Caspases: their intracellular localization and translocation during apoptosis.

The activation of the caspase family of proteases has been detected in numerous cell systems and appears to function as a common pathway through which apoptotic mechanisms may operate. Caspases are synthesized as precursors (pro-caspases) and are converted into mature enzymes by apoptotic signals. The effects of caspases in apoptosis are accomplished by the cleavage of numerous proteins located in different intracellular compartments. In the present study we have addressed the question of the subcellular localization of different pro- and active caspases as well as several other proteins, such as Apaf-1, calpain and DFF, which also play important roles in the apoptotic process. We found that at least three pro-caspases (pro-caspases-2, -3 and -9) were present in both the mitochondrial and cytosolic fractions of untreated Jurkat T lymphocytes. Only pro-caspase-2 was found in the nuclear fraction. Pro-caspases-7 and -8 were found only in the cytosolic fraction. In apoptotic cells, caspases-3, -8 and -9 were present in the cytosolic fraction, whereas caspases-3 and -9 were also found in the mitochondrial fraction and caspase-7 in the microsomal fraction. Caspases-2 and -3 were present in the nuclear fraction. The selective localization of pro-caspases in different subcellular compartments may play an important, but yet unknown, role in their activation. The translocation of active caspases to other subcellular compartments appears to be critical for the development of the apoptotic process.

Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis.

The release of two mitochondrial proteins, cytochrome c and apoptosis-inducing factor (AIF), into the soluble cytoplasm of cells undergoing apoptosis is well established. Using spectrophotometric determination of enzyme activity, the accumulation of adenylate kinase (AK) activity in the cytosolic fraction of apoptotic cells has also been observed recently. However, three isozymes, AK1, AK2 and AK3, have been characterized in mammalian cells and shown to be localized in the cytosol, mitochondrial intermembrane space and mitochondrial matrix, respectively, and it is unknown which one of these isozymes accumulates in the cytosol during apoptosis. We now demonstrate that in apoptotic cells only AK2 was translocated into the cytosol concomitantly with cytochrome c. The amount of AK1 in cytosol, as well as the amount of matrix-associated AK3, remained unchanged during the apoptotic process. Thus, our data suggest that only intermembrane proteins are released from mitochondria during the early phase of the apoptotic process.

Two different proteases are involved in the proteolysis of lamin during apoptosis.

To investigate the involvement of different proteases in the execution step of apoptosis and to determine their intracellular location, isolated rat thymocyte nuclei were incubated either in the presence of Ca2+ and Mg2+ or with cytosolic extract from Jurkat T lymphocytes treated with anti-Fas (APO-1, CD-95) antibody. Inhibitors of caspases, VADcmk and DEVDcho, were not effective in hindering Ca2+-induced apoptotic changes in isolated nuclei, but did prevent similar changes in nuclei treated with the cytosolic extract from apoptotic Jurkat cells. In contrast, the inhibitor of the Ca2+-regulated, nuclear scaffold-associated serine protease, AAPFcmk, was able to inhibit lamin B1 breakdown, as well as chromatin cleavage in nuclei incubated in the presence of Ca2+ and Mg2+, but only partially prevented the same changes induced with cytosolic extract. Our findings provide evidence for the involvement of at least two proteases in lamin cleavage. One belongs to the caspase family and to cleave lamins this enzyme must be translocated from the cytoplasm into the nucleus. The second protease has a nuclear location and is activated by Ca2+. Finally, neither of these two lamin-cleaving proteases is responsible for the cleavage of another nuclear target protein, poly(ADP-ribose)polymerase (PARP), during apoptosis.

The role of proteolysis in T cell apoptosis triggered by chelation of intracellular Zn2+.

Our previous work showed that chelation of intracellular Zn2+ with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) induces apoptosis in rat thymocytes. The molecular mechanism involved in TPEN-triggered apoptosis remains unknown, except that it is a Ca2+-independent process. In the present study, we show that TPEN is unable to induce DNA fragmentation when added to isolated thymocyte nuclei, indicating that activation of a cytoplasmic component is essential for TPEN-induced apoptosis. Since cytosolic proteases related to interleukin-1beta-converting enzyme (ICE) are implicated as key activators of apoptosis in many different systems, we investigated the possible involvement of such proteases in TPEN-induced apoptosis. We found that treatment of thymocytes with TPEN caused an early degradation of nuclear poly(ADP-ribose) polymerase (PARP) and lamin prior to DNA cleavage. This could be inhibited by Z-Val-Ala-Asp-chloromethylketone (VADcmk), an inhibitor of ICE-like proteases, but not by an inhibitor of Ca2+-regulated serine protease. Jurkat T cells also underwent extensive DNA fragmentation when incubated with TPEN. A cytosolic fraction, prepared from TPEN-treated Jurkat cells, produced extensive DNA fragmentation when applied to isolated thymocyte nuclei, whereas the cytoplasmic extract from untreated cells was ineffective either alone or together with TPEN. The apoptosis-inducing activity in cytosolic fraction from TPEN-treated Jurkat cells was blocked by incubating cells in the presence of VADcmk or another inhibitor of ICE-like proteases, Ac - Asp - Glu - Val - Asp-aldehyde (DEVD-CHO), which has been found to competitively inhibit CPP32/apopain. An increase in enzyme activity that cleaves Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC), a fluorogenic substrate of CPP32/apopain and Mch3alpha, was detected in TPEN-treated thymocytes and Jurkat cells. In addition, the proteolytic cleavage of CPP32 resulting in the formation of two active fragments (p17 and p12) was observed in cytosolic extracts from TPEN-treated Jurkat cells, but not in extracts which were prepared from cells treated with TPEN in the presence of VADcmk or DEVD-CHO. Our results suggest that activation of cytosolic ICE-like proteases is an essential step in TPEN-induced apoptosis, and that CPP32/apopain is critically involved in this process.

Multiple proteases are involved in thymocyte apoptosis.

To investigate the involvement of proteases in apoptosis, rat thymocytes were treated with the glucocorticoid hormone methylprednisolone or the topoisomerase II inhibitor etoposide in the presence of selective substrate inhibitors of either interleukin-1 beta-converting enzyme (ICE), (Z-Val-Ala-Asp-chloromethylketone, VADcmk) or Ca(2+)-regulated serine protease (Suc-Ala-Ala-Pro-Phe-chloromethylketone, AAPFcmk). VADcmk protected from lamin proteolysis, chromatin fragmentation, cell shrinkage, and formation of apoptotic nuclei in both methylprednisolone- and etoposide-treated thymocytes when present during the initiation of the apoptotic process. AAPFcmk prevented lamin breakdown, chromatin fragmentation, and apoptotic morphological changes in thymocytes treated with methylprednisolone, but not with etoposide. Both MPS- and etoposide-treated thymocytes exhibited enhanced ICE-like protease activity which was maximal 1 h after treatment. This increase in proteolytic activity was blocked by VADcmk, but not AAPFcmk. Our findings suggest that ICE-like protease activity is critically involved in the early phase of both methylprednisolone- and etoposide-induced apoptosis in thymocytes, whereas the Ca(2+)-regulated serine protease is an obligatory component of the proteolytic cascade in methylprednisolone-induced apoptosis.

Two separate plasma membrane Ca2+ carriers participate in receptor-mediated Ca2+ influx in rat hepatocytes.

The plasma membrane Ca2+ carrier system involved in receptor-mediated Ca2+ entry was studied. Using the Ca2+ readdition protocol, the rate of cytosolic free Ca2+ concentration ([Ca2+]i) increase in vasopressin-pretreated hepatocytes was significantly higher than in thapsigargin- or 2,5-di(tert-butyl)hydroquinone-pretreated cells. The addition of Mn2+ to unstimulated hepatocytes resulted in a biphasic quench of fura-2 fluorescence. After an initial phase that was fast in rate but of short duration, the rate of fura-2 quench by Mn2+ became much slower and lasted until all the cellular fura-2 was quenched. Pretreatment of the cells with vasopressin only accelerated the rate of the latter phase but not of the initial one. In agonist-stimulated cells, acidification of the extracellular medium or the presence of ruthenium red, econazole or SK&F 96365 decreased the rates of both [Ca2+]i increase and Mn2+ entry upon addition of the respective cation. By contrast, neomycin and N-tosyl-L-phenylalanine chloromethyl ketone markedly decreased the rate of [Ca2+]i increase upon Ca2+ readdition but had no effect on vasopressin-stimulated Mn2+ entry. None of the treatments affected the ability of vasopressin and thapsigargin to mobilize the internal Ca2+ store. It is concluded that in hepatocytes the two pathways of receptor-mediated Ca2+ entry control two distinct yet pharmacologically related cation carriers.

Formation of 50 kbp chromatin fragments in isolated liver nuclei is mediated by protease and endonuclease activation.

Isolated rat liver nuclei were incubated in the presence of divalent cations, and the mechanisms underlying the subsequent chromatin fragmentation were investigated. Either of the two cations, Ca2+ or Mg2+ was sufficient to produce chromatin fragments with sizes between 700 and 300 kbp. The formation of chromatin fragments of 50 kbp as well as the following internucleosomal DNA cleavage--which are characteristic of apoptosis--were markedly stimulated in the presence of Ca2+. Chromatin degradation to 50 kbp and smaller (oligonucleosome-size) fragments was prevented by inhibitors of endonucleases and serine proteases. We suggest a mechanism whereby the concerted activity of both proteases and endonucleases results in the widespread chromatin cleavage observed in cells undergoing apoptosis.

Cyclic AMP stimulates Ca2+ entry in rat hepatocytes by interacting with the plasma membrane carriers involved in receptor-mediated Ca2+ influx.

The regulation of Ca2+ influx in rat hepatocytes by glucagon and cyclic AMP (cAMP) was investigated. Exposing hepatocytes to glucagon resulted in an increase in the initial rate of Ca2+ entry. The concentrations of glucagon producing half-maximal and maximal stimulation of Ca2+ entry were 10(-10) and 10(-8) M, respectively. A similar stimulation of Ca2+ influx was obtained in cells exposed to cAMP analogues or to forskolin. Exposing hepatocytes suspended in nominally Ca(2+)-free medium to glucagon for 3 min produced a 9% decrease in the size of the vasopressin-sensitive Ca2+ pool; in contrast, N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (Bt2cAMP) slightly augmented the size of this pool. Glucagon and Bt2cAMP synergized the initial vasopressin-stimulated Ca2+ and Mn2+ influx rates, but only moderately increased the initial rate of Ca2+ entry after thapsigargin addition. The glucagon- and Bt2cAMP-stimulated Ca2+ influx was inhibited by the same antagonists of the plasma membrane Ca2+ carriers that mediate Ca2+ entry during stimulation by vasopressin. Thus, cAMP does not stimulate Ca2+ entry through either a capacitative type of mechanism or inositol phosphate turnover. The authors' findings instead suggest that cAMP acts directly, or through protein kinase A on the same Ca2+ carriers that are activated by phospholipase C-linked receptor agonists.

Eicosanoids released following inhibition of the endoplasmic reticulum Ca2+ pump stimulate Ca2+ efflux in the perfused rat liver.

In the isolated perfused rat liver 2,5-di(tert-butyl)hydroquinone (tBuHQ), a selective inhibitor of the endoplasmic reticulum Ca2+ pump, induces a prolonged glucose output and stimulates Ca2+ efflux. The present study shows that tBuHQ depleted the hormone-sensitive Ca2+ pool in the perfused liver, abolishing the vasopressin- or phenylephrine-induced Ca2+ efflux. The effects of tBuHQ were reversible, since the response to these agonists gradually returned within 1 hr of perfusion, and protein synthesis was not required for this recovery. Since tBuHQ does not cause Ca2+ efflux from isolated hepatocytes, we examined the mechanism responsible for the tBuHQ-induced Ca2+ efflux observed in the intact liver. The cyclooxygenase inhibitor indomethacin prevented the Ca2+ extrusion stimulated by tBuHQ, but not that induced by vasopressin. During infusion of tBuHQ there was a 9-fold increase in the concentration of thromboxane B2 in the perfusate. The Ca2+ efflux response to tBuHQ was inhibited by the thromboxane/prostaglandin endoperoxide receptor antagonist, L-655,240 (3-[1-(4-chlorobenzyl)-5-fluoro-3-methyl-indol-2-yl]2,2-dimethylpropa noic acid) in the absence of any effect on thromboxane B2 release. Thus, the inhibition of the endoplasmic reticulum Ca2+ pump by tBuHQ results in a rise in the cytosolic Ca2+ concentration in non-parenchymal cells, leading to the formation of cyclooxygenase products. The released eicosanoids, in turn, stimulate Ca2+ efflux from hepatocytes.

Evidence for two pathways of receptor-mediated Ca2+ entry in hepatocytes.

Receptor-mediated Ca2+ entry was studied in fura-2-loaded isolated hepatocytes. Emptying of internal Ca2+ stores by treatment with either the Ca(2+)-mobilizing hormone vasopressin or the inhibitors of the microsomal Ca2+ pump, 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ) or thapsigargin, stimulated Ca2+ entry, as indicated by a rise in the cytosolic free Ca2+ concentration after Ca2+ was added to cells suspended in nominally Ca(2+)-free medium. The enhancement of Ca2+ entry was proportional to the degree of depletion of the intracellular Ca2+ pool and occurred also after removal of vasopressin from its receptor. In contrast, the stimulation of Mn2+ entry by vasopressin required the continuous presence of the agonist, since it was prevented by the addition of vasopressin receptor antagonist. This effect was observed under conditions where refilling of the agonist-sensitive pool was prevented by using nominally Ca(2+)-free medium. Unlike vasopressin, tBuBHQ or thapsigargin did not stimulate Mn2+ entry. These results suggest the existence of two pathways for receptor-mediated Ca2+ entry in hepatocytes, a 'capacitative' pathway that is sensitive to the Ca2+ content in the Ins(1,4,5)P3-sensitive Ca2+ pool and does not allow Mn2+ entry, and a second pathway that depends on receptor occupation, seems to require a second messenger for activation, and permits influx of Mn2+.

Comparison between the effects of the microsomal Ca(2+)-translocase inhibitors thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone on cellular calcium fluxes.

The effects of two inhibitors of the microsomal Ca(2+)-ATPase, thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone, were compared in hepatocytes and in a T-cell line (JURKAT). Both compounds mobilized the same intracellular Ca2+ pool, which contained the Ins(1,4,5)P3-sensitive store, in hepatocytes and in JURKAT cells. The mobilization of the internal Ca2+ store with either compound activated Mn2+ entry in JURKAT cells, but not in hepatocytes. This suggests different properties of the bivalent-cation entry pathway between these cell types.

Mobilization of the hormone-sensitive calcium pool increases hepatocyte tight junctional permeability in the perfused rat liver.

Hepatocyte tight junctional permeability has been shown to be regulated by hormones that exert their effects via phospholipase C activation. However, the precise transduction pathway involved in this effect is not known. The present study has employed the selective inhibitor of microsomal Ca2+ sequestration, 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), to examine the effect of the mobilization of the endoplasmic reticular Ca2+ pool on tight junctional permeability in the perfused rat liver. Infusion of tBuBHQ followed by a bolus infusion of horseradish peroxidase (HRP) resulted in a significant increase in the first peak of biliary HRP, a measure of junctional permeability, whereas transcellular (vesicular) transport of HRP was not affected. Therefore, we conclude that the effect of hormones on tight junctional permeability is mediated, at least in part, by the mobilization of intracellular Ca2+.

Release of Ca2+ from the endoplasmic reticulum is not the mechanism for bile acid-induced cholestasis and hepatotoxicity in the intact rat liver.

The hypothesis that monohydroxy bile acids exert their cholestatic and hepatotoxic effects via a sustained elevation of cytosolic [Ca2+] was tested in the isolated perfused rat liver. Infusion of the specific inhibitor of microsomal Ca2+ sequestration, 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) (25 microM for 10 min) produced efflux of Ca2+ from the liver and a sustained (20 min) increase in cytosolic [Ca2+] as indicated by the threefold increase in hepatic glucose output. Release of the endoplasmic reticular Ca2+ pool was demonstrated by the complete abolition of vasopressin- and phenylephrine-induced Ca2+ exchange between the liver and perfusate. Despite the profound perturbation of intracellular Ca2+ homeostasis produced by tBuBHQ, there was no decrease in bile flow and no evidence of hepatocellular injury (for 60 min), as indicated by lactate dehydrogenase release. In contrast, lithocholic acid (25 microM for 10 or 30 min) or taurolithocholic acid (5 microM for 10 or 30 min) produced an 80-90% inhibition of bile flow and a progressive increase in perfusate lactate dehydrogenase activity. During and after bile acid infusion, there was no change in Ca2+ fluxes between liver and perfusate, no stimulation of glucose output from the liver, and hormone-stimulated Ca2+ responses were preserved. It is concluded that the mechanisms for bile acid-induced cholestasis and hepatotoxicity in the intact liver are not attributable to changes in intracellular Ca2+ homeostasis, and especially not to prolonged release or depletion of Ca2+ sequestered in the endoplasmic reticulum.