Canine Pulmonary Arterial Smooth Muscle Research Articles

Caffeine inhibits InsP 3 responses and capacitative calcium entry in canine pulmonary arterial smooth muscle cells

Caffeine is a well described and characterized ryanodine receptor (RyR) activator. Previous evidence from independent research studies also indicate caffeine inhibits InsP 3 receptor functionality, which is important to activation of capacitative Ca 2+ entry (CCE) in some cell types. In addition, RyR activation elicits excitatory-coupled Ca 2+ entry (ECCE) in skeletal muscle myotubes. Recent studies by our group show that canine pulmonary arterial smooth muscle cells (PASMCs) have functional InsP 3 receptors as well as RyRs, and that CCE is dependent on InsP 3 receptor activity. The potential for caffeine to activate ECCE as well as inhibit InsP 3 receptor function and CCE was examined using fura-2 fluorescent imaging in canine PASMCs. The data show caffeine causes transient as well as sustained cytosolic Ca 2+ increases, though this is not due to CCE or ECCE activity as evidenced by a lack of an increase in Mn 2+ quench of fura-2. The experiments also show caffeine reversibly inhibits 5-HT elicited—InsP 3 mediated Ca 2+ responses with an IC 50 of 6.87 × 10 − 4 M and 10 mM caffeine fully inhibits CCE. These studies provide the first evidence that caffeine is an inhibitor of InsP 3 generated Ca 2+ signals and CCE in PASMCs.

Cell culture alters Ca2+ entry pathways activated by store-depletion or hypoxia in canine pulmonary arterial smooth muscle cells

Previous studies have shown that, in acutely dispersed canine pulmonary artery smooth muscle cells (PASMCs), depletion of both functionally independent inositol 1,4,5-trisphosphate (IP(3))- and ryanodine-sensitive Ca(2+) stores activates capacitative Ca(2+) entry (CCE). The present study aimed to determine if cell culture modifies intracellular Ca(2+) stores and alters Ca(2+) entry pathways caused by store depletion and hypoxia in canine PASMCs. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured in fura 2-loaded cells. Mn(2+) quench of fura 2 signal was performed to study divalent cation entry, and the effects of hypoxia were examined under oxygen tension of 15-18 mmHg. In acutely isolated PASMCs, depletion of IP(3)-sensitive Ca(2+) stores with cyclopiazonic acid (CPA) did not affect initial caffeine-induced intracellular Ca(2+) transients but abolished 5-HT-induced Ca(2+) transients. In contrast, CPA significantly reduced caffeine- and 5-HT-induced Ca(2+) transients in cultured PASMCs. In cultured PASMCs, store depletion or hypoxia caused a transient followed by a sustained rise in [Ca(2+)](i). The transient rise in [Ca(2+)](i) was partially inhibited by nifedipine, whereas the nifedipine-insensitive transient rise in [Ca(2+)](i) was inhibited by KB-R7943, a selective inhibitor of reverse mode Na(+)/Ca(2+) exchanger (NCX). The nifedipine-insensitive sustained rise in [Ca(2+)](i) was inhibited by SKF-96365, Ni(2+), La(3+), and Gd(3+). In addition, store depletion or hypoxia increased the rate of Mn(2+) quench of fura 2 fluorescence that was also inhibited by these blockers, exhibiting pharmacological properties characteristic of CCE. We conclude that cell culture of canine PASMCs reorganizes IP(3) and ryanodine receptors into a common intracellular Ca(2+) compartment, and depletion of this store or hypoxia activates voltage-operated Ca(2+) entry, reverse mode NCX, and CCE.

Role of InsP3 and ryanodine receptors in the activation of capacitative Ca2+ entry by store depletion or hypoxia in canine pulmonary arterial smooth muscle cells

Experiments were performed to determine if capacitative Ca(2+) entry (CCE) in canine pulmonary arterial smooth muscle cells (PASMCs) is dependent on InsP(3) receptors or ryanodine receptors as induction of CCE is dependent on simultaneous depletion of the functionally separate InsP(3)- and ryanodine-sensitive sarcoplasmic reticulum (SR) Ca(2+) stores in these cells. Myocytes were isolated from canine pulmonary arteries using enzymatic procedures and were used within 8 h of preparation. Measurements of cytosolic Ca(2+) were made by imaging fura-2 loaded individual myocytes that were perfused with physiological buffered saline solution with or without Ca(2+). Treating myocytes with 10 microM cyclopiazonic acid (CPA), removing extracellular Ca(2+), and briefly applying 10 mM caffeine and 10 microM 5-hydroxytryptamine (5-HT) depleted SR Ca(2+) stores. Extracellular Ca(2+) reintroduction caused cytosolic [Ca(2+)] to elevate above baseline signifying CCE. The InsP(3) receptor inhibitors 2-aminobiphenylborate (50-75 microM; 2-APB) and xestospongin-C (20 microM; XeC) abolished CCE. Yet, CCE was unaffected by 10 microM or 300 microM ryanodine or 10 microM dantrolene, which modify ryanodine receptor activity. Higher dantrolene concentrations (50 microM), however, can inhibit both ryanodine receptors and InsP(3) receptors, did reduce CCE. In contrast, CCE activated by hypoxia was unaffected by XeC (20 microM). The results provide evidence that CCE activated by depletion of both InsP(3) and ryanodine SR Ca(2+) stores in canine PASMCs is dependent on functional InsP(3) receptors, whereas the activation of CCE by hypoxia appears to be independent of functional InsP(3) receptors.

Inhibition of Ryanodine Receptors by 4-(2-Aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365) in Canine Pulmonary Arterial Smooth Muscle Cells

Ryanodine is a selective ryanodine receptor (RyR) blocker, with binding dependent on RyR opening. In whole-cell studies, ryanodine binding can lock the RyR in an open-conductance state, short-circuiting the sarcoplasmic reticulum, which restricts studies of inositol-1,4,5-trisphosphate receptor (InsP3R) activity. Other RyR blockers have nonselective effects that also limit their utility. 4-(2-aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365) blocks RyR-elicited Ca2+ increases in skeletal and cardiac muscle; yet, its actions on smooth muscle are unknown. Canine pulmonary arterial smooth muscle cells (PASMCs) express both RyRs and InsP3Rs; thus, we tested the ability of FLA 365 to block RyR- and serotonin-mediated InsP3R-elicited Ca2+ release by imaging fura-2-loaded PASMCs. Acute exposure to 10 mM caffeine, a selective RyR activator, induced Ca2+ increases that were reversibly reduced by FLA 365, with an estimated IC50 of approximately 1 to 1.5 microM, and inhibited by 10 microM ryanodine or 10 microM cyclopiazonic acid. FLA 365 also blocked L-type Ca2+ channel activity, with 10 microM reducing Ba2+ current amplitude in patch voltage-clamp studies to 54 +/- 6% of control and 100 microM FLA 365 reducing membrane current to 21 +/- 6%. InsP3R-mediated Ca2+ responses elicited by 10 microM 5-hydroxytryptamine (serotonin) in canine PASMCs and 100 microM carbachol in human embryonic kidney (HEK)-293 cells were not reduced by 2 microM FLA 365, but they were reduced by 20 microM FLA 365 to 76 +/- 9% of control in canine PASMCs and 52 +/- 1% in HEK-293 cells. Thus, FLA 365 preferentially blocks RyRs with limited inhibition of L-type Ca2+ channels or InsP3R in canine PASMCs.

Reorganization of Intracellular Ca 2+ Stores Caused by Cell Culture Does Not Prevent Hypoxic‐Induced Rise in [Ca 2+ ] i in Canine Pulmonary Arterial Smooth Muscle Cells

We previously showed in acutely isolated canine pulmonary artery smooth muscle cells (PASMCs) that the IP3-sensitive and ryanodine-sensitive Ca2+ stores are functionally independent and hypoxia causes rise in [Ca2+]i through mobilization of the intracellular Ca2+ stores leads to consequent activation of voltage–operated Ca2+ entry and capacitative Ca2+ entry. The aim of the present study was to determine if cell culture changes organization of intracellular Ca2+ stores and whether the change could affect hypoxic-induced rise in [Ca2+]i in canine PASMCs. In acutely isolated PASMCs loaded with fura-2, CPA abolished 5HT-induced intracellular Ca2+ transient but had no effect on the initial caffeine-induced intracellular Ca2+ transient. In contrast, CPA significantly reduced 5HT- and caffeine-induced intracellular Ca2+ transient in cultured PASMCs. In 2mM Ca2+-solution, hypoxia caused a rise in [Ca2+]i but it was abolished when extracellular Ca2+ was removed. Subsequent addition of 2mM Ca2+ elicited a transient rise in [Ca2+]i that was partially inhibited by 10μM nifedipine, leaving a nifedipine-insensitive transient and nifedipine-insensitive sustained rise in [Ca2+]i. These results suggest that cell culture of PASMCs results in reorganization of IP3-sensitive and ryanodine-sensitive stores and this does not prevent hypoxic-induced rise in [Ca2+]i. (Supported by HL49254 and NCRR P20RR15581)

FLA 365 as an inhibitor of ryanodine receptors and L‐type Ca 2+ channels in arterial myocytes

Ryanodine (RY) is a highly selective ryanodine receptor (RyR) blocker, but RY binding is dependent on RyR opening. In whole-cell studies, RY binding can lock the RyR in an open-conductance state, short-circuiting the sarcoplasmic reticulum (SR). This restricts studies of InsP3 receptor (InsP3R) activity. Other RyR blockers also have non-selective effects that also limit their utility. FLA 365 ([2,6-dichloro-4-dimethyl-aminophenyl] isopropylamine) blocks RyR elicited Ca2+ increases in skeletal and cardiac muscle, yet its actions on smooth muscle are unknown. Canine pulmonary arterial smooth muscle cells (PASMCs) express both RyRs and InsP3Rs; thus, we tested the ability of FLA 365 (FLA) to block RyR and InsP3R elicited Ca2+ release by imaging fura-2 loaded PASMCs. Acute exposure to 10 mM caffeine, a selective RyR activator, induced Ca2+ increases that were significantly reduced by 20 μM FLA, which was reversible. 10–100 μM FLA 365 reduced Ba2+ currents through L-type Ca2+ channels in patch voltage-clamp studies, similar to phenylalkylamines. 10 μM 5-HT, which activates InsP3Rs, induced Ca2+ increases of equivalent amplitude in the absence or presence of FLA. Thus, FLA blocks RyRs and L-type Ca2+ channels with limited impact on InsP3R signaling in PASMCs. NIH P20RR15518 from NCRR & HL49254 (JRH), PO1ES11269 & 2PO AR17605 (INP), HL10476, AI55462 & UM faculty fellowship (SMW).

NO responsiveness in pulmonary artery and airway smooth muscle: the role of cGMP regulation

The purpose of this study was to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Canine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used to perform concentration response studies to an NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM exhibited a greater NO responsiveness whether PASM and TSM were contracted with receptor agonists, phenylephrine and acetylcholine, respectively, or with KCl. The >10-fold difference in NO sensitivity in PASM was observed with both submaximal and maximal contractions. This difference in NO responsiveness was not due to differences in endothelial or epithelial barriers, since these were removed, nor was it due to the presence of cGMP-independent NO-mediated relaxation in either tissue. At equal concentrations of NO, the intracellular cGMP concentration ([cGMP]i) was also greater in PASM than in TSM. Phosphodiesterase (PDE) inhibition using isobutylmethylxanthine indicated that the greater [cGMP]i in PASM was not due to greater PDE activity in TSM. Expression of soluble guanylate cyclase (sGC) subunit mRNA (2 +/- 0.2 and 1.3 +/- 0.2 attomol/microg total RNA, respectively) and protein (47.4 +/- 2 and 27.8 +/- 3.9 ng/mg soluble homogenate protein, respectively) was greater in PASM than in TSM. sGCalpha1 and sGCbeta1 mRNA expression was equal in PASM but was significantly different in TSM, suggesting independent regulation of their expression. An intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is greater sGC activity.

Mobilization of sarcoplasmic reticulum stores by hypoxia leads to consequent activation of capacitative Ca2+ entry in isolated canine pulmonary arterial smooth muscle cells

Capacitative Ca2+ entry (CCE) has been speculated to contribute to Ca2+ influx during hypoxic pulmonary vasoconstriction (HPV). The aim of the present study was to directly test if acute hypoxia causes intracellular Ca2+ concentration ([Ca2+]i) rises through CCE in canine pulmonary artery smooth muscle cells (PASMCs). In PASMCs loaded with fura-2, hypoxia produced a transient rise in [Ca2+]i in Ca2+-free solution, indicating Ca2+ release from the intracellular Ca2+ stores. Subsequent addition of 2 mm Ca2+ in hypoxia elicited a sustained rise in [Ca2+]i, which was partially inhibited by 10 microm nisoldipine. The dihydropyridine-insensitive rise in [Ca2+]i was due to increased Ca2+ influx, because it was abolished in Ca2+-free solution and hypoxia was shown to significantly enhance the rate of Mn2+ quench of fura-2 fluorescence. The dihyropyridine-insensitive rise in [Ca2+]i and the increased rate of Mn2+ quench of fura-2 fluorescence were inhibited by 50 microm SKF 96365 and 500 microm Ni2+, but not by 100 microm La3+ or 100 microm Gd3+, exhibiting pharmacological properties characteristic of CCE. In addition, predepletion of the intracellular Ca2+ stores inhibited the rise in [Ca2+]i induced by hypoxia. These results provide the first direct evidence that acute hypoxia, by causing Ca2+ release from the intracellular stores, activates CCE in isolated canine PASMCs, which may contribute to HPV.

Hypotonic activation of volume-sensitive outwardly rectifying chloride channels in cultured PASMCs is modulated by SGK.

The serum- and glucocorticoid-inducible kinase (SGK) is a serine/threonine protein kinase (PK) transcriptionally regulated by corticoids, serum, and cell volume. SGK regulates cell volume of various cells by effects on Na(+) and K(+) transport through membrane channels. We hypothesized a role for SGK in the activation of volume-sensitive osmolyte and anion channels (VSOACs) in cultured canine pulmonary artery smooth muscle cells (PASMCs). Intracellular dialysis through the patch electrode of recombinant active SGK, but not kinase-dead Delta60-SGK-K127M, heat-inactivated SGK, or active Akt1, partially activated VSOACs under isotonic conditions. Dialysis of active SGK before cell exposure to hypotonic medium significantly accelerated the activation kinetics and increased the maximal density of VSOAC current. Exposure of PASMCs to hypotonic medium (230 mosM) activated phosphatidylinositol 3-kinases (PI3Ks) and their downstream targets Akt/PKB and SGK but not PKC-epsilon. Inhibition of PI3Ks with wortmannin reduced the activation rate and maximal amplitude of VSOACs. Immunoprecipitated ClC-3 channels were phosphorylated by PKC-epsilon but not by SGK in vitro, suggesting that SGK may activate VSOACs indirectly. These data indicate that the PI3K-SGK cascade is activated on hypotonic swelling of PASMCs and, in turn, affects downstream signaling molecules linked to activation of VSOACs.

The Effect of Nickel and Manganese on Store-Operated Channels in Canine Pulmonary Arterial Smooth Muscle Cells

Background: Pulmonary vasoconstriction greatly contibutes to the elevated pulmonary vascular resistance in patients with pulmonary hypertension. A rise in cytosolic concentration in pulmonary arterial smooth muscle cells (PASMCs) is a major trigger for pulmonary vasoconstriction. Presently, little is known about the nature of the store-operated channels (SOCs) in PASMCs, even though store depletion-mediated capacitative entry (CCE) is a critical mechanism for refilling the empty sarcoplasmic reticulum (SR) with , and for maintaining a sustained increase in cyt. The goal of this in vitro study was to investigate the effects of nickel and manganese on store-operated channels in canine PASMCs. Methods: Isolated PASMCs were obtained from an enzymatically treated canine pulmonary artery. Currents were recorded at room temperature using the dialyzed whole cell recording technique. The protocol used to deplete the intracellular stores and to monitor the development of the store-operated currents, involved voltage-clamping cells at 0 mV to inactivate any voltage-dependent calcium currents. Crrents were recorded in response to a 200 ms voltage step from 120 to 40 mV in 20 mV increments every 15 seconds. Results: Simultaneous depletion of intracellular leads to a voltage-independent and linear store-operated current () reversal near 0 mV. Nickel and manganese inhibit . Conclusions: In canine PASMCs, the depletion of intracellular stores leads to the activation of Isoc, which is inhibited by nickel and manganese. These metals are non-specific inhibitors of non-selective cation channels. Our results indicate that - and -sensitive pathways may mediate entry, or that a class of non-selective cation channels may contribute to CCE in canine PASMCs.

Functional effects of novel anti-ClC-3 antibodies on native volume-sensitive osmolyte and anion channels in cardiac and smooth muscle cells.

Whether ClC-3 encodes volume-sensitive organic osmolyte and anion channels (VSOACs) remains controversial. We have shown previously that native VSOACs in some cardiac and vascular myocytes were blocked by a commercial anti-ClC-3 carboxy terminal antibody (Alm C592-661 antibody), although recent studies have raised questions related to the specificity of Alm C592-661 antibody. Therefore, we have developed three new anti-ClC-3 antibodies and investigated their functional effects on native VSOACs in freshly isolated canine pulmonary artery smooth muscle cells (PASMCs) and guinea pig cardiac myocytes. These new antibodies produced a common prominent immunoreactive band with an apparent molecular mass of 90-92 kDa in the guinea pig heart and PASMCs, and a similar molecular mass immunoreactive band was observed in the brain from homozygous Clcn3+/+ mice but not from homozygous Clcn3-/- mice. VSOACs elicited by hypotonic cell swelling in PASMCs and guinea pig atrial myocytes were nearly completely abolished by intracellular dialysis with two new anti-ClC-3 antibodies specifically targeting the ClC-3 carboxy (C670-687 antibody) and amino terminus (A1-14 antibody). This inhibition of native VSOACs can be attributed to a specific interaction with endogenous ClC-3, because 1) preabsorption of the antibodies with corresponding antigens prevented the inhibitory effects, 2) extracellular application of a new antibody raised against an extracellular epitope (Ex133-148) of ClC-3 failed to inhibit native VSOACs in PASMCs, 3) intracellular dialysis with an antibody targeting Kv1.1 potassium channels failed to inhibit native VSOACs in guinea pig atrial myocytes, and 4) anti-ClC-3 C670-687 antibody had no effects on swelling-induced augmentation of the slow component of the delayed rectifying potassium current in guinea pig ventricular myocytes, although VSOACs in the same cells were inhibited by the antibody. These results confirm that endogenous ClC-3 is an essential molecular entity responsible for native VSOACs in PASMCs and guinea pig cardiac myocytes.

The Effect of Nicardipine on Capacitative Calcium Entry in Canine Pulmonary Arterial Smooth Muscle Cells

Background: Capacitive calcium entry involves the influx of across the sarcolemma in response to the depletion of intracellular stores. Presently, little is known about the nature of the intracellular store (s) in pulmonary arterial smooth muscle cells (PASMCs), even though the unique contractile response of this tissue to hypoxia may at least partially involve the intracellular release of . The authors aimed to investigate the effects of nicardipine on capacitative calcium entry. Methods: Isolated pulmonary smooth muscle cells were obtained from enzymatically treated canine pulmonary artery. Currents were recorded at room temperature using the dialyzed whole cell recording technique. The protocol used to deplete intracellular stores and to monitor the development of the store-operated currents, involved cells being were voltage-clamped at 0 mv to inactivate any voltage-dependent calcium currents, which were recorded in response to a 200 ms voltage step from 120 to 40 mV in 20 mV increments. Results: Simultaneous depletion of intracellular leads to linear store-operated current (ISOC) reversal near 0 mV. Nicardipine does not affect ISOC. Conclusions: In canine PASMCs, the depletion of intracellular stores leads to the activation of ISOC, which is not inhibited by nicardipine, a voltage-dependent channel (VDCC) blocker, indicating that VDCC blocked by nicardipine does not contribute to CCE in canine PASMCs.

Regulation of volume-sensitive outwardly rectifying anion channels in pulmonary arterial smooth muscle cells by PKC.

We tested the possible role of endogenous protein kinase C (PKC) in the regulation of native volume-sensitive organic osmolyte and anion channels (VSOACs) in acutely dispersed canine pulmonary artery smooth muscle cells (PASMC). Hypotonic cell swelling activated native volume-regulated Cl(-) currents (I(Cl.vol)) which could be reversed by exposure to phorbol 12,13-dibutyrate (0.1 microM) or by hypertonic cell shrinkage. Under isotonic conditions, calphostin C (0.1 microM) or Ro-31-8425 (0.1 microM), inhibitors of both conventional and novel PKC isozymes, significantly activated I(Cl.vol) and prevented further modulation by subsequent hypotonic cell swelling. Bisindolylmaleimide (0.1 microM), a selective conventional PKC inhibitor, was without effect. Dialyzing acutely dispersed and cultured PASMC with epsilon V1-2 (10 microM), a translocation inhibitory peptide derived from the V1 region of epsilon PKC, activated I(Cl.vol) under isotonic conditions and prevented further modulation by cell volume changes. Dialyzing PASMC with beta C2-2 (10 microM), a translocation inhibitory peptide derived from the C2 region of beta PKC, had no detectable effect. Immunohistochemistry in cultured canine PASMC verified that hypotonic cell swelling is accompanied by translocation of epsilon PKC from the vicinity of the membrane to cytoplasmic and perinuclear locations. These data suggest that membrane-bound epsilon PKC controls the activation state of native VSOACs in canine PASMC under isotonic and anisotonic conditions.

Modulatory role of ERK MAPK-caldesmon pathway in PDGF-stimulated migration of cultured pulmonary artery SMCs.

Extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) phosphorylate caldesmon in vivo, but the function of caldesmon phosphorylation in smooth muscle physiology is controversial. We hypothesized that ERK MAPKs and caldesmon modulate chemotactic migration of cultured canine pulmonary artery smooth muscle cells (PASMCs). Platelet-derived growth factor (PDGF; 10 ng/ml) and endothelin-1 (ET-1; 100 nM) transiently activated ERK MAPKs: PDGF produced higher maximal and more potent activation of ERK MAPKs over 5 h. While both PDGF and ET-1 increased caldesmon phosphorylation, only PDGF stimulated migration of cultured cells (13 times over basal migration). At concentrations from 0.01 to 10 nM, ET-1 failed to enhance migration; 100 nM ET-1 produced only a slight increase (1.31 +/- 0.18 times basal migration). ET-1 (100 nM) did not potentiate migration triggered by 0.5 or 3 ng/ml PDGF. The MEK1 inhibitor PD-98059 (50 microM) abolished the PDGF-stimulated phosphorylation of ERK MAPKs and caldesmon and reduced cell migration by 50%. We conclude that while ERK MAPK activity is not required to initiate migration, an ERK MAPK-caldesmon pathway may modulate later events necessary for PDGF-stimulated migration of cultured PASMCs.

Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody.

Intracellular dialysis of NIH/3T3 cells with a commercially available anti-ClC-3 polyclonal antibody (Ab) for approximately 30 min completely inhibited expressed guinea-pig ClC-3 currents (IgpClC-3), while intracellular dialysis with antigen-preabsorbed anti-ClC-3 Ab failed to affect IgpClC-3. Anti-ClC-3 Ab was used as a selective probe to examine the relationship between endogenous ClC-3 expression and native volume-sensitive outwardly rectifying anion channels (VSOACs) in guinea-pig cardiac cells, canine pulmonary arterial smooth muscle cells (PASMCs) and Xenopus laevis oocytes. Intracellular dialysis or injection of anti-ClC-3 Ab abolished native VSOAC function in cardiac cells and PASMCs and significantly reduced VSOACs in oocytes. In contrast, native VSOAC function was unaltered by antigen-preabsorbed anti-ClC-3 Ab. It is suggested that endogenous ClC-3 represents a major molecular entity responsible for native VSOACs in cardiac and smooth muscle cells and Xenopus oocytes. Anti-ClC-3 Ab should be a useful experimental tool to directly test the relationship between endogenous ClC-3 expression and native VSOAC function, and help resolve existing controversies related to the regulation and physiological role of native VSOACs in a wide variety of different cells.

Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells.

To examine the nature of inositol 1,4,5-trisphosphate (IP(3))-sensitive and ryanodine (Ryn)-sensitive Ca(2+) stores in isolated canine pulmonary arterial smooth cells (PASMC), agonist-induced changes in global intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured using fura 2-AM fluorescence. Properties of elementary local Ca(2+) release events were characterized using fluo 3-AM or fluo 4-AM, in combination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca(2+) stores with Ryn (300 microM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-induced intracellular Ca(2+) transients but had little or no effect on the initial IP(3)-mediated intracellular Ca(2+) transient induced by ANG II (1 microM). Cyclopiazonic acid (CPA; 10 microM) abolished IP(3)-induced intracellular Ca(2+) transients but failed to attenuate the initial Caf-induced intracellular Ca(2+) transient. These results suggest that in canine PASMC, IP(3)-, and Ryn-sensitive Ca(2+) stores are organized into spatially distinct compartments while similar experiments in canine renal arterial smooth muscle cells (RASMC) reveal that these Ca(2+) stores are spatially conjoined. In PASMC, spontaneous local intracellular Ca(2+) transients sensitive to modulation by Caf and Ryn were detected, exhibiting spatial-temporal characteristics similar to those previously described for "Ca(2+) sparks" in cardiac and other types of smooth muscle cells. After depletion of Ryn-sensitive Ca(2+) stores, ANG II (8 nM) induced slow, sustained [Ca(2+)](i) increases originating at sites near the cell surface, which were abolished by depleting IP(3) stores. Discrete quantal-like events expected due to the coordinated opening of IP(3) receptor clusters ("Ca(2+) puffs") were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca(2+) stores and elementary Ca(2+) release events in isolated PASMC.

K+channel inhibition, calcium signaling, and vasomotor tone in canine pulmonary artery smooth muscle

We investigated the role of K(+) channels in the regulation of baseline intracellular free Ca(2+) concentration ([Ca(2+)](i)), alpha-adrenoreceptor-mediated Ca(2+) signaling, and capacitative Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs). Inhibition of voltage-gated K(+) channels with 4-aminopyridine (4-AP) increased the membrane potential and the resting [Ca(2+)](i) but attenuated the amplitude and frequency of the [Ca(2+)](i) oscillations induced by the alpha-agonist phenylephrine (PE). Inhibition of Ca(2+)-activated K(+) channels (with charybdotoxin) and inhibition (with glibenclamide) or activation of ATP-sensitive K(+) channels (with lemakalim) had no effect on resting [Ca(2+)](i) or PE-induced [Ca(2+)](i) oscillations. Thapsigargin was used to deplete sarcoplasmic reticulum Ca(2+) stores in the absence of extracellular Ca(2+). Under these conditions, 4-AP attenuated the peak and sustained components of capacitative Ca(2+) entry, which was observed when extracellular Ca(2+) was restored. Capacitative Ca(2+) entry was unaffected by charybdotoxin, glibenclamide, or lemakalim. In isolated pulmonary arterial rings, 4-AP increased resting tension and caused a leftward shift in the KCl dose-response curve. In contrast, 4-AP decreased PE-induced contraction, causing a rightward shift in the PE dose-response curve. These results indicate that voltage-gated K(+) channel inhibition increases resting [Ca(2+)](i) and tone in PASMCs but attenuates the response to PE, likely via inhibition of capacitative Ca(2+) entry.

Capacitative Ca(2+) entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells.

We investigated the role of capacitative Ca(2+) entry and tyrosine kinase activation in mediating phenylephrine (PE)-induced oscillations in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in canine pulmonary arterial smooth muscle cells (PASMCs). [Ca(2+)](i) was measured as the 340- to 380-nm ratio in individual fura 2-loaded PASMCs. Resting [Ca(2+)](i) was 96 +/- 4 nmol/l. PE (10 micromol/l) stimulated oscillations in [Ca(2+)](i), with a peak amplitude of 437 +/- 22 nmol/l and a frequency of 1.01 +/- 0.12/min. Thapsigargin (1 micromol/l) was used to deplete sarcoplasmic reticulum (SR) Ca(2+) after extracellular Ca(2+) was removed. Under these conditions, a nifedipine-insensitive, sustained increase in [Ca(2+)](i) (140 +/- 7% of control value) was observed when the extracellular Ca(2+) concentration was restored; i.e., capacitative Ca(2+) entry was demonstrated. Capacitative Ca(2+) entry also refilled SR Ca(2+) stores. Capacitative Ca(2+) entry was attenuated (32 +/- 3% of control value) by 50 micromol/l of SKF-96365 (a nonselective Ca(2+)-channel inhibitor). Tyrosine kinase inhibition with tyrphostin 23 (100 micromol/l) and genistein (100 micromol/l) also inhibited capacitative Ca(2+) entry to 63 +/- 12 and 85 +/- 4% of control values, respectively. SKF-96365 (30 micromol/l) attenuated both the amplitude (15 +/- 7% of control value) and frequency (50 +/- 21% of control value) of PE-induced Ca(2+) oscillations. SKF-96365 (50 micromol/l) abolished the oscillations. Tyrphostin 23 (100 micromol/l) also inhibited the amplitude (17 +/- 7% of control value) and frequency (45 +/- 9% of control value) of the oscillations. Genistein (30 micromol/l) had similar effects. Both SKF-96365 and tyrphostin 23 attenuated PE-induced contraction in isolated pulmonary arterial rings. These results demonstrate that capacitative Ca(2+) entry is present and capable of refilling SR Ca(2+) stores in canine PASMCs and may be involved in regulating PE-induced Ca(2+) oscillations. A tyrosine kinase is involved in the signal transduction pathway for alpha(1)-adrenoreceptor activation in PASMCs.

Atypical adrenoceptor-mediated relaxation of canine pulmonary artery through a cyclic adenosine monophosphate-dependent pathway.

To determine whether functional atypical beta-adrenoceptors (beta(3)-adrenoceptors) are present in pulmonary vascular smooth muscle, we studied isolated canine pulmonary arterial rings under isometric conditions in vitro. Addition of beta-adrenoceptor agonists produced a concentration-dependent relaxation of noradrenaline-precontracted tissues, a rank order potency being isoproterenol (1) > salbutamol (0.95) > selective beta(3)-adrenoceptor agonists, CL 316243 (0.85), and BRL 37344 (0. 83). A marked desensitization to salbutamol occurred by pretreatment with salbutamol but not with CL 316243. When beta(1)-adrenoceptors had been blocked, the relaxant responses to salbutamol were competitively antagonized by the beta(2)-adrenoceptor antagonist ICI 118551 with a pA(2) value of 7.67 +/- 0.21 (mean +/- S.E.), but the response to CL 316243 was weekly antagonized by ICI 118551 only at a high concentration of 10(-5) M, where an apparent pA(2) value was 5. 24. In contrast, cyanopindolol, a nonselective beta-adrenoceptor antagonist, antagonized CL 316243-induced relaxation in a competitive manner with a pA(2) of 6.10 +/- 0.11. This pA(2) value was lower than that when salbutamol was used as an agonist (6.69 +/- 0.14, p < 0.01). Intracellular 3',5'-cyclic adenosine monophosphate (cAMP) levels were increased by CL 316243 in a concentration-dependent fashion, an effect that was not altered by ICI 118551. These results suggest that beta(3)-adrenoceptors may exist in canine pulmonary artery smooth muscle and that stimulation of this atypical receptor causes vasodilation through a cAMP-dependent pathway.

Atypical Adrenoceptor-Mediated Relaxation of Canine Pulmonary Artery through a cAMP-Dependent Pathway

We studied the existence of β3-adrenoceptors in canine pulmonary artery smooth muscle under isometric conditionsin vitro.A rank order potency of vascular relaxation was isoproterenol > salbutamol > selective β3-adrenoceptor agonists, CL 316243 and BRL 37344. A marked desensitization to salbutamol occurred by pretreatment with salbutamol but not with CL 316243. When β1-adrenoceptors were blocked, the relaxant responses to salbutamol were competitively antagonized by the β2-adrenoceptor antagonist ICI 118551, whereas the response to CL 316243 was not. Cyanopindolol, a non-selective β-adrenoceptor antagonist, antagonized CL 316243-induced relaxation in a competitive manner with apA2of 6.10, and this value was lower than that when salbutamol was used as an agonist (6.69). Intracellular cAMP levels were increased by CL 316243, an effect that was not altered by ICI 118551. Therefore, β3-adrenoceptors may be present and functioning in canine pulmonary artery.