Release from the Endoplasmic Reticulum and Entry from the Extracellular Medium

In classical Ca2+ signal transduction, Ca2+ release from the endoplasmic reticulum is often followed by entry from the extracellular space according to the capacitative mechanism explained earlier (Section I,B 2). After treatment with reactive oxygen species, particularly hydroperoxides, several cell types have shown a response consisting of a transient increase in the [Ca2+]c that may be followed by a more permanent increase when the oxidative treatment reaches a threshold. In most cases, release from the endoplasmic reticulum and entry from the extracellular space have been found to be responsible for the transient and permanent increases, respectively. It has been shown that release from the endoplasmic reticulum may be due to decreased Ca2+ ATPase activity or to increased outflow through Ca2+ channels. Entry from the extracellular space may be due to capacitative entry in response to the depletion of the endoplasmic reticulum Ca2+ pool, but it also may be due to inhibition of plasma membrane Ca2+ ATPases or the Na+/Ca2+ exchanger.

A transient increase followed by a permanent elevation in the [Ca2+]c has been found in several cell types treated with reactive oxygen species and lipid peroxidation products (Fig. 2). The transient increase may occur in a matter of seconds and is due to release of Ca2+ from the IP3-sensitive Ca2+ pool. On the other side, the permanent increase proceeds gradually and may go on for many minutes, and is due to entry from the extracellular medium. The intracellular Ca2+ concentration rises from the basal level of 0.1-0.2 X 10 6 M to up to no more than 1 X 10"6M, thus creating changes in the Ca2+ concentration in the same order of magnitude, or at least not necessarily much higher, than those due to stimulation by classic agonists. This pattern of changes in the [Ca2+]c with initial release from the IP3-sensitive Ca2+ pool has been described in endothelial cells (Doan et al., 1994; Dreher et al., 1995; Volk et al, 1997), ventricular myocytes (Goldhaber and Liu, 1994), and smooth muscle cells (Roveri et al, 1992) treated with hydrogen peroxide or hydrogen peroxide generating systems, hepatocytes treated with 4-hydro-xynonenal (Carini et al, 1996), and chicken B cells treated with hydrogen peroxide (Qin et al 1996).

In some of the cases just mentioned, Ca2+ mobilization was found to be dependent on the activation of certain components of signal transduction pathways. Thus, Qin etal, (1996) found that the hydrogen peroxide-induced Ca2+ mobilization occurred after p53/p56lyn-dependent p72syk activation. p53/p56lyn and p72syk are nonreceptor protein kinases of the Src and Syk families that intervene in lymphocyte activation.

Carini et al, (1996) found that incubation of isolated hepatocytes with low micromolar concentrations of 4-hydroxynonenal caused first a transient increase in [Ca2+]c that peaked at 0.4 X 10 6 M 5 min after addition of the aldehyde. After that, a second rise went on until the end of the incubation (30 min) when [Ca2+]c was 0.9 X 10~6M. The first, transient increase, but not the second, permanent increase, was inhibited by U73122, an inhibitor

Figure 2 Changes in [Ca2+]c due to oxidative stress. Arrows represent net Ca2+ movement from different Ca2+ pools into the cytosol. The right-hand side of the figure is an attempt to summarize the results of the studies discussed in the text, in which [Ca2+]c was determined in a variety of cell types exposed to reactive oxygen species or lipid peroxidation products. These graphs do not represent any particular actual measurements. Transient Ca2+ increases like the one shown in graph A may be due to release of Ca2+ from the endoplasmic reticulum (2), occur in a matter of seconds or very few minutes after exposure, and are characteristic of low-level oxidative stress. Under relatively stronger oxidative stress, they are typically followed (graph B) or substituted (graph C) by a more sustained and gradual elevation of [Ca2+]c due to the opening of channels in the plasma membrane (1). This opening may be due to, or independent of, the capacitative mechanism in response to release from the endoplasmic reticulum, when the latter occurs. Release of Ca2+ from Ca2+-binding proteins (3) might explain increases in [Ca2+]c in alveolar macrophages treated with peroxides and may prove to be a novel mechanisms for Ca2+ signaling. All these changes in [Ca2+]c are of a magnitude similar to those caused by classic agonist stimulation, from basal levels of about 0.1-0.2 X 10"6 M to not more than 10"6M. They mimic the Ca2+ release by classic agonists and this way modulate cell responses to the agonists.

of phospholipase C, and also by thapsigargin. It was concluded that the first increase follows the phospholipase C/IP3 pathway for release of Ca2+ from the endoplasmic reticulum. The second increase could not be explained by capacitative influx in response to the depletion of the IP3-sensitive store, but had to be due to alteration of the signaling for the capacitative mechanism itself or of the influx channels.

Phospholipase C activation was also found to be involved in the increase of [Ca2+]c caused by the exposure of endothelial cells to hydrogen peroxide

(Volk et al., 1997). These cells were treated with hydrogen peroxide and superoxide generated by hypoxanthine/xanthine oxidase or with hydrogen peroxide generated by glucose/glucose oxidase. Relatively high rates of generation of the reactive oxygen species by these enzymatic systems led to a gradual increase of [Ca2+]c that went on for 40 min and peaked at 0.6 X 10"6 M. This kind of treatment was responsible for drastic decreases in viability, and the gradual increase in [Ca2+]c was found to be due to increased uptake from the extracellular medium. At lower concentrations of xanthine oxidase and glucose oxidase, no toxicity was observed and the elevation of [Ca2+]c occurred very rapidly and was transient. These transients lasted for not more than 15 sec and peaked at a similar concentration as before (~0.6 X 10"6M). In the case of the hydrogen peroxide and superoxide generating system hypoxanthine/xanthine oxidase, it was determined that hydrogen peroxide was responsible for the increase in [Ca2+]c. Interestingly, bolus addition of the peroxide had to be of a concentration much higher than the concentration achieved by the enzymatic systems in order to get the same effect. Transients were diminished by the phospholipase C inhibitor U73122 and were due to release from the endoplasmic reticulum. The effect of Ca2+ mobilization by these low levels of hydrogen peroxide on the Ca2+ signaling that occurs after stimulation with the agonist ATP and histamine was studied, but no alteration of the Ca2+ mobilization characteristic of these agonists was observed.

Elliott, Schilling, and co-workers described the effect of incubating vascular endothelial cells with 400 X 10"6 M i-butyl hydroperoxide on the subsequent Ca2+ mobilization caused by the physiological agonist bradykinin (Elliott et al., 1992; Schilling and Elliott, 1992). Treatment for 30 min caused inhibition of the Ca2+ influx provoked by bradykinin. Longer treatments caused inhibition of release from the IP3-sensitive intracellular pool and eventually a loss of responsiveness and a progressive increase in the basal Ca2+ level. This corresponds with initial inhibition of Ca2+ influx, followed in time by inhibition of efflux, as determined using 45Ca2+ to measure Ca2+ movement (Elliott and Schilling, 1991).

1. Ca2+ ATPase Pumps and IP3-Sensitive Channels in the Endoplasmic Reticulum

Reactive oxygen species are believed to be involved in ischemia-

reperfusion damage to cardiac muscle. Because Ca2+-ATPase pumps and

IP3-sensitive channels might be targets of this oxidative damage, preparations of sarcoplasmic reticulum have been used to study the susceptibility of these proteins to inactivation by reactive oxygen intermediates. Permeabilized hepatocytes and endothelial and smooth muscle cells have also been used for these studies.

Treatment of preparations of sarcoplasmic reticulum with hydrogen peroxide (Boraso and Williams, 1994) and with a singlet oxygen plus superoxide anion generating system (Holmberg et al., 1991; Xiong et al., 1992) casued an increase in the probability of the Ca2+ release channels being open. The same result was found when the channel protein reconstituted in phospholipid bilayers was treated with the ascorbate/iron couple (Stoya-novsky et al., 1994). In preparations treated with hypoxanthine/xanthine oxidase, both stimulation of IP3 release and inhibition of Ca2+ ATPases were found. Superoxide anion, not hydrogen peroxide, was responsible for this effect, and cysteine protected the Ca2+-ATPase from inhibition, suggesting the possibility of modification of sulfhydryl groups (Suzuki and Ford, 1991, 1992). Treatment with the hydroxyl radical generating system hydrogen peroxide/Fe3+/nitriloacetic acid caused inhibition of the Ca2+-ATPase as well. ATP exhibited a protective effect, suggesting that the target of the modification is localized in the ATP-binding site of the pump (Xu et al., 1997).

Treatment of permeabilized hepatocytes with i-butyl hydroperoxide caused both inhibition of uptake and sensitization of release of Ca2+ from the endoplasmic reticulum. Interestingly, these effects were also caused by glutathione disulfide and were reversed by the sulfhydryl-reducing agent dithiothreitol (Rooney etal., 1991). It was described later, also in permeabilized hepatocytes, that not only glutathione disulfide but also cystine increases the sensitivity of the IP3-dependent channels. Dithiothreitol, cysteine, and glutathione reversed the sensitization of the IP3-dependent channels, whereas sulfhydryl group-modifying agents did not have any effect on IP3-sensitive channels but inhibited Ca2+-ATPase pumps (Renard et al., 1992). Thus, the release of Ca2+ from the IP3-sensitive pool may be regulated by the formation of mixed disulfides. In the paper mentioned earlier, Rooney and co-workers (1991), using fluorescent microscopy, showed that the Ca2+ oscillatory waves caused by ¿-butyl hydroperoxide exhibited the same kinetic characteristics as those caused by stimulation with the receptor-mediated agonist phenylephrine. The latter, unlike i-butyl hydroperoxide, activates phospholipase C.

Grover and Samson (1997) have compared the inhibition by hydrogen peroxide of the Ca2+-ATPase pump in permeabilized endothelial and smooth muscle cells. They found that the pump is inhibited much more easily in smooth muscle than in endothelial cells, which may be of physiological relevance as the endothelium constitutes the first barrier of defense against attack by reactive oxygen species in the vasculature. Accordingly, in arterial rings the inhibition by hydrogen peroxide of the smooth muscle-dependent contraction induced by cyclopiazonic acid and angiotensin was much greater than in the endothelium-dependent, cyclopiazonic acid- and bradykinin-induced relaxation.

2. Ca2+ ATPase Pumps, Na+/Ca2+ Exchanger, and Ca2+ Channels in the Plasma Membrane

Influx of Ca2+ into isolated hepatocytes and a few other cell types due to alterations of Ca2+-ATPase pumps and the Na+/Ca2+ exchanger of the plasma membrane has been described. Lipid antioxidants and/or sulfhydryl-reducing agents have proved to be effective in preventing this inhibition in many cases.

Treatment of isolated hepatocytes with Fe3+ caused a vigorous lipid peroxidation that was associated with entry of Ca2+ into the cell. Lipid antioxidants prevented this influx, which otherwise went on for the duration of the incubations (180 min), and led to an increase in the concentration of Ca2+ in endoplasmic reticulum and mitochondria (Albano et al., 1991). In the same experimental system, Carini et al, (1995) described that the opening of Ca2+ channels, rather than the Na+/Ca2+ exchanger functioning in a reverse mode, was responsible for the increase in the intracellular Ca2+ concentration. Some reports had previously shown that treatment with the redox cycling quinone menadione (Carini et al, 1994) or hypoxia (Haigney et al, 1992) was able to convert the Na+/Ca2+ exchanger to a reverse mode that was the cause of net Ca2+ entry into the cells. In isolated hepatocytes, inactivation of the Ca2+-ATPase pump of the plasma membrane by treatment with cytotoxic doses of menadione was also reversed by the addition of dithiothreitol, showing that this activity may be regulated by the oxidation/ reduction of sulfhydryl groups (Nicotera et al, 1985). Inhibition of the Ca2+-ATPase was also found in red blood cells after treatment with Fe2+; this inhibition was prevented by lipid antioxidants (Rohn et al., 1996). In rat alveolar macrophages, a sustained elevation of the intracellular Ca2+ concentration due to treatment with i-butyl hydroperoxide could also be reversed by the addition of dithiothreitol (Hoyal et al, 1996b).

Treatment of cardiac myocytes with hydrogen peroxide caused entry of Ca2+ into the cells due to the opening of channels, which made the plasma membrane leaky (Wang et al, 1995). In these cells, treatment with rose bengal, a generator of singlet oxygen and superoxide anion, also induced an electrogenic Na+/Ca2+ exchange that was proposed as the cause of oxidative stress-induced arrhythmias (Matsuura and Shattock, 1991). A situation similar to the one described earlier in hepatocytes was found in smooth muscle cells treated with hydrogen peroxide (Roveri et al, 1992). After a rapid increase, [Ca2+]c went down but to a new steady-state level higher than before treatment with the peroxide. The first increase was due to release from the IP3-sensitive intracellular store, whereas the subsequently sustained increased steady-state level was due to uptake through plasma membrane channels. The sustained increase was inhibited by lipid antioxidants and sulfhydryl group reducing agents.

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