Analysis of the V2 Vasopressin Receptor (V2R) Mutations Causing Partial Nephrogenic Diabetes Insipidus Highlights a Sustainable Signaling by a Non-peptide V2R Agonist.

Disease-causing mutations in G protein-coupled receptor (GPCR) genes, including the V2 vasopressin receptor (V2R) gene, often cause misfolded receptors, leading to a defect in plasma membrane trafficking. A novel V2R mutation, T273M, identified in a boy with partial nephrogenic diabetes insipidus (NDI), shows intracellular localization and partial defects similar to the two mutants we described previously (10). Although non-peptide V2R antagonists have been shown to rescue the membrane localization of V2R mutants, their level of functional rescue is weak. Interestingly, it has been reported that a non-peptide agonist, OPC51803, activates misfolded V2R mutants intracellularly without degradation, thus potentially serving as a therapeutic agent against NDI (14). In our current experiments, however, a peptide antagonist blocked arginine vasopressin (AVP)- or OPC51803-stimulated cAMP accumulation both in COS-7 and MDCK cells, suggesting that OPC51803 mainly stimulates cell surface V2R mutants. In addition, our analyses revealed that OPC51803 works not only as a non-peptide agonist that causes activation/ß-arrestin-dependent desensitization of V2R mutants expressed at the plasma membrane but also as a pharmacochaperone that promotes the endoplasmic reticulum-retained mutant maturation and trafficking to the plasma membrane. The ratio of the pharmacochaperone effect to the desensitization effect likely correlates negatively with the residual function of the tested mutants, suggesting that OPC5 has a more favorable effect on the V2R mutants with a less residual function. We speculated that the canceling of the desensitization effect of OPC51803 by the pharmacochaperone effect after long-term treatment may produce sustainable signaling, and thus pharmacochaperone agonists such as OPC51803 may serve as promising therapeutics for NDI caused by misfolded V2R mutants.

Localization of pufferfish saxitoxin and tetrodotoxin binding protein (PSTBP) in the tissues of the pufferfish, Takifugu pardalis, analyzed by immunohistochemical staining.

Pufferfish saxitoxin and tetrodotoxin binding protein (PSTBP) was previously isolated from the plasma of the marine pufferfish, Takifugu pardalis. In this study, we investigated distribution pattern of PSTBP in intestine, liver, ovary, skin, and skeletal muscle of T. pardalis by immunohistochemical staining for the study of functions of this protein. In the skin, dermis around the tetrodotoxin secreting gland was positive, while this secreting gland itself was negative. In the ovary containing vitellogenic oocytes, ovarian wall and vitelline envelope were positive, while yolk and nucleus were negative. In the liver, hepatocytes with large fat droplets and capillaries were positive. In the intestine, the lamina propria mucosae were positive, while the mucosal epithelium was negative. In the skeletal muscle, only capillaries were positive. Furthermore, liver specific expression of PSTBP was confirmed by Northern blot analysis. Based on these results together with reported tetrodotoxin localization pattern in pufferfish, PSTBP was assumed to be a carrier protein to transfer tetrodotoxin among the tissues, especially liver, ovary, and skin.

Evaluation of methods for cell harvesting and the biological properties at successive passages of canine bone marrow stromal cells.

OBJECTIVE: To compare methods for harvesting canine bone marrow stromal cells (BMSCs) and determine the biological properties of canine BMSCs at successive passages in vitro. SAMPLE: BMSCs collected from the femurs of 9 Beagles. PROCEDURES: A fibroblast assay was performed to compare 2 methods for harvesting BMSCs: the aspiration and perfusion method. Flow cytometric analysis was performed to evaluate the cell surface markers. Changes in proliferative activity were analyzed by examining radioactivity of hydrogen 3-thymidine. Cell senescence was studied via senescence-associated ß-galactosidase staining, and differentiation properties (osteogenesis and adipogenesis) were estimated in association with passage. RESULTS: The aspiration method yielded significantly more fibroblasts than the perfusion method. The cells harvested by both methods gave positive results for CD44 and CD90 and negative results for CD34 and CD45. After induction, the cells had osteogenic and adipogenic phenotypes. The biological properties of BMSCs harvested by the aspiration method were estimated in association with passage. With increasing number of passages, the proliferative activity was reduced and the proportion of cells with senescence-associated ß-galactosidase staining was increased. The capacity of differentiation was reduced at passage 3. CONCLUSIONS AND CLINICAL RELEVANCE: The aspiration method was superior for collection of BMSCs. In early passages, canine BMSCs had the proliferative activity and potential of osteogenic and adipogenic differentiation, but this decreased with increased number of passages. Consideration of passage will be important to the success of any strategy that seeks to regenerate tissue though the use of BMSCs.

An acquired hypocalciuric hypercalcemia autoantibody induces allosteric transition among active human Ca-sensing receptor conformations.

The seven-spanning calcium-sensing receptor (CaSR) activates multiple G proteins including Gq and Gi, and thereby activates a variety of second messengers and inhibits parathyroid hormone (PTH) secretion. However, the exact signaling mechanisms underlying the functional activity of CaSR are not yet fully understood. The heterozygous inactivation of CaSR or its inhibition by antibody blocking results in either familial hypocalciuric hypercalcemia or acquired hypocalciuric hypercalcemia (AHH), respectively. Here, we report the identification of a unique CaSR autoantibody in an AHH patient. Paradoxically, we find that this autoantibody potentiates the Ca(2+)/Gq-dependent accumulation of inositol phosphates by slightly shifting the dose dependence curve of the Ca(2+) mediated activation of phosphatidylinositol turnover to the left, whereas it inhibits the Ca(2+)/Gi-dependent phosphorylation of ERK1/2 in HEK293 cells stably expressing human CaSR. Treatment of these same cells with a calcimimetic, NPS-R-568, augments the CaSR response to Ca(2+), increasing phosphatidylinositol turnover and ERK1/2 phosphorylation, and overcoming the autoantibody effects. Our observations thus indicate that a calcium-stimulated CaSR primed by a specific autoantibody adopts a unique conformation that activates Gq but not Gi. Our findings also suggest that CaSR signaling may act via both Gq and Gi to inhibit PTH secretion. This is the first report of a disease-related autoantibody that functions as an allosteric modulator and maintains G protein-coupled receptors (GPCRs) in a unique active conformation with its agonist. We thus speculate that physiological modulators may exist that enable an agonist to specifically activate only one signaling pathway via a GPCR that activates multiple signaling pathways.

Mutual binding inhibition of tetrodotoxin and saxitoxin to their binding protein from the plasma of the puffer fish, Fugu pardalis.

The mutual binding inhibition of tetrodotoxin and saxitoxin to their binding protein from the plasma of Fugu pardalis was investigated by HPLC. The values for the half inhibitory concentration of tetrodotoxin (1.6 microM) binding to this protein (1.2 microM) for saxitoxin, and of saxitoxin (0.47 microM) binding to that (0.30 microM) for tetrodotoxin were 0.35 +/- 0.057 microM and 81 +/- 16 microM (n = 2), respectively.