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. We show that: (i) The patch-pipette does not always impose its Cl- concentration. (ii) In rat lactotrope cells, membrane resting potential is partially determined by [Cl-]i. (iii) Besides ion channel activity, electroneutral ion transports (cotransports such as K(+)-Cl- and Na(+)-K(+)-2Cl-) participate actively in maintaining a high [Cl-]i. (iv) Finally, Cl- homeostasis is probably linked to cell energetics.

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-). The voltage-dependence of I(to) was not modified. However, of the two kinetic components of I(to) inactivation, the characteristics of the fast component differed significantly between BrdU+ and BrdU- cells. Recovery from inactivation of I(to) showed biexponential and monoexponential function in BrdU- and BrdU+ cells, respectively. This suggests that the molecular basis of this current varies during the cell cycle. We further demonstrated that 4-aminopyridine, which blocks I(to), inhibited GH3 cell proliferation without altering the membrane potential. These data suggest that I(to) may play a role in GH3 cell proliferation processes.

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. The first phase of increase which corresponded to the release of a mitochondrial CCCP-sensitive Ca2+ store was followed by a second increase phase consisting of Ca2+ influx through the plasma membrane. The relationship between the CCCP- and the InsP3-sensitive stores was investigated using thapsigargin (TG). The release part of the Ca2+ response to TG was reduced in a time-dependent manner by previous exposure of the cells to CCCP, suggesting that CCCP also acts on non-mitochondrial stores. Our results show that CCCP releases Ca2+ from both mitochondrial and non-mitochondrial stores in prostatic cells. The possible mechanisms of these effects are discussed.

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. The latter three substitutes induced a significant shift in the voltage dependence of T-type calcium channel activation towards hyperpolarized values. However, this shift alone cannot explain the increase in secretion. Anion permeability studies also demonstrated that the organic anion CH(3)SO(-3) was less permeant than Cl(-), whereas monovalent inorganic anions were more permeant, with the following anion permeability sequence: SCN(-) > ClO(-4) > NO(-3) > Br(-). In conclusion, deprivation of Cl(-) ions has converse consequences on basal and induced secretion; permeating anions result in a transient increase in intracellular Ca(2+) ions. This process involves voltage-dependent Ca(2+) channels. We propose that an alteration in intracellular anion concentrations may influence the activation of internal effectors such as G proteins or channel proteins and, therefore, interfere with exocytosis. These effects are correlated with an external action of lyotropic anions, particularly NO(-3), ClO(-4) and SCN(-), on the gating properties of T-type calcium channels, probably through changes in cell surface charges. The results demonstrate the modulatory effect of anions on the secretory activity of rat lactotropes and underline the specific role played by chloride in stimulus-secretion coupling.

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. The slope conductance of K+ channels in a symmetrical 140 mM K+ gradient was 78 pS. In excised inside-out patches, the channel was inhibited by increasing the cytoplasmic Ca2+ concentration (with half-block at 0.5 microM Ca2+) over a wide range of membrane potentials. The K+ channel had a high sensitivity to tetraethylammonium (TEA), that reduced the single channel conductance with Kd of 280 +/- 27 microM. The K+ channel open probability was inhibited by alpha-dendrotoxin (DTX) (with a half-blocking concentration of approximately 5 nM) and mast cell degranulating peptide (MCDP) (with half-blocking concentration of approximately 70 nM) at all membrane potentials and with very slow reversibility. In view of the biophysical and pharmacological properties of K+ channels in LNCaP cells, it is not possible to classify these channels as one of the previously characterized types of voltage- or ligand-gated K+ channels in other cell lines.

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. Our aims were: 1) to study the effect of K+ channel inhibition on the proliferation of GH3 cells; and 2) to investigate the putative intracellular signals involved in this inhibition. Tetraethylammonium chloride (TEA), a blocker of the calcium (Ca2+)-dependent K+ conductances of GH3, was found to reversibly inhibit cell proliferation, as measured by 3H-thymidine incorporation. Cell cycle block specifically occurred at the G1/S phase of the cell cycle. This inhibition of proliferation was observed for 1-4 mM TEA, which suppressed most of the Ca2+-activated K+ current and part of the inward rectifying K+ current, as shown by electrophysiological experiments. Increasing extracellular K+ concentrations with KCI also inhibited cell proliferation in a dose-dependent manner. Both TEA and KCl depolarized the cells and increased intracellular Ca2+ levels ([Ca2+]i), showing that, in this type of excitable cell, inhibition of cell proliferation can be associated with elevated Ca2+ levels. Ca2+ and membrane resting potential (MRP) were considered as possible messengers of this inhibition. Our results suggest that cell cycle arrest of GH3 cells by K+ channel block probably involves an additional pathway, distinct from those of Ca2+ and MRP.

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. RESULTS: Only one type of voltage-dependent ion conductance, a potassium K+ conductance, was identified. This current, which was depressed by a rise in intracellular Ca2+, had a high sensitivity to tetraethylammonium (TEA) (with half-block at 2 mM) and was also inhibited by 2 nM alpha-dendrotoxin (DTX) and 20 nM mast-cell degranulating peptide (MCDP). K+ channel inhibitors inhibited [3H]thymidine incorporation and protein content, in a dose-dependent fashion, indicating that K+ channels are involved in cell growth. CONCLUSIONS: We conclude from our findings that the human cancer prostate cell line LNCaP has a new type of K+ channel, likely to play an essential role in the physiology of these cells and, more specifically, in cell proliferation.

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. In these experiments, we investigated the specific role of each of these two phases in the hormone response to GHRP6. We found that inhibition by thapsigargin of the intracellular Ca2+ mobilization phase significantly inhibited the hormone response to the peptide during 30 min incubations. Inhibition of the extracellular Ca2+ influx phase by nifedipine, a blocker of voltage-sensitive Ca2+ channels, resulted in a 53 percent reduction of the secretory response to 10(-5)M GHRP6. Antagonism of PKC by phloretin, a flavonoid which prevents PKC activation, and PKC depletion induced by a 24 h treatment with 10(-6)M PMA, completely inhibited the response to GHRP6. Somatostatin, which also inhibits the second phase of the Ca2+ response, suppressed the secretory response to GHRP6. We conclude that, Ca2+ is the main second messenger and both Ca2+ mobilization and Ca2+ entry play a role in the response to GHRP6. However, experiments with PKC depletion and SRIF suggest that other messengers are implicated in GHRP6 signalling in somatotrophs.

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. For the same concentration of agonist, two distinct patterns of GnRH-GAP-induced Ca(2+) mobilization were observed (1) sinusoidal oscillations, and (2) monophasic transient. The latter is followed by a protein kinase C (PKC)-dependent increase in calcium influx through L-type channels. In type II adenomas (7 out of 20 adenomas), GnRH and GAP only stimulate calcium influx through dihydropyridine-sensitive Ca(2+) channels by a PKC-dependent mechanism. TG (1 µM) did not affect [Ca(2+)](i) in these cells, suggesting that they do not possess TG-sensitive Ca(2+) pools. All the effects of GnRH and GAP were blocked by an inhibitor of phospholipase C (PLC), suggesting that they were owing to the activation of the phosphoinositide turnover. Type I and type II adenoma cells showed spontaneous Ca(2+) oscillations that were blocked by dihydropyridines and inhibition of PKC activity. GnRH and GAP had no effect on the [Ca(2+)](i) of type III adenoma cells that were also characterized by a low resting [Ca(2+)](i) and by the absence of spontaneous Ca(2+) fluctuations. K(+)-induced depolarization provoked a reduced Ca(2+) influx, whereas TG had no effect on the [Ca(2+)](i) of type III adenoma cells. The variety of [Ca(2+)](i) response patterns makes these cells a good cell model for studying calcium homeostasis in pituitary cells.

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. We have now investigated these effects on decidual protein and dPRL synthesis after 24 h treatments. When Ca2+ channel blockers or EGTA (2 mM) were added to the culture medium, dPRL release and [3H] leucine incorporation into proteins decreased. Increasing external Ca2+ up to 2 mM instead of 0.8 mM or changing the external K+ concentration (30 mM instead of 5.6) had no consequence on dPRL release, whereas 2 mM of Ca2+ enhanced total protein synthesis. No toxicity was noted with these treatments. Finally a possible effect of Ca2+ modulation on dPRL synthesis was studied using [35S] methionine. The specific activity of [35S] methionine on dPRL was similar in control and treated cells (EGTA, 2 mM Ca2+, cobalt). These results support the idea that Ca2+ controls dPRL synthesis in decidual cells, acting only on general protein synthesis processes.

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. A short application of GnRH (1 to 100 nM) induced an increase in [Ca2+]i due to Ca2+ entry through plasma membrane voltage-sensitive L-type Ca2+ channels. Protein kinase C (PKC) depletion induced by a pretreatment with 1 microM PMA for 24 h abolished spontaneous Ca2+ transients and the action of GnRH on [Ca2+]i and Ca2+ channels. Phloretin (250 microM) and staurosporine (20 nM), two protein kinase C inhibitors, inhibited Ca2+ channel activity, thereby suppressing the effect of GnRH. On the other hand, activation of PKC by a short application of phorbol myristate acetate (10 nM) stimulated Ca2+ influx through Ca2+ channels. These findings demonstrate that, in human NS adenoma cells, GnRH (1 to 100 nM) induces an increase in [Ca2+]i, principally due to Ca2+ entry through L-type voltage-activated Ca2+ channels. PKC regulates this mechanism as well as basal ion channel activity, thus exerting both positive and negative control of [Ca2+]i in stimulated and unstimulated NS adenoma cells.

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. A short application of GHRP-6 (10(-5) M, 10 s) induced a biphasic Ca2+ response in most cells (44%), which consisted in a rapid and large rise in [Ca2+]i followed by sustained oscillations. This response is dose dependent in a range of concentrations from 10(-10) to 10(-5) M. The first phase of the GHRP-6 response persisted in the absence of Ca2+ in the extracellular medium, whereas the second phase was inhibited. The application of Ca2+ channel blockers like cadmium chloride (200 microM) or PN-200-110 (200 nM) also prevented the second phase. Conversely, when the cells were pretreated with thapsigargin (TG) (100 nM), the first phase of the GHRP-6 Ca2+ response was abolished, whereas the second phase alone was preserved. When the cells were depleted in PKC by incubation with 10(-6) M PMA for 24 h, the second phase of the GHRP-6 response was inhibited, and only the first phase was maintained. These results were corroborated by using phloretin, a PKC inhibitor. These data show that GHRP-6 induces a biphasic elevation of the [Ca2+]i in rat somatotrophs. The first phase is probably due to mobilization of the intracellular Ca2+ stores, whereas the second phase is a PKC-dependent process.

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. In these five cases, more than 68% of individual cells responded to TRH by an increase in [Ca2+]i. No significant increase in PRL secretion was found in another culture in which TRH increased [Ca2+]i in only 37% of the cells. The effect of GAP (10(-7) M) was studied in five cell populations. In three of them, a decrease of 20% to 51% of the PRL basal secretory rate was observed under GAP. GAP inhibited [Ca2+]i in respectively 59%, 46% and 94% of the cells from these cultures. The inhibitory effect of GAP was blocked by a pertussis toxin (PT) pretreatment which demonstrates the involvement of a PT-sensitive G-protein in GAP action. In two other cultures, GAP did not significantly alter PRL secretion or individual cell [Ca2+]i. These observations suggest that GAP might play a role in the control of PRL secretion in the human.

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. We used the microspectrofluorimetric method and indo-1 as the fluorescent probe. The cell populations cultured from the tumors of patients A and C showed increased hormone secretion in response to GRF in vitro, whereas cultures from patients B, D, and E were unresponsive to the peptide. Basal [Ca2+]i measured in the five cell populations ranged from 82 +/- 18 to 118 +/- 27 nM. A 10-sec application of 10 nM GRF induced an increase in [Ca2+]i in 60% and 54% of A and C cells, respectively. In the nonresponsive cell populations, the number of calcium responses to GRF was lower, 26% (B cells), 5% (D cells), and 10% (E cells). Two principal responses types were observed: 1) an initial increase in [Ca2+]i, followed by a sustained plateau phase lasting for more than 200 sec; and 2) a monophasic peak of increased [Ca2+]i lasting approximately 1 min before returning to baseline levels. GRF responses were totally suppressed in the absence of Ca2+ ions in the external medium. Sixteen to 30% of the cells cultured from four of the five tumors showed spontaneous fluctuations of [Ca2+]i. These spontaneous Ca2+ transients were suppressed in Ca(2+)-free medium. The number of cells exhibiting such Ca2+ transients decreased with time in culture. Basal hormone secretion was higher in cultures from patient D, in which no spontaneous Ca2+ transients were observed in any of the 72 studied cells, and in cultures from patients E, in which only 16% of cells were spontaneously active. We conclude that 1) in human responsive somatotrophs, the involvement of Ca2+ in GRF stimulus-secretion coupling mechanisms is apparently similar to that described in somatotrophs of other species; 2) the lack of a secretory response to GRF observed in some tumors may result from impairment of Ca2+ responsiveness in either cell recruitment or response amplitude and/or duration; and 3) spontaneous rhythmic Ca2+ activity is apparently dissociated from basal hormone secretion in some of these tumor cells.

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. Ca2+ ionophore A23187 and membrane depolarizations activated the Cl- conductance. However, a feedback effect of Cl- gradient modifications on Ca2+ movements was also observed in normal lactotrophs. In the latter, TRH (100 nM) mobilization of intracellular Ca2+ activated this Cl- conductance together with the potassium (K+) conductance when both ions were present in the intracellular medium (IM) or alone when K+ was absent. Chloride conductance was not activated in the GH3 cells, where mobilization of intracellular Ca2+ by TRH (100 nM) activated only Ca2(+)-dependent K+ conductance. It seems likely that the activation of Cl- conductance in these two different cell types involves different mechanisms.

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. This suggested that the GH3 cells may lack GHRF receptors. The present study investigates the effect of GHRF on free intracellular Ca2+ concentrations in GH3 cells. Cytosolic free calcium concentrations ([Ca2+]i) were monitored in individual cells by microspectrofluorimetry using the fluorescent dye indo 1. When the cells were challenged with a brief application of GHRF (100 nM; 15 sec), 36 out of 59 of these cells responded within a few seconds by a marked increase in [Ca2+]i. GHRF enhanced the frequency of [Ca2+]i oscillations in spontaneously active cells or triggered [Ca2+]i oscillations in inactive cells. The response to GHRF was totally blocked by external Ca2+ free solutions and Ca2+ channel blockers. Combined electrophysiological and fluorescent experiments were carried out in 16 cells. Eleven responded to GHRF. In all cases, the Ca2+ transients triggered by GHRF were associated with action potentials. The Ca2+ responses observed in our experiments clearly show that GH3 cells possess membrane receptors to GHRF. Thus, it is likely that the lack of secretory response observed in GH3 cells does not result from the absence of binding sites to the peptide. It is more likely to be related to alterations of transduction mechanisms resulting in uncoupling between stimulation and secretion.

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. This reduction of [Ca++]i results from coordinate actions of GAP on K+ and Ca++ conductances and may explain the inhibitory effect of GAP on hormonal secretion by PRL-secreting cells.

[Stimulation of calcium intake by growth-hormone-releasing-hormone in GH3 cells]. / Stimulation de l'entrée de calcium par le Growth-Hormone-Releasing-Hormone dans les cellules GH3.

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). This response which is blocked by calcium channel inhibitors results from an increased influx of Ca2+ ions from the external medium.

[Electrophysiological study of the action mechanism of somatoliberin (GH-RH) on hypophyseal GH3 tumor cells]. / Etude électrophysiologique du mécanisme d'action de la somatolibérine (GH-RH) sur des cellules hypophysaires tumorales GH3.

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. Voltage clamp studies reveal that GH-RH potentiates calcium inward currents and a calcium-dependent chloride current; transient outward current is diminished. These changes in membrane conductance account for the cytosolic free calcium rise shown by Indo-1 fluorescence measurements.

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. Membrane electrophysiological properties (resting potential and input resistance) of pituitary cells in primary culture varied widely. Under the recording conditions reported here, the mean resting potential of lactotrophs was about -30 mV. There were spontaneous fluctuations in membrane resting potential (10-15 mV) as well as of membrane input resistance, making these parameters difficult to evaluate accurately. Most of the cells exhibited spontaneous firing activity that was shown to be mainly calcium-dependent. There was no difference between L and D cells in resting membrane electrophysiological properties. TRH (10(-7) M) induced a transient hyperpolarization of the membrane similar to that previously described in the GH3 clonal pituitary cell line. Voltage-clamp studies showed that this hyperpolarization resulted from activation of an outward current, the reversal potential of which ranged from -48 to -86.5 mV. Experimental manipulations of the ionic composition of internal and external recording media suggested that both K+ and Cl- were involved. This hyperpolarizing response was observed both in D and L cells, although L cells had larger and faster responses than D cells. This observation may be of physiological significance because lactotrophs have been reported to exist in various subtypes.