centerpiece of your scrapbook page, making it the background where you could lay other photos on it. The best types or those which are pale bright in color, as well as suits the theme of the page. If the page is about your child's birthday, you might want to use vintage images that fits the situation.
Use vintage paper decorations as embellishments for your scrapbook. If you are unable to get your hands on those, what you could do is to look through your collection of vintage images and look for ones with pattern you could use. Simply cut and paste the desired patterns onto your scrapbook for that nostalgic feel. You could do the same to create borders and frames around your scrapbook pages. You could look at old postcards and greeting cards for vintage frame ideas.
Be sure that you set up your scrapbook so your personal photos "interact" with the vintage images that you choose to use. You do not want your scrapbook to look messy and dull. While vintage images could help a lot to enhance the looks of your scrapbook, you still need to be creative with planning the layouts and positioning.
List of online places to find vintage images
Here are links to several places online to find vintage images:
1. Images of vintage christmas and cherubs angels
2. Vintage Cherubs and angels clip art
4. Free Vintage Valentine Hearts Clip Art
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Comments 1 comment
You discus some good ideas to using the vintage images in scrapbooks. i really like this idea and vote up for useful hub.


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Banks is a 6-7 ½, 195-pound small forward at Bergen Catholic (N.J.) and has a similar body build to that of N.C. State freshman and former St. Raymond's star Julius Hodge.
Banks and his 'mates get going this Friday at Paramus and the junior will try and improve upon his numbers from a year ago – and not just the 17.1 points, five boards, four blocks and four steals per contest.
"I think we can honestly compete for the state title," he said.
Banks is wiry and active. He handles the ball well and is a confident scorer. However, he needs to make a better effort to hit the boards.
He said his current list includes UNC (which he listed first), Seton Hall, St. John's, Duke, Villanova, Syracuse and Texas. He added that Seton Hall, UNC, Duke and Louisville are the most aggressive.
"I really like Villanova so far," he added.
Here's an assessment from IC recruiting analyst Clint Jackson back from the Charlie Weber Tournament during the summer:
"He's a sleek wing player with a fine handle, and showed the capacity to put the ball on the floor and shed defenders. He utilizes a nice stutter step and changes speeds to keep defenders off balance. He showed nice range on his jump shot and canned some impressive three-balls in the two games that I saw him. Overall a nice high-major wing forward/shooting guard."
Read IC's September feature on Banks - Junior to Watch: Sean Banks


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Thanks Tony, good info.
Stop spamming this blog with your advertisement. There are plenty of good plans for making your own colloidal silver generator online, including the one above.View Instructable »
Apparently the human body has a way to take silver ions into the cells without them being modified in the digestive tract. Interesting info belowhttp://www.thesilveredge.com/whats-so-true-about-true-colloidal-silver.shtml#.WACJXn_TUdd
I finally found the answer to that question here after much research and disinformation. Hope it helps someone as much as it did help me. http://www.thesilveredge.com/whats-so-true-about-t...View Instructable »
Before you consider using this internally, do yourself a favour and leave the saline out! It will take longer as a result because the water resistance is higher. Using some from a previous batch would help speed up the process.Then you should get a current regulating diode of 1mA. This needs to be hooked into the positive lead after the batteries and before the electrode. This will ensure that the current does not rise when the resistance lowers as more silver ions enter the water. This is very important! This will keep the quality high and prevent large particles being formed which are harmful. If you look up Papa Smurf the blue man you will see that he used salt in the water and he did not restrict the current flow and he ended up blue/grey as a result.Having something to stir it would also be beneficial. You could make a magnetic stirrer from an old PC fan. See youtube for instructions on building one. They can be made fairly cheaply using a lunch box and a fan, two small rare earth magnets/Neodymium which are glued to the fan, preferably a variable resistor and a 12 volt power supply. A stir bar is needed which you put in the bottom of the glass flask. This helps prevent agglomeration of the silver.This does complicate it but it pays to do it safely rather than having regrets because you turned blue/grey.View Instructable »
DO NOT USE THIS RECIPE FOR INTERNAL COLLOIDAL SILVER. Do diligence. Read more. NEVER use saline in any CS. It turns it to silver salts. Very bad for you. If you follow this guide, eliminate the salts.View Instructable »
it's called a 1 troy oz.
i use got pure silver from coin shop. It's not a coin but it's about the same size and it last much longer.View Instructable »
your silver wires are good until they become brittle and break. your problem lies in one of your other parts. or a missing partView Instructable »
My silver wires seems to have run out of ions. There are no longer any silver ion that comes out of my silver when I put a charge between them and put the wires in water. Can anybody explain what I can do except for buying some new silver wires? Help is much appreciated.View Instructable »
Hi SofisintownI'm wondering if you know I have silver wound dressing. Is there any way I could use it to make liquid silver?View Instructable »


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Cars to take centre stage at Lydiard Park on Saturday 25 July
Car enthusiasts will be revved up by a special festival at Lydiard Park this weekend.
More than 100 classic, vintage and super cars will be on display in front of Lydiard House between 11am and 6pm as part of the Swindon Borough Council-owned park's Auto and Retro Festival, which has been organised in conjunction with RMJ Top Events.
Among the cars on display will be a Marcos Mantula, pictured, a Jaguar E Type, a Porsche 911 and a Triumph Spitfire. The collection of cars will also feature a number of MGs and Volkswagens.
There will also be car clubs attending including Six-Ex Mini Owners Club and the Cotswold Morris Minor Club.
As well as old classics, a range of modern cars from Swindon dealerships will also be on display including Ferrari, Maserati, Jaguar, Seat, Mazda and Volvo.
Music will be on the agenda throughout the day with Swingout Swindon performing in both the morning and afternoon, while they will also be offering a taster Lindy Hop dance class in the marquee.
Four piece traditional jazz band, Acoustic Jass will add further musical entertainment and there will be plenty of food available as well as a Pimms and champagne bar.
In keeping with the vintage theme, traders will be selling vintage toys and retro household items, while there will be hair braiding, face painting and vintage clothing on offer.
Cllr Garry Perkins, Swindon Borough Council's Cabinet Member responsible for Lydiard Park, said: "If you love cars then Lydiard really is the place to be on Saturday because our Auto and Retro Festival will have some stunning examples on display.
"We have really expanded the festival this year and we have cars from almost every era as well as a Retro focus which we hope will bring lots of fun for all the family."
Kat Dixon, Events Assistant at Lydiard Park, said "Even if cars aren't your thing there's plenty going on to entertain with the retro hair salon, clothing stands and live performances from Swindon Swingout and Acoustic Jass.
"The food on offer is incredible and all tying in with a vintage theme. It promises to be a really lovely event and I can't wait to see people enjoying it."
Tickets for the festival can be bought on the gate or in advance by visiting: www.swindontickets.co.uk and are priced at £8 for adults (includes free entry for one child), £4 for children aged between 3 and 16. Children under 3 go free.
For more information please visit www.lydiardpark.org.uk or email: email@example.com


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Started May 8, 2009 | Discussions thread
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Jack West • Senior Member • Posts: 1,144
May 8, 2009
i had the highlights blinking in playback mode a while back but now i can't get this feature working again on my d-300? i go to menu - playback - display mode - put a check on the highlight box then click ok and when the checkmark is in i click done. then i take a way overexposed shot and get no blinkies in the playbabk shot? what am i forgetting? thanks for any help here as i'm off on a nice trip soon. best regards, jack
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Sean Cook (seancook37)'s status on Friday, 21-Feb-14 22:51:35 PST
Word Unscrambler Online And Free Online #Puzzles Games http://bit.ly/MH1r3WFriday, 21-Feb-14 22:51:35 PST from web
Word Unscrambler Online And Free Online #Puzzles Games http://bit.ly/MH1r3W
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Previously, we showed that physiological functions of renal proximal tubular cells (RPTC) do not recover following S-(1,2-dichlorovinyl)-l-cysteine (DCVC)-induced injury. This study investigated the role of protein kinase C-α (PKC-α) in the lack of repair of mitochondrial function in DCVC-injured RPTC. After DCVC exposure, basal oxygen consumption (Qo2), uncoupled Qo2, oligomycin-sensitive Qo2, F1F0-ATPase activity, and ATP production decreased, respectively, to 59, 27, 27, 57, and 68% of controls. None of these functions recovered. Mitochondrial transmembrane potential decreased 53% after DCVC injury but recovered on day 4. PKC-α was activated 4.3- and 2.5-fold on days 2 and 4, respectively, of the recovery period. Inhibition of PKC-α activation (10 nM Go6976) did not block DCVC-induced decreases in mitochondrial functions but promoted the recovery of uncoupled Qo2, oligomycin-sensitive Qo2, F1F0-ATPase activity, and ATP production. Protein levels of the catalytic β-subunit of F1F0-ATPase were not changed by DCVC or during the recovery period. Amino acid sequence analysis revealed that α-, β-, and ε-subunits of F1F0-ATPase have PKC consensus motifs. Recombinant PKC-α phosphorylated the β-subunit and decreased F1F0-ATPase activity in vitro. Serine but not threonine phosphorylation of the β-subunit was increased during late recovery following DCVC injury, and inhibition of PKC-α activation decreased this phosphorylation. We conclude that during RPTC recovery following DCVC injury, 1) PKC-α activation decreases F0F1-ATPase activity, oxidative phosphorylation, and ATP production; 2) PKC-α phosphorylates the β-subunit of F1F0-ATPase on serine residue; and 3) PKC-α does not mediate depolarization of RPTC mitochondria. This is the first report showing that PKC-α phosphorylates the catalytic subunit of F1F0-ATPase and that PKC-α plays an important role in regulating repair of mitochondrial function.
- renal proximal tubular cells
- mitochondria
- F1F0-ATPase
s-(1,2-dichlorovinyl)-l-cysteine (DCVC), a nephrotoxic metabolite of the environmental contaminants trichloroethylene and dichloroacetylene, produces cell injury and death in renal proximal tubular cells (RPTC) (10, 14–18, 20, 26, 28, 29, 35, 36, 39). DCVC exposure induces mitochondrial dysfunction, depletion of cellular ATP, modification of thiols, and decreases in active Na+ transport, Na+-K+-ATPase activity, and Na+-dependent glucose uptake (12, 14, 16, 26, 28, 29). Mitochondrial dysfunction is a hallmark of DCVC-induced toxicity. DCVC exposure decreases mitochondrial respiration, total adenine nucleotide pool, and mitochondrial glutathione content, alters the concentration of several citric acid cycle intermediates, reduces the activity of isocitrate dehydrogenase and succinate:cytochrome c oxidoreductase, and inhibits succinate-linked state 3 respiration (1, 15). Furthermore, DCVC impairs the ability of mitochondria to retain Ca2+ and to generate the mitochondrial membrane potential (ΔΨm), which leads to extensive mitochondrial swelling, release of cytochrome c, and activation of caspase-3 (6, 7, 37).
The return of RPTC physiological functions is critical for the restoration of normal renal function. We showed that RPTC fully recover physiological functions following an oxidant injury but not following DCVC-induced injury, which suggests that DCVC inhibits regenerative responses in RPTC (29). For example, the mitochondrial function remains suppressed in DCVC-injured RPTC for 6 days following the treatment (29). Our previous studies reported that epidermal growth factor (EGF), pharmacological concentrations of ascorbic acid, collagen IV, and longer exposures to phorbol ester promote recovery of mitochondrial function in DCVC-injured RPTC (23, 24, 26, 28, 29). However, the mechanisms underlying the lack of mitochondrial repair and the pathways by which EGF, ascorbic acid, collagen, and phorbol ester promote the return of RPTC functions in DCVC-injured RPTC remain unknown.
Protein kinase C (PKC) is a family of serine/threonine protein kinases, which play a central role in cell signaling and the regulation of a variety of cellular functions, including growth, differentiation, motility, contraction, ion and macromolecule secretion, electrolyte transport, synaptic transmission, axonal regeneration, tumor promotion, cell aging, apoptosis, and survival (8). Each PKC isozyme is involved in the regulation of different functions and has a unique role in the cell. PKC-α, one of the classic PKC isozymes, has been implicated in cellular proliferation and differentiation (22, 33) as well as cell injury and death (25). A previous study by this laboratory showed that PKC-α mediates mitochondrial dysfunction, decreases in active Na+ transport, and apoptosis in cisplatin-injured RPTC, suggesting an important role of PKC-α in the regulation of mitochondrial function during cell injury and death (25). Specifically, cisplatin-induced activation of PKC-α inhibits oxidative phosphorylation by decreasing electron transport rate and F1F0-ATPase activity, suggesting an important role of PKC-α in the regulation of F1F0-ATPase activity (25).
F1F0-ATPase is localized in the inner mitochondrial membrane and is responsible for ATP synthesis under physiological conditions (2, 3). Under some pathological conditions leading to the loss of ΔΨm, F1F0-ATPase operates in a reverse mode and hydrolyzes ATP. The enzyme has multiple subunits forming two major complexes, F0 and F1 (3, 4). F1 is a water-soluble catalytic complex made up of five subunits (α3β3γδε), with the catalytic site located on the β-subunit. F0 is made up of several integral membrane proteins that form a proton channel (3, 4, 32, 34). The activity of this enzyme is tightly regulated to synchronize ATP synthesis with cellular energy expenditure. Protein phosphorylation, a posttranslational modification producing changes in enzymatic conformation and activity, may represent one of the mechanisms responsible for regulation of F1F0-ATPase activity. It has been shown that platelet-derived growth factor induces tyrosine phosphorylation of the δ-subunit of F1F0-ATPase (42). Indeed, our search of the amino acid sequence of F1F0-ATPase revealed several possible PKC phosphorylation sites on subunits α, β, and ε as well as on coupling factor 6.
PKC-α has also been implicated in cellular repair and regenerative processes. Renal regeneration following folic acid- or DCVC-induced injury in vivo is associated with the downregulation of PKC-α protein levels in kidney homogenates (9, 43). Our previous study also suggested that PKC-α is one of the PKC isozymes involved in the repair of mitochondrial function and active Na+ transport following DCVC injury in RPTC, but the mitochondrial targets of this PKC isozyme have not been examined (26). Therefore, the aim of the present study was to determine the role of PKC-α in the inhibition of the repair of mitochondrial function and to elucidate the mitochondrial targets of PKC-α in DCVC-injured RPTC.
Female New Zealand White rabbits (2.0–2.5 kg) were purchased from Myrtle's Rabbitry (Thompson Station, TN). DCVC was synthesized according to the method of Moore and Green (31). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and the antibodies against F1F0-ATPase subunits were obtained from Molecular Probes (Eugene, OR). The cell culture media (a 50:50 mixture of DMEM and Ham's F-12 nutrient mix without phenol red, pyruvate, and glucose) were purchased from MediaTech Cellgro (Herndon, VA). PKC-α inhibitor, Go6976, recombinant PKC-α, -ε, and -δ were supplied by Calbiochem (La Jolla, CA). Protease inhibitors and an ATP Bioluminescence Assay Kit HS II were obtained from Roche (Mannheim, Germany), and phosphatase inhibitors cocktail was obtained from Sigma (St. Louis, MO). Tris-glycine gels were supplied by BioWhittaker Molecular Applications (Rockland, ME). A Silver Stain Plus kit and nitrocellulose membranes were obtained from Bio-Rad (Hercules, CA). Phospho-PKC-α and PKC-α antibodies were purchased from Upstate Biotechnology (Lake Placid, NY) and BD Transduction Laboratory (San Diego, CA), respectively. Phosphoserine-PKC substrate antibody and phosphothreonine antibody were obtained from Cell Signaling Technologies (Beverly, MA) and Biomol (Plymouth Meeting, PA), respectively. Anti-mouse IgG coupled to horseradish peroxidase was supplied by Kirkegaard & Perry Laboratory (Gaithersburg, MD) and Supersignal Chemiluminescent Substrate by Pierce (Rockford, IL). Redivue [γ-32P]ATP (3,000 Ci/mmol) was purchased from Amersham Biosciences (Piscataway, NJ). Protein A/G agarose was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), an Amicon Diaflo apparatus with a PM-10 filter (10,000-molecular mass cutoff point) was obtained from Millipore (Bedford, MA), and phosphocellulose disks were from Whatman (Clifton, NJ). The sources of the other reagents and cell culture hormones have been described previously (25, 30).
Isolation and culture of RPTC.
Renal proximal tubules were isolated from rabbit kidneys by the iron-oxide perfusion method and cultured in 35-mm culture dishes in improved conditions as previously described (30). The culture medium was a 50:50 mixture of DMEM and Ham's F-12 nutrient mix without phenol red, pyruvate, and glucose, supplemented with 15 mM NaHCO3, 15 mM HEPES, and 6 mM lactate (pH 7.4, 295 mosmol/kgH2O). Human transferrin (5 μg/ml), selenium (5 ng/ml), hydrocortisone (50 nM), bovine insulin (10 nM), and l-ascorbic acid-2-phosphate (50 μM) were added to the media immediately before daily media change (2 ml/dish).
DCVC treatment of RPTC monolayer.
RPTC monolayers reached confluence within 6 days and were treated with DCVC (200 μM, 90 min) on day 7 of culture. After DCVC exposure, the monolayer was washed with fresh warm (37°C) medium and cultured for an additional 4 days. In experiments using the PKC-α inhibitor, RPTC were pretreated for 1 h with 10 nM Go6976 and then exposed to DCVC. Go6976 was added daily starting with the media change immediately following DCVC exposure. RPTC samples were taken at various time points after DCVC exposure for measurements of mitochondrial functions, biochemical analyses, immunoprecipitation, and immunoblotting.
Oxygen consumption.
RPTC monolayers were gently detached from the dishes using a rubber policeman and transferred to the oxygen consumption (Qo2) measurement chamber. Qo2 was measured using a Clark-type electrode as described previously (25, 29, 30). Oligomycin-sensitive Qo2 was used as a marker of oxidative phosphorylation and was calculated as a difference between basal Qo2 and oligomycin-insensitive Qo2, which was measured in the presence of oligomycin (0.6 μg/ml). Uncoupled Qo2 was used as a marker of the electron transfer rate and integrity of complexes of the respiratory chain and was measured in the presence of FCCP (2 μM).
Measurement of intracellular ATP content.
Intracellular ATP content in RPTC lysates was measured by the luciferase method using an ATP Bioluminescence Assay Kit HS II according to the manufacturer's protocol.
ATP production rate.
The assessment of state 3 respiration (the maximum rate of ATP synthesis) was carried out by a modified method of Borkan et al. (2). In brief, the culture media were aspirated and replaced with 1 ml of a buffer solution resembling an intracellular electrolyte milieu (120 mM KCl, 5 mM KH2PO4, 10 mM HEPES, 1 mM MgSO4, and 2 mM EGTA, adjusted to pH 7.4 with KOH), containing digitonin (0.1 mg/ml) and 5 mM glutamate + 5 mM malate as the substrates. The reaction was initiated by adding excess ADP (2 mM final concentration) and carried out for 5 min at 37°C. Initial experiments determined that ATP production in these conditions was linear for 10 min. The reaction was terminated by adding an aliquot of ice-cold perchloric acid (3% final concentration), and the suspension was snap-frozen in liquid nitrogen. After thawing, the suspension was spun down at 15,000 g × 1 min at 4°C. The supernatant was neutralized to pH 7.5 and centrifuged again at 15,000 g × 10 min at 4°C. The final supernatant was analyzed for ATP content using an ATP Bioluminescence Assay Kit HS II as described above. The initial pellet was assayed for protein content following solubilization in a buffer containing 100 mM Tris·HCl (pH 7.5), 150 mM NaCl, and 0.05% Triton X-100.
ΔΨm Was assessed as described previously (25) using JC-1, a cationic dye that exhibits potential-dependent accumulation and formation of red fluorescent J-aggregates in mitochondria, which is indicated by a fluorescence emission shift from green (525 nm) to red (590 nm). At different time points of the recovery period, RPTC monolayers were loaded with 10 μM JC-1 for 30 min at 37°C. After loading, media were aspirated and monolayers were put on ice, washed with ice-cold PBS, scraped off culture dishes, washed, and resuspended in PBS. Fluorescence was determined by flow cytometry (FACSCalibur, BD Biosciences) using excitation by a 488-nm argon-ion laser. The JC-1 monomer (green) and the J-aggregates (red) were detected separately in FL1 (525 nm) and FL2 (590 nm) channels, respectively. ΔΨm Is presented as JC-aggregates/JC-1 monomer ratio.
Isolation of RPTC mitochondria.
Mitochondria were isolated from RPTC by the method of Lash and Sall (19). RPTC were homogenized in ice-cold isolation buffer [225 mM sucrose, 10 mM Tris·HCl, 10 mM potassium phosphate (pH 7.4), 5 mM MgCl2, 20 mM KCl, 2 mM EGTA, protease and phosphatase inhibitors cocktail] using a Dounce homogenizer and centrifuged at 1,000 g for 5 min at 4°C. The supernatant was collected and centrifuged at 15,000 g for 10 min at 4°C. The pellet containing RPTC mitochondria was washed twice in the isolation buffer and spun down again at 15,000 g for 10 min at 4°C. The final mitochondrial pellet was resuspended in 10 mM Tris·HCl buffer (pH 7.4) containing 25 mM sucrose, 75 mM mannitol, and 100 mM KCl and used for measurement of F1F0-ATPase activity. In some experiments, the final mitochondrial pellet was resuspended in 50 μl of modified radioimmune precipitation assay (RIPA) buffer (50 mM Tris·HCl, 150 mM NaCl, 1 mM EGTA, 1% Triton X-100, 1 mM Na3VO4, 1 mM NaF, and the protease and phosphatase inhibitors cocktail; pH 7.4) and used for immunoblot analysis.
Measurement of F1F0-ATPase activity.
F1F0-ATPase activity was determined in freshly isolated RPTC mitochondria by measuring the release of Pi from ATP according to Law et al. (21) with modifications described previously (23).
Isolation of F1F0-ATPase.
Freshly isolated rabbit renal cortical mitochondria were used for the isolation of F1F0-ATPase using the method described by Catterall and Pedersen (5). Briefly, mitochondria were resuspended and homogenized in a homogenization buffer (3.0 mM Tris·HCl and 5.0 mM EDTA; pH 7.5) at a protein concentration of 50 mg/ml. After homogenization, the sample was sonicated using a Vibra Cell sonicator (Sonics and Materials, Danbury, CT) at maximum speed for 2 min on ice and then centrifuged at 150,000 g for 45 min at 0°C. The pellet was resuspended in a washing buffer (3.0 mM Tris·HCl and 50.0 mM EDTA; pH 7.5) at a protein concentration of 20 mg/ml and centrifuged at 150,000 g for 45 min at 0°C. The pellet was then washed five times in the washing buffer and centrifuged at 150,000 g for 45 min. The final pellet was resuspended in a washing buffer containing 10% ethylene glycol and 4.0 mM ATP, incubated for 16 h at room temperature, and centrifuged at 150,000 g for 45 min at room temperature. The pellet was resuspended in a buffer containing 250 mM sucrose, 3.0 mM Tris·HCl, and 5.0 mM EDTA (pH 7.5) and sonicated for 30 min (three 10-min intervals) in a 22°C water bath followed by immediate centrifugation at 150,000 g for 90 min at room temperature. The clear supernatant was applied to a DEAE cellulose column (2.0 × 8.0 cm) preequilibrated with 20 mM potassium phosphate, 5.0 mM EDTA (pH 7.5), and the enzyme was eluted with a linear gradient of potassium phosphate (50–250 mM; pH 7.5) containing 5.0 mM EDTA at a flow rate of 2 ml/min. The fractions eluted by 150–200 mM potassium phosphate were pooled and concentrated using an Amicon Diaflo apparatus with a PM-10 filter (10,000 cutoff point), and the concentrated sample was snap-frozen in liquid nitrogen and stored at −70°C until used in assays.
Phosphorylation of F1F0-ATPase by PKC-α.
In vitro phosphorylation of isolated F1F0-ATPase by recombinant PKC-α was performed using a modification of the assay described by Yasuda et al. (41). Briefly, experiments were carried out at 30°C in an incubation mixture (25 μl) containing 20 mM Tris, pH 7.5, 0.35 mM EDTA, 0.35 mM EGTA, 1 mM CaCl2, 20 mM MgCl2, 6 mM β-mercaptoethanol, 10 μM PMA, 280 μg/ml l-α-phosphatidyl-l-serine, 2.4 μg F1F0-ATPase, and 30 nM PKC-α. Histone type III phosphorylation by PKC-α was used as a positive control. Reaction was started by the addition of ATP (10 μM, final concentration) with trace amounts of [γ-32P]ATP (60,000 cpm/μl). Under these assay conditions, phosphotransferase activity of PKC-α was linear for at least 5 min. The reaction was stopped after 5 min by adding Laemmli sample buffer (60 mM Tris·HCl, pH 6.8, containing 2% SDS, 10% glycerol, 100 mM β-mercaptoethanol, and 0.01% bromophenol blue) and boiling samples for 5 min (13). Proteins were resolved by SDS-PAGE, gels were dried, and 32P incorporation was determined by autoradiography. In some experiments, the reaction was stopped by spotting the samples on P81 phosphocellulose disks, the disks were washed twice with 1% phosphoric acid and twice with deionized water, and 32P incorporation was quantified by liquid scintillation spectrometry.
To determine the identities of phosphorylated proteins, immunoblot analysis of the isolated F1F0-ATPase (2 μg) was performed using antibodies against various subunits of F1F0-ATPase. To visualize proteins resolved on gels, some gels were fixed and stained with silver stain using the Silver Stain Kit and the protocol provided by the manufacturer (Bio-Rad).
To examine the effect of phosphorylation by PKC-α on F1F0-ATPase catalytic activity, the phosphorylation assay was performed as described above using nonradioactive ATP and the enzymatic activity of F1F0-ATPase was determined in the reaction mixture by measuring the release of Pi from ATP (25).
Immunoblot analysis was used to assess protein levels of PKC-α, PKC-β, PKC-μ, phospho-PKC-α, phospho-PKC-β, phospho-PKC-μ, PKC-phosphorylated serine, phosphothreonine, and F1F0-ATPase subunits in total RPTC homogenates, mitochondria, and F1F0-ATPase preparations. Samples were lysed and boiled for 5 min in Laemmli sample buffer, and proteins were separated by SDS-PAGE and transferred electrophoretically to a nitrocellulose membrane. Blots were blocked in Tris-buffered saline (TBS) containing 0.5% casein and 0.1% Tween 20 (blocking buffer) and incubated overnight at 4°C in the presence of primary antibodies diluted in the blocking buffer. After being washed in TBS containing 0.05% Tween 20 (TBS-T), the membranes were incubated for 1 h in the presence of secondary IgGs coupled to horseradish peroxidase and washed again in TBS-T. The supersignal chemiluminescent system was used for protein detection. Quantification of the results obtained from immunoblotting and autoradiography was performed using scanning densitometry.
RPTC monolayers were washed twice with ice-cold PBS, scraped off the dishes, lysed in ice-cold modified RIPA buffer for 15 min at 4°C, and spun down at 14,000 rpm for 10 min at 4°C. The supernatants (500 μg protein) were precleared with a protein A/G agarose bead slurry for 1 h and incubated overnight (both at 4°C) with the monoclonal antibody specific to the β-subunit of the F1F0-ATPase (5 μg). Nonimmune mouse IgG was used as a negative control. The protein A/G agarose bead slurry was added to capture the immunocomplexes, and the incubation was continued for an additional 1 h at 4°C. Protein A/G agarose-attached immunocomplexes were harvested by centrifugation at 14,000 rpm at 4°C for 10 min and washed four times with ice-cold RIPA buffer. The final pellet was resuspended in Laemmli buffer, boiled for 5 min to dissociate immunocomplexes from protein A/G agarose beads, spun down at 14,000 rpm for 2 min at 4°C, and the supernatant was used for immunoblotting with antibodies specific to 1) PKC-phosphorylated serine, 2) phosphothreonine, and 3) the β-subunit of the F1F0-ATPase. Antibody against PKC-phosphorylated serine detects phosphorylated serine residues when they are within a PKC consensus motif.
DNA content was determined using a CyQUANT Cell Proliferation Assay Kit (Molecular Probes) and the manufacturer's protocol. RPTC samples were prepared for DNA assay as described previously (32). Protein concentration in all samples was determined using bicinchoninic acid assay with bovine serum albumin as the standard.
Statistical analysis.
Data are presented as means ± SE and were analyzed for significance by ANOVA. Multiple means were compared using Fisher's protected least significance difference test with a level of significance of P < 0.05. RPTC isolated from an individual rabbit represented one experiment (n = 1) consisting of data obtained from 2 to 10 culture plates.
DCVC-induced injury in RPTC.
Exposure of confluent RPTC monolayers to 200 μM DCVC for 90 min resulted in 30 and 43% cell loss at 4 and 24 h following the treatment, as indicated by the monolayer DNA content (26.3 ± 7.9 and 21.6 ± 3.0 μg/plate at 4 and 24 h, respectively, following DCVC treatment vs. 37.7 ± 3.0 μg/plate in controls). Monolayer DNA content in DCVC-injured RPTC did not recover during the 4 days following injury (23.3 ± 3.8 μg/plate in DCVC-injured RPTC vs. 40.8 ± 3.3 μg/plate in controls). However, visual inspection under a microscope showed that monolayer confluence recovered by day 4 following DCVC treatment (data not shown). PKC-α inhibitor Go6976 had no effect on DNA content in control or DCVC-injured RPTC (21 and 46% cell loss at 4 and 24 h, respectively).
Activation of PKC-α during RPTC repair following DCVC injury.
Figure 1 shows that the recovery of RPTC following DCVC exposure was associated with the activation of PKC-α. The ratio of phosphorylated PKC-α to total PKC-α in RPTC homogenates increased 4- and 2.5-fold on days 2 and 4, respectively, of the recovery period (Fig. 1D). Go6976 abolished the activation of PKC-α in DCVC-treated RPTC at all time points studied (Fig. 1A). DCVC-induced injury had no effect on phosphorylation of PKC-β (Fig. 1C) and PKC-μ (data not shown).
Activation of mitochondrial PKC-α during RPTC repair following DCVC-induced injury.
Our previous report demonstrated that PKC-α is present in RPTC mitochondria (25). The present data show that the lack of recovery of mitochondrial function following DCVC-induced injury is associated with increased levels of active PKC-α in RPTC mitochondria (Fig. 2A). The ratio of phosphorylated PKC-α to total PKC-α in the mitochondria isolated from DCVC-injured RPTC was not changed during the early recovery period (1–4 h) following the injury (Fig. 2). However, on day 4 of the recovery period, the ratio of phosphorylated PKC-α to total PKC-α was threefold higher in mitochondria of DCVC-injured RPTC than in controls (Fig. 2, B and C). Treatment of RPTC with Go6976 had no effect on the ratio of phosphorylated PKC-α to total PKC-α at 4 h following DCVC exposure but it prevented the activation of PKC-α on day 4 (Fig. 2, B and C). These results show that mitochondrial PKC-α is activated during the late recovery period following DCVC injury and that PKC-α inhibitor Go6976 prevents the activation of mitochondrial PKC-α.
Mitochondrial respiration.
Basal and uncoupled Qo2s were used as markers of mitochondrial respiration. Specifically, basal Qo2 reflects the overall function of the mitochondria. The basal Qo2 was decreased by 50% at 4 h following DCVC treatment and did not recover over time (Fig. 3A). The inhibition of PKC-α activation by Go6976 did not affect DCVC-induced decreases in basal Qo2 but promoted the recovery of basal Qo2 in DCVC-injured RPTC on day 4 (Fig. 3A).
Uncoupled Qo2 was used as a marker of the mitochondrial electron transfer rate and the integrity of complexes of the respiratory chain. Uncoupled Qo2 declined by 60% in DCVC-treated RPTC at 4 h following the exposure and remained decreased on day 4 of the recovery period (Fig. 3B). The inhibition of PKC-α activation by Go6976 promoted the recovery of uncoupled Qo2 in DCVC-injured RPTC (Fig. 3B).
These results demonstrate that basal respiration and the electron transfer rate in mitochondria do not recover in DCVC-injured RPTC and that inhibition of PKC-α activation promotes the return of these functions.
Mitochondrial respiration results in the generation of proton and pH gradients across the inner mitochondrial membrane and produces the transmembrane potential (ΔΨm), which represents most of the energy of the proton gradient. ΔΨm In RPTC was assessed by the measurement of changes in the amount of JC-1 aggregates (red fluorescence) and J-aggregate/JC-1 monomer ratio. The amount of JC-1 aggregates decreased in DCVC-injured RPTC at 24 h following the treatment (Fig. 4A). The J-aggregate/JC-1 monomer ratio in control RPTC was 1.29 ± 0.20, decreased by 52.8% in DCVC-injured RPTC at 24 h following the treatment, and returned to control levels by day 4 (Fig. 4B). Go6976 had no effects on ΔΨm in control or DCVC-injured RPTC (Fig. 4B). These results show that DCVC-induced decreases in ΔΨm recover over time and that the decrease and recovery of ΔΨm following DCVC injury are not mediated by PKC-α.
ATP production.
The rate of ATP synthesis was measured during state 3 (the maximal capacity of mitochondria to generate ATP) in digitonin-permeabilized RPTC incubated in the presence of ADP and metabolic substrates linked to the respiratory complex I (glutamate + malate). The rate of ATP production in control RPTC was 67.4 ± 10.6 nmol/mg protein. Treatment with Go6976 for 4 h and 4 days had no effect on the ATP production rate (Fig. 5A). ATP production was decreased 32% at 4 h following DCVC exposure and did not recover on day 4 (Fig. 5A). However, treatment with Go6976 during the recovery period promoted the return of the ATP synthesis rate (Fig. 5A). These results demonstrate that PKC-α activation following DCVC-induced injury in RPTC mediates decreases in ATP production.
Intracellular ATP.
As shown in Fig. 5B, intracellular ATP content decreased by 32 and 40%, respectively, at 4 and 24 h and did not recover on day 4 following DCVC treatment. Treatment with Go6976 during the recovery period did not promote the return of intracellular ATP in DCVC-injured RPTC (Fig. 5B). These results show that despite the return of the ATP synthesis rate, inhibition of PKC-α activation following DCVC-induced injury does not result in the recovery of intracellular ATP content.
Oxidative phosphorylation.
Oxidative phosphorylation and ATP synthesis are the fundamental functions of mitochondria. Oligomycin-sensitive Qo2 was used as a marker of oxidative phosphorylation in RPTC. Oligomycin-sensitive Qo2 declined 69% at 4 h following DCVC treatment and did not return throughout the recovery period (Fig. 6). Inhibition of PKC-α activation had no effect on the decreases in oligomycin-sensitive Qo2 at 4 h but promoted, in part, the return of oligomycin-sensitive Qo2 on day 4 following DCVC-induced injury (Fig. 6). These results show that oxidative phosphorylation does not recover in DCVC-injured RPTC and that PKC-α activation mediates the inhibition of the recovery of oxidative phosphorylation in this model.
F1F0-ATPase activity.
F1F0-ATPase catalyzes ATP synthesis using the energy of the proton gradient across the inner mitochondrial membrane. Exposure of RPTC to DCVC decreased activity of F1F0-ATPase by 41% at 4 h following the treatment (Fig. 7A). F1F0-ATPase activity remained decreased over the 4-day recovery period (Fig. 7A). Inhibition of PKC-α activation by Go6976 had no effect on the F1F0-ATPase activity at 4 h following DCVC exposure but promoted full recovery of the F1F0-ATPase activity on day 4 (Fig. 7A). These data demonstrate that the lack of recovery of F1F0-ATPase activity in DCVC-injured RPTC is mediated by PKC-α.
F1F0-ATPase protein levels.
Decreases in F1F0-ATPase activity can be due to reduced enzymatic activity or decreased protein levels of the F1F0-ATPase. F1F0-ATPase is composed of multiple subunits and several regulatory peptides associated with these subunits. Because the β-subunit is the catalytic subunit of F1F0-ATPase, we determined protein levels of the β-subunit in cell homogenates and mitochondria isolated from DCVC-injured RPTC during the recovery period. As demonstrated in Fig. 7, B and C, protein levels of the β-subunit of F1F0-ATPase in cell homogenates and mitochondria were not changed after DCVC-induced injury or throughout the recovery period. The inhibition of PKC-α by Go6976 had no effect on protein levels of the β-subunit of F1F0-ATPase in mitochondria isolated from control and DCVC-injured RPTC (Fig. 7C). These results demonstrate that DCVC does not decrease protein levels of the catalytic subunit of F1F0-ATPase and suggest that the decreases in F1F0-ATPase activity in DCVC-injured RPTC are due to the reduced catalytic activity of F1F0-ATPase and not the amount of the enzyme.
Phosphorylation of F1F0-ATPase by PKC-α in vitro.
PKC, a serine/threonine kinase, phosphorylates a wide range of intracellular substrates. Several F1F0-ATPase subunits, including the β-subunit (Fig. 8), have PKC consensus amino acid motifs. An in vitro assay was performed to determine whether F1F0-ATPase subunits are phosphorylated by major PKC isozymes present in RPTC. Phosphorylation of F1F0-ATPase by recombinant PKC isozymes was determined using an in vitro kinase assay followed by liquid scintillation spectrometry. Among the three isozymes tested in this study, PKC-α, PKC-δ, and PKC-ε, only PKC-α phosphorylated F1F0-ATPase in vitro (Fig. 9A).
To determine which subunits of F0F1-ATPase are phosphorylated by PKC-α, the reaction mixture was subjected to SDS-PAGE and autoradiography following the phosphorylation assay in vitro. The F1F0-ATPase isolated for this study consisted of three major (molecular mass ∼58, 56, and 26 kDa) and two minor (molecular mass ∼46 and 40 kDa) proteins as revealed by SDS-PAGE and silver staining (Fig. 9B). Autoradiography demonstrated that PKC-α phosphorylated two of these proteins (molecular mass ∼56 and 26 kDa; Fig. 9C). To determine whether the 56-kDa protein represented the β-subunit of F1F0-ATPase (molecular mass 56.6 kDa), the F1F0-ATPase preparation was subjected to SDS-PAGE simultaneously with the proteins following the phosphorylation assay and subjected to immunoblotting. As shown in Fig. 9D, the antibody to the β-subunit of F1F0-ATPase detected a 56-kDa protein corresponding to the radioactive 56-kDa band. The identity of the phosphorylated 26-kDa band is not known at present. Thus these data demonstrate that the β-subunit of F1F0-ATPase is a PKC-α but not PKC-δ or PKC-ε substrate.
An in vitro assay was carried out to determine whether phosphorylation of F1F0-ATPase by PKC-α alters F1F0-ATPase activity. Figure 9E shows that the activity of F1F0-ATPase decreased 53% in the presence of active PKC-α and ATP. These results suggest that phosphorylation by PKC-α has an inhibitory effect on F1F0-ATPase.
Phosphorylation of F1F0-ATPase by PKC-α in DCVC-injured RPTC.
Immunoprecipitation was used to determine 1) whether phosphorylation of the β-subunit of F1F0-ATPase occurs in DCVC-injured RPTC and 2) whether the β-subunit of F1F0-ATPase is phosphorylated by PKC-α following DCVC exposure. The β-subunit of F1F0-ATPase was immunoprecipitated from RPTC lysates and the serine and threonine phosphorylation of the β-subunit was examined at various time points following DCVC injury. Phosphothreonine levels in the β-subunit were not affected by DCVC and Go6976 treatments throughout the recovery period (Fig. 10B, data not shown). Phosphoserine PKC substrate antibody used for immunoblotting detects only phosphorylated serine residues that are within a PKC consensus motif. The phosphoserine levels on the β-subunit of F1F0-ATPase were not altered at 1, 2, and 4 h but decreased at 8 h of the recovery period in DCVC-injured RPTC (Fig. 10A, data not shown). However, on day 4 of the recovery period, phosphoserine levels on the β-subunit of F1F0-ATPase increased in DCVC-injured RPTC (Fig. 10A). PKC-α inhibitor Go6976 decreased the serine phosphorylation of the β-subunit of F1F0-ATPase on day 4 of the recovery period (Fig. 10A). Thus these results show that PKC-α activation in RPTC during late recovery following DCVC exposure increases phosphorylation of serine(s) but not threonine(s) on the β-subunit of F1F0-ATPase.
After nephrotoxicant exposure, the sublethally injured RPTC must undergo repair process to restore the physiological functions of the kidney. The repair process is composed of several stages including loss of the lethally injured cells, dedifferentiation of the sublethally injured cells, proliferation and morphological recovery of the damaged area, and differentiation to restore the physiological functions of renal proximal tubules (39). Despite the fact that numerous studies have been performed in the past to seek molecules that may accelerate regeneration following acute renal failure, the mechanisms regulating the recovery of renal functions are largely unknown. The aim of the present study was to determine whether PKC-α is involved in these mechanisms.
Previously, we showed that DCVC produces significant RPTC death and loss. The remaining sublethally injured RPTC survive the insult but neither proliferate nor regain differentiated functions (29). Our present and previous studies show that DCVC exposure produces sustained mitochondrial dysfunction in sublethally injured RPTC and that mitochondrial function does not recover within 6 days following DCVC exposure (23, 26, 28, 29). In contrast, RPTC injury induced by an oxidant is followed by full recovery of mitochondrial and transport functions (27).
The decrease in cellular functions of the surviving RPTC was accompanied by sustained activation of PKC-α on days 2 and 4 of the recovery period following DCVC treatment. The finding that the PKC-α inhibitor Go6976 (10 nM) blocked DCVC-induced activation of PKC-α but was not toxic in RPTC validated the use of this inhibitor in our study. It is known that, at higher concentrations, Go6976 can also inhibit other PKC isozymes such as PKC-β and PKC-μ. However, these isozymes were not activated in RPTC at any time point during the injury or the recovery of DCVC-injured RPTC. Inibition of PKC-α activation by Go6976 did not affect the DCVC-induced cell loss or decreases in mitochondrial functions, which showed that Go6976 is not toxic in RPTC. The initial (at 4 h) decreases in mitochondrial function in DCVC-injured RPTC were not accompanied by PKC-α activation, which suggests that PKC-α does not play a role in the initial decline of mitochondrial function. However, our results demonstrated that the lack of mitochondrial repair was associated with the activation of PKC-α and that attenuation of PKC-α activation during the 4-day recovery period promoted the return of overall mitochondrial function (improved basal Qo2). Specifically, the inhibition of PKC-α activation promoted recovery of the electron transfer rate and oxidative phosphorylation (improved uncoupled Qo2 and oligomycin-sensitive Qo2). These results suggest that PKC-α is involved in the inhibition of recovery of these functions in sublethally injured RPTC. Similar to the previous studies (7, 38), we show that DCVC decreases ΔΨm in RPTC. In contrast to other mitochondrial functions, ΔΨm recovered on day 4 following DCVC injury. Furthermore, inhibition of PKC-α activation did not affect the regain of ΔΨm, suggesting that PKC-α inhibits the repair of mitochondrial functions by mechanisms independent of ΔΨm.
Because the inhibition of PKC-α activation promoted the recovery of oligomycin-sensitive Qo2 in DCVC-injured RPTC, we hypothesized that PKC-α may target the F1F0-ATPase. Oligomycin-sensitive Qo2 and F1F0-ATPase activity decreased following DCVC treatment and remained at the reduced levels throughout the recovery period. Inhibition of PKC-α activation did not affect the initial reductions in F1F0-ATPase activity following DCVC treatment but promoted the recovery of the enzyme activity on day 4. Therefore, our data suggest that PKC-α does not play a role in the initial inhibition of F1F0-ATPase but negatively regulates the enzyme activity during the repair period after DCVC exposure. This conclusion is consistent with the observation that PKC-α activation in mitochondria does not occur during the early time points following DCVC exposure.
PKC-α has a variety of intracellular targets and regulates multiple processes including gene transcription and translation (11). It is likely that PKC-α inhibits transcription and translation of F1F0-ATPase subunits in DCVC-injured RPTC. Therefore, we examined whether DCVC injury alters the protein levels of the catalytic (β) subunit of F1F0-ATPase in RPTC. The protein levels of the β-subunit of F1F0-ATPase in both cell homogenates and mitochondria in DCVC-treated RPTC remained unchanged during the whole recovery period. This indicated that the decreases in F1F0-ATPase activity following DCVC injury were not due to decreased protein levels of this enzyme. Thus, our data suggested that F1F0-ATPase activity may be regulated by posttranslational modification of one or more subunits of F1F0-ATPase leading to alterations in catalytic activity. Protein phosphorylation, a posttranslational modification producing changes in conformation and enzymatic activity, may represent a mechanism regulating F1F0-ATPase activity. Indeed, tyrosine phosphorylation of the δ-subunit of F1F0-ATPase was described previously in cortical neurons following treatment with platelet-derived growth factor, but the functional consequences of this phosphorylation are unknown (42).
We performed analysis of amino acid sequences on several F1F0-ATPase subunits. The results suggested the existence of several PKC consensus motifs on yeast subunit ε, human subunit α, coupling factor 6, and β-subunit from Chaetosphaeridium globosum. Therefore, we tested whether PKC targets F1F0-ATPase directly by phosphorylating one or more subunits. Our results show that recombinant PKC-α catalyzed incorporation of 32P to the β-subunit of F1F0-ATPase under in vitro conditions. In contrast, PKC-δ and PKC-ε did not phosphorylate F1F0-ATPase under the same in vitro conditions. Thus our data suggest that PKC-α-mediated phosphorylation of the catalytic β-subunit plays a role in the regulation of F1F0-ATPase activity. To determine whether the β-subunit of F1F0-ATPase is phosphorylated by PKC-α following DCVC exposure in RPTC, we carried out an immunoprecipitation of this subunit from RPTC lysates and examined its phosphorylation status. Phosphoserine levels on the β-subunit of F1F0-ATPase were increased in DCVC-injured RPTC on day 4 but not during the early recovery period. PKC-α inhibition abolished phosphorylation of the β-subunit on day 4, which is consistent with the activation and involvement of PKC-α in the inhibition of F1F0-ATPase activity in the late recovery period. In contrast, the phosphothreonine levels remained unchanged in DCVC-injured RPTC. Thus our results show that PKC-α activation in RPTC during late recovery following DCVC exposure results in phosphorylation of serine(s) but not threonine(s) on the β-subunit of F1F0-ATPase. The data also suggest that PKC-α-mediated phosphorylation negatively regulates F1F0-ATPase activity. Indeed, catalytic activity of F1 portion of F1F0-ATPase is decreased following phosphorylation by PKC-α in vitro. However, the effect of phosphorylation on the activity of F1F0-ATPase in the cell is not known at present. Our data suggest that F1F0-ATPase phosphorylation decreases enzymatic activity and contributes to the lack of recovery of oxidative phosphorylation in DCVC-injured RPTC and that PKC-α is the isozyme responsible for the modification of F1F0-ATPase activity in our model. Because our results are based on the use of a pharmacological PKC-α inhibitor, another approach such as overexpression of dominant positive and negative PKC-α is necessary to confirm the role of PKC-α in phosphorylation and regulation of F1F0-ATPase activity in DCVC-injured RPTC.
The recovery of oxidative phosphorylation and overall mitochondrial function depends on the repair of electron transport. Previous reports demonstrated that DCVC alters the concentration of several citric acid cycle intermediates, reduces the activity of isocitrate dehydrogenase, and inhibits succinate:cytochrome c oxidoreductase and succinate-linked state 3 respiration, suggesting that DCVC targets electron transport in RPTC (1, 15). Furthermore, our previous data showed that electron transport rate does not recover in DCVC-injured RPTC (29). These results show that attenuation of PKC-α activation partially promotes the recovery of the electron transport rate in DCVC-injured RPTC. Thus our results suggest that, in addition to phosphorylating F1F0-ATPase, PKC-α inhibits the repair of mitochondrial function also by decreasing the electron transport rate. Although inhibition of PKC-α activation promoted the recovery of mitochondrial respiration and ATP production, it did not restore the intracellular ATP content in RPTC. The reason for this apparent dilemma is unknown at present. Because the intracellular ATP content reflects a net difference between synthesis and consumption of ATP, it is conceivable that the inhibition of PKC-α activation also promoted the repair of energy-consuming processes such as active ion transport and thus increased ATP consumption.
The present study shows that the inhibition of PKC-α only partially promoted the recovery of mitochondrial function, indicating that other mechanisms are also involved in the lack of the repair of mitochondrial functions in DCVC-injured RPTC. The other mechanisms may include the growth factor receptor-mediated mechanisms that result in activation of tyrosine kinase and phosphorylation of tyrosine residues in target proteins (40). Indeed, tyrosine phosphorylation of the δ-subunit of F0F1-ATPase was described in cortical neurons treated with platelet-derived growth factor (42). Furthermore, our previous studies demonstrating that epidermal growth factor promotes recovery of mitochondrial function and active Na+ transport following DCVC injury support the role of growth factor receptor- and tyrosine kinase-mediated mechanisms in the repair of mitochondrial function (29). Thus it is very likely that the recovery of mitochondrial function is under control of more than one signaling pathway.
In conclusion, the lack of repair of mitochondrial function following DCVC exposure is associated with activation of PKC-α, including mitochondrial PKC-α. PKC-α activation during the repair period in DCVC-injured RPTC mediates the inhibition of the recovery of the electron transfer rate, oxidative phosphorylation, ATP production, and F1F0-ATPase activity. Furthermore, our data show that PKC-α phosphorylates the β-subunit of F1F0-ATPase both under in vitro conditions and in recovering RPTC and that in vitro phosphorylation of F1F0-ATPase decreases its catalytic activity. These results suggest that phosphorylation of the catalytic β-subunit of F1F0-ATPase by PKC-α represents a mechanism regulating F1F0-ATPase activity and the repair of mitochondrial function following toxicant-induced injury in RPTC.
This work was supported by National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-59558.
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- Copyright © 2004 the American Physiological Society