Biomedical subjects

E Gozal

Publications and source records attributed to E Gozal.

29 records · Page 2Linked to original sources

PubMed1998

Protein kinase C modulation of ventilatory response to hypoxia in nucleus tractus solitarii of conscious rats.

This study aimed to determine the role of protein kinase C (PKC) in signal transduction mechanisms underlying ventilatory regulation in the nucleus tractus solitarii (NTS). Microinjection of phorbol 12-myristate 13-acetate into the commissural NTS of nine chronically instrumented, unrestrained rats elicited significant cardiorespiratory enhancements that lasted for at least 4 h, whereas administration of vehicle (n = 15) or the inactive phorbol ester 4alpha-phorbol 12,13-didecanoate (n = 7) did not elicit minute ventilation (VE) changes. Peak hypoxic VE responses (10% O2-balance N2) were measured in 19 additional animals after NTS microinjection of bisindolylmaleimide (BIM) I, a selective PKC inhibitor (n = 12), BIM V (inactive analog; n = 7), or vehicle (Con; n = 19). In Con, VE increased from 139 +/- 9 to 285 +/- 26 ml/min in room air and hypoxia, respectively, and similar responses occurred after BIM V. BIM I did not affect room air VE but markedly attenuated hypoxia-induced VE increases (128 +/- 12 to 167 +/- 18 ml/min; P < 0. 02 vs. Con and BIM V). When BIM I was microinjected into the cerebellum (n = 4), cortex (n = 4), or spinal cord (n = 4), VE responses were similar to Con. Western blots of subcellular fractions of dorsocaudal brain stem lysates revealed translocation of PKCalpha, beta, gamma, delta, epsilon, and iota isoenzymes during acute hypoxia, and enhanced overall PKC activity was confirmed in the particulate fraction of dorsocaudal brain stem lysates harvested after acute hypoxia. These studies suggest that, in the adult rat, PKC activation in the NTS mediates essential components of the acute hypoxic ventilatory response.

Animals↗

PubMed1998

NF-kappaB induction during in vivo hypoxia in dorsocaudal brain stem of rat: effect of MK-801 and L-NAME.

In the nucleus of the solitary tract, NMDA receptors are critical for the hypoxic ventilatory response while neuronal nitric oxide synthase (NOS) modulates the late component of this response. Nuclear factor (NF)-kappaB is a ubiquitous transcription factor that increases the expression of multiple stress-activated genes. We sought to examine temporal changes in expression of NF-kappaB within the dorsocaudal brain stem of conscious rats after exposures to 10% O2. Time-dependent increases in NF-kappaB occurred with hypoxia and peaked at 60 min. Pretreatment with the N-methyl-D-aspartate (NMDA)-receptor channel antagonist dizocilpine maleate (MK-801) markedly attenuated NF-kappaB complexes during hypoxia. In contrast, after NOS inhibition with NG-nitro-L-arginine methyl ester (L-NAME), although NF-kappaB was diminished in normoxia, increased NF-kappaB expression still occurred with hypoxia. Increased phosphorylation of the NF-kappaB regulatory unit [inhibitory (I)kappaB] was detected by immunoblotting and also peaked at 60 min. Phosphorylation of Ikappa-B during hypoxia was attenuated by MK-801 but not by L-NAME. Thus NMDA-receptor activation in the dorsocaudal brain stem during hypoxia elicits in NF-kappaB activity marked enhancements that are unaffected after NOS blockade.

Animals↗

PubMed1998

Nitric oxide modulates anoxia-induced gasping in the developing rat.

Gasping is an important mechanism for survival. Nitric oxide (NO) plays an excitatory role in brainstem regions mediating respiratory responses to hypoxia. We hypothesized that neural structures mediating anoxia-induced gasping would display NO dependency. Two- to 15-day-old rat pups underwent anoxic exposures with 100% N2 in a plethysmograph following administration of N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) blocker, L-arginine (L-Arg), a NO precursor, or normal saline. In general, gasp latencies were significantly shorter after L-Arg, and were prolonged with L-NAME. Furthermore, NOS inhibition prolonged gasping duration and reduced gasping frequency at all postnatal ages, although this effect was particularly increased with advancing postnatal age. NADPH-diaphorase staining and Western blots of protein lysates from the lateral tegmental field, the putative neural center underlying gasp generation, revealed progressively increased neuronal NOS abundance with animal maturation. We conclude that anoxia-induced gasping neurogenesis is modulated by NO mechanisms in neonatal pups. We postulate that higher NO brainstem concentrations may favor early autoresuscitation but be detrimental to overall survival during prolonged asphyxia.

Animals↗

PubMed1998

Modulation of the alveolar macrophage superoxide production by protein phosphorylation.

Stimulation of alveolar macrophages (AM) with adenosine-5-diphosphate (ADP) results in transient production of superoxide anion radical (O2.-; superoxide) and H2O2 in a metabolic event known as the respiratory burst. Initiation of the respiratory burst appears to depend on activation of protein kinase activity, whereas protein phosphatases might involved in termination of the burst. The involvement of protein kinase C was suggested by inhibition by bisindolylmaleimide I (GF 109203X), a relatively specific inhibitor. KN-62, an inhibitor of calcium-calmodulin protein kinase II, also partly inhibited the respiratory burst stimulated by ADP and phorbol esters. The role of protein phosphatases in termination of the ADP-stimulated respiratory burst of AM was examined with calyculin A (CA) (25-75 nM) or okadaic acid (OA) (1-5 microM), two inhibitors of protein phosphatase 1 and 2a (PP1;PP2a). A dose-dependent prolongation of the respiratory burst was observed in the presence of these inhibitors. CA and OA also markedly enhanced the rate of superoxide production stimulated by ADP, consistent with involvement of PP1/PP2a in regulating both the rate of activation and timing of termination. Treatment of AM with cyclosporin A (CsA) (1-50 microM), an inhibitor of the calcium-dependent protein phosphatase 2b (PP2b), stimulated superoxide production by itself and significantly prolonged the duration of ADP-stimulated superoxide production. CsA, however, did not increase the ADP-stimulated rate of superoxide production. Thus, PP1/PP2a appear to be the primary phosphatases for controlling the intensity of the respiratory burst during receptor-elicited superoxide production in AM, whereas PP1/PP2a and PP2b play a role in turning off the respiratory burst.

Adenosine Diphosphate↗

PubMed1997

Hypoxic ventilatory roll-off is associated with decreases in protein kinase C activation within the nucleus tractus solitarius of the rat.

Protein kinase C (PKC) inhibition attenuates ventilatory responses to acute hypoxia. Hypoxic ventilatory roll-off (VRO) could reflect underlying changes in PKC activity. Immunoblots of NTS lysates harvested at peak ventilation revealed subcellular translocations of particular PKC isoforms which coincided with PKC activity elevations. In contrast, reductions in PKC activity occurred during VRO, concomitant with selective decreases in PKCbeta and -delta translocation. Thus, alterations in PKC activity within the NTS which occur over time during hypoxia are isoform-selective and coincide with changes in ventilation.

Animals↗

PubMed1997

Nitric oxide modulates ventilatory responses to hypoxia in the developing rat.

Nitric oxide (NO) is an important excitatory neurotransmitter in the central nervous system. In the adult rat, both selective and nonselective blockers of constitutive nitric oxide synthase (NOS) induce marked ventilatory reductions during sustained hypoxia, thereby enhancing ventilatory roll-off. Since hypoxic ventilatory depression is greater in developing mammals during the late phases of hypoxic exposure, we hypothesized that limited NOS activity may play a role in the late arm of the ventilatory response. To test our hypothesis, 5-d-, 10-d-, and 15-d-old rat pups underwent a 30-min hypoxic challenge (10% O2) before and after administration of 100 mg/kg N-nitro-L-arginine methyl ester (L-NAME), a competitive NOS inhibitor. Minute ventilation (VE) was measured using whole-body plethysmography. In 5-d-old pups, early VE hypoxic responses were enhanced, and late VE were similar after administration of L-NAME. In contrast, in 15-d-old hypoxic pups, L-NAME administration was associated with smaller early VE increments and significantly larger VE reductions when compared with pretreatment conditions. The role of central nervous system NO in the development of these ventilatory changes was further assessed by Western blots of protein equivalents from the nucleus tractus solitarius (NTS), the first central relay for peripheral chemoreceptor afferent input, which revealed increasing neuronal NOS expression with age. Furthermore, NADPH-diaphorase immunohistochemical staining of neurons in the NTS revealed increased positively labeled neuronal populations within subnuclei of this structure with advancing postnatal age. Current findings suggest that NOS activity mediates both excitatory and inhibitory components of the hypoxic ventilatory response. Furthermore, in brainstem respiratory regions, NO may play a role in modulating the prominent second phase of the biphasic response to hypoxia typically seen in early postnatal life.

Aging↗

PubMed1996

Nitric oxide synthase isoforms and peripheral chemoreceptor stimulation in conscious rats.

To test the effect of nitric oxide synthase (NOS) blockade on the ventilatory responses to carotid body chemoreceptor stimulation in freely behaving animals, chronically instrumented adult Sprague-Dawley rats received increasing intravenous doses of sodium cyanide (NaCN; 0-300 micrograms kg-1) before and after i.v. administration of either 100 mg kg-1 N-nitro-L-arginine methyl ester (L-NAME), a non-specific NOS blocker, or 10 mg kg-1 S-methyl-L-thiocitrulline (SMTC), a selective neuronal NOS inhibitor. SMTC did not modify the NaCN dose-response curve. In contrast, L-NAME significantly enhanced the ventilatory responses to NaCN. Western blots of equivalent amounts of protein from carotid body tissue homogenates revealed higher levels of endothelial NOS than of neuronal NOS. We conclude that endothelial NOS provides the major source for NO within the carotid body, and exerts a down-regulatory effect upon peripheral chemoreceptor responsivity.

Animals↗

PubMed1996

Modulation of the rat alveolar macrophage respiratory burst by hydroperoxides is calcium dependent.

Sublethal concentrations of hydroperoxides (H2O2 or tert-butylhydroperoxide) produce a dual effect upon the respiratory burst of rat alveolar macrophages in which low concentrations (< 50 microM) enhance and higher concentrations (> 50 microM) produce inhibition (J. K. Murphy, et al., Free Radical. Biol. Med. 18, 37-45, 1995). These effects correlate with transient versus sustained elevation of [Ca2+]i caused by exposure to hydroperoxides prior to stimulation of the respiratory burst. In the present study changes in [Ca2+]i caused by exposure to sublethal levels of hydroperoxide were buffered by incubating macrophages with the acetoxy-methyl ester of BAPTA, an intracellular Ca2+ chelator. The enhancement of the phorbol ester-stimulated respiratory burst by tBOOH was abolished by BAPTA, while the inhibition was attenuated. Thus, the modulation by tBOOH appears to be largely dependent upon the changes in [Ca2+]i. Receptor mediated stimulation of the respiratory burst (ADP stimulation) involves release of Ca2+ from the inositol-1,4,5-triphosphate (IP3)-sensitive pool in the endoplasmic reticulum. Comparisons were made of the effects of thapsigargin (TG), an endoplasmic reticulum Ca-ATPase inhibitor, with tBOOH on release of intracellular Ca2+ and the respiratory burst. Treatment with TG did not affect changes in [Ca2+]i caused by tBOOH or vice versa. Although TG decreased the ADP-stimulated respiratory burst, it had no effect upon tBOOH modulation. Thus, the effect of tBOOH upon the respiratory burst is dependent upon the release of Ca2+ and the release of Ca2+ occurs from a non-IP3-dependent pool. This aberrant mimicry of normal signal transduction underlies oxidative modulation of the respiratory burst.

Animals↗

PubMed1994

gamma-Glutamyl transpeptidase is increased by oxidative stress in rat alveolar L2 epithelial cells.

The tripeptide glutathione (GSH) is used by cells to detoxify hydroperoxides, produced during oxidative stress, and is consumed in the process. Previous studies have indicated that cells can be protected against oxidative stress by extracellular GSH through its degradation catalyzed by the exoenzyme gamma-glutamyl transpeptidase (gamma GT) and its de novo synthesis within the cytosol. We hypothesized that gamma GT would be increased as part of the adaptation of cells to oxidative stress. We examined whether oxidative stress could increase gamma GT activity, protein, and mRNA in a lung epithelial cell line (L2). Cultures were subjected to H2O2-mediated toxicity by 15 min of exposure to the redox cycling quinone, menadione. Menadione (50 microM) caused an initial decrease (27 +/- 9% of baseline after 15 min) in intracellular GSH, followed by resynthesis to levels significantly higher than baseline (335 +/- 40% after 24 h, P < 0.001). This elevation was prevented by acivicin, a gamma GT inhibitor. Menadione also caused a dose-dependent increase in gamma GT enzymatic activity (715 +/- 125% of control at 24 h after 15 min of exposure to 100 microM menadione, P < 0.001) that was prevented by actinomycin D. Western blot analysis indicated increased levels of gamma GT protein with increasing menadione. A concentration-dependent increase in gamma GT-mRNA was also observed. Previous investigation has demonstrated that an increase in gamma GT activity enhances the capacity of cells to utilize extracellular GSH. The findings presented here are consistent with a role for gamma GT in cellular adaptation to oxidative stress.

Animals↗

PubMed1993

Extracellular glutathione and gamma-glutamyl transpeptidase prevent H2O2-induced injury by 2,3-dimethoxy-1,4-naphthoquinone.

Quinones are intracellular H2O2 generators that have been used extensively in models of oxidant injury; however, their toxicity is mediated partially through direct conjugation with glutathione (GSH). To focus upon the action of extracellular GSH in preventing H2O2-mediated toxicity, we used 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), which cannot conjugate with GSH but does continuously generate H2O2 through redox cycling. A eukaryotic cell line (3T3-GGT) stably overexpressing gamma-glutamyl transpeptidase (GGT) activity was used to study the role of GGT in utilizing extracellular GSH against DMNQ-induced oxidative stress. DMNQ (0 to 150 microM) caused a dose-dependent decrease of intracellular GSH and adenosine 5'-triphosphate (ATP) in both control and 3T3-GGT cells. The rate of H2O2 escape into the medium during DMNQ exposure was also the same in both cell lines. Administration of GSH helped to maintain intracellular GSH and supported resistance to ATP depletion caused by DMNQ in 3T3-GGT cells but not in control cells. The protective effect of extracellular GSH was completely prevented by acivicin, an inhibitor of GGT. Our results suggest that GGT-dependent breakdown of extracellular GSH for subsequent intracellular resynthesis helped to maintain cellular GSH levels and increased cellular resistance against DMNQ-induced oxidative injury.

3T3 Cells↗

PubMed1993

Stimulation of the rat alveolar macrophage respiratory burst by extracellular adenine nucleotides.

Exogenous nucleotides can serve as extracellular factors that cause significant functional changes in numerous cells, including phagocytes. In the current study, addition of ATP, ADP, and ATP gamma S directly stimulated the respiratory burst (superoxide production) by rat alveolar macrophages, whereas adenosine and AMP did not. The relative potency of these nucleotides at saturating concentration was ADP > or = ATP gamma S >> ATP; however, simultaneous addition of maximally stimulatory concentrations of ADP and ATP (100 microM of each) produced an additive effect suggesting involvement of two P2 receptors. Following addition of the nucleotides, an elevation of intracellular Ca2+ ([Ca2+]i) occurred within seconds, followed by a decline within 1 min but with a prolonged elevation above baseline for at least 5 min. Removal of extracellular Ca2+ only slightly attenuated the initial elevation, indicating that adenine nucleotides stimulate the rapid release of intracellular Ca2+ stores. Removal of extracellular Ca2+ also eliminated the sustained elevation in [Ca2+]i and markedly suppressed the respiratory burst. Incubation with verapamil, a Ca2+ channel blocker, also significantly inhibited the respiratory burst stimulated by ATP. These studies demonstrate that adenine nucleotides stimulate a Ca(2+)-dependent respiratory burst by rat alveolar macrophages, probably through purinergic receptors.

Adenine Nucleotides↗