Fuller Albright and our current understanding of calcium and phosphorus regulation and primary hyperparathyroidism.

The major contributions of Fuller Albright to our understanding of calcium and phosphorus regulation and primary hyperparathyroidism are highlighted. Albright was the first investigator to initiate a systematic study of mineral metabolism. With resources limited to the measurement of serum calcium and phosphorus and the infusion of parathyroid extract, Albright used balance studies to establish a framework for our understanding of calcium and phosphorus regulation and primary hyperparathyroidism. Albright was the first to show that the etiology of primary hyperparathyroidism could be from either an adenoma or hyperplasia of the parathyroid glands and stone disease was a separate manifestation of primary hyperparathyroidism. Albright also showed that: 1) a renal threshold for calcium excretion was present in hypoparathyroid patients; 2) correction of hypocalcemia in hypoparathyroid patients with vitamin D had a phosphaturic action; 3) renal failure reduced the intestinal absorption of calcium in primary hyperparathyroidism; 4) the ''hungry bone'' syndrome developed after parathyroidectomy in severe primary hyperparathyroidism; and 5) a target organ can fail to respond to a hormone. He also suggested that a malignant tumor could be responsible for ectopic hormone production. Finally, our review integrates the observations of Albright with our current knowledge of calcium regulation and disorders.

Effects of fasting, feeding, and bisphosphonate administration on serum calcitriol levels in phosphate-deprived rats.

BACKGROUND: In a recent study, we showed in phosphate-deprived rats that morning feeding decreased serum phosphate and increased serum calcium values as compared with similar rats fasted overnight, and high doses of bisphosphonates did not reduce the magnitude of hypercalcemia. In the present study, we evaluated in phosphate-deprived rats whether serum calcitriol values were: (1) affected by the differences in serum phosphate induced by morning feeding and overnight fasting, (2) correlated with changes in serum phosphate levels, and (3) influenced by bisphosphonate administration. METHODS: Four groups of rats were studied: (1) low-phosphate diet (LPD; P < 0.05%), (2) LPD + the bisphosphonate pamidronate (APD), (3) normal diet (ND; P 0.6%), and (4) ND + APD. Both diets contained 0.6% calcium. In rats receiving APD, high doses (0.8 mg/kg) were given subcutaneously four times during the study. On day 11, rats were sacrificed after an overnight fast or two to four hours after morning feeding. RESULTS: In the fed phosphate-deprived rats (LPD and LPD + APD), serum phosphate levels were less (P < 0.05) and serum calcium levels were greater (P < 0.05) than in similar rats fasted overnight. In rats on the ND (ND and ND + APD), no differences were observed between fed and fasted rats. In phosphate-deprived rats, serum calcitriol levels were greater (LPD, P < 0.05) or tended to be greater (LPD + APD, P = 0.10) in the fed than in the fasted groups. In APD-treated rats, serum calcitriol values were greater than in rats not given APD whether rats were (1) fed or fasted, or (2) on an LPD or ND. An inverse correlation was present between serum phosphate and serum calcitriol (r = -0.58, P = 0.001). In a stepwise regression model in which serum calcitriol was the dependent variable and independent variables were APD administration and serum calcium, phosphate, and PTH, serum phosphate (P = 0.003) had an inverse and APD (P < 0.001) administration a direct effect on serum calcitriol (r2 = 0.59). CONCLUSION: Calcitriol synthesis is rapidly inducible in rats during chronic phosphate deprivation, and the increase in serum calcitriol values is best attributed to feeding-induced decreases in serum phosphate. APD administration independently increases serum calcitriol levels in rats on normal and phosphate-deprived diets. Finally, whether our results in the rat are applicable to the clinical setting should be evaluated because in previous human studies of dietary phosphate restriction, serum calcitriol measurements were performed the morning after an overnight fast.

Chronic metabolic acidosis in azotemic rats on a high-phosphate diet halts the progression of renal disease.

BACKGROUND: Hyperphosphatemia and metabolic acidosis are general features of advanced chronic renal failure (RF), and each may affect mineral metabolism. The goal of the present study was to evaluate the effect of chronic metabolic acidosis on the development of hyperparathyroidism and bone disease in normal and azotemic rats on a high-phosphate diet. Our assumption that the two groups of azotemic rats (acid-loaded vs. non-acid-loaded) would have the same degree of renal failure at the end of the study proved to be incorrect. METHODS: Four groups of rats receiving a high-phosphate (1.2%), normal-calcium (0.6%) diet for 30 days were studied: (1) normal (N); (2) normal + acid (N + Ac) in which 1.5% ammonium chloride (NH4Cl) was added to the drinking water to induce acidosis; (3) RF, 5/6 nephrectomized rats; and (4) RF + acid (RF + Ac) in which 0.75% NH4Cl was added to the drinking water of 5/6 nephrectomized rats to induce acidosis. RESULTS: At sacrifice, the arterial pH and serum bicarbonate were lowest in the RF + Ac group and were intermediate in the N + Ac group. Serum creatinine (0.76 +/- 0.08 vs. 1.15 +/- 0.08 mg/dL), blood urea nitrogen (52 +/- 8 vs. 86 +/- 13 mg/dL), parathyroid hormone (PTH; 180 +/- 50 vs. 484 +/- 51 pg/mL), and serum phosphate (7.46 +/- 0.60 vs. 12.87 +/- 1.4 mg/dL) values were less (P < 0.05), and serum calcium (9.00 +/- 0.28 vs. 7.75 +/- 0.28 mg/dL) values were greater (P < 0.05) in the RF + Ac group than in the RF group. The fractional excretion of phosphate (FEP) was greater (P < 0.05) in the two azotemic groups than in the two nonazotemic groups. In the azotemic groups, the FEP was similar even though PTH and serum phosphate values were less in the RF + Ac than in the RF group. NH4Cl-induced acidosis produced hypercalciuria in the N + Ac and RF + Ac groups. When acid-loaded (N + Ac and RF + Ac) and non-acid-loaded (N and RF) rats were combined as separate groups, serum phosphate and PTH values were less for a similarly elevated serum creatinine value in acid-loaded than in non-acid-loaded rats. Finally, the osteoblast surface was less in the N + Ac group than in the other groups. However, in the acid-loaded azotemic group (RF + Ac), the osteoblast surface was not reduced. CONCLUSIONS: The presence of chronic metabolic acidosis in 5/6 nephrectomized rats on a high-phosphate diet (1) protected against the progression of RF, (2) enhanced the renal clearance of phosphate, (3) resulted in a lesser degree of hyperparathyroidism, and (4) did not reduce the osteoblast surface. The combination of metabolic acidosis and phosphate loading may protect against the progression of RF and possibly bone disease because the harmful effects of acidosis and phosphate loading may be counterbalanced.

Phosphate depletion in the rat: effect of bisphosphonates and the calcemic response to PTH.

BACKGROUND: The removal of phosphate from the diet of the growing rat rapidly produces hypercalcemia, hypophosphatemia, hypercalciuria, and hypophosphaturia. Increased calcium efflux from bone has been shown to be the important cause of the hypercalcemia and hypercalciuria. It has been proposed that the increased calcium efflux from bone is osteoclast mediated. Because bisphosphonates have been shown to inhibit osteoclast-mediated bone resorption, this study was performed to determine whether bisphosphonate-induced inhibition of osteoclast function changed the biochemical and bone effects induced by phosphate depletion. METHODS: Four groups of pair-fed rats were studied: (a) low-phosphate diet (LPD; phosphate less than 0.05%), (b) LPD plus the administration of the bisphosphonate Pamidronate (APD; LPD + APD), (c) normal diet (ND, 0.6% phosphate), and (d) ND + APD. All diets contained 0.6% calcium. A high dose of APD was administered subcutaneously (0.8 mg/kg) two days before the start of the study diet and on days 2, 6, and 9 during the 11 days of the study diet. On day 10, a 24-hour urine was collected, and on day 11, rats were either sacrificed or received an additional APD dose before a 48-hour parathyroid hormone (PTH) infusion (0.066 microgram/100 g/hr) via a subcutaneously implanted miniosmotic pump. RESULTS: Serum and urinary calcium were greater in the LPD and LPD + APD groups than in the ND and ND + APD groups [serum, 11.12 +/- 0.34 and 11.57 +/- 0.45 vs. 9.49 +/- 0.17 and 9.48 +/- 0.15 mg/dl (mean +/- SE), P < 0.05; and urine, 8.78 +/- 2.74 and 16.30 +/- 4.68 vs. 0.32 +/- 0.09 and 0.67 +/- 0.28 mg/24 hr, P < 0.05]. Serum PTH and serum and urinary phosphorus were less in the LPD and LPD + APD than in the ND and ND + APD groups (P < 0.05). The calcemic response to PTH was less (P < 0.05) in the LPD and LPD + APD groups than in the ND group and was less (P = 0.05) in the LPD + APD than in the ND + APD group. Bone histology showed that phosphate depletion increased the osteoblast and osteoclast surface, and treatment with APD reduced the osteoblast surface (LPD vs. LPD + APD, 38 +/- 4 vs. 4 +/- 2%, P < 0.05, and ND vs. ND + APD, 20 +/- 2 vs. 5 +/- 2%, P < 0.05) and markedly altered osteoclast morphology by inducing cytoplasmic vacuoles. CONCLUSIONS: (a) Phosphate depletion induced hypercalcemia and hypercalciuria that were not reduced by APD administration. (b) The calcemic response to PTH was reduced in phosphate-depleted rats and was unaffected by APD administration in normal and phosphate-depleted rats, and (c) APD administration markedly changed bone histology without affecting the biochemical changes induced by phosphate depletion.

Production of interleukin-6 by osteoblastic cells is independent of medium inorganic phosphate.

A state of severe bone loss is often observed in patients and animals suffering from phosphate (Pi) depletion. Conversely, Pi surfeit may have an anabolic effect on bone and may antagonize bone resorption. To study whether Pi has a direct effect on the production of the bone-resorbing interleukin-6 (IL-6) by osteoblasts, we cultured MC3T3-E1, UMR-106, and isolated rat calvaria cells in media containing varying concentrations of Pi (0-3 mM) and measured the production of IL-6 released into the media. IL-6 production was steady with time and was stimulated by parathyroid hormone, 1,25-dihydroxyvitamin D3, and interleukin-1 alpha. However, IL-6 production did not change with varying Pi concentrations. We concluded that the IL-6 production by osteoblastic cells is independent of the medium Pi.

Cell-matrix interaction in bone: type I collagen modulates signal transduction in osteoblast-like cells.

Cell interaction with extracellular matrix (ECM) modulates cell growth and differentiation. By using in vitro culture systems, we tested the effect of type I collagen (Coll-I) on signal transduction mechanisms in the osteosarcoma cell line UMR-106 and in primary cultures from neonatal rat calvariae. Cells were cultured for 72 h on Coll-I gel matrix and compared with control cells plated on plastic surfaces. Agonist-dependent and voltage-dependent rises in cytosolic Ca2+ concentration ([Ca2+]i; measured by fura 2 fluorometry) were significantly blunted in cells cultured on Coll-I compared with cells grown on plastic. In UMR-106 cells, the collagen matrix effect was mimicked by 24-h incubation with soluble Coll-I or short peptides containing the arginine-glycine-aspartate motif. Accumulation of cellular adenosine 3',5'-cyclic monophosphate (cAMP) stimulated by parathyroid hormone, cholera toxin, and forskolin was augmented (50-150%) in cells plated on Coll-I vs. control. The collagen effect on both [Ca2+]i- and adenylate cyclase-signaling pathways in UMR-106 cells was abrogated in the presence of protein kinase C (PKC) depletion or inhibition. Also, Coll-I induced a twofold increase in membrane-bound PKC without changing cytosolic PKC activity. Thus, by altering PKC activity, Coll-I modulates the [Ca2+]i- and cAMP-signaling pathways in osteoblasts. This, in turn, may influence bone remodeling processes.

Endothelin receptor in osteoblastic cells is coupled to multiple messenger signals.

We analyzed the functional characteristics of endothelin (ET) peptides in the osteoblastic UMR-106 cells by studying receptor binding as well as dose-response curves for ET-1 and ET-3 on two biological responses: 1) induction of Ca2+ transients and 2) activation of the Na(+)-H+ exchanger. ET specifically binds to a single class of receptor with a rank order of affinity ET-1 >> ET-3. ET-1 and ET-3 dose dependently stimulated a rise in intracellular Ca2+ ([Ca2+]i), with ET-1 being two orders of magnitude more potent than ET-3 [50% effective concentration (EC50) = 8 x 10(-10) and 9 x 10(-8) M for ET-1 and ET-3, respectively; P < 0.01]. The effect of ET-1 on [Ca2+]i was 90% inhibitable by the ETA antagonist BQ-123. The activity of Na(+)-H+ exchange was studied by using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein as well as by 22Na+ fluxes. ET-1 and ET-3 activated the exchange in a concentration-dependent manner and with similar potencies (EC50 approximately 10(-10) M). The action of ETs on Na(+)-H+ exchange was mimicked neither by phorbol esters nor by Ca2+ ionophores. It was, however, blocked by BQ-123 as well as by the protein tyrosine kinase inhibitor genistein. We conclude that in UMR-106 cells, a single ET receptor subtype is coupled to multiple effectors, a Ca2+ message system and a tyrosine-kinase system which, in turn, activates the Na(+)-H+ exchanger.

Interleukin-6 attenuates agonist-mediated calcium mobilization in murine osteoblastic cells.

Interleukin-6 (IL-6) is a multifunctional cytokine which is made by osteoblasts and has diverse effects on bone metabolism. We studied the interaction of IL-6 with the Ca2+ and cAMP signaling systems in the osteoblastic cell line UMR-106 and in primary osteoblastic cultures derived from neonatal rat calvariae. IL-6 did not alter basal intracellular calcium concentration ([Ca2+]i) but inhibited Ca2+ transients induced by parathyroid hormone (PTH), prostaglandin E2 (PGE2), and endothelin-1 in both dose- (100-400 U/ml) and time- (4-48 h) dependent manners. The effect of the cytokine was abolished by the tyrosine kinase inhibitor, herbimycin A (50 ng/ml). The IL-6 effect on the Ca2+ message system was related to suppressed production of hormonally induced inositol 1,4,5-triphosphate and inhibition of Ca2+ release from intracellular stores. Hormonally induced calcium entry pathways (estimated by using Mn2+ as a surrogate for Ca2+) were not, however, altered by the cytokine. IL-6 did not modulate cAMP generation in osteoblasts. With respect to osteoblast function, IL-6, although having no effect on cell proliferation by itself, greatly enhanced the antiproliferative effect of PGE2 and PTH. Because the production of IL-6 in osteoblasts is stimulated by calciotropic hormones (e.g., PTH and PGE2), the suppressive effect of the cytokine on hormonally induced Ca2+ transients may serve as an autocrine/paracrine mechanism for modulating the effect of hormones on bone metabolism.

The role of chronic anion gap and/or nonanion gap acidosis in the osteodystrophy of chronic renal failure in the predialysis era: a minority report.

Chronic renal failure (CRF) due to (1) glomerulopathies, vascular and tubulointerstitial disorders, and (2) chronic nonazotemic renal tubular disorders creates sustained acidosis in the untreated state. Number 1 represents a mixture of anion and nonanion gap acidosis and number 2 a pure nonanion gap acidosis. There remains significant uncertainty as to the role of the acidosis (CRF) in the associated osteodystrophy. In general, little attention has been given to this subject in recent monographs. It is the purpose of this review ('minority report') to 'reexamine' the information available on this subject in humans and animals. The author has concluded that the chronic metabolic acidosis of CRF may well contribute to the development and maintenance of the osteodystrophy, and that its treatment should be included along with the other modalities of therapy. The subject is not a 'dead issue' but one definitely deserving further investigation. The response of the skeleton to acid loads clearly represents another 'trade-off' in ion metabolism in CRF.

Acute phosphate depletion inhibits the Na+/H+ antiporter in a cultured renal cell line.

We studied the effect of acute Pi depletion on the regulation of intracellular pH (pHi) in the OK opossum kidney cell line by using the pH-sensitive dye 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Cell recovery from an NH4Cl acid load in HCO3-free buffer disclosed an Na(+)-dependent component blocked by amiloride and a smaller Na(+)-independent component that increased on exposure of the cells to a high-K+ buffer. After 24-h incubation of the cells in phosphate-free medium, pHi recovery by the Na+/H+ exchanger was markedly inhibited, whereas the Na(+)-independent pHi recovery was not affected. The inhibition of Na+/H+ exchange was reversible on correction of cellular Pi deficit. A similar phenomenon was observed when cellular Pi depletion was induced by acute exposure (min) to fructose. Pi depletion shifted the pHi dependence of the exchanger and also reduced its maximal activity. Time-course studies revealed that the effect of Pi depletion could not be attributed to attenuation of Na(+)-K(+)-adenosinetriphosphatase activity and resultant diminution of the transmembrane gradient for the Na+ influx. We conclude that acute Pi depletion in cultured proximal tubular cells leads to reversible inhibition of the Na+/H+ exchanger. This in vitro finding may relate to the in vivo observation of impaired HCO3 reabsorption and bicarbonaturia in acute Pi depletion.

1,25-Dihydroxyvitamin D3 inhibits Na(+)-dependent phosphate transport in osteoblastic cells.

In the present work we investigated the influence of vitamin D3 metabolites on Na(+)-dependent phosphate (Pi) transport in the clonal osteoblastic cell line UMR-106. The vitamin D3 metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] dose-dependently inhibited Pi transport with a half-maximal concentration of approximately 5 x 10(-11) M. The effect of 1,25(OH)2D3 was first observed after 8 h of preincubation period. Inhibition of phosphate uptake was relatively specific for the 1,25(OH)2D3 analogue of vitamin D3. The potency order was 1,25(OH)2D3 >> 24,25-dihydroxyvitamin D3 > 25-[3H]hydroxyvitamin D3. Kinetically, 1,25(OH)2D3 decreased the maximal velocity of the phosphate uptake system, whereas the affinity for phosphate was unaffected. Activation of protein kinase C (PKC) in UMR-106 cells stimulated Na(+)-dependent Pi transport. Nonetheless, the inhibitory effect of 1,25(OH)2D3 on Pi transport was not related to downregulation of PKC. Chemical determination of intracellular Pi showed a 50% reduction after 24-h preincubation with 10(-8) M 1,25(OH)2D3. We conclude that 1,25(OH)2D3 inhibits Na(+)-dependent phosphate transport in osteoblastic cells. This in turn leads to intracellular Pi depletion. The physiological implication of this phenomenon on the effects of vitamin D on osteoblasts in situ is discussed.

1,25(OH)2D3 blunts hormone-elevated cytosolic Ca2+ in osteoblast-like cells.

Cytosolic free calcium ([Ca2+]i) is an important regulator of bone cell physiology. We studied the interaction of vitamin D metabolites on the hormonal-activated Ca message system in the osteoblastic cell line UMR-106. The acute rise in [Ca2+]i induced by different calciotropic hormones [parathyroid hormone, prostaglandin E2 (PGE2)] was dose dependently blunted by 1,25-dihydroxyvitamin D [1,25(OH)2D3; half-maximal inhibitory concn approximately 5 x 10(-11) M] and was initially observed after 8 h of preincubation. The 1,25(OH)2D3 metabolite of vitamin D was two orders of magnitude more potent than 24,25(OH)2D3 and 25(OH)D3. To discern between an effect of 1,25(OH)2D3 on hormonal-induced Ca2+ entry through the plasma membrane channel vs. release of Ca2+ from internal stores, we suspended fura-2-loaded cells in Mn2+ rather than Ca2+ buffers. In cells preincubated with 1,25(OH)2D3, [Ca2+]i release (indicated by [Ca2+]i transient) was significantly blunted, whereas Mn2+ influx (indicating Ca2+ flux across the plasma membrane) was unaltered, suggesting a selective effect of 1,25(OH)2D3 on hormonal-activated release of Ca2+ from intracellular stores. 1,25(OH)2D3 also inhibited the PGE2-induced production of inositol 1,4,5-trisphosphate. We conclude that, in osteoblasts, chronic (hours) incubation with 1,25(OH)2D3 leads to attenuated stimulation of the [Ca2+]i transduction pathway by calciotropic hormones. This effect of 1,25(OH)2D3 may provide a cellular basis for the synergism between the effects of vitamin D and calciotropic hormones at the bone level.

Dissociation between parathyroid hormone-stimulated cAMP and calcium increase in UMR-106-01 cells.

We used the osteogenic sarcoma cell line, UMR-106-01, to determine whether the rise in free cytosolic Ca2+ concentration ([Ca2+]i) and cellular cAMP following PTH stimulation are able to be regulated independently. For this purpose, we compared the effect of a PTH antagonist, stimulation of protein kinase C, augmentation by prostaglandins, and the time course of desensitization of the two cellular responses. Two x 10(-7) M of the PTH antagonist 8,18Nle 34Tyr-bPTH(3-34) amide ([Nle,Tyr]bPTH(3-34)A) was required to inhibit 10(-9) M bPTH(1-34)-stimulated cAMP generation by 50%. 10(-7) M bPTH(1-34) completely overcame the inhibition induced by 10(-6) M [Nle,Tyr]bPTH(3-34)A. Only 7 x 10(-8) M and 2.7 x 10(-7) M [Nle,Tyr]bPTH(3-34)A were required to half maximally inhibit the [Ca2+]i increase evoked by 3 x 10(-8) and 10(-7) M bPTH(1-34), respectively. In addition, dissociation between [Ca2+]i and cAMP signals was observed when modulation by protein kinase C and prostaglandins was tested. Preincubation of the cells with 10 nM TPA for 5 minutes markedly inhibited the PTH-evoked [Ca2+]i increase. Short incubation with PGF2 alpha augmented the PTH-evoked [Ca2+]i increase. Similar pretreatments had no effect on the PTH-stimulated cAMP increase. Finally, preincubation with 1.5 x 10(-9) M bPTH(1-34) for 20 minutes almost completely blocked the effect of 10(-7) M bPTH(1-34) on [Ca2+]i, while preincubation with 5 x 10(-9) M bPTH(1-34) for 4 hours was required to inhibit the effect of 10(-8) M bPTH(1-34) on cAMP production by 50%. The differences in the regulation of the two PTH-stimulated cellular signaling systems, in particular, the response to antagonists and the time course of desensitization, could be at the level of the PTH receptor(s) or at a postreceptor domain.

Role of calcium and cAMP messenger systems in intracellular pH regulation of osteoblastic cells.

We have recently shown that two mechanisms are involved in the regulation of pHi in the osteoblastic phenotype cell line UMR-106 (Na(+)-H+ antiporter and a Na(+)-independent Cl(-)-HCO 3(-)-OH- exchanger). In the present work, we used the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein as well as isotope fluxes to investigate the influence of second messengers on the activity of these transporters. Elevation in intracellular calcium concentration [( Ca2+]in) in UMR-106 cells (measured by fura-2 fluorescence) is followed by stimulation of the Cl(-)-HCO3- exchanger, leading to cytosolic acidification. Subsequently, cell alkalinization, mediated by the Na(+)-H+ exchanger, restores pHi to its resting value. An acute reduction in [Ca2+]in abruptly stops the activity of the anion exchanger while having no influence on the activity of the Na(+)-H+ exchanger. The stimulatory effect of Ca2+in on the anion exchanger is dose dependent and is abrogated by the calmodulin inhibitors N-(6-aminohexyl)-5-chloro-naphthalenesulfonamide and calmidazolium. An increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) brought about by forskolin, 8-bromo-cAMP, or prostaglandin E2 leads to inhibition of activity of both the Na(+)-H+ antiporter and the anion exchanger. The suppressive effect of cAMP on Cl(-)-HCO3- exchange could be overcome by elevating [Ca2+]in. We conclude that 1) Ca2+in and cAMP can influence pHi in osteoblasts by altering the activities of pHi regulatory mechanisms and 2) the effect of Ca2+in is probably mediated by calmodulin.

Voltage-dependent phosphate transport in osteoblast-like cells.

Phosphate ion (Pi) in sufficient concentrations is crucial for bone mineralization. The osteoblast (OB) may be responsible for the transport of Pi into the bone interstitium, where mineralization occurs. We previously characterized a Na(+)-dependent Pi transporter (NaPi) in the osteoblastic UMR-106-01 cell line. In the present study, the alteration of Na(+)-dependent Pi transport by changes in membrane potential was investigated. Depolarizing the cells with increasing concentrations of ambient K+ and valinomycin resulted in a progressive decline in Na(+)-dependent Pi uptake to a maximum of 28% at a membrane potential of -18 mV compared to control Na(+)-dependent Pi uptake at a membrane potential of approximately -60 mV. Hyperpolarizing the cells with SCN- increased Na(+)-dependent Pi uptake over control by 50% at an SCN- concentration of 70 mM. Determination of membrane potential by using the fluorescent probe, DiSC3(5), showed that the addition of Pi to cells in Na(+)-containing medium resulted in a small depolarization. These data show that NaPi activity can be altered by membrane potential changes and that the initiation of Na(+)-dependent Pi uptake is associated with depolarization of the plasma membrane of UMR-106-01 cells. Taken together, the cotransport of Na+ and Pi results in the movement of a net positive charge into the cell.

Prostaglandins enhance parathyroid hormone-evoked increase in free cytosolic calcium concentration in osteoblast-like cells.

Prostaglandins (PGs) are autocrine or paracrine hormones that may interact with circulating hormones such as parathyroid hormone (PTH) in bone. We examined the interaction of the PGs, PGF2 alpha, PGE2, and 6-keto-PGF1 alpha with PTH to enhance the rapid, initial transient rise in free cytosolic calcium ([Ca2+]i) and cAMP levels stimulated by PTH. Pretreatment of UMR-106, MC3T3-E1, and neonatal rat calvarial osteoblast-like cells by PGs resulted in an enhancement of the early transient rise in [Ca2+]i stimulated by PTH. PGF2 alpha was approximately 100 times more potent than PGE2. PGE2 itself was more potent than 6-keto-PGF1 alpha in enhancing PTH-stimulated rise in [Ca2+]i. Near-maximal augmentation was achieved at PGF2 alpha doses of 10 nM and PGE2 of 1 microM. The degree of augmentation in [Ca2+]i by PGF2 alpha was independent of preincubation time. PGF2 alpha pretreatment did not alter the EC50 for the PTH-induced [Ca2+]i increase but only the extent of rise in [Ca2+]i at each dose of PTH. The augmented increase in [Ca2+]i was mostly due to enhanced PTH-mediated release of Ca2+ from intracellular stores. PGF2 alpha did not stimulate an increase in PTH receptor number as assessed by [125I]-PTH-related peptide binding. PG pretreatment partially reversed PTH inhibition of cell proliferation, suggesting that an increase in [Ca2+]i may play a role in tempering the anti-proliferative effect of PTH mediated by cAMP. These studies suggest a new mode by which PGs can affect cellular activity.

Acute phosphate depletion dissociates hormonal stimulated second messengers in osteoblast-like cells.

The acute effect (24 h) of either phosphate depletion or phosphate surfeit on hormonal stimulated signal transduction systems was studied in the osteoblastic cell line UMR-106. Elevation of intracellular Ca2+ ([Ca2+]in), induced by different calciotropic hormones (PTH, prostaglandin E2, endothelin) was blunted by acute phosphate depletion, whereas at high inorganic phosphate (Pi) concentrations the rise in [Ca2+]in was augmented. Basal [Ca2+]in was not altered by either Pi depletion or Pi excess. The effect of acute phosphate depletion on hormonal mediated [Ca2+]in rise was not observed in the absence of extracellular Ca2+ suggesting that under these conditions, the release of Ca2+ from intracellular stores, is not affected. Also, nonhormonal calcium entry pathways such as depolarization-activated calcium channels or protein kinase C-activated Ca2+ channels were not affected by acute phosphate depletion. cAMP accumulation in the cells, either through receptor or nonreceptor-mediated mechanisms, increased under low Pi conditions and decreased as Pi concentration in the culture media was progressively increased from 0 to 2 mM during 24 h of incubation. Changes in Pi concentration had no effect on basal cAMP generation by the cells. The facilitative effect of acute Pi depletion on agonist-induced cAMP accumulation could be demonstrated in both the presence and absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2 mM). PTH receptor binding assessed with [Nle8 Nle18 Tyr34] bovine PTH (1-34) NH2 was not altered by phosphate depletion. We conclude that exposure of osteoblasts to different Pi environments modulates the second messenger responses to hormones in a reciprocal fashion so that acute phosphate depletion down-regulates [Ca2+]in signals while augmenting cAMP generation and vice versa. Inasmuch as bone resorption processes can be modulated by Ca2+ and cAMP the data presented herein suggest that the altered bone resorptive response to calciotropic hormones (e.g. PTH), under surfeit or deficit of phosphate, is mediated by changes in [Ca2+]in and cAMP.