Effects of endothelin on cytosolic Ca2+ level and mechanical activity in rat uterine smooth muscle.

The effects of endothelin (ET) on cytosolic Ca2+ level ([Ca2+]i) and mechanical activity were examined in isolated rat uterine smooth muscle. ET-1, ET-2, ET-3 and sarafotoxin S6b (STX) induced rhythmic contractions superimposed on an increased muscle tone. The concentration needed to induce a half-maximum contraction (EC50) was 1.6-3.3 nM for ET-1, ET-2 and STX and higher than 200 nM for ET-3, suggesting that the ET(A) receptor is responsible for these contractions. The sensitivity to ET-1 of uterus at day 20 of gestation was higher than that of non-pregnant rat uterus. Contraction induced by ET-1 followed an increase in [Ca2+]i. The relation between [Ca2+]i and muscle tension, an an indicator of Ca2+ sensitivity of contractile elements, in the presence of ET-1 was identical to that in the presence of high K+ in non-pregnant and pregnant rat uteri. The ET-1-induced increases in [Ca2+]i and muscle tension were strongly inhibited by verapamil in non-pregnant rat uterus. In pregnant rat uterus, however, verapamil only partially inhibited the increases. The verapamil-insensitive portions of [Ca2+]i and contraction were inhibited by EGTA. In the absence of external Ca2+, ET changed neither [Ca2+]i nor muscle tension. These results suggest that ET-1 acts on ET(A) receptors, increase [Ca2+]i and induces contraction without changing Ca2+ sensitivity of contractile elements. The increase in [Ca2+]i seemed to be mediated by opening of L-type Ca2+ channels in non-pregnant rat uterus and also of non-L-type Ca2+ channels in pregnant rat uterus, but not by Ca2+ release from intracellular storage sites.     

Effects of sodium nitroprusside on cytosolic calcium level in vascular smooth muscle

The inhibitory effect of sodium nitroprusside on the cytosolic Ca2+ level ([Ca2+]cyt), measured simultaneously with contraction by means of a fluorescence dye, fura-2, and on the 45Ca2+ uptake was tested in the isolated rat aortic smooth muscle. Norepinephrine increased muscle tension, 45Ca2+ uptake and [Ca2+]cyt. In a Ca2+-deficient solution, norepinephrine transiently increased muscle tension and [Ca2+]cyt. Sodium nitroprusside inhibited all changes induced by norepinephrine although the inhibition of [Ca2+]cyt was less than that of muscle contraction. Sodium nitroprusside also inhibited the high K+-induced contraction at concentrations higher than those needed to inhibit norepinephrine-induced contraction. Inhibition of the high K+induced contraction was accompanied by a small decrease in [Ca2+]cyt and a smaller decrease in 45Ca2+ uptake. Methylene blue antagonized, and M&B 22,948 potentiated the inhibitory effect of sodium nitroprusside. These results suggest that sodium nitroprusside has multiple sites of action. At relatively low concentrations, sodium nitroprusside could inhibit the Ca2+ influx and Ca2+ release. At relatively high concentrations, this inhibitor could also augment Ca2+ sequestration and decrease the sensitivity of contractile elements to Ca2+.     

Effects of cyclopiazonic acid and ryanodine on cytosolic calcium and contraction in vascular smooth muscle.

1. In smooth muscle, both Ca2+ release from the sarcoplasmic reticulum (SR) and Ca2+ influx across the plasma membrane are responsible for the increase in the cytosolic Ca2+ level ([Ca2+]i). To understand further the role of SR on smooth muscle contraction, the effects of an inhibitor of the SR Ca2+ pump, cyclopiazonic acid (CPA 10 microM), an inhibitor of the Ca(2+) -induced Ca2+ release, ryanodine, (10 microM), and an activator of the Ca(2+) -induced Ca2+ release, caffeine (20 mM), on [Ca2+]i and contractile force were examined in the ferret portal vein loaded with a photoprotein, aequorin. 2. CPA induced a small increase in the aequorin signal reaching a maximum at 7 min. Several minutes after the increase in the aequorin signal, muscle tension increased reaching a maximum at 21.5 min. In contrast, ryanodine changed neither the aequorin signal nor contraction. In the presence of ryanodine, caffeine induced a sustained increase in the aequorin signal and transient contraction. After washing ryanodine and caffeine, the aequorin signal and muscle tone returned to their respective control levels. After treatment with ryanodine and caffeine, the second addition of caffeine was almost ineffective whereas CPA still increased the aequorin signal and muscle tension. 3. In the presence of external Ca2+, noradrenaline (NA, 10 microM) induced a transient increase followed by a sustained increase in the aequorin signal and sustained contraction. In contrast, KCl (70 mM) induced sustained increases in the aequorin signal and sustained contraction. In Ca(2+) -free solution, NA induced a small transient increase in the aequorin signal and a small transient contraction. These changes were inhibited in the presence of CPA or on pretreatment of the muscle with ryanodine and caffeine. These results suggest that CPA or ryanodine and caffeine depleted Ca2+ in SR. High K+ was ineffective in the absence of external Ca2+. 4. In the presence of external Ca2+ and CPA, NA and high K+ induced larger aequorin signals than in the absence of CPA, whereas the magnitude and shape of the contractions did not change. In contrast, pretreatment with ryanodine and caffeine did not have such an effect. In the muscle pretreated with ryanodine and caffeine, CPA changed the responses to high K+ and NA in a similar manner to that in the muscle without the pretreatment with ryanodine and caffeine. 5. Dissociation of contraction from [Ca2+]i as measured with aequorin suggests that NA and high K+ increase Ca2+ in two compartments: a compartment containing contractile elements (contractile compartment) and another compartment unrelated to contractile elements (non-contractile compartment). Because CPA augmented the stimulant-induced increase in aequorin signal without changing contraction, the non-contractile compartment may be located near the SR and the CPA-sensitive SR Ca2+ pump may regulate the Ca2+ level in this compartment. However, because CPA changed neither the magnitude nor shape of the contractions in the presence of external Ca2+, the SR Ca2+ pump may have little effect on regulation of Ca2+ level in the contractile compartment. Furthermore, the release of Ca2+ from SR seems to have little effect on the increase in the contractile Ca2+ because ryanodine and caffeine changed neither the aequorin signals nor contractions induced by NA and high K+ in the presence of external Ca2+ in the ferret portal vein.     

小檗胺对ROCC介导的血管平滑肌细胞内游离钙的影响

目的　研究小檗胺 (BA)对受体调控性Ca2 +通道介导的家兔胸主动脉血管平滑肌细胞内游离钙 ([Ca2 +]i)的影响。方法　家兔主动脉血管平滑肌以Fluo 3/AM负载 ,通过激光扫描共聚焦显微镜 (LSCM )测定 [Ca2 +]i。结果　在细胞外Ca2 +存在的条件下 ,BA 30 μmol·L-1不影响静息[Ca2 +]i;但对去甲肾上腺素 (NE) 30mmol·L-1、5 羟色胺 (5 HT) 1μmol·L-1诱导的 [Ca2 +]i 升高有明显的抑制作用。在无胞外钙时 ,对咖啡因 40mmol·L-1诱导的 [Ca2 +]i 升高没有作用。结论　BA对ROCC激活后的外钙内流有明显的抑制作用 ,对内钙释放没有影响。其作用与维拉帕米相似     

左旋千金藤定碱对培养牛主动脉血管平滑肌细胞胞内游离Ca~(2+)的影响

目的：研究左旋千金藤定碱（ｌ－ｓｔｅｐｈｏｌｉｄｉｎｅ，ＳＰＤ）对血管平滑肌的作用。方法：采用Ｆｕｒａ－２和ＡＲ－ＣＭ－ＭＩＣ阳离子测定系统测定培养牛主动脉血管平滑肌细胞内游离钙。结果：ＳＰＤ１～１００μｍｏｌ·Ｌ－１不影响静息［Ｃａ２＋］ｉ，但可剂量依赖地抑制高Ｋ＋引起的［Ｃａ２＋］ｉ增高，其ＩＣ５０为３９．６（９５％可信限２３．４～６７．１）μｍｏｌ·Ｌ－１，但其作用弱于尼群地平；ＳＰＤ１～１００μｍｏｌ·Ｌ－１对去甲肾上腺素、血管紧张素Ⅱ、５－ＨＴ、ＡＴＰ引起的［Ｃａ２＋］ｉ增高也有明显的抑制作用；高浓度ＳＰＤ对无外钙时去甲肾上腺素引起的［Ｃａ２＋］ｉ增高也有一定的抑制作用。结论：左旋千金藤定碱对培养血管平滑肌细胞电压依赖性钙通道和受体调控性钙通道均有抑制作用；其对电压依赖性钙通道的抑制作用弱于尼群地平。     

糖基化终产物对培养的大鼠主动脉平滑肌细胞增殖及胞浆游离钙含…

目的：研究糖基化终产物（ＡＧＥＰ）对主动脉平滑肌细胞增殖的影响及其与［Ｃａ＾２＋］ｉ的关系。方法：采用同位素掺入法分别测定ＤＮＡ和蛋白质合成；Ｆｕｒａ２－ＡＭ测定［Ｃａ＾２＋］ｉ。结果：ＡＧＥＰ以浓度、时间相关的方式促进［＾３Ｈ］ＴｄＲ与［＾Ｈ］Ｌｅｕ掺入细胞，随ＡＧＥＰ作用时间、糖化时间延长，掺入率增加明显，ＡＧＥＰ增加［Ｃａ＾２＋］ｉ，与时间、浓度相关，但随ＡＧＥＰ作用时间延长（４０分钟后）而     

Age-related decrease in the effect of parathyroid hormone-related protein on cytosolic free calcium level and tension in rat aortic smooth muscle

Age-related changes in the effect of parathyroid hormone-related protein (PTHrP) on the cytosolic free calcium level ([Ca⁺]_i) and on the tension in rat aortic smooth muscle were investigated. The possible involvement of cAMP, a second messenger of PTHrP, in such changes was also investigated. Spiral aortic strip preparations without endothelium from 8-weeks, 6-months, and 24-months old rats were treated with fura 2/AM, and the fluorescence ratio R340/380, an index of [Ca⁺]_i was measured. Simultaneously, the tension of the preparations was measured. PTHrP-(1–34) and dibutyryl cAMP produced concentration-dependent decreases in the tension and in R340/380 in aortas precontracted with phenylephrine (10^–7 M). These effects were significantly lower in the aortas of 24-months old rats than in the vessels of 8-weeks and 6-months old rats. The effects were similar in the aortas of 8-weeks and 6-months old rats. PTHrP-(1–34) concentration-dependently increased cAMP production in vascular smooth muscle cells (VSMCs) from 8-weeks old rats. However, the increase in cAMP production was significantly lower in cultured VSMCs from 6-months and 24-months old rats than in cells from 8-weeks old rats.These results suggest that the reduced cAMP production stimulated by PTHrP and the reduced effects of cAMP with aging might contribute to the age-related changes in the decreases in tension and [Ca⁺]_i in response to PTHrP in the rat aorta.     

Resveratrol decreases calcium sensitivity of vascular smooth muscle and enhances cytosolic calcium increase in endothelium

Resveratrol causes endothelium dependent and independent relaxation of vascular smooth muscle. This study investigated the mechanisms behind the effect of resveratrol on vascular tone. Resveratrol (0.1 mM) inhibited KCl-stimulated contractions in endothelium-denuded rat aorta and this inhibition was not reversed by tetraethylammonium (TEA) (5 mM), glyburide (3 microM), ouabain (0.1 mM), thapsigargin (1 microM), or indomethacin (10 microM). KCl (90 mM) increased the intracellular free calcium concentration ([Ca2+]i) in the isolated smooth muscle cells from the rat aorta and resveratrol (0.1 mM) did not inhibit the KCl-stimulated [Ca2+]i increase. The CaCl2 (0.1-100 microM) stimulated contractions were inhibited by resveratrol (0.1 mM) in the Triton X-100 skinned smooth muscle of the aorta. In heart valve endothelium, resveratrol (0.1 mM) augmented the acetylcholine (10 microM) stimulated [Ca2+]i increase. Resveratrol-induced augmentation of the acetylcholine-stimulated [Ca2+]i elevation was reversed by glyburide (3 microM), but not by TEA (5 mM). The present study indicated that resveratrol affected vascular smooth muscle and endothelium in different ways. Resveratrol decreased the Ca2+ sensitivity but did not affect the KCl-stimulated [Ca2+]i increase in the vascular smooth muscle. In the endothelial cells, resveratrol enhanced the agonist-stimulated [Ca2+]i increase that might trigger nitric oxide synthesis from endothelial cells.     

Effects of advanced glycosylation end products on proliferation and cytosolic free calcium in cultured rat aortic smooth muscle cells

目的：研究糖基化终产物（ＡＧＥＰ）对主动脉平滑肌细胞增殖的影响及其与［Ｃａ２＋］ｉ的关系．方法：采用同位素掺入法分别测定ＤＮＡ和蛋白质合成；Ｆｕｒａ２ＡＭ测定［Ｃａ２＋］ｉ．结果：ＡＧＥＰ以浓度、时间相关的方式促进［３Ｈ］ＴｄＲ与［３Ｈ］Ｌｅｕ掺入细胞，随ＡＧＥＰ作用时间、糖化时间延长，掺入率增加明显．ＡＧＥＰ增加［Ｃａ２＋］ｉ，与时间、浓度相关，但随ＡＧＥＰ作用时间延长（４０分钟后）而有所降低，ＢＳＡ修饰中葡萄糖浓度的增加，糖基化时间延长，［Ｃａ２＋］ｉ也呈上升趋势．结论：ＡＧＥＰ刺激平滑肌细胞增殖，并与细胞［Ｃａ２＋］ｉ浓度增加有关．     

Correlation between spontaneous electrical, calcium and mechanical activity in detrusor smooth muscle of the guinea-pig bladder

1. To investigate the cellular mechanisms underlying spontaneous excitation of smooth muscle of the guinea-pig urinary bladder, isometric tension was measured in muscle bundles while recording the membrane potential from a cell in the bundle with a microeletrode. Changes in the intracellular calcium concentration ([Ca(2+)](i); calcium transients) were recorded in strips loaded with the fluorescent dye, fura-PE3. 2. In 40% of preparations, individual action potentials and contractions, which were abolished by nifedipine (1 microm), were generated. In the remaining preparations, bursting action potentials and contractions were generated. Contractions were again abolished by nifedipine (1 microm), while higher concentrations of nifedipine (10-30 microm) were required to prevent the electrical activity. 3. Carbachol (0.1 microm) increased the frequency of action potentials and corresponding contractions. Apamin (0.1 microm) potentiated bursting activity and enhanced phasic contraction. Charybdotoxin (CTX, 50 nm) induced prolonged action potentials that generated enlarged contractions. In contrast, levcromakalim (0.1 microm) reduced the frequency of action potentials, action potential bursts and the size of the contractions. 4. Forskolin (0.1 microm), 8-bromoguanosin 3', 5' cyclic monophosphate (8Br-cGMP, 0.1 mm) and Y-26763 (10 microm) suppressed contractions without reducing the amplitude of either action potentials or Ca transients. 5. This paper confirms that action potentials and associated calcium transients are fundamental mechanisms in generating spontaneous contractions in smooth muscles of the guinea-pig bladder. However, in parallel with the excitation-contraction coupling, the sensitivity of the contractile proteins for Ca(2+) may play an important role in regulating spontaneous excitation and can be modulated by cyclic nucleotides and Rho kinase.     

Effects of endothelin on the cardiovascular system and on smooth muscle preparations in different species

Summary (1) The actions of porcine endothelin (ET), an endothelium-derived vasoconstrictor peptide, have been investigated in several in vitro smooth and cardiac muscle preparation as well as on the blood pressure of anaesthetized rats and rabbits. (2) In isolated visceral smooth muscles (guinea pig ileum, rat colon and uterus, rabbit jejunum) ET caused a long-lasting contraction which persisted after repeated rinsing. ET did not interfere with the spontaneous phasic activity of the rabbit jejunum or with contractions evoked by histamine or carbachol. (3) ET contracted isolated blood vessels (portal vein of guinea-pig and rat). In isolated perfused organs (rabbit ear, guinea-pig lung, rat mesentery and hindpaw) ET led to a long-lasting vasoconstriction. In the isolated perfused guinea-pig lung ET caused vaso- and bronchoconstriction. (4) ET produced a long-lasting positive inotropic effect in spontaneously beating isolated guinea-pig atria. The positive inotropic action of strophanthin was augmented in the presence of ET. (5) In the isolated perfused guinea-pig and rat hearts the prominent effect of ET was a long-lasting coronary vasoconstriction. (6) Pithed rats responded to i.v. injection of ET with a long-lasting increase in blood pressure. In pentobarbitone anaesthetized rats pretreated either with atropine, with guanethidine plus atropine, or with guanethidine plus atropine plus indomethacin, the long-lasting increase in blood pressure was smaller than in pithed rats whereas the initial short-lasting decrease in blood pressure was more pronounced; in pentobarbitone anaesthetized rabbits endothelin caused bronchoconstriction, a decrease in blood pressure and a pronounced increase in central venous pressure possibly resulting from pulmonary or coronary artery constriction or a combination of both effects. (7) Neither topical nor systemic application of ET influenced the bleeding time from small rat mesenteric arteries. (8) The general effect of ET on visceral, bronchial and vascular smooth muscles was a longlasting contraction. Despite its general constrictor potency seen in isolated vascular preparations ET can cause a transient decrease in arterial blood pressure in anaesthetized rats and rabbits. The role of ET when released from vascular endothelium remains a matter of speculation. Send offprint requests to F. Lembeck at the above address     

Effects of imatinib mesylate on spontaneous electrical and mechanical activity in smooth muscle of the guinea-pig stomach

BACKGROUND AND PURPOSE: Effects of imatinib mesylate, a Kit receptor tyrosine kinase inhibitor, on spontaneous activity of interstitial cells of Cajal (ICC) and smooth muscles in the stomach were investigated. EXPERIMENTAL APPROACH: Effects of imatinib on spontaneous electrical and mechanical activity were investigated by measuring changes in the membrane potential and tension recorded from smooth muscles of the guinea-pig stomach. Its effects on spontaneous changes in intracellular concentration of Ca(2+) ([Ca(2+)](i)) (Ca(2+) transients) were also examined in fura-2-loaded preparations. KEY RESULTS: Imatinib (1-10 microM) suppressed spontaneous contractions and Ca(2+) transients. Simultaneous recordings of electrical and mechanical activity demonstrated that imatinib (1 microM) reduced the amplitude of spontaneous contractions without suppressing corresponding slow waves. In the presence of nifedipine (1 microM), imatinib (10 microM) reduced the duration of slow waves and follower potentials in the antrum and accelerated their generation, but had little affect on their amplitude. In contrast, imatinib reduced the amplitude of antral slow potentials and slow waves in the corpus. CONCLUSIONS AND IMPLICATIONS: Imatinib may suppress spontaneous contractions of gastric smooth muscles by inhibiting pathways that increase [Ca(2+)](i) in smooth muscles rather than by specifically inhibiting the activity of ICC. A high concentration of imatinib (10 microM) reduced the duration of slow waves or follower potentials in the antrum, which reflect activity of ICC distributed in the myenteric layers (ICC-MY), and suppressed antral slow potentials or corporal slow waves, which reflect activity of ICC within the muscle bundles (ICC-IM), presumably by inhibiting intracellular Ca(2+) handling.     

Effects of a novel smooth muscle relaxant, KT-362, on contraction and cytosolic Ca2+ level in the rat aorta.

1. Inhibitory effects of a novel smooth muscle relaxant, KT-362 (5-[3-([2-(3,4-dimethoxyphenyl)-ethyl]amino)-1-oxopropyl]-2,3,4,5- tetrahydro-1,5-benzothiazepine fumarate), on contraction and the cytosolic Ca2+ level ([Ca2+]cyt) in isolated vascular smooth muscle of rat aorta were examined. 2. KT-362 inhibited the contractions induced by high K+ and noradrenaline. The inhibitory effect was antagonized by an increase in external Ca2+ concentration. A Ca2+ channel activator, Bay K 8644, did not change the effect of KT-362 on high K+-induced contraction. 3. [Ca2+]cyt, measured with fura-2-Ca2+ fluorescence, increased during the contractions induced by high K+ or noradrenaline. KT-362 decreased [Ca2+]cyt and muscle tension stimulated by high K+ or noradrenaline. By contrast, a Ca2+ channel blocker, verapamil, inhibited the noradrenaline-induced increase in [Ca2+]cyt with only partial inhibition of the noradrenaline-induced contraction and KT-362 inhibited the verapamil-insensitive portion of the contraction without changing [Ca2+]cyt. 4. In a Ca2(+)-free solution, noradrenaline and caffeine induced a transient contraction following a transient increase in [Ca2+]cyt. KT-362 inhibited the increments due to noradrenaline but not those induced by caffeine. 5. These results suggest that KT-362 inhibits vascular smooth muscle contraction by inhibiting Ca2+ channels, receptor-mediated Ca2+ mobilization, and receptor-mediated Ca2+ sensitization of contractile elements.     

Effect of palytoxin on the calcium current and the mechanical activity of frog heart muscle.

1. The effect of palytoxin (PTX) on the Ca current (ICa) and the mechanical activity of frog atrial fibres was studied by use of the double sucrose gap voltage clamp technique. 2. In normal Ringer solution, PTX transiently increased the electrically-evoked peak tension which then decreased while a major contracture developed. PTX slowed the time course of the relaxation phase of the evoked tension. 3. Evidence is presented which suggests that the toxin also increased the entry of Ca and Sr via the Na-Ca exchange mechanism. It also induced the development of a Ca-dependent outward current which was inhibited by Sr. 4. In Na-free solution, PTX increased ICa and shifted the reversal potential for Ca towards more negative membrane potentials, thus suggesting that the internal Ca concentration had increased. Current-voltage, tension-voltage, time to peak-voltage and inactivation time constant-membrane potential curves were all shifted towards more negative membrane potentials in the presence of PTX. 5. These effects of PTX are similar to those caused by the increase in internal Ca concentration induced by Na ionophores by way of voltage-dependent Ca influx of the Na-Ca exchange mechanism.     

Effects of procaine and lignocaine on electrical and mechanical activity of smooth muscle of sheep carotid arteries

1 Procaine in concentrations up to 20 mM facilitated or induced electrical and mechanical activity in arterial smooth muscle, even with tetrodotoxin present.2 Procaine (up to 20 mM) caused relaxation when electrical activity was blocked by prior potassium depolarization.3 Procaine (1 mM) reduced mechanical responses to noradrenaline (1 muM) which were not accompanied by action potentials. It generally reduced mechanical responses to noradrenaline (1 mM) while increasing electrical activity induced by this.4 Lignocaine (1 mM) did not facilitate electrical activity significantly; it relaxed arteries in saline or potassium-rich solution and reduced mechanical responses to noradrenaline.5 High concentrations of procaine (at least 80 mM) or lignocaine (at least 20 mM) blocked electrical activity and caused contraction followed by relaxation and complete unresponsiveness.     

The bradykinin potentiating activity of two pentapeptides on various isolated smooth muscle preparations.

Two bradykinin potentiating peptides A-VI5 (Val-Glu-Ser-Ser-Lys) and BPP5a (Pyr-Lys-Trp-Ala-Pro), were compared with respect to their potentiation of a number of different bradykinin effects on six isolated smooth muscle preparations. Apart from the considerable difference in effective concentrations, no essential qualitative difference was observed between these two peptides. Therefore the assumption of a different mechanism of action for the two peptides, which have a completely different structure, could not be substantiated.     

Effects of isoquinoline derivatives, HA1077 and H-7, on cytosolic Ca level and contraction in vascular smooth muscle

The effects of isoquinoline derivatives, HA1077 (1-[5-isoquinolinesulfonyl]-homopiperazine) and H-7 (1-[5-isoquinoline-sulfonyl]-2-methylpiperazine), on cytosolic Ca²⁺ levels ([Ca²⁺]_i) and muscle tension were examined in vascular smooth muscle of rat aorta. High K⁺ (72.7 mM) and norepinephrine (1 μM) induced a sustained contraction with a sustained increase in [Ca²⁺]_i. HA1077 and H-7 (3–10 μM) inhibited the increse in muscle tension more strongly than the increase in [Ca²⁺]_i. Verapamil (10 μM) completely inhibited the increase in [Ca²⁺]_i and the contraction induced by K⁺ whereas it inhibited the increase in [Ca²⁺]_i more strongly than the contraction due to norepinephrine. The verapamil-insensitive portion of the norepinephrine-induced contraction was inhibited by HA1077 or H-7. In Ca²⁺-free solution, 0.1 μM norepinephrine induced a transient increase in [Ca²⁺]_i and muscle tension. The transient contraction was inhibited by 10 μM HA1077 or 10 μM H-7 without inhibiting the increase in [Ca²⁺]_i. 12-Deoxyphorbol 13-isobutyrate (DPB) (1 μM) caused a sustained contraction, and this contraction was inhibited by HA1077 and H-7 at similar concentrations needed to inhibit the contractions induced by high K⁺ or norepinephrine. In rabbit mesenteric artery permeabilized with Staphylococcus aureus -toxin, 100 μM HA1077 and 100 μM H-7 inhibited the contraction induced by 0.3 μM Ca²⁺. These results suggest that the inhibitory effects of isoquinoline derivatives, HA1077 and H-7, are due to a decrease in [Ca²⁺]_i and in the Ca²⁺ sensitivity of contractile elemenst in vascular smooth muscle.     

Effect of endothelin on angiotensin converting enzyme activity in cultured vascular smooth muscle cells

We evaluated the effect of endothelin-1(ET) on the angiotensin converting enzyme (ACE) activity in rat aortic smooth muscle cells (VSMCs). ACE activity was determined by radioimmunoassay of the amount of angiotensin II generated after the addition of angiotensin I (500 pg/ml) to cultured VSMCs. The antibody used had less than 0.1% cross-reactivity with angiotensin 1. ACE activity increased 1.9-fold 5 hr after the addition of 10⁻⁶ M ET under serum-free conditions. This stimulatory effect of ET on ACE activity in VSMCs was completely inhibited by 10^-7 M captopril. Results suggested that the ACE present in SMCs is stimulated by ET.     

Effects of calcium antagonists on smooth muscle membranes of the canine stomach

In circular muscles of the canine stomach, diltiazem (greater than 3 X 10(-6)M) depolarized and nicardipine (greater than 10(-6)M) hyperpolarized the membrane. Diltiazem (greater than 5 X 10(-6)M) or nicardipine (greater than 3 X 10(-7M) inhibited the plateau potential of spontaneously generated or electrically evoked action potential. In Na-deficient solution (between 137 and 30 mM Na), diltiazem and nicardipine selectively inhibited the plateau potential. In Na- (15 mM Na) or Ca-deficient (0.25 mM Ca) solution, spontaneously generated action potential ceased, and only graded responses were evoked by electrical stimulation. These graded responses were inhibited by diltiazem (greater than 5 X 10(-6)M) or nicardipine (greater than 3 X 10(-7)M). The ionic contribution for generation of action potential in this muscle cell was discussed in relation to actions of Ca antagonists.     

The activity of phosphorothioate analogues of ATP in various smooth muscle systems.

Phosphorothioate analogues of adenosine 5'-triphosphate (ATP) have been tested on the rat and guinea-pig vas deferens, the guinea-pig taenia coli and urinary bladder. Adenosine 5'0-(2-thiotriphosphate) (ATP beta S) was more active than adenosine 5'0(1-thiotriphosphate) (ATP alpha S) and ATP in producing contractile responses on the vas deferens of rat and guinea-pig, and guinea-pig bladder, though the difference of potency was less marked for producing relaxation of the carbachol-contracted taenia coli. No differences were observed between the A and B diastereoisomers of ATP alpha S or ATP beta S. Contractions of the vas deferens produced by ATP alpha S were of much longer duration than those produced by ATP beta S. When tested against electrically-evoked twitch responses of the vas deferens the order of potencies was reversed with ATP being most active and ATP beta S least active. These inhibitory effects were blocked by 8-phenyl-theophylline. The calculated pA2 values for ATP, adenosine, beta, gamma-methylene ATP (APPCP) and ATP alpha S were similar, suggesting a common site of action. The results do not reveal any stereoselectivity among the tissues tested, for the diastereoisomers of ATP phosphorothioates; the observed differences of potency may be due to differences between ATP alpha S and ATP beta S in their rates of metabolism to adenosine. The different response profiles to the phosphorothioates may however reflect some differences of receptor mechanisms.