External calcium dependence of the uterine contraction induced by prostaglandins E2 and F2 alpha and its antagonism with natural progestins.

Prostaglandins (PGs) E2 and F2 alpha are strong inducers of uterine contraction by promoting a Ca2+ increase into the cell through specific receptors coupled with the calcium channels. On the contrary, progesterone and 5 beta-reduced progestins promote smooth muscle relaxation by blocking the ion calcium influx. Thus, this study was designed to emphasize the importance of external calcium in the PGs-induced rat uterus contraction. Likewise, also studied was the antagonism and the interaction between PGs and progestins (progesterone and its 5 alpha and 5 beta-reduced derivatives) in the myometrium. Results showed that uterine contraction induced by PGs depends on external calcium, since verapamil or extracellular calcium depletion abolished the PGs effect. Regarding the PGs-progestins antagonism, it was observed that pregnanedione, pregnanolone and epipregnanolone were quite effective for counteracting of PGs-induced contraction. However, progesterone was effective in a middle range, whereas 5 alpha-reduced progestins (allopregnanedione and allopregnanolone) were almost ineffective. It has been concluded that the participation of PGs and progestins in the modulation of uterine contraction might be achieved through the control of calcium influx by opening (PGs) or blocking (progestins) receptor-operated calcium channels.     

External calcium dependence of the uterine contraction induced by prostaglandins E2 and F2α and its antagonism with natural progestins

Prostaglandins (PGs) E₂ and F2α are strong inducers of uterine contraction by promoting a Ca²⁺ increase into the cell through specific receptors coupled with the calcium channels. On the contrary, progesterone and 5β-reduced progestins promote smooth muscle relaxation by blocking the ion calcium influx. Thus, this study was designed to emphasize the importance of external calcium in the PGs-induced rat uterus contraction. Likewise, also studied was the antagonism and the interaction between PGs and progestins (progesterone and its 5α and 5β-reduced derivatives) in the myometrium. Results showed that uterine contraction induced by PGs depends on external calcium, since verapamil or extracellular calcium depletion abolished the PGs effect. Regarding the PGs-progestins antagonism, it was observed that pregnanedione, pregnanolone and epipregnanolone were quite effective for counteracting of PGs-induced contraction. However, progesterone was effective in a middle range, whereas 5α-reduced progestins (allopregnanedione and allopregnanolone) were almost ineffective. It has been concluded that the participation of PGs and progestins in the modulation of uterine contraction might be achieved through the control of calcium influx by opening (PGs) or blocking (progestins) receptor-operated calcium channels.     

Dependence of Ca outflow and depression of frog myocardium contraction on ryodipine concentration

The effect of ryodipine on calcium outflow from tissues, on contraction force, the duration of action potentials and the relaxation phase time-constant in the contraction cycles of myocardial strips was studied using frog heart preparations. It was found that calcium outflow (delta Ca) as a function on ryodipine concentration can be represented as: (formula; see text) A linear correlation exists between Ca2+, contraction blocking and the shortening of the action potential in the presence of various ryodipine concentrations. Ryodipine (10(-5) mol/l) decreased the relaxation time-constant by about 20% as compared to controls. It was concluded that calcium outflow from myocardial tissues in response to ryodipine is due to blockade of calcium entry into the cells and their output through the Na+--Ca2+ exchange system. Frog heart myocardial contractions are essentially under the control of calcium entry through sarcolemmal calcium channels.     

Effects of temperature and Na0+ on the relaxation of phasic and tonic tension of guinea-pig ureter muscle

Effects of temperature and Na0+ on the relaxation of guinea-pig ureter smooth muscle were studied. Relaxation of phasic contraction was found to be highly temperature-dependent, practically independent of Na0+ and Ca02+, and resistant to vanadate. The relaxation of the tonic tension of both high-K and low-Na contracture was less temperature-dependent and affected by Na0+. The relaxation of tonic tension produced by introduction of Na0+ was about 3-5 times faster than that produced by Ca-free solution. La3+ ions were found to block the relaxation of the tonic component of the Na+-free contracture initiated by removal of Ca02+. Three systems of regulation of cell calcium are suggested to be operative in the ureter muscle: a fast one which is highly temperature-dependent and responsible for the relaxation of the phasic contraction (probably the sarcoplasmic reticulum), and two slow membrane-linked carriers, one of which is dependent on Na0+ (probably Na-Ca exchange) and another one which is independent of Na0+ and inhibited by La3+ (probably Ca-pump).     

Mechanism of the effect of dibasol on the contractile and electric activities of the smooth muscle of the portal vein

The effects of dibasol on spontaneous electrical and contractile activities as well as on the reactions evoked by hyperkalemic solution and noradrenaline were studied in smooth muscle of rabbit portal vein. It was shown that dibasol blocked the potential-operated influx Ca2+ into smooth muscle cells. The noninactivating calcium channels were found to be more sensitive to dibasol than inactivating ones. Significant part of the tonic contraction induced by noradrenaline was resistant to dibasol suggesting its weak effect on Ca2+ influx through calcium channels operated by alpha 1-adrenoceptors. It is supposed that vasodilative effect of dibasol is associated with blocking the influx Ca2+ through potential-operated noninactivating calcium channels into smooth muscle cells.     

The vasodepressor effect of androgens in pithed rats: potential role of calcium channels

Estrogens induce vasodilatation and/or hypotension in several experimental models, probably by a blockade of calcium currents. However, very little is known about the potential cardiovascular effects of androgens. We have previously shown that 5 beta-reduced androgens are more potent vasorelaxants than their precursors (delta 4-3 keto), 5-reduced progestins and 17beta-estradiol. The present study set out to investigate if this vasorelaxant effect of 5-reduced androgens is operative in vivo in the analysis of the potential vasodepressor effect of these compounds in vagosympathectomized, pithed rats. After increasing diastolic blood pressure (DBP) by a continuous infusion of norepinephrine (0.059 micromol x kg(-1)min(-1)), i.v. bolus injections of 3 alpha-hydroxy-5 beta-androstan-17-one (etiocholanolone), 5 beta-dihydrotestosterone (5 beta-DHT), and its isomer 5 alpha-dihydrotestosterone (5 alpha-DHT) (5-25 micromol x kg(-1) each) produced, separately, dose-dependent vasodepressor responses. These responses were biphasic: an immediate fall in DBP (reaching the nadir within 1.7 min) was followed by a further slow decrease that reached a maximum between 80 and 100 min after steroid administration. The order of potency of androgens in decreasing DBP was: 5 beta-DHT>5 alpha-DHT=etiocholanolone for the short-lasting response and 5 alpha-DHT>5 beta-DHT>or=etiocholanolone for the longer lasting response. Importantly, the same doses of these compounds produced no significant changes in heart rate. Moreover, 5 beta-DHT significantly antagonized the vasopressor responses to methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluromethylphenyl)-pyridine-5-carboxylate (Bay K 8644) with a blocking profile similar to that of nifedipine (NIF). This finding suggests that a blockade of voltage-operated calcium channels may be involved in androgen-induced hypotension.     

Influence of 5alpha- and 5beta-reduced progestins on the contractility of isolated human myometrium at term.

It is very well known that progesterone induces uterine relaxation on myometrium contractile activity. However, little attention has been paid to the effect induced by its metabolites on human uterine contractility. Therefore, we set out to analyze the potential relaxing effect of some 5alpha- and 5beta-reduced progesterone derivatives on the spontaneous contractility of myometrium from pregnant women. Samples were obtained by caesarian section at 38-40 weeks of pregnancy. Spontaneous uterine contractions were recorded in vitro in the presence of progesterone, or progestins independently, at different non-cumulative microM concentrations. The progestins elicited an immediate relaxing effect that was concentration-dependent. With the exception of two 5alpha-reduced progestins (5alpha and 3beta,5alpha), the remaining progestins used in the present study were more potent than progesterone. The potency order with respect to their IC50 values was: 3alpha,5alpha (35 microM) > 5beta (81 microM) > 3beta,5beta (156 microM) > 3alpha,5beta (205 microM) > P4 (225 microM) > 5alpha (19 mM) > 3beta,5alpha (28 mM). When tissues were washed, the contractile activity was recovered. This rapid and reversible relaxing effect was not blocking by antiprogestin RU 486, suggesting that is not through receptor-mediated genomic action. The metabolites from progesterone may also determine the pattern of motility, ensuring the necessary quiescent environment to prevent abortion during gestation.     

Vasodilatory action mechanisms of apigenin isolated from Apium graveolens in rat thoracic aorta.

The effect of apigenin, isolated from Apium graveolens, on the contraction of rat thoracic aorta was studied. Apigenin inhibited the contraction of aortic rings caused by cumulative concentrations of calcium (0.03-3 mM) in high potassium (60 mM) medium, with an IC50 of about 48 microM. After pretreatment it also inhibited norepinephrine (NE, 3 microM)-induced phasic and tonic contraction in a concentration (35-140 microM)-dependent manner with an IC50 of 63 microM. At the plateau of NE-induced tonic contraction, addition of apigenin caused relaxation. This relaxing effect of apigenin was not antagonized by indomethacin (20 microM) or methylene blue (50 microM), and still existed in endothelial denuded rat aorta or in the presence of nifedipine (2-100 microM). Neither cAMP nor cGMP levels were changed by apigenin. Both the formation of inositol monophosphate caused by NE and the phasic contraction induced by caffeine in the Ca(2+)-free solution were unaffected by apigenin. 45Ca2+ influx caused by either NE or K+ was inhibited by apigenin concentration-dependently. It is concluded that apigenin relaxes rat thoracic aorta mainly by suppressing the Ca2+ influx through both voltage- and receptor-operated calcium channels.     

17beta-estradiol inhibits calcium-dependent and -independent contractions in isolated human saphenous vein

Studies suggest that estrogen modulate vascular reactivity but at present its exact mechanism of action has yet to be clarified. The aim of this study was to evaluate the effect of 17beta-estradiol (E2) on calcium-dependent and -independent contractions induced in the human saphenous veins (HSVs). HSVs were obtained from patients undergoing coronary artery bypass graft surgery. The ability of E2 to modulate Ca(2+) entry was assessed by obtaining concentration-response curve to CaCl(2) in the absence or presence of E2. In other experiments intracellular Ca(2+) was depleted by repeated application of phenylephrine in the presence of cyclopiazonic acid (CPA). Then, at the plateau of PGF(2alpha) contraction, E2 or nifedipine (NIF) was added. Involvement of protein kinase C (PKC) in relaxant effect of E2 was evaluated by application of phorbol-12,13-dibutyrate (PDBu) in normal or Ca(2+)-free Krebs' solution. When the contraction was obtained, E2 or NIF was added. In Ca(2+)-free hyperpolarizing solution, pretreatment with E2, concentration dependently reduced contractions induced by cumulative addition of calcium chloride. Furthermore, E2 elicited relaxant effects on the PGF(2alpha)-induced contractions in Ca(2+)-free solution in the presence or absence of CPA. Both E2 and NIF produced significant relaxation in HSV rings contracted by direct activation of PKC in Krebs' solution. However, in Ca(2+)-free solution, NIF failed to induce relaxant effect but E2 kept its effect on the PDBu-induced contraction. These results suggest that the relaxant effect of E2 on HSV is elicited by calcium-dependent and -independent pathways. The calcium-independent pathway may involve PKC inhibition.     

Effect of beta-adrenergic stimulation on uterine contraction in response to arginine vasotocin and prostaglandin F2 alpha in the gecko Hoplodactylus maculatus.

The effects of beta-adrenergic stimulation on uterine contractions occurring in response to arginine vasotocin (AVT) and prostaglandin F2 alpha (PGF2 alpha) were compared during late pregnancy in the viviparous gecko Hoplodactylus maculatus. High doses of AVT (150 or 1,500 ng/g body weight) induced birth in vivo, but PGF2 alpha at doses of up to 2,000 ng/g did not induce birth. The effect of AVT (150 ng/g) on birth rate in vivo was not enhanced by pretreatment 20 min beforehand with the beta-adrenoreceptor antagonist dichloroisoproterenol (2 micrograms/g), whereas the effect of PGF2 alpha (200 ng/g) was markedly enhanced: geckos treated with dichloroisoproterenol and then with PGF2 alpha showed rapid birth-related behavior and gave birth. Isolated uteri showed a tonic contraction in response to AVT (100 ng/ml) and to PGF2 alpha (1,000 ng/ml). Pre-exposure of isolated uteri to the beta-adrenoreceptor agonist isoproterenol (1 microgram/ml) caused relaxation; this pre-exposure did not block the tonic contraction occurring in response to AVT, whereas it completely blocked the tonic contraction induced by PGF2 alpha. We conclude that in H. maculatus, beta-adrenergic stimulation inhibits uterine contractions induced by PGF2 alpha but not those induced by AVT. These data are the first to show that beta-adrenergic stimulation inhibits uterotonic responses to PGF2 alpha in a reptile, and they suggest that the cellular mechanisms by which AVT and PGF2 alpha induce contraction may differ in this species. They also provide further evidence for similarities between mammals and reptiles in the effects of beta-adrenergic stimulation on uterine relaxation.     

Effects of extracellular calcium on canine tracheal smooth muscle

Strips of canine tracheal smooth muscle were studied in vitro to determine the effects of changes in the extracellular calcium (Cao) concentration on tonic contractions induced by acetylcholine and 5-hydroxytryptamine. Strips were contracted with graded concentrations of the above agents in 2.4 mM Ca, after which CaCl2 was administered to achieve final concentrations of 5.0, 10.0, and 20.0 mM. Increases in Cao to 5 mM or above caused significant relaxation of muscles contracted with 5-hydroxytryptamine but did not significantly relax muscles contracted with acetylcholine. Increases in Cao also caused significant relaxation of muscles contracted with low concentrations of K+ (20 or 30 mM). However, in 60 or 120 mM K+, increases in Cao resulted predominantly in muscle contraction. Inhibition of the Na+-K+-ATPase by ouabain (10(-5) M) or K+ depletion reversed the effects of Cao from relaxation to contraction in tissues contracted with 5-hydroxytryptamine. Increases in Cao also caused contraction rather than relaxation in the presence of verapamil (10(-6) M). We conclude that calcium has both excitatory and inhibitory effects on the contractile responses of canine tracheal smooth muscle. The inhibitory effects of Ca2+ appear to be linked to the activity of the membrane Na+-K+-ATPase.     

Ca++ dependence of the contraction of coronary artery segments in response to norepinephrine after beta-adrenergic blockade

The effect of external calcium concentration on the NE-induced contraction after beta-adrenergic blocking was studied in vitro. It resulted that the effect of NE was enhanced by increase, or reduced by decrease of calcium concentration. NE-induced contraction was not abolished when the bathing fluid was Ca++-free. The disappearance of the NE effect was only obtained in preparations treated with EDTA and perfused with Ca++-free Ringer-Locke solution. It is concluded that NE induced contraction after beta-adrenergic blocking is Ca++-dependent and on the tissue bound Ca++.     

The influence of prostaglandin E2 (PGE2) on the acetylcholine-initiated contractions of isolated gastric muscularis muscle of Bufo marinus

Acetylcholine (ACh) (1.5 X 10(-5) M) elicited three different types of tonic and phasic contraction of muscularis muscle from different parts (cardiac, middle and pyloric) of the stomach of Bufo marinus. Prostaglandin E2 (PGE2) (10(-9)-10(-6) M) induced a concentration-dependent relaxation of tonic contractions elicited by ACh (1.5 x 10(-5) M) of strips from the cardiac part while potentiating the phasic contractions from the middle part of the stomach. PGE2 (10(-7) M) relaxed tonic contraction and potentiated phasic contraction concomitantly in preparations in which tonic and phasic contractions were elicited by ACh (1.5 x 10(-5) M). The effects of PGE2 on the preparation are related to the part of the stomach from where the strips are prepared and the muscle tone of the preparation.     

EDRF-release and Ca+(+)-channel blockade by magnolol, an antiplatelet agent isolated from Chinese herb Magnolia officinalis, in rat thoracic aorta

Magnolol is an antiplatelet agent isolated from Chinese herb Magnolia officinalis. It inhibited norepinephrine (NE, 3 microM)-induced phasic and tonic contractions in rat thoracic aorta. At the plateau of the NE-induced tonic contraction, addition of magnolol caused two phases (fast and slow) of relaxation. These two relaxations were concentration-dependent (10-100 micrograms/ml), and were not inhibited by indomethacin (20 microM). The fast relaxation was completely antagonized by hemoglobin (10 microM) and methylene blue (50 microM), and disappeared in de-endothelialized aorta while the slow relaxation was not affected by the above treatments. Magnolol also inhibited high potassium (60 mM)-induced, calcium-dependent (0.03 to 3 mM) contraction of rat aorta in a concentration-dependent manner. 45Ca(+)+ influx induced by high potassium or NE was markedly inhibited by magnolol. Cyclic GMP, but not PGI2, was increased by magnolol in intact, but not in de-endothelialized aorta. It is concluded that magnolol relaxed vascular smooth muscle by releasing endothelium-derived relaxing factor (EDRF) and by inhibiting calcium influx through voltage-gated calcium channels.     

Synthetic lapachol derivatives relax guinea-pig ileum by blockade of the voltage-gated calcium channels

The present study was designed to further evaluate a possible spasmolytic activity of synthetic lapachol derivatives, norlapachol, alpha-norlapachone, beta-norlapachone and hydro-hydroxy-norlapachol (HH-norlapachol), on guinea-pig ileum. In guinea-pig ileum, except for norlapachol, all naphthoquinones inhibited the phasic contractions induced by carbachol or histamine. Even when the ileum was pre-contracted with KCl, carbachol or histamine, all naphthoquinones induced relaxation, suggesting that these naphthoquinones could be acting on the voltage-gated calcium channels (Ca(V)). As the tonic component this contraction is maintained mainly by the opening of the Ca(V), we hypothesized that these naphthoquinones might be acting on these channels. This hypothesis was confirmed by the observation that norlapachol (pD'2 = 4.99), alpha-norlapachone (pD'2 = 4.49), beta-norlapachone (pD'2 = 6.33), and HH-norlapachol (pD'2 = 4.53) antagonized the contractions induced by CaCl2 in depolarizing medium nominally without Ca2+. As beta-norlapachone was the most potent we decided to continue the study of its action mechanism. The fact that this naphthoquinone has inhibited the tonic contractions induced by S-(-)-Bay K8644 [EC50 = (1.6 +/- 0.30) x 10(-5) M] suggests that the Ca2+ channel involved belongs to the type L (Ca(V)1.2). In addition, in the functional level, the spasmolytic effect of beta-norlapachone does not involve participation of free radicals, since its curve of relaxation was unchanged in the presence of glutathione, an antioxidant agent.     

Mechanisms involved in the relaxation of bovine aortic endothelial cells

The importance of endothelial cell contraction in the regulation of vascular biology is being increasingly recognized. Our group has demonstrated that reactive oxygen species, particularly hydrogen peroxide, which are released in pathological conditions such as ischemia-reperfusion, are able to induce contraction in bovine aortic endothelial cells (BAEC). The cGMP-dependent relaxation of contractile cells depends on the ability of the cyclic nucleotide to interfere with intracellular calcium; however, this is not the only mechanism involved. The present experiments were designed to analyse the mechanism by which cGMP induces relaxation in BAEC. Sodium nitroprusside (SNP), an activator of soluble guanylate cyclase, as well as atrial natriuretic (ANP) and C-type natriuretic (CNP) peptides, activators of particulate guanylate cyclase, blunted the hydrogen peroxide-induced contraction of BAEC and myosin light chain phosphorylation. The inhibitory effect was more marked with SNP and CNP than with ANP, and the action of SNP and CNP were partially reversed by blocking soluble and particulate guanylate cyclases, respectively. Dibutyryl cGMP (db-cGMP), a cGMP analogue, mimicked the effect of SNP and CNP. Cyclic GMP-dependent protein kinase (cGK) protein levels and activity were measured. Hydrogen peroxide induced a significant reduction in cGK activity without any change in protein level. This effect was completely reversed by preincubation with db-cGMP. Calyculin A, a myosin light chain phosphatase inhibitor, prevented the cGMP-induced relaxation of BAEC. SNP, CNP and db-cGMP also partially prevented the hydrogen peroxide-induced increase in intracellular calcium levels. Catalase completely blocked this effect. In summary, the present results support a role for those metabolites which activate guanylate cyclases in the relaxation of BAEC, and suggest that the cGMP-induced BAEC relaxation could be due, at least partially, to the stimulation of cGK and/or myosin light chain phosphatase activity, and to calcium blockade.     

Nipradilol induces vasodilation of canine isolated posterior ciliary artery via stimulation of the guanylyl cyclase-cGMP pathway

We examined the effect of nipradilol on contraction of the posterior ciliary artery induced by high potassium or norepinephrine and on cyclic GMP (cGMP) levels in the posterior ciliary artery of dogs. Nipradilol caused dose-dependent relaxation of KCl-and norepinephrine-induced contractions of posterior ciliary artery. The relaxant effect of nipradilol on norepinephrine-contracted ciliary artery was significantly greater than that on KCl-contracted ciliary artery. In KCl-contracted ciliary artery, N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 10(-4) M) did not alter the relaxant effect of nipradilol, whereas 1H-1,2,4-oxadiazolo-4,3-a-quinoxalin-1-one (ODQ, 10(-6) M) significantly inhibited this effect. Ethacrynic acid at 10(-5) M, sulfasalazine at 10(-4) M and S-decylglutathione at 10(-4) M (glutathione S-transferase inhibitors) did not inhibit the relaxant effect of nipradilol. In addition, nipradilol produced dose-dependent increases in cGMP levels in the canine posterior ciliary artery. These findings indicate that nipradilol-induced vasorelaxation in the canine posterior ciliary artery occurs via stimulation of the guanylyl cyclase-cGMP pathway.