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PubMed1986

In vitro effects of ionophores and inhibitors of main sodium and calcium movements on tyrosine and tryptophan transport by human erythrocytes.

Peripheral models using blood cells might be biochemical markers in various psychiatric illnesses. In previous papers we reported a deficit of tyrosine and tryptophan transport in red cells incubated in plasma from depressed patients. In the present study we investigated the role played by sodium and calcium in these transports by using inhibitors and ionophores of the main movements of these electrolytes. We also studied the contribution of phloretin-sensitive countertransport, which has been described as low in psychiatric conditions.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Antimicrobial strategies in the prevention of dental caries.

Antimicrobial agents, applied either professionally or delivered from dentifrices or mouthwashes, could reduce caries by controlling plaque formation, suppressing cariogenic species, or by inhibiting bacterial metabolism. Chlorhexidine has proven anticaries activity; other agents might also prevent caries by means of their antiplaque properties. Sugar substitutes stabilize the microflora by reducing the number of acid challenges to plaque and stimulating saliva flow; some also have antimicrobial properties, especially against mutans streptococci. Vaccines have been prepared from purified antigens of mutans streptococci. These vaccines confer protection in non-human primates, but have yet to be tested in a human clinical trial.

Alkaloids↗
PubMed1994

Effects of subinhibitory concentrations of chemical agents on hydrophobicity and in vitro adherence of Streptococcus mutans and Streptococcus sanguis.

The aim of this investigation was to determine whether sublethal concentrations of chlorhexidine (Cx), hexetidine (Hx), cetylpyridinium chloride (Cc), sodium dodecyl sulfate (SDS), sanguinarine (Sg), sodium fluoride (NaF), and ammonium fluoride (NH4F) could affect hydrophobicity and adhesion of Streptococcus sanguis and Streptococcus mutans to saliva-coated hydroxyapatite (S-HA). Determination of the minimum inhibitory concentrations (MICs) showed that both species were susceptible to all agents tested. Growth in the presence of sub-MIC concentrations of Cx, SDS, Cc, NaF, or NH4F did not change significantly the hydrophobicity of S. sanguis cells when compared to the control which lacked any agent. However, growth in the presence of Hx or Sg resulted in a significant reduction in their hydrophobicity. Sub-MIC levels of SDS or Sg in the growth medium resulted in S. mutans cells with increased affinity for hexadecane compared with the control. The adherence of S. sanguis was changed significantly only by Hx or Sg, resulting in less cells adhering to S-HA. However, S. mutans cells previously incubated with NaF, NH4F, or Sg showed a higher adherence to S-HA than the control. The mechanisms of interference with adherence are at present not completely understood. Thus, antimicrobial agents at sub-MIC concentrations can interfere selectively with hydrophobicity and/or adhesion of oral streptococci.

Adsorption↗
PubMed2003

Differential regulation of early growth response gene-1 expression by insulin and glucose in vascular endothelial cells.

OBJECTIVE: Early growth response gene (Egr)-1 is a key transcription factor involved in vascular pathophysiology. Its role in diabetic vascular complications, however, remains unclear. Because hyperinsulinemia and hyperglycemia are major risk factors leading to diabetic vascular complications, we examined the effect of insulin and glucose on Egr-1 expression in murine glomerular vascular endothelial cells. METHODS AND RESULTS: Insulin or glucose, when added separately, increased egr-1 mRNA levels and promoter activity, as well as Egr-1 protein levels in nuclear extracts. When insulin was added to cells preincubated with glucose, the two had an additive effect on Egr-1 expression. Furthermore, vascular endothelial growth factor receptor-1 (flt-1) and plasminogen activator inhibitor-1, two known Egr-1-responsive genes, were also upregulated in the presence of insulin or glucose. An investigation into the underlying molecular mechanisms demonstrated that insulin, but not glucose, increased Egr-1 expression through extracellular signal-regulated kinase 1/2 activation, which is consistent with our previous reports. In contrast, inhibition of protein kinase C by phorbol ester or by the specific protein kinase C inhibitor chelerythrine chloride downregulated glucose-induced, but not insulin-induced, Egr-1 expression. CONCLUSIONS: Differential regulation of Egr-1 expression by insulin and glucose in vascular cells may be one of the initial key events that plays a crucial role in the development of diabetic vascular complications.

Alkaloids↗
PubMed2004

Systemic regulation of vascular NAD(P)H oxidase activity and nox isoform expression in human arteries and veins.

OBJECTIVE: Impaired endothelial function, characterized by nitric oxide scavenging by increased superoxide production, is a hallmark of vascular disease states. However, molecular mechanisms regulating superoxide production in human blood vessels remain poorly defined. METHODS AND RESULTS: We compared endothelial function, vascular superoxide production, and the expression of NAD(P)H oxidase subunits in arteries and veins from patients undergoing coronary bypass surgery (n=86). Superoxide release was similar in arteries and veins. Inhibitor studies revealed that the NAD(P)H oxidase system was a quantitatively and proportionately greater source of superoxide in veins, whereas xanthine oxidase also contributed significantly to superoxide production in arteries. Moreover, NAD(P)H oxidase molecular composition differed in veins and arteries; veins expressed more nox2 and p22phox, whereas the relative expression of nox4 was greater in arteries. However, there were strong correlations between p22phox and nox4 expression and between superoxide production, NAD(P)H oxidase activity, and endothelial function in arteries and veins from the same patient. CONCLUSIONS: In individuals with coronary artery disease, changes in vascular superoxide production, endothelial function, and NAD(P)H oxidase activity and expression are related in veins and arteries. These findings highlight the importance of systemic effects on the molecular regulation of the NAD(P)H oxidases in human vascular disease. Endothelial dysfunction is characterized by increased superoxide production. NAD(P)H oxidase activity and endothelial function are correlated in veins and arteries in coronary artery disease, suggesting regulation by systemic factors. The expression of the NAD(P)H oxidase subunits p22phox and nox4, although different in veins and arteries, are also correlated.

Acetylcholine↗
PubMed2000

Proliferative effect of lipoprotein lipase on human vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) proliferation is a key event in the development and progression of atherosclerotic lesions. Accumulating evidence suggests that lipoprotein lipase (LPL) produced in the vascular wall may exert proatherogenic effects. The aim of the present study was to examine the effect of LPL on VSMC proliferation. Incubation of growth-arrested human VSMCs with purified endotoxin-free bovine LPL for 48 and 72 hours, in the absence of any added exogenous lipoproteins, resulted in a dose-dependent increase in VSMC growth. Addition of VLDLs to the culture media did not further enhance the LPL effect. Treatment of growth-arrested VSMCs with purified human or murine LPL (1 microg/mL) led to a similar increase in cell proliferation. Neutralization of bovine LPL by the monoclonal 5D2 antibody, irreversible inhibition, or heat inactivation of the lipase suppressed the LPL stimulatory effect on VSMC growth. Moreover, preincubation of VSMCs with the specific protein kinase C inhibitors calphostin C and chelerythrine totally abolished LPL-induced VSMC proliferation. In LPL-treated VSMCs, a significant increase in protein kinase C activity was observed. Treatment of VSMCs with heparinase III (1 U/mL) totally inhibited LPL-induced human VSMC proliferation. Taken together, these data indicate that LPL stimulates VSMC proliferation. LPL enzymatic activity, protein kinase C activation, and LPL binding to heparan sulfate proteoglycans expressed on VSMC surfaces are required for this effect. The stimulatory effect of LPL on VSMC proliferation may represent an additional mechanism through which the enzyme contributes to the progression of atherosclerosis.

Alkaloids↗
PubMed2003

Reactive oxygen species are involved in smoking-induced dysfunction of nitric oxide biosynthesis and upregulation of endothelial nitric oxide synthase: an in vitro demonstration in human coronary artery endothelial cells.

BACKGROUND: Our group has previously shown that human umbilical vein endothelial cells exposed to smokers' serum decreased nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) activity in the presence of increased eNOS expression. In the present study, we examined whether these observations extended to human coronary artery endothelial cells (HCAECs). In addition, the role of reactive oxygen species in the observed alterations was examined. METHODS AND RESULTS: HCAECs were incubated with serum from 10 nonsmokers and 15 smokers for 12 hours with or without the addition of either polyethylene glycol-superoxide dismutase (PEG-SOD, 300 U/mL), PEG-SOD+PEG-catalase (1000 U/mL), chelerythrine (3 micromol/L), or tetrahydrobiopterin (20 micromol/L). At the end of incubation, NO, eNOS protein, and eNOS activity were measured from the same culture. HCAECs incubated with smokers' serum alone showed significantly lower NO production (P<0.05) and eNOS activity (P<0.005) but higher eNOS expression (P<0.005) compared with nonsmokers. In smokers, addition of PEG-SOD, PEG-SOD+PEG-catalase, or tetrahydrobiopterin significantly (P<0.05) improved NO levels and eNOS activity. Interestingly, in the same smokers, a significant decrease in eNOS expression was only seen with the addition of PEG-SOD+PEG-catalase (P<0.05) and treatment with PEG-SOD alone insignificantly increased eNOS expression. CONCLUSIONS: The present study indicates that in vitro, HCAECs show similar changes in NO biosynthesis as human umbilical vein endothelial cells when exposed to smokers' serum and also confirms that oxidative stress plays a central role in smoking-mediated dysfunction of NO biosynthesis in endothelial cells. Furthermore, these data support other studies suggesting a role for hydrogen peroxide in the upregulation of eNOS.

Adult↗
PubMed2004

Regulation of proangiogenic factor CCN1 in cardiac muscle: impact of ischemia, pressure overload, and neurohumoral activation.

BACKGROUND: CCN1, a potent proangiogenic factor, is induced in the vasculature by tissue injury, angiotensin II (Ang II), and growth factor stimulation. Because these conditions occur in myocardial ischemia and pressure overload, we investigated the regulation of CCN1 in cardiomyocytes in vitro and in the heart in vivo. METHODS AND RESULTS: Ang II, signaling via the angiotensin type 1 (AT1) receptor, and alpha1-adrenergic stimulation with phenylephrine induced CCN1 expression in ventricular cardiomyocytes isolated from 1- to 3-day-old rats. Cell culture supernatant of Ang II-treated cardiomyocytes induced migration of smooth muscle cells, which was abolished by neutralizing antibody to CCN1. Ang II- and phenylephrine-mediated induction of CCN1 expression in cardiomyocytes was completely abolished by inhibition of MEK/extracellular signal-regulated kinases (ERK) or protein kinase C (PKC). Likewise, mechanical stretch induced CCN1 expression in cardiomyocytes, an effect that was prevented by AT1 receptor blockade or PKC inhibition. Similarly, pressure overload in vivo upregulated myocardial CCN1 expression levels via AT1 receptor- and PKC-dependent mechanisms. After myocardial infarction in mice, CCN1 expression was strongly induced in both ischemic and remote left ventricular myocardium. Marked CCN1 protein expression was noted in cardiomyocytes of patients with end-stage ischemic cardiomyopathy but was almost absent in nonfailing human myocardium. CONCLUSIONS: Pressure overload, ischemia, and neurohormonal factors, such as Ang II or alpha1-adrenergic stimuli, induce myocardial expression of CCN1, a potent proangiogenic factor, supporting the notion that CCN1 may play an important role in the adaptation of the heart to cardiovascular stress.

Alkaloids↗
PubMed2004

Gene therapy of endothelial nitric oxide synthase and manganese superoxide dismutase restores delayed wound healing in type 1 diabetic mice.

BACKGROUND: Nitric oxide (NO) deficiency contributes to diabetic wound healing impairment. The present study tested the hypothesis that increased cutaneous superoxide (O2-) levels in type 1 diabetic mice cause NO deficiency and delayed wound healing. METHODS AND RESULTS: Wound healing was markedly delayed in streptozotocin-induced type 1 diabetic mice compared with the normal controls. There were significantly reduced levels of endothelial NO synthase (eNOS) protein and constitutive NOS activity in diabetic wounds, whereas O2- levels were markedly increased. A single regimen of cutaneous gene therapy of eNOS or manganese superoxide dismutase (MnSOD) restored such healing delay, with a concomitant suppression of wound O2- levels and augmentation of both eNOS protein and constitutive NOS activity. Gene therapy of MnSOD also increased cutaneous MnSOD activity. Cutaneous O2- levels were also increased in Ins2(Akita) diabetic mice. In vitro glucose treatment of cutaneous tissues from normal mice for 24 hours increased O2- levels in a concentration-dependent manner. The enhanced cutaneous O2- levels induced by high glucose in both normal and diabetic mice were abolished by the NADPH oxidase inhibitor apocynin and the protein kinase C inhibitor chelerythrine. Furthermore, ex vivo gene transfer of dominant-negative HA-tagged N17Rac1, which inhibits NADPH oxidase subunit Rac1, significantly inhibited cutaneous O2- formation induced by high glucose in both normal and Ins2(Akita) diabetic mice. CONCLUSIONS: These results indicate that hyperglycemia augments cutaneous O2- levels, at least in part, via NADPH oxidase and protein kinase C pathways, resulting in impaired wound healing in type 1 diabetic mice. Gene therapy strategies aimed at restoring cutaneous NO bioavailability may provide an effective means to ameliorate delayed diabetic wound healing.

Acetophenones↗
PubMed1995

Regulation of endothelial constitutive nitric oxide synthase by protein kinase C.

Protein kinase C (PKC) plays a key role in a variety of signal transduction processes. The promoter region of the endothelial constitutive nitric oxide synthase (ecNOS) gene contains a transcriptional factor AP-1 binding element. In the present study, we sought to determine the effect of PKC inhibition on the expression of ecNOS in cultured bovine aortic endothelial cells (BAEC). The PKC inhibitor staurosporine (10 to 100 nmol/L) increased the expression of ecNOS mRNA, assessed by Northern analysis, in a dose-dependent manner. A newly developed, more specific PKC inhibitor, chelerythrine (1 to 3 mumol/L), also increased the level of ecNOS mRNA. Incubation of BAEC with phorbol 12-myristate 13-acetate (100 nmol/L) for 24 hours, which downregulates PKC, increased ecNOS mRNA expression. The protein content of ecNOS, assessed by Western analysis, was also increased in staurosporine-treated or chelerythrine-treated BAEC. The release of nitrogen oxides from staurosporine-treated or chelerythrine-treated cells both under basal conditions and in response to calcium ionophore A23187 was significantly increased (P < .05). In conclusion, the present study suggests that regulation of ecNOS is mediated by PKC. The increased release of nitric oxide induced by PKC inhibition may play a protective role against atherogenic process.

Alkaloids↗
PubMed1996

Chelerythrine inhibits Na(+)-H+ exchange in platelets from spontaneously hypertensive rats.

Hypertension has been associated with increased activity of the Na(+)-H+ exchanger. To study the role played by protein kinase C in this process, we used chelerythrine, a potent and specific inhibitor of the kinase. After an acid load by ammonium chloride preincubation, platelets isolated from spontaneously hypertensive rats showed a faster and larger increase in intracellular pH than platelets from Wistar-Kyoto rats. The initial rate of intracellular pH recovery was 2.46 +/- 0.26 pH units per minute in spontaneously hypertensive rats and 1.74 +/- 0.19 in Wistar-Kyoto rats. For protein kinase C inhibition, platelets were incubated for 30 minutes with 10 mumol/L chelerythrine. This treatment induced a significant reduction in the recovery rate only in spontaneously hypertensive rat platelets, indicating that a pathway involving protein kinase C participates in the prestimulation of the exchanger in cells from this rat strain. Addition of chelerythrine reduced the baseline intracellular pH of platelets. No significant difference was found between the decrease of steady-state intracellular pH induced by chelerythrine in either rat strain. These findings indicate that this model of hypertension is characterized by increased Na(+)-H+ activity mediated by protein kinase C stimulation.

Alkaloids↗
PubMed1997

Growth factors mediate intracellular signaling in vascular smooth muscle cells through protein kinase C-linked pathways.

Intracellular Ca2+ and pH are potent modulators of growth factor-induced mitogenesis and contraction. This study examined platelet-derived growth factor-(PDGF-BB) and insulin-like growth factor (IGF-1)-mediated signal transduction in primary cultured unpassaged vascular smooth muscle cells (VSMC) from mesenteric arteries of Sprague-Dawley rats. Intracellular free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) were measured by fluorescence digital imaging using fura-2 AM and 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Characteristics of [Ca2+]i transients were determined by pre-exposing cells to Ca2+-free buffer, and involvement of the Na+/Ca2+ exchanger was assessed by withdrawal of extracellular Na+ and by exposure to dimethylbenzamil (Na+/Ca2+ exchange blocker). To determine whether pHi responses were mediated via the Na+/H+ exchanger, cells were preincubated with 10(-5) mol/L 5-(N-ethyl-N-isopropyl)amiloride (a selective Na+/H+ exchange blocker). The role of protein kinase C (PKC) and tyrosine kinases in growth factor signaling was assessed by pre-exposing cells to calphostin C and chelerythrine chloride (selective PKC inhibitors; 10(-5) mol/L) and tyrphostin A23 (a selective tyrosine kinase inhibitor; 10(-5) mol/L). PDGF-BB and IGF-1 (1 to 10 ng/mL) increased [Ca2+]i and pHi in a dose-dependent manner. At concentrations greater than 1 ng/mL both growth factors induced a biphasic [Ca2+]i response with an initial transient peak followed by a sustained elevation. At 5 ng/mL PDGF-BB and IGF-1 significantly increased [Ca2+]i from 95+/-3 nmol/L to 328+/-28 and 251+/-18 nmol/L, respectively. Ca2+ withdrawal abolished the second phase of [Ca2+]i elevation. Agonist-induced [Ca2+]i responses were similarly altered by Na+ withdrawal, by Na+/ Ca2+ exchange blockade, and by PKC inhibition; latency, the period from stimulus application to the first [Ca2+]i peak, was increased, the initial [Ca2+]i peak was attenuated, and the sustained phase was prolonged. PDGF-BB and IGF-1 (10 ng/mL) significantly increased pHi from 6.89+/-0.04 nmol/L to 7.11+/-0.01 and 7.09+/-0.02 nmol/L, respectively. EIPA and calphostin C completely inhibited agonist-elicited alkalinization. Tyrphostin A-23 abolished second-messenger responses to PDGF-BB and IGF-1, whose receptors have tyrosine kinase activity. In conclusion, PDGF-BB and IGF-1 elicit significant [Ca2+]i and pHi responses in VSMC. The underlying pathways that mediate these responses are partially dependent on Na+/ Ca2+ transporters and the Na+/H+ exchanger, both of which are linked to PKC activation.

Alkaloids↗
PubMed2004

Reversible connexin 43 dephosphorylation during hypoxia and reoxygenation is linked to cellular ATP levels.

Altered gap junction coupling of cardiac myocytes during ischemia may contribute to development of lethal arrhythmias. The phosphoprotein connexin 43 (Cx43) is the major constituent of gap junctions. Dephosphorylation of Cx43 and uncoupling of gap junctions occur during ischemia, but the significance of Cx43 phosphorylation in this setting is unknown. Here we show that Cx43 dephosphorylation in synchronously contracting myocytes during ischemia is reversible, independent of hypoxia, and closely associated with cellular ATP levels. Cx43 became profoundly dephosphorylated during hypoxia only when glucose supplies were limited and was completely rephosphorylated within 30 minutes of reoxygenation. Similarly, direct reduction of ATP by various combinations of metabolic inhibitors and by ouabain was closely paralleled by loss of phosphoCx43 and recovery of phosphoCx43 accompanied restoration of ATP. Dephosphorylation of Cx43 could not be attributed to hypoxia, acid pH or secreted metabolites, or to AMP-activated protein kinase; moreover, the process was selective for Cx43 because levels of phospho-extracellular signal regulated kinase (ERK)1/2 were increased throughout. Rephosphorylation of Cx43 was not dependent on new protein synthesis, or on activation of protein kinases A or G, ERK1/2, p38 mitogen-activated protein kinase, or Jun kinase; however, broad-spectrum protein kinase C inhibitors prevented Cx43 rephosphorylation while also sensitizing myocytes to reoxygenation-mediated cell death. We conclude that Cx43 is reversibly dephosphorylated and rephosphorylated during hypoxia and reoxygenation by a novel mechanism that is sensitive to nonlethal fluctuations in cellular ATP. The role of this regulated phosphorylation in the adaptation to ischemia remains to be determined.

Adenosine Triphosphate↗
PubMed2004

Silent information regulator 2alpha, a longevity factor and class III histone deacetylase, is an essential endogenous apoptosis inhibitor in cardiac myocytes.

Yeast silent information regulator 2 (Sir2), a nicotinamide adenine dinucleotide-dependent histone deacetylase (HDAC) and founding member of the HDAC class III family, functions in a wide array of cellular processes, including gene silencing, longevity, and DNA damage repair. We examined whether or not the mammalian ortholog Sir2 affects growth and death of cardiac myocytes. Cardiac myocytes express Sir2alpha predominantly in the nucleus. Neonatal rat cardiac myocytes were treated with 20 mmol/L nicotinamide (NAM), a Sir2 inhibitor, or 50 nmol/L Trichostatin A (TSA), a class I and II HDAC inhibitor. NAM induced a significant increase in nuclear fragmentation (2.2-fold) and cleaved caspase-3, as did sirtinol, a specific Sir2 inhibitor, and expression of dominant-negative Sir2alpha. TSA also modestly increased cell death (1.5-fold) but without accompanying caspase-3 activation. Although TSA induced a 1.5-fold increase in cardiac myocyte size and protein content, NAM reduced both. In addition, NAM caused acetylation and increases in the transcriptional activity of p53, whereas TSA did not. NAM-induced cardiac myocyte apoptosis was inhibited in the presence of dominant-negative p53, suggesting that Sir2alpha inhibition causes apoptosis through p53. Overexpression of Sir2alpha protected cardiac myocytes from apoptosis in response to serum starvation and significantly increased the size of cardiac myocytes. Furthermore, Sir2 expression was increased significantly in hearts from dogs with heart failure induced by rapid pacing superimposed on stable, severe hypertrophy. These results suggest that endogenous Sir2alpha plays an essential role in mediating cell survival, whereas Sir2alpha overexpression protects myocytes from apoptosis and causes modest hypertrophy. In contrast, inhibition of endogenous class I and II HDACs primarily causes cardiac myocyte hypertrophy and also induces modest cell death. An increase in Sir2 expression during heart failure suggests that Sir2 may play a cardioprotective role in pathologic hearts in vivo.

Acetylation↗
PubMed1994

Protein kinase C. Its role in ischemic preconditioning in the rat.

The present study investigated whether protein kinase C (PKC) plays a role in ischemic preconditioning in the rat heart. Chelerythrine, a specific antagonist of PKC, and 1,2-dioctanoyl-sn-glycerol (DOG), a diacylglycerol analogue and specific antagonist of PKC, were used to determine whether preconditioning could be blocked or triggered, respectively. Sprague-Dawley rats were anesthetized and instrumented for coronary occlusion and reperfusion. All animals were subjected to 45 minutes of regional ischemia (ISC) followed by 2.5 hours of reperfusion. The preconditioning protocol consisted of 5 minutes of ischemia and then 10 minutes of reperfusion. There were six groups: (1) control (group C, n = 5), (2) preconditioned and ISC (group PC, n = 6), (3) chelerythrine given 2 minutes before ISC (group CC, n = 5), (4) preconditioned and chelerythrine given 2 minutes before ISC (group PCC, n = 6), (5) DOG (dissolved in dimethylsulfoxide [DMSO]) given 10 minutes before ISC (group CD, n = 5), and (6) DMSO given 10 minutes before ISC (group DMSO, n = 3). The end point was infarct size measured using triphenyl tetrazolium chloride and expressed as a percentage of the volume at risk (I/R), measured with fluorescent particles. I/R was significantly reduced by preconditioning (group C, 58.6 +/- 5.0%; group PC, 32.7 +/- 6.3%; P < .01) and by the PKC agonist DOG, which reduced I/R to a similar extent as preconditioning (group C, 58.6 +/- 5.0%; group CD, 28.0 +/- 7.0%; P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1994

Modulation of an inactivating human cardiac K+ channel by protein kinase C.

The transient outward current (ITO) is an important repolarizing component of the cardiac action potential. In native cardiac myocytes, ITO is modulated after activation of protein kinase C, although the molecular nature of this effect is not well understood. A channel recently cloned from human ventricular myocardium (Kv1.4, HK1) produces a rapidly inactivating K+ current, which has phenotypic similarities to the 4-aminopyridine-sensitive component of ITO. Therefore, we examined whether this recombinant channel was also modulated by protein kinase C activation by investigating the effects of the diacylglycerol analogue phorbol 12-myristate 13-acetate (PMA) on Kv1.4 K+ current expressed in Xenopus oocytes. At a concentration of 10 nmol/L, PMA caused a biphasic response with an initial increase (14 +/- 4%, mean +/- SEM) in current, which peaked in 14 minutes. This was followed by a significant reduction (40 +/- 11%) in the current within 30 minutes. There was no significant change in cell membrane electrical capacitance with 10 nmol/L PMA (1 +/- 1% decline in 30 minutes), demonstrating that loss of cell membrane surface area did not explain the reduction in K+ current, although cell capacitance did decrease when using a higher concentration of PMA (81 nmol/L). The inactive stereoisomer, 4 alpha-PMA, had no effect on Kv1.4 current, whereas preincubation with the protein kinase inhibitor staurosporine or protein kinase C-selective chelerythrine prevented the effects of PMA. When purified from a stably transfected mammalian cell line by using immunoprecipitation, the channel protein was readily phosphorylated in vitro by purified protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1995

Requirement for protein kinase C activation in basic fibroblast growth factor-induced human endothelial cell proliferation.

The intracellular signaling mechanisms that mediate basic fibroblast growth factor (bFGF)-induced angiogenesis have not been fully identified. In particular, whether activation of the intracellular enzyme protein kinase C (PKC) is necessary or sufficient for bFGF-induced mitogenesis of human endothelial cells is not clear. Accordingly, the effect of bFGF stimulation on the Ca2+ increase and PKC activity of normal human endothelial cells (HEC) was studied, as was the effect of inhibition of PKC and the distribution of PKC isoenzymes in these cells. The addition of bFGF to cultured HEC increased overall PKC activity in the absence of an increase in intracellular Ca2+ and markedly stimulated their proliferation, as did the addition of PKC-activating phorbol esters. bFGF-induced proliferation was prevented by the PKC inhibitors chelerythrine and H-7 and by downregulation of PKC after prolonged incubation with phorbol esters. In contrast, these inhibitors did not prevent HEC proliferation induced by epidermal growth factor. Because of the failure of bFGF to increase Ca2+, we determined whether bFGF-induced proliferation could be mediated by novel or atypical PKC isoenzymes (which are not regulated by Ca2+). Investigation of the isoenzyme distribution of confluent and subconfluent HEC by immunoblotting, Northern transfer analysis, and polymerase chain reaction of reverse-transcribed RNA revealed the presence of several novel and atypical isoenzymes (PKC-delta, -eta, -theta, and -zeta) as well as small amounts of the conventional (Ca(2+)-regulated) isoenzymes PKC-alpha and -beta. Activation of PKC by bFGF, in the absence of an increase in intracellular Ca2+, suggests that one or more of these Ca(2+)-independent PKC isoenzymes are both necessary and sufficient for HEC proliferation after bFGF.

Alkaloids↗