Ole Terland

Electron carriers of the bovine adrenal chromaffin granules.

Abstract Chromaffin granules of bovine adrenal medulla essentially free of contamination from mitochondria, lysosomes and fragments of endoplasmic reticulum, have been lysed by hypo-osmotic shock. The membrane fraction has been shown to contain a b -type cytochrome (cytochrome b 561 ), flavoprotein(s) and NADH: (acceptor) oxidoreductase (EC 1.6.99.3) with ferricyanide, 2,6-dichlorophenolindophenol (DCIP), bovine heart ferricytochrome c and the endogenous ferricytochrome b 561 as acceptors. Furthermore, the membrane fraction catalyzes the rapid oxidation of bovine heart ferrocytochrome c . This enzymic activity, which is completely inhibited by CN(3 · 10 −4 M) and CO, is not attributed to cytochrome a + a 3 (ferrocytochrome c : O 2 oxidoreductase, EC 1.9.3.1).

Cytochrome b561 of the bovine adrenal chromaffin granules. A high potential b-type cytochrome

Simultaneous potentiometric and spectrophotometric assays of cytochrome b561 oxidation-reduction in chromaffin granule membrane preparations (depleted of catecholamines and ATP) have been carried out under strictly anaerobic conditions. The cytochrome oxidation-reduction is described by a one-electron transition with an apparent midpoint potential at pH 7.0 (E′m7) of +140 mV. This value is in good agreement with the rate and extent of its aerobic reduction by ascorbate; the K′m for the reductant was found to be 0.34 mM. The available evidence therefore confirms our previous suggestion that cytochrome b561 is a unique mammalian b-type cytochrome of chromaffin tissues.

Oxidoreductase activities of chromaffin granule ghosts isolated from the bovine adrenal medulla

1. Based on estimated s-values of subpopulations of bovine adrenal chromaffin granules (Bødtker-Naess, V., Slinde, E., Terland, O. and Flatmark, T. (1978) Biochim. Biophys. Acta 541, 124--134) a new large-scale procedure is described for the isolation of the total population of chromaffin granules by differential centrifugation in 0.25 M sucrose. 2. Using the total population of chromaffin granules obtained by differential centrifugation, final purification was achieved by density-gradient centrifugation in either sucrose or Percoll-sucrose. In either case, the isolated granule fractions were contaminated with mitochondria to about the same degree. 3. Chromaffin granule ghosts, obtained by hypoosmotic lysis of granules isolated by sucrose density-gradient, centrifugation, were subjected to centrifugation on a discontinuous density gradient (buffer/0.9 M sucrose). By this procedure a substantial purification of the ghosts was achieved as determined from measurements of protein and various marker enzymes. 4. In contrast to preparations of chromaffin granule ghosts prepared by previous standard procedures, those purified by gradient centrifugation (on 0.9 M sucrose) did not reveal any NADH-linked cytochrome b-561 reductase activity. However, experimental evidence is presented for the existence of an intrinsic NADH-oxidizing enzyme system in the granule membrane. 5. No significant difference was observed in the specific content of cytochrome b.561 and NADH:(acceptor) oxidoreductase activities between ghost preparations obtained from populations of heavy and light chromaffin granules. 6. The functional significance of cytochrome b-561 and the NADH:(acceptor) oxidoreductase activities of the granule membrane remains to be determined.

Isolation and characterization of noradrenalin storage granules of bovine adrenal medulla.

A method is described for the preparation of (1) the heavy population of bovine adrenal chromaffin granules (SH (average sedimentation coefficient) = 12 400 S in 0.25 M sucrose) essentially free from contamination with mitochondria and other organelles, and (2) a subpopulation of this heavy population which is highly enriched in noradrenalin (greater than or approximately 95% of the total catecholamine is noradrenalin). The method is based on isopycnic gradient centrifugation using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.5 M sucrose medium. The isolated population of noradrenalin granules appeared highly electron dense in transmission electron microscopy and revealed a rather narrow size distribution. The specific content of amine and adenine nucleotides (with reference to total granule protein) was markedly higher than for the total population of heavy chromaffin granules. The molar ratio of amines to adenine nucleotides was, however, lower in the noradrenalin granules, i.e. 4.8 vs. 11.9.

Subcellular distribution of ascorbate in bovine adrenal medulla: Evidence for accumulation in chromaffin granules against a concentration gradient

The subcellular distribution of ascorbate and catecholamines has been studied in homogenates of bovine adrenal medulla and cortex. 1. The recovery of the vitamin was found to be 4.10 +/- 0.22 and 9.57 +/- 1.37 mumol/g wet weight for the medulla and cortex, respectively. A major fraction (34.4%) of the vitamin was recovered in the particulate fraction of the medulla as compared to about 8% in the corresponding fraction of the cortex. In comparison, 78.9% of the catecholamines were found in the particulate fraction of the medulla. 2. Analytical differential centrifugation of medulla homogenates revealed a sedimentation profile of ascorbate which was identical to that obtained for noradrenalin and adrenalin. The co-sedimentation of these compounds indicates that ascorbate is an essential component of the heavy as well as the light population of chromaffin granules. The stoichiometry of catecholamines to ascorbate was approx. 25:1 in both subpopulations. 3. Based on an estimated volume fraction of approximately 13% for the chromaffin granules, as determined morphometrically (Kryvi, H., Flatmark, T. and Terland, O. (1979) Eur. J. Cell Biol. 20, 76-82), a concentration gradient (chromaffin granules:cytosol) of approx. 4 was estimated for ascorbate in the cells of adrenal medulla. 4. No ascorbate 2-sulfate was detected in any of the subcellular fractions isolated, and the content of dehydroascorbate in isolated chromaffin granules was less than 1% of the total ascorbate value.

Drug-induced parkinsonism: cinnarizine and flunarizine are potent uncouplers of the vacuolar H-ATPase in catecholamine storage vesicles

Cinnarizine (1-diphenylmethyl-4-(3-phenyl-2-propenyl)piperazine) and its di-fluorinated derivative flunarizine inhibit the MgATP-dependent generation of a transmembrane proton electrochemical gradient in chromaffin granule ghosts. The concentrations giving 50% inhibition (IC50) of the MgATP-dependent generation of the pH-gradient were 5.9+/-0.6 microM (n = 6) and 3.0+/-0.3 microM (n = 5) for cinnarizine and flunarizine, respectively. The IC50 values for inhibiting the generation of the membrane potential were even lower, i.e. 0.19+/-0.06 microM (n = 6) and 0.15+/-0.01 microM (n = 4) for cinnarizine and flunarizine, respectively. Cinnarizine (10 microM) also inhibited the energy-dependent vesicular uptake of [14C]-dopamine (50 microM) by 76%, i.e. from 2.1+/-0.9 to 0.5+/-0.6 nmol/mg protein/min (n = 5, P < 0.002). Cinnarizine (10 microM) increased the MgATPase activity of the granule ghosts by 47+/-26% (n = 4) compatible with an uncoupling of the vacuolar H+-ATPase activity. The IC50-values observed for the two compounds are in the same range as their reported therapeutic plasma concentrations in vivo, suggesting that cinnarizine and flunarizine may well inhibit proton pumping and catecholamine uptake in storage vesicles also in vivo. This mechanism of action may contribute to the drug-induced parkinsonism seen as a side-effect of the two drugs.

NADH(NADPH): (acceptor) oxidoreductase activities of the bovine adrenal chromaffin granules.

Abstract 1. 1. An analysis of the NADH(NADPH):(acceptor) oxidoreductase activities of adrenal chromaffin granules depleted of low molecular weight substances (notably catecholamines and ATP), has been made in terms of the flavoprotein(s) and cytochrome b 561 previously reported (Flatmark, T., Terland, O. and Helle, K. B. (1971) Biochim. Biophys. Acta 226, 9–19). 2. 2. The overall NADH oxidation of freshly prepared granule preparations was rather low (0.06–0.32 nmole NADH oxidized per min per mg of protein; n = 21), but approximately 10 times higher than the NADPH oxidation (0-0.04 nmole NADPH oxidized per min per mg of protein; n = 6). 3. 3. Of the total NADH oxidase activity of the granule preparation approximately 45% was attributed to an oxidative sequence involving a p -hydroxymercuribenzoate (PHMB)-sensitive flavoprotein, cytochrome b 561 and a cyanide-sensitive oxidase. This NADH oxidase activity was greatly stimulated by mammalian cytochrome c ( K ′ m = 1.6 · 10 −6 M) and resembled the microsomal activity, e.g. of liver, in its insensitivity to antimycin A. Approximately 45% of the NADH oxidase activity was attributed to an oxidative sequence probably involving a flavoprotein and an endogenous electron acceptor. This activity was inhibited only at high concentrations of PHMB and was stimulated by fumarate. An additional 10% of the NADH oxidation was completely insensitive to PHMB as well as to cyanide and revealed the same specific activity with NADPH. 4. 4. The NADH(NADPH): (acceptor) oxidoreductase activities vary according to the structural state of the membrane phase. Thus, the oxidative reactions suffer profound changes upon disintegration of the membrane structure by various means, e.g. by dilution (particularly at higher temperatures) and by detergent (Triton X-100). One of the most remarkable features of this transition was the appearance of a fumarate-stimulated oxidation of NADH which did not require molecular oxygen, but could be accounted for on the basis of the presence of an endogenous electron acceptor of molecular weight

The effect of calcium channel blockers on the H+-ATPase and bioenergetics of catecholamine storage vesicles

A number of commonly used calcium channel blockers have been compared with respect to their effects on the bioenergetics of catecholamine storage vesicles. Chromaffin granule ghosts with a well-preserved ability to actively transport and store catecholamines, were used as a model for adrenergic synaptic vesicles due to their functional similarity. Nicardipine, verapamil, terodiline and diltiazem were found to have effects comparable to that of prenylamine (Grønberg, M., O. Terland, E.S. Husebye and T. Flatmark, 1990. Biochem. Pharmacol. 40, 351) by inhibiting the generation of a transmembrane proton electrochemical gradient driven by the vesicular H(+)-ATPase, mainly by loose-coupling/uncoupling of this ATPase. Amlodipine inhibited the internal acidification of the vesicles in a tyramine-like manner and increased the steady-state membrane potential (positive inside) generated by the MgATP-dependent proton translocation. Nifedipine and felodipine also inhibited the efficiency of the proton pump, but their mechanisms of action require further investigation. The concentrations giving 50% inhibition of the H(+)-ATPase-dependent generation of a pH-gradient were found to be: 12 microM felodipine, 16 microM nicardipine, 25 microM terodiline, 50 microM nifedipine, 60 microM verapamil, 65 microM amlodipine and 150 microM diltiazem. The effects of the calcium channel blockers on the bioenergetics of chromaffin granules explain the release of catecholamines from sympathetic nerves and ganglia in vitro by the calcium channel blockers.

Cytochrome b-561 as the single heme protein of the bovine adrenal chromaffin granule membrane

Based on difference spectroscopy of freshly prepared chromaffin granule membranes and assay of the total heme content, it is shown that all the heme (which is protoheme) is accounted for as cytochrome b-561. CO forms only a weak absorption band at 418 nm and a shoulder at 455 nm upon reduction by dithionite, probably due to the presence of a small fraction of cytochrome b-561 (denatured?) which binds CO. Thus, no evidence was obtained for the presence of cytochrome P-450 and P-420 in the granule membranes.

On the polydispersity of bovine adrenal chromaffin granules Analytical differential centrifugation in isotonic homogeneous medium

Abstract The polydispersity of chromaffin granules of bovine adrenal medulla homogenates has been analyzed by analytical differential centrifugation in homogeneous media. Adrenaline and noradrenaline were determined by automated ion-exchange column chromatographic preseparation using ninhydrin as the subsequent detection reagent. 1. 1. Two populations of storage granules were detected in the large granule fraction, containing about 95% of the catecholamines in the homogenate, by sedimentation in a medium containing 0.25 M sucrose whether adrenaline or noradrenaline was used as the specific marker. The average sedimentation coefficents at 4°C ( s 4, B ) were estimated to s H = 12 440 ± 1850 S and s L = 3910 ± 280 S for the heavy and light population, respectively. The relative proportion of the two populations was estimated to be 65 ± 9% ( s L and 35 ± 8% ( s H ) . 2. 2. Several lines of the chromaffin granules in both types of adrenal medullary cells, i.e., their degree of maturation.