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Parathyroid hormone (PTH) and PTH-related protein/peptide (PTHrP) bind to the same PTH/PTHrP receptor and stimulate osteoblasts to secrete pro-inflammatory cytokines like interleukin (IL)-6. In patients with primary hyperparathyroidism, elevation of plasma levels of tumor necrosis factor (TNF)-alpha and IL-6 was also described. We, therefore, postulated that PTHrP secreted from cancer cells stimulates the secretion of cytokines and causes increases in their blood levels. Blood concentrations of several cytokines (TNF-alpha, IL-1beta, IL-5, IL-6, IL-8, IL-11 and IL-12) in cancer-bearing patients with or without elevation of blood PTHrP were measured by ELISA. The patients with high plasma PTHrP levels (n=29, intact PTHrP: 8.5 +/- 1.4 pmol/l, normal: <1.1) had higher serum type 1 collagen C-telopeptide (ICTP). Twenty of the patients were hypercalcemic. Plasma concentrations of TNF-alpha, IL-6 and IL-8 were significantly increased in patients with high PTHrP, in either the presence or absence of hypercalcemia. The concentrations of TNF-alpha and IL-6 were also significantly correlated with those of PTHrP. Our observations indicate that high plasma levels of PTHrP in cancer-bearing patients contribute not only to the development of hypercalcemia, but also to the development of the syndrome caused by an excess of pro-inflammatory cytokines.

Sentinel node (SN) biopsy is a promising replacement for standard axillary lymph node dissection for the staging of early breast cancer, and various techniques have been studied to identify SNs with dye or radioactive colloid. This study assesses the effect of the dose of radioactivity and the time before biopsy in order to set standards for the use of 99mTc-rhenium sulphide for the detection of SNs in breast cancer patients. Sixty patients with stage T1-2 N0 M0 breast cancer underwent SN biopsy, which was immediately followed by standard axillary dissection to confirm the SN results. For SN biopsy, 99mTc-rhenium colloid was injected peritumorally. A 1 day (morning injection and afternoon surgery) or 2 day (day before afternoon injection and morning surgery) protocol was applied. A dose-finding study was performed simultaneously using 7.4-37 MBq for the 1 day protocol and 37-74 MBq for the 2 day protocol. A scintigram was taken at 2 h for the 1 day protocol and 16 h for the 2 day protocol. After the injection of blue dye, SN biopsy was performed with a gamma probe, followed by standard axillary node dissection. The radiation exposure received by the surgical team during the operation was monitored. Histopathological comparison between SNs and axillary nodes was performed. Patient characteristics that might affect the radiocolloid uptake by SNs were assessed. SNs were identified in all patients regardless of the dose or administration protocol used. Two patients showed false negative pathological SN results, and the negative predictive value was 96 and the positive predictive value was 100. In addition, radiation exposure to the surgical team and the amount of radioactive surgical waste were low, especially at lower doses. Two groups of patient characteristics were related to SN uptake. One was the body mass index (BMI) and the other was the age or menopausal status. Patients with a larger BMI tended to take up a smaller amount of 99mTc colloid. Older or post-menopausal patients showed lower SN uptake. 99mTc-rhenium sulphide colloid is an efficient radiopharmaceutical for SN detection. Both 1 day and 2 day protocols have equally good efficacy, and the recommended dose is 7.4 MBq for the 1 day protocol and 37 MBq for the 2 day protocol. Patients with larger BMI and older or post-menopausal patients tend to take up less 99mTc colloid.

The clinical usefulness of incadronate was compared with elcatonin in 26 patients with malignancy-associated hypercalcaemia. Data from 21 and 24 patients could be used to assess efficacy and safety, respectively. Eleven patients were given a single 10-mg intravenous infusion of incadronate and 10 received twice-daily intramuscular injections of 40 IU of elcatonin for 7 consecutive days. After treatment, corrected serum calcium levels decreased significantly in both groups. The anti-hypercalcaemic effect of elcatonin was characterized by its rapid onset, with serum calcium levels reduced 1 day after administration. In contrast, the anti-hypercalcaemic effect of incadronate was more sustained but only became apparent a few days after infusion. Evaluation of symptoms revealed significantly greater improvement rates in the incadronate group compared with the elcatonin group. Adverse drug reactions were observed in three patients in the incadronate group, i.e. mild and transient fever in two cases and exacerbation of disturbance of consciousness in one case. These findings suggest that incadronate produces more marked and sustained hypocalcaemic effects than elcatonin, and that co-administration of these two drugs may yield both rapid and sustained control of malignancy-associated hypercalcaemia.

Background/Aims:Although the PTH-suppressive effect of intravenous calcitriol has already been demonstrated by various studies, the precise dose-response to calcitriol has not been fully determined for uremic secondary hyperparathyroidism (2HPT). In order to investigate in detail the dose-response of intravenous calcitriol and the adequate initial dose against 2HPT, a randomized prospective double-blind study was conducted. Method:One-hundred and sixty-two patients with 2HPT undergoing hemodialysis three times per week were randomly assigned to four calcitriol (Ro21-5535) treatment groups, 0 (placebo), 1, 1.5 or 2 µg. Calcitriol or placebo was given intravenously after each dialysis for 12 weeks under double-blind conditions. Results:Calcitriol dose-dependently reduced both intact-PTH and high-sensitivity assay mid-terminal (HS)-PTH levels. The rate of per-week change in intact-PTH was 0.0% in the placebo group, –7.8% in the 1-µg group, –18.9% in the 1.5-µg group and –24.1% in the 2-µg group. Calcitriol dose-dependently increased the rate of increase in serum Ca adjusted by albumin level. The per-week increases in adjusted serum Ca were –0.01, 0.08, 0.23 and 0.35 mg/dl in the placebo, 1-, 1.5- and 2-µg groups, respectively. Although the degree of PTH suppression was correlated with the adjusted serum Ca increase, by-patients investigation revealed that the number of patients with suppression of PTH despite of no or slight elevation of adjusted serum Ca level was largest in the 1-µg group among the three calcitriol groups. Conclusion:Intravenous calcitriol was found to have a clear dose-dependent effect on PTH reduction in patients with 2HPT, and the appropriate initial dose of this agent was determined to be 1 µg per dialysis session.

Abstract: Background. Bisphosphonates are bone resorption inhibitors which are effective in the treatment of diseases of increased bone turnover, such as hypercalcemia of malignancy and osteolytic bone metastasis. The safety and efficacy of incadronate, a third-generation bisphosphonate, were evaluated in breast cancer patients with bone metastases. Methods. Fifteen breast cancer patients with bone metastasis were enrolled. Incadronate's safety, its effectiveness in relieving bone pain, and its effects on bone metabolic markers and a tumor marker were assessed in 8 patients treated with a 10-mg IV infusion once a week for 5 weeks (10 mg × 5), 3 patients treated with a single 20-mg IV infusion (20 mg × 1), and 4 patients treated with a 20-mg IV infusion once a week for 5 weeks (20 mg × 5). Pain assessment was performed only in the patients with the repeated infusion regimens. Results. All incadronate treatment regimens were administered without any serious adverse reactions. Minimal fever was noted in 6 patients, but it subsided without any treatment. Incadronate relieved bone pain in 10 of the 12 patients who received repeated infusions. Levels of bone resorption markers dropped transiently, but the decreases in the individual markers of bone resorption varied. Levels of bone formation markers did not change significantly. Levels of a tumor marker specific to breast cancer, carbohydrate antigen (CA)15-3 decreased in patients whose metastases were limited to bone. Conclusion. The third-generation bisphosphonate, incadronate, was administered safely at dosages of up to 20 mg once a week for 5 weeks. Incadronate reduced bone pain, bone resorption marker levels, and CA15-3 tumor marker levels in breast cancer patients with bone metastases.

Abstract: Background. The efficacy and cost-performance benefit of radionuclide bone scintigraphy in monitoring metastatic bone activity remain controversial. Bone metabolic markers are now expected to play a role in the diagnosis and follow-up of bone metastasis. Methods. We investigated several bone metabolic markers in patients with breast cancer. We measured three metabolic markers of bone resorption: pyridinoline cross-linked carboxy terminal telopeptide (ICTP), C-telopeptides of type I collagen (CTx), and the free form of deoxypyridinoline (fDPD), and four metabolic markers of bone formation: procollagen I carboxy terminal peptide (PICP), total alkaline phosphatase (Al-p), bone-specific alkaline phosphatase (BAl-p), and osteocalcin (BGP) in 210 patients without and 268 patients with bone metastasis. Patients without bone metastasis were analyzed in terms of menstruation status. Patients with bone metastasis were analyzed in terms of bone metastatic burden and tumor lesion "condition" (ie, determination by X-ray and/or computed tomography and bone scan findings of new lesion, progression of disease, no change, improvement, and complete remission, according to the criteria of the International Unite Against Cancer). Results. In patients without bone metastasis, ICTP did not change with menopause. All markers other than ICTP were significantly elevated with menopause. In patients with bone metastasis, all markers, except for BGP, were significantly elevated according to metastatic bone tumor burden. Among the seven markers, ICTP showed the best receiver operating characteristic curves. ICTP also showed the best correlation to bone metastatic burden among the markers by Spearman's rank correlation coefficient. In patients stratified by "condition", ICTP, CTx, fDPD, Al-p, and BAl-p showed significant elevation in patients with progression, new lesion, and no change, while PICP and BGP showed only minimal elevation in those patients. Conclusion. Bone metabolic markers, particularly ICTP, appear to be valuable for the diagnosis of bone metastasis from breast cancer.

Although alfacalcidol is widely used in the treatment of osteoporosis, its mechanism of action in bone is not fully understood. Alfacalcidol stimulates intestinal calcium (Ca) absorption, increases urinary Ca excretion and serum Ca levels, and suppresses parathyroid hormone (PTH) secretion. It remains to be clarified, especially under vitamin D-replete conditions, whether alfacalcidol exerts skeletal effects solely via these Ca-related effects, whether the resultant suppression of PTH is a prerequisite for the skeletal actions of alfacalcidol, and, by inference, whether alfacalcidol has an advantage over vitamin D in the treatment of osteoporosis. To address these issues, we (1) compared the effects of alfacalcidol p.o. (0.025–0.1 μg/kg BW) vis-à-vis vitamin D3(50–400 μg/kg BW) on bone loss in 8-month-old, ovariectomized (OVX) rats as a function of their Ca-related effects, and (2) examined whether the skeletal effects of alfacalcidol occur independently of suppression of PTH, using parathyroidectomized (PTX) rats continuously infused with hPTH(1–34). The results indicate that (1) in OVX rats, alfacalcidol increases BMD and bone strength more effectively than vitamin D3at given urinary and serum Ca levels: larger doses of vitamin D3are required to produce a similar BMD-increasing effect, in the face of hypercalcemia and compromised bone quality; (2) at doses that maintain serum Ca below 10 mg/dl, alfacalcidol suppresses urinary deoxypyridinoline excretion more effectively than vitamin D3; and (3) alfacalcidol is capable of increasing bone mass in PTX rats with continuous infusion of PTH, and therefore acts independently of PTH levels. It is suggested that alfacalcidol exerts bone-protective effects independently of its Ca-related effects, and is in this respect superior to vitamin D3, and that the skeletal actions of alfacalcidol take place, at least in part, independently of suppression of PTH. Together, these results provide a rationale for the clinical utility of alfacalcidol and its advantage over vitamin D3in the treatment of osteoporosis.

Insulin-like growth factor II (IGF II) is a member of somatomedin family and is a potent mitogen in various types of mammalian cells. We have recently reported that IGF II stimulates calcium influx in competent BALB/c 3T3 cells primed with epidermal growth factor (J. Biol. Chem. 262: 12120-12126, 1987). Using patch-clamp technique, we show here an IGF II-sensitive cation channel in BALB/c 3T3 cell plasma membrane. Calcium is permeable to this cation channel and its opening behavior is independent of membrane potential. IGF II increases the opening probability of the identical channel in cells pretreated sequentially with platelet-derived growth factor and epidermal growth factor, whereas IGF II does not affect the opening of the channel in G0-arrested cells. This cation channel activity is observed only when IGF II is included in the patch pipette, indicating direct regulation of the channel by IGF II. We suggest that IGF II stimulates calcium influx by opening this cation channel and that IGF II activates the channel in a unique cell cycle-dependent manner.

The effect of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] metabolism was examined in rats fed on a low-calcium diet. These rats exhibit hypocalcaemia, high urinary cyclic AMP excretion, a markedly elevated serum 1,25(OH)2D concentration and low serum concentrations of both 24,25(OH)2D and 25(OH)D. When the rats are treated orally with 1, 5 or 10 micrograms of 24,25(OH)2D3/100 g every day, there is a dramatic decrease in serum 1,25(OH)2D concentration in a dose-dependent manner concomitant with an increase in serum 24,25(OH)2D concentration. Serum calcium concentration and urinary cyclic AMP excretion are not significantly affected by the 24,25(OH)2D3 treatment, which suggests that parathyroid function is not affected by the 24,25(OH)2D3 treatment. The 25(OH)D3 1 alpha-hydroxylase activity measured in kidney homogenates is markedly elevated in rats on a low-calcium diet but is not affected by any doses of 24,25(OH)2D3. In contrast, recovery of intravenously injected [3H]1,25(OH)2D3 in the serum is decreased in 24,25(OH)2D3-treated rats. Furthermore, when [3H]1,25(OH)2D3 is incubated in vitro with kidney or intestinal homogenates of 24,25(OH)2D3-treated rats there is a decrease in the recovery of radioactivity in the total lipid extract as well as in the 1,25(OH)2D3 fraction along with an increase in the recovery of radioactivity in the water-soluble phase. These results are consistent with the possibility that 24,25(OH)2D3 has an effect on 1,25(OH)2D3 metabolism, namely that of enhancing the degradation of 1,25(OH)2D3. However, because a considerable proportion of the injected 24,25(OH)2D3 is expected to be converted into 1,24,25(OH)3D3 by renal 1 alpha-hydroxylase in 24,25(OH)2D3-treated rats, at least a part of the decrease in serum 1,25(OH)2D concentration may be due to a competitive inhibition by 24,25(OH)2D3 of the synthesis of 1,25(OH)2D3 from 25(OH)D3. Thus the physiological importance of the role of 24,25(OH)2D3 in regulating the serum 1,25(OH)2D concentration as well as the mechanism and metabolic pathway of degradation of 1,25(OH)2D3 remain to be clarified.

Renal cortical and medullary infarction are 2 of the severe complications of perinatal asphyxia and shock. The angiographic and ultrasonographic findings in these conditions have not been described previously. They demonstrate nephromegaly, a nonhomogeneous nephrogram and internal echoes. Similar findings may be seen in renal vein thrombosis, hydronephrosis, and polycystic kidneys.

Amyloid precursor protein (APP), a transmembrane precursor of beta-amyloid, possesses a function whereby it associates with G(o) through its cytoplasmic His657-Lys676. Here we demonstrate that APP has a receptor function. In phospholipid vesicles consisting of baculovirally made APP695 and brain trimeric G(o), 22C11, a monoclonal antibody against the extracellular domain of APP, increased GTP gamma S binding and the turnover number of GTPase of G(o) without affecting its intrinsic GTPase activity. This effect of 22C11 was specific among various antibodies and was observed neither in G(o) vesicles nor in APP695/Gi2 vesicles. In APP695/G(o) vesicles, synthetic APP66-81, the epitope of 22C11, competitively antagonized the action of 22C11. Monoclonal antibody against APP657-676, the G(o) binding domain of APP695, specifically blocked 22C11-dependent activation of G(o). Therefore, APP has a potential receptor function whereby it specifically activates G(o) in a ligand-dependent and ligand-specific manner.

The present study was undertaken to identify serum components other than vitamin D-binding proteins that bind to 1,25(OH)₂ D₃, and its analog. The binding rate of 1,25(OH)₂ D₃, 22-oxa-1,25(OH)₂D₃ (OCT) or 25(OH) D₃ to total lipoprotein(TLP) represented 16.7%, 4.65%, and 3.11% of total counts added, respectively. Polyacrylamide gel electrophoresis of the TLP revealed that 1,25(OH)₂ D₃ and OCT were associated with LDL. The binding studies of OCT-bound LDL to the fibroblasts showed specific pathway to the cells mediated by LDL-receptor. These findings may have important implications in understanding the mechanisms of the diverse biological actions of 1,25(OH)₂ D₃ and in designing a novel delivery system for vitamin D analogs.Copyright 1995, 1999 Academic Press, Inc.

1. Effects of activin A on ionic channels were examined in human FSH‐secreting tumour cells using electrophysiological techniques. 2. Under voltage clamp with the conventional whole‐cell clamp technique, the voltage‐gated Na+ channel, the T‐ and L‐type Ca2+ channels, the delayed K+ channel and the A‐channel were observed. 3. With the nystatin‐perforated whole‐cell clamp technique, the same voltage‐gated channels were recorded. Activin A (10(‐7) M) increased the amplitude of the L‐type Ca2+ current, whereas it decreased the amplitude of the delayed K+ current. 4. Under current clamp with the perforated whole‐cell clamp technique, more than 80% of the cells exhibited spontaneous action potentials. Application of 10(‐7) M activin A depolarized the membrane with a conductance increase and augmented action potential frequency. The reversal potential of the activin A‐induced current was ‐20 to 0 mV. The activin A‐induced current was abolished in a Na(+)‐free extracellular solution, indicating that the membrane depolarization caused by activin A was due to the conductance increase to Na+ ions through non‐selective cation channels.

Membrane electrical properties and the response to somatostatin were examined in dissociated human pituitary adenoma cells that secrete growth hormone (GH). Under current clamp condition with a patch electrode, the resting potential was -52.4 +/- 8.0 mV, and spontaneous action potentials were observed in 58% of the cells. Under voltage clamp condition an outward K+ current, a tetrodotoxin-sensitive Na+ current, and a Ca2+ current were observed. Cobalt ions suppressed the Ca2+ current. The threshold of Ca2+ current activation was about -60 mV. Somatostatin elicited a membrane hyperpolarization associated with increased membrane permeability in these cells. The reversal potential of somatostatin-induced hyperpolarization was -78.4 +/- 4.3 mV in 6 mM K+ medium and -97.2 +/- 6.4 mV in 3 mM K+ medium. These reversal potential values and a shift with the external K+ concentration indicated that membrane hyperpolarization was caused by increased permeability to K+. The hyperpolarized membrane potential induced by somatostatin was -63.6 +/- 5.9 mV in the standard medium. This level was subthreshold for Ca2+ and Na+ currents and was sufficient to inhibit spontaneous action potentials. Hormone secretion was significantly suppressed by somatostatin and cobalt ions. Therefore, we suggest that Ca2+ entering the cell through voltage-dependent channels are playing an important role for GH secretion and that somatostatin suppresses GH secretion by blocking Ca2+ currents. Finally, we discuss other possibilities for the inhibitory effect of somatostatin on GH secretion.

The present study was undertaken to determine whether an agonist-induced activation of C-kinase leads to an inhibition of phospholipase C in adrenal glomerulosa cells. When cells are treated with 100 nM-TPA (12-O-tetradecanoylphorbol 13-acetate), subsequent angiotensin (‘angiotensin II’)-induced aldosterone secretion is greatly inhibited. Treatment with TPA completely inhibits the angiotensin-induced increase in both inositol trisphosphate and the cytosolic Ca2+ concentration. The dose-response curve for TPA-induced inhibition reveals that quite a high concentration of TPA is necessary to block angiotensin action compared with that needed to stimulate aldosterone secretion. 1-Oleoyl-2-acetylglycerol has a weak inhibitory effect, whereas neither 4 alpha-phorbol 12,13-didecanoate or 4 beta-phorbol inhibits angiotensin action. When the time course of changes in inositol trisphosphate and diacylglycerol is measured, angiotensin action is sustained for up to 30 min. In addition, 100 nM-TPA added after 20 min of angiotensin addition attenuates production of both inositol trisphosphate and diacylglycerol. These results suggest that high dose of TPA inhibits angiotensin-induced activation of phospholipase C by acting, at least partly, on C-kinase, but that an inhibitory effect of TPA may be a pharmacological effect with little physiological significance in this system.

Phosphate uptake was studied in confluent monolayers of an epithelial-cell line (JTC-12) derived from monkey kidney. Phosphate uptake consisted of a saturable, Na+-dependent, component, which accounted for about 80% of the uptake, and a nonsaturable, Na+-independent, component. The saturable component was specifically dependent on the presence of extracellular Na+ and has an apparent Km value for phosphate of 0.12 mM at 137-mM-Na+, which is close to those reported in the brush-border membranes in mammalian kidneys. The presence of Na+ in the uptake solution decreased the Km for phosphate without affecting the Vmax. Phosphate uptake was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and ouabain, suggesting that phosphate transport is an active, energy-dependent, process and is dependent on an Na+ gradient across cell membranes. With respect to the effect of external Na+ concentration, a sigmoid relation was seen between the initial velocity of phosphate uptake and Na+ concentrations, and Hill analysis gave a Hill coefficient of 1.8. In the pH range 6.6-7.4, phosphate uptake declined with increasing pH. Phosphate uptake was stimulated when cells were cultured in the presence of insulin, and was also affected by changes in phosphate concentrations in cultured medium. These results indicate that JTC-12 cells have an Na+-dependent phosphate-transport system with many of the features of phosphate transport in the proximal tubule.

The effect of prostaglandin E2 on accumulation in plasma of 1 alpha,25-dihydroxy[3H]vitamin D3 from 25-hydroxy[3H]vitamin D3 was studied in vivo using vitamin D-deficient thyroparathyroidectomized rats. Intra-arterial infusion of 10-50 micrograms of prostaglandin E2/h caused a significant stimulation of 1 alpha,25-dihydroxy[3H]vitamin D3 production. No significant changes in plasma Ca2+ and Pi concentrations or urinary cyclic AMP excretion were observed after prostaglandin E2 infusion. Theophylline did not enhance the effect of a submaximal dose of prostaglandin E2 on 1 alpha,25-dihydroxy[3H]vitamin D3 production. These data indicate that prostaglandin E2 stimulates plasma accumulation of 1 alpha,25-dihydroxy[3H]vitamin D3 independent of the adenylate cyclase/cyclic AMP system, and suggest that prostaglandin E2 has a modulatory role in the regulation of 25-hydroxyvitamin D3 1 alpha-hydroxylase in the kidney.

The functional significance of the Na+K+-ATPase activity in defining the sensitivity of vascular smooth muscle response to pressor stimuli was studied in guinea pig aortic strips. Subthreshold doses of ouabain (10-8, 10-7, 10-6M), potentiated the norepinephrine- and angiotensin II-induced contractile responses, dose-dependently. Furthermore, in the presence of subthreshold dose of ouabain (10-6M), tension developments were observed with subthreshold doses of norepinephrine and angiotensin II. The mechanism by which subthreshold dose of ouabain potentiated the norepinephrine-induced contractile response was revealed to involve the enhancement both of sensitivity and contractile activity. Ouabain (10-6M) potentiated the norepinephrine- and A23187-induced contractile responses, even in the presence of verapamil. These facts indicate that suppression of the vascular Na+K+-ATPase activity could favor the development of hypertension through potentiating contractile responses to various stimuli and that the potentiation could be a reflection, at least partly, of the decrease in Ca2+-efflux

We found that the human parathyroid hormone-related peptide (hPTHrP) gene contained a DNA element (nVDREhPTHrP) homologous to a negative vitamin D response element in the human parathyroid hormone gene. It bound to vitamin D receptor (VDR) but not retinoic acid Xalpha receptor (RXRalpha) in the human T cell line MT2 cells. VDR binding to this element was confirmed by the Southwestern assay combined with immunodepletion using anti-VDR monoclonal antibody, and this binding activity was repressed by 1,25-dihydroxyvitamin D3. Such a repression was reversed by acid phosphatase treatment, suggesting that 1,25-dihydroxyvitamin D3 phosphorylates VDR to weaken its binding activity to nVDREhPTHrP. In electrophoretic mobility shift assay, we found anti-Ku antigen antibody specifically supershifted the MT2 nuclear proteinnVDREhPTHrP complex. The nVDREhPTHrP-bearing reporter plasmid produced vitamin D-dependent inhibition of the reporter activity in MT2 cells, which was markedly masked by the introduction of the Ku antigen expression vector in the antisense orientation. On the other hand, such a procedure did not perturb the vitamin D response element-mediated gene stimulation by vitamin D. These results indicate that nVDREhPTHrP interacts with Ku antigen in addition to VDR to mediate gene suppression by vitamin D.

Through the specific binding of a negative calcium-responsive element to its binding protein in response to extracellular Ca (Ca2+e), negative calcium-responsive element-bearing genes, such as the human parathyroid hormone gene, are negatively regulated by Ca2+e. The Ku antigen mediated negative gene regulation by Ca2+e by interacting with a redox factor protein, REF1. Although sequence-nonspecific DNA binding activity of the Ku antigen has been well characterized, the mechanism of its sequence-specific DNA binding remained obscure. Here, we report that the specific binding of the Ku antigen to another protein, REF1, leads to DNA-protein complex formation with a novel sequence specificity and thereby regulates gene expression.

Prostaglandin F2 alpha (PGF2 alpha) stimulates proliferation of clonal osteoblastic MC3T3-E1 cells mainly via the stimulation of phospholipase C. These cells constitutively produced and secreted insulin-like growth factor I (IGF-I). In addition, a neutralizing anti-IGF-I antibody completely abolished DNA synthesis stimulated by PGF2 alpha in MC3T3-E1 cells, suggesting that IGF-I indeed mediates the PGF2 alpha effect. However, PGF2 alpha decreased the expression of IGF-I mRNA and the secretion of immunoreactive IGF-I into the medium, whereas progression activity in the conditioned medium was not affected by PGF2 alpha. Although IGF-I alone did not stimulate DNA synthesis in MC3T3-E1 cells, when PGF2 alpha was added to the cultures, IGF-I stimulated their proliferation. Thus, PGF2 alpha may potentiate the action of IGF-I. At the same time, PGF2 alpha increased the number of high affinity binding sites (molecular mass of 130 kDa) for IGF-I in a dose-dependent manner. The increase in IGF-I-binding site number preceded the elevation of DNA synthesis by approximately 3 h. Furthermore, MC3T3-E1 cells secreted at least three species of IGF-binding proteins (IGFBPs) with molecular masses of 24, 30, and 34 kDa. In the early period of PGF2 alpha exposure, PGF2 alpha attenuated the secretion of all of these IGFBPs, whereas thereafter, it markedly increased their secretion, especially that of the 34-kDa IGFBP, suggesting a modulation of metabolism and action of IGF-I. These effects of PGF2 alpha on IGF-I receptor number and IGFBP secretion may play a role in the synergism between PGF2 alpha and IGF-I that results in the stimulation of DNA synthesis in MC3T3-E1 cells.

Accumulating evidence suggests that activin A/erythroid differentiation factor (EDF), a homodimer of beta A chain, is a physiologic hematopoietic factor, particularly of erythroid lineage. Media conditioned by phorbol myristate acetate (PMA)-stimulated murine bone marrow stromal cell lines, MC3T3-G2/PA6 and ST2, contained activin A/EDF, assayed as the activity inducing erythroid differentiation of an activin A/EDF-responsive murine erythroleukemia cell clone F5. Follistatin, a protein specifically binding to activin A/EDF, abolished this erythroid differentiation. Northern blot analysis showed that PMA rapidly increased beta A chain messenger RNA levels in MC3T3-G2/PA6 and ST2 cells. In a search for natural stimulators, we found that tumor necrosis factor-alpha, in itself and synergistically with interleukin-1 beta, induced activin A/EDF production in both cell lines. These results indicate that stromal cells produce activin A/EDF in bone marrow.

Osteoblast-like UMR106 cells secreted bone resorption-stimulating activity (BRSA) under stimulation by human parathyroid hormone 1-34. Most of BRSA was associated with cell/matrix surface and could be extracted by 2 M NaCl. Pretreatment with 17β-estradiol (E₂) reduced BRSA by enhancing the elaboration of an inhibitory factor of BRSA in a dose-dependent manner, and 10−9 M 17β -E₂ showed a significant effect. Neither 17α -E₂ nor dihydrotestosterone showed a similar effect. The inhibitory factor of BRSA was also bound to cell/matrix surface, showed affinity for heparin, and was trypsin- and heat-sensitive. Furthermore, this factor could also inhibit resorption pit formation stimulated by 1,25-dihydroxyvitamin D₃, interleukin (IL)-1α and IL-6. Elaboration of such an inhibitory factor of bone resorption from osteoblasts may play an important role in the protective effect of estrogen against bone resorption.Copyright 1993, 1999 Academic Press

G alpha i2 is a tissue-specific proto-oncogene product, whose activated mutant gip2 induces transformation through less defined downstream pathways. We found that c-fos promoter is a target of gip2 in multiple kinds of cells. Serum response element was shown to be the positive enhancer element that mediates gip2-induced c-fos expression. We further demonstrated that gip2 stimulates the negative silencer activity of the retinoblastoma (Rb) control element (RCE) and inhibits the c-fos promoter activity through RCE located in the c-fos promoter region. The effect of gip2 on RCE was shown to be mediated by the Rb gene product (pRb). Furthermore, gip2 augmented underphosphorylated active form of pRb by promoting pRb expression and by affecting the phosphorylation state of pRb. gip2 therefore propagates both positive and negative signals to the c-fos promoter through two different elements, and pRb mediates the negative signal of gip2. We conclude that gip2 has bifunctional roles in transformation which pRb critically regulates. Given that Rat-1 cells, which gip2 can transform, lack the sensitivity to the gip2/pRb-mediated negative pathway, this study provides a novel insight into oncogenesis by gip2 and its tissue specificity.

In BALB/c 3T3 cells pretreated with platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) (primed-competent cells), insulin-like growth factors I and II (IGF-I and IGF-II) bind to their own receptors (IGF-IR and IGF-IIR) and stimulate calcium influx and DNA synthesis by a mechanism involving a 40-kDa pertussis toxin substrate. In contrast, these IGFs do not act on unprimed quiescent cells. In this study, the 40-kDa pertussis toxin substrate was identified as Gi-2 alpha using anti-G protein antibodies. We analyzed the quality of signal transduction from IGF-II to Gi-2 alpha. There was no difference in the amount of Gi-2 alpha between quiescent and primed-competent cells, and both of these cells had similar Kd values and numbers of IGF-II-binding sites. Whereas IGF-II did not alter pertussis toxin-catalyzed ADP-ribosylation of Gi-2 alpha in quiescent cells, IGF-II reduced the pertussis toxin substrate activity by 35-50% via the IGF-IIR in primed-competent cells. The action of IGF-II lasted for up to 3 h when IGF-II was present in the medium, and it disappeared when IGF-II was removed. These results suggest that the signaling pathway triggered by IGF-II is uncoupled between the IGF-IIR and Gi-2 alpha in quiescent cells and that PDGF and EGF restore the IGF-IIR-Gi-2 coupling. This study also indicates that low concentrations of IGF-I reduce the pertussis toxin substrate activity of Gi-2 alpha in primed-competent cells in a time course slower than that of IGF-II, but not at all in quiescent cells. However, both of these cells had similar Kd values and numbers of IGF-I binding sites. Therefore, the IGF-I signaling pathway may also be uncoupled between the IGF-IR and Gi-2 alpha in quiescent cells and restored by PDGF and EGF. In BALB/c 3T3 cells transfected with temperature-sensitive Kirsten sarcoma virus bearing the v-Ki-ras gene (ts cells), a 40-kDa pertussis toxin substrate was also identified as Gi-2 alpha. In nonpermissive ts cells, IGF-II was without effect on the pertussis toxin substrate activity of Gi-2 alpha or on calcium influx.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of adrenocorticotropin (ACTH) on cytoplasmic free calcium concentration, [Ca2+]c, have been measured in adrenal glomerulosa cells using a calcium-sensitive photoprotein, aequorin. ACTH causes a rapid and transient increase in [Ca2+]c. Dose response study demonstrates that 1 pM ACTH induces an elevation of [Ca2+]c and that effect of ACTH appears to be saturated at 100 pM. ACTH action is greatly inhibited but not abolished by removal of extracellular calcium and is completely blocked in medium containing no added calcium and 1 mM EGTA. Under similar conditions, angiotensin II induces a remarkable rise in [Ca2+]c. ACTH action is not affected by pretreatment with dantrolene, which considerably decreases angiotensin II action on [Ca2+]c. One micromolar forskolin, which mimics 1 nM ACTH-mediated elevation of intracellular cAMP, does not increase [Ca2+]c nor modulates changes in [Ca2+] induced by a low dose of ACTH. One hundred micromolar forskolin or 1 mM 8-bromo-cAMP, however, increases [Ca2+]c even in calcium-free medium containing 1 mM EGTA. When glomerulosa cells are co-loaded with aequorin and quin2, angiotensin II-induced change in aequorin signal is greatly reduced, and ACTH-induced change is abolished. Quin2 loading results in accumulation of calcium in the cell under both unstimulated and stimulated conditions. These results indicate that ACTH increases [Ca2+]c by cAMP-independent mechanism, that ACTH action on [Ca2+]c is exclusively dependent on extracellular calcium, and that quin2 is unable to detect the rapid change in [Ca2+]c because of its calcium chelating activity.

We previously reported that insulin-like growth factor-I (IGF-I) stimulated calcium entry (Kojima, I., Matsunaga, H., Kurokawa, K., Ogata, E., and Nishimoto, I. (1988) J. Biol. Chem. 263, 16561-16567) and production of 1,2-diacylglycerol in IGF-responsive “primed competent” Balb/c 3T3 cells (Kojima, I., Kitaoka, M., and Ogata, E. (1990) J. Biol. Chem. 265, 16846-16850). The present study was conducted to determine a role of protein kinase C (PKC) in the progression activity of IGF-I. To monitor the activity of PKC in intact cells, we measured phosphorylation of a synthetic KRTLRR peptide, a substrate of PKC, immediately after the permeabilization of the cells with digitonin. When 1 nM IGF-I was added to primed competent cells, KRTLRR peptide phosphorylation was augmented. IGF-I induced more than 2-fold increase in KRTLRR peptide phosphorylation that was blocked by PKC19-36, a pseudosubstrate of PKC, which blocks the activity of the kinase, and Ro31-8220, an inhibitor of PKC. The phosphorylation remained elevated for up to 6 h. To assess the role of PKC in cell cycle progression, IGF-I-induced nuclear labeling was measured in the presence of Ro31-8220. Ro31-8220 reduced the rate of entrance into S phase when added in the first quarter of the G1 phase, but did not affect cell cycle progression when added at the second quarter or later. In contrast, reduction of extracellular calcium completely blocked cell cycle progression when done in the first, second, and third quarter but had no effect in the last quarter. These results indicate that IGF-I persistently activates PKC in primed competent cells, but the activation is required only for the initiation of progression. We conclude that IGF-I promotes cell cycle progression by calcium-dependent mechanisms that are largely independent of PKC.

The role of GTP on somatostatin-induced K+ current increase was examined in dissociated human pituitary tumor cells obtained from three acromegalic patients. Pituitary cells in culture were voltage-clamped by using the patch clamp technique in the whole-cell configuration. Somatostatin (100 nM) increased the membrane permeability to K+ ions and inhibited hormone secretion. A current-voltage relation of the somatostatin-induced K+ current showed an inward rectification when the concentration of extracellular K+ ions was increased. The amplitude of the somatostatin-induced K+ current decreased during recording when the patch pipette solution did not contain GTP; addition of 100 microM GTP to the patch pipette solution prevented this reduction. Intracellular application of 100 microM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] evoked an inward rectifying K+ conductance in the absence of somatostatin. After the GTP[gamma S]-induced K+ conductance reached a steady level, application of somatostatin did not further increase the K+ conductance. In pertussis toxin-treated cells GTP[gamma S] did not evoke K+ conductance. It was concluded that somatostatin-induced K+ channels were regulated by a GTP-binding protein.

In mouse Balb/c3T3 fibroblasts, insulin-like growth factor (IGF)-II activates a calcium-permeable cation channel through a cell surface IGF-II receptor (Kojima, I., Nishimoto, I., Iiri, T., Ogata, E., and Rosenfeld, R. G. (1988) Biochem. Biophys. Res. Commun. 154, 9-19; Matsunaga, H., Nishimoto, I., Kojima, I., Yamashita, N., Kurokawa, K., and Ogata, E. (1988) Am. J. Physiol. 255, C442-C446). In the action of IGF-II, a pertussis toxin (or islet-activating protein; IAP)-sensitive GTP-binding protein (G protein) is inferred to be involved (Nishimoto, I., Hata, Y., Ogata, E., and Kojima, I. (1987) J. Biol. Chem. 262, 12120-12126). In the present study, we examined the direct coupling of the IGF-II receptor with G proteins. In broken Balb/c3T3 cell membranes, 10 nMIGF-II rapidly attenuated the IAP-catalyzed ADP-ribosylation of a 40-kDa protein in a manner requiring magnesium ion. The IGF-II-mediated attenuation in the IAP substrate activity was 80% recovered after washing off IGF-II and inhibited by coexisting guanosine 5′-O-(2-thiodiphosphate), while either aluminum fluoride solution (10 mMNaF plus 100 εMAlCl3) or 100 εMguanosine 5′-O-(3-thiotriphosphate) (GTPγS) reproduced the action of IGF-II. When purified IAP substrate G proteins (Gi1, Gi2, Go) were incubated with IGF-II in the presence of membranes from IAP-treated Balb/c3T3 cells, the attenuation in the IAP substrate activity was evident in Gi2, but not in Gi1or Go. On the other hand, 10 nMinsulin had no effect on the modification of the 40-kDa IAP substrate in Balb/c3T3 cell membranes, whereas 10 nMIGF-I elicited a slow onset of the IAP sensitivity attenuation from the 40-kDa protein. However, the specific involvement of the IGF-II receptor in the modification of the IAP substrate induced by low concentrations of IGF-IIwas suggested by the observations that (i) IGF-I receptor-lacking cell membranes were effective for the Gi2modification by IGF-II, (ii) the ability of membranes to mediate the action of IGF-II was markedly attenuated in IGF-II receptor-lacking cell membranes, and (iii) agonistic anti-IGF-II receptor antibody mimicked the action of IGF-II on the 40-kDa protein in Balb/c3T3 cell membranes in a dose-dependent manner similar to that observed in the antibody-induced blocking of membrane IGF-II binding. Phospholipid vesicles reconstituted with purified rat IGFI1 receptor and Gi2demonstrated functional coupling between the receptor and Giz in two ways: (i) the addition of 100 ε GTPγS augmented the dissociation constant of the IGF-I1 receptor binding from 8.9 × 10−10to 7.0 × 10−9M, and (ii) IGF-I1 facilitated GTPγS binding to GiP in an IAP-sensitive manner. We conclude that the IGF-I1 receptor may interact with G proteins in cell membranes and is coupled to Gi2in phospholipid vesicles. This study elucidates the significant function of the IGF-I1 receptor in transmembrane signaling and provides evidence tah asti ngle-spanning receptor can be directlyc oupled to G protein

In adrenal glomerulosa cells, angiotensin II causes an immediate release of calcium from an intracellular trigger pool (Kojima, I., Kojima, K., and Rasmussen, H. (1985) Am. J. Physiol. 247, E36-E43). The present study was conducted to determine how the trigger pool of calcium is restored after cessation of the agonist action. Upon termination of angiotensin II action, calcium influx rate decreased immediately while total cell calcium increased rapidly. The increase in total cell calcium is not affected by 1 microM nitrendipine, which blocks angiotensin II-stimulated calcium influx without inhibiting basal influx of calcium. In contrast, total cell calcium did not increase in medium containing 1 microM calcium, in which basal calcium influx is negligible. A rapid increase in total cell calcium after an addition of the antagonist was not accompanied by changes in cytoplasmic free calcium concentration. A second stimulation of cells with either angiotensin II or carbachol did not cause calcium release when the interval of two stimulations was shorter than 20 min. The longer the interval, the greater the magnitude of calcium release in response to the second stimulator. The maximum response was obtained when the interval was 40 min or more. When exogenous arachidonic acid, which mobilized calcium by acting directly on the inositol trisphosphate-sensitive pool, was employed as a second stimulator, the magnitude of the decrease in total cell calcium was also dependent on the interval. These results suggest that, upon termination of angiotensin II action, calcium is rapidly accumulated first in an intracellular pool which is insensitive to either inositol 1,4,5-trisphosphate or arachidonic acid and that the trigger pool is restored gradually thereafter.

Proteoglycans in mineralized (0.5 M-EDTA/4 M-guanidinium chloride-extractable) and non-mineralized (4 M-guanidinium chloride-extractable) matrices synthesized by a mouse osteoblastic-cell line MC3T3-E1 were characterized at different phases of mineralization in vitro. Cell cultures were labelled with [35S]sulphate and either [3H]glucosamine or 3H-labelled amino acids. At the mineralization phase a large majority of proteoglycans were extracted with 4 M-guanidinium chloride (G extract), and at least five species of labelled proteoglycans were identified; dermatan sulphate proteoglycans (DSPG), apparent Mr approx. 120,000 and 70,000), heparan sulphate proteoglycans (HSPG, apparent Mr approx. 200,000 and 120,000) and DS chains with very little core protein. DSPGs weakly bound to an octyl-Sepharose CL-4B column and HSPGs bound more tightly, whereas DS chains did not bind to the column. Amounts of labelled proteoglycans extracted with 0.5 M-EDTA/4 M-guanidinium chloride (EDTA extract) were much less than those in G extract. Although the predominant species in the EDTA extract were comparable with the DS or DSPGs in the G extract, none of them bound to octyl-Sepharose CL-4B, indicating their lack of hydrophobicity. At the nonmineralizing phase a large chondroitin sulphate proteoglycan (Mr greater than 600,000) was found in the matrix in addition to the five proteoglycan species similar to those at the mineralization phase. Although DS chains at the early phase were similar in size to those at the mineralization phase, the ratio of 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulpho-D-galactose to 2-acetamido-2-deoxy-3-O-(beta-D-gluculo-4-enepyranosyluronic acid)-6-O-sulpho-D-galactose was less than that at the mineralization phase. These results agree with those of previous studies performed in vivo and suggest that alteration in the synthesis of proteoglycans is involved in the mineralization process. They also suggest that at the osteoblastic mineralization front proteoglycans undergo partial degradation and lose their hydrophobicity.

The synthetic 1-34 fragment of human parathyroid hormone (1-34hPTH) stimulated glucose production in isolated rat hepatocytes. The effect of 1-34hPTH was dose-dependent and 10(10) M-1-34 hPTH elicited the maximum glucose output, which was approx. 80% of that by glucagon. Although 1-34hPTH induced a small increase in cyclic AMP production at concentrations higher than 10(-9) M, 10(-10) M-1-34hPTH induced the maximum glucose output without significant elevation of cyclic AMP. This is in contrast to the action of forskolin, which increased glucose output to the same extent as 10(-10) M-1-34hPTH by causing a 2-fold elevation of cyclic AMP. In addition to increasing cyclic AMP, 1-34hPTH caused an increase in cytoplasmic free calcium concentration ([Ca2+]c). When the effect of 1-34hPTH on [Ca2+]c was studied in aequorin-loaded cells, low concentrations of 1-34hPTH increased [Ca2+]c: the 1-34hPTH effect on [Ca2+]c was detected at as low as 10(-12) M and increased in a dose-dependent manner. 1-34hPTH increased [Ca2+]c even in the presence of 1 microM extracellular calcium, suggesting that PTH mobilizes calcium from an intracellular pool. In line with these observations, 1-34hPTH increased the production of inositol trisphosphate. These results suggest that: (1) PTH activates both cyclic AMP and calcium messenger systems and (2) PTH stimulates glycogenolysis mainly via the calcium messenger system.

Previous results have shown that 1,25-dihydroxycholecalciferol [1,25(OH)2D3] enhances the synthesis of phosphatidylserine (PS) and suppresses the synthesis of phosphatidylethanolamine (PE) in osteoblast-like rat osteogenic sarcoma UMR 106 cells [Matsumoto, Kawanobe, Morita & Ogata (1985) J. Biol. Chem. 260, 13704-13709]. In the present study, the effect of parathyroid hormone (PTH) on phospholipid metabolism is examined by using these cells. Treatment of UMR 106 cells with human PTH-(1-34)-peptide suppresses the synthesis of phosphatidylethanolamine in a dose- and time-dependent manner without affecting the synthesis of PS. The maximal effect on PE synthesis is obtained with 2.4 nM-human PTH-(1-34)-peptide when the cells are treated for 48 h or longer. In addition, when human PTH-(1-34)-peptide is added together with the maximal dose of 1,25(OH)2D3, there is a further decline in PE synthesis, whereas the stimulation of PS synthesis by 1,25(OH)2D3 is not altered. Because methylation of PE is suggested to affect hormone receptor-adenylate cyclase coupling, the observed change in PE metabolism by PTH and 1,25(OH)2D3 may be, at least in part, involved in the development of desensitization phenomenon to PTH in these cells.

The action of insulin-like growth factor II (IGF-II) on calcium influx was studied in BALB/c 3T3 cells. IGF-II did not affect calcium influx rate in either quiescent or platelet-derived growth factor-treated “competent” cells. In contrast, IGF-II induced an approximately 2-fold sustained increase in calcium influx rate in competent cells briefly primed with epidermal growth factor (“primed competent” cells). The IGF-II-stimulated calcium influx was dependent on extracellular calcium and was inhibited by lanthanum, cobalt, and tetramethlin but not by nitrendipine. The IGF-II-stimulated [3H]thymidine incorporation was also dependent on extracellular calcium and was inhibited by cobalt and tetramethlin. A pharmacological stimulation of calcium influx by BAYK8644 resulted in an increase in [3H]thymidine incorporation in primed competent cells but not in either quiescent or competent cells. Pretreatment of primed competent cells with pertussis toxin completely abolished subsequent action of IGF-II on both calcium influx and [3H]thymidine incorporation. Inhibitory actions of pertussis toxin correlated well with toxin-induced ADP-ribosylation of a 41-kDa protein. The binding of 125I-IGF-II to membrane fraction was inhibited by guanosine 5'-O-(thiotriphosphate), and this inhibition was reversed by pretreatment of the cell with pertussis toxin. These results suggest that IGF-II stimulates calcium influx in primed competent BALB/c 3T3 cells by a mechanism involving G protein and that calcium influx may be a message of IGF-II action on cell proliferation.

We previously reported that insulin-like growth factor-I (IGF-I) induced sustained calcium cycling across the plasma membrane in primed competent Balb/c 3T3 cells (Kojima, I., Matsunaga, H., Kurokawa, K., Ogata, E., and Nishimoto, I. (1989) J. Biol. Chem. 263, 16561-16567). The present study was conducted to examine whether IGF-I affected cellular metabolism of 1,2-diacylglycerol (1,2-DAG). In primed competent cells prelabeled with [3H]myristate, 1 nM IGF-I caused a 50% increase in [3H]DAG within 10 min. This increase in [3H]DAG was accompanied by 1) a decrease in radioactivity in the glycosylphosphatidylinositol fraction in [3H]glucosamine-labeled cells and a concomitant increase in [3H]inositol-glycan, and 2) a decrease in [3H]phosphatidylcholine and a concomitant elevation of [3H]phosphorylcholine in [3H]choline-labeled cells. When [3H]choline-labeled cells were treated with 10 nM 12-O-tetradecanoylphorbol-4-acetate (TPA), [3H]phosphatidylcholine was reduced by 50%. The TPA-induced reduction of [3H]phosphatidylcholine was completely blocked by 50 microM sphingosine and 50 microM H-7, inhibitors of protein kinase C. Both sphingosine and H-7 attenuated IGF-I-mediated reduction of [3H]phosphatidylcholine. In addition, treatment with IGF-I for 3 h or more resulted in sustained increase in 1,2-DAG mass, which was attenuated by cycloheximide. The increase in DAG mass was accompanied by enhanced incorporation of [14C]glucose into 1,2-DAG. These results indicate that, in primed competent Balb/c 3T3 cells, IGF-I stimulates 1,2-DAG production via multiple pathways and that IGF-I may induce breakdown of phosphatidylcholine by a mechanism involving protein kinase C.

The 14-residue peptide (peptide 14) corresponding to Arg2410-Lys2423 of the insulin-like growth factor II receptor (IGF-IIR) can activate the adenylate cyclase-inhibitor guanine nucleotide-binding protein Gi, and the 15-residue beta III-2 peptide Arg259-Lys273 of the beta 2-adrenergic receptor (beta 2AR) can activate the stimulatory protein Gs. In phospholipid vesicles, IGF-IIR and beta 2AR activate Gi and Gs in response to IGF-II and isoproterenol, respectively. We constructed a chimeric IGF-II receptor (beta III-2/IGF-IIR) by converting its native peptide 14 sequence to the beta III-2 sequence. In cells expressing beta III-2/IGF-IIR, membrane adenylate cyclase activity markedly increased without IGF-II and was further promoted by IGF-II. This was verified by measuring chloramphenicol acetyltransferase (CAT) activity in beta III-2/IGF-IIR cells with cotransfection of a cAMP response element-CAT construct. This study shows not only the conversion of G-protein specificity of a receptor from Gi to Gs but also the simulation of G protein-coupled receptor signals by using a short receptor region and intact cells. These findings indicate that the G protein-activation signals are interchangeable, self-determined structural motifs that function in the setting of either a single-spanning or multiple-spanning receptor.

The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on phospholipid metabolism was examined in clonal rat osteogenic sarcoma cells, UMR 106, of osteoblastic phenotype. Treatment of UMR 106 cells with 10(-8)M 1,25-(OH)2D3 for 48 h caused an increase in [14C]serine incorporation into phosphatidylserine (PS) and a decrease in [3H]ethanolamine, [3H]linositol, and [14C]choline incorporation into phosphatidylethanolamine (PE), phosphatidylinositol, and phosphatidylcholine, respectively; the decrease in [3H]ethanolamine incorporation into PE was the largest. The total contents of phospholipids were similarly affected by 10(-8)M 1,25-(OH)2D3 treatment, suggesting that the effects of 1,25-(OH)2D3 are due largely to alterations in the synthesis of these phospholipids. The effects of 1,25-(OH)2D3 were evident at 10(-10) M 1,25-(OH)2D3, and 10(-8)M 1,25-(OH)2D3 caused a maximal stimulation of [14C]PS synthesis (167% of control) and a maximal reduction in the [3H]PE synthesis (41% of control). The [14C]PS/[3H]PE ratio increased gradually and reached a maximum after 70 h of treatment with 10(-8)M 1,25-(OH)2D3. When the cells were cultured in calcium-free medium containing 0.5 mM EGTA or when 5 microM cycloheximide was added to the medium, the effect of 1,25-(OH)2D3 on phospholipid metabolism was almost completely inhibited. Neither 25-hydroxyvitamin D3 nor 24,25-dihydroxyvitamin D3 caused significant changes in phospholipid metabolism. These results suggest that 1,25-(OH)2D3 alters phospholipid metabolism by enhancing PS synthesis through a calcium-dependent stimulation of the base exchange reaction of serine with other phospholipids and that the effect of 1,25-(OH)2D3 requires the synthesis of new proteins. Because PS is thought to be important for apatite formation and bone mineralization by binding calcium and phosphate to form calcium-PS-phosphate complexes, the present data suggest that 1,25-(OH)2D3 may stimulate bone mineralization by a direct effect on osteoblasts, stimulating PS synthesis.

1. Regulation of membrane potential by extracellular Ca2+ concentration ([Ca2+]o) was examined in freshly isolated rabbit osteoclasts. 2. The resting membrane potential of osteoclasts was close to the K+ equilibrium potential in 1 mM Ca2+ medium. An elevation of [Ca2+]o caused membrane depolarization, accompanied by a decrease in the membrane conductance. 3. The inwardly rectifying K+ current observed under voltage clamp was dose‐dependently inhibited by an elevation of [Ca2+]o, which explained the membrane depolarization caused by high [Ca2+]o. 4. Other divalent cations also inhibited the inwardly rectifying K+ current with the following order of potency: Ca2+ < Ni2+ < or = Co2+ < Cd2+. 5. In the presence of intracellular GTP gamma S the inwardly rectifying K+ current was irreversibly inhibited by [Ca2+]o, whereas the inhibition of the inwardly rectifying K+ current was greatly attenuated by intracellular application of GDP beta S. 6. Pertussis toxin (PTX) treatment did not abolish the inhibition of the inwardly rectifying K+ current caused by [Ca2+]o. 7. These results suggest that inwardly rectifying K+ channels in osteoclasts were regulated by a PTX‐insensitive G‐protein, which was coupled to the putative Ca2+ receptor or sensor on the cell membrane.