The intercellular adhesion molecule 1 (ICAM-1) is induced on endothelial cells by tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and lipopolysaccharide (LPS). We have reported the sensitivity of cytokine-induced ICAM-1 expression to protein kinase inhibitors, including inhibitors of protein kinase C (PKC) [C. L. Myers, S. N. Desai, J. Schembri-King, G. L. Letts, and R. W. Wallace. Am. J. Physiol. 262 (Cell Physiol. 31): C365-C373, 1992]. To directly investigate the role of PKC in ICAM-1 induction, we downregulated PKC by pretreatment of human umbilical vein endothelial cells with phorbol 12-myristate 13-acetate (PMA) and assessed ICAM-1 protein and mRNA induction elicited by subsequent exposure to inflammatory stimuli. PMA treatment results in ICAM-1 protein induction that declines to basal levels by 3 days. Western blots of endothelial cell lysates reveal a nearly complete loss of immunologically reactive PKC. Subsequent activation with cytokine or LPS leads to reinduction of ICAM-1 protein and mRNA; however, the cells no longer produced substantial amounts of ICAM-1 protein or mRNA in response to PMA stimulation. Cross desensitization is observed with phorbol dibutyrate, while 4 alpha-phorbol has no desensitizing effect. The data indicate that PKC activation, while capable of inducing ICAM-1 expression, is not essential for ICAM-1 induction by the inflammatory mediators TNF-alpha, IL-1 beta, or LPS.

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Acute exposure (30 min-1 h) of bovine pulmonary artery endothelial cells in culture (BPAEC) to phorbol myristate acetate (PMA 10(-10)-10(-6) M) resulted in concentration-dependent decrement in serotonin (5-HT) uptake. Neither cell viability (trypan blue exclusion or release of deoxyglucose) nor activity of another plasma membrane function, angiotensin-converting enzyme activity, were affected. A decrease in 5-HT uptake was also noted with phorbol 12,13 dibutyrate and mezerein, but not with 4-alpha-phorbol 12,13 didecanoate, which does not stimulate protein kinase C (PKC). Inhibition of 5-HT uptake by PMA (160 nM) was reversed in a concentration-dependent manner by pretreatment of cells with the PKC inhibitor staurosporine (3-100 nM). BPAEC were treated with PMA (160 nM) for 1 h, and activities of PKC in cytosolic and membrane compartments were determined. PMA did not significantly affect total cellular PKC activity but resulted in a translocation of activity from cytosol to membrane (control membrane activity 67 +/- 4%; PMA-treated membrane activity 97 +/- 1% of total cellular PKC). Thus we propose that translocation of PKC from cytosol to membrane results in inhibition of 5-HT uptake by BPAEC.

Allopurinol is thought to protect hearts against damage due to hypoxia or ischemia by inhibiting xanthine oxidase and oxygen radical generation. We subjected isolated rabbit hearts, equilibrated by perfusion at 37 degrees C, to 1 h of global ischemia at 27 or 37 degrees C with or without brief pretreatment with 100 microM allopurinol. The total absence of xanthine or uric acid in the coronary effluent following ischemia, the presence of hypoxanthine (25 +/- 4 microM peak concentration), and the failure of allopurinol to alter purine washout profiles or postischemic cardiac function suggest that rabbit myocardium lacks xanthine oxidase or dehydrogenase. Data obtained with a similar rat heart preparation showed appreciable formation of xanthine (12 +/- 2 microM peak) and uric acid (10 +/- 3 microM). Allopurinol pretreatment inhibited xanthine and uric acid formation and significantly improved key indicators of postischemic left ventricular function. We conclude that there is species dependency in the myocardial activity of xanthine oxidase or dehydrogenase, that when present it can be inhibited by acute allopurinol pretreatment, and that xanthine oxidase activity and its ability to generate oxygen radicals are not universal contributors to cardiac ischemic damage.