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Biomedical subjects

C D Brown

Publications and source records attributed to C D Brown.

At least 91 records · Page 5Linked to original sources
PubMed1987

Heterogeneity of cholesterol homeostasis in man. Response to changes in dietary fat quality and cholesterol quantity.

Studies were carried out to examine the effects of dietary fat and cholesterol on cholesterol homeostasis in man. 75 12-wk studies were carried out during intake of 35% of calories as either saturated or polyunsaturated fat, first low and then high in dietary cholesterol. Dietary fat and cholesterol intakes, plasma lipid and lipoprotein levels, cholesterol absorption and sterol synthesis in isolated blood mononuclear leukocytes were measured during each diet period. In 69% of the studies the subjects compensated for the increased cholesterol intake by decreasing cholesterol fractional absorption and/or endogenous cholesterol synthesis. When an increase in plasma cholesterol levels was observed there was a failure to suppress endogenous cholesterol synthesis. Plasma cholesterol levels were more sensitive to dietary fat quality than to cholesterol quantity. The results demonstrate that the responses to dietary cholesterol and fat are highly individualized and that most individuals have effective feedback control mechanisms.

Cells, Cultured↗
PubMed1986

Characterization of a Ca-dependent maxi K channel in the apical membrane of a cultured renal epithelium (JTC-12.P3).

A Ca and potential-dependent K channel of large unit conductance was detected in the apical membrane of JTC-12.P3 cells, a continuous epithelial cell line of renal origin. The open probability of the channel is dependent on membrane potential and cytoplasmic-free Ca concentration. At cell-free configuration of the membrane patch, the open probability shows a bell-shaped behavior as function of membrane potential, which decreases at larger depolarization. With increasing Ca concentration, the width of the bell-shaped curve increases and the maximum shifts into the hyperpolarizing direction. For the first time the kinetics of this channel was analyzed under cell-attached conditions. In this case the kinetics could sufficiently be described by a simple open-closed behavior. The channel has an extremely small open probability at resting potential, which increases exponentially with depolarization. The low probability induces an uncertainty about the actual number of channels in the membrane patch. The number of channels is estimated by kinetic analysis. It is discussed that this K channel is essential for the repolarization of the membrane potential during electrogenic sodium-solute cotransport across the apical membrane.

Animals↗
PubMed1986

Loop-diuretic inhibition of adrenaline-stimulated Cl- secretion in a cultured epithelium of renal origin (MDCK).

Loop diuretics (furosemide, bumetanide, piretanide) inhibit the adrenaline-stimulated short-circuit current due to transepithelial Cl- secretion in cultured renal epithelial layers (MDCK). The inhibition of Cl- secretion by loop diuretics is consistent with the presence of basal-lateral 'cotransport' since inhibition is observed only with the basal applications of loop diuretics, is of high potency (half-maximal bumetanide inhibition being observed at 0.8 microM, bumetanide being more potent than furosemide) and is without effect upon the adrenaline-stimulated increase in tissue conductance. Loop diuretics are also shown to inhibit a component of K+ efflux across the basal-lateral surfaces. Cellular uptake of [3H]bumetanide across both apical and basal surfaces of intact epithelial layers was measured in order to localize the cotransport system. A component of cellular [3H]bumetanide uptake sensitive to competition by 0.1 mM unlabelled loop diuretic is only observed from the basal-lateral cell surfaces. There is no evidence for transepithelial bumetanide secretion as is seen in renal cortex.

Animals↗
PubMed1986

NaCl transport stimulates prostaglandin release in cultured renal epithelial (MDCK) cells.

Prostaglandins (PGs) can modulate a variety of renal functions, including Na+ and Cl- reabsorption. However, it is not known if a direct interdependence exists between PG synthesis and transport activity. The present study was done to find out whether or not the rate of NaCl transport has an influence on PG synthesis in renal tubular cells. For our studies we used cultures of so-called high-resistance MDCK cells, which were originally derived from canine kidney. This cell type has a loop diuretic- and ouabain-sensitive NaCl transport that can be enhanced by activation of the adenylate cyclase (AC). In MDCK cell cultures we found that each state of increased NaCl transport during stimulation of AC by either epinephrine (10(-6) M), isoprenaline (10(-5) M), or forskolin (10(-5) M) was accompanied by a twofold increase in PG release. During inhibition of NaCl transport by furosemide (10(-4) M) or ouabain (2 X 10(-4) M), stimulation of AC failed to increase PGE2 release, whereas basal PG production was not inhibited by either furosemide or ouabain. Furthermore, PG formation during activation of AC was dependent on the concentration of extracellular Na+, whereas PG formation in the absence of activators of AC was independent of extracellular Na+. These results suggest that increased NaCl transport stimulates PG formation in cultures of high-resistance MDCK cells.

Adenylyl Cyclases↗
PubMed1985

Identification of a voltage-dependent anion channel in the apical membrane of a Cl(-)-secretory epithelium (MDCK).

An anion-selective channel of large unit conductance is present in apical membranes of a secretory epithelial cell. For cell-attached patch-clamp configuration on cultured MDCK cells channels with a conductance of about 460 pS are observed at 37 degrees C. The channel becomes spontaneously activated only in rare cases. In inside-out membrane patches the probability of observing this type of channel increased significantly suggesting that the channel is controlled by an up to now unknown mechanism. The channel shows a voltage-dependent burst kinetic.

Anions↗
PubMed1985

Characterization of a Na : K : 2C1 cotransport system in the apical membrane of a renal epithelial cell line (LLC-PK1).

86Rb uptake into LLC-PK1 cells (an established renal epithelial cell line) was found to be comprised of an active ouabain-sensitive component, a loop diuretic-sensitive component which was passive and strictly dependent upon the presence of extracellular Na+ and Cl- for activity, and a "leak" component. The diuretic-sensitive component of influx was investigated further in apical membrane vesicles derived from these cells. A large fraction of 86Rb, 22Na and 36Cl flux into these vesicles was sensitive to inhibition by furosemide and dependent upon the presence of the other two co-ions, in keeping with the presence of a loop diuretic-sensitive Na+ : K+ : Cl- cotransport system. The kinetic parameters for Na+ and K+ interaction have been analyzed under initial linear zero trans conditions. The following values were obtained KmNa+ = 0.42 +/- 0.05 mmol/liter, Vmax = 303 +/- 24 pmol/mg/6 sec; KmK+ = 11.9 +/- 1.0 mmol/liter, VmaxK+ = 307 +/- 27 pmol/mg/6 sec. For Cl- interaction evidence for two cooperative binding sites with different affinities and different specificities were obtained. Thus, a stoichiometry of 1Na+ : 1K+ :2Cl- can be calculated. It is concluded that the apical membrane of LLC-PK1 cells contains a Na+ : K+ : 2Cl- co-transport system with properties similar to those described for the thick ascending limb of the loop of Henle.

Animals↗
PubMed1985

Adaptation of phosphate transport in phosphate-deprived LLC-PK1 cells.

Sodium-dependent transport of phosphate was studied in LLC-PK1 cells that had been deprived of phosphate (Pi). Compared with control cells (fed with 2 mM Pi) a twofold increase in the rate of Na-Pi cotransport was observed in cells incubated for 15 h in a phosphate-free medium, whereas transport of L-alanine and the specific activity of alkaline phosphatase were not changed. The same adaptive response was observed with apical membrane vesicles isolated from Pi-deprived cells. In both experimental systems Pi deprivation caused a change in the Vmax but not in the apparent Km (for Pi) of the cotransport system. Adaptation of the Na-Pi cotransport was triggered by free phosphate concentrations between 0 and 100 microM. Over the first 20 h the adaptive response was found to be a linear process that could be prevented by 70 microM cycloheximide. Adapted cells that were re-treated with phosphate showed a rapid (less than 3 h) decrease in the Na-Pi transport. The data suggest that LLC-PK1 cells adapt to low Pi conditions by increasing the rate of the Na-Pi cotransport, which is located in the apical membrane. Two mechanisms may be involved in the adaptive response: a long-term process involving new protein synthesis, and a short-term response involving activation-inactivation of transport systems already existing.

Adaptation, Physiological↗
PubMed1984

The action of epinephrine on Madin-Darby canine kidney cells.

We have used cultured monolayers of Madin-Darby canine kidney (MDCK) cells, which form epithelial layers of high transepithelial resistance, grown on Millipore filters, for transport studies. In the absence of hormones net ion transport is of small magnitude and is consistent with a net absorptive flow (apical to basal) of Na+. Epinephrine, effective only from the basolateral cell surface, stimulates a net secretion (basal to apical) of Cl-. A substantial portion of net Cl- secretion is inhibited by loop diuretics such as furosemide applied to the basolateral cell aspects. The participation of a diuretic-sensitive cotransport system for Na+, K+, and Cl-, similar to that found in other cells, in transepithelial Cl- flux is postulated. The action of catecholamines on MDCK cell adenylate cyclase and on a Ca2+-activated K+ conductance is described.

Adenylyl Cyclases↗
PubMed1984

Sodium-dependent phosphate transport by apical membrane vesicles from a cultured renal epithelial cell line (LLC-PK1).

Apical membrane vesicles were prepared from confluent monolayers of LLC-PK1 cells grown upon microcarrier beads. The final membrane preparation, obtained by a modified divalent cation precipitation technique, was enriched in alkaline phosphatase, leucine aminopeptidase and trehalase (8-fold compared to the initial homogenate). Analysis of phosphate uptake into the vesicles identified a specific sodium-dependent pathway. Lithium and other cations were unable to replace sodium. At 100 mmol/l sodium and pH 7.4, an apparent Km for phosphate of 99 +/- 19 mumol/l and an apparent Ki for arsenate of 1.9 mmol/l were found. Analysis of the sodium activation of phosphate uptake gave an apparent Km for sodium of 32 +/- 12 mmol/l and suggested the involvement of two sodium ions in the transport mechanism. Sodium modified the apparent Km of the transport system for phosphate. The rate of sodium-dependent phosphate uptake was higher at pH 6.4 than at pH 7.4. At both pH values, an inside negative membrane potential (potassium gradient plus valinomycin) had no stimulatory effect on the rate of the sodium-dependent component of phosphate uptake. It is concluded that the apical membrane of LLC-PK1 cells contains a sodium-phosphate cotransport system with a stoichiometry of 2 sodium ions: 1 phosphate anion.

Animals↗
PubMed1984

Plasma mevalonate as a measure of cholesterol synthesis in man.

Measurement of mevalonic acid (MVA) concentrations in plasma or 24-h urine samples is shown to be useful in studies of the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol synthesis. Plasma MVA concentrations, measured either at 7-9 a.m. after an overnight fast, or throughout the 24-h cycle, were compared with cholesterol synthesis rates that were measured by the sterol balance method: plasma MVA concentrations were directly related to the rate of whole body cholesterol synthesis (r = 0.972; p less than 0.001; n = 18) over a tenfold range of cholesterol synthesis rates. Moreover, hourly examination of MVA concentrations throughout the day demonstrated that interventions such as fasting or cholesterol feeding cause suppression of the postmidnight diurnal rise in plasma MVA concentrations, with little change in the base-line of the rhythm. Thus, the daily rise and fall of plasma MVA appears to reflect changes in tissues and organs, such as the liver and intestine, that are known to be most sensitive to regulation by fasting or by dietary cholesterol. The hypothesis that short-term regulation of HMG-CoA reductase in tissues is quickly reflected by corresponding variations in plasma MVA was tested by using a specific inhibitor of HMG-CoA reductase, mevinolin, to block MVA synthesis. Mevinolin caused a dose-dependent lowering of plasma MVA after a single dose; and in patients who received the drug twice a day for 4 wk, it decreased 24-h urinary MVA output. Significant lowering of plasma cholesterol was achieved through administration of mevinolin at doses that only moderately limit MVA production.

Cholesterol↗
PubMed1983

Sodium-dependent transport of phosphate in LLC-PK1 cells.

Transport of phosphate has been studied in subconfluent monolayers of LLC-PK1 cells. It was found that this transport system shows similar characteristics to those observed in the kidney. Uptake of phosphate is mediated by a Na+-dependent, substrate-saturable process with an apparent Km value for phosphate of 96 +/- 15 mumol/l. Kinetic analysis of the effect of Na+ indicated that at (pH 7.4) two sodium ions are cotransported with one HOP4(2-) ion (Hill coefficient 1.5) with an apparent Km value for sodium of 56 mmol/l. Pi uptake is inhibited by metabolic inhibitors (ouabain and FCCP). In the pH range of 6.6 of 7.4 Pi uptake rate does not change significantly, indicating that both the monovalent and the divalent form of phosphate are accepted by the transport system. It is suggested that phosphate is transported by LLC-PK1 cells together with sodium (2 Na+:1 HPO4(2-) in an electroneutral manner down a favourable sodium gradient.

Animals↗
PubMed1983

Treatment of familial hypercholesterolemia by portacaval anastomosis: effect on cholesterol metabolism and pool sizes.

Measurements of the key parameters of cholesterol homeostasis and the mass of the body pools of cholesterol were carried out in two patients with familial hypercholesterolemia (FH), one homozygote and one heterozygote, before and 28 and 18 months, respectively, after portacaval anastomosis (PCA). In both patients the procedure significantly reduced the plasma concentrations of total and low density lipoprotein cholesterol and the daily rate of whole body cholesterol and bile acid synthesis. In addition, PCA caused a net efflux of accumulated tissue cholesterol as demonstrated by reductions in the rapidly exchangeable and total exchangeable masses of body cholesterol. Shunt patency was verified by demonstration of increased bile acids in serum from fasting patients and from patients 2 hr after a meal and by increased plasma glucagon before and after arginine infusion. Other than a persistently increased level of serum alkaline phosphatase, liver function tests have fallen within the normal range in both patients; there has been no evidence of hepatic encephalopathy. In the homozygous patient there has also been a striking resolution in xanthoma size and distribution. These multiple effects on cholesterol homeostasis and pool sizes strongly suggest that PCA can reverse the progressive accumulation of cholesterol in body tissues of FH patients.

Bile Acids and Salts↗
PubMed1982

K+ transport in 'tight' epithelial monolayers of MDCK cells. Evidence for a calcium-activated K+ channel.

Measurements of 86Rb efflux across the apical and basal-lateral aspects of intact monolayers of 'high-resistance' MDCK cells mounted in Ussing chambers have been made. A transient increase in 86Rb efflux across both epithelial borders upon stimulation with adrenalineeeeeee or ionophore A23187 is observed. The increased 86Rb across the basal cell aspects is of greatest quantitative importance. Measurements of total cellular K+ contents by flame photometry of tissue extracts indicate a net loss of K+ following adrenalin addition. The effects of adrenalin and ionophore A23187 upon 86Rb efflux are abolished in 'Ca2+ -free' media. The properties of the Ca2+ -dependent increase in 86Rb efflux show similarities to Ca2+ -activated K+ conductances in other tissues, notably human red cells, including inhibition by quinine (1 mM), tetraethylammonium (25 mM) and insensitivity to bee venom toxin (apamin) (25 nM). Adrenalin is only effective when applied to the basal bathing solution suggesting that the receptors mediating adrenalin action are located upon the basal-lateral membranes. Half maximal stimulation of 86Rb efflux by adrenalin is observed at 9.1 X 10(-7) M. The action of various adrenergic receptor agonists and antagonists are consistent with adrenalin action being mediated by an alpha-adrenergic receptor.

Animals↗
PubMed1982

K+ transport in "tight' epithelial monolayers of MDCK cells.

Bidirectional transepithelial K+ flux measurements across 'high-resistance' epithelial monolayers of MDCK cells grown upon millipore filters show no significant net K+ flux. Measurements of influx and efflux across the basal-lateral and apical cell membranes demonstrate that the apical membranes are effectively impermeable to K+. K+ influx across the basal-lateral cell membranes consists of an ouabain-sensitive component, an ouabain-insensitive component, an ouabain-insensitive but furosemide-sensitive component, and an ouabain- and furosemide-insensitive component. The action of furosemide upon K+ influx is independent of (Na+ - K+)-pump inhibition. The furosemide-sensitive component is markedly dependent upon the medium K+, Na+ and Cl- content. Acetate and nitrate are ineffective substitutes for Cl-, whereas Br- is partially effective. Partial Cl- replacement by NO3 gives a roughly linear increase in the furosemide-sensitive component. Na+ replacement by choline abolishes the furosemide-sensitive component, whereas Li+ is a partially effective replacement. Partial Na+ replacement by choline abolishes the furosemide-sensitive component, whereas Li+ is a partially effective replacement. Partial Na+ replacement with choline gives an apparent affinity of approximately 7 mM Na, whereas variation of the external K+ content gives an affinity of the furosemide-sensitive component of 1.0 mM. Furosemide inhibition is of high affinity (K1/2 = 3 micrometer). Piretanide, ethacrynic acid, and phloretin inhibit the same component of passive K+ influx as furosemide; amiloride, 4,-aminopyridine, and 2,4,6-triaminopyrimidine partially so. SITS was ineffective. Externally applied furosemide and Cl- replacement by NO3- inhibit K+ efflux across the basal-lateral membranes indicating that the furosemide-sensitive component consists primarily of K:K exchange.

Animals↗