Oleoylacetylglycerol Research Articles

Regulation of phosphatidylinositol 4-kinase activity in rat pancreatic acini.

Previous data showed that carbachol (CCh) elicits a concentration-dependent increase in phosphatidylinositol (PtdIns) 4-kinase activity in homogenates derived from agonist-stimulated pancreatic acini. In the present study CCh elicited a time-dependent increase in PtdIns 4-kinase activity, which was blocked by n-methylscopolamine and mimicked by muscarine. A membrane-associated PtdIns 4-kinase activity was identified that displayed maximal activity in the pH range of 5.5-8.5. The zymogen granule fraction possessed relatively high specific enzyme activity, which was sensitive to CCh stimulation. The enzyme had apparent Km values for PtdIns and ATP of 4 and 60 microM, respectively. CCh caused no discernible change in the Km for either PtdIns or ATP but increased Vmax. Dioctanoylglycerol and oleoyl acetylglycerol augmented PtdIns 4-kinase activity, which was blocked by staurosporine, thus suggesting a possible role for protein kinase C in the regulation of PtdIns 4-kinase. These collective findings demonstrate a muscarinic receptor-mediated regulation of PtdIns 4-kinase activity in exocrine pancreas, involving a protein kinase C-dependent process.

Activation of neurokinin 3 receptor increases Nav1.9 current in enteric neurons

The intrinsic primary afferent neurons (IPANs) of the guinea pig enteric nervous system express Na(v)1.9 sodium channels that produce a persistent TTX-resistant current having a low activation threshold and slow gating kinetics. These neurons receive slow EPSPs induced mainly by the activation of neurokinin 3 receptors (NK3r). Here, we demonstrate that senktide, a specific NK3r agonist, potentiates the Na(v)1.9 current (I(Nav1.9)) in IPANs. Using whole-cell patch-clamp recordings from IPANs in duodenum longitudinal muscle/myenteric plexus preparations, we show that short (1-5 s) and long (up to 1 min) applications of senktide, increase the I(Nav1.9) peak current up to 13-fold. The effect, blocked by a NK3r antagonist SB235375 is transient, lasting approximately 2 min and is due to a negative shift of the activation voltage by approximately 20 mV and of fast inactivation by approximately 10 mV. As a consequence, the window current resulting from the product of the activation and fast inactivation curves is shifted and enlarged. The transient effect of senktide is likely to be due to the fast desensitization of NK3r. Protein kinase C (PKC) activation with phorbol or oleoyl acetylglycerol also increases I(Nav1.9), although persistently, by inducing similar voltage-dependent changes. Current-clamp experiments showed that I(Nav1.9) modulation by senktide lowers action potential threshold and increases excitability. The increase in I(Nav1.9) by NK3r activation is also likely to amplify slow EPSPs generated in the IPANs. These changes in excitability potentially have a profound effect on the entire enteric synaptic circuit and ultimately on gut motility and secretion.

Specific Modulation of Na+Channels in Hippocampal Neurons by Protein Kinase Cϵ

Acetylcholine binding to muscarinic acetylcholine receptors activates G-proteins, phospholipase C, and protein kinase C (PKC), which phosphorylates brain Na+ channels and reduces peak Na+ current in hippocampal neurons. Because multiple PKC isozymes with different regulatory properties are expressed in hippocampal neurons, we investigated which ones are responsible for mediating this effect. The diacylglycerol analog oleoylacetylglycerol (OAG) reduced the amplitude of Na+ current in dissociated mouse hippocampal neurons by 28.5 +/- 5.3% (p < 0.01). The reduction of peak Na+ current was similar with Ca2+-free internal solution and in 92 nm internal Ca2+, suggesting that calcium-dependent, conventional PKC isozymes were unlikely to mediate this response. Gö6976, which inhibits conventional PKC isozymes, reduced the effect of PKC activators only slightly, whereas rottlerin, which inhibits PKCdelta preferentially at 5 microm, had no effect. Ro-31-8425 (20 nm), which inhibits conventional PKC isozymes, did not reduce the response to OAG. However, higher concentrations of Ro-31-8425 (100 nm or 1 microm) that inhibit novel PKC isozymes effectively blocked OAG inhibition of Na+ current. Inclusion of a selective PKCepsilon-anchoring inhibitor peptide (PKCepsilon-I) in the recording pipette prevented the reduction of peak Na+ current by OAG, whereas an anchoring inhibitor peptide specific for PKCbeta and an inactive scrambled PKCepsilon-I peptide had no effect. In addition, OAG had no effect on Na+ current in hippocampal neurons from PKCepsilon null mice. Overall, our data from four experimental approaches indicate that anchored PKCepsilon is the isozyme responsible for PKC-mediated reduction of peak Na+ currents in mouse hippocampal neurons.

εPKC Is Required for the Induction of Tolerance by Ischemic and NMDA-Mediated Preconditioning in the Organotypic Hippocampal Slice

Glutamate receptors and calcium have been implicated as triggering factors in the induction of tolerance by ischemic preconditioning (IPC) in the brain. However, little is known about the signal transduction pathway that ensues after the IPC induction pathway. The main goals of the present study were to determine whether NMDA induces preconditioning via a calcium pathway and promotes translocation of the protein kinase C epsilon (epsilonPKC) isozyme and whether this PKC isozyme is key in the IPC signal transduction pathway. We corroborate here that IPC and a sublethal dose of NMDA were neuroprotective, whereas blockade of NMDA receptors during IPC diminished IPC-induced neuroprotection. Calcium chelation blocked the protection afforded by both NMDA and ischemic preconditioning significantly, suggesting a significant role of calcium. Pharmacological preconditioning with the nonselective PKC isozyme activator phorbol myristate acetate could not emulate IPC, but blockade of PKC activation with chelerythrine during IPC blocked its neuroprotection. These results suggested that there might be a dual involvement of PKC isozymes during IPC. This was corroborated when neuroprotection was blocked when we inhibited epsilonPKC during IPC and NMDA preconditioning, and IPC neuroprotection was emulated with the activator of epsilonPKC. The possible correlation between NMDA, Ca2+, and epsilonPKC was found when we emulated IPC with the diacylglycerol analog oleoylacetyl glycerol, suggesting an indirect pathway by which Ca2+ could activate the calcium-insensitive epsilonPKC isozyme. These results demonstrated that the epsilonPKC isozyme played a key role in both IPC- and NMDA-induced tolerance.

Peptidergic modulation of insect voltage-gated Ca(2+) currents: role of resting Ca(2+) current and protein kinases A and C.

The modulation of voltage-gated Ca(2+) currents in isolated dorsal unpaired median (DUM) neurons of cockroach was investigated using whole cell patch clamp. The neuropeptide neurohormone D (NHD), a member of the adipokinetic hormone family, affected Ca(2+) currents at pico- to nanomolar concentrations. It strongly enhanced currents activating at lower depolarizations, whereas those activating at strong depolarizations were slightly attenuated. The first effect results from upregulation of a previously characterized omega-conotoxin MVIIC- and omega-agatoxin IVA-sensitive "mid/low voltage-activated" (M-LVA) Ca(2+) current. The cAMP-analogue 8-bromo-cAMP, forskolin, and the catalytic subunit of protein kinase A (PKA) mimicked the stimulating action of NHD. In addition, preincubation of neurons with the PKA inhibitor KT 5720 abolished the action of NHD. Thus NHD seems to upregulate the M-LVA current via channel phosphorylation by PKA. Activation of protein kinase C by oleoylacetylglycerol (OAG) mimicked the effect of NHD, and subsequent NHD application only enhanced the current to a moderate extent. On the other hand, inhibition of protein kinase C (PKC) by Gö 6976 abolished the NHD effect. These results indicate that also PKC, too, may play a role in the peptidergic modulation of the M-LVA Ca(2+) current. The reduction of Ca(2+) currents in the high-voltage-range is caused by the NHD-induced upregulation of a voltage-independent Ca(2+) resting current, I(Ca,R), which most probably leads to enhanced Ca(2+)-dependent inactivation of voltage-gated Ca(2+) currents. To assess the major consequences of the Ca(2+) current changes, current-clamp investigations were performed. Experiments with iberiotoxin, a specific blocker of BK-type Ca(2+)-dependent K(+) currents, and the M-LVA current-blocking omega-toxins suggested that NHD causes-via increasing Ca(2+)-dependent K(+) currents-a larger hyperpolarization of action potentials. The lowering in the action potential threshold produced by NHD, however, seems to be a direct consequence of the hyperpolarizing shift of the activation curve of total Ca(2+) current resulting from NHD-induced upregulation of the M-LVA current component.

Stimulation of C32 and G361 melanoma cells using oleoyl acetyl glycerol and its effect on sulphur mustard cytotoxicity

Epidermal melanocytes have a higher sensitivity to sulphur mustard (HD) compared with other skin cell types. This may be due to the enzymatic production of melanin precursors exerting an additional cytotoxic effect following HD depletion of the cellular protectant, GSH. Stimulation of the protein kinase C pathway in melanocytes is known to increase melanin production in melanocytes and melanoma cell lines. In order to investigate the role of pigment synthesis in HD toxicology, cultures of an unpigmented melanoma cell line (C32) and of a pigmented melanoma line (G361) were treated with the potent diacyl glycerol analogue, oleoyl acetyl glycerol (OAG), in order to determine if protein kinase C-mediated increases in pigment production could increase sensitivity to subsequent HD exposure. Stimulation of C32 cells with OAG exerted a significant protective effect against the cytotoxic effects of HD. However, this was not due to increased melanin synthesis because this cell line cannot synthesize melanin pigments. The protective action observed is postulated to be due to modulation of protein kinase C activity. In contrast, stimulation of G361 melanoma cells with OAG resulted in an increased level of cytotoxicity upon subsequent exposure to HD. Protein kinase C controls several cellular pathways including checkpoints in the cell cycle, stalling the cell in G and promoting transition through the G2/M boundary. Given the genotoxic properties of HD, these two points in the cell cycle are important in determining the overall cytotoxic effect of HD. Control of the cell cycle by protein kinase C modulation and manipulation of melanin synthetic pathways may have therapeutic benefits.

Peptidergic modulation of an insect Na(+) current: role of protein kinase A and protein kinase C.

The modulation of voltage-gated Na(+) currents in isolated somata of dorsal unpaired median (DUM) neurons of the cockroach Periplaneta americana was investigated using the patch-clamp technique. The neuropeptide Neurohormone D (NHD), which belongs to the family of adipokinetic hormones, reversibly reduced the Na(+) current in concentration-dependent manner (1 pM to 10 nM). At 10 nM, NHD caused an attenuation of the maximum of current-voltage (I-V) relation for peak currents by 23 +/- 6%. An analysis of NHD action on current kinetics in terms of the Hodgkin-Huxley formalism revealed that NHD reduces the time constant of inactivation, whereas steady-state activation and inactivation as well as the time constant of activation were not affected. In addition, NHD prolonged the recovery from inactivation. The cAMP analogue 8-bromo-cAMP, forskolin, and the catalytic subunit of protein kinase A mimicked the action of NHD. Furthermore, preincubation of cells with the protein kinase A inhibitor KT 5720 abolished the action of NHD. Thus NHD seems to modify the Na(+) current via channel phosphorylation by protein kinase A. Activation of protein kinase C by oleoylacetylglycerol (OAG) also reduced the Na(+) current, but it did not occlude the action of NHD. On the other hand, inhibition of protein kinase C by chelerythrine or Gö 6976 did not essentially impair the NHD effects.

Altered activation of phospholipase D by lysophosphatidic acid and endothelin-1 in mouse embryo fibroblasts lacking phospholipase C-γ1

Lysophosphatidic acid (LPA) and endothelin-1 (ET-1) activate phospholipase D (PLD) in many cell types. To see if phospholipase C-γ1 plays a role, we used embryonic fibroblasts from mice in which the PLCγ1 gene was disrupted. Surprisingly, the effect of LPA on inositol phosphate accumulation was increased in these PLCγ1 −/− cells, whereas that of ET-1 was completely abrogated. When PLD activity was measured, the response to LPA was also enhanced and the response to ET-1 lost in the PLCγ1 −/− cells. Treatment of these cells with ionomycin and oleoyl acetyl glycerol to mimic PLC stimulation restored PLD activity. Treatment of either PLCγ1 +/+ and PLCγ1 −/− cells with tyrosine kinase inhibitors did not inhibit LPA- or ET-1-induced PLD activity. Moreover, LPA and ET-1 treatment of PLCγ1 +/+ and PLCγ1 −/− cells did not cause tyrosine phosphorylation of PLC-γ1 or PLC-γ2. In summary, these results show that the altered PLD responses to LPA and ET-1 in PLCγ1 −/− are due to changes in PLC activity and do not involve tyrosine kinase activity.

Activity of protein kinase C is necessary for sustained thrombin-induced [Ca2+]i oscillations in rat glioma cells.

The aim of the present study was to examine the possible role of protein kinase C (PKC) in thrombin-induced Ca2+ signalling. As shown before, continuous superfusion of rat glioma cells with thrombin caused sustained [Ca2+]i oscillations through activation of cell surface receptors [Czubayko U, Reiser G (1995) Neuroreport 6: 1249]. These oscillations were inhibited by protease nexin-1. Addition of PKC inhibitors, i. e. staurosporine (0.2-20 microM), bisindolylmaleimide (1 microM) or chelerythrine (1 microM), irreversibly suppressed thrombin-induced [Ca2+]i oscillations. Thereafter application of 2,5-di(tert-butyl)-1,4-benzohydroquinone (t-BuBHQ, 20 microM) or thapsigargin (1 microM) (inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase) caused no [Ca2+]i response, indicating that intracellular Ca2+ stores were completely empty. We tested whether PKC affects the refilling of internal Ca2+ stores in thrombin-stimulated cells, by monitoring the amount of Ca2+ release caused by t-BuBHQ in the presence or absence of PKC inhibitors or activators. The amount of Ca2+ released by t-BuBHQ, which was normalized by comparison with the thrombin-induced Ca2+ response, was decreased by simultaneous incubation with staurosporine or chelerythrine, but enhanced with the PKC activator oleoyl acetyl glycerol. Furthermore, the capacitative Ca2+ entry was reduced by inhibition or downregulation, and increased by activation, of PKC. Capacitative Ca2+ entry was induced in these experiments by depletion of Ca2+ stores by the addition of thapsigargin or t-BuBHQ. In contrast, the inhibition of PKC during thrombin-induced depletion of intracellular stores did not influence the Ca2+ entry but nearly completely abolished the refilling of the internal stores. Thus we conclude that during thrombin receptor stimulation activation of PKC is required to maintain the refilling of intracellular Ca2+ stores for sustained [Ca2+]i oscillations. Thus, the control by PKC of the capacitative Ca2+ entry is apparently different depending on whether it is induced by sarco/endoplasmic reticulum Ca2+-ATPase inhibition or by activation of the thrombin receptor.

Separation of agonist‐stimulated arachidonate mobilization from subsequent leukotriene B4 synthesis in human neutrophils: Different effects of oleoylacetylglycerol and phorbol myristate acetate as priming agents

Preincubation of human neutrophils with phorbol esters or soluble diglycerides enhances subsequent f-Met-Leu-Phe (fMLP)-stimulated arachidonate mobilization and leukotriene B4 (LTB4) synthesis. We have recently reported that 1,3-dioctanoylglycerol (1,3-diC8) is equipotent with 1,2-sn-dioctanoylglycerol (1,2-diC8) as priming agent, thus suggesting that the priming effects of diacylglycerols are protein kinase C (PKC) independent (Rosenthal et al., 1993, Biochim. Biophys. Acta 1177:79-86). In order to further investigate this question, the present study has directly compared the effects of oleoylacetylglycerol (OAG) and the PKC activator, phorbol 12-myristate 13-acetate (PMA), on agonist-stimulated lipid metabolism. The results indicate that both OAG and PMA dose dependently enhance f-Met-Leu-Phe (fMLP)-stimulated release of [3H]arachidonate. Optimal concentrations of OAG (5 microns) and PMA (10 nM) are equipotent in increasing fMLP-stimulated arachidonate mobilization as quantitated either with total radioactivity or by mass measurements of free arachidonate. By contrast OAG is sixfold more effective than PMA in enhancing synthesis of 5-lipoxygenase (5-LO) metabolites by mass and two to threefold more effective than PMA in enhancing synthesis of [3H]eicosanoids. Furthermore, OAG, but not PMA, enhances fMLP-stimulated synthesis of platelet-activating factor. By contrast, PMA directly stimulates [3H]arachidonate mobilization, while OAG (20 microM) does not; despite these differences, the combined effects of PMA + OAG on subsequent agonist-stimulated arachidonate release are not greater than those of PMA alone. In cells challenged with subthreshold concentrations (< 0.1 microM) of the calcium ionophore A23187, both OAG and PMA stimulate [3H]arachidonate release but not [3H]LTB4 synthesis. These findings suggest that OAG does not directly activate 5-LO, but instead couples arachidonate mobilization to leukotriene synthesis in a PKC-independent manner.

Phorbol 12-myristate 13-acetate and 1,25-dihydroxyvitamin D 3 regulate 1,25-dihydroxyvitamin D 3 receptors synergistically in rat osteosarcoma cells

In this study, we examined the effect of activation of protein kinase C (PKC) pathways on the regulation of 1,25-dihydroxyvitamin D receptors (VDR) in rat osteosarcoma (ROS) 17/2.8 cells. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) resulted in a time- and dose-dependent increase in VDR expression in ROS cells. Treatment of ROS cells with 4α-phorbol 12,13-dedeconate, a PKC-inactive phorbol ester, had no effect on VDR expression. Oleoyl acetyl glycerol (OAG), a synthetic diacylglycerol, stimulated VDR up-regulation in ROS cells. The PKC inhibitors (H-7, staurosporin, and sphingosine) all blocked PMA-mediated up-regulation of VDR in a dose-dependent manner. We next examined the interaction of 1,25(OH) 2D 3 and PKC activation by PMA on the regulation of VDR in ROS cells. We found that PMA or 1,25(OH) 2D 3 treatment alone resulted in a 50 and 200% increase in VDR, respectively. PMA treatment alone resulted in a 50% increase in VDR protein and a marginal 20% increase in VDR mRNA. 1,25(OH) 2D 3 up-regulation of VDR was associated with a 2-fold increase in VDR mRNA. In contrast, co-treatment of ROS cells with PMA and 1,25(OH) 2D 3 resulted in a synergistic 10-fold induction of VDR mRNA and the appearance of a 7.2 kb VDR transcript. VDR protein was also synergistically up-regulated by combined PMA and 1,25(OH) 2D 3 treatment of ROS cells. Scatchard analysis demonstrated that the synergistic effect of PMA and 1,25(OH) 2D 3 on VDR protein expression was not associated with any change in the affinity of VDR for 1,25(OH) 2D 3. The synergistic effect of 1,25(OH) 2D 3 and PMA on VDR expression supports a link between PKC signal pathways and the function of VDR.

The glomerular mesangium in diabetes mellitus

Like the renal glomerular mesangium in patients with diabetic nephropathy, glomerular mesangial cell cultures grown in 30 mM glucose accumulate increased amounts of the extracellular matrix (ECM) proteins fibronectin, laminin, and type IV collagen. This is due to increased ECM protein synthesis and mRNA levels. Similar to other cells types that are affected by the diabetic state (such as, vascular cells and peripheral nerve), mesangial cells transport glucose by an insulin-independent, facilitated diffusion transport system. Kinetic studies reveal that intracellular glucose levels may reach the ambient glucose concentrations achieved in diabetes. Growth studies reveal that glucose does not exert its effect on mesangial cell ECM accumulation by affecting cell growth, but rather it causes an increase in diacylglycerol (DAG) mass and activates protein kinase C. Agents such as phorbol myristate acetate (PMA) and the cell permeable DAG analogue, oleoyl acetyl glycerol (OAG) which activate protein kinase C also increase ECM mRNAs. These results implicate protein kinase C activation in the increased ECM accumulation observed in mesangial cell cultures grown in high glucose.

Protein kinase C modulates the release of [3H]5-hydroxytryptamine in the spinal cord of the rat: the role of L-type voltage-dependent calcium channels.

The present studies examined the relationship between protein kinase C (PKC) and L-type voltage-dependent calcium channels in modulating the release of neurotransmitter from K(+)-depolarized rat spinal cord synaptosomes. Activators of PKC, such as phorbol 12-myristate 13-acetate (PMA), mezerein and oleoyl acetylglycerol produced a concentration-dependent potentiation of K(+)-induced release of [3H]5-hydroxytryptamine ([3H]5-HT). Enhanced release was dependent on the concentration of both Ca2+ and K+ in the superfusion medium. Calcium-independent release of [3H]5-HT or release induced by the Ca2+ ionophore were unaffected by PKC activators. Calcium-dependent release of [3H]5-HT, evoked by K+, was enhanced under similar conditions by the L-type Ca2+ channel agonists Bay K 8644 and (+)-SDZ 202-791. Nimodipine, an L-type Ca2+ channel antagonist, while having no independent effect on K(+)-induced release of [3H]5-HT, abolished the potentiative effects of Bay K 8644 and PMA. Similarly, the PKC inhibitors, polymyxin B and staurosporine, blocked effects of both PMA and Bay K 8644 on K(+)-stimulated release of [3H]5-HT. Neither PMA nor Bay K 8644 altered the uptake of [3H]5-HT. These results suggest that PKC-dependent mechanisms utilize calcium influx, via the L-type calcium channel, to modulate release of neurotransmitter and indicate a possible functional link between PKC and L-type voltage-dependent calcium channels in the spinal cord.

Rapid ultrastructural changes in the dense tubular system following platelet activation

The dense tubular system (DTS) functions to regulate platelet activation by sequestering or releasing calcium, similar to the sarcotubules of skeletal muscle. In resting platelets, the DTS exists as thin elongated membranes. Within 10 seconds of the addition of thrombin, platelets show a major ultrastructural change in their DTS: from the thin elongated form to a rounded vesicular form. These morphologic changes were demonstrated with two different stains and two different fixation methods. Platelets exposed to the calcium ionophore A23187 showed the same ultrastructural changes in the DTS. In contrast, the DTS remains in a thin elongated form when platelets are stimulated by the protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and oleoylacetylglycerol (OAG). These morphologic changes may be related to the discharge of calcium from the DTS because this is stimulated by thrombin and A23187, but not by PMA. Preincubation of the platelets with the intracellular calcium chelator 5,5′-dimethyl-bis-(0- aminophenoxy)-ethane-N,N,N′,N tetra acetic acid (BAPTA) largely prevented both the thrombin-induced rise in intracellular calcium and the changes in DTS morphology, suggesting that the changes in DTS morphology are secondary to the increase in cytosolic calcium. The results provide a morphologic correlate to existing biochemical evidence showing that the DTS is involved early during paltelet activation.

Rapid Ultrastructural Changes in the Dense Tubular System Following Platelet Activation

The dense tubular system (DTS) functions to regulate platelet activation by sequestering or releasing calcium, similar to the sarcotubules of skeletal muscle. In resting platelets, the DTS exists as thin elongated membranes. Within 10 seconds of the addition of thrombin, platelets show a major ultrastructural change in their DTS: from the thin elongated form to a rounded vesicular form. These morphologic changes were demonstrated with two different stains and two different fixation methods. Platelets exposed to the calcium ionophore A23187 showed the same ultrastructural changes in the DTS. In contrast, the DTS remains in a thin elongated form when platelets are stimulated by the protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and oleoylacetylglycerol (OAG). These morphologic changes may be related to the discharge of calcium from the DTS because this is stimulated by thrombin and A23187, but not by PMA. Preincubation of the platelets with the intracellular calcium chelator 5,5'-dimethyl-bis-(0-aminophenoxy)-ethane-N,N,N',N tetra acetic acid (BAPTA) largely prevented both the thrombin-induced rise in intracellular calcium and the changes in DTS morphology, suggesting that the changes in DTS morphology are secondary to the increase in cytosolic calcium. The results provide a morphologic correlate to existing biochemical evidence showing that the DTS is involved early during paltelet activation.

Activation of protein kinase-C inhibits vitamin D receptor gene expression.

The abundance of 1,25-dihydroxyvitamin D3 receptors (VDR) in cultured cells has been shown to vary in direct relation to the rate of cell proliferation. This study examines the question of whether the growth-factor mediated up-regulation of VDR is due to direct modulation of VDR gene expression or is secondary to the stimulation of cell cycle events. Mitogenic agents, such as basic fibroblast growth factor and phorbol esters, were found to cause significant decreases in VDR abundance, while substantially stimulating proliferation of NIH-3T3 cells. Potent phorbol esters, such as phorbol myristate acetate (PMA) and phorbol-12,13-dibutyrate, whose biological actions have been shown to be mediated through the activation of protein kinase-C, down-regulated VDR in a time- and dose-dependent manner. An inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate, which does not activate protein kinase-C, did not alter VDR levels. Desensitization of protein kinase-C by prolonged exposure of cells to phorbol esters eliminated the PMA-mediated down-regulation of VDR. Staurosporine, an inhibitor of protein kinase-C, blocked the actions of PMA. Oleoyl acetyl glycerol, a synthetic diacyl glycerol, and A23187, a calcium ionophore, were both able to suppress VDR abundance alone and were additive in combination. The results suggest that activation of the protein kinase-C pathway and elevation of intracellular Ca2+ lead to significant down-regulation of VDR. The inhibitory effect of PMA appears to be exerted at the level of VDR mRNA expression. Northern blot analysis revealed significant decreases in steady state levels of VDR mRNA species that qualitatively corresponded to the decrease in VDR protein concentration seen on a Western blot.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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

Positive inotropic effect of interleukin-2. Role of phospholipases and protein kinase C

Recently, we have shown that soluble factors released by human lymphocytes after lectin stimulation could increase the contractile tension of rat atria “ in vitro” and that interleukin-2 (IL-2) could be part of this reaction. The effect of IL-2 was potentiated by the Ca 2+ ionophore A23187 or free arachidonic acid (AA). In this study we demonstrate that the action of IL-2 can be prevented by pre-incubation of the heart tissue with monoclonal anti-IL-2 receptor (anti-p55), suggesting that binding to the IL-2 receptor is necessary for the induction of the biologic effect. In the presence of A23187 or AA, the effect of the synthetic diacylglyceride oleoyl-acetyl-glycerol (OAG) was similar to that of IL-2. Elimination of phospholipase C activity by pre-incubation of the atria with 2-nitro-carboxyphenyl, N, N-diphenylcarbamate (NCDC) abrogated the effects of IL-2 in the presence of A23187 or AA, but was ineffective when OAG + A23187 or OAG + AA was used. Inhibition of atrial phospholipase A 2 activity with p-bromo-phenacylbromide (BPB) blocked the response of atria to either IL-2 + A23187 or OAG + A23187 but was not effective when AA was used as second signal (IL-2 + AA or OAG + AA). Both the OAG and the IL-2 positive inotropic effects could be prevented by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H7) but were poorly inhibited by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), an inhibitor of the cyclic nucleotide-dependent protein kinases. The atrial cyclo-oxygenase and lipoxygenase pathways of AA metabolism were required for the development of the IL-2 or OAG effects, indicating a role for the oxidative metabolites of AA in the production of the positive inotropic effect. These observations suggest that in this experimental system, IL-2 may trigger phospholipid turnover, generating metabolites of the phospholipase A 2 and phospholipase C pathways that can in turn favor the activation of protein kinase C and the positive inotropic response of the heart.

Evidence for Protein Kinase C Involvement in the Regulation of Renal 25-Hydroxyvitamin D3-24-Hydroxylase*

Although calcium-activated, phospholipid-dependent protein kinase (protein kinase C) has been implicated in the regulation of various steroidogenic pathways, comparatively little is known of its role in the metabolism of vitamin D. The present study was undertaken to determine whether protein kinase C is involved in the regulation of renal mitochondrial 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), the first enzyme in the C-24 oxidation pathway, a major catabolic pathway for vitamin D metabolites in kidney and other target tissues. We examined the effect of phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C, on 24-hydroxylase activity in fresh mouse renal tubules and correlated the changes in 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] production with translocation of protein kinase C and phosphorylation of mitochondrial proteins. PMA stimulated 24,25-(OH)2D3 synthesis, protein kinase C translocation from the cytosolic to the mitochondrial fraction, and phosphorylation of 30-35 K, 40 K, and 50 K mitochondrial proteins derived from 32P-labeled tubules. 4 alpha-Phorbol 12,13 didecanoate, an insert analog of PMA, did not elicit any of these effects. The synthetic diacylglycerol, oleoylacetyl glycerol, also stimulated 24,25-(OH)2D3 synthesis, whereas the protein kinase C inhibitors, H-7 and staurosporine, inhibited 24-hydroxylase activity. PMA did not further stimulate 24,25-(OH)2D3 production in tubules derived from mutant (Hyp) mice in which 24-hydroxylase and protein kinase C activities are elevated relative to normal. However, after treatment with H-7, 24-hydroxylase activity was reduced in both strains, and genotype differences were no longer apparent. Finally, H-7 failed to inhibit the induced renal 24-hydroxylase in tubules isolated from 1,25-dihydroxyvitamin D3-treated mice. These findings suggest a role for protein kinase C in the regulation of constitutive renal 24-hydroxylase and implicate the kinase in the aberrant expression of the hydroxylase in the Hyp mouse.

Transfection-mediated expression of a dominant cAMP-resistant phenotype in the opossum kidney (OK) cell line prevents parathyroid hormone-induced inhibition of Na-phosphate cotransport. A protein kinase-A-mediated event.

Sodium-phosphate cotransport in the PTH-responsive opossum kidney (OK) cell line is inhibited by PTH, cAMP, and activators of protein kinase C. In order to probe the role of cAMP, we stably transfected OK cells with an expression vector for a cAMP-binding mutation of the murine protein kinase A regulatory subunit. Two-dimensional electrophoresis of cAMP-binding proteins from transfected cells indicated a 20-fold overexpression of the mutant regulatory unit. Protein kinase A from these cells had a 20-fold increase in the concentration of cAMP required for half-maximal activation, 2.8 microM vs. 0.15 microM for wild type cells. In the transfected cells, Na-phosphate cotransport was insensitive to up to 1 mM 8-Br-cAMP and 1 microM PTH, while these same agonists caused a significant inhibition of transport in the wild type cells. The effects on Na-phosphate cotransport of the protein kinase C activators oleoyl-acetyl glycerol and tetradecanoyl-phorbol acetate, which were marked in the wild type cells, were still present, although attenuated, in the transfected mutants. With prolonged passage, the cAMP-insensitive phenotype reverted to wild type cAMP sensitivity despite continued selection for the cotransfected neo marker. The revertant cells had a normal cAMP requirement for half-maximal activation of protein kinase A, 0.13 microM, and the PTH and cAMP-sensitive inhibition of Na-phosphate cotransport was restored. We suggest that an intact and normally cAMP-sensitive protein kinase A pathway is an absolute requirement for PTH inhibition of Na-phosphate cotransport in the OK cell.