Heterogeneity of hypoxia-mediated decrease in I(K(V)) and increase in [Ca2+](cyt) in pulmonary artery smooth muscle cells

Hypoxic pulmonary vasoconstriction is caused by a rise in cytosolic Ca(2+) ([Ca(2+)](cyt)) in pulmonary artery smooth muscle cells (PASMC) via multiple mechanisms. PASMC consist of heterogeneous phenotypes defined by contractility, proliferation, and apoptosis as well as by differences in expression and function of various genes. In rat PASMC, hypoxia-mediated decrease in voltage-gated K(+) (Kv) currents (I(K(V))) and increase in [Ca(2+)](cyt) were not uniformly distributed in all PASMC tested. Acute hypoxia decreased I(K(V)) and increased [Ca(2+)](cyt) in approximately 46% and approximately 53% of PASMC, respectively. Using combined techniques of single-cell RT-PCR and patch clamp, we show here that mRNA expression level of Kv1.5 in hypoxia-sensitive PASMC (in which hypoxia reduced I(K(V))) was much greater than in hypoxia-insensitive cells (in which hypoxia negligibly affected I(K(V))). These results demonstrate that 1) different PASMC express different Kv channel alpha- and beta-subunits, and 2) the sensitivity of a PASMC to acute hypoxia partially depends on the expression level of Kv1.5 channels; hypoxia reduces whole-cell I(K(V)) only in PASMC that express high level of Kv1.5. In addition, the acute hypoxia-mediated changes in [Ca(2+)](cyt) also vary in different PASMC. Hypoxia increases [Ca(2+)](cyt) only in 34% of cells tested, and the different sensitivity of [Ca(2+)](cyt) to hypoxia was not related to the resting [Ca(2+)](cyt). An intrinsic mechanism within each individual cell may be involved in the heterogeneity of hypoxia-mediated effect on [Ca(2+)](cyt) in PASMC. These data suggest that the heterogeneity of PASMC may partially be related to different expression levels and functional sensitivity of Kv channels to hypoxia and to differences in intrinsic mechanisms involved in regulating [Ca(2+)](cyt).     

Chronic hypoxia decreases K(V) channel expression and function in pulmonary artery myocytes

Activity of voltage-gated K+ (KV) channels regulates membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). A rise in ([Ca2+](cyt))in pulmonary artery (PA) smooth muscle cells (SMCs) triggers pulmonary vasoconstriction and stimulates PASMC proliferation. Chronic hypoxia (PO(2) 30-35 mmHg for 60-72 h) decreased mRNA expression of KV channel alpha-subunits (Kv1.1, Kv1.5, Kv2.1, Kv4.3, and Kv9.3) in PASMCs but not in mesenteric artery (MA) SMCs. Consistently, chronic hypoxia attenuated protein expression of Kv1.1, Kv1.5, and Kv2.1; reduced KV current [I(KV)]; caused E(m) depolarization; and increased ([Ca2+](cyt)) in PASMCs but negligibly affected KV channel expression, increased I(KV), and induced hyperpolarization in MASMCs. These results demonstrate that chronic hypoxia selectively downregulates KV channel expression, reduces I(KV), and induces E(m) depolarization in PASMCs. The subsequent rise in ([Ca2+](cyt)) plays a critical role in the development of pulmonary vasoconstriction and medial hypertrophy. The divergent effects of hypoxia on KV channel alpha-subunit mRNA expression in PASMCs and MASMCs may result from different mechanisms involved in the regulation of KV channel gene expression.     

Divergent contractile and structural responses of the murine PKC-epsilon null pulmonary circulation to chronic hypoxia

Loss of PKC-epsilon limits the magnitude of acute hypoxic pulmonary vasoconstriction (HPV) in the mouse. Therefore, we hypothesized that loss of PKC-epsilon would decrease the contractile and/or structural response of the murine pulmonary circulation to chronic hypoxia (Hx). However, the pattern of lung vascular responses to chronic Hx may or may not be predicted by the acute HPV response. Adult PKC-epsilon wild-type (PKC-epsilon(+/+)), heterozygous null, and homozygous null (PKC-epsilon(-/-)) mice were exposed to normoxia or Hx for 5 wk. PKC-epsilon(-/-) mice actually had a greater increase in right ventricular (RV) systolic pressure, RV mass, and hematocrit in response to chronic Hx than PKC-epsilon(+/+) mice. In contrast to the augmented PA pressure and RV hypertrophy, pulmonary vascular remodeling was increased less than expected (i.e., equal to PKC-epsilon(+/+) mice) in both the proximal and distal PKC-epsilon(-/-) pulmonary vasculature. The contribution of increased vascular tone to this pulmonary hypertension (PHTN) was assessed by measuring the acute vasodilator response to nitric oxide (NO). Acute inhalation of NO reversed the increased PA pressure in hypoxic PKC-epsilon(-/-) mice, implying that the exaggerated PHTN may be due to a relative deficiency in nitric oxide synthase (NOS). Despite the higher PA pressure, chronic Hx stimulated less of an increase in lung endothelial (e) and inducible (i) NOS expression in PKC-epsilon(-/-) than PKC-epsilon(+/+) mice. In contrast, expression of nNOS in PKC-epsilon(+/+) mice decreased in response to chronic Hx, while lung levels in PKC-epsilon(-/-) mice remained unchanged. In summary, loss of PKC-epsilon results in increased vascular tone, but not pulmonary vascular remodeling in response to chronic Hx. Blunting of Hx-induced eNOS and iNOS expression may contribute to the increased vascular tone. PKC-epsilon appears to be an important signaling intermediate in the hypoxic regulation of each NOS isoform.     

Deranged Kv channel regulation in fibroblasts from mice lacking the serum and glucocorticoid inducible kinase SGK1

Coexpression of the serum and glucocorticoid inducible kinase 1 (SGK1) up-regulates Kv channel activity in Xenopus oocytes and human embryonic kidney cells. To investigate the physiological impact of SGK1 dependent Kv channel regulation, we recorded whole-cell currents in lung fibroblasts from SGK1 knockout mice (sgk1-/-) and wild-type littermates (sgk1+/+). Serum-grown mouse lung fibroblasts (MLF) from both genotypes exhibited voltage-gated outwardly rectifying K(+)-currents with time-dependent activation (tau(act) approximately 3 msec), slow inactivation (tau(inact) approximately 700 msec), use-dependent inactivation, and (partial) inhibition by K(+) channel blockers TEA, 4-AP, and margatoxin. In serum grown MLF peak Kv current density at +100 mV was significantly lower in sgk1-/- (14 +/- 2 pA/pF, n = 13) than in sgk1+/+ (31 +/- 4 pA/pF, n = 16). PCR amplification of different Kv1 and Kv3 subunits from mouse fibroblasts demonstrated the expression of Kv1.1-1.7, Kv3.1, and Kv3.3 mRNA in both sgk1+/+ and sgk1-/- cells. Upon serum deprivation Kv currents almost disappeared in sgk1+/+ (4 +/- 1 pA/pF, n = 11) but not in sgk1-/- (10 +/- 1 pA/pF, n = 6) MLF. Accordingly, following serum deprivation Kv current density was significantly lower in sgk1+/+ than in sgk1-/-. Stimulation of serum-depleted cells with dexamethasone (dex) (1 microM, 1 day), IGF-1 (6.7 microM, 4-6 h) or both, significantly activated Kv currents in sgk1+/+ but not in sgk1-/- MLF. In the presence of both, dex and IGF-1, the Kv current density was significantly larger in sgk1+/+ (27 +/- 3 pA/pF, n = 12) than in sgk1-/- (13 +/- 3 pA/pF, n = 10) cells. Similar to MLF, Kv currents were significantly higher in sgk1+/+ mouse tail fibroblasts (MTF). In sgk1+/+ but not sgk1-/- MTF the Kv currents were inhibited upon serum deprivation and reincreased after stimulation of serum deprived MTF with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h). According to Fura-2-fluorescence capacitative Ca(2+) entry was lower in sgk1-/- MTF compared to sgk1+/+ MTF. Upon serum deprivation capacitative Ca(2+) entry decreased significantly in sgk1+/+ but not in sgk1-/- MTF. Stimulation of depleted cells with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h) reincreased capacitative Ca(2+) entry in sgk1+/+ MTF, whereas in sgk1-/- cells it remained unchanged. In conclusion, lack of SGK1 does not abrogate Kv channel activity but abolishes regulation of those channels by serum, glucocorticoids and IGF-1, an effect influencing capacitative Ca(2+) entry.     

Pulmonary vascular response to normoxia and K(Ca) channel activity is developmentally regulated

To address developmental regulation of pulmonary vascular O(2) sensing, we tested the hypotheses that 1) fetal but not adult pulmonary artery smooth muscle cells (PASMCs) can directly sense an acute increase in O(2), 2) Ca2+-sensitive K(+) (K(Ca)) channel activity decreases with maturation, and 3) PASMC K(Ca) channel expression decreases with maturation. We used fluorescence microscopy to confirm that fetal but not adult PASMCs are able to sense an acute increase in O(2) tension. Acute normoxia induced a 22 +/- 2% decrease in cytosolic Ca2+ concentration ([Ca2+](i)) in fetal PASMCs and no change in ([Ca2+](i)) in adult PASMCs (P < 0.01). The effects of K(+) channel antagonists were studied on fetal and adult PASMC ([Ca2+](i)). Iberiotoxin (10(-9) M) caused PASMC ([Ca2+](i)) to increase by 694 +/- 22% in the fetus and caused no change in adult PASMCs. K(Ca) channel expression and mRNA levels in distal pulmonary arteries from fetal and adult sheep were examined. Both K(Ca) channel protein and mRNA expression in the distal pulmonary vasculature decreased with maturation. We conclude that maturation-dependent changes in PASMC O(2) sensing render the fetal PASMCs uniquely sensitive to an acute increase in O(2) tension at a biologically critical time point.     

Voltage-gated K(+)-channel activity in ovine pulmonary vasculature is developmentally regulated

To examine mechanisms underlying developmental changes in pulmonary vascular tone, we tested the hypotheses that 1) maturation-related changes in the ability of the pulmonary vasculature to respond to hypoxia are intrinsic to the pulmonary artery (PA) smooth muscle cells (SMCs); 2) voltage-gated K(+) (K(v))-channel activity increases with maturation; and 3) O(2)-sensitive Kv2.1 channel expression and message increase with maturation. To confirm that maturational differences are intrinsic to PASMCs, we used fluorescence microscopy to study the effect of acute hypoxia on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in SMCs isolated from adult and fetal PAs. Although PASMCs from both fetal and adult circulations were able to sense an acute decrease in O(2) tension, acute hypoxia induced a more rapid and greater change in [Ca(2+)](i) in magnitude in PASMCs from adult compared with fetal PAs. To determine developmental changes in K(v)-channel activity, the effects of the K(+)-channel antagonist 4-aminopyridine (4-AP) were studied on fetal and adult PASMC [Ca(2+)](i). 4-AP (1 mM) caused PASMC [Ca(2+)](i) to increase by 94 +/- 22% in the fetus and 303 +/- 46% in the adult. K(v)-channel expression and mRNA levels in distal pulmonary arteries from fetal, neonatal, and adult sheep were determined through the use of immunoblotting and semiquantitative RT-PCR. Both Kv2.1-channel protein and mRNA expression in distal pulmonary vasculature increased with maturation. We conclude that there are maturation-dependent changes in PASMC O(2) sensing that may render the adult PASMCs more responsive to acute hypoxia.     

Contribution of the K(Ca) channel to membrane potential and O2 sensitivity is decreased in an ovine PPHN model

Ca2+-sensitive K+ (K(Ca)) channels play an important role in mediating perinatal pulmonary vasodilation. We hypothesized that lung K(Ca) channel function may be decreased in persistent pulmonary hypertension of the newborn (PPHN). To test this hypothesis, pulmonary artery smooth muscle cells (PASMC) were isolated from fetal lambs with severe pulmonary hypertension induced by ligation of the ductus arteriosus in fetal lambs at 125-128 days gestation. Fetal lambs were killed after pulmonary hypertension had been maintained for at least 7 days. Age-matched, sham-operated animals were used as controls. PASMC K+ currents and membrane potentials were recorded using amphotericin B-perforated patch-clamp techniques. The increase in whole cell current normally seen in response to normoxia was decreased (333.9 +/- 63.6% in control vs. 133.1 +/- 16.0% in hypertensive fetuses). The contribution of the K(Ca) channel to the whole cell current was diminished in hypertensive, compared with control, fetal PASMC. In PASMC from hypertensive fetuses, a change from hypoxia to normoxia caused no change in membrane potential compared with a -14.6 +/- 2.8 mV decrease in membrane potential in PASMC from control animals. In PASMC from animals with pulmonary hypertension, 4-aminopyridine (4-AP) caused a larger depolarization than iberiotoxin, whereas in PASMC from control animals, iberiotoxin caused a larger depolarization than 4-AP. These data confirm the hypothesis that the contribution of the K(Ca) channel to membrane potential and O2 sensitivity is decreased in an ovine model of PPHN, and this may contribute to the abnormal perinatal pulmonary vasoreactivity associated with PPHN.     

Acute hypoxia selectively inhibits KCNA5 channels in pulmonary artery smooth muscle cells

Acute hypoxia causes pulmonary vasoconstriction in part by inhibiting voltage-gated K⁺ (Kv) channel activity in pulmonary artery smooth muscle cells (PASMC). The hypoxia-mediated decrease in Kv currents [I_K(V)] is selective to PASMC; hypoxia has little effect on I_K(V) in mesenteric artery smooth muscle cells (MASMC). Functional Kv channels are homo- and/or heterotetramers of pore-forming -subunits and regulatory -subunits. KCNA5 is a Kv channel -subunit that forms functional Kv channels in PASMC and regulates resting membrane potential. We have shown that acute hypoxia selectively inhibits I_K(V) through KCNA5 channels in PASMC. Overexpression of the human KCNA5 gene increased I_K(V) and caused membrane hyperpolarization in HEK-293, COS-7, and rat MASMC and PASMC. Acute hypoxia did not affect I_K(V) in KCNA5-transfected HEK-293 and COS-7 cells. However, overexpression of KCNA5 in PASMC conferred its sensitivity to hypoxia. Reduction of PO₂ from 145 to 35 mmHg reduced I_K(V) by 40% in rat PASMC transfected with human KCNA5 but had no effect on I_K(V) in KCNA5-transfected rat MASMC (or HEK and COS cells). These results indicate that KCNA5 is an important Kv channel that regulates resting membrane potential and that acute hypoxia selectively reduces KCNA5 channel activity in PASMC relative to MASMC and other cell types. Because Kv channels (including KCNA5) are ubiquitously expressed in PASMC and MASMC, the observation from this study indicates that a hypoxia-sensitive mechanism essential for inhibiting KCNA5 channel activity is exclusively present in PASMC. The divergent effect of hypoxia on I_K(V) in PASMC and MASMC also may be due to different expression levels of KCNA5 channels. membrane potential; potassium channels; vascular smooth muscle     

Bone morphogenetic protein-2 upregulates expression and function of voltage-gated K+ channels in human pulmonary artery smooth muscle cells

Activity of voltage-gated K(+) (K(V)) channels in pulmonary artery smooth muscle cells (PASMC) plays an important role in control of apoptosis and proliferation in addition to regulating membrane potential and pulmonary vascular tone. Bone morphogenetic proteins (BMPs) inhibit proliferation and induce apoptosis in normal human PASMC, whereas dysfunctional BMP signaling and downregulated K(V) channels are involved in pulmonary vascular medial hypertrophy associated with pulmonary hypertension. This study evaluated the effect of BMP-2 on K(V) channel function and expression in normal human PASMC. BMP-2 (100 nM for 18-24 h) significantly (>2-fold) upregulated mRNA expression of KCNA5, KCNA7, KCNA10, KCNC3, KCNC4, KCNF1, KCNG3, KCNS1, and KCNS3 but downregulated (at least 2-fold) KCNAB1, KCNA2, KCNG2, and KCNV2. The most dramatic change was the >10-fold downregulation of KCNG2 and KCNV2, two electrically silent gamma-subunits that form heterotetramers with functional K(V) channel alpha-subunits (e.g., KCNB1-2). Furthermore, the amplitude and current density of whole cell K(V) currents were significantly increased in PASMC treated with BMP-2. It has been demonstrated that K(+) currents generated by KCNB1 and KCNG1 (or KCNG2) or KCNB1 and KCNV2 heterotetramers are smaller than those generated by KCNB1 homotetramers, indicating that KCNG2 and KCNV2 (2 subunits that were markedly downregulated by BMP-2) are inhibitors of functional K(V) channels. These results suggest that BMP-2 divergently regulates mRNA expression of various K(V) channel alpha-, beta-, and gamma-subunits and significantly increases whole cell K(V) currents in human PASMC. Finally, we present evidence that attenuation of c-Myc expression by BMP-2 may be involved in BMP-2-mediated increase in K(V) channel activity and regulation of K(V) channel expression. The increased K(V) channel activity may be involved in the proapoptotic and/or antiproliferative effects of BMP-2 on PASMC.     

BMP signaling controls PASMC KV channel expression in vitro and in vivo

Bone morphogenetic proteins (BMPs) have been implicated in the pathogenesis of familial pulmonary arterial hypertension. The type 2 receptor (BMPR2) is required for recognition of all BMPs. Transgenic mice with a smooth muscle cell-targeted mutation in this receptor (SM22-tet-BMPR2(delx4+)) developed increased pulmonary artery pressure, associated with a modest increase in arterial muscularization, after 8 wk of transgene activation (West J, Fagan K, Steudel W, Fouty B, Lane K, Harral J, Hoedt-Miller M, Tada Y, Ozimek J, Tuder R, and Rodman DM. Circ Res 94: 1109-1114, 2004). In the present study, we show that these transgenic mice developed increased right ventricular pressures after only 1 wk of transgene activation, without significant remodeling of the vasculature. We then tested the hypothesis that the increased pulmonary artery pressure due to loss of BMPR2 signaling was mediated by reduced K(V) channel expression. There was decreased expression of K(V)1.1, K(V)1.5, and K(V)4.3 mRNA isolated from whole lung. Western blot confirmed decreased K(V)1.5 protein in these lungs. Human pulmonary artery smooth muscle cells (PASMC) treated with recombinant BMP2 had increased K(V)1.5 protein and macroscopic K(V) current density, which was blocked by anti-K(V)1.5 antibody. In vivo, nifedipine, a selective L-type Ca(2+) channel blocker, reduced RV systolic pressure in these dominant-negative BMPR2 mice to levels seen in control animals. This suggests that activation of L-type Ca(2+) channels caused by reduced K(V)1.5 mediates increased pulmonary artery pressure in these animals. These studies suggest that BMP regulates K(V) channel expression and that loss of this signaling pathway in PASMC through a mutation in BMPR2 is sufficient to cause pulmonary artery vasoconstriction.     

Modulation of Kv3.1b potassium channel phosphorylation in auditory neurons by conventional and novel protein kinase C isozymes

In fast-spiking neurons such as those in the medial nucleus of the trapezoid body (MNTB) in the auditory brainstem, Kv3.1 potassium channels are required for high frequency firing. The Kv3.1b splice variant of this channel predominates in the mature nervous system and is a substrate for phosphorylation by protein kinase C (PKC) at Ser-503. In resting neurons, basal phosphorylation at this site decreases Kv3.1 current, reducing neuronal ability to follow high frequency stimulation. We used a phospho-specific antibody to determine which PKC isozymes control serine 503 phosphorylation in Kv3.1b-tranfected cells and in auditory neurons in brainstem slices. By using isozyme-specific inhibitors, we found that the novel PKC-delta isozyme, together with the novel PKC-epsilon and conventional PKCs, contributed to the basal phosphorylation of Kv3.1b in MNTB neurons. In contrast, only PKC-epsilon and conventional PKCs mediate increases in phosphorylation produced by pharmacological activation of PKC in MNTB neurons or by metabotropic glutamate receptor activation in Kv3.1/mGluR1-cotransfected cells. We also measured the time course of dephosphorylation and recovery of basal phosphorylation of Kv3.1b following brief high frequency electrical stimulation of the trapezoid body, and we determined that the recovery process is mediated by both novel PKC-delta and PKC-epsilon isozymes and by conventional PKCs. The association between Kv3.1b and PKC isozymes was confirmed by reciprocal coimmunoprecipitation of Kv3.1b with multiple PKC isozymes. Our results suggest that the Kv3.1b channel is regulated by both conventional and novel PKC isozymes and that novel PKC-delta contributes specifically to the maintenance of basal phosphorylation in auditory neurons.     

Pulmonary vascular K+ channel expression and vasoreactivity in a model of congenital heart disease

K+ channels play an important role in mediating pulmonary vasodilation caused by increased oxygen tension, nitric oxide, alkalosis, and shear stress. To test the hypothesis that lung K+ channel gene expression may be altered by chronic increases in pulmonary blood flow, we measured gene and protein expression of calcium-sensitive (K Ca ) and voltage-gated (Kv2.1) K+ channels, and a pH-sensitive K+ channel (TASK), in distal lung from fetal lambs in which an aortopulmonary shunt was placed at 139 days gestation. Under baseline conditions, animals with an aortopulmonary shunt showed elevated pulmonary artery pressure and pulmonary blood flow compared with twin controls. Hypoxia caused a greater increase in pulmonary vascular tone in shunt animals compared with controls. Alkalosis caused pulmonary vasodilation in control but not shunt animals. To determine lung K+ channel mRNA levels, we performed quantitative RT-PCR. In comparison with control animals, lung K Ca channel mRNA content was increased in shunt animals, whereas TASK mRNA levels were decreased. There was no difference in Kv2.1 mRNA levels. Channel protein expression was consistent with these findings. We conclude that, in the presence of elevated pulmonary blood flow, K Ca channel expression is increased and TASK is decreased.     

Inhibition of hypoxic pulmonary vasoconstriction by antagonists of store-operated Ca2+ and nonselective cation channels

Previous studies indicated that acute hypoxia increased intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) influx, and capacitative Ca(2+) entry (CCE) through store-operated Ca(2+) channels (SOCC) in smooth muscle cells from distal pulmonary arteries (PASMC), which are thought to be a major locus of hypoxic pulmonary vasoconstriction (HPV). Moreover, these effects were blocked by Ca(2+)-free conditions and antagonists of SOCC and nonselective cation channels (NSCC). To test the hypothesis that in vivo HPV requires CCE, we measured the effects of SOCC/NSCC antagonists (SKF-96365, NiCl(2), and LaCl(3)) on pulmonary arterial pressor responses to 2% O(2) and high-KCl concentrations in isolated rat lungs. At concentrations that blocked CCE and [Ca(2+)](i) responses to hypoxia in PASMC, SKF-96365 and NiCl(2) prevented and reversed HPV but did not alter pressor responses to KCl. At 10 microM, LaCl(3) had similar effects, but higher concentrations (30 and 100 microM) caused vasoconstriction during normoxia and potentiated HPV, indicating actions other than SOCC blockade. Ca(2+)-free perfusate and the voltage-operated Ca(2+) channel (VOCC) antagonist nifedipine were potent inhibitors of pressor responses to both hypoxia and KCl. We conclude that HPV required influx of Ca(2+) through both SOCC and VOCC. This dual requirement and virtual abolition of HPV by either SOCC or VOCC antagonists suggests that neither channel provided enough Ca(2+) on its own to trigger PASMC contraction and/or that during hypoxia, SOCC-dependent depolarization caused secondary activation of VOCC.     

Differences in STIM1 and TRPC expression in proximal and distal pulmonary arterial smooth muscle are associated with differences in Ca2+ responses to hypoxia

Hypoxic pulmonary vasoconstriction (HPV) requires Ca(2+) influx through store-operated Ca(2+) channels (SOCC) in pulmonary arterial smooth muscle cells (PASMC) and is greater in distal than proximal pulmonary arteries (PA). SOCC may be composed of canonical transient receptor potential (TRPC) proteins and activated by stromal interacting molecule 1 (STIM1). To assess the possibility that HPV is greater in distal PA because store-operated Ca(2+) entry (SOCE) is greater in distal PASMC, we measured intracellular Ca(2+) concentration ([Ca(2+)](i)) and SOCE in primary cultures of PASMC using fluorescent microscopy and the Ca(2+)-sensitive dye fura 2. Both hypoxia (4% O(2)) and KCl (60 mM) increased [Ca(2+)](i). Responses to hypoxia, but not KCl, were greater in distal cells. We measured SOCE in PASMC perfused with Ca(2+)-free solutions containing cyclopiazonic acid to deplete Ca(2+) stores in sarcoplasmic reticulum and nifedipine to prevent Ca(2+) entry through L-type voltage-operated Ca(2+) channels. Under these conditions, the increase in [Ca(2+)](i) caused by restoration of extracellular Ca(2+) and the decrease in fura 2 fluorescence caused by Mn(2+) were greater in distal PASMC, indicating greater SOCE. Moreover, the increase in SOCE caused by hypoxia was also greater in distal cells. Real-time quantitative polymerase chain reaction analysis of PASMC and freshly isolated deendothelialized PA tissue demonstrated expression of STIM1 and five of seven known TRPC isoforms (TRPC1 > TRPC6 > TRPC4 > TRPC3 approximately TRPC5). For both protein, as measured by Western blotting, and mRNA, expression of STIM1, TRPC1, TRPC6, and TRPC4 was greater in distal than proximal PASMC and PA. These results provide further support for the importance of SOCE in HPV and suggest that HPV is greater in distal than proximal PA because greater numbers and activation of SOCC in distal PASMC generate bigger increases in [Ca(2+)](i).     

Perinatal hypoxia triggers alterations in K+ channels of adult pulmonary artery smooth muscle cells

Adverse events during the perinatal period, like hypoxia, have been associated with adult diseases. In pulmonary vessels, K(+) channels play an important role in the regulation of vascular tone. In the fetus, Ca(2+)-activated K(+) channels (K(Ca)) are predominant, whereas from birth voltage-gated K(+) channels (K(V)) prevail in the adult. We postulated that perinatal hypoxia could alter this maturational shift and influence regulation of pulmonary vascular tone in relation to K(+) channels in adulthood. We evaluated the effects of perinatal hypoxia on K(V) and K(Ca) channels in the adult main pulmonary artery (PA) using a murine model. Electrophysiological measurements showed a greater outward current in PA smooth muscle cells of mice born in hypoxia than in controls. In controls, only K(V) channels contributed to this current, whereas in mice born in hypoxia both K(V) and K(Ca) channels were implicated. K(V) channel activity was even higher in mice born in hypoxia than in controls. Therefore, perinatal hypoxia results in increased K(Ca) and K(V) channel activity in adult PA. Moreover, PA of adults born in hypoxia displayed higher large-conductance K(Ca) alpha-subunit and K(V)1.5 alpha-subunit protein expression than controls. Interestingly, relaxation induced by nitric oxide (NO) donors [S-nitroso-N-acetyl-D,l-penicillamine, 2-(N,N-diethylamino)-diazenolate-2-oxide] in isolated PA of control mice was not mediated by K(Ca) channels and only slightly by K(V) channels, whereas following perinatal hypoxia both K(Ca) and K(V) channels contributed to this relaxation. Thus perinatal hypoxia results in altered expression and activity of different K(+) channels in the adult main PA, which could contribute to modifications of pulmonary vasoreactivity.