Arginine vasopressin (AVP) stimulates biphasic release of ACTH from anterior pituitary corticotrophs. The response consists of an initial transient spike phase lasting less than 3 min and a subsequent sustained plateau phase that persists for as long as AVP is present. AVP also acts synergistically with CRF on ACTH release. We have previously shown that the initial spike phase of the response mainly requires release of intracellular Ca2+ and is independent of calmodulin, whereas the sustained plateau phase, like the monophasic sustained response elicited by CRF, involves the influx of extracellular Ca²⁺ via L-type voltage-sensitive Ca²⁺ channels and activation of calmodulin. We have also shown that the synergism between AVP and CRF does not require extracellular Ca²⁺ influx.

In this study we examined the role of Ca²⁺/phospholipiddependent protein kinase-C (PKC) in the two phases of the response to AVP and in the synergism between AVP and CRF. We exploited the observation that prolonged exposure to phorbol esters down-regulates PKC. Dispersed adult male rat anterior pituitary cells were incubated in static suspension culture for 4- 5 days, 0.5 μM phorbol 12-myristate 13-acetate (PMA) or 0.0005% dimethylsulfoxide vehicle alone was added, and the incubation was continued for 24 h. The cells were preperifused with PMA-free perifusion medium for 3 h and then perifused with various agents for 10-20 min. Effluent fractions were collected every 30 sec or 1 min and subjected to ACTH RIA.

Pretreatment with PMA inhibited the subsequent response to 100 nM PMA and 100 μM dioctanoylglycerol, but not to 5 μM forskolin or to depolarization with 56 mM KC1, demonstrating specific down-regulation of PKC. PMA pretreatment had no effect on the initial spike phase of the response to AVP, but inhibited the sustained plateau phase by 57% (P < 0.005) and, consequently, the integrated total response by 33% (P < 0.05). Pretreatment had no effect on the response to CRF. However, pretreatment with PMA completely blocked both phases of the synergistic response to the combination of AVP and CRF. These results indicate that activation of PKC is required for the sustained phase of the response to AVP and both phases of its synergistic interaction with the protein kinase-A pathway, but is not involved in the initial spike phase of the response to AVP, which presumably is mediated by inositol 1,4,5-trisphosphatestimulated mobilization of intracellular Ca²⁺, or in the independent activation of the protein kinase-A pathway by CRF. (Endocrinology₁₂₇: 350–357,1990)