- Email: [email protected]
Evidence that cyclic GMP may regulate cyclic AMP metabolism in the isolated frog ventricle
Journal of Molecular
and Cellular
Evidence that Metabolism
Cardiology
( 198 1) 13, 963-979
Cyclic GMP may in the Isolated
Frederick
W. Flitney
Regulate Cyclic Frog Ventricle
and Jaipaul
AMP
Singh”
Department of Physiology and Pharmacology, University of St Andrews, Bute Medical Buildings, St Andrews, Fife X226 9TS, Scotland (Received 10 October 1980, accepted in revised form 1 August 1981) F. W. FLITNEY AND J. SINGH. Evidence that Cyclic GMP may Regulate Cyclic AMP Metabolism in the Isolated Frog Ventricle. Journal of Molecular and Cellular Cardiology ( 1981) 13, 963-979. Both acetylcholine and 8-bromo cyclic GMP depress the contractile response of the isolated frog ventricle. An investigation has been made of the effects of both substances on the metabolism of endogenous 3’, 5’ cyclic nucleotides. The levels of adenosine 3’, 5’ cyclic monophosphate (cyclic AMP) and guanosine 3’, 5’ cyclic monophosphate (cyclic GMP) were measured after superfusing preparations with varying concentrations (10-l” to lo-* M) of acetylcholine and 8-bromo cyclic GMP. The decline in contractile force was found to be accompanied by a progressive fall in intracellular cyclic AMP and a rise in cyclic GMP levels. Both the decline in contractility and the reduction in endogenous cyclic AMP are attenuated by 1Oe4 theophylline. The decline in isometric twitch tension was paralleled, under all conditions, by a quantitatively equivalent reduction in the ratio cyclic AMP: cyclic GMP. The possibility that endogenous cyclic GMP may accelerate the conversion of cyclic AMP to 5’ AMP, by stimulating a cyclic GMP-sensitive form of cyclic AMP phosphodiesterase, is discussed. KEY WORDS: 8-Bromo cyclic
Cyclic GMP;
AMP; Cyclic Theophylline;
GMP; Contractility; Phosphodiesterase.
Frog
ventricle;
Acetylcholine;
Introduction The possibility that cyclic AMP and cyclic GMP are together involved in regulating myocardial contractility, and that they function antagonistically, was first proposed several years ago by Goldberg and his colleagues [19]. This suggestion is substantiated by the results of more recent experiments, in which responses of the isolated frog ventricle to a range of pharmacological agents (adenosine 5’-triphosphate [lo], uridine 5’-triphosphate [ 151, isoprenaline [39], a d renaline (unpublished), dibutyryl cyclic AMP [38], adenosine [37], sodium nitroprusside [13], and 8-bromo cyclic GMP [14, 381) were studied: these all reveal a clear and consistent correlation between changes in ventricular contractility and changes in the ratio of endogenous cyclic AMP: cyclic GMP. Cyclic AMP is known to activate enzymes (cyclic AMP-dependent protein kinases) which catalyze the phosphorylation of important regulatory proteins, whose physiological activities are then altered as a result [9, 11, 22, 24, 32, 41, 43, 467. The role of cyclic GMP is much less clear, but two alternative suggestions have been made to account for the way in which it might act to oppose the effects of cyclic AMP: * Present 0022-2828/81/l
address: 10963+
Department 17
$02.00/O
of Physiology,
The
University, 0
1981
Dundee Academic
DDl
4HN,
Press
Inc.
Scotland. (London)
Limited
964
F. W. Flitney
and J. Singh
first, that it may stimulate cyclic AMP phosphodiesterase activity, reducing cyclic AMP levels and thereby suppressing cyclic AMP-dependent protein phosphorylation [16]; and secondly, that it may enhance protein phosphatase activity, and so promote protein de-phosphorylation [IS, 371. The experiments to be described were designed to investigate the first of these possibilities. The approach used was to examine the effect of elevating cyclic GMP levels on those of cyclic AMP. The experiments were made with the isolated frog ventricle and two different procedures were employed to increase tissue cyclic GMP levels: by superfusing preparations with S-bromo cyclic GMP, a lipophilic (and therefore membrane-permeant) analogue of cyclic GMP; or alternatively, by activating muscarinic receptors with acetylcholine.
Materials
and
Methods
Ventricle perfusion Isolated ventricles from frogs (R. temporaria) were divided into two halves, one of which served as a control for the other. Control half-ventricles were superfused with oxygenated Ringer’s solution (composition in mM : NaCl, 115 ; KCI, 2.5 ; CaCl,, 1; Na,HPO,, 2.15; NaH,PO,, 0.85; glucose, 5.6; pH 7.2) at a flow rate of lOOml* min-r in a closed circuit perfusion system [19, 261. The total volume of the recirculated perfusion fluid was 1 1. “Test” half-ventricles were superfused under identical conditions, but with Ringer’s solution containing either acetylcholine (lo-lo to 10~~ M) or 8-bromo cyclic GMP (IO-lo to 1O-4 M) . The responses to 8-bromo cyclic GMP developed more slowly than those for acetylcholine, reaching.a steady-state after approximately 25 min. At this time, the perfusion fluid was exchanged for Ringer’s solution alone, in order to remove traces of extracellular 8-bromo cyclic GMP which would otherwise have interfered with the cyclic GMP assay (see below). A “wash-out” period of 30 s was given prior to freezing the preparation. Lamb and McGuigan [26], who used similar perfusion conditions, found that changes in twitch tension produced by lowering extracellular [Ca2+] from 2 mM to 1 mM required only 1 s for completion, so the 30 s “wash” period allowed was considered adequate to remove 8-bromo cyclic GMP from the interfibre spaces. This was confirmed experimentally, by taking a sample of the perfusion fluid (after 25 s) immediately after it had passed over the preparation and assaying this’ for cyclic GMP (six experiments). The quantity of 8-bromo cyclic GMP in all such samples was less than the lower limit of detection of the assay method ((0.05 pmol*ml-l). All preparations were paced electrically, by stimulating through silver wire electrodes (10 V amplitude, 4 mA, 5-ms duration) at a frequency of 0.5 Hz. Isometric twitch tension was measured using a strain gauge (Devices, type 4157 ; compliance: 40 p-g-1) and the output recorded continuously on a chart recorder. The optimum length, giving a maximal contraction, was established for each half-ventricle at the start of the experiment. A more complete account of the experimental procedure used is given elsewhere [17]. The effect of theophylline (lo-4 M) was studied by superfusing preparations for 15 min prior to the addition of either acetylcholine or 8-bromo cyclic GMP, and then throughout the subsequent response.
Cyclic
GMP
may
Extraction
Regulate
Cyclic
AMP
in Frog
Ventricle
965.
of cyclic nucleotides and assay procedure
When the twitch had attained its new steady-state (after 25 min for 8-bromo cyclic GMP and 2 min for acetylcholine) both “test” and “control” preparations were frozen rapidly. This was done by compressing the tissue between broad-tipped forceps which had been cooled previously by immersion in liquid nitrogen. The frozen tissue was pulverized in a stainless steel mortar (also cooled in liquid nitrogen) and afterwards extracted with acidified ethanol (1 ml 1 NHCI; 100 ml ethanol). The solvent was blown-off in a stream of nitrogen gas, and the residue taken up in TrisEDTA buffer (0.05 M-Tris, pH 7.5; containing 4 mM EDTA). Cyclic AMP and cyclic GMP levels were then determined, using the Radiochemical Centre’s assay kits TRK 432 and TRK 500, respectively. Precise details of the procedure used are to be found in the publications which accompany the assay kits (Radiochemical Centre, Amersham, England). Total protein was estimated using the Biuret method [ZO] and cyclic nucleotide concentrations are expressed throughout in pmol . mg-l tissue protein. Control experiments established that the cyclic GMP assay procedure is equally sensitive to the 8-bromo derivative as to the parent nucleotide (to within f2%, in the sample range 0.5 to 10 pmol). Hence, the values listed in Table 2 for tissue concentrations of “cyclic GMP” actually represent the sum of endogenous cyclic GMP #us an amount of 8-bromo cyclic GMP which had entered the fibres from the extracellular fluid. Results
Efects of acetylcholine and 8-bromo cyclic GMP
on contractility
and cyclic nucleotide levels
Figures 1 and 2 ( 0) show that both acetylcholine and 8-bromo cyclic GMP depressed the twitch, in a dose-dependent manner, and that the concentrations required to produce a 50% reduction were comparable, around I x 1O-s M and 5 x 1O-8 M, respectively. However, there was a large difference in the rate at which the twitch declined towards its new steady state value, being at least 100 times greater for acetylcholine than for 8-bromo cyclic GMP. The results of experiments with pharmacological receptor antagonists show that this is due to qualitative differences in the mode of action of the two substances. The experiments in question showed (a) that responses to acetylcholine could be entirely inhibited by atropine ( 1O-6 M) but were unaffected by either phentolamine (lo-’ M, an M antagonist) or propranolol (lo+ M, a p antagonist); and (b) that neither phentolamine, propranolol nor atropine attenuated responses to 8-bromo cyclic GMP. It can therefore be concluded that 8-bromo cyclic GMP does not act in the same way as acetylcholine, by activating muscarinic receptors. The most likely explanation for its slower action is that it exerts its effects intracellularly, and so must first gain access to the fibre interior by diffusing across the surface membrane. The experiments with phentolamine and propranolol also demonstrate that the method of stimulation used to “pace” the ventricle (see Materials and Methods) did not lead to the release of significant amounts of endogenous catecholamines. This conclusion is based upon studies which show that the inotropic actions of acetylcholine on responses already potentiated by catecholamines are much greater than’ on responses recorded in the presence of tc and/or p antagonists [5, IO]. Hence, if appreciable quantities of endogenous catecholamines had been liberated during
966
F. W. Flitney
and
J. Singh
0 t
lo-‘0
10-g
lo-* Concentration
10-7
10-s of ACh
10-s
10-4
IO-J
[ml
FIGURE 1. Dose-response curves showing the effects of acetylcholine (10-l” twitch tension, in the presence (0) and absence (0) of the phosphodiesterase (IO+ M). Theophylline decreases the sensitivity of the ventricle to acetylcholine. as multiples of control values.
to lOA M) on isometric inhibitor theophylline All points expressed
electrical stimulation, phentolamine and/or propranolol would have attenuated responses to acetylcholine, but neither did. The effects of acetylcholine and 8-bromo cyclic GMP on the twitch were found to be accompanied by changes in intracellular cyclic GMP and cyclic AMP. The concentrations measured in test and control preparations are listed in Tables 1 (acetylcholine) and 2 (8-bromo cyclic GMP). Two important features of these results should be emphasized. First, it can be seen that the extent to which acetylcholine and 8-bromo cyclic GMP depresses the twitch is paralleled closely by quantitatively-equivalent reductions in the ratio cyclic AMP : cyclic GMP (compare columns 10 and 11 in Tables 1 and 2), a result which agrees with our previous findings using a variety of pharmacologically-distinct inotropic agents (see Introduction for references). Secondly, the responses to both substances are characterized by increased concentrations of cyclic GMP and by marked reductions in endogenous cyclic AMP levels. The reciprocal relationship between changes in cyclic AMP and cyclic GMP is shown graphically in Figures 3 and 4 ( 0). Here, the decrease in endogenous cyclic
Cyclic
GMP
I lo-‘0
may
I 10-s
Regulate
I 10-e Concentmtion
Cyclic
I 10-7
AMP
I 10-e
of 8 - bromo
in Frog
I 10-s cGMP
I to-4
967
Ventricle
I(
[M]
FIGURE 2. Log-dose response curves showing the effects of differing IO+ M) of 8-bromo cyclic GMP on twitch amplitude in the presence (0) theophylline. All values expressed as multiples of control levels.
concentrations and absence
(0)
(IO-lo to of 1O-4
AMP levels is plotted as a function of the increase in intracellular cyclic GMP. The relationship is approximately linear and quantitatively similar for both substances, yielding a slope of around - IO pmol cyclic AMP. pmol-r increment in cyclic GMP (regression data : acetylcholinz: -9.82 f 0.59 pmol cyclic AMP. pmol-l cyclic GMP; P < 0.001; n = 25; 8-bromo cyclic GMP: -10.79 f 0.60 pmol cyclic AMP. pmol-r cyclic GMP; P < 0.001; n = 18). The latter observation suggests a possible role for cyclic GMP in regulating cyclic AMP metabolism: namely, that under certain circumstances it may serve to su@ress endogenous cyclic AMP levels. The question which arises then is: does it do so by inhibiting adenylate cyclase activity or by enhancing cyclic AMP phosphodiesterase activity? Effects of theophylline on responses to acetylcholine and 8-bromo cyclic GMP In an attempt to investigate this question, responses to both acetylcholine and 8-bromo cyclic GMP were recorded in the presence of theophylline, a phosphodiesterase inhibitor. Theophylline was selected for study because previous experiments (unpublished) had shown that it produces only a transient effect on the frog ventricle
1
CAMP (control)
CAMP
8.06 8.82 7.69
8.66 8.69 7.89
2.01* 2.39 1.84
1.47" 1.40 1.18
10-7
10-s
CAMP ratio ACh
4
0.07 0.05
0.17 0.16 0.15
0.25 0.27 0.24
0.64 0.60 0.62
0.87 0.86 0.88
0.94 0.95 0.97
>
nucleotide
( control
on cyclic 5
-8.09 -8.36
-7.19 -7.29 -6.71
-6.05 -6.93 -5.85
-3.13 -3.38 -3.17
-1.05 -1.13 -1.02
-0.52 -0.41 -0.23
(2-3)
Change in CAMP
levels 6
1.98 1.92
1.81 1.77 2.21
1.91 1.98 2.13
1.84 1.94 1.66
1.50 1.34 1.66
1.31 1.38 1.58
cGMP ‘(ACh)
7
1.06 1.02
1.10 1.06 1.34
1.22 1.23 1.35
1.36 1.45 1.25
1.30 1.18 1.43
1.20 1.28 1.48
cGMP (control)
8
1.86 1.88
1.64 1.67 1.65
1.56 1.60 1.58
1.35 1.34 1.33
1.15 1.18 1.16
1.09 1.08 1.07
( control
cGMP ratio ACh >
9
0.03 0.03
0.11 0.10 0.09
0.16 0.16 0.15
0.47 0.45 0.47
0.75 0.73 0.76
0.86 0.88 0.91
R
10
0.04 0.05
0.10 0.09 0.11
0.15 0.18 0.17
0.49 0.46 0.48
0.76 0.75 0.78
0.88 0.90 0.92
P
11
nucleotide ratio [R = cyclic AMP: cyclic treated ventricle/twitch tension of control cyclic AMP levels decrease progressively were 8.33 f 0.10 and 1.25 f 0.04 pmol-
$0.91 +0.90
+0.71 +0.71 +0.87
+0.69 $0.75 +0.78
+0.48 +0.44 +0.41
$0.20 +0.20 +0.23
+0.11 +0.10 +0.10
(6-7)
Change in cGMP
Values in columns 2, 3, 5,6, 7 and 9 are pmol. mg-l total protein. The figures in columns 10 and 11 relate changes in cyclic GMP (treated ventricle)/cyclic AMP: cyclic GMP (control ventricle)] and changes in twitch tension (P = twitch tension of ventricle): note that both decrease in parallel as the concentration of acetylcholine is increased. Note also that endogenous with increasing cyclic GMP levels [see also Figure 3 (@)I. The levels of cyclic AMP and cyclic GMP in control preparations performed in presence of phentolamine (10-T M) and propranolol (10-s M). mg-l protein, respectively (n = 17). * Experiments
8.70 8.80
8.68 8.45 8.34
5.55" 5.07 5.17
10-s
0.56" 0.44
7.98 8.05 8.33
6.93” 6.92 7.31
10-s
10-s
8.71 8.20 7.65
WW
3
2
of acetylcholine
8.19” 7.79 7.42
1. Effects
10-1s
(M)
Concentration ACh
TABLE
1
8.03 8.08 8.70
7.75 7.76 9.01
8.10 9.13 9.00
7.20 8.50 8.07
7.86 10.46 9.83
5.54* 5.28 5.81
4.30" 4.59 4.94
2.75" 3.48 3.24
2.06" 2.20 2.26
1.10” 1.98 1.70
10-s
IO-'
10-s
10-h
IO-4
GMP
0.14 0.18 0.17
0.28 0.26 0.27
0.31 0.38 0.36
0.55 0.59 0.55
0.64 0.65 0.67
0.80 0.78 0.82
(8-::;:Fp)
CAMP ratio
on cyclic
cyclic AMP of phentolamine
and
cyclic (lo-’
M)
GMP
in control propranolol
preparations ( low6 M) and
5 (8-Br
6
decline
7
(8-::i::p)
in legend
9
to Table
+0.62 $0.79 +0.65
+0.43 +0.55 +0.53
+0.40 10.44 +0.40
+0.31 +0.30 +0.35
$0.21 +0.16 +0.23
+0.19 +0.14 +0.10
(6-7)
Change in cGMP
1) with
R
10
4 (a)]. performed
increasing
0.08 0.12 0.11
0.20 0.18 0.19
0.26 0.29 0.27
0.43 0.47 0.43
0.58 0.56 0.56
0.70 0.71 0.75
levels [compare columns 5 and 9, see also Figure protein, respectively (n = 18). * Experiments
1.55 1.55 1.52
1.41 1.44 1.45
1.30 1.32 1.33
1.29 1.27 1.27
1.20 1.17 1.19
1.13 1.10 1.09
cGMP ratio
in R and P (defined
1.13 1.43 1.25
1.05 1.25 1.16
1.25 1.65 1.18
1.06 1.10 1.30
1.05 0.94 1.18
1.40 1.36 1.15
cGMP (control)
cyclic GMP 0.04 pmol.mg-’
1.75 2.22 1.90
1.48 1.80 1.69
1.65 2.19 1.58
1.37 1.40 1.65
1.26 1.10 1.41
1.59 1.50 1.25
cGMP cGMP)
the progressive
levels
with increasing were 8.41 -& 0.19 and 1.22 i atropine (10~~ M).
Note
-6.76 -8.42 -8.13
-5.14 -6.30 -5.81
-5.35 -5.65 -5.76
-3.45 -3.17 -4.07,
-2.49 -2.80 -2.89
-1.66 -1.74 -1.36
Change in CAMP (2-3)
nucleotide
Values in columns 2, 3, 5, 6, 7 and 9 are pmol.mg-l protein. of 8-bromo cyclic GMP. Endogenous cyclic AMP falls progressively
8.26 7.94 7.66
6.60" 6.20 6.30
CAMP (control)
3
cyclic
10-g
(M)
2
of 8-bromo
CAMP (8-Br cGMP)
2. Effects
Concentration 8-Br cGMP
TABLE
The levels of in presence
concentrations
0.09 0.10 0.12
0.18 0.16 0.17
0.25 0.32 0.29
0.44 0.48 0.45
0.60 0.58 0.55
0.72 0.73 0.76
P
11
970
F. W; Flitney
and
J. Singh
-8-
-6 -
+I
, 0
1
+0.2 Change Test minus
I
+ 0.4
I
+ 0.6
in introcellulor control (pm4
I
+ 0.0
+I
5,s cGMP mg-’ protein)
FIGURE 3. Inverse relationship between changes in cyclic AMP and cyclic GMP ieds (treatedcontrol values) following treatment with acetylcholine (IO-lo to 10e5 M) in the presence (0) and absence (0) of theophylline (lo-* M). The slope of the line relating the reduction in cyclic AMP to the corresponding increase in cyclic GMP levels (fitted by linear regression analysis) is markedly reduced by theophylline, by a factor of around six.
when used at a concentration of lo-* M {see also [29, 341). Initially, it evokes a positive inotropic response but within 10 to 12 min the twitch returns to its control level. Hence, in the experiments now to be described, ventricles were first pre-treated with theophylline for 15 min, prior to the addition of either 8-bromo cyclic GMP or acetylcholine. Theophylline also remained in the superfusing solution throughout the remainder of the response. Figures 1 and 2 (0) show that the sensitivity of the ventricle to both acetylcholine and 8-bromo cyclic GMP is markedly depressed by theophylline-the dose-response curves are displaced to the right, towards higher agonist concentrations. This observation is of some interest, because phosphodiesterase inhibitors generally potentiate cyclic nucleotide mediated responses [28, 3.51, by decreasing the rate at which cyclic AMP and cyclic GMP are hydrolyzed to their non-cyclical (inactive) forms (5’ AMP and 5’ GMP, respectively). What, then, is the explanation for this apparently anomalous effect? The data presented in Figures 3 and 4 ( 0) reveal that the desensitizing effect of theophylline is associated with a significant reduction in the slopes of the lines relating changes in tissue cyclic AMP and cyclic GMP levels, from around -10 pmol cyclic AMP. pmol-l cyclic GMP to -1.64 f 0.34 pmol cyclic AMP. pmol-r cyclic GMP for acetylcholine (P < 0.001, n = 12) and -4.27 f 0.29 pmol cyclic AMP. pmol-r cyclic GMP for 8-bromo cyclic GMP (P < 0.001, n = 18). Thus, theophylline is able to reduce the degree of coupling between changes in cyclic GMP and cyclic AMP. Accordingly, exposure of the
Cyclic
GMP
may
Regulate
Cyclic
AMP
in
Frog
Ventricle
971
-lO-
-0-
.
/ .
,/
-6/
-4-
.
-2-
.
/*
..
.,?
.
0’ 0 -
..
A-
god00 -0-o 0 .o.oQ," 0 I 0
I + 0.2
0
+o.
I
O I + 0.4
I + 0.6
I + 0.0
+ I.0
+ 0.2
-I- 0.3
+ 0.4
+ 0.5
Change in intracellular Test minus (pool
5,s cGMP mg-’ protein)
FIGURE 4. Relationship between changes in endogenous cyclic AMP and cyclic GMP levels (treated-control values) following exposure of the frog ventricle to varying concentrations ( 10e9 to lo-* M) of exogenous 8-bromo cyclic GMP in the presence (0) and absence (0) of 1OW M theophylline. The slope of the line relating the reduction in cyclic AMP to the corresponding increase in cyclic GMP levels (fitted by linear regression analysis) is reduced by theophylline by a factor of about two and a half. Abscissa: upper values in the presence of 8-bromo cyclic GMP; lower values in the presence of theophylline and 8-bromo cyclic GMP.
theophylline-treated ventricle to a particular concentration of acetylcholine or S-bromo cyclic GMP reduces the relative proportion of cyclic AMP: cyclic GMP to a lesser extent than in preparations not treated with theophylline. This is illustrated by the data in Tables 3 and 4 (columns 10 and 11) which again demonstrate a parallel though now greatly reduced decline in the ratio of cyclic AMP : cyclic GMP and isometric twitch tension.
Discussion
The experiments described here show that the negative inotropic effect of acetylcholine on the frog ventricle is associated with increased accumulation of cyclic GMP and a simultaneous depression of endogenous cyclic AMP levels. The ability of S-bromo cyclic GMP to likewise depress endogenous cyclic AMP levels strongly suggests that this effect may actually be mediated by the increase in intracellular cyclic GMP. The striking quantitative agreement in the relationship between changes in. cyclic AMP and cyclic GMP, whether induced by acetylcholine, acting via muscarinic recefltors, or by 8-bromo cyclic GMP, makes this hypothesis seem all the more plausible. The most straightforward interpretation of the results is to postulate that cyclic GMP constitutes part of a feed-back control mechanism which serves to regulate th.e
1
3.78 3.69
2.00 2.22
10-G
10-S
5 and 9 plotted listed in Table
4.80 5.59
10-T
Data in columns control preparation
5.46 4.52
5.39 4.52
10-S
4
0.56 0.58
3.57 3.84
5
nucleotide
- 1.57 -1.62
1.89 1.96
1.56 1.76
-0.95 -0.81
1.02 1.04
0.92 1.05
0.95 1.07
1.44 1.60
-0.15 -0.12
1.06 1.01
0.96 0.91
cGMP (control)
7
in the presence
0.98 0.97
1.21 1.16
1.07 0.96
cGMP Wh)
6
levels
1.25 1.23
-0.07 0.00
0.00 0.00
+0.28 +0.33
(2-3)
Change in CAMP
and cyclic
of 10-a 8
theophylline 9
7)
AMP
+0.87 +0.92
+0.64 +0.71
+0.49 to.53
+0.27 +0.26
+0.15 +0.15
+0.06 +0.05
(6-
Change in cGMP
levels of cyclic
1.85 1.88
1.70 1.68
1.52 1.50
1.28 1.27
1.14 1.15
1.06 1.06
cGMP ratio
M
in Figure 3 (0). Note (a) the parallel decline in P and R; and (b) control 1, which were not exposed to theophylline. See text for further details.
0.80 0.82
0.97 0.98
0.99 1 .oo
1 .oo 1.00
1.05 1.07
CAMP ratio
4.73 4.50
4.95 5.71
5.72 5.38
5.72 5.38
10-S
CAMP (control)
3
on contractility
5.70 4.70
W-W
CAMP
2
of acetylcholine
5.98 5.03
M)
(M)
3. Effect
10-10
ACh + Theophylline ( 1k4
tration
Concen-
TABLE
and cyclic
0.30 0.31
0.47 0.48
0.64 0.65
0.77 0.79
0.88 0.87
0.99 1.01
R
10
GMP
are less than in
0.28 0.29
0.49 0.50
0.65 0.67
0.80 0.81
0.91 0.89
1.00 1.02
P
11
3.70 5.00 4.32 4.80 4.89 5.06 3.73 3.54 3.37 4.30 4.54 4.76 3.83 4.05 3.59 3.73 3.40
10-S
3.73 5.04 4.50 4.95 4.99 5.27 4.15 3.85 3.88 4.88 5.05 5.67 4.68 5.06 5.06 5.25 5.07
3 CAMP (control)
cyclic GMP
0.99 0.99 0.96 0.97 0.98 0.96 0.90 0.92 0.87 0.88 0.90 0.84 0.82 0.80 0.71 0.68 0.67
0.82 1.06 0.89 0.97 1.12 1.19 1.02 0.94 1.20 1.14 1.14 1.22 1.13 1.19 1.28 1.45 1.25
6 cGMP (8-Br cGMP)
levels and contractility
-0.03 -0.04 -0.18 -0.15 -0.10 -0.21 -0.42 -0.31 -0.51 -0.58 -0.51 -0.91 -0.85 - 1.01 -1.47 - 1.52 - 1.67
5 Change in CAMP (2-3)
on cyclic nucleotide
0.80 1.05 0.86 0.92 1.05 1.08 0.96 0.87 1.04 1.03 1.02 0.95 0.89 0.96 0.95 1.07 0.92
7 cGMP (control)
1.02 1.01 1.03 1.05 1.06 1.10 1.06 1.08 1.15 1.11 1.12 1.28 1.27 1.24 1.35 1.36 1.36
in presence of lo+ M
+0.02 +0.01 +0.03 +0.05 +0.07 +0.11 + 0.06 +0.07 +0.16 +0.11 -to.12 10.27 + 0.24 10.23 +0.33 +0.38 +0.33
9 Change in cGMP (6-7)
theophylline
0.97 0.98 0.93 0.92 0.92 0.87 0.85 0.85 0.75 0.80 0.80 0.66 0.64 0.65 0.53 0.52 0.49
10 R
0.96 0.99 0.94 0.93 0.91 0.85 0.84 0.86 0.79 0.79 0.81 0.67 0.66 0.64 0.54 0.51 0.50
11 P
Data in columns 5 and 9 plotted in Figure 4 (0). Note (a) parallel decline in P and R and (b) control levels of cyclic AMP and cyclic GMP are lower than those in Table 2 for ventricles not exposed to theophylline. See text for further details.
10-d
10-s
IO-”
IO-7
10-s
2 CAMP (8-Br cGMP)
4. Effects of 8-bromo
1 Concentration (M) 8-Br cGMP + Theophylline ( 10-4 M)
TABLE
974
F. W. Flitney
and
J. Singh
metabolism of cyclic AMP. The observations which have been made thus far are entirely consistent with the idea that it does so by stimulating a cyclic GMP-sensitive isozyme of cyclic AMP phosphodiesterase, thereby accelerating the hydrolysis of cyclic AMP to 5’ AMP. Three major forms of cyclic nucleotide phosphodiesterase have been isolated from a variety of tissues [I, 40, 44, 4.51 and of these, two are of relevance to the present discussion, since their ability to hydrolyze cyclic AMP is subject to allosteric control-in one case, by cyclic GMP [I, 3, 36, 40, 421 and in the other, by calcium, acting indirectly through the Ca 2+-dependent regulator protein, calmodulin [ 7, 30, 471.
Cyclic GMP-dependent
regulation
of cyclic AMP
hydrolysis
The kinetic properties of a cyclic GMP-dependent cyclic AMP phosphodiesterase from rat heart have been investigated by Teresaki and Appleman [42]. They found that the ability of the enzyme to hydrolyze cyclic AMP at physiological concentrations was markedly stimulated by low levels of cyclic GMP. Qualitatively similar observations have been made on phosphodiesterase preparations from other tissues too [3, 7, 22, 36, 411. Stimulation by cyclic GMP of the rat heart preparation appears to be due to a loss of enzyme co-operativity, rather than to any change in either V,,, or Krr, for cyclic AMP, and under optimal conditions it can effect a 6 to 10 fold increase in the rate of hydrolysis of cyclic AMP. If this enzyme, or one possessing similar kinetic properties, is also present in frog heart, then it would afford a biochemical basis for the regulation of cyclic AMP degradation by cyclic GMP. There is another reason why it appears likely that cyclic GMP acts by stimulating cyclic AMP breakdown. It will be recalled that the inotropic responses to acetylcholine and to 8-b-romo cyclic GMP, and the effects that both substances have on endogenous cyclic AMP levels, are attenuated by treatment with theophylline, a phosphodiesterase inhibitor. This is to be anticipated if stimulation by cyclic GMP of a phosphodiesterase is involved in the regulation of cyclic AMP metabolism, because a given increment in tissue cyclic GMP would then be less effective at reducing cyclic AMP levels. The above considerations can only be regarded as circumstantial evidence in support of a role for cyclic GMP in regulating cyclic AMP metabolism: it remains to be seen whether a phosphodiesterase isozyme with similar properties to the one found in the rat heart is also present in the frog heart; and the specificity of theophylline as a phosphodiesterase inhibitor has also been called into question (see below). It should also be noted that a preparation of bovine heart phosphodiesterase is reported to be inhibited by cyclic GMP [31].
Ca2+-dependent
regulation
of cyclic nucleotide metabolism
The existence of a reciprocal relationship between changes in tissue cyclic AMP and cyclic GMP levels, while consistent with the notion that cyclic GMP may serve can be interpreted differently. It has been to regulate cyclic AMP metabolism, and 8-bromo cyclic GMP [23] impede the reported that both acetylcholine [18] entry of Ca2+ into the fibres during the action potential. The quantity of Ca2+ which crosses the membrane during excitation is not considered sufficient to activate con-
Cyclic
GMP
may
Regulate
Cyclic
AMP
in Frog
Ventricle
975
traction directly [24], but there is evidence that it is involved in controlling the from storage sites within the fibres amount of Ca2+ which is released subsequently [12]. This is an important point to consider, because free Ca2+ not only activates contraction, by combining with troponin C, but also influences cyclic nucleotide metabolism, via its effects on calmodulin. The Ca2+-activated form of calmodulin is an allosteric effector of cyclic nucleotide phosphodiesterases and of adenylate cyclase too [4, 8, 471. It stimulates the hydrolysis of both cyclic GMP and cyclic AMP, the former more so than the latter, and also enhances the activity of adenylate cyclase, resulting in an increase in the relative proportion of cyclic AMP:cyclic GMP. It can therefore be inferred that de-activation of calmodulin, under conditions of diminished Ca2+ availability, might have the reverse effects on cyclic nucleotide levels, producing changes which are qualitatively simiIar to those seen in the present study. Microelectrode recordings were not made during this investigation and so it is not known whether the reported effects of acetylcholine and 8-bromo cyclic GMP on the action potential actually precede changes in cyclic nucleotide levels; if they do not, then it can be argued that they may arise as a consequence of the changes in cyclic AMP and/or cyclic GMP, a point which is taken up again later.
The ability of theophylline to antagonize
responses to ace2ylcholine and 8-bromo cyclic GA4P
The use of theophylline as a means of identifying a possible site of action for cyclic GMP in the regulation of cyclic AMP metabolism requires some comment, since a number of authors have questioned its specificity as an inhibitor of phosphodiesterase activity [Z, 251. There is no doubt that it does have other effects, but it is less clear whether these should be regarded as ‘Lnon specific”, or if they should instead be thought of as arising directly from its ability to inhibit phosphodiesterase activities. The positive inotropic action of theophylline on the frog heart has been attributed to increased accumulation of intracellular Ca2+ [29]; similarly, the inotropic effect of caffeine, a related methylxanthine, on the toad heart appears to be due to increased mobilization of cellular Ca2+ [34]. It is to be expected of course that inhibition of phosphodiesterase activity by theophylline would initially increase intracellular cyclic AMP and cyclic GMP levels. Cyclic AMP is known to activate protein kinases which phosphorylate several regulatory proteins, including two that control Ca2+ movements, across the fibre membrane [24, 261 and between the sarcoplasmic reticulum and the myoplasm [22, 461 and one which appears to modulate the sensitivity of the contractile system to Ca2+ [9, II, 32, 41, 431. Since the regulatory capacities of all three proteins depend critically upon their state of phosphorylation, it is not surprising to observe what may appear to be “non specific” effects, such as those described above, but which are nevertheless secondary to a specific inhibition by theophylline of cyclic nucleotide phosphodiesterases. The possible involvement of calmodulin in mediating the effects of acetylcholine and of S-bromo cyclic GMP on the ventricle (discussed above) may also be relevant in connection with the experiments using theophylline. On the one hand, theophylline would be expected to increase cellular cyclic AMP and cyclic GMP, but there are reasons to suppose that this might be offset later by the activation of calmodulin, resulting from enhanced Ca2+ entry. Activation of calmodulin by Ca2+ would stimulate phosphodiesterase activity, and result in a lowering of cyclic nucleotide levels.
976
F. W. Flitney
and J. Singh
Some evidence that this may be happening is shown by the results presented in Tables 3 and 4. Preparations exposed to theophylline for 15 min had levels of cyclic AMP and cyclic GMP which were significantly lower than those found in ventricles not treated with theophylline (see controls in Tables 1 and 2, columns 3 and 7). It is difficult to explain this observation, other than to suppose that theophylline has primary and secondary effects on cyclic nucleotide metabolism. The situation might be clarified by studying the time course of changes in cyclic nucleotide levels and in action potential parameters during responses to theophylline alone; such experiments are currently in progress. Relationship
between changes in contractility
and cyclic nucleotide levels
The results of this investigation (and of others published elsewhere; see Introduction for references) reinforce the view that the contractile performance of the frog ventricle may be regulated, at any given moment, by the relative amounts of cyclic AMP: cyclic GMP present in its fibres. If this hypothesis turns out to be correct, then it follows that the two cyclic nucleotides must function antagonistically, cyclic AMP acting to potentiate contraction, and cyclic GMP exerting a counter-effect. It was pointed out earlier (p. 975) that cyclic AMP stimulates the phosphorylation of several proteins thought to be involved in regulating contraction and that this then alters their physiological properties. The role of cyclic GMP is much less clear and the literature abounds with contradictory evidence (see, for example [27]}. Indeed, there is as yet no concensus of opinion concerning even its involvement in regulating contraction. This is clearly an important issue, and one which is unlikely to be resolved by considering the relative importance of the two cyclic nucleotides in isolation from each other. The danger of attempting to do so was clearly recognized by Goldberg and his colleagues [19] who write that “if cyclic AMP and cyclic GMP of one to the other act in opposition to one another then . . . the relative proportion under certain circumstances may be more important than the absolute change in the concentration of only one of the components”. The present study and other reports which have appeared in the literature, suggesting that there may be a degree of interaction between the metabolism of the two cyclic nucleotides [I, 3, 10, 17, 38, 40, 42, 441, serves to reinforce this view. The apparent ability of cyclic GMP to stimulate the degradation of cyclic AMP affords one means whereby it could exert a regulatory influence on contraction but there are grounds for arguing that this cannot be the sole basis for its antagonistic effects. The reason is that correlations between changes in contractility and in either cyclic AMP or cyclic GMP are invariably less precise than those obtained by considering changes in the ratio cyclic AMP:cyclic GMP. If the depressant effect of cyclic GMP upon cyclic AMP levels was the only basis for the antagonistic effects of the two cyclic nucleotides, then the contractile response would be expected to correlate most closely with changes in intracellular cyclic AMP alone. These considerations led us to postulate earlier that cyclic GMP may also be involved in modulating the de-phosphorylation of regulatory proteins, perhaps by enhancing protein phosphatase activity [16, 371. Acknowledgements The authors thank The Wellcome Trust, Medical Heart Foundation for grants (to FWF) in support
Research Council of this work.
and .the British
Cyclic
GMP
may Regulate
Cyclic
AMP
in Frog Ventricle
977
REFERENCES 1.
2.
AMER, M. S. & KREIGHBAUM, W. E. Cyclic nucleotide phosphodiesterases: properties, activators, inhibitors, structure-activity relationships, and possible role in drug development. Journal of Pharmaceutical Science 64, ,l-37 (1975). ARGEL, M. I., VITTONE, L., GRASSI, A. O., CHIAPPE, L. E., CHIAPPE, G. E. & CINGOLANI, H. E. Effects of phosphodiesterase inhibitors on heart contractile behaviour, protein kinase activity and cyclic nucleotide levels. Journal of Molecular and Cellular Cardiology 12,
939-954 3.
4.
5. 6.
7. 8.
9. 10.
11.
12.
13. 14.
15.
16.
17.
18. 19.
( 1980).
BEAVO, J. A., HARDMAN, J. G. & SUTHERLAND, E. W. Stimulation of adenosine 3’, 5’monophosphate hydrolysis by guanosine 3’, 5’-monophosphate. Journal of Biological Chemistry 246, 3841-3846 (1971). BR~STR~M, C. O., HUANG, Y. C., BRECKENRIDGE, G. McL. & WOLFF, D. J. Identification of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase. Proceedings of the National Academy of Sciences of the U.S.A. 72, 64-68 (1975). BROWN, J. H. Adrenergic inhibition of catecholamine-stimulable cyclic AMP accumulation in murine atria. .Journal of Cyclic Nxleotide Research 5, 423-433 (1979). BEVERS, M. M., SMITS, R. A. E., VAN RIJN, J. & VAN WIJK, R. Heat treatment of rat liver adenosine 3’5’ cyclic monophosphate phosphodiesterase. Biochimica et biophysics actu 341, 120-128 (1974). CHAPMAN, R. A. R. Excitation-contraction coupling in cardiac muscle. Progress in Biophysics and Molecular Biology 35, l-52 (1979). CHEUNG, W. Y., LYNCH, T. J. & WALLACE, R. W. An endogenous Ca2+-dependent activator protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase. Advances in Cyclic Nucleotide Research 9, 233-251 (1978). COLE, H. A. & PERRY, S. V. The phosphorylation of troponin I from cardiac muscle. Biochemical Journal 149, 525-533 (1975). ENDOH, M. Correlation of cyclic AMP and cyclic GMP levels with changes in contractile force of dog ventricular myocardium during cholinergic antagonism of positive inotropic actions of histamine, glucagon, theophylline and papaverine. Japanese Journal of Pharmacolopy 29, 855-864 (1979). ENGLAND, P. J. Studies on the phosphorylation of the inhibitory subunit of troponin during modification of contraction in perfused rat heart. Biochemical Journal 160, 295-304 (1976). FABIATO, A. & FABIATO, F. Calcium-induced release of calcium from the sarcoplasmic reticulum of skinned cells from adult human, dog, cat, rabbit, rat and frog hearts and from foetal hearts and new born rat ventricles. Annals qf the New York Academy of Sciences 307, 491-522 (1978). FLITNEY, F. W., MOSHIRI, M. & SINGH, J. Effects of sodium nitroprusside on frog ventricle. Journal qf Physiology 305, 25-26P Abstract (1980). FLITNEY, F. W. & SINGEI, J. Depressant effect of 8-bromo guanosine 3’, 5’-cyclic monophosphate on endogenous adenosine 3’, 5’-cyclic monophosphate levels in frog ventricle. Journal of Phvsiologv 302, 29-30P Abstract (1980). FLITNEY, F. W. & SINGH, J. Exogenous uridine 5’-triphosphate enhances contractility and stimulates 3’, 5’-cyclic nucleotide metabolism in the isolated frog ventricle. Journal of Physiology 291, 52-53P Abstract (1979). FLITNEY, F. W. & SINGH, J. Inotropic responses of the frog ventricle to adenosine triphosphate and related changes in endogenous cyclic nucleotides. Journal of Physiology 304, 2142 (1980). FLITNEY, F. W. & SINGH, J. Release of prostaglandins from the isolated frog ventricle and associated changes in endogenous cyclic nucleotide levels. Journal of Physiology 304, I-20 (1980). GILES, W. & NOBLE, S. J. Changes in membrane currents in bullfrog atrium produced by acetylcholine. Journal of Physiology 261, 103-123 (1976). GOLDBERG, N. C., HADDOX, M. K., NICOL, S. E., GLASS, G. B., SANDFORD, C. H., KUEHL, F. A. & ESTENSEN, R. Biological regulation through opposing influences of cyclic AMP and cyclic GMP: The Yin Yang hypothesis. Advances in Cyclic Nucleotide Research 5, 307-330 (1975).
978 20. 2 1. 22. 23.
24.
25. 26. 27. 28.
29.
30. 31.
32.
33. 34. 35.
36.
37.
38.
39.
40.
41. 42.
F. W. Flitney
and J. Singh
GORNALL, A. G., BARDAWILL, D. J. & DAVID, M. M. Determination of serum proteins by means of the Biuret reaction. Journal of Biological Chemistry 177, 751-766 (1949). HIDAKA, H. & ASANO, T. Human blood platelet 3’5’ cyclic nucleotide phosphodiesterase. Biochimica et biophysics acta 429, 485-497 (1976). KATZ, A. M., TADA, M. & KIRCHBERGER, M. A. Control of calcium transport by cyclic AMP-protein kin&e. Advances in Cyclic Jfucleotide Research 5, 453-472 (1975). KOHLHARDT, M. & HAAP, K. 8-Bromo-guanosine-monophosphate mimics the effects of acetylcholine on slow response action potential and contractile force in mammalian atria1 myocardium. Journal of Molecular and Cellular Cardiology 10, 573-586 (1978). KRAUSE, E. G., WILL, H., SCHIRPKE, B. & WOLLENBERGER, A. Cyclic AMP enhances protein phosphorylation and calcium binding in a cell membrane-enriched fraction from myocardium. Advances in Cyclic Nucleotide Research 5, 473490 (1975). KRAUSE, E. G. & WOLLENBERGER, A. Cyclic nucleotides and heart. In Cyclic Nucleotides: Mechanism of Action, H. Cramer & J. Schultz, Eds, pp. 229-250. New York: Wiley (1976). LAMB, J. F. & MCGUIGAN, J. A. S. Contractures in superfused frog ventricle. Journal of Physiology 186, 261-283 (1966). LINDEN, J. & BROOKER, G. The questionable role of cyclic guanosine 3’5’ monophosphate in heart. Biochemical Pharmacology 28, 3351-3360 (1979). MARCUS, M. L., SKELTON, C. L., PRINDLE, K. H. & EPSTEIN, S. E. Potentiation of the inotropic effects of glucagon by theophylline. Journal of Pharmacolo@ and Experimental Therapeutics 179, 331-337 (1971). MASSINGHAM, R. & NASMYTH, P. A. The nature of the positive inotropic response of the isolated frog heart to theophylline and to imidazole. British Journal of Pharmacology 45, 229-239 (1972). MEANS, A. R. & DEDMAN, J. R. Calmodulin-an intracellular calcium receptor. Nature 285, 73-77 (1980). MOPE, L., MCCLELLAN, G. B. & WINEGRAD, S. Calcium sensitivity of the contractile system and phosphorylation of troponin in hyperpermeable cardiac cells. Journal of General Physiology 75, 271-282 (1980). MILLER, J. P., BOSWELL, K. H., MUNEYAMA, K., SIMON, L. N., ROBINS, R. K. & SHUMAN, D. A. Synthesis and biochemical studies of various 8-substituted derivatives of guanosine 3’, 5’, cyclic phosphate, inosine 3’5’ cyclic phosphate and xanthesine 3’5’ cyclic phosphate. Biochemistry 12, 5310-5319 (1973). NAWRATH, H. Cyclic AMP and cyclic GMP play opposing roles in influencing force of contraction in mammalian myocardium. Nature 262, 509-511 (1976). NAYLER, N. G. Effect of caffeine on cardiac contractile activity and radiocalcium movement. American Journal of Physiology 204, 969-974 ( 1963). RALL, T. W. & WEST, T. C. The potentiation of cardiac inotropic responses to norepinephrine by theophylline. Journal of Pharmacology and Experimental Therapeutics 139, 269-274 (1963). RUSSELL, T. R., TERESAKI, W. L. & APPLEMAN, M. M. Separate phosphodiesterases for the hydrolysis of cyclic adenosine 3’5’ monophosphate and cyclic guanosine 3’5’ monophosphate in rat liver. Journal of Biological Chemistry 248, 1334-1340 (1973). SINGH, J. & FLITNEY, F. W. Adenosine depresses contractility and stimulates 3’, 5’cyclic nucleotide metabolism in the isolated frog ventricle. Journal of Molecular and Cellular Cardiology 12, 285-289 (1980). SINGH, J. & FLITNEY, F. W. Inotropic responses of the frog ventricle to dibutyryl cyclic AMP and 8-Bromo cyclic GMP and related changes in endogenous cyclic nucleotide levels. Biochemical Pharmacology 30, 1475-1481 (1981). SINGH, J., FLITNEY, F. W. & LAMB, J. F. Effects of isoprenaline on contractile force and intracellular cyclic 3’, 5’ nucleotide levels in the hypodynamic frog ventricle. FEBS Letters 91, 269-272 (1978). STEFANOWCH, V. Cyclic nucleotide phosphodiesterase inhibitors as potential therapeutic agents. In Advances in Biosciences 24, G. Cehovic & G. A. Robison, Eds. Oxford: Pergamon Press (1979). STULL, J. T. Phosphorylation of contractile proteins in relation to muscle function. Advances in Cyclic .Nrxleotide Research 13, 39-93 (1980). TERESAKI, W. L. & APPLEMAN, M. M. The role of cyclic GMP in regulation of cyclic AMP hydrolysis. Metabolism 24, 311-319 (1975).
Cyclic 43. 44.
45. 46.
47.
GMP
may
Regulate
Cyclic
AMP
in Frog
Ventricle
979
TSIEN, R. W. Cyclic AMP and contractile activity in the heart. Advances in Cyclic Nucleotide Research 8, 363-419 (1977). WEISS, B. & HAIT, W. N. Selective cyclic nucleotide phosphodiesterase inhibitors as potential therapeutic agents. Annual Review of Pharmacology and Toxicology 17, 441-477 (1977). WELLS, J. N. & HARDMAN, J. G. Cyclic nucleotide phosphodiesterases. Advances in Cyclic Nucleotide Research 8, 119-143 (1979). WILL, H., MISSELWITZ, H.J., LEUVHENKO, T. S. & WOLLENBERGER, A. Some characteristics of low molecular weight phosphoprotein constituents of cardiac sarcoplasmic reticulum and sarcolemma. In Advances in PharmacoloD and Therapeutics 3, J. C. Stoclet, Ed., pp. 161-170. Oxford: Pergamon Press (1978). WOLFF, D. J. & BROSTROM, C. 0. (1979). Properties and functions of the calciumdependent regulator protein. Advances in Cyclic Nucleotide Research 11, 28-88 (1979).
and Cellular
Evidence that Metabolism
Cardiology
( 198 1) 13, 963-979
Cyclic GMP may in the Isolated
Frederick
W. Flitney
Regulate Cyclic Frog Ventricle
and Jaipaul
AMP
Singh”
Department of Physiology and Pharmacology, University of St Andrews, Bute Medical Buildings, St Andrews, Fife X226 9TS, Scotland (Received 10 October 1980, accepted in revised form 1 August 1981) F. W. FLITNEY AND J. SINGH. Evidence that Cyclic GMP may Regulate Cyclic AMP Metabolism in the Isolated Frog Ventricle. Journal of Molecular and Cellular Cardiology ( 1981) 13, 963-979. Both acetylcholine and 8-bromo cyclic GMP depress the contractile response of the isolated frog ventricle. An investigation has been made of the effects of both substances on the metabolism of endogenous 3’, 5’ cyclic nucleotides. The levels of adenosine 3’, 5’ cyclic monophosphate (cyclic AMP) and guanosine 3’, 5’ cyclic monophosphate (cyclic GMP) were measured after superfusing preparations with varying concentrations (10-l” to lo-* M) of acetylcholine and 8-bromo cyclic GMP. The decline in contractile force was found to be accompanied by a progressive fall in intracellular cyclic AMP and a rise in cyclic GMP levels. Both the decline in contractility and the reduction in endogenous cyclic AMP are attenuated by 1Oe4 theophylline. The decline in isometric twitch tension was paralleled, under all conditions, by a quantitatively equivalent reduction in the ratio cyclic AMP: cyclic GMP. The possibility that endogenous cyclic GMP may accelerate the conversion of cyclic AMP to 5’ AMP, by stimulating a cyclic GMP-sensitive form of cyclic AMP phosphodiesterase, is discussed. KEY WORDS: 8-Bromo cyclic
Cyclic GMP;
AMP; Cyclic Theophylline;
GMP; Contractility; Phosphodiesterase.
Frog
ventricle;
Acetylcholine;
Introduction The possibility that cyclic AMP and cyclic GMP are together involved in regulating myocardial contractility, and that they function antagonistically, was first proposed several years ago by Goldberg and his colleagues [19]. This suggestion is substantiated by the results of more recent experiments, in which responses of the isolated frog ventricle to a range of pharmacological agents (adenosine 5’-triphosphate [lo], uridine 5’-triphosphate [ 151, isoprenaline [39], a d renaline (unpublished), dibutyryl cyclic AMP [38], adenosine [37], sodium nitroprusside [13], and 8-bromo cyclic GMP [14, 381) were studied: these all reveal a clear and consistent correlation between changes in ventricular contractility and changes in the ratio of endogenous cyclic AMP: cyclic GMP. Cyclic AMP is known to activate enzymes (cyclic AMP-dependent protein kinases) which catalyze the phosphorylation of important regulatory proteins, whose physiological activities are then altered as a result [9, 11, 22, 24, 32, 41, 43, 467. The role of cyclic GMP is much less clear, but two alternative suggestions have been made to account for the way in which it might act to oppose the effects of cyclic AMP: * Present 0022-2828/81/l
address: 10963+
Department 17
$02.00/O
of Physiology,
The
University, 0
1981
Dundee Academic
DDl
4HN,
Press
Inc.
Scotland. (London)
Limited
964
F. W. Flitney
and J. Singh
first, that it may stimulate cyclic AMP phosphodiesterase activity, reducing cyclic AMP levels and thereby suppressing cyclic AMP-dependent protein phosphorylation [16]; and secondly, that it may enhance protein phosphatase activity, and so promote protein de-phosphorylation [IS, 371. The experiments to be described were designed to investigate the first of these possibilities. The approach used was to examine the effect of elevating cyclic GMP levels on those of cyclic AMP. The experiments were made with the isolated frog ventricle and two different procedures were employed to increase tissue cyclic GMP levels: by superfusing preparations with S-bromo cyclic GMP, a lipophilic (and therefore membrane-permeant) analogue of cyclic GMP; or alternatively, by activating muscarinic receptors with acetylcholine.
Materials
and
Methods
Ventricle perfusion Isolated ventricles from frogs (R. temporaria) were divided into two halves, one of which served as a control for the other. Control half-ventricles were superfused with oxygenated Ringer’s solution (composition in mM : NaCl, 115 ; KCI, 2.5 ; CaCl,, 1; Na,HPO,, 2.15; NaH,PO,, 0.85; glucose, 5.6; pH 7.2) at a flow rate of lOOml* min-r in a closed circuit perfusion system [19, 261. The total volume of the recirculated perfusion fluid was 1 1. “Test” half-ventricles were superfused under identical conditions, but with Ringer’s solution containing either acetylcholine (lo-lo to 10~~ M) or 8-bromo cyclic GMP (IO-lo to 1O-4 M) . The responses to 8-bromo cyclic GMP developed more slowly than those for acetylcholine, reaching.a steady-state after approximately 25 min. At this time, the perfusion fluid was exchanged for Ringer’s solution alone, in order to remove traces of extracellular 8-bromo cyclic GMP which would otherwise have interfered with the cyclic GMP assay (see below). A “wash-out” period of 30 s was given prior to freezing the preparation. Lamb and McGuigan [26], who used similar perfusion conditions, found that changes in twitch tension produced by lowering extracellular [Ca2+] from 2 mM to 1 mM required only 1 s for completion, so the 30 s “wash” period allowed was considered adequate to remove 8-bromo cyclic GMP from the interfibre spaces. This was confirmed experimentally, by taking a sample of the perfusion fluid (after 25 s) immediately after it had passed over the preparation and assaying this’ for cyclic GMP (six experiments). The quantity of 8-bromo cyclic GMP in all such samples was less than the lower limit of detection of the assay method ((0.05 pmol*ml-l). All preparations were paced electrically, by stimulating through silver wire electrodes (10 V amplitude, 4 mA, 5-ms duration) at a frequency of 0.5 Hz. Isometric twitch tension was measured using a strain gauge (Devices, type 4157 ; compliance: 40 p-g-1) and the output recorded continuously on a chart recorder. The optimum length, giving a maximal contraction, was established for each half-ventricle at the start of the experiment. A more complete account of the experimental procedure used is given elsewhere [17]. The effect of theophylline (lo-4 M) was studied by superfusing preparations for 15 min prior to the addition of either acetylcholine or 8-bromo cyclic GMP, and then throughout the subsequent response.
Cyclic
GMP
may
Extraction
Regulate
Cyclic
AMP
in Frog
Ventricle
965.
of cyclic nucleotides and assay procedure
When the twitch had attained its new steady-state (after 25 min for 8-bromo cyclic GMP and 2 min for acetylcholine) both “test” and “control” preparations were frozen rapidly. This was done by compressing the tissue between broad-tipped forceps which had been cooled previously by immersion in liquid nitrogen. The frozen tissue was pulverized in a stainless steel mortar (also cooled in liquid nitrogen) and afterwards extracted with acidified ethanol (1 ml 1 NHCI; 100 ml ethanol). The solvent was blown-off in a stream of nitrogen gas, and the residue taken up in TrisEDTA buffer (0.05 M-Tris, pH 7.5; containing 4 mM EDTA). Cyclic AMP and cyclic GMP levels were then determined, using the Radiochemical Centre’s assay kits TRK 432 and TRK 500, respectively. Precise details of the procedure used are to be found in the publications which accompany the assay kits (Radiochemical Centre, Amersham, England). Total protein was estimated using the Biuret method [ZO] and cyclic nucleotide concentrations are expressed throughout in pmol . mg-l tissue protein. Control experiments established that the cyclic GMP assay procedure is equally sensitive to the 8-bromo derivative as to the parent nucleotide (to within f2%, in the sample range 0.5 to 10 pmol). Hence, the values listed in Table 2 for tissue concentrations of “cyclic GMP” actually represent the sum of endogenous cyclic GMP #us an amount of 8-bromo cyclic GMP which had entered the fibres from the extracellular fluid. Results
Efects of acetylcholine and 8-bromo cyclic GMP
on contractility
and cyclic nucleotide levels
Figures 1 and 2 ( 0) show that both acetylcholine and 8-bromo cyclic GMP depressed the twitch, in a dose-dependent manner, and that the concentrations required to produce a 50% reduction were comparable, around I x 1O-s M and 5 x 1O-8 M, respectively. However, there was a large difference in the rate at which the twitch declined towards its new steady state value, being at least 100 times greater for acetylcholine than for 8-bromo cyclic GMP. The results of experiments with pharmacological receptor antagonists show that this is due to qualitative differences in the mode of action of the two substances. The experiments in question showed (a) that responses to acetylcholine could be entirely inhibited by atropine ( 1O-6 M) but were unaffected by either phentolamine (lo-’ M, an M antagonist) or propranolol (lo+ M, a p antagonist); and (b) that neither phentolamine, propranolol nor atropine attenuated responses to 8-bromo cyclic GMP. It can therefore be concluded that 8-bromo cyclic GMP does not act in the same way as acetylcholine, by activating muscarinic receptors. The most likely explanation for its slower action is that it exerts its effects intracellularly, and so must first gain access to the fibre interior by diffusing across the surface membrane. The experiments with phentolamine and propranolol also demonstrate that the method of stimulation used to “pace” the ventricle (see Materials and Methods) did not lead to the release of significant amounts of endogenous catecholamines. This conclusion is based upon studies which show that the inotropic actions of acetylcholine on responses already potentiated by catecholamines are much greater than’ on responses recorded in the presence of tc and/or p antagonists [5, IO]. Hence, if appreciable quantities of endogenous catecholamines had been liberated during
966
F. W. Flitney
and
J. Singh
0 t
lo-‘0
10-g
lo-* Concentration
10-7
10-s of ACh
10-s
10-4
IO-J
[ml
FIGURE 1. Dose-response curves showing the effects of acetylcholine (10-l” twitch tension, in the presence (0) and absence (0) of the phosphodiesterase (IO+ M). Theophylline decreases the sensitivity of the ventricle to acetylcholine. as multiples of control values.
to lOA M) on isometric inhibitor theophylline All points expressed
electrical stimulation, phentolamine and/or propranolol would have attenuated responses to acetylcholine, but neither did. The effects of acetylcholine and 8-bromo cyclic GMP on the twitch were found to be accompanied by changes in intracellular cyclic GMP and cyclic AMP. The concentrations measured in test and control preparations are listed in Tables 1 (acetylcholine) and 2 (8-bromo cyclic GMP). Two important features of these results should be emphasized. First, it can be seen that the extent to which acetylcholine and 8-bromo cyclic GMP depresses the twitch is paralleled closely by quantitatively-equivalent reductions in the ratio cyclic AMP : cyclic GMP (compare columns 10 and 11 in Tables 1 and 2), a result which agrees with our previous findings using a variety of pharmacologically-distinct inotropic agents (see Introduction for references). Secondly, the responses to both substances are characterized by increased concentrations of cyclic GMP and by marked reductions in endogenous cyclic AMP levels. The reciprocal relationship between changes in cyclic AMP and cyclic GMP is shown graphically in Figures 3 and 4 ( 0). Here, the decrease in endogenous cyclic
Cyclic
GMP
I lo-‘0
may
I 10-s
Regulate
I 10-e Concentmtion
Cyclic
I 10-7
AMP
I 10-e
of 8 - bromo
in Frog
I 10-s cGMP
I to-4
967
Ventricle
I(
[M]
FIGURE 2. Log-dose response curves showing the effects of differing IO+ M) of 8-bromo cyclic GMP on twitch amplitude in the presence (0) theophylline. All values expressed as multiples of control levels.
concentrations and absence
(0)
(IO-lo to of 1O-4
AMP levels is plotted as a function of the increase in intracellular cyclic GMP. The relationship is approximately linear and quantitatively similar for both substances, yielding a slope of around - IO pmol cyclic AMP. pmol-r increment in cyclic GMP (regression data : acetylcholinz: -9.82 f 0.59 pmol cyclic AMP. pmol-l cyclic GMP; P < 0.001; n = 25; 8-bromo cyclic GMP: -10.79 f 0.60 pmol cyclic AMP. pmol-r cyclic GMP; P < 0.001; n = 18). The latter observation suggests a possible role for cyclic GMP in regulating cyclic AMP metabolism: namely, that under certain circumstances it may serve to su@ress endogenous cyclic AMP levels. The question which arises then is: does it do so by inhibiting adenylate cyclase activity or by enhancing cyclic AMP phosphodiesterase activity? Effects of theophylline on responses to acetylcholine and 8-bromo cyclic GMP In an attempt to investigate this question, responses to both acetylcholine and 8-bromo cyclic GMP were recorded in the presence of theophylline, a phosphodiesterase inhibitor. Theophylline was selected for study because previous experiments (unpublished) had shown that it produces only a transient effect on the frog ventricle
1
CAMP (control)
CAMP
8.06 8.82 7.69
8.66 8.69 7.89
2.01* 2.39 1.84
1.47" 1.40 1.18
10-7
10-s
CAMP ratio ACh
4
0.07 0.05
0.17 0.16 0.15
0.25 0.27 0.24
0.64 0.60 0.62
0.87 0.86 0.88
0.94 0.95 0.97
>
nucleotide
( control
on cyclic 5
-8.09 -8.36
-7.19 -7.29 -6.71
-6.05 -6.93 -5.85
-3.13 -3.38 -3.17
-1.05 -1.13 -1.02
-0.52 -0.41 -0.23
(2-3)
Change in CAMP
levels 6
1.98 1.92
1.81 1.77 2.21
1.91 1.98 2.13
1.84 1.94 1.66
1.50 1.34 1.66
1.31 1.38 1.58
cGMP ‘(ACh)
7
1.06 1.02
1.10 1.06 1.34
1.22 1.23 1.35
1.36 1.45 1.25
1.30 1.18 1.43
1.20 1.28 1.48
cGMP (control)
8
1.86 1.88
1.64 1.67 1.65
1.56 1.60 1.58
1.35 1.34 1.33
1.15 1.18 1.16
1.09 1.08 1.07
( control
cGMP ratio ACh >
9
0.03 0.03
0.11 0.10 0.09
0.16 0.16 0.15
0.47 0.45 0.47
0.75 0.73 0.76
0.86 0.88 0.91
R
10
0.04 0.05
0.10 0.09 0.11
0.15 0.18 0.17
0.49 0.46 0.48
0.76 0.75 0.78
0.88 0.90 0.92
P
11
nucleotide ratio [R = cyclic AMP: cyclic treated ventricle/twitch tension of control cyclic AMP levels decrease progressively were 8.33 f 0.10 and 1.25 f 0.04 pmol-
$0.91 +0.90
+0.71 +0.71 +0.87
+0.69 $0.75 +0.78
+0.48 +0.44 +0.41
$0.20 +0.20 +0.23
+0.11 +0.10 +0.10
(6-7)
Change in cGMP
Values in columns 2, 3, 5,6, 7 and 9 are pmol. mg-l total protein. The figures in columns 10 and 11 relate changes in cyclic GMP (treated ventricle)/cyclic AMP: cyclic GMP (control ventricle)] and changes in twitch tension (P = twitch tension of ventricle): note that both decrease in parallel as the concentration of acetylcholine is increased. Note also that endogenous with increasing cyclic GMP levels [see also Figure 3 (@)I. The levels of cyclic AMP and cyclic GMP in control preparations performed in presence of phentolamine (10-T M) and propranolol (10-s M). mg-l protein, respectively (n = 17). * Experiments
8.70 8.80
8.68 8.45 8.34
5.55" 5.07 5.17
10-s
0.56" 0.44
7.98 8.05 8.33
6.93” 6.92 7.31
10-s
10-s
8.71 8.20 7.65
WW
3
2
of acetylcholine
8.19” 7.79 7.42
1. Effects
10-1s
(M)
Concentration ACh
TABLE
1
8.03 8.08 8.70
7.75 7.76 9.01
8.10 9.13 9.00
7.20 8.50 8.07
7.86 10.46 9.83
5.54* 5.28 5.81
4.30" 4.59 4.94
2.75" 3.48 3.24
2.06" 2.20 2.26
1.10” 1.98 1.70
10-s
IO-'
10-s
10-h
IO-4
GMP
0.14 0.18 0.17
0.28 0.26 0.27
0.31 0.38 0.36
0.55 0.59 0.55
0.64 0.65 0.67
0.80 0.78 0.82
(8-::;:Fp)
CAMP ratio
on cyclic
cyclic AMP of phentolamine
and
cyclic (lo-’
M)
GMP
in control propranolol
preparations ( low6 M) and
5 (8-Br
6
decline
7
(8-::i::p)
in legend
9
to Table
+0.62 $0.79 +0.65
+0.43 +0.55 +0.53
+0.40 10.44 +0.40
+0.31 +0.30 +0.35
$0.21 +0.16 +0.23
+0.19 +0.14 +0.10
(6-7)
Change in cGMP
1) with
R
10
4 (a)]. performed
increasing
0.08 0.12 0.11
0.20 0.18 0.19
0.26 0.29 0.27
0.43 0.47 0.43
0.58 0.56 0.56
0.70 0.71 0.75
levels [compare columns 5 and 9, see also Figure protein, respectively (n = 18). * Experiments
1.55 1.55 1.52
1.41 1.44 1.45
1.30 1.32 1.33
1.29 1.27 1.27
1.20 1.17 1.19
1.13 1.10 1.09
cGMP ratio
in R and P (defined
1.13 1.43 1.25
1.05 1.25 1.16
1.25 1.65 1.18
1.06 1.10 1.30
1.05 0.94 1.18
1.40 1.36 1.15
cGMP (control)
cyclic GMP 0.04 pmol.mg-’
1.75 2.22 1.90
1.48 1.80 1.69
1.65 2.19 1.58
1.37 1.40 1.65
1.26 1.10 1.41
1.59 1.50 1.25
cGMP cGMP)
the progressive
levels
with increasing were 8.41 -& 0.19 and 1.22 i atropine (10~~ M).
Note
-6.76 -8.42 -8.13
-5.14 -6.30 -5.81
-5.35 -5.65 -5.76
-3.45 -3.17 -4.07,
-2.49 -2.80 -2.89
-1.66 -1.74 -1.36
Change in CAMP (2-3)
nucleotide
Values in columns 2, 3, 5, 6, 7 and 9 are pmol.mg-l protein. of 8-bromo cyclic GMP. Endogenous cyclic AMP falls progressively
8.26 7.94 7.66
6.60" 6.20 6.30
CAMP (control)
3
cyclic
10-g
(M)
2
of 8-bromo
CAMP (8-Br cGMP)
2. Effects
Concentration 8-Br cGMP
TABLE
The levels of in presence
concentrations
0.09 0.10 0.12
0.18 0.16 0.17
0.25 0.32 0.29
0.44 0.48 0.45
0.60 0.58 0.55
0.72 0.73 0.76
P
11
970
F. W; Flitney
and
J. Singh
-8-
-6 -
+I
, 0
1
+0.2 Change Test minus
I
+ 0.4
I
+ 0.6
in introcellulor control (pm4
I
+ 0.0
+I
5,s cGMP mg-’ protein)
FIGURE 3. Inverse relationship between changes in cyclic AMP and cyclic GMP ieds (treatedcontrol values) following treatment with acetylcholine (IO-lo to 10e5 M) in the presence (0) and absence (0) of theophylline (lo-* M). The slope of the line relating the reduction in cyclic AMP to the corresponding increase in cyclic GMP levels (fitted by linear regression analysis) is markedly reduced by theophylline, by a factor of around six.
when used at a concentration of lo-* M {see also [29, 341). Initially, it evokes a positive inotropic response but within 10 to 12 min the twitch returns to its control level. Hence, in the experiments now to be described, ventricles were first pre-treated with theophylline for 15 min, prior to the addition of either 8-bromo cyclic GMP or acetylcholine. Theophylline also remained in the superfusing solution throughout the remainder of the response. Figures 1 and 2 (0) show that the sensitivity of the ventricle to both acetylcholine and 8-bromo cyclic GMP is markedly depressed by theophylline-the dose-response curves are displaced to the right, towards higher agonist concentrations. This observation is of some interest, because phosphodiesterase inhibitors generally potentiate cyclic nucleotide mediated responses [28, 3.51, by decreasing the rate at which cyclic AMP and cyclic GMP are hydrolyzed to their non-cyclical (inactive) forms (5’ AMP and 5’ GMP, respectively). What, then, is the explanation for this apparently anomalous effect? The data presented in Figures 3 and 4 ( 0) reveal that the desensitizing effect of theophylline is associated with a significant reduction in the slopes of the lines relating changes in tissue cyclic AMP and cyclic GMP levels, from around -10 pmol cyclic AMP. pmol-l cyclic GMP to -1.64 f 0.34 pmol cyclic AMP. pmol-r cyclic GMP for acetylcholine (P < 0.001, n = 12) and -4.27 f 0.29 pmol cyclic AMP. pmol-r cyclic GMP for 8-bromo cyclic GMP (P < 0.001, n = 18). Thus, theophylline is able to reduce the degree of coupling between changes in cyclic GMP and cyclic AMP. Accordingly, exposure of the
Cyclic
GMP
may
Regulate
Cyclic
AMP
in
Frog
Ventricle
971
-lO-
-0-
.
/ .
,/
-6/
-4-
.
-2-
.
/*
..
.,?
.
0’ 0 -
..
A-
god00 -0-o 0 .o.oQ," 0 I 0
I + 0.2
0
+o.
I
O I + 0.4
I + 0.6
I + 0.0
+ I.0
+ 0.2
-I- 0.3
+ 0.4
+ 0.5
Change in intracellular Test minus (pool
5,s cGMP mg-’ protein)
FIGURE 4. Relationship between changes in endogenous cyclic AMP and cyclic GMP levels (treated-control values) following exposure of the frog ventricle to varying concentrations ( 10e9 to lo-* M) of exogenous 8-bromo cyclic GMP in the presence (0) and absence (0) of 1OW M theophylline. The slope of the line relating the reduction in cyclic AMP to the corresponding increase in cyclic GMP levels (fitted by linear regression analysis) is reduced by theophylline by a factor of about two and a half. Abscissa: upper values in the presence of 8-bromo cyclic GMP; lower values in the presence of theophylline and 8-bromo cyclic GMP.
theophylline-treated ventricle to a particular concentration of acetylcholine or S-bromo cyclic GMP reduces the relative proportion of cyclic AMP: cyclic GMP to a lesser extent than in preparations not treated with theophylline. This is illustrated by the data in Tables 3 and 4 (columns 10 and 11) which again demonstrate a parallel though now greatly reduced decline in the ratio of cyclic AMP : cyclic GMP and isometric twitch tension.
Discussion
The experiments described here show that the negative inotropic effect of acetylcholine on the frog ventricle is associated with increased accumulation of cyclic GMP and a simultaneous depression of endogenous cyclic AMP levels. The ability of S-bromo cyclic GMP to likewise depress endogenous cyclic AMP levels strongly suggests that this effect may actually be mediated by the increase in intracellular cyclic GMP. The striking quantitative agreement in the relationship between changes in. cyclic AMP and cyclic GMP, whether induced by acetylcholine, acting via muscarinic recefltors, or by 8-bromo cyclic GMP, makes this hypothesis seem all the more plausible. The most straightforward interpretation of the results is to postulate that cyclic GMP constitutes part of a feed-back control mechanism which serves to regulate th.e
1
3.78 3.69
2.00 2.22
10-G
10-S
5 and 9 plotted listed in Table
4.80 5.59
10-T
Data in columns control preparation
5.46 4.52
5.39 4.52
10-S
4
0.56 0.58
3.57 3.84
5
nucleotide
- 1.57 -1.62
1.89 1.96
1.56 1.76
-0.95 -0.81
1.02 1.04
0.92 1.05
0.95 1.07
1.44 1.60
-0.15 -0.12
1.06 1.01
0.96 0.91
cGMP (control)
7
in the presence
0.98 0.97
1.21 1.16
1.07 0.96
cGMP Wh)
6
levels
1.25 1.23
-0.07 0.00
0.00 0.00
+0.28 +0.33
(2-3)
Change in CAMP
and cyclic
of 10-a 8
theophylline 9
7)
AMP
+0.87 +0.92
+0.64 +0.71
+0.49 to.53
+0.27 +0.26
+0.15 +0.15
+0.06 +0.05
(6-
Change in cGMP
levels of cyclic
1.85 1.88
1.70 1.68
1.52 1.50
1.28 1.27
1.14 1.15
1.06 1.06
cGMP ratio
M
in Figure 3 (0). Note (a) the parallel decline in P and R; and (b) control 1, which were not exposed to theophylline. See text for further details.
0.80 0.82
0.97 0.98
0.99 1 .oo
1 .oo 1.00
1.05 1.07
CAMP ratio
4.73 4.50
4.95 5.71
5.72 5.38
5.72 5.38
10-S
CAMP (control)
3
on contractility
5.70 4.70
W-W
CAMP
2
of acetylcholine
5.98 5.03
M)
(M)
3. Effect
10-10
ACh + Theophylline ( 1k4
tration
Concen-
TABLE
and cyclic
0.30 0.31
0.47 0.48
0.64 0.65
0.77 0.79
0.88 0.87
0.99 1.01
R
10
GMP
are less than in
0.28 0.29
0.49 0.50
0.65 0.67
0.80 0.81
0.91 0.89
1.00 1.02
P
11
3.70 5.00 4.32 4.80 4.89 5.06 3.73 3.54 3.37 4.30 4.54 4.76 3.83 4.05 3.59 3.73 3.40
10-S
3.73 5.04 4.50 4.95 4.99 5.27 4.15 3.85 3.88 4.88 5.05 5.67 4.68 5.06 5.06 5.25 5.07
3 CAMP (control)
cyclic GMP
0.99 0.99 0.96 0.97 0.98 0.96 0.90 0.92 0.87 0.88 0.90 0.84 0.82 0.80 0.71 0.68 0.67
0.82 1.06 0.89 0.97 1.12 1.19 1.02 0.94 1.20 1.14 1.14 1.22 1.13 1.19 1.28 1.45 1.25
6 cGMP (8-Br cGMP)
levels and contractility
-0.03 -0.04 -0.18 -0.15 -0.10 -0.21 -0.42 -0.31 -0.51 -0.58 -0.51 -0.91 -0.85 - 1.01 -1.47 - 1.52 - 1.67
5 Change in CAMP (2-3)
on cyclic nucleotide
0.80 1.05 0.86 0.92 1.05 1.08 0.96 0.87 1.04 1.03 1.02 0.95 0.89 0.96 0.95 1.07 0.92
7 cGMP (control)
1.02 1.01 1.03 1.05 1.06 1.10 1.06 1.08 1.15 1.11 1.12 1.28 1.27 1.24 1.35 1.36 1.36
in presence of lo+ M
+0.02 +0.01 +0.03 +0.05 +0.07 +0.11 + 0.06 +0.07 +0.16 +0.11 -to.12 10.27 + 0.24 10.23 +0.33 +0.38 +0.33
9 Change in cGMP (6-7)
theophylline
0.97 0.98 0.93 0.92 0.92 0.87 0.85 0.85 0.75 0.80 0.80 0.66 0.64 0.65 0.53 0.52 0.49
10 R
0.96 0.99 0.94 0.93 0.91 0.85 0.84 0.86 0.79 0.79 0.81 0.67 0.66 0.64 0.54 0.51 0.50
11 P
Data in columns 5 and 9 plotted in Figure 4 (0). Note (a) parallel decline in P and R and (b) control levels of cyclic AMP and cyclic GMP are lower than those in Table 2 for ventricles not exposed to theophylline. See text for further details.
10-d
10-s
IO-”
IO-7
10-s
2 CAMP (8-Br cGMP)
4. Effects of 8-bromo
1 Concentration (M) 8-Br cGMP + Theophylline ( 10-4 M)
TABLE
974
F. W. Flitney
and
J. Singh
metabolism of cyclic AMP. The observations which have been made thus far are entirely consistent with the idea that it does so by stimulating a cyclic GMP-sensitive isozyme of cyclic AMP phosphodiesterase, thereby accelerating the hydrolysis of cyclic AMP to 5’ AMP. Three major forms of cyclic nucleotide phosphodiesterase have been isolated from a variety of tissues [I, 40, 44, 4.51 and of these, two are of relevance to the present discussion, since their ability to hydrolyze cyclic AMP is subject to allosteric control-in one case, by cyclic GMP [I, 3, 36, 40, 421 and in the other, by calcium, acting indirectly through the Ca 2+-dependent regulator protein, calmodulin [ 7, 30, 471.
Cyclic GMP-dependent
regulation
of cyclic AMP
hydrolysis
The kinetic properties of a cyclic GMP-dependent cyclic AMP phosphodiesterase from rat heart have been investigated by Teresaki and Appleman [42]. They found that the ability of the enzyme to hydrolyze cyclic AMP at physiological concentrations was markedly stimulated by low levels of cyclic GMP. Qualitatively similar observations have been made on phosphodiesterase preparations from other tissues too [3, 7, 22, 36, 411. Stimulation by cyclic GMP of the rat heart preparation appears to be due to a loss of enzyme co-operativity, rather than to any change in either V,,, or Krr, for cyclic AMP, and under optimal conditions it can effect a 6 to 10 fold increase in the rate of hydrolysis of cyclic AMP. If this enzyme, or one possessing similar kinetic properties, is also present in frog heart, then it would afford a biochemical basis for the regulation of cyclic AMP degradation by cyclic GMP. There is another reason why it appears likely that cyclic GMP acts by stimulating cyclic AMP breakdown. It will be recalled that the inotropic responses to acetylcholine and to 8-b-romo cyclic GMP, and the effects that both substances have on endogenous cyclic AMP levels, are attenuated by treatment with theophylline, a phosphodiesterase inhibitor. This is to be anticipated if stimulation by cyclic GMP of a phosphodiesterase is involved in the regulation of cyclic AMP metabolism, because a given increment in tissue cyclic GMP would then be less effective at reducing cyclic AMP levels. The above considerations can only be regarded as circumstantial evidence in support of a role for cyclic GMP in regulating cyclic AMP metabolism: it remains to be seen whether a phosphodiesterase isozyme with similar properties to the one found in the rat heart is also present in the frog heart; and the specificity of theophylline as a phosphodiesterase inhibitor has also been called into question (see below). It should also be noted that a preparation of bovine heart phosphodiesterase is reported to be inhibited by cyclic GMP [31].
Ca2+-dependent
regulation
of cyclic nucleotide metabolism
The existence of a reciprocal relationship between changes in tissue cyclic AMP and cyclic GMP levels, while consistent with the notion that cyclic GMP may serve can be interpreted differently. It has been to regulate cyclic AMP metabolism, and 8-bromo cyclic GMP [23] impede the reported that both acetylcholine [18] entry of Ca2+ into the fibres during the action potential. The quantity of Ca2+ which crosses the membrane during excitation is not considered sufficient to activate con-
Cyclic
GMP
may
Regulate
Cyclic
AMP
in Frog
Ventricle
975
traction directly [24], but there is evidence that it is involved in controlling the from storage sites within the fibres amount of Ca2+ which is released subsequently [12]. This is an important point to consider, because free Ca2+ not only activates contraction, by combining with troponin C, but also influences cyclic nucleotide metabolism, via its effects on calmodulin. The Ca2+-activated form of calmodulin is an allosteric effector of cyclic nucleotide phosphodiesterases and of adenylate cyclase too [4, 8, 471. It stimulates the hydrolysis of both cyclic GMP and cyclic AMP, the former more so than the latter, and also enhances the activity of adenylate cyclase, resulting in an increase in the relative proportion of cyclic AMP:cyclic GMP. It can therefore be inferred that de-activation of calmodulin, under conditions of diminished Ca2+ availability, might have the reverse effects on cyclic nucleotide levels, producing changes which are qualitatively simiIar to those seen in the present study. Microelectrode recordings were not made during this investigation and so it is not known whether the reported effects of acetylcholine and 8-bromo cyclic GMP on the action potential actually precede changes in cyclic nucleotide levels; if they do not, then it can be argued that they may arise as a consequence of the changes in cyclic AMP and/or cyclic GMP, a point which is taken up again later.
The ability of theophylline to antagonize
responses to ace2ylcholine and 8-bromo cyclic GA4P
The use of theophylline as a means of identifying a possible site of action for cyclic GMP in the regulation of cyclic AMP metabolism requires some comment, since a number of authors have questioned its specificity as an inhibitor of phosphodiesterase activity [Z, 251. There is no doubt that it does have other effects, but it is less clear whether these should be regarded as ‘Lnon specific”, or if they should instead be thought of as arising directly from its ability to inhibit phosphodiesterase activities. The positive inotropic action of theophylline on the frog heart has been attributed to increased accumulation of intracellular Ca2+ [29]; similarly, the inotropic effect of caffeine, a related methylxanthine, on the toad heart appears to be due to increased mobilization of cellular Ca2+ [34]. It is to be expected of course that inhibition of phosphodiesterase activity by theophylline would initially increase intracellular cyclic AMP and cyclic GMP levels. Cyclic AMP is known to activate protein kinases which phosphorylate several regulatory proteins, including two that control Ca2+ movements, across the fibre membrane [24, 261 and between the sarcoplasmic reticulum and the myoplasm [22, 461 and one which appears to modulate the sensitivity of the contractile system to Ca2+ [9, II, 32, 41, 431. Since the regulatory capacities of all three proteins depend critically upon their state of phosphorylation, it is not surprising to observe what may appear to be “non specific” effects, such as those described above, but which are nevertheless secondary to a specific inhibition by theophylline of cyclic nucleotide phosphodiesterases. The possible involvement of calmodulin in mediating the effects of acetylcholine and of S-bromo cyclic GMP on the ventricle (discussed above) may also be relevant in connection with the experiments using theophylline. On the one hand, theophylline would be expected to increase cellular cyclic AMP and cyclic GMP, but there are reasons to suppose that this might be offset later by the activation of calmodulin, resulting from enhanced Ca2+ entry. Activation of calmodulin by Ca2+ would stimulate phosphodiesterase activity, and result in a lowering of cyclic nucleotide levels.
976
F. W. Flitney
and J. Singh
Some evidence that this may be happening is shown by the results presented in Tables 3 and 4. Preparations exposed to theophylline for 15 min had levels of cyclic AMP and cyclic GMP which were significantly lower than those found in ventricles not treated with theophylline (see controls in Tables 1 and 2, columns 3 and 7). It is difficult to explain this observation, other than to suppose that theophylline has primary and secondary effects on cyclic nucleotide metabolism. The situation might be clarified by studying the time course of changes in cyclic nucleotide levels and in action potential parameters during responses to theophylline alone; such experiments are currently in progress. Relationship
between changes in contractility
and cyclic nucleotide levels
The results of this investigation (and of others published elsewhere; see Introduction for references) reinforce the view that the contractile performance of the frog ventricle may be regulated, at any given moment, by the relative amounts of cyclic AMP: cyclic GMP present in its fibres. If this hypothesis turns out to be correct, then it follows that the two cyclic nucleotides must function antagonistically, cyclic AMP acting to potentiate contraction, and cyclic GMP exerting a counter-effect. It was pointed out earlier (p. 975) that cyclic AMP stimulates the phosphorylation of several proteins thought to be involved in regulating contraction and that this then alters their physiological properties. The role of cyclic GMP is much less clear and the literature abounds with contradictory evidence (see, for example [27]}. Indeed, there is as yet no concensus of opinion concerning even its involvement in regulating contraction. This is clearly an important issue, and one which is unlikely to be resolved by considering the relative importance of the two cyclic nucleotides in isolation from each other. The danger of attempting to do so was clearly recognized by Goldberg and his colleagues [19] who write that “if cyclic AMP and cyclic GMP of one to the other act in opposition to one another then . . . the relative proportion under certain circumstances may be more important than the absolute change in the concentration of only one of the components”. The present study and other reports which have appeared in the literature, suggesting that there may be a degree of interaction between the metabolism of the two cyclic nucleotides [I, 3, 10, 17, 38, 40, 42, 441, serves to reinforce this view. The apparent ability of cyclic GMP to stimulate the degradation of cyclic AMP affords one means whereby it could exert a regulatory influence on contraction but there are grounds for arguing that this cannot be the sole basis for its antagonistic effects. The reason is that correlations between changes in contractility and in either cyclic AMP or cyclic GMP are invariably less precise than those obtained by considering changes in the ratio cyclic AMP:cyclic GMP. If the depressant effect of cyclic GMP upon cyclic AMP levels was the only basis for the antagonistic effects of the two cyclic nucleotides, then the contractile response would be expected to correlate most closely with changes in intracellular cyclic AMP alone. These considerations led us to postulate earlier that cyclic GMP may also be involved in modulating the de-phosphorylation of regulatory proteins, perhaps by enhancing protein phosphatase activity [16, 371. Acknowledgements The authors thank The Wellcome Trust, Medical Heart Foundation for grants (to FWF) in support
Research Council of this work.
and .the British
Cyclic
GMP
may Regulate
Cyclic
AMP
in Frog Ventricle
977
REFERENCES 1.
2.
AMER, M. S. & KREIGHBAUM, W. E. Cyclic nucleotide phosphodiesterases: properties, activators, inhibitors, structure-activity relationships, and possible role in drug development. Journal of Pharmaceutical Science 64, ,l-37 (1975). ARGEL, M. I., VITTONE, L., GRASSI, A. O., CHIAPPE, L. E., CHIAPPE, G. E. & CINGOLANI, H. E. Effects of phosphodiesterase inhibitors on heart contractile behaviour, protein kinase activity and cyclic nucleotide levels. Journal of Molecular and Cellular Cardiology 12,
939-954 3.
4.
5. 6.
7. 8.
9. 10.
11.
12.
13. 14.
15.
16.
17.
18. 19.
( 1980).
BEAVO, J. A., HARDMAN, J. G. & SUTHERLAND, E. W. Stimulation of adenosine 3’, 5’monophosphate hydrolysis by guanosine 3’, 5’-monophosphate. Journal of Biological Chemistry 246, 3841-3846 (1971). BR~STR~M, C. O., HUANG, Y. C., BRECKENRIDGE, G. McL. & WOLFF, D. J. Identification of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase. Proceedings of the National Academy of Sciences of the U.S.A. 72, 64-68 (1975). BROWN, J. H. Adrenergic inhibition of catecholamine-stimulable cyclic AMP accumulation in murine atria. .Journal of Cyclic Nxleotide Research 5, 423-433 (1979). BEVERS, M. M., SMITS, R. A. E., VAN RIJN, J. & VAN WIJK, R. Heat treatment of rat liver adenosine 3’5’ cyclic monophosphate phosphodiesterase. Biochimica et biophysics actu 341, 120-128 (1974). CHAPMAN, R. A. R. Excitation-contraction coupling in cardiac muscle. Progress in Biophysics and Molecular Biology 35, l-52 (1979). CHEUNG, W. Y., LYNCH, T. J. & WALLACE, R. W. An endogenous Ca2+-dependent activator protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase. Advances in Cyclic Nucleotide Research 9, 233-251 (1978). COLE, H. A. & PERRY, S. V. The phosphorylation of troponin I from cardiac muscle. Biochemical Journal 149, 525-533 (1975). ENDOH, M. Correlation of cyclic AMP and cyclic GMP levels with changes in contractile force of dog ventricular myocardium during cholinergic antagonism of positive inotropic actions of histamine, glucagon, theophylline and papaverine. Japanese Journal of Pharmacolopy 29, 855-864 (1979). ENGLAND, P. J. Studies on the phosphorylation of the inhibitory subunit of troponin during modification of contraction in perfused rat heart. Biochemical Journal 160, 295-304 (1976). FABIATO, A. & FABIATO, F. Calcium-induced release of calcium from the sarcoplasmic reticulum of skinned cells from adult human, dog, cat, rabbit, rat and frog hearts and from foetal hearts and new born rat ventricles. Annals qf the New York Academy of Sciences 307, 491-522 (1978). FLITNEY, F. W., MOSHIRI, M. & SINGH, J. Effects of sodium nitroprusside on frog ventricle. Journal qf Physiology 305, 25-26P Abstract (1980). FLITNEY, F. W. & SINGEI, J. Depressant effect of 8-bromo guanosine 3’, 5’-cyclic monophosphate on endogenous adenosine 3’, 5’-cyclic monophosphate levels in frog ventricle. Journal of Phvsiologv 302, 29-30P Abstract (1980). FLITNEY, F. W. & SINGH, J. Exogenous uridine 5’-triphosphate enhances contractility and stimulates 3’, 5’-cyclic nucleotide metabolism in the isolated frog ventricle. Journal of Physiology 291, 52-53P Abstract (1979). FLITNEY, F. W. & SINGH, J. Inotropic responses of the frog ventricle to adenosine triphosphate and related changes in endogenous cyclic nucleotides. Journal of Physiology 304, 2142 (1980). FLITNEY, F. W. & SINGH, J. Release of prostaglandins from the isolated frog ventricle and associated changes in endogenous cyclic nucleotide levels. Journal of Physiology 304, I-20 (1980). GILES, W. & NOBLE, S. J. Changes in membrane currents in bullfrog atrium produced by acetylcholine. Journal of Physiology 261, 103-123 (1976). GOLDBERG, N. C., HADDOX, M. K., NICOL, S. E., GLASS, G. B., SANDFORD, C. H., KUEHL, F. A. & ESTENSEN, R. Biological regulation through opposing influences of cyclic AMP and cyclic GMP: The Yin Yang hypothesis. Advances in Cyclic Nucleotide Research 5, 307-330 (1975).
978 20. 2 1. 22. 23.
24.
25. 26. 27. 28.
29.
30. 31.
32.
33. 34. 35.
36.
37.
38.
39.
40.
41. 42.
F. W. Flitney
and J. Singh
GORNALL, A. G., BARDAWILL, D. J. & DAVID, M. M. Determination of serum proteins by means of the Biuret reaction. Journal of Biological Chemistry 177, 751-766 (1949). HIDAKA, H. & ASANO, T. Human blood platelet 3’5’ cyclic nucleotide phosphodiesterase. Biochimica et biophysics acta 429, 485-497 (1976). KATZ, A. M., TADA, M. & KIRCHBERGER, M. A. Control of calcium transport by cyclic AMP-protein kin&e. Advances in Cyclic Jfucleotide Research 5, 453-472 (1975). KOHLHARDT, M. & HAAP, K. 8-Bromo-guanosine-monophosphate mimics the effects of acetylcholine on slow response action potential and contractile force in mammalian atria1 myocardium. Journal of Molecular and Cellular Cardiology 10, 573-586 (1978). KRAUSE, E. G., WILL, H., SCHIRPKE, B. & WOLLENBERGER, A. Cyclic AMP enhances protein phosphorylation and calcium binding in a cell membrane-enriched fraction from myocardium. Advances in Cyclic Nucleotide Research 5, 473490 (1975). KRAUSE, E. G. & WOLLENBERGER, A. Cyclic nucleotides and heart. In Cyclic Nucleotides: Mechanism of Action, H. Cramer & J. Schultz, Eds, pp. 229-250. New York: Wiley (1976). LAMB, J. F. & MCGUIGAN, J. A. S. Contractures in superfused frog ventricle. Journal of Physiology 186, 261-283 (1966). LINDEN, J. & BROOKER, G. The questionable role of cyclic guanosine 3’5’ monophosphate in heart. Biochemical Pharmacology 28, 3351-3360 (1979). MARCUS, M. L., SKELTON, C. L., PRINDLE, K. H. & EPSTEIN, S. E. Potentiation of the inotropic effects of glucagon by theophylline. Journal of Pharmacolo@ and Experimental Therapeutics 179, 331-337 (1971). MASSINGHAM, R. & NASMYTH, P. A. The nature of the positive inotropic response of the isolated frog heart to theophylline and to imidazole. British Journal of Pharmacology 45, 229-239 (1972). MEANS, A. R. & DEDMAN, J. R. Calmodulin-an intracellular calcium receptor. Nature 285, 73-77 (1980). MOPE, L., MCCLELLAN, G. B. & WINEGRAD, S. Calcium sensitivity of the contractile system and phosphorylation of troponin in hyperpermeable cardiac cells. Journal of General Physiology 75, 271-282 (1980). MILLER, J. P., BOSWELL, K. H., MUNEYAMA, K., SIMON, L. N., ROBINS, R. K. & SHUMAN, D. A. Synthesis and biochemical studies of various 8-substituted derivatives of guanosine 3’, 5’, cyclic phosphate, inosine 3’5’ cyclic phosphate and xanthesine 3’5’ cyclic phosphate. Biochemistry 12, 5310-5319 (1973). NAWRATH, H. Cyclic AMP and cyclic GMP play opposing roles in influencing force of contraction in mammalian myocardium. Nature 262, 509-511 (1976). NAYLER, N. G. Effect of caffeine on cardiac contractile activity and radiocalcium movement. American Journal of Physiology 204, 969-974 ( 1963). RALL, T. W. & WEST, T. C. The potentiation of cardiac inotropic responses to norepinephrine by theophylline. Journal of Pharmacology and Experimental Therapeutics 139, 269-274 (1963). RUSSELL, T. R., TERESAKI, W. L. & APPLEMAN, M. M. Separate phosphodiesterases for the hydrolysis of cyclic adenosine 3’5’ monophosphate and cyclic guanosine 3’5’ monophosphate in rat liver. Journal of Biological Chemistry 248, 1334-1340 (1973). SINGH, J. & FLITNEY, F. W. Adenosine depresses contractility and stimulates 3’, 5’cyclic nucleotide metabolism in the isolated frog ventricle. Journal of Molecular and Cellular Cardiology 12, 285-289 (1980). SINGH, J. & FLITNEY, F. W. Inotropic responses of the frog ventricle to dibutyryl cyclic AMP and 8-Bromo cyclic GMP and related changes in endogenous cyclic nucleotide levels. Biochemical Pharmacology 30, 1475-1481 (1981). SINGH, J., FLITNEY, F. W. & LAMB, J. F. Effects of isoprenaline on contractile force and intracellular cyclic 3’, 5’ nucleotide levels in the hypodynamic frog ventricle. FEBS Letters 91, 269-272 (1978). STEFANOWCH, V. Cyclic nucleotide phosphodiesterase inhibitors as potential therapeutic agents. In Advances in Biosciences 24, G. Cehovic & G. A. Robison, Eds. Oxford: Pergamon Press (1979). STULL, J. T. Phosphorylation of contractile proteins in relation to muscle function. Advances in Cyclic .Nrxleotide Research 13, 39-93 (1980). TERESAKI, W. L. & APPLEMAN, M. M. The role of cyclic GMP in regulation of cyclic AMP hydrolysis. Metabolism 24, 311-319 (1975).
Cyclic 43. 44.
45. 46.
47.
GMP
may
Regulate
Cyclic
AMP
in Frog
Ventricle
979
TSIEN, R. W. Cyclic AMP and contractile activity in the heart. Advances in Cyclic Nucleotide Research 8, 363-419 (1977). WEISS, B. & HAIT, W. N. Selective cyclic nucleotide phosphodiesterase inhibitors as potential therapeutic agents. Annual Review of Pharmacology and Toxicology 17, 441-477 (1977). WELLS, J. N. & HARDMAN, J. G. Cyclic nucleotide phosphodiesterases. Advances in Cyclic Nucleotide Research 8, 119-143 (1979). WILL, H., MISSELWITZ, H.J., LEUVHENKO, T. S. & WOLLENBERGER, A. Some characteristics of low molecular weight phosphoprotein constituents of cardiac sarcoplasmic reticulum and sarcolemma. In Advances in PharmacoloD and Therapeutics 3, J. C. Stoclet, Ed., pp. 161-170. Oxford: Pergamon Press (1978). WOLFF, D. J. & BROSTROM, C. 0. (1979). Properties and functions of the calciumdependent regulator protein. Advances in Cyclic Nucleotide Research 11, 28-88 (1979).