Contraction Of Porcine Coronary Arteries Research Articles

NH 4Cl-induced contraction of porcine coronary artery involves activation of dihydropyridine-sensitive Ca 2+ entry

The role of voltage-dependent, dihydropyridine-sensitive Ca 2+ channels in NH 4Cl-induced vasoconstriction was investigated in isolated porcine coronary arteries by measuring in parallel isometric tone and 45Ca 2+ uptake. NH 4Cl (10–80 mM) concentration dependently induced tonic contractions which were preceded by a time lag of several minutes. Contractile responses to high (60 mM) as well as low (25 mM) concentrations of NH 4Cl were markedly inhibited by 1 μM nifedipine or removal of extracellular Ca 2+. The contractile effect of 25 mM NH 4Cl was substantially enhanced by increasing extracellular K + to 14.7 mM or by pretreatment of coronary arteries with either 5 mM tetraethylammonium chloride or 0.1 μM 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester (BAY K8644). NH 4Cl (60 mM) significantly increased 45Ca 2+ uptake with a lag time of more than 5 min. The increase in 45Ca 2+ uptake induced by 60 mM NH 4Cl was abolished in the presence of 1 μM nifedipine. Although NH 4Cl (25 mM) did not detectably stimulate 45Ca 2+ uptake in normal K + solution, it significantly augmented 45Ca 2+ uptake when extracellular K + was increased to 14.7 mM. Furthermore, NH 4Cl (20 mM) potentiated histamine-induced contraction of coronary arteries. This potentiating effect of NH 4Cl was completely antagonized by nifedipine. Our results suggest an involvement of nifedipine-sensitive Ca 2+ channels in NH 4Cl-induced vasoconstriction of porcine coronary artery.

Phosphorylation of Calponin Mediated by Protein Kinase C in Association with Contraction in Porcine Coronary Artery

Calponin is an actin-associated regulatory protein in smooth muscle. We report that both endothelin-1 (ET-1) and phorbol 12,13-dibutyrate(PDBu) caused a significant increase in phosphorylation of calponin during contraction of porcine coronary artery, while high levels of KCl were ineffective. This phosphorylation was predominantly catalyzed by activation of protein kinase C(PKC). In addition, the level of phosphorylation of calponin increased closely in association with the size of the contractile force induced by PDBu. Thus, the phosphorylation of calponin in vivo by PKC might modulate in part the contraction of smooth muscle that occurs in response to ET-1 or PDBu.

血管収縮反応とカルポニンのリン酸化・脱リン酸化反応

Calponin is a thin filament-associated smooth muscle protein that has been implicated to play a role in the regulation of smooth muscle. It inhibits actomyosin ATPase but the capacity for such inhibition is lost upon phosphorylation of calponin by protein kinase C. Thus, it seems possible that phosphorylation of calponin might modulate the contraction of smooth muscle. The objective of the present investigation was to determine whether calponin is phosphorylated by protein kinase C in intact smooth muscle in response to a vasoconstrictor and whether such phosphorylation is of functional significance with respect to the contraction of smooth muscle. Endothelin-1 and phorbol 12, 13-dibutyrate (PDBu) caused 2.3-fold and 2.6-fold increases, respectively, in tthe extent of phosphorylation of calponin during contraction of porcine coronary artery. However, high levels of KCl were ineffective despite development of an identical contractile force. These results suggest that the phosphorylation of calponin in vivo by protein kinase C might play an important role in the contraction of smooth muscle that occurs in response to endothelin-1 and PDBu.

Vasospasmolytic effect of KRN2391 on 3,4-diaminopyridine-induced rhythmic contraction of porcine coronary artery

1. In the present study, we examined the vasospasmolytic effect of KRN2391 on rhythmic contractions of porcine coronary artery caused by 3,4-diaminopyridine (3,4-DAP) compared with cromakalim and nitroglycerin. 2. KRN2391 at 10 −7 showed a tendency to prolong the cycle length and at 10 −6 M completely eliminated rhythmic contractions in all preparations. The elimination by 10 −6 M KRN2391 was antagonized by either oxyhemoglobin (10 −5 M) or glibenclamide (3 × 10 −6) although not completely. 3. Cromakalim at 10 −5 M and nitroglycerin at 10 −7 M completely eliminated 3,4-DAP-induced rythmic contractions in all preparations. The elimination by cromakalim and nitroglycerin was completely antagonized by glibenclamide and oxyhemoglobin, respectively. 4. The present study suggests that the vasospasmolytic effect of KRN2391 on 3,4-DAP-induced rhythmic contractions is based on its nitrate action and K channel opening action.

Pharmacological analysis of the inhibitory effects of KRN2391 on endothelin-1-induced contraction in isolated large coronary artery of the pig

1. The relaxant effect of KRN2391, N-cyano- N′-(2-nitroxyethyl)-3-pyridine-carboximidamide-monomethanesulfonate (with both K + channel opener and nitrate actions), nifedipine (Ca 2+ channel blocker), nitroglycerin (nitrate) and cromakalim (K + channel opener) were investigated in isolated porcine large coronary arteries contracted by endothelin-1. These drugs inhibited endothelin-1-induced contraction in a concentration-dependent manner. 2. The relaxation induced by KRN2391 was nearly complete at their maximum effects, but nifedipine and cromakalim could not produce complete relaxation. 3. The concentration-relaxation curves for KRN2391 underwent a rightward shift in the presence of methylene blue or glibenclamide. The concentration ratios of KRN2391 calculated based on EC 50 values were 2.8 and 3.7 in the presence of methylene blue and glibenclamide, respectively. 4. The concentration-relaxation curves for nitroglycerin and cromakalin underwent a rightward shift in the presence of methylene blue and glibenclamide, respectively, and the concentration ratios of nitroglycerin and cromakalim were 12.0 and 6.3. 5. These relaxant effects of KRN2391 and nitroglycerin on endothelin-1-induced contraction of porcine coronary artery were greater than those of cromakalim and nifedipine. This potent relaxant action of KRN2391 on endothelin-induced contraction is thought to be based on both a nitrate action and a K + channel opening action.

Endothelial regulation of coronary vascular tone in vitro: contribution of endothelin receptor subtypes and nitric oxide

The functional effects of endothelin-1, endothelin-3 and the ET B receptor agonist [Ala 1,3,11,15]endothelin-1 on pig coronary arteries were characterized in vitro by using the ET A receptor antagonist BQ-123 and the nitric oxide synthesis inhibitor N-nitro- L-arginine. Endothelin-1 (EC 50 value 8.8 nM), endothelin-3 (EC 50 11.6 nM) and [Ala 1,3,11,15]endothelin-1 (EC 50 42 nM) evoked concentration-dependent contractions with maximal responses that were 151 ± 21, 85 ± 12 and 11 ± 2%, respectively, of contractions evoked by 127 mM K +. BQ-123 (0.1–10 μM) induced concentration-related rightward shift of the response to endothelin-1. The response to the highest concentration of endothelin-1 was reduced by 62% in the presence of 10 μM BQ-123. Application of BQ-123 to vessels precontracted with endothelin-1 caused relaxation by 53%. BQ-123 also inhibited the contractile effect off endothelin-3, whereas the contractile responses to [Ala 1,3,11,15]endothelin-1, serotonin or neuropeptide Y (Y 1 receptor-mediated) were unaffected. In the presence of N-nitro- L-arginine (50 μM) the responses to [Ala 1,3,11,15]endothelin-1 and low concentrations of endothelin-3 were significantly enhanced. The present results show that endothelin-induced contractions of porcine coronary arteries are efficiently prevented and reversed by BQ-123 indicating that the responses are evoked by ET A receptors. A portion of the contraction seems to be mediated by ET B receptors. The contractile response to ET B stimulation is in part counteracted by release of nitric oxide.

P-142 - No evidence for involvement of uptake inhibition into neuronal and non-neuronal components in the selective potentiation by endothelin-1 of 5-hydroxytryptamine-induced contraction in porcine coronary artery.

P-142 - No evidence for involvement of uptake inhibition into neuronal and non-neuronal components in the selective potentiation by endothelin-1 of 5-hydroxytryptamine-induced contraction in porcine coronary artery.

Effects of opioids on vasoresponsiveness of porcine coronary artery.

Myocardial ischemia during surgery can be caused by coronary vasospasm. Neurohumoral mechanisms are involved in this phenomenon, and various substances have been suggested as possible causes, including acetylcholine, histamine, and norepinephrine. The responses of isolated porcine coronary arteries (from 117 pig hearts) with (E+) and without (E-) endothelium to these agents were investigated in the presence of fentanyl, sufentanil, and morphine. Fentanyl significantly shifted to the right, in a concentration-dependent fashion, the concentration-response curve to acetylcholine. This effect was not different between E+ and E- rings. Neither sufentanil nor morphine altered acetylcholine-induced contraction of porcine coronary arteries. Naloxone did not antagonize the suppressive effect of fentanyl on acetylcholine-induced contraction. The response of porcine coronary arteries to norepinephrine was decreased only at very high concentrations of fentanyl. Neither sufentanil nor morphine altered norepinephrine-induced contraction of porcine coronary arteries. Fentanyl, sufentanil, and morphine had no effect on histamine-induced contraction. We conclude that fentanyl antagonized acetylcholine-induced contraction of porcine coronary arteries. This effect of fentanyl seems to be caused by a direct effect on smooth muscle cells and is not opioid-receptor mediated.

Characterization of porcine coronary muscarinic receptors

To determine the muscarinic receptor subtype involved in the contractile response of coronary smooth muscle, we investigated the profiles of various muscarinic receptor antagonists competing for [3H]N-methyl-scopolamine ([3H]NMS) binding to membrane preparations from porcine coronary arteries. [3H]NMS binds to a single population of muscarinic binding sites with a KD of 135 pM and a Bmax of 57 fmol/mg. The affinity profiles of AF-DX 116 [11-2((-((diethylamino)methyl)-1-piperidinyl)acetyl)-5,11-dihydro-6H- pyrido (2,3-b)(1,4)-benzo-diazepin-6-one], atropine, 4-DAMP [4-diphenylacetoxy-N-methylpiperidine methiodide], methoctramine [N,N'-bis(6-((2-methoxybenzyl)amino)hexyl)-1,8-octane-diamine tetrahydrochloride], HHSiD [hexahydrosiladifenidol] and pirenzepine are consistent with binding to a mixed population of muscarinic binding sites, namely of the M2 and M3 subtype. Binding curves for AF-DX 116 and methoctramine are shallow with Hill-coefficients significantly less than unity. Comparison of data from binding studies with results obtained in functional experiments, i.e. antagonism of methacholine induced contraction of porcine coronary artery rings, it was found that only the low-affinity pKi values of AF-DX 116 (6.26) and methoctramine (6.51) correlated well with functional pA2 values. It is concluded that a mixed population of the M2 and M3 muscarinic receptor subtypes is present in porcine coronary arteries. Functional experiments do not support the contribution of the M2 subtype to the contractile response. Cholinergic induced contractions of porcine coronary arteries appear to be evoked via stimulation of the muscarinic M3 receptor subtype. However, since the compounds investigated here do not markedly discriminate between cloned m3, m4 and m5 receptors the involvement of muscarinic receptors different from M1, M2 and M3 cannot be excluded.

O-149 - Ca-independent contraction of porcine coronary artery induced by endothelin.

O-149 - Ca-independent contraction of porcine coronary artery induced by endothelin.

A novel peptide vasoconstrictor, endothelin, is produced by vascular endothelium and modulates smooth muscle Ca2+ channels.

Since the discovery of endothelium-dependent vasodilation, vascular endothelium has been recognized as an important functional unit contributing to the regulation of vascular tonus. Recent purification, structure determination and molecular cloning of a novel peptidergic vasoconstrictor, endothelin, from the culture supernatant of porcine aortic endothelial cells has further substantiated this concept. Starting from a large quantity of serum-fee endothelial cell-conditioned medium, endothelin was purified to homogeneity after three steps of high performance liquid chromatography by collecting active fractions and constricting porcine coronary artery strips. A peptide sequence analysis showed that endothelin was a previously unknown 21-residue peptide with two intrachain disulphide bonds. The EC50 of endothelin for contraction of porcine coronary artery was 4.0 x 10(-10) mol/l, indicating that endothelin is one of the most potent vasoconstrictors known. The pharmacological and structural properties of endothelin suggest that the peptide acts by activating voltage-dependent Ca2+ channels in vascular smooth muscle cells. Cloning and sequence analysis of complementary (c)DNA encoding porcine and human endothelin precursors showed that endothelin was produced de novo in endothelial cells from an approximately 200-residue prepropeptide through an unusual proteolytic processing by a putative 'endothelin-converting enzyme'. Northern blot analysis showed that preproendothelin messenger (m)RNA was expressed not only in cultured endothelial cells but also in fresh intimal tissue from porcine aorta, and that mRNA can be induced markedly in response to several vasoactive agents in cultured endothelial cells, suggesting the existence of a novel endothelium-mediated cardiovascular control system.

３，４‐Ｄｉａｍｉｎｏｐｙｒｉｄｉｎｅによるブタ冠動脈標本の周期性収縮に対する血管拡張薬の作用

3,4-Diaminopyridine (3,4-DAP), which is known to decrease K conductance, produced spontaneous repetitive phasic contractions of a regular (28/60) and an irregular (15/60) cycle or tonic contraction (16/60) following a latent period of 5-100 min in isolated porcine coronary arteries. Effects of pinacidil, a newly-synthesized vasodilator, were investigated using the preparation in which 3,4-DAP produced phasic contractions of the regular cycle in comparison with those of various vasodilators. Pinacidil produced dose-dependent prolongation of the cycle and reduced the peak tension and the tension at the relaxation phase, a mode of action that closely resembles that of nicorandil, suggesting the increase in K conductance and hyperpolarization. Nifedipine (10(-8) M) and dilazep (10(-4) M) markedly reduced the peak tension, while adenosine, dipyridamole and nitroglycerin did not produce such effects. The latter three drugs produced a prolongation of the cycle and reduced the tension of the relaxation phase. These data suggest that reduction of K conductance and activation of the voltage-dependent Ca channel may play an important role in initiation of the spontaneous repetitive phasic contraction in porcine coronary artery.

Absence of role of endothelium in the response of isolated porcine coronary arteries to acetylcholine.

Isolated precontracted arteries of various vascular beds relax in response to acetylcholine only if the endothelium is present. One explanation for this is that this drug stimulates the endothelial cells to release a vasodilator substance that in turn relaxes the underlying smooth muscle. To determine whether this mechanism is concerned also in the acetylcholine contraction of isolated porcine coronary arteries transverse strips of the extramural part of the left circumflex artery were used for recording isometric tension in the muscle bath. Dose-response curves for acetylcholine showed no significant difference before and after removal of endothelium. As a functional check on the removal of endothelium, the responsiveness of each preparation to a known endothelium dependent dilator (substance P) was tested before and after removal. These findings suggest that endothelial cells are not concerned in the acetylcholine induced contraction of porcine coronary arteries. Acetylcholine appears to act directly on smooth muscle of the porcine artery.