Ca2+ sensitization of smooth muscle contractility induced by ruthenium red.
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The effects of ruthenium red (RuR) on contractility were examined in skinned fibers of guinea pig smooth muscles, where sarcoplasmic reticulum function was destroyed by treatment with A-23187. Contractions of skinned fibers of the urinary bladder were enhanced by RuR in a concentration-dependent manner (EC50 = 60 microM at pCa 6.0). The magnitude of contraction at pCa 6.0 was increased to 320% of control by 100 microM RuR. Qualitatively, the same results were obtained in skinned fibers prepared from the ileal longitudinal smooth muscle layer and mesenteric artery. The maximal contraction induced by pCa 4.5 was not affected significantly by RuR. The enhanced contraction by RuR was not reversed by the addition of guanosine 5'-O-(2-thiodiphosphate) or a peptide inhibitor of protein kinase C [PKC-(19-31)]. The application of microcystin, a potent protein phosphatase inhibitor, induced a tonic contraction of skinned smooth muscle at low Ca2+ concentration ([Ca2+]; pCa > 8.0). RuR had a dual effect on the microcystin-induced contraction-to- enhancement ratio at low concentrations and suppression at high concentrations. The relaxation following the decrease in [Ca2+] from pCa 5.0 to >8.0 was significantly slowed down by an addition of RuR. Phosphorylation of the myosin light chain at pCa 6.3 was significantly increased by RuR in skinned fibers of the guinea pig ileum. These results indicate that RuR markedly increases the Ca2+ sensitivity of the contractile system, at least in part via inhibition of myosin light chain phosphatase. (+info)
Protein kinase C reduces the KCa current of rat tail artery smooth muscle cells.
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The hypothesis that protein kinase C (PKC) is able to regulate the whole cell Ca-activated K (KCa) current independently of PKC effects on local Ca release events was tested using the patch-clamp technique and freshly isolated rat tail artery smooth muscle cells dialyzed with a strongly buffered low-Ca solution. The active diacylglycerol analog 1,2-dioctanoyl-sn-glycerol (DOG) at 10 microM attenuated the current-voltage (I-V) relationship of the KCa current significantly and reduced the KCa current at +70 mV by 70 +/- 4% (n = 14). In contrast, 10 microM DOG after pretreatment of the cells with 1 microM calphostin C or 1 microM PKC inhibitor peptide, selective PKC inhibitors, and 10 microM 1,3-dioctanoyl-sn-glycerol, an inactive diacylglycerol analog, did not significantly alter the KCa current. Furthermore, the catalytic subunit of PKC (PKCC) at 0.1 U/ml attenuated the I-V relationship of the KCa current significantly, reduced the KCa current at +70 mV by 44 +/- 3% (n = 17), and inhibited the activity of single KCa channels at 0 mV by 79 +/- 9% (n = 6). In contrast, 0.1 U/ml heat-inactivated PKCC did not significantly alter the KCa current or the activity of single KCa channels. Thus these results suggest that PKC is able to considerably attenuate the KCa current of freshly isolated rat tail artery smooth muscle cells independently of effects of PKC on local Ca release events, most likely by a direct effect on the KCa channel. (+info)
Contribution of protein kinase C to ET-1-induced proliferation in human myometrial cells.
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The role of protein kinase C (PKC) in endothelin-1 (ET-1)-induced proliferation of human myometrial cells was investigated. ET-1 dose dependently stimulated DNA synthesis and the number of cultured myometrial cells. Inhibition of PKC by calphostin C or Ro-31-8220 or downregulation of PKC eliminated the proliferative effects of ET-1. The failure of two protein tyrosine kinase (PTK) inhibitors (tyrphostin 51 and tyrphostin 23) to affect ET-1-induced proliferation supports the hypothesis of noninvolvement of the tyrosine kinase signaling pathway in this process. The expression and distribution of PKC isoforms were examined by Western blot analysis. The five PKC isoforms (PKC-alpha, -beta1, -beta2, -zeta, -epsilon) evidenced in human myometrial tissue were found to be differentially expressed in myometrial cells, with a predominant expression of PKC-alpha and PKC-zeta. Treatment with phorbol 12, 13-dibutyrate (PDBu) resulted in the translocation of all five isoforms to the particulate fraction, whereas ET-1 induced a selective increase in particulate PKC-beta1, PKC-beta2, and PKC-epsilon. Our findings that multiple PKC isoforms are differentially responsive to ET-1 or PDBu suggest that they play distinct roles in the myometrial growth process. (+info)
Protein kinase C mediates experimental colitis in the rat.
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Protein kinase C (PKC) plays an important role in the cell signal transduction of many physiological processes. In contrast to these physiological responses, increases in PKC activity have also been associated with inflammatory disease states, including ulcerative colitis. The objective of this study was to examine the role of PKC as a causative mediator in initiation of experimentally induced colitis in the rat. Colitis was induced in rats by intrarectal (0.6 ml) instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS; 75 mg/kg in 50% ethanol) or the PKC activator phorbol 12-myristate 13-acetate (PMA; 1.5-3.0 mg/kg in 20% ethanol). Gross and histological mucosal damage, mucosal neutrophil infiltration, mucosal PKC activity, and PKC protein content for PKC isoforms alpha, beta, delta, and epsilon were assessed 2 h to 14 days after an inflammatory challenge. Both PKC activity and mucosal injury increased significantly within 4 h of TNBS treatment. PKC activity was maximal at 7 days and declined at 14 days, whereas mucosal damage became maximal at 1 day and declined after 7 days. In contrast, neutrophil infiltration as assessed by myeloperoxidase activity only increased 12 h after TNBS treatment, became maximal 1 day after TNBS administration, and declined thereafter. PKCbeta, -delta, and -epsilon were increased in response to TNBS, whereas PKCalpha protein content was decreased. The PKC antagonists staurosporine and GF-109203X (25 ng/kg iv) reduced TNBS-induced changes in mucosal PKC activity and the degree of mucosal damage. In contrast, neutropenia induced by antineutrophil serum treatment did not significantly affect the degree of injury or mucosal PKC activity. Furthermore, activation of mucosal PKC activity with PMA also induced mucosal damage, which was also inhibited by pretreatment with a PKC antagonist. In conclusion, these results suggest that increases in PKC activity play a causative role in TNBS-induced colitis. The PKC-mediated response to TNBS does not appear to involve neutrophil infiltration. (+info)
Proapoptotic effects of ANG II in human coronary artery endothelial cells: role of AT1 receptor and PKC activation.
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Anoxia-reoxygenation, tumor necrosis factor-alpha (TNF-alpha), and angiotensin II (ANG II) have been shown to induce apoptosis in myocytes. However, the role of these mediators in causing apoptosis of human coronary artery endothelial cells (HCAEC) is not known. This study was designed to examine the interaction of these mediators in induction of apoptosis in HCAEC. Cultured HCAEC were exposed to anoxia-reoxygenation, TNF-alpha, and ANG II. TNF-alpha enhanced apoptosis of HCAEC (determined by DNA nick-end labeling in situ and DNA laddering) caused by anoxia-reoxygenation. ANG II increased apoptosis beyond that caused by anoxia-reoxygenation and TNF-alpha. Apoptosis caused by ANG II was reduced by losartan, a specific ANG II type 1 receptor (AT1R) blocker, whereas PD-123,177, a specific ANG II type 2 receptor blocker, under identical conditions had minimal effect. The proapoptotic effects of ANG II were associated with the activation of protein kinase C (PKC). The importance of PKC activation as a signal transduction mechanism became evident in experiments wherein treatment of HCAEC with a specific inhibitor of PKC activation decreased ANG II-mediated apoptosis. Thus AT1R activation appears to be responsible for apoptosis caused by ANG II in HCAEC, and AT1R activation-mediated apoptosis involves activation of PKC. (+info)
Protein kinase C isoforms in human airway smooth muscle cells: activation of PKC-zeta during proliferation.
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Protein kinase C (PKC) is implicated in the regulation of smooth muscle contractility and growth. We have previously described the pattern of isoform expression of PKC in canine airway smooth muscle. This study identified the isoforms present in human cultured airway smooth muscle cells and also addressed the question of whether mitogenesis in these cells is associated with changes in a specific isoform, PKC-zeta. Western blot analysis revealed the presence of PKC-alpha, -betaI, and -betaII of the conventional group; PKC-delta, -theta, -epsilon, and -eta of the novel group; and PKC-zeta, -mu, and -iota of the atypical group. There was a significant increase in density of the Western blot for PKC-zeta in cells proliferating in response to 10% fetal bovine serum (FBS) to 372 +/- 115% of control values (P < 0.05; n = 3 patients) in the cytosolic fraction. Platelet-derived growth factor (PDGF) produced increases in PKC-zeta in both the cytosolic and membrane fractions to 210 +/- 49 and 443 +/- 227%, respectively, of control values (P < 0.05; n = 4 patients). There was no change in expression of PKC-alpha, -betaI, -betaII, -theta, -epsilon, -eta, -delta, or -iota in response to the same stimuli. PGE2 (1 microM) added to the cells 30 min before PDGF reduced incorporation of [3H]thymidine from 5,580 +/- 633 (SE) to 3, 980 +/- 126 dpm (P < 0.05; n = 3 patients) and, in addition, reduced expression of PKC-zeta in the membrane fraction as determined by Western blotting from 266 +/- 66 to 110 +/- 4% of control values (P < 0.05; n = 3 patients). PKC-zeta activity in stimulated cells (10% FBS), as assessed by immunoprecipitation and phosphorylation of glycogen synthase peptide, was approximately 3-fold greater than that in unstimulated cells, and the amount of PKC-zeta protein correlated with isoenzyme activity (r2 = 0.91; P < 0.02; n = 4 patients). In conclusion, this study 1) provides the first description of which isoforms of PKC are present in human cultured airway smooth muscle cells and 2) shows that proliferation of these cells is associated with upregulation of PKC-zeta. Whether activation of PKC-zeta is a primary or secondary event in airway smooth muscle cell proliferation remains to be determined. (+info)
AVP inhibits LPS- and IL-1beta-stimulated NO and cGMP via V1 receptor in cultured rat mesangial cells.
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The present study examined how arginine vasopressin (AVP) affects nitric oxide (NO) metabolism in cultured rat glomerular mesangial cells (GMC). GMC were incubated with test agents and nitrite, and intracellular cGMP content, inducible nitric oxide synthase (iNOS) mRNA, and iNOS protein were analyzed by the Griess method, enzyme immunoassay, and Northern and Western blotting, respectively. AVP inhibited lipopolysaccharide (LPS)- and interleukin-1beta (IL-1beta)-induced nitrite production in a dose- and time-dependent manner, with concomitant changes in cGMP content, iNOS mRNA, and iNOS protein. This inhibition by AVP was reversed by V1- but not by oxytocin-receptor antagonist. Inhibition by AVP was also reproduced on LPS and interferon-gamma (IFN-gamma). Protein kinase C (PKC) inhibitors reversed AVP inhibition, whereas PKC activator inhibited nitrite production. Although dexamethasone and pyrrolidinedithiocarbamate (PDTC), inhibitors of nuclear factor-kappaB, inhibited nitrite production, further inhibition by AVP was not observed. AVP did not show further inhibition of nitrite production with actinomycin D, an inhibitor of transcription, or cycloheximide, an inhibitor of protein synthesis. In conclusion, AVP inhibits LPS- and IL-1beta-induced NO production through a V1 receptor. The inhibitory action of AVP involves both the activation of PKC and the transcription of iNOS mRNA in cultured rat GMC. (+info)
Effects of prostaglandin F2 alpha on intracellular pH, intracellular calcium, cell shortening and L-type calcium currents in rat myocytes.
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OBJECTIVE: We have studied the mechanisms underlying the positive inotropic action of prostaglandin F2 alpha (PGF2 alpha) by monitoring intracellular calcium transients, intracellular pH, L-type calcium currents and cell shortening in isolated ventricular myocytes. METHODS: Rat myocytes were loaded with fura-2AM for intracellular calcium measurements, or BCECF-AM for pH measurements. Cell shortening was recorded using an edge detection system, and L-type calcium currents measured using whole cell patch clamping. RESULTS: PGF2 alpha (3 nmol l-1-3 mumol l-1 increased single myocyte shortening and reduced resting cell length in a concentration-dependent manner. While myocyte shortening was increased by PGF2 alpha, this was not associated with any change in the amplitude of intracellular calcium transients, diastolic calcium, or L-type calcium currents. However, the same myocytes were capable of responding to catecholamines with increases in calcium transient amplitude and L-type calcium currents. PGF2 alpha (3 mumol l-1 caused a reversible rise in intracellular pH of 0.08 +/- 0.01 pH units (n = 5, p < 0.05). The Na(+)-H+ exchanger inhibitor, HOE 694 (10 mumol l-1, abolished the PGF2 alpha-induced rise in pH and the increase in cell shortening. PGF2 alpha-induced increases in cell shortening and intracellular pH were also attenuated by the protein kinase C (PKC) inhibitor, chelerythrine (2 mumol l-1. CONCLUSION: The positive inotropic action of PGF2 alpha appears to be mediated via activation of the Na(+)-H+ exchanger with the possible involvement of PKC. This suggests that PGF2 alpha-produces intracellular alkalosis, which then sensitizes cardiac myofilaments to calcium. (+info)