Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake,spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca²⁺ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca²⁺ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca²⁺ content loaded into the SR, (2) the spontaneous Ca²⁺ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca²⁺ sensitivity in Triton X-100-treated preparations. Inhibition of Ca²⁺ loading by TG and CPA increased with time of exposure to the inhibitor over 18–24 min. TG and CPA produced half inhibition of Ca²⁺ loading at 34.9 and 35.7 μM respectively, when 18–24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 μM TG and 30 μM CPA both abolished the oscillations in this time. The myofilament Ca²⁺ sensitivity was not affected by 10 and 300 μM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca²⁺ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation. Received: 28 July 1995/Received after revision: 11 December 1995/Accepted: 18 December 1995     

Effects of cyclopiazonic acid on Ca2+ regulation by the sarcoplasmic reticulum in saponin-permeabilized skeletal muscle fibres

 The effects of the sarcoplasmic reticulum (SR) Ca²⁺ pump inhibitor cyclopiazonic acid (CPA) were studied in saponin-permeabilized frog skeletal muscle fibres. Release of Ca²⁺ from the SR was triggered by brief (2 s) applications of 40 mM caffeine at 2-min intervals. Changes in [Ca²⁺] within the fibre were monitored continuously using Fura-2 fluorescence. At a bathing [Ca²⁺] of 100 nM, introduction of 20 μM CPA induced a slow release of Ca²⁺ from the SR. The following one to two caffeine-induced Ca²⁺ transients were markedly increased in amplitude and duration. Thereafter, the caffeine-induced Ca²⁺ transients decreased progressively and were barely detectable 6–7 min after introduction of CPA. However, increasing the bathing [Ca²⁺] or increasing the Ca²⁺ loading period resulted in a partial recovery of the caffeine-induced Ca²⁺ transients, suggesting that pump inhibition is incomplete, even in the presence of 100 μM CPA. The slow Ca²⁺ efflux induced by CPA was insensitive to ryanodine, but absent following abolition of SR Ca²⁺ pump activity by ATP withdrawal. These results suggest that the caffeine-induced Ca²⁺ transient reflects a balance between efflux via the SR Ca²⁺ channel and reuptake by the Ca pump. Ca²⁺ release upon addition of CPA may result from inhibition of SR Ca²⁺ uptake, which reveals a tonic Ca²⁺ efflux that is independent of the Ca²⁺ release channels. Received: 26 November 1997 / Received after revision: 12 January 1998 / Accepted: 13 January 1998     

Ca2+ uptake by cardiac sarcoplasmic reticulum ATPase in situ strongly depends on bound creatine kinase

The role of creatine kinase (CK) bound to sarcoplasmic reticulum (SR), in the energy supply of SR ATPase in situ, was studied in saponin-permeabilised rat ventricular fibres by loading SR at pCa 6.5 for different times and under different energy supply conditions. Release of Ca²⁺ was induced by 5 mM caffeine and the peak of relative tension (T/T _max) and the area under isometric tension curves, S _T, were measured. Taking advantage of close localisation of myofibrils and SR, free [Ca²⁺] in the fibres during the release was estimated using steady state [Ca²⁺]/tension relationship. Peak [Ca²⁺] and integral of free Ca²⁺ transients (S[Ca²⁺]_f) were then calculated. At all times, loading with 0.25 mM adenosine diphosphate, Mg²⁺ salt (MgADP) and 12 mM phosphocreatine (PCr) [when adenosine triphosphate (ATP) was generated via bound CK] was as efficient as loading with both 3.16 mM MgATP and 12 mM PCr (control conditions). However, when loading was supported by MgATP alone (3.16 mM), T/T _max was only 40% and S[Ca²⁺]_f 31% of control (P < 0.001). Under these conditions, addition of a soluble ATP-regenerating system (pyruvate kinase and phosphoenolpyruvate), did not increase loading substantially. Both S _T and S[Ca²⁺]_f were more sensitive to the loading conditions than T/T _max and peak [Ca²⁺]. The data suggest that Ca²⁺ uptake by the SR in situ depends on local ATP/ADP ratio which is effectively controlled by bound CK. Received: 23 January 1996/Received after revision: 19 April 1996/Accepted: 3 May 1996     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

Evidence for the existence of inositol (1,4,5)-trisphosphate- and ryanodine-sensitive pools in bovine endothelial cells. Ca2+ releases in cells with different basal level of intracellular Ca2+

In single bovine aortic endothelial (BAE) cells pre-loaded with Fura-2, Ca²⁺ transients in a Ca²⁺-free medium have been revealed, which evidently reflects Ca²⁺ release from intracellular stores. In cells with different levels of resting basal cytoplasmic Ca²⁺ ([Ca²⁺]_i) from about 50 to 110 nM, a biphasic dependence of the Ca²⁺ transients on resting [Ca²⁺]_i was shown and spontaneous Ca²⁺ oscillations were observed. At a [Ca²⁺]_i level over 110 nM, a pronounced rise in Ca²⁺ transients occurred and only single transients were observed. Ryanodine (10 μM) produced a transient [Ca²⁺]_i elevation, suggesting the presence of ryanodine receptors in intracellular store membranes. The results imply that both inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release (IICR) and Ca²⁺-sensitive Ca²⁺ release (CICR) take place in BAE cells. Only IICR seems to be sufficient for generating baseline Ca²⁺ oscillations in BAE cells, whereas the ATP-induced (5–100 μM) Ca²⁺ response involves the CICR set in motion by an oscillatory IICR of high frequency. The completion of both the spontaneous and ATP-induced Ca²⁺ transients was associated with a [Ca²⁺]_i decrease to a level below the initial resting [Ca²⁺]_i (undershoot). Its depth biphasically depended on the resting [Ca²⁺]_i from 50 to 110 nM, suggesting that the lack of a Ca²⁺ leak from inositol 1,4,5-trisphosphate-sensitive stores is responsible for the undershoot in this range. The Ca²⁺ leak is concluded to play a key role in the initiation and termination of regenerative IICR both in spontaneous oscillations and in ATP-induced transients. Received: 13 November 1995/Received after revision and accepted 27 March 1996     

The effect of extracellular Ca2+ concentration on the negative staircase of Ca2+ transient in field-stimulated rat ventricular cells

We performed experiments using the Ca²⁺ indicator dye, fura-2 to investigate the effect of extracellular Ca²⁺ concentration ([Ca²⁺]_o) on sarcoplasmic reticulum (SR) Ca²⁺ release and loading in single rat ventricular cells. In normal Tyrode solution (1.8 mM [Ca²⁺]_o) repetitive stimulation (0.5 Hz) resulted in a gradual decrease in calcium transients (the negative staircase phenomenon) without being accompanied by a gradual decrease in diastolic intracellular Ca²⁺ concentration. The rate of the slow decline in calcium transient was faster in lower [Ca²⁺]_o. However, the peak of the first calcium transient was relatively invariant over a wide range of [Ca²⁺]_o (0.5–5 mM). The size of the calcium transient elicited by field stimulation was proportional to that induced by 10 mM caffeine, applied following the field stimulation. These results suggest that the size of calcium transients depends mainly on the Ca²⁺ content of the SR. The quiescent period favoured the replenishment of the SR and this effect was promoted further by increasing the driving force for Ca²⁺ entry across the sarcolemma during this period. We conclude that in low [Ca²⁺]_o, short stimulation interval may limit Ca²⁺ influx across the sarcolemma during the quiescent period to cause a gradual reduction in calcium content of the SR and thus the calcium transient.     

Influence of caffeine,Ca2+, and Mg2+ on ryanodine depression of the tension transient in skinned myocardial fibers of the rabbit

Ryanodine, a blocker for Ca²⁺-release channels of the sarcoplasmic reticulum (SR Ca²⁺-release channels), induces depression of myocardial contraction in isolated intact muscle, which is consistent with depression of the caffeine-induced tension transient in skinned muscle fibers. In isolated SR, ryanodine binds to a specific receptor with high affinity, and this binding is enhanced by caffeine and increasing Ca²⁺ and decreased by increasing Mg²⁺. The aim of this study was to test the hypothesis that depression of myocardial contraction is mediated by changes in ryanodine-receptor binding properties. Accordingly, factors (caffeine, Ca²⁺, and Mg²⁺) affecting ryanodine-receptor binding properties in the isolated SR membrane were studied in skinned myocardial fibers from adult rabbits. The depression of the caffeine-induced tension transient by ryanodine (ryanodine depression) influenced by these three factors was measured. In a dose-dependent manner, increasing caffeine or Ca²⁺ concentrations enhanced the ryanodine depression. The concentrations for 50% ryanodine depression (IC₅₀) approximated 7mM for caffeine, and pCa 5.25 for Ca²⁺. When 1 M ryanodine and 25 mM caffeine were combined, ryanodine depression was independent of Ca²⁺ at low Ca²⁺ concentrations (20%–30% at pCa>8 and 7.5) and was a direct function of Ca²⁺ at higher concentrations (pCa 7.5–6.0 with IC₅₀ approx. pCa 6.75). In contrast, increasing Mg²⁺ reduced the ryanodine depression with IC₅₀ approximately equal to pMg 3.3. In conclusion, the caffeineor Ca²⁺-enhanced, and Mg²⁺-reduced ryanodine depression observed in this study is consistent with known ryanodinereceptor binding properties.     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pH_i) and intracellular Ca²⁺ activity ([Ca²⁺]_i) of HT₂₉ cells was investigated microspectrofluorimetrically using pH- and Ca²⁺- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (V _m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pK _a value. Trimethylamine (20 mmol/l) increased pH_i by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pH_i and a slow and incomplete recovery: pH_i stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pH_i. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca²⁺]_i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca²⁺]_i plateau. The initial Δ[Ca²⁺]_i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca²⁺]_i peak was independent of the Ca²⁺ activity in the bath solution, but the [Ca²⁺]_i plateau was significantly lower under Ca²⁺-free conditions and could be immediately interrupted by application of CO₂ (10%; n = 6), a manoeuvre to acidify pH_i in HT₂₉ cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca²⁺ stores completely abolished this [Ca²⁺]_i transient. Tetramethylamine led to higher [Ca²⁺]_i changes than the other amines tested and only this transient could be completely blocked by atropine (10⁻⁶ mol/l). Trimethylamine (20 mmol/l) hyperpolarized V _m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pH_i changes, release Ca²⁺ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca²⁺ stores and increase Ca²⁺ influx into HT₂₉ cells. The latter may be related to both the store depletion and the hyperpolarization. Received: 11 September 1995/Received after revision and accepted: 18 December 1995     

Time course of changes in in vitro sarcoplasmic reticulum Ca2+-handling and Na+-K+-ATPase activity during repetitive contractions

Time-dependent changes in sarcoplasmic reticulum (SR) Ca²⁺-handling and Na⁺-K⁺-ATPase activity, as assessed in vitro, were investigated in the superficial (GS) and deep regions (GD) of rat gastrocnemius muscles undergoing short-term (up to 30 min) electrical stimulation. There was a rapid and progressive loss of force output during the first 5 min of stimulation. For GS, significant depressions (P < 0.05) in SR Ca²⁺-uptake rate and Ca²⁺-ATPase activity were observed during only the first 1 min. No further reductions occurred with stimulation time. SR Ca²⁺-release rate was significantly (P < 0.05) decreased at 3 min. For GD, significant reductions (P < 0.05) in Ca²⁺-uptake rate, Ca²⁺-release rate and Ca²⁺-ATPase activity were manifested after 3, 5, and 5 min, respectively. A decay in Na⁺-K⁺-ATPase activity was found only in 1-min stimulated GD and 30-min stimulated GS. After 30 min, the depressed functions reverted to resting levels in GD but not in GS. The alterations in any variables examined were not parallel with changes in force output. These results suggest that, at least under the conditions used in this study, in vivo disruptions in cation regulation mediated by vigorous contractile activity would be attributable primarily to events other than structural alterations to the respective proteins.     

Sarcoplasmic reticulum Ca2+ loading in rabbits 8 and 15 weeks after coronary artery ligation

 Calcium uptake by cardiac sarcoplasmic reticulum (SR) is reported to be reduced in heart failure in the human and in a number of animal models. However, the majority of studies have examined end-stage heart failure in the human and few animal studies have taken account of the duration and severity of left ventricular dysfunction. In this study we have compared SR Ca²⁺ loading in a haemodynamically assessed, coronary artery ligation model of heart failure at 8 and 15 weeks after ligation. Trabeculae were isolated from the right ventricle and mounted for isometric tension measurement. They were treated with saponin to permeabilize the sarcolemma but retain SR function and bathed in a mock intracellular solution including adenosine triphosphate (ATP) and buffered Ca²⁺. Caffeine was used to release Ca²⁺ from the SR. The amplitude of the caffeine-induced contracture was used as a quantitative gauge of the Ca²⁺ content of the SR. Eight weeks after ligation, trabeculae demonstrated enhanced SR Ca²⁺ uptake as manifest by larger caffeine-induced contractures (e.g. 200 nM [Ca²⁺], 120 s loading – 38.2±9.2 versus 67.3±10.1% of maximum Ca²⁺-activated force, F _{Ca, max}, P=0.03). At 15 weeks, trabeculae from ligated hearts were not significantly different from controls with SR Ca²⁺ loading returning to control levels (e.g. 200 nM [Ca²⁺], 120 s loading – 47.3±9.6 versus 30.2±12.8% F _{Ca, max}, P=0.12). These data suggest that SR Ca²⁺ loading may increase in the early stages of heart failure and fall back to normal with an increasing duration of left ventricular dysfunction. Increased incidence of spontaneous Ca²⁺ release observed from the SR at 8 weeks and not at 15 weeks may represent an arrhythmogenic mechanism specific to the early phase of heart failure. Received: 21 January 1998 / Received after revision and accepted: 3 April 1998     

The role of sarcoplasmic reticulum and sarcoplasmic reticulum Ca2+-ATPase in the smooth muscle tone of the cat gastric fundus

Circular smooth muscle strips isolated from cat gastric fundus were studied in order to understand whether the sarcoplasmic reticulum (SR) and SR Ca²⁺-ATPase could play a role in the regulation of the muscle tone. Cyclopiazonic acid (CPA), a specific inhibitor of SR Ca²⁺-ATPase, caused a significant and sustained increase in muscle tone, depending on the presence of extracellular Ca²⁺. Nifedipine and cinnarizin only partially suppressed the CPA-induced tonic contraction. Bay K 8644 antagonized the relaxant effect of nifedipine in CPA-contracted fundus. Nitric-oxide-releasing agents sodium nitroprusside and 3-morpholino-sydnonimine completely suppressed the CPA-induced tonic contraction. The blockers of Ca²⁺-activated K⁺ channels, tetraethylammonium, charybdotoxin and/or apamin, decreased the contractile effect of CPA. Vanadate increased the tone but did not change significantly the effect of CPA. CPA exerted its contractile effect even when Ca²⁺ influx was triggered through the Na⁺/Ca²⁺ exchanger and the other Ca²⁺ entry pathways were blocked. Thapsigargin, another specific SR Ca²⁺-ATPase inhibitor, also increased the muscle tone. The effect of thapsigargin was completely suppressed by sodium nitroprusside and 3-morpholino-sydnonimine and partially by nifedipine. In conclusion, under conditions when the SR Ca²⁺-ATPase is inhibited, the tissue develops a strong tonic contraction and a large part of this is mediated by Ca²⁺ influx presumably via nifedipine-sensitive Ca²⁺ channels. This study suggests the important role of SR Ca²⁺-ATPase in the modulation of the muscle tone and the function of SR as a “buffer barrier” to Ca²⁺ entry in the cat gastric fundus smooth muscle. Received: 10 August 1995/Received after revision: 9 November 1995/Accepted: 10 November 1995     

Modulation of Ca2+ channels by intracellular Mg2+ ions and GTP in frog ventricular myocytes

Under conditions of low intracellular [Mg²⁺] ([Mg²⁺]_i), achieved by dialysis with pipette solutions containing ethylenediamine tetraacetic acid (EDTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and adenosine triphosphate (ATP) as chelator, calcium currents through the L-type calcium channels (I _Ca) were increased in frog ventricular myocytes. Total suppression of phosphorylation by depleting the cell of ATP with a cocktail of β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP) 2-deoxyglucose and carboxylcyanide-M-chlorophenylhydrazone (CCCP) did not inhibit the increase in I _Ca in the Mg²⁺-deficient medium. Thus, the involvement of phosphorylation process in the increase in I _Ca was not likely. Effective suppression of this enhancement of I _Ca was achieved by the application of guanosine triphosphate (GTP). From the dose-response curve for GTP, the GTP concentration required for half-maximal inhibition (IC₅₀) was estimated to be 4.0 μM at pMg 6. This GTP-induced suppression of I _Ca is not due to the guanine nucleotide binding protein (G-protein) cascade, because both activators and inhibitors of G-protein, which are structural analogues of GTP, suppressed I _Ca similarly. Treatment with pertussis toxin (PTX) did not affect the inhibitory action of Mg²⁺ and GTP on I _Ca. GTP is therefore assumed to bind directly to the Ca²⁺ channel. Interaction of Mg²⁺ and GTP with the Ca²⁺ channel activated in the Mg²⁺-deficient medium was examined by comparing the dose/response curves for GTP at two different [Mg²⁺]. The IC₅₀ for GTP suppression was estimated to be 5.7 μM at pMg 6 and 6.9 μM at pMg 5. The results suggest strongly that Mg²⁺ and GTP independently bind and control Ca²⁺ channels. Received: 22 December 1995/Received after revision and accepted: 11 March 1996     

Ca2+ store determines gating of store operated calcium entry in mammalian skeletal muscle

This work describes the gating of the store operated calcium entry (SOCE) in adult mammalian skeletal muscle. Flexor digitorum brevis fibers (FDB) were isolated from adult mice and exposed to conditions to deplete the sarcoplasmic reticulum (SR). A transient SR depletion caused either by repetitive depolarizations, chlorocresol (CMC) or, cyclopiazonic acid (CPA) induced a bell shaped calcium entry that raised the [Ca²⁺]_i to a maximum of 27.09 ± 4.35 nM from the resting value. The activation time to reach 10–90% of the maximum amplitude was 112 ± 10 s (n = 22). On the other hand, any mechanism that caused a permanent SR depletion (like thapsigargin, continuous CPA, or continuous CMC) triggered a calcium entry pathway that lasted 325 ± 23 s and raised the [Ca²⁺]_i to 129.50 ± 13.05 nM from the resting level (n = 28). Then, a prolonged depletion triggered an increase in [Ca²⁺]_i to higher values and for a longer time than when the SR is transiently depleted (p < 0.001). Our results, in skeletal muscle, showed that calcium store depletion was the signal for SOCE activation and how the SR got depleted was not relevant. Also, we found that SOCE deactivation was not caused by [Ca²⁺]_i but by the SR content. Our results suggest that the SR calcium content plays an important role in SOCE gating in mammalian skeletal muscle and a calcium sensor is located inside the SR.     

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     

Attenuation of stimulated Ca2+ influx in colonie epithelial (HT29) cells by cAMP

In HT₂₉ colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca²⁺ activity ([Ca²⁺]_i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P ₃-) mediated Ca²⁺ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca²⁺ influx. The present study was undertaken to examine the effect of increased adenosine 3′,5′-cyclic monophasphate (cAMP) (forskolin 10 μmol/l = FOR) on the Ca²⁺ transient in the presence of CCH (100 μmol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca²⁺ ([Ca²⁺]_i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K⁺ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca²⁺]_i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca²⁺]_i uniformly from a fluorescence ratio (345/380) of 2.9 ± 0.12 to 1.8 ± 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (V _m) of the patch-clamped cells significantly and reversibly from −54 ± 7.4 to −27 ± 5.9 mV (n = 6). In additional experiments V _m was depolarized by 15–54 mV by various increments in the bath K⁺ concentration. This led to corresponding reductions in [Ca²⁺]_i. Irrespective of the cause of depolarization (high K⁺ or FOR) there was a significant correlation between the change in V _m and change in [Ca²⁺]_i. These data indicate that the cAMP-mediated attenuation of Ca²⁺ influx is caused by the depolarization produced by this second messenger. Received: 12 March 1996/Accepted: 2 April 1996     

Effects of halothane on functionally skinned rabbit soleus muscle fibers: A correlation between tension transient and45Ca release

The mechanism(s) involved in the halothane-induced increase in skeletal muscle contraction was studied using functionally skinned soleus muscle fibers from rabbits: For the tension study, single functionally skinned fibers were individually mounted on two pairs of forceps, with one end attached to a photodiode tension transducer. Ca²⁺-activated tension development of the contractile proteins, and Ca²⁺ uptake and release from the sarcoplasmic reticulum (SR) using caffeine-induced tension transients were studied. To measure the amount of calcium, skinned fibers at 0.1 g/ml were used and 0.075 Ci⁴⁵Ca/ml was spiked in the solution 3 (pCa 6.5 and 1 mM [EGTA]) which promoted rapid loading of Ca²⁺. Halothane (1–3%) did not change the [Ca²⁺]-tension relationship; 2 and 3% halothane reduced the maximum Ca²⁺-activated tension by 6–7%. Halothane (1–3%) added to the solution 3, reduced⁴⁵Ca uptake by 3, 22 and 23%; however, the subsequent caffeine-induced tension transient and⁴⁵Ca release were increased by 10–40%. During the release phase only halothane increased both caffeine-induced tension transient and⁴⁵Ca release by 20–60%. The effects of halothane on the tension transient and on the⁴⁵Ca release were comparable. There was no dose-response relationship to the effects of halothane on the above parameters. It is concluded that halothane affects the SR by increasing its membrane permeability to Ca²⁺, resulting in an increase in myoplasmic [Ca²⁺] and thus in the twitch tension in skeletal muscle.     

Activation and inactivation kinetics of a Ca2+-activated Cl- current: photolytic Ca2+ concentration and voltage jump experiments

The activation kinetics of the endogenous Ca²⁺-activated Cl⁻ current (I _Cl,Ca) from Xenopus oocytes was investigated in excised “giant” membrane patches with voltage and Ca²⁺ concentration jumps performed by the photolytic cleavage of the chelator DM-nitrophen. Currents generated by photolytic Ca²⁺ concentration jumps begin with a lag phase followed by an exponential rising phase. Both phases show little voltage dependence but are Ca²⁺-dependent. The lag phase decreases from about 10 ms after a small Ca²⁺ concentration jump (0.1 μM) to less than 1 ms after a saturating concentration jump (55 μM). The rate constant of the rising phase is half-maximal at about 5 μM. At saturating Ca²⁺ concentrations, the rate constant is 400 to 500 s⁻¹. The Ca²⁺ dependence of the stationary current can be described by the Hill equation with n=2.3 and K _0.5=0.5 μM. The amplitude of the stationary current decreases after the excision of the membrane patch with t _1/2≈5 min (run-down). The activation kinetics of the current elicited by a Ca²⁺ concentration jump is not affected by the run-down phenomenon. At low Ca²⁺ concentration (0.3 μM), voltage jumps induce a slowly activating current with voltage-independent time-course. Activation is preceded by an initial transient of about 1-ms duration. At saturating Ca²⁺ levels (1 mM), the initial transient decays to a stationary current. The transient can be explained by a voltage-dependent inactivation process. The experimental data reported here can be described by a linear five-state reaction model with two sequential voltage-dependent Ca²⁺-binding steps, followed by a voltage-independent rate-limiting transition to the open and a voltage-dependent transition to a closed, inactivated state.     

Effects of cytosolic Ca2+ on the Ca2+ content of the sarcoplasmic reticulum in saponin-permeabilized rat ventricular trabeculae

 Rat ventricular trabeculae were mounted for isometric tension recording, and then permeabilized with saponin. The Ca²⁺ concentration ([Ca²⁺]) within the permeabilized preparation (cytosolic [Ca²⁺]) was monitored continuously using Indo-1 and the integrals of Ca²⁺ transients resulting from brief caffeine application used as an index of the sarcoplasmic reticulum (SR) Ca²⁺ content. The relationship between SR Ca²⁺ content and cytosolic [Ca²⁺] was studied within the reported physiological range (i.e. 50–250 nmol · l^–1 Ca²⁺). Increasing cytosolic [Ca²⁺] from 50 nmol · l^–1 to 250 nmol · l^–1 increased the steady-state SR Ca²⁺ content about threefold. However, increasing [Ca²⁺] above 250 nmol · l^–1 typically resulted in spontaneous SR Ca²⁺ release, with no further increase in SR Ca²⁺ content. The SR Ca²⁺ content increased only slowly when cytosolic [Ca²⁺] was increased; it was unchanged 20 s after a rapid increase in cytosolic [Ca²⁺], but increased progressively to a new steady-state level during the following 1–2 min. In a parallel series of experiments using intact papillary muscles, increasing extracellular [Ca²⁺] (from 0.5 to 5 mmol · l^–1) significantly increased twitch tension within 20 s of the solution change. These results support previous suggestions that the SR Ca²⁺ content may increase when diastolic cytosolic [Ca²⁺] rises during inotropic interventions such as increased stimulus rate or extracellular [Ca²⁺]. However, the rate at which SR Ca²⁺ responds to changes in cytoplasmic [Ca²⁺] within the diastolic range does not appear rapid enough to explain the early potentiation of twitch tension in intact preparations after an increase in extracellular [Ca²⁺]. Received: 26 August 1997 / Accepted: 28 October 1997     

Modulation of sarcoplasmic reticulum Ca2+-release channels by caffeine,Ca2+, and Mg2+ in skinned myocardial fibers of fetal and adult rats

Ryanodine causes depression of the caffeine-induced tension transient (ryanodine depression) in skinned muscle fibers, because it blocks the sarcoplasmic reticulum (SR) Ca²⁺-release channels [Su, J. Y. (1988) Pflügers Arch 411:132–136, 371–377; (1992) Pflügers Arch 421:1–6]. This study was performed to examine the sensitivity of SR Ca²⁺-release channels to ryanodine in fetal compared to adult myocardium and to investigate the influence of Ca²⁺, caffeine, and Mg²⁺ on ryanodine depression in skinned fibers. Ryanodine (0.3 nM–1 M) caused a dose-dependent depression in skinned myocardial fibers of the rat, and the fetal fibers (IC₅₀74 nM) were 26-fold less sensitive than those of the adult (IC₅₀2.9 nM). The depression induced by 0.1 M or 1 M ryanodine was a function of [caffeine], or [Ca²⁺] (pCa<6.0), which was potentiated by caffeine, and an inverse function of [Mg²⁺]. At pCa>8.0 plus 25 mM caffeine, a 20% ryanodine depression was observed in both the fetal and adult fibers, indicating independence from Ca²⁺. Ryanodine depression in skinned fibers of the fetus was less affected than that seen in the adult by pCa_i, [caffeine]_i, or 25 mM caffeine plus pCa_i or plus pMg_i (IC₅₀pCa 4.5 versus 5.1; caffeine 12.7 mM versus 2 mM; pCa 6.7 versus 7.3; and pMg 3.9 versus 3.3 respectively). The results show that the SR Ca²⁺-release channel in both fetal and adult myocardium is modulated by Ca²⁺, caffeine, and Mg²⁺. It is concluded that less ryanodine depression seen in the skinned fibers of the fetus, indicating a relatively insensitive SR Ca²⁺-release channel, could contribute to the resistance of intact myocardium to ryanodine.     

Acute atrial arrhythmogenesis in murine hearts following enhanced extracellular Ca2+ entry depends on intracellular Ca2+ stores

Aim: To investigate the effect of increases in extracellular Ca²⁺ entry produced by the L-type Ca²⁺ channel agonist FPL-64176 (FPL) upon acute atrial arrhythmogenesis in intact Langendorff-perfused mouse hearts and its dependence upon diastolic Ca²⁺ release from sarcoplasmic reticular Ca²⁺ stores. Methods: Confocal microscope studies of Fluo-3 fluorescence in isolated atrial myocytes were performed in parallel with electrophysiological examination of Langendorff-perfused mouse hearts. Results: Atrial myocytes stimulated at 1 Hz and exposed to FPL (0.1 μm ) initially showed (<10 min) frequent, often multiple, diastolic peaks following the evoked Ca²⁺ transients whose amplitudes remained close to control values. With continued pacing (>10 min) this reverted to a regular pattern of evoked transients with increased amplitudes but in which diastolic peaks were absent. Higher FPL concentrations (1.0 μm ) produced sustained and irregular patterns of cytosolic Ca²⁺ activity, independent of pacing. Nifedipine (0.5 μm ), and caffeine (1.0 mm ) and cyclopiazonic acid (CPA) (0.15 μm ) pre-treatments respectively produced immediate and gradual reductions in the F/F₀ peaks. Such nifedipine and caffeine, or CPA pre-treatments, abolished, or reduced, the effects of 0.1 and 1.0 μm FPL on cytosolic Ca²⁺ signals. FPL (1.0 μm ) increased the incidence of atrial tachycardia and fibrillation in intact Langendorff-perfused hearts without altering atrial effective refractory periods. These effects were inhibited by nifedipine and caffeine, and reduced by CPA. Conclusion: Enhanced extracellular Ca²⁺ entry exerts acute atrial arrhythmogenic effects that is nevertheless dependent upon diastolic Ca²⁺ release. These findings complement reports that associate established, chronic, atrial arrhythmogenesis with decreased overall inward Ca²⁺ current.