Alpha PDD Research Articles

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, 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD), 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.

Phorbol ester activation of chloride current in guinea-pig ventricular myocytes.

1. Although earlier studies with phorbol esters indicate that protein kinase C (PKC) may be an important regulator of Cl- current (Icl) in cardiac cells, there is a need for additional quantitative data and investigation of conflicting findings. Our objectives were to measure the magnitude, time course, and concentration-dependence of Icl activated in guinea-pig ventricular myocytes by phorbol 12-myristate 13-acetate (PMA), evaluate its PKC dependence, and examine its modification by external and internal ions. 2. The whole-cell patch clamp technique was used to apply short depolarizing and hyperpolarizing pulses to myocytes superfused with Na(+)-, K(+)-, Ca(2+)-free solution (36 degrees C) and dialysed with Cs+ solution. Stimulation of membrane currents by PMA (threshold < or = 1nM, EC50 approximately equal to 14 nM, maximal 40% increase with > or = 100 nM) plateaued within 6-10 min. 3. PMA-activated current was time-independent, and suppressed by l mM 9-anthracenecarboxylic acid (9-AC). Its reversal potential (Erev) was sensitive to changes in the Cl- gradient, and outward rectification of the current-voltage (I-V) relationship was more pronounced with 30 mM than 140 mM Cl- dialysate. 4. The relative permeability of PMA-activated channels estimated from Erev measurements was I- > Cl- > > aspartate. Channel activation was independent of external Na+. 5. PMA failed to activate Icl in myocytes pretreated with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or dialysed with pCa 10.5 solution. Lack of response to 4 alpha-phorbol 12, 13-didecanoate (alpha PDD) was a further indication of mediation by PKC. 6. Icl induced by 2 microM forskolin was far larger than that induced by PMA, suggesting that endogenous protein kinase A is a much stronger Cl- channel activator than endogenous PKC in these myocytes. 7. The macroscopic properties of PMA-induced Icl appear to be indistinguishable from those of PKA-activated Icl. We discount stimulation of PKA by PMA as an explanation, and conclude that endogenous PKC may activate PKA-regulated Cl- channels in these myocytes.

PMA and ionomycin differently affect atrial natriuretic peptide stimulated cyclic GMP production in rat mesangial cells.

How 4 beta-phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io), a calcium ionophore, affect on the atrial natriuretic peptide (ANP) stimulated cyclic-3',5'-guanosine monophosphate (cGMP) production in cultured rat mesangial cells was examined. Cultured mesangial cells were prepared by isolated glomeruli from Sprague Dawley rats employing the sieving method and were used between the 3rd and 15th passage for experiments. cGMP and protein contents were measured by radioimmunoassay and Lowry method. Incubations with effectors were carried out either in the presence or absence of 0.5 mM 1-methyl-3-isobutyl-xanthine (MIX). The intracellular concentration of calcium ([Ca2+]i) was determined by using the Fura-2 method. Pretreatment with PMA, an activator of protein kinase C (PKC), attenuated ANP stimulated cGMP production in a time- and dose-dependent fashion, while alpha PDD (an inactive analog of PMA) did not inhibit cGMP production. PMA inhibition was reversed by addition of staurosporine, a protein kinase C inhibitor. Io attenuated ANP stimulated cGMP production in the absence but not in the presence of MIX. These findings suggested that PMA acts on ANP receptor or guanylate cyclase via activation of PKC in rat mesangial cells. Io may inhibit ANP stimulated cGMP production via activation of cyclic nucleotide phosphodiesterase.

Second messenger signaling of c‐fos gene induction by parathyroid hormone (PTH) and PTH‐related peptide in osteoblastic osteosarcoma cells: Its role in osteoblast proliferation and osteoclast‐like cell formation

The present study was performed to clarify second messenger signaling in parathyroid hormone (PTH)-induced c-fos gene expression, to characterize the participation of the c-fos gene in the regulation of osteoblast proliferation and function as well as osteoclast-like cell formation by PTH and to compare these effects of PTH with those of PTH-related peptide (PTHrP). Both human (h) PTH-(1-34) and hPTHrP-(1-34) at 10(-8) M induced a transient c-fos gene expression to a similar degree in osteoblastic osteosarcoma cells, UMR-106. N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) as well as Sp-diastereoisomer of adenosine cyclic 3',5'-phosphorothioate (Sp-cAMPS), an activator of cAMP-dependent protein kinase (PKA), induced a weak c-fos gene expression. Although Rp-diastereoisomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), an inhibitor of PKA, almost completely antagonized dbCAMP- and Sp-cAMPS-induced expression of c-fos gene, it did not cause an obvious inhibition of PTH- or PTHrP-induced expression. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), induced an intense expression of the c-fos gene, while 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD), incapable of activating PKC, and calcium ionophores (A23187 and ionomycin) did not. Protein kinase C inhibitor (H-7, 50 microM) completely blocked the expression of the c-fos gene by PTH as well as by PTHrP). Antisense oligodeoxynucleotides (as-ODN) complementary to c-fos mRNA, which have been shown to inhibit its mRNA translation, at 1 microM significantly antagonized PTH- and PTHrP-induced inhibition of [3H] thymidine incorporation and stimulation of osteoclast-like cell formation in the presence of osteoblasts, but not an increase in alkaline phosphatase activity, compared to control oligodeoxynucleotides with same nucleotides as as-ODN but with a random sequence. The present study indicates the involvement of PKC system in c-fos gene expression by PTH as well as PTHrP and also indicates the involvement of the c-fos gene in the regulation of bone cell physiology by PTH and PTHrP.

Apoptosis in splenic B lymphocytes. Regulation by protein kinase C and IL-4.

Small dense splenic B lymphocytes from adult specific pathogen-free mice were shown to undergo apoptosis in vitro as indicated by internucleosomal DNA fragmentation, hypodiploid DNA content of isolated nuclei, and morphologic features by electron microscopy. Unstimulated cultures showed spontaneous apoptosis increasing gradually and monotonically from < 2 to 32% of B cells by 16 h. The rate of accumulation of apoptotic cells was reduced by the addition of IL-4 or PMA, but not by the inactive phorbol ester, 4 alpha PDD. In contrast, inhibitors of protein kinase C (H7 and staurosporine) increased the percentage of cells undergoing apoptosis to > 70% by 12 h; HA 1004, genistein, and herbimycin A all had no effect on apoptosis. Thus, protein kinase C activity regulates apoptosis, but there is no evidence that protein kinases A and G and tyrosine kinases are involved. Cycloheximide increased apoptosis, indicating that apoptosis may be restrained in B cells by the presence of one or more labile protective proteins. The percentage of apoptotic cells measured by flow cytometry and the percentage of fragmented DNA measured by the diphenylamine method were nearly equal, regardless of the method of apoptotic regulation. Together with the absence of nuclei with flow cytometric properties intermediate between normal and apoptotic, these results suggest that in individual B cells apoptosis progresses rapidly to completion. These data suggest a fundamental change in our concept of the life-style of the "resting" B cell: instead of a dormant cell remaining unchanged until it receives activation signals, the mature spleen B cell appears programmed to die by apoptosis unless rescued by specific agents, such protein kinase C activators or IL-4.

Modulation of activin A action and specificity in the rat gonadotrope by protein kinase C

This study was undertaken to assess the role of protein kinase C (PKC) in activin action in the rat pituitary. Pretreatment with 500 nM phorbol 12-myristate 13-acetate (PMA) for 22-24 h reduced subsequent FSH and LH release (percentage of total cellular FSH and LH released) in response to 100 nM PMA. This action persisted for 2 days after the pretreatment. Pretreatment with 500 nM 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD, a phorbol ester which does not activate PKC) did not affect cell responsiveness to 100 nM PMA. Both PKC-down-regulated cells and cells with a full complement of PKC responded similarly to 100 nM GnRH and 100 microM A23187 during this period. Incubation with 50 ng/ml activin A for 48 h significantly increased both FSH release and total FSH (extracellular plus intracellular) compared to corresponding basal values in PMA-pretreated cells, as well as in vehicle-or 4 alpha PDD-pretreated cells. Activin stimulation of basal FSH release and total FSH was significantly more potent in PMA-pretreated cells than in cells not pretreated with PMA. Activin did not alter basal LH release or total LH in vehicle- or 4 alpha PDD-pretreated cells but significantly increased both in PMA-pretreated cells. When PMA was present only during the initial 2 h of the 22- to 24-h pretreatment period at 50 nM, PKC was not down-regulated. In these cells, the potency of activin stimulation of basal FSH release was not affected, but stimulation of basal LH release by activin was still observed. These results suggest that PKC is not required for activin to stimulate FSH release but is involved as a modulator of potency and specificity of the activin action.

Modulation of activin A action and specificity in the rat gonadotrope by protein kinase C.

This study was undertaken to assess the role of protein kinase C (PKC) in activin action in the rat pituitary. Pretreatment with 500 nM phorbol 12-myristate 13-acetate (PMA) for 22-24 h reduced subsequent FSH and LH release (percentage of total cellular FSH and LH released) in response to 100 nM PMA. This action persisted for 2 days after the pretreatment. Pretreatment with 500 nM 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD, a phorbol ester which does not activate PKC) did not affect cell responsiveness to 100 nM PMA. Both PKC-down-regulated cells and cells with a full complement of PKC responded similarly to 100 nM GnRH and 100 microM A23187 during this period. Incubation with 50 ng/ml activin A for 48 h significantly increased both FSH release and total FSH (extracellular plus intracellular) compared to corresponding basal values in PMA-pretreated cells, as well as in vehicle-or 4 alpha PDD-pretreated cells. Activin stimulation of basal FSH release and total FSH was significantly more potent in PMA-pretreated cells than in cells not pretreated with PMA. Activin did not alter basal LH release or total LH in vehicle- or 4 alpha PDD-pretreated cells but significantly increased both in PMA-pretreated cells. When PMA was present only during the initial 2 h of the 22- to 24-h pretreatment period at 50 nM, PKC was not down-regulated. In these cells, the potency of activin stimulation of basal FSH release was not affected, but stimulation of basal LH release by activin was still observed. These results suggest that PKC is not required for activin to stimulate FSH release but is involved as a modulator of potency and specificity of the activin action.

Involvement of Protein Kinase C in the Coupling between the V1Vasopressin Receptor and Phospholipase C in Rat Glomerulosa Cells: Effects on Aldosterone Secretion

We have previously shown that arginine vasopressin (AVP) possesses specific binding sites on rat adrenal glomerulosa cells and stimulates phosphoinositide breakdown and accumulation of inositol phosphates (IP) and diacylglycerol. Kinetic experiments also revealed that the production of IP declines rapidly under hormonal stimulation, even in the presence of Ca2+ in the external medium. In the present investigation, we studied the effects of a protein kinase C (PKC) activator phorbol ester (PDBu) on AVP-sensitive accumulation of IP. Experiments were conducted on glomerulosa cells cultured for 3 days. Results show that short term preincubation (5-10 min) with PDBu inhibits AVP-stimulated IP accumulation by 50% (ED50 = 2.6 +/- 0.9 nM). PKC most likely acts on the coupling between AVP receptor and the G-protein since PDBu reduces AVP-sensitive phospholipase C but does not alter either NaF-sensitive phospholipase C, AVP binding, or inositol lipid pools. However, after a 1- or 2-h preincubation with AVP or PDBu, a decrease in both IP accumulation and AVP binding capacity is observed. With regard to aldosterone secretion, PDBu alone stimulates hormone output, but when added simultaneously with AVP, it inhibits AVP-stimulated aldosterone secretion by 70%. If cells are allowed a resting period of 14 h after AVP or PDBu treatment, the AVP response (IP accumulation, AVP binding, and aldosterone output) is recovered and even enhanced. All these effects are specific since the inactive phorbol ester 4 alpha PDD is inactive, and staurosporine (a PKC inhibitor) reverses the PDBu effect. AVP stimulates transiently the translocation of PKC from the cytosol to the membrane, suggesting that the effect observed with PDBu reflects the effect of endogenous PKC stimulated by AVP. These results outline the complexities involved during hormonal stimulation and, at the same time, homologous desensitization phenomena. On one hand, acute treatment with PDBu--which induces PKC activation--is able to stimulate aldosterone secretion but at the same time initiate desensitization, since phorbol ester uncouples the AVP receptor from the coupling G protein. This suggests that PKC may participate in the first step of homologous desensitization. On the other hand, a 2-h incubation with PDBu induces a loss of AVP binding sites. This may represent the second step of homologous desensitization. Finally, a long term treatment with PDBu completely inactivates PKC, hence enabling AVP to further stimulate aldosterone secretion.

Activation of protein kinase C by phorbol ester induces downregulation of the Na+/K+-ATPase in oocytes ofXenopus laevis

Full-grown prophase-arrested oocytes of Xenopus laevis were treated with 50 nM phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, or with 50 nM 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD) that does not activate protein kinase C. The effect on membrane currents and capacitance, inulin uptake and ouabain binding, and on membrane morphology were analyzed. (i) During application of PMA, current generated by the Na+/K+ pump decreases; in addition, Cl- and K+ channels become inhibited. This general decrease in membrane conductance reaches steady state after about 60 min. 4 alpha PDD was ineffective. (ii) Ouabain binding experiments demonstrate that PMA (K1/2 = 7 nM), but not 4 alpha PPD, induces a reduction of the number of pump molecules in the surface membrane. Permeabilization of oocytes by digitonin plus 0.02% SDS renders all binding sites present prior to PMA treatment again accessible for ouabain. The KD value for ouabain binding is not influenced. 4 alpha PDD was ineffective. (iii) Exposure of oocytes to PMA reduces membrane capacitance and stimulates uptake of inulin suggesting an increase in endocytosis. Electron micrographs show that PMA reduces the number and length of microvilli, leading finally to a smooth membrane surface with a reduced surface area. From these results we conclude that stimulation of protein kinase C leads to downregulation of the sodium pump. A major portion of this inhibition is brought about by reduction in area of surface membrane with a concomitant internalization of pump molecules. In addition to this mode of downregulation, a direct effect of stimulation of protein kinase C on the pump molecule cannot be excluded.

Phorbol ester modulates serotonin-stimulated phosphoinositide breakdown in cultured vascular smooth muscle cells

Stimulation of cultured rabbit aortic vascular smooth muscle cells (VSMC) with serotonin (5HT) induced a rapid generation of inositol phosphates from receptor-mediated hydrolysis of inositol phospholipids. Pretreatment of these cells with 500ng/ml of pertussis toxin for 24h prior to addition of 5HT reduced 5HT-induced formation of inositol phosphates. Phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol-12,13-dibutyrate (PDBu), are known to activate protein kinase C (PKC), but their role on cultured VSMC stimulated by 5HT has not been defined. TPA exhibited a rapid inhibition of 5HT-stimulated phosphoinositide breakdown, although 4 alpha-phorbol-12,13-didecanoate (4 alpha PDD), an inactive phorbol ester, did not inhibit it. These data suggest that a guanine nucleotide inhibitory (Gi) protein couples 5HT receptor to phospholipase C and TPA modulates 5HT-stimulated hydrolysis of inositol phospholipids in cultured VSMC through activation of PKC.

Phorbol ester does not mimic, but antagonizes the alpha-adrenoceptor-mediated positive inotropic effect in the rabbit papillary muscle.

The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) was used to examine the hypothesis that phosphoinositide turnover is involved in the regulation of myocardial contractility mediated by stimulation of alpha-adrenoceptors in the mammalian cardiac muscle. Exposure of the isolated rabbit papillary muscle electrically driven at a rate of 1 Hz at a temperature of 37 degrees C to TPA in concentrations of 10-1000 nmol/l for 30 min did not affect the basal force of contraction. The concentration-response curve for the positive inotropic effect of (-)-phenylephrine mediated by stimulation of alpha-adrenoceptors in the presence of (+/-)-bupranolol (100 nmol/l) was shifted to the right and downward by TPA in concentrations of 30-1000 nmol/l, while the effect of (-)-phenylephrine mediated by stimulation of beta-adrenoceptors in the presence of prazosin (100 nmol/l) was not decreased, but slightly enhanced by exposure of the muscle to relatively low concentrations of TPA (10-100 nmol/l). Incubation of the membrane fraction isolated from the rabbit ventricular muscle with TPA in vitro under the same condition as employed in the physiological experiments decreased the specific binding of [3H]prazosin but not that of [3H]CGP-12177, while the non-tumor promoting phorbol ester, alpha PDD, was ineffective. These results indicate that activation of protein kinase C by TPA does not mimic the positive inotropic effect of catecholamines mediated by activation of myocardial alpha-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

An activator of protein kinase C (phorbol dibutyrate) attenuates atrial-natriuretic-factor-stimulated cyclic GMP accumulation in smooth-muscle cells.

Rat thoracic aortic smooth-muscle cells (A-10; A.T.C.C. CRL 1476) displays a high density of vasopressin and atrial-natriuretic-factor (ANF) receptors and a low density of beta-adrenergic receptors. ANF stimulates cGMP (cyclic GMP) accumulation in a time- and dose-dependent fashion. Pretreatment of these cells with phorbol dibutyrate (PDBu), a known activator of protein kinase C, attenuated ANF-stimulated cGMP accumulation without affecting basal cGMP concentrations. This effect was concentration-dependent and was observed as early as 2 min after treatment. 4 alpha-Phorbol 12, 13-didecanoate (alpha PDD), which does not activate protein kinase C, did not inhibit the cGMP accumulation. PDBu pretreatment did not affect the density and affinity of ANF receptors. These data suggest that PDBu, presumably via activation of protein kinase C, might stimulate phosphorylation of a key regulatory protein in the ANF/cGMP pathway.

Some Effects of the Tumor Promoter 12-&lt;italic&gt;O&lt;/italic&gt;-Tetradecanoylphorbol 13-Acetate on Cell Interactions In Vitro&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;

Studies were made of the effects of the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), 4-O-methyl TPA (4-O-MeTPA), and 4 alpha phorbol 12,13-didecanoate (4 alpha PDD) on the aggregation of embryonic chick cells in gyratory shaker culture, a model system useful for the study of cell adhesion and cell interactions. TPA and, to a lesser extent, 4-O-MeTPA significantly reduced the neural retina aggregate size at concentrations as low as 10(-9) M and 10(-8) M, respectively. An inactive isomer, 4 alpha PDD, had no effect up to 10(-6) M. The reduction in aggregate size appeared related to promoter activity since dexamethasone, a steroid that inhibits tumor promotion by TPA, significantly reversed the inhibitory effect of TPA. None of the agents tested affected the sorting pattern in mixed neural retina and heart cultures. The results indicate that intercellular adhesion, as determined by extent of aggregation, is reduced in the presence of TPA. This inhibition is considered to be related to the tumor-promoting activity of TPA.