Regulation of intracellular chloride concentration in rat lactotrope cells and its relation to the membrane resting potential.

Rat lactotrope cells in primary culture exhibit physiological properties closely associated with chloride ions (Cl-) homeostasis. In this work, we studied the regulation of intracellular Cl- concentrations ([Cl-]i) and its relation to the membrane resting potential, using a combination of electrophysiology and spectrofluorimetry. Variations in [Cl-]i resulting from the patch clamp technique, pHi, antagonists of Cl(-)-Ca(2+)-dependent channels, an anion exchanger antagonist, and an antagonist of K(+)-Cl- cotransport were considered with respect to their involvement in membrane potential.

Potassium channel expression level is dependent on the proliferation state in the GH3 pituitary cell line.

Previously, we showed that the peak density of the transient outward K(+) current (I(to)) expressed in GH3 cells was different in the S phase than in other phases of the cell cycle. Using cell synchronization, we show here that I(to) drops precisely at the quiescent (G(0) phase)/proliferating transition. This change is not due to a modification in the voltage dependence of I(to), but rather to a modification in its inactivation kinetics.

Calcium store depletion induced by mitochondrial uncoupling in prostatic cells.

The effects of mitochondrial uncoupling on the calcium homeostasis of prostatic cells were investigated using the prostatic cancer cell line LNCaP and indo-1 spectrofluorimetry. Carbonyl cyanide m-chloro-phenylhydrazone (CCCP) was used as uncoupler. Resting LNCaP cells responded to CCCP by a biphasic increase in [Ca2+]i.

Cell cycle-related changes in transient K(+) current density in the GH3 pituitary cell line.

Our aim was to determine whether the expression of K(+) currents is related to the cell cycle in the excitable GH3 pituitary cell line. K(+) currents were studied by electrophysiology, and bromodeoxyuridine (BrdU) labeling was used to compare their expression in cells thereafter identified as being in the S or non-S phase of the cell cycle. We show that the peak density of the transient outward K(+) current (I(to)) was 33% lower in cells in S phase (BrdU+) than in cells in other phases of the cell cycle (BrdU-).

Effects of Cl(-) substitution on electrophysiological properties, Ca(2+) influx and prolactin secretion of rat lactotropes in vitro.

In this study, we compared the effects of different chloride (Cl(-)) substitutes - methane sulfonate (CH(3)SO(-)(3)), bromide (Br(-)), nitrate (NO(-)(3)), thiocyanate (SCN(-)) and perchlorate (ClO(-)(4)) - on the secretory activity and calcium current activation of rat lactotropes in primary culture. We observed that CH(3)SO(-)(3) decreased basal prolactin (PRL) secretion. Br(-) had no effect, whereas the more lyotropic anions, such as NO(-3), SCN(-) and C1O(-4), increased basal PRL secretion.

Characterization of Ca(2+)-inhibited potassium channels in the LNCaP human prostate cancer cell line.

Potassium plasma membrane channels have been studied in the LNCaP androgen-sensitive human prostate cancer cell line, derived from a lymph node of a subject with metastatic carcinoma of the prostate. Membrane currents were recorded by the patchclamp technique, using the cell-attached, cell-free and whole-cell mode. A voltage-dependent, non-inactivating potassium channel (delayed rectifier) was the most commonly observed ion channel in LNCaP cells.

Potassium channel inhibition reduces cell proliferation in the GH3 pituitary cell line.

Potassium (K+) conductances are known to be involved in cell proliferation of a number of nonexcitable cell types. The nature of the mechanism by which K+ channel inhibition reduces cell proliferation has remained elusive despite intensive search. We investigated whether such a phenomenon could be demonstrated in excitable cells, using the GH3 pituitary cell line as a cell model.

Potassium conductance in the androgen-sensitive prostate cancer cell line, LNCaP: involvement in cell proliferation.

Background: Very little is known about the expression of ion channels in prostate cells (both normal and malignant), and their possible role in physiological and pathological functions. We therefore studied ion conductances and their role in the proliferation of LNCaP cells, an androgen-sensitive human prostate cancer cell line.

Methods: We applied patch-clamp recording techniques for electrophysiological studies, and 3H-thymidine incorporation and protein content assays for cell growth studies.

Spontaneous and agonist-induced calcium oscillations in single human nonfunctioning adenoma cells.

The effects of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) on cytosolic free calcium concentration ([Ca(2+)](i)) were investigated in 20 human nonfunctioning pituitary adenomas. We divided these tumors into three classes according to their response pattern to hypothalamic peptides. In type I adenomas (8 out of 20 adenomas), GnRH and GAP mobilized intracellular calcium ions stored in a thapsigargin (TG)-sensitive store.

GHRP6-stimulated hormone secretion in somatotrophs: involvement of intracellular and extracellular calcium sources.

GHRP6 is a synthetic hexapeptide which stimulates growth hormone (GH) secretion from the pituitary in vivo and in vitro. We have previously shown that in identified somatotrophs, GHRP6 induces a biphasic increase in cytosolic Ca2+ concentration ([Ca2+]i) consisting of an abrupt increase (first phase) followed by a sustained plateau of elevated [Ca2+]i (second phase). The first phase corresponds to mobilization of intracellular Ca2+ pools and the second phase to influx of extracellular Ca2+ ions through voltage-sensitive Ca2+ channels.

Long-term effects of calcium availability on prolactin and protein synthesis in human decidual cells.

Ever since decidual cells were recognized as the source of decidual prolactin (dPRL), very few reports have dealt with the role of calcium (Ca2+) on dPRL synthesis and release. In a recent work, we described the presence of T-type Ca2+ channels in these cells, giving Ca(2+)-dependent action potentials. However, we failed to demonstrate any action of decidual cell Ca2+ modulation on acute dPRL release, but observed only long-term effects.

Gonadotropin-releasing hormone induced Ca2+ influx in nonsecreting pituitary adenoma cells: role of voltage-dependent Ca2+ channels and protein kinase C.

The action mechanism of gonadotropin-releasing hormone (GnRH) on the cytosolic free calcium concentration ([Ca2+]i) and high-threshold voltage-dependent Ca2+ channel activity was studied in human nonsecreting (NS) pituitary adenoma cells. [Ca2+]i was monitored in individual cells by dual emission microspectrofluorimetry using indo 1 as intracellular fluorescent Ca2+ probe. The whole-cell recording patch-clamp technique was used to study Ca2+ channels.

GHRP-6 induces a biphasic calcium response in rat pituitary somatotrophs.

The mechanism of action of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6), a synthetic peptide which specifically induces the secretion of growth hormone (GH) in rat somatotrophs, is still poorly understood. We have studied the effects of GHRP-6 on the cytosolic free calcium concentration ([Ca2+]i) of somatotrophs in primary culture. [Ca2+]i was monitored in individual somatotrophs by dual emission microspectrofluorimetry, using Indo-1 as the intracellular fluorescent Ca2+ probe.

Thyrotropin-releasing hormone and gonadotropin-releasing hormone-associated peptide modulation of [Ca2+]i in human lactotrophs.

The effect of thyrotropin-releasing hormone (TRH) and gonadotropin-releasing hormone-associated peptide (GAP) was studied on both secretion and intracellular free Ca2+ concentrations ([Ca2+]i) in human pituitary cells cultured from prolactin (PRL)-secreting tumors. Secretion was measured during a 30-min incubation period and we used a microspectrofluorimetric method in individual cells and indo-1 as the fluorescent probe. TRH (10(-8) M) significantly increased PRL release in five out of the six cell populations.

Calcium homeostasis in growth hormone (GH)-secreting adenoma cells: effect of GH-releasing factor.

Human GH-secreting tumors are heterogenous regarding their basal secretory activity and response to GH-releasing factor (GRF). We have investigated whether such different secretory properties could be accounted for by alterations of intracellular mechanisms occurring at the calcium level. Basal free intracellular calcium concentrations ([Ca2+]i) and Ca2+ responses to GRF were studied in single cells cultured from fragments of five GH-secreting pituitary adenomas.

Calcium-activated chloride conductance of lactotrophs: comparison of activation in normal and tumoral cells during thyrotropin-releasing-hormone stimulation.

We studied a chloride (Cl-) conductance activated by calcium (Ca2+) in normal rat lactotrophs and compared its activation during TRH stimulation in normal rat lactotrophs and in GH3 tumoral lactosomatotrophs cells, using the whole-cell configuration of the patch-clamp technique. The Cl- specificity of the conductance was assessed by manipulation of internal and external Cl- concentrations. The reversal potentials were in agreement with those predicted by the Nernst equation.

Growth hormone-releasing factor stimulates calcium entry in the GH3 pituitary cell line.

The GH3 pituitary cell line has been extensively used to study various aspects of the stimulus secretion coupling process. It is known that GH3 cells release PRL and GH in the basal state and in response to various secretagogues. However, this cell line was considered unsuitable as a model for studying the effects of GHRF since the neuropeptide did not affect GH secretion or gene expression.

The gonadotropin-releasing hormone associated peptide reduces calcium entry in prolactin-secreting cells.

The precursor molecule to the GnRH contains a peptide named GnRH-associated peptide (GAP) with PRL-inhibiting properties. In this work, we have studied the electrophysiological properties and responses to GAP of three different types of PRL-secreting cells: 1) the rat tumor cell line GH3, 2) normal rat pituitary cells in primary culture, and 3) human PRL-secreting adenoma cells. Using different but complementary techniques we show that GAP reduces intracellular Ca++ levels, [Ca++]i, and inhibits Ca++ transients in these cells.

[Electrophysiological study of the action mechanism of somatoliberin (GH-RH) on hypophyseal GH3 tumor cells].

We have investigated the electrical response of patched GH3 cells to Growth-Hormone Releasing-Hormone (GH-RH). GH-RH (100 nM) enhanced firing frequency of action potentials. This is accompanied by membrane depolarization (5-10 mV) and conductance increase.

[Stimulation of calcium intake by growth-hormone-releasing-hormone in GH3 cells].

The effect of GH-RH in the intra-cytosolic free Ca2+ concentration was studied in GH3 cells. To this end, we have used microspectrofluorimetry performed on single cells. We show that 60% of cells respond to a brief application of 100 nM GH-RH by an increase of their [Ca2+]i (mean increase 100% over basal values).

Electrophysiological response to thyrotropin-releasing hormone of rat lactotrophs in primary culture.

The response of rat pituitary cells to thyrotropin-releasing hormone (TRH) in primary culture was studied in the whole-cell configuration with the patch-clamp technique. Prolactin (PRL)-containing cells were identified in the culture with a peroxidase-antiperoxidase immunocytochemical method. The cells were cultured from the pituitaries of diestrous (D) and lactating (L) female rats.

The electrophysiological effects of thyrotropin-releasing hormone are similar in human TSH- and prolactin-secreting pituitary cells.

We studied the electrophysiological properties of individually characterized TSH-secreting cells cultured from pituitary fragments surgically removed from three patients, two who had primary TSH-secreting adenomas and one who had chronic TSH hypersecretion (hyperplasia) secondary to primary hypothyroidism. The TSH-secreting cells were excitable and had calcium-dependent action potentials. More than 80% of the cells cultured from the two patients with TSH-secreting adenomas were spontaneously active, whereas fewer cells (20%) cultured from the hypothyroid patient were spontaneously active.

Anti-prolactin cell-surface immunoreactivity identifies a subpopulation of lactotrophs from the rat anterior pituitary.

Suspensions of cells dissociated from the anterior pituitary of the adult rat include many that contain intracellular PRL. After fixation, these cells can be identified and the distribution of their PRL determined by immunocytochemistry with anti-PRL antibodies. We have found that approximately 50% of the cells that contain intracellular PRL also have PRL or PRL-like immunoreactive material on the outer cell surface.

[LHRH and LH release in the red fox Vulpes vulpes L].

Pituitary responsiveness to exogenous LHRH was studied in vivo and in vitro in the female red fox, a mono-oestrous species. In vivo, the ability of the pituitary to release LH in response to a single injection of LHRH (2 micrograms/kg) was determined at various stages of the reproductive cycle. The greatest responsiveness is observed during the preovulatory period, the lowest during the luteal phase.