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Characterization of norepinephrine accumulation by a crude synaptosomal-mitochondrial fraction isolated from rat heart
Life Sciences, Vol. 48, pp. 1317-1324 Printed in the U.S.A.
Pergamon Press
CHARACTERIZATION OF NOREPINEPHRINE ACCUMULATION BY A CRUDE SYNAPTOSOMAL-MITOCHONDRIAL FRACTION ISOLATED FROM RAT HEART V. J. Aloyo1, H. B. Mcllvain 1, V. H. Bhavsar 1'2 and J. Roberts ~ 1Department of Pharmacology, Medical College of Pennsylvania, 3200 Henry Ave., Philadelphia, PA 19129, USA and 2ECFMG Fellow, Department of Pharmacology, Government Medical College, Surat, India. (Recelved in final form January 24, 1991)
Summary Norepinephrine (NE) uptake into a heart synaptosomalmitochondrial fraction was assessed under conditions where neuronal uptake (type 1) was linear with respect to both time and protein concentration. The NE accumulation process was sensitive to incubation temperature, sodium ion concentration and medium osmolality. Furthermore, NE uptake was attenuated by the neuronal uptake inhibitor desmethylimipranune (DMI) m a concentration dependent manner; the ICso value was approximately 10 nM and maximum inhibition was obtained at 100 nM. In contrast, the extraneuronal uptake inhibitor, metanephrine did not sigmficantly attenuate NE uptake. Kinetic analysis demonstrated that the DMI sensitive NE accumulation is saturable with a KM of approximately 400 rLM and that NE uptake occurs wa a single uptake process. This demonstration of neuronal type NE uptake by a synaptosomal-mltochondrial fraction constitutes a successful demonstration of the preparation of a rat heart subcellular fraction containing functional synaptosomes Cellular accumulation of norepinephrine (NE) occurs via two processes (1,2). Type 1 uptake has a high affinity for NE and is selectively inhibited by DMI. In contrast, type 2 uptake has a much lower affinity for NE and is preferentially inhibited by metanephrine. Type 1 uptake is observed in adrenergic neurons derived from both brain and cardiac tissue, while type 2 represents NE uptake into non-neuronal tissue. Neuronal uptake is a saturable, energy dependent process which is critically dependent upon the concentration of extracellular sodium ions (3). The use of isolated subcellular fractions containing pinched off nerve endings (synaptosomes) from brain tissue has been widely used to characterize neuronal uptake of neurotransmlttors, amino acids and other substances (4). However, the application of siimlar techniques to other tissues has been linnted, in part, because the homogemzation of these tissues results in the destruction of nerve terminals rather than the formation ofsynaptosomes (4) In some cases, pretreatment of the tissue with 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
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NE Uptake by Heart Synaptosomes
Vol. 48, No. 13, 1991
proteolytic enzymes can facilitate homogenization and improve formation of synaptosomes (5,6). During the course of our studies on changes in neurotransmission in the heart as a functmn of age (7,8), we dec~ded to investigate NE uptake using a subcellular fraction prepared from rat heart containing synaptosomes. In the present study, we have employed collagenase pretreatment of rat heart in order to prepare a crude synaptosomal-mitochondrial fraction (P2) and have characterized NE accumulation by this fraction. Materials and Methods Preparation of crude synaptosomal-mitochondrial fraction (P~) from rat heart Immediately following sacrifice of the six month old male Fisher 344 rat, the heart was rapidly removed and minced in ice-cold 0 32 M sucrose containing 1 mM EGTA, pH 7.5 The mince was transferred to a HEPES buffered Krebs-Ringer solution (KRH; HEPES 50 mM, NaC1 144 mM, MgC12 1.2 mM, CaC12 1.2 mM, KC1 5 mM, glucose 10 mM, ascorbic acid 1 raM, and pargyline 1 mM, pH 7.4) containing 12 units of collagenase (class II, Worthington Blochemicals) per mg of tissue. This suspension was then incubated at 370 C for 40 rain with continuous bubbling with oxygen. Subsequent to collection of the resulting tissue fragments by low speed centrifugation (120xg), the tissue was disrupted in 10 volumes (by weight) of ice-cold 0 32 M sucrose with a teflon/glass homogemzer (clearance 0.25 mm). Cellular debris, nuclei and large tissue shards were removed by centrifugation at 650xg for 10 min at 4 o C. The resulting supernatant was centrifuged at 21,000xg for 20 min at 4o C The pellet (P2) was resuspended in oxygenated, ice-cold KRH using a teflon/glass homogemzer (clearance 0 25 mm) and immediately used for experimentation. Protein concentration was determined by the Bradford method (9) In some experiments the concentration of endogenous NE in rat heart homogenate and the resulting P2 fraction was deterlnined using HPLC-EC methodology as described by Kreider and coworkers (10). NE uptake studies Aliquots of P2 suspenmon were prelncubated in KRH (with or without the indicated drug) at 37°C for 6 min. The uptake reaction was imtiated by the addition of SH-NE (21 Ci/mmole, New England Nuclear; 100nM, unless otherwise indicated) and the incubation was continued at 370 C for the time indicated. The final reaction volume was 1 ml. In some experiments the reaction conditions were altered in the following manner: A carrying out the premcubation and incubation at 0°C, B. omitting the NaC1 from the KRH (hypotonic buffer) or C. replacing the NaCI with an equal molar concentration of choline chloride (isotomc low sodium medium) The reaction was terminated by the addition of 5ml room temperature KRH followed by vacuum (13psi) filtration through Whatman GF/B filters This stopping procedure included two additional washes (5 ml each) and was completed within 10 sec. The amount of radioactivity retained on each filter was determined by liquid scintillation counting. Each assay consisted of triplicate or quadruplicate determinations and each experiment was repeated at least twice Where appropriate (fig 1 and 4b) the data were analyzed by hnear regression analysis Group means were compared by ANOVA
Vol. 48, No. 13, 1991
NE Uptake by Heart Synaptosomes
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Results
Accumulation of SH-NE by cardiac P2 in the absence of DMI is time dependent and protein concentration dependent Addition of the neuronal uptake blocker, DMI (lpM), attenuates NE accumulation. The DMI-sensitive NE uptake (calculated as the difference in NE accumulation in the absence of DMI minus that in the drug's presence) is linear for at least 20 mm (fig la). Likewise, DMI-sensitive NE uptake is tissue concentration dependent, being hnear between 150 and 500 ug ofP 2 protein per tube (fig. lb). Thus, subsequent studies were carried out within these time and protein concentratmn ranges. 7
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PROTEIN(#g)
TIME (rain)
FIG. 1 Time and tissue dependence
of NE accumulation
NE accumulatmn was determined by incubating ahquots of P2 suspensmn at 37°C wlth SH-NE (100 nM) in the presence ( O ) or absence ( • ) of 1 pM DMI Net (neuronal) uptake ( ~ ) ]s defined as the difference m accumulation with or without DMI A To determine time dependence a constant amount of P2 (400 pg protein) was incubated for 2, 5, 10 and 20 mm before the reaction was terminated B To determine tissue dependence varying concentrations of P2 protein were incubated with 3H-NE for a constant time (10 ram) Each point represents the mean and standard devmtlon for three determinations The hne for DMi sensitive accumulatmn was determined by hnear regresmon analyms
As an index of ennchment of nerve terminals in the P2 fraction, the concentration of endogenous NE was determined m the homogenate and the resulting P2 from four independent hearts The NE concentration m the P~ (36 7 [+ 5 4] ng per mg protein) was significantly enriched (p< 0.01) compared to the concentration in the homogenate (11.0 [+. 1.5] ng per mg protein). To assess the reproducibihty of the functional characteristics of the cardiac P2, the initial rate of NE uptake (at 100 nM SH-NE) by P2 prepared from 10 separate hearts was determined; the rate of uptake was 0 17 (+ 0.02 SEM) pmoles/mm/mg protein. This small variation indicates that tins method yields a subcellular fraction with consistent properties.
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NE Uptake by Heart Synaptosomes
Vol. 48, No. 13, 1991
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HYPOTONIC
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FIG. 2 I n f l u e n c e o f v a r i o u s factors on cardiac Ps NE a c c u m u l a t i o n . Aliquote of P~ suspenmon were incubated at 37°C with 8H-NE (100 nM) for 10 mm at 370 C m KRH (control), m the presence of I pM DMI (DMI), in hypotomc buffer (hypotomc), in low sodium buffer (low Na*), or at 0 ° C in KRH (0° C). Each bar represents the mean and standard dewatlon for four determinations All treatments mgmficantiy attenuated NE accumulation relatwe to control (p<0 001 ANOVA), but were not s]gmficantiy different from each other
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FIG. 3 I n f l u e n c e o f DMI a n d m e t a n e p h r i n e on NE a c c u m u l a t i o n . Ahquote ofP 2 suspension were incubated at 37°C with SH-NE (100 nM) plus the indicated drug A. The dose effect curve tur DMI mhlbation of NE accumulation was calculated reiatave to the accumu|atlon observed m the presence of (I~M) DMI B Allquote of P2 were incubated with 8H-NE (100 nM) m the presence of 0, 1 or 10 ~Vl metanephrine with or w~thout DMI (I~M) Each point represents the mean and standard dewatlon for three determlnataons Metanephrine did not slgmficantly attenuate NE uptake
Vol. 48, No. 13, 1991
NE Uptake by Heart Synaptosomes
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Several experiments were performed to characterize NE accumulation into cardiac P2 NE uptake was markedly attenuated by reducing the incubation temperature from 37°C to 0°C (fig. 2) indicating that this uptake is a temperature sensitive process. Reduction of the sodium concentration to 28 mM while maintaining isotonicity by substitution of choline for sodium also markedly reduced NE uptake (fig. 2). Additionally, three experiments were performed to examine whether the SH-NE is accumulating within osmotically sensitive compartment(s) as is characteristic for a synaptosomal preparation (11). Following SH-NE accumulation under standard conditions, the stopping procedure was modified by washing the P2 fraction with dmtilled water instead of KRH. This modification resulted in an approximately 50% reduction of the amount of aH-NE retained on the filter (data not shown). Furthermore, perforrmng the uptake assay m hypotonic buffer (approximately 80 mOs) instead of the normal isotonic KRH buffer (360 mOs) also resulted in a significant attenuation of NE uptake (fig 2). Likewise, preincubation of the P~ fraction in hypotonic buffer, followed by assaying the NE uptake in normal isotonic KRH resulted m the same significant attenuatmn of NE uptake The sensitivity of cardiac P2 NE accumulation to inhibition by DMI and metanephrine was also examined Incubation of the P2 fraction in the presence of varying concentratmns of the neuronal uptake inhibitor, DMI resulted in a concentratmn dependent inhibition of accumulatmn; the ICso value is approximately 10nM and maximum inhibition occurs at 100 nM (fig 3a). However, the nonneuronal uptake2 inhibitor, metanephrine (up to 10 pM) with or without DMI (lpM) did not significantly attenuate SH-NE accumulation (fig 3b). '
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FIG. 4 K i n e t i c s of NE a c c u m u l a t i o n by c a r d i a c Ps.
A. The imtml rate of NE accumulation was determined at several NE concentrataons in the absence ( • ) and presence (C) ) of DMI (1 pM) The triangle represents the DMI senmtive uptake ( /x ) defined as the difference in accumulation with or without DMI Each point represents the mean of triphcate determinations for a typical experiment B The Hofstee plot transformation of the DMI sensitive uptake for the experiment shown m part A. The experiment was repeated five t~mes with similar results
~F~ ~ 30
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NE Uptake by Heart Synaptosomes
Vol. 48, No. 13, 1991
A kinetic analysis of the NE accumulation by rat heart P2 was performed by Incubating the P~ with varying concentrations of NE with and without DMI (1 ~M). Tlus supramaxlmal concentration of DMI (based on the results presented in fig. 3) was employed to determine the amount of SH-NE retained on the filter by mechanisms other than uptake 1 The amount of NE retained on the filter increased as a function of the NE concentration both in the presence and absence of DMI (fig. 4). However, the DMI sensitive NE uptake (calculated as the difference in NE accumulation in the absence of DMI minus that in the drug's presence) proved to be a saturable process (fig. 4) Hofstee transformation of this DMI-sensitive NE accumulation, demonstrates that the uptake fits a straight line suggesting a single uptake mechanism. Five independent experiments demonstrated that this uptake process has a KMvalue of 431 (+ 30 SEM) nM and a VM~ of 1 10 (+0 24 SEM) pmoles/min/mg protein (fig 4). Discussion
In the present study we have characterized the uptake of NE by a crude mitochondrial-synaptosomal fraction prepared from rat heart by a modification of procedures routinely used to obtain the corresponding fraction from rat brain (3). As an index of intact nerve terminals we have determined the enrichment of endogenous NE in the P2 relative to the initial homogenate. The rational for use of this index is that NE is concentrated within the nerve terminals and even a temporary disturbance of the integrity of the plasma membrane will result in the loss of endogenous NE. By use of this criteria, the nerve terminal in cardiac P2 fraction is enriched more than 3 fold. This enrichment is approximately the same as that obtained in brain P2 (12). The initial velocity of NE uptake, deternnned in 10 independent P2s vaned by less than 12%, demonstrating that this procedure ymlds a reproducible preparation The uptake observed in cardiac P2 satisfies several criteria for synaptosomal uptake and consists of a single uptake process These studies were vahdated, in part, by demonstrating that NE accumulation is dependent on the length ofmcubatlon and concentration of P2 Both uptake and binding, result in NE accumulation which is time and protein concentration dependent. However, uptake may be distinguished from binding by subjecting the tissue fraction to hypotonic medium (11). Since uptake results m accumulation of NE within a sealed membrane, exposure to hypotonic medium causes membrane disruption and thus release of the stored contents. In contrast, bound ligand would not be affected by hypotonic shock. Exposure of cardiac P2 to hypotonic medmm either before or after incubation with SH-NE markedly attenuated the retention of 3H-NE. Thus, the osmotically sensitive NE accumulation represents uptake into sealed compartment(s), while the osmotically insensitive NE may represent binding (fig 2) As has been reported for NE uptake by brain synaptosomes (3,13), NE uptake by rat heart P2 is temperature sensitive; incubation of the P2 at 0° C significantly mhibits uptake (fig. 2) This observation suggests that the uptake is an energy dependent process Furthermore, NE accumulation by rat brain synaptosomes is critically dependant upon the concentration of sodium lens in the mcubatlon medium (3,14). For cardiac P2, reduction of sodium in the medium to 28 mM (by the equal molar replacement of sodium by choline) markedly attenuates the uptake of NE. This observation suggests that the NE uptake process m cardiac P2 operates via sodmm exchange as has been demonstrated in other NE uptake systems (15) The uptake
Vol
48, No, 13, 1991
NE Uptake by Heart Synaptosomes
1323
observed under conditions of low temperature, hypotonicity and low sodium ion concentratmn reduced the level of NE accumulatmn to that observed in the presence of 1 ~M DMI (fig. 2) suggesting a single uptake process. Furthermore, neuronal uptake (as demonstrated by the use of a supramaximal concentration of DMI), is of equal magnitude suggesting that all these factors reflect the same neuronal-type uptake process. Rat heart NE uptake was further characterized by the use of specific blockers for uptake1 (DMI) and uptake2 (metanephrine) The observation that 100nM DMI completely inhibits NE accumulation while metanephrine (up to 10~M) does not significantly attenuate this process, suggests that in heart P~, NE is accumulated by a neuronal, uptake 1 mechamsm (1). The ICso value for DMI inhibition of NE accumulation obtained m our studies (fig 3a) is m agreement with the ICso values reported for DMI inhibition of SH-NE uptake in perfused rat heart and in rat brain P2 (1,16) Further evidence that NE uptake into rat heart P2 occurs by a single process was obtained by a kinetic analysis. The initial velocity of NE accumulation into rat heart P2 is dependent upon the concentration of NE in the medium. Furthermore, as the concentration of NE is increased, the DMI-sensit~ve uptake process exhibits saturation (fig 4a). Hofstee analyms of the change in uptake velocity as a function of NE concentration, indicates that a single uptake process accounts for the observed accumulation of NE in heart P~. The apparent KMof 431 nM is m agreement with the KM'S observed by Iversen (1) for uptake1 in whole heart ( KM = 270 nM) and by Wong and coworkers (17) using whole brain P2 (KM = 580 nM) From the Hofstee analysis, the maximum rate of NE uptake (on a per mg protein basis) is approximately 11 pmole per nnn per mg protein In comparison, the VM~ for NE uptake by brain P2 is approximately 8 pmole per min per mg protein (17). These differences in VM~ may reflect the relative enrichment of the uptake compartment (presumably synaptosomes) These data suggest the existence of neuronal (type1) NE uptake and the absence of significant non-neuronal (type2) uptake in tlns isolated subcellular fraction (P2) from rat heart This model, representing functional synaptosomes from rat heart, will be of future interest for the investigation of the possible changes m cardiac NE accumulation after pharmacological interventions or as a consequence of physiologmal changes such as aging. Acknowledgements This work was supported by a grant from the National Institute on Aging (AG03362) and by funds from the Commonwealth of Pennsylvania, Department of Public Health, Office of Mental Health. References
L.L IVERSEN, Br. J Pharmacol. 4_! 571-591 (1971). L L IVERSEN, Br. Med. Bull. 2._99130-135 (1973). R W. COLBURN, F K GOODWIN, D.L MURPHY, W E. BUNNEY, JR., J M DAVIS, Biochem Pharmacol 1__77957-964 (1968).
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NE Uptake by Heart Synaptosomes
Vol. 48, No. 13, 1991
V.P. WHITTAKER, The Synaptosomes. In Lajtha A, Ed. Handbook of Neurochemistry. 7 1-39 (1984). W.S WILSON, J R. COOPER, J Neurochem 1._992779-2790 (1972) F A. OPMEER, A. WITTER, J.M.V REE, Naunyn-Schnnedeberg's Arch Pharmacol. 321 1-6 (1982). R.N DALY, P.B GOLDBERG, J ROBERTS, J Pharm. Exp Ther. 245 798-803, (1988) R.N. DALY, P.B GOLDBERG, J. ROBERTS, J. Gerontol. 44 B59-B66 (1989) M BRADFORD Anal Blochem. 72 248 (1976). M.S. KREIDER, P B. GOLDBERG, J ROBERTS, J Pharm Exp Ther. 231:367372 (1984). G.M. JONAKAIT, A.R. GINTZLER, M D. GERSHON, J. Neurochem. 32 13871400 (1979). R SIMANTOV, A.M SNOWMAN, S H. SNYDER, Brain ires 107 650- 657 (1976). R M FERRIS, F.L M TANG, R A MAXWELL, J. Pharm. Exp Ther. 181 407416, (1972). R.P SHANK, C.R SCHNEIDER, J.J TIGHE, J. Neurochem. 49 381-388, (1987). D.F BOGDANSKE, T.P BLASZKOWSKI, A.H TISSARI, Blochm Biophys. Acta 211 521-532 (1970) R.A MAXWELL, R.M. FERRIS, J BURCSU, E C. WOODWARD, D TANG, K WILLIARD, J. Pharm Exp. Ther. 191 418-430, (1974). D T WONG, J.S HORNG, F P. BYMASTER, Life Sci 17 755-760 (1975)
Pergamon Press
CHARACTERIZATION OF NOREPINEPHRINE ACCUMULATION BY A CRUDE SYNAPTOSOMAL-MITOCHONDRIAL FRACTION ISOLATED FROM RAT HEART V. J. Aloyo1, H. B. Mcllvain 1, V. H. Bhavsar 1'2 and J. Roberts ~ 1Department of Pharmacology, Medical College of Pennsylvania, 3200 Henry Ave., Philadelphia, PA 19129, USA and 2ECFMG Fellow, Department of Pharmacology, Government Medical College, Surat, India. (Recelved in final form January 24, 1991)
Summary Norepinephrine (NE) uptake into a heart synaptosomalmitochondrial fraction was assessed under conditions where neuronal uptake (type 1) was linear with respect to both time and protein concentration. The NE accumulation process was sensitive to incubation temperature, sodium ion concentration and medium osmolality. Furthermore, NE uptake was attenuated by the neuronal uptake inhibitor desmethylimipranune (DMI) m a concentration dependent manner; the ICso value was approximately 10 nM and maximum inhibition was obtained at 100 nM. In contrast, the extraneuronal uptake inhibitor, metanephrine did not sigmficantly attenuate NE uptake. Kinetic analysis demonstrated that the DMI sensitive NE accumulation is saturable with a KM of approximately 400 rLM and that NE uptake occurs wa a single uptake process. This demonstration of neuronal type NE uptake by a synaptosomal-mltochondrial fraction constitutes a successful demonstration of the preparation of a rat heart subcellular fraction containing functional synaptosomes Cellular accumulation of norepinephrine (NE) occurs via two processes (1,2). Type 1 uptake has a high affinity for NE and is selectively inhibited by DMI. In contrast, type 2 uptake has a much lower affinity for NE and is preferentially inhibited by metanephrine. Type 1 uptake is observed in adrenergic neurons derived from both brain and cardiac tissue, while type 2 represents NE uptake into non-neuronal tissue. Neuronal uptake is a saturable, energy dependent process which is critically dependent upon the concentration of extracellular sodium ions (3). The use of isolated subcellular fractions containing pinched off nerve endings (synaptosomes) from brain tissue has been widely used to characterize neuronal uptake of neurotransmlttors, amino acids and other substances (4). However, the application of siimlar techniques to other tissues has been linnted, in part, because the homogemzation of these tissues results in the destruction of nerve terminals rather than the formation ofsynaptosomes (4) In some cases, pretreatment of the tissue with 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
1318
NE Uptake by Heart Synaptosomes
Vol. 48, No. 13, 1991
proteolytic enzymes can facilitate homogenization and improve formation of synaptosomes (5,6). During the course of our studies on changes in neurotransmission in the heart as a functmn of age (7,8), we dec~ded to investigate NE uptake using a subcellular fraction prepared from rat heart containing synaptosomes. In the present study, we have employed collagenase pretreatment of rat heart in order to prepare a crude synaptosomal-mitochondrial fraction (P2) and have characterized NE accumulation by this fraction. Materials and Methods Preparation of crude synaptosomal-mitochondrial fraction (P~) from rat heart Immediately following sacrifice of the six month old male Fisher 344 rat, the heart was rapidly removed and minced in ice-cold 0 32 M sucrose containing 1 mM EGTA, pH 7.5 The mince was transferred to a HEPES buffered Krebs-Ringer solution (KRH; HEPES 50 mM, NaC1 144 mM, MgC12 1.2 mM, CaC12 1.2 mM, KC1 5 mM, glucose 10 mM, ascorbic acid 1 raM, and pargyline 1 mM, pH 7.4) containing 12 units of collagenase (class II, Worthington Blochemicals) per mg of tissue. This suspension was then incubated at 370 C for 40 rain with continuous bubbling with oxygen. Subsequent to collection of the resulting tissue fragments by low speed centrifugation (120xg), the tissue was disrupted in 10 volumes (by weight) of ice-cold 0 32 M sucrose with a teflon/glass homogemzer (clearance 0.25 mm). Cellular debris, nuclei and large tissue shards were removed by centrifugation at 650xg for 10 min at 4 o C. The resulting supernatant was centrifuged at 21,000xg for 20 min at 4o C The pellet (P2) was resuspended in oxygenated, ice-cold KRH using a teflon/glass homogemzer (clearance 0 25 mm) and immediately used for experimentation. Protein concentration was determined by the Bradford method (9) In some experiments the concentration of endogenous NE in rat heart homogenate and the resulting P2 fraction was deterlnined using HPLC-EC methodology as described by Kreider and coworkers (10). NE uptake studies Aliquots of P2 suspenmon were prelncubated in KRH (with or without the indicated drug) at 37°C for 6 min. The uptake reaction was imtiated by the addition of SH-NE (21 Ci/mmole, New England Nuclear; 100nM, unless otherwise indicated) and the incubation was continued at 370 C for the time indicated. The final reaction volume was 1 ml. In some experiments the reaction conditions were altered in the following manner: A carrying out the premcubation and incubation at 0°C, B. omitting the NaC1 from the KRH (hypotonic buffer) or C. replacing the NaCI with an equal molar concentration of choline chloride (isotomc low sodium medium) The reaction was terminated by the addition of 5ml room temperature KRH followed by vacuum (13psi) filtration through Whatman GF/B filters This stopping procedure included two additional washes (5 ml each) and was completed within 10 sec. The amount of radioactivity retained on each filter was determined by liquid scintillation counting. Each assay consisted of triplicate or quadruplicate determinations and each experiment was repeated at least twice Where appropriate (fig 1 and 4b) the data were analyzed by hnear regression analysis Group means were compared by ANOVA
Vol. 48, No. 13, 1991
NE Uptake by Heart Synaptosomes
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Results
Accumulation of SH-NE by cardiac P2 in the absence of DMI is time dependent and protein concentration dependent Addition of the neuronal uptake blocker, DMI (lpM), attenuates NE accumulation. The DMI-sensitive NE uptake (calculated as the difference in NE accumulation in the absence of DMI minus that in the drug's presence) is linear for at least 20 mm (fig la). Likewise, DMI-sensitive NE uptake is tissue concentration dependent, being hnear between 150 and 500 ug ofP 2 protein per tube (fig. lb). Thus, subsequent studies were carried out within these time and protein concentratmn ranges. 7
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5
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15
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0
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60
PROTEIN(#g)
TIME (rain)
FIG. 1 Time and tissue dependence
of NE accumulation
NE accumulatmn was determined by incubating ahquots of P2 suspensmn at 37°C wlth SH-NE (100 nM) in the presence ( O ) or absence ( • ) of 1 pM DMI Net (neuronal) uptake ( ~ ) ]s defined as the difference m accumulation with or without DMI A To determine time dependence a constant amount of P2 (400 pg protein) was incubated for 2, 5, 10 and 20 mm before the reaction was terminated B To determine tissue dependence varying concentrations of P2 protein were incubated with 3H-NE for a constant time (10 ram) Each point represents the mean and standard devmtlon for three determinations The hne for DMi sensitive accumulatmn was determined by hnear regresmon analyms
As an index of ennchment of nerve terminals in the P2 fraction, the concentration of endogenous NE was determined m the homogenate and the resulting P2 from four independent hearts The NE concentration m the P~ (36 7 [+ 5 4] ng per mg protein) was significantly enriched (p< 0.01) compared to the concentration in the homogenate (11.0 [+. 1.5] ng per mg protein). To assess the reproducibihty of the functional characteristics of the cardiac P2, the initial rate of NE uptake (at 100 nM SH-NE) by P2 prepared from 10 separate hearts was determined; the rate of uptake was 0 17 (+ 0.02 SEM) pmoles/mm/mg protein. This small variation indicates that tins method yields a subcellular fraction with consistent properties.
1320
NE Uptake by Heart Synaptosomes
Vol. 48, No. 13, 1991
09
08
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07
06
05
DMI
CONTROL
HYPOTONIC
LOW No
O'C
FIG. 2 I n f l u e n c e o f v a r i o u s factors on cardiac Ps NE a c c u m u l a t i o n . Aliquote of P~ suspenmon were incubated at 37°C with 8H-NE (100 nM) for 10 mm at 370 C m KRH (control), m the presence of I pM DMI (DMI), in hypotomc buffer (hypotomc), in low sodium buffer (low Na*), or at 0 ° C in KRH (0° C). Each bar represents the mean and standard dewatlon for four determinations All treatments mgmficantiy attenuated NE accumulation relatwe to control (p<0 001 ANOVA), but were not s]gmficantiy different from each other
10
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FIG. 3 I n f l u e n c e o f DMI a n d m e t a n e p h r i n e on NE a c c u m u l a t i o n . Ahquote ofP 2 suspension were incubated at 37°C with SH-NE (100 nM) plus the indicated drug A. The dose effect curve tur DMI mhlbation of NE accumulation was calculated reiatave to the accumu|atlon observed m the presence of (I~M) DMI B Allquote of P2 were incubated with 8H-NE (100 nM) m the presence of 0, 1 or 10 ~Vl metanephrine with or w~thout DMI (I~M) Each point represents the mean and standard dewatlon for three determlnataons Metanephrine did not slgmficantly attenuate NE uptake
Vol. 48, No. 13, 1991
NE Uptake by Heart Synaptosomes
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Several experiments were performed to characterize NE accumulation into cardiac P2 NE uptake was markedly attenuated by reducing the incubation temperature from 37°C to 0°C (fig. 2) indicating that this uptake is a temperature sensitive process. Reduction of the sodium concentration to 28 mM while maintaining isotonicity by substitution of choline for sodium also markedly reduced NE uptake (fig. 2). Additionally, three experiments were performed to examine whether the SH-NE is accumulating within osmotically sensitive compartment(s) as is characteristic for a synaptosomal preparation (11). Following SH-NE accumulation under standard conditions, the stopping procedure was modified by washing the P2 fraction with dmtilled water instead of KRH. This modification resulted in an approximately 50% reduction of the amount of aH-NE retained on the filter (data not shown). Furthermore, perforrmng the uptake assay m hypotonic buffer (approximately 80 mOs) instead of the normal isotonic KRH buffer (360 mOs) also resulted in a significant attenuation of NE uptake (fig 2). Likewise, preincubation of the P~ fraction in hypotonic buffer, followed by assaying the NE uptake in normal isotonic KRH resulted m the same significant attenuatmn of NE uptake The sensitivity of cardiac P2 NE accumulation to inhibition by DMI and metanephrine was also examined Incubation of the P2 fraction in the presence of varying concentratmns of the neuronal uptake inhibitor, DMI resulted in a concentratmn dependent inhibition of accumulatmn; the ICso value is approximately 10nM and maximum inhibition occurs at 100 nM (fig 3a). However, the nonneuronal uptake2 inhibitor, metanephrine (up to 10 pM) with or without DMI (lpM) did not significantly attenuate SH-NE accumulation (fig 3b). '
B.
A. 12
Z L,a
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e,
3 E E 2 o
c E
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~o
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~
O4
I
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0 O0
02
04
06
08
10
0
I 10
[Nc] ~,u
I 20 VELOCITY/ENE] ~ M
FIG. 4 K i n e t i c s of NE a c c u m u l a t i o n by c a r d i a c Ps.
A. The imtml rate of NE accumulation was determined at several NE concentrataons in the absence ( • ) and presence (C) ) of DMI (1 pM) The triangle represents the DMI senmtive uptake ( /x ) defined as the difference in accumulation with or without DMI Each point represents the mean of triphcate determinations for a typical experiment B The Hofstee plot transformation of the DMI sensitive uptake for the experiment shown m part A. The experiment was repeated five t~mes with similar results
~F~ ~ 30
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NE Uptake by Heart Synaptosomes
Vol. 48, No. 13, 1991
A kinetic analysis of the NE accumulation by rat heart P2 was performed by Incubating the P~ with varying concentrations of NE with and without DMI (1 ~M). Tlus supramaxlmal concentration of DMI (based on the results presented in fig. 3) was employed to determine the amount of SH-NE retained on the filter by mechanisms other than uptake 1 The amount of NE retained on the filter increased as a function of the NE concentration both in the presence and absence of DMI (fig. 4). However, the DMI sensitive NE uptake (calculated as the difference in NE accumulation in the absence of DMI minus that in the drug's presence) proved to be a saturable process (fig. 4) Hofstee transformation of this DMI-sensitive NE accumulation, demonstrates that the uptake fits a straight line suggesting a single uptake mechanism. Five independent experiments demonstrated that this uptake process has a KMvalue of 431 (+ 30 SEM) nM and a VM~ of 1 10 (+0 24 SEM) pmoles/min/mg protein (fig 4). Discussion
In the present study we have characterized the uptake of NE by a crude mitochondrial-synaptosomal fraction prepared from rat heart by a modification of procedures routinely used to obtain the corresponding fraction from rat brain (3). As an index of intact nerve terminals we have determined the enrichment of endogenous NE in the P2 relative to the initial homogenate. The rational for use of this index is that NE is concentrated within the nerve terminals and even a temporary disturbance of the integrity of the plasma membrane will result in the loss of endogenous NE. By use of this criteria, the nerve terminal in cardiac P2 fraction is enriched more than 3 fold. This enrichment is approximately the same as that obtained in brain P2 (12). The initial velocity of NE uptake, deternnned in 10 independent P2s vaned by less than 12%, demonstrating that this procedure ymlds a reproducible preparation The uptake observed in cardiac P2 satisfies several criteria for synaptosomal uptake and consists of a single uptake process These studies were vahdated, in part, by demonstrating that NE accumulation is dependent on the length ofmcubatlon and concentration of P2 Both uptake and binding, result in NE accumulation which is time and protein concentration dependent. However, uptake may be distinguished from binding by subjecting the tissue fraction to hypotonic medium (11). Since uptake results m accumulation of NE within a sealed membrane, exposure to hypotonic medium causes membrane disruption and thus release of the stored contents. In contrast, bound ligand would not be affected by hypotonic shock. Exposure of cardiac P2 to hypotonic medmm either before or after incubation with SH-NE markedly attenuated the retention of 3H-NE. Thus, the osmotically sensitive NE accumulation represents uptake into sealed compartment(s), while the osmotically insensitive NE may represent binding (fig 2) As has been reported for NE uptake by brain synaptosomes (3,13), NE uptake by rat heart P2 is temperature sensitive; incubation of the P2 at 0° C significantly mhibits uptake (fig. 2) This observation suggests that the uptake is an energy dependent process Furthermore, NE accumulation by rat brain synaptosomes is critically dependant upon the concentration of sodium lens in the mcubatlon medium (3,14). For cardiac P2, reduction of sodium in the medium to 28 mM (by the equal molar replacement of sodium by choline) markedly attenuates the uptake of NE. This observation suggests that the NE uptake process m cardiac P2 operates via sodmm exchange as has been demonstrated in other NE uptake systems (15) The uptake
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NE Uptake by Heart Synaptosomes
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observed under conditions of low temperature, hypotonicity and low sodium ion concentratmn reduced the level of NE accumulatmn to that observed in the presence of 1 ~M DMI (fig. 2) suggesting a single uptake process. Furthermore, neuronal uptake (as demonstrated by the use of a supramaximal concentration of DMI), is of equal magnitude suggesting that all these factors reflect the same neuronal-type uptake process. Rat heart NE uptake was further characterized by the use of specific blockers for uptake1 (DMI) and uptake2 (metanephrine) The observation that 100nM DMI completely inhibits NE accumulation while metanephrine (up to 10~M) does not significantly attenuate this process, suggests that in heart P~, NE is accumulated by a neuronal, uptake 1 mechamsm (1). The ICso value for DMI inhibition of NE accumulation obtained m our studies (fig 3a) is m agreement with the ICso values reported for DMI inhibition of SH-NE uptake in perfused rat heart and in rat brain P2 (1,16) Further evidence that NE uptake into rat heart P2 occurs by a single process was obtained by a kinetic analysis. The initial velocity of NE accumulation into rat heart P2 is dependent upon the concentration of NE in the medium. Furthermore, as the concentration of NE is increased, the DMI-sensit~ve uptake process exhibits saturation (fig 4a). Hofstee analyms of the change in uptake velocity as a function of NE concentration, indicates that a single uptake process accounts for the observed accumulation of NE in heart P~. The apparent KMof 431 nM is m agreement with the KM'S observed by Iversen (1) for uptake1 in whole heart ( KM = 270 nM) and by Wong and coworkers (17) using whole brain P2 (KM = 580 nM) From the Hofstee analysis, the maximum rate of NE uptake (on a per mg protein basis) is approximately 11 pmole per nnn per mg protein In comparison, the VM~ for NE uptake by brain P2 is approximately 8 pmole per min per mg protein (17). These differences in VM~ may reflect the relative enrichment of the uptake compartment (presumably synaptosomes) These data suggest the existence of neuronal (type1) NE uptake and the absence of significant non-neuronal (type2) uptake in tlns isolated subcellular fraction (P2) from rat heart This model, representing functional synaptosomes from rat heart, will be of future interest for the investigation of the possible changes m cardiac NE accumulation after pharmacological interventions or as a consequence of physiologmal changes such as aging. Acknowledgements This work was supported by a grant from the National Institute on Aging (AG03362) and by funds from the Commonwealth of Pennsylvania, Department of Public Health, Office of Mental Health. References
L.L IVERSEN, Br. J Pharmacol. 4_! 571-591 (1971). L L IVERSEN, Br. Med. Bull. 2._99130-135 (1973). R W. COLBURN, F K GOODWIN, D.L MURPHY, W E. BUNNEY, JR., J M DAVIS, Biochem Pharmacol 1__77957-964 (1968).
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V.P. WHITTAKER, The Synaptosomes. In Lajtha A, Ed. Handbook of Neurochemistry. 7 1-39 (1984). W.S WILSON, J R. COOPER, J Neurochem 1._992779-2790 (1972) F A. OPMEER, A. WITTER, J.M.V REE, Naunyn-Schnnedeberg's Arch Pharmacol. 321 1-6 (1982). R.N DALY, P.B GOLDBERG, J ROBERTS, J Pharm. Exp Ther. 245 798-803, (1988) R.N. DALY, P.B GOLDBERG, J. ROBERTS, J. Gerontol. 44 B59-B66 (1989) M BRADFORD Anal Blochem. 72 248 (1976). M.S. KREIDER, P B. GOLDBERG, J ROBERTS, J Pharm Exp Ther. 231:367372 (1984). G.M. JONAKAIT, A.R. GINTZLER, M D. GERSHON, J. Neurochem. 32 13871400 (1979). R SIMANTOV, A.M SNOWMAN, S H. SNYDER, Brain ires 107 650- 657 (1976). R M FERRIS, F.L M TANG, R A MAXWELL, J. Pharm. Exp Ther. 181 407416, (1972). R.P SHANK, C.R SCHNEIDER, J.J TIGHE, J. Neurochem. 49 381-388, (1987). D.F BOGDANSKE, T.P BLASZKOWSKI, A.H TISSARI, Blochm Biophys. Acta 211 521-532 (1970) R.A MAXWELL, R.M. FERRIS, J BURCSU, E C. WOODWARD, D TANG, K WILLIARD, J. Pharm Exp. Ther. 191 418-430, (1974). D T WONG, J.S HORNG, F P. BYMASTER, Life Sci 17 755-760 (1975)