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Acetylcholinesterase in the rat neurohypophysis is decreased after dehydration and released by stimulation of the pituitary stalk
Neuroscience Vol. 21, No. 2, pp. 421427, Printed in Great Britain
1987
0306-4522/87
$3.00 + 0.00
Pergamon Journals Ltd 0 1987 IBRO
ACETYLCHOLINESTERASE IN THE RAT NEUROHYPOPHYSIS IS DECREASED AFTER DEHYDRATION AND RELEASED BY STIMULATION OF THE PITUITARY STALK M. ALEXANDROVA,* M. HOLZBAUER, K. RACKET and D. F. SHARMAN AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, U.K.; and TDepartment of Pharmacology, University of Mainz, F.R.G. acetylcholinesterase and non-specific cholinesterases are present in all three lobes of the rat pituitary gland. This paper describes two new observations on hypophyseal acetylcholinesterase. Firstly, a prolonged increase of neurohormone secretion evoked by dehydration and sodium loading was
Abstract-Specific
accompanied by a decrease in the acetylcholinesterase. activity localized to the neural lobe, where acetylcholinesterase has previously been demonstrated in fine nerve fibres. Secondly, electrical stimulation of the pituitary stalk in vitro elicited the release of acetylcholinesterase and non-specific cholinesterases from the combined neural and intermediate lobe indicating that the enzyme can be released from nerve endings in the hypophysis by action potentials. The observed loss of enzyme activity during dehydration may be the consequence of a prolonged activation of cholinergic nerves in the gland, leading to an increased release of acetylcholinesterase, which is not immediately replaced by fresh enzyme. The decrease in acetylcholinesterase in the neural lobe during dehydration may also be connected with its peptidase function and thus with the previously observed loss of substance P from the neural lobe during dehydration [Holzbauer et al. (1984) Neurosci. Left. 47, 23-281.
The pituitary gland is innervated by nerve fibres containing classical neurotransmitter substances such as dopamine (DA), noradrenaline (NA), 5-hydroxytryptamine (5-HT) or substance P (SP). The concentrations of these substances in the neural lobe (NL) and/or in the intermediate lobe (IL) changed when neurohormone secretion was increased over several days (see e.g. Refs 15, 19, 20). For DA and 5-HT it has been established that their synthesis and catabolism rates are altered under these conditions. Both amines could be released from the NL and IL in vitro by electrical stimulation of the pituitary stalk and they have been shown to affect hormone release (see Refs 16, 18). The present paper describes a study of cholinesterases in the rat pituitary gland. These enzymes occur in peripheral and central cholinergic neurons, in other neuronal or non-neuronal tissues and in blood, in several forms. The form usually referred to as specific acetylcholinesterase (E.C. 3.1.1.7), is especially enriched at cholinergic synapses where its main function is the inactivation of acetylcholine.26 *Pressent address: Institute of Experimental Endocrinology, Centre of Physiological Sciences, Slovak Academy of Sciences, Bratislava, Czechoslovakia. Address for correspondence: Margarethe Holzbauer, Department of Pharmacology, University of Cambridge, Hills Road, CB2 2QD, U.K. Abbreviations: ACh, acetylchohne; AChE, acetylcholinesterase; ACTH, adrenocorticotrophic hormone; AL, anterior lobe; DA, dopamine; S-HT, 5_hydroxytryptamine; IL, intermediate lobe; NA, noradrenaline; NIL, neurointermediate lobe; NL, neural lobe, ns-ChE, nonspecific cholinesterases; SP, substance P. 421
This process occurs on an anionic site of the enzyme molecule which binds to the quartemary ammonium of acetylcholine (ACh). Specific-acetylcholinesterase (specificAChE or AChE) and non-specific cholinesterase (ns-ChE) have been demonstrated in all three lobes of the hypophysis of several mammalian” and lower species23*24 with histochemical and biochemical methods. The enzymes were localized in non-neurosecretory nerve fibres and in pituicytes of the NL, and in glandular cells of the IL and the anterior lobe (AL).‘,’ Species differences in the proportions of specificAChE and ns-ChE in different lobes were reported.37 In the NL of the rat pituitary gland, AChE-containing nerve fibres were found to be concentrated in the region of the junction between the pituitary stalk and the NL.’ After subcellular fractionation of homogenates of NLs from bovine pituitaries, about 15-20% of AChE and of ns-ChE were found in the fraction containing the broken off nerve endings and neurohormones. About 60% of ns-ChE was in the fraction containing the cell nuclei which, in the case of the NL, will have been derived mainly from pituicytes. Most of the specific-AChE (about 30%) was in a soluble form in the high speed supernatant3’ With histochemical methods a similar distribution of AChE and of choline acetyltransferase in nonneurosecretory nerve fibres in the NL was demonstrated. This is indicative of a cholinergic innervation of this lobe.’ Acetylcholine (ACh) itself has been identified in pituitary extracts on several occasions, with higher concentrations in the NL than in the AL (e.g. Ref. 3). No direct effect of ACh on hormone
422
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ALEXANDROVA ct ul
release from isolated NLs has been reported.y,‘),‘4.?s.‘: ACh has, however, a strong stimulatory effect on the release of vasopressin and oxytocin in situations where the NL is in connection with hypothalamic tissue (see e.g. Refs 9, 28, 32). The evidence for an effect of ACh on adrenocorticotrophic hormone (ACTH) release from the IL is conflicting: Fischer and Moriarty’” observed a stimulating effect of ACh on ACTH release on in vitro incubated neurointermediate lobes (NILS), whereas Briaud et al.,* working with superfused rat NILS, did not observe a significant effect of ACh on ACTH release. A connection between a cholinergic neuronal system and hormone release from the AL has been indicated by several observations. Chronic exposure of rats to cold temperatures and immobilization led to a decrease in the AChE activity in the AL,‘* AChE activity in the AL changed during the oestrous cycle of the rat,27 after treatment with oestrogens** and after orchidectomy.*’ AChE can be released from peripheral tissues and in the central nervous system. This was first observed in experiments on the adrenal gland by Chubb and Smith.’ Skau and Brimijoin3’ demonstrated the release of the enzyme from the rat phrenic nerve. They suggested that AChE is normally released when action potentials invade cholinergic nerve terminals and that the enzyme release occurs in parallel with, but separate from, acetylcholine. The release of AChE from the central nervous system is indicated by the presence of the enzyme in cerebrospinal fluid.4 In experiments with push-pull cannulae Weston and Greenfield demonstrated AChE release in the nigrostriatal system of the rat after local application of drugs. In the present work, it was firstly investigated whether prolonged increased secretion of neurohormones, as elicited by dehydration and sodium loading, alters the activity of AChE in the three lobes of the rat hypophysis. Secondly, it was tested whether AChE can be released from the in vitro incubated, combined NL and IL (NIL) of the rat by electrical stimulation of the pituitary stalk using stimulation parameters known to result in propagated action potentials. Rats
EXPERIMENTAL
PROCEDURES
The rats used were male Wistar rats, Porton strain, Babraham colony, 300-400 g, which were kept at a constant temperature of 21°C and a constant light (14 h) and dark (IO h) cycle. Food (Oxoid 41B pellets) and water were available ud Iibitumunkss stated otherwise. All rats were killed during the light period. Tissue preparations Two sets of experiments were carried out in which the effect of&hydration on AChE activity in the pituitary gland was studied. Dehydration was induced by water withdrawal for 72 h followed by 24 h during which a 2.5% solntion of NaCl was offered instead of drinking water. The control rats had free access to water throughout the experiment. All rats were killed by rapid decapitation and the pituitary glands dissected immediately. In the first set of experiments the
anterior lobes were removed, put into small glass centrifuge tubes and frozen on solid CO,. The whole NIL was placed into a drop of 0.9% NaCl solution (saline) on a black glass plate under a dissecting microscope and the IL teased off the NL with a pair of fine forceps. The NL was then transferred into a small glass tube and put on solid COz. The drop of saline containing the IL fragments was sucked into a Pasteur pipette and transferred into another glass tube. The glass plate and the pipette were washed twice with small amounts of saline which were added to the IL fragments, the tissue suspension was centrifuged, the saline discarded and the tube containing the IL tissue placed on solid CO,. The whole procedure took about 10 min. By this technique, more than 90% of the IL tissue was usually removed from the NL as assessed by the amount of immunoassayable ACTH in the tissues.” The tissue size was not measured in these experiments. In the second set of experiments, which was carried out one month later, the NL and IL were not separated. The NIL and AL were put into pre-weighed glass tubes which were weighed again after the tissues had been put in, and then put on solid COj. In both sets of experiments the samples were stored at -80°C for not longer than one week before the enzyme was assayed. To investigate whether AChE can be released from the rat NIL by electrical stimulation of the pituitary stalk, the NIL together with the pituitary stalk was dissected out as described previously. ” The cranial portion of the intact pituitary stalk was placed between the two branches of a split platinum electrode and the preparation incubated for 15min in 5 ml of Krebs-HEPES buffer (pH 7.4) of the following composition (mM): NaCl 118.5; KC1 4.7; CaCI, 2.5; MgCI, 1.2;o-glucose 1I. 1;HEPES 20.0. The buffer was saturated with pure oxygen. The preparation was then transferred to a glass microbath which contained 60~1 incubation buffer. The bath was surrounded by a water jacket (37°C). A second platinum electrode was situated at the bottom of the bath. The medium was changed every IOmin. The first 10min were used to assess spontaneous enzyme release. During the following 10min the pituitary stalk was stimulated with biphasic pulses of 0.2 ms duration, 10 V, 15 Hz, 4 mA, 5 times for 1 min with 1 min intervals by means of a Grass S9 stimulator. These parameters are similar to those required to cause propagated action potentials.‘6 During the last 10 min period, post-stimulation release was measured. Five groups of stimulation experiments (Fig. 3 A-E) were carried out. Because of the small amounts of enzymes released relative to the sensitivity of the assay method samples from four NIL preparations were pooled in experiments A, B and C, from six preparations in experiments D and E. The samples were kept on ice until assay on the same day. All rats used in the stimulation experiments had free access to water. Enzyme assay The AChE and ns-ChE activity in the tissue extracts and in the incubation media were estimated as described by Sharman and Cooper.W Tbis assay is based on the hydrolysis of ACh by AChE. The choline formed is oxidized by the enzyme choline oxidase giving rise to hydrogen peroxide which is measured by the fluorescence developed from 4-methoxy-3-hydroxy-phenylacetic acid in the presence of horseradish p&oxid&. The standard enzymd used was Sigma Standard AChE: one unit will hvdrolvse 1.0 umol of ACh per min at pH 8 at 37°C. The &s&s weie homogenized in 100~1 sodium phosphate buffer (0.1 M, pH 7.5). When isolated NLs or ILs were analysed, tissues from two rats were pooled. Ten ~1 of homogenate was employed for each assay. Ns-ChE activity was determined by the inhibition of enzyme activity resulting from the addition of ethopropa&e (10e4 M). Total e&me activity was determined by the inhibition of activity resulting from the addition of physostigmine (5 x IO-’ M) or neostigmine (5 x 10m5M).
AChE in the rat neurohypophysis
423
munits
Specific-AChE/ IVlmg Protein*
AL
NL
3
30
** 6
20
10
AL
c
No. of samples: No. of lobes:
4 4
5 5
4 6
4 6
3 6
3 6
Fig. 1. Experiment 1. Effect of dehydration (72 h with no water followed by 24 h with 2.5% NaCl instead of drinking water) on the activity of specific acetylcholinesterase (SpecificAChE) in the pituitary gland of the rat. Open columns: controls; shaded columns: dehydrated rats. Column height: mean values + SEM. AL, anterior lobe; NL, neural lobe; IL, intermediate lobe.. Significance of difference between controls and dehydrated rats: **P < 0.01 (Student’s r-test); # : for this calculation the mean (M) protein contents of 50 ALs, 50 NLs and 50 ILs of rats of the same colony, sex and age estimated on previous occasions were used. To estimate the enzyme activity in incubation media of NILS 100 or 200~1 of the incubation medium were added to assay tubes containing evaporated buffer solution corresponding to the volume used.29 The assay for AChE and ns-ChE activity was then carried out on these solutions. Special chemicals used. HEPES: N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (Sigma); ethopropazine (May and Baker, gift to Dr Ann Silver); physostigmine sulphate (BDH); neostigmine methylsulphate (Koch-Light). RESULTS
Distribution of acetylcholinesterase in the rat hypophysis and the effect of dehydration on enzyme activity in different lobes of the hypophysis The results are summarized in Figs 1 and 2 and in Table 1. Specific-AChE activity per lobe was largest in the AL in both sets of experiments (Figs 1 and 2); so was the total enzyme activity (specific-AChE + ns-ChE) as shown in Table 1. Per unit tissue specificAChE activity was 2-3 times higher in the NL and in the IL than in the AL. Specific-AChE activity in the combined NILS of the second set of experiments was of a similar order of magnitude as the sum of activities in the NLs and ILs of the first set of experiments (cf. Figs 1 and 2). Dehydration did not significantly affect specific AChE activity in the AL in the first set of experiments (Fig. 1). In experiment 2, the fall in AChE activity in the AL, when expressed per lobe (Fig. 2, column 1
and 2), was due to a significant fall in the weight of the ALs during dehydration (Fig. 2, columns 9 and 10): there was no change in enzyme activity when expressed per mg tissue (Fig. 2, columns 5 and 6). In contrast, dehydration caused a significant decrease (-40%) in specific-AChE activity in the isolated NL (Fig. 1, columns 3 and 4), but not in the IL (Fig. 1, columns 5 and 6). In the second set of experiments the activity of this enzyme in the NILS of the dehydrated rats was decreased by 33% when expressed per lobe and by 29% when expressed per mg NIL tissue (Fig. 2). In Table 1, values for total enzyme activity (specific-AChE + ns-ChE) are given as estimated in experiment 1. About 80% of the total enzyme activity was due to specificAChE. Although the specificAChE activity in the AL and the NIL in experiment 2 was similar to that in experiment 1, the total enzyme activity in experiment 2 was 44% higher in the AL and 50% higher in the combined NL + IL. Dehydration affected the activity of AChE and ns-ChE in a similar manner. Release of acetylcholinesterase from the neurointer mediate lobe evoked by electrical stimulation of the pituitary stalk
Figure 3 shows the effect of electrical stimulation of the pituitary stalk on the outflow of AChE from
424
M. munits
specific-AChE/Lobe
ALEXANVROVArl al.
m units
specific-AChE/mg
rng ‘wet’
tissue weight
t
3
At.
tissue
NIL
AL
JIL
AL
NIL
2
t
6
Fig. 2. Experiment 2. Effect of dehydration (72 h with no water followed by 24 h with a 2.5% NaCi solution instead of drinking water) on Ihe activity of specific acetyichoiinesterase (speciftc-AChE) in the rat pituitary gland. Open columns: controls; shaded columns: dehydrated rats. Column heights: mean values + SEM, n = 12 for each group. AL, anterior lobe; NIL, combined neural and intermtiate lobe. Statistical significance of differences from corresponding controls: *P < 0.05; l*P < 0.02; ***P< 0.01. (Student’s r-test.)
the NIL of normal male rats. The mean release of choline&erases (specific-AChE + ns-ChE)/NIL/lQ min was 104 p units during the pre-stimulation period; during electrical stimulation of the stalk it was 179 p units and during the post-stimulation period 112 p units. These mean values were calculated from all five experiments and represent the enzyme outflow from 24 NILS. Statistical analysis of the results according to Friedman’s two-way analysis of variance by ranks” using a value of 5 for n, showed that the increase in enzyme release evoked by electrical stimulation of the pituitary stalk was significant at P
In the samples collected in experiments D and E (Fig. 3) the percentage of specific-AChE was also determined. In all six samples analysed specificAChE and ns-ChE were present in about equal quantities. In experiment D electrical stimulation of the stalk increased the release of specific AChE by 56%, that of ns-ChE by 44%. Electrical stimulation of the pituitary stalk was apparently without effect on the enzyme content of the NIL: the specific-AChE activity in two NILS only incubated for 45 min in Krebs-HEPES buffer was 7.3 and 7.3 m units/NIL, ns-ChE was 6.7 and 5.9 m units/NIL. In two NILS in which the pituitary stalk
Table 1. Specific-acetylcholine&erase + non-specific choiinesterase (= total enzyme) in the hypophysis of control rats and rats in which neurohormone secretion had been stimulated by &hydration and sodium loading Total enzyme activity (m units/lobe)
Lobe
Control
1 H,O
AL NL IL
11.55+ 1.15(4) 2.84 + 0.13 (4) 2.16& 1.80(3)
12.90 f 0.69 (5) 1.84+ 0.06(4) 1.80 f 0.03 (3)
Difference between Control and JH,O Significancet (%) +12 -35 -17
NS P < 0.01 NS
Specific-AChE activity as % of total enzyme activity* Control 80 k2.7 88 rt 4.5 75 & 2.6
lH,O ___87 f 2.3 82 f 2.6 ?6* 1.4
Results of experiment 1. (For specific-AChE activity see Fig. 1.) Abbreviations: AL, anterior lobe, NL, neural lobe; IL, intermediate lobe. AL, single lobes used for each assay; NL and IL, tissues from two rats pooled for each ashy. Mean values f SEM; *calculated from individual values; tStudent’s r-test; NS, not significant. Figures in pare&&s: number of samples anaiysed. JH@ no water for 72 h followed by 24 h with 2.5% NaCI. Enzyme activity: one unit AChE will hydrolyse I.Opmoi of ACh per min at pH 7 at 37°C (Sigma standard AChE).
AChE in the rat neurohypophysis
350
300.
250J
c
.E E o
200.
L
150.
2
o
100.
Sample: 1 Experiment:
I
2 B
3
1
2 D
1
2 E
Fig. 3. Effect of electrical stimulation of the pituitary stalk ( ze: l0 V, 15 Hz, 4 mA, 0.2 ms, 5 times for 1 min with i rain intervals) on the release of specific acetylcholinesterase +non-specific cholinesterase (total enzyme) from the combined neural and intermediate lobe (NIL) of the rat pituitary gland incubated in vitro. Each column represents enzyme release over 10 min. Sample 1: spontaneous release. Sample 2: release during electrical stimulation of the stalk. Sample 3: release after stimulation. In experiments A, B and C samples from four NIL preparations were pooled; in experiments D and E samples from six NIL preparations were pooled. Dark portions of columns: electrically evoked enzyme release. (For further details see Experimental Procedures.) had been stimulated the AChE activity was 7.7 and 6.8 m u n i t s / N I L the ns-ChE activity 5.7 and 5.3 m units/NIL The mean specific-AChE activity of all four incubated NILs was 7.28 + 0 . 1 8 m units/NIL This value was 77% higher than the AChE activity in unincubated NILs of the control rats in Fig. 2 observed 5 months earlier. The maximal amount of AChE released by electrical stimulation of the stalk during l0 min was less than 2% of the amount of enzyme contained in the NIL. DISCUSSION The present paper describes two new observations on AChE in the rat pituitary gland. Firstly, prolonged activation of neurohormone secretion by dehydration and sodium loading caused a decrease in the tissue content of AChE activity which was restricted to the NL (Fig. 1.). Secondly, electrical
425
stimulation of the pituitary stalk using stimulation parameters known to cause propagated action potentials, evoked the release of AChE from the in vitro incubated NIL. From histochemical observations and the position of the stimulating electrodes it can be presumed that the cholinergic nerves, which descend from the hypothalamus to the NL, have been stimulated. Dehydration has previously been shown to affect classical neurotransmitter systems in the NIL such as the DA, the NA, the 5-HT and also the SP system. Thus, a comparable activation of the cholinergic system in the NL during periods of increased hormone secretion is possible. If it is permissible to draw a parallel between observations on peripheral cholinergic nerves and the cholinergic system in the NL, one may reason that an increase in the release of ACh during dehydration could have occurred which was accompanied by an increase in the release of AChE (cf. Ref. 31). The observed fall in the AChE content in the NL under these conditions (Figs 1 and 2) could then be the result of the increased release of the enzyme which was not immediately replaced by fresh enzyme. In previous experiments on the effect of dehydration on neurotransmitters in the rat hypophysis a selective decrease in the NL of immunoassayable SP by more than 60% has been observed, is This observation points towards a connection between AChE and SP in the pituitary gland, a possibility which is supported by the recently observed peptidase function of AChE which was first demonstrated with SP.5 A physiological role for this peptidase action of AChE on SP was suggested by the coexistence of SP and AChE in the dorsal horn of the spinal cord, a region in which no cholinergic neurons have so far been demonstrated. There is evidence that, on the enzyme molecule, the reactive site responsible for the hydrolysis of ACh is different from the peptidase site) Furthermore, a possible importance of the peptidase function of AChE in the N L during dehydration is indicated by a comparison of the present findings with observations on the dark adapted retina of the chick. During dark adaptation there was a decrease in the SP and in the [Leu]enkephalin immunoreactivity of the retina which was accompanied by a decrease in AChE activity. 6 A functional connection between the neuropeptides and the enzyme was demonstrated by the fact that addition of purified AChE to in vitro incubated sections of dark adapted retinae restored the neuropeptide content of the tissues. From these observations Chubb and Millar 6 concluded that AChE may be involved in the formation of peptide neurotransmitters from precursor proteins, the final step in the provision of neuropeptides to the nerve endings. The fall in AChE in the NL after dehydration together with the previously observed decrease of the SP content of the NL under these conditions suggests,
M. ALEXANDROVA ct ul.
426
that also in the NL, AChE could be involved in the formation of neuropeptides from precursor proteins and that the enzyme may be released together with its reaction product. This co-release of a neurotransmitter substance and of the enzyme responsible for its formation resembles the release of catecholamines and of DA-/I-hydroxylase from sympathetic nerves and the adrenal gland. ” The peptidase activity of AChE is not substrate specific. SP, two enkephalins and a variety of di- and tripeptides were all found to be degraded by pure AChE in in oitro experiments.’ However, oxytocin, vasopressin and /I-endorphin were not affected by AChE,’ a finding which is of particular interest for the present results on the NL. The observed increase in the release of AChE from the NIL after electrical stimulation of the stalk is unlikely to be due to leakage across the nerve fibre as no increase in AChE outflow from desheathed, in
vitro
incubated stretches of the phrenic nerve” or the vagus nerve34 was observed after electrical stimulation of the proximal end of the nerve. Thus, it is reasonable to conclude that AChE release in the NIL occurred at the nerve endings, possibly in parallel to ACh release3’ or with SP. To study the release of AChE from the NIL in detail, future experiments with tetrodotoxin and other substances involved in transmitter release are planned. The possibility that protein synthesis, in general, is modified by dehydration cannot be excluded. However, in the hypophysis, any such interference with the synthesis of the AChE protein would have to be restricted to AChE in the NL. Acknowledgements-We
would like to thank Mr T. R. for his help with some of the AChE assays. M.A. was supported by a grant from the Wellcome Trust. K.R. was in receipt of a British Council travel grant.
Cooper
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24. Tsuneki K., Urano A. and Kobayashi H. (1974) Monoamine oxidase and acetylcholinesterase in the neurohypophysis of the hagfish, Eptatretus burgeri. Gen. camp. Endocr. 24, 249-256. 25. Kilbinger-H., Lohnes I. and Muscholl E. (1975) Absence of muscarinic modulation of vasopressin release from the isolated rat neurohypophysis. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 287, 391-397. 26. Mamay A. and Nachmansohn D. (1937) Cholinesterase in voluntary frog’s muscle. J. Physiol., Land. 89, 359-367. 27. Chvasoyo J. O., Adeyemo 0. and Iramain C. A. (1980) Acetylcholinesterase activity in seven brain areas and adenohypophysis during the estrous cycle. Neuroscience Lett. 19, 289-292. 28. Pickford M. The inhibitory effect of acetylcholine on water diuresis in the dog and its pituitary transmission. J. Physiol., Land. 95, 226-238. 29. Potter L. T. (1967) A radiometric microassay of acetylcholinesterase. J. Pharmac. exp. Ther. 156, 5-506. 30. Sharman D. F. and Cooper T. R. (1986) A semi-automated fluorimetric method for measuring acetylcholinesterase activity in small volumes of cerebrospinal fluid and tissue extracts using acetylcholine as substrate. J. Neurosci. Meth. 16, 301-308. 31. Skau K. A. and Brimijoin S. (1978) Release of acetylcholinesterase from rat hemidiaphragm preparations stimulated through the phrenic nerve. Nature 275, 224226. 32. Sladek C. D. and Joynt R. J. (1979) Characterization of cholinergic control of vasopressin release by the organ-cultured rat hypothalamo-neurohypophyseal system. Endocrinology 104, 659-663. 33. Vilhardt H. and Baker R. V. (1976) Subcellular distribution of acetylcholin-esterases in the neural lobe of the bovine pituitary. Experientia 32, 1154-I 156. 34. Vogt M., Smith A. D. and Fuenmayor L. D. (1984) Factors influencing the cholinesterases of cerebrospinal fluid in the anaesthetized cat. Neuroscience 12, 979-995. 35. Weinshilboum R. M. (1978) Serum dopamine /I-hydroxylase. Pharmac. Rev. 30, 133-166. 36. Weston J. and Greenfield S. A. (1986) Release of acetylcholinesterase in the rat nigrostriatal pathway: relation to receptor activation and firing rate. Neuroscience 17, 1079-1088. 37. Whitaker S. and LaBella F. S. (1973) Cholinesterase in the posterior and intermediate lobes of the pituitary. Species differences as determined by light and electron microscopic histochemistry. Z. Zellforsch. mikrosk. Anat. 142, 69-88. (Accepted 8 September
1986)
1987
0306-4522/87
$3.00 + 0.00
Pergamon Journals Ltd 0 1987 IBRO
ACETYLCHOLINESTERASE IN THE RAT NEUROHYPOPHYSIS IS DECREASED AFTER DEHYDRATION AND RELEASED BY STIMULATION OF THE PITUITARY STALK M. ALEXANDROVA,* M. HOLZBAUER, K. RACKET and D. F. SHARMAN AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, U.K.; and TDepartment of Pharmacology, University of Mainz, F.R.G. acetylcholinesterase and non-specific cholinesterases are present in all three lobes of the rat pituitary gland. This paper describes two new observations on hypophyseal acetylcholinesterase. Firstly, a prolonged increase of neurohormone secretion evoked by dehydration and sodium loading was
Abstract-Specific
accompanied by a decrease in the acetylcholinesterase. activity localized to the neural lobe, where acetylcholinesterase has previously been demonstrated in fine nerve fibres. Secondly, electrical stimulation of the pituitary stalk in vitro elicited the release of acetylcholinesterase and non-specific cholinesterases from the combined neural and intermediate lobe indicating that the enzyme can be released from nerve endings in the hypophysis by action potentials. The observed loss of enzyme activity during dehydration may be the consequence of a prolonged activation of cholinergic nerves in the gland, leading to an increased release of acetylcholinesterase, which is not immediately replaced by fresh enzyme. The decrease in acetylcholinesterase in the neural lobe during dehydration may also be connected with its peptidase function and thus with the previously observed loss of substance P from the neural lobe during dehydration [Holzbauer et al. (1984) Neurosci. Left. 47, 23-281.
The pituitary gland is innervated by nerve fibres containing classical neurotransmitter substances such as dopamine (DA), noradrenaline (NA), 5-hydroxytryptamine (5-HT) or substance P (SP). The concentrations of these substances in the neural lobe (NL) and/or in the intermediate lobe (IL) changed when neurohormone secretion was increased over several days (see e.g. Refs 15, 19, 20). For DA and 5-HT it has been established that their synthesis and catabolism rates are altered under these conditions. Both amines could be released from the NL and IL in vitro by electrical stimulation of the pituitary stalk and they have been shown to affect hormone release (see Refs 16, 18). The present paper describes a study of cholinesterases in the rat pituitary gland. These enzymes occur in peripheral and central cholinergic neurons, in other neuronal or non-neuronal tissues and in blood, in several forms. The form usually referred to as specific acetylcholinesterase (E.C. 3.1.1.7), is especially enriched at cholinergic synapses where its main function is the inactivation of acetylcholine.26 *Pressent address: Institute of Experimental Endocrinology, Centre of Physiological Sciences, Slovak Academy of Sciences, Bratislava, Czechoslovakia. Address for correspondence: Margarethe Holzbauer, Department of Pharmacology, University of Cambridge, Hills Road, CB2 2QD, U.K. Abbreviations: ACh, acetylchohne; AChE, acetylcholinesterase; ACTH, adrenocorticotrophic hormone; AL, anterior lobe; DA, dopamine; S-HT, 5_hydroxytryptamine; IL, intermediate lobe; NA, noradrenaline; NIL, neurointermediate lobe; NL, neural lobe, ns-ChE, nonspecific cholinesterases; SP, substance P. 421
This process occurs on an anionic site of the enzyme molecule which binds to the quartemary ammonium of acetylcholine (ACh). Specific-acetylcholinesterase (specificAChE or AChE) and non-specific cholinesterase (ns-ChE) have been demonstrated in all three lobes of the hypophysis of several mammalian” and lower species23*24 with histochemical and biochemical methods. The enzymes were localized in non-neurosecretory nerve fibres and in pituicytes of the NL, and in glandular cells of the IL and the anterior lobe (AL).‘,’ Species differences in the proportions of specificAChE and ns-ChE in different lobes were reported.37 In the NL of the rat pituitary gland, AChE-containing nerve fibres were found to be concentrated in the region of the junction between the pituitary stalk and the NL.’ After subcellular fractionation of homogenates of NLs from bovine pituitaries, about 15-20% of AChE and of ns-ChE were found in the fraction containing the broken off nerve endings and neurohormones. About 60% of ns-ChE was in the fraction containing the cell nuclei which, in the case of the NL, will have been derived mainly from pituicytes. Most of the specific-AChE (about 30%) was in a soluble form in the high speed supernatant3’ With histochemical methods a similar distribution of AChE and of choline acetyltransferase in nonneurosecretory nerve fibres in the NL was demonstrated. This is indicative of a cholinergic innervation of this lobe.’ Acetylcholine (ACh) itself has been identified in pituitary extracts on several occasions, with higher concentrations in the NL than in the AL (e.g. Ref. 3). No direct effect of ACh on hormone
422
M.
ALEXANDROVA ct ul
release from isolated NLs has been reported.y,‘),‘4.?s.‘: ACh has, however, a strong stimulatory effect on the release of vasopressin and oxytocin in situations where the NL is in connection with hypothalamic tissue (see e.g. Refs 9, 28, 32). The evidence for an effect of ACh on adrenocorticotrophic hormone (ACTH) release from the IL is conflicting: Fischer and Moriarty’” observed a stimulating effect of ACh on ACTH release on in vitro incubated neurointermediate lobes (NILS), whereas Briaud et al.,* working with superfused rat NILS, did not observe a significant effect of ACh on ACTH release. A connection between a cholinergic neuronal system and hormone release from the AL has been indicated by several observations. Chronic exposure of rats to cold temperatures and immobilization led to a decrease in the AChE activity in the AL,‘* AChE activity in the AL changed during the oestrous cycle of the rat,27 after treatment with oestrogens** and after orchidectomy.*’ AChE can be released from peripheral tissues and in the central nervous system. This was first observed in experiments on the adrenal gland by Chubb and Smith.’ Skau and Brimijoin3’ demonstrated the release of the enzyme from the rat phrenic nerve. They suggested that AChE is normally released when action potentials invade cholinergic nerve terminals and that the enzyme release occurs in parallel with, but separate from, acetylcholine. The release of AChE from the central nervous system is indicated by the presence of the enzyme in cerebrospinal fluid.4 In experiments with push-pull cannulae Weston and Greenfield demonstrated AChE release in the nigrostriatal system of the rat after local application of drugs. In the present work, it was firstly investigated whether prolonged increased secretion of neurohormones, as elicited by dehydration and sodium loading, alters the activity of AChE in the three lobes of the rat hypophysis. Secondly, it was tested whether AChE can be released from the in vitro incubated, combined NL and IL (NIL) of the rat by electrical stimulation of the pituitary stalk using stimulation parameters known to result in propagated action potentials. Rats
EXPERIMENTAL
PROCEDURES
The rats used were male Wistar rats, Porton strain, Babraham colony, 300-400 g, which were kept at a constant temperature of 21°C and a constant light (14 h) and dark (IO h) cycle. Food (Oxoid 41B pellets) and water were available ud Iibitumunkss stated otherwise. All rats were killed during the light period. Tissue preparations Two sets of experiments were carried out in which the effect of&hydration on AChE activity in the pituitary gland was studied. Dehydration was induced by water withdrawal for 72 h followed by 24 h during which a 2.5% solntion of NaCl was offered instead of drinking water. The control rats had free access to water throughout the experiment. All rats were killed by rapid decapitation and the pituitary glands dissected immediately. In the first set of experiments the
anterior lobes were removed, put into small glass centrifuge tubes and frozen on solid CO,. The whole NIL was placed into a drop of 0.9% NaCl solution (saline) on a black glass plate under a dissecting microscope and the IL teased off the NL with a pair of fine forceps. The NL was then transferred into a small glass tube and put on solid COz. The drop of saline containing the IL fragments was sucked into a Pasteur pipette and transferred into another glass tube. The glass plate and the pipette were washed twice with small amounts of saline which were added to the IL fragments, the tissue suspension was centrifuged, the saline discarded and the tube containing the IL tissue placed on solid CO,. The whole procedure took about 10 min. By this technique, more than 90% of the IL tissue was usually removed from the NL as assessed by the amount of immunoassayable ACTH in the tissues.” The tissue size was not measured in these experiments. In the second set of experiments, which was carried out one month later, the NL and IL were not separated. The NIL and AL were put into pre-weighed glass tubes which were weighed again after the tissues had been put in, and then put on solid COj. In both sets of experiments the samples were stored at -80°C for not longer than one week before the enzyme was assayed. To investigate whether AChE can be released from the rat NIL by electrical stimulation of the pituitary stalk, the NIL together with the pituitary stalk was dissected out as described previously. ” The cranial portion of the intact pituitary stalk was placed between the two branches of a split platinum electrode and the preparation incubated for 15min in 5 ml of Krebs-HEPES buffer (pH 7.4) of the following composition (mM): NaCl 118.5; KC1 4.7; CaCI, 2.5; MgCI, 1.2;o-glucose 1I. 1;HEPES 20.0. The buffer was saturated with pure oxygen. The preparation was then transferred to a glass microbath which contained 60~1 incubation buffer. The bath was surrounded by a water jacket (37°C). A second platinum electrode was situated at the bottom of the bath. The medium was changed every IOmin. The first 10min were used to assess spontaneous enzyme release. During the following 10min the pituitary stalk was stimulated with biphasic pulses of 0.2 ms duration, 10 V, 15 Hz, 4 mA, 5 times for 1 min with 1 min intervals by means of a Grass S9 stimulator. These parameters are similar to those required to cause propagated action potentials.‘6 During the last 10 min period, post-stimulation release was measured. Five groups of stimulation experiments (Fig. 3 A-E) were carried out. Because of the small amounts of enzymes released relative to the sensitivity of the assay method samples from four NIL preparations were pooled in experiments A, B and C, from six preparations in experiments D and E. The samples were kept on ice until assay on the same day. All rats used in the stimulation experiments had free access to water. Enzyme assay The AChE and ns-ChE activity in the tissue extracts and in the incubation media were estimated as described by Sharman and Cooper.W Tbis assay is based on the hydrolysis of ACh by AChE. The choline formed is oxidized by the enzyme choline oxidase giving rise to hydrogen peroxide which is measured by the fluorescence developed from 4-methoxy-3-hydroxy-phenylacetic acid in the presence of horseradish p&oxid&. The standard enzymd used was Sigma Standard AChE: one unit will hvdrolvse 1.0 umol of ACh per min at pH 8 at 37°C. The &s&s weie homogenized in 100~1 sodium phosphate buffer (0.1 M, pH 7.5). When isolated NLs or ILs were analysed, tissues from two rats were pooled. Ten ~1 of homogenate was employed for each assay. Ns-ChE activity was determined by the inhibition of enzyme activity resulting from the addition of ethopropa&e (10e4 M). Total e&me activity was determined by the inhibition of activity resulting from the addition of physostigmine (5 x IO-’ M) or neostigmine (5 x 10m5M).
AChE in the rat neurohypophysis
423
munits
Specific-AChE/ IVlmg Protein*
AL
NL
3
30
** 6
20
10
AL
c
No. of samples: No. of lobes:
4 4
5 5
4 6
4 6
3 6
3 6
Fig. 1. Experiment 1. Effect of dehydration (72 h with no water followed by 24 h with 2.5% NaCl instead of drinking water) on the activity of specific acetylcholinesterase (SpecificAChE) in the pituitary gland of the rat. Open columns: controls; shaded columns: dehydrated rats. Column height: mean values + SEM. AL, anterior lobe; NL, neural lobe; IL, intermediate lobe.. Significance of difference between controls and dehydrated rats: **P < 0.01 (Student’s r-test); # : for this calculation the mean (M) protein contents of 50 ALs, 50 NLs and 50 ILs of rats of the same colony, sex and age estimated on previous occasions were used. To estimate the enzyme activity in incubation media of NILS 100 or 200~1 of the incubation medium were added to assay tubes containing evaporated buffer solution corresponding to the volume used.29 The assay for AChE and ns-ChE activity was then carried out on these solutions. Special chemicals used. HEPES: N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (Sigma); ethopropazine (May and Baker, gift to Dr Ann Silver); physostigmine sulphate (BDH); neostigmine methylsulphate (Koch-Light). RESULTS
Distribution of acetylcholinesterase in the rat hypophysis and the effect of dehydration on enzyme activity in different lobes of the hypophysis The results are summarized in Figs 1 and 2 and in Table 1. Specific-AChE activity per lobe was largest in the AL in both sets of experiments (Figs 1 and 2); so was the total enzyme activity (specific-AChE + ns-ChE) as shown in Table 1. Per unit tissue specificAChE activity was 2-3 times higher in the NL and in the IL than in the AL. Specific-AChE activity in the combined NILS of the second set of experiments was of a similar order of magnitude as the sum of activities in the NLs and ILs of the first set of experiments (cf. Figs 1 and 2). Dehydration did not significantly affect specific AChE activity in the AL in the first set of experiments (Fig. 1). In experiment 2, the fall in AChE activity in the AL, when expressed per lobe (Fig. 2, column 1
and 2), was due to a significant fall in the weight of the ALs during dehydration (Fig. 2, columns 9 and 10): there was no change in enzyme activity when expressed per mg tissue (Fig. 2, columns 5 and 6). In contrast, dehydration caused a significant decrease (-40%) in specific-AChE activity in the isolated NL (Fig. 1, columns 3 and 4), but not in the IL (Fig. 1, columns 5 and 6). In the second set of experiments the activity of this enzyme in the NILS of the dehydrated rats was decreased by 33% when expressed per lobe and by 29% when expressed per mg NIL tissue (Fig. 2). In Table 1, values for total enzyme activity (specific-AChE + ns-ChE) are given as estimated in experiment 1. About 80% of the total enzyme activity was due to specificAChE. Although the specificAChE activity in the AL and the NIL in experiment 2 was similar to that in experiment 1, the total enzyme activity in experiment 2 was 44% higher in the AL and 50% higher in the combined NL + IL. Dehydration affected the activity of AChE and ns-ChE in a similar manner. Release of acetylcholinesterase from the neurointer mediate lobe evoked by electrical stimulation of the pituitary stalk
Figure 3 shows the effect of electrical stimulation of the pituitary stalk on the outflow of AChE from
424
M. munits
specific-AChE/Lobe
ALEXANVROVArl al.
m units
specific-AChE/mg
rng ‘wet’
tissue weight
t
3
At.
tissue
NIL
AL
JIL
AL
NIL
2
t
6
Fig. 2. Experiment 2. Effect of dehydration (72 h with no water followed by 24 h with a 2.5% NaCi solution instead of drinking water) on Ihe activity of specific acetyichoiinesterase (speciftc-AChE) in the rat pituitary gland. Open columns: controls; shaded columns: dehydrated rats. Column heights: mean values + SEM, n = 12 for each group. AL, anterior lobe; NIL, combined neural and intermtiate lobe. Statistical significance of differences from corresponding controls: *P < 0.05; l*P < 0.02; ***P< 0.01. (Student’s r-test.)
the NIL of normal male rats. The mean release of choline&erases (specific-AChE + ns-ChE)/NIL/lQ min was 104 p units during the pre-stimulation period; during electrical stimulation of the stalk it was 179 p units and during the post-stimulation period 112 p units. These mean values were calculated from all five experiments and represent the enzyme outflow from 24 NILS. Statistical analysis of the results according to Friedman’s two-way analysis of variance by ranks” using a value of 5 for n, showed that the increase in enzyme release evoked by electrical stimulation of the pituitary stalk was significant at P
In the samples collected in experiments D and E (Fig. 3) the percentage of specific-AChE was also determined. In all six samples analysed specificAChE and ns-ChE were present in about equal quantities. In experiment D electrical stimulation of the stalk increased the release of specific AChE by 56%, that of ns-ChE by 44%. Electrical stimulation of the pituitary stalk was apparently without effect on the enzyme content of the NIL: the specific-AChE activity in two NILS only incubated for 45 min in Krebs-HEPES buffer was 7.3 and 7.3 m units/NIL, ns-ChE was 6.7 and 5.9 m units/NIL. In two NILS in which the pituitary stalk
Table 1. Specific-acetylcholine&erase + non-specific choiinesterase (= total enzyme) in the hypophysis of control rats and rats in which neurohormone secretion had been stimulated by &hydration and sodium loading Total enzyme activity (m units/lobe)
Lobe
Control
1 H,O
AL NL IL
11.55+ 1.15(4) 2.84 + 0.13 (4) 2.16& 1.80(3)
12.90 f 0.69 (5) 1.84+ 0.06(4) 1.80 f 0.03 (3)
Difference between Control and JH,O Significancet (%) +12 -35 -17
NS P < 0.01 NS
Specific-AChE activity as % of total enzyme activity* Control 80 k2.7 88 rt 4.5 75 & 2.6
lH,O ___87 f 2.3 82 f 2.6 ?6* 1.4
Results of experiment 1. (For specific-AChE activity see Fig. 1.) Abbreviations: AL, anterior lobe, NL, neural lobe; IL, intermediate lobe. AL, single lobes used for each assay; NL and IL, tissues from two rats pooled for each ashy. Mean values f SEM; *calculated from individual values; tStudent’s r-test; NS, not significant. Figures in pare&&s: number of samples anaiysed. JH@ no water for 72 h followed by 24 h with 2.5% NaCI. Enzyme activity: one unit AChE will hydrolyse I.Opmoi of ACh per min at pH 7 at 37°C (Sigma standard AChE).
AChE in the rat neurohypophysis
350
300.
250J
c
.E E o
200.
L
150.
2
o
100.
Sample: 1 Experiment:
I
2 B
3
1
2 D
1
2 E
Fig. 3. Effect of electrical stimulation of the pituitary stalk ( ze: l0 V, 15 Hz, 4 mA, 0.2 ms, 5 times for 1 min with i rain intervals) on the release of specific acetylcholinesterase +non-specific cholinesterase (total enzyme) from the combined neural and intermediate lobe (NIL) of the rat pituitary gland incubated in vitro. Each column represents enzyme release over 10 min. Sample 1: spontaneous release. Sample 2: release during electrical stimulation of the stalk. Sample 3: release after stimulation. In experiments A, B and C samples from four NIL preparations were pooled; in experiments D and E samples from six NIL preparations were pooled. Dark portions of columns: electrically evoked enzyme release. (For further details see Experimental Procedures.) had been stimulated the AChE activity was 7.7 and 6.8 m u n i t s / N I L the ns-ChE activity 5.7 and 5.3 m units/NIL The mean specific-AChE activity of all four incubated NILs was 7.28 + 0 . 1 8 m units/NIL This value was 77% higher than the AChE activity in unincubated NILs of the control rats in Fig. 2 observed 5 months earlier. The maximal amount of AChE released by electrical stimulation of the stalk during l0 min was less than 2% of the amount of enzyme contained in the NIL. DISCUSSION The present paper describes two new observations on AChE in the rat pituitary gland. Firstly, prolonged activation of neurohormone secretion by dehydration and sodium loading caused a decrease in the tissue content of AChE activity which was restricted to the NL (Fig. 1.). Secondly, electrical
425
stimulation of the pituitary stalk using stimulation parameters known to cause propagated action potentials, evoked the release of AChE from the in vitro incubated NIL. From histochemical observations and the position of the stimulating electrodes it can be presumed that the cholinergic nerves, which descend from the hypothalamus to the NL, have been stimulated. Dehydration has previously been shown to affect classical neurotransmitter systems in the NIL such as the DA, the NA, the 5-HT and also the SP system. Thus, a comparable activation of the cholinergic system in the NL during periods of increased hormone secretion is possible. If it is permissible to draw a parallel between observations on peripheral cholinergic nerves and the cholinergic system in the NL, one may reason that an increase in the release of ACh during dehydration could have occurred which was accompanied by an increase in the release of AChE (cf. Ref. 31). The observed fall in the AChE content in the NL under these conditions (Figs 1 and 2) could then be the result of the increased release of the enzyme which was not immediately replaced by fresh enzyme. In previous experiments on the effect of dehydration on neurotransmitters in the rat hypophysis a selective decrease in the NL of immunoassayable SP by more than 60% has been observed, is This observation points towards a connection between AChE and SP in the pituitary gland, a possibility which is supported by the recently observed peptidase function of AChE which was first demonstrated with SP.5 A physiological role for this peptidase action of AChE on SP was suggested by the coexistence of SP and AChE in the dorsal horn of the spinal cord, a region in which no cholinergic neurons have so far been demonstrated. There is evidence that, on the enzyme molecule, the reactive site responsible for the hydrolysis of ACh is different from the peptidase site) Furthermore, a possible importance of the peptidase function of AChE in the N L during dehydration is indicated by a comparison of the present findings with observations on the dark adapted retina of the chick. During dark adaptation there was a decrease in the SP and in the [Leu]enkephalin immunoreactivity of the retina which was accompanied by a decrease in AChE activity. 6 A functional connection between the neuropeptides and the enzyme was demonstrated by the fact that addition of purified AChE to in vitro incubated sections of dark adapted retinae restored the neuropeptide content of the tissues. From these observations Chubb and Millar 6 concluded that AChE may be involved in the formation of peptide neurotransmitters from precursor proteins, the final step in the provision of neuropeptides to the nerve endings. The fall in AChE in the NL after dehydration together with the previously observed decrease of the SP content of the NL under these conditions suggests,
M. ALEXANDROVA ct ul.
426
that also in the NL, AChE could be involved in the formation of neuropeptides from precursor proteins and that the enzyme may be released together with its reaction product. This co-release of a neurotransmitter substance and of the enzyme responsible for its formation resembles the release of catecholamines and of DA-/I-hydroxylase from sympathetic nerves and the adrenal gland. ” The peptidase activity of AChE is not substrate specific. SP, two enkephalins and a variety of di- and tripeptides were all found to be degraded by pure AChE in in oitro experiments.’ However, oxytocin, vasopressin and /I-endorphin were not affected by AChE,’ a finding which is of particular interest for the present results on the NL. The observed increase in the release of AChE from the NIL after electrical stimulation of the stalk is unlikely to be due to leakage across the nerve fibre as no increase in AChE outflow from desheathed, in
vitro
incubated stretches of the phrenic nerve” or the vagus nerve34 was observed after electrical stimulation of the proximal end of the nerve. Thus, it is reasonable to conclude that AChE release in the NIL occurred at the nerve endings, possibly in parallel to ACh release3’ or with SP. To study the release of AChE from the NIL in detail, future experiments with tetrodotoxin and other substances involved in transmitter release are planned. The possibility that protein synthesis, in general, is modified by dehydration cannot be excluded. However, in the hypophysis, any such interference with the synthesis of the AChE protein would have to be restricted to AChE in the NL. Acknowledgements-We
would like to thank Mr T. R. for his help with some of the AChE assays. M.A. was supported by a grant from the Wellcome Trust. K.R. was in receipt of a British Council travel grant.
Cooper
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