Analysis of the mechanisms influencing the expression of blood pressure regulating systems

Abstract Zubakova Radka, pharmacist oral examination on 15th May 2007 ANALYSIS OF THE MECHANISMS INFLUENCING THE EXPRESSION OF BLOOD PRESSURE REGULATING SYSTEMS Referees: Prof. Dr. Ulrich Hilgenfeldt, Prof. Dr. Gert Fricker Several hormonal and autocrine systems are involved in the physiology and pathophysiology of blood pressure regulation and salt-water homeostasis including the renin-angiotensin-aldosterone system, the kallikrein-kinin system, nitric oxide and the adenosine system. Within the scope of this project, the influence of high salt diet and aldosterone on the regulation of NO, NO synthase, adenosine-, and bradykinin-receptors should be examined in kininogen-deficient BNK rats and in control BN rats. For 10 days BNK and BN rats were given a standard or high salt diet in presence or absence of the aldosterone antagonist, spironolactone. Gene expression analysis was performed in tissue of kidney, atria, and ventricles using real-time RT-PCR. The activity of NO synthase was elicited by measuring NO metabolites in urine and plasma. In addition in a HEC cell line we measured the effects of different kinins on the bradykinin B1- and B2-receptor of rat, mouse and human via analysing fluorometrically the intracellular calcium influx. We found lower NOx production in plasma and urine of BNK rats in almost all experimental groups. Moreover, we could determine that in the kidney of BNK but not of BN rats high salt diet down-regulated the expression of nNOS and iNOS. The decreased NO level might be one of the factors responsible for the salt-sensitive hypertension in kininogen-deficient BNK rats. Furthermore, expression analysis of the kidney showed, that aldosterone plays an important role in the regulation of A1AR, A3AR and of nNOS. An increased salt intake seems to have no significant effect on renal adenosine receptors, NOS and B2R. Nevertheless, we found that high salt intake significantly up-regulated B1R in BN, but not in BNK rats. The enhanced B1R expression after high salt diet in the kidney of BN rats might have a protective effect. Down-regulation of A3AR and nNOS in ventricles of BNK rats after increased salt intake in BNK rats is probably one of the reasons for the increased cardiac sensitivity towards ischemia. We conclude that the lower NO levels and altered gene expression in kininogen- deficient BNK rats might contribute to the development of hypertension and salt-sensitivity. Furthermore, we could demonstrate for the first time that in the kidney aldosterone plays an important role in the regulation of A1AR, A3AR and nNOS. In addition, we showed that the efficacy and potency of BK, KAL and KLP on the B2R is similar in rat, mouse and human. Moreover, we demonstrated that des-Arg9-BK, des-Arg10-KAL and des-Arg10-KLP display similar effects on the B1R of rat, mouse and human

A kallidin-like peptide is a protective cardiac kinin, released by ischaemic preconditioning of rat heart

1. Bradykinin is thought to play a major role among the endogenous cardioprotective candidates of ischaemic preconditioning (IPC). Little attention has been paid to the fact that in the tissue kallidin (KAL), rather than bradykinin might be the physiological mediator of the kallikrein–kinin system. In order to evaluate the importance of one or the other peptide the release and effect of both kinins has been investigated in isolated rat hearts following IPC. 2. Bradykinin- and a KAL-like peptide were measured in the effluent of the rat isolated Langendorff heart with two different specific radioimmunoassays. The creatine kinase activity in the effluent was judged as degree of cardiac injury caused by ischaemia. 3. During IPC, which consists of three 5 min no-flow and 5 min reperfusion cycles prior to the 30 min ischaemia, the bradykinin level in the effluent did not change significantly (15.4–19.4 pg ml(−1)). In the control group the bradykinin levels were 15.9–16.6 pg ml(−1). 4. During IPC KAL-like peptide (Arg(1)-, instead of Lys(1)-KAL), which has recently been verified by mass spectrometry, displays 5.8-fold higher levels in the effluent and significantly increases in the same time interval from 90.4 to 189 pg ml(−1). 5. After 30 min ischaemia the bradykinin levels in the IPC group were not significantly different to those of the control group (18.7 vs 14.4 pg ml(−1)). The KAL-like peptide levels in the IPC group vs the control group were 105 vs 86.1 pg ml(−1). 6. By the 30 min ischaemia the creatine kinase activity in the IPC group increased from 0.367 to 8.93 U l(−1) (before and 10–30 min after ischaemia). In the control group during the same time period the creatine kinase levels increased from 0.277 to 34.9 U l(−1). The low increase in creatine kinase activity following IPC was taken as equivalent of the cardioprotective action. A KAL antibody or HOE140 (kinin B(2)-receptor antagonist) completely abolished this beneficial effect of IPC (36.6 and 53.0 U l(−1)) when added to the perfusion medium during the reperfusion cycles of IPC prior to the 30 min ischaemia. 7. Our data suggest that in rat hearts KAL-like peptide rather than bradykinin is the physiological compound activated by IPC and acting via the cardiac kinin B(2)-receptor. Thus, endogenously generated KAL-like peptide seems to play a major role in the cardioprotection of IPC

Kallidin-like peptide mediates the cardioprotective effect of the ACE inhibitor captopril against ischaemic reperfusion injury of rat heart

1. The potential cardioprotective effect of ACE inhibitors has been attributed to the inhibition of bradykinin degradation. Recent data in rats documented a kallidin-like peptide, which mimics the cardioprotective effect of ischaemic preconditioning. This study investigates in isolated Langendorff rat heart the effect of the ACE inhibitor captopril, the role of bradykinin, kallidin-like peptide, and nitric oxide (NO). 2. The bradykinin level in the effluent of the control group was 14.6 pg ml(−1) and was not affected by captopril in the presence or absence of kinin B(2)-receptor antagonist, HOE140. 3. The kallidin-like peptide levels were approximately six-fold higher (89.8 pg ml(−1)) and increased significantly by treatment with captopril (144 pg ml(−1)), and simultaneous treatment with captopril and HOE140 (197 pg ml(−1)). 4. Following 30 min ischaemia in the control group, the creatine kinase activity increased from 0.4 to 53.4 U l(−1). In the captopril group and in the captopril+L-NAME group, the creatine kinase activity was significantly lower (18.5 and 22.8 U l(−1)). This beneficial effect of captopril was completely abolished by the kinin B(2)-receptor antagonist, HOE140, as well as by the kallidin antiserum. 5. Perfusion of the hearts with kallidin before the 30 min ischaemia, but not with bradykinin, yielded an approximately 50% reduction in creatine kinase activity after reperfusion. 6. Pretreatment with L-NAME alone and simultaneously with captopril, and with kallidin, respectively, suggests a kinin-independent action of NO before the 30 min ischaemia on coronary flow and a kinin-dependent action after ischaemia. 7. These data show that captopril increases kallidin-like peptide in the effluent. Kallidin-like peptide via kinin B(2) receptor seems to be the physiological mediator of cardioprotective actions of captopril against ischaemic reperfusion injury. HOE140 as well as the kallidin antiserum abolished the cardioprotective effects of captopril