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Effect of time and initial concentration of agonist on contractile responses in rat aorta
Effect of Time and Initial Concentration Contractile Responses in Rat Aorta
of Agonist on
S. A. DOCCRELL AND D. M. PATON
The effect of time and initial concentration of agonist used on contractile responses was studied in rat aorta. All responses to (-)-noradrenaline increase in magnitude with time. When different initial concentrations were used, the pDz values for (-)-noradrenaline were different and the slope of the dose-response curve for methoxamine was altered. These results indicate that it is necessary to provide controls for both time and initial concentration of agonist used when undertaking pharmacological studies with the rat aorta.
Key Words: Rat aorta; Noradrenaline; Effect of time; Effect of concentration.
Methoxamine;
Contractile
responses;
INTRODUCTION The sensitivity of isolated tissue preparations to agonists often changes during the course of an experiment. Thus, a marked increase in sensitivity to noradrenaline was observed during the first l-2 hr in guinea-pig vas deferens (Nedergaard and Westermann, 1968). Although Furchgott and Bhadrakom (1953) observed a similar increase in sensitivity to noradrenaline in rabbit aorta, Shibata, Briggs and Holland (1967) were unable to confirm these results. Thus, before a study of the effects of drugs on contractile responses is undertaken, it is necessary to design controls that permit measurements of, and corrections for, any spontaneous changes in the sensitivity of the tissue preparation to agonists during the course of an experiment; such changes in sensitivity are not due to the addition of a drug (Furchgott, 1972). As a preliminary study, we have investigated the effect of both time and initial concentration of agonist added on contractile responses to noradrenaline and methoxamine in rat aorta. METHODS Mature Wistar rats were sacrificed by a sharp blow at the base of the skull and exsanguinated. The thoracic aorta was then removed and helical strips prepared. Aortic strips were mounted vertically under 1.25 g tension in 5 ml organ baths containing a modified Krebs solution at 37”C, equilibrated with 95% Oz and 5% Accepted March 9, 1978. From the Department of Pharmacology and Clinical Pharmacology, School of Medicine, University of Aukland, New Zealand. Address reprint requests to: Dr. S. A. Doggrell, Department of Pharmacology and Clinical Pharmacology, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand. 115 @Elsevier North-Holland,
Inc., 1978, Journal of Pharmacological
Methods
1, 115-119 (1978)
01~540278/ow1-0115/$01.75
116
S. A. Doggrell and D. M. Paton
CO,. The tissues were allowed to recover for 60 min the resting tension being maintained throughout. Contractions were recorded isometrically with force displacement transducers (Grass model FT03.C) connected. to a polygraph (Grass model RPS 7C8A). Tissues were exposed to agonists until the maximum response to that concentration was attained. The agonist was then washed out and the tissue allowed to fully recover, which took from 5 to 45 min depending on the concentration of agonist added. The resting tension was readjusted to 1.25 g before further addition of agonist. For the time study of responses to (-)-noradrenaline the following procedure was used: 3 noncumulative dose-response curves were performed on each tissue starting at 60, 170, and 280 min after the tissues were mounted in the organ baths. The response to each concentration of (-)-noradrenaline were calculated as a percentage of the maximum response from the dose-response curve initiated at 60 min and were compared using Student’s paired t-test. The effect of the first concentration of (-)-noradrenaline or methoxamine used to elicit a contractile response on subsequent responses to higher concentrations of these agonists was also studied. On each tissue a noncumulative dose-response curve to increasing .concentrations of both (-)-noradrenaline and methoxamine was performed. The initial concentrations were 3~10-‘~ M or lop9 M for (-)-noradrenaline and 3~10-~ M, 3~10~~ M, or 10e6 M for methoxamine. Responses for each concentration of agonist were calculated as a percentage of the maximum response. Values for pD, (negative logarithm of molar concentrations of agonist producing 50% of the maximum response) were determined from individual doseresponse curves by regression line analysis (over the range 20-80% maximum response) using a computer (Amdahl 470). Each mean value was determined from a minimum of four separate preparations and is expressed ?SEM. Differences between means were analyzed using Student’s unpaired t-test and were regarded as significant when PcO.05. The drugs used were (-)-noradrenaline bitartrate (Sigma) and methoxamine hydrochloride (Burroughs-Wellcome). The modified Krebs solution had the following composition (mM): NaCI, 116; KCI, 5.4; CaCI,, 2.5; MgC&, 1.2; NaH,PO1, 1.2; NaHC03, 22.0; o-glucose, 11.2; NaEDTA, 0.04. RESULTS The responses to all concentrations of (-)-noradrenaline (3x10-* M-IO-’ M) were significantly greater when a dose-response curve was initiated at 170 min than at 60 min, and when initiated at 280 min as opposed to 170 min (Fig. 1, left). However, when responses were expressed as a percentage of the maximum response for each curve (i.e., at each time period), the responses to (-)-noradrenaline did not differ significantly (Fig. 1, right). Thus, the increasing sensitivity to (-)noradrenaline with time had no effect on the magnitude of responses when they were plotted as a percentage of the maximum response of the same curve. The dose-response curve to (-)-noradrenaline was completely different when
Time and Concentration
( - ) - Noradrenaline
Effects in Rat Aorta
(MI
FIGURE 1. Time study of contractile responses to (-)-noradrenaline in rat aortic strips. Dose-response curves were initiated at 60 min (O), 170 min (H), and 280 min (A) after tissues were suspended in the organ baths. Left, responses are expressed as a percentage of the maximum response in the dose-response curve initiated at 60 min; Right, responses are expressed as a percentage of the maximum response at each time period. Each value is the mean 2 SEA4from four preparations.
the initial concentration was 3x10-12 M as opposed to 10Pg M (Fig. 2, top). When the dose-response curve was initiated with 3~10~‘~ M, the maximum response to (-)-noradrenaline was obtained at 3x10-’ M, whereas when the first concentration used was 10mgM, the maximum response was observed at 3x10+ M. The slope of the dose-response curve to (-)-noradrenaline, within the 20-80% maximum response range, was similar whether the initial concentration of (-)-noradrenaline was 3x10-l2 M or 10Pg M. The pD, value for (-)-noradrenaline was 11.01 + 0.15 when the initial concentration was 3x10-I2 M; this was significantly different from the pD2 of 8.25 f 0.36 obtained when the initial concentration was 10Pg M. The initial concentration of methoxamine added also affected the subsequent dose-response curve (Fig. 2, bottom). A response within the range 30-40% maximum response was obtained with each of the initial concentrations of methoxamine used (3~10~~ M, 3x10-’ M, or 10e6 M). The maximum response to methoxamine was, however, always obtained with a concentration of low4 M. Thus, the slope of the dose-response curve was influenced by the first concentration of methoxamine added. As the slopes of the dose-response curves were different no valid comparison of pD, values could be undertaken.
1.17
118
S. A. Doggrell and D. M. Paton
100 80 _ 60 -$40_ s B ; 20E
g
01
E
300
’
IO _
1
-12
I
I
I
-10
IO -e IO ( - I- Noradrenaline (M 1
I
1
I
1O-6
a2 .E =: + 80s
Methoxamine
( Ml
FIGURE 2. Contactile responses to (-)-noradrenaline and methoxamine in rat aortic strips. Top, dose-response curves to (-)-noradrenaline were initiated with 3~10-‘~ M (0) and 10es M (m); Bottom, dose-response curves to methoxamine were initiated with 3~10~~ M (0). 3~10~’ M (m), and 10m6i (A). All responses are expressed as a percentage of the maximum response. Each value is the mean -C SEM from four preparations.
DISCUSSION The increasing sensitivity to all concentrations of noradrenaline with time in rat aorta continued thoughout the experiment (64 hr) and was not limited to the first l-2 hr as observed in guinea-pig vas deferens (Nedergaard and Westermann, 1968) or rabbit aorta (Furchgott and Bhadrakom, 1953). A similar phenomenon has been observed by Shibata et al. (1967), who demonstrated an increasing sensitivity to low concentrations of adrenaline following addition of a high concentration in rabbit aorta. However, in their study, increasing sensitivity did not extend to high con-
Time and Concentration
Effects in Rat Aorta
centrations of adrenaline or any concentration of noradrenaline following addition of a high concentration of that agonist. The response obtained with all the differing initial concentrations of noradrenaline or methoxamine added was of the same order (i.e., 20-40% maximal response). This does not exclude the possibility that different tensions were developed or that differing maximal tensions were obtained. Nevertheless, the initial concentration does alter the potency and/or slope of a normalized curve and, therefore, must be controled for when studying the effects of drugs on responses. Although dose-response curves to noradrenaline and to methoxamine were influenced by initial concentration added, the effects were dissimilar. Methoxamine is not a substrate for neuronal uptake (Trendelenburg, Maxwell, and Pluchino, 1970). However, it is unlikely that the difference in pattern of responses to noradrenaline and methoxamine is related to this as the rat aorta has a poor adrenergic innervation and cocaine is without effect on responses to noradrenaline (Maling, Fleisch, and Saul, 1971). The rat thoracic aorta contains /3-adrenoceptors which mediate relaxation (Fleisch, Maling, and Brodie, 1970). High concentrations or noradrenaline, but not methoxamine, will stimulate /3-adrenoceptors. Thus, it is possible, that at high concentrations noradrenaline, but not methoxamine, may stimulate both CX-and @adrenoceptors in rat aorta. This may account for the observed differences between the pattern of responses to noradrenaline and methoxamine. However, activation of /3-adrenoceptors cannot account for the effect of initial concentration on the shape of the dose-response curve to methoxamine. In conclusion, the studies have demonstrated that an increase in the magnitude of response with time must be taken into account when a study of the effect of drugs on responses is undertaken in rat aorta. In addition, the initial concentration of an agonist used may affect not only the slope of the dose-response curve but also the pDz values.
REFERENCES Fleisch JH, Maling HM, Brodie BB (1970) Beta-receptor activity in aorta. Variations with age and species. Circ Res 26: 151-162. Furchgott RF (1972) The classification of adrenoceptors (adrenergic receptors). An evaluation from the standpoint of receptor theory. In Handbook
of Experimental
Pharmacology
XXX/l/
Cat-
Eds., H Blaschko and E Muscholl. Berlin Heidelberg New York: Springer-Verlag, pp. 283-335.
echolamines.
Furchgott RF, Bhadrakom S (1953) Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrate and other drugs. / Pharmacol
Exp Ther 108: 129-143.
Maling HM, Fleisch JH, Saul WF (1971) Species differences in aortic responses to vasoactive
amines: The effects of compound 48/80, cocaine and 6-hydroxydopamine. / Pharmacol Exp Ther 176: 672-683.
Nedergaard OA, Westermann E (1968) Action of various sympathomimetic amines on the isolated vas deferens of the guinea-pig. Br / Pharmacol34:
475-485.
Shibata S, Briggs AH, Holland WC (1967) Epinephrine potentiation in isolated rabbit aortic strips. J Pharmacol Exp Ther 155: $22-427. Trendelenburg V, Maxwell RA, Pluchino S (1970) Methoxamine as a tool to assess the importance of intraneuronal uptake of (norepinephrine in the cat’s nictitating membrane. 1 Pharmacol Exp Ther 172: 91-99.
119
of Agonist on
S. A. DOCCRELL AND D. M. PATON
The effect of time and initial concentration of agonist used on contractile responses was studied in rat aorta. All responses to (-)-noradrenaline increase in magnitude with time. When different initial concentrations were used, the pDz values for (-)-noradrenaline were different and the slope of the dose-response curve for methoxamine was altered. These results indicate that it is necessary to provide controls for both time and initial concentration of agonist used when undertaking pharmacological studies with the rat aorta.
Key Words: Rat aorta; Noradrenaline; Effect of time; Effect of concentration.
Methoxamine;
Contractile
responses;
INTRODUCTION The sensitivity of isolated tissue preparations to agonists often changes during the course of an experiment. Thus, a marked increase in sensitivity to noradrenaline was observed during the first l-2 hr in guinea-pig vas deferens (Nedergaard and Westermann, 1968). Although Furchgott and Bhadrakom (1953) observed a similar increase in sensitivity to noradrenaline in rabbit aorta, Shibata, Briggs and Holland (1967) were unable to confirm these results. Thus, before a study of the effects of drugs on contractile responses is undertaken, it is necessary to design controls that permit measurements of, and corrections for, any spontaneous changes in the sensitivity of the tissue preparation to agonists during the course of an experiment; such changes in sensitivity are not due to the addition of a drug (Furchgott, 1972). As a preliminary study, we have investigated the effect of both time and initial concentration of agonist added on contractile responses to noradrenaline and methoxamine in rat aorta. METHODS Mature Wistar rats were sacrificed by a sharp blow at the base of the skull and exsanguinated. The thoracic aorta was then removed and helical strips prepared. Aortic strips were mounted vertically under 1.25 g tension in 5 ml organ baths containing a modified Krebs solution at 37”C, equilibrated with 95% Oz and 5% Accepted March 9, 1978. From the Department of Pharmacology and Clinical Pharmacology, School of Medicine, University of Aukland, New Zealand. Address reprint requests to: Dr. S. A. Doggrell, Department of Pharmacology and Clinical Pharmacology, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand. 115 @Elsevier North-Holland,
Inc., 1978, Journal of Pharmacological
Methods
1, 115-119 (1978)
01~540278/ow1-0115/$01.75
116
S. A. Doggrell and D. M. Paton
CO,. The tissues were allowed to recover for 60 min the resting tension being maintained throughout. Contractions were recorded isometrically with force displacement transducers (Grass model FT03.C) connected. to a polygraph (Grass model RPS 7C8A). Tissues were exposed to agonists until the maximum response to that concentration was attained. The agonist was then washed out and the tissue allowed to fully recover, which took from 5 to 45 min depending on the concentration of agonist added. The resting tension was readjusted to 1.25 g before further addition of agonist. For the time study of responses to (-)-noradrenaline the following procedure was used: 3 noncumulative dose-response curves were performed on each tissue starting at 60, 170, and 280 min after the tissues were mounted in the organ baths. The response to each concentration of (-)-noradrenaline were calculated as a percentage of the maximum response from the dose-response curve initiated at 60 min and were compared using Student’s paired t-test. The effect of the first concentration of (-)-noradrenaline or methoxamine used to elicit a contractile response on subsequent responses to higher concentrations of these agonists was also studied. On each tissue a noncumulative dose-response curve to increasing .concentrations of both (-)-noradrenaline and methoxamine was performed. The initial concentrations were 3~10-‘~ M or lop9 M for (-)-noradrenaline and 3~10-~ M, 3~10~~ M, or 10e6 M for methoxamine. Responses for each concentration of agonist were calculated as a percentage of the maximum response. Values for pD, (negative logarithm of molar concentrations of agonist producing 50% of the maximum response) were determined from individual doseresponse curves by regression line analysis (over the range 20-80% maximum response) using a computer (Amdahl 470). Each mean value was determined from a minimum of four separate preparations and is expressed ?SEM. Differences between means were analyzed using Student’s unpaired t-test and were regarded as significant when PcO.05. The drugs used were (-)-noradrenaline bitartrate (Sigma) and methoxamine hydrochloride (Burroughs-Wellcome). The modified Krebs solution had the following composition (mM): NaCI, 116; KCI, 5.4; CaCI,, 2.5; MgC&, 1.2; NaH,PO1, 1.2; NaHC03, 22.0; o-glucose, 11.2; NaEDTA, 0.04. RESULTS The responses to all concentrations of (-)-noradrenaline (3x10-* M-IO-’ M) were significantly greater when a dose-response curve was initiated at 170 min than at 60 min, and when initiated at 280 min as opposed to 170 min (Fig. 1, left). However, when responses were expressed as a percentage of the maximum response for each curve (i.e., at each time period), the responses to (-)-noradrenaline did not differ significantly (Fig. 1, right). Thus, the increasing sensitivity to (-)noradrenaline with time had no effect on the magnitude of responses when they were plotted as a percentage of the maximum response of the same curve. The dose-response curve to (-)-noradrenaline was completely different when
Time and Concentration
( - ) - Noradrenaline
Effects in Rat Aorta
(MI
FIGURE 1. Time study of contractile responses to (-)-noradrenaline in rat aortic strips. Dose-response curves were initiated at 60 min (O), 170 min (H), and 280 min (A) after tissues were suspended in the organ baths. Left, responses are expressed as a percentage of the maximum response in the dose-response curve initiated at 60 min; Right, responses are expressed as a percentage of the maximum response at each time period. Each value is the mean 2 SEA4from four preparations.
the initial concentration was 3x10-12 M as opposed to 10Pg M (Fig. 2, top). When the dose-response curve was initiated with 3~10~‘~ M, the maximum response to (-)-noradrenaline was obtained at 3x10-’ M, whereas when the first concentration used was 10mgM, the maximum response was observed at 3x10+ M. The slope of the dose-response curve to (-)-noradrenaline, within the 20-80% maximum response range, was similar whether the initial concentration of (-)-noradrenaline was 3x10-l2 M or 10Pg M. The pD, value for (-)-noradrenaline was 11.01 + 0.15 when the initial concentration was 3x10-I2 M; this was significantly different from the pD2 of 8.25 f 0.36 obtained when the initial concentration was 10Pg M. The initial concentration of methoxamine added also affected the subsequent dose-response curve (Fig. 2, bottom). A response within the range 30-40% maximum response was obtained with each of the initial concentrations of methoxamine used (3~10~~ M, 3x10-’ M, or 10e6 M). The maximum response to methoxamine was, however, always obtained with a concentration of low4 M. Thus, the slope of the dose-response curve was influenced by the first concentration of methoxamine added. As the slopes of the dose-response curves were different no valid comparison of pD, values could be undertaken.
1.17
118
S. A. Doggrell and D. M. Paton
100 80 _ 60 -$40_ s B ; 20E
g
01
E
300
’
IO _
1
-12
I
I
I
-10
IO -e IO ( - I- Noradrenaline (M 1
I
1
I
1O-6
a2 .E =: + 80s
Methoxamine
( Ml
FIGURE 2. Contactile responses to (-)-noradrenaline and methoxamine in rat aortic strips. Top, dose-response curves to (-)-noradrenaline were initiated with 3~10-‘~ M (0) and 10es M (m); Bottom, dose-response curves to methoxamine were initiated with 3~10~~ M (0). 3~10~’ M (m), and 10m6i (A). All responses are expressed as a percentage of the maximum response. Each value is the mean -C SEM from four preparations.
DISCUSSION The increasing sensitivity to all concentrations of noradrenaline with time in rat aorta continued thoughout the experiment (64 hr) and was not limited to the first l-2 hr as observed in guinea-pig vas deferens (Nedergaard and Westermann, 1968) or rabbit aorta (Furchgott and Bhadrakom, 1953). A similar phenomenon has been observed by Shibata et al. (1967), who demonstrated an increasing sensitivity to low concentrations of adrenaline following addition of a high concentration in rabbit aorta. However, in their study, increasing sensitivity did not extend to high con-
Time and Concentration
Effects in Rat Aorta
centrations of adrenaline or any concentration of noradrenaline following addition of a high concentration of that agonist. The response obtained with all the differing initial concentrations of noradrenaline or methoxamine added was of the same order (i.e., 20-40% maximal response). This does not exclude the possibility that different tensions were developed or that differing maximal tensions were obtained. Nevertheless, the initial concentration does alter the potency and/or slope of a normalized curve and, therefore, must be controled for when studying the effects of drugs on responses. Although dose-response curves to noradrenaline and to methoxamine were influenced by initial concentration added, the effects were dissimilar. Methoxamine is not a substrate for neuronal uptake (Trendelenburg, Maxwell, and Pluchino, 1970). However, it is unlikely that the difference in pattern of responses to noradrenaline and methoxamine is related to this as the rat aorta has a poor adrenergic innervation and cocaine is without effect on responses to noradrenaline (Maling, Fleisch, and Saul, 1971). The rat thoracic aorta contains /3-adrenoceptors which mediate relaxation (Fleisch, Maling, and Brodie, 1970). High concentrations or noradrenaline, but not methoxamine, will stimulate /3-adrenoceptors. Thus, it is possible, that at high concentrations noradrenaline, but not methoxamine, may stimulate both CX-and @adrenoceptors in rat aorta. This may account for the observed differences between the pattern of responses to noradrenaline and methoxamine. However, activation of /3-adrenoceptors cannot account for the effect of initial concentration on the shape of the dose-response curve to methoxamine. In conclusion, the studies have demonstrated that an increase in the magnitude of response with time must be taken into account when a study of the effect of drugs on responses is undertaken in rat aorta. In addition, the initial concentration of an agonist used may affect not only the slope of the dose-response curve but also the pDz values.
REFERENCES Fleisch JH, Maling HM, Brodie BB (1970) Beta-receptor activity in aorta. Variations with age and species. Circ Res 26: 151-162. Furchgott RF (1972) The classification of adrenoceptors (adrenergic receptors). An evaluation from the standpoint of receptor theory. In Handbook
of Experimental
Pharmacology
XXX/l/
Cat-
Eds., H Blaschko and E Muscholl. Berlin Heidelberg New York: Springer-Verlag, pp. 283-335.
echolamines.
Furchgott RF, Bhadrakom S (1953) Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrate and other drugs. / Pharmacol
Exp Ther 108: 129-143.
Maling HM, Fleisch JH, Saul WF (1971) Species differences in aortic responses to vasoactive
amines: The effects of compound 48/80, cocaine and 6-hydroxydopamine. / Pharmacol Exp Ther 176: 672-683.
Nedergaard OA, Westermann E (1968) Action of various sympathomimetic amines on the isolated vas deferens of the guinea-pig. Br / Pharmacol34:
475-485.
Shibata S, Briggs AH, Holland WC (1967) Epinephrine potentiation in isolated rabbit aortic strips. J Pharmacol Exp Ther 155: $22-427. Trendelenburg V, Maxwell RA, Pluchino S (1970) Methoxamine as a tool to assess the importance of intraneuronal uptake of (norepinephrine in the cat’s nictitating membrane. 1 Pharmacol Exp Ther 172: 91-99.
119