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.

Comparison of the ultrastructure of adrenaline and noradrenaline storage granules of bovine adrenal medulla.

The ultrastructure of the membranes of noradrenaline (NA) and adrenaline (A) granules of the bovine adrenal medulla (Terland, O., T. Flatmark, and H. Kryvi, Biochim, Biophys. Acta 553, 460--468 (1979)) was analyzed by transmission, negative staining and freeze-etch electron microscopy. The two types of storage granules can be distinguished mainly by two morphological criteria: (a) The NA-granules have a more electron dense matrix core than the A-granules, (b) the NA-granules revealed less asymmetry in the distribution of intramembrane particles (nPF:nEF = 4,5:1) than the A-granules (nPF:nEF = 9:1). Thus, the trilaminar structure, negative staining pattern and size distribution of the intramembrane particles of the two fracture faces on freeze-etch electron microscopy were very similar for the two types of granules. Freeze-etching revealed a wide range of the particle size distribution for both fracture faces in both types of granules, with an average diameter of 12.6 +/- 2.7 nm (A-granules) and 10.2 +/- 2.8 nm (NA-granules) for the E-fracture faces and 11.4 +/- 2.7 nm (A-granules) and 9.8 +/- 2.4 nm (NA-granules) for the P-fracture faces. Some of the particles on the P-fracture face (outer surface of the membrane) revealed a subunit structure, most clearly seen in the specimens of NA-granules. Morhpometric analyses of sectioned bovine adrenal medulla revealed that the chromaffin granules on an average account for approx. 13.5% of the cytoplasmic volume in the total population of chromaffin cells.

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.

Energy-dependent accumulation of calcium antagonists in catecholamine storage vesicles.

The calcium antagonists verapamil, nitrendipine, mibefradil, and amlodipine accumulate in chromaffin granule ghosts with apparent equilibrium partition coefficients [(mol/mg membrane lipid)/(mol/mg solvent water)] of 246 +/- 105 (N = 8), 2700 +/- 600 (N = 4), 7400 +/- 2200 (N = 4), and 8100 +/- 1100 (N = 5), respectively. In the presence of 1.2 mM MgATP, the partition coefficients were 854 +/- 206 (N = 10), 2300 +/- 600 (N = 4), 32,700 +/- 8,900 (N = 7), and 20,300 +/- 5,000 (N = 11) for verapamil, nitrendipine, mibefradil, and amlodipine, respectively. Except for nitrendipine, the apparent partition coefficients in the presence of MgATP were significantly different from the control (P < 0.001). For amlodipine and verapamil, the vacuolar H(+)-ATPase inhibitors bafilomycin A1 (30 nM) and N-ethylmaleimide (2 mM) and the protonophore (uncoupler) carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 microM) completely blocked the increase in partition coefficients in response to MgATP. The extra amlodipine, mibefradil, and verapamil that accumulated in response to MgATP were released into the medium by CCCP (10 microM) by 18% (N = 5), 30% (N = 5), and 88% (N = 5) for amlodipine, mibefradil, and verapamil, respectively. Thus, amlodipine, mibefradil, and verapamil, but not nitrendipine, accumulate in catecholamine storage vesicles in response to delta mu H+ generated by the endogenous V-type H(+)-ATPase, and are partially released by de-energetisation. Hence, these calcium antagonists can reach unexpectedly high concentrations in certain target cells, and give pharmacodynamic properties not shared by nitrendipine.

The effect of prenylamine and organic nitrates on the bioenergetics of bovine catecholamine storage vesicles.

We have compared the cardioprotective agents prenylamine and glyceryl trinitrate (GTN) with respect to their effects on the bioenergetics of catecholamine storage vesicles. Chromaffin granule ghosts, which have a well preserved ability to actively transport and store catecholamines, were used as a model for adrenergic synaptic vesicles due to their functional similarity. Prenylamine, which partially and reversibly deplete the endogenous stores of noradrenaline in adrenergic nerves and ganglia, was found to inhibit the generation of the transmembrane proton electrochemical gradient driven by a H(+)-ATPase, mainly by acting as an uncoupler of this ATPase. The inhibition of the energy dependent dopamine uptake (and noradrenaline biosynthesis) by prenylamine could be accounted for by its effect on the bioenergetics of the storage vesicles. The organic nitrates glyceryl trinitrate and isosorbide dinitrate also partly inhibited the catecholamine uptake in parallel with their effects on the proton electrochemical gradient. It is concluded that GTN is a weak catecholamine depletor. Experiments with 3-morpholinosydnonimin-hydrochloride, a source of nitric oxide (NO), opens up the possibility that the mechanism of inhibition of the bioenergetics of chromaffin granule ghosts by GTN is mediated by NO.

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.

[Furosemide capsules in the treatment of hypertension or heart failure in the elderly in general practice]. / Furosemidkapsler i behandlingen av hypertensjon eller hjertesvikt hos eldre i allmennpraksis.

The article reports the efficacy and tolerability of a new slow-release formulation of furosemide (Lasix Retard) given to an elderly (mean: 72 years, range 48-92) population (n = 115, 77 females) suffering from hypertension or heart failure. In 34 of 52 patients with heart failure the clinical condition improved. Lasix Retard reduced blood pressure significantly from 187/99 to 166/93 after one year on 30 mg/day (n = 19). It was found that Lasix Retard replaces thiazide diuretics without causing any significant change in blood pressure. There were no significant changes in metabolic indicators (S-potassium, S-urate, S-glucose). Side effects were in the same range as reported for thiazide diuretics. There were 33 (29%) drop-outs.

[Better compliance--the most important weapon of the general practitioner against stochastic medicine]. / Bedre legecompliance--allmennlegens viktigste våpen mot en stokastisk medisin.

The practice of general practitioners varies widely when handling different conditions, even when strict guidelines are available. This variation is probably no less in relation to persons without any specific disease, when both doctor and patient stress the somatic symptoms. For this group the final result is determined by factors beyond the doctor's control. Medicine may appear to be stochastic, which could mask the use of treatments of no proven value. In any medical situation, good quality in general practice must be associated with the treatment. A better understanding of the patient's situation is the means of raising quality in general practice.

[Catecholamine release--unpredictable side effects of calcium antagonists]. / Katekolaminfrisetting--kalsiumantagonistenes usikre følgesvenn.

Animal and in vitro studies have shown that calcium antagonists release noradrenaline from sympathetic nerves. Several calcium antagonists interact with the catecholamine storage vesicles in a way that reduces the ability of the vesicles to take up and store catecholamines. Clinical studies indicate that verapamil and nifedipine increase release of noradrenaline from the heart during ischemia. Calcium antagonists thus have a direct catecholamine-releasing effect that may be clinically relevant (may induce negative side effects).

Dopamine oxidation generates an oxidative stress mediated by dopamine semiquinone and unrelated to reactive oxygen species.

Dopamine (100 microM, 10-30 min) inhibits/inactivates the MgATP-dependent generation of a transmembrane proton electrochemical gradient in chromaffin granule ghosts. The dopamine dependent inhibition was enhanced by adding soluble dopamine beta-monooxygenase (DBM, 0.2 U/ml) and completely prevented by ascorbate (1 mM), dithiothreitol (2 mM) and approximately 80% by the DBM inhibitor fusaric acid (10 microM). This indicates that the inhibition is caused by the dopamine semiquinone free radical generated during DBM-dependent dopamine oxidation. Catalase, superoxide dismutase or both did not prevent the inhibition, and DBM-catalysed dopamine oxidation did not change the basal level of lipid peroxidation, excluding the involvement of reactive oxygen species as being responsible for the inhibition. N-ethylmaleimide-sensitive ATPase activity (i.e. the proton translocating ATPase) in the vesicle membranes was inhibited during dopamine incubation, indicating that the toxic metabolite (dopamine semiquinone) inhibits proton pumping by inhibiting the endogenous vacuolar H(+)-ATPase. As this proton pump represents the driving force for the vesicular uptake and storage of catecholamines, the dopamine dependent inhibition, if taking place in vivo, may inhibit dopamine uptake in storage vesicles in sympathetic neurons, e.g. as observed in the myopathic hamster heart.