Jan Häggendal

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.

The recovery of the capacity for uptake‐retention of [3H]noradrenaline in rat adrenergic nerves after reserpine

The uptake retention of [3H]noradrenaline (2.5 μg/kg, i.v., 30 min before death) in rat salivary glands was studied at different times after reserpine treatment (10 mg/kg, i.p.). The effect of removing the cervical superior ganglion 12 h before death on the recovery of the [3H]noradrenaline uptake‐retention capacity after reserpine was also investigated. The ganglionectomy was unilateral, and the contralateral side was always preganglionically denervated. In glands with uninterrupted postganglionic adrenergic nerves the onset of recovery of the [3H]noradrenaline retention capacity occurred 24–36 h after reserpine. Normal contents were found on the second to third days. Between day 3 and 6 a possible overshoot of [3H]nor‐ adrenaline content, followed by normal and subnormal contents (7–21 days) were recorded. Ganglionectomy, 12 h before death, markedly delayed the recovery of [3H]noradrenaline retention capacity. Both the recovery curve for [3H]noradrenaline retention in glands with intact postganglionic nerves, and the effect of ganglionectomy on the [3H]noradrenaline retention capacity, were clearly related to the relative number of new functioning amine granules that are transported via the axons to the nerve terminals at different times after the reserpine‐pretreatment. The results indicate that young amine granules, recently transported to the nerve terminals via the axons, have the greatest capacity to take up and retain [3H]noradrenaline. The half‐life of this capacity in the young granules appears to be about 12 h. Since published results indicate that [3H]noradrenaline is initially taken up in the “small easily releasable pool” of transmitter, we suggest that young amine granules are of the greatest importance for adrenergic function, i.e. that they are particularly active in taking up recaptured noradrenaline, in the synthesis, and in the release of this transmitter.

Recovery of noradrenaline levels after reserpine compared with the life‐span of amine storage granules in rat and rabbit

SIR,-After reserpine treatment the noradrenaline in peripheral tissues and in the central nervous system (CNS) rapidly decreases to very low levels, and the recovery takes place slowly over several weeks (Carlsson, Rosengren, Bertler & Nilsson, 1957). The mechanism appears to be a longlasting blockage of the storage mechanism in the amine granules (Bertler, Hillarp & Rosengren, 1961, Carlsson, Hillarp & Waldeck, 1963; Lundborg, 1963; Dahlstrom, Fuxe & Hillarp, 1965). However, the recovery time for the cell bodies is much shorter (24-48 hr) both centrally (Dahlstrom & Fuxe, 1964a) and peripherally (Norberg & Hamberger, 1964). Thus, a great difference exists between the recovery time after reserpine treatment in the adrenergic nerve cell bodies and in their terminals. This difference may be explained by the fact that the storage granules are formed in the pericarya of the neurones and transported down the axons to the terminals (see, inter alia, Dahlstrom, 1965). The recovery time for noradrenaline after reserpine has in this study been examined in different tissues of the rat and rabbit. Brain, heart and skeletal muscle (gastrocnemius) were examined. Male albino rats (Sprague-Dawley, 200 g) and male albino rabbits (14-2.0 kg) were injected with reserpine (Serpasil ampoules, 2.5 mg/ml, diluted with isotonic glucose solution) intraperitoneally (rats, 1 and 10 mg/kg) and intravenously (rabbits, 0.2 and 2 mg/kg). The animals were killed 48 hr, 1, 2, 3, 4, 5, and 6 weeks (rat) or 48 hr, 1, 2, 4 and 6 weeks (rabbit) after the injection. Noradrenaline was measured spectrophotofluorimetrically (Bertler, Carlsson & Rosengren, 1958 ; Haggendal, 1963). The recovery of noradrenaline after reserpine administration could be represented graphically as an approximately straight line for all the tissues examined. The time required for a total recovery was between 4 and 5 weeks for the rat after both 1 and 10 mg/kg doses. For the rabbit the corresponding time was about 7 weeks after the high dose (2 mg/kg); at this dose the amines decreased initially to very low levels. After the lower dose of 0.2 mg/kg, the initial decrease was less marked and the recovery time was about 6 weeks. However, if the recovery curve for this latter group of rabbits was extrapolated to zero level of noradrenaline the total time for recovery was about 7 weeks. These findings are supported by results obtained by Haggendal & Lindqvist (1964) on the effect of a single dose of reserpine (0.2 and 1 mg/kg) on the catecholamine levels in brain and heart of the albino rabbit. If the recovery curves of both brain and heart noradrenaline were extrapolated to zero and to normal levels the time required for a total recovery would be about 6 weeks. The storage granules being synthesised in the cell body and transported via the axons to the adrenergic terminals have a course of transport which has been found to be linear (Dahlstrom & Haggendal, 1966a, b). The adrenergic nerve terminals are thus supplied with newly formed granules at a rate which is in all probability fairly steady. The time required for a total exchange of granules in the terminals (the life-span of the granules) has been calculated for hind-leg skeletal muscle of rat to be about 5 weeks (Dahlstrom & Haggendal, 1966a), and for the rabbit about 7 weeks (Dahlstrom & Haggendal, 1966b). The straightness of the noradrenaline recovery curve of rat and rabbit after reserpine treatment and the fact that the time required for a total recovery for both species is close to the calculated life-span of the amine granules, indicate that the course of the noradrenaline recovery after reserpine reflects the downward-transport of the newly formed storage granules, unaffected by reserpine.

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.

The time course of noradrenaline decrease in rat spinal cord following transection

Abstract The time course of degeneration of bulbospinal monoamine neurones following spinal cord transection was studied biochemically and histochemically in 3 consecutive 1 cm parts distal to transection. In the part immediately below transection clear degenerative changes were found on day 2, in the more distal part on day 4, and latest in the most distal part. This time-sequence of nerve terminal degeneration is probably related to the interruption of the comparatively slow intra-axonal transport in these neurons. The results indicate a complex situation in the nerve terminal-receptor area in the caudal part of the spinal cord following transection.

Some Aspects of the Quantal Release of the Adrenergic Transmitter

In a recent study concerning the noradrenaline (NA) release at the vasoconstrictor nerve endings in the cat calf muscles, the results suggested that the transmitter amount released per stimulus was only about 1/50.000 of the total regional NA content (Folkow et al., 1967). Assuming that, first, by far the major part of tissue NA is present in the varicosity granules (vesicles) and, second, the majority of varicosities discharge transmitter when an impulse arrives, it was deduced that some 400 NA molecules would be released per varicosity and impulse. Such an amount, which creates a peak NA concentration of 0.5 to 1 μg/ml if evenly distributed in a junction gap having the size of 1 to 2 μ2 and a width of 1000 A, would correspond to only some 3% of the NA content of one granule, provided that the varicosity contains about 1000 granules and each granule about 15.000 NA molecules. These figures were based on calculations of the number of varicosities in the rat peripheral adrenergic neuron and the total NA content of the corresponding tissue (Dahlstbom et al., 1966).

Effects of phenoxybenzamine on transmitter release and effector response in the isolated portal vein

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The Importance of Axoplasmic Transport of Amine Granules for the Functions of Adrenergic Neurons

In the adrenergic neurons, the amine granules which are formed in the cell bodies and transported to the nerve terminals at a rate of several mm/h, probably play an important role for the functions of the nerve terminals. Results obtained with and without reserpine pre-treatment indicate that the average life-span of the granules with regard to their capacity to store endogenous noradrenaline (NA) is about 4 weeks. The capacity of the granules to store 3H-NA, on the other hand, appears to be rather short-lasting, in the order of a few days. This indicates that the new amine granules may be more active in storing 3H-NA than the older granules. Hypothetically, also the release of the transmitter may occur predominatly from the new granules.

The depletion and recovery of noradrenaline in the brain and some sympathetically innervated mammalian tissues after tetrabenazine.

The depleting effect of tetrabenazine on the monoamine levels appears generally to be less in peripheral tissues than in the central nervous system. The noradrenaline levels in brain and some sympathetically innervated tissues such as heart, submandibular glands, and skeletal muscle were examined in the rat after administration of the drug. The levels of 5‐hydroxytryptamine, dopamine and noradrenaline were also estimated in the rabbit brain after tetrabenazine and compared with the levels in the rabbit heart. In both the brain and peripheral tissues the monoamine levels were strongly reduced 4 hr after tetrabenazine and increased thereafter, reaching normal levels after about 36 to 48 hr. The site of action of tetrabenazine is briefly discussed and compared to the site of action of reserpine.

THE POSSIBLE IMPORTANCE OF YOUNG (LARGE) AMINE STORAGE GRANULES FOR ADRENERGIC NERVE TERMINAL FUNCTION

Publisher Summary This chapter elaborates possible importance of young (large) amine storage granules for adrenergic nerve terminal function. Following one large dose of reserpine, catecholamines and serotonin are depleted centrally and peripherally. The recovery of endogenous noradrenaline (NA) has been studied previously. The reserpine effect is considered to be because of an irreversible blockade of the uptake-storage mechanism of the granules. Because functioning amine granules are necessary for nerve terminal functions 3H-NA uptake-retention and transmission are also markedly depressed during the initial period after reserpine. Onset of recovery of the three different parameters occurs in the rat peripheral tissues 24–36 h after reserpine. Within the same period of time functioning, NA containing granules start to appear in the nerve terminals of long adrenergic neurons. Recovery to normal, however, proceeds differently, 3H-NA uptake-storage and transmission are normalized within 2–3 days, while endogenous NA reach normal levels after 3–5 weeks. These results indicate (1) that uptake-retention of 3H-NA occurs mainly in “young” amine granules recently arrived to the nerve terminals, and (2) that this capacity of the “young” granules is rather shortlasting. The T½ for this capacity has been estimated to around 12 h.