- Email: [email protected]
Increase in myocardial adenine nucleotides induced by adenosine: Dosage, mode of application and duration, species differences
Journal
of h4olecular
Increase Dosage,
and Cellular
Cardiolou
(1980)
12, 619-634
in Myocardial Adenine Nucleotides Mode of Application and Duration,
Induced Species
by Adenosine Differences*
:
WOLF ISSELHARD, JURGEN EITENMULLER, WOLFGANG MAURER, ASTRID DeVREESE, HELMUT REINEKE, ANDRZEJ CZERNIAK, JOSEF STURZ AND HANS-GUNTER HERB Institut
fiir
Experimentelle 5000
(Received
Medirin der Universitiit LU Kiln, Robert-Koch-StraJe Kiiln 41, Federal Republic of Germany
10 April
1979, accepted in revised form
23 November
10
1979)
W. ISSELHARD, J. EITENGLLER, W. MLURER, A. DEVREESE, H. REINEKE, A. CZERNIAK, J. STURZ AND H.-G. HERB. Increase in Myocardial Adenine Nucleotides Induced by Adenosine: Dosage, Mode of Application and Duration, Species Differences. Journal of Molecular and Cellular Cardiology ( 1980) 12, 6 19-634. Ad enosine or saline in control experiments were infused into the left ventricle, the left atrium and the venae cavae respectively in experiments with rabbits, rats, and dogs. The infusion volume was 4 ml/h x kg body weight in rabbits and rats, and 6 ml/h x kg in dogs. The adenosine concentrations varied between 0.125% and 1.5%, the duration of infusion varied between 1 and 5 h. The analyzes included the determination of the left ventricular tissue levels of the adenine nucleotides, creatine phosphate and total creatine, glycogen, glucose, lactate, and adenosine as well as the systemic.pressure and heart rate. Adenosine caused significantly increased myocardial tissue levels of adenine nucleotides with a maximal average rise in ATP above the mean control value of 3976 in rabbit hearts, 480/b in rat hearts, and 21% in canine hearts. A 30% increased tissue level of ATP and adenine nucleotides returned to the control ranges after 24 h. KEY
WORDS:
adenosine
Myocardial metabolic status; Myocardial adenine nucleotides; Prolonged infusion; Adenosine induced increase in myocardial adenine nucleotides.
1. Introduction After sL. ,re anaerobiosis, the restitution of normal tissue levels of adenine nucleotides (AN) in the myocardium is significantly accelerated by a continuous infusion of adenosine (Ado) [7, 12, 13, 16, 211. A decrease in the sum of AN (SAN) and especially in ATP results from an insufficient energy supply during anaerobiosis [ 10, 11, 13, 16, 17, 201. Further methods are needed to protect the myocardium during anaerobic situations such as induced cardiac arrest without coronary perfusion for cardiac surgery, to improve myocardial anoxic tolerance, or to improve and accelerate post-anaerobic recovery. In this context, it is of interest to evaluate whether a normal tissue level of AN can be substantially increased pre-anaerobically by a continuous administration of Ado. * This work was supported 68 “Cardiovascular Restitution OO22-2828/80/060619+
by the Deutsche Forschungsgemeinschaft, and Organ Substitution”.
16 $02.00/O
0
1980 Academic
Sonderforschungsbereich Press Inc.
(London)
Limited
620
\V.
ISSELHARD
ET
AL.
2. Methods The experiments were performed on Sprague-Dawley rats (200 to 250 g), rabbits (2.5 to 3.0 kg), and mongrel dogs (7.5 to 13.0 kg). Anaesthesia was induced with pentobarbital (Nembutal@) in rats (5 mg/lOO g) and dogs (17 to 23 mg/kg after premeditation with 20 mg morphine and 5 mg atropine), and with butyl-bromoallyl-barbiturate (Pernocton@) in rabbits (100 mg/kg). During the course of the experiments, the anaesthetics were re-applied in small doses if necessary. Rabbits and rats were tracheotomized, and the trachea was cannulated; dogs were intubated. The systemic pressure was monitored in a femoral artery in dogs and in a carotid artery in rabbits and rats respectively. The heart rate was calculated from the ECG or from the arterial pulse. Normal body temperature was maintained by the application of external warmth. Ado or saline in control experiments were administered in three variations: into the left ventricular cavity through a catheter installed via a carotid artery (groups LV), into the superior vena cava through a catheter via the right superficial jugular vein, the end of which was placed as close as possible to the right (groups LA). For the latter atrium (groups CV), and into the left atrium approach, a thoracotomy in the 5th intercostal space was performed, the left auricular appendix was cannulated, and the thoracotomy was superficially closed. Between the end of the surgical procedure and the start of infusions, there was a 30 min period to obtain steady state conditions. The animals breathed spontaneously except for rabbits in group LA and all dogs, which were artificially ventilated during the entire course of experiments with room air and N,O-0, (end-expiratory CO,: 4 Vol’;&) respectively. Deviations in the acid-base status were compensated only during the steady state period. Iso-osmolarity of the Ado solutions was achieved by addition of NaCl. The infusion volume was 4 ml/h x kg body weight in rabbits and rats, and 6 ml/h x kg in dogs. The infusates were kept at a temperature of 40°C. About 30 min before termination of the experiments, artificial respiration was resumed in all experiments, and the hearts were exposed. The operative procedure was finished 10 to 15 min before the removal of the heart by the freeze-stop technique [30]. In rabbits and rats, the whole hearts were frozen. From canine hearts, the apical part of the ventricular myocardium was grasped between large pre-cooled metal blocks of tongs with a sufficient cold capacity and severed from the base with a scalpel. Metal tongs and myocardial specimen were immediately immersed in liquid oxygen. The specimens were cleaned of blood and epicardium under liquid oxygen and weighed. Only left ventricular myocardium was used. Canine myocardium was differentiated into an epicardial and an endocardial half; the latter was discarded. The specimens were freeze-dried, re-weighed, and extracted. The preparation of perchlorid acid tissue extracts and the enzymatic analysis of the
ADENOSINE-INDUCED
myocardial
INCREASE
tissue levels of substrates
IN
ADENINE
621
NUCLEOTIDES
and metabolites
have been described
[Id, 18>
191. All data are presented as tissue levels in pmol/g frozen wet myocardial tissue with a dry matter content of 20% in the rabbit, 22.5O/, in the rat, and 22O, in the dog; experimentally induced variations in this ratio were considered. The data are presented as mean values i S.D. The t test was applied for statistical analysis. 3. Results Continuous infusion of Ado increased significantly the tissue levels of AN in the left ventricular myocardium of rabbits, rats and dogs (Figures 1 to 3 ; Table 1 ), while the infusion of saline did not cause significant alterations in the AN status (Figure 2; Table 1). Rabbits: Without infusion
Left
0.125%
(0)
odenosine
0.25%
b)
infusion
(4ml/kg,
ventricle
0.5%
(~1
(4
3h) Left
1.0%
(a)
atrium
0.5%
1.5%
(f)
into
(cl)
Vv.
1.0%
(h)
covoe
I .00/o
(i)
4 3 2 I 0 ECP: 0.931 n : 12
0.920
0.947
0,954
0.914
0.938
0.926
0.937
6
5
6
8
6
6
5
n
AMP
0
ADP
FIGURE 1. Tissue levels of adcninc nuclcoti(le potential (ECP (ATP 1 O..i hI>P)~SAN) control conditions without infusion and affcr trations into the left ventricle (group I-1.1, the CA7 for 3 h (means J: s.D.). Significance of Pi 0.001 (P .: 0.001); (b) vs (c) P : 0.05 (P ..; 0.05); (d) vs (e) n.s. (ns.); (e) vs (f) ig) vs (h) P .-c 0.02 (P -_ 0.05).
M.C.C.
q
ATP
N
0.923
5
SAN
~:ymol~g frwrn wet tissue) and rncrgy chargr 111 left vrntricular myocardium of rabbits under infusion of adcnosim solutions of diffcrcnt concrnIrft atrium (group L:\). and the vcnac cavac (group d~ffcrrnces in : ZTP (SW): (ai vs (b). (c) . (i) in.s.); (CI vs (d) n.s. (KU: (b) \‘s (d) P. 0.005 n.\. (P -: 0.05); (di v?, (fi P <: 0.023 (P 0.05 I ;
2c
622
W.
ISSELHARD
ET AL.
AN in rabbit myocardium Under control conditions, the tissue levels were 4.94 f 0.24 for ATP, 0.63 & 0.20 for ADP, 0.08 f 0.03 for AMP, and 5.65 5 0.32 pmol/g for SAN (Figure 1). ATP (SAN) increased to 137 (131)0/b of the control after infusion of 0.5”,, Ado for 3 h in group LV; Ado solutions of lower and higher concentrations were less effective, but also resulted in significant increases (Figure 1). In group CV, the infusion of 1 “,, Ado for 3 h caused ATP (SAN) to increase to 139 (139) I’,, (Figure 1). In group LA, ATP (SAN) reached 118 (119)“, when applying 0.5”,, Ado and 128 ( 126) (I,, when employing loi, Ado for 3 h (Figure 1). A prolongation of the Ado infusion from 3 to 5 h had no additional effect, except for group LA which received 0.5”,, Ado (F i g ure 2). But this measure only raised the AN levels to values achieved already after 3 h in the other experiments. An infusion of 1 h duration was not suflicient to raise maximally the levels of AN (Figure 2). The energy charge potential (ECP ATP + 0.5 ADP/ATP + ADP $ .4MP [I] remained unchanged during the infusion of Ado or saline (Figure I), indicating that the infusion of Ado increased the tissue levels of ATP, ADP, and AMP correspondingly. AN in rat m)locardium The infusion of Ado for 3 h under group CX conditions resulted in a marked increase in ATP and AN, which was more pronounced after the application of
r-,
Q
6.0
5.0
_ 1
J,
(41----
1(12)
I 4.5
I 0
I I
I 2
I 3
I 4
I 5
Before infusion Duration
of
infusion
(h)
FIGURE 2. Tiaauc Icvcl of :\TI’ ( !~mol/g frozen tissuej in left vcntrirular myocardium of rabbit> under control conditions before infusion and after infusion for 1, 3, and 5 h of adcnosine solutions (.kIo) of diHirrnt concentrations or saline into the left atrium (group IA) (mean> 1 s.ni.
CV cv
c N
Kal
Group
Duration infusion
from
13 4 3 6 6 6 6
t1
0 0 1 .o I .o 1.0 1.0 1 .o
of (h)
1-I 6 3 8
C values:
(00)
0 0 0.5 1.0
Adenosine concentration
5.73 5.78 7.02 6.40 6.95 6.77 6.70
4.59 4.29 6.18 6.78
* + + + * -7 -J;
* & + rf~
ATP
0.34 0.34 1.07t 0.4ot 0.24" 0.32* 0.35*
0.30 0.17 0.23* 0.63*
& & + &
0.13 0.17 0.02 O.lS§
0.68 t 0.22 0.81 * 0.05 I.11 + 0.23: 0.89 + 0.13' 1.07 -I 0.20t 1.00 11: 0.11* 0.94 .+y 0.1og
0.91 0.92 0.98 1.08
ADP
-& & f 3:
+ + & * 5 + +
0.22 0.23 0.31 0.32
0.13 0.20 0.21 0.19 0.23 0.23 0.19
AMP
0.08 0.04 0.08 0.07 O.OY.0.11 0.06
0.06 0.05 O.OOj/ 0.07
1. ‘I’issue levels of adeninr nucleotidrs (lkmol/g frozen wet tissue) in left ventricular myocardium c.ontrol conditions (C), after infusion for various periods of time of saline (W), or adenosine into I,\‘), the venar ravac (group C:Vl, or the left atrium (group L.4.)
Significance of differrnce * P < 0.001. t P < 0.005. r P i 0.001. g P < 0.002. j/ P < 0.02.5. f P < 0.05.
1’:\HL,I:
6.54 6.79 8.34 7.48 8.25 8.00 7.83
5.72 5.44 7.47 8.18
& -& & -j+ .+ +
-& & ~-fr 4
SAN
0.41 0.36 1.37* 0.37* 0.23* 0.53* O.&O*
0.39 0.22 0.24* 0.68*
of rats and dogs under the left ventricle (group
621 I”,, Ado than unchanged.
W.
after
ISSELHARD
the infusion
of 0.5”&
ET
AL.
Ado
(Table
1). The
ECP
remained
AN in canine myocardium The infusion of 1 O,, Ado resulted in significantly increased tissue levels of ATP, ADP, and SAN after 3 and 5 h (Table 1). The variations in duration and approach of infusion did not yield different results. The ECP remained within normal ranges. Duration
of eleuation of AN
Upon discontinuation of the Ado infusion, a normalization of the elevated AN tissue levels occurred slowly. A 30”” increased tissue level of SAN and ATP returned to the ranges of control hearts after 24 h (Figure 3). The slope of the decrease curve can be described by the equations y = 7.3524 x e-“.oo97~ for SAN and y = 6.3035 x e-0.0°g5* for ATP, the correlation coefficients being r = 0.7930 and Y = 0.7820 respectively. Supporting
data on myocardial metabolism
Ado was not detectable in control hearts. Its tissue level averaged 0.03 pmol/g in hearts of rabbits which had received an infusion of at least 1 y/, Ado. The glycogen level was not altered during the infusion periods. The control values were in rabbits 37.70 & 8.80 pmol/g, in rats 23.01 h 3.03 pmol/g, and in dogs 39.50 i 8.62 pmol/g.
(n; (12)
(8)(5)
(5)
(4)
16) ADP
I--+
/+-O-+------4 wt*
“7
AOI --
+ AMP
+ 3
15 Time
t 24
(h)
FIGURE 3. Adenine nucleotides in left ventricular conditions (C), at the end of a 3 h period (A) of infusion left ventricle (group LV), and during a 24 h post-infusion
myocardium of rabbits under control of 1 :,I adenosine (4 ml/kg x hi into the period (means J SD.).
N 0
i: a
3 5
I .OOo/0 Ado
LA
v, v = P < 0.05. w, w = P < 0.02. x x =- P < 0.01. Y , y = P < 0.005. z, z = P < 0.001.
N, n = ms.
to group
3 5
Ado
l.OOq/,
CV
of difference
3 3 3 5
0.25% 0.50% 1 .OO%
LV
D: Significance
3 5
Saline
LA
Ado Ado Ado
3
Saline
C (K, V-Z)
5 5
5
5 6 8 2
4 3
6
4
3
Saline
n
cv
of (h)
LV
Duration infusion 12
Infusate
1.36
2.02
2.11 0.97
2.64
.+ 4.25 .+ 2.80
f
+ 1.98 * 2.46 + 3.66
k f
-& 2.16
i
*
and between
8.61 9.92
6.89
6.10 7.46 9.36 8.47
3.00 2.63
5.19
3.97
5.48
Rabbit a -& S.D.
saline
8
14
n
3.67
2.01
+
f
1.53
0.48
S.D.
(n, v-z.).
jz +
Rat
myocardium (Ado) into
and Ado groups
N, v y, x
N, n
N,n N, v X, w
N V
N
N
D
2. Tissue level of glucose (pmol/g frozen wet tissue) in left ventricular conditions (C) and after infusion for 3 and 5 h of saline or adenosine (group CV), or the left atrium (LA)
C
Group
TABLE
Z
D
5 6
6 5
3
3
2
13
n
of rabbits, rats, the left ventricle
0.33
+
1.75
& 0.07
*
S.D.
1.78 & 0.82 1.57 * 0.29
1.47 f 0.98 1.63 & 0.41
4.04
1.01
1.38
0.90
fi *
Dog
Y z
N Y
Y
N
D
and dogs under control (group LV), the venae
626
W.
ISSELHARD
ITT AL.
The myocardial tissue level of glucose (Table 2) increased during the infusion of Ado. The rise increased with the Ado concentration (rabbits, group LV) and was independent of the duration of the Ado infusion. Upon cessation of the Ado infusion, the glucose level returned to the control range within 1 h. During the period of infusion of saline, the tissue glucose remained constant in canine myocardium and tended to decrease in the rabbit heart. The myocardial tissue level of lactate (Table 3) increased during the period of infusion of saline or Ado. This increase was slightly higher in animals which received Ado in concentrations of 0.5:~ or more than in experiments with saline infusion. However, this difference was not significant except for the rabbit group LA which received 1% Ado for 5 h. The myocardial lactate level returned to the control range 15 h after the discontinuation of the Ado infusion (rabbits, group LV). The myocardial tissue level of total creatine (TCr) amounted to 15.61 & 1.88 Fmol/g in rabbits, to 14.14 f 2.12 pmol/g in rats, and to 20.90 k 1.38 pmol/g in dogs. It was not markedly influenced by the infusion of saline or Ado. The myocardial tissue level of creatine phosphate (CrP) (Table 4) was not altered by the infusion of saline in dogs and rabbits of groups LV and CV; it increased in rabbits of group LA and in rats. In rabbits of group LV, infusion of 0.125% or 0.25% Ado for 3 h resulted in a tendency towards increased CrP levels, whereas the application of 0.5% of 1 y0 Ado caused depressed tissue levels of CrP. Also in group LA, the infusion of 0.5% and lo,; Ado for 3 or 5 h caused a decrease in the CrP level, which was particularly distinct in comparison to experiments with saline infusion. Upon discontinuation of the Ado infusion, the tissue level of CrP returned to the control range within few minutes. In rats (group CV), 0.5% Ado did not alter the myocardial CrP level, while ly/, Ado reduced CrP in comparison to the value obtained after saline infusion of 3 h duration. In the dog, the infusion of 1% Ado did not result in significantly altered CrP levels in groups LV and CV; however, CrP was significantly increased in group LA. Blood pressure Under control conditions, the mean systemic blood pressure (MSBP) averaged 86 f 12.3 mmHg in rabbits, 133 f 19.9 mmHg in rats, and 98 k 16.4 mmHg in dogs. The infusion of Ado resulted in a prompt reduction of MSBP. In rabbits of group LV, MSBP decreased in proportion to the dosage of Ado (Figure 4). However, in group CV the Ado induced drop in MSBP was slightly retarded; 5 min after the onset of the infusion of 1 o/0 Ado, MSBP had reached a range which on the average was 2 mmHg higher during the course of experiments than during the infusion of 1% Ado in group LV. In group LA, preparing the animals for the infusion resulted in a significant decrease in MSBP to 77 + 10.5 mmHg (P < 0.001). The infusion of both 0.5% and I”/$ Ado caused a
0.5% 1.0%
1 .O%
0.5% 1 .O%
LV
CV
LA
N, w, x, Y, z,
Ado Ado
of difference
n = n.s. w = P < 0.05. x = P < 0.02. y = P < 0.005. z = P < 0.001.
D: Significance
3 3 5
Saline
Ado
3
5
Saline
CV
LA
Ado Ado
4
C (N, W-Z)
5 5 5
5 3 5
to group
5
3 5
6 8 2
3
6
12
n
3
of (h)
Saline
Duration infusion
LV
Infusate
2.17
1.76 1.18
& 3.13 & 2.85 i. 3.83
& 2.13
i: +
& 2.58
& 1.99
f
5 0.91
S.D.
and between
8.16 5.78 11.62
4.55
6.14 5.01 6.85
5.53
4.41
5.65
+
Rabbit
2.58
ji
saline
z, w
Y
Z, n
8
6
14
n
2.87
2.73
2.05
1.33
(n, w-z).
-& 1.57
-& 1.73
f
Rat 5 + S.D.
myocardium (Ado) into
and Ado groups
X n
Z, n Z, n
Y
X
Y
D
3. Tissue level of lactate (kmol/g frozen wet tissue) in left ventricular conditions (C) and after infusion for 3 and 5 h of saline or adenosine cavae (group CV), or the left atrium (group LA)
C
Group
TABLE
N, n
N
D
6 6
6 5
3
3
2
13
n
of rabbits, rats, the left ventricle
3.19 3.86
2.71 3.76
2.33
1.94
2.66
I.141
2 &
+ 0.82 -& 2.40
& 0.80 + 1.26
& 0.51
-& 0.35
0.45
SD.
Dot-s
Z Z
Z,n Z
Y
x
D
and dogs under control (group LV), the venae
628
W.
ISSELHARD
ET AL.
decrease in MSBP similar in level and time course to that obtained after infusion of 1.59,; Ado in group LV. In rats of group CV, MSBP averaged 48 + 7.8 mmHg and 32 & 7.5 mmHg 5 min and 3 h after the start of the infusion of Ado respectively. There were no significant differences in rats which received 0.5 or 1 y;, Ado. In dogs, administration of lq,b Ado decreased MSBP to a stable level of 56 f 4.7 mmHg in group CV and of 45 & 6.6 mmHg in groups LA and LV during the first 3 h. During the following 2 h, MSBP tended to increase by 5 to 10 mmHg. Upon discontinuation of Ado infusions, MSBP returned to control ranges within few minutes in rabbits and dogs. Rats tolerated prolonged Ado infusion less well.
+“-t-Y~“+-)!“‘.f.,,+-+.*,+~T.t-t i I
1 100
70? E LEI
_
50-
30I -20
I
I -10
I
I 0
I 5
I IO
I 20 Time
1
, 60
,
,
, 120
,
1
( 180
._
(min)
FIGURE 4. Mean systemic blood pressure in rabbits before and during the infusion of saline or adenosine (Ado) in different concentrations into the left ventricle (group LV): (a) saline, n = 5, (b) Ado 0.125:,, n = 7, (c) Ado 0.250/,, n = 5, (d) Ado 0.5%, R = 7, (e) Ado l%, a = 35, (f) Ado l.Sq,, n = 8 (means i s.E.M.).
Heart rate The heart rate (HR) averaged under control conditions in rabbits 288 f 32.6 min-l, in rats 363 5 50.9 min-*, and in dogs 113 & 25.3-l. It was not changed significantly during the period of saline infusion. In rabbits, the infusion of Ado in concentrations up to 0.5q, did not remarkably influence HR. Infusion of lyb Ado reduced HR within few minutes to a quasi stable level of 258 & 38.7 min-l in group LV (P < 0.001) and to 262 + 24.8 min-l in group CV (P < 0.001). The increase in the Ado concentration to I.596 in group LV did not depress further HR. In group LA, 1 sb Ado decreased significantly HR to a range between 240 and 260 min-’ with a tendency to lower
Ado
of difference
u, u = P < 0.05. \', v P < 0.02. IV, w y P < 0.025. s, x -. P < 0.01. Y, y .L P L 0.00.5. z. z 7. P .' 0.001.
I): Significance N, n -- n.s.
0.500°’ ,o Ado
LA
1.OOO”o
0.500:& 1 .OOO:/,
CV
to group
3 5 3 5
3 3 5
3 3 3 3 5
3 5
C: (N, U-Z)
5
0.125% 0.250% 0.500% 1 .OOO:<
LV
Ado Ado
Saline
LA
Ado Ado Ado Ado
Saline
cv
f * f i
f
& f 5 &
f f
f
f-
1.11 0.85 1.53 1.63
0.46
1.48 1.08 0.96 1.15
0.85 0.96
0.48
1.51
1.02
S.D.
and between
4.35 4.93 4.99 4.24
5.09
5.97 6.64 4.60 3.92 4.35
a.27 7.38
5.02
6.02
3
5.62
f
Rabbit .? &
4
n 12
3
of (h)
Saline
Duration infusion
n n n w
saline
a
3
6
14
n
Rat
8.40 6.78
a.93
1.04
(n, u-z).
+ 0.73 f 1.61
f
+ 0.84
.% Ifr S.D.
7.12
and Ado groups
u, 2 N, x N, x U, w
N, n
N, N, N, Y,
z v
N
N
D
phosphate (pmol/g frozen wet tissue) in left ventricular (C) and after infusion for 3 and 5 h of saline or adenosine CV), or the left atrium (group LA)
LV
Infusate
4. Tissue level of creatine under control conditions the venae cavae (group
C
Croup
TABLE
U, n N, v
z
D
6 6
6 6
2
3
2
13
n
11.02 10.24
8.23 8.10
7.66
a.39
a.14
7.37
.? &
1.37
3 +
1.74 1.19
~-t 2.04 i 3.54
5 0.50
+
z z
N, n N, n
N
D
rats, and dogs (group LV),
S.D.
Dog
myocardium of rabbits, (Ado) in the left ventricle
630 rates Ado In 0.5% In
W.
ISSELHARD
ET AL.
with increasing duration of the infusion period. Upon discontinuation of the applications, HR returned to the control ranges within 5 min. rats, bradycardia was more pronounced than in rabbits. The infusion of or 1% Ado reduced HR to 273 & 23.6 min-‘. dogs, HR was not changed significantly in any of the experimental groups. 4. Discussion
The continuous infusion of Ado resulted in a significant increase in the myocardial tissue levels of AN above the normal range in rabbits, rats, and dogs. The extent of this increase was a function of the site and duration of application, and of the dose of Ado. The volume load of 4 ml/kg x h in rabbits and rats and of 6 ml/kg x h in dogs did not cause significant alterations in the myocardial AN. When a 1 y. Ado solution was applied, the supply of Ado amounted to 2.495 pmol/ min x kg body weight in rabbits and rats and to 3.742 pmol/min x kg in dogs respectively. However, only a part of the applied Ado reached the myocardium. The maximum average rise in ATP was 39% in the rabbit heart and 48% in the rat heart. The increase was less pronounced in canine hearts in spite of a 50% increased infusion rate. The increase observed under the various experimental conditions is more pronounced than that which was achieved in isolated hearts [Z, 21, 281. Both the differences in the animal species and the differences resulting from variations in the site of Ado application can be explained by the deamination of Ado in blood. The deaminating activity of canine blood is markedly higher than that of rabbit blood (unpublished data). This is in keeping with the result that the decrease in MSBP was more pronounced in rabbits and rats than in dogs. Rabbits [27] and rats [5] have been described to be particularly sensitive .to the blood pressure-lowering effect of Ado. In the rabbit, an increase in ATP and SAN of similar degree resulted from the infusion of 0.5% Ado in group LV and of 1 o/; Ado in group CV. The duration of passage from the right to the left heart was long enough to convert a considerable amount of Ado into inosine and other degradation products. A supply of inosine, however, failed to increase the myocardial AN [16]. In spite of a comparable short distance between the site of Ado . . in group apphcation and the myocardial tissue, the effects were less pronounced LA than in group LV for two reasons: (a) a greater stress of the animals resulting from the early thoracotomy and the long-term artificial ventilation; and (b) an apparently smaller net supply of Ado to the myocardium despite comparable doses due to a declining cardiac output resuiting from thoracotomy and long-term artificial ventilation. A reduced cardiac work in group LA is indicated by the drop in MSBP and is reflected in the higher myocardial tissue level of CrP in animals with a “sham infusion” of saline. The myocardial tissue level of CrP is inversely related to the myocardial 0, consumption [23, 2.51; it increases with a
ADENOSINE-INDUCED
INCREASE
IN
ADENINE
NUCLEOTIDES
631
reduction of the volume work and especially of the pressure work of the heart [8]. The site of application of Ado was of minor importance in dogs. The apparently much smaller net supply of Ado corresponding to the efficiency of an intraventricular infusion in rabbits of about 0.125% Ado possibly effaced differences in the AN increase. The differences in the increase in AN in the myocardium of dogs and rabbits or rats also might result from species differences in the myocardial uptake of Ado. Ado was taken up by hearts of rats and guinea-pigs, and was retained mainly in the form of its phosphate derivatives, but the kinetics of the uptake differed in each species [9]. With respect to a rapid and maximal increase in ATP and SAN, the application of 0.57; Ado in group LV and of l:tO Ado in the group CV for 3 h was optimal. Both the reduction of the Ado concentration and of the duration of application resulted in lesser increases, because the supply of Ado apparently was quantitatively insufficient. A prolonged infusion of “optimal” doses had no additional effect. The tissue levels of ATP and SAN reached in rabbits and rats seem to be a maximal elevation. A similar increase above normal ranges was seen in experiments in which post-anaerobic restoration of myocardial AN was accelerated by infusion of Ado [16], although synthesis is markedly enhanced post-anaerobically [3, 4, 311. While the absence of a further gain in AN in spite of a further increased Ado supply of 3.742 Fmol/min x kg could have been explained by a saturation kinetic of the Ado uptake, for which experimental evidence has been produced [Xl, the significantly lesser efficacy of a 1 .59j0 Ado solution in rabbits is difficult to explain. As MSBP was lowered in relation to the Ado dosage in group LV, an underperfusion of endocardial layers of the myocardium would have been conceivable. This interpretation, however, is contradicted by the observation that there was neither a decrease in the overall tissue levels of glycogen or TCr nor an increase in myocardial lactate as compared to animals which had received saline or Ado in lower concentration. Also, there were no signs of a functional myocardial insufficiency during and after the Ado infusion. The increase in AN is a metabolic effect of Ado. It does not result from an Ado-induced decrease in cardiac work. Under regular conditions of oxygen and substrate supply, the normally high tissue level of myocardial ATP changes only little with wide variations in myocardial oxygen consumption and cardiac work [23]. A decrease in MSBP for 3 h by phentolamine comparable to a hypotension induced by 0.5% Ado in rabbits (group LV) did not influence the normal myocardial tissue levels of ATP and AN [15]. On the other side, the application of Ado resulted in increased myocardial tissue levels of ATP and AN in experiments, in which the Ado-induced hypotension and bradycardia were counteracted by noradrenaline or xylometazoline [ 151. The rise in the myocardial tissue levels of glucose and lactate is to be attributed to elevated concentrations of these substances in the extracellular fluid. The Adoinduced rise in glucose could be explained by an increased production of cate-
632
W.
ISSELHARD
ET
AL.
cholamine in response to the hypotension. This interpretation is in keeping with the result, that the myocardial glucose level increased with the Ado concentration and the extent of hypotension, and returned to the control range within 1 h after restoration of a normal MSBP. The elevated lactate seems to result mainly from the prolonged period of anaesthesia and from the surgical instrumentation of the animals with tissue damage, since it developed to a similar extent in animals which received Ado or saline and outlasted the restoration of a regular circulatory situation by discontinuation of the Ado infusion for a rather long period of time. The changes in myocardial CrP remain open for discussion. The marked increase in CrP in rabbits of group LA which received saline, is to be seen in terms of a reduced pressure-volume-work of the heart in long-term anaesthetized, ventilated, and thoracotomized animals (see above). The tendency towards and increase in CrP during the infusion of Ado in low concentrations of 0.125% or 0.25% in rabbits of group LV can be interpreted in a similar way and attributed to the well established negative inotropic and chronotropic effects of Ado. Ado in higher concentrations depressed the tissue level of CrP in the myocardium of rabbits and rats, which cannot be finally explained. This depression is not likely to stem from a shift in intracellular equilibria resulting from the increased ATP tissue level, since the increase in ATP outlasted the CrP depression. It also cannot be attributed to an increased myocardial energy demand due to the infusion volume load or the possibly increased catecholamine output: (a) The infusion of similar volumes of saline did not result in decreased CrP levels. (b) During the infusion of 1% Ado into rabbits (group CV), the pressure-volume-work of the hearts dropped to 50 to SOT/, of the pre-infusion value. (c) In isolated supported hearts [S, 221 as well as in hearts in situ [24], the oxygen consumption decreased during the application of Ado. (d) Ado acts as an inhibitor of myocardial effects of catecholamines [29]. Up so far, a depressed CrP level in rabbit and rat heart muscle might be best interpreted as to result from a borderline perfusion of some myocardial portions. This assumption is supported by some arguments: (a) For reasons already mentioned, a partial underperfusion of myocardium is rather unlikely. (b) The tissue level of CrP is one of the most sensitive parameters indicating deficiencies earlier than other metabolites. (c) The CrP level returned immediately to the control range, if the Ado infusion was discontinued. (d) The depression in CrP was not so marked in experiments in which the Ado-induced hypotension was counteracted by the simultaneous application of xylometazoline [IS]. A more or less increased CrP level in dogs after infusion of Ado in high dosage does not contradict the findings in rabbits and rats, since apparently the net supply of Ado to the canine myocardium was much smaller (see above). It has to be investigated in further experiments whether this experimentally induced significant increase in an important energy-rich phosphate in the myocardium is of any “biological value”, e.g. during or after anaerobiosis.
ADENOSINE-INDUCED
INCREASE
IN ADENINE
NUCLEOTIDES
633
Acknowledgements The
skilful
technical
Mrs
Hanna
Schapeit,
assistance and
Mrs
of Christa
Mrs
Dorothee
Freiberg
Schwappach-Ammermann, is gratefully
acknowledged.
REFERENCES
9. 10. 11. 12. 13.
14.
15.
ATKINSON, D. E. The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7, 4030-4034 (1968). AUSSEDAT, J. & Rossr, A. Consequences de l’incorporation d’adtnosine sur la vitesse de renouvellement des nucltotides adtnyliques, dans le coeur isole et perfuse de lapin. Journal of Physiology (Paris) 73, 633-652 (1977). BERNE, R. M. & RUBIO, R. Adenine nucleotide metabolism in the heart. Circulation Research (Supplement III) 34,35, III-109 to 111-l 18 (1974). GERLACH, E., PECHAN, I., MARKO, P., TRENDELENBURG, CH. & KAMMERMEIER, H. Syntheseraten von Adeninnucleotiden, Ribonucleinsauren und Proteinen im Herzen soweie in Nierenund Gehirnschnitten wihrend postanoxischer Erholung. PJZugers Archiv fir die gesamte Physiologie des Men&en und der Tiere 300, R 12 (1968). GORDON, D. B. & HESSE, D. H. Blood pressure lowering action of adenosine diphosphate and related compounds. AmericanJournal of Physiology 201, 1123-l 125 (1961). GROSS, G. J., WARLTIER, D. C. & HARDMAN, H. F. Effect of adenosine on myocardial oxygen balance. Journal of Pharmacolopy and Experimental Therapeutics 196, 445-454 (1976). HINZEN, D., ISSELHARD, W., MKLJRER, W. & GEPPERT, E. EinfluB verschiedener Substrate auf die post-asphyktische Nucleotidresynthese im Kaninchenherzen in vivo. PJiigers Archiv. European Journal of Physiology 307, 21-22 (1969). HOCHREIN, H. & DURING, H. J. Die energiereichen Phosphate des Myocards bei Variation der Belastungsbedlingungen. Versuche am Hera-Lungen-Praparat des Meerschweinchens PJiigers Archiv ftir die gesamte Physiologie des Menscher und der ‘Tiere 271, 548-563 (1960). HOPKINS, S. V. & GOLDIE, R. G. A species difference in the uptake of adenosine by heart. Biochemical Pharmacology 20, 3359-3365 ( 197 1). ISSELHARD, W. Des Verhalten des Energiestoffwechsels im Warmbltiterhez bei ktinstlichem Herzstillstand PJliigers Archiv 271, 3477360 (1960). ISSELHARD, W. Acute anoxia and resuscitation. German Medical Monthly 10, 194-203 (1965). ISSELHARD, W. MaBnahmen zur Verbesserung der Erholung des Herzens nach Anaerobiose. Archiv fiir klinische Chirurgie 319, 665-672 (1967). ISSELHARD, W. Metabolism and function of the heart during acute asphyxia and in postasphyxial recovery. Aspects of resuscitation. Acta anaesthesiologica Scandinavia. (Supplement XXIX) 203-216 (1968). ISSELHARD, W., BERGER, M., DENECKE, H., WITTE, J., FISCHER, J. H. & MOLZBERGER, H. Metabolism of canine kidneys in anaerobic ischemia and in aerobic ischemia by persufflation with gaseous oxygen. PJhigers Archiv. European Journal of Physiology 337, 87-106 (1972). ISSELHARD, W., EITENM~LLER, J., MKURER, W., WELTER, H. & REINEKE, H. Myocardial adenine nucleotides after infusion of adenosine. Recent Advances in Studies on Cardiac Structure and Metabolism. Heart Function and Metabolism, Vol. 11, pp. 319-323. T. Kobayashi, T. Sano & N. S. Dhalla, Eds. Baltimore: University Park Press (1978).
634 16.
17.
18.
19.
20. 21.
22.
23. 24.
25.
26. 27. 28.
29.
30.
31.
W.
ISSELHARD
E’T AL.
ISSELHARD, W., HINZEN,D., GEPPERT, E. & M;~URER,W. Beeinflussung des postasphyktischen Wiederaufbaues der Adeninnucleotide im Kaninchenenherzen in vivo durch Substratangebot. PJiigers Archiv. European Journal of Physiology 320, 1955209 (1970). ISSELHARD, W., MAURER,W., STREMMEL, W., KREBS, J., SCHMITZ, H., NEUHOF, H. & ESSER, A. Stoffwechsel des Kaninchenherzens in situ wahrend Asphyxie und in der post-asphyktischen Erholung. PJiigers Archiv ftir die gesamte Physiologie des Menschen und der Tiere 316, 164-193 (1970). ISSELHARD, W. & MERGUET,H. Metabolite des Glykolyse-Cyclus und des Adenylsaure-Phosphokreatin-Systems in schlagenden und durchbluteten Warmbhiterherzen unter verschiedenen Versuchsbedingungen. PJugers Archiv 276, 21 l-235 (1962). ISSELHARD, W., MERGUET, H. & PALM,K. Bestimmung des Gesamtglykogens neben saureldslichen Metaboliten in Perchlorsaure-Organhomogenaten. zeitschrift fiir die gesamte experimentelle Medizin 136, 174- 182 (1962). ISSELHARD, W., MERGUET,H., SCHCLER, H. W. & POHL,W. Herzstoffwechsel bei ktinstlichem Herzstillstand und bei Reperfusion. Thoraxchirurgie 11, 2 1 l-2 16 (1963). ISSELHARD, W., POHL,W., BERGHOFF, W.J.W., SCHMERBAUCH, D. & SCH~?LER, H.W. Versuche zur Verbesserung der Energiebereitstellung im ktinstlich stillgestellten Verhandlungen der Deutschen Gesellschaft Herzen und in der Erholung bei Reperfusion. fdr Kreislaufirschung 30, 2 16-22 1 ( 1964). JESMOK, G. J., GROSS, G. J. & HARDMAN,H. F. The effect of adenosine on myocardial metabolism and oxygen consumption in the isolated dog heart preparation. Journal of Molecular and Cellular Cardiology 10, 249-26 1 (1978). KOBLER,W. Tierexperimentelle Untersuchungen zum Myokardstoffwechsel im Angina-pectoris-Anfall und beim Herzinfarkt. Bibliotheca Cardiologica NO. 22. (1969). LAMMERANT, J., BECSEI, I., MERTENS-STRIJTHAGEN, J. & DE SCHRYVER, C. Changesin the calculated myocardial oxygen consumption during adenosine infusion versus estimates of developed tension and velocity of contraction. Archives Internationales de Pharmacodynamie et de Therapie 186, 1666178 (1970). LOCHNER, W., ARNOLD,G., & MUELLER-RUCHHOLTZ, E. Metabolism ofthe artificially arrested heart and of the gas-perfused heart. American Journal of Cardiology 22, 299-311 (1968). NAMM, D. H. Myocardial nucleotides synthesis from purine bases and nucleotides. Circulation Research 33, 6866695 ( 1973). NICHOLS, R. E. & WALASZEK,E. J. Antagonism of the vasodepressor effect of ATP by caffeine. Federation Proceedings 22, 308 (1963). SCHMERBAUCH, D. Wiederaufbau der Adeninnucleotide im isolierten Herzen bei nach kiinstlichem Herzstillstand Angebot von Substrat und Katecholaminen Dissertationsschrift, Koln (1967). SCHRADER, J., BAUMANN,G. & GERLACH,E. Adenosine as inhibitor of myocardial effects of catecholamines. PJiigers Archiv European Journal of Physiology 372, 29-35 (1977). WOLLENBERGER, A., RISTAU,0. & SCHOFFA, G. Eine einfache Technik der extrem schnellen Abkiihlung grijl3erer Gewebestiicke. PJiigers Archiv fur die gesamte Physiologie des Menschen und der Tiere 270, 399-4 12 ( 1960). ZIMMER,H. G., TRENDELENBURG, C., KAMMERMEIER, H. & GERLACH,E. De novo synthesis of myocardial adenine nucleotides in the rat: Acceleration during recovery from oxygen deficiency. Circulation Research 32,635-642 (1973).
of h4olecular
Increase Dosage,
and Cellular
Cardiolou
(1980)
12, 619-634
in Myocardial Adenine Nucleotides Mode of Application and Duration,
Induced Species
by Adenosine Differences*
:
WOLF ISSELHARD, JURGEN EITENMULLER, WOLFGANG MAURER, ASTRID DeVREESE, HELMUT REINEKE, ANDRZEJ CZERNIAK, JOSEF STURZ AND HANS-GUNTER HERB Institut
fiir
Experimentelle 5000
(Received
Medirin der Universitiit LU Kiln, Robert-Koch-StraJe Kiiln 41, Federal Republic of Germany
10 April
1979, accepted in revised form
23 November
10
1979)
W. ISSELHARD, J. EITENGLLER, W. MLURER, A. DEVREESE, H. REINEKE, A. CZERNIAK, J. STURZ AND H.-G. HERB. Increase in Myocardial Adenine Nucleotides Induced by Adenosine: Dosage, Mode of Application and Duration, Species Differences. Journal of Molecular and Cellular Cardiology ( 1980) 12, 6 19-634. Ad enosine or saline in control experiments were infused into the left ventricle, the left atrium and the venae cavae respectively in experiments with rabbits, rats, and dogs. The infusion volume was 4 ml/h x kg body weight in rabbits and rats, and 6 ml/h x kg in dogs. The adenosine concentrations varied between 0.125% and 1.5%, the duration of infusion varied between 1 and 5 h. The analyzes included the determination of the left ventricular tissue levels of the adenine nucleotides, creatine phosphate and total creatine, glycogen, glucose, lactate, and adenosine as well as the systemic.pressure and heart rate. Adenosine caused significantly increased myocardial tissue levels of adenine nucleotides with a maximal average rise in ATP above the mean control value of 3976 in rabbit hearts, 480/b in rat hearts, and 21% in canine hearts. A 30% increased tissue level of ATP and adenine nucleotides returned to the control ranges after 24 h. KEY
WORDS:
adenosine
Myocardial metabolic status; Myocardial adenine nucleotides; Prolonged infusion; Adenosine induced increase in myocardial adenine nucleotides.
1. Introduction After sL. ,re anaerobiosis, the restitution of normal tissue levels of adenine nucleotides (AN) in the myocardium is significantly accelerated by a continuous infusion of adenosine (Ado) [7, 12, 13, 16, 211. A decrease in the sum of AN (SAN) and especially in ATP results from an insufficient energy supply during anaerobiosis [ 10, 11, 13, 16, 17, 201. Further methods are needed to protect the myocardium during anaerobic situations such as induced cardiac arrest without coronary perfusion for cardiac surgery, to improve myocardial anoxic tolerance, or to improve and accelerate post-anaerobic recovery. In this context, it is of interest to evaluate whether a normal tissue level of AN can be substantially increased pre-anaerobically by a continuous administration of Ado. * This work was supported 68 “Cardiovascular Restitution OO22-2828/80/060619+
by the Deutsche Forschungsgemeinschaft, and Organ Substitution”.
16 $02.00/O
0
1980 Academic
Sonderforschungsbereich Press Inc.
(London)
Limited
620
\V.
ISSELHARD
ET
AL.
2. Methods The experiments were performed on Sprague-Dawley rats (200 to 250 g), rabbits (2.5 to 3.0 kg), and mongrel dogs (7.5 to 13.0 kg). Anaesthesia was induced with pentobarbital (Nembutal@) in rats (5 mg/lOO g) and dogs (17 to 23 mg/kg after premeditation with 20 mg morphine and 5 mg atropine), and with butyl-bromoallyl-barbiturate (Pernocton@) in rabbits (100 mg/kg). During the course of the experiments, the anaesthetics were re-applied in small doses if necessary. Rabbits and rats were tracheotomized, and the trachea was cannulated; dogs were intubated. The systemic pressure was monitored in a femoral artery in dogs and in a carotid artery in rabbits and rats respectively. The heart rate was calculated from the ECG or from the arterial pulse. Normal body temperature was maintained by the application of external warmth. Ado or saline in control experiments were administered in three variations: into the left ventricular cavity through a catheter installed via a carotid artery (groups LV), into the superior vena cava through a catheter via the right superficial jugular vein, the end of which was placed as close as possible to the right (groups LA). For the latter atrium (groups CV), and into the left atrium approach, a thoracotomy in the 5th intercostal space was performed, the left auricular appendix was cannulated, and the thoracotomy was superficially closed. Between the end of the surgical procedure and the start of infusions, there was a 30 min period to obtain steady state conditions. The animals breathed spontaneously except for rabbits in group LA and all dogs, which were artificially ventilated during the entire course of experiments with room air and N,O-0, (end-expiratory CO,: 4 Vol’;&) respectively. Deviations in the acid-base status were compensated only during the steady state period. Iso-osmolarity of the Ado solutions was achieved by addition of NaCl. The infusion volume was 4 ml/h x kg body weight in rabbits and rats, and 6 ml/h x kg in dogs. The infusates were kept at a temperature of 40°C. About 30 min before termination of the experiments, artificial respiration was resumed in all experiments, and the hearts were exposed. The operative procedure was finished 10 to 15 min before the removal of the heart by the freeze-stop technique [30]. In rabbits and rats, the whole hearts were frozen. From canine hearts, the apical part of the ventricular myocardium was grasped between large pre-cooled metal blocks of tongs with a sufficient cold capacity and severed from the base with a scalpel. Metal tongs and myocardial specimen were immediately immersed in liquid oxygen. The specimens were cleaned of blood and epicardium under liquid oxygen and weighed. Only left ventricular myocardium was used. Canine myocardium was differentiated into an epicardial and an endocardial half; the latter was discarded. The specimens were freeze-dried, re-weighed, and extracted. The preparation of perchlorid acid tissue extracts and the enzymatic analysis of the
ADENOSINE-INDUCED
myocardial
INCREASE
tissue levels of substrates
IN
ADENINE
621
NUCLEOTIDES
and metabolites
have been described
[Id, 18>
191. All data are presented as tissue levels in pmol/g frozen wet myocardial tissue with a dry matter content of 20% in the rabbit, 22.5O/, in the rat, and 22O, in the dog; experimentally induced variations in this ratio were considered. The data are presented as mean values i S.D. The t test was applied for statistical analysis. 3. Results Continuous infusion of Ado increased significantly the tissue levels of AN in the left ventricular myocardium of rabbits, rats and dogs (Figures 1 to 3 ; Table 1 ), while the infusion of saline did not cause significant alterations in the AN status (Figure 2; Table 1). Rabbits: Without infusion
Left
0.125%
(0)
odenosine
0.25%
b)
infusion
(4ml/kg,
ventricle
0.5%
(~1
(4
3h) Left
1.0%
(a)
atrium
0.5%
1.5%
(f)
into
(cl)
Vv.
1.0%
(h)
covoe
I .00/o
(i)
4 3 2 I 0 ECP: 0.931 n : 12
0.920
0.947
0,954
0.914
0.938
0.926
0.937
6
5
6
8
6
6
5
n
AMP
0
ADP
FIGURE 1. Tissue levels of adcninc nuclcoti(le potential (ECP (ATP 1 O..i hI>P)~SAN) control conditions without infusion and affcr trations into the left ventricle (group I-1.1, the CA7 for 3 h (means J: s.D.). Significance of Pi 0.001 (P .: 0.001); (b) vs (c) P : 0.05 (P ..; 0.05); (d) vs (e) n.s. (ns.); (e) vs (f) ig) vs (h) P .-c 0.02 (P -_ 0.05).
M.C.C.
q
ATP
N
0.923
5
SAN
~:ymol~g frwrn wet tissue) and rncrgy chargr 111 left vrntricular myocardium of rabbits under infusion of adcnosim solutions of diffcrcnt concrnIrft atrium (group L:\). and the vcnac cavac (group d~ffcrrnces in : ZTP (SW): (ai vs (b). (c) . (i) in.s.); (CI vs (d) n.s. (KU: (b) \‘s (d) P. 0.005 n.\. (P -: 0.05); (di v?, (fi P <: 0.023 (P 0.05 I ;
2c
622
W.
ISSELHARD
ET AL.
AN in rabbit myocardium Under control conditions, the tissue levels were 4.94 f 0.24 for ATP, 0.63 & 0.20 for ADP, 0.08 f 0.03 for AMP, and 5.65 5 0.32 pmol/g for SAN (Figure 1). ATP (SAN) increased to 137 (131)0/b of the control after infusion of 0.5”,, Ado for 3 h in group LV; Ado solutions of lower and higher concentrations were less effective, but also resulted in significant increases (Figure 1). In group CV, the infusion of 1 “,, Ado for 3 h caused ATP (SAN) to increase to 139 (139) I’,, (Figure 1). In group LA, ATP (SAN) reached 118 (119)“, when applying 0.5”,, Ado and 128 ( 126) (I,, when employing loi, Ado for 3 h (Figure 1). A prolongation of the Ado infusion from 3 to 5 h had no additional effect, except for group LA which received 0.5”,, Ado (F i g ure 2). But this measure only raised the AN levels to values achieved already after 3 h in the other experiments. An infusion of 1 h duration was not suflicient to raise maximally the levels of AN (Figure 2). The energy charge potential (ECP ATP + 0.5 ADP/ATP + ADP $ .4MP [I] remained unchanged during the infusion of Ado or saline (Figure I), indicating that the infusion of Ado increased the tissue levels of ATP, ADP, and AMP correspondingly. AN in rat m)locardium The infusion of Ado for 3 h under group CX conditions resulted in a marked increase in ATP and AN, which was more pronounced after the application of
r-,
Q
6.0
5.0
_ 1
J,
(41----
1(12)
I 4.5
I 0
I I
I 2
I 3
I 4
I 5
Before infusion Duration
of
infusion
(h)
FIGURE 2. Tiaauc Icvcl of :\TI’ ( !~mol/g frozen tissuej in left vcntrirular myocardium of rabbit> under control conditions before infusion and after infusion for 1, 3, and 5 h of adcnosine solutions (.kIo) of diHirrnt concentrations or saline into the left atrium (group IA) (mean> 1 s.ni.
CV cv
c N
Kal
Group
Duration infusion
from
13 4 3 6 6 6 6
t1
0 0 1 .o I .o 1.0 1.0 1 .o
of (h)
1-I 6 3 8
C values:
(00)
0 0 0.5 1.0
Adenosine concentration
5.73 5.78 7.02 6.40 6.95 6.77 6.70
4.59 4.29 6.18 6.78
* + + + * -7 -J;
* & + rf~
ATP
0.34 0.34 1.07t 0.4ot 0.24" 0.32* 0.35*
0.30 0.17 0.23* 0.63*
& & + &
0.13 0.17 0.02 O.lS§
0.68 t 0.22 0.81 * 0.05 I.11 + 0.23: 0.89 + 0.13' 1.07 -I 0.20t 1.00 11: 0.11* 0.94 .+y 0.1og
0.91 0.92 0.98 1.08
ADP
-& & f 3:
+ + & * 5 + +
0.22 0.23 0.31 0.32
0.13 0.20 0.21 0.19 0.23 0.23 0.19
AMP
0.08 0.04 0.08 0.07 O.OY.0.11 0.06
0.06 0.05 O.OOj/ 0.07
1. ‘I’issue levels of adeninr nucleotidrs (lkmol/g frozen wet tissue) in left ventricular myocardium c.ontrol conditions (C), after infusion for various periods of time of saline (W), or adenosine into I,\‘), the venar ravac (group C:Vl, or the left atrium (group L.4.)
Significance of differrnce * P < 0.001. t P < 0.005. r P i 0.001. g P < 0.002. j/ P < 0.02.5. f P < 0.05.
1’:\HL,I:
6.54 6.79 8.34 7.48 8.25 8.00 7.83
5.72 5.44 7.47 8.18
& -& & -j+ .+ +
-& & ~-fr 4
SAN
0.41 0.36 1.37* 0.37* 0.23* 0.53* O.&O*
0.39 0.22 0.24* 0.68*
of rats and dogs under the left ventricle (group
621 I”,, Ado than unchanged.
W.
after
ISSELHARD
the infusion
of 0.5”&
ET
AL.
Ado
(Table
1). The
ECP
remained
AN in canine myocardium The infusion of 1 O,, Ado resulted in significantly increased tissue levels of ATP, ADP, and SAN after 3 and 5 h (Table 1). The variations in duration and approach of infusion did not yield different results. The ECP remained within normal ranges. Duration
of eleuation of AN
Upon discontinuation of the Ado infusion, a normalization of the elevated AN tissue levels occurred slowly. A 30”” increased tissue level of SAN and ATP returned to the ranges of control hearts after 24 h (Figure 3). The slope of the decrease curve can be described by the equations y = 7.3524 x e-“.oo97~ for SAN and y = 6.3035 x e-0.0°g5* for ATP, the correlation coefficients being r = 0.7930 and Y = 0.7820 respectively. Supporting
data on myocardial metabolism
Ado was not detectable in control hearts. Its tissue level averaged 0.03 pmol/g in hearts of rabbits which had received an infusion of at least 1 y/, Ado. The glycogen level was not altered during the infusion periods. The control values were in rabbits 37.70 & 8.80 pmol/g, in rats 23.01 h 3.03 pmol/g, and in dogs 39.50 i 8.62 pmol/g.
(n; (12)
(8)(5)
(5)
(4)
16) ADP
I--+
/+-O-+------4 wt*
“7
AOI --
+ AMP
+ 3
15 Time
t 24
(h)
FIGURE 3. Adenine nucleotides in left ventricular conditions (C), at the end of a 3 h period (A) of infusion left ventricle (group LV), and during a 24 h post-infusion
myocardium of rabbits under control of 1 :,I adenosine (4 ml/kg x hi into the period (means J SD.).
N 0
i: a
3 5
I .OOo/0 Ado
LA
v, v = P < 0.05. w, w = P < 0.02. x x =- P < 0.01. Y , y = P < 0.005. z, z = P < 0.001.
N, n = ms.
to group
3 5
Ado
l.OOq/,
CV
of difference
3 3 3 5
0.25% 0.50% 1 .OO%
LV
D: Significance
3 5
Saline
LA
Ado Ado Ado
3
Saline
C (K, V-Z)
5 5
5
5 6 8 2
4 3
6
4
3
Saline
n
cv
of (h)
LV
Duration infusion 12
Infusate
1.36
2.02
2.11 0.97
2.64
.+ 4.25 .+ 2.80
f
+ 1.98 * 2.46 + 3.66
k f
-& 2.16
i
*
and between
8.61 9.92
6.89
6.10 7.46 9.36 8.47
3.00 2.63
5.19
3.97
5.48
Rabbit a -& S.D.
saline
8
14
n
3.67
2.01
+
f
1.53
0.48
S.D.
(n, v-z.).
jz +
Rat
myocardium (Ado) into
and Ado groups
N, v y, x
N, n
N,n N, v X, w
N V
N
N
D
2. Tissue level of glucose (pmol/g frozen wet tissue) in left ventricular conditions (C) and after infusion for 3 and 5 h of saline or adenosine (group CV), or the left atrium (LA)
C
Group
TABLE
Z
D
5 6
6 5
3
3
2
13
n
of rabbits, rats, the left ventricle
0.33
+
1.75
& 0.07
*
S.D.
1.78 & 0.82 1.57 * 0.29
1.47 f 0.98 1.63 & 0.41
4.04
1.01
1.38
0.90
fi *
Dog
Y z
N Y
Y
N
D
and dogs under control (group LV), the venae
626
W.
ISSELHARD
ITT AL.
The myocardial tissue level of glucose (Table 2) increased during the infusion of Ado. The rise increased with the Ado concentration (rabbits, group LV) and was independent of the duration of the Ado infusion. Upon cessation of the Ado infusion, the glucose level returned to the control range within 1 h. During the period of infusion of saline, the tissue glucose remained constant in canine myocardium and tended to decrease in the rabbit heart. The myocardial tissue level of lactate (Table 3) increased during the period of infusion of saline or Ado. This increase was slightly higher in animals which received Ado in concentrations of 0.5:~ or more than in experiments with saline infusion. However, this difference was not significant except for the rabbit group LA which received 1% Ado for 5 h. The myocardial lactate level returned to the control range 15 h after the discontinuation of the Ado infusion (rabbits, group LV). The myocardial tissue level of total creatine (TCr) amounted to 15.61 & 1.88 Fmol/g in rabbits, to 14.14 f 2.12 pmol/g in rats, and to 20.90 k 1.38 pmol/g in dogs. It was not markedly influenced by the infusion of saline or Ado. The myocardial tissue level of creatine phosphate (CrP) (Table 4) was not altered by the infusion of saline in dogs and rabbits of groups LV and CV; it increased in rabbits of group LA and in rats. In rabbits of group LV, infusion of 0.125% or 0.25% Ado for 3 h resulted in a tendency towards increased CrP levels, whereas the application of 0.5% of 1 y0 Ado caused depressed tissue levels of CrP. Also in group LA, the infusion of 0.5% and lo,; Ado for 3 or 5 h caused a decrease in the CrP level, which was particularly distinct in comparison to experiments with saline infusion. Upon discontinuation of the Ado infusion, the tissue level of CrP returned to the control range within few minutes. In rats (group CV), 0.5% Ado did not alter the myocardial CrP level, while ly/, Ado reduced CrP in comparison to the value obtained after saline infusion of 3 h duration. In the dog, the infusion of 1% Ado did not result in significantly altered CrP levels in groups LV and CV; however, CrP was significantly increased in group LA. Blood pressure Under control conditions, the mean systemic blood pressure (MSBP) averaged 86 f 12.3 mmHg in rabbits, 133 f 19.9 mmHg in rats, and 98 k 16.4 mmHg in dogs. The infusion of Ado resulted in a prompt reduction of MSBP. In rabbits of group LV, MSBP decreased in proportion to the dosage of Ado (Figure 4). However, in group CV the Ado induced drop in MSBP was slightly retarded; 5 min after the onset of the infusion of 1 o/0 Ado, MSBP had reached a range which on the average was 2 mmHg higher during the course of experiments than during the infusion of 1% Ado in group LV. In group LA, preparing the animals for the infusion resulted in a significant decrease in MSBP to 77 + 10.5 mmHg (P < 0.001). The infusion of both 0.5% and I”/$ Ado caused a
0.5% 1.0%
1 .O%
0.5% 1 .O%
LV
CV
LA
N, w, x, Y, z,
Ado Ado
of difference
n = n.s. w = P < 0.05. x = P < 0.02. y = P < 0.005. z = P < 0.001.
D: Significance
3 3 5
Saline
Ado
3
5
Saline
CV
LA
Ado Ado
4
C (N, W-Z)
5 5 5
5 3 5
to group
5
3 5
6 8 2
3
6
12
n
3
of (h)
Saline
Duration infusion
LV
Infusate
2.17
1.76 1.18
& 3.13 & 2.85 i. 3.83
& 2.13
i: +
& 2.58
& 1.99
f
5 0.91
S.D.
and between
8.16 5.78 11.62
4.55
6.14 5.01 6.85
5.53
4.41
5.65
+
Rabbit
2.58
ji
saline
z, w
Y
Z, n
8
6
14
n
2.87
2.73
2.05
1.33
(n, w-z).
-& 1.57
-& 1.73
f
Rat 5 + S.D.
myocardium (Ado) into
and Ado groups
X n
Z, n Z, n
Y
X
Y
D
3. Tissue level of lactate (kmol/g frozen wet tissue) in left ventricular conditions (C) and after infusion for 3 and 5 h of saline or adenosine cavae (group CV), or the left atrium (group LA)
C
Group
TABLE
N, n
N
D
6 6
6 5
3
3
2
13
n
of rabbits, rats, the left ventricle
3.19 3.86
2.71 3.76
2.33
1.94
2.66
I.141
2 &
+ 0.82 -& 2.40
& 0.80 + 1.26
& 0.51
-& 0.35
0.45
SD.
Dot-s
Z Z
Z,n Z
Y
x
D
and dogs under control (group LV), the venae
628
W.
ISSELHARD
ET AL.
decrease in MSBP similar in level and time course to that obtained after infusion of 1.59,; Ado in group LV. In rats of group CV, MSBP averaged 48 + 7.8 mmHg and 32 & 7.5 mmHg 5 min and 3 h after the start of the infusion of Ado respectively. There were no significant differences in rats which received 0.5 or 1 y;, Ado. In dogs, administration of lq,b Ado decreased MSBP to a stable level of 56 f 4.7 mmHg in group CV and of 45 & 6.6 mmHg in groups LA and LV during the first 3 h. During the following 2 h, MSBP tended to increase by 5 to 10 mmHg. Upon discontinuation of Ado infusions, MSBP returned to control ranges within few minutes in rabbits and dogs. Rats tolerated prolonged Ado infusion less well.
+“-t-Y~“+-)!“‘.f.,,+-+.*,+~T.t-t i I
1 100
70? E LEI
_
50-
30I -20
I
I -10
I
I 0
I 5
I IO
I 20 Time
1
, 60
,
,
, 120
,
1
( 180
._
(min)
FIGURE 4. Mean systemic blood pressure in rabbits before and during the infusion of saline or adenosine (Ado) in different concentrations into the left ventricle (group LV): (a) saline, n = 5, (b) Ado 0.125:,, n = 7, (c) Ado 0.250/,, n = 5, (d) Ado 0.5%, R = 7, (e) Ado l%, a = 35, (f) Ado l.Sq,, n = 8 (means i s.E.M.).
Heart rate The heart rate (HR) averaged under control conditions in rabbits 288 f 32.6 min-l, in rats 363 5 50.9 min-*, and in dogs 113 & 25.3-l. It was not changed significantly during the period of saline infusion. In rabbits, the infusion of Ado in concentrations up to 0.5q, did not remarkably influence HR. Infusion of lyb Ado reduced HR within few minutes to a quasi stable level of 258 & 38.7 min-l in group LV (P < 0.001) and to 262 + 24.8 min-l in group CV (P < 0.001). The increase in the Ado concentration to I.596 in group LV did not depress further HR. In group LA, 1 sb Ado decreased significantly HR to a range between 240 and 260 min-’ with a tendency to lower
Ado
of difference
u, u = P < 0.05. \', v P < 0.02. IV, w y P < 0.025. s, x -. P < 0.01. Y, y .L P L 0.00.5. z. z 7. P .' 0.001.
I): Significance N, n -- n.s.
0.500°’ ,o Ado
LA
1.OOO”o
0.500:& 1 .OOO:/,
CV
to group
3 5 3 5
3 3 5
3 3 3 3 5
3 5
C: (N, U-Z)
5
0.125% 0.250% 0.500% 1 .OOO:<
LV
Ado Ado
Saline
LA
Ado Ado Ado Ado
Saline
cv
f * f i
f
& f 5 &
f f
f
f-
1.11 0.85 1.53 1.63
0.46
1.48 1.08 0.96 1.15
0.85 0.96
0.48
1.51
1.02
S.D.
and between
4.35 4.93 4.99 4.24
5.09
5.97 6.64 4.60 3.92 4.35
a.27 7.38
5.02
6.02
3
5.62
f
Rabbit .? &
4
n 12
3
of (h)
Saline
Duration infusion
n n n w
saline
a
3
6
14
n
Rat
8.40 6.78
a.93
1.04
(n, u-z).
+ 0.73 f 1.61
f
+ 0.84
.% Ifr S.D.
7.12
and Ado groups
u, 2 N, x N, x U, w
N, n
N, N, N, Y,
z v
N
N
D
phosphate (pmol/g frozen wet tissue) in left ventricular (C) and after infusion for 3 and 5 h of saline or adenosine CV), or the left atrium (group LA)
LV
Infusate
4. Tissue level of creatine under control conditions the venae cavae (group
C
Croup
TABLE
U, n N, v
z
D
6 6
6 6
2
3
2
13
n
11.02 10.24
8.23 8.10
7.66
a.39
a.14
7.37
.? &
1.37
3 +
1.74 1.19
~-t 2.04 i 3.54
5 0.50
+
z z
N, n N, n
N
D
rats, and dogs (group LV),
S.D.
Dog
myocardium of rabbits, (Ado) in the left ventricle
630 rates Ado In 0.5% In
W.
ISSELHARD
ET AL.
with increasing duration of the infusion period. Upon discontinuation of the applications, HR returned to the control ranges within 5 min. rats, bradycardia was more pronounced than in rabbits. The infusion of or 1% Ado reduced HR to 273 & 23.6 min-‘. dogs, HR was not changed significantly in any of the experimental groups. 4. Discussion
The continuous infusion of Ado resulted in a significant increase in the myocardial tissue levels of AN above the normal range in rabbits, rats, and dogs. The extent of this increase was a function of the site and duration of application, and of the dose of Ado. The volume load of 4 ml/kg x h in rabbits and rats and of 6 ml/kg x h in dogs did not cause significant alterations in the myocardial AN. When a 1 y. Ado solution was applied, the supply of Ado amounted to 2.495 pmol/ min x kg body weight in rabbits and rats and to 3.742 pmol/min x kg in dogs respectively. However, only a part of the applied Ado reached the myocardium. The maximum average rise in ATP was 39% in the rabbit heart and 48% in the rat heart. The increase was less pronounced in canine hearts in spite of a 50% increased infusion rate. The increase observed under the various experimental conditions is more pronounced than that which was achieved in isolated hearts [Z, 21, 281. Both the differences in the animal species and the differences resulting from variations in the site of Ado application can be explained by the deamination of Ado in blood. The deaminating activity of canine blood is markedly higher than that of rabbit blood (unpublished data). This is in keeping with the result that the decrease in MSBP was more pronounced in rabbits and rats than in dogs. Rabbits [27] and rats [5] have been described to be particularly sensitive .to the blood pressure-lowering effect of Ado. In the rabbit, an increase in ATP and SAN of similar degree resulted from the infusion of 0.5% Ado in group LV and of 1 o/; Ado in group CV. The duration of passage from the right to the left heart was long enough to convert a considerable amount of Ado into inosine and other degradation products. A supply of inosine, however, failed to increase the myocardial AN [16]. In spite of a comparable short distance between the site of Ado . . in group apphcation and the myocardial tissue, the effects were less pronounced LA than in group LV for two reasons: (a) a greater stress of the animals resulting from the early thoracotomy and the long-term artificial ventilation; and (b) an apparently smaller net supply of Ado to the myocardium despite comparable doses due to a declining cardiac output resuiting from thoracotomy and long-term artificial ventilation. A reduced cardiac work in group LA is indicated by the drop in MSBP and is reflected in the higher myocardial tissue level of CrP in animals with a “sham infusion” of saline. The myocardial tissue level of CrP is inversely related to the myocardial 0, consumption [23, 2.51; it increases with a
ADENOSINE-INDUCED
INCREASE
IN
ADENINE
NUCLEOTIDES
631
reduction of the volume work and especially of the pressure work of the heart [8]. The site of application of Ado was of minor importance in dogs. The apparently much smaller net supply of Ado corresponding to the efficiency of an intraventricular infusion in rabbits of about 0.125% Ado possibly effaced differences in the AN increase. The differences in the increase in AN in the myocardium of dogs and rabbits or rats also might result from species differences in the myocardial uptake of Ado. Ado was taken up by hearts of rats and guinea-pigs, and was retained mainly in the form of its phosphate derivatives, but the kinetics of the uptake differed in each species [9]. With respect to a rapid and maximal increase in ATP and SAN, the application of 0.57; Ado in group LV and of l:tO Ado in the group CV for 3 h was optimal. Both the reduction of the Ado concentration and of the duration of application resulted in lesser increases, because the supply of Ado apparently was quantitatively insufficient. A prolonged infusion of “optimal” doses had no additional effect. The tissue levels of ATP and SAN reached in rabbits and rats seem to be a maximal elevation. A similar increase above normal ranges was seen in experiments in which post-anaerobic restoration of myocardial AN was accelerated by infusion of Ado [16], although synthesis is markedly enhanced post-anaerobically [3, 4, 311. While the absence of a further gain in AN in spite of a further increased Ado supply of 3.742 Fmol/min x kg could have been explained by a saturation kinetic of the Ado uptake, for which experimental evidence has been produced [Xl, the significantly lesser efficacy of a 1 .59j0 Ado solution in rabbits is difficult to explain. As MSBP was lowered in relation to the Ado dosage in group LV, an underperfusion of endocardial layers of the myocardium would have been conceivable. This interpretation, however, is contradicted by the observation that there was neither a decrease in the overall tissue levels of glycogen or TCr nor an increase in myocardial lactate as compared to animals which had received saline or Ado in lower concentration. Also, there were no signs of a functional myocardial insufficiency during and after the Ado infusion. The increase in AN is a metabolic effect of Ado. It does not result from an Ado-induced decrease in cardiac work. Under regular conditions of oxygen and substrate supply, the normally high tissue level of myocardial ATP changes only little with wide variations in myocardial oxygen consumption and cardiac work [23]. A decrease in MSBP for 3 h by phentolamine comparable to a hypotension induced by 0.5% Ado in rabbits (group LV) did not influence the normal myocardial tissue levels of ATP and AN [15]. On the other side, the application of Ado resulted in increased myocardial tissue levels of ATP and AN in experiments, in which the Ado-induced hypotension and bradycardia were counteracted by noradrenaline or xylometazoline [ 151. The rise in the myocardial tissue levels of glucose and lactate is to be attributed to elevated concentrations of these substances in the extracellular fluid. The Adoinduced rise in glucose could be explained by an increased production of cate-
632
W.
ISSELHARD
ET
AL.
cholamine in response to the hypotension. This interpretation is in keeping with the result, that the myocardial glucose level increased with the Ado concentration and the extent of hypotension, and returned to the control range within 1 h after restoration of a normal MSBP. The elevated lactate seems to result mainly from the prolonged period of anaesthesia and from the surgical instrumentation of the animals with tissue damage, since it developed to a similar extent in animals which received Ado or saline and outlasted the restoration of a regular circulatory situation by discontinuation of the Ado infusion for a rather long period of time. The changes in myocardial CrP remain open for discussion. The marked increase in CrP in rabbits of group LA which received saline, is to be seen in terms of a reduced pressure-volume-work of the heart in long-term anaesthetized, ventilated, and thoracotomized animals (see above). The tendency towards and increase in CrP during the infusion of Ado in low concentrations of 0.125% or 0.25% in rabbits of group LV can be interpreted in a similar way and attributed to the well established negative inotropic and chronotropic effects of Ado. Ado in higher concentrations depressed the tissue level of CrP in the myocardium of rabbits and rats, which cannot be finally explained. This depression is not likely to stem from a shift in intracellular equilibria resulting from the increased ATP tissue level, since the increase in ATP outlasted the CrP depression. It also cannot be attributed to an increased myocardial energy demand due to the infusion volume load or the possibly increased catecholamine output: (a) The infusion of similar volumes of saline did not result in decreased CrP levels. (b) During the infusion of 1% Ado into rabbits (group CV), the pressure-volume-work of the hearts dropped to 50 to SOT/, of the pre-infusion value. (c) In isolated supported hearts [S, 221 as well as in hearts in situ [24], the oxygen consumption decreased during the application of Ado. (d) Ado acts as an inhibitor of myocardial effects of catecholamines [29]. Up so far, a depressed CrP level in rabbit and rat heart muscle might be best interpreted as to result from a borderline perfusion of some myocardial portions. This assumption is supported by some arguments: (a) For reasons already mentioned, a partial underperfusion of myocardium is rather unlikely. (b) The tissue level of CrP is one of the most sensitive parameters indicating deficiencies earlier than other metabolites. (c) The CrP level returned immediately to the control range, if the Ado infusion was discontinued. (d) The depression in CrP was not so marked in experiments in which the Ado-induced hypotension was counteracted by the simultaneous application of xylometazoline [IS]. A more or less increased CrP level in dogs after infusion of Ado in high dosage does not contradict the findings in rabbits and rats, since apparently the net supply of Ado to the canine myocardium was much smaller (see above). It has to be investigated in further experiments whether this experimentally induced significant increase in an important energy-rich phosphate in the myocardium is of any “biological value”, e.g. during or after anaerobiosis.
ADENOSINE-INDUCED
INCREASE
IN ADENINE
NUCLEOTIDES
633
Acknowledgements The
skilful
technical
Mrs
Hanna
Schapeit,
assistance and
Mrs
of Christa
Mrs
Dorothee
Freiberg
Schwappach-Ammermann, is gratefully
acknowledged.
REFERENCES
9. 10. 11. 12. 13.
14.
15.
ATKINSON, D. E. The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7, 4030-4034 (1968). AUSSEDAT, J. & Rossr, A. Consequences de l’incorporation d’adtnosine sur la vitesse de renouvellement des nucltotides adtnyliques, dans le coeur isole et perfuse de lapin. Journal of Physiology (Paris) 73, 633-652 (1977). BERNE, R. M. & RUBIO, R. Adenine nucleotide metabolism in the heart. Circulation Research (Supplement III) 34,35, III-109 to 111-l 18 (1974). GERLACH, E., PECHAN, I., MARKO, P., TRENDELENBURG, CH. & KAMMERMEIER, H. Syntheseraten von Adeninnucleotiden, Ribonucleinsauren und Proteinen im Herzen soweie in Nierenund Gehirnschnitten wihrend postanoxischer Erholung. PJZugers Archiv fir die gesamte Physiologie des Men&en und der Tiere 300, R 12 (1968). GORDON, D. B. & HESSE, D. H. Blood pressure lowering action of adenosine diphosphate and related compounds. AmericanJournal of Physiology 201, 1123-l 125 (1961). GROSS, G. J., WARLTIER, D. C. & HARDMAN, H. F. Effect of adenosine on myocardial oxygen balance. Journal of Pharmacolopy and Experimental Therapeutics 196, 445-454 (1976). HINZEN, D., ISSELHARD, W., MKLJRER, W. & GEPPERT, E. EinfluB verschiedener Substrate auf die post-asphyktische Nucleotidresynthese im Kaninchenherzen in vivo. PJiigers Archiv. European Journal of Physiology 307, 21-22 (1969). HOCHREIN, H. & DURING, H. J. Die energiereichen Phosphate des Myocards bei Variation der Belastungsbedlingungen. Versuche am Hera-Lungen-Praparat des Meerschweinchens PJiigers Archiv ftir die gesamte Physiologie des Menscher und der ‘Tiere 271, 548-563 (1960). HOPKINS, S. V. & GOLDIE, R. G. A species difference in the uptake of adenosine by heart. Biochemical Pharmacology 20, 3359-3365 ( 197 1). ISSELHARD, W. Des Verhalten des Energiestoffwechsels im Warmbltiterhez bei ktinstlichem Herzstillstand PJliigers Archiv 271, 3477360 (1960). ISSELHARD, W. Acute anoxia and resuscitation. German Medical Monthly 10, 194-203 (1965). ISSELHARD, W. MaBnahmen zur Verbesserung der Erholung des Herzens nach Anaerobiose. Archiv fiir klinische Chirurgie 319, 665-672 (1967). ISSELHARD, W. Metabolism and function of the heart during acute asphyxia and in postasphyxial recovery. Aspects of resuscitation. Acta anaesthesiologica Scandinavia. (Supplement XXIX) 203-216 (1968). ISSELHARD, W., BERGER, M., DENECKE, H., WITTE, J., FISCHER, J. H. & MOLZBERGER, H. Metabolism of canine kidneys in anaerobic ischemia and in aerobic ischemia by persufflation with gaseous oxygen. PJhigers Archiv. European Journal of Physiology 337, 87-106 (1972). ISSELHARD, W., EITENM~LLER, J., MKURER, W., WELTER, H. & REINEKE, H. Myocardial adenine nucleotides after infusion of adenosine. Recent Advances in Studies on Cardiac Structure and Metabolism. Heart Function and Metabolism, Vol. 11, pp. 319-323. T. Kobayashi, T. Sano & N. S. Dhalla, Eds. Baltimore: University Park Press (1978).
634 16.
17.
18.
19.
20. 21.
22.
23. 24.
25.
26. 27. 28.
29.
30.
31.
W.
ISSELHARD
E’T AL.
ISSELHARD, W., HINZEN,D., GEPPERT, E. & M;~URER,W. Beeinflussung des postasphyktischen Wiederaufbaues der Adeninnucleotide im Kaninchenenherzen in vivo durch Substratangebot. PJiigers Archiv. European Journal of Physiology 320, 1955209 (1970). ISSELHARD, W., MAURER,W., STREMMEL, W., KREBS, J., SCHMITZ, H., NEUHOF, H. & ESSER, A. Stoffwechsel des Kaninchenherzens in situ wahrend Asphyxie und in der post-asphyktischen Erholung. PJiigers Archiv ftir die gesamte Physiologie des Menschen und der Tiere 316, 164-193 (1970). ISSELHARD, W. & MERGUET,H. Metabolite des Glykolyse-Cyclus und des Adenylsaure-Phosphokreatin-Systems in schlagenden und durchbluteten Warmbhiterherzen unter verschiedenen Versuchsbedingungen. PJugers Archiv 276, 21 l-235 (1962). ISSELHARD, W., MERGUET, H. & PALM,K. Bestimmung des Gesamtglykogens neben saureldslichen Metaboliten in Perchlorsaure-Organhomogenaten. zeitschrift fiir die gesamte experimentelle Medizin 136, 174- 182 (1962). ISSELHARD, W., MERGUET,H., SCHCLER, H. W. & POHL,W. Herzstoffwechsel bei ktinstlichem Herzstillstand und bei Reperfusion. Thoraxchirurgie 11, 2 1 l-2 16 (1963). ISSELHARD, W., POHL,W., BERGHOFF, W.J.W., SCHMERBAUCH, D. & SCH~?LER, H.W. Versuche zur Verbesserung der Energiebereitstellung im ktinstlich stillgestellten Verhandlungen der Deutschen Gesellschaft Herzen und in der Erholung bei Reperfusion. fdr Kreislaufirschung 30, 2 16-22 1 ( 1964). JESMOK, G. J., GROSS, G. J. & HARDMAN,H. F. The effect of adenosine on myocardial metabolism and oxygen consumption in the isolated dog heart preparation. Journal of Molecular and Cellular Cardiology 10, 249-26 1 (1978). KOBLER,W. Tierexperimentelle Untersuchungen zum Myokardstoffwechsel im Angina-pectoris-Anfall und beim Herzinfarkt. Bibliotheca Cardiologica NO. 22. (1969). LAMMERANT, J., BECSEI, I., MERTENS-STRIJTHAGEN, J. & DE SCHRYVER, C. Changesin the calculated myocardial oxygen consumption during adenosine infusion versus estimates of developed tension and velocity of contraction. Archives Internationales de Pharmacodynamie et de Therapie 186, 1666178 (1970). LOCHNER, W., ARNOLD,G., & MUELLER-RUCHHOLTZ, E. Metabolism ofthe artificially arrested heart and of the gas-perfused heart. American Journal of Cardiology 22, 299-311 (1968). NAMM, D. H. Myocardial nucleotides synthesis from purine bases and nucleotides. Circulation Research 33, 6866695 ( 1973). NICHOLS, R. E. & WALASZEK,E. J. Antagonism of the vasodepressor effect of ATP by caffeine. Federation Proceedings 22, 308 (1963). SCHMERBAUCH, D. Wiederaufbau der Adeninnucleotide im isolierten Herzen bei nach kiinstlichem Herzstillstand Angebot von Substrat und Katecholaminen Dissertationsschrift, Koln (1967). SCHRADER, J., BAUMANN,G. & GERLACH,E. Adenosine as inhibitor of myocardial effects of catecholamines. PJiigers Archiv European Journal of Physiology 372, 29-35 (1977). WOLLENBERGER, A., RISTAU,0. & SCHOFFA, G. Eine einfache Technik der extrem schnellen Abkiihlung grijl3erer Gewebestiicke. PJiigers Archiv fur die gesamte Physiologie des Menschen und der Tiere 270, 399-4 12 ( 1960). ZIMMER,H. G., TRENDELENBURG, C., KAMMERMEIER, H. & GERLACH,E. De novo synthesis of myocardial adenine nucleotides in the rat: Acceleration during recovery from oxygen deficiency. Circulation Research 32,635-642 (1973).