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Influence of the endothelium on contractile responses of arteries from diabetic rats
European Journal of Pharmacology, 153 (1988) 55-64
55
Elsevier EJP 50387
Influence of the endothelium on contractile responses of arteries from diabetic rats 1 K i m H. H a r r i s a n d K a t h l e e n M. M a c L e o d 2,. Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver B. C., Canada Received 26 April 1988, accepted 17 M a y 1988
Contractile responses of aortas and mesenteric arteries from control and 3 month streptozotocin-diabetic rats to a-adrenoceptor agonists were compared in the presence and absence of endothelium. In the presence of endothelium, responses of both arteries from diabetic animals to norepinephrine and methoxamine were enhanced compared to control, although no response to clonidine could be detected in arteries from either control or diabetic animals. Following endothelium removal, no significant differences were found between control and diabetic arteries in maximum contractile responses to noradrenaline or methoxamine. However, the sensitivity (pD 2) of diabetic aortas to these two agonists was significantly increased, while maximum responses of diabetic aortas and mesenteric arteries to clonidine were much greater than control. In addition, no differences between control and diabetic aortas were detected when cGMP levels were measured in the absence and presence of acetylcholine. These results suggest that enhanced responsiveness of arteries from diabetic animals to c~-adrenoceptor stimulation is not the result of a decrease in endothelium-derived relaxing factor (EDRF) release in diabetic blood vessels. Vascular reactivity; Diabetes; Endothelium; a-Adrenoceptor agonists
1. Introduction It has been suggested that some of the cardiovascular disturbances which are known to occur in patients with diabetes mellitus are a consequence of alterations in the reactivity of blood vessels to neurotransmitters and circulating h o r m o n e s (Christlieb et al., 1976; Weidmann et al., 1979). This hypothesis has been extensively investigated in blood vessels from animals with chemically induced diabetes, but there has been little agreement between different studies on the effects of
i This work was supported by a grant from the Medical Research Council of Canada. 2 Canadian Heart Foundation Scholar. * To whom all correspondence should be addressed: Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver B.C. Canada V6T 1W5.
diabetes on vascular reactivity. The results of some studies have suggested that the influence of diabetes on vascular reactivity varies with the duration of the diabetic state (MacLeod and McNeill, 1985), the vascular preparation studied (MacLeod and McNeill, 1985; Ramanadham et al., 1985) and the sex of the animals used (MacLeod, 1985). Other contributing factors may be the nature of the diabetogenic agent (alloxan or streptozotocin), as well as its dose and route of administration. Recently a report from this laboratory (MacLeod, 1985) demonstrated that contractile responses of aortas and mesenteric arteries from male rats with streptozotocin-induced diabetes of 3 months duration to norepinephrine were enhanced. Although the threshold dose of norepinephrine was unchanged, responses of both arteries from diabetic animals to concentrations of norepinephrine greater than 3 × 10 -8 M were sig-
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
56 nificantly greater than control. The increased responsiveness of the diabetic blood vessels to norepinephrine could be prevented from occurring, orreversed once established, by treatment of diabetic animals with insulin (MacLeod, 1985), suggesting that it was a consequence of the diabetic state. However, the enhanced responsiveness did not appear to result from a generalized increase in the contractility of the preparations, since responses of K+-depolarized preparations from diabetic animals to increased extracellular Ca 2+ were not altered compared to control. Recently, it has become apparent that contractile responses of rat arteries to a-adrenoceptor agonists are reduced in the presence of an intact endothelium (Egleme et al., 1984; Lues and Schtimann, 1984; Murakami et al., 1985; Martin et al., 1986; MacLeod et al., 1987). Initially it was suggested that stimulation of a2-adrenoceptors located on the endothelium results in increased release of endothelium-derived relaxing factor (EDRF), which elevates c G M P levels, and inhibits contractile responses to stimulation of a-receptors located on vascular smooth muscle (Egleme et al., 1984; Miller et al., 1984). However, more recent studies have suggested that it is the spontaneous release of E D R F from the endothelium, and associated tonic elevation of c G M P levels, which results in inhibition of responses to a-adrenoceptor agonists and to serotonin (Martin et al., 1986; MacLeod et al., 1987), although E D R F is less effective at inhibiting contractile responses to K + depolarization (Godfraind, 1986). Both structural (Arbogast et al., 1984) and functional (reviewed in Colwell et al., 1979) deterioration of the vascular endothelium has been reported to occur in both clinical and experimental diabetes. Therefore, it seemed possible that the increased responsiveness of aortas and mesenteric arteries from diabetic rats to norepinephrine might be the result of a decrease in the spontaneous release of E D R F , resulting from damage to the endothelial cells. This was investigated in the present study by comparing contractile responses of blood vessels from diabetic rats and their agematched controls to c~-adrenoceptor stimulation in the presence and absence of endothelium. To further investigate the functional status of the endo-
thelium in diabetic arteries, the influence of the tonic and stimulated release of E D R F on c G M P levels was compared in aortas from control and diabetic rats.
2. Materials and methods
Male Wistar rats, weighing 190-215 g were used in the present investigation. Animals were lightly anesthetized with ether, and either streptozotocin (60 m g / k g ) or citrate buffer vehicle ( p H 4.5) were administered by injection into the lateral tail vein. Streptozotocin- and vehicle-treated animals were housed separately and given free access to food and water. Streptozotocin-treated animals were monitored periodically for the development of glycosuria using Lilly Testape. Three months after injection, animals were weighed and then killed by stunning followed by decapitation. Blood was collected for insulin and glucose assay, and the aorta and superior mesenteric artery were removed and placed in Krebs solution of composition (mM): NaC1 113, KC1 4.7, CaC12 2.5, K H 2 P O 4 1.2, MgSO 4 1.2, NaHCO3 25 and dextrose 11.5. Vessels were carefully cleaned of fat and connective tissue, and then cut into rings. Two 3 m m rings of mesenteric artery, and two 5 m m rings of aorta from each animal were used. One ring of each pair was left intact, while the other ring was gently rotated on a stainless steel rod in order to remove the endothelium. Each ring was then suspended between the bases of two triangular shaped wires. One wire was attached to a fixed tissue support in a 20 ml isolated tissue bath containing Krebs solution, maintained at 3 7 ° C and oxygenated with 95% 02 - 5 % CO 2. The other wire was attached by a silk thread to a Grass FT.03 force displacement transducer connected to a Beckman Model 611 dynograph. Rings of mesenteric artery were placed under a resting tension of 1.0 g, and rings of aorta under a resting tension of 2.0 g. These had been determined in preliminary experiments to be the optimal resting tensions for both control and diabetic arteries. Then tissues were allowed to equilibrate for 90-120 rain before experiments were begun. All experiments were done in the presence
57 of 1 /~M timolol, 0.1 /~M desipramine, and 1 /~M hydrocortisone, to eliminate the effects of fladrenoceptors, and neuronal and extraneuronal uptake respectively. 2.1. Contractility studies Cumulative dose-response curves to clonidine, followed by methoxamine and then norepinephfine were obtained in each preparation. This is in order of increasing efficacy of these agonists. In other experiments we have found that responses to methoxamine and noradrenaline are not affected by prior exposure of the blood vessels to less efficacious a-adrenoceptor agonists. Tissues were washed for 60 min between each dose-response curve. Following completion of the norepinephrine dose-response curve, tissues were washed for a further 60 min, pre-contracted with an EDT0 dose of norepinephrine (5 X 10 7 M), and then a cumulative dose-response curve to acetylcholine was obtained in each preparation. At the completion of each experiment tissues were lightly blotted, measured and weighed. The cross-sectional area of each preparation was calculated using the following formula: cross-sectional area (mm 2) = weight (mg)/length ( m m ) x density ( m g / m m 3). The density of the arteries was assumed to be 1.05 m g / m m 3. Responses of each preparation to clonidine, methoxamine and norepinephrine were then calculated as the increase in tension (g) in response to each concentration of agonist/cross-sectional area of tissue (mm 2). Responses to acetylcholine were calculated as the percent tension remaining in the presence of each concentration of acetylcholine, pD 2 ( - log EDs0) values were also calculated for each agonist. However, responses to clonidine in the presence of endothelium, and to acetylcholine in its absence, were too small for reliable calculation of pD 2 values. 2.2. Measurement of cGMP levels Rings of aorta, with and without endothelium, were set up and equilibrated in isolated tissue baths as described above. At various times after the addition of the agonists, tissues were frozen
with clamps cooled in liquid N2, and then were stored at - 8 0 ° C until assayed for cGMP. The frozen aortas were then homogenized in 1 ml of 6% trichloroacetic acid, and centrifuged at 6000 x g for 40 min. The supernatants were extracted four times in five volumes of water-saturated ether. Following acetylation, c G M P levels were determined using radioimmunoassay kits obtained from New England Nuclear. 2.3. Drugs and assays used Clonidine hydrochloride, ( - ) - n o r e p i n e p h r i n e hydrochloride and acetylcholine chloride were all obtained from Sigma while methoxamine hydrochloride was obtained from Burroughs Wellcome. Stock solutions of clonidine, methoxamine and norepinephrine were prepared fresh daily in distilled water containing 1 m g / m l ascorbic acid. Serum glucose levels were measured using an enzymatic colorimetric assay (Peridochrom ® glucose, Boehringer Mannheim). Serum insulin was measured using a radioimmunoassay kit (Amersham). 2.4. Statistics Results were compared for significant differences using one-way analysis of variance (ANOVA). When more than two groups of data were compared, results were further analyzed using the Newman-Keuls test. Results were considered to be significantly different if P < 0.05.
3. Results
Rats treated with streptozotocin gained weight at a much slower rate than non-diabetic rats, so that at the time of killing, streptozotocin-treated animals weighed significantly less than their agematched controls (table 1). Diabetic animals also exhibited significantly elevated serum glucose levels, and decreased serum insulin levels relative to controls (table 1). Aortas and mesenteric arteries from diabetic animals were significantly smaller in cross-sectional area than the corresponding arteries from control animals (table 1). Therefore, all con-
58 TABLE 1 Characteristics of rats 3 months after induction of diabetes with streptozotocin, compared to age-matched control rats.
Control Streptozotocin
12 12
Body weights
Serum glucose
Serum insulin
(g)
(mg/dl)
(kt U / ml )
501 _+21 308 _+18 ~
113.8 + 4.5 377.4 _+2.9 "
57.3 _+5.7 12.7 +_0.1 "
Cross-sectional areas (mm 2 ) Aortas
Mesenteric arteries
1.02 _+0.02 0.77 _+0.01 ~'
0.37 _+0.02 0.29 ± 0.01 "
a p < 0.05 compared to corresponding control value.
tractile responses were corrected for the cross-sectional areas of the tissues. 3.1. Aorta
As previously reported (MacLeod, 1985), aortas with intact endothelia from streptozotocin-treated rats were more responsive to the contractile effects of increasing concentrations of norepinephrine than were aortas with intact endothelia from agematched control animals (fig. 1). In these experiments, the m a x i m u m response to norepinephrine was significantly increased, from 2.30 _+ 0.09 g / r a m 2 (mean-t-S.E., n = 6) in control aortas to 2.90 + 0 . 1 4 g / r a m 2 ( n = 6) in diabetic aortas. In addition, the sensitivity (pD 2 value) of diabetic aortas to norepinephrine was significantly increased compared to control (table 2). Removal of the endothelium also resulted in a significant in-
crease in the m a x i m u m response (to 2.76 _+ 0.12 g / m m 2, n = 6) and sensitivity (table 2) of control aortas to norepinephrine. However, removal of the endothelium from diabetic aortas resulted only in a leftward shift in the norepinephrine dose-response curve (fig. 1), with a significant increase in the norepinephrine p D 2 value (table 2). The maxim u m response of diabetic aortas to norepinephrine was 2.82 + 0.24 g / m m 2 (n = 6) in the absence of endothelium, which was not significantly different from the value in the presence of endothelium. The magnitude of the increase in p D 2 value produced by removing the endothelium was very similar in control and diabetic aortas, and in the absence of endothelium the norepinephrine pD: was significantly greater in diabetic than in control aortas (table 2). The results obtained with methoxamine were similar to those obtained with norepinephrine. TA B LE 2
TENSION (g/mm 2)
Agonist pD 2 values in aortas from control and diabetic a n i m a l s in the presence and absence of endothelium. ND, not determined.
30
2.0
I 075
1.0 1.0-
9
8
7
6
S
NOREPINEPHRINE I-log M]
7
6
5
4
METHOXAMINE I - l o g M]
8
7
6
5
CLONIDINE [_-log M]
Fig. 1. Dose-response curves of aortas from rats with streptozotocin-induced diabetes of 3 months duration (n II) and agematched controls ( o O) to norepinephrine, methoxamine and clonidine, in the presence (© rn) and absence (O II) of endothelium. Each curve represents the mean of data obtained from six aortas. Only a single representative S.E. is shown in the midpoint of each curve, for the sake of the clarity.
Norepinephrine Control Streptozotocin Methoxamine Control Streptozotocin Clonidine Clonidine Streptozotocin Acetylcholine Control Streptozotocin
+Endothelium
n
-Endothelium
6.79_+0.11 a 7.10+0.07 "
6 6
7.45_+0.10 a 7.85_+0.08 a
4.63_+0.09 ~ 4.98+0.11 ~
6 6
5.35_+0.06 ~ 5.71 +0.12 ~
ND ND 7.07+0.14 6.63___0.19
n
6.75 _+0.08 ~ 7.18_+0.04 " 6 6
ND ND
" Significantly different from all other values for that agonist (P < 0.05, ANOVA, followed Newman-Keuls test).
59
although in the preparations used in this investigation, methoxamine behaved as a partial agonist relative to norepinephrine (fig. 1). The m a x i m u m response of control aortas with intact endothelia to methoxamine was 0.95 + 0.08 g / m m 2, which was not significantly different from the m a x i m u m response of diabetic aortas with intact endothelia to this agonist (1.27 +_ 0.18 g / m m 2 ) . However, the methoxamine p D 2 value was significantly increased in diabetic aortas under these conditions (table 2). Removal of the endothelium from control aortas resulted in a marked upward and leftward shift in the methoxamine dose-response curve (fig. 1), significantly increasing the m a x i m u m response to this agonist, to 1.75 -+ 0.10 g / m m 2, and significantly increasing the methoxamine p D 2 value (table 2). Removal of the endothelium from diabetic aortas resulted in an increase in the maxim u m response to methoxamine to 1.61_+0.16 g / m m 2, a value which was not significantly different from the m a x i m u m methoxamine response in the presence of endothelium. However, the methoxamine p D 2 value was significantly greater in the absence than in the presence of endothelium in diabetic aortas (table 2). As with norepinephrine, endothelium removal produced approximately the same degree of shift in the methoxamine dose-response curve in both control and diabetic aortas. In the absence of endothelium, the methoxamine p D 2 value was significantly greater in diabetic than in control aortas, although m a x i m u m responses of the two preparations to methoxamine were not significantly different (fig. 1, table 2). Almost no response to clonidine was detected in either control or diabetic aortas with intact endothelia (fig. 1). In the presence of endothelium, the m a x i m u m clonidine response was 0.04 +_ 0.02 g / m m 2 in control aortas, and 0.10 _+ 0.06 g / m m 2 in diabetic aortas. Removal of the endothelium resulted in an enhancement of responses to clonidine, which was more marked in diabetic than in control aortas (fig. 1). The m a x i m u m response of diabetic aortas to clonidine was increased to 1.05 _+ 0.17 g / m m 2 following endothelium removal; this value was significantly greater than the maxim u m response of 0.68 4- 0.09 g / m m 2 obtained in control aortas following removal of the endo-
TENSION (g/mm 2) 2.0
_* *
1.0
0
r
T
9
8
,
i
7
,
i
i
6
5
ACETYLCHOLINE
I-log M] Fig. 2. Responses of aortas from rats with streptozotocin-induced diabetes of 3 months duration (e) and age-matched controls ( o ) with intact endothelia to increasing concentrations of acetylcholine. Aortas were pre-contracted with 5 x 10 7 M norepinephrine which produced an increase in tension of 1.20 _+0.11 g / m m 2 in control aortas (n = 6), and 1.77 _+0.19 g/rfllTl 2 in diabetic aortas (n = 5). * P < 0.05 compared to corresponding control response.
thelium. In addition, the clonidine p D 2 value was significantly greater in diabetic than in control aortas in the absence of endothelium (table 2). The ability of acetylcholine to relax arteries pre-contacted with a sub-maximal dose of norepinephrine was also determined. Acetylcholine produced dose-dependent relaxation of both control and diabetic aortas with intact endothelia (fig. 2). This effect was abolished in both vessels by removal of the endothelium (data not shown). Although the degree of pre-contraction in response to norepinephrine was greater in diabetic than in control aortas, the m a x i m u m relaxation produced by acetylcholine in diabetic aortas was not significantly different from the m a x i m u m relaxation it produced in control aortas, nor was the acetylcholine p D 2 value different between the two preparations (table 2). In order to further establish the functional status of the endothelium in diabetic aortas, c G M P levels were measured in control and diabetic aortas in the absence and presence of endothelium, in the absence of drug, and following exposure to a m a x i m u m concentration of acetylcholine. There was no significant difference in basal c G M P levels between control and diabetic aortas, in either the
60 300 -
200-
75f£
® 100-
u
:~÷S{ CNTL +ENDO
CNTL -
ENDO
Ach + ENDO
Ach -
ENDO
Fig. 3. Cyclic GMP levels in aortas from rats with streptozotocin-induced diabetes of 3 months duration (shaded bars) and age-matched controls (open bars). All tissues were pretreated with 5 )<10-7 M norepinephrine, which has previously been shown to have no significant effect on cGMP levels in the absence or presence of endothelium in aortas (MacLeod et al., 1987). CNTL+Endo: tissues with intact endothelia, frozen after no further treatment. CNTL-Endo: aortas with endothelium removed, frozen after no further treatment. Ach+ Endo: aortas with intact endothelia, frozen 2 min after the addition of 1/~M acetylcholine. Ach-Endo: tissues with endothelium removed, frozen 2 min after the addition of 1 /~M acetylcholine. Each bar represents the mean+S.E, of data obtained from three to four tissues. * P < 0.05 compared to all other groups. absence or the presence of e n d o t h e l i u m (fig. 3). R e m o v a l of the e n d o t h e l i u m of vessels from b o t h groups of a n i m a l s resulted in a r e d u c t i o n of c G M P levels to a p p r o x i m a t e l y 50% of the levels f o u n d i n the presence of e n d o t h e l i u m , as previously reported ( M a c L e o d et al., 1987), a l t h o u g h with the statistics used i n the present investigation, these differences were n o t statistically significant. Acetylcholine p r o d u c e d a significant elevation of c G M P levels, the m a g n i t u d e of which was n o t different i n control a n d diabetic aortas. This effect of acetylcholine was abolished b y removal of the e n d o t h e l i u m (fig. 3).
3.2. Mesenteric artery Mesenteric arteries with intact e n d o t h e l i a from diabetic rats exhibited greater increases i n t e n s i o n in response to n o r e p i n e p h r i n e t h a n did their agem a t c h e d controls (fig. 4). The m a x i m u m increase
in t e n s i o n p r o d u c e d by n o r e p i n e p h r i n e in diabetic arteries was 3.77 _+ 0.44 g / m m 2, which was signific a n t l y greater t h a n the c o r r e s p o n d i n g value ol 2.53 _+0.22 g / m m 2 f o u n d in control arteries. However, the n o r e p i n e p h r i n e p D 2 values in control a n d diabetic mesenteric arteries did n o t differ significantly (table 3). Removal of the endothelium from control arteries resulted in an increase in the m a x i m u m response to n o r e p i n e p h rine, to 3.06 _+ 0.27 g / m m 2, (n = 6) a n d in a sign i f i c a n t increase in the n o r e p i n e p h r i n e p D 2 value (table 3). E n d o t h e l i u m removal had no effect on the m a x i m u m response of diabetic mesenteric arteries to n o r e p i n e p h r i n e , which was 3.71 _+ 0.38 g / m m 2 (n = 6) in the absence of e n d o t h e l i u m , but did p r o d u c e a significant increase in sensitivity to this agonist (table 3). I n the absence of endothelium, n o significant differences were f o u n d between control a n d diabetic arteries in response to norepinephrine. Responses of mesenteric arteries with intact e n d o t h e l i a from diabetic a n i m a l s to m e t h o x a m i n e were also increased c o m p a r e d to control (fig. 4). T h e m a x i m u m response of control arteries to m e t h o x a m i n e was 1.27 + 0.16 g / m m 2 (n = 6), while that of diabetic arteries was 1.93 ± 0.30 g / m m 2 (n = 6). However, m e t h o x a m i n e p D 2 values did n o t differ significantly between control a n d diabetic arteries (table 3). Removal of the e n d o t h e l i u m from control mesenteric arteries
TENSION (g/ram?)
i£
1° I 0.5j
9
8
7
6
5
NOREPINEPHRINE l-log M]
4
•
6
5
4
METHOXAMINE l-log M]
3
8
7
6
5
4
CLONIDINE l-log M]
Fig. 4. Dose-response curves of mesenteric arteries from rats with streptozotocin-induced diabetes of 3 months duration ([]11) and age-matched controls (©e) to norepinephrine, methoxamine and clonidine, in the presence (o D) and absence (e II) of endothelium. Each curve represents the mean of data obtained from six or seven preparations, with a representative S.E. shown around the midpoint of each curve.
61
resulted in a leftward shift in the methoxamine dose-response curve, with a significant increase in the methoxamine p D 2 value (table 3), and a small, but not significant increase in the m a x i m u m response to this agonist, to 1.69 _+ 0.21 g / m m 2 (n = 6). In diabetic arteries, removal of the endothelium also resulted in a leftward shift in the methoxamine dose-response curve (table 3), while having no effect on the m a x i m u m response to this agonist, which was 2.06 _+ 0.20 g / m m 2 (n = 6) in the absence of endothelium. As with norepinephrine, in the absence of endothelium no significant differences could be detected between control and diabetic arteries in response to methoxamine. In the presence of intact endothelia, responses of mesenteric arteries from both control and diabetic animals to clonidine were exceedingly small (fig. 4). The m a x i m u m response of control arteries to clonidine was 0.06 _+ 0.03 g / m m 2 (n = 6), while that of diabetic arteries was 0.05 _+ 0.02 g / r a m z (n = 6). Removal of the endothelium resulted in marked enhancement of responses of vessels from both groups of animals to this agonist, although to a greater extent in diabetic than in control arteries. The m a x i m u m response of control arteries to clonidine increased to 0.35 + 0.10 g / m m 2 (n = 6), and that of diabetic arteries increased to 0.76 _+ 0.16 g / r a m 2 ( n = 7) following removal of the endothelium. These values were significantly differTABLE 3 Agonist pD 2 values in mesenteric arteries from control and diabetic rats, in the presence and absence of endothelium. ND, not determined.
Norepinephrine Control Streptozotocin Methoxamine Control Streptozotocin Clonidine Control Streptozotocin Acetylcholine Control Streptozotocin
+ Endothelium
n
- Endothelium
n
6.59_+0.14 6.71+0.08
6 6
7.03+0.13 a 7.105:0.10 "
6 7
4.83-+0.07 5.05+0.04
6 6
5.32_+0.15 a 5.50+0.06 a
6 7
6.68 + 0.17 6.81_+0.14
4 7
TENSION (g/mm 2) 3.o- ~
~
± 2.0-
i
i-
T
1.0
0
, 9
,
, 8
,
i 7
.
i
'
6
i S
ACETYLCHOLINE
E-log M] Fig. 5. Responses of mesenteric arteries from rats with streptozotocin-induced diabetes of 3 months duration (e) and age-matched controls ( o ) with intact endothelia to increasing concentrations of acetylcholine. Mesenteric arteries were precontracted with 5 × 1 0 -7 M norepinephrine, which produced an increase in tension of 1.75_+0.19 g / r a m 2 in control mesenteric arteries (n = 6) and 2,75_+0.33 g / r a m 2 in diabetic mesenteric arteries (n = 6). * P < 0.05 compared to corresponding control response.
ent. However, no difference was detected between control and diabetic arteries in clonidine p D 2 values in the absence of endothelium (table 3). The ability of acetylcholine to relax mesenteric arteries pre-contracted with 5 × 10 -v M norepinephrine in the absence and presence of endothelium was also determined. Acetylcholine produced dose-dependent relaxation of arteries from both control and diabetic animals in the presence of endothelium (fig. 5). This effect of acetylcholine was lost following endothelium removal (data not shown). Despite the greater contractile response of diabetic arteries to norepinephrine, neither the m a x i m u m relaxation produced by acetylcholine, nor the acetylcholine pD2 value (table 3), were different between control and diabetic arteries.
4. Discussion ND ND 7.14_+0.25 6.95-+0.13
6 6
ND ND
a p < 0.05 compared to corresponding p D 2 value in the presence of endothelium.
The results of the present investigation confirm a previous report from this laboratory (MacLeod, 1985) that contractile responses of aortas and mesenteric arteries with intact endothelia from rats with streptozocin-induced diabetes of 3
62 months duration to norpinephrine are enhanced compared to responses of age-matched control arteries. In this study, we also found an increase in the sensitivity (pD 2 value) of diabetic aortas to norepinephrine. However, the magnitude of the increase in sensitivity, although significant, was very small, which may explain why it was not detected in the previous investigation. Responses to methoxamine, an agonist selective for C~l-adrenoceptors, which has been previously reported to act as a partial agonist relative to noradrenaline in rat aorta and mesenteric artery (Lues and Schtimann, 1985; MacLeod et al., 1987) were also enhanced in diabetic arteries with intact endothelia, compared to their age-matched controls. However, responses of aortas and mesenteric arteries with intact endothelia from both diabetic and control animals to clonidine were very small and not significantly different from each other. Removal of the endothelium from aortas and mesenteric arteries of control animals resulted in enhancement of responses to each of the c~-adrenoceptor agonists tested. Responses were shifted upward, and, at least in the case of norepinephrine and methoxamine, to the left of responses to these agonists in the presence of endothelium. The degree of enhancement of maximum responses appeared to be inversely related to the efficacy of the agonists tested, in that responses to clonidine were enhanced the most, and those to norepinephrine the least, by removal of the endothelium. These results are similar to those reported previously in aorta and mesenteric artery (Egleme et al., 1984; Lues and Schtimann, 1984; MacLeod et al., 1987). Recent studies have attributed the inhibition of c~-adrenoceptor-mediated responses in the presence of an intact endothelium to the spontaneous release of E D R F from the endothelium (Martin et al., 1986; MacLeod et al., 1987). The spontaneously released E D R F appears to result in tonic, low elevation of c G M P levels in the vascular smooth muscle, which exert an inhibitory effect against contractions elicited by stimulation of c~adrenoceptors and serotonin receptors (Martin et al., 1986; MacLeod et al., 1987). Evidence also suggests that the spontaneously released E D R F has a greater depressant effect against agonists of low efficacy, such as clonidine, than those of
higher efficacy, such as norepinephrine (Martin et al., 1986; MacLeod et al., 1987). The effects of endothelium removal on responses of diabetic aortas and mesenteric arteries to norepinephrine and methoxamine were less marked than in the corresponding blood vessels from control animals. However, the opposite was true of responses to clonidine. In diabetic aortas and mesenteric arteries, removal of the endothelium caused no further increase in maximum responses to norepinephrine, although the magnitude of the leftward shift in the norepinephrine dose-response curve was similar to that obtained in control preparations. The lack of further effect on m a x i m u m norepinephrine responses may have occurred because the tissues themselves were not able to respond with further increases in tension. However, endothelium removal resulted in loss of the differences in response to norepinephrine found between control and diabetic mesenteric arteries, although diabetic aortas still had a significantly elevated pD 2 value relative to control. Similar results were also obtained with methoxamine on removal of the endothelium from the diabetic arteries. These data are less easily explained, since in both diabetic and control arteries, methoxamine acted as a partial agonist relative to norepinephrine, even in the absence of endothelium. Thus, the inability of methoxamine to produce a further increase in tension in diabetic arteries following removal of the endothelium cannot be explained by an inability of the tissues to contract further. Since there were no significant differences between responses of diabetic and control mesenteric arteries to methoxamine in the absence of endothelium, and only a slight increase in the methoxamine p D 2 value in diabetic aortas compared to control under these conditions, it might be argued that these results support the hypothesis that ~-adrenoceptor-mediated responses are enhanced in diabetic blood vessels due to decreased release of E D R F . However, other evidence obtained in this investigation does not agree with this interpretation of the data. Firstly, clonidine responses were almost undetectable in diabetic aortas with intact endothelia; this would not be expected if there was decreased release of E D R F . Secondly, in the absence of
63 endothelium, clonidine responses were much greater in diabetic aortas and mesenteric arteries than in the corresponding control tissues. This suggests that some other change is responsible for the enhanced c~-adrenoceptor responsiveness in diabetes. Measurements of c G M P levels, in both the absence and presence of acetylcholine, also suggest that both the tonic and stimulated release of E D R F is similar in control and diabetic vessels. Basal c G M P levels were similar in control and diabetic aortas with intact endothelia, and were reduced to the same extent by endothelium removal. This reduction in c G M P levels on removal of the endothelium has previously been reported (Miller et al., 1984; Griffith et al., 1985; Martin et al., 1985; MacLeod et al., 1987) and has been taken as evidence in support of the tonic release of E D R F . Therefore, by this measure the spontaneous release of E D R F appears to be similar in control and diabetic aortas. In addition, the increase in c G M P levels produced by a m a x i m u m concentration of acetylcholine was very similar in control and diabetic aortas, indicating that the ability of the endothelium to respond to this agonist is not altered in diabetes. The relaxation of aortas and mesenteric arteries from diabetic and control animals in response to acetylcholine was also not significantly different, although in general the arteries from diabetic animals appeared to be slightly less sensitive to acetylcholine. This is probably related to the fact that relaxant responses to acetylcholine were measured in the presen.ce of a concentration of norepinephrine which produced a greater increase in tension in diabetic arteries than in controls. Responses to vasorelaxants are determined at least in part by the contractile tension attained prior to their addition (Furchgott, 1983). N o other more suitable contractile agonist was available for use in these experiments, as preliminary experiments indicated that contractile responses to serotonin were also enhanced in diabetic arteries, while responses to K + depolarization, which are not enhanced in diabetes (MacLeod, 1985), are resistant to relaxation by acetylcholine (Furchgott, 1983; unpublished observations). However, we found that when the concentration of norepinephrine was increased in control mesenteric arteries to 10 5
M, which produced the same increase in tension as that found in diabetic mesenteric arteries in response to 5 >< 10 .7 M norepinephrine, acetylcholine actually produced a greater maximum relaxation of diabetic arteries (78.9 + 6.7%, means + S.E., n = 8) than of control arteries (53.1 + 7.6%, n = 8). During the preparation of this manuscript, the results of two other less extensive investigations of endothelium function in aortas from chronic s t r e p t o z o t o c i n - d i a b e t i c rats were published (Oyama et al., 1986; Wakabayashi et al., 1987). In agreement with the results of the present investigation, one group reported that the endotheliumdependent relaxation of aortas in response to acetylcholine was unaffected by the diabetic state (Wakabayashi et al., 1987). However, this group also found that responses to clonidine were enhanced to a much greater extent in control than in diabetic aortas by removal of the endothelium, suggesting that the tonic release of E D R F from diabetic aortas may have been reduced in their experiments. In contrast, O y a m a et al. (1986) reported that the endothelium-dependent relaxation of diabetic aortas in response to both acetylcholine and histamine was attenuated compared to control. Although the reason for the discrepancy between our results and those of the other two groups is not clear, one possibility is that there was a difference in the intensity of the diabetic state between studies. Blood glucose values were much greater, and body weight gains were much less, in diabetic rats used in the other two investigations (Oyama et al., 1986; Wakabayashi et al., 1987) than those used in our study. Overall, the results of this investigation do not support a role for decreased release of E D R F in mediating enhanced responses of diabetic arteries to c~-adrenoceptor stimulation. Therefore, the mechanism for the enhanced reactivity to a-adrenoceptor agonist in diabetes is likely to involve some change at the level of the vascular smooth muscle. This could be either in the ch-adrenoce ptors themselves, or in the coupling of these receptors to the contractile response. Other reports have suggested that there is increased mobilization of extracellular Ca 2+ mediated by ch-adrenoceptors in aortas from streptozotocin-diabetic rats (Scar-
64
borough and Carrier, 1983; 1984). However, in the present investigation, responses to both the putative al-adrenoceptor agonist methoxamine and the a2-adrenoceptor agonist clonidine could be shown to be enhanced in arteries from diabetic animals, under different conditions. Therefore, these data do not support a role for either a-adrenoceptor subtype in selectively mediating the increased responsiveness of arteries from diabetic rats to c~-adrenoceptor agonists. In summary, these results confirm that a-adrenoceptor-mediated responses are enhanced in aortas and mesenteric arteries from rats with chronic STZ-induced diabetes. The enhanced responsiveness to a-receptor stimulation does not appear to result from a decrease in release of E D R F from the endothelium of these vessels. However, if the endothelium is damaged on removal of these arteries, some changes in responsiveness resulting from diabetes may be masked. It is possible that this has contributed to some of the controversy surrounding the influence of diabetes on vascular reactivity, and suggests that in future studies of this type, the status of the endothelium should be established.
References Arbogast, B.W., D.L. Berry and C.L. NeweU, 1984, Injury of arterial endothelial cells in diabetic, sucrose-fed and aged rats, Atherosclerosis 51, 31. Christlieb, A.R., H.U. Janka, B. Kraus, R.E. Gleason, E.A. lcasas-Cabral, L.M. Aiello, B.V. Cabral and A. Solano, 1976, Vascular reactivity to angiotensin I1 and to norepinephrine in diabetic subjects, Diabetes 25, 268. Colwell, J.A., P.V. Halushka, K.E. Sarji, M.F. Lopes-Virella and J. Segal, 1979, Vascular disease in diabetes, Arch. Int. Med. 139, 225. Egleme, C., T. Godfraind and R.C. Miller, 1984, Enhanced responsiveness of rat isolated aorta to clonidine after removal of the endothelial cells, Br. J. Pharmacol. 81,016. Furchgott, R.F., 1983, Role of endothelium in responses of vascular smooth muscle, Circ. Res. 53, 557. Godfraind, T., 1985, E D R F and cyclic G M P control gating of receptor-operated calcium channels in vascular smooth muscle, European J. Pharmacol. 126, 341. Griffith, T . M . D . H . Edwards, M.J. Lewis and A.H. Henderson,
1985, Evidence that cyclic guanosine monophosphate ( c G M P ) mediates e n d o t h e l i u m - d e p e n d e n t relaxation, European J. Pharmacol. 112, 195. Lues, I. and J.J. Schiimann, 1984, Effect of removing the endothelial cells on the reactivity of rat aortic segments to different alpha-adrenoceptor agonists, Naunyn-Schmiedeb. Arch. Pharmacol. 328, 160. MacLeod, K.M., 1985, The effect of insulin treatment on changes in vascular reactivity in chronic, experimental diabetes, Diabetes 34, 1160. MacLeod, K.M. and J.H. McNeill, 1985, The influence of chronic experimental diabetes on contractile responses of rat isolated blood vessels, Can. J. Physiol. Pharmacol. 63, 52. MacLeod, K.M., D.D.W. Ng, K.H. Harris and J. Diamond, 1987, Evidence that c G M P is the m e d i a t o r of endothelium-dependent inhibition of contractile responses of rat arteries to a-adrenoceptor stimulation, Mol. Pharmacol. 32, 59. Martin, W., R.F. Furchgott, G.M. Villani and D. Jothianandan, 1986, Depression of contractile responses in rat aorta by spontaneously released endothelium-derived relaxing factor, J. Pharmacol. Exp. Ther. 237, 529. Miller, R.C., M. Mony, V. Shini, P. Schoeffter and J.C. Stoclet. 1984, Endothelial mediated inhibition of contraction and increase in cyclic G M P levels evoked by the alpha-adrenoceptor agonist, B-HT 920 in rat isolated aorta, Br. J. Pharmacol. 83, 903. Murakami, K., H. Karaki and N. Urakawa, 1985, Role of endothelium in the contractions induced by norepinephrine and clonidine in rat aorta, Jap. J. Pharmacol. 39, 357. Oyama, Y., H. Kawasaki, Y. Hattori and M. Kanno, 1986, Attenuation of endothelium-dependent relaxation in aorta from diabetic rats, European J. Pharmacol. 131, 75. R a m a n a d h a m , S., W.H. Lyness and T.E. Tenner, 1984, Alterations in aorta and tail artery reactivity to agonists after streptozotocin treatment, Can. J. Physiol. Pharmacol. 62, 418. Scarborough, N.L. and G.O. Carrier, 1983, Increased alpha-2adrenoreceptor mediated vascular contraction in diabetic rats, J. Auton. Pharmacol. 3, 177. Scarborough, N.L. and G.O. Carrier, 1984, Nifedipine and alpha adrenoceptors in rat aorta. II Role of extracellular calcium in enhanced alpha-2-adrenoceptor-mediated contraction in diabetes, J. Pharmacol. Exp. Ther. 23, 603. Wakabayashi, I., K. Hatake, N. Kimura, E. Kakishita and K. Nagai, 1987, Modulation of the vascular tonus by the endothelium in experimental diabetes, Life Sci. 40, 643. Weidmann, P., C.B. Piccoli, G. Keusch, M. Mutagic, M. Grimm, A. Meier and W.H. Zeigler, 1979, Sodium-volume factor, cardiovascular reactivity and hypotensive mechanism of diuretic therapy in mild hypertension associated with diabetes mellitus, Am. J. Med. 67, 779.
55
Elsevier EJP 50387
Influence of the endothelium on contractile responses of arteries from diabetic rats 1 K i m H. H a r r i s a n d K a t h l e e n M. M a c L e o d 2,. Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver B. C., Canada Received 26 April 1988, accepted 17 M a y 1988
Contractile responses of aortas and mesenteric arteries from control and 3 month streptozotocin-diabetic rats to a-adrenoceptor agonists were compared in the presence and absence of endothelium. In the presence of endothelium, responses of both arteries from diabetic animals to norepinephrine and methoxamine were enhanced compared to control, although no response to clonidine could be detected in arteries from either control or diabetic animals. Following endothelium removal, no significant differences were found between control and diabetic arteries in maximum contractile responses to noradrenaline or methoxamine. However, the sensitivity (pD 2) of diabetic aortas to these two agonists was significantly increased, while maximum responses of diabetic aortas and mesenteric arteries to clonidine were much greater than control. In addition, no differences between control and diabetic aortas were detected when cGMP levels were measured in the absence and presence of acetylcholine. These results suggest that enhanced responsiveness of arteries from diabetic animals to c~-adrenoceptor stimulation is not the result of a decrease in endothelium-derived relaxing factor (EDRF) release in diabetic blood vessels. Vascular reactivity; Diabetes; Endothelium; a-Adrenoceptor agonists
1. Introduction It has been suggested that some of the cardiovascular disturbances which are known to occur in patients with diabetes mellitus are a consequence of alterations in the reactivity of blood vessels to neurotransmitters and circulating h o r m o n e s (Christlieb et al., 1976; Weidmann et al., 1979). This hypothesis has been extensively investigated in blood vessels from animals with chemically induced diabetes, but there has been little agreement between different studies on the effects of
i This work was supported by a grant from the Medical Research Council of Canada. 2 Canadian Heart Foundation Scholar. * To whom all correspondence should be addressed: Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver B.C. Canada V6T 1W5.
diabetes on vascular reactivity. The results of some studies have suggested that the influence of diabetes on vascular reactivity varies with the duration of the diabetic state (MacLeod and McNeill, 1985), the vascular preparation studied (MacLeod and McNeill, 1985; Ramanadham et al., 1985) and the sex of the animals used (MacLeod, 1985). Other contributing factors may be the nature of the diabetogenic agent (alloxan or streptozotocin), as well as its dose and route of administration. Recently a report from this laboratory (MacLeod, 1985) demonstrated that contractile responses of aortas and mesenteric arteries from male rats with streptozotocin-induced diabetes of 3 months duration to norepinephrine were enhanced. Although the threshold dose of norepinephrine was unchanged, responses of both arteries from diabetic animals to concentrations of norepinephrine greater than 3 × 10 -8 M were sig-
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
56 nificantly greater than control. The increased responsiveness of the diabetic blood vessels to norepinephrine could be prevented from occurring, orreversed once established, by treatment of diabetic animals with insulin (MacLeod, 1985), suggesting that it was a consequence of the diabetic state. However, the enhanced responsiveness did not appear to result from a generalized increase in the contractility of the preparations, since responses of K+-depolarized preparations from diabetic animals to increased extracellular Ca 2+ were not altered compared to control. Recently, it has become apparent that contractile responses of rat arteries to a-adrenoceptor agonists are reduced in the presence of an intact endothelium (Egleme et al., 1984; Lues and Schtimann, 1984; Murakami et al., 1985; Martin et al., 1986; MacLeod et al., 1987). Initially it was suggested that stimulation of a2-adrenoceptors located on the endothelium results in increased release of endothelium-derived relaxing factor (EDRF), which elevates c G M P levels, and inhibits contractile responses to stimulation of a-receptors located on vascular smooth muscle (Egleme et al., 1984; Miller et al., 1984). However, more recent studies have suggested that it is the spontaneous release of E D R F from the endothelium, and associated tonic elevation of c G M P levels, which results in inhibition of responses to a-adrenoceptor agonists and to serotonin (Martin et al., 1986; MacLeod et al., 1987), although E D R F is less effective at inhibiting contractile responses to K + depolarization (Godfraind, 1986). Both structural (Arbogast et al., 1984) and functional (reviewed in Colwell et al., 1979) deterioration of the vascular endothelium has been reported to occur in both clinical and experimental diabetes. Therefore, it seemed possible that the increased responsiveness of aortas and mesenteric arteries from diabetic rats to norepinephrine might be the result of a decrease in the spontaneous release of E D R F , resulting from damage to the endothelial cells. This was investigated in the present study by comparing contractile responses of blood vessels from diabetic rats and their agematched controls to c~-adrenoceptor stimulation in the presence and absence of endothelium. To further investigate the functional status of the endo-
thelium in diabetic arteries, the influence of the tonic and stimulated release of E D R F on c G M P levels was compared in aortas from control and diabetic rats.
2. Materials and methods
Male Wistar rats, weighing 190-215 g were used in the present investigation. Animals were lightly anesthetized with ether, and either streptozotocin (60 m g / k g ) or citrate buffer vehicle ( p H 4.5) were administered by injection into the lateral tail vein. Streptozotocin- and vehicle-treated animals were housed separately and given free access to food and water. Streptozotocin-treated animals were monitored periodically for the development of glycosuria using Lilly Testape. Three months after injection, animals were weighed and then killed by stunning followed by decapitation. Blood was collected for insulin and glucose assay, and the aorta and superior mesenteric artery were removed and placed in Krebs solution of composition (mM): NaC1 113, KC1 4.7, CaC12 2.5, K H 2 P O 4 1.2, MgSO 4 1.2, NaHCO3 25 and dextrose 11.5. Vessels were carefully cleaned of fat and connective tissue, and then cut into rings. Two 3 m m rings of mesenteric artery, and two 5 m m rings of aorta from each animal were used. One ring of each pair was left intact, while the other ring was gently rotated on a stainless steel rod in order to remove the endothelium. Each ring was then suspended between the bases of two triangular shaped wires. One wire was attached to a fixed tissue support in a 20 ml isolated tissue bath containing Krebs solution, maintained at 3 7 ° C and oxygenated with 95% 02 - 5 % CO 2. The other wire was attached by a silk thread to a Grass FT.03 force displacement transducer connected to a Beckman Model 611 dynograph. Rings of mesenteric artery were placed under a resting tension of 1.0 g, and rings of aorta under a resting tension of 2.0 g. These had been determined in preliminary experiments to be the optimal resting tensions for both control and diabetic arteries. Then tissues were allowed to equilibrate for 90-120 rain before experiments were begun. All experiments were done in the presence
57 of 1 /~M timolol, 0.1 /~M desipramine, and 1 /~M hydrocortisone, to eliminate the effects of fladrenoceptors, and neuronal and extraneuronal uptake respectively. 2.1. Contractility studies Cumulative dose-response curves to clonidine, followed by methoxamine and then norepinephfine were obtained in each preparation. This is in order of increasing efficacy of these agonists. In other experiments we have found that responses to methoxamine and noradrenaline are not affected by prior exposure of the blood vessels to less efficacious a-adrenoceptor agonists. Tissues were washed for 60 min between each dose-response curve. Following completion of the norepinephrine dose-response curve, tissues were washed for a further 60 min, pre-contracted with an EDT0 dose of norepinephrine (5 X 10 7 M), and then a cumulative dose-response curve to acetylcholine was obtained in each preparation. At the completion of each experiment tissues were lightly blotted, measured and weighed. The cross-sectional area of each preparation was calculated using the following formula: cross-sectional area (mm 2) = weight (mg)/length ( m m ) x density ( m g / m m 3). The density of the arteries was assumed to be 1.05 m g / m m 3. Responses of each preparation to clonidine, methoxamine and norepinephrine were then calculated as the increase in tension (g) in response to each concentration of agonist/cross-sectional area of tissue (mm 2). Responses to acetylcholine were calculated as the percent tension remaining in the presence of each concentration of acetylcholine, pD 2 ( - log EDs0) values were also calculated for each agonist. However, responses to clonidine in the presence of endothelium, and to acetylcholine in its absence, were too small for reliable calculation of pD 2 values. 2.2. Measurement of cGMP levels Rings of aorta, with and without endothelium, were set up and equilibrated in isolated tissue baths as described above. At various times after the addition of the agonists, tissues were frozen
with clamps cooled in liquid N2, and then were stored at - 8 0 ° C until assayed for cGMP. The frozen aortas were then homogenized in 1 ml of 6% trichloroacetic acid, and centrifuged at 6000 x g for 40 min. The supernatants were extracted four times in five volumes of water-saturated ether. Following acetylation, c G M P levels were determined using radioimmunoassay kits obtained from New England Nuclear. 2.3. Drugs and assays used Clonidine hydrochloride, ( - ) - n o r e p i n e p h r i n e hydrochloride and acetylcholine chloride were all obtained from Sigma while methoxamine hydrochloride was obtained from Burroughs Wellcome. Stock solutions of clonidine, methoxamine and norepinephrine were prepared fresh daily in distilled water containing 1 m g / m l ascorbic acid. Serum glucose levels were measured using an enzymatic colorimetric assay (Peridochrom ® glucose, Boehringer Mannheim). Serum insulin was measured using a radioimmunoassay kit (Amersham). 2.4. Statistics Results were compared for significant differences using one-way analysis of variance (ANOVA). When more than two groups of data were compared, results were further analyzed using the Newman-Keuls test. Results were considered to be significantly different if P < 0.05.
3. Results
Rats treated with streptozotocin gained weight at a much slower rate than non-diabetic rats, so that at the time of killing, streptozotocin-treated animals weighed significantly less than their agematched controls (table 1). Diabetic animals also exhibited significantly elevated serum glucose levels, and decreased serum insulin levels relative to controls (table 1). Aortas and mesenteric arteries from diabetic animals were significantly smaller in cross-sectional area than the corresponding arteries from control animals (table 1). Therefore, all con-
58 TABLE 1 Characteristics of rats 3 months after induction of diabetes with streptozotocin, compared to age-matched control rats.
Control Streptozotocin
12 12
Body weights
Serum glucose
Serum insulin
(g)
(mg/dl)
(kt U / ml )
501 _+21 308 _+18 ~
113.8 + 4.5 377.4 _+2.9 "
57.3 _+5.7 12.7 +_0.1 "
Cross-sectional areas (mm 2 ) Aortas
Mesenteric arteries
1.02 _+0.02 0.77 _+0.01 ~'
0.37 _+0.02 0.29 ± 0.01 "
a p < 0.05 compared to corresponding control value.
tractile responses were corrected for the cross-sectional areas of the tissues. 3.1. Aorta
As previously reported (MacLeod, 1985), aortas with intact endothelia from streptozotocin-treated rats were more responsive to the contractile effects of increasing concentrations of norepinephrine than were aortas with intact endothelia from agematched control animals (fig. 1). In these experiments, the m a x i m u m response to norepinephrine was significantly increased, from 2.30 _+ 0.09 g / r a m 2 (mean-t-S.E., n = 6) in control aortas to 2.90 + 0 . 1 4 g / r a m 2 ( n = 6) in diabetic aortas. In addition, the sensitivity (pD 2 value) of diabetic aortas to norepinephrine was significantly increased compared to control (table 2). Removal of the endothelium also resulted in a significant in-
crease in the m a x i m u m response (to 2.76 _+ 0.12 g / m m 2, n = 6) and sensitivity (table 2) of control aortas to norepinephrine. However, removal of the endothelium from diabetic aortas resulted only in a leftward shift in the norepinephrine dose-response curve (fig. 1), with a significant increase in the norepinephrine p D 2 value (table 2). The maxim u m response of diabetic aortas to norepinephrine was 2.82 + 0.24 g / m m 2 (n = 6) in the absence of endothelium, which was not significantly different from the value in the presence of endothelium. The magnitude of the increase in p D 2 value produced by removing the endothelium was very similar in control and diabetic aortas, and in the absence of endothelium the norepinephrine pD: was significantly greater in diabetic than in control aortas (table 2). The results obtained with methoxamine were similar to those obtained with norepinephrine. TA B LE 2
TENSION (g/mm 2)
Agonist pD 2 values in aortas from control and diabetic a n i m a l s in the presence and absence of endothelium. ND, not determined.
30
2.0
I 075
1.0 1.0-
9
8
7
6
S
NOREPINEPHRINE I-log M]
7
6
5
4
METHOXAMINE I - l o g M]
8
7
6
5
CLONIDINE [_-log M]
Fig. 1. Dose-response curves of aortas from rats with streptozotocin-induced diabetes of 3 months duration (n II) and agematched controls ( o O) to norepinephrine, methoxamine and clonidine, in the presence (© rn) and absence (O II) of endothelium. Each curve represents the mean of data obtained from six aortas. Only a single representative S.E. is shown in the midpoint of each curve, for the sake of the clarity.
Norepinephrine Control Streptozotocin Methoxamine Control Streptozotocin Clonidine Clonidine Streptozotocin Acetylcholine Control Streptozotocin
+Endothelium
n
-Endothelium
6.79_+0.11 a 7.10+0.07 "
6 6
7.45_+0.10 a 7.85_+0.08 a
4.63_+0.09 ~ 4.98+0.11 ~
6 6
5.35_+0.06 ~ 5.71 +0.12 ~
ND ND 7.07+0.14 6.63___0.19
n
6.75 _+0.08 ~ 7.18_+0.04 " 6 6
ND ND
" Significantly different from all other values for that agonist (P < 0.05, ANOVA, followed Newman-Keuls test).
59
although in the preparations used in this investigation, methoxamine behaved as a partial agonist relative to norepinephrine (fig. 1). The m a x i m u m response of control aortas with intact endothelia to methoxamine was 0.95 + 0.08 g / m m 2, which was not significantly different from the m a x i m u m response of diabetic aortas with intact endothelia to this agonist (1.27 +_ 0.18 g / m m 2 ) . However, the methoxamine p D 2 value was significantly increased in diabetic aortas under these conditions (table 2). Removal of the endothelium from control aortas resulted in a marked upward and leftward shift in the methoxamine dose-response curve (fig. 1), significantly increasing the m a x i m u m response to this agonist, to 1.75 -+ 0.10 g / m m 2, and significantly increasing the methoxamine p D 2 value (table 2). Removal of the endothelium from diabetic aortas resulted in an increase in the maxim u m response to methoxamine to 1.61_+0.16 g / m m 2, a value which was not significantly different from the m a x i m u m methoxamine response in the presence of endothelium. However, the methoxamine p D 2 value was significantly greater in the absence than in the presence of endothelium in diabetic aortas (table 2). As with norepinephrine, endothelium removal produced approximately the same degree of shift in the methoxamine dose-response curve in both control and diabetic aortas. In the absence of endothelium, the methoxamine p D 2 value was significantly greater in diabetic than in control aortas, although m a x i m u m responses of the two preparations to methoxamine were not significantly different (fig. 1, table 2). Almost no response to clonidine was detected in either control or diabetic aortas with intact endothelia (fig. 1). In the presence of endothelium, the m a x i m u m clonidine response was 0.04 +_ 0.02 g / m m 2 in control aortas, and 0.10 _+ 0.06 g / m m 2 in diabetic aortas. Removal of the endothelium resulted in an enhancement of responses to clonidine, which was more marked in diabetic than in control aortas (fig. 1). The m a x i m u m response of diabetic aortas to clonidine was increased to 1.05 _+ 0.17 g / m m 2 following endothelium removal; this value was significantly greater than the maxim u m response of 0.68 4- 0.09 g / m m 2 obtained in control aortas following removal of the endo-
TENSION (g/mm 2) 2.0
_* *
1.0
0
r
T
9
8
,
i
7
,
i
i
6
5
ACETYLCHOLINE
I-log M] Fig. 2. Responses of aortas from rats with streptozotocin-induced diabetes of 3 months duration (e) and age-matched controls ( o ) with intact endothelia to increasing concentrations of acetylcholine. Aortas were pre-contracted with 5 x 10 7 M norepinephrine which produced an increase in tension of 1.20 _+0.11 g / m m 2 in control aortas (n = 6), and 1.77 _+0.19 g/rfllTl 2 in diabetic aortas (n = 5). * P < 0.05 compared to corresponding control response.
thelium. In addition, the clonidine p D 2 value was significantly greater in diabetic than in control aortas in the absence of endothelium (table 2). The ability of acetylcholine to relax arteries pre-contacted with a sub-maximal dose of norepinephrine was also determined. Acetylcholine produced dose-dependent relaxation of both control and diabetic aortas with intact endothelia (fig. 2). This effect was abolished in both vessels by removal of the endothelium (data not shown). Although the degree of pre-contraction in response to norepinephrine was greater in diabetic than in control aortas, the m a x i m u m relaxation produced by acetylcholine in diabetic aortas was not significantly different from the m a x i m u m relaxation it produced in control aortas, nor was the acetylcholine p D 2 value different between the two preparations (table 2). In order to further establish the functional status of the endothelium in diabetic aortas, c G M P levels were measured in control and diabetic aortas in the absence and presence of endothelium, in the absence of drug, and following exposure to a m a x i m u m concentration of acetylcholine. There was no significant difference in basal c G M P levels between control and diabetic aortas, in either the
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Ach -
ENDO
Fig. 3. Cyclic GMP levels in aortas from rats with streptozotocin-induced diabetes of 3 months duration (shaded bars) and age-matched controls (open bars). All tissues were pretreated with 5 )<10-7 M norepinephrine, which has previously been shown to have no significant effect on cGMP levels in the absence or presence of endothelium in aortas (MacLeod et al., 1987). CNTL+Endo: tissues with intact endothelia, frozen after no further treatment. CNTL-Endo: aortas with endothelium removed, frozen after no further treatment. Ach+ Endo: aortas with intact endothelia, frozen 2 min after the addition of 1/~M acetylcholine. Ach-Endo: tissues with endothelium removed, frozen 2 min after the addition of 1 /~M acetylcholine. Each bar represents the mean+S.E, of data obtained from three to four tissues. * P < 0.05 compared to all other groups. absence or the presence of e n d o t h e l i u m (fig. 3). R e m o v a l of the e n d o t h e l i u m of vessels from b o t h groups of a n i m a l s resulted in a r e d u c t i o n of c G M P levels to a p p r o x i m a t e l y 50% of the levels f o u n d i n the presence of e n d o t h e l i u m , as previously reported ( M a c L e o d et al., 1987), a l t h o u g h with the statistics used i n the present investigation, these differences were n o t statistically significant. Acetylcholine p r o d u c e d a significant elevation of c G M P levels, the m a g n i t u d e of which was n o t different i n control a n d diabetic aortas. This effect of acetylcholine was abolished b y removal of the e n d o t h e l i u m (fig. 3).
3.2. Mesenteric artery Mesenteric arteries with intact e n d o t h e l i a from diabetic rats exhibited greater increases i n t e n s i o n in response to n o r e p i n e p h r i n e t h a n did their agem a t c h e d controls (fig. 4). The m a x i m u m increase
in t e n s i o n p r o d u c e d by n o r e p i n e p h r i n e in diabetic arteries was 3.77 _+ 0.44 g / m m 2, which was signific a n t l y greater t h a n the c o r r e s p o n d i n g value ol 2.53 _+0.22 g / m m 2 f o u n d in control arteries. However, the n o r e p i n e p h r i n e p D 2 values in control a n d diabetic mesenteric arteries did n o t differ significantly (table 3). Removal of the endothelium from control arteries resulted in an increase in the m a x i m u m response to n o r e p i n e p h rine, to 3.06 _+ 0.27 g / m m 2, (n = 6) a n d in a sign i f i c a n t increase in the n o r e p i n e p h r i n e p D 2 value (table 3). E n d o t h e l i u m removal had no effect on the m a x i m u m response of diabetic mesenteric arteries to n o r e p i n e p h r i n e , which was 3.71 _+ 0.38 g / m m 2 (n = 6) in the absence of e n d o t h e l i u m , but did p r o d u c e a significant increase in sensitivity to this agonist (table 3). I n the absence of endothelium, n o significant differences were f o u n d between control a n d diabetic arteries in response to norepinephrine. Responses of mesenteric arteries with intact e n d o t h e l i a from diabetic a n i m a l s to m e t h o x a m i n e were also increased c o m p a r e d to control (fig. 4). T h e m a x i m u m response of control arteries to m e t h o x a m i n e was 1.27 + 0.16 g / m m 2 (n = 6), while that of diabetic arteries was 1.93 ± 0.30 g / m m 2 (n = 6). However, m e t h o x a m i n e p D 2 values did n o t differ significantly between control a n d diabetic arteries (table 3). Removal of the e n d o t h e l i u m from control mesenteric arteries
TENSION (g/ram?)
i£
1° I 0.5j
9
8
7
6
5
NOREPINEPHRINE l-log M]
4
•
6
5
4
METHOXAMINE l-log M]
3
8
7
6
5
4
CLONIDINE l-log M]
Fig. 4. Dose-response curves of mesenteric arteries from rats with streptozotocin-induced diabetes of 3 months duration ([]11) and age-matched controls (©e) to norepinephrine, methoxamine and clonidine, in the presence (o D) and absence (e II) of endothelium. Each curve represents the mean of data obtained from six or seven preparations, with a representative S.E. shown around the midpoint of each curve.
61
resulted in a leftward shift in the methoxamine dose-response curve, with a significant increase in the methoxamine p D 2 value (table 3), and a small, but not significant increase in the m a x i m u m response to this agonist, to 1.69 _+ 0.21 g / m m 2 (n = 6). In diabetic arteries, removal of the endothelium also resulted in a leftward shift in the methoxamine dose-response curve (table 3), while having no effect on the m a x i m u m response to this agonist, which was 2.06 _+ 0.20 g / m m 2 (n = 6) in the absence of endothelium. As with norepinephrine, in the absence of endothelium no significant differences could be detected between control and diabetic arteries in response to methoxamine. In the presence of intact endothelia, responses of mesenteric arteries from both control and diabetic animals to clonidine were exceedingly small (fig. 4). The m a x i m u m response of control arteries to clonidine was 0.06 _+ 0.03 g / m m 2 (n = 6), while that of diabetic arteries was 0.05 _+ 0.02 g / r a m z (n = 6). Removal of the endothelium resulted in marked enhancement of responses of vessels from both groups of animals to this agonist, although to a greater extent in diabetic than in control arteries. The m a x i m u m response of control arteries to clonidine increased to 0.35 + 0.10 g / m m 2 (n = 6), and that of diabetic arteries increased to 0.76 _+ 0.16 g / r a m 2 ( n = 7) following removal of the endothelium. These values were significantly differTABLE 3 Agonist pD 2 values in mesenteric arteries from control and diabetic rats, in the presence and absence of endothelium. ND, not determined.
Norepinephrine Control Streptozotocin Methoxamine Control Streptozotocin Clonidine Control Streptozotocin Acetylcholine Control Streptozotocin
+ Endothelium
n
- Endothelium
n
6.59_+0.14 6.71+0.08
6 6
7.03+0.13 a 7.105:0.10 "
6 7
4.83-+0.07 5.05+0.04
6 6
5.32_+0.15 a 5.50+0.06 a
6 7
6.68 + 0.17 6.81_+0.14
4 7
TENSION (g/mm 2) 3.o- ~
~
± 2.0-
i
i-
T
1.0
0
, 9
,
, 8
,
i 7
.
i
'
6
i S
ACETYLCHOLINE
E-log M] Fig. 5. Responses of mesenteric arteries from rats with streptozotocin-induced diabetes of 3 months duration (e) and age-matched controls ( o ) with intact endothelia to increasing concentrations of acetylcholine. Mesenteric arteries were precontracted with 5 × 1 0 -7 M norepinephrine, which produced an increase in tension of 1.75_+0.19 g / r a m 2 in control mesenteric arteries (n = 6) and 2,75_+0.33 g / r a m 2 in diabetic mesenteric arteries (n = 6). * P < 0.05 compared to corresponding control response.
ent. However, no difference was detected between control and diabetic arteries in clonidine p D 2 values in the absence of endothelium (table 3). The ability of acetylcholine to relax mesenteric arteries pre-contracted with 5 × 10 -v M norepinephrine in the absence and presence of endothelium was also determined. Acetylcholine produced dose-dependent relaxation of arteries from both control and diabetic animals in the presence of endothelium (fig. 5). This effect of acetylcholine was lost following endothelium removal (data not shown). Despite the greater contractile response of diabetic arteries to norepinephrine, neither the m a x i m u m relaxation produced by acetylcholine, nor the acetylcholine pD2 value (table 3), were different between control and diabetic arteries.
4. Discussion ND ND 7.14_+0.25 6.95-+0.13
6 6
ND ND
a p < 0.05 compared to corresponding p D 2 value in the presence of endothelium.
The results of the present investigation confirm a previous report from this laboratory (MacLeod, 1985) that contractile responses of aortas and mesenteric arteries with intact endothelia from rats with streptozocin-induced diabetes of 3
62 months duration to norpinephrine are enhanced compared to responses of age-matched control arteries. In this study, we also found an increase in the sensitivity (pD 2 value) of diabetic aortas to norepinephrine. However, the magnitude of the increase in sensitivity, although significant, was very small, which may explain why it was not detected in the previous investigation. Responses to methoxamine, an agonist selective for C~l-adrenoceptors, which has been previously reported to act as a partial agonist relative to noradrenaline in rat aorta and mesenteric artery (Lues and Schtimann, 1985; MacLeod et al., 1987) were also enhanced in diabetic arteries with intact endothelia, compared to their age-matched controls. However, responses of aortas and mesenteric arteries with intact endothelia from both diabetic and control animals to clonidine were very small and not significantly different from each other. Removal of the endothelium from aortas and mesenteric arteries of control animals resulted in enhancement of responses to each of the c~-adrenoceptor agonists tested. Responses were shifted upward, and, at least in the case of norepinephrine and methoxamine, to the left of responses to these agonists in the presence of endothelium. The degree of enhancement of maximum responses appeared to be inversely related to the efficacy of the agonists tested, in that responses to clonidine were enhanced the most, and those to norepinephrine the least, by removal of the endothelium. These results are similar to those reported previously in aorta and mesenteric artery (Egleme et al., 1984; Lues and Schtimann, 1984; MacLeod et al., 1987). Recent studies have attributed the inhibition of c~-adrenoceptor-mediated responses in the presence of an intact endothelium to the spontaneous release of E D R F from the endothelium (Martin et al., 1986; MacLeod et al., 1987). The spontaneously released E D R F appears to result in tonic, low elevation of c G M P levels in the vascular smooth muscle, which exert an inhibitory effect against contractions elicited by stimulation of c~adrenoceptors and serotonin receptors (Martin et al., 1986; MacLeod et al., 1987). Evidence also suggests that the spontaneously released E D R F has a greater depressant effect against agonists of low efficacy, such as clonidine, than those of
higher efficacy, such as norepinephrine (Martin et al., 1986; MacLeod et al., 1987). The effects of endothelium removal on responses of diabetic aortas and mesenteric arteries to norepinephrine and methoxamine were less marked than in the corresponding blood vessels from control animals. However, the opposite was true of responses to clonidine. In diabetic aortas and mesenteric arteries, removal of the endothelium caused no further increase in maximum responses to norepinephrine, although the magnitude of the leftward shift in the norepinephrine dose-response curve was similar to that obtained in control preparations. The lack of further effect on m a x i m u m norepinephrine responses may have occurred because the tissues themselves were not able to respond with further increases in tension. However, endothelium removal resulted in loss of the differences in response to norepinephrine found between control and diabetic mesenteric arteries, although diabetic aortas still had a significantly elevated pD 2 value relative to control. Similar results were also obtained with methoxamine on removal of the endothelium from the diabetic arteries. These data are less easily explained, since in both diabetic and control arteries, methoxamine acted as a partial agonist relative to norepinephrine, even in the absence of endothelium. Thus, the inability of methoxamine to produce a further increase in tension in diabetic arteries following removal of the endothelium cannot be explained by an inability of the tissues to contract further. Since there were no significant differences between responses of diabetic and control mesenteric arteries to methoxamine in the absence of endothelium, and only a slight increase in the methoxamine p D 2 value in diabetic aortas compared to control under these conditions, it might be argued that these results support the hypothesis that ~-adrenoceptor-mediated responses are enhanced in diabetic blood vessels due to decreased release of E D R F . However, other evidence obtained in this investigation does not agree with this interpretation of the data. Firstly, clonidine responses were almost undetectable in diabetic aortas with intact endothelia; this would not be expected if there was decreased release of E D R F . Secondly, in the absence of
63 endothelium, clonidine responses were much greater in diabetic aortas and mesenteric arteries than in the corresponding control tissues. This suggests that some other change is responsible for the enhanced c~-adrenoceptor responsiveness in diabetes. Measurements of c G M P levels, in both the absence and presence of acetylcholine, also suggest that both the tonic and stimulated release of E D R F is similar in control and diabetic vessels. Basal c G M P levels were similar in control and diabetic aortas with intact endothelia, and were reduced to the same extent by endothelium removal. This reduction in c G M P levels on removal of the endothelium has previously been reported (Miller et al., 1984; Griffith et al., 1985; Martin et al., 1985; MacLeod et al., 1987) and has been taken as evidence in support of the tonic release of E D R F . Therefore, by this measure the spontaneous release of E D R F appears to be similar in control and diabetic aortas. In addition, the increase in c G M P levels produced by a m a x i m u m concentration of acetylcholine was very similar in control and diabetic aortas, indicating that the ability of the endothelium to respond to this agonist is not altered in diabetes. The relaxation of aortas and mesenteric arteries from diabetic and control animals in response to acetylcholine was also not significantly different, although in general the arteries from diabetic animals appeared to be slightly less sensitive to acetylcholine. This is probably related to the fact that relaxant responses to acetylcholine were measured in the presen.ce of a concentration of norepinephrine which produced a greater increase in tension in diabetic arteries than in controls. Responses to vasorelaxants are determined at least in part by the contractile tension attained prior to their addition (Furchgott, 1983). N o other more suitable contractile agonist was available for use in these experiments, as preliminary experiments indicated that contractile responses to serotonin were also enhanced in diabetic arteries, while responses to K + depolarization, which are not enhanced in diabetes (MacLeod, 1985), are resistant to relaxation by acetylcholine (Furchgott, 1983; unpublished observations). However, we found that when the concentration of norepinephrine was increased in control mesenteric arteries to 10 5
M, which produced the same increase in tension as that found in diabetic mesenteric arteries in response to 5 >< 10 .7 M norepinephrine, acetylcholine actually produced a greater maximum relaxation of diabetic arteries (78.9 + 6.7%, means + S.E., n = 8) than of control arteries (53.1 + 7.6%, n = 8). During the preparation of this manuscript, the results of two other less extensive investigations of endothelium function in aortas from chronic s t r e p t o z o t o c i n - d i a b e t i c rats were published (Oyama et al., 1986; Wakabayashi et al., 1987). In agreement with the results of the present investigation, one group reported that the endotheliumdependent relaxation of aortas in response to acetylcholine was unaffected by the diabetic state (Wakabayashi et al., 1987). However, this group also found that responses to clonidine were enhanced to a much greater extent in control than in diabetic aortas by removal of the endothelium, suggesting that the tonic release of E D R F from diabetic aortas may have been reduced in their experiments. In contrast, O y a m a et al. (1986) reported that the endothelium-dependent relaxation of diabetic aortas in response to both acetylcholine and histamine was attenuated compared to control. Although the reason for the discrepancy between our results and those of the other two groups is not clear, one possibility is that there was a difference in the intensity of the diabetic state between studies. Blood glucose values were much greater, and body weight gains were much less, in diabetic rats used in the other two investigations (Oyama et al., 1986; Wakabayashi et al., 1987) than those used in our study. Overall, the results of this investigation do not support a role for decreased release of E D R F in mediating enhanced responses of diabetic arteries to c~-adrenoceptor stimulation. Therefore, the mechanism for the enhanced reactivity to a-adrenoceptor agonist in diabetes is likely to involve some change at the level of the vascular smooth muscle. This could be either in the ch-adrenoce ptors themselves, or in the coupling of these receptors to the contractile response. Other reports have suggested that there is increased mobilization of extracellular Ca 2+ mediated by ch-adrenoceptors in aortas from streptozotocin-diabetic rats (Scar-
64
borough and Carrier, 1983; 1984). However, in the present investigation, responses to both the putative al-adrenoceptor agonist methoxamine and the a2-adrenoceptor agonist clonidine could be shown to be enhanced in arteries from diabetic animals, under different conditions. Therefore, these data do not support a role for either a-adrenoceptor subtype in selectively mediating the increased responsiveness of arteries from diabetic rats to c~-adrenoceptor agonists. In summary, these results confirm that a-adrenoceptor-mediated responses are enhanced in aortas and mesenteric arteries from rats with chronic STZ-induced diabetes. The enhanced responsiveness to a-receptor stimulation does not appear to result from a decrease in release of E D R F from the endothelium of these vessels. However, if the endothelium is damaged on removal of these arteries, some changes in responsiveness resulting from diabetes may be masked. It is possible that this has contributed to some of the controversy surrounding the influence of diabetes on vascular reactivity, and suggests that in future studies of this type, the status of the endothelium should be established.
References Arbogast, B.W., D.L. Berry and C.L. NeweU, 1984, Injury of arterial endothelial cells in diabetic, sucrose-fed and aged rats, Atherosclerosis 51, 31. Christlieb, A.R., H.U. Janka, B. Kraus, R.E. Gleason, E.A. lcasas-Cabral, L.M. Aiello, B.V. Cabral and A. Solano, 1976, Vascular reactivity to angiotensin I1 and to norepinephrine in diabetic subjects, Diabetes 25, 268. Colwell, J.A., P.V. Halushka, K.E. Sarji, M.F. Lopes-Virella and J. Segal, 1979, Vascular disease in diabetes, Arch. Int. Med. 139, 225. Egleme, C., T. Godfraind and R.C. Miller, 1984, Enhanced responsiveness of rat isolated aorta to clonidine after removal of the endothelial cells, Br. J. Pharmacol. 81,016. Furchgott, R.F., 1983, Role of endothelium in responses of vascular smooth muscle, Circ. Res. 53, 557. Godfraind, T., 1985, E D R F and cyclic G M P control gating of receptor-operated calcium channels in vascular smooth muscle, European J. Pharmacol. 126, 341. Griffith, T . M . D . H . Edwards, M.J. Lewis and A.H. Henderson,
1985, Evidence that cyclic guanosine monophosphate ( c G M P ) mediates e n d o t h e l i u m - d e p e n d e n t relaxation, European J. Pharmacol. 112, 195. Lues, I. and J.J. Schiimann, 1984, Effect of removing the endothelial cells on the reactivity of rat aortic segments to different alpha-adrenoceptor agonists, Naunyn-Schmiedeb. Arch. Pharmacol. 328, 160. MacLeod, K.M., 1985, The effect of insulin treatment on changes in vascular reactivity in chronic, experimental diabetes, Diabetes 34, 1160. MacLeod, K.M. and J.H. McNeill, 1985, The influence of chronic experimental diabetes on contractile responses of rat isolated blood vessels, Can. J. Physiol. Pharmacol. 63, 52. MacLeod, K.M., D.D.W. Ng, K.H. Harris and J. Diamond, 1987, Evidence that c G M P is the m e d i a t o r of endothelium-dependent inhibition of contractile responses of rat arteries to a-adrenoceptor stimulation, Mol. Pharmacol. 32, 59. Martin, W., R.F. Furchgott, G.M. Villani and D. Jothianandan, 1986, Depression of contractile responses in rat aorta by spontaneously released endothelium-derived relaxing factor, J. Pharmacol. Exp. Ther. 237, 529. Miller, R.C., M. Mony, V. Shini, P. Schoeffter and J.C. Stoclet. 1984, Endothelial mediated inhibition of contraction and increase in cyclic G M P levels evoked by the alpha-adrenoceptor agonist, B-HT 920 in rat isolated aorta, Br. J. Pharmacol. 83, 903. Murakami, K., H. Karaki and N. Urakawa, 1985, Role of endothelium in the contractions induced by norepinephrine and clonidine in rat aorta, Jap. J. Pharmacol. 39, 357. Oyama, Y., H. Kawasaki, Y. Hattori and M. Kanno, 1986, Attenuation of endothelium-dependent relaxation in aorta from diabetic rats, European J. Pharmacol. 131, 75. R a m a n a d h a m , S., W.H. Lyness and T.E. Tenner, 1984, Alterations in aorta and tail artery reactivity to agonists after streptozotocin treatment, Can. J. Physiol. Pharmacol. 62, 418. Scarborough, N.L. and G.O. Carrier, 1983, Increased alpha-2adrenoreceptor mediated vascular contraction in diabetic rats, J. Auton. Pharmacol. 3, 177. Scarborough, N.L. and G.O. Carrier, 1984, Nifedipine and alpha adrenoceptors in rat aorta. II Role of extracellular calcium in enhanced alpha-2-adrenoceptor-mediated contraction in diabetes, J. Pharmacol. Exp. Ther. 23, 603. Wakabayashi, I., K. Hatake, N. Kimura, E. Kakishita and K. Nagai, 1987, Modulation of the vascular tonus by the endothelium in experimental diabetes, Life Sci. 40, 643. Weidmann, P., C.B. Piccoli, G. Keusch, M. Mutagic, M. Grimm, A. Meier and W.H. Zeigler, 1979, Sodium-volume factor, cardiovascular reactivity and hypotensive mechanism of diuretic therapy in mild hypertension associated with diabetes mellitus, Am. J. Med. 67, 779.