Human GHRH reduces voltage-gated K⁺ currents via a non-cAMP-dependent but PKC-mediated pathway in human GH adenoma cells

1. Whole-cell voltage-gated K⁺ currents and the K⁺ current response to growth hormone-releasing hormone (GHRH) were characterised in primary cultures of human acromegalic somatotropes. 2. Both delayed rectifier (I(K)) and transient (I(A)) K⁺ currents were recorded from human somatotropes held at -80 mV and bathed in a solution containing Cd²⁺ (1 mM), TTX (1 μM) and a low concentration of Ca²⁺ (0.5 mM). Only I(K) was recorded, however, when a holding potential of -40 mV was used. 3. GHRH (10 nM) immediately and significantly reduced the amplitude of both I(A) and I(K). While the reduction in the amplitude of I(A) was fully reversed following the removal of GHRH, the amplitude of I(K) had only partially recovered 10 min after GHRH removal. In addition, GHRH shifted the voltage-dependent inactivation curve of I(A) by 13.5 mV in the negative direction. 4. In a low Ca²⁺ and Cd²⁺-containing solution, the Ca²⁺-activated K⁺ channel blockers apamin (100 nM and 1 μM) and charybdotoxin (1 μM) did not alter K⁺ currents or the effect of GHRH on the recorded K⁺ currents. 5. The whole-cell K⁺ currents and their responses to GHRH were unaffected by the application of 8-bromo-cAMP (100 μM), Rp-cAMP (100 μM) or the protein kinase A (PKA) inhibitor H₈₉ (1 μM). In addition, intracellular dialysis of the PKA inhibitory peptide PKI (10 μM) had no effect on the K⁺ current response to GHRH. 6. While the application of protein kinase C (PKC) inhibitors calphostin C (100 nM) or chelerythrine (1 μM) did not affect the amplitude of tile K⁺ currents, the K⁺ current response to GHRH M as significantly attenuated. Downregulation of PKC with phorbol 12,13-dibutyrate (PDBu, 0.5 μM for 16 h) also abolished the K⁺ current response to GHRH. In addition, intracellular dialysis of somatotropes with the PKC inhibitory peptide PKC₁₉₃₆, (1 μM) prevented the GHRH-induced decrease in the amplitude of the voltage-gated K⁺ currents. Local application of PDBu (1 μM) significantly reduced the amplitude of the voltage-gated K⁺ currents in a similar manner to that induced by GHRH, but without clear recovery upon removal. 7. This study demonstrates that GHRH decreases voltage-gated K⁺ currents via a PKC- mediated pathway in human adenoma somatotropes, rather than by the cAMP-PKA pathway that is usually implicated in the actions of GHRH.