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.     

Effect of morphine on some sympathetically innervated effectors

Morphine, in doses of 0.5 to 55 mg/kg, inhibited contraction of the nictitating membrane of the cat following stimulation of the postganglionic sympathetic nerve fibres. Morphine was more effective at low than at high frequencies of stimulation, independently of the size of contraction of the membrane; the speed of contraction was reduced at all frequencies. Cocaine potentiated the contraction of the nictitating membrane following nerve stimulation more at low than at high frequencies, and antagonized the action of morphine. These findings, and the absence of an effect of morphine on the action of injected noradrenaline, make it likely that morphine interferes with the release of noradrenaline from the postganglionic nerve endings in the nictitating membrane. Morphine had no effect on the cardioaccelerator action of the cardiac nerves and inconsistent results were obtained on the emptying of the spleen after stimulation of the splenic nerves.     

The isolated nictitating membrane of the cat. A new model for the study of narcotic analgesics.

Kinetic parameters of morphine and azidomorphine derivatives as well as methionine enkephalin methylester were determined on the electrically stimulated isolated medial smooth muscle of the cat nictitating membrane. The organ was found to be a promising in vitro model for testing the action of opiate agonists on opiate B receptors.     

The effects of morphine on the release of noradrenaline from the cat isolated nictitating membrane and the guinea-pig ileum myenteric plexus-longitudinal muscle preparation.

1. Electrical field stimulation of either the cat isolated nictitating membrane or the guinea-pig ileum myenteric plexus-longitudinal muscle preparation caused the release of noradrenaline into the bathing medium. 2. In the cat nictitating membrane, the output per pulse of noradrenaline was constant at frequencies of stimulation from 0.5 to 15 Hz. In the guinea-pig myenteric plexus preparation the output per pulse of noradrenaline increased as the frequency of stimulation was increased from 2 to 16 Hz. 3. Phenoxybenzamine (29.3 muM) caused a marked increase in the noradrenaline output from both the cat nictitating membrane and guinea-pig myenteric plexus preparations. 4. Morphine (0.13-8 muM) inhibited the contractions of the cat nictitating membrane caused by electrical stimulation. This effect was greater at low (1Hz) than at high (15Hz) frequencies of stimulat The site of action is at the nerve-smooth muscle junction. 5. The action of narcotic analgesic drugs on the cat nictitating membrane showed stereospecificity. Naloxone (0.1 muM) reversed the inhibition caused by normorphine (3.2 muM). 6. Morphine (3 muM) reduced the noradrenaline output from the cat nictitating membrane stimulated at 1 Hz but not at 15 Hz. At 1 Hz, the inhibition of noradrenaline output by normorphine (muM) was reversed by naloxone (0.25 muM). 7. Morphine (1.5 muM) did not alter the noradrenaline output from the guinea-pig myenteric plexus preparation stimulated at 2 or 16 Hz.     

The action of morphine on the superior cervical ganglion and on the nictitating membrane of the cat

The intravenous injection of 0.05 to 2.5 mg. of morphine reduced the response of the nictitating membrane in the cat to pre- and post-ganglionic stimulation. This inhibitory action of morphine was due neither to inhibition of ganglionic transmission nor to a depressant action on the smooth muscle of the nictitating membrane. It is suggested that morphine inhibits the release of the sympathetic transmitter from the postganglionic nerve endings.

Small amounts of morphine (5 to 20 μg.) injected intravenously reduced or abolished the contraction of the nictitating membrane due to the injection of histamine, pilocarpine and 5-HT into the arterial blood supply of the superior cervical ganglion. This inhibitory action of morphine was due to an action on the ganglion cells, since such small amounts of morphine did not reduce the response of the nictitating membrane to postganglionic stimulation. Similar amounts of morphine did not abolish the stimulation of the ganglion by nicotine, tetramethylammonium and potassium chloride.

The results provide further evidence for the view that histamine, pilocarpine, and 5-hydroxytryptamine have no “nicotine-like” properties but act on receptors of the ganglion cells different from the acetylcholine receptors.

     

Inhibitory effects of alpha, beta-methylene ATP on nerve-mediated contractions of the nictitating membrane in reserpinised cats

Residual responses of the cat nictitating membrane to nerve stimulation were obtained after reserpine pretreatment (40% of controls), in spite of a pronounced reduction in noradrenaline content. The putative ATP-receptor desensitising agent alpha, beta-methylene ATP (alpha, beta-MATP), administered intraarterially through the lingual artery produced a contraction of the nictitating membrane and subsequently inhibited the residual responses evoked by sympathetic nerve stimulation in reserpinised cats. These doses of alpha, beta-MATP did not modify the contractions evoked by exogenous noradrenaline (i.a.) but antagonized the contractions of the nictitating membrane elicited by beta, gamma-methylene ATP, which is an agonist at P2 receptors. These results are compatible with a co-transmitter role for ATP in the neurally mediated contractile responses of the nictitating membrane following depletion of endogenous noradrenaline stores by pretreatment with reserpine.     

Role of adrenergic neurone blockade in the hypotensive action of propranolol

1. Propranolol, in doses of 25-100 mug/kg, blocks contractions of the nictitating membrane to nerve stimulation but not to injected noradrenaline.2. This adrenergic neurone blocking action of propranolol is antagonized by amphetamine.3. It is also reversed by raising the dose of propranolol to amounts exceeding 0.5 mg/kg.4. Still larger amounts potentiate the responses of the nictitating membrane to both submaximal stimulation of the cervical sympathetic nerve and to injected noradrenaline.5. The (+) isomer of propranolol produced adrenergic nerve blockade and some degree of hypotension without blocking cardiac beta-adrenoceptors.6. The relevance of adrenergic neurone blockade to the hypotensive effect of propranolol is discussed.     

The actions of bretylium: adrenergic neurone blocking and other effects

Bretylium caused a specific and lasting depression of many excitatory and inhibitory responses evoked by electrical stimulation of the peripheral sympathetic nervous system, probably by impairing conduction of impulses in adrenergic neurones with consequent failure of noradrenaline and adrenaline release. This effect, which will be referred to as the adrenergic neurone blocking action, was preceded by weak sympathomimetic effects. In the presence of bretylium the effects of adrenaline and noradrenaline were increased, as after sympathectomy. Concentrations producing blocking of adrenergic neurones did not prevent the release of adrenaline and noradrenaline from the adrenal medulla by splanchnic nerve stimulation or by the injection of dimethylphenylpiperazinium iodide, nor did they cause antiparasympathetic or parasympathomimetic effects. No action on the central nervous system has been detected. Curare-like neuromuscular block occurred with 10 to 30 times the amount required to block the response to adrenergic nerve stimulation alone and was accompanied by signs of temporary synaptic block in autonomic ganglia. Adrenergic nerve trunks and sensory nerves in the skin were readily blocked for long periods by topical application of bretylium, whereas the phrenic nerve of the rat was not. Bretylium had little effect on gastrointestinal propulsion or on the sensitivity of smooth muscle to acetylcholine, 5-hydroxytryptamine, adrenaline, or noradrenaline, but moderate amounts depressed the peristaltic reflex and the sensitivity of the guinea-pig ileum to histamine. Bretylium caused postural hypotension in the cat in doses which had little effect on the supine blood pressure. Experiments on the nictitating membrane indicated that compensation for the effects of bretylium on low rates of stimulation of postganglionic sympathetic nerves could be attained by a small increase in the rate of stimulation, whereas compensation for its effects on high rates required an increase in the rate of stimulation beyond physiological limits.     

A quantitative study of the effect of cocaine on the response of the cat nictitating membrane to nerve stimulation and to injected noradrenaline

Results are reported of a quantitative study of the potentiating effect of cocaine on the responses of the cat nictitating membrane to intravenously and intra-arterially injected noradrenaline, as well as to different types of sympathetic nerve stimulation. Responses of the membrane to noradrenaline were potentiated more with intravenous than with close-arterial injections. From studies of the responses of the nictitating membrane to various forms of sympathetic nerve stimulation before and after injection of cocaine, conclusions are drawn as to the extent to which the transmitter amine liberated by nerve activity is normally removed and its effect thereby limited in duration and extent. This uptake was greatest at low stimulus frequencies. The mechanism by which cocaine potentiates sympathetic responses is discussed.     

Inhibition of the cardiac response to sympathetic nerve stimulation by opioid peptides and its potentiation by morphine and methadone

[D-Ala2,D-Leu5]enkephalin (1-10 microM) and [Met5]enkephalin-Arg-Phe (1-10 microM) produced concentration-dependent inhibition of the cardiac response to field stimulation of the adrenergic nerve terminals in preparations pretreated with peptidase inhibitors (captopril 10 microM, bestatin 10 microM, thiorphan 0.3 microM and L-leucyl-L-leucine 2 mM). The inhibitory response to the opioid agonists was evident in preparations superfused with solutions containing 1.8 mM calcium, but not in those containing 3.6 mM calcium. Moreover the inhibition was antagonized by naloxone 10 microM. [D-Ala2,Met5]enkephalinamide (1-3 microM) and beta-endorphin (1-3 microM) did not significantly affect the sympathetic response. The cardiac response to sympathetic stimulation was not inhibited but, on the contrary, was potentiated by morphine (3-10 microM) and methadone (3-10 microM). It is suggested that the depressant effect of the opioid peptides was due to stimulation of presynaptic inhibitory opiate receptors on adrenergic nerve terminals of the heart, and that the potentiation of the sympathetic response by morphine and methadone was probably attributable to an unspecific inhibitory effect on the neuronal uptake of noradrenaline.     

The influence of an extraneuronal compartment on the relaxation of the cat nictitating membrane in vivo.

1 Contractions of the cat nictitating membrane were elicited on stimulation of the internal carotid nerve, and the effects were studied of desipramine and two inhibitors of catechol-O-methyltransferase, U-0521 and pyrogallol, on the subsequent relaxation of the muscle. 2 The relaxation of the nictitating membrane occurred in at least two phases. The late phase of relaxation was prolonged after increase in the period of nerve stimulation and the duration of this phase was further prolonged after treatment with pyrogallol. 3 After inhibition of neuronal uptake of noradrenaline with desipramine both the early and late phases of relaxation were increased in duration, and subsequent administration of pyrogallol or U-0521 caused a further increase in the duration of the late phase of relaxation. 4 The results suggest that the late phase of relaxation of the nictitating membrane is influenced by efflux of noradrenaline from an extraneuronal pool.     

Inhibitory effects of α,β-methylene ATP on nerve-mediated contractions of the nictitating membrane in reserpinised cats

Residual responses of the cat nictitating membrane to nerve stimulation were obtained after reserpine pretreatment (40% of controls), in spite of a pronounced reduction in noradrenaline content. The putative ATP-receptor desensitising agent α,β-methylene ATP (α,β-MATP), administered intraarterially through the lingual artery produced a contraction of the nictitating membrane and subsequently inhibited the residual responses evoked by sympathetic nerve stimulation in reserpinised cats. These doses of α,β-MATP did not modify the contractions evoked by exogenous noradrenaline (i.a.) but antagonized the contractions of the nictitating membrane elicited by β,γ-methylene ATP, which is an agonist at P2 receptors. These results are compatible with a co-transmitter role for ATP in the neurally mediated contractile responses of the nictitating membrane following depletion of endogenous noradrenaline stores by pretreatment with reserpine.     

A new example of a morphine-sensitive neuro-effector junction: Adrenergic transmission in the mouse vas deferens

The isolated mouse vas deferens possesses an adrenergic excitatory motor innervation which can be inhibited by low concentrations of morphine (ID50=0.5 muM). This effect of morphine is mediated by specific receptors which are blocked by naloxone. Activation of the morphine receptors inhibits noradrenaline release. It is concluded that adrenergic neurotransmission in the mouse vas deferens differs in some important way from that at the more common, morphine-insensitive, adrenergic junctions.     

Comparison of bretylium and guanethidine: tolerance, and effects on adrenergic nerve function and responses to sympathomimetic amines

Bretylium depresses the slope of regression lines relating frequency of sympathetic nerve stimulation to magnitude of contractions of the cat nictitating membrane. In contrast, guanethidine and reserpine preferentially abolish responses to low rates of nerve stimulation and cause a roughly parallel shift of the regression lines. The hypersensitivity of the nictitating membranes of cats to intravenous adrenaline or noradrenaline is far greater after a series of small daily doses of bretylium or guanethidine than after single large doses. The maximal sensitivity produced was similar to that after postganglionic sympathetic nerve section and exceeded that produced by ganglion blockade. The development of hypersensitivity to catechol amines is accompanied by some return of responses of the nictitating membranes to sympathetic nerve stimulation despite continued daily administration of bretylium or guanethidine. In cats given bretylium daily, responses to low rates of nerve stimulation become greater than in controls unless the dose of bretylium given subcutaneously is 50 mg/kg or more. When marked hypersensitivity to catechol amines has been produced by giving bretylium or guanethidine daily for 7 or 14 days, the sympathomimetic effects of these compounds are greater. Responses to intravenous dimethylphenylpiperazinium are also increased and the results suggest that even large daily doses of adrenergic neurone blocking agents do not appreciably impair the functioning of the adrenal medulla. The pressor effects of intravenous adrenaline, noradrenaline and dimethylphenylpiperazinium iodide increase less than the corresponding nictitating membrane responses. These results are discussed in relation to tolerance to adrenergic neurone blockade, and differences between the effects of bretylium and guanethidine found in man. Bretylium and guanethidine depress the slopes of the dose-response curves for the pressor and nictitating membrane contracting effects of tyramine. When single doses or a short series of daily doses were given, guanethidine caused more depression of the slopes than did bretylium, but nevertheless large depressions of slope were found after giving bretylium daily for several weeks. The magnitude of the responses can be greater or less than in controls depending on the dose of the sympathomimetic amine, the dose of the adrenergic neurone blocking agent and the duration of its administration. The results suggest that injection of tyramine produces a progressively smaller release of adrenaline or noradrenaline during the daily administration of bretylium (or guanethidine) but that in some test situations this is more than compensated for by the development of hypersensitivity to the catechol amine released. Some corresponding changes in responses to amphetamine and ephedrine are also described.     

DUAL PRESYNAPTIC EFFECTS OF 5-HYDROXYTRYPTAMINE ON PERIPHERAL NORADRENERGIC SYNAPSES

• 1 The aim of the present experiments was to study the effects of 5-hydroxytryptamine (5-HT) on the responses to postganglionic stimulation of two models of the peripheral sympathetic nervous system: the isolated nictitating membrane of the cat and the guinea-pig isolated atria.
• 2 In the nictitating membrane of the cat, 5-HT (0.1 μM) shifted to the left the frequency-response curve to nerve stimulation. This potentiating effect was prevented by 5-HT receptor antagonists (0.1 μM. methysergide, 0.1 μM pizotifen and 0.1 μM morphine) and also by the β-adrenoreceptor blocker propranolol (0.1 μM). The α₂-adrenoreceptor antagonist yohimbine (0.1 μM) had no effect on the 5-HT-induced potentiation.
• 3 In the guinea-pig isolated atria the responses to cardioaccelerans nerve stimulation were diminished by 5-HT (0.1 to 1.0 μM). The shift to the right in the frequency-response curve induced by 5-HT (1.0 μM) was additive to the antagonism caused in the atria by propranolol (0.1 μM). The inhibitory effect of 5-HT on the pacemaker responses to nerve stimulation was prevented by the 5-HT receptor antagonists methysergide (1.0 and pizotifen (1.0 μM) and also by the α-adrenoreceptor antagonist phentolamine (0.1 μM).
• 4 The selective α₂-adrenoreceptor agonist clonidine (0.01 μM) reduced to the same extent as 5-HT (1.0 μM) the responses of the guinea-pig atria to nerve stimulation. The inhibitory effect of clonidine was prevented by the α-adrenoreceptor blocker phentolamine (0.1 μM) but not by the 5-HT receptor blocker pizotifen (1.0 μM).
• 5 With the exception of propranolol, which in the atria shifted to the right the concentration-response curve to exogenous noradrenaline (NA), neither 5-HT nor the different antagonists employed modified the sensitivity to NA in the tissues studied.
• 6 The present observations show that 5-HT can produce a dual effect on the sympathetic neurotransmission. It is proposed that a modification in the overflow of NA in response to nerve stimulation is caused by 5-HT and results from the interaction of 5-HT with specific receptors located on the sympathetic fibres. These presynaptic 5-HT receptors behave as excitatory (cat nictitating membrane) or inhibitory (guinea-pig atria) depending on the tissue studied.

     

Axoplasmic transport of [3H]ouabain binding sites and catecholamine secretion from an adrenergic nerve trunk.

The presence of a functional Na+/Ca2+ exchange system was explored in the ligated cat hypogastric nerve, a preparation that has been proposed as a model of giant noradrenergic nerve terminal free of effector cells. The rationale for this study was to monitor noradrenaline secretion from the ligated cat hypogastric nerve promoted by the increase in intracellular Ca2+ levels after ouabain blockade of Na+,K(+)-ATPase molecules present in the plasma membrane of the ligated cat hypogastric nerve. Such an increase in intracellular Ca2+ levels is achieved by activation, in "reverse mode," of the Na+/Ca2+ exchange system. In the present study, [3H]ouabain binding sites were identified on crude preparations of hypogastric nerve membranes. A single, high affinity (Kd around 10 nM), binding site was observed in both ligated and nonligated nerves. The number of binding sites increased with the time of ligation, reaching a peak of about 1 pmol/mg of protein 48 hr after ligation. Blockade of these binding sites by ouabain induced a dose-dependent, Ca(2+)-dependent release of noradrenaline, with an ED50 around 50 microM. The maximum release amounted to 9% of the total noradrenaline content in the cells. As would be expected for ouabain-induced noradrenaline secretion mediated by a Na+/Ca2+ exchange system working in reverse mode, the effect of ouabain was dependent upon the presence of Na+ in the incubation medium, reaching a plateau at an extracellular Na+ concentration of 100 mM. Calcium uptake after Ca2+ reintroduction in ouabain-treated nerves increased with time of ligation, suggesting the incorporation of Na+/Ca2+ exchange carrier molecules into the axolemma of hypogastric nerves. The similarity between ouabain-induced noradrenaline secretion from the ligated cat hypogastric nerve and from other adrenergic systems strongly supports the idea that the ligated cat hypogastric nerve is equipped with a functional Na+/Ca2+ exchange system that would contribute to the regulation of intracellular Ca2+ levels. Furthermore, these data, together with previously published reports, fully characterize, from a biochemical point of view, the ligated hypogastric nerve as a model of giant noradrenergic nerve terminal free of effector cells.     

Presynaptic opiate receptor- and alpha 2-adrenoceptor-mediated inhibition of noradrenaline release in the rat brain: role of hyperpolarization?

The inhibitory actions of exogenous noradrenaline (1 microM) and clonidine (1 microM) as well as well as of the opiate receptor agonists morphine (1 microM) and [D-Ala2,D-Leu5]enkephalin (DADLE, 1 microM) on the potassium-induced Ca2+-dependent release of [3H]noradrenaline from superfused rat brain cortex slices were independent of the degree of depolarization when release was effected by 15 or 56 mM K+ for 5 min. The non-depolarization-dependent release of [3H]noradrenaline induced by exposing the slices for 5 min to medium with NaCl replaced by LiCl was only partially Ca2+-dependent and was not inhibited by the Ca2+ antagonist Cd2+ (50 microM). This release was strongly inhibited by morphine, DADLE, exogenous noradrenaline and clonidine at 1 microM concentrations both in the presence and the absence of extracellular Ca2+. Together with other data in the literature these results strongly suggest that opiate receptors and alpha 2-adrenoceptors located on noradrenergic axonal varicosities, unlike those located on cell bodies, do not primarily mediate hyperpolarization of the neuronal membrane. Instead the activation of these presynaptic receptors causes a reduction of Ca2+ availability for, or the utilization of Ca2+ by the secretion process upon invasion of an action potential.     

Possible role of a beta-adrenoceptor in the regulation of noradrenaline release by nerve stimulation through a positive feed-back mechanism.

1 The effects of isoprenaline, propranolol and phentolamine, were studied on tritiated noradrenaline overflow elicited by postganglionic nerve stimulation in guinea-pig isolated atria. 2 Isoprenaline (1.2 times 10-minus 8M) increased while propranolol (1.0 times 10-minus 7M) reduced the overflow of tritiated noradrenaline evoked by nerve stimulation. These effects were less than those of phentolamine (3.1 times 10-minus 6M), which increased by approximately three-fold the overflow of [3H]-noradrenaline elicited by nerve stimulation. 3 Neuronal accumulation of tritiated noradrenaline in guinea-pig atria was not affected by isoprenaline, propranolol or phentolamine at the concentration employed in this study. 4 Isoprenaline (1.2 times 10-minus 8M) induced a positive chronotropic effect of about 80 percent of the maximum. On the other hand, propranolol produced a shift to the right in the frequency-response curve to nerve stimulation and in the concentration-response curve to exogenous noradrenaline in guinea-pig atria. 5 In the isolated nictitating membrane of the cat, the frequency-response curve to nerve stimulation was not modified by propranolol, while in the presence of 3.9 times 10-minus 6M of N,-2-(2,6-dimethylphenoxy)propyl-N,N,N-trimethylammonium (beta-methyl-TM 10) there was a shift to the right and a depression of slope. Neither propranolol nor beta-methyl-TM 10 affected responses to exogenous noradrenaline. 6. The effects of isoprenaline and of propranolol on transmitter release are compatible with the view that in addition to the presynaptic negative feed-back mechanism for noradrenaline release by nerve stimulation mediated via alpha-adrenoceptors a positive feed-back mechanism exists in adrenergic nerve endings which is triggered through the activation of presynaptic beta-adrenoceptors.     

Sympathetic postganglionic cholinergic fibres

When the postganglionic fibres to the nictitating membrane are stimulated in a cat treated with reserpine, the membrane contracts. The contraction is increased after the injection of eserine and is abolished with atropine. Thus the fibres stimulated appear to be cholinergic. When the splenic nerve is stimulated in a cat treated with reserpine there is similar evidence of the presence of cholinergic fibres; the spleen dilates, the dilatation is greater in the presence of eserine and is abolished by atropine. The hypogastric nerves when stimulated cause inhibition of the uterus of the virgin cat. When the cat is treated with reserpine, stimulation then causes contraction which is increased in the presence of eserine and abolished by atropine. There is also evidence of cholinergic fibres in the postganglionic supply to the vessels of the rabbit's ear. From this and other evidence it is suggested that a form of adrenergic mechanism may exist such that cholinergic fibres in the postganglionic sympathetic supply liberate acetylcholine; this in turn liberates noradrenaline from a store around the sympathetic nerve ending.     

Modification of some actions of guanethidine by the acute administration of reserpine

Summary The pressor action of guanethidine (4 mg/kg) is enhanced by previous administration of reserpine (2 mg/kg), when the time elapsing between the injections of the two drugs is varied between 2 min and 8 hr. This enhancement was observed in the dog (anaesthetized, or spinal or nialamide-pretreated animals) and in the cat. The pressor effects of adrenaline and noradrenaline were not modified by reserpine. Guanethidine caused the nictitating membrane of the dog and the cat, as well as isolated cat spleen strips, to contract and the contractions were enhanced by reserpine; however, the contractions of the nictitating membrane induced by electrical stimulation of the postganglionic sympathetic nerve were not changed by reserpine. Similarly, the pressor effect caused by electrical stimulation of the central end of the vagus nerve was not enhanced. Several mechanisms are discussed in order to explain the observed potentiation; the results are in good agreement with the hypothesis of the existence of different noradrenaline pools.Calouste Gulbenkian Foundation Scholar.