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

Ruwei Xu, Sang-Gun Roh, Kylie Loneragan, Michael Pullar * and Chen Chen

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.

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.

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.

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.

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.

While the application of protein kinase C (PKC) inhibitors calphostin C (100 nM) or chelerythrine (1 µM) did not affect the amplitude of the K⁺ currents, the K⁺ current response to GHRH was 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 PKC_19-36 (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.

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.

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