Stimulation of muscarinic receptor in hippocampal neuron induces characteristic increase in cytosolic free Ca2+ concentration

Changes in cytosolic free Ca2+ concentration ([Ca2+]i) in response to acetylcholine (ACh) were examined by fura-2 fluorometry in cultured rat hippocampal neurons. ACh (greater than or equal to 10(-5) M) induced an increase in [Ca2+]i composed of fast transient and slow long-lasting phases. Atropine (10(-8) M) abolished the fast component and greatly reduced the slow component. The slow component was selectively blocked by pirenzepine (10(-6) M). The effect of ACh remained partially in a Ca2+-deficient medium where effects of L-glutamate and KCl (50 mM) were abolished. Present results suggest that ACh elevates [Ca2+]i by activation of muscarinic receptor subtypes, one of which is coupled with ion channels and the other of which transduces the ACh binding to mobilization of intracellularly stored Ca2+.     

Increased contractile responses to endothelin-1 and U46619 via a protein kinase C-mediated nifedipine-sensitive pathway in diabetic rat aorta

To determine how diabetes alters vasocontractile responses to endothelin-1 (ET-1) and the thromboxane A2-mimetic U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxyprostaglandin F2alpha) and to explore the possible mechanisms of the altered responses, contractions produced by these agonists were examined in aortic rings from rats with 8- to 12-weeks streptozotocin-induced diabetes in comparison with those from age-matched control rats. ET-1 (> or = 1 nM) and U46619 (> or = 100 nM) induced significantly greater contractions in diabetic aorta. The enhanced contractile responses of diabetic aorta to these agonists were abolished in the presence of 1 microM nifedipine, resulting in no significant difference in the maximum responses between control and diabetic aortas. Pretreatment with 1 microM calphostin C or 20 nM staurosporine caused marked reductions in contractions induced by ET-1 and U46619 in both control and diabetic aortas, and the difference in the maximum contractile responses to these agonists between control and diabetic aortas were eliminated by their treatment. These results suggest that chronic diabetes enhances aortic contractions induced by ET-1 and U46619 and the enhanced contractions are possibly due to an increased Ca2+ influx through transmembrane Ca2+ channels resulting from increased protein kinase C-activated process.     

Modulation of vascular dynamics by spontaneous contraction of smooth muscle in the isolated carotid artery of the rat

Possible roles of smooth muscle contractions in the vascular dynamics were studied in the isolated rat large artery which has previously been shown to generate spontaneous rhythmic contractions depending upon the extracellular Ca2+. A cylindrical segment of the common carotid artery was superfused externally and perfused intraluminally by Tyrode solution, and the steady state intraluminal pressure (P)-volume (V) relationship of the vessel was obtained. The pressure buffering characteristic (Windkessel effect) of the vascular wall was evaluated by measuring the intraluminal pressure swing during application of external alternating pressure (semi-triangular pressure pulse, 0-120 mmHg, 300 cycle/min, "dynamic pressure load") to the intraluminal space. Effects on these vascular dynamics of inhibition of spontaneous smooth muscle contraction during superfusion of the preparation with nominally Ca2+-free Tyrode solution were investigated. Spontaneous rhythmic fluctuations of the intraluminal pressure (0.2-1.0 mmHg, 2-25 cycle/min), presumably due to contractions of vascular smooth muscles, were detected. The Ca2+-free solution abolished these spontaneous changes in the intraluminal pressure and reduced the baseline intraluminal pressure. The volume distensibility (delta V/(V X delta P] of the preparation at higher intraluminal fluid volumes (or pressures, higher wall stretch) was increased by Ca2+-free solution. The magnitude of intraluminal pressure swing and the maximum rate of changes in the pressure during application of "dynamic pressure load" were decreased in Ca2+-free solution. The results suggest that the inhibition of spontaneous contractions of smooth muscle by Ca2+-free solution may increase the volume distensibility and pressure-buffering function of the large elastic artery.     

Characterization of two different calcium entry pathways in small mesenteric arteries from rat

The effects of Ca2+ removal, nifedipine, and La3+ on contractions induced by 124 mM K+ and 10 microM noradrenaline (NA) were investigated in small mesenteric arteries from rat. Ring segments of the arteries were suspended between two steel wires in a 2.5 ml muscle bath, and the mechanical activity recorded "isometrically". The tonic components of the contractile responses to both K+ and NA were critically dependent on the presence of Ca2+ in the bath solution. Nifedipine effectively relaxed K+-contracted arteries, whereas those activated by NA were considerably less affected by the drug. Application of NA to arteries depolarized by K+ in the presence of nifedipine induced a sustained tonic contraction, which was only approximately 20% smaller than that elicited by NA in "standard" Krebs solution, implicating pharmacomechanical coupling. Unlike nifedipine, La3+ inhibited K+- and NA-induced contractions to approximately the same extent. Re-application of Ca2+ to "Ca2+-depleted" preparations exposed to K+ and/or NA induced concentration-dependent contractions. The experimental results suggested that the effects of K+ and NA on the membrane permeability to Ca2+ were additive. The Ca2+-induced contractions were more inhibited by nifedipine in K+-depolarized than in NA-exposed arteries. It is concluded that K+ and NA utilize partly different Ca2+ entry pathways to increase the myoplasmic Ca2+ concentration in rat mesenteric arteries. Whereas K+ seems to promote the influx Ca2+ by activation of CA2+ channels sensitive to the membrane potential, the nature of the receptor-operated Ca2+ entry pathway remains to be established.     

Intracellular calcium mobilization by muscarinic receptors is regulated by micromolar concentrations of external Ca2+

Carbachol-induced contractions of rat stomach fundus strips, obtained in a nutrient solution containing 1.8 mM Ca2+, were resistant to Ca2+ withdrawal, even after 1 h of bathing the tissues in a nominal 0 Ca2+ solution. This was not observed when K+ was used to evoke contractions, which were rapidly inhibited after Ca2+ removal (t1/2=2 min). The effect of carbachol in 0 Ca2+ solution was reduced by using drugs that reduce intracellular pools of Ca2+, such as caffeine (1-3 mM), ryanodine (30 microM) or thapsigargin (1 microM), corroborating the involvement of intracellular Ca2+ stores. On the other hand, when the 0 Ca2+ solution contained EGTA, a complete decline of carbachol effects was observed within about 8 min, indicating the involvement of extracellular Ca2+. Atomic absorption spectrometry showed that our 0 Ca2+ solution still contained 45 microM Ca2+, which was drastically reduced to 5.9 nM in the presence of EGTA. Taken together, our results indicate that the effects of carbachol are due to the mobilization of caffeine-, ryanodine- and thapsigargin-sensitive intracellular Ca2+ stores, and that these stores are not inactivated or depleted if micromolar concentrations (45 microM), but not nanomolar concentrations (5.9 nM) of Ca2+ are maintained in the extracellular milieu.     

Mechanism of acidic pH-induced contraction in spontaneously hypertensive rat aorta: role of Ca2+ release from the sarcoplasmic reticulum

AIM: This study was conducted to investigate the mechanism of acidic pH-induced contraction (APIC) with regard to Ca2+ handling using isometric tension recording experiments. RESULTS: Decreasing extracellular pH from 7.4 to 6.5 produced a marked and sustained contraction of spontaneously hypertensive rat (SHR) aorta, that was 128.7 +/- 2.0% of the 64.8 mm KCl-induced contraction. Verapamil, an inhibitor of voltage-dependent Ca2+ channels (VDCC) significantly inhibited the APIC. In Ca2+-deficient solution, sustained contraction induced by acidic pH was abolished completely, while a transient contraction was still observed suggesting the release of Ca2+ from intracellular site. Ryanodine (1 microm), a ryanodine receptor blocker, and 10 microm cyclopiazonic acid (CPA; a sarco/endoplasmic reticulum Ca2+ ATPase inhibitor) abolished the transient contraction induced by acidosis. In normal Ca2+-containing solution, ryanodine significantly decreased the rate of rise as well as maximum level of APIC. Interestingly, ryanodine and CPA showed an additive inhibitory effect with verapamil and the combined treatment of ryanodine or CPA with verapamil nearly abolished the APIC. CONCLUSIONS: It is concluded that acidic pH induces Ca2+ release from ryanodine/CPA-sensitive store of sarcoplasmic reticulum in SHR aorta. This Ca2+ plays an important role in the facilitation of the rate of rise of APIC, as well as contributing to the sustained contraction via a mechanism which is independent of Ca2+ influx through VDCC.     

Cyclic AMP-mediated inhibition of noradrenaline-induced contraction and Ca2+ influx in guinea-pig vas deferens

The relaxation effects of forskolin and methylxanthines on noradrenaline (NA)-induced contractions were investigated by measuring isotonic contraction and intracellular calcium concentration ([Ca2+]i) in the epididymal side of guinea-pig vas deferens. NA (100 microM) and high K+ (55 mM) induced a biphasic contraction; fast, transient (phasic) and slow, sustained (tonic) phases. Both phases in either NA or high K+ stimulation were abolished in Ca2+-free solution. Pretreatment with 10 microM nifedipine, an L-type Ca2+ channel blocker, reduced both phasic and tonic contractions induced by high K+. In the case of NA-induced contraction, however, nifedipine reduced the phasic contraction but not the tonic contraction. The nifedipine-insensitive tonic contraction was relaxed by the application of polyvalent cations (Mn2+, Co2+, Cd2+ and La3+). These findings indicate that NA-induced biphasic contraction is mainly due to nifedipine-insensitive Ca2+ influx, especially in the tonic phase. Cyclic AMP-increasing agents such as forskolin (0.5-10 microM), IBMX (5-500 microM) and caffeine (1-20 mM) relaxed the NA-induced contraction extensively in a concentration-dependent manner. However, these agents only partially relaxed the high K+-induced contraction. Forskolin (10 microM) and IBMX (100 microM) reduced the [Ca2+]i response to NA, but had no effect on the [Ca2+]i response to high K+. These results suggest that an increase in intracellular cAMP may relax the NA-induced contraction by attenuating a nifedipine-insensitive Ca2+ influx and by a mechanism independent of a reduction in [Ca2+]i.     

Ca2+ removal effects on bullfrog gastric mucosa in the presence of drugs

Bullfrog gastric mucosa can secrete acid in vitro for more than 24 hr. In Ca2+-deficient media, the acid secretion measured by pH stat method gradually decreased to cease about 200 min after the start of immersion in the media. When A23187 or quinidine, which are known to mobilize Ca2+ from the intracellular pools, was present on the submucosal side, a prolongation of the duration of acid secretion in the absence of Ca2+ was found. Dibutyl c-AMP also induced a similar prolongation. Histamine or theophylline not only prolonged the duration of acid secretion but also transiently increased the acid secretory rate over the control level. Based upon the known fact that there are different intracellular Ca2+ compartments, i.e, one related to the process of acid secretion and the other to the maintenance of junctional complexes, the present results of drug effects were discussed especially in regard to their Ca2+ mobilizing effects. Cessation of acid secretion in a K+- and Ca2+-deficient solution occurred much more quickly than that in a Ca2+-deficient or a K+-deficient medium.     

Interactions between magnesium and calcium in beta-cell-rich pancreatic islets

Calcium-magnesium interactions, total amounts of intracellular magnesium, and insulin release were studied in beta-cell-rich pancreatic islets from ob/ob mice. Mg2+ inhibited the uptake of intracellular 45Ca and insulin release induced by glucose or high concentrations of potassium. Omission of Mg2+ from a Ca2+-deficient medium resulted in an increased efflux of 45Ca, whereas the characteristic glucose inhibition of the efflux was diminished. After addition of Mg2+ to a Mg2+-depleted medium, the glucose-stimulated 45Ca efflux was markedly reduced. Mg2+ inhibited the basal efflux of 45Ca, and this effect was preceded by a transient stimulation. Ca2+ but not Mg2+ stimulated 45Ca efflux in a medium depleted of Ca2+, Mg2+, and Na+. The data indicate that Mg2+ interferes with Ca2+ entry through voltage-dependent Ca2+ channels. Mg2+ may also inhibit the outward transport of Ca2+ from the cells at a site different from the Na+-Ca2+ countertransport mechanism. The total amount of intracellular magnesium remained unaffected by glucose and was not changed unless the ionic composition of the mediums were changed grossly. Under physiological conditions it is therefore unlikely that fluctuations in the intracellular Mg2+ concentration are part of the mechanism by which the functionally important Ca2+ is regulated.     

Ca2+ influx, but not Ca2+ release from internal stores, is required for the PACAP-induced increase in excitability in guinea pig intracardiac neurons

Mechanisms modulating the pituitary adenylate cyclase activating polypeptide (PACAP)-induced increase in excitability have been studied using dissociated guinea pig intrinsic cardiac neurons and intact ganglion preparations. Measurements of intracellular calcium (Ca2+) with the fluorescent Ca2+ indicator dye fluo-3 indicated that neither PACAP nor vasoactive intestinal polypeptide (VIP) at either 100 nM or 1 microM produced a discernible elevation of intracellular Ca2+ in dissociated intracardiac neurons. For neurons in ganglion whole mount preparations kept in control bath solution, local application of PACAP significantly increased excitability, as indicated by the number of action potentials generated by long depolarizing current pulses. However, in a Ca2+ -deficient solution in which external Ca2+ was replaced by Mg2+ or when cells were bathed in control solution containing 200 microM Cd2+, PACAP did not enhance action potential firing. In contrast, in a Ca2+ -deficient solution with Ca2+ replaced by strontium (Sr2+), PACAP increased excitability. PACAP increased excitability in cells treated with a combination of 20 microM ryanodine and 10 mM caffeine to interrupt release of Ca2+ from internal stores. Experiments using fluo-3 showed that ryanodine/caffeine pretreatment eliminated subsequent caffeine-induced Ca2+ release from intracellular stores, whereas exposure to the Ca2+ -deficient solution did not. In dissociated intracardiac neurons voltage clamped with the perforated patch recording technique, 100 nM PACAP decreased the voltage-dependent barium current (IBa). These results show that, in the guinea pig intracardiac neurons, the PACAP-induced increase in excitability apparently requires Ca2+ influx through Cd2+ -sensitive calcium permeable channels other than voltage-dependent Ca2+ channels, but not Ca2+ release from internal stores.     

Fulminant type 1 diabetes mellitus observed in insulin receptor substrate 2 deficient mice

The objective of this study was to characterise the fulminant type 1 diabetes mellitus (DM) accompanying abrupt hyperglycaemia and ketonuria observed in insulin receptor substrate 2 (IRS2)-deficient mice. IRS2-deficient mice backcrossed onto the original C57BL/6J:Jc1 background (B6J-IRS2(-/-) mice) for more than 10 generations were used. Eight male IRS2-deficient mice with ketonuria and abrupt increase in plasma glucose concentrations over 25 mmol/l were used as the fulminant type 1 diabetic mice (diabetic mice) and 8 male IRS2-deficient mice (8 weeks old) without glycosuria were used as the control mice. Plasma metabolite, immunoreactive insulin (IRI) and C-peptide concentrations, hepatic energy metabolism related enzyme activities and histopathological change in pancreatic islets were investigated. The diabetic mice showed significantly higher plasma glucose and cholesterol concentrations and lower plasma IRI and C-peptide concentrations than the control mice. In livers of the diabetic mice, glycolytic and malate-aspartate shuttle enzyme activities decreased significantly and gluconeogenic, lipogenic and ketone body synthesis enzyme activities increased significantly compared to those in the control mice. The pancreatic islets of the diabetic mice decreased significantly in size and number of beta cells. The diabetic IRS2-deficient mice did not show the islet-related antibodies observed in the diabetic NOD mice in their sera. The characteristics of the diabetic IRS2-deficient mice resembled those of the human nonautoimmune fulminant type 1 DM. IRS2-deficient mice may be a useful animal model for studying the degradation mechanism of pancreatic beta cells in the process of development of fulminant type 1 DM.     

Role of Ca2+ in genesis of lower esophageal sphincter tone and other active contractions.

The effects of absent or low Ca2+ (0.5 mM), verapamil, nifedipine, Na nitroprusside, theophylline, La2+, and ethanol on basal active tension (tone), "off" contractions, and carbachol contractions were studied in opossum lower esophageal sphincter strips. Incubation in Ca2+-free Ringer (0.1 mM EGTA) abolished tone and contractions. Low Ca2+, verapamil, nifedipine, and theophylline depressed tone more rapidly than "off" contractions. Only verapamil and nifedipine depressed carbachol contractions. Na nitroprusside rapidly depressed tone but left contractions unchanged. La3+ at 1 X 10(-3) M behaved like Ca2+-free incubation but produced sustained contractions with muscle stimulation. Ethanol depressed "off" contractions more than tone and did not affect carbachol-induced contractions. These results suggest that tone probably results from inward leak of Ca2+, whereas "off" contractions depend on release of Ca2+ sequestered in the cell by a mechanism not immediately dependent on increased Ca2+ influx. Carbachol may increase Ca2+ influx as well as utilize sequestered Ca2+. Nifedipine and verapamil may act to block both resting and stimulated Ca2+ influx. Na nitroprusside may act by increasing Ca2+ efflux. Ethanol may act by decreasing the availability of sequestered Ca2+ or by inhibiting the function of a mediator responsible for "off" contractions.     

Effects of removal of Na(+) and Cl(-) on spontaneous electrical activity, slow wave, in the circular muscle of the guinea-pig gastric antrum

In the circular muscle of the guinea-pig gastric antrum, a decrease in the external Na(+) to less than 20 mM produced depolarization of the membrane with transient prolongation of the slow wave. This was followed by a high rhythmic activity. The activity was inhibited by reapplication of Na(+) before recovery. The depolarization in Na(+)-deficient solution was prevented and rhythmic activity continued at about 5/min for at least 6 min by simultaneous removal of K(+), Ca(2+), or Cl(-). After exposure to a Na(+)- and Cl(-)-deficient solution for a few minutes, reapplication of the Na(+) in Cl(-)-deficient solution inhibited generation of the slow wave until Cl(-) reapplication. Similar results were obtained when Na(+) and Cl(-) were reapplied in the absence of K(+) after exposure to a Na(+)-, K(+)-free, and Cl(-)-deficient solution, although the inhibition was weaker than Na(+) reapplication in a Cl(-)-deficient solution. In the presence of furosemide or bumetanide, a strong inhibition of activity was produced by the reapplication of Na(+) and Cl(-) after exposure to an Na(+)- and Cl(-)-deficient solution. A hypothesis is presented that intracellular Ca(2+) concentration ([Ca(2+)](i)) is the most important factor determining the generation and frequency of the slow wave and that [Ca(2+)](i) is regulated by the Na(+) concentration gradient across the plasma membrane. The recovery of the Na(+) concentration gradient by Na(+) reapplication after removal of Na(+) and Cl(-) is mainly controlled by a Na(+)-K(+)-Cl(-) co-transport.     

MaxiK channel-triggered negative feedback system is preserved in the urinary bladder smooth muscle from streptozotocin-induced diabetic rats.

MaxiK channel, the large-conductance Ca2+-sensitive K+ channel, facilitates a negative feedback mechanism to oppose excitation and contraction in various types of smooth muscles including urinary bladder smooth muscle (UBSM). In this study, we investigated how the contribution of MaxiK channel to the regulation of basal UBSM mechanical activity is altered in streptozotocin-induced diabetic rats. Although the urinary bladder preparations from both control and diabetic rats were almost quiescent in their basal mechanical activities, they generated spontaneous rhythmic contractions in response to a MaxiK channel blocker, iberiotoxin (IbTx). The effect of IbTx on the mechanical activity was significantly greater in diabetic rat than in control animal. Similarly, the basal mechanical activity was increased with apamin, an inhibitor for some types of small conductance Ca2+-sensitive K+ channels, and this effect was more pronounced for diabetic rat. However, in both control and diabetic animals, IbTx action was stronger than that of apamin. Diabetes also enhanced the responses to BayK 8644, an L-type Ca2+ channel agonist. The extent of this enhancement in diabetic bladder vs. control was, however, almost the same as that attained with IbTx. Expression levels for MaxiK channel as well as apamin-sensitive K+ channels and L-type Ca2+ channel were not altered by diabetes, when determined as their corresponding mRNA levels. These results indicate that diabetes can potentially increase the basal UBSM mechanical activity. However, in diabetic UBSM, the main negative-feedback system triggered by MaxiK channel is still preserved enough to counteract the possible enhancement of this smooth muscle mechanical activity.     

A novel pathway for Ca2+ signalling in neutrophils by immune complexes.

The data presented here demonstrates that immune complexes use novel routes for stimulating a two-phase rise in cytosolic-free Ca2+ concentration. The initial transient Ca2+ rise resulted from release of Ca2+ from intracellular stores, by a route which, unlike f-met-leu-phe, was inhibited by bromophenacyl bromide. The second phase resulted from transmembrane influx and occurred in the absence of store release and by Ca2+ channels that were inhibited by Ni2+ but not SKF 96365 or econazole. Stimulation by immune complexes therefore involves novel routes for both the release of stored Ca2+ and the opening of Ca2+ channels in the plasma membrane.     

Aconitine-induced delayed afterdepolarization in frog atrium and guinea pig papillary muscles in the presence of low concentrations of Ca2+

Aconitine will induce arrhythmias after the fiber has been completely repolarized. This arrhythmia is generally facilitated in the presence of high Ca2+ solution, yet the aconitine-induced arrhythmia occurs even in the presence of low Ca2+ solutions. We studied aconitine-induced arrhythmia (particularly the amplitude of delayed afterdepolarization) in the frog atrium or guinea-pig papillary muscles in Ca2+-free solution, in the presence or absence of Ca2+ channel blocking agents. In Ca2+-free solution, aconitine (10(-5) g/ml) decreased the resting potential, overshoot, Vmax, and shortened the duration of the 90% action potential, before the onset of delayed afterdepolarization in frog atrial preparations. Tetrodotoxin (TTX) (2 X 10(-7) g/ml) blocked these aconitine-induced electrical changes. Verapamil (10(-6) g/ml) in nominally Ca2+-free solution blocked neither the generation of delayed afterdepolarization nor the triggered activity, while LaCl3 (0.5 mM) or TTX halted it. Delayed afterdepolarization appeared following the aconitine-induced transient increase in twitch tension. This transient increase in twitch tension was blocked by LaCl3 and TTX but not by verapamil. Delayed afterdepolarization in Ca2+-free solution demonstrated the voltage dependence of a U shape between -40 and -80 mV and was inhibited by low Na+ and high K+. Under the influence of aconitine in the guinea pig papillary muscle exposed to the Ca2+-free solution, depolarizing clamp pulses produced a transient inward current, and here the sigmoid time- and voltage-dependent characteristics were similar to those seen in the case of digitalis intoxication. These results suggest that intracellular Na+ loading plays an important role in the aconitine-induced delayed afterdepolarization and transient inward currents in low Ca2+ solution.     

Extracellular magnesium deficiency induces contraction of arterial muscle: role of PI3-kinases and MAPK signaling pathways

The present study investigated the actions of extracellular Mg2+ ([Mg2+]o) deficiency on isolated rat aortae and rat aortic smooth muscle cells (RASMC). Exposure of isolated, intact rat aortic rings to Mg(2+)-free or Mg(2+)-deficient medium (0.15-0.6 mM) produced endothelium-independent, concentration-dependent contractions: the lower the [Mg2+]o, the stronger the contraction. Pre- or post-incubation of the vessels with low concentrations of U0126, SB-203580, PD-98059, wortmannin, LY-294002, or a SH2 domain inhibitor peptide suppressed [Mg2+]o deficiency-induced contractions significantly. The concentrations of these antagonists required for half-maximal inhibition (IC50) were not very different from the inhibitory constants (Ki) for these drugs. A variety of specific pharmacological antagonists of several known endogenously-formed vasoconstrictors did not inhibit or attenuate the contractions induced by low [Mg2+]o. Mg(2+)-free medium induced a 6- to 7-fold increase in intracellular Ca2+ ([Ca2+]i) in RASMC. Pre- or post-treatment of the cells with U0126, SB-203580, PD-98059, wortmannin, LY-294002, or a SH2 domain inhibitor peptide markedly inhibited the increments in ([Ca2+]i) in RASMC induced by exposure to Mg(2+)-free medium. The present findings suggest that Mg(2+)-deficiency-induced contractions of rat aortae are associated with activation of several cellular signal pathways, such as mitogen-activated protein kinase, phosphatidylinositol-3 (PI3) kinases, and SH2 domain-containing proteins.     

Functional properties of human embryonic stem cell-derived cardiomyocytes: intracellular Ca2+ handling and the role of sarcoplasmic reticulum in the contraction

Since cardiac transplantation is limited by the small availability of donor organs, regeneration of the diseased myocardium by cell transplantation is an attractive therapeutic modality. To determine the compatibility of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) (7 to 55 days old) with the myocardium, we investigated their functional properties regarding intracellular Ca2+ handling and the role of the sarcoplasmic reticulum in the contraction. The functional properties of hESC-CMs were investigated by recording simultaneously [Ca2+]i transients and contractions. Additionally, we performed Western blot analysis of the Ca2+-handling proteins SERCA2, calsequestrin, phospholamban, and Na+/Ca2+ exchanger (NCX). Our major findings are, first, that hESC-CMs displayed temporally related [Ca2+]i transients and contractions, negative force-frequency relations, and lack of post-rest potentiation. Second, ryanodine, thapsigargin, and caffeine did not affect the [Ca2+]i transient and contraction, indicating that at this developmental stage, contraction depends on transsarcolemmal Ca2+ influx rather than on sarcoplasmic reticulum Ca2+ release. Third, in agreement with the notion that a voltage-dependent Ca2+ current is present in hESC-CMs and contributes to the mechanical function, verapamil completely blocked contraction. Fourth, whereas hESC-CMs expressed SERCA2 and NCX at levels comparable to those of the adult porcine myocardium, calsequestrin and phospholamban were not expressed. Our study shows for the first time that functional properties related to intracellular Ca2+ handling of hESC-CMs differ markedly from the adult myocardium, probably due to immature sarcoplasmic reticulum capacity.     

Tension response and45Ca release in vascular smooth muscle incubated in Ca-free solution

The contractile response of arterial smooth muscles induced by agonists as noradrenaline or histamine in Ca-free solution consists of two phases: an initial phasic component, which is transient and accompanied by a release of cellular Ca, and a small tonic component, which persists as long as the agonist is present. A second admission of the agonist without reexposure to Ca elicits only the tonic component. This tonic contraction differs in several respects from the phasic response obtained in Ca-free solution: it is independent of the duration of exposure to the Ca-free solution, it can be elicited many times without reexposure to Ca, and it is not accompanied by a measurable release of Ca from the cells.During superfusion with Ca-free solution, a tonic contraction is also induced by fluoride ions at concentrations exceeding 4 mM. The amplitude of this contraction is maximal at about 12 mM. Increasing the fluoride concentration shortens the delay between the addition of the F^– and the onset of the contraction. As is the case for the tonic noradrenaline-response, the F^–-induced contractions can be clicited many times without reexposure to Ca. The tonic contractions evoked by noradrenaline or histamine and by fluoride ions are additive. Both contractions are reversibly inhibited by caffeine, theophylline, Na-nitroprusside, papaverine and by nitroglycerine.The possiblity that these tonic contractions are not accompanied by an increase of the cytoplasmic Ca²⁺ concentration is discussed.This work was supported by the grant of the Fonds voor Wetenschappelijk Geneeskundig Onderzoek, Belgium, nr. 3.0087.74     

2,3-Butanedione monoxime increases speed of relaxation in single muscle fibres of frog.

The effects of 2,3-butanedione monoxime (BDM) on intracellular Ca2+ transient and cross-bridge function were studied in frog single fibres from the anterior tibialis muscle of Rana temporaria (sarcomere length, 2.2 microm; temperature, 2-4 degrees C). The fluorescent dye fluo-3 was used to monitor the intracellular free calcium concentration ([Ca2+]i) during isometric contractions. BDM (1-5 mM) reduced the amplitude of the Ca2+ transient during twitches, but this effect was too small to explain the marked inhibition of BDM on twitch force. [Ca2+]i reached at the end of 1-s tetanic stimulation was not significantly affected by BDM (1.0 and 1.8 mM) while the maximum tetanic tension was substantially reduced. The rate of relaxation during isometric tetanus was increased by BDM whereas the rate of decay of the Ca2+ transient was reduced in the presence of BDM. The results strongly suggest that BDM, under the experimental conditions used, mainly affects the contractile machinery resulting in altered performance of the cross-bridges. These effects of BDM were evaluated in terms of the cross-bridge model of Huxley (1957) which was fitted to the experimental force-velocity data in the presence and absence of BDM.