α-Adrenoceptor responsiveness in the aged rat

European Journal of Pharmacology, 126 (1986) 75-80

75

Elsevier

a - A D R E N O C E P T O R R E S P O N S I V E N E S S IN T H E A G E D R A T JAMES R. DOCHERTY * and LISA HYLAND Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, Dublin 2, Ireland

Received 30 January 1986, revised MS received 18 March 1986, accepted 22 April 1986

J.R. DOCHERTY and L. HYLAND, a-Adrenoceptor responsiveness in the aged rat, European J. Pharmacol. 126 (1986) 75-80. The effects of ageing on vascular a 1- and a2-adrenoceptors were examined using anaesthetised and pithed young (3-7 months) and old (21-24 months) Sprague-Dawley rats. In pithed animals, the pressor and cardioinhibitory effects of the az-adrenoceptor agonist xylazine were significantly reduced in old animals (8- and 6-fold shift), but the pressor effects of the aa-adrenoceptor agonist amidephrine were not significantly altered by ageing. In anaesthetised rats, the pressor response to the mixed a-adrenoceptor agonist noradrenaline (NA) was not significantly altered, and the pressor potency of the al-adrenoceptor agonist amidephrine was only slightly reduced (1.7-fold shift) in old animals. In the presence of cocaine (1 mg kg-1) the pressor potency of NA was markedly reduced (13-fold shift) in old animals. In the presence of prazosin (1 mg kg-1) to eliminate al-adrenoceptor-mediated responses, the pressor potency of NA was markedly reduced in old as compared to young animals (16-fold shift). The neuronal uptake blocker cocaine (1 mg kg-1) significantly potentiated the pressor response to NA only in young. In summary, we have found a reduced a2-adrenoceptor-mediated pressor and cardioinhibitory responsiveness and a reduced neuronal uptake of NA in old animals with little change in aa-adrenoceptor-mediated vascular responsiveness. Vascular smooth muscle

al-Adrenoceptors

az-Adrenoceptors

I. Introduction There are a n u m b e r of conflicting reports of the effects of ageing on a-adrenoceptor-mediated vasoconstriction, showing decreased (Tuttle, 1966; Elliott et al., 1982) or unchanged (Stevens and Moulds, 1981; Scott and Reid, 1982; Buhler et al., 1980) responsiveness with increasing age (see D o c h e r t y and O'Malley, 1985). We have recently found that the az-adrenoceptor antagonist yohimbine, but not the al-adrenoceptor antagonist prazosin, was less potent with increasing age at inhibiting the stimulation-evoked isometric contraction in the h u m a n isolated saphenous vein ( H y l a n d and Docherty, 1985a). However, since a m-adrenoceptor-mediated contractions are of minor importance in the h u m a n saphenous vein (Docherty and Hyland, 1985a), we wished to ob* To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.

Cocaine

Ageing

tain further evidence of differential effects of ageing on a 1- and a2-adrenoceptor-mediated responses in a preparation in which both types of receptor were important. We chose to examine anaesthetised and pithed rats, since the rat vasculature contains both a t- and a2-adrenoceptors (Docherty et al., 1979). Some of these results have been published in abstract form (Docherty and Hyland, 1985b).

2. Materials and methods Old male Sprague-Dawley rats (21-24 months, 500-700 g) and y o u n g adult rats (5-7 months, 450-600 g; or 3 months, 250 g) were employed. 2.1. Anaesthetised rats

Animals were anaesthetised with pentobarbitone sodium, a tracheal cannula was placed and

76 animals were respired artificially with 100% 0 2 . Ventilation with 100% 02 was required to maintain old rats. Animals were heavily anaesthetised following intubation to suppress spontaneous breathing. The carotid artery was cannulated for blood pressure and heart rate recording, and the jugular vein was cannulated for injection of drugs.

(-)-noradrenaline bitartrate (Sigma); prazosin hydrochloride (gift: Pfizer); xylazine hydrochloride (gift: Bayer). Stock solutions of drugs were dissolved in saline (NaC1 0.9% w / v ) except for prazosin (distilled water) and dilutions were made up in saline. All stocks were made up just before administration.

2.2. Pithed rats

2.5. Statistics

Following anaesthesia (as described above) animals were pithed by the method of Gillespie et al. (1970). The pithing rod was used as an electrode to stimulate the cardioaccelerator nerves with a single stimulus every 2 min (supramaximal voltage, 0.5 ms).

Values are means + S.E. of mean or geometric mean and 95% confidence limits. Potency of drugs was expressed as an EDs0 value (dose producing 50% of maximum response) or an IDs0 value (dose producing 50% inhibition of the cardioacceleration to a single electrical stimulus), where appropriate. In pithed rats, the pressor potency of xylazine was expressed as an ED45 (dose producing a rise in DBP of 45 mm Hg, approximately 50% of the maximum pressor response to xylazine) and the pressor potency of amidephrine was expressed as an ED60 (dose producing a rise in DBP of 60 mm Hg, approximately 50% of the maximum pressor response to amidephrine). Differences between groups were assessed using an unpaired Student's t-test.

2. 3. Drug administration

Drugs were administered cumulatively in 0.5 log unit (noradrenaline (NA)) or 1 log unit (xylazine, amidephrine) increments at approximately 5 min intervals. In pithed rats, a2-adrenoceptor responsiveness was assessed as the inhibition by xylazine of the cardioacceleration to a single stimulus (presynaptic potency) or the pressor response to xylazine (postsynaptic potency), and al-adrenoceptor responsiveness was assessed as the pressor response to amidephrine. In anaesthetised rats, al-adrenoceptor responsiveness was assessed as the pressor response to amidephrine but, since selective a2-adrenoceptor agonists like xylazine have an ability to lower diastolic blood pressure (DBP) in anaesthetised rats by a central action, peripheral a2-adrenoceptor responsiveness was assessed as the pressor potency of NA in the presence of prazosin (1 mg kg -1) to eliminate al-adrenoceptor-mediated effects. In some experiments, the effect of the neuronal uptake blocker cocaine (1 mg kg -~) were assessed against the pressor response to NA. In these experiments, responses were obtained twice to N A (0.1, 0.3 and 1 /~g kg -1), and responses were repeated in the presence of cocaine or saline. 2.4. Drugs

( - ) - A m i d e p h r i n e hydrochloride (gift: Bristol Myers); cocaine hydrochloride (Cockburns);

3. Results 3.1. Pithed rats

In pithed rats, resting heart rate (HR) was 298 + 8 min -1 (n = 19) and 277 + 12 min -1 (n = 11) and resting DBP was 35.7 + 1.4 mm Hg (n = 19) and 27.0 + 1.2 mm Hg (n = 11) in young (3 and 6 months combined) and old, respectively (DBP was significantly lower in old rats; Student's t-test, P < 0.01). Xylazine was significantly more potent in young than in old rats at producing a pressor response, whereas amidephrine had similar potency in old and young rats (fig. 1 and table 1). Xylazine was significantly more potent in young than in old at producing an inhibition of the cardioacceleration to a single electrical stimulus given via the pithing rod (fig. 2 and table 1). The shift in potency of xylazine between young and old was similar to that found for the potency of

77 a

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Fig. 1. Pressor responses of xylazine (filled symbols) and amidephrine (unfilled symbols) in 3 months (*, zx), 6 months (11, D) and 24 months old (e, O) pithed rats. Vertical bars indicate S.E. of mean from at least 4 experiments.

xylazine at inhibiting the contraction of the epididymal portion of the rat vas deferens to a single stimulus (fig. 2, see Hyland and Docherty, 1985b). 3.2. Anaesthetised rats

In anaesthetised rats, resting H R was 420 _+ 10 min - I (n = 19) and 381 + 6 min - t ( n = 29) and resting DBP was 120.4 ___4.6 m m Hg (n = 19) and

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Fig. 2. Inhibition by xylazine of (a) the isometric contraction of epididymal portions of rat vas deferens to a single electrical stimulus in the presence of nifedipine (10 ttM), and (b) the cardioacceleration to a single stimulus in pithed rats. Symbols: young (6 months) ((3); old (24 months) (e). Vertical bars represent S.E. of mean from at least 4 experiments. Data for (a) taken from Hyland and Docherty (1985b).

98.1 + 5.6 m m Hg (n = 29) in young (6 months) and old, respectively (both H R and DBP were significantly lower in old rats; Student's t-test, P < 0.01). N A (0.1-1 /~g kg -1) produced pressor responses in young and old animals, but there was no significant difference between groups in the response to N A (fig. 3). The %-adrenoceptor agonist amidephrine produced pressor responses

TABLE 1 EDs0 values obtained in anaesthetised rats for NA in the presence of cocaine (1 mg kg-]), amidephrine, and NA in the presence of prazosin (1 mg kg -1) and ED45, ED6o or IDs0 values for xylazine and amidephrine in pithed rats. Values are geometric mean (p.g kg -1) and 95% confidence limits from at least 4 experiments. The dose ratio was obtained by dividing the mean value obtained in old animals by that obtained in young animals. Asterisks denote values obtained in old animals significantly different from those obtained in young (Student's t-test: a p < 0.05; b p < 0.01; c p < 0.001). ns indicates no significant shift in potency. 3 months (/~g kg -1)

6 months (~g kg -1)

24 months (/~g kg - l )

Dose ratio

A naesthetised

NA (cocaine) pressor ED5o Amidephrine pressor EDso NA (prazosin) pressor EDs0

0.12 (0.05-0.31) 4.40 (3.04--6.56) 0.61 (0.19--0.87)

1.55 b (0.33-7.24) 7.58 b (5.62--10.2) 10.0 c (5.01-20.0)

178 (120-263) 1.32 (0.28-6.30) 41.7 (26.9-64.6)

1 380 a (190-10000) 7.35 a (3.98-13.8) 63.1 (36.3-109)

12.6 1.7 16.4

Pithed

Xylazine pressor ED45 Xylazine cardioinhib. IDso Amidephrine pressor ED60

138 (69.2-275)

55.0 (22.9-132)

10/7.8 5.6 ns

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Fig. 3. Effects of N A and amidephrine on the DBP of the anaesthetised rat. Symbols: N A ( O , @); N A in the presence of prazosin (1 mg kg -1) (zx, A); amidephrine (O, B). Open symbols represent responses obtained in young animals (6 months) and filled symbols represent responses obtained in old animals (24 months). Vertical bars represent S.E. of mean from at least 4 experiments.

in young and old rats which did not differ in maximum effect (fig. 3), but there was a small but significant reduction of potency in old animals. The EDs0 value of amidephrine was significantly lower in young than in old animals, but the shift in potency was small (table 1). Prazosin (1 mg kg-1) lowered DBP to 47.6 + 5.4 mm Hg (n = 6) in young and 22.0 _+ 2.2 mm Hg

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Fig. 4. Potentiation by cocaine (1 mg kg -1) of the pressor response to NA (0.1-1 ~g kg - l ) in 6 months (unfilled symbols) and 24 months (filled symbols) anaesthetised rats. Effects of cocaine are shown in (a) and effects of saline vehicle in (b). Symbols: initial response (squares); response after cocaine or saline (circles). Vertical bars represent S.E. of mean from at least 4 experiments. Asterisks denote significance of difference between responses to N A in absence and presence of cocaine (Student's t-test for paired data: * P < 0.05).

(n -= 6) in old animals. In the presence of prazosin to eliminate al-adrenoceptor-mediated effects, NA was significantly more potent in young than in old animals (see fig. 3 and table 1). Note that, in young animals, NA is apparently more potent in the presence than in the absence of prazosin: this is at least partly due to the very low DBP in the presence of prazosin. Cocaine (1 mg kg -1) lowered DBP by 12.5 + 4.0 mm Hg (n = 5) in 6 months and by 9.0 + 8.2 mm Hg (n = 4) in 24 months old rats (measured 5 min after cocaine injection). There was no significant difference between young and old in the effects of cocaine on resting DBP. Cocaine (1 mg kg -1) significantly potentiated the pressor response to NA (0.1-1 #g kg -1) in young but not in old animals (fig. 4a). Saline vehicle had no significant effect on the pressor response to NA (fig. 4b). The maximum response to NA in the presence of cocaine was obtained (data not shown) and from this the pressor potency of NA was calculated: NA was significantly more potent in young than in old animals in the presence of cocaine (table 1).

4. Discussion

In this investigation of age-related changes in vascular a-adrenoceptor-mediated responsiveness in anaesthetised and pithed rats, we have obtained 3 main findings: a2-adrenoceptor-mediated vasoconstriction is reduced in the aged rat; aa-adrenoceptor-mediated vasoconstriction is unchanged in the aged rat; the neuronal uptake process for NA is reduced in the aged rat. These points are discussed below. We have recently reported that the potency of the az-adrenoceptor antagonist yohimbine at inhibiting stimulation-evoked contractions in the human saphenous vein declines with increasing age, but that there is no such decline in the potency of the al-adrenoceptor antagonist prazosin (Hyland and Docherty, 1985a). However, since the predominant receptor-mediating stimulationevoked contractions in the human saphenous vein is a2 (Docherty and Hyland, 1985a), we wished to investigate whether this selective loss of ch-adrenoceptors could be demonstrated in a preparation

79

in which a t- and ct2-adrenoceptors had roughly equal importance. We chose the rat vasculature in which both a 1- and a2-adrenoceptors are present and mediate vasoconstriction (Dew and Whiting, 1979; Docherty et al., 1979; Docherty and McGrath, 1980). Due to a high failure rate in pithing old rats, only a small number of results were obtained for xylazine and amidephrine in pithed rats. Xylazine and amidephrine are selective agonists at a 2- and al-adrenoceptors, respectively (see Docherty, 1984). The pressor potency of xylazine, but not of amidephrine, was significantly reduced in old pithed rats. Xylazine lowered DBP in anaesthetised rats by a central action and could not be used to examine vascular a2-adrenoceptor responsiveness in that preparation. However, the pithed rat, lacking central or baroreflex complications, is a more suitable preparation to examine peripheral vascular responses. In anaesthetised rats, there was only a very limited loss of potency for the cq-adrenoceptor agonist amidephrine in aged rats (1.7-fold shift), and no loss of potency for NA. However, in the presence of cocaine (1 mg kg -1) to eliminate neuronal uptake of NA, the potency of NA was significantly reduced in old (13-fold shift) and in the presence of prazosin (to eliminate al-adrenoceptor-mediated effects) the potency of NA was significantly reduced in old as compared with young animals (16-fold shift). The evidence is in favour of a major loss of vascular a2-adrenoceptor-mediated responsiveness with ageing in the rat: potency of xylazine (pithed rat) and of NA (in the presence of prazosin in the anaesthetised rat) markedly reduced with almost no change in the potency of amidephrine (pithed and anaesthetised rats). Hence, a selective loss of c~2-adrenoceptor responsiveness occurs with ageing in the rat vasculature (present data) and human saphenous vein (Hyland and Docherty, 1985a) with virtually no change in a~-adrenoceptor responsiveness in rat vasculature (present results) and rat vas deferens (Docherty and O'Malley, 1983). We have previously reported that the presynaptic inhibitory potency of xylazine is significantly reduced in the vas deferens of aged rats (Docherty and O'Malley, 1983; Hyland and Docherty,

1985b). We now have additional data that the same is true for the potency of xylazine at inhibiting the electrically evoked cardioacceleration in the pithed rat. This may be due to a loss of c~2-adrenoceptors on peripheral nerve terminals with ageing. While NA is a suitable peripherally acting agonist at both cq- and a2-adrenoceptors, it is taken up from the circulation by the neuronal amine uptake process. This is especially important since it has been reported that the neuronal uptake blocker cocaine (Trendelenburg, 1966) is more effective at potentiating the response to N A in isolated hearts from old than from young rats (Kreider et al., 1984). However, not only were we unable to demonstrate an increased importance of neuronal uptake in restricting pressor responses in aged rats, we were even unable to find any importance of the uptake process in aged rats, at least in terms of pressor responses to NA (admittedly, we did not measure neuronal uptake directly). A reduced removal of NA by uptake may explain why, despite reduced ct2-adrenoceptor-mediated responsiveness, the overall responsiveness to NA (in the absence of cocaine) is not reduced in old animals (present results). In conclusion, we have found evidence for a reduced responsiveness of vascular and neuronal a2-adrenoceptors, without change in the responsiveness of vascular al-adrenoceptors in aged rats. Neuronal uptake of NA is reduced in the vasculature of aged rats and this may partly counteract losses in receptor-mediated responsiveness.

Acknowledgements This work was supported by the Royal College of Surgeons in Ireland and by the Medical Research Council of Ireland. L.H. is an MRC(1) scholar.

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