No abstract

The role of the neuropeptide galanin in the regulation of anterior pituitary function was studied in vivo in conscious male rats and in vitro with cultured anterior pituitary cells. injected into the third cerebral ventricle of rats produced highly significant, doserelated increases of plasma growth hormone (GH) concentrations, whereas galanin increased prolactin (PRL) and decreased thyroid-stimulating hormone (TSH) levels only at the highest dose (60 pmol) tested. Intravenous galanin failed to alter PRL and TSH levels in these rats. In contrast with the results with intraventricular injection of the peptide, intravenous injection of 30 or 300 pmol of galanin produced small, brief, dose-related increases in plasma GH. The response to the 300-pmol dose was less than that induced by a factor-of-20-lower intraventricular dose, which establishes a central action of galanin. Galanin in concentrations ranging from 1 nM to 1 gAM failed to alter significantly GH, PRL, or TSH release from dispersed anterior pituitary cells. It also failed to alter GH secretion in response to 100 nM GH-releasing hormone; however, at this dose galanin did potentiate the effect of 100 nM TSH-releasing hormone on TSH and PRL release. Thus, the effects ofthird-ventricular injection ofthe peptide are mediated by the hypothalamus. To determine the physiological significance of galanin in control of pituitary hormone release, highly specific antiserum against galanin was injected intraventricularly. Third-ventricular injection of 3 ,ul of galanin antiserum resulted in a dramatic decrease in plasma GH values as compared with those of normal rabbit serum-injected controls within 15 min, which persisted until the end of the experiment (5 hr postinjection). Galanin antiserum did not decrease plasma PRL or TSH levels at any time period after its third-ventricular injection; however, a transient increase of plasma TSH levels occurred after 30 and 60 min in comparison with TSH levels in normal rabbit serum-injected controls.Since there was no effect of the antiserum on plasma PRL and only a transient elevation in TSH, galanin may not be physiologically significant enough during resting conditions to alter PRL and TSH release in the male rat. The results of the experiments with galanin antiserum indicate that endogenous galanin has a tonic action within the hypothalamus to stimulate GH release. The rapidity of onset of the effects of galanin and the antiserum directed against it suggest that it acts to stimulate release of GH-releasing hormone from periventricular structures, which then stimulates the release of GH.The gastrointestinal peptide galanin has been isolated from porcine intestinal extracts (1). Galanin has been shown to cause contraction of smooth muscle preparations (1, 2) and to inhibit both substance P-and acetylcholine-induced smooth muscle contraction (2). Furthermore, galanin causes hyperglycemia in dogs via inhibition of insulin release (1, 3).Galanin is widely distributed in the rat brain and intestine (4-8). In the central ...

Epoxyeicosatrienoic acids (EETs), lipid mediators synthesized from arachidonic acid by cytochrome P-450 epoxygenases, are converted by soluble epoxide hydrolase (SEH) to the corresponding dihydroxyeicosatrienoic acids (DHETs). Originally considered as inactive degradation products of EETs, DHETs have biological activity in some systems. Here we examined the capacity of EETs and DHETs to activate peroxisome proliferator-activated receptor-α (PPARα). We find that among the EET and DHET regioisomers, 14,15-DHET is the most potent PPARα activator in a COS-7 cell expression system. Incubation with 10 μM 14,15-DHET produced a 12-fold increase in PPARα-mediated luciferase activity, an increase similar to that produced by the PPARα agonist Wy-14643 (20 μM). Although 10 μM 14,15-EET produced a threefold increase in luciferase activity, this was abrogated by the SEH inhibitor dicyclohexylurea. 14-Hexyloxytetradec-5( Z)-enoic acid, a 14,15-EET analog that cannot be converted to a DHET, did not activate PPARα. However, PPARα was activated by 2-(14,15-epoxyeicosatrienoyl)glycerol, which was hydrolyzed and the released 14,15-EET converted to 14,15-DHET. COS-7 cells incorporated 14,15-[3H]DHET from the medium, and the cells also retained a small amount of the DHET formed during incubation with 14,15-[3H]EET. Binding studies indicated that 14,15-[3H]DHET binds to the ligand binding domain of PPARα with a Kd of 1.4 μM. Furthermore, 14,15-DHET increased the expression of carnitine palmitoyltransferase 1A, a PPARα-responsive gene, in transfected HepG2 cells. These findings suggest that 14,15-DHET, produced from 14,15-EET by the action of SEH, may function as an endogenous activator of PPARα.

We investigated the presence of nitric oxide (NO) synthase in ovarian follicular cells obtained from women undergoing in vitro fertilization procedures. Endothelial NO synthase messenger RNA was demonstrated by polymerase chain reaction amplification of reverse transcribed RNA. NO synthase was localized to granulosa-luteal cells by immunocytochemistry, using a monoclonal antibody. Ovarian follicular cell NO synthase enzyme activity was confirmed by measuring the conversion of L-arginine to citrulline. To investigate the effect of NO on granulosa-luteal cell steroidogenesis, NO synthase inhibitors and NO donors were added to cell cultures. NG-Monomethyl-L-arginine and N-nitro-arginase methyl ester, selective inhibitors of NO synthase, significantly increased estradiol secretion by granulosa-luteal cells. S-Nitroso-L-acetyl penicillamine (S-NAP) and S-nitroso glutathione, NO donors, caused a dose-dependent decrease in both estradiol and progesterone secretion. The decrease by S-NAP was reversed by hemoglobin, which binds free NO. Although S-NAP increased the concentration of cGMP in granulosa-luteal cells, cGMP analogs had no effect on steroidogenesis in cell cultures. S-NAP and native NO in solution decreased cellular and microsomal aromatase activities. We conclude that NO synthase is present in human granulosa-luteal cells and that NO inhibits estradiol secretion independent of cGMP by directly inhibiting aromatase.

Soluble epoxide hydrolase (sEH) plays a major role in regulating vascular epoxyeicosatrienoic acid metabolism and function, and substituted urea derivatives that inhibit sEH activity reduce blood pressure in hypertensive rats. We found that substituted urea derivatives containing a dodecanoic acid group, besides effectively inhibiting sEH, increased peroxisome proliferatoractivated receptor (PPAR) ␣ activity. In PPAR␣ transfected COS-7 cells, treatment with 10 M N-cyclohexyl-NЈ-dodecanoic acid urea (CUDA) or N-adamantanyl-NЈ-dodecanoic acid urea (AUDA) produced 6-and 3-fold increases, respectively, in PPAR␣ activation. Neither CUDA nor AUDA activated PPAR␦ or PPAR␥ directly, indicating selectivity for PPAR␣. CUDA did not alter PPAR␣ protein expression, and it competitively inhibited the binding of Wy-14643 (pirinixic acid) to the ligand binding domain of PPAR␣, suggesting that it functions as a PPAR␣ ligand. CUDA and AUDA were metabolized to chain-shortened ␤-oxidation products, a process that reduced their potency as sEH inhibitors and their ability to bind and activate PPAR␣. N,NЈ-Dicylclohexylurea and N-cyclohexyl-NЈ-dodecylurea, sEH inhibitors that do not contain a carboxylic acid group, did not activate PPAR␣. In HepG2 cells, CUDA increased the expression of the PPAR␣-responsive gene carnitine palmitoyltransferase 1A. We conclude that CUDA and AUDA, by virtue of their carboxylic acid substitution, activate PPAR␣ in addition to potently inhibiting sEH. Further development of these compounds could lead to a class of agents with hypotensive and lipid-lowering properties that may be valuable for the prevention and treatment of cardiovascular disease.