Release of noradrenaline from the hypothalamus in vivo

I"UROPI'~AN JOURNAL O1: PHARMACOLOGY 9 (1970) 52 58. NORTH-HOLLAND PUBLISHING COMP., AMSTt'RDAM

RELEASE OF NORADRENALINE FROM THE HYPOTHALAMUS IN VIVO A. PHILIPPU, G. HEYD and A. BURGER l~stitute of Pharmacology, Medical School of Ess'en. Ruhr University, Essen, Germany

Received 27 July 1969

Accepted 19 September 1969

A.PItlLIPPU, G.tIFYD and A.BURGER, Release o j noradrenaline ,[)'ore the hypothalamus in vivo. European J. Pharmacol. 9 (1970) 52 58. The cat hypothalanms was labelled with 14C-noradrenaline and perfused with artificial cerebrospinal fluid or with Ringer solution. Calcium ~5 X 10-3 to 1 X 10-2 M), acetylcholine (2.2 × 10-3 M) and electrical stimulation of the nuclei posterior, ventromedialis and anterior medialis caused an enhanced release of noradrenaline and its metabolites from the hypothalamus. The releasing effect of acetylcholine was dependent on the presence of calcium ions. The significance of calcium and acetyleholine for the release of noradrenaline from the hypothalamus in vivo is discussed. [typothalamus Catecholamines

Release Acetylcholine

1. INTRODUCTION It has been shown in a previous paper that calcium and acetylcholine accelerate th.: release of noradrenaline from isolated hypothalamic vesicles (Philippu and Przuntek, 1967). it has been suggested that both agents may also be responsible for the release of noradrenaline form the nerve endings of the hypothalamus under physiological conditions. In order to investigate this hypothesis the cat hypothalamus was perfused in viw) and the effects of calcium and acetylcholine and of electrical stimulation on the release of noradrenaline were studied.

2. METHODS Cats were anaesthetized with pentobarbitone sodium (36 mg/kg i.p.). The trachea was cannulated and the head of the cat fixed in a stereotaxic instrument. The third ventricle was then cannulated and 5 /~Ci 14C-noradrenaline (50 mCi/mmote) were injected * Suppmted by the Deutsche Forschungsgemeinschaft.

Calcium Electrical stimulation

intraventricularly. After 4 hr the aqueduct was cannulated and the third ventricle was perfused with artificial cerebrospinal fluid (Merlis, 1940) or Ringer solution containing tropolone (50 × 10-6 g/ml) and nialanfid (1.5 × 10.6 g/ml) at a rate of 0.15 ml/min. This technique enables the selective perfusion of tire hypothalaxnus (Feldberg and Myers, 1966). The effluent was collected in 20 rain samples in tubes containing 0.1 ml HC[O4 (70%). In order to determine total radioactivity, the samples were centrifuged and 0.2 ml of the supernatants were placed in scintillation vials containing 10 ml of scintillator (4 g PPO and 50 mg POPOP/1000 ml toluene) and 3 ml of ethanol. The released total radioactivity is expressed as a percentage of the first control effluent. The relative concentrations of txoradrenaline and its metabolites were determined after separation by paper chromatography [Vogt, 1952). The chromatogram was divided in strips 1.5 cnr wide and placed in vials containing 15 ml of scintillator. The sum of the recovered radioactivity of noradrenaline and its metabolites was set as 100%. In a number of experiments the brain was removed at the end of the perfusion, the hypothalamus sepa-

RELEASE OF NORADRENALINE FROM TIlE BRAIN rated, extracted with 0.4 N HCI04 and the total radioactivity was measured in an aliquot of the supernatant after centrifugation. The relative concentrations of the radioactive compounds were determined after separation of noradrenaline and its metabolites by paper chromatography as described above. In some experiments the perfusion o f the third ventricle began 20 hr after labelling the brain with noradrenaline. For this purpose a Collison cannula was implanted into the lateral ventricle under anaesthesia and after an interval o f 4 6 days the brain of the unanaesthetized cat was labelled with 5 or 10/2Ci 4 C-noradrenaline.

F o r electrical stimulation of the hypothalamus, monopolar stainless steel electrodes (0.2 mm diameter, 0,5 mm exposed tip) were used. The indifferent electrode was attached to the ear of the cat. The hypothalamus was stimulated with square wave pulses of 6 msec duration at 40 c/sec, 10 V. During a collection period o f 20 rain the hypothalamus was stimulated ten times for 60 sec. The perfusion of the hypothalamus was ascertained by adding methylene blue to the perfusing fluid at the end of experiment (Feldberg and Myers, 1966). The following substances were used: DL-7-~4C noradrenaline (NEN Chemicals, Dreieichenhain/ Frankfurt), nialamid (Pfizer GmbH, Karlsruhe), tropolone (Regis, Chicago), acetylcholine (Lematte et Boinot, Paris).

53

3. RESULTS 3.1. Relative conc'entration o f noradrenaline and its metabolites in the effluent. Effect o f enzyme inhibitors The perfusates contained 10.5% a4C-noradrenaline, 17.8% 14 C-3,4-dihydroxymandelic acid (DOMA), 17.1% 14C-normetanephrine (NM), 13.4% 14C3-methoxy-4-hydroxyphenylglycol (MOPEG) and 41.1% 14 C-3-me thoxy-4-hydroxymandelic acid (VMA). Perfusion of the hypothalamus with nialamid containing Ringer solution did not influence the relative concentration of the compounds (table 1). By perfusing with Ringer solution containing nialamid and tropolone the relative concentration of novadrenaline was increased (19.4%)while the concentration of VMA was reduced significantly (30.6%). The highest percentage concentrations o f noradrenaline (31.3%) and the lowest of VMA (23.0%) were achieved by perfusion of the third ventricle with artificial cerebrospinal fluid containing nialamid and tropolane. The following experiments were performed with Ringer solution containing both enzyme inhibitors. 3.2. Effect o f acetyleholine on the spontaneous release o f catecholamines from the hypothalamus During perfusion the release o f total radioactivity from the hypothalamus decreased gradually (fig. 1), the initial slope of the curve declining with a half-life

Table 1 14C-noradrenaline and its metabolites in the brain effluent by perfusion with Ringer solution or artificial cerebrospinal fluid (CSt:)

Ringer Ringer+ nialamid Ringer + nialamid + tropolone CSF + nialamid + tropolone

tl

NA (%)

18 16 11 7

10.5 13.8 19.4 31.3

+0.9 _+ 1.7 _+ 1.3 _+3.3

DOMA (%)

NM (%)

MOPEG (~)

VMA (%)

17.8 _+ 1.1 18.2 _+0.9 22.9 _4-1.5 21.2 _+ 1.4

17.1 _+ 1.0 19.3 _+ 1.1 15.4 _+ 1.4 12.0 _4-1.I

13.4+0.7 11.5 + 0.7 11.8 _+ 1.0 12.2 +_ 1.7

41.l 37.t 30.6 23.0

+ 1.4 _+2.0 _+ 1.2 _+ 1.0

The values are expressed as per cent of the total radioactivity recovered. Nialamid: 1.5 X 10-6 g/ml. Tropolone: 50 × 10-6 g/ml. Mean values 4- S.E.M.

54

A.PH1LIPPU, G.HEYD and A.BURGER

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}rig. l. Efflux of radioactivity from the cat hypothalamus. Between 20 and 320 min the regression coefficient is 0.005887 _+ O.000425/min (S.D.M.). Means of 22 experiments _+S.E.M.

(hA) of about 6.5 rain. The slope of the second part of the curve followed a single exponential decline with a half-life of 118 rain. The regression coefficient ,,f this part of tire curve was 0.005887 -+ 0.000425/rain. The exponential decline suggested that between 40 and 320 min catecholammes were released only from intracellular compartments. Hence, all experiments were performed during this period. Addition of acetylcholine (2.2 X 10 -3 M) to the perfusing solution increased the release of catecholamines. The releasing effect of acetylcholine could be demonstrated repeatedly in the same cat (fig. 2). Frequently a lower concentration of acetylcholine (2.2 × 10 -4 M) enhanced the release of catecholaillines. To study the influence of calcium on the catecholamine releasing effect of acetylcholine experiments were performed in the presence and in the absence of this ion. Ionized calcium in the hypothalamic region of the ventricle was removed by perfusion with

Ringer solution containing EDTA (l × 10-3 M) for 60 rain before the collection period. During perfusion of the third ventricle with Ringer solution containing calcium (2.5 × 10-~ to 1 X 10-2 M), acetylcholine ( 2 . 2 X 10-3M) increased the release of catecholamines from the hypothalamus (fig. 3). However acetylcholine was ineffective if the third ventricle was perfused with calcium free Ringer solutio,. Similar results were obtained if the experiments were performed 20 hr after labelling the hypothalamus with ~4 C-noradrenaline. 3.3. Effect of caleium on the release ~,f catecholamines from the hypothalamus The ventricle was perfused with Ringer solution containing EDTA (1 X 10-3 M) for 60 rain before the collection period. The hypothalamus was then perfused with calcium free Ringer solution. Perfusion with solution containing calcium (1 × 10-2 M) caused an increased release of radioactive compounds (fig. 4). A lower calcium concentration (5 × 10-3 M)

RELEASE OF NORADRENALINE FROM THE BRAIN

55

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Fig. 2. Effect of acetylcholine on the release of catecholamines from the cat hypothalamus. Ach = acetylcholine (2.2X10 -3 M). Total radioactivity of 0.2 ml of the perfusates, total volume of each sample 3 ml.

also enhanced significantly the release of catecholamines from the hypothalamus.

3.4. Release o f catecholamines f r o m the hypothalamus by electrical stimulation Electrical stimulation o f the nucleus anterior

medialis (10 V, 6 msec duration, 40 cps) enhanced the release of catecholamines (fig. 5) as did stimulation o f the nuclei posterior and ventromedialis (fig. 6). A similar releasing effect was obtained by stimulating the different hypothalamic nuclei with 5 V, 3 msec duration at 70 cps.

56

A.PHILIPPU, G.HEYD and A.BURGER

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Fig. 3. Effect of acetylcholine on the release of catecholamines from the cat h y p o t h a l a m u s in the presence and in the absence of calcium. Means + S.E.M.

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Fig. 5. Effect of electrical stimulation (nucleus anterior medialis) on the release of catecholamincs from tt]e cat hypothalamus. Electrical stimulation: 10 V, 6 reset., 40 cps. Means _+S.E.M.

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Fig. 6. Effect of electrical stimulation (nuclei posterior and ventromedialis) on the release of catecholamines from the cat hypot ha l a mus . Electrical stimulation: 10 V, 6 rnsec., 40 cps. Means + S.E.M.

RELEASE OF NORADRENALINE FROM THE BRAIN

57

Table 2 Release of noradrenaline and its metabolites by calcium, acetylcholine and electrical stimulation /7

NA ~%)

DOMA (%)

NM (%)

MOPEG (%)

VMA (%)

Controls Ca 2.

3 3

17.2 _+2.1 24.7_+4.8

19.6 _+2.3 19.5_+1.2

17.3 + 0.4 16.8_+1.6

11.2 _+2.5 11.0_+3.0

34.8 + 0.9 27.8_+5.6

Controls Ach

4 4

24.3 _+2.7 24.5 _+5.8

19.8 _+0.5 16.7 + 2.7

15.0 + 2.0 18.1 -+ 3.9

9.4 -+ 1.0 8.6 _+ 1.1

31.5 -+ 3.6 32.6 _+7.0

Controls Electrical stimulation

6 8

18.1 _+0.9 15.8 _+ 1.8

26.6 _+ 1.1 25.8 _+3.7

14.1 _+2.5 12.2 _+ 1.9

12.4 _+ 1.6 13.6 _+2.0

28.9 _+ 1.6 32.6 _+3.1

The third ventricle was perfused with Ringer solution containing nialamid (1.5 X l 0 -6 g/ml) and tropolone (50 × 10 -6 g/ml). Ca2÷ = 5 X.10-3 M, Ach = 2.2 × 10-3 M acetylcholine, electrical stimulation: 10 V, 6 msec, 40 cpm. Means + S.E.M.

3.5. Influence o f drugs and o f electrical stimulation on the relative concentrations o f noradrenaline and its metabolites Noradrenaline and its metabolites were determined before and during perfusion with calcium and acetylcholine or electrical stimulation of the nuclei posterior and anterior medialis (table 2). Neither the amine releasing agents nor electrical stimulation influenced the relative concentrations of the radioactive compounds.

3.6. Radioactive compounds in the hypothalamus In some experiments the brain was removed at the end of the perfusion and the radioactive compounds of the hypothalamus were determined (table 3). The relative concentration of noradrenaline in the hypothalamus was 48.6%, while its relative concentration in the perfusate was only 19.4% Table 3 14C-noradrenaline and its metabolites in the hypothalamus Radioactive compounds

Relative concentration (%)

NA DOMA NM MOPEG VMA

48.6 21.7 13.0 7.1 9.6

+ 4.8 _+3.6 + 1.2 + 1.5 + 1.5

The values are expressed as percentage of the total radioactivity recovered. Means of 9 experiments _+S.E.M.

(table I). Therefore noradrenaline was further metabolized after its release from the hypothalamic cells, although the perfusing fluid contained both nialamid and tropolone.

4. DISCUSSION A method for the perfusion of the whole rat brain

in vivo has been published by Palaic, Page and Khairallah (1967). The method described in the present paper enables the continuous determination of noradrenaline and its metabolites released from the cat hypothalamus. Perfusion of the third ventricle with solutions containing nialamid has practically no effect on the metabolism o f noradrenaline while the presence of nialamid and tropolone in the perfusing fluid evokes an increase in the relative concentration of noradrenaline and a decrease in that of VMA. The failure of nialamid to increase the percentage concentration of noradrenaline is not surprising, since MAO inhibitors do not affect the metabolism of noradrenaline in the cat brain (Vogt, 1954; Mannarino, Kirshner and Nashold, 1963). Perfusion of the third ventricle with acetylcholine or calcium ions causes an enhanced release of noradrenaline and its metabolites from the hypothalamus. However the effect of acetylcholine is dependent on the presence of calcium ions. This dependence of acetylcholine's action on the calcium ion has been observed in experiments with suprarenals of cat and cattle (Douglas and Rubin, 1961; Schi.imann and

58

A.PHIL1PPU, G.HEYD and A.BURGER

Philippu, 1963). It is therefore likely that in the central nervous system acetylcholine induces the release o f noradrenaline from noradrenergic nerve endings by increasing membrane permeability to the calcium ion, as in the adrenal gland (Douglas and Poisner, 1961). Since calcium ions increase the release of noradrenaline from the hypothalamic vesicles (Philippu and Przuntek, 1967) it may be postulated that in v i v o the calcium ions which enter the cell act on the subcellular particles and cause the release of noradrenaline. The release of catecholamines from the hypothalamus is also enhanced by electrical stimulation of the nuclei posterior, ventromedialis and anterior medialis. Although the differences were not significant, electrical stimulation of the nucleus posterior provoked the most pronounced release of noradrenaline and its metabolites. Therefore the releasing effect of electrical stimulation parallels noradrenaline concentration in different areas of the hypothalamus, since of the nuclei stimulated, the nucleus posterior contained the highest density o f noradrenergic nerve terminals (DahlstriSm and Fuxe, 1965). Acetylcholine may be a chemical transmitter in the central nervous system (Feldberg, 1945). On the other hand, the existence of nerve terminals containing catecholamines which are stored in subcelhdar particles (Dahlstr6m and Fuxe, t965; De Robertis et al., 1965; Philippu and Przuntek, 1967) and their release by electrical stimulation of the nuclei suggest that noradrenaline may also be a mediator in the hypothalamus. Recently Bloom, Oliver and Salmoiraghi (1963) have reported that all areas of the hypothalamus possess acetylcholine sensitive neurons which respond to acetylcholine either by increasing or by decreasing their spontaneous rate of discharge. These observations together with the above described finding that acetylcholine enhances the release of noradrenaline could support the hypothesis that in the hypothalamus the transmitter of cholinergic neurones influences the release of catecholamines from the noradrenergic nerve terminals, provided that calcium ions are present.

ACKNOWLEDGEMENT The authors wish to thank Pfizer GmbH, Karlsruhe, for nialamid and Farbenfabriken Bayer, Leverkusen, for supplying cats. They also thank Miss B. Piel and Miss T. Seeber for skilful technical assistance.

REFERENCES Bloom, F.E., A.P. Oliver and G.C. Salmoiraghi, 1963, The responsiveness of individual hypothalamic neurons to microelectrophoretically administered endogenous amines, Intern. J. Neuropharmacol. 2, 181. Dahlstr~im, A. and K. Fuxe, 1965, Evidence for the existence of monoamine neurons in the central nervous system, Acta Physiol. Scand. 64, Suppl. 247. Douglas, W.W. and A.M. Poisner, 1961, Stimulation of uptake of calcium-45 in the adrenal gland by acetylcholine, Nature 193, 1299. Douglas, W.W. and R.P. Rubin, 1961, The role of calcium in the secretory response of the adrenal medulla to acetylcholine, J. Physiol. 159, 40. Feldberg, W., 1945, Present views on the mode of action of acctylcholine in the central nervous system. Physiol. Rev. 25,596. l:eldberg, W. and R.D. Myers, 1966, Appearance of 5-hydroxy-tryptamine and an unindentified pharmacological active lipid acid in effluent from perfused cerebral ventricles, J. Physiol. 184,837. Mannarino, E., N. Kirshner and B.S. Nashold, 1963, The metabolism of 14C-noradrenaline by cat brain in vivo, J. Neurochem. 10, 373. Merlis, J.K., 1940, The effect of changes in the calcium content of the cerebrospinal fluid on spinal reflex activity in the dog. Am. J. Physiol. 131, 67. Palaic, J., I.H. Page and P.A. Khairallah, 1967, Uptake and metabolism of 14C-serotonin in rat brain, J. Neurochem. 14.63. Philippu, A. and H.Przuntek, 1967, Noradrenalinspeichemng im Hypothalamus und Wirkung yon Pharmaka auf die isolierten Hypothalamus-Vesikel. Arch. Exptl. Pathol. Pharmakol. 258,238. Robertis, E. de, A. Pellegrino de lraldi, G. Rodriguez de Lores Arnaiz and L.M. Zieher 1965, Synaptic vesicles from the rat hypothalamus, Life Sci. 4, 193. Schi_imann, l~I.J, and A. Philippu, 1963, Zum Mechanismus der durch Calcium and Magnesium verursachten Freisetzung der Nebennierenmarkhormone, Arch. Exptt. Pathol. Phaxmakol. 244,466. Vogt, M.. 1952, The secretion of the denervated adrenal medulla of the cat, Brit. J. Pharmacol. 7,325. Vogt, M., 1954, The concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs. J. Physiol. 123,451.