European Journal of Pharmacology, 229 (1992) 69-73
69
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52807
Ultradian variations in sensitivity of rat aorta rings to noradrenaline M i c h e l G o h a r , Pascal D a l e a u , J e f f r e y A t k i n s o n a a n d Y v e s - M i c h e l G a r g o u i l CEREP, B.P. 1, 86600 Celle l'Evescault, France and "Laboratoire de Pharmacologie Cardio-t~asculaire, 5 Rue Albert Lebrun, 54000 NanQ,, France
Received 14 October 1991, revised MS received 9 September 1992, accepted 22 September 1992
The in vitro sensitivity of the rat aorta to the vasoconstrictor effect of noradrenaline was found to vary according to the time the animals were killed, with a minimum at 10:00 h and a maximum at 16:00 h. This ultradian rhythm was not influenced by the presence of the endothelium but was modified by some, as yet unidentified, circulating factor(s). The results also strongly suggested an ultradian rhythm of in vitro sensitivity to the /3-adrenoceptor agonists, salbutamol and isoprenaline. This rhythm appears to be opposite to that for the sensitivity of aortic a-adrenoceptors to stimulation. In conclusion, our results demonstrate that the regulation of aortic tone follows an ultradian rhythm. Diurnal rhythm; Adrenoceptors; Noradrenaline; Aorta; (Rat)
1. Introduction Receptor binding studies show that /3-adrenoceptor density follows a diurnal rhythm in rat heart ventricles (Lemmer and Lang, 1984) and that both a- and /3adrenoceptor density in rat brain, follows a similar rhythm (Kafka et al., 1983). In order to investigate the possible existence and functional significance of such rhythms in blood vessels, we studied the diurnal changes in the in vitro vasoconstrictor responses of the rat aorta to noradrenaline. In aorta rings of the rat, noradrenaline induces contraction via stimulation of an adrenoceptor of the at-subtype (Cauvin and Malik, 1984; Macia et al., 1984; Alosachie and Godfraind, 1988). Thus diurnal variation in the in vitro vasoconstrictor responses may be interpreted as reflecting diurnal variabiality in a~-adrenoceptor affinity a n d / o r number, as the neuronal reuptake of noradrenaline presumably does not modify responses to exogenous noradrenaline in this sparsely innervated preparation. As the endothelium can modulate vasoconstrictor responses to noradrenaline (Miller and Stoclet, 1985), some experiments were performed with endotheliumdenuded aorta rings in order to investigate whether the diurnal variation in the in vitro vasoconstrictor responses to noradrenaline is due to a diurnal variation in vasorelaxant, endothelial activity. Finally, as nor-
Correspondence to: M. Gohar, Roussel-Uclaf, CRBA, Route de la Valentine, St. Marcel B.P. 1, 13367 Marseille Cedex 11, France. Tel. 33.91.35.90.35, fax 33.91.44.62.01.
adrenaline could stimulate both a 1- and /3-adrenoceptors in the rat aorta, we studied the diurnal changes in in vitro sensitivity of precontracted aorta rings to the /3-adrenoceptor agonists, isoprenaline and salbutamol. This provided us with insight into whether the diurnal variation in the in vitro vasoconstrictor responses to noradrenaline was due to diurnal variation in /3-adrenoceptor activity.
2. Materials and methods 2.1. A n i m a l s
Male Wistar rats (300-350 g, 5 month old, CERJ, France) were given food and water ad libitum and housed for at least 10 days at 20°C, under fixed lighting conditions (07:00 h to 19:00 h. 2.2. A o r t a ring preparation
Rats were killed by cervical dislocation. The thoracic aorta was removed and placed in a physiological salt solution (mM): NaCI 120, KCI 4.7, CaC12 1.8, N a H 2 P O 4 1.2, MgC12 1.2, N a H C O 3 25 and glucose 11, 37°C, pH 7.5, bubbled with 5% CO 2 and 95% 0 2. Eight rings were dissected from the same aortic segment, and in half of the rings the luminal surface was gently rubbed with a metal rod to remove the endothelium. Rings were mounted in 20-ml organ baths and contractions were measured with isometric transducers (CPOL25, Celaster, Poitiers, France). The rings were
70 allowed to equilibrate for 1 h and were then rinsed 4 times and stretched to a tension of 1.5 g. Drugs were then added to the organ bath. Concentrations are given as in terms of the base and cumulative dose-response curves were obtained by stepwise increases in the concentrations of the various drugs used. At the end of each experiment, acetylcholine (1 IxM) was added to the organ bath after the rings had been precontracted with noradrenaline in order to verify the presence or absence of the endothelium (Furchgott, 1983). Intact aorta rings that relaxed less than 20% were discarded.
N:22 8 8 I~ C~_
8
Variations in the in vitro sensitivity of the rat aorta to noradrenaline during the day were investigated in groups of rats (3-4) killed at different times between 08:30 and 18:30 h. In this protocol aorta rings with an intact endothelium were used. Variations in the in vitro sensitivity of the rat aorta to noradrenaline over a 2-day period were investigated in rats killed every 3 h in groups of 3 or 4 over a period of 48 h. Ex vivo changes in the in vitro sensitivity of the rat aorta to noradrenaline were investigated by killing the rats at 13:00 h (n = 2 rats). In half of the aorta rings prepared from any individual animal, the in vitro sensitivity to noradrenaline was determined immediately afterward the animals were killed, at 13:00 h. In the remaining 4 aorta rings the in vitro sensitivity to noradrenaline was determined at 16:00 h after storage at room temperature. The possible modulation of noradrenaline-induced contraction by /3-adrenoceptors was investigated in aorta rings prepared from two groups of rats (n = 4 rats per group) killed at 10:00 h and 16:00 h. Noradrenaline (0.03 txM) was added to the organ bath before and after incubation for 30 min with propranolol (1 txM). The diurnal rhythm of the in vitro sensitivity of /3-adrenoceptors was investigated in 2 groups of rats killed at 10:00 h and 16:00 h (n = 2 rats per group). Aorta rings were precontracted with PGF2~ (prostaglandin F2~) at a concentration of 2 /xM, which produces half-maximal contraction of this preparation (Suba and Roth, 1987), and then exposed to isoprenaline or salbutamol. In some experiments rings were incubated for 30 min with propranolol prior to the addition of PGF2~ followed by isoprenaline.
2.4. Analysis of results Results were stored on a computer and analyzed by using MOISE3 software (Celaster, Poitiers, France). All data are expressed as means + S.E.M. Statistical differences between means were determined with Student's t-test, pD 2 was calculated as -logl0(ECs0) after
~6 8
9
9.0
-,~ r---i
8.6
co (-.
8.4. 8.2-
c_ co c_ o Z
8.07.8. 7.67.4
9
1:1
13 Time
2.3. Specific protocols
11
9,2
:15 ( hours
17
19
)
Fig. 1. Sensitivity of rat aorta rings to noradrenaline (expressed as pD 2) during the day (from 10:00 h to 18:00 h). Values are expressed as means + S.E.M. N refer to the number of aorta rings.
least-squares linear regression analysis of transformed data.
2.5. Drugs Noradrenaline bitartrate, PGF2~ Tris salt, acetylcholine chloride, propranolol hydrochloride, salbutamol hemisulphate and isoprenaline hydrochloride were purchased from Sigma, St. Louis, Missouri, U.S.A.
3. Results
3.1. Variation in the in vitro sensitiuity of rat aorta rings to noradrenaline during the day A peak in sensitivity (pD 2 = 8.91 _+ 0.09, n = 8 rings) was observed at 16:00 h and a nadir (pD 2 = 7.60 _+ 0.12, n = 8 rings) at 10:30 h (fig. 1). The difference between these two values was highly significant (P < 0.001). Sensitivity was determined in the presence of the endothelium.
3.2. Variation in the in L'itro sensitivity of rat aorta rings to noradrenaline over a 2-day period During the first 24 h of the 2-day period, the diurnal rhythm of sensitivity to noradrenaline was similar to that described above, with a pD 2 = 7.86 _+ 0.05 (n = 15 rings) at 10:00 h (fig. 2). There was a pronounced leftward shift in the noradrenaline dose-response curve obtained at 16:00 h (pD z = 8.40 _+ 0.11, n = 9 rings, fig. 3) compared to that obtained at 10:00 h. The magnitude of the variation in pD z values was also statistically significant in the second 24-h period (16:00 h pD z = 8.23 _+ 0.10, n = 13 rings, versus 10:00 h pD 2 = 7.61 _+ 0.10, n = 7 rings, P < 0.005). Removal of the endothelium produced an overall increase in sensitivity but the ultradian rhythm remained.
71 n:
9
6
i5 19
12
17
15 19
8
8
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15
17 14
17
is
N: 15
7
9
20
15
i s 12
7
io
is
16
15 17
i5
i7
12
170 160
9.0
150 140 130
8.8
C o
8.6
o
120
8.4 8.2,
m
g
a. 100 ~g 90 ~ 807O
L 8.0 7.8 I
~
o z
7.6 7.4
-10 19
1
Time
7
i3
( hours
19
I
)
When rats were killed at 13:00 h and p D 2 values were determined immediately afterwards, a value of 7.34 _+ 0.04 (n = 8 rings) was found. The same series of rings tested at 16:00 h gave a pD 2 value of 7.16 _+ 0.02 (n = 8 rings). When a separate series of rings from the same animals were tested at 16:00 h a pD 2 value of 7.36 _+ 0.03 was found. Thus sensitivity to noradrenaline did not change after 3 h of storage at room temperature.
3.4. Modulation of noradrenaline-induced contractions by fl-adrenoceptors The presence of the fl-adrenoceptor antagonist propranolol did not modify the vasoconstrictor responses to noradrenaline. In rings tested at 10:00 h, responses to noradrenaline (30 nM) in the presence of propra-
E
iO0
80
-6
-5
Isoprenaline
-4 (M)
Fig. 4. C u m u l a t i v e i s o p r e n a l i n e d o s e - r e s p o n s e curves in intact aorta rings p r e p a r e d from rats killed at 10:00 h (circles, n = 13-8) and 16:00 h ( i n v e r t e d triangles, n = 1 0 - 8 ) and p r e c o n t r a c t e d with P G F 2 , (2 /xM). R e s u l t s are e x p r e s s e d as means_+S.E.M. ** P < 0 . 0 1 , *** P < 0.005.
nolol were 1 0 1 + 2% (n = 10 rings) of those in the absence of propranolol. At 16:00 h a value of 105 _+ 2% (n = 8 rings) was obtained.
3.5. Diurnal variations in the in vitro sensitivity of fladrenoceptors In intact aorta rings obtained from rats killed at 10:00 h and precontracted with PGF2~, lower concentrations of the /3-adrenoceptor agonist isoprenaline (0.1-1 /zM) caused relaxation whereas higher concentrations (1-100 /zM) caused contraction (fig. 4). In rings obtained from rats killed at 16:00 h, isoprenaline caused contractions only. Addition of the /3-adrenoceptor antagonist, propranolol, did not alter the responses obtained at 16:00 h but abolished the isoprenaline-induced responses obtained at 10:00 h (fig. 5). The flz-adrenoceptor agonist, salbutamol, caused a greater relaxant effect in aorta rings (precontracted with PGF2~) obtained from rats killed at 10:00 h than
g
~70
~
lg0
m
150
g
140
o
130 B0
120 m o
40
I00
x
E
-7
log
3.3. Ex vivo changes in the in vitro sensitivity of the rat aorta to noradrenaline
to
-8
7
Fig. 2. V a r i a t i o n s in n o r a d r e n a l i n e p D 2 in rat a o r t a rings over a 2-day period. I n t a c t a o r t a rings are r e p r e s e n t e d by s q u a r e s (N = n u m b e r of rings) a n d r u b b e d a o r t a rings by circles (n = n u m b e r of rings). R e s u l t s are e x p r e s s e d as m e a n s + S.E.M.
4J to (J
-9
. . . . . . . . . . . . . . . 13
=
** ***
***
90 -10
20
-9
-8 log
0
-10
-9 log
-8 Noradrenaline
-7
-6 (M)
Fig. 3. C u m u l a t i v e n o r a d r e n a l i n e d o s e - r e s p o n s e curves in intact a o r t a rings p r e p a r e d from rats k i l l e d at 10:00 h (circles, n = 15) a n d 16:00 h ( i n v e r t e d triangles, n = 9). ,4 p < 0.01, 44, p < 0.005.
-7
-6
Isoprenaline
-5
-4 (M)
Fig. 5. Effect of p r o p r a n o l o l (1 # M ) on r e s p o n s e s to i s o p r e n a l i n e in i n t a c t a o r t a rings p r e p a r e d from rats k i l l e d at 10:00 h (circles, n = 8) and 16:00 h ( i n v e r t e d triangles, n = 8) and p r e c o n t r a c t e d with P G F 2 , . C o n t r o l curves in the a b s e n c e of p r o p r a n o l o l are given in fig. 4. R e s u l t s are e x p r e s s e d as m e a n s + S . E . M . ** P < 0 . 0 1 , *** P < 0.005.
72
g
105
-~ 4-J
I00
•
CJ
m f_
c o u
95 90 85
u2 (-9
80
n
75 o
~
70, 65 -8
-7
-6
-5
-4
(M) Fig. 6. Cumulative salbutamol dose-response curves in intact aorta rings prepared from rats killed at 10:00 h (circles, n = 11) and at 16:00 h (inverted triangles, n = 8) and precontracted with PGF2~ (2 ~M). Results are expressed as means _+S.E.M. log
Salbutamol
in rings obtained at 16:00 h (fig. 6), but the differences were not significant. Salbutamol did not cause contraction.
4. D i s c u s s i o n
T h e p D 2 values for noradrenaline in the rat aorta r e p o r t e d in the literature vary from 6.7 to 8.5 (Godfraind, 1983; S c a r b o r o u g h and Carrier, 1984; Cauvin and Malik, 1984; Suba and Roth, 1987; Bevan et al., 1988; Alosachie and Godfraind, 1986; 1988). In these reports there is no indication of the time of day when the experiments were carried out, but it is possible that part of the reason why noradrenaline EDs0 values a p p e a r to vary some 100-fold is because the various authors did their experiments at different times of the day. W e f o u n d a slightly less than 100-fold change in EDs0 values when these were m e a s u r e d at 10:00 h and at 16:00 h. This change followed a bimodal diurnal rhythm, which could reflect an underlying ultradian rhythm with a 12-h periodicity. Endothelial factors do not apparently interfere with this rhythm as similar results were obtained in intact and d e n u d e d preparations. R e m o v a l of the e n d o t h e l i u m m o r e o v e r p r o d u c e d an overall increase in responses to noradrenaline, as has been already published (Alosachie and Godfraind, 1986; 1988; Miller and Stoclet, 1985). A l t h o u g h some preparations exhibit intrinsic circadian rhythms ex vivo (Kasal and Perez-Polo, 1982; Kadle and Folk, 1983), this does not a p p e a r to be the case for the aorta rings in our experiments. Thus the diurnal rhythm appears to d e p e n d on some in vivo factor. D e B o e r and Van G u g t e n (1987) have r e p o r t e d a diurnal variation in plasma corticosterone levels with a p e a k at the end of the light period. As corticosterone is an uptake-2 inhibitor, the diurnal rhythm in its
plasma concentration could cause a diurnal variation in the synaptic concentration of locally released noradrenaline. This would seem an unlikely explanation, however, as the aorta is poorly innervated in the rat (Patti et al., 1972; Burnstock, 1975) and its noradrenaline content is very low (Berkowitz et al., 1971). Thus the rate of uptake 2 is presumably low in the rat aorta. De Boer and Van G u g t e n (1987) also reported that circulating noradrenaline and adrenaline levels were higher at night than during the day. Furthermore, Westerink and Koolstra (1986) showed a bimodal rhythm in the urinary excretion of catecholamines. Thus, variations in plasma catecholamine concentration may modulate the sensitivity of the aorta to noradrenaline by receptor desensitization. As the variation in plasma catecholamines is limited, this is not a likely explanation. Experiments carried out with noradrenaline, which has low affinity for /3-adrenoceptors, and salbutamol and isoprenaline showed an opposite diurnal rhythm for /3-adrenoceptor sensitivity. However, it is unlikely that the diurnal rhythm of noradrenergic constrictor responses is caused by changes in the balance between a- and /3-adrenoceptor sensitivity. Moreover, blockade of /3-adrenoceptors had no effect. High doses of the non-selective /3-adrenoceptor agonist, isoprenaline, contracted the aorta, presumably via stimulation of a - a d r e n o c e p t o r s (Fleish et al., 1970). O n c e again, this a - a d r e n o c e p t o r - m e d i a t e d contraction revealed by high doses of isoprenaline was consistent with the previously found ultradian rhythm. In conclusion, we found that the sensitivity of rat aorta to noradrenaline showed an ultradian variation. A l t h o u g h further experiments are required to define the mechanism underlying this rhythm, we suggest that our results may explain the wide variation in p D 2 values for noradrenaline in aorta reported in the literature.
References
Alosachie, I. and T.G. Godfraind, 1986, Role of cyclic GMP in the modulation of the adrenolytic action of prazosin in the rat isolated aorta, Br. J. Pharmacol. 89, 525. Alosachie, I. and T.G. Godfraind, 1988, The modulatory role of vascular endothelium in the interaction of agonists and antagonists with a-adrenoceptors in the rat aorta, Br. J. Pharmacol. 95, 619. Bevan, J.A., R.D. Bevan, K. Kite and M.A. Oviowo, 1988, Species differences in sensitivity of aortae to norepinephrine are related to alpha-adrenoceptor affinity, Trends Pharmacol. Sci. 9, 87. Berkowitz, B.A., J.H. Tarver and S. Spector, 1971, Norepinephrine in blood vessels: concentration, binding, uptake and depletion, J. Pharmacol. Exp. Ther. 177, 119. Burnstock, G., 1975, Innervation of vascular smooth muscle: histochemistry and electron microscopy, Clin. Exp. Pharmacol. Physiol. 2, 7.
73 Cauvin, C. and S. Malik, 1984, Induction of Ca ++ influx and intracellular Ca ++ release in isolated aorta and mesenteric resistance vessels by norepinephrine activation of alpha, receptors, J. Pharmacol. Exp. Ther. 230, 413. De Boer, S.F. and J. Van der Gugten, 1987, Daily variations in plasma noradrenaline, adrenaline and corticosterone concentrations in rats, Physiol. Behav. 40, 323. Fleish, J.H., H.M. Maling and B.B. Brodie, 1970, Beta receptor activity in aorta - variation with age and species, Circ. Res. 26, 151. Furchgott, R.F., 1983, Role of endothelium in responses of vascular smooth muscle, Circ. Res. 53, 557. Godfraind, T., 1983, Actions of nifedipine on calcium fluxes and contraction in isolated rat arteries, J. Pharmacol. Exp. Ther. 224, 443. Kadle, R. and G.E. Folk, 1983, Importance of circadian rhythms in animal cell cultures, Comp. Biochem. Physiol. 76A, 773. Kafka, M.S., A. Wirz-Justice, D. Naber, R.Y. Moore and M.A. Benedito, 1983, Circadian rhythms in rat brain neurotransmitter receptors, Fed. Proc. 42, 2796. Kasal, C. and J.R. Perez-Polo, 1982, Circadian rhythms in vitro, Trends Biochem. Sci. 7, 59.
Lemmer, B. and P.H. Lang, 1984, Circadian stage dependency in antagonist binding of 3H-dihydroalprenolol to rat heart ventricular membranes, Ann. Rev. Chronopharmacol. 1,321. Macia, R.A., W.D. Matthew, J. Lafferty and R.M. DeMarinis, 1984, Assessment of alpha-adrenergic receptor subtypes in isolated rat aortic segments, Naunyn-Schmiedeb. Arch. Microbiol. Immunol. 90, 123. Miller, R.C. and J.-C. Stoclet, 1985, Modulation by endothelium of contractile response in rat aorta in absence and presence of flunarizine, Br. J. Pharmacol. 86, 655. Patil, P.N., K. Fudge and D. Jacobowitz, 1972, Steric aspects of adrenergic drugs. VIII. a-adrenergic receptors and mammalian aorta, Eur. J. Pharmacol. 19, 79. Scarborough, N.L. and G.O. Carrier, 1984, Nifedipine and alphaadrenoceptors in rat aorta. I. Role of extracellular calcium in alpha 1 and alpha 2 adrenoceptor-mediated contraction, J. Pharmacol. Exp. Ther. 231,597. Suba, E.A. and B.L. Roth, 1987, Prostaglandins activate phosphoinositide metabolism in rat aorta, Eur. J. Pharmacol. 136, 325. Westerink, B.H.C. and W. Koolstra, 1986, Circadian variation of catecholamine excretion in rats: correlation with locomotor activity and effects of drugs, Neuropharmacology 25, 1255.
69
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52807
Ultradian variations in sensitivity of rat aorta rings to noradrenaline M i c h e l G o h a r , Pascal D a l e a u , J e f f r e y A t k i n s o n a a n d Y v e s - M i c h e l G a r g o u i l CEREP, B.P. 1, 86600 Celle l'Evescault, France and "Laboratoire de Pharmacologie Cardio-t~asculaire, 5 Rue Albert Lebrun, 54000 NanQ,, France
Received 14 October 1991, revised MS received 9 September 1992, accepted 22 September 1992
The in vitro sensitivity of the rat aorta to the vasoconstrictor effect of noradrenaline was found to vary according to the time the animals were killed, with a minimum at 10:00 h and a maximum at 16:00 h. This ultradian rhythm was not influenced by the presence of the endothelium but was modified by some, as yet unidentified, circulating factor(s). The results also strongly suggested an ultradian rhythm of in vitro sensitivity to the /3-adrenoceptor agonists, salbutamol and isoprenaline. This rhythm appears to be opposite to that for the sensitivity of aortic a-adrenoceptors to stimulation. In conclusion, our results demonstrate that the regulation of aortic tone follows an ultradian rhythm. Diurnal rhythm; Adrenoceptors; Noradrenaline; Aorta; (Rat)
1. Introduction Receptor binding studies show that /3-adrenoceptor density follows a diurnal rhythm in rat heart ventricles (Lemmer and Lang, 1984) and that both a- and /3adrenoceptor density in rat brain, follows a similar rhythm (Kafka et al., 1983). In order to investigate the possible existence and functional significance of such rhythms in blood vessels, we studied the diurnal changes in the in vitro vasoconstrictor responses of the rat aorta to noradrenaline. In aorta rings of the rat, noradrenaline induces contraction via stimulation of an adrenoceptor of the at-subtype (Cauvin and Malik, 1984; Macia et al., 1984; Alosachie and Godfraind, 1988). Thus diurnal variation in the in vitro vasoconstrictor responses may be interpreted as reflecting diurnal variabiality in a~-adrenoceptor affinity a n d / o r number, as the neuronal reuptake of noradrenaline presumably does not modify responses to exogenous noradrenaline in this sparsely innervated preparation. As the endothelium can modulate vasoconstrictor responses to noradrenaline (Miller and Stoclet, 1985), some experiments were performed with endotheliumdenuded aorta rings in order to investigate whether the diurnal variation in the in vitro vasoconstrictor responses to noradrenaline is due to a diurnal variation in vasorelaxant, endothelial activity. Finally, as nor-
Correspondence to: M. Gohar, Roussel-Uclaf, CRBA, Route de la Valentine, St. Marcel B.P. 1, 13367 Marseille Cedex 11, France. Tel. 33.91.35.90.35, fax 33.91.44.62.01.
adrenaline could stimulate both a 1- and /3-adrenoceptors in the rat aorta, we studied the diurnal changes in in vitro sensitivity of precontracted aorta rings to the /3-adrenoceptor agonists, isoprenaline and salbutamol. This provided us with insight into whether the diurnal variation in the in vitro vasoconstrictor responses to noradrenaline was due to diurnal variation in /3-adrenoceptor activity.
2. Materials and methods 2.1. A n i m a l s
Male Wistar rats (300-350 g, 5 month old, CERJ, France) were given food and water ad libitum and housed for at least 10 days at 20°C, under fixed lighting conditions (07:00 h to 19:00 h. 2.2. A o r t a ring preparation
Rats were killed by cervical dislocation. The thoracic aorta was removed and placed in a physiological salt solution (mM): NaCI 120, KCI 4.7, CaC12 1.8, N a H 2 P O 4 1.2, MgC12 1.2, N a H C O 3 25 and glucose 11, 37°C, pH 7.5, bubbled with 5% CO 2 and 95% 0 2. Eight rings were dissected from the same aortic segment, and in half of the rings the luminal surface was gently rubbed with a metal rod to remove the endothelium. Rings were mounted in 20-ml organ baths and contractions were measured with isometric transducers (CPOL25, Celaster, Poitiers, France). The rings were
70 allowed to equilibrate for 1 h and were then rinsed 4 times and stretched to a tension of 1.5 g. Drugs were then added to the organ bath. Concentrations are given as in terms of the base and cumulative dose-response curves were obtained by stepwise increases in the concentrations of the various drugs used. At the end of each experiment, acetylcholine (1 IxM) was added to the organ bath after the rings had been precontracted with noradrenaline in order to verify the presence or absence of the endothelium (Furchgott, 1983). Intact aorta rings that relaxed less than 20% were discarded.
N:22 8 8 I~ C~_
8
Variations in the in vitro sensitivity of the rat aorta to noradrenaline during the day were investigated in groups of rats (3-4) killed at different times between 08:30 and 18:30 h. In this protocol aorta rings with an intact endothelium were used. Variations in the in vitro sensitivity of the rat aorta to noradrenaline over a 2-day period were investigated in rats killed every 3 h in groups of 3 or 4 over a period of 48 h. Ex vivo changes in the in vitro sensitivity of the rat aorta to noradrenaline were investigated by killing the rats at 13:00 h (n = 2 rats). In half of the aorta rings prepared from any individual animal, the in vitro sensitivity to noradrenaline was determined immediately afterward the animals were killed, at 13:00 h. In the remaining 4 aorta rings the in vitro sensitivity to noradrenaline was determined at 16:00 h after storage at room temperature. The possible modulation of noradrenaline-induced contraction by /3-adrenoceptors was investigated in aorta rings prepared from two groups of rats (n = 4 rats per group) killed at 10:00 h and 16:00 h. Noradrenaline (0.03 txM) was added to the organ bath before and after incubation for 30 min with propranolol (1 txM). The diurnal rhythm of the in vitro sensitivity of /3-adrenoceptors was investigated in 2 groups of rats killed at 10:00 h and 16:00 h (n = 2 rats per group). Aorta rings were precontracted with PGF2~ (prostaglandin F2~) at a concentration of 2 /xM, which produces half-maximal contraction of this preparation (Suba and Roth, 1987), and then exposed to isoprenaline or salbutamol. In some experiments rings were incubated for 30 min with propranolol prior to the addition of PGF2~ followed by isoprenaline.
2.4. Analysis of results Results were stored on a computer and analyzed by using MOISE3 software (Celaster, Poitiers, France). All data are expressed as means + S.E.M. Statistical differences between means were determined with Student's t-test, pD 2 was calculated as -logl0(ECs0) after
~6 8
9
9.0
-,~ r---i
8.6
co (-.
8.4. 8.2-
c_ co c_ o Z
8.07.8. 7.67.4
9
1:1
13 Time
2.3. Specific protocols
11
9,2
:15 ( hours
17
19
)
Fig. 1. Sensitivity of rat aorta rings to noradrenaline (expressed as pD 2) during the day (from 10:00 h to 18:00 h). Values are expressed as means + S.E.M. N refer to the number of aorta rings.
least-squares linear regression analysis of transformed data.
2.5. Drugs Noradrenaline bitartrate, PGF2~ Tris salt, acetylcholine chloride, propranolol hydrochloride, salbutamol hemisulphate and isoprenaline hydrochloride were purchased from Sigma, St. Louis, Missouri, U.S.A.
3. Results
3.1. Variation in the in vitro sensitiuity of rat aorta rings to noradrenaline during the day A peak in sensitivity (pD 2 = 8.91 _+ 0.09, n = 8 rings) was observed at 16:00 h and a nadir (pD 2 = 7.60 _+ 0.12, n = 8 rings) at 10:30 h (fig. 1). The difference between these two values was highly significant (P < 0.001). Sensitivity was determined in the presence of the endothelium.
3.2. Variation in the in L'itro sensitivity of rat aorta rings to noradrenaline over a 2-day period During the first 24 h of the 2-day period, the diurnal rhythm of sensitivity to noradrenaline was similar to that described above, with a pD 2 = 7.86 _+ 0.05 (n = 15 rings) at 10:00 h (fig. 2). There was a pronounced leftward shift in the noradrenaline dose-response curve obtained at 16:00 h (pD z = 8.40 _+ 0.11, n = 9 rings, fig. 3) compared to that obtained at 10:00 h. The magnitude of the variation in pD z values was also statistically significant in the second 24-h period (16:00 h pD z = 8.23 _+ 0.10, n = 13 rings, versus 10:00 h pD 2 = 7.61 _+ 0.10, n = 7 rings, P < 0.005). Removal of the endothelium produced an overall increase in sensitivity but the ultradian rhythm remained.
71 n:
9
6
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12
17
15 19
8
8
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15
17 14
17
is
N: 15
7
9
20
15
i s 12
7
io
is
16
15 17
i5
i7
12
170 160
9.0
150 140 130
8.8
C o
8.6
o
120
8.4 8.2,
m
g
a. 100 ~g 90 ~ 807O
L 8.0 7.8 I
~
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7.6 7.4
-10 19
1
Time
7
i3
( hours
19
I
)
When rats were killed at 13:00 h and p D 2 values were determined immediately afterwards, a value of 7.34 _+ 0.04 (n = 8 rings) was found. The same series of rings tested at 16:00 h gave a pD 2 value of 7.16 _+ 0.02 (n = 8 rings). When a separate series of rings from the same animals were tested at 16:00 h a pD 2 value of 7.36 _+ 0.03 was found. Thus sensitivity to noradrenaline did not change after 3 h of storage at room temperature.
3.4. Modulation of noradrenaline-induced contractions by fl-adrenoceptors The presence of the fl-adrenoceptor antagonist propranolol did not modify the vasoconstrictor responses to noradrenaline. In rings tested at 10:00 h, responses to noradrenaline (30 nM) in the presence of propra-
E
iO0
80
-6
-5
Isoprenaline
-4 (M)
Fig. 4. C u m u l a t i v e i s o p r e n a l i n e d o s e - r e s p o n s e curves in intact aorta rings p r e p a r e d from rats killed at 10:00 h (circles, n = 13-8) and 16:00 h ( i n v e r t e d triangles, n = 1 0 - 8 ) and p r e c o n t r a c t e d with P G F 2 , (2 /xM). R e s u l t s are e x p r e s s e d as means_+S.E.M. ** P < 0 . 0 1 , *** P < 0.005.
nolol were 1 0 1 + 2% (n = 10 rings) of those in the absence of propranolol. At 16:00 h a value of 105 _+ 2% (n = 8 rings) was obtained.
3.5. Diurnal variations in the in vitro sensitivity of fladrenoceptors In intact aorta rings obtained from rats killed at 10:00 h and precontracted with PGF2~, lower concentrations of the /3-adrenoceptor agonist isoprenaline (0.1-1 /zM) caused relaxation whereas higher concentrations (1-100 /zM) caused contraction (fig. 4). In rings obtained from rats killed at 16:00 h, isoprenaline caused contractions only. Addition of the /3-adrenoceptor antagonist, propranolol, did not alter the responses obtained at 16:00 h but abolished the isoprenaline-induced responses obtained at 10:00 h (fig. 5). The flz-adrenoceptor agonist, salbutamol, caused a greater relaxant effect in aorta rings (precontracted with PGF2~) obtained from rats killed at 10:00 h than
g
~70
~
lg0
m
150
g
140
o
130 B0
120 m o
40
I00
x
E
-7
log
3.3. Ex vivo changes in the in vitro sensitivity of the rat aorta to noradrenaline
to
-8
7
Fig. 2. V a r i a t i o n s in n o r a d r e n a l i n e p D 2 in rat a o r t a rings over a 2-day period. I n t a c t a o r t a rings are r e p r e s e n t e d by s q u a r e s (N = n u m b e r of rings) a n d r u b b e d a o r t a rings by circles (n = n u m b e r of rings). R e s u l t s are e x p r e s s e d as m e a n s + S.E.M.
4J to (J
-9
. . . . . . . . . . . . . . . 13
=
** ***
***
90 -10
20
-9
-8 log
0
-10
-9 log
-8 Noradrenaline
-7
-6 (M)
Fig. 3. C u m u l a t i v e n o r a d r e n a l i n e d o s e - r e s p o n s e curves in intact a o r t a rings p r e p a r e d from rats k i l l e d at 10:00 h (circles, n = 15) a n d 16:00 h ( i n v e r t e d triangles, n = 9). ,4 p < 0.01, 44, p < 0.005.
-7
-6
Isoprenaline
-5
-4 (M)
Fig. 5. Effect of p r o p r a n o l o l (1 # M ) on r e s p o n s e s to i s o p r e n a l i n e in i n t a c t a o r t a rings p r e p a r e d from rats k i l l e d at 10:00 h (circles, n = 8) and 16:00 h ( i n v e r t e d triangles, n = 8) and p r e c o n t r a c t e d with P G F 2 , . C o n t r o l curves in the a b s e n c e of p r o p r a n o l o l are given in fig. 4. R e s u l t s are e x p r e s s e d as m e a n s + S . E . M . ** P < 0 . 0 1 , *** P < 0.005.
72
g
105
-~ 4-J
I00
•
CJ
m f_
c o u
95 90 85
u2 (-9
80
n
75 o
~
70, 65 -8
-7
-6
-5
-4
(M) Fig. 6. Cumulative salbutamol dose-response curves in intact aorta rings prepared from rats killed at 10:00 h (circles, n = 11) and at 16:00 h (inverted triangles, n = 8) and precontracted with PGF2~ (2 ~M). Results are expressed as means _+S.E.M. log
Salbutamol
in rings obtained at 16:00 h (fig. 6), but the differences were not significant. Salbutamol did not cause contraction.
4. D i s c u s s i o n
T h e p D 2 values for noradrenaline in the rat aorta r e p o r t e d in the literature vary from 6.7 to 8.5 (Godfraind, 1983; S c a r b o r o u g h and Carrier, 1984; Cauvin and Malik, 1984; Suba and Roth, 1987; Bevan et al., 1988; Alosachie and Godfraind, 1986; 1988). In these reports there is no indication of the time of day when the experiments were carried out, but it is possible that part of the reason why noradrenaline EDs0 values a p p e a r to vary some 100-fold is because the various authors did their experiments at different times of the day. W e f o u n d a slightly less than 100-fold change in EDs0 values when these were m e a s u r e d at 10:00 h and at 16:00 h. This change followed a bimodal diurnal rhythm, which could reflect an underlying ultradian rhythm with a 12-h periodicity. Endothelial factors do not apparently interfere with this rhythm as similar results were obtained in intact and d e n u d e d preparations. R e m o v a l of the e n d o t h e l i u m m o r e o v e r p r o d u c e d an overall increase in responses to noradrenaline, as has been already published (Alosachie and Godfraind, 1986; 1988; Miller and Stoclet, 1985). A l t h o u g h some preparations exhibit intrinsic circadian rhythms ex vivo (Kasal and Perez-Polo, 1982; Kadle and Folk, 1983), this does not a p p e a r to be the case for the aorta rings in our experiments. Thus the diurnal rhythm appears to d e p e n d on some in vivo factor. D e B o e r and Van G u g t e n (1987) have r e p o r t e d a diurnal variation in plasma corticosterone levels with a p e a k at the end of the light period. As corticosterone is an uptake-2 inhibitor, the diurnal rhythm in its
plasma concentration could cause a diurnal variation in the synaptic concentration of locally released noradrenaline. This would seem an unlikely explanation, however, as the aorta is poorly innervated in the rat (Patti et al., 1972; Burnstock, 1975) and its noradrenaline content is very low (Berkowitz et al., 1971). Thus the rate of uptake 2 is presumably low in the rat aorta. De Boer and Van G u g t e n (1987) also reported that circulating noradrenaline and adrenaline levels were higher at night than during the day. Furthermore, Westerink and Koolstra (1986) showed a bimodal rhythm in the urinary excretion of catecholamines. Thus, variations in plasma catecholamine concentration may modulate the sensitivity of the aorta to noradrenaline by receptor desensitization. As the variation in plasma catecholamines is limited, this is not a likely explanation. Experiments carried out with noradrenaline, which has low affinity for /3-adrenoceptors, and salbutamol and isoprenaline showed an opposite diurnal rhythm for /3-adrenoceptor sensitivity. However, it is unlikely that the diurnal rhythm of noradrenergic constrictor responses is caused by changes in the balance between a- and /3-adrenoceptor sensitivity. Moreover, blockade of /3-adrenoceptors had no effect. High doses of the non-selective /3-adrenoceptor agonist, isoprenaline, contracted the aorta, presumably via stimulation of a - a d r e n o c e p t o r s (Fleish et al., 1970). O n c e again, this a - a d r e n o c e p t o r - m e d i a t e d contraction revealed by high doses of isoprenaline was consistent with the previously found ultradian rhythm. In conclusion, we found that the sensitivity of rat aorta to noradrenaline showed an ultradian variation. A l t h o u g h further experiments are required to define the mechanism underlying this rhythm, we suggest that our results may explain the wide variation in p D 2 values for noradrenaline in aorta reported in the literature.
References
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73 Cauvin, C. and S. Malik, 1984, Induction of Ca ++ influx and intracellular Ca ++ release in isolated aorta and mesenteric resistance vessels by norepinephrine activation of alpha, receptors, J. Pharmacol. Exp. Ther. 230, 413. De Boer, S.F. and J. Van der Gugten, 1987, Daily variations in plasma noradrenaline, adrenaline and corticosterone concentrations in rats, Physiol. Behav. 40, 323. Fleish, J.H., H.M. Maling and B.B. Brodie, 1970, Beta receptor activity in aorta - variation with age and species, Circ. Res. 26, 151. Furchgott, R.F., 1983, Role of endothelium in responses of vascular smooth muscle, Circ. Res. 53, 557. Godfraind, T., 1983, Actions of nifedipine on calcium fluxes and contraction in isolated rat arteries, J. Pharmacol. Exp. Ther. 224, 443. Kadle, R. and G.E. Folk, 1983, Importance of circadian rhythms in animal cell cultures, Comp. Biochem. Physiol. 76A, 773. Kafka, M.S., A. Wirz-Justice, D. Naber, R.Y. Moore and M.A. Benedito, 1983, Circadian rhythms in rat brain neurotransmitter receptors, Fed. Proc. 42, 2796. Kasal, C. and J.R. Perez-Polo, 1982, Circadian rhythms in vitro, Trends Biochem. Sci. 7, 59.
Lemmer, B. and P.H. Lang, 1984, Circadian stage dependency in antagonist binding of 3H-dihydroalprenolol to rat heart ventricular membranes, Ann. Rev. Chronopharmacol. 1,321. Macia, R.A., W.D. Matthew, J. Lafferty and R.M. DeMarinis, 1984, Assessment of alpha-adrenergic receptor subtypes in isolated rat aortic segments, Naunyn-Schmiedeb. Arch. Microbiol. Immunol. 90, 123. Miller, R.C. and J.-C. Stoclet, 1985, Modulation by endothelium of contractile response in rat aorta in absence and presence of flunarizine, Br. J. Pharmacol. 86, 655. Patil, P.N., K. Fudge and D. Jacobowitz, 1972, Steric aspects of adrenergic drugs. VIII. a-adrenergic receptors and mammalian aorta, Eur. J. Pharmacol. 19, 79. Scarborough, N.L. and G.O. Carrier, 1984, Nifedipine and alphaadrenoceptors in rat aorta. I. Role of extracellular calcium in alpha 1 and alpha 2 adrenoceptor-mediated contraction, J. Pharmacol. Exp. Ther. 231,597. Suba, E.A. and B.L. Roth, 1987, Prostaglandins activate phosphoinositide metabolism in rat aorta, Eur. J. Pharmacol. 136, 325. Westerink, B.H.C. and W. Koolstra, 1986, Circadian variation of catecholamine excretion in rats: correlation with locomotor activity and effects of drugs, Neuropharmacology 25, 1255.
