European Journal of Pharmacology, 37 ( 1976) 13--22
13
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
THE EFFECTS OF CHRONIC RESERPINE TREATMENT ON THE CONTRACTILE A C T I V I T Y O F T H E I S O L A T E D VAS D E F E R E N S O F T H E G U I N E A P I G S]~RGIO DE MORAES Laboratory of Therapeutics, Faculty o f Veterinary Medicine, University of SSo Paulo, S~oPaulo, Brasil
Received 18 June 1975, revised MS received 29 December 1975, accepted 6 January 1976
DE MORAES, S. The effects of chronic reserpine treatment on the contractile activity of the isolated vas deferens of the guinea pig, European J. Pharmacol. 37 (1976) 13--22. Chronic reserpine treatment of guinea pigs during 5 days (1 mg/kg/day) induces postjunctional supersensitivity in the isolated vas deferens. It has been previously proposed that postjunctional supersensitivity occurs as a result of an ionic and/or membrane mechanism. Contrasting with previous observations in vascular smooth muscle the present results demonstrate that chronic reserpine treatment did not increase the sensitivity of the depolarized vas deferens to calcium. Experiments on drug responsiveness show that the supersensitive depolarized tissues have a greater and slower rate of loss of responsiveness than do control vasa deferentia. However, in a Ca 2+-free Krebs solution responsiveness of supersensitive vasa deferentia did not differ from that of control tissues. These findings suggest that, in the guinea-pig vas deferens, reserpine-induced supersensitivity could be at least partially dependent on the increased availability of a calcium store(s) probably located at the cell membrane and/or cytoplasmic compartments. Supersensitivity
Reserpine
Isolated vas deferens
1. I n t r o d u c t i o n P o s t j u n c t i o n a l s u p e r s e n s i t i v i t y is c h a r a c t e r ized b y an increased responsiveness o f tissues to d r u g s t i m u l a t i o n ( F l e m i n g et al., 1973). R e s e r p i n e - i n d u c e d p o s t j u n c t i o n a l supersensitivity o f t h e isolated guinea pig vas d e f e r e n s is a w e l l - k n o w n p h e n o m e n o n (Westfall, 1 9 7 0 a ) . T h e r e is g r o w i n g r e c o g n i t i o n t h a t p o s t j u n c t i o n a l s u p e r s e n s i t i v i t y is p r o d u c e d b y s o m e a l t e r a t i o n i.n t h e p h y s i o l o g y o f t h e r e s p o n d i n g cells, b e y o n d the r e c e p t o r level ( F l e m i n g 1963; Lee et al., 1 9 7 5 ) . T h e m o s t r e c e n t evidence indicates t h a t c a l c i u m ions are involved in r e s e r p i n e - i n d u c e d s u p e r s e n s i t i v i t y in vascular tissues (Hudgins a n d Harris, 1 9 7 0 ; G a r r e t a n d Carrier, 1 9 7 1 ; Carrier and J u r e vics, 1 9 7 3 ; Hester, 1974). Nevertheless, Westfall ( 1 9 7 0 b ) f o u n d no changes in t h e electrolyte levels o f vasa d e f e r e n t i a f r o m reserpinep r e t r e a t e d g u i n e a pigs. As c a l c i u m p l a y s a k e y role in the c o n t r a c t i l e a c t i v i t y o f s m o o t h
Drug responsiveness
muscle cells it was o f interest to t e s t w h e t h e r an e n h a n c e d availability o f calcium f o r cont r a c t i o n c o u l d be r e s p o n s i b l e f o r reserpine-ind u c e d supersensitivity. I n a d d i t i o n to confirming t h e d e v e l o p m e n t o f p o s t j u n c t i o n a l supersensitivity in t h e guinea pig vas d e f e r e n s a f t e r c h r o n i c reserpine a d m i n i s t r a t i o n , it is suggested t h a t an increased m o b i l i z a t i o n o f c a l c i u m for c o n t r a c t i o n c o u l d be a k e y m e c h a n i s m f o r r e s e r p i n e - i n d u c e d supersensitivity.
2. Materials and m e t h o d s 2.1. A n i m a l s
T h e animals used in t h e s e e x p e r i m e n t s w e r e guinea pigs o f a b o u t 5 0 0 g weight. T h e a n i m a l s were killed b y a b l o w o n t h e head. T h e vasa d e f e r e n t i a w e r e r e m o v e d and carefully d e s h e a t h e d .
14
2. 2. Bathing solutions The following bathing media were used (mM concentration). Krebs solution: NaC1 115.0; KC1 4.6; CaC12 2.5; KH2PO4 1.2; MgSO4 1.2; NaHCO3 2.5 and glucose 25; Ca2+-free Krebs solution: Krebs solution with no added calcium and with disodium edetate added to give a final concentration of 5 × 10 -6 M; sodium- and calcium-free KC1--Ringer (Edman and Schild, 1962): KC1 165.0; KHCO3 3.6; glucose 5.5 and 5 × 10 .6 M disodium edetate.
2. 3. Concentration--effect curves The method of setting up the tissues has been previously described (de Moraes and Carvalho, 1973). Contractions were monitored with isotonic myograph transducers coupled to a Narco recorder. The vasa deferentia were allowed to equilibrate in the muscle chamber under an initial load of 900 mg for at least 60 min before any procedure or drug testing. The bathing solutions were kept at 31°C and constantly bubbled with 95% oxygen and 5% carbon dioxide. The muscle chambers had a 22 ml working volume. Full concentration--effect curves were obtained by applying norepinephrine, acetylcholine or histamine in separate concentrations. The interval between successive drug concentrations was 5 min and after 30 sec the agonists were washed out thoroughly. Cumulative concentration--effect curves to calcium were constructed from vasa deferentia depolarized by replacing normal Krebs solution by KC1--Ringer. Changes in drug sensitivities were evaluated by comparing the ECs0 's i.e., the drug concentrations producing a response which was 50% of m a x i m u m in individual experiments. The mean EC so's are presented as geometric means with 95% confidence intervals (Fleming et al., 1972).
2. 4. Loss o f responsiveness These experiments were carried out in order to study the smooth muscle dependence
s. DE MORAES on calcium for contraction in vasa deferentia removed from untreated and reserpine-pretreated animals. The basic experimental protocol was as follows. After 60 min equilibration period in Krebs solution the tissues were initially submitted to maximal concentrations of norepinephrine, acetylcholine or histamine to elicit standard reference responses. After 30 min for recovery the preparations were transferred to KC1--Ringer. After the tissues returned to the baseline, which took approximately 4 min, the depolarizing solution was immediately replaced by fresh KC1-Ringer. The washing process was repeated twice with 1 min. The preparations were then submitted to the same drug concentration which produced the standard reference response in Krebs solution and thereafter successively exposed to the drug at 5-min intervals. The same procedure was used for experiments in Ca2+-free Krebs solution except that the tissues did not contract after transfer to a Ca2÷-free solution. For comparison of drug responsiveness the standard reference responses in Krebs solution were designated as the 100% responses.
2.5. Reserpine pretreatment schedule All injections of reserpine were administered i.p. Reserpine powder was dissolved in a 20% solution of ascorbic acid. The animals were divided into two groups: a control group which was treated with 0.4 to 0.5 ml of the vehicle once a day during 5 d a y s and a chronic reserpine-pretreated group which was injected with reserpine 1 mg/kg/day, for 5 days.
2.6. Drugs and solutions All drugs and solutions were prepared in double-distilled water which had been passed through a cation-exchange column. The drugs used were (--)-norepinephrine bitartrate, acetylcholine iodide and histamine diphosphate. All drug solutions were prepared immediately before use and 5 mg/ml of ascorbic acid was
CHRONIC RESERPINE TREATMENT AND ISOLATED VAS DEFERENS added to the solution of the sympathomimetic amine in order to reduce metal-catalysed oxidation. A 10-' M stock solution of CaC12 was prepared and used throughout the experiments. 2. 7. Statistical methods The methods used for evaluation of the data presented in this paper included Student's t-test for paired and unpaired samples, linear regression by the method of least squares and analysis of covariance (Snedecor, 1956). The 0.05 level of probability was accepted as significant.
3. Results 3.1. Effects o f chronic reserpine treatment on the sensitivity o f the vas deferens to norepinephrine, acetylcholine and histamine in Krebs solution In order to demonstrate nonspecific supersensitivity after reserpine, concentration--effect curves were constructed for three ago-
15
nists with different mechanisms of action. Table 1 summarizes the effects of reserpine on sensitivity and maximum response of the isolated vas deferens to norepinephrine, histamine and acetylcholine. Comparisons of the change in sensitivity, made by evaluating the dose ratio (ECs0 control/ECs0 reserpinetreated), showed that sensitization was greater for norepinephrine than f o r . h i s t a m i n e and acetylcholine. As shown in table 1 the changes in the maximum response of the vas deferens to norepinephrine, histamine and acetylcholine were not statistically significant (p > 0.05). 3.2. Lack o f influence o f reserpine treatment on the sensitivity o f the vas deferens to calcium Cumulative concentration--effect curves to calcium were constructed from vasa deferentia immersed in KC1--Ringer. Fig. 1 illustrates that reserpine pretreatment failed to increase sensitivity of the depolarized smooth muscle to calcium, the ECs0 values being 5.8
TABLE 1 The effect of chronic reserpine treatment on sensitivity and maximum contractile response of the vas deferens of the guinea pig to norepinephrine, histamine and acetylcholine. Group
Histamine
Norepinephrine n *
ECs0 ** X 10 -7 M
Ratio Maximum of response E C s ' 0 ' s ~ ( m m ~+ S . E . M . )
n *
7
100.3 (72.2--146.8)
--
36.5 -+ 1.9
ii
Reserpine
9
7.5 ~ (5.4--10.6)
13.3
42.5 + 3.4
7
Acetylcholine
Control Reserpine (1 m g / k g / 5
days
11
22.3 (10.2--48.3)
--
37.3 + 2.7
6
6 . 8 {~ (1.1--10.3)
3.2
3 5 . 8 -+ 4 . 0
* N u m b e r of e x p e r i m e n t s . ** See Materials and methods. E C s 0 c o n t r o l / E C 5 0 reserpine-treated. Significantly different from c o n t r o l (p < 0 . 0 5 ) .
Ratio
Maximum
of
response
ECs0's
Control
( i mg/kg/5 days
E C s 0 ** X 10 -6 M
~ (mm + S.E.M.)
40.6 (17.9--91.2)
--
15.8 + 1.6
7.4 ~ (1.1--15.0)
5.4
1 9 . 7 -+ 2 . 1
16
S. DE MORAES
W
100-t - - - , - - e C 0 NTR 0 L W (,/3
z 80-o
e , , - - , - e R ESE R P I N E TREATED
control
/t /
O. U) LLI
i N[
N£
NE W
/
-
%
40--
W
W
reserpine
20--
treated
Ol
"m
4
3 -- LOG
2
1
M
Fig. 1. Mean concentration--effect curves for calcium in depolarized vasa deferentia isolated from control and reserpine-treated guinea pigs (1 mg/kg/day, 5 days). Each point is the mean of 8 experiments. Vertical bars represent S.E.
Fig. 2. Diagrammatic drawing of a typical experiment on drug responsiveness of the isolated guinea pig vas deferens. See Materials and methods for further details. NE = norepinephrine 10 -4 M. The smooth muscles were depolarized by immersion in KCl-Ringer. W indicates washing process. Means of the results are presented in fig. 3.
(3.6--24.7) × 10 -3 M in the control (8 experiments) and 8.4 (4.1--17.5) X 10 ~3 M in the reserpine-treated group (8 experiments).
acetylcholine or histamine between control and supersensitive vasa deferentia the following experimental approach was used. The experiment is presented schematically in fig. 2. Each vas serves as its own control i.e., the smooth muscle was stimulated in Krebs solution with concentrations of the agonists which were found to elicit a maximum contraction. After establishing a constant response the tissue was transferred to KC1-Ringer or to Ca2+-free Krebs solution and submitted to the same drug concentration. In KC1--Ringer, the responsiveness of control tissues to norepinephrine, acetylcholine or histamine exhibited the same degree of nonsignificant reduction when compared with responses observed in Krebs solution. However, in the supersensitive depolarized vasa
3. 3. Effects of chronic reserpine treatment on drug responsiveness of the vas deferens in KCI--Ringer and in Ca2*-free Krebs solution In sodium- and calcium-free KC1--Ringer solution or in Ca2*-free Krebs solution concentration--effect curves to norepinephrine, acetylcholine or histamine could not be constructed. In each bathing solution only the first drug addition produced a noticeable response. Further drug additions induced smaller responses until no effect was recorded. In order to investigate possible differential responsiveness to norepinephrine,
CHRONIC RESERPINE TREATMENT A N D ISOLATED VAS DEFERENS
17
3 0 0 --
2 0 0 -\ \ \ \ \\\\ \ \ \ \
N,E 10-4 l
=~ 1 8 0 -
Ach 3.10 -5 M
4(" 160\\\\ \\\\ \\\\, \\\\ \\\\
140I¢:
HIST 3.10 -4 II
\\\\ \\\\ \\\\.
120-
\~,\\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ ",,, \,\\\ \~\\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \
100=I¢
__=
80-¢:¢
==
R
60--
\\\ \ \ \ \ \ \\ \\ \ \ \ \ \ \ \ \ \ \\\ \ , \ \
40 20 10
10
CONTROL
10
18
13
20
REgF.RPINE-IRE AIEI)
Fig. 3. Comparison of the responsiveness to norepinephrine (NE), acetylcholine (Ach) and histamine (Hist.) of control and supersensitive vasa deferentia in KC1--Ringer. Numbers inside the bars refer to number of experiments and vertical bars represent S.E. Asterisks indicate a significant difference from the responsiveness in Krebs solution (p < 0.05).
deferentia the responsiveness to the three stimulatory agents was greater than that observed in Krebs solution (fig. 3). Fig. 4 shows that in Ca2÷-free Krebs solution the responsiveness o f control and supersensitive vasa deferentia to norepinephrine and acetylcholine did not differ from that monitored in Krebs solution. However, after replacement of Krebs solution by Ca2+-free solution the response to a test concentration of histamine in control and supersensitive vasa deferentia was significantly increased. As can be seen in figs. 5, 6 and 7 the rate of loss of responsiveness to norepinephrine, acetylcholine and histamine in the depolarizing solution was also affected by chronic reserpine administration. In KC1--Ringer or in
Ca2+-free Krebs solution the loss of drug responsiveness of control and supersensitive vasa deferentia exhibited a fast exponential rate when the tissues were successively stimulated at 5 min intervals. A regression analysis of the rate of loss of drug responsiveness vs. time indicates a significant correlation (p < 0.01) for all the agonists studied both in control or supersensitive tissues. However, vasa deferentia which were removed from reserpine-treated animals and were depolarized in KC1--Ringer showed a slow loss of responsiveness. When norepinephrine was the agonist, the ratio of the slopes of the regression line in control tissues to that in the supersensitive vasa deferentia was 17.31; this indicated that the rate of loss of responsive-
300
200-
/ / / / /
/I///
,//// /I111
180--
Ach 3.10-5 M
o., 160-
13
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
THE EFFECTS OF CHRONIC RESERPINE TREATMENT ON THE CONTRACTILE A C T I V I T Y O F T H E I S O L A T E D VAS D E F E R E N S O F T H E G U I N E A P I G S]~RGIO DE MORAES Laboratory of Therapeutics, Faculty o f Veterinary Medicine, University of SSo Paulo, S~oPaulo, Brasil
Received 18 June 1975, revised MS received 29 December 1975, accepted 6 January 1976
DE MORAES, S. The effects of chronic reserpine treatment on the contractile activity of the isolated vas deferens of the guinea pig, European J. Pharmacol. 37 (1976) 13--22. Chronic reserpine treatment of guinea pigs during 5 days (1 mg/kg/day) induces postjunctional supersensitivity in the isolated vas deferens. It has been previously proposed that postjunctional supersensitivity occurs as a result of an ionic and/or membrane mechanism. Contrasting with previous observations in vascular smooth muscle the present results demonstrate that chronic reserpine treatment did not increase the sensitivity of the depolarized vas deferens to calcium. Experiments on drug responsiveness show that the supersensitive depolarized tissues have a greater and slower rate of loss of responsiveness than do control vasa deferentia. However, in a Ca 2+-free Krebs solution responsiveness of supersensitive vasa deferentia did not differ from that of control tissues. These findings suggest that, in the guinea-pig vas deferens, reserpine-induced supersensitivity could be at least partially dependent on the increased availability of a calcium store(s) probably located at the cell membrane and/or cytoplasmic compartments. Supersensitivity
Reserpine
Isolated vas deferens
1. I n t r o d u c t i o n P o s t j u n c t i o n a l s u p e r s e n s i t i v i t y is c h a r a c t e r ized b y an increased responsiveness o f tissues to d r u g s t i m u l a t i o n ( F l e m i n g et al., 1973). R e s e r p i n e - i n d u c e d p o s t j u n c t i o n a l supersensitivity o f t h e isolated guinea pig vas d e f e r e n s is a w e l l - k n o w n p h e n o m e n o n (Westfall, 1 9 7 0 a ) . T h e r e is g r o w i n g r e c o g n i t i o n t h a t p o s t j u n c t i o n a l s u p e r s e n s i t i v i t y is p r o d u c e d b y s o m e a l t e r a t i o n i.n t h e p h y s i o l o g y o f t h e r e s p o n d i n g cells, b e y o n d the r e c e p t o r level ( F l e m i n g 1963; Lee et al., 1 9 7 5 ) . T h e m o s t r e c e n t evidence indicates t h a t c a l c i u m ions are involved in r e s e r p i n e - i n d u c e d s u p e r s e n s i t i v i t y in vascular tissues (Hudgins a n d Harris, 1 9 7 0 ; G a r r e t a n d Carrier, 1 9 7 1 ; Carrier and J u r e vics, 1 9 7 3 ; Hester, 1974). Nevertheless, Westfall ( 1 9 7 0 b ) f o u n d no changes in t h e electrolyte levels o f vasa d e f e r e n t i a f r o m reserpinep r e t r e a t e d g u i n e a pigs. As c a l c i u m p l a y s a k e y role in the c o n t r a c t i l e a c t i v i t y o f s m o o t h
Drug responsiveness
muscle cells it was o f interest to t e s t w h e t h e r an e n h a n c e d availability o f calcium f o r cont r a c t i o n c o u l d be r e s p o n s i b l e f o r reserpine-ind u c e d supersensitivity. I n a d d i t i o n to confirming t h e d e v e l o p m e n t o f p o s t j u n c t i o n a l supersensitivity in t h e guinea pig vas d e f e r e n s a f t e r c h r o n i c reserpine a d m i n i s t r a t i o n , it is suggested t h a t an increased m o b i l i z a t i o n o f c a l c i u m for c o n t r a c t i o n c o u l d be a k e y m e c h a n i s m f o r r e s e r p i n e - i n d u c e d supersensitivity.
2. Materials and m e t h o d s 2.1. A n i m a l s
T h e animals used in t h e s e e x p e r i m e n t s w e r e guinea pigs o f a b o u t 5 0 0 g weight. T h e a n i m a l s were killed b y a b l o w o n t h e head. T h e vasa d e f e r e n t i a w e r e r e m o v e d and carefully d e s h e a t h e d .
14
2. 2. Bathing solutions The following bathing media were used (mM concentration). Krebs solution: NaC1 115.0; KC1 4.6; CaC12 2.5; KH2PO4 1.2; MgSO4 1.2; NaHCO3 2.5 and glucose 25; Ca2+-free Krebs solution: Krebs solution with no added calcium and with disodium edetate added to give a final concentration of 5 × 10 -6 M; sodium- and calcium-free KC1--Ringer (Edman and Schild, 1962): KC1 165.0; KHCO3 3.6; glucose 5.5 and 5 × 10 .6 M disodium edetate.
2. 3. Concentration--effect curves The method of setting up the tissues has been previously described (de Moraes and Carvalho, 1973). Contractions were monitored with isotonic myograph transducers coupled to a Narco recorder. The vasa deferentia were allowed to equilibrate in the muscle chamber under an initial load of 900 mg for at least 60 min before any procedure or drug testing. The bathing solutions were kept at 31°C and constantly bubbled with 95% oxygen and 5% carbon dioxide. The muscle chambers had a 22 ml working volume. Full concentration--effect curves were obtained by applying norepinephrine, acetylcholine or histamine in separate concentrations. The interval between successive drug concentrations was 5 min and after 30 sec the agonists were washed out thoroughly. Cumulative concentration--effect curves to calcium were constructed from vasa deferentia depolarized by replacing normal Krebs solution by KC1--Ringer. Changes in drug sensitivities were evaluated by comparing the ECs0 's i.e., the drug concentrations producing a response which was 50% of m a x i m u m in individual experiments. The mean EC so's are presented as geometric means with 95% confidence intervals (Fleming et al., 1972).
2. 4. Loss o f responsiveness These experiments were carried out in order to study the smooth muscle dependence
s. DE MORAES on calcium for contraction in vasa deferentia removed from untreated and reserpine-pretreated animals. The basic experimental protocol was as follows. After 60 min equilibration period in Krebs solution the tissues were initially submitted to maximal concentrations of norepinephrine, acetylcholine or histamine to elicit standard reference responses. After 30 min for recovery the preparations were transferred to KC1--Ringer. After the tissues returned to the baseline, which took approximately 4 min, the depolarizing solution was immediately replaced by fresh KC1-Ringer. The washing process was repeated twice with 1 min. The preparations were then submitted to the same drug concentration which produced the standard reference response in Krebs solution and thereafter successively exposed to the drug at 5-min intervals. The same procedure was used for experiments in Ca2+-free Krebs solution except that the tissues did not contract after transfer to a Ca2÷-free solution. For comparison of drug responsiveness the standard reference responses in Krebs solution were designated as the 100% responses.
2.5. Reserpine pretreatment schedule All injections of reserpine were administered i.p. Reserpine powder was dissolved in a 20% solution of ascorbic acid. The animals were divided into two groups: a control group which was treated with 0.4 to 0.5 ml of the vehicle once a day during 5 d a y s and a chronic reserpine-pretreated group which was injected with reserpine 1 mg/kg/day, for 5 days.
2.6. Drugs and solutions All drugs and solutions were prepared in double-distilled water which had been passed through a cation-exchange column. The drugs used were (--)-norepinephrine bitartrate, acetylcholine iodide and histamine diphosphate. All drug solutions were prepared immediately before use and 5 mg/ml of ascorbic acid was
CHRONIC RESERPINE TREATMENT AND ISOLATED VAS DEFERENS added to the solution of the sympathomimetic amine in order to reduce metal-catalysed oxidation. A 10-' M stock solution of CaC12 was prepared and used throughout the experiments. 2. 7. Statistical methods The methods used for evaluation of the data presented in this paper included Student's t-test for paired and unpaired samples, linear regression by the method of least squares and analysis of covariance (Snedecor, 1956). The 0.05 level of probability was accepted as significant.
3. Results 3.1. Effects o f chronic reserpine treatment on the sensitivity o f the vas deferens to norepinephrine, acetylcholine and histamine in Krebs solution In order to demonstrate nonspecific supersensitivity after reserpine, concentration--effect curves were constructed for three ago-
15
nists with different mechanisms of action. Table 1 summarizes the effects of reserpine on sensitivity and maximum response of the isolated vas deferens to norepinephrine, histamine and acetylcholine. Comparisons of the change in sensitivity, made by evaluating the dose ratio (ECs0 control/ECs0 reserpinetreated), showed that sensitization was greater for norepinephrine than f o r . h i s t a m i n e and acetylcholine. As shown in table 1 the changes in the maximum response of the vas deferens to norepinephrine, histamine and acetylcholine were not statistically significant (p > 0.05). 3.2. Lack o f influence o f reserpine treatment on the sensitivity o f the vas deferens to calcium Cumulative concentration--effect curves to calcium were constructed from vasa deferentia immersed in KC1--Ringer. Fig. 1 illustrates that reserpine pretreatment failed to increase sensitivity of the depolarized smooth muscle to calcium, the ECs0 values being 5.8
TABLE 1 The effect of chronic reserpine treatment on sensitivity and maximum contractile response of the vas deferens of the guinea pig to norepinephrine, histamine and acetylcholine. Group
Histamine
Norepinephrine n *
ECs0 ** X 10 -7 M
Ratio Maximum of response E C s ' 0 ' s ~ ( m m ~+ S . E . M . )
n *
7
100.3 (72.2--146.8)
--
36.5 -+ 1.9
ii
Reserpine
9
7.5 ~ (5.4--10.6)
13.3
42.5 + 3.4
7
Acetylcholine
Control Reserpine (1 m g / k g / 5
days
11
22.3 (10.2--48.3)
--
37.3 + 2.7
6
6 . 8 {~ (1.1--10.3)
3.2
3 5 . 8 -+ 4 . 0
* N u m b e r of e x p e r i m e n t s . ** See Materials and methods. E C s 0 c o n t r o l / E C 5 0 reserpine-treated. Significantly different from c o n t r o l (p < 0 . 0 5 ) .
Ratio
Maximum
of
response
ECs0's
Control
( i mg/kg/5 days
E C s 0 ** X 10 -6 M
~ (mm + S.E.M.)
40.6 (17.9--91.2)
--
15.8 + 1.6
7.4 ~ (1.1--15.0)
5.4
1 9 . 7 -+ 2 . 1
16
S. DE MORAES
W
100-t - - - , - - e C 0 NTR 0 L W (,/3
z 80-o
e , , - - , - e R ESE R P I N E TREATED
control
/t /
O. U) LLI
i N[
N£
NE W
/
-
%
40--
W
W
reserpine
20--
treated
Ol
"m
4
3 -- LOG
2
1
M
Fig. 1. Mean concentration--effect curves for calcium in depolarized vasa deferentia isolated from control and reserpine-treated guinea pigs (1 mg/kg/day, 5 days). Each point is the mean of 8 experiments. Vertical bars represent S.E.
Fig. 2. Diagrammatic drawing of a typical experiment on drug responsiveness of the isolated guinea pig vas deferens. See Materials and methods for further details. NE = norepinephrine 10 -4 M. The smooth muscles were depolarized by immersion in KCl-Ringer. W indicates washing process. Means of the results are presented in fig. 3.
(3.6--24.7) × 10 -3 M in the control (8 experiments) and 8.4 (4.1--17.5) X 10 ~3 M in the reserpine-treated group (8 experiments).
acetylcholine or histamine between control and supersensitive vasa deferentia the following experimental approach was used. The experiment is presented schematically in fig. 2. Each vas serves as its own control i.e., the smooth muscle was stimulated in Krebs solution with concentrations of the agonists which were found to elicit a maximum contraction. After establishing a constant response the tissue was transferred to KC1-Ringer or to Ca2+-free Krebs solution and submitted to the same drug concentration. In KC1--Ringer, the responsiveness of control tissues to norepinephrine, acetylcholine or histamine exhibited the same degree of nonsignificant reduction when compared with responses observed in Krebs solution. However, in the supersensitive depolarized vasa
3. 3. Effects of chronic reserpine treatment on drug responsiveness of the vas deferens in KCI--Ringer and in Ca2*-free Krebs solution In sodium- and calcium-free KC1--Ringer solution or in Ca2*-free Krebs solution concentration--effect curves to norepinephrine, acetylcholine or histamine could not be constructed. In each bathing solution only the first drug addition produced a noticeable response. Further drug additions induced smaller responses until no effect was recorded. In order to investigate possible differential responsiveness to norepinephrine,
CHRONIC RESERPINE TREATMENT A N D ISOLATED VAS DEFERENS
17
3 0 0 --
2 0 0 -\ \ \ \ \\\\ \ \ \ \
N,E 10-4 l
=~ 1 8 0 -
Ach 3.10 -5 M
4(" 160\\\\ \\\\ \\\\, \\\\ \\\\
140I¢:
HIST 3.10 -4 II
\\\\ \\\\ \\\\.
120-
\~,\\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ ",,, \,\\\ \~\\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \
100=I¢
__=
80-¢:¢
==
R
60--
\\\ \ \ \ \ \ \\ \\ \ \ \ \ \ \ \ \ \ \\\ \ , \ \
40 20 10
10
CONTROL
10
18
13
20
REgF.RPINE-IRE AIEI)
Fig. 3. Comparison of the responsiveness to norepinephrine (NE), acetylcholine (Ach) and histamine (Hist.) of control and supersensitive vasa deferentia in KC1--Ringer. Numbers inside the bars refer to number of experiments and vertical bars represent S.E. Asterisks indicate a significant difference from the responsiveness in Krebs solution (p < 0.05).
deferentia the responsiveness to the three stimulatory agents was greater than that observed in Krebs solution (fig. 3). Fig. 4 shows that in Ca2÷-free Krebs solution the responsiveness o f control and supersensitive vasa deferentia to norepinephrine and acetylcholine did not differ from that monitored in Krebs solution. However, after replacement of Krebs solution by Ca2+-free solution the response to a test concentration of histamine in control and supersensitive vasa deferentia was significantly increased. As can be seen in figs. 5, 6 and 7 the rate of loss of responsiveness to norepinephrine, acetylcholine and histamine in the depolarizing solution was also affected by chronic reserpine administration. In KC1--Ringer or in
Ca2+-free Krebs solution the loss of drug responsiveness of control and supersensitive vasa deferentia exhibited a fast exponential rate when the tissues were successively stimulated at 5 min intervals. A regression analysis of the rate of loss of drug responsiveness vs. time indicates a significant correlation (p < 0.01) for all the agonists studied both in control or supersensitive tissues. However, vasa deferentia which were removed from reserpine-treated animals and were depolarized in KC1--Ringer showed a slow loss of responsiveness. When norepinephrine was the agonist, the ratio of the slopes of the regression line in control tissues to that in the supersensitive vasa deferentia was 17.31; this indicated that the rate of loss of responsive-
300
200-
/ / / / /
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,//// /I111
180--
Ach 3.10-5 M
o., 160-
