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

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 (IK) and transient (IA) 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 IK was recorded, however, when a holding potential of −40 mV was used. GHRH (10 nM) immediately and significantly reduced the amplitude of both IA and IK. While the reduction in the amplitude of IA was fully reversed following the removal of GHRH, the amplitude of IK had only partially recovered 10 min after GHRH removal. In addition, GHRH shifted the voltage-dependent inactivation curve of IA 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 PKC19–36 (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. Growth hormone-releasing hormone (GHRH) has been shown to stimulate growth hormone (GH) secretion in several species since it was identified more than a decade ago (Guillemin et al. 1982; Spiess et al. 1982; Heiman et al. 1984; Jansson et al. 1985). It is generally accepted that in somatotropes, GHRH increases Ca2+ influx via voltage-gated Ca2+ channels, and this leads to an increase in intracellular free Ca2+ concentration ([Ca2+]i) and subsequently to an increase in GH secretion (Lussier et al. 1991; Chen et al. 1993, 1994a; Naumov et al. 1994; Chen & Clarke, 1995a; Kwiecien et al. 1997). Activation of sodium currents by GHRH was reported in rat somatotropes (Kato & Sakuma, 1996, 1997), which was suggested to initiate membrane depolarisation. The main second-messenger system employed by GHRH is believed to be intracellular cAMP (Chen et al. 1994a, 1998; Sartin et al. 1996; Wu et al. 1996; Takei et al. 1996, 1998; Kato & Sakuma, 1997). Since most of the ionic currents across the membrane at the resting potential are carried by K+, the transmembrane K+ channels are thought to play an important role in GHRH-induced depolarisation (Mollard et al. 1988; Ohlsson & Lindstrom, 1989; Sikdar et al. 1989; Chen et al. 1993; Chen & Clarke, 1995a). GH-releasing peptide (GHRP), a synthetic GH secretagogue, has been shown to decrease the voltage-gated K+ currents in rat somatotropes (McGurk et al. 1993). GHRP also depolarises ovine somatotropes in a very similar way to that by GHRH (Chen & Clarke, 1995b). Somatostatin, a GH-release inhibitory factor, has the capacity to increase K+ currents in neurones (Yatani et al. 1987; Scheweizer et al. 1998) and the voltage-gated and inwardly rectifying K+ currents in rat, ovine and human somatotropes (Wang et al. 1989; Chen et al. 1990, 1994a; Sims et al. 1991; Takano et al. 1997; Bauer, 1998; Chen, 1998). GHRH may antagonise the inhibitory action of somatostatin partially by reducing K+ currents. This has raised the questions whether GHRH has the same effect on voltage-gated K+ currents in human somatotropes as that of GHRP in rat somatotropes, and what is the intracellular signalling pathway for such a possible reduction in K+ currents.