Continuous exposure of hemi-pituitary glands from intact female rats to LH releasing hormone (LH-RH) in vitro displayed three phases in the pattern of LH release: during the first hour release of LH was low (first phase response), then it increased to a higher level during the second hour and remained constant during the next 2 h (second phase response), after which there was a refractoriness of LH release (third phase response). The initial phase response of pituitary glands from intact rats was blocked by EGTA (a Ca2+ chelator) but there was a small but significant increase in the rate of LH release during the second phase response. This increase could be prevented by inhibition of protein synthesis by cycloheximide. Cycloheximide and EGTA did not affect basal release of LH by glands from intact rats, neither did EGTA affect the high basal release of LH by glands from ovariectomized rats. However, the LH-RH-induced release of LH from pituitary glands of ovariectomized rats, which did not show the initial phase of low LH release, was completely suppressed by EGTA throughout a 4-h incubation period.
The pattern of LH release stimulated by the combination of N6-monobutyryl cyclic AMP and theophylline (mbcAMP/theophylline) showed an initial phase of low LH release lasting 4 h after which it increased. The magnitude of the effect was small compared with the action of LH-RH. As it did with LH-RH, EGTA completely blocked the initial response, but allowed a small increase in the rate of LH release thereafter; this increase could also be blocked by inhibition of protein synthesis.
Addition of EGTA to media during pretreatment of pituitary glands from intact rats with either LH-RH or mbcAMP/theophylline did not impair the facilitatory effect of these secretagogues on the responsiveness of the glands to subsequent exposure to LH-RH and cycloheximide and normal Ca2+ levels. The restoration of Ca2+ levels after withdrawal neither affected basal nor LH-RH-induced release of LH.
Exclusion of Ca2+ from the media during a 6-h incubation of pituitary glands from intact rats with LH-RH prevented the glands from becoming refractory to subsequent stimulation by LH-RH, which occurs when normal Ca2+ concentrations are present.
The results suggested that extracellular Ca2+ is obligatory for LH release and the induction of refractoriness by LH-RH. In contrast, that part of the action of LH-RH which is cyclic AMP-mediated and protein synthesis-dependent is not affected by withdrawal of extracellular Ca2+.