Control of intracellular ionized calcium concentration by sarcolemmal and intracellular mechanisms

The regulation of the resting intracellular ionized calcium concentration [( Ca2+]i) has been studied in ferret papillary muscle using the photoprotein aequorin to measure [Ca2+]i. Elevating [Ca2+]o produced an initial rapid increase of [Ca2+]i and tension which then decayed to a steady level. This secondary fall of [Ca2+]i is attributed to a secondary decrease of Ca entry on Na-Ca exchange produced by the known fall of [Na+]i. Replacing external Na by K produced a large transient increase of both [Ca2+]i and tension which then decayed spontaneously to near the resting level. If Na was removed after metabolic inhibition with cyanide and deoxyglucose then neither tension nor [Ca2+]i recovered. The addition of the mitochondrial uncoupler FCCP to a muscle in Na-free solution produced a gradual rise of tension but only elevated [Ca2+]i after a delay of many minutes. Similarly caffeine did not elevate [Ca2+]i. These experiments do not support the hypothesis that the regulation of resting [Ca2+]i in Na-free solutions depends solely on intracellular sequestration of [Ca2+]i. The first twitch elicited in Na-containing solutions after exposure to Na-free solution was much larger than control and was associated with a large Ca transient attributed to increased loading of the sarcoplasmic reticulum with Ca in the Na-free solution. The elevation of [Ca2+]i in Na-free solutions was accompanied by spontaneous fluctuations of both [Ca2+]i and tension with a frequency of about 3 Hz. These fluctuations were abolished by drugs such as caffeine or ryanodine which interfere with sarcoplasmic reticulum function. These results provide direct evidence for the spontaneous release of Ca from the sarcoplasmic reticulum inferred from previous, less direct, work.     

PKC-dependent preconditioning with norepinephrine protects sarcoplasmic reticulum function in rat trabeculae following metabolic inhibition.

The authors have previously shown that norepinephrine (NE) pretreatment attenuates Ca2+ overloading in cardiac rat trabeculae during metabolic inhibition, and improves contractile function during a subsequent recovery period. The present study investigated: (i) whether protection of sarcoplasmic reticulum (SR) function during metabolic inhibition (MI) is involved in the preconditioning-like effect of NE-pretreatment, and (ii) whether or not this process is PKC-dependent. A 15 min preincubation period was used with 1 micromol/l exogenous NE to precondition isolated, superfused rat trabeculae against contractile dysfunctioning following 40 min of MI in 2 mmol/l NaCN containing Tyrode (gassed with 95% O2/5% CO2; pH 7.4, 24 degrees C) without glucose at 1-Hz stimulation frequency. Contractile recovery was studied during a subsequent 60 min recovery period (RP) in glucose containing Tyrode at 0.2 Hz. Force and intracellular free calcium ([Ca2+]ii) were monitored throughout the experimental protocol. Pretreatment of trabeculae with NE (group NE) substantially diminished the Ca2+ rise from the onset of rigor development during MI, compared to preparations which were pretreated with NE, in the presence of specific PKC blocker chelerythrine (2 micromol/l; group NE+CHEL). After 40 min of MI, resting [Ca2+]i in group NE and NE+CHEL was increased to 0.50+/-0.03 and 2.08+/-0.20 micromol/l, respectively (P<0.05), whereas total intracellular ATP levels were similar in both groups (approximately 0.20 micromol/g dry wt). This corresponded with an increase in active force development (119%) and a decrease in twitch force relaxation time (77%) during subsequent RP in group NE, compared to pre-MI values of the same group. In contrast, a significant decrease in force recovery (54%) and an increase in twitch force relaxation time (123%) was observed in group NE+CHEL. Values for [Ca2+]i, contractile recovery, and twitch force relaxation time in untreated controls as well as CHEL preparations corresponded to those measured in the NE+CHEL group. Rapid cooling contractures (RCCs), which provide information on both SR-Ca2+ loading and Ca2+ re-uptake activity, revealed a 2-fold higher SR Ca2+ content during RP in group NE compared to controls and group NE+CHEL. In addition, kinetic analysis of the RCC rewarming spike (RWS) showed that this was accompanied by greater than a 28% increase in the maximum rate of RWS relaxation (-dF/dt/rws) in group NE compared to group NE+CHEL. The change of -dF/dt/rws in the NE group during RP following MI persisted after SR Ca2+-release channel blockade by ryanodine treatment (100 micromol/l), which suggests involvement of NE-induced, PKC-dependent protection of SR Ca2+-ATPase activity. The results of the present study point to an inverse relationship between the Ca2+ rise during MI and SR functioning, in which PKC appears to play a key role. It is concluded that the preconditioning-like effect of NE-pretreatment on contractile recovery is at least partly mediated by protection of SR function.     

Abnormal platelet and lymphocyte calcium handling in prehypertensive rats

We have reported that the basal and stimulated cytosolic free calcium concentrations [( Ca2+]i) are elevated in platelets isolated from 12-14-week-old spontaneously hypertensive rats (SHR) as compared with normotensive Wistar-Kyoto (WKY) rats. To determine whether altered cell calcium metabolism precedes the development of overt hypertension, we measured [Ca2+]i under resting and stimulated conditions in blood platelets and thymic lymphocytes isolated from 4-week-old prehypertensive SHR and WKY rats. Blood pressure was similar in both groups (SHR 95 +/- 8 versus WKY rats 92 +/- 7 mm Hg). Basal [Ca2+]i in platelets was higher in SHR than WKY rats (63.4 +/- 3.9 versus 54.8 +/- 3.1 nM, p less than 0.003). Also the [Ca2+]i response to thrombin was greater in SHR than WKY rats in both the presence and absence of extracellular calcium. For lymphocytes, although no difference was detected in basal [Ca2+]i, the concanavalin A-induced peak [Ca2+]i was higher for SHR than WKY rats in both calcium-containing and calcium-free media. These results suggest that agonist-stimulated calcium influx and calcium discharge from intracellular stores are enhanced in both platelets and lymphocytes of 4-week-old SHR. We conclude that abnormalities in calcium metabolism in two different cell types precede the development of overt hypertension in the SHR.     

Alpha 1-adrenergic stimulation of in vitro growth hormone release and cytosolic free Ca2+ in rat somatotrophs

The effects of alpha 1-adrenergic agents on GH release and intracellular free Ca2+ concentration ([Ca2+]i) were investigated in purified rat somatotroph preparations. Phenylephrine (PHE) stimulated in vitro GH release; the maximal effect (2.5-fold stimulation) occurred at 1 microM PHE. The effect was completely blocked by the alpha-adrenergic antagonist phentolamine and partially counteracted by the beta-antagonist propranolol. Experiments with the fluorescent Ca2+ probe fura 2 show that PHE causes [Ca2+]i to rise from 178 +/- 31 nM (mean +/- SE; n = 25) to 370 +/- 55 nM (n = 9). This effect was complete within 20 sec and was maintained for at least 5-10 min. The rise was rapidly interrupted by administration of 1 microM phentolamine. The beta-receptor agonist isoproterenol caused a small [Ca2+]i rise due to action on alpha 1-adrenoreceptors. The PHE-induced [Ca2+]i rise showed two components: an initial peak due to Ca2+ mobilization from intracellular stores and a subsequent rise due to Ca2+ influx from the extracellular space. Somatostatin (SRIF) lowered both resting [Ca2+]i and Ca2+ influx stimulated by PHE. Pertussis toxin pretreatment did not modify PHE-induced [Ca2+]i changes, while it completely prevented the effect of SRIF on both resting and triggered [Ca2+]i, thus suggesting that a GTP-binding protein sensitive to the toxin is involved in the transduction of SRIF action. The increase in cAMP induced by cholera toxin pretreatment modified neither PHE nor SRIF action on [Ca2+]i. In conclusion, in rat somatotrophs Ca2+ mobilization and influx are stimulated by alpha 1-adrenergic agents, and this triggered [Ca2+]i rise results in a stimulation of GH release. In these cells SRIF is able to reduce both resting [Ca2+]i levels and [Ca2+]i increases induced by alpha 1-adrenergic activation.     

Ranolazine decreases diastolic calcium accumulation caused by ATX-II or ischemia in rat hearts

Cardiac pathologies are associated with increased late INa that contributes to the dysregulation of ion homeostasis and causes electrical and contractile dysfunction. This study was designed to test the hypothesis that an increased late sodium channel current (INa) leads to Ca2+ overload and left ventricular (LV) dysfunction, and thereby inhibition of late INa (e.g., by ranolazine) improves Ca2+ homeostasis and reduces LV dysfunction. Intracellular Ca2+ ([Ca2+]i) and LV function were measured simultaneously in rat isolated perfused hearts. Augmentation of late INa with sea anemone toxin-II (ATX-II, 12 nM) increased diastolic [Ca2+]i (d[Ca2+]i), and impaired LV mechanical function, but had no effect on [Ca2+]i transient amplitude. Although ranolazine (4 and 9 microM), an inhibitor of late INa, had no direct effects on d[Ca2+]i or LV function, it significantly reduced the deleterious effects of ATX-II. Global ischemia increased d[Ca2+]i and inhibited Ca2+ transient amplitude. During reperfusion, Ca2+ transient amplitude recovered fully, but d[Ca2+]i remained elevated and LV function was depressed, indicative of Ca2+ overload. Ranolazine (9 microM) reduced d[Ca2+]i accumulation during ischemia as well as reperfusion and improved recovery of LV function. These results show that augmentation of late INa with ATX-II or by ischemia is associated with diastolic Ca2+ overload and LV dysfunction. The beneficial effects of ranolazine in reducing Ca2+ overload and LV mechanical dysfunction during ischemia/reperfusion is consistent with the inhibition of late INa mechanism of action.     

Cytosolic free calcium of aorta in hypertensive rats. Chronic inhibition of angiotensin converting enzyme

Cytosolic free calcium concentration ([Ca2+]i) and muscle tension were simultaneously measured in aortic tissue isolated from spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto (WKY) rats, and SHR chronically treated with a novel angiotensin converting enzyme inhibitor, CS-622. In the presence of 2.5 mM Ca2+ in the bathing solution, aortic [Ca2+]i measured with fura-2 was higher in SHR than in WKY rats, and it was almost the same in CS-622-treated SHR and untreated WKY rats. Increase of external Ca2+ concentration from zero to 2.5 mM elicited a contraction in SHR aortas but not in aortas from both CS-622-treated SHR and untreated WKY rats. When the aortas were contracted by 60 mM K+, however, [Ca2+]i as well as developed tension was similar in the three groups. CGP-28392 (10(-6) M), a Ca2+ channel activator, induced a rhythmic activity superimposed on a gradual increase of [Ca2+]i and tension in SHR aortas but not in the aortas of CS-622-treated SHR or untreated WKY rats. Nicardipine (10(-7) M) decreased the resting [Ca2+]i and the resting tone in SHR aortas, but not in WKY rat aortas. These results suggest that SHR aortas have a higher myogenic tone due to increased [Ca2+]i than WKY rat aortas and that the increased [Ca2+]i is attributed to alterations of dihydropyridine-sensitive Ca2+ channels in SHR aortas. Further, the decrease of the vascular tone induced by long-term administration of the angiotensin converting enzyme inhibitor may be due to a reduction of increased [Ca2+]i in SHR.     

Lack of effect of parathyroid hormone on calcium homeostasis in rat platelets.

The in vitro effects of parathyroid hormone on Ca2+ handling by fura-2 loaded rat platelets were studied. The incubation of these platelets with rat parathyroid hormone (1-34) for 10 min had no effect on intracellular fura-2 metabolism or [Ca2+]i in the resting state. The [Ca2+]i response to 0.1 U/mL thrombin was unaffected by preincubation with parathyroid hormone in the presence or absence of extracellular Ca2+. Furthermore, the recovery rate of [Ca2+]i after the thrombin-induced peak in Ca(2+)-depleted media was not altered with parathyroid hormone. These data indicate that parathyroid hormone may not have a significant effect on Ca2+ homeostasis in rat platelets in unstimulated and stimulated conditions.     

Separation between cytosolic calcium and secretion in chromaffin cells superfused with calcium ramps.

This paper describes experiments in which cytosolic Ca2+ concentrations ([Ca2+]i) and catecholamine release were measured in two populations of chromaffin cells stimulated with a solution enriched in K+ (100 mM). Once depolarized, external Ca2+ or Ba2+ ions were offered to cells either as a single 2.5 mM step or as a ramp that linearly increased the concentration from 0 to 2.5 mM over a 10-min period. A clear separation between the changes of the [Ca2+]i and the time course of secretion was observed. Specifically, secretion and [Ca2+]i rose in parallel when a Ca2+ step was used to reach a peak in a few seconds; however, while secretion declined to the basal level, [Ca2+]i remained elevated at a plateau of 400 nM. With a Ca2+ ramp, only a transient small peak of secretion was observed, yet the [Ca2+]i remained elevated throughout the 10-min stimulation period. The separation between secretion and [Ca2+]i was observed even when voltage-dependent Ca2+ channels were expected to remain open (mild depolarization in the presence of 1 microM Bay K 8644). By using Ba2+ steps or ramps, sustained noninactivating secretory responses were obtained. The results suggest that the rate and extent of secretion are not a simple function of the [Ca2+]i at a given time; they are compatible with the following conclusions: (i) A steep extracellular-to-cytosolic Ca2+ gradient is required to produce a sharp increase in the [Ca2+]i at exocytotic sites capable of evoking a fast but transient secretory response. (ii) As a result of Cai(2+)-dependent inactivation of Ca2+ channels, those high [Ca2+]i are possible only at early times after cell depolarization. (iii) The Cai(2+)-dependent supply of storage granules to the secretory machinery cooperates with the supply of Ca2+ through Ca2+ channels to regulate the rate and extent of secretion.     

Energetic requirement of carbachol-induced Ca2+ signaling in single mouse beta-cells

Insulin secretion is under multifactorial control by glucose and neurohumoral factors like acetylcholine (ACH), which activate the Ca2+/phospholipase C signaling pathway. All insulin secretagogues elevate cytosolic free Ca2+ ([Ca2+]i) that is central to the stimulation of insulin secretion. The actions of ACH on [Ca2+]i are glucose dependent but the metabolic steps involved are only partly understood. Here we have characterized the metabolic steps by which glucose exerts its synergistic effects on ACH-linked Ca2+-signals. [Ca2+]i was measured in single fura-2 loaded beta-cells. The ACH analog carbachol (3 microM) caused rise in [Ca2+]i that was strongly dependent on the extracellular glucose concentration ranging from 0-10 mM. Iodoacetate, which blocks glycolysis, thereby preventing the generation of NADH and ATP from glucose metabolism, and rotenone or antimycin, which inhibit complex 1 and 2 of the mitochondrial respiratory chain, respectively, inhibited in glucose (6 mM) the carbachol-induced Ca2+ signal to a similar extent as glucose deprivation. This demonstrates that glucose metabolism and generation of ATP through oxidative phosphorylation of energy rich substrates like NADH and FADH2 are required for carbachol-induced Ca2+ signals. While sodium arsenate, which prevents net glycolytic production of ATP without inhibiting glycolysis, had no significant effect on the carbachol-induced Ca2+-signal, the mitochondrial pyruvate transport inhibitor alpha-cyano-4-hydroxycinnamate and the Krebs cycle inhibitor monofluoroacetate strongly suppressed the rise in [Ca2+]i elicited by carbachol. While pyruvate was ineffective, methyl pyruvate, a membrane-permeant pyruvate analog, and alpha-ketoisocaproate in combination with glutamine, which are both substrates for mitochondrial ATP production, could restore the carbachol-induced Ca2+ signal in glucose-free medium. These data demonstrate for the first time that Krebs cycle metabolism of glucose and ATP formation through oxidative phosphorylation is critical for the glucose dependency of ACH-linked Ca2+-signals in mouse beta-cells, and they suggest that mitochondrial metabolism plays a key role in the interactive regulation of beta-cells by neurohumoral factors activating the Ca2+/phospholipase C signaling pathway.     

Changes in intracellular free calcium concentration during long exposures to simulated ischemia in isolated mammalian ventricular muscle.

Intracellular free calcium concentration ([Ca2+]i) was measured in isolated ferret ventricular papillary muscles during and after long exposures to ischemia. All experiments were performed at 37 degrees C, and the muscles were stimulated at 1 Hz. Ischemia was simulated by changing from superfusion with oxygenated Tyrode's solution to superfusion with water-saturated gas (95% N2-5% CO2), thus simultaneously stopping oxygenation and restricting the extracellular space. [Ca2+]i was measured with aequorin, which was microinjected into superficial cells of the preparation. Exposure to ischemia caused a complex series of changes in [Ca2+]i. In the first few minutes the changes in [Ca2+]i were variable; however, after approximately 5 minutes all preparations exhibited a progressive increase in amplitude and duration of the stimulated rise in [Ca2+]i (the calcium transient). The amplitude of the calcium transients peaked after approximately 18 minutes of ischemia, when they were 339% of the control value. After this peak, the calcium transients progressively failed to occur in response to stimulation and declined in amplitude; simultaneously, spontaneous oscillations of [Ca2+]i appeared and increased in size and frequency. The oscillations in turn then gradually became less frequent until a large, prolonged (5-10 minute) increase in [Ca2+]i occurred, after which [Ca2+]i returned to a low level. There were no further oscillations after this event, which was seen on average after 37 minutes of ischemia. A slowly progressive contracture often began to develop at about this time. A gradual rise in resting [Ca2+]i occurred during the remainder of the exposure to ischemia. When muscles were reperfused after long exposures to ischemia, there was a very large and prolonged increase in [Ca2+]i, which was usually associated with a contracture and failure of recovery of developed tension. The large increase in [Ca2+]i could be reduced by the inclusion of 3 mM nickel chloride in the reperfusing solution. Comparison between reperfusion with O2 gas versus reperfusion with anoxic Tyrode's solution indicated that reoxygenation was more beneficial to the muscle than resumption of bulk flow. These results reveal the complex spectrum of changes in [Ca2+]i that occur during ischemia and on reperfusion. These changes in [Ca2+]i are likely to play an important role in the generation of ischemic arrhythmias and muscle damage.     

Growth hormone increases intracellular free calcium in rat adipocytes: correlation with actions on carbohydrate metabolism.

Adipocytes that have been preincubated for 3 h or more in hormone-free medium respond to GH with a transient insulin-like increase in glucose metabolism, followed by a period of refractoriness to further insulin-like stimulation. Adipocytes freshly isolated from normal rats and adipocytes that were exposed to GH in the first hour of a 4-h incubation period are refractory to this insulin-like effect. Because earlier studies revealed a relationship between refractoriness and cellular calcium, we examined the effects of GH on the intracellular calcium concentration ([Ca2+]i) under a variety of conditions in which sensitivity to insulin-like stimulation or refractoriness is known to be affected. A dual nitrogen laser imaging microscope with computer-assisted image processing to measure fluorescence changes in individual adipocytes loaded with fura-2 AM was used to measure [Ca2+]i. After prolonged incubation in vitro, resting [Ca+2]i was 120 +/- 6 nM and remained unchanged for more than 1 h after the addition of 100 ng/ml human GH (hGH), which doubled the rate of incorporation of [3-3H] glucose into triglycerides in this interval. Lipogenesis declined in the second hour, and [Ca+2]i slowly increased, reaching 324 +/- 49 nM by the end of the third hour (P less than 0.05). When added with GH, actinomycin-D, an inhibitor of RNA synthesis, caused the accelerated rate of lipogenesis to persist for at least 5 h and blocked the delayed increase in resting [Ca+2]i. Resting [Ca2+]i in freshly isolated, and hence refractory, adipocytes was 342 +/- 34 nM and declined to 112 +/- 11 nM concomitant with acquisition of insulin-like sensitivity to GH. The addition of 100 ng/ml hGH to these cells at the beginning of incubation, under conditions known to sustain refractoriness prevented the decline in resting [Ca2+]i and enabled them to exhibit a further acute increase in [Ca2+]i in response to a second exposure to hGH in the fourth hour. When added 60 min after GH, actinomycin-D blocked the ability to raise [Ca2+]i acutely in response to GH, but did not interfere with GH's action to sustain resting [Ca2+]i, which remained at about 300 nM. The concentration of GH needed to increase [Ca2+]i acutely in refractory cells is about 6-fold higher than that needed to maintain resting [Ca+2]i. Differences in the time of sensitivity to actinomycin-D and dose dependency suggest that sustaining resting [Ca2+]i and production of the acute increase in [Ca2+]i are separate phenomena.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of Ca2+ availability to myofilaments and their sensitivity to Ca2+ in myocyte contractile dysfunction in heart failure

OBJECTIVE: Contractile function is depressed at the isolated myocyte level in heart failure (HF), which could result from the decreased availability of intracellular calcium ([Ca2+]i) to the myofibrils and/or the depressed sensitivity of myofilaments to [Ca2+]i. However, the cellular basis of contractile dysfunction remains unestablished. METHODS: We isolated left ventricular myocytes from dogs with rapid pacing-induced HF. Cell shortening and [Ca2+]i transients were measured by indo-1 fluorescence and the myofilament Ca2+ sensitivity was analyzed by the shortening-[Ca2+]i relation in intact myocytes as well as by the pCa tension relation in skinned cells. RESULTS: Peak cell shortening magnitude was depressed in HF, associated with a parallel decrease of [Ca2+]i transient amplitude. There was a significant positive correlation between these two variables (r = 0.71, P < 0.01). In contrast, myofibrillar sensitivity to Ca2+, determined by both intact and skinned myocytes, was comparable between control and HF. Further, there was no significant difference in Ca2+ sensitivity between control and HF even at shorter (1.8 microns) or longer (2.2 microns) sarcomere length. CONCLUSIONS: Using both intact and skinned cellular preparations, a potential defect in myocyte contractile function in HF was a reduction in Ca2+ availability to the myofilaments, rather than the inherent defects in myofilament sensitivity to Ca2+.     

Passive electrical properties, mechanical activity, and extracellular potassium in arterially perfused and ischemic rabbit ventricular muscle. Effects of calcium entry blockade or hypocalcemia

The relation among passive electrical resistive properties, longitudinal conduction velocity, extracellular potassium concentration, [K+]o, and mechanical activity was investigated in the isolated rabbit papillary muscle during normal arterial perfusion and no-flow ischemia in the presence and absence of verapamil, or a reduced extracellular Ca2+ concentration [Ca2+]o. During normal arterial perfusion, verapamil (0.5 microM, free [Ca2+]o = 1.0 mM) and hypocalcemic blood perfusate (free [Ca2+]o = 0.4 mM) reduced the maximal isometric twitch tension by 48% and 78%, depolarized the resting membrane by +3 and +7 mV, decreased the extracellular longitudinal resistance (ro) by 15% and 26%, and increased conduction velocity by 4% and 6%, respectively. The changes in conduction velocity during these interventions were consistent with those predicted by linear cable theory (+3% and +9%) for the observed changes in ro. In contrast, verapamil shortened whereas a reduced [Ca2+]o lengthened action potential duration. Comparison of simultaneously measured longitudinal whole tissue resistance (rt), intracellular longitudinal resistance (ri), [K+]o, and resting tension during ischemia showed a close association between abrupt cell-to-cell electrical uncoupling, development of ischemic contracture, and the secondary rise of [K+]o, which all started to develop after approximately 15 minutes of ischemia. Electrical cell-to-cell uncoupling was completed within 15 minutes. In the presence of verapamil, the relation among the onset of electrical cell-to-cell uncoupling, secondary rise of [K+]o, and onset of ischemic contracture in ischemia was qualitatively the same as in its absence; however, these events were postponed by approximately 10 minutes, and the rates of contracture development and uncoupling were diminished. Conduction velocity decreased after 12 minutes of ischemia from 54 to 36 cm/sec in the absence of and from 61 to 46 cm/sec in the presence of verapamil. This slowing effect on impulse conduction could not be attributed to changes of electrical cell-to-cell coupling because at this time an increase in ri had not yet taken place. In the presence of a reduced [Ca2+]o, the resting tension and ri increased almost immediately after the onset of ischemia. Although the resting tension rose progressively throughout the course of ischemia, the ri showed a biphasic increase characterized by an early transient increase that reached a peak at 8 minutes (+87%) and a second, irreversible increase beginning at approximately 12 minutes. This final onset of electrical cell-to-cell uncoupling and the secondary rise of [K+]o were not different from the findings with a normal [Ca2+]o.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cellular calcium fluctuations in mammalian heart: direct evidence from noise analysis of aequorin signals in Purkinje fibers.

Indirect evidence suggests that fluctuations, or oscillations, in the intracellular free calcium concentration [( Ca2+]i) can occur spontaneously in intact cardiac preparations, but such [Ca2+]i fluctuations have never been demonstrated directly. We used the bioluminescent Ca2+-sensitive protein aequorin to detect fluctuations in the [Ca2+]i in canine cardiac Purkinje fibers. Noise analysis of the aequorin luminescence reveals prominent peaks of power density at frequencies of 1-4 Hz; these peaks become larger and shift to higher frequencies as the [Ca2+]i increases. Caffeine and ryanodine abolish the [Ca2+]i fluctuations, suggesting that Ca2+ release and uptake by the sarcoplasmic reticulum generate these events. When [Ca2+]i fluctuations are present, less tension is produced at any given level of mean aequorin luminescence. Thus, [Ca2+]i fluctuations will undermine attempts to relate [Ca2+]i and force in intact myocardium.     

Tri-iodothyronine increases intra-cellular calcium levels in single rat myocytes.

We have studied the effects of acute administration of triiodothyronine (T3) on cytosolic free calcium levels [Ca2+]i in single rat myocytes microinjected with aequorin. Ventricular myocytes were isolated by perfusing rat hearts with collagenase, and healthy, rod-shaped cells were injected to less than 1% of their volume with aequorin. The photons emitted from single cells were measured and a conversion to [Ca2+]i made on the basis of an in vitro calibration after the remaining aequorin had been discharged by cell lysis. Only cells that depolarized reversibly (showing elevated [Ca2+]i levels) when superfused with 80 mM KCl, and which gave a substantial signal on lysis with distilled water were used. The [Ca2+]i rose from a resting value of 150 +/- 56 nM (mean +/- SD, n = 14) by 127 +/- 47 nM on depolarization with 80 mM KCl. Application of T3 (1-100 nM) led to an increase (P less than 0.05) in [Ca2+]i (mean amplitude of 152 +/- 35 nM) before returning to baseline. The median duration of these events was 10 min (range = 1.4-34.4 min). The time to response was shorter when 100 nM T3 was applied (median and range; 6.8, 0-14 min) than when 1 nM T3 was used (16, 7.0-56.1 min) (P less than 0.05). To conclude, physiological concentrations of thyroid hormones caused rapid but transient stimulation of [Ca2+]i in single rat myocytes.     

Refractoriness to growth hormone is associated with increased intracellular calcium in rat adipocytes.

In adipocytes that have been deprived of growth hormone (GH) for at least 3 hr, GH elicits a transient insulin-like response that is followed by a period of refractoriness to further insulin-like stimulation. Exposure of adipocytes to GH in the first hour of a 3-hr incubation prevents the appearance of insulin-like sensitivity. Intracellular Ca2+ concentration [( Ca2+]i) was measured in individual adipocytes that were loaded with fura-2 hexakis(acetoxymethyl) ester after preincubation in the presence (refractory) or absence (sensitive) of recombinant human GH at 100 ng/ml. Using a dual nitrogen laser imaging microscope with computer-assisted image processing to measure fluorescence changes, we observed that resting [Ca2+]i was 220 +/- 10 nM in refractory adipocytes and 110 +/- 6 nM in sensitive adipocytes (P less than 0.001). GH had no acute effect on [Ca2+]i in sensitive adipocytes but caused a sustained 3-fold increase in [Ca2+]i in refractory cells within 3 min (P less than 0.001). Insulin did not change [Ca2+]i in either sensitive or refractory adipocytes. In refractory cells treated with insulin and GH simultaneously, insulin completely blocked the rise in [Ca2+]i due to GH. Oxytocin elicited a prompt increase in [Ca2+]i followed by a quick return to resting levels in both sensitive and refractory cells. These findings indicate that basal [Ca2+]i is increased in refractory cells and that GH produces a sustained rise in [Ca2+]i only in refractory adipocytes. We suggest that the sustained increase in [Ca2+]i produced by GH in refractory cells prevents the expression of the insulin-like response.     

Effects of endothelin-1 on hepatic stellate cell proliferation, collagen synthesis and secretion, intracellular free calcium concentration

AIM: To explore the effects of endothelin-1(ET-1) on hepatic stellate cells (HSCs) DNA uptake, DNA synthesis, collagen synthesis and secretion, inward whole-cell calcium concentration ([Ca2+]i) as well as the blocking effect of verapamil on ET-1-stimulated release of inward calcium (Ca2+) of HSC in vitro. METHODS: Rat hepatic stellate cells (HSCs) were isolated and cultivated. 3H-TdR and 3H-proline incorporation used for testing DNA uptake and synthesis, collagen synthesis and secretion of HSCs cultured in vitro; Fluorescent calcium indicator Fura-2/AM was used to measure [Ca2+]i inward HSCs. RESULTS: ET-1 at the concentration of 5X10(-8) mol/L, caused significant increase both in HSC DNA synthesis (2,247+/-344 cpm, P<0.05) and DNA uptake (P<0.05) when compared with the control group. ET-1 could also increase collagen synthesis (P<0.05 vs control group) and collagen secretion (P<0.05 vs control group). Besides, inward HSC [Ca2+]i reached a peak concentration (422+/-98 mol/L, P<0.001) at 2 min and then went down slowly to 165+/-51 mol/L (P<0.01) at 25 min from resting state (39+/-4 mol/L) after treated with ET-1. Verapamil (5 mol/L) blocked ET-1-activated [Ca2+]i inward HSCs compared with control group (P<0.05). Fura-2/AM loaded HSC was suspended in no Ca2+ buffer containing 1 mol/L EGTA, 5 min later, 10(-8) mol/L of ET-1 was added, [Ca2+]i inward HSCs rose from resting state to peak 399+/-123 mol/L, then began to come down by the time of 20 min. It could also raise [Ca2+]i inward HSCs even without Ca2+ in extracellular fluid, and had a remarkable dose-effect relationship(P<0.05). Meanwhile, verapamil could restrain the action of ET-1(P<0.05). CONCLUSION: Actions of ET-1 on collagen metabolism of HSCs may depend on the transportation of inward whole-cell calcium.     

Agonist-stimulated calcium entry in primary cultures of human cerebral microvascular endothelial cells.

Primary cultures of human cerebral microvascular endothelial cells (HCMEC) were loaded with fura-2. The intracellular free Ca2+ concentration ([Ca2+]i) was measured by digital imaging microscopy. Agonists ATP (100 micro), thrombin (10 units/ml), and histamine (25 microM) induced a transient [Ca2+]i increase. Histamine (100 microM) induced a biphasic [Ca2+]i increase with an initial [Ca2+]i peak followed by a [Ca2+]i plateau. The [Ca2+]i plateau was blocked by the receptor-operated Ca2+ channel (ROC) blockers SK&F 96365 and NCDC, indicating a contribution by Ca2+ influx through ROC to the [Ca2+]i plateau. However, this [Ca2+]i plateau was not blocked by the voltage-gated Ca2+ channel (VGC) blocker diltiazem (DTZ). Depolarization with 80K+ or application of the VGC agonist BAY K 8644 did not alter the resting [Ca2+]i; but 80K+ reduced the histamine (100 microM) induced [Ca2+]i plateau. These results show that HCMEC are devoid of functional VGC. Thus the membrane potential (Em) regulates Ca2+ entry mainly by enhancing the electrochemical Ca2+ gradient, such that hyperpolarization increases while depolarization decreases [Ca2+]i. Blockade of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) by CPA increased [Ca2+]i. This effect was dependent on extracellular Ca2+ and reduced by iberiotoxin (IBTX) blockade of Ca2+-activated K+ channels (Kca), suggesting a role for Kca in regulating Ca2+ influx. Ca2+ is the principal activator of endothelial nitric oxide synthase (eNOS), which stimulates cyclic GMP production. The final result that the eNOS inhibitor L-NAME enhanced the histamine (100 microM) induced [Ca2+]i plateau suggests a negative feedback loop (via cGMP) of endothelial NO on its own synthesis in the regulation of endothelial [Ca2+]i signal.     

Effects of calcium on shortening velocity in frog chemically skinned atrial myocytes and in mechanically disrupted ventricular myocardium from rat.

Effects of [Ca2+] on isometric tension and unloaded shortening velocity were characterized in single chemically skinned myocytes from frog atrium and in mechanically disrupted myocardium from rat ventricle. The preparations were attached to a force transducer and piezoelectric translator and were viewed with an inverted microscope to allow continuous monitoring of sarcomere length during mechanical measurements. Unloaded shortening velocity was determined by measuring the time required to take up various amounts of slack imposed at one end of each preparation. Ca2+ sensitivity of isometric tension was assessed as pCa50, i.e., the Ca2+ concentration at which tension was 50% maximal, and was greater for frog atrial myocytes (pCa50 6.17) than for rat ventricular myocytes (pCa50 6.06). This difference in Ca2+ sensitivity may be due to variations in myofibrillar protein isoform composition in the two preparations. Inclusion of caffeine in the activating solutions substantially increased the Ca2+ sensitivity of tension, which may be a manifestation of a direct effect of caffeine on the myofibrillar proteins. Unloaded shortening velocity during maximal activation averaged 4.32 muscle lengths per second in frog atrial myocytes and 4.46 muscle lengths per second in rat ventricular myocytes. When [Ca2+] was reduced, unloaded shortening velocity decreased substantially in both preparations. Possible mechanisms for the effect of Ca2+ on shortening velocity in myocardium include Ca2+ dependence of the rate of ADP dissociation from actomyosin complexes or a shortening-dependent internal load involving structures such as C protein or long-lived myosin cross-bridges.     

Intracellular calcium transients in myocardium from spontaneously hypertensive rats during the transition to heart failure

To investigate the mechanism of impaired myocardial function after long-term pressure overload, we studied cardiac muscle mechanical contraction and intracellular calcium transients using the bioluminescent indicator aequorin. Left ventricular papillary muscle preparations were examined from three groups of rats: 1) aging spontaneously hypertensive rats (SHR) with clinical and pathological evidence suggesting heart failure (SHR-F group), 2) age-matched SHRs with no evidence of heart failure (SHR-NF group), and 3) age-matched normotensive Wistar-Kyoto rats (WKY group). Isometric force development was depressed in both SHR groups relative to the WKY group. Resting [Ca2+]i was lower in the SHR-F group, and the time to peak [Ca2+]i was prolonged in this group. The relative increases in peak [Ca2+]i with the inotropic interventions of increased [Ca2+]o and the addition of isoproterenol were similar among groups. Although inotropy increased in all groups with increased [Ca2+]o, after isoproterenol, inotropy increased only in the WKY group. Thus, in SHR myocardium, [Ca2+]i increased after isoproterenol, but inotropy failed to increase. Myosin isozymes were shifted toward the V3 isoform in both SHR groups; the V3 isoform was virtually 100% in papillary muscles from the SHR-F group. These changes may reflect events directly contributing to the development of heart failure or represent adaptive changes to chronic pressure overload and heart failure.