Phorbol ester, prolactin, and relaxin cause translocation of protein kinase C from cytosol to membranes in human endometrial cells.

Incubation of endometrial cells with 100 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 2, 5, 10 and 30 min decreased cytosolic protein kinase C (PKC) activity to 80%, 68%, 66%, and 72% of the control values, while membrane-associated PKC increased to 116%, 168%, 154% and 134% of the control values, respectively. Long-term incubation of cells with TPA resulted in a loss in total PKC activity. Treatment of secretory endometrial cells with prolactin (100 ng/ml) decreased cytosolic PKC to 64% (10 min) and 72% (20 min) while membrane PKC increased to 133% (10 min) and 158% (20 min) compared to control values. Relaxin (100 ng/ml) also caused translocation of PKC in secretory endometrium. Neither hormone induced PKC translocation in proliferative endometrium. In intact endometrial cells TPA stimulated the phosphorylation of an 80 kDa protein. Cytosolic protein phosphorylation in the presence of EGTA resulted in phosphorylation of proteins of 68 kDa and 19 kDa which was increased by prolactin. Upon activation by calcium and phosphatidylserine, PKC phosphorylated a protein of 39 kDa, and prolactin did not further enhance its phosphorylation. The present results indicate that TPA induces an intracellular translocation and down-regulation of PKC. The translocation of PKC by prolactin and relaxin suggests an involvement of this enzyme in the action of these hormones in human endometrium.

Stimulation of pituitary hormone secretion by neurotransmitter amino acids in humans.

The effects of several neurotransmitter amino acids on pituitary hormone secretion were examined in normal humans. Oral administration of 10 g of glutamic acid stimulated the secretion of prolactin (PRL) and cortisol to approximately twice baseline values, with no effect on GH, TSH or LH. Aspartic acid (10 g), taurine (5 g), and cysteine (5 or 10 g) had no consistent effect on any hormone measured, although the lack of effect of aspartic acid may relate to the modest increments in serum concentration achieved. Glutamic acid may be an important modulator of PRL and ACTH secretion in humans.

Identification of calmodulin-sensitive and calmodulin-insensitive adenylate cyclase in rat kidney.

Adenylate cyclase from rat kidney membranes solubilized with Lubrol-PX, was resolved into calmodulin-insensitive and calmodulin-sensitive forms using DEAE-Sephacel and calmodulin-Sepharose affinity chromatography. The major fraction, 90% of the activity recovered, did not bind to the calmodulin-Sepharose in the presence of Ca2+, and was insensitive to activation by calmodulin. The calmodulin-sensitive enzyme, approximately 10% of the recovered activity, bound to the affinity column and was eluted with buffer containing 2 mM EGTA. In the presence of free Ca2+, calmodulin increased the specific activity of the calmodulin-sensitive adenylate cyclase from 15.2 to 60.4 pmol/mg protein-1 min-1. Maximum stimulation occurred at 0.035-0.076 mM Ca2+. The apparent Ka for calmodulin was 8 nM. The calmodulin-mediated increase in activity was inhibited by trifluoperazine, but not by its analog trifluoperazine-5-oxide. In contrast, trifluoperazine did not inhibit the calmodulin-insensitive activity. The GTP analog, guanyl-5'-yl imidodiphosphate, did not activate either fraction. Furthermore, activation by calmodulin did not require the presence of a guanyl nucleotide. The present finding of a calmodulin-sensitive form of adenylate cyclase in kidney raises the possibility that a calmodulin-mediated mechanism is involved in the formation of cAMP in this organ.

Involvement of calmodulin in the regulation of adenylate cyclase activity in guinea-pig enterocytes.

The involvement of calmodulin as an activator of adenylate cyclase activity was examined in isolated guinea-pig enterocytes and in a membrane preparation. In enterocytes, which responded to prostaglandin E1, vasoactive intestinal peptide and cholera toxin with a significant increase in the rate of cAMP formation trifluoperazine, a calmodulin antagonist, completely inhibited cAMP formation. In a membrane preparation adenylate cyclase activity was stimulated 10-20-fold by the GTP analog, guanosine 5'-[beta-imido]5'-triphosphate (Gpp[NH]p). Prostaglandin E1 and vasoactive intestinal peptide enhanced cAMP formation in this system by 2-3- and 1.2-1.6-fold. respectively. Addition of 200 nM calmodulin to membranes, in which endogenous calmodulin was decreased from 1.4 microgram/mg protein to 0.5 microgram/mg protein by washing with buffer containing EGTA and EDTA, resulted in a 3-4-fold increase of adenylate cyclase activity. The absolute increment in adenylate cyclase activity caused by calmodulin (10-15 pmol cAMP/min per mg protein) was approximately the same in the absence or presence of Gpp[NH]p. The apparent Ka for Gpp[NH]p (6 . 10-7 M) was not significantly changed by the addition of calmodulin. Although endogenous calcium (approx. 10 microM) in the enzyme assay was adequate to affect stimulation by calmodulin, a maximal effect was observed at a calcium concentration of 100 microM. These findings indicate that a calmodulin-sensitive form of adenylate cyclase is present in guinea-pig enterocytes, and that stimulation of cAMP formation in the intestinal mucosa may involve a calmodulin-mediated mechanism.

Hepatic metabolism of vitamin D3 in streptozotocin-induced diabetic rat.

The effect of streptozotocin-induced diabetes on the in vitro conversion of vitamin D3 to 25-hydroxy-vitamin D3 (25-OHD3) by isolated liver microsomes from rachitic rats was examined. Enzymic activity was significantly less than that observed in control animals (P less than 0.001). Administration of insulin restored activity almost to control values. These findings provide evidence that diabetes in this animal model produces alterations in the metabolism of vitamin D.

Serum thyroid hormones changes in patients undergoing Caesarean section under general or regional anaesthesia.

The effects of anaesthesia on serum thyroid hormones were studied in 32 pregnant young women undergoing Caesarean section at term. Eighteen patients received general anaesthesia and 14 lumbar extradural blockade. Maternal serum concentrations of thyrotrophin (TSH), thyroxine (T4), triiodothyronine (T3) and reverse triiodothyronine (rT3) were measured using radioimmunoassay at 0, delivery and 24 h. There were no significant changes in TSH in the two groups. T4 concentrations decreased significantly at 24 h in the general anaesthesia group but regional anaesthesia produced a significant decrease only at the time of delivery. T3 concentrations decreased with both techniques. Reverse T3 increased markedly with general anaesthesia only at 24 h.

Significance of serum level of 25-hydroxycholecalciferol in gastrointestinal disease.

The serum 25-OHCC concentration was measured in 151 patients in order to evaluate the potential use of this determination in the management of gastrointestinal disorders. Patients with functional bowel disease had lower serum mean 25-OHCC levels than normals. The results were divided into normal (greater than 21 ng/ml), low normal (12-21 ng/ml), and low levels (less than 21 ng/ml). Two thirds of patients with malabsorption had low serum 25-OHCC. Most patients following jejunoileal bypass surgery for morbid obesity had low levels despite supplemental oral calciferol therapy. In patients with chronic liver disease, cholestasis more than parenchymal cell disease appeared responsible for low serum 25-OHCC levels. Measurement of serum 25-OHCC may be an ancillary screening test for fat malabsorption and in patients with intraluminal bile salt deficiency. Furthermore, repeated measurements may be useful in monitoring therapy with vitamin D products in patients with chronic malabsorption and cholestasis.

Effect of human chorionic gonadotrophin on adenylate cyclase activity and testosterone content in rat testicular mitochondria.

The adneylate cyclase activity and the concentration of testosterone in testicular mitochondria from immature rats were measured after administration of human chorionic gonadotrophin (HCG) or dibutyryl cyclic AMP in vivo or in vitro. Intratesticular injection of HCG produced an increase in adenylate cyclase activity which preceded the rise in the level of testosterone, whereas addition of the trophic hormone in vitro resulted simultaneous increases. Administration of dibutyryl cyclic AMP in vivo enhanced the testosterone content of the mitochondria. However, the cyclic nucleotide added in vitro at concentrations up to 5 mmol/l had no effect. Cycloheximide injected intraperitoneally before the administration of HCG abolished the stimulatory effect of the trophic hormone on the level of testosterone in the mitochondria, whereas chloramphenicol had no effect. These results, although they confirm the role of cyclic AMP as an intermediate in the stimulatory effect of HCG on the concentration of testosterone in rat testis, do not support a role for mitochondrial adenylate cyclase in this action. A protein regulator(s) formed extramitochondrially appears to be involved in the stimulatory effect of gonadotrophins on steroidogenesis.

Amniotic fluid adenosine 3',5' - monophosphate in prostaglandin-induced midtrimester abortions.

Cyclic adenosine 3'5-monophosphate (cAMP) was serially measured in the amniotic fluid (AF) of 9 patients undergoing midtrimester abortions induced by intraamniotic prostaglandins. The results demonstrate an increase in AF cAMP ranging from 2- to 7-fold within the 6 hours of observation. The fetal heart tones were closely monitored by a Doppler instrument and the time from injection of abortifacient to fetal demise (IDT) and to fetal expulsion (IAT) was accurately recorded. No correlation between the rate of AF cAMP increase and IDT or IAT could be demonstrated. The concentration of cAMP in amniotic fluid obtained from patients with fetal demise in utero was lower than that found in AF of fetuses of corresponding gestational age where the fetus was alive. The significance of AF cAMP as a potential indicator of fetal distress is discussed.