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.     

Pituitary adenylate cyclase-activating polypeptide specifically increases cytosolic calcium ion concentration in rat gonadotropes and somatotropes.

The hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), is a potent stimulator of cAMP accumulation in the anterior pituitary gland, though its physiological function has yet to be defined. To establish the target cells of PACAP action we have measured PACAP-induced changes in cytosolic free calcium ion concentration ([Ca2+]i) in single identified anterior pituitary cells. This was achieved by combining fura-2 videomicroscopy, to measure [Ca2+]i, and reverse hemolytic plaque assays, to identify the secreted hormone. PACAP (100 nM) increased [Ca2+]i in 32% of all pituitary cells. These responses were predominantly seen in identified gonadotropes and somatotropes, but rarely in corticotropes or lactotropes. PACAP induced two forms of Ca2+ response in gonadotropes; a "Ca2+ spike" (independent of extracellular Ca2+) in 72% of responding gonadotropes, and an extracellular Ca(2+)-dependent "Ca2+ plateau" (28% of cells). In somatotropes, PACAP stimulated either Ca2+ plateau responses (58% of responding somatotropes) or repetitive "Ca2+ transients" (42% of cells), both of which were dependent upon extracellular Ca2+. PACAP, therefore, produces distinct changes in [Ca2+]i in gonadotropes and somatotropes, which may be related to distinct intracellular messenger pathways. The identification of these cell types as targets of PACAP action suggests a role in the regulation of reproduction and growth.     

Intracellular calcium concentration and growth hormone secretion in individual somatotropes: effects of growth hormone-releasing factor and somatostatin

The cytosolic free calcium concentration and cumulative GH release were measured simultaneously in normal pituitary cells. This was made possible by a novel combination of fluorescence microscopy using the calcium indicator fura-2 and a reverse hemolytic plaque assay. GRF (10 nM) rapidly increased the intracellular free calcium concentration ([ Ca2+]i) from a basal level of 234 +/- 17 nM (mean +/- SE) to a peak value of 480 +/- 61 nM 1 min after stimulation. This GRF-induced calcium rise was totally abolished in calcium-free medium or in the presence of calcium channel blockers cobalt chloride (2 mM) and verapamil (100 microM). When somatostatin (SRIF; 1 nM) was added after basal recordings, cytosolic calcium decreased to 96 +/- 23 nM in identified somatotropes. [Ca2+]i returned to baseline upon the removal of SRIF inhibition. This rebound was higher when a sequential treatment of SRIF followed by GRF was applied. Exposing cells to a combination of GRF (10 nM) plus SRIF (1 nM) resulted in a decrease in [Ca2+]i identical to that caused by SRIF treatment alone. Despite the 10-fold excess of GRF, SRIF not only inhibited hormone secretion, but also totally overcame the GRF-induced rise of [Ca2+]i. In summary, stimulation by GRF increases cytosolic calcium in normal somatotropes. This increase is proposed to be due to the influx of calcium through membrane ion channels. In contrast, SRIF decreases [Ca2+]i. This might explain the cAMP-independent effects of this peptide. The effect of SRIF dominates over that of GRF with respect to both changes in [Ca2+]i and hormone release. Changes in the GH secretory rate are, therefore, accompanied by parallel changes in [Ca2+]i, both of which are primarily regulated by SRIF.     

慢性乙型肝炎患者PMN内游离钙含量与PMN凋亡的研究

目的了解慢性乙型肝炎患者外周血多形核粒细胞(PMN)内游离钙含量变化、凋亡情况及其在慢性乙型肝炎发病机制中的作用。方法分离正常外周血PMN加入乙肝患者血清,在体外培养0、3、6、12小时后负载荧光染料用激光扫描共聚焦显微镜测定PMN内游离钙含量,同时应用流式细胞仪检测PMN凋亡情况。结果PMN内游离钙含量及凋亡率较正常对照均降低。结论慢性乙型肝炎患者外周血PMN凋亡延迟。     

Epoxyeicosatrienoic acids increase intracellular calcium concentration in vascular smooth muscle cells

Epoxyeicosatrienoic acids (EETs) are cytochrome P450-derived metabolites of arachidonic acid. They are potent endogenous vasodilator compounds produced by vascular cells, and EET-induced vasodilation has been attributed to activation of vascular smooth muscle cell (SMC) K(+) channels. However, in some cells, EETs activate Ca(2+) channels, resulting in Ca(2+) influx and increased intracellular Ca(2+) concentration ([Ca(2+)](i)). We investigated whether EETs also can activate Ca(2+) channels in vascular SMC and whether the resultant Ca(2+) influx can influence vascular tone. The 4 EET regioisomers (1 micromol/L) increased porcine aortic SMC [Ca(2+)](i) by 52% to 81%, whereas arachidonic acid, dihydroxyeicosatrienoic acids, and 15-hydroxyeicosatetraenoic acid (1 micromol/L) produced little effect. The increases in [Ca(2+)](i) produced by 14,15-EET were abolished by removal of extracellular Ca(2+) and by pretreatment with verapamil (10 micromol/L), an inhibitor of voltage-dependent (L-type) Ca(2+) channels. 14,15-EET did not alter Ca(2+) signaling induced by norepinephrine and thapsigargin. When administered to porcine coronary artery rings precontracted with a thromboxane mimetic, 14,15-EET produced relaxation. However, when administered to rings precontracted with acetylcholine or KCl, 14,15-EET produced additional contractions. In rings exposed to 10 mmol/L KCl, a concentration that did not affect resting ring tension, 14,15-EET produced small contractions that were abolished by EGTA (3 mmol/L) or verapamil (10 micromol/L). These observations indicate that 14,15-EET enhances [Ca(2+)](i) influx in vascular SMC through voltage-dependent Ca(2+) channels. This 14,15-EET-induced increase in [Ca(i)(2+)] can produce vasoconstriction and therefore may act to modulate EET-induced vasorelaxation.     

玉郎伞多糖对Aβ25-35所致PC12细胞损伤细胞内钙离子浓度的影响

目的观察Aβ25-35诱导PC12细胞凋亡损伤模型胞内钙浓度的变化及YLSP的影响。方法采用Aβ25-35诱导PC12细胞凋亡损伤模型,石杉碱甲作为对照药,给予YLSP预处理后,加入Flu-3/AM,用流式细胞仪（FCM）测定各组细胞的平均荧光强度。结果各组PC12细胞内钙离子含量存在显著性差异（F=14.051,P〈0.01）,其中,Aβ25-35作用PC12细胞后,PC12细胞内钙离子含量显著升高（P〈0.01）,经YLSP处理后,PC12细胞内钙离子含量较模型组细胞显著降低（P〈0.01）,其中,YLSP高剂量组的PC12细胞内钙离子含量显著低于石杉碱甲组（P〈0.01）。结论 YLSP能对抗Aβ引起的细胞内钙离子浓度升高,防止钙超载的发生。     

Heterogeneous intracellular free calcium responses to parathyroid hormone correlate with morphology and receptor distribution in osteogenic sarcoma cells.

The osteogenic sarcoma cell line UMR 106-01 exhibits heterogeneous morphology and hormone response in subconfluent monolayer cultures. In these studies we have explored the correlation between morphological profiles and patterns of cytosolic calcium [Ca2+]i response to PTH and other agonists in single UMR 106-01 cells loaded with the Ca(2+)-sensitive fluorescent indicator, fura-2. Realtime recording of [Ca2+]i revealed that PTH (10(-7) M) produced a transient [Ca2+]i rise in 19% of the cells studied. [Ca2+]i transients were also induced by prostaglandins E2 and F2 alpha, and fetal bovine serum, but with different response frequencies (20%, 12%, and 58%, respectively). Spatial resolution of changes in [Ca2+]i by video image analysis revealed that the response to PTH was more frequent in large polygonal cells with long cytoplasmic processes and less common in smaller cells growing in clusters, whereas there was no clear subtype specificity for the effects of epidermal growth factor and fetal bovine serum on [Ca2+]i. Autoradiographic analysis of cell monolayers demonstrated a higher density of PTH-binding sites on cells with cytoplasmic extensions, whereas epidermal growth factor-binding sites were largely on colony-forming cells. Thus, the [Ca2+]i response to hormonal stimulation is heterogeneous within UMR 106-01 cell populations and within single cells, and it correlates with receptor density. This suggests that osteoblastic cells respond to PTH by activation of changes in [Ca2+]i only at certain specific steps during osteoblast development or stages of the cell cycle.     

Effects of metabolic blockade on intracellular calcium concentration in isolated ferret ventricular muscle

Tension and intracellular free calcium concentration [( Ca2+]i) were measured in isolated ferret papillary muscles. When both anaerobic glycolysis and oxidative phosphorylation were prevented (metabolic blockade), there was a rapid decline of both developed tension and systolic [Ca2+]i signals. Subsequently, resting tension increased, and after a further delay, resting [Ca2+]i also rose. When oxidative metabolism was restarted after a period of metabolic blockade that was sufficient to elevate both resting tension and [Ca2+]i, a variable recovery of mechanical function occurred. In preparations that showed recovery, resting tension declined toward control level, and there was considerable recovery of developed tension. [Ca2+]i initially fell, but it then rose to a level similar to that at the end of the preceding period of metabolic blockade and exhibited large variations in amplitude with frequency components in the range 0.2-1 Hz. This elevated [Ca2+]i gradually declined. Arrhythmias were often present during this recovery period and appeared to be triggered by the spontaneous increases in [Ca2+]i. In preparations that failed to recover, resting tension remained elevated or increased, and developed tension showed little recovery. Such preparations showed larger rises in [Ca2+]i both during and after metabolic blockade, and [Ca2+]i continued to rise when oxidative metabolism was restarted. In experiments in which Na-Ca exchange was inhibited (by replacement of sodium by lithium or by the application of nickel), the rise of [Ca2+]i when oxidative metabolism was restarted was reduced, but recovery of mechanical function was improved. The correlation between elevated [Ca2+]i on reactivation of oxidative metabolism and failure of recovery of mechanical function suggests that elevated [Ca2+]i has a direct role in preventing the recovery of mechanical function.     

Endothelin does not affect intracellular calcium ion concentration in the platelets of rats

We recently demonstrated that porcine endothelin increases intracellular calcium ion concentration ([Ca2+]i) in monolayers of cultured vascular smooth muscle cells isolated from rat renal arteries. The present study was designed to examine the effect of porcine endothelin on [Ca2+]i in the platelets of rats. [Ca2+]i was serially measured with the fluorescent dye, fura 2, and the photoprotein, aequorin. Endothelin (10(-9) mol/L, 10(-8) mol/L, and 10(-7) mol/L) did not induce any change in [Ca2+]i in the platelets of rats while thrombin (2 U/mL) dramatically increased [Ca2+]i as measured by these methods. Our results indicate that [Ca2+]i in rat platelets is not influenced by porcine endothelin.     

Mechanical stretch increases intracellular calcium concentration in cultured ventricular cells from neonatal rats

Summary We investigated the effects of mechanical stretch on intracellular calcium concentration ([Ca²⁺]_i) of cultured neonatal rat ventricular cells using micro-fluorometry with fura-2. Myocytes were cultured on laminin-coated silicon rubber and stretched by pulling the rubber with a manipulator. Myocytes were either mildly stretched (to less than 115% of control length), moderately so (to 115%–125% of control length), or extensively (to over 125% of the control length). “Quick stretches” (accomplished within 10s) of moderate to extensive intensities produced a large transient increase of [Ca²⁺]_i in the early phase of stretch (30s-2 min), followed by a small but sustained increase during the late phase of stretch (5–10 min). The initial transient increase in [Ca²⁺]_i after the “quick stretch” was preserved in the presence of gallopamil (10⁻⁷M) or ryanodine (10⁻⁵ M), but was absent in Ca²⁺-free medium or in the presence of gadolinium (10⁻⁷M). The late or steady state [Ca²⁺]_i increase was observed in the presence of gadolinium, gallopamil, or ryanodine but was abolished in Ca²⁺-free medium. A steady-state increase in [Ca²⁺]_i was also evoked by “slow stretch” in which cells were slowly pulled to the final length within 1–2min. As the presence of external Ca²⁺ was indispensable, increased trans-sarcolemmal Ca²⁺ influx appears to be involved in both initial and steady-state increases in [Ca²⁺]_i. The initial increase in [Ca²⁺]_i after the “quick stretch” can be attributed to the activation of gadolinium-sensitive, stretch-activated channels.     

Delta cell secretory responses to insulin secretagogues are not mediated indirectly by insulin

Aims/hypothesis  

Somatostatin from islet delta cells inhibits insulin and glucagon secretion, but knowledge of the regulation of pancreatic somatostatin release is limited. Some insulin secretagogues stimulate somatostatin secretion, and here we investigated whether delta cell secretory responses are indirectly regulated in a paracrine manner by insulin released from beta cells.     

Ion transporters and cardiovascular diseases: pH control or modulation of intracellular calcium concentration

The regulation of the intracellular pH is under tight control by several ion transport systems including the sodium-proton exchanger, the sodium-bicarbonate cotransporter and the chlore-bicarbonate anion exchanger. While the activation of the anion exchange induces a cellular acidification, both the sodium-proton exchanger and the sodium-bicarbonate cotransporter are responsible for a protection against acidosis by extruding protons or importing bicarbonate. These transporters are transmembrane proteins whose activity is regulated by several mechanisms including phosphorylation, calcium binding and which are involved in several pathophysiologic processes such as ischemia, hypertrophy and arrhythmias. Recent studies suggest that the activation of these transporters during various diseases induces an increase in intracellular calcium concentration. Therefore, inhibiting these transporters could represent novel therapeutic strategies for the treatment of cardiovascular diseases.     

铝对大鼠海马细胞内钙水平和钙通道蛋白基因表达的影响

目的 观察铝对大鼠海马细胞内游离钙([Ca2+]i)水平和钙通道蛋白表达的影响.方法 以64只健康Wistar大鼠为研究对象,按体质量将大鼠分为16组,经口灌胃给予氯化铝(AlCl3),按AlCl3剂量(mg/kg)分为O(对照)、37.3、74.7、248.7组;染铝时间为45、75、120 d,其中120 d的大鼠再正常饲养30 d;分别在实验45、75、120、150 d处死大鼠,取脑分离海马.应用荧光分光光度计测定海马[Ca2+]i,应用RT-PCR方法检测海马中Ryanodine受体2(RyR2)和L-型钙通道α1C亚基(L-Ca2+α1C)mRNA表达情况.结果 AlCl3剂量和实验时间均可增加大鼠海马[Ca2+],(F值分别为23.136、19.089,P<0.01),二者具有交互作用(F=2.270,P<0.05);实验120、150 d时,大鼠海马[Ca2+]i,37.3、74.7、248.7 mg/kg组[(299.3±48.7)、(342.7±35.3),(391.2±47.9)、(408.1±42.8),(397.9±55.8)、(405.2±22.7)nmol/L]均较对照组[(195.1±29.9)、(209.1±30.6)nmol/L]明显升高(P<0.01).AlCl3可使大鼠海马RyR2、L-Ca2+α1C mRNA表达增加(F值分别为23.301、60.812,P<0.01).实验时间对大鼠海马中RyR2 mRNA表达无影响(F=1.361,P>0.05),但可下调L-Ca2+α1CmRNA的表达(F=6.088,P<0.01);AlCl3剂量和实验时间对L-Ca2+α1C mRNA表达有交互作用(F=5.876,P<0.01).实验75、120、150 d时,大鼠海马L-Ca2+α1C mRNA表达水平74.7、248.7 mg/kg组(1.03±0.16、1.18±0.18、0.92±0.11,1.89±0.26、1.25±0.10、1.07±0.14)均较同时间对照组(0.63±0.09、0.78±0.16、0.69±0.11)明显增加(P<0.05或<0.01).实验45、75、120、150 d时,大鼠海马RyR2 mRNA表达水平,74.7、248.7ms/kg组(0.49±0.06、0.51±0.07、0.57±0.11、0.47±0.11,0.47±0.03、0.52±0.09、0.70±0.10、0.78±0.09)均较同时间对照组组(0.24±0.07、0.32±0.04、0.30±0.06、0.27±0.06)明显升高(P<0.05或<0.01).结论 铝可以通过上调RyR2 mRNA和L-Ca2+α1C mRNA的表达而增加海马[Ca2+]i,发挥不可恢复性的神经毒性作用.     

Perturbation of intracellular calcium ion concentration in single rat granulosa cells by angiotensin II

A physiological role of angiotensin II (Ang II) on the ovary has been suggested recently, but the mechanism of action is not understood. In 18 out of 44 individual rat granulosa cells loaded with fura-2, Ang II caused a rapid and transient increase in intracellular free calcium ion concentration, [Ca2+]i. In 12 of these cells, 10(-5) M Ang II caused a 3.7 +/- 0.5-fold increase in [Ca2+]i. After 74 +/- 4 sec, [Ca2+]i returned to the resting levels (96.0 +/- 3.7 nM). Angiotensin I was without effect (n = 9). The effect of Ang II on [Ca2+]i changes could be completely blocked by a potent long-acting Ang II antagonist, [Sar1, Thr8]-angiotensin II, suggesting a receptor-mediated mechanism. The present results strongly indicate that rapid alterations in [Ca2+]i is an early step in the signal transduction of Ang II in a subpopulation of cells in the rat ovary.     

铝对大鼠海马细胞内钙水平和钙通道蛋白基因表达的影响

Objective To dynamically investigate the effects of aluminum on the concentration of free intracellular Ca2+([Ca2+]i) and the expression of calcium channels in the hippocampus of rats. Methods Healthy 64 Wistar rats were taken as the experimental objects. And these rats were randomly divided into 16 groups according to their weights, and were instilled with AlCl3 at 0(control),37.3,74.7 and 248.7 mg/kg respectively. The experimental time exposed to AlCl3 was 45,75,120 d, among which the rats were given AlCl3 for 120 d fed normally for 30 d. The hippoeampus were segregated on day 45,75,120 and 150 d and the[Ca2+]i of hippocampus of rats were detected by fluorospectrophotometer. The expression of Ryanodine receptor 2 (RyR2) mRNA and α1C ubunit of L-type calcium ehannels(L-Ca2+α1C) mRNA were detected by RT-PCR analysis. Results [Ca2+]i was increased by AlCl3 in a dose-and time-dependant manner(F=23.136 and 19.089, P<0.01). There was a synergistic effect between the dose and time in [Ca2+]i (F=2.270, P<0.05). In time of 120,150 days, the [Ca2+]i of rats hippocampus in 37.3[(299.3±48.7), (342.7±35.3)nmol/L], 74.7[(391.2±47.9), (408.1±42.8)nmol/L] and 248.7 mg/kg group[(397.9±55.8), (405.2±22.7)nmol/L] significantly increased compared with control group [(195.1±29.9), (209.1±30.6)nmol/L; P<0.01]. The expression of RyR2 mRNA and L-Ca2+α1C mRNA were increased by AlCl3(F=23.301 and 60.812, P<0.01). The experimental time could lower the expression of L-Ca2+ α1C mRNA (F=6.088, P<0.01), but had no influences on the expression of RyR2 mR NA (F=1.361, P>0.05). There was interaction between the dose of AlCl3 and the time in the expression of L-Ca2+α1C mRNA (F=5.876,P< 0.01). On day 75,120 and 150 of the experiment, the expression of L-Ca2+α1C mRNA in rat hippocampus of 74.7 (1.03±0.16,1.18±0.18,0.92±0.11) and 248.7 mg/kg group(1.89±0.26, 1.25±0.10, 1.07±0.14) also increased compared with control group(0.63±0.09,0.78±0.16,0.69±0.11; P<0.05 or <0.01). On day 45,75, 120 and 150 of the experiment, the expression of RyR2 mRNA in 74.7(0.49±0.06,0.51±0.07,0.57±0.11, 0.47±0.11), 248.7(0.47±0.03,0.52±0.09, 0.70±0.10, 0.78±0.09)mg/kg AlCl3 groups was highly increased compared with control group (0.24±0.07, 0.32±0.04, 0.30±0.06, 0.27±0.06; P<0.05 or<0.01). Conclusion Al increases [Ca2+]i by increasing the expression of the RyR2 mRNA and L-Ca2+α1C mRNA, thus exerts an irreversible neuronal toxicity.     

Mitochondrial responses to intracellular pulses of photosynthetic oxygen

When submitting anaerobic algal cells to a series of saturating flashes, transient absorption changes of mitochondrial origin were detected, showing the characteristic flash-number dependence of photosynthetic oxygen evolution. The faster kinetic event is the oxidation of heme a₃ of the cytochrome-c oxidase, which reaches a maximum at [unk]3.5 ms before again being reduced within 20 ms. The oxidation of cytochrome c involves an initial submillisecond lag, and its half-time is [unk]3.3 ms. Another component, probably indicating oxidation of heme a, is seen around 607 nm, with a kinetic behavior similar to that of cytochrome c. The fast time scale of these reactions excludes long-range diffusion and supports a direct intracellular trapping of O₂. It is estimated that, under appropriate conditions, the yield of this process is >30%. The linearity of these responses with respect to the amplitude of the oxygen pulse implies that a single turnover of the cytochrome oxidase is involved. These results suggest that the intracellular oxygen pathway may be of physiological importance in green algae. On the other hand, this technique seems promising both as an alternative to polarographic detection of photosynthetic oxygen and as a means of studying the cytochrome oxidase response in vivo to single-turnover oxygen pulses.     

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.