Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

During development, the innervation of rat sweat glands undergoes a striking change from noradrenergic to cholinergic function. The acquisition of secretory responsiveness by the glands is temporally correlated with the appearance of cholinergic properties. In addition, responsiveness fails to appear in the absence of innervation. To investigate the basis of the onset of functional transmission and secretory responsiveness and its possible relationship to innervation, we analyzed the development of muscarinic cholinergic receptors in sweat glands, examined their expression in the glands of adult rats sympathectomized at birth, and assayed the ability of muscarinic agonists to increase phosphoinositide (PI) turnover. Autoradiographic and in situ hybridization analysis revealed that muscarinic ligand binding sites were first detectable as glands begin to form on postnatal day 4 (P4). Between P4 and P14, receptor concentration increased in parallel with mRNA for the m3 receptor subtype. On P14, the concentration of ligand binding sites approached adult levels, although only a small proportion of glands at this age secrete in response to nerve stimulation or cholinergic agonists. When the pharmacological properties of muscarinic receptors in sweat glands of adult rats sympathectomized at birth were compared to those of normal glands, the concentration and affinity determined with [N-methyl-3H]-scopolamine and the Ki values determined with the subtype-selective muscarinic antagonists 4-DAMP, pirenzepine, and AF DX-116 were similar. In addition, the molecular subtype was unchanged as was the level of m3 message. Studies of PI turnover in response to muscarinic stimulation indicated that the receptors expressed in sweat glands isolated from sympathectomized and acutely denervated, as well as control, rats were functionally coupled to phospholipase C. The absence of sympathetic innervation therefore does not appear to influence either the development of muscarinic receptors or their coupling to PI turnover. Our results suggest that functional sympathetic cholinergic innervation plays a central role in the development and maintenance of secretory function at a step distal to signal transduction across the cell membrane.
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PMID:Developmental expression of muscarinic cholinergic receptors and coupling to phospholipase C in rat sweat glands are independent of innervation. 174 89

Pharmacological studies on pirenzepine (PZ), 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP) and AFDX-116 antagonism of carbachol (CCh)-induced contraction, inositol trisphosphate (IP3) production and cAMP formation revealed the involvement of M3 receptors in these responses. The PA2 values for PZ and 4-DAMP antagonism to CCh-induced contraction were 7.1 and 9.0, respectively, and AFDX-116 had no effect on these responses. Further, 4-DAMP was a much more potent inhibitor than PZ of CCh-stimulation of IP3 production and cAMP formation. Both L-type calcium channel blockers, which inhibit Ca2+ influx, and BAPTA, an intracellular calcium chelator, inhibited these biochemical and pharmacological responses due to CCh. It is concluded that both intracellular and extracellular Ca2+ mobilization are involved in muscarinic stimulation of cAMP production, and that M3 receptors are coupled to the activation of both phospholipase C and adenylate cyclase in this tissue. The data presented here are consistent with previous work that stimulation of muscarinic receptors in dog iris sphincter with CCh (> 5 microM) increases intracellular cAMP levels.
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PMID:M3 muscarinic receptors mediate an increase in both inositol trisphosphate production and cyclic AMP formation in dog iris sphincter smooth muscle. 820 21

This study was designed to characterize the muscarinic acetylcholine receptor (mAChR) subtype present in rat exorbital lacrimal gland as well as its biochemical coupling. The nonselective muscarinic antagonist [N-methyl-3H]scopolamine ([3H]NMS) binds with high affinity to a homogeneous population of binding sites in both membranes [dissociation constant (Kd) = 82.3 +/- 3.2 pM] and acinar cell (Kd = 170.3 +/- 20 pM) preparations. Muscarinic antagonist inhibition of [3H]NMS binding is homogeneous with the following order of potency: atropine > or = 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) > pirenzepine > 11-([2-(diethylamino)-ethyl]-1-piperidinyl)-acetyl- 5,11-dihydro-6H-pirido[2,3-b]1,4,benzo diazepine-6-one (AFDX 116). Both the affinity of the selective antagonists 4-DAMP, pirenzepine, and AFDX 116 and Northern blot analysis of lacrimal gland mRNAs show a single mAChR population of the M3 subtype. Muscarinic agonist inhibition of [3H]NMS binding displays both high (approximately 20%)- and low-affinity sites (approximately 80%). Both the receptor occupancy and the stimulation by agonists or the inhibition by antagonists of the accumulation of [3H]inositol phosphate were examined under identical conditions with respect to tissue preparations (acinar cells) and buffer (Krebs-Ringer). Results demonstrate 1) the efficient coupling of the M3 mAChR subtype with the phosphatidylinositol (4,5))bisphosphate-specific phospholipase C activity and 2) that the efficacy of a muscarinic agonist is dependent on its structure. Lastly, comparison of the agonists affinity and potency to trigger the [3H]inositol phosphate accumulation suggests that the occupation of the high-affinity agonist binding state of the M3 mAChR was involved in the cellular response.
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PMID:M3 muscarinic acetylcholine receptor coupling to PLC in rat exorbital lacrimal acinar cells. 833 5

Intracellular calcium measurements were performed in cultured human trabecular meshwork cells preloaded with the cell permeant dye fura 2-AM. Fluctuations in calcium levels were then monitored with microscope-based ratio fluorometry. Carbachol increased intracellular calcium in a dose-dependent manner; as did oxotremorine-M, aceclidine, and pilocarpine. Carbachol's effect was blocked by the non-selective muscarinic antagonist atropine, as well as by muscarinic receptor subtype-selective antagonists such as pirenzepine (M1-selective), p-fHHSiD (M3-selective), and 4-DAMP (M1, M3 subtypes). Rank order of potencies for the antagonists' effects was atropine = 4-DAMP > p-fHHSiD > pirenzepine, a profile suggesting that the M3 receptor subtype is essential in the carbachol effect. Phospholipase C activity was estimated via measurement of total production of inositol phosphates in cultured human trabecular meshwork cells pre-exposed to 3H-myoinositol. In these cells, carbachol also stimulated phosphoinositide production in a dose-dependent manner, and an antagonist profile similar to that seen for calcium response was obtained when carbachol was used as the effector. The data indicate that muscarinic effects on cultured human trabecular meshwork calcium mobilization and phospholipase C activity are mediated by an M3-like receptor subtype. Therefore, the muscarinic M3 receptor may play a role in trabecular meshwork cell function(s).
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PMID:Effects of muscarinic agents on cultured human trabecular meshwork cells. 869 29

1. Cytosolic Ca2+ concentration ([Ca2+]i) during exposure to acetylcholine or caffeine was measured in mouse duodenal myocytes loaded with fura-2. Acetylcholine evoked a transient increase in [Ca2+]i followed by a sustained rise which was rapidly terminated after drug removal. Although L-type Ca2+ currents participated in the global Ca2+ response induced by acetylcholine, the initial peak in [Ca2+]i was mainly due to release of Ca2+ from intracellular stores. 2. Atropine, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, a muscarinic M3 antagonist), pirenzepine (a muscarinic M1 antagonist), methoctramine and gallamine (muscarinic M2 antagonists) inhibited the acetylcholine-induced Ca2+ release, with a high affinity for 4-DAMP and atropine and a low affinity for the other antagonists. Selective protection of muscarinic M2 receptors with methoctramine during 4-DAMP mustard alkylation of muscarinic M3 receptors provided no evidence for muscarinic M2 receptor-activated [Ca2+]i increase. 3. Acetylcholine-induced Ca2+ release was blocked by intracellular dialysis with a patch pipette containing either heparin or an anti-phosphatidylinositol antibody and by external application of U73122 (a phospholipase C inhibitor). 4. Acetylcholine-induced Ca2+ release was insensitive to external pretreatment with pertussis toxin, but concentration-dependently inhibited by intracellular dialysis with a patch pipette solution containing an anti-alpha q/alpha 11 antibody. An antisense oligonucleotide approach revealed that only the Gq protein was involved in acetylcholine-induced Ca2+ release. 5. Intracellular applications of either an anti-beta com antibody or a peptide corresponding to the G beta gamma binding domain of the beta-adrenoceptor kinase 1 had no effect on acetylcholine-induced Ca2+ release. 6. Our results show that, in mouse duodenal myocytes, acetylcholine-induced release of Ca2+ from intracellular stores is mediated through activation of muscarinic M3 receptors which couple with a Gq protein to activate a phosphatidylinositol-specific phospholipase C.
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PMID:Specific Gq protein involvement in muscarinic M3 receptor-induced phosphatidylinositol hydrolysis and Ca2+ release in mouse duodenal myocytes. 917 86

Muscarinic m2 and m4 receptors couple preferentially to inhibition of adenylyl cyclase, whereas m1, m3, and m5 receptors couple preferentially to activation of phospholipase C-beta and in some cells to stimulation of cAMP. Smooth muscle cells were shown to express adenylyl cyclases types V and/or VI. Acetylcholine (ACh) stimulated the binding of [35S]GTPgammaS.Galpha complexes in smooth muscle membranes to Galphaq/11 and Galphai3 antibody. Binding to Galphaq/11 antibody was inhibited by the m3 receptor antagonist, 4-DAMP, and binding to Galphai3 antibody was inhibited by the m2 receptor antagonist, N,N'-bis[6[[(2-methoxyphenyl)methyl]amino]hexyl]-1,8-octanediamine tetrahydrochloride (methoctramine). The decrease in basal cAMP (35 +/- 5%) induced by ACh in dispersed muscle cells was accentuated by 4-DAMP or Gbeta antibody (55 +/- 8 to 63 +/- 6%). In contrast, methoctramine, pertussis toxin (PTx), or Galphai3 antibody converted the decrease in cAMP to increase above basal level (+28 +/- 5 to +32 +/- 6%); the increase in cAMP was abolished by 4-DAMP or Gbeta antibody. In muscle cells where only m3 receptors were preserved by selective receptor protection, ACh caused only an increase in cAMP that was abolished by 4-DAMP. Conversely, in muscle cells where only m2 receptors were preserved, ACh caused an accentuated decrease in cAMP that was abolished by methoctramine or PTx. In conclusion, m2 receptors in smooth muscle couple to inhibition of adenylyl cyclases V/VI via Galphai3, and m3 receptors couple to activation of the enzymes via Gbetagammaq/11.
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PMID:Differential coupling of muscarinic m2 and m3 receptors to adenylyl cyclases V/VI in smooth muscle. Concurrent M2-mediated inhibition via Galphai3 and m3-mediated stimulation via Gbetagammaq. 926 Nov 44

The effects of carbachol (CCh) on inositol 1,4,5-trisphosphate (IP3) production and intracellular calcium ([Ca2+]i) mobilization, and their regulation by cAMP-elevating agents were investigated in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. CCh produced time- and dose-dependent increases in IP3 production; the t1/2 and EC50 values were 68 s and 0.5 microM, respectively. The muscarinic agonist provoked a transient increase in [Ca2+]i which reached maximum within 77 s, and increased [Ca2+]i mobilization in a concentration-dependent manner with an EC50 of 1.4 microM. Thapsigargin, a Ca(2+)-pump inhibitor, caused a rapid rise in [Ca2+]i and subsequent addition of CCh was without effect. Both CCh-induced IP3 production and CCh-induced [Ca2+]i mobilization were more potently antagonized by 4-DAMP, an M3 muscarinic receptor antagonist, than by pirenzepine, an M1 receptor antagonist, suggesting that both responses are mediated through the M3 receptor subtype. Treatment of the cells with U73122, a phospholipase C (PLC) inhibitor, resulted in a concentration-dependent decrease in both CCh-stimulated IP3 production and [Ca2+]i mobilization. These data indicate close correlation between enhanced IP3 production and [Ca2+]i mobilization in these smooth muscle cells and suggest that the CCh-stimulated increase in [Ca2+]i could be mediated through increased IP3 production. Isoproterenol (ISO) inhibited CCh-induced IP3 production (IC50 = 80 nM) and [Ca2+]i mobilization (IC50 = 0.17 microM) in a concentration-dependent manner. Microsomal fractions isolated from SV-CISM-2 cells contained phospholipase C (PLC) which was stimulated by CCh (10 microM) and GTP gamma S (0.1 microM). Pretreatment of the cells with ISO or forskolin, 5 microM each, produced membrane fractions in which CCh-stimulated PLC activity was significantly attenuated. Furthermore, when microsomal fractions isolated from SV-CISM-2 cells were phosphorylated with Protein kinase A (PKA), the CCh- and GTP gamma S-stimulated IP3 production were significantly inhibited. It can be concluded from these studies that in SV-CISM-2 cells, activation of M3 muscarinic receptors results in stimulation of PLC-mediated PIP2 hydrolysis, generating IP3 which mobilizes [Ca2+]i. Furthermore, elevation of cAMP may inhibit IP3 production and [Ca2+]i mobilization through mechanisms involving PKA-dependent phosphorylation of PLC, G-proteins, IP3 receptor and/or IP3 metabolizing enzymes.
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PMID:Inhibition of muscarinic-stimulated polyphosphoinositide hydrolysis and Ca2+ mobilization in cat iris sphincter smooth muscle cells by cAMP-elevating agents. 937 22

A specific and saturable binding site for [3H]N-methyl-scopolamine ([3H]NMS) was observed in plasma membrane of Fischer rat thyroid (FRT) cells with an equilibrium dissociation constant (K(d)) of 0.11 +/- 0.02 nM and a concentration of receptor sites (B(max)) of 14.1 +/- 3.9 fmol/mg protein. Pharmacological characterization of this binding site using pirenzepine, himbacine, (11(2-diethyl-amino)methyl)-1-piperidinylacetyl-5-11-dihydro-6H-pyrido(14) benzodiazepine (AF-DX 116), dicyclomine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), and hexahydro-sila-difenidol (HHSD) showed clear differences, in terms of affinities, between these muscarinic receptor antagonists. The order of potency for inhibiting [3H]NMS binding was HHSD = dicyclomine > 4-DAMP > pirenzepine = himbacine > AF-DX 116. These findings suggest that the muscarinic receptors found in FRT cells belong to the M3 subtype. Stimulation of FRT cells with carbachol produced a biphasic and dose-dependent increase in the intracellular calcium concentration ([Ca2+]i), which was blocked in pretreated cells with atropine and almost abolished by a low concentration of 4-DAMP and HHSD. Removal of extracellular Ca2+ from the incubation medium reduced the initial transient peak and completely abolished the plateau phase, while the transient phase was markedly reduced by the phospholipase C inhibitor U73122. These data indicate that [Ca2+]i results from both Ca2+ influx across Ca2+ channels and mobilization of Ca2+ from intracellular Ca2+ stores. The present data showed the presence of the M3 muscarinic acetylcholine receptor subtype in plasma membrane of FRT cells, which may influence cellular function via modulation of [Ca2+]i.
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PMID:Muscarinic receptor subtypes and calcium signaling in Fischer rat thyroid cells. 1117 38

Regulation of glucose metabolism by cholinergic nervous activation has been demonstrated. In an attempt to evaluate the role of cholinergic receptor subtype in this regulation of glucose metabolism, we employed cultured myoblast C2C12 cells to investigate the glucose uptake in the present study. Acetylcholine (ACh) enhanced the uptake of radioactive glucose into C2C12 cells at the concentration range of 0.001 to 1.0 micromol/l. This effect was suppressed by the muscarinic antagonist atropine. Effect of ACh on muscarinic receptors was further supported by the blockade of scopolamine, another classical antagonist. Thus, activation of muscarinic receptors to enhance the radioactive glucose uptake into C2C12 cells can be considered. Moreover, pirenzepine, the antagonist of muscarinic M1 receptors, competitively antagonized the action of ACh in C2C12 cells. However, methoctramine at concentration sufficient to inhibit the muscarinic M2 receptors failed to produce similar effect. Similarly, 4-DAMP at effective concentration to block muscarinic M3 receptors lacked the influence. An activation of muscarinic M1 receptors seems responsible for the action of ACh in C2C12 cells. Pharmacological inhibition of phospholipase C by U73312 resulted in a concentration-dependent decrease in ACh-stimulated uptake of radioactive glucose into C2C12 cells. However, treatment with U73343, the inactive congener, failed to block the action of ACh. Moreover, both chelerythrine and GF 109203X diminished the action of ACh at concentrations sufficient to inhibit protein kinase C. Therefore, the obtained data suggest that increase of the glucose uptake evoked by ACh is mainly due to the activation of muscarinic M1 receptors in cultured myoblast C2C12 cells.
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PMID:Activation of muscarinic M1 receptors by acetylcholine to increase glucose uptake into cultured C2C12 cells. 1195 76

Depletion of phosphatidylinositol 4,5-bisphosphate (PIP(2)) induced by phenylephrine or endothelin causes the inhibition of acetylcholine-activated K(+) current (I(KACh)) in atrial myocytes. In the present study, we have investigated the hypothesis that muscarinic receptor induced PIP(2) depletion also causes inhibition of I(KACh), resulting in desensitization. We confirmed the expression of G(q)-coupled muscarinic receptors in mouse atrial myocytes using reverse transcriptase-polymerase chain reaction. The involvement of M(1) and M(3) receptors in desensitization is examined using specific antagonists, 4-DAMP and pirenzepine, but they significantly reduced peak I(KACh), implying nonspecific M(2) blockade. When ACh-induced phosphoinositide depletion was specifically inhibited using PLCbeta1 knock-out mice, the extent of desensitization during 4 min was 47.5 +/- 3.2%, which was not different from that in wild type (46.8 +/- 2.1%). Phenylephrine-induced phosphoinositide hydrolysis and phenylephrine-induced inhibition of I(KACh) were not affected by PLCbeta1 knock-out. To facilitate PIP(2) depletion, replenishment of PIP(2) was blocked by wortmannin. Wortmannin did not affect the desensitization and the recovery from desensitization. These results suggest that PIP(2) depletion by acetylcholine does not contribute to short-term desensitization of I(KACh). The differential regulation of I(KACh) by different phospholipase C-linked receptors may imply that receptor co-localization is required for PIP(2) to act as a signaling molecule.
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PMID:Acetylcholine-induced phosphatidylinositol 4,5-bisphosphate depletion does not cause short-term desensitization of G protein-gated inwardly rectifying K+ current in mouse atrial myocytes. 1201 67


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