Mechanism of inhibitory effects of azelastine on smooth muscle contraction.

The mechanism of inhibitory effects of azelastine, an antiallergic and antiasthmatic agent, on depolarization- and alpha-1 adrenergic agonist-induced contractions of intact smooth muscle was studied. The effects of azelastine on membrane currents were determined in isolated guinea pig ileum smooth muscle cells with the whole-cell clamp technique; the effects on contraction were evaluated in receptor- and G-protein-coupled, alpha-toxin-permeabilized rabbit femoral artery and portal vein smooth muscle strips. Azelastine (1-20 microM), like dihydropyridines, inhibited spontaneous rhythmic and high K(+)-induced contractions, mainly through inhibition of the voltage-dependent (L-type) Ca++ current. The tonic component of high K+ contractions was inhibited more than the phasic component, correlating to voltage-dependent inhibition of Ca++ current by the drug. Azelastine (IC50 of 0.25 microM), a known histamine blocker, also reversibly inhibited alpha-1 agonist-induced contractions in the presence and absence of extracellular Ca++. Both major pathways of pharmacomechanical coupling, agonist-induced Ca++ release from the sarcoplasmic reticulum and Ca++ sensitization of the regulatory/contractile apparatus were blocked by the same concentration of drug in permeabilized as in intact muscle. Inositol 1,4,5-trisphosphate-induced Ca++ release and guanosine 5'-O-(tau-thiotriphosphate)-induced Ca++ sensitization, however, were not inhibited. Azelastine at high (greater than 10 microM) concentrations reversibly inhibited Ca(++)-activated contraction, more potently at lower Ca++ concentration and in phasic smooth muscle, but inhibited neither adenosine 5'-O-(tau-thiotriphosphate)-induced, Ca(++)-independent nor phorbol ester-induced contractions. These results indicate that azelastine is a genuine Ca++ antagonist that inhibits voltage-gated Ca++ inward current and agonist-induced Ca++ release and Ca++ sensitization.     

Methylmercury blocks N- and L-type Ca++ channels in nerve growth factor-differentiated pheochromocytoma (PC12) cells.

Effects of methylmercury (MeHg) on whole-cell Ba++ currents in rat pheochromocytoma (PC12) cells were examined. Based on biophysical characteristics and sensitivity to omega-conotoxin GVIA and dihydropyridine agonists and antagonists, voltage-activated Ba++ currents (IBa) in PC12 cells were mediated by N- and L-type Ca++ channels. Addition of MeHg (10 microM) to the extracellular solution caused a rapid and complete block of current carried by 20 mM Ba++. The rate of block of IBa by MeHg increased in a concentration-dependent manner between 1 and 20 microM. Increasing the frequency of stimulation from 0.1 to 0.4 Hz facilitated block of IBa by MeHg. A 2-min application of 10 microM MeHg in the absence of stimulation also reduced IBa by approximately 80%. Thus, block of IBa by MeHg is not state-dependent. Additionally, MeHg blocked IBa when the membrane holding potential was -40, -70 and -90 mV, indicating that both N- and L-type Ca++ channels are blocked by MeHg. Block of IBa by MeHg was voltage-dependent at a membrane holding potential of -40 mV, but not at holding potentials of -70 and -90 mV. Decreasing the extracellular concentration of Ba++ ([Ba++]e) from 20 mM to 10 mM increased the magnitude of block by MeHg from 45.6 to 77.3%. Increasing [Ba++]e to 30 mM caused no further antagonism of block. Block of IBa by MeHg was not reversed by washing with MeHg-free solution. The ionic permeability of PC12 cell Ca++ channels was Ca++ = Sr++ greater than Ba++. In the presence of MeHg, all three divalent cations were equally permeant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dichlorobenzamil on calcium currents in clonal GH3 pituitary cells

Membrane currents were recorded from voltage-clamped clonal (GH3) pituitary cells, using the whole-cell patch clamp method. Under conditions in which currents through Na+ and K+ channels were abolished, two distinct Ca++ channel currents were identified. Dichlorobenzamil (DCB), an amiloride analog and potent inhibitor of Na-Ca exchange, inhibited both the T-type Ca++ current (ICa,t) and slowly-inactivating L-type current carried by either Ca++ (ICa,s) or Ba++ (IBa,s). The blockade was dose-dependent (1-25 microM) and ICa,t was more sensitive to inhibition by DCB than the L-type channel currents. Although the inhibition of ICa,t was not accompanied by changes in the time course of inactivation of the T-type channels, the blockade of L-type Ca++ channel currents was time-dependent, increasing throughout the depolarizing test pulse (300 msec). Repetitive stimulation at 1.0 Hz or the application of depolarizing prepulses augmented the blockade of ICa,s and IBa,s by DCB. It is proposed that the blockade is modulated by the functional state of the L-type channel, being enhanced by the channel opening. Currents conveyed by Na+ ions through both types of Ca++ channels in GH3 cells equilibrated with Ca++-free salines were inhibited by DCB concentrations (1-5 microM) comparable to those required for blocking the currents conveyed by Ca++ or Ba++ ions.     

Papaverine blocks hKv1.5 channel current and human atrial ultrarapid delayed rectifier K+ currents

Papaverine, 1-[(3,4-dimethoxyphenyl)methyl]-6,-7-dimethoxyisoquinoline, has been used as a vasodilator agent and a therapeutic agent for cerebral vasospasm, renal colic, and penile impotence. We examined the effects of papaverine on a rapidly activating delayed rectifier K(+) channel (hKv1.5) cloned from human heart and stably expressed in Ltk(-) cells as well as a corresponding K(+) current (the ultrarapid delayed rectifier, I(Kur)) in human atrial myocytes. Using the whole cell configuration of the patch-clamp technique, we found that papaverine inhibited hKv1.5 current in a time- and voltage-dependent manner with an IC(50) value of 43.4 microM at +60 mV. Papaverine accelerated the kinetics of the channel inactivation, suggesting the blockade of open channels. Papaverine (100 microM) also blocked I(Kur) in human atrial myocytes. These results indicate that papaverine blocks hKv1.5 channels and native hKv1.5 channels in a concentration-, voltage-, state-, and time-dependent manner. This interaction suggests that papaverine could alter cardiac excitability in vivo.     

Effect of endothelin-1 on guinea pig gallbladder smooth muscle in vitro.

The purpose of this study was to investigate the pharmacological activity of endothelin-1 (ET-1) on guinea pig gallbladder smooth muscle. Guinea pig gallbladder muscle strips were mounted in 10-ml siliconized organ baths containing Krebs' solution. After 1 hr of equilibration, ET-1 was added cumulatively. ET-1 induced slow-developing and long-duration contractile responses. The EC50 was approximately 10 nM. ET-1 was 5 times less potent than cholecystokinin (EC50, 2 nM), but 20 and 40 times more potent than carbachol (EC50, 200 nM) and histamine (EC50, 400 nM), respectively. The concentration-response curve to ET-1 was not affected by tetrodotoxin (0.1 microM) or by the muscarinic antagonist, atropine (10 microM). The neuronal N-type calcium channel blocker, omega-conotoxin (0.1 microM), had no significant effect on the ET-1 concentration-response curve. In contrast, the contractile effect to ET-1 was reduced markedly by removal of extracellular calcium or by the voltage-dependent calcium channel blockers nicardipine and diltiazem. Substitution of strontium (an inhibitor of intracellular calcium release) for Ca++ significantly reduced the response to ET-1. The cyclooxygenase inhibitor indomethacin had no significant effect on the contractile activity of ET-1. These finding suggest that ET-1 is a potent contractile stimulant of guinea pig gallbladder and that it acts directly on the smooth muscle. The activity depends on extracellular Ca++, suggesting involvement of Ca++ influx via the voltage-dependent Ca++ channel and on intracellular calcium.     

Ca++ inhibition of isoproterenol responses in mammalian skeletal muscle

In noncontracting mouse hemidiaphragms incubated in modified Krebs-Ringer--bicarbonate buffer with 10 mM Ca++, isoproterenol-stimulated phosphorylase a formation, conversion of phosphorylase kinase to the activated form, elevation of cyclic AMP-dependent protein kinase activity ratios and increase in cyclic AMP concentrations were reduced 35 to 50% over the responses in buffer with 2.5 mM Ca++. In buffer with 10 mM Ca++, the initial rate of isoproterenol-stimulated cyclic AMP accumulation was 59% of that in buffer with 2.5 mM Ca++. The inhibitory action of Ca++ on cyclic AMP accumulation was antagonized by verapamil, but not by inhibitors of cyclic nucleotide phosphodiesterase activity. In buffer with 2.5 mM Ca++, isoproterenol-stimulated cyclic AMP accumulation was inhibited by A23187 and caffeine, agents that can increase intracellular Ca++ concentrations. In addition to Ca++, high concentrations of Co++, Ni++, Mn++ and, to a lesser extent, Sr++ inhibited the isoproterenol response. The results of these studies indicate that high buffer Ca++ concentrations inhibit the response of the glycogenolytic pathway to isoproterenol by an action on cyclic AMP formation. We propose that the site of the inhibitory action of Ca++ is the divalent metal activator site associated with hormone-stimulated adenylate cyclase activity.     

Effects of McN-6186 on voltage-dependent Ca++ channels in heart and pituitary cells

McN-6186 (N-[2-(3,5-dimethoxyphenyl)ethyl]-5-methoxy-alpha-methyl-2 -(phenylethynyl) benzeneethanamine hydrochloride is a compound structurally distinct from other Ca++ channel ligands. McN-6186 showed some stimulation of 1,4-dihydropyridine-sensitive Ca++ uptake in neonatal rat ventricular cells at concentrations of 1 and 3 nM. At higher concentrations McN-6186 inhibited this uptake in rat ventricular cells at concentrations approximately 100-fold less than those needed to block the corresponding Ca++ uptake in rat anterior pituitary (GH3) cells. McN-6186 (2 microM) inhibited L-type Ca++ channel current in neonatal rat ventricular cells in a voltage-dependent manner while having little or no effect on this current in GH3 cells. In some ventricular cells tested, the T-type Ca++ current was also blocked by 2 microM McN-6186. McN-6186 inhibited (+)-[3H]PN200-110 binding in rat cardiac membranes with an IC50 value of 1.45 X 10(-7) M and a shallow Hill slope (nH = 0.42). It is concluded that McN-6186 blocks L-type Ca++ channels in heart cells preferentially to those found in GH3 cells. Furthermore, McN-6186 may have other sites and mechanisms of action in addition to L-type Ca++ channel blockade.     

A new site for the activation of cardiac calcium channels defined by the nondihydropyridine FPL 64176.

We examined the effects of a new ligand, FPLnM-64176, on L-type Ca++ channels in cardiac tissue. FPL 64176 (10-1 microM) enhanced Ca++ influx into neonatal rat ventricular myocytes, a response which was blocked by nifedipine. FPL 64176 had no effect on [3H]PN200-110 binding in rat ventricular membranes, but dramatically increased L-type Ca++ channel current amplitude. FPL 64176 (1 microM) slowed both the activation and the inactivation kinetics of the L-channel in neonatal rat ventricular cells. We also noted a hyperpolarizing shift in the threshold and peak potential of the Ca++ channel current-voltage relationship in response to the compound. Additionally, the binding site for FPL 64176 appeared to be located on the extracellular face of the channel. We conclude that FPL 64176 is a potent new activator of L-type Ca++ channels with a novel mechanism and site of action.     

Mechanism of inotropic action of xestoquinone, a novel cardiotonic agent isolated from a sea sponge.

Xestoquinone (XQN) isolated from the sea sponge Xestospongia sapra produced dose-dependent cardiotonic effects on guinea pig left and right atria. A direct action of XQN (1-30 microM) on the contractile machinery of cardiac myofilaments was demonstrated in chemically skinned fiber preparations from guinea pig papillary muscles. In atrial preparations, the XQN-induced inotropic effect was markedly inhibited by verapamil or nifedipine, but was not affected by practolol, chlorpheniramine, cimetidine, tetrodotoxin or reserpine. The Ca++ dependence curve for the contractile response of the atria was substantially shifted to the left by XQN (10 microM), and this XQN-induced shift was reversed by verapamil. The time-to-peak tension and relaxation times of the atrial contractions were shortened by XQN, and the action potential duration was markedly prolonged. Whole-cell patch clamp recordings in left atrial strips confirmed that XQN (30 microM) increased the slow inward current. However, there was a temporal dissociation between altered tension development and prolongation of the action potential duration. Cyclic AMP phosphodiesterase activity was inhibited and tissue cyclic AMP content of guinea pig left atria was increased by XQN (0.3-10 microM) in a concentration-dependent manner, but increases in cyclic AMP content did not occur in parallel with increases in contractile response. These observations suggest that an enhancement of intracellular cyclic AMP content and Ca++ influx across the cell membrane contribute to the late phase of XQN-caused cardiotonic responses, whereas the early phase may largely be elicited through direct activation of contractile elements. XQN may provide a novel leading compound for valuable cardiotonic agents.     

Effects on calmodulin of bepridil, an antianginal agent

Using biopharmacological techniques, we determined the effect on calmodulin of bepridil, a Ca++ channel blocker. We used two Ca++/calmodulin-dependent enzymes, Ca++/calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain and myosin light chain kinase from chicken gizzard. Bepridil inhibited the calmodulin-induced activation of Ca++/calmodulin-dependent cyclic nucleotide phosphodiesterase and the concentration of this drug producing 50% inhibition (IC50) of this enzyme was 8 microM. There was no significant effect on unactivated Ca++/calmodulin-dependent cyclic nucleotide phosphodiesterase (in the absence of Ca++-calmodulin), up to a concentration of 100 microM. Bepridil inhibited specifically Ca++/calmodulin-dependent phosphorylation of chicken gizzard myosin light chain with an IC50 value of 18 microM. Moreover, this agent produced a marked displacement of [3H]N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, an antagonist that interacts selectively with calmodulin. The influence of bepridil on the dose-response curves of mesenteric arterial strips for CaCl2, norepinephrine and serotonin differed from the influence seen with nifedipine, another Ca++ blocker. Bepridil (100 microM) suppressed the Ca++-induced contraction of saponin-skinned mesenteric arteries and calmodulin (26 microM) reversed partly the relaxant effect of this agent. These results suggest that the effect of bepridil on the cardiovascular system is due not only to its Ca++ channel blocking action but also to a calmodulin antagonistic action.     

Frequency modulation of the inotropic action of isoproterenol in mouse heart

In mouse right ventricular strips, field-stimulated to contract isometrically in an oxygenated bicarbonate-buffered physiological salt solution at 22--24 degrees C, the EC50 for the inotropic action of isoproterenol decreased from 37 nM in muscles stimulated at 0.2 Hz to 5 nM in muscles stimulated at 3.3 Hz. At higher rates of contraction, there was also an increased sensitivity to the inotropic actions of norepinephrine and epinephrine but not to those of Ca++ and N6,O2'-dibutyryl cyclic AMP. Increasing the Ca++ concentration further decreased the EC50 for isoproterenol at 3.3 Hz but had no effect on the EC50 at 0.2 Hz. The leftward shift of the contractile response curve at 3.3 Hz was inhibited by verapamil (0.6 microM) and Mn++ (0.25 mM). The stimulation of cyclic AMP accumulation was approximately 6-fold more sensitive to isoproterenol at 3.3 than at 0.2 Hz, but isoproterenol increased contractile force at concentrations two orders of magnitude lower than those that significantly increased cyclic AMP accumulation. Inhibition of cyclic AMP phosphodiesterase activity further increased the sensitivity to the inotropic actions of isoproterenol but did not attenuate the frequency difference. The results indicate that isoproterenol-stimulated Ca++ influx through the slow channel plays an important role in the mechanism of the increased sensitivity to the inotropic action of isoproterenol found at higher frequencies of contraction. Although cyclic AMP accumulation was also frequency dependent, its role in the inotropic action of isoproterenol in mouse heart is not clear.     

Mechanisms of the bepridil-induced vasodilation of the rabbit mesenteric artery

Bepridil, at a concentration over 10 microM, increased the membrane resistance and, over 20 microM, depolarized the smooth muscle membrane of the rabbit mesenteric artery. These changes in membrane properties may be due to inhibition of K and possibly Cl conductances, as examined by the current-voltage relationship. The spike potential evoked by current pulses in the presence of 3 mM tetraethylammonium or from the excitatory junction potential (e.j.p.) was somewhat inhibited by bepridil (over 10 microM). Bepridil (over 5 microM) reduced the amplitude of e.j.p. without changing the facilitation process of e.j.ps and, at concentrations beyond 20 microM, it inhibited the facilitation of e.j.ps. Bepridil (over 0.1 microM) inhibited and over 1 microM blocked completely the tonic response of the K-induced contraction. Thus, the voltage-dependent Ca channels activated by K-induced depolarization and during the spike generation differ in nature. Bepridil inhibited the caffeine-induced contraction in intact muscles and the Ca-induced contraction in skinned muscles prepared by saponin to the same extent, but more effectively inhibited the norepinephrine-induced contraction without altering the norepinephrine-induced depolarization. We conclude that bepridil mainly inhibits the voltage-dependent Ca channel (the K depolarization sensitive) in smooth muscle membrane. High concentrations of bepridil (over 10 microM) inhibit the Ca spike, the K and Cl conductances, contractile proteins and Ca release from intracellular stored sites of smooth muscle cells and Ca mobilization at nerve terminals. Actions of bepridil differ from those of other Ca antagonists.     

Calcium channel blockers: correlation among receptor binding, calcium uptake and contractility in vitro

Ten known calcium channel blockers were studied for inhibition of K+-induced 45Ca++ uptake into rabbit aortic smooth muscle cells in culture, and for displacement of [3H]nitrendipine [2,6-dimethyl-3-carbomethoxy-5-carbomethoxy-4-(3-nitro)phenyl-1, 4-dihydroxypyridine] binding to rat ventricular membrane preparations, in order to relate their effects on receptor binding with their inhibitory activities on 45Ca++ uptake and on contractile responses of vascular smooth muscle. Steady-state 45Ca++ uptake increased with K+ concentration in a dose-dependent manner. With 25 to 50 mM K+, Ca++ uptake was 0.6 nmol of Ca++ per one million cells. All calcium channel blockers inhibited K+-induced 45Ca++ uptake and [3H]nitrendipine binding in a dose-dependent fashion. The enatiomeric dihydropyridines 202-791 [isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2, 6-dimethyl-5-nitro-3-pyridinecarboxylate] exhibited marked stereoselectivity in both studies, the agonist (+)-202-791 significantly enhancing 45Ca++ uptake at 15 to 50 mM K+. The similarity between the order of potency in inhibiting 45Ca++ uptake and displacing [3H]nitrendipine resulted in a highly significant linear (1:1) correlation. An equally significant correlation was also established for the 10 blockers between their inhibitory potencies on 45Ca++ uptake and the contractile response of rabbit aortic strips as cited in the literature. These findings support the hypothesis that calcium channel blockers block contraction of vascular muscle by inhibiting cellular calcium uptake through voltage-dependent calcium channels as a result of binding to receptors associated with these channels. The aortic cells possess channels that are functionally similar to those found in intact vascular tissue.     

Bronchodilator mechanisms in bullfrog lung: differences in response to isoproterenol, theophylline and papaverine

The effects of bronchodilators and smooth muscle relaxants on mechanical responses and lung cyclic nucleotide levels in the isolated hemilung of Rana catesbeiana demonstrate striking differences in intensity and time course of drug action in an unstimulated preparation of airway smooth muscle. Isoproterenol, nitroprusside and nitroglycerin elicit a fast onset relaxation (minutes) with ceiling effects at 20, 22 and 43%, respectively, of maximal relaxation. Theophylline, dibutyryl cyclic AMP and papaverine produce maximal or near maximal relaxation, but require 8 to 32 hr for peak effect. Papaverine-induced relaxation is accompanied by a slow increase in lung cyclic AMP and cyclic GMP and is markedly accelerated by isoproterenol. Theophylline (10(-3) M) produces no change in cyclic nucleotide levels and its relaxant effect is not accelerated by isoproterenol. The hierarchy of relaxant responses suggests drug action at discrete loci in a highly compartmentalized effector chain, with cyclic AMP-dependent mechanisms separable into at least two components. The first is activated by isoproterenol and elicits a rapid, limited response, presumably reflecting an increase in cyclic AMP in a relatively restricted pool. The second is activated by papaverine and elicits a very slow, but complete relaxation, presumably reflecting a more pervasive or diffuse accumulation of cyclic AMP secondary to phosphodiesterase inhibition. The major portion of theophylline-induced relaxation in this preparation appears to be independent of changes in cyclic nucleotide metabolism.     

Guanine nucleotide binding proteins may modulate gating of calcium channels in vascular smooth muscle. II. Studies with guanosine 5'-(gamma)triphosphate

Our previous studies with fluoride have indicated that G-proteins may mediate the gating of Ca++ channels in vascular smooth muscle (VSM). We now present further studies on the relationship between G-proteins and Ca++ channels in VSM using guanosine-5'-(gamma-thio)triphosphate (GTP gamma S), a hydrolysis-resistant analog of GTP. Rat tail artery helical strips pretreated with GTP gamma S in a cytosol-like solution contracted in a Ca++-dependent manner in the absence of a depolarizing concentration of K+, hormones or any other Ca++ agonists. Contraction was dependent on the concentrations of applied GTP gamma S. The ability of strips pretreated with GTP gamma S to contract in response to Ca++ was not reversed by repeated washing. Incubation with 1 mM GTP applied extracellularly did not induce tension development. Treatment with a subthreshold concentration of GTP gamma S shifted the K+ concentration-related tension curve to the left but did not alter the maximum response. The contractions induced by GTP gamma S pretreatment and by submaximal (60 mM) KCI were additive at all levels of Ca++ tested. Extra tension development could be evoked from tissue maximally contracted with GTP gamma S by adding maximal K+ and norepinephrine. The relaxing sensitivity of the GTP gamma S-related contraction to reversal by nifedipine was between those for K+ depolarization and norepinephrine, and the GTP gamma S-induced rise in tension was partially inhibited by the Ca++ channel blocker nifedipine. Ca++-elicited contraction of the GTP gamma S-pretreated strips was relaxed by forskolin, an adenylate cyclase activator, 3-isobutyl-l-methyl-xanthanine, a cyclic nucleotide phosphodiesterase inhibitor, and dibutyryl cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of the low Km cyclic AMP phosphodiesterase and activation of the cyclic AMP system in vascular smooth muscle by milrinone

The purposes of the present study were to quantitate the effects of the cardiotonic/vasodilator milrinone on phosphodiesterase (PDE) isozymes isolated from vascular (aortic) smooth muscle from several species, and to quantitate changes in cellular cyclic AMP (cAMP) content, activation of cAMP protein kinase (cAPK) and vasorelaxation by milrinone in isolated guinea pig aortic smooth muscle. With PDE isozymes isolated from rat (Wistar-Kyoto or spontaneously hypertensive rats), guinea pig, monkey or canine aortic smooth muscle, milrinone is a potent (IC50 = 0.16-0.90 microM) and selective (100 times peak III relative to peak I) peak III inhibitor. The potency of milrinone and other vascular peak III PDE inhibitors parallels their potency as vasorelaxants in isolated guinea pig aortic rings (r = 0.86; P less than .01). Vasorelaxation of phenylephrine-contracted (3 microM) guinea pig aortic rings is accompanied by significant increases in cAMP content or cAPK activation with concentrations of milrinone greater than or equal to 10 microM. Temporally, significant increases in cAMP content accompany significant vasorelaxation; however, activation of cAPK is not significantly increased until at least 1 to 2 min after addition of milrinone. Similar concentration and temporal relationships are seen with the cAMP-related vasorelaxants papaverine and forskolin. As with milrinone, a temporal dissociation between increases in cAMP content and increases in cAPK activity ratio is evident.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antispasmodic and bronchodilator activities of St John's wort are putatively mediated through dual inhibition of calcium influx and phosphodiesterase

The crude extract of aerial parts of St John's wort (Hypericum perforatum) (Hp.Cr) and its fractions were studied in vitro for its possible spasmolytic and bronchodilator activities to rationalize some of its medicinal uses. In rabbit jejunum preparations, Hp.Cr caused a concentration-dependent relaxation of both spontaneous and K+ (80 mm)-induced contractions at a similar concentration range (0.1-1.0 mg/mL), similar to that produced by papaverine, whereas verapamil was relatively potent against K+-induced contractions. Hp.Cr shifted the Ca2+ concentration-response curves (CRCs) to the right, similar to that caused by papaverine or verapamil and also caused leftward shift of isoprenaline-induced inhibitory CRCs, similar to papaverine. In guinea-pig tracheal preparations, Hp.Cr caused relaxation of carbachol and K+-induced contractions at similar concentrations (0.01-0.3 mg/mL) and also shifted the isoprenaline-induced inhibitory CRCs to the left, similar to that caused by papaverine. In rabbit aorta preparations at rest, Hp.Cr produced a moderate vasoconstriction, while exhibited vasodilator effect against phenylephrine and K+-induced contractions. Papaverine and verapamil also produced similar non-specific vasodilation, but were devoid of any vasoconstrictor effect. Hp.Cr caused suppression of atrial force of contractions at concentrations about 20 times higher than those that produced inhibitory effect in smooth muscle preparations, similar to papaverine. These results suggest that the spasmolytic effects of Hp.Cr are mediated through dual inhibition of calcium influx and phosphodiesterase (PDE)-like mechanisms, which might explain the medicinal use of St John's wort in the disorders of gastrointestinal and respiratory tracts. Furthermore, the presence of Ca2+ antagonist and PDE inhibitory-like constituents might also be contributing to some extent in the well established use of plant in depression.     

Blocking action of terodiline on calcium channels in single smooth muscle cells of the guinea pig urinary bladder.

The blocking action of terodiline, a nonspecific organic Ca++ antagonist, on smooth muscle Ca++ channels of the guinea pig urinary bladder was investigated. Inward Ca++ currents were recorded from smooth muscle cells isolated from the urinary bladder using the whole-cell patch-clamp technique. In the absence of terodiline, a use-dependent reduction in the amplitude of inward Ca++ current was observed at a stimulus frequency of 0.2 Hz. When terodiline (1-10 microM) was applied, the use-dependent reduction was accelerated markedly, depending on the stimulus frequency. The blocking action of terodiline was also dose-dependent; the Kd value as measured at the end of 20 times repetitive stimulation at 0.2 Hz was 1.7 microM. In addition to such a use-dependent block, terodiline produced a hyperpolarizing shift in the steady-state inactivation curve. The results suggest that terodiline preferentially binds to the Ca++ channel in the open state and also in the inactivated state.     

Regulation of phosphorylase A formation and calcium content in aortic smooth muscle and smooth muscle cells: effects of atrial natriuretic peptide II

Atrial natriuretic peptide II (ANP II) raises cyclic GMP and relaxes vascular smooth muscle in vitro. The manner in which ANP II relaxes vascular smooth muscle is unknown but may involve alterations in the concentration of free intracellular Ca++. To examine this possibility, changes in intracellular Ca++ were monitored in rat aortic strips using the Ca++-dependent conversion of phosphorylase b to a, while Ca++ levels and phosphorylase were measured in cultured rat aortic smooth muscle cells. ANP II produced time- and concentration-dependent decreases in phosphorylase a and tension in norepinephrine-contracted aortic strips. The decrease in the formation of phosphorylase a was accompanied by an increase in cyclic GMP content. ANP II also decreased phosphorylase a formation in K+-depolarized tissues but to a lesser extent. Agonists such as angiotensin II and arginine vasopressin, and depolarizing concentrations of K+ elevated Ca++ levels in cultured aortic cells. ANP II inhibited Ca++ accumulation to either agonists or K+, but was more effective against agonists. Phosphorylase a formation which was increased by agonists and K+ in cultured cells was also inhibited by ANP II. We conclude that phosphorylase a formation can be a useful indicator of intracellular Ca++ concentrations in smooth muscle preparations and that ANP II regulates Ca++ levels in agonist and depolarized smooth muscle, suggesting that ANP II affects mainly Ca++ removal from the cytoplasm.     

Electrophysiological actions of the pimobendan metabolite, UD-CG 212 Cl, in guinea pig myocardium.

Pimobendan (UD-CG 115 BS), an inotropic agent and inhibitor of type III phosphodiesterase activity, is demethylated in vivo to form UD-CG 212 Cl, which is a more potent type III phosphodiesterase inhibitor. This study examined cyclic AMP (cAMP)-mediated actions of UD-CG 212 Cl. In guinea pig papillary muscles, UD-CG 212 Cl increased cAMP and stimulated Ca(++)-dependent slow action potentials (APs) in a dose-dependent manner. When compared to previous studies using pimobendan, UD-CG 212 Cl was approximately 100-fold more potent. UD-CG 212 Cl had no additional effects on slow APs in the presence of a maximal dose of isoproterenol (1 microM). Propranolol had little effect on UD-CG 212 Cl-induced slow APs. These results, along with previous studies, indicate that slow AP induction by UD-CG 212 Cl was cAMP-dependent, and the increase in cAMP levels was most likely due to phosphodiesterase inhibition and not beta receptor stimulation. Experiments with tetraethylammonium.Cl suggested that UD-CG 212 Cl probably did not induce slow APs by blocking K+ channels. In voltage-clamped ventricular myocytes UD-CG 212 Cl (100 microM) could stimulate Ca++ current (+21 +/- 5%) when basal cAMP levels were enhanced with a submaximal dose of isoproterenol (10(-9)-10(-8) M). Isoproterenol was not required to observe the stimulating effect of UD-CG 212 Cl on Ca++ current in intact, nondialyzed cells prepared using the nystatin-perforated patch method. Studies with the stereoisomers of UD-CG 212 Cl showed that the D-isomer was more potent than the L-isomer.(ABSTRACT TRUNCATED AT 250 WORDS)