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Liposomes are of great interest as drug delivery vehicles, and studies have focused on understanding how the physical and chemical characteristics of liposomes can be modified to improve their in vivo behavior. In a previous study, we found that the slightly negatively-charged liposomes aggregate only in the culture medium of human umbilical vein endothelial cells, whereas the liposomes modified with polyethylene glycol (PEG) (PEGylated) did not aggregate. In the present study, we investigated the underlying mechanism of this phenomenon. Firstly, it was found that heparin in the culture medium is one of the factors that cause aggregation of the non-PEGylated liposomes. Since the addition of ethylenediaminetetraacetic acid (EDTA) prevented the aggregation, metal ions, such as Ca(2+) and Mg(2+), in the culture medium could also be important in driving the aggregation. In the presence of heparin, higher concentrations of Ca(2+) or Mg(2+) increased the particle size of the non-PEGylated liposomes, although no change in the particle size of PEGylated liposomes was observed. Under conditions in which aggregation occurred, we measured the binding and uptake of liposomes by macrophages in vitro. The binding and uptake of non-PEGylated liposomes were significantly increased with increasing Ca(2+) concentrations, whereas those of PEGylated liposomes were unchanged. While the formation of aggregations of cationic or anionic liposomes has been reported previously, there are few reports addressing the aggregation of slightly negatively-charged or neutral liposomes. Thus, our data provide useful insights on the effect of PEGylation on liposomal aggregation and in vivo behavior.

The waveform of the myocardial action potential (AP) triggering contraction differs among the species, developmental stage, and pathological state. The species difference in heart rate, which inversely correlates with body size, originates in the ion-channel mechanisms responsible for diastolic depolarization of the sinoatrial node. In some cases, such as the chronically AV-blocked dog and 11- to 13-day chick embryo, the repolarization reserve is decreased making the heart useful for drug evaluation. The degree of dependence of contraction on sarcoplasmic reticulum (SR) function increases during development. The large SR dependence and short AP of the adult mouse and rat support their rapid contraction under high heart rate. The function of the Na(+)/Ca(2+) exchanger is affected by AP waveform and ion concentrations; its major role is Ca(2+) extrusion, but under pathological conditions such as ischemia-reperfusion, it allows Ca(2+) influx and leads to myocardial injury, including loss of mitochondrial function. The role of mitochondria in ATP supply is less in the fetus where glycolysis plays a greater role. The pharmacological properties of the myocardium are affected by all of these factors and also by autonomic innervation and the hormonal status. Such comprehensive understanding is indispensable for the development of novel therapeutic strategies.

We examined the source of Ca(2+) involved in the volume regulation of Madin-Darby canine kidney (MDCK) cells with confocal microscopy and fluoroprobes. Hyposmosis induced a transient increase in cell volume, as well as cytoplasmic Ca(2+), which peaked at 3 to 5 min and gradually decreased to reach the initial value within about 30 min. This late decrease in cell volume, as well as the transient rise in cytoplasmic Ca(2+), was reduced in Ca(2+)-free solution and was abolished by pretreatment with thapsigargin. In conclusion, Ca(2+) released from the intracellular store contributes to the regulatory volume decrease following hyposmotic swelling in MDCK cells.

The cardioprotective effects of SEA0400, a novel Na(+)-Ca(2+) exchanger inhibitor, were examined in isolated guinea pig myocardial tissue and ventricular myocytes. In a coronary-perfused right ventricular tissue preparation, SEA0400 had no cardiosuppressive effect during normoxia and experimental ischemia, but enhanced the recovery of contractile force during reperfusion. SEA0400 had no effect on tissue ATP content during normoxia, but attenuated its decrease during ischemia. Treatment of ventricular myocytes with an ischemia mimetic solution (high K(+), glucose free, pH 6.0, gassed with N(2)) resulted in the depolarization of the mitochondrial membrane potential and an increase in cytoplasmic and mitochondrial Ca(2+) concentration, which had a similar time course. SEA0400 significantly delayed these changes. These results suggest that SEA0400 maintains mitochondrial function and tissue ATP content during ischemia through the inhibition of cytoplasmic and mitochondrial Ca(2+) overload.

Rapid-scanning cofocal microscopy has been applied to the analysis of early phase Ca(2+) transients in ventricular and atrial cardiomyocytes. On electrical stimulation of ventricular myocytes, Ca(2+) concentration begins to rise earliest at the Z-line level and becomes uniform throughout the cytoplasm within about 10 ms after the onset of the action potential; transsarcolemmal Ca(2+) influx triggers Ca(2+) release from release sites on the junctional sarcoplasmic reticulum (SR) coupled to T-tubules at the Z-line throughout the cytoplasm. In atrial myocytes lacking the T-tubular network, transsarcolemmal Ca(2+) influx during an action potential triggers SR Ca(2+) release only at subsarcolemmal region. SR Ca(2+) release then spreads towards the central region of the cell through a propagated Ca(2+)-induced-Ca(2+) release mechanism. The atrio-ventricular difference in excitation-contraction coupling mechanisms underlies some of the atrio-ventricular difference in response to physiological and pharmacological stimuli.

We previously observed Ca2+ release from intracellular Ca2+ stores caused by reduction in extracellular Na+ concentration ([Na+]o). The purpose of this study was to determine whether lowering [Na+]o can elicit Ca2+ release from Ca2+ stores via the Na+/Ca2+ exchanger and to elucidate the mechanisms related to the Ca2+ release pathway in cultured longitudinal smooth muscle cells obtained from guinea pig ileum. Low [Na+]o-induced Ca2+ release was inhibited by antisense oligodeoxynucleotides for Na+/Ca2+ exchanger type 1 (anti-NCX). Application of anti-NCX to cells attenuated both the number of Ca2+ responding cells and the expression of the exchanger. Moreover, microinjection of heparin, a blocker of inositol 1,4,5-trisphosphate (IP3) receptors, into the cells inhibited low [Na+]o-induced Ca2+ release. These findings suggest that low [Na+]o-induced Ca2+ release occurs through an IP3-induced Ca2+ release mechanism due to changes in the Ca2+ flux regulated by the Na+/Ca2+ exchanger.

The effects of tri-n-butyltin chloride (TBT), an environmental pollutant, on the release of Ca(2+) from intracellular stores were investigated in isolated rat hepatocytes. Isolated hepatocytes permeabilized with digitonin were suspended in solution, and the concentration of extracellular Ca(2+) was measured, using a fluorescent Ca(2+) dye, fura-2. In the solution containing permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min, the extracellular Ca(2+) concentration was high, but the inositol 1,4,5-trisphosphate (InsP(3))-induced increase in Ca(2+) concentration was suppressed, suggesting that the extracellular release of Ca(2+) in response to TBT treatment was from intracellular stores. Images of the Ca(2+) concentration in the intracellular stores of primary cultured hepatocytes loaded with fura-2 were obtained after digitonin-permeabilization, using digitalized fluorescence microscopy. The permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min had a very low fura-2 fluorescence ratio (340/380 nm), suggesting that stored Ca(2+) was released. When the hepatocytes were treated with 4.0 microM TBT after digitonin-permeabilization, the decrease in the fura-2 fluorescence ratio was very small. However, when the permeabilized hepatocytes were incubated with 4.0 microM TBT and 2.0 microM NADPH, the decrease was enhanced, raising the possibility that TBT might be metabolized to the active form(s), thus releasing Ca(2+) from intracellular stores. When the hepatocytes were preincubated with 0.1 microM TBT for 30 min and then were permeabilized, the fura-2 fluorescence ratio was almost the same as that in the control permeabilized hepatocytes. However, the InsP(3)-induced decrease in the fluorescence ratio was suppressed significantly in the permeabilized hepatocytes. These results suggest that TBT released Ca(2+) from the intracellular stores at high concentrations, and suppressed the InsP(3)-induced Ca(2+) release at non-toxic low concentrations. It is probable that the latter effect was responsible for the previously reported suppression of Ca(2+) response induced by hormonal stimulations (Kawanish et al., Toxicol Appl Pharmacol 1999;155:54-61).

Recently, the technique for high time- and spatial-resolution imaging of intracellular calcium ion concentration ([Ca2+]i) has been developed using rapid scanning confocal microscopy to investigate calcium dynamics in restricted areas of cells. Here, the techniques are summarized and the images obtained are introduced. For such imaging, the development of fast scanning confocal microscopes was indispensable. Therefore, the UV-applicable video-rate (30 frames/sec) scanning confocal microscope in which a resonant glavanometer scanner is used for fast horizontal scans has been developed. The microscope enabled us to obtain ratio-images of [Ca2+]i using indo-1, a ratio-imaging probe. The key to success in high time- and temporal-resolution imaging is to find the best probe-loading condition, which depends on the probes and the cells. It is also very important to increase the signal-to-noise ratio value without any effects on the cells. Using the technique, we have succeeded in kinetic analysis of calcium waves in cultured hepatocytes. We have also succeeded in determining the relationship between calcium sparks to calcium transients in excitation-contraction coupling.

1. Line-scan analyses of spontaneous Ca2+ sparks, non-propagating local rises in Ca2+ concentration, and the early phase of Ca2+ transients in cardiomyocytes were performed with a rapid-scanning laser confocal microscope (Nikon RCM8000) and fluo-3. 2. On electrical stimulation, points at which rise in Ca2+ began earliest were observed at regular spacings of 1.82 +/- 0.26 micron (mean +/- S.D.) along the longitudinal axis of the cell. The points were heavily stained with di-2-ANEPEQ, which stains the T-tubules, indicating that they were at the Z-line. 3. The points where spontaneous Ca2+ sparks originated coincided with the points which showed faster Ca2+ elevation, i.e. the Z-line. 4. In some cases where a Ca2+ spark had occurred within about 30 ms before the evoked Ca2+ transient, fast elevation of Ca2+ was not observed at the corresponding Z-line, indicating the presence of a refractory period in Ca2+ release from the SR. 5. The present results provide visual evidence for Ca2+ release from the junctional sarcoplasmic reticulum in cardiomyocytes. The presence of a refractory period in Ca2+ release after Ca2+ sparks provided new evidence that the normal Ca2+ transient may be the summation of Ca2+ sparks.

We investigated changes in intracellular pH (pHi) in relation to intracellular Ca2+ concentration ([Ca2+]i) in primary cultured hippocampal neurons treated with glutamate. [Ca2+]i and pHi were imaged with fluorescent dyes and confocal microscopy. Exposure to 1 mM glutamate for 10 min increased [Ca2+]i and evoked acidosis. These changes persisted for at least 60 min, even after removal of glutamate. The increase in [Ca2+]i and the acidosis were not observed in Ca2+-free solution and were attenuated in the presence of MK-801, an NMDA receptor antagonist. We also found that the increase in [Ca2+]i and acidosis could be induced by addition of Ca2+ to the extracellular solution in the cells pretreated with glutamate in Ca2+-free solution, even if glutamate did not exist in the extracellular solution. On the other hand, ionomycin and Br-A23187, calcium ionophores, increased [Ca2+]i to almost the same level as glutamate and increased pHi. Extracellular Ca2+ was also indispensable for the increase in [Ca2+]i and the alkalosis. These results suggest the followings: 1) intracellular acidosis by glutamate is dependent on the presence of extracellular Ca2+; 2) the acidosis does not result from only the increase in [Ca2+]i; and 3) glutamate induces the irreversible disorder of regulatory mechanisms of [Ca2+]i not only by Ca2+-dependent process, but also by Ca2+-independent process.

Two dimensional images of myocardial Ca2+ sparks, non-propagating local rises in cytoplasmic Ca2+ concentration, were obtained at 4 msec intervals with a rapid-scanning confocal laser microscope, Nikon RCM 8000, and fluo-3. Spontaneous Ca2+ sparks were observed at apparently random sites throughout the cytoplasm of rat ventricular cells. The duration of sparks was 30 to 40 msec and the time to peak intensity about 10 msec. Ryanodine (1 microM) completely inhibited Ca2+ sparks while nicardipine (3 microM) had no effect. Isoproterenol (1 microM) had no effect on the frequency and distribution of Ca2+ sparks but significantly increased their amplitude. These results suggest that myocardial Ca2+ sparks are the result of spontaneous release of Ca2+ from the sarcoplasmic reticulum and that beta-adrenergic stimulation may result in functional modification of the ryanodine receptor channel.

The target cell-induced calcium response in natural killer (NK) cells was examined using indo-1 (a ratiometric fluorochrome), a newly developed video-rate ultraviolet laser-scanning confocal microscope system, and a human NK leukemia cell line, YTN. Susceptible target cells (JY cells), but not unsusceptible target cells (K562 cells), increased the intracellular calcium concentration ([Ca]i) in almost all indo-1-loaded YTN cells with a lag time, although the kinetics of increase in [Ca]i differed in individual YTN cells. The increase in [Ca]i was completely blocked by 2 mM EGTA and partially (around 50%) blocked by 10 microM nicardipine, the extents of inhibition of calcium response correlating well with those of inhibition of cytotoxic activity. Thus, the calcium response in YTN cells observed in this study may constitute a critical step in the manifestation of cytotoxic activity.

Two-dimensional images of intracellular free Ca2+ movements in cultured cardiac myocytes were obtained at 33-ms intervals with a Ca(2+)-sensitive fluorescence probe, fluo-3, and a rapid scanning confocal laser microscope, a prototype of Nikon RCM8000. The cells used were isolated from the ventricular myocardium of neonatal mice, cultured for approximately 72 h and loaded with fluo-3. One type of cytoplasmic Ca2+ movement observed was a simultaneous increase in [Ca2+] throughout the cytoplasm, termed a "spike"; another type was a local increase in [Ca2+] propagating in the cytoplasm, termed a "wave." Cells with either spike or wave or both types of movements were observed. Tetrodotoxin (TTX) 10(-5) M, nicardipine 10(-6) M, and increased extracellular potassium concentration (40 mM) selectively inhibited spike, and ryanodine 10(-6) M and cyclopiazonic acid (CPA) 3 x 10(-6) M selectively inhibited wave. These results indicate that spike was triggered by depolarization-induced Ca2+ influx across the sarcolemma, whereas wave was a propagating local increase in Ca2+ due to Ca2+ release from the sarcoplasmic reticulum (SR). On spike, nuclear [Ca2+] was shown to increase and decrease synchronously with cytoplasmic [Ca2+], with a delay and slower time course.

Two-dimensional images of cytoplasmic and nuclear free Ca2+ movements in cardiac myocytes were obtained at 67-msec intervals using a Ca(2+)-sensitive fluorescence probe, indo-1, and a rapid scanning confocal laser microscope, Nikon RCM8000. Isolated guinea pig ventricular cells were loaded with indo-1 and stimulated at 0.5 Hz through patch pipettes. On stimulation, nuclear Ca2+ concentration ([Ca2+]) was observed to rise and fall following cytoplasmic [Ca2+] with an obvious delay. Application of isoproterenol significantly increased the peak [Ca2+] on stimulation in both the cytoplasm and nucleus with no substantial change in the basal [Ca2+]; the increase in peak [Ca2+] produced by application of isoproterenol was larger in the cytoplasm than in the nucleus. Under a low [Na+] condition, the basal [Ca2+] was increased from the control values in both the cytoplasm and nucleus; no difference in basal [Ca2+] was observed between the two regions. The increase in peak [Ca2+] by low [Na+] in the cytoplasm was significantly larger than that in the nucleus. When the cells were voltage clamped at 0 mV for 3 sec, no difference in the steady state [Ca2+] was observed between the cytoplasm and nucleus. Nuclear [Ca2+] was also observed to increase following a Ca2+ wave, a local increase in [Ca2+] propagating within the cytoplasm, with a delay. Thus, we demonstrated in isolated myocardial cells that cytoplasmic Ca2+ movements, although hampered by the nuclear envelope, are propagated into the nucleus, a mechanism through which factors affecting cytoplasmic Ca2+ may influence intranuclear events.

The mechanism of increase in intracellular Ca2+ concentration ([Ca2+]i) by removal of extracellular Na+, which phenomena were reported previously (Japan. J. Pharmacol. 63 83-91 1993), was investigated in cultured guinea pig ileum longitudinal muscle cells loaded with a fluorescent Ca2+ indicator, fura-2, by digital ratio imaging microscopy. Isotonic substitution of choline chloride for NaCl induced a transient increase in [Ca2+]i. The pretreatment of thapsigargin (0.5 microM), but not nicardipine (10 microM), suppressed the transient increase completely. In solutions containing micromolar concentrations of free Ca2+ (nominally Ca2+-free solution), the Na+-free induced transient increase was observed, but neither the second cell exposure to the Na+-free solution nor the following application of histamine increased [Ca2+]i, indicating that removal of extracellular Na+ releases Ca2+ from intracellular stores including inositol 1,4,5-trisphosphate (IP3)-releasable pools. The Na+-free induced transient increase required the presence of more than micromolar concentrations of extracellular free Ca2+ and releasable Ca2+ within the stores, but ryanodine did not affect the transient increase. These results suggest that undetectable influx of Ca2+ by the reverse-mode action of the Na+/Ca2+ exchanger can release Ca2+ from the thapsigargin-sensitive intracellular stores including IP3-releasable pools.

Cytosolic Ca2+ transients induced by hepatocyte growth factor (HGF) were imaged in primary cultured rat hepatocytes using newly developed rapid scanning confocal microscopes and indo-1. HGF (40 ng/ml) increased cytosolic free Ca2+ concentration ([Ca2+]i) in about 60% of hepatocytes, in 45% of which the increases were oscillatory. In each of the oscillatory hepatocytes, the repetitive increases in [Ca2+]i originated from a specific same region adjacent to the cell membrane and propagated across the cell like waves. Phenylephrine (10 microM) also induced Ca2+ waves. The locus where HGF-induced Ca2+ waves and phenylephrine-induced Ca2+ waves were originated was the same, and there was a correlation in the peak height between HGF-induced Ca2+ waves and phenylephrine-induced Ca2+ waves in each cell, although the mechanisms of inositol 1,4,5-trisphosphate (ins(1,4,5)P3) formation induced by HGF should be different from those by phenylephrine. On the other hand, there was no correlation between sensitivity of each cell to HGF and that to phenylephrine which were measured as latent periods prior to Ca2+ rises after an addition of the agonists. These results suggested the following: the spatial patterns of Ca2+ waves were decided by a common mechanism, probably not the propagation of ins(1,4,5)P3 but the distribution of ins(1,4,5)P3-sensitive Ca2+ pools; sensitivities of each cell to the agonists did not mainly depend on the common mechanism.

ATP and thrombin both induced Ca2+ mobilization from intracellular Ca2+ store site of megakaryocyte, the progenitor cell of platelet (Uneyama C., Uneyama H. and Akaike N. (1993) J. Physiol. (Lond.), 470, 73-749). Since in platelet, thrombin is known as a strong agonist and ADP is known as a weak agonist, we further investigated the effect of these agonists on megakaryocyte. Thrombin induced Ca2+ mobilization, 5-hydroxy tryptamine (5-HT) release and aggregatory morphological changes in megakaryocyte, but ATP induced only Ca2+ mobilization. Thrombin-induced 5-HT release was inhibited by adenylate cyclase-activating drugs, and the morphological changes could be induced by H-8, an inhibitor of cAMP-dependent protein kinase. These results suggest that the Ca2+ mobilization is not sufficient to induce morphological changes, and the signal to cause morphological changes in megakaryocyte may be cAMP.

The relationship between spontaneous Ca2+ oscillations and cell proliferation was investigated in cultured longitudinal muscle cells from guinea pig ileum. BAPTA, caffeine, thapsigargine and La3+ suppressed the spontaneous Ca2+ oscillations and the DNA synthesis at 5.0 mM, 10.0 mM, 15.62 nM and 0.5 mM, respectively, whereas verapamil, nicardipine and ryanodine did not suppress either. However, BAPTA and La3+ did not inhibit the DNA synthesis at 1.25 mM and 0.13 mM, but did suppress the Ca2+ oscillations even at 1.0 mM and 0.1 mM, respectively. These results show that the spontaneous Ca2+ oscillations are not prerequisite for the cell proliferation.

The cytoplasmic free calcium ion concentration ([Ca2+]i) of cultured guinea pig ileum longitudinal muscle cells loaded with a fluorescent [Ca2+]i indicator, fura-2, was measured by digital ratio imaging microscopy. Spontaneous [Ca2+]i oscillations were observed in 25% to 80% of the cells, which differed with the batches of the cultured cells after 5 to 8 days in culture. The frequency and amplitude of the [Ca2+]i oscillations in each individual cell were usually regular, but heterogeneity between neighboring cells was observed. The spontaneous [Ca2+]i oscillations were also observed even after incubation of the cells under a serum-free condition for 72 hr. Exchange of extracellular solution to Ca(2+)-free solution containing EGTA or BAPTA immediately stopped the [Ca2+]i oscillations. The ratio of the oscillating cells was dependent on the extracellular calcium ion concentration ([Ca2+]o); and heterogeneity in the range of the [Ca2+]o to generate the [Ca2+]i oscillations was observed. An inorganic Ca(2+)-antagonist, LaCl3, immediately suppressed the [Ca2+]i oscillations, but the treatment with verapamil or nicardipine, Ca(2+)-channel blockers, did not have any effect on the [Ca2+]i oscillations. An inhibitor of the intracellular Ca2+ pump, thapsigargin, induced a transient increase in [Ca2+]i and then inhibited the spontaneous [Ca2+]i oscillations. Neomycin, a compound known to inhibit phosphoinositide turnover, inhibited the [Ca2+]i oscillations.(ABSTRACT TRUNCATED AT 250 WORDS)

1 alpha,25-Dihydroxy-21-norvitamin D3 (3) was synthesized from 1 alpha-hydroxydehydroepiandrosterone (4). Certain biological properties of 3 were examined in comparison with those of 1 alpha,25-dihydroxyvitamin D3 (1) and 1 alpha,25-dihydroxy-21-nor-20-oxavitamin D3 (2) to evaluate the effect of the 21-methyl substituent on biological activities. The differentiation-inducing activity of 3 towards human myeloid leukemia cells was approximately one-fifth of that of 1, while in the binding affinity with chick intestinal cytosolic receptor, 3 was about one-tenth of that of 1. The rather weak effect of 3 on serum calcium levels in normal mice at a dosage of 500 micrograms/kg (intravenous administration) indicates that the essential importance of the 21-methyl moiety may lie in its effect on the regulation of calcium metabolism.

The model of "chemical hypoxia" with KCN plus iodoacetic acid mimics the ATP depletion and reductive stress of hypoxia. Here, we examined the effects of chemical hypoxia on cytosolic free Na+ and Ca2+ in single cultured rat hepatocytes by multiparameter digitized video microscopy and ratio imaging of sodium-binding furan indicator (SBFI) and Fura-2. Intracellular Na+ increased from about 10 mM to more than 100 mM after 20 min of chemical hypoxia, whereas cytosolic free Ca2+ remained virtually unchanged. In normoxic hepatocytes, phenylephrine (50 microM) and Arg-vasopressin (20-40 nM) induced Ca2+ oscillations in 70 and 40% of cells, respectively. These Ca2+ oscillations were suppressed after one spike following the onset of chemical hypoxia. Phenylephrine and vasopressin also increased inositol phosphate formation by 22 and 147%, respectively. This effect was suppressed by KCN plus iodoacetate. Intracellular acidosis is characteristic of chemical hypoxia. Intracellular acidosis induced by 40 mM Na-acetate suppressed Ca2+ oscillations but did not inhibit hormone-induced inositol phosphate formation. Cytosolic alkalinization also suppressed Ca2+ oscillations. However, prevention of intracellular acidosis with monensin (10 microM) did not prevent suppression of Ca2+ oscillations during chemical hypoxia. Mitochondrial depolarization with uncoupler did not change free Ca2+ levels during chemical hypoxia, indicating that mitochondria do not regulate free Ca2+ during chemical hypoxia. From these results, we conclude: 1) chemical hypoxia does not block Na+ influx across the plasma membrane; 2) Chemical hypoxia inhibits hormone-stimulated Ca2+ flux pathways across cellular membranes by two different mechanisms: (a) by ATP depletion, which disrupts hormone-myo-inositol 1,4,5-triphosphate coupling, and (b) by intracellular acidosis, which inhibits myo-inositol 1,4,5-triphosphate-stimulated Ca2+ release from intracellular stores; 3) during ATP depletion by chemical hypoxia, mitochondria do not take up Ca2+ to maintain cytosolic free Ca2+ at low concentrations.

The critical events that lead to the transition from reversible to irreversible injury remain unclear. Studies are reviewed that have suggested that a rise in cytosolic free Ca2+ initiates plasma membrane bleb formation and a sequence of events that leads ultimately to cell death. In recent studies, we have measured changes in cytosolic free Ca2+, mitochondrial membrane potential, cytosolic pH, and cell surface blebbing in relation to the onset of irreversible injury and cell death following anoxic and toxic injury to single hepatocytes utilizing multiparameter digitized video microscopy (MDVM). MDVM is an emerging new technology that permits single living cells to be labeled with multiple probes whose fluorescence is responsive to specific cellular parameters of interest. Fluorescence images specific for each probe are collected over time, and then digitized and stored. Image analysis and processing then permits quantitation of the spatial distribution of the various parameters within the single living cells. Our results indicate the following: (1) formation of plasma membrane blebs accompanies all types of injury in hepatocytes; (2) cell death is a rapid event, initiated by rupture of a plasma membrane bleb, and is coincident with the onset of irreversible injury; (3) an increase of cytosolic free Ca2+ is not the stimulus for bleb formation or the final common pathway leading to cell death; (4) a decrease of mitochondrial membrane potential precedes loss of cell viability; (5) cytosolic pH falls by more than 1 pH unit during chemical hypoxia. This acidosis protects against the onset of cell death.

Isolated rat hepatocytes were loaded with the Ca2+ indicator fura-2 to measure cytosolic free Ca2+ concentrations ([Ca2+]i) in individual cells by digital ratio imaging microscopy. Stimulation with 0.1 nM vasopressin, 0.5 microM phenylephrine, or 0.5 microM ATP caused repetitive spikes of high [Ca2+]i in a high percentage of cells, in agreement with Woods et al. (Woods, N. M., Cuthbertson, K. S. R., and Cobbold, P. H. (1986) Nature 319, 600-602), but unlike the results of Monck et al. (Monck, J. R., Reynolds, E. E., Thomas, A. P., and Williamson, J. R. (1988) J. Biol. Chem. 263, 4569-4575). Reduction in extracellular [Ca2+] decreased the frequency but not the amplitude of the spikes, suggesting that the spikes result from dumping of intracellular stores and that the entry of extracellular Ca2+ affects only the rate of replenishment of those stores. Membrane depolarization failed to elevate [Ca2+]i and had an effect similar to removal of extracellular Ca2+ in decreasing the frequency of agonist-evoked [Ca2+]i oscillations or inhibiting them altogether, arguing against any significant role for voltage-operated Ca2+ channels.