We have combined patch clamp recording with simultaneous [Ca2+]i measurements in single LNCaP cells (a human prostate cancer cell line), to study the activation of Ca2+‐permeable channels by two different inducers of apoptosis, ionomycin and serum deprivation. In perforated patch recording, LNCaP cells had a membrane potential of ‐40 mV and a resting [Ca2+]i of 90 nM. Application of ionomycin at levels that induced apoptosis in these cells (10 μM) produced a biphasic increase in [Ca2+]i. The first rise in [Ca2+]i was due to release of Ca2+ from internal stores and it was associated with a membrane hyperpolarization to ‐77 mV. The latter was probably due to the activation of high conductance, Ca2+‐ and voltage‐dependent K+ channels (maxi‐K). Conversely, the second rise in [Ca2+]i was always preceded by and strictly associated with membrane depolarization and required external Ca2+. Serum deprivation, another inducer of apoptosis, unmasked a voltage‐independent Ca2+ permeability as well. A lower concentration of ionomycin (1 μM) did not induce apoptosis, and neither depolarized LNCaP cells nor produced the biphasic increase in [Ca2+]i. However, the first increment in [Ca2+]i due to release from internal Ca2+ stores was evident at this concentration of ionomycin. Simultaneous recordings of [Ca2+]i and ion channel activity in the cell attached configuration of patch clamp revealed a Ca2+‐permeable, Ca2+‐independent, non‐selective cation channel of 23 pS conductance. This channel was activated only during the second increment in [Ca2+]i induced by ionomycin. The absence of serum activated the 23 pS channel as well, albeit at a lower frequency than with ionomycin. Thus, the 23 pS channel can be activated by two unrelated inducers of apoptosis and it could be another Ca2+ influx mechanism in programmed cell death of LNCaP cells.

Apoptosis is an important mechanism of cell demise in multicellular organisms and Cl− transport has an important role in the progression of the apoptotic volume decrease (AVD). DIDS (4,4'-Diisothiocyanatostilbene-2,2'-disulfonate) is one of the most commonly used Cl− transport inhibitors that eliminates or reduces different apoptotic hallmarks such as AVD, caspase-3 activity and DNA fragmentation. DIDS is also a protein crosslinker that alkylates either amino or thiol groups. Since caspases are thiol proteases, our aim was to study whether DIDS could directly inhibit the activity of these proteases. Here, we show that caspase activity induced by 4 h incubation with staurosporine was inhibited by DIDS in HeLa cells that were maintained in the absence of serum for 24 h. Interestingly, the caspase-inhibitory effect of DIDS is downstream to the inhibition of cytochrome c release, suggesting that DIDS might be also acting at the apoptosome. Moreover, DIDS was able to inhibit capase-3, -9, and -8 activities in cell lysates, implying that DIDS can react with and directly block caspases. Our data suggest that antiapoptotic activity of DIDS involves not only inhibition of the voltage-dependent anion channel (VDAC) at the mitochondria and Cl− channels at the plasma membrane, but also a third mechanism based on the direct inhibition of caspases.

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Ion fluxes at the plasma membrane have an important role in early stages of apoptosis. Accordingly, plasma membrane depolarization and gain of Na+ and loss of K+ are initial events in apoptosis. We have studied the effect of staurosporine (STS), a well-established apoptosis inducer, on the membrane potential of HeLa cells to determine the nature of STS-activated ion conductances and their role in the activation of different caspases. We observed that STS can activate tetraethylammonium (TEA+) and 4-aminopyridine-sensitive K+ channels and flufenamic-sensitive cation channels as an early response. The combination of these ion channel inhibitors significantly reduced cytochrome c (cyt c) release and activation of caspase-9, -3 and -8. STS also induced a large reduction in the intracellular [K+] that was not blocked by the ion channel inhibitors. Our data suggest that reduction in the [K+]i is necessary but not sufficient and that ion channel inhibitors block activation of caspase-3 by two different mechanisms: the inhibitors of K+ channels by reducing cyt c release while flufenamic acid by a different, unrelated mechanism that does not involve cation channels at the plasma membrane. Our data also imply that these ion channels activated by STS are not responsible for the reduction in the [K+]i associated with apoptosis.

Acrosome reaction inactivation (ARI) is a process that renders sperm irreversibly refractory to the egg jelly (the natural inducer of the acrosome reaction, AR). This process triggered by the egg jelly, is associated with an increase in [Ca2+]i. However, we show here that a rise in [Ca2+]i alone is not sufficient to induce ARI, since artificially increasing [Ca2+]i with either an ionophore or rising external pH, does not trigger ARI. Contrary to the AR which strictly requires Ca2+, ARI can be triggered almost equally well by Sr2+. On the other hand, Mn2+ inhibits ARI and, as we showed earlier, does not affect AR. These observations indicate that the mechanisms involved in ARI differ from those leading to AR. In addition, we report here that high external pH (a non-physiological inducer of AR) triggers the AR in previously inactivated sperm by opening the same Ca2+ channels activated by the egg jelly. Considering that the opening of Ca2+ channels is one of the earliest responses triggered by the egg jelly and that ARI requires the egg jelly receptor to be activated, we have concluded that ARI involves the uncoupling between the egg jelly receptor and Ca2+ channels. Furthermore, intracellular pH (pHi) determinations, in the presence or absence of ionomycin to substitute for the uncoupled Ca2+ channels, indicate that pHi regulation is also impaired in inactivated sperm. In conclusion, ARI is a manifestation of the uncoupling of the egg jelly receptor from the different ion transport systems required for the acrosome reaction.