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iotmtal

1992 Elsevier

of P/tartttacul~~y, Science Publishes

220 ( 1992) 271-272 B.V. All rights reserved ~14-2~9/92/~05.~

EJP 0370R Rapid communication

Nitric oxide induces u~urotransmitt~r

release from ~~~~Qcarn~a~slices

Gyorgy Lonart, Jia Wang and Kenneth M. Johnson Dcparttnent

of Pitarntaco!ogv and Toxicology, Unitrrsity

Received

13

of Texas Medical Brartch, Galreston,

TX 77555-1031.

USA

August 1992,accepted 17August 1992

Hydr~~iamine (l-300 @MI, a nitric oxide generator, stimulated the release of [~H]norepineph~ne (13H]NE) and f’4C]acetylchoIine C[‘4ClACh) from rat hippocampal slices in a concentration-dependent manner (EC,,, = 30 PM). A maximally effcctivc concentration of hydroxylaminc (300 PM) produced a 24-fold incrcasc in the basal [‘HINE and 3.6-fold increase in the in the basal [“ClACh efflux. Sodium nitroprusside GNP), also stimulated the release of [‘H]NE, but only at high concentrations (lo-30 mM). Calcium-free experimental buffer (1 mM EGTA) abolished the response. Hemoglobin 10.3 PM) inhibited the effect of 100 PM hydroxylaminc in a manner which was specific for nitric oxide. In addition, 100 PM hydroxylamine incrcascd the cfflux of cndogcnous GABA and glutamate by 3- and 6-fold, rcspcctivcly. Hydr~~iamine;

Sodium nitroprusside;

Nitric axide (NO) has been suggested to potentiate neurotransmitter release in the hippocampus (e.g. O’Dell et al., 1991). In this study we show that generation of NO stimulates the release of f”H]norepinephrine ([“HJNE) and [‘4C]ace~lcho~ine [ti4C]ACh> as well as endogenous ~-aminob~tyric acid (GABA) and glutamate from hippocampal slices. Adult Sprague-Dawley rats were killed by decapitation, the hippocampus was dissected and placed in ice-cold Krebs bicarbonate buffer. In those experiments in which [“H]NE effiux was measured, the buffer also contained 10 FM pargyline. Cross-chopped slices (300 pm X 300 pm) were washed three times with ice-cold buffer, then incubated with [“HINE (60 nM), and in a series of experiments with [‘4C]choline (10 PM), for 20 min. Slices were placed into superfusion chambers and superfused at 0.3 ml/min. In those experiments where we measured 14C as an index of [‘4C]ACh release (Richardson and Szerb, 1974), the buffer contained 10 EM hemichoiinium to prevent the reuptake of ~14~]~holinc liberated by hydrolysis of released [‘4C]ACh. After ! h, three 5 min fractions were collected to determine basal [‘HINE and f”C]ACh efflux. Efflux of radioactivity was calculated as fractional release. The effect of drugs were expressed as the maximal fold increase over basal release. Hydroxyl-

Correspondence to: K.M. Johnson Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555-1031,USA. ‘ICI. 1 (409) 772.0623, fax 1 (409) 772-9042.

Neurotransmitter

release

amine or sodium nitroprusside (SNP) was introduced at the beginning of the fourth fraction and left in the superfusate until the end of the experiment. In another series of experiments, the effects of hydroxyfamine on the efflux of endogenous tyrosine, GABA and glutamate was estimated using HPLC with fluorometric detection according to De Montigny et al. (1987). The superfusion protocol was similar except that 9 min fractions were collected and the hydroxylamine exposure time was limited’to 20 min. Depending on the actuai concentration, superfusion with hydro~lamin~ stimulated the release of [“HINE and [‘4C]ACh in a biphasi~, time-dependent manner. For example, after 100 PM hydro~lamine was introduced, [“WINE efflux was significantly above baseline 10 min later, but did not reach its peak until 25 min. The peak response decayed by 50% by the end of the experiment (25 min after the peak). Hydroxylamine was equally potent in releasing [“HINE or E’4ClACh (ECs,, = 30 PM), but it was about 7 times more efficacious in releasing [“H]NE (fig. 1). The difference in efficacy suggests that hydro~iaminc-stimulated neurotransmitter release is not a consequence of a general neurotoxicity. The onset of the SNP action on basal [“H]NE efflux was even slower (lo-25 min, data not shown) and atso dependent on the concentration of SNP. The SNP response did not reach an apparent ma~mum at the highest concentration tested (fig. 1). When 100 PM hydro~lamine was introduced with 0.3 PM hemoglobin the increase in [“HINE efflux was reduced by 73% (fig. 1). Thirty minute superfusion of

increased

_

the cfflux

of glutamate

31.6 + 3.3 pmol/min pmol/min Glutamate

from a baseline

per mg to a peak

of

of

199 + 2X

per mg 27 min after introduction (n = 10). cfflux returned to baseline 63 min after

introduction of hydroxylaminc. GABA cfflux was similarly affected, except that the peak effect was smaller (baseline

of 36.7 + 5.9 pmol/min

min of I I3 * I4 pmol/min

:

i : t. -;

. : .. . .

to baseline

at 63-72 min). Hemoglobin (0.3 PM) had no effect on hydroxylamine-induced glutamate or GABA efflux, but

3

‘_

c

per mg. peak at 27

per mg, return

c’

IO PM hcmoglohin

:

induced GABA

. .. .

complctcly

blocked hydroxylaminc-

cfflux (unpublished

effect of hemoglobin

on glutamate

observation).

estimated because of the apparent high hemoglobin contamination with glutamate pmol of glutamate/nmol of sample

through

of hemoglobin

a Milliporc

The

efflux could not be lcvcl of (120 + 5

after filtration

polysulfone

filter,

30

kDa cutoff). These data support the recent finding that superfusion of the rat basal forchrain through a push pull cannula with the NO donor, 3-morpholino-sydnonimin with Il.2 PM hemoglobin prior to introduction of a mixture of 100 PM hydroxylamino and 0.3 ~Fvl hern~~g~~~binreduced the cffcct of hydroxylaminc the

slica

(SIN- 1) increased acetylcholinc release (Prast and Philippu, lYY2) and strongly suggest a potential role for NO in the regulation

of ncurotransmitter

release.

by 95% (fig. 1). The dcpcndcncc of inhibition on the length of incubation (which is probably due to the slow diffusion of hemoglobin into the intercellular space) suggests that hemoglobin is not acting by inactivation ydrokylamine but via binding NO generated by cellular metabolism. A 30 min prcincubation with tctrodotoxin

(TI’X.

0.5 PM)

halt no significant

IS.9 + %-fold increase over basal release in the ITXtrcatcd slices. n = 4. P > 0.05. Student’s t-test), suggcsting ,hat NO is acting directly at the noradrenergic terminals. Omission of CaCI, (and addition of I mM completely

inhibited

the hydroxylaminc

( 100

FM) effect on [‘HJNE rclcasc (31.X f 3.5fold increase over basal rclcasc in control; 1.2 & 0. I in c;L’+-free buffer. n = 3. P < 0.05. Student’s t-test). Since the formation of NO from hydroxylaminc is indcpcndent of the Ca” concentration (Pou et al.. 1991). the Ca’+ depcndcnce may reflect that NO relcascs [ ‘H]NE synaptic vesicles by cxocytosis. In a more iimiicd

series of cxperimcnts

This

work

was supported

hy Grant

DA-02073

from

the

U.S.

lI.t1.ti.s.

cffcct

on It!@ I+M hydrohylamine-induced [‘H]NE release CI!!).S+ 2.7-fold incrcasc over basal rcleasc in control;

EGTA)

Acknowledgement

from

using a 20

min pulse of 100 PM hydroxylaminc, WC obscrvcd that hydroxylamine had no effect on tyrwinc cftlux, hut

References Dc

Montigny.

P.. J.F.

Stoh;mgh.

R.S.

Givens.

Srinivas:~char. L.A. Sternson and T. Iliguchi. 2.3-dicarhoxy;lIdch:;dc/cy;lnidc gcnic reagent for primary O’Dell.

T.J.. R.D.

I Iuwkins. for

ion: a tdionally

dcsigrd

E.R. Kandel and 0. p rancio.

nitric

substances in :