Preparation of smooth muscle cell suspensions from the rabbit oviduct and prostaglandin binding analysis.

Abstract

The preparation, viability, and prostaglandin (PG) binding characteristics of dispersed smooth muscle cells from the rabbit oviduct have been determined. Cell suspensions were prepared by enzymatic degradation of the intracellular matrix and subsequent mechanical dispersion with a wide bore pipette. The digestion media consisted of a modified Hanks' Balanced Salt Solution, pH 7.1, containing 1.6 U/mg wet wt elastase and 8 U/mg wet wt collagenase. This method provided a yield of single smooth muscle cells of approximately 3 x 10(6) cells/100 mg wet wt within 3-4 h of organ removal. Cell viability was determined by trypan blue dye exclusion, retention of lactate dehydrogenase, absence of 57Co-EDTA uptake, and ouabain sensitivity of cationic transport. Roughly 80% of the isolated cells remained viable after the digestion procedure. The dispersed cells specifically bound [3H]PGE2 and [3H]PGF2 alpha. Scatchard analysis of the binding data revealed separate homogenous populations of high affinity sites for both PGE2 and PGF2 alpha. The equilibrium dissociation constants and total sites per cell were 0.55 nM and 11,332 for PGE2 and 0.19 nM and 5,154 for PGF2 alpha, respectively. Specific labeled PG binding was inhibited in a concentration-dependent manner by increasing amounts of unlabeled PG. Inhibition of labeled PGE2 binding by unlabeled PGF2 alpha, and vice versa were negligible, except at high concentrations. The results indicate that smooth muscle cells can be enzymatically dispersed from the rabbit oviduct with minimal damage. Also, these cells possess distinct specific binding sites for PGE2 and PGF2 alpha that differ in regard to affinity and total number of sites per cell.