Cyclic Nucleotide-dependent Protein Kinases Research Articles

Differential growth inhibition of isoquinolinesulfonamides H-8 and H-7 towards multidrug-resistant P388 murine leukaemia cells

The effects of N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) on the growth of P388 and its multidrug-resistant (MDR) variants were examined with the objective of assessing the possible changes in cyclic nucleotide-dependent protein kinases and protein kinase C-mediated pathways associated with MDR. H-8, an inhibitor of cyclic nucleotide-dependent protein kinases, inhibited the growth of the parental P388 murine leukaemic cells, but not that of MDR variants up to 200 microM. However the growth of both drug-sensitive and resistant cell lines were uniformly inhibited by H-7. Both the cytotoxic and cytokinetic results revealed that the growth-inhibition by H-8 of P388 cells is mainly due to a blockade of cell-cycle progression rather than due to a killing of cells. The degree of resistance to H-8 was directly proportional to their extent of resistance to vincristine, adriamycin, and 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene)-beta-D-gluco pyr anoside (VP-16) and to that of the expression of P-glycoprotein. These findings raised the possibility that P-glycoprotein might play a role in the cross-resistance to H-8. To test the hypothesis, we examined the effect of H-8 on the binding of 3H-vincristine to membrane fraction isolated from P388/VCR-600 cells and on the enhancement of cytotoxicity to anticancer drugs in MDR cells. H-8 did not have any influences on these reactions. Thus, the cross-resistance to H-8 may be mediated through a mechanism different from an overexpression of P-glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of ligand specificity in cyclic nucleotide-gated channels from rod photoreceptors and olfactory epithelium.

Cyclic nucleotide-gated ionic channels in photoreceptors and olfactory sensory neurons are activated by binding of cGMP or cAMP to a receptor site on the channel polypeptide. By site-directed mutagenesis and functional expression of bovine wild-type and mutant channels in Xenopus oocytes, we have tested the hypothesis that an alanine/threonine difference in the cyclic nucleotide-binding site determines the specificity of ligand binding, as has been proposed for cyclic nucleotide-dependent protein kinases [Weber, I.T., Shabb, J.B. & Corbin, J.D. (1989) Biochemistry 28, 6122-6127]. The wild-type olfactory channel is approximately 25-fold more sensitive to both cAMP and cGMP than the wild-type rod photoreceptor channel, and both channels are 30- to 40-fold more sensitive to cGMP than to cAMP. Substitution of the respective threonine by alanine in the rod photoreceptor and olfactory channels decreases the cGMP sensitivity of channel activation 30-fold but little affects activation by cAMP. Substitution of threonine by serine, an amino acid that also carries a hydroxyl group, even improves cGMP sensitivity of the wild-type channels 2- to 5-fold. We conclude that the hydroxyl group of Thr-560 (rod) and Thr-537 (olfactory) forms an additional hydrogen bond with cGMP, but not cAMP, and thereby provides the structural basis for ligand discrimination in cyclic nucleotide-gated channels.

Endogenous activity of cyclic nucleotide-dependent protein kinase in plasma membranes isolated from Strongylocentrotus purpuratus sea urchin sperm

Activity of cyclic nucleotide-dependent protein kinase was investigated in flagellar plasma membranes of sea urchin sperm (S. purpuratus). Membranes incubated with [gamma-32P]ATP showed in the presence of 1 microM cAMP an increased phosphorylation in multiple polypeptides. Half maximal response was seen at 0.6 microM of cAMP. In contrast, higher concentrations (100 microM) of cGMP were required to cause the same amount of protein phosphorylation. 80% of the protein kinase activity stimulatable by cAMP was resistant to extraction by 10 mM EGTA and sonication but it was entirely recovered in a detergent-solubilized fraction. Membranes pretreated with 200 microM cAMP, ultracentrifuged and resuspended in buffer solution did not undergo cAMP-stimulated phosphorylation in their polypeptides. This study demonstrates that flagellar plasma membranes isolated from S. purpuratus sea urchin sperm have an endogenous cAMP-dependent protein kinase, which may be bound to the membrane via its regulatory subunit.

Differential accumulation of transcripts encoding protein kinase homologs in greening pea seedlings.

Degenerate oligonucleotides, corresponding to conserved regions within the catalytic domain of known protein-serine/threonine kinases, were used as primers for the polymerase chain reaction to amplify cDNA synthesized from poly(A)+ RNA purified from the apical buds of 7-day-old pea seedlings. Five partial cDNAs were obtained and designated PsPK1 through PsPK5 (for Pisum sativum protein kinase) in order of decreasing length. The deduced amino acid sequences show that each member of the PsPK series is different in length, and, although their sequences are quite similar overall, each has a unique sequence. Moreover, each member of the PsPK series has structural features typical of members of the protein-serine/threonine kinase family of protein kinases. All are equally similar to cyclic nucleotide-dependent protein kinase and protein kinase C, suggesting that the pea homologs may be involved in signal transduction. DNA gel blots show that each PsPK cDNA is likely to be encoded by a single gene within the pea genome. RNA blot analyses show that the PsPK transcripts accumulate differentially during greening of etiolated seedlings. PsPK3 and PsPK5 transcripts show a large and rapid decline during deetiolation. In contrast, the level of PsPK4 RNA increases steadily during deetiolation whereas PsPK1 and PsPK2 transcripts show little change during the greening period. Thus light regulates changes in the levels of transcripts encoding putative protein kinases in plants.

Interrelationships between mevalonate metabolism and the mitogenic signaling pathway in T lymphocyte proliferation

Upon stimulation with antigen or antibodies directed at the CD3.T cell receptor complex, T lymphocytes undergo a series of biochemical events that result in DNA synthesis and cellular proliferation. The purpose of the current study was to explore the role of mevalonic acid and its metabolites in this process. Stimulation of freshly isolated human T cells with immobilized anti-CD3 monoclonal antibody (mAb) results in the induction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase message, with maximum induction occurring at 24 h of culture, approximately 12 h before the onset of DNA synthesis. Protein kinase C (PKC) probably mediates this induction, as H7, which inhibits PKC and cyclic nucleotide-dependent protein kinases, but not HA1004, which inhibits all of these protein kinases except PKC, completely abrogates the appearance of HMG-CoA reductase message. The importance of HMG-CoA reductase induction and mevalonate production in cell cycle progression was demonstrated by the observation that either 25-hydroxycholesterol, which inhibits this induction, or lovastatin, a competitive inhibitor of HMG-CoA reductase, inhibited anti-CD3-induced T cell mitogenesis in a dose-dependent manner. The presence of lovastatin during the first 24-36 h of culture results in a progressive delay of cell cycle progression, whereas this agent, when present only for the first 12 h of culture, had no effect on T cell proliferation. These results suggest that mevalonate is required for cell cycle progression from mid-G1 into late G1. Exogenous mevalonate overcomes the antiproliferative effect of lovastatin but not of 25-hydroxycholesterol. Since 25-hydroxycholesterol suppresses the metabolism of mevalonic acid at multiple points, this result suggests that one or more metabolites of mevalonate, rather than mevalonate itself, plays an essential role in cell cycle progression. One metabolite of mevalonate, farnesol pyrophosphate, may play such a role, since free farnesol suppresses anti-CD3 mAb-induced T cell proliferation in a concentration-dependent manner. In mAb is associated with PKC-dependent induction of HMG-CoA reductase which, in turn, leads to the generation of mevalonic acid and its metabolites, one or more of which play a requisite role in cell cycle progression.

Effect of cyclic AMP on ciliary activity of human respiratory epithelium

In order to investigate the effect of cyclic adenosine monophosphate (cAMP) on ciliary beat frequency (CBF) in human respiratory epithelium, cells were brushed from the inferior nasal turbinates of three groups of ten subjects: awake adults (aged 20-34 yrs), anaesthetized children (2-15 yrs) and anaesthetized adults (19-61 yrs). Cells from the awake adults were also studied after storage for 24 h in tissue culture medium. CBF was measured in vitro with a photometric technique at room temperature (22.0 +/- 1.5 degrees C). Samples were mounted in a perfusion chamber and challenged with either control solutions, dibutyryl cAMP (10(-4) or 10(-3) M), or the cyclic nucleotide-dependent protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H-7). Dibutyryl cAMP (10(-3) M) caused a significant increase in CBF in all groups studied: awake adults (+1.1 Hz; p less than 0.01), anaesthetized children (+1.2 Hz; p less than 0.01), anaesthetized adults (+1.2 Hz; p less than 0.01), stored cells (+1.1 Hz; p less than 0.01). This response was inhibited by preincubation with H-7 (10(-4) and 10(-3) M). It is concluded that cAMP is a regulator of ciliary activity in human respiratory epithelium.

Involvement of protein kinase C in competence induction of macrophages to generate T suppressor cells.

We have previously described an Ia-expressing macrophage hybridoma clone, termed clone 59, which attains the ability to induce Ts cells after activation with murine rIFN-gamma. In this report, we show that a protein kinase C (PKC) activator, PMA (10 ng/ml) can replace IFN-gamma in inducing this form of macrophage competence. IFN-gamma-induced cellular competence was abrogated specifically by a PKC inhibitor but not by inhibitors that have specificity for cyclic nucleotide-dependent protein kinases. Furthermore, PGE2 known to induce protein kinase A in murine macrophages also failed to induce competence. In contrast, the ability to induce Th responses was neither dependent on IFN-gamma nor inhibited by prior treatment with protein kinase inhibitors. Furthermore, PKC depletion of macrophages by treatment with high doses (100 ng/ml) of PMA abrogated their ability to induce Ts cells. In addition, PKC-depleted macrophages failed to regain the ability to stimulate Ts cells after further treatment with IFN-gamma. The ability of IFN-gamma to modulate macrophage-mediated induction of Ts cells does not clearly correlate with an increased Ia expression as inducible expression of Ia was not consistently abrogated by PKC inhibitor treatment. In addition, PKC inhibitors failed to prevent the production of the cytokines IL-1 and IL-6. However, incubation of IFN-gamma or PMA-treated macrophages with antibodies recognizing the putative IJ ligand blocked the ability to induce Ts cells, suggesting the expression of these determinants on accessory cells is responsible for Ts induction.

Progesterone Receptor Regulation in Uterine Cells:Stimulation by Estrogen, Cyclic Adenosine 3′,5′-Monophosphate, and Insulin-Like Growth Factor I andSuppression by Antiestrogens and Protein KinaseInhibitors*

Primary uterine cell cultures were used to study multifactor regulation of progesterone receptor (PR) and the signal transduction pathways which may serve to mediate that regulation. Increases in intracellular cAMP, brought about by treatment with cholera toxin plus isobutyl methyl xanthine or by addition of 8-bromo-cAMP, result in 6- to 7-fold increases in the intracellular content of PR as monitored by [3H]R5020 binding and by Western immunoblot using anti-PR antibodies. In these primary cultures of uterine cells isolated from 19-day-old immature rats, 8-bromo-cAMP evokes significant increases in PR by 8 h with maximal increases by 24 h. This time course and magnitude of PR stimulation are similar to those evoked by maximally effective concentrations of estradiol (3 x 10(-9) M) or IGF-I (20 ng/ml). Dose-response studies reveal that 10(-6) to 10(-4) M concentrations of 8-bromo-cAMP (8-Br-cAMP) elicit a maximal response. In contrast, 8-bromo-cGMP over a wide concentration range was unable to elevate cellular PR levels. Under these culture conditions, cell proliferation was not altered by treatment with any of these agents. Although estrogen, cAMP, and insulin-like growth factor I (IGF-I) may act via different pathways to increase PR, the effects evoked by maximally effective concentrations of these agents are not additive implying involvement of a common component. The increases in PR evoked by estradiol, cAMP, or IGF-I are markedly suppressed by treatment with antiestrogen (ICI 164,384) or the cyclic nucleotide-dependent protein kinase inhibitor H8 or the protein kinase A inhibitor PKI, indicating the involvement of the estrogen receptor and phosphorylation pathways in PR regulation by these three agents. The present studies identify cAMP, as well as estrogen and IGF-I, as important regulators of the level of PR in uterine cells and suggest that multiple factors, including those affecting intracellular cAMP levels, might influence responsiveness to progestins via regulation of the intracellular PR content.

ANP stimulates phospholipase C in cultured RIMCT cells: roles of protein kinases and G protein

We examined the possibility that, in addition to stimulation of guanylate cyclase (GC), atrial natriuretic peptide (ANP) also activates phospholipase C (PLC) in cultured rat inner medullary collecting tubule (RIMCT) cells. ANP (10(-12)M) causes marked release of inositol trisphosphate (IP3) at a concentration that does not stimulate GC. Concentrations of ANP that stimulate GC (greater than or equal to 10(-10) M) result in attenuated IP3 release. Similarly, exogenous dibutyryl guanosine 3',5'-cyclic monophosphate (10(-6) M) markedly inhibits the response to 10(-10) M ANP. Inhibition of cyclic nucleotide-dependent protein kinase by H 8, but not inhibition of protein kinase C by H 7, restores the response to 10(-8) and 10(-6) M ANP. Therefore, activation of cyclic nucleotide-dependent protein kinase inhibits ANP-stimulated PLC activity. Activation of protein kinase C by phorbol 12-myristate-13-acetate (PMA) decreases ANP-stimulated IP3 production. Pretreatment with H 7, but not H 8, prevents inhibition by PMA. To explore a potential role for G proteins, we examined the effect of guanine nucleotide analogues on ANP-stimulated IP3 production in saponin-permeabilized cells. ANP-stimulated IP3 production is enhanced by GTP gamma S and is inhibited by GDP beta S. Similarly, preincubation with pertussis toxin prevents ANP-stimulated IP3 release. We conclude that ANP stimulates PLC in RIMCT cells via a pertussis toxin-sensitive G protein. Stimulation of PLC is inhibited on activation of either cyclic nucleotide or Ca2+-phospholipid dependent protein kinases.

Schistosoma mansoni: Possible involvement of protein kinase C in linoleic acid-induced proteolytic enzyme release from cercariae

The possible involvement of protein kinase C and Ca 2+ metabolism in the proteolytic enzyme release from schistosome cercariae was studied. Cercariae were placed in dechlorinated tap water containing 0.37 m M calcium in the small glass petri dish and exposed to the stimuli-(linoleic acid, phorbol esters, and Ca 2+ ionophore) with or without inhibitors of protein kinase C or Ca 2+ metabolism. The proteolytic activity of incubation medium of cercariae thus treated was measured by the azocoll assay. The penetration response of cercariae induced by linoleic acid, a physiological stimulus, was mimicked by phorbol esters. When exposed to phorbol esters, 0.02 to 2 μM of 12- O-tetradecanoylphorbol-13-acetate (TPA) and 0.2 to 2 μM of phorbol-12,13-dibutyrate (PDBu), cercariae ceased the swimming movement, began a rhythmic thrusting of the anterior tip of the parasite, and released the proteolytic enzyme, but they did not shed the tails. Lowering Ca 2+ in water by addition of 5 m M ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), phorbol ester-induced release of enzyme was completely inhibited. Phorbol ester-induced release of enzyme was partially inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, at a concentration of 100 μ M. H-7 alone, at a concentration of 100 μ M, did not affect the swimming movement of cercariae. The cercariae were stimulated to release the enzyme by high concentrations (10 and 100 μ M) of the Ca 2+ ionophore, A23187, but enzyme was not released by low concentrations (0.5 and 1 μ M) of this drug. Cercariae exposed to A23187 behaved differently from those exposed to phorbol esters. They ceased swimming, showed strong muscle contraction, and shed their tail. A23187 stimulated cercariae to release the enzyme in the water containing 5 m M EGTA. A23187-induced enzyme release was not inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, trifluoperazine (TFP), a better calmodulin antagonist on schistosome, or by verapamil, a Ca 2+ channel blocker. Linoleic acid-induced release of enzyme was partially inhibited by 0.5 and 5 m M of EGTA and by 1 to 100 μ M of H-7. While it was not inhibited by N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), inhibitors of cyclic nucleotide-dependent protein kinase which were used as negative controls of H-7, W-7, TFP, 8-( N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), an intracellular Ca 2+ antagonist, and verapamil. These results strongly suggest the involvement of protein kinase C in the proteolytic enzyme release from cercariae which come into contact with skin lipid. The mechanisms by which phorbol esters and linoleic acid stimulate cercariae to release the proteolytic enzyme and the necessity of Ca 2+ in release of proteolytic enzyme were discussed.

Diacylglycerols induce both ion pumping in patch-clamped guard-cell protoplasts and opening of intact stomata.

Stomatal guard cells in leaves regulate the apertures of microscopic pores through which photosynthetic gas exchange and water vapor loss occur. Environmental signals, including light, high humidity, and low CO2 concentrations, open stomata by increasing the volume of guard cells. Activation of a plasma membrane H+ pump initiates K+ and Cl- influx, accompanied by malate synthesis, resulting in osmotic water flow into the guard cells, a bowing apart of the guard-cell pair, and consequent stomatal opening. Physiological and electrophysiological techniques were employed to investigate the possibility that a second-messenger lipid, 1,2-diacylglycerol, is involved in the transduction of opening stimuli. The synthetic diacylglycerols 1,2-dihexanoylglycerol and 1,2-dioctanoylglycerol enhanced light-induced stomatal opening in Commelina communis and induced stomatal opening under darkness, whereas an isomer with no known second-messenger role, 1,3-dioctanoylglycerol, did not affect stomatal responses. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, the enzyme typically activated by 1,2-diacylglycerol in animal cells, inhibited light-stimulated stomatal opening and enhanced dark-induced stomatal closure. N-[(2-Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), which inhibits cyclic nucleotide-dependent protein kinases preferentially over lipid-dependent protein kinases such as protein kinase C, had little effect on stomatal apertures. Whole-cell patch clamping of guard-cell protoplasts of Vicia faba revealed that 1,2-dihexanoylglycerol and 1-oleoyl-2-acetylglycerol activated an ATP-dependent, voltage-independent current, suggesting activation of an electrogenic ion pump such as the H+ pump. Diacylglycerol or functionally similar lipids may act through protein phosphorylation to provide the intracellular signals that mediate H+-ATPase activation and stomatal opening in response to light or other opening stimuli.

Decreased respiratory burst activity in neonatal bovine neutrophils stimulated by protein kinase C agonists

SUMMARY Newborn calves have a high susceptibility to bacterial infections, which may be related to the impaired neutrophil defense functions in newborns. The oxygen-dependent production of the free radical superoxide anion (O2-) represents an important part of the leukocyte respiratory burst central to neutrophil-directed defenses against bacterial infection. Because protein kinase C (pkc) activation is considered to be an important step in the signal transduction pathway for the O2- generating system, we compared O2- production by newborn and adult bovine neutrophils stimulated with 3 different pkc agonists. When the phorbol ester phorbol 12-myristate 13-acetate (pma) was used, pkc-dependent O2- generation from newborn neutrophils was significantly reduced (P < 0.01) for all concentrations of pma tested (10, 100, and 500 ng/ml). In addition, newborn neutrophils had a significantly (P < 0.01) reduced lag time for O2- generation. Similar significantly (P < 0.01) reduced O2- generation from newborn neutrophils was observed with an additional phorbol ester (phorbol 12, 13-dibutyrate); lag times were not calculated for phorbol 12, 13-dibutyrate. When O2- generation was stimulated with a synthetic diacylglycerol analogue (1,2-dioctanoyl-sn-glycerol), less O2- was generated from both adult and newborn neutrophils than was obtained with the phorbol esters, and newborn neutrophils produced significantly (P < 0.01) less O2- only at 50 μM 1,2-dioctanoyl-sn-glycerol. To assess the importance of pkc and cyclic nucleotide-dependent protein kinases in the O2--related signal-transduction pathways of bovine neutrophils, we determined the influence of a pkc inhibitor, H-7, and a cyclic nucleotide-dependent protein kinase inhibitor, HA-1004, on the respiratory burst of adult and newborn bovine neutrophils. Preincubation of neutrophils (15 minutes) with 100 or 500 μM H-7 inhibited subsequent pma-induced O2- generation from newborn and adult neutrophils in a dose-dependent fashion, but preincubation with HA-1004 did not affect subsequent pma-induced O2- generation from either newborn or adult neutrophils. The pkc inhibitor, H-7 (100 and 500 μM), induced a significantly (P < 0.01) prolonged lag time in adult, but not newborn, neutrophils; HA-1004 had no effect on lag time. These results indicate that pkc, but not cyclic nucleotide-dependent protein kinase, is important in the signal-transduction pathway leading to O2- generation in appropriately stimulated adult and newborn bovine neutrophils, and that deficient respiratory burst activity in newborn neutrophils may be explained by altered activation of pkc.

Cyclic nucleotide-dependent protein kinases in the neural tissue of the honeybee Apis mellifera

Two cyclic nucleotide-dependent protein kinases have been purified and characterized from neural tissue of the honeybees Apis mellifera. The first kinase ( M r = 180,000 ) has a tetrameric structure with two regulatory and two catalytic subunits that dissociate upon addition of cAMP. The regulatory subunits exist in phospho- and dephosphoform, which show electrophoretic mobilities in SDS-PAGE corresponding to M r = 50,000 and M r = 49,000 , respectively. The values for half-maximal stimulation using histone IIA as substrate is 3.3 nM cAMP or 33 nM cGMP. The second cyclic nucleotide-dependent protein kinase, with M r = 80,000 , is dimeric with a regulatory (phosphoform M r = 36,000 ) and a catalytic subunit. Detection of this regulatory subunit in heads and brains homogenized in presence of SDS, TCA and various protease inhibitors supports strong evidence that the dimeric kinase is no artefact but exists in vivo. In contrast to the regulatory subunit of the tetrameric kinase, the regulatory subunit of the dimeric kinase is localized only in the soluble fraction.

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

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

Protein Kinase Inhibitor H-7 Increases Lipoprotein Lipase Activity in Isolated Rat Fat Pads.

A protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) increased lipoprotein lipase (LPL) activity in isolated rat fat pads in a time- and dose-dependent manner. The incubation of H-7 with partially purified LPL did not affect its activity. Under the marked inhibition of protein synthesis by cycloheximide, H-7 still showed a full effect on the increase in LPL activity. A slight but significant increase in LPL activity in the fat pads was observed with inhibitors of cyclic nucleotide-dependent protein kinase. H-7, therefore, may increase LPL activity through processes other than the direct activation of the LPL molecule, or the stimulation of LPL molecule synthesis; probably through a decrease in the activity of protein kinases, especially protein kinase C.

Effect of H-8, an isoquinolinesulfonamide inhibitor of cyclic nucleotide-dependent protein kinase, on cAMP- and cGMP-mediated vasorelaxation.

We theorized that H-8, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cyclic nucleotide-dependent protein kinases, might be a useful probe to assess cyclic nucleotide-dependent relaxation of blood vessels. However, working in the rat caudal artery and aorta, we found that neither cyclic AMP- nor cyclic GMP-mediated relaxations were diminished by even large doses of H-8. For example, in the caudal artery, relaxation of a phenylephrine contraction by 8-bromo-cGMP (10(-5) M) was unaffected by H-8: control, 33 +/- 6.2%; 10 microM H-8, 41 +/- 12%; 30 microM H-8, 30 +/- 16% (p NS). The amount of relaxation by 8-bromo-cAMP (3 x 10(-4) M) was actually increased by H-8: control, 29 +/- 7.6%; 10 microM H-8, 34 +/- 7.4%; 30 microM H-8, 80 +/- 14%. In the rat aorta, H-8 also failed to diminish relaxation induced by 8-bromo-cGMP, or by atriopeptin II or sodium nitroprusside. In both caudal artery and aorta, H-8 of itself caused a dose-dependent suppression of alpha-adrenergic contraction: for example, in the caudal artery, with 10 or 30 microM H-8, peak contraction to phenylephrine was reduced to 70 (SEM) +/- 12% or 52 +/- 7% of control, respectively. The results suggest that the protein kinase inhibitor H-8 is not a useful probe to study cyclic nucleotide-dependent relaxation.

Pharmacologic evidence for the requirement of protein kinase C in IFN-induced macrophage Fc gamma receptor and Ia antigen expression.

Previous studies have implicated protein kinase C (PKC) as a mediator in the activation of macrophages by interferons. In order to probe further into the suspected role of protein kinase C in mouse peritoneal macrophage activation, the effects of protein kinase inhibitors in macrophage Fc gamma R and Ia Ag expression were studied. The protein kinase inhibitor, H7, reduced basal levels, and inhibited IFN-alpha-induced expression of Fc gamma R significantly. The concentration of H7 required to inhibit 50% of the Fc gamma R induction was approximately 12 microM, which reflects the previously reported affinity of this compound for PKC in vitro. H7 had only a minimal effect on IFN-gamma-induced Fc gamma R, suggesting different pathways of Fc gamma R induction by the two types of IFN. Ia induction by IFN-gamma was also inhibited by H7, indicating that both types of IFN can utilize PKC to mediate at least part of the signal required for Fc gamma R or Ia expression. HA-1004, a derivative of H7 which possesses high affinity for cyclic nucleotide-dependent protein kinases, but low affinity for PKC, did not alter induction, while H8, a slightly less effective PKC inhibitor than H7, was effective at higher concentrations. Another structurally distinct PKC antagonist, staurosporine, was also effective inhibiting IFN-alpha-induced Fc gamma R and IFN-gamma-induced Ia Ag expression, providing additional evidence that PKC is important. H7 was found to be effective when added as late as several hours after IFN treatment, indicating a prolonged or delayed requirement of PKC for optimal induction of Ia and Fc gamma R by IFN.

Does Protein Phosphorylation by Protein Kinase C Support Pseudopodial Cable Growth in Cultured Micromere-Derived Cells of the Sea Urchin, Hemicentrotus pulcherrimus?: (sea urchin/protein kinase C/spicule formation/phorbol ester/H-7).

In cultured cells derived from micromeres, H-7 strongly inhibited the outgrowth of pseudopodial cables and the formation of spicule rods at concentrations around the Ki values for protein kinases. HA1004 did not inhibit the cable growth and spicule rod formation in these cells at higher concentrations than the Ki values for cyclic nucleotide-dependent protein kinases. Pseudopodial cable growth was also inhibited by H-7 in furosemide-treated cells which were able to undergo normal growth of the cables without the formation of spicule rods. Protein phosphorylation, measured by 32 P incorporation into proteins in the cells exposed to 32 Pi, was inhibited by H-7 at the concentrations for the blockage of the cable growth but was hardly blocked by HA1004. The cable growth and protein phosphorylation were activated by phorbol 12-myristate 13-acetate. The activity of Ca2+ , phospholipid-dependent protein kinase (protein kinase C), which was inhibited by H-7, became appreciably high in micromere-derived cells at 16 hr of culture at 20°C, at which the outgrowth of pseudopodial cables was going to be initiated and gradually increased keeping pace with the cable growth. These suggest that the outgrowth of the cables is supported by protein phosphorylation catalyzed by protein kinase C.

Endotoxin-mediated bovine alveolar macrophage procoagulant induction is dependent on protein kinase C activation

The induction of pulmonary alveolar macrophage (PAM) tissue factor-dependent procoagulant activity is central to the deposition of inflammatory fibrin in the pulmonary alveolus. The presence of enhanced tissue factor activity is often associated with pulmonary fibrin deposition, an important pathogenetic event that can delay resolution of pulmonary inflammation and promote the induction of pulmonary fibrosis. Since tissue factor synthesis induction and activation pathways are potential therapeutic targets for modulation of alveolar macrophage tissue factor (procoagulant) activity, we examined the pathways through which endotoxin lipopolysaccharide (LPS) induces bovine PAM tissue factor-dependent procoagulant activity. PAM procoagulant activity was markedly enhanced to 10 times the levels of freshly isolated PAM after 8 h of culture in the presence of either the protein kinase C (PKC) agonist phorbol 12-myristate 13-acetate (PMA) or LPS. Both LPS-(P less than 0.002) and PMA-induced activity (P less than 0.007) was completely ablated by the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H 7,100 microM) but was unaffected by the cyclic nucleotide-dependent protein kinase inhibitor N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 100 microM). The arachidonate cyclooxygenase pathway inhibitor phenylbutazone (10(-4) M) had modest effects that were not statistically significant. The unstimulated increase of procoagulant activity in 8-h cultures was unaffected by the same inhibitory modulations.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased rate of phosphorylation-dephosphorylation of the translational initiation factor eIF-4E correlates with the induction of protein and glycoprotein biosynthesis in activated B lymphocytes.

A 10-50-fold, biphasic increase in the rate of 32Pi labeling of eIF-4E was closely correlated with the induction of protein and glycoprotein biosynthesis when resting murine splenic B lymphocytes (B cells) were activated by bacterial lipopolysaccharide or the combination of phorbol 12-myristate 13-acetate and ionomycin. The fraction of eIF-4E which was phosphorylated only increased from 46% in resting cells to 83% in lipopolysaccharide-activated cells. This discrepancy between the increase in the fraction of phosphorylated eIF-4E and the increase in 32Pi labeling suggested that the phosphoryl group of eIF-4E turns over slowly in resting B cells compared with activated cells. The turnover rate for the eIF-4E phosphate moiety in lipopolysaccharide-activated cells was rapid (t1/2 = 2 h) in comparison to the eIF-4E polypeptide chain, which did not turn over detectably in 6 h. Neither protein kinase C nor a cyclic nucleotide-dependent protein kinase appeared to be involved in eIF-4E phosphorylation in B cells, based on the observations that the metabolic labeling of eIF-4E by 32Pi was insensitive to the protein kinase inhibitors H-7 and HA1004, and that maximal labeling occurred after protein kinase C activity was "down-regulated" to very low levels in phorbol 12-myristate 13-acetate/ionomycin-activated cells. Dephosphorylation in vivo was blocked by okadaic acid (IC50 = 200 nM). These results indicate that a rapid phosphorylation-dephosphorylation of eIF-4E is associated with high translation rates during the activation of B cells, and implicate protein phosphatase-1 (or possibly-2A) in the dephosphorylation of the initiation factor.