Mary A. Robinson

Oxygen-dependent regulation of nitric oxide production by inducible nitric oxide synthase

Inducible nitric oxide synthase (iNOS) catalyzes the reaction that converts the substrates O(2) and l-arginine to the products nitric oxide (NO) and l-citrulline. Macrophages, and many other cell types, upregulate and express iNOS primarily in response to inflammatory stimuli. Physiological and pathophysiological oxygen tension can regulate NO production by iNOS at multiple levels, including transcriptional, translational, posttranslational, enzyme dimerization, cofactor availability, and substrate dependence. Cell culture techniques that emphasize control of cellular PO(2), and measurement of NO or its stable products, have been used by several investigators for in vitro study of the O(2) dependence of NO production at one or more of these levels. In most cell types, prior or concurrent exposure to cytokines or other inflammatory stimuli is required for the upregulation of iNOS mRNA and protein by hypoxia. Important transcription factors that target the iNOS promoter in hypoxia include hypoxia-inducible factor 1 and/or nuclear factor κB. In contrast to the upregulation of iNOS by hypoxia, in most cell types NO production is reduced by hypoxia. Recent work suggests a prominent role for O(2) substrate dependence in the short-term regulation of iNOS-mediated NO production.

Physiological and hypoxic O2 tensions rapidly regulate NO production by stimulated macrophages

Nitric oxide (NO) production by inducible NO synthase (iNOS) is dependent on O(2) availability. The duration and degree of hypoxia that limit NO production are poorly defined in cultured cells. To investigate short-term O(2)-mediated regulation of NO production, we used a novel forced convection cell culture system to rapidly (response time of 1.6 s) and accurately (+/-1 Torr) deliver specific O(2) tensions (from <1 to 157 Torr) directly to a monolayer of LPS- and IFNgamma-stimulated RAW 264.7 cells while simultaneously measuring NO production via an electrochemical probe. Decreased O(2) availability rapidly (<or=30 s) and reversibly decreased NO production with an apparent K(m)O(2) of 22 (SD 6) Torr (31 microM) and a V(max) of 4.9 (SD 0.4) nmol min(-1) 10(-6) cells. To explore potential mechanisms of decreased NO production during hypoxia, we investigated O(2)-dependent changes in iNOS protein concentration, iNOS dimerization, and cellular NO consumption. iNOS protein concentration was not affected (P = 0.895). iNOS dimerization appeared to be biphasic [6 Torr (P = 0.008) and 157 Torr (P = 0.258) >36 Torr], but it did not predict NO production. NO consumption was minimal at high O(2) and NO tensions and negligible at low O(2) and NO tensions. These results are consistent with O(2) substrate limitation as a regulatory mechanism during brief hypoxic exposure. The rapid and reversible effects of physiological and pathophysiological O(2) tensions suggest that O(2) tension has the potential to regulate NO production in vivo.

Pharmacokinetics of dexamethasone following intra‐articular, intravenous, intramuscular, and oral administration in horses and its effects on endogenous hydrocortisone

This study investigated and compared the pharmacokinetics of intra-articular (IA) administration of dexamethasone sodium phosphate (DSP) into three equine joints, femoropatellar (IAS), radiocarpal (IAC), and metacarpophalangeal (IAF), and the intramuscular (IM), oral (PO) and intravenous (IV) administrations. No significant differences in the pharmacokinetic estimates between the three joints were observed with the exception of maximum concentration (Cmax ) and time to maximum concentration (Tmax ). Median (range) Cmax for the IAC, IAF, and IAS were 16.9 (14.6-35.4), 23.4 (13.5-73.0), and 46.9 (24.0-72.1) ng/mL, respectively. The Tmax for IAC, IAF, and IAS were 1.0 (0.75-4.0), 0.62 (0.5-1.0), and 0.25 (0.08-0.25) h, respectively. Median (range) elimination half-lives for IA and IM administrations were 3.6 (3.0-4.6) h and 3.4 (2.9-3.7) h, respectively. A 3-compartment model was fitted to the plasma dexamethasone concentration-time curve following the IV administration of DSP; alpha, beta, and gamma half-lives were 0.03 (0.01-0.05), 1.8 (0.34-2.3), and 5.1 (3.3-5.6) h, respectively. Following the PO administration, the median absorption and elimination half-lives were 0.34 (0.29-1.6) and 3.4 (3.1-4.7) h, respectively. Endogenous hydrocortisone plasma concentrations declined from a baseline of 103.8 ± 29.1-3.1 ± 1.3 ng/mL at 20.0 ± 2.7 h following the administration of DSP and recovered to baseline values between 96 and 120 h for IV, IA, and IM administrations and at 72 h for the PO.

pO2-dependent NO production determines OPPC activity in macrophages

Stimulated macrophages produce nitric oxide (NO) via inducible nitric oxide synthase (iNOS) using molecular O(2), L-arginine, and NADPH. Exposure of macrophages to hypoxia decreases NO production within seconds, suggesting substrate limitation as the mechanism. Conflicting data exist regarding the effect of pO(2) on NADPH production via the oxidative pentose phosphate cycle (OPPC). Therefore, the present studies were developed to determine whether NADPH could be limiting for NO production under hypoxia. Production of NO metabolites (NOx) and OPPC activity by RAW 264.7 cells was significantly increased by stimulation with lipopolysaccharide (LPS) and interferon gamma (IFNgamma) at pO(2) ranging from 0.07 to 50%. OPPC activity correlated linearly with NOx production at pO(2)>0.13%. Increased OPPC activity by stimulated RAW 264.7 cells was significantly reduced by 1400 W, an iNOS inhibitor. OPPC activity was significantly increased by concomitant treatment of stimulated RAW 264.7 cells with chemical oxidants such as hydroxyethyldisulfide or pimonidazole, at 0.07 and 50% O(2), without decreasing NOx production. These results are the first to investigate the effect of pO(2) on the relationship between NO production and OPPC activity, and to rule out limitations in OPPC activity as a mechanism by which NO production is decreased under hypoxia.

AICAR administration affects glucose metabolism by upregulating the novel glucose transporter, GLUT8, in equine skeletal muscle

Equine metabolic syndrome is characterized by obesity and insulin resistance (IR). Currently, there is no effective pharmacological treatment for this insidious disease. Glucose uptake is mediated by a family of glucose transporters (GLUT), and is regulated by insulin-dependent and -independent pathways, including 5-AMP-activated protein kinase (AMPK). Importantly, the activation of AMPK, by 5-aminoimidazole-4-carboxamide-1-D-ribofuranoside (AICAR) stimulates glucose uptake in both healthy and diabetic humans. However, whether AICAR promotes glucose uptake in horses has not been established. It is hypothesized that AICAR administration would enhance glucose transport in equine skeletal muscle through AMPK activation. In this study, the effect of an intravenous AICAR infusion on blood glucose and insulin concentrations, as well as on GLUT expression and AMPK activation in equine skeletal muscle (quantified by Western blotting) was examined. Upon administration, plasma AICAR rapidly reached peak concentration. Treatment with AICAR resulted in a decrease (P <0.05) in blood glucose and an increase (P <0.05) in insulin concentration without a change in lactate concentration. The ratio of phosphorylated to total AMPK was increased (P <0.05) in skeletal muscle. While GLUT4 and GLUT1 protein expression remained unchanged, GLUT8 was increased (P <0.05) following AICAR treatment. Up-regulation of GLUT8 protein expression by AICAR suggests that this novel GLUT isoform plays an important role in equine muscle glucose transport. In addition, the data suggest that AMPK activation enhances pancreatic insulin secretion. Collectively, the findings suggest that AICAR acutely promotes muscle glucose uptake in healthy horses and thus its therapeutic potential for managing IR requires investigation.

Pharmacokinetics and pharmacodynamics of dermorphin in the horse.

Dermorphin is a μ-opioid receptor-binding peptide that causes both central and peripheral effects following intravenous administration to rats, dogs, and humans and has been identified in postrace horse samples. Ten horses were intravenously and/or intramuscularly administered dermorphin (9.3 ± 1.0 μg/kg), and plasma concentration vs. time data were evaluated using compartmental and noncompartmental analyses. Data from intravenous administrations fit a 2-compartment model best with distribution and elimination half-lives (harmonic mean ± pseudo SD) of 0.09 ± 0.02 and 0.76 ± 0.22 h, respectively. Data from intramuscular administrations fit a noncompartmental model best with a terminal elimination half-life of 0.68 ± 0.24 (h). Bioavailability following intramuscular administration was variable (47-100%, n = 3). The percentage of dermorphin excreted in urine was 5.0 (3.7-10.6) %. Excitation accompanied by an increased heart rate followed intravenous administration only and subsided after 5 min. A plot of the mean change in heart rate vs. the plasma concentration of dermorphin fit a hyperbolic equation (simple Emax model), and an EC(50) of 21.1 ± 8.8 ng/mL was calculated. Dermorphin was detected in plasma for 12 h and in urine for 48 or 72 h following intravenous or intramuscular administration, respectively.

Plasma interleukin-6 concentration in Standardbred racehorses determined by means of a novel validated ELISA

OBJECTIVE To evaluate plasma interleukin 6 (IL-6) concentration in Standardbred racehorses by means of a novel ELISA following validation of the assay for use with equine plasma samples. SAMPLE Plasma samples obtained from 25 Thoroughbreds for use in assay validation and from 319 Standardbred racehorses at rest 2 to 2.5 hours prior to warm-up and racing. PROCEDURES A sandwich ELISA was developed with equine anti-IL-6 polyclonal antibody and the biotin-streptavidin chemical interaction to enhance sensitivity. The assay was validated for specificity, sensitivity, precision, and accuracy by use of both recombinant and endogenous proteins. RESULTS For the assay, cross-reactivity with other human and equine cytokines was very low or absent. Serial dilution of plasma samples resulted in proportional decreases in reactivity, indicating high specificity of the method. Partial replacement of detection antibody with capture antibody or pretreatment of samples with capture antibody caused assay signals to significantly decrease by 55%. The inter- and intra-assay precisions were ≤ 13.6% and ≤ 9.3%, respectively; inter- and intra-assay accuracies were within ranges of ± 14.1% and ± 8.6%, respectively, at concentrations from 78 to 5,000 pg/mL, and the sensitivity was 18 pg/mL. Plasma IL-6 concentration varied widely among the 319 Standardbreds at rest (range, 0 to 193,630 pg/mL; mean, 6,153 pg/mL; median, 376 pg/mL). CONCLUSIONS AND CLINICAL RELEVANCE This ELISA method proved suitable for quantification of IL-6 concentration in equine plasma samples. Plasma IL-6 concentration was high (> 10,000 pg/mL) in 9.1% of the Standardbred racehorses, which warrants further investigation.