Cyclic GMP depresses hippocampal Ca2+ current through a mechanism independent of cGMP-dependent protein kinase

Cyclic GMP depresses Ba2+ current through high-voltage-activated Ca2+ channels (ICa) in acutely isolated hippocampal neurons. The effect is produced by intra-, but not extracellular, cGMP or by 5' GMP. The membrane-permeant derivative, 8-Br-cGMP, produces a reversible suppression. The effect of 8-Br-cGMP is similar to phorbol ester-induced ICa depression, except that ICa depression due to 8-Br-cGMP is not blocked by protein kinase inhibitors H-8 or H-7, whereas phorbol ester effects are. The data suggest that cGMP depresses ICa by a cGMP-kinase- and protein kinase C (PKC)-independent mechanism. Cyclic AMP, which enhances ICa, and the cyclic nucleotide phosphodiesterase inhibitor, IBMX, both antagonize ICa depression induced by 8-Br-cGMP, but not that due to phorbol esters. Cyclic IMP, a more potent activator of phosphodiesterase than of cGMP-dependent protein kinase, is also a powerful depressant of ICa. We conclude that cGMP-induced depression of ICa is mediated by activation of cyclic nucleotide phosphodiesterase with consequent reduction of intracellular cAMP.     

Arachidonic acid modulates hippocampal calcium current via protein kinase C and oxygen radicals

Arachidonic acid (AA) is a second messenger liberated via receptor activation of phospholipase A2 or diacylglycerol-lipase. We used whole-cell voltage clamp of acutely isolated hippocampal CA1 pyramidal cells to investigate the hypothesis that AA modulates Ca2+ channel current (ICa) via activation of protein kinase C (PKC) and generation of free radicals. AA depressed ICa in a dose- and time-dependent manner similar to that previously reported for the action of phorbol esters on ICa. A similar depression was seen with a xanthine-based free radical generating system. The specific PKC inhibitor PKCI (19-36), the protein kinase inhibitor H-7, and the superoxide free radical scavenger SOD each blocked ICa depression by 70%-80%. Complete block of the AA response occurred when SOD was used simultaneously with a PKC inhibitor. These data suggest that PKC and free radicals play a role in AA-induced suppression of ICa.     

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous protein kinase C

Single IK(Ca) channels of human erythrocytes were studied with the patch-clamp technique to define their modulation by endogenous protein kinase C (PKC). The perfusion of the cytoplasmic side of freshly excised patches with the PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited channel activity. This effect was blocked by PKC(19-31), a peptide inhibitor specific for PKC. Similar results were obtained by perfusing the membrane patches with the structurally unrelated PKC activator 1-oleoyl-2-acetylglycerol (OAG). Blocking of this effect was induced by perfusion with PKC(19-31) or chelerythrine. Channel activity was not inhibited by the PMA analog 4alpha-phorbol 12,13-didecanoate (4alphaPDD), which has no effect on PKC. Activation of endogenous cAMP-dependent protein kinase (PKA), which is known to up-modulate IK(Ca) channels, restored channel activity previously inhibited by OAG. The application of OAG induced a reversible reduction of channel activity previously up-modulated by the activation of PKA, indicating that the effects of the two kinases are commutative, and antagonistic. Kinetic analysis showed that down-regulation by PKC mainly changes the opening frequency without significantly affecting mean channel open time and conductance. These results provide evidence that an endogenous PKC down-modulates the activity of native IK(Ca) channels of human erythrocytes. Our results show that PKA and PKC signal transduction pathways integrate their effects, determining the open probability of the IK(Ca) channels.     

A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C

CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.     

IL-5-induced IgA synthesis by LPS-stimulated mouse B cells is prevented by protein kinase C inhibitors.

We have previously demonstrated that activation of protein kinase C (PKC) by phorbol esters induces selectively IgA synthesis by mouse B cells. In this study, we investigated the effects of a number of protein kinase inhibitors on IgA secretion induced by a recombinant murine IL-5 in LPS-stimulated mouse B cells. The results show that PKC inhibitors, such as sphingosine (SPH), staurosporine (STP) and H-7, blocked IL-5-induced IgA synthesis; the protein kinase A inhibitor HA-1004 and the inhibitor of calcium/calmodulin dependent protein kinase W-7 had no effect on IgA secretion induced by IL-5. The proliferation of the IL-5 sensitive B13 cell line in response to IL-5 was also inhibited by addition of SPH or STP or H-7. The data suggest an involvement of the PKC pathway in IL-5-induced B cell differentiation into IgA secreting cells.     

Selectivity of protein kinase inhibitors in human intact platelets

The specificity of commonly used protein kinase inhibitors has been evaluated in the intact human platelet. Protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) were activated selectively by treating platelets with phorbol dibutyrate (PDBu) or prostacyclin (PGl2). PKC activity was quantitated by measuring PDBu-specific phosphorylation of a 47,000 molecular weight protein, and PKA activity monitored by measuring prostacyclin-dependent phosphorylation of a 22,000 molecular weight protein. Staurosporine and 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7) were found to be non-specific inhibitors in the intact platelet, consistent with their effects on the isolated enzymes. Tamoxifen inhibited PKC activity (IC50 = 80 microM) but increased PKA-dependent protein phosphorylation. These results support the use of human platelets for measuring the specificity of protein kinase inhibitors and indicate that tamoxifen might have value for experimental purposes as a relatively selective PKC inhibitor.     

Glucocorticoid stimulation of amnion cell prostaglandin synthesis: suppression by protein kinase C inhibitors and independence of phorbol ester-sensitive protein kinase C.

Glucocorticoids stimulate the prostaglandin E2 production of confluent amnion cell cultures, but have no stimulatory effect on the PGE2 output of freshly isolated human amnion cells. Since protein phosphorylation may modify the responsiveness of target cells to steroids, and activators of protein kinase C (PKC), as well as corticosteroids, promote amnion cell PGE2 output by stimulating the synthesis of prostaglandin endoperoxide H synthase (PGHS), we investigated the possibility that PKC is involved in the glucocorticoid-induction of PGE2 synthesis in cultured amnion cells. The dexamethasone-induced PGE2 output of arachidonate-stimulated cells was blocked by the protein kinase inhibitors staurosporine, K-252a, H7, HA1004, and sphinganine, in a manner consistent with their effect on PKC. However, dexamethasone increased the PGE2 production of cultures treated with maximally effective concentrations of the PKC-activator compound TPA. Moreover, dexamethasone stimulated PGE2 synthesis in cultures which were desensitized to TPA-stimulation by prolonged phorbol ester treatment. Concentration-dependence studies showed that staurosporine completely (greater than 95%) blocked glucocorticoid-provoked PGE2 synthesis at concentrations which did not inhibit TPA-stimulated prostaglandin output, and that K-252a inhibited the effect of TPA by more than 95% at concentrations which decreased the effect of dexamethasone only moderately (approximately 40%). Dibutyryl cyclic AMP had no influence on the basal- or dexamethasone-stimulated PGE2 production, and on the staurosporine inhibition of the steroid effect. These results show that glucocorticoids and phorbol esters control amnion PGE2 production by separate regulatory mechanisms. It is suggested that the response of human amnion cells to glucocorticoids is modulated by protein kinase(s) other than phorbol ester-sensitive PKC and cyclic AMP-dependent protein kinase.     

Na+-H+ antiport during fertilization of the sea urchin egg is blocked by W-7 but is insensitive to K252a and H-7

Fertilization of the sea urchin egg initiates or accelerates a number of metabolic activities, which have been causally linked to a rise in cytoplasmic pH due to increased Na+-H+ antiport. Two possible regulatory pathways linking sperm-egg fusion to the activity of the antiporter are activation of protein kinase C (PKC) and Ca2+, calmodulin (CaM)-dependent kinase. This report presents the effects of protein kinase inhibitors on antiporter activation during fertilization and treatment with PKC agonists, dioctanoylglycerol or phorbol diester. Protein kinase inhibitors, K252a and H-7 blocked the action of PKC agonists, without inhibiting cytoplasmic alkalinization during fertilization. In contrast, W-7 blocked fertilization-induced rise in cytoplasmic pH, without altering the actions of PKC agonists. These results suggest that the Na+-H+ antiporter may be regulated by PKC or Ca2+, CaM-dependent kinase activities, but activation of the antiporter during fertilization is Ca2+, CaM-dependent, despite production of diacylglycerols by hydrolysis of phosphatidylinositols.     

The regulation of Na/K/2Cl cotransport and bumetanide binding in avian erythrocytes by protein phosphorylation and dephosphorylation. Effects of kinase inhibitors and okadaic acid

The Na/K/2Cl cotransport system in the avian erythrocyte can be activated by agents that raise intracellular cAMP suggesting the involvement of cAMP-dependent protein kinase (cAMP-PK) in its regulation. Another group of stimuli including fluoride and hypertonicity stimulate cotransport via cAMP-independent means. To further investigate the role of phosphorylation in these processes, we examined the effects of protein kinase inhibitors of 8 (p-Cl-phenylthio)-cAMP (cpt-cAMP), fluoride and hypertonic activation of cotransport in duck red cells, and [3H]bumetanide binding to isolated membranes. Preincubation of cells with the kinase inhibitors K-252a (Ki approximately 1.6 microM) and H-9 (Ki approximately 100 microM) blocked cpt-cAMP activation of bumetanide-sensitive 86Rb influx and bumetanide binding. These inhibitors also led to a rapid deactivation of cotransport and decrease in bumetanide binding when added to cells maximally stimulated by cpt-cAMP. K-252a and H-9 inhibited cotransport activation by cAMP-independent stimuli, but 10-fold higher concentrations were required, implying the involvement of a cAMP-independent phosphorylation process in the mechanism of action of these agents. Removal of stimuli that elevate cAMP leads to a rapid reversal of cotransport indicating the presence of active protein phosphatases in these cells. The protein phosphatase inhibitor okadaic acid (OA, EC50: 630 nM) stimulated both Na/K/2Cl cotransport and bumetanide binding to membranes. As with fluoride and hypertonic stimulation, the OA effect was inhibited only at relatively high concentrations of K-252a. Phosphorylation of the membrane skeletal protein goblin (Mr 230,000) at specific cAMP-dependent sites was used as an in situ marker for the state of activation of cAMP-PK. Goblin phosphorylation at these sites was increased by norepinephrine and cpt-cAMP and rapidly reversed by K-252a and H-9, confirming that both inhibitors do block cAMP-PK activity. While OA markedly increased overall phosphorylation of many erythrocyte membrane proteins, including goblin, it did not affect goblin phosphorylation at specific cAMP-dependent sites. These results implicate a cAMP-independent protein kinase in the mediation of the OA effect on cotransport and bumetanide binding. The bumetanide-binding component of the avian erythrocyte cotransporter, an Mr approximately 150,000 protein that can be photolabeled with the bumetanide analog [3H]4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)-benzoic acid was found to be a phosphoprotein. These results strongly support the hypothesis that phosphorylation and dephosphorylation, possibly of the Na/K/2Cl cotransporter itself, regulates the activity of     

Rapid effects of estrogen to modulate G protein-coupled receptors via activation of protein kinase A and protein kinase C pathways.

17Beta-estradiol (E2) rapidly (<20 min) attenuates the ability of mu-opioids to hyperpolarize guinea pig hypothalamic neurons. We have used intracellular recordings from female guinea pig hypothalamic slices to characterize the receptor and intracellular pathway(s) mediating E2's rapid effects. E2 acts stereospecifically with physiologically relevant concentration-dependence (EC50 = 8 nM) to cause a fourfold reduction in the potency of the mu-opioid agonist (D-Ala2-N-Me-Phe4-Gly5-ol)-enkephalin and the GABA(B) agonist baclofen to activate an inwardly rectifying K+ conductance in hypothalamic neurons. Both the nonsteroidal estrogen diethylstilbestrol and the anti-estrogen ICI 164,384 blocked E2 actions to uncouple mu-opioid receptors. Using a pharmacological Schild analysis, we found that ICI 164,384 competed for this E2 receptor with a Ke of approximately 0.3 nM. The protein synthesis inhibitor cycloheximide did not block the estrogenic uncoupling of the mu-opioid receptor from its K+ channel, implying a rapid, nongenomic mechanism of E2 action. The effects of E2 were mimicked by the bath application of the protein kinase A (PKA) activators, forskolin and Sp-cAMP, and the protein kinase C (PKC) activator phorbol-12,13-dibutyrate. Furthermore, the selective PKA antagonists Rp-cAMP and KT5720, which have different chemical structures and modes of action, both blocked the effects of E2. In addition, the actions of E2 were blocked by the selective PKC inhibitor Calphostin C. Therefore, it appears that E2 can activate both PKA and PKC to cause a heterologous desensitization of both mu-opioid and GABA(B) receptors, which has the potential to alter synaptic transmission in many regions of the CNS.     

Ca(2+)-independent, phospholipid-activated protein kinase in 3Y1 cells.

We obtained a Ca(2+)-independent but 12-O-tetradecanoyl phorbol ester (TPA).phospholipid-activated protein kinase from rat embryo fibroblast 3Y1 cells by succeeding steps of DEAE-cellulose, H-9 affinity, and hydroxylapatite chromatography. This kinase was separated chromatography. This kinase was separated from a conventional PKC (Type III), by H-9 affinity column chromatography. The major peak from H-9 affinity column was eluted at 0.4 M of arginine and on the following step of hydroxylapatite column chromatography, at the KPO4 concentration of 0.1 M. The enzyme could be stimulated by phospholipids and by the tumor promoter TPA, but did not respond to calcium. The Ca(2+)-independent, phospholipid-activated protein kinase activity was susceptible to the protein kinase C inhibitors H-7 and K252a, but showed a phospholipid dependency and substrate specificity distinct from the conventional types of PKC. This protein kinase did not react with monoclonal antibodies against Types I, II, and III PKC. The activity of this enzyme was specifically reduced by immunoprecipitation, depending on the concentration of the polyclonal antibody, PC-delta, which was raised against a peptide synthesized according to a sequence of rat brain nPKC delta. The enzyme had a Mr of 76,000 as estimated by Western blotting. These results provide evidence for a unique type of Ca(2+)-independent, phospholipid-activated kinase, as expressed in 3Y1 cells.     

Involvement of protein kinase C in translocation of desmoplakins from cytosol to plasma membrane during desmosome formation in human squamous cell carcinoma cells grown in low to normal calcium concentration

The intracellular signal transduction mechanism leading to desmosome formation in low-calcium-grown keratinocytes after addition of calcium to the medium was studied by immunofluorescence using antibodies to desmoplakins I and II (cytoplasmic desmosomal proteins) and by electron microscopy before and after addition of calcium; protein kinase C (PKC) activators 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibutyrate (PDBu), and 1,2-dioctanoylglycerol (DOG); calcium ionophore A23187; selective PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine; and a Ca2+/calmodulin-dependent kinase inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). In previous studies using a low-calcium-grown human epidermal squamous cell carcinoma, we have shown that an increase in extracellular Ca2+ caused a four-fold increase in PKC activity and addition of TPA (10 ng/ml) induced a transient increase in membrane-bound PKC activity in association with cell-cell contact formation. The present study showed that TPA (10 ng/ml). PDBu (10 ng/ml), and DOG (1 mg/ml) induced a rapid cell-cell contact and redistribution of desmoplakins from cytoplasm to the plasma membrane with desmosome formation within 60-120 min, which was similar, although less marked, to the effect of increased Ca2+. The TPA-induced desmosome formation was inhibited by selective PKC inhibitors, H-7 (20 microM) or staurosporine (100 nM). On the other hand, calcium ionophore A23187 induced only a temporary increase in the number of desmoplakin-containing fluorescent spots in the cytoplasm and a temporary cell-cell attachment without desmosome formation. The calcium-induced desmosome formation was partially inhibited by 20-100 microM H-7 or 100 nM staurosporine; however, it was not inhibited by W-7 at a concentration of 25 microM, at which this agent selectively inhibits calmodulin-dependent protein kinase. These results suggest that PKC activation plays an important role in desmoplakin translocation from the cytoplasm to the plasma membrane as one of the processes of calcium-induced desmosome formation.     

Effects of calcium, calmodulin, protein kinase C and protein tyrosine kinases on volume-activated taurine efflux in human erythroleukemia cells.

The effects of calcium, calmodulin, protein kinase C (PKC) and protein tyrosine kinase (PTK) modulators were examined on the volume-activated taurine efflux in the erythroleukemia cell line K562. Exposure to hypoosmotic solution significantly increased taurine efflux and intracellular calcium concentration ([Ca2+]i). The Ca2+ channel blockers La3+ (1 mM), verapamil (200 microM) and nifedipine (100 microM) inhibited the hypoosmotically-induced [Ca2+]i increase by more than 90%, while the volume-activated taurine efflux was inhibited by 61.3 +/- 9.5, 74.1 +/- 9.3 and 38.0 +/- 1.5%, respectively. Furthermore, the calmodulin inhibitors W7 (50 microM) and trifluoperazine (10 microM) and the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62 (2 microM) significantly blocked the volume-activated taurine efflux by 93.4 +/- 2.7, 77.9 +/- 3.5 and 61.3 +/- 15.8%, respectively. In contrast, the PKC inhibitor staurosporine (200 nM) or the PKC activator phorbol 12-myristate 13-acetate (100 nM) did not have significant effects on the volume-activated taurine efflux. However, pretreatment with PTK inhibitors genistein, tyrphostin A25, and tyrphostin A47 blocked the volume-activated taurine efflux. These results suggest that the volume-activated taurine efflux in K562 cells may not directly involve Ca2+, but may require the presence of calmodulin and/or PTK.     

Potent and preferential inhibition of Ca2+/calmodulin-dependent protein kinase II by K252a and its derivative, KT5926.

Effects of protein kinase inhibitors, K252a and its derivative KT5926, on Ca2+/calmodulin-dependent protein kinase II were examined. Both compounds potently inhibited Ca2+/calmodulin-dependent protein kinase II. Kinetic analyses indicated that the inhibitory effect of K252a and KT5926 was competitive with respect to ATP (Ki: 1.8 and 4.4 nM, respectively) and noncompetitive with respect to the substrates. Taken together with a previous report (Nakanishi et al. Mol. Pharmacol. 37, 482, 1990) concerning the Ki values of these compounds for ATP with various protein kinases, the results suggest that K252a and KT5926 are potent and preferential inhibitors of Ca2+/calmodulin-dependent protein kinase II.     

Amyloid beta protein (25-35) phosphorylates MARCKS through tyrosine kinase-activated protein kinase C signaling pathway in microglia

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid beta protein (A beta) (25-35) and A beta (1-40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with A beta (25-35) at 10 nM or 12-O-tetradecanoylphorbol 13-acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The A beta (25-35)-induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms alpha, delta, and epsilon were identified in microglia by immunocytochemistry and western blots using isoform-specific antibodies. PKC-delta was tyrosine-phosphorylated by the treatment of microglia for 10 min with A beta (25-35) at 10 nM. Other PKC isoforms alpha and epsilon were tyrosine-phosphorylated by A beta (25-35), but only to a small extent. We propose that a tyrosine kinase-activated PKC pathway is involved in the A beta (25-35)-induced phosphorylation of MARCKS in rat microglia.     

Effects of Protein Kinase Inhibitors on Morphology and Function of Cultured Bovine Adrenal Chromaffin Cells: KN-62 Inhibits Secretory Function by Blocking Stimulated Ca2+ Entry

Abstract: In cultured bovine adrenal chromaffin cells, a nonselective protein kinase inhibitor, staurosporine, inhibits secretory function and induces neurite outgrowth. In the present study, effects of other nonselective protein kinase inhibitors (K-252a, H-7, and H-8) and reportedly selective protein kinase inhibitors (KN-62 and chelerythrine chloride) were examined on bovine adrenal chromaffin cell morphology, secretory function, and ⁴⁵Ca²⁺ uptake. Treatment of chromaffin cells with 10 µ M K-252a, 50 µ M H-7, or 50 µ M H-8 induced changes in cell morphology within 3 h; these compounds also induced a time-dependent inhibition of stimulated catecholamine release. Chelerythrine chloride, a selective inhibitor of Ca²⁺/phospholipid-dependent protein kinase, did not induce outgrowth or inhibit secretory function under our treatment conditions. KN-62, a selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMK II), significantly inhibited stimulated catecholamine release (IC₅₀ value of 0.32 µ M ), but had no effect on cell morphology. The reduction of secretory function induced by 1 µ M KN-62 was significant within 5 min and rapidly reversible. Unlike H-7, H-8, and staurosporine, KN-62 significantly inhibited stimulated ⁴⁵Ca²⁺ uptake. KN-04, a structural analogue of KN-62 that does not inhibit CaMK II, inhibited stimulated ⁴⁵Ca²⁺ uptake and catecholamine release like KN-62. These studies indicate that KN-62 inhibits secretory function via the direct blockade of activated Ca²⁺ influx. The nonselective inhibitors, K-252a, H-7, H-8, and staurosporine, inhibit secretory function by another mechanism, perhaps one involving alterations in the cytoskeleton.     

Protein kinase C induces phosphorylation and desensitization of the human 5-HT1A receptor

The effects of short-term phorbol ester treatment of CHO cells that stably express 900 fmol of recombinant human serotonin 5-HT1A receptor/mg of protein on coupling to the inhibition of adenylyl cyclase and on phosphorylation of the receptor were studied. Pretreatment of cell monolayers with phorbol 12-myristate 13-acetate (PMA) caused a dose- and time-dependent shift of the half-maximal dose of serotonin (5-HT) required to inhibit membrane adenylyl cyclase (from IC50 approximately 100 nM to approximately 400 nM). This desensitization (shift in IC50) was rapid, occurring with 5 min of pretreatment and being maximal by 10-15 min; it was also dose-dependent, being half-maximal at approximately 300 nM PMA. Desensitization was also induced by sn-dioctanoylglycerol (DiC8) and blocked by the protein kinase C (PKC) inhibitors sphingosine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). In detached permeabilized cells, PMA pretreatment caused a rapid phosphorylation of immunoprecipitated 5-HT1A receptors, with an approximately 3-4-fold increase that was maximal after 15 min and persisted for 90 min. The phosphorylation occurred at a similar dose of PMA as that which induced desensitization (half-maximal at approximately 300 nM, maximal at 500 nM to 1 microM), could be reproduced by pretreatment with the PKC activators DiC8 or phorbol 12,13-dibutyrate (PDBu), and could be blocked by the PKC inhibitors sphingosine or H-7. The stoichiometry of the phosphorylation was approximately 2 mol of [32P]ATP/mol of receptor, suggesting the involvement at least two of three putative PKC sites within the 5-HT1A receptor. The close concordance between the PKC-induced desensitization and phosphorylation suggests a potential causative link between these two effects of PKC on the human 5-HT1A receptor.     

Role of protein kinase C in aldosterone-induced non-genomic inhibition of basolateral potassium channels in human colonic crypts

Aldosterone produces rapid, non-genomic, inhibition of basolateral intermediate conductance K(+) (IK(Ca)) channels in human colonic crypt cells but the intracellular second messengers involved are unclear. We therefore evaluated the role of protein kinase C (PKC) in aldosterone's non-genomic inhibitory effect on basolateral IK(Ca) channels in crypt cells from normal human sigmoid colon. Patch clamp studies revealed that in cell-attached patches, IK(Ca) channel activity decreased progressively to 38+/-8% (P<0.001) of the basal value 10 min after the addition of 1 nmol/L aldosterone, and decreased further to 23+/-6% (P<0.02) of the basal value 5 min after increasing the aldosterone concentration to 10 nmol/L. Pre-incubation of crypts with 1 micromol/L chelerythrine chloride or 1 micromol/L G? 6976 (PKC inhibitors) prevented the inhibitory effect of aldosterone. Conversely, channel activity decreased to 60+/-9% (P<0.02) of the basal value 10 min after the addition of 500 nmol/L PMA (a PKC activator), whereas 4alpha-PMA (an inactive ester) had no effect. When aldosterone (10 nmol/L) and PMA were added together, IK(Ca) channel activity was inhibited to the same extent as with aldosterone alone. These results indicate that aldosterone's non-genomic inhibitory effect on the macroscopic basolateral K(+) conductance in human colonic crypts reflects PKC-mediated inhibition of IK(Ca) channels.     

Regulation of a calcium-sensitive K+ channel (cIK1) by protein kinase C

Ca2+-sensitive K+ channels (IK1 channels) are required for many physiological functions such as cell proliferation, epithelial transport or cell migration. They are regulated by the intracellular Ca2+ concentration and by phosphorylation-dependent reactions. Here, we investigate by means of the patch-clamp technique mechanisms by which protein kinase C (PKC) regulates the canine isoform, cIK1, cloned from transformed renal epithelial (MDCK-F) cells. cIK1 elicits a K+-selective, inwardly rectifying, and Ca2+-dependent current when expressed in HEK293 or CHO cells. It is inhibited by charybdotoxin, clotrimazole, and activated by 1-ethyl-2-benzimidazolone. cIK1 is activated by intracellular application of ATP or ATP[gS]. ATP-dependent activation is reversed by PKC inhibitors (bisindolylmaleimide, calphostin C), while stimulation with ATP[gS] resists PKC inhibition. Stimulation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) leads to the acute activation of cIK1 currents, which are blocked by PKC inhibitors. In contrast, PKC depletion by overnight incubation with PMA prevents ATP-dependent cIK1 activation. Neither single mutations nor the simultaneous mutation of all PKC sites (T101, S178, T329) to alanine alter the acute regulation of cIK1 channels by PKC. However, current amplitudes of CIK1-T329A and the triple mutant are dramatically increased upon long-term treatment with PMA. These mutations thereby disclose an inhibitory effect on cIKl current of the PKC site at T329. Our results indicate that cIK1 channel activity is regulated in two ways. PKC-dependent activation of cIK1 channels occurs indirectly, while the inhibitory effect probably requires a direct interaction with the channel protein.     

Induction of differentiation of HL-60 cells by protein kinase C inhibitor, K252a

To clarify the role of protein kinase C and protein kinase A in cell proliferation and differentiation, the effects of K252a and its derivatives (K252b, KT5720), which have different inhibitory activity to these protein kinases, on the proliferation and differentiation of HL-60 cells were investigated. The proliferation and DNA synthesis of the HL-60 cells were inhibited by K252a in a dose dependent manner. However, K252b and KT5720 which are more specific inhibitors of protein kinase C or protein kinase A, respectively, had no observable effect on cell proliferation. K252a (40nM) enhanced the differentiation of HL-60 cells induced by 1,25(OH)2D3, retinoic acid and DMSO. K252b and KT5720 did not affect 1,25(OH)2D3-induced differentiation. K252a significantly inhibited the differentiation induced by PMA. These results demonstrate that K252a but not its derivatives can function as an antitumor drug and enhancer of the differentiation induced by various inducers.