Increased cyclic AMP response to forskolin in Epstein-Barr virus-transformed human B-lymphocytes derived from schizophrenics

Phorbol 12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator, elevated basal cyclic AMP levels and enhanced isoproterenol-, prostaglandin E₁- (PGE₁), forskolin- and cholera toxin-stimulated cyclic AMP accumulation in Epstein-Barr virus (EBV)-transformed human B-lymphocytes. Staurosporine, a PKC inhibitor, significantly antagonized the increase in cyclic AMP accumulation produced by PMA, whereas the inactive phorbol ester, 4α-phorbol 12,13-didecanoate (4αPDD), had no effect. Basal levels of cyclic AMP and the accumulation of cyclic AMP produced by PMA, isoproterenol, PGE₁, cholera toxin and the combination of these compounds with PMA were not significantly different between schizophrenics and controls. The cyclic AMP response to forskolin in the presence and absence of PMA was significantly greater in EBV-transformed human B-lymphocytes from schizophrenics. These results suggest that activation of adenylyl cyclase by forskolin is elevated in EBV-transformed B-lymphocytes derived from schizophrenics and that this elevation is further enhanced through a PKC-dependent phosphorylation mechanism. Received: 20 May 1996/Final version: 6 November 1996     

Regulation of cyclic AMP metabolism in bovine adrenal medullary cells.

The capacity of cultured bovine adrenal medullary cells to metabolize and export cyclic AMP has been studied. Basal cellular cyclic AMP levels were increased 50% by 100 microM 3-isobutyl-1-methylxanthine (IBMX) and rolipram, a class IV (cyclic AMP-specific) phosphodiesterase (PDE) inhibitor. They were not affected by inhibition of class I (Ca2+/calmodulin-dependent), class III (cyclic GMP-inhibited) or class V PDE (cyclic GMP-specific) with vinpocetine or 3-isobutyl-8-methoxymethyl-1-methylxanthine (8-methoxymethyl-IBMX), SK&F 94120, or MB 22,948, respectively, all at 100 microM. Furthermore, only IBMX and rolipram enhanced the cyclic AMP response to 0.3 microM forskolin. Rolipram had an EC50 of < or = 1 microM and was equally effective at 100 microM and 1 mM. IBMX enhanced cyclic AMP levels significantly more at 1 mM than at 100 microM. Neither vinpocetine nor 8-methoxymethyl-IBMX (100 microM) enhanced the Ca(2+)-dependent cyclic AMP response to K+ depolarization. Elevation of cyclic GMP levels with sodium nitroprusside (10 or 100 microM), to activate any cyclic GMP-stimulated class II PDE and to inhibit any cyclic GMP-inhibited class III PDE, also had no effect on basal or forskolin-stimulated cyclic AMP levels. In the presence of IBMX (1 mM), forskolin (5 microM) caused a rapid and large increase in cellular cyclic AMP levels which was maximal after about 5 min and declined slightly over 3 hr. Over this period, extracellular cyclic AMP levels rose almost linearly reaching levels 2-3 times those in the cells. The results indicate bovine adrenal medullary cells have a high capacity for sustained cyclic AMP export. Furthermore, two PDE isozymes appear to degrade cyclic AMP in these cells, a rolipram-sensitive, cyclic AMP-specific, class IV isozyme and a rolipram-insensitive isoform.     

Protein kinase C activation increases noradrenaline release from the rat hippocampus and modifies the inhibitory effect of α2-adrenoceptor and adenosine A1-receptor agonists

Summary We have studied the effect of stimulating protein kinase C with phorbol esters on the release of [³H]-noradrenaline (NA) in the absence or presence of presynaptic ₂-adrenoceptor blocking agents and compared that to the elevation of cyclic AMP levels more than 10-fold by a combination of rolipram and forskolin. 4--Phorbol 12,13-dibutyrate (PDiBu) increased stimulated (3 Hz) [³H]-NA release markedly and in a concentration dependent manner. 4--Phorbol-12,13-didecanoate was ineffective. The effect of PDiBu was not significantly reduced by nifedipine (1 M), but was proportionally less in the presence of an ₂-adrenoceptor antagonist, yohimbine. PDiBu inhibited the presynaptic effect of ₂-adrenoceptor agonists clonidine and UK 14304. By contrast, the presynaptic effect of the adenosine analogue R-PIA was not reduced by PDiBu. PDiBu caused an increase in cyclic AMP that depended on adenosine receptor stimulation. Elevation of cyclic AMP had a limited effect on NA release from rat hippocampus, and did not significantly decrease the presynaptic inhibitory effect of UK 14304 (0.1 M), of morphine (1 M) or of the adenosine A₁-receptor agonist CHA (1 M). The effect of phorbol esters and several presynaptic inhibitors of NA-release in the rat hippocampus cannot be explained by changes in cyclic AMP levels in the tissue. Phorbol esters that stimulate protein kinase C appear to interact with a target that is the site of action ₂-adrenoceptors in this tissue. This site is not a dihydropyridine sensitive Ca-channel and is also different from the target of presynaptic adenosine receptors. Thus, activation of protein kinase C discriminates between apparently similar presynaptic mechanisms. Send offprint requests to B. Fredholm at the above address     

Possible Involvement of Protein Kinase C in the Modulation of Inotropic and Chronotropic Effects Induced by Ouabain in Rat Right Atrial Muscles

Modulations of the inotropic and chronotropic effects of ouabain and protein kinase C (PKC) stimulation with phorbol esters in rat right atria were examined. Cumulative administration of ouabain (3–30 μm ) caused a positive inotropic effect in a concentration-dependent manner, but did not produce a chronotropic effect. A single administration of ouabain (30 μm ) also had similar effects: +744 ± 84% (n = 23, P < 0.01) in the contractile force and ?0.7 ± 1.3% (n = 23, P > 0.05) in the sinus rate. Addition of phorbol esters reinforced the ouabain-evoked positive inotropic effect: 26.5 ± 8.9% (n = 6, P < 0.05) with 100 μm 4-β-phorbol-12,13-dibutyrate (PDB), and 6.4 ± 3.3% (n = 6, P > 0.05) with 100 μm 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Simultaneously, the mixture of ouabain and phorbol ester raised the resting tension. Phorbol esters alone caused a positive inotropic effect (by about 21–27%). Non-PKC activating phorbol ester, 4-α-phorbol-12,13-didecanoate (PDD, 100 μm ), did not have any effect. Pretreatment with the PKC inhibitor (staurosporine 100 μm ) significantly decreased the ouabain-induced positive inotropic effect and caused a negative chronotropic effect, but H-7 (1-(5-isoquinolinylsulphonyl)-2-methylpiperazine dihydrochloride) (5 μm ) had no effect. These results suggest that PKC stimulation may be involved in the ouabain-evoked responses in the right atria of rat as seen by increased cellular Ca²⁺ concentration (and Ca²⁺-sensitivity); thus the positive inotropic effect may not be due only to modulation of Na⁺/K⁺ pump activity.     

Role of protein kinase C in bradykinin-induced prostaglandin formation in osteoblasts

Bradykinin (1 μM 5 min) induced translocation of protein kinase C (PKC) to the plasma membrane fraction in osteoblastic MC3T3-E1 cells. Bradykinin also enhanced the binding of phorbol 12,13-dibutyrate (PDBu) to intact cells, a measure of PKC activation. Addition of bradykinin (1 μM) to cells preincubated with [³H]PDBu (10 nM, 20 min) caused an increase in specific PDBu binding that was maximal after 5–10 min. The bradykinin-induced enhancement of PDBu binding was seen at 1 nM and was maximal at 10 nM. The bradykinin B1 receptor agonist des-Arg⁹-bradykinin (1 μM) did not enhance specific PDBu binding to intact MC3T3-E1 cells. PDBu at and above 3 nM stimulated the formation of prostaglandin E2 (PGE₂) in MC3T3-EI cells. This stimulatory effect was seen after 15–20 min incubation. The Ca²⁺ ionophore A23187 at and above 1 μM induced a rapid (within seconds) burst of PGE₂ formation in MC3T3-E1 cells. The effect of PDBu and A23187 on PGE₂ formation was synergistic. The PKC inhibitor staurosporine (200 nM) inhibited basal as well as bradykinin-induced prostaglandin-formation in MC3T3-E1 cells. In conclusion: bradykinin enhances PKC activation in osteoblastic MC3T3-E1 cells. This kinase activation may be involved in bradykinin-induced prostaglandin formation.     

Diverse effects of Ca2+ on the prostacyclin and corticotropin modulation of adenosine 3': 5'-monophosphate and steroid production in normal cat and mouse tumor cells of the adrenal cortex

Isolated cat adrenocortical cells exposed to corticotropin (ACTH) (2–50 μU) and prostacyclin (PGI₂) (10^?8 to 10^?4M) demonstrated dose- and time-dependent increases in cyclic AMP formation and steroid production. In the absence of extracellular Ca²⁺ ACTH (5 μU) did not augment cyclic AMP levels and steroid release. In contrast, Ca²⁺ deprivation did not affect the ability of PGI₂ (10^?6M) to augment cyclic AMP levels and only partially depressed PGI₂-induced steroid release, indicating that ACTH and PGI₂ affect the adenylate cyclase-cyclic AMP system differently. The distinctive effects of PGI₂ and ACTH were further demonstrated in mouse adrenal tumor (Y-1) cells, which responded to ACTH and cholera extertoxin (choleragen) (10^?9 M) with increases in cyclic AMP formation and steroidogenesis, whereas PGI₂ elicited a profound decrease in cyclic AMP levels and failed to enhance steroid production. The basis of these characteristic effects of ACTH and PGI₂on normal and adrenal tumor cells appears to be their particular actions on membrane receptors, which modulate cyclic AMP metabolism through different Ca²⁺ -mediated mechanisms.     

Involvement of protein kinase C in the UTP-mediated potentiation of cyclic AMP accumulation in mouse J774 macrophages

1. We have investigated the effects of nucleotide analogues on cyclic AMP formation in mouse J774 macrophages and the mechanisms involved.
2. UTP, in the concentration range 0.1–100 μM, induced concentration-dependent potentiation of prostaglandin E₁ (PGE₁)-induced cyclic AMP formation, but had no effect on basal cyclic AMP formation. UDP showed an equal potency, while 2-methylthio ATP, α,β-methylene ATP and β,γ-methylene ATP gave either a slight increase or had no effect at concentrations up to 100 μM. ATP, although 100 fold less effective than UTP, also caused cyclic AMP potentiation, but had no effect on agonist-stimulated or basal cyclic AMP levels.
3. The cyclic AMP potentiation effect of UTP correlated with increased [Ca²⁺]_i and inositol phosphate (IP) formation over the same concentration range.
4. Ionomycin, which evokes an increase in [Ca²⁺]_i without affecting IP formation, did not cause an increase in cyclic AMP content, indicating that UTP-induced cyclic AMP regulation is not due to activation of Ca²⁺-sensitive adenylyl cyclase isoforms.
5. Although reduced, UTP potentiation was seen in cells incubated in a Ca²⁺-free and/or BAPTA-containing medium. Under these conditions, the UTP-increased IP accumulation was similarly reduced.
6. Exposure of cells to phorbol 12-myristate 13-acetate (PMA) also increased PGE₁ stimulation of cyclic AMP levels, and the UTP-induced potentiation of cyclic AMP formation was inhibited by either staurosporine or Ro 31-8220. Pretreatment of cells with PMA for 4–24 h resulted in marked attenuation of UTP-stimulated cyclic AMP potentiation.
7. Pretreatment with pertussis toxin (24 h, 100 ng ml⁻¹) did not significantly affect UTP-induced cyclic AMP potentiation and IP formation, although it increased the cyclic AMP response to PGE₁.
8. Analysis of J774 cells by Western blotting with antibodies specific for different protein kinase C (PKC) isoforms shows the presence of the βI, βII, δ, ε, ζ, μ, λ and ζ isoforms. Moreover, UTP significantly increased the level of PKC βI, βII, δ, ε, μ, λ and ζ immunoreactivity in the membrane fraction and decreased the cytosolic reactivity of PKC βII, δ, ε and ζ.
9. Immunoblot studies also indicate the presence of type II adenylyl cyclase.
10. These results indicate that PKC is required for the potentiation of adenylyl cyclase activity by macrophage pyrimidinoceptors, which exhibit a higher specificity for UTP and UDP than for ATP.

     

Nicotine-like effects of cotinine on protein kinase C activity and noradrenaline release in bovine adrenal chromaffin cells

1 We studied the effect of cotinine, a slowly eliminated metabolite of nicotine, on protein kinase C (PKC) distribution and noradrenaline release in primary cultured bovine adrenal chromaffin cells. Changes in PKC activity were detected by [³H]-phorbol-12,13-dibutyrate binding, histone phosphorylation assay and by Western blot. 2 Cotinine (10–32 mm ) increased phorbol binding to chromaffin cells in response to 10 min but not to 24 h exposure. The increased binding was reversed by a nicotinic antagonist hexamethonium (10 μm ). 3 Cotinine (10 mm , 30 min) also increased membrane-associated PKC activity and membrane-associated PKC α and ε immunoreactivity. 4 Cotinine (0.1–32 mm for 10 s to 20 min) dose- and time-dependently increased the release of preloaded [³H]-noradrenaline from the cultured cells. The release increased with increasing duration of the contact period. In treatments lasting 1 min or longer, a peak effect was followed by a reduced response at higher concentrations. 5 We confirm the earlier findings that cotinine is biologically active, and conclude that its effects are at least partly mediated via nicotinic cholinergic receptors and through PKC.     

1-Ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, hydrochloride (SQ 20009)--a potent new inhibitor of cyclic 3',5'-nucleotide phosphodiesterases

The properties of SQ 20009 [1-ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, HCl] as a cyclic nucleotide phosphodiesterase inhibitor have been investigated. The phosphodiesterase preparations used in this study were ammonium sulfate-fractionated supernatants of homogenates of rat brain, rabbit brain, rat adrenal, rat lipocyte and cat heart; commercially available beef heart phosphodiesterase was also studied. The concentrations of SQ 200009 required to inhibit these phosphodiesterase activities 50 per cent were 2·0, 4·8, 20, 21, 27 and 60 μM, respectively, using 1·6 × 10⁻⁷ M cyclic AMP as substrate. SQ 20006 (the parent of SQ 20009 lacking the 4-isopropylidene moiety), theophylline and caffeine were also tested against all six enzyme preparations. Whereas SQ 20009 was more potent than SQ 20006 using the phosphodiesterase prepared from rat adrenal, the potencies were reversed when the lipocyte enzyme was used. SQ 20009 was approximately 60 and 75 times as potent an inhibitor of rat brain cyclic AMP phosphodiesterase as were theophylline and caffeine respectively. The kinetic properties of the phosphodiesterases prepared from rat brain, cat heart and beef heart were also investigated. Using the rat brain enzyme, two K_m values for cyclic AMP, 4·0 × 10⁻⁶ and 1·2× 10⁻⁴ M and a single K_m, 2·0 × 10⁻⁵ M, for cyclic GMP were confirmed. The K_i of SQ 20009 against the low K_m cyclic AMP phosphodiesterase was 2·0 × 10⁻⁶ M and that for cyclic GMP hydrolysis was 2·4 × 10⁻⁵ M. The inhibition by SQ 20009 of the hydrolysis of both cyclic nucleotides by both the rat brain and beef heart phosphodiesterases was competitive. The cat heart cyclic nucleotide phosphodiesterase was inhibited non-competitively by SQ 20009; the K_i for cyclic AMP hydrolysis was 6·4 × 10⁻⁵ M, and the K_i for cyclic GMP hydrolysis was 3·0 × 10⁻⁵ M. The inhibition by SQ 20009 of cyclic AMP hydrolysis by both the rat brain and cat heart preparations was reversible.     

Phorbol-12,13-dibutyrate enhances the cyclic AMP accumulation in rat hippocampal slices induced by adenosine analogues

Summary Phorbol-12,13-dibutyrate (PDiBu) augmented the accumulation of [³H]-cyclic AMP in rat hippocampal slices elicited by the adenosine receptor agonist 5-(N-ethyl)carboxamidoadenosine (NECA). A similar, but less pronounced, effect was observed when using tetradecanoylphorbol acetate (TPA) provided that the slices were preincubated with the phorbol ester before addition of NECA. PDiBu also induced cyclic AMP accumulation in the absence of NECA, but this effect could be markedly reduced or even abolished by the adenosine antagonist 8-para sulphophenyl theophylline (8-pst). Whereas no clearcut synergism was observed between isoprenaline and PDiBu under normal circumstances, such synergism was observed after elimination of the cyclic AMP accumulation caused by endogenous adenosine. PDiBu slightly enhanced the stimulatory effect of low (0.1–1 M) concentrations of forskolin, but did not enhance the effect of 10 M forskolin. In the presence of forskolin, another adenosine analogue, R-PIA, had a biphasic effect on cAMP accumulation. With PDiBu present the inhibitory phase was entirely eliminated and the stimulatory phase substantially enhanced. A synergistic interaction between PDiBu and NECA was found even after inhibition of the N_i protein by N-ethyl maleimide.It is concluded that the phorbol ester may enhance the stimulatory effect of adenylate cyclase stimulatory compounds, including endogenous adenosine. The effect may partly depend on protein kinase C-mediated inactivation of N-protein mediated adenylate cyclase inhibition, but other mechanisms also seem to be involved.Abbreviations PDiBu Phorbol-12,13-dibutyrate - TPA 12-0-tetradecanoyl-phorbol 13-acetate - OAG 1-oleyl-2-acetyl glycerol - 8-pst 8-parasulphophenyltheophylline - NA noradrenaline - NECA 5-N-ethylcarboxamido adenosine - R-PIA (–)-R-N⁶-phenylisopropyl adenosine Send offprint requests to B. B. Fredholm at the above address     

INVOLVEMENT OF PROTEIN KINASE C IN THE REGULATION OF Na+/Ca2+ EXCHANGER IN BOVINE ADRENAL CHROMAFFIN CELLS

• 1 The Na⁺/Ca²⁺ exchanger (NCX) exchanges Na+ and Ca²⁺ bidirectionally through the forward mode (Ca²⁺ extrusion) or the reverse mode (Ca²⁺ influx). The present study was undertaken to clarify the role of protein kinase C (PKC) in the regulation of NCX in bovine adrenal chromaffin cells. The Na⁺‐loaded cells were prepared by treatment with 100 µmol/L ouabain and 50 µmol/L veratridine. Incubation of Na⁺‐loaded cells with Na⁺‐free solution in the presence of the Ca²⁺ channel blockers nicardipine (3 µmol/L) and ω‐conotoxin MVIIC (0.3 µmol/L) caused Ca²⁺ uptake and catecholamine release.
• 2 The Na⁺‐dependent Ca²⁺ uptake and catecholamine release were inhibited by 2‐[4‐[(2,5‐difluorophenyl)methoxy]phenoxy]‐5‐ethoxyaniline (SEA0400; 1 µmol/L) and 2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]isothiourea (KB‐R7943; 10 µmol/L), both NCX inhibitors. These results indicate that the Na⁺‐dependent responses are mostly due to activation of the NCX working in the reverse mode.
• 3 In addition, we examined the effects of PKC inhibitors and an activator on the NCX‐mediated Ca²⁺ uptake and catecholamine release. Bisindolylmaleimide I (0.3–10 µmol/L) and chelerythrine (3–100 µmol/L), both PKC inhibitors, inhibited NCX‐mediated responses. In contrast, phorbol 12,13‐dibutyrate (0.1–10 µmol/L), a PKC activator, enhanced the responses. Bisindolylmaleimide I and chelerythrine, at effective concentrations for inhibition of Na⁺‐dependent catecholamine release, had a little or no effect on high K⁺‐induced catecholamine release in intact cells or on Ca²⁺‐induced catecholamine release in β‐escin‐permeabilized cells.
• 4 These results suggest that PKC is involved in the activation of NCX in bovine adrenal chromaffin cells.

     

P-glycoprotein phosphorylation/dephosphorylation and cellular accumulation of L-DOPA in LLC-GA5 Col300 cells

1 The present work was aimed to study the effect of PKC activation and protein-serine/threonine phosphatase (PP1/PP2 A) inhibition on P-glycoprotein (P-gp) mediated transport of L-DOPA in LLC-GA5 Col300 cells, a renal cell line expressing the human P-glycoprotein in the apical membrane. 2 L-DOPA accumulation was a time-and concentration-dependent process with the following kinetic characteristics: k_in, 57.3 ± 1.2 pmol mg protein^?1 min^?1; k_out, 3.3 ± 0.1 pmol mg^?1 protein min^?1; A_max, 10.6 ± 0.8; K_m, 198 ± 64 μm ; V_max, 5.2 ± 0.7 nmol mg protein^?1. 3 Verapamil (25 μm ), a P-glycoprotein inhibitor, markedly increased (≈ 40% increase) the accumulation of a non-saturating concentration of L-DOPA (2.5 μm ) at both initial rate of uptake (IRU, 6 min incubation) and at steady-state (SS, 30 min incubation). 4 PKC activation with phorbol 12,13-dibutyrate (PDBu, 1, 3 and 10 n m ) produced a concentration-dependent decrease in L-DOPA accumulation at SS, but not at IRU. The inactive phorbol ester, 4α-phorbol 12,13-didecanoate (100 n m ), produced no change in L-DOPA accumulation. The effect of PDBu was completely reverted by staurosporine (100 n m ). The phosphatase inhibitor okadaic acid (100 n m ) reduced by 20% the accumulation of L-DOPA at IRU, but not at SS. 5 It is suggested that P-glycoprotein plays a role in regulation of intracellular availability of L-DOPA in renal epithelial cells, and phosphorylation/dephosphorylation of P-glycoprotein may be involved in the regulation of the transporter.     

Role of a pertussis toxin sensitive G-protein in mediating the effects of phorbol esters on receptor activated cyclic AMP accumulation in Jurkat cells

Summary In the human T -cell line, Jurkat, the accumulation of cyclic AMP induced by adenosine is enhanced by tumor-promoting phorbol esters, whereas prostaglandin E₂ receptor-stimulated cAMP accumulation is antagonized (Nordstedt et al. 1989). In the present study we examine the involvement of pertussis toxin sensitive guanine nucleotide binding proteins (G-proteins) in producing the phorbol ester effects.Pertussis toxin pretreatment of the Jurkat cells invariably caused an ADP ribosylation of two G-proteins that inhibit adenylyl cyclase, tentatively identified as G_i2 and G_i3, using Western blots. Pertussis toxin treatment had little effect on basal cAMP accumulation, but sometimes inhibited, sometimes stimulated agonist and cholera toxin induced cAMP accumulation. The latter effect was not mimicked by the B-oligomer. Irrespective of whether pertussis toxin stimulated or inhibited NECA and cholera toxin-induced cAMP accumulation it could not block the effect of phorbol-12,13-dibutyrate (PDBu). The inhibitory effect of PDBu on prostaglandin E2-induced cAMP accumulation was, however, invariably eliminated by pertussis toxin treatment.In conclusion, activation of protein kinase C by phorbol esters reveals a G_i-mediated prostaglandin E receptor-induced inhibition of adenylate cyclase in addition to the prostaglandin E receptor-mediated stimulation of cAMP accumulation in Jurkat cells. The enhancement of adenosine A₂ receptor stimulated CAMP accumulation by PDBu, on the other hand, does not involve a PTX sensitive G_i-protein. Send offprint requests to I. van der Ploeg at the above address     

Polymyxin B,a selective inhibitor of protein kinase C,diminishes the release of noradrenaline and the enhancement of release caused by phorbol 12,13-dibutyrate

Summary Slices of the rabbit hippocampus were labelled with ³H-noradrenaline, superfused continuously with a modified Krebs-Henseleit medium containing the uptake inhibitor cocaine and stimulated electrically (2 ms, 3 Hz, 24 mA, 5 V/cm). Phorbol 12,13-dibutyrate (PDB), a potent activator of protein kinase C (PKC), strongly enhanced the electrically-evoked overflow of tritium. In contrast, polymyxin B, a relatively selective inhibitor of PKC, diminished the evoked tritium overflow in a time-and concentration-dependent manner. The enhancement of the evoked overflow of tritium caused by PDB was strongly reduced in the presence of polymyxin B (100 mol/l). These results suggest 1. that PKC may be involved in the physiological mechanism of action-potential-induced noradrenaline release from noradrenergic nerve terminals and 2. that the PDB-induced enhancement of noradrenaline release may be due to a direct activation of PKC.Abbreviations PKC protein kinase C - PDB phorbol 12,13-dibutyrate - TPA 12-O-tetradecanoyl 13-acetate     

Histamine-induced increases in cyclic AMP levels in bovine adrenal medullary cells.

1. The effect of histamine on cellular cyclic AMP levels in cultured bovine adrenal medullary cells has been studied. 2. Histamine (0.3-30 microM) increased cyclic AMP levels transiently, with a maximal response after 5 min, a smaller response after 20 min, and no increase seen after 80 or 180 min. The EC50 at 5 min was approximately 2 microM. Histamine had no effect on cyclic AMP release from the cells over 5 min, but increased it after 90 min. 3. The cyclic AMP response to 5 microM histamine was reduced by 45% by 1 microM mepyramine and by almost 30% by 1 microM cimetidine, and was abolished by the combination of both antagonists. Cimetidine at 100 microM did not inhibit the response to histamine more than 1 microM cimetidine. The H3-receptor antagonist, thioperamide (1 microM), had no effect on the response to histamine. 4. The H1-receptor agonist, 2-thiazolyethylamine (5-100 microM) and the H2-receptor agonist, dimaprit (5-100 microM), each induced a cyclic AMP response, and gave more-than-additive responses when combined. The H3 agonist (R) alpha-methylhistamine (100 microM) had no effect either on its own or in combination with either the H1 or the H2 agonist. The response to 100 microM 2-thiazolylethylamine was unaffected by cimetidine (100 microM). 5. The cyclic AMP responses to 5 microM histamine, 100 microM thiazolylethylamine and 100 microM dimaprit were each weakly enhanced in the presence of 1 mM 3-isobutyl-1-methylxanthine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol ester-induced contractions of mouse detrusor muscle are inhibited by nifedipine

The effects of phorbol esters on contractions of detrusor strips isolated from mouse urinary bladder were studied. β-Phorbol-12,13-dibutyrate (β-PDBu, 10 nM) significantly enhances both the neurogenic and myogenic detrusor contractions to a similar extent. By contrast, an inactive isoform of protein kinase C (PKC) stimulation, α- phorbol-12,13-dibutyrate (100 nM) has no such enhancing effect on the muscle contraction. The effect of β-PDBu was dependent on the extracellular Ca²⁺ concentration. Nifedipine (0.3 μM, a L-type Ca²⁺ channel blocker), staurosporine (1 μM) and bisindolylmaleimide I ( μM, a selective PKC inhibitor) but not ω-conotoxin GVIA (an N-type Ca²⁺ channel blocker) abolished the enhancing effect of β-PDBu. In other words, β-PDBu failed to augment the nifedipine-insensitive component of the muscle contraction. Moreover, β-PDBu not only enhances the muscle response induced by exogenous agonists (acetylcholine or ATP) and KCl but also increases the resting tone of detrusor muscle, an effect which is also inhibited by nifedipine and bisindolylmaleimide I. From these findings, it is concluded that the enhancing effect of β-PDBu is due to activation of the L-type Ca²⁺ channel through phosphorylation by protein kinase C. This allows more Ca²⁺ influx from the extracellular medium, leading to an increase in the contractions of the mouse detrusor muscle. Received: 29 October 1997 / Accepted; 17 February 1998     

Ketamine interacts with the noradrenaline transporter at a site partly overlapping the desipramine binding site

Effects of the intravenous anaesthetic ketamine on the desipramine-sensitive noradrenaline transporter (NAT) were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NAT (bNAT). Incubation (1–3 h) of adrenal medullary cells with ketamine (10–300 μM) caused an increase in appearance of catecholamines in culture medium. Ketamine (10–1000 μM) inhibited desipramine-sensitive uptake of [³H] _{noradrenaline} (NA) (IC₅₀=97 μM). Saturation analysis showed that ketamine reduced V _max of [³H]NA uptake without changing K _m, indicating a non-competitive inhibition. Other inhibitors of NAT, namely cocaine and desipramine, showed a competitive inhibition of [³H]NA uptake while a derivative of ketamine, phencyclidine, showed a mixed type of inhibition. Ketamine (10–1000 μM) also inhibited the specific binding of [³H]desipramine to plasma membranes isolated from bovine adrenal medulla. Scatchard analysis of [³H]desipramine binding revealed that ketamine increased K _d without altering B _max, indicating a competitive inhibition. In transfected Xenopus oocytes expressing the bNAT, ketamine attenuated [³H]NA uptake with a kinetic characteristic similar to that of cultured adrenal medullary cells. These findings are compatible with the idea that ketamine non-competitively inhibits the transport of NA by interacting with a site which partly overlaps the desipramine binding site on the NAT. Received: 18 December 1997 / Accepted: 17 June 1998     

Effects of protein kinase C activators upon the late stages of the ACTH secretory pathway of AtT-20 cells.

1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of phorbol 12-myristate 13-acetate (PMA)-mediated enhancement of calcium-evoked adrenocorticotrophin (ACTH) secretion. 2. PMA stimulated ACTH secretion from intact cells in a concentration-dependent manner. Other phorbol esters; phorbol 12,13-dibutyrate (PDBu) and phorbol 12,13-didecanoate (PDD) and diacylglycerol analogues; 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG) also stimulated ACTH release from intact AtT-20 cells. This would suggest that activation of protein kinase C (PKC) stimulates ACTH secretion from AtT-20 cells. 3. Calcium stimulated ACTH secretion from electrically-permeabilized cells over the concentration-range of 10(-7) M to 10(-5) M. PMA (10(-7) M) enhanced the amount of ACTH secreted at every concentration of calcium investigated. The PKC inhibitor, chelerythrine (10(-5) M) blocked the PMA (10(-7) M)-evoked enhancement of calcium (10(-5) M)-stimulated ACTH secretion but did not alter significantly the calcium (10(-5) M)-evoked secretion itself. This suggests that PKC modulates the secretory response to increases in intracellular calcium but does not mediate the effects of calcium. 4. Guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S, 10(-5) M) stimulated ACTH secretion from permeabilized cells in the absence of calcium and was additive with calcium-evoked ACTH secretion up to a maximum value which could be achieved by calcium acting alone. This suggests that a GTP-binding protein mediates the secretory response to increases in the intracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages

1. The modulatory effects of protein kinase C (PKC) on the activation of cytosolic phospholipase A₂ (cPLA₂) and adenylyl cyclase (AC) have recently been described. Since the signalling cascades associated with these events play critical roles in various functions of macrophages, we set out to investigate the crosstalk between PKC and the cPLA₂ and AC pathways in mouse RAW 264.7 macrophages and to determine the involvement of individual PKC isoforms. The cPLA₂ and AC pathways were studied by measuring the potentiation by the phorbol ester PMA of ionomycin-induced arachidonic acid (AA) release and prostagladin E₁ (PGE₁)-stimulated cyclic AMP production, respectively.
2. PMA at 1 μM caused a significant increase in AA release both in the presence (371%) and absence (67%) of ionomycin induction, while exposure of RAW 264.7 cells to PMA increased PGE₁ stimulation of cyclic AMP levels by 208%.
3. Treatment of cells with staurosporine and Ro 31-8220 inhibited the PMA-induced potentiation of both AA release and cyclic AMP accumulation, while Go 6976 (an inhibitor of classical PKC isoforms) and LY 379196 (a specific inhibitor of PKCβ) inhibited the AA response but failed to affect the enhancement of the cyclic AMP response by PMA.
4. Long term pretreatment of cells with PMA abolished the subsequent effect of PMA in potentiating AA release, but only inhibited the cyclic AMP response by 42%.
5. Neither PD 98059, an inhibitor of MEK, nor genistein, an inhibitor of tyrosine kinases, had any effect on the ability of PMA to potentiate AA or cyclic AMP production.
6. The potentiation of AA release, but not of cyclic AMP formation, by PMA was sensitive to inhibition by wortmannin. This effect was unrelated to the inhibition of PKC activation as deduced from the translocation of PKC activity to the cell membrane.
7. Western blot analysis revealed the presence of eight PKC isoforms (α, βI, βII, δ, ε, μ λ and ξ) in RAW 264.7 cells and PMA was shown to induce the translocation of the α, βI, βII, δ, ε and μ isoforms from the cytosol to the cell membrane within 2 min.
8. Pretreatment of cells with PMA for 2–24 h resulted in a time-dependent down-regulation of PKCα, βI, βII, and δ expression, while the levels of the other four PKC isozymes were unchanged after PMA treatment for 24 h. A decrease in the potentiation of AA release by PMA was observed, concomitant with the time-dependent down-regulation of PKC.
9. These results indicate that PKCβ has a crucial role in the mediation of cPLA₂ activation by the phorbol ester PMA, whereas PMA utilizes PKC ε and/or μ to up-regulate AC activity.

     

Stimulatory effect of ionophores on adenosine 3',5'-monophosphate content in human mononuclear leukocytes

Ionophores A23187 and bromo-lasalocid ethanolate enhanced the cyclic AMP content in human mononuclear leukocytes. The maximum effect of A23187 with a 10-min incubation was found with 0.3–1.0μM concentrations with or without l-isoproterenol (1 μM) or prostaglandin E ₁ (pge ₁) (0.3 μM). The maximum effect after 5 min of incubation at 37° was observed with 0.05, 0.2 and 1 μm A23187. The effect of ionophore A23187 was enhanced by both aminophylline (1 mM) and isobutyl-methylxanthine (1 mM). Calcium (1 mM). aspirin (1 mM) and indomethacin (100 μM) decreased the stimulatory action of A23187. Bromo-lasalocid ethanolate increased cyclic AMP content in cells maximally at a 3 μM concentration with or without 0.3 μM pge ₁.