After the initial signaling action of parathyroid hormone (PTH) on bone was shown to be activation of adenylyl cyclase, its target was found to be cells of the osteoblast lineage, to the exclusion of osteoclasts and their precursors. This led to the view that the osteoblast lineage regulated osteoclast formation, a proposal that was established when the molecular mechanisms of osteoclast formation were discovered. This is in addition to the effect of PTH1Rv signaling throughout the osteoblast differentiation process to favour the formation of bone-forming osteoblasts. Initial signaling in the PTH target cells through cAMP and protein kinase A (PKA) activation is extremely rapid, and marked by an amplification process in which the later event, PKA activation, precedes cAMP accumulation in time and is achieved at lower concentrations. All of this is consistent with the existence of "spare receptors", as is the case with several other peptide hormones. PTH-related protein (PTHrP), that was discovered as a cancer product, shares structural similarity with PTH in the amino-terminal domain that allows the hormone, PTH, and the autocrine/paracrine agent, PTHrP, to share actions upon a common G protein coupled receptor, PTH1R, through which they activate adenylyl cyclase with equivalent potencies. Studies of ligand-receptor kinetics have revealed that the PTH/PTH1R ligand-receptor complex, after initial binding and adenylyl cyclase activation at the plasma membrane, is translocated to the endosome, where adenylyl cyclase activation persists for a further short period. This behavior of the PTH1R resembles that of a number of hormones and other agonists that undergo such endosomal translocation. It remains to be determined whether and to what extent the cellular effects through the PTH1R might be influenced when endosomal is added to plasma membrane activation.

The stimulation of the production of osteocalcin by human osteoblast-like cells in response to 1,25(OH)2D3 is antagonized by several agents that induce the synthesis of prostaglandin E2 (PGE2) including interleukin 1 (IL-1), tumour necrosis factor (TNF) and parathyroid hormone (PTH). The mechanism whereby these agents inhibit the synthesis of osteocalcin is not known. In this report we show that exogenous PGE2 inhibits this stimulatory action of 1,25(OH)2D3 on human osteoblast-like cells in a dose-dependent manner, suggesting that PGE2 may contribute to the inhibition of osteocalcin synthesis in response to these agents. Assessment of the inhibitory role of endogenous PGE2 synthesis in the action of rhIL-1 alpha, rhIL-1 beta and rhTNF alpha on the production of osteocalcin demonstrated that the inhibition by these agents could be partially overcome by the addition of indomethacin, an inhibitor of PGE2 synthesis. In contrast, the inhibitory action observed with bPTH (1-84) was unaffected by indomethacin. These observations indicate that endogenous PGE2 synthesis mediates, in part, some of the inhibitory actions of the cytokines on the induction of osteocalcin synthesis in response to 1,25(OH)2D3, but not of PTH. Since the antagonism of the synthesis of osteocalcin by rhIL-1 alpha, rhIL-1 beta and rhTNF alpha was not completely abolished following the inhibition of PGE2 synthesis this would indicate that additional PGE2-independent mechanisms also account for the action of these cytokines on osteocalcin production. The nature of these mechanisms is currently not known.

We have developed a mouse bone marrow culture system in which multinucleated osteoclast (OC)-like cells are formed within 8 days. Using this culture system, we examined the effect of prostaglandins (PGs), potent bone-resorbing agents, on OC-like cell formation. Four PGs (PGE1 and PGE2 at 10(-8)-10(-5) M, 6-keto-PGF1 alpha at 10(-5) M, and PGF2 alpha at 10(-6)-10(-5) M) significantly stimulated the formation of OC-like cells. The potency of the PGs in inducing OC-like cell formation was the highest in PGE1 and PGE2, followed by PGF2 alpha and 6-keto-PGF1 alpha in that order, and the order was highly correlated with the order of the potency in increasing the production of cyclic adenosine 3',5'-monophosphate (cAMP) in bone marrow cells. Addition of dibutyryl-cAMP also induced OC-like cell formation. Moreover, isobutylmethylxanthine (IBMX), a potent inhibitor of phosphodiesterase, potentiated the OC-like cell formation induced by PGE2, whereas salmon calcitonin greatly inhibited it. Calcitonin induced cAMP production in cultures treated with PGE2, but not in cultures with vehicle. When bone marrow mononuclear cells were cultured on dentine slices in the presence of PGE2, multinucleated OC-like cells were similarly formed and they resorbed calcified dentine, resulting in so-called Howship's lacunae. These results suggest that PGs stimulate resorption of calcified tissues by promoting osteoclast formation. The activity of PGs in inducing OC-like cell formation is considered mediated mainly by a mechanism involving cAMP.

The biological properties of Thromboxane B2 (TXB2) on isolated rat heart were studied. Its actions were compared with U-46619 a Thromboxane A2 mimetic compound and with isoproterenol. TXB2 induced a concentration-dependent increase in contractility, that was non-competitively antagonized by propranolol. In addition TXB2 inhibited Na+ + K+-ATPase activity at the same concentrations that influenced the mechanical activity. Inhibition of beta-adrenoceptors efficiently blocked the inhibitory action of TXB2 upon Na+ + K+-ATPase-activity. Isoproterenol simulated the positive inotropic effect and the inhibitory action of TXB2 on Na+ + K+-ATPase-activity. In contrast, U-46619 did not alter the basal dF/dt, neither the enzyme activity. The foregoing results suggest that TXB2 resembles the biological effect of catecholamines-inducing stimulation of myocardial contractility and inhibition of Na+ + K+-ATPase activity.

1. Thromboxane (TX) B2 and epithiomethano (sTXA2), in concentrations that were insufficient to alter the basal tone, potentiated contractile responses of helical strips of dog mesenteric arteries to transmural electrical stimulation. The potentiating effect of TXB2 (up to 10(-6) M) was not abolished by diphloretin phosphate (DPP), a prostaglandin antagonist, whereas the potentiation by sTXA2 was abolished by the antagonist. 2. sTXA2 and TXB2 (3 x 10(-6) M or higher) potentiated the responses to noradrenaline, the potentiation being antagonized by DPP. 3. 3H-overflow evoked by transmural stimulation in superfused arterial strips previously soaked in medium containing [3H]-noradrenaline was increased by TXB2, but not altered by sTXA2. 4. TXB2 in low concentrations potentiated the contractile response to adrenergic nerve stimulation, possibly by increasing the release of noradrenaline, while the potentiation by the TXA2 analogue appears to be due to increased sensitivity of the arteries to noradrenaline. Prejunctional effects of TXB2 may be mediated by receptor sites functionally different from those located postjunctionally.

The effect of prostaglandin E2 (PGE2) on the kinetic of bone resorption in vitro was assessed by following the release of minerals and degradation of matrix in cultured mouse calvarial bones. PGE2 (1 and 3 mumol/liter) caused an initial inhibition of the release of 45Ca, stable calcium, and inorganic phosphate from unstimulated calvarial bones. The effect was transient and after 24 and 48 hours the release of 45Ca, stable calcium, and inorganic phosphate from PGE2-treated bones was enhanced. 0.3 mumol/liter of PGE2 stimulated the release of 45Ca after 24 hours, but at this concentration no initial inhibition was observed. The initial inhibitory effect of PGE2 (1 mumol/liter) could be further increased by three structurally different inhibitors of cyclic AMP breakdown. PGE2 (1 mumol/liter) caused not only an initial inhibition of mineral release but also an initial inhibition of matrix degradation, as assessed by the release of 3H from [3H]-proline labeled bones. In addition, PGE2 (3 mumol/liter), in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, caused a rapid (6 hours) inhibition of the release of the lysosomal enzymes beta-glucuronidase and beta-N-acetyl-glucosaminidase, without affecting the release of the cytosolic enzyme lactate dehydrogenase. Similar specific initial inhibition of lysosomal enzyme release was also seen in the presence of calcitonin and dibutyryl cyclic AMP, but not in the presence of parathyroid hormone (PTH). Neither PGE2 nor the phosphodiesterase inhibitors rolipram and Ro 20.1724, could inhibit the initial stages of PTH-induced 45Ca release. Nor did PGE2 inhibit the stimulation of radioactive calcium mobilization induced by 1 alpha (OH)-vitamin D3.(ABSTRACT TRUNCATED AT 250 WORDS)

The methods for establishing osteoblast-rich rat calvarial cell cultures have been described, together with methods for the use of clonal osteogenic sarcoma cells of osteoblast phenotype. The latter clonal lines are useful for several purposes, but all the precautions and quality control measures necessary in the study of clonal lines must be observed. Some of the techniques for studying biochemical responses to hormones in these cells have also been detailed, but clearly others are applicable, including studies of the synthesis of matrix constituents. Osteoclast-like cells have not been considered in this chapter, because osteoclast culture methods have not yet been developed to the degree of purity and reproducibility necessary for this type of biochemical approach.

The clonal cell line UMR 106, which was originally derived from a rat transplantable osteogenic sarcoma with an osteoblastic phenotype, was subcloned after the emergence of a calcitonin-responsive adenylate cyclase was noted in late passages. Detailed studies on the stimulation of adenylate cyclase and activation profile of the cyclic AMP-dependent protein kinase isoenzymes in response to parathyroid hormone (PTH) and salmon calcitonin (SCT) were conducted on two subclones (UMR 106-01 and UMR 106-06). Both subclones responded in an identical manner to PTH, which stimulated adenylate cyclase and activated both isoenzyme I and isoenzyme II of cyclic AMP-dependent protein kinase. In contrast, only UMR 106-06 cells responded to calcitonin. At 3 X 10(-8)M SCT, there was a sevenfold stimulation of adenylate cyclase, 84% activation of isoenzyme I, and 44% activation of isoenzyme II. The activation profiles of the isoenzymes to PTH and SCT in UMR 106-06 were similar. Furthermore, their response to SCT correlates with the presence of specific, saturable binding of 125I-labeled SCT. Binding parameters indicate apparent Kd = 0.8 nM and 6,000 receptors/cell. These data point to a significant phenotypic change having taken place in this clonal cell line with prolonged maintenance in culture, with the emergence of a calcitonin receptor linked to adenylate cyclase and protein kinase activation.

The culture media of three cell lines, a human prostate carcinoma (PC3), a rat Leydig cell tumor (Rice-500), and a rat carcinosarcoma (WRC-256), that were derived from tumors associated with humoral hypercalcemia of malignancy (HHM), were examined for stimulation of adenylate cyclase in ROS 17/2.8 osteoblastic cells and for bone resorptive activity in culture. Cells from a nonhypercalcemic variant of the WRC256 tumor served as control. Extracts from three solid human tumors, a lung adenocarcinoma from a patient with HHM and two adenocarcinoma from normocalcemic patients (lung and colon), were also examined for adenylate cyclase stimulation. We found excellent correlation between stimulation of cyclic AMP accumulation in ROS 17/2.8 cells and bone resorbing activity in culture, or production of HHM in vivo. Stimulation of adenylate cyclase by HHM factors was inhibited by the parathyroid hormone competitive inhibitor, [8norleucyl, 18norleucyl, 34tyrosinyl] bovine parathyroid hormone (3-34) amide.

The metabolism of arachidonic acid to its cyclo-oxygenase products was studied in monolayer cultures of osteoblast-rich rat calvarial cells and of clonal cell lines from a rat osteogenic sarcoma, enriched in the osteoblast phenotype. Prostanoids were measured by radioimmunoassay after extraction of media and fractionation by high pressure liquid chromatography. In both normal and malignant osteoblasts the major cyclooxygenase product was 6-oxo-prostaglandin F1 alpha, the hydration product of prostacyclin, with lesser amounts of prostaglandin E2 and prostaglandin F2 alpha. No significant thromboxane B2 was detected. Prostaglandins are thought to have a local role in the regulation of bone resorption. These results point to the possible importance of prostacyclin either in bone resorption or in some other local function, e.g., regulation of bone blood flow.

The number of agents and treatment regimens which can be used in the medical treatment of hypercalcemia has increased markedly over the last 5 yr. As this list has increased, clinicians are anxious to know more about the humoral and cellular mechanisms which are responsible for the hypercalcemia of malignancy and to understand how these drugs work. Unfortunately there is no treatment available presently which is uniformally safe and effective, and the potential pathogenetic mechanisms responsible for hypercalcemia are hotly debated. In this review, we plan to summarize current views of the pathogenesis, clinical features and treatment of hypercalcemia associated with malignant disease.

Intracerebroventricular administration of prostacyclin (PGI2) at room temperature (21 degrees C) induced dose-related hyperthermia in rabbits and also produced hyperthermia at low (4 degrees C) and high (30 degrees C) ambient temperatures. The PGI2-induced hyperthermia was not mediated by its stable metabolite 6-keto prostaglandin F1 alpha. Of the three anion transport systems (iodide, hippurate and liver-like) present in the choroid plexus, only the liver transport system seems to be important to central inactivation of pyrogen, prostaglandin E2 (PGE2) and the PGI2. Iodipamide (an inhibitor of the liver transport system) augmented the hyperthermia produced by PGI2, PGE2 and pyrogen. Phenoxybenzamine and pimozide had no thermolytic effect on PGI2-induced hyperthermia. After norepinephrine (NE) and dopamine levels were depleted by 6-hydroxydopamine, PGI2 still induced hyperthermia. Indomethacin and SC-19220 (a PG antagonist) did not antagonize PGI2-induced hyperthermia. Furthermore, the hyperthermia due to PGI2 was not accentuated by theophylline. In contrast, the hyperthermic response to PGI2 was attenuated by central administration of the protein synthesis inhibitor, anisomycin. These results indicate that PGI2-induced hyperthermia is not mediated by NE, dopamine, PGS, cyclic AMP, but, rather, that a protein mediator is implicated in the induction of fever by PGI2.

We have utilized ionophores to test whether stimulation of chondrocyte prostaglandin biosynthesis is accompanied by an increase in cyclic nucleotide levels in these cells. Radioimmunoassay of prostaglandin E2, 6-oxo-prostaglandin F1 alpha (the stable metabolite of prostaglandin I2) and prostaglandin F2 alpha showed that synthesis of each was stimulated by the divalent-cation ionophore, A23187 after short-term incubation (1-7 min) in serum-free medium. No stimulation of thromboxane B2 was detected. Two monovalent ionophores, lasalocid and monensin failed to stimulate prostaglandin biosynthesis after short-term incubation. Ionophore A23187-stimulated prostaglandin biosynthesis was variably and partially inhibited by sodium meclofenamate, indomethacin and aspirin, but not by sodium salicylate. Ionophore A23187-stimulated prostaglandin biosynthesis was accompanied by a 7.5-fold increase in cyclic AMP levels after 15 min. Sodium meclofenamate, indomethacin and aspirin which inhibited prostaglandin E2 biosynthesis also reduced cyclic AMP levels. Exogenous prostaglandin E2 (1 microgram/ml) stimulated cyclic AMP biosynthesis, which was not inhibited by aspirin. These results indicated that prostaglandins can be considered as one of the local effectors controlling cyclic AMP production in articular cartilage.

The products of endogenous and exogenous arachidonic acid metabolism via the cyclooxygenase pathway in mouse bone in organ culture were identified and quantitated by the use of high performance liquid chromatography and radioimmunoassay. Production of prostaglandins E2, F2 alpha, and I2 from endogenous substrate was stimulated by incubation of bone with epidermal growth factor and the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Addition of arachidonic acid to the culture medium not only resulted in the accumulation of prostaglandins E2, F2 alpha, and I2 but also thromboxane. Bone metabolized prostaglandins E2 and F2 alpha to their respective 13,14-dihydro-15-keto-derivatives. Prostaglandin I2, measured as 6-keto-prostaglandin F1 alpha was synthesized by bone, and metabolic products of prostaglandin I2 or 6-keto-prostaglandin F1 alpha, either 6,15-diketo-prostaglandin F1 alpha or 13,14-dihydro-6,15-diketo-prostaglandin F1 alpha, were also detected in the culture media. Formation of cyclooxygenase products of endogenous and exogenous arachidonic acid metabolism (both basal and stimulated) and bone resorption were inhibited by indomethacin. Bone as a tissue responded biochemically not only to exogenous prostaglandins and agents that enhance endogenous prostaglandin production but also to exogenous arachidonic acid by biosynthesis of prostaglandins, prostacyclin and thromboxane. Furthermore, bone metabolized these cyclooxygenase products to their more stable metabolites.

1. The addition of 50 000g cytosol preparations of isolated human platelets, cultured rat osteogenic sarcoma or cultured bone cells to particulate preparations of adenylate cyclase, from the same or unrelated tissues, caused marked enhancement of the hormone-stimulated enzyme activities. 2. The degree of enhancement obtained by addition of the cytosol preparations was similar to that observed on addition of GTP. 3. The enhancing activity of the three cytosol types was found to be sensitive to digestion by trypsin and alkaline phosphatase, partially heat-labile and partially inactivated by exposure to charcoal. 4. Gel filtration studies indicated an apparent molecular weight of 20 000--30 000. Further, the 20000-30000-mol.wt. fractions obtained by gel filtration could enhance the adenylate cyclase activity of particulate preparations derived from unrelated cell types. 5. The results suggest a common or similar adenylate-cyclase-enhancing factor or factors, protein in nature, present in the three cytosol types.

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF1 alpha, the hydrolysis metabolite of PGI2, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF1 alpha was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF2 alpha or PGE2. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI2 metabolite. Studies in vitro, using isolated cortical cells exposed to 6-keto-PGF1 alpha (10(-6)-10(-4)M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI2 (10(-6)-10(-4)M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering cortico-sterone production, ACTH (5-200 microU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI2 produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused in situ with PGI2 showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF1 alpha is present in the rat adrenal cortex, its precursor, PGI2, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.

Incubation of bovine coronary artery (BCA) rings with isobutylmethylxanthine (IBMX) resulted in a time-dependent increase of cAMP content. This effect was blocked, when the rings were preincubated with indomethacine or 15-hydroperoxy-arachidonic acid for 5 min, indicating that the IBMX-induced increase in cAMP content may depend on endogenous PGI2 formation. PGE2 did not increase the cAMP content in BCA rings. Dipyridamole did not effect cAMP content, when used as a substitute for IBMX. It is suggested that PGI2 stimulates cAMP formation in arterial walls, but that this effect only becomes visible in the presence of a phosphodiesterase inhibitor.

The actions of thromboxane B2 on various parameters of cardiac performance were studied using the isolated perfused rat heart model. In concentrations from 100 pg/ml to 1 microgram/ml TXB2 significantly reduced the total generated myocardial contractile force. These changes were usually associated with an increase in the coronary perfusion pressure indicating an elevated coronary vascular resitance. Significant coronary pressure alterations were seen with TXB2 concentrations between 1 ng/ml and 1 microgram/ml. No significant changes were seen in either the resting tension or heart rate after TXB2 administration. However TXB2 (10 pg/ml to 10 ng/ml,, significantly reduced the amplitude of the electrical activity as observed in R wave changes of the surface electrocardiogram recording. In another series of experiments the action of TXB2 on rat heart sarcolemmal ATPase activity was studied. TXB2 significantly reduced the activity of the MG++ dependent - Na+ - K+ stimulated ATPase (Na+ - K+ ATPase) in these membrane preparations in concentrations from 10 ng/ml to 1 microgram/ml. Kinetic studies demonstrated that TXB2 reduced Vmax and increased the concentration required of ATP, Na+ and K+ for half-maximal enzyme activity. TXB2 did not inhibit either Ca++ or ouabain-induced depression of Na+ - K+ ATPase activity. The activity of either Mg++ or Ca++ - stimulated ATPase was not affected by TXB2. These results suggest possible important actions of TXB2 on rat heart activity which may be related to Na+ - K+ ATPase inhibition.

Prostaglandin synthesis by fetal rat bones was examined by thin-layer chromatography of culture media after preincubation with labeled arachidonic acid. Cultures in rabbit complement (non-heat inactivated serum) were compared with cultures in heat-inactivated serum or cultures treated with indomethacin. The major complement-dependent products were PGE2, PGF2 alpha and 6-keto-PGF1 alpha, the metabolite of prostacyclin (PGI2). Since PGI2 had not been previously identified in bone its ability to stimulate bone resorption was tested. Repeated addition of PGI2 stimulated release of previously incorporated 45Ca from fetal rat long bones in both short-term and long-term cultures at concentrations of 10(-5) to 10(-9)M. Because of the short half life of PGI2 in solution at neutral pH, we tested a sulfur analog, thiaprostacyclin (S-PGI2) which was found to be a stimulator of bone resorption at concentrations of 10(-5) to 10(-6)M. These studies suggest that endogenous PGI2 production may play a role in bone metabolism. Since vessels produce PGI2 it is possible that PGI2 release may be responsible for the frequent association between vascular invasion and resorption of bone or calcified cartilage in physiologic remodeling and pathologic osteolysis.

Rat anterior pituitary explants were incubated with PGI2, PGH2 and PGE2 in the presence of theophylline (1mM) and the production of cyclic AMP was measured. PGE2 was found to be about 20 times more potent than PGI2 while PGH2 was slightly more effective than PGI2. The results suggest that PGI2 does not play a AMP was measured. PGE was found to be about 20 times more potent than physiological role in cyclic AMP mediated events in the rat anterior pituitary.