Characterisation of P2Y receptor subtypes mediating vasodilation and vasoconstriction of rat pulmonary artery using selective antagonists.

Pulmonary vascular tone is modulated by nucleotides, but which P2 receptors mediate these actions is largely unclear. The aim of this study, therefore, was to use subtype-selective antagonists to determine the roles of individual P2Y receptor subtypes in nucleotide-evoked pulmonary vasodilation and vasoconstriction. Isometric tension was recorded from rat intrapulmonary artery rings (i.d. 200-500 µm) mounted on a wire myograph. Nucleotides evoked concentration- and endothelium-dependent vasodilation of precontracted tissues, but the concentration-response curves were shallow and did not reach a plateau. The selective P2Y2 antagonist, AR-C118925XX, inhibited uridine 5'-triphosphate (UTP)- but not adenosine 5'-triphosphate (ATP)-evoked relaxation, whereas the P2Y6 receptor antagonist, MRS2578, had no effect on UTP but inhibited relaxation elicited by uridine 5'-diphosphate (UDP). ATP-evoked relaxations were unaffected by the P2Y1 receptor antagonist, MRS2179, which substantially inhibited responses to adenosine 5'-diphosphate (ADP), and by the P2Y12/13 receptor antagonist, cangrelor, which potentiated responses to ADP. Both agonists were unaffected by CGS1593, an adenosine receptor antagonist. Finally, AR-C118925XX had no effect on vasoconstriction elicited by UTP or ATP at resting tone, although P2Y2 receptor mRNA was extracted from endothelium-denuded tissues using reverse transcription polymerase chain reaction with specific oligonucleotide primers. In conclusion, UTP elicits pulmonary vasodilation via P2Y2 receptors, whereas UDP acts at P2Y6 and ADP at P2Y1 receptors, respectively. How ATP induces vasodilation is unclear, but it does not involve P2Y1, P2Y2, P2Y12, P2Y13, or adenosine receptors. UTP- and ATP-evoked vasoconstriction was not mediated by P2Y2 receptors. Thus, this study advances our understanding of how nucleotides modulate pulmonary vascular tone.

Dysfunctional bladder neurophysiology in urofacial syndrome Hpse2 mutant mice.

AIMS: Urofacial syndrome (UFS) is an autosomal recessive disease characterized by detrusor contraction against an incompletely dilated outflow tract. This dyssynergia causes dribbling incontinence and incomplete voiding. Around half of individuals with UFS have biallelic mutations of HPSE2 that encodes heparanase 2, a protein found in pelvic ganglia and bladder nerves. Homozygous Hpse2 mutant mice have abnormal patterns of nerves in the bladder body and outflow tract, and also have dysfunctional urinary voiding. We hypothesized that bladder neurophysiology is abnormal Hpse2 mutant mice. METHODS: Myography was used to study bladder bodies and outflow tracts isolated from juvenile mice. Myogenic function was analyzed after chemical stimulation or blockade of key receptors. Neurogenic function was assessed by electrical field stimulation (EFS). Muscarinic receptor expression was semi-quantified by Western blot analysis. RESULTS: Nitrergic nerve-mediated relaxation of precontracted mutant outflow tracts was significantly decreased vs littermate controls. The contractile ability of mutant outflow tracts was normal as assessed by KCl and the α1-adrenoceptor agonist phenylephrine. EFS of mutant bladder bodies induced significantly weaker contractions than controls. Conversely, the muscarinic agonist carbachol induced significantly stronger contractions of bladder body than controls. CONCLUSIONS: The Hpse2 model of UFS features aberrant bladder neuromuscular physiology. Further work is required to determine whether similar aberrations occur in patients with UFS.

Role of Kv7 channels in responses of the pulmonary circulation to hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is a beneficial mechanism that diverts blood from hypoxic alveoli to better ventilated areas of the lung, but breathing hypoxic air causes the pulmonary circulation to become hypertensive. Responses to airway hypoxia are associated with depolarization of smooth muscle cells in the pulmonary arteries and reduced activity of K(+) channels. As Kv7 channels have been proposed to play a key role in regulating the smooth muscle membrane potential, we investigated their involvement in the development of HPV and hypoxia-induced pulmonary hypertension. Vascular effects of the selective Kv7 blocker, linopirdine, and Kv7 activator, flupirtine, were investigated in isolated, saline-perfused lungs from rats maintained for 3-5 days in an isobaric hypoxic chamber (FiO2 = 0.1) or room air. Linopirdine increased vascular resistance in lungs from normoxic, but not hypoxic rats. This effect was associated with reduced mRNA expression of the Kv7.4 channel α-subunit in hypoxic arteries, whereas Kv7.1 and Kv7.5 were unaffected. Flupirtine had no effect in normoxic lungs but reduced vascular resistance in hypoxic lungs. Moreover, oral dosing with flupirtine (30 mg/kg/day) prevented short-term in vivo hypoxia from increasing pulmonary vascular resistance and sensitizing the arteries to acute hypoxia. These findings suggest a protective role for Kv7.4 channels in the pulmonary circulation, limiting its reactivity to pressor agents and preventing hypoxia-induced pulmonary hypertension. They also provide further support for the therapeutic potential of Kv7 activators in pulmonary vascular disease.

Human HPSE2 gene transfer ameliorates bladder pathophysiology in a mutant mouse model of urofacial syndrome.

Rare early-onset lower urinary tract disorders include defects of functional maturation of the bladder. Current treatments do not target the primary pathobiology of these diseases. Some have a monogenic basis, such as urofacial, or Ochoa, syndrome (UFS). Here, the bladder does not empty fully because of incomplete relaxation of its outflow tract, and subsequent urosepsis can cause kidney failure. UFS is associated with biallelic variants of HPSE2, encoding heparanase-2. This protein is detected in pelvic ganglia, autonomic relay stations that innervate the bladder and control voiding. Bladder outflow tracts of Hpse2 mutant mice display impaired neurogenic relaxation. We hypothesized that HPSE2 gene transfer soon after birth would ameliorate this defect and explored an adeno-associated viral (AAV) vector-based approach. AAV9/HPSE2, carrying human HPSE2 driven by CAG, was administered intravenously into neonatal mice. In the third postnatal week, transgene transduction and expression were sought, and ex vivo myography was undertaken to measure bladder function. In mice administered AAV9/HPSE2, the viral genome was detected in pelvic ganglia. Human HPSE2 was expressed and heparanase-2 became detectable in pelvic ganglia of treated mutant mice. On autopsy, wild-type mice had empty bladders, whereas bladders were uniformly distended in mutant mice, a defect ameliorated by AAV9/HPSE2 treatment. Therapeutically, AAV9/HPSE2 significantly ameliorated impaired neurogenic relaxation of Hpse2 mutant bladder outflow tracts. Impaired neurogenic contractility of mutant detrusor smooth muscle was also significantly improved. These results constitute first steps towards curing UFS, a clinically devastating genetic disease featuring a bladder autonomic neuropathy.

Neurogenic Defects Occur in LRIG2-Associated Urinary Bladder Disease.

Introduction: Urofacial, or Ochoa, syndrome (UFS) is an autosomal recessive disease featuring a dyssynergic bladder with detrusor smooth muscle contracting against an undilated outflow tract. It also features an abnormal grimace. Half of individuals with UFS carry biallelic variants in HPSE2, whereas other rare families carry variants in LRIG2.LRIG2 is immunodetected in pelvic ganglia sending autonomic axons into the bladder. Moreover, Lrig2 mutant mice have abnormal urination and abnormally patterned bladder nerves. We hypothesized that peripheral neurogenic defects underlie LRIG2-associated bladder dysfunction. Methods: We describe a new family with LRIG2-associated UFS and studied Lrig2 homozygous mutant mice with ex vivo physiological analyses. Results: The index case presented antenatally with urinary tract (UT) dilatation, and postnatally had urosepsis and functional bladder outlet obstruction. He had the grimace that, together with UT disease, characterizes UFS. Although HPSE2 sequencing was normal, he carried a homozygous, predicted pathogenic, LRIG2 stop variant (c.1939C>T; p.Arg647∗). Lrig2 mutant mice had enlarged bladders. Ex vivo physiology experiments showed neurogenic smooth muscle relaxation defects in the outflow tract, containing the urethra adjoining the bladder, and in detrusor contractility. Moreover, there were nuanced differences in physiological outflow tract defects between the sexes. Conclusion: Putting this family in the context of all reported UT disease-associated LRIG2 variants, the full UFS phenotype occurs with biallelic stop or frameshift variants, but missense variants lead to bladder-limited disease. Our murine observations support the hypothesis that UFS is a genetic autonomic neuropathy of the bladder affecting outflow tract and bladder body function.

Remodelling and dysfunction of the sinus node in pulmonary arterial hypertension.

Patients with pulmonary arterial hypertension (PAH) have a high burden of arrhythmias, including arrhythmias arising from sinus node dysfunction, and the aim of this study was to investigate the effects of PAH on the sinus node. In the rat, PAH was induced by an injection of monocrotaline. Three weeks after injection, there was a decrease of the intrinsic heart rate (heart rate in the absence of autonomic tone) as well as the normal heart rate, evidence of sinus node dysfunction. In the sinus node of PAH rats, there was a significant downregulation of many ion channels and Ca2+-handling genes that could explain the dysfunction: HCN1 and HCN4 (responsible for pacemaker current, If), Cav1.2, Cav1.3 and Cav3.1 (responsible for L- and T-type Ca2+ currents, ICa,L and ICa,T), NCX1 (responsible for Na+-Ca2+ exchanger) and SERCA2 and RYR2 (Ca2+-handling molecules). In the sinus node of PAH rats, there was also a significant upregulation of many fibrosis genes that could also help explain the dysfunction: vimentin, collagen type 1, elastin, fibronectin and transforming growth factor ß1. In summary, in PAH, there is a remodelling of ion channel, Ca2+-handling and fibrosis genes in the sinus node that is likely to be responsible for the sinus node dysfunction. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Identification of contractile P2Y1, P2Y6, and P2Y12 receptors in rat intrapulmonary artery using selective ligands.

ATP and UDP constrict rat intrapulmonary arteries, but which receptors mediate these actions is unclear. Here, we used selective agonists and antagonists, along with measurements of P2Y receptor expression, to characterize the receptor subtypes involved. Isometric tension was recorded from endothelium-denuded rat intrapulmonary artery rings (i.d. 200-500 µm) mounted on a wire myograph. Expression of P2Y receptor subtype expression was determined by using reverse transcription-polymerase chain reaction with receptor-specific oligonucleotide primers. The selective P2Y(1) agonist (N)-methanocarba-2-methylthioadenosine-5'-O-diphosphate (MRS2365) induced small, concentration-dependent contractions that were inhibited by the P2Y(1) antagonist N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179). Contractions evoked by ATP were unaffected by MRS2179, but inhibited by approximately one-third by the P2Y(12) antagonist N(6)-(2-methylthiomethyl)-2-(3,3,3-trifluoropropylthio)dichloro-methylene ATP (AR-C69931MX). Combined blockade of P2X1 and P2Y(12) receptors virtually abolished the response to ATP. ADP also evoked contractions that were abolished by AR-C69931MX. The selective P2Y(6) receptor agonist 3-(2-oxo-2-phenylethyl)-UDP (PSB 0474) evoked concentration-dependent contractions and was approximately three times more potent than UDP, but the P2Y(14) agonist UDP-glucose had no effect. Contractions evoked by UDP were inhibited by the P2Y(6) receptor antagonist N,N'-1,4-butanediylbis-N'-(3-isothiocyanatophenyl)thiourea (MRS2578), but not the cysteinyl leukotriene 1 (CysL(1)) antagonist 3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)((3-dimethylamino-3-oxopropyl)thio)methyl)thiopropanoic acid (MK571). Higher concentrations of MRS2578 inhibited contractions to KCl, so they were not studied further. mRNA for P2Y(1), P2Y(6), and P2Y(12) receptors was identified. Our working model is that P2Y(12) and P2X1 receptors are present in rat intrapulmonary arteries and together mediate ATP-induced vasoconstriction. Contractile P2Y(6), but not P2Y(14) or CysLT(1), receptors are also present and are a major site through which UDP evokes constriction.

Kv7 Channels in Cyclic-Nucleotide Dependent Relaxation of Rat Intra-Pulmonary Artery.

Pulmonary hypertension is treated with drugs that stimulate cGMP or cAMP signalling. Both nucleotides can activate Kv7 channels, leading to smooth muscle hyperpolarisation, reduced Ca2+ influx and relaxation. Kv7 activation by cGMP contributes to the pulmonary vasodilator action of nitric oxide, but its contribution when dilation is evoked by the atrial natriuretic peptide (ANP) sensitive guanylate cyclase, or cAMP, is unknown. Small vessel myography was used to investigate the ability of Kv7 channel blockers to interfere with pulmonary artery relaxation when cyclic nucleotide pathways were stimulated in different ways. The pan-Kv7 blockers, linopirdine and XE991, caused substantial inhibition of relaxation evoked by NO donors and ANP, as well as endothelium-dependent dilators, the guanylate cyclase stimulator, riociguat, and the phosphodiesterase-5 inhibitor, sildenafil. Maximum relaxation was reduced without a change in sensitivity. The blockers had relatively little effect on cAMP-mediated relaxation evoked by forskolin, isoprenaline or treprostinil. The Kv7.1-selective blocker, HMR1556, had no effect on cGMP or cAMP-dependent relaxation. Western blot analysis demonstrated the presence of Kv7.1 and Kv7.4 proteins, while selective activators of Kv7.1 and Kv7.4 homomeric channels, but not Kv7.5, caused pulmonary artery relaxation. It is concluded that Kv7.4 channels contribute to endothelium-dependent dilation and the effects of drugs that act by stimulating cGMP, but not cAMP, signalling.

Contractile and electrophysiological properties of pulmonary artery smooth muscle are not altered in TASK-1 knockout mice.

The acid-sensitive, two-pore domain K+ channel, TASK-1, contributes to the background K+ conductance and membrane potential (Em) of rat and human pulmonary artery smooth muscle cells (PASMCs), but its role in regulating tone remains elusive. This study aimed to clarify the role of TASK-1 by determining the functional properties of pulmonary artery (PA) from mice in which the TASK-1 gene was deleted (TASK-1/3 KO), in comparison with wild-type (WT) C57BL/6 controls. Small vessel wire myography was used to measure isometric tension developed by intact PA. Em and currents were recorded from freshly isolated PASMCs using the perforated patch-clamp technique. Reverse transcription-polymerase chain reaction (RT-PCR) was used to estimate K+ channel expression. We could find no difference between PA from WT and TASK-1/3 KO mice. They showed similar constrictor responses to a range of agonists and K+ concentrations, the K+ channel blockers 4-aminopyridine, tetraethylammonium ions and XE991. Treprostinil, proposed to dilate by activating TASK-1, was just as effective in TASK-1/3 KO arteries. Blocking Ca2+ influx with nifedipine (1 µM) or levcromakalim (10 µM) had no effect on resting tone in either strain. The resting Em of PASMCs and its responses to K+ channel blockers were unchanged in TASK-1/3 KO mice as were voltage-activated K+ currents, including the non-inactivating K+ current (IKN) measured at 0 mV. The Em was, however, depolarised in comparison with other species.Mouse IKN was much smaller than in rat and showed no sensitivity to pH. The results imply that TASK-1 does not form a functional channel in mouse PASMCs.

In situ characterization of CD4+ T cell behavior in mucosal and systemic lymphoid tissues during the induction of oral priming and tolerance.

The behavior of antigen-specific CD4+ T lymphocytes during initial exposure to antigen probably influences their decision to become primed or tolerized, but this has not been examined directly in vivo. We have therefore tracked such cells in real time, in situ during the induction of oral priming versus oral tolerance. There were marked contrasts with respect to rate and type of movement and clustering between naive T cells and those exposed to antigen in immunogenic or tolerogenic forms. However, the major difference when comparing tolerized and primed T cells was that the latter formed larger and longer-lived clusters within mucosal and peripheral lymph nodes. This is the first comparison of the behavior of antigen-specific CD4+ T cells in situ in mucosal and systemic lymphoid tissues during the induction of priming versus tolerance in a physiologically relevant model in vivo.

KCNQ potassium channels: new targets for pulmonary vasodilator drugs?

Smooth muscle cells regulate the diameter of pulmonary arteries and the resistance to blood flow in the pulmonary circulation. These cells are normally relaxed to maintain low intrinsic vessel tone, but are contracted in pulmonary arterial hypertension (PAH). Potassium channels in the smooth muscle cell help to maintain low tone by polarising the membrane and preventing Ca(2+) influx through voltage-operated Ca(2+) channels. There is a loss of K(+) channel activity in PAH, so drugs that open K(+) channels are predicted to have a beneficial effect, provided their action can be restricted to the pulmonary circulation. Here we review the myriad of K(+) channels that are expressed in pulmonary arteries and suggest the roles that each might play in regulating pulmonary artery tone. We conclude that members of the KCNQ family of K(+) channels, the most recent K(+) channels to be discovered in pulmonary artery, may be a useful therapeutic target for the treatment of PAH. KCNQ channels appear to be preferentially expressed in pulmonary arteries and drugs that modulate their activity have potent effects on pulmonary artery tone.

KCNQ modulators reveal a key role for KCNQ potassium channels in regulating the tone of rat pulmonary artery smooth muscle.

Potassium channels are central to the regulation of pulmonary vascular tone. The smooth muscle cells of pulmonary artery display a background K(+) conductance with biophysical properties resembling those of KCNQ (K(V)7) potassium channels. Therefore, we investigated the expression and functional role of KCNQ channels in pulmonary artery. The effects of selective KCNQ channel modulators were investigated on K(+) current and membrane potential in isolated pulmonary artery smooth muscle cells (PASMCs), on the tension developed by intact pulmonary arteries, and on pulmonary arterial pressure in isolated perfused lungs and in vivo. The KCNQ channel blockers, linopirdine and XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone], inhibited the noninactivating background K(+) conductance in PASMCs and caused depolarization, vasoconstriction, and raised pulmonary arterial pressure without constricting several systemic arteries or raising systemic pressure. The KCNQ channel openers, retigabine and flupirtine, had the opposite effects. PASMCs were found to express KCNQ4 mRNA, at higher levels than mesenteric artery, along with smaller amounts of KCNQ1 and 5. It is concluded that KCNQ channels, most probably KCNQ4, make an important contribution to the regulation of pulmonary vascular tone, with a greater contribution in pulmonary compared with systemic vessels. The pulmonary vasoconstrictor effect of KCNQ blockers is a potentially serious side effect, but the pulmonary vasodilator effect of the openers may be useful in the treatment of pulmonary hypertension.

Effects of streptozotocin-induced diabetes on the pharmacology of rat conduit and resistance intrapulmonary arteries.

BACKGROUND: Poor control of blood glucose in diabetes is known to promote vascular dysfunction and hypertension. Diabetes was recently shown to be linked to an increased prevalence of pulmonary hypertension. The aim of this study was to determine how the pharmacological reactivity of intrapulmonary arteries is altered in a rat model of diabetes. METHODS: Diabetes was induced in rats by the beta-cell toxin, streptozotocin (STZ, 60 mg/kg), and isolated conduit and resistance intrapulmonary arteries studied 3-4 months later. Isometric tension responses to the vasoconstrictors phenylephrine, serotonin and PGF2alpha, and the vasodilators carbachol and glyceryl trinitrate, were compared in STZ-treated rats and age-matched controls. RESULTS: STZ-induced diabetes significantly blunted the maximum response of conduit, but not resistance pulmonary arteries to phenylephrine and serotonin, without a change in pEC50. Agonist responses were differentially reduced, with serotonin (46% smaller) affected more than phenylephrine (32% smaller) and responses to PGF2alpha unaltered. Vasoconstriction caused by K+-induced depolarisation remained normal in diabetic rats. Endothelium-dependent dilation to carbachol and endothelium-independent dilation to glyceryl trinitrate were also unaffected. CONCLUSION: The small resistance pulmonary arteries are relatively resistant to STZ-induced diabetes. The impaired constrictor responsiveness of conduit vessels was agonist dependent, suggesting possible loss of receptor expression or function. The observed effects cannot account for pulmonary hypertension in diabetes, rather the impaired reactivity to vasoconstrictors would counteract the development of pulmonary hypertensive disease.

Simvastatin causes pulmonary artery relaxation by blocking smooth muscle ROCK and calcium channels: Evidence for an endothelium-independent mechanism.

Simvastatin reduces pulmonary arterial pressure and right ventricular hypertrophy in animal models of pulmonary arterial hypertension (PAH) and is thought to restore endothelial dysfunction. In vivo effects of drugs are complicated by several factors and little is known of the direct effects of statins on pulmonary arteries. This study investigated the direct effects of simvastatin on pulmonary arteries isolated from rats with or without monocrotaline-induced PAH. Simvastatin suppressed contractions evoked by the thromboxane A2 receptor agonist U46619 (30 nM), the α1-adrenergic agonist phenylephrine (5 µM) and KCl (50 mM) by ~50% in healthy and diseased arteries, but did not reduce contraction evoked by sarco/endoplasmic reticulum ATPase blockers. It relaxed hypertensive arteries in the absence of stimulation. Removing the endothelium or inhibiting eNOS did not prevent the inhibition by simvastatin. Inhibiting RhoA/rho kinase (ROCK) with Y27632 (10 µM) suppressed contractions to U46619 and phenylephrine by ~80% and prevented their inhibition by simvastatin. Y27632 reduced KCl-induced contraction by ~30%, but did not prevent simvastatin inhibition. Simvastatin suppressed Ca2+ entry into smooth muscle cells, as detected by Mn2+ quench of fura-2 fluorescence. The calcium antagonist, nifedipine (1 µM), almost abolished K+-induced contraction with less effect against U46619 and phenylephrine. We conclude that simvastatin relaxes pulmonary arteries by acting on smooth muscle to interfere with signalling through G-protein coupled receptors and voltage-dependent Ca2+ entry. Its actions likely include inhibition of ROCK-dependent Ca2+ sensitisation and voltage-gated Ca2+ channels. These are likely to contribute to the beneficial effects of simvastatin in animal models of PAH.

"A Step and a Ceiling": mechanical properties of Ca2+ spark vasoregulation in resistance arteries by pressure-induced oxidative activation of PKG.

We investigated the biomechanical relationship between intraluminal pressure within small mesenteric resistance arteries, oxidant activation of PKG, Ca2+ sparks, and BK channel vasoregulation. Mesenteric resistance arteries from wild type (WT) and genetically modified mice with PKG resistance to oxidative activation were studied using wire and pressure myography. Ca2+ sparks and Ca2+ transients within vascular smooth muscle cells of intact arteries were characterized using high-speed confocal microscopy of intact arteries. Arteries were studied under conditions of varying intraluminal pressure and oxidation. Intraluminal pressure specifically, rather than the generic stretch of the artery, was necessary to activate the oxidative pathway. We demonstrated a graded step activation profile for the generation of Ca2+ sparks and also a functional "ceiling" for this pressure --sensitive oxidative pathway. During steady state pressure - induced constriction, any additional Ca2+ sensitive-K+ channel functional availability was independent of oxidant activated PKG. There was an increase in the amplitude, but not the Area under the Curve (AUC) of the caffeine-induced Ca2+ transient in pressurized arteries from mice with oxidant-resistant PKG compared with wild type. Overall, we surmise that intraluminal pressure within resistance arteries controls Ca2+ spark vasoregulation through a tightly controlled pathway with a graded onset switch. The pathway, underpinned by oxidant activation of PKG, cannot be further boosted by additional pressure or oxidation once active. We propose that these restrictive characteristics of pressure-induced Ca2+ spark vasoregulation confer stability for the artery in order to provide a constant flow independent of additional pressure fluctuations or exogenous oxidants.

Pharmacological profile of store-operated Ca(2+) entry in intrapulmonary artery smooth muscle cells.

Store-operated Ca(2+) entry (SOCE) plays an important role in the contraction and proliferation of pulmonary artery smooth muscle cells (PASMCs). The aim of this study was to characterise the pharmacological properties of the SOCE pathway in freshly isolated PASMCs from rat lung and to determine whether this Ca(2+) entry pathway is sensitive to nitric oxide donor drugs. Following depletion of Ca(2+) from the sarcoplasmic reticulum, by treating cells with thapsigargin, re-addition of Ca(2+) produced an increase in cytosolic fluo-4 fluorescence that was sustained for the period that extracellular Ca(2+) was present. Thapsigargin also increased the rate of quench of fura-2 fluorescence, confirming that SOCE was activated. The SOCE pathway was not affected by nifedipine or verapamil; however, it was inhibited by the divalent cations Ni(2+) (10 microM) and Cd(2+) (10 microM) by 47+/-5% and 49+/-5% respectively. SOCE was also inhibited 42+/-5% by 2-aminoethoxydiphenyl borate (2-APB; 75 microM) and 58+/-4% by Gd(3+) (10 microM), although La(3+) (100 microM) had little effect. None of the NO donors examined, including sodium nitroprusside, glyceryl trinitrate, and 2-(N,N-diethylamino)-diazenolate-2-oxide had any effect on SOCE. Thus, the pulmonary vasorelaxation produced by NO does not involve direct inhibition of SOCE in PASMCs. Western blot and immunocytochemistry using antibodies directed against specific TRPC subunits detected the presence of TRPC1, 3, and 6 in pulmonary artery and the pharmacological profile of SOCE in PASMCs favours a role for TRPC1 in mediating the underlying channels that are activated by store depletion.

Zinc pyrithione activates K+ channels and hyperpolarizes the membrane of rat pulmonary artery smooth muscle cells.

The membrane potential helps determine pulmonary artery smooth muscle cell (PASMC) contraction. The Kv7 channel activators, retigabine and flupirtine, are thought to dilate pulmonary arteries by hyperpolarising PASMC. Zinc pyrithione activates Kv7 channels by a mechanism distinct from retigabine and with different Kv7 subunit selectivity. This study aimed to determine if zinc pyrithione selectively activates Kv7 channels in rat PASMC to evoke pulmonary artery dilation. Zinc pyrithione relaxed pulmonary arteries with half-maximal effect at 4.3µM. At 10µM it activated pronounced voltage-dependent K+ current and hyperpolarized PASMCs by around 10mV. Tetraethylammonium ions (TEA, 10mM) and paxilline (1µM) abolished both the current and hyperpolarisation. XE991 (10µM) blocked the hyperpolarization and reduced the current by 30%. Iberiotoxin (50nM) had no effect on the hyperpolarisation, but reduced the current by 40%. The XE991-sensitive current activated with an exponential time course (time constant 17ms), whereas the iberiotoxin-sensitive current followed a bi-exponential time course (time constants 6 and 57ms), suggesting that the drugs blocked different components of the zinc pyrithione-induced current. Zinc pyrithione therefore appears to activate at least two types of K+ channel in PASMC; an XE991, TEA and paxilline-sensitive Kv7 channel and a TEA, paxilline and iberiotoxin-sensitive BKCa channel. Both could contribute to the relaxing effect of zinc pyrithione on pulmonary artery.

Effects of aging on Ca2+ signaling in murine mesenteric arterial smooth muscle cells.

Pathophysiological changes in arterial smooth muscle structure and function occur with aging and there are a number of reports illustrating reductions in vascular responsiveness with aging. While much is known about arterial remodeling and functional adaptations with aging, very little is known about the biophysical adaptations in individual arterial myocytes. Cytosolic Ca2+ signaling, involving activation of L-type Ca2+ channels on the plasma membrane as well as InsP3 and ryanodine receptors on the sarcoplasmic reticulum, is integral to vascular tone and reactivity. Thus, we tested the hypothesis that aging results in reductions in the functional expression of L-type channels and temporal aspects of ryanodine receptor and InsP3 receptor Ca2+ signaling, in mesenteric arterial smooth muscle cells isolated from 6 and 30 months old C57Bl/6 mice. Comparisons of L-type current activity were made using dialyzed, whole-cell voltage-clamp techniques and Ba2+ as charge carrier. Ca2+ signaling was measured using fura-2 fluorescence microscopy techniques. Cell morphological changes were also investigated using electrophysiological and immunocytochemical approaches. The amplitudes of L-type Ca2+ currents were increased in older mice, but this was associated with membrane surface area increases of approximately 50%, due to increases in cell length not cell width. Consequently, L-type Ca2+ current densities were preserved with age, indicating functional channel expression was unchanged. In contrast, aging was associated with decrements in Ca2+ signaling in response to either ryanodine receptor stimulation by caffeine or InsP3 receptor activation with phenylephrine. These changes with aging may be related to the previously reported depression in myogenic reactivity.

The role of twin pore domain and other K+ channels in hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction (HPV) describes the vasoconstrictor response of pulmonary arteries to hypoxia, which directs blood flow towards better ventilated areas of the lung. Exactly how pulmonary arteries sense oxygen and mediate this response is widely debated and several hypotheses have emerged. One has smooth muscle K+ channels as the primary O2 sensor, hypoxia causing K+ channel inhibition, membrane depolarization and voltage-dependent Ca2+ influx. Even if this mechanism is not the primary response of pulmonary arteries to hypoxia, inhibition of K+ channel activity probably plays a role in HPV, due to enhanced membrane excitability and Ca2+ influx. Hypoxia inhibits several different K+ channels expressed in pulmonary artery smooth muscle, most from the K(v) class of voltage-gated K+ channels, but the properties of many K(v) channels are incompatible with a role in initiating HPV. Twin-pore domain K+ channels have emerged as prime candidates for controlling the resting membrane potential of cells. The identification of the twin-pore channel, TASK, in pulmonary artery smooth muscle, along with reports that it is inhibited by hypoxia, raises the possibility that a member of this family of channels acts as an O2 sensor in pulmonary artery. An unidentified low-threshold, voltage-dependent K+ channel might also contribute.

Pulmonary vasoconstrictor action of KCNQ potassium channel blockers.

BACKGROUND: KCNQ channels have been widely studied in the nervous system, heart and inner ear, where they have important physiological functions. Recent reports indicate that KCNQ channels may also be expressed in portal vein where they are suggested to influence spontaneous contractile activity. The biophysical properties of K+ currents mediated by KCNQ channels resemble a current underlying the resting K+ conductance and resting potential of pulmonary artery smooth muscle cells. We therefore investigated a possible role of KCNQ channels in regulating the function of pulmonary arteries by determining the ability of the selective KCNQ channel blockers, linopirdine and XE991, to promote pulmonary vasoconstriction. METHODS: The tension developed by rat and mouse intrapulmonary or mesenteric arteries was measured using small vessel myography. Contractile responses to linopirdine and XE991 were measured in intact and endothelium denuded vessels. Experiments were also carried out under conditions that prevent the contractile effects of nerve released noradrenaline or ATP, or block various Ca2+ influx pathways, in order to investigate the mechanisms underlying contraction. RESULTS: Linopirdine and XE991 both contracted rat and mouse pulmonary arteries but had little effect on mesenteric arteries. In each case the maximum contraction was almost as large as the response to 50 mM K+. Linopirdine had an EC50 of around 1 microM and XE991 was almost 10-fold more potent. Neither removal of the endothelium nor exposure to phentolamine or alpha,beta-methylene ATP, to block alpha1-adrenoceptors or P2X receptors, respectively, affected the contraction. Contraction was abolished in Ca2+-free solution and in the presence of 1 microM nifedipine or 10 microM levcromakalim. CONCLUSION: The KCNQ channel blockers are potent and powerful constrictors of pulmonary arteries. This action may be selective for the pulmonary circulation as mesenteric arteries showed little response. The results imply that the drugs act directly on smooth muscle cells and contraction requires voltage-dependent Ca2+ influx. It is concluded that the drugs probably act by blocking KCNQ channels in pulmonary artery myocytes, leading to membrane depolarization and Ca2+ influx through L-type Ca2+ channels. This implies a functional role for KCNQ channels in regulating the resting membrane potential of pulmonary artery myocytes.