Related Experiment Videos
Prostaglandin E2 and F2 alpha binding sites in the bovine iris ciliary body
S Csukas1, P Bhattacherjee, L Rhodes
1Kentucky Lions Eye Research Institute, University of Louisville School of Medicine 40292.
Insights
Prostaglandin E2 (PGE2) specific binding sites are concentrated in the bovine iris sphincter muscle. This suggests PGE2, not Prostaglandin F2 alpha (PGF2 alpha), mediates in vivo effects in the iris via these EP receptors.
Area of Science:
- Ocular pharmacology
- Prostanoid receptor research
- Biochemistry of ocular tissues
Background:
- Prostaglandins are lipid compounds with diverse physiological roles.
- Prostaglandin E2 (PGE2) and Prostaglandin F2 alpha (PGF2 alpha) are implicated in ocular function.
- Understanding their specific binding sites is crucial for ocular drug development.
Purpose of the Study:
- To map the distribution of PGE2 and PGF2 alpha binding sites in bovine iris and ciliary body membranes.
- To determine the selectivity of these binding sites for different prostanoids.
Main Methods:
- Preparation of membrane fractions from bovine iris sphincter, ciliary body, and iris.
- Radioligand binding assays using 3H-PGE2 and 3H-PGF2 alpha.
- Scatchard analysis to determine binding site characteristics and competition studies to assess selectivity.
Main Results:
- PGE2 specific binding sites were predominantly found in the sphincter muscle (65%), followed by iris (17%) and ciliary body (18%).
- PGF2 alpha specific binding sites were not measurable in the iris or ciliary body.
- Binding in the bovine sphincter was primarily to EP receptors, with a potency order of PGE2 > PGF2 alpha > PGD2 > Iloprost.
Conclusions:
- The distribution of PGE2 binding sites in the bovine sphincter correlates with its contractile responses to prostanoids.
- The limited PGF2 alpha binding suggests its in vivo ocular effects may be mediated through PGE2 specific binding sites.
Purpose:
To determine the regional distribution and selectivity of prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) specific binding sites in membrane preparations from bovine iris ciliary body.
Methods:
Bovine eyes were obtained fresh from a local abattoir. Whole irides were separated into sphincter muscle, ciliary body, and remaining iris tissue then homogenized in 50 mM tris buffer, pH 7.5, containing cyclooxygenase, protease, and soybean trypsin inhibitors. Membranes were obtained after three-stage high-speed centrifugation then reconstituted in buffer. Aliquot portions were incubated with 3H-PGE2 or 3H-PGF2 alpha at 37 degrees C for 30 min in a final volume of 500 microliters. Competition studies were performed in the presence of up to 1000-fold excess unlabeled ligand. At the end of a 30-min incubation period, free and membrane bound ligand were separated by rapid filtration through a type HA millipore filter preequilibrated with buffer. The radioactivity bound to the membranes retained on the filter was quantitated in a scintillation counter.
Results:
The equilibrium dissociation constant and maximum number 3H-PGE2 saturable binding sites were determined by Scatchard analysis. The data best fit to a single binding site model for all three membrane preparations. The majority of the 3H-PGE2 specific binding sites were in sphincter muscle (65%), followed by iris (17%), and ciliary body (18%). 3H-PGE2 alpha binding sites were not measurable in either iris or ciliary body and PGF2 alpha was less competitive than PGE2 in all tissues. The rank order of potency for agonist displacement by both 3H-PGE2 and 3H-PGF2 in sphincter muscle membrane was PGE2 > PGF2 alpha > PGD2 > Iloprost. It was determined that 3H-PGE2 and 3H-PGF2 alpha binding in the bovine sphincter was primarily to EP not FP, DP, or IP prostaglandin receptor types.
Conclusions:
The characteristics of PGE2 and PGF2 alpha specific binding sites in the bovine sphincter correlate with its in vitro contractile response to these prostanoids. The inability of PGF2 alpha to effectively compete with PGE2 for specific binding sites in the sphincter and lack of high affinity PGF2 alpha specific binding sites in the iris and ciliary body suggests that this prostaglandin may exert its in vivo effects through PGE2 specific binding sites.