Protection by phentolamine against the effects of phenoxybenzamine on transmitter release elicited by nerve stimulation in the perfused cat heart

1 The effects of cocaine, phentolamine and phenoxybenzamine on neuronal uptake of [(3)H]-noradrenaline and on (3)H-transmitter and noradrenaline overflow elicited by nerve stimulation were determined in the perfused heart of the cat.2 During perfusion with cocaine 3.4 x 10(-7)M, there was a 2-fold increase in transmitter overflow while neuronal uptake of [(3)H]-noradrenaline was inhibited by 31.3 +/- 2.1%.3 After exposure to phenoxybenzamine 8.7 x 10(-7)M for 20 min and washing with drug-free solution for 165 min there was an 8-fold increase in transmitter overflow during nerve stimulation. Under these conditions neuronal uptake of [(3)H]-noradrenaline was inhibited by only 17.5 +/- 5.4%.4 There was no significant change in transmitter overflow or in neuronal uptake of [(3)H]-noradrenaline, 155 min after a 30 min exposure to phentolamine (3.2 x 10(-5)M).5 Perfusion with phentolamine (3.2 x 10(-5)M) before and during exposure to phenoxybenzamine (8.7 x 10(-7)M), prevented the increase in transmitter overflow observed after perfusion with phenoxybenzamine alone.6 Protection by phentolamine against the effects of phenoxybenzamine supports the view that the effects on transmitter release obtained after perfusion with phenoxybenzamine are due to the blockade of presynaptic alpha-adrenoceptors which regulate transmitter release through a negative feed-back mechanism.     

Role of the α-adrenoceptor in regulating noradrenaline overflow by nerve stimulation

1. A study of the actions of phenoxybenzamine on transmitter overflow, neuronal and extraneuronal uptake of noradrenaline and in causing alpha-adrenoceptor blockade was carried out using the isolated cat nictitating membrane preparation.2. Phenoxybenzamine increased transmitter overflow elicited by nerve-stimulation at 10 Hz in a concentration dependent manner in the range 10(-8) to 10(-5) g/ml.3. Neuronal uptake of [(3)H]-noradrenaline was not inhibited by concentrations lower than 10(-6) g/ml of phenoxybenzamine. With 10(-7) g/ml of phenoxybenzamine a significant increase in transmitter overflow was obtained, although neuronal uptake of noradrenaline was not affected. Higher concentrations of phenoxybenzamine (10(-6) and 10(-5) g/ml) inhibited the neuronal uptake of noradrenaline and further increased transmitter overflow.4. Extraneuronal uptake of [(3)H]-noradrenaline was inhibited only with the highest concentration of phenoxybenzamine tested (10(-5) g/ml) and therefore appears to be unrelated to the effects on transmitter overflow.5. There was a significant correlation between the degree of alpha-adrenoceptor block produced by phenoxybenzamine and the increase in transmitter overflow obtained by nerve stimulation.6. These results indicate that phenoxybenzamine, in addition to increasing overflow by preventing reuptake of noradrenaline, may increase transmitter release.7. The possibility that phenoxybenzamine acts on alpha-adrenoceptors in the adrenergic nerve terminal is discussed. These receptors would be involved in a negative feedback mechanism regulating transmitter release.     

Lack of correlation between presynaptic inhibition of noradrenaline release and end organ responses during nerve stimulation

1 LD 3098 (cirazoline) is an imidazoline derivative, possessing agonist properties at alpha-adrenoceptor sites.2 When transmitter release was measured directly as tritium overflow from perfused cat spleen preparations, prelabelled with [(3)H]-noradrenaline, LD 3098 was found to be 10 times more selective for presynaptic than for postsynaptic alpha-adrenoceptors.3 In addition, in this preparation, LD 3098 appears to induce a postsynaptic sensitization to the transmitter released by nerve depolarization because under conditions in which [(3)H]-noradrenaline overflow decreased, there was a paradoxical potentiation in the response to nerve stimulation. This potentiation also occurred with a concentration of LD 3098 that did not per se affect stimulation-evoked [(3)H]-noradrenaline release or the basal perfusion pressure of the spleen.4 Both the reduction in (3)H-transmitter release induced through activation of alpha-presynaptic adrenoceptors and the potentiation of the responses to nerve stimulation were concentration-dependent phenomena.5 In pentobarbitone anaesthetized dogs, the heart rate response to low frequency ansa-subclavia stimulation was not affected by LD 3098. Whilst the alpha(1) mediated increase in blood pressure responses to injected noradrenaline and tyramine was significantly potentiated by LD 3098, the beta(1)-mediated heart rate responses to these injected amines were not modified in the presence of LD 3098.6 Thus it is possible that the failure to detect any presynaptic effects with LD 3098 when transmitter release is measured indirectly at the level of the postsynaptic responses is due to end organ sensitivity changes.7 These findings emphasize that caution is necessary when assessing presynaptic alpha-adrenoceptor effects through end organ responses to nerve stimulation both in vitro and in vivo and the need for measurements of transmitter overflow as well as adequate postsynaptic controls in such experiments.     

Yohimbine affects the evoked overflow of neurotransmitters from rat brain slices by more than one mechanism

Both yohimbine (0.1–10 μm) and phentolamine (10 μm) increased the tritium overflow evoked by electrical stimulation (3Hz, 2 ms, 18 mA for 120s every 20 min) of rat brain cortex slices previously incubated with [³H](?)-noradrenaline. At their maximally effective concentrations, neither of these compounds produced an effect which was fully maintained over the 1 h of the experiment, but the decline in effect of yohimbine (1.25 μM) was more marked, falling sharply to reach, after 1 h, 25% of the effect observed after 20 min, whereas phentolamine only declined to 60% of the effect after 20 min. In rat brain cortex slices previously incubated with [³H]5-hydroxytryptamine ([³H]5-HT), the tritium overflow evoked by electrical stimulation (3 Hz, 2 ms, 40 mA for 120 s) was increased by yohimbine (0.1–1 μM) and phentolamine (0.1–10 μM), but a higher concentration of yohimbine (10 μM) decreased evoked overflow below the levels seen in the absence of either drug. It is concluded that, at concentrations effective in inhibiting the presynaptic α-adrenoceptor-mediated mechanisms controlling transmitter release in rat brain slices, a non-a-adrenoceptor-mediated, possible local anaesthetic, action of yohimbine contributes to the overall effect of this drug on transmitter overflow.     

The effects of labetalol (AH 5158) on adrenergic transmission in the cat spleen.

1. The competitive alpha- and beta-adrenoceptor blocking agent labetalol, in concentrations up to 10(-4) M, produced dose-dependent increases in transmitter overflow from the isolated blood perfused spleen of the cat following nerve stimulation at 10 and 30 Hz. 2. At concentrations above 10(-4) M labetol produced a pronounced decrease in transmitter overflow. 3. Labetalol (1.5 X 10(-4) M) increased the recovery of 3H label in the venous blood following the close-arterial infusion of [3H]-(-)-noradrenaline indicating that the drug inhibits uptake of the amine. 4. Both labetalol (3.8 X 10(-5) M) and piperoxan (7.4 X 10(-6) M) produced parallel shifts to the right of the dose-response curves to noradrenaline and oxymetazoline in isolated strips of cat splenic capsule. In this preparation both drugs acted as competitive postsynaptic alpha-adrenoceptor blocking agents. 5. Labetalol (3.3 X 10(-5) M) increased the transmitter overflow following stimulation of the splenic nerves with 200 impulses at 10 Hz. The overflow could be further increased by subsequent addition of piperoxan (7.2 X 10(-6 M). Piperoxan (5.7 X 10(-6) M) alone produced a marked increase in transmitter overflow which could be further increased by subsequent addition of desmethylimipramine (DMI; 3.2 X 10(-5) M). Cocaine (1.5 X 10(-5) M) or DMI (5.4 X 10(-5 M) produced a small increase in transmitter overflow which was not further increased by addition of labetalol (2.8 X 10(-5) M). 6. Labetalol produced a biphasic effect on the responses of the isolated blood perfused spleen of the cat to nerve stimulation. With low doses (up to 10(-4) M) vascular responses were potentiated and with high doses (greater than 10(-4) M) inhibited. The potentiation was related to uptake blockade and the inhibition to decreased transmitter overflow and postsynaptic alpha-adrenoceptor blockade. 7. Labetalol appears to act as a postsynaptic alpha-adrenoceptor antagonist in the isolated blood perfused spleen of the cat with little effect on presynaptic alpha-adrenoceptors. The moderate elevation of transmitter overflow by the drug is related to the inhibitory effect of the drug on neuronal uptake rather than on presynaptic alpha-adrenoceptors.     

Influence of reserpine-induced depletion of noradrenaline on the negative feed-back mechanism for transmitter release during nerve stimulation

1. The effects of depletion of endogenous noradrenaline by reserpine-pretreatment on [(3)H]-noradrenaline overflow elicited by nerve stimulation were determined in the isolated nerve-muscle preparation of the cat's nictitating membrane.2. Reserpine pretreatment (0.3 mg/kg, s.c., 4 days prior to the experiment) reduced the noradrenaline levels in the smooth muscle of the nictitating membrane to about 10% of the control values while granular retention of [(3)H]-noradrenaline had recovered to nearly 40% of the controls.3. In the reserpine-pretreated tissue the fraction release per shock induced by nerve stimulation was 2.2-fold higher than the value obtained in the untreated tissues. This effect was correlated with the degree of depletion of the noradrenaline stores rather than with the decrease in the response of the effector organ.4. Phenoxybenzamine, 2.9 muM reduced the responses to nerve stimulation to the same extent in control and in reserpine-pretreated tissues. Yet, this concentration of phenoxybenzamine increased by 13-fold the overflow of the labelled transmitter in the controls and only by 3-fold in reserpine-pretreated tissues.5. The decrease in effectiveness of phenoxybenzamine in enhancing transmitter overflow after reserpine-pretreatment appears to be due to the decrease in the total release of the transmitter.6. The results obtained support the view that in reserpine-pretreated tissues decreased transmitter output reduces the activation of the presynaptic alpha-adrenoceptors which mediate the negative feed-back mechanism that regulates transmitter release by nerve stimulation.     

THE EFFECTS OF COCAINE AND LIGNOCAINE ON THE RAT RIGHT VENTRICLE IN VITRO

• 1 The effects of cocaine and lignocaine on the contractile responses to field stimulation and to exogenously applied agents, in the absence of other stimuli, have been investigated in the rat right ventricle using methods we have recently described (Doggrell & Vincent, 1981a). In addition the effects of ³H accumulation from (?)-[³H]-noradrenaline and on the spontaneous and field stimulation-induced overflow of ³H, following preloading of the tissue with (?)-[³H]-noradrenaline, are reported.
• 2 Cocaine, but not lignocaine, inhibited the accumulation of ³H from (?)-[³H]-noradrenaline. The spontaneous overflow of ³H, following preloading of the tissue with (?)-[³H]-noradrenaline was not altered by cocaine, 10μ, or lignocaine, 100μM. 10μM Lignocaine had no effect on the overflow of ³H evoked by field stimulation at 5Hz. Lignocaine, 100μM, increased and cocaine, 1 and 10μM, reduced the decline in evoked release of ³H. This effect of lignocaine probably represents a decrease in nerve excitability and that of cocaine inhibition of neuronal uptake of noradrenaline.
• 3 Cocaine, μM, reduced the rate of beat response to tyramine, 1μM, alone, probably by inhibiting the neuronal uptake process.
• 4 Cocaine, 1 and 10μM, had no effect on the contractile responses to field stimulation (at 2 and/or 5Hz). The rate of beat to (?)-noradrenaline or (?)-isoprenaline, 1μM, alone was decreased by cocaine, 10μM. Lignocaine, 10μM, reduced the force of contractions to field stimulation at 5 Hz and the responses to (?)-noradrenaline or (?)-isoprenaline alone. It is suggested that the inhibitory effects of cocaine and lignocaine on responses to (?)-isoprenaline in the rat right ventricle are due to a decreased postjunctional membrane excitability. The inability of 10μM cocaine to potentiate contractile responses to endogenous or exogenous (?)-noradrenaline as a consequence of the inhibition of neuronal uptake is also probably due in part to decreased post-junctional excitability of the right ventricle.

     

Muscarinic inhibition of [3H]-noradrenaline release on rabbit iris in vitro: effects of stimulation conditions on intrinsic activity of methacholine and pilocarpine.

1. Rabbit isolated irides were loaded with [3H]-noradrenaline and superfused with Tyrode solution. The inhibition by the muscarinic agonists (+/-)-methacholine and pilocarpine of the [3H]-noradrenaline overflow into the superfusate evoked by field stimulation (pulses of 1 ms duration, 75 mA) was measured as an index of activation of presynaptic muscarinic receptors. 2. The fractional rate of release per pulse during the first stimulation period (S1) was low with 360 pulses at 3 Hz, intermediate with 360 pulses at 10 Hz and high with 1200 pulses at 10 Hz. Upon repetitive stimulation (7 periods at 20 min intervals), the fractional rates of release per pulse during S7 no longer differed, suggesting a 'long-term' regulation of [3H]-noradrenaline release depending on the stimulation conditions. 3. The evoked [3H]-noradrenaline overflow was depressed by (+/-)-methacholine in a concentration-dependent manner. The EC50 ranged from 0.29 to 0.42 microM. Methacholine nearly abolished the transmitter release evoked at 3 Hz but reduced that induced at 10 Hz by only 50%. Under the latter condition the methacholine concentration-inhibition curve was bell-shaped and no muscarinic inhibition was observed in the presence of methacholine 30 microM. After washout of methacholine the evoked [3H]-noradrenaline release was temporarily enhanced. 4. Atropine 0.1 microM enhanced the [3H]-noradrenaline overflow (evoked by stimulation with 360 or 1200 pulses at 10 Hz), probably antagonizing a presynaptic inhibition by endogenous acetylcholine. The inhibition by methacholine was competitively antagonized by atropine 0.1 microM (apparent -log KB = 8.5-9.0). 5. Depending on the concentration, pilocarpine reduced the [3H]-noradrenaline overflow evoked by 360 pulses at 3 Hz up to 63%. However, at 10 Hz stimulation frequency the compound was inactive as an agonist but competitively antagonized the presynaptic inhibition induced by methacholine. The KB under the latter condition (0.95 microM) was very close to the EC50 value determined at 3 Hz (0.85 microM). 6. The results demonstrate a muscarinic inhibition of noradrenaline release from the rabbit isolated iris. The activation by pilocarpine of the presynaptic receptors provides an alternative explanation for the miosis induced in the rabbit in vivo, which might be the result of a decreased sympathetic tone in the iris dilator muscle.     

The effects of labetalol (AH 5158) on metabolism of 3H(-)-noradrenaline released from the cat spleen by nerve stimulation

The competitive α and β adrenoceptor antagonist labetalol, in concentrations up to 10^?4 M, produced a dose dependent increase in overflow of ³H and [³H]noradrenaline in the isolated blood perfused cat spleen following stimulation of the splenic nerves at a frequency of 10 Hz. Labetalol had no effect on the pattern of overflow of label following stimulation. In experiments in which the metabolism of [³H]noradrenaline released on nerve stimulation was examined, labetalol produced a concentration dependent increase in the percentage of [³H]noradrenaline and a decrease in the percentage of [³H]DOPEG in the venous blood following nerve stimulation. Production of [³H]COMT metabolites and [³H]DOMA was not affected. It is suggested that in the isolated blood perfused cat spleen labetalol produces the elevation of overflow and effects on noradrenaline metabolism by inhibition of neuronal uptake of noradrenaline. The drug has no detectable effects on the enzymes MAO or COMT or on extraneuronal uptake.     

Pharmacological differentiation of presynaptic inhibitory alpha-adrenoceptors and opiate receptors in the cat nictitating membrane.

1 The action of morphine, naturally occurring and synthetic opiate peptides on [3H]-noradrenaline release induced by nerve stimulation was studied in the isolated nerve muscle preparation of the cat nictitating membrane under experimental conditions in which the alpha-presynaptic receptors were blocked by phentolamine 1 microM. 2 Morphine and the naturally occurring peptides: [Met5]-enkephalin, [Leu5]-enkephalin and beta-endorphin reduced 3H-transmitter overflow and responses to nerve stimulation from the cat nictitating membrane, effects which were completely antagonized by naloxone 0.3 microM. The relative order of potency for the inhibition of the stimulation-induced 3H-transmitter overflow at the level of the IC50 (microM) was as follows: [Met5]-enkephalin (0.020 microM) greater than or equal to [Leu5]-enkephalin (0.036 microM) > morphine (0.3 microM) > beta-endorphin (1 microM). 3 The synthetic opiate pentapeptides: BW 180 C (Tyr-D-Ala-Gly-Phe-D-Leu), and BW834 C (Tyr-D-Ala-Gly-pClPhe-DLeu), which are resistant to enzymatic degradation were more potent than the enkephalins in reducing the stimulation-evoked transmitter overflow from the cat nictitating membrane. On the other hand, the tetrapeptide BW832 C, which lacks the D-leucine terminal of BW180 C l was less potent than the enkephalins in inhibiting neurotransmission. 4 In the presence of phenoxybenzamine 1 microM, 3H-transmitter overflow was increased 8 fold and the inhibition of neurotransmission by methionine-enkephalin was not affected. Exposure to phenoxybenzamine 10 microM increased [3H]-noradrenaline overflow 15 fold and antagonized the effects of methionine enkephalin on transmitter release. 5 In the cat nictitating membrane the inhibitory presynaptic opiate receptors are different from the presynaptic alpha-autoreceptors which regulate the release of noradrenaline elicited by nerve depolarization through a negative feed-back mechanism.     

The determination of presynaptic KA values of methacholine and pilocarpine and of a presynaptic receptor reserve in the rat perfused heart.

Rat isolated perfused hearts with the right sympathetic nerves attached were loaded with [3H]-noradrenaline. The nerves were stimulated with up to 11 trains of 10 pulses at 0.1 Hz. The evoked increases of [3H]-noradrenaline overflow into the perfusate were measured in the presence of cocaine, corticosterone and propranolol. Activation of presynaptic muscarinic receptors by methacholine or pilocarpine inhibited the evoked transmitter release in a reversible and concentration-dependent manner. Preperfusion with phenoxybenzamine (5 microM) for 15 min (followed by a washout of 35 min) changed neither resting nor evoked overflow of [3H]-noradrenaline. The concentration-response curve of methacholine was shifted to the right after exposure of the hearts to phenoxybenzamine (1 microM) without depression of the maximum effect. Pretreatment with phenoxybenzamine (5 microM) reduced the maximum inhibition of release by about 50%. Analysis of the data gave a dissociation constant for the agonist-receptor complex (KA) of 4.0 microM and a receptor reserve of roughly 70%. Half-maximal inhibition of [3H]-noradrenaline release occurred when about 2% of the total receptor population was occupied. Comparison of the concentration-response data for methacholine and pilocarpine revealed a relative efficacy (methacholine/pilocarpine) of 16, a KA of 10 microM for pilocarpine and no receptor reserve for this agonist. The results show that KA values for methacholine and pilocarpine obtained at presynaptic receptors are similar to those obtained at postsynaptic muscarinic receptors. This is in agreement with the idea that muscarinic receptors located on postganglionic adrenergic nerves are not different from those located on effector sites of non-neuronal tissue.     

The effects of piperoxan on uptake of noradrenaline and overflow of transmitter in the isolated blood perfused spleen of the cat.

1 The competitive alpha-adrenoceptor blocking agent, piperoxan, in concentrations up to 2 x 10(-4) M, produced large dose-dependent increases in transmitter overflow from the isolated blood perfused spleen of the cat following nerve stimulation at 10 hertz. 2 At concentrations greater than 2 x 10(-4) M, piperoxan produced a rise in perfusion pressure, a contraction of the splenic capsule, and a marked dose-dependent decrease in transmitter overflow. 3 Phenoxybenzamine (10(-4) M) and desmethylimipramine (3 x 10(-5) M) produced further increases in transmitter overflow when added after piperoxan. 4 Piperoxan (5.8 to 6.6 x 10(-6) M) had no effect on the recovery of 3H in the venous blood following the close arterial infusion or injection of (3H)-(--)-noradrenaline, indicating that the drug does not inhibit uptake of the amine. 5 Piperoxan produced dose-dependent inhibition of responses of the splenic vasculature to close arterial injection of 1 microgram of (--)-noradrenaline but was much less effective at inhibiting responses to nerve stimulation. At 2 x 10(-6) M piperoxan produced a considerable reduction of the response to injected noradrenaline but potentiated the response to nerve stimulation. 6 In isolated strips of cat splenic capsule, piperoxan produced a shift to the right of the dose-response curve to noradrenaline with no change of the maximum response. There was no evidence of a postsynaptic sensitizing effect of the type observed in the rat vas deferens.     

The determination of presynaptic pA2 values of yohimbine and phentolamine on the perfused rat heart under conditions of negligible autoinhibition.

1 Rat isolated perfused hearts with the right sympathetic nerves attached were loaded with [3H]-(-)-noradrenaline. The nerves were stimulated with up to 40 trains of 10 pulses every min at 1 Hz, and the evoked increases of [3H-]noradrenaline overflow into the perfusate, of right atrial tension development and ventricular beating frequency were measured. 2 Oxymetazoline inhibited the evoked transmitter overflow (IC50: 10 nM) and decreased the postsynaptic responses in a concentration-dependent manner. It behaved as a full against in abolishing the evoked transmitter overflow. 3 Yohimbine up to 1 microM neither enhanced the evoked [3H]-noradrenaline overflow nor the postsynaptic parameters. Phentolamine (1 microM) caused a transient, minor (less than 30%) increase in [3H]-noradrenaline overflow. 4 Yohimbine (0.03-1.0 microM) and phentolamine (0.1-5.0 microM) shifted to the right the concentration-response curve of oxymetazoline for the inhibition of [3H]-noradrenaline overflow in response to nerve stimulation without depressing the maxima. The pA2 values were 7.82 and 7.52, respectively. 5 Yohimbine (0.1 microM) also antagonized the decrease induced by oxymetazoline in the postsynaptic responses to nerve stimulation. 6 The results confirm the existence of presynaptic inhibitory alpha 2-adrenoceptors at the adrenergic nerve fibres of the rat heart in vitro. Under the stimulation and perfusion conditions selected, the released endogenous transmitter apparently does not activate a negative feedback mechanism, thus permitting the determination of pA2 values.     

Actions of bretylium and guanethidine on the uptake and release of [3h]-noradrenaline

The effect of bretylium and guanethidine has been studied on the uptake and the spontaneous and reserpine-induced release of [³H]-noradrenaline in the rat heart and in the splenic nerve endings of the cat. Bretylium and guanethidine inhibited the uptake by the heart of circulating [³H]-noradrenaline. Bretylium blocked spontaneous and reserpine-induced release of [³H]-noradrenaline, while guanethidine caused release and partially antagonized the reserpine-induced release. Both compounds produced a transient liberation of [³H]-noradrenaline from splenic nerves, but blocked the release of the [³H]-catechol amine following stimulation of the splenic nerve.     

Stimulation of presynaptic β-adrenoceptors enhances [3H]-noradrenaline release during nerve stimulation in the perfused cat spleen

1 The effects of isoprenaline, propranolol and phosphodiesterase inhibitors on ³H-transmitter overflow elicited by low frequency nerve stimulation were determined in the isolated perfused spleen of the cat.2 (-)-Isoprenaline (0.14, 1.4, and 14 nM) produced a concentration-dependent increase in [³H]-transmitter overflow evoked by nerve stimulation at 1 Hz and was more effective at 1 Hz than at 2 hertz.3 A concentration of propranolol (0.1 μM), devoid of neurone blocking activity, blocked this effect of (-)-isoprenaline. These results are compatible with the presence of β-adrenoceptors in the noradrenergic nerve endings of the cat spleen.4 (+)-Isoprenaline (140 nM) failed to increase the release of radioactivity induced by nerve stimulation, indicating that the β-adrenoceptor mediating the facilitation of transmitter release was stereospecific.5 The increase in ³H-transmitter overflow induced by nerve stimulation during exposure to the phosphodiesterase inhibitor, papaverine (27 μM) was more pronounced than that obtained in the presence of 3-isobutyl-1-methyl xanthine (IBMX) 0.5 mM. The facilitation in transmitter release induced by papaverine was not correlated with the granular effect produced by this drug.6 In the presence of papaverine, the concentration-effect curve for (-)-isoprenaline on transmitter release was shifted to the left and its maximum was increased. In addition, propranolol significantly reduced the enhancement in noradrenaline release obtained by exposure to papaverine under conditions in which the granular effect produced by the phosphodiesterase inhibitor was even greater than in the absence of the β-blocker.7 It is concluded that activation of presynaptic β-adrenoceptors in the perfused cat spleen leads to an enhancement in transmitter release which appears to be linked to an increase in cyclic adenosine 3′,5′-monophosphate levels in noradrenergic nerve endings.     

Mechanism of the enhancement in transmitter release from central and peripheral noradrenergic nerve terminals induced by the purified scorpion venom,tityustoxin

Summary In the isolated nerve-muscle preparation of the cat nictitating membrane exposure to 0.04 M of the scorpion venom tityustoxin (TsTX) increased significantly the overflow of ³H-noradrenaline and the responses elicited by postganglionic nerve stimulation (1200 pulses, 0.5 ms duration, supramaximal voltage). Concentration effect curves to exogenous (-)-noradrenaline were not affected in the presence of this concentration of TsTX.The enhanced release of ³H-noradrenaline obtained during nerve stimulation as well as the increase of the postsynaptic responses observed during exposure to TsTX were more pronounced at 4 Hz than at 20 Hz. The increase in the overflow of noradrenaline observed with the toxin was selective for nerve stimulation since the release evoked by tyramine was not affected by TsTX.TsTX did not increase further the enhancement of ³H-noradrenaline release obtained in the presence of 18 mM tetraethylammonium (TEA). On the other hand, both TsTX and TEA were able to increase further the overflow of ³H-noradrenaline after block of the presynaptic alpha-adrenoceptors with phenoxybenzamine 0.29 or 2.9 M.In slices of rat cerebral cortex, TsTX 0.04 M increased ³H-noradrenaline release induced by 10 mM and by 20 mM KCl. The increased release evoked by the toxin was more pronounced for the lower concentration of K⁺.An increased release of ³H-noradrenaline in the presence of the toxin was also observed in rat hypothalamic slices stimulated with 20 mM K⁺. The K⁺ stimulated induced release of ³H-noradrenaline was also increased by 1.8 mM TEA. As shown for the peripheral nervous, system the simultaneous addition of TEA and TsTX did not result in additive effects when compared with the effects of the two agents added separately. Tityustoxin did not modify the metabolic pattern of the neurotransmitter released by K⁺ from rat hypothalamic slices.It is concluded that TsTX increases the stimulation-induced release of ³H-noradrenaline from both peripheral and central noradrenergic nerve terminals. Tityustoxin appears to act on the nerve terminal by a mechanism similar to that of TEA, an agent known to enhance the amount of noradrenaline released by nerve stimulation by increasing the duration of the action potentials.     

Prejunctional alpha-adrenoceptor-mediated inhibition of norepinephrine release in blood-perfused skeletal muscle in situ

The influence of alpha-adrenoceptor antagonists on the overflow of endogenous norepinephrine (NE) and vasoconstrictor responses elicited by sympathetic nerve stimulation (1-4 Hz, 2 min) was investigated in desipramine-pretreated canine blood-perfused skeletal muscle in situ. The nonselective alpha-adrenoceptor antagonist phentolamine enhanced stimulation-evoked NE overflow and reduced vasoconstrictor responses concentration-dependently. Similar effects were obtained with phenoxybenzamine (irreversible alpha-adrenoceptor antagonist). Desipramine pretreatment attenuated the enhancement of stimulation-evoked NE overflow produced by phenoxybenzamine, indicating that phenoxybenzamine also inhibits neuronal uptake. The enhancement by phenoxybenzamine was independent of the stimulation frequency, suggesting a similar engagement of prejunctional alpha-adrenoceptor-mediated inhibition of transmitter release over the frequency range studied here. The alpha 2-selective adrenoceptor antagonist yohimbine enhanced nerve stimulation-evoked NE overflow at concentrations similar to those required to antagonize vasoconstrictor responses to exogenous NE; 10-fold higher concentrations were required, however, to antagonize nerve stimulation-induced vasoconstriction. The concept of a quantitatively important prejunctional alpha 2-adrenoceptor-mediated feedback inhibition of NE release in vivo is supported by our findings in the skeletal muscle vasculature. Postjunctional alpha 2-adrenoceptors appear to be preferentially activated by circulating catecholamines but also seem to be involved in the nervous control of vascular tone.     

Effect of 6-hydroxydopamine on [3H]-adenine nucleotide and [3H]-noradrenaline release from the guinea-pig taenia caecum evoked by electrical field stimulation, nicotine and perivascular nerve stimulation

1 The effects of pretreatment of guinea-pigs with 6-hydroxydopamine (6-OHDA) on the release of [(3)H]-noradrenaline and [(3)H]-adenine nucleotide following electrical field, nicotine and perivascular nerve stimulation of guinea-pig taenia caecum were studied.2 High frequency electrical field stimulation (15,30 Hz) released both [(3)H]-noradrenaline and [(3)H]-adenine nucleotide but low frequency (0.5, 5 Hz) stimulation, producing comparable muscle relaxation led only to [(3)H]-noradrenaline release.3 6-OHDA (50-200 mg/kg) pretreatment inhibited the muscle relaxation and [(3)H]-noradrenaline release with electrical stimulation or with nicotine in isolated taenia but did not affect the release of [(3)H]-nucleotide.4 A low dose of 6-OHDA (50 mg/kg), completely inhibited the muscle relaxation and [(3)H]-noradrenaline release elicited by perivascular nerve stimulation.5 Both tissue noradrenaline content and [(3)H]-noradrenaline uptake were decreased to the same extent by low as well as high doses of 6-OHDA: noradrenaline content was reduced to 20% and uptake to 30% of the control value.6 Catecholamine fluorescence disappeared from tissue layers of the taenia after treatment with a high dose of 6-OHDA.7 In these experiments the inhibitory action of electrical stimulation and nicotine on the taenia can be correlated better with noradrenaline than with nucleotide release.     

Effect of flow-stop on noradrenaline release from normal spleens and spleens treated with cocaine, phentolamine or phenoxybenzamine

1. Cat spleens were perfused with Krebs-bicarbonate solution, using a constant-flow pump at a rate of about 7 ml/min at 33-35 degrees C. Noradrenaline (NA) overflow by nerve stimulation at 10 Hz for 20 s was determined with or without flow-stop before and after treatment with cocaine, phentolamine or phenoxybenzamine. In order to determine the effect of flow-stop on overflow, the arterial and the venous flows were occluded by clamping the inflow and outflow tubes during the period of stimulation plus 30, 60 or 120 seconds.2. Without flow-stop, NA output was 0.93+/-0.25 ng/stimulus, which was significantly increased after cocaine (123+/-6.6%), phentolamine (415+/-93%) and phenoxybenzamine (578+/-107%). Phentolamine and phenoxybenzamine were much more effective than cocaine in enhancing overflow.3. Before treatment with drugs, flow-stops of 30, 60 and 120 s reduced NA outputs to 70+/-6.6, 27.5+/-2 and 7%, respectively, of the control outputs without flow-stop. None of the drugs significantly influenced the percentage reductions in NA outputs during a 30 s flow-stop. However, the percentage outputs after cocaine or phenoxybenzamine treatment during a 60 s flow-stop significantly increased to 45+/-2.5% and 57+/-6%, respectively, as compared to the percentage output of 27.5+/-2% from untreated spleens during a corresponding flow-stop period. During flow-stop, there was no appreciable metabolism of the released transmitter.4. Diffusion of the released transmitter from the site of liberation plays only a minor role in the removal of the released NA.5. It is suggested that the NA released by nerve stimulation acts on the presynaptic alpha sites to inhibit its own release by a negative feedback mechanism. Adrenoceptor blocking agents enhance the NA overflow from spleen because they remove this autoinhibition by blocking the presynaptic alpha sites.     

The role of calcium in the effects of noradrenaline and phenoxybenzamine on adrenergic transmitter release from atria: no support for negative feedback of release

1 The relation of calcium ion influx into nerve terminals to presynaptic adrenoceptor function and the possible masking, by desensitization due to intraneuronal calcium accumulation, of the effects of adrenoceptor agonists and antagonists on presynaptic alpha-adrenoceptors was investigated in guinea-pig atria previously incubated with [(3)H]-noradrenaline.2 Atria were stimulated with 100 pulses at various frequencies (1 to 15 Hz) in standard (2.3 mm), low (0.26 mm) and high (6.9 mm) calcium-Krebs solution in the absence and then the presence first of noradrenaline and subsequently phenoxybenzamine.3 The per pulse overflow of tritium was directly related to the calcium concentration of the Krebs solution, being much reduced and substantially increased in 0.26 and 6.9 mm calcium-Krebs solutions respectively.4 Noradrenaline inhibited the overflow of tritium in low calcium-Krebs solution, to a relatively constant extent, independently of frequency. In addition, the agonist had a greater maximal inhibitory effect in standard than in reduced calcium-Krebs. The catecholamine was as effective an inhibitor of overflow at the lowest and highest frequencies in high as it was in standard calcium-Krebs solution. Phenoxybenzamine invariably increased the tritium overflow but was generally less effective both in low and in high calcium-Krebs solution. The patterns of inhibition and enhancement of stimulation-induced tritium overflow by these two agents do not indicate an intimate relationship between calcium influx and adrenoceptor activation; nor does desensitization appear to be an adequate explanation of the relationship between frequency of stimulation and the intensity of agonist and antagonist effect in the three different calcium concentrations.5 It is concluded that the perineuronal levels of adrenergic transmitter do not establish the magnitudes of effect of exogenous adrenoceptor agonists and antagonists on tritium overflow and that a negative feedback regulation of release by transmitter is exceedingly unlikely under ordinary conditions of neurotransmission.