CHLOROTETRACYCLINE-ASSOCIATED FLUORESCENCE CHANGES DURING CALCIUM UPTAKE AND RELEASE BY RAT BRAIN SYNAPTOSOMES' WALTERT. SCHAFFER' and MERLES. O L S O N ~ Department of Biochemistry, College of Medicine, University of Arizona. Tucson, AZ 85724. U.S.A. (Receiwd 15 January 1976. Accepted 24 Mu!.1976) Abstract-The fluorescent divalent metal chelate-probe, chlorotetracycline (CTC), was used as a dynamic monitor of calcium association with rat brain snynaptosomes. The determined fluorescence excitation and emission maxima, 412 nm and 522 nm respectively, were used to monitor membrane-calcium interactions as a function of various parameters. Positive correlations were observed between increased or decreased fluorescence quantum yield and the uptake of both CTC and 45Ca by synpatosomes. The divalent metal ionophore A23187 enhanced fluorescence as well as probe and "Ca uptake. Whereas, the polar chelator, EGTA, markedly reduced fluorescence. and the synaptosomal bound CTC and 45Ca. The CTC fluorescence changes also demonstrated the saturable manner in which 45Ca bound synaptosomes. At concentrations greater than 100 pg/ml. CTC bound to the synaptosomes in a manner which quenched fluorescence at 522 nm. Also, CTC. at concentrations above I 5 &ml, enhanced the uptake of 4sCa. At CTC concentrations between 10 and 15 &rnl the quenching and ionophoretic properties of the probe were minimized without affecting the capability of using the probe to visualize calcium interactions with synaptosomal membranes. Also, at a low CTC concentration (12.5 pg/ml) the inhibition of calcium uptake by increasing monovalent ion concentrations was clearly demonstrated.
BECAUSEcalcium ions have been implicated in the process of neurotransmitter release from neurons (KATZ& MILEDI,1967; KATZ, 1969; REDBURN et al., 1976). various studies have been initiated to characterize calcium fluxes in isolated mammalian presynaptic nerve terminals (synaptosomes). The effects o n calcium uptake of internal and/or external monovalent ion concentrations (BLAUSTEIN, 1975 ; BLAUSTEIN & OBORN,1975; KAMINOet a/., 1974; KAMINOef a/., 1975; LAZAREWICZ et a/., 1974; SWANSON et al., 1974) numerous drugs (BLAUSTEIN, 1975; SWANSONet al., 1974) and metabolic substrates (LAZAREWICZ et al.,
NUSON. 1973 : BREWER, 1974). sarcoplasmic reticular and phospholipid vesicles (JILKA ef a/.. 1975) and neuronal systems (HALLETT et a/., 1972). The present experiments were performed to document the usefulness of the fluorescent chelate probe, chlorotetracycline, for monitoring calcium-membrane interactions In synaptosomal systems.
MATERIALS AND METHODS
Synaptosomes were isolated from the whole brains of male and female Sprague-Dawley Rats (300-500 g) using either the procedure described by GRAY& WHITTAKER 1974; LUST & ROBINSON,1970; SWANSON et al., 1974) (1962) or more recently that of HAJOS(1975).Both of these have been investigated. In each of the studies, refer- methods employ differential and gradient centrifugation enced above. rather laborious sampling and assay schemes in sucrose solutions. 4sCa and chlorotetracycline uptake under a variety of incubation conditions were techniques were employed t o measure changes in synaptosomal calcium levels under various exper- nearly identical in the Hajos and in the Gray-Whittaker synaptosomal preparations, justifying our use of the less imental conditions. Recently the fluorescent divalent time consuming Hajos preparation. The type of prepmetal chelate-probe, chlorotetracycline, has been aration used in individual experiments is indicated in the employed to provide a continuous monitor of calcium legends of the various figures. Following preparation, the ions associated with various types of cellular memsynaptosomes were stored at 4 C in isotonic sucrose. at branes in mitochondria1 (CASWELL& HUTCHISON, approx 20mg synaptosomal protein per ml for no more 1971; CASMLL, 1972; SCHUSTER & OLSON. 1973, than 4 h prior to use in the various experiments. 1967; DOCKER& MAG1974), bacterial (FRANKLIN. Timed incubations were performed at room temperature in isotonic solutions of sucrose and/or sodium chloride buffered with Tris-chloride, 20 mM. pH 7.4. Water used in I Supported by NIH Grant Number HL-16111 and a the preparation of the various isolation and incubation Training Grant from the U.S.P.H.S. solutions was distilled and deionized. For the uptake exA bhreuiation used: CTC, Chlorotetracycline. Present address: Department of Biochemistry, Univer- periments described in Figs. 4. 5 and 6 the incubations sity of Texas Health Science Center, 7703 Floyd Curl were initiated with the addition of synaptosomes to the incubation tncdium. Drive, San Antonio, TX 78284. U.S.A. 1319
W. T. SCHAFFFRand MERLES. OLsoN
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The association of calcium with the synaptosomal membranes was visualized using the lipophilic divalent metal & HUTCHISOX. chelate-probe, chlorotetracycline (CASWELL 1971; CASWELL, 1972: HALLETIef ol.. 1972: SCHUSTER& OLSON, 1973. 1974). The fluorescence changes were monitored with a Perkin-Elmer Model M PF-ZA spectrofluorometer with excitation and emission wavelengths of 412 nm and 522 nm respectively. These wabelengths were determined as described in the Results. Excitation and emission slits were no wider than 4 p m in order to minimize contributions from scattered light. The uptake of 45Ca was measured by adding 0.1 pCi,ml of incubation mixture. in which the total calcium chloride concentration was 0.5 mY. The incubation vessel was continuously stirred and samples were removed from the vessel at appropriate times using a 0.5 ml Eppendorf automatic pipet. The samples were centrifuged rapidly in an Eppendorf Microcentrifuge at approx. 9000 g for 1 min. The supernatant was aspirated rapidly and the pellet was dissolved in 0.5 ml of l " , sodium dodecyl sulfate. The solub i k e d pellet was transferred to a scintillation vial containing 10 ml of Aquasol (New England Nuclear. Boston. MA1 and counted using standard procedures. "5Ca uptake by the synaptosomal preparations was calculated as nmol per mg protein from the measured specific radioactivity of the added "Ca. It should be noted that the amount of adhering solution remaining in the pellet after aspiration of the supernatant was estimated, using C3H]inulin (Amersham: Searle, Amersham. England), to be less than I", of the total sample. Therefore, '5Ca and chlorotetracycline uptake data were not corrected for adherence. Protein concentrations of the synaptosomes were estimated by a biuret
procedure ( LAYNE.1957) using crystallized bovine serum albumin as the standard. Chlorotetracycline uptake by the synaptosomes was measured by removing samples from the incubation mixture. sedimenting the synaptosomal membranes by high speed centrifugation and dissolving the resultant pellet in I"(, sodium dodecyl sulfate. The solubilized pellet was diluted to 2 m l with l " , sodium dodecyl sulfate and the absorbance of the sample was measured at 388 nm. In data not shown an absorbance maximum of 388nm was observed for chlorotetracycline in 1"; sodium dodecyl sulfate. Using an appropriate standard curve chlorotetracycline uptake by the synaptosomes was reported as pg of chlorotetracycline per mg protein. Chlorotetracycline was purchased from Nutritional Biochemical Corporation, Cleveland, Ohio. The divalent metal ionophorous antibiotic A23187 was the generous gift of the Eli Lilly Laboratories. Indianapolis, Indiana. 45Ca chloride was purchased from New England Nuclear Corporation. Boston. MA, at a specific radioactivity of 11.6 mCi.'mg, All other chemicals and reagents were of the highest purity available and were purchased from common commercial suppliers.
RESULTS I n order to utilize the chelate p r o b e in a synaptosoma1 system, scans of t h e excitation a n d emission wavelengths of chlorotetracycline-treated synaptosomes were performed. Figure 1 illustrates t h e greatly increased fluorescence q u a n t u m yield associ-
Excitation
A
B
Emlssion
I
-
80
a, -
mu
0 -
3
60
C
P
a f
40
ar u
? I
3
20
300
400
500 400 Wavelength (nm)
500
600
FIG. 1. Fluorescence spectra of chlorotetracycline treated synaptosomes in the presence and absence of calcium chloride. Gray-Whittaker rat brain synaptosomes (0.95 mg protein/ml) were incubated in a medium containing sucrose. 310 mv. and Tris-chloride. 20 mM. pH 7.4 at X C . Chlorotetracycline (50 pg/ml) and calcium chloride (0.5 m u ) were added as indicated. The solid lines correspond to the spectral scans of a solution of chlorotetracycline plus calcium chloride. The dotted lines are the scans of synaptosomes incubated in the presence of chlorotetracycline. The dashed lines correspond to the scans of synaptosomes in the presence of both chlorotetracycline and calcium.
Calcium uptake and release by synaptosomes
1321
EGTA
I Chlorotetracycline Fluorescence 412nm -522nm
EGTA Added Without A23167
CaCI,
FIG.2. Chlorotetracycline-associated fluorescence changes in rat brain synaptosomes. Gray-Whittaker synaptosomes (0.95 mg protein/ml) were suspended in the sucrose-Tris+hloride buffer defined in the legend to Fig. 1. Additions of chlorotetracycline (CTC) (50 pgiml), calcium chloride (0.5 mM), A23187 (5 pg/ml). and EGTA (0.5 mM) were made as indicated. The fluorescence of the incubation was monitored using excitation and emission wavelengths of 412 and 3522 nm. respectively. ated with the interaction of chlorotetracycline, calcium chloride, and synaptosomes. Also shown in Fig. 1 are the fluorescence responses of the chelate probe to calcium chloride in the absence of synaptosomes and to synaptosomes in the absence of exogenous calcium. Scans of the excitation and emission wavelengths in a cuvette containing chlorotetracycline in the absence of both calcium and synaptosomal membranes showed no maxima whatever. In all cases, including various solutions containing buffers other than Tris-chloride, the monovalent metal cations sodium and potassium, different concentrations of probe or synaptosomal protein, or various concentrations of calcium chloride, the observed excitation maxima were 397 nm and 412 nm and the emission maximum was 522 nm. Differences in the emissive output of synaptosomal suspensions excited at 397nm or 412nm were undetectable in the experiments reported here. Although different wavelengths have been used in mitochondria1 systems (CASWELL & HUTCHISON, 1971; SCHUSTER & OLSON, 1973, 1974), the excitation-emission couple, 412 nm-522 nm was selected for this system. In Fig. 2 the addition of chlorotetracycline (CTC) to a synaptosomal suspension caused an initial decrease in fluorescence followed by a time-dependent return of the trace to slightly above the original baseline fluorescence level. Calcium chloride (0.5 mM) addition caused a rapid and extensive enhancement of fluorescence followed by a slower rate of fluorescence increase. Inclusion of the divalent metal cation specific ionophore A23187 in the incubation mixture caused a further increase in fluorescence. Addition of u c 2116-
BECAUSEcalcium ions have been implicated in the process of neurotransmitter release from neurons (KATZ& MILEDI,1967; KATZ, 1969; REDBURN et al., 1976). various studies have been initiated to characterize calcium fluxes in isolated mammalian presynaptic nerve terminals (synaptosomes). The effects o n calcium uptake of internal and/or external monovalent ion concentrations (BLAUSTEIN, 1975 ; BLAUSTEIN & OBORN,1975; KAMINOet a/., 1974; KAMINOef a/., 1975; LAZAREWICZ et a/., 1974; SWANSON et al., 1974) numerous drugs (BLAUSTEIN, 1975; SWANSONet al., 1974) and metabolic substrates (LAZAREWICZ et al.,
NUSON. 1973 : BREWER, 1974). sarcoplasmic reticular and phospholipid vesicles (JILKA ef a/.. 1975) and neuronal systems (HALLETT et a/., 1972). The present experiments were performed to document the usefulness of the fluorescent chelate probe, chlorotetracycline, for monitoring calcium-membrane interactions In synaptosomal systems.
MATERIALS AND METHODS
Synaptosomes were isolated from the whole brains of male and female Sprague-Dawley Rats (300-500 g) using either the procedure described by GRAY& WHITTAKER 1974; LUST & ROBINSON,1970; SWANSON et al., 1974) (1962) or more recently that of HAJOS(1975).Both of these have been investigated. In each of the studies, refer- methods employ differential and gradient centrifugation enced above. rather laborious sampling and assay schemes in sucrose solutions. 4sCa and chlorotetracycline uptake under a variety of incubation conditions were techniques were employed t o measure changes in synaptosomal calcium levels under various exper- nearly identical in the Hajos and in the Gray-Whittaker synaptosomal preparations, justifying our use of the less imental conditions. Recently the fluorescent divalent time consuming Hajos preparation. The type of prepmetal chelate-probe, chlorotetracycline, has been aration used in individual experiments is indicated in the employed to provide a continuous monitor of calcium legends of the various figures. Following preparation, the ions associated with various types of cellular memsynaptosomes were stored at 4 C in isotonic sucrose. at branes in mitochondria1 (CASWELL& HUTCHISON, approx 20mg synaptosomal protein per ml for no more 1971; CASMLL, 1972; SCHUSTER & OLSON. 1973, than 4 h prior to use in the various experiments. 1967; DOCKER& MAG1974), bacterial (FRANKLIN. Timed incubations were performed at room temperature in isotonic solutions of sucrose and/or sodium chloride buffered with Tris-chloride, 20 mM. pH 7.4. Water used in I Supported by NIH Grant Number HL-16111 and a the preparation of the various isolation and incubation Training Grant from the U.S.P.H.S. solutions was distilled and deionized. For the uptake exA bhreuiation used: CTC, Chlorotetracycline. Present address: Department of Biochemistry, Univer- periments described in Figs. 4. 5 and 6 the incubations sity of Texas Health Science Center, 7703 Floyd Curl were initiated with the addition of synaptosomes to the incubation tncdium. Drive, San Antonio, TX 78284. U.S.A. 1319
W. T. SCHAFFFRand MERLES. OLsoN
1320
The association of calcium with the synaptosomal membranes was visualized using the lipophilic divalent metal & HUTCHISOX. chelate-probe, chlorotetracycline (CASWELL 1971; CASWELL, 1972: HALLETIef ol.. 1972: SCHUSTER& OLSON, 1973. 1974). The fluorescence changes were monitored with a Perkin-Elmer Model M PF-ZA spectrofluorometer with excitation and emission wavelengths of 412 nm and 522 nm respectively. These wabelengths were determined as described in the Results. Excitation and emission slits were no wider than 4 p m in order to minimize contributions from scattered light. The uptake of 45Ca was measured by adding 0.1 pCi,ml of incubation mixture. in which the total calcium chloride concentration was 0.5 mY. The incubation vessel was continuously stirred and samples were removed from the vessel at appropriate times using a 0.5 ml Eppendorf automatic pipet. The samples were centrifuged rapidly in an Eppendorf Microcentrifuge at approx. 9000 g for 1 min. The supernatant was aspirated rapidly and the pellet was dissolved in 0.5 ml of l " , sodium dodecyl sulfate. The solub i k e d pellet was transferred to a scintillation vial containing 10 ml of Aquasol (New England Nuclear. Boston. MA1 and counted using standard procedures. "5Ca uptake by the synaptosomal preparations was calculated as nmol per mg protein from the measured specific radioactivity of the added "Ca. It should be noted that the amount of adhering solution remaining in the pellet after aspiration of the supernatant was estimated, using C3H]inulin (Amersham: Searle, Amersham. England), to be less than I", of the total sample. Therefore, '5Ca and chlorotetracycline uptake data were not corrected for adherence. Protein concentrations of the synaptosomes were estimated by a biuret
procedure ( LAYNE.1957) using crystallized bovine serum albumin as the standard. Chlorotetracycline uptake by the synaptosomes was measured by removing samples from the incubation mixture. sedimenting the synaptosomal membranes by high speed centrifugation and dissolving the resultant pellet in I"(, sodium dodecyl sulfate. The solubilized pellet was diluted to 2 m l with l " , sodium dodecyl sulfate and the absorbance of the sample was measured at 388 nm. In data not shown an absorbance maximum of 388nm was observed for chlorotetracycline in 1"; sodium dodecyl sulfate. Using an appropriate standard curve chlorotetracycline uptake by the synaptosomes was reported as pg of chlorotetracycline per mg protein. Chlorotetracycline was purchased from Nutritional Biochemical Corporation, Cleveland, Ohio. The divalent metal ionophorous antibiotic A23187 was the generous gift of the Eli Lilly Laboratories. Indianapolis, Indiana. 45Ca chloride was purchased from New England Nuclear Corporation. Boston. MA, at a specific radioactivity of 11.6 mCi.'mg, All other chemicals and reagents were of the highest purity available and were purchased from common commercial suppliers.
RESULTS I n order to utilize the chelate p r o b e in a synaptosoma1 system, scans of t h e excitation a n d emission wavelengths of chlorotetracycline-treated synaptosomes were performed. Figure 1 illustrates t h e greatly increased fluorescence q u a n t u m yield associ-
Excitation
A
B
Emlssion
I
-
80
a, -
mu
0 -
3
60
C
P
a f
40
ar u
? I
3
20
300
400
500 400 Wavelength (nm)
500
600
FIG. 1. Fluorescence spectra of chlorotetracycline treated synaptosomes in the presence and absence of calcium chloride. Gray-Whittaker rat brain synaptosomes (0.95 mg protein/ml) were incubated in a medium containing sucrose. 310 mv. and Tris-chloride. 20 mM. pH 7.4 at X C . Chlorotetracycline (50 pg/ml) and calcium chloride (0.5 m u ) were added as indicated. The solid lines correspond to the spectral scans of a solution of chlorotetracycline plus calcium chloride. The dotted lines are the scans of synaptosomes incubated in the presence of chlorotetracycline. The dashed lines correspond to the scans of synaptosomes in the presence of both chlorotetracycline and calcium.
Calcium uptake and release by synaptosomes
1321
EGTA
I Chlorotetracycline Fluorescence 412nm -522nm
EGTA Added Without A23167
CaCI,
FIG.2. Chlorotetracycline-associated fluorescence changes in rat brain synaptosomes. Gray-Whittaker synaptosomes (0.95 mg protein/ml) were suspended in the sucrose-Tris+hloride buffer defined in the legend to Fig. 1. Additions of chlorotetracycline (CTC) (50 pgiml), calcium chloride (0.5 mM), A23187 (5 pg/ml). and EGTA (0.5 mM) were made as indicated. The fluorescence of the incubation was monitored using excitation and emission wavelengths of 412 and 3522 nm. respectively. ated with the interaction of chlorotetracycline, calcium chloride, and synaptosomes. Also shown in Fig. 1 are the fluorescence responses of the chelate probe to calcium chloride in the absence of synaptosomes and to synaptosomes in the absence of exogenous calcium. Scans of the excitation and emission wavelengths in a cuvette containing chlorotetracycline in the absence of both calcium and synaptosomal membranes showed no maxima whatever. In all cases, including various solutions containing buffers other than Tris-chloride, the monovalent metal cations sodium and potassium, different concentrations of probe or synaptosomal protein, or various concentrations of calcium chloride, the observed excitation maxima were 397 nm and 412 nm and the emission maximum was 522 nm. Differences in the emissive output of synaptosomal suspensions excited at 397nm or 412nm were undetectable in the experiments reported here. Although different wavelengths have been used in mitochondria1 systems (CASWELL & HUTCHISON, 1971; SCHUSTER & OLSON, 1973, 1974), the excitation-emission couple, 412 nm-522 nm was selected for this system. In Fig. 2 the addition of chlorotetracycline (CTC) to a synaptosomal suspension caused an initial decrease in fluorescence followed by a time-dependent return of the trace to slightly above the original baseline fluorescence level. Calcium chloride (0.5 mM) addition caused a rapid and extensive enhancement of fluorescence followed by a slower rate of fluorescence increase. Inclusion of the divalent metal cation specific ionophore A23187 in the incubation mixture caused a further increase in fluorescence. Addition of u c 2116-
