No abstract

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 proteincoupled 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.allosteric modulation ͉ disease ͉ functional selectivity ͉ G protein-coupled receptors ͉ multiple active conformations

Some species of puffer fish have been reported to possess both of tetrodotoxin and saxitoxin, which share one binding site on sodium channels. We purified a novel soluble glycoprotein that binds to these toxins from plasma of the puffer fish, Fugu pardalis, and named puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP). PSTBP possessed a binding capacity of 10.6 ± 0.97 nmol·mg−1 protein and a Kd of 14.6 ± 0.33 nm for [3H]saxitoxin in equilibrium binding assays. [3H]Saxitoxin (10 nm) binding to PSTBPs was half‐inhibited by the presence of tetrodotoxin and saxitoxin at 12 µm and 8.5 nm, respectively. From the results of gel filtration chromatography (200 kDa) and SDS/PAGE (104 kDa), PSTBP was suggested to consist of noncovalently linked dimers of a single subunit. PSTBP was completely deglycosylated by glycopeptidase F, producing a single band at 42 kDa. Two highly homologous cDNAs to each other coding PSTBP (PSTBP1 and PSTBP2, the predicted amino‐acid identity 93%), were obtained from a cDNA library of F. pardalis liver. These proteins consisted to two tandemly repeated homologous domains. The predicted amino‐acid sequences of PSTBP1 and 2 were not homologous to that of saxiphilin, a reported saxitoxin binding protein, or sodium channels, but their N‐terminus sequences were homologous to that of the reported tetrodotoxin binding protein from plasma of Fugu niphobles, which has not been fully characterized. The partially homologous cDNA sequences to PSTBP1 and 2 were also found in expressed sequence tag clones of nontoxic flounders liver. Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish.

Previous studies have shown that serine proteases and Rho‐associated kinase contribute to carbon ion radiation‐enhanced invasion of the human pancreatic cancer cell line PANC‐1. The results presented here show that nitric oxide synthase (NOS) also plays a critical role in this process. Irradiation of PANC‐1 cells promoted invasion and production of nitric oxide (NO), which activated the PI3K–AKT signaling pathway, while independently activating RhoA. Inhibition of PI3K, Rho‐associated kinase, and serine protease alone or in conjunction with NOS suppressed the radiation‐enhanced invasion of PANC‐1 cells, suggesting that they could serve as possible targets for the management of tumor metastasis.

Colon smooth muscle electrical control (ECA) and response activities (ERA) were recorded for up to 4 wk postoperatively for 48 patients after major abdominal operations. Bipolar electrodes were implanted into right and left colon circular muscle and exteriorized through the flanks, and signals were tape recorded for 2-24 h daily beginning on the 1st postoperative day. A computer program was used for data reduction and analysis. Recorded signals were digitized and filtered. The ECA frequency components were identified by fast Fourier transformation, and their relative tenancy in low, mid, and high frequency ranges was determined. Short and long ERA burst duration and frequency and number and velocity of propagating long ERA bursts were determined. ECA was omnipresent and exhibited a downshift of the dominant frequency from the mid to the low range as recovery from postoperative ileus progressed. Concurrently, first in the right and then in the left colon, the frequency of long ERA bursts increased, followed by the appearance of propagating long ERA. After the 6th postoperative day, no further significant changes in parameters of colon electrical activity occurred with time.