Roles Played by the Na+/Ca2+ Exchanger and Hypothermia in the Prevention of Ischemia-Induced Carrier-Mediated Efflux of Catecholamines into the Extracellular Space: Implications for Stroke Therapy.

The release of [3H]dopamine ([3H]DA) and [3H]noradrenaline ([3H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca2+ further increased the release rates of [3H]DA and [3H]NA induced by ischemic conditions. This finding indicated that the Na+/Ca2+ exchanger (NCX), working in reverse in the absence of extracellular Ca2+, fails to trigger the influx of Ca2+ in exchange for Na+ and fails to counteract ischemia by further increasing the intracellular Na+ concentration ([Na+]i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca2+ was removed. Hypothermia inhibited the excessive release of [3H]DA in response to ischemia, even in the absence of Ca2+. These findings further indicate that the NCX plays an important role in maintaining a high [Na+]i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [3H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H2O2), a mediator of ischemic brain injury enhanced the striatal resting release of [3H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca2+]o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na+/Cl--dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects.

[Electrogenic Na+/Ca2+-exchange regulation by alpha2-receptor in peripheral sympathetic nerve]. / Elektrogén Na+/Ca2+-csere alpha2-receptor regulációja perifériás szimpatikus idegen.

Electrical depolarisation-(2 Hz, 1 ms)-induced [3H]noradrenaline ([3H]NA) release was measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(-5)M; corticosterone, 5 x 10(-5)M) and after blocking the MAO-enzyme by pargyline (1,2 x 10(-4)M). Substitution of most of the external Na+ by Li+ (113 mM; [Na+]0: 25 mM) slightly potentiated the stimulation-induced release of [3H]NA in a tetrodotoxin (TTX, 10(-7)M) sensitive manner. The reverse Na+/Ca2+-exchange inhibitor KB-R7943 (3 x 10(-5)M) failed to inhibit the stimulation-evoked release of [3H]NA, but increased the resting outflow of neurotransmitter. The 'N-type' voltage-sensitive Ca2+-channel (VSCC) blocker omega-conotoxin (omega-CgTx) GVIA (10(-8)M) significantly and irreversibly inhibited the release of [3H]NA on stimulation (approximately 60-70%). The 'residual release' of NA was abolished either by TTX or by reducing external Ca2+ from 2,5 to 0,25 mM. The 'residual release' of NA was also blocked by the non-selective VSCC-blocker neomycin (3 x 10(-3)M). Direct correlation was obtained between the extent of VSCC-inhibition and the transmitter release enhancing-effect of presynaptic alpha2-receptor blocker yohimbine (3 x 10(-7)M). When the release of [3H]NA was blocked by omega-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na+-pump by removal of K+ from the external medium increased both the resting and the stimulation-evoked release of [3H]NA in the absence of functioning VSCCs (i.e. in the presence of neomycin and after omega-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca2+-removal (+1 mM EGTA). Similarly, external Li+ (113 mM) or the reverse Na+/Ca2+ exchange blocker KB-R7943 (3 x 10(-5)M) significantly inhibited the nerve-evoked release of NA in 'K+-free' solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions, in which the Na+-pump was inhibited in the absence of functioning VSCCs, yohimbine (3 x 10(-7)M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10(-6)M), an agonist of presynaptic alpha2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li+-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs, brief depolarising pulses may reverse Na+/Ca2+-exchange and release neurotransmitter in Na+-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na+/Ca2+-exchange may also be inhibited by presynaptic alpha2-receptor activation.

Pre-junctional stimulatory and post-junctional inhibitory effects of pinacidil in the isolated main pulmonary artery of the rabbit under different experimental conditions.

Low frequency (2Hz) nerve-stimulation induced [3H]noradrenaline ([3H]NA) release has been measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(-5)M; corticosterone, 5 x 10(-5), with parallel measurements of post-junctional contractile responses. The K+ ATP-channel opener pinacidil (10(-6)-10(-4)M), slightly potentiated the nerve-evoked release of [3H]NA which failed to show close concentration-dependency. Large concentration of pinacidil (10(-4)M) increased the ratio of [3H]NA release from 0.99 +/- 0.02 to 1.29 +/- 0.05; P < 0.0005). On the other hand, pinacidil inhibited the nerve-evoked contractions in a concentration-dependent manner. 10(-4)M caused nearly 70% inhibition of contractile response. The pre- and post-junctional effects of pinacidil were studied under the following experimental conditions: (1) exogenously applied I-NA; (2) excess K+; (3) 'L-type' Ca(2+)-channel activation (BAY K 8644); (4) K(+)-channel inhibition (4-AP); and (5) Na(+)-pump inhibition/reactivation. Pinacidil (10(-4)M) retained its marginal NA-release stimulatory effect in all cases. However, pinacidil inhibited the contraction of smooth muscle, although to a different extent, in all of the experimental conditions used in our study.

Dependence of release of [3H]noradrenaline from rabbit pulmonary artery on internal sodium.

1. [3H]Noradrenaline ([3H]NA) release from the isolated main pulmonary artery of the rabbit has been measured in the presence of uptake blockers (cocaine, 3 x 10(-5) M, and corticosterone, 5 x 10(-5) M) and after blocking the monoamine oxidase enzyme by pargyline (1.2 x 10(-4) M). 2. In normal Krebs solution Mn2+ (2 mM) significantly inhibited both [3H]NA release (approximately 80%; P < 0.001) and the contraction following 2 Hz field stimulation. 3. In Ca(2+)-free, EGTA (1 mM)-containing solution, the Na+ pump was inhibited by removal of K+ from the external medium. In Na+ pump-inhibited arteries, 2 mM Mn2+ (free Mn2+, 1 mM) increased the spontaneous release of [3H]NA according to the time of Na+ loading. TTX (10(-7) M) did not inhibit significantly the Mn(2+)-induced [3H]NA release from Na(+)-loaded preparations (percentage inhibition, approximately 24; P > 0.30). 4. Without Na+ loading (Ca2+ free, EGTA alone), Mn2+ failed to promote 3H release from arteries. 5. With constant Na+ loading (120 min 'K(+)-free' perfusion in Ca(2+)-free, 1 mM EGTA-containing solution), the release of 3H was also directly dependent on free Mn2+ concentration (0.2, 0.6 and 1 mM). 6. The Mn2+ (2 mM; free Mn2+, 1 mM)-induced 3H release from Na(+)-loaded nerves (120 min 'K(+)-free', perfusion) was further enhanced, when external Na+ was simultaneously reduced from 139.2 to 26.2 mM (choline+ or sucrose substitution). 7. Diphenylhydantoin (DPH, 10(-4) M) significantly reduced the Mn(2+)-evoked 3H release (approximately 44%; P < 0.02) when it was present during 'K(+)-free', perfusion. 8. Mn2+ was ineffective in releasing 3H if the Na+ pump was previously reactivated by readmission of K+ to Na(+)-loaded arteries. 9. It is concluded that in Ca(2+)-free solution Mn2+ releases neurotransmitter in a manner which depends on the degree of loading with internal Na+. The results suggest this depends at least partly on a block of Ca2+ efflux.

Depolarization promotes caffeine induced [3H]-noradrenaline release in calcium-free solution from peripheral sympathetic nerves.

The transmitter releasing action of caffeine was studied in the absence of extracellular Ca2+ from the peripheral sympathetic nerves of the rabbit main pulmonary artery. Caffeine (10 mM) increased the release of [3H]-noradrenaline moderately, but not significantly in Ca2(+)-free (+1 mM EGTA) Krebs solution. When peripheral nerve endings/varicosities were depolarized by elevating extracellular K+ to 47.2 mM and 70.8 mM in Ca2(+)-free solution, the transmitter releasing effect of 10 mM caffeine became significant. Ca2+ removal itself transiently increased the [3H]-noradrenaline outflow. In the individual experiments the amount of the caffeine evoked transmitter release at 47.2 mM and 70.8 mM K(+)-depolarization was inversely correlated to the release evoked by Ca2(+)-removal. Our results suggest that caffeine-sensitive calcium stores are present in peripheral nerve terminals of rabbit pulmonary artery, and part of the caffeine sensitive calcium stores may discharge during Ca2(+)-removal from the extracellular solution.

Catecholamine release from bovine chromaffin cells: the role of sodium-calcium exchange in ouabain-evoked release.

Spontaneous catecholamine (CA) release from bovine chromaffin cells maintained in primary tissue culture has been measured after pre-loading the cells with [3H]noradrenaline. Ouabain inhibited 86Rb+ uptake and increased 3H release in a concentration-dependent manner during a 60 min incubation period. Low external Na+ (5 mM: Li+ substitution) also increased 3H release. Whereas the 3H-releasing action of ouabain was maintained, the Li(+)-evoked release decreased with time. The effects of both ouabain and low Na+ solution on 3H release were completely inhibited by removal of Ca2+ from the external medium even though in Ca2(+)-free solution ouabain further inhibited 86Rb+ uptake into the cells. Readmission of Ca2+ to Na(+)-loaded cells (10-4 M-ouabain in Ca2(+)-free-1 mM-EGTA solution for 60 min) markedly increased the release of 3H. In the additional presence of diphenylhydantoin (DPH, 10-4 M) 3H release was significantly less on Ca2+ readmission. The 3H release from Na(+)-loaded cells was proportional to the concentration of Ca2+ readmitted. The 3H release was further increased from Na(+)-loaded cells in response to Ca2+ readmission when [Na+]o was lowered from 149 to 5 mM (Li+, choline+, Tris+ or sucrose substitution) though Li+ was less effective than the other Na+ substitutes. Potassium removal from the external medium significantly inhibited the 3H release evoked by Ca2+ readmission to Na(+)-loaded cells, even when [Ca2+]o was greater than normal (7.5 mM) or if Ca2+ was readmitted in low [Na+]o solution. Rb+, Cs+ or Li+ could substitute for K+ with the order of potency: Rb+ greater than or equal to K+ greater than Cs+ greater than Li+. A slight increase of external K+ (10.8 mM) potentiated the 3H release from Na(+)-loaded cells on Ca2+ readmission, but a higher concentration of K+ (149.4 mM) had the opposite action. The data is consistent with the hypothesis that ouabain-evoked CA release from bovine chromaffin cells is, in part, a consequence of an internal Na(+)-dependent Ca2+ influx. The evidence also suggests that there is Na(+)-Ca2+ competition at the external arm of the exchanger together with a monovalent cation activation site.

Multiple effects of cocaine upon evoked secretion in bovine adrenal medullary chromaffin cells. Additional insight into the mechanism of action of cardiac glycosides.

Experiments to determine the effects of the catecholamine neuronal uptake blockers cocaine and desipramine, and of the cardiac glycoside, ouabain, upon 3H-(noradrenaline) efflux have been performed with bovine adrenal medullary chromaffin cells in tissue culture. Both cocaine and desipramine reduced 3H-noradrenaline uptake into chromaffin cells. Inhibitable uptake was 80% of total accumulation over 60 min; this degree of inhibition was produced by cocaine (30 mumol/l) or desipramine (1 mumol/l). Cocaine (30 mumol/l) had no effect upon spontaneous 3H-efflux measured over 60 min, but reduced that evoked over the same period by carbachol (EC50), veratridine (EC50) and by ouabain (100 mumol/l). Cocaine did not reduce that efflux evoked by raised levels of K+ (28 mmol/l; EC50). Desipramine (1 mumol/l), like cocaine, had no effect upon spontaneous efflux of 3H, but reduced that efflux evoked by carbachol, veratridine and ouabain. Tetrodotoxin (TTX) inhibited veratridine-evoked 3H efflux (IC50 0.2 mumol/l). The degree of inhibition caused by TTX (0.2 mumol/l) was not increased by cocaine (30 mumol/l). TTX also inhibited ouabain-evoked 3H efflux: this was reduced by 55% by a concentration of TTX (1 mumol/l) sufficient to virtually abolish veratridine-evoked efflux. Cocaine (30 mumol/l) in the presence of TTX (1 mumol/l) did not further inhibit ouabain-evoked efflux. Cocaine (30 mumol/l) did not alter 86Rb+ uptake into chromaffin cells, nor did it alter that inhibition of 86Rb+ uptake produced by ouabain (100 mumol/l) indicating that cocaine has no effect upon Na,K-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

A-23187 evoked transmitter release from rabbit pulmonary artery and its inhibition by reactivation of sodium-pump.

1. The spontaneous [3H]-release has been measured from the isolated main pulmonary artery of the rabbit preloaded with [3H]noradrenaline in the presence of uptake blockers (cocaine, 3 x 10(-5) M; corticosterone, 5 x 10(-5) M). 2. The Ca-ionophore A-23187 (3 x 10(-7)-3 x 10(-5) M) increased the outflow of [3H] by a concentration dependent manner. 3. Inhibition of Na+-pump by removal of K+ from the external medium also increased the release of labelled noradrenaline. 4. In the absence of external K+, the applied A-23187 (3 x 10(-6) M; EC50) further increased the release of [3H]. 5. Reactivation of Na+-pump by readmission of K+ (5.9 mM) to the external medium abolished the [3H]-release which had previously been increased in "K+-free" solution. 6. The reactivated Na+-pump significantly inhibited the transmitter releasing action of A-23187. 7. This latter was antagonized by an increase of external Ca2+ (7.5 mM). 8. It is concluded that the reactivated Na+-pump caused re-establishment of Na+-gradient is capable to counteract the Ca-ionophore facilitated Ca2+-influx and release from internal stores, which can be antagonized by excess Ca2+.

Sodium-azide-evoked noradrenaline and catecholamine release from peripheral sympathetic nerves and chromaffin cells.

1. The spontaneous release of [3H]noradrenaline [( 3H]NA) has been measured from rabbit pulmonary arteries and bovine chromaffin cells in the presence of neuronal uptake blocker cocaine (3 x 10(-5) M). 2. The Na+-pump inhibitor sodium-azide (NaN3, 2mM) produced a moderate increase of [3H]NA release from both preparations and relaxed the arteries. The [3H]releasing action of NaN3 was accompanied by a 30% inhibition of 86Rb-uptake into chromaffin cells. 3. In both preparations, ouabain (10(-4) M) markedly increased the release of [3H], contracted the arteries and inhibited the 86Rb-uptake of chromaffin cells by about 75%. A combined application of NaN3 and ouabain produced a similar inhibition of 86Rb-uptake of chromaffin cells and failed to increase further the release of [3H] in comparison to that found in response to ouabain alone. 4. Removal of K+ from the external medium increased both the release of [3H]NA and the tone of pulmonary arteries. NaN3 further increased the transmitter release in "K+-free" solution but relaxed the muscle. In the absence of external K+ and in the presence of azide, ouabain further enhanced the transmitter release but failed to produce significant contraction. 5. Reactivation of the Na+-pump by readmission of K+ (5.9 mM) to the external medium abolished the transmitter releasing action of NaN3 in arteries. 6. It is concluded that in peripheral sympathetic nerves and chromaffin cells, NaN3 inhibits the Na+-pump producing NA and CA release respectively and in nerves even if NA release had already been increased by K+-removal.(ABSTRACT TRUNCATED AT 250 WORDS)

Augmentation of the indirect sympathomimetic action of tyramine by cardioactive steroids is a consequence of elevated intracellular sodium.

The augmentation by the cardioactive steroid acetylstrophanthidin of neurotransmitter release evoked by tyramine, and the dependence of the augmentation upon Na+, has been investigated in dog saphenous vein rings in which monoamine oxidase activity and uptake2 had been inhibited with pargyline and corticosterone respectively. High extracellular Na+ (Nao; 263 mmol/l) reduced basal efflux of 3H-compounds from the rings and also reduced tyramine-evoked efflux. Low Nao (25 mmol/l) increased basal efflux of 3H but reduced tyramine-evoked efflux. The increment in basal 3H-efflux caused by low Nao was cocaine-sensitive. A presumed increase in intracellular Na+ (Nai), produced by preincubating rings with acetylstrophanthidin in normal (143 mmol/l) or high Nao, augmented 3H-efflux evoked by subsequent incubation with tyramine in normal Nao. Pre-incubating rings with acetylstrophanthidin in low Nao, conditions which would not be expected to increase Nai, did not cause augmentation of the subsequent tyramine-evoked 3H-efflux. An increase in Nai, produced either as above or by pre-incubating rings in high Nao alone, reduced subsequent neuronal 14C-tyramine uptake. Low Nao present only during incubation reduced neuronal 14C-tyramine uptake, but high Nao present only during incubation did not increase neuronal 14C-tyramine uptake from that measured in normal Nao. The data are consistent with the following hypotheses: that tyramine uptake is dependent upon the prevailing inwardly directed Na+-gradient, that consequent noradrenaline efflux is Na+-gradient dependent and that the enhancement by acetylstrophanthidin of tyramine-evoked 3H-efflux is a consequence of the raised Nai caused by Na+,K+-ATPase inhibition.

[3H]noradrenaline release from rabbit pulmonary artery: sodium-pump-dependent sodium-calcium exchange.

1. The release of [3H]noradrenaline ([3H]NA) from the isolated main pulmonary artery of the rabbit has been measured in the presence of neuronal (cocaine, 3 X 10(-5) M) and extraneuronal (corticosterone, 5 X 10(-5) M) uptake blockers. 2. K+ removal from the external medium increased the release of [3H]NA, an action transiently inhibited by Ca2+-free (+1 mM-EGTA) solution, i.e. after Ca2+ removal transmitter release was first abolished and then started to increase again after a delay lasting about 90-120 min. 3. Ca2+ readmission to arteries which had been kept in Ca2+- and 'K+-free' solution, markedly increased the [3H]NA release. The rate of transmitter release was dependent on the preceding perfusion period with 'K+-free' solution, being greater for longer exposure times. 4. When Ca2+ and K+ were readmitted together to K+-depleted and Na+-enriched preparations, the release of [3H]NA transiently increased. 5. If K+ was readmitted first, the subsequently applied Ca2+ was ineffective in producing transmitter release. 6. Different alkali metal ions (Rb+, Cs+ or Li+) were also readmitted as K+ substitutes together with Ca2+. In all cases the release of neurotransmitter transiently increased; however, the rate of release was dependent on the monovalent cation used. Thus, Rb+ ions were as effective as, Cs+ about one-third as effective as, and Li+ about one-fifth as effective as K+ in activating the Na+ pump. 7. It is concluded that in the absence of external Ca2+, and in response to Na+-pump inhibition, the release of Ca2+ from internal stores is responsible for the NA release observed. On readmission of Ca2+ the rate of transmitter release is dependent on the Na+ previously gained inside. Furthermore, the activity of the Na+ pump determines the rate of transmitter release through the Na-Ca exchange mechanism.

Transmitter releasing action of selegiline ((-)-deprenyl) from peripheral sympathetic nerves under different experimental conditions.

A high concentration of selegiline ((-)-deprenyl; 10(-4) M) potentiated low frequency (2 Hz) nerve stimulation-evoked release of [3H]noradrenaline from the isolated main pulmonary artery of the rabbit in the presence of neuronal (cocaine, 3 X 10(-5) M) and extraneuronal (corticosterone, 5 X 10(-5) M) uptake blockers, and inhibited the postsynaptic response. The transmitter-releasing action of 10(-4) M selegiline was inhibited by a moderate increase of external K+ (23.6 mM). Excess K+ by itself abolished the nerve-evoked release of [3H]noradrenaline but did not increase the resting outflow of radioactivity. Excess Ca2+ (7.5 mM) increased the stimulation-evoked transmitter release. In the presence of excess Ca2+, selegiline (10(-4) M) was effective in increasing the [3H]noradrenaline release in response to nerve-stimulation. Excess Ca2+ partly antagonized the postsynaptic inhibitory action of selegiline. In Ca2+-free, 1 mM EGTA-containing Krebs solution both the nerve-evoked 3H release and the transmitter releasing action of selegiline were abolished in agreement with the 'Ca-hypothesis'. The voltage-dependent K+-channel blocker, 4-aminopyridine (10(-5) M), increased the nerve-stimulation-evoked release of tritium from arteries. If selegiline was also present in the perfusion medium the nerve-evoked transmitter release further increased. 4-Aminopyridine completely antagonized the inhibitory action of selegiline on the postsynaptic contraction.

The role of internal calcium-stores in the termination of noradrenaline release during sodium-pump reactivation in peripheral nerves.

The release of [(3)H]noradrenaline has been measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(?5) M; corticosterone, 5 x 10(?5) M). K(+)-removal from Krebs solution increased the release of [(3)H]NA even in the absence of external calcium. K(+)-readmission to the external medium terminated the transmitter release. The rate of recovery was much faster than the rate of rise of transmitter release both in the presence and absence of external Ca(2+). The main aim of the present study was to examine whether the reactivated Na(+)-pump per se or the Na(+)-gradient dependent Ca(2+)-extrusion and -uptake into the internal calcium-stores is responsible for the termination of transmitter release. In Ca(2+)-free, 1 mM EGTA containing solution the "K-free" stimulated [(3)H]NA release was further enhanced by calcium-store releasers (CCCP, 10(?5) M; A23187, 3 x 10(?6) M). External K(+)-readmission was effective in abolishing the transmitter release evoked by CCCP and A23187. However when veratrine (10(?4) g/ml) was also present the readmitted K(+) was ineffective in inhibiting the [(3)H]NA release although the Na(+)-pump was fully activated by the elevated level of Na(+) inside. The results suggest that the Na(+)-gradient dependent Ca(2+)-metabolism of the nerves is responsible for the abolition of transmitter release rather than the Na(+)-pump reactivation per se.

The action of trelibet, a new antidepressive agent on [3H]noradrenaline release from rabbit pulmonary artery.

Trelibet, a new antidepressant, used at 10(-7)-10(-4) M failed to affect the [3H]noradrenaline ([3H]NA) release evoked from the isolated main pulmonary artery of the rabbit low frequency (2 Hz) nerve stimulation whether the neuronal uptake inhibitor cocaine (3 X 10(-5) M) was present or not. Its metabolite (EGYT-2760) however, potentiated the nerve-evoked release of [3H]NA. In the absence of cocaine both the resting and the stimulation-evoked release of 3H increased in response to EGYT-2760. These effect were accompanied by muscle contraction. The EGYT-2760-potentiated transmitter release was inhibited either by exogenously applied 1-noradrenaline (10(-6) M) or clonidine (10(-6) M), preferential agonists of presynaptic alpha 2-adrenoceptors. The 1-noradrenaline-induced inhibition of transmitter release potentiated by EGYT-2760 was antagonized by 3 X 10(-7) M yohimbine, a preferential alpha 2-adrenoceptor inhibitor. In the absence of cocaine, Ca2+ removal from the external medium failed to affect the 3H outflow-increasing effect of EGYT-2760 but abolished the nerve-evoked release-potentiating action of this compound. It is concluded that the metabolite of trelibet exerts a 'yohimbine-like' action, as well as a 'tyramine-like' effect in peripheral sympathetic nerve fibres.

[3H]noradrenaline-releasing action of vinpocetine in the isolated main pulmonary artery of the rabbit.

Vinpocetine (10(-6)-3 X 10(-5) M) increased both the resting and the nerve stimulation-evoked release of [3H]noradrenaline from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 X 10(-5) M; corticosterone, 5 X 10(-5) M), and inhibited the nerve stimulation-evoked postsynaptic response. The resting transmitter releasing action of vinpocetine increased in the absence of cocaine. Exogenously applied (-)noradrenaline [(-)NA] (10(-6) M) or clonidine (10(-6) M) inhibited the vinpocetine (3 X 10(-5) M)-potentiated [3H]NA release and contracted the circular muscle. The clonidine-induced contraction was abolished by 10(-7) M prazosin. The inhibitory action of (-)-NA on vinpocetine-potentiated [3H]NA release was partly antagonized by 3 X 10(-7) M yohimbine, a preferential alpha 2-adrenoceptor blocker. In Ca-free Krebs solution containing 1 mM EGTA the neurotransmitter releasing action of vinpocetine was abolished, however, its stimulating action on the resting [3H]NA outflow was not changed. In Na-pump-inhibited arteries (K-free solution), where both the resting and the nerve stimulation-evoked release of neurotransmitter had already been increased, vinpocetine further enhanced the nerve stimulation-evoked release of [3H]NA. It is concluded that vinpocetine may have alpha 2- and alpha 1-adrenoceptor blocking action, as well as a tyramine-like effect. The presynaptic neurotransmitter releasing action of vinpocetine is presumably the consequence of its inhibitory action on the Ca-pump which is suggested by the finding that in K-free solution vinpocetine was able to enhance further the release of neurotransmitter.

Ouabain-evoked [3H]noradrenaline release from the rabbit pulmonary artery in calcium-free solution.

[3H]noradrenaline ([3H]NA) release from the isolated main pulmonary artery of the rabbit has been measured in the presence of neuronal (cocaine, 3 X 10(-5) M) and extraneuronal (corticosterone, 5 X 10(-5) M) uptake blockers. 10(-4) M ouabain significantly increased the [3H]NA release in normal external ionic environments after an initial delay (20-30 min). Excess K (23.6 mM) failed to affect the resting [3H]NA outflow, significantly inhibited the ouabain-stimulated [3H]NA release and shortened the initial delay by about 10-20 min. Higher concentration of K (47 X 2 mM) enhanced the outflow of [3H]NA. In the absence of external Ca and in the presence of 1 mM EGTA, 23.6 mM-K failed to exert an inhibitory action on ouabain evoked transmitter release. Higher concentrations of K, however, significantly inhibited the [3H]NA-releasing effect of ouabain without changing the resting outflow of labelled neurotransmitter. The initial delay of ouabain-evoked [3H]NA release was shortened by increasing the concentration of K. Total substitution of external Na by K (143.3 mM) increased the [3H]NA release in the absence of external Ca. After the Na gradient was re-established in Ca-free solution the release of [3H]NA was terminated. Under these conditions the NA-releasing action of ouabain was dependent on the preceding perfusing period in Na-free solution, being smaller if longer exposure time was used. When external Na was substituted by Li (137.4 mM) in Ca-free solution the [3H]NA release was dramatically increased. After Na readmission ouabain was ineffective in producing transmitter release. It is suggested that in the main pulmonary artery of the rabbit, when the electrochemical gradient of Ca is reversed, ouabain is effective in producing transmitter release if the internally stored Ca has not been completely lost. Since in Ca-free solution the ouabain-evoked [3H]NA release can be inhibited by external K it seems that the transmitter release observed is due to a Na-dependent release of intracellular Ca rather than the penetration of ouabain into the cell followed by direct inhibition of the active transport of internal store membranes.