We previously reported the differential stimulation of stored and newly synthesized rat (r)GH release by human GH-releasing factor-44 (hGRF-44). Those studies were performed over a 3-h period in a static in vitro incubation system. The present experiments focus on hGRF-44 effects upon release of new hormone (synthesized during tissue stimulation by the secretagogue) and were performed in in vitro perifusion to study the time course of the response. A double label ([14C], [3H]), immunoprecipitation protocol defined hormone release in relation to time of synthesis: intracellular stores of hormone were prelabeled with [14C]; subsequent exposure of prelabeled tissue to continuous concurrent [3H]leucine and 3 nM hGRF-44 defined newly synthesized hormone and associated it with the secretagogue. In a parallel set of experiments, 1 mM (Bu)2cAMP was substituted f6r hGRF-44. Prolonged exposure to hGRF-44 in perifusion stimulated an initial surge of stored [14C]rGH release which was followed by a rate of [14C]rGH release which declined rapidly but remained suprabasal. [14C]rGH release in response to (Bu)2cAMP was also biphasic, but the initial surge was delayed and the later stimulatory period was better sustained in comparison to responses to hGRF-44. Stimulation of stored [14C] rPRL release by hGRF-44 was observed in perifusion, confirming our observation in the static system. Release of newly synthesized, 3H-labeled rGH was immediately stimulated by either hGRF-44 or (Bu)2cAMP, and that stimulation was maintained throughout exposure to either secretagogue. In contrast, whereas newly synthesized [3H]rPRL release was stimulated by (Bu)2cAMP, its release in response to hGRF-44 resembled that in control experiments. No effect upon hormone synthesis was observed during the 3 h of exposure to hGRF-44. In conclusion, these experiments confirm that hGRF-44 differentially stimulates release of both newly synthesized and stored rGH, and demonstrate differential dynamics in the response as well. Specifically, hGRF-44 stimulation of new rGH release is sustained while its effect on stored hormone simultaneously wanes. Further, when expressed as a percent of intracellular hormone available for release, new hormone is released more than 10 times faster than stored hormone. These observations argue for the existence of separate intracellular paths along which newly synthesized and stored hormone are released by the somatotroph. Finally, these data confirm that hGRF-44 stimulates release of stored rPRL without altering release of newly synthesized rPRL.
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