Characterization Of Alpha-Adrenergic Receptors In Neonatal Piglet Aortas By Radioligand Binding

Vascular smooth muscle cell membranes were prepared from neonatal piglet aortas by homogenization, low speed centrifugation to remove intact cells and ultracentrifugation. Aliquots of the membrane preparation containing 100 micrograms of membrane protein (Lowry assay) were then incubated in tris buffer pH 7.45 at 25°C with varying concentrations of either tritiated prazosin (selective alpha-1 antagonist) or tritiated yohimbine (selective alpha-2 antagonist). Incubation was stopped by addition of ice cold tris; radioligand bound to the membrane preparation was separated from free radioligand by rapid filtration. Non-specific binding (other than to alpha receptors) to membrane prep or filter was quantified by identical procedure with the addition of 10 mM 1-norepinephrine to the incubation medium to occupy alpha receptors. No specific binding of prazosin to membrane preparations was observed. Specific, saturable binding of yohimbine was observed and was analyzed by Scatchard plot giving a dissociation constant of 2.4 nM and a receptor content of 100 fmole/mg membrane protein. These preliminary observations suggest that the major alpha receptor subtype in the neonatal piglet aorta is the alpha-2 receptor, and there may be no alpha-1 receptors present.