Multiple P2Y receptors couple to calcium-dependent,chloride channels in smooth muscle cells of the rat pulmonary artery

Background

Uridine 5''-triphosphate (UTP) and uridine 5''-diphosphate (UDP) act via P2Y receptors to evoke contraction of rat pulmonary arteries, whilst adenosine 5''-triphosphate (ATP) acts via P2X and P2Y receptors. Pharmacological characterisation of these receptors in intact arteries is complicated by release and extracellular metabolism of nucleotides, so the aim of this study was to characterise the P2Y receptors under conditions that minimise these problems.

Methods

The perforated-patch clamp technique was used to record the Ca²⁺-dependent, Cl^- current (I_Cl,Ca) activated by P2Y receptor agonists in acutely dissociated smooth muscle cells of rat small (SPA) and large (LPA) intrapulmonary arteries, held at -50 mV. Contractions to ATP were measured in isolated muscle rings. Data were compared by Student''s t test or one way ANOVA.

Results

ATP, UTP and UDP (10^-4M) evoked oscillating, inward currents (peak = 13–727 pA) in 71–93% of cells. The first current was usually the largest and in the SPA the response to ATP was significantly greater than those to UTP or UDP (P < 0.05). Subsequent currents tended to decrease in amplitude, with a variable time-course, to a level that was significantly smaller for ATP (P < 0.05), UTP (P < 0.001) and UDP (P < 0.05) in the SPA. The frequency of oscillations was similar for each agonist (mean≈6–11.min^-1) and changed little during agonist application. The non-selective P2 receptor antagonist suramin (10^-4M) abolished currents evoked by ATP in SPA (n = 4) and LPA (n = 4), but pyridoxalphosphate-6-azophenyl-2'',4''-disulphonic acid (PPADS) (10^-4M), also a non-selective P2 antagonist, had no effect (n = 4, 5 respectively). Currents elicited by UTP (n = 37) or UDP (n = 14) were unaffected by either antagonist. Contractions of SPA evoked by ATP were partially inhibited by PPADS (n = 4) and abolished by suramin (n = 5). Both antagonists abolished the contractions in LPA.

Conclusion

At least two P2Y subtypes couple to I_Cl,Ca in smooth muscle cells of rat SPA and LPA, with no apparent regional variation in their distribution. The suramin-sensitive, PPADS-resistant site activated by ATP most resembles the P2Y₁₁ receptor. However, the suramin- and PPADS-insensitive receptor activated by UTP and UDP does not correspond to any of the known P2Y subtypes. These receptors likely play a significant role in nucleotide-induced vasoconstriction.     

Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) Activity and Sinoatrial Nodal Pacemaker Cell Energetics

Ca²⁺-activated basal adenylate cyclase (AC) in rabbit sinoatrial node cells (SANC) guarantees, via basal cAMP/PKA-calmodulin/CaMKII-dependent protein phosphorylation, the occurrence of rhythmic, sarcoplasmic-reticulum generated, sub-membrane Ca²⁺ releases that prompt rhythmic, spontaneous action potentials (APs). This high-throughput signaling consumes ATP.

Aims

We have previously demonstrated that basal AC-cAMP/PKA signaling directly, and Ca²⁺ indirectly, regulate mitochondrial ATP production. While, clearly, Ca²⁺-calmodulin-CaMKII activity regulates ATP consumption, whether it has a role in the control of ATP production is unknown.

Methods and Results

We superfused single, isolated rabbit SANC at 37°C with physiological saline containing CaMKII inhibitors, (KN-93 or autocamtide-2 Related Inhibitory Peptide (AIP)), or a calmodulin inhibitor (W-7) and measured cytosolic Ca²⁺, flavoprotein fluorescence and spontaneous AP firing rate. We measured cAMP, ATP and O₂ consumption in cell suspensions. Graded reductions in basal CaMKII activity by KN-93 (0.5–3 µmol/L) or AIP (2–10 µmol/L) markedly slow the kinetics of intracellular Ca²⁺ cycling, decrease the spontaneous AP firing rate, decrease cAMP, and reduce O₂ consumption and flavoprotein fluorescence. In this context of graded reductions in ATP demand, however, ATP also becomes depleted, indicating reduced ATP production.

Conclusions

CaMKII signaling, a crucial element of normal automaticity in rabbit SANC, is also involved in SANC bioenergetics.     

Nicotinic receptors on rat alveolar macrophages dampen ATP-induced increase in cytosolic calcium concentration

Background

Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine.

Methods

Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca²⁺]_i) were conducted.

Results

Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, β1, and β2, with most stable expression being noted for subunits α9, α10, β1, and β2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca²⁺]_i revealed changes in membrane current in response to ACh and in [Ca²⁺]_i in response to nicotine, respectively. However, nicotine (100 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca²⁺]_i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium.

Conclusions

Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca²⁺-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential.     

Cyclic Nucleotide-Gated Channels Contribute to Thromboxane A2-Induced Contraction of Rat Small Mesenteric Arteries

Background

Thromboxane A₂ (TxA₂)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can be partly attributed to TxA₂-induced Ca²⁺ influx, which resulted in vascular contraction via Ca²⁺-calmodulin-MLCK pathway. This study aims to identify the channels that mediate TxA₂-induced Ca²⁺ influx in vascular smooth muscle cells.

Methodology/Principal Findings

Application of U-46619, a thromboxane A₂ mimic, resulted in a constriction in endothelium-denuded small mesenteric artery segments. The constriction relies on the presence of extracellular Ca²⁺, because removal of extracellular Ca²⁺ abolished the constriction. This constriction was partially inhibited by an L-type Ca²⁺ channel inhibitor nifedipine (0.5–1 µM). The remaining component was inhibited by L-cis-diltiazem, a selective inhibitor for CNG channels, in a dose-dependent manner. Another CNG channel blocker LY83583 [6-(phenylamino)-5,8-quinolinedione] had similar effect. In the primary cultured smooth muscle cells derived from rat aorta, application of U46619 (100 nM) induced a rise in cytosolic Ca²⁺ ([Ca²⁺]_i), which was inhibited by L-cis-diltiazem. Immunoblot experiments confirmed the presence of CNGA2 protein in vascular smooth muscle cells.

Conclusions/Significance

These data suggest a functional role of CNG channels in U-46619-induced Ca²⁺ influx and contraction of smooth muscle cells.     

P2X receptors regulate adenosine diphosphate release from hepatic cells

Extracellular nucleotides act as paracrine regulators of cellular signaling and metabolic pathways. Adenosine polyphosphate (adenosine triphosphate (ATP) and adenosine diphosphate (ADP)) release and metabolism by human hepatic carcinoma cells was therefore evaluated. Hepatic cells maintain static nanomolar concentrations of extracellular ATP and ADP levels until stress or nutrient deprivation stimulates a rapid burst of nucleotide release. Reducing the levels of media serum or glucose has no effect on ATP levels, but stimulates ADP release by up to 10-fold. Extracellular ADP is then metabolized or degraded and media ADP levels fall to basal levels within 2–4 h. Nucleotide release from hepatic cells is stimulated by the Ca²⁺ ionophore, ionomycin, and by the P2 receptor agonist, 2′3′-O-(4-benzoyl-benzoyl)-adenosine 5′-triphosphate (BzATP). Ionomycin (10 μM) has a minimal effect on ATP release, but doubles media ADP levels at 5 min. In contrast, BzATP (10–100 μM) increases both ATP and ADP levels by over 100-fold at 5 min. Ion channel purinergic receptor P2X7 and P2X4 gene silencing with small interference RNA (siRNA) and treatment with the P2X inhibitor, A438079 (100 μM), decrease ADP release from hepatic cells, but have no effect on ATP. P2X inhibitors and siRNA have no effect on BzATP-stimulated nucleotide release. ADP release from human hepatic carcinoma cells is therefore regulated by P2X receptors and intracellular Ca²⁺ levels. Extracellular ADP levels increase as a consequence of a cellular stress response resulting from serum or glucose deprivation.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9419-2) contains supplementary material, which is available to authorized users.     

Involvement of RhoA-mediated Ca2+ sensitization in antigen-induced bronchial smooth muscle hyperresponsiveness in mice

Background

It has recently been suggested that RhoA plays an important role in the enhancement of the Ca²⁺ sensitization of smooth muscle contraction. In the present study, a participation of RhoA-mediated Ca²⁺ sensitization in the augmented bronchial smooth muscle (BSM) contraction in a murine model of allergic asthma was examined.

Methods

Ovalbumin (OA)-sensitized BALB/c mice were repeatedly challenged with aerosolized OA and sacrificed 24 hours after the last antigen challenge. The contractility and RhoA protein expression of BSMs were measured by organ-bath technique and immunoblotting, respectively.

Results

Repeated OA challenge to sensitized mice caused a BSM hyperresponsiveness to acetylcholine (ACh), but not to high K⁺-depolarization. In α-toxin-permeabilized BSMs, ACh induced a Ca²⁺ sensitization of contraction, which is sensitive to Clostridium botulinum C3 exoenzyme, indicating that RhoA is implicated in this Ca²⁺ sensitization. Interestingly, the ACh-induced, RhoA-mediated Ca²⁺ sensitization was significantly augmented in permeabilized BSMs of OA-challenged mice. Moreover, protein expression of RhoA was significantly increased in the hyperresponsive BSMs.

Conclusion

These findings suggest that the augmentation of Ca²⁺ sensitizing effect, probably via an up-regulation of RhoA protein, might be involved in the enhanced BSM contraction in antigen-induced airway hyperresponsiveness.     

Cardiomyogenesis of embryonic stem cells upon purinergic receptor activation by ADP and ATP

Purinergic signaling may be involved in embryonic development of the heart. In the present study, the effects of purinergic receptor stimulation on cardiomyogenesis of mouse embryonic stem (ES) cells were investigated. ADP or ATP increased the number of cardiac clusters and cardiac cells, as well as beating frequency. Cardiac-specific genes showed enhanced expression of α-MHC, MLC2v, α-actinin, connexin 45 (Cx45), and HCN4, on both gene and protein levels upon ADP/ATP treatment, indicating increased cardiomyogenesis and pacemaker cell differentiation. Real-time RT-PCR analysis of purinergic receptor expression demonstrated presence of P2X1, P2X4, P2X6, P2X7, P2Y1, P2Y2, P2Y4, and P2Y6 on differentiating ES cells. ATP and ADP as well as the P2X agonists β,γ-methylenadenosine 5′-triphosphate (β,γ-MetATP) and 8-bromoadenosine 5′-triphosphate (8-Br-ATP) but not UTP or UDP transiently increased the intracellular calcium concentration ([Ca²⁺]_i) as evaluated by the calcium indicator Fluo-4, whereas no changes in membrane potential were observed. [Ca²⁺]_i transients induced by ADP/ATP were abolished by the phospholipase C-β (PLC-β) inhibitor U-73122, suggesting involvement of metabotropic P2Y receptors. Furthermore, partial inhibition of [Ca²⁺]_i transients was achieved in presence of MRS2179, a selective P2Y1 receptor antagonist, whereas PPADS, a non-selective P2 receptor inhibitor, completely abolished the [Ca²⁺]_i response. Consequently, cardiomyocyte differentiation was decreased upon long term co-incubation of cells with ADP and P2 receptor antagonists. In summary, activation of purinoceptors and the subsequent [Ca²⁺]_i transients enhance the differentiation of ES cells toward cardiomyocytes. Purinergic receptor stimulation may be a promising strategy to drive the fate of pluripotent ES cells into a particular population of cardiomyocytes.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-015-9468-1) contains supplementary material, which is available to authorized users.     

Breast Feeding Increases Vasoconstriction Induced by Electrical Field Stimulation in Rat Mesenteric Artery. Role of Neuronal Nitric Oxide and ATP

Objectives

The aim of this study was to investigate in rat mesenteric artery whether breast feeding (BF) affects the vasomotor response induced by electrical field stimulation (EFS), participation by different innervations in the EFS-induced response and the mechanism/s underlying these possible modifications.

Methods

Experiments were performed in female Sprague-Dawley rats (3 months old), divided into three groups: Control (in oestrous phase), mothers after 21 days of BF, and mothers that had recovered their oestral cycle (After BF, in oestrous phase). Vasomotor response to EFS, noradrenaline (NA) and nitric oxide (NO) donor DEA-NO were studied. Neuronal NO synthase (nNOS) and phosphorylated nNOS (P-nNOS) protein expression were analysed and NO, superoxide anion (O₂ ^.–), NA and ATP releases were also determined.

Results

EFS-induced contraction was higher in the BF group, and was recovered after BF. 1 µmol/L phentolamine decreased the response to EFS similarly in control and BF rats. NA vasoconstriction and release were similar in both experimental groups. ATP release was higher in segments from BF rats. 0.1 mmol/L L-NAME increased the response to EFS in both control and BF rats, but more so in control animals. BF decreased NO release and did not modify O₂ ^.– production. Vasodilator response to DEA-NO was similar in both groups, while nNOS and P-nNOS expressions were decreased in segments from BF animals.

Conclusion

Breast feeding increases EFS-induced contraction in mesenteric arteries, mainly through the decrease of neuronal NO release mediated by decreased nNOS and P-nNOS expression. Sympathetic function is increased through the increased ATP release in BF rats.     

Activation of the SPHK/S1P signalling pathway is coupled to muscarinic receptor-dependent regulation of peripheral airways

Background

In peripheral airways, acetylcholine induces contraction via activation of muscarinic M2-and M3-receptor subtypes (M₂R and M₃R). Cholinergic hypersensitivity is associated with chronic obstructive pulmonary disease and asthma, and therefore the identification of muscarinic signaling pathways are of great therapeutic interest. A pathway that has been shown to be activated via MR and to increase [Ca²⁺]_i includes the activation of sphingosine kinases (SPHK) and the generation of the bioactive sphingolipid sphingosine 1-phosphate (S1P). Whether the SPHK/S1P signaling pathway is integrated in the muscarinic control of peripheral airways is not known.

Methods

To address this issue, we studied precision cut lung slices derived from FVB and M₂R-KO and M₃R-KO mice.

Results

In peripheral airways of FVB, wild-type, and MR-deficient mice, SPHK1 was mainly localized to smooth muscle. Muscarine induced a constriction in all investigated mouse strains which was reduced by inhibition of SPHK using D, L-threo-dihydrosphingosine (DHS) and N, N-dimethyl-sphingosine (DMS) but not by N-acetylsphingosine (N-AcS), a structurally related agent that does not affect SPHK function. The initial phase of constriction was nearly absent in peripheral airways of M₃R-KO mice when SPHK was inhibited by DHS and DMS but was unaffected in M₂R-KO mice. Quantitative RT-PCR revealed that the disruption of the M₂R and M₃R genes had no significant effect on the expression levels of the SPHK1-isoform in peripheral airways.

Conclusion

These results demonstrate that the SPHK/S1P signaling pathway contributes to cholinergic constriction of murine peripheral airways. In addition, our data strongly suggest that SPHK is activated via the M₂R. Given the important role of muscarinic mechanisms in pulmonary disease, these findings should be of considerable therapeutic relevance.     

Amiodarone Inhibits Apamin-Sensitive Potassium Currents

Background

Apamin sensitive potassium current (I _KAS), carried by the type 2 small conductance Ca²⁺-activated potassium (SK2) channels, plays an important role in post-shock action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (VF) in failing ventricles.

Objective

To test the hypothesis that amiodarone inhibits I _KAS in human embryonic kidney 293 (HEK-293) cells.

Methods

We used the patch-clamp technique to study I _KAS in HEK-293 cells transiently expressing human SK2 before and after amiodarone administration.

Results

Amiodarone inhibited I_KAS in a dose-dependent manner (IC₅₀, 2.67±0.25 µM with 1 µM intrapipette Ca²⁺). Maximal inhibition was observed with 50 µM amiodarone which inhibited 85.6±3.1% of I_KAS induced with 1 µM intrapipette Ca²⁺ (n = 3). I_KAS inhibition by amiodarone was not voltage-dependent, but was Ca²⁺-dependent: 30 µM amiodarone inhibited 81.5±1.9% of I _KAS induced with 1 µM Ca²⁺ (n = 4), and 16.4±4.9% with 250 nM Ca²⁺ (n = 5). Desethylamiodarone, a major metabolite of amiodarone, also exerts voltage-independent but Ca²⁺ dependent inhibition of I _KAS.

Conclusion

Both amiodarone and desethylamiodarone inhibit I _KAS at therapeutic concentrations. The inhibition is independent of time and voltage, but is dependent on the intracellular Ca²⁺ concentration. SK2 current inhibition may in part underlie amiodarone''s effects in preventing electrical storm in failing ventricles.     

Cardiac subsarcolemmal and interfibrillar mitochondria display distinct responsiveness to protection by diazoxide

Objective

Cardiac subsarcolemmal (SSM) and interfibrillar (IFM) mitochondrial subpopulations possess distinct biochemical properties and differ with respect to their protein and lipid compositions, capacities for respiration and protein synthesis, and sensitivity to metabolic challenge, yet their responsiveness to mitochondrially active cardioprotective therapeutics has not been characterized. This study assessed the differential responsiveness of the two mitochondrial subpopulations to diazoxide, a cardioprotective agent targeting mitochondria.

Methods

Mitochondrial subpopulations were freshly isolated from rat ventricles and their morphologies assessed by electron microscopy and enzymatic activities determined using standard biochemical protocols with a plate reader. Oxidative phosphorylation was assessed from State 3 respiration using succinate as a substrate. Calcium dynamics and the status of Ca²⁺-dependent mitochondrial permeability transition (MPT) pore and mitochondrial membrane potential were assessed using standard Ca²⁺ and TPP⁺ ion-selective electrodes.

Results

Compared to IFM, isolated SSM exhibited a higher sensitivity to Ca²⁺ overload-mediated inhibition of adenosine triphosphate (ATP) synthesis with decreased ATP production (from 375±25 to 83±15 nmol ATP/min/mg protein in SSM, and from 875±39 to 583±45 nmol ATP/min/mg protein in IFM). In addition, SSM exhibited reduced Ca²⁺-accumulating capacity as compared to IFM (230±13 vs. 450±46 nmol Ca²⁺/mg protein in SSM and IFM, respectively), suggestive of increased Ca²⁺ sensitivity of MPT pore opening. Despite enhanced susceptibility to stress, SSM were more responsive to the protective effect of diazoxide (100 μM) against Ca²⁺ overload-mediated inhibition of ATP synthesis (67% vs. 2% in SSM and IFM, respectively).

Conclusion

These results provide evidence for the distinct sensitivity of cardiac SSM and IFM toward Ca²⁺-dependent metabolic stress and the protective effect of diazoxide on mitochondrial energetics.     

Hepatitis B Virus S Protein Enhances Sperm Apoptosis and Reduces Sperm Fertilizing Capacity In Vitro

Objective

Studying the impact of Hepatitis B virus S protein (HBs) on early apoptotic events in human spermatozoa and sperm fertilizing capacity.

Methodology/Principal Findings

Spermatozoa were exposed to HBs (0, 25, 50, 100 µg/ml) for 3 h, and then fluo-4 AM calcium assay, Calcein/Co²⁺ assay, protein extraction and ELISA, ADP/ATP ratio assay, sperm motility and hyperactivation and sperm-zona pellucida (ZP) binding and ZP-induced acrosome reaction (ZPIAR) tests were performed. The results showed that in the spermatozoa, with increasing concentration of HBs, (1) average cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) rose; (2) fluorescence intensity of Cal-AM declined; (3) average levels of cytochrome c decreased in mitochondrial fraction and increased in cytosolic fraction; (4) ADP/ATP ratios rose; (5) average rates of total motility and mean hyperactivation declined; (6) average rate of ZPIAR declined. In the above groups the effects of HBs exhibited dose dependency. However, there was no significant difference in the number of sperms bound to ZP between the control and all test groups.

Conclusion

HBs could induce early events in the apoptotic cascade in human spermatozoa, such as elevation of [Ca²⁺]_i, opening of mitochondrial permeability transition pore (MPTP), release of cytochrome c (cyt c) and increase of ADP/ATP ratio, but exerted a negative impact on sperm fertilizing capacity.     

Elevation of Extracellular Ca2+ Induces Store-Operated Calcium Entry via Calcium-Sensing Receptors: A Pathway Contributes to the Proliferation of Osteoblasts

Aims

The local concentration of extracellular Ca²⁺ ([Ca²⁺]_o) in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca²⁺]_o induced store-operated calcium entry (SOCE) in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca²⁺]_o to osteoblastic proliferation.

Methods

Cytosolic Ca²⁺ concentration ([Ca²⁺]_c) of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR) as well as inhibitors of phospholipase C (PLC), SOCE and voltage-gated calcium (Cav) channels were applied to study the mechanism in detail.

Results

Our data showed that elevating [Ca²⁺]_o evoked a sustained increase of [Ca²⁺]_c in a dose-dependent manner. This [Ca²⁺]_c increase was blocked by TMB-8 (Ca²⁺ release inhibitor), 2-APB and BTP-2 (both SOCE blockers), respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist) or {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (a PLC inhibitor) strongly reduced the [Ca²⁺]_o-induced [Ca²⁺]_c increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca²⁺]_o resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca²⁺]_o significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM (an intracellular calcium chelator), 2-APB, BTP-2, TMB-8, NPS2143 and {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, respectively, but not affected by Cav channels antagonists.

Conclusions

Elevating [Ca²⁺]_o induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca²⁺ concentration.     

Acid-sensing ion channel 3 decreases phosphorylation of extracellular signal-regulated kinases and induces synoviocyte cell death by increasing intracellular calcium

Introduction

Acid-sensing ion channel 3 (ASIC3) is expressed in synoviocytes, activated by decreases in pH, and reduces inflammation in animal models of inflammatory arthritis. The purpose of the current study was to characterize potential mechanisms underlying the control of inflammation by ASIC3 in fibroblast-like synoviocytes (FLS).

Methods

Experiments were performed in cultured FLS from wild-type (WT) and ASIC3-/- mice, ASIC1-/- mice, and people with rheumatoid arthritis. We assessed the effects of acidic pH with and without interleukin-1β on FLS and the role of ASICs in modulating intracellular calcium [Ca²⁺]_i, mitogen activated kinase (MAP kinase) expression, and cell death. [Ca²⁺]_i was assessed by fluorescent calcium imaging, MAP kinases were measured by Western Blots; ASIC, cytokine and protease mRNA expression were measured by quantitative PCR and cell death was measured with a LIVE/DEAD assay.

Results

Acidic pH increased [Ca²⁺]_i and decreased p-ERK expression in WT FLS; these effects were significantly smaller in ASIC3-/- FLS and were prevented by blockade of [Ca²⁺]_i. Blockade of protein phosphatase 2A (PP2A) prevented the pH-induced decreases in p-ERK. In WT FLS, IL-1β increases ASIC3 mRNA, and when combined with acidic pH enhances [Ca²⁺]_i, p-ERK, IL-6 and metalloprotienase mRNA, and cell death. Inhibitors of [Ca²⁺]_i and ERK prevented cell death induced by pH 6.0 in combination with IL-1β in WT FLS.

Conclusions

Decreased pH activates ASIC3 resulting in increased [Ca²⁺]_i, and decreased p-ERK. Under inflammatory conditions, acidic pH results in enhanced [Ca²⁺]_i and phosphorylation of extracellular signal-regulated kinase that leads to cell death. Thus, activation of ASIC3 on FLS by acidic pH from an inflamed joint could limit synovial proliferation resulting in reduced accumulation of inflammatory mediators and subsequent joint damage.     

Oxidative stress disruption of receptor-mediated calcium signaling mechanisms

Background

Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [³H]N-methylscopolamine ([³H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca²⁺]_i and [Ca²⁺]_L, respectively) were measured by fluorescent imaging.

Results

Activation of M3 muscarinic receptors induced a biphasic increase in [Ca²⁺]_i: an initial, inositol trisphosphate (IP3)-mediated release of Ca²⁺ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca²⁺ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca²⁺]_i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca²⁺]_i from 26 to up to 127 nM and decreased [Ca²⁺]_L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca²⁺ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP.

Conclusions

Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca²⁺]_i, [Ca²⁺]_L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.     

Differential Regulation of Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Signaling and Membrane Trafficking by Multiple P2 Receptors in Brown Adipocytes

Extracellular ATP triggers changes in intracellular Ca²⁺, ion channel function, and membrane trafficking in adipocytes. The aim of the present study was to determine which P2 receptors might mediate the Ca²⁺ signaling and membrane trafficking responses to ATP in brown fat cells. RT-PCR was used to determine which P2 receptors are expressed in brown fat cells. Responses to nucleotide agonists and antagonists were characterized using fura-2 fluorescence imaging of Ca²⁺ responses, and FM 1-43 fluorescence imaging and membrane capacitance measurements to assess membrane trafficking. The pharmacology of the Ca²⁺ responses fits the properties of the P2Y receptors for which mRNA is expressed, but the agonist and antagonist sensitivity of the membrane-trafficking response was not consistent with any P2 receptor described to date. Brown adipocytes expressed mRNA for P2Y₂, P2Y₆, and P2Y₁₂ metabotropic receptors and P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, and P2X₇ ionotropic receptors. The agonists ATP, ADP, UTP, UDP and 2′, 3′-(benzoylbenzoyl) ATP (BzATP) increased intracellular Ca²⁺, while 100 μM suramin, pyridoxal-phosphate-6-azophenyl-2′ 4′-disulfonic acid (PPADS), or Reactive Blue 2 partially blocked Ca²⁺ responses. ATP, but not ADP, UTP, UDP or BzATP activated membrane trafficking. The membrane response could be blocked completely with 1 μM PPADS but not by the antagonist MRS2179. We conclude that multiple P2 receptors mediate the ATP responses of brown fat cells, and that membrane trafficking is regulated by a P2 receptor showing unusual properties.     

Hyperpolization-activated Ca channels in guard cell plasma membrane are involved in extracellular ATP-promoted stomatal opening in Vicia faba

Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5′-O-(3-thio) triphosphate (ATPγS), 3′-O-(4-benzoyl) benzoyl adenosine 5′-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5′-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca²⁺ channel blockers GdCl₃ and LaCl₃. A hyperpolarization-activated Ca²⁺ channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca²⁺ channel significantly. Extracellular ATP-promoted Ca²⁺ channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca²⁺ channels.     

Airway smooth muscle relaxation results from a reduction in the frequency of Ca<Superscript>2+</Superscript> oscillations induced by a cAMP-mediated inhibition of the IP<Subscript>3</Subscript> receptor

Background

It has been shown that the contractile state of airway smooth muscle cells (SMCs) in response to agonists is determined by the frequency of Ca²⁺ oscillations occurring within the SMCs. Therefore, we hypothesized that the relaxation of airway SMCs induced by agents that increase cAMP results from the down-regulation or slowing of the frequency of the Ca²⁺ oscillations.

Methods

The effects of isoproterenol (ISO), forskolin (FSK) and 8-bromo-cAMP on the relaxation and Ca²⁺ signaling of airway SMCs contracted with methacholine (MCh) was investigated in murine lung slices with phase-contrast and laser scanning microscopy.

Results

All three cAMP-elevating agents simultaneously induced a reduction in the frequency of Ca²⁺ oscillations within the SMCs and the relaxation of contracted airways. The decrease in the Ca²⁺ oscillation frequency correlated with the extent of airway relaxation and was concentration-dependent. The mechanism by which cAMP reduced the frequency of the Ca²⁺ oscillations was investigated. Elevated cAMP did not affect the re-filling rate of the internal Ca²⁺ stores after emptying by repetitive exposure to 20 mM caffeine. Neither did elevated cAMP limit the Ca²⁺ available to stimulate contraction because an elevation of intracellular Ca²⁺ concentration induced by exposure to a Ca²⁺ ionophore (ionomycin) or by photolysis of caged-Ca²⁺ did not reverse the effect of cAMP. Similar results were obtained with iberiotoxin, a blocker of Ca²⁺-activated K⁺ channels, which would be expected to increase Ca²⁺ influx and contraction. By contrast, the photolysis of caged-IP₃ in the presence of agonist, to further elevate the intracellular IP₃ concentration, reversed the slowing of the frequency of the Ca²⁺ oscillations and relaxation of the airway induced by FSK. This result implied that the sensitivity of the IP₃R to IP₃ was reduced by FSK and this was supported by the reduced ability of IP₃ to release Ca²⁺ in SMCs in the presence of FSK.

Conclusion

These results indicate that the relaxant effect of cAMP-elevating agents on airway SMCs is achieved by decreasing the Ca²⁺ oscillation frequency by reducing internal Ca²⁺ release through IP₃ receptors.

     

The functional expression of extracellular calcium-sensing receptor in rat pulmonary artery smooth muscle cells

Background

The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown.

Methods

The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca²⁺]_i) was detected by a laser-scanning confocal microscope.

Results

The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca²⁺]_o (extracellular calcium concentration) or Gd³⁺ (an agonist of CaSR) induced an increase of [Ca²⁺]_i and PAs constriction in a concentration-dependent manner_. In addition, the above-mentioned effects of Ca²⁺ and Gd³⁺ were inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP₃ receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase).

Conclusions

CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca²⁺]_i through G-PLC-IP₃ pathway.