scholarly journals Inositol lipid metabolism and signal transduction in clonal pituitary cells

1986 ◽  
Vol 124 (1) ◽  
pp. 337-358
Author(s):  
A. H. Drummond
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Prolactin Secretion ◽  
Free Calcium ◽  
Pituitary Cells ◽  
Cytosolic Free Calcium ◽  
Release Process ◽  
Cell Functions ◽  
Kinase C ◽  
Inositol 1,4,5 Trisphosphate

A number of clonal cell lines derived from a rat pituitary tumour, collectively termed GH cells, have retained a range of differentiated cell functions, including their ability to secrete the hormones prolactin and growth hormone in response to stimuli such as thyrotropin-releasing hormone (TRH). The mechanisms underlying this release process involve, at least in part, an increase in cytosolic free calcium levels, and the cells have proved useful as a model system in studies of receptor-controlled calcium mobilization. The initial response of the cells to the addition of TRH now appears to be the interaction of the occupied TRH receptor with a GTP-binding protein. A sophisticated signalling system is then activated which initially involves the phosphodiesteratic hydrolysis of phosphatidylinositol 4,5-bisphosphate to 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. Both of these products are important intracellular messengers, and their formation leads to a plethora of biochemical and electrical changes which culminate in the biphasic release of hormone from the cell. The changes in cytosolic free calcium that occur following TRH addition follow a complex temporal pattern. Within 1 s, the concentration starts to increase from a resting level, in the range 100–150 nmol l-1, to a peak value of around 1 mumol l-1 which is attained within 6–8 s. This ‘spike’ of calcium is almost exclusively derived from intracellular stores, probably the endoplasmic reticulum, in response to the formation of inositol 1,4,5-trisphosphate. With high concentrations of the peptide, the cytosolic free calcium concentration declines promptly, due to the activation of a protein kinase C-mediated extrusion and/or sequestration process. This inhibitory phase is less marked at low agonist concentrations but, in all cases, is superseded by a second increase in free calcium, which is due to the stimulated influx of the cation through dihydropyridine-sensitive calcium channels. These biphasic changes in calcium, in concert with the activation of protein kinase C, appear sufficient to regulate prolactin secretion.

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

1993 ◽  
Vol 264 (4) ◽  
pp. C1020-C1028 ◽  
Author(s):  
A. A. Haymes ◽  
P. M. Hinkle
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Prolactin Secretion ◽  
Secretory Response ◽  
Gh3 Cells ◽  
Free Calcium ◽  
Pituitary Cells ◽  
Acetoxymethyl Ester ◽  
Kinase C ◽  
High K

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.

Effects of withdrawal of dopamine on translocation of protein kinase C isozymes and prolactin secretion in rat lactotroph-enriched pituitary cells. Modulation of substance P-mediated responses

1997 ◽  
Vol 18 (3) ◽  
pp. 181-191 ◽  
Author(s):  
S E Mau
Keyword(s):  
Protein Kinase C ◽  
Catalytic Activity ◽  
Protein Kinase ◽  
Substance P ◽  
Prolactin Secretion ◽  
Particulate Fraction ◽  
Pituitary Cells ◽  
Resting Cells ◽  
Kinase C ◽  
Pkc Isozymes

ABSTRACT The present study deals with the effects of withdrawal of dopamine (DA) on the translocation of protein kinase C (PKC) isozymes and release of prolactin (Prl) in resting- and substance P (SP)-stimulated cultures of enriched rat pituitary lactotrophs. Following a brief tonic input (10 min), DA withdrawal induced a redistribution of PKC α- and β-immunoreactivity (IR) to the particulate fraction with maximal levels, attained after 5 min, remaining translocated for 20 min. DA withdrawal prolonged the effect of SP-induced translocation of PKC α-and β-IR. Similar effects were detected when the catalytic activity of PKC in response to DA withdrawal was evaluated. Thus, DA washout redistributed PKC catalytic activity and prolonged the effect of SP on catalytical PKC translocation to the particulate fraction. Pretreatment of cells with the protein kinase A inhibitor, rp-adenosine-3′,5′-cyclic monophosphothionate (rp-cAMP), reduced the amount of PKC α- and β-IR redistributed after DA withdrawal. Furthermore, this treatment also reduced the DA withdrawal effect on SP-mediated translocation of PKC α- and β-IR. Methoxyverapamil, a blocker of voltage-gated Ca2+ channels, completely inhibited the redistribution of PKC isozymes after DA withdrawal, but also reduced the potentiating effect of DA withdrawal on SP-induced redistribution of PKC isozyme-IR. In perifused enriched lactotrophs, DA withdrawal induced a release of Prl that lasted 45-55 min and prolonged the effect of SP on Prl secretion. rp-cAMP did not significantly affect Prl release due to DA removal, but the prolonging effect of DA withdrawal on SP-induced Prl secretion was abolished. Methoxyverapamil completely abolished the rebound release of Prl after DA withdrawal, and the potentiating effect of DA removal on SP-mediated Prl release was also diminished. Readdition of DA after DA withdrawal was able to suppress the translocation of PKC isozyme-IR and catalytic activity and to reduce the release of Prl to baseline levels. Moreover, readdition of DA reduced the potentiating effects of DA withdrawal on the same parameters after SP-stimulation of cells. On the basis of these results it is concluded that in resting cells following DA withdrawal prolactin is released and specific PKC isozymes and concomitant catalytic activity are translocated to the particulate fraction in enriched lactotrophs. While cAMP/PKA and influx of Ca2+ seem to work in concert in translocating PKC, influx of Ca2+ is the primary mechanism responsible for the rebound release of Prl after DA withdrawal. DA withdrawal exerts a potentiating effect on SP-induced PKC translocation and Prl release. It is suggested that the biochemical events involved in these processes are cAMP/PKA and Ca2+ influx.

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

1992 ◽  
Vol 262 (5) ◽  
pp. C1211-C1219 ◽  
Author(s):  
A. A. Haymes ◽  
Y. W. Kwan ◽  
J. P. Arena ◽  
R. S. Kass ◽  
P. M. Hinkle
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ion Concentration ◽  
Free Calcium ◽  
Pituitary Cells ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Bathing Medium ◽  
Channel Current ◽  
Kinase C

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)

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