Membrane effects of thyrotropin-releasing hormone and estrogen shown by intracellular recording from pituitary cells.

The effects of thyrotropin-releasing hormone and 17 beta-estradiol on the electrical membrane properties of a prolactin-secretin pituitary cell line (GH3/B6) were studied with intracellular microelectrode recordings. Of the cells tested, 50 percent were excitable and displayed calcium-dependent action potentials when depolarized. When injected directly on the membrane of an excitable cell, thyrotropin-releasing hormone and 17 beta-estradiol induced action potentials within 1 minute. The spiking activity was preceded by a progressive increase of the input resistance without any detectable change in the resting membrane polarization. The results reveal a rapid effect of both substances on the membrane of GH3/B6 cells. In the case of thyrotropin-releasing hormone, which has both a short-term effect on release of prolactin and a long-term effect on its synthesis, the induced electrical activity may be associated with the stimulation of prolactin production. The physiological implication of 17 beta-estradiol-induced, calcium-dependent spiking activity remains to be elucidated.

Interplay between Ca2+ release and Ca2+ influx underlies localized hyperpolarization-induced [Ca2+]i waves in prostatic cells.

Calcium seems to be a major second messenger involved in the regulation of prostatic cell functions, but the mechanisms underlying its control are poorly understood. We investigated spatiotemporal aspects of Ca2+ signals in the LNCaP cell line, a model of androgen-dependent prostatic cells, by using non-invasive external electric field pulses that hyperpolarize the anode facing membrane and depolarize the membrane facing the cathode. Using high-speed fluo-3 confocal imaging, we found that an electric field pulse (10-15 V/cm, 1-5 mA, 5 ms) initiated rapidly, at the hyperpolarized end of the cell, a propagated [Ca2+]i wave which spread through the cell with a constant amplitude and an average velocity of about 20 microns/s. As evidenced by the total wave inhibition either by the block of Ca2+ entry or the depletion of Ca2+ stores by thapsigargin, a specific Ca(2+)-ATPase inhibitor, the [Ca2+]i wave initiation may imply a localized Ca2+ influx linked to a focal auto-regenerative process of Ca2+ release. Using different external Ca2+ and Ca2+ entry blockers concentrations, Mn2+ quenching of fluo-3 and fura-2 fluorescence and inhibitors of InsP3 production, we found evidence that the [Ca2+]i wave progression required, in the presence of basal levels of InsP3, an interplay between Ca2+ release from InsP3-sensitive Ca2+ stores and Ca2+ influx through channels possibly activated by the [Ca2+]i rise.

Tamoxifen reduces calcium currents in a clonal pituitary cell line.

The effect of the anti-estrogen Tamoxifen (Tx) on membrane electrical properties and the underlying calcium (Ca2+) conductances was examined in the clonal pituitary cell line GH3/B6 which exhibits calcium action potentials at rest. Electrophysiological recordings (109 cells) were made using either high resistance intracellular microelectrodes or the whole-cell recording (WCR) patch-clamp technique. Electrical activities of 39 spontaneously active GH3/B6 cells were recorded with sharp microelectrodes filled with 3 M KCl. The spikes were Ca2+-dependent since they were blocked by Cobalt ions (Co2+, 5 mM). When applied directly to the recorded cell, Tx (10(-7) M) inhibited action potential firing. This blockade was accompanied by a discrete hyperpolarization of the membrane potential (-2.8 +/- 2 m V) from rest (-39.5 +/- 5 m V) and a 10% increase in the input membrane resistance. The effect stopped soon after Tx removal (mean 11.4 +/- 6 sec), and Tx solvent was unable to elicit the response. Current clamp WCR with pipettes containing potassium gluconate (KGlu, 140 mM) confirmed these results (30 cells), but the addition of cell extract to KGlu was necessary to prevent rundown of the response and to obtain reproducible action potential blockade. Short (1-5 min) or long term (48 h) pretreatment with estradiol (10(-7) to 10(-5) M) did not change the response to Tx, thus indicating that its effect was not mediated through estrogen receptors. Voltage clamp WCR study of the effect of Tx (10(-7) M) using pipettes filled with cesium chloride (140 mM) showed that both fast and slow inactivating calcium conductances were inhibited (38 cells). The fast inactivating Ca2+ current was reduced by about 60-80% whereas slow inactivating Ca2+ current was completely inhibited. This action may represent one way by which the antitumoral effect of this antiestrogen is mediated.

Intracellular Ca2+-dependent protein kinase C activation mimics delayed effects of thyrotropin-releasing hormone on clonal pituitary cell excitability.

Biochemical and spectrophotometric studies of second messenger pathways transducing TRH signals in clonal pituitary (GH) cells have shown that TRH induces rapid turnover of phosphoinositides and changes in cytoplasmic Ca2+ as well as activation of protein kinase C (PKC) and secretion of PRL. Here we have used classical microelectrode and contemporary patch pipette recording techniques under current-clamp conditions to compare the effects of TRH receptor-coupled stimulation with direct activation of PKC on the excitability of GH3/B6 cells. With high resistance microelectrodes TRH induced a complex sequence of changes in membrane properties consisting of an initial 20- to 30-mV hyperpolarization associated with an increase in membrane conductance lasting less than a minute, followed by several minutes of low amplitude fluctuations and action potential activity superimposed on a modest increase in input resistance. Active phorbol ester induced a slowly developing hyperpolarization of about 5 mV and a modest increase in input resistance, followed by several minutes of low amplitude fluctuations and spontaneous action potential activity. Both the peptide- and phorbol ester-evoked changes in excitability were attenuated or completely lost during patch recordings in the whole cell mode. Dilute aqueous lysates of the clone restored various phases of the electrical response. The low amplitude fluctuations and action potential activity phase could be induced by either TRH or phorbol ester if the cells were dialyzed with intracellular electrolyte containing PKC and at least 50 nM Ca2+. These results demonstrate that the phosphoinositide/PKC circuit activated by TRH in clonal pituitary cells has electrically detectable effects on cell excitability, and these help to explain TRH's actions on electrical activity.

Somatostatin blocks Ca2+ action potential activity in prolactin-secreting pituitary tumor cells through coordinate actions on K+ and Ca2+ conductances.

The hypothalamic peptide somatostatin (SRIF) suppresses secretory activity in phenotypically distinct pituitary endocrine cells. We have used tight-seal whole-cell recording techniques to study the peptide's effects on the electrical properties of tumor pituitary cells derived from rat (GH3/B6) and human adenomas that secrete human PRL in a SRIF-sensitive manner. Both cell types exhibited qualitatively similar electrophysiological properties and electrical responses to SRIF. Under the experimental conditions employed the majority of cells spontaneously generated Ca2+-dependent actions potentials. The actions of the peptide on cellular excitability were markedly affected by the presence of horse and fetal calf sera. Without these additives the electrical responses faded and could not be studied in detail. Therefore, recordings were conducted in media containing sera. In the presence of sera almost all cells spontaneously generated Ca2+ action potentials, and peptide-induced changes in excitability were well preserved. SRIF depressed spontaneous and evoked action potential activity in a dose-dependent manner at concentrations that reduced intracellular free calcium ([Ca2+]i) and suppressed basal PRL release. Current and voltage clamp experiments revealed coordinate actions of the peptide on excitable membrane properties. SRIF (1 nM) enhanced a depolarization-activated, rapidly inactivating outward K+ current, thereby effectively reducing the rate at which action potentials occurred. Over the 10-1000 nM range SRIF slowly activated a virtually noninactivating K+ conductance over a wide range of membrane potential. This effectively hyperpolarized cells away from the threshold for triggering Ca2+-dependent action potentials and shunted the membrane. The peptide induced K+ conductance activated at the level of the resting potential was progressively lost during the intracellular dialysis of whole-cell recording. Dilute aqueous lysates of cells included in the patch pipette prevented much of the rundown of this SRIF-induced electrical response while inclusion of an ATP-regenerating system preserved some of the peptide action. Over the 10-100 nM concentration range SRIF also reduced voltage-dependent Ca2+ current. Furthermore, pretreatment of cells with pertussis toxin abolished SRIF action on cellular excitability, suggesting that SRIF can regulate the function of ionic channels through GTP-binding proteins (G proteins). The results demonstrate that SRIF acts coordinately on the primary conductances expressed in tumor PRL cells to attenuate or block Ca2+ action potential generation and thus Ga2+ entry from extracellular sources.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrical properties of cultured human adrenocorticotropin-secreting adenoma cells: effects of high K+, corticotropin-releasing factor, and angiotensin II.

ACTH-secreting pituitary adenoma cells were cultured from specimens obtained by transphenoidal hypophysectomy in five patients with Cushing's disease. The majority of adenoma cells (90%) stained specifically with antiserum against human ACTH. The electrophysiological properties and response to hormones of these cells were studied with intracellular recording techniques under current clamp and voltage clamp conditions. Most (80%) of the cells fired action potentials that were Ca2+-dependent inasmuch as they were blocked by Co2+ (5 mM) and by removal of Ca2+ from the medium, but were unaffected by tetrodotoxin (0.3 mM) and by Na+ removal. The cells responded to factors known to stimulate ACTH release, including high K+, CRF, and angiotensin II (AII). High K+ (50 mM) induced a membrane depolarization in association with an increase in conductance. CRF (100 nM) produced a depolarization, a decrease in conductance, an increase in spike firing, and an increase in spike duration. Although AII was inactive in ordinary recordings, in cells loaded with lithium (Li+) to promote the phospholipid-dependent second messenger system, the peptide produced an increase in spike firing and spike duration with no change in membrane potential. The combination of CRF and AII (CRF + AII; 100 nM each) in Li+-loaded cells caused a greater excitatory effect than either peptide alone. Under voltage clamp, the response either to CRF or to CRF + AII could be attributed, at least in part, to the inhibition of a slow, voltage-dependent K+ current that is persistently active at resting potential. These results indicate that modulation of action potential firing may be an early step in the regulation of ACTH release from pituitary cells by known secretagogues. Since action potentials in these cells are associated with Ca2+ entry, the resulting changes in intracellular Ca2+ levels could mediate the effects of the hormones on secretion.

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.

Biphasic changes in intracellular pH induced by thyrotropin-releasing hormone in pituitary cells.

We studied the effects of TRH on intracellular pH (pHi) in individual cells of the GH3 pituitary clonal cell line using the seminaphtorhodafluor pH indicator. We show that, in a majority of cells, TRH action on pHi occurs in two phases: first acidification then alkalinization. Acidification and Ca2+ mobilization are related in time. K+ depolarization (KCl, 50 mM), and Ca2+ ionophores, A23187 (10 microM) or ionomycin (5 microM) lead to acidification. We conclude that a marked increase in [Ca2+]i can induce acidification and that the TRH-induced acidification is due to Ca2+ mobilization. TRH-induced alkalinization is due to Na+/H+ exchanger activation, since it is inhibited by amiloride (200 microM) and Na(+)-free medium. We show that this alkalinization does not occur after a 20-h pretreatment with phorbol myristate acetate (1 microM) which depletes protein kinase C. We also show that blocking Ca2+ entry does not affect the TRH-induced alkalinization, but an increase in [Ca2+]i concomitant with the activation of protein kinase C mimics TRH-induced alkalinization. We conclude that both Ca2+ mobilization and protein kinase C activation are necessary for TRH-induced alkalinization. Studies of secretion in Na(+)-free medium or with amiloride (200 microM) show that pHi does not seem to be involved in PRL short-term release (30 min) but suggest that activation of the Na+/H+ exchanger leading to cytoplasmic alkalinization may have an important role in PRL synthesis.

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.

Voltage-dependent calcium current in human decidual cells and its relation to prolactin secretion.

Human decidual cells synthesize and release decidual PRL (dPRL) immunologically and biochemically identical to human pituitary PRL. However, stimulators and inhibitors of PRL secretion such as TRH, bromocriptine or dopamine have no effect on dPRL release. The evidence for the involvement of Ca2+ in dPRL release is based on contradictory or unclear data. Since little is known about Ca2+ movement in human decidual cells we studied the membrane Ca2+ conductance of cultured decidual cells using the patch-clamp technique in the whole-cell configuration. We report the existence of Ca(2+)-dependent action potentials triggered by hyperpolarizing or depolarizing pulses and blocked by cobalt (Co2+; 5 mM). Spontaneous action potentials were observed in the cell-attached mode and found also to be Co(2+)-sensitive. A tetrodotoxin-insensitive and Ca(2+)-dependent rapidly inactivating inward current was investigated in voltage clamp. Its activation threshold was between -60 and -45 mV. Indo-1 measurements of free intracellular Ca2+ concentrations ([Ca2+]i, 169 +/- 14 nM and 141 +/- 8 nM in short-term culture vs. 149 +/- 5 nM in cells cultured for 3-6 days) showed that decidual cells have spontaneous transient fluctuations of [Ca2+]i and that [Ca2+]i was decreased by Ca2+ channel blockers. The existence of Ca2+ movements in decidual cells in culture is thus demonstrated. The occurrence of action potentials in decidual cells derived from fibroblasts, reputed to be inexitable cells, is an interesting biological observation. However, Ca2+ is not involved in the short-term release of PRL by decidual cells, and its effects on long-term secretion still requires further investigation.

Short term effect of prolactin on intracellular calcium in Chinese hamster ovary cells stably transfected with prolactin receptor complementary deoxyribonucleic acid.

The mechanism of transduction of the PRL signal in target cells is poorly understood. We examined the effects of PRL on the intracellular free Ca2+ concentration in Chinese hamster ovary cells overexpressing functional PRL receptors. [Ca2+]i was determined by dual emission microspectrofluorimetry using indo-1 as the Ca2+ fluorescent probe. We demonstrate that at physiological concentrations (0.5-5 nM), PRL stimulates Ca2+ entry (type I) and/or induces a mobilization of calcium ions stored in intracellular compartments (type II). Two types of Ca2+ mobilization, distinguishable by their onset kinetics, were observed, a slow mobilization (type IIa; transition time to peak, approximately 10 sec) and a fast mobilization (type IIb; transition time to peak, < 2 sec). PRL responses were delayed (15-120 sec) compared to the well known activation by phosphatidylinositol 4,5 bisphosphate hydrolysis-coupled receptors. This suggests that inositol trisphosphate is not involved in PRL response or that phosphatidylinositol 4,5 bisphosphate hydrolysis is not directly coupled to the PRL receptor. The amplitude of the PRL-induced Ca2+ increases (300-1400 nM) would be sufficient to provoke several physiological responses, such as stimulation of secretion, cell proliferation, or gene activation. However, the relation between the increase in Ca2+ and activation of milk protein genes remains to be established.

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.

Gonadotropin-releasing hormone-induced changes of intracellular pH in pituitary gonadotrophs: influence of estradiol.

Using the pH indicator, seminaphtorhodafluor, we studied the effects of GnRH on intracellular pH (pHi) in single gonadotroph cells, obtained from 3-week ovariectomized rats, treated or not with estradiol (E2) (OVX + E2, OVX). In a majority of cells (77.7% for OVX cells and 93.7% for OVX + E2 cells), GnRH induced acidification. A biphasic change of pHi, acidification followed by alkalinization, was observed in about 44% of the cells tested. In E2-treated cells, amplitude of acidification and duration of alkalinization were increased. Acidification and Ca2+ mobilization were related in time with a short delay (4-5 sec.). Depolarization with KCl and ionomycin, a Ca2+ ionophore, induced acidification. Taken together these observations suggest that acidification was caused by [Ca2+]i increase. When the Na+/H+ exchanger was blocked by amiloride or in Na(+)-free medium, GnRH-induced alkalinization was inhibited. Alkalinization disappeared completely when the cells were depleted in protein kinase C (PKC). Nevertheless, acute application of phorbol myristate acetate, known to activate PKC, was not sufficient to induce alkalinization. We conclude that PKC is necessary but not sufficient for alkalinization. In contrast, the GnRH response can be mimicked by a simultaneous application of phorbol myristate acetate and KCl. To further explore the putative role of pHi in the secretory process, LH release was studied. Using Na(+)-free medium or amiloride, we show that basal LH was not dependent upon the Na+/H+ exchanger activity. Conversely, GnRH-induced LH release was significantly decreased; this decrease was greater in E2-treated cells but prevented by bicarbonate. These data show that pHi and the Na+/H+ exchanger play an important role in the stimulus secretion coupling process of gonadotrophs. E2, which is an important factor in the regulation of gonadotropic hormone release, participates also in the pHi variations.

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. TRH (50 nmol/L) induced a complex pattern of electrical changes. The initial response was transient hyperpolarization (activation of potassium conductance), followed by increased low amplitude voltage fluctuations occasionally leading to action potentials. These TRH-induced electrophysiological changes were similar to those reported in rat and human PRL-secreting adenoma cells. These results suggest that TRH may have an identical mode of action in tumoral PRL and TSH cells. In the cells from the hypothyroid patient, the initial response to TRH cells was similar, but the second phase response was greater. The findings that the cells cultured from these patients behaved differently with regard to their electrophysiological characteristics (action potentials) and responses to TRH may reflect the different clinical conditions from which they were derived.

Voltage-dependent Ca2+ channels in Chinese hamster ovary (CHO) cells.

Voltage-dependent Ca2+ channels were identified in CHO-K1 cells, currently used in molecular biology studies. Experimental data obtained at both macroscopic and single-channel levels using the patch-clamp technique show that the Ca2+ current in CHO cells is similar to the high-threshold L-type of Ca2+ current previously observed in excitable cells. It can be carried by Ca2+ or Ba2+ ions, blocked by both inorganic (Co2+) and organic (nifedipine, isradipine) Ca2+ channel blockers. The unitary Ca2+ channel activity was characterized by a conductance of 19 pS in 60 mM Ca2+, by single exponential distribution of open times (t = 0.84 ms) and biexponential distribution of closed times (tf = 1.67 ms, ts = 7.9 ms). However, the functional role of these Ca2+ channels in CHO cells remains unclear.

Regulation of intracellular chloride concentration in rat lactotrophs: possible role of mitochondria.

Increasing evidence is accumulating for the involvement of chloride ions in the stimulus-secretion coupling of pituitary cells. We show that the mean intracellular chloride concentration [Cl-]i of rat lactotroph cells maintained in culture is high, close to 60 mM (59.4 mM), using the Cl- sensitive fluorescent probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), coupled with whole-cell patch-clamp recordings. We demonstrate that this high level is correlated with the presence of mitochondrial stores of Cl- as shown by the release of Cl- in response to various metabolic inhibitors. We determine that CCP (50 microM) induces a mean [Cl-]i increase of 15.8+/-5.8 mM, using combined electrophysiology and microspectrofluorimetry methods. These data strongly suggest that cell metabolism, including the mitochondrial function, modulate [Cl-]i.

Prolactin induces an inward current through voltage-independent Ca2+ channels in Chinese hamster ovary cells stably expressing prolactin receptor.

There is still only limited understanding of the early steps of prolactin (PRL) signal transduction in target cells. Recent studies have identified some of the essential first steps: these include the rapid association of the PRL receptor with JAK tyrosine kinases and tyrosine phosphorylation of a number of proteins, including members of the signal transducer and activator of transcription (Stats) family. On the other hand, binding of PRL to its receptor is rapidly followed by calcium influx. However, PRL-induced ionic events and the related ionic channels involved have not been clearly established. This work was undertaken to characterise the channels responsible for calcium influx and to obtain an insight into their activation processes. Using the patch-clamp technique in the cell-attached configuration, single Ca2+ channel currents were recorded following PRL application (10 nM) in Chinese hamster ovary (CHO) cells stably expressing PRL receptor (CHO-E32). Statistical analysis showed that the recorded currents were voltage-independent, with a slope conductance of 16 pS. Although these channels were present in excised patches, the fact that PRL was unable to activate them suggested that a soluble cytoplasmic component may be required. Application of the purified inositol phosphate, Ins(1,3,4,5)P4 (2 microM), to the inside of the excised patch membrane activated the voltage-independent 16 pS Ca2+ channel. The open probability (Popen) was enhanced. The inositol phosphates Ins(1,2,3,4,5)P5 and Ins(1,4,5)P3 did not affect channel activity while InsP6 (20 microM) had some effect, although less marked than that of Ins(1,3,4,5)P4. Using the anion-exchange HPLC technique, we then studied the effects of PRL (10 nM) on the turnover of inositol phosphates (InsPs) in CHO-E32. Our studies showed that PRL induces rapid increases in the production of Ins(1,3,4,5)P4 (207% at 30 s), InsP5 (171% at 30 s), and InsP6 (241% at 30 s). Conversely, Ins(1,4,5)P3 showed a transient decrease at 5 s, accompanied by a concomitant increase in Ins(1,3,4,5)P4, suggesting that the former could be transiently phosphorylated to produce the latter. Comparison of the production kinetics of Ins(1,4,5)P3, Ins(1,3,4,5)P4, InsP5, and InsP6 indicated the possibility of additional metabolic routes which have yet to be determined. This study suggests that PRL promotes Ca2+ entry through voltage-independent Ca2+ channels that may be activated by Ins(1,3,4,5)P4 and InsP6.

Signal transduction mechanisms in cultured CNS neurons and clonal pituitary cells.

The experimental accessibility of monolayer culture has been used to study signal transduction mechanisms in primary CNS neurons and clonal pituitary cells. Here we review results on two signals representative of the emerging diversity of mechanisms discovered in all species studied thus far. One is mediated by micromolar concentrations of the amino acid GABA at postsynaptic membranes throughout the mammalian CNS and involves transient activation of Cl- ion channels whose distribution of conducting periods accounts for the millisecond time course of the signal. This signal serves to depress the probability that the target cell will trigger an action potential. The signal intensifies as the postsynaptic membrane is depolarized and can be modulated by clinically important drugs, primarily through changes in channel kinetics. The other signal involves nanomolar concentrations of the peptide TRH, which stimulates secretion of prolactin from clonal "GH3" pituitary cells. Intracellular recordings of GH3B6 cells show that TRH triggers a complex electrical response lasting several minutes. The response consists of Ca2+-activated K+ conductance followed by Ca2+-action potential activity. Whole-cell patch recordings, which rapidly dialyze the cell, can eliminate the TRH-induced changes in membrane excitability. Inclusion of aqueous lysates of the GH3B6 clone or the soluble second messenger factors inositol trisphosphate (IP3) or protein kinase (PKC) can restore various aspects of the change in membrane excitability. Thus, TRH alters ion conductance mechanisms through a second messenger cascade likely to involve IP3-mediated mobilization of Ca2+ from the endoplasmic reticulum and transient translocation of PKC from cytoplasm to plasma membrane. These synaptic and extrasynaptic signals reflect some of the diversity of transduction mechanisms involved in intercellular communication.

Intracellular calcium concentration and hormone secretion are controlled differently by TRH in rat neonatal lactotrophs and somatotrophs.

We studied the effects of TRH on the cytosolic free calcium concentration ([Ca2+]i) of female rat pituitary prolactin-secreting (lactotroph) and GH-secreting (somatotroph) cells in the early postnatal period, i.e. at postnatal days 5 and 10. [Ca2+]i of single identified lactotrophs and somatotrophs was recorded by dual-emission microspectrofluorimetry using the intracellular fluorescent calcium probe indo 1. An application of TRH (100 nM, 10 s) induced a marked [Ca2+]i increase in 65% of neonatal lactotrophs and 34% of neonatal somatotrophs while the remaining cells were unaffected. Most of the responsive cells, both lactotrophs and somatotrophs, exhibited a similar biphasic Ca2+ response, made up of an initial rapid large increase in [Ca2+]i followed by sustained [Ca2+]i fluctuations. In both cell types, removal of Ca2+ from the extracellular medium or addition of the Ca2+ channel blocker, cadmium chloride (500 microM), inhibited the second phase whereas the first phase persisted. Furthermore, in both cell types, protein kinase C (PKC) depletion by incubation in phorbol myristate acetate (1 microM) for 24 h abolished the second phase but did not inhibit the first phase. Conversely, when cells were pretreated with the Ca(2+)-ATPase inhibitor, thapsigargin (100 nM), all TRH-induced [Ca2+]i changes in both cell types disappeared. TRH therefore induces a biphasic increase in [Ca2+]i involving intra- and extracellular Ca2+ in neonatal lactotrophs and somatotrophs as it does in adult lactotrophs. The first phase is presumably due to mobilization of Ca2+ from intracellular stores whereas the second phase presumably results from a PKC-sensitive influx of Ca2+. TRH action on membrane potential was then investigated using the patch-clamp technique in the whole-cell mode. TRH-induced changes in membrane potential consisted of an initial hyperpolarization followed by depolarization and action potential firing. We also investigated TRH action on prolactin and GH secretion by neonatal pituitary cells using RIA. Surprisingly, static assays of prolactin and GH revealed only stimulation of prolactin release by TRH but no effect on GH secretion, although, as expected, GH-releasing factor was a potent agonist of GH secretion. Our results suggest that TRH regulates neonatal lactotrophs and somatotrophs differently, in that the [Ca2+]i changes do not correlate with stimulation of exocytosis in the latter cell type.

Involvement of both calcium influx and calcium mobilization in growth hormone-induced [Ca2+]i increases in Chinese hamster ovary cells.

This study reports rapid effects of growth hormone (GH) on the intracellular free calcium concentration ([Ca2+]i) in Chinese hamster ovary (CHO) cells stably expressing rabbit GH receptor. [Ca2+]i was measured by spectrofluorimetric methods in single cells and membrane Ca2+ currents by patch clamp techniques in the whole-cell configuration. In individual CHO cells, bathed in a standard saline solution containing 2 mM Ca2+, basal [Ca2+]i was 191 +/- 27 nM (mean +/- S.D.; n=83). Short term administration of GH (100 ng/ml, 30 s) induced a [Ca2+]i increase in 54% of cells tested (n = 398 of 743). Responses were clearly heterogeneous. Maximum calcium increase varied from 16 to 853 nM and time to peak varied from 4 to 320 s. On examination of the [Ca2+]i increases, it was possible to define two different types of calcium responses to GH. Experimental manipulations of extracellular and intracellular calcium concentrations demonstrated that GH-induced calcium increases involved both calcium influx and calcium mobilization. Calcium influx, a long lasting, small amplitude (63 +/- 34 nM) response, was observed in 121 out of 398 cells (30%) whereas calcium mobilization, a transient, large amplitude (263 +/- 175 nM) response, was observed in 277 out of 398 cells (70%). Moreover, patch clamp data show that influx did not involve the dihydropyridine-sensitive calcium channels.