| Summary: | It is well established that LH action is mediated primarily by adenylate
cyclase/cAMP. Conversely, the role of inositol phosphate/calcium in LH
signalling has only recently been investigated. We examined the effects of
gonadotrophins on intracellular calcium mobilisation in HEK293 cells transiently
transfected with human wild type or chimeric gonadotrophs receptors
(n=3400). Intracellular free calcium concentration was measured using fura-2
microspectrofluorimetric techniques. Human LH (2-4 μg/ml) arid CG (10
IU/ml) consistently evoked oscillatory calcium signals in HEK293 cells transfected
with hLHr, whereas hFSH (2-4 μg/ml) failed to elicit any calcium responses.
Both hLH and hFSH failed to elicit a calcium response from HEK293
cells transfected with hFSHr. Pre-treatment of transfected HEK293 cells with
pertussis toxin (100 ng/ml) or with U-73122 (10 μM), a phospholipase C inhibitor,
negated all gonadotrophin-evoked calcium mobilisation. Our study of chimeric
gonadotrophin receptors show that the carboxy-terminal third of the
hLHr is crucial in evoking intracellular calcium changes. Although various
subdivisions of this region is capable of stimulating calcium transients, an
intact carboxy-terminal third of the receptor is required for normal and sustained
intracellular calcium profile. To our knowledge, this is the first demonstration of calcium oscillations in response to the activation of the hLH receptor,
and to unequivocally show that the hLH receptor is coupled to the inositol
phosphate/calcium signalling pathway via a pertussis toxin-sensitive G
protein. The role of extracellular ATP in the human ovary remains equivocal. We
demonstrated that P2 purinoreceptor agonists evoke oscillatory intracellular
calcium responses in hGLCs. The cells were responsive to ATP at concentrations
ranging from 1-100 μM. ATP and UTP were more effective in stimulating
calcium mobilisation than ADP. Neither adenosine nor AMP were capable of
inducing intracellular calcium responses. The positive responses to adenosine
thiotriphosphate, a non-hydrolysable ATP analogue, indicate that the calcium
responses were not due to by-products from ATP hydrolysis, and that hGLCs
possess P2U purinoreceptors. We have also demonstrated that these purinergic-mediated
intracellular calcium responses involve both Ca2 + influx; and Ca2+
mobilisation from intracellular stores. [Scientific formulae used in this abstract could not be reproduced.]
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