Acta physiol. scand. 1975. 94. 46-53 From the Department of Zoophysiology, University of Goteborg, Sweden
The Effect of ATP and Related Compounds on Spontaneous Mechanical Activity in the Rat Portal Vein BY BJORNSJOBERG and Bo A. WAHLSTROM
Received 26 November 1974
Abstract SJOBERG, B. and B. A. WAHLSTROM. The effect of ATPandrelated compounds on the spontaneous mechanical activity in the rat portal vein. Acta physiol. scand. 1975. 94. 46-53. The effects of ATP, ADP, A M P and adenosine were studied on the spontaneous mechanical activity of the rat portal vein. It was found that ATP and ADP, in concentrations higher than 300pM. caused a transient tetanus, followed by inhibition, and at lower concentrations an increase in the frequency and amplitude of the spontaneous contractions. A M P and adenosine on the other hand, inhibited spontaneous activity, by reducing the amplitude of contractions and increasing their frequency. The effects were dose-dependent. ATP was found to be 2.2 times more potent than ADP, while A M P and adenosine were equipotent. Weak inhibitory effects were obtained with GMP, guanosine and adenine, while GTP, 3'5'-cyclic A M P and guanine had n o effect. ATP and A D P increased the K-contracture, while A M P and adenosine relaxed it. The effects of A T P were augmented in Mg-free solutions and partially inhibited in Mg-high solutions in the normally polarized muscle, while Mg had no influence on the ATP-induced contraction in the depolarized muscle. Theophylline potentiated the inhibitory response to A M P and adenosine. Adrenergic and cholinergic blockers had no influence o n the response to ATP, ADP, A M P o r adenosine. It is suggested that the effects of ATP and A D P are linked with Ca++ movements across the membrane, while A M P and adenosine might stimulate intracellular metabolism causing increased intracellular Ca++ binding.
ATP and related compounds have been reported to cause relaxation or contraction in a wide variety of smooth muscles, the direction of the response depending on the type of tissue and the substance studied (Burnstock, Satchel1 and Smythe 1972). On intact circuiation ATP has been reported to produce fall in blood pressure (Drury and Scent-Gyorgyi 1928) increased coronary flow (Scholtholt, Bussman and Lochner 1965), and increased flow in skeletal muscle (Duff, Patterson and Shepherd 1954), while adenosine is a potent vasodilator of the coronary circulation (Berne, Rubio et al. 1971). Studies on isolated blood vessels have shown that adenosine relaxes coronary arteries in the rabbit (Norton, Gellai and Detar 1972). Both coronary and renal arteries in the mongrel dog are relaxed by ATP, AMP and adenosine (Walter and Bassenge 1968). Dog portal vein however, is contracted by adenosine (Walter and Bassenge 1968), while the rabbit portal vein relaxes in response to ATP, ADP and AMP (Hughes and Vane 1967). Daniel and Irwin (1965) showed that the effect of ATP on rat uterus was dependent upon the concentration of external Mg, and 46
ATP EFFECTS ON VASCULAR SMOOTH MUSCLE
47
could be potentiated by removing Mg++-ions from the bath. Similar results were obtained from the rat renal artery (Hrdina, Bonaccorsi and Benvenuti 1970). This study was made to investigate the effects of adenosine compounds on the rat portal vein, with regard to their possible interaction with Ca, Mg and exogenous acetylcholine and noradrenaline.
Methods White Sprague-Dawley rats of both sexes, weighing 200-250 g were used. They were stunned by a blow over the neck and bled out. The portal vein was rapidly dissected, tied to two hooks of stainless steel, connected via a plastic rod to a Grass FT03 transducer, mounted in an organ bath for recording of mechanical activity and stretched to a passive tension of 5.10-3 N. The preparation was left to recover for at least 60 min before any experiments were started. During this period the tension was adjusted a couple of times due to stress relaxation in the muscle. The spontaneous mechanical activity was continuously recorded and displayed on an inkwriting Gross Polygraph, together with the integrated activity, measured by an analogue electronic integrator. Excitatory effects of applied substances were calculated from the difference between the integrated mechanical activity during the drug application and the spontaneous activity preceding it and expressed in percent of maximal N A induced contraction. Inhibitory effects were expressed as percentage changes in amplitude and frequency of contractions, because, although the inhibitory response was always easily discernible to the naked eye, large increases in frequency combined with moderate decreases in amplitude often masked the effect on the integrated response. The physiological salt solution (PSS) used in these experiments was made up daily from isotonic stock solutions and had the following composition in mM: Na+ 137; K+ 6.0; Mg++ 1.2; Ca++ 2.5; Cl- 134; HCOT 15.5; H,PO? 1.2; Glucose 11.5. The temperature was 3 7 T , and all solutions were kept at pH 7.4 by constant bubbling with 97 % 0 , / 3 % CO,. Mg-free, Mg-low and Ca-low solutions were made by substituting MgCl, or CaCl, by NaCl on an equiosmolar basis. Mg-high, Ca-high and K-high solutions were made by substituting a proper amount of NaCl by MgCl,, CaCI, or KCl. The following substances were used: ATP, disodium salt (Sigma), ADP, disodium salt (Sigma), 5‘-AMP, monosodium salt (Sigma), 3’5’-cyclic AMP, acid (Sigma), Adenosine (Sigma), Adenine (Sigma), GTP, disodium salt (Sigma), GMP, monosodium salt (Sigma), Guanosine (Sigma), Guanine (Sigma), Theophylline (Sigma), Noradrenaline, free base (Sigma), Acetylcholine HCl (Sigma), Phenoxybenzamine HCI (Smith, Kline and French). Propranolol HCI (Sigma) and Atropine Sulfate (Sigma). Small amounts of concentrated stock solutions were made up each day as required and diluted to the final concentration in the organ bath. Numerical values are expressed as mean 1-2 S.E. and difference between means considered significant if p
The Effect of ATP and Related Compounds on Spontaneous Mechanical Activity in the Rat Portal Vein BY BJORNSJOBERG and Bo A. WAHLSTROM
Received 26 November 1974
Abstract SJOBERG, B. and B. A. WAHLSTROM. The effect of ATPandrelated compounds on the spontaneous mechanical activity in the rat portal vein. Acta physiol. scand. 1975. 94. 46-53. The effects of ATP, ADP, A M P and adenosine were studied on the spontaneous mechanical activity of the rat portal vein. It was found that ATP and ADP, in concentrations higher than 300pM. caused a transient tetanus, followed by inhibition, and at lower concentrations an increase in the frequency and amplitude of the spontaneous contractions. A M P and adenosine on the other hand, inhibited spontaneous activity, by reducing the amplitude of contractions and increasing their frequency. The effects were dose-dependent. ATP was found to be 2.2 times more potent than ADP, while A M P and adenosine were equipotent. Weak inhibitory effects were obtained with GMP, guanosine and adenine, while GTP, 3'5'-cyclic A M P and guanine had n o effect. ATP and A D P increased the K-contracture, while A M P and adenosine relaxed it. The effects of A T P were augmented in Mg-free solutions and partially inhibited in Mg-high solutions in the normally polarized muscle, while Mg had no influence on the ATP-induced contraction in the depolarized muscle. Theophylline potentiated the inhibitory response to A M P and adenosine. Adrenergic and cholinergic blockers had no influence o n the response to ATP, ADP, A M P o r adenosine. It is suggested that the effects of ATP and A D P are linked with Ca++ movements across the membrane, while A M P and adenosine might stimulate intracellular metabolism causing increased intracellular Ca++ binding.
ATP and related compounds have been reported to cause relaxation or contraction in a wide variety of smooth muscles, the direction of the response depending on the type of tissue and the substance studied (Burnstock, Satchel1 and Smythe 1972). On intact circuiation ATP has been reported to produce fall in blood pressure (Drury and Scent-Gyorgyi 1928) increased coronary flow (Scholtholt, Bussman and Lochner 1965), and increased flow in skeletal muscle (Duff, Patterson and Shepherd 1954), while adenosine is a potent vasodilator of the coronary circulation (Berne, Rubio et al. 1971). Studies on isolated blood vessels have shown that adenosine relaxes coronary arteries in the rabbit (Norton, Gellai and Detar 1972). Both coronary and renal arteries in the mongrel dog are relaxed by ATP, AMP and adenosine (Walter and Bassenge 1968). Dog portal vein however, is contracted by adenosine (Walter and Bassenge 1968), while the rabbit portal vein relaxes in response to ATP, ADP and AMP (Hughes and Vane 1967). Daniel and Irwin (1965) showed that the effect of ATP on rat uterus was dependent upon the concentration of external Mg, and 46
ATP EFFECTS ON VASCULAR SMOOTH MUSCLE
47
could be potentiated by removing Mg++-ions from the bath. Similar results were obtained from the rat renal artery (Hrdina, Bonaccorsi and Benvenuti 1970). This study was made to investigate the effects of adenosine compounds on the rat portal vein, with regard to their possible interaction with Ca, Mg and exogenous acetylcholine and noradrenaline.
Methods White Sprague-Dawley rats of both sexes, weighing 200-250 g were used. They were stunned by a blow over the neck and bled out. The portal vein was rapidly dissected, tied to two hooks of stainless steel, connected via a plastic rod to a Grass FT03 transducer, mounted in an organ bath for recording of mechanical activity and stretched to a passive tension of 5.10-3 N. The preparation was left to recover for at least 60 min before any experiments were started. During this period the tension was adjusted a couple of times due to stress relaxation in the muscle. The spontaneous mechanical activity was continuously recorded and displayed on an inkwriting Gross Polygraph, together with the integrated activity, measured by an analogue electronic integrator. Excitatory effects of applied substances were calculated from the difference between the integrated mechanical activity during the drug application and the spontaneous activity preceding it and expressed in percent of maximal N A induced contraction. Inhibitory effects were expressed as percentage changes in amplitude and frequency of contractions, because, although the inhibitory response was always easily discernible to the naked eye, large increases in frequency combined with moderate decreases in amplitude often masked the effect on the integrated response. The physiological salt solution (PSS) used in these experiments was made up daily from isotonic stock solutions and had the following composition in mM: Na+ 137; K+ 6.0; Mg++ 1.2; Ca++ 2.5; Cl- 134; HCOT 15.5; H,PO? 1.2; Glucose 11.5. The temperature was 3 7 T , and all solutions were kept at pH 7.4 by constant bubbling with 97 % 0 , / 3 % CO,. Mg-free, Mg-low and Ca-low solutions were made by substituting MgCl, or CaCl, by NaCl on an equiosmolar basis. Mg-high, Ca-high and K-high solutions were made by substituting a proper amount of NaCl by MgCl,, CaCI, or KCl. The following substances were used: ATP, disodium salt (Sigma), ADP, disodium salt (Sigma), 5‘-AMP, monosodium salt (Sigma), 3’5’-cyclic AMP, acid (Sigma), Adenosine (Sigma), Adenine (Sigma), GTP, disodium salt (Sigma), GMP, monosodium salt (Sigma), Guanosine (Sigma), Guanine (Sigma), Theophylline (Sigma), Noradrenaline, free base (Sigma), Acetylcholine HCl (Sigma), Phenoxybenzamine HCI (Smith, Kline and French). Propranolol HCI (Sigma) and Atropine Sulfate (Sigma). Small amounts of concentrated stock solutions were made up each day as required and diluted to the final concentration in the organ bath. Numerical values are expressed as mean 1-2 S.E. and difference between means considered significant if p
