Bovine iris-sphincter muscle lacks FP receptor binding sites.
OBJECTIVE
To determine the affinity, density, and specificity of the binding sites of tritium-labeled prostaglandin F2alpha in membrane preparations of bovine iris-sphincter muscle and corpus luteum.


METHODS
Membrane preparations were incubated with 0.312-40.0 nM 3H-prostaglandin F2alpha in saturation experiments, or 8 nM 3H-prostaglandin F2alpha in competition studies, in the presence or absence of varying concentrations of unlabeled prostaglandin F2alpha or other prostaglandin receptor agonists. The affinity (Kd) and the density of the binding sites (Bmax) of 3H-prostaglandin F2alpha in the bovine iris-sphincter muscle were determined by Scatchard analysis. Reverse transcription polymerase chain reaction was performed with bovine iris-sphincter muscle and corpus luteum total RNA and the PCR products were hybridized with specific 32P-labeled probe for further confirmation of FP receptor expression.


RESULTS
Specific binding sites of 3H-prostaglandin F2alpha in the membranes of bovine iris-sphincter muscle are saturable with an affinity of 9.5 nM and a density of 596 fmoles/mg of protein. Prostaglandin E2, 17-phenyl trinor prostaglandin E2, and GR63799 (EP, EP1, and EP3 receptor agonists, respectively) inhibited 3H-prostaglandin F2alpha binding with an IC50 of 0.0048 microM, 0.0038 microM, and 0.044 microM, respectively. Fluprostenol, a specific FP receptor agonist did not inhibit 3H-prostaglandin F2alpha binding. In contrast, prostaglandin F2alpha and fluprostenol effectively inhibited 3H-prostaglandin F2alpha binding in the bovine corpus luteum with an IC50 of 0.031 microM and 0.037 microM, respectively.


CONCLUSIONS
Our results demonstrate that in the bovine iris-sphincter muscle, FP receptors are not expressed and 3H-prostaglandin F2alpha binds to EP1 and EP3 receptor sites. RT-PCR results demonstrated that FP receptor mRNA, which is present in bovine corpus luteum, is probably absent in iris-sphincter muscle.
Department of Ophthalmology & Visual Sciences, University of Louisville, KY 40202, USA.
P
P
Bhattacherjee
B S
BS
Williams
P
P
Mukhopadhyay
C A
CA
Paterson
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D017931:DNA Primers / Q000737:chemistry; D015237:Dinoprost / Q000378:metabolism*; D007498:Iris / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D011982:Receptors, Prostaglandin / Q000819:agonists / Q000378:metabolism*; D018078:Receptors, Prostaglandin E / Q000819:agonists / Q000378:metabolism*; D058306:Receptors, Prostaglandin E, EP1 Subtype; D058308:Receptors, Prostaglandin E, EP3 Subtype; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D011982:Receptors, Prostaglandin; D018078:Receptors, Prostaglandin E; D058306:Receptors, Prostaglandin E, EP1 Subtype; D058308:Receptors, Prostaglandin E, EP3 Subtype; C034671:prostaglandin F2alpha receptor; D015237:Dinoprost