Two populations of nerve fibers in the adrenal nerve

Journal of the Autonomic Nervous 8yetem, 2 (1980) 81--86

~) El~vierlNorth-Holland B/omedical Preu

81

Short C o m m u n k ~ n s TWO POPULATIONS OF N E R V E FIBERS IN THE A D R E N A L NERVE

TETSUO HIRANO and AKIRA NIIJIMA

Department of Physiolo#y, NiiEata University School of Medicine, Nii~'ata (Japan)

(Received January 25th, 1980) (Accepted February Imt, 1980) Eeywords: rabbit --adrenal nerve activity -- adrenergic drugs -- adrenergic blocking agents

Efferent activity of the adrenal nerve was recorded in the rabbit with deafferentations of bazoreceptors. Two kinds of fibers were identified in the adrenal nerve, the activities of which were s u p p ~ by epinephrine injection. In one group of fibers, nerve activity was inhibited by isoproterenol (AD type) while in another group, the activity was suppressed by applications of norepinephrine and phenylephrine but not by isoproterenol (NA type). The suppreuive effect of adrenergic drugs was blocked by beta blocking agent in AD type fibers while it was blocked by alpha blocking agent in NA type fibers. The present results suggest two reflex routes for inhibition of adrenal nerve activity, each depending on a different central adrenergic receptor. Recent studies on secretion of catecholamines from the adrenal medulla suggest that there is a selective release of epinephrine and norepinephrine, and *.he differential responses partly depend on the nature of the stimuli. Insulin hypoglycemia, [3,13] carotid occlusion [2] and asphyxia [14] are shown to change the proportion of the two catecholamines in the circulating blood. Hypoglycemia leads to major secretion of epinephrine, depleting the adrenal medulla, w h i l e carotid occlusion produces a low percentage of epinephrine depletion. Following asphyxia, the main secretion is that of epinephrine. The presence of centers in the brain controlling adrenal medullary secretion has been shown by stimulation of the bulbar region [11] or hypothalamus [1]. Stimulation of different areas of hypothalamus release different proportions of epinephrine and norepinephrine [2,5,14]. It is suggested that secretion of epinephrine and norepinephrine is mediated by two pathways and that the two types of adrenal chromaffin cells [8], A cells (adrenaline~ecreting cells) and NA cells (noradrenaline4ecreting cells), are innervated by separate fibers with a different hypothalamic representation. A recent electron microscopic study has identified the different profiles

82 of nerve endings on the two types of chromaffin cells in hamster adrenals [6]. It would seem that the adrenal nerve also contains more than one type of fiber. However, little physiological and morphological work has been done to identify different types of nerve fibers in the adrenal nerve. The present experiments were designed to investigate this matter. Thirty~six adult rabbits (2.2--4.2 kg) of either sex were used. Under sodium pentobarbital anesthesia (25 mg/kg, i.v.) the trachea was intubated. Additional doses of the anesthetic were injected through a r~theter placed in the left jugular vein whenever necessary. Drug administrations were made into the cephalic end of the left carotid ~rtery (i.c.a.). The cardiac end was connected to a transducer to monitor blood pressure. The substances used were VL-epinephrine hydrochloride 0.1--5.0 #g, DL-norepinephrine hydrochloride 0.1--5.0 pg, phenylephrine hydrochloride 0.1--5.0 /~g, isoproterenol hydrochloride 0.1--5.0 /~g, propranolol hydrochloride 0.5 mg and phenoxybenzamine hydrochloride 0.5 mg (doses expressed in weight of the base). Drugs were dissolved in normal saline. Depressor nerves were cut bilaterally at the cervical level and the sinus nerves were crushed. Validity of complete baroreceptor deafferentation was confirmed by the absence of inhibition of adrenal nerve activity on rapid injection (i.c~.) of 0.5--1.0 ml of normal saline at body temperature. Efferent activity of single or multiple fiber preparations was recorded from dissected adrenal nerve fibers. Adrenal nerve activity was amplified with a differential amplifier. After converting impulses to standard pulses by a window discriminator, determinations of drug effects on adrenal nerve activity were performed by comparing mean spikes per 5 sec for over 100 sec (mean of 20 samples) just before and after the injections. Differences were evaluated by the Student's t-test. Values are given as mean ± standard errors of the mean. Effect of epinephrine and ~lorepinephrine on adrenal nerve activity. Epinephrine injection (5.0 ~g, i.c.a.) reduced the spontaneous activity (Fig. l ) from 23.2 spikes/5sec± 0.3 spike/5sec to 20.0 s p i k e s / 5 s e c ± 0.7 spike/5 sec thus leading to a reduction of about 14% (P<~ 0.001). A similar dose of norepinephrine injected i.c.a, also led to a significant ( P < 0.001) decrease in nerve activity, reducing the spike rate of 23.8 spikes/5 sec ± 0.3 spike/5 sec to 21.0 spikes/5 sec ± 0.5 spike/5 .~ec and thus showing a decrease of 12% (Fig. 1). The inhibition in adrenal rierve activity was immediate and preceded the norepinephrine- and epinephrine-induced increase in blood pressure by 20--30 sec. The magnitude of norepinephrine suppression in adrenal nerve activity varied from very little change to almost the same degree of suppression as that seen after epinephrine injection. The effects of norepinephrine (1.0--5.0 ~g) were observed in 19 out of 28 fibers (P < 0.05). Effect o f adrenergic drugs on adrenal nerve activity. Since epinephrine and norepinephrine have both alphs and beta actions, effects of other adrenergic drugs were examined to determine the major action that could have caused a suppression of adrenal nerve activity. Phenylephrine was

83 5n~n

*I

5O

E

f

N

Fill. 1. Epinephrine and norepinephflne idmilarly suppremed •drenal ~erve activity. Epinephrine (0.5 /~g) deereued the spontaneous activity to about 86% while the &~ne dose of norepinephfine decreased it to 88%. Suppressive effeet~ of theJe two catechoiamines w ~ blocked by phenoxybenzamine (0.5 rag). E, epinephrine; N, norepinephrine; POB, phenoxybenzamine.

used as an alpha adrenergic and isoproterenol as a beta adrenergic stimulant. In a previous study we reported that i.c.a, injection of isoproterenol led to a significant inhibition of adrenal nerve activity with a fall of blood pres. sure [9]. Suppressive effect of isoproterenol was observed in 11 out of 20 fibers in the present study ( P ~ 0.05). Out of these 11 fibers, significant suppression ( P < 0.05) was found in 3 fibers following norepinephrine injection (1.0--5.0 pg) (Fig. 2a), but showed no such suppression after phenylephrine injection. The remaining 9 fibers, in which isopz~terenol had no suppressive effect, showed suppression in all fibers after norepinephrine injection (P < 0.05). Phenylephrine also caused an inhibition of the activity (Fig. 2b). These observations thus suggest the presence of two types of fibers: (1) AD type fibers in which nerve activity is inhibited by epinephrine and isoproterenol injections but in which norepinephrine is les.c effective and phenylephrine is ineffective; and (2) NA type fibers in which nerve activity is t . ~ . tyO*

%l& lyDf'

10e j d

;

'. • L

.

P~,E

1111 LI --

~(

EP

'SP

o

OHE

-

N(

[ P

,(,

Fig. 2. Adrenal nerve activity in response to the injections of adrenergic druD (2/~g). a: AD type fiber, b: NA type fiber. White column: spontaneous activity; shaded column: nerve activity just after the injection of • eerUdn drug. Vertical bars indicate 8.E. of the mean. PHE, phenylephrinc; NE, nonepinephrtem; EP. ~.~inep&rime' ISP, lmproterenol. Decrease of AD type nerve activity is broulflbt •~,>l, ,~ . '~ep~rth, ~und il~oproterenol injection whilst that of NA type nerve ~tivP ,. wing the injection of phenylephrine, epinephrine~md norepinephrine. . . . . ttY~r~t from the ~pontaneous activity, (P < 0.01); ++ P < 0.001.

84

inhibited by epinephrine, norepinephrine and phenylephrine, but n o t by isoproterenol.

Effect of adrenergic blocking agents on suppressive effect of adrvnergic drugs. The presence of two types of fibers was further confirmed by using adrenergic blocking agents. ~4D type fibers. Six such fibers were tested with blocking agents. Administration of epinephrine and norepinephrine (1.0--5.0/~g) were effective in suppressing adrenal nerve activity even after the injection of an alpha blocker (phenoxybenzamine 0.5 rag). Injections of propranolo~ (0.5 rag), a beta blocker, on the other hand, clearly blocked the inhibitory effects of epinephrine, norepinephrine and isoproterenol in all the 6 fibers. NA type fibers. The effect c,f propranolol was determined in 3 NA type fibers of two single and one multiple fiber preparations. In these fibers propranolol (0.5 rag) did not block the suppression induced by epinephrine and norepinephrine injections. However, after phenoxybenzamine (0.5 rag) injection, epinephrine and norepinephrine injections produced no significant change in adrenal nerve activity (Fig. 1). Table I shows the number of fibers tested and classified. Out of 41 fibers 37 responded to adrenergic drugs. Twenty-one fibers were identified as AD type and 10 as NA type. In the 6 fibers mentioned as unclassified, only the suppressive effect of epinephrine was observed. Isoproterenol and adrenergic blocking agents were not tested. Four fibers were insensitive to adrenergic drugs. The present study demonstrates that two populations of nerve fibers are present in the adrenal nerve: A D type fibers, the activity of which was suppressed by beta stimulant and the effect of which was blocked by beta blocker, and N A type fibers, the activity of which was inhibited by alpha stimulant and the inhibitory effect of which was blocked by alpha blocker. This is in keeping with the view that two types of adrenal chromaffin cells in the adrenal medulla are innervated by different fibers. The present results further showed that norepinephrine injected i.c.a. suppressed adrenal nerve activity in baroreceptor deafferentated rabbit. TABLE [ NUMBER OF FIBERS CLASSIFIED IN THE PRESENT STUDY

AD type NA type Unclassified s Insensitive b

Single fiber preparations

Multiple fiber preparations

Total

3 5 1 0

18 5 5 4

21 10 6 4

• Unclassified: nerve activity was inhibited by epinephrine application but n o t identified by isoproterenol or blocking agents. b Insensitive: nerve activity was not inhibited by applications o f adrenergic agents.

85

The catecholamines of the adult rabbit adrenal gland are reported to consist entirely of epinephrine [11,12]. H~kfelt and McLean [11] showed that 87--100% of catecholamines were epinephrine in the adult rabbit. Since, however, the rabbit possesses a considerable volume of extra-adrenal chromafirm tissues with presence of norepinephrine as shown by a positive iodate reaction [4,6] and also the most superficial of the hilar medullary cells are positive (NA cells), it is likely that norepinephrine could be released from these tissues into the circulating blood. In previous studies [9,10] we have reported that catecholamines secreted from the adrenal medulla inhibited adrenal nerve activity through catecholamine receptors in the brain besides through action of baroreceptors. We have also observed that alpha and beta blocking agents, resp,:ctively, block the suppressive effect of epinephrine, suggesting two kinds of nerve fibers in the adrenal nerve [10]. The present results indentified the two kinds of nerve fibers in the adrenal nerve and showed that one third of the nerve fibers were NA type. In view of the existing morphological and biochemical reports [11,12] showing that the rabbit adrenal gland contains very few NA cells and very small amounts of norepinephrine, it may seem to disagree with the high proportion of NA type fibers found in the present study. However, the number of fibers in the adrenal nerve do not represent the population of medullary chromaffin cells. Hillarp [7] and Marley and Prout [13] proposed the concept of "secretory unit". According to these authors, each axon innervates a number of secretory cells and several neurones terminate on the secretory unit. Based on their studies on rat and hamster adrenal glands, Coupland [4] and Grynszpan-Winograd [6] objected to this concept of convergence of axons from several neurons on the same secretory unit as they found only one nerve terminal in the case of A cells. However, they did not mention the NA cells which receive multiple nerve terminals on the surface. In either case, it is reasonable to consider t h a t the number of fibers in the adrenal nerve does not represent the number of chromaffin cells in the adrenal medulla. We have reported earlier [9,10] that there exists a reflex inhibition of adrenal nerve activity through adrenergic receptors besides that obtained through activation of baroreceptors. The present results indicate that there are two such reflex routes, one is through alpha adrenergic receptors and the other through beta adrenergic receptors. The two reflex routes may act as follows: one through alpha adrenergic receptors to NA type fibers which terminate on NA cells in the hilus of the adrenal gland and/or in juxta adrenal or extra adrenal chromaffin tissue, and the other through beta adrenergic receptors to affect AD type fibers which terminate on A cells in the adrenal medulla. We wish to express our thanks to Prof. K.N. Sharma and Prof. Dua Sharma for their valuable suggestions in preparing the manuscript.

86 1 Brsuner, F., Brik:ke, I% Kalndl, F. and Neumayr. A., Quantltstivs BestimmunlPm iiber die Sekrmtlon dee Nebsnnierenmarkes bsi eiektrbeher Hypothaliunumeizunll, Arch. Intern. Pharmacodyn. Therap., 85 (1951) 419--430. 2 R~icke, F., KaJndl, F. and Mayer, H., Uber die Ver~nderunl in del" Zusammensetaung des Nebennierenmarklnkretes bei elektrischer Reizung des Hypothalamus, Arch. Intern. Pharmecodyn. Therap., 88 (1962) 407--412. 8 Burn, J.H., Huteheon, D.E. and Parker, R.H.O., Adrenaline and noradrenaline in the suprarenal medulla after insulin, Brit. J. Pharmacol., 5 (1950) 417--423. 4 Couplend, R.E., The Natural History of the Chromaffin Cell, Lonlmens, London 1965. 5 Folkow, B. and von Euler, U.S., Selective activation of nor~drenallne and adrensllne producing cells in the cat's adrenal gland by hypothalamic stimulation, Circular. Res., 2 (1954) 191--196. 6 Grynszpan-Winorrad, O., Adrenaline and noradrenaline ceils in the adrenal medulla of the hamster: a morphological study of their innervation, J. Neurocytol., 3 (1974) 341--361. 7 Hillarp, N-A., Structure of the synapse and the peripheral innervation apparatus of the autonomic nervous system, Acts Anat. (Lond.) (Suppl), 4 (1946) 1--153. 8 Hillarp, N-A., and H6kfelt, B., Evidenc~eof adrenaline and noradrenaline in separate adrenal medullary ceils, Acts physiol, scand., 30 (1953) 56--68. 9 Hirano, T. and Niijima, A., Bets adrene.'lic stimulant suppression of efferent activity of the adrenal nerve, Neurolci. Lett., 14 (1979) 67--70. I0 Hirano, T. and Niijima, A., Epinephrine suppression of efferent activity of the adrenal nerve, J. Auton. Nerv. Syst., 1 (1980) 283--290. 11 H6kfelt, B. and McLean, J., The adrenaline and noradrenaline content of the suprarenal glands of the rabbit under normal conditions and after various forms of stimulation, Acts physiol, scand., 21 (1950) 258--270. 12 Houssay, B.-A. et Molinelli, E.-A., Secretion de l'adrenaline pourdite pM la piq0re ou I'excitstion ~lectrique du bulbs, C.R. Soc. Biol. (Paris), 91 (1924) 1045--1049. 13 MaHey, E. and Prout, G.I., Physiology and pharmacolorY of the splanchnic---adrenal medullary junction, J. Physiol. (Lond.), 180 (1965) 483--613. 14 Outschoorn, A.S., The hormones of the adrenal medulla end their release, Brit. J. Pharmacol., 7 (1952) 605--615. 15 Redpte, .~:.S. and Gellhorn, E., Nature of sympathetico-adrenal discharge under conditions of excitation of central autonomic structures, Amer. J. Physiol., 174 (1953) 457--480. 16 Shephe;d, D.M. and West, G.B., Noredrensline and the suprarenal medulla, Brit. J. Pharmacol., 6 (1951) 665--672.