Asymmetric signal transduction in polarized ileal Na+-absorbing cells: carbachol activates brush-border but not basolateral-membrane PIP2-PLC and translocates PLC-γ1 only to the brush border

Abstract

In ileal Na+ absorptive cells, carbachol inhibits NaCl absorption and its component brush-border Na+/H+ exchanger, acting via basolateral membrane (BLM) receptors. This carbachol effect involves brush-border but not BLM protein kinase C. In the present work we describe another asymmetric aspect of signal transduction in these epithelial cells, this time involving phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC). Thirty seconds and 1 min after carbachol treatment, brush-border PIP2-specific PLC activity increased, returning to control levels by 2.5 min. Involvement of brush-border tyrosine kinase(s) in this effect was suggested by inhibition of the carbachol effect on NaCl absorption by the tyrosine kinase inhibitor genistein, added to the mucosal but not the serosal surface. Luminal genistein pretreatment also prevented the carbachol-induced increase in brush-border PLC activity. In contrast, carbachol exposure did not change the BLM PIP2-specific PLC activity. Western analysis and immunoprecipitation demonstrated that PLC-gamma 1 is present in the brush border and that carbachol increases the PLC-gamma 1 amount in the brush border. Both the brush border and BLM contain PLC-beta 3 and a small amount of PLC-delta 1 but no PLC-beta 1, whereas BLM lacks detectable PLC-gamma 1. No change in PLC-beta 3 or PLC-delta 1 amount in the brush border occurred with carbachol exposure. No change in tyrosine phosphorylation of brush-border PLC-gamma 1 occurred with carbachol treatment. The Ca2+ ionophore A23187 did not alter PIP2-specific PLC activity in either the brush border or the BLM. These studies demonstrate that carbachol but not Ca2+ ionophore effects on brush-border NaCl absorption are associated with increases in brush-border but not BLM PIP2-specific PLC activity and in the amount of brush-border PLC-gamma 1, and involve tyrosine phosphorylation. This asymmetric aspect of epithelial signal transduction, together with the previous demonstration of localization of high-sensitivity IP3 stores to the apical membrane area in intestinal epithelial cells, shows that different aspects of signal transduction occur at the apical and basolateral membranes in epithelial and requires studies in both domains to define mechanisms of intracellular signalling.

Phospholipase C-γ Binds Directly to the Na+/H+ Exchanger 3 and Is Required for Calcium Regulation of Exchange Activity

“…Moreover, these results imply a scaffolding role for PLC-␥ in mediating elevated [Ca 2ϩ ] i regulation of NHE3 activity. Previous studies in Caco-2BBe cells and intact rabbit ileum have demonstrated carbachol-increased BB diacylglycerol levels and PKC activity that are consistent with lipase-dependent functions of PLC-␥ (19). These data along with the results from this study suggest that the role of PLC-␥ in [Ca 2ϩ ] i regulation of NHE3 activity may involve both lipase-dependent and lipaseindependent functions.…”

“…Previous studies by our laboratories in intact rabbit ileum have suggested a potential role for PLC-␥ in calcium regulation of NHE3 activity (19). Carbachol treatment resulted in a transient increase of [Ca 2ϩ ] i , first at the apical membrane, followed by a general rise in [Ca 2ϩ ] i .…”

“…In addition, there was a rapid increase in BB PLC-␥ expression and activity, which was not tyrosine-phosphorylated, following carbachol treatment that also was associated with an increase in BB diacylglycerol levels and PKC activity. Moreover, the increase in BB PLC-␥ is not only due to increased [Ca 2ϩ ] i as this effect was not observed in rabbit ileum treated with A23187 (19). Although the results of these studies suggested a role for PLC-␥ in NHE3 regulation, this study was designed to determine whether a direct relationship exists between NHE3 and PLC-␥.…”

“…This increases brush border (BB) levels of diacylglycerol, protein kinase C (PKC␣), and activated brush border phospholipase D (19). Moreover, carbachol causes a rapid translocation of activated (but not tyrosine-phosphorylated) phospholipase C-␥1 (PLC-␥) to the apical membrane (19). Although previous studies have suggested a role for PLC in elevated [Ca 2ϩ ] i inhibition of NHE3, a molecular mechanism for this effect has not been defined.…”

“…The carbachol-induced increase in [Ca 2ϩ ] i occurs initially at the apical membrane, followed by increased intracellular calcium throughout the cytoplasm at later time points (18). This increases brush border (BB) levels of diacylglycerol, protein kinase C (PKC␣), and activated brush border phospholipase D (19). Moreover, carbachol causes a rapid translocation of activated (but not tyrosine-phosphorylated) phospholipase C-␥1 (PLC-␥) to the apical membrane (19).…”