Hypothalamic LHRH neurons form the final pathway for the central control of reproduction. Release of LHRH from hypothalamic neurons is regulated by the interaction of several neurotransmitter systems with gonadal steroids, which act at different levels of their synaptic network. We previously found that LHRH-producing GT1-7 cells respond to ACh with an increase in [Ca²⁺]_i through activation of muscarinic receptors. This effect is acutely modulated by 17β-oestradiol (E2) in a manner compatible with specific membrane binding sites (Morales et al. 2002). Increasing evidence suggests that second messengers are involved in the rapid action of oestrogen by interacting with membrane receptors (Nadal et al. 2000). The aim of this study was to characterize the effect of E2 on ACh-induced Ca²⁺ signals in GT1-7 cells and to identify the potential second messenger systems that might be involved.

[Ca²⁺]_i measurements were achieved in cells loaded with fluo-3 AM by confocal microscopy as described elsewhere (Nadal et al. 2000). Control cells were exposed to two 5 s pulses of 100 µM of ACh separated by 10 min. In oestrogen-treated cells, 2 min prior to the second application of ACh, the cells were exposed to different E2 concentrations from 10 pM to 10 µM. Peak parameters analysed were maximal amplitude (maximal [Ca²⁺]_i), area under the curve (total [Ca²⁺]_i), and t_1/2 (time at half-maximal height).

Doses of E2 as low as 10 pM reduced total [Ca²⁺]_i and t_1/2 of the rising phase, while doses over 1 µM were ineffective. Maximal [Ca²⁺]_i was also reduced by 10, 100 nM or 1 µM. To investigate the involvement of second messengers, dibutyryl cAMP (dB-cAMP, 100 µM), forskolin (FK, 100 nM), 8-bromoguanosine 3â,5â-cyclic monophosphate (8-Br-cGMP, 10 µM) and sodium nitroprusside (SNP, 10 µM) were tested. Application of 8-Br-cGMP also reduced ACh-induced maximal [Ca²⁺]_i, while neither dB-cAMP, nor FK, nor SNP exerted any effect (Fig. 1).

We conclude that physiological concentrations of E2 affect different mechanisms of ACh-induced Ca²⁺ transients in GT1-7 cells in a rapid manner, which might be related to cGMP production by a membrane-associated guanylyl cyclase.

This work was supported by SAF2001-3614-C03-01/02.