Effects of Growth Hormone-Releasing Peptide-2 (GHRP-2) on Membrane Ca2+ Permeability in Cultured Ovine Somatotrophs

The newly synthesized GH-releasing peptide, GHRP-2 (D-Ala-D-βNal-Ala-Trp-D-Phe-Lys-NH₂), was studied in somatotroph-enriched populations of ovine pituitary cells in primary culture. Nystatin-perforated whole-cell recordings were made on identified somatotrophs after 4–14 days of culture. Using a standard bath solution (containing Na⁺, Ca²⁺) and an electrode solution containing K⁺ in current-clamp recordings, GHRP-2 (10 nM) depolarized the membrane potential of the cells triggering a burst of action potentials. Voltage-clamp recordings indicated that GHRP-2 produced a slowly inactivated inward current with a slight reduction in outward current. The inward current was blocked by the Ca²⁺ channel blocker, Co²⁺ (0.5 mM). Ca²⁺ currents were then isolated using tetraethylammonium bath solution and an electrode solution containing Cs⁺. Ovine somatotrophs possess transient (T type) and long lasting (L type) Ca²⁺ currents. The L type current was abolished by addition of nifedipine (3 μM) to the bath solution and T type current was isolated on this basis. Current-voltage relationships indicated an increase in both T and L type Ca²⁺ currents in response to GHRP-2. The voltage-dependent inactivation curve for T type Ca²⁺ current was shifted towards a less negative level by the peptide. Intracellular free Ca²⁺ concentration (Ca²⁺]i) in somatotroph-enriched populations was specifically increased by GHRP-2 but this effect was totally abolished by blockade of membrane Ca²⁺ channels. These data show that GHRP-2 causes an influx of Ca²⁺ leading to an increase in Ca²⁺]i in ovine somatotrophs. The Ca²⁺ currents were both L type and T type with a shift in the inactivation curve of the latter by the releasing peptide.