TiPS - April 2990 [Vol. 2 11
Domenico Regoli, Nour-Eddine Rhaleb, Stephane Dion and Guy Drapeau progress Iras beerr r&e recerttl!l irt Ure field of kini!: pharmacology with t/w iderlGfication of sensitive bioassay organs and the discovery of Dradykirh E2 receptor arltagonists. Data obtained with such compounds in various laboratories supyort the hypofhis that kinins ncf on multiple (at least two) receptor hypes. Domenico Regoli and colleagues reriezv kere the basic criteria of rccrpfor c/rarac~cr~~~fioffas Frey apply fo kinins and present a criticaf a~ia~ysis of the ~~oassa!/ orgarzs and B2 receptor anfagof~isfs currently used in kih plmtwacolog!y. _ _
Srr&nrrM
Kallikreins are a group of serine proteases that are generally kept in an inactive state in blood and tissues. When activated in pathophysiological conditions such as inflammation, trauma, bums, shock, allergy and perhaps some cardiovascular diseases, they release bradykinin and kallidin, two potent arterial vasodiiators and venocons~icto~ that also evoke pain, favour oedema formation and are involved in regulation of renal functions’. Kinins act through at least two different receptors which have been named bradykinin B1 and B2 receptors2. This early classification was based on agonist order of potency and on specific B, receptor antagonists’. Lack of specific B1 receptor antagonists prevented characterization of B2 receptors until 1985, when Vavrek and Stewart3 first described various kinin analogues that block Bz receptors. Such compounds have now been extensively used in vivo and in vitro, and a complex picture of B1 receptor subtypes is emerging4,5. Bradykinin B2 receptor antagonists, especially the early compounds, have several limitations as pharmacological tools since: (1) they are partial agonists rather than antagonists in various bio1ogic.J system@ and thus pro-
mote release of histamine7 and prostaglandins; and (2) they are not selective for B2 receptors, having some affinity for Bt receptor.@. Moreover. early Bz receptor antagonists showed rather low affinities for B2 receptors, at least two log units lower than the kinin agonists. Attempts have been made to design new potent and specific antagonists with improved pharmacological profiles. New, sensitive bioassays are now also available. Pharmacological assay organs Kinins contract or relax smooth muscle of intestine, tracheobronchial tree, urinary tract, uterus, epididymus and a variety of arteries and veins from various species. In the majority of these organs, kinins act indirectly by promoting the release of other endogmous agents that modulate smooth muscle tone (Table I). For example:
e In large arteries, kinins act on the endothelium9 and promote the release of endothelium-derived relaxing factor (EDRF; nitric oxide), a potent relaxant of arterial smooth muscles. e Kinins contract the hamster urinary bladder by activating the production of stimulatory prostaglandins: such a mechanism is common to bladders and other tissues from various animals. 8 Kinins potentiate the effect of electrical stimulation in the rat vas deferens by favouring the release of neurotransmitters from sympathetic nerve endings; the same mechanisms occur in isolated large vessels and peripheral vascular trees. a Contractions and relaxations of respiratory smooth muscle (e.g. in the guinea-pig trachea) in response to kinins are due to either the release of contractile motabolites of the arachidonic acid cascade (pr~~gl~dins, leukotrienes) or the release of inhibitor prostaglandinslO. In all these tissues the pharmacology is very complex. Kinin receptors are not limited to smooth muscle cells, but may be located on other cell types. Biology is evoked indirectly, response thereby involving a receptor for the mediator or second messenger (in the case of EDRF the postinvolves mechanism receptor guanylyl cyclase). Thus the kininreceptor intera~on is remote from the biological response and the precision of the bioassay may be critically reduced. In other organs, such as the rabbit jugular vein, or the rabbit aorta devoid of endothelium (Table I), kinins act directly on smooth muscle receptors that mediate contraction. Such organs appear to be less complex than
TABLE I. Biological effects of kinins in isolated organs Tissue or organ
Target cdl
Mediator
Smooth muscle response
Dog carotid artery (and arteries of various species)
endothelium
EDRF
relaxation
various cells Hamster urinary bladder (and other bladders and tissues)
prostaglandins
contraction
Rat vas deferens (and other electrically stimulated organs)
autonomic nerve endings
noradrenaline; other transmitters
contraction
Guinea-pig trachea
variow ceils
prostagla~jns; leukotrienes
contraction
Rabbit jugular vein
smooth muscle
contraction
Rabbit aorta
smooth muscle
contraction
prostaglandins
relaxation
TiPS - April 1990 [Vol. 111
157 The orders of potency of agonists in all other tissues tested (guinea-pig ileum and trachea, the hamster urinary bladder and the rat vas deferens) indicated that they contained receptors of the B2 type, since all responded to kinins but were almost insensitive to the C-terminal desArg compounds (Table III). Apparent affinities of bradykinin, evaluated in terms of pD2 (-log of the molar concentration of agonist that produces 50% of the maximal response), are similar. The values of 8.46 (rabbit jugular vein), 8.10 (guinea-pig trachea), 7.90 (guinea-pig ileum), 7.70 (hamster urinarlr bladder) and 7.62 (rat vas defer.ns) are all within a range of on log unit. Such variations might be due to metabolic degradation of bradykinin, to diffusion barriers or to efficiency of the effector system. Results obtained with agonists are quite homogeneous and indicate that the kinin-receptor interaction is reflected reasonably well in the magnitude of the biological effects, despite the complexity of certain systems (for instance the guinea-pig trachea).
TABLE II. Order of potency of agonists in two isolated vessels Peptide
Rabbit aorta PDZ
Rabbit jugular vein
RA
PD,
RA 100
Bradykinin (BK) Kallidin
6.22 7.27
8 95
8.46 8.63
desArgg-BK
7.29
100
4.39
desArg”‘-kallidin
8.61
2100
5.33
[HYP~IBK
6.17
7
8.86
254
8.70
168
oArg-BK
6.39
13
[oPhe’]desA$-BK
7.82
339
(oPheg]desArg’o-kallidin
8.28
968
[PheBqArgs]BK
150 0.01 1.oo
c 0.01 < 0.01 65
8.29
i.
PD,. -log of the concentration of agonist required to evoke 50% of the maximum response;
RA. relative affinity expressed as percentage of that of desArg’-BK in the rabbit aorta and of BK in the rabbit jugular vein; w, CH,NH; I., inactive. Data from Ref. 21.
others (e.g. bladders, tracheae) and provide reliable bioassays for kinins. The characterization of kinin receptors presented below was carried out using the rabbit jugular vein for the Bz and the rabbit aorta for the 6, receptor. Data obtained in these preparations are used as reference for comparing the responses of the other tissues to kinin agonists and antagonists. Receptor characterization by agonists Two different patterns of agonist activities have been identified in the rabbit jugular vein and rabbit aorta, using kinins and their metabolites or analogues. Some tests on the rabbit aorta were carried out in the absence of mergetpa [an inhibitor of kininase I (carboxypeptidase N; EC3.4.17.3)J to allow the conversion of bradykinin and kallidin to C-terminal desArg metabolitess. Both rabbit aorta and jugular vein were treated with captopril to inhibit enzyme angiotensin-converting (kininase II). Bradykinin 13, receptors are activated by C-terminal desArg peptides, while B2 receptors are almost insensitive to these compounds (Table II). The presence of a C-terminal Arg is instrumental for interaction of the kinins with Bz receptors. Affinity for the B2 receptor is increased by replacing Pro3 with Hyp and extending the peptide chain at the N terminus with DArg. An N-terminal extension with Lys reduces selectivity, since Lys favours Bi receptor occupation (see desArgrO-kallidin). Replacing the C-terminal Phe
with the o-isomer prevents degradation of these potent and relatively selective B1 receptor agonists (Table II). Orders of potency of agonists are as follows: Bi
desArgrs-kallidin desArgs-BK = kallidin >> BK
>
Bz
BK = kallidin >> desArgiO-kallidin > desArgs-BK
RABBIT JUGULAR VEIN
2
--J*
r
indometacin
:d
All,
ciesArgg-Bt
,
,
_A-
I
anti-B,
anti-B,
fi,
q
indometacin 0) In d L_ 4 min
GUINEA-PIG TRACHEA
. .
BK
, T--,
+anti-B,
anti-B,
indometacir
Fig. 1. Myotropic effects of bradykinin (BK) and desArg?BK in the absence of inhibitors and in the presence of a bradykinin B, receptor antagonist (anti-B,: [Leus]desArgg-SK, 10e7M), a Bz receptor antagonist (anti-B*: oArg[tfyp3,Leu5?oPhe7/BK, lo-’ M), or indometacin (2.8 x lo-‘M), in isolated organs. Abscissa, time in min; ordinate, tension in g.
7’iPS - April 2990 [Vol. 111
15s TABLE III. Order of contractile potency of kmins in various isolated tissues Peptide
Guinea-pig ileum
Rabbit jugular vein ~4
PDZ
RA
RA
Hamster urinary bladder P&
Rat vas daferens
RA
PDZ
RA
P&
RA
100
7.62
100
8.10
100
7.93
68
BK
8.46
100
7.90
100
7.70
Kallidin desArQg-BK
8.63
140 0.01
7.88
05
Domenico Regoli, Nour-Eddine Rhaleb, Stephane Dion and Guy Drapeau progress Iras beerr r&e recerttl!l irt Ure field of kini!: pharmacology with t/w iderlGfication of sensitive bioassay organs and the discovery of Dradykirh E2 receptor arltagonists. Data obtained with such compounds in various laboratories supyort the hypofhis that kinins ncf on multiple (at least two) receptor hypes. Domenico Regoli and colleagues reriezv kere the basic criteria of rccrpfor c/rarac~cr~~~fioffas Frey apply fo kinins and present a criticaf a~ia~ysis of the ~~oassa!/ orgarzs and B2 receptor anfagof~isfs currently used in kih plmtwacolog!y. _ _
Srr&nrrM
Kallikreins are a group of serine proteases that are generally kept in an inactive state in blood and tissues. When activated in pathophysiological conditions such as inflammation, trauma, bums, shock, allergy and perhaps some cardiovascular diseases, they release bradykinin and kallidin, two potent arterial vasodiiators and venocons~icto~ that also evoke pain, favour oedema formation and are involved in regulation of renal functions’. Kinins act through at least two different receptors which have been named bradykinin B1 and B2 receptors2. This early classification was based on agonist order of potency and on specific B, receptor antagonists’. Lack of specific B1 receptor antagonists prevented characterization of B2 receptors until 1985, when Vavrek and Stewart3 first described various kinin analogues that block Bz receptors. Such compounds have now been extensively used in vivo and in vitro, and a complex picture of B1 receptor subtypes is emerging4,5. Bradykinin B2 receptor antagonists, especially the early compounds, have several limitations as pharmacological tools since: (1) they are partial agonists rather than antagonists in various bio1ogic.J system@ and thus pro-
mote release of histamine7 and prostaglandins; and (2) they are not selective for B2 receptors, having some affinity for Bt receptor.@. Moreover. early Bz receptor antagonists showed rather low affinities for B2 receptors, at least two log units lower than the kinin agonists. Attempts have been made to design new potent and specific antagonists with improved pharmacological profiles. New, sensitive bioassays are now also available. Pharmacological assay organs Kinins contract or relax smooth muscle of intestine, tracheobronchial tree, urinary tract, uterus, epididymus and a variety of arteries and veins from various species. In the majority of these organs, kinins act indirectly by promoting the release of other endogmous agents that modulate smooth muscle tone (Table I). For example:
e In large arteries, kinins act on the endothelium9 and promote the release of endothelium-derived relaxing factor (EDRF; nitric oxide), a potent relaxant of arterial smooth muscles. e Kinins contract the hamster urinary bladder by activating the production of stimulatory prostaglandins: such a mechanism is common to bladders and other tissues from various animals. 8 Kinins potentiate the effect of electrical stimulation in the rat vas deferens by favouring the release of neurotransmitters from sympathetic nerve endings; the same mechanisms occur in isolated large vessels and peripheral vascular trees. a Contractions and relaxations of respiratory smooth muscle (e.g. in the guinea-pig trachea) in response to kinins are due to either the release of contractile motabolites of the arachidonic acid cascade (pr~~gl~dins, leukotrienes) or the release of inhibitor prostaglandinslO. In all these tissues the pharmacology is very complex. Kinin receptors are not limited to smooth muscle cells, but may be located on other cell types. Biology is evoked indirectly, response thereby involving a receptor for the mediator or second messenger (in the case of EDRF the postinvolves mechanism receptor guanylyl cyclase). Thus the kininreceptor intera~on is remote from the biological response and the precision of the bioassay may be critically reduced. In other organs, such as the rabbit jugular vein, or the rabbit aorta devoid of endothelium (Table I), kinins act directly on smooth muscle receptors that mediate contraction. Such organs appear to be less complex than
TABLE I. Biological effects of kinins in isolated organs Tissue or organ
Target cdl
Mediator
Smooth muscle response
Dog carotid artery (and arteries of various species)
endothelium
EDRF
relaxation
various cells Hamster urinary bladder (and other bladders and tissues)
prostaglandins
contraction
Rat vas deferens (and other electrically stimulated organs)
autonomic nerve endings
noradrenaline; other transmitters
contraction
Guinea-pig trachea
variow ceils
prostagla~jns; leukotrienes
contraction
Rabbit jugular vein
smooth muscle
contraction
Rabbit aorta
smooth muscle
contraction
prostaglandins
relaxation
TiPS - April 1990 [Vol. 111
157 The orders of potency of agonists in all other tissues tested (guinea-pig ileum and trachea, the hamster urinary bladder and the rat vas deferens) indicated that they contained receptors of the B2 type, since all responded to kinins but were almost insensitive to the C-terminal desArg compounds (Table III). Apparent affinities of bradykinin, evaluated in terms of pD2 (-log of the molar concentration of agonist that produces 50% of the maximal response), are similar. The values of 8.46 (rabbit jugular vein), 8.10 (guinea-pig trachea), 7.90 (guinea-pig ileum), 7.70 (hamster urinarlr bladder) and 7.62 (rat vas defer.ns) are all within a range of on log unit. Such variations might be due to metabolic degradation of bradykinin, to diffusion barriers or to efficiency of the effector system. Results obtained with agonists are quite homogeneous and indicate that the kinin-receptor interaction is reflected reasonably well in the magnitude of the biological effects, despite the complexity of certain systems (for instance the guinea-pig trachea).
TABLE II. Order of potency of agonists in two isolated vessels Peptide
Rabbit aorta PDZ
Rabbit jugular vein
RA
PD,
RA 100
Bradykinin (BK) Kallidin
6.22 7.27
8 95
8.46 8.63
desArgg-BK
7.29
100
4.39
desArg”‘-kallidin
8.61
2100
5.33
[HYP~IBK
6.17
7
8.86
254
8.70
168
oArg-BK
6.39
13
[oPhe’]desA$-BK
7.82
339
(oPheg]desArg’o-kallidin
8.28
968
[PheBqArgs]BK
150 0.01 1.oo
c 0.01 < 0.01 65
8.29
i.
PD,. -log of the concentration of agonist required to evoke 50% of the maximum response;
RA. relative affinity expressed as percentage of that of desArg’-BK in the rabbit aorta and of BK in the rabbit jugular vein; w, CH,NH; I., inactive. Data from Ref. 21.
others (e.g. bladders, tracheae) and provide reliable bioassays for kinins. The characterization of kinin receptors presented below was carried out using the rabbit jugular vein for the Bz and the rabbit aorta for the 6, receptor. Data obtained in these preparations are used as reference for comparing the responses of the other tissues to kinin agonists and antagonists. Receptor characterization by agonists Two different patterns of agonist activities have been identified in the rabbit jugular vein and rabbit aorta, using kinins and their metabolites or analogues. Some tests on the rabbit aorta were carried out in the absence of mergetpa [an inhibitor of kininase I (carboxypeptidase N; EC3.4.17.3)J to allow the conversion of bradykinin and kallidin to C-terminal desArg metabolitess. Both rabbit aorta and jugular vein were treated with captopril to inhibit enzyme angiotensin-converting (kininase II). Bradykinin 13, receptors are activated by C-terminal desArg peptides, while B2 receptors are almost insensitive to these compounds (Table II). The presence of a C-terminal Arg is instrumental for interaction of the kinins with Bz receptors. Affinity for the B2 receptor is increased by replacing Pro3 with Hyp and extending the peptide chain at the N terminus with DArg. An N-terminal extension with Lys reduces selectivity, since Lys favours Bi receptor occupation (see desArgrO-kallidin). Replacing the C-terminal Phe
with the o-isomer prevents degradation of these potent and relatively selective B1 receptor agonists (Table II). Orders of potency of agonists are as follows: Bi
desArgrs-kallidin desArgs-BK = kallidin >> BK
>
Bz
BK = kallidin >> desArgiO-kallidin > desArgs-BK
RABBIT JUGULAR VEIN
2
--J*
r
indometacin
:d
All,
ciesArgg-Bt
,
,
_A-
I
anti-B,
anti-B,
fi,
q
indometacin 0) In d L_ 4 min
GUINEA-PIG TRACHEA
. .
BK
, T--,
+anti-B,
anti-B,
indometacir
Fig. 1. Myotropic effects of bradykinin (BK) and desArg?BK in the absence of inhibitors and in the presence of a bradykinin B, receptor antagonist (anti-B,: [Leus]desArgg-SK, 10e7M), a Bz receptor antagonist (anti-B*: oArg[tfyp3,Leu5?oPhe7/BK, lo-’ M), or indometacin (2.8 x lo-‘M), in isolated organs. Abscissa, time in min; ordinate, tension in g.
7’iPS - April 2990 [Vol. 111
15s TABLE III. Order of contractile potency of kmins in various isolated tissues Peptide
Guinea-pig ileum
Rabbit jugular vein ~4
PDZ
RA
RA
Hamster urinary bladder P&
Rat vas daferens
RA
PDZ
RA
P&
RA
100
7.62
100
8.10
100
7.93
68
BK
8.46
100
7.90
100
7.70
Kallidin desArQg-BK
8.63
140 0.01
7.88
05
