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.

NO causes perinatal pulmonary vasodilation through K+-channel activation and intracellular Ca2+ release.

Evidence suggests that nitric oxide (NO) causes perinatal pulmonary vasodilation through K+-channel activation. We hypothesized that this effect worked through cGMP-dependent kinase-mediated activation of Ca2+-activated K+ channel that requires release of intracellular Ca2+ from a ryanodine-sensitive store. We studied the effects of 1) K+-channel blockade with tetraethylammonium, 4-aminopyridine, a voltage-dependent K+-channel blocker, or glibenclamide, an ATP-sensitive K+-channel blocker; 2) cyclic nucleotide-sensitive kinase blockade with either KT-5823, a guanylate-sensitive kinase blocker, or H-89, an adenylate-sensitive kinase blocker; and 3) blockade of intracellular Ca2+ release with ryanodine on NO-induced pulmonary vasodilation in acutely prepared late-gestation fetal lambs. N-nitro-L-arginine, a competitive inhibitor of endothelium-derived NO synthase, was infused into the left pulmonary artery, and tracheotomy was placed. The animals were ventilated with 100% oxygen for 20 min, followed by ventilation with 100% oxygen and inhaled NO at 20 parts/million (ppm) for 20 min. This represents the control period. In separate protocols, the animals received an intrapulmonary infusion of the different blockers and were ventilated as above. Tetraethylammonium (n = 6 animals) and KT-5823 (n = 4 animals) attenuated the response, whereas ryanodine (n = 5 animals) blocked NO-induced perinatal pulmonary vasodilation. 4-Aminopyridine (n = 5 animals), glibenclamide (n = 5 animals), and H-89 (n = 4 animals) did not affect NO-induced pulmonary vasodilation. We conclude that NO causes perinatal pulmonary vasodilation through cGMP-dependent kinase-mediated activation of Ca2+-activated K+ channels and release of Ca2+ from ryanodine-sensitive stores.

A comparison of two fixatives on IFA HEp-2 slides for the detection of antinuclear antibodies.

A comparative evaluation of two fixatives on HEp-2 slides that detect antinuclear antibodies via indirect immunofluorescence was undertaken. The sensitivities of these two methods were compared to determine which of the two is more efficient in screening for anti-SS-A (Ro) antibodies. Fixing HEp-2 cells with a pure acetone solution resulted in a 97.5% sensitivity when anti-SS-A (Ro) positive samples were tested while only an 81.3% sensitivity was seen on HEp-2 cells fixed in an alcohol/acetone solution when detecting anti-SS-A (Ro) antibodies. In sera with only anti-SS-A (Ro) antibodies present, the fluorescence was more pronounced on the acetone fixed slides which made it easier to read than the alcohol/acetone fixed slides.