Exclusion of urate oxidase as a candidate gene for hyperuricosuria in the Dalmatian dog using an interbreed backcross

Hyperuricosuria, an autosomal recessive disorder, is characterized by high levels of uric acid in the urine of Dalmatian dogs. Whereas high levels of uric acid are known to be caused by the silencing of the urate oxidase (uox) gene in humans and higher primates, the molecular basis for the Dalmatian defect is unknown. Transplantation studies show that the organ responsible for the Dalmatian phenotype is the liver, which is where urate oxidase is exclusively expressed and uric acid is converted into allantoin. We cloned and sequenced the canine uox cDNA and compared the sequence between a Dalmatian and non-Dalmatian dog. No change in cDNA sequence was identified. A Dalmatian x pointer backcross family was used to track the segregation of microsatellite markers surrounding the urate oxidase locus. The uox gene was excluded for Dalmatian hyperuricosuria based on the cDNA sequence identity and negative LOD scores.     

The transport of sodium into human erythrocytes in vivo

The relative Na²⁴ specific activity of red cells and plasma was measured at periods up to 30 hours following a single intravenous injection of Na²⁴ in normal healthy young adults. The average specific activity of the red cells relative to that of the plasma at 24 hours and beyond was found to average 0.83 ± 0.05 in a series of five normal individuals, significantly different from 1.0. This indicates that all the intracellular Na is not exchangeable in 24 hours, and confirms earlier in vitro results. The red cell Na concentration in man was shown to be 12.1 ± 1.1 m.eq. Na/liter red cell, as measured in a series of nineteen normal healthy young adults. A theoretical analysis of the data on exchangeable cell Na suggests that the red cell Na (5.3 m.eq. Na/liter blood) is divided into a fast compartment comprising 4.25 m.eq. Na/liter blood, and a slow compartment comprising 1.07 m.eq. Na/liter blood. If these compartments are arranged in parallel, the flux between plasma and fast compartment is 1.32 m.eq. Na/liter blood hour, and that between plasma and slow compartment is 0.016 m.eq. Na/liter blood hour. Results of experiments on two patients with congenital hemolytic jaundice suggest that the fraction of slowly exchanging Na may increase with the age of the red cell.     

cGMP and glutathione-conjugate transport in human erythrocytes.

The nature of cGMP transport in human erythrocytes, its relationship to glutathione conjugate transport, and possible mediation by multidrug resistance-associated proteins (MRPs) have been investigated. MRP1, MRP4 and MRP5 are detected in immunoblotting studies with erythrocytes. MRP1 and MRP5 are also detected in multidrug resistant COR-L23/R and MOR/R cells but at greatly reduced levels in the parent, drug sensitive COR-L23/P cells. MRP4 is detected in MOR/R but not COR-L23/R cells. Uptake of cGMP into inside-out membrane vesicles prepared by a spontaneous, one-step vesiculation process is shown to be by a low affinity system that accounts for more than 80% of the transport at all concentrations above 3 micro m. This transport is reduced by MRP inhibitors and substrates including MK-571, methotrexate, estradiol 17-beta-d-glucuronide, and S(2,4-dinitrophenyl)glutathione (DNP-SG) and also by glibenclamide and frusemide but not by the monoclonal Ig QCRL-3 that inhibits high-affinity transport of DNP-SG by MRP1. It is concluded that the cGMP exporter is distinct from MRP1 and has properties similar to those reported for MRP4. Furthermore the evidence suggests that the protein responsible for cGMP transport is the same as that mediating low-affinity DNP-SG transport in human erythrocytes.     

Inhibition of choline transport into human erythrocytes by choline mustard aziridinium ion.

Acetylcholine mustard aziridinium ion inhibited the transport of [3H]choline into human erythrocytes. Treatment of the erythrocytes with 1 X 10(-4) M tetraethylpyrophosphate prevented the inhibition of [3H]choline transport by acetylcholine mustard aziridinium ion. Hydrolyzed acetylcholine mustard aziridinium ion inhibited choline transport both in the presence and absence of 1 X 10(-4) M tetraethylpyrophosphate. The product of hydrolysis was equipotent with acetylcholine mustard in its ability to inhibit choline transport; incubation of this product with sodium thiosulfate prevented inhibition of choline transport thereby indicating the presence of an aziridinium ion. The hydrolysis product is likely to be choline mustard aziridinium ion. Results on the efflux of [3H]choline from erythrocytes in the presence of the proposed choline mustard aziridinium ion showed that the mustard moiety was transported into the red cells on the choline carrier. The rate of efflux of [3H]choline produced by choline mustard aziridinium ion was 55% of that produced by the same concentration of choline. It is concluded that acetylcholinesterase (EC 3.1.1.7) of red cells rapidly hydrolyzes acetylcholine mustard aziridinium ion to acetate and choline mustard aziridinium and the latter compound can act as a potent inhibitor of choline transport. This finding would indicate that the hemicholinium-like toxicity of acetylcholine mustard in the mouse is due to the formation of choline mustard aziridinium ion.     

Exclusion of galectin 9 as a candidate gene for hyperuricosuria in the Dalmatian dog

All Dalmatian dogs have an inherited defect in purine metabolism leading to high levels of uric acid excretion in their urine (hyperuricosuria) rather than allantoin, the normal end product of purine metabolism in all other breeds of dog. Transplantation experiments have demonstrated that the defect is intrinsic to the liver and not the kidney. Uricase, the enzyme involved in the breakdown of urate into allantoin, has been shown to function in Dalmatian liver cells. Therefore, candidate genes for this defect include transporters of urate, a salt of uric acid, across cell membranes. We excluded one such urate transporter candidate, galectin 9, using a Dalmatian x Pointer backcross in which hyperuricosuria was segregating.     

Purine base transport in normal and mutant erythrocytes.

The uptake of adenine and hypoxanthine in HGPRT-deficient and normal human erythrocytes was measured using a rapid filtering centrifugation technique. The transport of hypoxanthine as well as of adenine is impaired in the mutant cells. The transport of hypoxanthine into HGPRT-deficient erythrocytes differs from that into normal cells with respect to a higher accumulation capacity, to lower initial velocities and to the kinetic properties of the translocator. In addition, a higher accumulation capacity and lower initial velocities of adenine uptake could be demonstrated in mutant cells. A linkage of the purine translocator with purine phosphoribosyltransferases associated with the erythrocyte membrane is discussed.     

Sugar transport in reversibly hemolyzed avian erythrocytes.

The technique of reversible hemolysis represents one approach which may be used to study transport regulation in nucleated red cells. After 1 h of incubation at 37 degrees C, 88% of the ghosts regained their permeability barrier to L-glucose. In these ghosts, the carrier-mediated rate of entry of 3-O-methylglucose was more than 10-fold greater than the rate in intact cells. Glyceraldehyde-3-phosphate dehydrogenase prevented ghosts from resealing when it was present at the time of hemolysis. Albumin, lactic dehydrogenase and peroxidase did not have this effect. Sugar transport rate could not be tested in the unsealed ghosts. Two possible mechanisms for the effect of hypotonic hemolysis on sugar transport rate were discussed: (1) altered membrane organization and (2) loss of intracellular compounds which bind to the membrane and inhibit transport in intact cells.     

Amino acid transport in normal and glutathione-deficient sheep erythrocytes.

1. Uptake rates for 23 amino acids were measured for both normal (high-GSH) and GSH-deficient (low-GSH) erythrocytes from Finnish Landrace sheep. 2. Compared with high-GSH cells, low-GSH cells had a markedly diminished permeability to D-alanine, L-alanine, alpha-amino-n-butyrate, valine, cysteine, serine, threonine, asparagine, lysine and ornithine. Smaller differences were observed for glycine and proline, whereas uptake of the other amino acids was not significantly different in the two cell types.     

Chemical instability of [2-14C] uric acid in alkaline solution: the effect on observed kinetics of urate transport in human erythrocytes.

[2-¹⁴C]Uric acid, in strongly alkaline solution, is chemically unstable when stored at +4°C or ?20°C. Two major degradation products occur, which co-chromatograph with allantoin and allantoic acid. The nonenzymic decomposition of [2-¹⁴C]uric acid markedly alters the observed kinetics of uric acid efflux from human erythrocytes.     

Mechanical properties of the dog renal capsule.

The renal capsule is an important determinant of whole kidney volume/pressure relationships. To gain further insights into its possible role we examined the mechanical properties of the dog renal capsule using standard materials testing procedures. From each of four locations on the kidney surface, the following mechanical properties of the renal capsule were determined: elastic modulus (force/unit of cross-sectional area theoretically required to double the length of the specimen), tensile stiffness (force/unit width theoretically required to double the length of the specimen), ultimate strength (stress at time of fracture of the specimen), and maximum strain (percent strain at time of the fracture of the specimen). We found that the elastic modulus of the renal capsule from all capsular sites was substantially greater than values previously reported for dog aorta. The stiffness of the capsule covering the anterior-posterior surface of the kidney was found to be about 50% greater than the stiffness of the capsule covering the lateral and polar surfaces of the kidney. The ultimate strength of the anterior-posterior capsule was significantly greater than that of the lateral or polar capsule. This finding may explain the clinical observation that the spontaneous rupture of the renal capsule and parenchyma associated with the acute swelling of transplant rejection is confined almost exclusively to the lateral and polar portions of the renal capsule and cortex. The mean maximum strain at each capsular site was about 35%. This degree of circumferential expansion corresponds to about a doubling of kidney volume. Thus, this observation suggests that the renal capsule is at risk to undergo spontaneous rupture when renal volume increases of this magnitude are observed.     

Glutamine transport and metabolism by mitochondria from dog renal cortex. General properties and response to acidosis and alkalosis.

Mitochondria from dog renal cortex were incubated with L-[14Cglutamine. Glutamate metabolism was prevented by inhibitors so that glutamate accumulated either in the mitochondrial matrix space or in the medium. The formation and accumuation of glutamate formed from glutamine and the distribution of glutamine in the mitochondrial fluid spaces were studied. In the matrix space glutamate rapidly reaches levels over 5 times that of glutamine in the medium. A more gradual accumulation occurs in the medium as glutamate is transported out of the mitochondria. Addition of an energy source such as succinate to the medium accelerates glutamate formation. A Km of 0.6 mM appears to govern the reaction at low concentrations of glutamine; at about 4 mM an abrupt change kinetics occurs with a Km of 5 mM above that level. Both NH4+ and glutamate inhibit glutamine metabolism and phosphate stimulates it, but little effect glutamate or phosphate occurs at low levels of these substances. The pH optimum of the reaction is between 7.4 and 7.8. Mersalyl and p-chloromercuribenzoate strongly inhibit glutamate formation; N-ethylmaleimide and bromcresol green have weaker inhibitory actions, and borate increases the reaction rate. In the presence of mersalyl, glutamine is striclly confined to the outer space of mitochondria and none is detectable in the matrix space. Similarly at ) degrees glutamine is confined to the simultaneously determined sucrose or mannitol spaces...     

alpha- and beta-monosaccharide transport in human erythrocytes

Equilibrative sugar uptake in human erythrocytes is characterized by a rapid phase, which equilibrates 66% of the cell water, and by a slow phase, which equilibrates 33% of the cell water. This behavior has been attributed to the preferential transport of beta-sugars by erythrocytes (Leitch JM, Carruthers A. Am J Physiol Cell Physiol 292: C974-C986, 2007). The present study tests this hypothesis. The anomer theory requires that the relative compartment sizes of rapid and slow transport phases are determined by the proportions of beta- and alpha-sugar in aqueous solution. This is observed with D-glucose and 3-O-methylglucose but not with 2-deoxy-D-glucose and D-mannose. The anomer hypothesis predicts that the slow transport phase, which represents alpha-sugar transport, is eliminated when anomerization is accelerated to generate the more rapidly transported beta-sugar. Exogenous, intracellular mutarotase accelerates anomerization but has no effect on transport. The anomer hypothesis requires that transport inhibitors inhibit rapid and slow transport phases equally. This is observed with the endofacial site inhibitor cytochalasin B but not with the exofacial site inhibitors maltose or phloretin, which inhibit only the rapid phase. Direct measurement of alpha- and beta-sugar uptake demonstrates that erythrocytes transport alpha- and beta-sugars with equal avidity. These findings refute the hypothesis that erythrocytes preferentially transport beta-sugars. We demonstrate that biphasic 3-O-methylglucose equilibrium exchange kinetics refute the simple carrier hypothesis for protein-mediated sugar transport but are compatible with a fixed-site transport mechanism regulated by intracellular ATP and cell shape.     

Calcium transport in chicken leukocytes and erythrocytes

1. In the present study, Ca2+ uptake and Ca2(+)-ATPase activity of two different chicken leukocyte populations and erythrocytes isolated from 1- to 6-week-old chickens were determined. 2. The Ca2(+)-ATPase activity of the two leukocyte populations significantly increased at 3 weeks of age. Erythrocyte Ca2(+)-ATPase activity significantly increased at 2 weeks of age. 3. Calcium transport activities into the two leukocyte populations did not differ significantly with age.