Extracellular Ca++-Independent and -Dependent Components of the Biphasic Release of LH in Response to Luteinizing Hormone-Releasing Hormone in Vitro*

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Abstract
Pituitary tissue blocks excised from diestrous II rats were exposed to continuous (4 h) or bolus (10 min) infusions of LHRH in Krebs Improved Ringer I medium using a perifusion system. Two different rates of LH release were obtained in response to continuous infusions of various concentrations of LHRH: an initial relatively low rate of LH release (phase I), followed approximately 60 min later by a greatly increased secretion rate (phase II). In the absence of extracellular Ca++, phase I release was completely inhibited while phase II release was only partially inhibited. The acute response (phase I) to a bolus infusion of LHRH was also inhibited by removing Ca++ from the medium, the inhibition being readily reversed by Ca++ replacement. During constant stimulation (4 h) with LHRH, normal medium was switched to Ca++-free medium after 1.5 h. This resulted in a steady decline in the rate of release of LH to the level previously obtained during LHRH stimulation in a Ca++-free medium. Cycloheximide (5 μM), which had no effect on phase I release of LH, completely inhibited the phase II release obtained in normal medium as well as the partial release obtained in Ca++-free medium. Finally, LH released in response to infusions of a nonspecific secretogogue (60 mM K+) was completely blocked in the absence of extracellular Ca++. These results indicate that phase I release of LH is a Ca++- dependent, protein synthesis-independent response. On the other hand, the second phase release is totally dependent on protein synthesis and can be dissociated into a Ca++-dependent component and an extracellular Ca++-independent component which is specific to LHRH.

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