It is proposed that the action of lh on oocyte maturation involves the mediation of the adenyl cyclase/cyclic AMP system and possibly of the prostaglandins. An action of steroids could not thus far be implicated. The experimental model described permits study ofthe mechanism of the meiosis-inducing action of lh under controlled conditions in vitro.

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We have previously shown that vanadate potentiates the activating effect of phorbol ester (TPA) on cellular phospholipase A2 (PLA2) in a pathway dependent on the formation of reactive oxygen species (ROS). Here we evaluate the chain of enzymes (protein kinases and phosphatases) that participate in this process. Treatment of macrophages with vanadate plus TPA led to activation of protein kinase C (PKC) and NADPH oxidase (O2- generation in intact cells), massive cellular protein tyrosine phosphorylation, suppression of protein tyrosine phosphatase (PTP) activity and a sustained activation of protein tyrosine kinase (PTK) and myelin basic protein kinase activity (the latter three enzyme activities were assessed in cell lysates). Inhibition of ROS formation by diphenyleneiodonium (DPI) prevented PTP inhibition, PTK activation and protein tyrosine phosphorylation by vanadate plus TPA. Vanadate plus H2O2 mimicked the effect of vanadate plus TPA on PKC activation, cellular protein tyrosine phosphorylation, PTP and PTK, but their effects were resistant to DPI. Suppression of PKC activity (down-regulation; selective inhibitors) prevented the above-mentioned effects of vanadate plus TPA, but not of vanadate plus H2O2. Collectively, the results show that ROS formation induced by TPA in association with vanadate is essential in the modulation of protein tyrosine phosphorylation and PLA2 activity.

Mechanical forces applied to cultured bone cells induce the production of cAMP via stimulation of the formation of prostaglandin E2 (PGE2) and its release into the medium, resulting in stimulation of adenylate cyclase. In this paper we show that either the antibiotic gentamycin (100 micrograms/ml) or antiphospholipid antibodies (0.1%) which bind to membrane phospholipids abolish cAMP formation induced by mechanical forces; exogenously added arachidonic acid or PGE2 stimulates cAMP formation, even in the presence of these agents. Addition of exogenous phospholipase A2 (but not phospholipase C) causes an increase in the formation of cAMP in bone cells, a response that is also inhibited by gentamycin or antiphospholipase antibodies. These observations suggest that mechanical forces exert their effect on bone cells via the following chain of events: (1) activation of phospholipase A2, (2) release of arachidonic acid, (3) increased PGE synthesis, (4) augmented cAMP production.

The actions of luteinizing hormone (LH) and of prostaglandin E2 (PGE2) on the intact ovary or isolated components of the ovary from adult or prepubertal rats were studied in vitro with respect to (1) the rate of incorporation of [3H]adenine via ATP into cyclic adenosine-3\m='\,5\m='\-monophosphate (cAMP), or the level of cAMP measured by a competitive protein-binding assay; (2) protein kinase activity in the 27000 g supernatant, with calf thymus histone as the substrate; and (3) rate of oxidation of d-[6-14C]glucose. In addition, ornithine decarboxylase activity was assayed in the ovary in vitro after hormone treatment in vivo.When ovaries from adult or pubertal rats were incubated in Krebs\p=m-\ Ringer bicarbonate buffer, addition of PGE2 to the medium resulted within 1 min in a doubling of the rate of cAMP formation; the rate was about fourfold after 5 min. PGE2 was 25 times more potent in this respect than prostaglandin F2\ g=a\ . Both isolated Graafian follicles and corpora lutea responded to either PGE2 or LH with increased cAMP production. During the perinatal period and until the age of 10 days the ovaries were unresponsive to LH, but responded to PGE2 with increased cAMP formation. Follicles cultured with LH for 18 h and then washed were refractory to subsequent stimulation by LH, yet remained fully responsive to PGE2.A prostaglandin analogue, 7-oxa-13-prostynoic acid, did not inhibit LH-stimulated or PGE2-stimulated cAMP formation in vitro in whole ovaries. Indomethacin, a substance reported to inhibit prostaglandin synthesis in other tissues, likewise failed to inhibit this action of LH. Simultaneous addition of LH and PGE2 to the incubation medium augmented cAMP production to a significantly greater extent than did either agonist when added at maximally effective concentrations on their own, though this augmentation was short of a full additive effect. The latter finding provides presumptive evidence against the view that PGE2 is an obligatory mediator of LH action on the ovary, but this question remains open.PGE2 mimicked the action of LH in stimulating glucose oxidation by ovaries in vitro, and in causing a 15-fold increase in ovarian ornithine decarboxylase activity within 4 h of injection into prepubertal rats.Ovarian protein kinase activity in pubescent (28-day-old) rats was markedly enhanced by incubation of intact ovaries with LH or PGE2 or by exogenous cAMP added to the 27000 g supernatant. The stimulatory action of LH or PGE2 on the enzyme was attended by a significant decrease (to 10% of control value) in the binding of exogenous cyclic [3H]AMP to protein in the 2000 g supernatant fraction of the ovary, presumably reflecting saturation of the cAMP-binding regulatory subunit of protein kinase by enhanced endogenous generation of the cyclic nucleotide.Stimulation of ovarian protein kinase by exogenous cAMP was demonstrable in 12-day-old rats, but insignificant at the age of 7 days. It thus appears that the competence of ovarian protein kinase to respond to cAMP, and of ovarian adenylate cyclase...

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