Phorbol esters induce oscillatory contractions of intestinal smooth muscles.

The actions of tumor-promoting phorbol esters in smooth muscle excitation-contraction coupling were studied in isolated guinea pig ileum in the presence of various contractile agents. Muscarinic agonists, histamine and bradykinin elicited an initial transient phasic contraction and a subsequent sustained tonic contraction in guinea pig ileum. The Ca2+ channel antagonist nifedipine selectively inhibited the tonic contraction. Phorbol esters, protein kinase C activators, induced immediate muscle relaxation followed by oscillatory contractions when added during the tonic phase of contraction. Phorbol esters, when added in advance, slightly altered the ligand-induced phasic contraction but converted tonic contractions into oscillatory spikes. The amplitude, frequency and shape of the oscillation induced by phorbol esters were dependent upon the dose of phorbol ester: amplitude was increased and frequency was decreased by increasing the doses of phorbol ester. In contrast, the phorbol ester potentiated the tonic contraction induced by high potassium chloride with little effect on the phasic component. It also sensitized the muscles to Bay K 8644. Bay K 8644, which was ineffective in stimulating muscle contraction at 1 nM, became a very effective stimulator in the presence of the phorbol ester. All of these phorbol ester-induced potentiations and oscillations were sensitive to inhibition by staurosporine or nifedipine. These data suggest that in guinea pig ileum, protein kinase C plays a positive regulatory role in Ca2+ channel activation and promotes a complex regulatory effect on Ca(2+)-mobilizing ligand-stimulated Ca2+ channel activity, which results in oscillatory contractile responses to carbachol, methacholine, histamine and bradykinin.     

Effects of calcium-entry modulators on the biphasic neurogenic contractions in the circular muscle of guinea pig vas deferens

Effects of nifedipine (10(-8)-10(-6) M), verapamil (10(-6)-3 x 10(-5) M) and Bay k 8644 (10(-7) M) on the contractions induced by transmural nerve stimulation or agonists were investigated in the circular smooth muscle of guinea pig vas deferens using a ring preparation. Transmural stimulation (20 Hz for 20 sec) produced biphasic contractions consisting of initial phasic and secondary tonic components. Nifedipine preferentially inhibited the tonic component and norepinephrine (NE)-induced contractions over the phasic component and beta, gamma-methylene ATP (mATP)-induced contractions. Nifedipine suppressed the enhancing effect of NE on the mATP-induced contractions. The tonic component and NE-induced contractions were inhibited by verapamil in a similar dose range. Verapamil slightly inhibited the phasic component, but rather enhanced mATP-induced contractions. Bay k 8644 slightly increased the phasic component and mATP-induced contractions in the presence of prazosin. Bay k 8644 slowed the relaxation of the tonic component, thereby prolonging the tonic component. Bay k 8644 also prolonged the duration of NE-induced contractions. These results, except for the contradictory effects of verapamil between the phasic component and mATP-induced contractions, are consistent with the claim that ATP and NE are responsible for the generation of phasic and tonic component, respectively, in the circular muscle of guinea pig vas deferens.     

The effects of Bay K 8644 and nifedipine on the neural responses of the rabbit ear artery.

The effects of Bay K 8644 and nifedipine on the electrical and mechanical responses of the distal ear artery of the rabbit to stimulation of the perivascular nerves were studied. Neural stimulation elicited excitatory junction potentials (e.j.ps) in the smooth muscle cells of the artery. An action potential was generated when the e.j.p. reached a threshold of about -53 mV. Contraction was always triggered by the action potential. Bay K 8644 at low concentrations (7 X 10(-8) M) had no significant effect on the resting membrane potential and the e.j.p. However the amplitudes of the action potential, and the contraction were potentiated by Bay K 8644. Multiple action potentials could also be generated from a single e.j.p. At higher concentrations, Bay K 8644 (3 X 10(-6) M) caused membrane depolarization, development of vascular tone and spontaneous action potentials. All the effects of Bay K 8644 could be reversed by exposure to light. Nifedipine (3 X 10(-7) M) inhibited the action potential and the resulting contraction. The effect of nifedipine could be reversed by Bay K 8644 but not by light. These results demonstrate that the excitatory and inhibitory effects of Bay K 8644 and nifedipine on vascular smooth muscle are associated with changes in the electrical responses.     

Interactions of calcium antagonists and the calcium channel agonist Bay K 8644 on neurotransmission of the mouse isolated vas deferens.

1. The present study compares the effects of verapamil and Bay K 8644 on twitches of the mouse vas deferens induced by field stimulation at 0.1 Hz. The influence of interactions between these drugs and nifedipine on neurotransmission was also investigated. 2. Bay K 8644 (0.1 nM-3 microM) and verapamil (1-100 microM) potentiated twitches maximally by about 1000% (EC50 17.3 nM) and 300% (EC50 17.5 microM), respectively. Nifedipine (0.1 nM-1 microM) only reduced twitch magnitude (IC50 7.7 nM). All effects were reversed following washout. 3. Yohimbine (1-100 microM) reversed twitch potentiation caused by verapamil but not by Bay K 8644. Prazosin (1 microM) did not reduce basal twitch tension nor antagonize twitch potentiation by verapamil. 4. Twitch inhibition by nifedipine was unaltered by previous incubation with verapamil (30 microM), but Bay K 8644 (1 microM) shifted the curve to nifedipine 120 fold to the right. Previous incubation with nifedipine (1 microM) blocked potentiation induced by verapamil but did not modify responsiveness to Bay K 8644. 5. Previous addition of verapamil (30 microM) markedly enhanced twitch potentiation caused by Bay K 8644 in a supra-additive fashion. In experiments conducted in the reversed condition, Bay K 8644 (1 nM but not 10 nM) potentiated the effect of verapamil in a similar manner but to a lesser extent. 6. It is concluded that verapamil, in contrast to nifedipine, markedly enhances neurally-evoked twitches of the mouse vas deferens. Bay K 8644 produces essentially the same effect as verapamil, but its potency is 1000 fold and its maximal effect about 3 fold greater than that observed for verapamil.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage and time dependency of calcium mediated phasic and tonic responses in rat vas deferens smooth muscle--the effect of some calcium agonist and antagonist agents

1. Phasic and tonic components of the K+-induced contracture response were found to be expressed to different degrees in the prostatic and epididymal portions of the rat vas deferens. 2. With elevation of external potassium greater than 25 mM, the mechanisms underlying the phasic component operate only transiently before inactivation and replacement with tonic tension. 3. Both phasic and tonic components of the vas deferens response to potassium were markedly dependent upon external calcium ions. 4. Nifedipine and verapamil equally inhibited the phasic and tonic components of the K+ response in the prostatic vas deferens. However, inhibition by these agents was far more pronounced in the phasic components than in the tonic component of the epididymal vas deferens. 5. BAY K 8644 potentiated, in a dose-dependent manner, the phasic components of the K+ response, particularly in the prostatic vas deferens. 6. Abscisic acid also potentiated, in a dose-dependent manner the K+ response of rat vas deferens, but this action was far more pronounced in the tonic component of the epididymal portion. 7. Papaverine abolished the BAY K 8644 potentiated epididymal K+ response but did not affect the BAY K 8644 potentiated prostatic K+ response. 8. It is concluded that abscisic acid potentiated responses associated with the activation of voltage-dependent, non-inactivating slow calcium channels. 9. Papaverine, nifedipine and verapamil appear to be less selective than abscisic acid in that they equally inhibit phasic and tonic responses in prostatic vas deferens, but these may be interdependent, yet they more strongly inhibit phasic responses of epididymal vas deferens.     

Bay K 8644-induced potentiation of the contractile response of rabbit iliac artery to caffeine in a Ca2+-free medium

1. In a Ca2+-free medium caffeine (10 mM) was still able to cause a phasic contraction in rabbit iliac arteries. 2. Bay K 8644 at 10(-6) but not at 10(-7) M potentiated the residual response to caffeine in a Ca2+-free medium. In a Ca2+-free medium with or without KCl (40 mM), Bay K 8644, however, caused no contraction. 3. Nifedipine (10(-6) M) did not affect the residual caffeine-response or the potentiating effect of Bay K 8644. Verapamil (10(-6) M), however, inhibited both the caffeine response and the potentiation. 4. Bay K 8644 (10(-6) M) potentiated the contractile response to Ca2+ (0.01-2.4 mM) in a Ca2+-free medium containing KCl. The potentiation was equally inhibited by nifedipine or verapamil. 5. La#+ (1 mM), EGTA (0.1 mM), or vanadate (10(-4) M) completely inhibited the Bay K 8644-induced potentiation without affecting the residual caffeine response. 6. These results suggest that the potentiating action of Bay K 8644 on the residual caffeine response in a Ca2+-deficient medium may not be related to voltage-dependent Ca2+ channels. In addition, the activity of Ca2+-ATPase in sarcolemmal membranes may be important in this potentiation.     

Effects of a novel calcium channel agonist dihydropyridine analogue, Bay k 8644, on pig coronary artery: biphasic mechanical response and paradoxical potentiation of contraction by diltiazem and nimodipine

Bay k 8644 is a structural analogue of the 1,4-dihydropyridines whose pharmacological actions on heart and vascular smooth muscle are opposite from those of nifedipine and other similar calcium antagonists. We have examined the action of Bay k 8644 ("calcium channel agonist") on isolated porcine coronary artery rings. The interactions between Bay k 8644 and the vasodilators isosorbide dinitrate (ISDN), diltiazem, and nimodipine were quantitated. Bay k 8644 produced a biphasic, dose-dependent mechanical response, with contraction occurring over the concentration range of 1-350 nM (ED50 = 11.4 nM) and relaxation observed at concentrations greater than 350 nM (IC50 = 5.7 microM). ISDN, diltiazem, and nimodipine relaxed, in a dose-dependent manner, maximal Bay k 8644-induced contractions. When the coronary rings were pretreated for 25-90 min with 80% inhibitory concentrations of these vasodilators, there was little or no effect by ISDN on Bay k 8644-induced contractions; however, there was a surprising potentiation by diltiazem and by nimodipine. Pretreatment of coronary rings with higher concentrations of ISDN or diltiazem caused an inhibition of Bay k 8644-induced contraction, while pretreatment with higher concentrations of nimodipine caused further potentiation of contraction elicited by Bay k 8644. Bay k 8644 increased the tension developed in response to high potassium (potential-operated channel activation) or histamine (receptor-operated channel activation). To account for the biphasic response to Bay k 8644 (dose-dependent contraction and relaxation), and the unexpected potentiation of Bay k 8644-induced contraction by nimodipine and by diltiazem, a molecular model is proposed for vascular smooth muscle in which Bay k 8644 functions as a partial calcium channel agonist at two functionally distinct 1,4-dihydropyridine "receptor sites."     

Electrical and mechanical properties of the capsular smooth muscles of the rabbit prostate in relation to the actions of the alpha 1-adrenoceptor blocker, YM-12617.

1. Electrical and mechanical properties of smooth muscle cells of the rabbit prostate capsule and the actions of the alpha 1-adrenoceptor blocker, YM-12617, were investigated using microelectrode and isometric tension recording methods. 2. The capsular muscles comprised thick and thin muscle bundles. In the former, noradrenaline (NA; 0.1-10 microM) provoked the phasic and tonic mechanical responses, with twitch contractions superimposed on the tonic response. YM-12617, in concentrations over 1 nM inhibited the contraction evoked by any given concentration of NA. Yohimbine (up to 10 microM) slightly inhibited the NA-induced contraction whilst clonidine (up to 10 microM) and acetylcholine (ACh; up to 10 microM) produced no mechanical response. 3. In thin muscle bundles, NA (0.1-10 microM) produced a contraction but the phasic response was small and the tonic response was negligible. These changes were blocked by YM-12617. In contrast, ACh (0.1-10 microM) produced atropine-sensitive, large phasic and tonic responses similar to those observed on application of NA to thick muscle bundles. 4. In thin and thick muscle bundles, the mean resting membrane potentials were -54 and -56 mV, respectively, values which were not statistically different. However, in thick muscle bundles, NA (over 0.1 microM) depolarized the membrane in a concentration-dependent manner and produced repetitive spike generation; ACh (up to 1 microM) did not modify the membrane potential. In thin muscle bundles, the above concentrations of NA hyperpolarized the membrane but ACh produced a large depolarization with repetitive spike generation. 5. In thick muscle bundles, nifedipine (0.3 microM) blocked twitch contractions generated spontaneously or provoked by application of NA with no effect on phasic and tonic responses. The NA-induced depolarization persisted after superfusion with nifedipine up to a concentration of 1.0 microM. In a Ca-free solution containing 2 mM EGTA, NA produced only the phasic responses, and re-addition of Ca (2.6 mM) restored the generation of a tonic response. 6. After application of 0.3 microM nifedipine, the effects of YM-12617 and prazosin were observed on the tonic component of the NA-induced contraction of thick muscle bundles. The ID50 values for YM-12617 and prazosin were 1 nM and 15 nM, respectively (n = 4). YM-12617 shifted the NA concentration-response curve to the right in a concentration-dependent and parallel manner. The Schild plot yielded a straight line with slope of 0.97 +/- 0.05, (n = 4). The pA2 value for YM-12617 was 10.4 +/- 0.05, (n = 4).(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of the dihydropyridine Bay K 8644 in guinea-pig isolated trachealis.

In trachea bathed by Krebs solution containing indomethacin 0.8 mumol l-1, Bay K 8644 (0.01-1 mumol l-1) evoked mild spasm. Peak tension was achieved after 10 min and was generally less than 20% of an acetylcholine (ACh) maximum. The effect of Bay K 8644 was not potentiated by addition of 2.5 mmol l-1 potassium chloride (KCl) to the Krebs solution. Bay K 8644 (1 mumol l-1) caused a small potentiation of KCl and tetraethylammonium (TEA). In contrast it did not modify the actions of ACh or histamine. Bay K 8644 (1 mumol l-1) caused a small potentiation of the effect of calcium chloride (CaCl2) tested in trachea bathed by a K+-rich, Ca2+-free, MOPS-buffered physiological salt solution. Organic inhibitors of calcium influx such as nifedipine (0.1 mumol l-1), verapamil (1 mumol l-1) or diltiazem (10 mumol l-1) each caused marked depression of concentration-effect curves to KCl. Bay K 8644 (0.01-1 mumol l-1) provided concentration-dependent protection against this effect in all three cases. Estimation of calcium influx by the lanthanum technique revealed that Bay K 8644 (1 mumol l-1) was able to promote the cellular influx of Ca2+. Intracellular electrophysiological recording showed that Bay K 8644 (1 mumol l-1) caused no change in the resting membrane potential of trachealis cells and no change in the properties of the spontaneous electrical slow waves. However, Bay K 8644 was able to delay the slow wave suppression evoked by 1 mumol l-1 nifedipine. The ability of Bay K 8644 to promote Ca2+ influx and its ability to protect against the effects of several structurally-unrelated inhibitors of Ca2+ influx are consistent with Bay K 8644 acting as an agonist at the dihydropyridine receptor associated with the voltage-operated Ca2+ channel (VOC) of trachealis muscle. By this action it potentiates those spasmogens (KCl, TEA) which act by permitting Ca2+ influx through VOCs. In contrast it has no effect on those spasmogens (ACh, histamine) which principally act to liberate Ca2+ from intracellular sites of sequestration.     

The effect of L-type calcium channel modulators on the mobilization of intracellular calcium stores in guinea-pig intestinal smooth muscle.

1. The action of Ca2+ channel modulators has been examined on the intracellular Ca2+ signal in the longitudinal smooth muscle cells of the guinea-pig intestine after exposure to histamine and to agents known to affect intracellular Ca2+ stores. Isometric contraction has been measured simultaneously with front-surface fluorometry of fura 2-loaded preparations. 2. Histamine (10 microM) evoked a phasic and tonic increase in [Ca2+]i and contraction which were both sensitive to the Ca2+ channel blockers, nimodipine and D600. 3. Caffeine (10 mM) evoked in rapid increase in [Ca2+]i which was sustained as long as the preparation was exposed to the drug, whereas the contractile response was only phasic. In the presence of nimodipine 1 microM, the phasic contraction was absent although the fura 2-Ca2+ signal amounted to 32% of the control. 4. Ryanodine (10 microM) evoked a slow increase in [Ca2+]i and a contraction, both of which were reversed after exposure to nimodipine (1 microM) or D600 (10 microM). In the presence of diazoxide (500 microM), a hyperpolarizing agent, the ryanodine-evoked increase in [Ca2+]i and in muscle tone were inhibited. 5. Thapsigargin (1 microM) also produced an increase in [Ca2+]i and a contraction both of which were blocked by nimodipine (1 microM). 6. In Ca2+-free solution, histamine 10 microM evoked non-reproducible phasic Ca2+ signal and contraction. This response was recovered after refilling in Ca2+ containing solution. The recovery was blocked by nimodipine, D600 or diazoxide and was facilitated by the Ca2+ channel activator, Bay K 8644. When the refilling medium was supplemented with thapsigargin, the recovered response was significantly reduced, but Bay K 8644 still had some action. 7. The present results show that blockage of L-type Ca2+ channels inhibited changes in [Ca2+]i evoked by histamine, caffeine and ryanodine which are generally attributed to Ca2+ mobilization from intracellular stores. They also show that when the tissue was exposed to nimodipine, D600 and diazoxide during the procedure of refilling after depletion of intracellular stores, the action of histamine on [Ca2+]i and contraction was blocked. Bay K 8644 had an opposite effect even when the Ca2+ pumping activity of the sarcoplasmic reticulum was reduced by thapsigargin. This indicates that refilling of intracellular Ca2+ stores depleted by histamine in guinea-pig intestine mainly occurred through L-type Ca2+ channels.     

Ca2+ buffering action of sarcoplasmic reticulum on Bay k 8644-induced Ca2+ influx in rat femoral arterial smooth muscle

We examined the Ca2+ buffering action of sarcoplasmic reticulum during the stimulation of arterial smooth muscle with Bay k 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyr idine-5-carboxylate]. The effects of Bay k 8644 on tension and cellular Ca2+ level were first determined in endothelium-denuded strips of rat femoral artery. The Ca2+ buffering action was examined by using cyclopiazonic acid and thapsigargin to inhibit Ca2+-ATPase of sarcoplasmic reticulum and ryanodine to deplete Ca2+ stored in sarcoplasmic reticulum. The addition of Bay k 8644 (0.3-300 nM) to the resting strips almost failed to cause a contraction. When the strips were preincubated with 10 microM cyclopiazonic acid, Bay k 8644 induced a concentration-dependent contraction that is antagonized by nifedipine. The maximum contraction induced by Bay k 8644 in the presence of cyclopiazonic acid was comparable to the maximum contraction induced by 65.9 mM K+-depolarization and the ED50 value for Bay k 8644 was around 5 nM. Similar results were obtained when the strips were preincubated with 30 nM thapsigargin or 10 microM ryanodine. Bay k 8644 also induced a strong contraction when the extracellular K+ concentration was elevated. During the stimulation with 100 nM Bay k 8644, the Ca2+ influx was increased. We conclude that in rat femoral arterial smooth muscle, (1) the Ca2+ influx induced by Bay k 8644 is completely buffered by Ca2+ uptake into the sarcoplasmic reticulum, and (2) this sarcoplasmic reticulum can buffer a large amount of Ca2+ that induces a maximum contraction.     

The positive inotropic action of the nifedipine analogue, Bay K 8644, in guinea-pig and rat isolated cardiac preparations.

The inotropic effect of Bay K 8644 has been studied in rat and guinea-pig atria and ventricular strips stimulated at 1 Hz, in a medium containing CaCl2 1.8 mM. The positive inotropic effect at maximal effective concentrations of Bay K 8644 was in the following order: guinea-pig ventricle greater than rat ventricle greater than guinea-pig atria greater than greater than rat atria. In rat preparations, the tension recorded at maximum effective concentrations of Bay K 8644 was similar at three different calcium concentrations (0.7, 1.8, 3.0 mM). The amplitude of the positive inotropic effect evoked by Bay K 8644 increased when atrial and ventricular contractions were reduced by lowering the external calcium concentration. The contractile tension reached in the presence of maximum effective concentrations of Bay K 8644 (3 X 10(-7) -1 X 10(-6) M) was greater than that produced by the maximum effective concentration of external calcium (3 mM) in rat ventricles but not in rat atria. High doses of nifedipine (3 X 10(-7) -1 X 10(-6) M) depressed the contraction of rat atria more than the contraction of rat ventricles. In rat ventricles, nifedipine shifted to the right the inotropic dose-effect curve of Bay K 8644. It is concluded that the interaction between nifedipine and Bay K 8644 occurred at the same binding sites. These sites have some characteristics of the low affinity binding sites of nifedipine and other related dihydropyridines.     

Regional differences in the mechanical properties of rabbit airway smooth muscle.

1. In studies of rabbit airway smooth muscle, differences in mechanical responses to acetylcholine, histamine and high K+ in intact muscles, and in Ca2+ sensitivity in skinned muscles, have been examined in tissue taken from 5 different regions of the airway. Interactions between prostaglandin F2 alpha and epithio-thromboxane A2 and the above spasmogenic agencies were also studied. 2. Mechanical responses to histamine (10 microM) and to 128 mM K+ were smallest in trachea and were largest in 3rd and 4th order bronchi. In all regions, spasm evoked by 10 microM acetylcholine was greater than that evoked by 10 microM histamine or 128 mM K+. 3. In the third and fourth branches of the rabbit right middle bronchus, contractions evoked by 10 microM acetylcholine, 10 microM histamine and 128 mM K+ showed similar amplitudes of phasic response. In Ca2+-free solution containing 2 mM EGTA, the phasic components of the acetylcholine- or histamine-induced contraction remained unchanged in comparison with that observed in Krebs solution, but the phasic and tonic components of the K+-induced contraction and the tonic changes induced by acetylcholine and histamine were abolished. 4. Two subtypes of the histamine receptor, excitatory H1- and inhibitory H2- receptors were detected on the bronchial smooth muscle. The H1-induced contraction was mediated by release of stored Ca2+ together with activation of Ca2+ influx relatively insensitive to Ca2+ antagonists. 5. The -log(EC50) values for acetylcholine and histamine (in the presence of cimetidine and atropine) were 6.11 +/- 0.11 and 5.33 +/- 0.08, respectively, in the third branch of right middle bronchus. These values were similar to those observed for trachea.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Bay K 8644 on cat adrenal catecholamine secretory responses to A23187 or ouabain

Calcium ionophore A23187 increases the rate of spontaneous catecholamine release from cat adrenal glands perfused at 37 degrees C with oxygenated Krebs bicarbonate solution, in a time- and Ca-concentration-dependent manner. The secretory profile obtained with the ionophore was not modified in the presence of the Ca channel activator Bay K 8644. Ouabain also enhanced the rate of spontaneous catecholamine outputs in a time- and concentration-dependent manner. The threshold ouabain concentration capable of producing a clear, yet delayed secretory response was 10(-6) M. Increasing ouabain concentrations up to 10(-4) M enhanced catecholamine release and shortened the time to peak release. The dihydropyridine Ca channel activator Bay K 8644 (10(-6) M) markedly potentiated the secretory effects of all ouabain concentrations used (10(-7)-10(-4) M). However, the most impressive potentiations were seen at 10(-5)M ouabain; while at this concentration ouabain alone released 2.6 +/- 0.07 micrograms catecholamines per 30 min, in the presence of Bay K 8644 the release was 73.4 +/- 5.7 micrograms per 30 min. Conversely, at a fixed ouabain concentration (10(-5) M), the potentiation was also dependent on the Bay K 8644 concentration (10(-8)-10(-5) M). Although K deprivation inhibits Na pumping as does ouabain, Bay K 8644 did not modify the rate of catecholamine release evoked by K removal from the perfusion medium. Potassium deletion, nimodipine or high Mg all reversed the fully developed secretory response evoked by ouabain plus Bay K 8644. In glands depolarized by continuous perfusion with high K solutions, once the secretory response was inactivated, the introduction of ouabain caused an enhancement of the catecholamine secretory rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological analysis of the effects of Bay K 8644 and organic calcium antagonists on the mouse isolated distal colon.

1. Bay K 8644 (10(-8) to 10(-6) M) induced concentration-related contractions of the longitudinal muscle of the mouse distal colon. The maximal responses were enhanced and the EC50 was lowered in the presence of tetrodotoxin (TTX; 1.5 x 10(-7) M). The responses were not affected by atropine (10(-7) M), mepyramine (2.5 x 10(-7) M), methysergide (5 x 10(-7) M), propranolol (10(-6) M), phentolamine (10(-6) M) or naloxone (4 x 10(-7) M). By contrast, the contractile responses were inhibited by Ca2+ entry blockers (verapamil, nifedipine) and abolished in Ca2+-free EGTA solution. These observations indicate that the contractile effects of Bay K 8644 are dependent on its ability to promote Ca2+ influx. 2. At 10(-4) M, Bay K 8644 provoked a slow relaxation of the preparation. Moreover, from 10(-5) M, Bay K 8644 markedly reduced the contractile responses to ACh and K+ depolarization. These inhibitory effects were comparable with those produced by nifedipine. Such data suggest that, at high concentrations, Bay K 8644 could act in part as a dihydropyridine Ca2+ channel antagonist. 3. Bay K 8644 (10(-9) M) preferentially enhanced, while nifedipine (10(-10) to 10(-8) M) as well as verapamil (3 x 10(-9) to 10(-6) M) preferentially inhibited, the tonic component of the contractile response evoked by K+ depolarizing solution. This may indicate that different populations of voltage-sensitive Ca2+ channels are involved in the biphasic response to K+ depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase A inhibitors selectively inhibit the tonic contraction of the guinea pig ureter to high potassium

• 1.1. We have investigated the effect of various protein kinase A (PKA) inhibitors on the phasic and tonic components of the response to potassium chloride (KCI) in the guinea pig ureter. All experiments were performed in ureters pretreated with capsaicin (10 μM for 15 min) to prevent the release of sensory neuropeptides and in the presence of 1 μM Bay K 8644 to maximize calcium (Ca) entry via voltage-sensitive channels. The addition of 80 mM hypertonic KCI produced maximal shortening of the ureter with distinct phasic and tonic components, the latter further showing a transient and a sustained component. Nifedipine (30 μM for 120 min) totally abolished all the responses to KCI.
• 2.2. The selective PKA inhibitor, H89 (10 μM), abolished the tonic response to KCI in about 30 min with minor inhibitory effect on the phasic contraction. This pattern was unchanged when extending the contact time to 120 min. When added 30 min before the next challenge, H89 (1–30 μM) concentration-dependently inhibited the responses to KCI with a preferential inhibitory effect on the tonic contraction. Another PKA inhibitor, H8, produced similar effects at tenfold higher concentrations (10–300 μM) than H89, consistent with the known potency ratio of these isoquinoline derivatives in inhibiting PKA.
• 3.3. The potent and nonselective protein kinase inhibitor, staurosporine (10–100 nM) produced an even depression of the various phases of the response to KCI. The selective protein kinase G inhibitor, KT 5823 (10 μM for 60 min) produced only a slight reduction of the sustained tonic response to KCI. The selective protein kinase C inhibitor GF 109,203X (1–3 μM) and the cAMP analog, Rp-cAMPS (300 μM for 60 min) had no effect on the three components of the response to KCI.
• 4.4. In the presence of Bay K 8644, electrical field stimulation (10 Hz for 1 sec, 60 V, pulse width 5 ms) produces direct myogenic phasic contractions (twitches) of the ureter which are suppressed by nifedipine (10–30 μM). H8 (up to 30 μM) and H89 (up to 300 μM) had minor effect on the amplitude of twitches, consistent with their poor inhibitory activity on the phasic responses to KCI.
• 5.5. In sucrose gap, superfusion with 80 mM hypertonic KCI produced action potentials followed by a sustained depolarization of the membrane: the two electrical responses underlie the phasic and tonic components of contraction to KCI, respectively. H89 (10 μM for 30 min) did not affect the resting membrane potential nor the KCl-evoked action potentials and sustained depolarization. H89 had no effect on the phasic contraction to KCl but markedly depressed (about 65% inhibition) the tonic contraction.
• 6.6. The present findings are consistent with the view that phosphorylation by PKA increases the availability of L-type Ca channels in the ureter smooth muscle. Blockade of PKA dissociates the electromechanical coupling between the sustained membrane depolarization produced by KCI and the corresponding sustained increase in tension. The L-type Ca channel responsible for generating action potentials and phasic contractions to KCI are less sensitive to PKA inhibitors than those responsible for the tonic contraction.

     

Cellular localization of the inhibitory action of relaxin against uterine spasm.

1. The aim of this study was to determine whether the site of action of relaxin as a relaxant of rat myometrium is at the cell membrane or at an intracellular-site. Therefore, the potency of relaxin was determined against spasms reliant predominantly upon either extracellular Ca2+ or intracellular Ca2+. Uterine spasms dependent upon extracellular Ca2+ were elicited by (i) oxytocin (0.2 nM) (ii) Bay K 8644 (1 microM) in 10 mM K(+)-rich PSS and (iii) KCl (80 mM). Uterine spasm dependent upon intracellular Ca2+ was elicited by oxytocin (20 nM) in the presence of nifedipine (500 nM). The effects of relaxin against these spasmogens were compared with those of levcromakalim, nifedipine and salbutamol. 2. Relaxin (0.2-6.3 nM), levcromakalim (25-800 nM), salbutamol (1-63 nM) and nifedipine (1-250 nM) caused concentration-dependent inhibition of the spasm evoked by oxytocin (0.2 nM) and relaxin was the most potent relaxant. 3. Relaxin and nifedipine were slightly less potent against the spasm induced by Bay K 8644 (1 microM) than against spasm induced by oxytocin (0.2 nM) (15 fold and 13 fold respectively). Levcromakalim and salbutamol were equipotent against the spasm evoked by Bay K 8644 (1 microM) and that evoked by oxytocin (0.2 nM). 4. Relaxin induced only 47 +/- 7% inhibition of the KCl (80 mM)-evoked spasm at a concentration of 0.8 microM. Levcromakalim was much less potent (427 fold) against the spasm evoked by KCl (80 mM) than against the spasm evoked by oxytocin (0.2 nM). The potency of salbutamol against the spasm evoked by KCl (80 mM) was modestly reduced (14 fold) compared to that against the spasm evoked by oxytocin (0.2 nM). The potency of nifedipine against the KCl (80 mM)-evoked spasm was not different from that against the oxytocin (0.2 nM)-evoked spasm. 5. The potencies of relaxin and levcromakalim against the spasm evoked by oxytocin (20 nM) + nifedipine (500 nM) were greatly reduced (74 fold and 234 fold respectively) compared to their potencies against the spasm evoked by oxytocin (0.2 nM). The potency of salbutamol against these two spasmogens was not different. 6. Relaxin was much less potent against the spasm dependent upon intracellular Ca2+ (that induced by oxytocin (20 nM) + nifedipine (500 nM)) than against the spasms dependent upon extracellular Ca2+, those induced by oxytocin (0.2 nM) and Bay K 8644 (1 microM). In this regard, relaxin resembled levcromakalim and nifedipine rather than salbutamol. Therefore, the major site of action of relaxin appears to be located at the plasma membrane rather than at an intracellular level. The observation that relaxin was less effective against the KCl (80 mM)-induced spasm than against the oxytocin (0.2 nM)-evoked spasm may indicate that relaxin has a minor action involving K(+)-channel opening. 7. High concentrations of relaxin (up to 1 microM) induced significant inhibition of the spasm dependent upon intracellular Ca2+. Thus at high concentrations relaxin also appears to have an additional intracellular action.     

Effects of Bay K 8644 on contraction of the human isolated bronchus and guinea-pig isolated trachea.

The effects of Bay K 8644, a dihydropyridine which increases calcium flux through the potential-operated channels were studied on the contractions induced by histamine, acetylcholine, KCl and Ca2+ on human isolated bronchial strips and the results were compared to those obtained on guinea-pig isolated tracheal spirals. Subsequently the contractant effects of Bay K 8644 in K+-enriched medium and in the presence of Ca2+ 0.03 mM were investigated. In Krebs normal calcium medium, Bay K 8644 did not significantly modify the EC50 of acetylcholine or histamine on the human bronchus, but in concentrations of 10(-7)-10(-6)M it potentiated the effects of KCl on that preparation. It did not modify the EC50 of acetylcholine, histamine or KCl on the guinea-pig trachea. In Ca2+-free Krebs medium with additional K+ (30 mM), Ca2+ concentration-response curves were displaced to the left by Bay K 8644 in the two preparations. Shifts were 0.52 +/- 0.11 and 0.72 +/- 0.16 log units respectively with Bay K 8644 10(-8) and 10(-7) M on human bronchus (n = 4) and 0.67 +/- 0.16 and 1.06 +/- 0.19 log units respectively with Bay K 8644 10(-7) and 10(-6) M on the guinea-pig trachea (n = 5). In Krebs medium with Ca2+ 0.03 mM and K+ 30 mM, Bay K 8644 (10(-8) to 10(-6) M) contracted both the human bronchus and the guinea-pig isolated trachea. This effect was competitively antagonized by nicardipine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Bay K 8644 and ryanodine on the refractory period,action potential and mechanical response of the guinea-pig ureter to electrical stimulation

We have investigated the effect of the dihydropyridine calcium channel agonist, Bay K 8644, and of the plant alkaloid blocker of calcium-induced calcium release (CICR) from the sarcoplasmic reticulum, ryanodine, on the refractory period, action potential and mechanical response of the guinea-pig isolated ureter to electrical stimulation. All experiments were performed in ureters pre-exposed to 10 M capsaicin to eliminate the inhibitory influence exerted by local release of sensory neuropeptides on ureteral excitability and contraction. In organ bath experiments, electrical field stimulation with parameters which produce direct excitation of ureteral smooth muscle (train of pulses at 10 Hz, 5 ms pulse width, 60 V for 1 s) produced tetrodotoxin- (1 M) resistant phasic contractions. The response to EFS was abolished by nifedipine (1 nM-3 M) and was enhanced by Bay K 8644 (1 nM-3 M). In the presence of Bay K 8644 (1 M), nifedipine (30 M) abolished the evoked contractions. Ryanodine (10–100 M) had no significant effect on the amplitude of evoked contraction. The response of the guinea-pig ureter to direct electrical stimulation of smooth muscle is characterized by a refractory period: at least 40 s interstimulus interval was required to produce a second response in all preparations tested. Bay K 8644 (1 M) markedly reduced the refractory period of the ureter and a similar effect was observed with ryanodine (100 M). To further analyze the effect of Bay K 8644 and ryanodine on the refractory period, the response of the ureter was investigated over a 10 s period of stimulation (other parameters as above). In control ureters, continuous stimulation for 10 s produced only one phasic contraction just after the beginning of the train of stimuli. In the presence of Bay K 8644 or ryanodine, more than one phasic contraction developed during a 10 s stimulation, i.e. the refractory period became shorter than the train duration. When both Bay K 8644 and ryanodine were tested on the same preparations, an additive excitatory effect was observed on the mechanical response to electrical stimulation. A slight elevation of KCI concentration (5–10 mM) reduced the refractory period of the ureter as observed with ryanodine or Bay K 8644. Application of KCI (80 mM) produced a biphasic contractile response of the ureter: a series of phasic contractions occurred first, which were then replaced by a slowly developing tonic response. Bay K 8644 (1 M) enhanced both components of the response to KCI. Ryanodine (10 and 100 M) markedly prolonged the duration of phasic contractions evoked by KCI and, at 100 M, slightly (about 25%) reduced the amplitude of tonic contraction.In sucrose gap experiments, electrical stimulation (single pulse, 40–130 V, 1–3 ms pulse duration) evoked an action potential and accompanying phasic contraction which were abolished by 1 M, nifedipine. Bay K 8644 (1 M) produced a marked prolongation of action potential duration, increased the number of spikes and enhanced contraction amplitude and duration. Ryanodine (100 M) depolarized the membrane, reduced the delay between stimulus application and onset of the action potential, shortened the action potential at 50% of repolarization and increased afterhyperpolarization, without producing marked effects on the accompanying mechanical response. KCI (5 mM) likewise produced a slight membrane depolarization and decreased latency between stimulus application and onset of the action potential but did not affect action potential duration. The combined administration of ryanodine and Bay K 8644 produced additive effects on action potential and contractions: furthermore, the contractile phase of the overall contraction-relaxation cycle was significantly prolonged by the combined administration of the two agents, an effect not observed with either drug alone. In the presence of both Bay K 8644 and ryanodine, multiple action potentials and contractions were observed during a train of pulses delivered at a frequency of 1 Hz for 12 s: when a second action potential was triggered before relaxation of the preceding contraction, a summation of the contractile response was observed. These findings demonstrate that availability of voltage-dependent L-type calcium channels is a major mechanism in determining the refractory period of the guinea-pig ureter and, consequently, can be considered as a limiting step in regulating the maximal frequency of ureteral peristalsis. Furthermore, a ryanodine-sensitive mechanism regulates the excitability and contraction-relaxation cycle of ureteral smooth muscle. The increased electrical excitability of the ureter observed in the presence of ryanodine may involve blockade of transient outward currents triggered by spontaneous calcium release from the store and consequent membrane depolarization.     

The stimulatory action of the calcium channel agonist Bay K 8644 on isolated duodenal muscle

Summary The action of the novel dihydropyridine analogue Bay K 8644 has been evaluated on the rat isolated duodenal muscle. Bay K 8644 (0.3 nmol/l to 1 pmol/l) increased both the tone and the phasic movements of the duodenum; the maximum response was about 60% of that to acetylcholine. Nifedipine (30 nmol/l) induced a parallel shift of the concentration-response curve to this compound to the right, without depressing the maximum response; conversely, verapamil (30 nmol/l) caused an unsurmountable antagonism. Incubation of the strips in Ca²⁺-free medium reduced the contractile response to the calcium agonist. The spasmogenic effect of Bay K 8644 was inhibited by atropine (0.1 pmol/l) which caused a significant reduction in the maximum response to the compound. The competitive interaction between Bay K 8644 and nifedipine is consistent with an action at the same dihydropyridine binding site in the calcium channel and suggests that these compounds could be selective probes for detecting the dihydropyridine receptor also in the intestinal smooth muscle. The sensitivity of the contractile effect of Bay K 8644 to atropine may indicate an action of this compound in the cholinergic system, probably mediated by a release of acetylcholine from the nerve terminal rather than due to a direct stimulatory action at muscarinic receptors in the smooth muscle.