No abstract

The effects of expression of the Drosophila melanogaster Trpl protein, which is thought to encode a putative Ca2+ channel [Phillips, Bull and Kelly (1992) Neuron 8, 631-642], on divalent cation inflow in Xenopus laevis oocytes were investigated. The addition of extracellular Ca2+ ([Ca2+]0) to oocytes injected with trpl cRNA and to mock-injected controls, both loaded with the fluorescent Ca2+ indicator fluo-3, induced a rapid initial and a slower sustained rate of increase in fluorescence, which were designated the initial and sustained rates of Ca2+ inflow respectively. Compared with mock-injected oocytes, trpl-cRNA-injected oocytes exhibited a higher resting cytoplasmic free Ca2+ concentration ([Ca2+]i), and higher initial and sustained rates of Ca2+ inflow in the basal (no agonist) states. The basal rate of Ca2+ inflow in trpl-cRNA-injected oocytes increased with (1) an increase in the time elapsed between injection of trpl cRNA and the measurement of Ca2+ inflow, (2) an increase in the amount of trpl cRNA injected and (3) an increase in [Ca2+]0. Gd3+ inhibited the trpl cRNA-induced basal rate of Ca2+ inflow, with a concentration of approx. 5 microM Gd3+ giving half-maximal inhibition. Expression of trpl cRNA also caused an increase in the basal rate of Mn2+ inflow. The increases in resting [Ca2+]1 and in the basal rate of Ca2+ inflow induced by expression of trpl cRNA were inhibited by the calmodulin inhibitors W13, calmodazolium and peptide (281-309) of (Ca2+ and calmodulin)-dependent protein kinase II. A low concentration of exogenous calmodulin (introduced by microinjection) activated, and a higher concentration inhibited, the trpl cRNA-induced increase in basal rate of Ca2+ inflow. The action of the high concentration of exogenous calmodulin was reversed by W13 and calmodazolium. When rates of Ca2+ inflow in trpl-cRNA-injected oocytes were compared with those in mock-injected oocytes, the guanosine 5'-[beta-thio]diphosphate-stimulated rate was greater, the onset of thapsigargin-stimulated initial rate somewhat delayed and the inositol 1,4,5-trisphosphate-stimulated initial rate markedly inhibited. It is concluded that (1) the divalent cation channel activity of the Drosophila Trpl protein can be detected in Xenopus oocytes: (2) in the environment of the Xenopus oocyte the Trpl channel admits some Mn2+ as well as Ca2+, is activated by cytoplasmic free Ca2+ (through endogenous calmodulin) and by a trimeric GTP-binding regulatory protein, but does not appear to be activated by depletion of Ca2+ in the endoplasmic reticulum; and (3) expression of the Trpl protein inhibits the process by which the release of Ca2+ from intracellular stores activates endogenous store-activated Ca2+ channels.

OBJECTIVES:To investigate (1) whether type 3 17b-hydroxysteroid dehydrogenase (17b-HSD), the enzyme which catalyzes the conversion of androstenedione to testosterone in the testis, is co-expressed with P450aromatase in the preadipocytes of women, and (2) whether the relative expression of type 3 17b-HSD and aromatase varies in subcutaneous abdominal vs intra-abdominal adipose tissue of women. SUBJECTS: Subcutaneous abdominal and intra-abdominal adipose tissue was obtained from women undergoing elective abdominal surgery (age 22 -78 y, body mass index (BMI) 22.4 -52.9 kg=m 2 ). MEASUREMENTS: Expression of type 3 17b-HSD in adipose cell fractions was determined using RT-PCR. Preadipocyte steroidogenesis was investigated in primary cultures using androstenedione as substrate. Messenger RNA levels for type 3 17b-HSD and aromatase were measured in adipose tissue from the subcutaneous abdominal and intra-abdominal depots using a quantitative multiplex competitive RT-PCR assay. RESULTS: Type 3 17b-HSD is co-expressed with aromatase in the abdominal preadipocytes of women. Cultured preadipocytes from both subcutaneous abdominal (n ¼ 5) and intra-abdominal (n ¼ 5) sites converted androstenedione to testosterone, and there was minimal conversion of androstenedione to estrone. Consistent with this, the levels of type 3 17b-HSD mRNA were significantly higher than aromatase mRNA at both sites (P < 0.05; n ¼ 8 subcutaneous abdominal, n ¼ 12 intra-abdominal adipose tissue). The ratio of levels of 17b-HSD mRNA to aromatase mRNA in intra-abdominal adipose tissue was positively correlated with BMI (n ¼ 11, r ¼ 0.61, P < 0.05) and waist circumference (n ¼ 10, r ¼ 0.65, P < 0.05). The converse was found in subcutaneous abdominal adipose tissue. CONCLUSION: The intra-abdominal adipose tissue of women may be substantially androgenic, increasingly so with increasing obesity, particularly central obesity. While androgen production by this adipose tissue deposit may not contribute to circulating testosterone levels due to hepatic clearance, it may have hitherto unrecognised local effects in the intra-abdominal adipose tissue and also on the liver via the hepatic portal system. These studies suggest a mechanism linking central obesity with insulin resistance and dyslipidaemia.

No abstract