Regulation of putative muscarinic cholinergic receptor subtypes in rat brain

Transection of the fimbria/fornix, producing a 75% reduction in the activity of the cholinergic marker choline-o-acetyltransferase (CAT EC. 2.3.1.6) in rat hippocampus, did not change the binding characteristics of the non-subtype selective, muscarinic cholinergic receptor antagonist ligand [³H](−)quinuclidinyl benzilate {[³H](−)QNB}. Pirenzepine competition for [³H](−)QNB binding in the hippocampus was best described by a computer derived model assuming two binding sites of high affinity (putative M₁ receptors) and low affinity (putative M₂ receptors). There was no change in the proportion of high and low affinity pirenzepine binding sites in the hippocampus following cholinergic deafferentation. Thus, these data provide no evidence for a discrete localization of either putative subtype of muscarinic receptor discriminated by pirenzepine restricted to the terminals of CAT containing neurons innervating the rat hippocampus.Chronic scopolamine treatment produced a 48% increase in the B_max of [³H](−)QNB binding in the hippocampus, but again there was no change in the proportions of the sites discriminated by pirenzepine demonstrating that both putative subtypes were regulated identically. Similarly, carbachol competition for [³H](−)QNB was unaltered following cholinergic deafferentation or chronic scopolamine treatment. Furthermore, similar guanylyl-5′-imidodiphosphate [Gpp(NH)p] modulation of the proportions of high and low affinity carbachol binding sites was found in the hippocampus following transection of the fimbria/fornix or chronic scopolamine treatment. Thus there is no adaptation of receptor-effector coupling following these treatments that is reflected by changes in receptor recognition site characteristics.Carbachol competition for [³H]pirenzepine binding to putative M₁ receptors in the hippocampus was biphasic and was also modulated by Gpp(NH)p. In the brainstem, there was a homogeneous population of putative M₂ [³H](−)QNB binding sites having low affinity for pirenzepine. Carbachol competition for these binding sites was also biphasic and modulated by guanine nucleotides. Thus, both putative M₁ and M₂ muscarinic receptors, as defined by high or low affinity for pirenzepine respectively, may mediate their effects in rat brain via a guanine nucleotide regulatory subunit.     

Central cholinergic signal-mediated neuroendocrine regulation of vasopressin and oxytocin in ovine fetuses

Background  

The hypothalamic-neurohypophysial system plays a fundamental role in the maintenance of body fluid homeostasis by secreting arginine vasopressin (AVP) and oxytocin (OT) in response to a variety of signals, including osmotic and nonosmotic stimuli. It is well established that central cholinergic mechanisms are critical in the regulation of cardiovascular responses and maintenance of body fluid homeostasis in adults. Our recent study demonstrated that intracerebroventricular (i.c.v.) injection of carbachol elicited an increase of blood pressure in the near-term ovine fetuses. However, in utero development of brain cholinergic mechanisms in the regulation of the hypothalamic neuropeptides is largely unknown. This study investigated AVP and OT neural activation in the fetal hypothalamus induced by central carbachol.     

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 responses of gastric parietal cells

Summary Isolated rabbit gastric glands were used to study the nature of the muscarinic cholinergic responses of parietal cells. Carbachol (CCh, 100 m) stimulation of acid secretion, as measured by the accumulation of aminopyrine, was inhibited by the M1 antagonist, pirenzepine, with an IC₅₀ of 13 m; by the M2 antagonist, 11,2-(diethylamino)methyl-1 piperidinyl acetyl-5,11-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepin-6-one (AF-DX 116), with an IC₅₀ of 110 m; and by the M1/M3 antagonist, diphenylacetoxy-4-methylpiperidinemethiodide (4-DAMP), with an IC₅₀ of 35nm. The three antagonists displayed equivalent IC₅₀ values for the inhibition of carbachol-stimulated production of¹⁴CO₂ from radiolabeled glucose, which is a measure of the turnover of the H,K-ATPase, the final step of acid secretion. Intracellular calcium levels were measured in gastric glands loaded with FURA 2. Carbachol was shown to both release calcium from an intracellular pool and to promote calcium entry across the plasma membrane. The calcium entry was inhibitable by 20 m La³⁺. The relative potency of the three muscarinic antagonists for inhibition of calcium entry was essentially the same as for inhibition of acid secretion or pump related glucose oxidation. Image analysis of the glands showed the effects of carbachol, and of the antagonists, on intracellular calcium were occurring largely in the parietal cell. The rise in cell calcium due to release of calcium from intracellular stores was inhibited by 4-DAMP with an IC₅₀ of 1,7nm, suggesting that the release pathway was regulated by a low affinity M3 muscarinic receptor or state; Ca entry and acid secretion are regulated by a high affinity M3 muscarinic receptor or state, inhibited by higher 4-DAMP concentrations (>30nm), suggesting that it is the steady-state elevation of Ca that is related to parietal cell function rather than the [Ca] _i transient. Displacement of³H N-methyl scopolamine (NMS) binding to purified parietal cells by CCh showed the presence of two affinities for CCh, but only a single affinity for 4-DAMP and lower affinity for pirenzepine and AFDX 116, providing further evidence for the parietal cell location of the [Ca] _i response. Elevation of steady-state [Ca] _i levels with either ionomycin or arachidonic acid did not replicate M3 stimulation of acid secretion or glucose oxidation, hence elevation of [Ca] _i is necessary but not sufficient for acid secretion.     

Cholinergic Dilatation and Constriction of Feline Cerebral Blood Vessels Are Mediated by Stimulation of Phosphoinositide Metabolism via Two Different Muscarinic Receptor Subtypes

Abstract: The muscarinic receptors involved in phosphoinositide (PI) hydrolysis have been pharmacologically characterized in cat cerebral blood vessels. Carbachol elicited a concentration-dependent increase in inositol phosphate accumulation [inositol monophosphate, bisphosphate, trisphosphate (IP₃) and tetrakisphosphate] in both major cerebral arteries and small pial vessels, which reached 140–280% of baseline at 10^?3M carbachol (referred to as maximal effect). However, the inositol phosphate accumulation response was found to be biphasic with a submaximal effect (30–50% of the maximal stimulation) obtained at low carbachol concentrations (<10^?5M). Endothelial denudation induced a virtual disappearance of the submaximal PI response without affecting that elicited by high concentrations of carbachol. The pharmacology of the two carbachol-induced PI responses was investigated by comparing the potency of selected muscarinic antagonists to block the IP₃ accumulation induced by 10^?7M (endothelium-dependent submaximal effect) and 10^?4M (endothelium-independent near-maximal effect) carbachol. In both major arteries and pial vessels, the activation of IP₃ production by 10^?4M carbachol was similarly inhibited by muscarinic antagonists with the following averaged rank order of potency (in -log IC₅₀): 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 8.65) > pirenzepine (8.28) > 6-chloro-5,10-dihydro-5-[(1-methyl-4-piperidinyl)acetyl]-11H-dibenzo[b,e][1,4]diazepine-11-one (UH-AH 371; 7.87) > 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,-11-dihydro-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116; 6.62), a pharmacological profile compatible with an M₁ receptor subtype. In contrast, the submaximal stimulation of the PI metabolism elicited by 10^?7M carbachol in major arteries was blocked by the same antagonists with the following order of potency (in -log IC₅₀): 4-DAMP (8.38) > pirenzepine (7.25) > UH-AH 371 (6.25) > AF-DX 116 (5.72), which was reminiscent of an M₃ pharmacological profile. These findings indicate that stimulation of cerebrovascular muscarinic receptors is accompanied by PI hydrolysis via two distinct receptors, most probably the M₁ and M₃ subtypes that have been associated with constriction and dilatation, respectively, of cat cerebral arteries. Furthermore, these results provide strong evidence for an endothelial localization of the M₃ dilatatory receptors within the vessel wall.     

Effect of monensin on osmotic water flow across the toad bladder and its stimulation by vasopressin and cyclic AMP

Summary The effects of the sodium ionophore monensin on osmotic water flow across the urinary bladder of the toadBufo marinus were studied. Monensin alone did not alter osmotic water flow; however, the ionophore inhibited the hydrosmotic response to vasopressin and cyclic AMP in a dose-dependent manner. The inhibitory effects of monensin were apparent when the ionophore was added to the serosal bathing solution but not when it was added to the mucosal bathing solution. The inhibitory effect of serosal monensin required the presence of sodium in the serosal bathing solution but not the presence of calcium in the bathing solutions. Thus, it appears that intracellular sodium concentration is a regulator of the magnitude of the hydrosmotic response to vasopressin and cyclic AMP.     

Muscarinic acetylcholine receptors of the chick amnion

The presence of muscarinic (M) acetylcholine receptors in the noninnervated chick amnion makes it possible to analyze their functioning with presynaptic effects excluded. The M receptors of the amnion mediating its contraction were identified by testing with selective antagonists: pirenzepine for M₁, methoctramine for M₂, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) for M₃, and tropicamide for M₄ receptor subtype. All antagonists acted as competitive inhibitors of M-acetylcholine receptors. With respect to cholinolytic activity estimated from the response to carbacholine (CBC) (-logIC₅₀), the antagonists could be arranged in the following series: 4-DAMP (8.29) > tropicamide (6.97) > pirenzepine (5.85) > methoctramine (5.63). In addition, the effect of forskolin (5 μM), activator of adenylate cyclase (AC), was unidirectional with ?-adrenergic agonists; it blocked CBC-induced contractile activity of the amnion, whereas phospholipase C (1.25 U/ml) stimulated this activity. These data suggest that CBC-or acetylcholine (ACh)-induced contractile activity of the amnion is mediated by M₃ acetylcholine receptors. Evaluation of contractile response to ACh by the tonic component usually revealed one pool of M₃ acetylcholine receptors. One pool was also revealed after treatment with 4-DAMP, with the Hill coefficient being increased (ACh, n = 1.07; ACh against the 4-DAMP background, n = 1.48). It is possible to detect two pools of M₃-acetylcholine receptors on the basis of either phase-frequency or tonic response, i.e., independently of the test parameter.     

Cholinergic agonists transactivate EGFR and stimulate MAPK to induce goblet cell secretion

Conjunctival goblet cells are the primarysource of mucins in the mucous layer, the innermost layer of the tearfilm. Conjunctival goblet cell mucin secretion is under neural controlbecause exogenous addition of parasympathetic agonists stimulatesgoblet cell secretion. To elucidate the intracellular signal pathwaysused by cholinergic agonists to stimulate goblet cell mucin secretion,we determined whether p42/p44 mitogen-activated protein kinase (MAPK)is activated during cholinergic agonist-stimulated mucin secretion. Ratconjunctiva was removed, preincubated with or without antagonists, andstimulated with the cholinergic agonist carbachol (10⁴M). Carbachol statistically significantly stimulated thephosphorylation of MAPK in a time- and concentration-dependent manner.U-0126, an inhibitor of MAPK activation, completely inhibited both the activation of MAPK and goblet cell secretion stimulated by carbachol. The M₁ muscarinic antagonist pirenzepine, theM₂ muscarinic antagonist gallamine, and theM₁/M₃ muscarinic receptor antagonistN-(3-chloropropyl)-4-piperidinyl diphenylacetate (4-DAMP)also inhibited carbachol-stimulated MAPK activation. Increasing theintracellular Ca²⁺ concentration with a Ca²⁺ionophore increased MAPK activation, and chelation of extracellular Ca²⁺ inhibited carbachol-stimulated activation. Carbacholalso increased tyrosine phosphorylation of Pyk2, p60Src, and theepidermal growth factor receptor (EGFR). The Src inhibitor PP1 and theEGFR inhibitor AG-1478 completely inhibited carbachol-stimulated MAPKactivation. AG-1478 also inhibited goblet cell secretion. We concludethat carbachol transactivates the EGFR to activate MAPK, leading to conjunctival goblet cell secretion. In addition, carbachol also activates Pyk2 and p60Src that could play a role in the transactivation of the EGFR.

     

Hormone-specific responses and biosynthesis of sulfolipids in cell lines derived from mammalian kidney

The established cell lines isolated from mammalian kidney were characterized by its receptor activities against hormones and the ability to synthesize sulfolipids localized in the renal tubule.The level of 3′: 5′-cyclic AMP in JTC-12.P3 (monkey kidney) cells increased in 2 min as much as 2.5–5-fold on activation with 1.0 unit/ml of bovine parathyroid hormone or 1.9 units/ml of synthetic parathyroid hormone (1–34) resulting in intracellular cyclic AMP concentration of more than 40 pmol/mg protein. Prostaglandin E₁ (14 μM) and isopropylnorepinephrine (10 μM) were also found to increase the concentration of cyclic AMP by more than 30- and 9-fold, respectively. Addition in medium of calcitonin, arginine vasopressin, adrenocorticotropic hormone and glucagon caused no significant changes of cyclic AMP level in the cell.In contrast, MDCK, a cell line isolated from canine kidney, reacted to arginine vasopressin, isopropylnorepinephrine and prostaglandin E₁ and only slightly to parathyroid hormone. MDBK cell line derived from bovine kidney or fibroblast cell lines from rat lung and guinea pig kidney did not react to any of the hormones specific to kidney, i.e. arginine vasopressin, calcitonin or parathyroid hormone in the presence of theophylline. However, in the presence of 2 mM isobutylmethylxanthine, small but significant elevation of cellular cyclic AMP levels in response to calcitonin, arginine vasopressin, isopropylnorepinephrine and prostaglandin E₁ was observed.The cell lines JTC-12, MDCK and MDBK, when incubated with H₂³⁵SO₄, incorporated the isotope into sulfolipids assigned as sulfatides and ceramide dihexoside sulfate or in MDCK also into cholesterol sulfate.The results suggested that JTC-12, MDCK and MDBK cell lines are epithelial origin and also JTC-12 and MDCK originated most probably from renal tubular cells of cortex and medulla, respectively.     

Effect of monensin on osmotic water flow across the toad bladder and its stimulation by vasopressin and cyclic AMP

The effects of the sodium ionophore monensin on osmotic water flow across the urinary bladder of the toad Bufo marinus were studied. Monensin alone did not alter osmotic water flow; however, the ionophore inhibited the hydrosmotic response to vasopressin and cyclic AMP in a dose-dependent manner. The inhibitory effects of monensin were apparent when the ionophore was added to th serosal bathing solution but not when it was added to the mucosal bathing solution. The inhibitory effect of serosal monensin required the presence of sodium in the serosal bathing solution but not the presence of calcium in the bathing solutions. Thus, it appears that intracellular sodium concentration is a regulator of the magnitude of the hydrosmotic response to vasopressin and cyclic AMP.     

Pharmacological Profiles of the Subtypes of Muscarinic Cholinoceptors That Mediate Aggregation of Pigment in the Melanophores of Two Species of Catfish

Using selective antagonists, including pirenzepine, adiphenine, AF-DX116, gallamine, and 4-DAMP we attempted to characterize the muscarinic cholinoceptors on the melanophores of the translucent glass catfish Kryptopterus bicirrhis and the mailed catfish Corydoras paleatus. The M₃ receptor-selective antagonist, 4-DAMP, potently inhibited the acetylcholine-induced aggregation of pigment in both species. It appeared, therefore, that the receptors that mediated the cholinergically evoked aggregation of melanosomes in these species were of the M₃ muscarinic subtype.     

Selective Inhibition of the Expression of Signal Transduction Proteins by Lithium in Nerve Growth Factor-Differentiated PC12 Cells

Abstract: Muscarinic receptor in human neuroblastoma SK-N-BE(2)C cells was identified and characterized. Treatment of the cells with carbachol evoked the generation of inositol 1,4,5-trisphosphate (IP₃) with a peak level reached at 1 min after stimulation. Carbachol increased intracellular Ca²⁺ ([Ca²⁺]_i) with an EC₅₀ value of 35 µM. In addition, carbachol produced a 1.3–3-fold increase in the cyclic AMP (cAMP) level compared with untreated control and elevated synergistically the cAMP level in the treatment with prostaglandin E₂ (PGE₂). The M₃ antagonist p-fluorohexahydrosiladifenidol (IC₅₀ = 0.5–0.8 µM) inhibited the increases in [Ca²⁺]_i, IP₃, and cAMP more effectively than the M₁ antagonist pirenzepine (IC₅₀ = 5–9 µM) and the M₂ antagonist methoctramine (IC₅₀ = 20–30 µM). The involvements of [Ca²⁺]_i elevation and protein kinase C activation induced by phospholipase C activation were tested in the carbachol-induced cAMP production. The calcium chelator BAPTA/AM (75 µM) inhibited significantly the synergistic effects of carbachol and PGE₂ on the production of cAMP, whereas the Ca²⁺ ionophore ionomycin (1 µM) clearly enhanced PGE₂-induced cAMP production. However, phorbol 12-myristate 13-acetate did not enhance PGE₂-stimulated cAMP production. These data suggest that phospholipase C-linked M₃ receptors are present and that stimulation of the receptors activates adenylyl cyclase, at least in part, by the Ca²⁺-dependent system in the neuronal cells.     

Muscarinic pharmacology of the inhibition of adenylate cyclase in N18TG2 neuroblastoma cells

This study characterizes the muscarinic cholinergic receptors associated with the inhibition of adenylate cyclase on N18TG2 neuroblastoma cell membranes. Agonists could be divided into two classes: oxotremorine, acetylcholine, carbachol and arecoline exerted the most efficacious and potent inhibition, while McN-A343, bethanechol and AHR-602 were partial agonists. Both quinuclidinyl benzilate and atropine maximally antagonized the inhibitory effect of McN-A343, carbachol and oxotremorine. Pirenzepine was almost as potent as atropine in reversing the inhibitory effect of McN-A343, but was 300 times less potent than atropine or quinuclidinyl benzilate in antagonizing the effects of either carbachol or oxotremorine. Gallamine was ineffective as an antagonist at concentrations up to 1 mM. These results suggest that the receptors that modulate this inhibition are of the M₂ type, since they were activated by carbachol, acetylcholine and oxotremorine, but much less by McN-A343 and AHR-602 (both M₁ selective agonists). The full agonists were blocked by atropine and quinuclidinyl benzilate but not by low concentrations of pirenzepine (M₁ selective antagonist).     

Reversible histochemical modifications of endoplasmic reticulum following arginine vasopressin stimulation of granular cells of toad bladder

The endoplasmic reticulum is generally absent from schematic representations of transport phenomena, although it shows a well-organized network in most transport epithelial cells. In order to examine the correlation between this organelle and cellular activity, bladders of Bufo marinus were studied under different experimental conditions and fixed by immersion in glutaraldehyde, followed by OsO₄ impregnation for 3 days. Normal granular and mitochondria-rich cells showed a rich cytoplasmic network of canaliculi, well-impregnated by osmium deposits. Following a 2 to 15-min stimulation (serosal bath) with arginine vasopressin, the V₂ receptor agonist dD-arginine-vasopressin or cyclic AMP (cAMP), the staining of endoplasmic reticulum in granular cells disappeared. After washing out of the hormone or the agonist, impregnation of the endoplasmic reticulum could be observed once again. Arginine vasopressin did not modify the impregnation of endoplasmic reticulum of either mitochondria-rich or basal cells. Our data indicate a correlation between the reactivity of endoplasmic reticulum to osmium, and a cAMP-dependent effect of arginine vasopressin through its V₂ receptors. Incubation of toad bladders carried out with agents interfering with cellular calcium (calcium ionophores, high or low bath calcium) or with calcium release from the endoplasmic reticulum (TMB-8, thapsigargin) suggested that an early step in the cAMP-dependent effect of arginine vasopressin must involve the release of intracellular calcium from the endoplasmic reticulum. However, calcium ATPases in this organelle do not seem to participate in the hormonal effect. The reversible loss of osmium impregnation induced by arginine vasopressin may represent protein changes in the endoplasmic reticulum accompanying a cAMP-dependent calcium release, from the organelle.     

Agonist activation of cytosolic Ca2+ in subfornical organ cells projecting to the supraoptic nucleus

The subfornical organ (SFO) is sensitive to both ANG II and ACh, and local application of these agents produces dipsogenic responses and vasopressin release. The present study examined the effects of cholinergic drugs, ANG II, and increased extracellular osmolarity on dissociated, cultured cells of the SFO that were retrogradely labeled from the supraoptic nucleus. The effects were measured as changes in cytosolic calcium in fura 2-loaded cells by using a calcium imaging system. Both ACh and carbachol increased intracellular ionic calcium concentration ([Ca2+]i). However, in contrast to the effects of muscarinic receptor agonists on SFO neurons, manipulation of the extracellular osmolality produced no effects, and application of ANG II produced only moderate effects on [Ca2+]i in a few retrogradely labeled cells. The cholinergic effects on [Ca2+]i could be blocked with the muscarinic receptor antagonist atropine and with the more selective muscarinic receptor antagonists pirenzepine and 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP). In addition, the calcium in the extracellular fluid was required for the cholinergic-induced increase in [Ca2+]i. These findings indicate that ACh acts to induce a functional cellular response in SFO neurons through action on a muscarinic receptor, probably of the M1 subtype and that the increase of [Ca2+]i, at least initially, requires the entry of extracellular Ca2+. Also, consistent with a functional role of M1 receptors in the SFO are the results of immunohistochemical preparations demonstrating M1 muscarinic receptor-like protein present within this forebrain circumventricular organ.     

Contribution of the vasopressin V1 receptor to its hydrosmotic response

Toad urinary bladder epithelial cells grown in culture (primary) show a significant increase in water-soluble inositol phosphates when treated with 10(-8) M vasopressin (AVP), but not with (1-deamino-8-D-arginine)vasopressin (dDAVP), a V2-agonist. The increase in inositol phosphates was blocked by the V1-antagonist, d(CH2)5Tyr(Me)AVP, suggesting a V1-coupled phosphoinositide breakdown. The V1-antagonist had no effect on basal adenylate cyclase activity nor on that stimulated by AVP. However, the V1-antagonist was found to attenuate the hydrosmotic response of AVP, suggesting some role of the V1-receptor cascade in the water flow response. Mezerein (MZ), a non-phorbol activator of protein kinase C (PKC) increased osmotic water flow when added to the mucosal surface. The response was less in magnitude and occurred over a longer period (90 min) than that observed with AVP. In an attempt to emulate the V1-response, activation of PKC, and an increase in intracellular calcium, toad bladders were incubated with MZ and the calcium ionophore A23187 (IP). It was found that IP enhanced the water flow response to MZ at all times measured. Mz and IP were also found to enhance cAMP-mediated water flow, suggesting that apical membrane permeability may be regulated in part through V1-receptor stimulation and its respective second messengers. Collectively, these observations suggest that the V1 receptor may play a role not only as part of a negative feedback system, but also as an integral component of the enhanced water permeability that occurs at the apical membrane.     

Cholinergic Receptors in the Human vas Deferens

Abstract

This study represents the first investigation demonstrating the contractile response to exogenous acetylcholine (ACh) in the isolated human vas deferens. Pharmacological characterization of cholinergic receptors was achieved using selective antagonists to define receptor subtypes. In the HVD the effect of exogenous ACh is revealed as a dose-dependent sudden increase in the basal tension of the vasa. The ACh receptors of the HVD were competitively antagonized by atropine (ATR) with a high pA₂ value (8.78). The main finding of this study is the presence of cholinergic receptors of the pharmacologically defined M₂-ACh subtype in the isolated HVD, according to the pA₂ values obtained with pirenzepine (PRZ) 7.39, AF-DX 116 (AF) 5.92 and 4-DAMP 5.65, M₁-ACh, _M2-ACh and M₃-ACh selective antagonists, respectively. Prazosin (PZ), a selective α₁-adrenergic antagonist, displayed a similar competitive antagonism for the contractile response evoked both by ACh (pA₂ = 8.69) and NE (pA₂ = 8.58) in the HVD. The antagonism exerted by PZ on the ACh-induced contractile response of the HVD, suggests that ACh probably acts at a presynaptic level stimulating the release of NE from an adrenergic neuron. According to these findings, the receptor involved in this action, located in the proximity of the nerve terminals, seems to be of the M₂-ACh subtype.     

Studies on Capacitative Calcium Entry in Vascular Smooth Muscle Cells by Measuring 45Ca2+ Influx

Abstract

Capacitative calcium entry was studied in the A7r5 vascular smooth muscle cell line by measuring ⁴⁵Ca²⁺ influx. Entry was induced by depletion of the Ca²⁺ pools by either the receptor agonist [Arg]⁸vasopressin (AVP) or the SR-Ca²⁺-ATPase inhibitor thapsigargin (TG). TG showed a higher efficacy for calcium influx than AVP. This is probably due to a larger Ca²⁺ release from the pools induced by TG compared to AVP and the irreversible inhibition of the SR-Ca²⁺-ATPase by TG causing influx to persist for a longer period of time. At maximally effective concentrations signals induced by AVP and TG were synergistic in the absence but not in the presence of the intracellular calcium chelator, 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Depolarisation with 55 mM KCl completely inhibited ⁴⁵Ca²⁺ influx induced by TG but only slightly the one induced by AVP, both effects being less pronounced in the presence of BAPTA. [Ca²⁺]_c signals induced by AVP and TG were both inhibited by depolarisation.

In conclusion, although our results show differences between AVP- and TG-induced Ca²⁺ influx, they can be explained by their different mechanism of action and are in accordance with an activation of the same capacitative entry pathway by both agents.     

Selective Alkylation of Rat Urinary Bladder Muscarinic Receptors with 4-DAMP Mustard Reveals a Contractile Function for the M2 Muscarinic Receptor

Abstract

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 β-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 KCI, 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.