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Characteristics of K+- and veratridine-induced release of ATP from synaptosomes prepared from dorsal and ventral spinal cord
372
&at,, Re~'earch ~ ?i (~!~,~5) ~72 ~ ;~i
BRE 20798
Characteristics of K +- and veratridine-induced release of ATP from synaptosomes prepared from dorsal and ventral spinal cord ITHOMAS D. WHITE, 1JOHN W. DOWNIE and 2RONALD A. LESLIE Departments of ~Pharmacology and 2Anatomy, Dalhousie University, Halifax, N.S. B3H 4H7 (Canada)
(Accepted December 18th. 1984) Key words: ATP release - - synaptosomes - - primary afferents - - spinal cord
K + and veratridine released 2-3 times more ATP from dorsal than from ventral spinal cord synaptosomes (P2). K+-induced release of ATP was Ca2+-dependent whereas veratridine-induced release was augmented in a Ca2+-free medium. Twenty-one to 24 days after section of the right sciatic nerve of the rat the evoked release of ATP from right dorsal synaptosomes was indistinguishable from release from left dorsal synaptosomes. Although these latter results suggest that ATP may not be a transmitter at primary afferent synapses in the spinal cord, it is possible that sciatic nerve section does not deplete the releasable pool of ATP in primary afferent terminals or that ATP is also released from interneurons in the dorsal spinal cord. In 1959, H o l t o n 2 showed that A T P was released from p e r i p h e r a l terminals when sensory nerves were stimulated antidromically, raising the possibility that this nucleotide might function as a sensory neurotransmitter. Interest has recently been r e k i n d l e d by the finding that pressure application of A T P excites a subpopulation of rat dorsal horn neurones grown in culture 3. Most n e u r o t r a n s m i t t e r s are not evenly distributed in the brain and where a relatively high concentration of a particular substance occurs, one might suspect a possible physiological role in that region. Unfortunately, total tissue contents of A T P are unlikely to reveal possible n e u r o t r a n s m i t t e r functions since only a small p r o p o r t i o n of total cellular A T P content is likely to be involved in neurotransmission. In the present study, we have characterized the e v o k e d release of A T P from synaptosomes p r e p a r e d from guinea pig spinal cord and c o m p a r e d release from dorsal and ventral cord synaptosomes. W e have also studied the effect of sciatic nerve section on e v o k e d release of A T P from rat spinal synaptosomes. U n i l a t e r a l section of rat sciatic nerve depletes the substance P content of the ipsilaterai dorsal horn 4, p r e s u m a b l y as a consequence of biochemical but n o n - d e g e n e r a t i v e changes in the sensory afferent terminals.
L u m b a r spinal cord was removed from two guinea pigs weighing 300-400 g which had been killed by decapitation. The cords were divided into dorsal and ventral halves with a scalpel and each half homogenized in 10 ml of ice-cold 0.32 M sucrose with 15 strokes of a T e f l o n - g l a s s h o m o g e n i z e r (clearance 0.25 mm). The h o m o g e n a t e s were p o o l e d and centrifuged at 1000 g for 10 min.. the supernatants decanted and saved, the pellets r e s u s p e n d e d with 7 ml of sucrose each and recentrifuged at 1000 g for 10 min. These supernatants were c o m b i n e d with the previous supernatants and centrifuged at 20,000 g for 30 min to yield a crude synaptosomal pellet (Pc)Each pellet was r e s u s p e n d e d up to 6.5 ml with Krebs-Henseleit medium containing 111 mM NaC12, 26.2 m M N a H C O 3, 1.2 m M NaHPO4, 4.7 m M KC1, 1.8 m M CaC12, 1.2 m M MgC12, 11 m M glucose and gassed with 95% 02, 5% C O 2 to maintain a p H of 7.4. In some cases the pellets were resusp e n d e d in a Ca2*-free m e d i u m otherwise identical to the above. The suspensions were p r e i n c u b a t e d for 30 min at 37 °C with periodic gassing before studies of A T P release were performed. R e l e a s e of A T P e v o k e d by elevated extracellular K ÷ or by veratridine was m o n i t o r e d directly and continuously using firefly luciferin-luciferase as described previously6.
Correspondence: T. D. White, Department of Pharmacology, Dalhousie University, Halifax, N,S. Canada B3H 4H7
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
373 Release of ATP was expressed as the maximum concentration of ATP achieved in the cuvette per mg protein, the latter determined according to Hartree~. Dorsal and ventral spinal preparations of synaptosomes from guinea pigs were fixed and examined electron-microscopically as described previously for myenteric synaptosomes 7 and occluded lactate dehydrogenase (LDH) was determined to give a measure of the relative yields of synaptosomes in dorsal and ventral P2 prep arati°nsv. In another series of 3 experiments, right sciatic nerves were sectioned 4 in 8 rats and the animals allowed to recover for 21-24 days, at which time they were decapitated, laminectomies performed and L5 and L6 dorsal roots traced to the spinal cord. Spinal cord sections consisting of segments L5 and L6 were removed to ice-cold 0.32 M sucrose and divided into left and right dorsal and left and right ventral quarters with a scalpel. The material from all 8 animals was pooled and synaptosomes (P2) prepared as described above. The P2 preparations were suspended to 3.5 ml with K r e b s Henseleit medium and preincubated for 30 min at 37 °C before studies of ATP release were performed. Both elevated extracellular K +- and the Na+-chan nel activator, veratridine, released ATP from spinal synaptosomes prepared from guinea pigs, release being 2-3 times greater from dorsal than from ventral cord preparations (Fig. la, Table I). The greater release of ATP from dorsal versus ventral preparations is not likely due to different yields of synaptosomes since the ventral preparation contained as much occluded L D H as the dorsal preparation (Table I). Occluded L D H is a measure of the amount of cytoplasm contained within intact cell membranes and gives an estimate of the number of synaptosomes present 7.
TABLE I
Evoked release of A TP from synaptosomes prepared from dorsal or ventral lumbar spinal cord of guinea pigs Material from 2 guinea pigs was pooled for each determination. Values shown are the means + S.E.M. from 5 experiments
Spinal cord
Dorsal Ventral
Evoked A TP release ( x lO~w M/mg protein) KCl (24 mM)
Veratridine (50 ~M)
12.5 + 1.5 3.92 _+ 0.95
21.0 + 1.4 9.24 + 2.47
Occluded LDH content (A OD at 340 nm/min/mg protein) 1.05 + 0.13 1,13 + 0.09
a
~-
o~ c,. I
2
DORSAL
VENTRAL
DORSAL
VENTRAL
1 rain
b
C
A
!i
~'\'v'
J I rain
Ca2+- FREE
Ca2+ FREE
Fig. 1. Evoked release of ATP from guinea pig spinal synaptosomes, a: synaptosomes were prepared from dorsal or ventral cord and depolarized with KCI or veratridine as described in text. b, e: synaptosomes from dorsal cord were prepared in Ca2+-free medium and the release of ATP by K + and veratridine determined for Ca2+-free and Ca2+-free preparations where the CaCI 2 in the medium was restored to 1.8~M.
Moreover, the dorsal preparation appeared morphologically indistinguishable from the ventral preparation under electron microscopic examination. The K+-induced release of ATP from dorsal spinal synaptosomes was Ca2+-dependent (Fig. lb) and therefore resembled the exocytotic release of putative neurotransmitters from presynaptic nerve terminals. In contrast, the veratridine-induced release of ATP was actually augmented in a CaZ+-free medium (Fig. lc), suggesting that this process could originate from other sites, or by another mechanism. The veratridineevoked release of amino acid transmitters from brain preparations is also increased in CaZ+-free medium s and similar Ca 2+ requirements have been reported previously for the K +- and veratridine-induced release of ATP from synaptosomes prepared from rat and guinea pig brains 6,v. In the rat as in the guinea pig, K + and veratridine released much more ATP from dorsal than from ventral spinal cord synaptosomes (Table II). These find-
374 TABLE II
The apparent slight increase in A T P release from
Effect of sciatic nerve section on the evoked release of A TP from synaptosomes prepared from dorsal and ventral spinal cord (L5-L6) of rats
dorsal synaptosomes prepared from the sectioned side, which was not statistically significant, could be
Right sciatic nerves were sectioned and the rats allowed to recover for 21-24 days. Material from 8 rats was pooled for each determination. Values shown are the means _+ S.E.M. from 3 experiments. Spinal cord
Dorsal left Dorsal right Ventral left Ventral right
Evoked A TP release (x lO-u M/mg protein)
Occluded L D H content (zlOD at 340 nm/min/mg protein)
KCl (24 mM)
Veratridine (50~M)
17.4 + 1.9
48.3 _+5.7
0.60 __+0.17
22.6 + 5.7
60.6 + 3.6
0.82 _+0.06
not detectable
21.2 -+ 1.8
0.62 _+0.22
not detectable
27.1 -+ 9.6
0.69 ___0.13
due to increased n u m b e r s of synaptosomes present since the occluded L D H was increased in these preparations. While these studies were in progress, Yoshioka and Jessel (personal communication) also observed that rat dorsal cord synaptosomes release more A T P than do ventral synaptosomes exposed to veratridine. Furthermore, neither dorsal rhizotomy nor spinal transection diminished the veratridine-evoked release of A T P from rat spinal synaptosomes. Although these results suggest that A T P may not be a transmitter at primary afferents, it is possible that A T P is released from intrinsic dorsal horn neurons as well as from primary afferents so that a reduction in release after dorsal rhizotomy may not be detectable. In any event, the observations that dorsal spinal synapto-
ings are consistent with the possibility that A T P
somes from both guinea pigs and rats release much
might be a n e u r o t r a n s m i t t e r in primary sensory after-
more A T P than do ventral synaptosomes and that A T P excites a subpopulation of dorsal horn n e u r o n s in culture 3 are consistent with the possibility that extraneuronal A T P may perform some function in the
ents to the spinal cord. O n the other hand, section of the right sciatic nerve, using a procedure previously shown to reduce dorsal horn substance P levels by 7 5 - 8 0 % 4, did not reduce the K ÷- or veratridineevoked release of A T P from right dorsal synaptosomes when compared with the corresponding unsectioned left dorsal synaptosomes as controls (Table II). It is possible that this t r e a t m e n t did not deplete
dorsal spinal cord. We thank Sharon Temple for her expert technical assistance. This research was supported by a grant to
releasable stores of A T P from afferent terminals.
T . D . W . from the Medical Research Council of Canada.
1 Hartree, E. F., Determination of protein: a modification of the Lowry method that gives a linear photometric response, Anal. Biochem., 48 (1972) 422-427. 2 Holton, P., The liberation of adenosine triphosphate on antidromic stimulation of sensory nerves, J. Physiol. (Lond.), 145 (1959) 494-504. 3 Jahr, C. E. and Jessel, T. M., ATP excites a subpopulation of rat dorsal horn neurones, Nature (Lond.), 304 (1983) 730-733. 4 Jessel, T., Tsunoo, A., Kanazawa, I. and Otsuka, M., Substance P: depletion in the dorsal horn of rat spinal cord after
section of the peripheral processes of primary sensory neurons, Brain Research, 168 (1979) 247-259. 5 Minchin, M. C. W., Veratrum alkaloids as transmitter-releasing agents, J. Neurosci. Meth., 2 (1980) 111-121. 6 White, T. D., Release of ATP from a synaptosomal preparation by elevated extracellular K÷ and by veratridine, J. Neurochem., 30 (1978) 329-336. 7 White, T. D. and Leslie, R. A., Depolarization-induced release of adenosine 5'-triphosphate from isolated varicosities derived from the myenteric plexus of the guinea pig small intestine, J. Neurosci., 2 (1982) 206-215.
&at,, Re~'earch ~ ?i (~!~,~5) ~72 ~ ;~i
BRE 20798
Characteristics of K +- and veratridine-induced release of ATP from synaptosomes prepared from dorsal and ventral spinal cord ITHOMAS D. WHITE, 1JOHN W. DOWNIE and 2RONALD A. LESLIE Departments of ~Pharmacology and 2Anatomy, Dalhousie University, Halifax, N.S. B3H 4H7 (Canada)
(Accepted December 18th. 1984) Key words: ATP release - - synaptosomes - - primary afferents - - spinal cord
K + and veratridine released 2-3 times more ATP from dorsal than from ventral spinal cord synaptosomes (P2). K+-induced release of ATP was Ca2+-dependent whereas veratridine-induced release was augmented in a Ca2+-free medium. Twenty-one to 24 days after section of the right sciatic nerve of the rat the evoked release of ATP from right dorsal synaptosomes was indistinguishable from release from left dorsal synaptosomes. Although these latter results suggest that ATP may not be a transmitter at primary afferent synapses in the spinal cord, it is possible that sciatic nerve section does not deplete the releasable pool of ATP in primary afferent terminals or that ATP is also released from interneurons in the dorsal spinal cord. In 1959, H o l t o n 2 showed that A T P was released from p e r i p h e r a l terminals when sensory nerves were stimulated antidromically, raising the possibility that this nucleotide might function as a sensory neurotransmitter. Interest has recently been r e k i n d l e d by the finding that pressure application of A T P excites a subpopulation of rat dorsal horn neurones grown in culture 3. Most n e u r o t r a n s m i t t e r s are not evenly distributed in the brain and where a relatively high concentration of a particular substance occurs, one might suspect a possible physiological role in that region. Unfortunately, total tissue contents of A T P are unlikely to reveal possible n e u r o t r a n s m i t t e r functions since only a small p r o p o r t i o n of total cellular A T P content is likely to be involved in neurotransmission. In the present study, we have characterized the e v o k e d release of A T P from synaptosomes p r e p a r e d from guinea pig spinal cord and c o m p a r e d release from dorsal and ventral cord synaptosomes. W e have also studied the effect of sciatic nerve section on e v o k e d release of A T P from rat spinal synaptosomes. U n i l a t e r a l section of rat sciatic nerve depletes the substance P content of the ipsilaterai dorsal horn 4, p r e s u m a b l y as a consequence of biochemical but n o n - d e g e n e r a t i v e changes in the sensory afferent terminals.
L u m b a r spinal cord was removed from two guinea pigs weighing 300-400 g which had been killed by decapitation. The cords were divided into dorsal and ventral halves with a scalpel and each half homogenized in 10 ml of ice-cold 0.32 M sucrose with 15 strokes of a T e f l o n - g l a s s h o m o g e n i z e r (clearance 0.25 mm). The h o m o g e n a t e s were p o o l e d and centrifuged at 1000 g for 10 min.. the supernatants decanted and saved, the pellets r e s u s p e n d e d with 7 ml of sucrose each and recentrifuged at 1000 g for 10 min. These supernatants were c o m b i n e d with the previous supernatants and centrifuged at 20,000 g for 30 min to yield a crude synaptosomal pellet (Pc)Each pellet was r e s u s p e n d e d up to 6.5 ml with Krebs-Henseleit medium containing 111 mM NaC12, 26.2 m M N a H C O 3, 1.2 m M NaHPO4, 4.7 m M KC1, 1.8 m M CaC12, 1.2 m M MgC12, 11 m M glucose and gassed with 95% 02, 5% C O 2 to maintain a p H of 7.4. In some cases the pellets were resusp e n d e d in a Ca2*-free m e d i u m otherwise identical to the above. The suspensions were p r e i n c u b a t e d for 30 min at 37 °C with periodic gassing before studies of A T P release were performed. R e l e a s e of A T P e v o k e d by elevated extracellular K ÷ or by veratridine was m o n i t o r e d directly and continuously using firefly luciferin-luciferase as described previously6.
Correspondence: T. D. White, Department of Pharmacology, Dalhousie University, Halifax, N,S. Canada B3H 4H7
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
373 Release of ATP was expressed as the maximum concentration of ATP achieved in the cuvette per mg protein, the latter determined according to Hartree~. Dorsal and ventral spinal preparations of synaptosomes from guinea pigs were fixed and examined electron-microscopically as described previously for myenteric synaptosomes 7 and occluded lactate dehydrogenase (LDH) was determined to give a measure of the relative yields of synaptosomes in dorsal and ventral P2 prep arati°nsv. In another series of 3 experiments, right sciatic nerves were sectioned 4 in 8 rats and the animals allowed to recover for 21-24 days, at which time they were decapitated, laminectomies performed and L5 and L6 dorsal roots traced to the spinal cord. Spinal cord sections consisting of segments L5 and L6 were removed to ice-cold 0.32 M sucrose and divided into left and right dorsal and left and right ventral quarters with a scalpel. The material from all 8 animals was pooled and synaptosomes (P2) prepared as described above. The P2 preparations were suspended to 3.5 ml with K r e b s Henseleit medium and preincubated for 30 min at 37 °C before studies of ATP release were performed. Both elevated extracellular K +- and the Na+-chan nel activator, veratridine, released ATP from spinal synaptosomes prepared from guinea pigs, release being 2-3 times greater from dorsal than from ventral cord preparations (Fig. la, Table I). The greater release of ATP from dorsal versus ventral preparations is not likely due to different yields of synaptosomes since the ventral preparation contained as much occluded L D H as the dorsal preparation (Table I). Occluded L D H is a measure of the amount of cytoplasm contained within intact cell membranes and gives an estimate of the number of synaptosomes present 7.
TABLE I
Evoked release of A TP from synaptosomes prepared from dorsal or ventral lumbar spinal cord of guinea pigs Material from 2 guinea pigs was pooled for each determination. Values shown are the means + S.E.M. from 5 experiments
Spinal cord
Dorsal Ventral
Evoked A TP release ( x lO~w M/mg protein) KCl (24 mM)
Veratridine (50 ~M)
12.5 + 1.5 3.92 _+ 0.95
21.0 + 1.4 9.24 + 2.47
Occluded LDH content (A OD at 340 nm/min/mg protein) 1.05 + 0.13 1,13 + 0.09
a
~-
o~ c,. I
2
DORSAL
VENTRAL
DORSAL
VENTRAL
1 rain
b
C
A
!i
~'\'v'
J I rain
Ca2+- FREE
Ca2+ FREE
Fig. 1. Evoked release of ATP from guinea pig spinal synaptosomes, a: synaptosomes were prepared from dorsal or ventral cord and depolarized with KCI or veratridine as described in text. b, e: synaptosomes from dorsal cord were prepared in Ca2+-free medium and the release of ATP by K + and veratridine determined for Ca2+-free and Ca2+-free preparations where the CaCI 2 in the medium was restored to 1.8~M.
Moreover, the dorsal preparation appeared morphologically indistinguishable from the ventral preparation under electron microscopic examination. The K+-induced release of ATP from dorsal spinal synaptosomes was Ca2+-dependent (Fig. lb) and therefore resembled the exocytotic release of putative neurotransmitters from presynaptic nerve terminals. In contrast, the veratridine-induced release of ATP was actually augmented in a CaZ+-free medium (Fig. lc), suggesting that this process could originate from other sites, or by another mechanism. The veratridineevoked release of amino acid transmitters from brain preparations is also increased in CaZ+-free medium s and similar Ca 2+ requirements have been reported previously for the K +- and veratridine-induced release of ATP from synaptosomes prepared from rat and guinea pig brains 6,v. In the rat as in the guinea pig, K + and veratridine released much more ATP from dorsal than from ventral spinal cord synaptosomes (Table II). These find-
374 TABLE II
The apparent slight increase in A T P release from
Effect of sciatic nerve section on the evoked release of A TP from synaptosomes prepared from dorsal and ventral spinal cord (L5-L6) of rats
dorsal synaptosomes prepared from the sectioned side, which was not statistically significant, could be
Right sciatic nerves were sectioned and the rats allowed to recover for 21-24 days. Material from 8 rats was pooled for each determination. Values shown are the means _+ S.E.M. from 3 experiments. Spinal cord
Dorsal left Dorsal right Ventral left Ventral right
Evoked A TP release (x lO-u M/mg protein)
Occluded L D H content (zlOD at 340 nm/min/mg protein)
KCl (24 mM)
Veratridine (50~M)
17.4 + 1.9
48.3 _+5.7
0.60 __+0.17
22.6 + 5.7
60.6 + 3.6
0.82 _+0.06
not detectable
21.2 -+ 1.8
0.62 _+0.22
not detectable
27.1 -+ 9.6
0.69 ___0.13
due to increased n u m b e r s of synaptosomes present since the occluded L D H was increased in these preparations. While these studies were in progress, Yoshioka and Jessel (personal communication) also observed that rat dorsal cord synaptosomes release more A T P than do ventral synaptosomes exposed to veratridine. Furthermore, neither dorsal rhizotomy nor spinal transection diminished the veratridine-evoked release of A T P from rat spinal synaptosomes. Although these results suggest that A T P may not be a transmitter at primary afferents, it is possible that A T P is released from intrinsic dorsal horn neurons as well as from primary afferents so that a reduction in release after dorsal rhizotomy may not be detectable. In any event, the observations that dorsal spinal synapto-
ings are consistent with the possibility that A T P
somes from both guinea pigs and rats release much
might be a n e u r o t r a n s m i t t e r in primary sensory after-
more A T P than do ventral synaptosomes and that A T P excites a subpopulation of dorsal horn n e u r o n s in culture 3 are consistent with the possibility that extraneuronal A T P may perform some function in the
ents to the spinal cord. O n the other hand, section of the right sciatic nerve, using a procedure previously shown to reduce dorsal horn substance P levels by 7 5 - 8 0 % 4, did not reduce the K ÷- or veratridineevoked release of A T P from right dorsal synaptosomes when compared with the corresponding unsectioned left dorsal synaptosomes as controls (Table II). It is possible that this t r e a t m e n t did not deplete
dorsal spinal cord. We thank Sharon Temple for her expert technical assistance. This research was supported by a grant to
releasable stores of A T P from afferent terminals.
T . D . W . from the Medical Research Council of Canada.
1 Hartree, E. F., Determination of protein: a modification of the Lowry method that gives a linear photometric response, Anal. Biochem., 48 (1972) 422-427. 2 Holton, P., The liberation of adenosine triphosphate on antidromic stimulation of sensory nerves, J. Physiol. (Lond.), 145 (1959) 494-504. 3 Jahr, C. E. and Jessel, T. M., ATP excites a subpopulation of rat dorsal horn neurones, Nature (Lond.), 304 (1983) 730-733. 4 Jessel, T., Tsunoo, A., Kanazawa, I. and Otsuka, M., Substance P: depletion in the dorsal horn of rat spinal cord after
section of the peripheral processes of primary sensory neurons, Brain Research, 168 (1979) 247-259. 5 Minchin, M. C. W., Veratrum alkaloids as transmitter-releasing agents, J. Neurosci. Meth., 2 (1980) 111-121. 6 White, T. D., Release of ATP from a synaptosomal preparation by elevated extracellular K÷ and by veratridine, J. Neurochem., 30 (1978) 329-336. 7 White, T. D. and Leslie, R. A., Depolarization-induced release of adenosine 5'-triphosphate from isolated varicosities derived from the myenteric plexus of the guinea pig small intestine, J. Neurosci., 2 (1982) 206-215.