The protein kinase C inhibitors H-7 and H-9 fail to inhibit human neutrophil activation

The protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) were examined for their ability to inhibit human neutrophil activation. At concentrations up to 100 micromolar, these compounds failed to inhibit either respiratory burst or the secretory response of neutrophils stimulated with particulate (serum-opsonized zymosan) or soluble (A23187, FMLP, PMA) stimuli. In contrast, the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) inhibited both oxygen radical generation and lysosomal enzyme release in response to the same stimuli. These results suggest that calmodulin-dependent enzymes, rather than protein kinase C, may be essential for neutrophil activation.     

Homologous Desensitization of Serotonin 5-HT2 Receptor-Stimulated Intracellular Calcium Mobilization in C6BU-1 Glioma Cells via a Mechanism Involving a Calmodulin Pathway

Abstract: Serotonin 5-HT₂ receptor-mediated intracellular Ca²⁺ mobilization was investigated in rat glioma C6BU-1 cells. The receptors became desensitized after previous exposure to 5-HT in a time-and concentration-dependent manner. The desensitization of 5-HT₂ receptor-mediated intracellular signaling appeared to be homologous because previous exposure to 5-HT did not alter the response to other transmitters such as thrombin or isoproterenol and because previous exposure to thrombin or isoproterenol did not diminish the response to 5-HT. The desensitization induced by pretreatment with 5-HT was potently prevented by the naphthalenesulfonamide derivative W-7, a calmodulin antagonist, when it was cosupplied with 5-HT. Furthermore, the preventive effect of W-7 was greater than that of W-5, a weak analogue of W-7, and than that of H-7, a nonselective inhibitor of protein kinases. These results suggest that 5-HT₂ receptor-mediated Ca²⁺ mobilization can be desensitized homologously after prolonged exposure to 5-HT in a calmodulin-dependent manner in rat glioma C6BU-1 cells.     

Comparison of the roles of calmodulin and protein kinase C in activation of the human neutrophil respiratory burst

The roles of calmodulin and protein kinase C in the activation of the human neutrophil respiratory burst were characterized pharmacologically. The protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) did not inhibit superoxide anion generation by neutrophils stimulated for 30 minutes with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). However, H-7 did depress superoxide production during the first 5 minutes following stimulation. In contrast, the specific calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and the dual calmodulin antagonist/protein kinase C inhibitor trifluoperazine (TFP) were potent inhibitors of the response throughout the 30 minute incubation. Stimulation of neutrophils with submaximal doses of FMLP or PMA failed to promote inhibition of the respiratory burst by H-7 or H-9, but did stimulate a respiratory burst response which was not inhibited by TFP or W-7. These results suggest that while protein kinase C may play a role in the initiation of the respiratory burst response, propagation of the response is dependent on calmodulin-dependent processes. The inability of TFP and W-7 to inhibit superoxide anion generation in response to submaximal stimulatory doses of FMLP or PMA suggests that calmodulin-independent processes may also be involved in activation of the respiratory burst.     

Effect of Desipramine on a Glycoprotein Sialyltransferase Activity in C6 Cultured Glioma Cells

The tricyclic antidepressant desipramine, when added to culture medium, gave rise in C6 rat glioma cells to a decrease of the activity of the enzyme asialofetuin sialyltransferase. The inhibition was dose and time dependent and was observed in both multiplying cells and cells blocked with 2 mM thymidine or depletion of amino acids. This inhibition was rather specific to the sialyltransferase, as under the conditions where this enzyme was inhibited up to 70%, other enzymes such as dolichol phosphate mannose synthetase, glutamine synthetase, and glycerol phosphate dehydrogenase remained unaffected. This inhibition was not reversed after removal of desipramine from the medium and was not observed by direct addition of desipramine to the sialyltransferase incubation assay. Under the same conditions, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], which is known to be a potent calmodulin antagonist and an inhibitor of calmodulin-dependent kinases, gave the same concentration-dependent inhibition profile of sialyltransferase as desipramine, whereas H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], which is an inhibitor of protein kinase C and cyclic nucleotide-dependent kinases, had no effect. So, it is suggested that desipramine inhibits the sialyltransferase activity in C6 glioma cells through a calmodulin-dependent system.     

Effects of the protein kinase C inhibitor H-7 and calmodulin antagonist W-7 on superoxide production in growing and resting human histiocytic leukemia cells (U937)

Serum, phorbol 12,13-didecanoate (PDD) and 1-oleoyl-2-acetoy-sn-glycerol (OAG) stimulated O2- release in human histiocytic leukemia U937 cells. The kinetics of O2- release caused by PDD but not by serum or OAG in growing cells differed from those in resting cells. Both the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl) 2-methylpiperidine (H-7) and calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) reduced the superoxide generation induced by these stimuli. H-7 inhibited the O2- release either from growing or resting cells but the effect of W-7 varied according to the growth phase. From these results, it is suggested that activation of protein kinase C and calmodulin-dependent process has an important role in O2(-)-release induced by serum, OAG and PDD, and that the mechanism for PDD-induced O2(-)-release is different in growing and resting cells.     

Phosphorylation of the CD20 phosphoprotein in resting B lymphocytes. Regulation by protein kinase C

CD20, a B cell integral membrane protein, regulates B cell activation and is differently phosphorylated in resting and activated cells. We have previously shown that CD20 phosphorylation is increased in activated cells and in phorbol ester-treated resting cells. Phosphorylation is also altered by agents which signal B cell proliferation, such as anti-IgM and a B cell growth factor. The present study was designed to address whether protein kinase C (PKC) or other kinases used CD20 as a substrate. When purified PKC was incubated with isolated CD20, both the 35- and 37-kDa CD20 proteins were phosphorylated in vitro. Intact resting B cells were next incubated with the protein kinase inhibitors H-7, H-8, and W-7. No change in basal CD20 phosphorylation was observed in the presence of W-7 and H-8, indicating that the protein cyclic nucleotide-dependent and calmodulin-dependent kinases were not actively phosphorylating CD20. Surprisingly, the PKC inhibitor H-7 increased CD20 phosphorylation at concentrations above 25-50 microM. To assess whether PKC either activated a phosphatase or inactivated a kinase affecting CD20 phosphorylation, tryptic phosphopeptide mapping of CD20 was performed. These studies revealed that addition of phorbol 12-myristate 13-acetate increased phosphorylation of some peptides differing from those which had increased phosphorylation following addition of H-7. Furthermore, signalling through surface immunoglobulin increased phosphorylation of CD20 peptides distinct from those hyperphosphorylated following addition of phorbol 12-myristate 13-acetate. These results demonstrate that 1) CD20 has multiple phosphorylation sites, as predicted from sequence data, and 2) whereas PKC can use CD20 as substrate, at least one other unidentified kinase phosphorylates CD20 in resting cells. Our data also predict that activation of B cells via the antigen receptor (surface IgM) may activate other protein kinases in addition to PKC.     

Inhibitors of IL-2 production and IL-2 receptor expression in human leukemic T-cell line, Jurkat

1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase inhibitor, suppressed interleukin 2 (IL-2) production and IL-2 receptor (IL-2R) expression of the human leukemic T-cell line, Jurkat, induced by 12-O-tetradecanoyl-phorbol-13-acetate and phytohemagglutinin-P. This effect was significant at 5 microM H-7 without loss of cell viability. Such activity was not observed with N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), a potent inhibitor of cGMP- and cAMP-dependent kinases, and a weak inhibitor of Ca2+-phospholipid-dependent protein kinase (protein kinase C). These findings suggest that protein kinase C is more closely associated with IL-2 receptor expression and IL-2 production of T cells than cGMP- or cAMP-dependent kinases. In addition, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor, suppressed both IL-2 production and IL-2R expression. Cycrosporin A (Cy A), a potent immunosuppressive drug, markedly inhibited IL-2 production of Jurkat cells whereas it did not affect the IL-2R expression. Thus, the mechanism of action of Cy A appears to differ from that of the protein kinase inhibitor, H-7, and the calmodulin inhibitor, W-7.     

Involvement of the protein kinase pathway in melanin synthesis by chick retinal pigment epithelial cells

Protein kinases are involved in a variety of cellular functions and cell proliferation in eyes. We have explored the involvement of protein kinase C (PKC) in cell proliferation and melanin synthesis by chick retinal pigment epithelial (RPE) cells in vitro. This was achieved by incubation of confluent RPE cells with known inhibitors of protein kinase, H-7, W-7, H-8, and staurosporine. Chick RPE cells were cultured in the presence or absence of the protein kinase inhibitors for a 10-day period. Effects of the inhibitors on cell proliferation and melanin synthesis, as an indication of cell differentiation, were assessed by counting the number of surviving cells and by measuring the melanin content in the cells, respectively. H-7, W-7, and staurosporine inhibited cell proliferation and increased melanin synthesis in a concentration-dependent manner during culture; however, H-8 did not produce these cellular effects. These findings indicate that PKC and calcium/calmodulin-dependent kinase pathways are involved in the proliferation and differentiation of chick RPE cells.     

Characterization of specific binding sites for [3H]-staurosporine on various protein kinases

Binding of [3H]-staurosporine to different protein kinases was time-dependent, reversible and saturable. Scatchard analysis of saturation isotherms indicated one class of binding sites for [3H]-staurosporine with dissociation constants (KD) of 9.6, 2.0, 3.0 and 7.4 nM for protein kinase C, cAMP-dependent protein kinase, tyrosine protein kinase and calcium/calmodulin-dependent protein kinase respectively. [3H]-staurosporine binding was fully displaced by unlabelled staurosporine or the related compound K-252a whereas other protein kinase inhibitors (H-7, H-8 and W-7) did not compete with [3H]-staurosporine. These data confirm that sataurosporine shows no selectivity for different protein kinases and suggest the putative existence of distinct, specific binding sites for [3H]-staurosporine on these enzymes.     

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.     

Activation process of calcium-dependent potassium channel in Euhadra neurons: involvement of calcium/calmodulin and subsequent protein phosphorylation.

1. The activation process of Ca(2+)-dependent potassium channel was studied electrophysiologically and pharmacologically using identified neurons of the land snail, Euhadra peliomphala. 2. Ca(2+)-mediated delayed outward K current (IKD) was dose-dependently reduced by the calmodulin inhibitors, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5, week) and N-(6-aminohexyl)-5-chloro-naphthalenesulfonamide (W-7, potent). These antagonists also caused a slight membrane depolarization and increase in impulse discharge frequency with decrease in the amplitude of both action potential and after hyperpolarization. 3. The cAMP-dependent protein kinase inhibitor N-[2-(methylamino) ethyl]-5-isoquinoline-sulfonamide (H-8) did not produce any significant effect on IKD and membrane potential. 4. Calmodulin, when injected into the neuron which had been treated with either W-5 or W-7, transiently restored the suppressed IKD nearly to the pretreatment level, and caused hyperpolarization of the cell. In contrast, calcium chloride, intracellularly injected in the same way, had little effect on both the IKD and the membrane potential shifted by these antagonists. 5. Intracellular injection of kinase II, a Ca2+/calmodulin-dependent protein kinase, caused an increase in the IKD and membrane hyperpolarization. Similar but weak effects were produced when a catalytic subunit (CS) of cAMP-dependent protein kinase was intracellularly injected. However, the neurons pretreated with W-7 no longer had any detectable increase in the IKD and hyperpolarization of the membrane. 6. These results suggest the possibility that Ca2+/camodulin-dependent protein phosphorylation may finally mediate the activation of a certain number of potassium channels.     

Involvement of Calmodulin and Calmodulin-Dependent Myosin Light Chain Kinase in Blue Light-Dependent H Pumping by Guard Cell Protoplasts from Vicia faba L

Signal transduction processes involved in blue light-dependent proton pumping were investigated using guard cell protoplasts from Vicia faba. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cyclic AMP- and cyclic GMP-dependent protein kinases, had no effect. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) and calphostin C, inhibitors of protein kinase C, produced slight inhibition of the blue light-dependent proton pumping. 1-[N, O-Bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl] -4-phenylpiperazine, a specific inhibitor of Ca²⁺/calmodulin (CaM)-dependent protein kinase II, did not inhibit the proton pumping, but 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine and 1-(5-chloro-naphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), inhibitors of Ca²⁺/CaM-dependent myosin light chain kinase, strongly suppressed the proton pumping. A CaM antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), inhibited blue light-dependent proton pumping, whereas its less active structural analog, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), had little effect on the response. Other CaM antagonists, trifluoperazine, compound 48/80, prenylamine, and 3-(2-benzothiazolyl)-4,5-dimethoxy-N-[3-(4-phenyl-piperidinyl)- propylbenzenesulfonamide inhibited the proton pumping. In accord with these results, light-induced stomatal opening in the epidermis of Commelina benghalensis ssp. was inhibited by ML-9 and W-7, but not by H-7 and W-5. Thus, it is concluded that CaM and Ca²⁺/CaM-dependent myosin light chain kinase are the components of the signal transduction process in blue light-dependent proton pumping in guard cells.     

Agonist stimulation of Na+/K+/Cl- cotransport in rat glomerular mesangial cells. Evidence for protein kinase C-dependent and Ca2+/calmodulin-dependent pathways

Studies were performed to investigate regulatory pathways of loop diuretic-sensitive Na+/K+/Cl- cotransport in cultured rat glomerular mesangial cells. Angiotensin II, alpha-thrombin, and epidermal growth factor (EGF) all stimulated Na+/K+/Cl- cotransport in a concentration-dependent manner. Pertussis toxin pretreatment reduced the effects of angiotensin II and alpha-thrombin but not that of EGF. Addition of the protein kinase C inhibitor staurosporine or down-regulation of protein kinase C by prolonged incubation with phorbol 12-myristate 13-acetate partially reduced the effects of angiotensin II and alpha-thrombin and completely blunted the phorbol 12-myristate 13-acetate-induced stimulation of Na+/K+/Cl- cotransport but did not affect EGF-induced stimulation. Exposure of cells to a calcium ionophore, A23187, resulted in a concentration-dependent stimulation of Na+/K+/Cl- cotransport, which was not significantly inhibited by down-regulation of protein kinase C but was completely inhibited by the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Stimulation of the cotransport by angiotensin II or alpha-thrombin was also partially inhibited by W-7. Inhibitory effects of protein kinase C down-regulation and W-7 were additive and, when combined, produced a complete inhibition of angiotensin II-induced stimulation of Na+/K+/Cl- cotransport. In saponin-permeabilized mesangial cells, phosphorylation of a synthetic decapeptide substrate for Ca2+/calmodulin-dependent kinase II, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-NH3, was demonstrated. Maximal activation of the decapeptide substrate phosphorylation required the presence of Ca2+ and calmodulin and was dependent on Ca2+ concentration. These findings indicate that stimulation of Na+/K+/Cl- cotransport by angiotensin II and alpha-thrombin is mediated by protein kinase C and Ca2+/calmodulin-dependent kinases whereas the action of EGF is mediated by other pathways.     

Inhibition of macrophage activation by isoquinolinesulfonamides, phenothiazines, and a napthalenesulfonamide

The influence of isoquinolinesulfonamides (H-7 and H-8), phenothiazines(trifluoperazine and fluphenazine), and a naphthalenesulfonamide (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on stimulated superoxide anion production and phosphatidyl inositol (PI) cycle activity was investigated in the guinea pig alveolar macrophage. All five drugs were able to inhibit superoxide anion production stimulated by n-formyl-nel-leu-phe (FNLP), leukotriene B4 (LTB4), and phorbol-12,13-dibutyrate (PDB). The order of potency was trifluoperazine greater than or equal to fluphenazine greater than H-7 = W-7 greater than H-8. The dose response curves could be shifted to less efficacy by increasing extracellular calcium. By itself, W-7 markedly stimulated 45Ca+2 efflux, fluphenazine and trifluoperazine slightly stimulated 45Ca+2 efflux, while H-7 and H-8 had no effect on 45Ca+2 efflux from macrophages preloaded with 45Ca+2. Consistent with these results, W-7 markedly stimulated PI cycle activity, fluphenazine and trifluoperazine slightly stimulated PI cycle activity, while H-7 and H-8 had no significant effects on PI cycle activity. In addition, W-7 by itself was able to stimulate a weak and short-lived "burst" of superoxide anion production. In order to evaluate whether a site of action of the inhibitors was at protein kinase C and whether protein kinase C was involved in terminating the normally short-lived FNLP- and LTB4-stimulated macrophage activation, fluphenazine and H-7 were used to evaluate the duration of FNLP- and LTB4-stimulated PI cycle activity, at concentrations of the inhibitors that significantly blocked stimulated superoxide anion production. In all cases, FNLP and LTB4 still stimulated PI cycle activity, which still terminated even though protein kinase C was inhibited. These results suggest that all five drugs block protein kinase C, but H-7 was the most specific in its action at protein kinase C, while the phenothiazines and W-7 have multiple sites of action. In addition, these results suggest that protein kinase C may not function to cause the termination of FNLP- and LTB4-stimulated PI cycle activity and subsequent superoxide anion production.     

(R,S)-α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid (AMPA) Receptors Mediate a Calcium-Dependent Inhibition of the Metabotropic Glutamate Receptor-Stimulated Formation of Inositol 1,4,5-Trisphosphate

Abstract: l -Glutamate (3-1,000 μ M ) and (1S,3R)-l-aminocyclopentane-1,3-dicarboxylic acid (1S.3R-ACPD; 10-1,000 μ M ), a selective agonist for the metabotropic glutamate receptor, stimulated the formation of inositol 1,4,5-trisphosphate in a concentration-dependent manner. l -Glutamate was half as efficacious as 1S,3R-ACPD. N -methyl- d -aspartate (nMDA; 1 n M to 1 m M ) did not significantly influence the response to a maximally effective concentration of 1S,3R-ACPD (100 μ M ). On the other hand, coapplication of (R,S)-α-amino-3-hydroxy-5-methylisoxa-zole-4-propionic acid (AMPA; 1-300 n M ) produced a concentration- and time-dependent inhibition of the 1S,3R-ACPD effect, with a maximal inhibition (97%) at 100 n M . Ten micromolar 6-cyano-7-nitroquinoxaline-2,3-dione. an antagonist of the AMPA receptor, blocked the inhibitory effect of AMPA. Reduced extracellular calcium concentration, as well as 10 μ M nimodipine, an l -type calcium channel antagonist, inhibited the AMPA influence on the 1S,3R-ACPD response. W-7, a calcium/calmodulin antagonist, prevented the inhibition by AMPA. whereas H-7. an inhibitor of protein kinase C, had no effect. These data suggest that activation of AMPA receptors has an inhibitory influence on inositol 1,4,5-trisphosphate formation mediated by stimulation of the metabotropic glutamate receptor. The mechanism of action involves calcium influx through l -type calcium channels and possible activation of calcium/calmodulin-dependent enzymes.     

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist,also inhibits phospholipid sensitive calcium-dependent protein kinase

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), commonly regared as a calmodulin antagonist, inhibted phospholipid-sensitive Ca²⁺-dependent protein kinase and to a lesser extent cyclic GMP- and cyclic AMP-dependent protein kinases. Kinetic studies of the inhibition of the homogenous spleen phospholipid-sensitive Ca²⁺-dependent protein kinase indicated that W-7 inhibited the enzyme activity competitively with respect to phospholipid (K_i = 60 μM). N-(6-Aminohexyl)-1-naphthalenesulfonamide (W-5) was found to be musch less potent than W-7. The findings indicate that W-6 was able to inhibit a variety of protein kinases, in addition to those requiring calmodulin previously reported.     

Sphingosine, W-7, and Trifluoperazine Inhibit the Elevation in Cytosolic Calcium Induced by High K+Depolarization in Synaptosomes

Abstract: A possible role for protein kinases in the regulation of free cytosolic Ca²⁺ levels in nerve endings was investigated by testing the effect of several kinase inhibitors on the increase in cytosolic Ca²⁺ (monitored with the Ca²⁺-sensitive dye fura-2) induced by depolarization with 15 or 30 mM K⁺. The ability of various drugs to inhibit the cytosolic Ca²⁺ response appeared to correlate with their reported mechanism of action in inhibiting protein kinases. W-7 and trifluoperazine, drugs reported to inhibit calmodulin-dependent events, were effective inhibitors of the increase in cytosolic Ca²⁺ induced by high K⁺ depolarization, as was sphingosine, a drug that inhibits protein kinase C by binding to the regulatory site, but which also inhibits calcium/calmodulin kinase. On the other hand, drugs that inhibit protein kinases by binding to the catalytic site, such as H-7 (1 m/W ), staurosporine (1μ M ), and K252_a(1μ M ), were ineffective. Activation of protein kinase C, which is blocked by each of these drugs, does not appear to be essential to the maintenance of elevated cytosolic Ca²⁺ in depolarized synaptosomes. All of the drugs, including sphingosine, that functionally inhibit the depolarization-induced elevation in cytosolic Ca²⁺ have in common the ability to bind to calmodulin. Because the drugs that inhibit protein kinases by competing with ATP binding at the active catalytic site did not block the response in this system, we suggest that a calmodulin or a calmodulin-like binding site participates in the regulation of Ca²⁺ increases after depolarization.     

Induction of ornithine decarboxylase in guinea-pig lymphocytes. Synergistic effect of diacylglycerol and calcium

Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5) and trifluoperazine inhibited ornithine decarboxylase induction in lymphocytes activated with phytohemagglutinin or inophore A23187. W-7, a more potent calmodulin antagonist than W-5, suppressed ornithine decarboxylase induction in a higher extent than did W-5. These results suggest that calmodulin may play an important role in ornithine decarboxylase induction in the activated lymphocytes. However, the extent of ornithine decarboxylase induction was greater in cells pretreated with Clostridium phospholipase C and then incubated with ionophore A23187 than in cells incubated with ionophore A23187 without the pretreatment. Moreover, combined treatment of cells with ionophore A23187 and tumor promotor, phorbol 12-myristate 13-acetate, caused synergistic induction of ornithine decarboxylase activity. These results, taken together, suggest that both activations of Ca2+-activated phospholipid-dependent protein kinase by diacylglycerol and of calmodulin-dependent function resulted from an elevation of cytosolic Ca2+ concentration may operate in the induction of ornithine decarboxylase in the activated lymphocytes.     

Clearer demonstration of calcium/calmodulin-dependent events in synaptosomes by use of the differential effects of two calmodulin antagonists, N-(aminohexyl)-5-chloro-1-naphthalenesulfonamide and N-(6-aminohexyl)-1-naphthalenesulfonamide

1. In order to demonstrate more clearly calcium/calmodulin-dependent events, the differential effects of two calmodulin antagonists, W-7 and W-5, on synapsin I phosphorylation and norepinephrine release associated with calcium influx, were investigated using 32Pi in synaptosomes derived from rat cerebral cortex. 2. The calcium ionophore (A23187)-stimulatory effect on synapsin I phosphorylation and norepinephrine release was markedly reduced by W-7 and slightly reduced by W-5; whereas neither the strong nor the weak calmodulin antagonist had an effect on A23187-stimulated synaptosomal uptake of calcium. 3. Preincubation with H-8 reduced both W-5- and W-7-inhibited A23187-stimulated synapsin I phosphorylation by the same amount but did not affect their inhibitory effect nor the ionophore-stimulated norepinephrine release, thereby suggesting that W-5 may serve as an appropriate control for non-calmodulin-mediated effect of both calmodulin antagonists.     

Comparative study on the stimulation of superoxide production in guinea-pig eosinophils by the calcium ionophore A23187

The effect of calcium and/or magnesium on O2- production by guinea-pig eosinophils stimulated by the calcium ionophore A23187 was studied in comparison to neutrophils. In the absence of calcium, A23187 did not stimulate O2- production in resting eosinophils and neutrophils, regardless of the presence of extracellular magnesium. The A23187-induced O2- production by both cells increased linearly with extracellular Ca2+ concentrations. However, the concentration of Ca2+ required for maximum O2- production in eosinophils was about 10-times lower than that required of neutrophils. The addition of Mg2+ strongly inhibited O2- production, especially in eosinophils at low Ca2+ concentrations. The intracellular Ca2+ concentration was lower in eosinophils than in neutrophils in the resting state, and the enhancement of the intracellular Ca2+ concentration in response to A23187 was much lower in eosinophils than in neutrophils. The activation of the NADPH-dependent O2(-)-forming enzyme (NADPH oxidase) in eosinophils depended on extracellular calcium, as observed in O2- production. However, the NADPH oxidase activity in the particulate fraction from A23187-stimulated eosinophils was only slightly affected by the addition of divalent cations or EDTA. The compound W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride), a calmodulin antagonist, significantly inhibited O2- production by both cells. On the other hand, the compound H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride), a protein kinase C antagonist, was less effective on O2- production than was W-7. H-7 had little effect on O2- production of eosinophils. These findings suggest that NADPH oxidase might be activated by a smaller Ca2+ concentration through the calmodulin-dependent reaction. This was not observed with protein kinase C, at least in eosinophils.