The anti-inflammatory action of heparin: Heparin as an antagonist to histamine, bradykinin and prostaglandin E1

THROMHOSIS @Pergamon

RESEARCH 16: 507-516 Press Ltd.1979. printed

in Great Britain

oo4g-~848/7g/llOl-o5o7 $02.00/0

TBE

ANTI-INFLANNA TORY ACTION OF EBPAIUN:

BBPAEINASANANTAGONIST To NISTANINE, BNADYEININANDPNOSTAGLANDINEl

Dr. Jean Carr, B.Sc., M.B.Ch.B. Assistant Professor,Departmentof Physiology College of Medicine, University of Saskatchewan Saskatoon, Saskatchewan,Canada S7NOW (Received 2.2.1979; in revised form 10.7.1979. Accepted by Editor M.I. Barnhart)

Beparin at a dose of 50-100 units/kg intravenouslywill suppreos increased vascular permeabilityinduced by logarithmicdoses of histamine (p
INTNDDUCTION A major feature of the anti-inflammatoryresponse is leakage of plasma proteins from the blood vessels. This increasedvascular permeabilityhas been extensively studied (1, 2, 3, 4, 5). It may be assessed by msasuring the accumulationof osdema fluid, (6, 7) by measuring the degree of joint swelling (a), or by measuring the amount of plasma protein bound dye leaking from cutaneous or mesenteric vessels (9, 10, 11, 12, 13). It is caused by various agents in various species, these include histamine, 5IiT,polypeptides,pjrostaglandins and their precursors or releasers. Under certain circumstanceswhere the injury is minor, eg. ultraviolet injury (14, 15) the leakage is biphasic. The primary phase is due to histamine release fraa mast cell granules. The latter also contain heparin which is released at the same time. In the course of evaluating the histologicalresponse to a minute ismnmologicalstimulus--repeatedchallengeby mosguito bites (16) it was shown in sections stained by Toluidine Blue, that where many metachromatic cells had shed their granules, there was a distinct area of metachromasia 507

HF.PARIN:ANTI-INFL.AMMATORY ACTION

508

vo1.16,No.3/4

of the adjacent collagen fibres. This occurred at a predictabletime followingthe challenge. It was postulated that initiationof the inflaPrnatory response would be triggeredby mast cell discharge due to sensitizationby IgE concentratedon the mast cell membrane. It seems reasonable to suppose that heparin might have some function in the inflammatory response. The present experimentswere designed to test the hypothesis that heparin might act as a feedback control mechanism in increased vascular permeability. These experimentsconsist of an analysis of the effects of heparin on increasedvascular permeabilityand on smooth muscle contraction,induced by histamine,bradykininand proetaglandinEl. Parallel experimentswere carried out on the effect of heparin as antagonist to the smooth muscle response to the same permeabilityfactors.

NATBRIUS

AND METHODS

Capillary penaeabilityexperiments Non-roultingmale albino rabbits weighing 2-5 kgs were used in groups of 4 or 5. The dorsal skin was shaved with clippers and completelydepilated with grey barium sulphide paste (12) at least 30 minutes before testing caamenced. Immediatelybefore injecting the permeabilityfactors (PFs) the animals were given Evans Blue (B.D.H.)30 mgs/kg as a 3% solution in 0.451 NaCl byintravenous injection. These animals are referred to as "blued." ??

Permeabilityfactors were given in threefold increasesof concentration on a weight basis, all were prepared in 0.9% sterile NaCl; all doses were given in 0.1 ml volume by tuberculinsyringe and 26 gauge needle. r,asiom due tc the saline vehicle never exceeded 3.0 lamin diameter. Lesions were measured 20 mins after injection. Heparin was given by intravenousinjection after the last control PF injectionhad been measured. It was allowed to equilibratefor 10 mins. The original PF dilutionswere again injected in the mirror image positions in the same animals to avoid variation in response at different sites (171, and variations due to minor inaccuraciesin dilution. AsmanY as 3 We can be assayed in each animal. Drugs Used Evans Blue (BDH) Histamine dihydrochloride(EastmanOrganic Chem. N.Y.) Lot no. 2533 cited as base. Bradykinin (BectonDickinson & Co., N.Y.) Lot V2075. ProstaglandinEl (Was a gift from Dr. J. Pike), (Upjohn)Lot No. 12851-VDV-75. Na Heparin ("Panheprin"Abbott) Lot No. D836 245 50 units/kg 100 units/kg 200 units/kg

Vo1.16,No.3/4

HEPARINrANTI-INKAMMA

TORY ACTION

509

All drugs were made up in chraned glassware,rinsed 10 times in double distilled water. In Vitro--Smoothmuscle experiments Female guinea pigs (350-450g body weight) were killed by cervical dislocation. The proximal duodenum or the urinary bladder was removed into cooled modified Erebs-Benseleitsolution. Fat and connective tissue were dissected away, and pieces of duodenum (20 mm) or urinary bladder (15 xanx 3 mm) were suspended under 1 g tension in 5 ml jacketedorgan baths maintained at 37T. The Erebs-Benseleitsolution bathing the tissue was changed by overflow at a rate of l-2 ml/rain. Isometric contractionswere measured using force displacementtransducers (Grassmodel FTQ3Cc)and displayed on a polygraph (E and M instrumentsCo., Inc., Texas type DMP4A). The modified Krebs-Benseleitsolution had the following composition (mWL) NaCl, 116; ECl, 5.4; NaB2PC4, 1.2; caC12, 2.5: MgCl2, 1.2; NaBCC3, 22.0; D glucose, 11.2: This solution when equilibratedwith 95% 02/5S CO2 gas mixture had a pIiof 7.4. All chemicals used were of analyticalgrade and were obtained from Fisher Scientific. Drugs used were as previously quoted. A 3-point dose response curve representing25%, 50% and 75% of the maximal response of the test tissue to the permeabilityfactor was used, i.e. on the straight-linearea of the dose-responsecurve. Test and control tissues from the same animal were tested simultaneously,under identical conditions. Guinea-pig duodenum (excludingthe most proximal section) was found to bs the best assay tissue for Bradykininand ProstaglandinE1, while guinea-pig urinary bladder was the best assay Doses were tested at 20 min. intervals. tissue for histamine. Doses used All given as final bath concentration. Histamine: (citedas base) 300 ng/ml, 1 ug/ml and 3 pg/ml Bradykinin: 2 ng/ml, 4 ng/ml and 8 ng/ml ProstaglandinEl:

1 ng/ml, 2 ng/ml and 4 ng/ml

a) Following the control assays, Heparin 0.5 unit/ml was Heparin: added to the perfusing solution for the remainder of the experiment in the test tissues. It was equilibratedfor 20 minutes before rs-tsating. b) in the multiple heparin dose assay, doses were added to the bath 1 minute before the agonist. Doses used were 0.05, 1.0 or 10 units/ml, 0.1, 2.5 or 25 units/ml, and 0.25, 5.0 and 50 units/ml. All were tested at 10 minute intervals as tachyphylaxisdoes not occur in response to histamine in the bladder. Experimentswere carried out on groups of 6 animals. The results were expressed as a percentage of the initial response of the tissues to the same concentrationof the drug. The responseswere considered significantlydifferent when the Student *t' test gave a probability p x 0.05. The results are graded as the mean t SBM of the given number of experiments.

510

HEPAFUNrANTI-INFLAMMATORY

ACTION

Vo1.16,No.y/4

RESULTS

PermeabilityF.xperiments Logarithmicallyincreasingdoses of histemine,bradykinin and proataglendinEl yield a straight line dose response at the doses used, when they are titrated in the intact non-anaesthetized"blued" animal. The doses of histamine and bradykinin used correspondwell with those used in the past in rabbits (185 (Figs. 1, 2). The appropriatersnge for prostaglandinwas found by trial in these animals. (Fig. 3). Histamine Histamine in 3-fold increasingdoses yielded a slope of 4.166 between doses of 0.022-5.4 Dg cited as histamine base. When Na Hepsrin 100 units/ kg was injected intravenouslyinto the animals following the initial titration,and the injectionsof histaminewere repeated after an interval of 10 mins, the dose response curve was moved to the right (Fig. 1). This is a 2.14 fold increase in dose to induce the sams response. The slope was 4.12 ma was almost pszallel. The probabilityof these results occurringby chance was p < 0.001 using an snalysis of variants, on a parallel line assay. When the &se of heparin was increased to 200 units/ kg, there was no significsntinhibitoryeffect on histamine. When the heparin dose was reduced to 50 units/kg the two plots coincided. There was no inhibitoryef feet. Thus heparin gave a statisticallysignificantinhibitionp < 0.001 at a single dose level of 100 units/kg but was ineffectiveat a higher aa a lower dose. Bradykinin Bradykininin 3-fold increasingdoses yielded a slope of 1.967 between doses of 0.8-67 ug (Fig. 2). Following the injectionof Na Heparin 50 units/kg intravenously,the response line was moved to the right with a slope of 1.678 indicatinga 4-5 fold suppressionof activity p < 0.001. It was alraostparallel with the control responses. When the dose of heparin was increasedtn 100 units/kg the response line was again moved to the right with a slope of 2.405 as against a test value of 2.151 indicatinga 3.2 fold increase in dose required to produce a lesion of comparablesize. The probabilityof this was marginal due to the small sample involved. ProetaqlandinEl: ProstaglandinEl in 3-fold increasingdoses yielded a slope of 1.356 of 0.067-0.6 pg. Only 3 of 4 animals responded. After Na Heparin 50 units per kg was injected intravenously,there was total suppressionof all increasedvascular permeabilityto PGEl in all animals.

between doses

Simultaneoustesting of other PFs in the same animals showed no diminutionof effect on increasedvascular permeability. This indicates that this is not a non-specificresponse. At this temperatureand timeinterval prostaglandinEl is stable in solution, thus there is no evidence to suggest that this was the reason for the lack of response. Analysis of variance on a parallel line assay was inappropriatein this case but a paired "t" test at each dose level showed that the probebilityof this occurring as a random chance varied from p < 0.01 at

vo1.16,~0.3/4

HEPA.RIN:ANTI-INFLAMMATORY

FIG. 1

FIG. 3

ACTION

FIG.

97 I

2

Figure 1: The capillary pernraability inducing potency of logarithmicdoses of histamine in rabbit skin. O----Q: control values in 5 animals. A---A: values in the same animals imediately afterwards,following the intravenous injection of heparin. Measurements of superficiallesion diameter were made in Wblueal' animals. Figure 2: The capillary permeability inducing potency of log8rithmicdoses of bradykinin in rabbit skin. O----O: control values in 4 animals. A----A: values in the same animals imediately afterwards,following the intravenous injection of heparin. Measurements of supsrficiallesion diaab3ter were made in "blued" animals. Figure 3: The capillary permeability inducing potency of logarithmicdoses of ProstaglandinEl in rabbit skin. O----O: control values in 3 of 4 animals which responded. A----L: values in the sams animals inmdiately afterwards, follouing the intravenous injectionof heparin. Measurements of superficiallesion diameter were made in "blued" animals.

312

JSEPARIN:ANTI-INl%AMMATORY

300ng/l1

‘nq/ml

1”ahl

FIG. 4

1nqh1

*"g/ml

FIG. 6

ACTION

vo1.16,No.3/4

4ng/m1

enq/ml

FIG. 5

4ng,m1

Figure 4: The muscle-contraction resoonse of guinea-pig urinary bladder to increasing-doses of histamine with and without heparin 0.5 unit/ml in the perfusing medium. Results are given as percentage of initial response of the same tissue to the same dose. white = control, black = with heparin. Figure 5: The muscle-contraction response of guinea-pig duodenum to increasing doses of bradykinin with and without heparin 0.5 unit/ml in the perfusing medium. Results are given as percentage of initial response of the same tissue to the same dose. white = control, black = with heparin. Figure 6: The muscle-contraction response of guinea pig duodenum to increasing doses of prostaglandin El with and without heparin 0.5 unit/ml in the perfusing medium. Results are given as percentage of initial response of the same tissue to the same dose. white = control. black = with heparin.

vo1.16,No.3/4

HEPARIN:ANTI-INlXAMMATORY

ACTION

513

a dose of 1.8 pg to < 0.001 at all other doses tested. Only 3 of the test animals showed increased vascular permeability to these doses of El, therefore the 4th animal was discarded in the calculationof the statistics. Thus, this is a very small sample. At a heparin dose of 100 units/kg there was a much Sadler degree of suppression,the probabilityof this occurring by chance was marginal p < 0.1 but > 0.05.

In vitro smooth muscle experiments Histamine: There was no inhibitionof the effect of histamine at the lowest dose level of 300 ng/ml, if anything, slight enhancementoccurred at 3 US/ml. At 1 pg/ml, where the greatest response was obtained, there was an enhancement,p < 0.03, which is statisticallysignificant (Fig. 4). In view of the fact that there was a narrow range of dosage-response in vivo, a wide range of heparin concentrationswas then tested in. Iieparinat a concentrationabove 25 units/ml significantlypotentiated the responses of the guinea-pigurinary bladder to all three concentrationsof histamine,added 1 minute after the given concentration of heparin. The range of values for p was < 0.05 to < 0.006, using the student t-test. Bradykinin: There was scme depressionof contractionto bracykininwith heparin at a bath concentrationof 0.5 units/ml (Fig. 5). This is not statistically significantby the Student "t" test, as the standard error of themean is too great. ProstaglanainEl: There was no obvious effect on the smooth muscle activity of Prostaglandin El (Fig. 6). In vivo, heparin acted as a modulator of increasedvascular permesbility induced by histamine, bradykinin and prostaglsndinEl in the skin of rabbits. It was particularly sensitive in its effective dose-range. The maximally effective &se-range varied between the permeability factors used. In vitro, heparin at a parallel dose-level did not have the ssme effects on these agents, when tested on smooth muscle activity.

DISCUSSIW These experimentssupport the hypothesis that heparin may act as a feedback control mechanism in increased vascular permeability. How this happens is not yet apparent. However, the in vitro experiment5on smooth muscle activity show that it does not directly inhibit all of the actions of these substances. Also heparin does not bind to and inactivate sub-

514

HEPARIN:ANTI-INFLAMMATORY

ACTION

vo1.16,No.3/4

stances, since there was no inhibitioneven at very high and nonphysiologicalconcentrationsof heparin. Following intravascularinjection,heparin is bound to the vascular endothelium (19). Heparin also binds to other cell surfaces and may preferentiallydisplace other mucopolysaccharides(20). Majno and Palade (21) showed that histamine and 5 HT caused the endothelialcells to draw apart briefly at their junctionsin the prevenular capillaries. They also showed venules 40 urndiameter constrict, with stasis and haemoconcentrationdistally. Thus heparin may act directly on the endothelialcell occupying cellular receptors for permeabilityfactors. In viva in the inflmtory reaction, heparin may be bound to the connective tissue after mast-cell disruption. The concentrationgradient between this depot and the plasma will lead to absorptionof heparin into the bloodstream. Gn the way it would necessarilycross the endothelirrm. The effect of histamine on increased vascular permeabilityis brief, about 5 minutes in the skin, while that of kinins and prostaglandinsis much more prolonged (1). Thus it is attractive to suggest that heparin released at the same time as histamine,might have a prolonged effect on the microcirculation. It would explain the lag phase between primary and secondaryresponses of increasedvascular permeabilitywith a timelag of up to 5 hours in ultra-violetinjury. The true physiological It is presently used as a pharmacofunction of heparinis not known. logical bludgeon to inhibit coagulationin viva as well as in vitro (22). Investigationof the fine structuraldetail of heparin however, has shown that-it is composed of up to 21 subfractions,& pairs of subfractionsunder suitable conditions (23) many of which have no anticoagulantor other known activity (24). Perhaps one of these subfractionsmay act on blood vessels without affectingclotting. This could be of clinical value.

ACXNDBTS

My thanks are due to Dr. A.J. Johns for help and advice with the smooth muscle assays and statistics,Dr. G.J. Millar for statistical advice, and Dr. L.B. Jagues for gifts of drugs. This research was carried out on funds from the Special Teaching and Research Fund of the College of Medicine, Universityof Saskatchewan, and from the SaskatchewanHeart Foundation.

REFERENCES 1. WILLIS, A.L. Release of histsmine,kinin and prostaglandinsduring carrageenininduced inflananation in the rat. InProstaglandins, Peptides and Amines. P. Mantegazza and E.W. Horton (Fds.1 London Academic Press, 1969.

vo1.16,No.3/4

HEPARIN:ANTI-INFLAMFUTORY

ACTION

515

2. FERREIRA, S.H. and VANE, J.R. New aspects of the mode of action of non-steroidanti-inflammatorydrugs. Ann. Rev. Phanuacol. 14, 57, 1974. 3.

GREAVES, M.W. Prostaglandinsand inflamation. In Prostaglandins, Physiological,Pharmacologicaland PathologicalAspr~ts. S.M.M. Rarim (Ed.1 Baltimore,University Park Press, 1976.

4.

LEWIS, G.P. (Ed.) The role of prostaglandinsin inflammation. Bern, Stuttgart,Vienna, Hans Huber, 1976.

5. WILLCUGBBY,D.A. Inflamnation. Bndeavour 2, 57, 1978. 6.

ltaEMEN, E. The inhibitionby sodim salicylateof oedema of the hind-paw of the rat induced by S-hydroxytryptamine. Brit. J. Pharmacol. 12, 28, 1957

7.

RISLEY, E.A., and NUSS, G.W. Carrageenininduced oedema WINTER, C.A., in hind-paw of the rat as an assay for anti-inflammatorydrugs. Proc. Sot. exp. Biol. Wed. 111, 544, 1962.

8. LABBLLE, A. and 'IDPWABBN,J.A. Effects of various analgesicson inflammatoryoedema resulting from silver nitrate injection. Sci. 114, 187, 1957. 9. MBNKIN, V. and MWICIN, M.F. Studies on inflammation.II. A measure of the permeabilityof capillariesin an inflamed area. J. Exper. Med. 51, 285, 1930. 10. JUDAH, J.D. and WILLCUGBBY,D.A. A quantitativemethod for the study of capillary permeability,extractionand determinationof trypan blue in tissues. J. Path. Bact. 83, 567, 1962. 11. NITTA, R., BAYASBI, H. and NCRIWATSU, K. Quantitativeextraction of Pontamine Blue fran skin, its application for measurementof increased capillarypermeability. Proc. Sot. exp. Biol. Med. 113, 185, 1963. 12. CARR, J. and WILBELW, D.L. The evaluationof increased vascular permeabilityin the skin of guinea-pigs. Aust. J. exp. Biol. Med. Sci. 42, 511, 1964. 13. CARR, J. The effect of anti-inflanmmtorydrugs on increasedvascular permeabilityinduced by chemical mediators. J. Path. 108, 1, 1972. 14. ICGAN, G.G. and WILHELM, D.L. Ultra-violetinjury as an experimental model of the inflawnatoryreaction. Nature (Land.)198, 968, 1963. 15.

G.G. and WILHELM, D.L. Vascular permeabilitychanges in inflarmnation.The role of endogenouspermeability factors in ultraviolet injury. Brit. J. exper. Path. 47, 300,.1966. LOGAN,

16. CARR, J.

Unpublished observations,1978.

HEPARINtAI'JTI-II'JF'LAMMATORY ACTION

516

17.

Vo1.16,No.3/4

MILES, A.A. and MILES, E.M. Vascular reactions to histamine, histamine liberator and leucotaxinein the skin of guinea-pigs. J. Physiol. 118, 228, 1952.

18. CARR, J. and WILHELM, D.L. Interspeciesdifferencesin response to polypeptidesas permeabilityfactors. Nature (Lond.1.208,653, 1965. 19. HIEBRRT, L.M. and JAQUES, L.B. The observationof heparin on endothelium after injection. ThrombosisResearch 8, 1?5, 1976. 20. laAEMER, P.M. Heparin releases heparan sulphate from the cell surface. Biochem. Biophys. Res. Cosmun. 78, 1334, 1977. 21. MAJNO, G., and PALADE, G.E. Studies on inflammation.I. The effect of histamine and serotoninon vascular permeability;an electron microscope study. J. Biophys. biochem. Cytol. 11, 571, 1961. 22. JAQURS, L.B. Heparins--LinearAnionic polyelectrolytedrugs. In press, Pharmacol. Revs., 1979. C-P., and NADER, H.B. Electrofocussingof 23. McDUPPIE, N.M., DIETRICH, Heparin; fractionationof heparin into 21 componentsdistinguishable from other acidic mucopolysaccharides. Biopolymers14, 1473, 1975.

24. COWIE, N.W. "Electrofocusingof Heparin" M.Sc. of Saskatchewan,1976.

Thesis. University