Regulation of L-type voltage-gated calcium channels by epidermal growth factor.

GH3 rat pituitary cells have L-type voltage-gated calcium channels, and both the rate of uptake of 45Ca2+ and the concentration of intracellular free calcium ion ([Ca2+]i) are increased by depolarization with high potassium. Cells incubated for several days with 10 nM epidermal growth factor (EGF) responded to depolarization with a 30-65% smaller increase in 45Ca2+ uptake than untreated cells. The inhibitory response to EGF developed slowly, with a maximal effect requiring 24-48 h. EGF exerted a half-maximal reduction in depolarization-stimulated 45Ca2+ uptake at 0.1 nM and a maximal effect at 1-10 nM. 45Ca2+ uptake was reduced by EGF at strongly depolarized potentials (added K+, > 25 mM) with or without the calcium channel agonist BAY K8644. [Ca2+]i was measured using fura-2 before and after depolarization in control cells and cells incubated for 48 h with 10 nM EGF. EGF-treated cells responded to the addition of 30-50 mM KCl with a smaller increase in [Ca2+]i than control cells. Digital fluorescence imaging of individual fura-2-loaded cells confirmed that the average [Ca2+]i response to depolarization was lower in cells that had been incubated with EGF for 36 h. EGF treatment increased the amount of PRL secreted basally, but inhibited the acute PRL secretory response to depolarization with 50 mM KCl and 1 microM BAY K8644 from 2.6- to 1.5-fold. The results indicate that EGF reduces the activity of voltage-gated dihydropyridine-sensitive calcium channels on pituitary cells, and that this reduction in L-channel activity is accompanied by a smaller secretory response to depolarization.

Activation of protein kinase C increases Ca2+ sensitivity of secretory response of GH3 pituitary cells.

The effect of protein kinase C on the secretory response of GH3 pituitary cells to Ca2+ was investigated. Activation of protein kinase C with 100 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 40 min reduced the rise in intracellular free calcium concentration ([Ca2+]i) stimulated by depolarization with high K+ but did not affect the threefold increase in prolactin secretion stimulated by 50 mM K+. Both [Ca2+]i and prolactin release were measured for control and TPA-treated cells over a range of [Ca2+]i values attained by adding the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)-ethane -N,N,N',N'- tetraacetic acid (BAPTA/AM) to reduce [Ca2+]i or high K+ with or without BAY K 8644 to increase [Ca2+]i. Half-maximal prolactin secretion occurred at lower [Ca2+]i concentrations for cells treated with TPA (approximately 160 nM) than for control cells (approximately 270 nM), but the rate of secretion at high [Ca2+]i was the same. GH3 cells also secreted more prolactin in response to thyrotropin-releasing hormone (TRH) after protein kinase C activation, although TRH evoked a smaller Ca2+ transient. Fluorescence ratio imaging revealed that GH3 cells undergo spontaneous [Ca2+]i oscillations (4-12/min) and that TPA nearly abolishes [Ca2+]i oscillations as well as inhibits the increase in [Ca2+]i stimulated by depolarization. These results demonstrate that activation of protein kinase C increases the Ca2+ sensitivity of the secretory response in GH3 cells, causing up to a twofold increase in the rate of secretion at typical intracellular Ca2+ concentrations.

Activation of protein kinase C reduces L-type calcium channel activity of GH3 pituitary cells.

These studies describe the effect of protein kinase C (PKC) activation on the activity of voltage-sensitive L-type Ca2+ channels of GH3 pituitary cells. The rate of 45Ca2+ uptake was stimulated greater than 25-fold by depolarization in the presence of BAY K 8644; the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) reduced this response by 70% in a concentration-dependent fashion. Phorbol 12,13-dibutyrate (PDBu) inhibited depolarization-induced 45Ca2+ uptake within 1 min and caused a nearly maximal reduction after 1 h; its effects were rapidly reversible. TPA decreased the high K(+)-stimulated increase in intracellular free calcium ion concentration ([Ca2+]i) from 8.5- to 3.2-fold by 5 min and to 2.0-fold after 18 h without altering the peak [Ca2+]i response to the peptide hormone TRH. Ca2+ channel current, measured directly using the whole cell configuration of the patch-clamp technique, declined an average of 6.4% over 5 min for control cells and 28.9% when TPA was added to the bathing medium for 5 min. Treatment with 100 nM TPA for 24 h dramatically reduced peak current without shifting the peak of the current-voltage relationship. The mean peak Ca2+ channel current was reduced from 423 to 128 pA, although a few cells seemed completely resistant. To determine whether the effects of phorbol esters were due to the activation of PKC we tested the potency of several drugs to inhibit L-channel activity and to shift the affinity of the epidermal growth factor (EGF) receptor, an established PKC response.(ABSTRACT TRUNCATED AT 250 WORDS)