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.     

Inhibition of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilisation by phorbol ester in rat cultured vascular smooth muscle cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 microM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+]i, were 7.8 +/- 0.3 M and 1 microM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 microM PMA for either 1 or 24 h did not significantly change the K(D) and Bmax of the BK receptor for binding (control: K(D) = 1.7 +/- 0.2 nM; Bmax = 47.3 +/- 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs.     

Sphingosine inhibits thyrotropin-releasing hormone binding to pituitary cells by a mechanism independent of protein kinase C

Sphingosine inhibited [3H]methylhistidine-thyrotropin-releasing hormone (MeTRH) binding to intact GH3 cells and to GH3 membranes. This inhibition was dependent on the concentration of sphingosine and on the ratio of sphingosine to cell number (or membrane protein) and was partly reversed by washing. In intact cells, the IC50 was 63 microM (1.8 X 10(6) cells/ml; 2 nM MeTRH), and 100 microM sphingosine was found, by Scatchard analysis, to increase the apparent dissociation constant (Kd) from 1.1 +/- 0.3 to 6.5 +/- 2.3 nM and to decrease the maximal binding capacity (Bmax) to 41 +/- 9.5% of control. Kinetic analysis showed that the major effect of sphingosine on Kd was due to a marked decrease in the apparent association rate constant for MeTRH from 2.5 +/- 0.4 X 10(5) M-1 s-1 to 0.10 +/- 0.015 X 10(5) M-1 s-1. At 100 microM, sterylamine was as effective as sphingosine in inhibiting MeTRH binding, whereas sphinganine was less effective, and psychosine and steroylsphingosine were without effect. The following observations show that sphingosine inhibition of MeTRH binding did not involve protein kinase C. The IC50 for sphingosine inhibition of MeTRH binding was the same in GH3 cells that had been incubated with 1 microM phorbol 12-myristate 13-acetate for 16 h, to "down-regulate" protein kinase C, as in control cells. Sphingosine inhibited MeTRH binding to membranes isolated from GH3 cells that contain very little protein kinase C activity. In GH3 membranes, 100 microM sphingosine increased the Kd for MeTRH from 3.4 +/- 0.1 to 13 +/- 3.1 nM but did not significantly decrease Bmax (12 +/- 5.0% of control, p greater than 0.05). And, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, an inhibitor of protein kinase C, failed to decrease MeTRH binding to intact GH3 cells or to membranes, and did not interfere with the effects of sphingosine. These data show that sphingosine and its analogs have complex actions to inhibit MeTRH binding to GH3 cells, at least some of which are independent of protein kinase C, and thereby demonstrate that sphingolipids cannot be used as specific inhibitors of protein kinase C.     

M3 muscarinic acetylcholine receptors regulate cytoplasmic myosin by a process involving RhoA and requiring conventional protein kinase C isoforms.

Although muscarinic acetylcholine receptors (mAChR) regulate the activity of smooth muscle myosin, the effects of mAChR activation on cytoplasmic myosin have not been characterized. We found that activation of transfected human M3 mAChR induces the phosphorylation of myosin light chains (MLC) and the formation of myosin-containing stress fibers in Chinese hamster ovary (CHO-m3) cells. Direct activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) also induces myosin light chain phosphorylation and myosin reorganization in CHO-m3 cells. Conventional (alpha), novel (delta), and atypical (iota) PKC isoforms are activated by mAChR stimulation or PMA treatment in CHO-m3 cells, as indicated by PKC translocation or degradation. mAChR-mediated myosin reorganization is abolished by inhibiting conventional PKC isoforms with Go6976 (IC50 = 0.4 microM), calphostin C (IC50 = 2.4 microM), or chelerythrine (IC50 = 8.0 microM). Stable expression of dominant negative RhoAAsn-19 diminishes, but does not abolish, mAChR-mediated myosin reorganization in the CHO-m3 cells. Similarly, mAChR-mediated myosin reorganization is diminished, but not abolished, in CHO-m3 cells which are multi-nucleate due to inactivation of Rho with C3 exoenzyme. Expression of dominant negative RhoAAsn-19 or inactivation of RhoA with C3 exoenzyme does not affect PMA-induced myosin reorganization. These findings indicate that the PKC-mediated pathway of myosin reorganization (induced either by M3 mAChR activation or PMA treatment) can continue to operate even when RhoA activity is diminished in CHO-m3 cells. Conventional PKC isoforms and RhoA may participate in separate but parallel pathways induced by M3 mAChR activation to regulate cytoplasmic myosin. Changes in cytoplasmic myosin elicited by M3 mAChR activation may contribute to the unique ability of these receptors to regulate cell morphology, adhesion, and proliferation.     

Tyrosine kinase and protein kinase C regulate L-type Ca(2+) current cooperatively in human atrial myocytes

The effects of tyrosine protein kinases (TK) on the L-type Ca(2+) current (I(Ca)) were examined in whole cell patch-clamped human atrial myocytes. The TK inhibitors genistein (50 microM), lavendustin A (50 microM), and tyrphostin 23 (50 microM) stimulated I(Ca) by 132 +/- 18% (P < 0.001), 116 +/- 18% (P < 0.05), and 60 +/- 6% (P < 0.001), respectively. After I(Ca) stimulation by genistein, external application of isoproterenol (1 microM) caused an additional increase in I(Ca). Dialyzing the cells with a protein kinase A inhibitor suppressed the effect of isoproterenol on I(Ca) but not that of genistein. Inhibition of protein kinase C (PKC) by pretreatment of cells with 100 nM staurosporine or 100 nM calphostin C prevented the effects of genistein on I(Ca). The PKC activator phorbol 12-myristate 13-acetate (PMA), after an initial stimulation (75 +/- 17%, P < 0.05), decreased I(Ca) (-36 +/- 5%, P < 0.001). Once the inhibitory effect of PMA on I(Ca) had stabilized, genistein strongly stimulated the current (323 +/- 25%, P < 0.05). Pretreating myocytes with genistein reduced the inhibitory effect of PMA on I(Ca). We conclude that, in human atrial myocytes, TK inhibit I(Ca) via a mechanism that involves PKC.     

Gender-related distinctions in protein kinase C activity in rat vascular smooth muscle

Gender differences in vascular reactivity have been suggested; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the gender differences in vascular reactivity reflect gender-related, possibly estrogen-mediated, distinctions in the expression and activity of specific protein kinase C (PKC) isoforms in vascular smooth muscle. Aortic strips were isolated from intact and gonadectomized male and female Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Isometric contraction was measured in endothelium-denuded aortic strips. PKC activity was measured in the cytosolic and particulate fractions, and the amount of PKC was measured using Western blots and isoform-specific anti-PKC antibodies. In intact male WKY rats, phenylephrine (Phe, 10(-5) M) and phorbol 12,13-dibutyrate (PDBu, 10(-6) M) stimulated contraction to 0.37 +/- 0.02 and 0.42 +/- 0.02 g/mg tissue wt, respectively. The basal particulate/cytosolic PKC activity ratio was 0.86 +/- 0.06, and Western blots revealed alpha-, delta-, and zeta-PKC isoforms. Phe and PDBu increased PKC activity and caused significant translocation of alpha- and delta-PKC from the cytosolic to particulate fraction. In intact female WKY rats, basal PKC activity, the amount of alpha-, delta-, and zeta-PKC, the Phe- and PDBu-induced contraction, and PKC activity and translocation of alpha- and delta-PKC were significantly reduced compared with intact male WKY rats. The basal PKC activity, the amount of alpha-, delta-, and zeta-PKC, the Phe and PDBu contraction, and PKC activity and alpha- and delta-PKC translocation were greater in SHR than WKY rats. The reduction in Phe and PDBu contraction and PKC activity in intact females compared with intact males was greater in SHR ( approximately 30%) than WKY rats ( approximately 20%). Phe and PDBu contraction and PKC activity were not significantly different between castrated males and intact males but were greater in ovariectomized (OVX) females than intact females. Treatment of OVX females or castrated males with 17 beta-estradiol, but not 17 alpha-estradiol, subcutaneous implants caused significant reduction in Phe and PDBu contraction and PKC activity that was greater in SHR than WKY rats. Phe and PDBu contraction and PKC activity in OVX females or castrated males treated with 17 beta-estradiol plus the estrogen receptor antagonist ICI-182,780 were not significantly different from untreated OVX females or castrated males. Thus a gender-related reduction in vascular smooth muscle contraction in female WKY rats with intact gonads compared with males is associated with reduction in the expression and activity of vascular alpha-, delta-, and zeta-PKC. The gender differences in vascular smooth muscle contraction and PKC activity are augmented in the SHR and are possibly mediated by estrogen.     

Ca2+/calmodulin mediated pathways regulate the uptake of L-DOPA in mouse neuroblastoma neuro 2A cells

The present study examined the involvement of protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), protein tyrosine kinase (PTK) and Ca2+/calmodulin mediated pathways on the uptake of L-DOPA through the L-type amino acid transporter in Neuro 2A cells, an in vitro model of neuronal cells. Non-linear analysis of the saturation curve for L-DOPA revealed a Km value (in microM) of 54+/-2 and a Vmax value (in nmol mg protein/6 min) of 34+/-1. L-DOPA uptake was a sodium-independent process and insensitive to N-(methylamino)-isobutyric acid (MeAIB, 1 mM), but sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC, IC50=82 microM). The Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA (2.5 microM) uptake with IC50's of 33 and 105 microM, respectively. The inhibitory effect of BHC on the accumulation of L-DOPA was of the competitive type, whereas that of calmidazolium and trifluoperazine was of the non-competitive type. Modulators of PKA (cyclic AMP, forskolin, isobutylmethylxanthine and cholera toxin), PKG (cyclic GMP, zaprinast, LY 83583 and sodium nitroprusside), PKC (phorbol 12,13-dibutirate, phorbol 12-myristate 13-acetate and chelerythrine) and PTK (genistein and tyrphostin 25) failed to affect the accumulation of a non-saturating (2.5 microM) concentration of L-DOPA. It is concluded that L-DOPA uptake in Neuro 2A cells is promoted through the L-type amino acid transporter and appears to be under the control of Ca2+/calmodulin mediated pathways.     

Cyclic AMP stimulates luteinizing-hormone (lutropin) exocytosis in permeabilized sheep anterior-pituitary cells. Synergism with protein kinase C and calcium.

Sheep anterior-pituitary cells permeabilized with Staphylococcus aureus alpha-toxin were used to investigate the role of cyclic AMP (cAMP) in exocytosis of luteinizing hormone (lutropin, LH) under conditions where the intracellular free Ca2+ concentration ([Ca2+]free) is clamped by Ca2+ buffers. At resting [Ca2+]free (pCa 7), cAMP rapidly stimulated LH exocytosis (within 5 min) and continued to stimulate exocytosis for at least 30 min. When cAMP breakdown was inhibited by 3-isobutyl-1-methylxanthine (IBMX), the concentration giving half-maximal response (EC50) for cAMP-stimulated exocytosis was 10 microM. cAMP-stimulated exocytosis required millimolar concentrations of MgATP, as has been found with Ca2(+)- and phorbol-ester-stimulated LH exocytosis. cAMP caused a modest enhancement of Ca2(+)-stimulated LH exocytosis by decreasing in the EC50 for Ca2+ from pCa 5.6 to pCa 5.9, but had little effect on the maximal LH response to Ca2+. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) dramatically enhanced cAMP-stimulated LH exocytosis by both increasing the maximal effect 5-7-fold and decreasing the EC50 for cAMP to 3 microM. This synergism between cAMP and PMA was further augmented by increasing the [Ca2+]free. Gonadotropin-releasing hormone (gonadoliberin, GnRH) stimulated cAMP production in intact pituitary cells. Since GnRH stimulation is reported to activate PKC and increase the intracellular [Ca2+]free, our results suggest that a synergistic interaction of the cAMP, PKC and Ca2+ second-messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis.     

Lysophosphatidylcholine-induced taurine release in HeLa cells involves protein kinase activity

It has recently been demonstrated that exogenous addition of low concentrations (< 15 microM) of lysophosphatidyl choline (LPC, palmitic acid in the sn-1 position) induces a transient increase in taurine efflux from HeLa cells in a process that seems to involve generation of reactive oxygen species (ROS) and tyrosine phosphorylation (J. Membrane Biol. 176 (2000) 175-185). We now demonstrate that LPC also induces release of taurine under isotonic conditions in mouse fibroblast (NIH/3T3) and Ehrlich ascites tumor cells. Furthermore, we show that in the case of HeLa cells addition of the calmodulin antagonist W-7 (50 microM) or the calmodulin-dependent kinase II (CaMKII) inhibitor KN-62 (10 microM) reduces the LPC-induced taurine release under isotonic conditions. Conversely, addition of a standard protein kinase C (PKC) inhibitor chelerythrine (10 microM) leads to a potentiation of the LPC-induced taurine efflux, whereas direct activation of PKC by the phorbol ester PMA has no effect. It is suggested that the putative generation of ROS following addition of LPC is modulated by calmodulin/CaMKII, and that the effect of chelerythrine is more likely related to the ROS production than to PKC inhibition.     

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.     

Desensitization of angiotensin II: effect on [Ca2+]i, inositol triphosphate, and prolactin in pituitary cells

Activation of pituitary angiotensin (ANG II) type 1 receptors (AT1) mobilizes intracellular Ca2+, resulting in increased prolactin secretion. We first assessed desensitization of AT1 receptors by testing ANG II-induced intracellular Ca2+ concentration ([Ca2+](i)) response in rat anterior pituitary cells. A period as short as 1 min with 10(-7) M ANG II was effective in producing desensitization (remaining response was 66.8 +/- 2.1% of nondesensitized cells). Desensitization was a concentration-related event (EC(50): 1.1 nM). Although partial recovery was obtained 15 min after removal of ANG II, full response could not be achieved even after 4 h (77.6 +/- 2.4%). Experiments with 5 x 10(-7) M ionomycin indicated that intracellular Ca2+ stores of desensitized cells had already recovered when desensitization was still significant. The thyrotropin-releasing hormone (TRH)-induced intracellular Ca2+ peak was attenuated in the ANG II-pretreated group. ANG II pretreatment also desensitized ANG II- and TRH-induced inositol phosphate generation (72.8 +/- 3.5 and 69.6 +/- 6.1%, respectively, for inositol triphosphate) and prolactin secretion (53.4 +/- 2.3 and 65.1 +/- 7.2%), effects independent of PKC activation. We conclude that, in pituitary cells, inositol triphosphate formation, [Ca2+](i) mobilization, and prolactin release in response to ANG II undergo rapid, long-lasting, homologous and heterologous desensitization.     

Participation of protein kinase C in the activation of human neutrophils by phorbol ester

The calmodulin antagonist N(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) has been examined as an inhibitor of superoxide anion production and granule exocytosis in phorbol ester (PMA)-activated neutrophils. Inhibition of the respiratory burst was observed at a concentration of W-7 identical to that required for inhibition of native protein kinase C (PKC), whereas the concentration required to inhibit the secretory response was found to correspond to that required for inhibition of the proteolytically converted fully active PKC. The IC50 of W-7 was in both cases 5 and 12 fold higher than that required for inhibition of calmodulin dependent kinases. The results confirm the essential role for the membrane-bound PKC in the production of O2- radicals and provide a clear evidence of the direct participation of the proteolytically activated cytosolic PKC to the secretory response of PMA activated neutrophils.     

Long-term phorbol ester treatment down-regulates protein kinase C and sensitizes the phosphoinositide signaling pathway to hormone and growth factor stimulation. Evidence for a role of protein kinase C in agonist-induced desensitization

Exposure of a nontransformed, continuous line of epithelial cells derived from rat liver (WB cells) to epidermal growth factor, angiotensin II, [Arg8]vasopressin, and epinephrine resulted in rapid accumulation of the inositol phosphates (InsP) InsP1, InsP2, and InsP3. Although short-term (5-60 min) pretreatment of WB cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly attenuated InsP accumulation in response to all agonists, the inhibitory effects on the InsP response were lost after 2 h incubation with PMA; and, with extended (6-24 h) preincubation, a time-dependent potentiation of the InsP response to angiotensin II, epidermal growth factor and [Arg8]vasopressin was observed. The InsP response during a 15-min challenge with angiotensin II in cells pretreated for 18 h with 600 nM and 10 microM PMA was increased by 2-3-fold and 4-6-fold, respectively. Long-term (18 h) treatment with 600 nM and 10 microM PMA caused a similar 90-100% loss of measurable Ca2+/phospholipid-dependent enzyme (protein kinase C) activity in cytosolic and soluble particulate fractions. The effects of long-term PMA pretreatment do not represent a general enhancement of hormone responsiveness since the InsP response to epinephrine was not affected. In control cells, the InsP response to angiotensin II and epinephrine desensitized very rapidly. Long-term pretreatment with PMA greatly reduced the contribution of agonist-induced desensitization to the angiotensin II response; in contrast, the extent of desensitization occurring during incubation of WB cells with epinephrine was unaltered by long-term treatment with PMA suggesting that an additional mechanism may be involved in alpha 1-adrenergic receptor desensitization. No PMA-induced change in resting levels of [3H]phosphoinositides or the metabolism of exogenous [3H]inositol 1,4,5-trisphosphate by WB homogenates occurred. Stimulation of InsP formation in intact cells by NaF and activation of phospholipase C by GTP gamma S in membranes both were unaltered by short-term or long-term PMA pretreatment. These data are consistent with the idea that following long-term treatment of WB cells with PMA, the occurrence of agonist-induced desensitization of receptors linked to the phosphoinositide/Ca2+ signaling system is reduced, apparently at least in part due to the loss of contribution of a negative feedback regulatory role of protein kinase C.     

Protein kinase C mediates the effect of vasopressin in pituitary corticotrophs

The role of protein kinase C (PKC) on vasopressin (VP) action was investigated by inhibition of endogenous PKC using prolonged incubation of the cells with phorbol ester, and by direct measurement of PKC activity in pituitary cells. Preincubation of the cells for 6 h with 100 nM TPA at 37 C resulted in a 90% decrease in total PKC activity. In the PKC-depleted cells, cAMP responses to stimulation with 100 nM CRF for 30 min were normal, but the potentiating effects of VP and PMA on CRF-stimulated cAMP production were abolished. The stimulation of ACTH secretion by VP and PMA alone was also abolished in PKC- depleted cells. PKC activity in cytosolic and detergent-solubilized membrane fractions from enriched pituitary corticotrophs obtained by centrifugal elutriation, was directly measured by enzymatic assays and by immunoblotting techniques. Basal PKC activity was higher in the cytosol than in the membranes (8.43 +/- 0.47 and 1.93 +/- 0.11 pmol 32P incorporated/10 min, respectively). After incubation of the cells with VP for 15 min or [3H] phorbol-12-myristate-13-acetate (PMA) for 30 min, PKC activity in cytosol was decreased by 40% and 89%, respectively, while the activity in the membrane was increased by 138% and 405%, respectively. Such VP- and PMA-induced translocation of PKC was also observed when the enzyme content in the cytosol and the membranes was measured by immunoblotting using a specific anti-PKC antibody and [125I]protein A. Autoradiographic analysis of immunoblots revealed an 80 kilodalton band characteristic of PKC, with OD higher in the cytosolic than in the membrane fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential involvement of protein kinase C in basal versus acetylcholine-regulated prolactin secretion in rat anterior pituitary cells during aging

Although it is well known that plasma concentration of prolactin (PRL) increases during aging in rats, how the anterior pituitary (AP) aging per se affects PRL secretion remains obscure. The objectives of this study were to determine if changes in the pituitary PRL responsiveness to acetylcholine (ACh; a paracrine factor in the AP), as compared with that to other PRL stimulators or inhibitors, contribute to the known age-related increase in PRL secretion, and if protein kinase C (PKC) is involved. We also determined if replenishment with aging-declined hormones such as estrogen/thyroid hormone influences the aging-caused effects on pituitary PRL responses. AP cells were prepared from old (23-24-month-old) as well as young (2-3-month-old) ovariectomized rats. Cells were pretreated for 5 days with diluent or 17beta-estradiol (E(2); 0.6 nM) in combination with or without triiodothyronine (T(3); 10 nM). Then, cells were incubated for 20 min with thyrotropin-releasing hormone (TRH; 100 nM), angiotensin II (AII; 0.2-20 nM), vasoactive intestinal peptide (VIP; 10(-9)-10(-5) M), dopamine (DA; 10(-9)-10(-5) M), or ACh (10(-7)-10(-3) M). Cells were also challenged with ACh, TRH, or phorbol 12-myristate 13-acetate (PMA; 10(-6) M) following PKC depletion by prolonged PMA (10(-6) M for 24 h) pretreatment. We found that estrogen priming of AP cells could reverse the aging-caused effects on pituitary PRL responses to AII and DA. In hormone-replenished cells aging enhanced the stimulation of PRL secretion by TRH and PMA, but not by AII and VIP. Aging also reduced the responsiveness of cells to ACh and DA in suppressing basal PRL secretion, and attenuated ACh inhibition of TRH-induced PRL secretion. Furthermore, ACh suppressed TRH-induced PRL secretion mainly via the PMA-sensitive PKC in the old AP cells, but via additional mechanisms in young AP cells. On the contrary, basal PRL secretion was PKC (PMA-sensitive)-independent in the old AP cells, but dependent in the young AP cells. Taken together, these results suggest differential roles of PMA-sensitive PKC in regulating basal and ACh-regulated PRL responses in old versus young AP cells. The persistent aging-induced differences in AP cell responsiveness to ACh, DA, TRH, and PMA following hormone (E(2)/T(3)) replenishment suggest an intrinsic pituitary change that may contribute, in part, to the elevated in vivo PRL secretion observed in aged rats.     

Inhibition of parasite protein kinase C by new antileishmanial imidazolidin-2-one compounds

The protein kinase C (PKC) family of isoenzymes mediate a wide range of signal transduction pathways in many different cells lines. Little is known regarding the presence and functional roles of PKC in Leishmania spp. Here we report the inhibition of parasite PKC by new imidazolidinone compounds. The most active derivative 7 showed an important activity (IC50 = 9.9 microM) against the clinical relevant stage of parasites in comparison with Glucantime (IC50 = 464.5 microM), without inducing toxicity on human fibroblast cells (IC50 = 102 microM). Pretreatment of intact parasites with 10 microM of compound 7 inhibited 80% of PKC activity. At the same concentration, this compound inhibited 70% of the parasite-host cell invasion process. An in vivo model showed that compound 7 reduced the liver parasite burden by 25% and spleen parasite burden by 44%. These results provide the first evidence that PKC plays a critical role in the invasion process. Thus Leishmania PKC activity could be a relevant therapeutic target and the imidazolidinones novel antileishmanial candidates.     

WS-1 human fibroblasts contain distinct calcium and protein kinase C-mediated pathways for activation of Na+/H+ exchange: contrasting effects of thrombin and PMA

PMA and thrombin were examined for their ability to activate Na+/H+ exchange in growth-arrested WS-1 human fibroblasts. PMA or thrombin caused a cytoplasmic alkalinization that required extracellular sodium and was sensitive to 1 mM amiloride, suggesting that the rise in pH was mediated by the Na+/H+ exchanger. However, PMA and thrombin activated Na+/H+ exchange by distinctly different mechanisms. The rate of cytoplasmic alkalinization caused by 30 nM PMA was slower than 10 nM thrombin. The PMA-induced pH change was sensitive to the protein kinase inhibitors staurosporine (50 nM) and H-7 (100 microM). No increase in intracellular calcium was observed after PMA treatment and the cytoplasmic alkalinization caused by PMA was not sensitive to the drug TMB8 (200 microM) or the intracellular calcium-chelator BAPTA. In contrast, the thrombin-induced rise in cytoplasmic pH was insensitive to 50 nM staurosporine and only partially reduced with 100 microM H-7. The thrombin-induced activation of Na+/H+ exchange was inhibited by 200 microM TMB8 or pretreatment with BAPTA. PMA caused translocation of PKC activity from a cytoplasmic to membrane fraction whereas thrombin did not. Pretreatment with 50 nM staurosporine significantly reduced measurable PKC activity with or without PMA treatment. PMA and thrombin were also examined for their ability to induce DNA synthesis in growth-arrested WS-1 human fibroblasts. Unlike thrombin, PMA did not stimulate [3H]-thymidine incorporation in cells serum-deprived for 48 hours. In addition, PMA inhibited thrombin-induced DNA synthesis when added at the same time or as late as 10 hours after thrombin addition. Therefore, thrombin and PMA activate Na+/H+ exchange by distinct pathways, but only the thrombin-induced pathway correlates with a mitogenic response.     

S100B(betabeta) inhibits the protein kinase C-dependent phosphorylation of a peptide derived from p53 in a Ca2+-dependent manner.

S100B(betabeta) is a dimeric Ca2+-binding protein that is known to inhibit the protein kinase C (PKC)-dependent phosphorylation of several proteins. To further characterize this inhibition, we synthesized peptides based on the PKC phosphorylation domains of p53 (residues 367-388), neuromodulin (residues 37-53), and the regulatory domain of PKC (residues 19-31), and tested them as substrates for PKC. All three peptides were shown to be good substrates for the catalytic domain of PKC. As for full-length p53 (Baudier J, Delphin C, Grunwald D, Khochbin S, Lawrence JJ. 1992. Proc Natl Acad Sci USA 89:11627-11631), S100B(betabeta) binds the p53 peptide and inhibits its PKC-dependent phosphorylation (IC50 = 10 +/- 7 microM) in a Ca2+-dependent manner. Similarly, phosphorylation of the neuromodulin peptide and the PKC regulatory domain peptide were inhibited by S100B(betabeta) in the presence of Ca2+ (IC50 = 17 +/- 5 microM; IC50 = 1 +/- 0.5 microM, respectively). At a minimum, the C-terminal EF-hand Ca2+-binding domain (residues 61-72) of each S100beta subunit must be saturated to inhibit phosphorylation of the p53 peptide as determined by comparing the Ca2+ dependence of inhibition ([Ca]IC50 = 29.3 +/- 17.6 microM) to the dissociation of Ca2+ from the C-terminal EF-hand Ca2+-binding domain of S100B(betabeta).     

Distinct mechanisms of regulation of protein kinase C epsilon by hormones and phorbol diesters

In this study, we examined the effects of T cell activators on the regulation of protein kinase C (PKC) isozymes present in thymocytes. Using affinity-purified anti-PKC antisera, we determined that the major PKC isoforms in murine thymocytes are PKC beta and PKC epsilon. The CD4+/CD8+ thymocyte subset expressed high levels of both PKC beta and PKC epsilon, whereas the CD4-/CD8- subset expressed much less of both. PKC beta was down-regulated following treatment of thymocytes with phorbol 12-myristate acetate (PMA) (2 x 10(-8) M) or ionomycin (0.4 microM). In contrast, PMA did not induce the down-regulation of PKC epsilon. Ionomycin alone, however, induced PKC epsilon down-regulation, similar to its effect on PKC beta. Similar observations were made on a promonocytic cell line, U937, which expresses PKC alpha, PKC beta (Strulovici, B., Daniel-Issakani, S., Oto, E., Nestor, J., Jr., Chan, H., and Tsou, A.-P. (1989) Biochemistry 28, 3569-3576), and PKC epsilon. To facilitate the study of PKC beta and PKC epsilon, we established a Chinese hamster ovary cell line which expresses murine PKC epsilon in addition to endogenous PKC alpha and PKC beta. Both PKC isoforms (beta and epsilon) were mostly in particulate form. PMA treatment left the majority of immunoreactive PKC epsilon intact. By contrast, thrombin treatment caused the disappearance of particulate and cytosolic PKC epsilon (60% by 10 min and 80% by 1 h). PMA and thrombin promoted the down-regulation of PKC beta with similar kinetics (100% down-regulation by 3 h). These results indicate that: 1) thymocytes express PKC epsilon; and 2) this isozyme exhibits a novel form of regulation distinct from the other PKC isozymes.     

A calmodulin antagonist inhibits histamine-stimulated acid production by isolated rat parietal cells

The role of calmodulin in the regulation of histamine-stimulated parietal cell function was studied in isolated rat parietal cells using [14C]aminopyrine uptake as a quantitative index of acid production. In enriched (77-87%) intact parietal cells the calmodulin antagonist naphthalene sulfonamide W 7 dose-dependently inhibited the response to 10(-4) M histamine (IC50: 2 X 10(-6) M). The mechanism of this inhibition was examined further with two other stimuli of H+-production: forskolin which directly activates the parietal cell adenylate cyclase without interacting at the histamine H2-receptor and dbcAMP which mimics the biological action of cAMP without preceding activation of adenylate cyclase. W 7 effectively inhibited the responses to 10(-4) M forskolin (IC50: 6 X 10(-7) M), 10(-3) M dbcAMP (IC50: 10(-6) M) and to 10(-2) M K+ (IC50: 3 X 10(-6) M). The action of W 7 followed non-competitive kinetics since the antagonist reduced the entire range of the concentration-response curves without shifting them rightwards towards higher concentrations of the respective stimulants. The effect of W 7 was reversed by washing the cells. ATP-induced [14C]aminopyrine uptake into digitonin-permeabilized oligomycin-inhibited parietal cells reflects H+-production independent of oxidative phosphorylation and was also inhibited by W 7 (IC50: 10(-5) M). Inhibition of K+-stimulated H+/K+-ATPase activity required even higher W 7-concentrations (IC50: 1.4 X 10(-4) M). Our data suggest that calmodulin might be involved in the intracellular mediation of the response to histamine. Between histamine-induced cAMP-generation and the H+-secreting tubulovesicular system W 7 seems to inhibit an intracellular step that finally activates the H+/K+-ATPase. Yet, direct inhibition of the ATPase requires W 7 concentrations of questionable specificity and is unlikely to be the mechanism behind the action of W 7 on the parietal cell response to histamine.