Porcine Coronary Artery Smooth Muscle Research Articles

P-343 - Effects of KRN2391 and KRN4884 on membrane potential in porcine coronary artery smooth muscle

P-343 - Effects of KRN2391 and KRN4884 on membrane potential in porcine coronary artery smooth muscle

O-131 - Activation of Ca2+-dependent K+ current by nordihydroguaiaretic acid in porcine coronary arterial smooth muscle cells

O-131 - Activation of Ca2+-dependent K+ current by nordihydroguaiaretic acid in porcine coronary arterial smooth muscle cells

Benzofused macrocyclic lactams as triple inhibitors of endothelin-converting enzyme, neutral endopeptidase 24.11, and angiotensin-converting enzyme.

The purpose of this study was to identify endothelin-converting enzyme (ECE) inhibitors that also possess inhibitory activity for neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE). The ortho-substituted benzofused macrocyclic lactams, such as CGS 26670, are generally potent NEP inhibitors but poor ACE inhibitors. CGS 26670 inhibited ECE activity with an IC50 of 600 nM, whereas it inhibited NEP and ACE activities with IC50 values of 0.9 and > 10,000 nM, respectively. This compound also prevented the conversion of big endothelin-1 (big ET-1) to ET-1 by denuded porcine coronary arterial smooth muscle with an IC50 of 200 nM. The ACE inhibitory activity is greatly is greatly improved in metasubstituted benzofused macrocyclic lactams. For example, CGS 26582 inhibited ECE, NEP, and ACE activities with IC50 values of 620, 4, and 175 nM, respectively. When injected at 30 mg/kg i.v. in conscious rats, followed by a challenge with big ET-1 at 1 nmol/kg i.v., this compound suppressed by 44% the increase in mean arterial blood pressure owing to the generation of ET-1 by ECE. Because ECE, NEP, and ACE play regulatory roles in cardiovascular and renal function, triple inhibitors of these enzymes may represent a novel class of agents for treatment of cardiovascular and renal diseases.

Angiotensin II activates phosphatidylinositol 3-kinase in vascular smooth muscle cells.

Phosphatidylinositol 3-kinase (PI3K) is an important component of the signal transduction systems activated by tyrosine kinase receptors. It has not been established, however, whether PI3K is also an essential mediator for G protein-coupled receptors. The potential involvement of PI3K in G protein-linked angiotensin II (Ang II)-dependent signaling was assessed in a primary cell culture system of porcine coronary artery smooth muscle cells (SMCs). Treatment of quiescent SMCs with Ang II (10(-5) to 10(-8) mol/L) resulted in a dose-dependent activation of PI3K when assayed in vivo and in vitro. The Ang II receptor antagonists losartan and PD123319 were used to establish that Ang II stimulates PI3K through the Ang II type-1 (AT1) receptor. Immunofluorescent microscopy revealed that Ang II (10(-6) mol/L) stimulated the translocation of p85, the regulatory subunit of PI3K, from the perinuclear region to distinct foci throughout the cell within 15 minutes. Western blot analysis of p85 subcellular distribution demonstrated that p85 concentrations were also increased within 15 minutes in the membrane fraction and concomitantly decreased in the cytoskeletal and nuclear fractions. These changes in PI3K location and activity were paralleled by increased tyrosine phosphorylation of p85. A potential correlation between angiotensin-mediated PI3K activation and SMC growth was found using LY294002, a specific inhibitor of PI3K, which blocked the increase in DNA and RNA synthesis as well as cellular hyperplasia generated by Ang II (10(-6) mol/L) stimulation of quiescent SMCs. These data indicate that PI3K may operate as a mediator of vascular SMC growth after stimulation with Ang II.

Activation of BK Ca channel via endothelin ET A receptors in porcine coronary artery smooth muscle cells

It has been demonstrated previously that endothelin-1 stimulates the Ca 2+-activated K + (BK Ca) channel activity in porcine coronary artery smooth muscle cells. The purpose of the present study was to delineate the endothelin receptor subtype involved in this action. In receptor binding studies, [ 125I]endothelin-1 was shown to bind to the homogenate of porcine primary coronary artery smooth muscle cells in a single class of binding sites with K D and B max values of 73 pM and 99 fmol/mg protein, respectively. Furthermore, endothelin-1 and endothelin-3 displaced the binding of [ 125I]endothelin-1 to these cells with respective IC 50 values of 70 and 17 000 pM, a 240-fold difference in potency. The effects of endothelin-3 on the activity of the BK Ca channel in porcine coronary artery smooth muscle cells were examined using the cell-attached patch-clamp technique. Similar to endothelin-1, endothelin-3 also exhibited a bell-shaped concentration-response curve. A maximal increase of 95% in channel open-state probability ( P o) was induced by 100 nM endothelin-3 as compared with the 320% increase in P o by 1 nM endothelin-1. Thus, endothelin-1 was about 100-fold more potent and 3.4-fold more efficacious than endothelin-3 in this action. Both the receptor binding and the electrophysiological results suggest that the effects of endothelins on the BK Ca channel are mediated through the endothelin ET A receptor subtype. © 1997 Elsevier Science B.V. All rights reserved.

Effect of ouabain on adenosine receptor-mediated hyperpolarization in porcine coronary artery smooth muscle

We investigated the effect of inhibitors of endothelium-derived nitric oxide and sodium-potassium (Na +-K +) pumps on adenosine receptor-mediated hyperpolarization of porcine coronary artery smooth muscle with and without endothelium. The average resting membrane potential (RMP) in porcine coronary artery smooth muscle was −51.5±0.2 and −50.7±0.2 mV, in the presence and absence of endothelium, respectively. Neither ouabain, N-nitro- l-arginine methyl ester ( l-NAME) nor ouabain and l-NAME in combination significantly affected the resting membrane potential in the absence of vasodilator agonists. Adenosine agonists, 2-chloroadenosine and 5′- N-ethylcarboxamidoadenosine at 10 −5 M caused a significant increase in RMP with intact endothelium and caused a smaller but significant increase in RMP in the absence of endothelium. Ouabain (10 −5 M) in the absence of l-NAME significantly reduced hyperpolarization due to 2-chloroadenosine and 5′- N-ethylcarboxamidoadenosine in the presence of endothelium. However, in the absence of endothelium, its inhibitory effect was not significant. When ouabain plus l-NAME (10 −5 M) were given simultaneously, the hyperpolarization caused by adenosine agonists was significantly further attenuated nearly to the RMP level. Attenuation of the response to 2-chloroadenosine and 5′- N-ethylcarboxamidoadenosine by ouabain was not reversed by the nitric oxide precursor, l-arginine (10 −4 M) both in the presence and absence of endothelium. These results suggest that hyperpolarization of vascular smooth muscle of the porcine coronary artery by adenosine agonists is at least partly endothelium dependent and possibly involves the Na +-K + pump and the release of nitric oxide. © 1997 Elsevier Science B.V. All rights reserved.

P-325 - Endothelin Converting Enzyme-1 in Porcine Coronary Arterial Smooth Muscle Cells: mRNA Expression and Function

P-325 - Endothelin Converting Enzyme-1 in Porcine Coronary Arterial Smooth Muscle Cells: mRNA Expression and Function

The effects of hypoxia on pHi in porcine coronary artery endothelium and smooth muscle. A novel method for measurements in endothelial cells in situ.

Endothelium-dependent relaxation of porcine coronary arteries is attenuated under hypoxic conditions. Recent evidence also indicates that pHi may modulate the release of the endothelium-derived relaxing factor. We tested the hypothesis that hypoxia-induced attenuation of endothelium-dependent relaxation is mediated by alterations in pHi. We developed a novel method for loading surface cells, whereby endothelial cell pHi could be measured in situ on the intact porcine coronary artery. Endothelial cells of arterial ring segments were selectively loaded with the fluorescent indicator BCECF-AM. Differential loading of the endothelial cell layer was verified by confocal microscopy. pHi of the endothelial cells in situ and of endothelium-denuded arteries was measured with a Photon Technology International spectrofluorimeter. The functional integrity of the endothelium was assessed by the endothelium-dependent relaxation to substance P in a paired adjacent ring. In the experimental protocol for pHi measurements, preparations were perfused with a physiological bicarbonate buffer (pH 7.4), stimulated with KCI (29 mmol/L), and then subjected to hypoxia and reoxygenation. The mean basal pHi in endothelial cells on the intact six arteries was 6.92 +/- 0.07. Addition of KCI to the perfusion medium decreased (P = .025) pHi to 6.79 +/- 0.07. Subsequent bubbling with N2 increased (P = .009) pHi to 7.00 +/- 0.06, which was reversed by reoxygenation. In contrast to the in situ endothelium, pHi of the smooth muscle was not significantly altered from its basal value of 7.24 +/- 0.06 (n = 5) by either KCI or hypoxia. This differential behavior corroborated the confocal data indicating differential dye loading. These data thus suggest that oxygen-sensitive alterations in pHi may be an important mechanism of signal transduction in endothelial cells.

Coronary vasodilation by adenosine: Receptor subtypes and mechanism(s) of action

This article briefly reviews our recent findings regarding the status and functions of the adenosine receptors in porcine coronary artery smooth muscle and endothelium. The adenosine receptors mediating vasorelaxations were characterized pharmacologically using isolated tissue bath experiments. The rank order of potency of a series of adenosine analogs for the vasorelaxations suggested the presence of multiple A 2 -type adenosine receptors both on the smooth muscle and endothelium of the coronary artery. The smooth muscle A 2 -type receptors were of two types: one being xanthine-sensitive and activated by N - and C2-substituted adenosine analogs, and the other xanthine-insensitive stimulated by 5'-uronamide derivatives. The endothelial A 2 adenosine receptors were xanthine-sensitive and were responsive to the 5'-uronamides. These receptors were associated with the release of nitric oxide. Radioligand binding studies using [ 125 I]-APE and photoaffinity labeling with azide derivative of [ 125 I]-APE revealed a binding site corresponding to A 2A receptor in membranes of porcine coronary artery smooth muscle. In addition, ligand binding with [ 3 H]-DPCPX demonstrated the existence of A 1 adenosine receptors in the porcine coronary artery smooth muscle. These receptors were found to be linked to adenylate cyclase in an inhibitory manner via pertussis toxin-sensitive G protein(s), and could induce attenuation of vasorelaxation. In conclusion, the information presented in this article generally suggests that the coronary artery contains various types of adenosine receptors that may play a role in the regulation of its function. However, additional studies are required to further characterize and delineate the roles of the various adenosine receptors, and the information given herewith can provide an impetus for this.

Effects of a physiological insulin concentration on the endothelin-sensitive Ca2+ store in porcine coronary artery smooth muscle.

The effect of insulin to attenuate the Ca2+ and contractile response of vascular smooth muscle to a number of agonists has been described previously, but the Ca2+ regulatory mechanisms of insulin action remain unclear. We determined the effect of a physiological insulin concentration (300 pmol/l) on the Ca2+ response of vascular smooth muscle cells of the porcine right coronary artery to endothelin 1 (ET-1); furthermore, we examined the cellular Ca2+ stores affected by insulin (i.e., Ca2+ stores releasable by inositol 1,4,5-trisphosphate, caffeine, and ionomycin). We measured the Ca2+ responses of acutely isolated single smooth muscle cells with the fluorescent Ca2+ indicator Fura-2. Acute insulin exposure (20 min) significantly attenuated the Ca2+ response of single smooth muscle cells to 10 nmol/l ET-1. This inhibitory effect of insulin was observed both in the presence and absence of extracellular Ca2+. In contrast with the effects on ET-1-induced Ca2+ responses, insulin did not inhibit the Ca2+ response to 5 mmol/l caffeine, an agent that directly releases sarcoplasmic reticulum Ca2+ stores. Insulin was also without effect on the total cellular Ca2+ store released by 1 micromol/l ionomycin, a Ca2+-transporting ionophore. When ET-1 and caffeine were given in succession, a sizable caffeine-sensitive Ca2+ store could be released from insulin-treated cells but not control cells, indicating that the sarcoplasmic reticulum Ca2+ store of insulin-treated cells was not depleted by ET-1. Generalized depletion of the sarcoplasmic reticulum Ca2+ store is not one of the cellular mechanisms involved in the effect of insulin on coronary smooth muscle; instead, the effect may be due to an inhibitory influence on transmembrane signal transduction, such as diminished ET-1-induced inositol 1,4,5-trisphosphate production or reduced ability of this phosphoinositol to release stored Ca2+.

Effects of a physiological insulin concentration on the endothelin-sensitive Ca2+ store in porcine coronary artery smooth muscle

Effects of a physiological insulin concentration on the endothelin-sensitive Ca2+ store in porcine coronary artery smooth muscle

Cyclic ADP-ribose stimulates sarcoplasmic reticulum calcium release in porcine coronary artery smooth muscle.

Cyclic ADP-ribose (cADPR) was shown to induce calcium release from the endoplasmic reticulum via ryanodine-sensitive pathways. In smooth muscle, two pathways for calcium release from the sarcoplasmic reticulum (SR) have been previously demonstrated: D-myo-inositol 1, 4, 5-trisphosphate-gated and ryanodine-gated. However, evidence for cADPR as a regulator for SR Ca2+ release in smooth muscle is lacking. We used permeabilized porcine coronary artery smooth muscle cells to directly examine the stimulation of SR Ca2+ release by cADPR. The results provide direct evidence that cADPR stimulates SR Ca2+ release and that this response is not inhibited by heparin, by depletion of the caffeine-sensitive Ca2+ pool, or by blockade or ryanodine receptors. These results indicate a novel mechanism for Ca2+ release from the SR of vascular smooth muscle.

Opening of Ca2+-Dependent K+ Channels by Nordihydroguaiaretic Acid in Porcine Coronary Arterial Smooth Muscle Cells

Effects of nordihydroguaiaretic acid (NDGA) on the Ca(2+)-dependent K+ channel (BK channel) were examined by the patch clamp technique in single smooth muscle cells of porcine coronary artery. The open probability of BK channels in inside-out patches increased by about 30 times at the holding potential of 0 mV, when 10 microM NDGA was added to the bathing solution (pCa 7.0). The effect of NDGA was concentration-dependent in the range of 1-100 microM and partly removed by washout. The enhancement of BK channels by NDGA was not observed when the cytosolic Ca2+ concentration was very low (pCa > 8.5). These results clearly indicate that NDGA possesses a BK channel opening property in coronary arterial myocytes.

Endothelin-induced changes in intracellular pH and Ca2+ in coronary smooth muscle: role of Na(+)-H+ exchange.

The relationship between endothelin-1 (ET-1)-induced stimulation of Na(+)-H+ exchange and intracellular free Ca2+ ([Ca2+]i) was examined in primary cultures of porcine coronary artery smooth muscle cells. Intracellular pH (pHi) and [Ca2+]i were measured using 2,7-bis-carboxyethyl-5(6)-carboxyfluorescein and the acetoxymethyl ester of fura-2 respectively. In HCO3(-)-free buffer (pH = 7.4), ET-1 (0.1-50 nM) induced a sustained, dose-dependent increase in pHi. ET-1 (10 nM) increased pHi from 6.83 +/- 0.01 to 6.93 +/- 0.02 (P < 0.01). The alkalinization was blocked by the Na(+)-H+ exchange inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 3 microM) or by substitution of Na+ with N-methylglucamine or choline chloride (P < 0.05). Recovery of pHi in response to acidification, induced by washout of a 20 mM NH4Cl prepulse, was > 90% inhibited by EIPA (3 microM), confirming the presence of an ET-1-responsive Na(+)-H+ exchanger. Coronary smooth muscle cells responded to ET-1 with a dose-dependent, biphasic increase in [Ca2+]i which was not inhibited by manipulations (EIPA pretreatment or Na(+)-free media) shown to block the Na(+)-H+ exchanger. The ET-1-mediated alkalinization was not inhibited by removal of extracellular Ca2+ ([Ca2+]o). However, complete blockade of the ET-1-mediated [Ca2+]i response using the intracellular Ca(2+)-chelator, [bis-(2-amino-5-methylphenoxy)ethane-NNN'N'-tetraacetic acid tetraacetoxymethyl ester] (MAPTAM), in [Ca2+]o-free media, demonstrated that an increment in [Ca2+]i is required for activation of the Na(+)-H+ exchanger by ET-1. The ET-1-induced rise in [Ca2+]i was not associated with a rise in pHi in the presence of CO2/HCO3-. We conclude that: (1) activation of Na(+)-H+ exchange by ET-1 requires an increment in [Ca2+]i; and (2) ET-1 stimulates EIPA-sensitive Na(+)-H+ exchange, but this stimulation does not modulate ET-1-induced changes in [Ca2+]i.

Bioactivation of nitroglycerin in vascular smooth muscle cells is different from that in non-vascular tissue

The mechanism of biotransformation of nitroglycerin into the pharmacologically active radical nitric oxide (NO) or a related compound is still unclear. Different enzymes have been discussed to be involved in the bioactivation process. The effects of inhibition of glutathione- S-transferase and cytochrome P-450 enzymes were investigated on nitroglycerin-induced relaxation of bovine and porcine coronary arteries and on nitroglycerin-induced activation of guanylyl cyclase in cultivated porcine aortic smooth muscle cells. The glutathione- S-transferase inhibitor sulfobromophthalein had no effect on nitroglycerin-induced vascular relaxation, nor on nitroglycerin-induced elevation of cGMP levels in porcine coronary artery smooth muscle cells. The modulation of cytochrome P-450 activity by selective inhibitors as well as inducers did not alter the bioactivity of nitroglycerin in both systems. The data demonstrate that the isoenzymes of both enzyme families, which have been shown to be involved in the metabolism of nitroglycerin in different non-vascular tissues, do not play a role in bioactivation of nitroglycerin in the vascular system.

Protein kinase c-independent inhibition of the Ca 2+-activated K + channel by angiotensin II and endothelin-1

We previously reported that the Ca 2+-activated K + channel (K ca-channel) in cultured smooth muscle cells from porcine coronary artery was inhibited by protein kinase C (C-kinase). In this study, inhibition of the K ca-channel by receptor-mediated vascular contractile agonists, such as angiotensin II (ANG II) and endothelin-1 (ET-1), was investigated by the patch-clamp technique. In cell-attached patches, addition of ANG II (500 nM) or ET-1 (50 nM) to the bath inhibited the K ca-channel activated by the calcium ionophore A23187 (10–20 μM). Phorbol 12-myristate 13-acetate (PMA, 1 μM), a C-kinase activator, also decreased the open probability of the K ca-channel. The PMA-induced decrease in the open probability was reversed by subsequent application of staurosporine (1 nM), a C-kinase inhibitor, but the ANG II- and ET-1-induced decreases were not reversed by subsequent application of staurosporine (> 30 nM). Pretreatment of smooth muscle cells with 30 nM staurosporine, a protein kinase inhibitor, or 1 mM neomycin, an inhibitor of phospholipase C, also did not abolish the inhibition of the K ca-channel by ANG II. Furthermore, ANG II inhibited the K ca-channel in cells in which C-kinase was down-regulated. These results indicate that, in porcine coronary artery smooth muscle cells, ANG II and ET-1 inhibit the K ca-channel by a C-kinase-independent mechanism.

Effects of metalloprotease inhibitors on smooth muscle endothelindashconverting enzyme activity

The enzyme responsible for the conversion of exogenous big endothelindash1 to endothelindash1 by porcine coronary arterial smooth muscle has been shown to be a metalloprotease. The potencies of eight metalloprotease inhibitors for this endothelindashconverting enzyme were determined. CGS 25015, CGS 26129, and thiorphan inhibited the enzyme activity monophasically with ic 50 values of 2.6, 2.4, and 190 μM, respectively. In contrast, the data obtained using phosphoramidon as an inhibitor were best fit by a two-site model. The biphasic concentrationdashresponse curve had ic 50 values of 4.6 μM and 2.2 mM. Three analogs of phosphoramidon were also tested for enzyme inhibition. Removal of the rhamnose moiety of phosphoramidon reduced the potency ( IC 50 = 15 μM), whereas substitution of the rhamnose by ndash[2-(2-naphthyl)ethyl] improved the potency ( IC 50 = 2.0 μM). These results identify a thiol and a phosphonyl series of compounds as smooth muscle endothelindashconverting enzyme inhibitors. The structure-activity relationships revealed that an aromatic or aliphatic group in the P 2′ position or an aromatic group in the P 1 position of the inhibitor significantly increased the potency.

P2-44 - Effects of quinidine on delayed rectifier K current in porcine coronary arterial smooth muscle cells

P2-44 - Effects of quinidine on delayed rectifier K current in porcine coronary arterial smooth muscle cells

Myosin phosphorylation and Ca 2+ sensitization in porcine coronary arterial smooth muscle stimulated with endothelin-1

We examined the mechanism of endothelin-1-induced contraction in the porcine coronary artery by assessing the changes in myosin light chain phosphorylation and cytosolic Ca 2+ concentration ([Ca 2+] i) determined by fura-2 and front-surface fluorometry. Endothelin-1 induced a rapid phosphorylation of myosin both in the presence and in the absence of extracellular Ca 2+. During the later phase of contraction (5–30 min), the tension and [Ca 2+] i were maintained, while the phosphorylated myosin decreased in the presence of extracellular Ca 2+. At a similar extent of [Ca 2+] i elevation, endothelin-1 induced a greater tension development than did 118 mM K +-depolarization and this was not attributed to a greater degree of phosphorylation of myosin. In the Ca 2+-depleted strips, endothelin-1 induced a slow and transient contraction without an increase in either [Ca 2+] i or myosin phosphorylation. Therefore, in addition to the activation of mechanisms of [Ca 2+] i-myosin phosphorylation, endothelin-1 may activate additional calcium regulatory mechanisms which may thus increase the Ca 2+ sensitivity of contractile elements while also enhance and prolong the contractions in the porcine coronary artery.

Effects of pHi on isometric force and [Ca2+]i in porcine coronary artery smooth muscle.

During ischemia or hypoxia, alterations in pHi may play a significant role in alteration of vessel wall function. We studied the effects of altering pHi on isometric force and [Ca2+]i in porcine coronary artery. pHi was altered at constant pHo by use of NH4Cl and measured with the fluorescent dye BCECF. [Ca2+]i was monitored with fura 2 and ratiometric fluorescence measurements. Addition of NH4Cl elicited a concentration-dependent (2 to 30 mmol/L) sustained increase in isometric force in unstimulated tissues. In tissues stimulated with KCl (29 mmol/L) or U46619 (1 mumol/L), addition of NH4Cl elicited a rapid but transient decrease followed by a sustained increase in force above the initial stimulated levels. Removal of NH4Cl was associated with a transient decrease and increase followed by a prolonged depression of force and slow recovery to initial levels. Addition of NH4Cl elicited a rapid monotonic increase in pHi and then a slow recovery toward initial levels; washout of NH4Cl led to a rapid acidification followed by recovery. In contrast to the steady state effects of NH4Cl on force, its effects on [Ca2+i were in the opposite direction. During the sustained increase in force elicited by NH4Cl alkalinization, [Ca2+]i was substantially decreased, whereas when force was depressed during the acidification elicited by NH4Cl washout, [Ca2+i was increased to values observed before addition of NH4Cl. The initial transients in force elicited by NH4Cl addition or washout were also associated with opposite changes in [Ca2+]i. Thus, the effects on force of the NH4Cl-induced changes in pHi are associated with changes in the Ca2+ sensitivity of the contractile apparatus rather than mediated through changes in [Ca2+]i.