Ca2+/Diacylglycerol-Activated, Phospholipid-Dependent Protein Kinase in the Hermissenda CNS

In mammalian systems, Ca2+/diacylglycerol-activated phospholipid-dependent protein kinase (C-kinase) appears to play an important role in regulating physiological responses that outlast the transient rise in cytosolic Ca2+. Electrophysiological experiments in neurons of the nudibranch mollusc, Hermissenda crassicornis, have suggested a role for C-kinase in the long-lasting reductions in early and late K+ currents that have been observed following associative learning. Accordingly, we have investigated the catalytic properties of C-kinase in Hermissenda CNS. Following homogenization in Ca2+-free buffer, C-kinase can be separated from Ca2+/calmodulin-dependent protein kinase by centrifugation; C-kinase activity is found in the supernatant whereas essentially all of the Ca2+/calmodulin-dependent protein kinase is found in the membrane fraction. Addition of Ca2+, phosphatidylserine, and diacylglycerol to the cytosol results in phosphorylation of at least eight endogenous proteins. The Hermissenda CNS C-kinase can also phosphorylate lysine-rich histone, a substrate for mammalian C-kinase. The molluscan enzyme exhibits phospholipid specificity in that phosphatidylserine is much more effective than phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, and phosphatidic acid. Addition of diacylglycerol, in the presence of Ca2+ and phosphatidylserine, increases the activity of the C-kinase. The percentage of activation by diacylglycerol is larger at lower Ca2+ concentrations. Enzyme activity is inhibited by trifluoperazine and polymixin B sulfate. These studies indicate that the Hermissenda C-kinase is catalytically similar to mammalian C-kinase.     

Thrombin and C-kinase activators potentiate calcium-stimulated arachidonic acid release in human platelets.

Human platelets were depleted of intracellular Ca2+ and then made selectively permeable to external Ca2+ by addition of the ionophore ionomycin. In this cell system a rapid release of arachidonic acid was seen in direct response to added Ca2+ at concentrations corresponding to cytosolic Ca2+ levels measured in thrombin-stimulated platelets. Thrombin and other activators of Ca2+/phospholipid-dependent protein kinase (C-kinase) potentiated the Ca2+-stimulated arachidonic acid release while exerting little or no effect in the absence of added Ca2+. Agents which increase (R59022) or decrease (isoquinolinesulphonylmethylpiperazine) the activation of C-kinase correspondingly enhanced or inhibited, respectively, the potentiation of arachidonic acid release caused by thrombin. These results support the hypothesis that arachidonic acid release in human platelets is regulated by a co-operative action between intracellular Ca2+ and C-kinase.     

Calcium, phospholipid turnover and transmembrane signalling

Turnover of phosphatidylinositol, which is provoked by various neurotransmitters, peptide hormones and many other biologically active substances, appears to serve as a signal for the transmembrane control of protein phosphorylation through activation of a novel protein kinase (C-kinase). The activation of this enzyme absolutely requires Ca2+ and phosphatidylserine. Diacylglycerol derived from the receptor-linked breakdown of phosphatidylinositol dramatically increases the affinity of C-kinase for Ca2+, and thereby renders this enzyme fully active without a net increase in the concentration of Ca2+. Under appropriate conditions synthetic diacylglycerol directly added to intact cell systems activates C-kinase fully without interaction with surface receptors. By using such synthetic diacylglycerol and the Ca2+ ionophore A23187, it is shown that either receptor-linked protein phosphorylation or Ca2+ mobilization alone is merely a prerequisite but not a sufficient requirement, and both are synergistically effective for causing a full physiological cellular response. In some tissues cyclic nucleotides, both cyclic AMP and cyclic GMP, may inhibit the receptor-linked breakdown of phosphatidylinositol, and appear to provide negative control that prevents over-response.     

Regulation of the phosphoinositide hydrolysis pathway in thrombin-stimulated platelets by a pertussis toxin-sensitive guanine nucleotide-binding protein. Evaluation of its contribution to platelet activation and comparisons with the adenylate cyclase inhibitory protein, Gi

In platelets activated by thrombin, the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C produces inositol 1,4,5-triphosphate (IP3) and diacylglycerol, metabolites which are known to cause Ca2+ release from the platelet dense tubular system and granule secretion. Previous studies suggest that phospholipase C activation is coupled to platelet thrombin receptors by a guanine nucleotide-binding protein or G protein. The present studies examine the contribution of this protein to thrombin-induced platelet activation and compare its properties with those of Gi, the G protein which mediates inhibition of adenylate cyclase by thrombin. In platelets permeabilized with saponin, nonhydrolyzable GTP analogs reproduced the effects of thrombin by causing diacylglycerol formation, Ca2+ release from the dense tubular system and serotonin secretion. In intact platelets, fluoride, which by-passes the thrombin receptor and directly activates G proteins, caused phosphoinositide hydrolysis and secretion. Fluoride also caused an increase in the platelet cytosolic free Ca2+ concentration that appeared to be due to a combination of Ca2+ release from the dense tubular system and increased Ca2+ influx across the platelet plasma membrane. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits G protein function, inhibited the ability of thrombin to cause IP3 and diacylglycerol formation, granule secretion, and Ca2+ release from the dense tubular system in saponin-treated platelets. Increasing the thrombin concentration overcame the effects of GDP beta S on secretion without restoring diacylglycerol formation. The effects of GDP beta S on platelet responses to thrombin which had been subjected to partial proteolysis (gamma-thrombin) were similar to those obtained with native alpha-thrombin despite the fact that gamma-thrombin is a less potent inhibitor of adenylate cyclase than is alpha-thrombin. Thrombin-induced diacylglycerol formation and 45Ca release were also inhibited when the saponin-treated platelets were preincubated with pertussis toxin, an event that was associated with the ADP-ribosylation of a protein with Mr = 41.7 kDa. At each concentration tested, the inhibition of thrombin-induced diacylglycerol formation by pertussis toxin paralleled the inhibition of thrombin's ability to suppress PGI2-stimulated cAMP formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blocking with phorbol ester, a protein kinase C activator, of receptor-dependent platelet calcium channels

The regulation of receptor-operated calcium channels of human platelets by phospholipid-dependent, Ca2+- and diacylglycerol-activated protein kinase C was studied. In order to induce the activation of endogenous protein kinase C, a cell-penetrable structural diacylglycerol analog, 4 beta-phorbol 12 beta-myristate-13 alpha-acetate (FMA), was used. Using two independent approaches, i. e., the fluorescent probe for Ca2+, quin-2, and 45Ca2+ absorption technique, it was demonstrated that FMA (10(-10) - 10(-8) g/ml) blocks Ca2+ influx into the platelets induced by aggregation factors, e. g., ADP, vasopressin, platelet activating factor, thrombin and thromboxane A2 receptor agonist U46619. The half-maximum inhibition of the receptor-sensitive influx of Ca2+ was observed at (3-6) X 10(-10) g/ml of FMA. Under physiological conditions, protein kinase C is activated with an increase in Ca2+ concentration in the cytoplasm in the presence of diacylglycerol. Since the above-mentioned inducers besides Ca2+ influx stimulate diacylglycerol synthesis, it was assumed that the activation of protein kinase C triggers a negative feedback mechanism which blocks the receptor-operated calcium channels.     

Evidence that treatment of platelets with phorbol ester causes proteolytic activation of Ca2+-activated, phospholipid-dependent protein kinase

Incubation of human platelets with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) caused the accumulation of a protein kinase in the particulate fraction which was not dependent on Ca2+ and phosphatidylserine (Ptd-Ser). The Ca2+/Ptd-Ser-independent kinase eluted from DEAE-cellulose at a NaCl concentration of 0.18-0.22 M compared with 0.08 - 0.16 M for Ca2+/Ptd-Ser-dependent protein kinase (C-kinase). Formation of the Ca2+/Ptd-Ser-independent kinase in 12-O-tetradecanoylphorbol-13-acetate-treated platelets was blocked by leupeptin, an inhibitor of Ca2+-dependent neutral proteases. The Ca2+/Ptd-Ser-independent kinase and C-kinase both catalysed the same pattern of phosphorylation of smooth muscle myosin light chains and histone H1 as detected by one-dimensional or two-dimensional peptide mapping after tryptic digestion. The phosphorylation sites were different from those obtained with myosin light chain kinase or cAMP-dependent kinase. The Ca2+/Ptd-Ser-independent kinase and C-kinase had Mr values of about 50 000 and 77 000 respectively as determined by sucrose-gradient centrifugation. It was concluded that 12-O-tetradecanoylphorbol-13-acetate induces the proteolytic cleavage of C-kinase to a Ca2+/Ptd-Ser-independent form.     

Elucidation of calpain dependent phosphorylation of myosin light chain in human platelets

Newly synthetized calpain inhibitors (CI-I approximately III) were used to prove potential participation of calpain in protein phosphorylation. CIs were about 1,000 times more potent against platelet calpain I than N-ethyl-maleimide (NEM) and an epoxy succinate derivative (E-64). CI-II inhibited 20K (myosin light chain) and 47K phosphorylation of Ca2+-stimulated lysed platelets as well as protein degradation (actin binding protein, P235). Both myosin light chain kinase (MLCK) and C-kinase dependent phosphorylation of 20K were inhibited by CI-II as demonstrated in phosphopeptides mapping. Electropermeabilized platelets (EP) were employed to examine the effects of CI-II on Ca2+ mediated reactions in non-lysed platelets. Phosphorylation of 20K and 47K induced by Ca2+ addition to EP was inhibited by CI-II, though secretory response was not modified. Only MLCK dependent phosphorylation of 20K was observed in Ca2+-activated EP, which was inhibited by CI-II. Collectively, the data indicated that calpain may activate both MLCK and C-kinase to phosphorylate 20K by partial proteolysis.     

Induction of the fibrinogen receptor on human platelets by intracellular mediators

We have used platelets permeabilized with saponin to examine the mechanism by which platelet activation causes the exposure of surface receptors for fibrinogen. Receptor exposure was detected using 125I-fibrinogen and 125I-PAC1, a monoclonal antibody specific for the activated form of the fibrinogen receptor. The potential mediators that were studied included guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and guanosine 5'O-(thiotriphosphate) (GTP gamma S), which cause G protein-dependent phospholipase C activation in platelets; inositol 1,4,5-triphosphate (IP3), which causes Ca2+ release from the platelet dense tubular system; and diacylglycerol and phorbol ester, which activate protein kinase C. Each of these molecules caused fibrinogen and PAC1 binding. The effect of IP3 was mimicked by raising the cytosolic free Ca2+ concentration in the permeabilized platelets. However, IP3 and Ca2+-induced PAC1 binding were abolished by indomethacin or aspirin, which had no effect on PAC1 binding caused by Gpp(NH)p, phorbol ester, or diacylglycerol. This suggests that the response to IP3 and Ca2+ is due to the formation of metabolites of arachidonic acid. One such metabolite, TxA2, is believed to activate platelets by stimulating G protein-dependent phosphoinositide hydrolysis. Indeed, we found that the G protein inhibitor guanyl-5'-yl thiophosphate (GDP beta S) inhibited PAC1 binding caused by a thromboxane A2 analog (U46619), IP3, and Ca2+, but had no effect on diacylglycerol or phorbol ester-induced PAC1 binding. Thrombin-induced PAC1 binding and phosphoinositide hydrolysis were also inhibited by GDP beta S and by pertussis toxin. Increasing the thrombin concentration overcame the inhibition of PAC1 binding caused by GDP beta S but did not overcome the inhibition of phosphoinositide hydrolysis. These observations demonstrate that fibrinogen receptor exposure occurs by at least two routes. One of these, in response to agonists such as thrombin and U46619, is initiated by G protein-dependent phosphoinositide hydrolysis and involves the formation of IP3 and diacylglycerol. IP3 appears to act by stimulating Ca2+-dependent arachidonic acid metabolism which, in turn, triggers further phosphoinositide hydrolysis. Diacylglycerol acts by stimulating protein kinase C. A second route is activated by high concentrations of thrombin and is independent of phosphoinositide hydrolysis.     

The temporal integration of the aldosterone secretory response to angiotensin occurs via two intracellular pathways

Angiotensin II (AII) regulates the secretion of aldosterone from adrenal glomerulosa cells by a calcium-dependent mechanism which involves both the uptake of calcium from the extracellular pool, and the release of calcium from a dantrolene-sensitive intracellular pool. In the present study, it was shown that AII induces the rapid (10 s) hydrolysis of phosphatidylinositol 4-phosphate and -4,5-bisphosphate, leading to the sustained production of inositol bis- and trisphosphate (Ins-P3), and diacylglycerol rich in arachidonic acid. Saponin-permeabilized glomerulosa cells accumulate calcium into a nonmitochondrial pool by an ATP-dependent manner. Ins-P3 (0.5-5 microM) induces a release of Ca2+ from this pool. This release was blocked by dantrolene (10 microM). Adrenal glomerulosa cells were shown to contain the calcium-activated, phospholipid-dependent protein kinase (C-kinase). Perfusion of glomerulosa cells with combined 12-O-tetradecanoyl phorbol 13-acetate and A23187 induced an immediately developing, sustained, maximal secretory response similar to that induced by AII. These data are interpreted in terms of a model in which, after AII addition, there is a flow of information through two separate branches of the calcium messenger system, each with its unique temporal role: a calmodulin branch activated by the transient rise in the [Ca2+] in the cell cytosol, which is largely responsible for the initial transient cellular response; and a C-kinase branch activated by the increase in both cytosolic [Ca2+] and the diacylglycerol content of the plasma membrane, which is largely responsible for the sustained phase of the cellular response. The temporal integration of these two phases underlies the observed pattern of cellular response.     

Stereospecificity of diacylglycerol for stimulus-response coupling in platelets

In intact platelets, a permeable diacylglycerol having a 1,2-sn- but not 2,3-sn- configuration activated protein kinase C directly. In the presence of Ca2+-ionophore this diacylglycerol caused full activation of platelet release reaction. 1,3-Isomer was inactive. Among these isomers only 1,2-sn-diacylglycerol was converted rapidly to the corresponding phosphatidic acid in both intact and broken cell preparations. Thus, the diacylglycerol which functions in stimulus-response coupling possesses a 1,2-sn-glycerol backbone, and other isomers are not involved in the signal transduction through the protein kinase C pathway.     

Trifluoperazine and chlorpromazine block secretion from human platelets evoked at basal cytoplasmic free calcium by activators of C-kinase

Trifluoperazine, chlorpromazine and other drugs to inhibit calmodulin-dependent processes are also known to inhibit protein kinase C. The effect of these agents on secretion evoked by known activators of C-kinase has been studied in human platelets loaded with the fluorescent Ca indicator, quin2 and preincubated with aspirin. The secretory response stimulated by phorbol ester and exogenous diacyglycerol, at basal levels of cytoplasmic free Ca²⁺, [Ca²⁺]_i, was suppressed by trifluoperazine, chlorpromazine and W-7, as was the secretion evoked by collagen that occurs without a change in [Ca²⁺]_i, The response to thrombin, which is accompanied by elevated [Ca²⁺]_i was barely affected. Modest elevation of [Ca²⁺]_i by Ca ionophore was able to overcome the inhibitory effect of these drugs on the response to phorbol ester, diacylglycerol, and collagen.     

Biochemical mechanisms of memory storage

A series of studies on Hermissenda classical conditioning has lead to a discovery that the biophysical events (accumulation of Ca2+ and depolarization in B cell) found during memory acquisition are clearly distinct from those (suppression of K-currents, IA and ICa2+K+) detected in the retention phase of memory. Biochemical analysis of eyes isolated shortly after (a few hours) training revealed increased phosphorylation of a 20,000 M.W. protein which is very likely one of the substrates for both Ca/CaM-dependent protein kinase and C-kinase and possibly a locus of convergence for conditioned stimulus and unconditioned stimulus pathways. Furthermore, conditioning-specific changes in the two K+ currents have been reproduced by simultaneous activation of the CaM-kinase pathway (via iontophoretic injection of CaM-kinase II plus Ca2+-load or IP3 injection) and the C-kinase pathway (via bath application of phorbol-ester or diacylglycerol analog plus Ca2+-load). Therefore, synergistic interaction between the two Ca2+-dependent phosphorylation systems in the identified B cell is considered to be critically important for acquisition of associative memory. Evidence also has been obtained for similar biophysical changes and molecular mechanisms during retention of classical conditioning in the mammalian brain. Further work will be needed to uncover the biochemical mechanism(s) responsible for transforming short-term into long-lasting memory.     

Activation of protein kinase C in human neutrophils attenuates agonist-stimulated rises in cytosolic free Ca2+ concentration by inhibiting bivalent-cation influx and intracellular Ca2+ release in addition to stimulating Ca2+ efflux.

Stimulation of fura-2-loaded human neutrophils with formylmethionyl-leucyl-phenylalanine (FMLP) or ionomycin elevated the cytosolic free Ca2+ concentration, [Ca2+], to a maintained elevated level. Activation of protein kinase C (C-kinase) with phorbol 12-myristate 13-acetate, 4 beta-phorbol 12,13-didecanoate or dioctanoylglycerol caused decreases in [Ca2+]i from this level. 4 alpha-Phorbol didecanoate, which does not activate C-kinase, had no effect. These results confirm previous reports that C-kinase activation decreases neutrophil [Ca2+]i by stimulating removal of Ca2+ from the cytosol. Further experiments showed that activation of C-kinase attenuated the component of the FMLP-stimulated [Ca2+]i rise that was dependent on external Ca2+. C-kinase activation also inhibited FMLP-stimulated entry of the quenching cation, Mn2+, used as an indicator of bivalent-cation entry. In contrast, C-kinase activation caused only a partial inhibition of FMLP-stimulated release of Ca2+ from intracellular stores. 4 alpha-Phorbol didecanoate was ineffective in inhibiting Ca2+ entry, Mn2+ entry and intracellular Ca2+ release. Addition of FMLP also stimulated a decrease in the ionomycin-elevated [Ca2+]i, and this effect was blocked by staurosporine, a protein kinase inhibitor. These results show that, in addition to stimulating Ca2+ efflux, C-kinase activation in neutrophils inhibits FMLP-stimulated entry of bivalent cations, and partially inhibits intracellular release of Ca2+. Further, FMLP itself can modulate [Ca2+]i by activation of C-kinase.     

Prostaglandin E1 and forskolin antagonize C-kinase activation in the human platelet.

In human platelets, prostaglandin E1 and forskolin inhibit 5-hydroxytryptamine-induced phospholipase C, C-kinase and myosin light-chain kinase activity in a concentration-dependent way. Phospholipase C activation, however, was only partly inhibited, and this at higher concentrations than the protein kinases. Direct activation of the C kinase either by exogenous synthetic diacylglycerol or by 12-O-tetradecanoylphorbol 13-acetate was antagonized by prostaglandin E1 and forskolin. Since C-kinase activation is one of the key events in excitatory signal transduction in the platelets, we suggest that the inhibitory effect of agents that increase platelet cyclic AMP on platelet secretion and aggregation might reside in their capacity to antagonize C-kinase activity.     

Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease

A Ca2+-dependent protease I), which hydrolyzes casein at Ca2+ concentrations lower than the 10(-5) M range, is purified roughly 4000-fold from the soluble fraction of rat brain. This protease is able to activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) by limited proteolysis analogously to the previously known Ca2+-dependent analogously to the previously known Ca2+-dependent protease (Ca2+ protease II) which is active at the millimolar range of Ca2+ (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616). The protein kinase fragment thus produced shows a molecular weight of about 5.1 X 10(4), and is significantly smaller than native protein kinase C (Mr = 7.7 X 10(4). Although protein kinase C may be normally activated in a reversible manner by the simultaneous presence of phospholipid and diacylglycerol at Ca2+ concentrations less than 10(-6) M, this enzyme fragment is fully active without any lipid fractions and independent of Ca2+. The limited proteolysis of protein kinase C is markedly enhanced in the velocity by the addition of phospholipid and diacylglycerol, which are both required for the reversible activation of the enzyme. However, casein hydrolysis by this protease is not affected by phospholipid and diacylglycerol. Available evidence suggests that, at lower concentrations of this divalent cation, Ca2+ protease I reacts preferentially with the active form of protein kinase C which is associated with membrane, and converts it to the permanently active form. In contrast, the inactive form of protein kinase C, which is free of membrane phospholipid, does not appear to be very susceptible to the proteolytic attack. It remains unknown, however, whether this mechanism of irreversible activation of protein kinase C does operate in physiological processes. It is noted that Ca2+ protease II, which is active at higher concentrations of Ca2+, proteolytically activates protein kinase C irrespective of the presence and absence of phospholipid and diacylglycerol.     

Nerve growth factor action is mediated by cyclic AMP- and Ca+2/phospholipid-dependent protein kinases

Nerve growth factor (NGF) mediates the phosphorylation of tyrosine hydroxylase in PC12 cells on two distinct peptide fragments, separable by two-dimensional tryptic phosphopeptide mapping (phosphopeptides T1 and T3). Phorbol diester derivatives capable of activating Ca+2/phospholipid-dependent protein kinase (C-kinase) cause a specific phosphorylation of peptide T3 in a dose-dependent, saturable manner. Derivatives of the endogenous C-kinase activator diacylglycerol, also cause the phosphorylation of tyrosine hydroxylase on peptide T3. The C-kinase inhibitors chlorpromazine and trifluoperazine inhibit the phorbol diester stimulated phosphorylation of site T3 in a dose-dependent manner. These agents inhibit the phosphorylation of T3 in response to NGF, but have no effect on NGF's ability to cause T1 phosphorylation. In a PC12 mutant deficient in cAMP-dependent protein kinase activity, NGF mediates the phosphorylation of tyrosine hydroxylase on peptide T3 but not on T1. We conclude that NGF mediates the activation of both the cAMP-dependent protein kinase and the C-kinase to phosphorylate substrate proteins. These kinases can act independently to phosphorylate tyrosine hydroxylase, each at a different site, and each of which results in the enzyme activation. A molecular framework is thus provided for events underlying NGF action.     

A role of calcium-activated phospholipid-dependent protein kinase in human platelet activation. Comparison of thrombin and collagen actions

In human platelets stimulated by thrombin and collagen, diacylglycerol is rapidly produced from phosphatidylinositol. Concurrently, an endogenous protein having a molecular weight of about 40,000 (40K protein) is phosphorylated, and serotonin is released. These reactions are all inhibited by a prior treatment of platelets with prostaglandin E1, dibutyryl cyclic AMP, sodium nitroprusside, or with 8-bromo-cyclic GMP, which are known as potent inhibitors for platelet activation. Ca2+-activated phospholipid-dependent protein kinase (protein kinase C) preferentially phosphorylates 40K protein. As judged by fingerprint analysis, the sites in 40K protein that are phosphorylated during the platelet activation appear to be identical with those phosphorylated by protein kinase C in a purified cell-free system. 12-O-Tetradecanoylphorbol-13-acetate, which directly activates protein kinase C by substituting for diacylglycerol, stimulates 40K protein phosphorylation and release reaction without inducing diacylglycerol formation. Tetracaine, which inhibits protein kinase C by competing with phospholipid, blocks 40K protein phosphorylation and serotonin release without inhibiting the receptor-linked diacylglycerol formation. The results indicate that thrombin and collagen activate platelets in almost similar mechanisms and that protein kinase C may lie on a common pathway which leads to the release of serotonin. However, analysis with indomethacin indicates that the role of thromboxane A2 appears to be more predominant for the action of collagen, and it is suggestive that this arachidonate metabolite activates platelets in an analogous mechanism to thrombin.     

Aequorin detects increased cytoplasmic calcium in platelets stimulated with phorbol ester or diacylglycerol

A biochemical pathway to platelet activation involving protein kinase C has been deemed "Ca2+-independent", because the intracellular fluorophore quin2 indicates no rise in cytoplasmic [Ca2+] in platelets stimulated by certain agonists. However, unlike quin2, the Ca2+-sensitive photoprotein aequorin demonstrates a rise in [Ca2+] when platelet aggregation is induced by phorbol ester or diacylglycerol. Aequorin and quin2 appear to report different aspects of Ca2+ homeostasis, and the absence of a quin2 signal may not be sufficient to establish that a metabolic pathway is "Ca2+-independent".     

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.     

Tumor promoters and diacylglycerol activate the Na+/H+ antiporter of sea urchin eggs

Various tumor promoters (TPA, lyngbyatoxin and aplysiatoxin) and diacylglycerol induced cytoplasmic alkalinization of sea urchin eggs independently of intracellular Ca2+ release. This response stimulated protein synthesis and was blocked by amiloride or a lack of extracellular Na+, procedures which inhibit the Na+/H+ antiporter. These results suggest that the antiporter which is responsible for cytoplasmic alkalinization in sea urchin eggs is activated directly or indirectly by protein kinase C in a Ca2+-independent manner.