Function of a STIM1 homologue in C. elegans: evidence that store-operated Ca2+ entry is not essential for oscillatory Ca2+ signaling and ER Ca2+ homeostasis

1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca(2+) entry (SOCE) is activated during endoplasmic reticulum (ER) Ca(2+) store depletion and is believed to be an essential and ubiquitous component of Ca(2+) signaling pathways. SOCE is thought to function to refill Ca(2+) stores and modulate Ca(2+) signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca(2+) sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with approximately 21% sequence identity to human STIM1. Green fluorescent protein (GFP)-tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca(2+) (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca(2+) signaling, in wild type and IP(3) signaling mutant worms, and has no effect on intestinal Ca(2+) oscillations and waves. Depletion of intestinal Ca(2+) stores by RNAi knockdown of the ER Ca(2+) pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca(2+) signaling processes and for maintenance of store Ca(2+) levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.     

Physiological roles of STIM1 and Orai1 homologs and CRAC channels in the genetic model organism Caenorhabditis elegans

The nematode Caenorhabditis elegans provides numerous experimental advantages for developing an integrative molecular understanding of physiological processes and has proven to be a valuable model for characterizing Ca(2+) signaling mechanisms. This review will focus on the role of Ca(2+) release activated Ca(2+) (CRAC) channel activity in function of the worm gonad and intestine. Inositol 1,4,5-trisphosphate (IP(3))-dependent oscillatory Ca(2+) signaling regulates contractile activity of the gonad and rhythmic posterior body wall muscle contraction (pBoc) required for ovulation and defecation, respectively. The C. elegans genome contains a single homolog of both STIM1 and Orai1, proteins required for CRAC channel function in mammalian and Drosophila cells. C. elegans STIM-1 and ORAI-1 are coexpressed in the worm gonad and intestine and give rise to robust CRAC channel activity when coexpressed in HEK293 cells. STIM-1 or ORAI-1 knockdown causes complete sterility demonstrating that the genes are essential components of gonad Ca(2+) signaling. Knockdown of either protein dramatically inhibits intestinal cell CRAC channel activity, but surprisingly has no effect on pBoc, intestinal Ca(2+) oscillations or intestinal ER Ca(2+) store homeostasis. CRAC channels thus do not play obligate roles in all IP(3)-dependent signaling processes in C. elegans. Instead, we suggest that CRAC channels carry out highly specialized and cell specific signaling roles and that they may function as a failsafe mechanism to prevent Ca(2+) store depletion under pathophysiological and stress conditions.     

On the Control of Oocyte Meiotic Maturation and Ovulation inCaenorhabditis elegans

Prior to fertilization, oocytes undergo meiotic maturation (cell cycle progression) and ovulation (expulsion from the ovary). To begin the study of these processes inCaenorhabditis elegans,we have defined a time line of germline and somatic events by video microscopy. As the oocyte matures, its nuclear envelope breaks down and its cell cortex rearranges. Immediately thereafter, the oocyte is ovulated by increasing contraction of the myoepithelial gonadal sheath and relaxation of the distal spermatheca. By systematically altering the germ cell contents of the hermaphrodite using mutant strains, we have uncovered evidence of four cell–cell interactions that regulate maturation and ovulation. (1) Both spermatids and spermatozoa induce oocyte maturation. In animals with a feminized germline, maturation is inhibited and oocytes arrest in diakinesis. The introduction of sperm by mating restores maturation. (2) Sperm also directly promote sheath contraction. In animals with a feminized or tumorous germline, contractions are infrequent, whereas in animals with a masculinized germline or with sperm introduced by mating, contractions are frequent. (3 and 4) The maturing oocyte both induces spermathecal dilation and modulates sheath contractions at ovulation; dilation of the distal spermatheca and sharp increases in sheath contraction rates are only observed in the presence of a maturing oocyte.     

Caenorhabditis elegans inositol 5-phosphatase homolog negatively regulates inositol 1,4,5-triphosphate signaling in ovulation

Ovulation in Caenorhabditis elegans requires inositol 1,4,5-triphosphate (IP(3)) signaling activated by the epidermal growth factor (EGF)-receptor homolog LET-23. We generated a deletion mutant of a type I 5-phosphatase, ipp-5, and found a novel ovulation phenotype whereby the spermatheca hyperextends to engulf two oocytes per ovulation cycle. The temporal and spatial expression of IPP-5 is consistent with its proposed inhibition of IP(3) signaling in the adult spermatheca. ipp-5 acts downstream of let-23, and interacts with let-23-mediated IP(3) signaling pathway genes. We infer that IPP-5 negatively regulates IP(3) signaling to ensure proper spermathecal contraction.     

IRI-1, a LIN-15B homologue, interacts with inositol-1,4,5-triphosphate receptors and regulates gonadogenesis, defecation, and pharyngeal pumping in Caenorhabditis elegans

Inositol-1,4,5-triphosphate receptors (IP(3)Rs) are ligand-gated Ca(2+) channels that control Ca(2+) release from intracellular stores. They are central to a wide range of cellular responses. IP(3)Rs in Caenorhabditis elegans are encoded by a single gene, itr-1, and are widely expressed. Signaling through IP(3) and IP(3)Rs is important in ovulation, control of the defecation cycle, modulation of pharyngeal pumping rate, and embryogenesis. To further elucidate the molecular basis of the diversity of IP(3)R function, we used a yeast two-hybrid screen to search for proteins that interact with ITR-1. We identified an interaction between ITR-1 and IRI-1, a previously uncharacterized protein with homology to LIN-15B. Iri-1 is widely expressed, and its expression overlaps significantly with that of itr-1. In agreement with this observation, iri-1 functions in known itr-1-mediated processes, namely, upregulation of pharyngeal pumping in response to food and control of the defecation cycle. Knockdown of iri-1 in an itr-1 loss-of-function mutant potentiates some of these effects and sheds light on the signaling pathways that control pharyngeal pumping rate. Knockdown of iri-1 expression also results in a sterile, evl phenotype, as a consequence of failures in early Z1/Z4 lineage divisions, such that gonadogenesis is severely disrupted.     

Oscillatory Ca2+ signaling in the isolated Caenorhabditis elegans intestine: role of the inositol-1,4,5-trisphosphate receptor and phospholipases C beta and gamma

Defecation in the nematode Caenorhabditis elegans is a readily observable ultradian behavioral rhythm that occurs once every 45-50 s and is mediated in part by posterior body wall muscle contraction (pBoc). pBoc is not regulated by neural input but instead is likely controlled by rhythmic Ca(2+) oscillations in the intestinal epithelium. We developed an isolated nematode intestine preparation that allows combined physiological, genetic, and molecular characterization of oscillatory Ca(2+) signaling. Isolated intestines loaded with fluo-4 AM exhibit spontaneous rhythmic Ca(2+) oscillations with a period of approximately 50 s. Oscillations were only detected in the apical cell pole of the intestinal epithelium and occur as a posterior-to-anterior moving intercellular Ca(2+) wave. Loss-of-function mutations in the inositol-1,4,5-trisphosphate (IP(3)) receptor ITR-1 reduce pBoc and Ca(2+) oscillation frequency and intercellular Ca(2+) wave velocity. In contrast, gain-of-function mutations in the IP(3) binding and regulatory domains of ITR-1 have no effect on pBoc or Ca(2+) oscillation frequency but dramatically increase the speed of the intercellular Ca(2+) wave. Systemic RNA interference (RNAi) screening of the six C. elegans phospholipase C (PLC)-encoding genes demonstrated that pBoc and Ca(2+) oscillations require the combined function of PLC-gamma and PLC-beta homologues. Disruption of PLC-gamma and PLC-beta activity by mutation or RNAi induced arrhythmia in pBoc and intestinal Ca(2+) oscillations. The function of the two enzymes is additive. Epistasis analysis suggests that PLC-gamma functions primarily to generate IP(3) that controls ITR-1 activity. In contrast, IP(3) generated by PLC-beta appears to play little or no direct role in ITR-1 regulation. PLC-beta may function instead to control PIP(2) levels and/or G protein signaling events. Our findings provide new insights into intestinal cell Ca(2+) signaling mechanisms and establish C. elegans as a powerful model system for defining the gene networks and molecular mechanisms that underlie the generation and regulation of Ca(2+) oscillations and intercellular Ca(2+) waves in nonexcitable cells.     

Necrotic cell death in C. elegans requires the function of calreticulin and regulators of Ca(2+) release from the endoplasmic reticulum

In C. elegans, a hyperactivated MEC-4(d) ion channel induces necrotic-like neuronal death that is distinct from apoptosis. We report that null mutations in calreticulin suppress both mec-4(d)-induced cell death and the necrotic cell death induced by expression of a constitutively activated Galpha(S) subunit. RNAi-mediated knockdown of calnexin, mutations in the ER Ca(2+) release channels unc-68 (ryanodine receptor) or itr-1 (inositol 1,4,5 triphosphate receptor), and pharmacological manipulations that block ER Ca(2+) release also suppress death. Conversely, thapsigargin-induced ER Ca(2+) release can restore mec-4(d)-induced cell death when calreticulin is absent. We conclude that high [Ca(2+)](i) is a requirement for necrosis in C. elegans and suggest that an essential step in the death mechanism is release of ER-based Ca(2+) stores. ER-driven Ca(2+) release has previously been implicated in mammalian necrosis, suggesting necrotic death mechanisms may be conserved.     

CLH-3, a ClC-2 anion channel ortholog activated during meiotic maturation in C. elegans oocytes

BACKGROUND: ClC anion channels are ubiquitous and have been identified in organisms as diverse as bacteria and humans. Despite their widespread expression and likely physiological importance, the function and regulation of most ClCs are obscure. The nematode Caenorhabditis elegans offers significant experimental advantages for defining ClC biology. These advantages include a fully sequenced genome, cellular and molecular manipulability, and genetic tractability. RESULTS: We show by patch clamp electrophysiology that C. elegans oocytes express a hyperpolarization- and swelling-activated Cl(-) current with biophysical characteristics strongly resembling those of mammalian ClC-2. Double-stranded RNA-mediated gene interference (RNAi) and single-oocyte RT-PCR demonstrated that the channel is encoded by clh-3, one of six C. elegans ClC genes. CLH-3 is inactive in immature oocytes but can be triggered by cell swelling. However, CLH-3 plays no apparent role in oocyte volume homeostasis. The physiological signal for channel activation is the induction of oocyte meiotic maturation. During meiotic maturation, the contractile activity of gonadal sheath cells, which surround oocytes and are coupled to them via gap junctions, increases dramatically. These ovulatory sheath cell contractions are initiated prematurely in animals in which CLH-3 expression is disrupted by RNAi. CONCLUSIONS: The inwardly rectifying Cl(-) current in C. elegans oocytes is due to the activity of a ClC channel encoded by clh-3. Functional and structural similarities suggest that CLH-3 and mammalian ClC-2 are orthologs. CLH-3 is activated during oocyte meiotic maturation and functions in part to modulate ovulatory contractions of gap junction-coupled gonadal sheath cells.     

铁对血管收缩活动的影响及其机制

动脉粥样硬化的发生和铁引起的氧化应激密切相关。铁对血管的直接效应及其对血管收缩功能的影响尚不明确。本文采用血管环灌流装置 ,观察铁对离体SD大鼠去内皮胸主动脉环的直接效应 ,及对去内皮主动脉环KCl和苯肾上腺素 (PE)引发的收缩效应的影响。结果显示 :( 1) 10 0 μmol/L枸橼酸铁 (FAC)引起大鼠血管环发生相位性收缩 ,最大收缩幅度可达KCl诱发的最大收缩的 2 4 0 2± 2 3 7%。当 [Ca2 +]o 增加 1倍时 ,铁所致的血管环收缩幅度明显增加 (P <0 0 1)。阻断L 型钙通道后 ,铁所致的血管环收缩幅度明显降低 (P <0 0 1)。在无钙液中 ,用佛波酯收缩血管环 ,待收缩稳定后给予FAC ,此时收缩幅度增加 49 18± 3 75 %。 ( 2 )铁孵育 3 0min后 ,KCl引起血管环收缩的幅度显著降低 (P <0 0 1)。铁孵育可使PE引起的收缩量 -效曲线右移 (P <0 0 5 )。 ( 3 )二甲基亚砜、过氧化氢酶和谷胱甘肽可明显降低铁对PE血管收缩反应的抑制作用 (P <0 0 5 )。从这些结果可得到以下结论 :铁可引起胸主动脉发生相位性收缩 ,其机制可能与L 型钙通道短暂开放导致钙离子内流 ,及平滑肌对钙的敏感性增加有关 ;较长时间与铁孵育后 ,可对血管收缩功能产生损伤 ,氧自由基的生成增加和细胞内GSH的水平降低可能参与铁对收缩功能的     

Phospholipase Cepsilon regulates ovulation in Caenorhabditis elegans

Phospholipase Cepsilon (PLCepsilon) is a novel class of phosphoinositide-specific PLC with unknown physiological functions. Here, we present the first genetic analysis of PLCepsilon in an intact organism, the nematode Caenorhabditis elegans. Ovulation in C. elegans is dependent on an inositol 1,4,5-trisphosphate (IP(3)) signaling pathway activated by the receptor tyrosine kinase LET-23. We generated deletion mutants of the gene, plc-1, encoding C. elegans PLCepsilon. We observed a novel ovulation phenotype whereby oocytes are trapped in the spermatheca due to delayed dilation of the spermatheca-uterine valve. The expression of plc-1 in the adult spermatheca is consistent with its involvement in regulation of ovulation. On the other hand, we failed to observe genetic interaction of plc-1 with let-23-mediated IP(3) signaling pathway genes, suggesting a complex mechanism for control of ovulation.     

Linking integrin to IP(3) signaling is important for ovulation in Caenorhabditis elegans

Signals from germ and myoepithelial sheath cells initiate ovulation in Caenorhabditis elegans. The coordinated dilation and contraction of spermatheca lead to subsequent fertilization of oocyte. Either the dominant negative mutant pat-3 beta integrin or disruption of talin expression block ovulation. Talin loss-of-function uncovers roles in cell contractility and migration in C. elegans. Role of beta pat-3 integrins in development and function of Caenorhabditis elegans muscles and gonads, suggesting that the interaction between the cell and the extracellular matrix (ECM) is also important for ovulation. Here, we report that integrin plays an essential role in fertility via IP(3) signaling. Sterility caused by RNAi of pat-3 and ECM molecules was suppressed by increased IP(3) signaling. Our data suggest that the cell-ECM interaction controls ovulation via IP(3) signaling.     

The contraction of smooth muscle cells of intrapulmonary arterioles is determined by the frequency of Ca2+ oscillations induced by 5-HT and KCl

Increased resistance of the small blood vessels within the lungs is associated with pulmonary hypertension and results from a decrease in size induced by the contraction of their smooth muscle cells (SMCs). To study the mechanisms that regulate the contraction of intrapulmonary arteriole SMCs, the contractile and Ca(2+) responses of the arteriole SMCs to 5-hydroxytrypamine (5-HT) and KCl were observed with phase-contrast and scanning confocal microscopy in thin lung slices cut from mouse lungs stiffened with agarose and gelatin. 5-HT induced a concentration-dependent contraction of the arterioles. Increasing concentrations of extracellular KCl induced transient contractions in the SMCs and a reduction in the arteriole luminal size. 5-HT induced oscillations in [Ca(2+)](i) within the SMCs, and the frequency of these Ca(2+) oscillations was dependent on the agonist concentration and correlated with the extent of sustained arteriole contraction. By contrast, KCl induced Ca(2+) oscillations that occurred with low frequencies and were preceded by small, localized transient Ca(2+) events. The 5-HT-induced Ca(2+) oscillations and contractions occurred in the absence of extracellular Ca(2+) and were resistant to Ni(2+) and nifedipine but were abolished by caffeine. KCl-induced Ca(2+) oscillations and contractions were abolished by the absence of extracellular Ca(2+) and the presence of Ni(2+), nifedipine, and caffeine. Arteriole contraction was induced or abolished by a 5-HT(2)-specific agonist or antagonist, respectively. These results indicate that 5-HT, acting via 5-HT(2) receptors, induces arteriole contraction by initiating Ca(2+) oscillations and that KCl induces contraction via Ca(2+) transients resulting from the overfilling of internal Ca(2+) stores. We hypothesize that the magnitude of the sustained intrapulmonary SMC contraction is determined by the frequency of Ca(2+) oscillations and also by the relaxation rate of the SMC.     

Two distinct myosin II populations coordinate ovulatory contraction of the myoepithelial sheath in the Caenorhabditis elegans somatic gonad

The myoepithelial sheath in the somatic gonad of the nematode Caenorhabditis elegans has nonstriated contractile actomyosin networks that produce highly coordinated contractility for ovulation of mature oocytes. Two myosin heavy chains are expressed in the myoepithelial sheath, which are also expressed in the body-wall striated muscle. The troponin/tropomyosin system is also present and essential for ovulation. Therefore, although the myoepithelial sheath has smooth muscle–like contractile apparatuses, it has a striated muscle–like regulatory mechanism through troponin/tropomyosin. Here we report that the myoepithelial sheath has a distinct myosin population containing nonmuscle myosin II isoforms, which is regulated by phosphorylation and essential for ovulation. MLC-4, a nonmuscle myosin regulatory light chain, localizes to small punctate structures and does not colocalize with large, needle-like myosin filaments containing MYO-3, a striated-muscle myosin isoform. RNA interference of MLC-4, as well as of its upstream regulators, LET-502 (Rho-associated coiled-coil forming kinase) and MEL-11 (a myosin-binding subunit of myosin phosphatase), impairs ovulation. Expression of a phosphomimetic MLC-4 mutant mimicking a constitutively active state also impairs ovulation. A striated-muscle myosin (UNC-54) appears to provide partially compensatory contractility. Thus the results indicate that the two spatially distinct myosin II populations coordinately regulate ovulatory contraction of the myoepithelial sheath.     

库容性Ca2+内流参与ACh诱导的大鼠远端结肠平滑肌收缩

应用生物换能技术和Ca^2＋通道特异性阻断剂观察并记录大鼠离体远端结肠平滑肌收缩张力的变化,分析库容性Ca^2＋内流（capacitative Ca^2＋ entry,CCE）是否与ACh诱导的离体远端结肠平滑肌收缩反应有关。结果表明,以无钙的Krebs液灌流或应用EGTA螯合细胞外Ca^2＋后,高K^＋及ACh引起的远端结肠平滑肌收缩几乎完全消失。电压操纵性Ca^2＋通道阻断剂verapamil也能减弱高K^＋及ACh引起的远端结肠平滑肌收缩,其减弱的程度分别为74％和41％。在无钙的Krebs液中,5μmol／LACh可引起离体肠管瞬时性收缩,这是由肌质网（sarcoplasmic reticulum,SR）释放钙所致：然后加入10μmol／L阿托品（atropine）,并在此基础上恢复细胞外Ca^2＋（2．5mmol／L）,结肠平滑肌则出现持续性收缩,待收缩反应达峰值时,加入5μmol／L verapamil,收缩无明显变化,且该收缩反应对钙库操纵性通道（store-operated Ca^2＋ channel,socc）阻断剂La^3＋敏感,20,50和100μmol／L的La^3＋使上述收缩张力分别降低15％,23％和36％,且呈浓度依赖性,但对Cd^2＋不敏感。研究结果提示,细胞外Ca^2＋内流对高K^＋及ACh介导的离体远端结肠平滑肌持续性收缩是必需的,由ACh诱导的远端结肠平滑肌收缩至少包括SR释放钙引起的短暂性收缩及受体操纵性Ca^2＋通道（receptor-operated Ca^2＋ channel,ROCC）、电压操纵性Ca^2＋通道（voltage-operated Ca^2＋ channel,VOCC）和CCE介导的胞外Ca^2＋ 内流等途径。这将从通道水平进一步分析消化管平滑肌收缩的机制和特征,亦将为预防和控制因胃肠动力紊乱所致的消化管疾病寻求有针对性的药物干预和治疗提供理论依据。     

Store depletion by caffeine/ryanodine activates capacitative Ca(2+) entry in nonexcitable A549 cells

Capacitative Ca(2+) entry is essential for refilling intracellular Ca(2+) stores and is thought to be regulated primarily by inositol 1, 4,5-trisphosphate (IP(3))-sensitive stores in nonexcitable cells. In nonexcitable A549 cells, the application of caffeine or ryanodine induces Ca(2+) release in the absence of extracellular Ca(2+) similar to that induced by thapsigargin (Tg), and Ca(2+) entry occurs upon the readdition of extracellular Ca(2+). The channels thus activated are also permeable to Mn(2+). The channels responsible for this effect appear to be activated by the depletion of caffeine/ryanodine-sensitive stores per se, as evidenced by the activation even in the absence of increased intracellular Ca(2+) concentration. Tg pretreatment abrogates the response to caffeine/ryanodine, whereas Tg application subsequent to caffeine/ryanodine treatment induces further Ca(2+) release. The response to caffeine/ryanodine is also abolished by initial ATP application, whereas ATP added subsequent to caffeine/ryanodine induces additional Ca(2+) release. RT-PCR analyses showed the expression of a type 1 ryanodine receptor, two human homologues of transient receptor potential protein (hTrp1 and hTrp6), as well as all three types of the IP(3) receptor. These results suggest that in A549 cells, (i) capacitative Ca(2+) entry can also be regulated by caffeine/ryanodine-sensitive stores, and (ii) the RyR-gated stores interact functionally with those sensitive to IP(3), probably via Ca(2+)-induced Ca(2+) release.     

The Rho/Rac-family guanine nucleotide exchange factor VAV-1 regulates rhythmic behaviors in C. elegans

Rhythmic behaviors are a fundamental feature of all organisms. Pharyngeal pumping, the defecation cycle, and gonadal-sheath-cell contractions are three well-characterized rhythmic behaviors in the nematode C. elegans. The periodicities of the rhythms range from subsecond (pharynx) to seconds (gonadal sheath) to minutes (defecation). However, the molecular mechanisms underlying these rhythmic behaviors are not well understood. Here, we show that the C. elegans Rho/Rac-family guanine nucleotide exchange factor, VAV-1, which is homologous to the mammalian Vav proto-oncogene, has a crucial role in all three behaviors. vav-1 mutants die as larvae because VAV-1 function is required in the pharynx for synchronous contraction of the musculature. In addition, ovulation and the defecation cycle are abnormal and arrhythmic. We show that Rho/Rac-family GTPases and the signaling molecule inositol triphosphate (IP(3)) act downstream of VAV-1 signaling and that the VAV-1 pathway modulates rhythmic behaviors by dynamically regulating the concentration of intracellular Ca(2+).     

Regulated disruption of inositol 1,4,5-trisphosphate signaling in Caenorhabditis elegans reveals new functions in feeding and embryogenesis

Inositol 1,4,5-trisphosphate (IP(3)) is an important second messenger in animal cells and is central to a wide range of cellular responses. The major intracellular activity of IP(3) is to regulate release of Ca(2+) from intracellular stores through IP(3) receptors (IP(3)Rs). We describe a system for the transient disruption of IP(3) signaling in the model organism Caenorhabditis elegans. The IP(3) binding domain of the C. elegans IP(3)R, ITR-1, was expressed from heat shock-induced promoters in live animals. This results in a dominant-negative effect caused by the overexpressed IP(3) binding domain acting as an IP(3) "sponge." Disruption of IP(3) signaling resulted in disrupted defecation, a phenotype predicted by previous genetic studies. This approach also identified two new IP(3)-mediated processes. First, the up-regulation of pharyngeal pumping in response to food is dependent on IP(3) signaling. RNA-mediated interference studies and analysis of itr-1 mutants show that this process is also IP(3)R dependent. Second, the tissue-specific expression of the dominant-negative construct enabled us to circumvent the sterility associated with loss of IP(3) signaling through the IP(3)R and thus determine that IP(3)-mediated signaling is required for multiple steps in embryogenesis, including cytokinesis and gastrulation.     

cAMP inhibits IP(3)-dependent Ca(2+) release by preferential activation of cGMP-primed PKG

The singular effects and interplay of cAMP- and cGMP-dependent protein kinase (PKA and PKG) on Ca(2+) mobilization were examined in dispersed smooth muscle cells. In permeabilized muscle cells, exogenous cAMP and cGMP inhibited inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release and muscle contraction via PKA and PKG, respectively. A combination of cAMP and cGMP caused synergistic inhibition that was exclusively mediated by PKG and attenuated by PKA. In intact muscle cells, low concentrations (10 nM) of isoproterenol and sodium nitroprusside (SNP) inhibited agonist-induced, IP(3)-dependent Ca(2+) release and muscle contraction via PKA and PKG, respectively. A combination of isoproterenol and SNP increased PKA and PKG activities: the increase in PKA activity reflected inhibition of phosphodiesterase 3 activity by cGMP, whereas the increase in PKG activity reflected activation of cGMP-primed PKG by cAMP. Inhibition of Ca(2+) release and muscle contraction by the combination of isoproterenol and SNP was preferentially mediated by PKG. In light of studies showing that PKG phosphorylates the IP(3) receptor in intact and permeabilized muscle cells, whereas PKA phosphorylates the receptor in permeabilized cells only, the results imply that inhibition of IP(3)-induced Ca(2+) release is mediated exclusively by PKG. The effect of PKA on agonist-induced Ca(2+) release probably reflects inhibition of IP(3) formation.     

Impairment of Ca(2+) mobilization in circular muscle cells of the inflamed colon

This study investigated whether inflammation modulates the mobilization of Ca(2+) in canine colonic circular muscle cells. The contractile response of single cells from the inflamed colon was significantly suppressed in response to ACh, KCl, and BAY K8644. Methoxyverapamil and reduction in extracellular Ca(2+) concentration dose-dependently blocked the response in both normal and inflamed cells. The increase in intracellular Ca(2+) concentration in response to ACh and KCl was significantly reduced in the inflamed cells. However, Ca(2+) efflux from the ryanodine- and inositol 1,4, 5-trisphosphate (IP(3))-sensitive stores, as well as the decrease of cell length in response to ryanodine and IP(3), were not affected. Heparin significantly blocked Ca(2+) efflux and contraction in response to ACh in both conditions. ACh-stimulated accumulation of IP(3) and the binding of [(3)H]ryanodine to its receptors were not altered by inflammation. Ruthenium red partially inhibited the response to ACh in normal and inflamed states. We conclude that the canine colonic circular muscle cells utilize Ca(2+) influx through L-type channels as well as Ca(2+) release from the ryanodine- and IP(3)-sensitive stores to contract. Inflammation impairs Ca(2+) influx through L-type channels, but it may not affect intracellular Ca(2+) release. The impairment of Ca(2+) influx may contribute to the suppression of circular muscle contractility in the inflamed state.     

Tropomyosin and troponin are required for ovarian contraction in the Caenorhabditis elegans reproductive system

Ovulation in the nematode Caenorhabditis elegans is coordinated by interactions between the somatic gonad and germ cells. Myoepithelial sheath cells of the proximal ovary are smooth muscle-like cells, but the regulatory mechanism of their contraction is unknown. We show that contraction of the ovarian muscle requires tropomyosin and troponin, which are generally major actin-linked regulators of contraction of striated muscle. RNA interference of tropomyosin or troponin C caused sterility by inhibiting ovarian contraction that is required for expelling mature oocytes into the spermatheca where fertilization takes place, thus causing accumulation of endomitotic oocytes in the ovary. Tropomyosin and troponin C were associated with actin filaments in the myoepithelial sheath, and the association of troponin C with actin was dependent on tropomyosin. A mutation in the actin depolymerizing factor/cofilin gene suppressed the ovulation defects by RNA interference of tropomyosin or troponin C. These results strongly suggest that tropomyosin and troponin are the actin-linked regulators for contraction of ovarian muscle in the C. elegans reproductive system.