Detailed kinetic analysis of adrenocorticotropin secretion by dispersed rat anterior pituitary cells in a microperifusion system: effects of ovine corticotropin-releasing factor and arginine vasopressin.

We have developed a microperifusion system in which we have examined the ACTH secretory responses of acutely dispersed normal rat anterior pituitary cells to ovine CRF (oCRF) and arginine vasopressin (AVP), alone and in combination. The system approached square-wave stimulus hydrodynamics. ACTH secretion was observed within 5 sec of exposure to either secretagogue and reached a maximum within 20-40 sec. ACTH secretion remained constant for as long as oCRF was perifused and then fell gradually toward the basal level. Persistent ACTH release after oCRF perifusion was stopped could not be explained by persistence of oCRF in the perifusion chamber. In contrast to the response to oCRF, ACTH secretion fell progressively toward basal despite continued AVP perifusion. AVP had a synergistic effect with oCRF only if it was perifused simultaneously with oCRF or within 30 sec after oCRF was stopped; it had no synergistic effect if perifused immediately before oCRF. Simultaneous perifusion of oCRF and AVP resulted in an oCRF-like response of greater magnitude, whereas sequential perifusion of oCRF followed by AVP resulted in the usual plateau response to oCRF followed by the initial spike response characteristic of very high concentrations of AVP alone and a subsequent rapid decrease in secretion despite continued perifusion of AVP. The different kinetic response profiles suggest that oCRF and AVP act via different intracellular signal transduction pathways, and the time and sequence dependency of their synergism suggests that the factors that mediate their interactions have different intracellular half-lives. The microperifusion system appears to be uniquely suited to detailed kinetic analysis of anterior pituitary hormone secretion and the intracellular pathways through which secretagogues act and interact.