bone is innervated by autonomic nerve terminals belonging to both the sympathetic and parasympathetic system . sympathetic activity leads to increased bone resorption and subsequent bone loss as observed in the pathophysiology of osteoporosis . osteoporosis is a systemic disease characterized by diminished bone mineral density and changes in bone architecture . inhibition of sympathetic signaling through the beta - adrenergic system by beta - blockers reduces ovariectomy - induced bone loss . this results in an accrual of bone mass through release of the parasympathetic neurotransmitter acetylcholine ( ach ) and binding to muscarinic acetylcholine receptors ( machr ) of the subtype m3 ( m3 machr ) . in addition to having a neuronal origin , ach can be secreted by a multiplicity of non - neuronal cells like endothelial cells , immune cells , and mesenchymal cells . it binds in an auto / paracrine manner ( like a hormone ) on acetylcholine receptors and enhances proliferation , differentiation , and maintenance of cell - cell contacts of many cell types , including osteoblasts . besides m3 machr , 4 other subtypes of machr were identified ( m1m5 machr )   and expressed in bone tissue , especially in osteoblasts [ 68 ] . upon activation , machr couple to g - proteins to regulate ion channel permeability and second messenger signaling pathways . m2 and m4 machr bind to gi / o and m1 , m3 , and m5 machr to gq/11 , thereby increasing intracellular calcium , whereas m2 and m4 machr have only small effects on calcium signaling . it is well known that ovariectomy ( ovx ) induces the state of postmenopausal osteoporosis [ 1117 ] . one cause of postmenopausal osteoporosis is estrogen deficiency , which increases osteoclast formation and therefore supports bone resorption . this leads to an imbalance between osteoblastic bone formation and osteoclastic bone resorption , favoring bone loss and resulting in a high risk for fractures . especially vitamin d and calcium are known to contribute to bone mineralization and lack of these factors decreases bone mass [ 1923 ] . acetylcholine binding to m3 machr results in an elevation of cytosolic calcium concentrations and proliferation of osteoblast - like cells , which were inhibited by the muscarinic receptor antagonist , atropine . mice with a homozygous gene deficient in m3 machr showed a significant reduction of bone bending stiffness , relative bone volume , and alterations of the trabecular microstructure . since loss of m3 machr leads to a decrease in bone mass , we hypothesized that m3 machr is involved in the regulation of bone mass during osteoporosis . a regulating role of m1 , m2 , and m4 machr in bone remodeling is somewhat unlikely because mice that are gene - deficient for these receptors do not show any bone phenotype . m3 machr and m5 machr belong together to gq/11 coupled proteins that enhance the intracellular calcium content and therefore are an interesting target for investigations . thus , we analyzed the expression of m3 machr and m5 machr in vertebra of rats with osteoporosis induced by ovariectomy and a deficient diet using real - time rt - pcr . our results confirmed the hypothesis that m3 machr is involved in osteoporotic bone loss , whereas m5 machr does not seem to participate in the regulation of bone remodeling under pathological conditions . the basis for our study was a well - established animal osteoporosis model developed by heiss et al . sprague - dawley rats were purchased from charles river at the age of 10 weeks . experiments complied with the german protection of animals act and ethical standards laid down in the 1964 declaration of helsinki , and were approved by the regional council of giessen the second group underwent ovariectomy and was fed a deficient diet ( n=27 , ovx+diet ) . the diet was deficient in calcium , vitamins c , d2 , and d3 , as well as in phosphorus ( c1034 , altromin spezialfutter gmbh , lage , germany ) . the osteoporotic bone status of rats was monitored with bone mineral density ( bmd ) measurement . after 3 , 12 , and 14 months , rats were sacrificed and bones dissected . for our study , thoracic vertebra 11 was manually extracted from the spine with a scalpel and surrounding tissue was completely removed . the dissected body of thoracic vertebra 11 was collected in an eppendorf tube containing rna later and stored at 80c before detecting m3 machr and m5 machr expressions by real - time rt - pcr . rna was isolated by homogenizing ~100 mg of thoracic vertebra 11 in 1 ml trizol ( invitrogen , darmstadt , germany ) . after 5 min of incubation at room temperature , 200 l of chloroform were added . samples were shaken and incubated for 23 min at room temperature and afterwards centrifuged for 15 min and 12000 g at 4c . supernatants were collected in a fresh tube and 0.5 ml isopropanol added and incubated for 10 min at room temperature before centrifugation at 4c and 12000 g for 10 min . supernatants were discarded , pellets washed in 1 ml of 75% ethanol and then centrifuged at 4c and 7500 g for 5 min . rna - pellets were dissolved in rnase - free water and the rna concentration measured by nanodrop at 230 nm . using the quantitect reverse transcription kit ( qiagen , hilden , germany ) , 500 ng of rna were reverse transcribed into cdna in a cycler ( tc-3000 , techne , bibby scientific , usa ) . according to the manufacturer s protocol , 2 l of gdna wipeout buffer were added to samples for 5 min at 42c to remove genomic dna . afterwards , 6 l of the respective rt master mix consisting of 1 l quantiscript reverse transcriptase , 4 l quantiscript rt buffer , 1 l rt primer mix or 1 l rnase - free water were added to sample tubes and incubated at 42c for 30 min . real - time rt - pcr was performed using the quantifast sybr green pcr kit ( qiagen ) in an i - cycler with an iqtm5 detection system ( bio - rad , munich , germany ) . one l cdna was added to 0.5 l primer ( table 1 ) , 11 l of rnase- and dnase - free h2o and 12.5 l quantifast sybr green pcr master mix . the cycling procedure started with heating for 5 min at 95c , followed by 40 cycles of denaturation for 10 sec at 95c and a combined step of annealing and elongation for 30 sec at 60c . finally , a melting curve was conducted to check purity of the rt - pcr product . samples that were not reverse transcribed or received water instead of cdna served as negative controls . they were used in the same runs as the probes and remained blank . for evaluation , delta cycle threshold ( ct ) values were determined using the reference gene beta-2-microglobulin ( b2 m ) . statistics was performed with the computer program spss ( version 21.0 ; spss institute inc , chicago , usa ) , which was also used for the generation of graphs in figures 1 and 2 . delta ct values were evaluated with kruskal - wallis and mann - whitney tests and considered to be significant at p0.05 . the basis for our study was a well - established animal osteoporosis model developed by heiss et al . sprague - dawley rats were purchased from charles river at the age of 10 weeks . experiments complied with the german protection of animals act and ethical standards laid down in the 1964 declaration of helsinki , and were approved by the regional council of giessen the second group underwent ovariectomy and was fed a deficient diet ( n=27 , ovx+diet ) . the diet was deficient in calcium , vitamins c , d2 , and d3 , as well as in phosphorus ( c1034 , altromin spezialfutter gmbh , lage , germany ) . the osteoporotic bone status of rats was monitored with bone mineral density ( bmd ) measurement . after 3 , 12 , and 14 months , rats were sacrificed and bones dissected . for our study , thoracic vertebra 11 was manually extracted from the spine with a scalpel and surrounding tissue was completely removed . the dissected body of thoracic vertebra 11 was collected in an eppendorf tube containing rna later and stored at 80c before detecting m3 machr and m5 machr expressions by real - time rt - pcr . rna was isolated by homogenizing ~100 mg of thoracic vertebra 11 in 1 ml trizol ( invitrogen , darmstadt , germany ) . after 5 min of incubation at room temperature , 200 l of chloroform were added . samples were shaken and incubated for 23 min at room temperature and afterwards centrifuged for 15 min and 12000 g at 4c . supernatants were collected in a fresh tube and 0.5 ml isopropanol added and incubated for 10 min at room temperature before centrifugation at 4c and 12000 g for 10 min . supernatants were discarded , pellets washed in 1 ml of 75% ethanol and then centrifuged at 4c and 7500 g for 5 min . rna - pellets were dissolved in rnase - free water and the rna concentration measured by nanodrop at 230 nm . using the quantitect reverse transcription kit ( qiagen , hilden , germany ) , 500 ng of rna were reverse transcribed into cdna in a cycler ( tc-3000 , techne , bibby scientific , usa ) . according to the manufacturer s protocol , 2 l of gdna wipeout buffer were added to samples for 5 min at 42c to remove genomic dna . afterwards , 6 l of the respective rt master mix consisting of 1 l quantiscript reverse transcriptase , 4 l quantiscript rt buffer , 1 l rt primer mix or 1 l rnase - free water were added to sample tubes and incubated at 42c for 30 min . real - time rt - pcr was performed using the quantifast sybr green pcr kit ( qiagen ) in an i - cycler with an iqtm5 detection system ( bio - rad , munich , germany ) . one l cdna was added to 0.5 l primer ( table 1 ) , 11 l of rnase- and dnase - free h2o and 12.5 l quantifast sybr green pcr master mix . the cycling procedure started with heating for 5 min at 95c , followed by 40 cycles of denaturation for 10 sec at 95c and a combined step of annealing and elongation for 30 sec at 60c . finally , a melting curve was conducted to check purity of the rt - pcr product . samples that were not reverse transcribed or received water instead of cdna served as negative controls . they were used in the same runs as the probes and remained blank . for evaluation , delta cycle threshold ( ct ) values were determined using the reference gene beta-2-microglobulin ( b2 m ) . rna was isolated by homogenizing ~100 mg of thoracic vertebra 11 in 1 ml trizol ( invitrogen , darmstadt , germany ) . after 5 min of incubation at room temperature , 200 l of chloroform were added . samples were shaken and incubated for 23 min at room temperature and afterwards centrifuged for 15 min and 12000 g at 4c . supernatants were collected in a fresh tube and 0.5 ml isopropanol added and incubated for 10 min at room temperature before centrifugation at 4c and 12000 g for 10 min . supernatants were discarded , pellets washed in 1 ml of 75% ethanol and then centrifuged at 4c and 7500 g for 5 min . rna - pellets were dissolved in rnase - free water and the rna concentration measured by nanodrop at 230 nm . using the quantitect reverse transcription kit ( qiagen , hilden , germany ) , 500 ng of rna were reverse transcribed into cdna in a cycler ( tc-3000 , techne , bibby scientific , usa ) . according to the manufacturer s protocol , 2 l of gdna wipeout buffer were added to samples for 5 min at 42c to remove genomic dna . afterwards , 6 l of the respective rt master mix consisting of 1 l quantiscript reverse transcriptase , 4 l quantiscript rt buffer , 1 l rt primer mix or 1 l rnase - free water were added to sample tubes and incubated at 42c for 30 min . real - time rt - pcr was performed using the quantifast sybr green pcr kit ( qiagen ) in an i - cycler with an iqtm5 detection system ( bio - rad , munich , germany ) . one l cdna was added to 0.5 l primer ( table 1 ) , 11 l of rnase- and dnase - free h2o and 12.5 l quantifast sybr green pcr master mix . the cycling procedure started with heating for 5 min at 95c , followed by 40 cycles of denaturation for 10 sec at 95c and a combined step of annealing and elongation for 30 sec at 60c . finally , a melting curve was conducted to check purity of the rt - pcr product . samples that were not reverse transcribed or received water instead of cdna served as negative controls . they were used in the same runs as the probes and remained blank . for evaluation , delta cycle threshold ( ct ) values were determined using the reference gene beta-2-microglobulin ( b2 m ) . statistics was performed with the computer program spss ( version 21.0 ; spss institute inc , chicago , usa ) , which was also used for the generation of graphs in figures 1 and 2 . delta ct values were evaluated with kruskal - wallis and mann - whitney tests and considered to be significant at p0.05 . m3 and m5 machr were detected in thoracic vertebra 11 of rats . in general , a decrease of m3 machr mrna expression was detectable in thoracic vertebra 11 of rats that were sacrificed at later time points of the investigation ( figure 1 ) . however , m3 machr mrna was generally expressed at higher levels than m5 machr mrna . the mrna expression of m5 machr was barely influenced by progression of the disease , since no overall significant differences in mrna expression of m5 machr within and between the rat groups could be detected ( figure 2 ) . we found that the ovx+diet group exhibited significantly lower mrna expressions of m3 machr in comparison to the sham group ( p=0.014 ) , indicating that the expression of this receptor decreased with severity of osteoporosis . the 3-month sham group showed higher m3 machr mrna expressions than the 3-month ovx+diet group . rats that underwent ovariectomy and were kept on diet for 3 months ( 3-month ovx+diet ) showed significantly higher m3 machr mrna expressions than the group of ovariectomized rats that received the diet for 14 months ( 14-month ovx+diet ) ( p=0.02 ) . m3 machr mrna expression of the 12-month sham group was significantly higher in comparison to 12 ( p=0.041 ) and 14 month ovx+diet groups ( p=0.013 ) ( figure 1 ) . our study verified the assumption that m3 machr is down - regulated in bone of rats of an osteoporosis model . recently , components of the cholinergic system ( e.g. , acetylcholine receptors , enzymes , and transporters ) were detected in bone   and it is known that machr play an important role in bone physiology . detected m3 and m5 machr in osteoblast - like cells and shi et al . moreover , it has been shown that m3 machr - knockout mice have bone structures similar to systemically diseased bone , as in osteoporosis . liu et al .   concluded that machr regulate cell proliferation and induce calcium signaling in bone and thus may be involved in bone remodeling because osteoblast proliferation contributes to this process . shi et al .   demonstrated that m3 machr increases bone formation and decreases bone resorption . they reasoned that the parasympathetic system affects bone remodeling by involving the central nervous system and thereby decreases the sympathetic tone of bone . the foundation of our study was a well - established model by heiss et al . ( 2013 ) in which osteoporosis was induced in rats by ovariectomy and a diet deficient in calcium and vitamin d. heiss et al . ( 2013 ) confirmed the osteoporotic bone status of rats by dual energy x - ray absorptiometry ( dexa ) . a significant down - regulation in bmd and content was determined for rats that were ovariectomized and received the deficient diet in comparison to the sham group . apart from the sham group , all rats were osteoporotic , thus demonstrating the basis for our interrogation regarding relative expression of m3 machr and m5 machr in osteoporotic bone . although the human situation is more closely represented by large animal osteoporosis models ( e.g. , sheep , pigs , or goats ) because comparable weight ratios and weight - bearing are important for the maintenance of the bone microstructure and mineral density , small animal models offer the advantage of shorter breeding cycles and less expensive animal housing , and thus the possibility to analyze a larger number of animals . therefore , it was possible to investigate and compare 2 experimental groups : a ) sham - operated and b ) ovx+diet animal groups at several time points ( 3 , 12 , and 14 months ) . thereby , a gradual down - regulation was measured for m3 machr expression with progression of the experiment and of osteoporosis . we suppose that the decline in relative m3 machr mrna expression is due to a reduced number of osteoblasts in osteoporosis . it is well - known that m3 machr is expressed by osteoblasts [ 68 ] . since calcium is the main element of the bone matrix , intracellular calcium content of osteoblasts is important for this process . stimulation of machr of the group of gq/11-coupled binding proteins is followed by a strong up - regulation of intracellular calcium concentrations . thus , m3 machr might be involved in delayed accumulation of calcium in the mineralized bone matrix in osteoporosis . animal models with initial ovariectomy mimic reduction of the estrogen content in postmenopausal women . estrogens however , functional studies showed that estrogen treatment increased the responsiveness to m3 machr in rat myometrium . in contrast to myometrium , ovariectomy of rats induced an increase in machr expression in the hippocampus , which can be prevented with estradiol replacement therapy starting directly after ovariectomy . thus , there is an organ- and duration - specific connection between estrogen and machr , which suggests the hypothesis that loss of estrogen in bone might be followed by a decrease in cholinergic components such as m3 machr , which subsequently causes loss of bone mass as shown in m3 machr knockout mice . in our study , m3 machr expression decreased in groups that were ovariectomized and received a diet deficient in calcium , vitamins c , d2 , d3 , and phosphorus , and that was soy- and phytoestrogen - free . relative m3 machr expression was lower in the ovx+diet group than in the sham group . since it is known that m3 machr is expressed by osteoblasts [ 68 ] , we assume that the decline in m3 machr mrna expression occurs as a result of reduced osteoblast numbers in osteoporosis . decrease of relative m3 machr expression correlates with progression of osteoporosis due to estrogen and subsequent osteoblast loss . further research is necessary to determine whether the stimulation of m3 machr can be used to decrease osteoporotic bone resorption .
backgroundcholinergic signaling via muscarinic acetylcholine receptors ( machr ) is known to influence various physiological functions . in bone , 
 m3 machr and m5 machr were identified on the membrane of osteoblast - like cells . 
 m3 machr seems to be particularly relevant for bone physiology , as signaling via this receptor was reported to increase bone formation and decrease bone resorption . 
 thus , in the present study we investigated the relative mrna expression of m3 and m5 machr in bones of a rat osteoporosis model.material/methodsosteoporosis was induced in sprague - dawley rats by bilateral ovariectomy and additional feeding of a diet deficient in calcium , vitamins c , d2 , d3 , and phosphorus , and free of soy and phytoestrogen . after a period of 3 , 12 , and 14 months , relative mrna expression of m3 machr and m5 machr 
 was analyzed in the 11th thoracic vertebra by real - time rt-pcr.resultsrelative mrna expression of m3 machr was significantly reduced in bones of osteoporotic rats compared to sham operated animals that served as controls . 
 further , m3 machr mrna expression was significantly down - regulated when comparing 14-month osteoporotic rats to 3-month osteoporotic rats . 
 relative m5 machr mrna was expressed to a lesser extent than m3 machr and did not show significant differences in mrna expression level between the experimental groups.conclusionsm3 machr mrna expression was reduced upon induction of osteoporosis and progression of disease was associated with further decrease of this receptor , indicating that m3 machr is involved in the development and regulation of osteoporosis .