The hypoxic constriction of isolated pulmonary vessels is composed of an initial transient phase (phase 1) followed by a slowly developing increase in tone (phase 2). We investigated the roles of the endothelium and of intracellular Ca2+ stores in both preconstricted and unpreconstricted intrapulmonary rabbit arteries when challenged with hypoxia (PO2 16-21 Torr). Removing the endothelium did not affect phase 1, but phase 2 appeared as a steady plateau. Removing extracellular Ca2+ had essentially the same effect as removing the endothelium. Depletion of sarcoplasmic reticulum Ca2+ stores with caffeine and ryanodine abolished the hypoxic response. Omitting preconstriction reduced the amplitude of the hypoxic response but did not qualitatively affect any of the above responses. We conclude that hypoxia releases intracellular Ca2+ from ryanodine-sensitive stores by a mechanism intrinsic to pulmonary vascular smooth muscle without the need for Ca2+ influx across the plasmalemma or an endothelial factor. Our results also suggest that extracellular Ca2+ is required for the release of an endothelium-derived vasoconstrictor.

We have examined the eects of Y-27632, a speci®c inhibitor of Rho-activated kinases (ROCK I and ROCK II) upon sustained hypoxic pulmonary vasoconstriction (HPV) in both rat isolated small intrapulmonary arteries (IPA) and perfused rat lungs in situ. Y-27632 (100 nM ± 3 mM) was found to cause a concentration-dependent inhibition of acute sustained HPV in rat IPA. Application of Y-27632 (10 ± 600 nM) in perfused rat lungs caused no change in basal perfusion pressure, but was found to inhibit HPV in a concentration-dependent manner, resulting in complete ablation of the pressor response to hypoxia at a concentration of 600 nM. Furthermore, addition of Y-27632 at any point during hypoxia caused a reversal of HPV in perfused rat lungs. These results suggest that activation of Rho-associated kinase may be a pivotal step in the generation of sustained HPV.

Abstract-Previous

Previous studies on pulmonary arterial smooth muscle cells have shown that nicotinic acid adenine dinucleotide phosphate (NAADP) evokes highly localized intracellular Ca 2؉ signals by mobilizing thapsigargininsensitive stores. Such localized Ca 2؉ signals may initiate global Ca 2؉ waves and contraction of the myocytes through the recruitment of ryanodine receptors on the sarcoplasmic reticulum via Ca 2؉ -induced Ca 2؉ release. Here we show that NAADP evokes localized Ca 2؉ signals by mobilizing a bafilomycin A1-sensitive, lysosome-related Ca 2؉ store. These lysosomal stores facilitate this process by co-localizing with a portion of the sarcoplasmic reticulum expressing ryanodine receptors to comprise a highly specialized trigger zone for NAADPdependent Ca 2؉ signaling by the vasoconstrictor hormone, endothelin-1. These findings further advance our understanding of how the spatial organization of discrete, organellar Ca 2؉ stores may underpin the generation of differential Ca 2؉ signaling patterns by different Ca 2؉ -mobilizing messengers.

The contributions of specific K+ currents to the resting membrane potential of rabbit isolated, pulmonary artery myocytes, and their modulation by hypoxia, were investigated by use of the whole‐cell, patch‐clamp technique. In the presence of 10 μm glibenclamide the resting potential (−50±4 mV, n=18) was unaffected by 10 μm tetraethylammonium ions, 200 nm charybdotoxin, 200 nm iberiotoxin, 100 μm ouabain or 100 μm digitoxin. The negative potential was therefore maintained without ATP‐sensitive (KATP) or large conductance Ca2+‐sensitive (BKCa) K channels, and without the Na+‐K+ATPase. The resting potential, the delayed rectifier current (IK(V)) and the A‐like K+ current (IK(A)) were all reduced in a concentration‐dependent manner by 4‐aminopyridine (4‐AP) and by quinine. 4‐AP was equally potent at reducing the resting potential and IK(V), 10 mm causing depolarization from −44 mV to −22 mV with accompanying inhibition of IK(V) by 56% and IK(A) by 79%. In marked contrast, the effects of quinine on resting potential were poorly correlated with its effects on both IK(A) and IK(V). At 10 mm, quinine reduced IK(V) and IK(A) by 47% and 38%, respectively, with no change in the resting potential. At 100 μm, both currents were almost abolished while the resting potential was reduced <50%. Raising the concentration to 1 mm had little further effect on IK(A) or IK(V), but essentially abolished the resting potential. Reduction of the resting potential by quinine was correlated with inhibition of a voltage‐gated, low threshold, non‐inactivating K+ current, IK(N). Thus, 100 μm quinine reduced both IK(N) and the resting potential by around 50%. The resting membrane potential was the same whether measured after clamping the cell at −80 mV, or immediately after a prolonged period of depolarization at 0 mV, which inactivated IK(A) and IK(V), but not IK(N). When exposed to a hypoxic solution, the O2 tension near the cell fell from 125±6 to 14±2 mmHg (n=20), resulting in a slow depolarization of the myocyte membrane to −35±3 mV (n=16). The depolarization occurred without a change in the amplitude of IK(V) or IK(A), but it was accompanied by 60% inhibition of IK(N) at 0 mV. Our findings suggest that the resting potential of rabbit pulmonary artery myocytes depends on IK(N), and that inhibition of IK(N) may mediate the depolarization induced by hypoxia. British Journal of Pharmacology (1997) 120, 1461–1470; doi:

Abstract-Hypoxic pulmonary vasoconstriction (HPV) is unique to pulmonary arteries, and it aids ventilation/perfusion matching. However, in diseases such as emphysema, HPV can promote hypoxic pulmonary hypertension. We recently showed that hypoxia constricts pulmonary arteries in part by increasing cyclic ADP-ribose (cADPR) accumulation in the smooth muscle and, thereby, Ca 2ϩ release by ryanodine receptors. We now report on the role of cADPR in HPV in isolated rat pulmonary arteries and in the rat lung in situ. In isolated pulmonary arteries, the membrane-permeant cADPR antagonist, 8-bromo-cADPR, blocked sustained HPV by blocking Ca 2ϩ release from smooth muscle ryanodine-sensitive stores in the sarcoplasmic reticulum. Most importantly, we showed that 8-bromo-cADPR blocks HPV induced by alveolar hypoxia in the ventilated rat lung in situ. Inhibition of HPV was achieved without affecting (1) constriction by membrane depolarization and voltage-gated Ca 2ϩ influx, (2) the release (by hypoxia) of an endothelium-derived vasoconstrictor, or (3) endothelium-dependent vasoconstriction. Our findings suggest that HPV is both triggered and maintained by cADPR in the rat lung in situ. Key Words: cADP-ribose Ⅲ pulmonary artery Ⅲ hypoxia S ince it was first described, hypoxic pulmonary vasoconstriction (HPV) has been recognized as the critical and distinguishing characteristic of pulmonary arteries 1 ; systemic arteries dilate in response to hypoxia. Physiologically, HPV contributes to ventilation-perfusion matching in the lung. However, when alveolar hypoxia is global, as it is in disease states such as emphysema and cystic fibrosis, it results in pulmonary hypertension and, eventually, right heart failure. 2 In isolated pulmonary arteries, HPV is biphasic. A transient constriction (phase 1) is followed by slow tonic constriction (phase 2). It was thought that phase 1 was initiated by a reduction in membrane K ϩ conductance in the smooth muscle 3,4 and voltage-gated Ca 2ϩ influx. 5,6 In addition, the primary mediator of phase 2 of HPV was thought to be an endothelium-derived vasoconstrictor. 7,8 Contrary to this, we and others discovered that hypoxia promotes phases 1 and 2 by releasing Ca 2ϩ from ryanodine-sensitive stores in the sarcoplasmic reticulum (SR) by a mechanism intrinsic to the smooth muscle. 9 -11 Recently, we showed that the ␤-NAD ϩ metabolite cyclic ADP-ribose (cADPR), 12,13 which increases Ca 2ϩ release by ryanodine receptors, 14 plays a role in this process. Thus, hypoxia increases cADPR accumulation and SR Ca 2ϩ release in pulmonary artery smooth muscle, leading to constriction in isolated rabbit pulmonary arteries. 15 In the present investigation, we demonstrate that HPV is triggered and maintained by cADPR in isolated rat pulmonary arteries and in the rat lung in situ. Materials and Methods DissectionMale Wistar rats (250 to 350 g) were anesthetized with 4% enflurane and exsanguinated. The heart and lungs were removed and placed in chilled physiological saline solution A containing (in mmol/L): 118 NaCl...

In a significant proportion of melanoma patients, CTL specific for the melan-A26/7–35 epitope can be detected in peripheral blood using HLA-A2/peptide tetramers. However, the functional capacity of these CTL has been controversial, since although they prove to be effective killers after in vitro expansion, in some patients they have blunted activation responses ex vivo. We used phenotypic markers to characterize melan-A tetramer+ cells in both normal individuals and melanoma patients, and correlated these markers with ex vivo assays of CTL function. Melanoma patients with detectable melan-A tetramer+ cells in peripheral blood fell into two groups. Seven of thirteen patients had a CCR7+ CD45R0− CD45RA+ phenotype, the same as that found in some healthy controls, and this phenotype was associated with a lack of response to melan-A peptide ex vivo. In the remaining six patients, melan-A tetramer+ cells were shifted toward a CCR7− CD45R0+ CD45RA− phenotype, and responses to melan-A peptide could be readily demonstrated ex vivo. When lymph nodes infiltrated by melan-A-expressing melanoma cells were examined, a similar dichotomy emerged. These findings demonstrate that activation of melan-A-specific CTL occurs in only some patients with malignant melanoma, and that only patients with such active immune responses are capable of responding to Ag in ex vivo assays.

1. An outward current (IK(N)) was identified in rabbit pulmonary artery myocytes, which persisted after Ca2+-activated and ATP-sensitive K+ currents were blocked by TEA (10 mM) and glibenclamide (10 /M), respectively, and after A-like (IK(A)) and delayed rectifier (IK(v)) K+ currents were inactivated by clamping the cell at 0 mV for > 10 min. It was found in smooth muscle cells at all levels of the pulmonary arterial tree. by 51 and 47 %, respectively. 5. Activation of IK(N) was detected at potentials close to the resting membrane potential of pulmonary artery smooth muscle cells, under physiological conditions. Thus it is likely to contribute to the resting membrane potential of these cells.