Evidence for a PTH-independent humoral mechanism in post-transplant hypophosphatemia and phosphaturia.

BACKGROUND: Patients undergoing successful kidney transplantation often manifest overt hypophosphatemia associated with exaggerated phosphaturia during the early post-transplant period (2 weeks to 3 months). The mechanism for this phenomenon has not been fully elucidated. We tested the hypothesis that a circulating serum factor [non-parathyroid hormone (non-PTH)], which operates during chronic renal failure (CRF) to maintain phosphate (Pi) homeostasis, can increase fractional excretion of Pi (FE(PO4)) in normal functioning kidney grafts during the early post-transplant period, thereby causing phosphaturia and hypophosphatemia. METHODS: Five groups of patients were studied: control subjects (group 1, N = 16), "early" (2 weeks to 1 month) post-transplant patients (group 2, N = 22), "late" (9 to 12 months) post-transplant patients (group 3, N = 14), patients with advanced CRF (glomerular filtration rate = 30 to 40 mL/min; group 4, N = 8), and patients who suffered from end-stage renal failure and were treated by chronic hemodialysis (group 5, N = 14). Group 2 manifested significant hypophosphatemia and phosphaturia when compared with groups 1 and 3 (Pi = 0.9 +/- 0.003 mg/dL, FE(PO4) = 68+/- 5%, P < 0.0005 vs. groups 1 and 3). Sera were taken from each of the five subject groups and applied to the proximal tubular opossum kidney (OK) cells. The activity of Na/Pi-type 4 (that is, OK-specific type II transporter) was evaluated by measuring Na(+)-dependent (32)Pi flux. The expression of Na/Pi type II mRNA and the abundance of Na/Pi protein were determined by Northern and Western blot assays, respectively. RESULTS: When compared with sera from groups 1 and 3, 10% sera taken from groups 2, 4, and 5 (incubated overnight with OK cells) inhibited (32)Pi flux by 25 to 30% (P < 0.0003). Both Na/Pi mRNA and the expression of Na/Pi protein were markedly augmented under the same conditions (P < 0.05 groups 2, 4, and 5 vs. groups 1 and 3). Time-course analysis revealed that the up-regulation of Na/Pi protein by sera from groups 2, 4, and 5 was observed as early as four hours of incubation, whereas augmented abundance of Na/Pi mRNA was only seen after eight hours of incubation. The addition of PTH (1-34) to sera from groups 2, 4, and 5 abolished the augmented expression of NaPi protein. We labeled OK cell surface membrane proteins with N-hydroxysuccinimide bound to biotin (NHS-SS-biotin). Biotinylated transporters incubated with the different sera were precipitated by strepavidin and identified by Western blot analysis. Cells incubated in sera from group 2 showed increased membrane bound transporter when compared with control sera, whereas the intracellular pool of the transporter was comparable between the two groups. CONCLUSION: A non-PTH circulating serum factor (possibly phosphatonin) that increases FE(PO4) during CRF is also responsible for phosphaturia and hypophosphatemia in the early period following successful kidney transplantation. The putative factor inactivates Na/Pi activity along with inhibition of the transporter trafficking from the cell membrane into the cytosol.

Regulation of cellular rabbit growth hormone (GH) receptor and GH-binding protein generation in vitro.

In rabbits and probably in man, GH-binding protein (GHBP) is generated from proteolysis of GH receptor (GHR). The present study describes the modulation of spontaneous release of GHBP into the culture medium in relation to cellular GH receptor (GHR) in Chinese hamster ovary cells transfected with rabbit GHR complementary DNA. Secretion of GHBP (approximately 50K protein) from these cells was dependent on time, percentage of FCS, temperature, and protein synthesis. GHBP was detected in the medium at 30 min, and a linear increase was observed over the next 4 h. GHBP release was reduced by low incubation temperature, suggesting that GHBP cleavage is an energy-requiring mechanism. N-Ethylmaleimide (500 microM for 30 min at 30 C) markedly increased GHBP secretion, matched by a corresponding decrease in GHR. However, the lack of effect of N-ethyl-maleimide observed at 4 C further confirms the temperature dependence of GHBP release. We have attempted to characterize the GHBP release protease with a number of recognized protease inhibitors. Benzamidine (10 mM) was the only protease inhibitor that reduced GHBP release; however, it also reduced the cellular GHR level. Cycloheximide (20 micrograms/ml) caused a parallel disappearance of cellular GHR and secreted GHBP with a half-life of about 50 min, but increased GHR messenger RNA expression (superinduction). Indeed, 4 h after removal of cycloheximide, GHR and GHBP were increased by 181% and 369%, respectively, compared to the control value. In summary, Chinese hamster ovary cells expressing rabbit GHR provide a useful cellular model system for studies on the mechanism of GHBP generation from GHR and its physiological importance.

In search of the cellular site of growth hormone (GH)-binding protein cleavage from the rabbit GH receptor.

Transfection of Chinese hamster ovary cells with rabbit GH receptor (GHR) complementary DNA resulted in high expression of cellular GHR as well as markedly time- and temperature-dependent secretion of soluble GH-binding protein (GHBP) into the culture medium. In the present study, these cells were used as an in vitro model system to examine GHBP secretion in relation to GHR internalization, degradation, recycling, and biosynthesis. Incubation for 20 h with the lysosomotropic agents NH4Cl and monensin inhibited GH internalization and reduced cell surface GHR, whereas no significant effect on the level of secreted GHBP was observed. Cytochalasin B, a microfilament-disrupting agent, reduced the GHR level, but GHBP was not affected. Colchicine, a microfilament depolymerization agent, had no effect on the GHR level; however, it stimulated GHBP secretion approximately 2-fold. Brefeldin A (5 micrograms/ml), a transport blocker, incubated for 15-180 min resulted in a time-dependent decline in GHR, whereas no significant modulation effect on GHBP was apparent. The capacity of these cells to synthesize and incorporate GHR at the plasma membrane in relation to the generation of soluble GHBP was obtained by destruction of cell surface GHR by mild trypsinization and subsequently monitoring the rate of recovered GHR and GHBP. The rate of reappearance of GHR and GHBP was rapid, being observed within 1 h, whereas full recovery occurred within 2 and 3 h, respectively. The recovery was completely blocked by cycloheximide and brefeldin A. NH4Cl and monensin reduced GHR restoration by about 50%, but the recovery of GHBP was not affected. These data emphasize the importance of lysosomes and vesicular traffic in the regulation of secreted GHBP that might be derived from the internalized GHR and may provide insight into a better understanding of the cleavage process of GHBP from GHR.

Overexpression of the short form of the growth hormone receptor in 3T3-L1 mouse preadipocytes.

In rodents, the gene for the growth hormone receptor (GHR) gives rise to two mRNA transcripts encoding two proteins: a larger membrane spanning receptor (GHRL) and a smaller isoform, GHRs that consists of the extracellular domain and a unique hydrophilic carboxyl terminus. We examined the hypothesis that GHRs may contribute to cellular binding of GH and play a role in growth hormone (GH) signaling. Rat cDNA encoding GHRs was ligated into the mammalian expression vector pcDNA-I/neo and stably transfected into mouse 3T3-L1 preadipocytes which have endogenous GH receptors and, when differentiated into adipocytes, have the biochemical machinery to express the various GH effects. Sixteen of 24 neomycin resistant clones secreted at least twice as much GHRs in the growth medium as cells transfected with the vector alone, and in nine of these, GH binding was increased 2- to 4-fold. The amount of GHRL in extracts of these cells was unchanged, indicating that increased binding could not be accounted for by effects on formation or degradation of GHRL. The transfected cDNA for GHRs directs the synthesis of a 50 kDa protein. Cross-linking of [125I]hGH to transfected 3T3-L1 cells indicated a 3.5-fold increase in a 72 kDa GHRs[125I]hGH complex. In the presence of 10% newborn calf serum, incorporation of [35S]methionine into cellular proteins was similar in transfected clones and control cells. Deprivation of serum for 2 hr decreased protein synthesis by approximately 70% in control cells, but in the transfected cells, protein synthesis was reduced only by approximately 50% or 30% in cells exhibiting 2x or 3x increases in GH binding. In all cells, 1 nM IGF-1 restored protein synthesis to the serum replete level. Similarly, although 3H-2-deoxy-D-glucose (2DG) uptake was comparable in all cells after 2 hr of serum deprivation, 18 hr of serum deprivation decreased uptake by approximately 70% in control cells, but only by approximately 30% in cells with increased GH binding. One nanomolar IGF-1 restored 2DG uptake to levels seen after 2 hr or serum deprivation. IGF-1 had no effect after only 2 hr of serum deprivation. Measurement of IGF-1 secreted into the medium, revealed that clones which overexpress GHRs produce greater amounts of IGF-1 than control cells or transfected clones that failed to overexpress GHRs. We conclude that GHRs contributes to GH binding and may therefore be a functional receptor. In addition, overexpression of GHRs in 3T3-L1 cells altered cell function in the absence of GH.

Stoichiometry of the pulsating growth hormone (GH) binding to the GH-binding protein and the turnover GH-receptor.

The pulsatile pattern of growth hormone (GH) secretion is synchronized with the GH-receptor (GHR) turnover and the ensuing GH-binding protein (GHBP). We investigated the effect of GH pulse frequency on the turnover of GHR, and tested the theoretical reciprocal impacts of GH, GHR, and GHBP in different species and clinical conditions. Male Sprague-Dawley rats were hypophysectomized (hypox) at 35 days of age. Two groups of 16 rats received a single or two iv injections of 10 micrograms human GH (hGH) at an interval of 45 min. They were killed 45, 90, or 135 min after the single or second injection. A third group of 25 hypox rats were given continuous sc infusion of hGH for 6 days. Liver membranes were prepared for hGH somatogenic binding. A bolus of GH at 45-min intervals expedited GHR turnover, and continuous GH resulted in faster turnover cycles of 90 min. The impact of GHBP on GH bioactivity was then calculated in human serum by a rabbit liver membrane displacement assay. Bioactivity was diminished by GHBP with increasing GH levels up to a point, within the physiological range, where GH bioactivity is gradually restored. Finally, simulation calculation of the bound and free fraction of GH over a typical pulse in man and male rat showed the changing relations of free/bound GH. In man free hormone predominates most of the pulse, whereas in rat free GH comprised a smaller fraction. Thus, the reciprocal effects of GH on GHR turnover and GHBP generation, and that of GHBP on GH t1/2 lead to self-perpetuation of high (mostly free) GH in downregulation of GHR and its turnover with resultant lower GHBP.

Roles of pulsatility and continuity of growth hormone (GH) administration in the regulation of hepatic GH-receptors, and circulating GH-binding protein and insulin-like growth factor-I.

GH secretion in the male rat is characterized by regular GH peaks of high amplitude, and their GH receptors and GH-binding protein (BP) are low. Female rats have a more continuous secretion, and their GH receptors and GH-BP are high. To determine how the pattern of GH delivery may affects the physiological responses to the hormone, hypophysectomized male rats were infused with human GH (1.5 U/kg.day) for 5 days through an implanted iv cannula in either a pulsatile manner (for 5 min every 3 h) or continuously. We then measured free and total (after MgCl2 treatment) somatogenic and lactogenic receptors in hepatic cell membranes, and GH-BP and immunoreactive insulin-like growth factor-I (IGF-I) in serum. The concentrations of occupied, but not free, somatogenic and lactogenic binding sites were higher 5 min after a 5-min infusion of human GH than 90 min after the infusion. Consequently, the total levels (free plus ligand binding) of somatogenic and lactogenic receptors were also higher 5 min after a GH pulse than after 90 min. The levels of circulating GH-BP were higher 90 min than 5 min after a GH pulse, and the serum IGF-I levels did not differ between these time points. The concentrations of free and total somatogenic and free lactogenic receptors in the liver, as well as GH-BP and IGF-I in circulation were higher and body weight gain was lower in rats given continuous GH infusion than after pulsatile infusions. It is concluded that the nature of GH-levels profile has a marked influence on the interrelationship of GH, GH receptors, GH-BP, serum IGF-I and growth, whereby the later is stimulated maximally by pulsatile GH-levels, while the receptor and GH-BP are enhanced maximally by continuous levels. The basis for this discrepancy may lay in the competitive power of GH-BP toward GH receptor binding. A pulsatile GH pattern induced cyclicity in the levels of hepatic cell membrane GH receptors and circulating GH-BP. It is suggested that these factors are regulated in a similar way by the plasma GH pattern in pituitary intact rats.

Evidence for the role of the secretory pattern of growth hormone in the regulation of serum concentrations of cholesterol and apolipoprotein E in rats.

Adult male Sprague-Dawley rats were hypophysectomized and connected to an automatic i.v. infusion system. The same daily dose of human GH (hGH) was given either as eight daily pulses (3-h intervals) to mimic the male specific secretory pattern of GH or as a continuous infusion of GH, to mimic the female secretory pattern. Hypophysectomized rats received i.v. replacement therapy with L-thyroxine and cortisol. The rats were treated for 5 days. The serum cholesterol concentration was higher when hGH was given continuously than when hGH was given as eight daily pulses. The concentration of high-density lipoprotein (HDL)-cholesterol was not influenced by intermittent GH treatment, but increased when hGH was given as a continuous infusion. The serum concentration of apolipoprotein (Apo) E increased following treatment with a continuous infusion of hGH, whereas eight daily pulses of hGH had no effect. The serum concentration of ApoA-I was unaffected by hGH treatment. The serum concentration of ApoB decreased to the same degree whether hGH was given as a continuous infusion or as eight daily pulses. The serum concentration of triglycerides was not affected by hGH treatment. These results indicate that the higher serum HDL-cholesterol and serum ApoE concentrations of female rats may be due to their more continuous secretion of GH. In contrast, the effects of GH on the serum concentration of ApoB, which is not sexually differentiated, may be independent of the mode of GH secretion.

Identification of growth hormone binding protein in rat serum.

The present report describes the initial characterization of a specific, high-affinity growth hormone binding protein (GH-BP) in adult male rat serum. GH-BP activity was measured by incubation of rat serum with [125I]hGH and [125I]rGH and separation of bound from free GH by dextran-coated charcoal. [125I]hGH binding to rat serum was dependent on serum concentration and incubation time, equilibrium being reached within 10 min both at 4 and 37 degrees C. Binding was rapidly and completely reversible and specific for somatogenic (but not lactogenic) hormones. Scatchard analysis yielded a linear plot with an affinity (Ka) of 1.51 +/- 0.63 x 10(8) M-1. Preliminary data obtained in various physiological conditions showed that GH-BP activity in adult male rats was 5.95 +/- 0.20%/0.1 ml serum. Significantly higher values were obtained in sera of female (21.66 + 0.79%/0.1 ml serum) and pregnant rats (23.02 +/- 1.15%/0.1 ml serum). A closer analysis of these binding values by Scatchard analysis revealed that the binding capacity in pregnant rats (50.5 +/- 5.8 pmol/0.1 ml serum) was significantly higher than in adult female estrous rats (19.2 +/- 6.5 pmol/0.1 ml serum), both being much higher than in adult male rats (2.5 +/- 0.6 pmol/0.1 ml serum). The GH-BP activity of 10-day-old rats was only approximately 63% of the adult male rat value. The presence of high-affinity GH-specific binding protein in rat serum suggests a probable action in regulation of GH activity. The detailed physiological role of rat serum GH-BP is currently being investigated.

The interrelationship of growth hormone (GH), liver membrane GH receptor, serum GH-binding protein activity, and insulin-like growth factor I in the male rat.

Indirect evidence suggests that the serum GH-binding protein (GH-BP) is related and possibly derived from the GH-receptor. GH, through its specific receptor, is the major regulator of insulin-like growth factor I (IGF-I) synthesis. The present study was undertaken to correlate serum GH-BP activity with liver plasma membrane GH receptors and their effects on serum IGF-I concentration during spontaneous pulsation of rat (r)GH in the normal male rat and after continuous delivery of human (h)GH to hypophysectomized male rats. In the first set of experiments, 45-day-old male rats were decapitated at 15 min intervals for 4 h. Serum GH-BP levels fluctuated with a 60 min lag behind the rGH levels. IGF-I pulsated over a 3-fold concentration range. IGF-I peak levels coincided with one of the rGH peaks, but its periodicity was longer than 3 h. Taken together with our previous studies on the turnover of the GH receptors, we suggest that each GH surge results in individual pulse-related turnover wave of receptor internalization and recycling. This is accompanied by a parallel increase in serum GH-BP activity. The GH and the receptor wave are responsible for an individual secretion pulse of IGF-I. In the second set of experiments male rats were hypophysectomized at 35 days of age. Four days later osmotic minipumps were implanted for continuous delivery of hGH. After 6 days of hGH treatment the rats were killed, blood was collected for hGH, GH-BP, and IGF-I determination, and the livers were removed. Plasma membranes were prepared, and lactogenic and somatogenic binding of [125I]hGH was evaluated. Removal of endogenous ligand was performed by exposing the membranes to 3 M MgCl2. Continuous administration of hGH induced a dose-dependent increase in liver membrane lactogenic and somatogenic binding. Parallel to that increase, serum GH-BP also increased in a dose-dependent manner, and the correlation between serum GH-BP and the liver membrane receptor was significant. Furthermore, hGH induced a dose-dependent increase in IGF-I concentration. There was a close correlation between IGF-I concentration and liver somatogenic receptors. It is concluded that up-regulation of the liver membrane GH receptors is accompanied by increased GH-BP and IGF-I. In both the pulsation experiment and the continuous infusion experiment, GH-BP closely correlated with the liver membrane GH receptor.

Alterations of human growth hormone binding by rat liver membranes during hypo- and hyperthyroidism.

Hypothyroid rats treated with human GH (hGH) were partially refractory to the latter's effects. The present study was undertaken to investigate the role of hypo- and hyperthyroidism on the GH receptor. Seven-week-old rats were rendered either hypothyroid, by methimazole, or hyperthyroid, by a daily overdose of T4, during weeks 7-14 of life. Livers were homogenized and overlaid on sucrose discontinuous density gradient. Removal of endogenous ligand from the receptor was performed by exposing the membranes to MgCl2. hGH was used with excess ovine PRL to characterize somatogenic specific binding. Lactogenic specific binding was calculated by subtracting somatogenic specific binding from the total specific binding. Creatine kinase was also measured in homogenized livers. Liver membranes of the hypothyroid rats showed a significant decline in somatogenic and lactogenic binding of hGH. This was true for both the free unoccupied binding sites and total binding after dissociation of the endogenous ligand. Replacement of T4 for 2 weeks restored hGH binding to control values. Hyperthyroid rats had high somatogenic and lactogenic hGH binding. Creatine kinase activity decreased significantly in liver homogenates of hypothyroid rats, was restored by T4 replacement, and increased significantly in hyperthyroid rats. Thus, lactogenic and somatogenic receptors are directly related to the thyroid status in vivo.

The role of catecholamines in estradiol and progesterone secretion by cultured explants and cells of human term placentae.

The effects of physiological concentrations of the native catecholamines norepinephrine and epinephrine upon term placental hormonal function were examined by measuring estradiol and progesterone secretion by organ and cell culture systems. Results show that, in explants, both catecholamines caused a significant increase in the secretion of both sex steroids, p less than 0.05. Estradiol secretion was blocked by the alpha and beta adrenergic receptors antagonists, phenoxybenzamine and propranolol, respectively, p less than 0.05. Norepinephrine but not epinephrine dependent progesterone secretion was blocked by propranolol. In cells, epinephrine stimulated cyclic AMP generation and caused a 30% increase in estradiol secretion, p less than 0.05. Both were abrogated by propranolol. Norepinephrine increased secretion by 25%, p less than 0.05. This was inhibited by yohimbin and prazosin, alpha-1 and -2 receptors antagonists, respectively. In conclusion, the placenta in vitro is a target organ for catecholamines. The marked response of the explant system as compared with the marginal response of the cell culture system indicates that cell to cell contact/communication is required for full expression of catecholamine effect.

Adaptation of liver membrane somatogenic and lactogenic growth hormone (GH) binding to the spontaneous pulsation of GH secretion in the male rat.

The secretory pattern of GH is pulsatile in both man and experimental animals. The present study was undertaken to investigate the effect of the endogenous pulsatility of serum GH on the dynamics of plasma membrane GH binding sites. In order to characterize somatogenic and total specific binding, male rats 45 days old were decapitated at 15-min intervals, and rat liver membranes were prepared. Binding studies were done in parallel with and without excess ovine PRL, 1 microgram/tube. Removal of endogenous ligand was performed by exposing the membranes to 3 M MgCl2. A composite picture of serum GH showed relatively good synchronization of the secretory episodes among the animals. Peak levels of the spontaneous secretion pulse of rat GH were followed by an immediate decrease in free somatogenic binding. Two hours later the binding activity returned to presecretion levels, essentially resetting the cell for another cycle. The kinetics of desaturated somatogenic binding were similar. The occupancy of liver GH somatogenic binding sites was maximal at the time of peak serum GH. High levels of the endogenous hormone caused an immediate sharp down-regulation of lactogenic desaturated binding. Up-regulation of the binding sites occurred about 90 min later. Scatchard analysis showed that binding activity of the membranes was a result of the altered number of binding sites, while the binding affinity remained fairly constant. Thus, the centrally regulated GH secretion in the male rat is complemented by appropriate dynamics of the GH receptor turnover, which in turn recognizes individual pulses and allows individual pulse-related responses to occur.

The dynamics of somatogenic and lactogenic growth hormone binding: internalization to Golgi fractions in the male rat.

Rapid turnover of the GH receptor has previously been shown, and its turnover (t1/2) has been estimated to be 30-85 min. In a companion study, we found that lactogenic and somatogenic GH binding sites on rat liver membranes were down-regulated immediately after an episode of GH secretion and reappeared at the plasma membrane in time for the next secretory surge of the hormone. In the present in vivo study, we followed the fate of the down-regulated membrane GH binding sites in the Golgi membranes. Male rats were killed at 1000, 1100, and 1200 h, and their livers were removed for preparation of Golgi membranes. Lactogenic and somatogenic [125I]human GH binding to Golgi membranes was measured. The results of the present study show that Golgi receptors are related to the endogenous pulsation of serum GH. After the GH surge, an increase in the capacity of the lactogenic and somatogenic receptors in the Golgi membranes takes place. Most of these receptors were occupied by ligand and represent, therefore, internalized receptors. Two hours after the GH secretory peak, the occupied receptors had disappeared from the Golgi membranes and appeared in the plasma membrane, suggesting that receptors were recycled to the plasma membrane, awaiting the next GH surge. The following model is proposed: A surge of GH secretion causes immediate down-regulation of the plasma membrane receptors to GH. The receptors that disappear from the plasma membrane are internalized to the Golgi complex. A fraction of the Golgi receptors is recycled to the membrane, along with newly synthesized receptors, awaiting the next GH pulse.

Effects of human growth hormone upon term placental hormone secretion in vitro.

The role of human growth hormone (hGH) on placental hormone secretion at term was investigated in two in vitro models: placental explants and cultured trophoblastic cells. Physiological concentrations of hGH caused a significant dose-dependent increase in placental lactogen and progesterone secretion. In the explant model it stimulated estradiol secretion. In order to determine whether this stimulatory effect on estradiol is exerted via aromatase, an isolated cell culture was utilized where androstenedione was supplied as substrate. In this model, hGH exerted a mild inhibitory effect. In conclusion, hGH at levels present in the fetal circulation exerts a significant stimulatory effect upon placental function as reflected by both peptide and steroid hormone production and secretion. The effect of estradiol secretion is the end result of an inhibitory effect on androgen aromatization and a stimulatory effect on earlier steps.

The human placenta as a target tissue for glucagon.

Glucagon's effect on the placenta was studied in cultured human term trophoblast and in homogenized term and first-trimester placentas. In studies with cultured term trophoblast, glucagon stimulated the generation of cyclic AMP and estradiol secretion and inhibited placental lactogen secretion. Incubation of homogenates of term and of first-trimester placenta with 0.5 mM dibutyryl cAMP revealed a marked decrease of pyruvate kinase activity. Glucagon produced a similar decrease in first-trimester homogenates, but failed to affect term placentas. The present demonstration of the placenta as a target tissue for glucagon suggests an active contribution of the trophoblast to energy metabolism during pregnancy.

Two pathways of placental lactogen secretion by cultured human trophoblast.

To assess the relative importance of regulated and of constitutive secretion of placental lactogen, a cell culture model of term human trophoblast was utilized. Time courses of secretion revealed a constant secretion rate over 9 days of culture, with relatively small constant intracellular hormone concentration. Potassium, 21 mM, produced a slight but significant increase in hormone secretion into the medium. Growth hormone-releasing hormone (5 X 10(-10)-5 X 10(-9)) stimulated a 27-48% increase in placental lactogen secretion. The data suggest a major process of constitutive secretion and a minor role for regulated secretion from a storage pool.

Interrelated calcium ion and cyclic AMP inhibition of placental lactogen secretion by cultured human term trophoblast.

Calcium ion has been shown to have a negative regulatory effect on the secretion of human placental lactogen (hPL). We report an inhibitory effect of cyclic AMP on hPL secretion in a model system of cultured human term trophoblast. Similar results were obtained by modulation of cyclic AMP-phosphodiesterase. The effects of ionophore A23187 and cyclic AMP were non-additive. In searching for the sequence of the common pathway, we found that the ionophore A23187 reduced cyclic AMP concentration and that the inhibitory effect of cyclic AMP was abolished by verapamil, a calcium channel blocker, and by trifluoperazine, a calcium-calmodulin inhibitor. Cyclic AMP seems to mobilize intracellular calcium, and the latter, by way of a calcium-calmodulin complex, modulates the secretion of hPL.

The dual effect of calcium on aromatization by cultured human trophoblast.

To study the effect of calcium ion on aromatization of an androgenic precursor to estradiol by the placenta, cultured term trophoblasts were used as a model system. Secretion of estradiol into the culture medium was regarded as indicating aromatization, since cells cultured with no androgenic precursors produced only insignificant amounts of estradiol. EGTA, verapamil and ionophore A23187 inhibited aromatization, while trifluoperazine, an inhibitor of the calcium-calmodulin complex, interfered with the stimulatory effect of cyclic AMP on aromatization. We conclude that calcium ion has an essential role in the aromatization of 4-androstene-3,17-dione to estradiol. The calcium-calmodulin complex is required for activation of aromatase by cyclic AMP. However, when flooded with calcium by ionophore A23187, the trophoblast is unable to effectively buffer calcium, and aromatization is inhibited.

The effect of inhibitors on insulin regulation of hormone secretion by cultured human trophoblast.

The effect of insulin in physiological concentrations on hormone secretion by human term trophoblast cell culture was studied in relation to insulin control of glucose transport and utilization. Specific inhibitors to these 2 functions were added to the culture, either alone to test for a dose response or with insulin. 2-Deoxy-D-glucose, which inhibits glycolysis, did not change the pattern of response of estradiol and placental lactogen (hPL) secretion to insulin. Phloridzin, an inhibitor of glucose transport, interfered with the stimulatory effect of insulin on hPL secretion, but stimulated insulin inhibition of estradiol secretion when added by itself. The results indicate that hPL response to insulin is dependent on glucose transport, but is independent of glycolysis. Estradiol response is dependent on glucose transport, but is independent of glycolysis.