Direct evidence that protein kinase C plays an essential role in the development of late preconditioning against myocardial stunning in conscious rabbits and that epsilon is the isoform involved

Brief ischemic episodes confer marked protection against myocardial stunning 1-3 d later (late preconditioning [PC] against stunning). The mechanism of this powerful protective effect is poorly understood. Although protein kinase C (PKC) has been implicated in PC against infarction, it is unknown whether it triggers late PC against stunning. In addition, the entire PKC hypothesis of ischemic PC remains controversial, possibly because the effects of PKC inhibitors on PC protection have not been correlated with their effects on PKC activity and/or translocation in vivo. Thus, conscious rabbits underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles for three consecutive days (days 1, 2, and 3). In the control group (group I, n = 7), the recovery of systolic wall thickening after the six O/R cycles was markedly improved on days 2 and 3 compared with day 1, indicating the development of late PC against stunning. Administration of the PKC inhibitor chelerythrine at a dose of 5 mg/kg before the first O on day 1 (group II, n = 10) abrogated the late PC effect against stunning, whereas a 10-fold lower dose (0.5 mg/kg; group III, n = 7) did not. Administration of 5 mg/kg of chelerythrine 10 min after the sixth reperfusion on day 1 (group IV, n = 6) failed to block late PC against stunning. When rabbits were given 5 mg/kg of chelerythrine in the absence of O/R (group V, n = 5), the severity of myocardial stunning 24 h later was not modified. Pretreatment with phorbol 12-myristate 13-acetate (4 microg/kg) on day 1 without ischemia (group VI, n = 11) induced late PC against stunning on day 2 and the magnitude of this effect was equivalent to that observed after ischemic PC. In vehicle-treated rabbits (group VIII, n = 5), the six O/R cycles caused translocation of PKC isoforms epsilon and eta from the cytosolic to the particulate fraction without significant changes in total PKC activity, in the subcellular distribution of total PKC activity, or in the subcellular distribution of the alpha, beta1, beta2, gamma, delta, zeta, iota, lambda, and mu isoforms. The higher dose of chelerythrine (5 mg/kg; group X, n = 5) prevented the translocation of both PKC epsilon and eta induced by ischemic PC, whereas the lower dose (0.5 mg/kg; group XI, n = 5) prevented the translocation of PKC eta but not that of epsilon, indicating that the activation of epsilon is necessary for late PC to occur whereas that of eta is not. To our knowledge, this is the first demonstration that a PKC inhibitor actually prevents the translocation of PKC induced by ischemic PC in vivo, and that this inhibition of PKC translocation results in loss of PC protection. Taken together, the results demonstrate that the mechanism of late PC against myocardial stunning in conscious rabbits involves a PKC-mediated signaling pathway, and implicate epsilon as the specific PKC isoform responsible for the development of this cardioprotective phenomenon.     

Nitric oxide and peroxynitrite released by ultraviolet B-irradiated human endothelial cells are possibly involved in skin erythema and inflammation

In this study we attempted to demonstrate whether endothelial cell nitric oxide synthase (eNOS) and xanthine oxidase (XO) could be activated to release nitric oxide (NO) and peroxynitrite (ONOO-) following exposure to ultraviolet B (UVB) radiation and to define whether this light-induced response could be involved in the pathogenesis of sunburn erythema and inflammation. Treatment of human endothelial cells with UVB (290-320 nm) radiation (up to 100 mJ/cm2) resulted in an increase of both NO and ONOO- release that was inhibited by NG-monomethyl-L-arginine (L-NMMA). Treatment of cell cytosol with various doses of UVB radiation (up to 20 mJ/cm2) resulted in a threefold increase of XO activity that was inhibited (approximately 90% by oxypurinol. In reconstitution experiments, when purified eNOS was added to purified XO, an almost fourfold increase in ONOO- production at 20 mj/cm2 UVB radiation was observed. UVB radiation (100 mg/cm2) decreased cell membrane fluidity, indicating changes in the physicochemical characteristics of the membranes. In in vivo experiments, when human volunteers were subjected to UVB light, a protection factor (PF) of 3.90 +/- 0.85 was calculated when an emulsified cream formulation containing nitro-L-arginine (L-NA; 2%) and L-NMMA (2%) was applied to their skin. The present studies indicate that UVB radiation acts as a potent stimulator of eNOS and XO in human endothelial cells. The cytotoxic effects of NO and ONOO- may be the main factors in the integrated response of the skin leading to vasodilatation, the first key event of erythema production and the inflammation process.     

Inhibition of host RNA polymerase II-dependent transcription by vesicular stomatitis virus results from inactivation of TFIID

The present study tested the hypothesis that one or more tyrosine kinase(s) are downstream of protein kinase C (PKC) in the signal transduction pathway responsible for the cardioprotective effect of ischemic preconditioning (PC). Isolated rabbit hearts were subjected to 30 min of regional ischemia followed by 2 h of reperfusion. Infarct size was measured by triphenyltetrazolium staining and expressed as a percentage of the area at risk. Infarction in control hearts was 32.9+/-1.8%. Ischemic PC with 5-min ischemia/10-min reperfusion reduced infarct size to 11.5+/-1.5% (P<0.05). Infusion of the tyrosine kinase inhibitors, genistein (50 microM) or lavendustin A (0.5 microM), alone did not affect the level of infarction. When infused around the 5-min PC ischemia genistein failed to block protection (13.7+/-1.0%). However, when present at the onset of the 30-min ischemia both genistein and lavendustin A completely aborted protection (31.4+/-2.0 and 28.1+/-1.5%, respectively). Activation of PKC by phorbol 12-myristate 13-acetate (PMA, 0.05 nmol) was as protective is ischemic PC (14.9+/-3.0%; P<0. 05). Similar to PC, PMA-induced protection was completely prevented by both genistein and lavendustin A. Conversely, anisomycin (50 ng/ml), an activator of MAP kinase kinases (dual tyrosine and threonine kinases), was very protective (7.5+/-1.6%; P<0.05) and this protection was still present when PKC was inhibited by 5 microM chelerythrine (12.1+/-1.6%; P<0.05). In conclusion, activation of a tyrosine kinase during the long ischemia appears to be required for cardioprotection in the rabbit heart. Furthermore, the ability of tyrosine kinase inhibitors to block PMA-induced protection in conjunction with the failure of PKC inhibition to prevent anisomycin-induced protection suggests that the tyrosine kinase is downstream of PKC and that the tyrosine kinase may be a MAP kinase kinase.     

Inducible and endothelial nitric oxide synthase expression during development of transplant arteriosclerosis in rat aortic grafts

In the vascular system, distinct isoforms of nitric oxide synthase (NOS) generate nitric oxide (NO), which acts as a biological messenger. Its role in the development of transplant arteriosclerosis (TA) is still unclear. To investigate whether NO is involved in TA, we studied the expression of NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), by immunohistochemistry and in situ hybridization during the first two post-transplantation months and their relation with cold ischemia (1 to 24 hours) and reperfusion injury using an aortic transplantation model in the rat. We found an increased iNOS expression in the intima and adventitia and a decreased expression in the media, whereas eNOS expression was not significantly altered during the development of TA. Co-localization studies suggested that iNOS-positive cells were vascular smooth muscle cells, monocyte-derived macrophages, and endothelial cells. Prolonged ischemic storage time resulted in an increase in eNOS expression in the neointima. In situ hybridization showed iNOS mRNA expression by vascular cells in the neointima and media. NO produced by iNOS and eNOS may be involved, at least in part, in the pathogenesis of TA in aortic grafts. Additional studies are needed to confirm the modulatory mechanism of NO during the development of TA.     

Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase

The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.     

Imaging of the buffering effect of insulin antibodies in the autoimmune hypoglycemic syndrome

The activity and protein expression of endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) were investigated during the development of hypertension in spontaneously hypertensive rats (SHR). SHR and Wistar-Kyoto rats (WKY) were studied at three different ages: 4, 14 to 17, and 63 weeks of age. After treatment with saline or lipopolysaccharide (LPS, 10 mg/kg IV) for 3 hours, the aortas were removed for measurement of NOS activity and protein expression assay by [3H]-L-citrulline formation method and Western blot analysis, respectively. Plasma levels of nitrite/nitrate (NO2-/NO3-) and tumor necrosis factor-alpha (TNF-alpha) were also determined. At 14 to 17 weeks and 63 weeks, the basal activity and protein expression of eNOS in the aortas were significantly lower in SHR than in WKY. In addition, the aged WKY exhibited lower eNOS activity than that of adult WKY, but this change was not seen in SHR. By comparison, the basal activity and protein expression of iNOS were only observed in SHR of the 14-to-17-week group and in the 63-week group; SHR still exhibited higher activities, and these differences were further exaggerated by treatment with LPS. The basal and LPS-induced NO2-/NO3- and TNF-alpha levels in the plasma were also higher in the SHR except the 4-week group. After treatment with quinapril, the basal and LPS-induced expressions of iNOS in SHR were significantly attenuated. Our results demonstrated that alterations of activity and protein expression of eNOS and iNOS occurred in SHR. In addition, aging may reduce the activity of eNOS in WKY but not in SHR. The decline of eNOS activity and/or expression may contribute to the development of hypertension, whereas the increase of iNOS expression may be a consequence of the pathological state of vessels associated with hypertension in SHR. However, the augmented expression of iNOS in SHR was attenuated by antihypertensive therapy, suggesting that the abnormal expression of iNOS is associated with hypertension.     

Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia

Focal cerebral ischemia is associated with expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), enzymes whose reaction products contribute to the evolution of ischemic brain injury. We tested the hypothesis that, after cerebral ischemia, nitric oxide (NO) produced by iNOS enhances COX-2 activity, thereby increasing the toxic potential of this enzyme. Cerebral ischemia was produced by middle cerebral artery occlusion in rats or mice. Twenty-four hours after ischemia in rats, iNOS-immunoreactive neutrophils were observed in close proximity (<20 micrometer) to COX-2-positive cells at the periphery of the infarct. In the olfactory bulb, only COX-2 positive cells were observed. Cerebral ischemia increased the concentration of the COX-2 reaction product prostaglandin E2 (PGE2) in the ischemic area and in the ipsilateral olfactory bulb. The iNOS inhibitor aminoguanidine reduced PGE2 concentration in the infarct, where both iNOS and COX-2 were expressed, but not in the olfactory bulb, where only COX-2 was expressed. Postischemic PGE2 accumulation was reduced significantly in iNOS null mice compared with wild-type controls (C57BL/6 or SV129). The data provide evidence that NO produced by iNOS influences COX-2 activity after focal cerebral ischemia. Pro-inflammatory prostanoids and reactive oxygen species produced by COX-2 may be a previously unrecognized factor by which NO contributes to ischemic brain injury. The pathogenic effect of the interaction between NO, or a derived specie, and COX-2 is likely to play a role also in other brain diseases associated with inflammation.     

Increased expression of inducible and endothelial constitutive nitric oxide synthases in rat colon tumors induced by azoxymethane

Nitric oxide (NO) is an important bioregulatory mediator involved in a variety of biological processes under both physiological and pathological conditions. To assess whether NO production is altered in colon carcinogenesis, the expression levels and localization of two isoforms of NO synthase, inducible NO synthase (iNOS) and endothelial constitutive NO synthase (eNOS), were examined by immunoblot and immunohistochemical methods in normal colonic mucosa and colon carcinomas induced by azoxymethane in male F344 rats. All colon carcinoma tissues examined were found to have an increased expression of iNOS and eNOS proteins as compared to normal colonic mucosa. In particular, the pronounced staining of iNOS protein localized to the luminal surface of carcinoma epithelial cells was not detectable in normal colon epithelium. The neovasculature in tumor tissues also demonstrated intense eNOS immunoreactivity in endothelial cells. These findings indicate that NO production is markedly elevated in azoxymethane-induced rat colon carcinomas, suggesting that regulatory pathways involving this mediator have some biological relevance to colon carcinogenesis in this model.     

Localization of nitric oxide synthases and nitric oxide production in the rat mammary gland

We investigated nitric oxide (NO) production and the presence of nitric oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the inguinal parts of female Wistar-MS rats primed by implantation with pellets of 17beta-estradiol and progesterone and were diced into approximately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/DMEM plus carboxy-PTIO (an NO scavenger, 100 microM) in the presence or absence of LPS (0.5 microgram/ml) for 2 days. The amount of NO produced spontaneously by the cultured mammary glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS. This enhancement of NO production was completely eliminated by addition of hydrocortisone (3 microM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyses with specific antisera against NOS isoforms such as iNOS, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 +/- 2 kD) and eNOS (152 +/- 3 kD) in extracts prepared from the mammary glands in the culture without LPS. The immunoreactive band of iNOS was highly intense after the treatment of mammary glands with LPS, whereas the corresponding eNOS immunoreactive band was faded. The immunohistochemical study of anti-iNOS antiserum on frozen sections of the cultured mammary glands showed that an immunoreactive substance with the antiserum was localized to the basal layer (composed of myoepithelial cells of alveoli and lactiferous ducts) of the mammary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the mammary glands. Similar observations were noted in the immunohistochemistry of eNOS. In contrast, the immunoreactive signal with the bNOS antiserum was barely detected in the epithelial parts of alveoli and lactiferous ducts of the mammary glands. These observations demonstrate that three isoforms of NOS are present not only in the endothelium of blood vessels but also in the parenchymal cells (the glandular epithelium) of the rat mammary gland, such as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands.     

The solution structure of human endothelin-2 a 1H-NMR and CD study

OBJECTIVE: Peroxynitrite (ONOO-) is an oxidant formed from the rapid reaction of superoxide and nitric oxide (NO) at sites of inflammation. The literature reports conflicting data on the effects of ONOO- in biological systems, with both NO- and oxidant-dependent effects having been demonstrated. The aim of this study was to investigate these distinct mechanisms through examining molecular aspects of the effects of ONOO- on human platelets, a system in which we have previously shown that ONOO- has both pro- and anti-aggregatory effects. METHODS: Platelet function was assessed by measuring platelet P-selectin expression flow cytometrically, intraplatelet Ca2+ concentrations, and by light aggregometry. A colorimetric method was used to measure extracellular platelet membrane thiols. The contribution of NO and cGMP to the pharmacological effects of ONOO- was investigated using an inhibitor of the soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), and the NO scavenger oxy-haemoglobin. RESULTS: Peroxynitrite (50-400 microM) caused a concentration-dependent increase in the number of platelets expressing P-selectin, an increase in intraplatelet Ca2+ concentrations and a decrease in platelet membrane thiols. Peroxynitrite-induced P-selectin expression was augmented by ODQ. In contrast, when P-selectin expression was elicited by collagen, ONOO- acted as an inhibitor of this process, an effect that was further enhanced by the addition of 1% plasma, ODQ or oxy-haemoglobin abolished this inhibitory effect. Finally, low concentrations (50-100 microM) of ONOO- inhibited collagen-induced platelet aggregation, an effect that was reversed by oxy-haemoglobin. CONCLUSIONS: Peroxynitrite exerts dual effects on platelets, which are either activating or inhibitory due to the conversion of ONOO- to NO or NO donors. Peroxynitrite-induced platelet activation seems to be due to thiol oxidation and an increase in intracellular Ca2+. It is important to note that inhibitory, NO-dependent effects occur at lower concentrations than the activating effects. These data are then consistent with the conflicting literature, showing both damaging and cytoprotective effects of ONOO- in biological systems. We hypothesize that the conversion of ONOO- to NO is the critical factor determining the outcome of ONOO- exposure in vivo.     

Immunohistochemical localization of nitric oxide synthase in normal human skin: expression of endothelial-type and inducible-type nitric oxide synthase in keratinocytes

Nitric oxide (NO) is a critical mediator of various biological functions. NO is generated from L-arginine by nitric oxide synthase (NOS), which has three isoforms; endothelial-type NOS (eNOS) and brain-type NOS (bNOS) are constitutive enzymes, and inducible-type NOS (iNOS) is expressed after stimulation. We investigated the expression of NOS in normal human skin by an immunohistochemical technique and western blotting analysis. In human skin, epidermal keratinocytes and the outer root sheath were labeled with not only eNOS antibody but also with iNOS antibody. Both eNOS and iNOS protein in epidermal keratinocytes were confirmed by western blotting. eNOS immunoreactivity was observed in endothelial cells, fibroblasts, the arrector pili muscle, apocrine secretory gland, eccrine coiled duct, and eccrine secretory gland. bNOS immunoreactivity was observed in mast cells. No staining with anti-bNOS antibody was observed in any other cell type. Our present findings suggest that epidermal keratinocytes in normal human skin contain both eNOS and iNOS.     

Calcium channel blockade enhances nitric oxide synthase expression by cultured endothelial cells

In a recent study, we found marked increases in nitric oxide (NO) production and endothelial and inducible NO synthase (eNOS and iNOS) expressions with calcium channel blockade in rats with chronic renal failure. This study was undertaken to determine whether enhanced NO production with calcium channel blockade is a direct effect of this therapy or a consequence of the associated hemodynamic and humoral changes. We tested the effects of a calcium channel blocker, felodipine (10(-5), 10(-6), and 10(-7) mol/L), on nitrate and nitrite (NOx) generation, Ca2+-dependent and -independent NOS activity, and eNOS and iNOS protein masses in proliferating and quiescent rat aortic endothelial cells in culture. Compared with vehicle alone, felodipine significantly increased NOx generation, Ca2+-dependent NOS activity, and eNOS protein mass in proliferating and quiescent endothelial cells. Felodipine did not modify the stimulatory action of 10% fetal calf serum on DNA synthesis (thymidine incorporation) and cell proliferation. Ca2+-independent NOS activity and iNOS protein expression were negligible and unaffected by calcium channel blockade. NOx production and NOS expression were greater in proliferating cells than in quiescent cells. Thus, calcium channel blockade upregulates endothelial NO production in vitro, confirming our previous in vivo study. This observation indicates that the reductions in cytosolic [Ca2+] and vasodilation with calcium channel blockade are not only due to inhibition of Ca2+ entry but also to an NO-cGMP mediated mechanism.     

Temporal profiles of the in vitro phosphorylation rate and immunocontent of glial fibrillary acidic protein (GFAP) after kainic acid-induced lesions in area CA1 of the rat hippocampus: demonstration of a novel phosphoprotein associated with gliosis

Kainate-induced seizure activity causes persistent changes in the hippocampus that include synaptic reorganization and functional changes in the mossy fibers. Using in situ hybridization histochemistry, the expression of PKC alpha, PKC beta, PKC gamma, PKC delta and PKC epsilon mRNAs was investigated in the hippocampus of adult rats following seizures induced by a s.c. injection of kainic acid. In CA1 and CA3, we found a significant decrease in PKC gamma mRNA 1 day after kainic acid which persisted for a 2nd day in CA1. None of the other PKC isoform mRNAs were altered in CA1 or CA3. In granule cells, a significant up-regulation specific to PKC epsilon mRNA was observed. One week after kainic acid administration, a marked increase in PKC epsilon immunoreactivity was found that persisted 2 months after kainic acid administration. PKC epsilon immunoreactivity was found associated with mossy fibers projecting to the hilus of the dentate gyrus and to the stratum lucidum of the CA3 field and presumably with the newly sprouted mossy fibers projecting to the supragranular layer. These data provide the first evidence for a long-lasting increase of the PKC epsilon in the axons of granule cells caused by kainate-induced seizures and suggest that PKC epsilon may be involved in the functional and/or structural modifications of granule cells that occur after limbic seizures.     

Expression of endothelial and inducible nitric oxide synthase in human glomerulonephritis

The presence of nitric oxide (NO) in the kidney has been implicated in the pathogenesis of human glomerulonephritis. However, the exact type of glomerular cells that express NO synthase (NOS) and the NOS isoform involved in the local production of NO has not been identified in the human diseased kidney. We examined the expression of three isoforms of NOS, inducible NOS (iNOS), endothelial NOS (eNOS) and brain NOS (bNOS) in the renal tissue of patients with IgA nephropathy (IgAN, N = 10), lupus nephritis (LN, N = 5), membranous nephropathy (MN, N = 5) and minimal change nephrotic syndrome (MCNS, N = 5). Sections were immunostained and the correlation between the expression of each NOS and the degree of glomerular injury in that section was also examined. Normal portions of surgically resected kidneys served as controls. eNOS was present in glomerular endothelial cells and endothelium of cortical vessels in the control and diseased kidneys. iNOS was localized in mesangial cells, glomerular epithelial cells and infiltrating cells in the diseased glomeruli, whereas immunostaining for iNOS was hardly detected in control kidneys. In addition, the expression pattern of eNOS in each glomerulus was the reverse of that of iNOS. In IgAN and LN, the extent of staining for eNOS correlated negatively with the degree of glomerular injury, while the extent of staining for iNOS correlated positively with the degree of glomerular injury in the same tissues. bNOS was not detected in normal or nephritic glomeruli. Our results indicate the presence of a NO pathway in human diseased kidney, and suggest that NO derived from eNOS and iNOS may be involved in the progression of renal diseases and that NO derived from each NOS may play an important role in different way in human inflamed glomeruli.     

Synergistic cooperation between phorbol ester and IFN-gamma for induction of nitric oxide synthesis in murine peritoneal macrophages

The role of protein kinase C (PKC) in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages was examined. Phorbol ester, a PKC activator, had no effect on NO synthesis by itself, whereas IFN-gamma alone had modest activity. When phorbol ester was used in combination with IFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. This increase in NO synthesis was reflected as increased amount of inducible NO synthase (iNOS) mRNA, as determined by Northern blotting. The optimal effect of phorbol ester was shown at 6 h after treatment with IFN-gamma. Phorbol ester also induced the release of NO to the incubation medium by bacillus Calmette-Guerin-infected peritoneal macrophages. Prolonged incubation of cells with phorbol ester, which down-regulates PKC activity, abolished the synergistic cooperative effect on NO production with IFN-gamma. In addition, such PKC inhibitors as staurosporin or polymyxin B reduced NO production induced by IFN-gamma plus phorbol ester. When the cells were treated with both actinomycin D and phorbol ester after IFN-gamma stimulation, more NO was produced and more iNOS mRNA was expressed than in the cells treated with actinomycin D alone. On the basis of these observations, we conclude that PKC might not be directly involved in the expression of NO synthase, but, instead, might be involved in the stabilization of the iNOS mRNA already expressed by the treatment of IFN-gamma.     

Role of nitric oxide and its interaction with superoxide in the suppression of cardiac muscle mitochondrial respiration. Involvement in response to hypoxia/reoxygenation

BACKGROUND: Nitric oxide (NO); superoxide anion (O2.d-); the reaction product of NO with O2.d-, peroxynitrite (ONOO-); and ischemia/reperfusion have all been reported to inhibit respiration in isolated mitochondria. However, the specific species involved in the inhibition of respiration in intact tissues are poorly understood. METHODS AND RESULTS: O2 consumption in isolated cardiac muscle from bovine calf hearts was quantified by use of a Clark-type electrode. Exogenous and endogenous sources of NO, from S-nitroso-N-acetylpenicillamine (SNAP) and bradykinin or carbachol, reversibly inhibited respiration, whereas the O2.- releasing agent, pyrogallol (PG), inhibited respiration in a manner that was only partially reversed when examined 15 minutes after the removal of PG. The generation of ONOO- with SNAP + PG caused a potentiation of the O2(-)-elicited inhibition of respiration when examined 15 minutes after the removal of the ONOO- generating system. Tiron (a scavenger of O2.-) did not alter the actions of SNAP, but it attenuated the direct inhibitory effects of PG +/- SNAP and essentially eliminated the suppression of respiration observed 15 minutes after removal of the O2.- or ONOO- generating system. Urate (a scavenger of ONOO-) antagonized only the actions of PG + SNAP. After exposure of muscle slices to a model of hypoxia (15 minutes) and reoxygenation (10 minutes), respiratory inhibition was observed. This reoxygenation-induced inhibition was potentiated by L-arginine, the substrate for NO biosynthesis, and was markedly blocked by nitro-L-arginine (an NO synthase inhibitor), Tiron, or urate. CONCLUSIONS: The potentially physiological reversible regulation of respiration in cardiac muscle by NO is converted to an effect that does not show rapid reversibility under conditions in which ONOO- forms, and this could contribute to cardiac dysfunction in situations such as hypoxia/reoxygenation.     

Effects of redox-related congeners of NO on apoptosis and caspase-3 activity

Nitric oxide has been shown to inhibit apoptosis of human umbilical venous endothelial cells (HUVEC). Therefore we investigated the effect of different NO donors, PAPA NONOate (NOC-15; NO.) and nitrosodium tetrafluoroborate (NOBF4, NO+), and the reaction product of NO and O2-, peroxynitrite (ONOO- ), on TNF-alpha- or serum depletion-induced apoptosis of HUVEC. TNF-alpha-induced DNA fragmentation, determined by ELISA, was inhibited by NOC-15, NOBF4, and ONOO- in a concentration-dependent manner (maximal effects with 10 microM NO. and ONOO- and 100 microM NO+). The inhibition of apoptosis correlated with a protective effect on cell viability. The caspases, a cysteine protease family, play an important role in apoptotic processes. To determine whether the different NO donors and ONOO- regulate this enzyme, caspase-3-like activity was measured in homogenates of TNF-alpha-treated HUVEC. The TNF-alpha-induced enzyme activity was abrogated by NO., NO+, and ONOO-. Furthermore, caspase-3 activity was determined in vitro by reconstitution of the separately cloned, bacterially expressed, and purified active p17 and p12 subunits. The reconstituted caspase-3 exhibited enzyme activity, which was suppressed by the different NO donors and ONOO- with an IC50 of 50 microM for NOC-15, 1 mM for NOBF4, and 50 microM for ONOO-. The inhibition of caspase-3 activity correlated with a S-nitrosylation of the reactive cysteine residue and was reversed by further addition of dithiothreitol. This study suggests that the cellular regulatory processes of NO to protect cells from apoptosis may be independent of the redox state and that low concentrations of NO and ONOO- inhibit the cellular suicide program in HUVEC via S-nitrosylation of members of the caspase family.     

Cyclosporin inhibits nitric oxide production in medullary ascending limb cultured cells

BACKGROUND: Nitric oxide (NO) has been shown to play a role in cyclosporin (CsA) nephrotoxicity, but its mechanism of action is still unclear. As inducible NO synthase (iNOS) mRNA has been found to be expressed in rat medullary thick ascending limb (mTAL) cells, we investigated the effects of CsA on NO production in a model of mouse cultured mTAL cells. MATERIALS AND METHODS: The experiments were carried out on sub-cultured cells derived from isolated mTAL microdissected from the kidney of C57BL/6 mice. The identification of the iNOS mRNA in mTAL microdissected segment and cultured cell was confirmed by RT PCR and RsaI digestion. Nitrite (NO2) released by mTAL cells was determined using the modified Griess reagent method and taken as an index of nitric oxide production. The cultured cells were treated with various concentrations of CsA and different signal transduction regulators to assess the effect and possible pathway(s) of action of CsA on NO production in mTAL cells. RESULTS: The basal production of NO by mTAL cells increased by 1.8-fold following incubation with bacterial lipopolysaccaride (LPS). Both aminoguanidine and L-NAME inhibited NO production. CsA (10-300 ng/ml) also inhibited NO production in a dose-dependent manner and prevented its increase induced by LPS. Phorbol 12-myristate 13-acetate (PMA), a PKC stimulator, enhanced slightly the production of NO under basal conditions and prevented the inhibitory action of CsA on NO production. These results suggest that the NO secreted by mouse cultured mTAL cells is dependent on the PKC pathway. CONCLUSION: These results show that CsA may down-regulate the production of NO by cultured mTAL cells expressing iNOS mRNA and that the PKC pathway is involved in this process.     

Effect of protein kinase C inhibitors on cardioprotection by ischemic preconditioning depends on the number of preconditioning episodes

OBJECTIVES: This study examined the possibility that the role of PKC in [corrected] PC, and thus the response to PFC inhibitors, may differ depending on how many ischemic episodes are employed to precondition the heart. METHODS: In the first series of experiments, myocardial infarct was induced by 30 min of coronary occlusion and 3 h of reperfusion in the rabbit. Infarct size was determined by tetrazolium staining and expressed as a percentage of area at risk (%IS/AR). Prior to the 30-min ischemia, rabbits were subjected to no PC, single PC (i.e., PC with an episode of 5 min ischemia/5 min reperfusion), and repetitive PC (2 cycles of 5 min ischemia/5 min reperfusion) with or without one of three treatments: polymyxin B (PolyB), staurosporine (Stauro), and 8-sulfophenylthephylline (SPT). In the second series of experiments, the rabbits received 5 min of coronary occlusion after repetitive PC with or without PolyB or Stauro treatment. Then, myocardial tissue in the ischemic region was sampled for assay of PKC activity. Untreated rabbits served as controls. RESULTS: Single and repetitive PC limited %IS/AR to the same extent (%IS/AR = 9.8 +/- 1.9 and 10.4 +/- 2.3, both p < 0.05, vs. the control value of 44.5 +/- 3.4), and single PC was blocked by PolyB (%IS/AR = 43.9 +/- 2.7) and Stauro (%IS/AR = 31.5 +/- 3.2). Although the protocol of PolyB injection maintained the plasma PolyB level during sustained ischemia well above its Ki for PKC, this agent and also Stauro failed to abolish the protection by repetitive PC (%IS/AR = 21.6 +/- 3.0 and 11.4 +/- 4.3, respectively). SPT, an adenosine receptor antagonist, not only blocked single PC (%IS/AR = 44.4 +/- 4.4) but also attenuated protection by repetitive PC (%IS/AR = 28.3 +/- 3.6). Infarct sizes in non-preconditioned hearts were not modified by PolyB, Stauro, or SPT. The ratio of membrane fraction PKC activity to cytosolic fraction PKC activity was elevated by repetitive PC plus 5 min ischemia, and this change in PKC was inhibited in hearts given PolyB and Stauro. CONCLUSIONS: In contrast to single PC, repetitive PC protects the heart against infarction even when PolyB and Stauro are administered to inhibit PKC during ischemic insult. This difference may be attributable to a PKC-independent mechanism, in which the adenosine receptor may be partly involved.     

Upregulation of renal and vascular nitric oxide synthase in young spontaneously hypertensive rats

The available data on the role of the L-arginine/nitric oxide (NO) pathway in the genesis of hypertension in spontaneously hypertensive rats (SHR) are limited and contradictory. In an attempt to address this issue, male SHR were studied during the early phase of evolution of hypertension (age 8 to 12 weeks) to distinguish the primary changes of NO metabolism from those caused by advanced hypertension, vasculopathy, and aging late in the course of the disease. A group of age-matched male Wistar-Kyoto rats (WKY) served as controls. The SHR exhibited a marked rise in arterial blood pressure and a significant increase in urinary excretion and plasma concentration of NO metabolites (nitrite/nitrate [NOx]). Likewise, the SHR showed a significant elevation of thoracic aorta NO synthase (NOS) activity coupled with significant increases of kidney, aorta, inducible NOS (iNOS), and endothelial NOS (eNOS) proteins. In an attempt to determine whether the enhanced L-arginine/NO pathway is a consequence of hypertension, studies were repeated using 3-week-old animals before the onset of hypertension. The study revealed significant increases in urinary NOx excretion as well as vascular eNOS and renal iNOS proteins. In conclusion, the L-arginine/NO pathway is upregulated in young SHR both before and after the onset of hypertension. Thus, development of hypertension is not due to a primary impairment of NO production in SHR. On the contrary, NO production is increased in young SHR both before and after the onset of hypertension.