Expression of gastric and colonic H(+)-K(+)-ATPase in the rat kidney.

Enzymatic and microperfusion studies have indicated that an ATP-dependent H+/K+ exchange process is present in the collecting duct of the mammalian kidney. Immunochemical staining has also provided evidence for expression of a gastric-type H(+)-K+ adenosine triphosphatase (H(+)-K(+)-ATPase). Rat kidney mRNA was probed with use of the polymerase chain reaction (PCR) to determine the presence of an H(+)-K(+)-ATPase. cDNA made with mRNA isolated from the kidneys of rats maintained on a low-K diet was used as template in PCR reactions with primers encompassing the cDNA sequence of the alpha-subunit of the gastric H(+)-K(+)-ATPase and the 5' and 3' ends of the colonic H(+)-K(+)-ATPase. The resulting products, 300-700 bp in size, hybridized with probes directed against either the gastric or colonic sequences of the H(+)-K(+)-ATPase. Sequencing of the individual PCR products showed identity with the appropriate regions of the alpha-subunits of the gastric H(+)-K(+)-ATPase and colonic H(+)-K(+)-ATPase. These data indicate that the rat kidney expresses mRNAs encoding both gastric and colonic H(+)-K(+)-ATPases.

Temafloxacin pharmacokinetics in subjects with normal and impaired renal function.

The pharmacokinetics of temafloxacin were investigated following oral administration of single 400-mg doses to 6 normal subjects and 18 subjects with various degrees of impaired renal function. Renal impairment did not significantly affect the peak concentration, time to peak concentration, or the nonrenal clearance of temafloxacin. Both renal clearance (CLR) and total apparent clearance (CLT/F, where F represents the fraction of dose absorbed) of temafloxacin were highly correlated with creatinine clearance (CLCR). The regression equations were as follows: CLR = 0.85.CLCR, with R2 = 0.907, and CLT/F = 56.0 + 0.92.CLCR, with R2 = 0.656. The half-life (mean +/- standard deviation) increased from 10.6 +/- 2.4 h in the normal volunteers to 24.6 +/- 7.3 h in the subjects with a CLCR of less than 10 ml/min; the respective CLT/F decreased from 169 +/- 58 to 70 +/- 27 ml/min. Compared with the CLT/F in the subjects with normal renal function, CLT/F was reduced 60% in subjects with a CLCR of less than 40 ml/min, indicating that the dosage should be reduced by at least one-half for patients with comparable impairment. For the subjects on chronic hemodialysis, most of the variability in the nonrenal clearance and the terminal-phase rate constant of temafloxacin was associated with the quantity of calcium carbonate and related medication taken for the treatment of hyperphosphatemia. Supplemental dosage is not required for patients undergoing hemodialysis, since the distribution of temafloxacin in tissue is extensive and the recoveries from 4-h dialysis sessions accounted for less than 10% of the drug present at the start of the dialysis.

Use of calcium carbonate as a phosphate binder in dialysis patients.

Aluminum-containing phosphate (Al-binders) employed to control serum phosphorus in patients with chronic renal failure can be associated with the development of aluminum toxicity. To obviate the need for Al-binders, we examined the effectiveness of CaCO3 as a phosphate binder in 31 hemodialysis and 8 CAPD patients followed for 2 months while receiving Al-binders, and then, for 3-14 months while receiving CaCO3 (5.8 +/- 0.4 g/day). Monthly serum phosphorus averaged 5.4 +/- 0.2 mg/dl with Al-binders and 5.1 +/- 0.3 to 5.7 +/- 0.4 mg/dl with CaCO3 (p = NS). There were 25.2 episodes of hyperphosphatemia (serum phosphorus greater than 6.5 mg/dl) per 100 treatment months with Al-binders and 19.2 episodes/100 treatment months with CaCO3 (p = NS). Plasma aluminum levels, 105 +/- 21 micrograms/l during ingestion of Al-binders, fell to 34 +/- 11 micrograms/l after 8 months of therapy with CaCO3 (p less than 0.01). Monthly serum Ca averaged 9.5 +/- 0.1 mg/dl during Al administration and was 8.9 +/- 0.8 to 10.0 +/- 0.2 mg/dl with CaCO3 (p = NS). Thirty-four episodes of hypercalcemia (serum Ca greater than 11.0 mg/dl) occurred in 14 patients ingesting CaCO3, but hypercalcemia did not occur with ingestion of Al-binders. Al-related bone disease was found on bone biopsy in 11 of 13 patients who developed hypercalcemia, compared to only 5 of the 11 biopsied patients who remained normocalcemic (p less than 0.01 by chi 2 analysis). Other side effects included diarrhea in 1 patient and constipation in 3 patients.(ABSTRACT TRUNCATED AT 250 WORDS)

The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450.

The crystal structure of Pseudomonas putida cytochrome P-450cam in the ferric, camphor bound form has been determined and partially refined to R = 0.23 at 2.6 A. The single 414 amino acid polypeptide chain (Mr = 45,000) approximates a triangular prism with a maximum dimension of approximately 60 A and a minimum of approximately 30 A. Twelve helical segments (A through L) account for approximately 40% of the structure while antiparallel beta pairs account for only approximately 10%. The unexposed iron protoporphyrin IX is sandwiched between two parallel helices designated the proximal and distal helices. The heme iron atom is pentacoordinate with the axial sulfur ligand provided by Cys 357 which extends from the N-terminal end of the proximal (L) helix. A substrate molecule, 2-bornanone (camphor), is buried in an internal pocket just above the heme distal surface adjacent to the oxygen binding site. The substrate molecule is held in place by a hydrogen bond between the side chain hydroxyl group of Tyr 96 and the camphor carbonyl oxygen atom in addition to complementary hydrophobic contacts between the camphor molecule and neighboring aliphatic and aromatic residues. The camphor is oriented such that the exo-surface of C5 would contact an iron bound, "activated" oxygen atom for stereoselective hydroxylation.

Effect of metabolic acidosis in intestinal absorption of calcium and phosphorus.

To investigate the effect of metabolic acidosis on intestinal calcium (Ca) and phosphorus (P) absorption and vitamin D metabolism, metabolic balance studies and in vitro gut sac uptake of 45Ca and [32P]phosphate were performed in rats maintained on low-Ca and moderately low-P diet and fed NH4Cl for 3 or 9 days and pair-fed controls. Plasma 1,25(OH)2D concentration was measured in the rats fed NH4Cl for 9 days and their controls. Net Ca and P absorption was 87-92% in the acidotic rats and did not differ from control. Moreover, gut sac uptakes of 45Ca and [32P]phosphate were not different from control. Plasma 1,25(OH)2D was higher in the ammonium chloride-fed rats than in controls (213 +/- 44 vs. 110 +/- 12 pg/ml), and serum P was lower in the acidotic animals (4.6 +/- 0.7 vs. 7.6 +/- 0.3 mg/dl). These data indicate that metabolic acidosis does not depress the augmented intestinal absorption of calcium and phosphorus noted during their dietary deprivation nor reduce the plasma level of 1,25(OH)2D.

A hypothetical model of the cytochrome c peroxidase . cytochrome c electron transfer complex.

A hypothetical three-dimensional model of the cytochrome c peroxidase . tuna cytochrome c complex is presented. The model is based on known x-ray structures and supported by chemical modification and kinetic data. Cytochrome c peroxidase contains a ring of aspartate residues with a spatial distribution on the molecular surface that is complementary to the distribution of highly conserved lysines surrounding the exposed edge of the cytochrome c heme crevice, namely lysines 13, 27, 72, 86, and 87. These lysines are known to play a functional role in the reaction with cytochrome c peroxidase, cytochrome oxidase, cytochrome c1, and cytochrome b5. A hypothetical model of the complex was constructed with the aid of a computer-graphics display system by visually optimizing hydrogen bonding interactions between complementary charged groups. The two hemes in the resulting model are parallel with an edge separation of 16.5 A. In addition, a system of inter- and intramolecular pi-pi and hydrogen bonding interactions forms a bridge between the hemes and suggests a mechanism of electron transfer.