Ca2+-cyclic AMP interaction in chemically skinned smooth muscle.

Using guinea-pig taenia coli smooth muscle that is chemically skinned with Triton X-100 to functionally destroy plasmalemma as well as sarcoplasmic reticulum, we demonstrate that cAMP can enhance the extent as well as the rate of relaxation produced by lowering free [Ca2+] in a skinned fiber bundle precontracted with maximal [Ca2+]. However, these actions of cAMP are strongly dependent on the level of free [Ca2+] in the bathing medium, the effect of cAMP being greatly attenuated at higher [Ca2+]. These data support and further extend the results of our previous study indicating that modulations in [Ca2+] can have a strong influence on the ability of cAMP to produce a direct inhibitory effect on the contractile machinery in smooth muscle.

High-K+-induced inhibition of cAMP accumulation in smooth muscle.

This study examines a possibility that high-K+ depolarization induced inhibition of cAMP accumulation by beta-adrenoceptor stimulation (observed in our previous studies) is mediated by depolarization induced release of acetylcholine. Rat uterine strips were depolarized by high-K+ solution with or without atropine. Isoproterenol produced similar degrees of relaxation and increases in cAMP levels in depolarized smooth muscle with or without atropine. It is suggested that the release, if any, of acetylcholine from smooth muscle depolarization is not the primary cause of the observed inhibition of cAMP accumulation.

Effect of extracellular Na on isoproterenol-induced cAMP levels in depolarized uterus.

In the present study, the absence of a quantitative correlation between isoproterenol induced relaxation of uterine strips and tissue cAMP levels was demonstrated by using three depolarizing media: 127 mM KCl (0 NaCl), 47.5 mM KCl (NaCl), and 47.5 mM KCl (80 mM NaCl). While the degree of relaxation by isoproterenol was similar in all three media, isoproterenol (10(-4)M) increased cAMP by 100% in Na+ free depolarizing media, by 337% in Na+ containing depolarizing medium and by 600% in normal non-depolarizing medium. After pretreatment of the tissue with 10(-5) M D-600, 10(-4) M isoproterenol increased cAMP levels by 600% in all three depolarzing media. Studies using 10(-8) M isoproterenol produced qualitatively similar results. cGMP levels did not change significantly in any of the above studies. Na+ appears to be producing its effect on isoproterenol induced increase in cAMP levels indirectly by reducing the increase in intracellular Ca2+ concentration known to occur with depolarization.

Effect of isoproterenol on the isolated pregnant rat myometrium.

In pregnant rat myometrium, isoproterenol (10(-8) M) inhibited spontaneous contractions without causing hyperpolarization. Isoproterenol (10(-6) M) relaxed the depolarized muscle without affecting the membrane potential. The presence of 80 mM Na+ did not affect the degree of high-K+ depolarization. It was also without any influence on the effects of isoproterenol on the depolarized uterus. The results are consistent with the concept that hyperpolarization is not a prerequisite for beta-adrenoceptor induced relaxation of uterine smooth muscle.

The effects of amrinone on contractility, Ca2+ uptake and cAMP in smooth muscle.

Amrinone, a known positive inotropic agent in the heart, was found to cause a dose-dependent (10--100 micrograms/ml) inhibition of norepinephrine (NE) or high-K+-induced contractions of rabbit aorta. Amrinone also inhibited carbachol or high-K+-induced contractions of guinea-pig taenia coli. Neither total tissue 45Ca uptake nor the rate of 45Ca uptake induced by 80 mM K+ in rabbit aorta was altered by pretreatment with amrinone. On the other hand, a similar pretreatment with amrinone inhibited NE (10(-6) or 10(-5) M) induced tissue 45Ca uptake. Amrinone (100 micrograms/ml) caused about a 70% increase in cAMP concentration over resting levels. It is concluded that amrinone causes a nonspecific inhibition of smooth muscle contractility by acting probably at multiple sites to decrease the availability of Ca2+ required for activation. One or more of these mechanisms may involve cAMP.

Cardiovascular effects of the K-ATP channel blocker U-37883A and structurally related morpholinoguanidines.

The cardiovascular effects of the K-ATP channel blocker U-37883A and 5 related morpholinoguanidines were determined in 6 experimental preparations. In anesthetized dogs, U-37883A (0.5-8.0 mg/kg i.v.) increased mean arterial pressure (MAP; +18%) and left ventricular (LV) effective refractory period (ERP; +35%), and decreased LV contractility (-41%). Higher doses of U-37883A (16-32 mg/kg) fatally reduced MAP (-84%), heart rate (HR; -57%) and LV contractility (-72%). In anesthetized rats, U-37883A (1.0-50 mg/kg i.v.) also maximally reduced MAP, HR and LV contractility by 68, 77 and 48%, respectively. U-37883A and its analogs were diuretic in conscious rats (1.5-15 mg/kg i.v.) and blocked pinacidil in rabbit mesenteric artery (EC50 = 0.5-50 microM). In rabbit papillary muscle, 50 microM U-37883A significantly reduced force of contraction (-33%) and prolonged conduction time (+244%). Milder papillary effects were seen with the N'-OH analog U-45194A, which did not depress LV contractility in intact rats. In conscious dogs, oral U-45194A (50 mg/kg) was diuretic but reduced LV stroke volume and increased peripheral vascular resistance. These studies characterize U-37883A's systemic cardiovascular and direct myocardial effects, and identify U-45194A as a less cardiac depressant analog having U-37883A-like diuretic and functional K-ATP channel blocking activities.

Dependence of cyclic-AMP induced relaxation on Ca2+ and calmodulin in skinned smooth muscle of guinea pig Taenia coli.

cAMP (10(-6) - 10(-4) M) produced a dose-dependent relaxation of Ca2+-induced contraction in the guinea-pig taenia coli skinned with 1% Triton X-100. At 0.53 microM Ca2+ and 0.05 microM calmodulin (CaM), cAMP (10(-4) M) produced a maximal relaxation of 75% (pH 6.7; 25 degrees C). Increasing Ca2+ (0.8 microM) or CaM (0.37 microM) reduced cAMP-induced relaxation to 25 and 5% respectively. At high CaM (5 microM), cAMP-induced relaxation could be completely inhibited by as low as 0.25 microM Ca2+. Furthermore, small increases in Ca2+ or CaM could effectively reverse the cAMP-induced relaxation in the continuous presence of cAMP. These results demonstrate that small modulations in the Ca2+-calmodulin activity have a strong effect on the ability of cAMP to produce a direct relaxing effect on the contractile proteins in skinned fiber. It is suggested that the effects of cAMP on the cellular mechanisms that lower cytoplasmic free Ca2+ concentration may act as the important determinants of the extent of the direct inhibitory effect of cAMP on the contractile elements. These two mechanisms may act in concert in this fashion to effect cAMP-induced relaxation in smooth muscle during beta-adrenergic stimulation.

Biochemical mechanisms by which minoxidil sulfate influences mammalian cells.

The antihypertensive effect of minoxidil has been shown to occur via direct vasodilation induced by its active metabolite, minoxidil sulfate (MxSO4). Thus, an in vitro vascular smooth muscle preparation was used as a model to investigate cellular biochemical mechanisms influenced by MxSO4. In rabbit isolated superior mesenteric artery, the contractions produced by receptor agonists such as norepinephrine (NE) were relaxed by about 85% with MxSO4. In contrast, MxSO4 was without any effect on 80 mM KCl-induced contraction. Furthermore, pretreatment of the tissues with tetraethlammonium (TEA, a K+ channel blocker), or ouabain, or 10-25 mM KCl, all caused pronounced inhibition of MxSO4-induced relaxation of NE-induced contraction. These investigations revealed that MxSO4-induced relaxation could be effectively inhibited by treatments that interfere with plasmalemmal K+ permeability. It is proposed that MxSO4 acts like a K+ channel agonist to enhance K+ permeability; this should result in hyperpolarization and cause reduction in agonist-stimulated Ca2+ influx. The end result is a decrease in cytoplasmic free Ca2+ concentration, and thus relaxation. Consistent with this, we could also demonstrate enhancement of 42K efflux as well as inhibition of NE-stimulated 45Ca influx. The above-proposed mechanism appears specific for MxSO4 since it was not shared by forskolin (a cyclic-AMP-increasing agent), sodium nitroprusside (a cyclic-GMP-increasing agent) or D600 (a Ca2+ antagonist). The relevance of the proposed unique cellular mechanism (i.e. K+ permeability) of MxSO4 to its hair growth effect remains to be examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effects of a synthetic atrial peptide on contractions and 45Ca fluxes in vascular smooth muscle.

The mechanism of a synthetic atrial peptide (APII)-induced inhibition of smooth muscle contractility was investigated by studying its effects on tension development and 45Ca fluxes in isolated rabbit aorta. APII (10(-9) to 10(-7) M) produced a dose-dependent relaxation of contractions produced by alpha adrenoceptor activation with norepinephrine (NE; 10(-6) M). APII was a potent relaxant of NE contraction with an IC50 = 1.1 X 10(-8) M, with 10(-7) M APII causing a 97% relaxation. APII also produced a dose-dependent inhibition of NE contraction when added to the resting muscle before the exposure to NE. The relaxing effects of APII were found to be endothelium independent. In contrast, APII was only marginally effective in relaxing high-K+ contraction, with 10(-7) M APII causing only 17% relaxation. Furthermore, when a NE contraction was obtained on top of a high-K+ contraction, APII was still capable of relaxing the NE component. APII was similarly more effective in inhibiting NE-stimulated 45Ca influx than high-K+-stimulated 45Ca influx, indicating selective action of APII on the receptor-operated Ca++ channels. This was in contrast to D600, a well known Ca++ antagonist, which had a more selective inhibitory effect on the potential-operated Ca++ channels. The data presented indicate that APII is a potent relaxant of contractions produced by receptor-agonists involving 45Ca influx through receptor-operated Ca++ channels. APII may also prove to be a very useful tool to further distinguish and define receptor-operated Ca++ channels and potential-operated Ca++ channels in vascular smooth muscle.

Mg2+--Ca2+--Na+ interactions in uterine smooth muscle: studies with cAMP.

The influence of variation in the extracellular concentrations of Na+, Mg2+, and Ca2+ in the depolarizing medium on isoproterenol-induced increases in cAMP levels and relaxation was studied in rat uterus. Isoproterenol (10(-8) M) failed to increase cAMP levels in the high-K+ medium containing no Na+. When 80 mM Na+ was present in the medium, isoproterenol caused increases in cAMP levels similar to those observed in nondepolarized uterus. A similar effect of 2.5 mM Mg2+ was observed on the cAMP response. These effects of Na+ and Mg2+ were antagonized by increasing the extracellular concentration of Ca2+. The simultaneous presence of 80 mM Na+ and 2.5 mM Mg2+ did not produce an additive effect on the cAMP responses.

Role of Ca in isoproterenol-induced increases in cAMP levels in rat uterus.

The influence of alteration in the Ca2+ environment of the tissue on isoproterenol-induced increases in cAMP levels and relaxation was studied in rat uterus. In muscles depolarized with 47.5 mM K+ (with or without Na+), the ability of isoproterenol to increase cAMP levels and to produce relaxation was found to be inversely related to external calcium concentration. The pretreatment of the muscle with D600 or EGTA restored the cAMP response to isoproterenol in the depolarized uterus to a level observed in nondepolarized muscle. The study with Ro 20-1724, a phosphodiesterase (PDE) inhibitor indicated that the failure of isoproterenol to elevate cAMP levels in the depolarized uterus could not be related to the activation of PDE by Ca2+. The exposure of rat uterus to a zero-Ca2+ solution accentuated the increases in cAMP levels produced by isoproterenol. These results have raised the question of a possible regulatory role of Ca2+ in beta-adrenoceptor-induced increases in cAMP levels in uterine smooth muscle.

Influence of immune stimulation and suppression on morphine physical dependence and tolerance.

The role of the immune system in the development of physical dependence and tolerance to morphine was studied in mice in which the immune response was either stimulated or suppressed. Immunization of mice against morphine increased the blood and brain levels of morphine as compared to controls. However, the development of physical dependence and tolerance was decreased. The chronic responses to morphine were also decreased by nonspecific immunosuppression (vincristine-cyclophosphamide treatment and gamma-irradiation exposure) and specific immunosuppression (antithymocyte and antilymphocyte sera treatment). Immunosuppressive treatments did not alter the rate of morphine absorption from the subcutaneous depot used to induce chronic exposure to the drug. However, the blood and brain levels of morphine were higher than control after 72 hours of morphine pellet implantation. It is apparent that manipulation of the immune system can alter the physical dependence and tolerance development to morphine.

Effects of atrial natriuretic factor on isolated arteries and perfused organs of trout.

Atrial natriuretic factor (ANF) increases blood pressure when injected into conscious trout [Duff and Olson, Am. J. Physiol. 251 (Regulatory Integrative Comp. Physiol. 20): R639-R642, 1986]. The effects and mechanisms of action of ANF on trout vessels in vitro were examined in the present study using isolated celiacomesenteric arterial rings, perfused gill arches, and a two-artery perfused trunk preparation. In all experiments, ANF alone either relaxed trout vessels or had no effect. ANF inhibited contractions of arterial rings produced by a variety of agonists in part through inhibition of intracellular calcium release. In perfused gills, ANF attenuated epinephrine (EPI) alpha-mediated vasoconstriction but had no effect on beta-stimulated increases in water permeability. ANF only slightly lowered an EPI-induced increase in resistance in either pathway of the perfused trunk. These results indicate that the vascular effects and mechanism of action of ANF are similar in fish and mammals and that the pressor response to ANF injection in vivo is mediated through central or secondary effects.

Effects of beta-adrenergic stimulation on calcium movements in rabbit aortic smooth muscle: relationship with cyclic AMP.

1. The effects of isoprenaline (10(-6) M) on relaxation, unidirectional as well as net Ca(2+) fluxes, and cyclic AMP levels were investigated in rabbit aorta under the condition of high-K(+) depolarization in the presence of phentolamine (10(-5) M).2. Isoprenaline (10(-6) M) caused significant inhibition of Ca(2+) influx stimulated by 145 mM-K(+) (0 Na(+)) solution. The time courses of Ca(2+) influx inhibition and relaxation by isoprenaline were parallel. Isoprenaline also caused a significant inhibition of high-K(+)-induced gain in net Ca(2+) content.3. Ro 20-1724 (1 mM), a phosphodiesterase inhibitor, also caused relaxation and Ca(2+) influx inhibition in high-K(+)-depolarized rabbit aorta. Pre-treatment with Ro 20-1724 potentiated isoprenaline-induced Ca(2+) influx inhibition and relaxation.4. Isoprenaline and Ro 20-1724 each alone increased cyclic AMP levels. Furthermore pre-treatment with Ro 20-1724 caused potentiation of isoprenaline-induced increases in cyclic AMP levels.5. At submaximal concentration, D600 (10(-7) M) caused partial inhibition of high-K(+)-stimulated Ca(2+) influx and produced relaxation. However, unlike Ro 20-1724, it did not potentiate isoprenaline-induced Ca(2+) influx inhibition and relaxation. D600 does not increase cyclic AMP levels in smooth muscle.6. Dibutyryl cyclic AMP (1 mM), a lipid-soluble analogue of cyclic AMP, caused relaxation and inhibited high-K(+)-stimulated Ca(2+) influx.7. Isoprenaline failed to cause stimulation of Ca(2+) efflux in high-K(+)-depolarized rabbit aorta.8. It is concluded that the inhibition of Ca(2+) influx may be one of the mechanisms by which beta-receptor stimulation can reduce intracellular free Ca(2+) to promote relaxation of smooth muscle. The data support the involvement of cyclic AMP in this action of the beta-agonist.9. Since the experiments were conducted in 145 mM-K(+) (0 Na(+)) depolarizing conditions, the role of hyperpolarization or of a Na(+)-Ca(2+) exchange mechanism in isoprenaline-induced Ca(2+) influx inhibition and/or relaxation can be excluded.

Synthetic atrial peptide inhibits intracellular calcium release in smooth muscle.

The effects of a synthetic atrial peptide (atriopeptin II; AP II) on the agonist-induced intracellular Ca2+ release was examined in the isolated rabbit aorta. The agonist-induced phasic contraction in a Ca2+-free physiological salt solution containing 2 mM ethyleneglycol-bis(beta-aminoethyl-ether)-N,N'-tetraacetic acid (EGTA-PSS) was used as an indicator of the intracellular Ca2+ release. The addition of AP II (10(-9)-10(-7) M) for 15 min to the tissue during the EGTA-PSS exposure caused a dose-dependent inhibition of norepinephrine (NE; 10(-6) M)-induced phasic contraction. The half-maximal inhibiting concentration of AP II was 3 X 10(-9) M, with 10(-7) M AP II causing 91% inhibition. This was confirmed by studying the inhibitory effect of AP II (10(-7) M) on NE-stimulated 45Ca efflux. Furthermore, the internal Ca2+ release by histamine (10(-5) M) and caffeine (25 mM), both of which share this internal Ca2+ pool with NE, was also inhibited by AP II. Thus AP II appears to be a potent inhibitor of the intracellular Ca2+ release that is utilized by various agonists for the activation of vascular smooth muscle. This may be an important mechanism by which AP II produces relaxation of blood vessels.

Mechanism of action of minoxidil sulfate-induced vasodilation: a role for increased K+ permeability.

The mechanism of smooth muscle relaxing effect of minoxidil sulfate (MxSO4) was investigated in isolated rabbit superior mesenteric artery. MxSO4 (5 X 10(-6) M) was found to effectively relax maximal norepinephrine (NE; at 5 X 10(-6) M) contraction, but failed to relax 80 mM K+-induced contraction. MxSO4-induced relaxation was endothelium independent. When the tissues were exposed to increased extracellular K+ (10-25 mM), and then contracted with NE, the relaxation response to MxSO4 was significantly attenuated. Tetraethylammonium (5-10 mM) pretreatment caused pronounced inhibition of MxSO4-induced relaxation. Pretreatment with ouabain (0.5-5 microM) also significantly inhibited MxSO4 relaxation. This effect of ouabain was found to be due to its effect on K+ gradient. These data suggested a role of K+ permeability during MxSO4 relaxation which was further confirmed when it was found that MxSO4 can cause a significant stimulation of 42K efflux from the mesenteric artery preloaded with 42K. It is suggested that MxSO4 may act as a K+ channel agonist to affect the plasmalemmal Ca++ permeability during agonist activation. Consistent with this, MxSO4 was demonstrated to cause an inhibition of NE-stimulated 45Ca influx in this tissue. Such a strong dependence on K+ permeability makes MxSO4 a unique vasodilator among the clinically used vasodilators.

The variable relation between myosin light-chain phosphorylation and actin-activated ATPase activity in chicken gizzard smooth muscle. Modulation by tropomyosin.

Using pure myosin light-chain kinase and myosin from chicken gizzard and rabbit skeletal actin, we have established the relationship between myosin phosphorylation and actin-activated ATPase activity by varying the concentration of Ca2+ or calmodulin (CaM). By increasing Ca2+ or CaM the extent of myosin phosphorylation increased from 10% to about 100%, the requirements for half-maximal activation being 0.17 microM Ca2+ and 1 nM CaM. ATPase was activated only when a phosphorylation 'threshold' of about 60% was surpassed. By increasing phosphorylation from 60% to 100%, ATPase activity was further stimulated, the relationship between myosin phosphorylation and ATPase activity being curvilinear. Addition of 1 microM tropomyosin lowered the Ca2+ requirement for half-maximal ATPase activity in the prescence of 0.05 microM CaM from 0.4 microM to 0.2 microM but did not affect the maximal ATPAse activity. In addition, the CaM requirement of ATPase activity was also lowered by gizzard tropomyosin at 9 microM Ca2+. The Ca2+/CaM requirement for myosin phosphorylation remained unaffected by tropomyosin. Thus, tropomyosin altered the relationship between myosin phosphorylation and ATPase activity: the phosphorylation 'threshold' was lowered from 60% to about 10%, hence causing a leftward shift of the relationship which becomes similar to that in the case of native actomyosin.