Biomedical subjects

Robert J Walker

Publications and source records attributed to Robert J Walker.

26 records · Page 2Linked to original sources

PubMed2003

Effects of bicarbonate buffered dialysate on human peritoneal mesothelial cell intracellular calcium homeostasis.

This study compares the biocompatibility of two bicarbonate-based peritoneal dialysis (PD) solutions using the measurement of intracellular free calcium (Ca(i)2+)) as a sensitive parameter of cell function in human peritoneal mesothelial cells (hPMC). Fura-2-loaded hPMC suspensions were exposed to bicarbonate (38 mmol/L) and bicarbonate (25 mmol/L), lactate-buffered PD (15 mmol/L) solutions at pH 7.4 and compared with Krebs-Ringer physiological saline (KRS; pH = 7.4). Resting Ca(i)2+ values and 4br-A23187 (1.0 micro mol/L) induced transients were compared in treatment and control groups. In separate studies, the effect that low saline pH had on Ca(i)(2+) homeostasis was examined. Suspended cells or cells attached to coverslips were bathed in citric acid-phosphate (McIllvaine's) buffered saline (MBS, pH = 7.4). Cells were acidified (pH = 5.3) with citric acid and then challenged with ionophore. Ionophore challenge produced a significantly reduced Ca(i)2+ transient response in cells exposed to the bicarbonate/lactate fluid compared with bicarbonate or KRS. Acidified cell suspensions produced a small monophasic Ca(i)2+ transient rise that was short lived. Gradual recovery of MBS to pH 7.4 produced no changes to Ca(i)2+ homeostasis of cell monolayers. Ionophore treatment produced a biphasic response identical to cells bathed in KRS. This study has demonstrated that short-term exposure to bicarbonate did not alter Ca(i)2+ homeostasis directly, or subsequent modulation of intracellular pH. The MBS system provided a reliable method of modifying the external pH during continuous Ca(i)2+ measurement.

Bicarbonates↗

PubMed2003

Urinary aquaporin-2 levels in healthy volunteers.

Renal water handling is regulated by the release of arginine vasopressin (AVP) and the subsequent insertion of aquaporin 2 (AQP2) in the apical membrane of collecting duct cells. This in turn increases the membrane permeability to water and the passive reabsorption of water down the concentration gradient present in the medulla. Aquaporin 2 can be detected in the urine under conditions of antidiuresis. We wish to validate an assay for urinary AQP2. Fourteen volunteers participated in studies of water loading and water deprivation followed by the administration of 1-deamino-8-D-arginine vasopressin (dDAVP). Urine osmolality was measured by vapour pressure osmometry. Urinary AQP2 was measured by using a chemiluminescent assay. Baseline correlations between serum AVP levels, urinary osmolality and urinary AQP2 levels were not significant. Following the administration of dDAVP, a positive correlation between urine osmolality and urinary AQP2 was evident (r = 0.762). For specific conditions where renal water retention is stimulated via AVP, urinary AQP2 measurements provide a reproducible measurement of the renal actions of AVP.

Administration, Intranasal↗

PubMed2003

Serum protein-bound 3,4-dihydroxyphenylalanine and related products of protein oxidation and chronic hemodialysis.

INTRODUCTION: Protein-bound dihydroxyphenylalanine (PB-DOPA) and its oxidation products are formed by free radical and oxidative attack on proteins. Hemodialysis and uremic toxins can activate leukocytes leading to overproduction of reactive oxygen species such as hydrogen peroxide and hypochlorous acid (HOCl) that increases protein oxidation. METHODS: We have used a sensitive fluorometric method to measure PB-DOPA and its oxidation products in proteins after gamma-irradiation and incubation with HOCl and in serum from hemodialysis patients and healthy controls. These PB-DOPA concentrations were compared with those measured by HPLC (PB-DOPAHPLC). RESULTS: Fluorescent PB-DOPA increased linearly with increasing amounts of human serum and with increasing amounts of gamma-irradiated bovine serum albumin. Concentrations of fluorescent PB-DOPA paralleled PB-DOPAHPLC levels but were approximately 60-70 times higher. Incubation of BSA and human serum albumin (HSA) with HOCl (39.4 mM) significantly (P < 0.0001) increased fluorescent PB-DOPA by 5 fold and 10 fold respectively and PB-DOPAHPLC by 6-fold for both proteins Fluorescent PB-DOPA concentration increased significantly (P < 0.0001) by 16-fold in human serum incubated with HOCl (39.4 mM). Mean serum fluorescent PB-DOPA was significantly (P < 0.0001) higher in 19 hemodialysis patients (57.7 +/- 16.1 microM) compared with 21 healthy controls (33.5 +/- 3.7 microM). Mean PB-DOPAHPLC was 4.45 +/- 1.63 microM in the healthy controls and 12 hemodialysis patients had values within the range of values in these controls while five patients had values that were outside eight SDs of the mean for healthy subjects. Serum fluorescent PB-DOPA was not correlated significantly with PB-DOPAHPLC in these subjects. CONCLUSIONS: The results of this study suggest that fluorophores of the type, which are derived from DOPA can be reproducibly measured in delipidated serum protein and that HOCl can increase levels of these fluorophores-generating proteins and may potentially contribute to the high levels in serum from hemodialysis patients. This high level of fluorescent PB-DOPA compounds is only partially due to authentic PB-DOPA and might also be derived from other related protein oxidation products including those from DOPA oxidation.

Adult↗

PubMed2003

Aquaporin expression in normal human kidney and in renal disease.

Aquaporins (AQPs), membrane-inserted water channel proteins, play a highly important role in the reabsorption of water from the renal tubular fluid. Experimentally, both in rats and mice, failure to insert functional AQP molecules into renal tubular membranes leads to nephrogenic diabetes insipidus. In humans, most forms of renal disease lead to a reduction in the water handling capacity of the kidney. AQP distribution in various forms of human renal disease has not been documented. Immunohistochemical studies of biopsy samples from a wide range of renal diseases revealed a substantial and striking upregulation of AQP-1 in the glomeruli of most diseased kidneys. AQP-1 expression remained prominent in proximal tubules in all lesions. In contrast, there was judged qualitatively to be a reduction in the amounts of AQP-2 and AQP-3 expression, especially in lesions with substantial interstitial fibrosis and nephron loss, as compared with a healthy region of normal kidneys. The results were quantitatively confirmed by real-time reverse transcriptase-PCR. This is the first documentation of altered AQP expression in human renal disease. The significance of the increased AQP-1 expression requires further studies.

Animals↗

PubMed2002

Effect of meals rich in heated olive and safflower oils on oxidation of postprandial serum in healthy men.

The present randomised, crossover study sought to determine the effect of meals rich in safflower oil and olive oil (60 g) which had been heated for 8 h at 210 degrees C and the corresponding unheated oils on copper ion oxidation of dilute serum from 16 healthy men. Four hours after the meals rich in the heated oils, there were significant decreases of similar magnitude (-12%) in the lag time in conjugated diene formation during diluted serum oxidation. In the 12 subjects who consumed meals containing unheated oils, the lag time also decreased (-11%) significantly after the meal rich in unheated safflower oil (US) and did not change significantly after the unheated olive oil (UO) meal and these changes were different between the meals at a marginal level of significance (P=0.05). Our data suggest that susceptibility to oxidation of lipoproteins in low antioxidant environments similar to dilute serum may be increased in the postprandial period following meals rich in heat-modified vegetable oils and unheated oils rich in polyunsaturated fatty acids but not following meals rich in native olive oil. These findings may be relevant to the choice of fat to replace saturated fats in lipid-lowering diets and to low risk of coronary heart disease in communities which have a high consumption of olive oil.

Adult↗

PubMed2002

Release of cholesterol from cell membranes to postprandial plasma from mildly hypercholesterolemic subjects: the effect of meals rich in olive and safflower oils.

Triglyceride-rich lipoproteins increase net transport of cell cholesterol to postprandial plasma from healthy subjects after a meal rich in fat and cholesterol. The aim of the present study was to determine the effect of meals rich in polyunsaturated fats (PUFA) and monounsaturated fats (MUFA) and low in cholesterol on net in vitro transport of cholesterol from red blood cells (RBCs) to postprandial plasma from 21 men with mild to moderate hypercholesterolemia in a randomized, crossover trial. Cholesterol concentration increased by 12% due to accumulation of cell cholesterol in fasted hypercholesterolemic plasma incubated with a 2/1 (vol/vol) excess of RBCs at 37 degrees C for 18 hours. The increase in cell cholesterol in plasma was mainly localized in the low-density lipoprotein (LDL) fraction (64%) and the remainder was approximately equally divided between the very-low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) fractions. Accumulation of cell cholesterol in the LDL fraction prevented the significant decrease in LDL cholesterol in plasma incubated alone. When RBCs were incubated with postprandial plasma isolated 4 hours and 6 hours after liquid meals rich in safflower and olive oils, the accumulation of cell cholesterol in plasma increased significantly (11%, P <.004) above values for fasted plasma and irrespective of the type of fat in the meal. Also, the content of cell cholesterol increased significantly (70%, P <.001) in triglyceride (TG)-rich lipoproteins and decreased significantly (P =.006) in the LDL fraction, which remained the main ultimate destination of cell cholesterol in postprandial plasma. The increased loss of cell cholesterol to fasted and postprandial plasma was closely correlated (r > 0.823, P <.001) with the concomitant increase in plasma cholesteryl esters (CE) generated by lecithin cholesterol acyltransferase (LCAT) activity. There was a small (5%), significant (P <.001) increase in plasma cholesterol esterification in postprandial plasma. These data suggest that high-fat meals rich in MUFA and PUFA and low in cholesterol may produce a small postprandial increase in the capacity of plasma to accept cell membrane cholesterol that is limited by a concomitant small increase in plasma cholesterol esterification, in hypercholesterolemic subjects. Thus, low-fat, lipid-lowering diets may have a minimal effect on this capacity but will reduce levels of atherogenic LDL cholesterol that appear to be maintained by diffusion of cell cholesterol to plasma.

Adult↗

PubMed2002

Ionic basis of the resting membrane potential and action potential in the pharyngeal muscle of Caenorhabditis elegans.

The pharynx of C. elegans is a rhythmically active muscle that pumps bacteria into the gut of the nematode. This activity is maintained by action potentials, which qualitatively bear a resemblance to vertebrate cardiac action potentials. Here, the ionic basis of the resting membrane potential and pharyngeal action potential has been characterized using intracellular recording techniques. The resting membrane potential is largely determined by a K(+) permeability, and a ouabain-sensitive, electrogenic pump. As previously suggested, the action potential is at least partly dependent on voltage-gated Ca(2+) channels, as the amplitude was increased as extracellular Ca(2+) was increased, and decreased by L-type Ca(2+) channel blockers verapamil and nifedipine. Barium caused a marked prolongation of action potential duration, suggesting that a calcium-activated K(+) current may contribute to repolarization. Most notably, however, we found that action potentials were abolished in the absence of external Na(+). This may be due, at least in part, to a Na(+)-dependent pacemaker potential. In addition, the persistence of action potentials in nominally free Ca(2+), the inhibition by Na(+) channel blockers procaine and quinidine, and the increase in action potential frequency caused by veratridine, a toxin that alters activation of voltage-gated Na(+) channels, point to the involvement of a voltage-gated Na(+) current. Voltage-clamp analysis is required for detailed characterization of this current, and this is in progress. Nonetheless, these observations are quite surprising in view of the lack of any obvious candidate genes for voltage-gated Na(+) channels in the C. elegans genome. It would therefore be informative to re-evaluate the data from these homology searches, with the aim of identifying the gene(s) conferring this Na(+), quinidine, and veratridine sensitivity to the pharynx.

4-Aminopyridine↗

PubMed2002

Renal PGE2 production in the human and rat following phenacetin, acetaminophen and p-aminophenol.

Co-oxidation of phenacetin, acetaminophen (APAP) and p-aminophenol (p-AP) by prostaglandin H synthase (PHS) was investigated in human and rat renal microsomes. The formation of prostaglandin E2 (PGE2) was assessed in cortex, outer and inner medulla following phenacetin, APAP and p-AP (0-5 mM) incubations. For all compounds and concentrations tested, a significantly higher PGE2 production was observed in inner medulla compared to cortex. Rat inner medulla incubated with phenacetin resulted in an increased formation of PGE2 at all concentrations compared to control (1 mM phenacetin increased production by 243%). Human inner medulla demonstrated an increased PGE2 production at 1, 3 and 5 mM phenacetin versus control (136% increase at 1 mM). An increase in PGE2 formation in rat and human inner medulla was observed at low APAP concentrations (0.1, 0.3, 0.5 and 1 mM) compared to control (216% and 396% in human and rat respectively following 1 mM APAP). 5 mM APAP inhibited PGE2 formation in the rat inner medulla but not in human inner medulla. An inhibition of PGE2 production by 5 mM p-AP was observed in both the rat and human inner medulla. In the rat PGE2 production was inhibited 69% by 5 mM, whereas in the human the inhibition was 76% at 5 mM. These studies demonstrate a species-specific PHS-mediated renal metabolism of APAP, with the human kidney demonstrating a continous formation of reactive metabolites at high concentrations of APAP. However, phenacetin and p-AP are metabolized in a similar manner in these 2 species.

Acetaminophen↗