Caffeine-induced Contraction Research Articles

GTP requirement for inositol-1,4,5-trisphosphate-induced Ca 2+ release from sarcoplasmic reticulum in smooth muscle

We have examined inositol-1,4,5-trisphosphate (IP 3)-induced Ca 2+ release from the sarcoplasmic reticulum (SR) in the skinned vascular smooth muscle. The amount of Ca 2+ in the SR was estimated indirectly by caffeine-induced contraction of the skinned preparation. The Ca 2+ release from the SR by IP 3 required GTP. A non-hydrolyzable analogue of GTP, guanosine 5′-(β′γ-imido) triphosphate (GppNHp) could substitute for GTP in the IP 3-induced Ca 2+ release. These results suggest an involvement of GTP-binding protein in the mechanism of Ca 2+ release from the SR by IP 3 in smooth muscle.

Stimulation frequency and external ionic composition control the repriming of caffeine-induced contractures in frog skeletal muscle.

The effect of stimulation rate and of external ionic composition on the repriming period of contractures induced by 6 mM caffeine was tested on isolated skeletal muscle fibres of the frog (Rana ridibunda). The repriming period, which was 11.2 +/- 0.1 min (mean +/- SEM, n = 9) on quiescent fibres, was shortened in fibres stimulated at a frequency ranging from 3 to 12 min-1 (optimal rate, 8 min-1; full repriming 5.7 +/- 0.2 min; n = 10). A 10-fold increase in the extracellular calcium concentration shortened the repriming period on both stimulated and quiescent fibres, whereas decreasing external calcium (1/10) delayed it. In a Na+-free solution (Li+ substituted) the repriming period of stimulated fibres was markedly delayed (14 min), whereas quiescent fibres never recover more than 10% of their ability to develop subsequent caffeine contractures. In contrast, with a 35% Na solution, the repriming period was greatly shortened (stimulated, 5.4 +/- 0.2 min, n = 7; quiescent, 6.2 +/- 0.5 min, n = 8). It is concluded that repriming depends on three mechanisms that seem to refill a calcium store and trigger recovery: the slow inward calcium current, a Na+-Ca2+ exchange, and perhaps a passive Ca2 influx.

Effects of Nicorandil on Electromechanical Activity of Frog Atrial Muscle

The effects of the antianginal drug nicorandil on electrical and mechanical activity of bullfrog atria were examined. The application of nicorandil at concentrations from 0.3 to 10 mM caused a negative inotropic effect without any appreciable change in the resting membrane potential. The amplitude of action potentials was attenuated. In the voltage clamp experiments, the slow inward current was slightly inhibited, but the background inward current and delayed outward current were not affected in the presence of nicorandil. The inward rectifying properties of time-independent outward current were slightly inhibited by 10 mM nicorandil. Nicorandil inhibited the caffeine-induced contractures elicited in the depolarized preparation, but had no effect on the contracture mediated through the Na-Ca exchange mechanism. These results suggest that the negative inotropic action of nicorandil is caused by inhibition of Ca influx and intracellular mobilization of Ca. Nicorandil had little effect on the potassium conductance in this preparation.

Mechanisms of the Ba2+-induced contraction in smooth muscle cells of the rabbit mesenteric artery.

The mechanism of the Ba2+-induced contraction was investigated using intact and saponin-treated skinned smooth muscle (skinned muscle) strips of the rabbit mesenteric artery. After depletion of Ca2+ stored in the caffeine-sensitive site, greater than 0.65 mM Ba2+ evoked contraction in muscle strips depolarized with 128 mM K+ in Ca2+-free solution in a dose-dependent fashion, and the ED50 values for Ca2+ and Ba2+ were 0.5 mM and 1.2 mM in intact muscle strips, respectively. Nisoldipine (10 nM) blocked the contraction evoked by high K+ or 10 microM norepinephrine (NE) in the presence of 2.6 mM Ba2+, but did not block the contraction evoked in the presence of 2.6 mM Ca2+. These results may indicate that Ba2+ permeates the voltage-dependent Ca2+ channel. In skinned muscle strips, the ED50 values for Ca2+ and Ba2+ were 0.34 and 90 microM, respectively, as estimated from the pCa- and pBa-tension relationships. Calmodulin enhanced and trifluoperazine inhibited the Ba2+- and Ca2+-induced contractions. After the application of Ba2+ or Ca2+ with ATP gamma S in rigor solution, myosin light chain (MLC) was irreversibly thiophosphorylated, as estimated from the Ba2+- or Ca2+-independent contraction. Furthermore, both divalent cations phosphorylated MLC, as measured using two-dimensional gel electrophoresis, to the extent expected from the amplitudes of the contraction evoked by these cations. Thus, Ba2+ is capable of activating the contractile proteins as Ca2+ does. The amount of Ca2+ or Ba2+ stored in cells was estimated from the caffeine response evoked in Ca2+-free solution in intact and skinned muscle strips. After the application of 0.3 microM Ca2+ or 0.1 mM Ba2+ for 60 s to skinned muscle strips after the depletion of Ca2+ stored in cells, caffeine produced a contraction only upon pretreatment with Ca2+ but not with Ba2+. When Ba2+ was applied successively just after the application of Ca2+, the subsequently evoked caffeine-induced contraction was much smaller than that evoked by pretreatment with Ca2+ alone. The above results indicate that Ba2+ permeates the voltage-dependent Ca2+ channel but may not permeate the receptor-operated Ca2+ channel, it releases Ca2+ from store sites but is not accumulated into the store site, and it directly activates the contractile proteins via formation of a Ba2+-calmodulin complex.

Modulation of ryanodine-induced Ca 2+ release in amphibian skeletal muscle

We examined effects of ryanodine on tension in intact and skinned amphibian skeletal muscle. 100 μM ryanodine (RY) alone in the frog Ringer's solution (FR) produced tension in the intact muscle reaching its peak by 1 h: 10 min treatment with RY augmented depolarization-induced tension and prevented a subsequent caffeine-induced contraction. In contrast, RY in Ca 2+-free FR was unable to produce tension, after which caffeine produced irreversible tension. In skinned fibers. RY at pCa 6.5 produced tension and abolished a subsequent caffeine-induced contraction: while RY in 2 mM EGTA did not produce tension. These data indicate that RY, in the presence of Ca 2+, releases Ca 2+ from the SR resulting in subsequent depletion of Ca in the SR.

Ca Mobilization from the Intracellular Ca Store in Spontaneously Hypertensive Rats

To evaluate the size of the intracellular Ca store in spontaneously hypertensive rats (SHR), caffeine-induced contractions were investigated in mesenteric artery preparation. Twelve-week-old SHR and age-matched Wistar-Kyoto (WKY) rats were used. The isolated mesenteric artery was prepared by the method of McGregor. Caffeine (2 mg)- and norepinephrine (2 micrograms)-induced pressor responses were measured in both normal (Ca, 2 mM) and Ca-free solution (1 mM EGTA solution). In the presence of Ca, pressor responses to exogenous norepinephrine in SHR were higher than those in WKY rats. In Ca-free solution, pressor responses to norepinephrine were markedly reduced in SHR compared to WKY rats and caffeine-induced contractions in SHR were lower than those in WKY rats. These results suggest that pressor responses to norepinephrine in SHR may be more dependent on extracellular Ca than in WKY rats and the caffeine-sensitive intracellular Ca store might be less in SHR.

The effects of hyperosmolar solutions on cerebral arterial smooth muscle.

The present study was conducted to clarify the effect of hyperosmolar solutions on the constrictor responses of cerebral arteries to vasoactive agents, in vitro. The canine basilar arteries under resting tension were slightly relaxed with both mannitol (0.5, 1 and 2%) and sucrose (1, 2, and 4%). Constrictor responses of canine basilar arteries to 40 mM K+, 10(-7) M serotonin or 10(-6) M prostaglandin F2 alpha (PGF2 alpha) were markedly suppressed by pretreatment with either mannitol or sucrose. The rate of suppression correlated well to osmolarity changes in the Kreb's solution. When the specimens were incubated in Ca++-free medium, 10(-6) M PGF2 alpha elicited small contractions. Addition of 1 mM Ca++ to the bath promptly elicited larger contractions. The large contractions in response to the influx of extracellular Ca++ were markedly suppressed by pretreatment with mannitol or sucrose, while the small contractions induced by intracellular Ca++ were not inhibited. In addition, the contractions induced by the addition of Ca++ to the specimens depolarized with 80 mM K+ in Ca++-free medium were dose-dependently inhibited with either mannitol or sucrose, while the caffeine-induced contractions in Ca++-free medium were not altered by mannitol. These results suggest that hyperosmolar solutions produce non-specific vasodilation of cerebral arteries by inhibiting the influx of external Ca++ rather than the release of intracellularly stored Ca++. This direct vasodilatory effect may account in part for the transient increase of cerebral blood flow following administration of hyperosmolar mannitol.

Chemical modification of calcium release from the sarcoplasmic reticulum of mechanically skinned skeletal bullfrog muscle fibers.

Several types of reagents that react with amino acid side chains induced repetitive phasic contracture of skinned skeletal muscle from frogs. The presence of 10 mM procaine or 5 mM magnesium in the medium or disruption of the sarcoplasmic reticulum (SR) eliminated this contracture, indicating that the calcium-induced calcium-release mechanism of SR is involved in the contraction. Dithiothreitol inhibited the contracture induced by chloramine T, N-acetylimidazole, or p-chloromercuriphenylsulfonic acid (pCMPS) but not in the case of carbodiimide, phenylglyoxal, trinitrobenzenesulfonic acid, diethylpyrocarbonate (DEP), or N-chlorosuccinimide (NCS). Therefore, modification of groups other than the sulfhydryl ones seems to induce contractures under such conditions. The amplitude of the caffeine-induced contracture decreased after treatment with pCMPS, DEP, or NCS. NCS shifted the pCa-tension curve toward low pCa in the SR-disrupted fibers. This shift would explain the decrease in the caffeine contracture. It is tentatively concluded that pCMPS and DEP release a large amount of calcium from SR.

ACTION OF HALOTHANE ON RAT SOLEUS MUSCLE: TEMPERATURE DEPENDENT INDUCTION OF CONTRACTURES AND MODIFICATION OF CAFFEINE AND POTASSIUM CONTRACTURES

Halothane induced calcium-dependent contractures in rat soleus muscle at temperatures between 4 degrees C and 13 degrees C, but not at higher temperatures. The peak tension of the contractures increased as temperature was decreased below 13 degrees C. The magnitudes of the contractures were reduced by the addition of cobalt or lanthanum ions to the medium in the water bath. The anaesthetic also potentiated caffeine-induced contractures in muscle at 22 degrees C and 37 degrees C in a calcium-dependent manner. The contractures at 37 degrees C were more sensitive to the concentration of this ion than were contractures at 22 degrees C. Halothane increased peak tension and prolonged the time course of potassium-induced contractures, but did not alter their threshold values at either 22 degrees C or 37 degrees C. It was concluded that, in rat muscle, halothane may act at a site on the plasmalemma, retard the onset of inactivation and move the inactivation curve further from the resting membrane potential.

A phorbol ester has dual actions on the mechanical response in the rabbit mesenteric and porcine coronary arteries.

To clarify the role of protein kinase C in the mechanical response, the effects of 12-o-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, were investigated on intact and skinned smooth muscle preparations of the rabbit mesenteric artery. TPA (0.1 microM) showed dual actions (initial enhancement followed by inhibition during long exposure) on the K+-induced contraction. The enhancement was marked in the presence of 39 mM-K+ but inhibition was the predominant effect in the presence of 128 mM-K+. Addition of 2.6 mM-Ca2+ to a Ca2+-free solution containing 2 mM-EGTA following application of A23187 (1 microM), produced contraction. TPA showed the same dual actions on this Ca2+-induced contraction. In chemically skinned muscles, TPA increased the amplitude of Ca2+-induced contractions evoked by low concentrations of Ca2+ (0.1-0.3 microM), but reduced those evoked by high concentrations of Ca2+ (1-10 microM). Both actions of TPA were facilitated in the presence of phosphatidylserine (PS). TPA with PS had no effect on the Ca2+-independent contraction evoked in relaxing solution containing 10 mM-EGTA and 4 mM-Mg ATP following application of adenosine-5-o-3-thiotriphosphate (ATP gamma S) and 0.3 microM-Ca2+. The amount of Ca2+ stored in cells estimated from the amplitude of the caffeine-induced contraction was not modified by application of TPA with PS in skinned or intact muscle tissues. The effects of TPA were investigated on the Ca2+ transient measured from the intensity of fluorescence of quin-2 in dispersed cell suspensions prepared from the porcine coronary artery. TPA had no effect on the Ca2+ transient in high K+ but enhanced the amplitude of the contraction. Amplitudes of the tonic response evoked by 39 mM-K+ in intact muscle tissues and the contraction induced by 0.3 microM-Ca2+ in skinned muscle were much the same. TPA with PS enhanced the amplitudes of both contractions to the same extent. From the above results, we concluded that TPA shows dual actions on the contractile machinery and may act on the regulatory systems of contractile proteins. Both excitatory and inhibitory actions of TPA depended on the concentration of Ca2+. However, the physiological action of protein kinase C as estimated from the action of TPA seems to be related to an excitatory action on the contractile machinery.

Verapamil and zero Ca2+ alter responses of cat muscle to halothane and caffeine.

Strips of soleus (slow twitch, oxidative) and gracilis (fast-twitch, glycolytic) muscle were obtained from 27 anesthetized cats and mounted in organ baths filled with oxygenated Krebs-Ringer solution (37 degrees C). The responses to caffeine, halothane (1%), caffeine in the presence of halothane, and electrical stimulation in the presence of halothane were examined in the two fiber types. These responses were compared with those observed in paired strips of muscle that had been treated with verapamil (10 or 28 microM), a slow calcium (Ca2+) channel blocker, with zero Ca2+, or with zero Ca2+ where magnesium (3.7 mM Ca2+) was added to replace the Ca2+. Halothane-induced contractures in the soleus were blocked by verapamil and zero Ca2+. Caffeine-induced contractures and tetanic contractions were attenuated in zero Ca2+ and by verapamil in both fiber types. Halothane overcame verapamil-induced reductions of caffeine contractures and tetanic contractions in both fiber types. In contrast, halothane did not overcome zero Ca2+-induced reductions in caffeine contractures or tetanic contractions in either fiber type. Furthermore, the addition of Mg2+ to the zero Ca2+ did not restore the responses. The findings with verapamil indicate that in cat muscle, both halothane- and caffeine-induced contractures and tetanic contractions are dependent on the influx of extracellular Ca2+. This extracellular Ca2+ may enter through the slow Ca2+ channels. However, because halothane in combination with caffeine or electrical stimulation overcame the effects of verapamil, there may be other sites involved.

Modification by dantrolene, procaine and suxamethonium of caffeine-induced changes in aequorin luminescence transients and twitch tensions of directly-stimulated diaphragm muscle of mouse.

A convenient method is described for measuring simultaneously Ca2+-related aequorin luminescence and twitch tension in the isolated diaphragm muscle of the mouse. Forty to fifty fibres were injected intracellularly with aequorin solution and the mechanical and luminescence responses to direct stimulation were recorded. The replacement of Na+ by K+ (to obtain 59 or 143.4 mM K+) in the nutrient solution decreased both aequorin luminescence and twitch tensions, but after a time lag, it produced a contracture. Caffeine (5 or 10 mM) increased both aequorin luminescence and twitch tensions, and after a time lag, it also produced a contracture. Dantrolene (1 and 30 microM) and procaine (10 microM, 300 microM and 1 mM) decreased aequorin luminescence transients and twitch tension. In addition procaine inhibited the caffeine-induced increase of aequorin luminescence, but dantrolene did not have this effect. At concentrations causing neuromuscular block, suxamethonium (130 microM) decreased aequorin luminescence transients and twitch tension. By contrast, (+)-tubocurarine (6.5 microM) did not affect the aequorin luminescence in directly stimulated muscles. These results suggest that Ca+-related aequorin luminescence transients accompanied by twitch tensions reflect the intracellular fast mobilization of compartmentalized Ca2+ from plasma membrane or sarcoplasmic reticulum, and that the increase in resting luminescence caused by a K+- or caffeine-induced contracture may be produced by the slow mobilization of Ca2+ from sarcoplasmic reticulum.

Nitroglycerine- and isoprenaline-induced vasodilatation: assessment from the actions of cyclic nucleotides.

To investigate the vasodilator actions of nitroglycerine and isoprenaline, the effects of these agents, dibutyryl cyclic AMP (db cyclic AMP) and 8-bromo cyclic GMP (8-Br cyclic GMP) on intact muscle tissue, and cyclic AMP and cyclic GMP on skinned muscle of the rabbit mesenteric artery were investigated. In porcine coronary artery, nitroglycerine (greater than 0.1 microM) increased the production of cyclic GMP with no change in the amount of cyclic AMP, while isoprenaline (greater than 0.1 microM) significantly increased the production of cyclic AMP with no change in the amount of cyclic GMP. In the rabbit mesenteric artery, nitroglycerine or isoprenaline inhibited the tonic component of the 39 mM [K]o-induced contraction to a greater extent than the phasic component. Nitroglycerine and 8-Br cyclic GMP showed a stronger inhibitory action on the K-induced contraction than did isoprenaline and db cyclic AMP. The sources of Ca utilized for the generation of contraction by noradrenaline and caffeine were estimated to be the same as those determined from the amplitudes of contractions evoked in Ca-free solution by various concentrations of noradrenaline or caffeine. In intact muscle tissues, the effects of nitroglycerine or 8-Br cyclic GMP on the amount of Ca stored in cells were estimated from the caffeine-induced contraction in Ca-free solution. Both agents inhibited the contractions due to a reduction in the amount of Ca in the cells. When the effects of isoprenaline or db cyclic AMP were observed, both agents inhibited the caffeine-induced contraction but the accumulation of Ca into cells was greater than the control. In saponin skinned muscles, the pCa-tension relationship in the presence of cyclic AMP and cyclic AMP-dependent protein kinase (cyclic AMP-PK) shifted to the right and to a lower level in comparison with the control. Applications of cyclic GMP with cyclic GMP-dependent protein kinase (cyclic GMP PK) also inhibited the contraction induced by low concentrations of Ca. In skinned muscles, cyclic AMP exhibited dual actions on Ca store sites, i.e. in the presence of high concentrations of Ca or prolonged superfusion of Ca, cyclic AMP reduced the amount of Ca due to activation of the Ca-induced Ca release mechanism by excess accumulation of Ca. On the other hand, cyclic GMP consistently inhibited the amplitude of the caffeine-induced contraction due to a reduction in the amount of Ca in the store sites. 8 These results indicate that nitroglycerine and isoprenaline increase the amount of cyclic GMP and cyclic AMP, respectively. The main effect of cyclic GMP is activation of Ca extrusion, thus reducing the amount of Ca stored in the cell, while the main effect of cyclic AMP is to increase the amount of Ca stored in the cell. Both cyclic AMP with cyclic AMP-PK and cyclic GMP with cyclic GMP-PK inhibit the phosphorylation of myosin. Consequently both cyclic nucleotides reduce the free Ca in the myoplasm and promote relaxation, but by different mechanisms.

In vitro responses of cat skeletal muscle to halothane and caffeine.

Strips of soleus (100% type I) and gracilis (90% type II) muscle were obtained from anesthetized cats and mounted in organ baths filled with aerated Krebs-Ringer solution (37 degrees C). The contractile patterns in response to electrical stimulation (0.1 Hz, 25 V, 5 ms), caffeine, halothane, and caffeine in the presence of halothane were examined in the two fiber types. The ability of 25 microM dantrolene to alter the contractile patterns was also evaluated. In vitro contractile properties in response to electrical stimulation were similar to properties observed in situ, except that twitch tension in soleus muscle was significantly less in vitro than in situ. In the presence of halothane, type I soleus muscle developed a rapid contracture. The contracture was blocked by pretreatment with dantrolene and was reversed by addition of dantrolene at the peak of the response. Halothane-induced contractures were not observed at any time in type II gracilis. Type I soleus was also significantly more sensitive both to caffeine alone and to caffeine in the presence of halothane than was type II gracilis. In both fiber types, halothane increased the sensitivity of the muscles to caffeine. Dantrolene attenuated caffeine-induced contractures in both fiber types, but the attenuating effect was less in the presence of halothane. The findings of a halothane-induced contracture in the cat soleus and differential sensitivities of the two muscle fiber types to caffeine indicate that further studies in these two muscles may be useful for delineating the mechanisms inducing contracture in muscle from individuals susceptible to malignant hyperthermia.

Depolarizing Neuromuscular Blocking Action Induced by Electropharmacological Coupling in the Combined Effect of Paeoniflorin and Glycyrrhizin

Twitch tensions of indirectly stimulated diaphragm muscles of mice were blocked by a combination of paeoniflorin (PF) and glycyrrhizin (GLR). The mechanism of this effect was studied electropharmacologically. When twitch responses were completely prevented, miniature end-plate, end-plate, and muscle action potentials were still observed when PF and GLR were combined, suggesting that the mechanism is postsynaptic. Potential amplitudes induced by acetylcholine (ACh), which was injected iontophoretically, were inhibited by about 70% by PF (25 μg/ml) plus GLR (75 μg/ml), although neither agent alone caused an inhibition. The combined ratio (PF:GLR = 1:3) by concentrations (g/ml) potentiated both the inhibition of ACh potential amplitudes and the depolarization of resting membrane potentials. These results indicate that the effect of combined PF and GLR is to depolarize the muscle membrane and to block ACh-receptor-linked processes. In chemically skinned (saponin-treated) muscles, the tension induced by 0.39 μM of free calcium was inhibited by PF (300 μg/ml), but it tended to be increased by GLR (300 μg/ml). Caffeine-induced contractures in the skinned muscles was not influenced by PF, and they tended to be decreased with GLR treatment. Thus, in muscles with sustained depolarization, these combined compounds seem to block intracellular Ca2+ movement.

Electrophysiological studies of myotoxin a, isolated from prairie rattlesnake ( Crotalus viridis viridis) venom, on murine skeletal muscles

S. J. Hong and C. C. Chang. Electrophysiological studies of myotoxin a, isolated from prairie rattlesnake ( Crotalus viridis viridis) venom, on murine skeletal muscles. Toxicort 23, 927–937, 1985. — Myotoxin a reduced the resting membrane potential of mouse and rat diaphragms from about −80 mV to −60 mV, induced spontaneous repetitive firing and enhanced the contractile force in response to single stimulations. The depolarizing effect was reversed noncompetitively by tetrodotoxin, local anesthetics or low Na + solution, but was augmented by ouabain or low Cl − solution while being unaffected by high K + solution or electrical stimulation of the muscle. The duration of muscle action potential was prolonged by only 20–30%, whereas the rate of rise (d V/ d t) was unaffected. About a 40% increase of membrane conductance was observed, which could be abolished by the Na +-channel blocker tetrodotoxin. By contrast, K + conductance was unaffected. Effects on caffeine-induced contracture, quantal release of neurotransmitter and the amplitude of miniature endplate potential were not appreciably affected. These effects of myotoxin a indicate that the toxin affects the muscle, but not the nerve, by acting specifically on the Na +-channel of the sarcolemma or T-tubule, like crotamine, rather than on the sarcoplasmic reticulum. The effects of sea anemone toxin II on the Na +-channel (marked depolarization and prolongation of action potential) could not be prevented by saturating the muscle with myotoxin a. On the other hand, the effect of veratridine, a member of another group of toxins acting on the Na +-channel, was enhanced. These results suggest that myotoxin a acts on the Na −-channel at a site which is discrete from those of tetrodotoxin, veratridine and sea anemone toxin II.

Changes in cyclic AMP and cyclic GMP levels in the contraction-relaxation cycle of isolated smooth muscle cells from guinea-pig taenia caeci.

The present study was undertaken to investigate the role of cyclic AMP and cyclic GMP in the regulation of the contraction-relaxation cycle in isolated smooth muscle cells from guinea pig taenia caeci. The contraction of isolated smooth muscle cells induced by both caffeine (4 X 10(-3) M) and carbachol (10(-4) M) consisted of an initial activation phase and a following spontaneous relaxation phase. Isoproterenol (10(-4) M) inhibited the carbachol-induced contraction and raised intracellular cyclic AMP level of isolated smooth muscle cells. Both the inhibition of the carbachol-induced contraction and the rise in cyclic AMP level elicited by isoproterenol were blocked by propranolol (10(-4) M). Caffeine increased intracellular cyclic AMP level significantly and contracted isolated smooth muscle cells. Caffeine and carbachol contracted isolated smooth muscle cells and increased intracellular cyclic GMP level. Cyclic GMP was significantly increased in spontaneous relaxation phase. The data suggest that beta-adrenergic inhibitory effect on cholinergic excitation in the mammalian isolated smooth muscle cells is mediated by the change in intracellular cyclic AMP level and that cyclic GMP related to the relaxation in these single cells. Thus, it appears that caffeine-induced contraction might be due to the stimulation of Ca2+-induced Ca2+ release by cyclic AMP.

The effects of caffeine on the noradrenaline-sensitive calcium store in rabbit aorta.

The effects of caffeine on the noradrenaline-sensitive Ca store in rabbit aorta have been studied measuring tension development and stimulated 45Ca efflux in Ca-free solution. In addition, the inhibitory effects of caffeine on transmembrane Ca movements and on intracellular Ca release have been investigated, again comparing contractile force and 45Ca fluxes. The maximally effective caffeine concentration fully depletes the noradrenaline-sensitive Ca store as found from both contraction and 45Ca efflux experiments. Within the concentration ranges used in this study for either caffeine and noradrenaline, a linear relation exists between the amplitude of contraction and the amount of stimulated 45Ca efflux. The Ca content of the store (presumably sarcoplasmic reticulum, s.r.) is 74 mumol/kg aorta, corresponding to an s.r. Ca concentration of 5.3 mM. After exposure of the cells to a high-K solution the s.r. Ca concentration reached a value of 8.5 mM. At 37 degrees C the Ca is lost from the store in Ca-free solution with a half-time of 34 min. Although a comparable amount of 45Ca is released by the maximal doses of caffeine and noradrenaline, the caffeine-induced contraction is much smaller than the noradrenaline-induced one. The increase in adenosine 3',5'-cyclic monophosphate (cyclic AMP) after caffeine was less than after noradrenaline, so could not account for the small size of the caffeine-induced contraction. Asynchrony in response to caffeine by successive layers of smooth muscle, and a separate inhibitory action of caffeine, may contribute to the small size of caffeine-induced contractions in thick muscle preparations. Caffeine inhibits high-K-induced contractions by inhibition of Ca influx. Caffeine also inhibits the noradrenaline-induced contractions in Ca-free solution. These inhibitory actions could be dissociated from caffeine-induced release of intracellular Ca.

Effects of isoprenaline on the contraction-relaxation cycle in the cat trachea.

Effects of isoprenaline (Isop) on the contractile properties of the smooth muscle cells of cat trachea were investigated using intact and chemically skinned muscle preparations and an isometric tension recording method. In the intact muscle preparations, Isop 3 X 10(-10) or 3 X 10(-9) M significantly suppressed the amplitude of tonic contractions evoked by acetylcholine (ACh) 10(-7) M or 10(-5) M, respectively. Following treatment of the tissue with Ca2+-free 2 mM EGTA-containing solution after depletion of stored Ca2+ with caffeine, 2.5 mM Ca2+ was applied for 5 min (procedure 1), and subsequently 10 mM caffeine was applied in Ca2+-free 2 mM EGTA containing solution. The object was to estimate the amount of stored Ca2+ during procedure 1 from the amplitude of the caffeine (10 mM)-induced contraction (procedure 2). Isop, applied during procedure 1, did not affect the amplitude of the caffeine-induced contraction; however, when applied during procedure 2, this agent (10(-8)M) significantly suppressed the amplitude of the caffeine-induced contraction to about 90% of the control value. ACh (10(-5)M), applied during procedure 1, evoked phasic and tonic contractions. Isop (10(-8)M), applied simultaneously with ACh (10(-5)M), suppressed the amplitude of the ACh-induced contraction yet increased the amplitude of contraction evoked by the subsequent application of caffeine 10 mM (procedure 2). Effects of conditioning application of ACh (10(-7) or 10(-5)M) on the caffeine-induced contraction were observed in the presence or absence of Isop during procedure 2. When ACh 10(-5)M was used, subsequent application of caffeine 10 mM evoked no mechanical response, in control conditions. However, after the pretreatment of the tissue with Isop during procedure 2, the amplitude of the ACh (10(-5)M)-induced contraction was not affected, yet the subsequent application of caffeine (10 mM) evoked minute but discrete contractions, indicating that Isop did enhance the sequestration of free Ca2+ into the storage sites. In the saponin-treated skinned muscles, the minimum concentration of Ca2+ required to produce contraction was 1 X 10(-7)M, and the maximum contraction was obtained with 1 X 10(-5)M Ca2+. Isop (10(-6)M) had no effect on the relationship between free-Ca2+ and the amplitude of the contraction. However, simultaneous application of high concentrations of cyclic AMP (10(-4)M) and cyclic AMP-dependent protein kinase (50 micrograms ml-1) significantly suppressed contractions evoked by 3 X 10(-7) or 10(-5)M Ca2+. 8 These results indicate that Isop suppresses the contraction evoked by various agonists in the cat trachea, mainly through sequestration of Ca2+ into the intracellular storage sites, rather than by direct or indirect (through cyclic AMP) actions on the contractile proteins.

The roles of stored calcium in contractions of cat tracheal smooth muscle produced by electrical stimulation, acetylcholine and high K+.

Effects of direct or indirect (nerve-mediated) muscle stimulation, acetylcholine (ACh), caffeine and procaine on the membrane and mechanical properties of smooth muscle cells of the cat trachea were investigated by means of double sucrose-gap and isometric tension recording methods. Outward current pulses (2 s in duration) applied to the muscle tissue in the presence of tetrodotoxin (10(-7)M), atropine (10(-6)M) and propranolol (10(-6)M) evoked no action potential (spike); however, when the depolarization exceeded 9 mV, a contraction was evoked. The spike and contraction evoked by outward current pulses in the presence of tetraethylammonium (TEA, 10 mM) were suppressed by treatment of the tissue with either Ca2+-free EGTA (2 mM) containing solution or Mn2+ (5 mM). In the presence of procaine (10 mM), outward current pulses evoked an action potential but no contraction. Field stimulation of short duration (50 microseconds) applied to the whole tissue produced an excitation of the intrinsic nerves and evoked excitatory junction potentials (e.j.ps), and when the amplitude of e.j.ps exceeded 4 mV, a twitch contraction occurred. E.j.p. was more effective in producing a contraction than was the membrane depolarization evoked by outward current pulses. Amplitudes of contractions evoked by exogenous ACh (10(-5)M) were much larger than those evoked by 128 mM-[K]0 or caffeine (10 mM), in normal Krebs solution. When the amplitudes of the contractions produced by 128 mM [K]0 were defined as a relative amplitude of 1.0, the mean amplitudes of contraction produced by ACh (10(-5)M) or caffeine were 2.5 +/- 0.20 or 1.2 +/- 0.26, respectively. In Ca2+-free EGTA (2 mM)-containing solution, the contraction induced by 128 mM-[K]0 was rapidly abolished, whereas the contractions evoked by caffeine (10 mM) or the initial phasic contraction produced by ACh (10(-5)M) were largely unaffected. When the amount of Ca2+ stored in the muscle cell was estimated from the amplitude of caffeine-induced contraction evoked in Ca2+-free solution, procaine (10 mM) applied simultaneously with Ca2+, after depletion of Ca2+ from the cells by means of caffeine, increased the amount of Ca2+ stored to 1.31 +/- 0.14 (n = 6) times the control value. However, ACh (10(-7)M) or excess concentrations of [K]0 applied with Ca2+ did not increase the amount of Ca2+ stored in the caffeine-sensitive intracellular compartment. 8 These results indicate that the amount of Ca2 + stored in the smooth muscle cells of the cat trachea may be larger than other visceral smooth muscle and plays an important role in the initiation of contraction, in response to endogenous or exogenous ACh.