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Changes in sympathetic and endothelium-mediated responses in the rabbit central ear artery after acrylamide treatment
Journal of the Autonomic Nervous System, 36 (1991) 55-64 © 1991 Elsevier Science Publishers B.V. All rights reserved 0165-1838/91/$03.50
55
JANS 01205
Changes in sympathetic and endothelium-mediated responses in the rabbit central ear artery after acrylamide treatment K.I. Maynard, J. Lincoln, P.
Milner and G. Burnstock
Department of Anatomy and Developmental Biology and Centrefor Neuroscience, University College London, Gower Street, London, U.K. (Received 3 December 1990) (Revision received 24 June 1991) (Accepted 1 July 1991)
Key words: R a b b i t ear artery; Acrylamide; Sympathetic transmission; E n d o t h e l i u m - m e d i a t e d responses
Abstract The effect of acrylamide intoxication on the innervation and local control of the rabbit central ear artery was investigated. There was no difference in the noradrenaline, neuropeptide Y and calcitonin gene-related peptide tissue content between control and experimental animals. There was, however, a slight reduction in catecholamine histofluorescence. Although the contractile efficiency of the rabbit central ear artery as measured by responses to potassium chloride was unchanged, nerve-mediated contractile responses were significantly attenuated in acrylamide-treated animals. Contractile responses induced by exogenous t~,/3-methylene ATP were markedly increased after acrylamide treatment, in contrast to contractions induced by exogenous noradrenaline which were attenuated at maximal concentrations. Modulatory effects of nerve-mediated contractile responses by neuropeptide Y were unaffected by acrylamide intoxication. It therefore appears that acrylamide intoxication damages sympathetic cotransmission, perhaps with preferential action on the purinergic component. Endothelium-dependent relaxant responses to acetylcholine and substance P were attenuated in acrylamide-treated animals, whereas relaxant responses mediated by calcitonin gene-related peptide (endothelium independent) were unaffected. The question of whether the damage to the endothelial cell action is a primary effect, or a secondary consequence of sympathetic nerve damage, is discussed.
Introduction A c r y l a m i d e is a plastic m o n o m e r which produces a dying-back type of d e g e n e r a t i o n in distal m o t o r a n d sensory nerves in m a n a n d a n i m a l s [15,21,30]. It is well established that large-diameter a f f e r e n t nerves are m o r e sensitive to acryla m i d e t h a n small e f f e r e n t a n d u n m y e l i n a t e d fibres [11,22-24,26], which are only affected after
Correspondence: G. Burnstock, Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, U.K.
p r o l o n g e d intoxication. T h e earliest d e t e c t a b l e structural a n d f u n c t i o n a l c h a n g e s r e p o r t e d in acrylamide n e u r o p a t h y occur in sensory receptors [27]. It has t h e r e f o r e b e e n suggested, a n d r e c e n t evidence has s u p p o r t e d , that the m a j o r effect of acrylamide intoxication might b e p e r i p h e r a l sensory n e r v e i m p a i r m e n t [1,8,14]. T h e s e n s o r i m o t o r n e u r o p a t h y p r o d u c e d by acrylamide is of clinical a n d e x p e r i m e n t a l import a n c e b e c a u s e it has m a n y pathophysiological features in c o m m o n with h u m a n n e u r o p a t h i e s caused by alcohol or v i t a m i n deficiency, a n d clinical similarities with the n e u r o p a t h y associated with diabetes [26]. R e s e a r c h has t h e r e f o r e c o n c e n t r a t e d
56 on large-diameter myelinated somatic and sensory nerves. Nonetheless, there have been a few studies on the effect of acrylamide on the sympathetic nervous system [22,23,26,30]. These reports have shown that acrylamide intoxication damages sympathetic fibres in rat, cat and dog, and impairs the neural control of the cat mesenteric vascular bed. There is now considerable evidence that noradrenaline (NA), adenosine 5'-triphosphate (ATP) and neuropeptide Y (NPY) coexist in sympathetic nerves, and that NA and ATP act as cotransmitters, while NPY is a neuromodulator [4]. However, studies which reported damage to sympathetic nerve fibres due to acrylamide intoxication, did not distinguish between noradrenergic and purinergic components of sympathetic cotransmission, nor investigate neuromodulation. We have shown that the rabbit central ear artery is innervated by both sympathetic (noradrenergic and purinergic) [14,28,29] and sensory (calcitonin gene-related peptide (CGRP)- and substance P (SP)-containing) nerves [20], and that both are involved in the control of local blood flow [13,19,20,28,29]. In addition, endothelial cells are now recognised as being important in local vascular control due to the synthesis and release of endothelium-derived relaxing factor ( E D R F ) [7,10], which interacts with neural control [16]. This study was therefore aimed at examining the effect of chronic acrylamide treatment on the innervation, sympathetic cotransmission and its neuromodulation, and endothelium-dependent responses of the rabbit ear artery.
Materials and Methods
Animals Male New Zealand White rabbits (2.0-3.5 kg) were treated with acrylamide (40 m g / k g ) which was dissolved in distilled water and administered intraperitoneally. The animals (n = 10) were treated daily for up to 14 days. Two rabbits were killed earlier (day 7 and day 9) as they developed hind limb paralysis accompanied by significant weight loss, i.e. signs of severe acrylamide poisoning, which all other animals exhibited by day 14.
There were no obvious differences between these 2, and the other 8 animals that survived full treatment. Control animals (n = 10) were treated with saline. Animals were subsequently killed by an overdose of sodium pentobarbitone (Sagatal) followed by exsanguination. The central ear artery was then dissected out and divided into portions for the various techniques described below. Proximal segments were used for in vitro pharmacological experiments, whilst mid-segments were used for histofluorescence and bioassay techniques.
Histofluorescence Segments of rabbit ear artery were stained for catecholamine histofluorescence by the method of glyoxylic acid fluorescence [17] as previously described [28]. Selected areas were photographed using Kodak T M A X P3200 film. NA assay Mid-segments of the rabbit ear artery from control (n = 7) and treated (n = 6) animals were cleaned and frozen in liquid nitrogen until assay. NA tissue content was quantified by a high-performance liquid chromatography assay with electrochemical detection as previously described [9]. Immunoassay Segments of the mid-portion of the rabbit ear artery were measured, weighed and the peptides extracted. NPY and C G R P levels were quantitated by an inhibition enzyme-linked immunosorbent assay as previously described [2]. In l,itro pharmacological studies The preparations were mounted as previously detailed [12], and changes in isometric tension were measured in each segment of the artery after they had been equilibrated for 1 h under resting tension (500-1000 mg). The modified Krebs' solution [12] contained bacitracin (30 m g / l , a peptide antibiotic), and bovine serum albumin (50 mg/1) to prevent the peptides sticking to the glassware. Electrical field stimulation (EFS) at supramaximal voltage, 0.3 ms pulse width and 1 s train duration was delivered every 2 rain via two platinum wire electrodes placed on either side of the preparation. A frequency-response curve for 4, 8,
57 16, 30 and 60 Hz was obtained and the preparations were washed and left for at least 10 min to recover. NA (0.1-300/xM) was then added cumulatively over a 20 min period and washed out after a maximum contraction had been recorded. Once the preparations had returned to baseline (after a series of washes) they were again allowed at least 10 min to recover before construction of the frequency-response curve was repeated. The effect of NPY (10 nM) was investigated on the responses to EFS at 16 and 60 Hz. Once consistent responses had been obtained at a particular frequency, NPY was added to the organ bath during a 2 min interval between periods of stimulation. The periods of stimulation were continued for 8-10 rain after the addition of the peptide. The effect of NPY (10 nM) was measured at the point of maximum potentiation. The effects of C G R P (26.3 nM) and SP (10 nM) were tested on preconstricted preparations. Concentrations used for both C G R P and SP were near the ECs0 value for each peptide and therefore sensitive to changes that might occur [20]. The tone was raised by the addition and maintained presence of NA (0.3 #M). Once a steady tone had been established, the peptide was added. The preparations were subsequently washed once a maximum relaxant response had been obtained. Concentration-response curves were also obtained for cumulative addition of acetylcholine (ACh, 0.1-300 /zM) on preconstricted preparations as above. a,/3-Methylene ATP (1 /~M) (a,fl-mATP, a potent, stable analog of ATP and P2x-purinoceptor agonist) and potassium chloride (KC1, 120 mM) were administered separately. The concentrations used were near the ECs0 value for each substance and therefore sensitive to any changes that might occur [13]. They were left in contact with the tissue until the ensuing contractile responses had reached a peak, after which they were washed out. Responses of the vascular smooth muscle to EFS and drugs were recorded with a Grass FTO3C force-displacement transducer and were displayed on a Grass 79D ink writing polygraph. EFS was supplied by a Grass SD9 stimulator controlled via a Digitimer D100.
Frequency-response curves were evaluated in terms of (mg) tension. The modulatory action of NPY (10 nM) on contractile responses to EFS, was evaluated as the percentage change. Responses to ACh (0.1-300 /zM), SP (10 nM) and C G R P (26.3 nM) on preconstricted preparations have been expressed as the percentage relaxation, using the raised level of tone as the baseline. Responses to NA (0.1-300/.~M) and a,/3-mATP (1 t~M) have been expressed as a percentage of the contractile response to KCI (120 mM). pD z values were calculated as - l o g [ECs0] values for NA and ACh responses.
Drugs and compounds used Acrylamide Grade 1 (99.9%) was ~obtained from B D H Chemicals Ltd. ACh, a,/3-mATP (lithium salt), bacitracin, bovine serum albumin, ( - ) - n o r a d r e n a l i n e bitartrate, and glyoxylic acid monohydrate were all obtained from the Sigma Chemical Company. Sodium pentobarbitone (Sagatal) was obtained from RMB Animal Health Ltd. a C G R P , SP and NPY peptides and C G R P antiserum raised in rabbit were obtained from Cambridge Research Biochemicals. NPY antiserum raised in rabbit was obtained from Immunodiagnostic Laboratories. NA was made up fresh each day, dissolved and diluted in 100/xM ascorbic acid, while a,/3-mATP was made up in distilled water and kept frozen at a stock concentration of 0.1 mM.
Statistical analysis Student's paired t-test was used to analyse any significance in the responses to EFS elicited before and after NA incubation in both groups of animals. All other data were analysed using Student's unpaired t-test. P < 0.05 was regarded as being significant. All data are expressed as the mean + SEM.
Results
Histofluorescence There was a dense network of catecholamine fluorescence in the control rabbit ear artery segments (n = 7) examined. The network consisted
58
amount of C G R P or NPY between the two groups of samples (Table I). Pharmacological int, estigations Contractile responses There was no significant difference between the contractile responses to KCI (120 mM) in control compared with treated animals.
)
Fig. I. Catecholamine fluorescence in whole-mount stretch preparations of the rabbit central ear artery from (a) saline-injected control and (b) chronic acrylamide-treated animals. Note the reduction in the n u m b e r of catecholamine fluorescent single a n d / o r small bundle perivascular nerve fibres in (b). Reduction of intensity was also sometimes seen but not illustrated here. Calibration bar = 20 ~ m .
of nerve bundles as well as single nerve fibres (Fig. la). Segments from treated animals showed either a reduction in intensity of catecholaminefluorescence (n = 4) a n d / o r a noticeable loss of single a n d / o r small bundles of catecholaminelabelled fluorescent fibres (n = 5 ) with nerve bundles intact (Fig. lb). NA assay There was no significant difference in N A tissue content between control (0.550 _+ 0.113 n g / m m , n = 7) and treated (0.538 + 0.077 rig/ram, n = 6) rabbit ear artery segments examined. lmmunoassays Immunoassays were performed on rabbit ear artery segments from control and treated animals. T h e r e was no significant difference in the
Frequency-response curues EFS of the perivascular nerve fibres of the rabbit ear artery produced rapid, frequency-dependent (4-60 Hz) contractions which were blocked by tetrodotoxin (1 p.M). In treated animals, responses to EFS were markedly attenuated at all frequencies, but were more significantly reduced at the lower frequencies (Fig. 2). After cumulative administration and washing out of N A (0.1-300/.~M), the responses to EFS at all frequencies examined in segments from acryiamide-treated animals were not significantly different from those in segments from control animals, before or after cumulative administration of NA (Fig. 2). Responses to exogenous NA NA (0.1-300 p.M) added cumulatively produced c o n c e n t r a t i o n - d e p e n d e n t contractions which were maintained in both control and treated animals. There was no significant difference between the pD 2 values in control (5.97 ± 0.06, n = 10) versus treated (6.09 _+ 0.06, n = 10) animals. The contractions elicited by N A (10-300 /xM), however, were significantly attenuated in TABLE 1 Tissue content of CGRP and N P Y in rabbit central ear arteo' segments of acrylamide-treated and control animals CGRP (pmol/cm) Control segments Treated segments
NPY (pmol/cm)
0.014 _+0.004
0.268 _+0.086
(6)
(6)
0.014 4- 0.003
0.247 _+0.062
(6)
(6)
Results are expressed as mean + SEM. Numbers in parentheses indicate the number of animals.
59 Tension [rag) 2000
Effect of NPY on the responses to EFS In the presence of NPY (10 nM) the contractile responses to EFS (16 and 60 Hz) were enhanced. The maximal effect was observed after 4 - 6 min; the responses then returned to control levels. The potentiation mediated by NPY was not significantly different in control compared with treated animals (Table II).
- -
1500
Relaxant responses Well-maintained contractile responses were observed in both control and treated preparations after addition of a sub-maximal concentration of NA (0.3 /~M). This concentration was used because we had already established that there was
1000 - -
500 - -
~+ee
% KCI (120 m M )
200 - -
o 4
I
I
I
I
8
16
30
60
Frequency [ H z )
Fig. 2. Frequency-response curves for rabbit isolated ear artery preparations. Preparations were stimulated at supramaximal voltage, 0.3 ms pulse width and 1 s train duration. Curves represent saline-injected control (circles) and chronic acrylamide-treated (squares) animals before (open symbols) and after (closed symbols) incubation with NA. Statistical analysis was performed using Student's t-test for unpaired observations i.e. saline-injected control animals versus acrylamidetreated animals. P < 0.05 was considered significant, n > 8 for each group of observations. *, P < 0.05; * * , P < 0.01. Symbols represent the m e a n and vertical lines show the SEM.
150 ~
.
*
100
50'
the treated compared with control animals (Fig. 3). 0--
Responses to exogenous a,fl-mA TP Application of a sub-maximal concentration of o~,/3-mATP (1 izM) produced a rapid contraction that was not maintained. There was a marked increase in the response to exogenous a,fl-mATP (1 /xM) in treated (44.7 + 4.9% response to KCI (120 mM), n = 10) compared with control (17.3 + 3.8% response to KCI (120 mM), n = 9) animals (Fig. 4).
I
1
I
I
I
0.1
0.3
1.0
3.0
10.0
N A Concentration
I 30.0
I
I
100.0
300.0
(pM]
Fig. 3. Concentration-response curves for the action of exogenous N A (0.1-300 ~ M ) on rabbit isolated central ear artery preparations from saline-injected control (open circles) and chronic acrylamide-treated (filled circles) animals. Results are calculated for each observation as a percentage of the contraction obtained by KC1 (120 mM). Student's unpaired t-test was used to calculate statistical significance, n = 10 for each concentration, and P < 0.05 was considered significant. *, P < 0.05; * * , P < 0.01; * * * , P < 0.001. Symbols represent the m e a n and vertical lines show the SEM.
60
% KCI 0 2 0 mM)
% Relaxation
100 --
50--
75--
25-50--
25--
0
Control Acrylamide treated Fig. 4. Effect of c~,/3-methylene ATP (1 /zM) on the rabbit isolated central ear artery in saline-injected control and chronic acrylamide-treated animals. Results are calculated for each observation as a percentage of the contraction obtained by potassium chloride (KCI, 120 raM). Statistical significance was calculated using Student's t-test for unpaired observations, and P < 0.05 was considered significant. * * *, P < 0.001. n = 9 for control animals, and 10 for acrylamide-treated animals. Bars represent the mean and vertical lines show the SEM.
0--
I
I
I
T
~
I
I
7.0
6.5
6.0
5.5
5.0
4.5
4.0
- log A c e t y l c h o l i n e C o n c e n t r a t i o n
Fig. 5. Concentration-response curves for the endothelium-dependent relaxant effect of ACh on preconstriced (NA, 0.3 /zM) rabbit isolated central ear artery preparations. Curves represent saline-injected control (open circles) and chronic acrylamide-treated (filled circles) animals. Statistical analysis was performed using Student's t-test for unpaired observations, n >_ 6 for each group of observations. P < 0.05 was considered significant. *, P < 0.05; * * , P < 0.01; * * * , P < 0.001. Symbols represent the mean and vertical lines show the SEM.
T A B L E II
The effect of acrylamide treatment on the potentiating action of N P Y (10 riM) on the neurogenic, contractile responses elicited by electrical field stimulation (supramaximal ~'oltage, 0.3 ms pulse width and 1 s train duration) in the rabbit central ear artery Frequency
% Change
Significance
(Hz)
Control
Treated
16
43.7 ± 19.9 (8) 16.2± 6.6 (7)
41.1 ± 16.7 (10) 16.0± 6.6 (10)
60
NS NS
NS: not significant. Numbers in parentheses indicate number of animals investigated.
no significant difference in contractile responses elicited by NA at this concentration in either group (Fig. 3). The preconstricted preparations were used to examine relaxant responses.
(a) Endothelium-dependent relaxant responses. ACh: ACh (0.1-300 /zM) added cumulatively produced a concentration-dependent relaxation on preconstricted preparations. Although the pD 2 values were not significantly different between control (6.71 + 0.13, n = 6) and treated (6.63 4-_ 0.05, n = 9) animals, responses to ACh were significantly attenuated at every concentration tested, except 10/xM (Fig. 5).
61 % Relaxation 90--
Key
iII
Control ~ Acrylamide treated
60--
30-
0-CGRP [26.3 nM)
/I
SP [10.0 nM]
Fig. 6. Effect of CGRP (26.3 nM, endothelium-independent relaxation) and substance P (SP, 10.0 nM, endothelium-dependent relaxation) on preconstricted (NA, 0.3 /~M) rabbit isolated central ear artery preparations. Statistical analysis was performed using Student's t-test for unpaired observations, n = 8 for each group of observations. P < 0.05 was considered significant. *, P < 0.05. Bars represent the mean and vertical lines show the SEM.
Substance P: Relaxant responses elicited by SP (10 nM) added to preconstricted preparations were significantly reduced in treated (34.7 + 5.6% relaxation, n = 8) compared with control (57.1 + 8.0% relaxation, n = 8) animals (Fig. 6). (b) Endothelium-independent relaxant responses. CGRP: Relaxations induced by CGRP (26.3 nM) on preconstricted preparations were not significantly affected in the treated (80.0 + 5.6%, n = 8) compared with control (89.5 + 4.1%, n = 8) animals (Fig. 6).
Discussion There was no loss of NA or NPY content in the rabbit ear artery with acrylamide treatment, although a reduction in the number of single a n d / o r small-bundle catecholamine fluorescent perivascular nerve fibres was observed. This is indicative of sympathetic nerve damage, which is
reported to occur with acrylamide intoxication [12,22-24,30]. The reduced fluorescence in single a n d / o r small bundles of fibres in the ear artery was probably not enough to make a significant difference in the total tissue content of NA, since there remained many large intensely fluorescent bundles of perivascular nerve fibres in ear artery preparations from experimental animals. The tissue content of the CGRP-containing (sensory) perivascular nerves, and the modulatory action of NPY on the sympathetic co-transmission in the ear artery were also unaffected by acrylamide treatment. The recovery of EFS-induced contractile responses to control levels at all frequencies after exposure to exogenous NA in preparations from acrylamide-treated animals, may be due to the fact that there was a shortage of NA in the terminal varicosities that was reversed by NA incubation. This possibility is supported by the reduced fluorescence in the single and small-bundle catecholamine-containing perivascular fibres. In the early stages of sympathetic nerve degeneration, leakage of transmitter occurs [25]. This leakage leads to a gradual disappearance of transmitter, perhaps reflected in this study by the reduced catecholamine histofluorescence, in addition to a degree of subsensitivity to the transmitter, as observed in the attenuated responsiveness to exogenous NA in preparations from acrylamide-treated animals. In the later stages sympathetic nerve degeneration can lead to postjunctional receptor supersensitivity [25], as observed in the increased responsiveness to a,/3-mATP after acrylamide treatment. The purinergic component of sympathetic cotransmission in the rabbit ear artery is favoured at low frequencies of stimulation [13]. In the present study, the significantly greater reduction of the EFS-induced contractile responses at the lower frequencies after acrylamide treatment could be taken to suggest that the purinergic component of sympathetic cotransmission may be more severely impaired than the noradrenergic component. To determine whether acrylamide poisoning preferentially affects the purinergic component of sympathetic transmission requires additional experimentation, including measure-
62 ment of the contractile response to electrical field stimulation after P2x-purinoceptor desensitization, a n d / o r using a~-adrenoceptor antagonists. It is interesting to note that we have previously shown a similar pattern of changes in the rabbit ear artery after acute exposure to a single dose of x-radiation. As in acrylamide intoxication, there was a decrease in the nerve-mediated responses, accompanied by a marked increase in responsiveness to c~,/3-mATP, but not to N A [31]. Further experimentation is needed to clarify the mechanism of action involved in these pathological changes. The endothelium-dependent responses examined by the application of exogenous ACh and SP on preconstricted ear artery preparations were both significantly attenuated in acrylamidetreated animals, whereas the endothelium-independent relaxation mediated by C G R P [5,20] was unaffected. The efficiency of the vascular smooth muscle of the ear artery was not affected by acrylamide intoxication, since there was no change in either the contractile responses to KC1, or CGRP-induced vasodilatation from control and treated animals. Acrylamide treatment may have caused primary damage to the mechanism of endothelium-dependent vaso0ilatation by affecting ACh and SP receptors on the endothelial cells of the ear artery. Other possible explanations are that there may be an impairment of E D R F synthesis, release, a n d / o r its action on the smooth muscle [7,10,16]. Acrylamide may also be acting at the molecular level [15] to inhibit the increase of cGMP, but not cAMP, since the former is reported to be the second messenger in E D R F mediated relaxant responses, whilst the latter is thought to be involved in C G R P - m e d i a t e d relaxation [6]. An alternative possibility is that acrylamide may also be exerting its effect of attenuating endothelium-dependent relaxant responses as a secondary consequence of sympathetic nerve damage. There is evidence that substances present in nerves may act as trophic factors [3]. It was shown that after chronic denervation of the rabbit ear artery, methacholine (endothelium-dependent)-induced relaxation was significantly reduced, but sodium nitroprusside (endothelium-in-
dependent)-induced relaxation was unchanged [18]. It is possible, therefore, that sympathetic nerve damage, induced by acrylamide intoxication in this study, may have affected endotheliummediated responses via trophic interactions [16]. In conclusion, our results show that although acrylamide intoxication attenuates sympathetic cotransmission, perhaps with preferential action on the purinergic component, neuromodulation mediated by NPY is unaffected by this treatment. Endothelium-dependent relaxant responses induced by ACh and SP are also attenuated in the a c r y l a m i d e - t r e a t e d animals, in contrast to CGRP-induced, endothelium-independent relaxant responses which are normal. Thus it appears that acrylamide does not exclusively damage nerves as has been generally assumed, but also affects the endothelial component of local mechanisms controlling blood flow.
Acknowledgements K.M. was a recipient of an Overseas Research Student Award and Departmental Research Studentship. This work was partly supported by the British H e a r t Foundation. The authors would like to express their gratitude to Dr. C.H.V. Hoyle for discussion of various aspects of this work, and Mrs. Philippa Charatan for her editorial assistance.
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63 5 Dogramatzis, D., Steinsland, O.S., Nickols, G.A. and Cooper, C.W., Mechanisms of the vasodilatory action of calcitonin gene-related peptide, Abstr. X Int. Conf. Pharmacol. (1987) p. 931. 6 Edvinsson, L. Fredholm, B.B., Hamel, E., Jansen, I. and Verrecchia, A.C., Perivascular peptides relax cerebral arteries concomitant with stimulation of cyclic adenosine monophosphate accumulation or release of an endothelium-derived relaxing factor in the cat, Neurosci. Lett., 58 (1985) 213-217. 7 Furchgott, R.F. and Zawadzki, J.V., The oligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature, 288 (1980) 373-376. 8 Goldstein, B.D., Acrylamide preferentially affects slowly adapting cutaneous mechanoreceptors, Tox. Appl. Pharmacol., 80 (1985) 527-533. 9 Griffith, S.G., Crowe, R., Lincoln, J., Haven, A.J. and Burnstock, G., Regional differences in the density of perivascular nerves and varicosities, noradrenaline content and responses to nerve stimulation in the rabbit ear artery, Blood Vessels, 19 (1982) 41-52. 10 Griffith, T.M., Edwards, D.H., Davies, R. L., Harrison, T.J. and Evans, K.T., Endothelium-derived relaxing factor (EDRF) and resistance vessels in an intact vascular bed: a microangiographic study of the rabbit isolated ear., Br. J. Pharmacol., 93 (1988) 654-662. 11 Hersch, M.I., McLeod, J.G., Satchell, P.M., Early, R.G. and Sullivan, C.E., Breathing pattern, lung inflation reflex and airway tone in acrylamide neuropathy, Respiratory Physiol., 76 (1989) 257-276. 12 Kennedy, C. and Burnstock, G., ATP produces vasodilation via Pl-purinoceptors and vasoconstriction via P2purinoceptors in the isolated rabbit central ear artery, Blood Vessels, 22 (1985) 145-155. 13 Kennedy, C., Saville, V.L. and Burnstock, G., The contributions of noradrenaline and ATP to the responses of the rabbit central ear artery to sympathetic nerve stimulation depend on the parameters of stimulation, Eur. J. Pharmacol., 122 (1986) 291-300. 14 Kudlacz, E.M., Gerald, M.C. and Wallace, L.J., Sensory nerves and urinary bladder function: effect of diabetes, capsaicin and acrylamide treatment, Gen. Pharmacol., 20 (1989) 31-34. 15 Lapadula, D.M., Bowe, M., Carrington, C.D., Dulak, L., Friedman, M. and Abou-Donia, M.B., In vitro binding of [14C]acrylamide to neurofilament and microtubule proteins of rats, Brain Res., 481 (1989) 157-161. 16 Lincoln, J. and Burnstock, G., Neural-endothelial interactions in control of local blood flow, In J. Warren (Ed.), The Endothelium: An Introduction to Current Research, Wiley-Liss Inc., New York, 1990, pp. 21-32.
17 Lindvall, O. and Bj6rklund, A., The glyoxylic acid fluorescence histochemical method: a detailed account of the methodology for the visualisation of central catecholaminergic neurons, Histochemistry, 39 (1974) 97-127. 18 Mangiarua, E.I. and Bevan, R.D., Altered endotheliummediated relaxation after denervation of growing rabbit ear artery, Eur. J. Pharmacol., 122 (1986) 149-152. 19 Maynard K.I. and Burnstock, G., Effect of CGRP on 3H-NA release in the rabbit ear artery, Regul. Pept., 26 (1989) 82. 20 Maynard, K.I., Saville, V.L. and Burnstock, G., Sensorymotor neuromodulation of sympathetic vasoconstriction in the rabbit central ear artery, Eur. J. Pharmacol., 187 (1990) 171-182. 21 Miller, M.S. and Spencer, P.S., The mechanisms of acrylamide axonopathy, Ann. Rev. Pharmacol. Toxicol., 25 (1985) 643-666. 22 Post, E.J. and McLeod, J.G., Acrylamide autonomic neuropathy in the cat. Part 1. Neurophysiological and histological studies, J. Auton. Nerv. Syst., 33 (1977) 353-374. 23 Post, E.J. and McLeod, J.G., Acrylamide autonomic neuropathy in the cat. Part 2. Effects on mesenteric vascular control, J. Auton. Nerv. Syst. 33 (1977) 375-385. 24 Post, E.J., Unmyelinated nerve fibres in feline acrylamide neuropathy, Acta Neuropathol., 42 (1978) 19-24. 25 Reas, H.W. and Trendelenburg, U., Changes in the sensitivity of the sweat glands of the cat after denervation, J. Pharmacol. Exp. Ther., 156 (1967) 126-136. 26 Satchell, P.M., Circulatory control in canine acrylamide neuropathy, J. Auton. Nerv. Syst., 10 (1984) 93-106. 27 Satchell, P.M. and McLeod, J.G., Abnormalities of oesophageal mechanoreceptors in canine acrylamide neuropathy, J. Neurol. Neurosurg. Psychiatry, 47 (1984) 692698. 28 Saville, V. and Burnstock, G., Use of reserpine and 6-hydroxydopamine support evidence for purinergic cotransmission in the rabbit central ear artery, Eur. J. Pharmacol., 155 (1988) 271-277. 29 Saville, V.L., Maynard, K.I. and Burnstock, G., Neuropeptide Y potentiates purinergic as well as adrenergic responses of the rabbit ear artery, Eur. J. Pharmacol., 176 (1990) 117-125. 30 Schmidt, R.E., Plurad, S.B. and Clark, H.B., Acrylamideinduced sympathetic autonomic neuropathy resulting in pineal denervation, Lab. Invest., 56 (1987) 505-517. 31 Stewart-Lee, A.L., Maynard, K.I., Lincoln, J. and Burnstock, G., Sympathetic neurotransmission in the isolated rabbit central ear artery is affected as early as one week following a single dose of x-irradiation, Br. J. Pharmacol., 102 (1991) 23-26.
55
JANS 01205
Changes in sympathetic and endothelium-mediated responses in the rabbit central ear artery after acrylamide treatment K.I. Maynard, J. Lincoln, P.
Milner and G. Burnstock
Department of Anatomy and Developmental Biology and Centrefor Neuroscience, University College London, Gower Street, London, U.K. (Received 3 December 1990) (Revision received 24 June 1991) (Accepted 1 July 1991)
Key words: R a b b i t ear artery; Acrylamide; Sympathetic transmission; E n d o t h e l i u m - m e d i a t e d responses
Abstract The effect of acrylamide intoxication on the innervation and local control of the rabbit central ear artery was investigated. There was no difference in the noradrenaline, neuropeptide Y and calcitonin gene-related peptide tissue content between control and experimental animals. There was, however, a slight reduction in catecholamine histofluorescence. Although the contractile efficiency of the rabbit central ear artery as measured by responses to potassium chloride was unchanged, nerve-mediated contractile responses were significantly attenuated in acrylamide-treated animals. Contractile responses induced by exogenous t~,/3-methylene ATP were markedly increased after acrylamide treatment, in contrast to contractions induced by exogenous noradrenaline which were attenuated at maximal concentrations. Modulatory effects of nerve-mediated contractile responses by neuropeptide Y were unaffected by acrylamide intoxication. It therefore appears that acrylamide intoxication damages sympathetic cotransmission, perhaps with preferential action on the purinergic component. Endothelium-dependent relaxant responses to acetylcholine and substance P were attenuated in acrylamide-treated animals, whereas relaxant responses mediated by calcitonin gene-related peptide (endothelium independent) were unaffected. The question of whether the damage to the endothelial cell action is a primary effect, or a secondary consequence of sympathetic nerve damage, is discussed.
Introduction A c r y l a m i d e is a plastic m o n o m e r which produces a dying-back type of d e g e n e r a t i o n in distal m o t o r a n d sensory nerves in m a n a n d a n i m a l s [15,21,30]. It is well established that large-diameter a f f e r e n t nerves are m o r e sensitive to acryla m i d e t h a n small e f f e r e n t a n d u n m y e l i n a t e d fibres [11,22-24,26], which are only affected after
Correspondence: G. Burnstock, Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, U.K.
p r o l o n g e d intoxication. T h e earliest d e t e c t a b l e structural a n d f u n c t i o n a l c h a n g e s r e p o r t e d in acrylamide n e u r o p a t h y occur in sensory receptors [27]. It has t h e r e f o r e b e e n suggested, a n d r e c e n t evidence has s u p p o r t e d , that the m a j o r effect of acrylamide intoxication might b e p e r i p h e r a l sensory n e r v e i m p a i r m e n t [1,8,14]. T h e s e n s o r i m o t o r n e u r o p a t h y p r o d u c e d by acrylamide is of clinical a n d e x p e r i m e n t a l import a n c e b e c a u s e it has m a n y pathophysiological features in c o m m o n with h u m a n n e u r o p a t h i e s caused by alcohol or v i t a m i n deficiency, a n d clinical similarities with the n e u r o p a t h y associated with diabetes [26]. R e s e a r c h has t h e r e f o r e c o n c e n t r a t e d
56 on large-diameter myelinated somatic and sensory nerves. Nonetheless, there have been a few studies on the effect of acrylamide on the sympathetic nervous system [22,23,26,30]. These reports have shown that acrylamide intoxication damages sympathetic fibres in rat, cat and dog, and impairs the neural control of the cat mesenteric vascular bed. There is now considerable evidence that noradrenaline (NA), adenosine 5'-triphosphate (ATP) and neuropeptide Y (NPY) coexist in sympathetic nerves, and that NA and ATP act as cotransmitters, while NPY is a neuromodulator [4]. However, studies which reported damage to sympathetic nerve fibres due to acrylamide intoxication, did not distinguish between noradrenergic and purinergic components of sympathetic cotransmission, nor investigate neuromodulation. We have shown that the rabbit central ear artery is innervated by both sympathetic (noradrenergic and purinergic) [14,28,29] and sensory (calcitonin gene-related peptide (CGRP)- and substance P (SP)-containing) nerves [20], and that both are involved in the control of local blood flow [13,19,20,28,29]. In addition, endothelial cells are now recognised as being important in local vascular control due to the synthesis and release of endothelium-derived relaxing factor ( E D R F ) [7,10], which interacts with neural control [16]. This study was therefore aimed at examining the effect of chronic acrylamide treatment on the innervation, sympathetic cotransmission and its neuromodulation, and endothelium-dependent responses of the rabbit ear artery.
Materials and Methods
Animals Male New Zealand White rabbits (2.0-3.5 kg) were treated with acrylamide (40 m g / k g ) which was dissolved in distilled water and administered intraperitoneally. The animals (n = 10) were treated daily for up to 14 days. Two rabbits were killed earlier (day 7 and day 9) as they developed hind limb paralysis accompanied by significant weight loss, i.e. signs of severe acrylamide poisoning, which all other animals exhibited by day 14.
There were no obvious differences between these 2, and the other 8 animals that survived full treatment. Control animals (n = 10) were treated with saline. Animals were subsequently killed by an overdose of sodium pentobarbitone (Sagatal) followed by exsanguination. The central ear artery was then dissected out and divided into portions for the various techniques described below. Proximal segments were used for in vitro pharmacological experiments, whilst mid-segments were used for histofluorescence and bioassay techniques.
Histofluorescence Segments of rabbit ear artery were stained for catecholamine histofluorescence by the method of glyoxylic acid fluorescence [17] as previously described [28]. Selected areas were photographed using Kodak T M A X P3200 film. NA assay Mid-segments of the rabbit ear artery from control (n = 7) and treated (n = 6) animals were cleaned and frozen in liquid nitrogen until assay. NA tissue content was quantified by a high-performance liquid chromatography assay with electrochemical detection as previously described [9]. Immunoassay Segments of the mid-portion of the rabbit ear artery were measured, weighed and the peptides extracted. NPY and C G R P levels were quantitated by an inhibition enzyme-linked immunosorbent assay as previously described [2]. In l,itro pharmacological studies The preparations were mounted as previously detailed [12], and changes in isometric tension were measured in each segment of the artery after they had been equilibrated for 1 h under resting tension (500-1000 mg). The modified Krebs' solution [12] contained bacitracin (30 m g / l , a peptide antibiotic), and bovine serum albumin (50 mg/1) to prevent the peptides sticking to the glassware. Electrical field stimulation (EFS) at supramaximal voltage, 0.3 ms pulse width and 1 s train duration was delivered every 2 rain via two platinum wire electrodes placed on either side of the preparation. A frequency-response curve for 4, 8,
57 16, 30 and 60 Hz was obtained and the preparations were washed and left for at least 10 min to recover. NA (0.1-300/xM) was then added cumulatively over a 20 min period and washed out after a maximum contraction had been recorded. Once the preparations had returned to baseline (after a series of washes) they were again allowed at least 10 min to recover before construction of the frequency-response curve was repeated. The effect of NPY (10 nM) was investigated on the responses to EFS at 16 and 60 Hz. Once consistent responses had been obtained at a particular frequency, NPY was added to the organ bath during a 2 min interval between periods of stimulation. The periods of stimulation were continued for 8-10 rain after the addition of the peptide. The effect of NPY (10 nM) was measured at the point of maximum potentiation. The effects of C G R P (26.3 nM) and SP (10 nM) were tested on preconstricted preparations. Concentrations used for both C G R P and SP were near the ECs0 value for each peptide and therefore sensitive to changes that might occur [20]. The tone was raised by the addition and maintained presence of NA (0.3 #M). Once a steady tone had been established, the peptide was added. The preparations were subsequently washed once a maximum relaxant response had been obtained. Concentration-response curves were also obtained for cumulative addition of acetylcholine (ACh, 0.1-300 /zM) on preconstricted preparations as above. a,/3-Methylene ATP (1 /~M) (a,fl-mATP, a potent, stable analog of ATP and P2x-purinoceptor agonist) and potassium chloride (KC1, 120 mM) were administered separately. The concentrations used were near the ECs0 value for each substance and therefore sensitive to any changes that might occur [13]. They were left in contact with the tissue until the ensuing contractile responses had reached a peak, after which they were washed out. Responses of the vascular smooth muscle to EFS and drugs were recorded with a Grass FTO3C force-displacement transducer and were displayed on a Grass 79D ink writing polygraph. EFS was supplied by a Grass SD9 stimulator controlled via a Digitimer D100.
Frequency-response curves were evaluated in terms of (mg) tension. The modulatory action of NPY (10 nM) on contractile responses to EFS, was evaluated as the percentage change. Responses to ACh (0.1-300 /zM), SP (10 nM) and C G R P (26.3 nM) on preconstricted preparations have been expressed as the percentage relaxation, using the raised level of tone as the baseline. Responses to NA (0.1-300/.~M) and a,/3-mATP (1 t~M) have been expressed as a percentage of the contractile response to KCI (120 mM). pD z values were calculated as - l o g [ECs0] values for NA and ACh responses.
Drugs and compounds used Acrylamide Grade 1 (99.9%) was ~obtained from B D H Chemicals Ltd. ACh, a,/3-mATP (lithium salt), bacitracin, bovine serum albumin, ( - ) - n o r a d r e n a l i n e bitartrate, and glyoxylic acid monohydrate were all obtained from the Sigma Chemical Company. Sodium pentobarbitone (Sagatal) was obtained from RMB Animal Health Ltd. a C G R P , SP and NPY peptides and C G R P antiserum raised in rabbit were obtained from Cambridge Research Biochemicals. NPY antiserum raised in rabbit was obtained from Immunodiagnostic Laboratories. NA was made up fresh each day, dissolved and diluted in 100/xM ascorbic acid, while a,/3-mATP was made up in distilled water and kept frozen at a stock concentration of 0.1 mM.
Statistical analysis Student's paired t-test was used to analyse any significance in the responses to EFS elicited before and after NA incubation in both groups of animals. All other data were analysed using Student's unpaired t-test. P < 0.05 was regarded as being significant. All data are expressed as the mean + SEM.
Results
Histofluorescence There was a dense network of catecholamine fluorescence in the control rabbit ear artery segments (n = 7) examined. The network consisted
58
amount of C G R P or NPY between the two groups of samples (Table I). Pharmacological int, estigations Contractile responses There was no significant difference between the contractile responses to KCI (120 mM) in control compared with treated animals.
)
Fig. I. Catecholamine fluorescence in whole-mount stretch preparations of the rabbit central ear artery from (a) saline-injected control and (b) chronic acrylamide-treated animals. Note the reduction in the n u m b e r of catecholamine fluorescent single a n d / o r small bundle perivascular nerve fibres in (b). Reduction of intensity was also sometimes seen but not illustrated here. Calibration bar = 20 ~ m .
of nerve bundles as well as single nerve fibres (Fig. la). Segments from treated animals showed either a reduction in intensity of catecholaminefluorescence (n = 4) a n d / o r a noticeable loss of single a n d / o r small bundles of catecholaminelabelled fluorescent fibres (n = 5 ) with nerve bundles intact (Fig. lb). NA assay There was no significant difference in N A tissue content between control (0.550 _+ 0.113 n g / m m , n = 7) and treated (0.538 + 0.077 rig/ram, n = 6) rabbit ear artery segments examined. lmmunoassays Immunoassays were performed on rabbit ear artery segments from control and treated animals. T h e r e was no significant difference in the
Frequency-response curues EFS of the perivascular nerve fibres of the rabbit ear artery produced rapid, frequency-dependent (4-60 Hz) contractions which were blocked by tetrodotoxin (1 p.M). In treated animals, responses to EFS were markedly attenuated at all frequencies, but were more significantly reduced at the lower frequencies (Fig. 2). After cumulative administration and washing out of N A (0.1-300/.~M), the responses to EFS at all frequencies examined in segments from acryiamide-treated animals were not significantly different from those in segments from control animals, before or after cumulative administration of NA (Fig. 2). Responses to exogenous NA NA (0.1-300 p.M) added cumulatively produced c o n c e n t r a t i o n - d e p e n d e n t contractions which were maintained in both control and treated animals. There was no significant difference between the pD 2 values in control (5.97 ± 0.06, n = 10) versus treated (6.09 _+ 0.06, n = 10) animals. The contractions elicited by N A (10-300 /xM), however, were significantly attenuated in TABLE 1 Tissue content of CGRP and N P Y in rabbit central ear arteo' segments of acrylamide-treated and control animals CGRP (pmol/cm) Control segments Treated segments
NPY (pmol/cm)
0.014 _+0.004
0.268 _+0.086
(6)
(6)
0.014 4- 0.003
0.247 _+0.062
(6)
(6)
Results are expressed as mean + SEM. Numbers in parentheses indicate the number of animals.
59 Tension [rag) 2000
Effect of NPY on the responses to EFS In the presence of NPY (10 nM) the contractile responses to EFS (16 and 60 Hz) were enhanced. The maximal effect was observed after 4 - 6 min; the responses then returned to control levels. The potentiation mediated by NPY was not significantly different in control compared with treated animals (Table II).
- -
1500
Relaxant responses Well-maintained contractile responses were observed in both control and treated preparations after addition of a sub-maximal concentration of NA (0.3 /~M). This concentration was used because we had already established that there was
1000 - -
500 - -
~+ee
% KCI (120 m M )
200 - -
o 4
I
I
I
I
8
16
30
60
Frequency [ H z )
Fig. 2. Frequency-response curves for rabbit isolated ear artery preparations. Preparations were stimulated at supramaximal voltage, 0.3 ms pulse width and 1 s train duration. Curves represent saline-injected control (circles) and chronic acrylamide-treated (squares) animals before (open symbols) and after (closed symbols) incubation with NA. Statistical analysis was performed using Student's t-test for unpaired observations i.e. saline-injected control animals versus acrylamidetreated animals. P < 0.05 was considered significant, n > 8 for each group of observations. *, P < 0.05; * * , P < 0.01. Symbols represent the m e a n and vertical lines show the SEM.
150 ~
.
*
100
50'
the treated compared with control animals (Fig. 3). 0--
Responses to exogenous a,fl-mA TP Application of a sub-maximal concentration of o~,/3-mATP (1 izM) produced a rapid contraction that was not maintained. There was a marked increase in the response to exogenous a,fl-mATP (1 /xM) in treated (44.7 + 4.9% response to KCI (120 mM), n = 10) compared with control (17.3 + 3.8% response to KCI (120 mM), n = 9) animals (Fig. 4).
I
1
I
I
I
0.1
0.3
1.0
3.0
10.0
N A Concentration
I 30.0
I
I
100.0
300.0
(pM]
Fig. 3. Concentration-response curves for the action of exogenous N A (0.1-300 ~ M ) on rabbit isolated central ear artery preparations from saline-injected control (open circles) and chronic acrylamide-treated (filled circles) animals. Results are calculated for each observation as a percentage of the contraction obtained by KC1 (120 mM). Student's unpaired t-test was used to calculate statistical significance, n = 10 for each concentration, and P < 0.05 was considered significant. *, P < 0.05; * * , P < 0.01; * * * , P < 0.001. Symbols represent the m e a n and vertical lines show the SEM.
60
% KCI 0 2 0 mM)
% Relaxation
100 --
50--
75--
25-50--
25--
0
Control Acrylamide treated Fig. 4. Effect of c~,/3-methylene ATP (1 /zM) on the rabbit isolated central ear artery in saline-injected control and chronic acrylamide-treated animals. Results are calculated for each observation as a percentage of the contraction obtained by potassium chloride (KCI, 120 raM). Statistical significance was calculated using Student's t-test for unpaired observations, and P < 0.05 was considered significant. * * *, P < 0.001. n = 9 for control animals, and 10 for acrylamide-treated animals. Bars represent the mean and vertical lines show the SEM.
0--
I
I
I
T
~
I
I
7.0
6.5
6.0
5.5
5.0
4.5
4.0
- log A c e t y l c h o l i n e C o n c e n t r a t i o n
Fig. 5. Concentration-response curves for the endothelium-dependent relaxant effect of ACh on preconstriced (NA, 0.3 /zM) rabbit isolated central ear artery preparations. Curves represent saline-injected control (open circles) and chronic acrylamide-treated (filled circles) animals. Statistical analysis was performed using Student's t-test for unpaired observations, n >_ 6 for each group of observations. P < 0.05 was considered significant. *, P < 0.05; * * , P < 0.01; * * * , P < 0.001. Symbols represent the mean and vertical lines show the SEM.
T A B L E II
The effect of acrylamide treatment on the potentiating action of N P Y (10 riM) on the neurogenic, contractile responses elicited by electrical field stimulation (supramaximal ~'oltage, 0.3 ms pulse width and 1 s train duration) in the rabbit central ear artery Frequency
% Change
Significance
(Hz)
Control
Treated
16
43.7 ± 19.9 (8) 16.2± 6.6 (7)
41.1 ± 16.7 (10) 16.0± 6.6 (10)
60
NS NS
NS: not significant. Numbers in parentheses indicate number of animals investigated.
no significant difference in contractile responses elicited by NA at this concentration in either group (Fig. 3). The preconstricted preparations were used to examine relaxant responses.
(a) Endothelium-dependent relaxant responses. ACh: ACh (0.1-300 /zM) added cumulatively produced a concentration-dependent relaxation on preconstricted preparations. Although the pD 2 values were not significantly different between control (6.71 + 0.13, n = 6) and treated (6.63 4-_ 0.05, n = 9) animals, responses to ACh were significantly attenuated at every concentration tested, except 10/xM (Fig. 5).
61 % Relaxation 90--
Key
iII
Control ~ Acrylamide treated
60--
30-
0-CGRP [26.3 nM)
/I
SP [10.0 nM]
Fig. 6. Effect of CGRP (26.3 nM, endothelium-independent relaxation) and substance P (SP, 10.0 nM, endothelium-dependent relaxation) on preconstricted (NA, 0.3 /~M) rabbit isolated central ear artery preparations. Statistical analysis was performed using Student's t-test for unpaired observations, n = 8 for each group of observations. P < 0.05 was considered significant. *, P < 0.05. Bars represent the mean and vertical lines show the SEM.
Substance P: Relaxant responses elicited by SP (10 nM) added to preconstricted preparations were significantly reduced in treated (34.7 + 5.6% relaxation, n = 8) compared with control (57.1 + 8.0% relaxation, n = 8) animals (Fig. 6). (b) Endothelium-independent relaxant responses. CGRP: Relaxations induced by CGRP (26.3 nM) on preconstricted preparations were not significantly affected in the treated (80.0 + 5.6%, n = 8) compared with control (89.5 + 4.1%, n = 8) animals (Fig. 6).
Discussion There was no loss of NA or NPY content in the rabbit ear artery with acrylamide treatment, although a reduction in the number of single a n d / o r small-bundle catecholamine fluorescent perivascular nerve fibres was observed. This is indicative of sympathetic nerve damage, which is
reported to occur with acrylamide intoxication [12,22-24,30]. The reduced fluorescence in single a n d / o r small bundles of fibres in the ear artery was probably not enough to make a significant difference in the total tissue content of NA, since there remained many large intensely fluorescent bundles of perivascular nerve fibres in ear artery preparations from experimental animals. The tissue content of the CGRP-containing (sensory) perivascular nerves, and the modulatory action of NPY on the sympathetic co-transmission in the ear artery were also unaffected by acrylamide treatment. The recovery of EFS-induced contractile responses to control levels at all frequencies after exposure to exogenous NA in preparations from acrylamide-treated animals, may be due to the fact that there was a shortage of NA in the terminal varicosities that was reversed by NA incubation. This possibility is supported by the reduced fluorescence in the single and small-bundle catecholamine-containing perivascular fibres. In the early stages of sympathetic nerve degeneration, leakage of transmitter occurs [25]. This leakage leads to a gradual disappearance of transmitter, perhaps reflected in this study by the reduced catecholamine histofluorescence, in addition to a degree of subsensitivity to the transmitter, as observed in the attenuated responsiveness to exogenous NA in preparations from acrylamide-treated animals. In the later stages sympathetic nerve degeneration can lead to postjunctional receptor supersensitivity [25], as observed in the increased responsiveness to a,/3-mATP after acrylamide treatment. The purinergic component of sympathetic cotransmission in the rabbit ear artery is favoured at low frequencies of stimulation [13]. In the present study, the significantly greater reduction of the EFS-induced contractile responses at the lower frequencies after acrylamide treatment could be taken to suggest that the purinergic component of sympathetic cotransmission may be more severely impaired than the noradrenergic component. To determine whether acrylamide poisoning preferentially affects the purinergic component of sympathetic transmission requires additional experimentation, including measure-
62 ment of the contractile response to electrical field stimulation after P2x-purinoceptor desensitization, a n d / o r using a~-adrenoceptor antagonists. It is interesting to note that we have previously shown a similar pattern of changes in the rabbit ear artery after acute exposure to a single dose of x-radiation. As in acrylamide intoxication, there was a decrease in the nerve-mediated responses, accompanied by a marked increase in responsiveness to c~,/3-mATP, but not to N A [31]. Further experimentation is needed to clarify the mechanism of action involved in these pathological changes. The endothelium-dependent responses examined by the application of exogenous ACh and SP on preconstricted ear artery preparations were both significantly attenuated in acrylamidetreated animals, whereas the endothelium-independent relaxation mediated by C G R P [5,20] was unaffected. The efficiency of the vascular smooth muscle of the ear artery was not affected by acrylamide intoxication, since there was no change in either the contractile responses to KC1, or CGRP-induced vasodilatation from control and treated animals. Acrylamide treatment may have caused primary damage to the mechanism of endothelium-dependent vaso0ilatation by affecting ACh and SP receptors on the endothelial cells of the ear artery. Other possible explanations are that there may be an impairment of E D R F synthesis, release, a n d / o r its action on the smooth muscle [7,10,16]. Acrylamide may also be acting at the molecular level [15] to inhibit the increase of cGMP, but not cAMP, since the former is reported to be the second messenger in E D R F mediated relaxant responses, whilst the latter is thought to be involved in C G R P - m e d i a t e d relaxation [6]. An alternative possibility is that acrylamide may also be exerting its effect of attenuating endothelium-dependent relaxant responses as a secondary consequence of sympathetic nerve damage. There is evidence that substances present in nerves may act as trophic factors [3]. It was shown that after chronic denervation of the rabbit ear artery, methacholine (endothelium-dependent)-induced relaxation was significantly reduced, but sodium nitroprusside (endothelium-in-
dependent)-induced relaxation was unchanged [18]. It is possible, therefore, that sympathetic nerve damage, induced by acrylamide intoxication in this study, may have affected endotheliummediated responses via trophic interactions [16]. In conclusion, our results show that although acrylamide intoxication attenuates sympathetic cotransmission, perhaps with preferential action on the purinergic component, neuromodulation mediated by NPY is unaffected by this treatment. Endothelium-dependent relaxant responses induced by ACh and SP are also attenuated in the a c r y l a m i d e - t r e a t e d animals, in contrast to CGRP-induced, endothelium-independent relaxant responses which are normal. Thus it appears that acrylamide does not exclusively damage nerves as has been generally assumed, but also affects the endothelial component of local mechanisms controlling blood flow.
Acknowledgements K.M. was a recipient of an Overseas Research Student Award and Departmental Research Studentship. This work was partly supported by the British H e a r t Foundation. The authors would like to express their gratitude to Dr. C.H.V. Hoyle for discussion of various aspects of this work, and Mrs. Philippa Charatan for her editorial assistance.
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