cGMP-independent inotropic effects of nitric oxide and peroxynitrite donors: potential role for nitrosylation

Nitric oxide (NO) has concentration-dependent biphasic myocardial contractile effects. We tested the hypothesis, in isolated rat hearts, that NO cardiostimulation is primarily non-cGMP dependent. Infusion of 3-morpholinosydnonimine (SIN-1, 10(-5) M), which may participate in S-nitrosylation (S-NO) via peroxynitrite formation, increased the rate of left ventricular pressure rise (+dP/dt; 19 +/- 4%, P < 0.001, n = 11) without increasing effluent cGMP or cAMP. Superoxide dismutase (SOD; 150 U/ml) blocked SIN-1 cardiostimulation and led to cGMP elaboration. Sodium nitroprusside (10(-10)-10(-7) M), an iron nitrosyl compound, did not augment +dP/dt but increased cGMP approximately eightfold (P < 0.001), whereas diethylamine/NO (DEA/NO; 10(-7) M), a spontaneous NO. donor, increased +dP/dt (5 +/- 2%, P < 0.05, n = 6) without augmenting cGMP. SIN-1 and DEA/NO +dP/dt increase persisted despite guanylyl cyclase inhibition with 1H-(1,2,4)oxadiazolo-(4,3,-a)quinoxalin-1-one (10(-5) M, P < 0.05 for both donors), suggesting a cGMP-independent mechanism. Glutathione (5 x 10(-4) M, n = 15) prevented SIN-1 cardiostimulation, suggesting S-NO formation. SIN-1 also produced SOD-inhibitable cardiostimulation in vivo in mice. Thus peroxynitrite and NO donors can stimulate myocardial contractility independently of guanylyl cyclase activation, suggesting a role for S-NO reactions in NO/peroxynitrite-positive inotropic effects in intact hearts.     

Investigation of the vasorelaxant effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) and diethylamine/nitric oxide (DEA/NO) on the human radial artery used as coronary bypass graft

The radial artery (RA) is used as a spastic coronary bypass graft. This study was designed to investigate the mechanism of vasorelaxant effects of YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole), a nitric oxide (NO)-independent soluble guanylate cyclase (sGC) activator, and DEA/NO (diethylamine/nitric oxide), a NO-nucleophile adduct, on the human RA. RA segments (n = 25) were obtained from coronary artery bypass grafting patients and were divided into 3-4 mm vascular rings.Using the isolated tissue bath technique, the endothelium-independent vasodilatation function was tested in vitro by the addition of cumulative concentrations of YC-1 (10-10 to 3 x 10-7 mol/L) and DEA/NO (10-8 to 3 x 10-5 mol/L) following vasocontraction by phenylephrine in the presence or absence of 10-5 mol/L ODQ (1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one), the selective sGC inhibitor, 10-7 mol/L iberiotoxin, a blocker of Ca2+-activated K+ channels, or 10-5 mol/L ODQ plus 10-7 mol/L iberiotoxin. We also evaluated the effect of YC-1 and DEA/NO on the cGMP levels in vascular rings obtained from human radial artery (n = 6 for each drug). YC-1 (10-10 to 3 x 10-7 mol/L) and DEA/NO (10-8 to 3 x 10-5 mol/L) caused the concentration-dependent vasorelaxation in RA rings precontracted with phenylephrine (10-5 mol/L) (n = 20 for each drug). Pre-incubation of RA rings with ODQ, iberiotoxin, or ODQ plus iberiotoxin significantly inhibited the vasorelaxant effect of YC-1, but the inhibitor effect of ODQ plus iberiotoxin was significantly more than that of ODQ and iberiotoxin alone (p < 0.05). The vasorelaxant effect of DEA/NO almost completely abolished in the presence of ODQ and iberiotoxin plus ODQ, but did not significantly change in the presence of iberiotoxin alone (p > 0.05). The pEC50 value of DEA/NO was significantly lower than those for YC-1 (p < 0.01), with no change Emax values in RA rings. In addition, YC-1-stimulated RA rings showed more elevation in cGMP than that of DEA/NO (p < 0.05). These findings indicate that YC-1 is a more potent relaxant than DEA/NO in the human RA. The relaxant effects of YC-1 could be due to the stimulation of the sGC and Ca2+-sensitive K+channels, whereas the relaxant effects of DEA/NO could be completely due to the stimulation of the sGC. YC-1 and DEA/NO may be effective as vasodilator for the short-term treatment of perioperative spasm of coronary bypass grafts.     

Adenosine signaling in outer medullary descending vasa recta

We tested whether dilation of outer medullary descending vasa recta (OMDVR) is mediated by cAMP, nitric oxide (NO), and cyclooxygenase (COX). Adenosine (A; 10(-6) M)-induced vasodilation of ANG II (10(-9) M)-preconstricted OMDVR was mimicked by the cAMP analog 8-bromoadenosine 3',5'-cyclic monophosphate (10(-10) to 10(-4) M) and reversed by the adenylate cyclase inhibitor SQ-22536. Adenosine (10(-4) M) stimulated OMDVR cAMP production greater than threefold. NO synthase blockade with N(G)-nitro-L-arginine methyl ester and N(G)-monomethyl-L-arginine (10(-4) M) did not affect adenosine vasodilation. Adenosine induced endothelial cytoplasmic calcium transients that were small. Indomethacin (10(-6) M) reversed adenonsine-induced dilation of OMDVR preconstricted with ANG II, endothelin, 4-bromo-calcium ionophore A23187, or carbocyclic thromboxane A(2). In contrast, selective A(2)-receptor activation dilated endothelin-preconstricted OMDVR even in the presence of indomethacin. We conclude that OMDVR vasodilation by adenosine involves cAMP and COX but not NO. COX blockade does not fully inhibit selective A(2) receptor-mediated OMDVR dilation.     

Prostaglandin inhibition of testosterone production induced by luteinizing hormone, dibutyryl cyclic AMP or 3-isobutyl-1-methyl xanthine in dispersed rat testicular interstitial cells.

Testicular interstitial cells were utilized to study the effects of prostaglandins (PG) on in vitro testosterone production and to examine the role of cyclic adenosine-3',5'-monophosphate (cAMP) in this process. Testosterone production was assessed after 3 hour incubations while cAMP accumulation was examined after a 0.5 hour incubation period. Testosterone and cAMP were measured by radioimmunoassay. None of the PGs tested (PGA, PGA2, PGB1, PGE1, PGE2, PGF1alpha PGF2alpha) altered basal testosterone production when present in incubates at concentrations of 1.3 X 10(-8) M to 1.3 X 10(-4). However, at concentrations of 1.3 X 10(-4) M all of these PGs were capable of decreasing Luteinizing Hormone (LH; 100ng)-induced testosterone production. The inhibition of LH-induced testosterone production by the B, E and F series PGs was less pronounced than that for the A series. PGA1 and PGA2 exhibited 80% and 95% inhibition, respectively, at 1.3 X 10(4) M. The inhibitory action of 4 X 10(5) M PGA1 or PGA2 was evident within 30 minutes. Preincubation of interstitial cells with indomethacin (10(-5) or 10(-6) M) for 30 minutes did not alter subsequent basal or LH (100ng)-induced testosterone production. Accumulation of cAMP was stimulated by LH (10 microgram) or by PGs (1.3 X 10(-4) M PGA1, PGA2, PGB1, PGE1 or PGF2alpha). The PG-induced cAMP accumulation thus occurred at concentrations where LH-stimulated testosterone production was inhibited. Furthermore, PGA1 and PGA2 (1.3 X 10(-4) M) inhibited testosterone production induced by either 3-isobutyl-1-methyl xanthine (MIX; 10(-4) M or 10(-3) M) or dibutyryl cAMP (dbcAMP; 10(-4) M or 10(-3) M). These results indicate that PGs can block testosterone production by a direct effect on testicular interstitial cells and suggest that PGs exert their inhibitory action distal to stimulation of cAMP formation. PGs do not appear to play a role in the mechanism of LH action.     

Regulation of flagellar bending by cAMP and Ca2+ in hamster sperm

Hamster sperm were immotile in the medium at free Ca2+ concentrations ([Ca2+]) below 1 x 10(-4) M. The flagellum was acutely bent in the opposite direction to the curve of the hook-shaped heads. This phenomenon seemed to be caused by the decrease in the intracellular cAMP concentration, since the cAMP concentration was low at [Ca2+] below 1 x 10(-4) M and increased abruptly at 1 x 10(-3) M, at which sperm were swimming actively. In addition, sperm became motile due to treatment with 8-bromo-cAMP, a membrane permeable analogue of cAMP, in a medium without Ca2+. These results suggested that extracellular Ca2+ is involved in the regulation of flagellar movement via increasing intracellular cAMP concentration. By the treatment with W-13, a calmodulin inhibitor, sperm also became motile, although cAMP concentration remained at a low level. These results suggested that cAMP is not always required for the flagellar movement when the function of calmodulin is depressed.     

Effect of time and vascular pressure on permeability and cyclic nucleotides in ischemic lungs

We previously found that increased intravascular pressure decreased ischemic lung injury by a nitric oxide (NO)-dependent mechanism (Becker PM, Buchanan W, and Sylvester JT. J Appl Physiol 84: 803-808, 1998). To determine the role of cyclic nucleotides in this response, we measured the reflection coefficient for albumin (sigma(alb)), fluid flux (), cGMP, and cAMP in ferret lungs subjected to either 45 min ("short"; n = 7) or 180 min ("long") of ventilated ischemia. Long ischemic lungs had "low" (1-2 mmHg, n = 8) or "high" (7-8 mmHg, n = 6) vascular pressure. Other long low lungs were treated with the NO donor (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium -1, 2-diolate (PAPA-NONOate; 5 x 10(-4) M, n = 6) or 8-bromo-cGMP (5 x 10(-4) M, n = 6). Compared with short ischemia, long low ischemia decreased sigma(alb) (0.23 +/- 0.04 vs. 0.73 +/- 0.08; P < 0.05) and increased (1.93 +/- 0.26 vs. 0.58 +/- 0.22 ml. min(-1). 100 g(-1); P < 0.05). High pressure prevented these changes. Lung cGMP decreased by 66% in long compared with short ischemia. Lung cAMP did not change. PAPA-NONOate and 8-bromo-cGMP increased lung cGMP, but only 8-bromo-cGMP decreased permeability. These results suggest that ischemic vascular injury was, in part, mediated by a decrease in cGMP. Increased vascular pressure prevented injury by a cGMP-independent mechanism that could not be mimicked by administration of exogenous NO.     

Insulin-like stimulation of cardiac fuel metabolism by physiological levels of glucagon: involvement of PI3K but not cAMP

At concentrations around 10(-9) M or higher, glucagon increases cardiac contractility by activating adenylate cyclase/cyclic adenosine monophosphate (AC/cAMP). However, blood levels in vivo, in rats or humans, rarely exceed 10(-10) M. We investigated whether physiological concentrations of glucagon, not sufficient to increase contractility or ventricular cAMP levels, can influence fuel metabolism in perfused working rat hearts. Two distinct glucagon dose-response curves emerged. One was an expected increase in left ventricular pressure (LVP) occurring between 10(-9.5) and 10(-8) M. The elevations in both LVP and ventricular cAMP levels produced by the maximal concentration (10(-8) M) were blocked by the AC inhibitor NKY80 (20 microM). The other curve, generated at much lower glucagon concentrations and overlapping normal blood levels (10(-11) to 10(-10) M), consisted of a dose-dependent and marked stimulation of glycolysis with no change in LVP. In addition to stimulating glycolysis, glucagon (10(-10) M) also increased glucose oxidation and suppressed palmitate oxidation, mimicking known effects of insulin, without altering ventricular cAMP levels. Elevations in glycolytic flux produced by either glucagon (10(-10) M) or insulin (4 x 10(-10) M) were abolished by the phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 (10 microM) but not significantly affected by NKY80. Glucagon also, like insulin, enhanced the phosphorylation of Akt/PKB, a downstream target of PI3K, and these effects were also abolished by LY-294002. The results are consistent with the hypothesis that physiological levels of glucagon produce insulin-like increases in cardiac glucose utilization in vivo through activation of PI3K and not AC/cAMP.     

Effect of some new cAMP analogs on cAMP-dependent protein kinase isoenzymes.

1. Ten new cAMP analogs were synthesized by replacing the purine ring with with indazole, benzimidazole or benztriazole and/or their nitro and amino derivatives. 2. Each analog proved effective in activating cAMP-dependent protein kinase I (PK-I) purified from rabbit skeletal muscle and cAMP-dependent protein kinase II (PK-II) from bovine heart and chasing 8-[3H]cAMP bound to regulatory subunits in the half-maximal effective concentrations of 2 x 10(-8)-8 x 10(-6) M. 3. The N-1-beta-D-ribofuranosyl-indazole-3'5'-cyclophosphate(I) proved a very poor chaser and activator of both isoenzymes, but when indazole was attached at its N-2 to ribose (IV) or when its H at C-4 (equivalent to the position of amino-group in adenine) was substituted by an amino-(III) or especially nitro-group (II) its efficiency was dramatically increased. 4. Analogs containing benztriazole ring proved as powerful as cAMP irrespective of the presence of substituents (VII-X). 5. Benzimidazole derivatives with amino-(VI) or nitro-group (V) activated PK-II 3 and 20 times better than PK-I. 6. Attaching of ribose to N-2 of indazole or benztriazole increased the affinity to PK-II 10 and 4 times, respectively. 7. Chasing efficiency of cAMP analogs at half-saturating [3H]cAMP tended to correlate with activating potency only for PK-I but at saturating [3H]cAMP concentration for both isoenzymes. 8. On the basis of synergistic activation with 8-Br-cAMP a site 2-selective binding of nitro-benzimidazole (V) and unsubstituted benztriazole (VII) derivatives to PK-II is suggested.     

Suppression of insulin-stimulated glucose transport in L6 myocytes by calcitonin gene-related peptide

The binding of calcitonin gene-related peptide (CGRP) to L6 myocytes, the coupling of this receptor to adenylyl cyclase and the resultant effects on insulin-stimulated 2-deoxyglucose uptake were examined. L6 cells express specific binding sites for CGRP. Binding of human [125I]CGRP was inhibited by rat CGRP with an IC50 of approximately 10(-9) M. Synthetic human calcitonin at concentrations up to 10(-6) M had no effect on the binding of CGRP, suggesting that L6 cells express CGRP receptors, rather than calcitonin receptors which are also capable of binding CGRP. The CGRP receptor appeared to be coupled to adenylyl cyclase. Concentrations of CGRP greater than 3 x 10(-9) M increased the cellular content of cAMP. At 3 x 10(-8) M, CGRP increased cAMP 500-fold. CGRP at 10(-10) M and above suppressed the stimulation of 2-deoxyglucose uptake by insulin. Acute incubation of L6 cells with insulin stimulated 2-deoxyglucose uptake 1.6-fold, which was inhibited up to 70% by CGRP. Our results demonstrate that the specific binding of CGRP to L6 cells causes large increase in the cellular content of cAMP - and inhibition of insulin-stimulated 2-deoxyglucose uptake, but the differences in the dose-response curves suggest that the suppression of insulin action by CGRP cannot be solely explained by the increase in cAMP.     

Pendrin protein abundance in the kidney is regulated by nitric oxide and cAMP

Pendrin is a Cl(-)/HCO(3)(-) exchanger, expressed in the apical regions of some intercalated cell subtypes, and is critical in the pressor response to angiotensin II. Since angiotensin type 1 receptor inhibitors reduce renal pendrin protein abundance in mice in vivo through a mechanism that is dependent on nitric oxide (NO), we asked if NO modulates renal pendrin expression in vitro and explored the mechanism by which it occurs. Thus we quantified pendrin protein abundance by confocal fluorescent microscopy in cultured mouse cortical collecting ducts (CCDs) and connecting tubules (CNTs). After overnight culture, CCDs maintain their tubular structure and maintain a solute gradient when perfused in vitro. Pendrin protein abundance increased 67% in CNT and 53% in CCD when NO synthase was inhibited (N(G)-nitro-l-arginine methyl ester, 100 μM), while NO donor (DETA NONOate, 200 μM) application reduced pendrin protein by ～33% in the CCD and CNT. When CNTs were cultured in the presence of the guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (10 μM), NO donors did not alter pendrin abundance. Conversely, pendrin protein abundance rose when cAMP content was increased by the application of an adenylyl cyclase agonist (forskolin, 10 μM), a cAMP analog (8-bromo-cAMP, 1 mM), or a phosphodiesterase inhibitor (BAY60-7550, 50 μM). Since NO reduces cellular cAMP in the CNT, we asked if NO reduces pendrin abundance by reducing cAMP. With blockade of cGMP-stimulated phosphodiesterase II, NO did not alter pendrin protein abundance. We conclude that NO acts through cAMP to reduce pendrin total protein abundance by enhancing cAMP degradation.     

Effect of ovarian steroids on cyclic adenosine 3':5'-monophosphate production stimulated by arginine vasopressin in rat renal monolayer cultured cells

Renal resistance to antidiuretic hormone (ADH) has been speculated to be a mechanism of transient nephrogenic diabetes insipidus occurring during late pregnancy. In order to study possible involvement of ovarian steroids in this mechanism, their effect on cyclic adenosine 3':5'-monophosphate (cAMP) response to arginine vasopressin (AVP) was examined utilizing rat and human renal medullary cells in monolayer culture. In both rat and human cells, estradiol significantly reduced cAMP response to AVP; estradiol at 1.84 x 10(-8) M, 1.84 x 10(-7) M and 1.84 x 10(-6) M decreased cAMP production stimulated by 10(-8) M AVP to 78 +/- 5%, 67 +/- 2% (P less than 0.05) and 52 +/- 1% (P less than 0.001) of the control in rat renal cells, respectively, and in human renal cells the effect of estradiol was comparable to that in rat cells. In rat renal cells, progesterone also reduced cAMP response to AVP dose-dependently; progesterone at 1.59 x 10(-7) M, 1.59 x 10(-6) M and 1.59 x 10(-5) M decreased cAMP production stimulated by 10(-8) M AVP to 87 +/- 1%, 72 +/- 5% (P less than 0.001) and 37 +/- 5% (P less than 0.001) of the control, respectively. On the other hand, corticosterone and dexamethasone at concentrations ranging from 10(-8) M to 10(-5) M and aldosterone at concentrations ranging from 10(-9) M to 10(-5) M did not alter cAMP response to AVP significantly. The suppressive effect of estradiol increased with time until six hours and thereafter it reached a plateau.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of human platelet aggregation by nitric oxide donor drugs: relative contribution of cGMP-independent mechanisms

Inhibition of platelet activation by nitric oxide (NO) is not exclusively cGMP-dependent. Here, we tested whether inhibition of platelet aggregation by structurally distinct NO donors is mediated by different mechanisms, partly determined by the site of NO release. Glyceryl trinitrate (GTN), sodium nitroprusside (SNP), S-nitrosoglutathione (GSNO), diethylamine diazeniumdiolate (DEA/NO), and a novel S-nitrosothiol, RIG200, were examined in ADP (8 microM)- and collagen (2.5 microgram/ml)-activated human platelet rich plasma. GTN was a poor inhibitor of aggregation whilst the other NO donors inhibited aggregation, irrespective of agonist. These effects were abolished by the NO scavenger, hemoglobin (Hb; 10 microM, P < 0.05, n = 6), except with high concentrations of DEA/NO, when NO concentrations exceeded the capacity of Hb. However, experiments with the soluble guanylate cyclase inhibitor, ODQ (100 microM), indicated that only SNP-mediated inhibition was exclusively cGMP-dependent. Furthermore, the cGMP-independent effects of S-nitrosothiols were distinct from those of DEA/NO, suggesting that different NO-related mediators (e.g., nitrosonium and peroxynitrite, respectively) are responsible for their actions.     

Changes in the activity and tertiary structure of beta-amylase from wheat seeds under the effect of salts]

Changes are observed of 1-anylinonaphtalene-8-sulphonate probe fluorescence intensity, connected with beta-amylase in the presence of Ca(NO3)2, Mg(NO3)2, KNO3, NH4NO3 and (NH4)2SO4 at a concentration range within 10(-4)--1 M. Considerable decrease of the fluorescence intensity was observed under the addition of all the salts mentioned at concentration of 10(-3)--10(-4) M. A quantum yield increase of probe fluorescence was noted for Mg(NO3)2 (10(-4) M). High concentrations of Ca(NO3)2 and Mg(NO3)2 (0.25--1 M) also resulted in a sharp increase of the fluorescence intensity. Changes of beta-amylase activity took place simultaneously. The changes of the enzyme activity are suggested to be due to changes in the conformation of the enzyme protein under the effect of salts.     

The inotropic effect of nitric oxide on mammalian papillary muscle is dependent on the level of beta1-adrenergic stimulation

We tested the hypothesis that nitric oxide has a positive inotropic effect on mammalian cardiac muscle contractility and that this effect sums with the positive inotropic effect of beta1-adrenergic agonists when both are present. Feline right ventricular papillary muscles were stimulated to contract isometrically at 0.2 Hz in Krebs-Henseleit bicarbonate buffer (KREBS) gassed with 95% O2 and 5% CO2 (26 degrees C; pH 7.34). The nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine (SNAP, 10(-5) M), and the membrane permeable cGMP analog 8-bromoguanosine-3',5'-cyclophosphate sodium (Br-cGMP, 10(-5) M), significantly increased developed force by 13.3+/-1.5% (n = 11) and 7.8+/-2.8% (n = 7), respectively. SNAP, at 10(-5) M, significantly increased the force developed by papillary muscle treated with 10(-11) M or 10(-9) M dobutamine hydrochloride (a beta1-adrenergic agonist) (n = 25, 11.3+/-2.9% and 10.0+/-3.6%, respectively) when compared with the addition of KREBS (n = 27, 2.6+/-0.9% and 5.5+/-0.9%), but the increase was less than predicted by the sum of inotropic effects of SNAP and dobutamine. SNAP at 10(-5) M did not change developed force in muscles treated with 10(-7) M dobutamine but it significantly decreased developed force in muscles challenged with 10(-5) M dobutamine (n = 18, 29.3+/-5.0%) when compared with KREBS (n = 10, 41.5+/-6.8%). Similarly, 10(-4) M 8-bromo-adenosine cyclic 3',5'-hydrogen phosphate monosodium (a membrane permeable cAMP analog) increased developed force 14.9+/-3.3% and the addition of 10(-5) M Br-cGMP to those muscles significantly reduced developed force by 3.5%+/-1.1% (n = 7). Thus, the positive inotropic effect of NO decreased and ultimately became an attenuation as the level of beta1-adrenergic stimulation increased due at least in part, to an interaction between the cAMP and cGMP second messenger pathways.     

Impact of nitric oxide on adrenomedullin- and proadrenomedullin N-terminal 20 peptide-induced cardiac responses: action by alone and combined administration

The cardiac effects of adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) as well as the possible signaling pathways were investigated. In the isolated perfused rat heart, infusion of AM (10(-11) to 10(-8) M) and PAMP(10(-11) to 10(-8) M) for 10 min, alone or in combination, induced concentration-dependent decreases in the left ventricular pressure (LVP), LVP +/- dp/dtmax of the hearts. The effects were attenuated by Nomega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase. ADM and PAMP alone or in combinations increased the coronary fluid (CF), which could be antagonized by L-NAME. Pretreatment of H89, an inhibitor of protein kinase A (PKA), failed to alter the AM- or PAMP-induced decreases in LVP and LVP +/- dp/dtmax, but further promoted the AM or PAMP increased CF. The cAMP content in left cardiac ventricle was increased significantly by ADM infusions but not by PAMP. There was no statistical difference in cAMP contents with ADM administrated alone from those combined with ADM and PAMP. In conclusion, this study reveals that ADM and PAMP infused alone or in combinations inhibited the function of rat hearts in vitro, which may be partly involved with the NOS/NO pathway, rather than cAMP/PKA.     

Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite: mechanisms and relative contributions to aconitase inactivation

Using highly purified recombinant mitochondrial aconitase, we determined the kinetics and mechanisms of inactivation mediated by nitric oxide (*NO), nitrosoglutathione (GSNO), and peroxynitrite (ONOO(-)). High *NO concentrations are required to inhibit resting aconitase. Brief *NO exposures led to a reversible inhibition competitive with isocitrate (K(I)=35 microM). Subsequently, an irreversible inactivation (0.65 M(-1) s(-1)) was observed. Irreversible inactivation was mediated by GSNO also, both in the absence and in the presence of substrates (0.23 M(-1) s(-1)). Peroxynitrite reacted with the [4Fe-4S] cluster, yielding the inactive [3Fe-4S] enzyme (1.1 x 10(5) M(-1) s(-1)). Carbon dioxide enhanced ONOO(-)-dependent inactivation via reaction of CO(3)*(-) with the [4Fe-4S] cluster (3 x 10(8) M(-1) s(-1)). Peroxynitrite also induced m-aconitase tyrosine nitration but this reaction did not contribute to enzyme inactivation. Computational modeling of aconitase inactivation by O(2)*(-) and *NO revealed that, when NO is produced and readily consumed, measuring the amount of active aconitase remains a sensitive method to detect variations in O(2)*(-) production in cells but, when cells are exposed to high concentrations of NO, aconitase inactivation does not exclusively reflect changes in rates of O(2)*(-) production. In the latter case, extents of aconitase inactivation reflect the formation of secondary reactive species, specifically ONOO(-) and CO(3)*(-), which also mediate m-aconitase tyrosine nitration, a footprint of reactive *NO-derived species.     

Up-regulation of thrombomodulin in human umbilical vein endothelial cells in vitro

Previous studies have shown that thrombomodulin (TM) on endothelial cells is down-regulated by endotoxin, interleukin-1 beta (IL-1 beta), and tumor necrosis factor (TNF). This loss of anti-coagulant potential is thought to be related to the hypercoagulable state in sepsis, inflammation, and cancer. The current studies describe up-regulation of TM in human umbilical vein endothelial cells (HUVECs) by several compounds as judged by increased surface cofactor activity, surface TM antigen, and TM mRNA levels. Surface TM activity was increased by active phorbol esters (10(-8) M, 24-48 h), analogs of cAMP (1-10 mM, 4 h), and forskolin (10(-5) M, 24-48 h). Up-regulation of TM in HUVECs by 4 beta-phorbol 12-myristate 13-acetate (PMA) and dibutyryl cAMP (dBcAMP) was due to de novo synthesis of TM protein resulting from increased TM mRNA levels. The results suggest that protein kinase C and protein kinase A may be involved in cellular regulatory mechanisms for TM expression. In addition, PMA effects on surface TM activity are biphasic, with an initial reduction followed by a significant enhancement. Hence, we propose that compounds capable of increasing intracellular cAMP concentrations in HUVECs may be useful in preventing thrombosis by increasing the anti-thrombotic properties of endothelial cells.     

Activators of Protein Kinase C Act at a Postreceptor Site to Amplify Cyclic AMP Production in Rat Pinealocytes

Activation of alpha 1-adrenoceptors appears to amplify beta-adrenergic stimulation of cyclic AMP (cAMP) accumulation in rat pinealocytes severalfold by a mechanism involving activation of a Ca2+-, phospholipid-dependent protein kinase (protein kinase C). The mechanism of action of protein kinase C was investigated in this report using intact cells. Activation of protein kinase C with 4 beta-phorbol 12-myristate 13-acetate (PMA; 10(-7) M) or the alpha 1-adrenergic agonist phenylephrine (PE; 10(-6) M) did not inhibit cAMP efflux in beta-adrenergically stimulated cells. The amplification of the beta-adrenergic cAMP response by these agents also occurred in the presence of isobutylmethylxanthine (10(-3) M) and Ro 20-1724 (10(-4) M), an observation suggesting that inhibition of cAMP phosphodiesterase activity is not the mechanism of action. Furthermore, although PMA (10(-7) M) caused a sixfold increase in the magnitude of the cAMP response to isoproterenol, it did not alter the EC50 of the response (1.7 X 10(-8) M), a result indicating that protein kinase C activation does not alter beta-adrenoceptor sensitivity. The cAMP response following cholera toxin pretreatment (60-120 min) was rapidly and markedly enhanced by alpha 1-adrenergic agonists (cirazoline greater than PE greater than methoxamine), by phorbol esters (PMA greater than 4 beta-phorbol 12,13,-dibutyrate much greater than 4 alpha-phorbol 12,13-didecanoate), and by synthetic diacylglycerols (1,2-dioctanoylglycerol greater than 1-oleoyl 2-acetylglycerol much greater than diolein). The cAMP response to forskolin (10(-5)-10(-3) M) was also increased by PE (3 X 10(-6) M) and PMA (10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of Cloudman melanoma tyrosinase activity occurs predominantly in G2 phase of the cell cycle

A widely accepted notion is that an increasing cellular cyclic AMP (cAMP) concentration is prerequisite for increasing tyrosinase activity and melanin synthesis and for regulating proliferation of pigment cells. alpha-Melanocyte stimulating hormone (alpha-MSH) increases cAMP and tyrosinase activity in Cloudman melanoma cells. Prostaglandins (PGs) E1 and E2 increase melanoma cell tyrosinase activity and inhibit proliferation. Both PGs, but not alpha-MSH, block the progression of Cloudman melanoma cells from G2 phase of the cell cycle into M or G1. Only PGE1 and not PGE2 causes an elevation of cellular cAMP concentrations. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) at 5 x 10(-4) M effectively blocks the increased cAMP synthesis by cells treated with 10 micrograms/ml PGE1. The addition of DDA, however, enhances the melanogenic response of melanoma cells to 10 micrograms/ml PGE1 or PGE2, 10(-7) M alpha-MSH, 10(-4) M isobutylmethylxanthine, 10(-4) M dibutyryl cyclic AMP. DDA also augments the effects of PGE1 or PGE2 on the melanoma cell cycle. Moreover, when DDA is added concomitantly with alpha-MSH, more cells are recruited into G2 than observed in untreated controls. Neither alpha-MSH nor DDA alone has any effect on the cell cycle. These findings undermine the role of cAMP in the melanogenic process and suggest that blocking melanoma cells in G2 may be required for the remarkable stimulation of tyrosinase activity observed with PGE1 or PGE2 alone or in combination with DDA. The observed block in G2 may be essential for the synthesis of sufficient mRNA, which is required for stimulation of tyrosinase activity.     

Effect of nitroxyl on the hamster retinal nitridergic pathway

There is a growing body of evidence on the role of nitric oxide (NO) in retinal physiology. Recently, interest has developed in the functional role of an alternative redox form of NO, namely nitroxyl (HNO/NO(-)), because it is formed by a number of diverse biochemical reactions. The aim of the present report was to comparatively analyze the effect of HNO and NO on the retinal nitridergic pathway in the golden hamster. For this purpose, sodium trioxodinitrate (Angeli's salt) and diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) were used as HNO and NO releasers, respectively. Angeli's salt and DEA/NO significantly decreased nitric oxide synthase activity. In addition, Angeli's salt (but not DEA/NO) significantly decreased l-arginine uptake. DEA/NO significantly increased cGMP accumulation at low micromolar concentrations, while Angeli's salt affected this parameter with a threshold concentration of 200muM. Although Angeli's salt and DEA/NO significantly diminished reduced glutathione and protein thiol levels in a similar way, DEA/NO was significantly more effective than AS in increasing S-nitrosothiol levels. None of these compounds increased retinal lipid peroxidation. These results suggest that HNO could regulate the hamster retinal nitridergic pathway by acting through a mechanism that only partly overlaps with that involved in NO response.