Cadmium-induced neurotoxic effects on rat basal forebrain cholinergic system through thyroid hormones disruption.

Cadmium (Cd) single and repeated exposure produces cognitive dysfunctions. Basal forebrain cholinergic neurons (BFCN) regulate cognitive functions. BFCN loss or cholinergic neurotransmission dysfunction leads to cognitive disabilities. Thyroid hormones (THs) maintain BFCN viability and functions, and Cd disrupts their levels. However, Cd-induced BFCN damages and THs disruption involvement was not studied. To research this we treated male Wistar rats intraperitoneally with Cd once (1 mg/kg) or repetitively for 28 days (0.1 mg/kg) with/without triiodothyronine (T3, 40 µg/kg/day). Cd increased thyroid-stimulating-hormone (TSH) and decreased T3 and tetraiodothyronine (T4). Cd altered cholinergic transmission and induced a more pronounced neurodegeneration on BFCN, mediated partially by THs reduction. Additionally, Cd antagonized muscarinic 1 receptor (M1R), overexpressed acetylcholinesterase S variant (AChE-S), downregulated AChE-R, M2R, M3R and M4R, and reduced AChE and choline acetyltransferase activities through THs disruption. These results may assist to discover cadmium mechanisms that induce cognitive disabilities, revealing a new possible therapeutic tool.

Long-term effects of early postnatal nicotine exposure on cholinergic function in the mouse hippocampal CA1 region.

In rodent models of smoking during pregnancy, early postnatal nicotine exposure results in impaired hippocampus-dependent memory, but the underlying mechanism remains elusive. Given that hippocampal cholinergic systems modulate memory and rapid development of hippocampal cholinergic systems occurs during nicotine exposure, here we investigated its impacts on cholinergic function. Both nicotinic and muscarinic activation produce transient or long-lasting depression of excitatory synaptic transmission in the hippocampal CA1 region. We found that postnatal nicotine exposure impairs both the induction and nicotinic modulation of NMDAR-dependent long-term depression (LTD). Activation of muscarinic receptors decreases excitatory synaptic transmission and CA1 network activity in both wild-type and α2 knockout mice. These muscarinic effects are still observed in nicotine-exposed mice. M1 muscarinic receptor activity is required for mGluR-dependent LTD. Early postnatal nicotine exposure has no effect on mGluR-dependent LTD induction, suggesting that it has no effect on the function of m1 muscarinic receptors involved in this form of LTD. Our results demonstrate that early postnatal nicotine exposure has more pronounced effects on nicotinic function than muscarinic function in the hippocampal CA1 region. Thus, impaired hippocampus-dependent memory may arise from the developmental disruption of nicotinic cholinergic systems in the hippocampal CA1 region.

Muscarinic M1 receptor partially modulates higher sensitivity to cadmium-induced cell death in primary basal forebrain cholinergic neurons: A cholinesterase variants dependent mechanism.

Cadmium is a toxic compound reported to produce cognitive dysfunctions, though the mechanisms involved are unknown. In a previous work we described how cadmium blocks cholinergic transmission and induces greater cell death in primary cholinergic neurons from the basal forebrain. It also induces cell death in SN56 cholinergic neurons from the basal forebrain through M1R blockage, alterations in the expression of AChE variants and GSK-3ß, and an increase in Aß and total and phosphorylated Tau protein levels. It was observed that the silencing or blockage of M1R altered ChAT activity, GSK-3ß, AChE splice variants gene expression, and Aß and Tau protein formation. Furthermore, AChE-S variants were associated with the same actions modulated by M1R. Accordingly, we hypothesized that cholinergic transmission blockage and higher sensitivity to cadmium-induced cell death of primary basal forebrain cholinergic neurons is mediated by M1R blockage, which triggers this effect through alteration of the expression of AChE variants. To prove this hypothesis, we evaluated, in primary culture from the basal forebrain region, whether M1R silencing induces greater cell death in cholinergic neurons than cadmium does, and whether in SN56 cells M1R mediates the mechanisms described so as to play a part in the cadmium induction of cholinergic transmission blockage and cell death in this cell line through alteration of the expression of AChE variants. Our results prove that M1R silencing by cadmium partially mediates the greater cell death observed on basal forebrain cholinergic neurons. Moreover, all previously described mechanisms for blocking cholinergic transmission and inducing cell death on SN56 cells after cadmium exposure are partially mediated by M1R through the alteration of AChE expression. Thus, our results may explain cognitive dysfunctions observed in cadmium toxicity.

Cadmium-induced cell death of basal forebrain cholinergic neurons mediated by muscarinic M1 receptor blockade, increase in GSK-3ß enzyme, ß-amyloid and tau protein levels.

Cadmium is a neurotoxic compound which induces cognitive alterations similar to those produced by Alzheimer's disease (AD). However, the mechanism through which cadmium induces this effect remains unknown. In this regard, we described in a previous work that cadmium blocks cholinergic transmission and induces a more pronounced cell death on cholinergic neurons from basal forebrain which is partially mediated by AChE overexpression. Degeneration of basal forebrain cholinergic neurons, as happens in AD, results in memory deficits attributable to the loss of cholinergic modulation of hippocampal synaptic circuits. Moreover, cadmium has been described to activate GSK-3ß, induce Aß protein production and tau filament formation, which have been related to a selective loss of basal forebrain cholinergic neurons and development of AD. The present study is aimed at researching the mechanisms of cell death induced by cadmium on basal forebrain cholinergic neurons. For this purpose, we evaluated, in SN56 cholinergic mourine septal cell line from basal forebrain region, the cadmium toxic effects on neuronal viability through muscarinic M1 receptor, AChE splice variants, GSK-3ß enzyme, Aß and tau proteins. This study proves that cadmium induces cell death on cholinergic neurons through blockade of M1 receptor, overexpression of AChE-S and GSK-3ß, down-regulation of AChE-R and increase in Aß and total and phosphorylated tau protein levels. Our present results provide new understanding of the mechanisms contributing to the harmful effects of cadmium on cholinergic neurons and suggest that cadmium could mediate these mechanisms by M1R blockade through AChE splices altered expression.

Ovulation requires the activation on proestrus of M1 muscarinic receptors in the left ovary.

We analyzed the effects of chemically blocking type 1 muscarinic receptors (M1R) on either the left or right ovary on ovulation rate, number of ova shed and steroid hormones levels. M1R were unilaterally blocked in ovary with the M1R selective antagonist pirenzepine (PZP). PZP was delivered into the bursa ovarica of the left or right ovary of adult rats at 13:00 h on proestrus day. PZP treatment in the left but not in the right ovary blocked ovulation. PZP did not modify the number of ova shed, nor progesterone or 17ß-estradiol serum levels. The surge of luteinizing hormone levels was diminished while that of follicle-stimulating hormone did not change in animals treated with PZP in the left ovary. Interestingly, treatment with either synthetic luteinizing hormone-releasing hormone or human chorionic gonadotropin 1 h after PZP administration in the left ovary restored ovulation in both ovaries. The presence of M1R protein in the theca cells of the ovarian follicles as well as in cells of the corpus luteum was detected on proestrus day. These results suggest that M1R activation in the left ovary is required for pre-ovulatory gonadotropin-releasing hormone (GnRH) secretion and ovulation. Furthermore, these results also suggest that M1R in the left ovary might be regulating ovulation asymmetrically through a stimulatory neural signal relayed to the hypothalamus via the vagus nerve to induce the GnRH secretion which then triggers ovulation.

Time-dependent modulation of muscarinic m1/m3 receptor signalling by local anaesthetics.

BACKGROUND: Signalling of several G-protein-coupled receptors of the Gq/11 family is time-dependently inhibited by local anaesthetics (LAs). Since LA-induced modulation of muscarinic m1 and m3 receptor function may explain their beneficial effects in clinical practice, such as decreased postoperative cognitive dysfunction or less bronchoconstriction, we studied how prolonged exposure affects muscarinic signalling (Wang D, Wu X, Li J, Xiao F, Liu X, Meng M. The effect of lidocaine on early postoperative cognitive dysfunction after coronary artery bypass surgery. Anesth Analg 2002; 95: 1134-41; Groeben H, Silvanus MT, Beste M, Peters J. Combined lidocaine and salbutamol inhalation for airway anesthesia markedly protects against reflex bronchoconstriction. Chest 2000; 118: 509-15). METHODS: A two-electrode voltage clamp was used to assess the effects of lidocaine or its permanently charged analogue QX314 on recombinantly expressed m1 and m3 receptors in Xenopus oocytes. Antisense knock-down of functional Gαq-protein and inhibition of protein kinase C (PKC) served to define mechanisms and sites of action. RESULTS: Lidocaine affected muscarinic signalling in a biphasic way: an initial decrease in methylcholine bromide-elicited m1 and m3 responses after 30 min, followed by a significant increase in muscarinic responses after 8 h. Intracellularly injected QX314 time-dependently inhibited muscarinic signalling, but had no effect in Gαq-depleted oocytes. PKC-antagonism enhanced m1 and m3 signalling, but completely abolished the LA-induced increase in muscarinic responses, unmasking an underlying time-dependent inhibition of m1 and m3 responses after 8 h. CONCLUSIONS: Lidocaine modulates muscarinic m1 and m3 receptors in a time- and Gαq-dependent manner, but this is masked by enhanced PKC activity. The biphasic time course may be due to interactions of LAs with an extracellular receptor domain, modulated by PKC activity. Prolonged exposure to LAs may not benefit pulmonary function, but may positively affect postoperative cognitive function.

Activation of M1 mAChRs by lesatropane rescues glutamate neurotoxicity in PC12 cells via PKC-mediated phosphorylation of ERK1/2.

Lesatropane, a synthesized chiral tropane (3S, 6S-isomer of satropane), is a novel muscarinic agonist, and is being under preclinical development in China for the treatment of primary glaucoma. The reports concerning that activation of muscarinic acetylcholine receptors (mAChRs) could protect cells against apoptosis prompted us to study the neuroprotective effects of lesatropane and the mechanism. We found that lesatropane could protect PC12 cells from glutamate-induced neurotoxicity and reverse the decreased ERK1/2 activation caused by glutamate. Atropine or pirenzepine, antagonist of mAChR or M1 mAChR, antagonized the protective effects of lesatropane respectively and suppressed the lesatropane's effects on ERK1/2. Furthermore, chelerythrine, a PKC inhibitor, partially suppressed ERK1/2 activation induced by lesatropane. The results indicated that the specific M1 mAChR via PKC-ERK1/2 pathway might be involved in the neuroprotective effects of lesatropane. While M1 mAChR is a therapeutic target of Alzheimer's disease (AD), the results of this paper contribute to further information concerning the activation of M1 mAChR as a therapeutic target in AD.

Use of allosteric targets in the discovery of safer drugs.

The need for drugs with fewer side effects cannot be overemphasized. Today, most drugs modify the actions of enzymes, receptors, transporters and other molecules by directly binding to their active (orthosteric) sites. However, orthosteric site configuration is similar in several proteins performing related functions and this leads to a lower specificity of a drug for the desired protein. Consequently, such drugs may have adverse side effects. A new basis of drug discovery is emerging based on the binding of the drug molecules to sites away (allosteric) from the orthosteric sites. It is possible to find allosteric sites which are unique and hence more specific as targets for drug discovery. Of many available examples, two are highlighted here. The first is caloxins - a new class of highly specific inhibitors of plasma membrane Ca²âº pumps. The second concerns the modulation of receptors for the neurotransmitter acetylcholine, which binds to 12 types of receptors. Exploitation of allosteric sites has led to the discovery of drugs which can selectively modulate the activation of only 1 (M1 muscarinic) out of the 12 different types of acetylcholine receptors. These drugs are being tested for schizophrenia treatment. It is anticipated that the drug discovery exploiting allosteric sites will lead to more effective therapeutic agents with fewer side effects.

Effects of paraoxon on neuronal and lymphocytic cholinergic systems.

The cholinergic system in lymphocytes is hypothesized to be a key target for neurotoxic organophosphates (OPs). The present study determined the comparative effects of paraoxon, the active metabolite of OP-parathion, which is detected in the human neuroblastoma line, SH-SY5Y, and leukemic T-lymphocytes, MOLT-3, in vitro. Paraoxon induced cytotoxic effects in a dose- and time-dependent manner in both cells. Further, the paraoxon-induced modulatory effects were comparable despite different cell types, including over-expression of N-terminus acetylcholinesterase (N-AChE) protein, a marker of apoptosis, down-regulations of mRNA encoding M1, M2, and M3 muscarinic acetylcholine receptors (mAChRs), and induction in expression of c-Fos gene, an indication of certain mAChR subtype(s) activation. Furthermore, the non-selective cholinergic antagonist atropine partially attenuated the paraoxon-induced N-AChE and c-Fos activations in both types of cells. These results provide initial and additional information that OPs may similarly induce neuro- and immuno-toxic effects through mAChRs activation, and they underline the potential of using lymphocytes for assessing OPs-induced neurotoxicity.

A multiplex calcium assay for identification of GPCR agonists and antagonists.

Activation of G(q) protein-coupled receptors can be monitored by measuring the increase in intracellular calcium with fluorescent dyes. Recent advances in fluorescent kinetic plate readers and liquid-handling technology have made it possible to follow these transient changes in intracellular calcium in a 1,536-well plate format for high-throughput screening (HTS). Here, we have applied the latest generation of fluorescence kinetic plate readers to multiplex the agonist and antagonist screens of a G protein-coupled receptor (GPCR). This multiplexed assay format provides an efficient and cost-effective method for HTS of G(q)-coupled GPCR targets.

Presynaptic M1, M2, and A1 receptors play roles in tetanic fade induced by pancuronium or cisatracurium.

PURPOSE: We investigated whether presynaptic facilitatory M1 and/or inhibitory M2 muscarinic receptors contributed to pancuronium- and cisatracurium-induced tetanic fade. METHODS: Phrenic nerve-diaphragm muscle preparations of rats were indirectly stimulated with tetanic frequency (75 +/- 3.3 Hz; mean +/- SD). Doses of pancuronium, cisatracurium, hexamethonium, and d-tubocurarine for producing approximately 25% fade were determined. The effects of pirenzepine and methoctramine, blockers of presynaptic M1 and M2 receptors, respectively, on the tetanic fade were investigated. RESULTS: The concentrations required for approximately 25% fade were 413 microM for hexamethonium (26.8 +/- 2.4% 4% fade), 55 nM for d-tubocurarine (28.7 +/- 2.55% fade), 0.32 microM for pancuronium (25.4 +/- 2.2% fade), and 0.32 microM for cisatracurium (24.7 +/- 0.8% fade). Pirenzepine or methoctramine alone did not produce the fade. Methoctramine, 1 microM, attenuated the fade induced by hexamethonium (to 16.0 +/- 2.5% fade), d-tubocurarine (to 6.0 +/- 1.6 fade), pancuronium (to 8.0 +/- 4.0% fade), and cisatracurium (to 11.0 +/- 3.3% fade). 10 nM pirenzepine attenuated only the fades produced by pancuronium (to 5.0 +/- 0.11% fade) and cisatracurium (to 13.3 +/- 5.3% fade). Cisatracurium (0.32 microM) showed antiacetylcholinesterase activity (in plasma, 14.2 +/- 1.6%; 6%; in erythrocyt 17.2 +/- 2.66%) similar to that of pancuronium (0.32 microM). The selective A1 receptor blocker, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 2.5 nM), also attenuated the fades induced by pancuronium and cisatracurium. CONCLUSION: The tetanic fades produced by pancuronium and cisatracurium depend on the activation of presynaptic inhibitory M2 receptors; these agents also have anticholinesterase activities. The fades induced by these agents also depend on the activation of presynaptic inhibitory A1 receptors through the activation of stimulatory M1 receptors by acetylcholine.

Spinal cholinergic mechanism of the relieving effects of electroacupuncture on cold and warm allodynia in a rat model of neuropathic pain.

This study was performed to determine whether spinal cholinergic systems mediate the relieving effects of electroacupuncture (EA) on cold and warm allodynia in a rat model of neuropathic pain. For neuropathic surgery, the right superior caudal trunk was resected at the level between the S1 and S2 spinal nerves innervating the tail. Two weeks after the injury, the intrathecal (i.t.) catheter was implanted. Five days after the catheterization, the rats were injected with atropine (non-selective muscarinic antagonist, 30 microg), mecamylamine (non-selective nicotinic antagonist, 50 microg), pirenzepine (M(1) muscarinic antagonist, 10 microg), methoctramine (M(2) antagonist, 10 microg) or 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (M(3) antagonist, 10 microg). Ten minutes after the injection, EA was applied to the ST36 acupoint for 30 min. The cold and warm allodynia were assessed by the tail immersion test [i.e., immersing the tail in cold (4 degrees C) or warm (40 degrees C) water and measuring the latency of an abrupt tail movement] before and after the treatments. The i.t. atropine, but not mecamylamine, blocked the relieving effects of EA on cold and warm allodynia. Furthermore, i.t. pirenzepine attenuated the antiallodynic effects of EA, whereas methoctramine and 4-DAMP did not. These results suggest that spinal muscarinic receptors, especially M(1) subtype, mediate the EA-induced antiallodynia in neuropathic rats.

Alterations of M2,3-muscarinic receptor protein and mRNA expression in the bladder of the fructose fed obese rat.

PURPOSE: The effects of metabolic syndrome on bladder function and M2,3-muscarinic receptor expression were studied using the fructose fed obese rat. MATERIALS AND METHODS: Male Wistar rats were divided into 2 groups, including group 1-normal control rats and group 2-6-week fructose fed rats. In vivo cystometry using anesthesia was performed. In vitro the bladder was divided into the urothelium and muscle layer by microdissection. Tissue M2,3-muscarinic receptor protein levels were measured by Western blotting. Expression of the mRNAs that encode M2,3-muscarinic receptors was estimated using reverse transcriptase-polymerase chain reaction. RESULTS: Premicturition unstable bladder contractions suggestive of detrusor overactivity were noted in 62.5% of fructose fed rats but in no controls. M2,3-muscarinic receptor protein and mRNA expression was noted in the urothelium and muscle layer of the rat bladder. In control rats the M2-to-M3-muscarinic receptor protein expression ratio was 1:0.68 in urothelium and 1:0.28 in the muscle layer, and that for mRNA expression was 1:0.32 and 1:0.36, respectively. Compared to controls bladder M2,3-muscarinic receptor protein expression in the urothelium and muscle layer of 8 preparations each was significantly increased in fructose fed rats by 89% and 90% for M2-muscarinic receptor (each p <0.001), and by 35% and 93% for M3-muscarinic receptor (p <0.01 and <0.001, respectively). Correspondingly M2,3-muscarinic receptor mRNA expression in the fructose fed rat bladder urothelium and muscle layer of 8 preparations each was also significantly increased by 31% and 49% for M2-muscarinic receptor (each p <0.01), and by 121% and 117% for M3-muscarinic receptor (each p <0.001, respectively). CONCLUSIONS: Metabolic syndrome induces increased expression of M2,3-muscarinic receptor mRNA and protein in the urothelium as well as the muscle layer of the bladder in 6-week fructose fed rats. The receptor alterations are associated with functional evidence of detrusor overactivity.

Muscarinic cholinoceptor activation modulates DNA synthesis and CD40 expression in fibroblast cells.

1 The aim of the present work was to examine the role of muscarinic acetylcholine receptors (mAChR) on DNA synthesis and CD40 expression in human fibroblast cells. Neonatal human skin fibroblast cultures were stimulated with carbachol in presence or absence of specific antagonists and the following parameters were measured: identification of mAChR subtypes, DNA synthesis, inositol phosphates (InsP) production and CD40 expression. 2 Human fibroblasts express mAChR with Kd 0.47 +/- 0.11 nm and Bmax 236 +/- 22 fmol mg protein(-1). Carbachol stimulates DNA synthesis, InsP and the expression of CD40. All these effects were inhibited by atropine, mustard hydrochloride (4-DAMP) and pirenzepine but not by AF-DX 116 and tropicamide, indicating that M3 and M1 mAChR are implicated in carbachol action. The relative Ki of the antagonists obtained by competition binding assay was in parallel to the relative potency for blocking both carbachol-stimulated InsP accumulation and DNA synthesis. 3 The intracellular pathway leading to carbachol-induced biological effects involved phospholipase C and calcium/calmodulin, as U-73122 and trifluoroperazine blocked carbachol effects, respectively. Calphostin C, a protein kinase C inhibitor, had no effect, indicating that this enzyme does not participate in the system. 4 These results may contribute to a better understanding of the modulatory role of the parasympathetic muscarinic system on normal human fibroblast function.

Function-specific blockage of M(1) and M(3) muscarinic acetylcholine receptors by VX and echothiophate.

Certain organophosphate (OP) cholinesterase inhibitors (ChEIs) are also known to bind to the muscarinic acetylcholine receptor (mAChR). The functional consequences of such binding were investigated here using the following OP compounds: VX, echothiophate, sarin, and soman. VX (charged at physiological pH) and echothiophate (formally charged) inhibited a specific signal transduction pathway in CHO cells expressing either the M(1) or M(3) mAChR. Hence, they blocked carbamylcholine (CCh)-induced cyclic adenosine monophosphate (cAMP) synthesis (muM) and had almost no effect on CCh-induced phosphoinositide (PI) hydrolysis. These substances were inactive on forskolin-induced cAMP inhibition signaling in CHO cells expressing M(2) mAChR. In binding studies, using [(3)H]-N-methyl scopolamine ([(3)H]NMS) as the competitor ligand, the ChEIs, VX and echothiophate exhibited binding to rat cortical mAChR with K(i) values in the muM range. The non-charged compounds, sarin and soman, were inert in modulating both cAMP metabolism and PI hydrolysis in CHO cells expressing M(1), M(2), and M(3) mAChRs, and no binding was observed in presence of [(3)H]NMS. These data suggest that VX and echothiophate act as function-specific blockers via a non-classical path of antagonistic activity, implying the involvement of allosteric/ectopic-binding site in M(1) and M(3) mAChRs. The functionally selective antagonistic behavior of echothiophate and VX makes them potential tools for dissecting the interactions of the mAChR with different G proteins.

Effects of pirenzepine on Dai-kenchu-to-induced elevation of the plasma neuropeptide levels in humans.

Dai-kenchu-to has been used for the treatment of abdominal obstructions, including bowel obstructions and a feeling of coldness in the abdomen. We reported that Dai-kenchu-to increases plasma neuropeptide [motilin, vasoactive intestinal polypeptide (VIP), serotonin, calcitonin gene-related peptide (CGRP), and substance P]-like immunoreactive substances (IS) levels and that its pharmacologic effects on the gastrointestine are due to changes in gastrointestinal mucosa-regulatory peptide levels. We examined the effects of the selective M(1) muscarinic receptor antagonist pirenzepine on the elevation of Dai-kenchu-to-induced plasma neuropeptide (gastrin, motilin, somatostatin, VIP, CGRP, substance P)-IS levels in human volunteers and the area under the plasma neuropeptide concentration-time curve from 0 to 240 min (AUC(0-->240 min)), which were calculated from the plasma neuropeptide concentration-time curves from each volunteers. Oral pretreatment with pirenzepine reduced the Dai-kenchu-to-induced elevation of plasma motilin and VIP-IS levels and AUC(0-->240 min). Combined treatment with Dai-kenchu-to and pirenzepine increased plasma somatostatin-IS levels and decreased plasma gastrin-IS levels and had no effects on plasma CGRP- and substance P-IS levels and AUC(0-->240 min) compared with administration of Dai-kenchu-to alone. Dai-kenchu-to appeared to induce the release of motilin and VIP into plasma mainly through the activation of M(1) muscarinic receptors, and pirenzepine may affect the pharmacologic action of Dai-kenchu-to by elevation of plasma motilin and VIP levels.

Protein kinase A and Ca(v)1 (L-Type) channels are common targets to facilitatory adenosine A2A and muscarinic M1 receptors on rat motoneurons.

At the rat motor endplate, pre-synaptic facilitatory adenosine A2A and muscarinic M1 receptors are mutually exclusive. We investigated whether these receptors share a common intracellular signalling pathway. Suppression of McN-A-343-induced M1 facilitation of [3H]ACh release was partially recovered when CGS21680C (an A2A agonist) was combined with the cyclic AMP antagonist Rp-cAMPS. Forskolin, rolipram and 8-bromo-cyclic AMP mimicked CGS21680C blockade of M1 facilitation. Both Rp-cAMPs and nifedipine reduced augmentation of [3H]ACh release by McN-A-343 and CGS21680C. Activation of M1 and A2A receptors enhanced Ca2+ recruitment through nifedipine-sensitive channels. Nifedipine inhibition revealed by McN-A-343 was prevented by chelerythrine (a PKC inhibitor) and Rp-cAMPS, suggesting that Ca(v)1 (L-type) channels phosphorylation by PKA and PKC is required. Rp-cAMPS inhibited [3H]ACh release in the presence of phorbol 12-myristate 13-acetate, but PKC inhibition by chelerythrine had no effect on release in the presence of 8-bromo-cyclic AMP. This suggests that the involvement of PKA may be secondary to M1-induced PKC activation. In conclusion, competition of M1 and A2A receptors to facilitate ACh release from motoneurons may occur by signal convergence to a common pathway involving PKA activation and Ca2+ influx through Ca(v)1 (L-type) channels.

The effects of the neurosteroids: pregnenolone, progesterone and dehydroepiandrosterone on muscarinic receptor-induced responses in Xenopus oocytes expressing M1 and M3 receptors.

The neurosteroids pregnenolone, progesterone, and dehydroepiandrosterone (DHEA) occur naturally in the nervous system. They act on neural tissues, participate in neuronal signaling, and are reported to alter neuronal excitability via nongenomic mechanisms. Muscarinic receptors have important roles in neuronal functions in the brain and autonomic nervous system. In this study, we investigated the effects of pregnenolone, progesterone, and DHEA on M(1) and M(3) muscarinic receptors using the Xenopus oocyte expression system. Pregnenolone and progesterone inhibited the acetylcholine (ACh)-mediated responses of M(1) and M(3) receptors expressed in Xenopus oocytes, whereas DHEA did not. The half-maximal inhibitory concentrations (IC(50)) for pregnenolone inhibition of M(1) receptor- and M(3) receptor-mediated currents were 11.4 and 6.0 microM respectively; the IC(50) values for progesterone inhibition of M(1) receptor- and M(3) receptor-mediated currents were 2.5 and 3.0 microM respectively. The selective protein kinase C (PKC) inhibitor GF109203X had little effect on the pregnenolone or progesterone inhibition of the ACh-induced currents in Xenopus oocytes expressing M(1) or M(3) receptors. The inhibitory effects of pregnenolone and progesterone were overcome at higher concentrations of ACh. Pregnenolone and progesterone inhibited the [(3)H]quinuclidinyl benzilate (QNB) binding to M(1) and M(3) receptor expressed in Xenopus oocytes, and Scatchard plot analysis of [(3)H]QNB binding revealed that pregnenolone and progesterone altered the K(d) value and the B(max), indicating noncompetitive inhibition. In conclusion, pregnenolone and progesterone inhibited M(1) and M(3) receptor functions noncompetitively by the mechanism independent of PKC and by interfering with ACh binding to the receptors.

Cholinergic and purinergic responses in isolated human detrusor in relation to age.

PURPOSE: We investigated whether the contractility of isolated human detrusor muscle, responsiveness to commonly used spasmolytic drugs, and expression of selected muscarinic and purinergic (P2X) receptor subtypes (M2, M3, P2X1 and P2X3) change with age. MATERIALS AND METHODS: Tissues were taken from 63 patients 37 to 84 years old undergoing radical cystectomy. Specimens from 49 patients were used for contractility studies and those from 50 were used for mRNA analysis. RESULTS: Propiverine, oxybutynin, tolterodine and atropine decreased contractions evoked by electric field stimulation to different degrees. However, neither the efficacy nor potency of the drugs showed age related changes. Since human detrusor muscle shows atropine resistant noncholinergic responses, we also studied the putative age dependence of concentration-response curves to the muscarinic agonist carbachol, and the purinergic agonists adenosine triphosphate (ATP) and alpha-beta-methylene-ATP. Sensitivity to alpha-beta-methylene-ATP increased with age, while the efficacy and potency of spasmolytic drugs did not depend on age. In addition, mRNA detected for M2, M3, P2X1 and P2X3 receptors did not change with age. CONCLUSIONS: Our results do not provide evidence for age related contractile deterioration in human detrusor muscle strips, nor do they suggest that responses to anticholinergic spasmolytic drugs change substantially with age.