Enzymes forming kinin-like peptides in cardiovascular tissues.

Abstract

That kinins are released during myocardial ischaemia has been known for sometime now but it is only recently they have been shown to improve metabolic and haemodynamic parameters following an ischaemic insult. This work was an attempt to investigate the possibility that the heart contains kinin-forming enzymes which become active during ischaemia. Dog ventricles and thoracic aorta and rat ventricles were used for this study. Aqueous extracts of dog ventricles released rat uterus contracting substance (UCS) optimally at pH 5.2 - 5.4. It showed no obvious optimum at neutral pH. Release of UCS was completely inhibited by pepstatin. It was inhibited 32.8 % by aprotinin and 13.5 % by lima bean trypsin inhibitor but not by SBTI. Gel filtration (Sephacryl S-300) showed that this enzymic activity was related to a fraction, molecular weight 42.6 ± 0.9 kDa which at pH 5.2, cleaved D-Val-Leu-Arg-pNA and released bradykinin-like immunoreactivity (BKIR) from a plasma kinin-forming substrate. Aqueous extracts of dog aorta released UCS optimally at both acid (pH 5.4) and neutral (pH 8.0) pH and cleaved D-Val-Leu-Arg-pNA optimally at pH 5.5 and pH 7.5. Release of UCS at acid pH was completely inhibited by pepstatin; 38.9 % by aprotinin and 33.3 % by LBTI. Gel filtration produced two acid optimum peaks, molecular weights 42.0 ± 4.6 kDa and 252.0 ± 39.0 kDa. Both cleaved D-Val-Leu-Arg-pNA and released BKIR from plasma kininogen. It also showed one neutral enzyme peak, molecular weight 48.5 ± 4.0 kDa, which released BKIR (pH 8.0) from plasma kininogen. The release of BKIR by the neutral enzyme peak was inhibited 94 % by aprotinin.

Aqueous extracts of rat ventricles showed one pH-related peak of UCS formation, at pH 4.7 - 5.3. There was no release of UCS at neutral pH. Release of UCS was completely inhibited by pepstatin. It was inhibited 20 % by aprotinin, 33.1 % by SBTI but not by LBTI. The extract released BKIR (pH 5.0) from a plasma kinin-forming substrate and from L-kininogen but not from H-kininogen. Gel filtration revealed one enzyme peak, molecular weight 42.8 ± 4.9 kDa, which cleaved D-Val-Leu-Arg-pNA optimally at pH 4.5 and released BKIR (pH 5.0) from L-kininogen and not from H-kininogen. Ligation of the left main coronary artery for 10 and 30 minutes reduced BKIR- releasing activity in extracts of rat left ventricles by 30 and 48.1 %, respectively. It also increased levels of free kinin in mixed venous blood from the right atrium by up to 7 fold after ligation for 10 minutes. We have shown that dog and rat ventricles contain one cathepsin D-like aspartic proteinase which releases immunoreactive kinin from human L- kininogen. This enzyme in the rat is released from the ventricles during ischaemia and concomitantly cleaves L-kininogen which results in elevation of kinins in the coronary effluent. Activation and or release of this enzyme and the release of kinins may be an adaptive mechanism for both haemodynamic and metabolic readjustment by the ischaemic myocardium.