T. Babila et Dc. Klein, CHOLERA TOXIN-INDUCED G(S)ALPHA DOWN-REGULATION IN NEURAL TISSUE - STUDIES ON THE PINEAL-GLAND, Brain research, 638(1-2), 1994, pp. 151-156
Cholera toxin (CT) treatment (50 mu g/ml) was used to down regulate th
e alpha subunit of the stimulatory guanine nucleotide binding protein
(Gs alpha) in pineal glands in organ culture, as has been seen in non-
neural tissue. A 15 h treatment reduces G(s) alpha by similar to 75% a
s measured using semi-quantitative Western blot technology. In contras
t, this treatment does not alter the abundance of G beta, G(i) alpha o
r G(0) alpha. This effect on G(5) alpha was still apparent following a
36-h washout period. The 48-h CT treatment increased cyclic AMP accum
ulation 10- to 17-fold but blocked the norepinephrine (NE)-induced inc
rease in cyclic AMP accumulation, presumably reflecting the loss of G(
s) alpha. This treatment did not, however, inhibit protein synthesis o
r stimulation of arylalkylamine N-acetyltransferase (NAT) activity pro
duced by treatment with either DB-cyclic AMP (N-6,2'-O-dibutyryl adeno
sine 3',5'-monophosphate) or 8 Br-cyclic AMP, stable cyclic AMP deriva
tives. This indicates that a 48-h CT treatment was not generally toxic
. In contrast, this treatment blocked subsequent CT stimulation of NAT
. The effects of CT treatment on the adrenergic stimulation of NAT was
examined using treatments which selectively produced alpha- or beta-a
drenergic stimulation. alpha(1)-Adrenergic activation of the pineal gl
and elevates [Ca2+](i), which potentiates effects of cyclic AMP; in th
ese studies the response to alpha-adrenergic activation was markedly i
ncreased in 48-h CT-treated glands, reflecting Ca2+ potentiation of th
e effects of elevated levels of cyclic AMP. In contrast, the effects o
f the selective beta-adrenergic agonist isoproterenol was reduced by s
imilar to 75%. These studies not only establish CT-induced G(s) alpha
down-regulation as a new tool for the study of adrenergic signal trans
duction in the pineal gland, but indicate that this paradigm is probab
ly useful in all neural tissue.