Vitamin D3 restores altered cholinergic and insulin receptor expression in the cerebral cortex and muscarinic M3 receptor expression in pancreatic islets of streptozotocin induced diabetic rats

Nutritional therapy is a challenging but necessary dimension in the management of diabetes and neurodegenerative changes associated with it. The study evaluates the effect of vitamin D₃ in preventing the altered function of cholinergic, insulin receptors and GLUT3 in the cerebral cortex of diabetic rats. Muscarinic M3 acetylcholine receptors in pancreas control insulin secretion. Vitamin D₃ treatment in M3 receptor regulation in the pancreatic islets was also studied. Radioreceptor binding assays and gene expression was done in the cerebral cortex of male Wistar rats. Immunocytochemistry of muscarinic M3 receptor was studied in the pancreatic islets using specific antibodies. Y-maze was used to evaluate the exploratory and spatial memory. Diabetes induced a decrease in muscarinic M1, insulin and vitamin D receptor expression and an increase in muscarinic M3, α7 nicotinic acetylcholine receptor, acetylcholine esterase and GLUT3 expression. Vitamin D₃ and insulin treatment reversed diabetes-induced alterations to near control. Diabetic rats showed a decreased Y-maze performance while vitamin D₃ supplementation improved the behavioural deficit. In conclusion, vitamin D₃ shows a potential therapeutic effect in normalizing diabetes-induced alterations in cholinergic, insulin and vitamin D receptor and maintains a normal glucose transport and utilisation in the cortex. In addition vitamin D₃ modulated muscarinic M3 receptors activity in pancreas and plays a pivotal role in controlling insulin secretion. Hence our findings proved, vitamin D₃ supplementation as a potential nutritional therapy in ameliorating diabetes mediated cortical dysfunctions and suggest an interaction between vitamin D₃ and muscarinic M3 receptors in regulating insulin secretion from pancreas.     

M1 Muscarinic Receptors Contribute to, whereas M4 Receptors Inhibit, Dopamine D1 Receptor-Induced [3H]-Cyclic AMP Accumulation in Rat Striatal Slices

In rat striatal slices labelled with [³H]-adenine and in the presence of 1 mM 3-isobutyl-1-methylxantine (IBMX), cyclic [³H]-AMP ([³H]-cAMP) accumulation induced by the dopamine D₁ receptor agonist SKF-81297 (1 μM; 177±13% of basal) was inhibited by the general muscarinic agonist carbachol (maximum inhibition 72±3%, IC₅₀ 0.30±0.06 μM). The muscarinic toxin 7 (MT-7), a selective antagonist at muscarinic M₁ receptors, reduced the effect of SKF-81297 by 40±7% (IC₅₀ 251±57 pM) and enhanced the inhibitory action of a submaximal (1 μM) concentration of carbachol (69±4% vs. 40±7% inhibition, IC₅₀ 386±105 pM). The toxin MT-1, agonist at M₁ receptors, stimulated [³H]-cAMP accumulation in a modest but significant manner (137±11% of basal at 400 nM), an action additive to that of D₁ receptor activation and blocked by MT-7 (10 nM). The effects of MT-7 on D₁ receptor-induced [³H]-cAMP accumulation and the carbachol inhibition were mimicked by the PKC inhibitors Ro-318220 (200 nM) and Gö-6976 (200 nM). Taken together our results indicate that in addition to the inhibitory role of M₄ receptors, in rat striatum acetylcholine stimulates cAMP formation through the activation of M₁ receptors and PKC stimulation.     

M1 and M3 muscarinic antagonists inhibit human nasal glandular secretion in vitro.

Mucus glycoproteins (MGP) are high-molecular-weight glycoconjugates that are released from submucosal glands and epithelial goblet cells in the respiratory tract. Muscarinic receptors have an important role in the regulation of human nasal glandular secretion and mucus production, but it is not known which of the five muscarinic receptor subtypes are involved. The effect of nonselective and M1-, M2-, and M3-selective muscarinic antagonists on methacholine (MCh)-induced MGP secretion from human nasal mucosal explants was tested in vitro. MGP was assayed by enzyme-linked immunosorbent assay using a specific anti-MGP monoclonal antibody (7F10). MCh (100 microM) induced MGP secretion up to 127% compared with controls. MCh-induced MGP release was significantly inhibited by atropine (100 microM), the M, receptor antagonist pirenzepine (10-100 microM), and the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 1-100 microM). 4-DAMP significantly inhibited MCh-induced MGP release at a lower concentration (1 microM) than pirenzepine (10 microM). The M2 receptor antagonists AF-DX 116 and gallamine (both at 100 microM) had no effect. No antagonist alone had a significant effect on MGP release. These results indicate that the M1 and M3 muscarinic receptor subtypes regulate MGP secretion from human nasal mucosa and suggest that the M3 receptor has the predominant effect.     

Ringing the dinner bell for insulin: muscarinic M3 receptor activity in the control of pancreatic beta cell function

In this issue of Cell Metabolism, Gautam et al. (2006) show that pancreatic beta cell M3 muscarinic acetylcholine receptors control insulin secretion. Their results highlight the role of the M3 receptor subtype in integrating nervous stimuli with metabolic control of insulin secretion and glucose homeostasis.     

Muscarinic receptor subtypes mediate stimulatory and paradoxical inhibitory effects on an insulin-secreting β cell line

Acetylcholine (ACh), a major neurotransmitter from the autonomic nervous system, regulates the cholinergic stimulation of insulin secretion, through interactions with muscarinic receptors. The present study has characterised the individual involvement of muscarinic receptor subtypes in ACh-induced insulin secretion, using clonal β cells and selective muscarinic receptor antagonists. BRIN BD11 cells clearly expressed mRNA encoding m1–m4 whereas m5 was not detected by RT-PCR. Insulin release was measured from BRIN BD11 cells treated with ACh in the presence of muscarinic receptor antagonists at concentrations ranging from 3 nM to 1 μM. 300 nM of muscarinic toxin-3 (M4 antagonist) and 1 μM of methoctramine (M2 antagonist) increased ACh (100 μM) stimulated insulin secretion by 168% and 50% respectively (ANOVA, P<0.05). The antagonists alone had no effect on insulin secretion. In contrast, 300 nM of pirenzepine (M1 antagonist) and 30 nM of hexahydro-sila-difenidol p-fluorohydrochloride (M3 antagonist) inhibited ACh stimulation by 91% and 84% respectively (ANOVA, P<0.01). It is concluded that ACh acts on different receptor subtypes producing both a stimulatory and an inhibitory action on insulin release.     

Autoantibodies against Muscarinic Receptors in Breast Cancer: Their Role in Tumor Angiogenesis

The presence of autoantibodies in cancer has become relevant in recent years. We demonstrated that autoantibodies purified from the sera of breast cancer patients activate muscarinic acetylcholine receptors in tumor cells. Immunoglobulin G (IgG) from breast cancer patients in T1N0Mx stage (tumor size≤2 cm, without lymph node metastasis) mimics the action of the muscarinic agonist carbachol stimulating MCF-7 cell proliferation, migration and invasion. Angiogenesis is a central step in tumor progression because it promotes tumor invasion and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) is the main angiogenic mediator, and its levels have been correlated with poor prognosis in cancer. The aim of the present work was to investigate the effect of T1N0Mx-IgG on the expression of VEGF-A, and the in vivo neovascular response triggered by MCF-7 cells, via muscarinic receptor activation. We demonstrated that T1N0Mx-IgG (10⁻⁸ M) and carbachol (10⁻⁹ M) increased the constitutive expression of VEGF-A in tumor cells, effect that was reverted by the muscarinic antagonist atropine. We also observed that T1N0Mx-IgG and carbachol enhanced the neovascular response produced by MCF-7 cells in the skin of NUDE mice. The action of IgG or carbachol was reduced in the presence of atropine. In conclusion, T1N0Mx-IgG and carbachol may promote VEGF-A production and neovascularization induced by breast tumor cells via muscarinic receptors activation. These effects may be accelerating breast tumor progression.     

Choline increases serum insulin in rat when injected intraperitoneally and augments basal and stimulated aceylcholine release from the rat minced pancreas in vitro.

Intraperitoneal injection of choline (30-90 mg.kg-1) produced a dose-dependent increase in serum insulin, glucose and choline levels in rats. The increase in serum insulin induced by choline (90 mg.kg-1) was blocked by pretreatment with the muscarinic acetylcholine receptor antagonists, atropine (2 mg.kg-1), pirenzepine (2 mg.kg-1) and 4-diphenylacetoxy-N-methylpiperidine (2 mg.kg-1) or the ganglionic nicotinic receptor antagonist, hexamethonium (15 mg.kg-1). The effect of choline on serum insulin and glucose was enhanced by oral glucose administration (3 g.kg-1). Choline administration was associated with a significant (P < 0.001) increase in the acetylcholine content of pancreatic tissue. Choline (10-130 microm) increased basal and stimulated acetylcholine release but failed to evoke insulin release from the minced pancreas at considerably higher concentrations (0.1-10 mm). Hemicholium-3, a choline uptake inhibitor, attenuated the increase in acetylcholine release induced by choline augmentation. Choline (1-32 mm) inhibited [3H]quinuclidinyl benzilate binding to the muscarinic receptors in the pancreatic homogenates. These data show that choline, a precursor of the neurotransmitter acetylcholine, increases serum insulin by indirectly stimulating peripheral acetylcholine receptors through the enhancement of acetylcholine synthesis and release.     

Differential effects of paraoxon on the M3 muscarinic receptor and its effector system in rat submaxillary gland cells

The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M₃ subtype of the muscarinic receptor and receptor-coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [³H]quinuclidinyl benzilate ([³H]QNB) was most sensitive to atropine and the M₃-specific antagonist 4-DAMP followed by pirenzepine and least sensitive to the cardioselective M₂ antagonist AFDX116. This, and the binding characteristics of [³H]4-DAMP, confirmed that the muscarinic receptors in this preparation are of the M₃ subtype. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems. Paraoxon (100 μM) reduced B_max of [³H]4-DAMP binding from 27 ± 4 to 13 ± 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolininduced cAMP formation in SMG cells with an EC₅₀ of 200 nM, but paraoxon was > 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 μM atropine, that by paraoxon was unaffected by up to 100 μM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect β-adrenoreceptor function in the preparation, since it did not affect the 10 μM isoproterenol-induced cAMP synthesis, which was inhibited totally by 10 μM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC-stimulated PI metabolism in the SMG cells. It is suggested that paraoxon binds to two different sites in these SMG cells. One is an allosteric site on the M₃ muscarinic receptor which affects ligand binding and may modulate receptor function. The other site may be on the G_i proteinadenylyl cyclase system, and produces CBC-like action, that is, inhibition of the forskolin-stimulated [³H]cAMP synthesis, and is unaffected by atropine inhibition of the muscarinic receptor. This adds to the complexity of paraoxon actions on muscarinic receptors and their effector systems.     

Enhanced muscarinic M1 receptor gene expression in the corpus striatum of streptozotocin-induced diabetic rats

Acetylcholine (ACh), the first neurotransmitter to be identified, regulate the activities of central and peripheral functions through interactions with muscarinic receptors. Changes in muscarinic acetylcholine receptor (mAChR) have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). Previous reports from our laboratory on streptozotocin (STZ) induced diabetic rats showed down regulation of muscarinic M1 receptors in the brainstem, hypothalamus, cerebral cortex and pancreatic islets. In this study, we have investigated the changes of acetylcholine esterase (AChE) enzyme activity, total muscarinic and muscarinic M1 receptor binding and gene expression in the corpus striatum of STZ – diabetic rats and the insulin treated diabetic rats. The striatum, a neuronal nucleus intimately involved in motor behaviour, is one of the brain regions with the highest acetylcholine content. ACh has complex and clinically important actions in the striatum that are mediated predominantly by muscarinic receptors. We observed that insulin treatment brought back the decreased maximal velocity (V_max) of acetylcholine esterase in the corpus striatum during diabetes to near control state. In diabetic rats there was a decrease in maximal number (B_max) and affinity (K_d) of total muscarinic receptors whereas muscarinic M1 receptors were increased with decrease in affinity in diabetic rats. We observed that, in all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.     

A critical role for beta cell M3 muscarinic acetylcholine receptors in regulating insulin release and blood glucose homeostasis in vivo

One of the hallmarks of type 2 diabetes is that pancreatic β cells fail to release sufficient amounts of insulin in the presence of elevated blood glucose levels. Insulin secretion is modulated by many hormones and neurotransmitters including acetylcholine, the major neurotransmitter of the peripheral parasympathetic nervous system. The physiological role of muscarinic acetylcholine receptors expressed by pancreatic β cells remains unclear at present. Here, we demonstrate that mutant mice selectively lacking the M₃ muscarinic acetylcholine receptor subtype in pancreatic β cells display impaired glucose tolerance and greatly reduced insulin release. In contrast, transgenic mice selectively overexpressing M₃ receptors in pancreatic β cells show a profound increase in glucose tolerance and insulin release. Moreover, these mutant mice are resistant to diet-induced glucose intolerance and hyperglycemia. These findings indicate that β cell M₃ muscarinic receptors play a key role in maintaining proper insulin release and glucose homeostasis.     

Impaired muscarinic type 3 (M3) receptor/PKC and PKA pathways in islets from MSG-obese rats

Monosodium glutamate-obese rats are glucose intolerant and insulin resistant. Their pancreatic islets secrete more insulin at increasing glucose concentrations, despite the possible imbalance in the autonomic nervous system of these rats. Here, we investigate the involvement of the cholinergic/protein kinase (PK)-C and PKA pathways in MSG β-cell function. Male newborn Wistar rats received a subcutaneous injection of MSG (4 g/kg body weight (BW)) or hyperosmotic saline solution during the first 5 days of life. At 90 days of life, plasma parameters, islet static insulin secretion and protein expression were analyzed. Monosodium glutamate rats presented lower body weight and decreased nasoanal length, but had higher body fat depots, glucose intolerance, hyperinsulinemia and hypertrigliceridemia. Their pancreatic islets secreted more insulin in the presence of increasing glucose concentrations with no modifications in the islet-protein content of the glucose-sensing proteins: the glucose transporter (GLUT)-2 and glycokinase. However, MSG islets presented a lower secretory capacity at 40 mM K⁺ (P < 0.05). The MSG group also released less insulin in response to 100 μM carbachol, 10 μM forskolin and 1 mM 3-isobutyl-1-methyl-xantine (P < 0.05, P < 0.0001 and P < 0.01). These effects may be associated with a the decrease of 46 % in the acetylcholine muscarinic type 3 (M3) receptor, and a reduction of 64 % in PKCα and 36 % in PKAα protein expressions in MSG islets. Our data suggest that MSG islets, whilst showing a compensatory increase in glucose-induced insulin release, demonstrate decreased islet M3/PKC and adenylate cyclase/PKA activation, possibly predisposing these prediabetic rodents to the early development of β-cell dysfunction.     

Muscarinic Control of MIN6 Pancreatic β Cells Is Enhanced by Impaired Amino Acid Signaling

We have shown recently that the class C G protein-coupled receptor T1R1/T1R3 taste receptor complex is an early amino acid sensor in MIN6 pancreatic β cells. Amino acids are unable to activate ERK1/2 in β cells in which T1R3 has been depleted. The muscarinic receptor agonist carbachol activated ERK1/2 better in T1R3-depleted cells than in control cells. Ligands that activate certain G protein-coupled receptors in pancreatic β cells potentiate glucose-stimulated insulin secretion. Among these is the M3 muscarinic acetylcholine receptor, the major muscarinic receptor in β cells. We found that expression of M3 receptors increased in T1R3-depleted MIN6 cells and that calcium responses were altered. To determine whether these changes were related to impaired amino acid signaling, we compared responses in cells exposed to reduced amino acid concentrations. M3 receptor expression was increased, and some, but not all, changes in calcium signaling were mimicked. These findings suggest that M3 acetylcholine receptors are increased in β cells as a mechanism to compensate for amino acid deficiency.     

Selective alkylation of rat urinary bladder muscarinic receptors with 4-DAMP mustard reveals a contractile function for the M2 muscarinic receptor.

Our previous data indicate that M3 muscarinic receptors mediate carbachol induced bladder contractions. The data presented here were obtained by selective alkylation of M3 receptors with 4-DAMP mustard and suggest that the M2 receptor subtype may be involved in inhibition of beta-adrenergic receptor induced relaxation, therefore, allowing recontraction. Alkylation resulted in 85% of M3 receptors and 65% of M2 receptors unable to bind radioligand as demonstrated by subtype selective immunoprecipitation. Rat bladder strips subjected to our alkylation procedure contracted submaximally, and direct carbachol contractions were inhibited by antagonists with affinities consistent with M3 receptor mediated contraction. In contrast, the affinities of antagonists for inhibition of carbachol induced recontractions following isoproterenol stimulated relaxation in the presence of 90 mM KCl, indicated a contractile function for the M2 receptor that was not observed in control strips. In conclusion, these studies demonstrate a possible role for the M2 subtype in bladder smooth muscle contraction.     

Expression of Cholinergic,Insulin, Vitamin D Receptors and GLUT 3 in the Brainstem of Streptozotocin Induced Diabetic Rats: Effect of Treatment with Vitamin D3

Complications arising from diabetes mellitus include cognitive deficits, neurophysiological and structural changes in the brain. The current study investigated the expression of cholinergic, insulin, Vitamin D receptor and GLUT 3 in the brainstem of streptozotocin-induced diabetic rats. Radioreceptor binding assays and gene expression were done in the brainstem of male Wistar rats. Our results showed that B_max of total muscarinic, muscarinic M3 receptors was increased and muscarinic M1 receptor was decreased in diabetic rats compared to control. A significant increase in gene expression of muscarinic M3, α7 nicotinic acetylcholine, insulin, Vitamin D₃ receptors, acetylcholine esterase, choline acetyl transferase and GLUT 3 were observed in the brainstem of diabetic rats. Immunohistochemistry studies of muscarinic M1, M3 and α7 nicotinic acetylcholine receptors confirmed the gene expression at protein level. Vitamin D₃ and insulin treatment reversed diabetes-induced alterations to near control. This study provides an evidence that diabetes can alter the expression of cholinergic, insulin, Vitamin D receptors and GLUT 3 in brainstem. We found that Vitamin D₃ treatment could modulate the Vitamin D receptors and plays a pivotal role in maintaining the glucose transport and expressional level of cholinergic receptors in the brainstem of diabetic rats. Thus, our results suggest a therapeutic role of Vitamin D₃ in managing neurological disorders associated with diabetes.     

Pharmacology of the isolated foregut of the locust Schistocerca gregaria—IV. Characterization of a muscarinic acetylcholine receptor

1. Acetylcholine (ACh; 10⁻⁶ M—7 × 10⁻⁵ M), in the presence of neostigmine (10⁻⁵ M), caused contraction of the locust isolated foregut.2. The effect of ACh was mimicked by carbachol, propionylcholine (PCh), butyrylcholine (BCh), nicotine, SD35651, oxotremorine and muscarine.3. The contractions caused by ACh, BCh and carbachol were abolished by atropine (10⁻⁶M) and reduced by d-tubocurarine (10⁻⁵ M) and decamethonium (5 × 10⁻⁵ M). Hexamethonium and α-bungaro-toxin had no effect on contractions caused by the above agonists.4. None of the antagonists used in this study blocked the contractile effects of nicotine.5. It is concluded that the foregut contains a neuronal nicotinic receptor which, when activated, causes release of ACh which acts on a neuromuscular muscarinic receptor.     

Characterization of Muscarinic Receptors: Type M2 (Subtype B) on Neuro-2A Neuroblastoma Cells

Abstract

The binding of the nonselective muscarinic antagonist, [³H]N-methylscopolamine (NMS) to a mouse neuroblastoma cell line (Neuro-2A) and its coupling to the inhibition of adenylate cyclase were characterized. Specific [³H]NMS binding to membrane preparations was rapid, saturable, and of high affinity. Saturation experiments revealed a single class of binding sites for the radioligand. Competition experiments with the muscarinic drugs pirenzepine, AF DX 116, dicyclomine and atropine revealed that the muscarinic receptors present on these cells are predominantly of a single class, subtype B (M2). In addition, agonist binding demonstrated existence of a GTP-sensitive high affinity binding state of the receptors. Coupling of these muscarinic receptors to the adenylate cyclase system was investigated using the muscarinic agonist carbachol which was able to inhibit the prostaglandin (PGE₁)-stimulated activation of adenylate cyclase. The agonist carbachol did not stimulate the formation of IP₃ above basal levels, which indicated that the receptors are not coupled to phosphatidylinositol metabolism. In conclusion, we show that possessing predominantly one subtype of muscarinic receptor, the Neuro-2A cells provide a useful model for the investigation of the heterogeneity of muscarinic receptors and the relationship of subtype to the coupling of different effectors.     

Muscarinic receptors are involved in LMM3 tumor cells proliferation and angiogenesis

Angiogenesis is a process of new blood vessel development from pre-existing vasculature and it plays an essential role in tumor growth and metastases. Here, we investigate the expression of muscarinic acetylcholine receptors (mAchR) and their participation in tumor cell proliferation and angiogenesis ability. Saturation binding assays with the tritiated muscarinic antagonist quinuclidinyl benzilate indicate that LMM3 cells derived from a murine mammary adenocarcinoma express a single class of functional mAchR. Competition binding assays with selective muscarinic antagonists indicate a predominance of M3 receptor subtype. The muscarinic agonist carbachol (CARB) stimulates LMM3 cell proliferation in a concentration dependent manner. The maximal effect induced by 10(-9)M CARB was totally blunted by atropine and by the selective M3 and M1 antagonists, para-fluoro hexahydro sila-difenidol (pf-HHSiD) and pirenzepine, respectively. In addition, pf-HHSiD completely blocked in vivo CARB-induced neovascular formation and vascular endothelial growth factor-A in LMM3 tumor cells. We can conclude that mAchR expressed in LMM3 mammary tumor cells positively regulate proliferation and angiogenesis required for tumor progression.     

Diadenosine tetraphosphate contributes to carbachol-induced tear secretion

The purpose of this study is to investigate if the cholinergic stimulation by carbachol on tear secretion is a direct process or if it is also mediated by purinergic mechanisms. Experiments were performed in New Zealand male rabbits. The amount of tear secretion was measured with Schirmer’s test and then analyzed by a HPLC protocol in order to study the nucleotide levels. Animal eyes were instilled with carbachol (a cholinergic agonist), pirenzepine, gallamine and 4-DAMP (muscarinic antagonists), PPADS, suramin and reactive blue 2 (purinergic antagonists), and a P2Y₂ receptor small interfering RNA (siRNA). Tear secretion increased with the instillation of carbachol, approximately 84 % over control values 20 min after the instillation and so did Ap₄A and ATP release. When we applied carbachol in the presence of muscarinic antagonists, tear volume only increased to 4 % with atropine, 12 % in the case of pirenzepine, 3 % with gallamine, and 8 % with 4-DAMP. In the presence of carbachol and purinergic antagonists, tear secretion was increased to 12 % (all values compared to basal tear secretion). By analyzing tear secretion induced with carbachol in presence of a P2Y₂ receptor siRNA, we found that tear secretion was diminished to 60 %. The inhibition of tear secretion in the presence of carbachol and purinergic antagonists or P2Y₂ siRNA occurred with no apparent change in the tear amount of Ap₄A. These experiments demonstrated the participation of Ap₄A in lacrimal secretion process.     

Muscarinic cholinoceptor agonists and antagonists are modulators of the activity of α<Subscript>1</Subscript>-adrenoceptors in the membranes of rat cerebral cortex

The effects of activation and inhibition of muscarinic cholinoceptors by carbachol and atropine on the binding of specific nonselective α₁-antagonist [³H]prazosine in synaptosomal membranes of rat cerebral cortex have been studied. It has been shown that the ligand-receptor interaction of α₁-adrenoceptors corresponds to the model suggesting the presence of a single receptor pool and the binding of two ligand molecules to the receptor. The parameters of [³H]prazosine binding to α₁-adrenoceptors were as follows: K _d = 1.56 ± 0.17 nM, B _max = 30.25 ± 1.78 fmol/mg protein, n = 2. Upon inhibition of muscarinic cholinoceptors by atropine or their activation by carbachol, the radiolabelled ligand is bound to α₁-adrenoceptors according to the same model but at n = 1. In the presence of atropine, the sensitivity of α₁-adrenoceptors to [³H]prazosine decreases more than twofold (K _d = 3.52 ± 0.36 nM) and the concentration of the active receptors is 36% lower (B _max = 19.45 ± 1.46 fmol/mg protein). Carbachol does not reduce the affinity of adrenoceptors to the ligand, while the concentration of active receptors decreases like in the case of atropine. It is supposed that α₁-adrenoceptors in the membranes of rat cerebral cortex exist as dimers. The modulating effects of atropine and carbachol on the binding of specific antagonist by α₁-adrenoceptors are exhibited as changes in the general character of binding (monomerization of α₁-adrenoceptors) and as inhibitory effect on the [³H]prazosine binding parameters.     

Evidence for the possible involvement of the P2Y<Subscript>6</Subscript> receptor in Ca<Superscript>2+</Superscript> mobilization and insulin secretion in mouse pancreatic islets

Subtypes of purinergic receptors involved in modulation of cytoplasmic calcium ion concentration ([Ca²⁺]_i) and insulin release in mouse pancreatic β-cells were examined in two systems, pancreatic islets in primary culture and beta-TC6 insulinoma cells. Both systems exhibited some physiological responses such as acetylcholine-stimulated [Ca²⁺]_i rise via cytoplasmic Ca²⁺ mobilization. Addition of ATP, ADP, and 2-MeSADP (each 100 μM) transiently increased [Ca²⁺]_i in single islets cultured in the presence of 5.5 mM (normal) glucose. The potent P2Y₁ receptor agonist 2-MeSADP reduced insulin secretion significantly in islets cultured in the presence of high glucose (16.7 mM), whereas a slight stimulation occurred at 5.5 mM glucose. The selective P2Y₆ receptor agonist UDP (200 μM) transiently increased [Ca²⁺]_i and reduced insulin secretion at high glucose, whereas the P2Y_2/4 receptor agonist UTP and adenosine receptor agonist NECA were inactive. [Ca²⁺]_i transients induced by 2-MeSADP and UDP were antagonized by suramin (100 μM), U73122 (2 μM, PLC inhibitor), and 2-APB (10 or 30 μM, IP₃ receptor antagonist), but neither by staurosporine (1 μM, PKC inhibitor) nor depletion of extracellular Ca²⁺. The effect of 2-MeSADP on [Ca²⁺]_i was also significantly inhibited by MRS2500, a P2Y₁ receptor antagonist. These results suggested that P2Y₁ and P2Y₆ receptor subtypes are involved in Ca²⁺ mobilization from intracellular stores and insulin release in mouse islets. In beta-TC6 cells, ATP, ADP, 2-MeSADP, and UDP transiently elevated [Ca²⁺]_i and slightly decreased insulin secretion at normal glucose, while UTP and NECA were inactive. RT-PCR analysis detected mRNAs of P2Y₁ and P2Y₆, but not P2Y₂ and P2Y₄ receptors.