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C R Ross

Publications and source records attributed to C R Ross.

At least 55 records · Page 3Linked to original sources
PubMed1989

Operational modes of the organic anion exchanger in canine renal brush-border membrane vesicles.

This study delineates the various operational modes catalyzed by the organic anion exchanger present in the canine renal brush-border membrane. The experiments examined the carrier-mediated effects of various organic and inorganic anions on the transport of either p-[3H]aminohippuric acid ([3H]PAH) or 36Cl-. [3H]PAH countertransport was significantly stimulated by PAH, urate, Cl-, Br-, HCO3-, and by a pH gradient. This pH stimulation remained in the absence of HCO3- (i.e., under N2), implying PAH-OH- exchange. Furosemide, bumetanide, penicillin, and probenecid inhibited countertransport of [3H]PAH. Likewise, the above anions produced cis inhibition of [3H]PAH transport. The cis and trans effects of SO4(-2) and formate were minimal. 36Cl- countertransport was stimulated by PAH, Cl-, Br-, HCO3-, formate, and by a pH gradient that was effective even in the absence of HCO3- (i.e., under N2), implying Cl- -OH- exchange. Cl- -OH- and Cl- -Cl- exchange was inhibited by PAH. In each instance, the trans-stimulation of 36Cl- efflux was insensitive to maneuvers that created an inside-positive membrane potential, demonstrating electroneutral mediated exchange. We conclude that the organic anion transporter can operate in three distinct exchange modes: organic-organic, organic-inorganic, and inorganic-inorganic.

Animals↗
PubMed1989

Gentamicin and verapamil compete for a common transport mechanism in renal brush border membrane vesicles.

The effects of gentamicin, an aminoglycoside antibiotic, and verapamil, a calcium channel blocker, on the transport systems in canine renal brush border membrane vesicles were examined. They were tested for their effects on the transport of either a prototypic organic cation, N1-[3H]methylnicotinamide or a prototypic organic anion p-[3H]aminohippurate. They were classified as a substrate for either transport system if the following two criteria were fulfilled: 1) cis inhibition and 2) trans stimulation with concentrative uptake of the prototypic ion. Both gentamicin and verapamil cis inhibited the uptake of the prototypic organic cation N1[3H]methylnicotinamide, with Ki values of 450 and 5 microM, respectively. Similarly, both drugs trans-stimulated N1-methylnicotinamide influx and produced concentrative uptake over the equilibrium value. The results demonstrate that gentamicin and verapamil are substrates for the renal organic cation transport system. We postulate that high-affinity substrates should protect against gentamicin-induced nephrotoxicity.

Animals↗
PubMed1988

Arginyl and histidyl groups are essential for organic anion exchange in renal brush-border membrane vesicles.

The effect of side chain modification on the organic anion exchanger in the renal brush-border membrane was examined to identify what amino acid residues constitute the substrate binding site. One histidyl-specific reagent, diethyl pyrocarbonate (DEPC), and 2 arginyl-specific reagents, phenylglyoxal and 2,3-butanedione, were tested for their effect on the specifically mediated transport of p-amino[3H]hippurate (PAH), a prototypic organic anion. The specifically mediated transport refers to the difference in the uptake of [3H]PAH in the absence and presence of a known competitive inhibitor, probenecid, and was examined in brush-border membrane vesicles isolated from the outer cortex of canine kidneys. The experiments were performed utilizing a rapid filtration assay. DEPC, phenylglyoxal, and 2,3-butanedione inactivated the specifically mediated PAH transport, i.e. probenecid inhibitable transport with IC50 values of 160, 710, and 1780 microM, respectively. The rates of PAH inactivation by DEPC and phenylglyoxal were suggestive of multiple pseudo first-order reaction kinetics and were consistent with a reaction mechanism whereby more than 1 arginyl or histidyl residue is inactivated. Furthermore, PAH (5 mM) did not affect the rate of phenylglyoxal inactivation. In contrast, PAH (5 mM) affected the rate of DEPC inactivation. The modification by DEPC was specific for histidyl residues since transport could be restored by treatment with hydroxylamine. The results demonstrate that histidyl and arginyl residues are essential for organic anion transport in brush-border membrane vesicles. We conclude that the histidyl residue constitutes the cationic binding site for the anionic substrate, whereas the arginyl residue(s) serves to guide the substrate to or away from the histidyl site.

Aldehydes↗
PubMed1988

Proton-coupled organic cation transport in renal brush-border membrane vesicles.

We had previously proposed that organic cations are transported across the brush-border membrane in the canine kidney by a H+ exchange (or antiport) system (Holohan, P.D. and Ross, C.R. (1981) J. Pharmacol. Exp. Ther. 216, 294-298). In the present report, we demonstrate that in brush-border membrane vesicles the transport of organic cations is chemically coupled to the countertransport of protons, by showing that the uphill or concentrative transport of a prototypic organic cation, N1-methylnicotinamide (NMN), is chemically coupled to the flow of protons down their chemical gradient. In a reciprocal manner, the concentrative transport of protons is coupled to the counterflow of organic cations down their concentration gradient. The transport of organic cations is monitored by measuring [3H]NMN while the transport of protons is monitored by measuring changes in acridine orange absorbance. The functional significance of the coupling is that a proton gradient lowers the Km and increases the Vmax for NMN transport.

Acridine Orange↗
PubMed1988

Secreted growth factors from estrogen receptor-negative human breast cancer do not support growth of estrogen receptor-positive breast cancer in the nude mouse model.

Estrogen receptor (ER)-negative MDA-231 human breast cancer cells have been shown to secrete high concentrations of several growth factors including transforming growth factor-alpha and insulin-like growth factor I, which could have important autocrine or paracrine growth regulatory functions and, additionally, could explain the rapid autonomous growth of these cells. In contrast, the hormone-responsive, ER-positive MCF-7 cells secrete low levels of these factors constitutively. Since estrogen treatment increases secretion of these growth factors in MCF-7 cells, it has been postulated that these growth factors mediate estrogen's growth effects through an autocrine mechanism. To test this hypothesis we reasoned that growth factors supplied by MDA-231 cells should support growth of MCF-7 cells in an estrogen-depleted environment. Inoculation of castrated female athymic nude mice with MDA-231 cells resulted in rapid tumor growth. However, MDA-231 tumors did not support growth of MCF-7 cells inoculated on the opposite flank by an endocrine mechanism; MCF-7 tumors required estrogen supplementation for growth. To determine if MDA-231 cells could support MCF-7 growth by a paracrine mechanism, various mixtures of the two cell lines were coinoculated at the same site in castrated or in estrogen-supplemented mice. ER was not detectable in tumors derived from a mixed inoculum, indicating the absence of MCF-7 cell growth. Furthermore, DNA flow cytometry of these tumors revealed only a single G1 peak representative of MDA-231 cells in estrogen-deprived mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Mechanism of ochratoxin A transport in kidney.

The effect of the fungal metabolite (mycotoxin) Ochratoxin A (OTA) on the transport of p-amino[3H]hippurate (PAH), a prototypic organic anion, was examined in renal brush border (BBMV) and basolateral membrane vesicles (BLMV). OTA was as effective an inhibitor of PAH uptake in both membranes as probenecid. The dose response curves for OTA in BBMV and BLMV gave IC50 values of 20 +/- 6 and 32 +/- 7 microM, respectively. The effect was specific since the transport of the organic cation N1-methylnicotinamide was not affected. The phenomenon of counterflow was studied to establish that OTA is translocated. OTA produced trans stimulation of PAH transport in both BBMV and BLMV, demonstrating that OTA is transported across both these membranes. The data suggest that OTA interacts with the PAH transport system in both BBMV and BLMV. We conclude that OTA transport in the kidney is mediated via the renal organic anion transport system.

Animals↗
PubMed1988

Gentamicin-induced increases in cytosolic calcium in pig kidney cells (LLC-PK1).

LLC-PK1 cells, an established epithelial cell line derived from pig kidney, were tested as a model system for assessing the role of calcium in gentamicin-induced nephrotoxicity. Cell viability was evaluated by a vital dye exclusion procedure, and intracellular free calcium [Ca2+]i was measured employing Fura-2 fluorescence. Exposing cell suspensions (10(6)/ml) to concentrations of the drug, which had no apparent effect on viability, produced a rapid and prolonged increase in intracellular [Ca2+]. The perturbation of calcium homeostasis could be blocked by the addition of mepiperphenidol, an inhibitor of the organic cation transport system. We propose that LLC-PK1 cells are an appropriate model to study drug-induced nephrotoxicity. Gentamicin disrupts calcium homeostasis and causes plasma membrane alterations. Since mepiperphenidol blocked the gentamicin-induced Ca2+ increases, the data suggest that aminoglycosides enter the cell via the organic cation transporter.

Animals↗
PubMed1987

Cl(-)-HCO3- exchange in rat renal basolateral membrane vesicles.

Pathways for HCO3- transport across the basolateral membrane were investigated using membrane vesicles isolated from rat renal cortex. The presence of Cl(-)-HCO3- exchange was assessed directly by 36Cl- tracer flux measurements and indirectly by determinations of acridine orange absorbance changes. Under 10% CO2/90% N2 the imposition of an outwardly directed HCO3- concentration gradient (pHo 6/pHi 7.5) stimulated Cl- uptake compared to Cl- uptake under 100% N2 in the presence of a pH gradient alone. Mediated exchange of Cl- for HCO3- was suggested by the HCO3- gradient-induced concentrative accumulation of intravesicular Cl-. Maneuvers designed to offset the development of ion-gradient-induced diffusion potentials had no significant effect on the magnitude of HCO3- gradient-driven Cl- uptake further suggesting chemical as opposed to electrical Cl(-)-HCO3- exchange coupling. Although basolateral membrane vesicle Cl- uptake was observed to be voltage sensitive, the DIDS insensitivity of the Cl- conductive pathway served to distinguish this mode of Cl- translocation from HCO3- gradient-driven Cl- uptake. No evidence for K+/Cl- cotransport was obtained. As determined by acridine orange absorbance measurements in the presence of an imposed pH gradient (pHo 7.5/pHi 6), a HCO3- dependent increase in the rate of intravesicular alkalinization was observed in response to an outwardly directed Cl- concentration gradient. The basolateral membrane vesicle origin of the observed Cl(-)-HCO3- exchange activity was verified by experiments performed with purified brush-border membrane vesicles. In contrast to our previous observations of the effect of Cl- on HCO3- gradient-driven Na+ uptake suggesting a basolateral membrane Na+-HCO3- for Cl- exchange mechanism, no effect of Na+ on Cl-HCO3- exchange was observed in the present study.

Animals↗
PubMed1987

HCO3- transport in basolateral membrane vesicles isolated from rat renal cortex.

The mechanism of HCO3- translocation across the proximal tubule basolateral membrane was investigated by testing for Na+-HCO3- cotransport using isolated membrane vesicles purified from rat renal cortex. As indicated by 22Na+ uptake, imposing an inwardly directed HCO3- concentration gradient induced the transient concentrative accumulation of intravesicular Na+. The stimulation of basolateral membrane vesicle Na+ uptake was specifically HCO3(-)-dependent as only basolateral membrane-independent Na+ uptake was stimulated by an imposed hydroxyl gradient in the absence of HCO3-. No evidence for Na+-HCO3- cotransport was detected in brush border membrane vesicles. Charging the vesicle interior positive stimulated net intravesicular Na+ accumulation in the absence of other driving forces via a HCO3(-)-dependent pathway indicating the flow of negative charge accompanies the Na+-HCO3- cotransport event. Among the anion transport inhibitors tested, 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid demonstrated the strongest inhibitor potency at 1 mM. The Na+-coupled transport inhibitor harmaline also markedly inhibited HCO3- gradient-driven Na+ influx. A role for carbonic anhydrase in the mechanism of Na+-HCO3- cotransport is suggested by the modest inhibition of HCO3- gradient driven Na+ influx caused by acetazolamide. The imposition of Cl- concentration gradients had a marked effect on HCO3- gradient-driven Na+ influx which was furosemide-sensitive and consistent with the operation of a Na+-HCO3- for Cl- exchange mechanism. The results of this study provide evidence for an electrogenic Na+-HCO3- cotransporter in basolateral but not microvillar membrane vesicles isolated from rat kidney cortex. The possible existence of an additional basolateral membrane HCO3(-)-translocating pathway mediating Na+-HCO3- for Cl- exchange is suggested.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1987

N,N'-dicyclohexylcarbodiimide inactivates organic cation transport in renal brush border membranes.

The molecular mechanism of the electroneutral organic cation/H+ antiporter in renal brush border membrane vesicles was studied utilizing the prototypic organic cation N1-methylnicotinamide. The hydrophobic carbodiimide, N,N'-dicyclohexylcarbodiimide (DCCD), inactivated organic cation transport irreversibly with an IC50 of 2.6 microM at pH 7.5 and 40 nM at pH 6.0. On the other hand, the hydrophilic reagents, 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, did not affect organic cation transport. Substrate did not affect the rate of the DCCD inactivation which followed pseudo-first-order-kinetics. A double logarithmic plot of the apparent rate constants vs. the DCCD concentration gave a straight line with a slope of 0.8. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of DCCD inactivates one carboxylate group per active transport unit and that the carboxylate group is critical for transport.

Animals↗
PubMed1987

The neurotoxins 1-methyl-4-phenylpyridinium and 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine are substrates for the organic cation transporter in renal brush border membrane vesicles.

We examined the effects of the neurotoxins 1-methyl-4-phenyl-pyridinium (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on the transport of the prototypic organic cation, N1-[3H]methylnicotinamide ([3H]NMN), in canine renal brush border membrane vesicles. A dose-response curve for MPTP and its oxidized metabolite, MPP+, revealed IC50 values of 160 and 16 microM, respectively. MPTP (5 mM) and MPP+ (5 mM), maximally inhibited H+-driven NMN (50 microM) uptake by 86.0 and 86.6%, respectively. Additionally, serotonin (0.5 mM) and harmaline (0.5 mM) inhibited NMN transport by 65.8 and 87.1%, respectively. Mepiperphenidol (Darstine) (0.5 mM), a classical organic cation competitor, inhibited NMN transport by 80.6%. However, dopamine (0.5 mM) was not as effective and resulted in only 24.9% inhibition. The cationic specificity was demonstrated by showing that MPP+ and MPTP had no effect on the transport of the organic anion, p-aminohippurate. Additionally, the effect of MPP+ on NMN was not due to either a voltage effect or vesicle disruption. In countertransport studies, MPTP acted as an elicitor of NMN counterflow and produced trans stimulation whereas MPP+ did not and resulted in trans inhibition. Inasmuch as transport involves both binding and translocation we speculate that MPTP binds and is translocated; in contrast MPP+ binds and is not translocated. These reagents may serve as a basis for elucidating the structure-activity requirements of the organic cation/H+ antiporter.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1987

Effect of parathyroid hormone on gentamicin plasma membrane binding and tissue accumulation.

Reportedly, the initiating event in the renal uptake of gentamicin is its binding to anionic, plasma membrane phospholipids. Because parathyroid hormone is known to affect phospholipid metabolism, the plasma membrane binding and tissue accumulation of gentamicin were examined as a function of the parathyroid hormone status of the animal. The experiments were conducted by evaluating the parameters in isolated brush border and basolateral membranes from control, hyper- and hypoparathyroid rats. Scatchard analysis revealed that [125I]gentamicin bound with equal affinity to either membrane to a single class of noninteracting sites. The basolateral membrane had more binding sites than did the brush border, 28 +/- 0.5 vs. 1.8 +/- 0.3 nmol/mg of protein, respectively. Neither the affinity constants nor the number of binding sites were affected by the parathyroid hormone status of the donor animal. On the other hand, in the hypoparathyroid state the amount and the rate of gentamicin accumulation were less than in the hyperparathyroid state. The difference in accumulation cannot be explained on the basis of a change in the number or affinity of the putative receptor. Therefore, the alteration must reflect some difference subsequent to the binding site.

Animals↗
PubMed1986

Sulfhydryl groups are essential for organic anion exchange in canine renal brush-border membranes.

The effect of several sulfhydryl-modifying reagents (HgCl2, p-chloromercuribenzenesulfonic acid (PCMBS), N-ethylmaleimide) on the renal organic anion exchanger was studied. The transport of p-amino[3H]hippurate, a prototypic organic anion, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. HgCl2, PCMBS and N-ethylmaleimide inactivated p-aminohippurate transport with IC50 values of 38, 78 and 190 microM. The rate of p-aminohippurate inactivation by N-ethylmaleimide followed apparent pseudo-first-order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the N-ethylmaleimide concentration with a slope of 0.8. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of N-ethylmaleimide inactivates one essential sulfhydryl group per active transport unit. Substrate (1 mM p-aminohippurate) affected the rate of the N-ethylmaleimide (1.3 mM) inactivation: the t1/2 values for inactivation in the presence and absence of p-aminohippurate were 7.4 and 3.7 min, respectively. The results demonstrate that there are essential sulfhydryl groups for organic anion transport in the brush-border membrane. Moreover, the ability of substrate to alter sulfhydryl reactivity suggests that the latter may play a dynamic role in the transport process.

4-Chloromercuribenzenesulfonate↗
PubMed1986

Essential disulfide and sulfhydryl groups for organic cation transport in renal brush-border membranes.

The disulfide reducing agent, dithiothreitol (DTT) and the sulfhydryl-modifying reagents p-chloromercuribenzenesulfonic acid and N-ethylmaleimide (NEM) were employed to assess the role of disulfide and sulfhydryl groups in organic cation transport. The transport of N1-[3H]methylnicotinamide (NMN), a prototypic organic cation, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. DTT inhibited NMN transport reversibly with an IC50 of 250 microM/mg of protein. 5 mM NMN protected against DTT inactivation. The specificity of substrate protection was demonstrated by showing that D-glucose had no effect on the DTT inactivation of NMN transport and conversely that NMN had no effect on the DTT inactivation of D-glucose transport. Disulfide bonds reduced by DTT could be reoxidized by washing with excess buffer or by addition of 0.02% H2O2 thereby restoring NMN transport. p-Chloromercuribenzenesulfonic acid reversibly inactivated NMN transport with an IC50 of 25 microM/mg of protein. 5mM NMN protected against inactivation. NEM irreversibly inactivated transport with an IC50 of 250 microM/mg of protein. The rate of NMN inactivation by NEM followed pseudo-first order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the NEM concentration with a slope of 1.3. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of NEM inactivates 1 sulfhydryl group/active transport unit. The presence of 5 mM NMN affected the rate of NEM (2.5 mM) inactivation: the t1/2 values for inactivation in the presence and absence of substrate were 7.3 and 2.0 min, respectively. The results demonstrate an essential requirement for disulfide and sulfhydryl groups.

Animals↗
PubMed1985

Electroneutral transport of organic cations in canine renal brush border membrane vesicles (BBMV).

The effect of potential difference on the organic cation/H+ antiport system located in brush border membrane vesicles was examined. Potential difference was generated using K+ gradients and a K+-specific ionophore, valinomycin. Transport of a prototypic organic cation, 1N-[3H]methylnicotinamide (NMN), was assessed under conditions where K+ diffusion potentials generated transient intravesicular negative and positive states. The results demonstrate that NMN transport is independent of potential difference. The organic cation/H+ coupling ratio was studied by imposing transmembrane H+ and NMN gradients of varying magnitudes and measuring net flux of NMN for a 5-sec period. Consistent with an equilibrium thermodynamics model, no net NMN flux is observed when the NMN gradient from out to in is equal to the H+ gradient from out to in ([NMN]o/[NMN]i equals [H+]o/[H+]i). This suggests that the carrier is at equilibrium and that the stoichiometry for the organic cation/H+ antiport is 1:1.

Animals↗
PubMed1984

Inhibition of renal Na+, K+-adenosine triphosphatase by gentamicin.

Inhibition of renal Na+,K+-adenosine triphosphatase is an early biochemical manifestation of gentamicin treatment in rats. Studies with isolated, perfused rat kidneys in filtering and nonfiltering modes indicate that gentamicin is transported across the brush border membrane before enzyme inhibition. The drug caused enzyme inhibition (42%) only in filtering kidneys, and this inhibition was blocked by spermine, an inhibitor of gentamicin binding. In purified rat renal basolateral membranes, bound [3H]gentamicin was displaced 88% by unlabeled gentamicin. After in vivo exposure to [3H]gentamicin, the radioactivity associated with the isolated basolateral membranes was displaced only 46% by unlabeled drug. These results suggest that inhibition of renal Na+,K+-adenosine triphosphatase by gentamicin is probably due to an interaction at the cytoplasmic face of the basolateral membrane. Scatchard plots of [3H]gentamicin binding to basolateral and brush border membranes revealed a single class of noninteracting sites in each membrane. Gentamicin did not change the bulk membrane lipid fluidity, as estimated by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene.

Animals↗
PubMed1983

Computed tomography of the diaphragm: normal anatomy and variants.

Computed tomographic examinations in 75 normal individuals are analyzed to define the cross-sectional anatomy of the diaphragm. Anatomic relationships among the inferoposterior portions of the lungs, the pleura, and the hemidiaphragms are clarified. The posterior pleural recess, an important potential space, is described and illustrated. The relationships of the crura, arcuate ligaments, and the hemidiaphragms are discussed as well. Awareness of these anatomic relationships forms an important basis in analyzing peridiaphragmatic fluid collections.

Diaphragm↗