Intracellular mechanisms involved in short-term regulation of net protein synthesis in pancreatic acini.

The mechanisms regulating the net synthesis of digestive enzymes during short-term stimulation by agonists were examined in pancreatic acini isolated from the rat. Dispersed pancreatic acini were stimulated for up to 60 min with various concentrations of cholecystokinin octapeptide (CCK-OP), carbachol, A23187, 4 beta-phorbol 12-myristate 13-acetate (PMA). The effects of these agonists on net protein synthesis was determined by measuring the incorporation of [3H]leucine or [35S]methionine into protein. Carbachol, PMA, A23187 and concentrations of CCK-OP of 100 pM and greater caused inhibition of protein synthesis. Fluorography of [35S]methionine labeled acinar cell proteins separated by one-dimensional SDS-polyacrylamide gel electrophoresis demonstrated that the agonists inhibited the synthesis of the digestive enzymes. Northern blot analysis using cDNA probes revealed that CCK-OP, carbachol and PMA did not alter the cellular content of amylase, lipase and elastase mRNA. The protein kinase C inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and staurosporine failed to reverse the inhibitory effects of CCK-OP, carbachol and PMA on protein synthesis. CCK-OP and PMA activated phospholipase A (PLA) which liberated lysophosphatidylcholine (LPC) and free fatty acids from membrane phosphatidylcholine. Exogenously added PLA2 (Naja naja venom) inhibited protein synthesis and increased LPC to a similar extent as CCK and PMA. The results suggest that the inhibitory effects of CCK and carbachol on net protein synthesis are due to their effects on intracellular calcium and PLA-mediated breakdown of phosphatidylcholine rather than protein kinase C activation.     

Crosstalk of sterol-dependent and non-sterol-dependent signaling in human monocytes after in vitro addition of LDL

The aim of the present study was to investigate low density lipoprotein (LDL)-induced, non-sterol-dependent signaling and its possible role in cholesterol balance. LDL in 10 microg ml(-1) concentration could induce inositol trisphosphate (IP3) and Ca2+ signal generation through a pertussis toxin (PT) sensitive G protein in human monocytes. The increase in [Ca2+]i was derived from the intracellular pools. LDL also induced activation and translocation of protein kinase C (PKC) into the cell membrane, by processes, which were significantly inhibited in the first 20 min by preincubation with PT and PKC-inhibitor H-7. The PKC-activating phorbol-12-myristate-13-acetate (PMA), differently from LDL, enhanced the LDL-receptor (LDL-R)-mediated binding and degradation of [125I]LDL, but inhibited endogenous cholesterol synthesis, and both effects were inhibited by H-7. The LDL-induced inhibition of binding and degradation of [125I]LDL was not affected by H-7, whereas decreased cholesterol synthesis was counteracted by H-7. These results suggest the existence of a non-sterol-dependent signal pathway of LDL-Rs, by which endogenous cholesterol synthesis, that is, the [14C]acetate incorporation, is regulated through PKC activation.     

Effect of protein kinase C on endoplasmic reticulum cholesterol.

Plasma membrane cholesterol both regulates and is regulated by effector proteins in the endoplasmic reticulum (ER) through a feedback system that is poorly understood. We now show that ER cholesterol varies over a fivefold range in response to experimental agents that act upon protein kinase C (PKC). Agents that activate Ca(2+)-dependent PKC [phorbol-12-myristate-13-acetate (PMA) and bryostatin 1] increased the level of ER cholesterol; inhibitors such as staurosporine and calphostin C decreased it. Rottlerin, a selective inhibitor of the PKC-delta isoform, also increased ER cholesterol. The esterification of plasma membrane cholesterol was altered by protein kinase C-directed agents in a corresponding fashion. Furthermore, the regulatory effect of plasma membrane cholesterol on the esterification of ER cholesterol was blocked by PKC-directed agents. These findings suggest that multiple protein kinase C isoforms participate in the regulation of ER cholesterol and therefore in cholesterol homeostasis.     

Reversal by protein kinase C inhibitor of suppressive actions of phorbol-12-myristate-13-acetate on polyphosphoinositide metabolism and cytosolic Ca2+ mobilization in thrombin-stimulated human platelets

In the presence of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C in vitro, phorbol-12-myristate-13-acetate (PMA) did not suppress the thrombin-induced increase of cytosolic Ca2+ concentration in human platelets. The H-7 reversal of the inhibitory action of PMA was also observed in thrombin-induced polyphosphoinositide breakdown by phospholipase C. These results provide additional support to the developing theory that the inhibition of PMA on Ca2+ mobilization and phosphoinositide turnover may be mediated by protein kinase C activation.     

Stimulation of dopamine synthesis and activation of tyrosine hydroxylase by phorbol diesters in rat striatum

In rat striatal synaptosomes, 4 beta-phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-dibutyrate (PDBu), two activators of Ca2+-phospholipid-dependent protein kinase (protein kinase C) increased dopamine (DA) synthesis measured by following the release of 14CO2 from L-[1-14C] tyrosine. Maximal stimulation (21-28% increase of basal rate) was produced by 0.5 microM PMA and 1 microM PDBu. 4 beta-Phorbol and 4 beta-phorbol 13-acetate, which are not activators of protein kinase C, were ineffective at 1 microM. PMA did not change the release of 14CO2 from L-[1-14C]DOPA. Addition of 1 mM EGTA to a Ca2+-free incubation medium failed to affect PMA stimulation. KC1 (60 mM) enhanced DA synthesis by 25%. Exposure of synaptosomes to either PMA or PDBu prior to KC1 addition resulted in a more than additive increase (80-100%) of DA synthesis. A similar synergistic effect was observed when the phorbol diesters were combined with either veratridine or d-amphetamine but not with forskolin and dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with phorbol diesters produced an activation on of tyrosine hydroxylase (TH) associated with a 60% increase of the Vmax and a decrease of the Km for the pterine cofactor 6-methyl-5,6,7,8-tetrahydropterin. These results indicate that protein kinase C participates in the regulation of striatal TH in situ and that its activation may act synergistically with DA releasing agents in stimulating DA synthesis.     

Modulation of Ca(v)3.2 T-type Ca2+ channels by protein kinase C

Although T-type Ca2+ channels have been implicated in numerous physiological functions, their regulations by protein kinases have been obscured by conflicting reports. We investigated the effects of protein kinase C (PKC) on Ca(v)3.2 T-type channels reconstituted in Xenopus oocytes. Phorbol-12-myristate-13-acetate (PMA) strongly enhanced the amplitude of Ca(v)3.2 channel currents (approximately 3-fold). The augmentation effects were not mimicked by 4alpha-PMA, an inactive stereoisomer of PMA, and abolished by preincubation with PKC inhibitors. Our findings suggest that PMA upregulates Ca(v)3.2 channel activity via activation of oocyte PKC.     

Possible involvement of protein kinase C in mediating gastrin-induced response in rat colonic epithelium.

We examined the potential role of protein kinase C in signal transduction induced by gastrin's stimulation of rat colonic epithelium. Protein synthesis ([35S]methionine incorporation into protein) and enzyme activity (decrease in the cytosolic activity) were measured following epithelial stimulation with gastrin. Gastrin (10 nM) increased [35S]methionine incorporation into protein to 265% above maintenance level. The effect of gastrin was comparable to the stimulation induced by phorbol 12-myristate, 13-acetate (PMA), a strong activator of protein kinase C. The increase in protein synthesis induced by gastrin was totally abolished by 1-(5-isoquinolinyl)-2-methylpiperazine, an inhibitor of protein kinase C activity. Gastrin also decreased the cytosolic activity of the enzyme, an index of its activation and subsequent translocation to other cellular compartments. Therefore, we conclude that gastrin may be acting through a protein kinase C mechanism.     

Effects of elevated cytoplasmic calcium and protein kinase C on endoplasmic reticulum structure and function in HEK293 cells

In human embryonic kidney (HEK) cells stably transfected with green fluorescent protein targeted to the endoplasmic reticulum (ER), elevation of intracellular Ca2+ ([Ca2+]i) altered ER morphology, making it appear punctate. Electron microscopy revealed that these punctate structures represented circular and branched rearrangements of the endoplasmic reticulum, but did not involve obvious swelling or pathological fragmentation. Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA), prevented the effects of ionomycin on ER structure without affecting the elevation of [Ca2+]i. These results suggest that protein kinase C activation alters cytoplasmic or ER components underlying the effects of high [Ca2+]i on ER structure. Treatment of HEK cells with PMA also reduced the size of the thapsigargin-sensitive Ca2+ pool and inhibited Ca2+ entry in response to thapsigargin. Thus, protein kinase C activation has multiple actions on the calcium storage and signalling function of the endoplasmic reticulum in HEK cells: (1) reduced intracellular Ca2+ storage capacity, (2) inhibition of capacitative Ca2+ entry, and (3) protection of the endoplasmic reticulum against the effects of high [Ca2+]i.     

Evidence for increased synthesis as well as increased degradation of protein kinase C after treatment of human osteosarcoma cells with phorbol ester

Phorbol 12-myristate 13-acetate (PMA) induces time-dependent changes in protein kinase C subcellular distribution and enzymatic activity in the human osteosarcoma cell line SaOS-2. Short (less than 60 min) incubations with PMA caused decreased cytosolic enzyme activity and a concomitant increase in particulate protein kinase; after 3 h, particulate protein kinase C activity also declined to reach less than 10% of basal activity by 24 h (Krug, E., and Tashjian, Jr., A. H., (1987) Cancer Res. 47, 2243-2246). In order to determine whether the loss in enzyme activity was due to decreased enzyme protein, Western blot analyses were performed using a polyclonal antibody against protein kinase C raised in rabbits. This approach confirmed the previously reported time-related changes: 80-kDa immunoreactive protein kinase C initially translocated from the cytosol to the particulate cell fraction and later disappeared completely from the particulate fraction. Loss of protein kinase C enzymatic activity thus results from actual loss of the 80-kDa protein; we found no evidence for generation of a calcium/phospholipid-independent protein kinase C-like form of the enzyme. Membrane association was confirmed by immunoprecipitation experiments using [35S]methionine-labeled cells. Brief exposure to PMA caused a marked loss in the [35S]methionine-labeled cytosolic protein kinase C band and an increase in the labeled particulate band. Protein kinase C immunoprecipitated from cells treated with PMA for 14 h displayed an increase in [35S]methionine label despite a greater than 80% loss of enzyme activity. The high specific radioactivity of the remaining 80-kDa protein leads us to conclude that long term treatment with PMA causes an increase in the rate of protein kinase C synthesis accompanied by a still greater increase in the rate of enzyme degradation in SaOS-2 cells.     

Effects of phorbol esters on steroidogenesis in small bovine luteal cells

The possible influence of an activator of protein kinase C, the tumor-promoting phorbol ester, PMA (phorbol-12-myristate-13-acetate), upon small bovine luteal cell steroidogenesis was investigated in vitro, PMA had no significant effect on basal and dibutyryl cyclic AMP (dbcAMP)-stimulated progesterone production but markedly modulated the LH-stimulated progesterone and cAMP productions. PMA potentiated the LH-stimulated cAMP accumulation whatever the dose of LH used. It also potentiated the LH-induced progesterone production in the presence of low doses of LH. Paradoxically, in the presence of maximal or submaximal effective doses of LH, PMA exerted a time- and dose-dependent inhibition of progesterone synthesis. Diacylglycerol was able to mimic the effects of PMA on LH-induced steroidogenesis. These observations suggest that the Ca2+- and phospholipid-dependent protein kinase C can modulate the regulation by LH of small bovine luteal cell steroidogenesis at a step before the synthesis of cAMP. They also suggest that the interaction between LH and its receptor is able to trigger a negative regulatory signal which would be only expressed for high doses of LH and in the presence of an activator of PKC.     

Study on the mechanism of Giardia lamblia induced diarrhoea in mice.

The transmucosal fluxes of Na+ and Cl- were studied in Giardia lamblia infected mice in the presence or absence of phorbol-12-myristate-13-acetate (PMA), the activator of protein kinase C (PKC) or 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H-7), the inhibitor of PKC or Ca(2+)-calmodulin. There was net secretion of Na+ and Cl- in infected animals, while in control animals there was net absorption of these ions. The addition of ionophore or PMA resulted in net secretion of Na+ and Cl- in the control group while in the infected group there was no change in the fluxes of these ions. The selective potent inhibitor of protein kinase C, H-7, reversed the secretion of Na+ and Cl- in infected group to absorption. The addition of PMA and Ca(2+)-ionophore together in the infected group had a partial additive effect. This study suggests that G. lamblia induced fluid secretion involves protein kinase C and further protein kinase C acts in synergism with calcium.     

Xanthine oxidase-catalysed oxidation of paracetamol.

[3H]Arachidonic acid is released after stimulation of rabbit neutrophils with fMet-Leu-Phe or platelet-activating factor (PAF). The release is rapid and dose-dependent, and is inhibited in phorbol 12-myristate 13-acetate (PMA)-treated rabbit neutrophils. The protein kinase C (PKC) inhibitor 1-(5-isoquinoline-sulphonyl)-2-methylpiperazine (H-7) prevents this inhibition. In addition, PMA increases arachidonic acid release in H-7-treated cells stimulated with fMet-Leu-Phe. [3H]Arachidonic acid release, but not the rise in the concentration of intracellular Ca2+, is inhibited in pertussis-toxin-treated neutrophils stimulated with PAF. The diacylglycerol kinase inhibitor R59022 increases the concentration of diacylglycerol and potentiates [3H]arachidonic acid release in neutrophils stimulated with fMet-Leu-Phe. This potentiation is not inhibited by H-7. These results suggest several points. (1) A rise in the intracellular concentration of free Ca2+ is not sufficient for arachidonic acid release in rabbit neutrophils stimulated by physiological stimuli. (2) A functional pertussis-toxin-sensitive guanine nucleotide regulatory protein and/or one or more of the changes produced by phospholipase C activation are necessary for arachidonic acid release produced by physiological stimuli. (3) Agents that stimulate PKC potentiate arachidonic acid release, and this potentiation is not inhibited by H-7. These agents produce their actions in part by direct membrane perturbation.     

Differential effects of phorbol ester on phenylephrine and vasopressin-induced Ca2+ mobilization in isolated hepatocytes

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.     

Regulation of N-acetylaspartate and N-acetylaspartylglutamate biosynthesis by protein kinase activators

The neuronal dipeptide N-acetylaspartylglutamate (NAAG) is thought to be synthesized enzymatically from N-acetylaspartate (NAA) and glutamate. We used radiolabeled precursors to examine NAA and NAAG biosynthesis in SH-SY5Y human neuroblastoma cells stimulated with activators of protein kinase A (dbcAMP; N6,2'-O-dibutyryl cAMP) and protein kinase C (PMA; phorbol-12-myristate-13-acetate). Differentiation over the course of several days with dbcAMP resulted in increased endogenous NAA levels and NAAG synthesis from l-[(3)H]glutamine, whereas PMA-induced differentiation reduced both. Exogenously applied NAA caused dose dependent increases in intracellular NAA levels, and NAAG biosynthesis from l-[(3)H]glutamine, suggesting precursor-product and mass-action relationships between NAA and NAAG. Incorporation of l-[(3)H]aspartate into NAA and NAAG occurred sequentially, appearing in NAA by 1 h, but not in NAAG until between 6 and 24 h. Synthesis of NAAG from l-[(3)H]aspartate was increased by dbcAMP and decreased by PMA at 24 h. The effects of PMA on l-[(3)H]aspartate incorporation into NAA were temporally biphasic. Using short incubation times (1 and 6 h), PMA increased l-[(3)H]aspartate incorporation into NAA, but with longer incubation (24 h), incorporation was significantly reduced. These results suggest that, while the neuronal production of NAA and NAAG are biochemically related, significant differences exist in the regulatory mechanisms controlling their biosynthesis.     

Glycoprotein biosynthesis in B lymphocytes: induction of protein N-glycosylation, RNA synthesis, and DNA synthesis by phorbol ester plus ionomycin is blocked by protein kinase inhibitors

The combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin produces a dramatic increase in the incorporation of [2-3H]mannose into Glc3Man9GlcNAc2-P-P-dolichol and glycoprotein, and the induction of RNA and DNA synthesis in murine splenic B lymphocytes (B cells). The kinetics of the induction processes and the concentrations of PMA and ionomycin required for the optimal response have been defined. While the levels of induction of RNA and DNA synthesis by PMA + ionomycin were similar to the mitogenic response to bacterial lipopolysaccharide, activation by PMA and the calcium ionophore resulted in a threefold higher stimulation in dolichol-linked oligosaccharide biosynthesis and protein N-glycosylation. These results indicate that all signalling mechanisms that trigger RNA and DNA synthesis may not be sufficient to produce maximal induction of the N-glycosylation apparatus. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C inhibitor, prevented the induction of protein N-glycosylation activity (IC50 = 11 microM), as well as RNA (IC50 = 18 microM) and DNA synthesis (IC50 = 12 microM), two common indices of B cell activation. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) also inhibited the induction of oligosaccharide-lipid intermediate, glycoprotein, RNA, and DNA synthesis, but required higher concentrations than H-7 for 50% inhibition. N-(2-Guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a potent inhibitor of cyclic nucleotide-dependent protein kinases, had little effect on the activation of the B cell metabolic processes. The H-7-sensitive reactions involved in the induction of RNA and DNA synthesis occurred within 4 h, but induction of lipid intermediate and glycoprotein biosynthesis remained sensitive to H-7 for 10 h after exposure to PMA and ionomycin. Direct in vitro assays in the presence of 0.6% Brij 58 reveal that a cytosolic, phospholipid-dependent protein kinase activity is translocated to a membrane site(s) after treatment with PMA and ionomycin, and the translocated protein kinase is sensitive to H-7. The relative order of potency of the protein kinase inhibitors on the metabolic processes strongly supports the hypothesis that protein kinase C, acting synergistically with Ca2+ mobilization, plays a key regulatory role in the early stages of B cell activation. The synthesis of oligosaccharide-lipid intermediates and protein N-glycosylation are also shown to be induced in B cells activated by PMA + ionomycin.     

Phorbol ester stimulates the synthesis and phosphorylation of a 48 kDa-intracellular protein in plasminogen activator secreting melanoma cells

Phorbol ester (12-O-tetradecanoyl-phorbol 13-acetate) stimulates the secretion of tissue-type plasminogen activator by the melanoma cell line, Bowes. This effect is associated with increased levels of mRNAs for both tissue-type plasminogen activator and a 48 kDa-protein. Labelling of melanoma cells with L-[35S]methionine allowed to identify an intracellular protein which, by 3 criteria, was identical with the in vitro translation product of the 48kDa-protein mRNA: a Mr of 48,000 on electrophoresis in the presence of sodium dodecyl sulphate; inducibility by phorbol ester and failure of reducing agents to affect electrophoretic mobility. As detectable by L-[35S]methionine labelling, the protein was mainly localized in the cytosol. In vitro phosphorylation reactions, carried out on subcellular fractions revealed a membrane-associated protein which also had the three characteristics of the aforementioned 48 kDa-protein. Phosphorylation did not require Ca2+-ions. Addition of phorbol ester to the reaction mixtures increased the phosphorylation. Reconstitution experiments between membrane and cytosol fractions of phorbol ester-treated and untreated cells showed that the 48kDa protein occurs in a cytosolic, unphosphorylated and a membrane-bound, phosphorylated form and that the former is converted to the latter by a phorbol ester activated, membrane-associated protein kinase.     

Ca2+ and phorbol ester activation of protein kinase C at intracellular Ca2+ concentrations and the effect of TMB-8

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from human polymorphonuclear leukocytes and shown to be identical to bovine protein kinase C. The Ca2+ activation of the enzyme was studied and the Ca2+ concentrations required to activate the enzyme were compared to free cytosolic Ca2+ concentrations in resting and activated polymorphonuclear leukocytes. The free calcium concentrations in the cytosol and in the enzyme assay mixture were determined using the calcium indicator quin 2. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range, but was not activated by phosphatidylserine at Ca2+ concentrations corresponding to the intracellular free Ca2+ concentration under resting conditions. However, at similar Ca2+ concentrations (less than 2.5 X 10(-7) M) the enzyme was highly activated by phorbol 12-myristate 13-acetate (PMA) or diolein in the presence of phosphatidylserine. It was demonstrated that PMA stimulation of human polymorphonuclear leukocytes did not induce any increase in the level of the intracellular free calcium concentration. It was concluded that PMA activation of protein kinase C occurred independently of a rise in the intracellular Ca2+ concentration. K0.5 (half-maximal activation) for the PMA activation of purified protein kinase C was shown to be equivalent to the K0.5 for PMA stimulation of superoxide (O-2) production in human polymorphonuclear leukocytes, suggesting that protein kinase C is involved in activation of the NADPH oxidase. The presumed intracellular Ca2+ antagonist TMB-8 inhibited the PMA-induced superoxide production, but neither by an intracellular Ca2+ antagonism nor by a direct inhibition of protein kinase C activity.     

Regulation of cAMP-activated apical membrane chloride conductance in gallbladder epithelium

Regulation of the cAMP-activated apical membrane Cl- conductance (GaCl) in Necturus gallbladder (NGB) epithelial cells was investigated with intracellular-microelectrode techniques. GaCl was increased by exposure to 8-Br-cAMP, theophylline or forskolin. Neither 8-Br-cGMP nor elevation of intracellular [Ca2+] using ionomycin had effects on GaCl or interfered with activation of GaCl by forskolin. N-(2- [methylamino]ethyl)-5-isoquinolinesulfonamide (H8), an inhibitor of cAMP-dependent protein kinase (PKA), slowed but did not prevent the GaCl response to 8-Br-cAMP. Phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), stimulated GaCl but had no effects on intracellular [cAMP]. GaCl was unaffected by 4 alpha- phorbol, a PMA analog which does not activate PKC. Okadaic acid (OA), an inhibitor of protein phosphatases (PP) types 1 and 2A, slowed the activation of GaCl by 8-Br-cAMP, hastened the return of GaCl to basal values following removal of 8-Br-cAMP, and significantly reduced the elevation in intracellular [cAMP] produced by forskolin. OA had no effects on the GaCl changes elicited by theophylline. We conclude that: (a) NGB GaCl can be activated by PKA-mediated phosphorylation of apical membrane Cl- channels or a regulatory protein, (b) GaCl can also be activated via PKC, by a cAMP-independent mechanism, (c) OA-sensitive PP are not required for inactivation of GaCl; OA appears to stimulate phosphodiesterase, which lowers intracellular [cAMP] and affects GaCl activation, and (d) the apical membrane of NGB epithelium lacks a Ca(2+)-activated Cl- conductance.     

Stimulation of heparan sulfate proteoglycan synthesis and secretion during G1 phase induced by growth factors and PMA

Fetal calf serum (FCS) and PMA (phorbol 12-myristate-13-acetate) specifically stimulate the synthesis of heparan sulfate proteoglycan in endothelial cells. Staurosporine and n-butanol, kinase inhibitors, abolish the PMA effect. Forskolin and 8-bromo adenosine 3′:5′-cyclic monophosphate, activators of, respectively, adenylate cyclase and protein kinase A cannot reproduce the PMA effect. The kinetics of cell entry into S phase of the endothelial cells was determined by DNA synthesis ([³H]-thymidine and Br-dU incorporation), and flow cytometry. The mitogenic effect of fetal calf serum is abolished by PMA. Also, PMA pre-treatment inhibits the enhanced synthesis of heparan sulfate proteoglycan after a second PMA exposure. Remarkably, the stimulation of heparan sulfate proteoglycan synthesis by fetal calf serum and PMA seems to be mainly restricted to G₁ phase. Therefore fetal calf serum and PMA cause an enhanced synthesis of heparan sulfate proteoglycan, and PMA causes a cell cycle block at G₁ phase. J. Cell. Biochem. 70:563–572, 1998. © 1998 Wiley-Liss, Inc.     

Phorbol ester stimulates catecholamine synthesis in isolated bovine adrenal medullary cells

In isolated bovine adrenal medullary cells, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C, stimulated [14C]catecholamine synthesis from [14C]tyrosine, but not from [14C]DOPA. This stimulatory effect of TPA on [14C]catecholamine synthesis was not dependent upon extracellular Ca2+, and TPA did not affect the uptake of 45Ca2+ or the release of catecholamine by the cells. TPA also did not affect the intracellular cyclic AMP (cAMP) level. 4 alpha-Phorbol 12, 13-didecanoate, which is not an activator of protein kinase C, did not stimulate the synthesis of [14C]catecholamine from [14C]tyrosine. The stimulatory effect of TPA on [14C]catecholamine synthesis was additive with that of carbamylcholine, but not with that of dibutyryl cAMP (DB-cAMP). From these results, it was suggested that protein kinase C is involved in the regulation of tyrosine hydroxylase activity and that this regulatory mechanism might be similar to that involving cAMP.