The signaling pathway for aldosterone-induced mitochondrial production of superoxide anion in the myocardium.

Mineralocorticoid receptor (MR) antagonists decrease morbidity and mortality in heart failure patients for whom oxidative stress is usual; however, the underlying mechanism for this protection is unclear. Since aldosterone stimulates reactive oxygen species (ROS) production in several tissues, we explored its effect and the intracellular pathway involved in the rat myocardium. Aldosterone dose-dependently increased O2(-) production in myocardial slices. At 10 nmol/L, aldosterone increased O2(-) to 165 ± 8.8% of control, an effect prevented not only by the MR antagonists eplerenone and spironolactone (107 ± 7.8 and 103 ± 5.3%, respectively) but also by AG1478 (105 ± 8.0%), antagonist of the EGF receptor (EGFR). Similar results were obtained by silencing MR expression through the direct intramyocardial injection of a lentivirus coding for a siRNA against the MR. The aldosterone effect on O2(-) production was mimicked by the mKATP channel opener diazoxide and blocked by preventing its opening with 5-HD and glibenclamide, implicating the mitochondria as the source of O2(-). Inhibiting the respiratory chain with rotenone or mitochondrial permeability transition (MPT) with cyclosporine A or bongkrekic acid also canceled aldosterone-induced O2(-) production. In addition, aldosterone effect depended on NADPH oxidase and phosphoinositide 3-kinase activation, as apocynin and wortmannin, respectively, inhibited it. EGF (0.1 µg/mL) similarly increased O2(-), although in this case MR antagonists had no effect, suggesting that EGFR transactivation occurred downstream from MR activation. Inhibition of mKATP channels, the respiratory chain, or MPT did not prevent Akt phosphorylation, supporting that it happened upstream of the mitochondria. Importantly, cardiomyocytes were confirmed as a source of aldosterone induced mitochondrial ROS production in experiments performed in isolated cardiac myocytes. These results allow us to speculate that the beneficial effects of MR antagonists in heart failure may be related to a decrease in oxidative stress.

Beta-adrenergic receptors and adenylate cyclase activity in diabetic rat fat cells.

Our study describes the effect of norepinephrine on adenylate cyclase activity in adipocytes from control and diabetic rats. The results show that diabetes induced an increase in both basal and norepinephrine-stimulated adenylate cyclase activity. This higher activity was not suppressed when the animals were treated for 2 days with the beta-blocking agent propranolol. On the other hand, adipocytes from control animals treated with propranolol showed a higher adenylate cyclase activity (basal and in response to norepinephrine). beta-Adrenergic receptors were examined in adipocytes from control and diabetic rats with and without treatment with propranolol. The results show a higher beta-receptor density in adipocytes from diabetic animals. When the animals were treated with propranolol, the beta-blocker induced a higher receptor density in adipocytes from control animals without affecting the already increased receptor density in diabetic preparations. The data suggest that adenylate cyclase activity in response to norepinephrine in adipose tissue is increased during at least a certain period of the diabetic state. This increase in adenylate cyclase activity is accompanied with an increase in beta-receptor density, but in contrast to control animals, this receptor density is not further increased with propranolol treatment.

Adipocyte responsiveness to norepinephrine in spontaneously hypertensive rats.

The present study describes the effect of norepinephrine on lipolysis and adenylate cyclase activity in adipocytes from Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats. The adipocytes were incubated in the presence of norepinephrine (10(-7) to 10(-4) mol/L) and the lipolytic activity was measured according to the accumulation of glycerol after one hour incubation. The results showed that norepinephrine induced a lower lipolytic activity in adipocytes from SH rats. cAMP-phosphodiesterase activities in adipocyte homogenates from WKY and SH rats were the same for both preparations. The effect of norepinephrine (10(-7) to 10(-4) mol/L) on adenylate cyclase activity in fat cell membranes from SH rats was decreased compared with WKY fat cell membranes. Adenylate cyclase activities in the presence of 10 mmol/L NaF were the same in both preparations. beta-receptor characteristics were examined, and the data demonstrate a statistically significant decrease in beta-receptor density in fat cell membranes from SH rats. The dissociation constants (Kd) were the same for WKY and SH preparations. This article suggests that adipocyte responsiveness to norepinephrine is decreased in SH rats. The decreased response to norepinephrine may be explained by a lower beta-receptor density in fat cell membranes from SH rats.

Phospholipid methyltransferase activity in diabetic rat fat cells: effect of isoproterenol and insulin.

The effects of isoproterenol and insulin on phospholipid methyltransferase (PLMT) activity were investigated in adipocytes from control and streptozotocin-diabetic rats. PLMT activity was assayed by measuring the rate of incorporation of 3H-methyl groups from S-adenosyl-L-[methyl-3H] methionine into phospholipids. Basal PLMT activity was higher in adipocytes from diabetic animals. Treatment of adipocytes with isoproterenol induced a concentration-dependent stimulation of PLMT activity. In control adipocytes, the maximal effect was obtained at 100 nM isoproterenol with 2.3 fold increase in PLMT activity and a half maximal effect at 25 nM. In adipocytes from diabetic rats, a lower dose of isoproterenol (10 nM), caused 1.2 fold increase with a half maximal effect at 4 nM. Addition of 100 nM insulin inhibited basal PLMT activity and the stimulatory effect of isoproterenol in both types of adipocytes. The beta-adrenergic blocking agent propranolol inhibited the stimulatory effect of isoproterenol on PLMT activity in control and diabetic adipocytes. Intracellular concentration of cAMP was higher in diabetic adipocytes but decreased to normal values after incubation in the presence of insulin.

Cyclic AMP, cyclic GMP and glucose utilization by diabetic rat fat cells.

Fat cells from epididymal adipose tissue from normal and streptozotocin-diabetic rats were studied to determine glucose utilization and cyclic nucleotide levels. Diabetic rat fat cells present a higher cAMP content (P less than 0.05) compared with controls. Addition of insulin decreases within 10-min incubation the cAMP content in both normal and diabetic cells (P less than 0.05). However, the value obtained in the latter remains by 25% higher than that of normal cells not exposed to insulin. No changes in cGMP were detected. Pretreatment of the diabetic animals during two days with propranolol (1 mg kg body wt-1 day-1) induces the decrease to normal levels of the fat cell cAMP content. However, it persists the impairment on glucose utilization observed in fat cells from diabetic animals. It seems that the increase in the intracellular amount of cAMP found in fat cells from diabetic rats is not involved, at least directly, to the impaired glucose utilization found in the diabetic state. Furthermore, through an unknown mechanism, pretreatment with propranolol can induce a drop in fat tissue cAMP toward normal values without normalizing glucose utilization.

Cyclic AMP, adenylate cyclase and cyclic AMP-phosphodiesterase activities in diabetic rat adipocytes.

The effect of streptozotocin-induced diabetes on cyclic AMP content, adenylate cyclase and cyclic-AMP phosphodiesterase activities in rat adipocytes was investigated. The results show that diabetes induced an increase in intracellular cyclic AMP. Basal adenylate cyclase activity and in response to norepinephrine were higher in fat cell membranes from diabetic rats. Adenylate cyclase activity stimulated by fluoride was the same in both normal and diabetic preparations. Low and high Km phosphodiesterase activities in adipocytes from diabetic rats were higher than the controls. The results suggest that the deficiency of insulin present in the diabetic state induces an increase in adenylate cyclase activity which increases the cyclic AMP production. This increase in cyclic AMP promotes a higher cyclic AMP-phosphodiesterase activity which is not sufficient to hydrolyze all the newly formed cyclic AMP.

Effect of pH on lipolysis, cAMP and cAMP-dependent protein kinase activity in isolated rat fat cells.

The effect of acidosis and alkalosis on lipolysis, cAMP production and cAMP-dependent protein kinase activity in isolated rat fat cells incubated in the presence of norepinephrine and norepinephrine plus theophylline has been investigated. The pH of the incubation medium was adjusted to 6.8, 7.4 and 7.8 respectively. Acidosis inhibited both norepinephrine- and norepinephrine plus theophylline-induced release of glycerol whereas alkalosis led to slight stimulation. Norepinephrine produced an increase in cAMP and cAMP-dependent protein kinase activity. However, comparison of both parameters in acidosis and alkalosis with those at pH 7.4 indicates that they were higher at pH 7.8 and lower at pH 6.8. Addition of theophylline in combination with norepinephrine increases cAMP production within 5 min, under acidosis to values similar to those obtained at pH 7.4 with norepinephrine. The same effect on protein kinase activity was obtained. In spite of this increment in cAMP and protein kinase activity produced by addition of norepinephrine plus theophylline, lipolysis remains inhibited by acidosis. Addition of theophylline at pH 7.4 and 7.8 induced a much higher cAMP production and cAMP-dependent protein kinase activity although at pH 7.8 there was a statistically significant increase in protein kinase activity at 10 min it did not induce a significant increase in lipolysis. This is discussed and possible mechanisms are suggested to explain the effect of acidosis and alkalosis on the lipolysis induced by norepinephrine in rat fat cells.

Chronic administration of nifedipine induces upregulation of dihydropyridine receptors in rabbit heart.

In rabbits chronically pretreated with nifedipine (20 mg/day for 25 days), we demonstrate upregulation of Ca2+ channels in mammalian heart (maximal binding capacity = 222 +/- 14 and 421 +/- 55 fmol/mg protein for control and treated, respectively; P < 0.05) without significant changes in dissociation constant. No changes in calcium sensitivity or maximal force were detected by pretreatment with nifedipine in chemically skinned fibers. Cardiac contractility at different extracellular Ca2+ concentrations was similar in control and pretreated animals. This upregulation of Ca2+ channels in mammalian heart offers an explanation for the lack of significant negative inotropic effect after chronic administration of nifedipine.

Effect of acidosis on heart cAMP-dependent protein kinase.

The effect of acidosis on cAMP-dependent protein kinase activity in perfused hearts from normal and reserpinized rats has been investigated. The results were compared to the effect of acidosis on myocardial contractility under the same conditions. The results showed that acidosis increases the cAMP-dependent protein kinase activity in normal hearts. This increase was abolished when the hearts were depleted of norepinephrine by previous treatment with reserpine. As regards myocardial contractility, there was a similar decrease by acidosis either in normal hearts with increased cAMP-dependent protein kinase activity or in reserpinized hearts in which the increase in protein kinase activity was prevented. Two alternative hypotheses are suggested: (1) a dissociation between contractility and cAMP levels, or (2) a "blockade" by acidosis of the mechanical effect of increasing cAMP-dependent protein kinase activity.

Glucose metabolism by isolated fat cells from sloth.

Glucose metabolism by sloth fat cells with and without addition of insulin was investigated. The data were compared to the results obtained with rat fat cells incubated under the same experimental conditions. Sloth fat cells showed a very low glucose oxidation to 14CO2 and incorporation into total lipids. The glucose incorporated into lipids is mainly in the glyceride-glycerol moiety. Addition of insulin did not produce an increase on glucose oxidation and a slight increase in the incorporation into total lipids was observed. Since it has been reported that sloths have a very low rate on thyroxine secretion, the results are discussed in relation to data in the literature on carbohydrate and lipid metabolism in hypothyroid animals.

Chronic administration of nifedipine induces up-regulation of functional calcium channels in rat myocardium.

Previous studies from our laboratory demonstrated the up-regulation of cardiac dihydropyridine (DHP) receptors in rabbits chronically treated with nifedipine (NIFE). The goal of the present study was to further examine the functionality of this increased number of receptors by analysing different steps of excitation contraction coupling mechanism in adult rats chronically treated with NIFE (a single 10-mg oral dose/kg/day for 28 days). Ca2+ channel density was assessed by specific binding at the DHP receptors with [methyl-(3)H]PN 200-110 in rat ventricular membranes. Chronic NIFE treatment produced up-regulation of Ca2+ channels, being the maximal binding capacities 222+/-19 fmol/mg protein (n=14) and 310+/-21 fmol/mg protein (n=11) in untreated and treated animals, respectively (P<0.05). The functional consequences of this up-regulation of Ca2+ channels were determined in isolated ventricular myocytes by measuring L-type Ca2+ currents (I(Ca)) with the whole-cell configuration of patch-clamp technique and by intracellular Ca2+ (Ca2+(i)) transients estimated by the Indo-1/AM fluorescence ratio (410/482) simultaneously monitored with cell shortening. Peak I(Ca) density recorded at 0 mV was 32% greater in myocytes isolated from the treated group than in those obtained from the untreated group (-10.43+/-0.73 pA/pF (n=13) vs-7.10+/-0.59 pA/pF (n=12) P<0.05). Ca2+(i) transient amplitude and cell shortening, explored at 1 and 2 mM extracellular calcium ([Ca]0) were significantly higher in ventricular myocytes obtained fom NIFE-treated rats than in myocytes isolated from untreated animals. At 2 mM [Ca]0, the values of Ca2+(i) transient and shortening were 460+/-61 nM and 11+/-1 % of resting length (L(0)) in myocytes from treated rats (n=9) and 212+/-22 nM and 5.3+/-0.5% of L(0) in myocytes from control rats (n=6, P<0.05). The results demonstrate an up-regulation of functionally-active cardiac Ca2+ channels after NIFE treatment, and offer a possible explanation for a "withdrawal effect" at myocardial level after the suppression of the treatment with this drug.