Thrombin and Phorbol Esters Potentiate Gs-Mediated cAMP Formation in Intact Human Erythroid Progenitors via Two Synergistic Signaling Pathways Converging on Adenylyl Cyclase Type VII
Abstract
In intact, but not in permeabilized, human erythroid progenitor cells, thrombin and phorbol esters potentiate cellular cAMP formation in response to Gs-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 Gs-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 Gs-coupled receptor agonists.
Footnotes
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Send reprint requests to: Dr. Hartmut Porzig, Pharmakologisches Institut der Universität Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland. E-mail:hartmut.porzig{at}pki.unibe.ch
- Abbreviations:
- PGE1
- prostaglandin E1
- PKC
- protein kinase C
- GF-109203X
- bisindolylmaleimide I
- HEL
- human erythroleukemia cell line
- PMA
- phorbol-12-myristate-13-acetate
- CD
- cluster of differentiation (surface antigen classification of blood cells)
- IDMEM
- Iscove’s modified Dulbecco’s modified Eagle’s medium
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- BCECF
- 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein
- EIPA
- 5-(N-ethyl-N-isopropyl)amiloride
- DMB-forskolin
- 7β-deacetyl-7β-[γ-(morpholino)butyryl]-forskolin hydrochloride
- FCS
- fetal calf serum
- PAF
- platelet activating factor
- AM
- acetoxymethyl ester
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- Received September 8, 1997.
- Accepted February 4, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
