Differential regulation of smooth muscle contraction in rabbit internal anal sphincter by substance P and bombesin.

Substance P and bombesin induce contraction of isolated IAS smooth muscle cells by different intracellular mechanisms. The cells contracted in a dose dependent manner to both peptides. The kinetics of contraction were different. Substance P induced contraction peaked at 30 seconds and declined in a time dependent manner while bombesin induced contraction peaked at 30 seconds and was maintained for up to 8 minutes. The absence of extracellular calcium in the medium (0 calcium and 2 mM EGTA) had no affect on substance P induced contraction while it blocked bombesin induced contraction. Substance P induced contraction was blocked by the calmodulin antagonist W7 (10(-9)M) and was not affected by the PKC antagonist H7 (10(-6)M). Bombesin induced contraction was blocked by the PKC antagonist H7 and was not affected by the calmodulin antagonist W7. Our data indicate that substance P induces a transient contraction utilizing intracellular calcium and a calmodulin dependent pathway, while bombesin induces a sustained contraction utilizing calcium from extracellular sources and a calmodulin independent pathway.     

The source of calcium for CCK-induced contraction of the guinea-pig gall bladder.

The sources of calcium for cholecystokinin octapeptide (CCK-OP)-induced gallbladder smooth muscle contraction are considered both extracellular and intracellular, but the relative need for intracellular calcium especially at low, physiological concentrations is not clear. To better define the calcium sources responsible for guinea-pig gallbladder contractions in vitro, we inhibited calcium influx using the calcium channel blocker, methoxyverapamil, and a calcium-free Krebs' solution. Availability and release of intracellular calcium stores were depleted by strontium substitution and ryanodine. CCK-OP was compared to bethanechol and potassium chloride (KCl). Preventing calcium influx with 10(-5) M methoxyverapamil depressed the responses to CCK-OP, bethanechol and KCl. Methoxyverapamil, however, had little effect on the time-dependent generation of tension to CCK-OP, but significantly reduced the response to bethanechol and KCl, each at ED50. The duration of the contractile response in the calcium-free Krebs' solution to CCK-OP was longer than that for bethanechol. Strontium (2.5 mM) significantly attenuated the response to CCK-OP and bethanechol, but not to KCl. Ryanodine significantly reduced contractions induced by CCK-OP but not for bethanechol, both at low dose ED25. These results indicate that contraction of the guinea-pig gallbladder induced by CCK-OP, bethanechol and KCl requires extracellular calcium influx. Further, the initiation and maintenance of contraction by CCK-OP and bethanechol necessitates calcium mobilisation from intracellular stores. CCK-OP may have a greater penchant for these calcium stores, particularly at physiological doses.     

Poloxamer 188 reduces the contraction-induced force decline in lumbrical muscles from mdx mice

Duchenne Muscular Dystrophy is a genetic disease caused by the lack of the protein dystrophin. Dystrophic muscles are highly susceptible to contraction-induced injury, and following contractile activity, have disrupted plasma membranes that allow leakage of calcium ions into muscle fibers. Because of the direct relationship between increased intracellular calcium concentration and muscle dysfunction, therapeutic outcomes may be achieved through the identification and restriction of calcium influx pathways. Our purpose was to determine the contribution of sarcolemmal lesions to the force deficits caused by contraction-induced injury in dystrophic skeletal muscles. Using isolated lumbrical muscles from dystrophic (mdx) mice, we demonstrate for the first time that poloxamer 188 (P188), a membrane-sealing poloxamer, is effective in reducing the force deficit in a whole mdx skeletal muscle. A reduction in force deficit was also observed in mdx muscles that were exposed to a calcium-free environment. These results, coupled with previous observations of calcium entry into mdx muscle fibers during a similar contraction protocol, support the interpretation that extracellular calcium enters through sarcolemmal lesions and contributes to the force deficit observed in mdx muscles. The results provide a basis for potential therapeutic strategies directed at membrane stabilization of dystrophin-deficient skeletal muscle fibers.     

Extracellular calcium transients at single excitations in rabbit atrium measured with tetramethylmurexide

Extracellular calcium transients were resolved within the time course of single contraction cycles in rabbit left atrium using tetramethylmurexide (2 mM) as the calcium-sensitive dye (150-250 microM total calcium, 80-150 microM free calcium). Net extracellular calcium depletion began within 2-4 ms upon excitation; over the following 5-20 ms, depletion continued steeply and amounted to 0.2 mumol/kg wet weight X 10 ms (135 microM free extracellular calcium). In regularly excited muscles (0.5-2 Hz), net depletion slowed rapidly and stopped early during the rise of contractile motion monitored by transmitted light. Maximum depletions amounted to 0.2-0.5% of total extracellular calcium (0.2-0.5 mumol/kg wet weight with 135 microM free calcium). Replenishment of extracellular calcium began at the latest midway to the peak of the motion signal. Calcium replenishment could be complete for the most part by an early phase of relaxation or could take place continuously through relaxation. The maximal net depletion per beat decreased manyfold with a decrease of frequency from 1 to 0.05 Hz. During paired pulse stimulation (200-300-ms twin pulse separation at basal rates of 0.3-1 Hz), extracellular calcium accumulation was enhanced at the initial potentiated contraction; extracellular calcium depletion was prolonged at the low-level premature contraction. With quadruple stimulation (three premature excitations), the apparent rate of net extracellular calcium accumulation at potentiated contractions approached or exceeded the apparent rate of early net calcium depletion. Under the special circumstance of a strongly potentiated post-stimulatory contraction after greater than 5 s rest, repolarization beyond -40 mV occurred within 10 ms, net extracellular calcium accumulation began with the onset of muscle motion, and net extracellular calcium accumulation (1-3 microM/kg wet weight) coincided with a more positive late action potential in comparison with subsequent action potentials. Consistent changes of the apparent rate of early net calcium depletion were not found with any of the simulation patterns examined. In ryanodine-pretreated atria, the duration of depletion was clearly limited by action potential duration at post-rest stimulations; in the presence of 4-aminopyridine (2 mM), depletion continued essentially undiminished for up to 200 ms. The resulting net depletion magnitudes were greater than 10 times larger than the transient depletions found during steady stimulation.     

Absence of staircase following disuse in rat gastrocnemius muscle

Repetitive stimulation of mammalian fast-twitch skeletal muscles will normally result in a positive staircase response. This phenomenon was investigated in the rat gastrocnemius muscle following a 2-week period of tetrodotoxin-induced disuse. Muscle inactivity was imposed by superfusing tetrodotoxin in saline over the left sciatic nerve via an implanted osmotic pump. In situ isometric contractile responses to double pulse stimulation and repetitive stimulation at 10 Hz were determined the day after removal of the pump. Two weeks of disuse resulted in 40% muscle weight loss. A twitch contraction gave the same force when expressed per gram of wet muscle weight in control muscles, 317 +/- 24.6 (means +/- SE) g/g, as compared with tetrodotoxin-treated muscles, 328 +/- 24.2 g/g. Both contraction time and half-relaxation time were prolonged following treatment with tetrodotoxin. Repetitive stimulation at 10 Hz resulted in a positive staircase response in the control muscles, but not in muscles of the tetrodotoxin-treated rats. The observed changes in the time course of the twitch contraction with repetitive stimulation following tetrodotoxin-induced disuse are consistent with alterations in sarcoplasmic reticulum handling of calcium. It is not certain if there is a change following disuse in the mechanism normally associated with staircase or if this mechanism is merely opposed by an early fatigue.     

Calcium dependency and the effect of calcium antagonists on molluscan proboscis smooth muscles from the whelk,Buccinum undatum

In all four proboscis muscles of the whelk Buccinum undatum, the potassium-induced depolarization response was acutely dependent upon extracellular calcium, being eliminated in calcium-free conditions. The responses to acetylcholine were found to be partly dependent upon intracellular calcium. Responses to the peptides phenylalanine-methionine-arginine-phenylalanine-NH₂ and phenylalanine-leucine-arginine-phenylalanine-NH₂ were much more resistant to calcium-free conditions and appeared to engage the excitation-contraction coupling mechanism by mobilizing stored intracellular calcium. Sucrose-gap studies of radular retractor muscles showed that the organic calcium “antagonist” nifedipine enhanced potassium-induced depolarization responses, initiating spike-like action potentials and associated fast twitch activity. The inorganic calcium antagonist gadolinium exerted concentration-dependent inhibitory actions on these muscles. Basal tonus and fast twitch activity in response to potassium-induced depolarization were eliminated as were the spike-like action potentials of the membrane electrical response. The inorganic calcium “antagonist” cadmium greatly enhanced potassium-induced contractures in all four muscles, and on its own it induced tonic force and fast twitches in all the muscles. It seems likely that cadmium may have displaced stored intracellular calcium to induce myofilament activation. While these molluscan smooth muscles appear to possess calcium channels with fast and slow characteristics, their behaviour and pharmacological manipulation is very different from their more well known mammalian transient and long-lasting channel counterparts.     

Effects of extracellular calcium on canine tracheal smooth muscle

Strips of canine tracheal smooth muscle were studied in vitro to determine the effects of changes in the extracellular calcium (Cao) concentration on tonic contractions induced by acetylcholine and 5-hydroxytryptamine. Strips were contracted with graded concentrations of the above agents in 2.4 mM Ca, after which CaCl2 was administered to achieve final concentrations of 5.0, 10.0, and 20.0 mM. Increases in Cao to 5 mM or above caused significant relaxation of muscles contracted with 5-hydroxytryptamine but did not significantly relax muscles contracted with acetylcholine. Increases in Cao also caused significant relaxation of muscles contracted with low concentrations of K+ (20 or 30 mM). However, in 60 or 120 mM K+, increases in Cao resulted predominantly in muscle contraction. Inhibition of the Na+-K+-ATPase by ouabain (10(-5) M) or K+ depletion reversed the effects of Cao from relaxation to contraction in tissues contracted with 5-hydroxytryptamine. Increases in Cao also caused contraction rather than relaxation in the presence of verapamil (10(-6) M). We conclude that calcium has both excitatory and inhibitory effects on the contractile responses of canine tracheal smooth muscle. The inhibitory effects of Ca2+ appear to be linked to the activity of the membrane Na+-K+-ATPase.     

Regulation of calcium influx into buccal muscles of Aplysia by acetylcholine and serotonin

Acetylcholine (ACh) causes contraction of Aplysia buccal muscles E1 and I5, and serotonin (5-hydroxytryptamine, 5-HT) enhances ACh-elicited contractions of these muscles. Possible roles of calcium influx in mediating these responses were examined by studying influx of 45Ca++. 5-HT increased calcium influx into both I5 and E1. Maximal influx occurred at 10(-6) M 5-HT and the increased influx could be sustained in the presence of 5-HT for at least 10 min. ACh also caused calcium influx, and calcium influx increased approximately in proportion to log[ACh] from 10(-5) M to 10(-3) M ACh. 5-HT and ACh probably bring about calcium influx by different mechanisms since the effect of ACh was additive to a maximal 5-HT response, and 10(-4) M hexamethonium bromide inhibited the increased influx caused by ACh but did not affect influx caused by 5-HT. Cyclic AMP analogues and forskolin neither caused an increase in calcium influx nor an increase in the influx caused by ACh. The data support a model in which ACh-elicited contractions of I5 and E1 are due primarily to calcium entry across the extracellular membrane, and 5-HT can "load" an intracellular site by a mechanism different from that activated by ACh. The data do not support a role for cyclic AMP in mediating the calcium influx response to 5-HT.     

Effect of low extracellular calcium and ryanodine on muscle contraction of the mouse during postnatal development.

We have examined the effects of low Ca2+ solutions, Co2+, and ryanodine on the isometric tension and contraction speed of isolated, developing mouse EDL muscles. Twitch responses of young muscles (7-14 days postnatal) were more sensitive to lowered [Ca2+]o than those of more fully developed muscles (22-35 days postnatal). Responses of EDL muscles from a middle-aged group (15-21 days postnatal) were intermediate between the two other groups. Overall, the time course of contraction in a single twitch was accelerated by low [Ca2+]o. Ca(2+)-free solution induced a 7.95 and 9.25 mV depolarization in young and "old" muscle fibres, respectively. The presence of cobalt ions (5 mM) in the Krebs solution had a similar effect as Ca(2+)-free Krebs in terms of reduction of the isometric twitch and tetanic tensions of EDL muscles from the various age groups. In contrast, the shortening of the contraction time seen with Ca(2+)-free solution did not take place following exposure to Co(2+)-containing solutions. Finally, young (7-14 days postnatal) muscles were less sensitive to the inhibitory action of ryanodine on the twitch compared with more fully developed muscles (22-35 days postnatal). Taken together, our results indicate that from birth to maturity, there is a gradual change in the spectrum of calcium utilization for the contractile process.     

Calcium dependence of effects of endothelin on rat mesenteric microvessels

We investigated the calcium dependence of the effects of endothelin (ET) on resistance vessels (less than 300 microns lumen diameter) from the mesenteric vascular bed of the rat, mounted on a wire myograph. ET-1 induced a potent sustained contraction with an ED50 of 12 nmol/L. The response to ET-3 and big ET at the maximum concentrations used (100 nmol/L) was less than 40% of that to ET-1, with an estimated ED50 of 45 nmol/L. Relaxation of the ET-1-induced contraction was slow, and resulted in a reduction of the maximum response to a second challenge with ET-1 to 60% of the initial contraction after 3 h. Long-lasting tachyphylaxis to arginine vasopressin (AVP) induced contraction also occurred. The response to 100 nmol/L ET-1 produced an active tension 88% greater than that induced by 124 mmol/L KCl, and similar to that produced by norepinephrine and AVP. The response to 100 nmol/L ET-1 in the absence of calcium + 1 mmol/L EGTA in the medium for 30 min resulted in a maximum contraction of 43% of the response in the presence of calcium, followed by a faster relaxation rate. The addition of calcium produced a further contraction, and stimulation with 100 nmol/L ET-1 at this point did not result in further response. The calcium channel blocker nitrendipine in concentrations of 1-10 mumol produced increasing reductions of the responses to 100 nmol/L ET-1 to 35% at the higher concentration. Nitrendipine (3 mumol/L) partially blocked the response to calcium after ET-1 was added in the absence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of cold-shock protein RBM3 as a possible regulator of skeletal muscle size through expression profiling

Changes in gene expression associated with skeletal muscle atrophy due to aging are distinct from those due to disuse, suggesting that the response of old muscle to inactivity may be altered. The goal of this study was to identify changes in muscle gene expression that may contribute to loss of adaptability of old muscle. Muscle atrophy was induced in young adult (6-mo) and old (32-mo) male Brown Norway/F344 rats by 2 wk of hindlimb suspension (HS), and soleus muscles were analyzed by cDNA microarrays. Overall, similar changes in gene expression with HS were observed in young and old muscles for genes encoding proteins involved in protein folding (heat shock proteins), muscle structure, and contraction, extracellular matrix, and nucleic acid binding. More genes encoding transport and receptor proteins were differentially expressed in the soleus muscle from young rats, while in soleus muscle from old rats more genes that encoded ribosomal proteins were upregulated. The gene encoding the cold-shock protein RNA-binding motif protein-3 (RBM3) was induced most highly with HS in muscle from old rats, verified by real-time RT-PCR, while no difference with age was observed. The cold-inducible RNA-binding protein (Cirp) gene was also overexpressed with HS, whereas cold-shock protein Y-box-binding protein-1 was not. A time course analysis of RBM3 mRNA abundance during HS showed that upregulation occurred after apoptotic nuclei and markers of protein degradation increased. We conclude that a cold-shock response may be part of a compensatory mechanism in muscles undergoing atrophy to preserve remaining muscle mass and that RBM3 may be a therapeutic target to prevent muscle loss.     

Role of mitochondrial superoxide dismutase in contraction-induced generation of reactive oxygen species in skeletal muscle extracellular space

Contractions of skeletal muscles produce increases in concentrations of superoxide anions and activity of hydroxyl radicals in the extracellular space. The sources of these reactive oxygen species are not clear. We tested the hypothesis that, after a demanding isometric contraction protocol, the major source of superoxide and hydroxyl radical activity in the extracellular space of muscles is mitochondrial generation of superoxide anions and that, with a reduction in MnSOD activity, concentration of superoxide anions in the extracellular space is unchanged but concentration of hydroxyl radicals is decreased. For gastrocnemius muscles from adult (6–8 mo old) wild-type (Sod2^+/+) mice and knockout mice heterozygous for the MnSOD gene (Sod2^+/-), concentrations of superoxide anions and hydroxyl radical activity were measured in the extracellular space by microdialysis. A 15-min protocol of 180 isometric contractions induced a rapid, equivalent increase in reduction of cytochrome c as an index of superoxide anion concentrations in the extracellular space of Sod2^+/+ and Sod2^+/- mice, whereas hydroxyl radical activity measured by formation of 2,3-dihydroxybenzoate from salicylate increased only in the extracellular space of muscles of Sod2^+/+ mice. The lack of a difference in increase in superoxide anion concentration in the extracellular space of Sod2^+/+ and Sod2^+/- mice after the contraction protocol supported the hypothesis that superoxide anions were not directly derived from mitochondria. In contrast, the data obtained suggest that the increase in hydroxyl radical concentration in the extracellular space of muscles from wild-type mice after the contraction protocol most likely results from degradation of hydrogen peroxide generated by MnSOD activity. hydroxyl radicals; microdialysis     

Characteristics of functioning of electromechanical coupling in striated muscles of higher and lower vertebrates

A comparative pharmacological analysis of relative contributions of different signal transduction pathways in the activation of contraction (excitation-contraction coupling, ECC) in intact fast striated muscles of frog and lamprey was performed. It was found that the major mechanism responsible for the ECC in muscles of both animals is Ca2+ release from the sarcoplasmic reticulum through the ryanodine-sensitive channels. However, the ECC in lamprey muscle displays some important differences in the units of electromechanical coupling, which precede the calcium release from sarcoplasmic reticulum. The maximum contraction force in frog muscle develops during caffeine-induced contracture, which indicates that all Ca2+ stored in sarcoplasmic reticulum is released through ryanodine-sensitive channels. In contrast, in lamprey muscle, the maximum force develops not in response to high caffeine concentration, but in response to repetitive electrical stimulation. Hence, in addition to stores liberated by ryanodine-sensitive channels, some other sources of calcium ions should exist, which contribute to the contraction activation. A source of this additional Ca2+ ions can be external medium, because acetylcholine contracture is abolished in a calcium-free medium. In frog muscle, the acetylcholine contracture was abolished in a Na(+)-free solution. It was concluded that in frog muscle ECC can be triggered by changes in the transmembrane potential (depolarization-induced calcium release), while in lamprey muscle the entry of calcium ions into myoplasm as the trigger in ECC (calcium-induced calcium release). The lamprey muscle was found to be more resistant to tetrodotoxin and tetracaine, which is indicative of a role in the activation of contraction of tetrodotoxin-resistant Na+ and/or Ca2+ channels. It was concluded, that ECC mechanism in striated muscles of low vertebrates is not limited by the generally accepted scheme of depolarization-induced calcium release but can include some other schemes, which require the Ca2+ influx into the cell.     

Effect of D20 on contractility and excitation contraction coupling in the rat papillary muscle]

D2O decreases the isometric contraction force of papillary muscles from the left ventricle of rats. This effect can be compensated only partially by increasing the calcium content in the perfusion solution. Differences in the effect of D2O under different calcium concentrations point also to an action site other than at the contractile proteins. The enlargement of the restitution of contraction 300 msec after the basic beat, the diminution of the postextrasystolic tiation and the disappearance of postextrasystolic depressions because of high calcium potencontent in the bathing solution make it more probable that the action site lies in cell compartments which may be involved into the supply of activator calcium. The increasing occurrence of aftercontractions alredy at slightly enhanced extracellular calcium is in good agreement with this fact.     

Loss of the Inducible Hsp70 Delays the Inflammatory Response to Skeletal Muscle Injury and Severely Impairs Muscle Regeneration

Skeletal muscle regeneration following injury is a highly coordinated process that involves transient muscle inflammation, removal of necrotic cellular debris and subsequent replacement of damaged myofibers through secondary myogenesis. However, the molecular mechanisms which coordinate these events are only beginning to be defined. In the current study we demonstrate that Heat shock protein 70 (Hsp70) is increased following muscle injury, and is necessary for the normal sequence of events following severe injury induced by cardiotoxin, and physiological injury induced by modified muscle use. Indeed, Hsp70 ablated mice showed a significantly delayed inflammatory response to muscle injury induced by cardiotoxin, with nearly undetected levels of both neutrophil and macrophage markers 24 hours post-injury. At later time points, Hsp70 ablated mice showed sustained muscle inflammation and necrosis, calcium deposition and impaired fiber regeneration that persisted several weeks post-injury. Through rescue experiments reintroducing Hsp70 intracellular expression plasmids into muscles of Hsp70 ablated mice either prior to injury or post-injury, we confirm that Hsp70 optimally promotes muscle regeneration when expressed during both the inflammatory phase that predominates in the first four days following severe injury and the regenerative phase that predominates thereafter. Additional rescue experiments reintroducing Hsp70 protein into the extracellular microenvironment of injured muscles at the onset of injury provides further evidence that Hsp70 released from damaged muscle may drive the early inflammatory response to injury. Importantly, following induction of physiological injury through muscle reloading following a period of muscle disuse, reduced inflammation in 3-day reloaded muscles of Hsp70 ablated mice was associated with preservation of myofibers, and increased muscle force production at later time points compared to WT. Collectively our findings indicate that depending on the nature and severity of muscle injury, therapeutics which differentially target both intracellular and extracellular localized Hsp70 may optimally preserve muscle tissue and promote muscle functional recovery.     

Further investigation of a potential calcium channel modulator isolated from rat erythrocytes

The contractile effects of a peptide isolated from rat erythrocytes were further studied in rat aortic rings. Previous data showed that preincubation of aortic tissue with the peptide had no effect on resting tension, but significantly enhanced K+ and norepinephrine (NE) induced contraction. The calcium channel antagonist verapamil noncompetitively blocked the effect of the peptide, whereas nifedipine blockage appeared to be competitive. In the present study the peptide enhanced K+, NE, and phenylephrine (PE) induced contraction in a concentration-dependent manner, with a maximum enhancement at peptide concentrations of 10(-7)-10(-6) M. At a concentration as low as 10(-9) M, the peptide significantly enhanced K(+)-induced, but not NE- or PE-induced, contraction. The magnitude of maximal enhancement was greater for K(+)-induced contraction than that for NE- or PE-induced contraction. Preincubation of the tissues with the peptide caused a leftward shift of cumulative concentration-response curves to K+ and NE. The peptide enhancement of contraction increased with increasing K+ and NE concentration. The peptide potentiated the contractile response to Ca2+ in K(+)-depolarizing medium. It also enhanced the contractile response to NE in intracellular Ca2(+)-pool-depleted tissue following the replenishment of extracellular Ca2+, but had no apparent effect on the mobilization of intracellular calcium. Addition of nifedipine caused a rightward shift of both the peptide and Bay K 8644 concentration-response curves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Static contraction increases arachidonic acid levels in gastrocnemius muscles of cats

Static contraction of hind-limb muscles is well known to increase reflexly cardiovascular function. Recently, blockade of cyclooxygenase activity has been reported to attenuate the reflex pressor response to contraction, a finding which suggests that working skeletal muscle releases arachidonic acid metabolites. Therefore, we measured the effects of static contraction and ischemia on arachidonic acid levels in the gastrocnemius muscles of barbiturate-anesthetized cats treated with indomethacin. Unesterified arachidonic acid levels were measured by high-pressure liquid chromatography. We found that static contraction of freely perfused gastrocnemius muscles increased arachidonic acid levels from 4.4 +/- 1.0 to 10.3 +/- 2.2 nmol/g wet wt (n = 12; P less than 0.005). Likewise, static contraction of gastrocnemius muscles made ischemic for 2 min before the onset of the contraction period increased arachidonic acid levels from 12.6 +/- 2.3 to 21.0 +/- 2.0 nmol/g wet wt (n = 12; P less than 0.01). Lastly, 2 min of ischemia with the gastrocnemius muscles at rest increased arachidonic acid levels from 5.9 +/- 1.1 to 10.5 +/- 3.0 nmol/g wet wt (n = 18; P less than 0.02). We conclude that both static contraction and ischemia increase arachidonic acid levels in working hindlimb muscle.     

Potential-dependent calcium channels in rabbit jejunum

Potential-dependent calcium channels have been studied in the isolated rabbit jejunum. A biphasic response was observed, a transient and rapid contraction followed by partial relaxation and a sustained contraction attaining a plateau state. These are similar to the phasic and tonic responses observed in the isolated smooth muscles of other species. Both the responses are susceptible to blockade by the calcium antagonists bepridil, diltiazem, nifedipine, PN 200-110 (isradipine), and verapamil. Two calcium pools or the presence of two channels affecting the two responses are proposed.     

Deuterium oxide reduces agonist and depolarization-induced contraction of rat aortic rings.

The influence of deuterium oxide (D2O) on calcium-dependent vascular smooth muscle contraction was investigated. The effect of D2O on receptor-operated calcium channels was investigated with phenylephrine-induced contraction in the rat aortic ring preparation. D2O depressed the contraction response in a dose-dependent manner with 50% inhibition of maximum contraction observed with 60% D2O. The effect of 60% D2O on phenylephrine-induced contraction was reversible and not dependent on an intact endothelium. Sixty percent D2O also reduced potassium chloride induced contractions by 50%, indicating an effect on voltage-operated calcium channels. Studies with Bay K 8644, and L-type calcium channel activator, confirm an effect on utilization of extracellular calcium sources and on the voltage-operated calcium channel. Sixty percent D2O also depressed a calcium contraction dose-response curve by approximately 25%. Likewise, a change in the pD2' for nifedipine in the presence of D2O may indicate an effect on the nifedipine binding site and (or) the voltage-dependent calcium channel. Further studies were performed to determine whether the D2O effects were nonspecific or selective effects on the receptor- and voltage-operated calcium channels. Sucrose-induced contaction in the presence of 60% D2O was found to be inhibited by approximately 50%. D2O similarly affected isoprenaline relaxation, which would suggest a nonspecific D2O effect on the vascular smooth muscle contractile process.     

Signaling in Muscle Contraction

Signaling pathways regulate contraction of striated (skeletal and cardiac) and smooth muscle. Although these are similar, there are striking differences in the pathways that can be attributed to the distinct functional roles of the different muscle types. Muscles contract in response to depolarization, activation of G-protein-coupled receptors and other stimuli. The actomyosin fibers responsible for contraction require an increase in the cytosolic levels of calcium, which signaling pathways induce by promoting influx from extracellular sources or release from intracellular stores. Rises in cytosolic calcium stimulate numerous downstream calcium-dependent signaling pathways, which can also regulate contraction. Alterations to the signaling pathways that initiate and sustain contraction and relaxation occur as a consequence of exercise and pathophysiological conditions.