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Plasticity of function is required for each of the anterior pituitary endocrine axes to support alterations in the demand for hormone with physiological status and in response to environmental challenge. This plasticity is mediated at the pituitary level by a change in functional cell mass resulting from a combination of alteration in the proportion of responding cells, the amount of hormone secreted from each cell, and the total number of cells within an endocrine cell population. The functional cell mass also depends on its organization into structural and functional networks. The mechanisms underlying alteration in gland output depend on the strength of the stimulus and are axis dependent but in all cases rely on sensing of output of the functional cell mass and its regulation. Here, we present evidence that the size of pituitary cell populations is constrained and suggest this is mediated by a form of quorum sensing. We propose that pituitary cell quorum sensing is mediated by interactions between the networks of endocrine cells and hormone-negative SOX2-positive (SOX2+ve) cells and speculate that the latter act as both a sentinel and actuator of cell number. Evidence for a role of the network of SOX2+ve cells in directly regulating secretion from multiple endocrine cell networks suggests that it also regulates other aspects of the endocrine cell functional mass. A decision-making role of SOX2+ve cells would allow precise coordination of pituitary axes, essential for their appropriate response to physiological status and challenge, as well as prioritization of axis modification.

The secretion of endocrine hormones from pituitary cells finely regulates a multitude of homeostatic processes. To dynamically adapt to changing physiological status and environmental stimuli, the pituitary gland must undergo marked structural and functional plasticity. Endocrine cell plasticity is thought to primarily rely on variations in cell proliferation and size. However, cell motility, a process commonly observed in a variety of tissues during development, may represent an additional mechanism to promote plasticity within the adult pituitary gland. To investigate this, we used multiphoton time-lapse imaging methods, GH-enhanced green fluorescent protein transgenic mice and sexual dimorphism of the GH axis as a model of divergent tissue demand. Using these methods to acutely (12 h) track cell dynamics, we report that ovariectomy induces a dramatic and dynamic increase in cell motility, which is associated with gross GH-cell network remodeling. These changes can be prevented by estradiol supplementation and are associated with enhanced network connectivity as evidenced by increased coordinated GH-cell activity during multicellular calcium recordings. Furthermore, cell motility appears to be sex-specific, because reciprocal alterations are not detected in males after castration. Therefore, GH-cell motility appears to play an important role in the structural and functional pituitary plasticity, which is evoked in response to changing estradiol concentrations in the female.

During gestation, parturition, and lactation, the endocrine axis of the dam must continually adapt to ensure the continual and healthy development of offspring. The anterior pituitary gland, which serves as the endocrine interface between the brain and periphery, undergoes adaptations that contribute to regulation of the reproductive axis. Growth factors and their receptors are potential candidates for intrapituitary and paracrine factors to participate in the functional and anatomical plasticity of the gland. We examined the involvement of the growth factor glial cell-derived neurotrophic factor (GDNF) and its receptor tyrosine kinase rearranged during transfection (Ret) in the physiological functional and anatomical plasticity of the anterior pituitary gland. We found that variations in both expression and subcellular localization of Ret during gestation and lactation are temporally correlated with changes in pituitary gland function. We showed that Ret/GDNF signaling could endorse two different functional roles depending on the physiological status. At the end of lactation and after weaning, Ret was colocalized with markers of apoptosis. We found that Ret could therefore act as a physiological dependence receptor capable of inducing apoptosis in the absence of GDNF. In addition, we identified the follicullostellate cell as a probable source for intrapituitary GDNF and proposed GDNF as a potential physiological modulator of endocrine cell function. During all stages studied, we showed that acute application of GDNF to pituitary slices was able to modulate both positively and negatively intracellular calcium activity. Altogether our results implicate Ret/GDNF as a potent pleiotropic factor able to influence pituitary physiology during a period of high plasticity.

The hypothalamic GHRH neurons secrete pulses of GHRH to generate episodic GH secretion, but little is known about the mechanisms involved. We have made transgenic mice expressing enhanced green fluorescent protein (eGFP) specifically targeted to the secretory vesicles in GHRH neurons. GHRH cells transported eGFP from cell bodies in the arcuate nucleus to extensively arborized varicose fiber terminals in the median eminence. Patch clamp recordings from visually identified GHRH cells in mature animals showed spontaneous action potentials, often firing in short bursts up to 10 Hz. GHRH neurons received frequent synaptic inputs, as demonstrated by the recording of abundant inward postsynaptic currents, but spikes were followed by large after-hyperpolarizations, which limited their firing rate. Because many GHRH neurons lie close to the ventral hypothalamic surface, this was examined by wide-field binocular epifluorescence stereomicroscopy. This approach revealed an extensive horizontal network of GHRH cells at low power and individual fiber projections at higher power in the intact brain. It also showed the dense terminal projections of the GHRH cell population in the intact median eminence. This model will enable us to characterize the properties of individual GHRH neurons and their structural and functional connections with other neurons and to study directly the role of the GHRH neuronal network in generating episodic secretion of GH.

Endocrine cells isolated from the anterior pituitary fire intracellular Ca2+ ([Ca2+]i) transients due to voltage-gated Ca2+ entry. However, the patterns of [Ca2+]i transients within the glandular parenchyma of the anterior pituitary are unknown. Here we describe, using real-time confocal laser microscopy, several spontaneous patterns of calcium signaling in acute pituitary slices prepared from male as well as cycling and lactating female rats. Forty percent of the cells demonstrated a spontaneous bursting mode, consisting of an active period of [Ca2+]i transients firing at a constant frequency, followed by a rest period during which cells were either silent or randomly active. The remaining recordings from endocrine cells either demonstrated random [Ca2+]i transients or were silent. These rhythmic bursts of [Ca2+]i transients, which required extracellular calcium, were detected in lactotrophs, somatotrophs, and corticotrophs within the acute slices. Of significance was the finding that the bursting mode could be adjusted by hypothalamic factors. In slices prepared from lactating rats, TRH recruited more bursting cells and finely adjusted the average duty cycle of [Ca2+]i bursts such that cells fired patterned bursts for approximately 70% of the recording period. Eighty-six percent of these cells were lactotrophs. Thus, the rhythmic [Ca2+]i bursts and their tuning by secretagogues may provide timing information that could encode for one or more cellular functions (e.g. exocytosis and/or gene expression) critical for the release of hormones by endocrine cells in the intact gland.

International audience; Growth hormone (GH) is the key hormone involved in the regulation of growth and metabolism, two functions that are highly modulated during infancy. GH secretion, controlled mainly by GH releasing hormone (GHRH), has a characteristic pattern during postnatal development that results in peaks of blood concentration at birth and puberty. A detailed knowledge of the electrophysiology of the GHRH neurons is necessary to understand the mechanisms regulating postnatal GH secretion. Here, we describe the unique postnatal development of the electrophysiological properties of GHRH neurons and their regulation by gonadal hormones. Using GHRH-eGFP mice, we demonstrate that already at birth, GHRH neurons receive numerous synaptic inputs and fire large and fast action potentials (APs), consistent with effective GH secretion. Concomitant with the GH secretion peak occurring at puberty, these neurons display modifications of synaptic input properties, decrease in AP duration, and increase in a transient voltage-dependant potassium current. Furthermore, the modulation of both the AP duration and voltage-dependent potassium current are specifically controlled by gonadal hormones because gonadectomy prevented the maturation of these active properties and hormonal treatment restored it. Thus, GHRH neurons undergo specific developmental modulations of their electrical properties over the first six postnatal weeks, in accordance with hormonal demand. Our results highlight the importance of the interaction between the somatotrope and gonadotrope axes during the establishment of adapted neuroendocrine functions.

We have generated transgenic mice with somatotroph-specific expression of a modified influenza virus ion channel, (H37A)M2, leading to ablation of GH cells with three levels of severity, dependent on transgene copy number. GH-M2(low) mice grow normally and have normal-size pituitaries but 40-50% reduction in pituitary GH content in adult animals. GH-M2(med) mice have male-specific transient growth retardation and a reduction in pituitary GH content by 75% at 42 d and 97% by 100 d. GH-M2(high) mice are severely dwarfed with undetectable pituitary GH. The GH secretory response of GH-M2(low) and GH-M2(med) mice to GH-releasing peptide-6 and GHRH was markedly attenuated. The content of other pituitary hormones was affected depending on transgene copy number: no effect in GH-M2(low) mice, prolactin and TSH reduced in GH-M2(med) mice, and all hormones reduced in GH-M2(high) mice. The effect on non-GH hormone content was associated with increased macrophage invasion of the pituitary. Somatotroph ablation affected GH cell network organization with limited disruption in GH-M2(low) mice but more severe disruption in GH-M2(med) mice. The remaining somatotrophs formed tight clusters after puberty, which contrasts with GHRH-M2 mice with a secondary reduction in somatotrophs that do not form clusters. A reduction in pituitary beta-catenin staining was correlated with GH-M2 transgene copy number, suggesting M2 expression has an effect on cell-cell communication in somatotrophs and other pituitary cell types. GH-M2 transgenic mice demonstrate that differing degrees of somatotroph ablation lead to correlated secondary effects on cell populations and cellular network organization.

In stable transfection experiments in the GH-producing GC cell line, a construct containing the entire signal peptide and the first 22 residues of human GH linked in frame with enhanced green fluorescent protein (eGFP), produced brightly fluorescent cells with a granular distribution of eGFP. This eGFP reporter was then inserted into a 40-kb cosmid transgene containing the locus control region for the hGH gene and used to generate transgenic mice. Anterior pituitaries from these GH-eGFP transgenic mice showed numerous clusters of strongly fluorescent cells, which were also immunopositive for GH, and which could be isolated and enriched by fluorescence-activated cell sorting. Confocal scanning microscopy of pituitary GH cells from GH-eGFP transgenic mice showed a markedly granular appearance of fluorescence. Immunogold electron microscopy and RIA confirmed that the eGFP product was packaged in the dense cored secretory vesicles of somatotrophs and was secreted in parallel with GH in response to stimulation by GRF. Using eGFP fluorescence, it was possible to identify clusters of GH cells in acute pituitary slices and to observe spontaneous transient rises in their intracellular Ca 21 concentrations after loading with Ca 21 sensitive dyes. This transgenic approach opens the way to direct visualization of spontaneous and secretagogue-induced secretory mechanisms in identified GH cells. (Endocrinology 141: 4681‐ 4689, 2000)

Spontaneous and CRF-stimulated changes in the cytosolic free calcium concentration ([Ca2+]i) were studied in two types of corticotrophs: 1) cultured human ACTH-secreting pituitary adenoma cells (hACTH cells), and 2) identified small ovoid corticotrophs cultured from normal rat pituitaries. [Ca2+]i was monitored in individual corticotrophs by dual emission microspectrofluorimetry using indo-1 as the intracellular fluorescent Ca2+ probe. In hACTH cells, [Ca2+]i measurements were carried out in combination with electrophysiological recordings obtained using whole cell patch-clamp techniques. It was shown that a single spontaneous Ca(2+)-dependent action potential led to a marked transient increase in [Ca2+]i in human tumoral corticotrophs. Spontaneous fluctuations in [Ca2+]i were also observed in unpatched corticotrophs whether derived from human pituitary tumors or normal rat tissue. Based on their striking kinetic features and their sensitivity to external Ca2+, we suggest that these spontaneous [Ca2+]i transients were the consequence of action potential firing. Under separate voltage-clamp (patch-clamp) conditions, tumor corticotrophs showed two Ca2+ current components: a low threshold, rapidly inactivating (T-type) current, and a higher threshold, slowly inactivating (L-type) current. The dihydropyridine Ca2+ channel blocker PN 200-110 (100 nM) abolished the L-type current without affecting the T-type current, while the inorganic Ca2+ channel blocker Cd2+ (200 microM) suppressed both Ca2+ currents. The Na+ channel blocker tetrodotoxin (5 microM) did not affect inward currents in tumor corticotrophs. Both L- and T-type voltage-gated Ca2+ channels were involved in controlling [Ca2+]i transients in both tumor and normal corticotrophs, inasmuch as Cd2+ (200 microM) abolished [Ca2+]i) transients, while PN 200-110 (100 nM) greatly diminished, but did not completely abolish, [Ca2+]i transients. The latter did not appear to depend on a voltage-dependent Na+ influx, since they were unaffected by tetrodotoxin (5 microM). Corticotrophs generate [Ca2+]i transients in response to the hypothalamic secretagogue CRF by acting on their membrane excitability. Indeed, we demonstrated in combined fluorescent and electrophysiological experiments that CRF (100 nM) had a coordinate action on human tumoral corticotrophs comprised of a modest depolarization and an increase in the frequency of both action potentials and subsequent [Ca2+]i transients. A coincident increase in the peak amplitude of the [Ca2+]i transient and after hyperpolarization was also observed in some CRF-stimulated cells. CRF (100 nM) evoked qualitatively similar [Ca2+]i patterns in human tumoral and normal rat corticotrophs not subjected to patch-clamping.(ABSTRACT TRUNCATED AT 400 WORDS)

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)

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.

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.