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Analysis of Relaxin Release by Cultured Porcine Luteal Cells Using a Reverse Hemolytic Plaque Assay: Effects of Arachidonic Acid, Cyclo- and Lipooxygenase Blockers, Phospholipase A₂, and Melittin^*

MICHAEL J. TAYLOR and CHERYL L. CLARK

Department of Veterinary Physiology and Pharmacology, Iowa State University College of Veterinary Medicine Ames, Iowa 50011

Abstract

The effect of the obligatory precursor of prostaglandin biosynthesis, arachidonic acid, on the release of relaxin by porcine luteal cells was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis, a plaque, developed around relaxin-releasing luteal cells, identified as large luteal cells (LLCs). The rate of development of plaques in time-course studies and the area of plaques were then used as an index of the rate of relaxin release and cumulative amount of hormone released, respectively. Incubation of collagenase-dispersed luteal cells derived from early pregnant pigs with 0.1-100 µM arachidonic acid (AA) resulted in dose-dependent increases in the rate of plaque formation. Despite AA stimulation, however, only 55–65% of all LLCs formed plaques during the experimental incubation period (up to 12 h). Minimally and maximally effective doses were about 1 and 10 µM, respectively. In the presence of 10 µ AA, maximal plaque formation occurred significantly faster (1–2 h) than in controls (4–8 h; P < 0.05). The percentage of plaqueforming cells (plaque-forming LLCs) was, likewise, significantly greater in 10 µM AA-treated monolayers than in controls during the first 3–4 h of incubation. Similarly, agents that liberate endogenous AA (phospholipase A₂ and melittin) also stimulated relaxin release. The stimulatory effect of AA (10 µM) on relaxin release was almost wholly blocked by a cyclooxygenase inhibitor (ibuprofen; 20 µM), but not by a lipooxygenase inhibitor (nordihydroguaretic acid; 20 µM). However, the same dose of ibuprofen (20 µM) failed to modulate the stimulatory effect of prostaglandin E₂ (1 µM) or phorbol diester (4β-phorbol 12β-myristate 13-acetate; 50 nM) on the rate of relaxin release. These results indicate that a product(s) of the cyclooxygenase pathway of AA metabolism participates in the control of relaxin release, but that this metabolite(s) is not essential to the biological action of at least one stimulatory secretagogue. Moreover, this metabolite failed to influence a subpopulation of nonresponsive LLCs. These data taken in association with our previous demonstration that the pathways of both calcium mobilization and protein kinase-C activation are implicated in the regulation of relaxin release, are consistent with the view that AA liberation may amplify the actions of other signalling mechanisms.

Footnotes

^* This work was supported by Department of Agriculture Formula Funds and NIH Grant HD 22786. A preliminary report of this work was presented at the 71st Annual Meeting of the Endocrine Society, Seattle WA, 1989.

To whom all correspondence and requests for reprints should be addressed.

Received April 27, 1989.