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Diminished contractile responses to neuropeptide Y of arteries from diabetic rabbits
Journal of the Autonomic Nervous System, 37 (1992) 215-222
215
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00 JANS 01244
Diminished contractile responses to neuropeptide Y of arteries from diabetic rabbits David A n d e r s s o n 1,2, Jan Brunkwall
2,3, David
Bergqvist 2,3 and Lars Edvinsson 1,2
1Department of Internal Medicine, 2 Department of Experimental Research and 3 Department of Surgery, University of Lund, Sweden (Received 30 July 1991) (Revision received and accepted 11 November 1991)
Key words: A l l o x a n ; D i a b e t e s m e l l i t u s ; H i s t a m i n e ; N e u r o p e p t i d e Vasomotor
Y; N o r a d r e n a l i n e ;
Serotonin;
reactivity
Abstract The vascular smooth muscle contractile response to neuropeptide Y (NPY), potassium, noradrenaline, histamine and serotonin was studied in circular segments of isolated vessels in vitro from rabbits with alloxan-induced diabetes mellitus. The injection of alloxan resulted in a marked and maintained increase in serum glucose as early as 1 week after treatment. Four vessel types were examined: abdominal aorta, and renal, left anterior descending coronary and middle cerebral arteries. There was no difference in the contractile response to histamine or serotonin between control and diabetic vessels. However, in the cerebral artery the contractile response to noradrenaline was reduced in the diabetic group, while in the aorta and the renal artery no significant differences were seen. Noradrenaline failed to evoke any contractile response in the coronary arteries in either group. NPY induced strong, concentration-dependent contractions of coronary and cerebral arteries, but did not have any contractile effect per se in aorta or renal arteries, either in control or in alloxan-treated rabbits. The maximal contractile effect and the sensitivity to NPY was significantly less in diabetic coronary and cerebral vessels as compared to control. There was no difference in dilator effect of acetylcholine and substance P between the diabetic animals and the control group in any of the vessel types, indicating that the changed vascular responses to NPY and noradrenaline were not endothelium-dependent. In conclusion, the present study has shown that the postjunctional effects of NPY and noradrenaline in the peripheral sympathetic nervous system are selectively attenuated in this model of chronic diabetes.
Introduction Differences in vasomotor responses to vasoactive a g e n t s in d i a b e t i c a n i m a l s h a v e b e e n e x t e n sively s t u d i e d i n s e v e r a l r e p o r t s [1,2,4,10,14,16, 20,21,23,24,27]. N o r a d r e n a l i n e is o n e o f t h e m o s t e x a m i n e d s u b s t a n c e s a n d still a m a t t e r o f s o m e
Correspondence: D. Andersson, University of Lund, Department of Experimental Research, Malm6 General Hospital, S-214 01 Maim6, Sweden.
c o n t r o v e r s y s i n c e i n c r e a s e d [1,2,4,14,20,21], d e c r e a s e d [10,16,24,27] o r u n c h a n g e d [10,20,21,23, 27] r e s p o n s i v e n e s s h a s b e e n f o u n d . T h e r e a s o n for this might reside in differences in species, vascular region and mode of inducing diabetes m e l l i t u s as w e l l as t i m e a f t e r d i a b e t e s i n d u c t i o n . In addition to the classical transmitter noradrenaline, the sympathetic neuronsalso synthesize a n d r e l e a s e t h e b i o l o g i c a l l y a c t i v e p e p t i d e n e u r o p e p t i d e Y ( N P Y ) . T h e 36 a m i n o a c i d p e p tide NPY has been demonstrated in p o s t g a n glionic nerves supplying arteries and veins, coex-
216
isting with noradrenaline and being released together with noradrenaline upon sympathetic nerve activation [17,18]. NPY has four major effects: a contractile effect of its own, potentiation of noradrenaline-induced contraction, inhibition of noradrenaline release, and inhibition of, e.g., acetylcholine- and substance P-induced relaxation [6]. Local administration of exogenous NPY to the human forearm increases regional vascular resistance with a resulting reduction in local blood flow as a result of vasoconstriction [22]. Since the sympathetic nervous system is of importance in cardiovascular disease in diabetics, previous studies have focused on the effects of noradrenaline [1,2,4,10,14,16,20,21,23,24,27]. The aim of the present study was to examine the sympathetic nervous system co-transmitter NPY in chronic diabetes mellitus. This was tested in a rabbit model with alloxan-induced chronic diabetes. Five to 7 months after inducing the diabetic condition, the rabbits were sacrificed and the vasomotor responses of ring preparations of aorta, coronary, cerebral and renal arteries were studied.
Materials and Methods
Induction of diabetes mellitus and control animals Female, New Zealand White rabbits weighing 2 - 3 kg at the time of induction of diabetes were used. Control rabbits were weight-matched at the time of death. The diabetic rabbits were significantly older than the controls ( P < 0.05, Table I). All animals were allowed free access to water and standard laboratory diet. Rabbits were rendered diabetic by injection of alloxan (150 m g / k g ) into the lateral ear vein,
which resulted in a marked and maintained increase of serum glucose seen already after one week (Table I). In two rabbits this was only achieved after a second alloxan injection. In order to prevent hypoglycemia during the first 24 h after the injection of alloxan the rabbits were given 60-80 ml 5% glucose subcutaneously. The experiment was approved by the local ethical committee.
Preparation of tissues The rabbits were sacrificed with an overdose of mebumal (NordVacc, Sweden). The abdominal aorta, the renal artery, the middle cerebral artery and the left anterior coronary artery were dissected out under a microscope and immersed in a cold oxygenated buffer solution. The vessels were cut into cylindrical segments (2-3 m m long) which were immediately used in the experiments. Each cylindrical segment was mounted on two L-shaped metal prongs, one of which was connected to a force displacement transducer (FT03C) attached to a Grass polygraph for continuous recording of the isometric tension, and the other to a displacement device [11]. The position of the holder could be changed by means of a movable unit allowing fine adjustments of the vascular tension by varying the distance between the metal prongs. The mounted specimens were immersed in temperature controlled (37°C) tissue baths containing a buffer solution of the following composition (raM): NaCI 119, N a H C O 3 15, KCI 4.6, MgC12 1.2, N a H 2 P O 4 1.2, CaCI 2 1.5 and glucose 11. The solution was continuously gassed with 5% CO 2 in 0 2 giving a pH of 7.4. Special care was taken to ensure that the endothelial layer was not damaged. This was checked by monitoring dilatatory responses to
TABLE I
Serum glucose ~alues, boo6, weight, age and duration of diabetes in diabetic and control rabbits Values shown are the mean _+ SEM for 7 experiments. The diabetic rabbits were significantly older than the controls and had higher serum glucose levels (P < 0.05). Treatment
Serum glucose ( m m o l / l )
Weight (kg)
Age (Months)
Duration of diabetes
Diabetic Control
25.7 _+ 2.3 6.9 ÷ 0.9
4.4 + 0.15 4.6 +_ 0.18
9.8 _+ 0.3 7.0 + 0.9
171 ÷ 15 days
217 acetylcholine and substance P in arteries precontracted with histamine 10 -5 M. Acetylcholine induced dilator responses in all four vessel types, with a maximal effect of 3 0 - 6 0 % of precontraction. Substance P induced dilator responses with a maximal effect of 3 0 - 4 0 % of precontraction in all vessel types but the aorta ( < 10%). There was no difference in dilator effect of acetylcholine and substance P between the diabetic animals and the control group in any of the vessel types. A tension of 4 mN was applied to the arterial segments and they were allowed to stabilize at this level of tension for 1.5 h. The contractile capacity of each vessel segment was examined by exposure to a potassium-rich (60 mM) buffer solution which had the same composition as the standard solution, except that some of the NaC1 was exchanged for an equimolar concentration of KC1. When two reproducible contractions had been achieved the vessels were used for further studies (variation less than 10%).
Drugs Cocaine, serotonin, noradrenaline, acetylcholine, substance P, histamine, NPY (Sigma, U.S.A.) and propranolol (ICI, U.K.), were dissolved in 0.9% saline containing 1% BSA to avoid possible breakdown or absorption to walls of containers, further diluted in 0.9% saline and
A
used in the experiments immediately. All concentrations given are final molar concentration in the tissue bath during the experiments. Alloxan was purchased from Sigma, U.S.A. Results are given below as percentage of the potassium-induced contraction. Maximum amount of contraction = Ema x. The potency is expressed as pD 2 ( - l o g concentration of agonist inducing half maximum contraction). The data are expressed as mean values + SEM.
Statistics Differences between values were evaluated with a Mann-Whitney U-test on a Macintosh II SI using Statview 512 + graphics software.
Results
Potassium Potassium (60 mM) induced strong contractile responses in all four vessel types tested. There was no difference between diabetic animals and controls ( P > 0.20).
NPY NPY induced strong concentration-dependent contractions in coronary and cerebral arteries in
B
100
lOO
,- 80
c 80
O .
"6
,0
E 60 "E
~t _ 60
O o
+
v
cO
40
o 40 +
v
"6 o~ 20 0
"6 20 i
-10
=
-9
i
i
-8
-7
Log conc NPY (M)
, -6
0
i
-10
-9
-8 -7 Log cone N P Y (M)
-6
Fig. 1. A. The contractile response to NPY in diabetic coronary arteries (o) compared with weight-matched controls (e). B. The contractile response to NPY in diabetic cerebral arteries (o) compared with weight-matched controls (e). n = 5-7. Values represent the mean + SEM. * P < 0.05, * * P < 0.01.
218
A 100
e--
.o_
90 8O 70
~ 60 c
50
0
~
40
~
20 10 i
i
-9
!
i
i
i
-8 -7 -6 -5 Log conc Noradrenaline (M)
-4
Noradrenaline T h e n o r a d r e n a l i n e r e s p o n s e was t e s t e d in the p r e s e n c e of cocaine (10 v M) a n d p r o p r a n o l o l (10 -~' M) to block n e u r o n a l u p t a k e a n d the p o s t j u n c t i o n a l d i l a t o r effect of /3-adrenoceptors, respectively. In the c e r e b r a l a r t e r y the c o n t r a c t i l e r e s p o n s e to n o r a d r e n a l i n e was r e d u c e d in the d i a b e t i c g r o u p (Fig. 2A), while in a o r t a and the renal artery no significant d i f f e r e n c e was seen (Fig. 2B, C). T h e p D 2 values did not differ between diabetics and controls for any vessel type tested. N o r a d r e n a l i n e failed to evoke any contractile r e s p o n s e in the c o r o n a r y a r t e r i e s in e i t h e r group. In the a b s e n c e of cocaine a n d p r o p r a n o l o l , n o r a d r e n a l i n e had a d i l a t o r effect in the c o r o n a r y arteries.
B 120 100 c 0
80
o m
c:
60
8 40 20 0
A w
w i
i
-9
-8
F
i
-7 -6 -5 Log conc Noradrenaline (M)
i
i
-4
the c o n t r o l rabbits. T h e c o n t r a c t i l e effect to N P Y was significantly less in d i a b e t i c c o r o n a r y a n d c e r e b r a l vessels as c o m p a r e d to control (Fig. 1A, B), with a rightward shift o f the c o n c e n t r a t i o n - r e s p o n s e curves of N P Y in both c o r o n a r y and c e r e b r a l arteries. C o r o n a r y artery: p D 2 (cont r o d = 8.35 +_ 0.17, p D 2 ( d i a b e t e s ) = 7.16 _+ 0.16 ( P < 0 . 0 5 ) . C e r e b r a l arteries: p D 2 ( c o n t r o l ) = 8.94_+ 0.31, p D 2 ( d i a b e t e s ) = 7.88_+ 0.22 ( P < 0.05). N P Y did not have any c o n t r a c t i l e effect p e r se in a o r t a or renal arteries, n e i t h e r in control nor in a l l o x a n - t r e a t e d rabbits.
C 200
Serotonin S e r o t o n i n had a contractile effect in all four vessel types, t h o u g h w e a k in the c e r e b r a l arteries. T h e r e s p o n s e s did not differ significantly b e t w e e n the g r o u p s a l t h o u g h t h e r e was a t e n d e n c y of a t t e n u a t i o n in d i a b e t i c c o r o n a r y artery. T h e p D 2 values did not differ b e t w e e n d i a b e t i c s and controls for any vessel type tested.
0
~
100
+
0
,
-9
,
-8
,
,
I
-7 -6 -5 Log conc Noradrenaline (M)
i
-4
Fig. 2. A. The contractile response to noradrenaline in diabetic cerebral arteries (©) compared with weight-matched controls (e). B. The contractile response to noradrenaline in diabetic renal arteries (©) compared with weight-matched controls (e). C. The contractile response to noradrenaline in diabetic aorta (©) compared with weight-matched controls (e). n = 5-7. Values represent the mean_+SEM. * P < 0.05, • * P < 0.01.
Histamine H i s t a m i n e i n d u c e d a strong c o n c e n t r a t i o n - d e p e n d e n t c o n t r a c t i o n in all four vessel types (Fig. 3). T h e c o n c e n t r a t i o n - r e s p o n s e curves did not differ b e t w e e n d i a b e t i c s a n d controls n e i t h e r in terms of Era, x n o r in p D 2 values. A n initial dose of h i s t a m i n e (10 -5 M) was given to all vessel s e g m e n t s as a r e f e r e n c e . This did not differ be-
219
A
B
200
300
c
.9
._o 200 =,.
100
tO
v+ 100
"6
i
,
-9
,
.
-8
r
.
i
.
i
-7 -6 -s Log cone Histamine (M)
,
i
-;
-9
-7
-6
-5
-4
-3
Log eonc Histamine (M)
D
C
200 -
150 "
e-
e. o
-8
.o
100"
C
8
O O
100
+
v
~* 50"
"6
0
0
i
-9
.
i
-8
,
i
.
i
i
-7 -6 -5 Log cone Histamine (M)
0
i
-4
-3
A w
v i
-9
-8
,
-7 -6 -5 Log cone Histamine (M)
i
-4
-3
Fig. 3. A. The contractile response to histamine in diabetic coronary arteries (©) compared with weight-matched controls (e). B. The contractile response to histamine in diabetic cerebral arteries (©) compared with weight-matched controls (e). C. The contractile response to histamine in diabetic renal arteries (©) compared with weight-matched controls (e). D. The contractile response to histamine in diabetic aorta (<3) compared with weight-matched controls (e). n = 5-7.
tween diabetics and controls in any subgroup, e.g. the segments tested on NPY.
Discussion
Diabetes mellitus is often accompanied by cardiovascular complications [9,13], which are responsible for the higher morbidity and mortality of diabetic patients [3,8]. Autonomic dysfunction with sympathetic nerve failure causes loss of peripheral and splanchnic vasoconstriction and may be responsible for orthostatic hypotension and skin ulceration [5]. Previous studies on contractile responses to various vascular substances have given contradic-
tory results. This might in part be explained by the use of different references for the studied contraction. We have used an initial KC1 (60 mM)-induced contraction of each studied vessel segment as reference. Differences caused by structural changes, general conditions and initial tension are thereby eliminated. The contractile response to potassium is dependent mainly on depolarization of the smooth muscle cell membrane followed by the passage of extracellular calcium into the cell [12,23]. There was no difference between diabetic and control animals in our study, but the potassium response in diabetes has been reported to be enhanced [2,20], unchanged [10,20] and decreased [10,16,23,24] in previous studies. Our choice of reference is therefore not
220 appropriate to study general, non-selective changes in vasomotor reactivity, but it is well suited to show selective changes of the vascular responses in different transmitter-receptor systems. Diabetic rabbits failed to gain weight at the same rate as non-diabetics, as shown previously [10]. This may result in differences in size of the vessels [10]. To avoid such structural changes the controls were weight-matched and thus somewhat younger than the diabetics. This might influence the results but in a previous study age-matched and weight-matched animals did not show any difference [23]. The NPY-induced contractile response (Emax) of both coronary and cerebral arteries was markedly reduced in the diabetic rabbits. Also the sensitivity to NPY (pD 2) was lower in alloxan-induced diabetes. This has not been reported before. One probable explanation for the reduced response is the sympathetic neuropathy that is regularly observed in diabetes. Depletion of cutaneous nerves containing NPY-like immunoreactivity has been observed in patients with chronic diabetes while a decreased axonal transport of neuropeptides (substance P) has been shown in diabetic rats [15,25]. These alterations may cause diminished NPY release in the peripheral vasculature. It is possible that this could lead to denervation supersensitivity in an earlier stage of the diabetes, as has been demonstrated for noradrenaline after surgical sympathectomy [26], but not for NPY [6]. Later in chronic disease (as in our study) the loss of noradrenaline and NPY release may lead to a down-regulation of receptors. Since both the maximum contractile effect and the pD 2 values are markedly altered, we interpret the data as indicating that chronic diabetic disease cause both decreased receptor affinity and reduced numbers of postjunctional receptors for NPY. NPY had no effect on renal arteries or aorta in either group. This is in agreement with previous experience, that NPY is most potent as a constrictor in smaller vessels. Often there is no contractile effect at all on larger vessels where NPY seems to act preferably by potentiating the noradrenaline-induced contractions [7]. This type
of response was not studied in the present experiments. The changes in contractile response to noradrenaline in diabetes mellitus have varied considerably in previous studies, being either increased [1,2,4,14,20,21], decreased [10,16,24,27] or unchanged [10,20,21,23,27]. This might be due to several factors; consequently we focused our study on the postjunctional changes in the contractile c~-adrenoceptor response. Since decreased neuronal uptake has been shown to cause supersensitivity [4], cocaine was used to block the noradrenaline reuptake and propranolol to block the /3-adrenoceptor response. Although diabetes mellitus is believed to be a general disease with systemic effects, the maxim u m response to noradrenaline was attenuated in the diabetic cerebral arteries but unchanged in aorta and renal arteries. The reason for this is not clear, but the size of the vessel might be of importance; smaller vessels being more vulnerable. Other local variations such as the extent of sympathetic innervation and different a-adrenoceptor subpopulations might also influence the results, e.g., in coronary arteries noradrenaline had dilatory and no contractile effect in diabetic or control animals. In contrast, the contractile responses to histamine and serotonin were unchanged in all four vessel types although there was a non-significant tendency for an attenuation of the serotonin response in the coronary artery. These findings show that the effects of diabetes mellitus on the NPY and noradrenaline responses are selective and not part of a general depressant effect on the vascular contractility. There was no difference in dilator effect of acetylcholine and substance P between the diabetic animals and the control group in any of the vessel types. Since the dilator effect of both acetylcholine and substance P is dependent on an intact endothelium, this indicates that the changed vascular responses to NPY and noradrenaline were not endothelium-dependent. In conclusion, this model of long term (chronic) diabetic disease potently attenuates the NPY-, and in the cerebral artery the noradrenaline-induced contractile response. This is of great ira-
221
portance since NPY is a widespread co-transmitter of the sympathetic nervous system with the ability to potentiate noradrenaline and to control vascular tone by itself. Diabetic patients with postural hypotension are unable to keep up vascular tone. If the dysfunction of the N P Y / noradrenaline system described in this study for cerebral and coronary arteries also represents what happens in the control of regional blood flow in vivo, it may be an important factor in the pathogenesis of skin ulcers and postural hypotension associated with diabetes mellitus.
Acknowledgements The skilled technical assistance of Mr. Ulf Stjernqvist is gratefully acknowledged. Supported by the Medical Research council (Grant nos. 5958 and 00759), the Medical Faculty of Lund University and the Royal Physiographic Society, Lund, and the Swedish Foundation against Heart and Chest Diseases.
References 1 Abebe, W., Harris, K.H. and McLeod, K.M., Enhanced contractile responses of arteries from diabetic rats to alphal-adrenoceptor stimulation in the absence and presence of extracellular calcium, J. Cardiovasc. Pharmacol., 16 (1990) 239-248. 2 Agrawal, D.K., Bhimji, S. and McNeill, J.H., Effect of chronic experimental diabetes on vascular smooth muscle function in rabbit carotid artery, J. Cardiovasc. Pharmacol., 9 (1987) 584-593. 3 Christlieb, A.R., Diabetes and cardiovascular disease. Mechanisms and treatment, Am. J. Cardiol., 32 (1973) 595-606. 4 Cohen, R.A., Tesfamariam, B., Weisbrod, R.M. and Zitnay, K.M., Adrenergic denervation in rabbits with diabetes mellitus, Am. J. Physiol., 259 (1990) H55-H61. 5 Edmonds, M.E., Nicolaides, K.H. and Watkins, P.J., Autonomic neuropathy and foot ulceration, Diabetic Med., 3 (1986) 56-59. 6 Edvinsson, L., Copeland, J.R., Emson, P.C., McCulloch, J. and Uddman, R., Nerve fibers containing neuropeptide Y in the cerebrovascular bed: Immunocytochemistry, radioimmunoassay and vasomotor effects. (1987) J. Cereb. Blood Flow Metab., 7 (1987) 45-57. 7 Edvinsson, L., Hfikansson, R., Wahlestedt, C. and Ud-
dman, R., Effects of neuropeptide Y on the cardiovascular system, Trends Pharmacol. Sci., 8 (1987) 231-235. 8 Ellenberg, M., Diabetic autonomic neuropathy: role in vascular events, N.Y. St. Med., 78 (1978) 2214-2218. 9 Garcia, M.J., McNamara, P.M., Gordon, T. and Kannel, W.B., Morbidity and mortality in diabetics in the Framingham population, Diabetes, 23 (1974) 105-111. 10 Head, R.J., Longhurst, P.A., Panek, R.L. and Stitzel, R.E., A contrasting effect of the diabetic state upon the contractile responses of aortic preparations from the rat and rabbit, Br. J. Pharmacol., 91 (1987) 275-286. 11 H6gestedt, E.D., Andersson, K.-E. and Edvinsson, L., Mechanical properties of rat cerebral arteries as studied by a sensitive device for recording of mechanical activity in isolated small blood vessels, Acta Physiol. Scand., 117 (1983) 449-461. 12 H6gestedt, E.D. and Andersson, K-E., Mechanisms behind the biphasic contractile response to potassium depolarization in isolated rat cerebral arteries, J. Pharmacol. Exp. Ther., 228 (1984) 187-195. 13 Kannel, W.B. and McGee, D.L., Diabetes and cardiovascular disease: the Framingham study, J. Am. Med. Assoc., 241 (1979) 2035-2038. 14 Koltai, M.Z., R6sen, P., Ballagy-Pordany, G., Hadhazy, P. and Pogatsa, G., Increased vasoconstrictor response to noradrenaline in femoral vascular bed of diabetic dogs. Is tbromboxane A 2 involved?, Cardiovasc. Res., 24 (1990) 707-710. 15 Levy, D.M., Karanth, S.S., Springall, D.R. and Polak, J.M., Depletion of cutaneous nerves and neuropeptides in diabetes mellitus: an immunocytochemical study, Diabetologia, 32 (1989) 427-433. 16 Longhurst, P.A. and Head, R.J., Responses of isolated perfused mesenteric vasculature from diabetic rats: The significance of appropriate control tissues, J. Pharmacol. Exp. Ther., 235 (1985) 1, 45-49. 17 Lundberg, J.M., Terenius, L., H6kfelt, T., Martling, CR., Tatemoto, K., Mutt, V., Polak, JM., Bloom, S.R. and Goldstein, M., Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function, Acta Physiol. Scand., 116 (1982) 477-480. 18 Lundberg, J.M., Angg~rd, A., Theodorsson-Norheim, E. and Pernow, J., Guanethidine-sensitive release of neuropeptide Y-like immunoreactivity in the cat spleen by sympathetic nerve stimulation, Neurosci. Lett., 52 (1984) 175-180. 19 Lundberg, J.M., Torsell, L., Sollevi, A., Pernow, J., Theodorsson-Norheim, E., ,g,ngghrd, A. and Hamberger, B., Neuropeptide Y and sympathetic vascular control in man, Regul. Peptides, 13 (1985) 41-52. 20 McLeod, K.M. and McNeill, J.H., The influence of chronic diabetes on contractile responses of rat isolated blood vessels, Can. J. Physiol. Pharmacol., 63 (1984) 52-57. 21 Morff, R.J., Microvascular reactivity to norepinephrine at different arteriolar levels and durations of streptozocin-induced diabetes, Diabetes, 39 (1990) 354-360.
222 22 Pernow, J., Lundberg, J.M. and Kaijser, L., Vasoconstrictor effects in vivo and plasma disappearence of neuropeptide Y in man, Life Sci., 40 (1987) 47-54. 23 Sj6gren, A. and Edvinsson, L., Vasomotor changes in isolated coronary arteries from diabetic rats, Acta Physiol. Scand., 134 (1988) 429-436. 24 Sullivan, S. and Sparks, H.V., Diminished contractile response of aortas from diabetic rabbits, Am. J. Physiol., 236(2) (1979) H301-H306. 25 Tatemoto, K., Neuropeptide Y: complete amino acid sequence of the brain peptide, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 5485-5489.
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215
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00 JANS 01244
Diminished contractile responses to neuropeptide Y of arteries from diabetic rabbits David A n d e r s s o n 1,2, Jan Brunkwall
2,3, David
Bergqvist 2,3 and Lars Edvinsson 1,2
1Department of Internal Medicine, 2 Department of Experimental Research and 3 Department of Surgery, University of Lund, Sweden (Received 30 July 1991) (Revision received and accepted 11 November 1991)
Key words: A l l o x a n ; D i a b e t e s m e l l i t u s ; H i s t a m i n e ; N e u r o p e p t i d e Vasomotor
Y; N o r a d r e n a l i n e ;
Serotonin;
reactivity
Abstract The vascular smooth muscle contractile response to neuropeptide Y (NPY), potassium, noradrenaline, histamine and serotonin was studied in circular segments of isolated vessels in vitro from rabbits with alloxan-induced diabetes mellitus. The injection of alloxan resulted in a marked and maintained increase in serum glucose as early as 1 week after treatment. Four vessel types were examined: abdominal aorta, and renal, left anterior descending coronary and middle cerebral arteries. There was no difference in the contractile response to histamine or serotonin between control and diabetic vessels. However, in the cerebral artery the contractile response to noradrenaline was reduced in the diabetic group, while in the aorta and the renal artery no significant differences were seen. Noradrenaline failed to evoke any contractile response in the coronary arteries in either group. NPY induced strong, concentration-dependent contractions of coronary and cerebral arteries, but did not have any contractile effect per se in aorta or renal arteries, either in control or in alloxan-treated rabbits. The maximal contractile effect and the sensitivity to NPY was significantly less in diabetic coronary and cerebral vessels as compared to control. There was no difference in dilator effect of acetylcholine and substance P between the diabetic animals and the control group in any of the vessel types, indicating that the changed vascular responses to NPY and noradrenaline were not endothelium-dependent. In conclusion, the present study has shown that the postjunctional effects of NPY and noradrenaline in the peripheral sympathetic nervous system are selectively attenuated in this model of chronic diabetes.
Introduction Differences in vasomotor responses to vasoactive a g e n t s in d i a b e t i c a n i m a l s h a v e b e e n e x t e n sively s t u d i e d i n s e v e r a l r e p o r t s [1,2,4,10,14,16, 20,21,23,24,27]. N o r a d r e n a l i n e is o n e o f t h e m o s t e x a m i n e d s u b s t a n c e s a n d still a m a t t e r o f s o m e
Correspondence: D. Andersson, University of Lund, Department of Experimental Research, Malm6 General Hospital, S-214 01 Maim6, Sweden.
c o n t r o v e r s y s i n c e i n c r e a s e d [1,2,4,14,20,21], d e c r e a s e d [10,16,24,27] o r u n c h a n g e d [10,20,21,23, 27] r e s p o n s i v e n e s s h a s b e e n f o u n d . T h e r e a s o n for this might reside in differences in species, vascular region and mode of inducing diabetes m e l l i t u s as w e l l as t i m e a f t e r d i a b e t e s i n d u c t i o n . In addition to the classical transmitter noradrenaline, the sympathetic neuronsalso synthesize a n d r e l e a s e t h e b i o l o g i c a l l y a c t i v e p e p t i d e n e u r o p e p t i d e Y ( N P Y ) . T h e 36 a m i n o a c i d p e p tide NPY has been demonstrated in p o s t g a n glionic nerves supplying arteries and veins, coex-
216
isting with noradrenaline and being released together with noradrenaline upon sympathetic nerve activation [17,18]. NPY has four major effects: a contractile effect of its own, potentiation of noradrenaline-induced contraction, inhibition of noradrenaline release, and inhibition of, e.g., acetylcholine- and substance P-induced relaxation [6]. Local administration of exogenous NPY to the human forearm increases regional vascular resistance with a resulting reduction in local blood flow as a result of vasoconstriction [22]. Since the sympathetic nervous system is of importance in cardiovascular disease in diabetics, previous studies have focused on the effects of noradrenaline [1,2,4,10,14,16,20,21,23,24,27]. The aim of the present study was to examine the sympathetic nervous system co-transmitter NPY in chronic diabetes mellitus. This was tested in a rabbit model with alloxan-induced chronic diabetes. Five to 7 months after inducing the diabetic condition, the rabbits were sacrificed and the vasomotor responses of ring preparations of aorta, coronary, cerebral and renal arteries were studied.
Materials and Methods
Induction of diabetes mellitus and control animals Female, New Zealand White rabbits weighing 2 - 3 kg at the time of induction of diabetes were used. Control rabbits were weight-matched at the time of death. The diabetic rabbits were significantly older than the controls ( P < 0.05, Table I). All animals were allowed free access to water and standard laboratory diet. Rabbits were rendered diabetic by injection of alloxan (150 m g / k g ) into the lateral ear vein,
which resulted in a marked and maintained increase of serum glucose seen already after one week (Table I). In two rabbits this was only achieved after a second alloxan injection. In order to prevent hypoglycemia during the first 24 h after the injection of alloxan the rabbits were given 60-80 ml 5% glucose subcutaneously. The experiment was approved by the local ethical committee.
Preparation of tissues The rabbits were sacrificed with an overdose of mebumal (NordVacc, Sweden). The abdominal aorta, the renal artery, the middle cerebral artery and the left anterior coronary artery were dissected out under a microscope and immersed in a cold oxygenated buffer solution. The vessels were cut into cylindrical segments (2-3 m m long) which were immediately used in the experiments. Each cylindrical segment was mounted on two L-shaped metal prongs, one of which was connected to a force displacement transducer (FT03C) attached to a Grass polygraph for continuous recording of the isometric tension, and the other to a displacement device [11]. The position of the holder could be changed by means of a movable unit allowing fine adjustments of the vascular tension by varying the distance between the metal prongs. The mounted specimens were immersed in temperature controlled (37°C) tissue baths containing a buffer solution of the following composition (raM): NaCI 119, N a H C O 3 15, KCI 4.6, MgC12 1.2, N a H 2 P O 4 1.2, CaCI 2 1.5 and glucose 11. The solution was continuously gassed with 5% CO 2 in 0 2 giving a pH of 7.4. Special care was taken to ensure that the endothelial layer was not damaged. This was checked by monitoring dilatatory responses to
TABLE I
Serum glucose ~alues, boo6, weight, age and duration of diabetes in diabetic and control rabbits Values shown are the mean _+ SEM for 7 experiments. The diabetic rabbits were significantly older than the controls and had higher serum glucose levels (P < 0.05). Treatment
Serum glucose ( m m o l / l )
Weight (kg)
Age (Months)
Duration of diabetes
Diabetic Control
25.7 _+ 2.3 6.9 ÷ 0.9
4.4 + 0.15 4.6 +_ 0.18
9.8 _+ 0.3 7.0 + 0.9
171 ÷ 15 days
217 acetylcholine and substance P in arteries precontracted with histamine 10 -5 M. Acetylcholine induced dilator responses in all four vessel types, with a maximal effect of 3 0 - 6 0 % of precontraction. Substance P induced dilator responses with a maximal effect of 3 0 - 4 0 % of precontraction in all vessel types but the aorta ( < 10%). There was no difference in dilator effect of acetylcholine and substance P between the diabetic animals and the control group in any of the vessel types. A tension of 4 mN was applied to the arterial segments and they were allowed to stabilize at this level of tension for 1.5 h. The contractile capacity of each vessel segment was examined by exposure to a potassium-rich (60 mM) buffer solution which had the same composition as the standard solution, except that some of the NaC1 was exchanged for an equimolar concentration of KC1. When two reproducible contractions had been achieved the vessels were used for further studies (variation less than 10%).
Drugs Cocaine, serotonin, noradrenaline, acetylcholine, substance P, histamine, NPY (Sigma, U.S.A.) and propranolol (ICI, U.K.), were dissolved in 0.9% saline containing 1% BSA to avoid possible breakdown or absorption to walls of containers, further diluted in 0.9% saline and
A
used in the experiments immediately. All concentrations given are final molar concentration in the tissue bath during the experiments. Alloxan was purchased from Sigma, U.S.A. Results are given below as percentage of the potassium-induced contraction. Maximum amount of contraction = Ema x. The potency is expressed as pD 2 ( - l o g concentration of agonist inducing half maximum contraction). The data are expressed as mean values + SEM.
Statistics Differences between values were evaluated with a Mann-Whitney U-test on a Macintosh II SI using Statview 512 + graphics software.
Results
Potassium Potassium (60 mM) induced strong contractile responses in all four vessel types tested. There was no difference between diabetic animals and controls ( P > 0.20).
NPY NPY induced strong concentration-dependent contractions in coronary and cerebral arteries in
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, -6
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Fig. 1. A. The contractile response to NPY in diabetic coronary arteries (o) compared with weight-matched controls (e). B. The contractile response to NPY in diabetic cerebral arteries (o) compared with weight-matched controls (e). n = 5-7. Values represent the mean + SEM. * P < 0.05, * * P < 0.01.
218
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Noradrenaline T h e n o r a d r e n a l i n e r e s p o n s e was t e s t e d in the p r e s e n c e of cocaine (10 v M) a n d p r o p r a n o l o l (10 -~' M) to block n e u r o n a l u p t a k e a n d the p o s t j u n c t i o n a l d i l a t o r effect of /3-adrenoceptors, respectively. In the c e r e b r a l a r t e r y the c o n t r a c t i l e r e s p o n s e to n o r a d r e n a l i n e was r e d u c e d in the d i a b e t i c g r o u p (Fig. 2A), while in a o r t a and the renal artery no significant d i f f e r e n c e was seen (Fig. 2B, C). T h e p D 2 values did not differ between diabetics and controls for any vessel type tested. N o r a d r e n a l i n e failed to evoke any contractile r e s p o n s e in the c o r o n a r y a r t e r i e s in e i t h e r group. In the a b s e n c e of cocaine a n d p r o p r a n o l o l , n o r a d r e n a l i n e had a d i l a t o r effect in the c o r o n a r y arteries.
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the c o n t r o l rabbits. T h e c o n t r a c t i l e effect to N P Y was significantly less in d i a b e t i c c o r o n a r y a n d c e r e b r a l vessels as c o m p a r e d to control (Fig. 1A, B), with a rightward shift o f the c o n c e n t r a t i o n - r e s p o n s e curves of N P Y in both c o r o n a r y and c e r e b r a l arteries. C o r o n a r y artery: p D 2 (cont r o d = 8.35 +_ 0.17, p D 2 ( d i a b e t e s ) = 7.16 _+ 0.16 ( P < 0 . 0 5 ) . C e r e b r a l arteries: p D 2 ( c o n t r o l ) = 8.94_+ 0.31, p D 2 ( d i a b e t e s ) = 7.88_+ 0.22 ( P < 0.05). N P Y did not have any c o n t r a c t i l e effect p e r se in a o r t a or renal arteries, n e i t h e r in control nor in a l l o x a n - t r e a t e d rabbits.
C 200
Serotonin S e r o t o n i n had a contractile effect in all four vessel types, t h o u g h w e a k in the c e r e b r a l arteries. T h e r e s p o n s e s did not differ significantly b e t w e e n the g r o u p s a l t h o u g h t h e r e was a t e n d e n c y of a t t e n u a t i o n in d i a b e t i c c o r o n a r y artery. T h e p D 2 values did not differ b e t w e e n d i a b e t i c s and controls for any vessel type tested.
0
~
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+
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,
-9
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-7 -6 -5 Log conc Noradrenaline (M)
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Fig. 2. A. The contractile response to noradrenaline in diabetic cerebral arteries (©) compared with weight-matched controls (e). B. The contractile response to noradrenaline in diabetic renal arteries (©) compared with weight-matched controls (e). C. The contractile response to noradrenaline in diabetic aorta (©) compared with weight-matched controls (e). n = 5-7. Values represent the mean_+SEM. * P < 0.05, • * P < 0.01.
Histamine H i s t a m i n e i n d u c e d a strong c o n c e n t r a t i o n - d e p e n d e n t c o n t r a c t i o n in all four vessel types (Fig. 3). T h e c o n c e n t r a t i o n - r e s p o n s e curves did not differ b e t w e e n d i a b e t i c s a n d controls n e i t h e r in terms of Era, x n o r in p D 2 values. A n initial dose of h i s t a m i n e (10 -5 M) was given to all vessel s e g m e n t s as a r e f e r e n c e . This did not differ be-
219
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B
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Fig. 3. A. The contractile response to histamine in diabetic coronary arteries (©) compared with weight-matched controls (e). B. The contractile response to histamine in diabetic cerebral arteries (©) compared with weight-matched controls (e). C. The contractile response to histamine in diabetic renal arteries (©) compared with weight-matched controls (e). D. The contractile response to histamine in diabetic aorta (<3) compared with weight-matched controls (e). n = 5-7.
tween diabetics and controls in any subgroup, e.g. the segments tested on NPY.
Discussion
Diabetes mellitus is often accompanied by cardiovascular complications [9,13], which are responsible for the higher morbidity and mortality of diabetic patients [3,8]. Autonomic dysfunction with sympathetic nerve failure causes loss of peripheral and splanchnic vasoconstriction and may be responsible for orthostatic hypotension and skin ulceration [5]. Previous studies on contractile responses to various vascular substances have given contradic-
tory results. This might in part be explained by the use of different references for the studied contraction. We have used an initial KC1 (60 mM)-induced contraction of each studied vessel segment as reference. Differences caused by structural changes, general conditions and initial tension are thereby eliminated. The contractile response to potassium is dependent mainly on depolarization of the smooth muscle cell membrane followed by the passage of extracellular calcium into the cell [12,23]. There was no difference between diabetic and control animals in our study, but the potassium response in diabetes has been reported to be enhanced [2,20], unchanged [10,20] and decreased [10,16,23,24] in previous studies. Our choice of reference is therefore not
220 appropriate to study general, non-selective changes in vasomotor reactivity, but it is well suited to show selective changes of the vascular responses in different transmitter-receptor systems. Diabetic rabbits failed to gain weight at the same rate as non-diabetics, as shown previously [10]. This may result in differences in size of the vessels [10]. To avoid such structural changes the controls were weight-matched and thus somewhat younger than the diabetics. This might influence the results but in a previous study age-matched and weight-matched animals did not show any difference [23]. The NPY-induced contractile response (Emax) of both coronary and cerebral arteries was markedly reduced in the diabetic rabbits. Also the sensitivity to NPY (pD 2) was lower in alloxan-induced diabetes. This has not been reported before. One probable explanation for the reduced response is the sympathetic neuropathy that is regularly observed in diabetes. Depletion of cutaneous nerves containing NPY-like immunoreactivity has been observed in patients with chronic diabetes while a decreased axonal transport of neuropeptides (substance P) has been shown in diabetic rats [15,25]. These alterations may cause diminished NPY release in the peripheral vasculature. It is possible that this could lead to denervation supersensitivity in an earlier stage of the diabetes, as has been demonstrated for noradrenaline after surgical sympathectomy [26], but not for NPY [6]. Later in chronic disease (as in our study) the loss of noradrenaline and NPY release may lead to a down-regulation of receptors. Since both the maximum contractile effect and the pD 2 values are markedly altered, we interpret the data as indicating that chronic diabetic disease cause both decreased receptor affinity and reduced numbers of postjunctional receptors for NPY. NPY had no effect on renal arteries or aorta in either group. This is in agreement with previous experience, that NPY is most potent as a constrictor in smaller vessels. Often there is no contractile effect at all on larger vessels where NPY seems to act preferably by potentiating the noradrenaline-induced contractions [7]. This type
of response was not studied in the present experiments. The changes in contractile response to noradrenaline in diabetes mellitus have varied considerably in previous studies, being either increased [1,2,4,14,20,21], decreased [10,16,24,27] or unchanged [10,20,21,23,27]. This might be due to several factors; consequently we focused our study on the postjunctional changes in the contractile c~-adrenoceptor response. Since decreased neuronal uptake has been shown to cause supersensitivity [4], cocaine was used to block the noradrenaline reuptake and propranolol to block the /3-adrenoceptor response. Although diabetes mellitus is believed to be a general disease with systemic effects, the maxim u m response to noradrenaline was attenuated in the diabetic cerebral arteries but unchanged in aorta and renal arteries. The reason for this is not clear, but the size of the vessel might be of importance; smaller vessels being more vulnerable. Other local variations such as the extent of sympathetic innervation and different a-adrenoceptor subpopulations might also influence the results, e.g., in coronary arteries noradrenaline had dilatory and no contractile effect in diabetic or control animals. In contrast, the contractile responses to histamine and serotonin were unchanged in all four vessel types although there was a non-significant tendency for an attenuation of the serotonin response in the coronary artery. These findings show that the effects of diabetes mellitus on the NPY and noradrenaline responses are selective and not part of a general depressant effect on the vascular contractility. There was no difference in dilator effect of acetylcholine and substance P between the diabetic animals and the control group in any of the vessel types. Since the dilator effect of both acetylcholine and substance P is dependent on an intact endothelium, this indicates that the changed vascular responses to NPY and noradrenaline were not endothelium-dependent. In conclusion, this model of long term (chronic) diabetic disease potently attenuates the NPY-, and in the cerebral artery the noradrenaline-induced contractile response. This is of great ira-
221
portance since NPY is a widespread co-transmitter of the sympathetic nervous system with the ability to potentiate noradrenaline and to control vascular tone by itself. Diabetic patients with postural hypotension are unable to keep up vascular tone. If the dysfunction of the N P Y / noradrenaline system described in this study for cerebral and coronary arteries also represents what happens in the control of regional blood flow in vivo, it may be an important factor in the pathogenesis of skin ulcers and postural hypotension associated with diabetes mellitus.
Acknowledgements The skilled technical assistance of Mr. Ulf Stjernqvist is gratefully acknowledged. Supported by the Medical Research council (Grant nos. 5958 and 00759), the Medical Faculty of Lund University and the Royal Physiographic Society, Lund, and the Swedish Foundation against Heart and Chest Diseases.
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