Role of calcium in stretch-induced release and mRNA synthesis of natriuretic peptides in isolated rat atrium

 To investigate the role of Ca²⁺ in stretch-induced synthesis and release of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) isolated superfused rat atria were stretched by raising intra-atrial pressure. The immunoreactive (ir-) ANP and BNP concentrations were analysed by radioimmunoassay and the corresponding mRNA levels were quantified by Northern blot and dot blot analyses. Stretch-induced ir-ANP release and a rise in BNP mRNA levels increased at high (3.0 mM) compared to low (0.5 mM) extracellular Ca²⁺ concentration ([Ca²⁺]_o). Moreover, the adaptation of stretch-induced ir-ANP release was dependent on [Ca²⁺]_o. Atrial BNP mRNA levels were increased by stretch also in non-paced, electrically silent atria, where voltage-activated Ca²⁺ channels are not activated. The stretch-induced rise in BNP mRNA was blocked by gadolinium (80 μM), but not by the L-type channel blocker diltiazem (3.0 μM). This study indicates that both the stretch-secretion coupling of ir-ANP release and the pressure-stimulated synthesis of BNP mRNA are Ca²⁺-dependent processes. Gadolinium inhibits the stretch-stimulated rise in BNP mRNA levels in contracting and non-contracting atria, which is similar to its ability to block stretch-activated ir-ANP release, suggesting the involvement of Ca²⁺-permeable stretch-activated channels. Received: 29 April 1996 / Received after revision and accepted: 17 June 1996     

Glibenclamide suppresses stretch-activated ANP secretion: involvements of K+ ATP channels and L-type Ca2+ channel modulation

 The mechanism by which glibenclamide regulates mechanically activated atrial natriuretic peptide (ANP) secretion was investigated using isolated perfused rat atria. A reduction in atrial pressure from an experimentally imposed distending pressure stimulated the secretion of ANP and caused concomitant translocation of extracellular fluid (ECF) into the atrial lumen. The activation of ANP secretion and ECF translocation were closely correlated with atrial volume changes and the increase in ANP secretion was a function of the ECF translocation. Glibenclamide (1, 10, 100 μM), an ATP-sensitive K⁺ (K⁺ _ATP) channel blocker, had no effect on the basal secretion of ANP, suppressed the stimulation of stretch-activated ANP secretion in a dose-dependent manner, but not the translocation of the ECF. Glipizide (100 μM) and tolbutamide (100 μM), other K⁺ _ATP channel blockers, had similar effects to those of glibenclamide. Suppression by glibenclamide (100 μM) of the stretch-induced ANP secretion was not observed in atria that had been pretreated with pinacidil (200 μM), an ATP-sensitive K⁺ channel opener: pinacidil alone had no effect on ECF translocation and ANP secretion. Furthermore, blocking Ca²⁺ influx by using the Ca²⁺ channel blocker diltiazem (10 nM), or a Ca²⁺-depleted medium prevented the suppression of stretch-induced ANP secretion by glibenclamide. In other experiments, atrial distension produced a slight membrane depolarization of cardiomyocytes; this was accentuated in the presence of glibenclamide. Furthermore, in single cardiomyocytes, glibenclamide increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner. From these results, we suggest that glibenclamide suppresses atrial release of ANP by blocking K⁺ _ATP channels and increasing Ca²⁺ influx and that the K⁺ _ATP channels are associated with the regulation of the mechanically activated ANP secretion from the atria. Received: 13 May 1996 / Received after revision: 10 February 1997 / Accepted: 5 March 1997     

Bell-shaped activation of inositol-1,4,5-trisphosphate-induced Ca2+ release by thimerosal in permeabilized A7r5 smooth-muscle cells

There is no consensus about the different types of Ca²⁺ transport processes in the endoplasmic reticulum that are targeted by the sulphydryl reagent thimerosal. We have therefore investigated how thimerosal affects the various Ca²⁺ transport processes in permeabilized A7r5 smooth-muscle cells, using an unidirectional ⁴⁵Ca²⁺ flux technique. Thimerosal up to a concentration of 32 M did not have an effect on the passive ⁴⁵Ca²⁺ leak from the stores, while higher concentrations increased this aspecific leak. Thimerosal inhibited the endoplasmic reticulum Ca²⁺ pump with an EC⁵⁰ of 9 M. Thimerosal exerted a biphasic effect on the Ca²⁺ release induced by inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] with a stimulation of the release at thimerosal concentrations below 10 M, and an inhibitory effect at higher concentrations. Thimerosal (2.5–250 M) did not exert an effect on the specific binding of [³H]Ins(1,4,5)P ₃ to its receptor, indicating that it probably did not act at the level of the binding site. This finding contrasts with the effect of the closely related sulphydryl reagent parachloromercuriphenylsulphonate, which, at high concentrations, inhibited [³H]Ins(1,4,5)P ₃ binding. The effects of thimerosal were largely prevented by the sulphydryl reducing agent dithiothreitol (3 mM). We conclude that thimerosal concentrations ranging from 0.32 to 1 M can stimulate the Ins(1,4,5)P ₃-induced Ca²⁺ release without inhibiting the Ca²⁺ pumps or without increasing the passive Ca²⁺ permeability of the endoplasmic reticulum.     

The dual effects of ouabain on45Ca2+ transport and contractility in adult rat ventricular myocytes

We used isolated ventricular myocytes to study⁴⁵Ca²⁺ transport in the presence of three concentrations of ouabain (10 nM, 1 M, and 100 M) in Tyrode solution containing 1 mM CaCl₂. The cells were quiescent and during⁴⁵Ca²⁺ uptake and⁴⁵Ca²⁺ efflux experiments 10 nM ouabain decreased Ca²⁺ content, 1 M, didn't change it appreciably, and 100 M increased it significantly. Qualitatively, the same results were obtained at 22°C and 35°C. Ouabain did not significantly affect the electrical activity of isolated, electrically stimulated myocytes, but it increased the amplitude of shortenings of these myocytes in a dose-dependent manner. Thus, the positive inotropic effect of ouabain at therapeutic doses (10 nM) occurs in spite of decreased Ca²⁺ content, while at high toxic doses the positive inotropic effect is accompanied by an increment in Ca²⁺ content. These data support the hypothesis that the mechanisms of positive inotropy of ouabain are different at therapeutic and toxic concentrations of this drug. Finally, our study demonstrates that the effects of low doses of ouabain are independent of the release of endogenous catecholamines.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Effects of the PKA inhibitor H-89 on excitation–contraction coupling in skinned and intact skeletal muscle fibres

This study investigated the effects of the protein kinase A (PKA) inhibitor, H-89, in mechanically-skinned muscle fibres and intact muscle fibres, in order to determine whether PKA phosphorylation is essential for normal excitation–contraction (E–C) coupling. In skinned EDL fibres of the rat, force responses to depolarization (by ion substitution) were inhibited only slightly by 10M H-89, a concentration more than sufficient to fully inhibit PKA. Staurosporine (1 M), a potent non-specific kinase inhibitor, also had little if any effect on depolarization-induced responses. At 1–2 M, H-89 significantly slowed the repriming rate in rat skinned fibres, most likely due to it deleteriously affecting the T-system potential. With 100 M H-89, the force response to depolarization by ion substitution was completely abolished. This inhibitory effect was reversed by washout of H-89 and was not due to block of the Ca²⁺ release channel in the sarcoplasmic reticulum (SR). In intact single fibres of the flexor digitorum longus (FDB) muscle of the mouse, 1–3 M H-89 had no noticeable effect on action-potential-mediated Ca²⁺ transients. Higher concentrations (4–10 M) caused Ca²⁺ transient failure in fibres stimulated at 20 Hz in a manner indicative of action-potential failure. At 10–100 M, H-89 also inhibited net Ca²⁺ uptake by the SR and affected the Ca²⁺-sensitivity of the contractile apparatus in rat skinned fibres. All such effects were proportionately greater in toad muscle fibres. These results do not support the hypothesis that phosphorylation is essential for the Ca²⁺ release channel to open in response to voltage-sensor activation in skeletal muscle fibres.     

Effects of intracellular Ca2+ chelating compounds on inward currents caused by Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum (SR) of mammalian cardiac myocytes occuring either due to activation by a depolarization or the resulting transmembrane Ca²⁺ current (I _Ca), or spontaneously due to Ca²⁺ overload has been shown to cause inward current(s) at negative membrane potentials. In this study, the effects of different intracellular Ca²⁺ chelating compounds on I _Ca-evoked or spontaneous Ca²⁺-release-dependent inward currents were examined in dialysed atrial myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. As compared to dialysis with solutions containing only a low concentration of a high affinity ethylene glycol-bis(-aminoethylether) N,N,N,N-tetraacetic acid (EGTA) like chelator (50–200 M), inward membrane currents (at –50 mV) due to evoked Ca²⁺ release, spontaneous Ca²⁺ release or Ca²⁺ overload following long-lasting depolarizations to very positive membrane potentials are prolonged if the dialysing fluid contains a high concentration of a low affinity Ca²⁺ chelating compound such as citrate or free adenosine 5-triphosphate (ATP). Without such a non-saturable Ca²⁺ chelator in the dialysing fluid, Ca²⁺-release-dependent inward currents are often oscillatory and show an irregular amplitude. With a low affinity chelator in a non-saturable concentration, discrete inward currents with constant properties can be recorded. We conclude that the variability in Ca²⁺-release-dependent inward current seen in single cells arises from spatial inhomogeneities of intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to localized saturation of endogenous and exogenous high affinity Ca²⁺ buffers (e.g. [2]). This can be avoided experimentally by addition of a non-saturable buffer to the intracellular solution. This condition might be useful, if properties of Ca²⁺ release from the SR and/ or the resulting membrane current, like for example arrhythmogenic transient inward current, are to be investigated on the single cell level.     

Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum ca2+ pump blockers: thapsigargin and cyclopiazonic acid

The effect of thapsigargin (TG) and cyclopiazonic acid (CPA) on the mechanical activity of the rat pulmonary artery were investigated. In chemically (-escin)-skinned arterial strips, application of TG (0.1–1 M) or CPA (0.5–10 M) prior and throughout the loading procedure of the internal Ca²⁺ stores (0.3 M free Ca²⁺ ions for 8–10 min) concentration dependently inhibited the subsequent contractile response induced by noradrenaline (NA, 10 M) or caffeine (25 mM). In intact strips repeatedly incubated in a Ca²⁺-containing solution (2.5 mM for 10 min), followed by incubation in a Ca²⁺-free solution 12 min before NA-stimulation, TG and CPA not only inhibited the NA-induced contraction but also increased the tension which appeared during the exposure time to Ca²⁺. The two phenomena developed with similar time courses. The increase in tension during the readmission of Ca²⁺ ions was not antagonized by verapamil (10 M) or nifedipine (1 M) but was blocked by La³⁺ (50 M) and Co²⁺ (1 mM) ions. The amplitude of the verapamil-insensitive TG (or CPA)-induced contraction was dependent on the external [Ca²⁺] [0.1–10 mM, concentration for half maximal effect (EC₅₀) =0.85 mM], not modified by the reduction of the external [Na⁺] (from 130 to 10 mM) and decreased by depolarization of the strip using K⁺-rich (30–120 mM) solutions. Under the latter condition, 38±9 and 83±4% reduction (n=5) was observed in the presence of 60 and 120 mM K⁺ respectively. This contraction was also concentration dependently inhibited by the tyrosine kinase inhibitors genistein (0.5–50 M) and tyrphostin (2–50 M). Sr²⁺ ions, which contracted both depolarized intact and skinned strips, failed to replace Ca²⁺ ions in the verapamil-insensitive contraction induced by TG or CPA (n=4). Finally, TG (1 M) and CPA (10 M) did not modify the pCa tension relationship in skinned strips (n=5). These results show that the main action of TG and CPA in rat pulmonary artery is to prevent the refilling of the internal Ca²⁺ store. TG and CPA also seem to facilitate a Ca²⁺ influx through a specific verapamil-insensitive pathway. The biophysical and molecular characteristics of this pathway remain to be elucitated, although it appears to involve a tyrosine kinase activity.     

Effects of tyramine and calcium on the kinetics of secretion in intact and electroporated chromaffin cells superfused at high speed

Fast superfusion of electroporated bovine adrenal chromaffin cells with a K⁺ glutamate-based solution containing 50 nM free Ca²⁺ and 2 mM adenosine 5-triphosphate, dipotassium salt (K₂ATP), produced a steady-state low catecholamine secretion, measured on-line with an electrochemical detector (about 20 nA). Rapid switching to electroporation solutions containing increasing Ca²⁺ concentrations ([Ca²⁺]) produced a rapid increase in the rate and peak secretion, followed by a decline. At intermediate [Ca²⁺] (3–100 M), a fast peak and a slow secretory plateau were distinguished. The fast secretory peak identifies a readily releasable catecholamine pool consisting of about 200–400 vesicles per cell. Pretreatment of cells with tyramine (10 M for 4 min before electroporation) supressed the initial fast secretory peak, leaving intact the slower phase of secretion. With [Ca²⁺] in the range of 0.1–3 M, the activation rate of secretion increased from 2.3 to 35.3 nA · s^–1, reached a plateau between 3–30 M and rose again from 100 to 1000 M [Ca²⁺] to a maximum of 91.9 nA · s^–1. In contrast, total secretion first increased (0.1–1 M Ca²⁺), then plateaud (1–100 M Ca²⁺) and subsequently decreased (100–1000 M Ca²⁺). At 30 and 1000 M extracellular [Ca²⁺] or [Ca²⁺]_o, the activation rates of secretion from intact cells depolarised with 70 mM K⁺were close to those obtained in electroporated cells. However, secretion peaks were much lower in intact (93 nA at 30 M Ca²⁺) than in electroporated cells (385 nA). On the other hand, inactivation of secretion was much faster in intact than in electroporated cells; as a consequence, total secretion in a 5-min period was considerably smaller in intact (10.6 A · s at 1000 M Ca²⁺) than in electroporated cells (42.4 A,s at 1 M Ca²⁺). Separation of the time-courses of changes in intracellular [Ca²⁺] or [Ca²⁺]_i and secretion in intact chromaffin cells depolarised with 70 mM K⁺was demonstrated at different [Ca²⁺]_o. The increase in the rate of catecholamine release was substantially higher than the increase of the average [Ca²⁺]_i. In contrast, the decline of secretion was faster than the decline of the peak [Ca²⁺]_i. The results are compatible with the idea that the peak and the amount of catecholamine released from depolarised intact cells is determined essentially by plasmalemmal factors, rather than by vesicle supply from reserve pools. These plasmalemmal factors limit the supply of Ca²⁺ by the rates of opening and closing of voltage-dependent Ca²⁺ channels of the L-and Q-subtypes, which control the local [Ca²⁺]_i near to exocytotic sites.     

The hypocalciuric effect of thiazides: subcellular localization of the action

The acute administration of thiazides results in a decrease in the urinary Ca²⁺/Na⁺ ratio, whereas chronic administration of these diuretics decreases calciuria. In both situations, Ca²⁺ transport is enhanced in the early part of the distal tubule. The purpose of our study was to determine whether the hypocalciuric action of thiazides was due to a change in the active transport of Ca²⁺ through the basolateral membrane of the nephron or to an effect (direct or indirect) on the permeability of the distal tubule luminal membrane to calcium. In order to detect intrinsic differences between membranes of the proximal and distal tubules, the effect of the diuretic was examined in proximal and distal tubule preparations, and in basolateral and luminal membranes from the two segments separately.Preincubation of microdissected distal tubules in hypotonic solution containing 500 M hydrochlorothiazide (HCTZ) did not influence the Ca²⁺-dependent ATP hydrolysis (Ca²⁺=1 M) nor the Mg²⁺-dependent ATP hydrolysis (Mg²⁺=100 M). Similarly 100 M HCTZ did not change the Ca²⁺ ATPase activity in intact proximal and distal tubule suspensions, at Ca²⁺ concentrations ranging from 0.05 M to 1 M.ATP-dependent Ca²⁺ transport was present in basolateral membrane vesicles from proximal and distal tubule suspensions. Preincubation of the membranes with 100 M HCTZ did not influence this transport. A Na⁺/ Ca²⁺ exchanger, present in the basolateral membranes from the distal tubule, was also insensitive to HCTZ. In contrast, preincubation of luminal membranes from the distal tubules (but not proximal tubules) with 500 M HCTZ significantly increased the Ca²⁺ uptake by these membranes. This increase in Ca²⁺ uptake, in the presence of Na⁺, was dose-dependent; the minimal and the maximal effects of the diuretic were observed at concentrations of 25 M and 100 M respectively. HCTZ increased the V _maxCa²⁺ from2.5±0.3 pmol g^–1 (10 s^–1) to 3.7±0.6 pmol g^–1 (10 s^–1) (P<0.01), but did not influence the K _m (1.43±0.25 mM and 1.37±0.1 mM Ca²⁺ in experimental and control membranes, respectively). Na⁺ was necessary for this effect. Na⁺ per se decreased Ca²⁺ uptake in a concentration-dependent manner and HCTZ partially reestablished Ca²⁺ uptake to the levels observed in a Na⁺-free medium. The anion of the Na⁺ salt also modulated the effect of HCTZ on Ca²⁺ transport. While Cl^– and SCN^– permitted HCTZ to enhance Ca²⁺ uptake, the SO ₄ ^2– anion did not. It is therefore concluded that (a) the hypocalciuric effect of thiazides is primarily due to an increase in the Ca²⁺ uptake of the luminal membrane from the distal tubule, (b) Na⁺ and Ca²⁺ transports are tightly related in the distal luminal membrane, (c) HCTZ modulates this interrelationship by decreasing the inhibitory effect of Na⁺ on Ca²⁺ uptake. Whether the Ca²⁺ and Na⁺ carriers are the same molecule or different entities needs further investigation.     

Permeation by zinc of bovine chromaffin cell calcium channels: relevance to secretion

Zn²⁺ increased the rate of spontaneous release of catecholamines from bovine adrenal glands. This effect was Ca²⁺ independent; in fact, in the absence of extracellular Ca²⁺, the secretory effects of Zn²⁺ were enhanced. At low concentrations (3–10 M), Zn²⁺ enhanced the secretory responses to 10-s pulses of 100 M 1,1-dimethyl-4-phenylpiperazinium (DMPP, a nicotinic receptor agonist) or 100 mM K⁺. In the presence of DMPP, secretion was increased 47% above controls and in high-K⁺ solutions, secretion increased 54% above control. These low concentrations of Zn²⁺ did not facilitate the whole-cell Ca²⁺ (I _Ca) or Ba²⁺ (I _Ba) currents in patch-clamped chromaffin cells. Higher Zn²⁺ concentrations inhibited the currents (IC₅₀ values, 346 M for I _Ca and 91 M for I _Ba) and blocked DMPP- and K⁺-evoked secretion (IC₅₀ values, 141 and 250 M, respectively). Zn²⁺ permeated the Ca²⁺ channels of bovine chromaffin cells, although at a much slower rate than other divalent cations. Peak currents at 10 mM Ba²⁺, Ca²⁺, Sr²⁺ and Zn²⁺ were 991, 734, 330 and 7.4 pA, respectively. Zn²⁺ entry was also evidenced using the fluorescent Ca²⁺ probe fura-2. This was possible because Zn²⁺ causes an increase in fura-2 fluorescence at the isosbestic wavelength for Ca²⁺, i.e. 360 nm. There was a slow resting entry of Zn²⁺ which was accelerated by stimulation with DMPP or high-K⁺ solution. The entry of Zn²⁺ was concentration dependent, slightly antagonized by 1 mM Ca²⁺ and completely blocked by 5 mM Ni²⁺. The entry of Ca²⁺ evoked by depolarization with high-K⁺ solution was antagonized by Zn²⁺. We conclude that inhibition by Zn²⁺ of evoked catecholamine secretion is associated with blockade of Ca²⁺ entry through Ca²⁺ channels recruited by DMPP or K⁺. However, the facilitation of secretion observed at low Zn²⁺ concentrations, or in the absence of Ca²⁺, may be exerted at an intracellular site on the secretory machinery. This is plausible because Zn²⁺ permeates the bovine chromaffin cell Ca²⁺ channels and in this way gains access to the cytosol. In addition, we have established conditions for measuring Zn²⁺ transients in fura-2-loaded cells with a very high sensitivity, taking advantage of the high-affinity binding of Zn²⁺ to fura-2 and the modification of its fluorescence spectrum.     

A long lasting Ca2+ -activated outward current in guinea-pig atrial myocytes

Among other characteristics, the steady-state current-voltage relationship of patch-clamped single atrial myocytes from guinea-pig hearts is defined by an outward current hump in the potential region –15 to +40mV. This hump was reversibly suppressed by Co²⁺ (3 mM) or nitrendipine (5 M) and enhanced by Bay K 8644 (5 M). The maintained outward current component suppressed by Co²⁺ extended between –15.2±1.9 mV and +39.5 ±1.7 mV (mean±SEM of 14 cells) and has an amplitude of 95.7±9.4 pA at +10 mV. In isochronal I-V curves, the hump was already visible at 400 ms with essentially the same amplitude as at 1500 ms. The Co²⁺ -sensitive outward current underlying the hump was poorly time-dependent during 1.5 s voltage pulses but slowly relaxed upon repolarization. Tail currents reversed near the K⁺ equilibrium potential under our experimental conditions. The current hump of the steady-state I-V curve was also abolished by caffeine (10 mM) or ryanodine (3 M), both drugs that interfere with sarcoplasmic reticulum function. Apamin (1 M) or quinine (100 M) but not TEA (5–50 mM) markedly reduced its amplitude. However, at similar concentrations as required to inhibit the hump, both apamin and quinine appeared to be poorly specific for Ca²⁺ -activated K⁺ currents in heart cells since they also inhibited the L-Type Ca²⁺ current. It is concluded that a long lasting Ca²⁺ -activated outward current, probably mainly carried by K⁺ ions but not sensitive to TEA, exists in atrial myocytes which is responsible for the current hump of the background I-V curve.     

Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle

Thapsigargin has been reported to inhibit ATP-dependent Ca²⁺ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca²⁺ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca²⁺ content of the SR. The SR was first depleted of Ca²⁺ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 M was required to inhibit Ca²⁺ loading by 50%. In contrast, another SR Ca²⁺ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 M and total inhibition at 50 M. When cyclopiazonic acid (100 M) was applied after, rather than during, Ca²⁺ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 M), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca²⁺ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.     

Effects of ryanodine on skinned skeletal muscle fibers of the rabbit

The mechanism(s) of ryanodine-induced contracture of skeletal muscle were studied in skinned fibers from soleus (SL) and adductor magnus (AM) (slow- and fast-twitch skeletal muscles) of rabbits. Pieces of SL or AM were homogenized (sarcolemma disrupted). Single fibers were dissected from the homogenate and mounted on photodiode force transducers. At concentrations 1–50 M, ryanodine slightly but significantly increased the submaximal Ca²⁺-activated tension development of the contractile proteins in skinned fibers of AM but not of SL. Ryanodine in uptake phase or release phase increased caffeine-induced tension transients in the SR of both muscle types; however, no dose-response relation was found. Ryanodine 1 M decreased, however, the second control tension transients in a dose-dependent manner. The depression was nearly irreversible and activity-dependent. The concentrations of ryanodine that inhibited the second control tension transients by 50% were 10 M and 5 M for SL and AM, respectively, following ryanodine administration in the release phase, and 100 M and 30 M, respectively, for these preparations after the drug was present in the uptake phase. The quantity of calcium released from the SR by Triton X-100 and caffeine in the second control tension transient was unchanged by ryanodine at all concentrations tested when compared with that of the absence of ryanodine. The present findings suggest that the ability of ryanodine to increase immediate calcium release from the SR, and in AM but not SL, to increase the sensitivity of the contractile proteins to Ca²⁺ underlies the contracture caused by this agent in intact skeletal muscles. The delayed decreased Ca²⁺ efflux by caffeine, as evidenced by depression of tension transient with no change in the calcium content may be responsible for the decreased twitch tension caused by this agent.     

Caffeine and histamine-induced oscillations of K(Ca) current in single smooth muscle cells of rabbit cerebral artery

In the present experiment, we characterized the intracellular Ca²⁺ oscillations induced by caffeine (1 mM) or histamine (1–3 M) in voltage-clamped single smooth muscle cells of rabbit cerebral (basilar) artery. Superfusion of caffeine or histamine induced periodic oscillations of large whole-cell K⁺ current with fairly uniform amplitudes and intervals. The oscillatory K⁺ current was abolished by inclusion of ethylenebis(oxonitrilo)tetraacetate (EGTA, 5 mM) in the pipette solution. Caffeine- and histamine-induced periodic activation of the large-conductance Ca²⁺-activated K⁺ [K_(Ca)] channel was recorded in the cell-attached patch mode. These results suggest that the oscillations of K⁺ current are carried by the K_(Ca) channel and reflect the oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Ryanodine (1–10 M) abolished both caffeine- and histamine-induced oscillations. Caffeine- induced oscillations were abolished by the sarcoplasmic reticulum Ca²⁺-adenosine 5-triphosphatase (Ca²⁺-ATPase) inhibitor, cyclopiazonic acid (10 M), and a high concentration of caffeine (10 mM). Inclusion of heparin (3 mg/ml) in the pipette solution blocked histamine-induced oscillations, but did not block caffeine-induced oscillations. By the removal of extracellular Ca²⁺, but not by the addition of verapamil and Cd²⁺, the caffeine-induced oscillations were abolished. Increasing Ca²⁺ influx rate increased the frequencies of caffeine-induced oscillations. Spontaneous oscillations were also observed in cells that were not superfused with agonists, and had similar characteristics to the caffeine-induced oscillations. From the above results, it is concluded, that in smooth muscle cells of the rabbit cerebral (basilar) artery, ryanodine-sensitive Ca²⁺-induced Ca²⁺ release pools play key roles in the generation of caffeine- and histamine-induced intracellular Ca²⁺ oscillations.     

Extracellular heparin inhibits Ca2+ transients and contraction in mammalian cardiac myocytes

The effect of heparin on Ca²⁺ transients and cell shortening was studied in isolated cardiac myocytes from rat and guinea-pig ventricles. Ca²⁺ signals were measured with the fluorescent indicator fura-2. Heparin reversibly decreased Ca²⁺ transients and cell shortening in a dose-dependent manner. Half and complete blockade were obtained with 50 g/ml and 200 g/ml heparin, respectively. The dihydropyridine agonist BAY K 8644 (50 nM) antagonized the effects of heparin. However, Ca²⁺ release elicited by caffeine (10 mM) was not affected by heparin. The actions of heparin were also studied in multicellular preparations. In papillary muscle, heparin (5 mg/ml) reversibly reduced the amplitude of the plateau of the action potential and the associated peak tension. BAY K 8644 (500 nM) also antagonized these effects. It is proposed that heparin interacts with dihydropyridine-sensitive Ca²⁺ channels to cause a decrease of Ca²⁺ transients and contractility in heart.     

Effects of ryanodine on acetylcholine-induced Ca2+ mobilization in single smooth muscle cells of the porcine coronary artery

To study the essential features of acetylcholine (ACh)-and caffeine-sensitive cellular Ca²⁺ storage sites in single vascular smooth muscle cells of the porcine coronary artery, the effects of ryanodine on both ACh- and caffeine-induced Ca²⁺ mobilization were investigated by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) using Fura 2 in Ca²⁺-containing or Ca²⁺-free solution. The resting [Ca²⁺]_i of the cells was 122 nM in normal physiological solution and no spontaneous activity was observed. In a solution containing 2.6 mM Ca²⁺, 10 M ACh or 128 mM K⁺ produced a phasic, followed by a tonic, increase in [Ca²⁺]_i but 20 mM caffeine produced only a phasic increase. In Ca²⁺-free solution containing 0.5 mM ethylenebis(oxonitrilo)tetraacetate (EGTA), the resting [Ca²⁺]_i rapidly decreased to 102 nM within 5 min, and 10 M ACh or 20 mM caffeine (but not 128 mM K⁺) transiently increased [Ca²⁺]_i. Ryanodine (50 M) greatly inhibited the phasic increase in [Ca²⁺]_i induced by 10 M ACh or 5 mM caffeine and increased the time to peak and to the half decay after the peak in the presence or absence of extracellular Ca²⁺. By contrast, ryanodine (50 M) enhanced the tonic increase in [Ca²⁺]_i induced by 128 mM K⁺ and also by 10 M ACh in Ca²⁺-containing solution. In Ca²⁺-free solution containing 0.5 mM EGTA, ACh (10 M) failed to increase [Ca²⁺]_i following application of 20 mM caffeine. The level of [Ca²⁺]_i induced by 20 mM caffeine was greatly reduced, but not abolished, following application of 10 M ACh in Ca²⁺-free solution. These results suggest that both ACh and caffeine release Ca²⁺ from the ryanodine-sensitive sarcoplasmic reticulum (SR) in smooth muscle cells of the porcine coronary artery. The finding that ryanodine significantly increased the resting [Ca²⁺]_i and inhibited the rate of decline of [Ca²⁺]_i following wasthout of high K⁺ or ACh in Ca²⁺-containing solution suggests that SR may negatively regulate the resting [Ca²⁺]_i in smooth muscle cells of the porcine coronary artery.     

Sodium-dependent recovery of ionised magnesium concentration following magnesium load in rat heart myocytes

Our objectives were to investigate regulation of intracellular ionised Mg²⁺ concentration ([fMg²⁺]_i) in cardiac muscle and cardiac Na⁺/Mg²⁺ antiport stoichiometry. [fMg²⁺]_i was measured at 37°C in isolated rat ventricular myocytes with mag-fura-2. Superfusion of myocytes with Na⁺ and Ca²⁺ free solutions containing 30 mM Mg²⁺ for 15 min more than doubled [fMg²⁺]_i from its basal level (0.75 mM). Re-addition of Na⁺ caused [fMg²⁺]_i to fall exponentially with time to basal level, the rate increasing linearly with [Na⁺]. Log(recovery rate) increased linearly with log([Na⁺]), the slope of 1.06 (95% confidence limits, 0.94–1.17) suggesting one Na⁺ ion is exchanged for each Mg²⁺. [fMg²⁺]_i recovery was complete even if the membrane potential was depolarised to 0 mV or if superfusate [Mg²⁺] was increased to 3 mM. Recovery was rapid in normal Tyrode (0.3 min^–1) with a Q₁₀ of 2.2. It was completely inhibited by 200 M imipramine but was unaffected by 20 M KB-R7943 or 1 M SEA0400, suggesting the Na⁺ /Ca²⁺ antiporter is not involved. Membrane depolarisation by increasing superfusate [K⁺] to 70 mM, or voltage clamp to 0 mV, increased recovery rate in Na⁺ containing solutions more than threefold. We conclude [fMg²⁺]_i recovery is by Mg²⁺ efflux on a 1 Na⁺:1 Mg²⁺ antiport.     

Suppression of Ca2+ oscillations induced by cholecystokinin (CCK) and its analog OPE in rat pancreatic acinar cells by low-level protein kinase C activation without transition of the CCK receptor from a high- to low-affinity state

Cholecystokinin (CCK) analogs, JMV-180 and OPE, release Ca²⁺ from intracellular stores and induce oscillations in the concentration of cytosolic Ca²⁺ ([Ca²⁺]_i), but do not generate a detectable rise in inositol 1,4,5-trisphosphate (InsP ₃) levels. In contrast, high concentrations of CCK elevate InsP ₃, as well [Ca²⁺]_i, to a peak which decreases to near basal levels without oscillations. The mechanisms which underlie inhibition of [Ca²⁺]_i oscillations observed with high CCK concentrations are unclear, but are believed to involve a low-affinity CCK receptor state. Alternately, CCK analogs may be weak partial agonists of the phospholipase C pathway, whereas native CCK, as a full agonist of this pathway, stimulates low levels of protein kinase C (PKC) activity. Preincubation of acini with 1 nM 12 O-tetradecanoyl-phorbol 13-acetate (TPA) for 15 min at 37°C did not affect OPE binding to acini, but abolished OPE-induced (at 1 M) [Ca²⁺]_i oscillations without affecting the initial [Ca²⁺]_i spike. These transformed OPE-induced [Ca²⁺]_i responses mimicked those induced by supramaximal CCK octapeptide (CCK-8) concentrations. Inhibition of [Ca²⁺]_i oscillations by 1 nM TPA was reversed by the PKC inhibitor staurosporine (0.2 M). After [Ca²⁺]_i oscillations were induced with OPE or low concentrations of CCK-8 (20 pM), 1 nM TPA caused a gradual slowing of oscillation frequency over 15–20 min without affecting [Ca²⁺]_i spike amplitude. In contrast, 1 M TPA inhibited OPE binding and caused a more generalized inhibition of OPE- and CCK-evoked Ca²⁺ signals. These data suggest that inhibitory effects of low-level PKC activation on agonist-evoked Ca²⁺ signalling are distinct from the effects of high-level PKC activation by 1 M TPA, and do not require the transition of the CCK receptor from a high-affinity to a low-affinity state.     

Ryanodine reduces the amount of calcium in intracellular stores of smooth-muscle cells of the rabbit ear artery

We have investigated the mechanism of action of ryanodine on intact and skinned smooth-muscle cells of the rabbit ear artery. The amplitude of the phasic response induced by low noradrenaline (NA) concentrations (<30 nM) was inhibited by 10 M ryanodine, while that elicited by high NA concentrations (>100 nM) was not affected. The phasic contractions induced by both low and high NA concentrations in Ca²⁺-free solution containing 2 mM EGTA were suppressed by 10 M ryanodine. The rate of⁴⁵Ca efflux in Krebs solution was enhanced by 10 M ryanodine, while the increased⁴⁵Ca efflux induced by 10 M NA was inhibited by ryanodine. 10 M ryanodine did not affect the contractile proteins in saponin-treated smoothmuscle cells. The intracellular Ca²⁺ stores of these skinned cells could be filled by exposing these cells to a solution containing 0.6 M Ca²⁺. After a wash in a Ca²⁺-free solution, a contraction due to a release of the accumulated Ca²⁺ could be induced by either 25 mM caffeine or 20 M inositol 1,4,5-trisphosphate (InsP₃) or 10 M A23187. These contractions did not occur if 10 M ryanodine was present during Ca²⁺ loading. The addition of ryanodine during the Ca²⁺-free wash did not affect the subsequent force development. These observations indicate that ryanodine, in the presence of Ca²⁺, depletes the intracellular Ca²⁺ stores, and that this depletion is responsible for the inhibition of the component of the NA-induced contraction which depends on the release of Ca²⁺ from intracellular stores.