Stereoconfiguration requirement for central activity of isoproterenol

Life Scíeacas Vol . 17, pp .

Printed in the II .S .A .

Pergamon Press

583-590

STEREOCONFIGURATION REQUIREMENT FOR CENTRAL ACTIVITY OF ISOPROTERENOL Gad Shtacher* and Lawrence Klevans Hoffmann-La Roche,

Inc ., Nutley, N . J . 07110

(Received in final form July 14, 1975) Summary The affect of intracerebroventricular (i .c .v .) administration of d and dl-tsoproterenol on heart rate and blood pressure was studied In chloralosa anesthetized vagotomt :ed cats . Both the racemic and dextrorotatory forms produced an immediate dose-dependent fall to diastolic blood pressure and an increase in heart rate . These changes were abolished by pretreatment with a ganglionic blocking agent administered peripherally . The cardiovascular response exhibited a marked degree of stereospecificity ; the isomeric activity ratios (equlpotent dose of d/dl tsoproterenol) for the heart rata and blood pressure responses were 32 and 28 . Systemic administration of d and dl-tsoproterenol yielded isomeric activity ratios on the order of two log units . These data favor the existence of central stereospectftc beta -adrenergic receptors that modify cardiovascular function . Tha existents of central alpha - and beta -adrenocaptors that control heart rate and blood pressure have been postulated by a number of investigators (1,2,3,4) . The concept of central alpha -receptors influencing the cardiovascular system is generally accepted ; however, evidence for the existence of central beta adrenoceptors Is still fragmentary and somewhat conflicting (5) . Although central beta -receptors have been characterized in terms of the cardiovascular response produced by tntracerebroventricularly (Icv) administered tsoproterenol, the results have been variable . Isoproterenol has been shown to decrease systemic arterial blood pressure and increase heart rate (1,2,6,7,8), decrease blood pressure and heart rate (9) . or increase blood pressure and heart raise (10) . This variability tends to challenge the existence of central beta -receptors . Therefore, to the present study, we tested for specific beta reeeptors which modify w rdlovascular function . To this end, the Isoprotarenol Isomeric activity ratio (equipotent dose of d/dl tsoproterenol) served as the maJor criterlan for establishing beta -receptor mediated mechanisms to the brain . Methods Cats of either sex weighing 2 .Sí0 .5 k9 were anesthetized with alpha -chloralose (65 mg/kg t .p .), plus urethane (200 mg/kg IV) . Body temperature was *Present address :

Tel-Aviv University, Sackler School of Medicine, Department of Physiology and Pharmacology, Ramat-Avtv, Israel .

Reprint requests :

Dr . L . R . Klevans Hoffmann-La Roche Nutley, N . J . 07110 583

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maintained at 37 °C with a heating lamp . Bilateral midcervical transectton of the vague nerves was performed to each animal . A femoral artery was cannulated for recording blood pressure, and a femoral vein for administering drugs. The animals were placed to a stereotaxic apparatus and the left lateral cerebral ventricle was calculated with 24-gauge stainless steel tubing at coordinates anterior 13 mm, lateral 3 "5 mn . Penetration of the cannula into the ventricle was wrried out according to the method of Goodrich et al . (11) . Drugs were dissolved in normal saline containing 5 mg $ ascorbic acid, adJusted to pH 7 .4, and infected in volumes of 0.1-0 .2 cc over a 30-second period . The dura overlying the spinal cord was pierced between L7 and S1 vertebrae to avoid intraventrlcular pressure build-up following repeated Icv infections . Blood pressure and heart rate were recorded in each experiment . Femoral arterial pressure was measured with a Statham P23 AA pressure transducer and displayed on a Beckman Dynograph . Heart rate was recorded continuously with the aid of a cardtotachometer which was triggered by lead II ECG . Three to five doses of d and dl-Isoproterenol were infected Icv In 10 animals . Equipotent doses of the two forms were used to calculate the tsameric activity Several doses of d and dl-tsoproterenol were also infected IV in six ratios . of these 10 animals, and an isomeric activity ratio was calculated for this route of administration . The following drugs were used : dl-Isoproterenol hydrochloride (Wtnthrop Laboratories, New York, N .Y .), d-Isoproterenol d-bitartrate (Starling-Wtnthrop Research Institute, Rensselanr, N .Y .) and hexamethonium bromide (Pfaltz and Bauer, Flushing, N .Y .) . Results Icv administration of dl-Isoproterenol produced an immediate decrease in diastolic blood pressure and increase in heart rate . The response lasted 515 minutes and was blocked by systemically administered hexamethonium (5 mg/ kg, IV) (Fig . 1) . Although both d and dl-Isoproterenol were active, a considerable difference in potency existed between the two forms . An equipotent concentration of d-isoproterenol was 28 times greater than the racemtc mixture ; that ts, the isomeric activity ratio was 28 (Table I) . Stnee the sensitivity to centrally administered Isoproterenol varied from cat to cat, dose schedules were adJusted for each animal so as to elicit a threshold and maximal response . The standard errors of the concentrations shown in Table I under tntracerebroventricular reflect this variability in dose regimens . Although a fixed dose schedule was not used, threshold concentrations were significantly less than maximal concentrations because a dose-response relationship was established for each animal (except for d-Isoproterenol) . In flue of ten experiments d-Tsoproterenol was Inactive, even at concentrations that approached the solubility limits of the drug . These experiments were excluded from the calculation of the isaneric activity ratio . Intravenous administration of d and dl-isoproterenol produced the typical fall in diastolic blood pressure . Equipotent concentrations differed by a factor of 341 (Table I) . Intravenously administered dl-Isoproterenol was at least 30 times more potent by thn IV as compared to the Icv route and, as a result, the isomeric activity ratios differed by one log unit .

Vol. 17, No . 4

Central Activity of Isoproterenol

; p~~: : =.~rR~~ n~: ;~

" =s~-r°_

d~~~,~

BP . . .-r.~,p ~

y!Y

. ~: :~.~ -- ~~~~+ :

HR

585

y~'~~~~~~

e~~~~

=x?~~il~i~il~~i~® ;~~~~~I~~l~l~ibl1~

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FIG . 1 Effect of hexamathonium bromide on the change to blood pressure (BP) and heart rate (HR) produced by Intracerebroventricularly (Icv) administered dl-Isoproterenol . Panel A :

Control ; InJectlon of 50 ug Isoproterenol at marker ; bottom line is one minute t(mer .

Panel B :

Ten minutas after hexamethonium (5 mg/kg, IV) ; first panel, 3 ug tsoproterenol IV ; second panel, 500 pg Isoproterenol Icv; third panel, 1000 yg Isoproterenol Icv .

The increase to heart rate produced by tsoproterenol administered Icv or IV followed the same profile as the blood pressure response (Table II) . dlIsoproterenol administered centrally or systemically was more potent than the d form ; the corresponding Isomeric actlvtty ratios were 32 and 436 . In addition, the isomeric actlvtty ratio was approximately one log unit greater for systemically, as compared to centrally administered Isoproterenol . Discussion Results from the present study demonstrate stereospecificity In the central action of tsoproterenol, and therefore strengthens previous indications for the existence of central beta -adrenerglc receptors that modify cardiovascular function . Since beta-agonists Interact optimally with their receptors in only one possible configuration [R, .L], a high isomeric actlvtty ratio serves as one of the maJor indices identifying a steraospecific or direct interaction with The high isomeric activity ratios for the cardiovascular beta -adrenoceptors . changes produced by centrally administered d and dl-isoproterenol suggests that such a direct interaction between beta -agonist and receptor occurred . Furthermore, the data suggest that the Interaction occurred at sites controlling heart rate and blood pressure, because their isomeric actlvtty ratios ware similar . It should be emphasized, however, that the ratios are only estimates, because they were not calculated from complete dose-response curves, but rather from two data points on the curve (see Tables I, II) . Data for

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Central Activity of Ieoproterenol

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TABLE 1 Effect of d and dl-Ieoprotereaol Administered Iatracarebroventricularly and Intravenously on Diastolic Blood Pressure INTRACEBEBROVENTRICIJI~AR

N

dl-Isoproterenol Response Dosa (Y~ (~ Ha)_

N

d-Isoprotereaol Response Doae (uI~ (mm Hg) .49 t 0 .11

- 6 t 1

4.43 3 0 .67*

-13 t 2*

10 0 .16 ± 0 .04 1

-12 ± 1

5a

10

-19 t 1**

5

0 .35 t 0.08**

Isomeric Activity Ratio (d/dl) :

4.43/ .16 = 28

INTRAVENOUS

N

dl-Isoproterenol Reaponae Doae (uiS) (mm Hg)

d-Isoprotereaol Response - N - Dose (ut~ (mm HgL

6

.0043 t .001

-15 ± 2

6

0.51 ± 0 .18

-10 ± 1

6

.0113 ± .002**

-27 t 2**

6

1 .47 # 0 .29**

-19 t 2**

Isomeric Activity Ratio (d/dl) :

1.47/4 .3 a 10-3 ~ 341

aIn 5 of 10 ezperimeata, the doses between 1.49 and 4 .43 1ùS did not produce a response and therefore were not included in the calculation . *p <0 .05 **p <0 .01; higher dose and response significantly greater than lower. N - number of experiments . Values are mean changea from control levels (vehicle injection) with standard errors of mesas .

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587

Central Activity of Iaoprotereaol

TA87,S II 8ffect of d and dl-Isoproteranol Administered Iatracarebroveatricularly and Iatravenousl~ on Heart Rate INTRACSRESGOVSIiTRICIIhAR

N

dl-Isonrotareaol Response Dose (1~-~ _Sb/mint

d-Ieovroterenol - N -

Dose (u?fL

Gesponse (b/minim

9

0.11 t 0 .04

+13 t 2

58

1 .21 t 0.37

+5 ± 1

9

0 .29 t 0.08**

+21 f 4*

5

3.60 t 0 .82*

+10 ± 2*

Isomeric Activity Gatio (d/dl) :

3 .60/ .11 ~ 32

INTRAVGNOIIS

N

dl-Ieoprotareaol Gesponse _Dose (1~ _~)

d-Isoproteranol N

Dose (~

6

.0038 t .001

+10 t 3

6

0.66 t 0 .17

6

.0113 t .002**

+19 t 4*

6

1.66 t 0 .27**

Isomeric Activity Ratio (d/dl) :

Gesponse (b/mint

~+ 5 t 1 +10 t 1**

1.66/3.84 z 10-3 = 436

aIn 4 of 9 ezperimeate, the doses batweea 1.49 and 4 .42 uI3 did not produce a response and therefore ware sot included in the calculation . *p <0 .05 **p <0 .01; higher dose and response significantly greater rhea lover . N ~ number of azperiments . Values are mesa changes from control levels (vehicle injection) with standard errors of means.

588

Central Activity of Ieoproterenol

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complete dose-response curves could not be obtained for two reasons : fixed doses could not be used because the sensitivity of the animals to d and dlisoproterenol varied widely ; and responses to d-isoproterenol often could not be elicited because the necessary concentrations often exceeded the solubility limits of the drug . Nevertheless, the 32 and 28-fold difference In potency between the d and dl forms for heart rate and blood pressure responses suggests the existence of stereospecificity . Furthermore, since racemic dl-isoproterenol has only about one-half the potency of the .~ enantlomer (12), our data underestimate the Isomeric activity ratio, thus making the point of stereospecifictty even more convincing . Tha Isomeric activity ratios for heart rate and blood pressure were also determined for intravenously administered isoproterenol and compared to centrally administered drug . The IV ratios were approximately 10 times greater than the Icv ratios . This difference occurred because the racemic form was at least 30 times more active by the IV compared to the Icv route . Such a dependence of an isomeric activity ratio on the more active isomer has previously been reported (13) . Isomeric activity ratios 2 log units in magnitude for systemically administered d and .C-isoproterenol have been reported by other Investigators (12,14) . The lower potency of dl-isoproteranol infected Icv could be the result either of inadequate distribution of the drug to those brain areas that control heart rate and blood pressure or a differential sensitivity between direct activation of target organ receptors via systemic administration and indirect activation via neuronal pathways . Alternatively, these data may also be explained by structural differences between central and peripheral beta receptors . The results reported in the present study are to agreement with some of the earlier observations (1,2,6,7,8) on the mode of action of centrally (Icv) infected dl-isoproterenol : the drug consistently caused a fall In diastolic blood pressure and an increase In heart rate . The purely central nature of the response was deduced from the observations that the wrdiovascular change occurred immediately following central administration of isoproterenol, and the response was abolished following intravenous administration of hexamethonium . These criteria have been used by other investigators to show that the cardiovascular response produced by Icv Isoproterenol is not due to leakage into the periphery (1,15) " There are no studies we are aware of on the mode of action of centrally administered d-tsoproterenol . References 1. 2.

D. J . GAGNON and K. I . MELVILLE : Int . J. Neuropharmacol . 6 :245-251 (1967) . K. P . BHARGAVA, N . MISHRA, AND K. K . TANGRI : Brit . J . Pharmacol . x:596602 (1972) " 3. N. E . ANDEN, H . CORRODI, K. FUXE, B . HOKFELT, T . HOKFELT, T. RYDIN, and T. SVE.NSSON : Life Sci . x:513-523 (1970) . 4. H . SCHMITT, H . SCHMITT, and S . FENARD : Europ . J . Pharmacol . _14:98-100 (1971) . M. D . DAY and A . G . ROACH : Clin . Exptl . Pharmacol . Physiol . _1 :347-360 5. (1974) " 6. N . TODA, Y . MATSUDA, and K. SHIMAMOTO : Int . J . Neuropharmacol . _8 :451 - 461 (1969) . 7. J . L. REID, P . J . LEWIS, M . G. MYERS, and C. T. DOLLERY : J . Pharmacol . Exp . Therap . 188 :394-399 (1974) " 8. E . L . CONWAY and W. J . LANG : Clin . Exptl . Pharmacol . Physiol . 1 :59-64 (1974) . 9. H . SCHMITT and S . FENARD : Arch . Intern . Pharmaeodyn . Therap . 10 :229-240 (1971) . 10 . M. D. DAY and A. G . ROACH : Brit . J . Pharmacol . x:325 - 333 (1974) "

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11 . 12 . 13 . 14 . 15 .

Central Activity of Isoproterenol

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J. C . A . GOODRICH, B . GREEHEY, T. B. MILLER, and J . R. PAPPENHEIMER : Appl . Physiol . 26 :137-140 (1969) " C . K . BUCKNER and P . N . PATIL : J . Pharmacol . Exp . Therap . x:634-649 (1971) . "Drug Design" (E . J . ARIENS, editor), Vol . 1, Academic E . J . ARIENS : Press, N. Y . (1971), pp . 149-157 " D . J . TRIGGLE : "Neurotransmitter-Receptor Interactions", Academic Press, London (1971), p . 220. M. D . DAY and A . G . ROACH : Nature New Biology, 242:30-31 (1973) "