Evidence for the convergence of β-adrenergic and muscarinic signalling systems at a post-receptor site

AbstractThe β-adrenergic agonist isoproterenol stimulates inositol trisphosphate (IP3) formation and cytosolic Ca2+ ([Ca2+]i) mobilization in rat parotid acini via a cAMP-dependent process. Atropine, a muscarinic antagonist, inhibited these isoproterenol responses without affecting isoproterenol-induced amylase secretion or peak [Ca2+]i and IP3 responses elicited by α1-adrenergic stimulation with epinephrine. Atropine had no effect on isoproterenol-induced [Ca2+]i responses in a cell line which lacked muscarinic receptors and did not alter β-adrenoreceptor ligand binding. These results suggest that the inhibition by atropine results from a post-receptor effect on cAMP-mediated stimulation of phosphatidylinositol 4,5 bisphosphate (PIP2) hydrolysis

TRPC1 participates in the HSV-1 infection process by facilitating viral entry

Mammalian transient receptor potential (TRP) channels are major components of Ca2+ signaling pathways and control a diversity of physiological functions. Here, we report a specific role for TRPC1 in the entry of herpes simplex virus type 1 (HSV-1) into cells. HSV-1-induced Ca2+ release and entry were dependent on Orai1, STIM1, and TRPC1. Inhibition of Ca2+ entry or knockdown of these proteins attenuated viral entry and infection. HSV-1 glycoprotein D interacted with the third ectodomain of TRPC1, and this interaction facilitated viral entry. Knockout of TRPC1 attenuated HSV-1-induced ocular abnormality and morbidity in vivo in TRPC1−/− mice. There was a strong correlation between HSV-1 infection and plasma membrane localization of TRPC1 in epithelial cells within oral lesions in buccal biopsies from HSV-1-infected patients. Together, our findings demonstrate a critical role for TRPC1 in HSV-1 infection and suggest the channel as a potential target for anti-HSV therapy.Fil: He, DongXu. Jiangnan University; ChinaFil: Mao, AiQin. Jiangnan University; ChinaFil: Li, YouRan. Jiangnan University; ChinaFil: Tam, SiuCheung. Chinese University Of Hong Kong; Hong KongFil: Zheng, YongTang. Kunming Institute Of Zoology Chinese Academy Of Sciences; ChinaFil: Yao, XiaoQiang. Chinese University Of Hong Kong; Hong KongFil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Ambudkar, Indu S.. National Institute Of Dental And Craniofacial Research ; Estados UnidosFil: Ma, Xin. Jiangnan University; Chin

Hydrogen Sulfide Maintains Mesenchymal Stem Cell Function and Bone Homeostasis via Regulation of Ca2+ Channel Sulfhydration

Gaseous signaling molecules such as hydrogen sulfide (H2S) are produced endogenously and mediate effects through diverse mechanisms. H2S is one such gasotrasmitter which regulates multiple signaling pathways in mammalian cells, and abnormal H2S metabolism has been linked to defects in bone homeostasis. Here, we demonstrate that bone marrow mesenchymal stem cells (BMMSCs) produce H2S to regulate their self-renewal and osteogenic differentiation, and H2S deficiency results in defects in BMMSC differentiation. H2S deficiency causes aberrant intracellular Ca2+ influx, due to reduced sulfhydration of cysteine residues on multiple Ca2+ TRP channels. This decreased Ca2+ flux downregulates PKC/Erk-mediated Wnt/β-catenin signaling which controls osteogenic differentiation of BMMSCs. Consistently, H2S-deficient mice display an osteoporotic phenotype, which can be rescued by small molecules which release H2S. These results demonstrate H2S regulates BMMSCs, and restoring H2S levels via non-toxic donors may provide treatments for diseases such as osteoporosis which can arise from H2S deficiencies

Local Ca2+ Entry Via Orai1 Regulates Plasma Membrane Recruitment of TRPC1 and Controls Cytosolic Ca2+ Signals Required for Specific Cell Functions

Store-operated Ca2+ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca2+ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent ISOC, activated in response to Ca2+ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated ICRAC; the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(684EE685). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca2+ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd3+, removal of extracellular Ca2+, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca2+-containing, but not Ca2+-free, medium. Consistent with this, ICRAC is activated in cells pretreated with thapsigargin in Ca2+-free medium while ISOC is activated in cells pretreated in Ca2+-containing medium. Significantly, TRPC1 function is required for sustained KCa activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca2+ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca2+ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE

Adenovirus-mediated Expression of Aquaporin-5 in Epithelial Cells

A recombinant adenovirus coding for rat aquaporin-5 was constructed and plaque purified. The recombinant adenovirus (AdrAQP5) mediated the expression of aquaporin-5 in rat and human salivary cell lines and in dog kidney cells in vitro as demonstrated by Northern blot and Western blot analyses, and by confocal microscopy after immunofluorescent labeling. In kidney cells, expression of the transgene was optimal if cells were infected at their basolateral surface, a phenomenon associated with the distribution of integrin receptors on these cells. The expressed aquaporin-5 protein was functionally active because viral-mediated gene transfer resulted in a significant increase in the osmotically directed net fluid secretion rate across monolayers of kidney cells. AdrAQP5 should provide an efficient and useful means to impart facilitated water permeability to cells lacking such a pathway

Recent Progress in the Regulation of TRPC1 by Store Depletion

Store-operated Ca entry (SOCE) is activated in response to depletion of the ER-Ca stores. The ER-Ca 2+ sensor protein, STIM1, oligomerizes when [Ca ] in the store is decreased and moves to ER/PM junctional domains where it interacts with and activates channels involved in SOCE, namely Orai and TRPC channels. 2+ Orai1 is the primary pore-forming component of the highly Ca selective CRAC channel. It is recruited to ER/ PM junctional domains by STIM1 where it is gated via interaction with a specific C-terminal domain of STIM1. Thus Orai1 and STIM1 are sufficient for generation of functional CRAC channels. Store depletion also leads to activation of relatively non-selective cation channels, referred to as SOC channels that contribute to SOCE in several other cell types. TRPC1 has been proposed as a possible candidate component of SOC channels. 2+ TRPC1 contributes to endogenous SOCE in many cells types. In these cells, TRPC1-mediated Ca entry and 3+ cation currents are stimulated with either agonist or thapsigargin, and inhibited by low [Gd ] and 10-20 µM 2APB (conditions that block SOCE). STIM1 also associates with and gates TRPC1 via electrostatic interaction between STIM1 (684KK685) and TRPC1 (639DD640). Further, functional Orai1 is required for activation of TRPC1-SOCE and this has been associated with recruitment of a TRPC1/STIM1/Orai1 complex. However, there is ongoing debate regarding the activation of TRPC1 by store depletion as well as the role of Orai1 and STIM1 in regulating its function. This chapter will summarize recent studies and concepts regarding the contri- butions of Orai1 and TRPC1 to SOCE. We will discuss major unresolved questions regarding functional inter- action between Orai1 and TRPC1 as well as possible mechanisms involved in the regulation of TRPC channels