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R C Harris

Publications and source records attributed to R C Harris.

At least 127 records · Page 7Linked to original sources
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↗
PubMed1991

Acetyl group accumulation and pyruvate dehydrogenase activity in human muscle during incremental exercise.

The changes in the muscle contents of CoASH and carnitine and their acetylated forms, lactate and the active form of pyruvate dehydrogenase complex were studied during incremental dynamic exercise. Eight subjects exercised for 3-4 minutes on a bicycle ergometer at work loads corresponding to 30, 60 and 90% of their VO2max. Muscle samples were obtained by percutaneous needle biopsy technique at rest, at the end of each work period and after 10 minutes of recovery. During the incremental exercise test there was a continuous increase in muscle lactate, from a basal value of 4.5 mmol kg-1 dry weight to 83 mmol kg-1 at the end of the final period. The active form of pyruvate dehydrogenase complex increased from 0.37 mmol acetyl-CoA formed per minute per kilogram wet weight at rest to 0.80 at 30% VO2max, 1.28 and 1.25 at 60 and 90% VO2max, respectively. Both acetyl-CoA and acetylcarnitine increased at the two highest work loads. The increase of acetyl-CoA was from 12.5 mumol kg-1 dry weight at rest to 27.3 after the highest work load and for acetylcarnitine from 6.0 mmol kg-1 dry weight to 15.2. The CoASH and free carnitine contents fell correspondingly. There was a close relationship between acetyl-CoA and acetylcarnitine accumulation in muscle during exercise, with a binding of approximately 500 mol acetyl groups to carnitine for each mole of acetyl-CoA accumulated. The results imply that the carnitine store in muscle functions as a buffer for excess formation of acetyl groups from pyruvate catalyzed by the pyruvate dehydrogenase complex.

Acetyl Coenzyme A↗
PubMed1991

Titrimetric determination of muscle buffering capacity (beta mtitr) in biopsy samples.

In vitro titration of muscle homogenates has been used to assess muscle buffering capacity (beta mtitr) in a variety of species. In the present study, factors likely to affect the estimation of beta mtitr were investigated. Also, values of beta mtitr from normal Thoroughbred horses are presented. A non-linear titration curve was obtained with addition of HCl to muscle homogenates. As a result, beta mtitr is expressed as the mumol H+ required to change the pH of 1g of dry muscle or wet muscle from 7.1 to 6.5. An effect of dilution on the initial pH was found below 40 mg wet muscle per ml homogenising reagent (10 mg dry muscle per ml) and on beta mtitr below 10 mg wet muscle. As a result, 40 mg wet muscle or 10 mg dry muscle per ml was chosen as the minimum concentration for determination of beta mtitr. Incubation of homogenates up to 60 mins did not affect beta mtitr significantly. As a mean, beta mtitr in wet muscle was approximately 25 per cent higher compared to dry muscle. The beta mtitr of dry muscle was increased by approximately 18 per cent when HCO3- was added in an amount equivalent to the calculated HCO3- content of wet muscle at rest. The homogenisation process resulted in complete loss of adenosine triphosphate and phosphocreatine with only small changes in adenosine diphosphate and adenosine monophosphate. It was concluded that the estimates of beta mtitr did not include any contribution from 'dynamic' buffering via rephosphorylation of adenosine diphosphate by phosphocreatine, and in dry muscle it was accounted for mainly through physico-chemical buffering by phosphates, proteins and dipeptides. beta mtitr determined in biopsy samples of muscle from 20 Thoroughbred horses ranged from 100.8 to 131.8 mumol H+/g dry muscle pH 7.1 to 6.5 (mean 121.2, sd +/- 7.4).

Adenosine Diphosphate↗
PubMed1991

Renal sympathetic nerves modulate glomerular ANP receptors and filtration.

We examined characteristics of atrial natriuretic peptide (ANP) receptors in glomeruli isolated from subacutely (3-5 days) denervated (DNX) and contralateral nondenervated (non-DNX) kidneys of normal rats (NL) and rats subjected to water deprivation for 48 h (WD). Total ANP receptor density in DNX kidneys of WD rats, measured by competitive inhibition binding between 125I-labeled ANP and ANP, was twofold higher than non-DNX kidneys (726 +/- 96 vs. 384 +/- 32 fmol/mg protein, P less than 0.05). Equilibrium association constant (Ka) was not significantly different (2.33 +/- 0.43 vs. 3.34 +/- 0.78 x 10(9) M-1). In NL rats, there was no difference in ANP receptor density between DNX and non-DNX kidneys (244 +/- 20 and 264 +/- 16 fmol/mg protein). Production of guanosine 3',5'-cyclic monophosphate (cGMP), a putative second messenger of ANP, in response to ANP (10(-7) M) in glomeruli isolated from DNX was significantly larger than non-DNX kidneys of WD rats. To determine whether these changes in ANP receptors have functional consequences in vivo, glomerular capillary ultrafiltration coefficient (Kf) was assessed by micropuncture technique in WD Munich-Wistar rats. In DNX kidneys, ANP infusion (4 micrograms.kg-1.h-1) significantly increased whole kidney glomerular filtration rate (GFR) and single-nephron (SN) GFR (0.64 +/- 0.06 to 0.89 +/- 0.17 ml/min and 25 +/- 2 to 33 +/- 2 nl/min, respectively; n = 7) and Kf (1.26 +/- 0.29 to 2.18 +/- 0.41 nl.min-1.mmHg-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Regulation of Na(+)-H+ exchange by ATP depletion and calmodulin antagonism in renal epithelial cells.

The present studies examined effects of ATP depletion and calmodulin antagonism on stimulation of Na(+)-H+ exchange by cytosolic acidification in renal epithelial cells (LLC-PK1). ATP depletion significantly inhibited both amiloride-sensitive 22Na+ uptake (P less than 0.001; n = 12) and Na(+)-dependent intracellular pH (pHi) recovery in 2',7'-bis (carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester (BCECF/AM)-loaded cells. Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) and calmidazolium, both caused a concentration-dependent inhibition of Na(+)-H+ exchange activity. The W-7-induced inhibition of Na(+)-H+ exchange occurred in cells incubated for 24 h with phorbol 12-myristate 13-acetate, indicating that the effect of W-7 was not mediated by protein kinase C inhibition. Both W-7 and ATP depletion shifted the pHi dependence of the antiporter, and ATP depletion also reduced the maximal activity. In LLC-PK1/CL4 cells grown on permeable filters, W-7 inhibited the cytosolic acidification-stimulated basolateral exchanger by 54 +/- 5% (P less than 0.005; n = 7) and, in contrast, stimulated the apical exchanger by 28 +/- 13% (P less than 0.05; n = 6). ATP depletion significantly inhibited apical Na(+)-H+ exchange. These results suggest that an ATP-Ca(2+)-calmodulin-dependent process is involved in regulation of Na(+)-H+ exchange in LLC-PK1 cells. A Ca(2+)-calmodulin-dependent process activated the amiloride-sensitive basolateral Na(+)-H+ exchanger and inhibited the amiloride-resistant apical antiporter. Phosphorylation of these two Na(+)-H+ exchangers or regulatory proteins by a Ca(2+)-calmodulin-dependent protein kinase may mediate this differential regulation.

Adenosine Triphosphate↗
PubMed1991

Cytosolic acidification stimulates a calcium influx that activates Na(+)-H+ exchange in LLC-PK1.

We have previously shown that cytosolic acidification-stimulated Na(+)-H+ exchange in LLC-PK1 cells is inhibited by calmodulin antagonists. To investigate further the role of Ca(2+)-calmodulin-dependent processes in intracellular pH (pHi) regulation in these cells, we studied the effects of pHi changes on cytosolic Ca2+ concentration ([Ca2+]i). In fura-2/acetoxymethylester (fura-2/AM)-loaded cells maintained in isotonic Na(+)-free buffer containing 1.8 mM CaCl2, [Ca2+]i was 168 +/- 59 nM (n = 5). After NH4Cl-induced alkalinization, [Ca2+]i decreased to 83 +/- 28 nM and partially recovered to 126 +/- 42 nM. Cytosolic acidification, after NH4Cl washout, caused an increase in [Ca2+]i to 481 +/- 166 nM (P less than 0.05; n = 5) that was dependent on extracellular Ca2+. An increase in [Ca2+]i was also observed in cells acidified with KCl-nigericin, with a return of [Ca2+]i to baseline with cell alkalinization. No increase in 45Ca2+ efflux occurred in association with initial NH4Cl-induced [Ca2+]i decrease, suggesting Ca2+ flux into an intracellular store during alkalinization. Membrane depolarization did not alter [Ca2+]i. The acidification-induced [Ca2+]i increase was inhibited by preincubation with verapamil or the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Na(+)-dependent pHi recovery in 2,'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester (BCECF/AM)-loaded LLC-PK1 cells and cytosolic acidification-stimulated basolateral Na(+)-H+ exchange activity in LLC-PK1/CL4 cells were both attenuated in the absence of extracellular Ca2+. The results indicate that cytosolic acidification activates an influx of extracellular Ca2+ in LLC-PK1 cells. Furthermore, in the absence of Ca2+ influx, Na(+)-H+ exchange is inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Chloride↗
PubMed1990

Agonist stimulation of Na+/K+/Cl- cotransport in rat glomerular mesangial cells. Evidence for protein kinase C-dependent and Ca2+/calmodulin-dependent pathways.

Studies were performed to investigate regulatory pathways of loop diuretic-sensitive Na+/K+/Cl- cotransport in cultured rat glomerular mesangial cells. Angiotensin II, alpha-thrombin, and epidermal growth factor (EGF) all stimulated Na+/K+/Cl- cotransport in a concentration-dependent manner. Pertussis toxin pretreatment reduced the effects of angiotensin II and alpha-thrombin but not that of EGF. Addition of the protein kinase C inhibitor staurosporine or down-regulation of protein kinase C by prolonged incubation with phorbol 12-myristate 13-acetate partially reduced the effects of angiotensin II and alpha-thrombin and completely blunted the phorbol 12-myristate 13-acetate-induced stimulation of Na+/K+/Cl- cotransport but did not affect EGF-induced stimulation. Exposure of cells to a calcium ionophore, A23187, resulted in a concentration-dependent stimulation of Na+/K+/Cl- cotransport, which was not significantly inhibited by down-regulation of protein kinase C but was completely inhibited by the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Stimulation of the cotransport by angiotensin II or alpha-thrombin was also partially inhibited by W-7. Inhibitory effects of protein kinase C down-regulation and W-7 were additive and, when combined, produced a complete inhibition of angiotensin II-induced stimulation of Na+/K+/Cl- cotransport. In saponin-permeabilized mesangial cells, phosphorylation of a synthetic decapeptide substrate for Ca2+/calmodulin-dependent kinase II, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-NH3, was demonstrated. Maximal activation of the decapeptide substrate phosphorylation required the presence of Ca2+ and calmodulin and was dependent on Ca2+ concentration. These findings indicate that stimulation of Na+/K+/Cl- cotransport by angiotensin II and alpha-thrombin is mediated by protein kinase C and Ca2+/calmodulin-dependent kinases whereas the action of EGF is mediated by other pathways.

Alkaloids↗
PubMed1990

Mediation of renal vascular effects of epidermal growth factor by arachidonate metabolites.

In the rat, intrarenal infusion of epidermal growth factor decreases renal blood flow and glomerular filtration rate, and epidermal growth factor (EGF) induces contraction of cultured rat mesangial cells. The present studies examined the role of arachidonic acid metabolites in this response. Intrarenal EGF infusion increased urinary iPGF2 alpha by 300%, and in isolated glomeruli EGF stimulated iPGF2 alpha by 38%, but did not affect thromboxane B2 production. Furthermore, the thromboxane A2 receptor antagonist, SQ29548, did not block EGF's vasoconstrictive effects. After selective cyclooxygenase inhibition with ibuprofen, intrarenal EGF infusion no longer produced local vasoconstriction but instead led to systemic vasodilation (SBP: 117 +/- 10 vs. 98 +/- 7; n = 5; P less than 0.05) that was accompanied by significant increases in RPF (3.8 +/- 0.4 vs. 5.6 +/- 0.2; P less than 0.01) and glomerular filtration rate (0.9 +/- 0.1 vs. 1.1 +/- 0.1; P less than 0.05). When total arachidonate metabolism was inhibited by the additional administration of 5,8,11,14-eicosatetraynoic acid, the EGF-induced vasodilation observed during cyclooxygenase inhibition alone was abolished, and vasoconstrictor responses to EGF were again noted. Similar effects were noted with concomitant administration of the c-P450 inhibitor ketoconazole. EGF's vasoconstrictive effects were unaltered by the simultaneous administration of the angiotensin II antagonist saralasin. Thus, the renal hemodynamic responses to EGF are mediated in part by arachidonic acid metabolites. Cyclooxygenase inhibition unmasks a potent renal and systemic vasodilator action of EGF owing to its stimulation of systemic release of noncyclooxygenase arachidonate metabolites.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1990

Epoxyeicosatrienoic acids activate Na+/H+ exchange and are mitogenic in cultured rat glomerular mesangial cells.

The present study examined responses of cultured rat glomerular mesangial cells to exogenous exposure of epoxyeicosatrienoic acids (EET's), products of cytochrome P450 epoxygenase. One day after administration of 8,9- or 14,15-EET, cultured rat mesangial cells demonstrated significant increases in [3H]thymidine incorporation (10(-7) M 14,15-EET: 120 +/- 7% of control; n = 6; P less than 0.025; 10(-6) M 14,15-EET: 145 +/- 10%; n = 20; P less than 0.0005; 10(-6) M 8,9-EET: 167 +/- 31%; n = 9; P less than 0.05), which was not affected by addition of the cyclooxygenase inhibitor indomethacin. In addition to stimulation of [3H]thymidine incorporation, the epoxides stimulated mesangial cell proliferation. 14,15-EET administration induced intracellular alkalinization of 0.2-0.3 pH units, which was prevented by extracellular Na+ removal and blunted by amiloride (0.5 mM). Following intracellular acidification with NH4Cl addition and removal, greater than 85% of 3 mM 22Na uptake into mesangial cells was inhibited by 1 mM amiloride, indicating Na+/H+ exchange. Under these conditions, 14,15-EET stimulated Na+/H+ exchange by 42% and 8,9-EET stimulated Na+/H+ exchange by 59%. Neither protein kinase C depletion nor addition of the protein kinase C inhibitor, staurosporine, affected this stimulation. In [3H]myo-inositol loaded mesangial cells, no significant stimulation of phosphoinositide hydrolysis was detected in response to administration of 14,15-EET. Twenty-four hours after addition of [14C]14,15-EET, greater than 90% was preferentially esterified to cellular lipids, with predominant incorporation into phosphatidylinositol, phosphatidylethanolamine, and diacylglycerol. Thus, these results demonstrate epoxyeicosatrienoic acids stimulate Na+/H+ exchange and mitogenesis in mesangial cells. These effects do not appear to be mediated via phospholipase C activation. In addition, 14,15-EET was selectively incorporated into cellular lipids known to mediate signal transduction. These observations extend the potential biologic roles of c-P450 arachidonate metabolites to include stimulation of cell proliferation and suggest a role for these compounds in vascular and renal injury.

8,11,14-Eicosatrienoic Acid↗
PubMed1990

Changes in muscle free carnitine and acetylcarnitine with increasing work intensity in the Thoroughbred horse.

Treadmill exercise in Thoroughbred horses of 2 min duration and increasing intensity resulted in increased formation and accumulation of acetylcarnitine in the working middle gluteal muscle. At high work intensities a plateau in acetylcarnitine formation was reached corresponding to approximately 70% of the total carnitine pool (approx. 30 mmol.kg-1 dry muscle). Formation of acetylcarnitine was mirrored by an equal fall in the free carnitine content, which stabilised, at the highest work intensities, at around 8 mmol.kg-1 dry muscle. Acetylcarnitine and carnitine reached their point of maximum change at a work intensity just below that resulting in the rapid production and accumulation of lactate and glycerol 3-phosphate. It is possible that the formation of acetylcarnitine is important in the regulation of the intramitochondrial acetyl CoA/CoA ratio; equally these changes may represent a blocking mechanism aimed at preventing the transfer of unwanted free fatty acids (as acylcarnitines) into the mitochondria at work intensities where they could contribute little to energy production.

Acetylcarnitine↗
PubMed1990

Recovery of prostaglandin synthesis in rat glomerular mesangial cells after aspirin inhibition: induction of cyclooxygenase activity by serum and epidermal growth factor.

We assessed cyclooxygenase activity in cultured rat mesangial cells by measuring prostaglandin production with reverse phase HPLC upon addition of exogenous 14C arachidonic acid. The profile of prostaglandins produced was PGE2 greater than PGF2a much greater than 6-keto PGF1a much greater than thromboxane and PGD2. In quiescent mesangial cells, exposure to 300 microM aspirin for 30 minutes irreversibly inhibited cyclooxygenase activity; after 5 hours, cyclooxygenase activity was only 19 +/- 3% of control. Addition of 10% fetal bovine serum after aspirin inactivation stimulated time-dependent recovery of cyclooxygenase activity to 118 +/- 30% of control by 5 hours. Serum induced-recovery was significantly inhibited by the simultaneous administration of the protein kinase C inhibitor, staurosporine. Phorbol myristate acetate also induced recovery of cyclooxygenase activity, suggesting that protein kinase C may be involved in the signaling process. In addition to serum, epidermal growth factor was also found to lead to partial recovery of cyclooxygenase activity. The serum and EGF-induced recoveries were inhibitable by cycloheximide and actinomycin D. These results suggest that recovery of cyclooxygenase activity in mesangial cells is stimulated by EGF and other components of serum, is dependent upon new protein synthesis and appears to be transcriptionally regulated.

Animals↗
PubMed1990

Muscle buffering capacity and dipeptide content in the thoroughbred horse, greyhound dog and man.

1. Muscle buffering capacity (beta m) and dipeptide content were measured in locomotory muscles of the Thoroughbred horse, Greyhound dog and Man. 2. Beta m and carnosine contents were highest in the horse. Anserine was only found in dog muscle. 3. The higher beta m in horse and dog muscle, compared with man, appears to be predominantly due to higher muscle contents of histidine containing dipeptides in these species.

Animals↗
PubMed1990

Hypoxanthine phosphoribosyltransferase activity in tissues and hypoxanthine concentrations in plasma and CSF of the horse in comparison with other species.

1. Plasma hypoxanthine and xanthine concentrations are very low in the horse and low in rat, mouse and greyhound compared to concentrations in beagles, man, sheep and rabbit. 2. Activities in erythrocytes of the main enzyme metabolizing hypoxanthine, hypoxanthine phosphori-bosyltransferase, show a similar pattern (Tax et al., 1976, Comp. Biochem. Physiol. 54B, 209-212); thus low activities have been found where plasma concentrations were low. 3. Hypoxanthine phosphoribosyltransferase activities in horse tissue other than erythrocytes are similar to those in man and rabbit with high activities in brain; this enzyme may therefore be functionally important in equine brain.

Adenosine Triphosphate↗
PubMed1990

Changes in gene expression after temporary renal ischemia.

Temporary renal ischemia is followed by increased DNA synthesis and cell division as the kidney restores the continuity of the renal epithelium. We sought to characterize some of the changes in proto-oncogene and growth factor expression during this proliferative response. Northern analysis of polyadenylated RNAs of kidney cortical and outer stripe of outer medullary tissue from male Sprague-Dawley rats was performed following release of renal hilar clamping of 50 minutes duration. Ischemia produced an increase in c-fos mRNA that reached a peak at one hour and declined rapidly to control levels by four hours after release of the clamp. A similar rapid increase and decrease in early growth response 1 (Egr 1) mRNA was noted. The response of these immediate early genes was typical of their response to mitogens, suggesting that they served a similar role in renal cell regeneration. Levels of c-Ki-ras and glyceraldehyde phosphate dehydrogenase mRNA were unchanged. Renal preproEGF mRNA decreased at two hours, was virtually absent by 24 hours and remained low for at least four days after ischemia. Urinary excretion of EGF fell immediately after release of ischemia and before the decline in preproEGF mRNA or SNGFR, suggesting post-transcriptional affects of ischemia on renal EGF production. EGF excretion returned to only 50% of control by day 21. Specific 125I-EGF binding increased in membrane fractions of cortex, outer medulla and inner medulla as early as 24 hours after release of the clamp. Cortical 125I-EGF binding increased in the proximal tubule but not in the glomerulus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗