Cytoplasmic lipid bodies of neutrophils: formation induced by cis- unsaturated fatty acids and mediated by protein kinase C

Lipid bodies, nonmembrane-bound cytoplasmic inclusions, serve as repositories of esterified arachidonate and are increased in cells associated with inflammatory reactions. We have evaluated stimuli and mechanisms responsible for lipid body formation within human polymorphonuclear leukocytes (PMNs). Arachidonic acid and oleic acid stimulated dose-dependent formation of lipid bodies over 0.5-1 h. Other C20 and C18 fatty acids were less active and demonstrated rank orders as follows: cis-unsaturated fatty acids were much more active than trans-fatty acids, and activity diminished with decreasing numbers of double bonds. Lipid bodies elicited in vitro with cis-fatty acids were ultrastructurally identical to lipid bodies present in PMNs in vivo. Lipid body induction was not because of fatty acid-elicited oxidants or fatty acid-induced ATP depletion. Cis-fatty acid-induced activation of protein kinase C (PKC) was involved in lipid body formation as evidenced by the capacity of other PKC activators, 1-oleoyl-2-acetyl-glycerol and two active phorbol esters, phorbol myristate acetate, and phorbol 12,13 dibutyrate, but not an inactive phorbol, to induce lipid body formation. The PKC inhibitor, 1-O-hexadecyl-2-O-methyl-glycerol, inhibited PMN lipid body formation induced by oleic and arachidonic acids and by 1-oleoyl-2-acetyl-glycerol and phorbol myristate acetate. Other PKC inhibitors (staurosporine, H-7) also inhibited lipid body formation. Formation of lipid bodies in PMNs is a specific cellular response, stimulated by cis-fatty acids and diglycerides and apparently mediated by PKC, which results in the mobilization and deposition of lipids within discrete, ultrastructurally defined cytoplasmic domains.     

Modulation of voltage-dependent Ca channel current by arachidonic acid and other long-chain fatty acids in rabbit intestinal smooth muscle.

The effects of arachidonic acid (AA) and other long-chain fatty acids on voltage-dependent Ca channel current (ICa) were investigated, with the whole cell patch clamp method, in longitudinal smooth muscle cells of rabbit ileum. 10-30 microM AA caused a gradual depression of ICa. The inhibitory effect of AA was not prevented by indomethacin (10 microM) (an inhibitor of cyclooxygenase) or nordihydroguaiaretic acid (10 microM) (an inhibitor of lipoxygenase). 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7; 25-50 microM) or staurosporine (2 microM) (inhibitors of protein kinase C) did not block the AA-induced inhibition of ICa, and application of phorbol ester (a protein kinase C activator) (phorbol-12,13-dibutyrate, 0.2 microM) did not mimic the AA action. Some other cis-unsaturated fatty acids (palmitoleic, linoleic, and oleic acids) were also found to depress ICa, while a trans-unsaturated fatty acid (linolelaidic acid) and saturated fatty acids (capric, lauric, myristic, and palmitic acids) had no inhibitory effects on ICa. Myristic acid consistently increased the amplitude of ICa at negative membrane potentials. The present results suggest the possible role of AA, and perhaps other fatty acids, in the physiological and/or pathological modulation of ICa in smooth muscle.     

Effect of cis-unsaturated fatty acids on aortic protein kinase C activity.

Long-chain cis-unsaturated fatty acids could substitute for phosphatidylserine and activate bovine aortic protein kinase C in assays with histone as substrate. The optimal concentration was 24-40 microM for oleic, linoleic and arachidonic acids. With arachidonic acid, the Ka for Ca2+ was 130 microM and kinase activity was maximal at 0.5 mM-Ca2+. Diolein only slightly activated the oleic acid-stimulated enzyme at low physiological Ca2+ concentrations (0.1 and 10 microM). Oleic acid also stimulated kinase C activity, determined with a Triton X-100 mixed-micellar assay. Under these conditions, the fatty acid activation was absolutely dependent on the presence of diolein, but a Ca2+ concentration of 0.5 mM was still required for maximum kinase C activity. The effect of fatty acids on protein kinase C activity was also investigated with the platelet protein P47 as a substrate, since the properties of kinase C can be influenced by the choice of substrate. In contrast with the results with histone, fatty acids did not stimulate the phosphorylation of P47 by the aortic protein kinase C. Activation of protein kinase C by fatty acids may allow the selective phosphorylation of substrates, but the physiological significance of fatty acid activation is questionable because of the requirement for high concentrations of Ca2+.     

A rice membrane calcium-dependent protein kinase is induced by gibberellin.

A rice (Oryza sativa) seed plasma-membrane calcium-dependent serine/threonine protein kinase (CDPK) has been partially purified. Comparing results in seeds that were treated with and without the plant hormone gibberellin (GA) for 10 min showed that rice CDPK was highly induced by GA. After separating solubilized membrane proteins by sodium dodecyl sulfate-gel electrophoresis, followed by renaturation, a radiolabeled phosphoprotein band of approximately 58 kD was detected, and it was apparently produced by autophosphorylation. There are five aspects of the rice CDPK that show similarity to mammalian protein kinase C (PKC) and to other plant CDPKs: (a) Histone IIIS and PKC peptide-ser25 (19-31) are phosphorylated by rice CDPK. (b) The phosphorylation reaction is strictly dependent on calcium. (c) The activity of the rice CDPK is inhibited by either staurosporine or the PKC inhibitory peptide (19-36). (d) Addition of calmodulin has no effect on the activity of the enzyme; however, the CDPK is inhibited by the calmodulin antagonists trifluoperazine and W-7. (e) The rice CDPK reacts with a mammalian anti-PKC antibody in immunoblotting analysis. However, there is one major difference between the rice CDPK and other CDPKs: the rice CDPK is induced by GA, whereas no mammalian PKC or other plant CDPKs are known to be induced by any hormone.     

Cis-unsaturated fatty acids induce both lipogenesis and calcium binding in adipocytes

The addition of 0.4-3 mM of cis-unsaturated fatty acids such as oleic acid (18:1) or linoleic acid (18:2) to intact rat adipocytes stimulated lipogenesis at 37 degrees C. Saturated or trans-unsaturated fatty acids were ineffective. Fluorescence photobleaching recovery studies performed under similar conditions indicated that the cis-unsaturated fatty acids do not alter lateral mobility of either a lipid probe or a general protein marker in the plasma membrane. A high concentration (7 mM) of Ca2+, which by itself has some stimulatory effect on lipogenesis, significantly potentiated the effect of oleic acid on this insulin-like activity. Measurement of 45Ca2+ binding by fat cells has indicated that cis-unsaturated (but not saturated) fatty acids increased 12- to 20-fold the amount of Ca2+ associated with the cells. The dependence of this effect on the fatty acid concentration correlates well with the effect of the fatty acid on the induction of lipogenesis. Our results suggest that cis-unsaturated fatty acids affect membrane organization in a manner which induces a significant increase in membrane associated or intracellular Ca2+. This increase may be responsible for inducing exocytotic-like processes which facilitate translocation of glucose transport activity from storage sites to the plasma membrane and thus produce an insulin-like effect.     

Constitutively and autonomously active protein kinase C associated with 14-3-3 zeta in the rodent brain

Persistent activation of protein kinase C (PKC) is required for the expression of synaptic plasticity in the brain. There are several mechanisms proposed that can lead to the prolonged activation of PKC. These include long lasting production of lipid activators (diacylglycerol and fatty acid) through mitogen-activated protein (MAP) kinase pathway, and a modification of PKC by reactive oxygen species. In nerve growth factor (NGF)-differentiated PC12 cells, we found that constitutive and autonomous Ca2+-independent PKC activity is associated with 14-3-3 zeta. Because PKC and 14-3-3 zeta are both involved in synaptic plasticity and learning and memory, we examined whether PKC interacts with 14-3-3 zeta in the brain and whether the PKC/14-3-3 zeta complex has autonomous activity. Here we show that three subclasses of PKC, Ca2+-dependent classical PKC, Ca2+-independent novel PKC, and Ca2+-independent and diacylglycerol-insensitive atypical PKC, all interact with 14-3-3 zeta in the rodent brain. The pool size of 14-3-3 zeta bound form of PKC is small (1-4% of each PKC isoform), but they show constitutive and autonomous activity. Our study indicates that the binding of PKC with 14-3-3 zeta is at least in part independent of phosphorylation of PKC and that the C1 domain of PKC is involved in the binding. As both molecules are enriched in synaptic locus, the constitutive PKC activity and its interaction with 14-3-3 zeta could be a mechanism for the persistent PKC activation in the brain.     

Arachidonic and cis-unsaturated fatty acids induce selective platelet substrate phosphorylation through activation of cytosolic protein kinase C

The ability of arachidonic acid and other fatty acids to induce phosphorylation of endogenous substrates and the role of protein kinase C in mediating these effects were examined. In a cell-free cytosolic system derived from human platelets, arachidonic, oleic, and other cis-unsaturated fatty acids induced a dose-dependent phosphorylation of several endogenous substrates. These substrates form a subset of phorbol ester-induced phosphorylations. Multiple lines of evidence suggested the direct involvement of protein kinase C in mediating fatty acid-induced phosphorylations. These observations suggest that arachidonic acid and other unsaturated fatty acids are capable of activating protein kinase C in a physiologic environment resulting in the phosphorylation of multiple endogenous substrates.     

Cyclic AMP-dependent protein kinase (PKA) and protein kinase C phosphorylate sites in the amino acid sequence corresponding to the M3/M4 cytoplasmic domain of alpha4 neuronal nicotinic receptor subunits

To determine whether alpha4 subunits of alpha4beta2 neuronal nicotinic receptors are phosphorylated within the M3/M4 intracellular region by cyclic AMP-dependent protein kinase A (PKA) or protein kinase C (PKC), immunoprecipitated receptors from Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of alpha4 (alpha4(336-597)) were incubated with ATP and either PKA or PKC. Both alpha4 and alpha4(336-597) were phosphorylated by PKA and PKC, providing the first direct biochemical evidence that the M3/M4 cytoplasmic domain of neuronal nicotinic receptor alpha4 subunits is phosphorylated by both kinases. When the immunoprecipitated receptors and the alpha4(336-597) fusion protein were phosphorylated and the labeled proteins subjected to phosphoamino acid analysis, results indicated that alpha4 and alpha4(336-597) were phosphorylated on the same amino acid residues by each kinase. Furthermore, PKA phosphorylated serines exclusively, whereas PKC phosphorylated both serines and threonines. To determine whether Ser(368) was a substrate for both kinases, a peptide corresponding to amino acids 356-371 was synthesized (alpha4(356-371)) and incubated with ATP and the kinases. The phosphorylation of alpha4(356-371) by both PKA and PKC was saturable with K(m)s of 15.3 +/- 3.3 microM and 160.8 +/- 26.8 microM, respectively, suggesting that Ser(368) was a better substrate for PKA than PKC.     

In Vitro Activation of Rat Brain Protein Kinase C by Polyenoic Very-Long-Chain Fatty Acids

Abstract: A variety of fatty acids including the cis -polyunsaturated very-long-chain fatty acids (VLCFA) (>22 carbon atoms) common in retina, spermatozoa, and brain were examined for their ability to activate protein kinase C (PKC) purified from rat brain. Arachidonic [20:4(n-6)], eicosapentaenoic [20:5(n-3)], and docosahexaenoic [22:6(n- 3)] acids as well as the VLCFA dotriacontatetraenoic [32:4(n-6)] and tetratriacontahexaenoic [34:6(n-3)] were equally capable of activating PKC in vitro with maximal activity being between 25 and 50 μ M. The phorbol ester 12- O -tetradecanoylphorbol 13-acetate further enhanced the in vitro activation of PKC when added to the protein kinase assay system with the fatty acids. The fully saturated arachidic acid (20:0) was inactive in both assay systems. The potential significance of the in vitro activation of PKC by the VLCFA is discussed.     

Efficient synthesis of 3-trifluoromethylphenyldiazirinyl oleic acid derivatives and their biological activity for protein kinase C

3-trifluoromethylphenyldiazirine based oleic acids derivatives are synthesized to elucidate the functions of specific activation of protein kinase C (PKC) with oleic acid. The synthetic route is based on the alkylation of phenolic derivative with oleic acid equivalent and the post-functionalization of the compound to achieve radiolabeling. Several compounds have biological activity for PKC with similar efficacy with that of oleic acid. The results indicated that the diaizinyl oleic acid derivatives should be useful to study the specific functions of oleic acid for PKC.     

Inhibition of insulin sensitivity by free fatty acids requires activation of multiple serine kinases in 3T3-L1 adipocytes

Insulin receptor substrate (IRS) has been suggested as a molecular target of free fatty acids (FFAs) for insulin resistance. However, the signaling pathways by which FFAs lead to the inhibition of IRS function remain to be established. In this study, we explored the FFA-signaling pathway that contributes to serine phosphorylation and degradation of IRS-1 in adipocytes and in dietary obese mice. Linoleic acid, an FFA used in this study, resulted in a reduction in insulin-induced glucose uptake in 3T3-L1 adipocytes. This mimics insulin resistance induced by high-fat diet in C57BL/6J mice. The reduction in glucose uptake is associated with a decrease in IRS-1, but not IRS-2 or GLUT4 protein abundance. Decrease in IRS-1 protein was proceeded by IRS-1 (serine 307) phosphorylation that was catalyzed by serine kinases inhibitor kappaB kinase (IKK) and c-JUN NH2-terminal kinase (JNK). IKK and JNK were activated by linoleic acid and inhibition of the two kinases led to prevention of IRS-1 reduction. We demonstrate that protein kinase C (PKC) theta is expressed in adipocytes. In 3T3-L1 adipocytes and fat tissue, PKCtheta was activated by fatty acids as indicated by its phosphorylation status, and by its protein level, respectively. Activation of PKCtheta contributes to IKK and JNK activation as inhibition of PKCtheta by calphostin C blocked activation of the latter kinases. Inhibition of either PKCtheta or IKK plus JNK by chemical inhibitors resulted in protection of IRS-1 function and insulin sensitivity in 3T3-L1 adipocytes. These data suggest that: 1) activation of PKCtheta contributes to IKK and JNK activation by FFAs; 2) IKK and JNK mediate PKCtheta signals for IRS-1 serine phosphorylation and degradation; and 3) this molecular mechanism may be responsible for insulin resistance associated with hyperlipidemia.     

Linoleic acid is a potent activator of protein kinase C type III-alpha isoform in pancreatic acinar cells; its role in amylase secretion

Linoleic acid, an unsaturated-long chain fatty acid, was found to maximally activate protein kinase C (PKC) more effectively than arachidonic or linolenic acid, while the saturated fatty acids palmitic or arachidic had no stimulatory effect. Treatment of intact pancreatic acinar cells with linoleic acid resulted in dose-dependent phosphorylation of endogenous substrate proteins for this kinase and simultaneously stimulated amylase secretion in a dose- and time-dependent fashion. During chromatographic separation of pancreas protein kinase C activity, utilizing hydroxylapatite (HTP), Type III-alpha PKC isoform was detected. These data are consistent with a role for PKC in the regulation of pancreatic exocrine secretion.     

IL-3-induced activation of protein kinases in the mast cell/megakaryocyte R6-XE.4 line

A role for second messenger-regulated protein kinases in the early post-IL-3 receptor signal transduction pathway was investigated in the mast cell/megakaryocyte line R6-XE.4. The activity of the calcium- and phospholipid-dependent protein kinase C (PKC) was assessed by the ability of the enzyme to phosphorylate histone H1 in the presence of calcium, diacylglycerol, and phosphatidylserine or after proteolytic activation of PKC with trypsin. In high serum-supplemented cells, but not in cells that were preincubated in serum-deficient media for 6 h, subsequent treatment for 15 min with synthetic IL-3 (10 micrograms/ml) caused up to a sixfold increase in the calcium- and lipid-stimulated histone H1 phosphorylating activity of particulate-associated PKC after fractionation on MonoQ. However, there was no corresponding reduction of cytosolic PKC activity. Therefore, IL-3 appeared to modify the activity of preexisting membrane-associated PKC rather than eliciting its recruitment from the cytoplasm in R6-XE.4 cells. This was in contrast to the situation with FDC-P1 cells, where IL-3 induced PKC translocation. IL-3 also stimulated a cytosolic protein kinase that phosphorylated a synthetic peptide patterned after a phosphorylation site in ribosomal protein S6, but this IL did not alter the activity of cAMP-dependent protein kinase.     

A synthetic peptide derived from the non-structural protein 3 of hepatitis C virus serves as a specific substrate for PKC

A synthetic peptide corresponding to the amino acid sequence Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 of the hepatitis C virus (HCV) polyprotein was found to be a selective substrate for protein kinase C (PKC). In the presence of Ca2+, TPA and phospholipid, PKC phosphorylates the peptide [termed HCV(1487-1500)] with a Km of 11 microM and Vmax of 24 micromol x min(-1) x mg(-1). HCV(1487-1500) acts as a competitive inhibitor of PKC towards other peptide or protein substrates and inhibits the kinase activity with an IC50 corresponding to the Km values measured for the substrates. N- or C-terminally deleted analogs of HCV(1487-1500) did not show inhibitory effects and were only marginally or not phosphorylatable. We designed an additional peptide in which the tyrosine residue was replaced by phenylalanine ([Phe1499]HCV(1487-1500)). This peptide was neither phosphorylated by other serine/threonine kinases tested nor by whole cell extracts prepared from PKC-depleted cells. [Phe1499]HCV(1487-1500) was used to monitor the TPA-induced translocation of PKC activity to the particulate fraction in JB6 cells. The use of SDS/PAGE to separate the peptide from ATP and Pi allowed to monitor simultaneously PKC autophosphorylation and phosphorylation of the peptide. The data presented here show that[Phe1499]HCV(1487-1500) can serve as a convenient tool for investigations of PKC activity also in the presence of other kinases in tissues or in crude cell extracts.     

Lovastatin inhibits the extracellular-signal-regulated kinase pathway in immortalized rat brain neuroblasts

Arachidonic acid is a potential paracrine agent released by the uterine endometrial epithelium to induce PTGS2 [PG (prostaglandin)-endoperoxide synthase 2] in the stroma. In the present study, bovine endometrial stromal cells were used to determine whether PTGS2 is induced by arachidonic acid in stromal cells, and to investigate the potential role of PPARs (peroxisome-proliferator-activated receptors) in this effect. Arachidonic acid increased PTGS2 levels up to 7.5-fold within 6 h. The cells expressed PPARalpha and PPARdelta (also known as PPARbeta) (but not PPARgamma). PTGS2 protein level was increased by PPAR agonists, including polyunsaturated fatty acids, synthetic PPAR ligands, PGA1 and NSAIDs (non-steroidal anti-inflammatory drugs) with a time course resembling that of arachidonic acid. Use of agonists and antagonists indicated PPARalpha (but not PPARdelta or PPARgamma) was responsible for PTGS2 induction. PTGS2 induction by arachidonic acid did not require PG synthesis. PTGS2 levels were increased by the PKC (protein kinase C) activators 4beta-PMA and PGF(2alpha), and the effects of arachidonic acid, NSAIDs, synthetic PPAR ligands and 4beta-PMA were blocked by PKC inhibitors. This is consistent with PPAR phosphorylation by PKC. Induction of PTGS2 protein by 4beta-PMA in the absence of a PPAR ligand was decreased by the NF-kappaB (nuclear factor kappaB) inhibitors MG132 and parthenolide, suggesting that PKC acted through NF-kappaB in addition to PPAR phosphorylation. Use of NF-kappaB inhibitors allowed the action of arachidonic acid as a PPAR agonist to be dissociated from an effect through PKC. The results are consistent with the hypothesis that arachidonic acid acts via PPARalpha to increase PTGS2 levels in bovine endometrial stromal cells.     

Protein kinase inhibition by omega-3 fatty acids

Recent data suggest that omega-3 fatty acids may be effective in epilepsy, cardiovascular disorders, arthritis, and as mood stabilizers for bipolar disorder; however, the mechanism of action of these compounds is unknown. Based on earlier studies implicating omega-3 fatty acids as inhibitors of protein kinase C activity in intact cells, we hypothesized that omega-3 fatty acids may act through direct inhibition of second messenger-regulated kinases and sought to determine whether the omega-3 double bond might uniquely confer pharmacologic efficacy and potency for fatty acids of this type. In our studies we observed that omega-3 fatty acids inhibited the in vitro activities of cAMP-dependent protein kinase, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, and the mitogen-activated protein kinase (MAPK). Our results with a series of long-chain fatty acid structural homologs suggest an important role for the omega-3 double bond in conferring inhibitory efficacy. To assess whether omega-3 fatty acids were capable of inhibiting protein kinases in living neurons, we evaluated their effect on signal transduction pathways in the hippocampus. We found that omega-3 fatty acids could prevent serotonin receptor-induced MAPK activation in hippocampal slice preparations. In addition, we evaluated the effect of omega-3 fatty acids on hippocampal long-term potentiation, a form of synaptic plasticity known to be dependent on protein kinase activation. We observed that omega-3 fatty acids blocked long-term potentiation induction without inhibiting basal synaptic transmission. Overall, our results from both in vitro and live cell preparations suggest that inhibition of second messenger-regulated protein kinases is one locus of action of omega-3 fatty acids.     

Effect of growth temperature on cellular fatty acids in sulphate-reducing bacteria

The effect of growth temperature on the cellular fatty acid composition of sulphate-reducing bacteria (SRB) was studied in 12 species belonging to eight genera including psychrophiles and mesophiles. Most of these species were of marine origin. The investigated SRB with the exception of four Desulfobacter species exhibited only a minor increase in the proportion of cis-unsaturated fatty acids (by < or = 5% per 10 degrees C) when the growth temperature was decreased; psychrophiles maintained their typically high content of cis-unsaturated fatty acids (around 75% of total fatty acids) nearly constant. The four Desulfobacter species, however, increased the proportion of cis-unsaturated among total fatty acids significantly (by > or =14% per 10 degrees C; measured in late growth phase) with decreasing growth temperature. The ratio between unsaturated and saturated fatty acids in Desulfobacter species changed not only with the growth temperature, but also with the growth state in batch cultures at constant temperature. Changes of cellular fatty acids were studied in detail with D. hydrogenophilus, the most psychrotolerant (growth range 0-35 degrees C) among the mesophilic SRB examined. Desulfobacter hydrogenophilus also formed cis-9,10-methylenehexadecanoic acid (a cyclopropane fatty acid) and 10-methylhexadecanoic acid. At low growth temperature (12 degrees C), the relative amount of these fatty acids was at least threefold lower; this questions the usefulness of 10-methylhexadecanoic acid as a reliable biomarker of Desulfobacter in cold sediments.     

Phosphorylation of the acetylcholine receptor by protein kinase C and identification of the phosphorylation site within the receptor delta subunit

Purified acetylcholine receptor is rapidly and specifically phosphorylated by partially purified protein kinase C, the Ca2+/phospholipid-dependent enzyme. The receptor delta subunit is the major target for phosphorylation and is phosphorylated on serine residues to a final stoichiometry of 0.4 mol of phosphate/mol of subunit. Phosphorylation is dose-dependent with a Km value of 0.2 microM. Proteolytic digestion of the delta subunit phosphorylated by either protein kinase C or the cAMP-dependent protein kinase yielded a similar pattern of phosphorylated fragments. The amino acids phosphorylated by either kinase co-localized within a 15-kDa proteolytic fragment of the delta subunit. This fragment was visualized by immunoblotting with antibodies against a synthetic peptide corresponding to residues 354-367 of the receptor delta subunit. This sequence, which contains 3 consecutive serine residues, was recently shown to include the cAMP-dependent protein kinase phosphorylation site (Souroujon, M. C., Neumann, D., Pizzighella, S., Fridkin, M., and Fuchs, S. (1986) EMBO J. 5, 543-546). Concomitantly, the synthetic peptide 354-367 was specifically phosphorylated in a Ca2+- and phospholipid-dependent manner by protein kinase C. Furthermore, antibodies directed against this peptide inhibited phosphorylation of the intact receptor by protein kinase C. We thus conclude that both the cAMP-dependent protein kinase and protein kinase C phosphorylation sites reside in very close proximity within the 3 adjacent serine residues at positions 360, 361, and 362 of the delta subunit of the acetylcholine receptor.     

Effects of protein phosphatase and kinase inhibitors on the cardiac L- type Ca current suggest two sites are phosphorylated by protein kinase A and another protein kinase

We previously showed (Frace, A.M. and H.C. Hartzell. 1993. Journal of Physiology. 472:305-326) that internal perfusion of frog atrial myocytes with the nonselective protein phosphatase inhibitors microcystin or okadaic acid produced an increase in the L-type Ca current (ICa) and a decrease in the delayed rectifier K current (IK). We hypothesized that microcystin revealed the activity of a protein kinase (PKX) that was basally active in the cardiac myocyte that could phosphorylate the Ca and K channels or regulators of the channels. The present studies were aimed at determining the nature of PKX and its phosphorylation target. The effect of internal perfusion with microcystin on ICa or IK was not attenuated by inhibitors of protein kinase A (PKA). However, the effect of microcystin on ICa was largely blocked by the nonselective protein kinase inhibitors staurosporine (10- 30 nM), K252a (250 nM), and H-7 (10 microM). Staurosporine and H-7 also decreased the stimulation of ICa by isoproterenol, but K252a was more selective and blocked the ability of microcystin to stimulate ICa without significantly reducing isoproterenol-stimulated current. Internal perfusion with selective inhibitors of protein kinase C (PKC), including the autoinhibitory pseudosubstrate PKC peptide (PKC(19-31)) and a myristoylated derivative of this peptide had no effect. External application of several PKC inhibitors had negative side effects that prevented their use as selective PKC inhibitors. Nevertheless, we conclude that PKX is not PKC. PKA and PKX phosphorylate sites with different sensitivities to the phosphatase inhibitors calyculin A and microcystin. In contrast to the results with ICa, the effect of microcystin on IK was not blocked by any of the kinase inhibitors tested, suggesting that the effect of microcystin on IK may not be mediated by a protein kinase but may be due to a direct effect of microcystin on the IK channel.     

Diacylglycerol and fatty acids synergistically increase cardiomyocyte contraction via activation of PKC

Lipid signaling pathways are thought to play a prominent role in transducing extracellular signals into contractile responses in cardiac muscle. Two putative lipid messengers, diacyglycerol and arachidonic acid, can be generated via distinct phospholipases in separate signaling pathways, but certain stimuli cause them to be elevated in parallel. We tested the hypothesis that these lipids function as comessengers in ventricular myocytes by activating protein kinase C (PKC). In previous work, we demonstrated that the diacylglycerol analog dioctanoylglycerol (diC(8)) can be stimulatory or inhibitory toward myocyte twitches depending on how it is applied. Here we report that arachidonic acid and other cis-unsaturated fatty acids (UFA), at concentrations too low for direct effects, synergistically enhance the stimulatory effects of diC(8) and convert inhibitory effects of diC(8) into stimulation of myocyte twitches. Intracellular Ca(2+) transients changed in parallel with twitch amplitude, suggesting regulation of Ca(2+) homeostasis by these lipids. cis-UFA also interacted synergistically with the PKC activator phorbol 12-myristate 13-acetate to promote positive inotropic responses. Responses were blocked by the PKC antagonists chelerythrine chloride, bisindolylmaleimide, and G?-6976. DiC(8) and arachidonic acid also synergistically translocated PKC-epsilon and PKC-alpha in intact myocytes. We propose that PKC integrates diacylglycerol and cis-UFA signals in the heart, resulting in preferential activation of positive inotropic mechanisms.