Acta physiol. scand. 1975. 93. 77-84 From the Department of Surgery 11, Sahlgrenska sjukhuset, University of Goteborg, Sweden
Comparative Studies on the Effects of Bradykinin and Vagal Stimulation on Motility in the Stomach and Colon BY S. FASTH, L. H U L T ~ NT., JAHNBERGand J. MARTINSON Received 31 May 1974
Abstract FASTH,S., L. HULTEN,T. JAHNBERGand J. MARTINSON. Comparative studies on the effects of bradykinin and vagal stimulation on motility in the stomach and colon. Acta physiol. scand. 1975. 93. 77-84. The effect of bradykinin on gastric and colonic motility was studied in anaesthetized cats with volume recording devices and compared with the effects of vagal nerve stimulation. When administered intraarterially bradykinin caused a profound and prolonged gastric relaxation. Simultaneously there was a marked and likewise prolonged colonic contraction. The gastric relaxation closely mimicked the atropine resistant relaxation elicited by vagal nerve stimulation. These effects could not be blocked by antiadrenergic drugs and it is suggested that bradykinin and the unknown transmittor substance(s) released on vagal stimulation act in a similar way on the gastric smooth muscles and that a kinin mechanism may be involved in the vagal response. As regards the colonic motor response it was shown that bradykinin does not reproduce the vagal motility effects on colon smooth muscle but mimicks closely the atropine resistant expulsive contraction elicited by activation of the pelvic nerves.
Studies on the extrinsic nervous control of the motility in the stomach and colon indicate that specific parasympathetic fibre sets are involved in the receptive relaxation of the stomach and the expulsive colonic contraction (Martinson 1965, HultCn 1969). These motility responses elicited after vagal and pelvic nerve stimulation respectively appear to be neither purecholinergic nor adrenergic. After cessation of the stimulation the receptive relaxation and the colonic contraction are extremely slowly eliminated in contrast to most other types of nervous effector responses. The transmittor mechanisms involved in these longlasting motor responses are obscure. The gastric receptive relaxation and the colonic expulsive contraction represents a specialized reservoir function in both organs. I n the stomach and the colon the respective responses are closely linked to a regional blood flow increase occurring concomitantly. The transmission mechanism responsible for this functional hyperemia is also largely unknown. A plasma kinin has turned out to be involved in the atropine-resistant functional vasodilatation of the cat’s submandibular gland (Hilton and Lewis 1955, 1956, Gautvik 1970, Gautvik, Nystad and Vystad 1972) and it is tempting to believe that a similar mecha77
78
s. FASTH,
L. H U L T ~ N ,T. JAHNBERC AND J. MARTINSON
nism might be involved in functional hyperemia in the glandular tissue of the stomach and colon. It has in fact recently been shown that bradykinin (Bk) can reproduce in detail not only the effect of pelvic nerve stimulation on the consecutive vascular sections of the colon but also the motor contraction which is closely linked to the vascular response (Fasth and Hulttn 1973 a, b). The present study was undertaken to see whether Bk could also reproduce the receptive gastric relaxation occurring after efferent vagal stimulation.
Material and Methods Experiments were performed on 18 cats weighing 2.4-5.3 kg and fasted for 24 h. The animals were anaesthetized intravenously with chloralose (50 mg/kg b.w.) after induction with ether. A tracheal cannula was inserted to allow a free airway. The right femoral artery was connected to a mercury manometer for recording of arterial pressure. Recording of gastric and colonic oofume. Since abdominal surgery interferes considerably with the gastric responses on vagal stimulation (Jansson 1969) another approach was preferred. The gastric motility was recorded by means of a large thinwalled rubber balloon introduced in the stomach via esophagus. The balloon was connected to a water-filled reservoir of wide dimensions coupled to a piston recorder or to a force transducer (Grass force displacement transducer FT 10) operating a potentiometer writer (Servogor 2 C).By means of such an arrangement intragastric pressure could be kept constant at about 4-5 cm H,O. In 7 expts. colonic motility was simultaneously recorded by means of a similar volume recording device. A plastic tube was introduced into the distal colon via anus and connected to a waterfilled pressure reservoir. The intraluminal pressure was kept constant at about 10 cm H,O. The pressure reservoir was coupled to a piston recorder operating on the kymograph (Fig. 1). Administration of drugs. T o allow for close intraarterial BK-administration a thin polyethylene catheter was inserted into the left femoral artery and passed upwards the aorta to a level some 3 4 cm above the celiac artery. Bk (kindly supported by Bofors Nobel-Pharma) was dissolved in 0.9% saline in concentrations between 1.0-100 pg/rnl and administered as bolus injections via the indwelling catheter. In some expts. Bk was also administered as i.v. injections. Other drugs used were: Atropine sulphate (Merck), guanethidine (Ismelin", Ciba), hexamethoniumchloride (Merck), phenoxybenzamine hydrochloride (Dibenzyline", Smith Kline & French), propranolol chloride (Inderal", Scanmeda) and histamine-chloride (ACO). In some experiments gallamine triethiodide (Flaxedil@, May and Baker) was used to eliminate somatomotor reflex activity. Artificial respiration was then maintained by a respiration pump. Neroous stimulation. The vagal nerves were in all experiments dissected free at the cervical level for subsequent stimulation. The peripheral cut ends were then mounted on silver ring electrodes. Stimulation parameters were usually 5 imp./s, 5 ms, 8 V. delivered from a Grass stimulator model 5 SE.
Results I. The effect of intraaortic Bk injections and vagal stimulation on gastric and colonic volume. As is shown in Fig. 2 intraaortic injection of Bk (10 yg) gave rise to a characteristic increase of gastric volume occurring within 5-10 s. A slight relaxation was sometimes observed after 1 pg Bk. In a dose of 100 pg Bk caused a somewhat more powerful response than did 10 pg Bk. The higher dose was followed by a profound decrease in systemic arterial blood pressure however, and was therefore avoided. The relaxatory response always occurred rapidly and reached its maximum within about 10-20 s. The gastric volume then decreased slowly and did not reach control level until 15-20 min had elapsed. Stimulation of the vagal nerves at high rates (usually 5 Hz 5 ms 8V) caused sometimes
79
BRADYKlNlN ON STOMACH MOTILITY
Fig. 1. Schematic illustration of the technique used for studying gastric and colonic volume changes.
a slight immediate gastric contraction. After cessation of the stimulation there was a powerful relaxation which in all respects closely mimicked the response observed on Bk injection (Fig. 2). Quantitatively the change in gastric volume varied between 20-70 ml in different cats but was in a given animal always of the same magnitude as that induced by subsequent vagal nerve stimulation. The relaxatory response was not abolished after Flaxedil@. If anyARTERAL BLOOD PRESSURE mmHg
;
,Lo
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y
~
~ ~~~
01
BRADYKlNlN 10 ug VAGAL NERVE STlM 5/sec. Smscc.BV TIME min
1
-
1
3
5
7
9
11
13
15
25
27 29
31
33
35
37
39
M
L3
Fig. 2. Cat 3.1 kg. The effect of intraaortic administration of bradykinin (left panel) and vagal stimulation on gastric volume (right panel). Note the identical responses which are characterized by a rapid, powerful relaxation and an extremely slow elimination.
s. FASTH,
80
GASTRIC VOLUME (mll
L.
HULTEN,
T. JAHNBERG AND J . MARTINSON
5D
--L-
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I
COLONIC VOLUME
(ml) BRAOYKININ 10 ua
& -1
Fig. 3. Cat 5.3 kg. The effect of bradykinin and vagal stimulation on gastric and colonic volume after atropine ( I mg/ kg i.v.) and hexamethonium (5 mg/kg i.v.). Note that the gastric relaxation contrary to the colonic response is not abolished after atropine (left panel). Note also that hexamethonium eliminates the vagal effects whereas the effects of bradykinin are unaffected (middle and right panel).
thing the Bk-response was more powerful after vagotomy partly due to the considerable decrease in gastric volume often seen after this procedure. concomitant with the gastric relaxation the colon responded to Bk injection by a powerful contraction which disappeared slowly within 5-1 5 min. Vagal stimulation produced an immediate increase of colonic motility which rapidly returned to “resting” level on cessation of the stimulation. There was no poststimulatory relaxation like that regularly observed in the stomach. After atropine ( I mg/kg) the gastric response to intraaortal Bk injection appeared unchanged whereas vagal stimulation gave rise to an immediate relaxation. The initial decrease in gastric volume was not observed indicating that the gastric motor response to Bk and vagal stimulation was completely identical after atropine (Fig. 3). The colonic contraction as elicited by Bk was not affected by atropine, whereas the increased motility regularly seen after vagal stimulation was completely abolished. Ganglionic blockade by means of hexamethonium ( 5 mg/kg b.w.) failed to affect the gastric and colonic response to intraaortic Bk injections. I n contrast the response to vagal stimulation were reduced or completely abolished. Guanethidine ( 5 mg/kg b.w.), known to abolish adrenergic nervous transmission, did not interfere with the gastric responses to intraaortic Bk injections and vagal stimulation (Fig. 4) Phenoxybenzamine ( 5 mg/kg b.w.1 and propranolol (3 mg/kg b.w.), well-known in blocking alfa- and betaadrenergic receptors respectively were also ineffective in this respect (Fig. 5 ) . The magnitude of the realxation was often less pronounced, however, mainly due to the lowered gastric smooth muscle tone produced by these antiadrenergic drugs. The effects of Bk on colonic motility also appeared unchanged after adrenergic blockade. 11. The effect of intravenous Bk injection on gustric volume. Intravenous Bk injection in
doses exceeding 10 pg caused only a moderate gastric relaxation appearing after some 30-50 s. This relaxation differed in many respects from that obtained after intraaortic Bk injection, however. Thus the volume increase which was considerably less pronounced,
81
BRADYKININ ON STOMACH MOTILITY
1
25 GASTRIC VOLUME
(rnll
L VAGAL NERVE SiIM 5/sec,Srnsec,BV
3
1
TIME rnin
5
7
15
17
19 ZI
23 25 27 29
3l
7,S,?l',?,?l
Fig. 4. Cat 2.3 kg. The effect of bradykinin and vagal stimulation o n gastric volume after guanethidine ( 5 mg/kg i.v.). The gastric relaxation elicited by bradykinin (middle panel) and vagal stimulation (right panel) are both unaffected.
amounting to at the most 5 ml as compared with 70 ml on intraaortic administration developed more slowly and ceased within 3 4 min. Furthermore it could be abolished after pretreatment with alfa- and betaadrenergic blocking drugs. Concomitant with the gastric relaxation there was an inhibition of the colonic motility.
05 ARTERIAL BLOOD PRESSURE mmHg
65 45
.
~
25
~
GASTRIC VOLUME (ml)
BRADYKININ 10 pg VAGAL NERVE STlM 5lsec,0msec,0V TIME min
t
1
3
5
17
19 21
23
25
33
35
37
39 41
L?
, 47 , L?
Fig. 5 . Cat 2.8 kg. The effect of bradykinin and vagal stimulation on gastric volume after propranolol (3 mg/kg i.v.) and phenoxybenzamine ( 5 mg/kg i.v.). Note that the relaxatory effects are still elicited after propranolol (middle panel) and phenoxybenzamine (right panel).
6 755871 -
s. FASTH,
L.
HULTBN,
T. JAHNBERG AND J. MARTINSON
ARTERIAL BLOOD PRESSURE mmHg
GASTRIC VOLUME (ml)
Fig. 6. Cat 5.3 kg, atropinized ( I mg/kg i.v.). The effect of intraaortic injection of histamine and bradykinin on gastric and colonic volume. Note the insignificant motor effects after histamine, compared to the protracted gastric relaxation and colonic contraction after bradykinin.
COLONIC VOLUME lrnll BRADYKlNlN 10 p g
HISTAMINE 10 pg VAGAL NERVE STlM 5kc.8rnsec. B V
TIME rnin
III. The effects of bradykinin and histamine on the motility in the stomach and colon. For comparison the effects of intraaortic histamine injections were studied in 5 expts. Histamine (10 pg) elicited a very moderate gastric relaxation and colonic contraction appearing within some 5-10 s. As is shown in Fig. 6 the responses differed also in many other respects from that following Bk. Thus the gastric relaxation after histamine developed more slowly and returned to control level within 2-3 min. The shortlasting decrease in colonic volume also differed considerably from that induced by Bk. The gastric response to histamine was on the whole similar to that occurring after intravenously administered Bk.
Discussion In the present study it has been shown that Bk causes a gastric relaxation and colonic expulsive contraction which are qualitatively and quantitatively almost identical to the m3tor effects elicited by vagal and pelvic nerve stimulation respectively. These nervous responses are considered to be involved in the gastric receptive relaxation and colonic expulsive contraction respectively (Martinson 1965, Hultkn 1969). Intraaortic Bk injections might hypothetically bring about gastric relaxation i n different ways. In most intestinal in vitro preparations Bk causes contraction, sometimes preceded by shortlasting relaxation (Rocha e Silva, Beraldo and Rosenfeld 1949, Konzett and Sturmer 1960, Turker, Kiran and Kaymalacan 1964, Fishloch 1966). In these studies Bk is supposed to exert its effects by a direct action on the smooth muscles. This is also in accordance with results presented by Gershon (1967). Bk has also been shown to inhibit even other smooth muscle preparations among other strips from the rat gastric fundus (Rocha e Silva 1963). Such a sympathicomimetic effect is neither caused by a local catecholamine release nor by interaction with the adrenergic receptors but may rather be dependent on the actual tone of smooth muscles (Antonia 1968, Hall and Bonta 1972). Studies in uiuo suggest that Bk can stimulate nociceptive paravascular nerve endings evoking reflex inhibition either relaying at supraspinal or at ganglionic level with subsequent release into the blood stream of catecholamines from the adrenal medulla (Della Bella, Benelli and Paoli 1972). It has also
BRADYKININ O N STOMACH MOTILITY
83
been shown that Bk can inhibit smooth muscles byadirect effect on the ganglioniccells(Lewi-, and Reit 1965). Furthermore Bk has turned out to be a potent releaser of catecholamines by a direct action on the adrenal medulla (Feldberg and Lewis 1964). In fact it has recently been shown that Bk, when administered intravenously causes a profound intestinal inhibition which is abolished after adrenalectomy (Fasth and Hultkn 1973 a). In the present study the gastric relaxation on intraaortic Bk injection which occurred within some 5-10 s was not abolished by antiadrenergic drugs and differed largely from that evoked by adrenaline and noradrenaline (Martinson 1965). It therefore appears unlikely that the response is caused by an indirect reflex release of catecholamines from the adrenal medulla. As compared with the effects of intraaortal administration the moderate gastric relaxatory effect of Bk injected intravenously occurred after a long latency and the colonic motility recorded simultaneously was then slightly inhibited. It therefore appears likely that this response was caused by an adrenergic mechanism. This is in accordance with a previous study (Fasth and Hulten 1973 a) which showed evidence that Bk administered intravenously inhibitis intestinal motility while intraarterial administration regularly gives rise to increased motility. The gastric relaxation and colonic contraction elicited by Bk could not be blocked by hexamethonium, indicating that Bk does not exert its effects by a direct action on the ganglionic cells. In this context it should be mentioned that the contraction of the nictitating membrane in cats in response to Bk, though unaffected by hexamethonium, was nevertheless shown to be caused by a direct stimulation of the superior cervical ganglion (Lewis and Reit 1965). The concentration of Bk exceeded considerably that in the present study and the motor response differed largely both qualitatively and quantitatively. The results of the present investigation therefore indicate that the gastric relaxation elicited by intraaortic Bk injection is neither caused by an adrenergic mechanism nor by a direct ganglion stimulating effect but is rather due to a direct effect on the gastric smooth muscles. The transmittor substance released and responsible for the potent motor effects elicited via vagal and pelvic nerves is, as mentioned still unknown. In view of the present results it may be tempting to believe that a kinin-like substance is involved. Since the motor responses are characteristically protracted local release of such specific transmittor agents must obviously be eliminated and/or inactivated at a very slow rate. Kinins are rapidly inactivated in the organism by peptidases, however (Erdos 1966). It therefore appearsless likely that the longlasting motor responses observed in the stomach and colon are due to continous action of the peptide per se unless some unknown mechanism interferes with its inactivation or elimination. Another explanation might be that these prolonged motor effects are induced along other pathways outside or within the smooth muscle cells. As regards the stomach it has been suggested that intramural nonadrenergic inhibitory neurons or purinergic nerves are involved in the vagal gastric relaxation (Burnstock 1972). As regards the longlasting colonic contraction such a mechanism is less likely however, since the purinergic nerves exert a similar relaxatory effect on the colonic smooth muscles and lack extrinsic nervous influence. It has previously been shown that Bk can in detail reproduce the effect of pelvic nerve stimulation on colonic motility and blood flow (Fasth and HultCn 1973 a, b) and even the
84
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T. JAHNBERG AND J. MARTINSW
effect of vagal stimulation on gastric circulation at least partly (Fasth and Martinson 1973). In the present series of experiments Bk was also shown to reproduce the vagally induced gastric relaxation. Further investigations are necessary to elucidiate whether a kinin-like substance is in fact involved in the nervous regulation of the stomach and colon. This research has been sponsored from the Swedish Research Council (872-17X-3126-02).from the Faculty of Medicine, University of Goteborg and from Goteborgs Lakaresallskap.
References ANTONIO, A., The relaxing effect of bradykinin on intestinal smooth muscle. Brir. J . Pliarmacol. 1968. 32. 78-86. BURNSTOCK, G., Purinergic nerves. Pharmacol. Reo. 1972. 24. 509-58 I . DELLABELLA.D., G. BENELLI and A. M. DEPAOLI,Indirect nervous mechanism in some effects of bradykinin. Arclr. in/. Pharmacoclyn. Suppl. 196: 50. 50-63. ERDOS,E. G., Hypotensive peptides Bradykinin, kallidin and eledosin. Adiianc. Pharmacol. 1966. 4. I .89. FASTH,S . and L. H U L T ~ N The , effect of bradykinin o n the motility and blood flow in the small and large intestine. Acta chir. scand. 1973 a. 139. 699-705. FASTH,S. and L. H U L T ~ N Neurohumoral , regulation of motility and blood flow in the colon. Expcvkvlia (Basel) 1973 b. 29: 3. 296-297. FASTH,S. and J. MARTINSON, O n the possible role of bradykinin in functional hyperemia of cats stomach. Acm physiol. scand. 1973. 89. 334-341. FELDBERG, W. and G. P. LEWIS,The action of peptides on the adrenal medulla. Release of adrenaline by bradykinin and angiotensin. J . Physiol. (Lond.). 1964. 171. 98-108. FISHLOCH, D. J., Effect of bradykinin o n the human isolated small and large intestine. Noritre (Lond.) 1966. 212. 1533-1535. GAUTVIK, K., Studies o n kininformation in functional vasodilatation of the submandibular salivary gland in cats. Acta physiol. scnnd. 1970. 79. 174-187. GAUTVIK, K., K. NYSTADand J. VYSTAD,Kininogenase activity in the stimulated submandibular salivary gland in cats. Acta physiol. scanrl. 1972. 85. 438-445. GERSHON,M. D., Effects of tetrodotoxin o n innervated smooth muscle preparations. Bri/. J. Pharmacol. 1967. 29. 259-279. HALL,D. W. R. and J. L. BONTA,Neurogenic factors involved in the relaxing effect of bradykinin on the isolated guinea-pig ileum. Arch. in/. Pharmarodyn. 1972. 197. 380-381. HILTON, S . M . and G. P. LEWIS.The mechanism of the functional hyperaemia in the submandibular salivary gland. J. Physiol. (Lond.). 1955. 129. 253-271. HILTON,S. M. and G. P. LEWIS,The relationship between glandular activity, bradykinin formation and functional vasodilatation in the submandibular salivary gland. J. Physiol. (Lond.). 1956. 134. 471-483. H U L T ~ NL., , Extrinsic nervous control of colonic motility and blood flow. Aria physiol. .wand. 1969. Suppl. 335. JANSSON, G., Extrinsic nervous control of gastric motility. An experimental study in the cat. Aria physiol. s c a d 1969. Suppl. 326. KONZETT, M. and E. STURMER,Biological activity of synthetic, polypeptides with bradykinin properties. Brit. J. Pharmacol. 1960. 15. 544-55 I. LEWIS,G. P. and E. REIT,The action of angiotensin and bradykinin on the superior cervival ganglion of the cat. J. Physiol. (Lond.). 1965. 179. 538-553. MARTINSON, J., Studies o n the efferent vagal control of the stomach. Acm physiol. scant/. 1965. 65. Siippl. 255. ROCHAE SILVA,R. M., W. E. BERALDO and G. ROSENFELD, Bradykinin, hypotensive and smooth muscle stimulating factor released from plasma globulin by snake venon and by trypsin. Anier. J. Physiol. 1949. I56. 261-273. ROCHAE SILVA.R. M., Acquisitions recentes sur la bradykinine. Acrualires phaniiacol. 1963. 15. 163-198. TURKER,K., B. K. KIRANand S . KAYMALACAN, The effects of synthetic bradykinin on intestinal motility i n different laboratory animals and in relation t o catecholamines. Arch. in/. Pharmacol. 1964. 151. 260-268.
Comparative Studies on the Effects of Bradykinin and Vagal Stimulation on Motility in the Stomach and Colon BY S. FASTH, L. H U L T ~ NT., JAHNBERGand J. MARTINSON Received 31 May 1974
Abstract FASTH,S., L. HULTEN,T. JAHNBERGand J. MARTINSON. Comparative studies on the effects of bradykinin and vagal stimulation on motility in the stomach and colon. Acta physiol. scand. 1975. 93. 77-84. The effect of bradykinin on gastric and colonic motility was studied in anaesthetized cats with volume recording devices and compared with the effects of vagal nerve stimulation. When administered intraarterially bradykinin caused a profound and prolonged gastric relaxation. Simultaneously there was a marked and likewise prolonged colonic contraction. The gastric relaxation closely mimicked the atropine resistant relaxation elicited by vagal nerve stimulation. These effects could not be blocked by antiadrenergic drugs and it is suggested that bradykinin and the unknown transmittor substance(s) released on vagal stimulation act in a similar way on the gastric smooth muscles and that a kinin mechanism may be involved in the vagal response. As regards the colonic motor response it was shown that bradykinin does not reproduce the vagal motility effects on colon smooth muscle but mimicks closely the atropine resistant expulsive contraction elicited by activation of the pelvic nerves.
Studies on the extrinsic nervous control of the motility in the stomach and colon indicate that specific parasympathetic fibre sets are involved in the receptive relaxation of the stomach and the expulsive colonic contraction (Martinson 1965, HultCn 1969). These motility responses elicited after vagal and pelvic nerve stimulation respectively appear to be neither purecholinergic nor adrenergic. After cessation of the stimulation the receptive relaxation and the colonic contraction are extremely slowly eliminated in contrast to most other types of nervous effector responses. The transmittor mechanisms involved in these longlasting motor responses are obscure. The gastric receptive relaxation and the colonic expulsive contraction represents a specialized reservoir function in both organs. I n the stomach and the colon the respective responses are closely linked to a regional blood flow increase occurring concomitantly. The transmission mechanism responsible for this functional hyperemia is also largely unknown. A plasma kinin has turned out to be involved in the atropine-resistant functional vasodilatation of the cat’s submandibular gland (Hilton and Lewis 1955, 1956, Gautvik 1970, Gautvik, Nystad and Vystad 1972) and it is tempting to believe that a similar mecha77
78
s. FASTH,
L. H U L T ~ N ,T. JAHNBERC AND J. MARTINSON
nism might be involved in functional hyperemia in the glandular tissue of the stomach and colon. It has in fact recently been shown that bradykinin (Bk) can reproduce in detail not only the effect of pelvic nerve stimulation on the consecutive vascular sections of the colon but also the motor contraction which is closely linked to the vascular response (Fasth and Hulttn 1973 a, b). The present study was undertaken to see whether Bk could also reproduce the receptive gastric relaxation occurring after efferent vagal stimulation.
Material and Methods Experiments were performed on 18 cats weighing 2.4-5.3 kg and fasted for 24 h. The animals were anaesthetized intravenously with chloralose (50 mg/kg b.w.) after induction with ether. A tracheal cannula was inserted to allow a free airway. The right femoral artery was connected to a mercury manometer for recording of arterial pressure. Recording of gastric and colonic oofume. Since abdominal surgery interferes considerably with the gastric responses on vagal stimulation (Jansson 1969) another approach was preferred. The gastric motility was recorded by means of a large thinwalled rubber balloon introduced in the stomach via esophagus. The balloon was connected to a water-filled reservoir of wide dimensions coupled to a piston recorder or to a force transducer (Grass force displacement transducer FT 10) operating a potentiometer writer (Servogor 2 C).By means of such an arrangement intragastric pressure could be kept constant at about 4-5 cm H,O. In 7 expts. colonic motility was simultaneously recorded by means of a similar volume recording device. A plastic tube was introduced into the distal colon via anus and connected to a waterfilled pressure reservoir. The intraluminal pressure was kept constant at about 10 cm H,O. The pressure reservoir was coupled to a piston recorder operating on the kymograph (Fig. 1). Administration of drugs. T o allow for close intraarterial BK-administration a thin polyethylene catheter was inserted into the left femoral artery and passed upwards the aorta to a level some 3 4 cm above the celiac artery. Bk (kindly supported by Bofors Nobel-Pharma) was dissolved in 0.9% saline in concentrations between 1.0-100 pg/rnl and administered as bolus injections via the indwelling catheter. In some expts. Bk was also administered as i.v. injections. Other drugs used were: Atropine sulphate (Merck), guanethidine (Ismelin", Ciba), hexamethoniumchloride (Merck), phenoxybenzamine hydrochloride (Dibenzyline", Smith Kline & French), propranolol chloride (Inderal", Scanmeda) and histamine-chloride (ACO). In some experiments gallamine triethiodide (Flaxedil@, May and Baker) was used to eliminate somatomotor reflex activity. Artificial respiration was then maintained by a respiration pump. Neroous stimulation. The vagal nerves were in all experiments dissected free at the cervical level for subsequent stimulation. The peripheral cut ends were then mounted on silver ring electrodes. Stimulation parameters were usually 5 imp./s, 5 ms, 8 V. delivered from a Grass stimulator model 5 SE.
Results I. The effect of intraaortic Bk injections and vagal stimulation on gastric and colonic volume. As is shown in Fig. 2 intraaortic injection of Bk (10 yg) gave rise to a characteristic increase of gastric volume occurring within 5-10 s. A slight relaxation was sometimes observed after 1 pg Bk. In a dose of 100 pg Bk caused a somewhat more powerful response than did 10 pg Bk. The higher dose was followed by a profound decrease in systemic arterial blood pressure however, and was therefore avoided. The relaxatory response always occurred rapidly and reached its maximum within about 10-20 s. The gastric volume then decreased slowly and did not reach control level until 15-20 min had elapsed. Stimulation of the vagal nerves at high rates (usually 5 Hz 5 ms 8V) caused sometimes
79
BRADYKlNlN ON STOMACH MOTILITY
Fig. 1. Schematic illustration of the technique used for studying gastric and colonic volume changes.
a slight immediate gastric contraction. After cessation of the stimulation there was a powerful relaxation which in all respects closely mimicked the response observed on Bk injection (Fig. 2). Quantitatively the change in gastric volume varied between 20-70 ml in different cats but was in a given animal always of the same magnitude as that induced by subsequent vagal nerve stimulation. The relaxatory response was not abolished after Flaxedil@. If anyARTERAL BLOOD PRESSURE mmHg
;
,Lo
g
y
~
~ ~~~
01
BRADYKlNlN 10 ug VAGAL NERVE STlM 5/sec. Smscc.BV TIME min
1
-
1
3
5
7
9
11
13
15
25
27 29
31
33
35
37
39
M
L3
Fig. 2. Cat 3.1 kg. The effect of intraaortic administration of bradykinin (left panel) and vagal stimulation on gastric volume (right panel). Note the identical responses which are characterized by a rapid, powerful relaxation and an extremely slow elimination.
s. FASTH,
80
GASTRIC VOLUME (mll
L.
HULTEN,
T. JAHNBERG AND J . MARTINSON
5D
--L-
,,I:,
I
COLONIC VOLUME
(ml) BRAOYKININ 10 ua
& -1
Fig. 3. Cat 5.3 kg. The effect of bradykinin and vagal stimulation on gastric and colonic volume after atropine ( I mg/ kg i.v.) and hexamethonium (5 mg/kg i.v.). Note that the gastric relaxation contrary to the colonic response is not abolished after atropine (left panel). Note also that hexamethonium eliminates the vagal effects whereas the effects of bradykinin are unaffected (middle and right panel).
thing the Bk-response was more powerful after vagotomy partly due to the considerable decrease in gastric volume often seen after this procedure. concomitant with the gastric relaxation the colon responded to Bk injection by a powerful contraction which disappeared slowly within 5-1 5 min. Vagal stimulation produced an immediate increase of colonic motility which rapidly returned to “resting” level on cessation of the stimulation. There was no poststimulatory relaxation like that regularly observed in the stomach. After atropine ( I mg/kg) the gastric response to intraaortal Bk injection appeared unchanged whereas vagal stimulation gave rise to an immediate relaxation. The initial decrease in gastric volume was not observed indicating that the gastric motor response to Bk and vagal stimulation was completely identical after atropine (Fig. 3). The colonic contraction as elicited by Bk was not affected by atropine, whereas the increased motility regularly seen after vagal stimulation was completely abolished. Ganglionic blockade by means of hexamethonium ( 5 mg/kg b.w.) failed to affect the gastric and colonic response to intraaortic Bk injections. I n contrast the response to vagal stimulation were reduced or completely abolished. Guanethidine ( 5 mg/kg b.w.), known to abolish adrenergic nervous transmission, did not interfere with the gastric responses to intraaortic Bk injections and vagal stimulation (Fig. 4) Phenoxybenzamine ( 5 mg/kg b.w.1 and propranolol (3 mg/kg b.w.), well-known in blocking alfa- and betaadrenergic receptors respectively were also ineffective in this respect (Fig. 5 ) . The magnitude of the realxation was often less pronounced, however, mainly due to the lowered gastric smooth muscle tone produced by these antiadrenergic drugs. The effects of Bk on colonic motility also appeared unchanged after adrenergic blockade. 11. The effect of intravenous Bk injection on gustric volume. Intravenous Bk injection in
doses exceeding 10 pg caused only a moderate gastric relaxation appearing after some 30-50 s. This relaxation differed in many respects from that obtained after intraaortic Bk injection, however. Thus the volume increase which was considerably less pronounced,
81
BRADYKININ ON STOMACH MOTILITY
1
25 GASTRIC VOLUME
(rnll
L VAGAL NERVE SiIM 5/sec,Srnsec,BV
3
1
TIME rnin
5
7
15
17
19 ZI
23 25 27 29
3l
7,S,?l',?,?l
Fig. 4. Cat 2.3 kg. The effect of bradykinin and vagal stimulation o n gastric volume after guanethidine ( 5 mg/kg i.v.). The gastric relaxation elicited by bradykinin (middle panel) and vagal stimulation (right panel) are both unaffected.
amounting to at the most 5 ml as compared with 70 ml on intraaortic administration developed more slowly and ceased within 3 4 min. Furthermore it could be abolished after pretreatment with alfa- and betaadrenergic blocking drugs. Concomitant with the gastric relaxation there was an inhibition of the colonic motility.
05 ARTERIAL BLOOD PRESSURE mmHg
65 45
.
~
25
~
GASTRIC VOLUME (ml)
BRADYKININ 10 pg VAGAL NERVE STlM 5lsec,0msec,0V TIME min
t
1
3
5
17
19 21
23
25
33
35
37
39 41
L?
, 47 , L?
Fig. 5 . Cat 2.8 kg. The effect of bradykinin and vagal stimulation on gastric volume after propranolol (3 mg/kg i.v.) and phenoxybenzamine ( 5 mg/kg i.v.). Note that the relaxatory effects are still elicited after propranolol (middle panel) and phenoxybenzamine (right panel).
6 755871 -
s. FASTH,
L.
HULTBN,
T. JAHNBERG AND J. MARTINSON
ARTERIAL BLOOD PRESSURE mmHg
GASTRIC VOLUME (ml)
Fig. 6. Cat 5.3 kg, atropinized ( I mg/kg i.v.). The effect of intraaortic injection of histamine and bradykinin on gastric and colonic volume. Note the insignificant motor effects after histamine, compared to the protracted gastric relaxation and colonic contraction after bradykinin.
COLONIC VOLUME lrnll BRADYKlNlN 10 p g
HISTAMINE 10 pg VAGAL NERVE STlM 5kc.8rnsec. B V
TIME rnin
III. The effects of bradykinin and histamine on the motility in the stomach and colon. For comparison the effects of intraaortic histamine injections were studied in 5 expts. Histamine (10 pg) elicited a very moderate gastric relaxation and colonic contraction appearing within some 5-10 s. As is shown in Fig. 6 the responses differed also in many other respects from that following Bk. Thus the gastric relaxation after histamine developed more slowly and returned to control level within 2-3 min. The shortlasting decrease in colonic volume also differed considerably from that induced by Bk. The gastric response to histamine was on the whole similar to that occurring after intravenously administered Bk.
Discussion In the present study it has been shown that Bk causes a gastric relaxation and colonic expulsive contraction which are qualitatively and quantitatively almost identical to the m3tor effects elicited by vagal and pelvic nerve stimulation respectively. These nervous responses are considered to be involved in the gastric receptive relaxation and colonic expulsive contraction respectively (Martinson 1965, Hultkn 1969). Intraaortic Bk injections might hypothetically bring about gastric relaxation i n different ways. In most intestinal in vitro preparations Bk causes contraction, sometimes preceded by shortlasting relaxation (Rocha e Silva, Beraldo and Rosenfeld 1949, Konzett and Sturmer 1960, Turker, Kiran and Kaymalacan 1964, Fishloch 1966). In these studies Bk is supposed to exert its effects by a direct action on the smooth muscles. This is also in accordance with results presented by Gershon (1967). Bk has also been shown to inhibit even other smooth muscle preparations among other strips from the rat gastric fundus (Rocha e Silva 1963). Such a sympathicomimetic effect is neither caused by a local catecholamine release nor by interaction with the adrenergic receptors but may rather be dependent on the actual tone of smooth muscles (Antonia 1968, Hall and Bonta 1972). Studies in uiuo suggest that Bk can stimulate nociceptive paravascular nerve endings evoking reflex inhibition either relaying at supraspinal or at ganglionic level with subsequent release into the blood stream of catecholamines from the adrenal medulla (Della Bella, Benelli and Paoli 1972). It has also
BRADYKININ O N STOMACH MOTILITY
83
been shown that Bk can inhibit smooth muscles byadirect effect on the ganglioniccells(Lewi-, and Reit 1965). Furthermore Bk has turned out to be a potent releaser of catecholamines by a direct action on the adrenal medulla (Feldberg and Lewis 1964). In fact it has recently been shown that Bk, when administered intravenously causes a profound intestinal inhibition which is abolished after adrenalectomy (Fasth and Hultkn 1973 a). In the present study the gastric relaxation on intraaortic Bk injection which occurred within some 5-10 s was not abolished by antiadrenergic drugs and differed largely from that evoked by adrenaline and noradrenaline (Martinson 1965). It therefore appears unlikely that the response is caused by an indirect reflex release of catecholamines from the adrenal medulla. As compared with the effects of intraaortal administration the moderate gastric relaxatory effect of Bk injected intravenously occurred after a long latency and the colonic motility recorded simultaneously was then slightly inhibited. It therefore appears likely that this response was caused by an adrenergic mechanism. This is in accordance with a previous study (Fasth and Hulten 1973 a) which showed evidence that Bk administered intravenously inhibitis intestinal motility while intraarterial administration regularly gives rise to increased motility. The gastric relaxation and colonic contraction elicited by Bk could not be blocked by hexamethonium, indicating that Bk does not exert its effects by a direct action on the ganglionic cells. In this context it should be mentioned that the contraction of the nictitating membrane in cats in response to Bk, though unaffected by hexamethonium, was nevertheless shown to be caused by a direct stimulation of the superior cervical ganglion (Lewis and Reit 1965). The concentration of Bk exceeded considerably that in the present study and the motor response differed largely both qualitatively and quantitatively. The results of the present investigation therefore indicate that the gastric relaxation elicited by intraaortic Bk injection is neither caused by an adrenergic mechanism nor by a direct ganglion stimulating effect but is rather due to a direct effect on the gastric smooth muscles. The transmittor substance released and responsible for the potent motor effects elicited via vagal and pelvic nerves is, as mentioned still unknown. In view of the present results it may be tempting to believe that a kinin-like substance is involved. Since the motor responses are characteristically protracted local release of such specific transmittor agents must obviously be eliminated and/or inactivated at a very slow rate. Kinins are rapidly inactivated in the organism by peptidases, however (Erdos 1966). It therefore appearsless likely that the longlasting motor responses observed in the stomach and colon are due to continous action of the peptide per se unless some unknown mechanism interferes with its inactivation or elimination. Another explanation might be that these prolonged motor effects are induced along other pathways outside or within the smooth muscle cells. As regards the stomach it has been suggested that intramural nonadrenergic inhibitory neurons or purinergic nerves are involved in the vagal gastric relaxation (Burnstock 1972). As regards the longlasting colonic contraction such a mechanism is less likely however, since the purinergic nerves exert a similar relaxatory effect on the colonic smooth muscles and lack extrinsic nervous influence. It has previously been shown that Bk can in detail reproduce the effect of pelvic nerve stimulation on colonic motility and blood flow (Fasth and HultCn 1973 a, b) and even the
84
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T. JAHNBERG AND J. MARTINSW
effect of vagal stimulation on gastric circulation at least partly (Fasth and Martinson 1973). In the present series of experiments Bk was also shown to reproduce the vagally induced gastric relaxation. Further investigations are necessary to elucidiate whether a kinin-like substance is in fact involved in the nervous regulation of the stomach and colon. This research has been sponsored from the Swedish Research Council (872-17X-3126-02).from the Faculty of Medicine, University of Goteborg and from Goteborgs Lakaresallskap.
References ANTONIO, A., The relaxing effect of bradykinin on intestinal smooth muscle. Brir. J . Pliarmacol. 1968. 32. 78-86. BURNSTOCK, G., Purinergic nerves. Pharmacol. Reo. 1972. 24. 509-58 I . DELLABELLA.D., G. BENELLI and A. M. DEPAOLI,Indirect nervous mechanism in some effects of bradykinin. Arclr. in/. Pharmacoclyn. Suppl. 196: 50. 50-63. ERDOS,E. G., Hypotensive peptides Bradykinin, kallidin and eledosin. Adiianc. Pharmacol. 1966. 4. I .89. FASTH,S . and L. H U L T ~ N The , effect of bradykinin o n the motility and blood flow in the small and large intestine. Acta chir. scand. 1973 a. 139. 699-705. FASTH,S. and L. H U L T ~ N Neurohumoral , regulation of motility and blood flow in the colon. Expcvkvlia (Basel) 1973 b. 29: 3. 296-297. FASTH,S. and J. MARTINSON, O n the possible role of bradykinin in functional hyperemia of cats stomach. Acm physiol. scand. 1973. 89. 334-341. FELDBERG, W. and G. P. LEWIS,The action of peptides on the adrenal medulla. Release of adrenaline by bradykinin and angiotensin. J . Physiol. (Lond.). 1964. 171. 98-108. FISHLOCH, D. J., Effect of bradykinin o n the human isolated small and large intestine. Noritre (Lond.) 1966. 212. 1533-1535. GAUTVIK, K., Studies o n kininformation in functional vasodilatation of the submandibular salivary gland in cats. Acta physiol. scnnd. 1970. 79. 174-187. GAUTVIK, K., K. NYSTADand J. VYSTAD,Kininogenase activity in the stimulated submandibular salivary gland in cats. Acta physiol. scanrl. 1972. 85. 438-445. GERSHON,M. D., Effects of tetrodotoxin o n innervated smooth muscle preparations. Bri/. J. Pharmacol. 1967. 29. 259-279. HALL,D. W. R. and J. L. BONTA,Neurogenic factors involved in the relaxing effect of bradykinin on the isolated guinea-pig ileum. Arch. in/. Pharmarodyn. 1972. 197. 380-381. HILTON, S . M . and G. P. LEWIS.The mechanism of the functional hyperaemia in the submandibular salivary gland. J. Physiol. (Lond.). 1955. 129. 253-271. HILTON,S. M. and G. P. LEWIS,The relationship between glandular activity, bradykinin formation and functional vasodilatation in the submandibular salivary gland. J. Physiol. (Lond.). 1956. 134. 471-483. H U L T ~ NL., , Extrinsic nervous control of colonic motility and blood flow. Aria physiol. .wand. 1969. Suppl. 335. JANSSON, G., Extrinsic nervous control of gastric motility. An experimental study in the cat. Aria physiol. s c a d 1969. Suppl. 326. KONZETT, M. and E. STURMER,Biological activity of synthetic, polypeptides with bradykinin properties. Brit. J. Pharmacol. 1960. 15. 544-55 I. LEWIS,G. P. and E. REIT,The action of angiotensin and bradykinin on the superior cervival ganglion of the cat. J. Physiol. (Lond.). 1965. 179. 538-553. MARTINSON, J., Studies o n the efferent vagal control of the stomach. Acm physiol. scant/. 1965. 65. Siippl. 255. ROCHAE SILVA,R. M., W. E. BERALDO and G. ROSENFELD, Bradykinin, hypotensive and smooth muscle stimulating factor released from plasma globulin by snake venon and by trypsin. Anier. J. Physiol. 1949. I56. 261-273. ROCHAE SILVA.R. M., Acquisitions recentes sur la bradykinine. Acrualires phaniiacol. 1963. 15. 163-198. TURKER,K., B. K. KIRANand S . KAYMALACAN, The effects of synthetic bradykinin on intestinal motility i n different laboratory animals and in relation t o catecholamines. Arch. in/. Pharmacol. 1964. 151. 260-268.
