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

---

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

1. The relationship between muscarinic receptor-mediated phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown and the increase of intracellular Ca2+ ([Ca2+])i has been examined in canine cultured tracheal smooth muscle cells (TSMCs). 2. Addition of acetylcholine (ACh) and carbachol led to a 2-3 fold increase in [Ca2+]i over the resting level as determined by fura-2, with half-maximal stimulation (EC50) obtained at concentrations of 97 and 340 nM, respectively. Addition of the partial agonist, bethanechol, showed a smaller increase in PIP2 turnover and [Ca2+]i than did ACh or carbachol. 3. Addition of ACh or carbachol to TSMCs that had been prelabelled with [3H]-inositol led to the rapid (5-15 s) release of inositol mono, bis and trisphosphates IP1, IP2 and IP3. The time course of IP3 accumulation is correlated with the time course of the peak rise in [Ca2+]i. 4. Inclusion of EGTA lowered the resting [Ca2+]i and markedly reduced the extent of the agonist-induced rise in [Ca2+]i. When assayed under conditions similar to those used for the [Ca2+]i measurements, EGTA reduced the muscarinic agonist-stimulated inositol phosphates (IPs) accumulation. Conversely, ionomycin could stimulate IPs accumulation and elevate [Ca2+]i. The addition of Ca2+ (2.7-617 nM) to digitonin-permeabilized TSMCs directly stimulated IPs accumulation. 5. Both Ca2+ and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) stimulated the formation of IPs in digitonin-permeabilized TSMCs prelabelled with [3H]-inositol. A further calcium-dependent increase in IPs accumulation was obtained by inclusion of either GTP gamma S or carbachol. The combined presence of carbachol and GTP gamma S elicited a synergistic effect on IPs accumulation, with half-maximal stimulation observed at approximately 8 nM free Ca2+.6. These results indicate that (i) the magnitude of the initial rise in [Ca2+], is directly related to the production of IPs and (ii) the phospholipase C-mediated PIP2 breakdown in TSMCs is sensitive to regulation by physiologically relevant concentrations of free Ca2+ ([Ca2+]f).
...
PMID:Muscarinic regulation of cytosolic free calcium in canine tracheal smooth muscle cells: Ca2+ requirement for phospholipase C activation. 829 14

Previous studies had shown that MyoD promoted nicotinic acetylcholine subunit gene expression; the present experiments were done to determine whether this subsequently led to the development of functional nicotinic acetylcholine receptors. Transfection of C3H 10T1/2 cells with MyoD cDNA resulted in the appearance of [125I]alpha-bungarotoxin binding sites; radiolabelled alpha-toxin binding was not observed in cells transfected with a plasmid that lacked MyoD cDNA. Receptor development plateaued over a time course of several days with maximal binding seven and 11 days after exposure to fusion medium. [125I]alpha-bungarotoxin binding was of high affinity (Kd = 1 nM), saturable and was inhibited by nicotinic but not muscarinic receptor ligands, with IC50s of 1-3 nM for alpha-bungarotoxin, 1-3 microM for d-tubocurarine and 3-10 microM for nicotine. Not only did the cells exhibit a cell surface nicotinic receptor but they also expressed a nicotinic receptor mediated functional response. Carbachol resulted in uptake of 22Na into the cells at concentrations similar to those required for receptor activation at a muscle type nicotinic receptor; furthermore, the functional response was effectively blocked by nicotinic receptor ligands, including alpha-bungarotoxin (IC50 = 2 to 6 nM) and d-tubocurarine (IC50 = 0.1 to 0.4 microM); muscarinic receptor ligands had no effect. A time course study showed that alpha-bungarotoxin binding and carbachol stimulated 22Na uptake developed in parallel, suggesting that the observed functional response was mediated through an interaction at the alpha-bungarotoxin recognition site.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Functional nicotinic receptor expression in mesodermal cells transfected with MyoD cDNA. 830 37

alpha 1B-Adrenergic receptor mRNA was injected into Xenopus oocytes, resulting in a norepinephrine-evoked Cl- current. The response was proportional to norepinephrine concentration, blocked by prazosin, and dependent on intracellular Ca2+ derived from inositol trisphosphate-sensitive stores. Oocytes treated with 2 micrograms/ml pertussis toxin showed a time-dependent decrease of the norepinephrine response, taking up to 72 h to show an 80% decrease. Overnight treatment with 10 micrograms/ml pertussis toxin also resulted in 80% reduction. Responses to two other cloned receptors (M1-muscarinic and serotonin-1c) expressed in oocytes were also reduced 50% or more by 72 h of pertussis toxin treatment. Pertussis toxin labeling of the cloned Xenopus alpha o-subunit translated in vitro showed that it was a significantly poorer substrate for pertussis toxin than the two mammalian alpha o-subunits expressed and assayed under identical conditions. This unexpected biochemical behavior of the Xenopus alpha o-subunit is in agreement with the rather unusual treatment conditions required to observe the effects of pertussis toxin on the receptor-evoked Cl- current in the oocyte. Injection of mammalian heterotrimeric G(o) but not Gi3 significantly enhanced the norepinephrine-evoked Cl- current in oocytes. Injection of mixtures of anti-sense oligonucleotides to the Xenopus alpha o-subunit reduced the norepinephrine-evoked Cl- current by 60% within 24 h, compared with oocytes injected with the oligonucleotides encoding sense sequences. These studies indicate that the expressed alpha 1B-adrenergic receptor, like the native muscarinic receptor, utilizes G(o) to couple to the phospholipase C-mediated Cl- current in Xenopus oocytes.
...
PMID:Coupling of the expressed alpha 1B-adrenergic receptor to the phospholipase C pathway in Xenopus oocytes. The role of Go. 838 10

Muscarinic acetylcholine receptors in the embryonic chicken heart undergo agonist-induced internalization followed by decreases in both receptor number and mRNA expression. Muscarinic agonists cause both inhibition of adenylyl cyclase and activation of phospholipase C in chick heart cells. Treatment of cells with islet activating protein, which blocks coupling of muscarinic receptors to adenylyl cyclase but not phospholipase C, blocks muscarinic receptor-mediated regulation of receptor mRNA levels. Incubation of cells with the partial agonist pilocarpine, which causes inhibition of adenylyl cyclase but not stimulation of phospholipase C, induces less down-regulation of receptor mRNA levels than agonist which regulate both second-messenger systems. Thus, both second-messenger pathways are required for maximal regulation of muscarinic receptor mRNA levels in response to receptor activation. We also demonstrate that the regulation of receptor mRNA by agonist plays an important role in modulating the rate of recovery of muscarinic acetylcholine receptor number following agonist-induced down-regulation.
...
PMID:Multiple second-messenger pathways mediate agonist regulation of muscarinic receptor mRNA expression. 838 52

We examined the rate and extent of labeling of total cellular phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol (PI) in canine tracheal smooth muscle in response to maximum levels of two different contractile agonists, carbachol (5.5 microM) and serotonin (5-hydroxytryptamine, 5-HT) (47 microM) and when a second agonist was given during a maximal contraction evoked by the first agonist. Unstimulated tracheal smooth muscle strips were incubated with [3H]-myo-inositol (MI) to label tissue MI without much labeling of inositol phospholipids. With carbachol, there was a 20-fold increase in the [3H]-MI incorporation rate into inositol phospholipids, decreases in PI and PIP2 contents, and increases in phosphatidic acid and diacylglycerol contents. PI and PIP2 specific radioactivities reached plateaus, 0.90 +/- 0.03 and 0.80 +/- 0.04, respectively, compared with [3H]-MI specific radioactivity. 5-HT at 47 microM evoked smaller changes including force development, [3H]-MI incorporation rate and lipid mass changes. However, the plateau of PI and PIP2 labeling reached levels similar to that determined during carbachol-evoked force, 0.90 +/- 0.06 and 0.82 +/- 0.04, respectively. Carbachol (55 microM) addition after incubation with 5-HT did not significantly alter the plateau levels of the specific radioactivities of PI or PIP2, although force and lipid mass changes were significantly changed. We conclude that 5-HT and muscarinic receptor coupling mechanisms utilize the same pool of PIP2 as a substrate for phospholipase C activation and the same PI pool for conversion to PIP and PIP2.
...
PMID:Common phosphatidylinositol 4,5-bisphosphate pools are involved in carbachol and serotonin activation of tracheal smooth muscle. 839 64

Carbachol stimulation of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by rat parotid gland membranes is dependent on the presence of GTP gamma S and is a result of m3-muscarinic receptor regulation of G-protein coupled, PIP2-specific phospholipase C (PLC). The PLC activity (> 80%) was solubilized with 1% Na-cholate but the solubilized enzyme was not stimulated by GTP gamma S and carbachol. Immunoblotting of rat parotid membranes with polyclonal antiserum, which recognizes alpha-subunits of the Gq/11 family, indicated the presence of two immunoreactive proteins of approximate molecular weights 41 and 42 kDa. Incubation of membranes with the common G alpha q/11 antiserum attenuated the stimulation of PIP2 hydrolysis, induced by GTP gamma S alone and by carbachol, in the presence of GTP gamma S. The antiserum had no effect on PIP2 hydrolysis in unstimulated membranes or in the cholate extract, where it is uncoupled from the G-protein. Antiserum against G alpha i, which is also coupled to the m3-muscarinic receptor in this tissue, had no effect on either basal or stimulated PIP2 hydrolysis. These results demonstrate that in rat parotid gland, activation of PIP2-specific PLC by m3-muscarinic receptor stimulation is mediated via alpha-subunits of the Gq/11 family of G-proteins.
...
PMID:Involvement of G alpha q/11 in m3-muscarinic receptor stimulation of phosphatidylinositol 4,5 bisphosphate-specific phospholipase C in rat parotid gland membranes. 839 94

To extend previous observations on the role of polyamines in insulin production, metabolism, and replication of insulin-secreting pancreatic beta cells, we have studied the role of polyamines in the regulation of the stimulus-secretion coupling of clonal rat insulinoma cells (RINm5F). For this purpose, RINm5F cells were partially depleted in their polyamine contents by use of the specific ornithine decarboxylase inhibitor difluoromethylornithine (DFMO), which led to an increase in cellular insulin and ATP contents. Analysis of different parts of the signal transduction pathway revealed that insulin secretion and the increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) after K(+)-induced depolarization were markedly enhanced in DFMO-treated cells. These effects were paralleled by increased voltage-activated Ca2+ currents, as judged by whole-cell patch-clamp analysis, probably reflecting increased channel activity rather than elevated number of channels per cell. DFMO treatment also rendered phospholipase C in these cells more sensitive to the muscarinic receptor agonist carbamylcholine, as evidenced by enhanced generation of inositol phosphates, increase in [Ca2+]i and insulin secretion, despite an unaltered ligand binding to muscarinic receptors and lack of effect on protein kinase C activity. In addition, the tumor promoter 12-O-tetradecanoylphorbol 13-acetate, at concentrations suggested to be specific for protein kinase C activation, evoked an increased insulin output in polyamine-deprived cells compared to control cells. The stimulatory effects of glucose or the cyclic AMP raising agent theophylline on insulin release were not increased by DFMO treatment. In spite of increased binding of sulfonylurea in DFMO-treated cells, there was no secretory response or altered increase in [Ca2+]i in response to the drug in these cells. It is concluded that partial polyamine depletion sensitizes the stimulus-secretion coupling at multiple levels in the insulinoma cells, including increased voltage-dependent Ca2+ influx and enhanced responsiveness to activators of phospholipase C and protein kinase C. In their entirety, our present results indicate that the behavior of the stimulus-secretion coupling of polyamine-depleted RINm5F insulinoma cells changes towards that of native beta cells, thus improving the usefulness of this cell line for studies of beta cell insulin secretion.
...
PMID:Enhanced stimulus-secretion coupling in polyamine-depleted rat insulinoma cells. An effect involving increased cytoplasmic Ca2+, inositol phosphate generation, and phorbol ester sensitivity. 840 43

Carbachol, a full muscarinic receptor agonist, stimulated [3H]inositol phosphate accumulation in both the ventral and dorsal hippocampus, but its efficacy and affinity were higher in the former area. The partial agonist oxotremorine had a weak stimulatory effect in both regions. The affinity profiles of pirenzepine and AF-DX 116 in antagonizing carbachol-stimulated [3H]inositol phosphate accumulation indicated that M1 and M3 receptors contributed equally to the response in either region. On the other hand, there were no differences in the receptor density, or in the distribution of muscarinic receptor subtypes between the two regions of the hippocampus which could account for the effect as determined in binding experiments with selective antagonists. Analysis of carbachol binding curves did, instead, indicate a difference in the way the agonist interacted with the receptors within the hippocampus, i.e., carbachol recognized three agonist affinity states (superhigh, high and low) in the ventral hippocampus, and only two (high and low) in the dorsal part. The findings thus suggested that the regional diversity in the efficacy of carbachol in stimulating phosphoinositide turnover was related to the complexity with which it bound to muscarinic receptors. Transduction processes that intervene between changes in the muscarinic receptors' conformation and activation of phospholipase C might be relevant to these differences.
...
PMID:Differences between rat dorsal and ventral hippocampus in muscarinic receptor agonist binding and interaction with phospholipase C. 843 9

The regulation of expression and function of the muscarinic acetylcholine receptor has been studied using several different systems. The role of glycosylation of the m2 receptor was examined by removal of glycosylation sites using site-directed mutagenesis followed by expression in stably transfected cells. The results demonstrated that glycosylation was not required for the synthesis and appearance of the receptors on the cell surface or for the coupling of the receptors to inhibition of adenylyl cyclase activity. Site-directed mutagenesis also was used to demonstrate that the single cysteine in the carboxy terminal domain of the m2 receptor was not required for receptor function, thus rendering unlikely a model suggesting a requirement for palmitoylation of this cysteine in receptor function. The muscarinic receptors expressed in embryonic chick heart were identified by molecular cloning. Two genes were initially identified which are expressed in chick heart and correspond to the chick m2 and m4 receptors. Experiments using the polymerase chain reaction to identify low abundance mRNAs indicate that at least one addition receptor gene is expressed in chick heart. In cell culture, activation of the muscarinic receptors decreases the levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription due to both mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. The elucidation of the factors which regulate the expression and function of muscarinic acetylcholine receptors (mAChR) is of obvious importance in understanding the mechanisms underlying cholinergic transmission. In this chapter, we will describe studies on the expression and function of wild type and mutant muscarinic receptors, the molecular characterization of mAChR expressed in chick heart, and the regulation of mAChR gene expression in response to muscarinic receptor activation.
...
PMID:Regulation of expression and function of muscarinic receptors. 844 24

Most isolated smooth muscle cells from guinea pig taenia caecum responded repeatedly, showing an all-or-none response to acetylcholine (ACh). However, the average responses of all the cells were graded owing to the difference in the threshold concentration of ACh, like that of whole tissue. The sensitivity of the muscarinic receptor on individual cells was the same as that of whole tissue and ACh bound to the receptor concentration-dependently. The shortening of the cells in response to ACh depended upon the influx of extracellular Ca2+ through the Ca channel. 45Ca2+ influx stimulated by ACh was very sharp and highly correlated with the shortening of the cells. The shortening of alpha-toxin-permeabilized smooth muscle cells was induced by increasing free Ca2+. The concentration-response relationship to free Ca2+ had a very steep slope, and the shortening appeared to be an all-or-none response rather than a graded response. In conclusion, it is suggested that isolated smooth muscle cells show an all-or-none response as a result of a slight increase in the intracellular free Ca2+ level over the threshold concentration when early signalling coupled to ACh-receptor stimulation reaches the threshold to evoke Ca2+ influx and Ca2+ release.
...
PMID:[All-or-none response of isolated smooth muscle cells from guinea pig taenia caecum to acetylcholine]. 848 19


<< Previous 1 2 3 4 5 6 7 8 9 10

---