Determinant roles of dendritic cell-expressed Notch Delta-like and Jagged ligands on anti-tumor T cell immunity.

BACKGROUND: Notch intercellular communication instructs tissue-specific T-cell development and function. In this study, we explored the roles of dendritic cell (DC)-expressed Notch ligands in the regulation of T-cell effector function. METHODS: We generated mice with CD11c lineage-specific deletion of Notch Delta-like ligand (Dll)1 and Jagged (Jag)2. Using these genetically-ablated mice and engineered pharmacological Notch ligand constructs, the roles of various Delta-like and Jagged ligands in the regulation of T-cell-mediated immunity were investigated. We assessed tumor growth, mouse survival, cytokine production, immunophenotyping of myeloid and lymphoid populations infiltrating the tumors, expression of checkpoint molecules and T-cell function in the experimental settings of murine lung and pancreatic tumors and cardiac allograft rejection. Correlative studies were also performed for the expression of NOTCH ligands, NOTCH receptors and PD-1 on various subsets of myeloid and lymphoid cells in tumor-infiltrating immune cells analyzed from primary human lung cancers. RESULTS: Mice with CD11c lineage-specific deletion of Notch ligand gene Dll1, but not Jag2, exhibited accelerated growth of lung and pancreatic tumors concomitant with decreased antigen-specific CD8+T-cell functions and effector-memory (Tem) differentiation. Increased IL-4 but decreased IFN-γ production and elevated populations of T-regulatory and myeloid-derived suppressor cells were observed in Dll1-ablated mice. Multivalent clustered DLL1-triggered Notch signaling overcame DC Dll1 deficiency and improved anti-tumor T-cell responses, whereas the pharmacological interference by monomeric soluble DLL1 construct suppressed the rejection of mouse tumors and cardiac allograft. Moreover, monomeric soluble JAG1 treatment reduced T-regulatory cells and improved anti-tumor immune responses by decreasing the expression of PD-1 on CD8+Tem cells. A significant correlation was observed between DC-expressed Jagged and Delta-like ligands with Tem-expressed PD-1 and Notch receptors, respectively, in human lung tumor-infiltrates. CONCLUSION: Our data show the importance of specific expression of Notch ligands on DCs in the regulation of T-cell effector function. Thus, strategies incorporating selectively engineered Notch ligands could provide a novel approach of therapeutics for modulating immunity in various immunosuppressive conditions including cancer.

De novo triiodothyronine formation from thyrocytes activated by thyroid-stimulating hormone.

The thyroid gland secretes primarily tetraiodothyronine (T4), and some triiodothyronine (T3). Under normal physiological circumstances, only one-fifth of circulating T3 is directly released by the thyroid, but in states of hyperactivation of thyroid-stimulating hormone receptors (TSHRs), patients develop a syndrome of relative T3 toxicosis. Thyroidal T4 production results from iodination of thyroglobulin (TG) at residues Tyr5 and Tyr130, whereas thyroidal T3 production may originate in several different ways. In this study, the data demonstrate that within the carboxyl-terminal portion of mouse TG, T3 is formed de novo independently of deiodination from T4 We found that upon iodination in vitro, de novo T3 formation in TG was decreased in mice lacking TSHRs. Conversely, de novo T3 that can be formed upon iodination of TG secreted from PCCL3 (rat thyrocyte) cells was augmented from cells previously exposed to increased TSH, a TSHR agonist, a cAMP analog, or a TSHR-stimulating antibody. We present data suggesting that TSH-stimulated TG phosphorylation contributes to enhanced de novo T3 formation. These effects were reversed within a few days after removal of the hyperstimulating conditions. Indeed, direct exposure of PCCL3 cells to human serum from two patients with Graves' disease, but not control sera, led to secretion of TG with an increased intrinsic ability to form T3 upon in vitro iodination. Furthermore, TG secreted from human thyrocyte cultures hyperstimulated with TSH also showed an increased intrinsic ability to form T3 Our data support the hypothesis that TG processing in the secretory pathway of TSHR-hyperstimulated thyrocytes alters the structure of the iodination substrate in a way that enhances de novo T3 formation, contributing to the relative T3 toxicosis of Graves' disease.

Glucose, amino acids and fatty acids directly regulate ghrelin and NUCB2/nesfatin-1 in the intestine and hepatopancreas of goldfish (Carassius auratus) in vitro.

Ghrelin and nesfatin-1 are two peptidyl hormones primarily involved in food intake regulation. We previously reported that the amount of dietary carbohydrates, protein and lipids modulates the expression of these peptides in goldfish in vivo. In the present work, we aimed to characterize the effects of single nutrients on ghrelin and nesfatin-1 in the intestine and hepatopancreas. First, immunolocalization of ghrelin and NUCB2/nesfatin-1 in goldfish hepatopancreas cells was studied by immunohistochemistry. Second, the effects of 2 and 4hour-long exposures of cultured intestine and hepatopancreas sections to glucose, l-tryptophan, oleic acid, linolenic acid (LNA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on ghrelin and nesfatin-1 gene and protein expression were studied. Co-localization of ghrelin and NUCB2/nesfatin-1 in the cytoplasm of goldfish hepatocytes was found. Exposure to glucose led to an upregulation of preproghrelin and a downregulation of nucb2/nesfatin-1 in the intestine. l-Tryptophan mainly decreased the expression of both peptides in the intestine and hepatopancreas. Fatty acids, in general, downregulated NUCB2/nesfatin-1 in the intestine, but only the longer and highly unsaturated fatty acids inhibited preproghrelin. EPA exposure led to a decrease in preproghrelin, and an increase in nucb2/nesfatin-1 expression in hepatopancreas after 2h. These results show that macronutrients exert a dose- and time-dependent, direct regulation of ghrelin and nesfatin-1 in the intestine and hepatopancreas, and suggest a role for these hormones in the digestive process and nutrient metabolism.

Alternagin-C (ALT-C), a disintegrin-like protein from Rhinocerophis alternatus snake venom promotes positive inotropism and chronotropism in fish heart.

Alternagin-C (ALT-C) is a disintegrin-like protein purified from the venom of the snake, Rhinocerophis alternatus. Recent studies showed that ALT-C is able to induce vascular endothelial growth factor (VEGF) expression, endothelial cell proliferation and migration, angiogenesis and to increase myoblast viability. This peptide, therefore, can play a crucial role in tissue regeneration mechanisms. The aim of this study was to evaluate the effects of a single dose of alternagin-C (0.5 mg kg(-1), via intra-arterial) on in vitro cardiac function of the freshwater fish traíra, Hoplias malabaricus, after 7 days. ALT-C treatment increased the cardiac performance promoting: 1) significant increases in the contraction force and in the rates of contraction and relaxation with concomitant decreases in the values of time to the peak tension and time to half- and 90% relaxation; 2) improvement in the cardiac pumping capacity and maximal electrical stimulation frequency, shifting the optimum frequency curve upward and to the right; 3) increases in myocardial VEGF levels and expression of key Ca(2+)-cycling proteins such as SERCA (sarcoplasmic reticulum Ca(2+)-ATPase), PLB (phospholamban), and NCX (Na(+)/Ca(2+) exchanger); 4) abolishment of the typical negative force-frequency relationship of fish myocardium. In conclusion, this study indicates that ALT-C improves cardiac function, by increasing Ca(2+) handling efficiency leading to a positive inotropism and chronotropism. The results suggest that ALT-C may lead to better cardiac output regulation indicating its potential application in therapies for cardiac contractile dysfunction.

TLR activation regulates damage-associated molecular pattern isoforms released during pyroptosis.

Infection of macrophages by bacterial pathogens can trigger Toll-like receptor (TLR) activation as well as Nod-like receptors (NLRs) leading to inflammasome formation and cell death dependent on caspase-1 (pyroptosis). Complicating the study of inflammasome activation is priming. Here, we develop a priming-free NLRC4 inflammasome activation system to address the necessity and role of priming in pyroptotic cell death and damage-associated molecular pattern (DAMP) release. We find pyroptosis is not dependent on priming and when priming is re-introduced pyroptosis is unaffected. Cells undergoing unprimed pyroptosis appear to be independent of mitochondrial involvement and do not produce inflammatory cytokines, nitrous oxide (NO), or reactive oxygen species (ROS). Nevertheless, they undergo an explosive cell death releasing a chemotactic isoform of the DAMP high mobility group protein box 1 (HMGB1). Importantly, priming through surface TLRs but not endosomal TLRs during pyroptosis leads to the release of a new TLR4-agonist cysteine redox isoform of HMGB1. These results show that pyroptosis is dominant to priming signals and indicates that metabolic changes triggered by priming can affect how cell death is perceived by the immune system.

Agonist-activated Ca2+ influx occurs at stable plasma membrane and endoplasmic reticulum junctions.

Junctate is a 33 kDa integral protein of sarco(endo)plasmic reticulum membranes that forms a macromolecular complex with inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptors and TRPC3 channels. TIRF microscopy shows that junctate enhances the number of fluorescent puncta on the plasma membrane. The size and distribution of these puncta are not affected by the addition of agonists that mobilize Ca(2+) from Ins(1,4,5)P(3)-sensitive stores. Puncta are associated with a significantly larger number of peripheral junctions between endoplasmic reticulum and plasma membrane, which are further enhanced upon stable co-expression of junctate and TRPC3. The gap between the membranes of peripheral junctions is bridged by regularly spaced electron-dense structures of 10 nm. Ins(1,4,5)P(3) inhibits the interaction of the cytoplasmic N-terminus of junctate with the ligand-binding domain of the Ins(1,4,5)P(3) receptor. Furthermore, Ca(2+) influx evoked by activation of Ins(1,4,5)P(3) receptors is increased where puncta are located. We conclude that stable peripheral junctions between the plasma membrane and endoplasmic reticulum are the anatomical sites of agonist-activated Ca(2+) entry.

CAR and PXR expression and inducibility of CYP2B and CYP3A activities in rat and rabbit lungs.

Several CYP enzymes are expressed in the lung of mammals but studies on their regulation have been rather neglected. In this study, the CAR and PXR expression and the inducibility of CYP 2B and CYP 3A isoforms in the lung rats and rabbits were investigated. Rats were treated with phenobarbital, clotrimazole or a mixture of dexametasone plus pregnenolone 16alpha-carbonitrile, whereas rabbits were treated with phenobarbital or rifampicin. A low constitutive expression of CAR mRNA was demonstrated by RT-PCR analysis in the lung of rat but not in rabbit. Phenobarbital treatment did not change the CAR expression profiles and did not induce in either rats and rabbits the pulmonary CYP 2B isoforms, as judged by western blot analysis and the marker pentoxyresorufin O-dealkylase and 7-ethoxy-4-trifluoroethylcoumarin O-deethylase activities. On the contrary, these marker activities were strongly induced by phenobarbital in the liver of both species. A low constitutive level of PXR mRNA was also detected by RT-PCR in the lung of rabbit but not in rat. However, also in this case, their expressions were not altered by the administration of strong CYP 3A inducers such as clotrimazole or a mixture of dexametasone plus pregnenolone 16alpha-carbonitrile for the rat and rifampicin or phenobarbital for the rabbit. For the first time, it was demonstrated by RT-PCR that rat lung expresses CYP 3A2, 3A9, 3A18 and 3A23 whereas the rabbit lung expresses the CYP 3A6, the only CYP 3A isoform identified in the rabbit so far. However, notwithstanding the differences observed in the constitutive presence of PXR and CYP 3A transcripts in both species, the above mentioned treatments did not affect in their lungs, unlike their livers, neither the anti-rat 3A immunoreactive proteins nor the CYP 3A marker 7-benzyloxyquinoline O-debenzylase and the 6beta-testosterone hydroxylase activities. The results obtained indicate that the role of CAR and PXR in the lung of rat and rabbit is different from that observed in the liver or other extrahepatic tissues where the induction of the CYP 2B and CYP 3A isoforms is regulated by these receptors.

Soluble guanylate cyclase activator reverses acute pulmonary hypertension and augments the pulmonary vasodilator response to inhaled nitric oxide in awake lambs.

BACKGROUND: Inhaled nitric oxide (NO) is a potent and selective pulmonary vasodilator, which induces cGMP synthesis by activating soluble guanylate cyclase (sGC) in ventilated lung regions. Carbon monoxide (CO) has also been proposed to influence smooth muscle tone via activation of sGC. We examined whether direct stimulation of sGC by BAY 41-2272 would produce pulmonary vasodilation and augment the pulmonary responses to inhaled NO or CO. METHODS AND RESULTS: In awake, instrumented lambs, the thromboxane analogue U-46619 was intravenously administered to increase mean pulmonary arterial pressure to 35 mm Hg. Intravenous infusion of BAY 41-2272 (0.03, 0.1, and 0.3 mg x kg(-1) x h(-1)) reduced mean pulmonary arterial pressure and pulmonary vascular resistance and increased transpulmonary cGMP release in a dose-dependent manner. Larger doses of BAY 41-2272 also produced systemic vasodilation and elevated the cardiac index. N(omega)-nitro-l-arginine methyl ester abolished the systemic but not the pulmonary vasodilator effects of BAY 41-2272. Furthermore, infusing BAY 41-2272 at 0.1 mg x kg(-1) x h(-1) potentiated and prolonged the pulmonary vasodilation induced by inhaled NO (2, 10, and 20 ppm). In contrast, inhaled CO (50, 250, and 500 ppm) had no effect on U-46619-induced pulmonary vasoconstriction before or during administration of BAY 41-2272. CONCLUSIONS: In lambs with acute pulmonary hypertension, BAY 41-2272 is a potent pulmonary vasodilator that augments and prolongs the pulmonary vasodilator response to inhaled NO. Direct pharmacological stimulation of sGC, either alone or in combination with inhaled NO, may provide a novel approach for the treatment of pulmonary hypertension.

Control of parathyroid cell growth by calcimimetics.

Parathyroid cell hyperplasia is commonly observed in patients with chronic renal insufficiency and largely accounts for refractory secondary hyperparathyroidism. Calcimimetics are newly synthesized compounds that activate a calcium receptor on the parathyroid cell and can suppress parathyroid hormone secretion. The calcimimetic compound AMG 073 has been examined in clinical trials, and the data obtained so far demonstrate that the compound can lower the circulating levels of parathyroid hormone and calcium-phosphorus product in patients with secondary hyperparathyroidism. Furthermore, experimental evidence indicates that calcimimetics have the potential to inhibit parathyroid cell proliferation and block the progression of parathyroid hyperplasia. These beneficial effects, especially the potential to control parathyroid cell proliferation, would place calcimimetics among the essential therapeutic agents for treating secondary hyperparathyroidism.

[Parathyroid gland, calcium receptor and calcimimetics]. / Glandes parathyroïdes, récepteur du calcium et calcimimétiques.

Parathyroid glands, bones, intestines and kidneys closely regulate ionized extracellular calcium concentration and thereby bone mineral density. This regulation is accomplished by a membrane associated receptor that responds to small changes in the extracellular calcium concentration (Ca2+)ec. The activation of this receptor regulates the secretion of PTH and the urinary excretion of calcium. The cloning of this calcium sensing receptor (RCa) in 1993 has allowed the identification of several hereditary disorders characterized by either a hyperparathyroidism or a hypoparathyroidism, as well as the development of pharmaceutical compounds potentially of interest for the treatment of these diseases. The calcimimetics are able either to directly stimulate the RCa or to make the RCa more sensitive to the effects of (Ca2+)ec. By this way, they decrease the secretion of PTH, but they also stimulate the secretion of calcitonin. The first clinical studies with a first-generation calcimimetic have demonstrated their efficacy lowering plasma intact PTH concentration in uremic patients with secondary hyperparathyroidism. However, the low biodisponibility of these first calcimimetics predict a difficult clinical utilization. The preliminary results obtained with a second-generation calcimimetic, the AMG-073, are very promising and await long term evaluation. The goal of this manuscript is to review the physiological, biological and clinical bases for the development of calcimimetic. Moreover, to try to replace the potential use of these drugs in the context of chronic renal failure and in the treatment of secondary hyperparathyroidism.

Role of RyRs and IP3 receptors after traumatic injury to spinal cord white matter.

Calcium influx and elevation of intracellular free calcium (Ca2+i), with subsequent activation of degenerative enzymes is hypothesized to cause cell injury and death after trauma. We examined the effects of traumatic compressive injury on (Ca2+)i dynamics in spinal cord white matter. We conducted electrophysiological studies with ryanodine and inositol (1,4,5)-triphosphate (IP3) receptor agonists and antagonists in an in vitro model of spinal cord injury (SCI). A 25-30-mm length of dorsal column was isolated from the spinal cord of adult rats, pinned in an in vitro recording chamber (37 degrees C) and injured with a modified clip (2-g closing force) for 15 sec. The functional integrity of the dorsal column was monitored electrophysiologically by quantitatively measuring the compound action potential (CAP) with glass microelectrodes. The CAP decreased to 55.2+/-6.8% of control (p < 0.05) after spinal cord injury (SCI). Chelation of Ca2+i with BAPTA-AM (a high-affinity calcium chelator) promoted significantly greater recovery of CAP amplitude (83.2+/-4.2% of control; p < 0.05) after injury. Infusion of caffeine (1 and 10 mM) exacerbated CAP amplitude decline (45.1+/-5.9% of control; p < 0.05; 44.6+/-3.1% of control; p < 0.05) postinjury. Blockade of Ca2+i release through ryanodine-sensitive receptors (RyRs) with dantrolene (10 microM) and ryanodine (50 microM), conferred significant (p < 0.05) improvement in CAP amplitude after injury. On the other hand, blockade of Ca2+i with inositol (1,4,5)-triphosphate receptor (IP3Rs) blocker 2APB (10 microM) also conferred significant improvement in CAP amplitude after injury (82.9+/-7.9%; p < 0.05). In conclusion, the injurious effects of Ca2+i in traumatic central nervous system (CNS) white matter injury appear to be mediated both by RyRs and through IP3Rs calcium-induced calcium release receptors (CICRs).

Expansion of the antigenic repertoire of a single T cell receptor upon T cell activation.

Activated T cells and their naive precursors display different functional avidities for peptide/MHC, but are thought to have identical antigenic repertoires. We show that, following activation with a cognate mimotope (NRP), diabetogenic CD8(+) T cells expressing a single TCR (8.3) respond vigorously to numerous peptide analogs of NRP that were unable to elicit any responses from naive 8.3-CD8(+) T cells, even at high concentrations. The NRP-reactive, in vivo activated CD8(+) cells arising in pancreatic islets of nonobese diabetic mice are similarly promiscuous for peptide/MHC, and paradoxically this promiscuity expands as the aviditiy of the T cell population for NRP/MHC increases with age. Thus, activation and avidity maturation of T lymphocyte populations can lead to dramatic expansions in the range of peptides that elicit functional T cell responses.

The calcium receptor modulates the hyperpolarization-activated current in subfornical organ neurons.

Here we report that neurons of the subfornical organ (SFO), a circumventricular structure devoid of a blood-brain barrier, show time-dependent, inward rectification indicative of the presence of a subthreshold, hyperpolarization-activated inward current (Ih). In whole-cell patch clamp experiments of isolated SFO neurons, we observed a Cs+-sensitive Ih in 47% of cells tested. Furthermore, we show that Ih is involved in the generation of evoked bursts in SFO neurons. An allosteric agonist of the extracellular calcium-sensing receptor (CaR) was found to potentiate Ih consistent with our previous observations of CaR-mediated bursting in SFO neurons. These studies indicate that a proportion of SFO neurons express Ih, and this may be one ionic mechanism through which bursting is regulated by various extracellular messengers.

Ethanol, Zn2+ and insulin interact as progression factors to enhance DNA synthesis synergistically in the presence of Ca2+ and other cell cycle initiators in fibroblasts.

In serum-starved NIH 3T3 fibroblasts, ethanol (30-80 mM) promoted the effects of insulin and insulin-like growth factor I (IGF-I) on DNA synthesis in a Zn(2+)-dependent manner. Ethanol and Zn(2+) were most effective when added shortly before or after insulin, indicating that all these agents facilitated cell cycle progression. The synergistic effects of ethanol, Zn(2+) and insulin (or IGF-I) on DNA synthesis required 1.1-2.3 mM Ca(2+), which seemed to act as the cell cycle initiator. When serum-starved cells were pretreated for 2 h with other cell cycle initiators such as 10% (v/v) serum, 50 ng/ml platelet-derived growth factor or 2 ng/ml fibroblast growth factor, subsequent co-treatments with 60 mM ethanol, Zn(2+) and insulin for an 18 h period again synergistically increased DNA synthesis. Of the various signal transducing events examined, ethanol stimulated cellular uptake of (45)Ca and it enhanced the stimulatory effects of insulin on p70 S6 kinase activity in a Zn(2+)-dependent manner. In contrast, ethanol inhibited insulin-induced activating phosphorylation of p42/p44 mitogen-activated protein kinases; these inhibitory ethanol effects were prevented by Zn(2+). The results show that, in NIH 3T3 fibroblasts, ethanol can promote cell cycle progression in the presence of a cell cycle initiator as well as Zn(2+) and insulin (or IGF-I).

Thimerosal increases the responsiveness of the calcium receptor in human parathyroid and rMTC6-23 cells.

Parathyroid cells express a plasma membrane calcium receptor (CaR), which is stimulated by a rise in extracellular calcium concentration ([Ca2+]ext). A decreased sensitivity to [Ca2+]ext occurs in adenomatous parathyroid cells in patients with primary hyperparathyroidism, but the underlying functional mechanism is not yet fully understood. This study explored whether CaR responsiveness is influenced by increasing the affinity of IP3 receptors--a major signalling component of other G-protein-coupled receptors. The sulphydryl reagent thimerosal was used to increase the responsiveness of IP3-receptors. Quantitative fluorescence microscopy in Fura-2-loaded cells was used to investigate the effects of thimerosal on the cytoplasmic calcium concentrations ([Ca2+]i) in human parathyroid cells and to compare its effects in a rat medullary thyroid carcinoma cell line (rMTC6-23) also expressing CaR. During incubation in Ca(2+)-free medium, thimerosal 5 microM induced a rapid sustained rise in [Ca2+]i in human parathyroid cells and no further [Ca2+]i increase appeared in response to the CaR agonist Gd3+ (100 microM). Thimerosal 1 microM induced only slow and minimal changes of basal [Ca2+]i and allowed a rapid response to Gd3+ 20 nM (a concentration without effect in control cells). The slope of the thimerosal-induced [Ca2+]i responses was steeper following exposure to CaR agonists. In the presence of 1 mM [Ca2+]ext, thimerosal (0.5 microM) induced a sharp increase in [Ca2+]i to a peak (within 60 s), followed either by return to basal [Ca2+]i or by a plateau of slightly higher amplitude. Similar results were obtained using rMTC6-23 cells. Thimerosal increases the responsiveness to CaR agonists through modulation of the sensitivity of the IP3 receptor in both parathyroid and rMTC6-23 cells.

Expression and signal transduction of calcium-sensing receptors in cartilage and bone.

We previously showed that Ca2+-sensing receptors (CaRs) are expressed in chondrogenic RCJ3.1C5.18 (C5.18) cells and that changes in extracellular [Ca2+]([Ca2+]o) modulate nodule formation and chondrogenic gene expression. In the present study, we detected expression of CaRs in mouse, rat, and bovine cartilage and bone by in situ hybridization, immunocytochemistry, immunoblotting, and RT-PCR; and we tested the effects of CaR agonists on signal transduction in chondrogenic and osteogenic cell lines. In situ hybridization detected CaR transcripts in most articular chondrocytes and in the hypertrophic chondrocytes of the epiphyseal growth plate. Expression of CaR transcripts was weak or absent, however, in proliferating and maturing chondrocytes in the growth plate. In bone, CaR transcripts were present in osteoblasts, osteocytes, and bone marrow cells, but rarely in osteoclasts. A complementary DNA was amplified from mouse growth plate cartilage, which was highly homologous to the human parathyroid CaR sequence. Immunocytochemistry of cartilage and bone with CaR antisera confirmed these findings. Western blotting revealed specific bands (approximately 140-190 kDa) in membrane fractions isolated from growth plate cartilage, primary cultures of rat chondrocytes, and several osteogenic cell lines (SaOS-2, UMR-106, ROS 17/2.8, and MC3T3-E1). InsP responses to high [Ca2+]o were evident in C5.18 cells and all osteogenic cell lines tested except for SaOS-2 cells. In the latter, high [Ca2+]o reduced PTH-induced cAMP formation. Raising [Ca2+]o also increased intracellular free [Ca2+] in SaOS-2 and C5.18 cells. These studies confirm expression of CaRs in cartilage and bone and support the concept that changes in [Ca2+]o may couple to signaling pathways important in skeletal metabolism.

The calcium receptor and calcimimetics.

Parathyroid cells can sense small changes in plasma Ca2+ levels by virtue of a cell surface Ca2+ receptor. Calcimimetics are newly synthesized compounds that act as agonists or positive allosteric modulators at the Ca2+ receptor and can suppress parathyroid hormone secretion. The first-generation calcimimetic, NPS R-568, has undergone clinical trials in primary hyperparathyroidism and in hyperparathyroidism secondary to chronic renal insufficiency. The data accumulated so far demonstrate that calcimimetics have potential as therapeutic agents for hyperparathyroidism and related bone diseases such as osteitis fibrosa.

Stimulatory effect of regucalcin on proteolytic activity in rat renal cortex cytosol: involvement of thiol proteases.

The effect of regucalcin, a calcium-binding protein, on neutral proteolytic activity in the cytosol of rat kidney cortex was investigated. Proteolytic activity was significantly increased in the presence of regucalcin (0.01-0.25 microM) in the enzyme reaction mixture. This increase was not significantly altered by the addition of CaCl, (0.01 and 1.0 mM) or EGTA (1.0 mM), indicating that the effect of regucalcin was independent on Ca2+. The effect of regucalcin to increase proteolytic activity was completely prevented in the presence of N-ethylmaleimide (5 mM), a modifying reagent of thiol groups. Proteolytic activity was clearly elevated by dithiothreitol (5 mM). This elevation was further enhanced by regucalcin (0.1 microM). Meanwhile, the stimulatory effect of regucalcin on proteolytic activity was not significantly altered in the presence of diisopropylfluorophosphate (2.5 mM), an inhibitor of serine proteases. Also, the regucalcin effect was not appreciably changed by the addition of EDTA (2.5 mM), a chelator of metal ions, indicating that it is not involved in metal-related proteases. These results demonstrate that regucalcin can increase proteolytic activity in the cytosol of rat kidney cortex. Regucalcin may activate thiol proteases independent on Ca2+.

Domains determining ligand specificity for Ca2+ receptors.

The Ca2+ receptor is a G protein-coupled receptor that enables parathyroid cells and certain other cells in the body to respond to changes in the level of extracellular Ca2+. The Ca2+ receptor is a member of a family of G protein-coupled receptors that includes metabotropic glutamate receptors (mGluRs), gamma-aminobutyric acidB receptors, and putative pheromone receptors. As a family, these receptors are characterized by limited sequence homology and an unusually large putative extracellular domain (ECD). The ECD of the mGluRs is believed to determine agonist selectivity, but the functions of the structural domains of the Ca2+ receptor are not known. To identify structural determinants for cation recognition and activation of the Ca2+ receptor (and to further study the mGluRs), two chimeric receptors were constructed in which the large ECD of the Ca2+ receptor and the mGluR1 were interchanged. When expressed in Xenopus laevis oocytes, one of these chimeras, named CaR/mGluR1 [ECD of the Ca2+ receptor and transmembrane domain (TMD) of the mGluR1], responded to cation agonists (Gd3+, Ca2+, neomycin) of the Ca2+ receptor at concentrations similar to those necessary for activation of the native Ca2+ receptor. A reciprocal construct, named mGluR1/CaR (ECD of the mGluR1 and TMD of the Ca2+ receptor), was responsive to mGluR agonists but was much less sensitive to two of three cation agonists known to activate the Ca2+ receptor. A deletion construct of the Ca2+ receptor (DeltantCaR), which lacked virtually the entire ECD, was only activated by one of three agonists tested. These results suggest that the primary determinants for agonist activation of both the Ca2+ receptor and the mGluRs are found in the large ECD and that the Ca2+ receptor is possibly distinguished from the mGluRs in that it may contain sites in the TMD that permit activation by certain cation agonists.

Adenosine 5'-triphosphate, uridine 5'-triphosphate, bradykinin, and lysophosphatidic acid induce different patterns of calcium responses by human articular chondrocytes.

Small calcium-mobilizing inflammatory mediators have been implicated in joint pathology. Here we demonstrate that bradykinin, adenosine 5'-triphosphate, uridine 5'-triphosphate, and lysophosphatidic acid raise the intracellular calcium concentration ([Ca2+]i) in human articular chondrocytes. Heterologous cross-desensitization experiments showed that the uridine 5'-triphosphate response was abolished by prior treatment with adenosine 5'-triphosphate and, conversely, that the adenosine 5'-triphosphate response was abolished by prior treatment with uridine 5'-triphosphate; this indicated competition for the same receptor site, whereas bradykinin and lysophosphatidic acid did not compete with other ligands. Pretreatment with thapsigargin abolished ligand-mediated Ca2+ responses but not vice versa; this confirmed that Ca2+ release occurred from intracellular stores. Single-cell analysis of Fura-2 acetoxymethyl ester loaded chondrocytes showed mediator-dependent patterns of oscillatory Ca2+ changes in a subset of cells when challenged with submaximal concentrations of bradykinin, adenosine 5'-triphosphate, or uridine 5'-triphosphate in the presence of extracellular Ca2+. However, no oscillatory responses were seen after a challenge with lysophosphatidic acid. Therefore, although a number of different Ca2+-mobilizing ligands activate chondrocytes, the differences that occur in the temporal patterning of Ca2+ responses may result in unique mediator-dependent changes in cellular activity.