The role and regulation of Ca²⁺ and other ion channels in the in vitro adrenocorticotropin (ACTH) response to arginine vasopressin (AVP), were investigated in static cultures of ovine anterior pituitary cells. Previous evidence suggests that the action of AVP in ACTH secreting (corticotroph) cells involves the activation of the polyphosphoinositide-derived (PI) second-messenger system, and has also been shown to be dependent on Ca²⁺ influx. In this report, a variety of chemically distinct blockers of Ca²⁺ influx, including the organic agents (methoxyverapamil (D600), nifedipine and diltiazem) and the inorganic ions (Cd²⁺ and Co²⁺) were all found to cause large reductions in the AVP-stimulated ACTH response, providing further evidence that the AVP-induced response is dependent on Ca²⁺ influx to a large degree. However, the entire AVP-induced response was not inhibited by the blockers, suggesting that other factors, such as release of intracellularly stored Ca²⁺ also participates in this response. The blocking agents used in this study are all classified as blockers of L-type (L-) voltage-sensitive Ca²⁺ channels (VSCC), and thus the results suggest that L-VSCC are responsible for the bulk of the Ca²⁺ influx that underlies the AVP-induced response. The inorganic blocking ions also inhibit T-type (T-) VSCC, and thus it is possible that these channels also contribute to the response.
In cells that possess voltage-activated Ca²⁺ channels, raising the extracellular K⁺ concentration ([K⁺]e) typically evokes hormone secretion, due to depolarisation-induced Ca²⁺ influx via the voltage-sensitive channels. Raising [K⁺]e caused ACTH secretion from ovine corticotrophs, and this response was also sensitive to VSCC blockers. These results provide further support for the presence of VSCC in ovine corticotrophs.
Simultaneous stimulation with AVP and raised [K⁺]e caused a level of ACTH secretion that was less than the sum of the individual responses, when the concentrations of the secretagogues were moderate to high. This result is consistent with the hypothesis that both secretagogues activate, to some extent, the same population of Ca²⁺ channels during their respective responses. At low concentrations of the secretagogues, a synergistic response was observed. Further experimentation and analysis suggested that this response may be generated at the level of Ca²⁺ influx, and thus raised the possibility that the VSCC may be subject to dual voltage and voltage-independent regulation.
The possibility that voltage-independent regulation of VSCC activity by protein kinase C (PKC), part of the PI second-messenger system, occurred during the response to AVP was explored by down-regulating PKC activity. This was achieved by chronic exposure of pituitary cells to the PKC-activating phorbol ester, 12-0-tetradecanoylphorbol 13 -acetate (TPA). This treatment totally inactivated PKC, reduced the responses to AVP, but not K⁺e and abolished the synergistic interaction between AVP and K⁺e. Thus these results are consistent with a role for PKC in the AVP-induced ACTH response, and with the hypothesis that the synergistic response occurs due to voltage-independent (chemical) regulation of VSCC. This hypothesis was further supported by the finding that simultaneous stimulation with TPA plus raised [K⁺]e caused synergistic ACTH responses.
The possibility that PKC activates VSCC was investigated by examining the effects of VSCC blockers on TPA-stimulated ACTH release. The organic blocker, D600, and the inorganic ion, Co²⁺, both reduced the TPA-induced response. However, the patterns of inhibition were not entirely consistent with those previously observed for inhibition of AVP-induced secretion. Thus an additional protocol, reducing or removing Ca²⁺ from the extracellular medium, was employed to investigate the involvement of Ca²⁺ influx during the response to TPA. This protocol reduced both AVP- and TPA-stimulated ACTH release, and thus provided additional evidence that the AVP-induced response is dependent on Ca²⁺ influx, and further suggested that PKC can activate Ca²⁺ influx in ovine corticotrophs. Thus the possibility that AVP-activated PKC can affect VSCC activity in corticotroph cells is a viable hypothesis.
Voltage regulation of VSCC by AVP, and the effects on ACTH secretion, were also investigated. The possibility that AVP may create a depolarisation stimulus via PKC-mediated inhibition of a K⁺ current that is active at rest, was examined. Exposure of cells to the K⁺ channel blocker tetraethylammonium (TEA), stimulated a small increase in ACTH release that was sensitive to Ca²⁺ channel blockers. These findings are consistent with the hypothesis under investigation.
Exposure of cells to TEA in the presence of AVP or TPA enhanced the responses to these secretagogues, suggesting that a (possibly Ca²⁺-activated) K⁺ current (distinct from the one discussed in the previous paragraph) is present in ovine corticotrophs, and may act to regulate the cellular response to these agents. Removal of external Na⁺ caused a small reduction in AVP-stimulated ACTH release, suggesting that Na+ channels may play a minor role in the response to AVP. These investigations extend the current knowledge regarding the regulation of the AVP-induced ACTH response, particularly with respect to ovine cells
