Gerald O. Carrier

Endothelium-independent effect of estrogen on Ca2+-activated K+ channels in human coronary artery smooth muscle cells

OBJECTIVE Postmenopausal estrogen replacement therapy lowers the incidence of cardiovascular disease, suggesting that estrogens support cardiovascular function. Estrogens dilate coronary arteries; however, little is known about the molecular basis of how estrogen affects the human coronary circulation. The cellular/molecular effects of estrogen action on human coronary smooth muscle were investigated in the present study. METHODS Patch-clamp and fluorescent microscopy studies were performed on human coronary myocytes in the absence of endothelium. RESULTS Estrogen increased whole-cell currents over a range of membrane potentials, and further studies indicated that the large-conductance (186.5 +/- 3 pS), calcium- and voltage-activated potassium (BK(Ca)) channel was the target of estrogen action. Channel activity was stimulated approximately 15-fold by nanomolar concentrations of 17 beta-estradiol, and this stimulation was reversed >90% by inhibiting cGMP-dependent protein kinase activity with 300 nM KT5823. 17 beta-Estradiol increased the level of cGMP and nitric oxide in human myocytes, and the stimulatory effect of estrogen on channel activity and NO production was reversed by inhibiting NO synthase with 10 microM N(G)-monomethyl-L-arginine. CONCLUSIONS Our cellular and molecular studies identify the BK(Ca) channel as a target of estrogen action in human coronary artery smooth muscle. This response to estrogen involves cGMP-dependent phosphorylation of the BK(Ca) channel or a closely associated regulatory molecule, and further evidence suggests involvement of the NO/cGMP signaling system in coronary smooth muscle. These findings are the first to provide direct evidence for a molecular mechanism that can account for endothelium-independent effects of estrogen on human arteries, and may also help explain why estrogens reduce myocardial ischemia and stimulate coronary blood flow in patients with diseased coronary arteries.

Angiotensin II Relaxes Microvessels Via the AT2 Receptor and Ca2+-Activated K+ (BKCa) Channels

Angiotensin II (Ang II) is one of the most potent vasoconstrictor substances, yet paradoxically, Ang II may dilate certain vascular beds via an undefined mechanism. Ang II–induced vasoconstriction is mediated by the AT1 receptor, whereas the relative expression and functional importance of the AT2 receptor in regulating vascular resistance and blood pressure are unknown. We now report that Ang II induces relaxation of mesenteric microvessels and that this vasodilatory response was unaffected by losartan, an AT1 receptor antagonist, but was inhibited by PD123,319, a selective antagonist of AT2 receptors. In addition, reverse transcriptase–polymerase chain reaction studies revealed high amounts of AT2 receptor mRNA in smooth muscle from these same microvessels. Ang II–induced relaxation was inhibited by either tetraethylammonium or iberiotoxin, suggesting involvement of the large-conductance, calcium- and voltage-activated potassium (BKCa) channel. Subsequent whole-cell and single-channel patch-clamp studies on single myocytes demonstrated that Ang II increases the activity of BKCa channels. As in our tissue studies, the effect of Ang II on BKCa channels was inhibited by PD123,319, but not by losartan. In light of these consistent findings from tissue physiology, molecular studies, and cellular/molecular physiology, we conclude that Ang II relaxes microvessels via stimulation of the AT2 receptor with subsequent opening of BKCa channels, leading to membrane repolarization and vasodilation. These findings provide evidence for a novel endothelium-independent vasodilatory effect of Ang II.

A novel transduction mechanism mediating dopamine-induced vascular relaxation: opening of BKCa channels by cyclic AMP-induced stimulation of the cyclic GMP-dependent protein kinase.

Dopamine dilates the coronary, renal and other vascular beds; however, the signaling pathway underlying this effect is unclear. In this study the signal-transduction process mediating dopamine-induced relaxation of porcine coronary arteries was investigated in isolated vessels and single arterial myocytes. Dopamine-induced relaxation of arteries was mediated through the DA- receptor and involved K+ efflux, and subsequent patch-clamp studies demonstrated that either dopamine or fenoldopam, a selective DA-1 agonist, increased the opening probability of the large-conductance, calcium- and voltage-activated K+ (BKCa) channel in coronary myocytes. Moreover, blockade of this channel by iberiotoxin prevented dopamine-induced coronary relaxation. Dopamine stimulation of BKCa channels was completely prevented by a DA-1-receptor antagonist, but was unaffected by propranolol. Furthermore, inhibiting adenylyl cyclase activity prevented stimulation of BKCa channel activity, whereas chlorophenylthio (CPT)-cyclic adenosine monophosphate (AMP), a membrane-permeable analog of cyclic AMP, mimicked the effects of dopamine. Interestingly, inhibiting the cyclic AMP-dependent protein kinase (PKA) did not affect the response to dopamine, whereas dopamine-induced channel activity was completely blocked by inhibiting the activity of the cyclic guanosine monophosphate (GMP)-dependent protein kinase (PKG). These findings demonstrate that activation of DA-1 receptors causes stimulation of BKCa channel activity by a mechanism involving cyclic AMP-dependent stimulation of PKG, but not PKA, and further suggest that this cross-reactivity mediates dopamine-induced coronary vasodilation.

Supersensitivity and Endothelium Dependency of Histamine-induced Relaxation in Mesenteric Arteries Isolated from Diabetic Rats

Diabetes mellitus is known to produce alterations in vascular reactivity. In the present study we have examined the effects of short-term diabetes on histamine-induced relaxation of isolated mesenteric arteries, and the role of the endothelial cell layer in this response. Removal of the endothelium completely abolished the histamine relaxation effect in both diabetic and age-matched control rats. In contrast, vessels isolated from streptozotocin-diabetic rats were supersensitive to histamine, and this relaxation was mediated only through the H1-receptors. The present findings suggest that histamine-induced relaxation of rat mesenteric arteries is dependent upon endothelial cell processes which are enhanced in arteries from STZ-diabetic rats.

α1- and α2-adrenoceptor agonist-induced contraction in rat mesenteric artery upon removal of endothelium

Abstract Contractile response to norepinephrine, methoxamine and clonidine were determined in rat mesenteric arteries with and without an intact endothelium. Removal of the endothelial cell layer markedly enhanced the maximum contractile effect of norepinephrine (2.8 fold), methoxamine (4.0 fold) and clonidine (13.0 fold). Furthermore, there was a significant decrease in the EC 50 values for these agonists. These findings indicate that both α 1 - and α 2 -adrenoceptor agonists can induce contraction of mesenteric arteries which is modulated by the endothelial cell layer.

INFLUENCE OF N‐ETHYLMALEIMIDE ON CHOLINOCEPTORS AND RESPONSES IN LONGITUDINAL MUSCLES FROM GUINEA‐PIG ILEUM

1 The binding of carbamylcholine to membranes prepared from the longitudinal muscle of guinea‐pig ileum was determined from its inhibition of the binding of [3H]‐3‐quinuclidinyl benzilate. Carbamylcholine binding was resolved into high and low affinity components with apparent dissociation constants of 0.11 ± 0.02 and 11 ± 1 μm 42% of the receptors displayed high affinity carbamylcholine binding. 2 Alkylation of longitudinal muscle membranes with N‐ethylmaleimide increased muscarinic receptor affinity for carbamylcholine in a manner consistent with a conversion of low affinity to high affinity receptors. After exposure the muscle membrane fragments to 1 mm N‐ethylmaleimide for 20 min at 35°C, carbamylcholine binding was resolved into two components with apparent dissociation constants of 0.11 ± 0.01 and 9 ± 2 μm, with 74% of the receptors displaying the higher affinity. 3 Exposure of longitudinal membranes mounted in an organ chamber to 1 mm N‐ethylmaleimide for 30 s depressed isometric contractions in response to acetylcholine by 80%, while contractions induced by K+ and Ba2+ were reduced by less than 20% and 10%, respectively. Acetylcholine dose‐response curves were shifted to the right while Ba2+ curves were unaffected. 4 It is suggested that N‐ethylmaleimide has a selective effect on muscarinic responses in the longitudinal muscle by disrupting processes occurring after receptor occupancy but before the induction of phospholipid turnover or calcium influx in the postsynaptic membrane.

TTX-sensitive Na+ channels and Ca2+ channels of the Land N-type underlie the inward current in acutely dispersed coeliac-mesenteric ganglia neurons of adult rats

Inward membrane currents of sympathetic neurons acutely dispersed from coeliac-superior mesenteric ganglia (C-SMG) of adult rats were characterized using the whole-cell variant of the patch-clamp technique. Current-clamp studies indicated that C-SMG neurons retained electrical properties similar to intact ganglia. Voltage-clamp studies designed to isolate Na+ currents revealed that tetrodotoxin (TTX, 1 μM) completely inhibited the large transient inward current. Half activation potential (Vh) and slope factor (K) were −26.8 mV and 6.1 mV, respectively. Inactivation parameters for Vh and K were −65 mV and 8.2 mV, respectively. Voltage-clamp studies also revealed a high-voltage-activated sustained inward Ca2+ current which was blocked by the removal of external Ca2+ or the presence of Cd2+ (0.1 mM). The dihydropyridine agonist, (+)202–791 (1 μM), caused a small increase (20%) in the amplitude of the Ca2+ current at more negative potentials and markedly prolonged the tail currents. ω-Conotoxin GIVA (ω, CgTX, 15 μM) caused a 66% inhibition of the high-voltage-activated Ca2+ current amplitude. Norepinephrine (1 μM) caused a 49% reduction in the peak Ca2+ current. This study is the first demonstration that dispersed C-SMG neurons from adult rats retain electrical characteristics similar to intact ganglia. A TTX-sensitive Na+ current as well as a high voltage-activated sustained Ca2+ current underlie the inward current in C-SMG neurons. The macroscopic Ca2+ current is composed of a small dihydropyridinesensitive (L-type current) and a large ω-CgTx-sensitive (N-type current) component. Thus, acutely dispersed CSMG neurons are suitable for examining the biophysical properties and modulation of membrane currents of adult prevertebral sympathetic neurons in normal and diseased states.

Cholinergic supersensitivity and decreased number of muscarinic receptors in atria from short-term diabetic rats

Autonomic neuropathy is a major complication of chronic diabetes and is responsible for disturbances in the cardiovascular system and other organs. Early cardiac disturbances have been attributed to defective vagal control of the heart. The heart rates of rats with chemically-induced diabetes are depressed and an increase in blood pressure produces a greater reflex bradycardia in diabetic rats. Tomlinson and Yusof found that isolated, stimulated left atria from rats made diabetic with alloxan are supersensitive to the negative inotropic influence of acetylcholine. On the other hand, Rao, et al. found that perfused working-heart preparations from streptozotocin- and alloxan-diabetic rats have a reduced sensitivity to carbamylcholine. In the present study, we measured chronotropic responses to cholinergic agonists of isolated, spontaneously-beating atria, as well as muscarinic receptor populations, in cardiac tissue from short-term (8 to 9 weeks) diabetic and age-matched control rats.

Insulin Prevention of Altered Muscarinic Receptor–G Protein Coupling in Diabetic Rat Atria

Right atria from rats rendered diabetic by injection of streptozocin (STZ-D) for 8–10 wk are supersensitive to the negative chronotropic effects of muscarinic agonists but have decreased levels of muscarinic receptors and acetylcholinesterase activity. Insulin treatment completely prevents the development of these changes. The proportion of atrial muscarinic receptors displaying high-affinity agonist binding is lower in STZ-D rats; however, the sensitivity of high-affinity agonist binding to regulation by a guanine nucleotide (5′-guanylylimidodiphosphate) is greater in atria from diabetic rats. Again, insulin treatment eliminates these differences. These findings indicate that alterations in atrial muscarinic systems in STZ-D rats are a consequence of the elaboration of the diabetic state and suggest that an alteration of functional muscarinic receptor–G protein coupling contributes to the altered physiological responsiveness of the heart in diabetes.

Estradiol Relaxes Rat Aorta via Endothelium-Dependent and -Independent Mechanisms

The effects of estrogen on arterial function are heterogeneous with respect to vessel and/or species. We have investigated 17β-estradiol-induced relaxation in isolated rat aorta with regard to the role of the vascular endothelium and ionic mechanisms. Estrogen induced a concentration-dependent relaxation of 46.5 ± 7.9% and 70.1 ± 12.2% (10–8 and 10–7M), which was reduced by endothelial denudation. Furthermore, L-nitroarginine methyl ester completely abrogated this effect; however, estradiol did not relax KCl-contracted rings. Tetraethyl ammonium (1 mmol/l) completely blocked estradiol-induced relaxation. Estradiol increased [cGMP] in isolated aortic rings via NO, but did not significantly affect NOS activity in endothelial cells. Thus, estrogen can relax rat aorta in vitro via both endothelium-dependent and -independent mechanisms involving the NO/cGMP and potassium channel signaling system.