tol 9. pp 403 to 40q P(,rtl~t))lt))) Pre,, l,nl It)TN Pru)tcd ttl Grval BrltaO)
(h.)) Phmma(
MINIREVIEW P R O S T A G L A N D I N S AS M O D U L A T O R S O F PAIN HEINZ
JUAN
Institut Cdr E x p e r i m e n t e l l e u n d K l i n i s c h e P h a r m a k o l o g i e . U n i v e r s i t i i t s p l a t z 4. A - 8 0 1 0 G r a z , A u s t r i a (Rec('ired
7 March
INTRODUCTION
"Pain" is a phenomenon well known as an indicator of injury or tissue damage to inflammation, ischemia, mechanical irritation or other irritation. Pain has been studied intensively with regard to central and peripheral mechanisms including the generation of pain induced by stimulation of nociceptors. Our knowledge of pain-mechanisms, based mainly on experimental work, is demonstrated by the great number of original and review articles available. Therefore, the minireview presented here will be limited only to one point of the peripheral pathway of pain : the stimulation of sensory nerve endings serving as paravascular pain receptors (Lim, 1966) by algesic agents and the interaction of these agents with prostaglandins. EVALUATION O F "'PAIN M E D I A T O R S "
The introduction of analgesic drugs brought the necessity of developing models for nociceptive stimulation and an appropriate response to these stimuli. Mechanical, electrical and thermal stimulations of the skin of animals have been used as summarized e.g. by Lim & Guzman (1968). The defence reactions of the conscious animals to such stimuli indicate the perception of pain and, therefore, experimental evaluation of pain in the conscious animal has to bc undertaken very cautiously especially from the ethical point. Also in man the evaluation of pain has become a point of interest. A few tests for investigating the pain-eliciting action of various substances have been used (see Lim & Guzman, 1968; Armstrong, 1970). Pain in inflammation has been supposed to be due to the release of chemical pain mediators. A series of substances serving as candidates for this function, such as lactic acid, hypertonic saline, histamine, 5-hydroxytryptamine, potassium chloride, acetylcholine, purines, bradykinin and substance P have been tested by injecting them mainly intra-arterially in lightly anesthetized dogs or via other routes in other animals or in man. These substances are algesic agents and produce a "pseudo-affective" response, characterized by vocalization, struggling, biting, hyperpnea and a rise in arterial blood pressure (see summarizing report by Armstrong, 1970). To get more detailed information, most chemical irritants have been investigated electrophysiologically, too. Close arterial injections of acetylcholine, histamine, 5-hydroxytryptamine or bradykinin resulted in G.P 9 6 II
1978)
an activation of action potentials in the afferent nerves via stimulation of paravascular pain receptors (Fjiillebrant & Iggo, 1961; Mense & Schmidt, 1974; Beck & Handwerker, 1974; Franz & Mense, 1975: Chahl & lggo, 1977). Paravascular pain receptors ,seem to be identified by electron-microscopic studies (Stockinger & Pritz, 1970; Pritz & Stockinger, 1971). They have thin myelinated or non-myelinated afferent fibers (Zottermann, 1959) and are thought to exist in two subdivisions, the mechanical and the thermal nociceptors (lggo, 1976). From all the algogens cited here, bradykinin has been found to be the most active substance, and a great number of papers describing the algesic effect of bradykinin, following administration by various routes are summarized by Armstrong (1970). Bradykinin is generated in inflammation and present in inflammatory exudates (Rocha e Silva & RosenthaL 1961; Eisen & Keele, 1966; Melmon et al., 1967; Di Rosa et al., 1971). Furthermore, it produces pain in low concentrations and may, therefore, be considered probably as the main factor responsible for the generation of pain in inflammation of various origin. Factors other than bradykinin can, however, not be excluded. Furthermore, it could be observed that some substances such as 5-hydroxytryptamin may be presensitizing the tissue so that bradykinin becomes more effective (Sicuteri, 1968).
403
N E W M O D E L S FOR M E A S U R E M E N T O F EFFECTS O F A L G O G E N S
To allow a better quantitative evaluation of pain by algogens and the measurement of interactions of pain-producing substances, several new methods have been introduced. The function of all these models is based on a reflex answer given by the anaesthetized animal. In the first model to be described, injections of bradykinin were made intra-arterially into the spleen of anaesthetized dogs. Injections were followed by a reflex rise in arterial blood pressure (Guzman et al., 1962; Hashimoto et al., 1964). This method was later used for investigating the pain-enhancing action of prostaglandins for the attack of bradykinin by Ferreira et al. (1973). The success of these experiments, however, strongly depends on the depth of the anaesthesia. In anaesthesia which is too deep, bradykinin was unable to elicit pressor responses and in anaesthesia which is too light there were no graded responses (Ferreira et al. 1973). A reflex rise in blood
404
HIrnz jl,an
pressure is also elicited following the injection of brad~kinin into the dog knee joint {Ferreira ~'t al.. 1974a: Moncada el al., 1974: 1975) or on the surface of the dog heart (Vane, 1976) or into the carotid artery of the dog {Riccioppo-Neto et a/., 1974). Whereas the dog responds to painful stimuli with a rise in arterial blood pressure, the anaesthetized rabbit shows a reflex fall in blood pressure. Mirzojan & Doblatian (1955)and M ietkiewski (1956)developed a mcthod for measuring the stimulation of "'chemoreceptors'" in the isolated pcrfused ear connected to the body b) the great auricular nerve onl.',. Acctylcholine, injcctcd into the artcrial inflow cannula of the car perfused with Tyrode's solution in single doses elicited a brief rcflcx fall in blood prcssurc. Lembcck (1957) used this method in order to investigate the action of crude extracts of the pcptide substance P on afferent nerve endings. EI"FE('T O F A I , G O G E N S IN T H E P A I N REFI,EX EAR
For a quantitative evaluation of pain in our experiments, the method of the "'pain reflex ear" was adapted. The animals were anaesthetized with pentobarbitonc. Anacsthesia was kept constant by repeated injections of 1-3 mg/kg in the course of the experiment. Thus. the responses usually became reproducible for up to 10 hr or morc. In the first investigations, the action of a series of substances on pain receptors was measured using the shortlasting reflex fall in blood pressure and also recording the asynchronous action potentials of the car nerve. Both methods showed identical results: the doses for activating action potentials and cliciting the reflex rcsponse were the same and both reactions wcrc dependent on the dose. In the later investigations only the "'reflex ear" method was used. Thc results clearl) showcd that bradykinin and crude extracts of substance P revealcd as the most activc substances (Juan & Lembeck, 1973, 1974b). The equieffective dose of acetylcholine was about 100 times that of the peptides on a weight basis which means about 7(X) times more on a molar basis. All other algesic substances tested (histamine. ATP, 5-hydroxytryptamin. potassium chloridc) were still much less active, the equieffective doses of them being in a range of about 10- 100 times the dose of acetylcholine. These findings correspond with the experiments of Potter et al. (1962) and Lim & Guzman (1968) who compared bradykinin with substance P, acetylcholine, serotonin, histamine and potassium chloride by intra-arterial administration in dogs. ATP was found to bc algesic following intra-peritoneal injection into mice but also only in much higher doses than bradykinin (Collier et al., 1966). In contrast to substance P preparations from beef gut, synthetic substance P did not elicit pain just as physalacmin and eledoisin, two pcptides closely related to substance P (Juan & kembeck. 1974b). Further investigations showed that a kinin-like material in substance P extracts from beef gut was responsible for its algesic effect and high potassium contents in beef brain extracts (Lembeck et al.. 1977; Lembeck & Gamsc, 1977). Other painful stimuli such as squeezing the car or heat also produced the reflcx fall in
blood pressure. Strong vasoconstriction by vasoprcssin, angiotensin or adrenaline did not causc pain and adenosin or ocytocin were ineffective, too. INTERACTION OF" PGE w'ITII ALGESI(" A(;ENTS
Surprisingly, prostaglandin E~ up to the high dose of 100pg was devoid of any algesic effect. Prostaglandins (PGs) had been found to cxcrt pain producing properties in rather high concentrations (Fcrrcira, 1972: Collier & Schneider, 1972: Karim, 1971: Willis et al., 1972: Collier et al., 1972; Gillespie, 1972) or. on thc other hand, failed to produce pain in other preparations IHorton, 1963: Crunkhorn & Willis. 1971: Fcrreira et al., 1973j. Whereas PGE~ by itself was inactive in the rabbit car, small amounts of PGE~ strongly enhanced the effect of all algesic substances but most that of bradykinin. A similar lowcring of the pain threshold to chemical or mechanical stimuli has been described earlier by scvcral authors (Fcrreira, 1972: Ferreira et al.. 1973; Kuhn & Willis. 1973: Willis & Cornelscn, 1973) and was confirmed by Moncada et al. (1975). In the isolated pain reflex car, a quantitative evaluation of the pain receptor sensitizing activity of PGE~ was possible: PGE~ shifted the dose-rcsponse curves of the algogens to the left (Figs 1 and 2)in the following ordcr: bradykinin, substance P (from becf gut) > 5-HT > histamine > acetylcholine > ATP > KCI. PGE 2 had a weakcr activity in enhancing algesic effects. The facilitation b',, PGE~ is cumulative in character and persists after thc cessation of its influence. Thcse results confirmed similar observations obtained by Fcrreira (1972), Fcrrcira et al. (1973). Kuhn & Willis (1973) and Willis & Cornelsen (1973) and support the conclusion of these authors that E-PGs do not exert pain by themselves but sensitize pain receptors to chemical stimuli in inflammation and cause a long lasting hyperalgesia. It has not yet been established how E-PGs facilitate pain effects. O
~
20
1:1
E
~
, 4C-
! C
I
I
I C
I k;'~
/.z¢~
Fig. 1. Pain reflex ear. Dosc-response curves of the reflex fall in mean arterial blood pressure after injection of substance P from gut (~, III), bradykinin (O, O) and acctylcholine (A. A). Open symbols: before infusion, n = 20. Closed symbols: during infusion, more than 20 rain after the start. of 50ng;ml PGEt, n = 8 12. Vertical bars represent s.e. mean. The amount of substance P from gut was expressed in weight units (ng) according to the comparison with synthetic substance P in the bioassay (Juan & Lembeck. 1974b).
Prostaglandins as modulators of pain
al., 1972) and spleen (Ferreira et al., 1973) of dogs. the lung of guinea-pigs (Palmer et al., 1973), the heart (Needleman et al., 1975) and also the ear of rabbits (Juan & Lembeck, 1975a, 1976a; Lembeck et al.,
O-
T
E E
Q.
2o
\
E o
n~
401
mOO
nO
I BOO0
! BOO00
pg
Fig. 2. Pain reflex ear. Dose-response curves of the reflex fall in mean arterial blood pressure after injection of ATP O. ~P), histamine (rq,, III). serotonin (O. @) and KCI (~, &). Open symbols: before infusion, n = 20. Closed symbols: during infusion, more than 20rain after the start. of 50ng/ml PGEt. n = 8-12. Vertical bars represent s.c. mean (Juan & Lembeck, 1974h).
I N H I B I T I O N O F P G R E L E A S E A N D PAIN
It has been described by Lim et al. (1964) that the algesic effect of bradykinin was inhibited by "aspirinlike" drugs peripherally. This effect could not be interpreted. Further investigations by Ferreira et al. 0973) also demonstrated a reduction of the algesic effect of bradykinin by indomethacin in the dog spleen. With our method of the pain reflex ear we found that indomethacin strongly reduced but did not abolish the effect of bradykinin. The effect of acetylcholine was hardly reduced (Lembeck & Juan, 1974). The reduction of the bradykinin effect can be explained on the basis of the inhibition of PG biosynthesis (Vane, 1971; Smith & Willis, 1971; Ferreira et al., 1971). PGs of the E-type are released by bradykinin from several organs such as the kidney (McGiff et O-
20-
c
o
g 40-
1976). The released PGE sensitizes the pain receptors for the attack of the algogen or other t~ainful stimuli. Inhibition of PGE release by indomethacin abolishes the pain enhancing action of the released PGE and the relatively weak proper effect of bradykinin remains. In contrast to bradykinin, acetyleholine did not release PGs or only when injected in high doses into the rabbit ear (Juan & Lembeck, 1975a. 1976a: Lembeck et al., 1976). Therefore. no sensitization can occur and indomethacin will be ineffective. A small part of the indomethacin effect may be mediated via a direct antagonistic action, especially when used in concentrations higher than necessary for inhibiting PGE release (Lembeck & Juan, 1974, Juan & Lembeck, 1976a; Lembeck et al., 1976). SELECTIVE INHIBITION OF BRADYKININ AND A C E T Y L C H O L I N E EFFECTS
Not only indomethacin but also a few other drugs showed analgesic properties in the ear. These could be used in order to find out whether bradykinin and acetylcholine exert their action via different nerve fiber types or different receptor sites. Using tetrodotoxin it was found that bradykinin and acetylcholine act probably via the same nerve fiber types (Juan & Lembeck, 1975b). However, distinct receptor types for bradykinin and acetylcholine have to be assumed since the bradykinin effect is selectively reduced by papaverine, NaNO2 and aminophylline (Juan & Lembeck, 1974a; Juan et al., 1977) whereas that of acetylcholine is selectively reduced by hexamethonium and high concentrations of atropine (Juan & Lembeck, 1974a). Furthermore, a high calcium concentration strongly reduced the algesic effect of bradykinin but facilitated that of acetylcholine (Lembeck & Juan, 1977). PROSTAGLANDIN
O.
~Z E E
~E
405
BroO.
I~ I n f of 5/,.zg/mt. PPP I
t
0 I
I
~g
ACh I n f of ..5,~glmt PPP
,'o
IO0 ' Ng
Fig. 3. Pain reflex ear. Influence of infusion of 5,ug/ml polyphloretin phosphate on reflex fall in mean arterial blood pressure (dose-response curves) elicited by bradykinin (left)and acetylcholine (rightL Controls: @. 10min ('-I), 20min (
(h.)) Phmma(
MINIREVIEW P R O S T A G L A N D I N S AS M O D U L A T O R S O F PAIN HEINZ
JUAN
Institut Cdr E x p e r i m e n t e l l e u n d K l i n i s c h e P h a r m a k o l o g i e . U n i v e r s i t i i t s p l a t z 4. A - 8 0 1 0 G r a z , A u s t r i a (Rec('ired
7 March
INTRODUCTION
"Pain" is a phenomenon well known as an indicator of injury or tissue damage to inflammation, ischemia, mechanical irritation or other irritation. Pain has been studied intensively with regard to central and peripheral mechanisms including the generation of pain induced by stimulation of nociceptors. Our knowledge of pain-mechanisms, based mainly on experimental work, is demonstrated by the great number of original and review articles available. Therefore, the minireview presented here will be limited only to one point of the peripheral pathway of pain : the stimulation of sensory nerve endings serving as paravascular pain receptors (Lim, 1966) by algesic agents and the interaction of these agents with prostaglandins. EVALUATION O F "'PAIN M E D I A T O R S "
The introduction of analgesic drugs brought the necessity of developing models for nociceptive stimulation and an appropriate response to these stimuli. Mechanical, electrical and thermal stimulations of the skin of animals have been used as summarized e.g. by Lim & Guzman (1968). The defence reactions of the conscious animals to such stimuli indicate the perception of pain and, therefore, experimental evaluation of pain in the conscious animal has to bc undertaken very cautiously especially from the ethical point. Also in man the evaluation of pain has become a point of interest. A few tests for investigating the pain-eliciting action of various substances have been used (see Lim & Guzman, 1968; Armstrong, 1970). Pain in inflammation has been supposed to be due to the release of chemical pain mediators. A series of substances serving as candidates for this function, such as lactic acid, hypertonic saline, histamine, 5-hydroxytryptamine, potassium chloride, acetylcholine, purines, bradykinin and substance P have been tested by injecting them mainly intra-arterially in lightly anesthetized dogs or via other routes in other animals or in man. These substances are algesic agents and produce a "pseudo-affective" response, characterized by vocalization, struggling, biting, hyperpnea and a rise in arterial blood pressure (see summarizing report by Armstrong, 1970). To get more detailed information, most chemical irritants have been investigated electrophysiologically, too. Close arterial injections of acetylcholine, histamine, 5-hydroxytryptamine or bradykinin resulted in G.P 9 6 II
1978)
an activation of action potentials in the afferent nerves via stimulation of paravascular pain receptors (Fjiillebrant & Iggo, 1961; Mense & Schmidt, 1974; Beck & Handwerker, 1974; Franz & Mense, 1975: Chahl & lggo, 1977). Paravascular pain receptors ,seem to be identified by electron-microscopic studies (Stockinger & Pritz, 1970; Pritz & Stockinger, 1971). They have thin myelinated or non-myelinated afferent fibers (Zottermann, 1959) and are thought to exist in two subdivisions, the mechanical and the thermal nociceptors (lggo, 1976). From all the algogens cited here, bradykinin has been found to be the most active substance, and a great number of papers describing the algesic effect of bradykinin, following administration by various routes are summarized by Armstrong (1970). Bradykinin is generated in inflammation and present in inflammatory exudates (Rocha e Silva & RosenthaL 1961; Eisen & Keele, 1966; Melmon et al., 1967; Di Rosa et al., 1971). Furthermore, it produces pain in low concentrations and may, therefore, be considered probably as the main factor responsible for the generation of pain in inflammation of various origin. Factors other than bradykinin can, however, not be excluded. Furthermore, it could be observed that some substances such as 5-hydroxytryptamin may be presensitizing the tissue so that bradykinin becomes more effective (Sicuteri, 1968).
403
N E W M O D E L S FOR M E A S U R E M E N T O F EFFECTS O F A L G O G E N S
To allow a better quantitative evaluation of pain by algogens and the measurement of interactions of pain-producing substances, several new methods have been introduced. The function of all these models is based on a reflex answer given by the anaesthetized animal. In the first model to be described, injections of bradykinin were made intra-arterially into the spleen of anaesthetized dogs. Injections were followed by a reflex rise in arterial blood pressure (Guzman et al., 1962; Hashimoto et al., 1964). This method was later used for investigating the pain-enhancing action of prostaglandins for the attack of bradykinin by Ferreira et al. (1973). The success of these experiments, however, strongly depends on the depth of the anaesthesia. In anaesthesia which is too deep, bradykinin was unable to elicit pressor responses and in anaesthesia which is too light there were no graded responses (Ferreira et al. 1973). A reflex rise in blood
404
HIrnz jl,an
pressure is also elicited following the injection of brad~kinin into the dog knee joint {Ferreira ~'t al.. 1974a: Moncada el al., 1974: 1975) or on the surface of the dog heart (Vane, 1976) or into the carotid artery of the dog {Riccioppo-Neto et a/., 1974). Whereas the dog responds to painful stimuli with a rise in arterial blood pressure, the anaesthetized rabbit shows a reflex fall in blood pressure. Mirzojan & Doblatian (1955)and M ietkiewski (1956)developed a mcthod for measuring the stimulation of "'chemoreceptors'" in the isolated pcrfused ear connected to the body b) the great auricular nerve onl.',. Acctylcholine, injcctcd into the artcrial inflow cannula of the car perfused with Tyrode's solution in single doses elicited a brief rcflcx fall in blood prcssurc. Lembcck (1957) used this method in order to investigate the action of crude extracts of the pcptide substance P on afferent nerve endings. EI"FE('T O F A I , G O G E N S IN T H E P A I N REFI,EX EAR
For a quantitative evaluation of pain in our experiments, the method of the "'pain reflex ear" was adapted. The animals were anaesthetized with pentobarbitonc. Anacsthesia was kept constant by repeated injections of 1-3 mg/kg in the course of the experiment. Thus. the responses usually became reproducible for up to 10 hr or morc. In the first investigations, the action of a series of substances on pain receptors was measured using the shortlasting reflex fall in blood pressure and also recording the asynchronous action potentials of the car nerve. Both methods showed identical results: the doses for activating action potentials and cliciting the reflex rcsponse were the same and both reactions wcrc dependent on the dose. In the later investigations only the "'reflex ear" method was used. Thc results clearl) showcd that bradykinin and crude extracts of substance P revealcd as the most activc substances (Juan & Lembeck, 1973, 1974b). The equieffective dose of acetylcholine was about 100 times that of the peptides on a weight basis which means about 7(X) times more on a molar basis. All other algesic substances tested (histamine. ATP, 5-hydroxytryptamin. potassium chloridc) were still much less active, the equieffective doses of them being in a range of about 10- 100 times the dose of acetylcholine. These findings correspond with the experiments of Potter et al. (1962) and Lim & Guzman (1968) who compared bradykinin with substance P, acetylcholine, serotonin, histamine and potassium chloride by intra-arterial administration in dogs. ATP was found to bc algesic following intra-peritoneal injection into mice but also only in much higher doses than bradykinin (Collier et al., 1966). In contrast to substance P preparations from beef gut, synthetic substance P did not elicit pain just as physalacmin and eledoisin, two pcptides closely related to substance P (Juan & kembeck. 1974b). Further investigations showed that a kinin-like material in substance P extracts from beef gut was responsible for its algesic effect and high potassium contents in beef brain extracts (Lembeck et al.. 1977; Lembeck & Gamsc, 1977). Other painful stimuli such as squeezing the car or heat also produced the reflcx fall in
blood pressure. Strong vasoconstriction by vasoprcssin, angiotensin or adrenaline did not causc pain and adenosin or ocytocin were ineffective, too. INTERACTION OF" PGE w'ITII ALGESI(" A(;ENTS
Surprisingly, prostaglandin E~ up to the high dose of 100pg was devoid of any algesic effect. Prostaglandins (PGs) had been found to cxcrt pain producing properties in rather high concentrations (Fcrrcira, 1972: Collier & Schneider, 1972: Karim, 1971: Willis et al., 1972: Collier et al., 1972; Gillespie, 1972) or. on thc other hand, failed to produce pain in other preparations IHorton, 1963: Crunkhorn & Willis. 1971: Fcrreira et al., 1973j. Whereas PGE~ by itself was inactive in the rabbit car, small amounts of PGE~ strongly enhanced the effect of all algesic substances but most that of bradykinin. A similar lowcring of the pain threshold to chemical or mechanical stimuli has been described earlier by scvcral authors (Fcrreira, 1972: Ferreira et al.. 1973; Kuhn & Willis. 1973: Willis & Cornelscn, 1973) and was confirmed by Moncada et al. (1975). In the isolated pain reflex car, a quantitative evaluation of the pain receptor sensitizing activity of PGE~ was possible: PGE~ shifted the dose-rcsponse curves of the algogens to the left (Figs 1 and 2)in the following ordcr: bradykinin, substance P (from becf gut) > 5-HT > histamine > acetylcholine > ATP > KCI. PGE 2 had a weakcr activity in enhancing algesic effects. The facilitation b',, PGE~ is cumulative in character and persists after thc cessation of its influence. Thcse results confirmed similar observations obtained by Fcrreira (1972), Fcrrcira et al. (1973). Kuhn & Willis (1973) and Willis & Cornelsen (1973) and support the conclusion of these authors that E-PGs do not exert pain by themselves but sensitize pain receptors to chemical stimuli in inflammation and cause a long lasting hyperalgesia. It has not yet been established how E-PGs facilitate pain effects. O
~
20
1:1
E
~
, 4C-
! C
I
I
I C
I k;'~
/.z¢~
Fig. 1. Pain reflex ear. Dosc-response curves of the reflex fall in mean arterial blood pressure after injection of substance P from gut (~, III), bradykinin (O, O) and acctylcholine (A. A). Open symbols: before infusion, n = 20. Closed symbols: during infusion, more than 20 rain after the start. of 50ng;ml PGEt, n = 8 12. Vertical bars represent s.e. mean. The amount of substance P from gut was expressed in weight units (ng) according to the comparison with synthetic substance P in the bioassay (Juan & Lembeck. 1974b).
Prostaglandins as modulators of pain
al., 1972) and spleen (Ferreira et al., 1973) of dogs. the lung of guinea-pigs (Palmer et al., 1973), the heart (Needleman et al., 1975) and also the ear of rabbits (Juan & Lembeck, 1975a, 1976a; Lembeck et al.,
O-
T
E E
Q.
2o
\
E o
n~
401
mOO
nO
I BOO0
! BOO00
pg
Fig. 2. Pain reflex ear. Dose-response curves of the reflex fall in mean arterial blood pressure after injection of ATP O. ~P), histamine (rq,, III). serotonin (O. @) and KCI (~, &). Open symbols: before infusion, n = 20. Closed symbols: during infusion, more than 20rain after the start. of 50ng/ml PGEt. n = 8-12. Vertical bars represent s.c. mean (Juan & Lembeck, 1974h).
I N H I B I T I O N O F P G R E L E A S E A N D PAIN
It has been described by Lim et al. (1964) that the algesic effect of bradykinin was inhibited by "aspirinlike" drugs peripherally. This effect could not be interpreted. Further investigations by Ferreira et al. 0973) also demonstrated a reduction of the algesic effect of bradykinin by indomethacin in the dog spleen. With our method of the pain reflex ear we found that indomethacin strongly reduced but did not abolish the effect of bradykinin. The effect of acetylcholine was hardly reduced (Lembeck & Juan, 1974). The reduction of the bradykinin effect can be explained on the basis of the inhibition of PG biosynthesis (Vane, 1971; Smith & Willis, 1971; Ferreira et al., 1971). PGs of the E-type are released by bradykinin from several organs such as the kidney (McGiff et O-
20-
c
o
g 40-
1976). The released PGE sensitizes the pain receptors for the attack of the algogen or other t~ainful stimuli. Inhibition of PGE release by indomethacin abolishes the pain enhancing action of the released PGE and the relatively weak proper effect of bradykinin remains. In contrast to bradykinin, acetyleholine did not release PGs or only when injected in high doses into the rabbit ear (Juan & Lembeck, 1975a. 1976a: Lembeck et al., 1976). Therefore. no sensitization can occur and indomethacin will be ineffective. A small part of the indomethacin effect may be mediated via a direct antagonistic action, especially when used in concentrations higher than necessary for inhibiting PGE release (Lembeck & Juan, 1974, Juan & Lembeck, 1976a; Lembeck et al., 1976). SELECTIVE INHIBITION OF BRADYKININ AND A C E T Y L C H O L I N E EFFECTS
Not only indomethacin but also a few other drugs showed analgesic properties in the ear. These could be used in order to find out whether bradykinin and acetylcholine exert their action via different nerve fiber types or different receptor sites. Using tetrodotoxin it was found that bradykinin and acetylcholine act probably via the same nerve fiber types (Juan & Lembeck, 1975b). However, distinct receptor types for bradykinin and acetylcholine have to be assumed since the bradykinin effect is selectively reduced by papaverine, NaNO2 and aminophylline (Juan & Lembeck, 1974a; Juan et al., 1977) whereas that of acetylcholine is selectively reduced by hexamethonium and high concentrations of atropine (Juan & Lembeck, 1974a). Furthermore, a high calcium concentration strongly reduced the algesic effect of bradykinin but facilitated that of acetylcholine (Lembeck & Juan, 1977). PROSTAGLANDIN
O.
~Z E E
~E
405
BroO.
I~ I n f of 5/,.zg/mt. PPP I
t
0 I
I
~g
ACh I n f of ..5,~glmt PPP
,'o
IO0 ' Ng
Fig. 3. Pain reflex ear. Influence of infusion of 5,ug/ml polyphloretin phosphate on reflex fall in mean arterial blood pressure (dose-response curves) elicited by bradykinin (left)and acetylcholine (rightL Controls: @. 10min ('-I), 20min (
