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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)

The characteristics of the specific binding of labelled insulin to turtle thyroid microsomes were investigated. Binding experiments were performed in Krebs-Ringer bicarbonate buffer (pH 7.4) at 25 or 4 degrees C for different periods of time. Dissociation of the labelled insulin from the binding sites was also evaluated. It was found that the binding is dependent on time, temperature and microsomal protein concentration, with an optimum pH of 8.0. Unlabelled insulin and pro-insulin competed with the labelled insulin, binding in direct proportion to their biological activities, while glucagon and growth hormones did not compete for the binding sites. Scatchard plot analysis established the presence of binding sites of high and low affinities, and the rate of dissociation of bound insulin was considerably increased by the addition of unlabelled insulin. Both results are compatible with a negative co-operativity site-site interaction model. Trypsin abolished the insulin binding. These findings indicate that the microsomes from the turtle thyroid gland contain specific binding sites for insulin. However, pre-incubation of microsomes with phospholipase C or S-adenosyl-L-methionine (SAM), or incubation in the presence of 2 mol NaCl/l did not increase the specific insulin binding. Therefore, the binding properties are similar to those observed in mammalian insulin-responsive tissues except for the absence of the effects of 2 mol NaCl/l, phospholipase C or SAM, which suggests the absence of masked insulin-binding sites.
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PMID:Characterization of the insulin-binding sites in turtle thyroid microsomes. 351 Nov 68

Vasopressin induced a transient increase of 50% in the total concentration of diacylglycerols (determined by g.l.c.) in isolated hepatocytes. The increase was maximal at 0.25 min, and the concentration of diacylglycerols in cells treated with vasopressin had returned to the basal value by 4 min. No change in the concentration of diacylglycerols was observed after the treatment of cells with glucagon. The dependency of this effect on the concentration of vasopressin was similar to that of the effect of the hormone on 45Ca2+ efflux measured at 0.1 mM extracellular Ca2+. Vasopressin increased the proportion of arachidonic acid and stearic acid and decreased the proportion of oleic acid present in the diacylglycerols. In hepatocytes prelabelled with [14C]arachidonic acid, vasopressin increased the amount of [14C]diacylglycerol. The effects of vasopressin on the total concentration of diacylglycerols and [14C]diacylglycerol were mimicked by an exogenous phospholipid phosphodiesterase (phospholipase C) from Clostridium perfringens. The results are consistent with the conclusion that the transient increase in diacylglycerols induced by vasopressin is caused by the rapid hydrolysis of both the phosphoinositides and one or more other phospholipids.
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PMID:A transient increase in diacylglycerols is associated with the action of vasopressin on hepatocytes. 647 30

Receptors for regulatory peptides (hormones or neurotransmitters) play a pivotal role in the ability of cells to taste the rich neuroendocrine environment of the gut. Recognition of low concentration of peptides with a high specificity and translation of the peptide-receptor interaction into a biological response through different signalling pathways (adenylyl cyclase-cAMP or phospholipase C-phosphatidylinositol) are crucial properties of receptors. While many new receptors have been identified and thereafter characterized functionally during the 1980s, molecular biology now emerges as the privileged way for the structural characterization and discovery of receptors. Different strategies of receptor cloning have been developed which may or may not require prior receptor purification. Among cloning strategies that do not require receptor purification, homology screening of cDNA libraries, expression of receptor cDNA or mRNA in Xenopus laevis oocytes or in COS cells, and the polymerase chain reaction method achieved great success, e.g. cloning of receptors for cholecystokinin, gastrin, glucagon-like peptide 1, gastrin-releasing peptide/bombesin, neuromedin K, neuropeptide Y, neurotensin, opioids, secretin, somatostatin, substance K, substance P and vasoactive intestinal peptide. All these receptors belong to the superfamily of G-protein-coupled receptors which consist of a single polypeptide chain (350-450 amino acids) with seven transmembrane segments, an N-terminal extracellular domain and a C-terminal cytoplasmic domain. In this chapter, we have detailed the properties of three receptors which play an important role in digestive tract physiology and illustrate various signal transduction pathways: pancreatic beta-cell galanin receptors which mediate inhibition of insulin release and intestinal epithelial receptors for vasoactive intestinal peptide and peptide YY, which mediate the stimulation and inhibition of water and electrolyte secretion, respectively.
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PMID:Receptors for gut regulatory peptides. 751 Sep 49

Rat islets respond to glucose stimulation with a marked first and second phase increase in insulin secretion. In contrast, mouse islets have a similar first phase response but little second phase secretion. In these studies, we determined if activation of phospholipase C (PLC) accounts for these differences in second phase insulin secretion in these two species. Stimulation of freshly isolated mouse and rat islets with 15 mM glucose resulted in comparable first phase insulin secretion; however, the second phase response from mouse islets was only doubled from 28 +/- 6 to 60 +/- 7 pg/islet.min compared with an increase from 24 +/- 4 to 1064 +/- 93 pg/islet.min from rat islets. The addition of the muscarinic agonist carbachol (100 microM) in the presence of 15 mM glucose, however, markedly increased second phase insulin release from mouse islets to 801 +/- 80 pg/islet.min. Similar increases in second phase insulin release from mouse islets were obtained with the addition of 500 nM of the protein kinase C activator tetradecanoyl phorbol acetate in the presence of 15 mM glucose. However, the incretin factor glucagon-like peptide-1, which elevates islet cAMP levels, had little effect on second phase insulin release in the mouse. An analysis of PLC-mediated phosphoinositide (PI) hydrolysis revealed that 15 mM glucose increased inositol phosphate (IP) accumulation 0.5-fold above baseline in mouse islets compared with 3.7-fold in rat islets. In contrast, carbachol stimulated IP accumulation 3.5-fold in both mouse and rat islets. Analysis of PLC isozymes with isozyme specific monoclonal antibodies, demonstrated that mouse islets express 14 +/- 4% of PLC-delta 1 and 18 +/- 6% of PLC-beta 1 compared with rat islets but similar amounts of the PLC-gamma 1 (117 +/- 16%). These findings suggest that the decreased second phase insulin secretory response in mouse compared with rat islets results, at least in part, from an inability of high glucose to stimulate comparable increments in PI hydrolysis. This lack of glucose responsiveness may be due to the pronounced underexpression of specific PLC isozymes in the mouse.
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PMID:Regulation of insulin release by phospholipase C activation in mouse islets: differential effects of glucose and neurohumoral stimulation. 758 23

Prostaglandins, released from Kupffer cells, have been shown to mediate the increase in hepatic glycogenolysis by various stimuli such as zymosan, endotoxin, immune complexes, and anaphylotoxin C3a involving prostaglandin (PG) receptors coupled to phospholipase C via a G(0) protein. PGs also decreased glucagon-stimulated glycogenolysis in hepatocytes by a different signal chain involving PGE2 receptors coupled to adenylate cyclase via a Gi protein (EP3 receptors). The source of the prostaglandins for this latter glucagon-antagonistic action is so far unknown. This study provides evidence that Kupffer cells may be one source: in Kupffer cells, maintained in primary culture for 72 hours, glucagon (0.1 to 10 nmol/L) increased PGE2, PGF2 alpha, and PGD2 synthesis rapidly and transiently. Maximal prostaglandin concentrations were reached after 5 minutes. Glucagon (1 nmol/L) elevated the cyclic adenosine monophosphate (cAMP) and inositol triphosphate (InsP3) levels in Kupffer cells about fivefold and twofold, respectively. The increase in glycogen phosphorylase activity elicited by 1 nmol/L glucagon was about twice as large in monocultures of hepatocytes than in cocultures of hepatocytes and Kupffer cells with the same hepatocyte density. Treatment of cocultures with 500 mumol/L acetylsalicylic acid (ASA) to irreversibly inhibit cyclooxygenase (PGH-synthase) 30 minutes before addition of glucagon abolished this difference. These data support the hypothesis that PGs produced by Kupffer cells in response to glucagon might participate in a feedback loop inhibiting glucagon-stimulated glycogenolysis in hepatocytes.
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PMID:Feedback-inhibition of glucagon-stimulated glycogenolysis in hepatocyte/Kupffer cell cocultures by glucagon-elicited prostaglandin production in Kupffer cells. 759 Jun 78

The two forms of pituitary adenylate cyclase-activating polypeptide (PACAP), PACAP27 and PACAP38, are neuropeptide hormones related to the vasoactive intestinal peptide/secretin/glucagon family of peptides. PACAP receptors that are positively coupled to adenylyl cyclase and phospholipase C have been recently identified. We have investigated the expression of PACAP-Rs in undifferentiated and differentiated PC-12 cells. PACAP27 and PACAP38 failed to significantly increase cAMP or [3H]inositol monophosphate levels in undifferentiated PC-12 cells treated with vehicle, insulin-like growth factor I, or epidermal growth factor but greatly elevated levels after differentiation with nerve growth factor (NGF) or basic fibroblast growth factor. PACAP responsiveness increased significantly after 24 hr of NGF treatment, reaching a maximum within 4 days. At this time of differentiation, the effect of PACAP was dose dependent between 1 nM and 0.1 microM, whereas vasoactive intestinal peptide, at the maximal dose of 10 microM, slightly increased cAMP formation and failed to affect [3H]inositol monophosphate content. Radioreceptor assays, performed with 125I-PACAP27, revealed the induction of high affinity type I PACAP receptors in differentiated PC-12 cells. Using reverse transcription-polymerase chain reaction methodology, we showed the absence of type I PACAP receptor mRNAs in undifferentiated PC-12 cells and the expression of PACAP-R-hop mRNA after NGF or basic fibroblast growth factor treatment. The increased PACAP responsiveness induced by these growth factors in PC-12 cells may therefore result from the expression of the PACAP-R-hop isoform, positively coupled to both adenylyl cyclase and phospholipase C.
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PMID:Differentiation induces pituitary adenylate cyclase-activating polypeptide receptor expression in PC-12 cells. 762 75

While many observations indicate that prostaglandins may act as positive regulators of hepatocyte proliferation, the underlying mechanisms are not known. We have examined some of the signal pathways in the growth response induced by prostaglandins in hepatocytes, with particular focus on adenylyl cyclase and phosphoinositide-specific phospholipase C. Adult rat hepatocytes were cultured as primary monolayers in serum-free medium in the presence of EGF and insulin. PGE2 or PGF2 alpha (added 0-3 h after plating) enhanced the incorporation of [3H]-thymidine into DNA (measured at 50 h); at 100 microM the stimulation was about threefold PGI2 and PGD2 also showed significant but smaller stimulatory effects. No significant increase in the level of cyclic AMP (cAMP) was detected in response to any of the prostaglandins. Low concentrations of glucagon (0.1-10 nM), a potent activator of hepatic adenylyl cyclase, or 8-bromo-cAMP (0.1-10 microM) enhanced the DNA synthesis. When 8-bromo-cAMP was used in maximally effective concentrations, no further stimulation was obtained by combining it with glucagon, whereas the effects of PGE2 and 8-bromo-cAMP were completely additive. All the prostaglandins also showed additivity with the effect of glucagon on the DNA synthesis. PGE2, PGF2 alpha, PGI2, and PGD2 increased intracellular inositol-1,4,5-trisphosphate (InsP3), with a relative order of efficacy roughly corresponding to their activity as stimulators of DNA synthesis. Increases in cytosolic free Ca2+, as measured in single cells, were elicited in a majority of the hepatocytes by all these prostaglandins at 1 microM. Supramaximal concentrations of vasopressin, a strong activator of phospholipase C in hepatocytes, acted additively with PGE2 on the DNA synthesis. Pretreatment of the hepatocytes with a concentration of pertussis toxin that prevented the inhibitory effect of PGE2 on glucagon-induced cAMP accumulation did not abolish the ability of PGE2 to stimulate the DNA synthesis. The results do not support a role for adenylyl cyclase activation in the stimulatory effect of prostaglandins on hepatocyte growth. While the data are compatible with an involvement of phosphoinositide-specific phospholipase C in the growth-promoting effect of prostaglandins in cultured rat hepatocytes, they suggest this may not be the sole mechanism.
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PMID:On the mechanisms of the growth-promoting effect of prostaglandins in hepatocytes: the relationship between stimulation of DNA synthesis and signaling mediated by adenylyl cyclase and phosphoinositide-specific phospholipase C. 765 56

The regulation of Ca2+ influx in rat hepatocytes by glucagon and cyclic AMP (cAMP) was investigated. Exposing hepatocytes to glucagon resulted in an increase in the initial rate of Ca2+ entry. The concentrations of glucagon producing half-maximal and maximal stimulation of Ca2+ entry were 10(-10) and 10(-8) M, respectively. A similar stimulation of Ca2+ influx was obtained in cells exposed to cAMP analogues or to forskolin. Exposing hepatocytes suspended in nominally Ca(2+)-free medium to glucagon for 3 min produced a 9% decrease in the size of the vasopressin-sensitive Ca2+ pool; in contrast, N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (Bt2cAMP) slightly augmented the size of this pool. Glucagon and Bt2cAMP synergized the initial vasopressin-stimulated Ca2+ and Mn2+ influx rates, but only moderately increased the initial rate of Ca2+ entry after thapsigargin addition. The glucagon- and Bt2cAMP-stimulated Ca2+ influx was inhibited by the same antagonists of the plasma membrane Ca2+ carriers that mediate Ca2+ entry during stimulation by vasopressin. Thus, cAMP does not stimulate Ca2+ entry through either a capacitative type of mechanism or inositol phosphate turnover. The authors' findings instead suggest that cAMP acts directly, or through protein kinase A on the same Ca2+ carriers that are activated by phospholipase C-linked receptor agonists.
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PMID:Cyclic AMP stimulates Ca2+ entry in rat hepatocytes by interacting with the plasma membrane carriers involved in receptor-mediated Ca2+ influx. 781 85

Calcium and phosphorus metabolism is mainly regulated by PTH through its actions on kidney and bone. PTHrP, which is associated with the hypercalcemia of malignancy syndrome, binds to and activates the same receptor that PTH does. cDNA clones of PTH/PTHrP receptors from rat osteosarcoma (ROS 17/2.8) and opossum kidney (OK) cells are highly homologous and are members of a novel G protein-linked receptor family that includes calcitonin, glucagon, GLP-1, GHRH, VIP, and secretin receptors. Analysis of the protein sequence predicts a receptor with 7 transmembrane domains, a 155 amino acids (aa) extracellular (EC) N-terminal, and 130aa intracellular C-terminal domaina. The extracellular domain has 6 conserved cysteines and 4 potential glycosylation sites. When transfected in COS cells, both receptors are able to bind PTH and PTHrP active fragments with equal affinity. Likewise, agonists activate both adenylate cyclase and phospholipase C efficiently. The N-terminal EC domain and the first EC loop seem to determine the receptor binding capacity with the agonists. Activation of adenylate cyclase and phospholipase C might involve multiple sites between the 3rd helix and the C-terminal tail. Partial characterization of the rat PTH/PTHrP receptor gene demonstrates the existence of at least 15 exons. The first six transmembrane domains are encoded by separated exons. The PTH/PTHrP receptor mRNA is expressed mainly in kidney and bone, and also is widely expressed in many tissues, but not all. A major 2.3-2.5 kb transcript is observed in all these tissues. Nevertheless, 2 larger transcripts are observed in kidney and liver, and multiple smaller mRNA species are observed in kidney, skin, and testis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Mode of action of parathyroid hormone (PTH) and PTH-related peptide (PTHrP) in target organs]. 785 77

Measurements of the cytosolic calcium concentration in single cells of the clonal endocrine pancreatic glucagon-secreting cell line INR1 G9 revealed the existence of spontaneous calcium oscillations in 20-70% of these cells. Inhibition of these spontaneous oscillations by thapsigargin as well as the phospholipase C inhibitor U 73122 demonstrated involvement of calcium release from intracellular stores, probably mediated by a high basal activity of phospholipase C. Removal of extracellular calcium but not the L-type calcium channel antagonists verapamil or nifedipine terminated the spontaneous oscillations, suggesting that calcium influx by a pathway distinct from L-type channels contributed to the oscillations. Similar spontaneous calcium oscillations could be the pacemaker of pulsatile glucagon release in endocrine pancreatic A-cells.
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PMID:Spontaneous calcium oscillations in clonal endocrine pancreatic glucagon-secreting cells. 799 60


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