Abstract
The role of diacylglycerol (DG) as a source of arachidonic acid during gonadotropin-releasing hormone (GnRH) stimulation of gonadotropin secretion was analyzed in primary cultures of rat anterior pituitary cells. An inhibitor of DG lipase (RHC 80267, RHC) caused dose-dependent blockade of GnRH-stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. The DG lipase inhibitor did not alter gonadotropin responses to arachidonic acid, and addition of arachidonic acid reversed its inhibition of GnRH-stimulated LH and FSH release. In [3H]arachidonic acid-prelabeled cells, incubation with RHC increased the accumulation of [3H]DG. These results suggest that DG lipase participates in GnRH action and that arachidonic acid mobilization from DG is involved in the mechanism of gonadotropin release. Gonadotropin responses to tetradecanoyl phorbol acetate and dioctanoyl glycerol were not altered by RHC, and the addition of these activators of protein kinase C (Ca2+- and phospholipid-dependent enzyme) did not prevent the inhibition of GnRH-induced gonadotropin release by RHC. Activation of phospholipase A2 by melittin increased LH and FSH secretion, whereas blockade of this enzyme by quinacrine reduced GnRH-stimulated hormone release. However, RHC did not diminish the gonadotropin response to melittin. The inhibitory actions of RHC and quinacrine were additive and were reversed by concomitant treatment with arachidonic acid. Ionomycin also increased LH and FSH release, and the gonadotropin responses to the ionophore were unaltered by RHC but were reduced by quinacrine. Incubation of cells in Ca2+-depleted (+/- [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) medium reduced but did not abolish the LH and FSH releasing activity of GnRH. Treatment with RHC also reduced the gonadotropin responses to GnRH under Ca2+-depleted conditions. These observations indicate that RHC inhibition of GnRH action is not due to nonspecific actions on Ca2+ entry, protein kinase C activation and actions, nor phospholipase A2 enzyme activity. The results of this study provide further evidence for an extracellular Ca2+-independent mechanism of GnRH action, and suggest that GnRH causes mobilization of arachidonic acid by two distinct lipases, namely, phospholipase A2 and DG lipase, during stimulation of gonadotropin secretion.
Highlights
From theEndocrinoloav and Rewroduction Research Branch, NationalInstitute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland20892
Arachidonic acid was less than that to 10 nM Gonadotropin-releasing hormone (GnRH), and the General-Anterior pituitaries were removed from sexually mature female rats at various stages of the estrous cycle(200-250g) and addition of arachidonic acid did not further enhance GnRHstimulatedLH release (Fig. 1, left panel).Incontrast,the dispersed by controlled trypsinization as previously described [27]
Addition of 10-100 p~ of the DG lipase gonadotrope preparations.' The luteinizing hormone (LH) response to 300 nM melinhibitor, RHC, did not alter basal gonadotropin release but ittin was similar to thatproduced by 10 nM GnRH (p > 0.05; completely inhibited GnRH-stimulated LH and FSH secre- Fig. 4), but the FSH response to melittin was greater than tion in a dose-dependent manner (p < 0.05)
Summary
Vol 263, No 35,Issue of December 15, pp. 18614-18620,1988 Printed in U.S.A. Dependence of Secretory Responses to Gonadotropin-releasing Hormone onDiacylglycerol Metabolism. GnRH-stimulated LH and FSH release.In r3H]arachi- mechanisms influence the secretion of the two gonadotropins donic acid-prelabeled cells, incubation with RHC in- in uiuo. The mechanisms by which GnRH that arachidonic acid mobilization froDmG is involved elicits LH secretion have been extensively studied using rat in themechanism of gonadotropin release. Activation of phospholipase AZ by melittin increased LH and FSH secretion, whereas blockaodfe this enzyme by quinacrine reduced GnRH-stimulated hormone release. The involvement of arachidonic acid and protein kinase C results of this study provide further evidence for an (Ca2+- andphospholipid-dependent enzyme) in the action of extracellular Ca2+-independent mechanism of GnRH GnRH has been proposed. $ Supported by a fellowshipfrom the Alberta Heritage Foundation erol; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonaic~id; for Medical Research.
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