Growth hormone-releasing peptide-2 reduces inward rectifying K+ currents via a PKA-cAMP-mediated signalling pathway in ovine somatotropes

摘要

Inward-rectifying potassium (Kir) channels are essential for maintaining the resting membrane potential near the K⁺ equilibrium and they are responsible for hyperpolarisation-induced K⁺ influx. We characterised the Kir current in primary cultured ovine somatotropes and examined the effect of growth hormone-releasing peptide-2 (GHRP-2) on this current and its related intracellular signalling pathways. The Kir current was, in most cases, isolated using nystatin-perforated patch-clamp techniques. In bath solution containing 5 mm K⁺, the Kir current was composed of both transient (fast activated) and delayed (slowly activated) components. An increase in the external K⁺ concentration from 5 to 25 mm induced an augmentation of ≈4-fold in the delayed part of the Kir current and both BaCl2 and CsCl dose-dependently inhibited this current, confirming the presence of the Kir current in ovine somatotropes. Moreover, this specific effect of high K⁺ on the Kir current was only observed in the cells that showed positive staining with anti-growth hormone (GH) antibodies, or in GC cells that belong to a rat somatotrope cell line. Application of GHRP-2 (100 nm) reversibly and significantly reduced the Kir current in bath solutions with 5 or 25 mm K⁺ in ovine somatotropes. In addition, we found that the reduction in the Kir current mediated by GHRP-2 was totally abolished by the pretreatments with H89 (1 μm) or Rp-cAMP (100 μm) or by intracellular dialysis of a specific protein kinase A (PKA) inhibitory peptide PKI (10 μm). The specific PKC blocker chelerythrine (1 μm) or inhibitory peptide PKC19-36 (10 μm) did not show any effects on the GHRP-2-induced decrease in the Kir current. These results suggest that the inhibition of Kir current through PKA-cAMP pathways may play an integral role in GHRP-2-induced depolarisation and GH release in ovine somatotropes.