Regulation of a swelling‐activated chloride current in bovine endothelium by protein tyrosine phosphorylation and G proteins

Abstract

The role of protein tyrosine phosphorylation and of G proteins in the activation of a swelling‐activated Cl− current (ICl,swell) in calf pulmonary artery endothelial (CPAE) cells was studied using the whole‐cell patch clamp technique. ICl,swell was activated by reducing the extracellular osmolality by either 12.5 % (mild hypotonicity) or 25 % (strong hypotonicity). The protein tyrosine kinase (PTK) inhibitors tyrphostin B46, tyrphostin A25 and genistein inhibited ICl,swell with IC50 values of, respectively, 9.2 ± 0.2, 61.4 ± 1.7 and 62.9 ± 1.3μM. Tyrphostin A1, a tyrphostin analogue with little effect on PTK activity, and daidzein, an inactive genistein analogue, were without effect on ICl,swell. The protein tyrosine phosphatase (PTP) inhibitors Na3VO4 (200 μM) and dephostatin (20 μM) potentiated ICl,swell activated by mild hypotonicity by 47 ± 9 and 69 ± 15 %, respectively. Intracellular perfusion with GTPγS (100 μM) transiently activated a Cl− current with an identical biophysical and pharmacological profile to ICl,swell. This current was inhibited by the tested PTK inhibitors and potentiated by the PTP inhibitors. Hypertonicity‐induced cell shrinkage completely inhibited the GTPγS‐activated Cl− current. Intracellular perfusion with GDPβS (1 mM) caused a time‐dependent inhibition of ICl,swell, which was more pronounced when the current was activated by mild hypotonicity. Our results demonstrate that the activity of endothelial swelling‐activated Cl− channels is dependent on tyrosine phosphorylation and suggest that G proteins regulate the sensitivity to cell swelling.

Role of Rho and Rho kinase in the activation of volume‐regulated anion channels in bovine endothelial cells

“…This activation is transient and occurs without any change in cell volume. Moreover, activation of ICl,swell is significantly impaired in the presence of intracellular GDPâS (Doroshenko & Neher, 1992;Voets et al 1998). However, these effects of GTPãS and GDPâS did not give any clue to the kind of G proteins that are involved.…”

“…Intracellular application of GTPãS has been shown to activate ICl,swell in several cell types (e.g. Doroshenko et al 1991;Tilly, 1991;Nilius et al 1994;Mitchell et al 1997;Voets et al 1998). This activation is transient and occurs without any change in cell volume.…”

“…The nature of the cellular volume sensor, as well as the transduction apparatus that controls the gating of VRACs, is unknown. The cell swelling-induced activation of VRACs requires one or more tyrosine phosphorylation steps, since inhibitors of protein tyrosine kinases inhibit ICl,swell whereas inhibitors of protein tyrosine phosphatases potentiate ICl,swell (Tilly et al 1993;Voets et al 1998). It was recently shown that p56 lck , a member of the src protein tyrosine kinase family, mediates activation of ICl,swell in lymphocytes (Lepple-Wienhues et al 1998).…”

Reduced intracellular ionic strength as the initial trigger for activation of endothelial volume-regulated anion channels

“…A direct effect of ⌫ i on VRAC seems unlikely, as activation of the current by lowered ⌫ i remains dependent on protein tyrosine kinase activity (6,30). We favor the idea that ⌫ i acts similar to a second messenger, and directly or indirectly regulates the activity of the intracellular enzyme(s) that mediate the activation of I Cl,swell .…”

Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance

“…20,28 In addition, signaling pathways contributing to VRAC activity include phosphatidyl-inositol-3-phosphate kinase (PI3K), NADH-dependent oxidases and reactive oxygen species (ROS) as well as tyrosine kinases. [29][30][31][32][33][34][35][36] Recently it was demonstrated that knockdown of integrin b 1 reduces RVD in adherent Ehrlich Lettre ascites cells (ELA) cells 37 and as integrin b 1 stretch in Figure 1. VRAC characteristics.…”