adrenaline , glucagon , glp-1 , 25-dideoxyadenosine ( dda ) , egta , poly - l - lysine , 3-isobutylmethylxanthine ( ibmx ) , somatostatin , and hepes were from sigma . catechol estrogen ( ce ) [ 1,3,5(10)-estratrien-2,3,17--triol ] for ac inhibition and the inactive control compound 1,3,5(10)-estratrien-3,17--triol were from makaira limited ( london , u.k . ) . j. buck and l. levin , weill medical college of cornell university , new york , ny . the acetoxymethyl esters of the ca indicators fura - pe3 and fura red were obtained from teflabs ( austin , tx ) and invitrogen , respectively . when the pancreas was excised , the lower duodenal part was omitted to reduce contribution of islets with cells producing pancreatic polypeptide ( 37 ) . after isolation , the islets were cultured for 14 days in rpmi-1640 medium containing 5.5 mmol / l glucose , 10% fetal calf serum , 100 g / ml penicillin , and 100 g / ml streptomycin at 37c in an atmosphere of 5% co2 in humidified air . some experiments were performed on single cells prepared by shaking the freshly isolated islets in a ca - deficient medium followed by attachment to coverslips during 25 days of culture . if not otherwise stated , data were obtained with cells from at least three independent islet isolations . where indicated , experiments were performed on insulin - secreting min6 -cells cultured as previously described ( 12 ) . for [ camp]pm measurements , the islets or cells were infected with camp biosensor - encoding adenoviruses using 10 fluorescence forming units ( ffu)/islet or 60 ffu / cell as previously described ( 12 ) . it was verified that the virus infection did not affect the islet [ ca]i response to glucose stimulation . the islets or cells were transferred to a buffer containing ( in mmol / l ) 125 nacl , 4.8 kcl , 1.3 cacl2 , 1.2 mgcl2 , 3 glucose , and 25 hepes ( ph adjusted to 7.40 with naoh ) and incubated for 30 min at 37c . in some experiments , this medium was supplemented with 20 mmol / l nahco3 after equimolar reduction of nacl . for ca recordings , the cells were preincubated in the presence of 1 mol / l of the acetoxymethyl ester of fura - pe3 and islets with 10 [ ca]i imaging with fura - pe3 was performed at 37c with an epifluorescence microscope setup as previously described ( 5 ) . measurements of the cytoplasmic ca concentration beneath the plasma membrane ( [ ca]pm ) were made with fura red in a total internal reflection fluorescence ( tirf ) setup consisting of an eclipse ti microscope with a 60 1.45 na - objective ( nikon ) , using 488 nm excitation with fluorescence detection at > 630 nm . using the same tirf system , [ camp]pm was measured in intact islets or cells expressing the camp translocation biosensor as previously described ( 12 ) . image pairs were acquired every 5 s using a back - illuminated emccd camera ( du-887 ; andor technology , belfast , northern ireland ) controlled by metafluor software ( molecular devices , downingtown , pa ) . the camp concentration was expressed as the background - corrected cyan fluorescent protein  to  yellow fluorescent protein ( cfp - to - yfp ) ratio . the basal ratio was normalized to unity for compensation for the variability in expression levels . individual cells in the experimental chamber were identified by immunostaining for insulin or glucagon . following the fluorescence recordings , the cells were rinsed with 25 , 50 , and 75% ethanol and eventually fixed in 95% ethanol for 5 min . after sequential rinsing with 3% h2o2 and tris buffer ( 0.05 mol / l , ph 7.4 ) , serum - free protein block ( dako ) was added to reduce background staining . polyclonal rabbit anti - glucagon or guinea pig anti - insulin ( 1:100 ; dako ) was added after 10 min and allowed to incubate for 30 min . after rinsing with tris buffer , the mach 3 rabbit probe alkaline phosphatase polymer kit ( biocare medical , concord , ca ) was used for visualization according to the manufacturer s instructions . after rinsing with distilled water , cell nuclei were stained with hematoxylin ( histolab ) for 0.52 min . hepes buffer ( 100 l ) supplemented with 3.3 mmol / l ( 94 gbq / mol ) or 11.1 mmol / l ( 28 gbq / mol ) d-[u - c]glucose , were incubated with or without test compounds for 90 min at 37c under an atmosphere of 95/5% o2/co2 during slow shaking . metabolism was then stopped by addition of 17 mol / l antimycin a ( sigma - aldrich ) in ethanol . the generated co2 was trapped in 250 l hyamine 10 ( perkin - elmer ) during incubation at 37c for 2 h as previously described ( 38 ) . radioactivity was measured in a liquid scintillation counter after addition of 5 ml ultima gold scintillation fluid ( perkin - elmer ) . [ camp]pm and [ ca]i response magnitudes were calculated from time - average data obtained before and during the stimulation period . when the pancreas was excised , the lower duodenal part was omitted to reduce contribution of islets with cells producing pancreatic polypeptide ( 37 ) . after isolation , the islets were cultured for 14 days in rpmi-1640 medium containing 5.5 mmol / l glucose , 10% fetal calf serum , 100 g / ml penicillin , and 100 g / ml streptomycin at 37c in an atmosphere of 5% co2 in humidified air . some experiments were performed on single cells prepared by shaking the freshly isolated islets in a ca - deficient medium followed by attachment to coverslips during 25 days of culture . if not otherwise stated , data were obtained with cells from at least three independent islet isolations . where indicated , experiments were performed on insulin - secreting min6 -cells cultured as previously described ( 12 ) . for [ camp]pm measurements , the islets or cells were infected with camp biosensor - encoding adenoviruses using 10 fluorescence forming units ( ffu)/islet or 60 ffu / cell as previously described ( 12 ) . it was verified that the virus infection did not affect the islet [ ca]i response to glucose stimulation . the islets or cells were transferred to a buffer containing ( in mmol / l ) 125 nacl , 4.8 kcl , 1.3 cacl2 , 1.2 mgcl2 , 3 glucose , and 25 hepes ( ph adjusted to 7.40 with naoh ) and incubated for 30 min at 37c . in some experiments , this medium was supplemented with 20 mmol / l nahco3 after equimolar reduction of nacl . for ca recordings immediately after preincubation , the islets were applied onto a poly - l - lysine  coated 25-mm coverslip and allowed to attach . [ ca]i imaging with fura - pe3 was performed at 37c with an epifluorescence microscope setup as previously described ( 5 ) . measurements of the cytoplasmic ca concentration beneath the plasma membrane ( [ ca]pm ) were made with fura red in a total internal reflection fluorescence ( tirf ) setup consisting of an eclipse ti microscope with a 60 1.45 na - objective ( nikon ) , using 488 nm excitation with fluorescence detection at > 630 nm . using the same tirf system , [ camp]pm was measured in intact islets or cells expressing the camp translocation biosensor as previously described ( 12 ) . image pairs were acquired every 5 s using a back - illuminated emccd camera ( du-887 ; andor technology , belfast , northern ireland ) controlled by metafluor software ( molecular devices , downingtown , pa ) . the camp concentration was expressed as the background - corrected cyan fluorescent protein  to  yellow fluorescent protein ( cfp - to - yfp ) ratio . the basal ratio was normalized to unity for compensation for the variability in expression levels . individual cells in the experimental chamber were identified by immunostaining for insulin or glucagon . following the fluorescence recordings , the cells were rinsed with 25 , 50 , and 75% ethanol and eventually fixed in 95% ethanol for 5 min . after sequential rinsing with 3% h2o2 and tris buffer ( 0.05 mol / l , ph 7.4 ) , serum - free protein block ( dako ) was added to reduce background staining . polyclonal rabbit anti - glucagon or guinea pig anti - insulin ( 1:100 ; dako ) was added after 10 min and allowed to incubate for 30 min . after rinsing with tris buffer , the mach 3 rabbit probe alkaline phosphatase polymer kit ( biocare medical , concord , ca ) was used for visualization according to the manufacturer s instructions . the reaction was stopped by addition of tris buffer . after rinsing with distilled water , cell nuclei hepes buffer ( 100 l ) supplemented with 3.3 mmol / l ( 94 gbq / mol ) or 11.1 mmol / l ( 28 gbq / mol ) d-[u - c]glucose , were incubated with or without test compounds for 90 min at 37c under an atmosphere of 95/5% o2/co2 during slow shaking . metabolism was then stopped by addition of 17 mol / l antimycin a ( sigma - aldrich ) in ethanol . the generated co2 was trapped in 250 l hyamine 10 ( perkin - elmer ) during incubation at 37c for 2 h as previously described ( 38 ) . radioactivity was measured in a liquid scintillation counter after addition of 5 ml ultima gold scintillation fluid ( perkin - elmer ) . [ camp]pm and [ ca]i response magnitudes were calculated from time - average data obtained before and during the stimulation period . figure 1a illustrates tirf measurements on peripheral mouse islet cells expressing the camp biosensor , and fig . the cfp - to - yfp ratio was usually stable in peripheral islet cells exposed to 3 mmol / l glucose ( fig . cell size and adrenaline responses have previously been used to discriminate between - and -cells because only the relatively small -cells expressing 1- and -adrenoceptors ( 24 ) respond with intracellular mobilization of ca ( 39 ) . because the -adrenoceptors mediate camp elevation in -cells and the larger -cells express camp - lowering 2-adrenoceptors ( 40 ) , we tested whether the camp response to adrenaline could be used for cell identification . this was indeed the case , and all of 25 cells with small tirf footprints that responded to adrenaline with [ camp]pm elevation stained positively for glucagon . in contrast , all of 30 cells with large footprint and negative adrenaline response were insulin positive . figure 1c and d shows that an immune - identified -cell responded to adrenaline with elevation of [ camp]pm , whereas a -cell ( immunostaining not shown ) did not respond under these conditions with basal [ camp]pm levels . the [ camp]pm - lowering effect of adrenaline on -cells became apparent under conditions where the levels of camp were elevated ( fig . tirf imaging of intact pancreatic islets shows that adrenaline induces differential effects on [ camp]pm in - and -cells . a : principle for tirf imaging of an intact pancreatic islet attached to a coverslip . reflection of the laser excitation light at the coverglass - medium interface generates an evanescent field within an 100 nm zone above the interface , which will excite fluorescent molecules near the plasma membrane of the various islet cells that adhere to the coverslip . b : tirf microscopy images of a mouse pancreatic islet expressing the camp translocation biosensor composed of a pka catalytic c subunit fused to yfp ( c-yfp ) and a truncated pka regulatory rii subunit fused to a membrane - anchored cfp ( rii-cfp - caax ) . c : tirf microscopy recordings of [ camp]pm in isolated immunoidentified - and -cells exposed to 3 mmol / l glucose and 5 mol / l adrenaline . adrenaline increases [ camp]pm in the -cell but does not affect basal [ camp]pm in the -cell . d : immunostaining of dispersed islet cells showing that a small cell responding to adrenaline with [ camp]pm elevation in panel c is a glucagon - positive -cell . ( a high - quality digital representation of this figure is available in the online issue . ) tirf microscopy recordings of [ camp]pm in individual cells within an intact mouse pancreatic islet exposed to 3 mmol / l glucose . a : oscillations of [ camp]pm induced by 10 nmol / l glucagon in a -cell identified by the [ camp]pm - lowering effect of adrenaline ( adr ) . b : concentration - dependence of the glucagon - induced time - average [ camp]pm elevation in islet -cells . means c : [ camp]pm oscillations induced by 10 nmol / l glucagon in an islet -cell identified by the [ camp]pm - elevating effect of adrenaline . d : suppression of glucagon - induced [ camp]pm elevation in an adrenaline - identified -cell exposed to 100 nmol / l somatostatin ( sst ) . e and f : effect of 0.1 mol / l clonidine on [ camp]pm elevation induced by 50 nmol / l glucagon in a -cell ( e ) and -cell ( f ) identified by their responses to adrenaline . when mouse islets were exposed to glucagon in the presence of 3 mmol / l glucose , there was a prompt increase in [ camp]pm with sustained elevation or oscillations in -cells ( 36 of 41 [ fig . , the integrated response was half - maximal and maximal at 80 and 1,000 nmol / l glucagon , respectively ( fig . 2b ) . similar responses were seen with the 2-adrenoceptor agonist clonidine , which lowered [ camp]pm in all of 11 -cells ( fig . 2f ) , indicating that both cell types express 2-receptors . however , adrenaline still caused elevation of [ camp]pm in the -cells , consistent with domination of -adrenoceptors in this cell type ( 40 ) . at concentrations of 0.510 3a and d ) . the oscillatory pattern varied between different cells with frequencies ranging from 0.18 to 2.1 min and amplitudes from 0.06 to 1.5 normalized ratio units . the amplitude and duration of individual oscillations tended to increase with glp-1 concentration , as illustrated in fig . high concentrations of glp-1 ( > 50 nmol / l ) induced prompt and sustained [ camp]pm elevation in -cells ( 19 of 23 ) that rapidly reversed upon omission of the peptide ( fig . figure 3c shows the concentration dependence of the integrated [ camp]pm response to glp-1 , with half - maximal and maximal effects at 30 and 300 nmol / l , respectively . the [ camp]pm oscillations induced by glp-1 were strikingly well synchronized among -cells in the islet even between those lacking direct contact ( fig . 3d ) . only occasional -cells responded to glp-1 with elevation of [ camp]pm ( 5 of 48 ) , and fig . 3e shows a graded response to 550 nmol / l glp-1 in one of these -cells identified with adrenaline . to investigate whether the lack of response in many -cells was because camp did not increase above the detection threshold , we exposed the cells to 5 mol / l ibmx , which is at the border to give a detectable increase of [ camp]pm . of 21 adrenaline - identified -cells , 10 responded to 5 mol / l ibmx . only two of those plus two of the ibmx - unresponsive -cells reacted to 100 nmol / l glp-1 , and the [ camp]pm increase was of a magnitude similar to that induced by adrenaline ( fig . tirf microscopy recordings of [ camp]pm in individual cells within an intact mouse pancreatic islet exposed to 3 mmol / l glucose . a : [ camp]pm oscillations in a -cell exposed to increasing concentrations of glp-1 ( the trace was interrupted between changes in glp-1 concentration ) . the amplitude and duration of individual oscillations tended to increase with the glp-1 concentration . b : stable and reversible [ camp]pm elevation in response to 100 nmol / l glp-1 in a -cell identified by the lack of adrenaline ( adr ) effect . c : dose dependence of the glp-1 induced time - average [ camp]pm elevation in islet -cells . means d : [ camp]pm oscillations induced by 1 nmol / l glp-1 are synchronized between -cells lacking apparent direct contact . the recordings are from the two cells in the tirf image encircled with broken lines . e : example of a rare -cell with dose - dependent [ camp]pm elevations in response to glp-1 . f : glp-1 response in an adrenaline - identified -cell exposed to 5 mol / l ibmx . ( a high - quality digital representation of this figure is available in the online issue . ) when the glucose concentration was elevated from 3 to 11 , 20 , or 30 mmol / l , most islet -cells responded with a pronounced rise of [ camp]pm followed by a stable plateau ( 16 of 41 ) or slow oscillations with frequencies in the 0.050.4/min range and amplitudes of 0.050.6 ratio units ( 21 of 41 ) ( fig . the remaining cells showed a single [ camp]pm peak followed by return to the baseline ( not shown ) . as previously described in isolated -cells ( 12 ) , the glucose - induced elevation of [ camp]pm was preceded by a small reduction in many cells . 4c . figure 4a  d also shows that adrenaline abolished the glucose - induced elevation of [ camp]pm . like the glp-1triggered oscillations of [ camp]pm , those induced by glucose were often synchronized among -cells within an islet ( fig . 4d ) . the glucose and hormone effects were additive , and the [ camp]pm elevation induced by a combination of 20 mmol / l glucose and 10 nmol / l glucagon or 100 nmol tirf microscopy recordings of [ camp]pm in individual cells within an intact mouse pancreatic islet . a and b : stable elevation ( a ) and oscillations ( b ) of [ camp]pm in response to a step increase of the glucose concentration from 3 to 11 or 20 mmol / l in -cells identified by the [ camp]pm - lowering effect of adrenaline ( adr ) . c : [ camp]pm recording showing that the initial glucose - induced [ camp]pm elevation in -cells is preceded by a slight lowering . d : synchronization of the glucose - induced [ camp]pm oscillations in two islet -cells lacking apparent direct contact . e : additive effect of 10 nmol / l glucagon and 20 mmol / l glucose ( 20 g ) on [ camp]pm in an islet -cell . f : means  sem for the average increases of [ camp]pm induced by 20 mmol / l glucose alone and in combination with 10 nmol / l glucagon or 100 nmol / l glp-1 . the amplitudes of the [ camp]pm oscillations were significantly suppressed , but the oscillations often persisted after removal of ca and addition of 2 mmol / l egta to prevent changes in [ ca]i ( 4 of 6 cells ) ( fig . the [ ca]i response of intact islets differs from that of isolated cells in displaying a pattern of rapid ( 24/min ) regular oscillations that depends not only on ca influx but also on release from intracellular stores ( 2 ) . corresponding rapid oscillations of [ camp]pm were never observed , even after elevating the extracellular ca concentration from 1.3 to 2.6 mmol / l ( data not shown ) , which typically promotes the appearance of the fast ca pattern . simultaneous measurements of [ camp]pm and [ ca]pm in islet -cells loaded with the fluorescent indicator fura red demonstrated that [ camp]pm and [ ca]pm elevations were synchronized ( fig . 5b ) , but the temporal relationship of the initial glucose - induced [ camp]pm and [ ca]pm elevations varied with [ camp]pm , sometimes increasing before ( fig . 5b ) [ ca]pm . together , these findings indicate that ca amplifies the [ camp]pm response but that [ ca]i oscillations are not essential for glucose - induced oscillations of [ camp]pm . glucose - induced [ camp]pm oscillations in islet -cells are not strictly dependent on ca . a : tirf microscopy recording from a mouse islet -cell showing that glucose - induced [ camp]pm oscillations persist in ca - deficient medium containing 2 mmol / l egta . b and c : simultaneous recordings of [ camp]pm ( black trace ) and [ ca]pm ( gray trace ) in islet -cells following a step increase of the glucose concentration from 3 to 20 mmol / l . cells typically exhibited coordinated oscillations of the two messengers ( b ) . [ ca]pm sometimes increased before ( b ) and sometimes after ( c ) [ camp]pm . islets expressing a modified camp sensor based on c-cfp translocation and nonfluorescent rii-caax were loaded with the ca indicator fura red . the fluorescence traces have been inverted to show increase of [ camp]pm and [ ca]pm as upward deflections . in both -cells within islets 6b ) , the glucose - induced [ camp]pm oscillations were completely inhibited by 30 mol / l kh7 , which has been described as a specific sac inhibitor ( 41 ) . similar inhibition was obtained with 100 mol / l of the tmac inhibitor dda in clonal ( fig . / l glp-1 was inhibited by both dda and kh7 ( n = 12 ) ( fig . however , whereas dda was without effect on glucose - induced [ ca]i oscillations , kh7 completely abolished this response ( n = 8) ( fig . 6e ) , indicating that the compound might interfere with upstream effects in glucose stimulus - secretion coupling . indeed , kh7 ( 30 mol / l ) markedly suppressed glucose oxidation in min6 cells . this effect could not be attributed to lowering of camp because the effect was not reversed by addition of 10 mmol / l of the membrane - permeable analog 8-br - camp . in contrast , a slight tendency of dda to inhibit glucose oxidation was completely reversed by 8-br - camp ( fig . glucose - induced [ camp]pm formation in min6 and islet -cells is mediated by transmembrane adenylyl cyclases . c : tirf recordings from mouse islet -cells ( a and c ) and a min6 cell ( b ) showing inhibition of glucose - induced [ camp]pm oscillations by 30 mol / l kh7 ( a and b ) and 100 mol d : tirf recording from an islet -cell showing that 100 mol / l dda and 30 mol / l kh7 suppresses the [ camp]pm response to 50 nmol / l glp-1 . e : epifluorescence recording of [ ca]i in a single mouse -cell showing that oscillations triggered by 20 mmol / l glucose are unaffected by 20 mol / l of the catechol estrogen compound [ 1,3,5(10)-estratrien-2,3,17 -triol ] ( ce ) and 100 mol / l dda but suppressed by 30 mol / l kh7 . f : glucose oxidation in min6 -cells exposed to 3 or 11 mmol / l glucose in the absence or presence of 30 mol / l kh7 , 10 mmol / l 8-br - camp , and 100 mol / l dda . means * p < 0.001 for difference between 3 and 11 mmol / l glucose . # p < 0.001 for difference from 11 mmol / l glucose . g : tirf recording from a single min6 -cell showing the effect of 20 mol / l ce on [ camp]pm oscillations induced by 11 mmol / l glucose . h : tirf recording of glucose - induced [ camp]pm oscillations in a single min6 -cell maintained in hco3-buffered medium and exposed to 20 mol / l ce and 100 mol / l dda . ce is an alternative inhibitor of sac ( 42 ) , and this compound was without effect on glucose - induced [ ca]i oscillations ( fig . in 60% of the min6 -cells , ce caused temporary interruption of the [ camp]pm oscillations , but after a few minutes , there were [ camp]pm oscillations indistinguishable from those under control conditions ( fig . the interruption probably was not mediated by changes in camp , given that it was observed also with the inactive control compound 1,3,5(10)-estratrien-3,17--triol ( data not shown ) . similar results were obtained when the experiments were performed in medium containing 20 mmol / l nahco3 , which promotes sac activity ( fig . [ camp]pm was low and stable in -cells identified by the adrenaline - induced [ camp]pm elevation . however , like in -cells , 1130 mmol / l glucose triggered [ camp]pm oscillations in many -cells ( 3 of 11 at 11 mmol / l , 13 of 28 at 20 mmol / l , and 8 of 17 at 30 mmol / l glucose ) ( fig . the nadirs and peaks of the oscillations reflected inhibition and stimulation , respectively , in relation to the baseline ( fig . 7b ) . in the few islets that permitted analysis of the phase relationship of [ camp]pm oscillations between different -cells ( n = 1 ) and between -cells and -cells ( n = 4 ) , there was no apparent synchronization of the oscillations ( fig . . a : oscillations of [ camp]pm in response to a step increase of the glucose concentration from 3 to 30 mmol / l in an islet -cell identified by [ camp]pm elevation in response to adrenaline ( adr ) . b : example of an islet -cell responding to a step increase of the glucose concentration from 3 to 20 mmol / l with [ camp]pm oscillations showing both stimulatory and inhibitory components . c and d : glucose - induced [ camp]pm oscillations recorded from two individual -cells ( c ) and one - and one -cell ( d ) within the same islet . figure 1a illustrates tirf measurements on peripheral mouse islet cells expressing the camp biosensor , and fig . the cfp - to - yfp ratio was usually stable in peripheral islet cells exposed to 3 mmol / l glucose ( fig . cell size and adrenaline responses have previously been used to discriminate between - and -cells because only the relatively small -cells expressing 1- and -adrenoceptors ( 24 ) respond with intracellular mobilization of ca ( 39 ) . because the -adrenoceptors mediate camp elevation in -cells and the larger -cells express camp - lowering 2-adrenoceptors ( 40 ) , we tested whether the camp response to adrenaline could be used for cell identification . this was indeed the case , and all of 25 cells with small tirf footprints that responded to adrenaline with [ camp]pm elevation stained positively for glucagon . in contrast , all of 30 cells with large footprint and negative adrenaline response were insulin positive . figure 1c and d shows that an immune - identified -cell responded to adrenaline with elevation of [ camp]pm , whereas a -cell ( immunostaining not shown ) did not respond under these conditions with basal [ camp]pm levels . the [ camp]pm - lowering effect of adrenaline on -cells became apparent under conditions where the levels of camp were elevated ( fig . tirf imaging of intact pancreatic islets shows that adrenaline induces differential effects on [ camp]pm in - and -cells . a : principle for tirf imaging of an intact pancreatic islet attached to a coverslip . reflection of the laser excitation light at the coverglass - medium interface generates an evanescent field within an 100 nm zone above the interface , which will excite fluorescent molecules near the plasma membrane of the various islet cells that adhere to the coverslip . b : tirf microscopy images of a mouse pancreatic islet expressing the camp translocation biosensor composed of a pka catalytic c subunit fused to yfp ( c-yfp ) and a truncated pka regulatory rii subunit fused to a membrane - anchored cfp ( rii-cfp - caax ) . c : tirf microscopy recordings of [ camp]pm in isolated immunoidentified - and -cells exposed to 3 mmol / l glucose and 5 mol / l adrenaline . adrenaline increases [ camp]pm in the -cell but does not affect basal [ camp]pm in the -cell . d : immunostaining of dispersed islet cells showing that a small cell responding to adrenaline with [ camp]pm elevation in panel c is a glucagon - positive -cell . ( a high - quality digital representation of this figure is available in the online issue . ) tirf microscopy recordings of [ camp]pm in individual cells within an intact mouse pancreatic islet exposed to 3 mmol / l glucose . a : oscillations of [ camp]pm induced by 10 nmol / l glucagon in a -cell identified by the [ camp]pm - lowering effect of adrenaline ( adr ) . b : concentration - dependence of the glucagon - induced time - average [ camp]pm elevation in islet -cells . means c : [ camp]pm oscillations induced by 10 nmol / l glucagon in an islet -cell identified by the [ camp]pm - elevating effect of adrenaline . d : suppression of glucagon - induced [ camp]pm elevation in an adrenaline - identified -cell exposed to 100 nmol / l somatostatin ( sst ) . e and f : effect of 0.1 mol / l clonidine on [ camp]pm elevation induced by 50 nmol / l glucagon in a -cell ( e ) and -cell ( f ) identified by their responses to adrenaline . when mouse islets were exposed to glucagon in the presence of 3 mmol / l glucose , there was a prompt increase in [ camp]pm with sustained elevation or oscillations in -cells ( 36 of 41 [ fig . the integrated response was half - maximal and maximal at 80 and 1,000 nmol / l glucagon , respectively ( fig . similar responses were seen with the 2-adrenoceptor agonist clonidine , which lowered [ camp]pm in all of 11 -cells ( fig . 2f ) , indicating that both cell types express 2-receptors . however , adrenaline still caused elevation of [ camp]pm in the -cells , consistent with domination of -adrenoceptors in this cell type ( 40 ) . at concentrations of 0.510 3a and d ) . the oscillatory pattern varied between different cells with frequencies ranging from 0.18 to 2.1 min and amplitudes from 0.06 to 1.5 normalized ratio units . the amplitude and duration of individual oscillations tended to increase with glp-1 concentration , as illustrated in fig . high concentrations of glp-1 ( > 50 nmol / l ) induced prompt and sustained [ camp]pm elevation in -cells ( 19 of 23 ) that rapidly reversed upon omission of the peptide ( fig . figure 3c shows the concentration dependence of the integrated [ camp]pm response to glp-1 , with half - maximal and maximal effects at 30 and 300 nmol / l , respectively . the [ camp]pm oscillations induced by glp-1 were strikingly well synchronized among -cells in the islet even between those lacking direct contact ( fig . 3d ) . only occasional -cells responded to glp-1 with elevation of [ camp]pm ( 5 of 48 ) , and fig . 3e shows a graded response to 550 nmol / l glp-1 in one of these -cells identified with adrenaline . to investigate whether the lack of response in many -cells was because camp did not increase above the detection threshold , we exposed the cells to 5 mol / l ibmx , which is at the border to give a detectable increase of [ camp]pm . of 21 adrenaline - identified -cells , 10 responded to 5 mol / l ibmx . only two of those plus two of the ibmx - unresponsive -cells reacted to 100 nmol / l glp-1 , and the [ camp]pm increase was of a magnitude similar to that induced by adrenaline ( fig . tirf microscopy recordings of [ camp]pm in individual cells within an intact mouse pancreatic islet exposed to 3 mmol / l glucose . a : [ camp]pm oscillations in a -cell exposed to increasing concentrations of glp-1 ( the trace was interrupted between changes in glp-1 concentration ) . the amplitude and duration of individual oscillations tended to increase with the glp-1 concentration . b : stable and reversible [ camp]pm elevation in response to 100 nmol / l glp-1 in a -cell identified by the lack of adrenaline ( adr ) effect . c : dose dependence of the glp-1 induced time - average [ camp]pm elevation in islet -cells . means d : [ camp]pm oscillations induced by 1 nmol / l glp-1 are synchronized between -cells lacking apparent direct contact . the recordings are from the two cells in the tirf image encircled with broken lines . e : example of a rare -cell with dose - dependent [ camp]pm elevations in response to glp-1 . f : glp-1 response in an adrenaline - identified -cell exposed to 5 mol / l ibmx . ( a high - quality digital representation of this figure is available in the online issue . ) when the glucose concentration was elevated from 3 to 11 , 20 , or 30 mmol / l , most islet -cells responded with a pronounced rise of [ camp]pm followed by a stable plateau ( 16 of 41 ) or slow oscillations with frequencies in the 0.050.4/min range and amplitudes of 0.050.6 ratio units ( 21 of 41 ) ( fig . the remaining cells showed a single [ camp]pm peak followed by return to the baseline ( not shown ) . as previously described in isolated -cells ( 12 ) , the glucose - induced elevation of [ camp]pm was preceded by a small reduction in many cells . 4c . figure 4a  d also shows that adrenaline abolished the glucose - induced elevation of [ camp]pm . like the glp-1triggered oscillations of [ camp]pm , those induced by glucose 4d ) . the glucose and hormone effects were additive , and the [ camp]pm elevation induced by a combination of 20 mmol / l glucose and 10 nmol / l glucagon or 100 nmol tirf microscopy recordings of [ camp]pm in individual cells within an intact mouse pancreatic islet . a and b : stable elevation ( a ) and oscillations ( b ) of [ camp]pm in response to a step increase of the glucose concentration from 3 to 11 or 20 mmol / l in -cells identified by the [ camp]pm - lowering effect of adrenaline ( adr ) . c : [ camp]pm recording showing that the initial glucose - induced [ camp]pm elevation in -cells is preceded by a slight lowering . d : synchronization of the glucose - induced [ camp]pm oscillations in two islet -cells lacking apparent direct contact . e : additive effect of 10 nmol / l glucagon and 20 mmol / l glucose ( 20 g ) on [ camp]pm in an islet -cell . f : means  sem for the average increases of [ camp]pm induced by 20 mmol / l glucose alone and in combination with 10 nmol / l glucagon or 100 nmol / l glp-1 . the amplitudes of the [ camp]pm oscillations were significantly suppressed , but the oscillations often persisted after removal of ca and addition of 2 mmol / l egta to prevent changes in [ ca]i ( 4 of 6 cells ) ( fig . the [ ca]i response of intact islets differs from that of isolated cells in displaying a pattern of rapid ( 24/min ) regular oscillations that depends not only on ca influx but also on release from intracellular stores ( 2 ) . corresponding rapid oscillations of [ camp]pm were never observed , even after elevating the extracellular ca concentration from 1.3 to 2.6 mmol / l ( data not shown ) , which typically promotes the appearance of the fast ca pattern . simultaneous measurements of [ camp]pm and [ ca]pm in islet -cells loaded with the fluorescent indicator fura red demonstrated that [ camp]pm and [ ca]pm elevations were synchronized ( fig . 5b ) , but the temporal relationship of the initial glucose - induced [ camp]pm and [ ca]pm elevations varied with [ camp]pm , sometimes increasing before ( fig . 5b ) [ ca]pm . together , these findings indicate that ca amplifies the [ camp]pm response but that [ ca]i oscillations are not essential for glucose - induced oscillations of [ camp]pm . glucose - induced [ camp]pm oscillations in islet -cells are not strictly dependent on ca . a : tirf microscopy recording from a mouse islet -cell showing that glucose - induced [ camp]pm oscillations persist in ca - deficient medium containing 2 mmol / l egta . b and c : simultaneous recordings of [ camp]pm ( black trace ) and [ ca]pm ( gray trace ) in islet -cells following a step increase of the glucose concentration from 3 to 20 mmol / l . cells typically exhibited coordinated oscillations of the two messengers ( b ) . [ ca]pm sometimes increased before ( b ) and sometimes after ( c ) [ camp]pm . islets expressing a modified camp sensor based on c-cfp translocation and nonfluorescent rii-caax were loaded with the ca indicator fura red . the fluorescence traces have been inverted to show increase of [ camp]pm and [ ca]pm as upward deflections . 6b ) , the glucose - induced [ camp]pm oscillations were completely inhibited by 30 mol / l kh7 , which has been described as a specific sac inhibitor ( 41 ) . similar inhibition was obtained with 100 mol / l of the tmac inhibitor dda in clonal ( fig . / l glp-1 was inhibited by both dda and kh7 ( n = 12 ) ( fig . however , whereas dda was without effect on glucose - induced [ ca]i oscillations , kh7 completely abolished this response ( n = 8) ( fig . 6e ) , indicating that the compound might interfere with upstream effects in glucose stimulus - secretion coupling . indeed , kh7 ( 30 mol / l ) markedly suppressed glucose oxidation in min6 cells . this effect could not be attributed to lowering of camp because the effect was not reversed by addition of 10 mmol / l of the membrane - permeable analog 8-br - camp . in contrast , a slight tendency of dda to inhibit glucose oxidation was completely reversed by 8-br - camp ( fig . glucose - induced [ camp]pm formation in min6 and islet -cells is mediated by transmembrane adenylyl cyclases . c : tirf recordings from mouse islet -cells ( a and c ) and a min6 cell ( b ) showing inhibition of glucose - induced [ camp]pm oscillations by 30 mol / l kh7 ( a and b ) and 100 mol d : tirf recording from an islet -cell showing that 100 mol / l dda and 30 mol / l kh7 suppresses the [ camp]pm response to 50 nmol / l glp-1 . e : epifluorescence recording of [ ca]i in a single mouse -cell showing that oscillations triggered by 20 mmol / l glucose are unaffected by 20 mol / l of the catechol estrogen compound [ 1,3,5(10)-estratrien-2,3,17 -triol ] ( ce ) and 100 f : glucose oxidation in min6 -cells exposed to 3 or 11 mmol / l glucose in the absence or presence of 30 mol / l kh7 , 10 mmol / l 8-br - camp , and 100 mol / l dda . means g : tirf recording from a single min6 -cell showing the effect of 20 mol / l ce on [ camp]pm oscillations induced by 11 mmol / l glucose . h : tirf recording of glucose - induced [ camp]pm oscillations in a single min6 -cell maintained in hco3-buffered medium and exposed to 20 mol / l ce and 100 mol / l dda . ce is an alternative inhibitor of sac ( 42 ) , and this compound was without effect on glucose - induced [ ca]i oscillations ( fig . in 60% of the min6 -cells , ce caused temporary interruption of the [ camp]pm oscillations , but after a few minutes , there were [ camp]pm oscillations indistinguishable from those under control conditions ( fig . the interruption probably was not mediated by changes in camp , given that it was observed also with the inactive control compound 1,3,5(10)-estratrien-3,17--triol ( data not shown ) . similar results were obtained when the experiments were performed in medium containing 20 mmol / l nahco3 , which promotes sac activity ( fig . in the presence of 37 mmol / l glucose , [ camp]pm was low and stable in -cells identified by the adrenaline - induced [ camp]pm elevation . however , like in -cells , 1130 mmol / l glucose triggered [ camp]pm oscillations in many -cells ( 3 of 11 at 11 mmol / l , 13 of 28 at 20 mmol / l , and 8 of 17 at 30 mmol / l glucose ) ( fig . the nadirs and peaks of the oscillations reflected inhibition and stimulation , respectively , in relation to the baseline ( fig . 7b ) . in the few islets that permitted analysis of the phase relationship of [ camp]pm oscillations between different -cells ( n = 1 ) and between -cells and -cells ( n = 4 ) . a : oscillations of [ camp]pm in response to a step increase of the glucose concentration from 3 to 30 mmol / l in an islet -cell identified by [ camp]pm elevation in response to adrenaline ( adr ) . b : example of an islet -cell responding to a step increase of the glucose concentration from 3 to 20 mmol / l with [ camp]pm oscillations showing both stimulatory and inhibitory components . c and d : glucose - induced [ camp]pm oscillations recorded from two individual -cells ( c ) and one - and one -cell ( d ) within the same islet . the current study uses a tirf microscopy approach to investigate camp signaling in intact pancreatic islets . the method images the plasma membrane and submembrane cytoplasm of the different types of cells in the islet mantle in direct contact with the coverslip . although innervation and normal microcirculation is lacking , the preparation allows analysis of camp signaling in cells able to interact within the micro - organ . unfortunately , immunostaining could not be used to identify particular cells within an islet after the tirf recording because the islets already detach from the coverslip during attempted fixation . free islet cells attach more firmly , and it became apparent that immune - identified - and -cells displayed distinct [ camp]pm responses to adrenaline , reflecting differences in adrenoceptor expression ( 40 ) , and these responses were used for identification of the two cell types in the islet . islets were isolated only from the parts of the pancreas where pp - cells are scarce ( 37 ) , and it seems unlikely that -cells responding to adrenergic receptor activation with lowering of [ camp]pm incorrectly have been taken for -cells . small cells and cells with small footprints were not taken as -cells irrespective of response . however , whereas glp-1 triggered [ camp]pm oscillations in most -cells , only occasional -cells responded to the hormone with elevation of [ camp]pm . some studies of rat -cells have failed to demonstrate glp-1 receptor expression ( 43,44 ) while others have shown that glp-1 increases the camp content ( 35 ) and exocytosis ( 35,45 ) . the present data from mouse -cells is in line with observations that glp-1 receptors are expressed only in a small subpopulation of rat ( 44 ) and mouse ( 36 ) -cells . it is well accepted that glp-1 is an inhibitor of glucagon secretion , but the underlying signaling is unclear . it was recently suggested that very small camp elevations inhibit glucagon secretion by a protein kinase a ( pka)-dependent mechanism ( 36 ) . however , from the present data , glp-1 inhibition of glucagon secretion seems to involve mechanisms other than changes of [ camp]pm . given that camp is a positive modulator of glucagon secretion ( 23,24,34,35 ) , our observations support a positive autocrine feedback effect of the hormone . the reason for the absence of [ camp]pm oscillations in low glucose when glucagon secretion is stimulated is currently unknown . in islet -cells , the camp responses to glucagon and glp-1 were significantly enhanced by high glucose , as expected from earlier biochemical studies ( 17 ) . glucose alone also induced pronounced changes in [ camp]pm , reminiscent of those of [ ca]i , with an initial lowering followed by a prominent rise . this rise may reflect both direct effects of glucose on the -cell and an early peak of glucagon secretion within the islet ( 32,33 ) . the period of the subsequent oscillations was usually several minutes , as in isolated cells ( 12 ) , and faster oscillations typical for islet recordings of membrane potential and [ ca]i were not observed . because ca is known to increase islet camp via ca / calmodulin - sensitive acs ( 17 ) , it seems possible that the changes of [ camp]pm are driven by those of [ ca]i ( 46 ) . however , like in isolated -cells ( 12 ) , [ camp]pm oscillations in intact islets often persisted when glucose - stimulated ca entry was prevented , and the camp signal sometimes preceded that of ca during the initial glucose - induced response . comparable findings were reported using transgenic mouse islets expressing another camp sensor , with glucose - induced stable camp elevation preceding the increase of [ ca]i and persisting in the absence of extracellular ca ( 13 ) . there are mutual interactions between camp and ca , and both signals depend on intracellular atp ( 12 ) . atp concentrations are in turn affected by ca , and interestingly , oscillations in -cell metabolism ( 47 ) show a ca dependence reminiscent of that of the presently observed camp oscillations . in light of these interactions , it is not surprising that there are slight variations in timing between the ca and camp signals . because the tmacs expressed in islets have in vitro km values for atp in the submillimolar range ( 48 ) , it is not clear how they would be regulated by the approximately 10-fold higher concentrations of atp in the cytoplasm . we now considered the possibility that glucose stimulates camp formation via sac , which is regulated by bicarbonate , ca , and millimolar concentrations of atp ( 18,19,49 ) and which was recently proposed to be present in insulin - secreting cells ( 20 ) . however , the small molecule inhibitor kh7 that is often used to define sac activity ( 20,41 ) showed deleterious effects on -cell metabolism , limiting its usefulness for studies of sac in intact cells . the less selective sac inhibitor ce failed to provide evidence that sac mediates glucose - induced camp formation in the subplasma membrane space of insulin - secreting cells . future studies will have to clarify which particular isoforms of tmacs sense changes in -cell metabolism and how the additive effects of glucose and hormones on [ camp]pm are mediated . interestingly , although glucose is an inhibitor of glucagon secretion and camp stimulates exocytosis , glucose also triggered camp oscillations in -cells . however , glucose - inhibited glucagon secretion is pulsatile , and the reduced time - average secretion in both rat ( 32 ) and human ( 33 ) islets is composed of alternating periods of inhibition and stimulation . the present findings are consistent with camp being involved in the stimulatory component , perhaps also explaining how very high glucose concentrations can paradoxically increase glucagon release ( 31 ) . pulsatile hormone secretion from the intact islet obviously requires synchronization of the secretory activity among the cells within the islet . functional coupling was indeed observed in glp-1stimulated -cells , but the underlying mechanism is unknown . however , gap junction  mediated diffusional exchange and electrical coupling and the stimulatory action of ca on camp formation are factors favoring camp signal propagation and synchronization between cells within functional islet subdomains . the synchronization was less striking during glucose stimulation , possibly because paracrine influences become perturbed by insufficient exchange of superfusion medium around the islet cells adhering to the coverslip . not only do glucose and glucagon independently evoke [ camp]pm oscillations in - and -cells , but somatostatin , whose secretion is stimulated by glucose and glucagon ( 50 ) , suppresses camp levels in these cells . in summary , we found that the camp signaling properties of isolated -cells are maintained when they are located within the intact pancreatic islet . clarification of the temporal relationship of camp signaling in - and -cells will help to explain the generation of antisynchronous pulses of insulin and glucagon underlying the 20- to 100-fold changes in insulin - to - glucagon ratio ( 33 ) that determine glucose homeostasis in the liver .
objectivecamp is a critical messenger for insulin and glucagon secretion from pancreatic - and -cells , respectively . 
 dispersed -cells show camp oscillations , but the signaling kinetics in cells within intact islets of langerhans is unknown.research design and methodsthe subplasma - membrane camp concentration ( [ camp]pm ) was recorded in - and -cells in the mantle of intact mouse pancreatic islets using total internal reflection microscopy and a fluorescent translocation biosensor . 
 cell identification was based on the opposite effects of adrenaline on camp in - and -cells.resultsin islets exposed to 3 mmol / l glucose , [ camp]pm was low and stable . 
 glucagon and glucagon - like peptide-1(7 - 36)-amide ( glp-1 ) induced dose - dependent elevation of [ camp]pm , often with oscillations synchronized among -cells . whereas glucagon also induced [ camp]pm oscillations in most -cells , < 20% of the -cells responded to glp-1 . 
 elevation of the glucose concentration to 1130 
 mmol / l in the absence of hormones induced slow [ camp]pm oscillations in both - and -cells . 
 these camp oscillations were coordinated with those of the cytoplasmic ca2 + concentration ( [ ca2+]i ) in the -cells but not caused by the changes in [ ca2+]i . 
 the transmembrane adenylyl cyclase ( ac ) inhibitor 25-dideoxyadenosine suppressed the glucose- and hormone - induced [ camp]pm elevations , whereas the preferential inhibitors of soluble ac , kh7 , and 1,3,5(10)-estratrien-2,3,17--triol perturbed cell metabolism and lacked effect , respectively.conclusionsoscillatory [ camp]pm signaling in secretagogue - stimulated -cells is maintained within intact islets and depends on transmembrane ac activity . 
 the discovery of glucose- and glucagon - induced [ camp]pm oscillations in -cells indicates the involvement of camp in the regulation of pulsatile glucagon secretion .