Effects of 2-nicotinamidoethyl nitrate on agonist-sensitive Ca++ release and Ca++ entry in rabbit aorta

The effects of 2-nicotinamidoethyl nitrate (SG-75) on norepinephrine (NE)- and KCI-induced responses in rabbit aorta were quantitated, correlated with 45Ca studies and compared with the effects of nifedipine (NIF) on similar parameters. NE- and KCI-induced dose-response relationships were differentially depressed by SG-75 (NE much greater than KCI) and NIF (KCI much greater than NE). Responses to KCI were relatively insensitive to prior SG-75, yet moderately relaxed by subsequent SG-75. Conversely, NIF markedly inhibited and completely relaxed similar responses. Responses to NE were relaxed and inhibited with SG-75, but unaffected by NIF. Responses to NE in La or O-Ca++ + ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid plus D600 (with and without KCI) solutions were phasic, reduced by SG-75 and insensitive to NIF. NE-dependent, Ca++-induced responses in a O-Ca++ + ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid plus D600 solution (with and without KCI) were attenuated by SG-75. Equilibrated (60 min) La -resistant (residual), high apparent affinity Ca++ binding was increased 26% with SG-75 and decreased 34% with NIF, yet neither altered the rate of exchange (10 min). Rate of exchange at low apparent affinity, residual sites was increased 21% by SG-75 without altering equilibrated values, whereas NIF reduced equilibrated values 11%, without affecting rate. NE reduced, SG-75 + NE augmented and NIF + NE decreased, in an additive fashion, high apparent affinity, residual bound Ca++. Residual Ca++ binding at low apparent affinity sites was increased with 160 mM substituted KCI (380%). This increase was only partially inhibited with SG-75, and eliminated by NIF. Net Ca++ efflux was persistently slowed by SG-75 and unaltered by NIF. The primary effects of SG-75 appear to be depression of Ca++ release and inhibition of receptor-operated (potential-independent) Ca++ entry, with limited attenuation of voltage-dependent Ca++ entry. NIF primarily inhibits voltage-dependent Ca++ entry.     

Differential effects of carbachol on cytosolic calcium levels in vascular endothelium and smooth muscle

Effects of norepinephrine (NE), carbachol (CCh) and histamine (HIS) on vascular tone and the endothelial and smooth muscle cytosolic C++ levels ([Ca++]i) were examined in rat aorta. The fura-2-Ca++ fluorescence emitted from endothelial and smooth muscle cells was detected at the endothelial surface. In the aorta with endothelium, NE increased both [Ca++]i and muscle tension whereas CCh slightly relaxed the muscle and increased [Ca++]i. The CCh-stimulated [Ca++]i was partially inhibited by verapamil. Addition of CCh to the NE-stimulated aorta relaxed the muscle with additional increase in [Ca++]i and positive correlation was obtained between the increase in [Ca++]i and relaxation. In the aorta without endothelium, NE increased both [Ca++]i and tension although CCh was ineffective. When endothelium was removed only from a small area from where the fura-2-Ca++ fluorescence was detected, CCh relaxed the muscle without changing [Ca++]i. In this preparation, NE increased both [Ca++]i and muscle tension and sequential addition of CCh relaxed the muscle with a small decrease in [Ca++]i, suggesting that Ca++ sensitivity of contractile elements is decreased. In Ca+(+)-free solution, CCh induced a transient increase in [Ca++]i and a decrease in muscle tension only in the presence of endothelium. HIS showed similar effects as CCh. By contrast, sodium nitroprusside decreased [Ca++]i and relaxed the muscle in NE-stimulated aorta with or without endothelium. These results suggest that CCh and HIS increase [Ca++]i in the endothelial cells which regulates the synthesis and/or release of endothelium-derived relaxing factor. Endothelium-derived relaxing factor may decrease [Ca++]i in the smooth muscle cells and also decrease Ca++ sensitivity of contractile elements resulting in vasodilatation.     

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)     

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

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

Mode of vasorelaxing action of 5-[3-[[2-(3,4-dimethoxyphenyl)-ethyl]amino]-1-oxopropyl]-2,3,4,5- tetrahydro-1,5-benzothiazepine fumarate (KT-362), a new intracellular calcium antagonist

In rabbit aorta, pretreatment with KT-362 (KT; 10(-6) and 10(-5) M) inhibited contractile responses to norepinephrine (NE; 3 X 10(-9)-10(-5) M) and methoxamine (10(-7)-10(-4) M) but failed to affect responses to potassium (10-70 mM). KT (10(-5) M) partially inhibited Ca++-induced contractions in K+-depolarized aorta pre-equilibrated in a Ca++-free medium. After incubation of tissues for 30 min in a Ca++-free medium containing EGTA (0.2 mM), residual responses to NE and methoxamine were inhibited by KT (10(-6)-10(-4) M) and nitroglycerin (10(-5) M), but not by nifedipine, verapamil or diltiazem (all 10(-5) M). The inhibitory action of a combined treatment with KT and nitroglycerin (both 10(-5) M) on the residual response to NE was also much greater than that of either agent alone. In a Ca++-free medium, the residual caffeine-induced contraction of rabbit iliac artery was inhibited by KT (10(-5)-10(-4) M) but not by nifedipine (10(-5) M). The inhibitory action of KT on the residual responses to methoxamine and caffeine in a Ca+-free medium was much greater than that of nitroglycerin. In a Ca++-free medium with low EGTA (0.01 mM), D600 (10(-5) M) and NE (3 X 10(-7) M), the addition of Ca++ (2 mM) resulted in a tonic contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of hydralazine and hydrazone derivatives with contractile mechanisms in rabbit aortic smooth muscle

The mechanism of action and relative potency of hydralazine (H) and tow hydrazone derivatives were investigated using isolated rabbit aortic strips. H, hydralazine acetone hydrazone (HA) and hydralazine butanone hydrazone (HBH) relaxed established K+ and norepinephrine (NE) contractures, and inhibited the development of contractures to these two agents on preincubation. H, HA and HBH increased the threshold to Ca++ and decreased the maximum tension responses during K+-Ca++-contractures (HA greater than H, P less than .05; HBH greater than H P less than .01). The Ca++-dependent and Ca++-independent components of NE contractures were both inhibited by H, HA and HBH. NE contractures were more sensitive to the effects of H than K+ contractures. These results are consistent with the conclusion that H and hydrazone derivatives produce effects on vascular muscle both by interactions with the fluxes of Ca++ from the extracellular space and effects on release from cell stores. However, other possibilities need to be assessed experimentally.     

Energetic aspects of the regulation of Ca++ sensitivity of permeabilized rabbit mesenteric artery: possible involvement of a second Ca++ regulatory system in smooth muscle contraction

The effects of phosphagen concentrations and adenosine-5'-O-(2-thiodiphosphate)(ADP beta S), a nonhydrolyzable ADP analog, on the pCa++ tension relationships were investigated, using alpha-toxin permeabilized rabbit mesenteric artery. The removal of creatine phosphate (CP) greatly affected the Ca++ sensitivity and induced a leftward shift of the pCa++ tension curve. Addition of ADP beta S (10-300 microM) also caused a leftward shift of the pCa++ tension curve. Ca++ solutions (0.3-10 microM) containing 0.1 mM ATP did not induce contraction. However, the addition of CP in the presence of 0.1 mM ATP dose-dependently increased force development which reached a maximum around 3 mM CP. A 10 microM Ca++ solution containing 0.1 mM ATP and 1 mM CP was much more effective in inducing contraction than a 10 microM Ca++ solution containing 1.1 mM ATP alone, although the total concentration of phosphagen (ATP + CP) was the same. Application of 0.1 mM ATP solution containing various concentrations of Ca++ after the maximal Ca+(+)-induced contraction relaxed the tissue, with the higher Ca++ concentrations inducing the faster relaxation. The same pattern of the relaxation was seen when the tissue was pretreated with adenosine-5'-O-(3-thiotriphosphate) beforehand. The contractile state observed in the Ca+(+)-free solution containing 0.1 mM ATP and 0.1 mM CP was completely relaxed by 1 mM vanadate, consistent with the idea that the sustained contraction was due to accumulation of the actomyosin-ADP complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific actions of gallium on norepinephrine-induced tension and associated 45Ca movements in rabbit aortic smooth muscle

Gallium ion (Ga) dose-dependently (60-360 microM) inhibited contractions induced by norepinephrine (NE, 1 microM) in rabbit aortic (and media intimal) strips, but did not affect contractions elicited with high K+ (80 mM) solution. The initial phasic portion of the NE-induced response was either unaffected or only slightly (less than 10%) reduced, but the tonic portion of the response was inhibited completely by higher concentrations (greater than or equal to 300 microM) of Ga . In resting muscles, the equilibrated (90 min) 45Ca uptake was not altered by Ga (360 microM). Also, 45Ca efflux from either high- or low-affinity Ca++ binding sites was unaltered by Ga . The effects of Ga (360 microM) on 45Ca retained after a subsequent 60-min washout at 0.5 degrees C in an isosmotic (80.8 mM) La solution were also examined. High affinity La -resistant 45Ca released by NE (1 microM) was not altered by Ga . Under conditions favoring low affinity Ca++ uptake, 45Ca retention in control and K+-treated muscles was not changed by Ga , but the additional incremental 45Ca uptake associated with NE (in the presence of high K+) was blocked. Thus, Ga appears to have a selective inhibitory action on NE-associated 45Ca uptake without affecting either resting and high K+-induced 45Ca uptake or that 45Ca fraction released by NE. This action may result from a selective blockade by Ga of receptor-linked Ca++ channels in rabbit aortic smooth muscle.     

Inhibition of norepinephrine and acetylcholine release from human neocortex by omega-conotoxin GVIA

Superfused slices of human neocortex, prepared from surgically removed tissue (to gain access to subcortical tumors) and prelabeled selectively with [3H]norepinephrine (NE) or [3H]choline, were stimulated electrically to evoke tritium overflow. This tritium overflow was abolished by the sodium channel blocker tetrodotoxin and by withdrawal of extracellular Ca++. Thus, the action potential-induced, exocytotic tritium overflow supports the assumption of a quasiphysiological release of NE from noradrenergic and of acetylcholine (ACh) from cholinergic nerve terminals, respectively. In addition, the modulation of NE release by adrenoceptor ligands displayed the appropriate pharmacology of alpha-2 autoreceptors; ACh release was modulated by muscarinic ligands. Both NE and ACh release decreased with the age of the patients. The effects of drugs on NE and ACh release were not age-related. The peptide modulator of the N-type voltage sensitive Ca++ channel, omega-conotoxin GVIA, inhibited NE release with an IC50 of about 14 nM and ACh release with an IC50 of about 3 nM, whereas L-type modulators were ineffective. The binding of [125I]omega-conotoxin GVIA to human neocortical membranes was of high affinity (KD = 1.3 pM) to one site (nH = 0.97) of substantial density (maximum binding = 878 fmol/mg of protein); the binding of the L-type modulator [3H]isradipine to these membranes was also of high affinity (KD = 89 pM) to one site (nH = 1.03) of lesser density (maximum binding = 429 fmol/mg of protein). In conclusion, Ca++ entry through N-type Ca++ channels, rather then L-type Ca++ channels, predominates in subserving NE and ACh release from noradrenergic and cholinergic nerve terminals, respectively, of human neocortex.     

Effects of ethanol on neurotransmitter release and intracellular free calcium in PC12 cells

The effect of ethanol on muscarine-stimulated release of l-[3H]norepinephrine ([3H]NE) was studied using the rat pheochromocytoma cell line, PC12. At concentrations of 25 mM and above, ethanol produced a dose-dependent inhibition of muscarine-stimulated release of [3H]NE. The inhibition of muscarine-stimulated transmitter release occurred in the absence of any detectable effect of ethanol on [3H]NE uptake or on muscarinic binding to the cells. However, ethanol produced an inhibition of muscarine-stimulated elevation of intracellular free Ca++ which corresponded with the inhibition of transmitter release. At concentrations greater than 100 mM, ethanol produced an increase in the basal release of [3H]NE. Intracellular free Ca++ also was increased by ethanol concentrations greater than 100 mM. The elevation of basal transmitter release and intracellular free Ca++ by concentrations of ethanol greater than 100 mM occurred independently of the inhibition by ethanol of muscarine-stimulated elevation of intracellular free Ca++ and transmitter secretion. These results suggest that the effects of ethanol on neurotransmitter release are associated with the effects of ethanol on intracellular free Ca++.     

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)     

Norepinephrine and adenosine triphosphate release in different ratio from guinea pig vas deferens by high potassium chloride, ouabain and monensin

Release of norepinephrine (NE) and ATP from the guinea pig vas deferens evoked by ouabain in combination with monensin or by high KCl was measured by a high-pressure liquid chromatography-ECD and luciferin-luciferase assay, respectively. Ouabain (10-100 microM) induced a concentration-dependent liberation of NE, which was enhanced by 10 microM monensin, a Na+-ionophore. The marked NE release elicited by the combined administration of both the drugs was unaffected by Ca++-removal but was reduced by lowering Na+ from the medium. This NE release in the Ca++-free medium was diminished markedly after treatment with 6-hydroxydopamine or reserpine and in low-temperature (25 degrees C) medium. This release was also decreased by ruthenium red (10-30 microM), an uptake inhibitor of Ca++ to mitochondria, and carbonyl cyanide-m-chlorophenyl hydrazone (10 microM), a metabolic inhibitor. On the other hand, 100 mM KCl caused a moderate, extracellular Ca++-dependent release of NE. ATP-outflow from the tissue evoked by 100 microM ouabain plus 10 microM monensin was almost unaltered by Ca++-removal but was inhibited by 6-hydroxydopamine or prazosin (0.3 microM), whereas release induced by high KCl was reduced by 6-hydroxydopamine and Ca++-free medium but was unaffected by prazosin. ATP/NE ratios at respective maximum effluxes evoked by 100 mM KCl and ouabain plus monensin were 6.59 and 0.22, respectively. These findings suggest that there may be more than one site of corelease for NE and ATP. Ouabain plus monensin seems to produce an extracellular Ca++-independent neuronal release of NE and ATP from the cytoplasmic and vesicular storage sites which predominantly release NE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protective effect of intrarenal calcium membrane blockers before or after renal ischemia. Functional, morphological, and mitochondrial studies

The present study examined whether a pre- or postischemic infusion of verapamil (V) or a postischemic infusion of nifedipine (N), drugs which block calcium (Ca++) influx across plasma membranes, provides protection against ischemic acute renal failure (ARF) in dogs. Renal hemodynamics and excretory function were examined 1 h (initiation phase) and 24 h (maintenance phase) after a 40-min intrarenal infusion of norepinephrine (NE). In each case, the uninfused contralateral kidney served as control. Four groups were studied: (a) dogs receiving NE alone; (b) dogs receiving an intrarenal infusion of V for 30 min before NE (V + NE); (c) dogs in which intrarenal V was infused for 2 h, beginning immediately after completion of NE infusion (NE + V); and (d) dogs in which intrarenal N was infused for 2 h, beginning immediately after completion of NE infusion (NE + N). Glomerular filtration rate (GFR) in the NE kidneys, as assessed by inulin clearance, at 1 and 24 h averaged 2.4 +/- 1.1 and 5.0 +/- 2.0 ml/min, respectively, as compared with control kidney GFRs of 28.0 +/- 3.5 and 43.8 +/- 5.0 ml/min, respectively (both at least P less than 0.01). In the V + NE group, GFR at 1 and 24 h averaged 15.0 +/- 5.5 and 31.0 +/- 4.5 ml/min, respectively, both at least P less than 0.05 as compared with values from NE kidneys. GFRs in the NE + V group averaged 15.0 +/- 2.4 and 16.3 +/- 3.6 ml/min at 1 and 24 h, both at least P less than 0.02 as compared with values from NE kidneys. GFR in the NE + N group averaged 18.6 +/- 6.0 ml/min at 24 h (P less than 0.05 as compared with GFRs in the NE kidneys). In addition, function of cortical mitochondria (Mito) was examined at the end of the 40-min NE infusion and after 1 and 24 h of reperfusion in the NE alone and NE + V groups. Mito respiration, assessed by acceptor control ratios, was reduced at each period in the NE alone kidneys. After 24 h, these Mito had accumulated Ca++ and exhibited reduced Ca++ uptake and increased Ca++ release rates. Mito from NE + V kidneys respired normally, did not accumulate Ca++, and exhibited no alterations in Ca++ uptake or release. Light and electron microscopy also demonstrated morphological protection of V against tubular necrosis and cell injury. Mito from the NE + N kidneys also respired normally and did not accumulate significant amounts of Ca++. The results of the present studies therefore demonstrated that chemically dissimilar calcium entry blockers exert substantial functional, cellular, and morphological protection against experimental ischemic ARF. These findings are compatible with the hypothesis that increased cytosolic Ca++ is critically important in the maintenance of renal vasoconstriction and the development of cellular necrosis with subsequent tubular obstruction in NE-induced ischemic ARF. V or N may provide protection against renal injury by retarding any increase in cytosolic Ca++ in renal vasculature and epithelium.     

The influence of ketamine on inotropic and chronotropic responsiveness of heart muscle.

The influence of ketamine on the inotropic and chronotropic responsiveness of heart muscle was examined in spontaneously beating right atrial preparations and in electrically driven left atrial preparations of guinea pigs. Ketamine (2.63 X 10(-5) to 4.2 X 10(-4) M) decreased heart rate of right atria and decreased contractile tension and its maximum rate of increase in both right and left atrial preparations (right atria greater than left atria). Ketamine did not prevent the heart rate increase produced by norepinephrine (NE; 1 X 10(-8) to 1 X 10(-4) M) in right atria; however, the maximum heart rate was consistently lower in ketamine-treated than in control muscles even after exposure to NE. Although contractile tension was decreased by ketamine, the maximum inotropic response to NE was consistently greater in ketamine-treated atria than in control atria. An inhibitor of the slow Ca++ current in heart muscle, D600, depressed the contractile effects of NE but did not prevent the positive inotropic interaction of ketamine and NE. Ketamine similarly enhanced the inotropic responses to norepinephrine (1 X 10(-6) M), epinephrine (1 X 10(-6) M), isoproterenol (1 X 10(-7) M) and dibutyryl cyclic adenosine 3':5'-monophosphate (AMP; 4 X 10(-3) M) in left atria electrically paced at a constant frequency of contraction of 1 Hz; however, ketamine inhibited the positive inotropic response to increased frequency of stimulation (0.1-3.0 Hz) and to ouabain (3 X 10(-7) M). These findings demonstrate that ketamine can exert a selective positive inotropic influence in heart muscle independent of heart rate or direct or reflexogenic autonomic nervous system changes, and suggest that this activity could in some way be associated with an alteration of the intracellular disposition of cyclic AMP.     

Cardiotoxic effects of maitotoxin, a principal toxin of seafood poisoning, on guinea pig and rat cardiac muscle

Maitotoxin (MTX), a principal toxin of seafood poisoning, produced powerful cardiotoxic effects on guinea pig isolated left atria at concentrations of 5 X 10(-9) to 3 X 10(-8) g/ml. The MTX-induced increase in resting tension of atria was abolished by Co++, D600 or Ca++-free solution. The tissue Ca content and 45Ca uptake of guinea pig atria were increased by MTX (5 X 10(-9) to 3 X 10(-8) g/ml), and these increases were inhibited markedly by Co++. In isolated rat cardiac myocytes, irreversible contracture was produced by MTX (10(-8) g/ml), and this effect of MTX was suppressed by verapamil or Ca++-free solution. The intracellular free Ca++ concentration of isolated rat myocytes was increased greatly by MTX (10(-9) to 3 X 10(-8) g/ml). Furthermore, the myocardial cells sampled from guinea pig left atria were characterized ultrastructurally by severely overcontracted sarcomeres, swollen mitochondria, peripheral aggregation of nuclear chromatin and loss of granular glycogen, and these morphological changes were abolished by omitting Ca++ from the medium. These results suggest that MTX increases the Ca++ influx through the cardiac muscle membrane to create the Ca-overloaded state and thus caused the cardiotoxic effects.     

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.     

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.     

Relationship between 45Ca movements, different calcium components and responses to acetylcholine and potassium in tracheal smooth muscle

Correlations between tension responses elicited with acetylcholine (ACh) and high K+ and corresponding alterations in Ca++ mobilization were obtained in rabbit and canine tracheal smooth muscle. Removal of Ca++ or preincubation with D-600 (50 microM) inhibited responses to K+ (50 or 80 mM) and low ACh (89 nM) and had only a small effect on responses to high ACh (8.9 microM). Conversely, solutions containing Sr++ instead of Ca++ inhibited responses to both concentrations of ACh to a greater degree than were those to K+. Washout of slow component 45Ca into a O-Ca solution was more rapid in rabbit trachea than reported previously for rabbit aorta. Washout of tracheal smooth muscle into an 80.8 mM La -substituted solution at 0.5 degrees C removed superficial (La -accessible) 45Ca and blocked both 45Ca uptake and most 45Ca efflux. D-600, which had no significant effect on control 45Ca uptake in rabbit aortic smooth muscle, decreased 45Ca uptake by 33% in rabbit tracheal smooth muscle. The uptake of 45Ca from the Ca++ binding sites with low affinity for Ca++ was increased by 80 mM K+, 50 mM K+ or 8.9 microM ACh, and the accumulation of Ca++ from the Ca++ binding sites with high affinity for 45Ca was inhibited by Sr++. The stronger effect of either Ca++ removal or D-600 on responses to K+ and the correspondingly greater effect of Sr++ on responses to ACh indicate that different Ca++ stores are present in tracheal smooth muscle. These Ca++ components appear to be qualitatively similar to those present in aortic smooth muscle but they differ quantitatively and are not as readily dissociated as are aortic Ca++ components.     

9-methyl-7-bromoeudistomin D, a powerful radio-labelable Ca++ releaser having caffeine-like properties, acts on Ca(++)-induced Ca++ release channels of sarcoplasmic reticulum.

9-Methyl-7-bromoeudistomin D (MBED), a derivative of eudistomin D isolated from a marine tunicate, induced Ca++ release from the heavy fraction of fragmented sarcoplasmic reticulum (HSR) in the same way as that of caffeine, followed by spontaneous Ca++ reuptake in the Ca++ electrode experiment. The rate of 45Ca++ efflux from HSR vesicles was accelerated markedly by MBED or caffeine in a concentration-dependent manner. The 50% effective concentrations of MBED and caffeine were approximately 1 microM and 1 mM, respectively, indicating that MBED is 1000 times more potent than caffeine in HSR. Procaine, ruthenium red or Mg++ caused concentration-dependent inhibition of MBED-triggered Ca++ release from HSR. The bell-shaped profile of Ca++ dependence for MBED is very similar to that of caffeine. The caffeine-produced maximum response of 45Ca++ efflux was increased further by adenosine-5'-(beta, gamma-methyl-ene)triphosphate, whereas that was not changed by MBED. MBED also caused Ca++ release from sarcoplasmic reticulum (SR) of chemically skinned fibers. These stimulatory effects of MBED on the Ca++ release from skeletal muscle SR were almost indistinguishable from those of caffeine except the difference in potencies. The [3H]ryanodine binding to junctional terminal cisternae membranes was not inhibited by MBED or caffeine. MBED did not cause Ca++ release from the light fraction of fragmented SR and turbidity change of mitochondrial suspension. These observations suggest a most likely idea that MBED binds to the caffeine-binding site in the Ca channel protein and thus produces the potentiation of Ca(++)-induced Ca++ release from SR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase in Ca++ sensitivity of the contractile system by MCI-154, a novel cardiotonic agent, in chemically skinned fibers from the guinea pig papillary muscles

The effects of MCI-154, a novel cardiotonic agent, on the contractile protein system and the sarcoplasmic reticulum (SR) were investigated by using thin bundles of chemically skinned fibers from the guinea pig papillary muscles. In the skinned muscle fibers treated with 50 micrograms/ml of saponin, MCI-154 shifted the -log[Ca++]M-tension relation curve to the left and upward in the concentration-dependent manner (10(-7) to 10(-4) M). This was confirmed also in the skinned muscle fibers treated with 250 micrograms/ml of saponin which destroyed not only the surface membrane but also the function of SR. Sulmazole (10(-4) M) shifted the -log[Ca++]M-tension relation curve to the left but the effect was about 100 times less potent than that of MCI-154. Unlike MCI-154, sulmazole had little effect on the maximum tension development induced by -log[Ca++]M 4.4. Milrinone did not affect the Ca++-induced tension development in the skinned cardiac fibers. Higher concentration of MCI-154 (10(-4) M) also increased amplitude of -log[Mg-ATP]M-tension-curve in the absence of free Ca++ ion (bell-shaped curve) to the upward. Initial rate and plateau phase of Ca++ uptake by the SR in the skinned fibers treated with 50 micrograms/ml of saponin was increased slightly by MCI-154 at the concentrations of 10(-6) and 10(-4) M. MCI-154 had no effect on the Ca++-induced Ca++ release mechanism in the SR. These results suggest that an increase in Ca++ sensitivity of the contractile protein system is responsible for, at least in part, the mechanism of the positive inotropic action of MCI-154.(ABSTRACT TRUNCATED AT 250 WORDS)