Rationale A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. Increased resting [Ca2+]cyt and enhanced Ca2+ influx have been implicated in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH). Objective We examined whether the extracellular Ca2+-sensing receptor (CaSR) is involved in the enhanced Ca2+ influx and proliferation in IPAH-PASMC and whether blockade of CaSR inhibits experimental pulmonary hypertension. Methods and Results In normal PASMC superfused with Ca2+-free solution, addition of 2.2 mM Ca2+ to the perfusate had little effect on [Ca2+]cyt. In IPAH-PASMC, however, restoration of extracellular Ca2+ induced a significant increase in [Ca2+]cyt. Extracellular application of spermine also markedly raised [Ca2+]cyt in IPAH-PASMC, but not in normal PASMC. The calcimimetic R568 enhanced, whereas the calcilytic NPS 2143 attenuated, the extracellular Ca2+-induced [Ca2+]cyt rise in IPAH-PASMC. Furthermore, the protein expression level of CaSR in IPAH-PASMC was greater than in normal PASMC; knockdown of CaSR in IPAH-PASMC with siRNA attenuated the extracellular Ca2+-mediated [Ca2+]cyt increase and inhibited IPAH-PASMC proliferation. Using animal models of pulmonary hypertension, our data showed that CaSR expression and function were both enhanced in PASMC, whereas intraperitoneal injection of the calcilytic NPS 2143 prevented the development of pulmonary hypertension and right ventricular hypertrophy in rats injected with monocrotaline and mice exposed to hypoxia. Conclusions The extracellular Ca2+-induced increase in [Ca2+]cyt due to upregulated CaSR is a novel pathogenic mechanism contributing to the augmented Ca2+ influx and excessive PASMC proliferation in patients and animals with pulmonary arterial hypertension.

Corals are known to contain a diverse microbiota; however, few studies have explicitly addressed the spatial variability of bacterial communities across individual, healthy coral colonies. This study applied culture-based and culture-independent methods to examine the spatial heterogeneity in bacterial communities in the mucus of 3 healthy Montastraea annularis colonies from Looe Key Reef, Florida Keys. Automated ribosomal intergenic spacer analysis (ARISA) results showed significant variability (up to 61% dissimilarity) in the composition of the total bacterial community at different locations only centimeters apart on individual coral colonies. Abundances of culturable Vibrio spp. determined by TCBS plating were highly variable across individual coral colonies, differing by up to 100-fold between different locations on the same colony. ARISA profiles indicated that intracolony variation rivaled intercolony differences in the composition of the culturable Vibrio community (i.e. types of culturable Vibrio spp. and their relative abundances). The high degree of spatial heterogeneity in coral-associated bacteria observed across individual colonies has implications for coral microbiology studies and coral restoration projects.KEY WORDS: Community profiling · ARISA · Bacteria · Coral · Spatial heterogeneity · Vibrio Resale or republication not permitted without written consent of the publisherMar Ecol Prog Ser 426: [29][30][31][32][33][34][35][36][37][38][39][40] 2011 in bacterial community composition have been observed in bleached and diseased corals, with opportunists and pathogens present in these health-compromised states (Ritchie & Smith 1995, Kushmaro et al. 1997, Frias-Lopez et al. 2002, Pantos et al. 2003, Pantos & Bythell 2006, Ritchie 2006.The coral surface layers are extremely complex and dynamic (Ainsworth et al. 2006, Johnston & Rohwer 2007; however, few studies have examined spatial heterogeneity in coral-microbe associations across individual coral colonies. In diseased coral colonies, significant differences have been observed between the bacteria found in the healthy versus diseased portions of the colony (Frias-Lopez et al. 2002, Pantos et al. 2003, Breitbart et al. 2005, Gil-Agudelo et al. 2006, Pantos & Bythell 2006, Sekar et al. 2006. A limited number of studies have also demonstrated spatial variation in the bacterial community associated with healthy branching corals. Rohwer et al. (2002) demonstrated that bacterial communities were spatially structured on the branching coral Porites furcata, with specific bacterial taxa only found at the branch tips. In addition, bacterial community analysis in 6 replicate tissue samples from healthy Pocillopora damicornis colonies revealed spatial heterogeneity across some coral colonies (Bourne & Munn 2005), although some bacteria were uniformly found throughout an individual coral colony. Hansson et al. (2009) also demonstrated that the cold water coral Madrepora oculata exhibited spatial variation of bacterial communities within and among co...

Intracellular Ca2+ plays a fundamental role in regulating cell functions in pulmonary arterial smooth muscle cells (PASMCs). A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) triggers pulmonary vasoconstriction and stimulates PASMC proliferation. [Ca2+]cyt is increased mainly by Ca2+ release from intracellular stores and Ca2+ influx through plasmalemmal Ca2+-permeable channels. Given the high concentration of intracellular Cl- in PASMCs, Ca2+-activated Cl-(ClCa) channels play an important role in regulating membrane potential and cell excitability of PASMCs. In this study, we examined whether activity of ClCa channels was involved in regulating [Ca2+]cyt in human PASMCs via regulating receptor- (ROCE) and store- (SOCE) operated Ca2+ entry. The data demonstrated that an angiotensin II (100 nM)-mediated increase in [Ca2+]cyt via ROCE was markedly attenuated by the ClCa channel inhibitors, niflumic acid (100 μM), flufenamic acid (100 μM), and 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid (100 μM). The inhibition of ClCa channels by niflumic acid and flufenamic acid significantly reduced both transient and plateau phases of SOCE that was induced by passive depletion of Ca2+ from the sarcoplasmic reticulum by 10 μM cyclopiazonic acid. In addition, ROCE and SOCE were abolished by SKF-96365 (50 μM) and 2-aminoethyl diphenylborinate (100 μM), and were slightly decreased in the presence of diltiazem (10 μM). The electrophysiological and immunocytochemical data indicate that ClCa currents were present and TMEM16A was functionally expressed in human PASMCs. The results from this study suggest that the function of ClCa channels, potentially formed by TMEM16A proteins, contributes to regulating [Ca2+]cyt by affecting ROCE and SOCE in human PASMCs.