Pharmacology 18: 149-154 (1979)
Effect of Clonidine on the Contractile Responsiveness of Aortic Smooth Muscle to Norepinephrine1 R.J. Ress, F.P. Field. O.E. Lockley2 and M.J. Fregly Department o f Physiology, Colleges o f Pharmacy and Medicine, University o f Florida, Gainesville, Fla.
Key Words. Antihypertensive agent • Aortic rings • Rats ■Vascular reactivity
The tension developed during experimental ly-induced contractions of vascular smooth muscle is known to be a function of its initial tension (6). Previous studies from this labora tory (4) have equilibrated circular aortic smooth muscle under an initial tension calcu lated by LaPlace’s law (tension = pressure X radius). Estimation of the radius of the artery from the arterial segment is subject to an error ' Supported by grant HL 14526-07 from the Nation al Heart and Lung Institute. 2 Minority Summer Hypertension Research Trainee supported by Grant 5T 32HL07298.
introduced by the contraction of aortic circular smooth muscle during preparation. Further more, differences in the mean blood pressure to which the aortic segment has been subjected may introduce another significant variable. Therefore, the initial objective of the studies described below was to determine the lengthtension relationship for aortic rings from ageand weight-matched normotensive rats. When this basic information was available, studies were carried out to test the responsive ness of aortic smooth muscle rings from rats to the antihypertensive agent, clonidine, alone and
Downloaded by: Nagoya University 133.6.82.173 - 8/1/2018 7:13:32 AM
Abstract. A length-tension relationship was established for aortic smooth muscle of rats by means of membrane depolarization with KC1. Maximal tension was developed by aortic rings when the preload initial tension was 8 -9 g. Two aortic rings (4 mm thick) were removed from segments of the aorta cut 1.5 (upper) and 2.4 cm (lower) below the aortic arch. Upper rings appeared to develop a greater tension than lower rings during depolarization with KC1. However, in spite of this quantitative difference between rings from upper and lower segments of the aorta, there were no qualitative differences observed. Clonidine, an antihypertensive agent, induced a contraction in aortic smooth muscle, apparently by way of a-adrenoreceptor stimulation. Clonidine also attenu ated significantly the development of active tension by norepinephrine but did not inhibit significantly the development of active tension following membrane depolarization with KC1. This suggests that there is a partial antagonism between norepinephrine and clonidine, presumably at the site of the receptors on smooth muscle, and that clonidine does not interfere with the contractile mechanism, per se.
Ress/Field/Lockley/Fregly
150
in combination with norepinephrine. Since clonidine was observed to decrease the respon siveness of aortic rings to norepinephrine, an additional study was carried out to determine whether clonidine affected the responsiveness of aortic rings to depolarization by KC1, and therefore, the contractile mechanism. The re sults of these studies are described below. Methods
allowed to equilibrate for 15 min before being ex posed to an 80 mM KCl solution. This procedure was repeated through 12 g o f resting tension at 1-gram increments. In this study two segments o f the thoracic aorta from each rat were removed. The segments were cut 1.5 (upper) and 2.5 cm (lower) below the aortic arch. Upper and lower segments from the same rat were studied simultaneously.
Experiment 2: Effect o f Clonidine on Tension Developed by Aortic Segments
6 male Sprague-Dawley derived rats weighing 345 t 22 g were used in this experiment. The aortic rings were removed 1.5 cm from the aortic arch and mount ed as described above. The passive tension was main 6 male adult Spraguc-Dawley derived rats weighingtained at 9 g throughout this experiment, as a result o f 362 ± 18 g were used. Tire animals were anesthetized the data obtained in experiment 1. The bath solutions with ether and sacrificed by cervical disclocation. The were changed at least once every 15 min and between aortae were excised and immediately placed into an dose-response determinations. aerated, modified Krebs solution at 26 °C (4). After After a 2-hour equilibration period, a cumulative adherent fat and loose connective tissue were re KCl ( 8 - 8 0 mAf) dose-response curve was generated to moved, two 4-mm rings were cut from the aortae by be certain that the aortic segment was active. The KCl means o f two surgical blades mounted 4 mm apart on was then washed out and the tension allowed to return a stainless steel block. The aortic rings were suspended to baseline level. A cumulative dose-response curve for between two stainless steel hooks inserted through the clonidine (1 0 ~ 13 to 1 0 '* M) was then determined. lumen to record circular smooth muscle contraction Freshly prepared solutions o f clonidine (kindly sup (11). The isolated tissue was mounted in a 20-ml plied by Dr. Stanley B. Garbus, Boehringer-lngelheim temperature-controlled (water-jacketed) muscle bath Corp.) were used in each study. and equilibrated for 2 h under 1 g o f tension which was maintained throughout the equilibration period. Experiment 3: Interaction between the Effects o f Contractions were recorded isometrically employing an Clonidine and Norepinephrine on Development o f
Experiment 1: Length-Tension Relationship for Smooth Muscle o f Rat Aorta Stimulated to Contract following Depolarization by KCl
Starting with an initial tension o f 1 g, the aortic segment was contracted maximally by the addition o f sufficient KCl to bring the bath solution to a concen tration o f 80 m M KC1/1. The tissue was allowed to contract for 15 min before the KCl was washed away with a change in the bath solution. The tension generated at the end o f the 15-min period was re corded in all cases. H ie initial (baseline) tension was then increased by 1 g and the aortic segment was
Active Tension by Aortic Smooth Muscle The objective o f this experiment was to determine whether clonidine and norepinephrine interacted at the receptor site on aortic smooth muscle. To ac complish this objective 6 adult male Sprague-Dawley derived rats weighing 320 ± 17 g were used. After a 2-hour equilibration period, a cumulative KCl ( 8 - 8 0 mM) dose-response curve was generated as de scribed above. The baseline tension on the aortic ring was set at 9 g. After washing out the KCl and the return to baseline tension, a cumulative norepineph rine dose-response curve was generated as described above. After completion o f the dose-response relation ship, the incubation medium was changed every 15 min for a minimum o f 1 h, at which time tension had returned to baseline level. At this time a cumu lative clonidine dose-response curve ( 1 0 ' 13 to 10"* M) was determined. The bath solution was then changed
Downloaded by: Nagoya University 133.6.82.173 - 8/1/2018 7:13:32 AM
F-50 microdisplacement myographic transducer with a model DMP-4B physiograph recorder (Narco Bio-Systems). The solution bathing the aortic rings was main tained at 37.5 °C and aerated with an 0 2:C 0j mixture (95:5%) which maintained the pH o f the solution at 7.3. The bath solutions were changed every 15 min during the equilibration period and between doseresponse determinations.
Clonidine on Responsiveness to Norepinephrine
and a cumulative norepinephrine ( 1 0 ' 10 to 10'* M) dose-response curve was generated in the presence o f clonidine (1 0 ‘ 4 M). Following this, the tissue was washed and allowed to return to baseline. At this time a second clonidine ( 1 0 ' 13 to 1 0 '4 M) dose-response curve was generated.
Experiment 4: Effect o f Clonidine on Responsive ness o f Aortic Smooth Muscle to KCl An experiment was designed to determine whether clonidine affected the responsiveness o f aortic smooth muscle to KCl. Aortic rings removed 1.5 cm from the aortic arch were prepared from 6 male Sprague-Dawley derived rats weighing 335 ± 21 g as described above and incubated under 9 g o f resting tension. After a 2-hour equilibrium period, a cumulative KCl dose-response curve ( 8 - 8 0 m M) was determined. The bath solutions were then changed and the tissues were allowed to return to baseline tension. After 15 min at baseline tension, the bath solutions were again changed and clonidine (1 0 ‘ 13 to 1 0 ' 4 M) was added cumulatively. Following this, the bath was again changed and an other KCl dose-response curve was determined imme
151
rings were compared at the same preload ten sions, significant (p
Effect of Clonidine on the Contractile Responsiveness of Aortic Smooth Muscle to Norepinephrine1 R.J. Ress, F.P. Field. O.E. Lockley2 and M.J. Fregly Department o f Physiology, Colleges o f Pharmacy and Medicine, University o f Florida, Gainesville, Fla.
Key Words. Antihypertensive agent • Aortic rings • Rats ■Vascular reactivity
The tension developed during experimental ly-induced contractions of vascular smooth muscle is known to be a function of its initial tension (6). Previous studies from this labora tory (4) have equilibrated circular aortic smooth muscle under an initial tension calcu lated by LaPlace’s law (tension = pressure X radius). Estimation of the radius of the artery from the arterial segment is subject to an error ' Supported by grant HL 14526-07 from the Nation al Heart and Lung Institute. 2 Minority Summer Hypertension Research Trainee supported by Grant 5T 32HL07298.
introduced by the contraction of aortic circular smooth muscle during preparation. Further more, differences in the mean blood pressure to which the aortic segment has been subjected may introduce another significant variable. Therefore, the initial objective of the studies described below was to determine the lengthtension relationship for aortic rings from ageand weight-matched normotensive rats. When this basic information was available, studies were carried out to test the responsive ness of aortic smooth muscle rings from rats to the antihypertensive agent, clonidine, alone and
Downloaded by: Nagoya University 133.6.82.173 - 8/1/2018 7:13:32 AM
Abstract. A length-tension relationship was established for aortic smooth muscle of rats by means of membrane depolarization with KC1. Maximal tension was developed by aortic rings when the preload initial tension was 8 -9 g. Two aortic rings (4 mm thick) were removed from segments of the aorta cut 1.5 (upper) and 2.4 cm (lower) below the aortic arch. Upper rings appeared to develop a greater tension than lower rings during depolarization with KC1. However, in spite of this quantitative difference between rings from upper and lower segments of the aorta, there were no qualitative differences observed. Clonidine, an antihypertensive agent, induced a contraction in aortic smooth muscle, apparently by way of a-adrenoreceptor stimulation. Clonidine also attenu ated significantly the development of active tension by norepinephrine but did not inhibit significantly the development of active tension following membrane depolarization with KC1. This suggests that there is a partial antagonism between norepinephrine and clonidine, presumably at the site of the receptors on smooth muscle, and that clonidine does not interfere with the contractile mechanism, per se.
Ress/Field/Lockley/Fregly
150
in combination with norepinephrine. Since clonidine was observed to decrease the respon siveness of aortic rings to norepinephrine, an additional study was carried out to determine whether clonidine affected the responsiveness of aortic rings to depolarization by KC1, and therefore, the contractile mechanism. The re sults of these studies are described below. Methods
allowed to equilibrate for 15 min before being ex posed to an 80 mM KCl solution. This procedure was repeated through 12 g o f resting tension at 1-gram increments. In this study two segments o f the thoracic aorta from each rat were removed. The segments were cut 1.5 (upper) and 2.5 cm (lower) below the aortic arch. Upper and lower segments from the same rat were studied simultaneously.
Experiment 2: Effect o f Clonidine on Tension Developed by Aortic Segments
6 male Sprague-Dawley derived rats weighing 345 t 22 g were used in this experiment. The aortic rings were removed 1.5 cm from the aortic arch and mount ed as described above. The passive tension was main 6 male adult Spraguc-Dawley derived rats weighingtained at 9 g throughout this experiment, as a result o f 362 ± 18 g were used. Tire animals were anesthetized the data obtained in experiment 1. The bath solutions with ether and sacrificed by cervical disclocation. The were changed at least once every 15 min and between aortae were excised and immediately placed into an dose-response determinations. aerated, modified Krebs solution at 26 °C (4). After After a 2-hour equilibration period, a cumulative adherent fat and loose connective tissue were re KCl ( 8 - 8 0 mAf) dose-response curve was generated to moved, two 4-mm rings were cut from the aortae by be certain that the aortic segment was active. The KCl means o f two surgical blades mounted 4 mm apart on was then washed out and the tension allowed to return a stainless steel block. The aortic rings were suspended to baseline level. A cumulative dose-response curve for between two stainless steel hooks inserted through the clonidine (1 0 ~ 13 to 1 0 '* M) was then determined. lumen to record circular smooth muscle contraction Freshly prepared solutions o f clonidine (kindly sup (11). The isolated tissue was mounted in a 20-ml plied by Dr. Stanley B. Garbus, Boehringer-lngelheim temperature-controlled (water-jacketed) muscle bath Corp.) were used in each study. and equilibrated for 2 h under 1 g o f tension which was maintained throughout the equilibration period. Experiment 3: Interaction between the Effects o f Contractions were recorded isometrically employing an Clonidine and Norepinephrine on Development o f
Experiment 1: Length-Tension Relationship for Smooth Muscle o f Rat Aorta Stimulated to Contract following Depolarization by KCl
Starting with an initial tension o f 1 g, the aortic segment was contracted maximally by the addition o f sufficient KCl to bring the bath solution to a concen tration o f 80 m M KC1/1. The tissue was allowed to contract for 15 min before the KCl was washed away with a change in the bath solution. The tension generated at the end o f the 15-min period was re corded in all cases. H ie initial (baseline) tension was then increased by 1 g and the aortic segment was
Active Tension by Aortic Smooth Muscle The objective o f this experiment was to determine whether clonidine and norepinephrine interacted at the receptor site on aortic smooth muscle. To ac complish this objective 6 adult male Sprague-Dawley derived rats weighing 320 ± 17 g were used. After a 2-hour equilibration period, a cumulative KCl ( 8 - 8 0 mM) dose-response curve was generated as de scribed above. The baseline tension on the aortic ring was set at 9 g. After washing out the KCl and the return to baseline tension, a cumulative norepineph rine dose-response curve was generated as described above. After completion o f the dose-response relation ship, the incubation medium was changed every 15 min for a minimum o f 1 h, at which time tension had returned to baseline level. At this time a cumu lative clonidine dose-response curve ( 1 0 ' 13 to 10"* M) was determined. The bath solution was then changed
Downloaded by: Nagoya University 133.6.82.173 - 8/1/2018 7:13:32 AM
F-50 microdisplacement myographic transducer with a model DMP-4B physiograph recorder (Narco Bio-Systems). The solution bathing the aortic rings was main tained at 37.5 °C and aerated with an 0 2:C 0j mixture (95:5%) which maintained the pH o f the solution at 7.3. The bath solutions were changed every 15 min during the equilibration period and between doseresponse determinations.
Clonidine on Responsiveness to Norepinephrine
and a cumulative norepinephrine ( 1 0 ' 10 to 10'* M) dose-response curve was generated in the presence o f clonidine (1 0 ‘ 4 M). Following this, the tissue was washed and allowed to return to baseline. At this time a second clonidine ( 1 0 ' 13 to 1 0 '4 M) dose-response curve was generated.
Experiment 4: Effect o f Clonidine on Responsive ness o f Aortic Smooth Muscle to KCl An experiment was designed to determine whether clonidine affected the responsiveness o f aortic smooth muscle to KCl. Aortic rings removed 1.5 cm from the aortic arch were prepared from 6 male Sprague-Dawley derived rats weighing 335 ± 21 g as described above and incubated under 9 g o f resting tension. After a 2-hour equilibrium period, a cumulative KCl dose-response curve ( 8 - 8 0 m M) was determined. The bath solutions were then changed and the tissues were allowed to return to baseline tension. After 15 min at baseline tension, the bath solutions were again changed and clonidine (1 0 ‘ 13 to 1 0 ' 4 M) was added cumulatively. Following this, the bath was again changed and an other KCl dose-response curve was determined imme
151
rings were compared at the same preload ten sions, significant (p
