The relation between the subcellular distribution of [3H]resperpine and its proposed site of action

The amount of [³H]reserpine retained after injection is greater in the heart, a tissue with a high density of adrenergic innervation than in femoral muscle. Subcellular distribution studies emphasized that 15 and 60 min after injection of [³H]reserpine it could not be shown to be uniquely associated with the noradrenaline-containing granules. The possibility existed that the large amount of reserpine bound to cellular lipids at these times might mask a more specific binding. A reduction in the dose and a lengthening of the time between drug administration and tissue measurement to 18 h revealed a small amount of [³H]reserpine to be bound more firmly to the microsomal or amine granule-containing fraction. Prior saturation of tissue binding sites by treatment with large amounts of unlabelled reserpine or tetrabenazine decreased the retention of subsequently injected [³H]reserpine. This decreased tissue retention resulted in an increase in the formation of [³H]trimethoxybenzoic acid. A model is proposed relating tissue distribution and binding to reserpine's mechanism of action.     

Subcellular distribution of [3H]amphetamine and [3H]guanethidine and their interaction with adrenergic neurons

The subcellular distribution of [³H]amphetamine and [³H]guanethidine and their interaction with each other and with noradrenaline binding sites have been examined. The ratio p/(p + s) × 100, an indication of affinity for noradrenaline storage particles, for [³H]amphetamine and [³H]guanethidine was 12% and 57% respectively. Protriptyline, a substance which inhibits amine transport mechanism at the level of the cell membrane, i.e. the membrane pump, and reserpine, an agent which impairs incorporation of amines into the storage particles in the adrenergic nerve fibre, inhibited the uptake and storage respectively, of [³H]guanethidine more than that of [³H]amphetamine. Retention of [³H]guanethidine by rat salivary glands was markedly decreased by sympathetic denervation of the glands while that of [³H]amphetamine was not. The results suggest that guanethidine possesses a much higher affinity for noradrenaline binding sites than amphetamine.     

Correlation of the recovery of the granular uptake-storage mechanism and the nerve impulse induced release of ( 3 H)noradrenaline after reserpine

Rats were treated intraperitoneally with 10 mg/kg reserpine. After various time intervals 1 μg/kg ± [³H]noradrenaline (³H-NA) was administered intravenously, and the uptake into subcellular fractions of the submaxillary gland was measured or, in some of the rats, the sympathetic chain of the neck was electrically stimulated with 10 impulses/s for 30 min. The release of ³H-NA and the contraction response of the lower eye-lid were measured. A striking parallel was observed between the recovery of the ³H-NA uptake into the amine storage particles and the nerve impulse-induced release of ³H-NA, and also the recovery of the functional response. The somewhat earlier recovery of the ³H-NA uptake into the coarse fraction might reflect the existence of another type of amine storage granule or might represent granules present near the nerve cell membrane. A possible increase in the turnover of the adrenergic transmitter during the period of recovery after reserpine is discussed.     

Uptake and retention of H-noradrenaline in adrenergic nerve terminals after reserpine and axotomy

The onset of recovery of endogenous noradrenaline levels and of the capacity of the adrenergic neuron to take up and retain ³H-noradrenaline both occurred between 24 and 36 hr after reserpine treatment. Axotomy delayed the increase in ³H-noradrenaline retention. The importance of axonal transport, in particular of amine storage granules, for recovery after reserpine is discussed.     

Subcellular distribution of [3H]-epsilon-aminocaproic acid and its effects on amine storage mechanisms

ε-Aminocaproic acid (EACA) is an amino-acid reported to cause almost complete depletion of cardiac noradrenaline stores. The present report indicates that this compound can be found in both the particulate and supernatant fractions derived from heart homogenates. The [³H]EACA in the supernatant probably represents a mixture of intra- and extraneuronally located drug. Protriptyline pretreatment decreases the uptake of [³H]EACA suggesting that the amino-acid probably utilizes the amine membrane transport system, EACA, like reserpine, can both impair the retention of exogenously administered amines by adrenergic storage particles and cause the release of amines previously stored in such particles.     

Recovery of noradrenaline in adrenergic axons of rat sciatic nerves after reserpine treatment

The recovery of noradrenaline in adrenergic axons of the rat sciatic nerve after a single dose of reserpine (10 mg/kg i.p.) has been studied in unligated nerves and nerves ligated for 6 h. In unligated nerves the recovery at 24 h after reserpine was about 14% of normal. The noradrenaline content then slowly rose to reach about normal concentrations 6–7 days after reserpine injection. In nerves ligated 6 h before death, about 8·0 ng of noradrenaline accumulated proximal to the ligation in normal animals. At 6 and 12 h after reserpine about 4% of normal amounts of noradrenaline were found. Thereafter the amount of accumulated noradrenaline rapidly increased to about normal levels on day 2 after reserpine. At this time the content in unligated nerves was only about 45% of normal unligated nerve. On days 3–5 after reserpine, supranormal accumulations of noradrenaline were found (statistically highly significant), having a maximum at day 4 of about 145% of normal. At this time the noradrenaline content in unligated nerve was only about 80% of normal. The results may indicate an increased synthesis and increased rate of downtransport of amine storage granules during the early recovery phase after reserpine. This phenomenon may be part of a feed-back mechanism operating after depletion of the transmitter in the nerve terminals.     

On the formation of adrenergic amine storage granules as measured by reserpine labeling

Summary Reserpine appears to be bound specifically and irreversibly to amine storage granules in the adrenergic neuron. The presence of reserpine persistently attached to amine granule should reflect the life span of the vesicles, and any additional binding of reserpine should then measure the rate of appearance of newly formed amine granules.Measurement of the persistent binding of tritiated reserpine (³H-Reserpine) in hearts of rats given labeled compound at various times after a saturating dose of unlabeled reserpine showed that the recovery of ³H-reserpine binding capacity occurred rapidly. Four days after reserpine the ³H-reserpine binding was 30% of control rats. The rate of return of binding capacity was then slowed being 65% of normal in 24 days.Calculation of the mean daily increment of binding capacity showed that the rate was an inverse linear function of the binding capacity existing in the neurone at an early phase (1–8 days) but became constant at a later interval (8–24 days).It is suggested that the return of reserpine binding capacity reflects the appearance of new storage granules and that the rate of storage granule synthesis in the cell body is under feedback control.The signal for enhanced granule formation does not appear to be related to the intensity of nerve traffic not to the concentration of transmitter in the neuron since maneuvers which alter these parameters did not change the rate of reserpine binding recovery.Supported by U.S. Public Health Service Grant MH-05831.     

EFFECTS OF RESERPINE ON THE CONTENT AND UPTAKE OF DOPAMINE AND NORADRENALINE IN RABBIT ARTERIES

1. Change with time of the content and uptake of dopamine (DA) and noradrenaline (NA) in the renal, superior mesenteric and femoral arteries and abdominal aorta of rabbit after reserpine administration was examined. Endogenous DA and NA were measured by high performance liquid chromatography coupled with electrochemical detector. 2. A single dose of reserpine (3 mg/kg, i.p.) maximally depleted the endogenous DA and NA contents in the four blood vessels 24 h after the administration; the ratios of reductions were 70–90% and approximately 90% of the normal levels, respectively. The DA contents in all four vessels recovered to the normal level within 4 days after reserpine. However, NA content did not recover to the normal levels within 30 days after reserpine except in the mesenteric artery. 3. The activity of dopamine β-hydroxylase (DBH) significantly increased in all four blood vessels 1 h after reserpine. Although the DBH activity returned to the normal level after 3 days in the mesenteric artery, it returned within 24 h in the other three vessels. 4. [³H]-Dopamine and [³H]-NA uptake were almost completely depressed 1 h after reserpine. The [³H]-NA uptake in four vessels recovered to the normal level 2–14 days after reserpine, and [³H]-DA uptake recovered after 30–45 days. Thus, the endogenous DA content in blood vessels was completely restored although DA uptake and NA content were still affected. 5. These results suggested that the recovery of stored DA after reserpine was faster than that of stored NA and the recovery of DA uptake after reserpine was slower than NA uptake. This indicates a possibility that a part of DA pool may be different from NA pool in adrenergic nerve terminals in the blood vessels.     

The recovery of noradrenaline in adrenergic nerve terminals of the rat after reserpine treatment

Tissue concentrations of endogenous noradrenaline in heart, submaxillary gland, and gastrocnemic muscle have been examined after one large dose of reserpine (10 mg/kg) to rats. After the initial depletion of the amine, the concentration started to rise between 24 and 36 h. For about one week thereafter the amine recovery proceeded comparatively fast, then the rate of the recovery slowed. Between the 4th and the 6th weeks there was a pronounced drop in the noradrenaline concentration in all three tissues, apparently beginning in the 4th week with a maximal decrease of about 20% in the 5th week after reserpine. Thereafter the concentrations increased to approach normal about 6 weeks after reserpine. These results are discussed in relation to the axonal down-transport of newly formed amine storage granules and to the life-span of these granules in the nerve terminals. The different parts of the noradrenaline recovery curve appeared to reflect the axonal down-flow of granules. A theoretical recovery curve was calculated, based on granular transport. This curve was similar to the observed recovery curve. The claim is made that the recovery of adrenergic function and noradrenaline levels after reserpine is due to a down-transport of newly formed, amine storage granules to the nerve terminals. There seems little need for the theory that the storage function reappears in old, reserpine-blocked granules, as a mechanism for noradrenaline recovery after a large dose of reserpine.     

Accumulation of amantadine by isolated chromaffin granules

The accumulation of amantadine and the effect of this pharmacologic amine upon the magnitude of the transmembrane proton gradient (ΔpH) and of the transmembrane potential gradient (ΔΨ) were investigated in bovine adrenal chromaffin granules isolated in isotonic sucrose. Freshly isolated chromaffin granules have an intragranular pH of 5.5, as measured by [^14C]methylamine distribution [R. G. Johnson and A. Scarpa, J. gen. Physiol.68, 601 (1976)]. The addition of amantadine (1–50 mM) to well-buffered suspensions of granules at pH 7.0 resulted in a dose-related alkalinization of the granule interior. Similar results were obtained with equivalent external concentrations of ammonia. When the time-resolved influx of labeled amines into the granules was studied radiochemically, using low external amine concentrations, the accumulation of [³H]amantadine was quite similar to that of [¹⁴C]methylamine with regard to rate and extent over a wide range of magnitudes of the transmembrane proton gradient. However, unlike biogenic amine accumulation into the chromaffin granule, which is driven by both transmembrane proton and potential gradients, the accumulation of [³H] amantadine was not stimulated by the existence of a transmembrane potential, nor was it inhibited by reserpine. Moreover, low concentrations of amantadine did not competitively inhibit biogenic amine accumulation in the isolated granules. These results indicate that amantadine can distribute across the membrane of chromaffin granules according to the magnitude of the endogenous ΔpH, and suggest that in vivo amantadine may be concentrated and stored as a pharmacologic agent in amine containing granules.     

Fate of [3H]amezinium in sympathetically innervated rabbit tissues

Perfused rabbit hearts accumulated intra-aortically infused [³H]amezinium. At concentrations of 1 or 10 nM, the accumulation proceeded at a constant rate for at least 60 min. After 60 min, tissue medium ratios were between 6 (1 μM) and approx. 100 (1 or 10 nM [³H]amezinium). Cocaine or pretreatment with 6-hydroxydopamine abolished, and pretreatment with reserpine reduced the accumulation of [³H]amezinium (1nM). Kinetic analysis yielded a K_m value of 0.9 μM and a V_max of 1.2 nmole g^?1 min^?1. When hearts had been labelled with 1 or 10 nM [³H]amezinium, the fractional rate of the subsequent efflux was very low (0.001 min^?1). It was greatly increased when the animals had been pretreated with reserpine. Electrical stimulation of the sympathetic nerves released [³H] amezinium from pre-labelled rabbit pulmonary artery strips. The electrically evoked overflow was abolished by tetrodotoxin or omission of Ca²⁺; it was enhanced by cocaine, desipramine and yohimbine and decreased by clonidine. The results show that amezinium, at least at low concentrations, is selectively taken up into postganglionic sympathetic neurones, that it is partly sequestered in the vesicles, and that it is released by action potentials. Amezinium is a structurally novel substrate of both the noradrenaline transport mechanism of the axolemma and the transport mechanism of the noradrenaline-storing synaptic vesicles.     

Uptake and storage of catecholamines in rabbit brain after chronic reserpine treatment

The brains of rabbits treated chronically with small doses of reserpine have been examined by the histochemical fluorescence method for dopamine, noradrenaline and 5-hydroxytryptamine. Measurements were made 4 and 24 hr after the last reserpine injection both in vivo and in vitro. Evidence has been obtained that the small functionally-important pool of amine can be directly visualized in the fluorescence microscope. It was found to be present intraneuronally, probably localized to amine storage granules. The degree of functional recovery was correlated with the recovery, as shown by the increased fluorescence, of a small intraneuronal pool of amine and with the ability of amine storage granules to take up monoamines.     

A note on the blockade of uptake of noradrenaline by 4-chloro-alpha,alpha-dimethylphenethylaminopropan-2-one in rodents

4-Chloro-αα-dimethylphenethylaminopropan-2-one blocked uptake of [³H]noradrenaline in the heart, had no effect on the endogenous levels of catecholamines or 5-hydroxytryptamine in the heart, brain or adrenals and decreased the [³H]noradrenaline-depleting activity of metaraminol, but not reserpine, in rodents. The compound appears to act by interfering with the active transport of noradrenaline through the nerve cell membrane.     

On the reduced retention of extravesicular noradrenaline in rabbit atria produced by potassium

The effects of KCl on the retention of extravesicular (?)-[³H]noradrenaline were investigated. Atria, from reserpine-pretreated rabbits, were exposed to tropolone and pargyline before incubation with (?)-[³H]noradrenaline. After efflux for 50–60 min, exposure of tissues to media containing 65 mM KCl resulted in increased efflux of (?)-[³H]noradrenaline. This effect was not Ca²⁺-dependent and was not altered by oxymetazoline, phentolamine, indomethacin or methacholine. The KCl-induced reduction in retention of (?)-[³H]noradrenaline was, however, inhibited by cocaine and desipramine, but not by lidocaine. Efflux was not increased by RbCl and CsCl. It is suggested that KCl may have accelerated the efflux of extravesicular (?)-[³H]noradrenaline by increasing a cocaine- and desipramine-sensitive carrier-mediated efflux process.     

The uptake and retention of [3H]noradrenaline in rat sciatic nerves after ligation

The uptake and retention of [³H]noradrenaline (³H-NA) was examined in sciatic nerves of albino rats. In the 1 cm part of nerve proximal to a 12 hr ligation the uptake and retention of exogenous noradrenaline was about 4 times the uptake in 1 cm of normal unligated nerves. Treatment with reserpine 10 hr before killing caused a marked decrease in the estimated amount of ³H-NA, while injection of nialamide 15 min before ³H-NA administration in ligated, reserpine-treated animals caused a somewhat larger uptake and retention of ³H-NA in the nerve part above the ligation. Protriptyline, a blocker of the “membrane pump”, was approximately 3 times less effective in 12 hr-ligated nerves than in unligated nerves, indicating a reduced efficiency of the “membrane pump” in the distended axons above a ligation.     

Inhibition of amine uptake in the mouse heart by some new "thymoleptic" drugs

A number of new and established thymoleptic drugs were given to mice. Their inhibitory action on the uptake of [³H]metaraminol and [³H]noradrenaline in the heart was investigated. Among the compounds tested a monomethylamino-derivative was in general 2–3 times more potent than the corresponding dimethyl-amino-derivative. The phthalan derivative 3,3-dimethyl-1-(3-methylaminopropyl)-1-phenylphthalan (Lu 3–010) was as efficient as protriptyline in all the tests performed. Changes in the substitution of the phthalan skeleton influenced the activity critically. As it is devoid of anticholinergic activity Lu 3–010 appears to be the most specific inhibitor of the amine transport mechanism of the adrenergic cell membrane found so far.     

Effects of S9977 on adrenergic neurotransmission

1. Experiments were designed to determine whether or not the putative promnesic drug S9977 (1,3,7-trimethyl 8-[3-(4-diethylaminocarbonyl-1-piperazinyl) 1-propyl]-3,7-dihydro (1H)2,6-purinedione hydrochloride) affects peripheral adrenergic neurotransmission.2. Rings of canine saphenous veins (without endothelium) were suspended for isometric tension recording in conventional organ chambers filled with modified Krebs-Ringer bicarbonate solution. The adrenergic nerve endings were activated with electrical impulses (9 V, 2 msec, 0.25–8 Hz).3. At 10⁻⁵ M, S9977 significantly reduced the contraction to 0.25, 0.5 and 1 Hz. The compound did not affect the response to higher stimulation frequencies or to exogenous noradrenaline. The inhibitory effect of S9977 was prevented by methiothepin, and not affected by atropine or 8-phenyltheophylline.4. Helical strips of canine saphenous veins were incubated with [³H]noradrenaline and suspended for superfusion and isometric tension recording. Under basal conditions, S9977 (10⁻⁴ M) augmented, the total ³H-overflow which was due mainly to an augmented overflow of [³H]deoxyphenylglycol (DOPEG); the extraneuronal metabolites 3,4-dihydromandelic acid (DOMA) and 3-methoxy-4-hydroxymandelic acid (VMA) were reduced.5. During electrical stimulation of the adrenergic nerves, S9977 (10⁻⁴ M) augmented the total ³H-overflow but reduced the contractile response; the evoked overflow of [³H]noradrenaline was not significantly affected.6. These experiments suggest that S9977 the displacement of noradrenaline from the adrenergic varicosities; most of the displaced transmitter is metabolized by intraneuronal monoamine oxidase before reaching the junctional cleft. In addition, S9977 exerts an inhibitory effect on the extraneuronal metabolism of catecholamines. S9977 does not inhibit the exocytotic release of the adrenergic neurotransmitter.     

The effect of corticosterone on extraneuronal amine uptake and effector response in rat salivary glands

The effect of corticosterone on effector cell response to noradrenaline in vivo and on extraneuronal amine uptake in vitro has been investigated in rat submaxillary glands. When tissue slices were incubated with [³H]noradrenaline the level of extraneuronally retained radioactive material was found to be markedly reduced at a concentration of 10 μg ml^?1 of corticosterone after inhibition of neuronal uptake by protriptyline. Corticosterone in a dose of 10 mg kg^?1 was found to markedly potentiate the secretory response to noradrenaline in vivo, when the neuronal uptake of noradrenaline was blocked by protriptyline (10 mg kg^?1, i.p.). Inhibition of neuronal uptake alone by protriptyline or of the extraneuronal uptake alone by corticosterone in the doses used here did not affect the dose-response curve for noradrenaline, at least not in its lower part. The data thus clearly show that the extraneuronal amine uptake of rat salivary glands is blocked by corticosterone and that this extraneuronal uptake might be regarded as a mechanism of importance for the inactivation of the adrenergic transmitter.     

Effect of reserpine on adenosine uptake and metabolism,and subcellular transport of platelet adenosine triphosphate in washed rabbit platelets

There is a slow exchange of adenine nucleotides between the metabolically active (cytoplasmic) pool and the releasable amine storage organelle pool of blood platelets. Reserpine is known to inhibit serotonin uptake into platelet storage organelles. Therefore, we have determined whether reserpine also inhibits the uptake of adenine nucleotides from the cytoplasm into the storage organelles of rabbit platelets. Transport of adenine nucleotides from the metabolically active pool into the releasable amine storage granule pool was followed by labeling the metabolically active pool of adenine nucleotides by incubating the platelets with [¹⁴C]adenosine or [¹⁴C]adenine. Practically complete release of amine storage granule constituents was induced at various times in aliquots of the labeled platelet suspensions by treatment with a high concentration of thrombin (0.45 units/ml. The fraction of the total labeled [¹⁴C]ATP released was taken as a measure of ATP transport from the metabolically active pool into the releasable pool. Reserpine (0.2 and 2 μM) decreased the rate of ATP transport into the storage granules by about 50 per cent. Platelets obtained from rabbits that had received 5 mg/kg of reserpine intraperitoneally 18 hr prior to the collection of blood released less ATP and ADP than control platelets from animals that had not received any drugs. This was not due to inhibition of the release reaction by reserpine. Since reserpine reduces the amount of adenine nucleotides in the storage granules, we conclude that if it affects the rate of efflux of adenine nucleotides from the granules at all, this effect must be slight compared with the inhibition of the uptake into the granules. Reserpine was also found to decrease the incorporation of [8-¹⁴C]adenosine into platelet adenine nucleotides by inhibiting adenosine uptake into the platelets noncompetitively (K_i = 2 μM). Inosine uptake was also inhibited by reserpine. The effect of reserpine on adenosine uptake was reversible. In contrast, the effect of reserpine on ATP transfer from the metabolically active pool into the releasable pool was irreversible. This is in keeping with earlier observations that some reserpine binds to platelets reversibly and some binds irreversibly.     

In vitro synthesis of noradrenaline in ganglia and salivary glands after in vivo preganglionic stimulation

The extent of synthesis of [¹⁴C]noradrenaline from [¹⁴C]tyrosine and from [¹⁴C]dopamine was assessed in slices of superior cervical ganglia, representing cell bodies, and in submaxillary salivary glands, representing terminals of noradrenergic neurons of the cat. Immediately and 6 h after preganglionic stimulation for 3 h the rate of synthesis of noradrenaline from tyrosine and dopamine was not altered in ganglia. In salivary glands, however, synthesis of noradrenaline from both tyrosine and dopamine was increased at both times. These results suggest that acute periods of increased neural activity results in the acceleration of noradrenaline synthesis in the terminals but not in the cell bodies of noradrenergic neurons.