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Dopamine 4-sulfate : Effects on isolated perfused rat heart and role of atria
Life Sciences, Vol. 43, pp. 1599-1606
Pergamon Press
Printed in the U.S.A.
DOPAMINE 4-SULFATE : EFFECTS ON ISOLATED PERFUSED RAT HEART AND ROLE OF ATRIA A.H.M. Mahbubul Hut? Suquru Matsuoka: Yoshida Kurahashi: Yasuhiro Kuroda', Sbok Je Mat Takeshi Ohuchi? Motoo Dkaa
1. Department of Pediatrics, School of Medicine, University
of Tokushima, Japan. 2. Department of Pediatrics, School of Medicine, Zennan University, Korea. 3. Department of Pharmacology, School of Medicine, University of Tokushima, Japan. (Received in final form September 15, 1988) Summary
We studied the effects of sulfate conjugate of dopamine on the isolated perfused rat heart (Langendorff preparation). In the experimental group, we removed atria from half number of the hearts. In the hearts with intact atria, dopamine 4-sulfate significantly improved the DT (devoloped tension), +dT/dt max (maximal rate of contraction), -dT/dt max (maximum rate of relaxation) over baseline values. But when atria were removed, dopamine 4-sulfate had no effect on the mechanical functions of heart. We analysed the effluent perfusate for the free and conjugated catecholamines. In the control group (no drug), and when atria were excised, the free catecholamine levels were negligible. But when the atria were kept intact, the effluent contained significant amount of free dopamine (DA), and norepinephrine (NE). These data suggested that dopamine sulfate had no direct effect on the ventricular muscle of rat heart, but was converted within the atria1 tissues into free catecholamines which might be responsible for the positive inotropic actions. Sulfoconjugation is a major pathway in the metabolism of catecholamines, but its exact significance is not known. Dopamine undergoes sulfoconjugation in the mammalian tissues to produce two sulfate esters : dopamine 3-sulfate & dopamine 4-sulfate. Oopmine sulfate conjugates have been proposed as transport forms of dopamine (l), as metabolic intermediates and as end products (2). There is evidence that dopamine sulfate might be a biologically active compound. In rat brain, dopamine sulfate conjugates produce bicuculline like convulsions which can be partially blocked by propanolol (3, 4). During excercise and also postsurgically the plasma concentration of dopamine sulfate changes (5, 6). In anesthesized dogs, i.v. administration of dopmine sulfate lowers blood pressure (7). However Ackerman et al., found no effect on blood pressure in the dogs receiving upto 34 pgs of dopamine sulfate /kg of body weight (8). In cultured bovine adrenal cells dopamine sulfate inhibits angiotensin II stimulated aldosterone release (9). Inspite of the large number of studies the physiological effects of dopamine sulfate are still far from clear. No study about the effect of conjugated dopamine on the heart function has been reported yet. We studied the effects of dopamine 4-sulfate on the isolated perfused rat heart. We had also 0024-3205/88 $3.00 + .OO Copyright (c) 1988 Pergamon Press plc
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evaluated the physiological and biochemical interactions of dopamine sulfate with atria & ventricle. In vitro conversion of dopamine sulfate into free catecholamines by the enzyme dopaminep-hydroxylase has been reported (10). This conversion might also be possible by arylsulfatase enzymes (11). In dog arylsulfatase enzymes are present mainly in atria but not in ventricles (Ohuchi unpublished). We decided to see the effect of excision of atria from isolated rat hearts. Materials and Methods Chemicals : Dopamine 4-sulfate was prepared from dopamine-HCl and concentrated HzSOq according to a previous method (1). The purity & identity of dopmine 4-sulfate were verified by high performance liquid chromatography. The contamination of sulfate ester by free dopamine did not exceed 100 ppm. Preparation of Isolated Heart and Establishment of Coronary Perfusion : Male rats of Sprague-Dawly strains, weighing about 180-200 gms. were used. The animals were heparinized (150 U/kg) & anesthesized by intraperitoneal injection of pentobarbital (40 mg/kg body wt.,iv.). Hearts (wet weight: 0.9 - 1.1 gm) were rapidly excised, & rinsed briefly with cold perfusion medium. Ascending aorta was canulated, & an arterially perfused isolated heart preparation (12, 13) was established within 30 seconds. Hearts were perfused with KrebsHenseleit (K-H) HC03-buffer solution (pH=7.4) containing 5 mM Glucose/litre. The perfusion medium was bubbled with 95% O2 -5% C02gas mixture. The temperature was kept at 37'C. We paced the hearts at the rate of 200 beats/min & perfused the hearts through the coronary arteries at a flow rate of 5 ml/min. We excised the atria from half of the hearts. Recording The Mechanical Functions : The mechanical effects were stable for 30 minutes after the establishment of perfusion and also after a single dose of dopamine or dopamine sulfate. After determining the time course of the effects of dopamine & dopamine sulfate (Fig l), we studied the effects of different doses (2, 20, 100 pgs/minute) of dopamine sulfate at 20 minutes intervals. We measured the muscle tension and its first derivative by a force transducer (Statham model UC4). By a multichannel polygraph, we continuously monitored the developed tension (DT), the resting tension (RT), the maximum rate of contraction (+dT/dt max), the maximum rate of relaxation (-dT/dt max), & the half relaxation time (T) (Fig 2). Measurement of Free Catecholamines in the Effluent : We stabilized the hearts (both with & without atria), by perfusing them with K-H solution. Samples of effluent perfusate were collected at timed intervals during perfusion with dopamine or dopamine sulfate at 20 Pg/min (4Pg/ml) and stored at -8OOC. Effluent was analysed for dopamine, dopamine sulfate, norepinephrine , epinephrine by HPLC (14, 15). Before injecting into HPLC, the effluent was filtered by a microfilter. (Chromatographic condition: Yanapak ODS-A (particle size 5pg) 4.6mm X 250mm; solvent: 0.05M NaH1P04 pH= 3.1. Flow rate: 1.0 ml/min; Temperature: ambient; Detections: amperometertic at tO.8OOV vs Ag/Agcl). As we stored the effluent at -8O'C immedietly after collection, we did not add any antioxidant to protect dopamine & dopamine sulfate. Statistical analysis was done by ANOVA. Results Mechanical functions : After stabilization with K-H solution, the DT, +dT/dt max, -dT/dt max, were constant for 2 hours unless dopamine or dopamine sulfate was added. When dopamine or dopamine sulfate was added, there was a dose dependent effect which reached maximum in 15 minutes (Tab 1, 2)(Fig 1).
Vol. 43, No. 20, 1988
t
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Dopamine 4-Sulfate on Rat Heart Functions
-1 0 Dopamlne Sulfate
1
f0
20
Tlme (Mb)
30
Fig 1 Time course of the mechanical function of the rat heart after a single dose of dopamine'sulfate. Atria were kept intact. +dTJdt max: maximal rate of contraction; -dT/dt max: maximal rate of relaxation. When dopamine was added in differnt doses (2, 20, 100 pg/min) there were positive inotropic effects in all the hearts, whether atria were removed or not (Tab l).OT,+dT/dt max,& -dT/dtmax, were initially 6.5 f 2 gms,115 t, 36 gms/sec, & 53 +- 16 gms/sec & increased 18 f 10 %, 44 ?r 11 X, & 119 + 36 % respectively, at a dose of 100 pg/min (Fig 3).
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Fig 2 Effects of dopamine sulfate on the mechanical functions of isolated rat heart (15 min after the dose). RT: Resting Tension; DT: Devoloped Tension; dT/dt: the first derivative of Developed Tension; +dT/dt max: maximum rate of contraction; -dT/dt max: maximum rate of relaxation; T: half relaxation time. DT & tdT/dt max were used as indices of contractility, while -dT/dt max & T were used as indices of relaxation. TABLE 1 : Effects of dopamine on the mechanical functions of heart. n=5. Values are mean +- SE ____________________~~~~~~~~~_~~~~~~__~~~~~__________________________________ 100 2 20 0 Dose (pg/min) ____________________-~~~~~~~~~~~~~~~__~~~~~~~~~~~~___________________________ 7.4 + 1.8 7.4 t 1.8 6.5 + 2.0 7.9 + 2.7 DT (gm) 164 + 50 162 + 55 115 f 36 140 + 60 +dT/dtmax (gm/sec) 100 f 40 100 +- 39 53 k 16 73 f 27 -dT/dtmax (gm/sec) 5.9 + 1.3 5.9 f 1.3 6.8 f 0.8 6.1 f 1.5 RT (gm) 62 f 10 62 t 10 94 f 16 68 t 6 T (msec) ___________________--~~~~~~~~~~~~~~~_~~~~~~~~~~~~~___~__~_~~~~~~_____________
TABLE 2 : Effects of dopamine 4-sulfate on the mechanical functions of heart. n=5. Atria were intact. Values are mean t SE ------~_____--_____--~~~~~~~~~~~~~~~__~~~~~~~~~~~~______~_~~~~~~_____________ 100 2 20 0 Dose (ug/min) -_----_____________--~~~~~~~~~~~~~~~~~~~~~~~~~~~~~____~_~_~~~~~~____~~~______ 6.9 f 1.2 6.7 t 1.2 5.7 f 0.8 6.3 t 1.0 DT (gm) 138 + 40 130 + 35 107 f 28 119 t 33 +dT/dtmax (gm/sec) 65 t 24 63 +- 24 47 f 14 57 + 17 -dT/dtmax (gm/sec) 5.3 t 1.3 6.1 f 1.1 6.3 f 0.9 6.4 + 1.3 RT (gm) 82 + 12 87 f: 16 98 + 13 87 + 16 T (msec) ___________________--~~~~~~~~~~~~~~~__~~~~~~~~~~~~___________________________
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TABLE 3 : Effects of dopamine 4-sulfate. n=5. Atria were excised. Values are mean + SE
____________________~__~~~___~~___~~___~~__~~~__~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 100 2 20 0 Dose (yg/min) ____________________~___~~___~~___~~_~~~~__~~~_~~~~~_~~~~_~~~~_~~~~_~~~~~~~~~ 3.8 f 1.3 3.8 f 1.3 3.7 + 0.9 3.7 +, 0.9 DT (gm) 59 + 21 60 f 21 58 !: 17 58 f 17 +dT/dtmax (gm/sec) 27 + 6 29 f 7 24 + 5 24 f 5 -dT/dtmax (gm/sec) 5.6 t 0.9 5.6 + 0.9 6.0 f 0.6 6.0 f 0.7 RT (gm) 68 * 8 68 f.8 78 f 16 75 * 19 T (msec) ____________________~___~~___~~__~~~___~~___~~___~~__~~~___~~~__~~~___~~___~~ When atria were kept intact, dopamine sulfate increased the TIT,+dT/dt max, & -dT/dt max dose dependently. DT, +dT/dt max, & -dT/dt max were initially 5.7 I+ 0.8 gms, 107 + 28 gmslsec & 47 f. 14 gms/sec respectively & increased 21 f 7 %, 28 f 16 %, & 49 +- 29 % respectivelyata dose of 100 pg/min (Fig 3). When the atria were removed, the effects were either abolished or much diminished. DT, +dT/dt max, & -dT/dt max were initially 37 * 0.9 gms, 58 f 11 gms/sec & 24 f 5 gms/sec. Even at a dose of 100 pg/min there was no significant change over baseline values. 150 0
n q
Dopamine
sulfate
(Atrium
Dopamine
sulfate
[Intact
Dopamine
(Atrium
removed) atrium)
removed)
100 3 4 Ip t : ._ L 3 5 it 2
50
0 +dT Idt
max.
-dTldt
nkx.
Fig 3 Effects of dopamine & dopamine 4-sulfate (100 ug/min) on DT, p ( 0.05. +dT/dt max & -dT/dt max of rat heart. Chemical data: When the hearts were perfused with K-H solution only, the effluent contained very little catecholamines. The concentration of dopamine, norepinephrine and epinephrine were 2 t 1, 40 +, 6, 4 f 2 ngs/ml respectively. There was no difference between with & without atria groups. When hearts with
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intact atria were perfused with K-H solution containing 4 gs/ml dopamine sulfate (20 pgs/min at the coronary flow rate of 5 ml/min r, the free catecholamine concentrations were significantly raised over control. Dopamine, norepinephrine & epinephrine concentrations were 200 -+ 12, 100 & 12 & 4 + 2 ngs/ml. Specially, the free dopamine level was greatly raised (100 times). When hearts without atria were perfused with K-H solution containing 4 pgs/ml dopmine 4-sulfate, there was no change over baseline values. Dopamine, norepinephrine & epinephrine concentrations were 8 -+ 3, 38 -+ 6, 6 f 1 ngs/ml respectively. (Fig 4). Discussion
In our study we found that dopamine 4-sulfate definitely improved the mechanical functions of the isolated rat heart. It increased the DT, +dT/dt max & -dT/dt max in the hearts with intact atria but not when the atria were removed. The positive inotropic actions of the dopmine 4-sulfate could be due to its effect on the ventricular muscle, or indirectly due to its conversion into some other active compounds, or indirectly due to its chronotropic action on the conducting tissues. But as there was no effect, when the atria were removed, direct effect on the ventricle seems unlikely. Also we kept the heart rate fixed by pacing. So it did not act indirectly via the chronotropic action on the conducting tissues. On chemical analysis we found significant amount of free dopamine & norepinephreine in the effluent perfusate, when the atria were kept intact but not when the atria were excised. It indicates that dopamine sulfate might be converted to free catecholamines within the atria1 tissues. In dog, atria1 tissues were found to be rich in arylsulfatase enzymes while ventricle contained very little amount (Ohuchi, unpublished). It is possible that dopamine sulfate might be converted by arylsulfatase enzymes into free catecholamines which then exerted positive inotropic effect on the heart. Another possibilty is the action of dopamine fl-hydroxylase which has been reported to convert conjugated dopamine into free catecholamines in vitro (IO). Arylsulfatase Dopamine sulfate ---------------3
Dopamine + Sulfate
hydroxylase-3
Norepinephrine t ( Sulfate ) Possible fate of dopmine sulfate within atria. Though sulfoconjugation is a major pathway of dopamine metabolism, for a long time, it was generally considered as an end metabolite, since sulfation is generally a method of detoxification (1). Owing to its ability to form internal salts which might confer a great facility for penetrating biological membranes, it had been proposed as a transport form of dopamine (1). Finally there is evidence that dopamine sulfate might have direct actions or it could be a metabolic intermediate. Small doses of 14C dopamine sulfate administered i.v. to guinea pigs or rats, were excreted rapidly, mainly as DOPAC (3,4 hydroxy-phenylacetic acid) & HVA (homovanilic acid) in guinea pigs & as glucuronides in rats (16). In rats, dopamine glucuronide, the major metabolite of dopamine decreases with stress (17). When adreanalectized rats were given ?'H dopamine sulfate, Buu et al., reported the presence of 3H-
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epinephrine in the kidney and 3H norepinephrine in urine (18). These studies indicate that dopamine conjugates are converted in vivo to free dopamine prior to excretion or conversion to various forms.
a : Control : DA Sulfate(Atris
: DA Sulfate[intact
Fig 4 Free catecholamines in the effluent. atria were excised. Hearts were then buffer solution only or K-H solution dopamine sulfate was added to adjust (4 ugs/ml). n=5
Removed) Atria)
From half of the hearts, perfused either with K-H with dopamine 4-sulfate. a dose of 20 ugs/min.
Buu & Kuchel in 1978 showed direct conversion in vitro of dopamine sulfate into free catecholamines by the enzyme dopamine P-hydroxylase, indicating that dopmine sulfate could be a substrate of catecholamine biosynthesis (10). that in vitro, dopamine sulfate also acted as a In 1983, QU. et al., reported substrate for catechol-O-methyl transferase (19). However Demassieux et al., reported in 1987, that catecholamine sulfates were not substrates for catecho-
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No. 20,
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lamine synthesis (11). They suggested that catecholamine sulfates represented excretion products, as generally accepted, but might also constitute a form of storage products for further biotransformation into free amines by means of an arylsulfatase system. They had found that catecholamine sulfate conjugates were hydrolysed by arylsulfatase A, B & C in vitro with Km in the mM range. Our study supports the latter concept that dopamine sulfate can be a storage form, from which free amines are formed in tissues. Our data showed that dopamine sulfate was converted into free catecholamines in coronary perfusate from an isolated rat heart with intact atria but not when atria were excised and this conversion of dopamine sulfate could be due to the arylsulfatase or dopamine p-hydroxylase enzymes present in atria. Further studies using dopamine glucuronide, which is the major dopamine conjugate in rat, would be interesting. References
:: 3. 4. :: :: 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Q. N. JENNER, & F. A. ROSE, Biochem J 135 109-114 (1973). R. L. BRONAUGH, S. E. HATLOX, M. M. HmN, R. C. MURPHY, & C. 0. RUTLAGE, J Pharmacol Exp Ther, 195 441-452 (1975). N. T. BUU, J. DUHAIME, 0. KUCHELTJ. GENEST, Life Sci., -29 23112316 (1981). N. T. BUU & J. DUHAIME, & 0. KUCHEL, Life Sci., 35 1083-1090 (1984). M. LEHMANN UND J. KEUL, Klin wochenschr 63 37-4T(1985). PharmaT. UNGER, N.T.BUU, 0. KUCHEL, & W. SCHURCKCan. J. Physiol. col. 58, 22-27 (1980). M. C.xOTT & M. A. ELCHISAK, Fed proc., 42 1363 (1983) D. M. ACKERMAN, J. P. HIEBLE, H. M. SARAV/ T. C. JAIN, Arch. Int. Pharmacodym. Ther., 276 241-248 (1984). & A. DELEAN, Am. J. Physiol. 247, 431K. RACZ , N. T. BUU ,rKUCHEL 435(1984). N. T. BUU & 0. KUCHEL, Life Sci. 24 783-790 (1979). S. DEMASSIEX, L. BORDELEAU, D. GRAVEL & S. CARRIERE, Life Sci. -40 183191 (1987). J.M. JARMAKANI. M. NAKAZAWA. T. NAGATOMO. G. A. LANGER, Am. J. Physiol. 235 (Heart Circ Physiol) H469-H474 (1978): T. NAKANISHI, S. MATSUOKA, S. UEMURA, T. SHIMIZU, K. NISHIOKA, N.D. NEUFIELD, J. M. JARMAKANI, Pediatric Research 18(12) 1344-1349 (1984). M. A. ELCHISAK, J. H. CARLSON, Life Sci. 30 2325-36, (1982). N. S. SHARPLESS, G. M. TYCE, L. J. THAL, J;-M. WALTZ, K. TABADDOR, AND L. I. WOLFSON, Brain Research 217, 107-118, (1981). J. MERITS, Biochem. Pharmacol., 25 829-833 (1976). N. ALEXANDER, S. YONEDA, N. 0. VLACHAKIS & R. F. MARONDE Am. J. Physiol. 247(pt 2) R203-R207 (1984). N. T. BUU, G. NAIR, 0. KUCHEL & J. GENEST, J. Lab. Clin. Med., -98 527535 (1981). Y. Q. QU., K. IMAI, Z. TAMURA, Y. HASHIMOTO & H. MIYAZAKI, Life Sci. -32 1811-1818 (1983).
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DOPAMINE 4-SULFATE : EFFECTS ON ISOLATED PERFUSED RAT HEART AND ROLE OF ATRIA A.H.M. Mahbubul Hut? Suquru Matsuoka: Yoshida Kurahashi: Yasuhiro Kuroda', Sbok Je Mat Takeshi Ohuchi? Motoo Dkaa
1. Department of Pediatrics, School of Medicine, University
of Tokushima, Japan. 2. Department of Pediatrics, School of Medicine, Zennan University, Korea. 3. Department of Pharmacology, School of Medicine, University of Tokushima, Japan. (Received in final form September 15, 1988) Summary
We studied the effects of sulfate conjugate of dopamine on the isolated perfused rat heart (Langendorff preparation). In the experimental group, we removed atria from half number of the hearts. In the hearts with intact atria, dopamine 4-sulfate significantly improved the DT (devoloped tension), +dT/dt max (maximal rate of contraction), -dT/dt max (maximum rate of relaxation) over baseline values. But when atria were removed, dopamine 4-sulfate had no effect on the mechanical functions of heart. We analysed the effluent perfusate for the free and conjugated catecholamines. In the control group (no drug), and when atria were excised, the free catecholamine levels were negligible. But when the atria were kept intact, the effluent contained significant amount of free dopamine (DA), and norepinephrine (NE). These data suggested that dopamine sulfate had no direct effect on the ventricular muscle of rat heart, but was converted within the atria1 tissues into free catecholamines which might be responsible for the positive inotropic actions. Sulfoconjugation is a major pathway in the metabolism of catecholamines, but its exact significance is not known. Dopamine undergoes sulfoconjugation in the mammalian tissues to produce two sulfate esters : dopamine 3-sulfate & dopamine 4-sulfate. Oopmine sulfate conjugates have been proposed as transport forms of dopamine (l), as metabolic intermediates and as end products (2). There is evidence that dopamine sulfate might be a biologically active compound. In rat brain, dopamine sulfate conjugates produce bicuculline like convulsions which can be partially blocked by propanolol (3, 4). During excercise and also postsurgically the plasma concentration of dopamine sulfate changes (5, 6). In anesthesized dogs, i.v. administration of dopmine sulfate lowers blood pressure (7). However Ackerman et al., found no effect on blood pressure in the dogs receiving upto 34 pgs of dopamine sulfate /kg of body weight (8). In cultured bovine adrenal cells dopamine sulfate inhibits angiotensin II stimulated aldosterone release (9). Inspite of the large number of studies the physiological effects of dopamine sulfate are still far from clear. No study about the effect of conjugated dopamine on the heart function has been reported yet. We studied the effects of dopamine 4-sulfate on the isolated perfused rat heart. We had also 0024-3205/88 $3.00 + .OO Copyright (c) 1988 Pergamon Press plc
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Vol. 43, No. 20, 1988
evaluated the physiological and biochemical interactions of dopamine sulfate with atria & ventricle. In vitro conversion of dopamine sulfate into free catecholamines by the enzyme dopaminep-hydroxylase has been reported (10). This conversion might also be possible by arylsulfatase enzymes (11). In dog arylsulfatase enzymes are present mainly in atria but not in ventricles (Ohuchi unpublished). We decided to see the effect of excision of atria from isolated rat hearts. Materials and Methods Chemicals : Dopamine 4-sulfate was prepared from dopamine-HCl and concentrated HzSOq according to a previous method (1). The purity & identity of dopmine 4-sulfate were verified by high performance liquid chromatography. The contamination of sulfate ester by free dopamine did not exceed 100 ppm. Preparation of Isolated Heart and Establishment of Coronary Perfusion : Male rats of Sprague-Dawly strains, weighing about 180-200 gms. were used. The animals were heparinized (150 U/kg) & anesthesized by intraperitoneal injection of pentobarbital (40 mg/kg body wt.,iv.). Hearts (wet weight: 0.9 - 1.1 gm) were rapidly excised, & rinsed briefly with cold perfusion medium. Ascending aorta was canulated, & an arterially perfused isolated heart preparation (12, 13) was established within 30 seconds. Hearts were perfused with KrebsHenseleit (K-H) HC03-buffer solution (pH=7.4) containing 5 mM Glucose/litre. The perfusion medium was bubbled with 95% O2 -5% C02gas mixture. The temperature was kept at 37'C. We paced the hearts at the rate of 200 beats/min & perfused the hearts through the coronary arteries at a flow rate of 5 ml/min. We excised the atria from half of the hearts. Recording The Mechanical Functions : The mechanical effects were stable for 30 minutes after the establishment of perfusion and also after a single dose of dopamine or dopamine sulfate. After determining the time course of the effects of dopamine & dopamine sulfate (Fig l), we studied the effects of different doses (2, 20, 100 pgs/minute) of dopamine sulfate at 20 minutes intervals. We measured the muscle tension and its first derivative by a force transducer (Statham model UC4). By a multichannel polygraph, we continuously monitored the developed tension (DT), the resting tension (RT), the maximum rate of contraction (+dT/dt max), the maximum rate of relaxation (-dT/dt max), & the half relaxation time (T) (Fig 2). Measurement of Free Catecholamines in the Effluent : We stabilized the hearts (both with & without atria), by perfusing them with K-H solution. Samples of effluent perfusate were collected at timed intervals during perfusion with dopamine or dopamine sulfate at 20 Pg/min (4Pg/ml) and stored at -8OOC. Effluent was analysed for dopamine, dopamine sulfate, norepinephrine , epinephrine by HPLC (14, 15). Before injecting into HPLC, the effluent was filtered by a microfilter. (Chromatographic condition: Yanapak ODS-A (particle size 5pg) 4.6mm X 250mm; solvent: 0.05M NaH1P04 pH= 3.1. Flow rate: 1.0 ml/min; Temperature: ambient; Detections: amperometertic at tO.8OOV vs Ag/Agcl). As we stored the effluent at -8O'C immedietly after collection, we did not add any antioxidant to protect dopamine & dopamine sulfate. Statistical analysis was done by ANOVA. Results Mechanical functions : After stabilization with K-H solution, the DT, +dT/dt max, -dT/dt max, were constant for 2 hours unless dopamine or dopamine sulfate was added. When dopamine or dopamine sulfate was added, there was a dose dependent effect which reached maximum in 15 minutes (Tab 1, 2)(Fig 1).
Vol. 43, No. 20, 1988
t
1601
Dopamine 4-Sulfate on Rat Heart Functions
-1 0 Dopamlne Sulfate
1
f0
20
Tlme (Mb)
30
Fig 1 Time course of the mechanical function of the rat heart after a single dose of dopamine'sulfate. Atria were kept intact. +dTJdt max: maximal rate of contraction; -dT/dt max: maximal rate of relaxation. When dopamine was added in differnt doses (2, 20, 100 pg/min) there were positive inotropic effects in all the hearts, whether atria were removed or not (Tab l).OT,+dT/dt max,& -dT/dtmax, were initially 6.5 f 2 gms,115 t, 36 gms/sec, & 53 +- 16 gms/sec & increased 18 f 10 %, 44 ?r 11 X, & 119 + 36 % respectively, at a dose of 100 pg/min (Fig 3).
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Fig 2 Effects of dopamine sulfate on the mechanical functions of isolated rat heart (15 min after the dose). RT: Resting Tension; DT: Devoloped Tension; dT/dt: the first derivative of Developed Tension; +dT/dt max: maximum rate of contraction; -dT/dt max: maximum rate of relaxation; T: half relaxation time. DT & tdT/dt max were used as indices of contractility, while -dT/dt max & T were used as indices of relaxation. TABLE 1 : Effects of dopamine on the mechanical functions of heart. n=5. Values are mean +- SE ____________________~~~~~~~~~_~~~~~~__~~~~~__________________________________ 100 2 20 0 Dose (pg/min) ____________________-~~~~~~~~~~~~~~~__~~~~~~~~~~~~___________________________ 7.4 + 1.8 7.4 t 1.8 6.5 + 2.0 7.9 + 2.7 DT (gm) 164 + 50 162 + 55 115 f 36 140 + 60 +dT/dtmax (gm/sec) 100 f 40 100 +- 39 53 k 16 73 f 27 -dT/dtmax (gm/sec) 5.9 + 1.3 5.9 f 1.3 6.8 f 0.8 6.1 f 1.5 RT (gm) 62 f 10 62 t 10 94 f 16 68 t 6 T (msec) ___________________--~~~~~~~~~~~~~~~_~~~~~~~~~~~~~___~__~_~~~~~~_____________
TABLE 2 : Effects of dopamine 4-sulfate on the mechanical functions of heart. n=5. Atria were intact. Values are mean t SE ------~_____--_____--~~~~~~~~~~~~~~~__~~~~~~~~~~~~______~_~~~~~~_____________ 100 2 20 0 Dose (ug/min) -_----_____________--~~~~~~~~~~~~~~~~~~~~~~~~~~~~~____~_~_~~~~~~____~~~______ 6.9 f 1.2 6.7 t 1.2 5.7 f 0.8 6.3 t 1.0 DT (gm) 138 + 40 130 + 35 107 f 28 119 t 33 +dT/dtmax (gm/sec) 65 t 24 63 +- 24 47 f 14 57 + 17 -dT/dtmax (gm/sec) 5.3 t 1.3 6.1 f 1.1 6.3 f 0.9 6.4 + 1.3 RT (gm) 82 + 12 87 f: 16 98 + 13 87 + 16 T (msec) ___________________--~~~~~~~~~~~~~~~__~~~~~~~~~~~~___________________________
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TABLE 3 : Effects of dopamine 4-sulfate. n=5. Atria were excised. Values are mean + SE
____________________~__~~~___~~___~~___~~__~~~__~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 100 2 20 0 Dose (yg/min) ____________________~___~~___~~___~~_~~~~__~~~_~~~~~_~~~~_~~~~_~~~~_~~~~~~~~~ 3.8 f 1.3 3.8 f 1.3 3.7 + 0.9 3.7 +, 0.9 DT (gm) 59 + 21 60 f 21 58 !: 17 58 f 17 +dT/dtmax (gm/sec) 27 + 6 29 f 7 24 + 5 24 f 5 -dT/dtmax (gm/sec) 5.6 t 0.9 5.6 + 0.9 6.0 f 0.6 6.0 f 0.7 RT (gm) 68 * 8 68 f.8 78 f 16 75 * 19 T (msec) ____________________~___~~___~~__~~~___~~___~~___~~__~~~___~~~__~~~___~~___~~ When atria were kept intact, dopamine sulfate increased the TIT,+dT/dt max, & -dT/dt max dose dependently. DT, +dT/dt max, & -dT/dt max were initially 5.7 I+ 0.8 gms, 107 + 28 gmslsec & 47 f. 14 gms/sec respectively & increased 21 f 7 %, 28 f 16 %, & 49 +- 29 % respectivelyata dose of 100 pg/min (Fig 3). When the atria were removed, the effects were either abolished or much diminished. DT, +dT/dt max, & -dT/dt max were initially 37 * 0.9 gms, 58 f 11 gms/sec & 24 f 5 gms/sec. Even at a dose of 100 pg/min there was no significant change over baseline values. 150 0
n q
Dopamine
sulfate
(Atrium
Dopamine
sulfate
[Intact
Dopamine
(Atrium
removed) atrium)
removed)
100 3 4 Ip t : ._ L 3 5 it 2
50
0 +dT Idt
max.
-dTldt
nkx.
Fig 3 Effects of dopamine & dopamine 4-sulfate (100 ug/min) on DT, p ( 0.05. +dT/dt max & -dT/dt max of rat heart. Chemical data: When the hearts were perfused with K-H solution only, the effluent contained very little catecholamines. The concentration of dopamine, norepinephrine and epinephrine were 2 t 1, 40 +, 6, 4 f 2 ngs/ml respectively. There was no difference between with & without atria groups. When hearts with
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intact atria were perfused with K-H solution containing 4 gs/ml dopamine sulfate (20 pgs/min at the coronary flow rate of 5 ml/min r, the free catecholamine concentrations were significantly raised over control. Dopamine, norepinephrine & epinephrine concentrations were 200 -+ 12, 100 & 12 & 4 + 2 ngs/ml. Specially, the free dopamine level was greatly raised (100 times). When hearts without atria were perfused with K-H solution containing 4 pgs/ml dopmine 4-sulfate, there was no change over baseline values. Dopamine, norepinephrine & epinephrine concentrations were 8 -+ 3, 38 -+ 6, 6 f 1 ngs/ml respectively. (Fig 4). Discussion
In our study we found that dopamine 4-sulfate definitely improved the mechanical functions of the isolated rat heart. It increased the DT, +dT/dt max & -dT/dt max in the hearts with intact atria but not when the atria were removed. The positive inotropic actions of the dopmine 4-sulfate could be due to its effect on the ventricular muscle, or indirectly due to its conversion into some other active compounds, or indirectly due to its chronotropic action on the conducting tissues. But as there was no effect, when the atria were removed, direct effect on the ventricle seems unlikely. Also we kept the heart rate fixed by pacing. So it did not act indirectly via the chronotropic action on the conducting tissues. On chemical analysis we found significant amount of free dopamine & norepinephreine in the effluent perfusate, when the atria were kept intact but not when the atria were excised. It indicates that dopamine sulfate might be converted to free catecholamines within the atria1 tissues. In dog, atria1 tissues were found to be rich in arylsulfatase enzymes while ventricle contained very little amount (Ohuchi, unpublished). It is possible that dopamine sulfate might be converted by arylsulfatase enzymes into free catecholamines which then exerted positive inotropic effect on the heart. Another possibilty is the action of dopamine fl-hydroxylase which has been reported to convert conjugated dopamine into free catecholamines in vitro (IO). Arylsulfatase Dopamine sulfate ---------------3
Dopamine + Sulfate
hydroxylase-3
Norepinephrine t ( Sulfate ) Possible fate of dopmine sulfate within atria. Though sulfoconjugation is a major pathway of dopamine metabolism, for a long time, it was generally considered as an end metabolite, since sulfation is generally a method of detoxification (1). Owing to its ability to form internal salts which might confer a great facility for penetrating biological membranes, it had been proposed as a transport form of dopamine (1). Finally there is evidence that dopamine sulfate might have direct actions or it could be a metabolic intermediate. Small doses of 14C dopamine sulfate administered i.v. to guinea pigs or rats, were excreted rapidly, mainly as DOPAC (3,4 hydroxy-phenylacetic acid) & HVA (homovanilic acid) in guinea pigs & as glucuronides in rats (16). In rats, dopamine glucuronide, the major metabolite of dopamine decreases with stress (17). When adreanalectized rats were given ?'H dopamine sulfate, Buu et al., reported the presence of 3H-
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epinephrine in the kidney and 3H norepinephrine in urine (18). These studies indicate that dopamine conjugates are converted in vivo to free dopamine prior to excretion or conversion to various forms.
a : Control : DA Sulfate(Atris
: DA Sulfate[intact
Fig 4 Free catecholamines in the effluent. atria were excised. Hearts were then buffer solution only or K-H solution dopamine sulfate was added to adjust (4 ugs/ml). n=5
Removed) Atria)
From half of the hearts, perfused either with K-H with dopamine 4-sulfate. a dose of 20 ugs/min.
Buu & Kuchel in 1978 showed direct conversion in vitro of dopamine sulfate into free catecholamines by the enzyme dopamine P-hydroxylase, indicating that dopmine sulfate could be a substrate of catecholamine biosynthesis (10). that in vitro, dopamine sulfate also acted as a In 1983, QU. et al., reported substrate for catechol-O-methyl transferase (19). However Demassieux et al., reported in 1987, that catecholamine sulfates were not substrates for catecho-
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lamine synthesis (11). They suggested that catecholamine sulfates represented excretion products, as generally accepted, but might also constitute a form of storage products for further biotransformation into free amines by means of an arylsulfatase system. They had found that catecholamine sulfate conjugates were hydrolysed by arylsulfatase A, B & C in vitro with Km in the mM range. Our study supports the latter concept that dopamine sulfate can be a storage form, from which free amines are formed in tissues. Our data showed that dopamine sulfate was converted into free catecholamines in coronary perfusate from an isolated rat heart with intact atria but not when atria were excised and this conversion of dopamine sulfate could be due to the arylsulfatase or dopamine p-hydroxylase enzymes present in atria. Further studies using dopamine glucuronide, which is the major dopamine conjugate in rat, would be interesting. References
:: 3. 4. :: :: 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Q. N. JENNER, & F. A. ROSE, Biochem J 135 109-114 (1973). R. L. BRONAUGH, S. E. HATLOX, M. M. HmN, R. C. MURPHY, & C. 0. RUTLAGE, J Pharmacol Exp Ther, 195 441-452 (1975). N. T. BUU, J. DUHAIME, 0. KUCHELTJ. GENEST, Life Sci., -29 23112316 (1981). N. T. BUU & J. DUHAIME, & 0. KUCHEL, Life Sci., 35 1083-1090 (1984). M. LEHMANN UND J. KEUL, Klin wochenschr 63 37-4T(1985). PharmaT. UNGER, N.T.BUU, 0. KUCHEL, & W. SCHURCKCan. J. Physiol. col. 58, 22-27 (1980). M. C.xOTT & M. A. ELCHISAK, Fed proc., 42 1363 (1983) D. M. ACKERMAN, J. P. HIEBLE, H. M. SARAV/ T. C. JAIN, Arch. Int. Pharmacodym. Ther., 276 241-248 (1984). & A. DELEAN, Am. J. Physiol. 247, 431K. RACZ , N. T. BUU ,rKUCHEL 435(1984). N. T. BUU & 0. KUCHEL, Life Sci. 24 783-790 (1979). S. DEMASSIEX, L. BORDELEAU, D. GRAVEL & S. CARRIERE, Life Sci. -40 183191 (1987). J.M. JARMAKANI. M. NAKAZAWA. T. NAGATOMO. G. A. LANGER, Am. J. Physiol. 235 (Heart Circ Physiol) H469-H474 (1978): T. NAKANISHI, S. MATSUOKA, S. UEMURA, T. SHIMIZU, K. NISHIOKA, N.D. NEUFIELD, J. M. JARMAKANI, Pediatric Research 18(12) 1344-1349 (1984). M. A. ELCHISAK, J. H. CARLSON, Life Sci. 30 2325-36, (1982). N. S. SHARPLESS, G. M. TYCE, L. J. THAL, J;-M. WALTZ, K. TABADDOR, AND L. I. WOLFSON, Brain Research 217, 107-118, (1981). J. MERITS, Biochem. Pharmacol., 25 829-833 (1976). N. ALEXANDER, S. YONEDA, N. 0. VLACHAKIS & R. F. MARONDE Am. J. Physiol. 247(pt 2) R203-R207 (1984). N. T. BUU, G. NAIR, 0. KUCHEL & J. GENEST, J. Lab. Clin. Med., -98 527535 (1981). Y. Q. QU., K. IMAI, Z. TAMURA, Y. HASHIMOTO & H. MIYAZAKI, Life Sci. -32 1811-1818 (1983).