Exercise-induced translocation of protein kinase C and production of diacylglycerol and phosphatidic acid in rat skeletal muscle in vivo. Relationship to changes in glucose transport

Contraction-induced translocation of protein kinase C (Richter E.A., Cleland, P.J.F., Rattigan, S., and Clark, M.G. (1987) FEBS Lett. 217, 232-236) implies a role for this enzyme in muscle contraction or the associated metabolic adjustments. In the present study, this role is further examined particularly in relation to changes in glucose transport. Electrical stimulation of the sciatic nerve of the anesthetized rat in vivo led to a time-dependent translocation of protein kinase C and a 2-fold increase in the concentrations of both diacylglycerol and phosphatidic acid. Maximum values for the latter were reached at 2 min and preceded the maximum translocation of protein kinase C (10 min). Stimulation of muscles in vitro increased the rate of glucose transport, but this required 20 min to reach maximum. There was no reversal of translocation or decrease in the concentrations of diacylglycerol and phosphatidic acid even after 30 min of rest following a 5-min period of stimulation in vivo. Translocation was not influenced by variations in applied load at maximal fiber recruitment but was dependent on the frequency of nontetanic stimuli, reaching a maximum at 4 Hz. The relationship between protein kinase C and glucose transport was also explored by varying the number of tetanic stimuli. Whereas only one train of stimuli (200 ms, 100 Hz) was required for maximal effects on protein kinase C, diacylglycerol, and phosphatidic acid, more than 35 trains of stimuli were required to activate glucose transport. It is concluded that the production of diacylglycerol and the translocation of protein kinase C may be causally related. However, if the translocated protein kinase C is involved in the activation of glucose transport during muscle contractions, an accumulated exposure to Ca2+, resulting from multiple contractions, would appear to be necessary.     

Stimulation of protein kinase C and insulin release by 1-oleoyl-2-acetyl-glycerol

The membrane-accessible diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol (OAG, 5-500 microM) caused a dose-related activation of protein kinase C in rat islet homogenates. In islet cell membranes exposed to [gamma-32P]ATP, OAG (100 microM) stimulated the net production of labelled phosphatidate and inhibited that of labelled phosphatidylinositol 4-phosphate. In intact islets exposed to 5.6 mM D-glucose, OAG (100 microM) decreased the outflow of 86Rb, increased that of 45Ca and caused a rapid stimulation of insulin release. The secretory response to OAG was dose-related in the 50-500 microM range, being most marked, in relative terms, at a glucose concentration close to the threshold value for stimulation of insulin release by this hexose. It was decreased but not abolished in the absence of CaCl2 and presence of EGTA. At variance with tumor-promoting phorbol esters, OAG failed to potentiate insulin release stimulated by a hypoglycaemic sulphonylurea. Although these findings support the view that activation of protein kinase C by diacylglycerol represents an efficient modality for stimulation of insulin release, they suggest that the effect of OAG upon islet function may not be solely attributable to such an activation.     

Substrate-dependent proton load and recovery of stunned hearts during pyruvate dehydrogenase stimulation

Stimulation of pyruvate dehydrogenase (PDH) improves functional recovery of postischemic hearts. This study examined the potential for a mechanism mediated by substrate-dependent proton production and intracellular pH. After 20 min of ischemia, isolated rabbit hearts were reperfused with or without 5 mM dichloroacetate (DCA) in the presence of either 5 mM glucose, 5 mM glucose + 2.5 mM lactate, or 5 mM glucose + 2.5 mM pyruvate. DCA inhibits PDH kinase, increasing the proportion of dephosphorylated, active PDH. Unlike pyruvate or glucose alone, lactate + glucose did not support the effects of DCA on the recovery of rate-pressure product (RPP) (without DCA, RPP = 14,000 +/- 1,200, n = 6; with DCA, RPP = 13,700 +/- 1,800, n = 9). Intracellular pH, from (31)P nuclear magnetic resonance spectra, returned to normal within 2.1 min of reperfusion with all substrates except for lactate + glucose + DCA or lactate + DCA, which delayed pH recovery for up to 12 min (at 2.1 min pH = 6. 00 +/- 0.08, lactate + glucose + DCA; pH = 6.27 +/- 0.34, for lactate + DCA). Hearts were also reperfused after 10 min of ischemia with 0.5 mM palmitate + 5 mM DCA and either 2.5 mM pyruvate or 2.5 mM lactate. Again, intracellular pH recovery was delayed in the presence of lactate. PDH activation in the presence of lactate also decreased coupling of oxidative metabolism to mechanical work. These findings have implications for therapeutic use of stimulated carbohydrate oxidation in stunned hearts.     

Stimulation of phosphatidylcholine hydrolysis, diacylglycerol release, and arachidonic acid production by oncogenic ras is a consequence of protein kinase C activation

Swiss-3T3 cells were scrape-loaded with oncogenically activated p21ras protein. 10-20 min after introducing Val12p21ras into the cell, diacylglycerol levels were increased, but levels of inositol phosphates were unaltered. However, cellular choline and phosphocholine levels were increased with a similar time course to that observed for diacylglycerol production, suggesting that ras increases phosphatidylcholine turnover but not phosphatidylinositol turnover. Down-regulation of protein kinase C (by prolonged exposure to phorbol esters prior to scrape loading) blocked the ability of ras protein to elevate the levels of diacylglycerol, choline, and phosphocholine. Oncogenic ras can, therefore, cause a substantial increase in diacylglycerol (which correlates with increased phosphatidylcholine breakdown) in a protein kinase C-dependent fashion. Val12p21ras also increased arachidonic acid release, which was also dependent on protein kinase C activation. Induction of DNA synthesis by oncogenic ras was unaffected by inhibitors of prostaglandin synthesis, indicating that conversion of the released arachidonic acid to various prostaglandins is not required for stimulation of DNA synthesis by ras. We suggest that ras rapidly activates protein kinase C, which in turn activates a number of cellular signalling systems, leading to a sustained increase in diacylglycerol levels. This elevation of diacylglycerol could sustain protein kinase C activation over the 12-15 h required for initiation of DNA synthesis.     

Phosphatidylethanol biosynthesis in ethanol-exposed NG108-15 neuroblastoma X glioma hybrid cells. Evidence for activation of a phospholipase D phosphatidyl transferase activity by protein kinase C

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulates the release of free choline from intact NG108-15 cells into the medium, without affecting the release of phosphocholine (Liscovitch, M., Blusztajn, J.K., Freese, A., and Wurtman, R.J. (1987) Biochem. J. 241, 81-86). To test the hypothesis that this response reflects activation of cellular phospholipase D, via protein kinase C (Ca2+/phospholipid-dependent enzyme), I examined in NG108-15 cells the biosynthesis of the abnormal phospholipid phosphatidylethanol, produced by phospholipase D in the presence of ethanol by transphosphatidylation. Phosphatidylethanol production was quantitated by measuring the incorporation of phosphatidyl moieties (prelabeled metabolically with [3H]oleic acid) into phosphatidylethanol. The production of phosphatidylethanol in NG108-15 cells was virtually dependent on stimulation by TPA, in a time- and concentration-dependent manner (EC50 = 18 nM). The rate of 3H-phosphatidylethanol formation reached a peak after 10 min of incubation with TPA and declined gradually thereafter. The levels of 3H-phosphatidylethanol in TPA-treated cells were directly related to ethanol concentration in the physiologically attainable range (20-80 mM). Phosphatidylethanol production was activated only by phorbol derivatives that are activators of protein kinase C (i.e. TPA, 4 beta-phorbol-12,13-dibutyrate, and 4 beta-phorbol-12,13-didecanoate) and could be mimicked by a cell-permeant diacylglycerol, 1,2-dioctanoyl-sn-glycerol, in a nonadditive manner. The effect of TPA was inhibited by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (0.1 mM) by 70% but not by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide. Phosphatidylethanol formation was completely abolished in cells in which protein kinase C was down-regulated by pretreatment of the cells with TPA. These results indicate that phosphatidylethanol biosynthesis in NG108-15 cells depends largely on activation of protein kinase C. In contrast to its effects on the release of free choline and on the accumulation of phosphatidylethanol, TPA did not affect the levels of phosphatidic acid in NG108-15 cells. It is therefore proposed that protein kinase C selectively activates the phosphatidyl transferase activity of phospholipase D, reflecting a signal termination mechanism which may be operative in phospholipase D-mediated signal transduction cascades.     

Alpha 1-adrenergic receptors promote phosphatidylcholine hydrolysis in MDCK-D1 cells. A mechanism for rapid activation of protein kinase C

Hydrolysis of phosphoinositides can lead to mobilization of calcium and production of diacylglycerol, which together are proposed to activate protein kinase C. We have shown that phosphoinositide hydrolysis mediated by alpha 1-adrenergic receptors on Madin-Darby canine kidney cells (MDCK-D1) occurred with an early lag and increased over a prolonged time course (Slivka, S.R., and Insel, P.A. (1987) J. Biol. Chem. 262, 4200-4207). In this study we characterize another type of receptor-mediated phospholipid hydrolysis in MDCK-D1 cells, alpha 1-adrenergic receptor-mediated hydrolysis of phosphatidylcholine. The predicted products of this hydrolysis, phosphorylcholine and diacylglycerol, were detectable as early as 0.5 min after alpha 1-adrenergic receptor stimulation by epinephrine. This hydrolysis appears to be a primary event after receptor occupancy because it occurred in the presence of neomycin, an inhibitor of polyphosphoinositide hydrolysis, and the protein kinase C inhibitors, sphingosine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). In addition, we demonstrate alpha 1-adrenergic receptor-mediated activation of protein kinase C in MDCK-D1 cells. This activation was measured as a rapid translocation (0.5 min) of protein kinase C activity from the cytosolic fraction to the membrane fraction. This translocation also was not inhibited by neomycin. The time course and agonist concentration dependence of both phosphatidylcholine hydrolysis and protein kinase C activation by alpha 1-adrenergic receptors were similar. Thus, we propose that agonists acting at alpha 1-adrenergic receptors promote hydrolysis of phosphatidylcholine which results in rapid generation of diacylglycerol for the activation of protein kinase C.     

Insulin increases membrane and cytosolic protein kinase C activity in BC3H-1 myocytes

Insulin treatment stimulated the activity of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in both cytosolic and membrane fractions of BC3H-1 myocytes. Within 60 s of insulin treatment, membrane protein kinase C activity increased 2-fold, diminished toward control levels transiently, and then increased 2-fold again after 15 min. Cytosolic protein kinase C activity increased more gradually and steadily up to 80% over a 20-min period. Increases in protein kinase C activity were dose-dependent and were not simply a result of translocation of cytosolic enzyme (although this may have occurred), as total activity was also increased. The increase in protein kinase C activity was not inhibited by cycloheximide (which also increased protein kinase C activity and 2-deoxyglucose transport) and was still evident following anion exchange chromatography. The insulin effect was decidedly different from those of 12-O-tetradecanoylphorbol-13-acetate and phenylephrine using histone III-S as substrate. Phenylephrine decreased cytosolic protein kinase C activity while increasing membrane activity; 12-O-tetradecanoylphorbol-13-acetate only decreased cytosolic protein kinase C activity. The early insulin-induced increases in membrane protein kinase C activity may be related to increased diacylglycerol generation from de novo phosphatidic acid synthesis, as there were rapid increases in [3H]glycerol incorporation into diacylglycerol, and transient increases in phospholipid hydrolysis, as there were transient rapid increases in [3H]diacylglycerol in cells prelabeled with [3H]arachidonate. Later, sustained increases in membrane and cytosolic protein kinase C activity may reflect the continuous activation of de novo phospholipid synthesis, as there were associated increases in [3H]glycerol incorporation into diacylglycerol at later, as well as very early time points.     

Effects of phorbol ester and cholecystokinin on the intracellular distribution of protein kinase C in rabbit pancreatic acini.

Treatment of rabbit pancreatic acini with the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), resulted in a time- and dose-dependent decrease of soluble protein kinase C activity coinciding with an increase of protein kinase C activity in the particulate fraction. After 5 min, soluble protein kinase C activity had decreased to almost 10% of the corresponding control. Total extractable protein kinase C activity, however, remained unchanged, indicating that the decrease of soluble protein kinase C activity was not due to TPA-induced inactivation of the enzyme. The biologically inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, did not induce such a translocation of protein kinase C. The half-maximal concentration for TPA-induced translocation of protein kinase C was 40 nM, and was equal to that for TPA-induced amylase secretion from isolated acini. This suggests that translocation of protein kinase C to the particulate fraction is an important step in TPA-induced activation of protein kinase C and enzyme secretion. On the other hand, cholecystokinin, a secretagogue of the calcium-mobilizing type, whose secretory action is thought to be mediated, at least in part, by protein kinase C, did not change the subcellular distribution of protein kinase C. In the presence of R59022 6-(2-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl ) ethyl-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one, an inhibitor of diacylglycerol kinase activity, cholecystokinin produced a small but significant translocation of protein kinase C, suggesting that the inability of the hormone to induce translocation is not due to a rapid conversion of the diacylglycerol formed into phosphatidic acid.     

Insulin and glucose modulate protein kinase C activity in rat adipocytes

In the presence of 1 mM glucose, insulin (10 ng/ml) increases both catalytic and receptor-binding properties of adipocyte cytosolic protein kinase C (PKC). Preincubation of adipocytes with 10 mM glucose raises basal PKC catalytic activity and prevents further stimulation of this enzyme by insulin. The effect of hyperglycemia is likely to be mediated by direct conversion of glucose into diacylglycerol. Thus, an incorporation of 14C-glucose into diacylglycerol is enhanced 10-fold in the presence of 10 mM glucose. These observations indicate that, in normal adipocytes, both insulin and glucose activate PKC; hyperglycemia eliminates the ability of insulin to stimulate this enzyme, thereby interfering with insulin action.     

Attenuation of hen granulosa cell steroidogenesis by a phorbol ester and 1-oleoyl-2-acetylglycerol

A series of studies was conducted to evaluate the effects of phorbol esters and a diacylglycerol analog on basal and hormone-stimulated steroidogenesis in granulosa cells from the largest preovulatory follicle of the domestic hen. Agents that previously have been shown to activate protein kinase C, such as the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), and the synthetic diacylglycerol analog, 1-oleoyl-2-acetylglycerol (OAG), suppressed luteinizing hormone (LH)-induced progesterone (PMA at levels of 10 and 100 ng/tube; OAG at levels of 10 and 25 micrograms/tube), and androgen (10 and 100 ng PMA; 25 micrograms OAG) production, but had no effect on basal levels of either steroid. Furthermore, PMA decreased the ability of vasoactive intestinal peptide to induce steroidogenesis, suggesting that protein kinase C activation may generally modulate the activity of hormones that act via the adenylyl cyclase/cyclic 3',5'-adenosine monophosphate (cAMP) second messenger system. In further support of this proposal was the finding that PMA and OAG decreased the production of cAMP in response to LH, and attenuated the steroidogenic response in granulosa cells exposed to 10 mM 8-bromo-cAMP. By contrast, the induction of calcium mobilization using a calcium ionophore (A23187; 0.5-2.0 microM) stimulated progesterone and androgen production without increasing intracellular levels of cAMP, and this stimulatory effect on steroidogenesis was not inhibited by the presence of 100 ng PMA/tube. From these data, we suggest that the activation of protein kinase C in granulosa cells of the hen may provide a physiological mechanism by which receptor-mediated steroidogenesis, involving the adenylyl cyclase second messenger system, is modulated.     

Response of cardiac myocytes to a ramp increase of diacylglycerol generated by photolysis of a novel caged diacylglycerol.

To test the responsiveness of living cells to the intracellular messenger diacylglycerol, we developed a prototype caged diacylglycerol compound, 3-O-(alpha-carboxyl-2,4-dinitrobenzyl)-1 ,2-dioctanoyl-rac-glycerol (designated alpha-carboxyl caged diC(8)), that produces dioctanoylglycerol (diC(8)) on photolysis. Alpha-Carboxyl caged diC(8) is biologically inert toward diacylglycerol kinase and protein kinase C in vitro and is readily incorporated into cardiac myocyte membranes, where it has no effect before irradiation. Exposure to near-UV light releases biologically active diC8 in good yield (quantum efficiency = 0.2). Here we examine a cellular response to controlled elevation of diC8 within single cardiac myocytes. Twitch amplitude was monitored in electrically stimulated myocytes, and a ramp increase in the concentration of diC(8) was generated by continuous irradiation of cells loaded with the caged compound. The myocyte response was biphasic with a positive inotropic phase (39% increase in twitch amplitude), followed by a large negative inotropic phase (>80% decrease). The time to peak inotropy for both phases depended on the light intensity, decreasing from 376 +/- 51 S to 44 +/- 5 s (positive phase) and 422 +/- 118 S to 51 +/- 9 S (negative phase) as the light intensity was increased eightfold. Both phases were inhibited by the protein kinase C inhibitor chelethyrine chloride. An increase in extracellular K+ from 5 mM to 20 mM to partially depolarize the cell membrane eliminated the positive inotropic phase, but the negative inotropic response was largely unaltered. The results reveal new features in the response of cardiac muscle to diacylglycerol, including a positive inotropic phase and a complex responsiveness to a simple linear increase in diacylglycerol. The effects of photoreleased diC(8) were similar to the effects of opiate agonists selective for kappa receptors, consistent with a major role for diacylglycerol in these responses.     

Phorbol ester-induced activation of protein kinase C leads to increased formation of diacylglycerol in human neutrophils

Human neutrophils stimulated with a phorbol ester (phorbol 12-myristrate 13-acetate or phorbol 12,13-dibutyrate) responded with an increase in diacylglycerol, considered the natural activator of protein kinase C. The amounts of diacylglycerol formed were considerable, reaching 700-900% of basal after 20 min. In contrast, 4-alpha-phorbol 12-myristate 13-acetate did not induce any detectable formation of diacylglycerol. Simultaneously, phorbol 12-myristate 13-acetate exposure caused increased breakdown of both phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. These results suggest that once activated, protein kinase C can positively modulate its own activity by inducing additional formation of diacylglycerol from at least two different sources.     

Effects of insulin secretagogues on protein kinase C-catalyzed phosphorylation of an endogenous substrate in isolated pancreatic islets

The influence of the insulin secretagogues, carbachol and glucose, on protein kinase C activation in isolated pancreatic islets has been examined by determination of the phosphorylation state of an endogenous 80-kDa protein substrate of protein kinase C. The islet 80-kDa protein was identified as the myristoylated alanine-rich C kinase substrate previously described (Stumpo D. J., Graff, J. M., Albert, K. A., Greengard, P., and Blackshear, P. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4012-4016) by immunoprecipitation studies. The muscarinic agonist, carbachol (500 microM), induced insulin secretion and a time-dependent increase in the phosphorylation state of this protein in islets. This phosphorylation was maximal (220 +/- 24% of control) at 5 min and was suppressed by the protein kinase C inhibitor, staurosporine. Concentrations of glucose (28 mM) which induce maximal insulin secretion did not induce a statistically significant increase in 80-kDa phosphorylation. The combination of carbachol and a submaximally stimulatory concentration of glucose (10 mM), when added simultaneously, exerted a marked synergistic effect on insulin secretion and a synergistic effect on the phosphorylation of the 80-kDa protein kinase C substrate. These data suggest that the activation of protein kinase C may play an important role in carbachol-induced insulin secretion and in the potentiation by carbachol of insulin secretion induced by glucose. However, the activation of protein kinase C does not appear to be a primary determinant of insulin secretion induced by glucose alone.     

Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor beta1 (TGF-beta1), and addition of TGF-beta1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-beta1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFbeta1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFbeta1-driven matrix production within a diabetic milieu.     

Protein kinase C from chicken gizzard: characterization and detection of an inhibitor and endogenous substrates

Protein kinase C was purified from the cytosolic fraction of chicken gizzard by Ca2+ -dependent hydrophobic interaction chromatography, anion-exchange chromatography, and hydrophobic chromatography. The molecular weight was estimated as 61,500 by gel filtration and 80,000 by denaturing gel electrophoresis, indicating that the native enzyme is a monomer. Using the mixed micellar assay, with histone III-S as the substrate, protein kinase C required Ca2+, phospholipid, and diacylglycerol for activity, with half-maximal activation at approximately 5 x 10(-7) M Ca2+ in the presence of L-alpha-phosphatidyl-L-serine and 1,2-diolein. No activation by Ca2+ was observed in the absence of diacylglycerol. Protein kinase C requires free Mg2+, in addition to the MgATP2- substrate, for activity. The Km for ATP was determined to be 20 microM. Activity was sensitive to ionic strength, with half-maximal inhibition at 70 mM NaCl. Using the liposomal assay, phosphorylation of platelet P47 protein and smooth muscle vinculin was more strongly dependent on Ca2+ and lipids than was histone phosphorylation. Partial digestion of protein kinase C with trypsin yielded a constitutively active fragment. A heat-stable inhibitor and three major endogenous protein substrates of protein kinase C were also detected in chicken gizzard smooth muscle.     

Link between free radicals and protein kinase C in glucose-induced alteration of vascular dilation

Development of vascular complications in diabetes has been linked to the quality of glucose regulation and characterized by endothelial dysfunction. The exact mechanism behind vascular complications in diabetes is poorly understood. However, alteration of nitric oxide (NO) biosynthesis or bioactivity is strongly implicated and the mechanism behind such alterations is still a subject for research investigations. In the present study, we tested the hypothesis that glucose-induced attenuation of vascular relaxation involves protein kinase C (PKC)-linked generation of free radicals. Vascular relaxation to acetylcholine (ACh; 10(-9)-10(-5) M), isoproterenol (10(-9)-10(-5) M), or NO donor, sodium nitropruside (SNP; 10(-9)-10(-6) M) was determined in phenylephrine (PE, 10(-7) M) pre-constricted aortic rings from Sprague-Dawley rats in the presence or absence of 30 mM glucose (30 min), L-nitro-arginine methyl ester (L-NAME; 10(-4) M for 15 min), a NO synthase inhibitor, or xanthine (10(-5) M), a free radical generator. ACh dose-dependently caused relaxation that was attenuated by L-NAME, glucose, or xanthine. Pre-incubation (15 min) of the rings with vitamin C (10(-4) M), an antioxidant or calphostin C (10(-6) M), a PKC inhibitor, restored the ACh responses. However, high glucose had no significant effects on SNP or isoproterenol-induced relaxation. ACh-induced NO production by aortic ring was significantly reduced by glucose or xanthine. The reduced NO production was restored by pretreatment with vitamin C or calphostin C in the presence of glucose, but not xanthine. These data demonstrate that oxidants or PKC contribute to glucose-induced attenuation of vasorelaxation which could be mediated via impaired endothelial NO production and bioavailability. Thus, pathogenesis of glucose-induced vasculopathy involves PKC-coupled generation of oxygen free radicals which inhibit NO production and selectively inhibit NO-dependent relaxation.     

Thyrotropin releasing hormone action in pituitary cells. Protein kinase C-mediated effects on the epidermal growth factor receptor

Thyrotropin releasing hormone (TRH) causes phosphatidylinositol bisphosphate hydrolysis to form inositol trisphosphate and diacylglycerol. Since diacylglycerol activates protein kinase C (Ca2+/phospholipid-dependent enzyme), this enzyme may be involved in mediating the physiological response to TRH. Activation of protein kinase C leads to phosphorylation of receptors for epidermal growth factor (EGF) and decreased EGF affinity. The present study examined the effect of TRH on EGF binding to intact GH4C1 rat pituitary tumor cells to test whether TRH activates protein kinase C. Cells were incubated with TRH at 37 degrees C and specific 125I-EGF binding was then measured at 4 degrees C. 125I-EGF binding was decreased by a 10-min treatment with 0.1-100 nM TRH to 30-40% of control in a dose-dependent manner. 125I-EGF binding was not altered if cells were incubated at 4 degrees C, although TRH receptors were saturated or in a variant pituitary cell line without TRH receptors. TRH (10 min at 37 degrees C) decreased EGF receptor affinity but caused little change in receptor density, 125I-EGF internalization, or degradation. When cells were incubated continuously with TRH, there was a recovery of 125I-EGF binding after 24 h. Incubation with the protein kinase C activating phorbol ester TPA caused an immediate (less than 10 min) profound (greater than 85%) decrease in 125I-EGF binding followed by partial recovery at 24 h. Maximally effective doses of TRH and TPA decreased EGF receptor affinity with half-times of 3 min. EGF treatment (5 min) caused an increase in the tyrosine phosphate content of several proteins; prior incubation with TRH resulted in a small decline in the EGF response. GH4C1 cells were incubated with 500 nM TPA for 24 h in order to down-regulate protein kinase C. Protein kinase C depletion was confirmed by immunoblots and the effects of TRH and TPA on 125I-EGF binding were tested. TRH and TPA were both much less effective in cells pretreated with phorbol esters. TRH increased cytoplasmic pH measured with an intracellularly trapped pH sensitive dye after mild acidification with nigericin. This TRH response is presumed to be the result of protein kinase C-mediated activation of the amiloride-sensitive Na+/H+ exchanger and was blunted in protein kinase C-depleted cells. All of these results are consistent with the view that TRH acts rapidly in the intact cell to activate protein kinase C and that a consequence of this activation is EGF receptor phosphorylation and Na+/H+ exchanger activation.     

Carnitine stimulation of glucose oxidation in the fatty acid perfused isolated working rat heart.

The effects of L-carnitine on myocardial glycolysis, glucose oxidation, and palmitate oxidation were determined in isolated working rat hearts. Hearts were perfused under aerobic conditions with perfusate containing either 11 mM [2-3H/U-14C]glucose in the presence or absence of 1.2 mM palmitate or 11 mM glucose and 1.2 mM [1-14C]palmitate. Myocardial carnitine levels were elevated by perfusing hearts with 10 mM L-carnitine. A 60-min perfusion period resulted in significant increases in total myocardial carnitine from 4376 +/- 211 to 9496 +/- 473 nmol/g dry weight. Glycolysis (measured as 3H2O production) was unchanged in carnitine-treated hearts perfused in the absence of fatty acids (4418 +/- 300 versus 4547 +/- 600 nmol glucose/g dry weight.min). If 1.2 mM palmitate was present in the perfusate, glycolysis decreased almost 2-fold compared with hearts perfused in the absence of fatty acids. In carnitine-treated hearts this drop in glycolysis did not occur (glycolytic rates were 2911 +/- 231 to 4629 +/- 460 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively. Compared with control hearts, glucose oxidation rates (measured as 14CO2 production from [U-14C]glucose) were unaltered in carnitine-treated hearts perfused in the absence of fatty acids (1819 +/- 169 versus 2026 +/- 171 nmol glucose/g dry weight.min, respectively). In the presence of 1.2 mM palmitate, glucose oxidation decreased dramatically in control hearts (11-fold). In carnitine-treated hearts, however, glucose oxidation was significantly greater than control hearts under these conditions (158 +/- 21 to 454 +/- 85 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively). Palmitate oxidation rates (measured as 14CO2 production from [1-14C]palmitate) decreased in the carnitine-treated hearts from 728 +/- 61 to 572 +/- 111 nmol palmitate/g dry weight.min. This probably occurred secondary to an increase in overall ATP production from glucose oxidation (from 5.4 to 14.5% of steady state myocardial ATP production). The results reported in this study provide direct evidence that carnitine can stimulate glucose oxidation in the intact fatty acid perfused heart. This probably occurs secondary to facilitating the intramitochondrial transfer of acetyl groups from acetyl-CoA to acetylcarnitine, thereby relieving inhibition of the pyruvate dehydrogenase complex.     

High glucose inhibits fructose uptake in renal proximal tubule cells: involvement of cAMP, PLC/PKC, p44/42 MAPK, and cPLA2

The precise signal that regulates fructose transport in renal proximal tubule cells (PTCs) under high glucose conditions is not yet known although fructose has been recommended as a substitute for glucose in the diets of diabetic people. Thus, we investigated that effect of high glucose on fructose uptake and its signaling pathways in primary cultured rabbit renal PTCs. Glucose inhibited the fructose uptake in a time- and dose-dependent manner. A maximal inhibitory effect of glucose on fructose uptake was observed at 25 mM glucose after 48 h, while 25 mM mannitol and l-glucose did not affect fructose uptake. Indeed, 25 mM glucose for 48 h decreased GLUT5 protein level. Thus, the treatment of 25 mM glucose for 48 h was used for this study. Glucose-induced (25 mM) inhibition of fructose uptake was blocked by pertussis toxin (PTX), SQ-22536 (an adenylate cyclase inhibitor), and myristoylated amide 14-22 (a protein kinase A inhibitor). Indeed, 25 mM glucose increased the intracellular cAMP content. Furthermore, 25 mM glucose-induced inhibition of fructose uptake was prevented by neomycin or U-73122 (phospholipase C inhibitors) and staurosporine or bisindolylmaleimide I (protein kinase C inhibitors). In fact, 25 mM glucose increased the total PKC activity and translocation of PKC from the cytosolic to membrane fraction. In addition, PD 98059 (a p44/42 mitogen-activated protein kinase (MAPK) inhibitor) but not SB 203580 (a p38 MAPK inhibitor) and mepacrine or AACOCF3 (phospholipase A2 inhibitors) blocked 25 mM glucose-induced inhibition of fructose uptake. Results of Western blotting using the p44/42 MAPK and GLUT5 antibodies were consistent with the results of uptake experiments. In conclusion, high glucose inhibits the fructose uptake through cAMP, PLC/PKC, p44/42 MAPK, and cytosolic phospholipase A2 (cPLA2) pathways in the PTCs.     

Does protein kinase C activation mediate thrombin-induced arachidonate release in human platelets?

Thrombin stimulated rapid formation of diacylglycerol, inositol 1,4,5-trisphosphate (IP3) and thromboxane B2 (TXB2) in human platelets. Formation of diacylglycerol and IP3 appeared to precede that of TXB2. Activation of protein kinase C by diacylglycerol combining with Ca+2 mobilization by IP3 has been implicated in mediating arachidonate release. However, addition of the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) to platelet suspension did not inhibit thrombin-stimulated arachidonate release and TXB2 synthesis, whereas addition of the Ca+2 antagonist, 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8) or the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) abolished arachidonate release. The correlation of IP3 production with arachidonate release on increasing the concentrations of thrombin was further examined. IP3 production reached near maximum at 0.2 U/ml, whereas TXB2 synthesis continued to increase at 1 U/ml. These results suggest that protein kinase C activation may not mediate arachidonate release and that Ca+2 mobilization by IP3 may only partially account for arachidonate release in platelets stimulated with relatively high concentrations of thrombin.