Effects of K+ channel blockers and cromakalim (BRL 34915) on the mechanical activity of guinea pig detrusor smooth muscle.

There is strong evidence that cromakalim (BRL 34915) relaxes smooth muscle by opening cell membrane K+ channels. The aim of this study was to use relatively selective K+ channel blockers to investigate 1) the K+ channel type(s) opened by cromakalim in guinea pig detrusor and 2) the role of different K+ channel types in the control of basal tension. Cromakalim produced a concentration-related relaxation (IC50 = 0.50 +/- 0.03 microM, n = 42) of 15 mM K(+)-evoked mechanical activity. The ATP-sensitive K+ channel blocker glyburide (0.3-3 microM) antagonized the effects of cromakalim in an apparently competitive manner (pA2 = 6.76). Charybdotoxin and iberiatoxin (3-30 nM), blockers of the large conductance, Ca(++)-activated K+ channel, appeared to functionally antagonize cromakalim. Apamin (1 microM) and leiurotoxin I (0.3 microM), blockers of the small conductance, Ca(++)-activated K+ channel, and noxiustoxin (0.3 microM), a blocker of squid axon delayed rectifer K+ channels, all failed to antagonize cromakalim. Cumulative administration of charybdotoxin and iberiatoxin produced marked, concentration-related stimulation of mechanical activity per se whereas glyburide, noxiustoxin, apamin and leiurotoxin I had no effect. Apamin and leiurotoxin I did stimulate mechanical activity to a small extent when administered noncumulatively, however. The results suggest that cromakalim opens ATP-sensitive K+ channels in detrusor and suggest that cromakalim does not open CA(++)-activated K+ channels and noxiustoxin-sensitive, delayed rectifier K+ channels. The marked stimulatory effects of charybdotoxin and iberiatoxin per se suggest an important role for large conductance, Ca(++)-activated K+ channels in the control of basal tension and, presumably, membrane potential in detrusor smooth muscle cells.     

Modulation of ATP-sensitive K+ channel activity and contractile behavior in mammalian ventricle by the potassium channel openers cromakalim and RP49356

We have investigated the effects of potassium channel opening drugs on the ATP-dependence of ATP-sensitive K+ channel activity and on contractile activity in rat and guinea pig ventricular myocytes. The results show that cromakalim (BRL34915), and RP49356, agents reported to open ATP-sensitive K+ channels, do so by shifting the intracellular [ATP] required to cause half-maximal inhibition of channel activity (ki) to higher [ATP]. In guinea pig ventricular myocytes at 37 degrees C, the ki was shifted from 79 to 152 microM by 40 microM cromakalim and, in rat myocytes at room temperature, the ki was also shifted to higher [ATP] by 50 microM RP49356. The effect of externally applied RP49356 on the contractile activity of intact rat ventricular myocytes was investigated. At 100 microM the drug was without effect in the presence of normal bathing solution containing 10 mM glucose. When glucose in the bathing medium had been replaced by 2-deoxyglucose for 84 +/- 2 min, 100 microM RP49356 decreased the twitch amplitude to 23 +/- 4% of control. The negative inotropic effect of 100 microM RP49356 increased with time after perfusion with 2-deoxyglucose, and the negative inotropic effect diminished on reperfusing with glucose; 83 +/- 3 min after reperfusing with glucose, twitch amplitude was decreased by only 52 +/- 6% on exposure to 100 microM RP49356. These results suggest that the effect of the potassium channel opening drugs on contractility and electrical behavior will depend critically on the intracellular [ATP]. The results provide an explanation of how potassium channel openers may become clinically useful as cardioprotective agents without interfering with normal function.     

SR 33557, a novel calcium entry blocker. I. In vitro isolated tissue studies.

The effects of SR 33557 on isolated cardiovascular preparations were compared to those of nifedipine, verapamil and diltiazem. In rat aortic strips, SR 33557, like nifedipine, verapamil and diltiazem, caused a significant and simultaneous inhibition of potassium-induced 45Ca++ influx and contractile responses (nifedipine greater than SR 33557 greater than verapamil greater than diltiazem). SR 33557 also antagonized Ca(++)-induced contractions in K(+)-depolarized aorta preparations (pA2:9.08 +/- 0.03) and is the first calcium channel antagonist, structurally not related to 1,4-dihydropyridines, to inhibit competitively contractions induced by BAY K8644. In spike-generating vascular smooth muscle (rat portal vein), contractures evoked by noradrenaline (4 microM) or KCl (100 mM) were reduced by all four antagonists, the pharmacological potency being nifedipine greater than SR 33557 greater than verapamil greater than diltiazem. Unlike SR 33557, nifedipine, verapamil and diltiazem showed a parallel enhancement of frequency of spontaneous contractions in rat portal vein in spite of a concentration-related reduction in amplitude. By using rabbit atrial preparations, spontaneous right atrial rate and electrically stimulated (120/min) basal contractions of left atria were used as indices of chronotropy and inotropy. The potency series for negative chronotropic effects was nifedipine greater than SR 33557 greater than verapamil greater than diltiazem. For negative inotropic effects the potency order was verapamil greater than nifedipine greater than SR 33557 greater than diltiazem, respectively. Thus, SR 33557 should depress heart rate to a greater extent than ventricular contractility. These results suggest that SR 33557 is a potent calcium entry blocker that (unlike verapamil and diltiazem) is particularly selective for vascular smooth muscle and devoid of any potent negative inotropic actions.     

Potassium channel openers: pharmacological and clinical aspects

Opening of plasmalemmal K+ channels leads to cellular hyperpolarization which, in excitable tissues possessing voltage-dependent Ca2+ channels, prevents the opening of such channels and thus prevents excitation. In the last few years, an increasing number of compounds have been identified which elicit their effects by opening K+ channels, preferentially in smooth muscle, but also in other excitable tissues. These include the novel benzpyrans, cromakalim and bimakalim, the thioformamide aprikalim, and also well known antihypertensives such as minoxidil sulphate, diazoxide and pinacidil. After a short overview of the various families of K+ channel openers (KCOs), their basic pharmacological properties, including inhibition by the sulfonyl ureas (such as glibenclamide) are presented. The actual discussion concerning the type of K+ channel(s) opened by these compounds and their mechanism(s) of vasorelaxation will be reported. The therapeutic potential of these compounds in the cardiovascular field (as antihypertensives and, in particular, as anti-ischemic agents in heart and skeletal muscle), and in asthma (where they reverse established airway hyperreactivity) will also be discussed. Improved tissue selectivity may be the essential pre-requisite for true clinical success of this class of compounds.     

Comparison of effects of cromakalim and pinacidil on mechanical activity and 86Rb efflux in dog coronary arteries

Effects of two K+ channel openers, cromakalim and pinacidil, on mechanical activity and on 86Rb efflux were compared in strips of dog coronary arteries. Cromakalim and pinacidil produced the relaxation in 20.9 mM K(+)-contracted strips with a pD2 of 6.53 and 5.95, respectively. In 65.9 mM K(+)-contracted strips, high concentrations of pinacidil, but not cromakalim, produced relaxation. Ca+(+)-induced contractions in 80 mM K(+)-depolarized strips were also inhibited by pinacidil but not by cromakalim. Glibenclamide, a blocker of ATP-regulated K+ (KATP) channels, competitively antagonized the relaxant responses to cromakalim with a pA2 value of 7.62. However, the antagonism by glibenclamide of the relaxant responses to pinacidil was not a typical competitive type, suggesting the contribution of other effects than the KATP channel opening activity to the relaxant effects of pinacidil. In resting strips preloaded with 86Rb, cromakalim and pinacidil increased the basal 86Rb efflux in a dose-dependent manner. The increase in the 86Rb efflux induced by cromakalim was greater than that by pinacidil. When the effects of cromakalim and pinacidil on the 86Rb efflux were determined in the 20.9 or 65.9 mM K(+)-contracted strips, both drugs increased the 86Rb efflux. Under the same conditions nifedipine, a Ca(+)+ channel blocker, produced the relaxation that is accompanied by the decrease in 86Rb efflux. The increase in the 86Rb efflux induced by cromakalim was much greater than that by pinacidil.(ABSTRACT TRUNCATED AT 250 WORDS)     

Paradoxical inhibitory effect of cromakalim on 86Rb outflow from pancreatic islet cells

Cromakalim appears to be the most potent pharmacologic agent belonging to the new class of smooth muscle relaxants: the "K+ channel openers." The present study aimed at characterizing the effects of cromakalim on 86Rb outflow, 45Ca outflow and insulin release from prelabeled and perifused rat pancreatic islets. Cromakalim provoked a concentration-dependent reduction in 86Rb outflow. This inhibitory effect was attenuated in islets exposed throughout to glibenclamide or to a Ca+(+)-free medium. In islets exposed to glucose and extracellular Ca++, cromakalim induced a dose-dependent reduction in 45Ca outflow. The drug also inhibited the increase in 45Ca outflow mediated by K+ depolarization. Lastly, cromakalim elicited a concentration-dependent inhibition of insulin release from islets perifused in the presence of glucose and extracellular Ca++. The present data suggest that the paradoxical inhibitory effect of cromakalim on 86Rb outflow probably reflects the capacity of the drug to reduce the activity of the ATP-sensitive K+ channels and to indirectly inhibit the Ca+(+)-activated K+ channels. Furthermore, the cromakalim-induced changes in 45Ca outflow are compatible with an inhibitory effect of the drug on the voltage-dependent Ca++ channels.     

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.     

Inhibition of tracheal smooth muscle contraction and myosin phosphorylation by ryanodine

Previous studies have shown that muscarinic activation of airway smooth muscle in low Ca++ solutions increases myosin phosphorylation without increasing tension. Blocking Ca++ influx reduced phosphorylation, but not to basal levels. It was proposed that release of intracellular Ca++ contributed to dissociation of phosphorylation and contraction. To test this hypothesis the effects of ryanodine were studied under similar conditions. Ryanodine (10(-7) to 10(-5) M) antagonized caffeine-induced contraction of canine tracheal smooth muscle. Ryanodine also reduced carbachol-induced contractions and carbachol-induced myosin phosphorylation. The effect of ryanodine on potassium and serotonin-induced contractions was also investigated to test for a nonspecific inhibitory effect. In contrast to the effect on carbachol responses, ryanodine (10(-5) M) potentiated the contractile response to low concentrations of serotonin and potassium, but had no effect on the maximum response to either stimulant. Carbachol (10(-6) M) and ryanodine (10(-5) M) both significantly decreased 45Ca++ content of tracheal muscle. The effect of ryanodine and carbachol together on 45Ca++ content was not greater than either drug alone suggesting that ryanodine reduces the caffeine and carbachol responses by depleting releaseable Ca++ stores. Ryanodine significantly reduced Ca++-induced contraction and myosin phosphorylation in carbachol-stimulated muscle, suggesting that some of the Ca++ responsible for elevated phosphorylation is released from the sarcoplasmic reticulum.     

Possible involvement of ATP-sensitive K+ channels in the relaxant response of dog middle cerebral artery to cromakalim

To determine the functions of ATP-sensitive K+ (KATP) channels in cerebral arterial smooth muscle, the effects of cromakalim, an opener of these channels, on tension and 86Rb efflux were investigated in endothelium-removed strips of dog middle cerebral arteries (MCAs). Cromakalim relaxed the strips that were precontracted with 20.9 mM K+ with a small maximum response. The relaxant responses to cromakalim were competitively antagonized by glibenclamide, a blocker of KATP channels. In strips precontracted with 65.9 mM K+, cromakalim failed to relax the strips. The addition of cromakalim to a resting strip caused a dose-dependent relaxation. In the resting strips of MCAs preloaded with 86Rb, cromakalim did not increase the 86Rb efflux. With 42K as the tracer ion, cromakalim still had no effect on the efflux from the resting strips. On the other hand, cromakalim increased the 86Rb and 42K efflux from the strips of dog coronary arteries (CAs). In 20.9 mM K(+)-contracted strips of MCAs, cromakalim significantly decreased the 86Rb efflux. However, after the inactivation of Ca(++)-activated K+ channels by the addition of 1 x 10(-7) M nifedipine to the 20.9 mM K(+)-contracted strips of MCAs, cromakalim produced a small but significant increase in the 86Rb efflux. Similarly, when the resting strips of MCAs were placed in the Ca(++)-free 12 mM-Mg(+)+ solution, cromakalim increased the 86Rb efflux. In 65.9 mM K(+)-contracted strips, cromakalim increased the 86Rb efflux from both arteries. However, the extent of the increase in 86Rb efflux was significantly smaller in the MCA than in the CA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca++ utilization in the constriction of rat aorta to stimulation of protein kinase C by phorbol dibutyrate

To determine the role of activated protein kinase C in vascular smooth muscle contraction, phorbol dibutyrate was used to stimulate this enzyme in order to evaluate the source(s) of Ca++ (10(-8) to 3 X 10(-6) M) elicited a concentration-dependent sustained contraction which was slow in onset but progressive in developed tension. The maximal contractile response induced by phorbol dibutyrate was only partly dependent on influx of extracellular Ca++ as shown by similar reductions (40%) produced by Ca++-free buffer, LaCl3 (1 mM) or nifedipine (10(-6) M). These data suggest that phorbol dibutyrate is able to open Ca++ channels which are sensitive to nifedipine blockade. However, unlike norepinephrine or K+-depolarization, phorbol dibutyrate evoked a slow 45Ca++ influx which occurred only after extended contact time. Pretreatment with nifedipine again abolished this response. In contrast to norepinephrine, phorbol dibutyrate did not cause 45Ca++ efflux indicating that intracellular Ca++ was not mobilized. It is concluded that the residual 60% contraction to phorbol dibutyrate most likely occurs via a mechanism independent of the Ca-calmodulin pathway.     

Peripheral type benzodiazepine receptor and airway smooth muscle relaxation

It has been postulated that a benzodiazepine receptor with a micromolar affinity may be associated with Ca++ channels in peripheral organs. We examined the actions of Ro5-4684 (parachlorodiazepam) and midazolam on guinea pig tracheal smooth muscle contraction. Binding studies using [3H]Ro5-4684 indicate the presence of a "peripheral" type binding site with a Kd of approximately 4 nM and maximum binding of 1 pmol/mg of protein. Midazolam did not displace radioligand. In tension studies no activity was seen for Ro5-4684 or midazolam at concentrations below 1 microM. Higher concentrations relaxed the airway smooth muscle under basal tone, the effect was augmented significantly by epithelium removal. Similar results were obtained in tissues precontracted with methacholine or KCl. Midazolam (1 or 100 microM) significantly (P less than .05) attenuated the response to Ca++ in K+-depolarized tracheal strips, the effect was greater at low Ca++ concentrations. The compounds appear to function as Ca++ antagonists in airway smooth muscle but ar not typical as shown by their ability to reduce basal tone in airway smooth muscle.     

Mast cell tryptase causes airway smooth muscle hyperresponsiveness in dogs.

Supernatants obtained by degranulation of dog mastocytoma cells greatly increased the sensitivity and the magnitude of the contractile response of isolated dog bronchial smooth muscle to histamine. The enhanced contractile response was reversed completely by H1-receptor antagonists and was prevented by an inhibitor of tryptase (a major protease released with histamine from secretory granules of mast cells). The potentiation of histamine-induced contractions was reproduced by active tryptase in pure form. The contractions due to the combination of histamine and purified tryptase were abolished by the Ca2+ channel blockers nifedipine and verapamil. The bronchoconstricting effects of KCl and serotonin, which, like histamine, contract airway smooth muscle by a mechanism predominantly involving membrane potential-dependent Ca2+ transport, were also potentiated by tryptase. However, the contractile effects of acetylcholine, which contracts dog airway smooth muscle by a mechanism independent of Ca2+ channels, were unaffected by tryptase. These findings show a striking promotion of agonist-induced bronchial smooth muscle contraction by mast cell tryptase, via direct or indirect effects on Ca2+ channels, and the findings therefore suggest a novel potential mechanism of hyperresponsiveness in dog bronchi.     

Factors affecting rabbit mesenteric artery smooth muscle sensitivity to calcium antagonists

The sensitivity of rabbit isolated superior mesenteric artery to Ca++ antagonists was examined under various conditions. Relaxation dose-response curves for D600 or nifedipine were generated, and IC50 values were calculated. In the first series of experiments, D600 or nifedipine IC50 was found to be 20-25-fold greater for norepinephrine (NE, 5 microM) contraction than for 80 nM K+ contraction. Even when the tissues were depolarized with 80 mM K+ before NE contraction, D600 or nifedipine IC50 still remained significantly greater compared with 80 mM K+ alone and remained closer to that during NE alone. Also a protocol was designed to study NE-induced phasic contraction in EGTA-physiological salt solution (a functional indicator of intracellular Ca++ release) as well as NE-induced sustained contraction after readdition of Ca++. The effects of varying [K+]ex (0-80 nM range) on NE-induced [Ca++]i release as well as on the D600 IC50 for NE contraction was studied. Increasing [K+]ex was found to enhance NE-sensitive [Ca++]i release and lower the D600 IC50 for NE contraction. Thus, conditions causing an increase in the ability of NE to cause [Ca++]i release were associated with an increase in the sensitivity of NE contraction to D600. These data provide functional evidence that the receptor-agonist sensitive Ca++ influx process in vascular smooth muscle is not solely regulated by changes in membrane potential. Additional mechanisms, such as a modulatory role of [Ca++]i release, in this process are implicated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca++ utilization in the constriction of rat aorta to full and partial alpha-1 adrenoceptor agonists

l-Norepinephrine and l-phenylephrine were full agonists and cirazoline, SKF d-89748, Sgd 101/75 and SKF l-89748 were partial agonists in contracting rat isolated aortic rings. Clonidine, l-amidephrine and St 587 were found ineffective. Nifedipine (10(-8)-10(-6) M) abolished the contractions to Sgd 101/75 and to high K+ with similar potency but only partially inhibited the contractions to the other alpha adrenoceptor agonists. Norepinephrine, phenylephrine, cirazoline and Sgd 101/75 were full agonists in stimulating 45Ca++ influx, which amounted to 50% of the maximal influx produced by high K+. SKF d- and l-89748 behaved as partial agonists, whereas St 587, amidephrine and clonidine were virtually inactive. Nifedipine was equally effective in blocking the influx of 45Ca++ produced by K+ and the alpha adrenoceptor agonists. Norepinephrine stimulated 45Ca++ efflux to an extent similar to that for high K+. In the following order of decreasing efficacy, phenylephrine, cirazoline and SKF d- and l-89748 caused significant stimulation of 45Ca++ efflux. Sgd 101/75, amidephrine, clonidine and St 587 were without effect. However, Sgd 101/75 (10(-5) M) antagonized the 45Ca++ efflux of norepinephrine. Nifedipine (3 X 10(-7) M) completely suppressed the K+-induced 45Ca++ efflux but only partly affected the 45Ca++ efflux caused by the alpha adrenoceptor stimulants. A highly significant (r = 0.975) linear relationship was found between the nifedipine-resistant contractile response and the 45Ca++ efflux obtained in the presence of nifedipine. The data suggest that the stimulation of alpha-1 adrenoceptors in rat aorta can activate two distinct processes of Ca++ utilization for contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro and in vivo comparison of two K+ channel openers, diazoxide and cromakalim, and their inhibition by glibenclamide

Diazoxide caused an increase in 86Rb+ efflux from the rat aorta and portal vein and inhibited spontaneous activity of the latter at concentrations 100 times higher than the K+ channel opener cromakalim. In the rabbit aorta both drugs inhibited vasoconstrictor responses to angiotensin II, noradrenaline and low concentrations (less than or equal to 30 mM) of KCl in a similar manner, the antivasoconstrictor activities being abolished in vessels depolarized with greater than or equal to 35 mM K+. In vivo cromakalim was about 100 times more potent than diazoxide at lowering blood pressure in rats. Diazoxide (30 mg/kg) caused a more than 2-fold increase in plasma glucose in rats and prevented any return toward base line within 1.5 hr after a glucose load. Cromakalim had minimal effects upon glucose homeostasis at equihypotensive doses. Glibenclamide, a potent blocker of ATP-dependent K+ channels, inhibited the stimulation by cromakalim and diazoxide of 86Rb+ efflux from the portal vein and aorta (IC50 approximately 0.1 microM), antagonized their vasorelaxant effects in vitro and in vivo (20-30 mg/kg i.v.) and reversed the diazoxide-induced changes in plasma glucose and insulin levels. These results provide evidence that diazoxide, like cromakalim, is able to open 86Rb+-permeable K+ channels in vascular smooth muscle. This action is likely to be responsible for the in vitro and in vivo vasodilator activity of these two drugs. However, there would seem to be pharmacological differences between the K+ channels affected by these drugs in vascular smooth muscle and the (ATP-sensitive) K+ channels of pancreatic beta-cells, which are thought to be responsible for the effects of diazoxide on plasma glucose.     

Agonist actions of Bay K 8644, a dihydropyridine derivative, on the voltage-dependent calcium influx in smooth muscle cells of the rabbit mesenteric artery

Actions of methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2- trifluoromethylphenyl)-pyridine-5-carboxylate (Bay K 8644) on the mechanical response evoked in intact and skinned mesenteric artery of the rabbit were investigated. The data were compared to that of nisoldipine, another dihydropyridine derivative Bay K 8644 increased the amplitudes of both the phasic and tonic components of the K+-induced contraction which is due to an increase in the voltage-dependent influx of Ca ion. Bay K 8644 antagonized competitively the actions of nisoldipine (a Ca antagonist) on the tonic but not on the phasic component of the K+-induced contraction. The contractions caused by high concentrations of norepinephrine were enhanced to a greater extent by Bay K 8644 than that evoked by lower concentrations of norepinephrine. Bay K 8644 had no effect on Ca++ extrusion from cells, which was estimated from the change in amplitudes of the norepinephrine-induced contractions in Na+- and Ca++-free solutions. This agent had no effect on the contractile proteins and Ca storage sites, as estimated from the Ca++- or caffeine-induced contraction observed in skinned muscles. The results suggested that Bay K 8644 acts primarily on the voltage-dependent Ca++ channel, presumably the same site at which other dihydropyridine derivatives (Ca antagonists) act, and that the influx of Ca++ is accelerated.     

KATP channel openers: structure-activity relationships and therapeutic potential

ATP-sensitive potassium channels (K(ATP) channels) are heteromeric complexes of pore-forming inwardly rectifying potassium channel subunits and regulatory sulfonylurea receptor subunits. K(ATP) channels were identified in a variety of tissues including muscle cells, pancreatic beta-cells, and various neurons. They are regulated by the intracellular ATP/ADP ratio; ATP induces channel inhibition and MgADP induces channel opening. Functionally, K(ATP) channels provide a means of linking the electrical activity of a cell to its metabolic state. Shortening of the cardiac action potential, smooth muscle relaxation, inhibition of both insulin secretion, and neurotransmitter release are mediated via K(ATP) channels. Given their many physiological functions, K(ATP) channels represent promising drug targets. Sulfonylureas like glibenclamide block K(ATP) channels; they are used in the therapy of type 2 diabetes. Openers of K(ATP) channels (KCOs), for example, relax smooth muscle and induce hypotension. KCOs are chemically heterogeneous and include as different classes as the benzopyrans, cyanoguanidines, thioformamides, thiadiazines, and pyridyl nitrates. Examples for new chemical entities more recently developed as KCOs include cyclobutenediones, dihydropyridine related structures, and tertiary carbinols.     

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)     

Cellular mechanisms of somatostatin inhibition of calcium influx in the anterior pituitary cell line AtT-20.

The cellular mechanisms by which the hypothalamic peptide somatostatin (SRIF) inhibits Ca+(+) influx were investigated in the pituitary cell line AtT-20. Cytosolic Ca+(+) levels were measured using the fluorescent probe Quin 2. Calcium influx was stimulated by the Ca+(+) channel agonist Bay K 8644. Bay K 8644 increased Ca+(+) influx in a concentration-dependent manner and the stimulation of Ca+(+) influx was blocked by the Ca+(+) channel antagonists nifedipine and nitrendipine. SRIF analogs also blocked Bay K 8644-stimulated Ca+(+) influx. The rank order of potency of the analogs (SRIF-28 greater than D-Trp8-SRIF greater than SRIF) suggests that the effects of SRIF are mediated by SRIF-28 preferring receptors. Pretreatment of AtT-20 cells with pertussis toxin abolished SRIF's inhibition of Bay K 8644-evoked Ca+(+) influx suggesting that G proteins mediate the inhibitory effects of SRIF on Ca+(+) influx. The K+ channel antagonists tetraethylammonium, 4-aminopyridine and CsCl all stimulated Ca+(+) influx into AtT-20 cells. These agents did not alter Bay K 8644-evoked Ca+(+) influx or did they affect the ability of SRIF to inhibit Ca+(+) influx. Tetrodotoxin, the sodium channel blocker which inhibits action potential generation in AtT-20 cells, lowered basal Ca+(+) levels in AtT-20 cells but did not modify SRIF's inhibition of Bay K 8644-stimulated Ca+(+) influx. These findings suggest that SRIF receptors, linked directly to Ca+(+) channels via G proteins, may mediate SRIF's inhibition of Ca+(+) influx.     

Effects of Bay k 8644 and nifedipine on isolated dog cerebral, coronary and mesenteric arteries

Vasoconstrictor effects of Bay k 8644, a dihydropyridine Ca++ agonist, and vasorelaxant effects of nifedipine were investigated in helical strips of dog cerebral (basilar, posterior cerebral and middle cerebral) and peripheral (coronary and mesenteric) arteries. The addition of Bay k 8644 produced a dose-dependent contraction in the absence of any contractile agent in the basilar artery with a pD2 value of 8.53. Similar sensitivity to Bay k 8644 was observed in the posterior cerebral, middle cerebral or coronary artery. Bay k 8644 was much less effective in producing a contraction in the mesenteric artery. An elevation of the concentration of extracellular K+ eliminated the difference between the responses to Bay k 8644 in the basilar and mesenteric artery. Contractile responses of the basilar artery to Bay k 8644 were antagonized competitively by nifedipine (pA2 = 8.17), but non-competitively by diltiazem. The pA2 values for nifedipine antagonism of Bay k 8644 responses with the elevated K+ were the same between the basilar and mesenteric arteries. Increased sensitivity to exogenously added K+ also was observed in cerebral and coronary arteries when compared with the mesenteric artery. The addition of nifedipine to an unstimulated strip produced a dose-dependent relaxation in cerebral and coronary arteries, but not in the mesenteric artery. When the cerebral and peripheral arteries were contracted with K+ to the same magnitude, nifedipine produced similar relaxations among these arteries. Nifedipine was less efficacious in antagonizing the contractile response to Bay k 8644 compared with the contractile response to K+ in cerebral arteries. These results suggest that 1) the voltage-dependent Ca++ channels in the cerebral and coronary arteries are in different states of activation from those in the mesenteric artery, 2) Bay k 8644 contracts the cerebral and coronary arteries by acting primarily on the same site, presumably dihydropyridine receptors of the voltage-dependent Ca++ channels at which nifedipine acts, 3) the dihydropyridine receptors were the same between the basilar and mesenteric arteries and 4) there may be a difference in the state of the Ca++ channel in the arteries between the stimulation with Bay k 8644 and K+-depolarization.