Liraglutide ameliorates glycometabolism and insulin resistance through the upregulation of GLUT4 in diabetic KKAy mice.

Liraglutide, a long-lasting glucagon­like peptide­1 analogue, has been used for the treatment of patients with type 2 diabetes mellitus since 2009. In this study, we investigated the anti-diabetic effects and mechanisms of action of liraglutide in a spontaneous diabetic animal model, using KK/Upj-Ay/J (KKAy) mice. The KKAy mice were divided into 2 groups, the liraglutide group (mice were treated with 250 µg/kg/day liraglutide) and the model group (treated with an equivalent amount of normal saline). C57BL/6J mice were used as the controls (treated with an equivalent amount of normal saline). The treatment period lasted 6 weeks. During this treatment period, fasting blood glucose (FBG) levels and the body weight of the mice were measured on a weekly basis. Our results revealed that liraglutide significantly decreased FBG levels, the area under the curve following a oral glucose tolerance test and insulin tolerance test, increased serum insulin levels, reduced homeostasis model assessment of insulin resistance and increased the insulin sensitivity index. Furthermore, liraglutide ameliorated glycometabolism dysfunction by increasing glycolysis via hexokinase and glycogenesis via pyruvate kinase activation. An ultrastructural examination of the pancreas revealed that liraglutide improved the damaged state of islet ß cells and increased the number of insulin secretory granules. The real-time PCR results revealed that the gene expression of glucose transporter 4 (GLUT4) increased following treatment with liraglutide. Liraglutide also upregulated the protein expression of GLUT4 in liver tissue and skeletal muscle. Our results suggest that liraglutide ameliorates glycometabolism and insulin resistance in diabetic KKAy mice by stimulating insulin secretion, increasing glycogenesis and glycolysis and upregulating the expression of GLUT4.

Optimizing the parameters of vagus nerve stimulation by uniform design in rats with acute myocardial infarction.

Vagus nerve stimulation (VNS) has been shown to improve left ventricular function and survival in rats with acute myocardial infarction (AMI), and this maneuver has also been adopted clinically for the treatment of patients with chronic heart failure (CHF). Recent in vitro and in vivo studies have suggested that VNS can modulate the level of pro-inflammatory factors. Despite the beneficial effects of VNS, the stimulation parameters for obtaining favorable outcomes appear highly variable. To optimize VNS parameters, we set up different stimulation protocols with different pulse width (1-2 ms), frequency (1-6 Hz), voltage (1-6 V) and duration (40-240 min) of VNS by uniform design (UD). Rats were divided into seven groups with (Group1-Group6) or without VNS (MI group). Our results demonstrate that (1) the parameter sets in Group1, Group2 and Group3 yield the best post-MI protection by VNS, while the protective role were not observed in Group4, Group5 and Group6; (2) baroreflex sensitivity and the α7 nicotinic acetylcholine receptor level were also increased in Group1, Group2 and Group3. (3) the parameter set in Group1 (G1:1 ms, 2 Hz, 3 V, 240 min) is judged the most optimal parameter in this study as rats in this group not only showed a reduced myocardial injury with better-preserved cardiac function compared with other groups, more important, but also exhibited minimal heart rate (HR) reduction. (4) the duration of VNS plays an important role in determining the protection effect of VNS. In conclusion, VNS displays a beneficial role in Group1, Group2 and Group3. Of note, the parameter set in Group1 provides the most optimal cardioprotective effect. These results may provide insight into development of novel treatment for ischemic heart diseases.

[Effect of combination of insulin and selenium on insulin signal transduction in cardiac muscle of STZ-induced diabetic rats].

This study is to investigate the effect of low doses of insulin (1 u x kg(-1)) and selenium (180 microg x kg(-1)) in combination on general physiological parameters and insulin signal molecules in cardiac muscle of STZ-induced diabetic rats. The levels of blood glucose were estimated using One Touch SureStep Blood Glucose meter. HbA1c levels were estimated using microcolumn assay. TG and TC were estimated using enzymatic assay. The levels of PI3K and GLUT4 in cardiac muscle were examined by immunoblotting and immunohistochemistry. The result showed that insulin in combination with selenium could significantly lower blood glucose and blood lipid levels and markedly restored the PI3K and GLUT4 levels in cardiac muscle. It could be concluded that there was cooperation between insulin and selenium, and that treatment of diabetic rats with combined doses of insulin and selenium increased cardiac glucose uptake by upregulating the level of PI3K-mediated GLUT4 in cardiac muscle, eventually ameliorating myocardial dysfunction.

Transfusion of autologous late-outgrowth endothelial cells reduces arterial neointima formation after injury.

AIMS: Late-outgrowth endothelial cells (OECs) exist in blood and other organs. We aimed to explore whether and how OECs participate in re-endothelialization and prevent vascular neointima formation after injury. METHODS AND RESULTS: Rabbit bone marrow OECs were cultured for 4 weeks to increase their numbers. Transfusion of autologous OECs (2 × 106-1 × 107/kg) soon after rabbit ear central artery injury reduced the increase in intima area and the decrease in lumen area observed at days 14 and 28. Transfusion of autologous OECs (1 × 107/kg) ameliorated some early (days 2 and 7) inflammatory and angiogenic responses (local and systemic) to the injury. Red fluorescence was seen within 7 days after transfusion of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labelled acetylated low-density lipoprotein (Dil-acLDL)-incorporated OECs, and 1 h after perfusion of the isolated rabbit ear with Ringer-Locke solution containing Dil-acLDL-incorporated OECs, in the injured rabbit ear central artery. After transfusion of 5-bromo-2'-deoxyuridine (BrdU) incorporated autologous OECs, BrdU-positive cells appeared in the injured artery intima at day 3 and were present in the rescued artery endothelium at day 28. The OECs, ranging from 5%-15% of vascular smooth muscle cells (VSMCs), and the OEC-conditioned medium (5-15%) both inhibited VSMC proliferation and migration in vitro and regulated the arrangement of VSMCs. The VSMCs were helpful for OECs to form tubes in vitro. CONCLUSION: Circulating OECs participate in re-endothelialization directly and inhibit VSMC migration and proliferation by a paracrine pathway; transfusion of large numbers of autologous OECs soon after vascular injury may prevent neointima formation.

Lipid soluble smoke particles upregulate endothelin receptors in rat basilar artery.

The present study examines the effect of dimethylsulphoxide-soluble particles (DSP) from cigarette smoke on endothelin (ET) receptors in the basilar artery. The contractile responses to ET-1 (ET(A) and ET(B) receptors agonist) and sarafotoxin 6c (ET(B) receptor agonist) were studied using a sensitive myograph. The mRNA levels of ET receptors were determined with real-time PCR, while the protein level was evaluated by immunohistochemistry. The results showed that a DSP concentration of 0.4 microl/ml increased the contractile responses induced by sarafotoxin 6c and ET-1 and the mRNA and protein levels of the ET receptors. Inhibitor SB203580 (a p38 inhibitor), staurosporine (a PKC inhibitor) or wedelolactone (a NF-kappaB inhibitor) attenuated the elevated sarafotoxin 6c-induced contraction, the increased mRNA expression and protein levels of the ET(B) receptor induced by DSP. The effects on the ET(A) receptor induced by DSP 0.4 microl/ml were inhibited by co-incubation with PD98059 (an ERK1/2 inhibitor) or SP600125 (a JNK inhibitor) and were further enhanced by SB203580. The results indicate that DSP 0.4 microl/ml upregulates the ET(B) receptor of basilar arterial smooth muscle cells via activation of the p38 pathway and transcriptional factor NF-kappaB, while also upregulating the ET(A) receptor via activation of the ERK1/2 and JNK pathways. Additionally, the p38 pathway seems to be involved in the feedback regulation of the ET(A) receptor.

Experimental diabetes mellitus down-regulates large-conductance Ca2+-activated K+ channels in cerebral artery smooth muscle and alters functional conductance.

Cerebral vascular dysfunction and associated vascular complications often develop over time in type-2 diabetes, but the underlying mechanisms are not wholly understood. The aim of the present study was to investigate whether large-conductance Ca(2+)-activated K(+) (BKCa) channels in cerebral artery smooth muscle cells (CASMCs) were impaired in experimental model of type-2 diabetes, and the changes could account for cerebral vascular complication in type-2 diabetes. Sprague-Dawley rats were fed with high fat and glucose diet for 8 weeks and then injected with streptozotocin (STZ/30 mg/kg i.p.). Three months after injection of STZ, the alterations of BKCa channels were assessed by using multi-myograph system, patch-clamp, RT-PCR and Western blot. Our results show that the model is characterized by insulin resistance, hyperglycaemia, hyperlipidemia and moderate hypertension, which resembles the clinical manifestation of patients with typre-2 diabetes. Inhibition of BKCa channels with 1 mM tetraethylammonium (TEA) or 1 microM paxilline (PAX) causes smaller constriction in type-2 diabetic cerebral basilar arteries than control arteries. The contractile efficacy of 5-Hydroxytryptamine (5-HT) is substantially reduced by TEA or PAX pretreatment in control > diabetic basilar artery rings. The response to 5-HT in diabetic basilar artery rings is higher than that of control artery rings after activation of BKCa channels with NS1619. The whole-cell K(+) currents are significantly decreased in type-2 diabetic CASMCs compared to control, and the sensitivity of BKCa channels to voltage, the specific inhibitor and opener are all diminished in diabetic CASMCs. The expression of BKCa channel beta1, but not alpha-subunits is markedly reduced at both of mRNA and protein levels in endothelial-denudated cerebral arteries. In conclusion, type-2 diabetes downregulates BKCa channel beta1-subunits in CASMCs, resulting in reduced activity of BKCa channel, increased vascular tone and blood pressure, thereby contributing to cerebral vascular complication in type-2 diabetes.

The effect of insulin in combination with selenium on blood glucose and GLUT4 expression in the cardiac muscle of streptozotocin-induced diabetic rats.

We evaluated the effect of a combination of low doses of insulin (1 U/kg/day) and selenium (180 microg/kg/day) on general physiological parameters and the level of glucose transporter (GLUT4) in the cardiac muscle of streptozotocin-induced diabetic rats. Diabetic rats were treated with insulin, selenium and a combination of insulin and selenium for 4 weeks. The levels of blood glucose and hemoglobin A1c were estimated; the level of the GLUT4 in the cardiac muscle was examined by immunoblotting and immunohistochemistry. Insulin in combination with selenium could significantly lower blood glucose and HbA1c levels and could restore disturbances in GLUT4 level in the cardiac muscle. The treatment with insulin was only partially effective in the restoration of diabetic alterations. We conclude that there was cooperation between insulin and selenium, and that the treatment of diabetic rats with combined doses of insulin and selenium was effective in the control of blood glucose and correction of altered GLUT4 distribution in diabetic rat hearts.

[A method for improving the accuracy and sensitivity of cell membrane chromatography].

OBJECTIVE: To improve the accuracy and sensitivity of cell membrane chromatography (CMC) and evaluate the feasibility of CMC in the study of subtype receptors. METHODS: Plasmids were used to transfer alpha(1B)-AR cDNA into human embryonic kidney 293 (HEK293) cell lines to obtain cell lines stably overexpressing the subtype receptors. HEK293 alpha(1B) cell membrane stationary phase (CMSP) was prepared by immobilizing the cell membrane on silica. The retention time of 9 alpha(1)-adrenoceptor ligands and capacity factors(kappa'(HEK293 alpha1B)) were calculated. The capacity factors of rat liver tissue and primary cultured rat hepatocytes were also calculated for a correlation analysis. RESULTS: The calculated capacity factors (kappa') were positively correlated to the published pKi values. The affinity rank orders were identical. The longest retention of the 9 alpha(1)-adrenoceptor ligands occurred on CMSP prepared with HEK293 alpha(1B) cell lines, while CMSP obtained from rat liver tissue showed the shortest retention of the ligands. CONCLUSION: CMC proves practical in the study of the subtype adrenoceptors. The accuracy and sensitivity of CMC can be improved using HEK293 alpha(1B) cell membrane.

Characterization of calcium signaling pathways in human preadipocytes.

Intracellular free Ca2+ (Ca(i)2+) is an important regulator of many cellular activities; however, Ca2+ signaling is not well studied in human preadipocytes. The purpose of the present study was to characterize Ca2+ signal pathways using a confocal scanning technique and RT-PCR. It was found that spontaneous Ca(i)2+ oscillations were observed in 12.1% preadipocytes, and number of cells with Ca2+ oscillations was increased to 47.9% by 1% fetal bovine serum. Ca(i)2+ oscillations were dependent on Ca2+ entry mainly via stored-operated Ca2+ (SOC) entry. They were suppressed by the SOC entry channel blocker La3+, the phospholipase C (PLC) inhibitor U73122, the inositol trisphosphate receptor (IP3R) blocker 2-amino-ethoxydiphenyl borate, or the sarcoplasmic/endoplasmic reticulum Ca2+ pump (SERCA) inhibitors thapsigargin and cyclopiazonic acid, but not by ryanodine. The IP3R activator thimerosal increased Ca(i)2+ oscillations. In addition, the plasma membrane Ca2+ pump (PMCA) inhibitor carboxyeosin and Na+--Ca2+ exchanger (NCX) inhibitor Ni2+ both suppressed Ca2+ oscillations. RT-PCR revealed that the mRNAs for IP3R1-3, SERCA1,2, NCX3 and PMCA1,3,4, Ca(V)1.2, and TRPC1,4,6, STIM1 and Orai1 (for SOC entry channels) were significant in human preadipocytes. The present study demonstrates that multiple Ca2+ signal pathways are present in human preadipocytes, and provides a basis for investigating how Ca2+ signals regulate biological and physiological activities of human preadipocytes.

Characterization of ion channels in human preadipocytes.

Ion channels participate in regulation of cell proliferation. However, though preadipocyte (the progenitor of fat cell) is a type of highly proliferating cells, ion channel expression and their role in proliferation is not understood in human preadipocytes. The present study was designed to characterize ion channels using whole-cell patch clamp technique, RT-PCR, and Western blotting. It was found that a 4-aminopyridine- (4-AP) sensitive transient outward K(+) current (I(to)) was present in a small population of (32.0%) cells, and an outward "noisy" big conductance Ca(2+)-activated K(+) current (I(KCa)) was present in most (92.7%) preadipocytes. The noisy current was inhibited by the big conductance I(KCa) channel blocker paxilline (1 microM), and enhanced by the Ca(2+) ionophore A23187 (5 microM) and the big conductance I(KCa) channel activator NS1619 (10 microM). RT-PCR and Western blot revealed the molecular identities (i.e., KCa1.1 and Kv4.2) of the functional ionic currents I(KCa) and I(to). Blockade of I(KCa) or I(to) with paxilline or 4-AP reduced preadipocyte proliferation, and similar results were obtained with specific siRNAs targeting to KCa1.1 and Kv4.2. Flow cytometric analysis showed ion channel blockade or knockdown of KCa1.1 or Kv4.2 with specific siRNA increased the cell number of G0/G1 phase. The present study demonstrates for the first time that two types of functional ion channel currents, I(to) and big conductance I(KCa), are present in human preadipocytes and that these two types of ion channels participate in regulating proliferation of human preadipocytes.

[Sedative, hypnotic and antiseizure effects of compound gardenia oil and jujube seed oil in mice].

OBJECTIVE: To study the sedative, hypnotic and antiseizure effects of the compound preparation of gardenia oil and jujube seed oil in mice and investigate the interaction of the two drugs in this preparation. METHODS: The compound preparation was administered intragastrically in mice, whose spontaneous activity was observed along with their tolerance of the preparation after long-term administration. The hypnotic effect of the compound was assessed by investigating the changes in the pentobarbital sodium-induced sleeping. The compound was tested for its antiseizure effect in mice with pentetrazole-induced clonic and tonic convulsion. Diazepam was used as the standard control in all experiments. RESULTS: The jujube seed oil, the gardenia oil and their compound all inhibited spontaneous activities of the mice. Compared with diazepam, the compound showed slow action in producing the sedative effect, which increased gradually with prolonged drug administration without obvious drug tolerance responses. The compound and the two oils all showed synergistic action with pentobarbital sodium in inducing sleeping of the mice. Prescription study showed that the compound produced stronger sedative and hypnotic effects than either of the oils. The two oils and the compound did not show significant antiseizure effects in mice. CONCLUSION: The compound of jujube seed oil and gardenia oil has sedative and hypnotic effects in mice, and the two oils in the compound show obvious synergistic effect.

Tanshinone IIA downregulates the CD40 expression and decreases MMP-2 activity on atherosclerosis induced by high fatty diet in rabbit.

Tanshinone IIA (Tan IIA) is a member of the major lipophilic components abstracted from the root of Salvia miltiorrhiza Bunge and has the capacity of anti-atherosclerosis. To investigate the potential mechanism, we established an animal model by giving high fatty diet to rabbits and Tan IIA was given in different dose. Then, superoxide dismutase (SOD) activity and the malondialdehyde (MDA) level in serum were detected using spectrophotometry; cluster of differentiation 40 (CD40) expression of cellular membrane fraction of aortas and matrix metalloproteinase-2 (MMP-2) activity of total protein extract of aortas were detected by Western Blotting and Zymography, respectively. Compared with the control group, the level of MDA, the expression of CD40 and the MMP-2 activity were increased while the SOD activity was decreased significantly in model group. After Tan IIA administration, the SOD activity was significantly increased while the level of MDA was decreased; both the expression of CD40 and the activity of MMP-2 were decreased. It is suggested that Tan IIA not only inhibits the oxidation but also suppresses the inflammation in atherosclerotic lesion. Our data suggest that not only anti-oxidation but also anti-inflammation by decrease the expression of CD40 and MMP-2 activity maybe the potential mechanisms by which Tan IIA anti-atherosclerosis.

[Pentoxifylline promotes learning and memory function of aging rats and mice with induced memory impairment].

OBJECTIVE: To investigate the effects of pentoxifylline on rats and mice with learning and memory dysfunctions. METHODS: Morris water maze test was used to observe the effects of pentoxifylline on learning and memory of naturally aging rats, and jumping stand test was performed to examine its effects in promoting the learning and memory functions in mice with scopolamine- and ethanol-induced memory dysfunctions. RESULTS: In aging rats, pentoxifylline at high, moderate and low doses all significantly reduced the latency of platform finding in the place navigation test (P<0.01 or P<0.05 ), and increased the quadrant searching frequency in the spatial probe test (P<0.05). Pentoxifylline at the 3 doses significantly increased the latency of electrification (P<0.01 or P<0.05) and decreased the times of error (P<0.05) of the mice as compared with scopolamine- treated group. Pentoxifylline also improved ethanol-induced memory dysfunction in the mice, but the changes in the performance of the mice were not statistically significant. CONCLUSION: Pentoxifylline can improve the learning and memory abilities of rats and mice.

Tanshinone IIA inhibits atherosclerotic plaque formation by down-regulating MMP-2 and MMP-9 expression in rabbits fed a high-fat diet.

Tanshinone IIA (Tan IIA) is one of the major lipophilic components of Salvia miltiorrhiza Bunge (SM) and has an anti-atherosclerotic effect. To investigate the potential mechanism of this effect, we established an atherosclerotic animal model by feeding rabbits a high-fat diet, and Tan IIA was given at different doses. Intimal area of the aorta was measured by image analysis, serum levels of vascular adhesion molecule-1 (VCAM-1) and interleukin (IL-1beta) were measured by ELISA, while matrix metalloproteinase-2 and-9 (MMP-2, MMP-9) expression and their activities in atherosclerotic lesions were assessed by Western blotting and zymography respectively. Compared with the control group, the intimal area, serum levels of VCAM-1 and IL-1beta, the expression of MMP-2 and MMP-9 as well as their activities were increased significantly in the high-fat fed rabbit group. After Tan IIA administration, all of these parameters were decreased in a dose-dependent manner. Our results show that Tan IIA can inhibit atherosclerotic lesion formation in aorta and down-regulate protein expression and activities of MMP-2 and MMP-9 as well as serum VCAM-1 and IL-1beta in rabbits fed a high-fat diet.

A non-steroidal anti-inflammatory agent provides significant protection during focal ischemic stroke with decreased expression of matrix metalloproteinases.

The present study was designed to investigate whether the neuroprotective effect of nimesulide was mediated by inhibiting expression of matrix metalloproteinase-9 (MMP-9) and/or matrix metalloproteinase-2 (MMP-2) in a rat model of thrombolytic reperfusion after the embolic focal cerebral ischemia (FCI). It was found that nimesulide at therapeutically relevant doses (3, 6 and 12 mg/kg) decreased neurological deficits, infarct volume, brain index and brain water content in a dose-dependent manner. Hemorrhagic transformation was reduced by 64% with treatment of 12 mg/kg nimesulide. Quantitative analysis of immunohistochemical staining of brain slices showed that the neuron number expressing MMP-9 and MMP-2 increased in the model animals treated with vehicle (p<0.01 vs sham group), and significantly decreased in nimesulide-treated animals (p<0.05 or p<0.01 vs vehicle group). Our results demonstrate that nimesulide significantly reduces the degree of neuronal injury and hemorrhage transformation caused by thrombolytic reperfusion after the embolic FCI, and that inhibition of MMP-9 and MMP-2 expression contributes at least in part to the neuroprotection.

[Experimental study on the effect of tiangui gengnian capsule on the aged female rats ostepoprosis].

OBJECTIVE: To study the therapeutic action of Seabuckthorn Fatty Acid on the aged female rats osteoporosis, and explore the corresponding mechanism related in hormone and cytokine. METHODS: The change of bone density (BMD), biomechanics and bone morphology were measured and E2, TGF-beta1, IGF-1 level in plasma were determined respectively for the young female rats, aged female rats (Seabuckthorn Fatty Acid 0.72, 1.8, 4.5 g/kg, E2 0.02 g/kg) after they were administrated corresponding drugs for 45 days. RESULTS: The bone density (BMD), bone biomechanics, bone morphology for female rats aged 22-months all decreased. The BMD, bone biomechanics, the number of bone trabeculas and cortical thickness in Seabuckthorn Fatty Acid treated groups (1.8 g/kg, 4.5 g/kg) were upgraded (P < 0.05). The TGF-beta1, IGF-1 level in plasma were increased, too (P < 0.05). CONCLUSION: Seabuckthorn Fatty Acid can meliorate bone metabolism in post-menopause and heighten the bone density. The adjustment mechanism may be based on the function of estrogen and cell cytokine.

[Experimental study on effect of tiangui gengnian soft capsule on aged female rats with osteoporosis].

OBJECTIVE: To investigate the effect of Tiangui Gengnian soft capsule (TGC), which mainly consists of seabuckthorn fatty acid, on serum estrogen and estrogen receptor (ER) in multiple target tissues as uterus, liver and bone, in aged female rats, in order to explore its mechanism from the aspects of receptor and cytokines. METHODS: Low (0.72 g/kg), middle (1.80 g/kg) and high (4.50 g/kg) dose of TGC were administered by gastrogavage to young and aged (22 months old) female rats with osteoporosis for 45 days, and diethylstilbestrol (0.02 mg/kg) was used as a positive control. The levels of serum estradiol (E2), transforming growth factor beta1 (TGF-beta1), insulin-like growth factor-1 (IGF-1) were measured by radioimmunoassay and ELISA method, the protein expression of their receptor in bone, uterus and liver was detected by SABC immunohistochemistry, and the mRNA expression of E2 in uterus and liver detected by in situ hybridization with digoxin probe. RESULTS: Intervention of TGC could cause increase of serum E2, IGF-1 and TGF-beta1 levels, the TGF-beta1 reached 90.63 +/- 18.71 pg/L in the group administered with high dose, which was significantly different to that in the aged group (P < 0.01). There was no obvious effect of the mRNA expression of E2 in uterus and liver, and no effect of TGF-beta1 and IGF-1 in liver in rats. CONCLUSION: TGC could improve the postmenopausal bone metabolism, alleviate and correct the bone loss, it is possibly realized by way of side-secreting/auto-secreting of E2 receptor and cytokines (TGF-beta1 and IGF-1) to improve the osteogenesis and inhibit the destruction of bone.

Evaluation of drug-muscarinic receptor affinities using cell membrane chromatography and radioligand binding assay in guinea pig jejunum membrane.

AIM: To study if cell membrane chromatography (CMC) could reflect drug-receptor interaction and evaluate the affinity and competitive binding to muscarinic acetylcholine receptor (mAChR). METHODS: The cell membrane stationary phase (CMSP) was prepared by immobilizing guinea pig jejunum cell membrane on the surface of a silica carrier, and was used for the rapid on-line chromatographic evaluation of ligand binding affinities to mAChR. The affinity to mAChR was also evaluated from radioligand binding assays (RBA) using the same jejunum membrane preparation. RESULTS: The capacity factor (k') profiles in guinea pig jejunum CMSP were: (-)QNB (15.4)>(+)QNB (11.5)>atropine (5.35)>pirenzepine (5.26)>4-DAMP (4.45)>AF-DX116 (4.18)>pilocarpine (3.93)>acetylcholine (1.31). These results compared with the affinity rank orders obtained from radioligand binding assays indicated that there was a positive correlation (r2= 0.8525, P<0.0001) between both data sets. CONCLUSION: The CMC method can be used to evaluate drug-receptor affinities for drug candidates.