Life Scieaces ool . 17, pp . 1837-1842 Printed in the U.S .A .

Pergamon Press

EFFECTS OF THE "CALCIUM IONOPHORE" A-23187 ON TRANSMITTER RELEASE AT THE FROG NEUROMOSCULAR JUNCTION Hiroshi Rita, Rathleea Madden, and William Vaa der Rloot Department of Physiology and Bíophyeics State University of New York at Stony Brook Stony Brook, New York 11794 (Received in final form November 17, 1975) Summary The ionophore A-23187 when added to the usual Ca t+-Ringer at the frog neuromuscular junction has almost no effect on the frequency of miniature end-plate poteatials (min .e .p .p .s) . The ionophore does íncrease the rate of CaZ+ efflua from frog muscle, eo ít ís ín affective concentrations in the Ringer . When added to Ringer containing N1 2+ instead of CaZ+, the ionophore increases the min.e .p .p, frequency, We suggest that the ionophore can carry divalent canons into the terminal, but there are mechanisms to keep the CaZ+ low, Apparently these mechanisms are unable to rapidly eject or sequester Ni ,

We have shown that the "calciua ionophore" %-537A causes a transitory increase in the min.e .p .p . frequency and as increase in e.p .p, amplitude when applied to the frog neuromuscular junction in the presence of a suitable di valent cation (1) . The results fit wall with the idea that the drug acts by transporting divalent canons from the eatracellular solution into the nerve terminals. Therefore we were interested in testing the effects of another ionophore, A-23187, which appears to have a higher specificity for divalent cations (Z) . METHODS Min,e.p .p .s were recorded from eartorius muscles of Rang i iens with intracellular electrodes using convenkiac~el techaiqueer, The usual Ringer contained 100 mM NaCl, 2.0 mM RC1, 2 .5 mM CaC12, and 8 .0 mM tris maleate pH ~ 7 .4 . 'Ií2+-Ringer" contained 2.5 mM NiC12 in place of the CaCl2 When min .e .p .p,s were recorded the Ringer also contained 10-óg/ml neostigmine Br . Two batches of A-23187 were used (gifts from the E1í Lilly Corp .) ; stock

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A-23197 and ACh Release

Vol . 17, No . 12

solutions were made up in ethanol (3) or in DTLSO (4), The solvents were also added to the Ringer so there was ao chaage in their level when A-23187 was added . In efflux experiments eartbriua muscles were first soaked overnight is Ringer containing 2 .0 uc/ml 45 Ca2+ " The next morning the efflux to ordinary Ringer was measured during successive ten minute time periods. RESULTS A-23187 in Ca t+-Ringer has little consistent effect on min.e .p .p, frequency (Table I) . In some cases there was a brief period of enhanced release following the application of the drug, but similar bursts also occurred in ordinary Ringer . Increasing the Cat+ in the Ringer had no effect . A possible explanation is that the A-23187 is not working ae an ionophore in our solutions . This is unlikely because it enhances Cat+ efflux from frog skeletal muscles (Fig . 1) .

:G

E

w

Fig . 1 Efflux of 45 Ca 2+ from frog sartorius muscles to Ringer . During the interval indicated by the arrows one muscle (~) was exposed to 25 uM A-23187 . The control (o) was kept in ordinary Ringer .

Why then is A-23187 without effect on the motor nerve terminals? One possibility is that A-23187 increâses Cat+ influx into the nerve terminal, but the usual cellular mechanisms are able to keep the free concentration low . This possiblity was tested by exposing neuromuscular junctions to A-23187 in Nit+-Ringer, on the grounds that a rise in intracellular N1 2+ appears to

A-23187 and ACh Release

Vol . 17, No . 12

1839

stimulate transmitter release (6), but we know of no evidence that N$+ is In Nib-Ringer, efficiently sequestered within or rapidly pumped out of cells . A-23187 causes a steady rise in min.e .p .p . frequency (Fig . 2), suggesting that Nit+ is accumulating within the nerve terminal . TABLE I EFFECT OF A-23187 ON MIN.E .P .P . FREQIIENCY A-23187 (uM)

Min.e .p .p .s/se c fo f

fc

fe

10* 15* 25*

0" 18 0" 18 0" 18

0 " 18 0 "32 0 " 32

0'25 0" 20 0"40

0 "20 0 " 27 0 " 28

0" 15 0" 15 0 " 22

--0 "13

25**

0" 74

0 " 88

2" 28***

1 " 18

0" 38

0 "47

50

0" 35

0 " 57

0"52

0 " 42

0 " 28

0'15

50

0'36

0 " 68

0" 55

0 " 45

-

75

0" 46

0 " 48

0'55

0'47

0" 32

100

0" 44

0 " 95

0"95

0'68

0 " 32

--

100

2" 02

3 " 10

3" 00

2'87

1'90

1 " 78

3

f10

f30

--

*Same end-plate **7 .5 mM [Cam]

o ***contained burst. The frequency of the following 1 min bin was 0.95 . fc : control frequency. Min.e .p .p . a were counted for 5 min just before the start of solution change . fe : frequency during solution change . Count was made for 1 min beginning at 2 min after the start of solution change . fo : frequency during the 1 min period just after the completion of solution change . í3 ,f 10 and f30 : frequency during the 1 min period 3, 10 and 30 min after the completion of solution change .

In the eaampla shown in the fi#ure the ionophore produced a marked increase in the rate constant for Cat efflux . In five additional eaperimente the effects of A-23187 were less dramatic, but in each instance there was a significant increase in the efflux raté constant (determined by an analysis of covariance for the data from 120 to 180 minutes ; p< .05) . It should be noted however that equimolar amounts of R-537A are more effective in increasing Cam efflux rate constants . Besides acting as a divalent metal ionophore, R-537A depolarized skeletal muscle fibers, perhaps because it carries univalent ions across the membrane (5) . " A--23187 does not depolarize (Table II) . Hwever, depolarization by R-537A cannot account for the increased quantal output from stimulated nerve terminals or for the increase in min .e .p .p . frequency observed in Bat+-Ringer

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Vol. 17, No . 12

A-23187 and ACh Release TABLE II EFFECTS OF A-23187 ON TAE RESTING POTENTIAL OF FROG SKELETAL MUSCLE

A.

SOLUTION

Em(mV) + S .D .

Cat+-Ringer

88 .7 + 2 .4

Ca

B.

2+

-Ringer +

25 uM A-23187

(n-21)

89 .2 ± 3 .3 (n-40)

Cat+-Ringer

88 .6 + 4 .5 (n-26)

Ca t+-Ringer + 50 uM A-23187

88 .6 + 5 .2 (n-20)

Fig . 2 The effects of A-23187 on min.e .p .p . frequency in N1 2+-Ringer . At the start of the measurements the muscle was in Ní 2+-Ringer (filled bars) . At the arrow A-23187 was added to bring the concentration to 0.3 vM " No measurements were made between 33 and 45 minutes . The remainder of the experiment is now shown by the figure . At 46 minutes the A-23187 concentration was raised to 8.0 uM . Ten minutes later the min.e .p .p . frequenc y was 21 .6/sec . containing concentrations of %-537A too low to depolarize muscle fibers (Kits, Therefore it seems Madden, and Van der Rloot, unpublished observations) . likely that %-537A produces its effects on transmitter release by acting as a divalent cation ionophore, not by depolarizing .

Vol . 17, No . 12

A-23187 and ACh Release

1841

DISCIISSION The effects of A-23187 are quite consistent with the concept that a rise in divalent cation concentration within the nerve terminal triggers quantal release At the frog neuromuscular junction this action cannot be demonstrated in Ca d+-Ringer, perhaps because the Cat+ brought by the ioaophore into the cell is rapidly pumped out or is sequestered within the mitochondria or other structures . Thda effect may also account for other reported differences in the effects of A-23187, like its ability to release catecholamines from the cat adrenal (7) but not from the frog adrenal (8) . The effectiveness of A-23187 was shown by using the ioaophore in N1 2+-Ringer . Simply soaking a nerve-muscle preparation in the Ni +-Ringer does not lead to an increase in min.e .p .p . fréquency . But a marked increase follows tetanic stimu latioa of the motor serve or depolarization of the terminal by elevated extracellular R+ (6) . R-537A in Ni -Ringer also produces a substantial increase in min.e .p .p . frequency (1) . The reasons for the difference is behavior between R-537A and A-23187 remain to be discovered . Perhaps R-537A is able to move Cat+ more rapidly, and therefore can overwhelm the mechanisms in the terminal for removing free Cat+ . Or the ability to move H+, R+, and Na+ across cellular membranes may be responsible for part of its activity on the terminal . ACRNOWLEDGEMENTS Supported by Grant 10320 from the NINDS . the experiments by Julia Portmore .

We were assisted in some of

REFERENCES (1) (2) (3) (4) (5) (6) (7) (8)

H. RITA, and W. VAN DER RLOOT, Nature, 250, 658-660 (1974) . P.W . REED, and B .D . LARRY, J . Biol . Chem ., _247, 6970 .6977 (1972) . N.B . THOA, J .L . COSTA, J . BOSS, aad I.B . ROFIA, Life _Sci ., 14, 1705-1719 (1974) . D.E . COCHRANE, and W.W . DOUGLAS, Proc . Nat . Acad . Sçi . IISA, 71, 408-412 (1974) . D.I . DÉVORE, and W.L . NASTUR, Nature , _253, 644-646 (1975) . H. RITA, and W. VAN DER RLOOT, Nature ,~5, 52-53 (1973) . A.G . GARCIA, S .M . RIItPERAR, and J .C . PRAM, J . Physiol . (Lond,)~244, 253-262 (1975) . A. RICCI, JR ., R.M . SANDERS, J . PORTI~DRE, and W. VAN DER RLOOT, Life Sçi ., 16, 177-184 (1975) .

Effect of the "calcium ionophore" A-23187 on transmitter release at the frog neuromuscular junction.

Life Scieaces ool . 17, pp . 1837-1842 Printed in the U.S .A . Pergamon Press EFFECTS OF THE "CALCIUM IONOPHORE" A-23187 ON TRANSMITTER RELEASE AT T...
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