Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle

Mammalian homologs of transient receptor potential (TRP) genes in Drosophila encode TRPC proteins, which make up cation channels that play several putative roles, including Ca2+ entry triggered by depletion of Ca2+ stores in endoplasmic reticulum (ER). This capacitative calcium entry (CCE) is thought to replenish Ca2+ stores and contribute to signaling in many tissues, including smooth muscle cells from main pulmonary artery (PASMCs); however, the roles of CCE and TRPC proteins in PASMCs from distal pulmonary arteries, which are thought to be the major site of pulmonary vasoreactivity, remain uncertain. As an initial test of the possibility that TRPC channels contribute to CCE and Ca2+ signaling in distal PASMCs, we measured [Ca2+]i by fura-2 fluorescence in primary cultures of myocytes isolated from rat intrapulmonary arteries (>4th generation). In cells perfused with Ca2+-free media containing cyclopiazonic acid (10 microM) and nifedipine (5 microM) to deplete ER Ca2+ stores and block voltage-dependent Ca2+ channels, restoration of extracellular Ca2+ (2.5 mM) caused marked increases in [Ca2+]i whereas MnCl2 (200 microM) quenched fura-2 fluorescence, indicating CCE. SKF-96365, LaCl3, and NiCl2, blocked CCE at concentrations that did not alter Ca2+ responses to 60 mM KCl (IC50 6.3, 40.4, and 191 microM, respectively). RT-PCR and Western blotting performed on RNA and protein isolated from distal intrapulmonary arteries and PASMCs revealed mRNA and protein expression for TRPC1, -4, and -6, but not TRPC2, -3, -5, or -7. Our results suggest that CCE through TRPC-encoded Ca2+ channels could contribute to Ca2+ signaling in myocytes from distal intrapulmonary arteries.     

Reversibility of Cisternal Stack Formation During Hypoxic Hypoxia and Subsequent Reoxygenation in Cerebellar Purkinje Cells

Cisternal stacks are induced during hypoxia, which may be associated with intracellular Ca²⁺ regulation. Although neurons are divided internally in different compartments, little is known about regional differences in cisternal stack formation. We investigated the effects of hypoxic hypoxia and later reoxygenation on cisternal stack formation and other ultrastructual changes in the proximal dendrite, dendritic spine, and cell body of cerebellar Purkinje cells in rats. After brief hypoxic events, cisternal stacks appeared predominantly in the proximal dendrites and after longer hypoxic events in dendritic spines and cell body. Following reoxygenation, cisternal stacks disappeared first in the cell body, followed by the dendritic spines, then the proximal dendrites. These results showed that stack formation occurred at different degrees and time courses among the three regions, and the effect was reversible, which suggests that these compartments are differentially sensitive to hypoxia.     

Hypoxic constriction and reactive oxygen species in porcine distal pulmonary arteries

To determine whether reactive oxygen species (ROS) play an essential role in hypoxic pulmonary vasoconstriction (HPV) and the cellular locus of ROS production and action during HPV, we measured internal diameter (ID) at constant transmural pressure, lucigenin-derived chemiluminescence (LDCL), and electron paramagnetic resonance (EPR) spin adduct spectra in small distal porcine pulmonary arteries, and dichlorofluorescein (DCF) fluorescence in myocytes isolated from these arteries. Hypoxia (4% O2) decreased ID, increased DCF fluorescence, tended to increase LDCL, and in some preparations produced EPR spectra consistent with hydroxyl and alkyl radicals. Superoxide dismutase (SOD, 150 U/ml) or SOD + catalase (CAT, 200 U/ml) did not alter ID during normoxia but reduced or abolished the constriction induced by hypoxia. SOD also blocked HPV in endothelium-denuded arteries after restoration of the response by exposure to 10-10 M endothelin-1. Confocal fluorescence microscopy demonstrated that labeled SOD and CAT entered pulmonary arterial myocytes. SOD, SOD + CAT, and CAT blocked the increase in DCF fluorescence induced by hypoxia, but SOD + CAT and CAT also caused a stable increase in fluorescence during normoxia, suggesting that CAT diminished efflux of DCF from cells or oxidized the dye directly. We conclude that HPV required increased concentrations of ROS produced by and acting on pulmonary arterial smooth muscle rather than endothelium.     

Partial HIF-1alpha deficiency impairs pulmonary arterial myocyte electrophysiological responses to hypoxia

Chronic hypoxia depolarizes and reduces K+ current in pulmonary arterial smooth muscle cells (PASMCs). Our laboratory previously demonstrated that hypoxia-inducible factor-1 (HIF-1) contributed to the development of hypoxic pulmonary hypertension. In this study, electrophysiological parameters were measured in PASMCs isolated from intrapulmonary arteries of mice with one null allele at the Hif1a locus encoding HIF-1alpha [Hif1a(+/-)] and from their wild-type [Hif1a(+/+)] littermates after 3 wk in air or 10% O2. Hematocrit and right ventricular wall and left ventricle plus septum weights were measured. Capacitance, K+ current, and membrane potential were measured with whole cell patch clamp. Similar to our laboratory's previous results, hypoxia-induced right ventricular hypertrophy and polycythemia were blunted in Hif1a(+/-) mice. Hypoxia increased PASMC capacitance in Hif1a(+/+) mice but not in Hif1a(+/-) mice. Chronic hypoxia depolarized and reduced K+ current density in PASMCs from Hif1a(+/+) mice. In PASMCs from hypoxic Hif1a(+/-) mice, no reduction in K+ current density was observed, and depolarization was significantly blunted. Thus partial deficiency of HIF-1alpha is sufficient to impair hypoxia-induced depolarization, reduction of K+ current density, and PASMC hypertrophy.     

Hypoxic constriction of porcine distal pulmonary arteries: endothelium and endothelin dependence

To determine the role of endothelium in hypoxic pulmonary vasoconstriction (HPV), we measured vasomotor responses to hypoxia in isolated seventh-generation porcine pulmonary arteries < 300 microm in diameter with (E+) and without endothelium. In E+ pulmonary arteries, hypoxia decreased the vascular intraluminal diameter measured at a constant transmural pressure. These constrictions were complete in 30-40 min; maximum at PO(2) of 2 mm Hg; half-maximal at PO(2) of 40 mm Hg; blocked by exposure to Ca(2+)-free conditions, nifedipine, or ryanodine; and absent in E+ bronchial arteries of similar size. Hypoxic constrictions were unaltered by indomethacin, enhanced by indomethacin plus N(G)-nitro-L-arginine methyl ester, abolished by BQ-123 or endothelial denudation, and restored in endothelium-denuded pulmonary arteries pretreated with 10(-10) M endothelin-1 (ET-1). Given previous demonstrations that hypoxia caused contractions in isolated pulmonary arterial myocytes and that ET-1 receptor antagonists inhibited HPV in intact animals, our results suggest that full in vivo expression of HPV requires basal release of ET-1 from the endothelium to facilitate mechanisms of hypoxic reactivity in pulmonary arterial smooth muscle.     

Regulated ectopic expression and allelic-replacement mutagenesis as a method for gene essentiality testing in Staphylococcus aureus

Conditional expression systems were utilized for the ectopic induction of essential genes in Staphylococcus aureus. Resulting strains were then subjected to allelic-replacement mutagenesis of the native allele under inducing conditions for expression of the ectopic copy of the gene. This strategy produced test strains whereby cellular viability was uniquely dependent on the presence of inducer and provided a direct and absolute confirmation of genetic essentiality for each locus. The procedure is particularly useful for genes that are difficult to analyze by conventional inactivation strategies due to either small size or complex genomic organization.     

A global approach to identify novel broad-spectrum antibacterial targets among proteins of unknown function

Attempted allelic replacement of 144 Streptococcus pneumoniae open reading frames of previously uncharacterized function led to the identification of 36 genes essential for growth under laboratory conditions. Of these, 14 genes (obg, spoIIIJ2, trmU, yacA, yacM, ydiC, ydiE, yjbN, yneS, yphC, ysxC, ytaG, yloI and yxeH4) were also essential in Staphylococcus aureus and Haemophilus influenzae or Escherichia coli, 2 genes (yrrK and ydiB) were only essential in H. influenzae as well as S. pneumoniae and 8 genes were necessary for growth of S.pneumoniae and S. aureus and did not have a homolog in H. influenzae(murD2, ykqC, ylqF, yqeH, ytgP, yybQ) or were not essential in that organism (yqeL, yhcT). The proteins encoded by these genes could represent good targets for novel antibiotics covering different therapeutic profiles. The putative functions of some of these essential proteins, inferred by bioinformatic analysis, are presented. Four mutants, with deletions of loci not essential for in vitro growth, were found to be severely attenuated in a murine respiratory tract infection model, suggesting that not all targets for antibacterial therapeutics are revealed by simple in vitro essentiality testing. The results of our experiments together with those collated from previously reported studies including Bacillus subtilis, E. coli and Mycoplasma sp. demonstrate that gene conservation amongst bacteria does not necessarily indicate that essentiality in one organism can be extrapolated to others. Moreover, this study demonstrates that different experimental procedures can produce apparently contradictory results.     

High-salt diet impairs hypoxia-induced cAMP production and hyperpolarization in rat skeletal muscle arteries

This study determined the effects of hypoxia on diameter, vascular smooth muscle (VSM) transmembrane potential (E(m)), and vascular cAMP levels for in vitro cannulated skeletal muscle resistance arteries (gracilis arteries) from Sprague-Dawley rats fed a low-salt (LS) or a high-salt (HS) diet. Arterial diameter and VSM E(m) were measured in response to hypoxia, iloprost, cholera toxin, forskolin, and aprikalim. In HS rats, arterial dilation and VSM hyperpolarization after hypoxia, iloprost, and cholera toxin were impaired versus responses in LS rats, whereas responses to forskolin and aprikalim were unaltered. Blockade of prostaglandin H(2) and thromboxane A(2) receptors had no effect on responses to hypoxia or iloprost in vessels from both rat groups, suggesting that inappropriate activation of these receptors does not contribute to the impaired hypoxic dilation with HS. Hypoxia, cholera toxin, and iloprost increased vascular cAMP levels in vessels of LS rats only, whereas forskolin increased cAMP levels in all vessels. These data suggest that reduced hypoxic dilation of skeletal muscle microvessels in rats on a HS diet may reflect an impaired ability of VSM to produce cAMP after exposure to prostacyclin.