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The effect of trypsin on rate, force and cyclic AMP in guinea pig atria
European Journal of Pharmacology, 74 ( 1981) 95- 99 Elsevier/North-Holland Biomedical Press
95
Short communication T H E E F F E C T OF TRYPSIN ON RATE, FORCE AND CYCLIC AMP IN GUINEA PIG ATRIA * G E R A R D H. CROS *, JEAN-JACQUES SERRANO * and JOHN H. MCNEILL **
Division of Pharmacologl~ and Toxicology, Faculty of Pharma~'eutical Sciences, University of British Columbia, Vancouver, Canada VrT 1W5 Received 12 March 1981, revised MS received 16 June 1981, accepted 6 July 198]
G.H. CROS, J.J. SERRANO and J.H. MCNEILL, The effect of trypsin on rate, force and o'clic AMP in guinea pig atria, European J. Pharmacol. 74 (1981) 95-99. Trypsin (10-6 M) produced a positive inotropic and chronotropic effect in left and right atria respectively and an increase in cyclic AMP. The effects were blocked by aprotinine while propranolol, phentolamine, promethazine and cimetidine and reserpine pretreatment did not alter trypsin activity. Trypsin effects were potentiated by RO 20,1724, a phosphodiesterase inhibitor. The cardiac effects of trypsin may be due to increases in cyclic AMP and are in agreement with previous work indicating that trypsin can activate cardiac adenylate cyclase.
Trypsin
Aprotinine
Heart
Cyclic AMP
1. Introduction
2. Materials and methods
Cardiac levels of cyclic AMP have been shown to increase prior to, or concomitant with, an increase in force of contraction following the administration of various cardiotonic agents such as adrenergic amines, histamine and glucagon in various species (see McNeill, 1979). Recent work from our laboratory has demonstrated that trypsin is capable of stimulating adenylate cyclase prepared from dog ventricle (Cros et al., 1980). In the present study we have examined the effects of trypsin on rate and force in guinea pig right and left atria and have attempted to Correlate these changes with the tissue levels of cyclic AMP.
2.1. Preparation of atria
t Supported by grants from MRC and CHF. * NSERC, Canada-France Exchange Scientist; present address: Laboratoire de Pharmacodynamie, Facult6 de Pharmacie, Universit6 de Montpellier, 34060 Montpellier, France. ** To whom all correspondence should be addressed.
Guinea pigs of either sex weighing between 300 and 500 g were killed by a blow on the head and the heart rapidly removed and placed in a beaker containing Chenoweth-Koelle (CKS) solution bubbled with 95% 02 and 5% CO 2 of the following composition (mM): NaCI 120; KC1 5.63; CaC12 2.0; dextrose 9.7; MgC12 2.0; N a H C O 3 26.0. Right and left atria were removed from the heart and mounted as described previously (Tenner and McNeill, 1978). Right atria were allowed to beat spontaneously, while left atria were paced at a frequency of 1.8 Hz, with a duration of 5 msec and a voltage twice the threshold by means of a Grass stimulator. Responses were recorded on a Grass polygraph. The tissues were allowed to equilibrate for 45 min after the basal tension had been adjusted to 1 g and were washed every 15 min. Drugs were added directly to the bath. Right atria were used to measure the effects of drugs on rate and left atria used to measure the effects of drugs on tension.
0014-2999/81/0000-0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press
96
2.2. Experimental design
2.3. Cycfic A M P measurements
For the study of the effects of trypsin alone, the tension and rate were recorded for 10 min after trypsin administration. For the experiment concerning aprotinine and trypsin, aprotinine was first administered and left in the bath for 10 min. It produced no effect. Trypsin was then added to the bath and left for 5 min. The preparation was then washed twice at intervals of 10 min and trypsin was tested again. The influence of trypsin on histamine and isoprenaline activities on left atria was measured as follows: after having tested histamine (3 X 10 -6 M) and isoprenaline (10 -8 M), the preparation was washed several times for 30 min and then trypsin (10-6 M) was tested. When the trypsin effect was maximum the preparation was washed and histamine or isoprenaline were tested again. Reserpinetreated animals received a single intraperitoneal injection of reserpine (5 mg/kg), 2 4 h before sacrifice. The influence of different antagonists on trypsin activity was tested with propranolol (10-7 M) on left and right atria, phentolamine (10 6 M) on left atria, cimetidine (3X 10 -5 M) on right atria and promethazine (3 x 10-5 M) on left atria. The efficiency of blockade of fl-, a-, H 2- and H~receptors was checked with two injections of the corresponding agonist, i.e. isoprenaline (10 -8 M), methoxamine (10 -6 M), and histamine (3X 10 - 6 M ) , 30 min before and 10 min after the administration of blocking agent. In each case the blockade was complete. The preparation was washed after the first agonist administration, but not after the second one. Trypsin (10 - 6 M) was administered immediately after the second administration of agonist and the effect was recorded for 10 min. RO-20,1724 (10 -5 M) was added to the preparation and led to an increase of both tension and rate. When the increases had been stabilized for 10 min trypsin (10 - 6 M) was added without washing the bath and the further increase in activity was measured.
Trypsin was added to the bath containing the paced left atria. At the time of the maximum effect, the atria were frozen with clamps that had been previously cooled in liquid nitrogen. Control atria, without any drug added, were frozen under the same experimental conditions. The atria thus obtained were stored at - 8 0 ° C until analyzed for cyclic AMP. Cyclic AMP was measured by radioimmunoassay using a cyclic AMP kit obtained from Beckton Dickinson. Drug effects were measured as percent of increase above basal activity and are expressed as mean__+S.E.M. The Student's t-test for unpaired data was used to test significance. Differences were considered significant when P < 0.05.
3. Results
Different doses of trypsin were tested on right and left atria in an attempt to determine a doseeffect relationship. However a dose-effect relationship could not be established. Concentrations of trypsin up to 1 0 - 6 M did not produce an effect while 10 -6 M and 3 X 10 -6 M produced the same response. Trypsin could not be dissolved at concentrations higher than 3 X 1 0 - 6 M . A further problem was encountered due to trypsin desensitization. Following a trypsin response the tissue either did not respond to a second exposure to trypsin or responded to a much lower extent. Therefore cumulative dose response curves could not be done. Thus all results presented will be those obtained following the administration of a single 1 0 - 6 M dose of trypsin with the exception of the trypsin-aprotinine experiments. Trypsin possesses both a positive chronotropic and inotropic effect. The effects started after a latency period of 20-40 sec and the maximum effect was obtained between 90-150 sec after trypsin administration. After a short plateau, force and rate decreased gradually to reach the basal levels within approximately 8 min after trypsin administration. Under similar conditions drugs such as histamine or isoprenaline produced effects within 10 sec with maximum responses occurring in 30-40
97 TABLE 1 Effect of trypsin on force and rate in isolated guinea pig left and right atria. Results are expressed as % increase over control. N u m b e r s in parentheses indicate the number of atria tested. Trypsin treatment plus:
% Increase in force
% Increase in rate
0 Cimetidine Propranolol Phentolamine Reserpine pretreatment Promethazine Aprotinine Aprotinine-wash, plus trypsin RO-20,1724
26.5 33.6 30.0 23.2 32.7 2.0
2.3 (3) 8.7 (3) 4.4 (5) 2.6 (4) 1.2 (5) i
18.0 16.3 18.7 13.5 2.8
2.0 (6) 1.4 (3) 1.1 (4)
24.8 55.0
4.4 (5) 2 6.1 (6)~
15.5 25.0
3.3 (4) 2 1.9(4) i
2.0 (15)
1.2 (4) 1.0 (5) t
1 Significantly different from trypsin alone P<0.05. 2 Significantly different from trypsin plus aprotinine P<0.05.
sec. In 50% of the experiments the rate and tension stabilized at less than the basal level. If the preparation was washed within 3 min following trypsin administration, the rate and tension stabilized at the basal level. The trypsin-induced increase, expressed in percent increase above the basal levels of rate was 18.0-q-2.0 (n = 6) and the increase of tension was 26.5 ± 2.0 (n = 15). In order to analyze the mechanism of action of trypsin a natural protease inhibitor, aprotinine, as well as several receptor blocking agents were tested for their ability to modify trypsin activity (table 1). The chronotropic or inotropic effects of trypsin were blocked only by aprotinine ( 2 X 1 0 - 6 M ) . When the preparation containing both aprotinine and trypsin was washed, a second administration of trypsin was found to be as potent as the first one without aprotinine. The blocking agents propranolol ( 1 0 - 7 M ) , phentolamine ( 1 0 - 6 M ) and promethazine (3 X 10-5 M) were tested on the left atria because of the known presence of fl-, a- and H l-receptors mediating an inotropic response on left atria (Tenner and McNeill, 1978; McNeill, 1979) in order to determine if the inotropic action of trypsin could be decreased. None of the antagonists was effective. Similarly, the blocking agents propranolol (10 7 M) and cimetidine (3× 10 5 M) were tested on right atria because of the known presence of fl- and H2-receptors mediating chronotropic responses (McNeill, 1979) to see if
the trypsin response was affected. Neither agent decreased the trypsin response. Reserpinization of the animals also did not alter the chronotropic or inotropic effect of trypsin. RO-20,1724, a phosphodiesterase inhibitor, enhanced both the inotropic and chronotropic response to trypsin. In order to check the specificity of trypsin and to determine if trypsin had impaired the ability of the atria to respond, isoprenaline (10 -8 M) and histamine (3X10 -6 M) were tested on left atria before and after exposure to trypsin ( 1 0 - 6 M ) . Trypsin did not alter the inotropic response to either amine. Before trypsin histamine increased force 54+__17% while isoprenaline increased force by 143__+13%. After trypsin the corresponding changes were 5 3 ~ 11% and 150 + 15% (n=3). Since trypsin had been shown to stimulate cardiac adenylate cyclase the effect of trypsin on left atrial cyclic AMP levels was determined. Trypsin nearly doubled the concentration of cyclic AMP in left atria. Values were 390 ± 18 f m o l / m g wet weight in control atria and 730___48 f m o l / m g wet weight in trypsin treated atria, a significant difference.
4. Discussion
In the present study the effect of trypsin on isolated guinea pig atria was studied in order to
98
determine if it could produce a positive inotropic and chronotropic effect. Earlier work from our laboratory had established that trypsin could stimulate adenylate cyclase prepared from dog heart (Cros et al., 1980) and it was reasoned that trypsin should increase cyclic AMP in isolated heart preparations. Trypsin was shown to produce both an increase in rate in right atria and in force in left atria (table 1). The results indicated that the response was direct and relatively specific since it was not blocked by a-, fl-, H 1- or H2-antagonists and was not prevented by reserpine pretreatment. Trypsin therefore did not directly activate any of the above receptors and also did not produce its cardiac effects through the release of adrenergic amines or histamine. Evidence was obtained that the cardiac actions of trypsin were mediated through the stimulation of adenylate cyclase. We have previously shown that trypsin will stimulate the enzyme in a dog heart preparation (Cros et al., 1980) and in the present study it was found that trypsin could increase cyclic AMP levels in atria and that the cardiac effects of trypsin were enhanced by pretreatment with the phosphodiesterase inhibitor, RO-20,1724. Trypsin effects were blocked by aprotinine a natural, non-specific protease inhibitor. Rapid desensitization to trypsin occurred and tachyphylaxis was essentially complete after on exposure unless the effect of the first exposure was blocked by aprotinine. The possibility that the effect of trypsin was related to proteolysis was considered because of the above findings. It should be noted that the desensitization effect is quite specific since the response to both histamine and isoprenaline was not affected after exposure to trypsin. The effect of trypsin on dog heart adenylate cyclase is biphasic on both washed particle preparations (Cros et al., 1980) and on purified sarcolemma preparations (Cros and Katz, unpublished observations). The effect is dependent on both trypsin concentration and length of incubation. At relatively low concentrations of trypsin for short incubation times a stimulatory effect on adenylate cyclase is noted. Increasing the concentration or time of incubation results in enzyme inhibition. It has been suggested that proteases
may produce these actions in one or two ways. The first is by a non-specific limited proteolysis of receptors or of the catalytic subunit (GuiraudSimplot and Colobert, 1966; Hanoune et al., 1977). The second hypothesis suggests a more specific mechanism such as (a) the destruction of a regulatory protein such as GTP dependent regulatory protein (Anderson et al., 1977), (b) modification of the catalytic unit or a protein closely associated with this unit (Stengel et al., 1980) or (c) the activation of membrane proteases resulting in a link between the regulatory and catalytic subunits of adenylate cyclase resulting in activation by exogenous proteases (Richert and Ryan, 1977). Our results do not permit us to choose between the various hypotheses. The data do indicate, however, that the effect of trypsin is a relatively specific one and that other receptors are not inactivated by exposure to the protease. The inhibitory effect of trypsin on adenylate cyclase is probably a nonspecific effect perhaps due to destruction of membrane proteins, particularly those of the catalytic subunit (Wallach et al., 1977). It is also possible that trypsin initially stimulates and then destroys a site on the regulatory subunit.
References Anderson, W.B., C.J. Jaworski and G. Vlahakis, 1979, Proteolyric activation of adenylate cyclase from cultured fibroblasts, J. Biol. Chem. 253, 2921. Cros, G., S. Katz and J.H. McNeill, 1980, Inhibition of cyclic AMP accumulation in dog heart washed particle preparations by aprotinine, Res. Commun. Chem. Pathol. Pharmacol. 28, 255. Guiraud-Simplot, A. and L. Colobert, 1977, Adenylate cyclase activation by trypsin in KB cell cultures, Experientia 33, 899. Hanoune, J., D. Stengel, M.L. Lacombe, G. Feldmann and E. Coudrier, 1977, Proteolytic activation of rat liver adenylate cyclase by a concomitant of crude collagenase from clostridium histolyticum, J. Biol. Chem. 252, 2039. McNeill, J.H., 1979, Cyclic AMP and myocardial contraction, in: Trends in Automatic Pharmacology (S. Kalsner, ed.) 1, 421. Richert, N.D. and R.J. Ryan, 1977, Protease inhibitors block activation of adenylate cyclase, Biochem. Biophys. Res. Commun. 78, 799. Stengel, D., P.M. Lad, T.B. Nielsen, M. Rodbell and J. Hanoune, 1980, Proteolysis activates adenylate cyclase in rat liver and A c - lymphoma cell independently of the
99 guanine nucleotide regulatory site, FEBS Letters 115, 260. Tenner, T.E. and J.H. McNeill, 1978, Characterization of the inotropic response induced by stimulation of/3 adrenergic and H 1 histaminergic receptors in guinea pig left atria, Can. J. Physiol. Pharmacol. 56, 926.
Wallach, D., W. Anderson and I. Pastan, 1978, Activation of adenylate cyclase in cultured fibroblasts by trypsin, J. Biol. Chem. 253, 24.
95
Short communication T H E E F F E C T OF TRYPSIN ON RATE, FORCE AND CYCLIC AMP IN GUINEA PIG ATRIA * G E R A R D H. CROS *, JEAN-JACQUES SERRANO * and JOHN H. MCNEILL **
Division of Pharmacologl~ and Toxicology, Faculty of Pharma~'eutical Sciences, University of British Columbia, Vancouver, Canada VrT 1W5 Received 12 March 1981, revised MS received 16 June 1981, accepted 6 July 198]
G.H. CROS, J.J. SERRANO and J.H. MCNEILL, The effect of trypsin on rate, force and o'clic AMP in guinea pig atria, European J. Pharmacol. 74 (1981) 95-99. Trypsin (10-6 M) produced a positive inotropic and chronotropic effect in left and right atria respectively and an increase in cyclic AMP. The effects were blocked by aprotinine while propranolol, phentolamine, promethazine and cimetidine and reserpine pretreatment did not alter trypsin activity. Trypsin effects were potentiated by RO 20,1724, a phosphodiesterase inhibitor. The cardiac effects of trypsin may be due to increases in cyclic AMP and are in agreement with previous work indicating that trypsin can activate cardiac adenylate cyclase.
Trypsin
Aprotinine
Heart
Cyclic AMP
1. Introduction
2. Materials and methods
Cardiac levels of cyclic AMP have been shown to increase prior to, or concomitant with, an increase in force of contraction following the administration of various cardiotonic agents such as adrenergic amines, histamine and glucagon in various species (see McNeill, 1979). Recent work from our laboratory has demonstrated that trypsin is capable of stimulating adenylate cyclase prepared from dog ventricle (Cros et al., 1980). In the present study we have examined the effects of trypsin on rate and force in guinea pig right and left atria and have attempted to Correlate these changes with the tissue levels of cyclic AMP.
2.1. Preparation of atria
t Supported by grants from MRC and CHF. * NSERC, Canada-France Exchange Scientist; present address: Laboratoire de Pharmacodynamie, Facult6 de Pharmacie, Universit6 de Montpellier, 34060 Montpellier, France. ** To whom all correspondence should be addressed.
Guinea pigs of either sex weighing between 300 and 500 g were killed by a blow on the head and the heart rapidly removed and placed in a beaker containing Chenoweth-Koelle (CKS) solution bubbled with 95% 02 and 5% CO 2 of the following composition (mM): NaCI 120; KC1 5.63; CaC12 2.0; dextrose 9.7; MgC12 2.0; N a H C O 3 26.0. Right and left atria were removed from the heart and mounted as described previously (Tenner and McNeill, 1978). Right atria were allowed to beat spontaneously, while left atria were paced at a frequency of 1.8 Hz, with a duration of 5 msec and a voltage twice the threshold by means of a Grass stimulator. Responses were recorded on a Grass polygraph. The tissues were allowed to equilibrate for 45 min after the basal tension had been adjusted to 1 g and were washed every 15 min. Drugs were added directly to the bath. Right atria were used to measure the effects of drugs on rate and left atria used to measure the effects of drugs on tension.
0014-2999/81/0000-0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press
96
2.2. Experimental design
2.3. Cycfic A M P measurements
For the study of the effects of trypsin alone, the tension and rate were recorded for 10 min after trypsin administration. For the experiment concerning aprotinine and trypsin, aprotinine was first administered and left in the bath for 10 min. It produced no effect. Trypsin was then added to the bath and left for 5 min. The preparation was then washed twice at intervals of 10 min and trypsin was tested again. The influence of trypsin on histamine and isoprenaline activities on left atria was measured as follows: after having tested histamine (3 X 10 -6 M) and isoprenaline (10 -8 M), the preparation was washed several times for 30 min and then trypsin (10-6 M) was tested. When the trypsin effect was maximum the preparation was washed and histamine or isoprenaline were tested again. Reserpinetreated animals received a single intraperitoneal injection of reserpine (5 mg/kg), 2 4 h before sacrifice. The influence of different antagonists on trypsin activity was tested with propranolol (10-7 M) on left and right atria, phentolamine (10 6 M) on left atria, cimetidine (3X 10 -5 M) on right atria and promethazine (3 x 10-5 M) on left atria. The efficiency of blockade of fl-, a-, H 2- and H~receptors was checked with two injections of the corresponding agonist, i.e. isoprenaline (10 -8 M), methoxamine (10 -6 M), and histamine (3X 10 - 6 M ) , 30 min before and 10 min after the administration of blocking agent. In each case the blockade was complete. The preparation was washed after the first agonist administration, but not after the second one. Trypsin (10 - 6 M) was administered immediately after the second administration of agonist and the effect was recorded for 10 min. RO-20,1724 (10 -5 M) was added to the preparation and led to an increase of both tension and rate. When the increases had been stabilized for 10 min trypsin (10 - 6 M) was added without washing the bath and the further increase in activity was measured.
Trypsin was added to the bath containing the paced left atria. At the time of the maximum effect, the atria were frozen with clamps that had been previously cooled in liquid nitrogen. Control atria, without any drug added, were frozen under the same experimental conditions. The atria thus obtained were stored at - 8 0 ° C until analyzed for cyclic AMP. Cyclic AMP was measured by radioimmunoassay using a cyclic AMP kit obtained from Beckton Dickinson. Drug effects were measured as percent of increase above basal activity and are expressed as mean__+S.E.M. The Student's t-test for unpaired data was used to test significance. Differences were considered significant when P < 0.05.
3. Results
Different doses of trypsin were tested on right and left atria in an attempt to determine a doseeffect relationship. However a dose-effect relationship could not be established. Concentrations of trypsin up to 1 0 - 6 M did not produce an effect while 10 -6 M and 3 X 10 -6 M produced the same response. Trypsin could not be dissolved at concentrations higher than 3 X 1 0 - 6 M . A further problem was encountered due to trypsin desensitization. Following a trypsin response the tissue either did not respond to a second exposure to trypsin or responded to a much lower extent. Therefore cumulative dose response curves could not be done. Thus all results presented will be those obtained following the administration of a single 1 0 - 6 M dose of trypsin with the exception of the trypsin-aprotinine experiments. Trypsin possesses both a positive chronotropic and inotropic effect. The effects started after a latency period of 20-40 sec and the maximum effect was obtained between 90-150 sec after trypsin administration. After a short plateau, force and rate decreased gradually to reach the basal levels within approximately 8 min after trypsin administration. Under similar conditions drugs such as histamine or isoprenaline produced effects within 10 sec with maximum responses occurring in 30-40
97 TABLE 1 Effect of trypsin on force and rate in isolated guinea pig left and right atria. Results are expressed as % increase over control. N u m b e r s in parentheses indicate the number of atria tested. Trypsin treatment plus:
% Increase in force
% Increase in rate
0 Cimetidine Propranolol Phentolamine Reserpine pretreatment Promethazine Aprotinine Aprotinine-wash, plus trypsin RO-20,1724
26.5 33.6 30.0 23.2 32.7 2.0
2.3 (3) 8.7 (3) 4.4 (5) 2.6 (4) 1.2 (5) i
18.0 16.3 18.7 13.5 2.8
2.0 (6) 1.4 (3) 1.1 (4)
24.8 55.0
4.4 (5) 2 6.1 (6)~
15.5 25.0
3.3 (4) 2 1.9(4) i
2.0 (15)
1.2 (4) 1.0 (5) t
1 Significantly different from trypsin alone P<0.05. 2 Significantly different from trypsin plus aprotinine P<0.05.
sec. In 50% of the experiments the rate and tension stabilized at less than the basal level. If the preparation was washed within 3 min following trypsin administration, the rate and tension stabilized at the basal level. The trypsin-induced increase, expressed in percent increase above the basal levels of rate was 18.0-q-2.0 (n = 6) and the increase of tension was 26.5 ± 2.0 (n = 15). In order to analyze the mechanism of action of trypsin a natural protease inhibitor, aprotinine, as well as several receptor blocking agents were tested for their ability to modify trypsin activity (table 1). The chronotropic or inotropic effects of trypsin were blocked only by aprotinine ( 2 X 1 0 - 6 M ) . When the preparation containing both aprotinine and trypsin was washed, a second administration of trypsin was found to be as potent as the first one without aprotinine. The blocking agents propranolol ( 1 0 - 7 M ) , phentolamine ( 1 0 - 6 M ) and promethazine (3 X 10-5 M) were tested on the left atria because of the known presence of fl-, a- and H l-receptors mediating an inotropic response on left atria (Tenner and McNeill, 1978; McNeill, 1979) in order to determine if the inotropic action of trypsin could be decreased. None of the antagonists was effective. Similarly, the blocking agents propranolol (10 7 M) and cimetidine (3× 10 5 M) were tested on right atria because of the known presence of fl- and H2-receptors mediating chronotropic responses (McNeill, 1979) to see if
the trypsin response was affected. Neither agent decreased the trypsin response. Reserpinization of the animals also did not alter the chronotropic or inotropic effect of trypsin. RO-20,1724, a phosphodiesterase inhibitor, enhanced both the inotropic and chronotropic response to trypsin. In order to check the specificity of trypsin and to determine if trypsin had impaired the ability of the atria to respond, isoprenaline (10 -8 M) and histamine (3X10 -6 M) were tested on left atria before and after exposure to trypsin ( 1 0 - 6 M ) . Trypsin did not alter the inotropic response to either amine. Before trypsin histamine increased force 54+__17% while isoprenaline increased force by 143__+13%. After trypsin the corresponding changes were 5 3 ~ 11% and 150 + 15% (n=3). Since trypsin had been shown to stimulate cardiac adenylate cyclase the effect of trypsin on left atrial cyclic AMP levels was determined. Trypsin nearly doubled the concentration of cyclic AMP in left atria. Values were 390 ± 18 f m o l / m g wet weight in control atria and 730___48 f m o l / m g wet weight in trypsin treated atria, a significant difference.
4. Discussion
In the present study the effect of trypsin on isolated guinea pig atria was studied in order to
98
determine if it could produce a positive inotropic and chronotropic effect. Earlier work from our laboratory had established that trypsin could stimulate adenylate cyclase prepared from dog heart (Cros et al., 1980) and it was reasoned that trypsin should increase cyclic AMP in isolated heart preparations. Trypsin was shown to produce both an increase in rate in right atria and in force in left atria (table 1). The results indicated that the response was direct and relatively specific since it was not blocked by a-, fl-, H 1- or H2-antagonists and was not prevented by reserpine pretreatment. Trypsin therefore did not directly activate any of the above receptors and also did not produce its cardiac effects through the release of adrenergic amines or histamine. Evidence was obtained that the cardiac actions of trypsin were mediated through the stimulation of adenylate cyclase. We have previously shown that trypsin will stimulate the enzyme in a dog heart preparation (Cros et al., 1980) and in the present study it was found that trypsin could increase cyclic AMP levels in atria and that the cardiac effects of trypsin were enhanced by pretreatment with the phosphodiesterase inhibitor, RO-20,1724. Trypsin effects were blocked by aprotinine a natural, non-specific protease inhibitor. Rapid desensitization to trypsin occurred and tachyphylaxis was essentially complete after on exposure unless the effect of the first exposure was blocked by aprotinine. The possibility that the effect of trypsin was related to proteolysis was considered because of the above findings. It should be noted that the desensitization effect is quite specific since the response to both histamine and isoprenaline was not affected after exposure to trypsin. The effect of trypsin on dog heart adenylate cyclase is biphasic on both washed particle preparations (Cros et al., 1980) and on purified sarcolemma preparations (Cros and Katz, unpublished observations). The effect is dependent on both trypsin concentration and length of incubation. At relatively low concentrations of trypsin for short incubation times a stimulatory effect on adenylate cyclase is noted. Increasing the concentration or time of incubation results in enzyme inhibition. It has been suggested that proteases
may produce these actions in one or two ways. The first is by a non-specific limited proteolysis of receptors or of the catalytic subunit (GuiraudSimplot and Colobert, 1966; Hanoune et al., 1977). The second hypothesis suggests a more specific mechanism such as (a) the destruction of a regulatory protein such as GTP dependent regulatory protein (Anderson et al., 1977), (b) modification of the catalytic unit or a protein closely associated with this unit (Stengel et al., 1980) or (c) the activation of membrane proteases resulting in a link between the regulatory and catalytic subunits of adenylate cyclase resulting in activation by exogenous proteases (Richert and Ryan, 1977). Our results do not permit us to choose between the various hypotheses. The data do indicate, however, that the effect of trypsin is a relatively specific one and that other receptors are not inactivated by exposure to the protease. The inhibitory effect of trypsin on adenylate cyclase is probably a nonspecific effect perhaps due to destruction of membrane proteins, particularly those of the catalytic subunit (Wallach et al., 1977). It is also possible that trypsin initially stimulates and then destroys a site on the regulatory subunit.
References Anderson, W.B., C.J. Jaworski and G. Vlahakis, 1979, Proteolyric activation of adenylate cyclase from cultured fibroblasts, J. Biol. Chem. 253, 2921. Cros, G., S. Katz and J.H. McNeill, 1980, Inhibition of cyclic AMP accumulation in dog heart washed particle preparations by aprotinine, Res. Commun. Chem. Pathol. Pharmacol. 28, 255. Guiraud-Simplot, A. and L. Colobert, 1977, Adenylate cyclase activation by trypsin in KB cell cultures, Experientia 33, 899. Hanoune, J., D. Stengel, M.L. Lacombe, G. Feldmann and E. Coudrier, 1977, Proteolytic activation of rat liver adenylate cyclase by a concomitant of crude collagenase from clostridium histolyticum, J. Biol. Chem. 252, 2039. McNeill, J.H., 1979, Cyclic AMP and myocardial contraction, in: Trends in Automatic Pharmacology (S. Kalsner, ed.) 1, 421. Richert, N.D. and R.J. Ryan, 1977, Protease inhibitors block activation of adenylate cyclase, Biochem. Biophys. Res. Commun. 78, 799. Stengel, D., P.M. Lad, T.B. Nielsen, M. Rodbell and J. Hanoune, 1980, Proteolysis activates adenylate cyclase in rat liver and A c - lymphoma cell independently of the
99 guanine nucleotide regulatory site, FEBS Letters 115, 260. Tenner, T.E. and J.H. McNeill, 1978, Characterization of the inotropic response induced by stimulation of/3 adrenergic and H 1 histaminergic receptors in guinea pig left atria, Can. J. Physiol. Pharmacol. 56, 926.
Wallach, D., W. Anderson and I. Pastan, 1978, Activation of adenylate cyclase in cultured fibroblasts by trypsin, J. Biol. Chem. 253, 24.