Increased calcium absorption in nephrolithiasis explained by uptake studies in ileal brush border membrane vesicles.

We previously showed that recurrent calcium renal stone formers have enhanced urinary excretions of calcium and oxalate resulting from malabsorption of citrate. In the present investigation, the mechanism of the citrate-induced increased calcium uptake was studied using guinea pig ileal brush border membrane vesicles. In this model, calcium is absorbed in a concentration dependent, single mechanism uptake with a Km of 275 +/- 30 umol/liter (SD) and a Vmax of 4.0 +/- 0.5 nmol/min.mg protein. Under conditions of maximal calcium uptake, both citrate and phosphate inhibited calcium absorption into brush border membrane vesicles (BBMVs). In contrast, when phosphate and citrate were added together, calcium absorption normalized. Citrate inhibition of calcium absorption appeared to be due to free citrate ions, and phosphate ions overcame this inhibition. Phosphate inhibition was mostly due to decreased concentrations of ionized calcium and partly to precipitation of insoluble calcium phosphate. These studies confirm that the effects of citrate in humans in enhancing calcium absorption occur in the lumen of the gut and are not related to further biochemical conversions of citrate by the gut cells, to effects of citrate on calcium-related hormones, or to the renal handling of calcium. Also, the effects of citrate on increasing calcium absorption should be increased or attenuated in patients who malabsorb citrate, and this explains the increased urinary calcium and oxalate excretions reported for recurrent calcium stone formers.

Interactions with hemoglobin: a source of error in measurements of transketolase activity in hemolysates.

Measurements of the activity of transketolase in human erythrocyte lysates by an assay coupled to NADH oxidation indicate that interactions of assay substrates with hemoglobin can give rise to overestimations of transketolase activity. Three potential sources of error are identified. Thus, in lysates containing methemoglobin, NADH oxidation can be due firstly to methemoglobin reductase activity or secondly to the monooxygenase activity of methemoglobin, for which the substrate can be ribose 5-phosphate, a substrate also of transketolase. Thirdly, the addition of high concentrations of the transketolase cofactor, TDP, to an insufficiently buffered reaction mixture can cause the aggregation and precipitation of hemoglobin: a phenomenon that may be misconstrued as an enhanced increase in absorbance at 340 nm and hence as additional transketolase activity. Although the present study concentrates on these potential artefacts in assays of transketolase activity, the findings may well be relevant to the measurement of other enzyme activities in hemolysates by procedures based ultimately on the rate of consumption or production of NAD(P)H.

Tetrandrine and transmembrane signal transduction: effect on phosphoinositide metabolism, calcium flux and protein kinase C translocation in human lymphocytes.

Time-course and dose-dependent studies showed consistent suppression of phosphoinositide turnover in Con A-stimulated human lymphocytes in the presence of the plant alkaloid, tetrandrine. Significant inhibition of Con A-stimulated calcium flux was also observed. Furthermore, protein kinase C activity was also significantly inhibited by tetrandrine irrespective of whether Con A or phorbol myristate acetate was the stimulant. These results suggest that the immunosuppressive properties of tetrandrine are in part mediated by the capacity of tetrandrine to interfere with transmembrane signalling.

Suppression by tetrandrine of human platelet aggregation induced by platelet-activating factor and other stimulants.

Tetrandrine was found to have inhibitory effects on platelet-activating factor induced platelet aggregation in a dose-dependent manner. There was preferential inhibition of platelet aggregation induced by agents such as collagen, thrombin, adrenaline, and adenosine diphosphate. No inhibitory effect of tetrandrine was observed on platelet aggregation induced by adenosine diphosphate, arachidonic acid, and the calcium ionophore A23187. These results support our previous findings of interference with the phosphatidylinositol second-messenger system as one of the sites of action of tetrandrine. Since platelets may have an important role in the pathogenesis of asthma and other allergic diseases, these findings suggest that tetrandrine may have clinical application as a non-steroidal broad-spectrum anti-allergic drug.

1-Methyl-3-phenyl-1,2,4-triazinium-5-olate: a new zwitterion with cytotoxic activity against human cancer cell-lines.

One of 6 newly synthesized triazinium zwitterions (JR-1--JR-6) was shown to induce 51Cr-release from leukemic (HL60 and CEM) and solid tumor (MM170 and HeLa) cell-lines. Leukemic cells were more sensitive to this compound than solid tumors as demonstrated by dose-response and time-course studies. Other experiments showed that JR-6 (1-methyl-3-phenyl-1,2,4-triazinium-5-olate) significantly suppressed protein, RNA and DNA synthesis at tumoricidal concentrations.

Identification and quantitation in human liver of cytochromes P-450 analogous to rabbit cytochromes P-450 forms 4 and 6.

1. Polyclonal antibodies raised against rabbit liver cytochrome P-450 isozymes form 4 and 6 have been used to probe human liver microsomes for analogous proteins using the Western blot technique. 2. Anti-Form 4 IgG recognized a protein in human liver microsomes from six subjects of identical molecular weight to purified rabbit liver cytochrome P-450 Form 4. 3. The equivalent content of cytochrome P-450 Form 4 in the same microsomes ranged from 1.1 to 9.1 pmol per mg protein. 4. Anti-Form 6 IgG recognized a protein in human liver microsomes from the same six subjects of slightly higher molecular weight than purified rabbit cytochrome P-450 Form 6. 5. The equivalent content of cytochrome P-450 Form 6 in the above microsomes ranged from 1.6 to 3.8 pmol per mg protein. 6. No significant correlations were observed between equivalent cytochrome P-450 Forms 4 and 6 content and 2-acetylaminofluorene N-hydroxylase, aminopyrine N-demethylase, benzyprene and aniline hydroxylase activities in liver microsomes from the six subjects tested.

Inhibition of histamine release from rat mast cells by the plant alkaloid tetrandrine.

The plant alkaloid tetrandrine was shown to have significant inhibitory effects on receptor-ligand-mediated histamine release from rat mast cells at concentrations similar to or lower than that observed with theophylline and sodium cromoglycate. Inhibition of histamine release did not occur when non-specific stimulants such as aspirin, A23187 or adenosine triphosphate were used. Inhibition of ovalbumin-IgE and concanavalin A-mediated histamine release was reversible by washing the cells, showing that tetrandrine does not bind tightly to the cell membrane or cytoplasmic components. These results, taken together with previous reports of its anti-phagocytic, anti-oxidant and immunosuppressive properties, suggest that tetrandrine may be a broad spectrum non-steroidal drug of potential value in the treatment of allergic diseases.

The nature of microsomal monooxygenase inhibition by cimetidine.

A kinetic investigation of the inhibitory effect of cimetidine on the O-dealkylation of 7-ethoxyresorufin by rat liver microsomes has yielded linear Lineweaver-Burk plots which intersect in the second quadrant. Though technically compatible with non-competitive inhibition, the results are shown to be readily explained by the more realistic molecular concept of competitive inhibition by invoking the involvement of cytochrome P-450 isoenzymes with widely different KI values. Microsomal monooxygenase heterogeneity is also shown to provide a plausible explanation of other published results signifying the departure of chloramphenicol and phenacetin from the concept of competitive inhibition despite competition with substrate for the active-site haem group of cytochrome P-450.

Evidence for induction of hepatic microsomal cytochrome P-450 by cimetidine: binding and kinetic studies.

The interaction of cimetidine with liver microsomes has been examined by spectral and equilibrium partition studies. First, difference spectroscopy has been used to evaluate the proportion of cytochrome P-450 in rat liver microsomes that exhibits an affinity for cimetidine in the pharmacologically relevant, low micromolar range of drug concentration. The value of 0.45 so obtained has confirmed that a substantial proportion of rat liver cytochrome P-450 has a high binding affinity for this drug. Second, a study of the binding of cimetidine to human liver microsomes by difference spectroscopy and partition equilibrium has detected a similar interaction, thus providing direct support for the postulate that the clinically observed impairment of oxidative drug metabolism may be due in part to inhibition of cytochrome P-450 monooxygenase by cimetidine. Hepatic microsomes from cimetidine-pretreated rats have been shown to exhibit elevated cytochrome P-450 specific content but a decreased proportion of sites with high affinity for the drug; this finding has been shown not to be the consequence of cimetidine-mediated, time-dependent, irreversible monooxygenase inhibition. Although cimetidine pretreatment caused enhanced specific activity of 7-ethoxyresorufin O-dealkylation, the specific activities for O-dealkylation of 7-ethoxycoumarin and 4-nitroanisole were decreased, as were those for the N-dealkylation of morphine, ethylmorphine, aminopyrine, and dimethylnitrosamine. Since cimetidine pretreatment was shown to cause no change in the Michaelis constants for oxidation of morphine or 7-ethoxyresorufin, it is argued that these results provide strong presumptive evidence for changes in the relative abundance of isoenzymes catalyzing these various oxidations. Thus, a dual role of cimetidine, acting both as inhibitor and inducer of the cytochrome P-450 system, is proposed to account for the impaired oxidative metabolism of some drugs that occurs during coadministration with this H2-receptor antagonist.

Evidence for lipid involvement in the high-affinity interaction between cimetidine and rat liver cytochrome P-450.

The spectral interaction between cimetidine and a cytochrome P-450 fraction isolated from liver of untreated rats has been shown to be markedly affected by dilauroylphosphatidylcholine. In the absence of the lipid the pigment preparation yielded a binding curve characteristic of a single isoenzyme species with low affinity for the drug, whereas its inclusion led to the observation of a much stronger interaction with a dissociation constant close to that obtained for the high-affinity component(s) of the parent microsomes; material with lower affinity was also observed. Gel chromatography and partition equilibrium studies yielded results which precluded interpretation of this finding either in terms of incomplete incorporation of the pigment into the phospholipid or of disproportionate solvation of cimetidine into the lipid phase of the reconstituted phospholipid complex. In contrast, phospholipid caused only a minor change in the strength of cimetidine binding by the predominant liver cytochrome P-450 from phenobarbitone-pretreated rats. Pronounced lipid sensitivity of cimetidine-binding affinity is thus not a general feature of the microsomal cytochrome P-450 system but rather a specific characteristic of individual isoenzyme species.

Evaluation of monooxygenase induction as a means of enhancing the yield of the rat liver cytochrome P-450 isoenzyme with high affinity for cimetidine.

The binding of cimetidine to liver microsomes prepared from untreated rats and rats pretreated with phenobarbitone or 3-methylcholanthrene has been investigated by difference spectroscopy and equilibrium partition methods. In M/15 phosphate buffer, pH 7.9, microsomes from each group of rats yielded markedly biphasic spectral binding curves, which have been interpreted in terms of two independent classes of cytochrome P-450 site with widely differing binding affinities for the drug. Support for such interpretation was provided by the finding that the spectral binding curve for a purified sample of the principal cytochrome P-450 isoenzyme from liver microsomes of phenobarbitone-pretreated rats could be described adequately by a single rectangular hyperbolic relationship, the spectral dissociation constant being indistinguishable experimentally from that for the weaker class of cytochrome P-450 binding site in the corresponding microsomes. The spectral dissociation constants were 2 microM and 80 microM for microsomes from untreated rats; 44 microM and 540 microM for those from phenobarbitone-pretreated rats; and 34 microM and 540 microM for microsomes from rats pretreated with 3-methylcholanthrene. On this basis, both classes of P-450 site in the microsomes from rats subjected to either pretreatment exhibited lower affinity for cimetidine than their counterparts in microsomes from untreated rats. Equilibrium partition studies of the higher-affinity class of microsomal binding site for cimetidine showed that the twofold increase in the cytochrome P-450 content of microsomes effected by 3-methylcholanthrene pretreatment was more than offset by a diminished proportion of the total cimetidine-binding capacity present as the higher-affinity, pharmacologically significant, receptor (18%, cf. 48% in control microsomes); and that phenobarbitone pretreatment resulted in replacement of the high-affinity receptor by one with a threefold weaker cimetidine-binding affinity. Thus the use of these monooxyginase inducers to enhance the cytochrome P-450 content of liver microsomes would seem to offer little potential in the isolation of the isoenzyme with high affinity for cimetidine.