Volume-sensitive Tyrosine Kinases Regulate Liver Cell Volume through Effects on Vesicular Trafficking and Membrane Na+ Permeability

Abstract

In liver cells, the influx of Na؉ mediated by nonselective cation (NSC) channels in the plasma membrane contributes importantly to regulation of cell volume. Under basal conditions, channels are closed; but both physiologic (e.g. insulin) and pathologic (e.g. oxidative stress) stimuli that are known to stimulate tyrosine kinases are associated with large increases in membrane Na ؉ permeability to ϳ80 pA/pF or more. Consequently, the purpose of these studies was to evaluate whether volumesensitive tyrosine kinases mediate cell volume increases through effects on the activity or distribution of NSC channel proteins. In HTC hepatoma cells, decreases in cell volume evoked by hypertonic exposure increased total cellular tyrosine kinase activity ϳ20-fold. Moreover, hypertonic exposure (320 -400 mosM) was followed after a delay by NSC channel activation and partial recovery of cell volume toward basal values (regulatory volume increase (RVI)). The tyrosine kinase inhibitors genistein and erbstatin prevented both NSC channel activation and RVI. Similarly, hypertonic exposure resulted in an increase in p60 c-src activity, and intracellular dialysis with recombinant p60 c-src led to activation of NSC currents in the absence of an osmolar gradient. Utilizing FM1-43 fluorescence, exposure to hypertonic media caused a rapid increase in the rate of exocytosis of ϳ40% (p < 0.01), and genistein inhibited both exocytosis and channel activation. These findings indicate that volume-sensitive increases in p60 c-src and/or related tyrosine kinases play a key role in the regulation of membrane Na ؉ permeability, suggesting that increases in the NSC conductance may be mediated in part through rapid recruitment of a distinct pool of channelcontaining vesicles.

The Src Family Kinase Yes Triggers Hyperosmotic Activation of the Epidermal Growth Factor Receptor and CD95

“…Multiple receptor pathways can couple with Src kinases, but Src family kinases are also activated in response to several forms of cell stress (for review see Refs. 13 and 14), such as UV radiation (29), hydrogen peroxide (30,31), or anisoosmolarity (11,12,32). c-Src was implicated in both hepatocellular volume increase and decrease in response to hyper-or hypoosmolarity, respectively (8,32).…”

“…13 and 14), such as UV radiation (29), hydrogen peroxide (30,31), or anisoosmolarity (11,12,32). c-Src was implicated in both hepatocellular volume increase and decrease in response to hyper-or hypoosmolarity, respectively (8,32). In Chinese hamster ovary cells, however, hyperosmolarity induced Src inhibition, but not Fyn stimulation, which participates in the hyperosmotic phosphorylation of cortactin and reorganization of the cytoskeleton (12).…”

Role of dipeptidyl peptidase IV in regulating activity of Na+/H+ exchanger isoform NHE3 in proximal tubule cells

“…Tyrosine kinase activity was measured in the OKP-DPPIV immune complex by Western blotting using an anti-phosphotyrosine antibody (17). OKP cells grown to confluence in six-well plates were incubated at 37°C for 20 min with NH 4 ϩ loading buffer.…”

Differential Regulation of Cell Volume and Shape in Confluent Rat Hepatocytes Under Hypertonic Stress

“…Recently, a hypertonic activation of non-selective cation channels was reported from rat hepatoma HTC cells [23]. Interestingly, in this system, PI 3-kinase appears to be one of the mediators of channel activation in addition to (and independent of) Src kinase.…”