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Biomedical subjects

R C Harris

Publications and source records attributed to R C Harris.

At least 73 records · Page 4Linked to original sources
PubMed1997

High-performance liquid chromatographic determination of imidazole dipeptides, histidine, 1-methylhistidine and 3-methylhistidine in equine and camel muscle and individual muscle fibres.

The combined solid-phase extraction (Isolute PRS columns) and reversed-phase gradient HPLC method presented provides a sensitive, reproducible and selective quantification of carnosine, balenine, homocarnosine, histidine, 1-methylhistidine and 3-methylhistidine in equine and camel muscle and individual muscle fibres. Recoveries were 91-115%. Lower limits of detection were 0.005-0.010 mmol kg-1 dry muscle. The compounds were isolated from other physiological amino acids and small peptides and resolved within a single chromatographic run of 55 min. Concentrations of these compounds in equine myocardium, diaphragm, skeletal muscle, camel muscle and individual muscle fibres of both species are presented for the first time.

Animals↗
PubMed1997

Effect of glucose, pyruvate, and insulin on type 1 angiotensin II receptor expression in SV40-immortalized rabbit proximal tubule epithelial cells.

Ambient glucose concentrations alter type 1 angiotensin II receptor (AT1R) expression in renal tissues. The direction of change in AT1R density may depend on the specific cell type and the capacity for that cell type to use glucose as an energy substrate. Given the effects of angiotensin II (Ang II) in proximal tubule epithelia, glucose-mediated fluctuations in AT1R expression could significantly alter tubular Na(+)-H+ exchange and volume reabsorption. To determine if glucose influenced AT1R expression in cultured proximal tubule epithelial cells, SV40-immortalized rabbit proximal tubule epithelial cells (RPTEC) were exposed to 25 mmol (hi-glc) or 5 mmol glucose-containing serum-free medium (lo-glc) for seven to nine days, with or without an alternative energy substrate, pyruvate. AT1R expression, assessed by quantitative reverse-transcription polymerase chain reaction and specific 125I-Ang II binding, decreased in lo-glc medium (% reduction AT1R mRNA expression: 52 +/- 8%; N = 6; P < 0.005 vs. hi-glc; % reduction specific 125I-Ang II binding: 48 +/- 12%; N = 12; P < 0.03 vs. hi-glc). AT1R mRNA expression and specific 125I-Ang II binding recovered to hi-glc levels following the addition of pyruvate [60 mmol] to lo-glc cells. To ascertain if a growth factor that increases glucose uptake in vivo also altered AT1R expression, RPTEC were cultured in hi-glc medium with or without exogenous insulin [100 nM]. Insulin addition increased AT1R mRNA expression and specific 125I-Ang II binding in a concentration-dependent manner. However, insulin (100 nM) addition to lo-glc cells did not significantly increase specific 125I-Ang II binding. These results suggest that AT1R expression in SV40-immortalized rabbit proximal tubule cells is significantly affected by the availability of energy substrate. Ultimately, changes in proximal tubule AT1R expression, mediated by elevated glucose concentrations and insulin, could contribute to sodium-dependent hypertension in the setting of hyperinsulinemia and hyperglycemia.

Angiotensin II↗
PubMed1997

A comparison of the lipolytic and anticoagulative properties of heparin and pentosan polysulphate in the thoroughbred horse.

The aim of the study was to develop a model for the pre-exercise elevation of plasma free fatty acids in the horse, with a view to its future use in investigations of fat metabolism during exercise. A comparison of the lipase releasing and anticoagulative effects of heparin and a related substance pentosan polysulphate was investigated. Furthermore, the ability of heparin and pentosan polysulphate to affect an increase in plasma free fatty acid concentration, when co-administered with-a triglyceride emulsion, was quantified. Doses of 0.39 and 1.3 mg kg-1 body wt of heparin and pentosan polysulphate respectively, administered intravenously, resulted in a significant increase in plasma total lipase activity (P < 0.001). There was, however, no significant difference in plasma lipase activity between treatments. Heparin resulted in a mean 14 +/- 6.5-fold increase in activated partial thromboplastin time compared with a mean 1.6 +/- 0.1-fold increase with pentosan polysulphate. Both heparin and pentosan polysulphate when coadministered with a triglyceride emulsion (lverlip 20) resulted in a significant increase in plasma free fatty acid concentration (P < 0.001), although there was no significant difference between treatments. Thus, whereas a higher dose of pentosan polysulphate elicited a comparable lipolytic effect to heparin, including significant elevation of plasma free fatty acids, this was associated with a much reduced effect upon clotting function. Pentosan polysulphate, therefore, represents a suitable alternative to heparin for the elevation of plasma free fatty acids before exercise when used in conjunction with a triglyceride emulsion.

Animal Husbandry↗
PubMed1997

Metabolic adaptation to fat-supplemented diet by the thoroughbred horse.

Following 10 weeks of fat supplementation a group of aerobically trained thoroughbred horses exhibited a significant decrease in postprandial plasma triacylglycerol concentration. This decrease was associated with a mean 50% increase in plasma total lipase activity following pentosan polysulfate administration and an increase in postprandial plasma cholesterol concentration. A significant increase in the activity of muscle citrate synthase (EC 4.1.3.7), expressed as a ratio to the total fractional area occupied by type I and type IIa muscle fibres, was also observed. No significant change in the concentration of resting muscle glycogen or triacylglycerol occurred as a result of fat supplementation. These results suggest that there was improved management of the fat load and that the triacylglycerol-clearing capacity of the horses was increased as a result of fat supplementation. It is suggested that the increase in plasma total lipase activity following pentosan polysulfate administration may have reflected an increase in muscle lipoprotein lipase (EC 3.1.1.34) activity, which would increase the capacity of muscle for free fatty acid uptake from circulating triacylglycerol-rich plasma lipoproteins. Fat supplementation may also enhance the oxidative capacity of muscle, as suggested by the significant increase in muscle citrate synthase and the trend towards an increase in beta-hydroxyacyl CoA dehydrogenase (EC 1.1.1.35) following 10 weeks of fat supplementation.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1997

Apical ANG II-stimulated PLA2 activity and Na+ flux: a potential role for Ca2+-independent PLA2.

Type 1 angiotensin II (ANG II) receptors (AT1R), which mediate proximal tubule (PT) salt and water reabsorption, undergo endocytosis and recycling. Prior studies in a PT-like model (LLC-PKcl4 cells expressing rabbit AT1R) (LLC-PK-AT1R cells) determined that quinacrine, a nonspecific phospholipase A2 (PLA2) inhibitor, and the haloenol lactone suicide substrate (HELSS), a Ca2+-independent PLA2 inhibitor, attenuated apical (AP) AT1R recycling. Further studies were undertaken to examine the association between AT1R endocytotic movement and PLA2 activity in this model. AP ANG II (100 nM) increased [3H]arachidonic acid ([3H]AA) release 4.4 +/- 0.38-fold in LLC-PK-AT1R cells cultured on permeable supports. Basolateral (BL) ANG II had no significant effect. Reversed-phase high-performance liquid chromatography confirmed that AP ANG II stimulated free [3H]AA release. Quinacrine, HELSS, and palmitoyl trifluoromethyl ketone, another Ca2+-independent PLA2 inhibitor, inhibited AP ANG II-stimulated [3H]AA release, as did inhibiting AP AT1R internalization with phenylarsine oxide. The role of HELSS-inhibitable AA release in ANG II-mediated 22Na flux was examined, given the effects of AT1R-mediated PLA2 activity on salt and water reabsorption. AP ANG II (100 nM) stimulated 22Na flux (AP--> BL), a response inhibited by HELSS. Thus, in this model, AP AT1R activated PLA2 with concomitant 22Na flux (AP --> BL), suggesting a link between AP AT1R endocytotic movement, AT1R-stimulated PLA2 activity, and 22Na flux in this model. The effects of HELSS suggest that Ca2+-independent PLA2 activity may be involved in this AP ANG II response.

Angiotensin II↗
PubMed1997

Cyclooxygenase-2 in rat nephron development.

The inducible second isoform of cyclooxygenase (COX-2) that mediates inflammation also is expressed at low levels in normal adult rat kidneys and is upregulated in response to noninflammatory stimuli (R. C. Harris, J. A. McKanna, Y. Akai, H. R. Jacobson, R. N. DuBois, and M. D. Breyer, J. Clin. Invest. 94: 2504-2510, 1994). Roles in morphogenesis are indicated by reported teratogenicity of COX inhibitors and renal dysgenesis in COX-2 knockout mice (J. E. Dinchuk, B. D. Car, R. J. Focht, J. J. Johnston, B. D. Jaffee, M. B. Covington, N. R. Contel, V. M. Eng, R. J. Collins, P. M. Czerniak, A. G. Stewart, and J. M. Trzaskos, Nature 378: 406-409, 1995; S. G. Morham, R. Lagenbach, C. D. Loftin, H. F. Tiano, N. Vouloumanos, J. C. Jennette, J. F. Mahler, K. D. Kluckman, A. Ledford, C. A. Lee, and O. Smithies. Cell 83: 473-482, 1995). Blots from developing rat kidneys demonstrated that COX-2 mRNA and immunoreactive protein were present in neonates, peaked in the 2nd and 3rd postnatal weeks and declined to adult levels by the 3rd month. Immunolocalization and in situ hybridization detected intense COX-2 immunoreactivity and mRNA in a subset of thick ascending limb epithelial cells near the macula densa in each developing nephron; after 2 wk the COX-2 gradually waned. These data demonstrate that COX-2 expression is subject to normal developmental regulation and can be sustained over extended periods; they also support the conclusion that metabolites of COX-2 play important roles in the differentiation and early functions of mammalian nephrons.

Aging↗
PubMed1997

Growth factors and cytokines in acute renal failure.

The mammalian kidney is susceptible to injury by ischemia/reperfusion and toxins, and regeneration after injury is characterized by hyperplasia and recovery of the damaged epithelial cells that line the tubules. Locally produced growth factors may serve as mediators of nephrogenesis and differentiation during renal development and of renal regeneration after acute injury. In cultured cells, administration of one or a mixture of growth factors to quiescent cells will initiate progression through the cell cycle and cell division. In the adult kidney, cell division normally is very low, but will increase up to 10-fold after acute injury. In addition to proliferation after lethal injury, there also is cellular repair in cells that have undergone sublethal injury. Recent studies indicate that growth factors inhibit programmed cell death in response to acute injury. Growth factors also may initiate or promote protein and lipid biosynthesis and provide an intracellular milieu that promotes cellular repair. In addition to cellular repair, growth factors also may be involved in the re-establishment of cell-extracellular matrix and cell-cell integrity. Finally, growth factors may limit injury by decreasing the factors that induce damage. Increased local renal expression of growth factors in response to acute injury include heparin binding epidermal growth factor (HB-EGF), hepatocyte growth factor (HGF), insulin-like growth factor-I (IGF-I), transforming growth factor-beta, parathyroid hormone-related peptide, and acidic fibroblast growth factor. In a number of experimental models of acute renal injury, administration of exogenous growth factors has been shown to accelerate both structural and functional recovery. Specifically, EGF, IGF-1, and HGF all have been shown to be effective in this regard. These studies are reviewed and potential therapeutic uses of growth factors and cytokines will be discussed.

Acute Kidney Injury↗
PubMed1996

Regulation of extracellular matrix by mechanical stress in rat glomerular mesangial cells.

Increases in intraglomerular pressure are known to predispose to the development of glomerular sclerosis, which is characterized by accumulation of extracellular matrix within the glomerulus. Glomerular mesangial cells are exposed to pulsatile capillary pressures and are a potential target for mechanical stress. In the present studies, we subjected cultured rat mesangial cells to continuous cycles of stretching and relaxation (stretch/relaxation) and examined alterations in extracellular matrix gene expression. After 48 h of stretch/relaxation, immunofluorescent localization of matrix accumulation indicated increases in types I, III, and IV collagens, fibronectin, and laminin, with the greatest increases seen at the periphery of the culture dish, at the point of the greatest deformation. Northern blot analysis of total RNA revealed time-dependent induction of alpha1(I) collagen, alpha1(III) collagen, alpha1(IV) collagen, fibronectin, and laminin by stretch/relaxation, with maximal increases occurring between 12 and 24 h. Transient transfection of reporter gene constructs of the 5' flanking region of alpha1(I) collagen gene indicated that stimulation of gene transcription was involved in the increased expression of matrix mRNA. Gelatinolytic activity in conditioned media was decreased at 24 and 48 h of stretch/relaxation, in association with a significant decrease in levels of mRNA for matrix metalloproteinase-2 (68-72 kD type IV collagenase) occurring within 6 h of stretch/relaxation. In contrast, expression of tissue inhibitor of metalloproteinase-2 was increased within 12 h of stretch/relaxation. Stretch/relaxation increased immunoreactive TGF-beta at 48 but not 12 h. TGF-beta1 mRNA levels remained unchanged during the initial 12 h of stretch/relaxation, but were significantly elevated at 48 h, and no differences in TGF-beta bioactivity could be detected in conditioned media for up to 12 h of stretch/relaxation. These findings demonstrate that in glomerular mesangial cells, repeated cycles of stretching and relaxation lead to matrix accumulation by stimulating production of extracellular matrix and decreasing activity of degradative enzymes. The observed induction of TGF-beta1 suggests a role in matrix accumulation occurring in response to continued mechanical deformation.

Animals↗
PubMed1996

Dopamine decreases expression of type-1 angiotensin II receptors in renal proximal tubule.

Systemic and/or locally produced angiotensin II stimulates salt and water reabsorption in the renal proximal tubule. In vivo, dopamine (DA) may serve as a counterregulatory hormone to angiotensin II's acute actions on the proximal tubule. We examined whether dopamine modulates AT1 receptor expression in cultured proximal tubule cells (RPTC) expressing DA1 receptors. Dopamine decreased basal RPTC AT1 receptor mRNA levels by 67 +/- 7% (n = 10; P < 0.005) and decreased 125I-angiotensin II binding by 41 +/- 7% (n = 4; P < 0.05). The DA1-specific agonist, SKF38393 decreased basal AT1 receptor mRNA levels (65 +/- 5% inhibition; n = 5; P < 0.025), and the DA1-specific antagonist, SCH23390 reversed dopamine's inhibition of AT1 receptor mRNA expression (24 +/- 10% inhibition; n = 8; NS) and angiotensin II binding (5 +/- 15%; n = 4; NS). DA2-specific antagonists were ineffective. In rats given L-DOPA in the drinking water for 5 d, there were decreases in both proximal tubule AT1 receptor mRNA expression (80 +/- 5%; n = 6; P < 0.005) and specific [125I] Ang II binding (control: 0.74 +/- 0.13 fmol/mg pro vs. 0.40 +/- 0.63 fmol/mg pro; n = 5; P < 0.05). In summary, dopamine, acting through DA1 receptors, decreased AT1 receptor expression in proximal tubule, an effect likely mediated by increased intracellular cAMP levels. Local dopamine production also led to decreased AT1 receptor expression, suggesting dopamine may reset sensitivity of the proximal tubule to angiotensin II.

Angiotensin II↗
PubMed1996

Effects of exercise intensity and environmental stress on indices of oxidative stress and iron homeostasis during exercise in the horse.

The effects of prolonged variable-intensity and short-term high-intensity exercise on indices of oxidative stress and iron homeostasis were compared in six fit horses under cool [20 degrees C, 40% relative humidity (RH)] or hot/humid (30 degrees C, 80% RH) environmental conditions. The exercise protocols were designed to simulate equine competition, including racing (intense exercise) or the speed and endurance phase of a 3-day event (prolonged exercise). Increased plasma concentrations of lipid hydroperoxides and haemolysate concentrations of oxidised glutathione (GSSG) were measured within 30 min of the completion of exercise, indicating production of reactive oxygen species (ROS) and lipid membrane peroxidation. The horses were unable to complete the prolonged exercise protocol at high temperature and humidity. This coincided with higher maximal values of lipid hydroperoxides [138.2 (17.7) microM and GSSG [110.6 (18.2) microM], compared to high-intensity [105.2 (14.9) microM and 63.6 (8.6) microM, respectively] or prolonged [100.7 (18.7) microM and 86.2 (9.1) microM, respectively] exercise performed under cooler environmental conditions. Significant correlations were found between the duration of the final stage of exercise during hot/humid environmental conditions and increased levels of lipid hydroperoxides (r = 0.85), GSSG (r = 0.94), xanthine (r = 0.92) and uric acid (r = 0.96). Exercise also decreased the iron (Fe)-binding antioxidant activity of the plasma and increased the total plasma Fe levels, although this was only significant for prolonged exercise in ambient conditions. There was no detectable free Fe in the plasma at any stage of exercise. Other changes in biochemical parameters had returned to pre-exercise levels within 24 h after exercise. The results show that exercise can induce changes in biochemical parameters that are indicative of oxidative stress in the fit horse and that this was, exacerbated during exercise at high temperature and humidity.

Animals↗
PubMed1996

Physiological responses in nonheat acclimated horses performing treadmill exercise in cool (20 degrees C/40% RH), hot dry (30 degrees C/40% RH) and hot humid (30 degrees C/80% RH) conditions.

The aim of the present study was to determine the effect of different environmental conditions on physiological response to exercise. Four winter acclimatised, nonheat acclimated horses of different breeds were exercised at 20 degrees C/40% RH (CD), 30 degrees C/40% RH (HD) and 30 degrees C/80% RH (HH). The exercise test was designed to represent the structure and intensity of a One star Speed and Endurance test (competition exercise test [CET]). All 4 horses were able to complete the full CET (60 min + 30 min active recovery) in CD and HD, but only one horse completed the CET in HH. Two horses were stopped because of pronounced general fatigue and one because of a right atrial temperature (TRA) of 43 degrees C. Oxygen uptake on each phase was not different between CD and HD, but was higher during Phases B, C and D in HH. Mean peak TRA at the end of Phase D was 40.3 +/- 0.2, 41.6 +/- 0.4 and 42 +/- 0.3 degrees C for CD, HD and HH, respectively. Corresponding, mean peak rectal temperatures (TREC) following Phase D were 39.5 +/- 0.1, 40.6 +/- 0.1 and 41.5 +/- 0.1 degree C for CD, HD and HH, respectively. Mean time to peak TREC was 9.3 +/- 1.1 (CD), 7.3 +/- 1.8 (HD) and 10.8 +/- 2.3 (HH) min and was not significantly different between conditions (P > 0.05). Heat dissipation amounted to 83 +/- 1, 73 +/- 2 and 70 +/- 1% of heat production in CD, HD and HH, respectively. Weight loss was significantly correlated with both body surface area (CD r = 0.85; HD r = 0.87; HH r = 0.81) and bodyweight (CD r = 0.97; HD r = 0.93; HH r = 0.94). The greatest weight loss recorded was 4.6% bodyweight in one horse in HD. The mean increase in exercise intensity over the whole CET (in terms of VO2) of HD and HH and HH compared with CD was 5 +/- 3 and 14 +/- 3% higher, respectively. The exercise induced hyperthermia and the reduced capacity for heat dissipation produced partial compensatory responses in minute ventilation (VE), particularly during Phase C, when the horses were trotting. In HD, the increase in VE was achieved mainly through an increase in frequency, whilst in HH it was achieved through an increase in tidal volume (VT). The horses demonstrated a high degree of tolerance to environmental heat load, suggesting a high thermoregulatory capacity. However, for unacclimatised animals exercising in severely hot and humid conditions, performance may be limited.

Acclimatization↗
PubMed1996

A potential mechanism for proximal tubule angiotensin II-mediated sodium flux associated with receptor-mediated endocytosis and arachidonic acid release.

Angiotensin II (Ang II) receptors in the proximal nephron significantly affect renal salt and water reabsorption. The predominant tubular-epithelial cell Ang II receptor, type 1 Ang II receptors (AT1R), is a member of the superfamily of G-protein-coupled receptors. Tubular cell AT1R are unusual as they are polarized to apical (AP) and basolateral (BL) cell membranes. In a proximal tubule cell model that mimics the in vivo polar distribution of AT1R (LLC-PKC14 cells transfected with rabbit AT1R), we have determined that AP and BL AT1R display differential rates of endocytosis and recycling. In addition, AP AT1R recycling was selectively inhibited by quinacrine, a non-specific inhibitor of phospholipase A2 (PLA2) activity and HELSS, a mechanism-based inhibitor of calcium-independent PLA2 activity. PLA2 catalyzes the hydroysis of sn-2 fatty acyl bonds, liberating free fatty acids and lysophosphoslipids, and proximal tubule AT1R are known to couple to PLA2 as a signaling pathway. In this model, AP Ang II treatment selectively stimulated [3H]-arachidonic acid ([3H]-AA) release. Furthermore, inhibiting AP AT1R endocytosis+recycling with either phenylarsine oxide (PAO) or HELSS, decreased AP Ang II-stimulated [3H]-AA release. In other studies, Ang II internalization also has been linked to vectorial sodium (Na+) flux. Preliminary studies in our model also suggested that HELSS inhibited AP Ang II-stimulated Na+ flux (AP --> BL), raising the possibility that AP AT1R targeting in proximal tubular epithelium, the endocytic profile of these receptors, and AP AT1R-mediated arachidonic acid release combine to serve as additional contributors to Ang II-mediated Na+ flux in the nephron.

Angiotensin II↗
PubMed1996

The intrarenal renin-angiotensin system: a paracrine system for the local control of renal function separate from the systemic axis.

The kidney serves an integral role in the systemic renin-angiotensin system (RAS) both as an effector and as a target organ. Increasing evidence also accumulates that local intrarenal RAS serve as important regulators of renal function. Evidence is reviewed concerning the expression and potential role of components of the RAS in glomeruli, proximal tubule and renal interstitium. In addition to regulating normal renal function, local renal RAS may be triggered in response to renal injury and may contribute to structural and functional abnormalities, including increased glomerulosclerosis and tubulointerstitial fibrosis.

Animals↗
PubMed1995

Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype.

Gene targeting was used to create a null allele at the epidermal growth factor receptor locus (Egfr). The phenotype was dependent on genetic background. EGFR deficiency on a CF-1 background resulted in peri-implantation death due to degeneration of the inner cell mass. On a 129/Sv background, homozygous mutants died at mid-gestation due to placental defects; on a CD-1 background, the mutants lived for up to 3 weeks and showed abnormalities in skin, kidney, brain, liver, and gastrointestinal tract. The multiple abnormalities associated with EGFR deficiency indicate that the receptor is involved in a wide range of cellular activities.

Abnormalities, Multiple↗