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Effects of desipramine on cardiovascular responses of rats to stimulation of the baroreceptor reflex and of central adrenoceptors
NeuropharmacologyVol. 24, No. 9, pp. 839-844, 1985 Printed in Great Britain,
0028~3908/85 $3.00 + 0.00 Pergamon Press Ltd
EFFECTS OF DESIPRAMINE ON CARDIOVASCULAR RESPONSES OF RATS TO STIMULATION OF THE BARORECEPTOR REFLEX AND OF CENTRAL ADRENOCEPTORS S. POOLE* Department
of Pharmacology,
and J. D.
STEPHENSON
Institute of Psychiatry, DeCrespigny London SE5 8AF, England (Accepfed
15 January
Park,
Denmark
Hill,
1985)
Summary-Phenylephrine (0.42.0 pg 300 g- ‘), injected intravenously, evoked similar dose-dependent increases in blood pressure in untreated rats and in rats treated with desipramine (10 mg kg-’ day-’ for 4 weeks). The (dose-dependent) reflex fall in heart rate to the blood pressure responses were smaller in the rats treated with desipramine. Treatment with desipramine did not affect the bradycardia evoked by intrahypothalamic injection of phenylephrine (10 p(g). After treatment with desipramine, the hypotension evoked by intrahypothalamic injection of isoprenaline (IOpg) was enhanced whereas the evoked tachycardia was diminished. Key words: desipramine, central adrenoceptors, rats
Chronic
treatment
with
various
isoprenaline,
antidepressant
phenylephrine,
repeated
electroconvulsive
*Present address: Hormones
N.P. 24,%-B
baroreceptor
reflex,
METHODS
The rats used were male Wistars, received at 150-250 g, housed singly in plastic cages (335 x 210 x 170mm) at 23 f 1.5”C with water and food (Diet 4lB, Dixons Foods Ltd) ad libitum, and subject to a 12 hr light-dark cycle (lights on 06.00, off 18.00). Materials
Intra-arterial catheters were made from 100mm lengths of polyethylene tubing (Portex 800/100/160, i.d. 0.5 mm, o.d. 1.O mm, Portland Plastics Ltd) coated with dichloromethyl silane (DCMS: 2% in
Division, National Institute
for Biological Standards and Control, pstead, London NW3 6RB, England.
responses,
1960). The baroreceptor reflex includes among its central structures the anterior hypothalamus, electrical stimulation of which evoked a depressor response that was sensitive to r,-adrenoceptor blockade (Evans, 1980). Local injection of isoprenaline also produced cardiovascular changes that were blocked by B-adrenoceptor antagonists (Day and Roach, 1974). Therefore the effects of treatment with desipramine on cardiovascular responses to intrahypothalamic injections of phenylephrine and isoprenaline were also investigated. The dose of desipramine used was 10 mg kg-’ day-’ for 4 weeks since this dose schedule evoked a marked reduction in fi-adrenoceptors and in the activity of adenyl cyclase in the rat forebrain, whereas a dose of 2 mg kg-’ day-’ desipramine for 4 weeks did not affect these variables (Garcha, Smokcum, Stephenson and Weeramanthri, 1985).
drugs
shock has been reported to reduce the number of p-adrenoceptors in the CNS, the sensitivity of the noradrenaline-sensitive CAMP generating system and the responsiveness of single neurones to fi-adrenoceptor stimulation (see Charney, Menkes and Heninger, 1981 for references). Less consistent changes in cc,-adrenoceptors were reported after such (antidepressant) treatment: z,-adrenoceptor binding studies have yielded conflicting results (Rehavi, Ramot, Yavetz and Sokolovsky, 1980; Peroutka and Snyder, 1980) while electrophysiological studies have indicated enhanced responsivecess (Menkes, Aghajanian and McCall, 1980) and a behavioural model, no change (Heal, 1983). Although modification of adrenergic transmission is thought to be important to the mechanism of action of antidepressant drugs and of electroconvulsive shock (see Charney et al., 1981 for references), the net effects of such treatment on the functional states of adrenoceptors in uiuo is not clear. Rats were treated chronically with the antidepressant drug desipramine and functional changes at central adrenoceptors deduced from altered cardiovascular responses to activation of the baroreceptor reflex, a predominantly noradrenergic reflex (Bousquet and Schwartz, 1981), by systemic injection of phenylephrine (Varma, Johnsen, Sherman and Youmans, or
cardiovascular
Holly Hill, Ham-
839
840
S. PWLE and J. D.
heptane, Aldrich Chemical Co. Ltd) and filled with a solution of heparin (100 U ml-‘, Evans Medical Ltd) in pyrogen-free sterile saline (I 50 mM NaCl). Plugs for the cathethers were 8 mm lengths of 22 gauge stainless-steel wire. Intravenous catheters were made from 100 mm lengths of polyethylene tubing (Portex 8OO/lOO/lOO, id. 0.28 mm, o.d. 0.61 mm) filled with a solution of heparin (20 U ml-‘) in pyrogen-free sterile saline (150 mM NaCl).
Forty-five rats (275-325 g) were anaesthetized with halothane and a 23 gauge (o.d. 0.64 mm) stainlesssteel guide cannula with stylette was aseptically implanted into the left or right anterior hypothalamus. The stereotactic coordinates were 0.2 mm posterior to bregma, OSmm lateral to the sagittal suture and at a depth of 8.5 mm below the surface of the skull. After further growth to 325-350g (about 2 weeks) the rats were anaesthetized with halothane and. under aseptic conditions, a 30mm incision was made along the midline of the throat and upper thorax. An Intel-arterial catheter was inserted 25 mm into the right carotid artery so that its tip lay close to the junction of the carotid artery with the aorta; the catheter was secured with ligatures and the free end drawn subcutaneously to the skull. exteriorized immediately behind the existing implant and ciosed with a plug. The portion of the catheter immediately distal to the plug was attached with acrylic cement to the implant on the skull.
STEPHENSON
Twenty-six rats (weighing 320.-380 g) were prepared with catheters in the right carotid artery and left external jugular vein. The catheters were exteriorized at the nape of the neck, closed with plugs and secured with “purse-string” sutures.
Consciom ruts. Four hours after implantation of systemic catheters and 24 hr after ~jthdrawal from desipramine, the rats were placed singly in a perspex experimental chamber (335 x 290 x 280 mm) at 23 f 0.5 ‘C through which air flowed at 2 I min-‘. The intra-arterial catheter was connected via a length of polyethylene tubing (Portex 800/100/120, i.d. o.d. 1.09 mm) containing heparin 0.38 mm, (100 U ml -‘) in saline to a blood pressure transducer. For intrahypothalamic injections a 30 gauge (o.d. 0.3 mm) stainless-steel injection cannula was inserted so that its tip protruded 0.2 mm below the tip of the cannula. guide Polyethylene tubing (Portex 800~100~100, i.d.0.28 mm, o.d. 0.61 mm) connected the injection cannula to a 10 ~1 syringe. The volume of injection was 0.5 ~1 and the injections were made over 1 sec. For intravenous injections, polyethylene tubing (Portex 800/100/120, i.d. 0.38mm, o.d. 1.09 mm), connected the intravenous catheter to a 500 ~1 syringe. The volume of injection was 2&16Opl and the injections were made over 1 sec. Anue~~t~et~~e~ruts. Experiments were performed as in conscious animals except that rats were ana-
400 %
300
T&E x-
200 100
m
0 ~
111 1
2
3
4
5
6
7
8
Weeks Fig. I. The changes (mean + SEM, n = 13) in fluid intake, intake of drug and body weight of rats taking desipramine (DMI. 3-I 5 mg/ 100 ml, closed columns) in their drinking water compared to a control group drinking tap water (open columns).
DMI and cardiovascular
responses
841
26b
26b
I
lmm
Fig. 2. The intrahypothalamic injection sites of the 45 rats injected. Each symbol represents the injection site of one rat: the centres of the symbols represent the centres of the lesions made by the injection cannulae. (The size of the symbols do nof indicate the size of the lesions made or the spread of injectate.} The triangles represent rats pretreated with desipramine (IOmg kg-‘day-‘, n = 25) and the circles represent untreated animals (n = 20). III V = third ventricle, F = columna fornicis, AH = anterior hypothalamus, LH = lateral hypothalamus, FMP = fasciculus medialis prosencephali, TO = tractus opticus.
esthetized by intraperitoneal drate (100mg ml-‘, 0.4ml
injection of chloral lOOg-‘, BDH).
hy-
Drugs Concentrations of drugs are given in terms of the base, dissolved in pyrogen-free sterile saline (150 mM NaCI). Oral udmi~istr~t~o#. Desipramine hydrochloride (Ciba-Geigy Ltd) was dissolved in the drinking water. The concentration of drug was adjusted between 0.1 I and 0.56 mM (3-15 mg/lOO ml) to ensure a daily intake of 0.038 mmol kg-’ day-’ for 4 weeks prior to injections of amines (Fig. 1). ~~~frav~no~s injections. Pyrogen-free sterile saline (150 mM NaCl), L-phenylephrine hydrochloride (0.12 mM, BDH) or aeetylcholine chloride (3.42 mM, Sigma). Intrahypothalamic injections. Pyrogen-free sterile saline (150mM NaCl, pH 5.0; osmotic pressure 0.31 osmoi I-‘), L-phenylephrine hydrochloride (I 18.93 mM, pH 6; osmotic pressure 0.47 osmol 1-l) or isoprenaline hydrochloride (94.68 mM, pH 3.3; osmotic pressure 0.47 osmol 1-l). The positions of the cannulae were subsequently contirmed histologically and the data described below relate to rats with intracerebral guide cannulae tips in the anterior hypothalamus (Fig. 2). Results are expressed as the mean rfr 1 standard error of the mean (SEM) of the number of determinations given in the text. Carotid mean arterial blood pressure was calculated from the electricallydamped blood pressure trace. The undamped output
from the blood pressure transducer was fed into a Devices heart rate meter which displayed heart rate derived from the interpulse interval (equivalent to the R-R interval of the electrocardiogram). Changes in blood pressure and heart rate (HR) following intravenous or intrahypothalamic injection of saline were ir 10 beats min-’ reand typically f 5 mm Hg spectively. Significance of the differences between results was determined using Student’s t-test and regression analysis. RESULTS The intake of water and gain in weight of rats receiving desipramine in their drinking water were reduced in comparison with control groups (Fig. 1). Intake of fluid was reduced by about 30% in the rats drinking the 15 mg/ 100 ml desipramine solution required to ensure a daily intake of drug of 10 mg kg-‘. Upon te~ination of desipramine treatment, the implanted rats weighted 12% less and the nonimplanted rats 14% less than the corresponding control groups (P < 0.01). The mean arterial blood pressure of the rats treated with desipramine (10 mg kg-’ day-’ DMI orally for 4 weeks) was elevated when compared with that of age-matched untreated control rats (138 + 3 mmHg vs 123 f 3 mmHg, n = 13, P < 0.05). In contrast, the mean heart rates of the 2 groups of rats were similar (432 + 8 beats min-’ for the DMI-treated group versus 427 + 9 beats min- ’ for controls). Phenylephrine (PE, 0.4-2.0 pg 300 pg-‘, 2O&lOO ~1 of 118.93 mM)
S. PCKILE and J D.
842
shifted the slope of AHR/AMAP to the right as can be seen from Fig. 4. The slope of the regression line for AHR on AMAP was 1.41 in controls and the y-intercept 11.11; in rats treated with desipramine the slope of the regression line was similar, 1.25, but the -3.87. The mean value of y-intercept was AHR/AMAP for 1.2pg of phenylephrine was 1.91 + 0.26 in untreated rats compared with I .I6 +- 0.11 in animals treated with desipramine (P < 0.05). The values of AHR/AMAP for the other doses of phenylephrine were, in untreated versus rats treated with desipramine: 1.5 f 0.28 vs 1.11 f 0.15 (0.4pg); 2.OkO.3 vs 1.18kO.15 (0.8pg); 1.87kO.26 vs 1.43_+0.14(1.6~g)and2.18+0.32~~ 1.49_+0.21 (2.0 pg). Since regression analysis showed that these values of AHR/AMAP were independent of the dose of phenylephrine (Fig. 3c), the mean values of AHR/AMAP for all 5 doses of phenylephrine were calculated for untreated and for rats treated with desipramine and were 2.0 f 0.2 and 1.3 k 0.1, respectively (n = 60, P < 0.01). The subsequent intravenous injection of acetylcholine (ACh, 20-80 pg 300 gg’) in 5 rats from each of the above groups (untreated and DMI-treated), anaesthetized with chloral hydrate, evoked similar falls in mean arterial pressure in treated and in untreated rats (Table 1).
(a)
/I
IOO-
2
90 -
5
80 -
E \ In
3 al
70
E
60-
t ; L
50-
STEPHENSON
(b)
2 5r
Intrahypothalamic
I 04
I
0.8 Dose
PE
(pg/
I
,
12
16
I
20
3OOg-‘)
Fig. 3. (a) Increases in mean arterial blood pressure (MAP), (b) reflex falls in heart rate (HR) and (c) values for the ratio AHR/AMAP in untreated rats (circles) and in rats treated with desipramine (triangles; 1Omg kgg day-’ DMI orally for 4 weeks, n = I l-13) after intravenous injection of phenylephrine (PE. 0.42.0 pg 300 gg’). Vertical bars are SE means.
injected intravenously evoked dose-dependent increases in mean arterial pressure in untreated and in rats treated with desipramine although the increases were slightly larger in the animals treated with desipramine (Fig. 3a). The (dose-dependent) reflex falls in heart rate to the blood pressure increases were smaller in the rats treated with desipramine, the difference being most pronounced with doses in the middle of the dose range for phenylephrine, i.e. 0.8 and 1.2 pg (Fig. 3b). From a plot of the changes in blood pressure (AMAP) evoked by 1.2 pg of phenylephrine against reflex changes in heart rate (AHR, Fig. 4), it can be seen that the magnitude of AMAP determined the value of AHR. The correlation coefficient for AHR vs AMAP was 0.73 in controls and 0.55 in rats treated with desipramine. Pretreatment with desipramine
injections
In rats with hypothalamic guide cannulae, both mean arterial pressure and heart rate of animals treated with desipramine (10 mg kg-’ day-‘) were slightly elevated (P > 0.05) when compared with age-matched untreated controls. The mean arterial pressure of control rats was 112 f 3 mmHg (n = 20) vs 120 f 2 mmHg (n = 25) in animals treated with desipramine, the respective values for heart rate were 386 t_ I I and 424 k 7 beats min’. In untreated rats (n = 9) intrahypothalmic injection of isoprenaline
150
r
b 2
60
0
c
I 10
I 20 Increase
I 30 in MAP
I 40
I 50 (mm
I 60
Hg)
Fig. 4. Reflex fall in heart rate to increases in mean arterial pressure (MAP) evoked by phenylephrine (1.2pg 300 g-‘) in untreated rats (circles) and in rats treated with desipramine (triangles; IOmg kg-’ day-’ DMI orally for 4 weeks).
DMI
and cardiovascular
Table I. Fall (means f SEM. n = 5) in mean arterial blood pressure (-AMAP) evoked by intravenous injection of acetylcholine (ACh) in untreated rats and in rats pretreated with desipramine (IOmgkg-‘day-’ DMI orally for 4 weeks) Dose ACh (pg 300g-‘) 10 20 40 80
-AMAP (mmHg) in untreated rats 17+4 20 + 3 27 + 2 33 + 3
-AMAP (mmHg) in DMI-treated rats 17+4 17f I 2s + 2 34 + 2
-
i.e. 0.5 ~1 of 94.68 mM) reduced the mean arterial pressure by 26 + 6 to 91 f 4 mmHg and increased heart rate by 105f13 to 519 + 10 beatsmin’. In rats treated with desipramine (n = 11) the mean arterial pressure fell by 37 + 4 to 8 1 f 4 mmHg and heart rate increased by 72 rfr 10 to 512 k 7 beats min-’ following a similar injection. The more pronounced hypotension and diminished tachycardia in animals treated with desipramine were statistically significant (P < 0.02). Intrahypothalamic injection of phenylephrine (10 p g PE, i.e. 0.5 ~1 of 118.93 mM) reduced the heart rate by 55 f 8 beats min-’ but did not affect the mean arterial pressure in untreated rats (n = 11). In rats treated with desipramine (n = 14) phenylephrine similar evoked bradycardia (1Opg) (63 f 9 beats min’) butaincreased mean arterial pressure by 16 f 1 mmHg. (IOpg,
DISCUSSION
The reduced body weight of the rats treated with desipramine, compared with controls, 12-14x in the present study, was slightly less than the 16% reduction reported after 4 weeks of daily intraperitoneal injections of the same dose (10 mg kg-‘) of desipramine (Garcha et al., 1985) although different experimental protocols and different initial weights of the rats used in the two studies precluded detailed comparison of the two routes of administration. The similar pressor responses to intravenous injection of phenylephrine in untreated and in rats treated with desipramine (24 hr after withdrawal) indicates that chronic exposure to the antidepressant drug did not cause a functional change at peripheral cc,-adrenoceptors, despite the known blocking activity of desipramine at these receptors and its inhibition of re-uptake of noradrenaline. Likewise, the similar depressor responses to intravenous injection of acetylcholine in untreated and in rats treated with desipramine suggests that the antidepressant drug did not cause a functional change at peripheral muscarinic cholinoceptors. This finding confirms the weak nature of the cholinoceptor blocking activity of desipramine (Richelson, 198 l), an important consideration because the vagus nerve forms the efferent arc of the baroreceptor reflex. In contrast to the above, desipramine reduced the reflex bradycardia to intravenous injection of phenylephrine. Since antagonism of (muscarinic) cholinoceptors was excluded, this
responses
843
reduction was probably caused by inhibition within central elements of the baroreceptor reflex, although an effect of desipramine on sensory afferents was possible. The bradycardia evoked by intrahypothalamic injection of phenylephrine was unaffected by pretreatment with desipramine. Since injection of noradrenaline into the anterior hypothalamus of rats also evoked bradycardia, bradycardia that was antagonized by intrahypothalamic injection of phentolamine and therefore attributed to activation of r,-adrenoceptors (Struyker-Boudier, Smeets, Brouwer and Van Rossum, 1974; Poole, 1983), it is likely that chronic treatment with desipramine did not cause a functional change at these receptors. This conclusion agrees with the observations of Heal (1983) that chronic treatment with desipramine did not alter the cc,-adrenoceptor-mediated behavioural activity observed after intracerebroventricular injection of phenylephrine in mice. The lack of effect of intrahypothalamic injections of phenylephrine on the blood pressure of control rats was evidence against any leakage of the injection into the systemic circulation and eliminates this as a cause of the small but significant rise in blood pressure consistently evoked by phenylephrine in rats treated with desipramine. An explanation that hypothalamic cc,-adrenoceptors became more sensitive or increased in number after treatment with desipramine is inconsistent with the unchanged bradycardia and with the expectation that stimulation of hypothalamic cc,-adrenoceptors would lead to a vasodepressor response as described in cats (Nashold, Mannarino and Wunderlich, 1962; Share and Melville, 1963; Gagnon and Melville, 1966; Day and Roach, 1974) and in dogs (McCubbin, Kaneko and Page, 1960; Bhargava, Mishra and Tangri, 1972). A possible explanation for the hypertensive response is that it was due to release by phenylephrine of the antidiuretic hormone (ADH, Hoffman, 1979) attenuation of the baroreceptor reflex by desipramine permitting the emergence of a pressor response to ADH. The effects of pretreatment with desipramine on cardiovascular responses to intrahypothalamic injection of isoprenaline were complex. After chronic treatment with desipramine, hypotension evoked by isoprenaline was more pronounced whereas the tachycardia was diminished. Tachycardia is the characteristic fi-adrenoceptor response to the central injection of isoprenaline in several species (Toda, Matsuda and Schimamoto, 1969; Kimura, Share, Wang and Crofton, 1981), it being accompanied by either hypertension or hypotension (Day and Roach, 1974). It is therefore tempting to attribute the reduced tachycardia to down-regulation of /I-adrenoceptors. However, because pre-injection values for blood pressure and heart rate were greater in rats treated with desipramine, the absolute values for blood pressure and heart rate after intrahypothalamic injection of isoprenaline were similar in untreated and desipramine-treated rats.
S. POOLEand J. D. STEPHENSON
844
In summary, while providing evidence that central elements of the baroreceptor reflex, in which noradrenergic mechanisms predominate, were inhibited after chronic treatment with desipramine, the present study has not demonstrated a functional change at either cr,-adrenoceptors or /I-adrenoceptors within the CNS after treatment with desipramine.
vasopressin release and blood pressure. Endocrino/ogy 198: 182991836. MacCubbin J. W.. Kaneko Y. and Page 1. H. (1960) Ability of serotonin and norepinephrine to mimic the central effects of reserpine on vasomotor activity. Circulation Res. 8: 849-858.
Menkes J. B., Aghajanian G. K. and McCall R. B. (1980) Chronic antidepressant treatment enhances rl-adrenergic and serotonergic responses in the facial nucleus. Life Sci. 27: 45-55.
Nashold B. S., Mannarino E. and Wunderlich M. (1962) Presser-depressor blood pressure responses in the cat after intraventricular injection of drugs. Nature, Lond.
REFERENCES Bhargava K. P., Mishra N. and Tangri K. K. (1972) An analysis of central adrenoceptors for control of cardiovascular function. Br. J. Pharmac. 45: 596-602. Bousquet P. and Schwartz J. (198 1) Alpha-adrenergic drugs. Pharmacological tools for the study of the central vasomotor control. Biochem. Pharmac. 32: 1459-1465. Charney D. S., Menkes D. B. and Heninger G. R. (1981) Receptor sensitivity and the mechanism of action of antidepressant. Archs gen. Psychiat. 38: 116&l 180. Day M. D. and Roach A. G. (1974) Central I- and /I-adrenoceptors modifying arterial blood pressure and heart rate in conscious cats. Br. J. Pharmac. 51: 325-333. Evans M. H. (1980) Vasoactive sites in the diencephalon of the rabbit. Brain Res. 183: 329-340. Gagnon D. J. and Melville K. L. (1966) Further observations on the possible role of norddrenahne in centrally mediated cardiovascular responses. Rev. Canad. Biol. 25: 99-105.
Garcha G., Smokcum R. W. J., Stephenson J. D. S. and Weeramanthri T. B. (1985) Effects of some atypical antidepressants on b-adrenoceptor binding and adenylate cyclase activity in the rat forebrain. Eur. J. Pharmac. In press. Heal D. J. (1983) Phenylephrine-induced activity in mice as a model of /I,-adrenoceptor function: studies of various antidepressant treatments. Br. J. Pharmac. 79: 372P. Hoffman W. E. (1979) Central cardiovascular and antidiuretic action of adrenergic drugs. Neuropharmucolog) 18: 7-12. Kimura T., Share L., Wang B. C. and Crofton J. T. (1981) The
role
of central
adrenoceptors
in the
control
of
193: 1297-1298. Peroutka S. J. and Synder S. H. (1980) Chronic antidepressant treatment decreases spiroperidol-labelled serotonin receptor binding. Science~210~ 88-90. Poole S. (1983) Cardiovascular resnonses of rats to intrahypothalamic injection of carbachol and noradrenaline. Br. J. Pharmac. 79: 693-700. Rehavi M., Ramot O., Yavetz B. and Sokolovsky M. (1980) Amitriptyline: long term treatment elevates a-adrenergic and muscarinic receptor binding in mouse brain. Brain Res. 194: 443-453. Richelson E. (1981) Tricyclic antidepressants: Interactions with histamine and muscarinic acetylcholine receptors. In: Anlidepressants: Neurochemical, Behauioural and Clinical Perspectioes (Nenna S. J., Malick J. B. and Richelson E.. Eds), pp. 53-73. Raven Press, New York. Share N. N. and Melville K. I. (1963) Centrally mediated sympathetic cardiovascular responses induced by intraventricular norepinephrine. J. Pharmac. exp. Ther. 141: 15-21.
Struyker-Boudier H. A. J., Smeets G. W. M., Brouwer G. M. and Van Rossum J. M. (1974) Hypothalamic alpha adrenergic receptors in cardiovascular regulation. Neuropharmacology 13: 837-846. Toda N., Matsudsa Y. and Shimamoto K. (1969) Cardiovascular effects of sympathomimetic amines injected into the cerebral ventricles of rabbits. Int. J. Neuropharmac. 8: 451461. Varma S., Johnsen S. D., Sherman D. E. and Youmans W. B. (1960) Mechanisms of inhibition of heart rate by phenylephrine. Circulation Res. 8: 1182-l 186.
0028~3908/85 $3.00 + 0.00 Pergamon Press Ltd
EFFECTS OF DESIPRAMINE ON CARDIOVASCULAR RESPONSES OF RATS TO STIMULATION OF THE BARORECEPTOR REFLEX AND OF CENTRAL ADRENOCEPTORS S. POOLE* Department
of Pharmacology,
and J. D.
STEPHENSON
Institute of Psychiatry, DeCrespigny London SE5 8AF, England (Accepfed
15 January
Park,
Denmark
Hill,
1985)
Summary-Phenylephrine (0.42.0 pg 300 g- ‘), injected intravenously, evoked similar dose-dependent increases in blood pressure in untreated rats and in rats treated with desipramine (10 mg kg-’ day-’ for 4 weeks). The (dose-dependent) reflex fall in heart rate to the blood pressure responses were smaller in the rats treated with desipramine. Treatment with desipramine did not affect the bradycardia evoked by intrahypothalamic injection of phenylephrine (10 p(g). After treatment with desipramine, the hypotension evoked by intrahypothalamic injection of isoprenaline (IOpg) was enhanced whereas the evoked tachycardia was diminished. Key words: desipramine, central adrenoceptors, rats
Chronic
treatment
with
various
isoprenaline,
antidepressant
phenylephrine,
repeated
electroconvulsive
*Present address: Hormones
N.P. 24,%-B
baroreceptor
reflex,
METHODS
The rats used were male Wistars, received at 150-250 g, housed singly in plastic cages (335 x 210 x 170mm) at 23 f 1.5”C with water and food (Diet 4lB, Dixons Foods Ltd) ad libitum, and subject to a 12 hr light-dark cycle (lights on 06.00, off 18.00). Materials
Intra-arterial catheters were made from 100mm lengths of polyethylene tubing (Portex 800/100/160, i.d. 0.5 mm, o.d. 1.O mm, Portland Plastics Ltd) coated with dichloromethyl silane (DCMS: 2% in
Division, National Institute
for Biological Standards and Control, pstead, London NW3 6RB, England.
responses,
1960). The baroreceptor reflex includes among its central structures the anterior hypothalamus, electrical stimulation of which evoked a depressor response that was sensitive to r,-adrenoceptor blockade (Evans, 1980). Local injection of isoprenaline also produced cardiovascular changes that were blocked by B-adrenoceptor antagonists (Day and Roach, 1974). Therefore the effects of treatment with desipramine on cardiovascular responses to intrahypothalamic injections of phenylephrine and isoprenaline were also investigated. The dose of desipramine used was 10 mg kg-’ day-’ for 4 weeks since this dose schedule evoked a marked reduction in fi-adrenoceptors and in the activity of adenyl cyclase in the rat forebrain, whereas a dose of 2 mg kg-’ day-’ desipramine for 4 weeks did not affect these variables (Garcha, Smokcum, Stephenson and Weeramanthri, 1985).
drugs
shock has been reported to reduce the number of p-adrenoceptors in the CNS, the sensitivity of the noradrenaline-sensitive CAMP generating system and the responsiveness of single neurones to fi-adrenoceptor stimulation (see Charney, Menkes and Heninger, 1981 for references). Less consistent changes in cc,-adrenoceptors were reported after such (antidepressant) treatment: z,-adrenoceptor binding studies have yielded conflicting results (Rehavi, Ramot, Yavetz and Sokolovsky, 1980; Peroutka and Snyder, 1980) while electrophysiological studies have indicated enhanced responsivecess (Menkes, Aghajanian and McCall, 1980) and a behavioural model, no change (Heal, 1983). Although modification of adrenergic transmission is thought to be important to the mechanism of action of antidepressant drugs and of electroconvulsive shock (see Charney et al., 1981 for references), the net effects of such treatment on the functional states of adrenoceptors in uiuo is not clear. Rats were treated chronically with the antidepressant drug desipramine and functional changes at central adrenoceptors deduced from altered cardiovascular responses to activation of the baroreceptor reflex, a predominantly noradrenergic reflex (Bousquet and Schwartz, 1981), by systemic injection of phenylephrine (Varma, Johnsen, Sherman and Youmans, or
cardiovascular
Holly Hill, Ham-
839
840
S. PWLE and J. D.
heptane, Aldrich Chemical Co. Ltd) and filled with a solution of heparin (100 U ml-‘, Evans Medical Ltd) in pyrogen-free sterile saline (I 50 mM NaCl). Plugs for the cathethers were 8 mm lengths of 22 gauge stainless-steel wire. Intravenous catheters were made from 100 mm lengths of polyethylene tubing (Portex 8OO/lOO/lOO, id. 0.28 mm, o.d. 0.61 mm) filled with a solution of heparin (20 U ml-‘) in pyrogen-free sterile saline (150 mM NaCl).
Forty-five rats (275-325 g) were anaesthetized with halothane and a 23 gauge (o.d. 0.64 mm) stainlesssteel guide cannula with stylette was aseptically implanted into the left or right anterior hypothalamus. The stereotactic coordinates were 0.2 mm posterior to bregma, OSmm lateral to the sagittal suture and at a depth of 8.5 mm below the surface of the skull. After further growth to 325-350g (about 2 weeks) the rats were anaesthetized with halothane and. under aseptic conditions, a 30mm incision was made along the midline of the throat and upper thorax. An Intel-arterial catheter was inserted 25 mm into the right carotid artery so that its tip lay close to the junction of the carotid artery with the aorta; the catheter was secured with ligatures and the free end drawn subcutaneously to the skull. exteriorized immediately behind the existing implant and ciosed with a plug. The portion of the catheter immediately distal to the plug was attached with acrylic cement to the implant on the skull.
STEPHENSON
Twenty-six rats (weighing 320.-380 g) were prepared with catheters in the right carotid artery and left external jugular vein. The catheters were exteriorized at the nape of the neck, closed with plugs and secured with “purse-string” sutures.
Consciom ruts. Four hours after implantation of systemic catheters and 24 hr after ~jthdrawal from desipramine, the rats were placed singly in a perspex experimental chamber (335 x 290 x 280 mm) at 23 f 0.5 ‘C through which air flowed at 2 I min-‘. The intra-arterial catheter was connected via a length of polyethylene tubing (Portex 800/100/120, i.d. o.d. 1.09 mm) containing heparin 0.38 mm, (100 U ml -‘) in saline to a blood pressure transducer. For intrahypothalamic injections a 30 gauge (o.d. 0.3 mm) stainless-steel injection cannula was inserted so that its tip protruded 0.2 mm below the tip of the cannula. guide Polyethylene tubing (Portex 800~100~100, i.d.0.28 mm, o.d. 0.61 mm) connected the injection cannula to a 10 ~1 syringe. The volume of injection was 0.5 ~1 and the injections were made over 1 sec. For intravenous injections, polyethylene tubing (Portex 800/100/120, i.d. 0.38mm, o.d. 1.09 mm), connected the intravenous catheter to a 500 ~1 syringe. The volume of injection was 2&16Opl and the injections were made over 1 sec. Anue~~t~et~~e~ruts. Experiments were performed as in conscious animals except that rats were ana-
400 %
300
T&E x-
200 100
m
0 ~
111 1
2
3
4
5
6
7
8
Weeks Fig. I. The changes (mean + SEM, n = 13) in fluid intake, intake of drug and body weight of rats taking desipramine (DMI. 3-I 5 mg/ 100 ml, closed columns) in their drinking water compared to a control group drinking tap water (open columns).
DMI and cardiovascular
responses
841
26b
26b
I
lmm
Fig. 2. The intrahypothalamic injection sites of the 45 rats injected. Each symbol represents the injection site of one rat: the centres of the symbols represent the centres of the lesions made by the injection cannulae. (The size of the symbols do nof indicate the size of the lesions made or the spread of injectate.} The triangles represent rats pretreated with desipramine (IOmg kg-‘day-‘, n = 25) and the circles represent untreated animals (n = 20). III V = third ventricle, F = columna fornicis, AH = anterior hypothalamus, LH = lateral hypothalamus, FMP = fasciculus medialis prosencephali, TO = tractus opticus.
esthetized by intraperitoneal drate (100mg ml-‘, 0.4ml
injection of chloral lOOg-‘, BDH).
hy-
Drugs Concentrations of drugs are given in terms of the base, dissolved in pyrogen-free sterile saline (150 mM NaCI). Oral udmi~istr~t~o#. Desipramine hydrochloride (Ciba-Geigy Ltd) was dissolved in the drinking water. The concentration of drug was adjusted between 0.1 I and 0.56 mM (3-15 mg/lOO ml) to ensure a daily intake of 0.038 mmol kg-’ day-’ for 4 weeks prior to injections of amines (Fig. 1). ~~~frav~no~s injections. Pyrogen-free sterile saline (150 mM NaCl), L-phenylephrine hydrochloride (0.12 mM, BDH) or aeetylcholine chloride (3.42 mM, Sigma). Intrahypothalamic injections. Pyrogen-free sterile saline (150mM NaCl, pH 5.0; osmotic pressure 0.31 osmoi I-‘), L-phenylephrine hydrochloride (I 18.93 mM, pH 6; osmotic pressure 0.47 osmol 1-l) or isoprenaline hydrochloride (94.68 mM, pH 3.3; osmotic pressure 0.47 osmol 1-l). The positions of the cannulae were subsequently contirmed histologically and the data described below relate to rats with intracerebral guide cannulae tips in the anterior hypothalamus (Fig. 2). Results are expressed as the mean rfr 1 standard error of the mean (SEM) of the number of determinations given in the text. Carotid mean arterial blood pressure was calculated from the electricallydamped blood pressure trace. The undamped output
from the blood pressure transducer was fed into a Devices heart rate meter which displayed heart rate derived from the interpulse interval (equivalent to the R-R interval of the electrocardiogram). Changes in blood pressure and heart rate (HR) following intravenous or intrahypothalamic injection of saline were ir 10 beats min-’ reand typically f 5 mm Hg spectively. Significance of the differences between results was determined using Student’s t-test and regression analysis. RESULTS The intake of water and gain in weight of rats receiving desipramine in their drinking water were reduced in comparison with control groups (Fig. 1). Intake of fluid was reduced by about 30% in the rats drinking the 15 mg/ 100 ml desipramine solution required to ensure a daily intake of drug of 10 mg kg-‘. Upon te~ination of desipramine treatment, the implanted rats weighted 12% less and the nonimplanted rats 14% less than the corresponding control groups (P < 0.01). The mean arterial blood pressure of the rats treated with desipramine (10 mg kg-’ day-’ DMI orally for 4 weeks) was elevated when compared with that of age-matched untreated control rats (138 + 3 mmHg vs 123 f 3 mmHg, n = 13, P < 0.05). In contrast, the mean heart rates of the 2 groups of rats were similar (432 + 8 beats min-’ for the DMI-treated group versus 427 + 9 beats min- ’ for controls). Phenylephrine (PE, 0.4-2.0 pg 300 pg-‘, 2O&lOO ~1 of 118.93 mM)
S. PCKILE and J D.
842
shifted the slope of AHR/AMAP to the right as can be seen from Fig. 4. The slope of the regression line for AHR on AMAP was 1.41 in controls and the y-intercept 11.11; in rats treated with desipramine the slope of the regression line was similar, 1.25, but the -3.87. The mean value of y-intercept was AHR/AMAP for 1.2pg of phenylephrine was 1.91 + 0.26 in untreated rats compared with I .I6 +- 0.11 in animals treated with desipramine (P < 0.05). The values of AHR/AMAP for the other doses of phenylephrine were, in untreated versus rats treated with desipramine: 1.5 f 0.28 vs 1.11 f 0.15 (0.4pg); 2.OkO.3 vs 1.18kO.15 (0.8pg); 1.87kO.26 vs 1.43_+0.14(1.6~g)and2.18+0.32~~ 1.49_+0.21 (2.0 pg). Since regression analysis showed that these values of AHR/AMAP were independent of the dose of phenylephrine (Fig. 3c), the mean values of AHR/AMAP for all 5 doses of phenylephrine were calculated for untreated and for rats treated with desipramine and were 2.0 f 0.2 and 1.3 k 0.1, respectively (n = 60, P < 0.01). The subsequent intravenous injection of acetylcholine (ACh, 20-80 pg 300 gg’) in 5 rats from each of the above groups (untreated and DMI-treated), anaesthetized with chloral hydrate, evoked similar falls in mean arterial pressure in treated and in untreated rats (Table 1).
(a)
/I
IOO-
2
90 -
5
80 -
E \ In
3 al
70
E
60-
t ; L
50-
STEPHENSON
(b)
2 5r
Intrahypothalamic
I 04
I
0.8 Dose
PE
(pg/
I
,
12
16
I
20
3OOg-‘)
Fig. 3. (a) Increases in mean arterial blood pressure (MAP), (b) reflex falls in heart rate (HR) and (c) values for the ratio AHR/AMAP in untreated rats (circles) and in rats treated with desipramine (triangles; 1Omg kgg day-’ DMI orally for 4 weeks, n = I l-13) after intravenous injection of phenylephrine (PE. 0.42.0 pg 300 gg’). Vertical bars are SE means.
injected intravenously evoked dose-dependent increases in mean arterial pressure in untreated and in rats treated with desipramine although the increases were slightly larger in the animals treated with desipramine (Fig. 3a). The (dose-dependent) reflex falls in heart rate to the blood pressure increases were smaller in the rats treated with desipramine, the difference being most pronounced with doses in the middle of the dose range for phenylephrine, i.e. 0.8 and 1.2 pg (Fig. 3b). From a plot of the changes in blood pressure (AMAP) evoked by 1.2 pg of phenylephrine against reflex changes in heart rate (AHR, Fig. 4), it can be seen that the magnitude of AMAP determined the value of AHR. The correlation coefficient for AHR vs AMAP was 0.73 in controls and 0.55 in rats treated with desipramine. Pretreatment with desipramine
injections
In rats with hypothalamic guide cannulae, both mean arterial pressure and heart rate of animals treated with desipramine (10 mg kg-’ day-‘) were slightly elevated (P > 0.05) when compared with age-matched untreated controls. The mean arterial pressure of control rats was 112 f 3 mmHg (n = 20) vs 120 f 2 mmHg (n = 25) in animals treated with desipramine, the respective values for heart rate were 386 t_ I I and 424 k 7 beats min’. In untreated rats (n = 9) intrahypothalmic injection of isoprenaline
150
r
b 2
60
0
c
I 10
I 20 Increase
I 30 in MAP
I 40
I 50 (mm
I 60
Hg)
Fig. 4. Reflex fall in heart rate to increases in mean arterial pressure (MAP) evoked by phenylephrine (1.2pg 300 g-‘) in untreated rats (circles) and in rats treated with desipramine (triangles; IOmg kg-’ day-’ DMI orally for 4 weeks).
DMI
and cardiovascular
Table I. Fall (means f SEM. n = 5) in mean arterial blood pressure (-AMAP) evoked by intravenous injection of acetylcholine (ACh) in untreated rats and in rats pretreated with desipramine (IOmgkg-‘day-’ DMI orally for 4 weeks) Dose ACh (pg 300g-‘) 10 20 40 80
-AMAP (mmHg) in untreated rats 17+4 20 + 3 27 + 2 33 + 3
-AMAP (mmHg) in DMI-treated rats 17+4 17f I 2s + 2 34 + 2
-
i.e. 0.5 ~1 of 94.68 mM) reduced the mean arterial pressure by 26 + 6 to 91 f 4 mmHg and increased heart rate by 105f13 to 519 + 10 beatsmin’. In rats treated with desipramine (n = 11) the mean arterial pressure fell by 37 + 4 to 8 1 f 4 mmHg and heart rate increased by 72 rfr 10 to 512 k 7 beats min-’ following a similar injection. The more pronounced hypotension and diminished tachycardia in animals treated with desipramine were statistically significant (P < 0.02). Intrahypothalamic injection of phenylephrine (10 p g PE, i.e. 0.5 ~1 of 118.93 mM) reduced the heart rate by 55 f 8 beats min-’ but did not affect the mean arterial pressure in untreated rats (n = 11). In rats treated with desipramine (n = 14) phenylephrine similar evoked bradycardia (1Opg) (63 f 9 beats min’) butaincreased mean arterial pressure by 16 f 1 mmHg. (IOpg,
DISCUSSION
The reduced body weight of the rats treated with desipramine, compared with controls, 12-14x in the present study, was slightly less than the 16% reduction reported after 4 weeks of daily intraperitoneal injections of the same dose (10 mg kg-‘) of desipramine (Garcha et al., 1985) although different experimental protocols and different initial weights of the rats used in the two studies precluded detailed comparison of the two routes of administration. The similar pressor responses to intravenous injection of phenylephrine in untreated and in rats treated with desipramine (24 hr after withdrawal) indicates that chronic exposure to the antidepressant drug did not cause a functional change at peripheral cc,-adrenoceptors, despite the known blocking activity of desipramine at these receptors and its inhibition of re-uptake of noradrenaline. Likewise, the similar depressor responses to intravenous injection of acetylcholine in untreated and in rats treated with desipramine suggests that the antidepressant drug did not cause a functional change at peripheral muscarinic cholinoceptors. This finding confirms the weak nature of the cholinoceptor blocking activity of desipramine (Richelson, 198 l), an important consideration because the vagus nerve forms the efferent arc of the baroreceptor reflex. In contrast to the above, desipramine reduced the reflex bradycardia to intravenous injection of phenylephrine. Since antagonism of (muscarinic) cholinoceptors was excluded, this
responses
843
reduction was probably caused by inhibition within central elements of the baroreceptor reflex, although an effect of desipramine on sensory afferents was possible. The bradycardia evoked by intrahypothalamic injection of phenylephrine was unaffected by pretreatment with desipramine. Since injection of noradrenaline into the anterior hypothalamus of rats also evoked bradycardia, bradycardia that was antagonized by intrahypothalamic injection of phentolamine and therefore attributed to activation of r,-adrenoceptors (Struyker-Boudier, Smeets, Brouwer and Van Rossum, 1974; Poole, 1983), it is likely that chronic treatment with desipramine did not cause a functional change at these receptors. This conclusion agrees with the observations of Heal (1983) that chronic treatment with desipramine did not alter the cc,-adrenoceptor-mediated behavioural activity observed after intracerebroventricular injection of phenylephrine in mice. The lack of effect of intrahypothalamic injections of phenylephrine on the blood pressure of control rats was evidence against any leakage of the injection into the systemic circulation and eliminates this as a cause of the small but significant rise in blood pressure consistently evoked by phenylephrine in rats treated with desipramine. An explanation that hypothalamic cc,-adrenoceptors became more sensitive or increased in number after treatment with desipramine is inconsistent with the unchanged bradycardia and with the expectation that stimulation of hypothalamic cc,-adrenoceptors would lead to a vasodepressor response as described in cats (Nashold, Mannarino and Wunderlich, 1962; Share and Melville, 1963; Gagnon and Melville, 1966; Day and Roach, 1974) and in dogs (McCubbin, Kaneko and Page, 1960; Bhargava, Mishra and Tangri, 1972). A possible explanation for the hypertensive response is that it was due to release by phenylephrine of the antidiuretic hormone (ADH, Hoffman, 1979) attenuation of the baroreceptor reflex by desipramine permitting the emergence of a pressor response to ADH. The effects of pretreatment with desipramine on cardiovascular responses to intrahypothalamic injection of isoprenaline were complex. After chronic treatment with desipramine, hypotension evoked by isoprenaline was more pronounced whereas the tachycardia was diminished. Tachycardia is the characteristic fi-adrenoceptor response to the central injection of isoprenaline in several species (Toda, Matsuda and Schimamoto, 1969; Kimura, Share, Wang and Crofton, 1981), it being accompanied by either hypertension or hypotension (Day and Roach, 1974). It is therefore tempting to attribute the reduced tachycardia to down-regulation of /I-adrenoceptors. However, because pre-injection values for blood pressure and heart rate were greater in rats treated with desipramine, the absolute values for blood pressure and heart rate after intrahypothalamic injection of isoprenaline were similar in untreated and desipramine-treated rats.
S. POOLEand J. D. STEPHENSON
844
In summary, while providing evidence that central elements of the baroreceptor reflex, in which noradrenergic mechanisms predominate, were inhibited after chronic treatment with desipramine, the present study has not demonstrated a functional change at either cr,-adrenoceptors or /I-adrenoceptors within the CNS after treatment with desipramine.
vasopressin release and blood pressure. Endocrino/ogy 198: 182991836. MacCubbin J. W.. Kaneko Y. and Page 1. H. (1960) Ability of serotonin and norepinephrine to mimic the central effects of reserpine on vasomotor activity. Circulation Res. 8: 849-858.
Menkes J. B., Aghajanian G. K. and McCall R. B. (1980) Chronic antidepressant treatment enhances rl-adrenergic and serotonergic responses in the facial nucleus. Life Sci. 27: 45-55.
Nashold B. S., Mannarino E. and Wunderlich M. (1962) Presser-depressor blood pressure responses in the cat after intraventricular injection of drugs. Nature, Lond.
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Garcha G., Smokcum R. W. J., Stephenson J. D. S. and Weeramanthri T. B. (1985) Effects of some atypical antidepressants on b-adrenoceptor binding and adenylate cyclase activity in the rat forebrain. Eur. J. Pharmac. In press. Heal D. J. (1983) Phenylephrine-induced activity in mice as a model of /I,-adrenoceptor function: studies of various antidepressant treatments. Br. J. Pharmac. 79: 372P. Hoffman W. E. (1979) Central cardiovascular and antidiuretic action of adrenergic drugs. Neuropharmucolog) 18: 7-12. Kimura T., Share L., Wang B. C. and Crofton J. T. (1981) The
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in the
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193: 1297-1298. Peroutka S. J. and Synder S. H. (1980) Chronic antidepressant treatment decreases spiroperidol-labelled serotonin receptor binding. Science~210~ 88-90. Poole S. (1983) Cardiovascular resnonses of rats to intrahypothalamic injection of carbachol and noradrenaline. Br. J. Pharmac. 79: 693-700. Rehavi M., Ramot O., Yavetz B. and Sokolovsky M. (1980) Amitriptyline: long term treatment elevates a-adrenergic and muscarinic receptor binding in mouse brain. Brain Res. 194: 443-453. Richelson E. (1981) Tricyclic antidepressants: Interactions with histamine and muscarinic acetylcholine receptors. In: Anlidepressants: Neurochemical, Behauioural and Clinical Perspectioes (Nenna S. J., Malick J. B. and Richelson E.. Eds), pp. 53-73. Raven Press, New York. Share N. N. and Melville K. I. (1963) Centrally mediated sympathetic cardiovascular responses induced by intraventricular norepinephrine. J. Pharmac. exp. Ther. 141: 15-21.
Struyker-Boudier H. A. J., Smeets G. W. M., Brouwer G. M. and Van Rossum J. M. (1974) Hypothalamic alpha adrenergic receptors in cardiovascular regulation. Neuropharmacology 13: 837-846. Toda N., Matsudsa Y. and Shimamoto K. (1969) Cardiovascular effects of sympathomimetic amines injected into the cerebral ventricles of rabbits. Int. J. Neuropharmac. 8: 451461. Varma S., Johnsen S. D., Sherman D. E. and Youmans W. B. (1960) Mechanisms of inhibition of heart rate by phenylephrine. Circulation Res. 8: 1182-l 186.