A multi-column perifusion system was used to investigate the dynamics of the dose–response relationships of ACTH release by ovine pituitary cells when stimulated by both corticotrophin-releasing hormone (CRF) and arginine vasopressin (AVP) given alone and in combination. A dose–response relationship was obtained when 10-min pulses were given at 60-min intervals over the range of 0·002–2000 nmol CRF/l and 1–2000 nmol AVP/l, with a minimum effective concentration of 0·02 nmol CRF/l or 1 nmol AVP/l. When AVP was given together with CRF, the expected potentiation of the ACTH response occurred when compared with the summed response of these secretagogues given separately. At the higher concentrations of CRF and AVP used, the ACTH responses to repeated pulses decreased with time during the experiment. The rate of this loss of responsiveness was significantly correlated to the size of the response to the first pulse (for CRF: r = 0·89, P<0·01; for AVP: r = 0·95, P < 0·01), being greatest when the response was potentiated by adding the secretagogues together (for CRF plus AVP: r = 0·95, P <0·01). Reduced availability of receptors or changes in intracellular transduction processes may contribute to this desensitization. Reduced levels of secretable ACTH do not appear to be implicated because desensitization to pulses of one secretagogue did not cause equivalent desensitization to the other. In addition, cells stimulated continuously with submaximal levels of either secretagogue showed desensitization while more ACTH was still available for release to higher levels of stimulant. The potentiation of CRF plus AVP resulted primarily from a rapid increase in the height of the response since the width of the response (at one-third maximum height) was significantly (P < 0·001) less after AVP plus CRF than after CRF alone. Also the rapidity with which ACTH concentrations fell after removal of the stimulus from the perifusion medium was significantly (P <0·01) faster following AVP, and CRF plus AVP (P <0·05), than after CRF alone.
It is concluded that reduced ACTH responsiveness following repeated stimulation is dependent upon the type and concentration of the secretagogue. AVP had a shorter duration of action than CRF in vitro and potentiated the initial response to CRF rather than prolonging its action. Desensitization to each stimulant appeared to act by a mechanism independent of the other and therefore appeared to occur at or near the receptor level and be unrelated to the availability of ACTH.