Staurosporine inhibits the anaphylactic reaction of the isolated guinea-pig heart

ELSEVIER

European Journal of Pharmacology 258 (1994) 247-251

Staurosporine inhibits the anaphylactic reaction of the isolated guinea-pig heart Susanne Sch~ifer, Alexander Walland * Department of Pharmacology, Boehnnger Ingelhetm KG, 55216 Ingelhelm, Germany (Received 30 September 1993, revised MS recewed 23 March 1994, accepted 25 March 1994)

Abstract

Isolated hearts from ovalbumln sensitized guinea-pigs were perfused according to Langendorff Ovalbumtn rejection decreased coronary flow Left ventricular pressure amphtude and heart rate increased lnltmlly and decreased thereafter Concomitantly, the hberatlon of histamine, prostaglandln F2,, as well as the sum of leukotrienes C 4 / D 4 / E 4 / F 4 , measured m the perfusate by radlolmmunoassay, was augmented Staurosporine (1 ~M), an inhibitor of protein kmases, did not influence the liberation of mediators in response to antigen challenge, but inhibited all mechamcal responses Infusion of phorbol mynstate acetate, an activator of protein kmase C, into non-sensitized hearts decreased coronary flow and left ventncular pressure amphtude, but did not liberate mediators Staurosporme (1 ~M) abolished these mechanical responses The results indicate that staurosporlne suppresses cardiac anaphylaxls by blockade of mediator effects rather than by inhibition of liberation or formation of mediators Key words Cardiac anaphylaxis; Langendorff heart, Staurosporxne, Phorbol ester, (Guinea-pig)

I. Introduction

Experimental anaphylactic reactions m gumea-p~gs have been studied since the b e g m n m g of this century (see Felgen and Prager, 1969). The anaphylactlc reaction can also be reduced by antigen in isolated hearts from sensitized ammals and is characterized by an mltml and a later long-lasting phase of coronary flow reduction, an m~tml posltwe lnotrop~c effect followed by a lasting depression of contractihty, and transient increases and decreases m heart rate (Capurro and Levi, 1975) These reactions are due to hberatlon of preformed and de novo synthesized medmtors, e.g hmtamlne, leukotrtenes, prostaglandins and thromboxanes (see Lleblg et al. 1975). Staurosporlne, a potent but non-selectwe inhibitor of protein kmase C (Tamaokl et al., 1986, Hldaka and Kabayashl, 1992), has been shown to inhibit the hberatlon of medmtors from mast cells (Gdfillan et al., 1990, A m o n and Dietz, 1992; White et al., 1990) and to block the signal transductlon process at the leukotnene D 4

* Corresponding author Tel 06132/77-2973, fax 06132/77-3016 0014-2999/94/$07 00 © 1994 Elsevier Science B V All rights reserved SSDI 0 0 1 4 - 2 9 9 9 ( 9 4 ) 0 0 1 9 4 - C

receptor (Crooke et al., 1990) Staurosporme is also known to prevent vasoconstriction reduced by various agomsts (Merkel et al, 1991; Sh~mamoto et al., 1993). Therefore, we wanted to investigate m ~solated hearts from sensitized guinea-pigs whether staurosporme ~s able to prevent ovalbumm-mduced anaphylaxls. The attenuation of anaphylactlc shock m consc~ous guinea-pigs by K-252a, a congener of staurosporme, supports our assumption (Paz et a l , 1991) As staurosporme indeed inhibited the anaphylacttc reaction, medmtors were assayed m the perfusate m order to detect contingent effects on their liberation and formation

2. Materials and methods

2 1 Sensitization Guinea-pigs (Chbb D H P , Dr. K Thomae, Blberach, F R G ) of both sexes weighing 300-350 g were sensitized by subcutaneous and mtraperltoneal rejection of ovalbumm at a dose of 100 m g / k g , ovalbumm was dissolved m 0 9% NaC1

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2 2 Heart preparatton attd per¢uston Three weeks after sensitization, the animals were stunned by a blow to the neck and exsangulnated by carotid transection The heart was removed and transferred Immediately into warm (37°C) and gassed Tyrode solution (see below), where a cannula was inserted into the aorta This cannula was used for perfuslon of the coronaries with Tyrode solution at a pressure of 60 cm H 2 0 , using a Langendorff apparatus (Kuschlnsky et al, 1974) The Tyrode solution had the following mllhmolar composition NaCI 137 0, KCI 27, CAC12 18, MgC12 03, NaHCO~ l l 9 , NaH2PO4 04, glucose 15 0 and was equlhbrated with 95% 02 and 5% CO~ at a temperature of 37°C The chemicals were d~ssolved in Ion-exchanged water taken from a metal pipe system and 0 1 mM ethylenedlamlnetetraacetic acid-calcium complex was added for binding of heavy metal ions As dlmethyl sulfoxlde was necessary for solublhzatlon of staurosporlne and phorbol esters, this solvent was added to all solutions at a concentration of 0 025%

2 3. Instrumentation Several minutes after the start of perfusion, the left atrium was opened for introduction of a plastic cannula with a latex balloon at the tip, filled with water, into the left ventricle The cannula was fixed in place and connected to a pressure transducer (P23 AC, Statham Instruments, Oxnard, CA, USA) for isometric recording of lntravenmcular pressure on a polygraph (P7 C, Grass Instruments C o , Quincy, MA, USA) The heart rate was recorded with a tachograph (P7 4K, Grass Instruments C o , Quincy, MA, USA), which was triggered by the lntraventrlcular pressure The coronary perfusate was collected in vials at l-rain intervals for determination of coronary flow and for measurement of the different mediators

2 4 Experimental protocol Anaphylactlc reactions were induced by bolus injection of 1 mg ovalbumln, dissolved in 0 1 ml Tyrode solution, into the aortic cannula 30 min after the beginning of the perfuslon In a group of six control hearts perfuslon was continued for 15 mln In another group of six hearts the Tyrode solution was switched to a medium containing 1 /xM staurosporlne 15 rain after the start of perfuslon For the experiments with phorbol esters, hearts from non-sensitized guinea-pigs were used Twenty-five minutes after the beginning of the experiments, in six hearts each the perfuslon was switched for 30 mln to a medium containing 2 × 10 -9 m o l / l phorbol-12-myrlstate-13-acetate (PMA) or phorbol-12-myrlstate-13-

acetate-O-methyl ether (PME) In another group of six hearts staurosporlne (1 /xM) was added to the perfuslon medium 20 rain before the switch to PMA Cardiac variables were monitored before and for 15 mln after antigen challenge and for 30 mln under phorbol ester perfuslon

2 5 Radlolmmunoassay Samples of coronary perfusate were collected for 1 mln, 5 and 1 mln before as well as 1, 2, 3 and 10 rain after the challenge with antigen or phorbol ester, and deep frozen Commercially available assays were used for evaluation of the concentrations of histamine, prostaglandln F . . and the sum of leukotrlenes C a, D4, E 4 and F4 in the perfusate

2 6 Materials The following drugs and radlolmmunoassays were used ovalbumln (Serva, Heidelberg, FRG), staurosporlne (Kamlya Biochemical C o , Thousand Oaks, CA, USA), &methyl sulfoxlde (Rledel-de Haen, Seelze, FRG), phorbol- 12-myrlstate- 13-acetate-O-methyl-ether (PME, Sigma Chemical C o , St. Louis, MO, USA), phorbol-12-mynstate-13-acetate (PMA, Sigma Chemical C o , St Louis, MO, USA), leukotrienes C 4 / D 4 / E 4 / F 4 [3H]-klt (Adv Magnetics, Cambridge, USA), histamine [12sJ]-klt (Blermann, Bad Nauheim, FRG), prostaglandln F2. [3H]-klt (Amersham I n t , Buckinghamshire, UK)

2 7 Calculatton and statlsttcs Mediator release was calculated by multlphcatlon of the coronary flow and the concentration of the mediator at corresponding times All variables are expressed as means _+ S E M The influence of staurosporlne on basal mechanical function Immediately before ovalbumin injection was evaluated by t-test The comparison between the albumin response of control and staurosporlne-treated hearts was assessed by analysis of variance with the factors group, organ within group and time, using the individual percentages of the pre-challenge value before ovalbumln Injection of mechanical parameters and the difference in absolute mediator release, respectively. Analysis of variance was followed by a t-test [with 'MS (within cell)' - compare Wlner (1971) - as an estimate for the variance, using Satterwalte's approximates for the degree of freedom] comparing each tlme separately between groups The total amounts of the mediators released during 10 rain after ovalbumln Injection were determined from the individual differences with respect to the pre-challenge values (time 0) and compared with a Wilcoxon-

S Schafer, A WaUand/European Journal of Pharmacology 258 (1994) 247-251

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(15 8 + 0 89 versus 9 0 + 0 48 ml). Left v e n t n c u l a r pressure a m p l i t u d e (30 8 + 3 52 versus 42.5 + 2.50 m m Hg) a n d h e a r t r a t e (206 + 3.8 versus 222 + 4.6 b e a t s / m m ) w e r e at t h e s a m e t i m e slgmficantly ( P = 0 0145 a n d 0 0150) l o w e r m t h e t r e a t m e n t g r o u p (F~g 1) P r e t r e a t m e n t wtth s t a u r o s p o r l n e i n f l u e n c e d the o v a l b u m m r e s p o n s e s u b s t a n t m l l y (Fig 1). A s a c o n c e n t r a t i o n of 1 /xM was o p t t m a l m p r e h m l n a r y experiments, th~s c o n c e n t r a t i o n was u s e d t h r o u g h o u t T h e solvent, d i m e t h y l sulforade, at the c o n c e n t r a t i o n u s e d d i d not influence t h e p a r a m e t e r s m e a s u r e d T h e dec r e a s e s in c o r o n a r y flow m t h e early as well as m t h e late p h a s e w e r e slgmficantly s m a l l e r m t h e p r e s e n c e o f staurosporme Treated hearts showed a much smaller m~tlal i n c r e a s e m left v e n t r l c u l a r p r e s s u r e a m p h t u d e , with only o n e p e a k a n d n o d e c r e a s e b e l o w t h e prec h a l l e n g e value. Slmdarly, t h e h e a r t r a t e r e s p o n s e was stgmflcantly r e d u c e d to a small m l t m l t a c h y c a r d l c effect

3 1 2 Medtators m the perfusate 15

I m m e d i a t e l y after t h e tnlectlon of o v a l b u m m into the perfusate of sensitized control hearts, histamine,

Fig 1 Effect of antsgen challenge m ,solated perfused hearts from senslUzed gumea-p,gs on coronary flow (CF), left ventncular pressure amphtude (LVPA) and heart rate (HR) 1 mg ovalbumm was injected at time 0 mm m stx hearts, which were perfused with Tyrode containing no (©) or 1 ~M staurosporme (o) Ovalbumm-mduced changes are expressed as percentages + S E M of the values at 0 mm Statistics analys~sof variance of the differences to the value at 0 mm and t-test,* 001 < P < 0 0 5 , * * 0001 < P_<001,*** P_<0001

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3. Results 3.1 Anaphylacttc reactton to oualbumm 3 1 1. Mechantcal functton T h e mject~on o f 1 m g o v a l b u m m into t h e p e r f u s a t e o f sensitized i s o l a t e d h e a r t s c a u s e d p r o n o u n c e d anaphylactic r e a c t i o n s which w e r e a b s e n t in t h e s~x nonsensitized h e a r t s (results n o t shown). C o r o n a r y flow d e c r e a s e d r a p i d l y a f t e r a n t i g e n c h a l l e n g e a n d rem a i n e d at a low level e x c e p t for a s h o r t p e r i o d of recovery L e f t v e n t r i c u l a r p r e s s u r e a m p h t u d e i n c r e a s e d imtially, showing two p e a k s , a n d d e c r e a s e d t h e r e a f t e r b e l o w p r e - c h a l l e n g e values. O v a l b u m i n i n d u c e d short p e r i o d s of t a c h y c a r d m a n d b r a d y c a r d i a . F i g 1 shows t h e relative c h a n g e s m c o r o n a r y flow, left v e n t n c u l a r p r e s s u r e a m p h t u d e a n d h e a r t r a t e r e d u c e d by ovalbumin. A c o m p a r i s o n of t h e v a l u e s i m m e d i a t e l y b e f o r e a n t i g e n c h a l l e n g e s h o w e d a significantly ( P = 0.0047) h i g h e r c o r o n a r y flow m t h e s t a u r o s p o r m e - t r e a t e d h e a r t s

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Fig 2 (a)-(c) Llberatton of histamine (a), prostaglandm F2a (b) and leukotnenes C 4 / / D 4 / / E 4 / / F 4 (c) from the sensitized guinea pig hearts, referred to m Fig 1, before and after ovalbumm mjectton at 0 mm Presented are mean values + S E M, calculated from coronary flow and mediator concentration m the perfusate assayed by rad~olmmunoassay in controls (open bars) and m the presence of 1 /xM staurosporme (hatched bars) Statistics see Fig 1

5 Sctmiet, A Walland / European Journal o/Pharrnacolog~ 258 (1004) 247 2ql

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prostaglandln F2. and leukomenes C 4 / D 4 / E 4 / F 4 became detectable in the effiuate or appeared in much greater amounts than before, with a maximum after 2 mln However, 10 rain after the challenge, values were already close to the control level (Fig 2a-c) The amount of histamine liberated in the staurosporlne group 1 mm after ovalbumln was greater than m the control group (Fig 2a) However, the total amounts of h~stamlne released over 10 rain mn the two groups were not significantly different (control range = 8526-27538, median 11668 ng, staurosporlne range = 10096-13607, median 11181 ng, P > 0 1) A statistical comparison between the amounts of prostaglandln F2. liberated from control and treated hearts revealed a significant inhibition by staurosporlne 3 mln after the challenge only (Fig. 2b) No statistically significant effect was obtained when considering the total amount released (control range 96-146, median = 112 ng, staurosporme range 78-155, median = 87 ng, P > 005) Staurosporlne did not cause significant changes m the leukotrlenes C 4 / D 4 / E 4 / F 4 (Fig 2c) released at a particular t~me or in the total amount hberated over 10

mln (control range = 60-319, median : 133 ng, staurosporme range = 34-456, median = 252 ng, P > 0 1)

3 2 Per Cuslon with phorbol esters and 9taurosponne Isolated hearts from non sensitized guinea pigs reacted to perfuslon with 20 nM P M A with slowly developing decreases in coronary flow and left ventricular pressure amplitude while heart rate was not affected Infusion of the non-active congener, PME, in another group of six hearts did not induce any changes (Fig 3) Perfuslon of non-sensitized hearts with 1 /xM staurosporlne resulted m a significant decrease ( P < 0 001) in left ventrlcular pressure amplitude (34 7 _+ 1 05 mm Hg) when compared with that of the PMA (61 3 __+2 3 mm Hg) or PME group (58 5 + 2 88 mm Hg) at 0 mln, while coronary flow and heart rate were not affected significantly Infusion of P M A an the presence of 1 /x M staurosporlne did not cause any changes (Fig 3) A statistical comparison of the areas under the curves of the changes induced by PMA infusion alone and in the presence of staurosporlne revealed significant differences for both coronary flow ( P = 0.005) and left ventrlcular pressure amplitude ( P = 0 031) The infusion of P M A did not increase the concentrations of histamine, prostaglandln F2~, or leukotrlenes C4/D4/E4/F 4 and B 4 i n the perfusate above the detection level

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F~g 3 Effects of infusions starting at 0 m m of 2 × 1 0 - s M P M E in s~x(/x),2×10 ~ MPMAlnseven(©)and2×10-8 MPMAlnthe presence of 1 /xM staurosporlne m six hearts (o) from non-sensitized guinea pigs on coronary flow (CF), left ventrlcular pressure amplitude (LVPA) and heart rate (HR), which are expressed as percenta g e s _ + S E M of the values at 0 r a m

The present results obtained with ovalbumin in senSltlzed isolated hearts from guinea-pigs confirm the full range of anaphylactlc reactions known from the literature (see Capurro and Levi, 1975, Aehrlnghaus et al., 1983) These reactions are mediated by a number of endogenous substances (see" Introduction), some of which were also detected in this study. The release of histamine seems to be responsible for the positive inotroplc and chronotroplc reactions, as they are abolished by histamine H 2 receptor antagonists (Levi, 1972, Capurro and Levi, 1973) Histamine receptors of the Hi-subtype might contribute to the late negative lnotropmc reaction (Hahn and Bernauer, 1972, Jolly et al, 1989) Platelet-activating factor has been claimed to make a contribution to the decrease In coronary flow, as indicated by the results of experiments with a specific receptor antagonist (Piper et al, 1990) Products of cyclooxygenase seem to be responsible for the early phase of coronary constriction which is blocked by indomethacln (Levi et al, 1976; EzeamouzIe and Assem, 1983, Yacoob and Piper, 1988) Products of hpoxygenase appear to play the most important role in coronary constriction and negative lnotropy These effects of the anaphylactlc reaction

s Schafer, A Walland/European Journal of Pharmacology 258 (1994) 247-251 d~sappear u p o n p r e t r e a t m e n t wtth mhtbttors of hpoxyg e n a s e (Jolly et a l , 1989; Y a c o o b a n d Piper, 1988) or blockers o f l e u k o t n e n e C 4 a n d D 4 r e c e p t o r s ( E z e a mouz~e and A s s e m , 1983, A e h r m g h a u s et al., 1983) In c o n t r a s t to t h e a b o v e - m e n t i o n e d mhib~tors and antagonists, wh i ch abolish only s o m e of t h e c o m p o ne nt s of the a n a p h y l a c t l c reaction, s t a u r o s p o r m e supp r e s s e d all o v a l b u m m - m d u c e d c h a n g e s substantmlly (F~g 1). S t a u r o s p o r i n e e x e r t e d th~s effect by b l o c k a d e of t he m e d i a t o r act,ons a n d not by mhib~hon o f m e d m tor synthes~s and r e l e a s e (F~g. 2 a - c ) . T h e lack o f effect of s t a u r o s p o r m e on a n t ~ g e n - m d u c e d m e d m t o r r e l e a s e m the ~solated h e a r t is in c o n t r a s t wtth results for ~solated cells (see" I n t r o d u c t i o n ) . M o r e o v e r , th e results ~mply that a c t w a h o n of p r o t e i n k m a s e C does not play an ~mportant role in th e c a r d m c r e l e a s e of m e d m t o r substances. Th~s c o n c l u s i o n ~s s u p p o r t e d by the abse nc e of m e d m t o r s m t h e p e r f u s a t e o f h e a r t s after a c t w a t l o n o f p r o t e i n k m a s e C w~th t h e p h o r b o l ester, P M A (Fig. 3). T h e c o n c e n t r a t i o n o f 1 /xM staur o s p o r i n e used sufficed to suppress the m a s s w e reach o n o f the h e a r t to P M A m a r k e d l y (Fig. 3) H o w e v e r , o u r results do n o t allow us to dec~de w h e t h e r staur o s p o r m e (H~daka and Kabayash~, 1992) a n t a g o m z e d the c a r d m c anaphylact~c r e a c t i o n by inhibition o f protern k m a s e C or o t h e r p h o s p h o r y l a t l n g enzymes. A s t h e long lasting constriction o f th e c o r o n a r y vascular b ed by l e u k o t n e n e s C 4 and D 4 d u r i n g the anaphylact~c r e s p o n s e s e e m s to t h r e a t e n c a r d m c function most, t h e i n t e r a c t i o n o f t h e s e lipoxygenase p r o d ucts w~th s t a u r o s p o r m e was t e s t e d m tissues f r o m n o n sensitized g u i n e a p~gs and r e v e a l e d a n ta g o n i s m , which wdl b e r e p o r t e d e l s e w h e r e

Acknowledgements We are obhged to Dr Inge Letmer for her adv,ce and help in stahst~cal evaluahon The excellent techmcal assistance of Mrs Manelle Le~belt and Mrs Manuela Schuhn ~s gratefully acknowledged

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