Mitochondrial membrane modifications induced by adriamycin-mediated electron transport

Adriamycin (ADM) was found to have a two-step mode of action on the cardiac mitochondrial membrane. (1) An interaction with cardiolipin (CL) resulted in the formation of an ADM-CL complex able to transfer electrons from NADH to cytochrome c (cyt.c) as well as coenzyme Q (CoQ). This complex formation stimulates an increased activity of NADH-CoQ oxidoreductase (complex I) and CoQ-cyt.c oxidoreductase (complex III). (2) Transfer of electrons through ADM resulted in the formation of a very strong complex between ADM and CL. This new complex is different and much stronger than the already known ADM-CL complex.     

Drug membrane transport enhancement using high energy drug polyvinylpyrrolidone (PVP) co-precipitates

Dissolution rate data obtained with sulfathiazole-povidone(PVP) co-precipitates and hydrocortisone-povidone co-precipitates were compared to cellophane membrane diffusion data obtained with the co-precipitates. The hypothesis, that the rate-limiting drug phases in the co-precipitate dissolution experiments were high energy amorphous phases of the drug, was tested. In regions of the dissolution rate studies where the carrier effects due to the PVP-drug complexes were small, the dissolution rates for the two PVP-drug systems agreed well with the results of the membrane diffusion experiments. The normalized fluxes were found to be about 3.5 for the high energy phase of sulfathiazole and 14–18 for that of hydrocortisone.     

Phenolphthalein- and harmaline-induced disturbances in the transport functions of isolated brush border and basolateral membrane vesicles from rat jejunum and kidney cortex

Phenolphthalein and harmaline were examined with respect to their effects on the transport functions of purified brush border and basolateral membrane vesicles from rat jejunum and kidney cortex. Phenolphthalein (0.5 mM) inhibited Na⁺-coupled d-glucose uptake by intestinal brush border membrane vesicles without affecting Na⁺-coupled l-alanine transport, Na⁺ transport or Na⁺-independent d-glucose transport. In renal brush border membrane vesicles, the same concentration of this drug did not even affect Na⁺-coupled d-glucose uptake. At a concentration of 1 mM or higher, however, Phenolphthalein rendered both intestinal and renal vesicles leaky to these solutes. In intestinal and renal basolateral membrane vesicles, phenolphthalein at a concentration of 0.5 mM noticeably inhibited (Na⁺-K⁺)-ATPase activity, but showed no effect on phloretin-sensitive Na⁺-independent d-glucose uptake. At 1 mM this drug also inhibited ouabain-insensitive ATPase activity. Harmaline, at concentrations greater than 2 mM, inhibited not only Na⁺-coupled d-glucose and l-alanine uptake by both intestinal and renal brush border membrane vesicles, but also Na⁺ translocation. The drug, however, affected neither Na⁺-independent d-glucose uptake nor the general permeability of these membranes. Harmaline also inhibited (Na⁺-K⁺)-ATPase activity of intestinal and renal basolateral membrane vesicles without affecting ouabain-insensitive ATPase. It did not influence, however, phloretin-sensitive Na⁺-independent d-glucose uptake by these vesicles. These observations suggest that harmaline acts as an inhibitor of Na⁺ and Na⁺-dependent transport mechanisms in intestinal as well as renal brush border membranes. Phenolphthalein at the lower concentration selectively inhibited certain transport processes in these membranes as well as in basolateral membranes, whereas at the higher concentration it caused widespread structural disturbances, possibly through its chaotropic action on membranes.     

Percutaneous absorption: transport in the dermis

Results are presented for the percutaneous absorption of 3 different esters of nicotinic acid from circular patches; the values of the diffusion coefficients and triggering concentrations for visible erythema are in good agreement with previous work. For methyl nicotinate the radial spread of the erythema has been followed as a function of time; the results are shown to fit a radial diffusion equation. However, the value of the diffusion coefficient is found to be a thousand times larger than the expected value for molecular diffusion. A model is presented in which the radial transport is caused by uptake in the capillaries. Reasonable values are found for the rate constants describing uptake and for the geometry of the capillaries. This geometry is shown to be close to the optimum for efficient transport to and from the dermis.     

The tensile strength and consolidation of lactose coated with non-ionic surfactants II. Tablets

The tensile strengths of tablets made from fine lactose powder coated with increasing amounts of non-ionic surfactants (Spans) have been measured using the diametral compression test, and their compression characteristics have been investigated. The tensile strengths of the tablets decreased, and their packing fractions increased at any compression pressure as the amount of the coding material was increased. The results have been analyzed using Cheng's equat on for the tensile strength and the compression equation of Heckel. The terms in these equations varied systematically with the amounts of the surfactant in the powders used for tabletting.     

The tensile strength and consolidation of lactose coated with non-ionic surfactants I. Powder

The tensile strength and the resistance to consolidation by pressure or tapping of fine lactose powder coated with increasing amounts of a series of non-ionic surfactants were examined.At a constant packing fraction, both the tensile strength and the resistance to consolidation initially decreased to minima when the coating was monomolecular and then increased as the additive formed pendular bonds between the particles.The packing fraction of the powder after 100 taps increased to a maximum, and then decreased as the amount of the coating material present was increased.Cheng's expression for tensile strength has been used to compare the forces that operate between the particles of the coated and the uncoated powders.     

Conjugation of 1-naphthol and transport of 1-naphthol-conjugates in the vascularly perfused small intestine of the mouse

A method is described which allows the simultaneous vascular and luminal perfusion of the murine small intestine. This preparation was used for the investigation of 1-naphthol conjugation in the gut and the sidedness of conjugate release. The viability of this preparation can be maintained for more than 1 hr as indicated by morphological controls, measurement of tissue metabolism and the transport of 3-O-methyl-glucose against a concentration gradient. When 100 μM 1-naphthol was administered on the luminal side, it was conjugated at a constant rate, yielding 1-naphthyl-glucuronide and 1-naphthyl-sulfate in a molar ratio of 1:2. Both metabolites were excreted into the blood at the contraluminal side of the epithelium. The results are discussed with respect to the sidedness of intestinal transport systems for anionic conjugates of xenobiotics and drugs.     

The effects of phenformin on the transport and metabolism of sugars by the rat small intestine

The effects of 0.25–10 mM phenformin on sugar transport and metabolism have been studied in a preparation for the combined perfusion of the vascular bed and the lumen. At all concentrations the effects of vascular phenformin were more pronounced than those of luminal phenformin. Phenformin inhibited galactose transport across the intestine, the pattern of inhibition depending on whether the phenformin was added to the luminal or vascular compartments. The active accumulation of galactose in the mucosal epithelial cells was also abolished. There was a linear relationship between the percentage reduction in mucosal ATP levels and vascular phenformin concentration. Phenformin reduced the rate of glucose uptake from the lumen, and the proportion of this glucose which reached the vascular effluent. Most of the glucose which did not reach the vascular side could be accounted for by the formation of lactic acid. Vascular phenformin increased glucose uptake from the vascular medium by ca 88%, 97% of which could be accounted for by lactate formation. Phenformin was sequestered by the mucosa when added to the vascular, but not the luminal, perfusates. There was very little translocation of intact phenformin across the gut in either the mucosal or serosal directions. It is suggested that the effects of phenformin on the gut mainly derive from an inhibition of mitochondrial oxidative phosphorylation, with a small contribution from a direct effect on the brush border, more pronounced at high phenformin concentrations. The results are consistent with the idea that phenformin delays sugar absorption in man, and that the intestine may be a significant source of lactate production in lactic acidosis     

Altered retrograde axonal transport of nerve growth factor after single and repeated doses of acrylamide in the rat

The retrograde axoplasmic transport of iodinated nerve growth factor (125I-NGF) was markedly altered by systemic acrylamide treatment. Single doses of acrylamide (25 to 100 mg/kg, ip) inhibited the retrograde transport of 125I-NGF in a dose-dependent manner within 12 hr after administration. A dose of acrylamide (15 mg/kg, ip) which did not alter retrograde transport when given as a single dose, markedly inhibited retrograde transport when given repeatedly. Alterations in retrograde transport appeared prior to detectable alterations in peripheral nerve function and correlated with cumulative dose of acrylamide. These data suggest that single doses of acrylamide produce a biochemical lesion which accumulates to produce neuropathy. Altered retrograde axoplasmic transport may reflect the primary biochemical event responsible for the induction of neuropathy by acrylamide.     

Hepatic transport of indocyanine green in dogs chronically intoxicated with dimethylnitrosamine

Hepatic transport of indocyanine green (ICG) was examined in dogs chronically intoxicated with dimethylnitrosamine (DMN) (2 mg/kg) intraportally once a week for 6 weeks. In pathophysiological consequences, significant increases (p less than 0.05) were shown in both glutamic-pyruvic transaminase (GPT) and total plasma bile acids, but no significant difference was shown in body weight, liver wet weight, glutamic-oxaloacetic transaminase (GOT), plasma alkaline phosphatase activity, total plasma protein, and total plasma bilirubin. By histologic examination of livers from intoxicated dogs, increased fibrosis in periportal, perisinusoidal, and especially pericentral areas, with loss of normal architecture, was observed. Partial fibrous bridging between periportal and pericentral areas was also demonstrated, but extensive pseudolobulation with regenerative nodules was not observed. The portal venous pressure of the intoxicated dogs was increased by approximately 50% of that of control dogs. In intoxicated dogs, delays were shown in both plasma disappearance and biliary excretion of ICG and significant decreases were observed in the pharmacokinetic parameters k12 (plasma to liver transfer rate constant), V2 (distribution volume of liver compartment), and CLtot (total body-plasma clearance), while a significant increase was observed in k23 (intrahepatic diffusion and transport rate constant); the V1 (distribution volume of plasma compartment) was not altered. From these findings, it is suggested that the decrease in the intrinsic clearance of ICG for the hepatic uptake process might explain the decrease in ICG uptake rate into the liver which was observed in the DMN-intoxicated dogs. Dogs chronically intoxicated with DMN might be a good model for studying hepatic dysfunction.     

Phenothiazine transport across liquid-lipid,phospholipid and soft polymer membranes

The transport of phenothiazines across phospholipids, isopropyl myristate (IPM) and polydimethylsiloxane (PDMS) membranes has been investigated using the rotating diffusion cell. The aqueous diffusion coefficients and the diffusivities across the PDMS membranes have been determined for the same phenothiazines. With phospholipids, the transport rate constants for mequitazine decreased in the following order: phospholipid free interface > DSPC > DPPC > DMPC. On comparing the transport rate constants across egg phosphatidylcholine liposomes, IPM and PDMS, no simple correlation was found between any two of the three model membranes. The structure of the membrane phase thus appears to be critical in determining the transport characteristics of complex molecules.     

The inhibition of Ca uptake in cardiac membrane vesicles by verapamil

Cardiac membrane vesicles take up Ca²⁺ in response to Na⁺ gradient (high inside) and negative inside membrane potential. Both components of the Ca²⁺ uptake, the Na⁺ gradient dependent uptake and the membrane potential dependent uptake are inhibited by verapamil; the action is dose-dependent and the concentrations of verapamil required to inhibit the Ca²⁺ uptake to 50% of its maximal value are 50 and 60 μM respectively. In the concentration ranges tested (50–750 μM Ca²⁺), the inhibitory effect of verapamil could not be antagonized by increasing the Ca²⁺ concentration of the medium. Introducing verapamil into the vesicles by rapid freezing and slow thawing of the vesicles had the same inhibitory effect as adding the same concentration of verapamil on the outside of the vesicles. Adding verapamil to both sides of the vesicle membrane led to higher inhibition of Ca²⁺ uptake. It is proposed that addition of verapamil can cause a change in cardiac membranes which is manifested by a decrease in the driving membrane potential and Ca²⁺ transport.     

Effects of decoquinate and clopidol on electron transport in mitochondria of Eimeria tenella (Apicomplexa: coccidia)

Resistance of the chicken coccidium Eimeria tenella to the anticoccidial agents decoquinate and clopidol, and the synergistic activity of mixtures of these compounds have been confirmed in vivo. Inhibition of electron transport by decoquinate and clopidol has been studied in mitochondria isolated from unsporulated oocysts of the same lines of E. tenella as those used in the in vivo studies. Electron transport in mitochondria of a drug sensitive line was susceptible to inhibition by both decoquinate and clopidol, and mitochondria isolated from lines made resistant to one or the other of these compounds showed a corresponding resistance at the level of electron transport. Combinations of low concentrations of decoquinate and clopidol caused a greater inhibition of electron transport than expected from summation of their individual actions. Isobolograms showed that decoquinate and clopidol in fact potentiated each other's effect on electron transport. Induced resistance to either decoquinate or clopidol resulted in an increased sensitivity of electron transport to inhibition by the other drug. Cytochrome spectra of E. tenella mitochondria and a biphasic response of NADH-oxidase and terminal oxidase activity to inhibition by cyanide or azide suggest the presence of two functional terminal oxidases. There is a correlation between the resistance of electron transport to inhibition by decoquinate or clopidol and the susceptibility to inhibition by cyanide or azide. Mitochondrial electron transport that is more resistant to inhibition by decoquinate exhibits greater sensitivity to cyanide and azide; electron transport that is more resistant to inhibition by clopidol exhibits a decreased sensitivity to cyanide and azide. Resistance to decoquinate and clopidol is discussed in view of a possibly branched electron transport chain in mitochondria of E. tenella.     

Inhibitory effect of triethyltin on taurine transport by glioma cells

The effects of triethyltin (TET) on the transport of taurine, glutamate, lysine, Na⁺, K⁺ (using ⁸⁶Rb⁺ as tracer), and Cl^? by LRM55 glioma cells were examined. Taurine transport was inhibited by TET at much lower concentrations (IC50 = 2.5 μM) than either glutamate or lysine transport (135 and 110 μM, respectively). TET had no significant effect on Na⁺, Cl^?, or ⁸⁶Rb⁺ influx at the low concentrations sufficient to inhibit taurine transport, but it enhanced Na⁺ and Cl^? uptake at concentrations > 100 μM. The failure of low concentrations (≤10 μM) of TET to affect ion transport indicated that inhibition of taurine transport was not secondary to effects of TET on ion movements or gradients. This conclusion was supported by the observation that neither ouabain nor furosemide, which do affect ion movement and gradients, strongly inhibited taurine transport. Uncouplers and inhibitors of oxidative phosphorylation (cyanide, 2,4-dinitrophenol, and carbonyl cyanide-m-chlorophenyl hydrazone) also had only small effects on taurine transport, suggesting that inhibition by TET was not secondary to possible effects on oxidative phosphorylation. TET had no effect on the efflux of taurine from LRM55 cells at the low concentrations that inhibit uptake, but it induced a nonspecific increase in membrane permeability at much higher concentrations (> 100 μM). Tri-n-propyltin and tri-n-butyltin were also potent inhibitors of taurine transport (IC50 = 2.3 and 11 μM, respectively), but trimethyltin was much less potent (144 μM).     

Interactions of vasopressin agonists and antagonists with membrane receptors

Plasma membranes containing one class of non-cooperative binding sites for tritium-labelled [8-arginine]vasopressin were isolated from bovine kidney inner medulla and from rat liver. By using a weighted, non-linear least squares fit to logistic curves, the binding parameters of eight vasopressin agonists and antagonists were determined in competition experiments. Vasopressin analogues with sarcosine or N-methyl-L-alanine in position 7 instead of proline showed a high ratio of antidiuretic to vasopressor activity. These analogues retained a high binding affinity to the renal vasopressin receptor with apparent dissociation constants KD in the order proline less than sarcosine less than methylalanine . In contrast, the affinity to the hepatic vasopressin receptor, which shares characteristics with vasopressor receptors, was drastically reduced with KD values being in the order proline much less than N- methylalanine less than sarcosine. By combining the substitutions at position 7 with substitutions of cysteine in position 1 by either deaminopenicillamine or beta-mercapto-beta, beta-cyclopentamethylenepropionic acid, inhibitors of the oxytocoic and vasopressor responses were obtained. These additional substitutions at position 1 led to a drastic decrease in the binding affinity to the vasopressin receptor in bovine kidney. The intrinsic activity of these analogues to stimulate the renal vasopressin sensitive adenylate cyclase was strongly reduced or completely lost. In the rat liver system, however, these vasopressin antagonists showed a remarkably increased affinity to vasopressin receptors as compared to analogues substituted only at position 7. GTP reduced the binding affinity of all analogues to the hepatic receptor. The results show that these structural modifications which influence both the conformational properties of the vasopressin molecule and the biological activities of the hormone had strikingly different effects on the interactions of the resulting analogues with physiologically important receptors in the kidney and the liver. These studies may lead to the development of more specific vasopressin agonists and antagonists.     

Nature of Na+-independent stimulation of renal transport of p-aminohippurate by exogenous metabolites

The relationship between p-aminohippurate (PAH) and Na⁺ transport in rabbit kidney cortical slices was examined under optimal metabolic conditions. Addition of lactate, pyruvate and acetate to the incubation medium stimulated PAH transport and accumulation, but had no effect on active Na⁺ efflux from the slices. Conversely, small concentration of F^?, in the presence of acetate, decreased PAH transport and accumulation, but had no effect on Na⁺ efflux. These observations constitute evidence that, in addition to Na⁺ cotransport [M. I. Sheikh and J. V. Møller Biochem. J.208, 243 (1982)], PAH uptake is metabolically stimulated by a Na⁺-independent pathway. This could occur either by a direct metabolic effect on the PAH carrier, or by exchange with intracellular anions that are generated in the presence of exogenous substrate. In support of the latter possibility the PAH carrier is demonstrated to function as an anion exchanger of PAH and fumurate after preloading of the slices with fumurate under anaerobic conditions.     

Opposite effects of tolbutamide and diazoxide on 86Rb+ fluxes and membrane potential in pancreatic B cells

The effects of tolbutamide and diazoxide on ⁸⁶Rb⁺ fluxes, ⁴⁵Ca²⁺ uptake, insulin release and B cell membrane potential have been studied in rat or mouse islets. In the presence of 3 mM glucose, tolbutamide rapidly and reversibly decreased Rb⁺ efflux from perifused islets and depolarised B cells. The effect on Rb⁺ efflux was paradoxically more marked with 20 than 100 μg/ml tolbutamide, at least in the presence of extracellular calcium. Addition of tolbutamide to a medium containing 6 mM glucose and calcium increased Rb⁺ efflux transiently with 20 μg/ml and permanently with 100 μg/ml. The drug also inhibited Rb⁺ influx in islet cells, but had little effect on Rb⁺ net uptake. Diazoxide rapidly, steadily and reversibly increased Rb⁺ efflux in a dose-dependent manner (20–100 μg/ml). When 20 μg/ml tolbutamide and diazoxide were combined in the presence of 3mM glucose, only a slight decrease in Rb⁺ efflux was observed. The depolarisation of B cells normally produced by tolbutamide was markedly reduced and the electrical activity completely suppressed by diazoxide. In the presence of 10 mM glucose, diazoxide increased Rb⁺ efflux from the islets and hyperpolarised B cells. Tolbutamide, tetraethylammonium and quinine reversed the increase in Rb⁺ efflux, the inhibition of Ca²⁺ uptake and the suppression of insulin release produced by diazoxide. Tolbutamide rapidly reversed the hyperpolarisation and restored electrical activity. It is suggested that the stimulation and inhibition of insulin release by tolbutamide and diazoxide are due to their respective ability to decrease and to increase the K permeability of the B cell membrane. This change in K permeability leads either to depolarisation and stimulation of Ca²⁺ influx or to hyperpolarisation and inhibition of Ca²⁺ influx.     

Effects of morphine and pentobarbital on mouse and rat renal transport systems.

The effects of morphine and pentobarbital administered by pellet implantation have been examined for possible nephrotoxic effects in the rat and the mouse. In particular, the effects of these drugs on various renal transport mechanisms were examined. Morphine was found to disrupt renal transport, tissue electrolytes, but not tissue oxygen consumption 24 hr after pellet implantation in the mouse. By 72 hr, however, all parameters examined had returned to control levels despite the continued presence of morphine. The specificity of this effect was demonstrable through the use of naloxone. This antagonist reversed completely all of the morphine effects in the mouse. In the rats, morphine altered only p-aminohippurate (PAH) transport. Pentobarbital reduced PAH and tetraethylammonium (TEA) transport in the rat. Kidney slices from mice implanted with pentobarbital showed significant increases in the uptake of PAH and α-aminoisobutyrate (AIB). No disruption of tissue electrolytes or water was observed in either species with pentobarbital administration.     

Effect of the organic acid transport inhibitor probenecid on renal cortical uptake and proximal tubular toxicity of hexachloro-1,3-butadiene and its conjugates

Hexachloro-1,3-butadiene (HCBD), its glutathione conjugate (HCBD-GSH), cysteine conjugate (HCBD-CYS), and mercapturic acid derivative (HCBD-NAC) all produce acute necrosis of the pars recta of the proximal renal tubule in the rat. Previous studies have shown that radiolabel from administered HCBD appears to concentrate in the pars recta region. Renal uptake of radioactivity from HCBD-NAC was studied in rats by giving a single ip injection of the chemical and measuring its concentration in plasma and renal cortex 4 hr later. Cortex/plasma ratios (C/P) of HCBD-NAC were 4.35 +/- 0.21 (8 animals) at a dose of 64 mumol/kg and 10.4 +/- 0.55 (5) at a dose of 16 mumol/kg. These ratios were greater than that of inulin [C/P inulin = 1.5 +/- 0.2 (4)]. Thus cortical HCBD-NAC content was significantly greater than can be accounted for by glomerular filtration alone. Prior administration of probenecid (500 mumol/kg), a competitive inhibitor of organic acid transport, to animals receiving 16 or 64 mumol/kg of HCBD-NAC reduced the C/P to 1.03 +/- 0.09 (5) and 0.81 +/- 0.05 (8), respectively. Administration of probenecid in increasing doses (100, 200, 300, and 400 mumol/kg) to animals receiving 64 mumol/kg HCBD-NAC resulted in decreases of the C/P (2.59, 2.29, 1.35, and 0.84, respectively), suggesting a competitive inhibition of cortical HCBD-NAC uptake. The extent of covalently bound radioactivity from 64 mumol/kg HCBD-NAC was significantly greater in the renal cortex (1.11 +/- 0.2 nmol eq/mg protein) than in the liver (0.19 +/- 0.01 nmol eq/mg protein). Prior administration of probenecid (500 mumol/kg) reduced the renal cortical concentration of HCBD-NAC to 0.25 +/- 0.02 nmol eq/mg protein. Increasing doses of probenecid resulted in a progressive decrease in renal cortical covalent binding. When treatment with probenecid led to renal cortical concentrations of less than 120 nmol eq HCBD-NAC/g and an amount of covalently bound material less than 0.4 nmol eq/mg protein the animals were completely protected against the nephrotoxicity, as assessed by plasma urea and histopathological examination 24 hr after dosing. Prior administration of probenecid (500 mumol/kg) also protected rats against the nephrotoxicity produced by HCBD (192 mumol/kg), HCBD-GSH (47 mumol/kg), and HCBD-CYS (36 mumol/kg). It is suggested that the renal cortical accumulation and selective proximal tubular toxicity of HCBD and its conjugates is related to a carrier-mediated transport system.