endothelin-1 and pervanadate

The activation of phospholipase D (PLD) is a common response to mitogenic stimuli in various cell types. As PLD-mediated signaling is known to be disrupted in the presence of ethanol, we tested whether PLD is involved in the ethanol-induced inhibition of cell proliferation in rat cortical primary astrocytes. Readdition of fetal calf serum (FCS) to serum-deprived astroglial cultures caused a rapid, threefold increase of PLD activity and a strong mitogenic response; both effects were dependent on tyrosine kinases but not on protein kinase C. Ethanol (0.1-2%) suppressed the FCS-induced, PLD-mediated formation of phosphatidic acid (PA) as well as astroglial cell proliferation in a concentration-dependent manner. Moreover, exogenous bacterial PLD increased astroglial proliferation in an ethanol-sensitive manner, whereas exogenous PA or lysophosphatidic acid was less effective. Formation of PA and astroglial proliferation were strongly inhibited by 1-butanol (0.1-1%), a substrate of PLD, but were unaffected by t-butanol, a non-substrate; 2-butanol had intermediate effects. Platelet-derived growth factor and endothelin-1 mimicked the mitogenic effect of FCS; their effects were also inhibited by the butanols in the potency order 1-butanol > 2-butanol > tert-butanol. Our results, in particular, the differential effects of 1-, 2-, and tert-butanol with respect to PA formation and astroglial proliferation, strongly suggest that the antiproliferative effects of ethanol in glial cells are due to the disruption of the PLD signaling pathway. This mechanism may also contribute to the inhibition of astroglial growth and brain development observed in alcoholic embryopathy.

Topics: 1-Butanol; Animals; Astrocytes; Becaplermin; Butanols; Cell Division; Culture Media, Serum-Free; DNA; Endothelin-1; Ethanol; Fetal Alcohol Spectrum Disorders; Genistein; Growth Inhibitors; Indoles; Nerve Tissue Proteins; Phosphatidic Acids; Phospholipase D; Phosphorylation; Platelet-Derived Growth Factor; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-sis; Rats; Signal Transduction; tert-Butyl Alcohol; Vanadates

Endothelin (ET-1), a contractor and mitogen in the vasculature, enhanced cAMP production (t1/2, 2.2 min; EC50, 89 +/- 6.3 nM) and stimulated activity of the cAMP-dependent protein kinase (PKA) in pig coronary arteries. These responses were blunted by the protein tyrosine kinase (PTK) inhibitors genistein and herbimycin-A, but not by inhibitors of protein kinase C or cyclooxygenase. In contrast, forskolin-stimulated cAMP production was unaffected by PTK inhibition. Immunoblot analysis revealed that ET-1 induced a concentration-dependent protein tyrosine (PT) phosphorylation. Sarafotoxin-c, a selective ETB receptor agonist, had no effect on either cAMP levels or PT phosphorylation. Moreover, pervanadate (PV), a potent inhibitor of PT phosphatases, enhanced both cAMP formation and PT phosphorylation, both of which were blocked by PTK inhibitors. The effects of ET-1 and PV were not additive, suggesting a similar mode of activation, whereas responses to ET-1 and forskolin were synergistic. These findings indicate that AC and PKA are activatable via a nonreceptor PTK-dependent pathway downstream from the G-protein-linked ETA receptor. Because cAMP is a dilator and antimitogen in smooth muscle, stimulation of AC activity may be a negative feedback mechanism regulating ET-1-induced vasoconstriction and/or mitogenesis.

Topics: Adenylyl Cyclases; Animals; Benzoquinones; Colforsin; Coronary Vessels; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Genistein; GTP-Binding Proteins; In Vitro Techniques; Kinetics; Lactams, Macrocyclic; Muscle, Smooth, Vascular; Peptides; Protein-Tyrosine Kinases; Quinones; Receptor, Endothelin A; Receptors, Endothelin; Rifabutin; Signal Transduction; Swine; Vanadates; Viper Venoms