Forskolin fails to activate L-type calcium current in hypertrophied cardiomyocytes of chronically hypoxic rats

We have shown that the contractile, cytosolic calcium ([Ca2+]i) and cyclic AMP (cAMP) responses to beta-adrenoceptor stimulation are attenuated in ventricular myocytes of chronically hypoxic (CH) rats. The aim of this study was to examine the effect of forskolin on the L-type Ca2+ current in CH hypertrophied ventricular myocytes. Patch-clamp recording of the L-type Ca2+ current was measured in right ventricular myocytes of normoxic control and CH rats exposed to 10% inspired oxygen for 4 weeks. The breadth, but not the length, of CH myocytes was significantly greater than that of the control group. Activation of beta-adrenoceptor with isoproterenol (0.1 microM) increased the peak Ca2+ current by 83% in the normoxic control but the increase of peak Ca2+ current was not significant in the CH myocytes. Forskolin (0.1 - 1 microM), an activator of adenylyl cyclase, increased the peak Ca2+ current by 49% - 102% in the normoxic controls but it did not cause significant change of the peak Ca2+ current in CH myocytes. These results suggest an absence of forskolin-induced activation of Ca2+ current in hypertrophied ventricular myocytes during chronic hypoxia. The failure of activation of the Ca2+ current is consistent with the idea that adenylyl cyclase function is down-regulated in CH hypertrophied myocytes.     

Role of cAMP in the functional interaction of carbachol with different cAMP elevating agents in rabbit atrium.

The muscarinic agonist carbachol antagonized positive inotropic responses of rabbit left atria to the beta-adrenoceptor agonist isoproterenol, the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor IBMX. Carbachol also reduced cAMP levels elevated by isoproterenol, but had no significant effect on cAMP levels in the presence of either forskolin or IBMX. Pre-treatment of rabbits with a dose of pertussis toxin which completely blocked the reduction by carbachol of isoproterenol-induced increases in cAMP, also blocked the reversal by carbachol of positive inotropic responses to isoproterenol, but only partially attenuated the antagonism by carbachol of inotropic responses to forskolin and IBMX. These data suggest that antagonism by carbachol of forskolin and IBMX-induced increases in cAMP levels does not play an important role in the functional interaction of carbachol with these cAMP-elevating agents.     

Reduced levels of cyclic AMP contribute to the enhanced oxidative stress in vascular smooth muscle cells from spontaneously hypertensive rats

We have earlier shown that aortic vascular smooth muscle cells (VSMC) from 12-week-old spontaneously hypertensive rats (SHR) exhibited enhanced production of superoxide anion (O(2)(-)) compared with Wistar-Kyoto (WKY) rats. This production was attenuated to control levels by losartan, an angiotensin II (Ang II) AT(1)-receptor antagonist, suggesting that the AT(1) receptor is implicated in enhanced oxidative stress in SHR. Since AT(1) receptor activation signals via adenylyl cyclase inhibition and decreases cAMP levels, it is possible that AT(1) receptor-mediated decreased levels of cAMP contribute to the enhanced production of O(2)(-) in SHR. The present study was undertaken to investigate this possibility. The basal adenylyl cyclase activity as well as isoproterenol and forskolin-mediated stimulation of adenylyl cyclase was significantly attenuated in VSMC from 12-week-old SHR compared with those from WKY rats, whereas Ang II-mediated inhibition of adenylyl cyclase was significantly enhanced by about 70%, resulting in decreased levels of cAMP in SHR. NADPH oxidase activity and the levels of O2- were significantly higher (about 120% and 200%, respectively) in VSMC from SHR than from WKY rats. In addition, the levels of p47(phox) and Nox4 proteins, subunits of NADPH oxidase, were significantly augmented about 35%-40% in VSMC from SHR compared with those from WKY rats. Treatment of VSMC from SHR with 8Br-cAMP, as well as with cAMP-elevating agents such as isoproterenol and forskolin, restored to control WKY levels the enhanced activity of NADPH oxidase and the enhanced levels of O(2)(-), p47(phox), and Nox4. Furthermore, in the VSMC A10 cell line, 8Br-cAMP also restored the Ang II-evoked enhanced production of O(2)(-), NADPH oxidase activity, and enhanced levels of p47(phox) and Nox4 proteins to control levels. These data suggest that decreased levels of cAMP in SHR may contribute to the enhanced oxidative stress in SHR and that increasing the levels of cAMP may have a protective effect in reducing oxidative stress and thereby improve vascular function.     

Modification of beta-adrenoceptors and adenylyl cyclase in hearts perfused with hypochlorous acid

It is now well known that the signal transduction pathway involving beta-adrenoceptors and adenylyl cyclase is altered in ischemic heart disease. Since leukocytes accumulate in the ischemic heart and produce hypochlorous acid (HOCl), we investigated the effects of HOCl upon beta-adrenoceptors and adenylyl cyclase activities by perfusing rat hearts with 0.1 mM HOCl for 10 min and isolating cardiac membranes. Marked depressions in both the density and affinity of beta1-adrenoceptors were observed, whereas no significant change in the affinity or density of beta2-adrenoceptors was seen in hearts perfused with HOCl. After treatment of hearts with HOCl, competition curves using isoproterenol, a beta-adrenoceptor agonist, revealed a decrease in the proportion of high affinity binding sites. The adenylyl cyclase activities in the absence and presence of forskolin, NaF, Gpp(NH)p, or isoproterenol were depressed in hearts perfused with HOCl; however, the stimulatory effects of these agents on adenylyl cyclase were either unaltered or augmented. The presence of methionine in the perfusion medium prevented the HOCl-induced changes in beta1-adrenoceptors and adenylyl cyclase activity. These results suggest that HOCl may produce a defect in the beta-adrenoceptor linked signal transduction mechanism by affecting both beta1-adrenoceptors and adenylyl cyclase enzyme in the myocardium.     

Characterization of myometrial desensitization to beta-adrenergic agonists

Continuous exposure of ovine myometrial strips exposed to isoproterenol (10 microM) resulted in only transient inhibition with contractions returning within 60 min. Rechallenging these strips with isoproterenol failed to induce inhibition, confirming the occurrence of desensitization. In contrast, exposure of myometrial tissue to isoproterenol for only 5 min did not result in desensitization. Myometrial strips exposed to isoproterenol demonstrated a significant increase in cAMP content associated with inhibition of contractile activity and a subsequent fall in cAMP content upon desensitization. Elevation of endogenous cAMP levels by either inhibition of cAMP-dependent phosphodiesterase activity (0.5 mM isobutylmethylxanthine, in ovine strips) or direct activation of adenylyl cyclase (10 microM forskolin, in rat strips) induced a rapid and significant inhibition of myometrial contractile activity in desensitized tissue. Scatchard analysis of the binding of the beta-adrenoceptor antagonist, [125I]iodocyanopindolol, revealed a significant reduction in the concentration of beta-adrenergic receptors (but no change in binding affinity) in desensitized myometrial tissue. Incubation of desensitized tissue with fresh buffer for 3 h induced only a partial recovery in responsiveness to isoproterenol. These data suggest that prolonged, but not acute, exposure of the myometrium to beta-adrenergic agonists induces a state of desensitization that is associated with a down-regulation of beta-adrenoceptors but maintenance of postreceptor function.     

Regulation of glutamatergic neurotransmission in the striatum by presynaptic adenylyl cyclase-dependent processes

The aim here was to examine the possible roles of adenylyl cyclase- and protein kinase A (PKA)-dependent processes in ionotropic glutamate receptor (iGluR)-mediated neurotransmission using superfused mouse striatal slices and a non-metabolized L-glutamate analogue, D-[3H]aspartate. The direct and indirect presynaptic modulation of glutamate release and its susceptibility to changes in the intracellular levels of cyclic AMP (cAMP), Ca(2+) and calmodulin (CaM) and in protein phosphorylation was characterized by pharmacological manipulations. The agonists of iGluRs, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate, stimulated the basal release of D-[3H]aspartate, while N-methyl-D-aspartate (NMDA) was without effect. Both the AMPA- and kainate-mediated responses were accentuated by the beta-adrenoceptor agonist isoproterenol. These facilitatory effects were mimicked by the permeable cAMP analogue dibutyryl-cAMP. The beta-adrenoceptor antagonist propranolol, the adenylyl cyclase inhibitor MDL12,330A, the inhibitor of PKA and PKC, H-7, and the PKA inhibitor H-89 abolished the isoproterenol effect on the kainate-evoked release. The dibutyryl-cAMP-induced potentiation was also attenuated by H-7. Isoproterenol, propranolol and MDL12,330A failed to affect the basal release of D-[3H]aspartate, but dibutyryl-cAMP was inhibitory and MDL12,330A activatory. In Ca(2+)-free medium, the kainate-evoked release was enhanced, being further accentuated by the CaM antagonists calmidazolium and trifluoperazine, though these inhibited the basal release. The potentiating effect of calmidazolium on the kainate-stimulated release was counteracted by both MDL12,330A and H-7.We conclude that AMPA- and kainate-evoked glutamate release from striatal glutamatergic terminals is potentiated by beta-adrenergic receptor-mediated adenylyl cyclase activation and cAMP accumulation. Glutamate release is enhanced if the Ca(2+)- and CaM-dependent, kainate-evoked processes do not prevent the excessive accumulation of intracellular cAMP.     

Nitric oxide-dependent and -independent mechanisms are involved in TNF-alpha -induced depression of cardiac myocyte contractility

Previous studies have demonstrated the presence of myocardial depression in clinical and experimental septic shock. This response is mediated, in part, through circulating TNF-alpha-induced, nitric oxide-dependent, depression of basal myocyte contractility. Other mechanisms of early myocardial dysfunction involving decreased response to adrenergic stimulation may exist. This study evaluated the presence and nitric oxide dependence of impaired adrenergic response to TNF-alpha in in vitro cardiac myocytes. The contraction of electrically paced neonatal rat cardiac myocytes in tissue culture was quantified using a closed-loop video tracking system. TNF-alpha induced depression of baseline contractility over the first 20 min of cardiac myocyte exposure. This effect was blocked by N-methyl-arginine (NMA), a nitric oxide synthase inhibitor, in all studies. Contractile and cAMP response to increasing concentrations of isoproterenol was deficient in cardiac myocytes exposed to TNF-alpha regardless of the presence of NMA. In contrast, increasing concentrations of forskolin (a direct stimulant of adenylate cyclase) and dibutyryl cAMP (a metabolically active membrane-soluble analog of cAMP) completely reversed TNF-alpha-mediated depression, though only in the presence of NMA. Forskolin-stimulated cAMP generation remained intact regardless of NMA. Increasing concentrations of exogenous calcium chloride, unlike other inotropic agents, corrected TNF-alpha-mediated defects of contractility independent of the presence of NMA. These data suggest that TNF-alpha exposure is associated with a second nitric oxide-independent but calcium-dependent early depressant mechanism that is manifested by reduced contractile and cAMP response to beta-adrenergic stimulation.     

Regulation of the adenylyl cyclase signaling system in various types of cultured endothelial cells

We studied the effects of modulators of the adenylyl cyclase pathway on the accumulation of cAMP in endothelial cells isolated from bovine aortas, pig pulmonary arteries, human umbilical veins, and human subcutaneous adipose microvessels. In addition to quantitative differences in the basal levels, cAMP stimulation in different endothelial cell types varied in sensitivity and magnitude in response to both the direct adenylyl cyclase activator forskolin and the β-adrenergic receptor agonist isoproterenol. Furthermore, the ubiquitous phosphodiesterase inhibitor IBMX differentially enhanced both the basal and the stimulated cAMP levels in the various cell types. Histamine caused an elevation of cAMP only in bovine aortic endothelial cells and in human umbilical vein endothelial cells. Treatment of the cells with cholera and pertussis toxins, which uniquely affect G-protein subunits, resulted in divergent elevation of cAMP in the various cells. Thus, in each cell type, a distinct profile of regulation of the cAMP levels was found. Our results suggest that the adenylyl cyclase signaling system in various types of endothelial cells can be differentially regulated at the levels of receptors, G-proteins, adenylyl cyclase, and phosphodiesterase.     

Effects of streptozotocin-induced diabetes on Kv channels in rat small coronary smooth muscle cells

Diabetes impairs endothelium dependent vasodilation, but the mechanism of endothelium independent dilation is not well understood. In the present study, we examined the effect of streptozotocin (STZ)-induced diabetes on the vasomotor of small coronary artery and the activity of voltage-dependent K+ channel of vascular smooth muscle cells in STZ rats [corrected] using the videomicroscopy and patch clamp method. STZ-induced diabetes appeared to [corrected] reduce the vasodilation induced by beta-adrenoceptor agonist, isoproterenol (10(-9)-10(-5) mol/l), and adenylyl cyclase activator forskolin (10(-9)-10(-5) mol/l) respectively (isoproterenol: 44.2 +/- 6.7% vs. 82.5 +/- 4.8%, and forskolin: 54.4 +/- 4.5% vs. 94.3 +/- 2.4%). 4-AP, a Kv channel blocker of VSMC, further decreased dilation to isoproterenol (44.2 +/- 6.7% vs. 10.2 +/- 3.5%) and forskolin (54.4 +/- 4.5% vs. 13.8 +/- 11.0%) significantly. Whole cell K+ current recording demonstrated that STZ-induced diabetes decreased isoproterenol and forskolin-induced K+ current (ISO: 55.6 +/- 7.8 pA/pF vs. 28.4 +/- 3.4 pA/pF, forskolin: 61.3 +/- 9.8 pA/pF vs. 32.4 +/- 3.4 pA/pF). 4-AP further reduced the decreased K+ current (ISO: 28.4 +/- 3.4 pA/pF vs. 14.3 +/- 2.1 pA/pF, forskolin: 32.4 +/- 3.4 pA/pF vs. 14.8 +/- 2.9 pA/pF). These results indicated that STZ-induced diabetes impaired cAMP mediated dilation of small coronary artery and suppressed the Kv channel activity of vascular smooth muscle cells. Kv channel of VSMC was shown to play a determinate role reducing dilation of small coronary artery in STZ rats.     

Vasoconstrictive effect of hydrogen sulfide involves downregulation of cAMP in vascular smooth muscle cells

Hydrogen sulfide (H(2)S), a new endogenous mediator, produces both vasorelaxation and vasoconstriction. This study was designed to examine whether cAMP mediates the vasoconstrictive effect of H(2)S. We found that NaHS at a concentration range of 10-100 microM (yields approximately 3-30 microM H(2)S) concentration-dependently reversed the vasodilation caused by isoprenaline and salbutamol, two beta-adrenoceptor agonists, and forskolin, a selective adenylyl cyclase activator, in phenylephrine-precontracted rat aortic rings. Pretreatment with NaHS (10-100 microM) for 5 min also significantly attenuated the vasorelaxant effect of salbutamol and forskolin. More importantly, NaHS (5-100 microM) significantly reversed forskolin-induced cAMP accumulation in vascular smooth muscle cells. However, NaHS produced significant, but weaker, vasoconstriction in the presence of N(G)-nitro-l-arginine methyl ester (100 microM), a nitric oxide synthase inhibitor, or in endothelium-denuded aortic rings. Blockade of ATP-sensitive potassium channels with glibenclamide (10 microM) failed to attenuate the vasoconstriction induced by H(2)S. Taken together, we demonstrated for the first time that the vasoconstrictive effect of H(2)S involves the adenyly cyclase/cAMP pathway.     

Functional changes in adenylyl cyclases and associated decreases in relaxation responses in mesenteric arteries from diabetic rats

To assess the functional change in adenylyl cyclases (AC) associated with the diabetic state, we investigated AC-mediated relaxations and cAMP production in mesenteric arteries from rats with streptozotocin (STZ)-induced diabetes. The relaxations induced by the water-soluble forskolin (FSK) analog NKH477, which is a putative AC5 activator, but not by the beta-adrenoceptor agonist isoproterenol (Iso) and the AC activator FSK, were reduced in intact diabetic mesenteric artery. In diabetic rats, however, Iso-, FSK-, and NKH477-induced relaxations were attenuated in the presence of inhibitors of nitric oxide synthase and cyclooxygenase. To exclude the influence of phosphodiesterase (PDE), we also examined the relaxations induced by several AC activators in the presence of 3-isobutyl-1-methylxanthine (IBMX; a PDE inhibitor). Under these conditions, the relaxation induced by Iso was greatly impaired in STZ-diabetic rats. This Iso-induced relaxation was significantly attenuated by pretreatment with SQ-22536, an AC inhibitor, in mesenteric rings from age-matched controls but not in those from STZ-diabetic rats. Under the same conditions, the relaxations induced by FSK or NKH477 were impaired in STZ-diabetic rats. Neither FSK- nor A-23187 (a Ca2+ ionophore)-induced cAMP production was significantly different between diabetics and controls. However, cAMP production induced by Iso or NKH477 was significantly impaired in diabetic mesenteric arteries. Expression of mRNAs and proteins for AC5/6 was lower in diabetic mesenteric arteries than in controls. These results suggest that AC-mediated relaxation is impaired in the STZ-diabetic rat mesenteric artery, perhaps reflecting a reduction in AC5/6 activity.     

Modulation of monocarboxylic acid transporter-1 kinetic function by the cAMP signaling pathway in rat brain endothelial cells

MCT1 (monocarboxylic acid transporter 1) facilitates bidirectional monocarboxylic acid transport across membranes. MCT1 function and regulation have not been characterized previously in cerebral endothelial cells but may be important during normal cerebral energy metabolism and during brain diseases such as stroke. Here, by using the cytoplasmic pH indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-acetoxymethyl ester, the initial rates of monocarboxylate-dependent cytoplasmic acidification were measured as an indication of MCT1 kinetic function in vitro using the rat brain endothelial cell (RBE4) model of blood-brain transport. The initial rate of L-lactate-dependent acidification was significantly inhibited by 5-10-min incubations with agonists of intracellular cAMP-dependent cell signaling pathways as follows: dibutyryl cAMP, forskolin, and isoproterenol. Isoproterenol reduced V(max) but did not affect K(m) values. The effects of forskolin were completely reversed by the protein kinase A inhibitor H89, whereas H89 alone increased transport rates. Cytoplasmic cAMP levels, measured by radioimmunoassay, were increased by forskolin or isoproterenol, and the effect of isoproterenol was inhibited by propranolol. MCT1-independent intracellular pH control mechanisms did not contribute to the forskolin or H89 effects on MCT1 kinetic function as determined with amiloride, monocarboxylate-independent acid loading, or the transport inhibitor alpha-cyano-4-hydroxycinnamate. The data demonstrate the direct modulation of MCT1 kinetic function in cerebral endothelial cells by agents known to affect the beta-adrenergic receptor/adenylyl cyclase/cAMP/protein kinase A intracellular signaling pathway.     

Cell-specific properties of type V and type IX adenylyl cyclase isozymes in 293T cells and embryonic chick ventricular myocytes

The cDNAs for types V and IX adenylyl cyclases were cloned from a chicken heart library and expressed in 293T cells (plasmid transfection) and in embryonic chick ventricular myocytes (adenovirus infection). Expression of type V or IX cyclases in 293T cells resulted in increases in basal and isoproterenol (ISO)-stimulated cAMP levels, whereas the expression of type V, but not type IX, cyclase increased forskolin (FK)-stimulated cAMP levels. Expression of type V cyclase in cardiac myocytes increased basal and FK-stimulated cAMP levels, variably increased ISO-stimulated cAMP levels, and decreased the content of beta-adrenergic receptors (betaARs). The expression of type IX cyclase in cardiac myocytes increased basal and ISO-elevated cAMP levels and, surprisingly, increased the cAMP-elevating effect of FK. The finding that FK responses are increased in cardiac myocytes but not in 293T cells expressing the type IX cyclase suggests that the host cell influences the properties of the type IX isozyme.     

Conditional stimulation of type V and VI adenylyl cyclases by G protein betagamma subunits

In a yeast two-hybrid screen of mouse brain cDNA library, using the N-terminal region of human type V adenylyl cyclase (hACV) as bait, we identified G protein beta2 subunit as an interacting partner. Additional yeast two-hybrid assays showed that the Gbeta(1) subunit also interacts with the N-terminal segments of hACV and human type VI adenylyl cyclase (hACVI). In vitro adenylyl cyclase (AC) activity assays using membranes of Sf9 cells expressing hACV or hACVI showed that Gbetagamma subunits enhance the activity of these enzymes provided either Galpha(s) or forskolin is present. Deletion of residues 77-151, but not 1-76, in the N-terminal region of hACVI obliterated the ability of Gbetagamma subunits to conditionally stimulate the enzyme. Likewise, activities of the recombinant, engineered, soluble forms of ACV and ACVI, which lack the N termini, were not enhanced by Gbetagamma subunits. Transfection of the C terminus of G protein receptor kinase 2 to sequester endogenous Gbetagamma subunits attenuated the ability of isoproterenol to increase cAMP accumulation in COS-7 cells overexpressing hACVI even when G(i) was inactivated by pertussis toxin. Therefore, we conclude that the N termini of human hACV and hACVI are necessary for interactions with, and regulation by, Gbetagamma subunits both in vitro and in intact cells. Moreover, Gbetagamma subunits derived from a source(s) other than G(i) are necessary for the full activation of hACVI by isoproterenol in intact cells.     

Alterations in cardiac adrenergic terminal function and beta-adrenoceptor density in pacing-induced heart failure

Congestive heart failure is associated with cardiac adrenergic nerve terminal changes and beta-adrenoceptor density downregulation. To study the temporal sequence of these changes, we performed studies in rabbits at 2, 4, and 8 wk of cardiac pacing (360 beats/min) and at 1, 2, and 4 wk after cessation of pacing. Rapid pacing produced left ventricular (LV) dysfunction and an increase in plasma norepinephrine (NE) in 1-2 wk. At week 2, NE uptake activity, NE uptake-1 density, and adenylyl cyclase responses to isoproterenol, 5'-guanylyl imidodiphosphate [Gpp(NH)p], and forskolin reduced. However, immunostained tyrosine hydroxylase profile, beta-adrenoceptor density, and NE histofluorescence did not reduce until 4-8 wk of pacing. After cessation of cardiac pacing, LV function normalized quickly, followed by return of tyrosine hydroxylase and NE profiles in 1 wk and adenylyl cyclase responses to agonists and NE uptake activity in 2 wk. Myocardial beta-adrenoceptor density returned to normal by 4 wk after cessation of pacing. Our results suggest that there is no permanent structural neuronal damage in the myocardium within the first 8 wk of rapid cardiac pacing. Abnormal myocardial NE reuptake mechanism may play an important pathophysiological role in heart failure.     

Decreased left ventricular function, myocarditis, and coronary arteriolar medial thickening following monocrotaline administration in adult rats.

Decreased right as well as left ventricular function can be associated with pulmonary hypertension (PH). Numerous investigations have examined cardiac function following induction of pulmonary hypertension with monocrotaline (MCT) assuming that MCT has no direct cardiac effect. We tested this assumption by examining left ventricular function and histology of isolated and perfused hearts from MCT-treated rats. Experiments were performed on 50 male Sprague-Dawley rats [348 +/- 6 g (SD)]. Thirty-seven rats received MCT (50 mg/kg sc; MCT group) while the remainder did not (Control group). Three weeks later, pulmonary artery pressure was assessed echocardiographically in 20 MCT and 8 Control rats. The hearts were then excised and perfused in the constant pressure Langendorff mode to determine peak left ventricular pressure (LVP), the peak instantaneous rate of pressure increase (+dP/dtmax) and decrease (-dP/dtmax), as well as the rate pressure product (RPP). Histological sections were subsequently examined. Pulmonary artery pressure was higher in the MCT-treated group compared with the Control group [12.9 +/- 6 vs. 51 +/- 35.3 mmHg (P < 0.01)]. Left ventricular systolic function and diastolic relaxation were decreased in the MCT group compared with the Control group (+dP/dtmax 4,178 +/- 388 vs. 2,801 +/- 503 mmHg/s, LVP 115 +/- 11 vs. 83 +/- 14 mmHg, RPP 33,688 +/- 1,910 vs. 23,541 +/- 3,858 beats x min(-1) x mmHg(-1), -dP/dtmax -3,036 +/- 247 vs. -2,091 +/- 389 mmHg/s; P < 0.0001). The impairment of cardiac function was associated with myocarditis and coronary arteriolar medial thickening. Similarly depressed ventricular function and inflammatory infiltration was seen in 12 rats 7 days after MCT administration. Our findings appear unrelated to the degree of PH and indicate a direct cardiotoxic effect of MCT.     

Nitric oxide modulates Gi-protein expression and adenylyl cyclase signaling in vascular smooth muscle cells

We have previously shown that treatment of rats with the nitric oxide (NO) synthase inhibitor N6-nitro-L-arginine methyl ester for 4 weeks resulted in the augmentation of blood pressure and enhanced levels of Gialpha proteins. The present studies were undertaken to investigate if NO can modulate the expression of Gi proteins and associated adenylyl cyclase signaling. A10 vascular smooth muscle cells (VSMC) and primary cultured cells from aorta of Sprague-Dawley rats were used for these studies. The cells were treated with S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) for 24 h and the expression of Gialpha proteins was determined by immunobloting techniques. Adenylyl cyclase activity was determined by measuring [32P]cAMP formation for [alpha-32P]ATP. Treatment of cells with SNAP (100 microM) or SNP (0.5 mM) decreased the expression of Gialpha-2 and Gialpha-3 by about 25-40% without affecting the levels of Gsalpha proteins. The decreased expression of Gialpha proteins was reflected in decreased Gi functions (receptor-independent and -dependent) as demonstrated by decreased or attenuated forskolin-stimulated adenylyl cyclase activity by GTPgammaS and inhibition of adenylyl cyclase activity by angiotensin II and C-ANP4-23, a ring-deleted analog of atrial natriuretic peptide (ANP) that specifically interacts with natriuretic peptide receptor-C (NPR-C) in SNAP-treated cells. The SNAP-induced decreased expression of Gialpha-2 and Gialpha-3 proteins was not blocked by 1H[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase, or KT5823, an inhibitor of protein kinase G, but was restored toward control levels by uric acid, a scavenger of peroxynitrite and Mn(111)tetralis (benzoic acid porphyrin) MnTBAP, a peroxynitrite scavenger and a superoxide dismutase mimetic agent that inhibits the production of peroxynitrite, suggesting that NO-mediated decreased expression of Gialpha protein was cGMP-independent and may be attributed to increased levels of peroxynitrite. In addition, Gsalpha-mediated stimulation of adenylyl cyclase by GTPgammaS, isoproterenol, and forskolin was significantly augmented in SNAP-treated cells. These results indicate that NO decreased the expression of Gialpha protein and associated functions in VSMC by cGMP-independent mechanisms. From these studies, it can be suggested that NO-induced decreased levels of Gi proteins and resultant increased levels of cAMP may be an additional mechanism through which NO regulates blood pressure.     

Zinc inhibition of adenylyl cyclase correlates with conformational changes in the enzyme

We have previously demonstrated that Zn(2+) inhibits hormone and forskolin stimulation of cAMP synthesis in intact N18TG2 cells, corresponding plasma membranes, and of recombinant adenylyl cyclase isoforms. If, however, the enzyme is pre-activated by hormone or forskolin, Zn(2+) inhibition is attenuated [J. Biol. Chem. 277 (2002) 11859]. We have extended our analyses of this inhibition to investigations of soluble adenylyl cyclase, composed of the CI and CII domains of the full-length protein. The properties of Zn(2+) inhibition of the soluble enzyme parallel that of the full-length protein, including the fact that inhibition is not competitive with Mg(2+). By monitoring intrinsic and extrinsic fluorescence, we demonstrate changes in enzyme conformers in response to the addition of varied effectors. The data suggest a possible mechanism by which Zn(2+) inhibits adenylyl cyclase activity.