Protein kinase C activation allows pulmonary artery smooth muscle cells to proliferate to hypoxia

E. C. Dempsey, I. F. McMurtry and R. F. O'Brien Department of Medicine, University of Colorado Health Sciences Center, Denver 80262. Pulmonary artery (PA) smooth muscle cell (SMC) proliferation occurs with hypoxic pulmonary hypertension in vivo. However, proliferation of cultured PA SMC to hypoxia has not been demonstrated, and thus the mechanism by which these cells respond to hypoxia is unknown. Because protein kinase C (PKC) plays a role in intracellular transduction of proliferative signals, we asked whether PKC activation 1) causes proliferation of bovine PA SMC and 2) is important in PA SMC proliferative response to hypoxia. By measuring [3H]thymidine incorporation and cell counts, we found that quiescent PA SMC from four different cows proliferated with the PKC activator, phorbol 12-myristate 13-acetate (PMA), in a concentration-dependent manner. The proliferation was blocked with a PKC inhibitor, dihydrosphingosine, or by downregulating SMC PKC. We tested whether "priming" PA SMC by PKC activation was required for in vitro SMC proliferative response to hypoxia. Each SMC population was treated with PMA and then exposed for 24 h to 20, 10, 7, 3 or 0% O2. These cells proliferated with hypoxia reaching a peak response at 3% O2. The magnitude of the response to PMA and hypoxia was different for each cell population tested. No hypoxic proliferation occurred in control cells (no PMA). Dihydrosphingosine blocked the hypoxic response to the same extent that it inhibited the initial PMA conditioning stimulus. PKC-downregulated PA SMC did not proliferate to PMA or to subsequent hypoxia. The hypoxic response was not due to a reduction in O2 radical-mediated antiproliferative effect; rather, the PMA-primed cells seemed to "acquire" the ability to directly sense hypoxia and proliferate. In summary, PKC activation caused proliferation of PA SMC in vitro and allowed an additional proliferative response to hypoxia. Activation of PKC may be a requisite step for PA SMC to respond directly to hypoxia.