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.     

Caffeine-induced calcium release from isolated sarcoplasmic reticulum of rabbit skeletal muscle

The essential conditions for the Ca²⁺ releasing action of caffeine from isolated sarcoplasmic reticulum (SR) of rabbits were evaluated by an investigation into the effects of Ca²⁺, Mg²⁺, MgATP^2–, and ATP concentration, ionic strength, and degree of loading. The heavy fraction (4,500×g) of the reticulum was used. Except for the study on degree of loading, 0.2 mg protein·ml^–1 SR was loaded actively with 0.02 mM⁴⁵CaCl₂, resulting in >90 nmol·mg protein^–1 at steady state, and then the effects of various parameters with or without (control) caffeine were tested.It was found that (1) caffeine induces a transient, dosedependent release of Ca²⁺, (2) the absolute amount of Ca²⁺ released by caffeine increases with the Ca²⁺ load of the SR, (3) increasing the ionic strength () from 0.09 to 0.3 lowers the threshold concentration of caffeine, (4) the SR is refractory to a repeated challenge by a caffeine concentration causing maximal effect, (5) caffeine-induced Ca²⁺ release increases with increasing (a) external Ca²⁺ concentrations up to 5 M total Ca²⁺ (or 3 M free Ca²⁺) and (b) free ATP concentrations up to 0.45 mM, and (6) caffeine-induced Ca²⁺ release is not affected by changes of either the Mg²⁺ or the MgATP^2– concentration.     

Agonist concentration influences the pattern and time course of intracellular Ca2+ oscillations in human arterial smooth muscle cells

Oscillations in intracellular Ca²⁺ were recorded in cultured human uterine artery vascular smooth muscle cells. In the absence of external Ca²⁺, prolonged application of 3 M histamine activated a large transient increase in Ca²⁺ followed by a burst of Ca²⁺ spikes. The time course and frequency of the spikes were approximately constant until the last two to three spikes, when the inter-spike interval progressively increased. At 30 M histamine the response was different; the amplitude of the spikes decreased rapidly to zero, the rate of rise of successive transients fell and the time between spikes increased. The cessation of oscillatory activity was not associated with the depletion of intracellular Ca²⁺ stores, since increased doses of agonist or the sulphydryl reagent thimerosal could reactivate Ca²⁺ release. The changes in the pattern of intracellular Ca²⁺ spikes seen with increasing agonist concentration may reflect the involvement of different inactivation mechanisms in the termination of Ca²⁺ transients. In the presence of external Ca²⁺, histamine (3–30 M) activated regular Ca²⁺ oscillations. The frequency, but not the amplitude, of the oscillations was dependent on agonist concentration, the highest frequency of spiking was observed at 30 M histamine. In cells depolarised with 30 mM K⁺, histamine was still able to activate Ca²⁺ oscillations, but the dependence of spike frequency upon agonist concentration was abolished. Ca²⁺ oscillations could be activated in the presence of verapamil and nifedipine (10 M). These data suggest that in human uterine artery vascular smooth muscle cells histamine-induced Ca²⁺ oscillations are generated largely by a cytosolic oscillator and are modified by the influx of Ca²⁺ across the surface membrane.     

ATP suppresses activity of Ca2+ -activated K+ channels by Ca2+ chelation

Ca²⁺-activated maxi K⁺ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pflügers Arch 417:517, 1990), channel activity (NP _o) was stimulated by 3 M [Ca²⁺]_c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na₂ATP. The following results suggest that suppression of NP _o by Na₂ATP is due to Ca²⁺ chelation and hence reduction of [Ca²⁺]_c and reduced Ca²⁺ activation of the channel. The effect was absent when Mg ATP was used instead of Na₂ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca²⁺]_c was buffered with 10 mM HDTA (apparent pK _Ca 5.58) instead of EGTA (pK _Ca 6.8). A Ca²⁺-sensitive electrode system indicated that 1 mM Na₂ATP reduced [Ca²⁺]_c in 1 mM Ca-EGTA from 3 M to 1.4 M. Na₂ATP, Na₂GTP, Li₄AMP-PNP and NaADP reduced measured [Ca²⁺]_c in parallel with their suppression of NP _o. After the Na₂ATP-induced reduction of [Ca²⁺]_c was re-adjusted by adding either CaCl₂ or MgCl₂, the effect of Na₂ATP on NP _o disappeared. In vivo, intracellular [Mg²⁺] exceeds free [ATP^4–], hence ATP modulation of maxi K⁺ channels due to Ca²⁺ chelation is without biological relevance.     

Effect of chloride on Ca2+ release from the sarcoplasmic reticulum of mechanically skinned skeletal muscle fibres

 The effect of intracellular Cl^– on Ca²⁺ release in mechanically skinned fibres of rat extensor digitorum longus (EDL) and toad iliofibularis muscles was examined under physiological conditions of myoplasmic [Mg²⁺] and [ATP] and sarcoplasmic reticulum (SR) Ca²⁺ loading. Both in rat and toad fibres, the presence of 20 mM Cl^–in the myoplasm increased Ca²⁺ leakage from the SR at pCa (i.e. –log₁₀ [Ca²⁺]) 6.7, but not at pCa 8. Ca²⁺ uptake was not significantly affected by the presence of Cl^–. This Ca²⁺-dependent effect of Cl^– on Ca²⁺ leakage was most likely due to a direct action on the ryanodine receptor/Ca²⁺ release channel, and could influence channel sensitivity and the resting [Ca²⁺] in muscle fibres in vivo. In contrast to this effect, acute addition of 20 mM Cl^– to the myoplasm caused a 40–50% reduction in Ca²⁺ release in response to a low caffeine concentration both in toad and rat fibres. One possible explanation for this latter effect is that the addition of Cl^– induces a potential across the SR (lumen negative) which might reduce Ca²⁺ release via several different mechanisms. Received: 20 October 1997 / Received after revision: 1 December 1997 / Accepted: 2 December 1997     

Ruthenium red affects the contractile apparatus but not sarcoplasmic reticulum Ca2+ release of skinned papillary muscle

Ruthenium red has been shown to have a positive inotropic effect on isolated perfused hearts. The cellular mechanism of this action is not clear. Ruthenium red is able to block the Ca²⁺ release channel in isolated sarcoplasmic reticulum (SR) vesicle and reconstituted channel preparations. However, the effect of ruthenium red on SR Ca²⁺ release has not been studied in skinned cardiac muscle preparations. In the present study we investigated the actions of ruthenium red on both the characteristics of force generation by the contractile apparatus and Ca²⁺ release from the SR in chemically skinned rat papillary muscle. Ruthenium red (2 and 10 M) significantly increased the Ca²⁺ sensitivity of the contractile apparatus (decreasing Ca²⁺ required for the half-maximal response from 1.56±0.04 M to 1.46±0.05 M) but had no effect on the maximal Ca²⁺-activated force in triton X-100 treated fibers. This result may suggest one explanation for the positive inotropic effect of ruthenium red on the heart. On the other hand, ruthenium red had no significant effect on either caffeine-induced Ca²⁺ release or Ca²⁺-induced Ca²⁺ release from the SR in saponin-skinned muscle fibers. Lack of a blocking effect on SR Ca²⁺ release by ruthenium red in skinned fibers suggests that the SR Ca²⁺ channels in intact preparations have characteristics that are different from those of either vesicular or reconstituted channel preparations.     

Prolonged exercise reduces Ca2+ release in rat skeletal muscle sarcoplasmic reticulum

Prolonged exercise decreased the rate of Ca⁺ release in sarcoplasmic reticulum (SR) vesicles isolated from rat muscle by 20–30% when release was initiated by 5, 10, and 20 M AgNO₃. [³H]Ryanodine binding was also depressed by 20% in SR vesicles isolated from the exercised animals. In contrast, the maximum amount of Ca²⁺ released by Ag⁺ remained unaffected by exercise. The passive permeability of SR vesicles and the rate of Ca²⁺ release in the presence of ruthenium red, a known inhibitor of the Ca²⁺ release mechanism, was not affected by prolonged exercise. These results suggest that exercise depressed Ca²⁺ release from SR by directly modifying the Ca²⁺ release channel. Current address: Department of Physics, Portland State University, Portland, OR 97207, USA     

Properties of two calcium transport systems of isolated rat ileal epithelial cells: effects of Ca2+ channel modulators and membrane potential examined with fluorescent dye,fura-2

Calcium transport systems of isolated ileal epithelial cells were investigated. The concentration of cytosolic free calcium ions, [Ca²⁺]_i, was monitored with a fluorescent Ca²⁺ dye, fura-2. The fluorescence intensity ratio (I ₃₄₀/I ₃₈₀) was used as an index of [Ca²⁺]_i. [Ca²⁺]_i of the cells suspended in the nominally Ca²⁺-free solution was estimated at 52±3 nM. Ca²⁺ uptake was followed for as long as 5 min in the presence of 100–1000 M added CaCl₂. Most of the experiments were performed at 200 M CaCl₂. The Ca²⁺ uptake was abolished by 0.8 mM Ni²⁺ and 50 M Mn²⁺ and partitally antagonized by 50 M verapamil and 50 M diltiazem but not affected by 20 M nifedipine. The Ca²⁺ entry was reduced by increasing concentrations of extracellular K⁺ in the presence of valinomycin, suggesting a voltage-dependent nature of the uptake. On the other hand, the Ca²⁺ transport doubled in the presence of Bay K8644 (8 M), a Ca²⁺ channel agonist. The Bay-K-8644-induced uptake was inhibited by either 10 M nifedipine, 10 M verapamil or 10 M diltiazem and was relatively independent of extracellular K⁺ concentration. These results suggest that there are at least two distinct Ca²⁺ transport systems in the rat ileal epithelial cells, one resistant to organic Ca²⁺ channel blockers but relatively sensitive to membrane potential (basal uptake) and another inducible by Bay K 8644 and sensitive to the channel blockers but relatively independent of membrane potential.     

Mechanism of the ATP-induced rise in cytosolic Ca2+ in freshly isolated smooth muscle cells from human saphenous vein

Effects of exogenous adenosine 5-triphosphate (ATP) were studied by measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and membrane currents in myocytes freshly isolated from the human saphenous vein. At a holding potential of –60 mV, ATP (10 M) elicited a transient inward current and increased [Ca²⁺]_i. These effects of ATP were inhibited by ,-methylene adenosine 5-triphosphate (AMPCPP, 10 M). The ATP-gated current corresponded to a non-selective cation conductance allowing Ca²⁺ entry. The ATP-induced [Ca²⁺]_i rise was abolished in Ca²⁺-free solution and was reduced to 30.1±5.5% (n=14) of the control response when ATP was applied immediately after caffeine, and to 23.7±3.8% (n=11) in the presence of thapsigargin. The Ca²⁺-induced Ca²⁺ release blocker tetracaine inhibited the rise in [Ca²⁺]_i induced by both caffeine and ATP, with apparent inhibitory constants of 70 M and 100 M, respectively. Of the ATP-induced increase in [Ca²⁺]_i 29.3±3.9% (n=8) was tetracaine resistant. It is concluded that the effects of ATP in human saphenous vein myocytes are only mediated by activation of P_2x receptor channels. The ATP-induced [Ca²⁺]_i rise is due to both Ca²⁺ entry and Ca²⁺ release activated by Ca²⁺ ions that enter the cell through P_2x receptor channels.     

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.     

Direct measurements of Ca2+-activated K+ currents in inner hair cells of the guinea-pig cochlea using photolabile Ca2+ chelators

Intracellular photorelease of Ca²⁺ from caged Ca²⁺ (DM-nitrophen or nitr5) and the patch-clamp technique in the whole-cell configuration were used to investigate Ca²⁺-activated currents in inner hair cells (IHCs) of the mammalian cochlea. Photoliberation of intracellular Ca²⁺ activated outward currents with a mean amplitude of 260±110 pA when IHCs were voltage-clamped, near the resting membrane potential, at –50 mV. The photoactivated currents were reversibly blocked by extracellular application of tetraethylammonium (TEA, 10 mM), neomycin (1 mM) and charybdotoxin (1 M), but not by apamin. The voltage dependence of membrane currents activated by photolysis of DM-nitrophen demonstrated a reversal potential near the K⁺ equilibrium potential (E _k) and saturation near 0 mV. The presence of Ca²⁺-activated currents was further confirmed by the effects of extracellular adenosine 5-triphosphate (ATP, 10 M) and the Ca²⁺ ionophore ionomycin (10 M). Both agents raised intracellular Ca²⁺ and simultaneously activated outward currents when IHCs were voltage-clamped near the resting membrane potential. In experiments where currents were activated by depolarizing voltage steps, nifedipine (50 M) and Cd²⁺ (1 mM) reduced significantly (20–50%) the whole-cell outward currents, suggesting the presence of L-type Ca²⁺ currents activating K⁺ currents. These results are the first direct evidence for Ca²⁺-activated K⁺ currents in mammalian IHCs, these currents being potentially important for cell repolarization during sound-induced depolarization and synaptic transmission.     

Mechanisms of antagonistic action of internal Ca2+ on serotonin-induced potentiation of Ca2+ currents in Helix neurones

The influence of internal Ca²⁺ ions has been investigated during intracellular perfusion of isolated neurones from pedal ganglia of Helix pomatia in which serotonin (5-HT) induces a cyclic-adenosine-monophosphate-(cAMP)-dependent enhancement of high-threshold Ca²⁺ current (I _Ca). Internal free Ca²⁺ ([Ca²⁺]_i) was varied between 0.01 and 10 M by addition of Ca²⁺-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer. Elevation of [Ca²⁺]_i depressed the 5-HT effect. The dose/ effect curve for the Ca²⁺ blockade had a biphasic character and could be described by the sum of two Langmuir's isotherms for tetramolecular binding with dissociation constants K _d1=0.063 M and K _d2=1 M. Addition of calmodulin (CM) antagonists (50 M trifluoperazine or 50 M chlorpromazine), phosphodiesterase (PDE) antagonists [100 M isobutylmethylxanthine (IBMX) or 5 mM theophylline] and protein phosphatase antagonists [2 M okadaic acid (OA)] in the perfusion solution caused anticalcium action and modified the Ca²⁺ binding isotherm. Using the effect of OA and IBMX, two components of the total Ca²⁺ inhibition were separated and evaluated. In the presence of one of these blockers tetramolecular curves with K _d1=0.04 M and K _d2=0.69 M were obtained describing the activation of the retained unblocked enzyme — PDE or calcineurin (CN) correspondingly. The sum of these isotherms gave a biphasic curve similar to that in control. Leupeptin (100 M), a blocker of Ca²⁺-dependent proteases did not influence the amplitude of 5-HT effect, indicating that channel proteolysis is not involved in the depression. Our findings show that the molecular mechanism of Ca²⁺-induced suppression of the cAMP-dependent upregulation of Ca²⁺ channels is due to involvement of two Ca²⁺-CM-dependent enzymes: PDE reducing the cAMP level, and CN causing channel dephosphorylation. No other processes are involved in the investigated phenomenon at a Ca²⁺ concentration of less than or equal to 10 M.     

Ca2+ channel activities in the limb bud of early embryonic chick

Ca²⁺ channel activities were recorded in the limb bud of embryonic day 4 chick with Ca²⁺ sensitive fluorescence (Fura-2) measurements and patch clamp techniques. Rises in intracellular Ca²⁺ concentrations were evoked by depolarization with the application of 100 mM K⁺ and this Ca²⁺ response was abolished by removing extracellular Ca²⁺. The Ca²⁺ response was blocked by 10 M nifedipine and enhanced by 5 M Bay K 8644. Long-lasting inward currents were revealed by whole-cell patch clamp recordings from dissociated cells of the limb bud. The inward current was also blocked by 10 M nifedipine. Our study suggested the presence of L-type Ca²⁺ channels in the limb bud cells.     

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.     

Effects of adenosine on Ca2+ transients and tension in aequorin-injected ferret papillary muscles

The effects of adenosine on Ca²⁺ transients and tension in ferret papillary muscles were investigated using the aequorin method. Adenosine (0.01–1 mM) reduced the peak of Ca²⁺ transients and caused a slight concentration-dependent decrease in tension. Adenosine prolonged the decay time of Ca²⁺ transients but did not alter the time course of tension. In isoproterenol (0.1 M)-treated preparations, adenosine decreased the peak of Ca²⁺ transients but did not alter the peak of tension. Adenosine prolonged the isoproterenol-shortened decay time of Ca²⁺ transients. The effects of adenosine on Ca²⁺ transients were antagonized by the selective A₁ receptor antagonist 8-cyclopentyl-1,3,-dipropylxanthine. In the presence of isoproterenol, adenosine (0.1 mM) shifted the intracellular [Ca²⁺]/tension relation to the left. These results can be explained by the hypothesis that adenosine inhibits the activity of adenylate cyclase via stimulation of the A₁ receptor, other mechanisms however cannot be overlooked. The prolongation of the decay time of Ca²⁺ transients and the increase in the Ca²⁺ sensitivity of the contractile elements are the underlying mechanisms of adenosine which maintain developed tension in twitch response, although adenosine decreases the peak of Ca²⁺ transients.     

Activity of cardiac L-type Ca2+ channels is sensitive to cytoplasmic calcium

Ca²⁺ -induced inactivation of L-type Ca²⁺ channels is proposed as an important negative feedback mechanism regulating Ca²⁺ entry. Here, for the first time, evidence for modification of heart L-type Ca²⁺ channel activity by cytoplasmic calcium is provided from excised insideout membrane patches. Ba²⁺ currents through cardiac L-type Ca²⁺ channels exhibited only modest inactivation in the absence of cytoplasmic Ca²⁺. Elevation of cytoplasmic Ca²⁺ to micromolar concentrations strikingly affected L-type Ca²⁺ channel activity as evaluated from ensemble average Ba²⁺ currents. Inactivation was markedly increased concomitant with a reduction of peak inward current, which was almost completely eliminated at about 15 M cytoplasmic Ca²⁺ concentration. Half maximal suppression of Ba²⁺ currents was observed at 2.3 M Ca²⁺. The observed modifications of L-type Ca²⁺ channel activity show that cytoplasmic Ca²⁺ induces channel closure. Below 4 M Ca²⁺, channels can be reversibly reactivated during repetitive depolarizations, while at high Ca²⁺ concentrations (15 M) most Ca²⁺ channels reside in a closed state. This may allow for a delicate regulation of Ca²⁺ entry, and consequently of heart contraction.     

The effect of L-type Ca2+ channel blockers on anoxia-induced increases in intracellular Ca2+ concentration in rabbit proximal tubule cells in primary culture

Ca²⁺ channel blockers (CCB) have been shown to be protective against ischaemic damage of the kidney, suggesting an important role for intracellular Ca²⁺ ([Ca²⁺]_i) in generating cell damage. To delineate the mechanism behind this protective effect, we studied [Ca²⁺]_i in cultured proximal tubule (PT) cells during anoxia in the absence of glycolysis and the effect of methoxyverapamil (D600) and felodipine on [Ca²⁺]_i during anoxia. A method was developed whereby [Ca²⁺]_i in cultured PT cells could be measured continuously with a fura-2 imaging technique during anoxic periods up to 60 min. Complete absence of O₂ was realised by inclusion of a mixture of oxygenases in an anoxic chamber. [Ca²⁺]_i in PT cells started to rise after 10 min of anoxia and reached maximal levels at 30 min, which remained stable up to 60 min. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for normoxic and anoxic [Ca²⁺]_i were 118±2 (n=98) and 662±22 (n=160) nM, respectively. D600 (1 M), but not felodipine (10 M), significantly reduced basal [Ca²⁺]_i in normoxic incubations. During anoxia 1 M and 100 M D 600 significantly decreased anoxic [Ca²⁺]_i levels by 22 and 63% respectively. Felodipine at 10 M was as effective as 1 M D600. Removal of extracellular Ca²⁺ and addition of 0.1 mM La³⁺ completely abolished anoxia-induced increases in [Ca²⁺]_i. We conclude that anoxia induces increases in [Ca²⁺]_i in rabbit PT cells in primary culture, which results from Ca²⁺ influx. Since this Ca²⁺ influx is partially inhibited by low doses of CCBs, Ltype Ca²⁺ channels may be involved.     

Differential,direct effects of H+ on Ca2+-activated force of Skinned fibers from the soleus,cardiac and adductor magnus muscles of rabbits

The effect of acidosis on Ca²⁺-activated force generation was studied in rabbit soleus, left ventricular, and adductor magnus muscles. Fibers were skinned (sarcolemma peeled off or mechanico-chemically disrupted) to facilitate direct manipulation and standardization of their intracellular ionic milieus according to bathing solution composition. Skinned single skeletal and small bundles of cardiac fibers were mounted in a photodiode force transducer and activated by immersion in buffered-Ca²⁺ bathing solutions. The magnitude of steady state isometric force at each [Ca²⁺] was determined at pH 7.0 and 6.5 (paired data) at both 1 mM and 10 mM Mg²⁺ in order to detect artifacts of errors in calculated [Ca²⁺]. All bathing solutions contained: 7 mM total EGTA [ethyleneglycol-bis-(-amino-ethylether)-N,N tetra-acetic acid], 70 mM (Na⁺+K⁺), 2 mM MgATP^2– (Mg adenosine triphosphate), 15 mM CP^2– (creatine phosphate), 15 units/ml CPK (creatine phosphokinase), imidazole (adjusted ionic strength to 0.15 M), and propionate anion at 23±1° C. Maximum tensions were similar at both [Mg²⁺]s but less at pH 6.5 than at pH 7.0, with the following order of mean magnitude of acidotic depression adductor>cardiac>soleus. The proportionately greater acidotic depression of submaximum (relative to maximum) forces that occurred only at 1 mM Mg²⁺ (cardiac>adductor>soleus) implicates acidotic depression of Ca²⁺-activated force as a major cause of decreased cardiac contractility.Supported by National Institute of Health grants HL 17373 and RR00374. Preliminary report: Biophysical J. (abs)17, 201a (1977)Dr. Hermansen was a visiting scientist supported by Fogarty International Fellowship Grant TWO2230 from the National Institutes of Health and by the Perkins Fund of the American Physiological Society. Present Address: Institute of Work Physiology, Oslo, Norway     

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.     

A rapidly inactivating Ca2+-dependent K+ current in pheochromocytoma cells (PC12) of the rat

The membrane electrical properties of undifferentiated pheochromocytoma cells of the rat (PC12) were studied using both current-and voltage-clamp techniques with the use of low-resistance blunt-tipped micropipettes (patch electrodes). In the presence of tetrodotoxin (TTX, 2–3 M), a spike-like wave form with a prominent after-hyperpolarization (AHP) was recorded following brief (< 10 ms) depolarizing current pulses. The inorganic divalent cations, Cd²⁺ (0.5 mM), Mn²⁺ (4mM), and 0 mM Ca²⁺/4 mM Mg²⁺ solution prolonged the duration, attenuated the AHP, slowed the rate of repolarization, and slightly enhanced the amplitude of this wave form. A rapidly inactivating outward current was recorded in over 70% of the cells under voltage-clamp conditions. This transient current was elicited at about ±30 mV, and was blocked by tetraethylammonium (5 mM), inorganic divalent cations (Cd²⁺, 0.5 mM; Mn²⁺, 4 mM; Ba²⁺, 3 mM), and removal of Ca²⁺ (0 mM Ca²⁺/4 mM Mg²⁺) from the local perfusion medium. In addition, 4-aminopyridine (5 mM), which blocks the transient outward K⁺ current I_A in a variety of excitable cells, did not have any appreciable effect on this rapidly inactivating current. Moreover, it was possible to elicit the current at a holding potential of ±40 mV. The reversal potential of this current was ±90 mV, and shifted positively when extracellular K⁺ concentrations were elevated. It is concluded that PC12 cells have a rapidly inactivating Ca²⁺ -dependent K⁺ current. A possible explanation for the transient nature of this current may be the presence of an effective intracellular Ca²⁺ buffering (uptake or extrusion) system.