Desensitization of the isolated guinea pig ileum to antihistaminic agents

Desensitization of the isolated guinea pig ileum to antihistaminic agents

European Journal of Pharmacology, 90 (1983) 185- 191 Elsevier DESENSITIZATION 185 OF THE ISOLATED GUINEA PIG ILEUM TO ANTIHISTAMINIC AGENTS IGOR ...

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European Journal of Pharmacology, 90 (1983) 185- 191 Elsevier

DESENSITIZATION

185

OF THE ISOLATED GUINEA PIG ILEUM TO ANTIHISTAMINIC

AGENTS

IGOR ONNEN * and GEORGES OLIVE **

Laboratotre de Phystologte, Facult$ de M~decme (Umversttb Parts Nord), Boblgny, France and ** Laboratmre de Pharmacologw, Umverstte Ren~ Descartes, Paris, France Received 3 January 1983, accepted 17 February 1983

I. ONNEN and G OLIVE, Desensmzatlon of the tsolated gumea ptg deum to antzhlstarnmw agents, European J Pharmacol 90 (1983) 185-191 Guinea p~g ileum developed in xatro a progressive desensltlzauon to the antihistaminic agents mepyramane and benadryl This desensmzatmn had uncommon characteristics (1) at the 6 5-9 h period of the experiments, it consisted only of a decreased rate of actmn of the drugs (1 66 fold and 2 fold increases m the 2 nun Kb value of mepyramlne and benadryl respectively), without variation in the equlhbrlum Kb values, (2) it was not a drug effect since control strips (straps exposed to the first dose of antlhlstanumc agent after 5 5 h m wtro) also showed a decreased rate of action of the drugs (1 58 fold and 1.6 fold increases m the 2 nun Kb value of mepyramlne and benadryl respectively), w~thout varlatmn in the Kbe values The desens~t~zatmn also &d not depend on the bathing medium or on the amount of avmlable tustarmne receptors The only explanatmn of the desensmzat~on ~s a slowing m the antdustanumc agent-receptor reactmn either m its access stage or less probably m its mteractmn stage G u i n e a pig d e u m , isolated H l-receptor blockers

Klnettcs of d r u g action

1. Introduction D e s e n s i t i z a t i o n is a state of decreased responsiveness to drugs which results f r o m either a m u t a t i o n (resistance of e.g. m i c r o o r g a n i s m s ) o r p r e v i o u s c o n t a c t with the drug (tolerance). T h e r e are two m a i n m e c h a n i s m s of desensitization ( G o l d s t e l n et al., 1974) Firstly. a biochermcal r e d u c t i o n in the drug c o n c e n t r a t i o n in the r e c e p t o r region (e.g. P s e u d o m o n a s resistance to arsenltes, P e p p u a n d A r l m a (1964); e t h a n o l tolerance, M e n d e l s o n et al (1965)). Secondly: a r e d u c t i o n in the affinity of the r e c e p t o r s for the d r u g G u i n e a pig ileum develops in vitro desensitization to a variety of drugs, e.g. m o r p h i n e (Paton, 1957), s e r o t o m n ( H u l d o b r o - T o r o a n d Force, 1980), a n d a t r o p i n e ( O n n e n a n d Olive, 1981) W h i l e the * To whom all correspondence should be addressed Laboratolre de Physiologic, Facult~ de Medecme, 74 Avenue Marcel Cachm, 93000 Bobigny, France 0014-2999/83/$03 00 © 1983 Elsevier Soence Pubhshers B V

Drug desensitization

Mepyramane

Benadryl

m o r p t u n e a n d s e r o t o n l n desensitizations are true tolerances with d e c r e a s e d responsiveness resulting f r o m a p r e w o u s contact, the a t r o p i n e desensitization has u n c o m m o n characteristics since it consists o f a slowed (instead of decreased) responsiveness resulting f r o m the in vitro c o n d i t i o n s (instead of f r o m a previous contact). Because of ItS in vitro nature, the a t r o p i n e desensitization rmght pres u m a b l y n o t be restricted to this drug, in other w o r d s to the chollnergac receptors. The present s t u d y mined to explore this state of desensitization in the h i s t a m i n e receptors of t h e guinea pig ileum T h e rate of a c t i o n of the a n t i h i s t a m i n i c agent m e p y r a n u n e was thus m e a s u r e d at the 1st h a n d at the 6th h of e x p e n m e n t s , 9 h in d u r a t i o n , on isolated guinea pig ileum, and, to test the effect of m vitro c o n d m o n s , after 5.5 h in each e x p e r i m e n t on a p m r e d c o n t r o l strip, i.e a strip e x p o s e d to the first dose of m e p y r a r m n e after 5 5 h in vitro In s o m e experiments, b e n a d r y l was studied i n s t e a d of m e p y r a r m n e . Both drugs allow accurate m e a s u r e -

186 ments of their rate of action, a rate which is synonymous of the rate at which a drug reaches and occupws its specific receptors First. these drugs (like lustamine) have only one type of receptor in the guinea pig ileum (the H~-receptors, Ash and Sctuld, 1966) excluding H E- (Bertaccini et al., 1979) and nervous (Brownlee and Johnson, 1963) receptors. Second: their effects do not develop too fast (mepyran~ne In about 10 nun; benadryl in about 5 mln; Scbald, 1947) and are thus easy to follow.

2. M a t e r i a l s a n d m e t h o d s

2 1 Alteratwns of the affmtty constants (Kb and 7"1/2 values) of mepyramme m strtps bathed m Tyrode solutwn In each of 16 experiments, two adjacent 2 cm strxps of terminal ileum were obtained from an adult guinea pig and immediately mounted, as described by the Edinburgh staff (1968), m two separate organ baths (Vlckers J33) containing Tyrode solution (NaC1 8 g/l, CaC12 0.2 g/l, KC1 0.2 g/I, MgCI 2 0.1 g / l , N a H 2 P O 4 0.05 g / I , N a H C O 3 0 5 g / I , glucose 1 g / l , p H 8) bubbled with air and warmed at 37°C. One strip served as the test strip, the other as the control. The experiment started (zero time) 40 nun later.

2 1.1 Affmtty constants of the test strtp durmg the tmttal (0-2.5 h) pertod Two-min Kb value, the ECs0 values of tustamine (HA) were measured every 3 5 nun on cumulatwe concentraUon-response curves; each curve lasted 1 nun. Curves to 2 and 6 n g / m l were usually alternated with curves to 4 and 8 n g / m l The responses, isotonic contractions between 30 and 70% of the maximum (the maxamum was measured at zero time), were read 30 s after the addition (with a pipette) of HA. Whenever 4 constant ECs0 values had been obtained (the ECs0 value was 110_+ 25 nmol), the bath fluid was replaced by a fluid containing mepyranune. After 2 nun, the effect of mepyramine (dose ratio for H A = ratio of equiactlve doses of H A after and before mepyranune) was measured. Three mepyra-

mine concentrations were tested successively 2 5, 5, and l0 nmol/1 and their effects were plotted in a reference (0-2.5 h period) graph: log (dose ratio - 1) versus log concentration of mepyranune (Arunlakshana and Schlld, 1959). The 2 m m Kb value (concentration producing a dose ratio of 2 / 1 after 2 mIn exposure) corresponded to y = log (2 - 1)= 0. Kb value at equilibrium (Kbe) and time to reach half of the equlhbrmm occupancy (onset T1/2): Kbe is the concentraUon producing a dose ratio of 2 / 1 after e q m h b n u m exposure The method of Furchgott (1967) was used the 2 mln exposure to 2 5 nmol/1 mepyranune used m the 2 nun Kb measurement was prolonged for 15-18 mln and dose ratios were measured every 3 nun K b e was 2.5 n m o l / ( D R e - 1) where D R e was the equihbrmm dose raUo usually reached at the 10th min. The onset T1/2 value 0 . 6 9 3 / K + was solved from the equation log ( o e - o t ) = l o g oe-K+t (Paton and Rang, 1965) where ot and oe are the receptor occupanoes (o = 100 ( ( D R - 1 ) / D R ) by 2 5 nmol m e p y r a m m e at t 0, 2, 5 and 8 mln (ot) and at equdlbrlum (oe). The dose ratxos needed no correction (Furchgott, 1967) since the control strips showed no sensitxwty changes to H A during these 15-18 nun testing periods. After D R e had been obtained, m e p y r a m m e was washed out and the disappearance of its effect was deternuned by the residual effect (ratio of equlactlve doses of H A at the end of washout and just before the exposure to mepyranune) and by the end T1/2 value 0 . 6 9 3 / K solved from the equation log ot = log oe - K - t

2 1.2 Alterattons m the affmtty constants for the test strip over the ensuing (2.5-9 h) pertod The sensitivity to H A was determined from sequential concentration-response curves by alternating the responses to x, 2x and 4x n g / m l at 3 rmn time intervals H A curves were sequential instead of cumulative since the need for rapid ECs0 measurements was not so great as in the initial (0-2 5 h) period. The 2 nun Kb value was reevaluated for the (3-5 h) and (6.5-9 h) periods The Kbe and T~/2 values were reevaluated at 6 5 h. Each mepyramlne solution was made up just before use.

187

2 1 3 Afftmty constants for the control strtp m the (5 5-8 h) pertod Sequential concentration response curves to H A were repeated until the 5.5th h as described m (2 1.2 ) and no mepyranune was given The ECs0 value (120 _+ 25 nmol) of H A was strongly correlated (P < 0001, r---0 83) with that of the test strips) and showed a 1 53 + 0.3 fold increase (control H A ratxo) at 5.5 h The 2 rain Kb value of m e p y r a m m e was measured for the (5 5-8 h) period, the Kbe and the TI/2 values were measured at 5 5 h as had been done at 1 h on the test strap 2.2 Alterations m the affmtty constants of mepyramme for strtps bathed m Krebs Henselelt solutton, effect of the previous trreverstble receptor blockade Eleven additional experiments were performed as described (in 2 1) but with Krebs Henselelt solution (Edinburgh staff, 1968) bubbled with 5% CO 2 in O 2, and w~thout Kb measurements at t 3-5 h. The ECs0 value of H A (120 _+ 30 nmol) was the same as In the Tyrode solution. In 7 of these 11 experiments, the first exposure to mepyranune was preceded by an irreversible subtotal receptor blockade. To obtain thas blockade, the test strip underwent at the 20th nun (and the control strip at 4.5 h) one single 20 nun exposure to 800 nmol/1 of the irreversible unspecific receptor blocker dlbenanune (DB) DB decreased the m a x i m u m effect of H A by 20% and multiplied the ECs0 value of H A by a factor f of 25 + 15 (f = 32 + 18 m control strips). This corresponded to the blockade (% of receptors blocked = 100 ( ( f - l / f ) , Paton and Rang, 1965) of about 95% of the receptors and indicated that the HA contractaons were mainly due to prewously sdent spare receptors (NIckerson, 1956) After the blockade had been obtained, the affinity constants of mepyramine were measured (test strips dunng t 0.45-2.5 h and 6.5-9 h, control strips during 5.5-8 h) The effects of mepyranune (rauos of equlactwe doses of H A after and before mepyranune) were not corrected for sensltw~ty changes to HA. the DB blockade was very slowly reversible so that the ECs0 value of H A varied (decreased) by only 7 _ 5% per 20 mln in the 1st h after DB and by 5% per h thereafter

2 3 Alterations m the afftmty constants of benadryl Three experiments were done as descrabed m (2 1) but with the fast-acting antihistaminic agent benadryl instead of mepyramlne

3. Results

3 1 Alterattons m the affmtty constants of mepyramme (Tyrode solution) 3 1 1 lmtlal (0-2 5 h) pertod test stops The 2 man Kb value was 1.9 + 0.3 nmol, with a slope of 1 28 + 0 12 (fig 1, table 1), the Kbe value was 0 54 + 0.05 nmol The l-ugh 2 nun K b / K b e ratio (3.4) indicates that the HA mepyranune antagonism was slow 3 1 2 Ensumg (3-9 h) pertods test strips The mepyrarmne antagonism settled at a slower rate (fig. 1, table 1). First the 2 nun Kb values increased; the 2 nun Kb ratio was above unity as soon as the (3-5 h) period (P < 0 01, paired t-test) and increased to 1.66 at the (6.5-9 h) period (P < 0 001, paired t-test). Second the onset T~/2 values increased (P < 0.001, paired t-test) Third the Kbe values remained unchanged 3 1 3 Ensuing (5 5-9 h) pertod control strtps A slowing in the mepyramine antagonism similar to that shown by the test strips occurred (fig 1, table 1). First: the 2 man Kb values were greater (P < 0 01, Student's t-test) than for the lnltxal (0-2.5 h) peraod; the 2 man Kb ratxo (1.58 at the (5.5-8 h) period) was strongly correlated (P < 0.001, r = 0 7) wath the 2 nun K b ratto of the test strips (1 66 at the (6 5-9 h) period). Second: the onset T~/z values were greater (P < 0 01, Student's t-test) than at the 1st h, which shows clearly (TI/2 was measured at 5 5 h l.e at the 1st exposure to mepyramine) that the slowang occurred an the absence of previous contact w~th the drug Third' the Kbe value were the same as at 1 h (P < 0 2, Student's t-test). 3 2 Alterattons in the affmtty constants of mepyramme for strtps bathed tn Krebs-Henselelt solutton, effect of prevtous wreverstble receptor blockade 3.2 1 No previous receptor blockade The mepyramlne antagomsm decreased

as

188 TABLE i Alterations m the affinity constants (Kb and onset Tl/2 values) and m the constants for the d~sappearance (residual effects and end Tt/2 values) of the mepyranune effect on gumea pig ileum bathed in Tyrode solution Kb and onset Tt/2 values were obtamed from the data in fig 1 The constants for the disappearance of the effect were evaluated d u n n g the 20-40 m m washout period succeedmg the 16-18 n u n e q m h b r m m exposure to 2 5 nmol mepyramlne The residual effect is the ratio of equmctwe doses of HA at the end of washout and just before the exposure to mepyrarmne (this ratio was umty when washout is complete) The end Tj/2 value is the time elapsed between the onset of washout and the time at which receptor occupancy (o = ( D R - I ) / D R ) was equal to o e / 2 (oe = e q m h b n u m occupancy) Each value is the mean _+S E M of 16 measurements Test strips

2mmKbvalue(nmol) 2nunKbratio Kbevalue(nmol) Onset T1/2 value (s) Resldualeffectofmepyranune End T1/2 value (s)

Control strips

0-25 h

3-5 h

6 5-9 h

5 5-8 h

19 + 0 3 Umty 054-+ 005 75 _+22 1 2 8 + 0 13 504 4- 91

24-+05 13_+02 -

3 2 -+ 0 6 166_+ 0 4 053-+ 0 0 8 132 _+ 22 1 53+ 032 888 -+ 161

31 -+ 0 8 158+ 04 047_+ 01 127 _+ 25 1 57_+ 0 2 2 850 -+ 185

TABLE 2 Changes in the a f h m t y constants (Kb and onset TI/2 values) of mepyranune for guinea pig ileum bathed m Krebs Henselelt solution, effect of previous irreversible subtotal receptor blockade Data from 11 experiments performed w~th Krebs Hensele~t solution Two rain Kb and Kbe (2 5 n m o l / ( D R e - l)) values of mepyrarmne were measured at each period by testmg successively 2 5. 5 and l0 nmol/1 The onset TI/2 values correspond to the onset of the effect of 2 5 n m o l / l Receptor blockade (by a smgle 20 m m exposure to 800 n m o l / l DB) was applied to the test strips at the 20th man and to the pa~red control strips at 4 5 h, that ~s in both cases prior to the first exposure to mepyramine Each value ~s the mean 4- S E M of n measurements No prewous blockade (n = 4)

2mmKbvalue(nmol) 2mmKbratlo Kbevalue(nmol) Onset T1/2 value (s)

Previous receptor blockade by DB (n = 7)

Test strips 0-25h

Control strips 55-8h

Test strips

65-9h

0-25h

65-9h

Control strips 55-8h

19 + 0 2 Unity 056_+ 01 83 + 27

34 + 07 174+ 03 057_+ 011 175 _+68

34 + 06 174_+ 0 6 050_+ 01 150 _+30

2 + 035 Unity 096_+ 025 61 + 10

2 8 + 053 139+ 017 080_+ 0 2 0 99 -+ 26

3 + 11 147_+ 031 067+ 014 109 -+ 35

TABLE 3 Changes in the affimty constants (Kb and onset Tl/2 values) of benadryl for guinea pig deum Data from 3 experiments performed w~th Krebs Henselelt soluUon Two n u n Kb, Kbe (40 n m o l / D R e - 1) and onset Ti/2 (of the onset of 40 nmol) values were evaluated at each pertod by testmg successively (1) 40 nmol (2 nun exposure prolonged until equlhbnum), (2) 80 nmol (2 nun exposure) and (3) 160 nmol (2 rain exposure) In ad&tlon, the 2 n u n Kb value of benadryl was evaluated at the (3-5 h) period by testing successtvely 40, 80 and 80 nmol At each period, the Kb ratio is the ratio of the 2 rain K b values at flus period and at the tmtlal (0-2 5 h) period Each n u m b e r is the mean of 3 measurements (range m parentheses) Test strips

2 m m Kb value(nmol) 2 rain Kb ratio K b e value (nmol) Onset T1/2 value(s)

Control strips

0-25h

3-5h

65-9h

55-8h

16 (15-17) Unity 12 3 (10-14) 30 (19-39)

27 (22-29) 1 6 (1 2-2) -

32 (23-40) 2 (1 5-2 4) 12 (12-13) 60 (41-84)

26 16 12 60

(22-29) (1 4-1 8) (11-14) (39-78)

189

1.2! ~" rT=test strips q , " I=control strips f

1 A

I 0

~0.5

Z'

°

/

,¢; I

I

I

2.5

5

lO

nmol mepyramlne (Iogscale) Fig 1 Alterations m the antilustamlmc effects of mepyralmne (2 mln and e q u i h b n u m exposure) on guinea pig ileum bathed in Tyrode solution (A) Concentration-effect curves for mepyrarmne (2 man exposures) obtained (1) from the test strips in the initial (0.2 5 h) period and in the (6 5-9 h) periods (open circles joined by continuous lines), and (2) from the paired control strips m the (5 5-8 h) periods (closed circles joined by dotted lines) The 2 n u n Kb values (intercepts of these curves with y = 0) are shown in table 1 The effect of 2 5 nmol at 3 h ( ~ ) and of 5 nmol at 4 h (~) and at 5 h (zx) is also shown (B) Effects of e q u d i b n u m exposures to 2 5 nmol m e p y r a m m e ((2 test strips at 1 h and at 6 5 h, • parred control strips at 5 5 h) The K b e values 2 5 n m o l / ( D R e - 1) are shown m table 1 (C) Effects of e q u l h b n u m exposures to 10 nmol m e p y r a m m e ([2 test strips at the 9th h, • paired control strips at 8 h) In each period, the strips (test strips at t 0-2 5 h and at t 6 5-9 h, control strips at t 5 5-8 h) underwent three successive exposures to mepyramine to 2 5 nmol (2 man prolonged until equihbrium), to 5 nmol (2 man) and to l0 nmol (2 mm) Each symbol is the mean + S E M of 16 measurements The concentrations correspond to the base of m e p y r a m m e

shown by the increased 2 mln Kb and T~/2 values (table 2)

3 2 2 Effects ofprewous wreverstble receptor blockade In the (0-2.5 h) period, DB increased (P = 0 05, Student's t-test) the Kbe value of mepyramlne (table 2) Le. the spare H~-receptors had less affinity for m e p y r a m m e than the normal H r r e c e p t o r s . This is reminiscent of the 3 2 fold increase in the

Kbe value of phentolamme produced by the DB analog tetracaine (Flelsch and Titus, 1973) The m e p y r a m m e antagonism was decreased This slowing was paradoxically associated with supersensltizatlon (table 2) the 2 nun Kb values increased (test strips P < 0 02, paired t-test; control strips. P = 0.05, Student's t-test) as well as the T1/2 values (test strips: P < 0 02, paired t-test, control stnps P = 0.02, Student's t-test), while the K b e values decreased, but only significantly (P = 0 05, Student's t-test) in the control strips (P = 0 1 in the test strips). However this slowing with supersensmzatlon (1 e with a greater equlhbrlum effect and thus a smaller Kbe value) was a true one since the greater the e q m h b n u m effect the faster the onset of the effect (Roberts and Stephenson, 1976, Kenalon, 1980)

3 3 Alteratzons m the affmtty constants of benadryl If benadryl differs from mepyramlne in being a fast-acting (the 2 mln K b / K b e ratio is 1.3 instead of 3.4) and weak (the Kbe value is 12 3 nmol instead of 0.54) antllustamlmc agent, it nonetheless shares with mepyramlne a great s l o w i n g i n itS rate of action" the 2 mln Kb and Z l / 2 increased slgmficantly in both test and control strips, whde the Kbe values remained unchanged (table 3).

4. Discussion The results of this study can be stated as follows. First: after several hours in vitro and whatever the bathing medium and the amount of receptors, the mepyramlne-receptor reaction m the guinea pig deum was slowed in Its time of onset as well (end Tl/2 values were increased, table 1) as in its disappearance but its equdlbrmm state was not altered (Kbe values remained unchanged; moreover, in the case of the spare receptors, the eqmh b r m m state was enhanced). Second: this slowing was not a drug effect since the control strips showed this slowing at the first exposure to mepyranune, and in 6 experiments (2 mln Kb ratio = 1.7_+ 0.4) with no spontaneous H A desensitization of the control strips at 5.5 h thus excluding a cross-desensmzatlon to mepyramlne

190 and HA. This in vitro slowing also shown by benadryl is not pecuhar to the ileum. Two strips of guinea pig taenia caecl (instologlcal controls showed that they consisted of isolated longitudinal muscle for half of their length) showed an increase in the onset Tl/2 value from 105 s at 1 h to 240 s at 6.5 h (test strips) and to 198 s at 5.5 h (control strips) (Onnen, unpublished) Furthermore, the slowing was not peculiar to mepyramine or to the guinea pig: a 1.5 fold increase in the 2 mln Kb value occurred in 3 experiments on 5 HT-methyserglde antagonism in the rat uterus (Onnen, unpubhshed). Before touchmg on the possible mechanisms of the antlinstamine agent desensitization, two consequences of using 2 mln exposures must be discussed. First: the slope of the dose-effect graph was 1.28 (fig. 1) instead of umty winch is the slope for mepyramine at eqmhbrium (Marshall, 1955) Tins is easdy explained" the rate of action increases with the concentration (Roberts and Stephenson, 1976, Kenakln, 1980) so that after 2 rain the effect of 2.5 nmol is farther from equihbrlum than that of l0 nmol Second. effects were less than after equdibrmm exposures (fig. 1) so that the recovery times and the amount of mepyramine ' M O ' remamlng m the tissues after washout were reduced: at 6.5 h, the cumulated residual effect ' C R E ' of mepyramlne on the test strips (CRE in each experiment was the ratio of eqmactive doses of H A at 6 h and at zero time &vided by the control H A ratio at 6 h so as to take into account the spontaneous HA desensitization of the control strips) was only 1.45 _+ 0.3 Tins ' C R E value' suggests that ' M O ' is small, about 0 3 nmol/1 if the equation of Furchgott (1967) Kbe = M / ( D R e - l) is solved for M = MO, Kbe = 0.55 nmol (table l) and D R e = CRE. Five possible mechanisms of the desensitization reduced by the antlinstamimc are now discussed. First" the ileum might undergo fatigue, as suggested by the slight increase in the fading of the contractions. The fading (decline of each contraction between its peak, attained 4-8 s after the addition of H A and 30 s thereafter) was 20 _+ 12% at 1 h and 33 _+ 18% at 8 h. However, this mechanism is very unlikely since the data of e g. table l did not change (2 min Kb ratio = 1.6 +_ 0 2 in the

test strips; 1.57 _ 0 3 in the control strips; no Kbe variation) when the contractions were measured at their peaks. Second" the binding of mepyramlne by non-receptor sites, shown to be present in the guinea pig ileum by Hall and Young (1981), might increase after 5 h in vitro. This might explain the slower effect and the greater residual effects of mepyramlne (table 1) which are rermnlscent of the slow effect and the very great residual effect of drugs acting on tissues with a great proportion of non-receptor sites (e.g. atropine, cocaine and the pigmented iris, Salazar et al., 1976). This binding mechanism is unlikely since mepyramlne desensitization occurred In the five experiments (table 1) with slnular residual effects at 1 and at 5 5 h (the 2 mln Kb ratio was l 8_+ 0.5 at 5 5 h), and when most of these non-receptor sites were blocked by DB (table 2) which is known to have such a blocking effect (Uchida, 1978). Third" enhanced enzynuc destruction is unlikely: this should slow the onset, and, at the same time (which was not the case: table l) accelerate the end of the effect of mepyramlne. Fourth: mepyrarmne might trigger H A silent receptors with as result greater responses to HA, and this potentiating effect of mepyramine might be enhanced after 5 h in vitro. Tins mechanism is unhkely because the potentiation should be effective only in the 1 mln of exposure to mepyramine since otherwise the Kbe values should increase like the 2 mln Kb values, and because slowing of the mepyramine antagonism also occurs when the silent receptors have become barely available (table 2). Fifth in the absence of the previously mentioned mechanisms, the only explanation is intrinsic slowing in the antlinstamine agent-receptor reaction, either in its first stage (access) or in i t s second stage (interaction) This point can only be elucidated with a technique winch reduces the access factors to a minimum Such a reduction was not obtained with the isolated longitudinal muscle in tins preparation, the onset of the benadryl effect was considerably slower (5 rain K b / K b e ratio = about 4, Kenakin, 1980) than in the intact ileum where the 2 iron K b / K b e ratio was only 1 3 (table 3) The only adequate technique, drug administration by iontophoresis shows that access lasts much longer than interaction. Among other examples, lachesine

191

has a Tl/2 values of 4 mm in the intact deum and of 5.7 mln in the isolated longitudinal muscle (Roberts and Stephenson, 1976) and of less than 15 s when it is apphed by lontophoresls (Bolton, 1977); slmdar observations were reported for a variety of drugs (curare, Armstong and Lester, 1979; naloxone, Neal and Keane, 1978). However, there is mdlrect evidence that with mepyramlne access lasts longer than interaction (Roberts and Stephenson, 1976) Under these con&tlons, the 1.7 fold increase m the onset TI/2 value (table 1) can be explained by a shght slowing in the access stage whxch ~s more probable than great slowing of the interaction

Acknowledgements We are greatly mdebted to Prof Roger Verley for helpful crmclsm We also thank Dr Jean Louis Kemenl and Mme Beatrice Quenette for the histological work Mepyramme maleate and methysergide were given by Laboratoire Sandoz Benadryl chlorhydrate was given by Laboratolre Substantia

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