Effect of human parathyroid hormone on the cAMP production and the endocrine functions of trophoblast cells from first trimester placenta.

Our previous study on teratocarcinoma cells suggested the role of human parathyroid hormone (hPTH) in early development of the placenta. The purpose of this study was to evaluate the possible role of hPTH on the functions of first trimester trophoblast cells. Adenylate cyclase activity in crude membranes from first trimester human placental villous tissue is stimulated 2-fold by hPTH (1-34) (10(-6) mol.l-1) from 265 +/- 32 to 532 +/- 80 pmol of cAMP/mg protein/15 min. A similar stimulation of adenylate cyclase is observed in human term placental villous tissue but not in 3 different choriocarcinoma cell lines. In order to evaluate the possible role of hPTH on the functions of first trimester human trophoblast cells, these cells were isolated by dispase and cultured (2 x 10(5) cells per plate) in DMEM supplemented with 20% fetal calf serum with or without 100 ng/ml of epidermal growth factor (EGF), for 4 d. On d 2 of culture, hPTH (10(-7) mol.l-1) stimulates cAMP production of these cells from 0.52 +/- 0.2 to 2.58 +/- 0.57 pmol.h-1 per 10(6) cells (mean +/- SEM). As compared to control (30 ng/ml), the output of hCG is increased by 1.5- (NS), 2- (P less than 0.01) and 3- (P less than 0.01) fold by EGF, hPTH, and hPTH added with EGF, respectively. Dibutyryl cAMP (10(-3) mol.l-1) increased hCG secretion by 3-fold (P less than 0.05). EGF and hPTH added separately or together significantly stimulated (P less than 0.01) the secretion of free alpha subunit 2-fold from 35 ng/ml to 70 ng/ml. In contrast, hPTH and EGF added separately did not change the secretion of free beta hCG. However, added together, they significantly increased (P less than 0.01) the secretion of free beta hCG after 48 h of culture, maximal stimulation (2.5 fold) being observed at d 4 of culture. In conclusion, human trophoblast cells are target cells for hPTH. hPTH acts in association with EGF in promoting expression of endocrine activity of these cells, such as hCG secretion. Trophoblast cells provide a model for the study of the cooperative effect between a peptide hormone and a growth factor in the regulation of endocrine function.     

Human parathyroid hormone (1-34) increases urinary excretion of lysosomal enzymes in rats

The kidney is the major target of parathyroid hormone (PTH), and PTH influences the urinary excretion of calcium, phosphate and hydrogen ions. It was previously reported that the urinary, excretion of N-acetyl-beta-D-glucosaminidase (NAG), a lysosomal enzyme, transiently increases after human PTH (hPTH) (1-34) infusion in normal subjects and idiopathic hypoparathyroidism patients, but not in pseudohypoparathyroidism type I patients. Here we report that intravenous infusion of hPTH(1-34) to rats transiently increased the urinary excretion of various lysosomal enzymes, such as beta-glucuronidase and acid phosphatase as well as NAG. However, it did not affect the urinary excretion of tubular brush border membrane enzymes, i.e. alkaline phosphatase, leucine aminopeptidase and gamma-glutamyl transpeptidase. Human PTH(1-34) dose-dependently increased the urinary excretion of NAG in rats with a peak at 30 min, which returned to a baseline within 60 min. The increase in the urinary NAG excretion caused by hPTH(1-34) positively correlated with the increase in the urinary cAMP excretion (r = 0.844, p < 0.01), and infusion of dibutyryl cAMP at a dose of 20 mg/kg similarly increased the urinary excretion of NAG. These results suggested that the increase in the urinary excretion of lysosomal enzymes caused by hPTH(1-34) may be a functional response to hPTH(1-34) occurring in the renal tubules via PTH signaling pathway.     

Progressive weakening of the response of key enzymes of liver glycogen metabolism to permanently increased plasma epinephrine levels

In adult male rats anaesthetized with pentobarbital the intravenous infusion of 0.5 micrograms.kg-1.min-1 of epinephrine increased liver phosphorylase a activity within 5 min, whereas later a weakening of the hormone effect was observed. After increasing the infusion rate to 1.0 micrograms.kg-1.min-1 and extending the study to more parameters, the diminishing effect on phosphorylase was confirmed and a similar response was established for liver cAMP. Concomitantly, a decrease and recovery of liver glycogen synthase a activity was observed. In rats with permanent catheters in one of their tail arteries for obtaining blood samples, the plasma epinephrine levels were shown to be permanently increased (from cca 1 pmol.ml-1 before infusion of 1.0 micrograms.kg-1.min-1 to more than 30 pmol.ml-1 during infusion) and remained at steady levels throughout the infusion. Therefore, the weakening of the epinephrine effect should be ascribed to changes at (or beyond) the catecholamine receptor level. A hitherto undescribed decrease of total glycogen synthase activity was observed during the infusions.     

Rat renal cell monolayer culture: a sensitive method for investigating ADH and PTH actions on the kidney by determining adenosine 3' :5'-cyclic monophosphate

The response of cAMP to antidiuretic hormone (ADH) was studied using rat renal medullary cells in a monolayer culture. In addition, cAMP response to parathyroid hormone (PTH) was studied in renal cortical cells. As the culture aged, an increase in basal cAMP content and a gradual decrease in the cAMP responsiveness to arginine vasopressin (AVP) were observed. After 2 days of culture, AVP and hPTH-(1-34) produced a rapid increase in intracellular cAMP with single peaks, after 10 min and 5 min, respectively. Extracellular cAMP was increased linearly by both AVP and hPTH-(1-34). The response of cAMP to AVP was markedly greater in the medulla than in the cortex, while the response to hPTH-(1-34) was remarkable only in the cortex. Outstanding sensitivity of cAMP responsiveness was observed in this system, i.e., 10(-12) M AVP (1 pg/ml) and 2.43 X 10(-10) M hPTH-(1-34) (1 ng/ml) provoked significant increases in cAMP from the basal level of 0.31 +/- 0.04 and 0.59 +/- 0.05 pmol/dish to 0.79 +/- 0.03 and 1.07 +/- 0.13 pmol/dish, respectively (P less than 0.001). In the medulla, potencies of lysine vasopressin (LVP), DDAVP and oxytocin at a concentration of 10(-9) M were 76.1%, 154.2% and 8.1% of that of AVP, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation of cAMP accumulation and phosphate uptake in opossum kidney (OK) cells with parathyroid hormone (PTH) and parathyroid hormone related protein (PTHrP)

Bovine parathyroid hormone (PTH) 1-34 [bPTH(1-34)] and human PTH related protein [hPTHrP(1-34)] stimulated cAMP accumulation in opossum kidney (OK) cells with Km of 5 x 10(-9) M, but inhibition of phosphate uptake was obtained with 17-fold lower Km of 3 x 10(-10) M. Phosphate uptake was partially inhibited with [Nle8.18Tyr34]bPTH(3-34)NH2 without concomitant cAMP stimulation. With hPTHrP(7-34)NH2, cAMP accumulation was increased in parallel to inhibition of phosphate uptake. [D-Trp12Tyr34]bPTH(7-34)NH2 and [Tyr34]hPTH(7-34)NH2 had no agonist activity on cellular cAMP and inhibition of phosphate uptake. bPTH(1-34)-stimulated cAMP accumulation was antagonized by [Nle8.18Tyr34]bPTH(3-34)NH2, [D-Trp12Tyr34]bPTH(7-34)NH2, hPTHrP(7-34)NH2 and [Tyr34]hPTH(7-34)NH2 with Ki of 1.4 x 10(-7), 2 x 10(-7), 4.7 x 10(-7) and 3.7 x 10(-6) M, respectively. But [Nle8.18Tyr34]bPTH(3-34)NH2 and [D-Trp12Tyr34]bPTH(7-34)NH2 reversed the inhibition of phosphate uptake only marginally, and hPTHrP(7-34)NH2 and [Tyr34]hPTH(7-34)NH2 were inactive. With hPTHrP(1-34) the Ki for cAMP accumulation of [Nle8,18Tyr34]bPTH(3-34)NH2 and hPTHrP(7-34)NH2 were 1.9 x 10(-7) and 7.2 x 10(-7) M, and inhibition of phosphate uptake was partially reversed with [Nle8,18Tyr34]bPTH(3-34)NH2, but not with hPTHrP(7-34)NH2. The present results indicate that truncated hPTHrP(7-34)NH2, unlike [Tyr34]hPTH(7-34)NH2 and [D-Trp12Tyr34]bPTH(7-34)NH2, elevates cellular cAMP and inhibits phosphate uptake. bPTH(1-34)- and hPTHrP(1-34)-evoked cAMP accumulation is suppressed by PTH and PTHrP fragments while inhibition of phosphate uptake remains largely unaltered.     

Human parathyroid hormone (1-34) transiently increases the excretion of lysosomal enzymes into urine and the size of renal lysosomes.

It has been reported that the urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG), a lysosomal enzyme, transiently increases in human after treatment with human parathyroid hormone (hPTH)(1-34). We report here that hPTH(1-34) caused transient changes in the size and density of rat renal lysosomes following urinary excretion of NAG and other lysosomal enzymes tested. Percoll density gradient centrifugation revealed that hPTH(1-34) slightly but significantly increased the fraction of high density lysosomes (around 1.12 g/ml) 5-10 min after the treatment with hPTH(1-34), with a concomitant decrease in the fraction of intermediate density lysosomes (1.07-1.08 g/ml). On electron micrographs, some lysosomes in proximal tubules but not in distal tubules showed a change in morphology from circular to oval, and became enlarged and electron-dense 5-10 min after the treatment with hPTH(1-34). These responses to hPTH(1-34) were also reversible and transient. NAG excreted in urine after treatment with hPTH(1-34) had the molecular mass of a mature form in lysosomes and/or endosomes and was not a prepro-and/or pro-form of the enzyme. Thus, the changes in the density and size of renal lysosomes appear to be associated with the exocytosis of lysosomal enzymes by hPTH(1-34).     

Fetal asphyxia stimulates an increase in fetal plasma catecholamines and [Met]-enkephalin-arg6-phe7 in the late-gestation sheep fetus

We have investigated whether enkephalin-containing peptides and catecholamines are increased in fetal plasma during periods of reduced uterine blood flow which produce moderate fetal asphyxia (i.e. hypoxemia, hypercapnia and acidemia). Experiments (n = 16) were performed in 11 ewes between 121-139 days gestation. In 8 experiments a clamp placed around the common iliac artery of the ewe was adjusted to produce a 50% reduction in the partial pressure of arterial oxygen (PO2) in fetal plasma for 30 min between 121-125 days gestation (n = 4) and between 131-139 days gestation (n = 4). Control (n = 8) experiments were performed when the arterial clamp was not adjusted. There was no significant effect of asphyxia on fetal plasma noradrenaline concentrations before 126 days gestation. After 130 days gestation during asphyxia, fetal plasma noradrenaline concentrations increased significantly from 2.20 +/- 0.72 pmol/ml (-15 min) to 14.06 +/- 0.75 pmol/ml (+5 min). The fetal adrenaline response to asphyxia did not change with increasing gestational age and after 130 days gestation fetal plasma adrenaline increased significantly from 1.48 +/- 0.46 pmol/ml (-15 min) to 4.05 +/- 1.22 pmol/ml (+10 min). Met-enkephalin-arg6-phe7 immunoreactivity was measurable (25-117 pg/ml) in all pre-experimental fetal sheep plasma samples collected between 121-139 days gestation. There was no specific effect of asphyxia on fetal plasma [Met]-enkephalin-arg6-phe7-IR before 130 days gestation. However after 130 days gestation, there was a significant increase in fetal plasma (Met-enkephalin Arg-6-phe7-IR above baseline values, when compared to control experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of alkaline phosphatase in the modulation of receptor signaling in osteoblasts: Evidence for a difference between human parathyroid hormone-related protein and human parathyroid hormone

We have previously reported that alkaline phosphatase (ALPase) is functionally involved in calcium uptake by several osteoblast-like cell lines. We have extended these studies to investigate the actions of ALPase on the cAMP response to and the receptor binding of human parathyroid hormone (hPTH) and human parathyroid hormone-related protein (hPTHrP). Pretreatment of human osteoblast-like SaOS-2 cells with human placental ALPase (hpALPase) inhibited the cAMP response to hPTH(1-34) but had no effect on the actions of hPTHrP(1-34) or vasoactive intestinal peptide. The inhibitory effect was reversed by L-Phe-Gly-Gly, an inhibitor of hpALPase. Treatment of SaOS-2 cells with hpALPase modestly reduced the binding of hPTH to 70% of control values, with little or no effect on the binding of hPTHrP. Bovine kidney and calf intestine ALPases were without effect on either the cAMP response or binding of hPTH or hPTHrP in SaOS-2 cells. In rat osteoblast-like ROS 17/2.8 cells, hpALPase had no effect on cAMP production stimulated by hPTH(1-34) or hPTHrP(1-34), arguing against a nonspecific effect of hpALPase. We suggest that, in SaOS-2 cells, the common PTH/PTHrP receptor can differentiate between the agonist activities of hPTH and hPTHrP by a mechanism that is sensitive to hpALPase. © 1994 Wiley-Liss, Inc.     

Dose-dependent effects of a pyridoquinazoline thromboxane synthetase inhibitor on arachidonic acid metabolites and hemodynamics during E. coli endotoxemia in anesthetized sheep

We investigated the effects of a new pyridoquinazoline thromboxane synthetase inhibitor infused before administering Escherichia Coli endotoxin into 18 anesthetized sheep with lung lymph fistulas. In normal sheep increasing plasma Ro 23-3423 concentrations were associated with increased plasma levels of 6-keto-PGF1 alpha, a reduced systemic vascular resistance (SVR, r = -0.80) and systemic arterial pressure (SAP, r = -0.92), the mean SAP falling from 80 to 50 mm Hg at the 20 and 30 mg/kg doses. Endotoxin infused into normal sheep caused transient pulmonary vasoconstriction associated with increased TxB2 and 6-keto-PGF1 alpha levels while vasoconstriction and TxB2 increase were significantly inhibited by pretreatment with Ro 23-3423 in a dose-dependent manner. When compared to controls, plasma and lymph levels of 6-keto-PGF1 alpha, PGF2 alpha and PGE2 after endotoxin infusion were increased several-fold by administering Ro 23-3423 up to plasma levels of 10 micrograms/ml. Doses over 30 mg/kg with blood levels above 10 micrograms/ml reduced plasma and lymph levels of 6-keto-PGF1 alpha, PGF2 alpha and PGE2, suggesting cyclooxygenase blockade at this dose. The peak 6-keto-PGF1 alpha levels at 60 min after endotoxin infusion in sheep with Ro-23-3423 levels below 10 micrograms/ml were associated with the greatest systemic hypotension due to a reduced SVR (r = -0.86). After endotoxin infusion the leukotrienes B4, C4, D4 and E4 in lung lymph were assayed by radioimmunoassay and high pressure liquid chromatography and remained at baseline values.     

The effect of atropine on bombesin and gastrin releasing peptide stimulated gastrin, pancreatic polypeptide and neurotensin release in man

The effects of 1-h infusions of bombesin and gastrin releasing peptide (GRP) at 50 pmol/kg per h and neurotensin at 100 pmol/kg per h on gastrin, pancreatic polypeptide (PP) and neurotensin release in man were determined following either saline or atropine infusion (20 micrograms/kg). Bombesin produced a rise in plasma neurotensin from 32 +/- 6 to 61 +/- 19 pmol/l and of PP from 26 +/- 8 to 36 +/- 7 pmol/l. There was a further rise of plasma PP to 50 +/- 13 pmol/l after cessation of the infusion. GRP had no significant effect on plasma neurotensin, but compared to bombesin, produced a significantly greater rise in plasma PP from 34 +/- 6 to 66 +/- 19 pmol/l during infusion. There was no post-infusional increase. At this dose, GRP was as effective as bombesin in releasing gastrin, although unlike bombesin its effect was enhanced by atropine. Neurotensin produced a rise in plasma PP from 17 +/- 4 to 38 +/- 8 pmol/l. Atropine blocked the release of PP during GRP and neurotensin infusion. Atropine had no effect on neurotensin or PP release during bombesin infusion, but did block the rise in plasma PP following bombesin infusion. We conclude that, in contrast to meal-stimulated neurotensin release, bombesin-stimulated neurotensin release is cholinergic independent. Despite structural homology, bombesin and GRP at the dose used are dissimilar in man in their actions and sensitivity to cholinergic blockade.     

Liberation of cyclic AMP and catecholamine from the heart during left stellate stimulation in the anesthetized dog

In open-chest pentothal-chloralose anesthetized dogs, plasma catecholamine and cyclic AMP levels were evaluated in the aortic and coronary sinus blood, during stimulations of the left ansa subclavia (1, 2, and 4 Hz). Basal aortic and coronary sinus catecholamine levels were respectively 0.373 +/- 0.090 and 0.259 +/- 0.048 ng/mL and cyclic AMP levels averaged 21.4 +/- 1.4 and 20.9 +/- 1.6 pmol/mL. Statistically significant increases in cyclic AMP levels were induced by sympathetic stimulations at 1 Hz (2.0 +/- 0.6 pmol/mL, 2 Hz (2.5 +/- 1.2 pmol/mL) and 4 Hz (6.5 +/- 1.5 pmol/mL), concomitantly with elevations of coronary sinus catecholamine levels. Sotalol (5 mg/kg) abolished the increases in coronary sinus cyclic AMP levels induced in coronary sinus cyclic AMP output averaged 282 +/- 30 pmol/min (1 Hz), 662 +/- 160 pmol/min (2 Hz), and 1679 +/- 242 pmol/min (4 Hz). Sympathetically induced cyclic AMP output (4Hz) was blunted by sotalol (-81 +/- 14 pmol/min). Aortic cyclic AMP levels were not significantly influenced by stellate stimulation. Intense correlations were found between increased in coronary sinus plasma catecholamines and cyclic AMP concentration levels (r = 0.81, slope - 1.45, ordinate = -1.42, n = 15) as well as between delta cyclic AMP output versus delta catecholamine output values in the coronary sinus (r = 0.93. slope output levels. Intracoronary infusion of phenylephrine (10 micrograms/min) or nitroprusside (200 micrograms/min) had no influence on cyclic AMP plasma levels whereas aortic and coronary sinus levels were respectively increased 5.5 +/- 1.9 and 7.3 +/- 1.4 pmol/mL during the administration of isoproterenol (5 micrograms/min). These data suggested that plasma cyclic AMP constitutes a sensitive index of cardiac beta-adrenergic activity elicited by the release of endogenous catecholamine during stellate stimulations.     

Evidence for direct inhibitory effects of dopamine on zona glomerulosa secretion of 18-hydroxycorticosterone in rhesus monkeys

This study was designed to more selectively investigate the dopaminergic regulation of 18-hydroxycorticosterone (18-OHB) and aldosterone production by the adrenal zona glomerulosa. Mature rhesus monkeys received either an infusion of dopamine (2 micrograms/kg/min) or 5% dextrose (0.2 ml/min) over a 60 min period (N=6). Dopamine had no effect on plasma levels of renin activity, cortisol, corticosterone, aldosterone or blood pressure. However, dopamine suppressed (p less than 0.05) plasma 18-OHB levels from a baseline of 31.6 +/- 3.5 ng/dl to 23.6 +/- 2.1 ng/dl at 60 min after onset of infusion. This observation is in agreement with some studies in humans but differs from others in which no depression in 18-OHB was observed following dopamine infusion. Dopamine infusion markedly (p less than 0.001) suppressed plasma PRL levels by 30 min after onset of infusion. Corticosteroid responses to metoclopramide (200 micrograms/kg) after dexamethasone 1 mg im every 6 h X 5 days or placebo treatment (vehicle im every 6 h X 5 days) was then evaluated. Dexamethasone significantly suppressed basal cortisol, corticosterone, 18-OHB and aldosterone. Although dexamethasone blunted the prolactin response, it did not inhibit the aldosterone response to metoclopramide. The 18-OHB response to metoclopramide was increased (p less than 0.01) following dexamethasone treatment. Following dexamethasone suppression, 18-OHB levels were still lowered (p less than 0.05) by dopamine infusion. These results suggest that dopamine selectively inhibits zona glomerulosa production of 18-OHB and aldosterone in rhesus monkeys.     

Preferential release of oxytocin in response to vasoactive intestinal polypeptide in rats

Vasoactive intestinal polypeptide (VIP) was infused into the aorta of pentobarbitone-anesthetized rats (n = 12) in stepwise increasing doses of 0.001 to 10 micrograms/rat at rates varying from 0.3 pmol/min/kg to 3000 pmol/min/kg over 3 min. Blood was withdrawn from the vena cava inferior for the measurement of oxytocin (OT) and vasopressin (AVP) by RIA. The loss of blood was compensated for by infusion of isotonic saline (0.9% NaCl with 0.5% human serum albumin). Control rats received this solution only (n = 11). VIP infusions resulted in a dose-dependent increase in plasma OT which was significantly greater than the slight rise observed in the controls. The difference from controls was significant at infusion rates of 3 pmol/min/kg and more. Plasma AVP, on the other hand, did not rise in response to VIP infusions until the infusion rate was increased to 300 and 3000 pmol/min/kg. At these infusion rates, the increments in AVP were much smaller than those of OT, the levels during the highest infusion rates rising to 8.6 +/- 2.8 and 27.2 +/- 4.8 microU/ml, respectively (log normal means). The preferential release of OT in response to exogenous VIP in rats differs from the response in cats where intracarotid administration of VIP resulted in the release of proportionately more AVP than OT. Immunoreactive VIP is found in the hypothalamo-neurohypophyseal system of rats in close proximity of some of the magnocellular neurons as well as within the nerve terminals. This, together with our data, suggests that endogenous VIP may participate in the release mechanism for OT in rats.     

Reversal of increased microvascular permeability associated with ischemia-reperfusion: role of cAMP.

Ischemia-reperfusion (IR) is a form of oxidant injury known to increase microvascular permeability in the lung. Agents that increase adenosine 3',5'-cyclic monophosphate (cAMP) levels have been shown to have beneficial effects in several models of oxidant lung injury associated with increased microvascular permeability. We investigated the role of adenylate cyclase activation with isoproterenol (ISO) or forskolin (FSK) in reversing the increased microvascular permeability associated with IR. ISO or FSK administered after 45 min of ischemia and 46 min of reperfusion caused a reduction in the capillary filtration coefficient (Kfc) from 1.25 +/- 0.13 to 0.53 +/- 0.08 and 0.55 +/- 0.10 ml.min-1.cmH2O-1.100 g tissue-1, respectively, at 90 min of reperfusion. This reduction in Kfc was accompanied by a rise in perfusate cAMP levels from 16.5 +/- 4.9 and 31.2 +/- 11.9 pmol/ml at 45 min of reperfusion to 444.2 +/- 147.8 and 276.1 +/- 91.0 pmol/ml at 105 min of reperfusion in lungs treated with ISO or FSK, respectively, at 46 min of reperfusion. Dibutyryl cAMP (DBcAMP), a membrane-permeable cAMP analogue, mimicked the permeability effect by reducing Kfc to 0.67 +/- 0.15 at 90 min of reperfusion. Significant hemodynamic changes occurred but were small and cannot explain the observed effect on Kfc. Photomicrographs from lungs treated with ISO or FSK revealed a reversal of the morphological manifestations of increased microvascular permeability. We conclude that the increased microvascular permeability associated with IR can be reversed by ISO, FSK, and DBcAMP and that cAMP produced by the lung contributes to the observed reversal.     

The effect of relaxin on cyclic adenosine 3',5'-monophosphate concentrations in human endometrial glandular epithelial cells

The effects of relaxin (RLX), forskolin (Fk), and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro, a phophodeisterase inhibitor) on the accumulation of cyclic adenosine 3',5'-monophosphate (cAMP) in human endometrial glandular epithelial cells were studied. Epithelial glands were isolated from the endometrium by digesting the viable tissue fragments with collagenase. The epithelial glands were incubated with Ro, RLX, and Fk separately or in combination. The amount of cAMP was determined at the end of incubation. A moderate increase in cAMP content was observed in epithelial glands incubated with Ro alone. Accumulation of cAMP after incubation with RLX was observed only in the presence of Ro. Increase of cAMP content in response to RLX and Ro was time- and dose-dependent. The accumulation of cAMP was apparent in 5 min, reached the maximum after 15 min, and remained elevated for 17 h incubation. One nanogram per millileter RLX was effective to increase the cAMP content, with a maximal response at 100 ng/ml. The effect of Ro and the combined effect of Ro and RLX on cAMP accumulation were studied in epithelial glands of 20 endometrial specimens obtained during different stages of the menstrual cycle. When epithelial glands were incubated with Ro alone, the cAMP concentration in glands from proliferative endometria was 120 +/- 67 pmol/mg protein (n = 6, means +/- SD), significantly higher than that of secretory endometria, 42 +/- 37 (n = 14, p = 0.007). RLX and Ro caused an additional increase of cAMP accumulation, 2- to greater than 10-fold increase over the sample incubated with Ro alone. There was no significant difference between proliferative and secretory phases (500 +/- 410, n = 6, and 470 +/- 300, n = 14, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma growth hormone secretion during constant growth hormone-releasing factor infusion

Synthetic human pancreatic growth hormone-releasing factor (hpGRF-44) was infused intravenously at a constant rate of 2.5 micrograms/min for 180 minutes in 3 normal boys of short stature. Plasma GH levels reached a peak at 60-120 min with a mean value (+/- SEM) of 69.1 +/- 14.3 ng/ml, and then, declined gradually in spite of continuous hpGRF-44 infusion up to 180 minutes. Similarly, constant infusion of hpGRF-44 at a rate of 2.5 micrograms/min in 5 normal but short boys for 90 minutes, together with an iv bolus injection of hpGRF-44 (2 micrograms/kg) administered at 0 and 90 minutes, elicited a prompt rise in plasma GH 15-30 minutes after the first bolus but no significant elevation of GH was observed after the second bolus. In contrast, when two iv bolus injections of hpGRF-44 (2 micrograms/kg) were given in 4 normal boys with short stature at 0 and 90 minutes, respectively, significant elevation of plasma GH was found after each bolus. These results suggest that under constant infusion of GRF the pituitary experiences a down-regulation after the initial peak of GH response, possibly due to desensitization to GRF.     

Properties of the oscillatory cAMP binding component of Dictyostelium discoideum cells and isolated plasma membranes.

The cAMP receptor on the surface of aggregation competent Dictyostelium discoideum cells specifically binds [3H]cAMP in an oscillatory manner with a periodicity of 2 min. The oscillatory cAMP-binding component is developmentallly regulated and has the nucleotide specificity expected for recognition of chemotactic signals. The concentration dependence of the peak amplitudes of cAMP binding exhibit an apparent threshold at 10(-8) M cAMP. The threshold concentration for cAMP binding that we measure is consistent with the concentration dependence of signal relay (cAMP secretion) and the chemotactic response. The kinetic data of binding and dissociation are very rapid, consistent with the time course of oscillations in receptor capacity (affinity). Specific binding oscillations are destroyed by heat or chymotrypsin but are insensitive to trypsin or glycosidase. A plasma membrane localization of receptor is supported by enrichment of cAMP binding in a plasma membrane preparation from differentiated cells. Receptor oscillations with a 2-min period are preserved in the membrane preparations, and the peak amplitudes are increased about 10-fold consistent with the enrichment of other plasma membrane markers. The alternating change in the receptor's binding capacity for cAMP may be the basis of the relay refractory period as well as the primary oscillator involved in the generation of postreceptor events such as stimulation of adenylate cyclase, cAMP secretion, and cellular movement, all of which have been previously shown to oscillate.     

Serum glucose and free fatty acids modulate growth hormone and luteinizing hormone secretion in the pig

Two experiments were conducted to determine the effect of free fatty acids (FFA) and glucose treatment on growth hormone (GH) and luteinizing hormone secretion in the pig. In Experiment (Exp) 1, 15 prepuberal gilts received an intravenous infusion of FFA (n = 5; 3 ml of 10% Liposyn II/kg), glucose (n = 5; 1 g/kg), or saline (n = 5; 3 ml of 0.9%/kg). Jugular blood samples were collected every 15 min for 2 hr before and 3 hr after intravenous infusion of saline, FFA, and glucose. Synthetic [Ala15]-h growth hormone-releasing factor-(1-29)NH2 (1 microgram/kg) and gonadotropin-releasing hormone (0.2 micrograms/kg) were administered 30 min after infusion (Time 0 = infusion). In Exp 2, eight prepuberal gilts received either FFA (n = 4) or saline (n = 4) as described in Exp 1, except that treatments were given every hour ove a 10-hr period. Blood samples were collected every 15 min from 1 hr before to 10 hr after the start of FFA or saline infusion. In Exp 1, the peak GH response to growth hormone-releasing factor was delayed by 45 min (P less than 0.01) by glucose treatment and suppressed (P less than 0.01) by FFA treatment. The luteinizing hormone response to gonadotroph-releasing hormone was suppressed (P less than 0.03) by glucose and enhanced (P less than 0.03) by FFA. In Exp 2, the number of GH pulses was increased (P less than 0.05) by FFA infusion and GH concentrations were positively correlated (r = 0.58, P less than 0.0003) with FFA concentrations, while luteinizing hormone pulse amplitude was greater (P less than 0.01) in FFA gilts than in saline gilts. These results indicate that FFA are more effective modulators of GH secretion than acute hyperglycemia, while metabolic status can alter pituitary responsiveness to gonadotropin-releasing hormone.     

Differential effects of parathyroid hormone and its analogues on cytosolic calcium ion and cAMP levels in cultured rat osteoblast-like cells

While the stimulatory effect of parathyroid hormone (PTH) on osteoblast-like cell adenylate cyclase is well known, the effect of PTH on cytosolic calcium ion ([Ca2+]i) mobilization is controversial, one group finding no effect but others reporting various increases. We investigated the effects on [Ca2+]i of synthetic rat PTH fragment 1-34 (rPTH(1-34)) and two bovine PTH analogues that inhibit PTH's stimulation of adenylate cyclase (bovine 8,18Nle, 34Tyr-PTH(3-34) and 34Tyr-PTH(7-34]. [Ca2+]i was measured before, during, and after exposure to PTH analogues in perifused, attached osteoblast-like rat osteosarcoma cells (ROS 17/2.8) that had been scrape-loaded with the luminescent photoprotein aequorin. Resting [Ca2+]i was 0.094 +/- 0.056 microM (mean +/- S.D., n = 103) and rose in a time- and dose-specific way after exposure to rPTH(1-34). At 10(-10) M rPTH(1-34), [Ca2+]i rose 100% within 30 s to a plateau; higher concentrations of PTH yielded increasing initial peaks of [Ca2+]i followed by lower plateaus. At 10(-6) M, the initial peak was 5-fold basal, or 0.64 +/- 0.07 microM. Both analogues of PTH were at least partial agonists for [Ca2+]i mobilization and did not reduce peak [Ca2+]i when co-perifused with rPTH(1-34). However, the analogues did reduce significantly rPTH(1-34)-induced cAMP accumulation and did not increase cAMP accumulation by themselves. Thus, rPTH(1-34) strongly mobilizes [Ca2+]i in ROS 17/2.8 cells, at near-physiologic concentrations. Failure of the PTH analogues to block the effect of PTH on [Ca2+]i while inhibiting the effect on cAMP accumulation suggests separate pathways for PTH activation of adenylate cyclase and mobilization of calcium.     

Effects of dibutyryl cAMP on pulmonary air embolism-induced lung injury in awake sheep

To assess the role of intracellular adenosine 3',5'-cyclic monophosphate (cAMP), we tested the effects of dibutyryl cAMP (DBcAMP), an analogue of cAMP, on lung injury induced by pulmonary air embolism in awake sheep with chronic lung lymph fistula. We infused air (1.23 ml/min) in the pulmonary artery for 2 h in untreated control sheep. In DBcAMP-pretreated sheep DBcAMP was infused (1 mg/kg bolus and 0.02 mg.kg-1.min-1 constantly for 5 h); after 1 h from beginning of DBcAMP administration the air infusion was started. After the air infusion, pulmonary arterial pressure (Ppa) and lung lymph flow rate (Qlym) significantly increased in both groups. DBcAMP-pretreated sheep showed significantly lower responses in Qlym (2.7 X base line) compared with untreated control sheep (4.6 X base line); however, Ppa, left atrial pressure, and lung lymph-to-plasma protein concentration ratio were not significantly different between the two groups. Although plasma and lung lymph thromboxane B2 and 6-ketoprostaglandin F1 alpha concentrations increased significantly during the air infusion, DBcAMP-pretreated sheep showed significantly lower responses. Thus DBcAMP infusion attenuated pulmonary microvascular permeability induced by air embolism. We conclude that pulmonary vascular permeability is in part controlled by the intracellular cAMP level.