Voltage-Dependent Potassium Currents in Ovine Somatotrophs and Their Function in Growth Hormone Secretion

Abstract

Sheep somatotroph-enriched cultures were obtained by means of enzyme dissociation and Percoll gradient separation. Nystatin-perforated-whole-cell recordings were performed on post-recording-identified somatotrophs after 4–14 days in vitro. Using Ca²⁺-free, tetrodotoxin-containing (1 µM) bath solution and K⁺ electrode solution, three types of voltage-dependent K⁺ currents were recorded as inward rectifying, outward transient and outward delayed rectifying K⁺ currents. The inward rectifying K⁺ current was very small at physiological extracellular K⁺ concentrations (5 mM) and enhanced by increasing the K⁺ concentration in the bath to 55 mM; it was blocked by tetraethylammonium (2 mM) but not by 4-aminopyridine (5 mM). A transient outward K⁺ current appeared at –50 mV and was selectively diminished by 4-aminopyridine (2 or 4 mM). A delayed rectifying outward K⁺ current was observed when the membrane potential was depolarized to -20 mV and was blocked by tetraethylammonium (2 mM) but not 4-aminopyridine (4 mM). Application of 4-aminopyridine but not tetraethylammonium (up to 5 mM) depolarized the cell membrane potential recorded under current clamp conditions and triggered action potentials when the bath solution contained Ca²⁺ (2 mM) but not tetrodotoxin. The intracellular Ca²⁺ concentration was increased by 4-aminopyridine as was growth hormone release. Therefore, the 4-aminopyridine-sensitive transient outward K⁺ current appears to be important in the determining the resting potential of ovine somatotrophs and plays a major role in regulating basal intracellular Ca²⁺ concentration and growth hormone secretion.

Electrical membrane properties and ionic currents in cultured goldfish gonadotrophs

“…The I A -type currents found in these cells had kinetics and sensitivity to 4-AP and TEA similar to those reported in neurons (Rudy 1988), other anterior pituitary cells (Lledo et al 1990;Bosma and Hille 1992;Chen et al 1994), and trout oligodendrocytes (Glassmeier et al 1992). Delayed rectifier currents similar to those reported here were also identified in other pituitary cell types (Lledo et al 1990;Chen et al 1994), as well as in general populations of dispersed goldfish pituitary cells (Price et al 1993).…”

“…The transient membrane hyperpolarizations are usually followed by a few Na + -and Ca 2+ -dependent action potentials (Tse and Hille 1993;Stojilkovic et al 1994). Other K + conductances were also shown to be involved in the neuroendocrine regulation of rat lactotrophs (Lledo et al 1990), amphibian melanotrophs (Valentijn et al 1991;Vaudry et al 1994), and ovine somatotrophs (Chen et al 1994). Therefore, a variety of ion channels are involved in the regulation of pituitary cell function.…”

The in vitro Effect of Leptin onBasal and Growth Hormone-Releasing Hormone-StimulatedGrowth Hormone Secretion from theOvine Pituitary Gland

“…Dispersed ovine pitutitary cells were subjected to Percoll gradient centrifugation to enrich the somatotrope population to 60–80% of cells [11]. The cells were then cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% sheep serum and 2% fetal calf serum in 48-well culture dishes (1–2 × 10 5 cells/well) and the culture medium was changed every 2–3 days.…”

G(o)‐2 protein mediates the reduction in Ca2+ currents by somatostatin in cultured ovine somatotrophs.

“…The yield was usually more than 107 cells per pituitary gland, with greater than 90% viability (Trypan Blue exclusion test). The cell suspension (3-5 ml) was placed, under sterile conditions, above a layered column of Percoll solutions of increasing density and centrifuged as described previously (Chen et al 1994a). Fractions which contained up to 85% of somatotrophs (1 and 2) were used in these experiments (Chen et al 1994a (ai-3 AS; 3'-CAG TAC CCG ACG TGC AAC-5'), anti-ao-1 (ao-1 AS; 3'-TTG TGG ATA CTT CTA CGT CGA CGG A-5') and anti-ao-2 (ao-2 AS; 3'-TCA CGG AAG TGT CTT CGA CAC CGA G-5') which have been reported previously (Hsu et al 1990;Kleuss et al 1991;Baertschi, Audigier, Lledo, Israel, Bockaert & Vincent, 1992).…”