pituitrin and 1-oleoyl-2-acetylglycerol

Physiologically relevant concentrations of [Arg(8)]-vasopressin (AVP) induce repetitive action potential firing and Ca(2+) spiking responses in the A7r5 rat aortic smooth muscle cell line. These responses may be triggered by suppression of KCNQ potassium currents and/or activation of non-selective cation currents. Here we examine the relative contributions of KCNQ5 channels and TRPC6 non-selective cation channels to AVP-stimulated Ca(2+) spiking using patch clamp electrophysiology and fura-2 fluorescence measurements in A7r5 cells. KCNQ5 or TRPC6 channel expression levels were suppressed by short hairpin RNA constructs. KCNQ5 knockdown resulted in more positive resting membrane potentials and induced spontaneous action potential firing and Ca(2+) spiking. However physiological concentrations of AVP induced additional depolarization and increased Ca(2+) spike frequency in KCNQ5 knockdown cells. AVP activated a non-selective cation current that was reduced by TRPC shRNA treatment or removal of external Na(+). Neither resting membrane potential nor the AVP-induced depolarization was altered by knockdown of TRPC6 channel expression. However, both TRPC6 shRNA and removal of external Na(+) delayed the onset of Ca(2+) spiking induced by 25pM AVP. These results suggest that suppression of KCNQ5 currents alone is sufficient to excite A7r5 cells, but AVP-induced activation of TRPC6 contributes to the stimulation of Ca(2+) spiking.

Topics: Animals; Calcium Signaling; Cell Line; Culture Media; Diglycerides; Ion Channel Gating; KCNQ Potassium Channels; Membrane Potentials; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Protein Subunits; Rats; RNA, Small Interfering; Sodium; TRPC Cation Channels; Vasopressins

The ubiquitously expressed canonical transient receptor potential (TRPC) ion channels are considered important in Ca2+ signal generation, but their mechanisms of activation and roles remain elusive. Whereas most studies have examined overexpressed TRPC channels, we used molecular, biochemical, and electrophysiological approaches to assess the expression and function of endogenous TRPC channels in A7r5 smooth muscle cells. Real time PCR and Western analyses reveal TRPC6 as the only member of the diacylglycerol-responsive TRPC3/6/7 subfamily of channels expressed at significant levels in A7r5 cells. TRPC1, TRPC4, and TRPC5 were also abundant. An outwardly rectifying, nonselective cation current was activated by phospholipase C-coupled vasopressin receptor activation or by the diacylglycerol analogue, oleoyl-2-acetyl-sn-glycerol (OAG). Introduction of TRPC6 small interfering RNA sequences into A7r5 cells by electroporation led to 90% reduction of TRPC6 transcript and 80% reduction of TRPC6 protein without any detectable compensatory changes in the expression of other TRPC channels. The OAG-activated nonselective cation current was similarly reduced by TRPC6 RNA interference. Intracellular Ca2+ measurements using fura-2 revealed that thapsigargin-induced store-operated Ca2+ entry was unaffected by TRPC6 knockdown, whereas vasopressin-induced Ca2+ entry was suppressed by more than 50%. In contrast, OAG-induced Ca2+ transients were unaffected by TRPC6 knockdown. Nevertheless, OAG-induced Ca2+ entry bore the hallmarks of TRPC6 function; it was inhibited by protein kinase C and blocked by the Src-kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Importantly, OAG-induced Ca2+ entry was blocked by the potent L-type Ca2+ channel inhibitor, *nimodipine. Thus, TRPC6 activation probably results primarily in Na ion entry and depolarization, leading to activation of L-type channels as the mediators of Ca2+ entry. Calculations reveal that even 90% reduction of TRPC6 channels would allow depolarization sufficient to activate L-type channels. This tight coupling between TRPC6 and L-type channels is probably important in mediating smooth muscle cell membrane potential and muscle contraction.

Topics: Animals; Blotting, Western; Calcium; Calcium Channels; Cations; Diglycerides; DNA Primers; Electrophysiology; Electroporation; Fura-2; Ions; Membrane Potentials; Models, Biological; Myocytes, Smooth Muscle; Nimodipine; Oligonucleotides; Patch-Clamp Techniques; Pyrimidines; Rats; Receptors, Vasopressin; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Signal Transduction; Sodium; Thapsigargin; Time Factors; TRPC Cation Channels; TRPC6 Cation Channel; Type C Phospholipases; Vasopressins

The role of protein kinase C activation and carboxyl-terminal region in rapid desensitization of the vasopressin V1a receptor was investigated in Xenopus oocytes. Preincubation of the oocytes with vasopressin or with the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG), or direct injection of active protein kinase C, all blunted the calcium response of the V1a receptor. Truncation of the 51 terminal amino acids (S374STOP) modified neither the intracellular calcium response to vasopressin nor its desensitization by vasopressin or OAG. These data suggest that desensitization of the V1a receptor is mediated by PKC activation and that its carboxyl-terminal domain is not required for signal transduction and rapid desensitization.

Topics: Animals; Calcium; Cell Membrane; Diglycerides; Enzyme Activation; Oocytes; Protein Kinase C; Rats; Receptors, Vasopressin; RNA, Complementary; Signal Transduction; Vasopressins; Xenopus laevis

We showed previously that glomerular mesangial cells displayed increased fibronectin, laminin, and type IV collagen synthesis and mRNA levels when grown in medium containing 30 mM glucose compared with those cells grown in 10 mM glucose [S. H. Ayo, R. A. Radnik, W. F. Glass II, J. A. Garoni, E. R. Rampt, D. R. Appling, and J. I. Kreisberg. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F185-F191, 1990]. However, total protein synthesis and actin mRNA were unchanged. In this report, we show that an increase in medium glucose concentration resulted in an increase in diacylglycerol (DAG) mass and transiently increased protein kinase C (PKC) activity as assessed by the translocation of PKC from the soluble to the particulate fraction. Effects of increased glucose on DAG were evident at 30 min and were maintained through 1 wk of growth in medium containing 30 mM glucose. Although total PKC activity (i.e., soluble plus particulate fractions) did not change with high-glucose treatment, the percent activity associated with the particulate fraction (i.e., activated PKC) increased significantly after 60 min in RPMI 1640 medium with 30 mM glucose. The distribution of PKC returned to control values by 24 h. High glucose did not stimulate phosphoinositide hydrolysis, as evidenced by the absence of an increase in the water-soluble inositol phosphates, indicating that DAG was not generated through the action of a phosphoinositide-specific phospholipase C. Cells treated with the cell-permeable DAG analogue 1-oleoyl-2-acetyl glycerol to activate PKC displayed approximately two-fold increases of fibronectin, laminin, and type IV collagen mRNA levels after normalization against actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cells, Cultured; Culture Media; Diglycerides; Enzyme Activation; Extracellular Matrix Proteins; Glomerular Mesangium; Glucose; Inositol; Inositol Phosphates; Protein Kinase C; RNA, Messenger; Vasopressins

The ability of Ca2(+)-mobilizing hormones to promote changes in the subcellular distribution of protein kinase C (PKC) was studied in isolated hepatocytes. In recently isolated cells the distribution of PKC between the soluble and particulate fractions was 47 and 53% respectively. Exposure of the hepatocytes to 100 nM-vasopressin produced an increased phosphoinositide turnover, as reflected by the changes in the concentrations of inositol trisphosphate and Ca2+, and in glycogen phosphorylase a activity. However, the distribution of both PKC activity and [3H]phorbol dibutyrate binding between the cytosol and the membranes remained unchanged under these conditions. To determine the threshold values of the concentrations of Ca2+ and diacylglycerol required to produce a redistribution of PKC, the hepatocytes were treated with the Ca2+ ionophore ionomycin, and with permeant diacylglycerol derivatives. Hepatocytes incubated in the presence of 100 nM-vasopressin required concentrations of Ca2+ 2.5 times those produced physiologically by the hormone to produce translocation of PKC from the cytosol to the membranes. These studies suggest that, at least in hepatocytes, activation of PKC in response to Ca2(+)-mobilizing hormones involves only the pre-existent membrane-bound enzyme without affecting the soluble enzyme.

Topics: Animals; Calcium; Cell Membrane; Cytosol; Diglycerides; Ionomycin; Kinetics; Liver; Male; Phorbol 12,13-Dibutyrate; Phosphatidylinositols; Phosphorylase a; Protein Kinase C; Rats; Rats, Inbred Strains; Vasopressins

Studies were conducted to see whether exogenous phospholipase C from Clostridium perfringens, phospholipase A2 from Crotalus adamanteus venom, arachidonic acid and 1-oleoyl-2-acetyl-sn-glycerol (OAG) mimic the anti-ketogenic action of vasopressin in isolated rat hepatocytes. Exogenous phospholipase C inhibited ketogenesis in the presence of 0.5 mM oleate. Experiments employing [1-14C]oleate, however, indicated that the mechanism involved in the anti-ketogenic action of exogenous phospholipase C is distinct from that of vasopressin. The decreased rate of the production of acid-soluble products from [1-14C]oleate in response to vasopressin could be explained by the sum of the increased rates of 14CO2 formation and [1-14C]oleate esterification. By contrast, exogenous phospholipase C suppressed not only the formation of acid-soluble products but also 14CO2 production and [1-14C]oleate esterification. Indeed, phospholipase C greatly inhibited [1-14C]oleate uptake into hepatocytes. It is suggested that the alteration of the architecture of plasma membrane by exogenous phospholipase C may lead to the disturbance of oleate uptake and consequent general suppression of oleate metabolism. Exogenous phospholipase A2, arachidonic acid and OAG increased ketogenesis regardless of the presence of oleate. The ketogenic effects may be attributed to the supply of fatty acids by these agents to hepatocytes.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Carbon Dioxide; Diglycerides; Dose-Response Relationship, Drug; Esterification; Glucose; Glycerides; Glycolysis; Ketone Bodies; Kinetics; Liver; Male; Oleic Acid; Oleic Acids; Oxidation-Reduction; Phospholipases; Phospholipases A; Phospholipases A2; Rats; Rats, Inbred Strains; Type C Phospholipases; Vasopressins

The putative roles for the second messenger, diacylglycerol, were investigated in intact platelets using a novel diacylglycerol kinase inhibitor, R 59 949, or (3-[2-[4-[bis(4-fluorophenyl)methylene]-1-piperidinyl]ethyl]-2,3- dihydro-2-thioxo-4(1H)-quinazolinone). The compound inhibited the diacylglycerol kinase in a concentration-dependent manner (10(-8) to 10(-5) M) in isolated platelet membranes and in intact platelets. When platelets were stimulated with vasopressin in the presence of the compound, protein kinase C activity was markedly increased; the formation of inositol phosphates, the increase in intracellular Ca2+ and shape-change reaction were antagonized while the vasopressin-induced polyphosphoinositide synthesis was amplified, and this in a distinct inositolphospholipid pool. In the presence of R 59 949, vasopressin- as well as collagen-induced release reaction and aggregation was strongly increased, independently of the formation of arachidonate metabolites. It is concluded that diacylglycerol formed after receptor activation, likely by activating the protein kinase C, plays an important role in the propagation of platelet functional responses in casu aggregation and secretion and controls the termination of the primary receptor coupled responses.

Topics: Blood Platelets; Calcium; Cell Membrane; Collagen; Cyclic AMP; Diacylglycerol Kinase; Diglycerides; Glycerides; Humans; Phosphates; Phosphatidylinositols; Phospholipids; Phosphoproteins; Phosphorylation; Phosphotransferases; Piperidines; Platelet Aggregation; Protein Kinase C; Quinazolines; Quinazolinones; Second Messenger Systems; Thromboxane B2; Vasopressins

Two activators of protein kinase C, phorbol 12,13-dibutyrate (PDBu) and 1-oleoyl-2-acetylglycerol (OAG), augment electrically stimulated vasopressin and oxytocin secretion from the nerve terminals of the isolated rat neurointermediate lobe. The increased hormone release produced by PDBu is specific to the beta-phorbol conformation, and is dependent upon electrical stimulation in the presence of calcium. Furthermore, the potentiation of release was evident during low frequency stimulation (4 Hz) but not when the same number of pulses were applied at 20 Hz. This occlusion of the phorbol ester's effect by high-frequency stimulation suggests that activation of protein kinase C may play a role in the normal process of frequency-dependent facilitation of secretion in the neurohypophysial system.

Topics: Animals; Diglycerides; Electric Stimulation; Glycerides; In Vitro Techniques; Male; Oxytocin; Phorbol 12,13-Dibutyrate; Phorbol Esters; Pituitary Gland, Posterior; Protein Kinase C; Rats; Rats, Inbred Strains; Vasopressins

Activators of protein kinase C, a calcium- and phospholipid-dependent protein kinase, inhibit vasopressin-stimulated water flow in toad bladder. To determine the biochemical mechanisms of this inhibition, we examined the effects of activators of protein kinase C on arginine vasopressin (AVP)-stimulated adenylate cyclase activity in cultured rabbit cortical collecting tubular cells. The phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA), the diacylglycerol, 1-oleyl-2-acetyl glycerol (OAG), and the diacylglycerol kinase inhibitor, R59022, all rapidly activate protein kinase C in collecting tubular cells. Pretreatment with PMA produces a delayed inhibition (greater than or equal to 4 h) of AVP-stimulated adenylate cyclase activity. The 4-h time lag suggests that the effects of protein kinase C are mediated indirectly, possibly as a consequence of stimulating cell proliferation. PMA does not inhibit cholera toxin- or forskolin-stimulated adenylate cyclase activity, suggesting an effect on the vasopressin receptor or coupling of the receptor to the stimulatory guanine nucleotide regulatory protein. Neither prostaglandins nor the inhibitory guanine nucleotide regulatory protein appear to mediate this effect. In contrast, treatment with either OAG or R59022 produces a rapid inhibition of both AVP- and forskolin-stimulated adenylate cyclase activity suggesting a prominent distal site of action, presumably at the catalytic subunit of adenylate cyclase. The results demonstrate that different activators of protein kinase C inhibit AVP-stimulated adenylate cyclase activity by distinctly different mechanisms possibly by altering the substrate specificity or activating multiple forms of the kinase. These results have important implications when using different activators to study the biological effects of protein kinase C.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Cells, Cultured; Diglycerides; Kidney Tubules; Kidney Tubules, Collecting; Phorbol Esters; Protein Kinase C; Pyrimidinones; Rabbits; Stimulation, Chemical; Tetradecanoylphorbol Acetate; Thiazoles; Vasopressins

To evaluate a possible modulation by protein kinase C of hormonal, cAMP-mediated effects on renal epithelial cells, we studied the effect of protein kinase C activators and of bradykinin on intracellular cAMP accumulation in MDCK cells. A 15-min pretreatment of cells with phorbol 12-myristate 13-acetate or 1-oleoyl-2-acetylglycerol induced a dose-dependent inhibition of vasopressin-stimulated cAMP synthesis, but not of basal or glucagon-, prostaglandin E2-, and forskolin-stimulated cAMP generation. 4 alpha-Phorbol 12,13-didecanoate, inactive on protein kinase C, did not affect cAMP accumulation. Bradykinin (0.1-10 microM) also inhibited the stimulatory effect of vasopressin on cAMP synthesis in a concentration-dependent manner, but affected neither basal cAMP content, nor its stimulation by glucagon, prostaglandin E2 and forskolin. The effect of activators of protein kinase C and of bradykinin occurred while renal prostaglandin synthesis was blocked with indomethacin. The inhibitory effect of protein kinase C activators and bradykinin on cAMP generation was reversed by the protein kinase C inhibitor H7, was enhanced by monensin, one effect of which is to block the recycling of membrane receptors, and persisted when the GTP-binding protein N1 was blocked with 1 mM Mn2+. Our data suggest that: protein kinase C can modulate the tubular effects of vasopressin by inhibiting cAMP generation; this effect is not mediated by renal prostaglandins, and might result from a direct action on the vasopressin receptor, or on its coupling with Ns; the modulation by bradykinin of vasopressin effects are likely to be exerted, at least partly, through activation of protein kinase C.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Bradykinin; Cell Line; Colforsin; Cyclic AMP; Diglycerides; Dinoprostone; Enzyme Activation; Glucagon; Isoquinolines; Piperazines; Prostaglandins E; Protein Kinase C; Tetradecanoylphorbol Acetate; Vasopressins

The regulatory mechanism of hepatic palmitate oxidation into ketone bodies by c-kinase has been studied in isolated hepatocytes. Glucagon and epinephrine stimulated [U-14C]palmitate oxidation to ketone bodies by 60 and 25% as early as at 1 h. The stimulatory effects were almost totally prevented by the simultaneous presence of vasopressin, phorbol 12-tetradecanoate 13-acetate (TPA), or diacylglycerol (1-oleoyl-2-acetylglycerol). When hepatocytes were treated with glucagon or epinephrine, carnitine palmitoyltransferase (CPT), a key regulatory enzyme of palmitate oxidation, was activated. This hormone-induced activation of CPT was not observed in the presence of TPA. These observations suggest that c-kinase inhibits glucagon- or epinephrine-stimulated palmitate oxidation to ketone bodies, and that this inhibition may be mediated through a covalent modification of CPT.

Topics: Animals; Carnitine O-Palmitoyltransferase; Diglycerides; Enzyme Activation; Epinephrine; Glucagon; Ketone Bodies; Liver; Male; Palmitic Acid; Palmitic Acids; Protein Kinase C; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate; Vasopressins

WRK-1 rat mammary tumour cells respond to vasopressin with increased accumulation of inositol phosphates as well as increased precursor incorporation into phosphatidylinositol. The phorbol ester, phorbol 13-myristate 12-acetate (PMA) inhibits by 80% both inositol phosphate accumulation and increased precursor incorporation. This inhibition is much less evident at early times (2 min) than at later times (25 min). The vasopressin-induced rise in cytosolic free Ca2+ is inhibited in a similar manner. Oleoylacetylglycerol is inactive with respect to inhibition of vasopressin-induced increases in incorporation of 32P into phosphoinositides. PMA has no effect on vasopressin binding at saturating concentrations of the hormone and does not affect the binding affinity.

Topics: Adenosine Triphosphate; Animals; Calcium; Cells, Cultured; Diglycerides; Inositol Phosphates; Mammary Neoplasms, Experimental; Phosphates; Phosphatidic Acids; Rats; Tetradecanoylphorbol Acetate; Vasopressins

The role of the phosphoinositide turnover-protein kinase C pathway in mediating PDGF-stimulated c-myc expression and cell proliferation was studied. Both direct activators of kinase C (e.g. phorbol ester analogues) and hormones that activate kinase C via receptor-mediated phosphoinositide turnover (e.g. PDGF, bradykinin, or vasopressin) elicited a rapid increase in c-myc mRNA expression. Desensitization of the kinase C pathway by prolonged exposure to phorbol abolished the induction of c-myc by subsequent phorbol challenge and attenuated c-myc induction by PDGF and bradykinin, but did not affect PDGF-stimulated mitogenesis. Bradykinin and phorbol esters stimulated the same magnitude of c-myc expression as PDGF but elicited less than one-tenth the PDGF-induced mitogenic response. We conclude that stimulation of c-myc expression is a common response to a diverse group of agents that elicit phosphoinositide turnover and activate protein kinase C, and that neither activation of protein kinase C nor enhanced c-myc expression is sufficient for the mitogenic action of PDGF.

Topics: Animals; Bradykinin; Cell Division; Cell Line; Diglycerides; DNA; Enzyme Activation; Epidermal Growth Factor; Humans; Mitogens; Phosphatidylinositols; Platelet-Derived Growth Factor; Protein Kinase C; Proto-Oncogenes; RNA, Messenger; Tetradecanoylphorbol Acetate; Thymidine; Vasopressins

Receptor-mediated breakdown of PtdIns(4,5)P2 produces two cellular signals, Ins(1,4,5)P3, which can release intracellular Ca2+, and diacylglycerol, which activates a Ca2+- and phospholipid-dependent protein kinase (protein kinase C). This study assesses the significance of protein kinase C in relation to phenylephrine- and vasopressin-induced Ca2+ mobilization in hepatocytes. Phorbol ester (4 beta-phorbol-12-myristate-13-acetate), which can directly activate protein kinase C, had no effect either on Ca2+ efflux from the cell (measured with arsenazo III) or on Ca2+ influx (measured with Quin-2), processes which are inhibited and stimulated, respectively, by both phenylephrine and vasopressin. No evidence of synergism between phorbol ester pretreatment of hepatocytes and the Ca2+ ionophore (ionomycin)-mediated effects on the increase of cytosolic free Ca2+ and phosphorylase activation could be obtained. These findings suggest that protein kinase C is not obligatorily involved in the regulation of hepatocyte Ca2+ fluxes. Pretreatment of hepatocytes with phorbol ester (PMA) or 1-oleoyl-2-acetylglycerol totally inhibited the effects of phenylephrine in elevating the cytosolic free Ca2+; half-maximal inhibitory effects occurred at PMA and 1-oleoyl-2-acetylglycerol concentrations of 1 ng/ml and 12 micrograms/ml, respectively. In contrast, pretreatment with PMA had a much smaller effect on Ca2+ mobilization induced by vasopressin. These observations suggest that protein kinase C may be involved in "down-regulation" of the alpha 1-receptor in hepatocytes and may thus exert a negative influence on the Ca2+-signalling pathway.

Topics: Aminoquinolines; Animals; Calcimycin; Calcium; Diglycerides; Dose-Response Relationship, Drug; Drug Synergism; Ethers; Ionomycin; Liver; Male; Phenylephrine; Phorbols; Phosphorylases; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate; Vasopressins

The tetradecapeptide bombesin converts epidermal growth factor (EGF) receptors on Swiss 3T3 cells from a high affinity state (KD = 9.8 X 10(-11)M) to a lower affinity state (KD = 1.8 X 10(-9)M). This conversion occurs when the cells are incubated with bombesin at 37 degrees C but not when incubated at 4 degrees C. Previously, a number of other (chemically unrelated) cell growth-promoting peptides and polypeptides have been shown to induce a similar indirect, temperature-dependent reduction of EGF receptor affinity. We have now demonstrated that hormones and growth factors which cross-regulate EGF receptor affinity in Swiss 3T3 cells have a common ability to stimulate the breakdown of phosphoinositides in these cells. We propose that the reduction of EGF receptor affinity is a consequence of the activation of protein kinase C by the diacylglycerol generated by this breakdown. In support of this proposal we have found that exogenously added diacylglycerol reduces the affinity of the Swiss 3T3 cell EGF receptor.

Topics: Animals; Bombesin; Cells, Cultured; Diglycerides; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Kinetics; Mice; Models, Biological; Phosphatidylinositols; Platelet-Derived Growth Factor; Protein Kinase C; Protein Kinases; Receptors, Cell Surface; Vasopressins