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ATPase activity of pharmacological preparations
EUROPEAN JOURNAL OF PHARMACOLOGY 19 (1972) 137-139. NORTH-HOLLANDPUBLISHINGCOMPANY
Short communication
ATPASE ACTIVITY OF PHARMACOLOGICAL PREPARATIONS G.S. HARRIS Pharmacology Unit, Department of Physiology, University of Auckland, New Zealand
Accepted 19 April 1972
Received 20 March 1972
G.S. HARRIS, ATPase activity of pharmacological preparations, European J. Pharmacol. 19 (1972) 137-139. The ATPase activity of pharmacological preparations was tested in phosphate buffer to determine if the breakdown products of ATP contribute significantly to the responses observed to externally applied ATP. Large masses of tissues rapidly broke down ATP whereas the arterial preparations were the most active per unit mass. ATP ATPase activity
Arteries Atria
Ileum Vas deferens
1. INTRODUCTION
2. MATERIALS AND METHODS
The role of the plasma membrane ATPase has been generally accepted as related to cation transport (Skou, 1965), and a family of ATPase enzyme systems appear to be involved in the regulation of ion transport across cell membranes (Allen and Daniel, 1970). ATPase-dependent systems have also been suggested as being involved in the adrenergic receptor (Bloom and Goldman, 1965) and calcium control of membrane permeability (Abood and Matsubara, 1968). ATP, the substrate of the ATPase system has a variety of actions when applied externally to smooth muscle containing tissues, contracting vascular and uterine smooth muscle and relaxing intestinal smooth muscle (Hrdina et al., 1967; Daniel and Irwin, 1965; Axelsson and Holmberg, 1969). In view of these differences the activity of the ATPase system in tissues used as routine pharmacological preparations was studied in a physiological phosphate buffer using 32p-labelled ATP, since most previous studies have used phosphate free systems (Allen and Daniel, 1970).
2.1. General Tissues were isolated as for routine pharmacological preparations and suspended in McEwen (1956) solution. Aerated with 95% 02 and 5% CO2 and maintained at 37°C. 2.2. A TPase activity using 32p A TP ATPase acitivity was determined by the displacement of the 32p-ATP, to inorganic phosphate. A variety of tissues, guinea-pig vas deferens, rabbit central ear artery and aorta, guinea-pig ileum and rabbit left atria weighing 2 0 - 7 0 0 mg were prepared, as for an in vitro pharmacological preparation. Each tissue mass being approximately the mass used in in vitro pharmacological preparations. The tissues were incubated in McEwen buffer solution for 60 min, then added to buffer containing 20 ml 2/aCi (127/~Ci/mM) of 32p-isotopically-labelled ATP. Aliquots of 1.0 ml were taken before the addition of the tissue and after 1, 2, 4, 8, 16 and 32 min. Inorganic phosphate in the 1.0-ml aliquot was
138
G.S.Harris, A TPase activity of pharmacological preparations
separated from 3~ P-ATP by shaking with 2 ml of a vanadomolybdate complex the resulting phosphate complex being extracted into 10ml of butanol (Parvin and Smith, 1969). The butanol phase was separated, dried by gentle heating, 10 ml of scintillation mixture added and the radioactivity measured in a Packard Tricarb Scintillation Spectrometer Model 3384. In control studies isotopically labelled 32p sod i u m orthophosphate and 32p-labelled ATP were added to aliquots of McEwen solution and subjected to the extraction procedure. The recovery of 32p.phosphate and efficiency of separation from 32 P-ATP were estimated.
3. RESULTS The recovery of 32p-phosphate added to McEwen solution was incomplete and varied in different experiments from 15-32%. However, in the control solutions the recovery at each experiment only varied from 2.5 to 3.7% and the recovery was independent of the phosphate concentration of the buffer. When 32p-ATP was added to McEwen solution at the concentration used in the study 6.2% of the
100 -
Table 1 ATPase activity determined by 32p-release from 32P-3,-ATP for a series of tissues in phosphate buffer. Tissues
Artery
tlA ATP (min) hA ATP (min/ 100g)
118 26
Was
deferens 138 77
Atria
Ileum
30 152
10.2 69
added 32P-ATP was extracted and incubation for 30 min in buffer solution increased this to 11.7% 32 P-release from 32p.AT P by tissues. Incubation of tracer quantities of 32p-ATP with tissues in a series of 5 experiments increased the rate of release of isotopically labelled phosphate, fig. 1. Tissues of small mass, vas deferens and arterial segments released phosphate from ATP at a constant rate over the 30 min studied. Atria and ileum which have a mass 2 0 - 2 5 greater initially released a large amount of labelled phosphate. However, a progressive reduction in rate then occurred. Using the initial linear portion of the curves of ATP breakdown an approximate t~/2for ATP in these preparations was determined (table 1). When ATP breakdown per unit mass of tissue was determined arterial segments were seen to have the greatest ability to split ATP.
80
4. DISCUSSION
m ILEUM
60• 32
/• P. ATP.
BREAKDOWN
40 IA
20
~
/
10
E
20
NS
30
Min.
Fig. l. The release of 32p from 32p-q,-ATP by a series of unstimulated pharmacological preparations in phosphate
buffer.
The physiological responses of tissues in vitro to ATP is of current interest and a variety of actions have been reported in response to externally applied ATP. ATP dilates most vascular beds (Haddy and 'Scott, 1968) but constricts the pulmonary vascular bed (Emmelin and Feldberg, 1948) and arterial segments in vitro (Hrdina et al., 1967). Adenosine by contrast does not constrict isolated vessels but constricts the vessels in the rat kidney, and both ATP and adenosine relax intestinal smooth muscle. Since both adenosine and ATP are vasoactive the rapidity with which ATP was broken down in vitro was studied in vitro to determine whether externally applied ATP was stable for long enough to exert an action or whether the obsserved effect were due to breakdown
G.S.Harr&, A TPase activity of pharmacological preparations products of ATP. The release of the terminal phosphate from ATP with the formation of inorganic phosphate has been shown to occur briskly in phosphate buffer even though the high phosphate content might have induced end product inhibition. When tissues were used in masses consistent with pharmacological preparations the half life of ATP in all preparations was greater than 5 min and with the smaller preparations the half life was greater than 20 rain. Since the response to ATP in vitro occurs over a period of less than 1 rain it may be assumed that the observed responses in vitro are due to ATP and not breakdown products. The findings that arterial segments had the highest activity is of interest since increases in ATPase activity is one of the enzymatic changes observed early in the development of atheroma and in hypertension.
139
REFERENCES Abood, L.G. and A. Matsubara, 1968, Biochim. Biophys. Acta 163,539. Allen, J.G. and E.E. Daniel, 1970, Arch. Intern. Pharmacodyn. 188, 213. Axelsson, J. and B. Holmberg, 1969, Acta Physiol. Scand. 75, 149. Bloom, M.B. and I.M. Goldman, 1966, Advan. Drug Res. 3, 121. Daniel, E.E. and J. Irwin, 1965, Can. J. Physiol. Pharmacol. 43, 89. Emmelin, N. and W. Feldberg, 1948, Brit. J. Pharmacol. 3, 273. Haddy, F.J. and J.B. Scott, 1968, Physiol. Rev. 48, 688. Hrdina, P., A. Bonaccorsi and S. Garattini, 1967, European J. Pharmacol. 1, 99. McEwen, L.M., 1956, J. Physiol. (London), 131,678. Parvin, R. and R.A. Smith, 1969, Anal. Biochem. 27, 65.
Short communication
ATPASE ACTIVITY OF PHARMACOLOGICAL PREPARATIONS G.S. HARRIS Pharmacology Unit, Department of Physiology, University of Auckland, New Zealand
Accepted 19 April 1972
Received 20 March 1972
G.S. HARRIS, ATPase activity of pharmacological preparations, European J. Pharmacol. 19 (1972) 137-139. The ATPase activity of pharmacological preparations was tested in phosphate buffer to determine if the breakdown products of ATP contribute significantly to the responses observed to externally applied ATP. Large masses of tissues rapidly broke down ATP whereas the arterial preparations were the most active per unit mass. ATP ATPase activity
Arteries Atria
Ileum Vas deferens
1. INTRODUCTION
2. MATERIALS AND METHODS
The role of the plasma membrane ATPase has been generally accepted as related to cation transport (Skou, 1965), and a family of ATPase enzyme systems appear to be involved in the regulation of ion transport across cell membranes (Allen and Daniel, 1970). ATPase-dependent systems have also been suggested as being involved in the adrenergic receptor (Bloom and Goldman, 1965) and calcium control of membrane permeability (Abood and Matsubara, 1968). ATP, the substrate of the ATPase system has a variety of actions when applied externally to smooth muscle containing tissues, contracting vascular and uterine smooth muscle and relaxing intestinal smooth muscle (Hrdina et al., 1967; Daniel and Irwin, 1965; Axelsson and Holmberg, 1969). In view of these differences the activity of the ATPase system in tissues used as routine pharmacological preparations was studied in a physiological phosphate buffer using 32p-labelled ATP, since most previous studies have used phosphate free systems (Allen and Daniel, 1970).
2.1. General Tissues were isolated as for routine pharmacological preparations and suspended in McEwen (1956) solution. Aerated with 95% 02 and 5% CO2 and maintained at 37°C. 2.2. A TPase activity using 32p A TP ATPase acitivity was determined by the displacement of the 32p-ATP, to inorganic phosphate. A variety of tissues, guinea-pig vas deferens, rabbit central ear artery and aorta, guinea-pig ileum and rabbit left atria weighing 2 0 - 7 0 0 mg were prepared, as for an in vitro pharmacological preparation. Each tissue mass being approximately the mass used in in vitro pharmacological preparations. The tissues were incubated in McEwen buffer solution for 60 min, then added to buffer containing 20 ml 2/aCi (127/~Ci/mM) of 32p-isotopically-labelled ATP. Aliquots of 1.0 ml were taken before the addition of the tissue and after 1, 2, 4, 8, 16 and 32 min. Inorganic phosphate in the 1.0-ml aliquot was
138
G.S.Harris, A TPase activity of pharmacological preparations
separated from 3~ P-ATP by shaking with 2 ml of a vanadomolybdate complex the resulting phosphate complex being extracted into 10ml of butanol (Parvin and Smith, 1969). The butanol phase was separated, dried by gentle heating, 10 ml of scintillation mixture added and the radioactivity measured in a Packard Tricarb Scintillation Spectrometer Model 3384. In control studies isotopically labelled 32p sod i u m orthophosphate and 32p-labelled ATP were added to aliquots of McEwen solution and subjected to the extraction procedure. The recovery of 32p.phosphate and efficiency of separation from 32 P-ATP were estimated.
3. RESULTS The recovery of 32p-phosphate added to McEwen solution was incomplete and varied in different experiments from 15-32%. However, in the control solutions the recovery at each experiment only varied from 2.5 to 3.7% and the recovery was independent of the phosphate concentration of the buffer. When 32p-ATP was added to McEwen solution at the concentration used in the study 6.2% of the
100 -
Table 1 ATPase activity determined by 32p-release from 32P-3,-ATP for a series of tissues in phosphate buffer. Tissues
Artery
tlA ATP (min) hA ATP (min/ 100g)
118 26
Was
deferens 138 77
Atria
Ileum
30 152
10.2 69
added 32P-ATP was extracted and incubation for 30 min in buffer solution increased this to 11.7% 32 P-release from 32p.AT P by tissues. Incubation of tracer quantities of 32p-ATP with tissues in a series of 5 experiments increased the rate of release of isotopically labelled phosphate, fig. 1. Tissues of small mass, vas deferens and arterial segments released phosphate from ATP at a constant rate over the 30 min studied. Atria and ileum which have a mass 2 0 - 2 5 greater initially released a large amount of labelled phosphate. However, a progressive reduction in rate then occurred. Using the initial linear portion of the curves of ATP breakdown an approximate t~/2for ATP in these preparations was determined (table 1). When ATP breakdown per unit mass of tissue was determined arterial segments were seen to have the greatest ability to split ATP.
80
4. DISCUSSION
m ILEUM
60• 32
/• P. ATP.
BREAKDOWN
40 IA
20
~
/
10
E
20
NS
30
Min.
Fig. l. The release of 32p from 32p-q,-ATP by a series of unstimulated pharmacological preparations in phosphate
buffer.
The physiological responses of tissues in vitro to ATP is of current interest and a variety of actions have been reported in response to externally applied ATP. ATP dilates most vascular beds (Haddy and 'Scott, 1968) but constricts the pulmonary vascular bed (Emmelin and Feldberg, 1948) and arterial segments in vitro (Hrdina et al., 1967). Adenosine by contrast does not constrict isolated vessels but constricts the vessels in the rat kidney, and both ATP and adenosine relax intestinal smooth muscle. Since both adenosine and ATP are vasoactive the rapidity with which ATP was broken down in vitro was studied in vitro to determine whether externally applied ATP was stable for long enough to exert an action or whether the obsserved effect were due to breakdown
G.S.Harr&, A TPase activity of pharmacological preparations products of ATP. The release of the terminal phosphate from ATP with the formation of inorganic phosphate has been shown to occur briskly in phosphate buffer even though the high phosphate content might have induced end product inhibition. When tissues were used in masses consistent with pharmacological preparations the half life of ATP in all preparations was greater than 5 min and with the smaller preparations the half life was greater than 20 rain. Since the response to ATP in vitro occurs over a period of less than 1 rain it may be assumed that the observed responses in vitro are due to ATP and not breakdown products. The findings that arterial segments had the highest activity is of interest since increases in ATPase activity is one of the enzymatic changes observed early in the development of atheroma and in hypertension.
139
REFERENCES Abood, L.G. and A. Matsubara, 1968, Biochim. Biophys. Acta 163,539. Allen, J.G. and E.E. Daniel, 1970, Arch. Intern. Pharmacodyn. 188, 213. Axelsson, J. and B. Holmberg, 1969, Acta Physiol. Scand. 75, 149. Bloom, M.B. and I.M. Goldman, 1966, Advan. Drug Res. 3, 121. Daniel, E.E. and J. Irwin, 1965, Can. J. Physiol. Pharmacol. 43, 89. Emmelin, N. and W. Feldberg, 1948, Brit. J. Pharmacol. 3, 273. Haddy, F.J. and J.B. Scott, 1968, Physiol. Rev. 48, 688. Hrdina, P., A. Bonaccorsi and S. Garattini, 1967, European J. Pharmacol. 1, 99. McEwen, L.M., 1956, J. Physiol. (London), 131,678. Parvin, R. and R.A. Smith, 1969, Anal. Biochem. 27, 65.