Cultured vascular smooth muscle cells: an in vitro system for study of alpha-adrenergic receptor coupling and regulation.
Cultured vascular smooth muscle cells derived by enzymatic dissociation of rabbit aortic media were used for study of alpha 1-adrenergic receptors (AAR) and receptor-coupled calcium flux. AAR were characterized by binding of the alpha 1-selective radioligand [3H]prazosin, and norepinephrine-stimulated 45calcium efflux was measured as an index of AAR-mediated calcium flux. The binding of [3H]prazosin to a crude cellular homogenate was to a single, saturable site of high affinity (Kd = 0.15 nM, Bmax = 75-125 fmol/mg protein), which was stereo-specific and exhibited the appropriate potency order for competition by agonists. Prazosin (Kd = 0.07 nM) was approximately 3000-fold more potent than the alpha 2-selective antagonist yohimbine (Kd = 222 nM), both of which exhibited Hill coefficients of one, indicative of binding to a single receptor type. In intact cells, norepinephrine caused a concentration-related (EC50 = 100 nM) increase in 45calcium efflux which was blocked by low concentrations of prazosin (IC50 approximately equal to 0.1 nM), but not yohimbine (IC50 greater than 100 nM). An alpha 1-selective concentration of prazosin (100 nM) fully blocked norepinephrine-stimulated 45calcium efflux, and therefore, it appears that this effect does not involve alpha 2-adrenergic receptors. Treatment of cultured cells with 1-norepinephrine for 48 h caused a concentration-related decrease in both AAR density and maximum norepinephrine-stimulated 45calcium efflux. This cultured vascular smooth muscle cell system provides several advantages for the study of alpha-adrenergic receptor coupling and regulation.
W S
WS
Colucci
T A
TA
Brock
W J
WJ
Atkinson
R W
RW
Alexander
M A
MA
Gimbrone
D000818:Animals; D001011:Aorta / Q000166:cytology; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D066298:In Vitro Techniques; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000502:physiology; D009638:Norepinephrine / Q000494:pharmacology; D011224:Prazosin; D011817:Rabbits; D011942:Receptors, Adrenergic, alpha / Q000502:physiology*; D015016:Yohimbine / Q000494:pharmacology
D011942:Receptors, Adrenergic, alpha; D015016:Yohimbine; D002118:Calcium; D009638:Norepinephrine; D011224:Prazosin