Chronic blockade of class I PI3-kinase attenuates Ang II-induced cardiac hypertrophy and autophagic alteration.

OBJECTIVE: Chronic Ang II stimulation is linked to cardiac remodeling characterized by fibrosis and cardiac hypertrophy. However, the underlying cellular mechanisms involved are not yet fully known. Here, we studied the molecular mechanisms underlying the chronic effect of Ang II on cardiac hypertrophy, fibrosis, and autophagy. MATERIALS AND METHODS: The role of class I PI3-kinase in these actions of Ang II was studied using lentiviral vector-mediated expression of a dominant negative form of PI3-kinase subunit p85α (Lv-DNp85) in the heart. Ang II was infused subcutaneously for 4 weeks on rats using osmotic pumps. Cardiac hypertrophy, fibrosis, reactive oxygen species (ROS), and autophagy were examined in four groups of rats (Ang II+Lv-DNp85, Ang II+Lv-GFP, Saline+Lv-DNp85, Saline+Lv-GFP). RESULTS: Chronic infusion of Ang II induced severe cardiac hypertrophy and perivascular fibrosis in the heart. These effects were associated with a significant reduction in LC3 II and elevation in ROS levels, suggesting marked impairment of cardiac autophagy and increased generation of ROS. Cardiac transduction of Lv-DNp85 significantly attenuated Ang II-induced impairment of autophagy and elevation of ROS, as well as Ang II-induced cardiac hypertrophy and perivascular fibrosis. To study the cellular mechanisms underlying those actions of Ang II, phosphorylated Akt and mTOR were measured in hearts from these rats. Ang II increased phosphorylation of Akt and mTOR; and cardiac transduction of Lv-DNp85 significantly abolished Ang II-induced phosphorylation of Akt and mTOR, a signaling pathway inhibiting autophagy. CONCLUSIONS: These results demonstrate that class I PI3-kinase, via activation of the Akt-mTOR pathway, is involved in Ang II-induced impairment of autophagy, elevation of ROS, cardiac hypertrophy, and fibrosis, suggesting a novel target for cardiac protection.

Effect of maternal nutrient restriction and melatonin supplementation from mid to late gestation on vascular reactivity of maternal and fetal placental arteries.

INTRODUCTION: Maternal nutrient restriction and decreased scotophase concentrations of melatonin have been associated with severely compromised pregnancies. We hypothesized that melatonin supplementation in a compromised pregnancy enhances the bradykinin (BK)-induced relaxations of placental arteries thereby ensuring sufficient umbilical blood flow to the developing fetus. METHODS: Pregnant ewes (n = 31) were fed an adequate (ADQ) or nutrient restricted (RES) diet supplemented with 5 mg of melatonin (MEL) or without melatonin (CON) from day 50 to 130 of gestation. On day 130 of gestation, the maternal (caruncular; CAR) and fetal (cotyledonary; COT) placental arteries were suspended in organ chambers for isometric tension recording. RESULTS: There were no treatment or dietary effects on CAR arteries for any vasoactive agent. However, in COT arteries, MEL ewes were more sensitive (P < 0.01) to bradykinin-induced relaxation than CON ewes. There was a melatonin by nutritional level interaction (P < 0.01) with sodium nitroprusside-induced relaxation of COT arteries where CON-RES were more sensitive to sodium nitroprusside compared to CON-ADQ, which was in contrast to when ewes were fed MEL. There was a significant melatonin by nutritional interaction (P = 0.04) for responsiveness to norepinephrine. The sensitivity of the COT arteries to norepinephrine in CON-RES ewes was decreased compared to CON-ADQ. Melatonin supplementation, regardless of maternal dietary intake, resulted in COT arteries having similar responsiveness to CON-RES ewes. CONCLUSION: An increase in placental vessel sensitivity to bradykinin-induced relaxation may contribute to melatonin-induced increases in umbilical artery blood flow.

2011 and 2012 Early Careers Achievement Awards: Placental programming: how the maternal environment can impact placental function.

Proper establishment of the placenta is important for fetal survival; however, placental adaptations to inadequate maternal nutrition or other stressors are imperative for fetal growth to be optimal. The effects of maternal nutritional status and activity level on placental vascular function and uteroplacental blood flows are important to understand as improper placental function leads to reduced growth of the fetus. In environments where fetal growth can be compromised, potential therapeutics may augment placental function and delivery of nutrients to improve offspring performance during postnatal life. Factors that could enhance placental function include supplementation of specific nutrients, such as protein, hormone supplements, such as indolamines, and increased activity levels of the dam. To understand the mechanism of how the maternal environment can impact uterine or umbilical blood flows, assessment of placental vascular reactivity has been studied in several large animal models. As we begin to understand how the maternal environment impacts uterine and umbilical blood flows and other uteroplacental hemodynamic parameters, development of management methods and therapeutics for proper fetal growth can be achieved.

Melatonin potentiates contractile responses to serotonin in isolated porcine coronary arteries.

The present study was designed to determine the effects of melatonin on coronary vasomotor tone. Porcine coronary arteries were suspended in organ chambers for isometric tension recording. Melatonin (10(-10)-10(-5) M) itself caused neither contraction nor relaxation of the tissues. Serotonin (10(-9)-10(-5) M) caused concentration-dependent contractions of coronary arteries, and in the presence of melatonin (10(-7) M) the maximal response to serotonin was increased in rings with but not without endothelium. In contrast, melatonin had no effect on contractions produced by the thromboxane A(2) analog U-46619 (10(-10)-10(-7) M). The melatonin-receptor antagonist S-20928 (10(-6) M) abolished the potentiating effect of melatonin on serotonin-induced contractions in endothelium-intact coronary arteries, as did treatment with 1H-[1, 2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10(-5) M), methylene blue (10(-5) M), or N(G)-nitro-L-arginine (3 x 10(-5) M). In tissues contracted with U-46619, serotonin caused endothelium-dependent relaxations that were inhibited by melatonin (10(-7) M). Melatonin also inhibited coronary artery relaxation induced by sodium nitroprusside (10(-9)-10(-5) M) but not by isoproterenol (10(-9)-10(-5) M). These results support the hypothesis that melatonin, by inhibiting the action of nitric oxide on coronary vascular smooth muscle, selectively potentiates the vasoconstrictor response to serotonin in coronary arteries with endothelium.

Dexfenfluramine-induced contraction of human and rat isolated pulmonary arteries.

Mechanisms of dexfenfluramine-induced vasoconstriction were studied in isolated pulmonary arteries suspended in organ baths for isometric tension recording. Dexfenfluramine (10(-7)-10(-4) M) caused concentration-dependent contractions in rat and human pulmonary arteries with and without endothelium. In pulmonary arteries of the rat, the response to dexfenfluramine was nearly abolished by treatment with the alpha-adrenoceptor antagonists, phentolamine (10(-6) M) or prazosin (10(-7) M). In human pulmonary arteries, the concentration-response curve to dexfenfluramine was unaltered by the presence of phentolamine (10(-6) M), prazosin (10(-7) M), ketanserin (10(-6) M), or indomethacin (3x10(-6) M). The results suggest that dexfenfluramine causes contraction of pulmonary vascular smooth muscle by multiple mechanisms, one of which involves activation of alpha-adrenoceptors within the blood vessel wall. The mechanisms by which dexfenfluramine causes pulmonary vasoconstriction may differ between rat and human pulmonary arteries.

Clamikalant (Aventis).

Clamikalant is a cardioselective blocker of the ATP-dependent potassium channel (KATP) which is under development by Aventis Pharma (formerly Hoechst Marion Roussel) for the potential treatment of cardiac arrhythmia. The sodium salt, HMR-1098, is in phase II trials. Aventis plans for an iv preparation of the drug to be launched in 2004, and an oral preparation to be available in 2005. Clamikalant prevented ischemia-induced reductions in refractory period in dogs with ventricular fibrillation without significant hemodynamic effects or alteration in blood glucose levels. HMR-1883 exerted an anti-arrhythmic effect in a model of isolated hearts from male White New Zealand rabbits, and indicated and did not interfere with post-ischemic hyperemia.

KATP channel activation mediates nicorandil-induced relaxation of nitrate-tolerant coronary arteries.

We compared the tolerance-inducing effects of nitroglycerin (NTG) and nicorandil (NIC) in porcine isolated coronary arteries and assessed the role of KATP channels in the response to NIC in nitrate-tolerant and nontolerant preparations. In coronary arteries contracted with U46619 (1-3 x 10(-9) M), NTG, NIC, sodium nitroprusside (SNP), and cromakalim produced concentration-dependent relaxations. The rank order of potency was NTG > or = SNP > cromakalim > nicorandil. Exposure of the rings to NTG (10(-4) M) for 90 min, followed by repeated rinsing for 1 h, produced a parallel, rightward shift of the subsequent concentration-response curves to NTG and SNP; a slight but significant reduction in the maximal response to NTG was also observed. Previous exposure to NTG had no effect on the NIC or cromakalim concentration-response curves. When the tissues were exposed to NIC (3 x 10(-4) M) for 90 min, followed by repeated rinsing for 1 h, there was no effect on the subsequent concentration-response curves to NTG, NIC, SNP, or cromakalim. In both nitrate-tolerant and nontolerant coronary arteries, glibenclamide (GLI 10(-6) M), a selective KATP channel blocker, caused a parallel rightward shift in the concentration-response curve to cromakalim, but had no effect on responses to NTG or SNP. In nontolerant coronary arteries, GLI had no effect on NIC-induced relaxation, but in nitrate-tolerant preparations, GLI produced a significant rightward shift in the NIC concentration-response curve. The results demonstrate that prolonged exposure to NTG, but not NIC, causes tolerance in isolated porcine coronary arteries and that the response to NIC is not affected by nitrate tolerance. The data also suggest that NIC-induced relaxation of nitratetolerant, but not nontolerant, coronary arteries is mediated by activation of KATP channels.

Effects of potassium channel blockers on resting tone in isolated coronary arteries.

The effects of several potassium channel blockers on resting vasomotor tone were studied in porcine isolated coronary arteries. Coronary artery rings were suspended in organ baths for isometric tension recording. The nonselective potassium channel blockers tetraethylammonium (TEA 10(-5)-3 x 10(-2) M) and 4-aminopyridine (4-AP 10(-5)-10(-2) M) caused concentration-dependent contractions that were similar in rings with and without endothelium. The concentration-response curves to TEA and 4-AP were unaffected by treatment with phentolamine (3 x 10(-6) M),propranolol (10(-6) M), or atropine (10(-6) M). Diltiazem (10(-6) M) almost abolished the contractions evoked by TEA and 4-AP. Charybdotoxin (10(-9)-10(-7) M) and apamin (10(-8)-10(-6) M), selective blockers of large and small calcium-activated potassium channels, respectively, and glyburide (10(-8)-10(-6) M), a selective blocker of ATP-sensitive potassium channels, caused little or no contraction in rings with or without endothelium. Therefore, in isolated coronary arteries, TEA and 4-AP caused contractions that were independent of the release of vasoactive mediators from the endothelium or perivascular nerves. These effects are not mediated by ATP-sensitive potassium channels or by large and small conductance calcium-activated potassium channels. The data are consistent with an effect of TEA and 4-AP on resting membrane potassium conductance in coronary arteries, resulting in contractions that are sensitive to inhibition by diltiazem. This pattern of responsiveness of isolated coronary arteries to potassium channel blockers differs from that observed in vessels from other vascular beds.

Analogies between oxytocin systems of the uterus and brain.

In this brief review we have compared OT systems in the brain with those of the uterus and ovary particularly with respect to interactions with steroids. We have presented evidence of heterogeneous OTR and 125I-P-3-BSA binding sites in the MPOA as well as evidence of extensive interactions of steroids and OT in the MPOA, that cannot be adequately explained by genomic effects of steroids. We also discuss a putative analogue between steroid control of OTR stimulation of intracellular calcium levels, phospholipase C activity and prostaglandins in the uterus and steroid effects on OT systems in brain. We have developed a model for steroid control of both OT release and OTR in which we suggest that steroids and OT bind to membrane receptors coupled to G proteins. This model may prove useful in understanding the interactive central actions of steroids and OT systems in regulating the endocrinology and behaviors associated with reproduction.

Inhibition of hypoxic coronary vasoconstriction by pinacidil.

Acute hypoxia causes constriction of isolated coronary arteries from several species. The present study was designed to test whether pinacidil, a potassium channel opener, inhibits hypoxia-induced contraction of porcine isolated coronary arteries. Coronary arterial rings were suspended in organ baths for isometric tension recording. Hypoxic contractions were evoked by rapidly changing gas mixture from 95% O2/5% CO2 to 95% N2/5% CO2 in preparations partially contracted with KCl. Pretreatment with pinacidil (10(-6) to 10(-4) M) caused concentration-dependent inhibition of the contractile response to hypoxia. The inhibitory effect of pinacidil was attenuated by the K ATP channel blocker, glibenclamide (10(-6) M). In rings contracted with acetylcholine, glibenclamide caused a rightward shift in the concentration-response curve to pinacidil while having no effect on the vasorelaxant responses to sodium nitroprusside and diltiazem, thus confirming the specificity of glibenclamide for potassium channel opener-mediated responses, Taken together, the data indicate that pinacidil prevents hypoxia-induced contraction of porcine coronary arteries, and that the effect of pinacidil may be mediated by the opening of glibenclamide-sensitive potassium channels.

Myocardial and endothelial dysfunction after multiple, brief coronary occlusions: role of oxygen radicals.

The major objective of the present study was to determine the effect of multiple, brief periods of coronary artery occlusion and reperfusion on postischemic contractile function (sonomicrometry) and endothelium-dependent vasodilator responses in isolated conduit coronary artery rings obtained from anesthetized dogs. The role of oxygen-derived free radicals was also investigated. Dogs were subjected to four 5-min episodes of left anterior descending coronary occlusion interspersed with 5 min of reperfusion followed by a final 60-min reperfusion period. The multiple occlusion-reperfusion protocol resulted in regional segment dysfunction (37 +/- 15% of preocclusion values at 60 min of reperfusion) and attenuated endothelium-dependent responses to acetylcholine, bradykinin, and the calcium ionophore, A23187. Responses to the endothelium-independent vasodilator, sodium nitroprusside, were unaffected. Infusion of superoxide dismutase (5,000 U/kg) and catalase (55,000 U/kg) markedly improved the recovery of myocardial function at 30 and 60 min of reperfusion and completely protected against vascular endothelial damage. These results suggest an important role for oxygen-derived free radicals in the myocardial and endothelial injury that occurs in this model of multiple stunned myocardium.

Adrenergic and cholinergic regulation of bronchial vascular tone.

The autonomic nervous system plays a vital role in the regulation of vasomotor tone. Previous studies of systemic arteries and veins have shown that the classic autonomic neurotransmitters, norepinephrine and acetylcholine, act on smooth muscle, adrenergic nerve endings, and endothelial cells in the blood vessel wall to regulate vasomotor tone. Similar studies with isolated tissues indicate that bronchial arteries are innervated by adrenergic neurons and that norepinephrine activates postjunctional alpha 1-adrenoceptors to cause smooth muscle contraction. Isolated canine bronchial arteries fail to relax in response to beta-adrenoceptor agonists, and they do not contract when exposed to acetylcholine. This lack of responsiveness may be species-specific, however, since isolated bronchial arteries from other species respond to these agonists. Acetylcholine causes endothelium-dependent relaxation of bronchial arteries in vitro; this response is mediated by endothelial M3-muscarinic receptors. The role of the endothelium in mediating responses to adrenergic agonists, as well as the prejunctional effects of norepinephrine and acetylcholine on adrenergic nerve endings, remain to be explored in isolated bronchial arteries.

Bioassay of endothelium-derived relaxing factor in diabetic rat aorta.

The bioassay technique was utilized to quantitate endothelium-derived relaxing factor (EDRF) released from perfused donor segments of control and diabetic rat aorta. In the presence of indomethacin, perfusates of donor segments with endothelium were allowed to superfuse recipient detector rings of normal rat aorta without endothelium. Under basal conditions, relaxations of the bioassay rings to perfusates of control and diabetic donor segments were similar. Perfusion of donor segments with acetylcholine produced relaxation of bioassay rings, which was decreased from endothelial perfusion of diabetic donor segments. These relaxations were inhibited by addition of methylene blue to the detector ring or by perfusion of donor segments with nitro-L-arginine. Infusion of superoxide dismutase (SOD) at a site proximal to the donor segment normalized relaxations induced by acetylcholine addition to diabetic donors. In contrast, infusion of SOD distal to the donor had no effect on acetylcholine-stimulated relaxations of detector rings from control donors while attenuating, paradoxically, the relaxations of detector rings from diabetic donors. These results suggest that diabetic rat aortas release similar levels of EDRF in response to acetylcholine, but the action of EDRF arising from diabetic donors is attenuated by enhanced release of oxygen-derived free radicals, which limits EDRF-mediated relaxation of vascular smooth muscle.

Adrenergic and cholinergic responsiveness of isolated canine bronchial arteries.

The purpose of this study was to characterize the adrenergic and cholinergic responsiveness of isolated rings of canine bronchial arteries. Electrical stimulation of the vessels produced frequency-dependent contractions that were inhibited by phenoxybenzamine, tetrodotoxin, and phentolamine but not by atropine. Norepinephrine caused concentration-dependent contractions of the rings; the concentration-response curve to norepinephrine was shifted to the right by phentolamine and prazosin, whereas rauwolscine had no effect. Isoproterenol, norepinephrine, tyramine, and electrical stimulation (in the presence of phentolamine) failed to elicit relaxation in rings contracted with prostaglandin F2 alpha. Contracted rings relaxed to acetylcholine in an endothelium- and concentration-dependent manner. At concentrations up to 10(-4) M, acetylcholine did not evoke contractions in these tissues. Endothelium-dependent relaxation to acetylcholine was inhibited by atropine and 4-diphenyl-acetoxy-N-methyl piperidine methiodine but not by pirenzepine. These results suggest that alpha 1-adrenoceptors mediate contraction to norepinephrine, and M3-muscarinic receptors mediate endothelium-dependent relaxation to acetylcholine in the canine bronchial artery. Moreover, the smooth muscle of these vessels does not respond directly to beta-adrenergic- or cholinergic-receptor stimulation.

Celiprolol has no direct or indirect relaxing effects in isolated arteries and veins.

Experiments were designed to study the potential mechanisms underlying the vasodilator effect of celiprolol. Rings of canine left circumflex coronary artery and rat mesenteric artery, with and without endothelium, were suspended in organ chambers for isometric tension recording. In both blood vessels, celiprolol (10(-9)-10(-4) M) failed to produce relaxation in rings with and without endothelium; these same tissues relaxed in an endothelium-dependent manner to acetylcholine (10(-6) M). All tissues relaxed completely in the presence of papaverine (10(-4) M). In the coronary artery, isoproterenol (10(-9)-10(-4) M) produced endothelium-independent relaxations which were inhibited in a competitive fashion by celiprolol (pA2 = 7.52 +/- 0.14; slope = 0.98, 95% confidence limits = 0.80-1.15). In other experiments, strips of canine saphenous veins were incubated with [3H]norepinephrine [( 3H]NE) and suspended for superfusion. Electrical stimulation (2 Hz, 4 V, 2 ms for 6 min) produced an increase in [3H]NE overflow. Isoproterenol (2 X 10(-6) M) augmented the evoked release of [3H]NE. Treatment of the strips with celiprolol (up to 5 X 10(-6) M) did not inhibit isoproterenol-induced facilitation of [3H]NE release. Thus, although celiprolol is a potent antagonist of postjunctional beta-adrenoceptors in the coronary artery, no evidence was obtained for a direct or indirect vasodilator effect of celiprolol on isolated blood vessels.

Differential inhibition by ergonovine of norepinephrine release in the coronary artery and saphenous vein of the dog.

Experiments were performed to determine the effect of ergonovine on adrenergic neurotransmission in the blood vessel wall. Strips of canine left circumflex coronary arteries and saphenous veins were incubated with [3H]norepinephrine and suspended for superfusion. Ergonovine (10(-6) M) decreased the overflow of [3H] norepinephrine to a modest degree in the coronary artery, whereas a profound inhibition of overflow was observed in the saphenous vein. Methiothepin, but not rauwolscine, reversed the inhibitory effect of ergonovine in both blood vessels. Treatment with atropine or droperidol did not alter the response to ergonovine in the coronary artery. The results demonstrate that ergonovine, by activating prejunctional serotonergic receptors, inhibits adrenergic neurotransmission to a greater extent in the saphenous vein than in the coronary artery. The differential effect of ergonovine on norepinephrine release in these vessels may provide evidence for heterogeneity in the prejunctional modulation of adrenergic neurotransmission.

Characterization of muscarinic receptors in canine bronchial smooth muscle.

Rings of canine bronchi were suspended for isometric tension recording. Contractions produced by exogenously added acetylcholine were inhibited by pirenzepine and pancuronium. The pKB values were 6.76 for pirenzepine (calculated at 10(-6) M) and 5.30 and 5.13 for pancuronium (calculated at 10(-5) and 3 X 10(-5) M, respectively). Contractile responses evoked by cholinergic nerve stimulation (0.2-16 Hz, 9 V) were depressed by pancuronium in a concentration-dependent manner, while concentrations of pirenzepine selective for M1-muscarinic receptors were without effect. The results indicate that exogenous and nerve-released acetylcholine activate a homogenous population of M2-muscarinic receptors in isolated preparations of canine bronchial smooth muscle.

Subtypes of muscarinic receptors on adrenergic nerves and vascular smooth muscle of the canine saphenous vein.

Experiments were designed to characterize pre- and postjunctional muscarinic receptors in the canine saphenous vein. In rings contracted by 2-Hz electrical stimulation, acetylcholine produced concentration-dependent relaxations that result from prejunctional inhibition of norepinephrine release from adrenergic nerve endings (prejunctional effect). In quiescent preparations, acetylcholine caused concentration-dependent contractions (postjunctional effect). Both responses to acetylcholine were inhibited in a competitive manner by atropine, pirenzepine and gallamine. The affinity of atropine for pre- and postjunctional muscarinic receptors was similar (pKB = 8.7 and 9.2, respectively). However, gallamine displayed higher affinity for the prejunctional than for the postjunctional receptor (pKB = 6.1 and 4.7, respectively), whereas the converse was true for pirenzepine (pKB = 6.5 and 8.1). The data support the presence in the canine saphenous vein of two muscarinic receptor subtypes, M1 postjunctionally and M2 prejunctionally, which are distinguishable by selective antagonists.

Inhibition of canine platelet aggregation by barbiturates.

The ability of barbiturates to affect in vitro platelet aggregation of canine platelets was examined. Platelet-rich plasma was incubated with thiamylal, pentobarbital, and barbital for 10 minutes before the addition of the aggregating stimulus. All three barbiturates produced a concentration-related inhibition of platelet aggregation induced by adenosine diphosphate (ADP) and collagen. The inhibitory effect of the barbiturates could not be overcome by increasing the concentration of extracellular calcium. In contrast to ADP- and collagen-induced aggregation, no inhibitory effect was observed on aggregation initiated by A23187, 12-O-tetradecanoylphorbol-13-acetate, or phospholipase C. In further studies, the ADP-induced rise in free cytosolic calcium was blocked by the barbiturates. These findings suggest that barbiturates may interfere with the rise in internal calcium associated with agonist-receptor stimulation of platelets.