Gen Pharmac Vol 15, No 3, pp 233-237, 1984 0306-3623/84 $3 00+000 Printed m Great Britain Pergamon Press Ltd

ASSESSMENT OF ADRENERGIC fl-BLOCKADE IN THE ANESTHETIZED RAT AT DIFFERENT

BASELINE VALUES

FRIEDRICH BRUNNER and GERALD POCH Instltut fur Pharmakodynamlk und Toxlkologle der Umversltat, Umversltatsplatz 2,

A-8010, Graz, Austrm (Tel (0316) 3800)

(Recewed 8 November 1983)

Al~traet--The influence of different basehne values (blood pressure, heart rate) on EDs0- and Erda-values of xsoprenahne dose-response curves (DRC) in the presence and absence of/~-blockmg drugs was assessed in urethane-anesthetxzed rats The results show that

1 Urethane is a suitable anestheUc in th~s ammal model 2 Different baselines have no influence m EDs0-values 3 Assessment of potency of competitive antagonists (pA2) from complete DRC appears unaffected by

differences in baseline or m E~x 4 Pharmacologic effects (heart rate, blood pressure) are better deplclted as absolute effect values than

A-values

INTRODUCTION

In one way or another , a blologacal response to receptor agomsts or drugs appears dependent on the m m a l value of a certain parameter , eather m agree- men t with the "basehne law" or in dasagreement (Poch and Brunner , 1984) whach poses certain prob- lems m pharmacologacal experiments Quate often, drug-reduced changes are more p ronounced at low baseline values than at higher initial values (Wilder, 1967), whereas EDs0- values of dose- response curves (DRC) may or may not vary with the baseline (Poch and Brunner , 1984)

Hence, estamataon of EDs0 f rom complete D R C may offer a d d m o n a l advantages besides those gener- ally accepted over sangle dose studaes with presentataon of changes an pa ramete r values (A-values) m the case of varying baseline levels In o ther words, the assessment of EDs0-values may under certain carcumstances not depend on the base° lane, whereas A-values do

In a recent article (Maggx and Meh, 1982) it was shown tha t A-values of response to a fixed dose of l soprenalme were dependent on the pretest ing value m hear t rate Since t h e ~ values for rest ing hear t rate varaed between /200 and 300/mm m ure thane- anesthetazed rats, thas p repara t ion was though t un- s tatable for test ing a n t a g o m s m o f ~ -b lockmg agents against 0 15/~g/kg asoprenalme i v (Maggl and Meh, 1982)

On the o ther hand , under samllar condmons , the EDs0 for the stamulant effect of l soprenahne on hear t rate and the depressant effect of this /~-receptor agomst on b lood pressure appeared virtually inde- penden t of baseline levels an a dose- response study (Brunner , 1983)

The results of the lat ter study w~ll be shown here and d~scussed wath respect to the possibility to test for compe tmve an tagon i sm under dafferent anmal values which do not affect EDs0-values of the receptor agomst used

METHODS

Ammals

Albino rats, Sprague-Dawley strain, weighing 270-350 g, were anesthetized with urethane (0 75 g/kg i p and 0 5 g/kg s c ) The left carotid artery and the right jugular vein were cannulated for blood pressure monitoring, Isoprenahne injection and blocker Infusion, respectively Respiration was assisted by a tracheal cannula Blood pressure was monitored with a Statham P 2 3 D b transducer and Statham SP 1400 amplifier For heart rate recording, a rate meter (EKA-Puls, HSE Elektronik, Frelburg, Germany) was employed Both signals were recorded by a two-channel potentlometer (RE 571, BBC, GOERZ) Following surgery, the animals were heparlnlzed (100-200units/animal) and several ml of physiological saline Injection If changes of basal blood pressure and cardiac frequency were observed upon adrmnlstratIon of 0 2 ml saline, routinely given before any Isoprenahne stimulation, 1-3 ml of saline were slowly refused In the Jugular vein, whereupon such nonspeclfiC lowenng of blood pressure and increase m heart rate were no longer observed

lsoprenahne dose-response curves and fl-blockade

Isoprenaline was administered In increasing doses of 0 05, 0 5, 5, 50 and 500 ng/g (reJection volume 0 15-0 2 ml) m each rat and the dose-response curves plotted m terms of absolute values, i e mmHg and beats/mm When testing ItS effect on the parameters measured, blockers were slowly infused intravenously and lsoprenahne stimulation resumed immediately, if the parameters were stable dunng infusion, or after their stabilization (usually 10-15 min post infusion)

The lsoprenahne-induced fall m arterial mean blood pressure was interpreted as a measure of stimulation of vascular fl2-receptors while changes in heart rate were attnbuted to activation of cardiac flj-receptors

The animals of all experimental series within a gwen treatment (lsoprenahne DRC with or without //-blocker) were dwlded into 2-3 groups with different control (basal) values and EDs0-values determined from anthmeuc means of basal and maxamum effect values (Emax) attained in that gwen expenment All DRC were estabhshed from absolute values (mmHg, beats/min)

Another group of animals was treated with guanethldme (20mg/kg, s c ) the day before being used Each dose of

233

234 FRIEDRICH BRUNNER and GERALD POCH

\

i , i I -

r r

I-- r e

[&l " r

DOSE 1 2 2 3 3 4 4 5 5

Fig 1 Scheme of apphcatlon of and response m heart rate to lsoprenahne in the urethane-anesthetized rat Doses 1-5

0 05, 0 5, 5, 50 and 500,ug/kg

~soprenallne was given twice successively m such a way (Fig 1) that dose 1 was apphed, the effect allowed to develop and dose 2 gwenjust after the effect of dose 1 began to subside Upon complete subsidence of the effects of dose 2, the same dose (2) was given a second time (but now from a baseline ldenUcal with or similar to the value prior to application of dose 1) and the effect registered Just as before, dose 3 was given a httle after the maximum effects of dose 2 were registered and so forth, until dose 5

By this procedure, a possible influence of the dosing procedure on lsoprenahne effect values (mmHg, beats/mln) could be detected

RESULTS

Reproduczbthty of tsoprenahne effects

To judge the reproduclb lh ty of hear t rates and b lood pressure values upon l soprenahne a lone and under /~-b lockade , animals were given the same dose two to three t imes successively and the effects com- pared Absolu te effect values were very reproducible the coefficients of var ia t ion were 1 3% (hear t rate) and 2 9% (blood pressure) (N = 57)

Dose-response curves m presence and absence of fl-blockers

M e a n arterial b lood pressure pr ior to appl icat ion of l soprenahne ranged between 95 and 120 m m H g in the absence of 3-blockers , and between 80 and 1 2 0 m m H g in the presence of 3-blockers The re- spective hear t rates were 300-400 or 200-400 bea ts /mln Figure 2 shows the effects of Boprenal lne on b lood pressure and hear t rate in the absence of /~-blockers ( = cont ro l DRC) EDs0-values are 2 5 / t g /kg (b lood pressure) and 0 20 #g /kg (heart rate), respectively If the individual D R C are grouped according to basehne values, EDs0-values of 2 8 and 2 5 #g /kg (blood pressure) and 0 19 and 0 22/~g/kg are obta ined (Fig 2) The same cons tancy of EDs0-values irrespective of baseline values is seen unde r /~ -b lockade

Figure 3 shows l soprenahne D R C under 14 mg/kg practolol , again grouped according to mean initial values 100 and l l 0 m m H g and 250, 355 and 380 beats /mln, respecUvely The cor responding EDs0-values are identical or very close to one ano ther Practolol infusion did not by itself lower initial b lood pressure or hear t rate values, controls (107 mmHg, 352bea t s /min) are no t significantly different f rom values under practolol b lockade ( 1 1 2 m m H g , 340 beats/man) This is not the case for met ropro lo l as well as for o ther blockers tested Infusion of, e g 14 mg/kg metoprolo l lowers initial hear t rate (before lsoprenal lne apphca t lon) to 2 5 4 + 9 ( . ~ - t - S E M , N = 7) beats /mln, b lood pressure is only slightly reduced In addi t ion, hear t rate and b lood pressure do not reach cont ro l values (434bea t s /min , 52 m m H g ) W h e n the individual curves are separated according to baseline values and EDs0-values deter- mined, their constancy is again appa ren t (Fig 4) These results indicate tha t in spate of slgmficantly different control values, the respective EDs0 values differ by less than a factor of 2 and are often identical, irrespective of a concomi tan t depression of the max- imum response obta ined in a gwen exper iment In order to verify the lack of influence of baseline values on the de te rmina t ion of an tagonis t potency

40

50

60

7O

8O

9O

1 0 0

1~0

1 2 0

Blood pressure 5 0 0 mmHg

,'' 4 5 0

2 8 . 400

~~ 2 5 300

O• I i I I I I 0 0 5 0 5 5 5 0 5 0 0

Heort rote - beGfs/mlr ]

/ 4

+.~ .~," j f l y

o 20 + / , o22

~41 _ 4 t I t o ' 005 0 5 5 50 500

Isoprenahne ( # g / k g )

Fig 2 Response in blood pressure (left) and heart rate (right) to rejection of 0 05-500 pg/kg lsoprenahne at different basehnes DRC /k - /k overall mean (N = 12), 0 0 and [] [] grouped according

to baseline values (N = 6 each) Mean values + SEM EDs0-values are indicated

fl-Blockade at different baseline values

Blood pressure Heart rote N = 4 mmHg 500 beots/mm ~ N ~ 6

/ / 40 N : 2

50 ,50 ' 2 / / '

. o .__,,+., 1 6 350

90 N = 2

100 ad ~.-~, / 300 -

110

25O 120

130

__L4t i I i i t t ll i 1 I l t i 0 005 05 5 50 500 0 005 0 5 5 50 500 5000

Isoprenahne ( t z g / k g )

Fig 3 Response m blood pressure (left) and heart rate (right) to injection of 0 05-500 #g/kg isoprenahne under 14mg/kg practolol DRC are grouped according to baseline values (control curves not shown)

Mean values 4- SEM of N experiments as shown EDs0-values are m&cated

Blood pressure Heart rote mmHg 500 beats/ram

40 N : 3

Oo o,/? 80 O 5

90 + . ~ / ~ , / 10 - / : 7 1 oo : / / 110 ~"4, ,~

12o 25° I ~ _ _ ~ _ - ~ " 1 3 0

~-II L L i I I I | l r l I I I I L L 0 0 0 5 0 5 5 50 500 5000 0 0 0 5 0 5 5 50 500 5000

Isoprenahne ( t~g/kg)

Fxg 4 Response in blood pressure (left) and heart rate (right) to injection of 0 05-5000/~ g/kg lsoprenahne under 14 mg/kg metoprolol DRC are grouped according to baseline values (control curves not shown)

Mean values ___ SEM EDs0-values are ln&cated

500

4 5 0

4 0 0

350

300

2 5 0

Heart fete beets/mln

I " " • / / I •

i 252,, f DR L:2- - °+ . . . . . I T

L .__/_ 8o~//

i / / /

I rr

o ~

I I I I 0[11015 015 5 5O 5OO5OOO

Isoprenahne ( ~ g / k g )

Metoprolol (mg/kg) EOso (/~g/kg)

0 A O 25

0 4 3 * 2 8 4 0 • 20 14 • 63

/ / "

• r = l O 0 slope = 0 93

p A z ~ 71

7 6 5 4 -- log m o l / k g Metoprolol

Fig 5 (Left) Response in heart rate to rejection of 0 5-500 (5000) #g/kg moprenahne in absence (A A) and presence of 0 43 (O Q), 4 0 ( I I ) and 14 0 (V V) mg/kg metoprolol Dose ratios are indicated (Rxght) Corresponding Schlld-Plot Note that pA 2 is expressed as - l o g mol/kg rather

than - l o g mol/l

235

236 FRIEDR1CH BRUNNER and GERALD POCH

40

50

60

70

80

90

100

110

Blood pressure mrn PIcJ

500

450

4 0 0

350

300

Be(]ts/mln

o o o

i-ll I I I I 1 o'lq I I 015 I I I 0 005 05 5 50 500 005 5 50 500

Isoprenahne ( p g / k g )

Fig 6 Response m blood pressure (left) and heart rate (nght) to reJection of 0 05-500 #g/kg lsoprenahne apphed m cumulatwe (A A, N = 4--6) and non-cumulatwe (© O, N = 4--6) manner (see Rg 1) Mean values + SEM The unconnected symbols mdlcate the basehnes at whmh moprenahne mjectaon took

place m cumulatwe (A) and m non-cumulatwe ((3) fashion

(pA2-value), three different concentrations of meto- prolol have been used, EDs0-values for heart rate determined and the corresponding Schlld-Piot con- structed (Fig 5) A slope of umty was obtained, m&catmg that a purely competmve antagonistic ac- tion of a fl-blocker drug can be estabhshed despite the fact that (in this case) mmal heart rates are lowered by more than 100 beats/mm under the doses chosen, and that the maximum heart rates do not reach the control value (Fig 5, left)

Cumulatwe vs non-cumulatwe dosmg

In another group of ammals pretreated w~th gua- nethldme, lsoprenallne DRC were estabhshed as be- fore, but each dose was gwen twice as described m Methods (Fig 1) This procedure allowed a com- parison between cumulatwe and non-cumulatwe ap- plication of ~soprenahne. Results are gtven in Fig 6

Virtually identical absolute values of blood pres- sure and heart rate were reached w~th ~soprenahne irrespective of the mode of moprenahne apphcanon EDs0-values were 0 40 and 0 63 #g/kg (blood pressure m Fig 6, left) and 0 25 or 0 30 #g/kg (heart rate m Fig 6, right), respectwely

DISCUSSION

Besides ~solated organs, anesthetized ammais have become ~mportant models for the testing of dose-effect relationships of new drugs In the charac- terization of new fl-adrenoceptor blocking drugs we have used urethane-anesthetmed rats m whmh the effects of lsoprenahne on heart rate (fit) and on blood pressure (f12) were evaluated m the absence or pres- ence of a fl-blockmg drug Isoprenahne was tested over a wide dose range m order to estabhsh a complete dose-response relataonshlp and we found this model statable for such testing Several obser- vations substantmte this contention first, despite matlal heart rate and mean blood pressure values ranging from 95 to 120mmHg and 300-

400beats/mm, respectwely, EDs0-values of DRC grouped according to mmal values are almost identi- cal Receptor affinity of ~soprenahne therefore ap- pears unaffected by natural variation m parameter values prior to agomst testing Furthermore, EDs0-values characterize agonlst affimty properly when virtually the same maximum effect values are reached (Fig 2, left), as well as when maximum effects are dependent on mmal values (Fig 2, right) It ~s, however, necessary that complete DRC are obtained and that agomst affimty ~s characterized by EDs0-values and not by the change of a parameter value ehclted by a single agomst dose

Under fl-blockade we have made the followmg observanons Either the blocking drug does not by itself influence basehne values, as is the case for blood pressure with almost all blockers tested The chro- notroplc responses, however, were found to be &- versely affected (Table 1) In a few ammals basehnes as well as maximum heart rates of ~soprenahne were markedly depressed after 14 mg/kg practolol, m other

Table 1 Heart rate (beats/mm) prtor to (basehne) and following 500-5000#g/kg lsoprenahne (maximum response) m the absence (control) and presence of practolol or metoprolol as mdmated

Mean values 4- SD

Maximum EDs0 Drugs Basehne response (,u g/kg)

Isoprenahne (overall mean = control) 353 4- 46 472 4- 44 0 20

(N = 12)

+Practolol (14mg/kg)

+Metoprolol (14mg/kg)

f 250 4- 28 332 4- 46 112 (N = 2) 355 4- 8 505 4- 19 12 0 (N = 6) 3804-12 508_+10 71 (U = 4)

237 ___ 5 426 _ 5 50 (N = 4) 277+_21 4454-15 71 (N = 3)

fl-Blockade at different basehne values 237

animals, both basehne- and maximum values were not affected by practolol In all three groups, how- ever, EDs0-values were almost identical (Table 1)

Under metoprolol-treatment (14mg/kg), initial heart rates were drastically lowered and maximum heart rates moderately depressed The corresponding EDs0-values were 50 and 71/~g/kg (Fig 4, Table 1), a difference of only 30~

The possible influence of baseline values on the determination of blocker potencies (pA2-values) was stu&ed with metoprolol (Fig 5) Doses of 0 43, 4 0 and 14/~g/kg gave dose ratios of 11, 80 and 252 and a slope of unity m the Schlld-plot Thus a purely competltwe interaction of fl-receptors appears un- perturbed by a concomitant negative chronotrop~c action of the fl-blockmg drug

In all DRC discussed above we have used direct reading values (mmHg, beats/min), and this should finally be discussed, specifically m relation to the work recently published by Maggl and Meh (1982), and already mentioned in the Introduction These authors found the same animal model (rats of com- parable weight, urethane anesthesia) unsuitable for the testing of fl-blockers They showed that the increases in heart rate ehclted by lsoprenaline over basal resting values were dependent on the latter which vaned between 200 and 400 beats/mm

Although we have not observed such large van- atlons in control values of heart rate before the first dose of isoprenahne rejected, we have also seen that ~soprenallne-induced increases, 1 e A-values were de- pendent on the pre-drug baseline m accordance with Maggl and Meh (1982)

On the other hand, we found that under urethane anesthesia wrtually the same absolute reading values of heart rate were reached by isoprenahne which were independent of differences or variations m baseline As a matter of fact, from the data of Maggl and Meh (1982), it is evident that 0 15/~g/kg lsoprenaline increased the heart rate from 306 9 to 363 6 beats/mm m urethane-anesthetlzed rats, and from 265 7 to 365 4 m reserpmxzed barbiturate-anesthetized rats Hence, the same drug-value of about 365 beats/mm was reached by ~soprenahne m both preparations

The conclusions of Maggi and Meh (1982) as to the unsuitability of urethane anesthesia therefore do not seem to be tenable A direct dependence of A-values on pre-drug baseline values will be encountered for mere theoretical reasons m all cases where an abso- lute maximum biological response (e g a heart rate of about 600 beats/min in rats) cannot he surpassed This consideration extends to cumulative testing of a drug

We have examined the usefulness of assessing DRC using reading values vs A-values of changes in the same ammal model, with the notable difference that the rats were pretreated with guanethldlne in order to exaggerate the response of heart rate and blood pressure to ~soprenallne A comparison of iso- prenahne effects in terms of reading values and A-values was possible following the injection schedule of lsoprenaline as depicted in Fig 1 Each dose of isoprenahne changed reading values of blood pres- sure (mmHg) or heart rate (beats/mln) to the same value, irrespective of the foregoing "control" value By a slight variation of the cumulative-type applica- tion of lsoprenahne (see Fig 1) it was possible to compare the effect of lsoprenahne "on top" of the 1/10 smaller dose with its effect at a baseline value Virtually no differences in reading values reached by lsoprenahne could be seen, nor major differences in the EDs0-values derived from the respective DRC (Fig 6)

Figure 6 further indicates the respective baseline values pnor to apphcatlon of each dose of lso- prenahne When only considering those values after complete subsidence of lsoprenallne effects (circles) one notices a gradual fall in blood pressure and rise in heart rate These baseline changes would influence the calculation of A-values, but are of no consequence to reading values Th~s latter phenomenon is certainly frequently encountered experimentally

In conclusion, the variation of basehne values in the ammal model used in the study obviously does not impair the correct determination of EDs0-values of agonists and pA2-values of receptor antagonists provided that complete DRC are established from direct reading values of heart rate and blood pressure

Acknowledgements--We wish to thank Mrs Betty Oberer, Ms Annemane Buchmann and Mr Thomas Markovlc for their valuable techmcal assistance

REFERENCES

Brunner F (1983) EDs0-values for effects oflsoprenahne m the anaesthetized rat are independent of basehnes Naunyn-Schmtedeberg's Arch Pharmac (Suppl) 322, Abstract 13 (R4)

Maggl C A and Meh A (1982) Unsuitability of urethane anaestheuzed rats for testing potential fl-adrenoceptor blockers Expertentta 38, 517-519

Poch G and Brunner F (1984) The underlying bases of pharmacological results m agreement or disagree- ment with the law of initial value Gen Pharmac 15, 231-234

Wdder J (1967) Sttmulus and Response The Law of Initial Value Wright, Bristol