Retinoic acid-induced tissue transglutaminase and apoptosis in vascular smooth muscle cells.

Retinoids exert antiproliferative and prodifferentiating effects in vascular smooth muscle cells (SMCs) and reduce neointimal mass in balloon-injured blood vessels. The mechanisms through which retinoids carry out these effects are unknown but likely involve retinoid receptor-mediated changes in gene expression. Here we report the cloning, chromosomal mapping, and biological activity of the retinoid-response gene rat tissue transglutaminase (tTG). Northern blotting studies showed that tTG is rapidly and dose-dependently induced in a protein synthesis-independent manner after stimulation with the natural retinoid all-trans retinoic acid (atRA). The induction of tTG was selective for atRA and its stereoisomers 9-cis and 13-cis RA, because little or no elevation in mRNA expression was observed with a panel of growth factors. Western blotting and immunofluorescence confocal microscopy showed an accumulation of cytosolic tTG protein after atRA stimulation. Radiolabeled cross-linking studies revealed a corresponding elevation in in vitro tTG activity. The increase in tTG activity was reduced in the presence of 2 distinct inhibitors of tTG (monodansylcadaverine and cystamine). atRA-induced tTG mRNA and protein expression were followed by a significant elevation in SMC apoptosis. Such retinoid-induced programmed cell death could be partially inhibited with each tTG inhibitor and was completely blocked when both inhibitors were used simultaneously. These results establish a role for atRA in the sequential stimulation of tTG and apoptosis in cultured SMCs. atRA-mediated apoptosis in SMCs seems to require the participation of active tTG, suggesting a potential mechanistic link between this retinoid-inducible gene and programmed cell death.

Cat cerebral arterial smooth muscle cells express cytochrome P450 4A2 enzyme and produce the vasoconstrictor 20-HETE which enhances L-type Ca2+ current.

1. Cerebral arteries express cytochrome P450 4A enzymes (P450 4A) and produce 20- hydroxyeicosatetraenoic acid (20-HETE), a potent constrictor of pial arterioles. It is not known which cell type in the vessel wall is responsible for the formation of 20-HETE. We examined whether freshly isolated cerebral arterial muscle cells (VSMCs) express P450 4A and produce 20-HETE. We also studied the effect of 20-HETE on pressurized cerebral arteries and on whole-cell L-type Ca2+current (ICa) recorded in cat cerebral VSMCs. 2. Cat cerebral VSMCs incubated with [14C]arachidonic acid ([14C]AA) produced 20-HETE (3.9 +/- 1.1 pmol min-1 (mg protein)-1). 3. Reverse transcription-polymerase chain reaction studies revealed that cat cerebral VSMCs express mRNA for P450 4A which metabolizes AA to 20-HETE. Cloning and sequencing of the cDNA amplified from mRNA isolated from VSMCs showed > 96 % amino acid homology to the rat and human P450 4A2 and 4A3. 4. 20-HETE (1-300 nM) induced a concentration-dependent constriction of cat cerebral arteries, which was inhibited by nifedipine. 5. Addition of 10 and 100 nM 20-HETE to the bath increased peak ICa by 50 +/- 3 and 100 +/- 10 %, respectively. This effect was not influenced by altering the frequency of depolarization. 20-HETE (100 nM) failed to increase ICa in the presence of nifedipine. 6. These results demonstrate that cat cerebral VSMCs express P450 4A enzyme, and produce 20-HETE which activates L-type Ca2+ channel current to promote cerebral vasoconstriction.