Agonist-induced Intracellular Ca2 Research Articles

Aging increases senescence, calcium signaling, and extracellular matrix deposition in human airway smooth muscle.

Lung function declines as people age and their lungs become stiffer. With an increasing elderly population, understanding mechanisms that contribute to these structural and functional changes in the aging lung is important. Part of the aging process is characterized by thicker, more fibrotic airways, and senile emphysema caused by changes in lung parenchyma. There is also senescence, which occurs throughout the body with aging. Here, using human airway smooth muscle (ASM) cells from patients in different age groups, we explored senescence pathways and changes in intracellular calcium signaling and extracellular matrix (ECM) deposition to elucidate potential mechanisms by which aging leads to thicker and stiffer lungs. Senescent markers p21, γH2AX, and β-gal, and some senescence-associated secretory proteins (SASP) increased with aging, as shown by staining and biochemical analyses. Agonist-induced intracellular Ca2+ responses, measured using fura-2 loaded cells and fluorescence imaging, increased with age. However, biochemical analysis showed that expression of the following markers decreased with age: M3 muscarinic receptor, TRPC3, Orai1, STIM1, SERCA2, MMP2 and MMP9. In contrast, collagen III, and fibronectin deposition increased with age. These data show that senescence increases in the aging airways that is associated with a stiffer but surprisingly greater intracellular calcium signaling as a marker for contractility. ASM senescence may enhance fibrosis in a feed forward loop promoting remodeling and altered calcium storage and buffering.

TRPP2 and STIM1 form a microdomain to regulate store-operated Ca2+ entry and blood vessel tone

BackgroundPolycystin-2 (TRPP2) is a Ca2+ permeable nonselective cationic channel essential for maintaining physiological function in live cells. Stromal interaction molecule 1 (STIM1) is an important Ca2+ sensor in store-operated Ca2+ entry (SOCE). Both TRPP2 and STIM1 are expressed in endoplasmic reticular membrane and participate in Ca2+ signaling, suggesting a physical interaction and functional synergism.MethodsWe performed co-localization, co-immunoprecipitation, and fluorescence resonance energy transfer assay to identify the interactions of TRPP2 and STIM1 in transfected HEK293 cells and native vascular smooth muscle cells (VSMCs). The function of the TRPP2-STIM1 complex in thapsigargin (TG) or adenosine triphosphate (ATP)-induced SOCE was explored using specific small interfering RNA (siRNA). Further, we created TRPP2 conditional knockout (CKO) mouse to investigate the functional role of TRPP2 in agonist-induced vessel contraction.ResultsTRPP2 and STIM1 form a complex in transfected HEK293 cells and native VSMCs. Genetic manipulations with TRPP2 siRNA, dominant negative TRPP2 or STIM1 siRNA significantly suppressed ATP and TG-induced intracellular Ca2+ release and SOCE in HEK293 cells. Inositol triphosphate receptor inhibitor 2-aminoethyl diphenylborinate (2APB) abolished ATP-induced Ca2+ release and SOCE in HEK293 cells. In addition, TRPP2 and STIM1 knockdown significantly inhibited ATP- and TG-induced STIM1 puncta formation and SOCE in VSMCs. Importantly, knockdown of TRPP2 and STIM1 or conditional knockout TRPP2 markedly suppressed agonist-induced mouse aorta contraction.ConclusionsOur data indicate that TRPP2 and STIM1 are physically associated and form a functional complex to regulate agonist-induced intracellular Ca2+ mobilization, SOCE and blood vessel tone.D6Kq4S1Z2iQsuoA7W_wV84Video abstract

Expression, Regulation, and Function of the Calmodulin Accessory Protein PCP4/PEP-19 in Myometrium.

Calmodulin (CaM) plays a key role in the orchestration of Ca2+ signaling events, and its regulation is considered an important component of cellular homeostasis. The control of uterine smooth muscle function is largely dependent on the regulation of Ca2+ and CaM signaling. The objective of this study was to investigate the expression, function, and regulation of CaM regulatory proteins in myometrium during pregnancy. Myometrium was obtained from nonpregnant women and 4 groups of pregnant women at the time their primary cesarean delivery: (i) preterm not in labor, (ii) preterm in labor with clinical and/or histological diagnosis of chorioamnionitis, (3) term not in labor; and (4) term in labor. The effect of perinatal inflammation on pcp4/pep-19 expression was evaluated in a mouse model of Ureaplasma parvum-induced chorioamnionitis. Human myometrial cells stably expressing wild-type and mutant forms of PCP4/PEP-19 were used in the evaluation of agonist-induced intracellular Ca2+ mobilization. Compared to other CaM regulatory proteins, PCP4/PEP-19 transcripts were more abundant in human myometrium. The expression of PCP4/PEP-19 was lowest in myometrium of women with preterm pregnancy and chorioamnionitis. In the mouse uterus, pcp4/pep-19 expression was lower in late compared to mid-gestation and decreased in mice injected intra-amniotic with Ureaplasma parvum. In myometrial smooth muscle cells, tumor necrosis factor alpha and progesterone decreased and PCP4/PEP-19 promoter activity increased. Finally, the overexpression of PCP4/PEP-19 reduced agonist-induced intracellular Ca2+ levels in myometrial cells. The decreased expression of PCP4/PEP-19 in myometrium contributes to a loss of quiescence in response to infection-induced inflammation at preterm pregnancy.

Abnormal Regulation of Intracellular Calcium in Human Megakaryocytes Contributes to the Pathophysiology of Calr-Mutant Myeloproliferative Neoplasms

The BCR-ABL-negative Myeloproliferative Neoplasms (MPNs), are a group of clonal hematopoietic malignancies, that consist of three disorders: Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). Approximately one quarter of patients with ET or PMF carry a somatic mutation of CALR, the gene encoding for the endoplasmic reticulum (ER) chaperone calreticulin. A 52-bp deletion (Type I) and a 5-bp insertion (Type II mutation) are the most frequent lesions. Both generate a new carboxy-terminus of the mutant proteins, in which the ER retention signal KDEL is lost, causing abnormal interaction of the mutants with the thrombopoietin (TPO) receptor (MPL), thus constitutively activating its downstream signaling in megakaryocytes. CALR Type I mutation causes important changes in the charge of the carboxyl-terminal tail, which is responsible for calcium (Ca2+) binding activity. As a consequence, megakaryocytes from CALR Type I patients present enhanced activation of Store-Operated Ca2+ Entry (SOCE), the mechanisms that trigger extracellular Ca2+ inflow upon agonist-induced intracellular Ca2+ mobilization from the ER. A fine control of SOCE and overall Ca2+ homeostasis is fundamental to ensure proper megakaryocyte function. Despite this knowledge, the role of a crosstalk among MPL, CALR and SOCE in regulating megakaryopoiesis has never been hypothesised.Our research analysed the activation of MPL downstream pathways and Ca2+ signalling in human cord blood and peripheral blood-cultured megakaryocytes, upon stimulation with recombinant human TPO (rhTPO). Additionally, we cultured megakaryocytes from the peripheral blood of patients carrying the CALR Type I mutation to investigate whether CALR and SOCE alterations determine abnormal activation of the MPL downstream pathway in MPNs.We demonstrated that binding of rhTPO to MPL induces a series of downstream signaling events that lead to the activation of STAT5, AKT and ERK1/2 in human megakaryocytes. This activation relies on significant accumulation of inositol triphosphate (IP3), and consequent intracellular Ca2+ release from the ER followed by extracellular Ca2+ entry, indicative of activated SOCE. Pharmacologic inhibition of the IP3 receptor or SOCE, with 2-APB or BTP-2 respectively, resulted in the complete absence of rhTPO-induced MPL signaling activation. SOCE activation is thus dependent on the dissociation of CALR and STIM1, a protein of the SOCE machinery, which normally forms a complex in un-stimulated megakaryocytes. Importantly, we verified that in human megakaryocytes in rhTPO-starved conditions, CALR forms a molecular complex with its co-chaperone, ERp57, and STIM1, while they dissociate upon rhTPO stimulation. Conversely, we observed that CALR, ERp57 and STIM1 are constitutively dissociated in megakaryocytes from CALR Type I patients, even in complete absence of rhTPO. This dissociation resulted in markedly increased TPO-induced cytosolic Ca2+ flows with a consequent abnormal megakaryocyte proliferation that could be counteracted by pharmacological inhibition of SOCE with BTP-2.Our findings provide the first evidence that CARL Type I, in addition to MPL activation, concurs to the alteration of SOCE, which sustains the phosphorylation of STAT5, AKT and ERK1/2, and induces megakaryocyte proliferation. Further investigation is needed to understand whether this altered megakaryocyte function is determined by the decrease in CALR levels or by the presence of CALR mutants in the ER. Nevertheless, the abnormal regulation of Ca2+ flows may represent a potentially actionable target. DisclosuresNo relevant conflicts of interest to declare.

Formaldehyde Induces Rho-Associated Kinase Activity to Evoke Airway Hyperresponsiveness.

Formaldehyde, a common indoor air pollutant, exacerbates asthma and synergizes with allergen to induce airway hyperresponsiveness (AHR) in animal models. The mechanisms mediating formaldehyde-induced AHR remain poorly understood. We posit that formaldehyde modulates agonist-induced contractile response of human airway smooth muscle (HASM) cells to elicit AHR. HASM cells were exposed to formaldehyde or vehicle and agonist-induced intracellular Ca2+ ([Ca2+]i) and myosin light-chain phosphatase (MYPT1) phosphorylation were determined. Air-liquid interface-differentiated human bronchial epithelial (HBE) cells were exposed to formaldehyde or vehicle and cocultured with HASM cells. Agonist-induced [Ca2+]i and MYPT1 phosphorylation were determined in the cocultured HASM cells. Precision-cut human lung slices were exposed to PBS or varying concentrations of formaldehyde, and then carbachol-induced airway narrowing was determined 24 hours after exposure. HASM cells were transfected with nontargeting or nuclear factor erythroid-derived 2, like 2 (Nrf-2)-targeting small interfering RNA and exposed to formaldehyde or vehicle, followed by determination of antioxidant response (quinone oxido-reductase 1 and thioredoxin 1) and basal and agonist-induced MYPT1 phosphorylation. Formaldehyde enhanced the basal Rho-kinase activity and MYPT1 phosphorylation with little effect on agonist-induced [Ca2+]i in HASM cells. Formaldehyde induced Nrf-2-dependent antioxidant response in HASM cells, although the MYPT1 phosphorylation was independent of Nrf-2 induction. Although HBE cells exposed to formaldehyde had little effect on agonist-induced [Ca2+]i or MYPT1 phosphorylation in cocultured HASM cells, formaldehyde enhanced carbachol-induced airway responsiveness in precision-cut human lung slices. In conclusion, formaldehyde induces phosphorylation of the regulatory subunit of MYPT1, independent of formaldehyde-induced Nrf-2 activation in HASM cells. The findings suggest that the Rho kinase-dependent Ca2+ sensitization pathway plays a role in formaldehyde-induced AHR.

The role of TRPP2 in agonist-induced gallbladder smooth muscle contraction.

TRPP2 channel protein belongs to the superfamily of transient receptor potential (TRP) channels and is widely expressed in various tissues, including smooth muscle in digestive gut. Accumulating evidence has demonstrated that TRPP2 can mediate Ca(2+) release from Ca(2+) stores. However, the functional role of TRPP2 in gallbladder smooth muscle contraction still remains unclear. In this study, we used Ca(2+) imaging and tension measurements to test agonist-induced intracellular Ca(2+) concentration increase and smooth muscle contraction of guinea pig gallbladder, respectively. When TRPP2 protein was knocked down in gallbladder muscle strips from guinea pig, carbachol (CCh)-evoked Ca(2+) release and extracellular Ca(2+) influx were reduced significantly, and gallbladder contractions induced by endothelin 1 and cholecystokinin were suppressed markedly as well. CCh-induced gallbladder contraction was markedly suppressed by pretreatment with U73122, which inhibits phospholipase C to terminate inositol 1,4,5-trisphosphate receptor (IP3) production, and 2-aminoethoxydiphenyl borate (2APB), which inhibits IP3 recepor (IP3R) to abolish IP3R-mediated Ca(2+) release. To confirm the role of Ca(2+) release in CCh-induced gallbladder contraction, we used thapsigargin (TG)-to deplete Ca(2+) stores via inhibiting sarco/endoplasmic reticulum Ca(2+)-ATPase and eliminate the role of store-operated Ca(2+) entry on the CCh-induced gallbladder contraction. Preincubation with 2 μmol L(-1) TG significantly decreased the CCh-induced gallbladder contraction. In addition, pretreatments with U73122, 2APB or TG abolished the difference of the CCh-induced gallbladder contraction between TRPP2 knockdown and control groups. We conclude that TRPP2 mediates Ca(2+) release from intracellular Ca(2+) stores, and has an essential role in agonist-induced gallbladder muscle contraction.

Mechanisms of Cigarette Smoke Effects on Human Airway Smooth Muscle.

Cigarette smoke contributes to or exacerbates airway diseases such as asthma and COPD, where airway hyperresponsiveness and airway smooth muscle (ASM) proliferation are key features. While factors such as inflammation contribute to asthma in part by enhancing agonist-induced intracellular Ca2+ ([Ca2+]i) responses of ASM, the mechanisms by which cigarette smoke affect ASM are still under investigation. In the present study, we tested the hypothesis that cigarette smoke enhances the expression and function of Ca2+ regulatory proteins leading to increased store operated Ca2+ entry (SOCE) and cell proliferation. Using isolated human ASM (hASM) cells, incubated in the presence and absence cigarette smoke extract (CSE) we determined ([Ca2+]i) responses and expression of relevant proteins as well as ASM proliferation, reactive oxidant species (ROS) and cytokine generation. CSE enhanced [Ca2+]i responses to agonist and SOCE: effects mediated by increased expression of TRPC3, CD38, STIM1, and/or Orai1, evident by attenuation of CSE effects when siRNAs against these proteins were used, particularly Orai1. CSE also increased hASM ROS generation and cytokine secretion. In addition, we found in the airways of patients with long-term smoking history, TRPC3 and CD38 expression were significantly increased compared to life-long never-smokers, supporting the role of these proteins in smoking effects. Finally, CSE enhanced hASM proliferation, an effect confirmed by upregulation of PCNA and Cyclin E. These results support a critical role for Ca2+ regulatory proteins and enhanced SOCE to alter airway structure and function in smoking-related airway disease.

The adaptor protein Swiprosin-1/EFhd2 is dispensable for platelet function in mice.

BackgroundPlatelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity, but may also cause pathological vessel occlusion. Reorganizations of the platelet cytoskeleton and agonist-induced intracellular Ca2+-mobilization are crucial for platelet hemostatic function. EF-hand domain containing 2 (EFhd2, Swiprosin-1) is a Ca2+-binding cytoskeletal adaptor protein involved in actin remodeling in different cell types, but its function in platelets is unknown.ObjectiveBased on the described functions of EFhd2 in immune cells, we tested the hypothesis that EFhd2 is a crucial adaptor protein for platelet function acting as a regulator of Ca2+-mobilization and cytoskeletal rearrangements.Methods and ResultsWe generated EFhd2-deficient mice and analyzed their platelets in vitro and in vivo. Efhd2-/- mice displayed normal platelet count and size, exhibited an unaltered in vivo life span and showed normal Ca2+-mobilization and activation/aggregation responses to classic agonists. Interestingly, upon stimulation of the immunoreceptor tyrosine-based activation motif-coupled receptor glycoprotein (GP) VI, Efhd2-/- platelets showed a slightly increased coagulant activity. Furthermore, absence of EFhd2 had no significant impact on integrin-mediated clot retraction, actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo EFhd2-deficiency resulted in unaltered hemostatic function and unaffected arterial thrombus formation.ConclusionThese results show that EFhd2 is not essential for platelet function in mice indicating that other cytoskeletal adaptors may functionally compensate its loss.

Dysfunction of Vascular Smooth Muscle and Vascular Remodeling by Simvastatin

Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, are widely prescribed for hypercholesterolemia. With the increasing use of statins, numerous reports demonstrated that statins can cause damage to skeletal muscles. However, the toxicities of statins on vascular smooth muscle, which are essential to cardiovascular homeostasis, have not been previously described. Here, we examined the effects of simvastatin on the contractile function and the integrity of vascular smooth muscle in isolated rat thoracic aortic rings, primary cultured vascular smooth muscle cells (VSMCs) in vitro and rats in vivo. In aortic rings, simvastatin suppressed the normal agonist-induced contractile responses in time- and concentration-dependent manners (0.86 g ± 0.11 at 10 μM simvastatin for 24 h compared with 1.89 g ± 0.11 at control). The suppression persisted in the endothelium-denuded aortic rings and was irreversible even after wash-out of simvastatin. Simvastatin suppressed the contraction induced by Bay K8644, an activator of voltage-operated Ca²⁺ channel (VOCC) in rat aortic rings and abolished agonist-induced intracellular Ca²⁺ increase in VSMCs. The simvastatin-induced contractile dysfunction was reversed by the supplementation of mevalonate and geranylgeranylpyrophosphate, precursors for protein isoprenylation. Consistently, activation of RhoA, a representative isoprenylated protein, was disrupted by simvastatin in VSMCs and RhoA-mediated phosphorylation of MYPT1 and CPI-17, and tonic tension were also suppressed. Notably, prolonged treatment of simvastatin up to 48 h induced apoptosis of vascular smooth muscle in aortic rings. Most importantly, simvastatin treatment in vivo significantly attenuated the agonist-induced vasoconstriction in rats ex vivo and induced a decrease in luminal area of the vascular wall. Collectively, these results demonstrate that simvastatin can impair the normal vascular contractility by disturbing Ca²⁺ influx and RhoA activity, ultimately leading to apoptosis and structural remodeling.

Inflammation, caveolae and CD38-mediated calcium regulation in human airway smooth muscle

The pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) increases expression of CD38 (a membrane-associated bifunctional enzyme regulating cyclic ADP ribose), and enhances agonist-induced intracellular Ca2+ ([Ca2+]i) responses in human airway smooth muscle (ASM). We previously demonstrated that caveolae and their constituent protein caveolin-1 are important for ASM [Ca2+]i regulation, which is further enhanced by TNFα. Whether caveolae and CD38 are functionally linked in mediating TNFα effects is unknown. In this regard, whether the related cavin proteins (cavin-1 and -3) that maintain structure and function of caveolae play a role is also not known. In the present study, we hypothesized that TNFα effects on CD38 expression and function in human ASM involve caveolae. Caveolar fractions from isolated human ASM cells expressed CD38 and its expression was upregulated by exposure to 20ng/ml TNFα (48h). ASM cells expressed cavin-1 and cavin-3, which were also upregulated by TNFα. Knockdown of caveolin-1, cavin-1 or cavin-3 (using siRNA) all significantly reduced CD38 expression and ADP-ribosyl cyclase activity in the presence or absence of TNFα. Furthermore, caveolin-1, cavin-1 and cavin-3 siRNAs reduced [Ca2+]i responses to histamine under control conditions, and blunted the enhanced [Ca2+]i responses in TNFα-exposed cells. These data demonstrate that CD38 is expressed within caveolae and its function is linked to the caveolar regulatory proteins caveolin-1, cavin-1 and -3. The link between caveolae and CD38 is further enhanced during airway inflammation demonstrating the important role of caveolae in regulation of [Ca2+]i and contractility in the airway.

P41 Hydrogen sulfide induces myosin light chain de-phosphorylation and smooth muscle relaxation in small intrapulmonary airways

Hydrogen sulfide (H 2 S) relaxes airway smooth muscle and it has been suggested to have a protective role during asthma and other obstructive lung diseases. However, the mechanism of H 2 S-induced airway relaxation is unknown. Smooth muscle cell (SMC) contraction depends on the phosphorylation state of the regulatory myosin light chain (rMLC) which is determined by the relative activities of the myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Changes in intracellular Ca 2+ concentration in SMCs regulate MLCK activity whereas Ca 2+ independent mechanisms regulate MLCP activity. In an accompanying abstract, we report that H 2 S inhibits intracellular Ca 2+ release through InsP 3 receptors and agonist-induced intracellular Ca 2+ oscillations in airway SMCs. Here we investigated the effect of H 2 S on rMLC phosphorylation in small intrapulmonary airways. We hypothesize that H 2 S-induced airway relaxation is accompanied by rMLC de-phosphorylation that results from a decrease in MLCK activity due to the inhibition of agonist-induced Ca 2+ oscillations. We used mouse lung slices in combination with phase-contrast- and confocal- video microscopy to measure contraction and Ca 2+ signaling in SMCs of small airways and used Western blot analysis to measure protein phosphorylation in airways micro-isolated from lung slices. Methacholine (MCh, 0.3 μM) induced strong and sustained airway contraction. This response was accompanied by the generation of intracellular Ca 2+ oscillations in airway SMCs that persisted in time and by a sustained increase in rMLC phosphorylation. Subsequent addition of 100 μM Na 2 S (a H 2 S donor) resulted in a strong and sustained airway relaxation. This relaxation was accompanied by a decrease in the frequency and amplitude of Ca 2+ oscillations and by rMLC de-phosphorylation. Washout of H 2 S in the continuous presence of MCh resulted in the increase in Ca 2+ oscillation frequency and amplitude, rMLC phosphorylation, and airway re-contraction. Our results suggest H 2 S induces relaxation of small intrapulmonary airways by inhibiting agonist-induced Ca 2+ oscillations which in turn induces rMLC de-phosphorylation in airway SMCs.

A Novel Function of Noc2 in Agonist-Induced Intracellular Ca2+ Increase during Zymogen-Granule Exocytosis in Pancreatic Acinar Cells

Noc2, a putative Rab effector, contributes to secretory-granule exocytosis in neuroendocrine and exocrine cells. Here, using two-photon excitation live-cell imaging, we investigated its role in Ca2+-dependent zymogen granule (ZG) exocytosis in pancreatic acinar cells from wild-type (WT) and Noc2-knockout (KO) mice. Imaging of a KO acinar cell revealed an expanded granular area, indicating ZG accumulation. In our spatiotemporal analysis of the ZG exocytosis induced by agonist (cholecystokinin or acetylcholine) stimulation, the location and rate of progress of ZG exocytosis did not differ significantly between the two strains. ZG exocytosis from KO acinar cells was seldom observed at physiological concentrations of agonists, but was normal (vs. WT) at high concentrations. Flash photolysis of a caged calcium compound confirmed the integrity of the fusion step of ZG exocytosis in KO acinar cells. The decreased ZG exocytosis present at physiological concentrations of agonists raised the possibility of impaired elicitation of calcium spikes. When calcium spikes were evoked in KO acinar cells by a high agonist concentration: (a) they always started at the apical portion and traveled to the basal portion, and (b) calcium oscillations over the 10 µM level were observed, as in WT acinar cells. At physiological concentrations of agonists, however, sufficient calcium spikes were not observed, suggesting an impaired [Ca2+]i-increase mechanism in KO acinar cells. We propose that in pancreatic acinar cells, Noc2 is not indispensable for the membrane fusion of ZG per se, but instead performs a novel function favoring agonist-induced physiological [Ca2+]i increases.

P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells

Extracellular ATP and ADP exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC), however the mechanisms of nucleotide-mediated angiogenesis remain not fully elucidated. This study was undertaken to delineate the purinergic receptor subtypes involved in extracellular nucleotide-mediated Ca2+ signaling and mitogenic responses in VVEC. Stimulation of VVEC with ATP resulted in the elevation of intracellular Ca2+ ([Ca2+]i) via Ca2+influx through plasma membrane channels as well as Ca2+ mobilization from intracellular stores; moreover, simultaneous Ca2+ responses were observed in both cytosolic and nuclear compartments. An increase in [Ca2+]i was observed in response to some P2, but not P1 purinergic receptor agonists. Using RT-PCR, we identified mRNA for the P2Y1,2,4,13,14, P2X2,5,7 and A1,2b,3 purinergic receptors in VVEC. Preincubation of VVEC with P2Y1 selective antagonist, MRS2179 and P2Y13 selective antagonist, MRS2211, as well as with Pertussis toxin, attenuated at varying degrees, agonist-induced intracellular Ca2+ responses and activation of ERK1/2, Akt and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a predominant role in VVEC growth responses. Together, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathologic vascular remodeling involving vasa vasorum expansion.

SERCA2a controls the mode of agonist-induced intracellular Ca 2+ signal, transcription factor NFAT and proliferation in human vascular smooth muscle cells

In blood vessels, tone is maintained by agonist-induced cytosolic Ca 2+ oscillations of quiescent/contractile vascular smooth muscle cells (VSMCs). However, in synthetic/proliferative VSMCs, Gq/phosphoinositide receptor-coupled agonists trigger a steady-state increase in cytosolic Ca 2+ followed by a Store Operated Calcium Entry (SOCE) which translates into activation of the proliferation-associated transcription factor NFAT. Here, we report that in human coronary artery smooth muscle cells (hCASMCs), the sarco/endoplasmic reticulum calcium ATPase type 2a (SERCA2a) expressed in the contractile form of the hCASMCs, controls the nature of the agonist-induced Ca 2+ transient and the resulting down-stream signaling pathway. Indeed, restoring SERCA2a expression by gene transfer in synthetic hCASMCs 1) increased Ca 2+ storage capacity; 2) modified agonist-induced IP 3R Ca 2+ release from steady-state to oscillatory mode (the frequency of agonist-induced IP 3R Ca 2+ signal was 11.66 ± 1.40/100 s in SERCA2a-expressing cells (n = 39) vs 1.37 ± 0.20/100 s in control cells (n = 45), p < 0.01); 3) suppressed SOCE by preventing interactions between SR calcium sensor STIM1 and pore forming unit ORAI1; 4) inhibited calcium regulated transcription factor NFAT and its down-stream physiological function such as proliferation and migration. This study provides evidence for the first time that oscillatory and steady-state patterns of Ca 2+ transients have different effects on calcium-dependent physiological functions in smooth muscle cells.

P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells

Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca(2+) signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of [Ca(2+)](i) via Ca(2+) influx through plasma membrane channels as well as Ca(2+) mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca(2+) responses in both cytosolic and nuclear compartments. An increase in [Ca(2+)](i) was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPβS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αβMeATP, and βγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca(2+) responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion.

Polycystin-2 Activation by Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Requires Its Direct Association with the Inositol 1,4,5-Trisphosphate Receptor in a Signaling Microdomain

Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca(2+) signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP(3)R), an intracellular Ca(2+) channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP(3)R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2(-/-) mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca(2+) release in intact cells and IP(3)-induced Ca(2+) release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca(2+) signal by a local Ca(2+)-induced Ca(2+)-release mechanism, which only occurred in the presence of the TRPP2-IP(3)R interaction, and not via altered IP(3)R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca(2+) microdomains necessary for initiating Ca(2+)-induced Ca(2+) release. The data strongly suggest that defects in this mechanism may account for the altered Ca(2+) signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.

Regulation of store-operated Ca2+entry by CD38 in human airway smooth muscle

The ectoenzyme CD38 catalyzes synthesis and degradation of cyclic ADP ribose in airway smooth muscle (ASM). The proinflammatory cytokine TNFalpha, which enhances agonist-induced intracellular Ca(2+) ([Ca(2+)](i)) responses, has been previously shown to increases CD38 expression. In the present study, we tested the hypothesis that the effects of TNFalpha on CD38 expression vs. changes in [Ca(2+)](i) regulation in ASM cells are linked. Using isolated human ASM cells, CD38 expression was either increased (transfection) or knocked down [small interfering RNA (siRNA)], and [Ca(2+)](i) responses to sarcoplasmic reticulum depletion [i.e., store-operated Ca(2+) entry (SOCE)] were evaluated in the presence vs. absence of TNFalpha. Results confirmed that TNFalpha significantly increased CD38 expression and ADP-ribosyl cyclase activity, an effect inhibited by CD38 siRNA, but unaltered by CD38 overexpression. CD38 suppression blunted, whereas overexpression enhanced, ACh-induced [Ca(2+)](i) responses. TNFalpha-induced enhancement of [Ca(2+)](i) response to agonist was blunted by CD38 suppression, but enhanced by CD38 overexpression. Finally, TNFalpha-induced increase in SOCE was blunted by CD38 siRNA and potentiated by CD38 overexpression. Overall, these results indicate a critical role for CD38 in TNFalpha-induced enhancement of [Ca(2+)](i) in human ASM cells, and potentially to TNFalpha augmentation of airway responsiveness.

Memantine Potentiates Agonist-Induced Ca2+ Responses in HEK 293 Cells

Background/Aims: The Alzheimer drug memantine (1-amino-3,5-dimethyl-adamantane) blocks the pore channel of the NMDA receptor. Since memantine also blocks the 5-HT₃ receptor, neuronal nicotinic receptor, and voltage-activated Na⁺ channels, the purpose of our study was to verify whether memantine could influence other types of channels involved in the regulation of Ca²⁺. Methods: Free intracellular Ca²⁺ concentrations in whole cells and in saponin-permeabilized cells were monitored spectrofluorometrically in HEK-293 cells stably expressing TRPC6. Results: Memantine decreased the basal level of intracellular Ca²⁺, increased the content of the intracellular Ca²⁺ store, which in turn increased the agonist-induced intracellular Ca²⁺ release, and increased the store-operated Ca²⁺ entry. Conclusion: In addition to blocking the NMDA receptor, memantine also decreases the basal level of intracellular Ca²⁺ and increases the sensitivity of cells to extracellular stimuli. All these effects may be of benefit in the treatment of Alzheimer''s disease.

Propofol Inhibits Phosphorylation of N -methyl-d-aspartate Receptor NR1 Subunits in Neurons

Anesthetics may interact with ionotropic glutamate receptors to produce some of their biologic actions. Cellular studies reveal that the ionotropic glutamate receptors, N-methyl-D-aspartate receptors (NMDARs), can be phosphorylated on their NR1 subunits at the C-terminal serine residues, which is a major mechanism for the regulation of NMDAR functions. It is currently unknown whether anesthetics have any modulatory effects on NMDAR NR1 subunit phosphorylation. The possible effect of a general anesthetic propofol on phosphorylation of NR1 subunits at serine 897 (pNR1S897) and 896 (pNR1S896) was detected in cultured rat cortical neurons. Propofol consistently reduced basal levels of pNR1S897 and pNR1S896 in a concentration-dependent manner. This reduction was rapid as the reliable reduction of pNR1S896 developed 1 min after propofol administration. Pretreatment of cultures with the protein phosphatase 2A inhibitors okadaic acid or calyculin A blocked the effect of propofol on the NR1 phosphorylation, whereas okadaic acid or calyculin A alone did not alter basal pNR1S897 and pNR1S896 levels. In addition, propofol decreased tyrosine phosphorylation of protein phosphatase 2A at tyrosine 307, resulting in an increase in protein phosphatase 2A activity. In the presence of propofol, the NMDAR agonist-induced intracellular Ca2+ increase was impaired in neurons with dephosphorylated NR1 subunits. Together, these data indicate an inhibitory effect of a general anesthetic propofol on NMDAR NR1 subunit phosphorylation in neurons. This inhibition was mediated through a signaling mechanism involving activation of protein phosphatase 2A.

Role of Transient Receptor Potential C3 in TNF-α–Enhanced Calcium Influx in Human Airway Myocytes

Previous studies have suggested that the proinflammatory cytokine, TNF-alpha, contributes to airway hyperresponsivness by altering airway smooth muscle (ASM) Ca(2+) responses to agonist stimulation. The present study examined the effects of TNF-alpha on Ca(2+) influx pathways in cultured human ASM cells (HASMCs). Proteins encoded by the transient receptor potential (TRP) gene family function as channels through which receptor-operated and store-operated Ca(2+) entry (SOCE) occur. In the present study, the presence of TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 mRNA and protein expression was confirmed in cultured HASMCs using RT-PCR and Western blot analysis. TNF-alpha treatment significantly increased TRPC3 mRNA and protein levels in HASMCs as well as SOCE. TNF-alpha treatment also increased both the peak and plateau intracellular Ca(2+) concentration responses in HASMCs elicited by acetylcholine and bradykinin. The effects of TNF-alpha treatment on SOCE and agonist-induced intracellular Ca(2+) concentration responses were attenuated using small interfering RNA transfection, which knocked down TRPC3 expression. Thus, in inflammatory airway diseases, TNF-alpha treatment may result in increased myocyte activation due to altered Ca(2+) influx pathways. These results suggest that TRPC3 may be an important therapeutic target in inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease.