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P D Holohan

Publications and source records attributed to P D Holohan.

At least 37 records · Page 2Linked to original sources
PubMed1987

Organic cation secretion in flounder renal tissue.

In the winter flounder, Pseudopleuronectes americanus, renal clearance experiments showed that the model organic cations, tetraethylammonium (TEA) and N'-methylnicotinamide (NMN), were strongly secreted; organic cation-to-polyethylene glycol (glomerular filtration rate marker) clearance ratios averaged 130 and 30, respectively. TEA uptake by isolated renal tubular masses was concentrative and saturable. Transport was inhibited by competitor organic cations and reduced by exposure to NaCN,2,4-dinitrophenol, ouabain, and HgCl2. Organic anions did not reduce TEA uptake. NMN was the poorest inhibitor of TEA uptake of all the organic cations tested. In addition, the rate of NMN uptake was slower than that of TEA, and the steady-state tissue-to-medium ratio was lower (5 for NMN vs. 10 for TEA; both at 25 microM). The data show the presence of an organic cation secretory system in flounder tissue that resembles the mammalian systems in several respects.

2,4-Dinitrophenol↗
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

Effect of cephaloridine on the transport of organic ions in dog kidney plasma membrane vesicles.

The cephalosporin antibiotics cephaloridine and cefazolin were examined for their effects on the transport of a prototype anion, [3H]p-aminohippurate (PAH) and a prototype cation, N1-[3H] methylnicotinamide (NMN) in basolateral membrane vesicles and brush border membrane vesicles (BBMV). Cefazolin inhibited transport of 50 microM PAH in both membranes and had no effect on transport of 50 microM NMN transport. Under identical conditions, cephaloridine, a zwitterion, inhibited PAH transport in both membranes and NMN transport in BBMV but not in basolateral membrane vesicles. Cephaloridine was less effective than cefazolin in inhibiting PAH transport in BBMV. The results demonstrate the organic transport systems in the basolateral membrane vesicles differ from those in the BBMV. Furthermore, a zwitterionic drug can interact with both systems in the BBMV. An explanation for the intracellular accumulation of cephaloridine is presented.

Aminohippuric Acids↗
PubMed1983

Na+ gradient-dependent p-aminohippurate (PAH) transport in rat basolateral membrane vesicles.

The relationship between the transmembrane Na+ gradient and p-aminohippurate (PAH) transport was examined in isolated rat basolateral membrane vesicles. A 100 mM Na+ gradient (o leads to i) accelerated the influx of 50 microM [3H]PAH whereas similar gradients of choline+, K+, or Li+ did not. The sodium effect was not due to a diffusion potential. The Na+ gradient (o leads to i) decreased the apparent Michaelis constant for PAH from 0.167 +/- 0.016 to 0.054 +/- 0.016 mM and increased the maximum flux rate from 116.00 +/- 13.50 to 427.34 +/- 98.96 pmol/mg/min. An "overshoot" of [3H]PAH influx (159 +/- 4% of the equilibrium value) could be demonstrated only in the presence of a Na+ gradient (o leads to i) plus an opposing gradient of unlabeled PAH (i leads to o). These results suggest that PAH transport and the Na+ gradient are functionally related. A model for cellular uptake of PAH by a Na+ gradient-dependent anion exchange mechanism is presented.

Aminohippuric Acids↗
PubMed1981

Mechanisms of organic cation transport in kidney plasma membrane vesicles: 2. delta pH studies.

The active step in the renal secretion of the organic cation N1-methylnicotinamide has been proposed to be the translocation across the luminal membrane. The presence of a H+:N1-methylnicotinamide antiport system has been found in luminal membrane vesicles isolated from dog kidney cortex but not in the antiluminal membrane. The conclusion reached is that N1-methylnicotinamide secretion is "secondarily active" and is driven by a proton gradient (urine to cell). The ph gradient in turn is created by the Na+:H+ antiport which is present in the luminal membrane. Therefore, two functionally linked antiport systems supply the energy for the active secretion of cationic drugs.

Animals↗
PubMed1980

Mechanisms of organic cation transport in kidney plasma membrane vesicles: 1. Countertransport studies.

Transport for organic cations has been described in both luminal and antiluminal membranes. The two transport systems difer from one another on the basis of kinetic parameters and countertransport. By using N1-[3H]methylnicotinamide as the indication cation, a series of organic cations (which were known to be secreted) were tested for their capacity to stimulate countertransport. The phenomenon of countertransport was symmetrical in that it was observed for both influx and efflux in both membranes. However, under appropriate conditions, certain organic cations were effective in producing "uphill" transport of N1-[3H]methylnicotinamide in the luminal membrane and the effect was limited to that membrane exclusively. Detailed analysis of countertransport in the luminal membrane showed quantitative differences in the maximal stimulation produced by the various cations: giving rise to the relationship that the concentration of organic cation which gave one-half maximal N1-[3H]methylnicotinamide countertransport approximated its affinity constant. The data were interpreted to suggest that all the members of the organic cation series tested are translocated across the luminal membrane by the same carrier but that they are moved at different rates.

Animals↗
PubMed1979

Isolation of luminal and antiluminal membranes from dog kidney cortex.

Luminal (brush border) and antiluminal (basal-lateral) membranes were isolated from canine renal cortex. The enzyme marker for luminal membrane, alkaline phosphatase was enhanced 19-fold and the antiluminal enzyme marker, (Na+ + K+)-ATPase, was enhanced 22-fold in their respective membrane preparation, while the amount of cross contamination was minimal. Contamination of these preparations by enzyme markers for lysosomes, endoplasmic reticulum and mitochondria was also low. Routinely, more than 50 mg membrane protein was isolated for each membrane. Electron micrographs showed that the membranes were uniform in size, appearance, and vesicular in nature. An examination of the orientation of these membranes showed that 76.5% of the antiluminal membranes and 86% of the luminal membranes were right-side out.

Animals↗
PubMed1979

Transport of organic ions in renal cortical luminal and antiluminal membrane vesicles.

Based upon saturability, kinetic analysis and specific inhibition, specific transport for the organic anion, p-aminohippurate was found in isolated luminal and antiluminal membrane vesicles from dog kidney cortex. The transport for organic anions in the two membranes differed from one another on the basis of kinetic parameters ((Km, Vmax and Ki for probenecid) and counterflow phenomenon (only the antiluminal membrane displayed the capacity to accelerate the exchange of p-aminohippurate). Also described is specific transport for the organic cation N1 methylnicotinamide in each membrane. The luminal and antiluminal transport of N1-methylnicotinamide are distinguished from one another by kinetic parameters (Km, Vmax and Ki for Darstine) and counterflow data (counterflow of N1-methylnicotinamide was observed only in the luminal membrane). The results are discussed in view of a proposed model to explain transepithelial transport of these organic ions.

Animals↗