Immunoglobulin G-induced single ionic channels in human alveolar macrophage membranes.

While it is well known that the engagement of IgG Fc receptors on the macrophage surface triggers a number of cellular responses, including particle ingestion, secretion, and respiratory burst activity, the mechanism of signal transmission following ligand binding remains poorly understood. To acquire more data in this area, we studied the electrical properties of the macrophage membrane and its response to oligomeric immunoglobulin G (IgG) using the patch-clamp technique on human alveolar macrophages that were obtained by bronchoalveolar lavage and maintained in short-term tissue culture. The results showed that cell resting potentials, as determined from whole-cell tight seal recordings, increased from -15 mV on the day of plating to -56 mV after the first day in culture and remained stable at this hyperpolarized level. Macrophages revealed an input resistance of 3.3 G omega, independent of age in culture. Extracellular application of heat-aggregated human IgG to cells voltage-clamped at -70 mV resulted in peak inward currents of approximately 470 pA. We identified an IgG-dependent, nonselective channel in both cell-attached and isolated membrane patches, with a unitary conductance of approximately 350 pS and a predominant subconductance level of 235 pS in symmetrical NaCl solutions. Single channel open times were observed to be in the range of seconds and, in addition, were dependent upon membrane voltage. Channel opening involved transitions between a number of kinetic states and subconductance levels. Channel events recorded in cell-attached patches showed characteristic exponential relaxations, which implied a variation in membrane potential as a result of a single ion channel opening. These data suggest that the IgG-dependent nonselective cation channel that we have characterized may provide the link between Fc receptor engagement and subsequent cellular activation.

Ibuprofen in canine endotoxin shock.

The participation of prostaglandins in the physiologic alterations of endotoxin shock has been well established with the aid of prostaglandin synthetase inhibitors. Our study was designed to investigate the potential of ibuprofen, a highly specific cyclooxygenase inhibitor, to reverse the hemodynamic and acid base abnormalities of canine endotoxin shock. Mean blood pressure fell to 49.8 +/- 6.6 mm Hg in dogs given endotoxin by 5 min after injection, and remained below 83 mm Hg for the duration of the 120-min observation period. In animals given endotoxin followed by ibuprofen, a similar initial drop of systemic blood pressure was seen, but it subsequently recovered to 150.2 +/- 4.1 mm Hg by 120 min (P less than 0.001). Cardiac index increased in animals given ibuprofen (2.3 +/- 0.28 liter/m2 per min) compared with animals given endotoxin alone (1.0 +/- 0.09 liter/m2 per min) by termination of the experiment. The arterial pH dropped in endotoxin treated animals to 7.18 +/- 0.03 by 120 min. Ibuprofen prevented the acidosis, the final pH in ibuprofen and endotoxin treated animals measuring 7.36 +/- 0.01. We conclude that ibuprofen protects against the hypotension, acidosis, and depression of cardiac index of canine endotoxin shock.

Contribution of epoxyeicosatrienoic acids to the hypoxia-induced activation of Ca2+ -activated K+ channel current in cultured rat hippocampal astrocytes.

Brief hypoxia differentially regulates the activities of Ca(2+)-activated K(+) channels (K(Ca)) in a variety of cell types. We investigated the effects of hypoxia (<2% O(2)) on K(Ca) channel currents and on the activities of cytochrome P450 2C11 epoxygenase (CYP epoxygenase) in cultured rat hippocampal astrocytes. Exposure of astrocytes to hypoxia enhanced macroscopic outward K(Ca) current, increased the open state probability (NPo) of 71 pS and 161 pS single-channel K(Ca) currents in cell-attached patches, but failed to increase the NPo of both the 71 pS and 161 pS K(Ca) channel currents recorded from excised inside-out patches. The hypoxia-induced enhancement of macroscopic K(Ca) current was attenuated by pretreatment with tetraethylammonium (TEA, 1 mM) or during recording using low-Ca(2+) external bath solution. Exposure of astrocytes to hypoxia was associated with generation of superoxide as detected by staining of cells with the intracellular superoxide detection probe hydroethidine (HE), attenuation of the hypoxia-induced activation of unitary K(Ca) channel currents by superoxide dismutation with tempol, and as quantitated by high-pressure liquid chromatography/fluorescence assay using HE as a probe. In cultured astrocytes in which endogenous CYP epoxygenase activity has been inhibited with either miconazole or N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MSPPOH) hypoxia failed to increase the NPo of both the 71 pS and 161 pS K(Ca) currents and generation of superoxide. Hypoxia increased the level of P450 epoxygenase protein and production of epoxyeicosatrienoic acids (EETs) from cultured astrocytes, as determined by immunohistochemical staining and LC/MS analysis, respectively. Exogenous 11,12-EET increased the NPo of both the 71 pS and 161 pS K(Ca) single-channel currents only in cell-attached but not in excised inside-out patches of cultured astrocytes. These findings indicate that hypoxia enhances the activities of two types of unitary K(Ca) currents in astrocytes by a mechanism that appears to involve CYP epoxygenase-dependent generation of superoxide and increased production or release of EETs.

Research on ibuprofen for sepsis and respiratory failure.

According to an experimental study in 26 dogs, ibuprofen reversed the hypotension, increased the cardiac index, prevented the acidosis associated with endotoxic shock, and apparently improved survival. In animals given endotoxin followed by ibuprofen, an initial decrease in systemic blood pressure subsequently recovered to 150.2 +/- 4.1 mm Hg in 120 minutes (p less than 0.001). Cardiac index increased in ibuprofen-treated animals (2.3 +/- 0.28 1/m2 per minute), compared with animals given endotoxin alone (1.0 +/- 0.09 1/m2 per minute) by termination of the experiment. In addition, although arterial pH decreased to 7.18 +/- 0.03 by 120 minutes in animals given only endotoxin, final pH was 7.36 +/- 0.01 in the ibuprofen-treated group.

Response of the lungs to aspiration.

Aspiration of acid from the stomach and water from the mouth can cause significant lung injury. Animal experiments suggest that acid entering the lungs is normally neutralized by bicarbonate derived from the plasma. It is hypothesized that this process may be impaired in patients with cystic fibrosis and that some of the airway injury that they experience may be related to this defect. This disease is characterized by abnormalities in the cystic fibrosis transmembrane conductance regulator, which normally conducts bicarbonate and chloride exchange. Evidence is discussed regarding the role of water channels (aquaporins) in transporting water from the airspaces into the vasculature.

Water transport and the distribution of aquaporin-1 in pulmonary air spaces.

Recent evidence suggests that water transport between the pulmonary vasculature and air spaces can be inhibited by HgCl2, an agent that inhibits water channels (aquaporin-1 and -5) of cell membranes. In the present study of isolated rat lungs, clearances of labeled (3HOH) and unlabeled water were compared after instillation of hypotonic or hypertonic solutions into the air spaces or injection of a hypotonic bolus into the pulmonary artery. The clearance of 3HOH between the air spaces and perfusate after intratracheal instillation and from the vasculature to the tissues after pulmonary arterial injections was invariably greater than that of unlabeled water, indicating that osmotically driven transport of water is limited by permeability of the tissue barriers rather than the rate of perfusion. Exposure to 0.5 mM HgCl2 in the perfusate and air-space solution reduced the product of the filtration coefficient and surface area (PfS) of water from the air spaces to the perfusate by 28% after instillation of water into the trachea. In contrast, perfusion of 0.5 mM HgCl2 in air-filled lungs reduced PfS of the endothelium by 86% after injections into the pulmonary artery, suggesting that much of the action of this inhibitor is on the endothelial surfaces. Confocal laser scanning microscopy demonstrated that aquaporin-1 is on mouse pulmonary endothelium. No aquaporin-1 was found on alveolar type I cells with immunogold transmission electron microscopy, but small amounts were present on some type II cells.

Stop-flow studies of solute uptake in rat lungs.

Stop-flow studies were used to characterize solute uptake in isolated rat lungs. These lungs were perfused at 8 or 34 ml/min for 10-28 s with solutions containing 125I-albumin and two or more of the following diffusible indicators: [3H]mannitol, [14C]urea, 3HOH, 201Tl+, or 86Rb+. After this loading period, flow was stopped for 10-300 s and then resumed to flush out the perfusate that remained in the pulmonary vasculature during the stop interval. Concentrations of 201Tl+ and 86Rb+ in the venous outflow decreased after the stop interval, indicating uptake from exchange vessels during the stop interval. The amount of these K+ analogs lost from the circulation during the stop interval was greater when the intervals were longer. However, losses of 201T1+ at 90 s approached those at 300 s. Because extraction continued after the vasculature had been flushed, vascular levels had presumably fallen to negligible levels during the stop interval. By 90 s of stop flow the vascular volume that was cleared of 201T1+ averaged 0.657 +/- 0.034 (SE) ml in the experiments perfused at 8 ml/min and 0.629 +/- 0.108 ml in those perfused at 34 ml/min. Increases in perfusate K+ decreased the cleared volumes of 201T1+ and 86Rb+. Uptake of [3H]mannitol, [14C]urea, and 3HOH during the stop intervals was observed only when the lungs were loaded at high flow for short intervals. Decreases in 201T1+ and 86Rb+ concentrations in the pulmonary outflow can be used to identify the fraction of the collected samples that were within exchange vessels of the lung during the stop interval and may help determine the distribution of solute and water exchange along the pulmonary vasculature.

Effects of chronic pulmonary overcirculation on pulmonary vasomotor tone.

BACKGROUND: A model of shunt-induced pulmonary hypertension was used to study the effects of pulmonary overcirculation on endothelial nitric oxide synthase (eNOS) and cytochrome P450-4A (cP450-4A) vasodilatory mechanisms and related hemodynamic responses. METHODS: An aortopulmonary shunt was constructed in 6-week-old piglets (n = 7, sham-operated controls n = 8). Hemodynamic measurements were made 4 weeks later under serial experimental conditions: baseline (fractional concentration of oxygen, 0.4); inhaled nitric oxide, 25 ppm (INO); hypoxia (fractional concentration of oxygen, 0.14); hypoxia + INO; N(omega)-nitro-L-arginine methylester (L-NAME 30 mg/kg intravenously, competitive NOS inhibitor); and L-NAME + INO. Lung protein levels of eNOS and cP450-4A and NOS activity were compared between groups. RESULTS: Shunted animals had a higher baseline pulmonary artery pressure (p < 0.05). L-NAME resulted in a greater increase in pulmonary vascular resistance in shunted animals (150% +/- 26% shunt versus 69% +/- 14% control; p = 0.01). The INO administered during baseline conditions decreased pulmonary vascular resistance only in control animals (p < 0.05). Protein levels of eNOS and NOS activity were similar in both groups; however, cP450-4A protein levels were decreased in the shunted group (p = 0.02). CONCLUSIONS: The NO production was preserved in shunted animals but they demonstrated greater vasodilatory dependence on NO, evidenced by an exaggerated increase in pulmonary vascular resistance after NOS inhibition. Loss of the cP450-4A vasodilatory system may be the driving force for NO dependency in the shunted pulmonary circulation.

Clinical indicators in sepsis and septic adult respiratory distress syndrome.

Sepsis and septic ARDS remain clinical problems of great significance because of the numbers of patients affected each year and the high mortality associated with development of the syndrome. The standard therapies for these conditions, judicious antibiotic administration and supportive care, continue to be the mainstays of treatment for these patients, but mortality even with optimal conventional therapy is between 50% and 90% for septic ARDS. The mortality for an individual patient may be anticipated to be substantially higher or lower than these average reported values, based on the presence or absence of several clearly identified risk factors, such as advanced age, shock, evidence of multiorgan system failure, and others discussed above. Similarly, the likelihood that the septic patient will develop ARDS is increased by the appearance of shock and thrombocytopenia. Two therapies that are used extensively in the intensive care unit today--corticosteroid administration and PEEP--have not been shown to reduce the overall mortality of sepsis or septic ARDS. Newer therapeutic modalities, designed to protect against or reverse cardiovascular consequences of sepsis, reduce the incidence of multiorgan system failure, and diminish the high incidence of uncontrolled infections in these patients, are needed; investigations of these interventions are in progress.

Mediators of septic lung injury.

Septic pulmonary injury remains a significant cause of morbidity and mortality among hospitalized patients today and is likely to increase in prevalence as advances in medical technology allow the salvage of more critically ill and immunocompromised hosts. Treatment of the host's underlying disease and even of the infection itself has appeared to redeem septic patients only to have them succumb in increasing numbers to the pulmonary injury reaction. Our understanding of the mechanisms and mediators of lung dysfunction in sepsis is in a rapidly expanding phase. Currently we recognize the contributions of several blood elements, lipids, and peptides to pulmonary injury, although the relative importance and points of interaction and interdependence of these mediators remain to be established. It is hoped that a more complete understanding of the process of pulmonary injury in sepsis will suggest effective means of intervention at a stage in which damage may be reversed or minimized.

Ion channels in the metabolic regulation of respiratory cells.

This article has focused on the characteristics of ion channels in cells of the respiratory system. Ion channels and their role in transepithelial fluid movement are best understood in tracheal epithelial cells. The bulk of the evidence indicates that a Cl- channel abnormality is etiologically involved in CF. In other cell types, such as isolated type II alveolar epithelial and vascular endothelial cells, ion channels have been described, but their functional significance is only incompletely understood. Finally, the majority of cells in the lungs has yet to be studied electrophysiologically. It is hoped that eventually studies of channel properties may enable investigators to determine how the channels affect cell function.

Serotonin and serotonin receptor expression in the aorta of carotid intact and denervated newborns.

Pharmacological blocking of serotonin (5-HT) 5A receptors abolishes aortic ventilatory chemosensitivity of carotid body denervated (CBD) piglets [J. Appl. Physiol. 92 (2002) 893]. Accordingly, the purpose of the present study was to determine whether 5-HT and 5-HT receptors exist at aortic sites that are chemosensitive after CBD. Aortas from CBD and sham CBD rats and piglets and from aortic denervated (AOD) and combined AOD+CBD piglets were harvested, sectioned and then studied using immunohistochemistry and western blot techniques. 5-HT immunoreactivity in piglets and rats was concentrated in the endothelium and sub-endothelial areas in several aortic regions studied, and in some areas also in the adventitia. At the aortic chemosensitive site (descending aorta in CBD piglets and the ascending aorta in CBD rats), the immunoreactivity was greater (P < 0.05) than in other aortic regions and greater than in other groups studied. The 5-HT(5a) receptor was expressed only at the chemosensitive sites and only in aortic innervated piglets. We conclude that the data from this and a previous study [J. Appl. Physiol. 92 (2002) 893] suggest that a serotonergic mechanism contributes to the aortic ventilatory chemoreflex after CBD.

Therapeutic implications of acute lung injury.

Although there are no specific therapies for septic shock or acute lung injury that have proven efficacy in humans, a growing understanding of mechanisms of tissue injury has suggested interventions that may prevent or treat this injury. These therapies range from immunization against the glycopolysaccharide core of endotoxin to cyclooxygenase inhibitors to specific oxygen radical scavengers. Each of these treatments is effective in ameliorating at least one of the pathophysiologic manifestations of acute lung injury, although the effect of these agents in the prevention of the sequelae of fibrosis is unknown. Interaction between several factors and mediators is likely necessary for the development of acute lung injury. It is hoped that with additional knowledge regarding mechanisms of injury gained through basic science and clinical research, we can apply definitive therapy that may salvage patients who now die with sepsis and acute lung injury.

Novel Flurometric Tool to Assess Mitochondrial Redox State of Isolated Perfused Rat Lungs after Exposure to Hyperoxia.

Recently we demonstrated the utility of optical fluorometry to detect a change in the redox status of mitochondrial autofluorescent coenzymes NADH (Nicotinamide Adenine Dinucleotide) and FAD (oxidized form of Flavin Adenine Dinucleotide (FADH2,)) as a measure of mitochondrial function in isolated perfused rat lungs (IPL). The objective of this study was to utilize optical fluorometry to evaluate the effect of rat exposure to hyperoxia (>95% O2 for 48 hours) on lung tissue mitochondrial redox status of NADH and FAD in a nondestructive manner in IPL. Surface NADH and FAD signals were measured before and after lung perfusion with perfusate containing rotenone (ROT, complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor), and/or pentachlorophenol (PCP, uncoupler). ROT- or KCN-induced increase in NADH signal is considered a measure of complex I activity, and KCN-induced decrease in FAD signal is considered a measure of complex II activity. The results show that hyperoxia decreased complex I and II activities by 63% and 55%, respectively, as compared to lungs of rats exposed to room air (normoxic rats). Mitochondrial complex I and II activities in lung homogenates were also lower (77% and 63%, respectively) for hyperoxic than for normoxic lungs. These results suggest that the mitochondrial matrix is more reduced in hyperoxic lungs than in normoxic lungs, and demonstrate the ability of optical fluorometry to detect a change in mitochondrial redox state of hyperoxic lungs prior to histological changes characteristic of hyperoxia.

Acute effects of prostaglandin E1 and E2 on vascular reactivity and blood flow in situ in the chick chorioallantoic membrane.

The chick chorioallantoic membrane (CAM) subserves gas exchange in the developing embryo and shell-less culture affords a unique opportunity for direct observations over time of individual blood vessels to pharmacologic interventions. We tested a number of lipids including prostaglandins PGE(1&2) for vascular effects and signaling in the CAM. Application of PGE(1&2) induced a decrease in the diameter of large blood vessels and a concentration-dependent, localized, reversible loss of blood flow through small vessels. The loss of flow was also mimicked by misoprostol, an agonist for 3 of 4 known PGE receptors, EP(2-4), and by U46619, a thromboxane mimetic. Selective receptor antagonists for EP(3) and thromboxane each partially blocked the response. This is a first report of the effects of prostaglandins on vasoreactivity in the CAM. Our model allows the unique ability to examine simultaneous responses of large and small vessels in real time and in vivo.

Vascular injury after whole thoracic x-ray irradiation in the rat.

PURPOSE: To study vascular injury after whole thoracic irradiation with single sublethal doses of X-rays in the rat and to develop markers that might predict the severity of injury. METHODS AND MATERIALS: Rats that received 5- or 10-Gy thorax-only irradiation and age-matched controls were studied at 3 days, 2 weeks, and 1, 2, 5, and 12 months. Several pulmonary vascular parameters were evaluated, including hemodynamics, vessel density, total lung angiotensin-converting enzyme activity, and right ventricular hypertrophy. RESULTS: By 1 month, the rats in the 10-Gy group had pulmonary vascular dropout, right ventricular hypertrophy, increased pulmonary vascular resistance, increased dry lung weights, and decreases in total lung angiotensin-converting enzyme activity, as well as pulmonary artery distensibility. In contrast, irradiation with 5 Gy resulted in only a modest increase in right ventricular weight and a reduction in lung angiotensin-converting enzyme activity. CONCLUSION: In a previous investigation using the same model, we observed that recovery from radiation-induced attenuation of pulmonary vascular reactivity occurred. In the present study, we report that deterioration results in several vascular parameters for

The lung HETEs (and EETs) up.

Arachidonic acid metabolites of the cyclooxygenase and lipoxygenase pathways have a variety of important lung functions. Recent observations indicate that cytochrome P-450 (P-450) monooxygenases are also expressed in the lung, localized to specific pulmonary cell types (e.g., epithelium, endothelium, and smooth muscle), and may modulate critical lung functions. This review summarizes recent data on the presence and biological activity of P-450-derived eicosanoids in the pulmonary vasculature and airways, including effects on pulmonary vascular and bronchial smooth muscle tone and airway epithelial ion transport. We hypothesize a number of potential functions of P-450-derived arachidonate metabolites in the lungs such as contribution to hypoxic pulmonary vasoconstriction, regulation of bronchomotor tone, control of the composition of airway lining fluid, and limitation of pulmonary inflammation. Finally, we describe a number of emerging technologies, including congenic and transgenic strains of experimental animals, P-450 isoform-specific inhibitors and inhibitory antibodies, eicosanoid analogs, and vectors for delivery of P-450 cDNAs and antisense oligonucleotides. These tools will facilitate further studies on the contribution of endogenously formed P-450 eicosanoid metabolites to lung function, under both normal and pathological conditions.