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

Open Access
Publisher Website
Google Scholar
Abstract
1 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). 2 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. 3 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. 4 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. 5 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. 6 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.