Increased Diacylglycerol Levels Inhibit [20-3H]Phorbol 12, 13-Dibutyrate Binding and the Glucocorticoid-Mediated Increase in Glycerol Phosphate Dehydrogenase Levels in C6 Rat Glioma Cells

I examined whether the phorbol ester-mediated inhibition of glycerol 3-phosphate dehydrogenase (GPDH) induction could be mimicked by raising the cellular diacylglycerol levels. Phorbol ester tumor promoters and diacylglycerols activate protein kinase C. An increase in radiolabeled diacylglycerol levels in C6 rat glioma cells was observed when cells were prelabeled overnight with [3H]arachidonic acid and treated with either phospholipase C (Clostridium perfringens) or 2-bromooctanoate. The increase was dose dependent. The diacylglycerols competed with [20-3H]phorbol 12,13-dibutyrate in binding to the phorbol ester receptor. A Scatchard analysis of the binding of cells treated with 0.1 unit/ml of phospholipase C demonstrated that the inhibition was mainly due to a decrease in binding affinity and not in the total number of binding sites. 2-Bromooctanoate and phospholipase C, but not the synthetic diacylglycerol 1-oleoyl 2-acetyl glycerol, inhibited the glucocorticoid induction of GPDH levels. Boiled phospholipase C, phospholipase A2, or phospholipase D was ineffective in inhibiting induction, a result suggesting that the inhibition was not due to nonspecific membrane perturbation. Thus, inhibition of the glucocorticoid-mediated increase in GPDH induction is most likely mediated by protein kinase C, and not by an alternate phorbol ester receptor.     

Phorbol ester-induced translocation of diacylglycerol kinase from the cytosol to the membrane in Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-sn-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent, starved Swiss 3T3 fibroblasts. We utilized exogenous dioleoylglycerol as substrate for the kinase. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C (Ca2+/phospholipid-dependent enzyme) by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on Swiss 3T3 membrane-bound diacylglycerol kinase nor does it directly effect cytosolic diacylglycerol kinase. When phorbol ester is added to Swiss 3T3 membranes in the presence of ATP, magnesium, and calcium, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Reconstitution studies show that the soluble rat brain diacylglycerol kinase binds to diacylglycerol-enriched membranes, produced by treatment of red cell ghosts with phospholipase C or calcium, suggesting that cytosolic diacylglycerol kinase may be capable of translocation to the membrane in response to elevated substrate concentration in the intact cell. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, by 5 min, also suggesting that the translocation of diacylglycerol kinase activity is regulated primarily by substrate concentration.     

Interactions Among Lithium, Calcium, Diacylglycerides, and Phorbol Esters in the Regulation of Adrenocorticotropin Hormone Release from AtT-20 Cells

Interactions among lithium, calcium, and phorbol esters in the regulation of adrenocorticotropin hormone (ACTH) release were examined in a tumor cell line (AtT-20) of the anterior pituitary. Lithium, which blocks the phosphatase that converts inositol phosphates (IPs) to inositol, increases the levels of IPs in these cells and stimulates ACTH release. This ion potentiates the ability of calcium, an activator of phospholipase C, to raise levels of IPs in these cells and to stimulate ACTH secretion. Pretreatment of AtT-20 cells with calcium specifically abolishes the ACTH release response to lithium or calcium, a result suggesting that these secretagogues may act through a common mechanism to induce hormone secretion. Prior exposure of AtT-20 cells to either lithium or calcium also attenuates the ACTH release induced by phorbol ester, an activator of protein kinase C. To examine the link among lithium, calcium, phosphatidylinositol (PI) turnover, and phorbol ester-evoked ACTH secretion, AtT-20 cells were treated with 1-oleoyl-2-acetoyl-sn-3-glycerol (OAG), an analogue of the diacylgylcerols that are formed by phospholipase C during PI metabolism and that also activate protein kinase C. OAG itself does not alter ACTH release or the levels of IPs in AtT-20 cells. Pretreatment of AtT-20 cells with OAG, however, selectively blocks the ACTH release response to lithium, calcium, or phorbol ester. Furthermore, such pretreatment reduced the ability of lithium to increase levels of IPs. The results suggest that one mechanism of action of lithium is to potentiate selectively an action of calcium, possibly the stimulation of phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The alpha 1-adrenergic transduction system in hamster brown adipocytes. Release of arachidonic acid accompanies activation of phospholipase C.

Previous studies of brown adipocytes identified an increased breakdown of phosphoinositides after selective alpha 1-adrenergic-receptor activation. The present paper reports that this response, elicited with phenylephrine in the presence of propranolol and measured as the accumulation of [3H]inositol phosphates, is accompanied by increased release of [3H]arachidonic acid from cells prelabelled with [3H]arachidonic acid. Differences between stimulated arachidonic acid release and formation of inositol phosphates included a requirement for extracellular Ca2+ for stimulated release of arachidonic acid but not for the formation of inositol phosphates and the preferential inhibition of inositol phosphate formation by phorbol 12-myristate 13-acetate. The release of arachidonic acid in response to phenylephrine was associated with an accumulation of [3H]arachidonic acid-labelled diacylglycerol, and this response was not dependent on extracellular Ca2+ but was partially prevented by treatment with the phorbol ester. The release of arachidonic acid was also stimulated by melittin, which increases the activity of phospholipase A2, by ionophore A23187, by lipolytic stimulation with forskolin and by exogenous phospholipase C. The arachidonic acid response to phospholipase C was completely blocked by RHC 80267, an inhibitor of diacylglycerol lipase, but this inhibitor had no effect on release stimulated with melittin or A23187 and inhibited phenylephrine-stimulated release by only 40%. The arachidonate response to forskolin was additive with the responses to either phenylephrine or exogenous phospholipase C. These data indicate that brown adipocytes are capable of releasing arachidonic acid from neutral lipids via triacylglycerol lipolysis, and from phospholipids via phospholipase A2 or by the sequential activities of phospholipase C and diacylglycerol lipase. Our findings also suggest that the action of phenylephrine to promote the liberation of arachidonic acid utilizes both of these reactions.     

Regulation of platelet phospholipase C

We have investigated factors affecting the activation of phospholipase C in human platelets. Prior exposure of platelets to phorbol esters that stimulated protein kinase C inhibits the activation of phospholipase C in response to a variety of receptor-directed agonists, including alpha- and gamma-thrombin and thromboxane A2 analogues. Such activation has been assayed by measurements of accumulated InsP3 (including Ins(1,4,5)P3 and Ins(1,3,4)P3) and PtdOH. Inhibition is not overcome by Ca2+ ionophores, and substances that block or mimic Na+-H+ exchange neither block nor mimic these inhibitory effects. Cyclic AMP and cyclic GMP, other agents known to inhibit phospholipase C activation, do not accumulate in platelets exposed to phorbol esters. Although a portion of the effects of phorbol ester on InsP3 accumulation may be explained by 5-phosphomonoesterase activity, it is likely that more direct effects on phospholipase C are being exerted as well, and contribute the major inhibitory route. We have examined the susceptibility of adenylyl cyclase-associated Gi and 'Gp'-activated phospholipase C to inhibitory ADP-ribosylation by pertussis toxin-derived enzyme (S1 protomer) administered to saponin-permeabilized platelets. The effects of alpha-thrombin on adenylyl cyclase can be inhibited by up to 50% by S1, at which point inhibition of phospholipase C is barely detectable. Thromboxane A2 analogues, which do not affect adenylyl cyclase (Gi), stimulate phospholipase C; this effect is not impaired by S1. We therefore propose that the inhibitory effects of phorbol esters on the activation of phospholipase C are not mediated primarily by effects on Gi.     

Protein Kinase C and Phospholipase C Mediate α- and β-Adrenoceptor Intercommunication in Rat Hypothalamic Slices

These experiments examined the mechanism by which phenylephrine enhances beta-adrenoceptor-stimulated cyclic AMP formation in rat hypothalamic and preoptic area slices. To this end we manipulated phospholipase C. phospholipase A2, and protein kinase C activity in slices and assessed the effects of these manipulations on phenylephrine augmentation of isoproterenol-stimulated cyclic AMP generation. Since previous work indicated that estrogen enhances the alpha 1-component of cyclic AMP formation, we examined slices from both gonadectomized and estrogen-treated animals. The alpha 1-antagonist prazosin eliminated phenylephrine augmentation of the beta-response, suggesting that alpha 1-adrenergic receptors mediate the potentiation of cyclic AMP formation. Inhibition of protein kinase C by H7 attenuated the alpha 1-augmentation of beta-stimulated cyclic AMP formation. Staurosporine, a more potent protein kinase C inhibitor, completely abolished the alpha 1-augmenting response. In addition, phenylephrine potentiation of the isoproterenol response was not observed if protein kinase C was first stimulated directly with a synthetic diacylglycerol (1-oleoyl-2-acetyl-sn-glycerol) or phorbol ester (phorbol 12,13-dibutyrate). Neomycin, an inhibitor of phospholipase C, decreased alpha 1-receptor enhancement of beta-stimulated cyclic AMP formation, whereas quinacrine, an inhibitor of phospholipase A2, did not. The data suggest that the postreceptor mechanism involved in alpha 1-adrenergic receptor potentiation of cyclic AMP generation in hypothalamic and preoptic area slices includes activation of phospholipase C and protein kinase C.     

Activation of protein kinase C by a tumor-promoting phorbol ester in pancreatic islets

Rat pancreatic islet homogenates display protein kinase C activity. This phospholipid-dependent and calcium-sensitive enzyme is activated by diacylglycerol or the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In the presence of TPA, the Ka for Ca2+ is close to 5 microM. TPA does not affect phosphoinositide turnover but stimulates [32P]- and [3H]choline-labelling of phosphatidylcholine in intact islets. Exogenous phospholipase C stimulates insulin release, in a sustained and glucose-independent fashion. The secretory response to phospholipase C persists in media deprived of CaCl2. It is proposed that protein kinase C participates in the coupling of stimulus recognition to insulin release evoked by TPA, phospholipase C and, possibly, those secretatogues causing phosphoinositide breakdown in pancreatic islets.     

Rapid effects of phorbol esters on isolated rat adipocytes. Relationship to the action of protein kinase C

Treatment of isolated rat adipocytes with tumor-promoting phorbol esters, caused a fivefold stimulation of glucose oxidation, determined as 14CO2 production from [1-14C]glucose and a fivefold increase in the rate of lipid synthesis from [14C]glucose. Treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate increased the rate of 86Rb+ uptake into the cells. Also phospholipase C was able to stimulate the rate of glucose oxidation; phospholipase C and 12-O-tetradecanoylphorbol 13-acetate stimulated glucose oxidation in a non-synergistic fashion, indicating a common mechanism for their action. Active phorbol esters and, in part, also phospholipase C, caused a translocation of protein kinase C activity from the soluble to the particulate fraction of the adipocytes. This process was rapid, being complete 30 s after the addition of phorbol ester, and resulted in the appearance of the kinase mainly in the mitochondrial and plasma membrane fractions. A comparison between the binding characteristics of adipocyte protein kinase C and the metabolic effects of the phorbol esters on the adipocytes revealed that the dose-response relationship did not correlate with binding of the phorbol esters, but, rather, a correlation was observed between the dose of phorbol esters required for translocation of protein kinase C and the intracellular effects. The results indicate that the intracellular translocation of protein kinase C might be a trigger for the effects of phorbol esters on the adipocyte and that binding of the esters to protein kinase C is not a sufficient event to cause this effect. Furthermore, it is suggested that activation of protein kinase C might be partly the action of hormones, such as insulin, on the fat cells.     

Phorbol ester-induced surface transferrin receptor modulation. No correlation with decreased cell proliferation

Both phorbol ester or diacylglycerol (DAG) reduce cell surface transferrin receptor (TFR) number in CEM cells (a human T-cell acute lymphoblastic leukemia line) and HL-60 cells (a human promyelocytic leukemia cell line). This effect occurs with a t1/2 of approx. 30 min and is mimicked by addition of phospholipase C to cell cultures. Although cell surface TFR number is reduced to 25-30% of the control level 5 h after phorbol ester administration, apparent cell proliferation (as measured by [3H]thymidine incorporation) remains unaffected. Although independent of extracellular calcium (EGTA is slightly enhancing), the phenomenon is completely blocked by 30-min pretreatment with the calcium channel blocker diltiazem. Dilitazem pretreatment, while preventing receptor redistribution, does not completely block the phorbol ester-induced increase in TFR phosphorylation thought to be associated with receptor redistribution. Thus, calcium channel blockade effectively dissociates the effects of tetradecanoylphorbol acetate (TPA) on TFR internalization and phosphorylation. Our results also demonstrate that phorbol ester-induced effects on the TFR can be mimicked by the endogenous stimulator of protein kinase C, DAG, whether added directly to cultures or produced by the cells in response to exogenous phospholipase C. Furthermore, the phenomenon of TFR redistribution here described is not associated with a decreased proliferative capacity.     

Effect of cellular fatty acid composition on the phospholipase A2 activity of bone marrow-derived macrophages, and their ability to induce lucigenin-dependent chemiluminescence

Mouse bone marrow macrophages were obtained by cultivation in serum-free medium. Addition of specific fatty acids to the medium leads to macrophage populations which differ in their fatty acid composition. The fatty acid composition of the cellular membranes directly modulates functional abilities of the macrophages such as the generation of superoxide anion and phospholipase A2 activity in response to phorbol ester and zymosan. Both capacities were lowest in macrophages cultured serum-free without lipids. Incorporation of unsaturated fatty acids into macrophage phospholipids leads to an increase of O2- production as measured by lucigenin-dependent chemiluminescence and to an increased phospholipase A2 activity after challenge with phorbol ester or zymosan.     

Involvement of protein kinase C in prostaglandin D2 synthesis by cultured astrocytes

The role of protein kinase C (PKC) and calcium in the stimulation of prostaglandin D₂ (PGD₂) synthesis was investigated in primary rat astroglial cultures using the phorbol esters phorbol 12-myristate, 13-acetate (PMA), phorbol 12,13-dibutyrate (PDB) and the calcium ionophore A₂₃₁₈₇. Both phorbol esters and the ionophore were able to stimulate PGD₂ synthesis in a concentration dependent manner. The inactive stereoisomers of PMA and PDB had no significant effect. Combinations of subthreshold concentrations of phorbol esters (10 nM PMA or 10 nM PBD) potentiated PG formation induced by 100 nM A₂₃₁₈₇. An even more pronounced effect was observed when phorbol ester concentrations were increased to 100nM. The contribution of extra- and intracellular calcium in phorbol ester or A₂₃₁₈₇ stimulated PGD₂ synthesis was evaluated by carrying out experiments with calcium-free media plus EGTA or with the intracellular calcium-chelating agent TMB-8. Ionophore stimulated PGD₂ release was shut down to basal values upon removal of extracellular calcium, whereas phorbol ester stimulated PGD₂ formation persisted at a reduced level. It was unabated also upon further addition of EGTA. In the presence of TMB-8, however, phorbol ester stimulated PGD₂ synthesis was completely suppressed. These data strongly suggest that PKC has an additional effect on the activation of phospholipase A₂ and subsequent prostanoid synthesis, which is independent from extracellular calcium and, thus, support the concept of more than one metabolic pathway in astrocytes that synergistically regulate phospholipase A₂ activity.     

Phosphatidylcholine synthesis in platelets is stimulated by diacylglycerol but not by phorbol ester

The biosynthesis of phosphatidylcholine (PC) in platelets was followed by measuring the incorporation of 32Pi. Incorporation into PC was stimulated by treatment with Clostridium perfringens phospholipase C or with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol. However, neither the phorbol ester tumour promoter 12-O-tetradecanoylphorbol-13-acetate or thrombin stimulated 32Pi incorporation into PC. We conclude that phorbol ester does not stimulate the hydrolysis of PC to diacylglycerol in platelets.     

Production of phosphatidylethanol by phospholipase D phosphatidyl transferase in intact or dispersed pancreatic islets: evidence for the in situ metabolism of phosphatidylethanol

To determine if phospholipase D is present in intact adult islets, we took advantage of the fact that, in the presence of ethanol, this enzyme generates phosphatidylethanol via transphosphatidylation. Extracts of cells prelabeled with [14C]arachidonate, [14C]myristate, or [14C]stearate were analyzed via three TLC systems; the identify of phosphatidylethanol was further confirmed via incorporation of [14C]ethanol into the same phospholipid bands. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate stimulated phosphatidylethanol (to 603% of basal by 60 min) both in intact adult islets and in dispersed neonatal islet cells. A nonphorbol activator of protein kinase C (mezerein) also stimulated phospholipase D, whereas a phorbol which does not activate protein kinase C (4 alpha-phorbol-12,13-didecanoate) was virtually inactive. The effects of the active phorbol ester or of mezerein were reduced by the protein kinase C inhibitor H-7 and were virtually eliminated by prior down-regulation of that enzyme. In addition, a calcium-selective ionophore (ionomycin) or fluoroaluminate also activated the islet phospholipase D. When accumulation of phosphatidylethanol (labeled with any of three fatty acids) was induced by a preincubation in the presence of ethanol plus agonist, which then were removed, phosphatidylethanol declined by 34-47% over a subsequent 60-min incubation. Thus, while phosphatidylethanol is relatively stable metabolically, it is detectably degraded (a variable overlooked in previous studies). In the absence of ethanol, stimulated islet cells generated phosphatidic acid, although such hydrolysis was less evident than transphosphatidylation. Ethanol provision distinguished phosphatidate formed via phospholipase D (inhibition, via phosphatidylethanol formation) from that due predominantly to phospholipase C (phosphatidate not inhibited). In view of our recent findings that phosphatidic acid (or exogenous phospholipase D) has potent insulinotropic effects, this pathway could play a role in stimulus-secretion coupling; conversely, stimulation of transphosphatidylation at the expense of hydrolysis could contribute to the inhibition of secretion caused by ethanol.     

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.     

Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells.

Protein tyrosine phosphorylation has not been considered to be important for cellular activation by phospholipase C-linked vasoactive peptides. We found that endothelin, angiotensin II, and vasopressin (AVP), peptides that signal via phospholipase C activation, rapidly enhanced tyrosine phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat renal mesangial cells. The phosphorylated proteins were cytosolic or membrane-associated, and none were integral to the membrane, suggesting that the peptide receptors are not phosphorylated on tyrosine. Epidermal growth factor (EGF), which does not activate phospholipase C in these cells, induced the tyrosine phosphorylation of its own 175-kDa receptor, in addition to five proteins of identical molecular mass to those phosphorylated in response to endothelin, AVP, and angiotensin II. This suggests that in mesangial cells there is a common signaling pathway for phospholipase C-coupled agonists and agonists classically assumed to signal via receptor tyrosine kinase pathways, such as EGF. The phorbol ester, phorbol 12-myristate 13-acetate, and the synthetic diacylglycerol, oleoyl acetylglycerol, stimulated the tyrosine phosphorylation of proteins identical to those phosphorylated by the phospholipase C-linked peptides, suggesting that protein kinase C (PKC) activation is sufficient to active tyrosine phosphorylation. However, the PKC inhibitor, staurosporine, and down-regulation of PKC activity by prolonged exposure to phorbol esters completely inhibited tyrosine phosphorylation in response to PMA but not to endothelin, AVP, or EGF. In conclusion, endothelin, angiotensin II, and AVP enhances protein tyrosine phosphorylation via at least two pathways, PKC-dependent and PKC-independent. Although activation of PKC may be sufficient to enhance protein tyrosine phosphorylation, PKC is not necessary and may not be the primary route by which these agents act. At least one of these pathways is shared with the growth factor EGF, suggesting not only common intermediates in the signaling pathways for growth factors and vasoactive peptides but also perhaps common cellular tyrosine kinases which phosphorylate these intermediates.     

Retinoids antagonize the induction of ornithine decarboxylase activity by phorbol esters and phospholipase C in rat tracheal epithelial cells

In this study we examined the action of phorbol esters, several phospholipases and retinoids on the induction of ornithine decarboxylase (ODC) activity in rat tracheal epithelial cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induces ODC activity in these cells in a dose-and time-dependent manner. This induction is inhibited by cycloheximide indicating a requirement for protein synthesis. Tracheal epithelial 2C5 cells contain two binding sites for phorbol esters, one with a high affinity KD,1 = 4.58 nM and one with a low affinity KD,2 = 344.8 nM. The ability of several phorbol esters to induce ODC correlates well with the described efficacy with which they bind to the receptor and is in agreement with the concept that phorbol ester receptors are involved in the induction of ODC. There is strong evidence that the phorbol ester receptor is the protein kinase C for which diacylglycerol is the physiological ligand. Treatment of cells with phospholipase C generates diacylglycerol and induces ODC activity in a dose- and time-dependent manner. Treatment with phospholipase A2 or D has no effect on ODC activity. These results support the concept that activation of protein kinase C is related to the induction of ODC activity. The induction of ODC by TPA as well as by phospholipase C is inhibited by retinoids. Specific cytosolic binding proteins for retinoids might be involved in at least some of the responses to these compounds. To examine whether the binding proteins are involved in the inhibition of ODC we determined the presence of these binding proteins and the structure-activity relationship of retinoids. Both retinol and retinoic acid-binding proteins can be detected in 2C5 cells, their levels are 1.06 and 3.36 pmoles/mg protein, respectively. The ability of several retinoids to inhibit ODC induction correlates well with their binding activity and support a role for these binding proteins in the action of retinoids on ODC induction.     

The effects of phorbol esters on fluid transport and blood flow in the small intestine

Studies were designed to examine the effects of phorbol esters on intestinal fluid transport and blood flow in the anesthetized cat and enteropooling in the conscious rat. Intraluminal administration of phorbol ester into a segment of isolated small bowel produced a copious intestinal secretion and a concomitant mesenteric hyperemia in the cat. Net fluid movement in the intestine was converted from absorption in the control state to secretion following phorbol ester administration. Intravenous atropine reduced the phorbol ester-induced secretion by 56%; clonidine abolished the remaining secretory response. In the rat, intragastric administration of phorbol ester produced enteropooling comparable to that of other potent intestinal secretagogues. Since phorbol esters are known to activate protein kinase C, these studies suggest that activation of protein kinase C in the small intestine may lead to a full secretory response. The evidence suggests that this secretion is accompanied by a metabolic hyperemia. These results suggest that protein kinase C plays an important role in the regulation of intestinal fluid transport.     

A transfected m1 muscarinic acetylcholine receptor stimulates adenylate cyclase via phosphatidylinositol hydrolysis

The m1 muscarinic acetylcholine receptor gene was transfected into and stably expressed in A9 L cells. The muscarinic receptor agonist, carbachol, stimulated inositol phosphate generation, arachidonic acid release, and cAMP accumulation in these cells. Carbachol stimulated arachidonic acid and inositol phosphate release with similar potencies, while cAMP generation required a higher concentration. Studies were performed to determine if the carbachol-stimulated cAMP accumulation was due to direct coupling of the m1 muscarinic receptor to adenylate cyclase via a GTP binding protein or mediated by other second messengers. Carbachol failed to stimulate adenylate cyclase activity in A9 L cell membranes, whereas prostaglandin E2 did, suggesting indirect stimulation. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated arachidonic acid release yet inhibited cAMP accumulation in response to carbachol. PMA also inhibited inositol phosphate release in response to carbachol, suggesting that activation of phospholipase C might be involved in cAMP accumulation. PMA did not inhibit prostaglandin E2-, cholera toxin-, or forskolin-stimulated cAMP accumulation. The phospholipase A2 inhibitor eicosatetraenoic acid and the cyclooxygenase inhibitors indomethacin and naproxen had no effect on carbachol-stimulated cAMP accumulation. Carbachol-stimulated cAMP accumulation was inhibited with TMB-8, an inhibitor of intracellular calcium release, and W7, a calmodulin antagonist. These observations suggest that carbachol-stimulated cAMP accumulation does not occur through direct m1 muscarinic receptor coupling or through the release of arachidonic acid and its metabolites, but is mediated through the activation of phospholipase C. The generation of cytosolic calcium via inositol 1,4,5-trisphosphate and subsequent activation of calmodulin by m1 muscarinic receptor stimulation of phospholipase C appears to generate the accumulation of cAMP.     

Bradykinin and Phorbol Dibutyrate Activate Phospholipase D in PC12 Cells by Different Mechanisms

Bradykinin is known to activate phospholipase D in PC12 cells. Because bradykinin may also activate protein kinase C in these cells, the possible role of this kinase in mediating the action of bradykinin was investigated. Phospholipase D activity in PC12 cells was assayed by measuring the formation of [3H]phosphatidylethanol in cells prelabeled with [3H]palmitic acid and incubated in the presence of ethanol. The phorbol ester phorbol dibutyrate mimicked the effect of bradykinin on [3H]phosphatidylethanol formation. The protein kinase C inhibitor staurosporine (1 microM) significantly attenuated the effect of phorbol dibutyrate (35-70%) but did not block bradykinin-stimulated [3H]phosphatidylethanol formation. In addition, the effect of phorbol dibutyrate was additive with that of bradykinin. Prolonged treatment of PC12 cells with phorbol dibutyrate (24 h), which depletes cells of protein kinase C, greatly attenuated bradykinin-stimulated [3H]phosphatidylethanol accumulation in intact cells. This treatment caused a 55% decrease in both fluoride-stimulated [3H]phosphatidylethanol production in the intact cell and phospholipase D activity as assessed by an in vitro assay using an exogenous substrate. Therefore, the effect of prolonged phorbol dibutyrate pretreatment on bradykinin-stimulated [3H]phosphatidylethanol production could not be attributed exclusively to the depletion of protein kinase C. Thus, although the data with phorbol ester suggest that activation of protein kinase C leads to an increase in phospholipase D activity, this kinase probably does not play a role in mediating the effect of bradykinin. Finally, although pretreatment with phorbol dibutyrate completely blocked bradykinin-stimulated [3H]phosphatidylethanol production in the intact cell, it only partially (approximately 50%) inhibited bradykinin-stimulated [3H]diacylglycerol formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of a keratinocyte phospholipase A2 by bradykinin and 4 beta-phorbol 12-myristate 13-acetate. Evidence for a receptor-GTP-binding protein versus a protein-kinase-C mediated mechanism.

The release of arachidonic acid from cellular phospholipids and its subsequent conversion to eicosanoids is the common early response of skin keratinocytes to a wide variety of exogenous or endogenous agonists including irritant skin mitogens such as the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA) or the inflammatory peptide bradykinin. In mouse keratinocytes labeled with [14C]arachidonic acid, both PMA and bradykinin induced the release of the fatty acid in a dose-dependent and time-dependent manner. Three lines of evidence indicate phospholipase A2 activity to be involved in arachidonic acid release: (a) its inhibition by mepacrine, (b) the concomitant generation of lysophosphatidylcholine from [3H]choline-labeled cells and (c) an increase in arachidonic acid release from 14C-labeled phosphatidylcholine in particulate fractions from PMA-treated and bradykinin-treated keratinocytes. Inhibition or down regulation of protein kinase C (PKC) led to a suppression of PMA-induced but not bradykinin-induced arachidonic acid release, indicating a critical involvement of this kinase in phorbol-ester-induced activation of epidermal phospholipase A2 activity. Bradykinin-induced activation of phospholipase A2 was however, shown to be mediated by specific B2 receptors coupled to GTP-binding proteins (G protein). In support of this mechanism it was demonstrated that the bradykinin-induced phospholipase A2 activity was increased in the presence of non-hydrolysable GTP but decreased upon addition of non-hydrolysable GDP analogues. Moreover, cholera toxin stimulated both basal and bradykinin-induced phospholipase A2 activity in a cAMP-independent manner, whereas pertussis toxin was found to be inactive in this respect. The data suggest that epidermal phospholipase A2 activity can be stimulated by bradykinin via a B2 receptor-G-protein-dependent pathway, which is independent of PKC and a PKC-dependent pathway which is activated by phorbol esters such as PMA.