John R. Michael

Cytokine-induced expression of a nitric oxide synthase in rat renal tubule cells.

Nitric oxide (NO.) has been implicated in the regulation of renal vascular tone and tubular sodium transport. While the endothelial cell is a well known source of NO(.), recent studies suggest that tubular epithelial cells may constitutively generate NO(.). An inducible isoform of nitric oxide synthase which produces far greater quantities of NO. exists in some cell types. We sought to determine whether kidney epithelial cells exposed to cytokines could express an inducible nitric oxide synthase. Primary cultures of rat proximal tubule and inner medullary collecting duct cells generated NO. on exposure to TNF-alpha and IFN-gamma. NO. production by both cell types was inhibited by NG-monomethyl-L-arginine; this inhibition was partially reversed by the addition of excess L-arginine. Stimulation of kidney epithelial cells with TNF-alpha and IFN-gamma dramatically increased the level of inducible nitric oxide synthase mRNA. In summary, renal proximal tubule and inner medullary collecting duct cells can produce NO. via expression of an inducible isoform of nitric oxide synthase.

Regulation of xanthine dehydrogenase and xanthine oxidase activity and gene expression in cultured rat pulmonary endothelial cells.

The central importance of xanthine dehydrogenase (XDH) and xanthine oxidase (XO) in the pathobiochemistry of a number of clinical disorders underscores the need for a comprehensive understanding of the regulation of their expression. This study was undertaken to examine the effects of cytokines on XDH/XO activity and gene expression in pulmonary endothelial cells. The results indicate that IFN-gamma is a potent inducer of XDH/XO activity in rat lung endothelial cells derived from both the microvasculature (LMVC) and the pulmonary artery. In contrast, interferon-alpha/beta, tumor necrosis factor-alpha, interleukin-1 or -6, lipopolysaccharide and phorbol myristate acetate have no demonstrable effect. The increase in XDH/XO activity requires new protein synthesis. By Northern analysis, IFN-gamma markedly increases the level of the 5.0-kb XDH/XO mRNA in LMVC. The increase is due, in part, to increased transcription rate of the XDH/XO gene. Transcriptional activation does not require new protein synthesis. The physiologic relevance of these observations was evaluated by administering IFN-gamma to rats. Intraperitoneal administration leads to an increased XDH/XO activity and XDH/XO mRNA level in rat lungs. In sum, IFN-gamma is a potent and biologically relevant inducer of XDH/XO expression; the major site of upregulation occurs at the transcriptional level.

Targets of oxidative stress in cardiovascular system

Although oxidants such as superoxide (O2.-) and hydrogen peroxide (H2O2) play a role in host-mediated destruction of foreign pathogens yet excessive generation of oxidants may lead to a variety of pathological complications in the cardiovascular system. An important mechanism by which oxidants cause dysfunction of the cardiovascular system appears to be due to the increase in intracellular free Ca2+ concentration. Oxidants cause cellular Ca2+ mobilization by modulating activities of a variety of regulators such as Na+/H+ and Na+/Ca2+ exchangers, Na+/K+ ATPase and Ca2+ ATPase and Ca2+ channels that are associated with Ca2+ transport in the plasma membrane and the sarco(endo)plasmic reticular membrane of myocardial cells. Recent research have suggested that the increase in Ca2+ level by oxidants plays a pivotal role in indicing several protein kinases such as protein kinase C, tyrosine kinase and mitogen activated protein kinases. Oxindant-mediated alteration of different signal transduction systems and their interations eventually regulate a variety of pathological conditoins such as atherosclerosis, apoptosis and necrosis in the myocardium

Ibuprofen prevents oxidant lung injury and in vitro lipid peroxidation by chelating iron.

Because ibuprofen protects from septic lung injury, we studied the effect of ibuprofen in oxidant lung injury from phosgene. Lungs from rabbits exposed to 2,000 ppm-min phosgene were perfused with Krebs-Henseleit buffer at 50 ml/min for 60 min. Phosgene caused no increase in lung generation of cyclooxygenase metabolites and no elevation in pulmonary arterial pressure, but markedly increased transvascular fluid flux (delta W = 31 +/- 5 phosgene vs. 8 +/- 1 g unexposed, P less than 0.001), permeability to albumin (125I-HSA) lung leak index 0.274 +/- 0.035 phosgene vs. 0.019 +/- 0.001 unexposed, P less than 0.01; 125I-HSA lavage leak index 0.352 +/- 0.073 phosgene vs. 0.008 +/- 0.001 unexposed, P less than 0.01), and lung malondialdehyde (50 +/- 7 phosgene vs. 24 +/- 0.7 mumol/g dry lung unexposed, P less than 0.01). Ibuprofen protected lungs from phosgene (delta W = 10 +/- 2 g; lung leak index 0.095 +/- 0.013; lavage leak index 0.052 +/- 0.013; and malondialdehyde 16 +/- 3 mumol/g dry lung, P less than 0.01). Because iron-treated ibuprofen failed to protect, we studied the effect of ibuprofen in several iron-mediated reactions in vitro. Ibuprofen attenuated generation of .OH by a Fenton reaction and peroxidation of arachidonic acid by FeCl3 and ascorbate. Ibuprofen also formed iron chelates that lack the free coordination site required for iron to be reactive. Thus, ibuprofen may prevent iron-mediated generation of oxidants or iron-mediated lipid peroxidation after phosgene exposure. This suggests a new mechanism for ibuprofens action.

Efficacy and safety of naloxone in septic shock

We evaluated the effectiveness and safety of iv naloxone in 12 septic patients who remained hypotensive despite volume replacement, appropriate antibiotics, and vasopressor therapy. Only four patients responded positively to naloxone, by increases in mean arterial pressure of between 10 to 15 mm Hg that lasted for 15 to 60 min. These patients could not be distinguished from the others on the basis of underlying illness, laboratory or physical findings, length of preceding hypotension, or glucocorticoid therapy. Four patients had adverse reactions: one developed pulmonary edema, one patient had a grand-mal seizure, and two patients became severely hypotensive. We conclude that in patients with well-established septic shock, naloxone does not reliably improve mean arterial pressure or other physiologic variables, and may cause severe adverse reactions.

Oxidant stress regulates basal endothelin-1 production by cultured rat pulmonary endothelial cells

Endothelin-1 (ET-1) is a pluripotent mediator that modulates vascular tone and influences the inflammatory response. Patients with inflammatory lung disorders frequently have elevated circulating ET-1 levels. Because these pathophysiological conditions generate reactive oxygen species that can regulate gene expression, we investigated whether the level of oxidant stress influences ET-1 production in cultured rat pulmonary arterial endothelial cells (RPAEC). Treatment with the antioxidant 1,3-dimethyl-2-thiourea (10 mM) or the iron chelator deferoxamine (1.8 μM) doubles basal ET-1 release. Conversely, exposing cells to H2O2generated by glucose and glucose oxidase (0.1-10 mU/ml) for 4 h causes a concentration-dependent decrease in ET-1 release. This effect occurs at concentrations of glucose oxidase that do not affect [3H]leucine incorporation or specific 51Cr release from RPAEC. Catalase prevents the decrease in ET-1 synthesis caused by glucose and glucose oxidase. Glucose and glucose oxidase decrease not only ET-1 generation but also ET-1 mRNA as assessed by semiquantitative polymerase chain reaction. Our results indicate that changes in oxidative stress can either up- or downregulate basal ET-1 generation by cultured pulmonary endothelial cells.

Regulation of endothelin-1 synthesis in human pulmonary arterial smooth muscle cells effects of transforming growth factor-β and hypoxia

OBJECTIVE Endothelin-1 (ET-1) potently regulates pulmonary vascular tone and promotes vascular smooth muscle cell growth. Clinical and animal studies implicate increased ET-1 production in the pathogenesis of primary and secondary pulmonary hypertension. Although pulmonary arterial smooth muscle cells (PASMCs) synthesize ET-1 under basal conditions, it is unknown whether factors that may be important in pulmonary hypertension, such as transforming growth factor-beta (TGF-beta) or hypoxia, augment ET-1 production by these cells. METHODS We determined the effect of TGF-beta and hypoxia on ET-1 release and preproET-1 mRNA from cultured rat and human PASMCs. RESULTS In the basal state, rat and human PASMCs synthesize, on average (mean+/-S.E.M.), 872+/-114 and 563+/-57 pg ET-1/mg cell protein over 24 h, respectively, a level that causes autocrine and paracrine effects in other tissues. TGF-beta significantly increases the expression of preproET-1 mRNA and ET-1 production by both rat and human PASMCs. Hypoxia for 24 h, however, does not affect ET-1 release from rat or human PASMCs. CONCLUSIONS Cultured rat and human PASMCs are a source of ET-1 production. Enhanced ET-1 release from PASMCs may contribute to the pathophysiology of TGF-beta-induced pulmonary hypertension. ET-1 production by PASMCs is unlikely to contribute to the role of ET-1 in hypoxia-induced pulmonary vasoconstriction.

Role of protein kinase C in oxidant — mediated activation of phospholipase A2 in rabbit pulmonary arterial smooth muscle cells

The present study was undertaken to test the hypothesis that activation of cell membrane associated protein kinase C (PKC) plays a role in stimulating cell membrane associated phospholipase A2 (PLA2) activity, and subsequent liberation of arachidonic acid (AA) under exposure of rabbit pulmonary arterial smooth muscle cells to the oxidant hydrogen peroxide (H2O2). Exposure of the smooth muscle cells to H2O2 dose-dependently stimulates [14C] AA release, and enhances the cell membrane associated PLA2 activity. Pretreatment of the cells with protein kinase C (PKC) inhibitors H7 and sphingosine prevent the cell membrane associated PLA2 activity, and AA release caused by H2O2. Treatment of the smooth muscle cells with H2O2 stimulates the cell membrane associated PKC activity. Pretreatment of the cells with an antioxidant vitamin E prevents H2O2 caused stimulation of the cell membrane associated PKC activity. The cell membrane associated PLA2 and PKC activities correlate linearly. These results suggest that H2O2 caused stimulation of the smooth muscle cell membrane associated PLA2 activity, and subsequent liberation of AA can occur through an increase in the activity of the cell membrane associated PKC. (Mol Cell Biochem122: 9–15, 1993)

ET-1 modulates KCa-channel activity and arterial tension in normoxic and hypoxic human pulmonary vasculature

The molecular mechanisms by which endothelin (ET)-1 induces pulmonary hypertension are poorly understood. We investigated the effects of ET-1 on outward K+ currents of normoxic and chronically hypoxic human pulmonary arterial (PA) smooth muscle cells (HPSMCs). In normoxic HPSMCs, ET-1 has dual effects. In intact cells, 5 nM ET-1 activates the large-conductance and Ca2+-activated K+ (KCa)-channel current [IK(Ca)] by increasing intracellular Ca2+ concentration, whereas it directly inhibits IK(Ca) in isolated membrane patches. At a higher concentration (10 nM), ET-1-induced IK(Ca) inhibition predominates. In hypoxic HPSMCs, ET-1 at 5 nM significantly reduces IK(Ca). The ETA-receptor antagonist BQ-123 reverses the ET-1-induced decrease in IK(Ca). Chronic BQ-123 treatment also prevents the hypoxia-induced decrease in IK(Ca). In PA rings obtained from human organ donors, ET-1 causes a concentration-dependent increase in tension. The ET-1-mediated increase in tension is reversed by a KCa-channel agonist. The increase in tension at the highest concentration studied (9 nM) was more pronounced in PA rings obtained from patients with chronic obstructive pulmonary disease. These results imply that an ET-1-induced decrease in IK(Ca) contributes to chronic hypoxia-induced pulmonary hypertension.The molecular mechanisms by which endothelin (ET)-1 induces pulmonary hypertension are poorly understood. We investigated the effects of ET-1 on outward K+ currents of normoxic and chronically hypoxic human pulmonary arterial (PA) smooth muscle cells (HPSMCs). In normoxic HPSMCs, ET-1 has dual effects. In intact cells, 5 nM ET-1 activates the large-conductance and Ca2+-activated K+(KCa)-channel current [ I K(Ca)] by increasing intracellular Ca2+concentration, whereas it directly inhibits I K(Ca) in isolated membrane patches. At a higher concentration (10 nM), ET-1-induced I K(Ca) inhibition predominates. In hypoxic HPSMCs, ET-1 at 5 nM significantly reduces I K(Ca). The ETA-receptor antagonist BQ-123 reverses the ET-1-induced decrease in I K(Ca). Chronic BQ-123 treatment also prevents the hypoxia-induced decrease in I K(Ca). In PA rings obtained from human organ donors, ET-1 causes a concentration-dependent increase in tension. The ET-1-mediated increase in tension is reversed by a KCa-channel agonist. The increase in tension at the highest concentration studied (9 nM) was more pronounced in PA rings obtained from patients with chronic obstructive pulmonary disease. These results imply that an ET-1-induced decrease in I K(Ca)contributes to chronic hypoxia-induced pulmonary hypertension.

Protein kinase C dependent and independent activation of phospholipase A2 under calcium ionophore (A23187) exposure in rabbit pulmonary arterial smooth muscle cells

Exposure of rabbit pulmonary arterial smooth muscle cells to the calcium ionophore A23187, dose‐dependently stimulates arachidonic acid (AA) release and phospholipase A2 (PLA2) activity. The protein kinase C (PKC) inhibitor, sphingosine does not prevents AA release and PLA2 activity caused by low doses of A23187. In contrast, sphingosine markedly prevents AA release and PLA2 activity caused by higher doses of A23187. PKC activity profile indicates that treatment of the cells with low doses of A23187 does not cause significant alteration of PKC translocation from cytosol to membrane whereas higher concentrations of the ionophore dose‐dependently enhance PKC translocation from Cytosol to membrane in the smooth muscle cells.