Comparison of lipolytic and antilipolytic activities of lower vertebrate growth hormones on chicken adipose tissue in vitro

Mammalian and avian growth hormones (GH) (pituitary derived or biosynthetic) exert two effects on chicken adipose tissue explants in vitro. They (i) increase the basal rate of glycerol release a lipolytic effect) and (ii) inhibit glucagon-stimulated glycerol release (an antilipolytic effect). The ability of lower vertebrate GH preparations to exert lipolytic and antilipolytic effects was examined and biological activity was compared to differences in amino-acid residue sequences and to predicted structure. Irrespective of species origin (blue shark, sturgeon, bonito, yellow tail, salmon, bullfrog, sea turtle), all lower vertebrate GH preparations showed very weak (less than 5% the potency of bovine GH), if any, lipolytic activity, but retained strong antilipolytic activity. The present data indicate that the structural requirements for lipolytic and antilipolytic activities of GH differ in chicken adipose tissue. Despite the high sequence homology (88%) between chicken and sea turtle GH, the latter preparation did not stimulate lipolysis. It is suggested that Pro132, conserved only in lipolytically active GH species (human, bovine, and chicken), represents a major determinant of lipolytic activity in chicken adipose tissue. The structural determinants for antilipolytic activity may comprise any or all of residues 3, 17, 64, 108, 109, and 152.     

Signaling pathways in ascidian oocyte maturation: the roles of cAMP/Epac, intracellular calcium levels, and calmodulin kinase in regulating GVBD

Most mature ascidian oocytes undergo germinal vesicle breakdown (GVBD) when released by the ovary into sea water (SW). Acidic SW blocks this but they can be stimulated by raising the pH, increasing intracellular cAMP levels by cell permeant forms, inhibiting its breakdown or causing synthesis. Boltenia villosa oocytes undergo GVBD in response to these drugs. However, the cAMP receptor protein kinase A (PKA) does not appear to be involved, as oocytes are not affected by the kinase inhibitor H-89. Also, the PKA independent Epac agonist 8CPT-2Me-cAMP stimulates GVBD in acidic SW. GVBD is inhibited in calcium free sea water (CaFSW). The intracellular calcium chelator BAPTA-AM blocks GVBD at 10?μM. GVBD is also inhibited when the ryanodine receptors (RYR) are blocked by tetracaine or ruthenium red but not by the IP(3) inhibitor D-609. However, dimethylbenzanthracene (DMBA), a protein kinase activator, stimulates GVBD in BAPTA, tetracaine or ruthenium red blocked oocytes. The calmodulin kinase inhibitor KN-93 blocks GVBD at 10?μM. This and preceding papers support the hypothesis that the maturation inducing substance (MIS) produced by the follicle cells in response to increased pH causes activation of a G protein which triggers cAMP synthesis. The cAMP then activates an Epac molecule, which causes an increase in intracellular calcium from the endoplasmic reticulum ryanodine receptor. The increased intracellular calcium subsequently activates calmodulin kinase, which causes an increase in cdc25 phosphatase activity, activating MPF and the progression of the oocyte into meiosis.     

Maitotoxin stimulates Cd influx in Madin-Darby kidney cells by activating Ca-permeable cation channels

This study examined the role of calcium channels for the uptake of cadmium (Cd) into Madin-Darby canine kidney (MDCK) cells. Maitotoxin, an activator of different types of calcium channels, increased accumulation of 109Cd and 45Ca in MDCK cells. We found that maitotoxin increased accumulation by stimulating 109Cd influx because it did not affect efflux. An inhibitor of store-operated Ca channels, SKF96365, partially blocked 45Ca influx but did not affect 109Cd influx. Ni and Mn, and loperamide and proadifen (SKF 525a), inhibited 45Ca and 109Cd influx in cells stimulated with maitotoxin, but La and nifedipine did not. Overnight treatment with phorbol 12, 13-ibutyrate (PDBu) to activate protein kinase C resulted in a decrease in the concentration of maitotoxin needed to stimulate 45Ca and 109Cd influx. The effect of PDBu was blocked by treating cells with the protein kinase C inhibitor GF109203X. Additionally, the effect of PDBu was lost in cells treated with an inhibitor of RNA synthesis actinomycin D. These results suggest that a Ca permeable cation channel different from voltage-dependent and store-operated Ca channels mediates the uptake of Cd in MDCK cells. The expression of this channel is regulated by protein kinase C.     

Signal transduction pathways in the induction of HLA class I antigen expression on Huh 6 cells by interferon-gamma.

This study investigated the intracellular signal transduction regulating the appearance of HLA class I antigens on Huh 6 cells induced by interferon-gamma. The expression was blocked by a protein kinase C inhibitor, H-7, but not by a calmodulin antagonist, W-7, nor by a protein kinase A inhibitor, H-8, at low dose. The antigen expression was induced by a direct activator of protein kinase C, phorbol myristate acetate, but not by calcium ionophore A23187 nor an analog of cAMP, dbcAMP. Therefore, we concluded that protein kinase C is involved in the expression of HLA class I antigens on Huh 6 cells induced by interferon-gamma but Ca(2+)-calmodulin and cAMP are not.     

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.     

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.     

Phorbol myristate acetate-induced down-modulation of CD4 is dependent on calmodulin and intracellular calcium

PMA causes rapid down-modulation of CD4 molecules on murine immature thymocytes, human PBL, and CD4-positive human tumor cell lines, but not on murine peripheral lymphocytes. The mechanisms of phorbol ester-induced down modulation of CD4 molecules, however, have not been elucidated. To determine how PMA down-modulates CD4 expression by T lymphocytes, we studied the ability of inhibitors of protein kinase C, calmodulin, actin, and tubulin to block PMA-induced modulation of CD4 in several murine and human cell types. We also tested the ability of intracellular and extracellular calcium chelators to block CD4 internalization. There was marked variability in the degree of PMA-induced down-modulation of CD4 among various cell types. The effects of PMA on CD4 expression were greater for murine thymocytes, for human PBL, and for the human lymphoblastic leukemia cell line, MOLT-3, than for any of the other cell types studied. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, blocked phosphorylation but not internalization of CD4 molecules induced by PMA. Therefore, phosphorylation of CD4 molecules by protein kinase C is not required for the internalization of the molecules. Internalization was blocked by both inhibitors of calmodulin, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide, and trifluoperazine. PMA-induced internalization of CD4 was blocked by Quin-2 AM, which chelates intracellular calcium. EGTA, which chelates extracellular calcium, did not block internalization. Inhibitors of actin or tubulin did not block internalization. These results suggest that PMA-induced modulation of CD4 can occur in the absence of phosphorylation of the CD4 molecules and is calmodulin and intracellular calcium dependent.     

Protein phosphorylation in rat cardiac microsomes: Effects of inhibitors of protein kinase A and of phosphatases

The phosphorylation of rat cardiac microsomal proteins was investigated with special attention to the effects of okadaic acid (an inhibitor of protein phosphatases), inhibitor 2 of protein phosphatase 1 and inhibitor of cyclic AMP-dependent protein kinase (protein kinase A). The results showed that okadaic acid (5 µM) modestly but reproducibly augmented the protein kinase A-catalyzed phospholamban (PLN) phosphorylation, although exerted little effect on the calcium/calmodulin kinase-catalyzed PLN phosphorylation. Microsomes contained three other substrates (M_r 23, 19 and 17 kDa) that were phosphorylated by protein kinase A but not by calcium/calmodulin kinase. The protein kinase A-catalyzed phosphorylation of these three substrates was markedly (2-3 fold) increased by 5 µM okadaic acid. Calmodulin was found to antagonize the action of okadaic acid on such phosphorylation. Protein kinase A inhibitor was found to decrease the protein kinase A-catalyzed phosphorylation of microsomal polyp eptides. Unexpectedly, inhibitor 2 was also found to markedly decrease protein kinase A-catalyzed phosphorylation of phospholamban as well these other microsomal substrates. These results are consistent with the views that protein phosphatase 1 is capable of dephosphorylating membrane-associated phospholamban when it is phosphorylated by protein kinase A, but not by calcium/calmodulin kinase, and that under certain conditions, calcium/calmodulin-stimulated protein phosphatase (protein phosphatase 2B) is also able to dephosphorylate PLN phosphorylated by protein kinase A. Additionally, the observations show that protein phosphatase 1 is extremely active against the three protein kinase A substrates (M_r 23, 19 and 17 kDa) that were present in the isolated microsomes and whose state of phosphorylation was particularly affected in the presence of dimethylsulfoxide. Protein phosphatase 2B is also capable of dephosphorylating these three substrates. (Mol Cell Biochem 175: 109–115, 1997     

17alpha-estradiol-induced VEGF-A expression in rat pituitary tumor cells is mediated through ER independent but PI3K-Akt dependent signaling pathway

17alpha-E(2), a weak estrogen exhibited both agonistic and antagonistic effects, and caused a time- and dose-dependent induction of VEGF-A mRNA expression in GH3 rat pituitary tumor cells. This effect was unaffected by the presence of the pure estrogen receptor antagonist ICI 182,780 but was specifically blocked by a protein synthesis inhibitor puromycin. Inhibition of phosphatidylinositol-3 kinase (PI3K) activity by wortmannin decreased the effect of 17alpha-E(2) on VEGF-A mRNA expression. This inhibitor also blocked the increase in phosphorylation of Akt induced by exposure to 17alpha-E(2). In contrast, exposure to the MAP kinase inhibitor, U0126, had no impact on 17alpha-E(2)-induced VEGF-A mRNA expression. Taken together, these studies indicate that like potent estrogens 17alpha-E(2) up-regulates VEGF-A mRNA expression in estrogen responsive GH3 rat pituitary tumor cells, but this induction is not mediated through a classical estrogen receptor pathway. PI3K-Akt signaling pathway is required for the induction of VEGF-A mRNA in GH3 cells by 17alpha-E(2).     

Thrombin signal transduction mechanisms in human glomerular epithelial cells.

We have previously shown that alpha-thrombin exerted a mitogenic effect on human glomerular epithelial cells and stimulated the synthesis of urokinase-type (u-PA) and tissue-type plasminogen activator (t-PA) and of their inhibitor, plasminogen activator inhibitor 1 (PAI-1). In the present study, we investigate the signal transduction mechanisms of thrombin in these cultured cells. Thrombin induced an increase in intracellular free calcium concentrations ([Ca2+]i) in a dose-dependent manner, a plateau being reached at 1 U/ml thrombin. A 60% inhibition of this effect was produced by 300 nM nicardipine, a dihydroperidine agent, or by 4 mM EGTA, indicating that increase in [Ca2+]i was due in part to extracellular Ca2+ entry through L-type voltage-sensitive calcium channels. Thrombin also induced an increase in inositol trisphosphate (IP3), suggesting that phospholipase C activation and phosphatidylinositides breakdown were stimulated. Interestingly thrombin-stimulated cell proliferation measured by 3H thymidine incorporation was inhibited by 300 nM nicardipine, and restored by addition of 10(-8) M ionomycin, indicating that calcium entry was critical for the mitogenic signal of thrombin. Conversely, nicardipine did not modify thrombin-stimulated synthesis of u-PA, t-PA, and PAI-1. Both thrombin-stimulated cell proliferation and protein synthesis required protein kinase C activation since these effects were blocked by 10 microM H7, an inhibitor of protein kinases, and by desensitization of protein kinase C by phorbol ester pretreatment of the cells. Interestingly, DFP-inactivated thrombin which binds the thrombin receptor and gamma-thrombin, which has some enzymatic activity but does not bind to thrombin receptor, had no effect when used alone. Simultaneous addition of these two thrombin derivatives had no effect on [Ca2+]i, and 3H thymidine incorporation but stimulated u-PA, t-PA, and PAI-1 synthesis although to a lesser extent than alpha-thrombin. This effect also required protein kinase C activation to occur, presumably by a pathway distinct from phosphoinositoside turnover since it was not associated with IP3 generation. In conclusion, multiple signalling pathways can be activated by alpha-thrombin in glomerular epithelial cells: 1) Ca2+ influx through a dihydroperidine-sensitive calcium channel, which seems critical for mitogenesis; 2) protein kinase C activation by phosphoinositide breakdown, which stimulates both mitogenesis and synthesis of u-PA, t-PA, and PAI-1; 3) protein kinase C activation by other phospholipid breakdown can stimulate u-PA, t-PA, and PAI-1 synthesis but not mitogenesis.     

Mitochondrial,but not peroxisomal, β-oxidation of fatty acids is conserved in coenzyme A-Deficient rat liver

Growth hormone (GH) exerts acute insulin-like effects, such as increased lipogenesis and inhibition of catecholamine-induced lipolysis, in rat adipocytes that have not been exposed to GH during the preceding three hours. We found that OPC3911, a highly specific inhibitor of the cGMP-inhibited cAMP phosphodiesterase, completely blocked the antilipolytic but not the lipogenic effect of GH. This indicates that the antilipolytic effect of GH is mediated through activation of this phosphodiesterase leading to reduction of cAMP levels in the same manner as has been shown for insulin.     

Searching for mechanisms of N-methyl-D-aspartate-induced glutathione efflux in organotypic hippocampal cultures

N-Methyl-d-aspartate (NMDA)-receptor stimulation evoked a selective and partly delayed elevated efflux of glutathione, phosphoethanolamine, and taurine from organotypic rat hippocampus slice cultures. The protein kinase inhibitors H9 and staurosporine had no effect on the efflux. The phospholipase A₂ inhibitors quinacrine and 4-bromophenacyl bromide, as well as arachidonic acid, a product of phospholipase A₂ activity, did not affect the stimulated efflux. Polymyxin B, an antimicrobal agent that inhibits protein kinase C, and quinacrine in high concentration (500 µM), blocked efflux completely. The stimulated efflux after but not during NMDA incubation was attenuated by a calmodulin antagonist (W7) and an anion transport inhibitor (DNDS). Omission of calcium increased the spontaneous efflux with no or small additional effects by NMDA. In conclusion, NMDA receptor stimulation cause an increased selective efflux of glutathione, phosphoethanolamine and taurine in organotypic cultures of rat hippocampus. The efflux may partly be regulated by calmodulin and DNDS sensitive channels.     

Growth hormone-induced alteration in ErbB-2 phosphorylation status in 3T3-F442A fibroblasts

The growth hormone receptor (GHR), a cytokine receptor superfamily member, requires the JAK2 tyrosine kinase for signaling. We now examine functional interactions between growth hormone (GH) and epidermal growth factor (EGF) in 3T3-F442A fibroblasts. Although EGF enhanced ErbB-2 tyrosine phosphorylation, GH, while causing retardation of its migration on SDS-polyacrylamide gel electrophoresis, decreased ErbB-2's tyrosine phosphorylation. GH-induced retardation was reversed by treatment of anti-ErbB-2 precipitates with both alkaline phosphatase and protein phosphatase 2A, suggesting that GH induced serine/threonine phosphorylation of ErbB-2. Both GH-induced shift in ErbB-2 migration and GH-induced MAP kinase activation were unaffected by a protein kinase C inhibitor but were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1) inhibitor, PD98059. Notably, leukemia inhibitory factor, but not interferon-gamma, also promoted ErbB-2 shift and mitogen-activated protein kinase activation. Cotreatment with EGF and GH versus EGF alone resulted in a 35% decline in acute ErbB-2 tyrosine 1248 autophosphorylation, a marked decline (approximately 50%) in DNA synthesis, and substantially decreased cyclin D1 expression. We conclude that in 3T3-F442A cells, 1) the GH-induced decrease in ErbB-2 tyrosine phosphorylation correlates with MEK1/mitogen-activated protein kinase activity and 2) GH antagonizes EGF-induced DNA synthesis and cyclin D1 expression in a pattern consistent with its alteration in ErbB-2 phosphorylation status.     

Evidence for a role of calmodulin in the regulation of prolactin gene expression

Epidermal growth factor (EGF) stimulates prolactin (PRL) gene expression in GH3 cells in a Ca2+-dependent manner (White, B. A., and Bancroft, F. C. (1983) J. Biol. Chem. 258, 4618-4622). The present report shows that the phenothiazine, calmidazolium (compound R 24571), blocks the ability of EGF plus Ca2+ to increase levels of PRL mRNA. Calmidazolium inhibition of this response is dose dependent in the range of 0.05-1.00 microM. Total inhibition of the response was consistently obtained at a level of calmidazolium (0.5 microM) that had no effect on total cytoplasmic RNA synthesis, total cytoplasmic protein synthesis, cell viability, or extent of EGF plus Ca2+-induced cell aggregation. The drug inhibited the increase in PRL mRNA when given immediately before or 48 h after treatment with EGF plus Ca2+. Another calmodulin inhibitor, W13, similarly blocked the ability of EGF plus Ca2+ to stimulate PRL mRNA, whereas the less active analog, W12, had little effect. These results implicate Ca2+-binding proteins such as calmodulin in the mechanism of action of EGF in GH3 cells, and, therefore, provide further evidence for a role of intracellular Ca2+ in the regulation of the expression of a specific eukaryotic gene, the PRL gene.     

A novel protein kinase from Brassica juncea stimulated by a protozoan calcium binding protein. Purification and partial characterization.

A novel protein kinase (BjCCaBPk) from etiolated Brassica juncea seedlings has been purified and partially characterized. The purified enzyme migrated on SDS/PAGE as a single band with an apparent molecular mass of 43 kDa. The optimum pH for the kinase activity was 8.0. It was stimulated more than sixfold by the protozoa Entamoeba histolytica calcium binding protein EhCaBP (10.5 nM) but not by calmodulin (CaM) when used at equimolar concentration. Moreover the kinase also did not bind CaM-Sepharose. There was neither inhibition of the kinase activity in the presence of W-7 (a CaM antagonist), KN-62 (a specific calcium/CaM kinase inhibitor) and anti-CaM Ig, nor any effect on BjCCaBPk activity of staurosporine (a protein kinase C inhibitor). Furthermore a CaM-kinase specific substrate, syntide-2, proved to be a poor substrate for the BjCCaBPk compared with histone III-S. The phosphorylation of histone III-S involved serine residues. Southern and Northern blot analysis showed the presence of EhCaBP homologues in Brassica. The data suggest that BjCCaBPk may be a novel protein kinase with an affinity towards a calcium binding protein like EhCaBP.     

Involvement of proteolytic enzymes in the lipotropic effect of the pituitary polypeptide hormones

The influence of proteinase inhibitors on the lipolytic effect of the pituitary polypeptide hormones and epinephrine in an isolated adipose tissue of rabbits and rats has been studied. Neither of proteinase inhibitors changed the basal rate of lipolysis. Trasylol, a serine proteinase inhibitor, suppressed completely growth hormone (GH) effect and partially reduced the effect of adrenocorticotropin (ACTH) and beta-lipotropin (beta-LPH) but did not change the effect of epinephrine. Bacitracin proved ineffective with regard to the effect of polypeptide hormones. Pepstatin, an acid proteinase inhibitor, partially blocked the stimulation of lipolysis by ACTH without affecting the effect of GH and beta-LPH. The influence of proteinase inhibitors on the ACTH effect in rat adipose tissue was similar to that found in rabbit tissue. The Trasylol-induced inhibition of the hormone-stimulated lipolysis decreased to a considerable extent after GH or ACTH incubation with rabbit plasma or partial GH digestion with pepsin. This decrease was not observed when plasma serine proteinases were blocked during GH incubation with plasma. The results demonstrate an involvement of some proteolytic enzymes in the realization of the polypeptide hormone lipolytic effect and permit to suppose the requirement of preliminary activation of the hormones by means of proteolytic modification.     

Berberine attenuates cAMP-induced lipolysis via reducing the inhibition of phosphodiesterase in 3T3-L1 adipocytes

Berberine, a hypoglycemic agent, has been shown to decrease plasma free fatty acids (FFAs) level in insulin-resistant rats. In the present study, we explored the mechanism responsible for the antilipolytic effect of berberine in 3T3-L1 adipocytes. It was shown that berberine attenuated lipolysis induced by catecholamines, cAMP-raising agents, and a hydrolyzable cAMP analog, but not by tumor necrosis factor α and a nonhydrolyzable cAMP analog. Unlike insulin, the inhibitory effect of berberine on lipolysis in response to isoproterenol was not abrogated by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but additive to that of PD98059, an extracellular signal-regulated kinase kinase inhibitor. Prior exposure of adipocytes to berberine decreased the intracellular cAMP production induced by isoproterenol, forskolin, and 3-isobutyl-1-methylxanthine (IBMX), along with hormone-sensitive lipase (HSL) Ser-563 and Ser-660 dephosphorylation, but had no effect on perilipin phosphorylation. Berberine stimulated HSL Ser-565 as well as adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. However, compound C, an AMPK inhibitor, did not reverse the regulatory effect of berberine on HSL Ser-563, Ser-660, and Ser-565 phosphorylation, nor the antilipolytic effect of berberine. Knockdown of AMPK using RNA interference also failed to restore berberine-suppressed lipolysis. cAMP-raising agents increased AMPK activity, which was not additive to that of berberine. Stimulation of adipocytes with berberine increased phosphodiesterase (PDE) 3B and PDE4 activity measured by hydrolysis of ³[H]cAMP. These results suggest that berberine exerts an antilipolytic effect mainly by reducing the inhibition of PDE, leading to a decrease in cAMP and HSL phosphorylation independent of AMPK pathway.     

K+-Dependent Stimulation of Dopamine Synthesis in Striatal Synaptosomes Is Mediated by Protein Kinase C

Dopamine synthesis rate was measured in striatal synaptosomes. Removal of Na⁺ increased synthesis rate; this was blocked in Ca²⁺-free medium and by addition of the Ca²⁺/calmodulin inhibitor N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide (W7). The increase in dopamine synthesis rate caused by the addition of the phorbol ester 12-O-tetradecanoylphorboI-13-acetate (TPA) was blocked by the protein kinase C inhibitor polymyxin B. K⁺-stimulated synthesis was unchanged in Ca²⁺-free medium or by addition of W7; it was blocked by polymyxin B. The effect of 50 mM K⁺ was additive with that of 8-Br cyclic AMP and of Na⁺ removal; the combined effect of 50 mM K⁺ and TPA was no greater than that of either alone. These results suggest that stimulation of dopamine synthesis in striatal synaptosomes by 50 mM K⁺ is mediated by protein kinase C.