Dehydroepiandrosterone prevents the aggregation of platelets obtained from postmenopausal women with type 2 diabetes mellitus through the activation of the PKC/eNOS/NO pathway.

The steroid hormone dehydroepiandrosterone (DHEA), suggested to be a cardioprotector, prevents platelet aggregation in healthy humans. This hormone is reduced in postmenopausal women by 60% of its normal value. Platelets in patients with type 2 diabetes (T2D) are more sensitive to aggregation, which has been attributed to a reduced ability to produce nitric oxide (NO). In light of these precedents and considering that DHEA is able to increase the production of NO in cultured endothelial cells, we suggest that DHEA prevents the aggregation of platelet from postmenopausal women with T2D through the activation of PKC/eNOS/NO/cGMP pathway. To determine the effect of DHEA in platelet aggregation, platelet-rich plasma (PRP) obtained from postmenopausal women with T2D was preincubated with DHEA, and aggregation induced by ADP was determined in the presence or absence of L-NNA (LNG-nitroarginine), Rottlerin, NOS, or PKC delta inhibitors, respectively. Platelet NO production was measured with the fluorescent probe DAF2DA and eNOS activation was determined by Western blot, using an anti-p-eNOS (ser 1177) antibody. DHEA 1) prevented platelet aggregation by 40% compared to control, 2) increased NO production by 63%, 3) increased p-eNOS (phosphorylated endothelial nitric oxide synthase) levels, and 4) increased cGMP production. These effects were reduced in the presence of L-NNA or Rottlerin. DHEA prevents platelet aggregation induced by ADP. This effect is mediated by the activation of the PKCδ/eNOS/NO/cGMP pathway. Our results suggest that DHEA could be considered to be a potential therapeutic tool in the prevention of atherothrombotic processes in postmenopausal women with T2D.

Melatonin and testicular function: characterization of binding sites for 2-[125I]-iodomelatonin in immature rat testes.

Melatonin-binding sites in membrane preparation immature rat testes were demonstrated by utilizing 2-[125I]-iodomelatonin as a radioligand. Binding at these sites was found to be reversible, saturable, specific and of, high affinity. Scatchard analysis of the specific binding revealed an equilibrium binding constant (kd) of 215 +/- 23 pmol/L and a total number of binding sites (Bmax) of 0.94 +/- 0.1 fmol/mg protein. The Hill coefficient of 1.0 suggests a single class of 2-[125I]-iodomelatonin-binding site in the rat testes. The Kd value determined from kinetic analysis was 179 pmol/L, which is in close agreement with the value determined from equilibrium studies. In competition studies, the order of pharmacological affinity for 2-[125I]-iodomelatonin binding sites in the rat membrane testes was: melatonin > 6-hydroxymelatonin > N-acetylserotonin > 5-hydroxyindole-3-acetic acid > 5-hydroxytryptamine > 5-hydroxy-L-tryptophan > tryptamine > > 5-methoxytryptamine, 5-methoxyl-DL-tryptophan, D-L-tryptophan. The 2-[125I]-iodomelatonin binding was markedly reduced by guanine nucleotides; treatment with nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) caused a 10-fold decrease in receptor affinity. In this paper, we report evidence indicating the presence of binding sites in immature rat tests, suggesting a possible direct role of melatonin on testicular steroidogenesis.

Melatonin binding sites in interstitial cells from immature rat testes.

Melatonin binding sites in rat testes interstitial cells were identified using 2-[125I]-iodomelatonin. Saturation studies of cells revealed a single class of high affinity binding sites, with an apparent equilibrium dissociation constant (Kd) of 100 +/- 20 pM, and a total binding capacity (B max) of 3.0 +/- 0.2 x 10(3) melatonin molecules per cell. Binding was reversible and inhibited by non radioactive melatonin. These results suggest that interstitial cells from immature rat testes have specific receptors for melatonin.

Melatonin binding sites in interstitial cells from immature rat testes

Melatonin binding sites in rat testes interstitial cells were identified using 2-[125I]-iodomelatonin. Saturation studies of cells revealed a single class of high affinity binding sites, with an apparent equilibrium dissociation constant (Kd) of 100 +/- 20 pM, and a total binding capacity (B max) of 3.0 +/- 0.2 x 10(3) melatonin molecules per cell. Binding was reversible and inhibited by non radioactive melatonin. These results suggest that interstitial cells from immature rat testes have specific receptors for melatonin

Testicular function of sexually immature rats chronically treated with melatonin.

Melatonin disturbs the reproductive process in seasonal but not in continuous breeders: however, it delays sexual development in the rat. The testicular function of rats injected daily with melatonin from 20 up to 25, 30, 35 or 40 days of age was analyzed. The spermatogenic and androgenic activity of testes was assessed by light microscopy and by the capacity for binding hCG and producing testosterone in vitro, respectively. In addition, LH, FSH and testosterone plasma levels were measured and 3B-hydroxysteroid dehydrogenase activity of Leydig cells was assessed histochemically to aid in the interpretation of results. Rats treated with melatonin for 15 or 20 days presented at the end of the juvenile period, abnormal progression of spermatogenesis and decreased ability of their Leydig cells to produce testosterone both in vivo and in vitro. This was associated with a lower number of binding sites for hCG and diminished production of testosterone in response to receptor and post-receptor mediated stimulation of steroidogenesis. Melatonin caused a marked decrease in LH serum levels. The diminished LH supply to the testis probably interfered with differentiation or impaired the functional ability of Leydig cells. As a consequence, testicular testosterone production was insufficient to support normal spermatogenic progression and growth and development of the sexual accessory organs.