Estimate of the number of urea transport sites in erythrocyte ghosts using a hydrophobic mercurial.

In this paper a variety of mercurials, including a pCMB-nitroxide analogue, were used to study urea transport in human red cell ghosts. It was determined that the rate of inhibition for pCMBS, pCMB, pCMB-nitroxide, and chlormerodrin extended over four orders of magnitude consistent with their measured oil/water partition coefficients. From these results, we concluded that a significant hydrophobic barrier limits access to the urea inhibition site, suggesting that the urea site is buried in the bilayer or in a hydrophobic region of the transporter. In contrast, the rate of water inhibition by the mercurials ranged by only a factor of four and did not correlate with their hydrophobicities. Thus, the water inhibition site may be more directly accessible via the aqueous phase. Under conditions that leave water transport unaffected, we determined that < or = 32,000 labeled sites per cell corresponded to complete inhibition of urea transport. This rules out major transmembrane proteins such as band 3, the glucose carrier, and CHIP28 as candidates for the urea transporter. In contrast, this result is consistent with the Kidd (Jk) antigen being the urea transporter with an estimated 14,000 copies per cell. From the experimental number of urea sites, a turnover number between 2-6 x 10(6) sec-1 at 22 degrees C is calculated suggesting a channel mechanism.

The action of organic mercury compounds on the function of isolated mammalian heart muscle.

The effects of four organic mercury compounds (methylmercuric chloride; bromomercurihydroypropane, BMHP; chlormerodrin; p-chloromercuribenzoic acid, PCMB) on mechanical and electrical functions of guinea-pig papillary muscles were investigated. An initial decline in contraction force was followed by a transient positive inotropic response. The first was accompanied by a shortening of the action-potential duration and by a reduction of the depolarization velocity and the duration of the Ca2+-dependent slow response. The latter was characterized by an indirect component (release of noradrenaline) and by a direct component, which was dependent on the stimulation rate and on the extracellular concentration of Na+ and K+. The direct positive effect, therefore, was likely to have resulted from inhibition of the sarcolemmal Na+ + K+-ATPase. This notion was confirmed by experiments with isolated membrane particles. The prevalence of the negative or positive inotropic action of these compounds could be ascribed to their lipophilic or hydrophilic properties, respectively.

Contraluminal transport of hexoses in the proximal convolution of the rat kidney in situ.

In order to study contraluminal hexose transport, concentration and time-dependent influx of 3H-2-deoxy-D-glucose from the interstitium into cortical tubular cells has been measured. The influx curves fit to a two parameter kinetics (Km 1.3 +/- 0.2 mmol/l, Jmax 0.67 +/- 0.16 pmol/s X cm) plus an additional diffusion term (with P = 6 X 10(-8) cm2/s) and a distribution ratio extracellular to intracellular amount of 2-deoxy-D-glucose of 1:0.6. Since the extracellular to intracellular free water space as estimated from morphological data was 1:2, one must conclude that glucose has only free access to 1/3 of the cell water. The intracellularly accessible space was augmented when the tubules were preperfused for 10 s with hypotonic saline. Thereby an increase of the compartment into which diffusion occurs was revealed and a final rupture of this intracellular compartment at 1/4 isotonic solutions was observed. Total replacement of ions in the peritubular perfusate by mannitol did not change 2-deoxy-D-glucose influx, indicating that it is Na+-independent. By adding isotonic concentrations of the respective sugars to the capillary perfusate, three degrees of inhibition of 2-deoxy-D-glucose influx could be revealed: strong inhibition by D-glucose, methyl-beta-D-glucoside, D-mannose, 3-O-methyl-D-glucose, 2-deoxy-D-galactose, methyl-beta-D-galactoside and 6-deoxy-D-glucose, moderate inhibition by D-galactose, L-glucose, L-mannose and D-fructose, no or borderline inhibition by methyl alpha-D-glucoside, 2-deoxy-methyl-alpha-D-galactoside, 1-thio-beta-D-glucose, 1-thio-beta-D-galactose, 5-thio-alpha-D-glucose, myo-inositol and mannitol.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective effects of organic mercurials on the GTP-regulatory proteins of adenylate cyclase systems.

Treatment of membranes from HeLa cells, rat adipocytes, and rat liver with organic mercurials results in complex effects on adenylate cyclase activity that are not mimicked by the reversible sulfhydryl reagent, tetrathionate. At low concentrations (0.1 mM or less 1 mercurials inactivate the enzyme; inactivation is reversed by the thiol-reducing agent, dithiothreitol. Treatment with higher concentrations of organic mercurials (1 mM and above) results in a time-dependent, irreversible change in the ability of guanine nucleotides and fluoride ion to stimulate adenylate cyclase activity. The irreversible changes are blocked by treatment of membranes with cholera toxin and NAD, suggesting that the GTP-regulatory component is the site of mercurial action. This is further suggested by the lack of irreversible effects of mercurials on adenylate cyclase activity in membranes from mouse lymphoma cells that lack this component. Irreversible effects of mercurials on the adipocyte cyclase system also include enhancement of basal activity and potentiation of the inhibitory effects of GTP on cyclase activity; the latter effects of GTP are mediated through a process independent from that mediating stimulation of activity by GTP. It is concluded that the GTP-regulatory proteins responsible for the modulation of adenylate cyclase activity by hormones and neurotransmitters contain the sites of action of organic mercurials. Their possible mode of action is discussed.

Antimicrobial activity of chlormerodrin.

A radiochemical study for antimicrobial activity of chlormerodrin was performed using some commonly occurring nonpathogenic and pathogenic microorganisms. Chlormerodrin concentrations of 15-35 and 20-45 microgram/ml of the culture medium were microbiostatic and microbicidal, respectively. The microbiostatic effect was reversible in the presence of cysteine, an amino acid containing a sulfhydryl group.

[Histoenzymatic studies and internal irradiation. III. Histoenzymatic changes of the kidney treated with chloromeridrin Hg 197]. / Ricerche istoenzimatiche ed irradiazione interna. III) Modificazioni istoenzimatiche del rene di ratto trattato con cloromeridrin Hg 197

The histochemical behaviour of alkaline phosphatase, adenosintriphosphatase, acid phosphatase and acetylglucosaminidase activities was studied in rat kidney treated with Chloromeridrin Hg. In these experimental conditions it was found that the nephrocytes in the proximal convoluted tubule present changes in the enzymatic activities examined, generally proportional to the doses employed. With lower doses of radionuclides, the histoenzymatic changes have a local character and are completely reversible after a month. With higher doses they take on a more extensive character and persis to some extent one month after treatment. These findings suggesti that Hg could be used clinically with due precautions.