Abstract

In intact, but not in permeabilized, human erythroid progenitor cells, thrombin and phorbol esters potentiate cellular cAMP formation in response to G_s-coupled receptor agonists such as prostaglandin E₁ (PGE₁). We show here that the two agonists achieve their phenotypically similar effects by using distinctly different signaling pathways, both of which require protein kinase C (PKC) activation. After short term exposure (11 min), phorbol esters caused an alkaline shift of cellular pH by ∼0.1 unit, resulting in a 1.5–2-fold increase in PGE₁-induced cAMP formation. The effect of phorbol esters was inhibited by 5-(N-ethyl-N-isopropyl)amiloride, a specific inhibitor of the Na⁺/H⁺ exchanger, and by the PKC inhibitors GF 109203X, Gö 6976, and staurosporine. Thrombin increased cellular pH by only 0.02–0.05 unit but seemed to potentiate PGE₁-stimulated cAMP formation by an effect on the G_s-activated adenylyl cyclase involving a Ca²⁺-independent (novel) PKC. This effect was inhibited by GF 109203X and staurosporine but was resistant to 5-(N-ethyl-N-isopropyl)amiloride or Gö 6976. Inactivation of PKC by incubation of the cells in the presence of 10 nm phorbol-12-myristate-13-acetate for 18 hr completely abolished the potentiating effect of thrombin on cyclase activity, whereas the pH-dependent stimulation was fully retained. Northern blots with specific cDNA probes and a lack of Ca²⁺sensitivity indicate that progenitor cells predominantly express adenylyl cyclase type VII. Our results suggest that in normal human erythroid progenitors, thrombin can activate pH-dependent and -independent, PKC-linked pathways converging on adenylyl cyclase type VII to potentiate cAMP formation in response to G_s-coupled receptor agonists.