Release and Inactivation of Kinins

GASTROENTEROLOGY

Copyright

Vol. 51, No.5, Part 2 Printed in U.S.A.

© 1966 by The Williams & Wilkins Co.

RELEASE AND INACTIVATION OF KININS ERVIN

G.

ERDOS

Department of Pharmacology, University of Oklahoma School of Medicine

I believe that I was asked to contribute to this symposium because I have neither prestige in the gastrointestinal field which could be damaged by hasty suggestions nor is my vision blurred by any depth of knowledge about ulcerative colitis. Being in this fortunate position, I should like to draw your attention to the role a group of agents may play in physiological and pathological processes related to the topic of this meeting. These agents are peptides of fairly simple structure named, collectively, plasma kinins. Currently we have three naturally occurring peptides which belong here: bradykinin, a nonapeptide, kallidin, a decapeptide, and an undecapeptide, methionyllysyl-bradykinin. Most of the published information deals with either bradykinin or kallidin. Table 1 shows the structure of the peptides. The large number of names used to describe the three synthetic peptides reflect the confusion and the lack of agreement in nomenclature. During a recent international symposium a committee dealt with the nomenclature. The report was compiled by M. E. Webster. 2 These kinins occur in blood plasma and possibly in lymph as inactive precursor, kininogen. Kininogen is a plasma globulin. Various proteolytic enzymes can release a kinin from this precursor (scheme 1). The most important naturally occurring enzymes are the kallikreins. Kallikreins Address requests for reprints to: Dr. Ervin G. Erdos, Department of Pharmacology, University of Oklahoma School of Medicine, Oklahoma City, Oklahoma 73104. Some of these studies were supported in part by Grant HE08764 from the National Institutes of Health, the United States Public Health Service. The data presented in this paper were based on experiments done in many institutions including our laboratories.'

893

belong to a group of enzymes which are found mostly in blood plasma, glandular tissues, and urine. Plasma kallikrein liberates bradykinin while the other kallikreins release kallidin. Kallikrein in the blood plasma and in the tissues frequently exists as an inactive zymogen, kallikreinogen. Many of the components of the blood coagulation system could participate in the activation of kallikrein. The release of kallikrein in the pancreas and in the submandibular gland is connected with an exocrine and not with an endocrine function of the glands. Kallikrein was also found in the intestinal wall, mostly as kallikreinogen. Its level is higher in the colon than elsewhere in the gut; cat colon has the highest kallikreinogen content. Carnivora have more kallikrein in the intestinal wall than other animals. Gut usually contains more kallikreinogen than kallikrein. Blood plasma and many tissues contain proteolytic inhibitors which inhibit kallikrein. Parotid gland and lung are rich sources for one of these peptides. For clinical use the inhibitor called Trasylol is manufactured from bovine lung. Bradykinin and kallidin, after being released from kininogen, are rapidly inactivated by blood plasma. The inactivating enzyme in human blood plasma was characterized in our laboratory as a carboxypeptidase and named carboxypeptidase N. Many of the protein constituents of the complex system which lead to the release of kinins have been highly purified. The kinin peptides are available in synthetic form. Bradykinin and its analogues are quite active in the various biological tests in vitro and in vivo. Bradykinin contracts many isolated smooth muscle preparations in vitro. It is among the strongest pain-

894

ERDOS T.\BLE 1. Structure of ki nins Compound

Struc ture

Bradykinin (kallidin I , kinin-9, kallidin-9, non apeptide)

H-ArgLPro 2- ProL GJyLPheLSerLProL PheL ArgLOH

K allidin (kallidin II , lysyl-bradykinin, kinin-10, decapeptide)

H-LysLArg 2-ProL ProLGJyLPhe•-SerL ProLPheLArg 1 o_OH

?llethionyl-lysyl -brady - H-MetLLys'-ArgL ProLPro L GJy 6-PheL kinin (methionyl-kalSers_ ProL PheiOlidin, kinin 11, undecapeptide, hendecaArg'LOH p eptide) Act ivato r

4-------Inhibitor (e. g., hexadimethrine) ~ Preenzyme (e.g., kallikreinogen)

l

Kinin ogenase (e .g., kallikrein )

j

1

<-- --Inhibitor (e.g. , kallikrein inhibitor, Trasylol)

Vol. 51, No.5, Part 2

healthy man may have 4 to 11 mp; of bradykinin in inactive form in every liter of circulating blood plasma. In addition, bradykinin is active in some assay systems at lower concentration than other substances which occur in the body. For example, in contracting isolated rabbit or guinea pig intestine, the peptide is much more effective than histamine. Although there is no general agreement yet about the normal physiological role of bradykinin, kallidin, or kallikrein, the importance of these substances in various pathological processes has been frequently mentioned. These cases range from Addison's disease to venoconstriction. A few selected pathological conditions of clinical importance are shown in table 2. The role bradykinin may play in infl ammation is of particular interest here. The known properties of the peptide would make it an excellent candidate for the TABLE 2. Selected pathological conditions where kinins may be of im portance

Kininogen

Condition

l

Anaphylact ic shock

Kinin 4-- - --4P==lo -+----

1

Inactive split product

I

Kin inase (e.g., carboxypeptidase?\)

Inhibitor (e.g., chelating agents, h eavy metals)

ScHEME 1. Simplified mecha nism of release and inactivation of kinins. (From Erdos.')

producing agents known. Bradykinin mcreases capillary permeability. Kinins are hypotensive in most animals, and they dilate arteries. In relatively high concentrations, they promote the migration of leukocytes. Although kinins have a variety of actions in vivo and in vitro, none of these properties is unique because the actions are shared with other pharmacologically active agents. The distinction of bradykinin rests upon the abundant presence of its precursor in blood plasma (and possibly in extracellular fluid), and upon the high releasing enzyme (kininogenase) content of blood and tissues. Potentially a

Finding

Level of bradykinin increases; brady kininogen dec reases in blood. Kininogenase appears in perfu sed lung and skin. Kallikrein \Yas found in heCarcinoid patic metastases of carcinoid tumor , kinin in hepatic venous blood during flushes. K allikrein content of intesIleus tina! wall increases. Infection by patho- Level of brady kininogen degenic organisms creases in blood . Kinin appears in urin e, plasma , and tissues. Inflammation Brad ykinin causes pain, edem a, hyperemia, and migration of leukocytes. P ermeabili ty globulins release kinins . Intestinal ischemia K allikrein type substance appears in blood . Pancreatitis Level of brady kininogen decreases in blood. Beneficial effe cts of kallikrein inhibitor Trasylol have been reported .

RELEASE AND I NACTIVATION OF KININS

November 1966

Injury

I

Inflammation

Toxic met abol ites

Bacteri al toxins

I

I

~

1 \ Cells

I~

Venoms

895 H eating

I

Irradiation

I

I

Activation and liberation of protenses and kinin ogenases

l

Secretory cells-+----- - Interstitial space

~

.-\ct iva t ion of

kalliki~in l

Blood vessels

and re lease of kinins /

Shock ScHEME 2. Some possible pathways of the liberation of kinin in pathological conditions. (According t o Werle. 3 R eproduced with permission.)

function of a mediator in tissue injuries. It is very potent in increasing t he capillary permeability, causing pain, and vasodil ation. In high concentration it promotes the migration of leukocytes. The situation is, however, more complex. Tissue injuries can lead to changes in capillary permeability with the subsequent leakage of the components of the plasma kallikrein-kininogen system. This may result in the secondary appearance of kinins. Antiphlogistic drugs block the effects of bradykinin on the capillary permeability only in some of the laboratory animals tested. In addit ion, although a number of agents can be made responsible for the first, initial increase in capillary permeability in inflammation , none of them seems to be the mediator in the second, delayed phase according to Miles. 1 Scheme 2 compiled according to vV erle 3 suggests some possible pathways for t he release of kininogenases and proteases. The liber ated enzymes then would activate t he kinin system intravascul arly, in the tissues, and in the interstitial space. The end result could be pancreatitis, vasodilation, edema, hemorrhage, and shock. Kinin would cause or contribute to these symptoms. . Since such large quantities of bradykinin are potentially available in the circulation, we may assume that a rapid liberation of substantial amounts of the peptide would have tragic consequences. One factor which contributes t o the homeost atic balance of the body is the rapid en-

zymatic hydrolysis of kinins in blood and in ti ssues. The importance of this reaction is also indicated by the fact that, among biologically active peptides, bradykinin is inactivated fastest in blood serum. A good part of our laboratory vvork was centered around this enzymatic process. IVc intended to characterize the enzyme in blood and in tissues which inactivate these peptides, to find compounds which inhibit t his enzymatic hydrolysis in vitro and in vivo, and finally to develop a method to block the effect of injected or endogenous peptides. I should like to touch upon the first two points very briefly. First of all, an enzyme \\·as characterized in human plasma fr action IV -1 which inactivates bradykinin and kallidin. It is a carboxypeptidase which contains a metal cofactor. It inacti~ vates bradykinin and kallidin by cleaving the bond of the C-terminal arginine of the pepticles. The enzyme is inhibited ; by chelating agents, heavy metals, €:..amino-ncaproic acid, and other agents. Red blood cells also contain a metal dependent enzyme vvhich inactivates bradykinin and kallidin very rapidly. The enzyme in erythrocytes is different from the plasma enzyme. Plasma of various laboratory ani,. mals, however, hydrolyzes bradykinin similarly to human plasma. Scheme 3 shows the hydrolysis of various bonds in the peptide chain of kini~s. (The data were collected from t he publications of numerous investigators.) The figure indicates t hat although bradykinin is

896

ERDOS

Vol. 51, No. 5, Part 2

Met-Lys-bradykinin 1 2 3 4 5 6 7 8 9 10 I II = H-Met-Lys-Arg-Pro-Pro-G ly-Phe-Ser-Pro-Phe-Arg-OH II =

1

2

3

12

I=

4

5 6 Kallidin

7

8

9

10

3 4 5 67 Bradykinin

8

9

Enzyme

Substrate

Trypsin Chymotrypsin Carboxypeptidase A Carboxypeptidase B (pancreas) Carboxypepti dase N (plasma) and carboxypeptidase in kidney Catheptic carboxypeptidase (spleen) Enzyme in kidney cortex Aminopeptidase (purified and plasma) Prolidase (purified and erythrocytes) Snake venom (Crotalus adamanteus) (Agkistrodon h.b .)

i i

i

rx jx

i i

i i

i i

III II !,III I I I,II

i

I

i

I

III II I

i

III

i i

I

ScHEME 3. Enzymatic hydrolysis of kinins (x = ) after removal of C-terminal arginine. (From Erdos.')

very active biologically, owing to its structure it is quite vulnerable to enzymatic attack, since breaking any of the bonds within the bradykinin peptide chain inactivates the peptide. The scheme also indicates that some enzymes can convert the longer kinins to bradykinin. It was also of interest to search for compounds which could potentiate the actions of kinins in vivo presumably by inhibiting their enzymatic inactivation. Nine compounds were found which potentiate the cardiovascular effects of bradykinin in the guinea pig. Since most of these agents inhibit the in vitro inactivation of bradykinin as well, it is assumed that the potentiation of bradykinin effect in the first place rests upon the inhibition of enzymatic inactivation in blood plasma. Similar experiments were carried out in various other laboratory animals. This potentiation is particularly noticeable in the dog when the peptides are injected into the mesenteric vein. Bradykinin has to be given in doses 2 to 5 times higher in the mesenteric vein than in the

femoral vein for an equal hypotensive response. In the particular experiment shown in figure 1, 1 p.g per kg of bradykinin lowered the systemic arterial blood pressure about as much as 0.2 p.g per kg of bradykinin did after injecting it in the femoral vein of the same dog. A likely explanation for this discrepancy in the required doses of the peptide would be the increased inactivation of the peptide in the splanchnic circulation. When the animals were pretreated with 2 intravenous injections of BAL (23 mg per kg) the effect of bradykinin was greatly enhanced. This was particularly noticeable after bradykinin was injected in the mesenteric vein. The drop in mean systemic arterial blood pressure increased and was longer lasting than before BAL was given. It was shown by others that, in the rat, infused labeled bradykinin which escapes inactivation in blood accumulates in the kidney, where it is subsequently inactivated. This finding indicates that the kidney also is an important source of enzymes that hydrolyze bradykinin.

November 1966

RELEASE AND INACTIVATION OF KIN I N S

897

B

A 100

ci

:I:

::;i

50

::=;: ....... 0::

:::> C/) C/)

LI.J

0::

a.. 0

100

0 0

....J

aJ

50

60 SEC.

Fra. 1. Potentiation of the effect of bradykinin by BAL in a dog. A, before and B, after BAL; 1, 0.2 p.g per kg of bradykinin injection via femoral vein. 2, 1 p.g per kg bradykinin inj ection via mesenteric vein. (From Erdos et al?)

Figure 2 shows the subcellular distribution of enzymes that inactivate bradykinin in the homogenized rat kidney. The inactivation of bradykinin was measured on the isolated rat uterus. The various subcellular fractions were obtained in the preparative ultracentrifuge. As shown in figure 2, most of the enzymatic activity sedimented with a fraction, which is considered to contain microsomes. The relatively low activity in the fraction which has lysosomes and light mitochondria probably was caused by microsomal contamination. Further characterization of the enzymes in kidney was carried out with chemical and biological techniques. The hydrolysis of the various peptide bonds of bradykinin and of various synthetic peptide fragments of bradykinin was shown by the sequential use of paper chromatography and electrophoresis. Scheme 4 summarizes the results.

Bradykinin in the swine kidney can be inactivated by at least three enzymes. One is a prolidase which attacks the Nterminal end, the other a carboxypeptidase which hydrolyzes the bond of the C-terminal arginine, and finally, the third enzyme inactivates bradykinin by removing two amino acids from the C-terminal end; thus it breaks a prolyl-phenylalanine bond. This latter enzyme acts as an endopeptidase (peptidyl peptide hydrolase), but it is inhibited by chelating agents just as the exopeptidases are. A number of inhibitors were found which inhibit the in vitro inactivation of the biological effects of bradykinin by the kidney enzymes. It can be stated, in conclusion, that although kininogen occurs in fair quantities in blood and many tissues are rich in kininogenases (kallikreins) , the body is well equipped to handle the liberated

898

ERDOS

., ...,

-

400

+

-

..,

..!!

::::0

-

c

"' 1V E

0

"'c.

.....

z: ~ >

-

., "'E ., .,0

-8

Vol. 51, No.5, Part 2

::::0

en

300

<..>

"'C:

.!::!~

~e ..,c.

.,c. tOo cuE .::_.., "'Q.

a; 0:::

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.=

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" of total protein FIG. 2. Inactivation of bradykinin by rat kidney subcellular fractions. (From Erdos et al?)

123456789 Arg-Pro-Pro-G ly- Phe-Ser- Pro-Phe-Arg Prolidase i Carboxypeptidase Enzyme in cortex

i i

ScHEME 4. Inactivation of bradykinin by enzymes in swine kidney.

kinins by inactivating them in the blood stream or in various organs such as the kidney.

REFERENCES 1. Erdos, E. G. 1966. Hypotensive peptides: Bradykinin, kallidin, and eledoisin. In S. Garrattini and P. A. Shore [eds.], Advances in pharmacology, Vol. 4, p. 1. Academic Press, New York. 2. Erdos, E. G., N. Back, and F. Sicuteri [eds.]. 1966. Hypotensive peptides, pp. 1-660. Springer Verlag, New York. 3. Werle, E. 1964. Biochemische Betrachtungen zur Regulierung der Gewebsdurchblutung. Arztliche Forschung 18: 281-292.

November 1966

DISCUSSION

899

DISCUSSION OF PAPER PRESENTED BY DR. ERDOS DR. S. C. SoMMERS (New York): As a general pathologist and not an enzymologist or pharmacologist, I admire what Dr. Erdos has done and would like to try and translate this into what may occur in ulcerative cohtis. Enzymatically, we have evidence of mediation of the inflammatory response in ulcerative colitis by histamine and 5-hydroxytryptamine. Dr. Lagunoff has reassured us that trypsin, histamine, 5-hydroxytryptamine (serotonin), and other vasoactive substances released from the mast cells are of increasing importance in inflammatory responses. If trypsin is released into the connective tissue of the lamina propria in ulcerative colitis, then it should activate bradykinin. It is stated in the literature that bradykinin has at least 10 or 15 times the activity of histamine so that we may assume that it could produce necrosis. There are other kinins, like kallidin (lysyl-bradykinin), which I understand is less active in vitro as an inflammatory inducer. However, there is some evidence that the in vivo effect of kallidin may last longer. Do you agree that kallidin has a longer in vivo response than other kinins? Another action of trypsin in the connective tissue is to release 5-hydroxytryptamine from other sites, such as platelets. Platelets normally carry 5-hydroxytryptamine, and trypsin is one of the very few agents known to release it. Furthermore, in continued inflammation there is something called "induced histamine." The site of origin is certainly not the mast cell, and it may be the endothelial cell. One wonders if the combination of bradykinin, kallidin, "induced histamine," "released histamine," and 5-hydroxytryptamine from other sites like platelets may participate in the prolonged and abnormal inflammatory response. It is clear that the inflammatory reaction in ulcerative colitis is abnormal. For example, lymphocyte diapedesis is inhibited both in skin windows and in the lamina propria. It would be interesting if

someone would comment on these observations. DR. ERDOS: Kallidin in some animals is more potent than bradykinin; in others it is less active. The effects of the two peptides on capillary permeability is about the same. Bradykinin and 5-hydroxytryptamine can act synergistically in some tests, as shown by Sicuteri. I believe that the possibility exists that the release of trypsin may trigger the activation of kallikrein in the intestinal wall. DR. CHARLES C. NoRLAND (Chicago): First, I would like to make a comment about some work by Landerman, who studied a family with angioneurotic edema. He found these people had an abnormality in a blood inhibitor of the kallikrein system. vVhen the skin of these people was scratched, kallikreins would be released but would not be inactivated, so the vasodilation from the release of kinins was prolonged. This is an intriguing observation, and I think similar studies should be carried out in patients with ulcerative colitis to determine if there are abnormalities in the kinin system. My question for Dr. Erdos is, where are the kallikreins located in the cat colon? Are they in the argentaffin cells? DR. ERDOS: Crude tissue homogenates were the source of kallikrein in the experiments which showed presence of the enzyme in the colon. I do not know where kallikrein would occur in the intestinal wall, but glandular tissues are usually rich in kallikrein. The inhibitor, the level of which is lower in hereditary angioneurotic edema, is not specific for kallikrein. It also inhibits the C'l esterase and the permeability factor. DR. P. EKDAHL (Gothenberg, Sweden): Is the inactivation of bradykinin in lysosomal and microsomal fractions similar? DR. ERDOS: The relatively low activity in the lysosomal fraction was probably due to microsomal contamination. Most of the enzymatic activity in the kidney is local-

900

DISCUSSION

ized in the microsomal fraction, while in the liver it was found in the supernatant. DR. B. J. HAVERBACK (Los Angeles): The question of whether trypsin or other proteolytic enzymes are present in normal or diseased colon has arisen. Although tryptic activity is found in normal colon as well as in diseased colon in amounts that vary from 2 to 4 p.g of trypsin activity per g of mucosa tissue, it does not mean that the enzyme being measured is trypsin. The synthetic substrate, toluenesulfonyl-Larginine methyl ester, is used for these measurements. One of the reasons that this enzyme may not be trypsin is the fact that it cannot be inhibited by the usual trypsin inhibitors. Using the synthetic substrate N-acetyl-tyrosine ethyl ester, a small amount of chymotrypsin activity is found in the colonic mucosa of normal and diseased tissue, the significance of which currently remains unknown. Once again, the enzyme may not be chymotrypsin. An-

Vol. 51, No. 5, Part 2

other point concerning enzymes and tissue is whether or not the enzyme is free or is bound. If, for example, a small amount of trypsin is added to serum in an amount that is not sufficient to neutralize the large amount of trypsin inhibitor in the a 1globulin fraction, a significant amount of tryptic activity remains. The likely explanation for this is that the trypsin now is bound to an a 2 -macroglobulin and the trypsin a 2 -macroglobulin or chymotrypsin a 2 -macroglobulin complex maintains its proteolytic effect but cannot be inhibited by the usual trypsin inhibitors. The question that I would like to ask Dr. Erdos is whether or not he has studied the kallikreins in blood with reference to their binding with the o:2 -macroglobulins, as this globulin binds a number of other proteolytic enzymes? DR. ERDOS: We did not study the problem.