Caffeine-induced Contraction Research Articles

Negative inotropic effect of heparin on tension development in rat skinned skeletal muscle fibres

Heparin inhibits inositol trisphosphate receptors, particularly in smooth muscle, but its effect on skeletal muscle is controversial. Our study showed that heparin induced a decrease in the amplitude of 10 mM caffeine-induced contracture in slow and fast saponin-skinned fibres. Moreover, measurements on Triton X-100-skinned fibres in soleus muscle showed that heparin alone decreased maximal Ca 2+-activated tension and Ca 2+ sensitivity of contractile proteins, whereas no significant effect was observed in extensor digitorum longus muscle. However, in the presence of caffeine, heparin decreased maximal Ca 2+-activated tension in both muscles. It would appear that the heparin-induced decrease in the amplitude of caffeine contracture in rat skeletal muscle was not related to a direct inhibition of Ca 2+ release from sarcoplasmic reticulum but to a desensitising effect of heparin and caffeine on myofilaments.

Different effects of halothane, isoflurane and sevoflurane on sarcoplasmic reticulum of vascular smooth muscle in dog mesenteric artery

The direct effect of halothane on vascular smooth muscle is mediated in part via its effects on the sarcoplasmic reticulum (SR). Little information is available concerning the effects of other volatile anesthetics including isoflurane and sevoflurane, whose vascular effects differ from those of halothane. The aim of the present study was to compare the effects of halothane, isoflurane and sevoflurane on the SR by testing the contraction induced by caffeine in vascular smooth muscle. Rings without endothelium from isolated canine mesenteric artery were mounted in physiological saline solution (PSS) for isometric tension recording. After complete depletion of Ca2+ from the SR by adding 35 mM caffeine, the rings were exposed to normal Ca2+ containing PSS (Ca2+ loading), to Ca(2+)-free PSS for 10 min, and then to 15 mM caffeine to induce contraction. Anesthetics were administered during Ca2+ loading, the Ca(2+)-free phase and simultaneously with caffeine administration. Halothane (0.5-2%) attenuated the caffeine-induced contraction of canine mesenteric artery when administered during Ca2+ loading in the SR (P < 0.001), whereas isoflurane and sevoflurane (1-4%) failed to affect the contraction. When given simultaneously with caffeine, halothane (1-2%) potentiated the caffeine-induced contraction (P < 0.05), but isoflurane and sevoflurane had no effect. When given before caffeine administration, halothane (0.5-2%), isoflurane (2-4%) and sevoflurane (4%) all potentiated the caffeine-induced contraction (P < 0.05). It has been shown that halothane not only potentiates caffeine-induced Ca2+ release from the SR, but also induces contraction by releasing Ca2+ from the SR. We conclude that halothane decreases Ca2+ accumulation in the SR while exerting facilitative and additive effects on caffeine-induced Ca2+ release from the SR when applied before caffeine administration and simultaneously with caffeine, respectively, whereas isoflurane and sevoflurane lack both the ability to decrease Ca2+ accumulation and an additive effect on caffeine-induced Ca2+ release from the SR, but are able to facilitate Ca2+ release by caffeine.

Susceptibility of caffeine- and Ins(1,4,5)P 3-induced contractions to oxidants in permeabilized vascular smooth muscle

Two principal pathways of Ca 2+ release from the sarcoplasmic reticulum of excitable and non-excitable cells have been described: one pathway dependent on the second messenger d- myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3), and a second pathway sensitive to Ca 2+ and regulated by caffeine and ryanodine. It was found that the Ca 2+-pump activity of vascular smooth muscle sarcoplasmic reticulum is inhibited by superoxide anion radicals (O 2 ⋅−); however, the effects of reactive oxygen intermediates on sarcoplasmic reticulum Ca 2+ release in vascular muscle cells are not well defined. The purpose of the present study was to evaluate the effects of reactive oxygen intermediates generated from the hypoxanthine/xanthine oxidase reaction system on contractions induced by caffeine, Ins(1,4,5)P 3 and norepinephrine in staphylococcal α-toxin-permeabilized rabbit mesenteric arteries. This system generates O 2 ⋅−, H 2O 2, and hydroxyl radicals. We wished to identify which class of reactive oxygen intermediates is responsible for the associated loss of vascular smooth muscle contractile function. Caffeine and Ins(1,4,5)P 3 produced a transient contraction when the sarcoplasmic reticulum of the permeabilized preparations was preloaded with pCa 7.0 solution for 5 min before washing with 0.5 mM EGTA solution; norepinephrine also produced a transient contraction. Exposure of the preparations to hypoxanthine/xanthine oxidase (for 30 min) attenuated caffeine-induced contraction, but was without effect on Ins(1,4,5)P 3-induced contraction. The observed effect of hypoxanthine/xanthine oxidase exposure was superoxide dismutase-inhibitable, suggesting O 2 ⋅− involvement. Hypoxanthine/xanthine oxidase also inhibited norepinephrine-induced contraction. The effect of hypoxanthine/xanthine oxidase on norepinephrine contraction was protected by catalase, but not by superoxide dismutase and dimethyl sulfoxide; exogenously added H 2O 2 mimicked the effect of hypoxanthine/xanthine oxidase exposure. H 2O 2, added exogenously, was without effect on Ins(1,4,5)P 3-induced contraction. It is suggested that the pathway of Ca 2+ release from the sarcoplasmic reticulum dependent on Ins(1,4,5)P 3 is insensitive to O 2 ⋅−. Instead, caffeine-induced Ca 2+ release mechanisms may be susceptible to O 2 ⋅− and H 2O 2, rather than O 2 ⋅− and hydroxyl radicals, may be the active agent in the norepinephrine-induced contraction. Our results are also consistent with the view that the attenuation by H 2O 2 of the norepinephrine-induced contraction may be linked to the receptor-associated pathway of Ins(1,4,5)P 3 formation, but not to degradation processes of Ins(1,4,5)P 3.

Propofol attenuates the contractile response induced by release of cellularly sequestered calcium isolated from human omental arteries and veins

A side effect of the intravenous propofol is hypotension. The underlying mechanism has been suggested to be a decrease in sympathetic activity and/or a direct effect on vascular smooth muscle. In the present study, the direct effect of propofol on human vascular smooth muscle was investigated, especially concerning the modulation of propofol on muscle contraction involving extra- or intracellular calcium. Isolated human omental arteries and veins were obtained from patients undergoing abdominal surgery and mounted in organ baths containing aerated modified Krebs-Ringer buffer solution. Changes in circular isometric smooth muscle tension in response to the administration of propofol were measured. Contractile responses induced by the stable thromboxane A2 analogue U46619, caffeine and KCI in the presence or absence of propofol were also measured. U46619 elicits its contractile response via IP3-mediated release of cellularly sequestered calcium and caffeine elicits its contractile response predominantly via ryanodine receptor-mediated release of cellularly sequestered calcium. On the other hand, KCI elicits via influx of extracellular calcium through voltage-dependent calcium channels. A Ca2+-free buffer solution was used for the experiments with U46619 and caffeine, whereas a normal buffer solution was used for the experiments with KCI. Propofol (10−3 M) induced a contraction in both arteries and veins. Conversely, propofol concentration-dependently attenuated the contraction elicited by U46619 and caffeine in the absence of extracellular calcium and the contraction elicited by KCI in the presence of extracellular calcium. The threshold concentration for the effect of propofol was lower for the U46619-induced contraction (10−5 M) and the caffeine-induced contraction (10−5 M and 10−4.5 for artery and vein respectively) than for the KCI-induced contraction (10−4 M). The present results suggest that propofol, at concentrations likely to occur clinically, attenuates contraction involving release of cellularly sequestered calcium. At higher concentrations of propofol, a contraction-attenuating effect of a different nature appears, which seems not solely to involve effects on intracellular calcium fluxes.

Effects of midazolam on contractions in smooth muscle of the rabbit mesenteric artery.

We investigated the undetermined effects of midazolam on agonist-induced contraction in vascular smooth muscle strips from the rabbit mesenteric resistance artery. Midazolam, in concentrations more than 10 microM, attenuated norepinephrine ([NE] 0.3-10 microM)-induced contractions in Krebs solution. The attenuating effect was more potent on the tonic and oscillatory responses than on the rapid phasic response. When voltage-operated Ca2+ channels (VOC) were blocked by nifedipine, midazolam, in concentrations more than 1 microM, attenuated both phasic and tonic responses. In Ca(2+)-free solution, midazolam, in concentrations more than 1 microM, attenuated NE-induced contractions, but not caffeine-induced contractions. When NE and caffeine were applied successively, midazolam attenuated NE-induced contractions, but enhanced caffeine-induced contractions. Because the attenuating effect of midazolam on NE-induced contractions in high K+, Ca(2+)-free solution were not different from the effect in normal Ca(2+)-free solution, the attenuating effects of midazolam could not have been induced via membrane hyperpolarization. These results indicate that midazolam attenuated the agonist-induced contractions by inhibition of Ca2+ influx occurring not only through VOC, but also through agonist-mediated Ca2+ channels and by the inhibition of Ca2+ release from intracellular store sites.

Developmental changes in the contractile system of the mesenteric small artery of rabbit.

This study investigated developmental changes in the contractile system of the mesenteric small arteries of the rabbit. Arteries about 1 mm in length and 100-250 microns in internal diameter were dissected out from the mesenteric bed of the fetus (29 d of gestation), newborn (3-5 d old), and adult rabbit. Vascular contraction was induced by high KCl concentration, and contractile force was measured using a tension transducer. The sensitivity of vascular contraction to high KCl was similar in the three age groups. To determine the role of Ca influx across the sarcolemma in vascular contraction, the vasorelaxant effect of diltiazem was studied in the artery precontracted with high KCl concentration. The vasorelaxant effect of diltiazem in the fetus and newborn was less than in the adult. To estimate the size of the intracellular Ca pool, caffeine-induced and noradrenaline-induced contraction were measured in the Ca- and Na-free solution. In the fetus and newborn, both the caffeine-induced contraction and noradrenaline-induced contraction were greater than in the adult. The ultrastructural study showed that the endoplasmic reticulum was abundant in the fetus and newborn, and it was scarce in the adult. These data indicate that the dependency of vascular contraction on Ca influx across the sarcolemma increases and the intracellular Ca store decreases with development in the mesenteric resistance arteries.

Effects of FK-506 on contraction and Ca2+ transients in rat cardiac myocytes.

FK-506 binding protein (FKBP) has been reported to be closely associated with the ryanodine receptor in skeletal and cardiac muscle and to modulate sarcoplasmic reticulum (SR) Ca2+ release channel gating in isolated channels. FK-506 can inhibit the activity of FKBP, thereby reversing its effects on SR Ca2+ release. We investigated the function of FKBP during normal contractions and Ca2+ transients in intact rat ventricular myocytes loaded with fluorescent Ca2+ indicators. FK-506 significantly increased steady state twitch Ca2+ transients and contraction amplitudes even under conditions in which the SR Ca2+ load and Ca2+ current were unaltered, suggesting that FK-506 increases the fraction of SR Ca2+ released during excitation-contraction (E-C) coupling. Action potentials were somewhat prolonged, consistent with the larger Ca2+ transients causing greater inward Na(+)-Ca2+ exchange current. FK-506 did not affect SR Ca2+ uptake but modestly decreased Ca2+ extrusion via Na(+)-Ca2+ exchange in intact cells (although no effect on Na(+)-Ca2+ exchange was seen in sarcolemmal vesicles). In most cells, FK-506 caused an increase in SR Ca2+ content during steady state stimulation, as assessed by caffeine-induced contractures. This was probably due to the inhibition of Ca2+ efflux via Na(+)-Ca2+ exchange. FK-506 also accelerated the rest decay of SR Ca2+ content and increased the frequency of resting Ca2+ sparks about fourfold. The increase in frequency of these basic Ca2+ release events was not associated with changes in the amplitude or duration of the Ca2+ sparks. We conclude that FK-506 increases the fraction of SR Ca2+ released during normal twitches and enhances the rate of SR Ca2+ release during rest. FK-506 also inhibits Na(+)-Ca2+ exchange, although this effect may be indirect. These effects are consistent with an important SR-stabilizing effect of FKBP in intact rat ventricular myocytes.

The effect of S-(+)-boldine on the alpha 1-adrenoceptor of the guinea-pig aorta.

1. The cardiovascular activity of S-(+)-boldine, an aporphine alkaloid structurally related to papaverine, was determined. The work includes functional studies on guinea-pig isolated aorta contracted with noradrenaline, caffeine, KCl or Ca2+, and on guinea-pig trachea contracted with acetylcholine or histamine. 2. S-(+)-boldine inhibited in a concentration-dependent manner the contractile response evoked by noradrenaline (10 microM) in guinea-pig aorta (IC50 = 1.4 +/- 0.2 microM) while the KCl depolarizing solution (60 mM)- or the Ca2+ (1 mM)-induced contractions were only partially affected by boldine up to 300 microM. In contrast, papaverine relaxed noradrenaline (NA), KCl or Ca2+ induced contractions showing similar IC50 values in all cases. S-(+)-boldine had a greater potency on the contraction elicited by NA whereas papaverine acted in a non-selective manner. 3. S-(+)-boldine was found to be an alpha 1-adrenoceptor blocking agent in guinea-pig aorta as revealed by its competitive antagonism of noradrenaline-induced vasoconstriction (pA2 = 5.64 +/- 0.08), and its potency was compared with that of prazosin (pA2 = 8.56 +/- 0.24), a known potent alpha 1-adrenoceptor antagonist. In contrast, papaverine caused rightward shifts of the NA concentration-response curves with depression of maximal response indicating that it acts as a non-competitive antagonist. 4. Contraction of guinea-pig aorta induced by caffeine (60 mM) in a Ca(2+)-containing Krebs solution was not affected by a 60 min incubation period with different doses of S-(+)-boldine (1-300 microM). Papaverine inhibited partially this caffeine-induced contraction at the maximal dose used (100 microM). 5. Inositol phosphates formation induced by noradrenaline (10 microM) in guinea-pig thoracic aorta was inhibited by S-(+)-boldine (30 microM) but not by papaverine (10 microM). 6. Contractions of guinea-pig trachea caused by acetylcholine (100 microM) or histamine (10 microM) were not modified by S-(+)-boldine (0.1-100 microM). 7. These results provide evidence that S-(+)-boldine, an aporphine alkaloid, has interesting properties as an alpha 1-adrenoceptor blocker in vascular smooth muscle, and acts as a competitive antagonist of the alpha 1-adrenoceptor present in the guinea pig aorta.

Effects of 8-(N-N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) on skinned myocardial fibres of the rat: reversible inhibition of calcium release from the sarcoplasmic reticulum.

The effects of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-3), which is reported to inhibit the release of intracellularly stored Ca2+ in skeletal and smooth muscles, were examined in ventricular myocardia of the adult rat. In skinned papillary muscle fibres with functional sarcoplasmic reticulum (SR) preserved, application of 100 or 300 microM TMB-8 during the Ca2+ loading period had no significant effect on the peak tension of subsequent caffeine-induced contraction, but when applied during exposure to caffeine, concentration-dependent reduction of the peak tension was observed. At 1000 microM, TMB-8 reduced the peak tension of caffeine-induced contraction when applied either during Ca2+ loading or during exposure to caffeine. TMB-8 had no substantial influence on the Ca(2+)-tension of skinned fibres without functional SR. In isolated papillary muscle preparations, TMB-8 prolonged the action potential duration and decreased the maximum rate of rise of potential, leading to abolition of contraction at 100 microM. In conclusion, TMB-8 may be a useful pharmacological tool for inhibiting Ca2+ release from SR, but only in skinned myocardial preparations.

Caffeine and micromolar Ca2+ concentrations can release Ca2+ from ryanodine-sensitive stores in crab and lobster striated muscle fibres

Ca2+ release mechanisms were studied in striated muscle from the walking legs of crabs using isometric tension recordings from isolated myofibrillar bundles. Caffeine-induced phasic contractions had properties consistent with Ca2+ release from a sarcoplasmic store, which could be optimally loaded in the presence of ATP at pCa 6.4&shy;6.1. Ryanodine (10 &micro;mol l-1) abolished the caffeine-induced contractions and in solutions with low Ca2+ buffering (0.1 mmol l-1 EGTA) itself caused phasic contractions, indicative of Ca2+ release. Ca2+-induced Ca2+ release (CICR) was observed in a pCa 5.8 solution (buffered by 1 mmol l-1 EGTA) as a phasic contraction of variable nature, inhibited by ryanodine (10 &micro;mol l-1), procaine (10 mmol l-1) or benzocaine (5 mmol l-1). Ca2+ release was measured as a function of releasing pCa by using the force&shy;time integral of the caffeine-induced contraction as an estimate of the Ca2+ remaining in the store. After the Ca2+ store had been loaded for 2 min at pCa 6.6, CICR was measured in the presence of 1 mmol l-1 Mg2+, 1 mmol l-1 EGTA and 5 mmol l-1 ATP. The threshold pCa for CICR was 6.0&shy;6.4 under these conditions and more than 90 % of stored Ca2+ was released in 1 min by pCa values in the range 3.5&shy;5.3. Benzocaine totally inhibited the release and promoted extra Ca2+ loading. Preliminary experiments showed a similar caffeine-releasable store in lobster abdominal muscle, which was slightly less sensitive to free [Ca2+]. It is concluded that in crustacean muscle caffeine and micromolar [Ca2+] can release Ca2+ from a ryanodine-sensitive store, which in many respects is similar to the sarcoplasmic reticulum of vertebrate skeletal and cardiac muscle.

Enhanced SR function in saponin-treated ventricular trabeculae from rabbits with heart failure.

Cardiac sarcoplasmic reticulum (SR) Ca(2+)-loading ability was assessed in a coronary artery ligation model of heart failure. Heart failure was produced in New Zealand White rabbits by ligation of the left marginal coronary artery. Sham-operated animals were used as controls. After hemodynamic and echocardiographic assessment 8 wk after coronary ligation, a free-running trabecula was isolated from the left or right ventricle, mounted for isometric tension measurement, and permeabilized with the chemical skinning agent saponin, leaving the SR functionally intact. The SR was Ca2+ loaded by exposure of the preparation to a mock intracellular solution with a Ca2+ concentration ([Ca2+]) of 150-300 nM. The amplitude of the caffeine-induced contracture was used as a measure of Ca2+ loaded by the SR. The same preparation was then treated with Triton X-100 to disrupt all cell membranes, and Ca2+ sensitivity {expressed as [Ca2+] required to produce 50% of maximal activation (pCa50)} of isometric tension production and maximum Ca2+ activated force (Cmax) were measured. Ligated animals demonstrated enhanced SR Ca(2+)-loading ability that correlated with the degree of left ventricular dysfunction. Enhanced SR Ca2+ loading was associated with evidence of SR Ca2+ overload revealed as spontaneous tension oscillations. Cmax and pCa50 were not significantly different from controls. Increased SR Ca(2+)-loading ability may predispose the SR to Ca2+ overload and could contribute to both contractile dysfunction and arrhythmogenesis in heart failure.

Characteristics of Ca2+ release for activation of K+ current and contractile system in some smooth muscles.

Characteristics of Ca2+ release from stores were investigated in strips from ileum and portal vein and in isolated myocytes from ileum and urinary bladder of the guinea pig with use of caffeine and 9-methyl-7-bromoeudistomin D (MBED), a potent releaser of Ca2+ from skeletal muscle sarcoplasmic reticulum. In skinned strips, 1-30 mM caffeine elicited a transient contraction, but 10-300 microM MBED did not. Pretreatment with 100 microM MBED did not affect the subsequent caffeine-induced contraction. In single cells loaded with indo 1-acetoxymethyl ester, 10 mM caffeine increased cytoplasmic Ca2+ concentration, whereas 100 microM MBED elicited a small or no increase. Under whole cell clamp, spontaneous transient outward currents through Ca(2+)-dependent K+ (BK) channels were first enhanced and then suppressed by 30 microM MBED or 5 mM caffeine. The amplitude of Ca(2+)-dependent transient K+ current on depolarization was reduced by MBED and caffeine (50% inhibitory concentrations = 20 microM and 1 mM, respectively). Other currents and single BK channel activity were not significantly affected by MBED. The Ca2+ release from stores responsible for BK channel activation may be resolved from that for the activation of the contractile system by MBED in these smooth muscle cells.

Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake, spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae.

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca2+ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca2+ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca2+ content loaded into the SR, (2) the spontaneous Ca2+ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca2+ sensitivity in Triton X-100-treated preparations. Inhibition of Ca2+ loading by TG and CPA increased with time of exposure to the inhibitor over 18-24 min. TG and CPA produced half inhibition of Ca2+ loading at 34.9 and 35.7 microM respectively, when 18-24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 microM TG and 30 microM CPA both abolished the oscillations in this time. The myofilament Ca2+ sensitivity was not affected by 10 and 300 microM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca2+ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation.

Dantrolene blocks the tonic contraction and calcium influx evoked by K + in real longitudinal smooth muscle

1. 1. Dantrolene (10 −8-10 −4M) reduced the K + (60 mM) tonic response in accordance with decreased Ca 2+ uptake, as determined by La method, more than the phasic in ileal muscle. 2. 2. After saponin-treatment of the fibers, which leaves the Ca 2+ storage sites intact, dantrolene had only a slight effect on the caffeine-induced contraction. In Triton-X-100-treated fibers, in which the Ca 2+ release sites are destroyed, dantrolene did not affect the Ca 2+-induced contraction. 3. 3. The results suggest that dantrolene inhibited the high-K +-induced tonic response mainly by inhibiting Ca 2+ influx via voltage-dependent Ca 2+ channels in ileum without affecting contractile elements. In contrast, dantrolene had only a slight effect on the intracellular Ca 2+ release from caffeine-sensitive release sites.

BAY K 8644 depresses excitation-contraction coupling in cardiac muscle

We determined the effect of the dihydropyridine L-type Ca channel agonist BAY K 8644 (BAY) on excitation-contraction (E-C) coupling in isolated ferret ventricular myocytes using whole cell voltage clamp. The sarcoplasmic reticulum (SR) Ca load during the test pulses, assessed by caffeine-induced contractures, was similar in the presence and absence of BAY, with extracellular Ca concentration lowered from 3 to 1 mM in BAY. The relationship between L-type Ca current (ICa) and contraction was assessed, with current and contractions measured during depolarizations from -40 to between -30 and +50 mV after a conditioning train (to ensure constant SR Ca load). BAY shifted the current-contraction relationship downward, such that, for a given ICa and SR Ca load, the contraction elicited was much smaller in the presence of BAY. BAY also induced a characteristic negative shift in the the current-voltage relationship. We conclude that BAY decreases the efficacy of a given Ca current to induce SR Ca release during E-C coupling in ferret cardiac tissue (in contrast to the BAY-induced increase of resting SR Ca release). This may reflect an alteration in the state of the SR Ca release channel due to BAY binding to dihydropyridine receptors.

Calcium movement of sarcoplasmic reticulum from hindlimb suspended muscle

The function of the sarcoplasmic reticulum (SR) was examined in the slow soleus and fast extensor digitorum longus (EDL) muscles of rats submitted to 14 days of weightlessness produced by hindlimb suspension (HS). Ca 2+ uptake, Ca 2+ release and passive Ca 2+ leakage through the SR membrane were investigated using a method of caffeine-induced contracture on the single mechanically skinned fibers. In the SR of suspended soleus muscles, the rate of Ca 2+ uptake was higher than in the control muscles. However, there was no difference between the suspended and control muscles in the rate of Ca 2+ uptake of the SR in EDL after HS. In soleus muscles, Ca 2+ movements of the SR from the suspended muscle acquired the properties that were similar to those of the control fast muscle. The study of Ca 2+ leakage showed that the velocity and amount of passive Ca 2+ leakage from SR in soleus and EDL were apparently increased after HS. The results suggested that the functional properties of the SR membrane in slow and fast muscles were changed after HS.

New intracellular calcium antagonists. I. Synthesis and pharmacological evaluation of 2,3,4,5-tetrahydro-1,5-benzothiazepine analogs

A series of 2,3,4,5-tetrahydro-1,5-benzothiazepine and related compounds were prepared, and the intracellular Ca2+ inhibitory effects were examined using methoxamine- or caffeine-induced contraction of isolated rabbit arteries. Structure-activity relationship studies of these compounds are discussed and the results suggest that novel 5-[3-[2-(3,4-dimethoxyphenyl)ethyl]aminopropionyl]-2,3,4,5- tetrahydro-1,5-benzothiazepine fumarate (20d) showed the most potent inhibitory action on the intracellular Ca2+ release.

Modulation of caffeine contractures in mammalian skeletal muscles by variation of extracellular potassium.

Caffeine contractures were induced after K(+)-conditioning of skeletal muscles from pigs and mice. K(+)-conditioning is defined as the partial depolarization caused by increasing external potassium (K+0) with [K+]x[Cl-] constant. Conditioning depolarizations that rendered muscles refractory to brief electrical stimulation still enhanced the contracture tension elicited by subsequent direct caffeine stimulation of sarcoplasmic reticulum (SR) calcium release. The effects of K(+)-conditioning on caffeine-induced contractures of intact cell bundles reached a maximum at 15-30 mM K+0 and then progressively declined at higher [K+]0. Conditioning with 30 mM K+ for 5 min, which inactivates excitation-contraction (EC) coupling in response to action potentials, both increased the magnitude of caffeine contractures 2-10-fold and shifted the contracture threshold toward lower caffeine concentrations. Enhanced sensitivity to caffeine was inhibited by dantrolene (20 microM) and its watersoluble analogue azumolene (150 microM). These drugs decreased caffeine-induced contractures following depolarization with 4-15 mM K+ to 25-50% of control tension. The inorganic anion perchlorate (CIO-4), which like caffeine potentiates twitches, increased caffeine-induced contractures approximately twofold after K(+)-conditioning (> 4 mM). The results suggest that CIO-4 and dantrolene, in addition to caffeine, also influence SR calcium release either directly or by mechanism(s) subsequent to depolarization of the sarcolemma. Moreover, since CIO-4 is known to shift the voltage-dependence of intramembrane charge movement, CIO-4 may exert effects on the transverse-tubule voltage sensors as well as the SR.

Increased calcium sequestration by sarcoplasmic reticulum in small muscular arteries in young spontaneously hypertensive rats.

1. Sarcoplasmic reticulum (SR) in small resistance arteries plays a role in the regulation of the cytosolic free calcium concentration by sequestration of calcium from cytoplasm. 2. To examine the contribution of calcium (Ca2+) sequestration by the SHR to both contraction and relaxation in young spontaneously hypertensive rats (SHR), we measured evoked tension before and after depletion of SR Ca2+ stores in the rings of the first branch of superior mesenteric artery in 5 week old SHR and age-matched Wistar-Kyoto (WKY) rats. Contractile responses were induced by 40 mmol/L potassium and 10 mmol/L caffeine before and after the treatment with ryanodine or thapsigargin, which depletes SR calcium stores. 3. The magnitude of potassium-induced contraction was almost the same in both strains. 4. Ryanodine and thapsigargin did not affect the resting tension and almost decreased caffeine-induced contraction in both strains. 5. After the treatment with ryanodine or thapsigargin, the magnitude and the rate of potassium-induced contraction were augmented greatly in SHR, but not in WKY. 6. The relative relaxation rate after exposure of potassium was significantly slowed in SHR by ryanodine or thapsigargin, but only slightly in WKY. 7. These results suggest that Ca2+ sequestration by the SR in SHR was greater than in WKY. Therefore, it is concluded that SR plays an important role in preventing the development of hypertension in SHR via a buffering effect on the elevation of cytosolic free Ca2+.

Effects of propofol on isolated rabbit mesenteric arteries and veins

We have investigated the effect of propofol on isolated rabbit mesenteric arteries and veins. Isometric tension was measured in rings of arteries (with or without endothelium) or veins in organ chambers. The preparation was stimulated with noradrenaline 10(-6) mol litre-1, K+ 50 mmol litre-1 and caffeine 20 mmol litre-1 in the presence or absence of propofol. Propofol potentiated noradrenaline-induced contractions at lower concentrations (3 x 10(-5) mol litre-1) and attenuated them at greater concentrations (10(-4) and 3 x 10(-4) mol litre-1) in arteries with endothelium. Propofol inhibited noradrenaline-induced contractions in arteries without endothelium. In contrast, propofol produced venodilatation in a concentration-dependent manner (10(-5) to 3 x 10(-4) mol litre-1) of significantly greater magnitude than that in arteries. Propofol inhibited K+-induced contraction of both arteries and veins. It decreased the relaxation induced by acetylcholine (3 x 10(-8), 10(-7) and 3 x 10(-7) mol litre-1) of noradrenaline-induced contractions of arteries. Propofol did not affect caffeine-induced contractions after pretreatment with increased Ca2+. We conclude that propofol has a more potent vasodilator effect on veins than on arteries. Vasoconstriction induced by propofol may be associated with inhibition of endothelium-derived relaxing factor, whereas vasodilatation induced by propofol may be associated with block of voltage-gated influxes of extracellular Ca2+.