Fink, Edwin

Korth, Petra

Linz, Wolfgang

Schölkens, Bernward A.

Wiemer, Gabriele

Wohlfart, Paulus

Universität München: Elektronischen Publikationen

396 Hypertension Voi 2 4 , N o 3 September 1 9 9 4 
P55 
Inhibition o f Aminopeptidase P Potentiates the Vasodepressor Response to Bradykinin. 
Shin-ichi Kitamura, Luis A . Carbini, Oscar A- Carretero, William R Simmons*, A. 
Guillermo Scicli. Henry Ford Hospital, Detroit, MI and *Loyola Univ., Maywood, IL 
A C E is an important kininase, and recent data suggest that some of the effects o f A C E 
inhibitors (ACEi) are kinin-mediated Another peptidase which may be involved in kinin 
metabolism is aminopeptidase P (Am P). We examined whether inhibition of A m P with 
apstatin (Aps) alters mean blood pressure (BP) responses to bradykinin (Bk). Anesthetized 
rats were divided into 4 groups: 1 ) vehicle; 2) Aps, 800 pg/kg; 3) lisinopril (Lis), 40 pg/kg: 
and 4) Aps+Lis. Five minutes after treatment with the inhibitors) or vehicle, we studied: 
I) the dose response to Bk (50, 100, 200, 400 ng/kg iv bolus) and 2) the BP response to 
Bk infusion (400 ng/kg/min for 5 min). Aps did not affect BP responses to angiotensin I 
or acetylcholine. Aps increased the ABP response to bolus Bk, i.e., for 200 ng Bk/kg: 
control -2.3 ± 0.6, Aps -5.7 ± 0.4 mm Hg (pO.01 vs control), Lis -19.0 ± 2.0 and 
Aps+Lis -27.3 ± 2.8 mm Hg (pO.01 vs Lis). Aps also increased the duration of the 
response (area under the curve): Lis 0.55 ± 0.06 and Aps+Lis 0.91+D.05 sec (pO.01). In 
the Bk infusion studies, BP tended to return to baseline with Lis, whereas it remained 
decreased with Aps+Lis as shown below. (n=l 1; *pO.001 vs Lis). 
Time of Bk infasion (mini Lfl ifî 2JL 
Lis, ABP(mm Hg) -17.612.2 -10.9±2.2 -8.7±1.5 
Aps+Lis ABP(mm Hg) -30.0±1.9* -29.6±1.9* -24.5±2.4* 
These data demonstrate that A m P is an important kininase in vivo. Inhibitors of A m P 
may be a new tool to further increase the contribution of kinins to the effects of A C E i . 
P56 
T i s s u e K a l l i k r e i n G e n e T h e r a p y R e d u c e s B l o o d Pressure i n 
Spontaneously Hypertensive Rats 
Lee C h a o , W i l l i a m X i o n g , Jul ie Chao . M e d i c a l Un ive r s i t y o f South 
Caro l ina , Char les ton, S C 
Extensive studies indicate that the tissue ka l l i k re in -k in in system is 
i n v o l v e d in b l o o d pressure homeostasis . W e recent ly deve loped 
transgenic mice expressing human tissue k a l l i k r e i n . These transgenic 
mice are hypotensive and their b lood pressure can be reversed by the 
inject ion o f aprot in in ( k a l l i k r e i n inhibi tor) or H o e 140 (b radykin in 
receptor b locke r ) . W e further investigated the potent ial o f human 
tissue k a l l i k r e i n gene therapy by injecting the k a l l i k r e i n gene into the 
skeletal muscle o f spontaneously hypertensive rats ( S H R ) . Expression 
o f the human tissue ka l l i k r e in gene in S H R was ident if ied by reverse 
t r a n s c r i p t i o n - P C R f o l l o w e d by Southern blot ana lys i s , E L Ì S A and 
immunohis tochemis t ry . Injection o f the human k a l l i k r e i n gene into 
S H R caused a reduct ion o f systemic b lood pressure one week post-
injection and this effect continued for two months. The blood pressure 
reduct ion was more than 20 m m H g compared to the controls w h i c h 
were injected w i t h vector D N A alone. The differences are statistically 
s ign i f ican t i nd i ca t i ng that somat ic de l ive ry o f the human tissue 
k a l l i k r e i n gene induces a sustained reduct ion o f sys temic b l o o d 
pressure in S H R . These results raise the p o s s i b i l i t y o f app ly ing 
ka l l ik re in gene therapy for treating human hypertensive diseases. 
P58 
Bradykinin Induces Nitric Oxide Production by Rat Carotid Smooth Muscle Cells in vitro. 
Luis A . Carbini and A. Guillermo Scicli. Henry Ford Hospital, Detroit, MI , USA. 
Bradykinin (BK) and nitric oxide (NO) may mediate part of the protective effect of 
angiotensin-converting enzyme inhibitors (ACEi) . Some of these effects cannot be 
explained by BK-induced formation of N O by endothelial cells. We hypothesized that B K 
can increase N O generation by vascular smooth muscle cells (VSMC). Carotid V S M C from 
Sprague-Dawley rats were plated (passages 4 to 8); 24 hr later, fresh medium, alone or 
containing either B K (1fr6 M), ramiprilat (R. 10 ug/ml) or BK+R. was added. At 5 and 20 
hr after treatment nitrite (NO,) was measured in the medium. Treatment for 5 hr resulted 
in no detectable increase in NO,*, while in cells treated 20 hr with BK+R NCV production 
(n = 23) increased by 213% (from 3.1±D.7 to 6.7 ± 1.0 nmol/mg protein, p < 0.004). In 
dose response studies B K at 10"* M , plus R, still induced significant increases in N O . 
(Control, 0.11±0.02; 10* M B K , 3.18+D.l 1; IO"7 M B K , 3.02±0.30; 1 0 - 8 M B K , 1.16±0.09 
[n - 3, p < 0.0001]). B K or R alone did not induce changes in NO,". In contrast, B K alone 
increased the production of prostacyclin and PGE, (6-keto P G F l a : Control= l.4±£>.2, B K = 
23.3±3.5, BK+R= 29.514.1 ng/mg protein [n = 4 , p < 0.001 ]; PGE,: C o n t r o l 7.0±1.4. B K = 
47.2±19.8, BK+R=44.2±12.3 [n = 4,p < 0.03]). Treatment with dexamethasone ( 1 Of5 M), 
which inhibits expression of inducible nitric oxide synthase (ìNOs), or with the N O 
synthase inhibitor N^-nitro-L-arginine methyl ester (840 jig/ml), abolished responses to 
BK+R (n = 3).. The increase in N Q f and prostanoids was completely blocked by Hoe 140 
(10~* M), a B K B , receptor antagonist. We conclude that stimulation of B K B ; receptors 
induces N O generation in V S M C , likely by increasing iNOs. Inhibition of B K degradation 
by A C E is needed to reveal the effects of B K on N O formation. The present data suggest 
that in addition to the known ability of kinins to release factors such as NO, prostacyclins 
and hyperpolarizing factor from endothelial cells, in the presence of an A C E i kinins can 
also increase production of vasodilatory and anti-mitogenic factors from V S M C . 
P59 
Intramuscular Del ivery o f Rat Ka l l i k r e in -b ind ing Protein Gene Reverses 
Hypotension in Transgenic M i c e Express ing Human Tissue K a l l i k r e i n 
Ju l ie C h a o , J i a n - x i n g M a , L e e C h a o . M e d i c a l U n i v e r s i t y o f Sou th 
C a r o l i n a , Char les ton, S C 
T h e tissue k a l l i k r e i n - k i n i n system has been postulated to play an 
important role in b lood pressure regu la t ion . Rat k a l l i k r e i n - b i n d i n g 
protein ( R K B P ) is a serine proteinase inh ib i to r w h i c h b inds to and 
inhibits tissue kallikxein's act ivi ty . W e recently developed several lines 
o f human tissue ka l l i k r e in transgenic mice w h i c h are hypotensive. In 
order to investigate the role o f R K B P in b lood pressure regulat ion, we 
del ivered the R K B P gene into these transgenic mice by intramuscular 
inject ion. Expres s ion o f the R K B P gene was detected in the skeletal 
muscle by reverse t ranscr ipt ion-PCR fo l lowed by Southern blot analysis 
at 10, 20, 30 and 40 days post-injection. Immunoreact ive R K B P levels 
in the musc le and serum o f these mice were quant if ied by a R K B P -
specific E L I S A and Western blot analysis . R K B P m R N A and protein 
levels were l o w at 10 days post-injection but increased s ignif icant ly at 
20 and 30 days . D u r i n g this t ime p e r i o d , R K B P gene d e l i v e r y 
s i g n i f i c a n t l y increased sys temic b l o o d pressure i n the k a l l i k r e i n 
transgenic mice to a level comparable to that o f normotensive control 
mice. Nei ther the R K B P gene nor vector D N A del ivery had an effect on 
the b lood pressure o f normotensive control mice. These results suggest 
that the increase o f systemic b lood pressure by R K B P gene de l ivery in 
these hypotensive transgenic mice might be mediated by inhibit ing tissue 
kal l ikrein activity. 
P57 
Early Blockade of Bradykinin Receptors Increases Blood Pressure in Rats. 
Paolo Madeddu, Paolo Pinna Parpaglia, Maria Vittoria Varoni, Nicola Glorioso. 
Clinica Medica and Farmacologia, University of Sassari, Sassari, Italy. 
We evaluated if chronic inhibition of B2-bradykinin receptors by the long-
acting antagonist Hoe 140 affects blood pressure (BP) of normotensive rats. Neither 
Hoe 140, at 25 nmol/day per kg body weight (BW) i.p. for 4 weeks, nor its vehicle 
altered systolic BP (plethysmography) of 9 week-old rats on normal (0.3%) or 
high (2%) sodium intake. In further experiments, pairs of Hoel40-treated rats were 
mated and their pups were maintained on Hoe 140 and normal sodium diet. 
Controls were given vehicle instead of Hoe 140. Rats given Hoc 140 when in utero 
and then during early phases of the life showed greater systolic BP, heart rate (HR) 
and B W values than controls at 7 weeks as well as at 9 weeks of age (122±3 vs 
113±3 mmHg, 444±6 vs 395±8 b/m, 258±7 vs 213±3 g, p<0l) . These differences 
remained unaltered when the rats were fed a high sodium diet while still continuing 
Hoel40 or vehicle for further 3 weeks (121±1 vs U2±2 mmHg, 399±11 vs 362±4 
b/m, 349±5 vs 307±6 g, p<0l). By contrast, low sodium diet (0.05 %) nullified the 
difference in systolic BP (116±2 vs 120±2 mmHg in controls, N.S.). Mean BP 
(direct measurement) was higher in HoeÌ40-treated rats on high sodium (115±2 vs 
99±2 mmHg in controls, p<0l). Propranolol (2 mg/kg, i.v. bolus) induced a greater 
bradycardic effect in Hoel40-treated rats on high sodium than in controls (105±8 
vs 6 l±10 b/m, p<05), without affecting mean BP. Heart weight was greater in 
Hoel40-treated rats than in controls (3.00±0.07 vs 2.75±0.04 g/kg BW, p<05). In 
conclusion, early blockade of bradykinin receptors increases BP and HR, while 
inhibition in adult life is ineffective. Our data suggest that endogenous kinins may 
play a role in the regulation of cardiovascular function by influencing the activity of 
sympathetic nervous system and/or renal excretory function in early phases of life. 
P60 
Blockade of ANG II (Subtyp« AT^) Receptors induces K i n i n aud 
P r o s t a c y c l i n Release from i s o l a t e d perfused Rat Hearts 
Gabriele Wiener, Edwin F i n k 1 , Petra Korth, Paulus Wohlfart, 
Bernward A. Schölkens, Wolfgang L i n z . Hoechst AG, Frankfurt/M, 
Germany, ^University München, Germany 
We have recently shown that reduction of postischemic 
reperfusion arrhythmias i n i s o l a t e d rat hearts by AT^ receptor 
antagonists seems to be mediated v i a endogenous ANG I I , 
probably through stimulation of cardiac e n d o t h e l i a l AT 2 
receptors. Since t h i s cardioprotective e f f e c t i s prevented by 
coperfusion with the B2~kinin receptor antagonist i c a t i b a n t we 
investigated the possible contribution of cardiac 
kininogenase-kinin pathways during AT^ receptor blockade. In 
normoxic with Krebs-Henseleit b u f f e r perfused hearts ATj 
receptor antagonists s i g n i f i c a n t l y enhanced the outflow of 
kinins i n t o the venous e f f l u e n t (from 166.4±32 to 518.0±42 
fmol/min). In p a r a l l e l an increased p r o s t a c y c l i n outflow was 
observed. In ischemic hearts the decrease in reperfusion 
arrhythmias by AT^ receptor antagonists was suppressed by 
coperfusion with either the serine or the cysteine protease 
i n h i b i t o r s dichloroisocoumarin and E 64 re s p e c t i v e l y . The data 
suggest that the cardioprotective action by AT^ receptor 
blockade i s accompanied by an enhanced synthesis and release 
of endogenous kinins. By promoting NO and p r o s t a c y c l i n 
synthesis k i n i n s are suggested to protect i s o l a t e d r a t hearts 
against endothelial dysfunction and myocardial ischemia. 


Blockade of ANG II (Subtype AT1) Receptors induces Kinin and Prostacyclin Release from isolated perfused Rat Hearts

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Blockade of ANG II (Subtype AT1) Receptors induces Kinin and Prostacyclin Release from isolated perfused Rat Hearts

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