Calcium-induced Contractions Research Articles

Calcium-induced contraction and contractile protein content of gallbladder smooth muscle after high-cholesterol feeding of prairie dogs

Feeding a high-cholesterol diet to prairie dogs causes a reduction in contractile responses of gallbladder smooth muscle from these animals. In this study, the influence of cholesterol feeding on the contractile response to calcium and on the concentration of the contractile proteins actin and myosin was determined. Strips of gallbladder smooth muscle, at their optimal length for tension development, were stimulated maximally with carbachol. Then the muscle cell membranes were made permeable and the strips were exposed to a maximally effective concentration of calcium. Strips from cholesterolfed animals developed less stress than those from control animals under all conditions. The concentration of actin and myosin was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There were no differences in the concentrations of either protein between the cholesterol-fed and control animals. Our results indicate that the decreased contractile response seen in muscle from cholesterol-fed animals is not due to alterations in membrane excitation, excitation-contraction coupling, or in total contractile proteins.

Calcium antagonists inhibit positive chronotropic responses to α1-adrenoceptor activation in rat isolated atria

Positive chronotropic responses of rat isolated atria to phenylephrine were reduced by propranolol (0.3 μM) and the residual response was further depressed by the selective α 1-adrenoceptor antagonist prazosin (1 nM) but not yohimbine (10 nM), confirming that a component of the response to phenylephrine was due to activation of α 1-adrenoceptors. When β-madrenoceptors were blocked by propranolol, the positive chronotropic response to phenylephrine was enhanced by increasing the calcium concentration and by the calcium channel activator Bay K 8644 (0.1 μM), whereas the response was decreased by lowering the calcium concentration and by the calcium antagonists verapamil (10 nM), nifedipine (10 nM) and diltiazem (100 nM). In the presence of prazosin, when phenylephrine acts only on β-adrenoceptors, calcium antagonists had no effect on the response. In rat isolated aortic strips in a calcium-free, high K + (40 mM) solution, verapamil (10 nM), nifedipine (10 nM) and diltiazem (100 nM) shifted the calcium-induced contraction curves to the right, but prazosin (10 nM) had no effect, indicating that it is not a calcium antagonist. The calcium antagonists in the concentrations stated above had no effect on phenylephrine-induced contractions of rat aortic strips in normal Krebs-Henseleit solution, indicating thay they did not block α 1-adrenoceptors in these concentrations. Taken together, these data suggest that the positive chronotropic effect of phenylephrine resulting from activation of α 1-adrenoceptors involves an increased influx of calcium through channels that are sensitive to organic calcium antagonists.

Dependence on ionic strength of the calcium-induced contraction of glycerinated stalk of the peritrich ciliate, Vorticella convallaria

1. 1. The dependence on ionic strength of the calcium-induced contraction of glycerinated stalks of the protozooan peritrich ciliate, Vorticella convallaria, has been investigated. 2. 2. With increasing ionic strength (KC1) in the reaction solution, the calcium concentration (Ca 2+) m, at which the fractional length of the stalk becomes 0.5, increases monotonically. 3. 3. The logarithm of (Ca 2+) m, can be expressed as a linear function of the logarithm of the activity coefficient of KC1 over a wide range of KC1 concentrations. 4. 4. The Hill constant, n, which is the number of calcium ions bound simultaneously and cooperatively to a contractile element for spasmoneme contraction, is about 2, independent of ionic strength. 5. 5. Our observations are consistent with the suggestion that the calcium binding site in a contractile element of the spasmoneme is negatively charged and is probably composed of carboxylic amino acid residues of an acidic calcium-binding protein.

Effect of nifedipine on calcium-induced contractions to potassium in the aorta and mesenteric arteries of spontaneously hypertensive and normotensive rats.

Graded contractions to cumulative additions of calcium in the presence of KCl were obtained in strips of aorta and mesenteric arteries of normotensive (WKY) and spontaneously hypertensive (SHR) rats. In calcium-free medium, a maximally effective concentration of KCl produced a response that was larger in the mesenteric arteries (43-51% of control) than in the aorta (12-14% of control). The calcium channel blocker nifedipine (NFD, up to 10(-7) M) did not significantly alter these calcium-insensitive responses. The Ca2+-induced responses were inhibited by NFD, in a concentration-dependent fashion, in both vessel types of WKY and SHR rats. The aortic responses were more sensitive to inhibition by NFD than the responses of mesenteric arteries. Moreover, the aortic responses of WKY were inhibited to a greater extent than those of the SHR. The results suggest: (a) a differential calcium dependence of contractions to KCl in the vessels studied; (b) that aortic responses are dependent on NFD-sensitive voltage-sensitive Ca2+ channels to a greater extent than the responses of mesenteric arteries; and (c) that hypertension results in a decreased sensitivity of the aorta Ca2+ channels to NFD.

Pharmacological actions of tannic acid. I. Effects on isolated smooth and cardiac muscles and on blood pressure.

Tannins are common constituents of medicinal plant crude extracts and have been reported to have pharmacological actions of their own. Studies on these actions are likely to be helpful in interpreting data obtained with tannin-rich crude plant extracts. In this context, the present report analyses the effects of tannic acid on responses of the isolated rat uterus and vas deferens to agonists and left atrium to electrical stimulation, as well as on blood pressure in anesthetized rats. Tannic acid dose-dependently and non-competitively antagonized contractions to different agonists in rat uterus and vas deferens. The compound produced a biphasic effect in the atrium. Whereas lower concentrations (30 to 100 µg/ml) enhanced inotropism, higher concentrations (> 300 µg/ml) promoted depression of contractility. Intravenous tannic acid led to hypotension, which was partially reduced by antihistamine pretreatment, but did not affect heart rate. The depressant effect of tannic acid in uterine fragments was potentiated by increasing calcium concentration in the bathing solution, although in the depolarized strips the compound non-competitively antagonized calcium-induced contractions. The results suggest that tannic acid can affect calcium availability for contraction of smooth and cardiac muscles. This action could well mask the effects of other active constituents of tannic-rich plant extracts.

Some properties of calcium-induced contraction in the isolated human and guinea-pig tracheal smooth muscle

We studied the effects of the cyclooxygenase inhibitors, indomethacin and aspirin, the leukotriene antagonist, FPL 55712 and the Ca antagonist, verapamil on basal tone and Ca-induced contractions in the human and guinea-pig tracheal muscle. Indomethacin and aspirin usually increased spontaneous tone or Ca-induced contractions in human strips, while consistently decreased tension development in the guinea-pig muscle. FPL 55712 strongly reduced contractions in the human, whereas it had a very weak effect on the guinea-pig muscle. Verapamil had a small inhibitory effect in the human trachea, either at 5.9 or 40 mM K, but markedly suppressed Ca-induced contractions of guinea-pig trachea in 40 mM K and had little effect on contractions in 5.9 mM K in this tissue. It is concluded that active tone in the two different tracheal muscles were controlled by different processes; that different arachidonate by-products may exert an effect on basal tone in the two different muscles; and that different plasma membrane Ca channels may be operative under basal conditions in the different muscles.

Characterization of the prostanoid receptors and of the contractile effects of prostaglandin F2 alpha in human pial arteries.

The contractile and relaxant effects of various prostanoids were studied on isolated human pial arteries. Contractions were elicited with the following order of potency: U46619 approximately equal to U44069 greater than PGB2 greater than PGF2 alpha greater than PGE2 approximately equal to PGD2 approximately equal to PGF1 alpha greater than or equal to TXB2, indicating that prostanoid-induced contractions probably are mediated by a thromboxane-sensitive receptor. Relaxation of PGF2 alpha-contracted arteries was induced with the order of potency: PGE2 greater than PGE1 greater than PGD2 approximately equal to PGD1. Vessels contrated by K+ were relaxed only by PGE1. Since PGI2 was previously found to be more potent than all the prostanoids tested in the present study, relaxant responses are probably mediated via a PGI2-sensitive receptor. The role of free extracellular and cellularly bound calcium for the contractile effects of PGF2 alpha and K+ were estimated by incubating the arteries for various times in calcium-free medium containing 10(-5) M EGTA. Incubation for 5-10 min abolished K+-induced contractions, whereas after 40 min of incubation PGF2 alpha still induced contractions that reached 70% of control. The PGF2 alpha-induced contraction was biphasic in 8 out of 10 preparations. The second phase could be eliminated by increasing the EGTA-concentration to 10(-4) M, as well as by nifedipine pretreatment. In calcium-free, high K+ medium calcium-induced contractions were elicited at lower concentrations in the presence of PGF2 alpha. The results suggest that PGF2 alpha-induced contractions in human pial arteries are relatively independent of free extracellular calcium. PGF2 alpha may promote trans-membrane influx of calcium, as well as release calcium from seemingly superficially located cellular stores.

Role of soluble myosin in cortical contractions of Xenopus eggs.

The layer of cytoplasm underlying the plasmalemma of Xenopus eggs has contractile activity which is of vital importance in fertilization and early development, being involved in such processes as sperm engulfment, cortical granule exocytosis, development of the axes of embryonic symmetry and cleavage. In amphibian eggs this layer is also involved in wound healing and changes of cellular shape at gastrulation. Two kinds of contractile structures can be distinguished near the surface of Xenopus eggs. To characterize the mechanism and regulation of this contractile activity, we have experimentally induced cortical contractions in bisected living Xenopus eggs. We have shown previously that cortical contractions are induced by calcium ions in the bisected egg. Here we show that extraction of soluble cytoplasmic components prevents the calcium-induced contractions, but that addition of exogenous soluble myosin restores them. In oocytes, both soluble and insoluble components of the cortical cytoplasm are unable to support contraction. Thus, during meiotic maturation of oocytes into eggs, both of the components of the cortical cytoplasm must change so as to become competent for contraction.

PN 200–110, A New Calcium Antagonist

The compound isopropyl 4-(2,1,3- benzoxadiazol -4-yl)-1,4-dihydro-5-methoxycarbonyl-2,6-dim ethyl-3- pyrid inecarboxylate (code name PN 200-110 [PN]) was investigated for calcium antagonistic effects in experiments in vitro. Action potentials recorded with intracellular microelectrodes in guinea pig papillary muscles were changed little by PN, 10(-7) M, except for a slight shortening of the duration of the plateau phase. Slow action potentials elicited in partially depolarized papillary muscles were gradually diminished and finally blocked by this concentration of PN. Contractile force was diminished in normal and partially depolarized muscles. The rate of spontaneously beating guinea pig right atria was decreased dose dependently, and the EC25 was 4.5 x 10(-10) M. The EC25 for the negative inotropic effects measured on paced guinea pig left atria was 1.5 x 10(-8) M. No membrane-stabilizing effects were found. Calcium-induced contraction of rabbit aorta in depolarizing bath solution was inhibited with an apparent pA2 of 10.3. Contraction elicited by graded depolarization at a constant calcium concentration was inhibited with an EC50 of 1.4 x 10(-9) M. Under resting conditions PN did not alter net uptake of 45Ca2+. KCl-stimulated uptake was inhibited with an EC50 of 3.6 x 10(-9) M. Neither noradrenaline-induced contractions nor noradrenaline-stimulated net uptake of 45Ca2+ were inhibited by a concentration of PN as high as 10(-5) M. PN thus is selective on cardiac tissue with respect to negative chronotropic versus inotropic activity and on rabbit aorta with respect to potential-operated versus receptor-operated channels.

Skinned smooth muscle: time course of force and ATPase activity during contraction cycle.

The time course of ATPase activity and force has been determined during contraction and relaxation in skinned (hyperpermeable) anterior byssus retractor muscle, ABRM, of Mytilus edulis and compared with corresponding measurements on skinned taenia coli of guinea-pigs. Following a calcium-induced contraction, lowering the [Ca++] to 10(-8) M rapidly reduces ATPase activity within 2 min to resting levels while force declines only to about 30-50% of maximal tension within the same time. Thus slow relaxation is due to a 'catch-like-state' which is common to different kinds of smooth muscles and can be reduced with cAMP in ABRM and by Pi in taenia coli.

Relaxation of rabbit aorta by chlordimeform.

Chlordimeform N'-(4-choro-O-tolyl)-N,N-dimethyl-formamidine; CDF), an acaracide-insecticide, relaxed helically cut strips of rabbit thoracic aorta and deadventitiated aorta strips. Relaxations by CDF of aorta strips contracted by 5 x 10(-7) M prostaglandin F2 alpha were not altered by the antimuscarinic agent atropine (10(-5) M), the beta-adrenergic antagonist propranolol (10(-5) M), the histamine H2 antagonist cimetidine (2 x 10(-4) M), the dopamine antagonist haloperidol (10(-5) M) or the Na+-K+-ATPase inhibitor ouabain (5 x 10(-6) M). CDF further relaxed deadventitiated strips contracted by 10(-6) M NE or 40 mM potassium and then partially relaxed by 10(-5) M verapamil. CDF (10(-5) M) potentiated contractions caused by the addition of calcium to potassium depolarized strips in zero calcium media, but higher concentrations reduced calcium-induced contractions. The highest CDF concentration (10(-2) M) not only prevented tension increase on calcium addition, but also progressively decreased tension to below baseline levels. The addition of 10(-2) M CDF to aorta strips not contracted by a vasoactive agent decreased resting tension. This relaxation was usually maximal at 10(-3) M and less at 10(-2) M. Thus, CDF relaxes vascular smooth muscle, not by antagonism at the usual vascular relaxant receptors, but by interference with calcium utilization.

Effects of propranolol on coronary arteries

Possible pharmacological characteristics of propranolol in coronary arteries were investigated. Propanolol produced dose-dependent relaxations of isolated dog coronary arterial strips under potassium-contracture, which were restored or prevented by additional calcium. Propranolol-induced relaxations were similar to those by its isomers and significantly greater than those by procaine and lidocaine. Atenolol and practolol had no relaxant effect. Propranolol relaxed the isolated dog coronary arterial and colon preparations under potassium-contracture, but not under lanthanum-contracture. Propranolol dose-dependently inhibited calcium-induced contractions of isolated coronary arterial strips in high potassium medium. In isolated and perfused hearts of rats and guinea-pigs under pacing, propranolol induced dose-dependent decreases in coronary perfusion pressure. These findings suggest that propranolol has a possible calcium antagonistic action on arterial smooth muscles, resulting in a dilatation of coronary arteries.

Calcium-Induced Contraction and Effect of Calcium Antagonist on Arterial Smooth Muscle of Spontaneously Hypertensive Rats

1. Calcium reactivity of arterial smooth muscle and the effect of diltiazem (calcium antagonist) were investigated in helical strips of mesenteric arteries from 6-week-old male spontaneously hypertensive rats (SHR) and age-and sex-matched normotensive Wistar-Kyoto (WKY) rats. Blood pressure was 130 ± 5 mmHg for SHR and 110 ± 5 mmHg for WKY rats (means ± sd). 2. At high Ca2+ concentration (3 × 10−4 to 3 × 10−3 mol/l) the calcium-induced tension of mesenteric arterial strips per cross-sectional area was significantly greater in SHR than in WKY rats in noradrenaline (10−5 mol/l)-containing solution or in high K+ (121 mmol/l) solution (P < 0.001). At low Ca2+ concentration (10−5 mol of noradrenaline/l and 3 × 10−5 mol/l in high K+), the tension was smaller in SHR strips than in WKY rat strips (P < 0.05). 3. The Ca2+ sensitivity of SHR strips was significantly lower than that of WKY strips in noradrenaline-containing solution or in high K+ solution. 4. Diltiazem, a calcium antagonist, shifted the dose-response curve of Ca2+-induced tension to the right equally in SHR and WKY rat strips. The inhibition of the maximum tension by the drug was greater in SHR strips than in WKY rat strips in noradrenaline-containing solution, but there was no difference in high K+ solution. 5. The results suggest that the contractile abnormality might be a possible cause of hypertension in SHR.

PY 108-068, a new, potent, and selective inhibitor of calcium-induced contraction of rabbit aortic rings.

PY 108-068 (PY) is a new benzoxadiazolyle dihydropyridine derivative. It potently antagonizes calcium-induced contractions of rabbit aorta in depolarizing solution (PD'2:8.8). This antagonism is selective, since no relevant antagonistic effects against noradrenaline (NA)-, serotonin (5-HT)-, and angiotensin II (AII)-induced contractions are found at the highest concentration of PY (10(-5) M). Verapamil (V), examined for comparison, was less selective with respect to its antagonism: pA2 value against calcium-induced contractions in depolarizing solution: 7.6, pA2 against serotonin-induced contractions: 6.9. In calcium-free Krebs--Henseleit solution tachyphylaxis occurred after three to four administrations of NA, 5-HT, or AII. When calcium was added in the presence of one of these agonists, the slow phase of concentration, which depends on extracellular calcium, developed. This slow phase of contraction was not inhibited to a relevant extent by a 10(-5) M concentration of PY. Verapamil inhibited the tonic contraction induced by serotonin (pD'2: 5.5) but not to a relevant extent the contractions induced by the two other agonists. PY therefore, inhibits calcium-induced contractions of rabbit aorta in depolarizing solution very selectively, and shows hardly any effects on receptor-operated channels.

The effects of chronic morphine administration to mice on the contractile activity in vitro of the vas deferens without and with morphine

Chronic treatment of mice with morphine enhances maximal responses to noradreneline in isolated vasa deferentia. Addition of morphine to the bath further increases the responses to noradrenaline (facilitatory effect). Vasa deferentia from reserpine-pretreated mice showed increased responses to noradrenaline, but upon addition of morphine to the bath no further facilitatory response to noradrenaline. The facilitatory response was not inhibited by naloxone and it was not elicited by narcotic analgesics other than morphine. High calcium in the medium (3.8 mM) suppressed the enhanced maximal responses to noradrenaline of vasa deferentia from mice chronically treated with morphine and also suppressed the facilitatory response to noradrenaline. Low calcium in the medium (1.43 mM) had the opposite effects. Reserpine-induced changes of noradrenaline responses were not affected by alteration of the calcium concentration in the medium. In the K-depolarized vas deferens, a reduction of the depressant effect of morphine on the calcium-induced contractions was observed after chronic administration of morphine.

Pharmacological studies on supersensitization. VI. Effect of cocaine on the utilization of calcium in acetylcholine-induced contraction of isolated vas deferens of guinea pig.

Effects of cocaine on the isotonic contractions of isolated intact and glycerinated vas deferens of guinea pig were examined. Cocaine potentiated the contractile responses of vas deferens to acetylcholine and potassium chloride in standard and calcium-free Tyrode solution. Cocaine also augmented the maximum response of calcium-induced contraction of partially depolarized vas deferens. The contractile responses to potassium chloride in standard Tyrode solution were completely abolished by lanthanum chloride, while the contractile responses to acetylcholine in standard Tyrode solution were attenuated by lanthanum chloride and the residual contractions were not affected by cocaine. The half-time of decrease in acetylcholine-induced contraction in calcium-free Tyrode solution was significantly different from that of potassium-induced contraction in calcium-free Tyrode solution. Cocaine did not affect the contractile response of the glycerinated muscle piece of vas deferens. These results suggest that cocaine facilitates the transmembrane influx of calcium from extracellular fluid and/or superficial calcium-binding sites without affecting the release of calcium from intracellular calcium-binding sites.

Overview on the pharmacological and toxicological characterization of the calcium antagonist tiapamil.

The results obtained in a variety of isolated organ preparations characterize tiapamil as a calcium antagonist or calcium entry blocker. Tiapamil inhibited in a concentration-dependent manner calcium-induced contractions in isolated, potassium-depolarized preparations of rat renal artery, dog coronary artery and rabbit main pulmonary artery and reduced ⁴⁵Ca influx into depolarized vascular smooth muscle cells. The inhibitory effects of tiapamil were surmountable by an elevation of the extracellular calcium concentration. Calcium-mediated, slowly rising potentials in partially depolarized guinea pig papillary muscle, evoked by electrical stimulation in the presence of isoprenaline, were also antagonized by tiapamil. As in vascular muscle, the tiapamil effects in cardiac muscle were reduced by elevation of external calcium.Tiapamil altered haemodynamic parameters in a characteristic sequence, with an increase in coronary flow appearing at the lowest effective doses, followed by decreases in blood pressure, total peripheral vascular resistance and, eventually, heart rate. A depression of myocardial contractility was not found at doses which are considered to be therapeutic. After cardiac autonomic denervation, tiapamil produced a dose-dependent bradycardia. However, cardiac denervation did not alter the extent of tiapamil-induced decreases in coronary vascular resistance and did not unmask a potential negative inotropic effect of the drug. A comparison of various vascular preparations suggested a certain degree of selectivity for coronary arteries in the calcium antagonistic action of tiapamil. The haemodynamic findings are in line with these in vitro results.A coronary segmental analysis revealed that tiapamil acts on small coronary arteries. It differs in this respect from nitroglycerin which dilates large coronary arteries. Tiapamil increased pO₂ in the subendocardial layers of the myocardium percentagewise more than in the subepicardial layers. Furthermore, it reduced experimentally-induced myocardial ischaemia as determined by a reduction of the S-T segment elevation of the epicardial ECG after ligation of a coronary artery.

Interactions of magnesium and verapamil on tone and contractility of vascular smooth muscle

Previous studies on isolated blood vessels indicate that (1) withdrawal of magnesium ([Mg 2+] 0) induces calcium-dependent contractile responses, and (2) [Mg 2+] 0 and verapamil (VE) inhibit calcium influx across the cell membrane. The present study, using isolated rat aortic strips and portal veins, was designed to assess the interactions of [Mg 2+] 0 and VE on increases in active tension and contractility induced by withdrawal of [Mg 2+] 0. [Mg 2+] 0 was found to: (1) enhance VE-induced inhibition of portal vein amplitude, and (2) antagonize VE-induced enhanced frequency of spontaneous phasic contractions in portal veins. Both [Mg 2+] 0 and VE could prevent and reverse the increases in active tension developed in aortic smooth muscle upon removal of [Mg 2+] 0. [Mg 2+] 0 markedly potentiated the inhibitory effect of verapamil on calcium-induced contractions of K +-depolarized aorta but not in K +-depolarized portal vein. This synergistic effect in aorta could be due to the influence of Mg and VE at the membrane and/or the influence of Mg at intracellular sites resulting in decreased levels of functional (activator) cytoplasmic Ca 2+. On the other hand, the lack of synergism in portal vein could indicate that both of these agents might be inactivating the same membrane channels involved in calcium influx.

Effects of Ad Libitum, Maintenance and Sub-Maintenance Feeding and of Compensatory Growth on some Biochemical Properties of Muscle from Weanling Rabbits

Weanling male rabbits were fed either: 1) a complete diet ad libitum (control); 2) a maintenance diet; or 3) a sub-maintenance diet. After 20 days, half of each group was slaughtered and the remainder was placed on treatment 1 until they achieved the same body weights as the controls (about 30 more days), when they were also killed. Body organs, including livers, hearts, lungs and kidneys, were significantly reduced in weight by nutritional stress. Maintenance and sub-maintenance feeding resulted in increases in ultimate muscle pH, water content and alkali-soluble and -insoluble stromal proteins. The intracellular proteins, especially the myofibrillar fraction, decreased markedly, depending on the degree of nutritional stress. Swelling of the stromal proteins was less for the underfed rabbits, indicating that nutritional stress increased the number of acid-stable cross-linkages. Calcium-induced contraction was not effected by any of the nutritional treatments. Blood enzymes were not affected by nutritional stress from the standpoint of their multiple molecular forms, mobility or band profiles. All differences in muscle characteristics disappeared during compensatory growth except for the increased amount of alkali-insoluble stroma protein. The concepts of labile, mobilizable and fixed body proteins are discussed.sub-maintenance maintenance protein depletion muscle protein

Antagonism of calcium-induced contraction in potassium-depolarized rabbit detrusor muscle strips by dicyclomine hydrochloride and rociverine.

The musculotropic action of dicyclomine hydrochloride in bladder muscle has been attributed to its local anesthetic activity. In this study rabbit detrusor strips were depleted of calcium by incubation in Ca2+-free solution containing ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). Contractions elicited by replacement of Ca2+ during depolarization with 80 mM K+ were only slightly affected by atropine or scopolamine but were antagonized in a noncompetitive manner by dicyclomine and rociverine (pD2' = 4.89 and 4.61, respectively). These contractions were also blocked with greater potency by nifedipine (pD2' = 8.50) and with lesser potency by lidocaine (pD2' = 2.52). Procaine was ineffective up to 1 mM. Comparison of the antagonism produced by dicyclomine and rociverine with that produced by nifedipine or lidocaine did not help to define the mechanism of the musculotropic drugs. However, as rociverine is not a local anesthetic and procaine was not effective, it appears that the "local anesthetic" property is not sufficient to explain the action of these musculotropic drugs.