Release of noradrenaline from isolated rat tail artery induced by bafilomycin A1.

The macrolide antibiotic bafilomycin A1, a selective inhibitor of the vesicular H(+)-transporting ATPase, increased irreversibly the overflow of 3,4-dihydroxyphenylethylene glycol from isolated segments of the rat tail artery. Maximum increase in the overflow was produced by exposing the tissues to 0.5 mumol/l bafilomycin A1. Unless the Na(+)-dependent neuronal amine carrier (uptake1) was inhibited, overflow of noradrenaline was below the detection limit. The bafilomycin A1-induced increase in overflow of noradrenaline from tissues with inhibited uptake1 was accompanied by a significant decrease in the (noradrenaline overflow:glycol overflow) ratio. Unlike reserpine and tetrabenazine, the antibiotic did not alter the (noradrenaline overflow:glycol overflow) ratio in arteries incubated in Ca(2+)-free, 120 mmol/l K+ medium. Bafilomycin A1 increased overflow of noradrenaline and normetanephrine from tissues with inhibited monoamine oxidase. Inhibitors of extraneuronal catecholamine transport (uptake2), corticosterone, 3-O-methylisoprenaline and 1,1'-diethyl-2,2'-cyanine, suppressed overflow of normetanephrine while increasing that of noradrenaline. Further increase in overflow of noradrenaline was produced by concomitant inhibition of uptake1. A similar effect was observed in tissues previously exposed to phenoxybenzamine. After exposure to bafilomycin A1, tyramine and (+) amphetamine (10 mumol/l) were equally effective in increasing overflow of noradrenaline from tissues with inhibited monoamine oxidase into corticosterone-containing medium. Bafilomycin A1 promotes leakage of noradrenaline from storage vesicles without affecting its conversion to 3,4-dihydroxyphenylethylene glycol. When uptake1 is inhibited, axoplasmic noradrenaline can be translocated effectively across the axonal membrane by the 'diffusional efflux'. When uptake1 is inhibited, spontaneous quantal release contributes significantly to overflow of noradrenaline into normal media. The 'diffusional efflux' of noradrenaline is unaffected by inhibitors of uptake2. Even at highly elevated concentrations of axoplasmic noradrenaline, the uptake1-mediated influx of noradrenaline exceeds the uptake1-mediated efflux. Enhancement of noradrenaline overflow from tissues with inhibited monoamine oxidase by indirectly acting sympathomimetic amines depends primarily on their ability to induce leakage of the transmitter from storage vesicles rather than its translocation across the axonal membrane.

The cocaine-insensitive component of non-exocytotic efflux of noradrenaline from adrenergic axons in the isolated rat tail artery.

The working hypothesis was that the cocaine-insensitive component of non-exocytotic efflux of noradrenaline represents diffusion of the unprotonated amine across the axonal membrane. It was tested by examination of the effect of changing axoplasmic pH--and thus the fraction of extravesicular noradrenaline in the unprotonated form--on the overflows of endogenous noradrenaline and 3,4-dihydroxyphenylethylene glycol from rat tail arteries. The catechols were assayed by liquid chromatography with amperometric detection. To dissipate the H+ gradient across the axonal membrane, the tissues were incubated in media of different pH, in which Na+ was completely replaced with K+ and which were HCO3(-)-(and Ca(2+)-)free. Exposure of the tissues to these media produced substantial, but reversible increases in the overflow of noradrenaline. Subsequently, the overflows of both noradrenaline and the glycol kept rising, but their ratio did not change. Cocaine (0.1 mmol/l) lowered the (noradrenaline overflow: glycol overflow) ratio significantly. The ratio observed in its presence increased steeply with decreasing external and, presumably, axoplasmic pH. Addition of valinomycin and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (1 mumol/l each) to the cocaine-containing media more than doubled the overflows without altering significantly the ratio. Under identical conditions, the overflow of noradrenaline from preparations with inactive neuronal monoamine oxidase did not decrease with decreasing pH. Since, in the presence of cocaine, the overflow ratio increased--rather than decreased--with decreasing pH, and because the overflow or noradrenaline from preparations with inactive monoamine oxidase did not decline with pH, the cocaine-insensitive component of noradrenaline efflux does not seem proportional to the axoplasmic concentration of the unprotonated amine.(ABSTRACT TRUNCATED AT 250 WORDS)

Swelling of the small adrenergic storage vesicles and the loss of vesicular ATP induced by cocaine in the isolated rat tail artery.

Exposure of the isolated rat tail artery to cocaine (greater than 0.03 mmol/l) was found to cause release of noradrenaline from the small (40 nm) storage vesicles in adrenergic varicosities (Palatý 1988). In the present study transmission electron microscopy was used to determine whether or not cocaine also affects the volume and the ATP content of the vesicles in situ. 1. Evaluation of electron micrographs of arterial segments fixed in a buffered mixture of paraformaldehyde and glutaraldehyde showed that cocaine (3 mmol/l; 15 min) caused significant, though non-uniform, swelling of the small vesicles. 2. To confirm the selectivity of the uranaffin method of ATP localization with respect to noradrenaline, the latter was depleted by prior injection of the rats with reserpine. As a result the fraction of the small vesicles containing electron-dense cores decreased from 55% in control specimens to 29%. 3. In the specimens processed by the uranaffin method, cocaine (3 mmol/l; 30 min) lowered the fraction of the vesicles containing electron-dense cores by about 80%. 4. After having been lowered by cocaine, the fractions of vesicles containing electron-dense cores in specimens processed by the chromaffin and uranaffin methods were restored during incubation of the tissue in cocaine-free medium supplemented with dopamine (7 mumol/l) and L-ascorbate (0.3 mmol/l). There was no restoration of the cores in the specimens processed by the uranaffin method when the tissue had been incubated in the absence of dopamine. 5. These observations support the hypothesis that unprotonated cocaine diffuses into and accumulates in the small vesicles in situ. They also suggest that, as in chromaffin granules (Caughey and Kirshner 1987), interaction of ATP and noradrenaline may be essential for effective storage of the nucleotide in the small vesicles.

Inhibition of monoamine oxidase by analogues of amiloride.

Amiloride analogues with nonaromatic substituents on the 5-amino group or different substituents on carbon-6 of the pyrazine ring were tested as inhibitors of monoamine oxidase A and B in rat brain homogenate. The inhibition was competitive and reversible. 5-(N,N-Tetramethylene)amiloride protected the A type in the homogenate against irreversible inhibition by clorgyline. A reciprocal relation was found to exist between inhibitory constants of 5-N-substituted amiloride analogues for monoamine oxidase A and the ratio of overflows of endogenous noradrenaline and 3,4-dihydroxyphenylethylene glycol from the isolated rat tail artery incubated in the presence of a 50 microM concentration of the analogue, when the tissue was exposed to 10 microM tyramine. The 5-amino group appeared to be essential for inhibition of the A but not of the B type. Bell-shaped relations between inhibitory constants of 5-(N-alkyl)- and 5-(N,N-dialkyl)-substituted analogues and lengths of alkyl chains were different for each type. The presence of a methyl group in the alpha-position of the chain increased substantially the inhibitory constant for the A type. Halogen atoms as substituents on carbon-6 increased inhibitory constants for both types of the enzyme in the sequence: I less than Br less than Cl less than F. These findings are consistent with the existence of hydrophobic binding sites of restricted dimensions in both types of the enzyme.

Release of vesicular noradrenaline in the rat tail artery induced by cocaine.

The effects of cocaine on overflows of endogenous noradrenaline and DOPEG from isolated rat tail arteries were examined. 1. Both overflows increased progressively with increasing concentration of cocaine, while the (NA overflow)/(DOPEG overflow) ratio first increased and then decreased. The changes in the overflows induced by cocaine (0.1 mmol/l) appeared reversible. 2. Exposure of the tissue for 30 min to cocaine, 1 mmol/l, resulted in a significant decrease in the proportion of storage vesicles containing electron-dense cores. 3. The changes in overflows of noradrenaline and DOPEG induced by cocaine (0.1 mmol/l were unaffected by the presence of desipramine (0.1 mumol/l) or removal of extracellular Ca2+. The effect of cocaine on the overflow of noradrenaline was potentiated by prior inhibition of MAO with clorgyline. 4. Exposure of segments to a Ca2+-free, high K, low Na incubation medium was accompanied by increased overflow of noradrenaline. Cocaine (0.1 mmol/l) reduced the overflow of noradrenaline to about a half, and substantially increased the overflow of DOPEG. 5. The increase in the overflow of DOPEG from segments bathed in HEPES-buffered solutions, the pH of which ranged from 6.80 to 7.38, was approximately proportional to the calculated concentration of unprotonated (uncharged) cocaine. 6. Quantitatively similar changes in the overflows were observed when norcocaine was substituted for cocaine. Ecgonine methyl ester was much less potent than cocaine, and O-benzoyl ecgonine was ineffective. 7. The small increases in the overflow of noradrenaline observed at relatively low concentration (less than 30 mumol/l) of cocaine can be attributed primarily to inhibition of reuptake of the released transmitter by the cocaine- and desipramine-sensitive amine carrier.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride acts as an alpha-adrenergic antagonist in the isolated rat tail artery.

In the final concentration of 100 microM, amiloride increased substantially the overflow of endogenous noradrenaline and decreased that of 3,4-dihydroxyphenylethylene glycol from the rat tail artery into Krebs solution supplemented with 10 microM veratridine. The overflow of the amine into a 120 mM-K version of Krebs solution was unaffected by amiloride, while that of the glycol was reduced. Abolition of the contractile response to 10 microM veratridine by 2 microM phentolamine indicated that the response was due to release of endogenous noradrenaline. Addition of amiloride in the final concentrations of 10 and 100 microM caused relaxation of strips contracted by the alkaloid. The dose-response relations for exogenous noradrenaline measured in the absence or presence of 50 microM amiloride indicated that the drug acted as a reversible competitive alpha-adrenergic antagonist. The phentolamine-resistant component of the contractile response to the 120 mM-K solution was unaffected by 100 microM amiloride. Although the exact site of action of amiloride remains to be determined, it can be concluded that amiloride inhibits adrenergic transmission at a postsynaptic site at a step preceding elevation of myoplasmic Ca2+.

Inhibition of monoamine oxidase by amiloride.

Amiloride was found to lower the overflow of 3,4-dihydroxyphenylethylene glycol from isolated rat tail artery. The overflow was reduced to about 50% in the presence of 10(-5) M concentration of the drug. Reduced overflows of the glycol were observed also under conditions when the nonexocytotic release of endogenous noradrenaline was enhanced by tyramine, reserpine, or by the elevation of external K+ in the absence of extracellular Ca2+. They were accompanied by increased overflows of the amine. Amiloride inhibited monoamine oxidase activity (E.C. 1.4.3.4) of the A form in rat brain homogenate by acting as a competitive inhibitor.

Release of 3,4-dihydroxyphenylglycol from the rat tail artery induced by veratridine.

The overflow of endogenous 3,4-dihydroxyphenylethylene glycol and noradrenaline from the isolated rat tail artery was measured by high pressure liquid chromatography with amperometric detection. Veratridine, but not elevated external K, caused a significant increase in the overflow of the glycol under conditions when formation of the latter from released noradrenaline was prevented by blockade of neuronal uptake with desipramine. The results support the hypothesis that, in addition to evoking exocytotic release of noradrenaline into Ca-containing solution, the alkaloid also increases leakage of noradrenaline from storage vesicles. The latter process seems responsible for veratridine-induced release of noradrenaline into Ca2+-free solution.

Release of endogenous noradrenaline from the rat tail artery induced by veratridine.

The overflow of endogenous noradrenaline from the isolated rat tail artery was measured using a radioenzymatic method. Veratridine increased the overflow markedly even in the absence of external Ca2+. Modifications of the effect of 5 microM veratridine by tetrodotoxin, pargyline, cocaine, lidocaine, and phenoxybenzamine indicated that interaction of the alkaloid with the sodium channel induces primarily nonexocytotic release of noradrenaline. Ouabain inhibited the effect of 5 microM veratridine on the overflow into Ca2+-free solution, but it greatly potentiated the effect if external Ca2+ was present. Potentiation of the effect of veratridine in Ca2+-free solution by cyanide was ouabain sensitive. These observations are consistent with the hypothesis that, at low concentrations of veratridine such as 5 microM, the initial cause of enhanced release of noradrenaline may be a consequence of increased activity of the sodium pump, namely increased consumption of ATP by the pump.

Release of noradrenaline from the rat tail artery induced by inhibition of the sodium pump in calcium-free solution.

The release of noradrenaline from the isolated rat tail artery into Ca2+- and K-free, 1 mM ouabain containing solution was measured by means of radioenzymatic method. The rate of noradrenaline release increased gradually reaching a maximum of measured by means of radioenzymatic method. The rate of noradrenaline release increased gradually reaching a maximum of ca. 2.30 nmol.g-1.h-1 after 100 min. The enhancement of noradrenaline release could be inhibited by cocaine and phenoxybenzamine but not by desipramine. The rate of noradrenaline release could be approximately doubled by prior inhibition of monoamine oxidase with pargyline. The release was accompanied by a decline in the proportion of storage vesicles containing an electron-dense core. These observations indicate that, in the absence of external Ca2+, inhibition of the sodium pump causes nonexocytotic release of endogenous noradrenaline.

The transient contractile response of the isolated rat tail artery to inhibition of the sodium pump.

The isolated rat tail artery responds to incubation in 1 mM ouabain containing, K-free physiological salt solution by transient contraction which is due to release of endogenous catecholamines. The eventual decline in active tension cannot be attributed solely to the decreasing rate of release of endogenous catecholamines, for the latter remains quite high even after the preparation has relaxed completely. It seems, therefore, that the relaxation is due also to the substantial decrease in the responsiveness of smooth muscle cells to (-)-norepinephrine that accompanies dissipation of the transmembrane gradients of Na+ and K+.

Some effects of the ionophore X-537A on the isolated rat tail artery.

The effects of micromolar concentrations of the ionophore X-537A (RO 2-2985) were studied using isolated preparations of the rat tail artery. The ionophore causes complete release of catecholamines from adrenergic nerves, which is the sole cause of the transient contractile response. The amines are released by a nonexocytotic process which seems to be related to the ability of X-537A to act as an efficient transmembrane carrier of Na+, k+, and H+. The ionophore also causes an almost complete and irreversible loss of the cocaine-sensitive component of metaraminol uptake by the tissue. X-537A dissipates the transmembrane concentration gradients of Na and K in the smooth muscle component of the preparation. This effect is unrelated to the release of endogenous catecholamines, and it can also be observed after the Na pump has been inhibited with ouabain. It is fully reversible, though not readily, and it can be induced repeatedly. In catecholamine-depleted strips, X-537A dissipates the transmembrane Na+ and K+ gradients without causing any change in tension. Stimulation of the rate of O2 consumption by X-537A in catecholamine-depleted tissue is reversible, and it is unaffected by ouabain and (or) removal of external Ca2+.

Pyruvate dehydrogenase activity in liver and brown fat of the developing rat.

The total activity of pyruvate dehydrogenase (EC 1.2.4.1) and the fraction of the enzyme in the active form were assayed in brown fat and liver throughout the development of the rat. In brown adipose tissue, the total activity increased until the late suckling period. After weaning, a decrease was noted. The fraction of the enzyme in the active form did not increase until after 10 days of age, reached its highest level in the late suckling period and remained at this level after weaning. Pyruvate dehydrogenase in liver decreased in both total activity and percentage activity in the early neonatal period. Both parameters increased after this period, reaching their highest levels in the late suckling period. In both fetal liver and fetal brown fat, the total activity of pyruvate dehydrogenase was increased by in vitro incubation with insulin.

Regulation of the cell magnesium in vascular smooth muscle.

1. The relation between [Mg](o) and the Mg content of the rat tail artery incubated in Ca-free solutions was studied.2. Variation of [Mg](o) in Ca-free Krebs solution between 0.6 and 2.4 mm did not affect the cell Mg. In ATP-depleted arteries, a sizeable fraction of the cell Mg was found to be proportional to [Mg](o).3. An even larger fraction of the cell Mg became proportional to [Mg](o) in metabolically active arteries in which the transmembrane gradient of Na had been dissipated as a result of inhibition of the Na pump. In contrast to the extracellular Mg, the fraction responded to a change in [Mg](o) at a very slow rate. The size of the fraction was reflected in net uptake of Mg if [Mg](o) was higher than 1.2 mm. The rate of the uptake was lowered markedly by external Ca. All the Mg taken up by the cells was extruded again after restoration of the Na gradient.4. The uptake of Mg in Ca-free, ouabain-containing solution took place even if the concomitant swelling of cells was prevented by substitution of isethionate for external Cl.5. Under steady-state conditions and at const. [Mg](o) and [Na](o), [Mg](i) increased with increasing [Na](i).6. The results are consistent with the hypothesis that the outwardly directed Mg pump in rat vascular smooth muscle utilizes the energy released in the course of spontaneous influx of Na.

Distribution of magnesium in the arterial wall.

1. The Mg, Ca, Na and K content were determined in the rat tail artery in vitro under various experimental conditions.2. The arterial Mg could be varied selectively at a constant [Mg](o) by altering the content of intracellular compounds capable of forming complexes with Mg(2+) such as nucleoside-5'-triphosphates or citrate.3. The release of tissue Mg during incubation in Mg-free solutions was found to consist of a fast and a slow component. The size of the fast component was dependent on the original [Mg](o). The size of the slow component was dependent on the content of intracellular compounds forming complexes with Mg(2+). The rate of efflux of the slow component could be increased by increasing [Mg(2+)](i), by removing external Ca, or by making the artery metabolically inactive.4. The results seem to indicate that [Mg(2+)](i) in metabolically active vascular smooth muscle cells is maintained at a level of ca. 10(-4)M.