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Biomedical subjects

R C Harris

Publications and source records attributed to R C Harris.

At least 19 recordsLinked to original sources
PubMed1992

Activation of S6 kinase by repeated cycles of stretching and relaxation in rat glomerular mesangial cells. Evidence for involvement of protein kinase C.

Quiescent rat glomerular mesangial cells were exposed to repeated cycles of stretching and relaxation, and the effects on the rate of collagen production, proliferation, and S6 kinase activity were investigated. Stretch/relaxation induced increases in production of both collagen and non-collagenous proteins. Proliferation of mesangial cells was stimulated by stretch/relaxation and epidermal growth factor, but not by angiotensin II; however, administration of angiotensin II augmented stretch/relaxation-induced cell proliferation. Cytosolic S6 kinase activity was stimulated by stretch/relaxation, angiotensin II, epidermal growth factor, or phorbol 12-myristate 13-acetate. The increased S6 kinase activity was detectable within 30 min after initiation of stretch/relaxation and was blocked by either inhibitors of protein kinase C or prior down-regulation of protein kinase C following prolonged incubation with phorbol 12-myristate 13-acetate. Both translocation of protein kinase C from the cytosolic to the membrane fraction and phosphorylation of an endogenous 80-kDa protein were observed within 5 min of initiation of stretch/relaxation. These results demonstrate that in mesangial cells, mechanical factors alone can induce increases in production of collagen and non-collagenous proteins and in cell proliferation. The observation that stretch/relaxation induced stimulation of S6 kinase activity through protein kinase C-dependent mechanisms suggests that activation of protein kinase C may be a key event in initiating adaptive responses of mesangial cells to increased workload.

Adenosine Triphosphate↗
PubMed1992

Determination of carnosine and other biogenic imidazoles in equine plasma by isocratic reversed-phase ion-pair high-performance liquid chromatography.

The isocratic reversed-phase ion-pair high-performance liquid chromatographic technique presented provides a sensitive, rapid and reproducible analytical method for the selective determination of carnosine and other biogenic imidazoles in equine plasma. Plasma was deproteinized with 5-sulphosalicylic acid and the compounds of interest were isolated by sorbent extraction on Bond Elut PRS cartridges. Recoveries were 97-105% and the lowest limits of detection were 58.3-80.1 nM. All compounds of interest were well resolved within a maximum retention time of 9.2 min. The mean equine plasma carnosine level determined by this method was 11.31 microM. Comparative determinations were made in canine and human plasma. Carnosine was not detected in human plasma. Concentrations of imidazole in canine plasma are reported here for the first time.

Animals↗
PubMed1992

Localization of p35 (annexin I, lipocortin I) in normal adult rat kidney and during recovery from ischemia.

The 35-kDa protein (p35, lipocortin I, annexin I), originally discovered as a Ca(++)-dependent substrate for the EGF receptor tyrosine kinase, binds Ca++ and phospholipids, is developmentally regulated in embryos and has restricted expression in adults. Immunohistochemistry of normal rat kidney shows that p35 is enriched in epithelia of Bowman's capsule, the macula densa, and medullary/papillary collecting ducts, suggesting that p35 is related to specialized renal functions. Light staining is observed in the thick ascending limb; elsewhere, immunoreactivity is nil. Since renal recovery from ischemia involves both hyperplasia and hypertrophy and reportedly is accelerated by EGF, we examined p35 distribution during this process. After 48 hours of recovery, both the distribution and amount of renal p35 are altered. Immunoblots show p35 levels increased at least threefold in whole-kidney homogenates. The expression of p35 is still highly restricted in recovering kidneys; however, the thick ascending limb now stains heavily. This is the first documentation of alterations in annexin levels during a pathophysiologic response. However, our attempts to discern effects of exogenous EGF on the recovery from ischemia were negative for both mitotic index and renal function assays.

Animals↗
PubMed1992

Carnosine and anserine concentrations in the quadriceps femoris muscle of healthy humans.

The content of anserine and carnosine in the lateral portion of the quadriceps femoris muscle of 50 healthy, human subjects has been studied. Anserine was undetectable in all muscle samples examined. Muscle carnosine values for the group conformed to a normal distribution with a mean (SD) value of 20.0 (4.7) mmol.kg-1 of dry muscle mass. The concentration of carnosine was significantly higher in the muscle of male subjects (21.3, 4.2 mmol.kg-1 dry mass) than in females of a similar age and training status (17.5, 4.8 mmol.kg-1 dry mass) (P less than 0.005). The test-retest reliability of measures was determined on a subgroup of 17 subjects. No significant difference in mean carnosine concentration was found between the two trials [21.5 (4.0) and 22.0 (5.2) mmol.kg-1 dry muscle mass; P greater than 0.05]. The importance of carnosine as a physicochemical buffer within human muscle was examined by calculating its buffering ability over the physiological pH range. From the range of carnosine concentrations observed (7.2-30.7 mmol.kg-1 dry muscle mass), it was estimated that the dipeptide could buffer between 2.4 and 10.1 mmol H+.kg-1 dry mass over the physiological pH range 7.1-6.5, contributing, on average, approximately 7% to the total muscle buffering. This suggests that in humans, in contrast to many other species, carnosine is of only limited importance in preventing the reduction in pH observed during high intensity exercise.

Adolescent↗
PubMed1992

Adenine nucleotide degradation in the thoroughbred horse with increasing exercise duration.

Adenine nucleotide (AN) degradation has been shown to occur during intense exercise in the horse and in man, at or close to the point of fatigue. The aim of the study was to compare the concentrations of muscle inosine 5'-monophosphate (IMP) and plasma ammonia (NH3) during intense exercise with the concentrations of muscle and blood lactate. Seven trained thoroughbred horses were used in the study. Each exercised on a treadmill for periods of between 30 s and 150 s, at 11 and/or 12 m.s-1. Blood and muscle samples were taken and analysed for lactate and NH3 and adenosine 5'-triphosphate (ATP), phosphorylcreatine (PCr), IMP, creatine, lactate and glycerol-3-phosphate respectively. Horses showed varying degrees of AN degradation as indicated by plasma [NH3] and muscle [ATP] and [IMP]. Comparisons of [IMP] with muscle [lactate], and plasma [NH3] with that of blood [lactate] indicated a threshold to the start of AN degradation. This threshold corresponded to a lactate content of around 80 mmol.kg-1 dry muscle and 15 mmol.l-1 in blood. We discuss the mechanisms which have been proposed to account for AN degradation and suggest that IMP formation occurs as a result of a sudden rise in the concentration of adenosine 5'-diphosphate (ADP) and consequently the concentration of adenosine 5'-monophosphate. The data suggest a critical pH below which there may be a substantial reduction in the kinetics of ADP rephosphorylation provided by PCr resulting in an increase in [ADP], which is the stimulus to AN degradation during intense exercise.

Adenine Nucleotides↗
PubMed1992

Elevation of creatine in resting and exercised muscle of normal subjects by creatine supplementation.

1. The present study was undertaken to test whether creatine given as a supplement to normal subjects was absorbed, and if continued resulted in an increase in the total creatine pool in muscle. An additional effect of exercise upon uptake into muscle was also investigated. 2. Low doses (1g of creatine monohydrate or less in water) produced only a modest rise in the plasma creatine concentration, whereas 5g resulted in a mean peak after 1h of 795 (SD 104) mumol/l in three subjects weighing 76-87 kg. Repeated dosing with 5g every 2h sustained the plasma concentration at around 1000 mumol/l. A single 5g dose corresponds to the creatine content of 1.1 kg of fresh, uncooked steak. 3. Supplementation with 5g of creatine monohydrate, four or six times a day for 2 or more days resulted in a significant increase in the total creatine content of the quadriceps femoris muscle measured in 17 subjects. This was greatest in subjects with a low initial total creatine content and the effect was to raise the content in these subjects closer to the upper limit of the normal range. In some the increase was as much as 50%. 4. Uptake into muscle was greatest during the first 2 days of supplementation accounting for 32% of the dose administered in three subjects receiving 6 x 5g of creatine monohydrate/day. In these subjects renal excretion was 40, 61 and 68% of the creatine dose over the first 3 days. Approximately 20% or more of the creatine taken up was measured as phosphocreatine. No changes were apparent in the muscle ATP content.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1992

Total carnitine content of the middle gluteal muscle of thoroughbred horses: normal values, variability and effect of acute exercise.

There was no detectable loss of total carnitine associated with intense exercise from the middle gluteal muscle of Thoroughbred horses. Measurements made on a single biopsy obtained during the course of a normal training and exercise programme may, therefore, be considered representative of the normal content at rest. The variability in total carnitine content within the normal muscle biopsy area amounted to 13.2 per cent of the normal mean content. Approximately 50 per cent of this variability could be attributed to covariation with citrate synthase, to which it was highly significantly correlated. The muscle carnitine content of yearling Thoroughbred horses ranged from 10.5 to 18.8 mmol/kg dry muscle (dm); the ranges in untrained, lightly trained and fully trained two-year-olds were 18.5 to 34.7, 14.1 to 24.2 and 22.9 to 26.9 mmol/kg dm, respectively. In horses aged over three years total carnitine ranged from 21.3 to 35.5 mmol/kg dm. A trend toward higher contents of total carnitine with age and training appeared to be largely a consequence of underlying changes in mitochondria density as indicated by differences in levels of citrate synthase activity. There was a marked difference in ratios of total carnitine to citrate synthase activity between training yards, reflecting possible differences in management regimens and/or bloodstock selection.

Animals↗
PubMed1992

Effects of high-intensity exercise on plasma catecholamines in the thoroughbred horse.

In Study 1, a single speed test of 6 to 12 m/sec was performed for 2 mins at an incline of 5 degrees on a high-speed treadmill (single-step test). Only one speed was performed per session and blood samples were taken before and after the test. In Study 2 horses cantered for 1 min at increasing speeds of 6 to 13 m/sec on an incline of 3 degrees (multiple-step test). Blood samples were taken before exercise, throughout the test and during recovery. In the single-step test plasma concentrations of adrenaline and noradrenaline both increased at speeds of 9 m/sec, as did blood lactate. Mean concentrations of adrenaline and noradrenaline at the end of the 12 m/sec test were 153 and 148 nmol/litre, respectively. Plasma concentrations were similar over all speeds although there was a tendency for the increase in noradrenaline to be greater than that of adrenaline at the lower speeds. The multiple-step test resulted in smaller increases in both adrenaline and noradrenaline. Although again closely correlated, increases in adrenaline were 20-30% greater than those for noradrenaline. In both exercise models, changes in plasma adrenaline and noradrenaline values with exercise showed an exponential relationship to plasma lactate. A plasma half-life of less than 30 secs was indicated during recovery from the multiple-step test. Changes in adrenaline and noradrenaline were much greater than previously recorded in man and emphasise the importance of catecholamines in mediating the physiological response of the horse to exercise.

Animals↗
PubMed1992

Estimation of the carnosine content of different fibre types in the middle gluteal muscle of the thoroughbred horse.

1. Skeletal muscle samples were obtained by needle biopsy from one of two depths of the m. gluteus medius in a group of twenty race-trained thoroughbred horses. 2. The content of carnosine was determined in each muscle sample, part of which was used for histochemical analysis. Fibres were classified as type I, type IIA or type IIB on the basis of the pH dependent lability of the myosin ATPase reaction. 3. Muscle samples with a higher type II fibre section area (FSA) have a higher carnosine content than those with a higher type I FSA. 4. Multiple linear regression analysis was used to estimate the mean carnosine content of individual fibre types. The results estimated a mean carnosine content in type I fibres of 54 mmol (kg dry muscle (DM))-1, in type IIA fibres 85 mmol (kg DM)-1 and in type IIB fibres 180 mmol (kg DM)-1. 5. Based on the estimated values of single fibre carnosine content, there was close concordance between the estimated and the measured carnosine content of mixed fibre samples. 6. It would appear from this and other studies that carnosine has an important role as a physico-chemical buffer in equine middle gluteal muscle and that this is greatest in type IIB fibres, where it may account for up to 50% of physico-chemical buffering of H+ produced by muscle in the pH range 7.1-6.5.

Animals↗
PubMed1992

Regulation of S6 kinase activity in renal proximal tubule.

The proximal tubule undergoes hypertrophy in response to loss of functioning renal mass and hyperplasia following injury by ischemia or nephrotoxins. Both hypertrophic growth and cell proliferation are characterized by increases in the rate of protein synthesis. To investigate regulation of protein synthesis in mammalian proximal tubule cells, potential peptide mediators of proximal tubule growth, epidermal growth factor (EGF) and angiotensin II, were studied in cultured rabbit proximal tubule cells. Although only EGF stimulated DNA synthesis, both agonists stimulated protein synthesis. One potential regulatory mechanism of eukaryotic protein synthesis involves phosphorylation of ribosomal protein S6 by activation of a specific serine/threonine kinase (S6 kinase). Both EGF and angiotensin II stimulated S6 kinase activity and S6 phosphorylation. Phorbol 12-myristate 13-acetate was also found to activate S6 kinase, and 24 h of pretreatment to deplete protein kinase C inhibited subsequent S6 kinase activation by a high concentration (10(-6) M) of angiotensin II. To determine whether S6 kinase was also activated in the kidney in vivo, S6 kinase activity was examined after ablation of renal mass. Within 1 h after contralateral nephrectomy, S6 kinase activity increased in rat renal cortex. In summary, both EGF and angiotensin II stimulated protein synthesis and S6 kinase activity in cultured proximal tubule cells, and S6 kinase activity also increased in renal cortex after contralateral nephrectomy.

Angiotensin II↗
PubMed1992

Taurine content and distribution in equine skeletal muscle.

Taurine (TAU) is found in large but variable amounts in the skeletal muscles of many species. It has been reported that slow twitch muscles in the rat exhibit higher TAU levels than fast twitch muscles. Variation in muscle taurine content may be attributable to differences in the fibre type composition of different muscles. TAU content (mmol kg-1 dry muscle) and percentage type-1, type-2A, and type-2B fibre section area (f.s.a.) were measured in muscle samples taken from up to six sites in the middle gluteal muscle of four horses and one pony at post mortem and in biopsy samples taken from twenty Thoroughbred horses in race training. TAU was positively correlated to type-1 f.s.a. (r = 0.94, p < 0.001) in both post mortem samples and biopsies from horses in race-training. Multiple linear regression analysis was used to estimate the TAU content of individual fibre types when present at 100%. TAU is almost exclusively localized in type-1 fibres. The TAU content of type-1 and type-2A fibres was estimated to be 45.4 mmol kg-1 d.m. and 4.5 mmol kg-1 d.m. respectively in the post mortem horses, and 32.4 mmol kg-1 d.m. and 7.9 mmol kg-1 d.m. respectively in the horses in training. TAU was estimated to be absent from type-2B fibres in both horse groups.

Animals↗
PubMed1992

Continuous stretch-relaxation in culture alters rat mesangial cell morphology, growth characteristics, and metabolic activity.

Mesangial cells serve many functions in the glomerulus, including regulation of glomerular ultrafiltration coefficient, matrix production, and eicosanoid generation. The glomerulus is a vascular bed, and the mesangial cell is continually exposed to rhythmic alterations in intraglomerular pressure. Since increased intraglomerular pressure has been implicated as a potential causative agent in the ultimate development of nephrosclerosis, we sought to determine the effect of continuous stretch-relaxation upon parameters of mesangial cell growth and function. Early passage (2-4) cultured rat mesangial cells were plated onto either rigid-bottom or flexible-bottom culture plates coated with type I collagen. After cell attachment, the cells on flexible supports were exposed to continuous stretch-relaxation for 72 to 96 hours at a rate of 100 cycles/minutes at an applied pressure of 7 to 8 KPa (53 to 61 mm Hg). Cellular morphology was altered by continuous stretch-relaxation, with the majority of mesangial cells presenting stellate or straplike morphology. Fluorescein isothiocyanate-labeled phalloidin staining indicated an increase in density of actin filaments running the long axis of the cell. Stretch-relaxation resulted in an approximately 50% increase in cell number. Prostaglandin production, assessed as irPGE2 production, was increased by stretching in mesangial cells from 28 +/- 1 to 49 +/- 4 pg/10(6) cells (N = 12; p less than 0.005). Mechanical stretch/relaxation increased the percentage of protein representing collagenous proteins from 47 +/- 6% to 70 +/- 4%, as assessed by collagenase susceptibility (p less than 0.025). Analysis of pepsin-resistant proteins synthesized indicated that stretch/relaxation resulted in increases in the relative amounts of types I and III collagens produced/cell. Additionally, stretch/relaxation selectively increased the relative amount of type I-homotrimers produced. Thus, when mesangial cells are exposed to cyclic stretch/relaxation, they exhibit significant alterations in morphology, growth, prostaglandin and collagen production.

Animals↗
PubMed1991

Time-dependent biphasic regulation of Na+/K+/Cl- cotransport in rat glomerular mesangial cells.

Time-dependent regulation of loop diuretic-sensitive Na+/K+/Cl- cotransport and [3H]bumetanide binding was investigated in cultured rat glomerular mesangial cells. Angiotensin II or epidermal growth factor induced stimulation of Na+/K+/Cl- cotransport within 5 min, with a return to the control values by 30 min. Treatment of cells with phorbol 12-myristate 13-acetate (0.1 microM) (PMA), the calcium ionophore A23187 (1 microM), or the combination of 5 mM NaF and 10 microM AlCl3 produced a transient stimulation of Na+/K+/Cl- cotransport in 5-10 min to 148, 135, and 163% of control, respectively, which was followed by a progressive decrease to 34, 64, and 20% of the base-line activity, respectively, by 60 min. Exposure to cyclic 8-bromo-AMP (0.1 mM) or to forskolin (1 microM) and isobutylmethylxanthine (0.1 mM) caused a maximal inhibition of the cotransport in 5 min to 79 and 60% of control, respectively, with a subsequent gradual increase to 137 and 164% of the base-line activity, respectively, by 60 min. The effects of PMA, forskolin, and cyclic 8-bromo-AMP were concentration-dependent. In order to characterize further the alterations in the cotransport activity, binding of [3H]bumetanide was determined. Saturation binding analyses showed that the late inhibition of the cotransport by PMA and stimulation by forskolin were associated with a significant decrease and increase, respectively, in Bmax, with no significant changes in binding affinity. Correlations between changes in the cotransport activity and [3H]bumetanide binding were also observed in cells treated with cyclic 8-bromo-AMP or with NaF and AlCl3. Incubation of cells in Cl- or Na+ free solution greater than or equal to 60 min resulted in an increase in both the cotransport activity and [3H]bumetanide binding. These observations indicate that, in glomerular mesangial cells, persistent stimulation of second messengers that regulate the cotransporter induces a time-dependent, biphasic regulation of Na+/K+/Cl- cotransport and that the regulation occurring after greater than or equal to 60 min of treatment is primarily due to changes in the number of the active cotransport sites. Because long term removal of the transported ions also increases the number of active cotransport sites, these results suggest that alterations in intracellular ionic homeostasis may also mediate cotransport activity.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

The influence of metabolic alkalosis upon exercise metabolism in the thoroughbred horse.

Six thoroughbred horses exercised on a motorised treadmill on two separate occasions at a speed of 11 or 12 m.s-1 for up to 2 min. 4 h prior to exercise each horse was given a 21 test solution of sodium bicarbonate (NaHCO3; 0.6 g.kg-1 body mass) or a control solution of water by nasogastric intubation, the order of administration of the two solutions was randomised. Blood samples (n = 15) were obtained before and during the 4 h after intubation, during exercise and for 30 min after exercise. NaHCO3 ingestion resulted in changes in pre-exercise acid-base status. The changes in blood lactate and base excess with exercise were greater after NaHCO3 administration; after 1 min of exercise in the case of lactate (P less than 0.05) and immediately after exercise in the case of base excess (P less than 0.05). Plasma ammonia levels were lower during (P less than 0.05) and immediately after (P less than 0.05) exercise following NaHCO3 ingestion. The peak change in plasma ammonia with exercise was also lower after NaHCO3 ingestion (P less than 0.05). Following exercise after NaHCO3 ingestion, five horses demonstrated lower muscle adenosine 5-triphosphate loss (P less than 0.05) and inosine 5-monophosphate formation (P = 0.05) and higher glycerol 3-phosphate formation (P less than 0.05). There is evidence to suggest that metabolic alkalosis may delay the onset of fatigue by decreasing the extent of adenine nucleotide loss during high-intensity exercise.

Acid-Base Equilibrium↗
PubMed1991

Muscle ATP loss and lactate accumulation at different work intensities in the exercising Thoroughbred horse.

The effect of 2 min treadmill exercise, at speeds of 6-12 m.s-1 on an incline of 5 degrees, upon muscle adenine nucleotide loss and lactate accumulation was studied in six Thoroughbred horses. Minimal change occurred in the adenosine triphosphate (ATP) content of the middle gluteal muscle at speeds of 10 m.s-1 or less, but significant loss (up to 47%) had occurred in all horses by 12 m.s-1. The decline in ATP significantly correlated with the accumulation of muscle lactate, beginning shortly after the accumulation of 40 mmol.kg-1 dry muscle lactate. Decline in muscle ATP was mirrored closely by the appearance of ammonia, and to a lesser extent, hypoxanthine and uric acid in plasma. The results suggest that peak accumulation of any of these, or simply the concentration at a specified recovery time, may be used as a measure of ATP loss in the musculature as a whole. This was not so in the case of xanthine, which may also be formed from the degradation of guanidine nucleotides. An In-In plot of plasma ammonia against treadmill speed indicated a break point in accumulation between 8 and 9 m.s-1. The kinetics of ammonia accumulation with speed differed from those of lactate.

Adenosine Triphosphate↗
PubMed1991

Potential physiologic roles for epidermal growth factor in the kidney.

Epidermal growth factor (EGF) is a 53-amino acid polypeptide that is known to produce a number of biologic effects both in vitro and in vivo. High concentrations of EGF are found in urine, and high concentrations of prepro-EGF mRNA have been detected in kidney, localized to thick ascending limb of Henle (TALH) and distal convoluted tubule. Specific high-affinity EGF receptors have been demonstrated in mesangial cells, proximal tubule, and cortical and inner medullary collecting duct, as well as in medullary interstitial cells. In the proximal tubule, EGF binding and EGF receptor-associated tyrosine kinase activity are localized to basolateral membrane, and functional responses in collecting duct are observed only with basolateral administration of EGF. A number of renal responses to administration of EGF have recently been described, including modulation of glomerular hemodynamics, renal metabolism, tubular transport functions, and eicosanoid synthesis. In addition, EGF has been shown to be a potent mitogen in vitro for a variety of cell types in the kidney and may be an important mediator of renal repair following injury.

Animals↗
PubMed1991

Reverse-phase ion-pairing high-performance liquid chromatography of phosphocreatine, creatine and creatinine in equine muscle.

A simple, robust and reproducible analytical method for the determination of phosphocreatine (PCr), creatine (Cr) and creatinine (Cn) in equine skeletal muscle is presented. The technique used isocratic reverse-phase ion-pairing high-performance liquid chromatography. Neutralized perchloric acid extracts of equine muscle biopsies were analysed and the values obtained were compared with determinations from an established enzymic procedure. Good resolution of all three metabolites was achieved within a retention time of less than 11 min. Linearity for each metabolite within the concentration range in the samples was demonstrated. Peak purity was specifically addressed. The abolition of each creatine in a pooled extract by enzymic incubation showed no underlying peaks. It was concluded that peaks were free of co-eluents which would otherwise lead to an overestimation of PCr, Cr and Cn concentrations.

Animals↗