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.     

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.     

Effects of hypoxia in porcine pulmonary arterial myocytes: roles of K(V) channel and endothelin-1

Effects of acute hypoxia on intracellular Ca(2+) concentration ([Ca(2+)](i)) and cell length were recorded simultaneously in proximal and distal pulmonary (PASMCs) and femoral (FASMCs) arterial smooth muscle cells. Reducing PO(2) from normoxia to severe hypoxia (PO(2) < 10 mmHg) caused small but significant decreases in length and a reversible increase in [Ca(2+)](i) in distal PASMCs and a small decrease in length in proximal PASMCs but had no effect in FASMCs, even though all three cell types contracted significantly to vasoactive agonists. Inhibition of voltage-dependent K(+) (K(V)) channel with 4-aminopyridine produced a greater increase in [Ca(2+)](i) in distal than in proximal PASMCs. In distal PASMCs, severe hypoxia caused a slight inhibition of K(V) currents; however, it elicited further contraction in the presence of 4-aminopyridine. Endothelin-1 (10(-10) M), which itself did not alter cell length or [Ca(2+)](i), significantly potentiated the hypoxic contraction. These results suggest that hypoxia only has small direct effects on porcine PASMCs. These effects cannot be fully explained by inhibition of K(V) channels and were greatly enhanced via synergistic interactions with the endothelium-derived factor endothelin-1.     

Effect of normoxia and hypoxia on K(+) current and resting membrane potential of fetal rabbit pulmonary artery smooth muscle

At birth, the increase in O(2) tension (pO(2)) is an important cause of the decrease in pulmonary vascular resistance. In adult animals there are impressive interspecies differences in the level of hypoxia required to elicit a pulmonary vasoconstrictor response and in the amplitude of the response. Hypoxic inhibition of some potassium (K(+)) channels in the membrane of pulmonary arterial smooth muscle cells (PASMCs) helps to initiate hypoxic pulmonary vasoconstriction. To determine the effect of the change in pO(2) on fetal rabbit PASMCs and to investigate possible species-dependent differences, we measured the current-voltage relationship and the resting membrane potential, in PASMCs from fetal resistance arteries using the amphotericin-perforated patch-clamp technique under hypoxic and normoxic conditions. Under hypoxic conditions, the K(+) current in PASMCs was small, and could be inhibited by 4-aminopyridine, iberiotoxin and glibenclamide, reflecting contributions by Kv, K(Ca) and K(ATP) channels. The average resting membrane potential was -44.3+/-1.3 mV (n=29) and could be depolarized by 4-AP (5 mM) and ITX (100 nM) but not by glibenclamide (10 microM). Changing from hypoxia, that mimicked fetal life, to normoxia dramatically increased the K(Ca) and consequently hyperpolarized (-9.3+/-1.7 mV; n=8) fetal rabbit PASMCs. Under normoxic conditions K(+) current was reduced by 4-AP with a significant change in resting membrane potential (11.1+/-1.7 mV; n=8). We conclude that resting membrane potential in fetal rabbit PASMCs under both hypoxic and normoxic conditions depends on both Kv and K(Ca) channels, in contrast to fetal lamb or porcine PASMCs. Potential species differences in the K(+) channels that control resting membrane potential must be taken into consideration in the interpretation of studies of neonatal pulmonary vascular reactivity to changes in O(2) tension.     

Inhibition of hypoxia-induced calcium responses in pulmonary arterial smooth muscle by acetazolamide is independent of carbonic anhydrase inhibition

Hypoxic pulmonary vasoconstriction (HPV) occurs with ascent to high altitude and can contribute to development of high altitude pulmonary edema (HAPE). Vascular smooth muscle contains carbonic anhydrase (CA), and acetazolamide (AZ), a CA inhibitor, blunts HPV and might be useful in the prevention of HAPE. The mechanism by which AZ impairs HPV is uncertain. Originally developed as a diuretic, AZ also has direct effects on systemic vascular smooth muscle, including modulation of pH and membrane potential; however, the effect of AZ on pulmonary arterial smooth muscle cells (PASMCs) is unknown. Since HPV requires Ca2+ influx into PASMCs and can be modulated by pH, we hypothesized that AZ alters hypoxia-induced changes in PASMC intracellular pH (pH(i)) or Ca2+ concentration ([Ca2+](i)). Using fluorescent microscopy, we tested the effect of AZ as well as two other potent CA inhibitors, benzolamide and ethoxzolamide, which exhibit low and high membrane permeability, respectively, on hypoxia-induced responses in PASMCs. Hypoxia caused a significant increase in [Ca2+](i) but no change in pH(i). All three CA inhibitors slightly decreased basal pH(i), but only AZ caused a concentration-dependent decrease in the [Ca2+](i) response to hypoxia. AZ had no effect on the KCl-induced increase in [Ca2+](i) or membrane potential. N-methyl-AZ, a synthesized compound lacking the unsubstituted sulfonamide group required for CA inhibition, had no effect on pH(i) but inhibited hypoxia-induced Ca2+ responses. These results suggest that AZ attenuates HPV by selectively inhibiting hypoxia-induced Ca2+ responses via a mechanism independent of CA inhibition, changes in pH(i), or membrane potential.     

Protein kinase C-epsilon-null mice have decreased hypoxic pulmonary vasoconstriction

PKC contributes to regulation of pulmonary vascular reactivity in response to hypoxia. The role of individual PKC isozymes is less clear. We used a knockout (null, -/-) mouse to test the hypothesis that PKC-epsilon is important in acute hypoxic pulmonary vasoconstriction (HPV). We asked whether deletion of PKC-epsilon would decrease acute HPV in adult C57BL6xSV129 mice. In isolated, salt solution-perfused lung, reactivity to acute hypoxic challenges (0% and 3% O(2)) was compared with responses to angiotensin II (ANG II) and KCl. PKC-epsilon -/- mice had decreased HPV, whereas responses to ANG II and KCl were preserved. Inhibition of nitric oxide synthase (NOS) with nitro-l-arginine augmented HPV in PKC-epsilon +/+ but not -/- mice. Inhibition of Ca(2+)-gated K(+) channels (K(Ca)) with charybdotoxin and apamin did not enhance HPV in -/- mice relative to wild-type (+/+) controls. In contrast, the voltage-gated K(+) channel (K(V)) antagonist 4-aminopyridine increased the response of -/- mice beyond that of +/+ mice. This suggested that increased K(V) channel expression could contribute to blunted HPV in PKC-epsilon -/- mice. Therefore, expression of the O(2)-sensitive K(V) channel subunit Kv3.1b (100-kDa glycosylated form and 70-kDa core protein) was compared in whole lung and pulmonary artery smooth muscle cell (PASMC) lysates from +/+ and -/- mice. A subtle increase in Kv3.1b was detected in -/- vs. +/+ whole lung lysates. A much greater rise in Kv3.1b expression was found in -/- vs. +/+ PASMC. Thus deletion of PKC-epsilon blunts murine HPV. The decreased response could not be attributed to a general loss in vasoreactivity or derangements in NOS or K(Ca) channel activity. Instead, the absence of PKC-epsilon allows increased expression of K(V) channels (like Kv3.1b) to occur in PASMC, which likely contributes to decreased HPV.     

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).     

Hypoxia-induced migration in pulmonary arterial smooth muscle cells requires calcium-dependent upregulation of aquaporin 1

Pulmonary arterial smooth muscle cell (PASMC) migration is a key component of the vascular remodeling that occurs during the development of hypoxic pulmonary hypertension, although the mechanisms governing this phenomenon remain poorly understood. Aquaporin-1 (AQP1), an integral membrane water channel protein, has recently been shown to aid in migration of endothelial cells. Since AQP1 is expressed in certain types of vascular smooth muscle, we hypothesized that AQP1 would be expressed in PASMCs and would be required for migration in response to hypoxia. Using PCR and immunoblot techniques, we determined the expression of AQPs in pulmonary vascular smooth muscle and the effect of hypoxia on AQP levels, and we examined the role of AQP1 in hypoxia-induced migration in rat PASMCs using Transwell filter assays. Moreover, since the cytoplasmic tail of AQP1 contains a putative calcium binding site and an increase in intracellular calcium concentration ([Ca(2+)](i)) is a hallmark of hypoxic exposure in PASMCs, we also determined whether the responses were Ca(2+) dependent. Results were compared with those obtained in aortic smooth muscle cells (AoSMCs). We found that although AQP1 was abundant in both PASMCs and AoSMCs, hypoxia selectively increased AQP1 protein levels, [Ca(2+)](i), and migration in PASMCs. Blockade of Ca(2+) entry through voltage-dependent Ca(2+) or nonselective cation channels prevented the hypoxia-induced increase in PASMC [Ca(2+)](i), AQP1 levels, and migration. Silencing AQP1 via siRNA also prevented hypoxia-induced migration of PASMCs. Our results suggest that hypoxia induces a PASMC-specific increase in [Ca(2+)](i) that results in increased AQP1 protein levels and cell migration.     

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).     

Intracellular alkalinization induces Ca2+ influx via non-voltage-operated Ca2+ channels in rat aortic smooth muscle cells

In smooth muscle, the cytosolic Ca2+ concentration ([Ca2+](i)) is the primary determinant of contraction, and the intracellular pH (pH(i)) modulates contractility. Using fura-2 and 2',7'-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH(i) on [Ca2+](i). The application of the NH(4)Cl induced concentration-dependent increases in both pH(i) and [Ca2+](i). The extent of [Ca2+](i) elevation induced by 20mM NH(4)Cl was approximately 50% of that obtained with 100mM K(+)-depolarization. The NH(4)Cl-induced elevation of [Ca2+](i) was completely abolished by the removal of extracellular Ca2+ or the addition of extracellular Ni2+. The 100mM K(+)-induced [Ca2+](i) elevation was markedly inhibited by a voltage-operated Ca2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca2+ channel blocker, SKF96365. On the other hand, the NH(4)Cl-induced [Ca2+](i) elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca2+ influx via non-voltage-operated Ca2+ channels and thereby increases [Ca2+](i) in the vascular smooth muscle cells. The alkalinization-induced Ca2+ influx may therefore contribute to the enhancement of contraction.     

Chronic intrauterine pulmonary hypertension increases capacitative calcium entry in fetal pulmonary artery smooth muscle cells

Oxygen causes perinatal pulmonary dilatation. Although fetal pulmonary artery smooth muscle cells (PA SMC) normally respond to an acute increase in oxygen (O2) tension with a decrease in cytosolic calcium ([Ca2+]i), an acute increase in O2 tension has no net effect on [Ca(2+)](i) in PA SMC derived from lambs with chronic intrauterine pulmonary hypertension (PHTN). The present experimental series tests the hypothesis that an acute increase in O2 tension decreases capacitative calcium entry (CCE) in normal, but not hypertensive, fetal PA SMC. PA SMC were isolated from late-gestation fetal lambs after either ligation of the ductus arteriosus (PHTN) or sham (control) operation at 127 days gestation. PA SMC were isolated from the distal PA (>or=4th generation) and maintained under hypoxic conditions ( approximately 25 Torr) in primary culture. After fura 2 loading, apparent [Ca2+]i in PA SMC was determined as the ratio of 340- to 380-nm fluorescence intensity. Under both hypoxic and normoxic conditions, cyclopiazonic acid (CPA) increased [Ca2+]i more in PHTN than in control PA SMC. CCE was determined in PA SMC under hypoxic and normoxic conditions, after superfusion with zero extracellular Ca2+ and intracellular store depletion with CPA, followed by superfusion with Ca2+-containing solution, in the presence of the voltage-operated calcium channel blockade. CCE was increased in PHTN compared with control PA SMC under conditions of both acute and sustained normoxia. Transient receptor potential channel gene expression was greater in control compared with PHTN PA SMC. PHTN may compromise perinatal pulmonary vasodilation, in part, by modulating PA SMC CCE.     

Cerebral artery K(ATP)- and K(Ca)-channel activity and contractility: changes with development

The present study was designed to test the hypothesis that in cerebral arteries of the fetus, ATP-sensitive (K(ATP)) and Ca(2+)-activated K(+) channels (K(Ca)) play an important role in the regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) and that this differs significantly from that of the adult. In main branch middle cerebral arteries (MCA) from near-term fetal ( approximately 140 days) and nonpregnant adult sheep, simultaneously we measured norepinephrine (NE)-induced responses of vascular tension and [Ca(2+)](i) in the absence and presence of selective K(+)-channel openers/blockers. In fetal MCA, in a dose-dependent manner, both the K(ATP)-channel opener pinacidil and the K(Ca)-channel opener NS 1619 significantly inhibited NE-induced tension [negative logarithm of the half-maximal inhibitory concentration (pIC(50)) = 5.0 +/- 0.1 and 8.2 +/- 0.1, respectively], with a modest decrease of [Ca(2+)](i). In the adult MCA, in contrast, both pinacidil and NS 1619 produced a significant tension decrease (pIC(50) = 5.1 +/- 0.1 and 7.6 +/- 0.1, respectively) with no change in [Ca(2+)](i). In addition, the K(Ca)-channel blocker iberiotoxin (10(-7) to 10(-6) M) resulted in increased tension and [Ca(2+)](i) in both adult and fetal MCA, although the K(ATP)-channel blocker glibenclamide (10(-7) to 3 x 10(-5) M) failed to do so. Of interest, administration of 10(-7) M iberiotoxin totally eliminated vascular contraction and increase in [Ca(2+)](i) seen in response to 10(-5) M ryanodine. In precontracted fetal cerebral arteries, activation of the K(ATP) and K(Ca) channels significantly decreased both tension and [Ca(2+)](i), suggesting that both K(+) channels play an important role in regulating L-type channel Ca(2+) flux and therefore vascular tone in these vessels. In the adult, K(ATP) and the K(Ca) channels also appear to play an important role in this regard; however, in the adult vessel, activation of these channels with resultant vasorelaxation can occur with no significant change in [Ca(2+)](i). These channels show differing responses to inhibition, e.g., K(Ca)-channel inhibition, resulting in increased tension and [Ca(2+)](i), whereas K(ATP)-channel inhibition showed no such effect. In addition, the K(Ca) channel appears to be coupled to the sarcoplasmic reticulum ryanodine receptor. Thus differences in plasma membrane K(+)-channel activity may account, in part, for the differences in the regulation of contractility of fetal and adult cerebral arteries.     

Fetal rabbit pulmonary artery smooth muscle cell response to ryanodine is developmentally regulated

To study developmental changes in intracellular calcium handling in pulmonary artery smooth muscle cells (PASMCs), cells were isolated from distal and proximal pulmonary arteries from rabbits at different developmental stages: juvenile (4-6 wk old), newborn (<48 h), and full-term fetal. Isolated PASMCs were studied using the calcium-sensitive dye fura 2. Cells from each age group responded to caffeine with an increase in calcium; however, ryanodine (50 microM) only increased calcium in fetal distal PASMCs. The ryanodine-induced increase was due to influx of extracellular calcium because it was blocked by removal of extracellular calcium or by diltiazem. The calcium-sensitive potassium (K(Ca)) channel blocker iberiotoxin produced a transient increase in calcium in the fetal distal PASMCs, which could be inhibited by prior application of ryanodine. Conversely, the ryanodine response was inhibited if iberiotoxin was given first. With the use of electrophysiology and confocal microscopy, fetal PASMCs were shown to exhibit spontaneous transient outward currents and calcium sparks, respectively. These observations suggest that ryanodine-sensitive release of calcium from the sarcoplasmic reticulum and K(Ca) channels act together to control intracellular calcium only in fetal distal PASMCs.     

Fetal and adult cerebral artery K(ATP) and K(Ca) channel responses to long-term hypoxia.

High-altitude long-term hypoxia (LTH) alters cerebral vascular contractile and relaxation responses in both fetus and adult. We tested the hypotheses that LTH-mediated vascular responses were secondary to altered K+ channel function and that in the fetus these responses differ from those of the adult. In middle cerebral arteries (MCA) from both nonpregnant adult and fetal (approximately 140 days gestation) sheep, which were either acclimatized to high altitude (3,820 m) or sea-level controls, we measured norepinephrine (NE)-induced contractions and intracellular Ca2+ concentration ([Ca2+]i) simultaneously, in the presence or absence of different K+ channel openers or blockers. In adult MCA, LTH was associated with approximately 20% decrease in NE-induced tension and [Ca2+]i, with a significant increase in Ca2+ sensitivity. In contrast, in fetal MCA, LTH failed to affect significantly NE-induced contraction or [Ca2+]i but significantly decreased the ATP-sensitive K+ (K(ATP)) channel and Ca2+-activated K+ (K(Ca)) channel-mediated relaxation. The significant effect of K(ATP) and K(Ca) channel activators on the relaxation responses and the fact that K+ channels play a key role in myogenic tone support the hypotheses that K+ channels play an important role in hypoxia-mediated responses. These results also support the hypothesis of significant developmental differences with maturation from fetus to adult.     

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.     

Ca2+-RhoA signaling pathway required for polyamine-dependent intestinal epithelial cell migration

Expression of voltage-gated K(+) (Kv) channel genes is regulated by polyamines in intestinal epithelial cells (IEC-6 line), and Kv channel activity is involved in the regulation of cell migration during early restitution by controlling membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). This study tests the hypothesis that RhoA of small GTPases is a downstream target of elevated ([Ca2+](cyt)) following activation of K(+) channels by increased polyamines in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced whole cell K+ currents [I(K(v))] through Kv channels and caused membrane depolarization, which was associated with decreases in ([Ca2+](cyt)), RhoA protein, and cell migration. Exogenous polyamine spermidine reversed the effects of DFMO on I(K(v)), E(m), ([Ca2+](cyt)), and RhoA protein and restored cell migration to normal. Elevation of ([Ca2+](cyt)) induced by the Ca2+ ionophore ionomycin increased RhoA protein synthesis and stimulated cell migration, while removal of extracellular Ca2+ decreased RhoA protein synthesis, reduced protein stability, and inhibited cell motility. Decreased RhoA activity due to Clostridium botulinum exoenzyme C(3) transferase inhibited formation of myosin II stress fibers and prevented restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These findings suggest that polyamine-dependent cell migration is partially initiated by the formation of myosin II stress fibers as a result of Ca2+-activated RhoA activity.     

Maturation of intracellular calcium homeostasis in sheep pulmonary arterial smooth muscle cells

Cytosolic Ca(2+) signaling dynamics are important to pulmonary arterial reactivity, and alterations are implicated in pulmonary vascular disorders. Yet, adaptations in cellular Ca(2+) homeostasis and receptor-mediated Ca(2+) signaling with maturation from fetal to adult life in pulmonary arterial smooth muscle cells (PASMCs) are not known. The present study tested the hypothesis that cytosolic Ca(2+) homeostasis and receptor-generated Ca(2+) signaling adapt with maturation in sheep PASMCs. Digitalized fluorescence microscopy was performed using isolated PASMCs from fetal and adult sheep that were loaded with the Ca(2+) indicator fura 2. The results show that basal cytosolic and sarcoplasmic reticulum Ca(2+) levels are attained before birth. Similarly, Ca(2+) efflux pathways from the cytosol and basal as well as capacitative Ca(2+) entry (CCE) are also developed before birth. However, receptor-mediated Ca(2+) signaling adapts with maturation. Prominently, serotonin stimulation elicited Ca(2+) elevations in very few fetal compared with adult PASMCs; in contrast, phenylephrine elevated Ca(2+) in a similar percentage of fetal and adult PASMCs. Serotonin and phenylephrine elicited Ca(2+) increases of a similar magnitude in reactive cells of fetus and adult, supporting the assertion that inositol trisphosphate signaling is intact. Caffeine and ATP elevated Ca(2+) in equivalent numbers of fetal and adult PASMCs. However, the caffeine-induced cytosolic Ca(2+) increase was significantly greater in fetal PASMCs, whereas the ATP-elicited increase was greater in adult cells. Overall, the results of this study demonstrate selective adaptations in receptor-mediated Ca(2+) signaling, but not in cellular Ca(2+) homeostasis.