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Release of 3H-metaraminol by adrenergic nerve stimulation in reserpine-treated rats
EUROPEAN JOURNAL OF PHARMACOLOGY 9 (1970) 344-346. NORTH-HOLLAND PUBLISHING COMP., AMSTERDAM
RELEASE OF 3H-METARAMINOL STIMULATION
BY A D R E N E R G I C
IN RESERPINE-TREATED
NERVE
RATS
Olle ALMGREN and Per G. LUNDBORG Department of Pharmacology, University of Goteburg, Goteburg, Sweden Received 22 April 1969
Accepted 24 November 1969
O. ALMGREN and P.G. LUNDBORG, Release of 3H-metaraminol by adrenergic nerve stimulation in reserpinetreated rats, European J. Pharmacol. 9 (1970) 344-346. Ten rats were injected with reserpine and 4 hr later with 3H-metaraminol. Stimulation of the cervical sympathetic chain for 30 rain with 10 impulses per second produced a slight but significant decrease in the metaraminol content of the particulate (granular) fraction of the salivary glands. The metaraminol content of the supernatant fraction was unaffected. The data give additional support for the view, that granular uptake of an amine must precede release by nerve stimulation. Metaraminol Nerve stimulation
Subcellular distribution Monoamines
Reserpine
1. INTRODUCTION
2. MATERIALS AND METHODS
In a previous communication it was shown that most of the 3H-metaraminol (MA) released from adrenergic nerves following stimulation came from the amine storage granules (Almgren, Lundborg and Stitzel, 1969). This supports the hypothesis that incorporation of the transmitter into the particle bound store must occur before it is available for release by nerve impulses. However, the abovementioned experiments did not completely exclude the possibility that some release might occur directly from the extragranular cytoplasm. Reserpine has been shown to inhibit the uptake of catecholamines and related amines by the storage granules of adrenergic nerves in vitro (Euler and Lishajko, 1963) and in vivo (Lundborg and Stitzel, 1967). In the present paper, animals treated with reserpine were used for investigating the availability of different subcellular fractions for release by the nerve impulse.
Male Sprague-Dawley rats, weighing between 1 5 0 - 3 3 0 g, were injected with 10 mg/kg reserpine intraperitoneally and kept at an ambient temperature of about 18°C for three hours. They were then transferred to an environmental temperature of 29°C to prevent further hypothermia. Four hours after the administration of reserpine the rats received 40/~g/kg of all-labelled (+)MA by injection into a tail vein. About 5 rain after the injection of the labelled amine the rats were anaesthesized with 1 g/kg urethane injected intraperitoneally and a tracheal cannula was inserted. The right cervical sympathetic trunk together with the vagus nerve was isolated under a dissecting microscope, ligated and cut at the level of the clavicle. By careful dissection the nerve trunks were freed from the carotid artery up to the level of the bifurcation, where the vagus nerve was again cut. The cervical sympathetic trunk of the other side was also cut to provide a control deprived of nervous activity.
RESERPINE AND NERVE STIMULATION
The rats were then fLxed on a heated table at a constant temperature of 37°C. The isolated nerve trunks were placed on a bipolar platinum electrode and immersed in paraffin oil. Stimulation with rectangular monophasic pulses from a Grass $4 stimulator was begun 30 min after the MA injection. Stimulation at a frequency of 10 impulses per second was continued for 30 min. In some experiments contractions of the lower eye-lid were recorded as described by Obianwu (1967). Previous experiments have shown that the stimulation parameters, a strength of 5 V and a duration of 5 msec, were supramaximal. Immediately after termination of the stimulation the submaxillary and sublingual glands of both sides were taken out and homogenized in icecold 0.25 M sucrose. A coarse fraction was obtained by centrifugation of the homogenates at 2000g for 10min. The supernatant was then centrifuged at 100,000g in a Spinco Model L ultracentrifuge providing a semident (particulate) fraction and a high speed supernatant. The 3H-MA content of the various fractions was estimated by liquid scintillation counting after ion exchange chromatography. The method has previously been described in detail (Almgren, Lundborg and Stitzel, 1969; Lundborg, 1967; Stitzel and Lundborg, 1967). Statistical analysis of the results presented was based upon the difference between the pair of gland from each rat. Substances used: Reserpine (Serpasil®) was generously supplied by Swedish Ciba Ltd; tritium labelled (+)-metaraminol was prepared in cooperation with Research Laboratories, Hassle Ltd. (Hallhagen and Waldeck, 1968).
3. RESULTS 3.1. The uptake of 3H-MA in subcellular fractions of rat salivary glands The results are presented in fig. I together with results from corresponding experiments with animals pretreated with reserpine (Almgren, Lundborg and Stitzel, 1969) for comparison. In the rats pretreated with reserpine the mean total a H-MA content (i.e. the sum of the fractions) of the unstimulated salivary gland one hour after the injection of the labelled amine was 46.4 -+ 4.27
345
~ Control L~ Stimulated ~1 Diff Contr- Stim. ± se(diff.} 50
Untreated n=lO
40
30 20 I0
Fraction:
0
N rn Coarse
Particulate Supernatax~t
30 Reserpine 2o f J~ n=lO ,o
Fraction:o
§
treated
~~ §
Q" Coarse ParticulateSupernatont
Fig. 1. Content of 3H-metaraminol in ng/g in coarse, particulate and supernatant fractions of stimulated (right) and unstimulated (left) submaxillary + sublingual glands 1 hr after the injection of the labelled amine (40 ug/kg). The statistical analysis was based upon the difference between matched pairs. For comparative purposes, results from corresponding experiments with animals nor pretreated with reserpine (Almgren, Lundborg and Stitzel, 1969) are included in the figure.
(S.E.M.) ng/g. About 35% of the aH-MA was recovered from the course fraction, 10% from the particulate and 55% from the supernatant fraction. The P/(P+S) ratio, i.e. the amount of labelled amine in particulate fraction as a proportion of that found in the particulate + supernatant fractions, amounted to 15.0 + 1.06%. In agreement with earlier observations (Lundborg and Stitzel, 1967; Lundborg, unpublished) the subcellular distribution of 3H-MA at this short time interval after its injection did not differ very much from that observed in animals not pretreated with reserpine. 3.2. Effect of sympathetic nerve stimulation on the release of 3H-MA from subcellular fractions of salivary glands of rats treated with reserpine Stimulation of the cervical sympathetic chain with
346
O.ALMGREN and P.G.LUNDBORG
a frequency of 10 impulses per second for 30 min induced no observable effects on pupil diameter, bulb protrusion, eye-lid contraction or salivation. Neither was there any reduction ( p > 0 . 1 0 ) in the total 3H-MA content o f the stimulated gland. There was a slight but significant decrease, however, in the 3H-MA content o f the particulate fraction from about 4.4 ng/g to about 3.6 ng/g (p<0.005). The 3H-MA content o f the supernatant was not influenced by nerve stimulation. The P/(P+S) ratio was thus significantly reduced from 15.0 to 11.9 percent (mean difference 3.2 + 0.67; p < 0 . 0 0 5 ) .
limited extent the reserpine-induced blockade of granular uptake and thus reachs positions available for release by the nerve impulse. Possibly the resistance o f 3 H-MA to monoamine oxidase may facilitate such a process. In support of the hypothesis the observations o f Haggendal and Malmfors (1969) may be quoted. These authors observed that in rats treated with reserpine and a monoamine oxidase inhibitor, dopamine given in large dosage was capable o f restoring noradrenaline release by nerve stimulation.
ACKNOWLEDGEMENTS 4. DISCUSSION In our earlier study in animals not treated with reserpine, nerve stimulation caused a marked decrease in 3H-MA in the granular fraction and a slight decrease in the supernatant fraction. In the present study with reserpine-treated animals, nerve stimulation caused a slight, though significant decrease o f the 3H-MA in the granular fraction but had no effect on that in the supernatant fraction. It is suggested therefore that nerve stimulation primarily affects the granular fraction and it may have no direct effect on the supernatant fraction. Experiments with 3 H-noradrenaline indicate that about half o f the amine originally stored in the granules is released into the supernatant during the fractionation procedure (Stitzel and Lundborg, 1967). The slight decrease in supernatant S H-MA following nerve stimulation in non-reserpinized rats may thus very well be an artefact due to release during fractionation. The lack of a decrease o f supernatant 3H-MA in reserpine-treated animals supports this interpretation: in this case, the effect o f nerve stimulation on granular 3H-MA was not of sufficient magnitude to make such an artefact detectable. It is remarkable that the large dose o f reserpine used did not completely inhibit the release o f 3H-MA b y nerve stimulation from the granular fraction. Probably the concomitant release o f noradrenaline was very slight or absent, judging by the complete absence of physiological responses. It may be speculated that 3H-MA is capable of circumventing to a
The research reported in this manuscript has been supported by the Swedish State Medical Research COuncil (B69-14X-155-05A, B69-14X-2157-03) and the faculty of Medicine, University of Goteborg, Sweden. For skilful technical assistance we are indebted to Mrs Marianne Olovsson and Miss Lena Ramstedt.
REFERENCES Almgren, O., P. Lundborg and R. Stitzel, 1969, Release of 3H-metaraminol from subcellular fractions of rat salivary glands by nerve stimulation, European J. Pharmacol. 6 (1969) 109. Euler, U.S.v. and F. Lishajko, 1963, Effect of adenine nucleotides on catecholamine release and uptake in isolated adrenergic nerve granules, Acta Physiol. Scand. 59, 454. Hallhagen, G. and B. Waldeck, 1968, Synthesis of tritium labelled metaraminol, ~-methylnoradrenaline and their corresponding f3-desoxy-derivatives, J. Labelled Compounds 4, 72. Haggendal, J. and T. Malmfors, 1969, The effect of nerve stimulation on catecholamines taken up in adrenergic nerves after reserpine pretreatment, Acta Physiol. Scand. 75, 33. Lundborg, P., 1967, Studies on the uptake and subceUular distribution of catecholamines and their Ot-methylated analogues, Acta Physiol. Scand. Suppl. 302. Lundborg, P. and R. Stitzel, 1967, Uptake of biogenic amines by two different mechanisms present in adrenergic granules, Brit. J. Pharmacol. 29, 342. Obianwu, H., 1967, Sympathetic and receptor blockade after prenylamine (Segontin(~), Acta Pharmacol. Toxicol. 25, 141. Stitzel, R.E. and P. Lundborg, 1967, Effect of reserpine and monoamine oxidase inhibition on the uptake and subcellular distribution of 3H-noradrenaline, Brit. J. Pharmacol. 29, 99.
RELEASE OF 3H-METARAMINOL STIMULATION
BY A D R E N E R G I C
IN RESERPINE-TREATED
NERVE
RATS
Olle ALMGREN and Per G. LUNDBORG Department of Pharmacology, University of Goteburg, Goteburg, Sweden Received 22 April 1969
Accepted 24 November 1969
O. ALMGREN and P.G. LUNDBORG, Release of 3H-metaraminol by adrenergic nerve stimulation in reserpinetreated rats, European J. Pharmacol. 9 (1970) 344-346. Ten rats were injected with reserpine and 4 hr later with 3H-metaraminol. Stimulation of the cervical sympathetic chain for 30 rain with 10 impulses per second produced a slight but significant decrease in the metaraminol content of the particulate (granular) fraction of the salivary glands. The metaraminol content of the supernatant fraction was unaffected. The data give additional support for the view, that granular uptake of an amine must precede release by nerve stimulation. Metaraminol Nerve stimulation
Subcellular distribution Monoamines
Reserpine
1. INTRODUCTION
2. MATERIALS AND METHODS
In a previous communication it was shown that most of the 3H-metaraminol (MA) released from adrenergic nerves following stimulation came from the amine storage granules (Almgren, Lundborg and Stitzel, 1969). This supports the hypothesis that incorporation of the transmitter into the particle bound store must occur before it is available for release by nerve impulses. However, the abovementioned experiments did not completely exclude the possibility that some release might occur directly from the extragranular cytoplasm. Reserpine has been shown to inhibit the uptake of catecholamines and related amines by the storage granules of adrenergic nerves in vitro (Euler and Lishajko, 1963) and in vivo (Lundborg and Stitzel, 1967). In the present paper, animals treated with reserpine were used for investigating the availability of different subcellular fractions for release by the nerve impulse.
Male Sprague-Dawley rats, weighing between 1 5 0 - 3 3 0 g, were injected with 10 mg/kg reserpine intraperitoneally and kept at an ambient temperature of about 18°C for three hours. They were then transferred to an environmental temperature of 29°C to prevent further hypothermia. Four hours after the administration of reserpine the rats received 40/~g/kg of all-labelled (+)MA by injection into a tail vein. About 5 rain after the injection of the labelled amine the rats were anaesthesized with 1 g/kg urethane injected intraperitoneally and a tracheal cannula was inserted. The right cervical sympathetic trunk together with the vagus nerve was isolated under a dissecting microscope, ligated and cut at the level of the clavicle. By careful dissection the nerve trunks were freed from the carotid artery up to the level of the bifurcation, where the vagus nerve was again cut. The cervical sympathetic trunk of the other side was also cut to provide a control deprived of nervous activity.
RESERPINE AND NERVE STIMULATION
The rats were then fLxed on a heated table at a constant temperature of 37°C. The isolated nerve trunks were placed on a bipolar platinum electrode and immersed in paraffin oil. Stimulation with rectangular monophasic pulses from a Grass $4 stimulator was begun 30 min after the MA injection. Stimulation at a frequency of 10 impulses per second was continued for 30 min. In some experiments contractions of the lower eye-lid were recorded as described by Obianwu (1967). Previous experiments have shown that the stimulation parameters, a strength of 5 V and a duration of 5 msec, were supramaximal. Immediately after termination of the stimulation the submaxillary and sublingual glands of both sides were taken out and homogenized in icecold 0.25 M sucrose. A coarse fraction was obtained by centrifugation of the homogenates at 2000g for 10min. The supernatant was then centrifuged at 100,000g in a Spinco Model L ultracentrifuge providing a semident (particulate) fraction and a high speed supernatant. The 3H-MA content of the various fractions was estimated by liquid scintillation counting after ion exchange chromatography. The method has previously been described in detail (Almgren, Lundborg and Stitzel, 1969; Lundborg, 1967; Stitzel and Lundborg, 1967). Statistical analysis of the results presented was based upon the difference between the pair of gland from each rat. Substances used: Reserpine (Serpasil®) was generously supplied by Swedish Ciba Ltd; tritium labelled (+)-metaraminol was prepared in cooperation with Research Laboratories, Hassle Ltd. (Hallhagen and Waldeck, 1968).
3. RESULTS 3.1. The uptake of 3H-MA in subcellular fractions of rat salivary glands The results are presented in fig. I together with results from corresponding experiments with animals pretreated with reserpine (Almgren, Lundborg and Stitzel, 1969) for comparison. In the rats pretreated with reserpine the mean total a H-MA content (i.e. the sum of the fractions) of the unstimulated salivary gland one hour after the injection of the labelled amine was 46.4 -+ 4.27
345
~ Control L~ Stimulated ~1 Diff Contr- Stim. ± se(diff.} 50
Untreated n=lO
40
30 20 I0
Fraction:
0
N rn Coarse
Particulate Supernatax~t
30 Reserpine 2o f J~ n=lO ,o
Fraction:o
§
treated
~~ §
Q" Coarse ParticulateSupernatont
Fig. 1. Content of 3H-metaraminol in ng/g in coarse, particulate and supernatant fractions of stimulated (right) and unstimulated (left) submaxillary + sublingual glands 1 hr after the injection of the labelled amine (40 ug/kg). The statistical analysis was based upon the difference between matched pairs. For comparative purposes, results from corresponding experiments with animals nor pretreated with reserpine (Almgren, Lundborg and Stitzel, 1969) are included in the figure.
(S.E.M.) ng/g. About 35% of the aH-MA was recovered from the course fraction, 10% from the particulate and 55% from the supernatant fraction. The P/(P+S) ratio, i.e. the amount of labelled amine in particulate fraction as a proportion of that found in the particulate + supernatant fractions, amounted to 15.0 + 1.06%. In agreement with earlier observations (Lundborg and Stitzel, 1967; Lundborg, unpublished) the subcellular distribution of 3H-MA at this short time interval after its injection did not differ very much from that observed in animals not pretreated with reserpine. 3.2. Effect of sympathetic nerve stimulation on the release of 3H-MA from subcellular fractions of salivary glands of rats treated with reserpine Stimulation of the cervical sympathetic chain with
346
O.ALMGREN and P.G.LUNDBORG
a frequency of 10 impulses per second for 30 min induced no observable effects on pupil diameter, bulb protrusion, eye-lid contraction or salivation. Neither was there any reduction ( p > 0 . 1 0 ) in the total 3H-MA content o f the stimulated gland. There was a slight but significant decrease, however, in the 3H-MA content o f the particulate fraction from about 4.4 ng/g to about 3.6 ng/g (p<0.005). The 3H-MA content o f the supernatant was not influenced by nerve stimulation. The P/(P+S) ratio was thus significantly reduced from 15.0 to 11.9 percent (mean difference 3.2 + 0.67; p < 0 . 0 0 5 ) .
limited extent the reserpine-induced blockade of granular uptake and thus reachs positions available for release by the nerve impulse. Possibly the resistance o f 3 H-MA to monoamine oxidase may facilitate such a process. In support of the hypothesis the observations o f Haggendal and Malmfors (1969) may be quoted. These authors observed that in rats treated with reserpine and a monoamine oxidase inhibitor, dopamine given in large dosage was capable o f restoring noradrenaline release by nerve stimulation.
ACKNOWLEDGEMENTS 4. DISCUSSION In our earlier study in animals not treated with reserpine, nerve stimulation caused a marked decrease in 3H-MA in the granular fraction and a slight decrease in the supernatant fraction. In the present study with reserpine-treated animals, nerve stimulation caused a slight, though significant decrease o f the 3H-MA in the granular fraction but had no effect on that in the supernatant fraction. It is suggested therefore that nerve stimulation primarily affects the granular fraction and it may have no direct effect on the supernatant fraction. Experiments with 3 H-noradrenaline indicate that about half o f the amine originally stored in the granules is released into the supernatant during the fractionation procedure (Stitzel and Lundborg, 1967). The slight decrease in supernatant S H-MA following nerve stimulation in non-reserpinized rats may thus very well be an artefact due to release during fractionation. The lack of a decrease o f supernatant 3H-MA in reserpine-treated animals supports this interpretation: in this case, the effect o f nerve stimulation on granular 3H-MA was not of sufficient magnitude to make such an artefact detectable. It is remarkable that the large dose o f reserpine used did not completely inhibit the release o f 3H-MA b y nerve stimulation from the granular fraction. Probably the concomitant release o f noradrenaline was very slight or absent, judging by the complete absence of physiological responses. It may be speculated that 3H-MA is capable of circumventing to a
The research reported in this manuscript has been supported by the Swedish State Medical Research COuncil (B69-14X-155-05A, B69-14X-2157-03) and the faculty of Medicine, University of Goteborg, Sweden. For skilful technical assistance we are indebted to Mrs Marianne Olovsson and Miss Lena Ramstedt.
REFERENCES Almgren, O., P. Lundborg and R. Stitzel, 1969, Release of 3H-metaraminol from subcellular fractions of rat salivary glands by nerve stimulation, European J. Pharmacol. 6 (1969) 109. Euler, U.S.v. and F. Lishajko, 1963, Effect of adenine nucleotides on catecholamine release and uptake in isolated adrenergic nerve granules, Acta Physiol. Scand. 59, 454. Hallhagen, G. and B. Waldeck, 1968, Synthesis of tritium labelled metaraminol, ~-methylnoradrenaline and their corresponding f3-desoxy-derivatives, J. Labelled Compounds 4, 72. Haggendal, J. and T. Malmfors, 1969, The effect of nerve stimulation on catecholamines taken up in adrenergic nerves after reserpine pretreatment, Acta Physiol. Scand. 75, 33. Lundborg, P., 1967, Studies on the uptake and subceUular distribution of catecholamines and their Ot-methylated analogues, Acta Physiol. Scand. Suppl. 302. Lundborg, P. and R. Stitzel, 1967, Uptake of biogenic amines by two different mechanisms present in adrenergic granules, Brit. J. Pharmacol. 29, 342. Obianwu, H., 1967, Sympathetic and receptor blockade after prenylamine (Segontin(~), Acta Pharmacol. Toxicol. 25, 141. Stitzel, R.E. and P. Lundborg, 1967, Effect of reserpine and monoamine oxidase inhibition on the uptake and subcellular distribution of 3H-noradrenaline, Brit. J. Pharmacol. 29, 99.