ELSEVIER lmmunopharmacology 33 (1996) 299-300

Immunopharmacology

The release of kininase from rat isolated hearts during myocardial ischaemia

Maqsood Ahmad, Isaac J. Zeitlin *, James R. Parratt Department q['Physiology and Pharmacology, UniL'ersity of Strathclyde, George Street, Glasgow, Scotland, UK

Abstract

BK destroying activity was observed in rat isolated heart perfusates. BK was optimally degraded at pH 8.4 in rat heart. The results indicated that myocardial kinin degradation was due to ACE and a serine protease. These results suggest that bradykinin may have some cardioprotective role during myocardial ischaemia at acidic pH.

Keywords: Myocardial ischaemia; Kininase; Bradykinin

1. Introduction

Bradykinin is continuously released by Sprague- Dawley rat isolated hearts during occlusion and reperfusion (Ahmad et al., 1995). Bradykinin is rapidly inactivated by carboxypeptidase-N in human plasma (Erd~Ss and Sloane, 1962). It is destroyed by carboxypeptidase-M and NEP in rat lung (Dragovic et al., 1993). In cultured human endothelial cells it is degraded by NEP and ACE (Graf et al., 1992). Little is known about its destruction in heart.

2. Method

Sprague-Dawley rats (300 + 50 g) were anaes- thetised (pentobarbitone sodium 60 mg/kg). Heparin (500 IU) was injected in the dorsal penis vein. Hearts

Abbreviations: BK, bradykinin; EDTA, ethylenediaminete- traacetic acid; NEP, neutral endopeptidase

* Corresponding author.

were removed under deep anaesthesia and immedi- ately perfused via the aorta with Krebs solution (37°C, pH 7.4 bubbled with O2/CO 2 (95:5%) on a Langendorff apparatus. After 25 min of stabilisation, the left descending coronary artery was occluded for 30 min. Perfusion of Krebs continued for a further 30 min after removing the ligature, Perfusates were collected before and 10 min after occlusion and 5 min after reperfusion, in chilled polypropylene tubes. Samples were stored at -20°C until used for en- zyme assay. Degradation of bradykinin (8.6 b~M) was measured by incubation (37°C, 3 h, pH 7.4). The reaction was stopped by boiling (10 rain). Bradykinin was extracted by using Sep-Pak Vac C18 cartridges. Eluents were dried under oxygen free N 2 and as- sayed by RP-HPLC (C18, acetonitrile, 25-70%, 0.09% TFA). Blanks contained only Krebs.

In other studies bradykinin (8.6 >M) was incu- bated with rat heart homogenates at pH 4.4-9.4 and also at pH 8.4 with different inhibitors. Bradykinin was assayed by RP-HPLC as described above. Data are presented as mean _+ SEM or Median and Q1 and Q3 as appropriate (n = 5 or 8). Group differences

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300 M. Ahmad et al. / lmmunopharmacology 33 (1996) 299-300

are compared using the Mann-Whitney U-test. P _< 0.05 was considered significant.

3. Results

3.1. Kininase release

Percentage degradation of BK in control, occlu- sion and reperfusion samples were 4.4 + 1.6, 9.6 + 2.6 and 16 + 3.1, respectively. Degradation was sig- nificantly greater during reperfusion ( P < 0 . 0 1 ) compared with control.

3.2. pH optimum for kinin

Destroying activity was maximum at pH 8.4 (73 ___ 4%), being significantly greater (P < 0.05) than at pH 7.4 (64 + 1.3%) and pH 9.4 (53 + 2%).

3.3. Inhibitor study

Destruction of BK was inhibited (P < 0.05) 33% by EDTA (40 raM), 40% by Captopril (10 mM) and 42% by aprotinin (15 000 klU). phosphoramidon (200 IxM) caused only 3% inhibition.

4. Discussion

Bradykinin (BK) is metabolised by various exo- and endopeptidases (Skidgel, 1992). Rat isolated heart perfusates contained BK-destroying activity. The BK-destroying enzymes in rat myocardial ho-

mogenates were pH dependent, destroying BK opti- mally at pH 8.4. After 10 rain ischaemia, rat myocar- dial tissue pH falls to pH 6.26, falling below pH 6 after 30 min (Khandoudi et al., 1990). At such acid pH there is minimal BK destruction. We have previ- ously reported that myocardium contains kinin form- ing enzymes optimally active at acid pH (Zeitlin et al., 1989). Kinins are known to protect the my- ocardium against ischaemic damage (Linz et al., 1993). Preliminary characterisation studies show that the myocardial kininase activity is inhibited by cap- topril and EDTA. It also cleaves des-Phe-Arg-BK indicating the involvement of ACE. Although NEP also cleaves the same BK peptide bond, phospho- ramidon, an NEP inhibitor did not inhibit the my- ocardial kininase. Aprotinin, a serine protease in- hibitor also significantly inhibited this enzyme indi- cating the probable involvement of at least one other kinin destroying enzyme in the extract. However, its further characterisation is required.

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