From INSERM U469, Centre CNRS/INSERM de Pharmacologie et d'Endocrinologie, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France
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ABSTRACT |
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We investigated the organization of spontaneous rises in cytosolic free Ca2+ concentration ([Ca2+]i) due to electrical activity in acute pituitary slices. Real time confocal imaging revealed that 73% of the cells generated fast peaking spontaneous [Ca2+]i transients. Strikingly, groups of apposing cells enhanced their [Ca2+]i in synchrony with a speed of coactivation >1,000 µm/s. Single-cell injection of Neurobiotin or Lucifer yellow labeled clusters of cells, which corresponded to coactive cells. Halothane, a gap junction blocker, markedly reduced the spread of tracers. Coupling between excitable cells was mainly homologous in nature, with a prevalence of growth hormone-containing cells. We conclude that spontaneously active endocrine cells are either single units or arranged in synchronized gap junction-coupled assemblies scattered throughout the anterior pituitary gland. Synchrony between spontaneously excitable cells may help shape the patterns of basal secretion.
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INTRODUCTION |
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Endocrine pituitary cells that release hormones from large dense core vesicles (LDCV)1 by calcium-mediated exocytosis exhibit spontaneous firing of action potentials. In cultured cell preparations, individual cells present asynchronous activity with different firing patterns (pace-making or bursting mode). When electrical recordings are combined with fluorescent monitoring of cytosolic free Ca2+ concentration ([Ca2+]i), single spontaneous spikes trigger transient rises in [Ca2+]i with characteristic features; a time to peak of less than 1 s and a return within a couple of seconds (1). The lag between the onset of the [Ca2+]i rise and exocytosis is also within a subsecond range (2, 3), sharing common features of stimulus-secretion coupling with neuroendocrine cells (e.g. chromaffin cells) and neurons, which release peptides from LDCV (4-6).
Although electrical activity has already been detected in the anterior pituitary gland, both in vivo and in tissue preparations (7-9), the dynamics and organization of [Ca2+]i rises associated with spontaneous action potentials have not yet been investigated at the tissue level. Based on the heterogeneous distribution of the five secretory cell types throughout the tissue (10), endocrine cells would display asynchronous firing so that the overall activity of each secretory type would simply reflect the average of single cell events. Cell regulation should mainly depend on the input of hypothalamic clocks, such as sequential release of growth hormone-releasing factor and somatostatin, which have been shown to pace growth hormone (GH) release (11). However, the gland disconnected from the hypothalamic inputs still shows pulsatile GH release (12), suggesting a synchronization of cellular signals within the tissue. With regard to the mechanisms accounting for synchronization in other tissues (13-18), two sources of cell-to-cell communication, not mutually exclusive, could be proposed. First, both endocrine and non-endocrine (folliculostellate) pituitary cells release various products (ATP, dopamine, and so forth), which locally act on neighboring cells (19-21). Second, gap junctions present in the anterior pituitary (9, 22-24) may allow both metabolic and electrical coupling between connected cells.
To study the behavior of spontaneously active cells within the adenohypophysis, we measured the multicellular patterns of spontaneous [Ca2+]i rises in acute slices of guinea pig pituitary, which preserved tissue structure (25). Real time confocal laser microscopy with the Ca2+-sensitive fluorescent dye fluo-3 offers a sensitive method for optical recording of the fast peaking [Ca2+]i transients due to spontaneous action potentials. By visualizing the multicellular profiles of [Ca2+]i activity in these slices, we detected clusters of spontaneously coactive endocrine cells, which were scattered throughout the anterior pituitary. An abstract of a preliminary account of these results has already been presented (51).
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EXPERIMENTAL PROCEDURES |
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Tissue Slice Preparation-- Acute pituitary slices were prepared according to previously reported methods (26). Briefly, the pituitary gland was removed from 4-8-week-old female guinea pigs (OCF-DH albinos) that had been killed by decapitation after pentobarbital anesthesia. After keeping the gland in ice-cold saline for 2 min, it was glued onto an agarose cube and transferred to the stage of a vibratome (Microslicer®, DTK-1000, D.S.K, Dosaka EM Co. Ltd., Kyoto, Japan). Coronal slices of 150-µm thickness were then cut with a razor blade and transferred to a storage chamber thermostated at 32 °C, containing Ringer's saline (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 12 glucose, and buffered to pH 7.4. The saline was continuously bubbled with carbogen (95% O2, 5% C02). As reported for the slices of the intermediate lobe (26), slices of the anterior lobe were suitable for patch-clamp recordings and Ca2+ signal measurements immediately after cutting. Slices were viable up to 8 h, as seen by the presence of spontaneous [Ca2+]i elevations. To achieve optical and/or electrophysiological recordings, pituitary slices were transferred to a recording chamber attached to the stage of an upright microscope fitted with differential interference contrast optics (Axioskop FS, Zeiss, Le Pecq, France) and continuously superfused with Ringer's saline at 30 °C.
Confocal Microscopy-- Fast spontaneous [Ca2+]i transients were routinely measured by a real time (30-480 frames/s) confocal laser scanning microscope equipped with an Ar/Kr laser (Odyssey XL with InterVision 1.4.1 software, Noran Instruments Inc., Middleton, WI). Cells were viewed with a 63 × 0.9 numerical aperture achroplan water immersion objective lens (Zeiss). Various thicknesses of confocal images was obtained by selecting different detection slits. The larger slit (100 µm) was used for [Ca2+]i signals, giving bright images with a 3.1-µm axial resolution. When cells were subsequently loaded with Lucifer yellow (see below), confocal images were acquired with a 25-µm slit, which provided an axial resolution of ~1.3 µm. Slices were loaded with the Ca2+-sensitive fluorescent probe fluo-3 by exposure to 10 µM fluo-3 acetoxymethyl ester (fluo-3/AM, Molecular Probes, Eugene, OR) for 20-30 min at 32 °C (27). Fluo-3 was excited through a 488-nm band pass filter, and the emitted fluorescence was collected through a 515-nm barrier filter. Transmitted images were acquired using the longer wavelength of the laser beam (647 nm), which penetrated deeply into pituitary slices. To follow the time course of fluo-3 emission changes, the bright over time tool of the InterVision 1.4.1 software package was applied to areas that surrounded cells either on live images or following capture of sequential images into memory of an Indy R4600SC/133 MHz Silicon Graphics station equipped with a Cosmo compress JPEG board. To ensure that the image rate acquisition was adequate to resolve the time delay between [Ca2+]i changes measured in cells of the same field, the fluorescence changes were also acquired in the line scanning mode at a rate of 4.4 ms/line (6400 ns sample time, 480 lines). In some experiments, the line trigger (TTL signal) from the Odyssey XL was used to trigger a voltage pulse that helped synchronize its timing with the line 100. A voltage stimulator delivered these depolarizing pulses (2 ms, 6 V) to a patch pipette filled with Ringer's saline and positioned on the cell (28). Because fluo-3 is a single-wavelength dye, its emission is a function of both intracellular Ca2+ and dye concentrations. [Ca2+]i changes were therefore expressed as the F/Fmin ratio where Fmin was the minimum fluorescence intensity measured during the recording (27). No detectable difference was noted between slices used just after cutting or after spending several hours in the storage chamber. Acquired data were then processed for analysis using either the Indy station (InterVision 1.4.1, two-dimensional analysis module) or a PowerPC 8100/100 MHz (NIH Image 1.6.0, Adobe Photoshop 3.0.5 or Igor Pro 2.03).
Electrophysiology-- Membrane potential was recorded in the whole-cell configuration of the patch-clamp technique (29). Patch pipettes were pulled to a resistance of 4-8 megohms from borosilicate glass (1.5-mm outer diameter, 1.17-mm inner diameter) and filled with the following internal solution (in mM): 140 potassium gluconate, 10 KCl, 2 MgCl2, 1.1 EGTA, 5 HEPES, that was titrated to pH 7.2 with KOH. For simultaneous recordings of membrane potential and [Ca2+]i, the perforated whole-cell patch-clamp technique was preferred to the conventional patch-clamp technique. In this case, the internal pipette solution was composed of (in mM): 10 KCl, 10 NaCl, 70 K2SO4, 7 MgCl2, 5 HEPES, and 100 µg/ml nystatin (Sigma). Nystatin was added to the electrode solution before filling the patch pipettes. Perforation was usually achieved within 10 min after seal formation. Cells with an access resistance >30 megohms were discarded. Membrane potential was recorded under current-clamp conditions using a List EPC-9 patch-clamp amplifier (HEKA Electronik, Lambrecht/Pfalz, Germany) and filtered at 3 kHz. Patch-clamp signals were acquired and analyzed using Pulse + PulseFit softwares (version 7.86, HEKA Electronik) on a PowerPC 8100/100.
Cell-to-Cell Communication and Dye Coupling-- The fluorescent dye Lucifer yellow (LY, 4% in 150 mM LiCl) was introduced into cells through a sharp microelectrode. The cells were impaled and filled for a few minutes, before image acquisition with the confocal microscope. When LY was injected into cells not subjected to [Ca2+]i imaging, the cells were selected on the basis of their round or oval shape and the presence of dense vesicles, which resembled hormone-containing vesicles. In addition, the hormonal content of impaled cells was routinely characterized by immunofluorescence after formaldehyde fixation of slices.
Cells were also loaded with the NeurobiotinTM (N-(2-aminoethyl)-biotinamide hydrochloride) tracer (1% in the internal solution) by diffusion through the patch pipette for 10-30 min. To increase the rate of Neurobiotin dialysis, depolarizing pulses (500-ms duration, 0.5 Hz) were periodically applied. In some cells, the presence of gap junctional communication was assessed by using the size exclusion properties of dextran conjugates (9). Cells were dialyzed with a patch pipette containing 1% Neurobiotin plus 2 mg/ml dextran Texas Red (lysine fixable, Mr 3,000) for at least 10 min. After fixation (see below), slices were rinsed in phosphate-buffered saline (PBS, pH 7.4) plus 0.1% bovine serum albumin (BSA) and then incubated in PBS + BSA + 0.8% saponin for 1 h at room temperature. After several rinses in PBS + BSA, Neurobiotin staining was revealed using fluorescein- or Texas Red-labeled avidin D (1:200 dilution, room temperature, 5 or 1 h, respectively).Immunohistofluorescence-- After optical/electrophysiological recordings, pituitary slices were fixed prior to immunohistofluorescence as follows. The tissue was uniformly fixed by two successive formaldehyde solutions as described previously (30). Slices were incubated firstly in formaldehyde 3% pH 6.5 (8 mM PIPES, 0.5 mM EGTA, 0.2 mM MgCl2) for 10 min and secondly in formaldehyde 3%, pH 11, (100 mM sodium borate) for 1 h at room temperature. They were then rinsed in 0.1% sodium borohydride (PBS, pH 8), permeabilized in 0.8% saponin (PBS, pH 7.4) for 45 min and washed with Tris-buffered saline (TBS) (10 mM TRIZMA® base, 150 mM NaCl) plus 1% BSA (pH 7.6).
Slices were incubated overnight at 4 °C in the presence of antibodies raised against human GH (developed in rabbit, used at 1:2500 dilution) with 0.05% BSA in TBS. The antiserum was kindly donated by Dr. Tillet (INRA, Nouzilly, France) and the NIDDK-human GH-B-1 was used as an antigen. Incubation was followed by several washes with TBS plus 0.05% BSA. The primary antibodies were localized by a 1-hr incubation with a Cy5- or fluorescein-conjugated anti-rabbit IgG developed in donkey (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), used at 1:250 dilution in TBS plus 0.05% BSA. After washes, slices were postfixed in formaldehyde 3% (PBS, pH 7.4) and rinsed in 50 mM NH4Cl before mounting in SlowfadeTM Light (Molecular Probes). Images were acquired with the confocal microscope as described above. Cells were viewed with a 63 × 1.4 numerical aperture plan-apochromat with a 15-µm detection slit (~0.65-µm axial resolution).Test Substances-- Drugs were pressure-ejected from an extracellular micropipette (tip diameter 2-5 µm), the tip of which was positioned in the vicinity of the recorded cells. The concentration reported are those in the pressure pipette. Lucifer yellow, somatostatin-14, and the calcium channel blocker CdCl2, were purchased from Sigma. The gap junction blocker halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) was from Fluka. To obtain Ca2+-free solution, CaCl2 was omitted from, and 5 mM EGTA was added to modified Ringer's saline. The Neurobiotin tracer was purchased from Vector Laboratories (Biosys S.A., Compiègne, France).
Statistics-- Numerical data are expressed as the mean ± S.E. Student's t test was used to compare means when appropriate. Differences between groups were assessed by using the non-parametric Mann-Whitney U test. Differences with p < 0.05 were considered significant.
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RESULTS |
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Experiments were performed in coronal slices (150-µm thickness) from pituitary of 4-8-week-old female guinea pigs. Slices were loaded with fluo-3/AM by bath application of the Ca2+ indicator, which produced widespread staining of the first and second layers of cells on the slice surface. When visualized with epifluorescence under the upright microscope, numerous cells within each field exhibited spontaneous changes in fluo-3 emission, reflecting rises in [Ca2+]i (31). Time-lapse optical sequences of the cells showing spontaneous [Ca2+]i rises were then recorded with fast scanning confocal imaging (120 images/s with averaging 4 frames) during different experimental protocols.
Presence of Spontaneously Coactive Cells in Acute Pituitary Slices-- In most slices, one to several groups of adjacent active cells which fired synchronously were observed, as visualized at first using the epifluorescent port of the microscope. Real time optical imaging revealed then that these clusters of synchronous cells coexisted with asynchronous neighboring cells as illustrated in Fig. 1A. The top left image shows a field in which four fluo-3-loaded cells could be observed in the same optical section. The montage of consecutive optical slices depicts a time series of fluo-3 emission frames encoded in pseudocolors (from blue to red with [Ca2+]i increasing). The three bottom cells fired spontaneous fast-peaking [Ca2+]i transients. The plots of relative fluo-3 emission changes show that two cells paced their [Ca2+]i in synchrony, whereas the third one had its own rhythm. With only the first pair of synchronized [Ca2+]i transients, it seems likely that the red-circled cell became active before the green-circled one while the lag between the following pairs of [Ca2+]i transients was indistinguishable under the time resolution used in these experiments.
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Dye Diffusion between Spontaneously Coactive Cells--
Different
communication mechanisms, either electrical or biochemical, could
explain the synchronization of spontaneously active cells. Since
coactivation always occurred between apposing cells of finite clusters,
the synchronization signal should be restricted to coactive cell-cell
boundaries, but not extensively diffused to neighboring asynchronous
cells. Since gap junctions were described in the anterior pituitary
(22), we carried out dye coupling experiments with a low molecular
weight fluorescent dye LY (457 Da). The tracer was injected through a
sharp microelectrode into single cells belonging to synchronized
clusters. Fig. 2A illustrates an example of two neighboring cells exhibiting synchronized spontaneous [Ca2+]i transients. After fluo-3 emission
measurements, cell labeled 2 was impaled with LY (4% in 150 mM LiCl). A few seconds later, cell labeled 1 was also stained with LY, indicating the LY diffusion from the impaled
cell to the coupled partner. No diffusion was observed in other
adjacent cells (n = 21). Moreover, LY diffusion was
restricted to single impaled cells, which spontaneously fired with
their own rhythm (Fig. 2B, n = 14). These
data strongly suggest that gap junctions could ensure the spread of
coactivation between excitable pituitary cells. Since large molecules
(1,000 Da) usually do not permeate through gap junctions, further
experiments were conducted with large molecular weight dextran
conjugates. Cells were randomly loaded with Neurobiotin, a low
molecular mass dye (323 Da, 1% in internal patch pipette solution) and
dextran Texas Red (3,000 Da, 2 mg/ml), by diffusion for 10-30 min. In two out of seven clusters, patched cells contained both markers, whereas coupled cells were labeled with Neurobiotin only (data not
shown). In the others, no dye diffusion was observed.
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Speed of Coactivation Is Higher than 1,000 µm/s-- Spread of coactivation through gap junctions can be due to either simple diffusion of Ca2+ from a trigger cell to coupled cells or electrical coupling between these cells. Since these two mechanisms are associated with distinct speeds of propagation, x-t-series line scans were performed to estimate the speed of the wave of coactivation. A single horizontal line crossing synchronized cells was continuously scanned over 2,133 ms (4.4 ms/line, 480 lines). Fig. 3A shows each line displayed in time along the y axis. The time course of the fluorescence changes in synchronized cells revealed a 5.6-ms delay between the onset of spontaneous [Ca2+]i transients. Taking into account the distance between cell centers, the average speed of the coactivation wave from the trigger cell to the other was 1,056 ± 112 µm/s (n = 40, 5 different coactive clusters). Interestingly, the calculation of the delay between [Ca2+]i transients within coactive cell assembly revealed that the trigger cell could become the responding cell and vice versa during recordings (four out of five clusters, Fig. 3B).
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Prevalence of Homologous Coupling between GH-containing Coactive Cells-- Earlier studies carried out in pituitary cells isolated from the tissue have revealed that spontaneous electrical activity and ensuing [Ca2+]i transients were mainly observed in cells containing either GH or prolactin (35). Since somatotrophs are the dominant secretory cell type in the anterior pituitary, we therefore investigated whether coactive cells could contain GH. To conduct this experiment, the hormonal content of cells was identified by immunohistofluorescence following formaldehyde fixation of slices. In 50% of cell clusters (14 out of 28), spontaneously coactive cells were GH-containing cells as illustrated in Fig. 4 (mid top frame). As expected from previously described results, the two synchronized cells were stained following LY injection (cells that turned green, second top frame). Interestingly, other apposing cells were also immunoreactive to GH but not dye-coupled to coactive cells. This suggests that homologous coupling could at least involve subsets of spontaneously active somatotrophs. Somatostatin, a native inhibitor of GH release, reversibly blocked spontaneous [Ca2+]i transients issued from both synchronous and asynchronous somatotrophs (n = 6 and 13, respectively, data not shown). It should be noted that clusters of synchronized cells composed of both GH-positive and -negative cells were also encountered (5 out of 28 clusters). The remainders were GH-negative (9 out of 28).
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DISCUSSION |
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Our experiments describe for the first time the multicellular [Ca2+]i rises, which spontaneously occur in acute slices from anterior pituitary. This spontaneous [Ca2+]i activity is spatially organized within the tissue into small groups of excitable cells that pace their [Ca2+]i synchronously. Combination of real time optical imaging with dye-coupling studies enabled us to observe a fast speed of coactivation (>1,000 µm/s) that involves cell-to-cell communication via gap junctions. In contrast, other cells which spontaneously display asynchronous [Ca2+]i transients are never dye-coupled to neighboring cells.
The discovery of coactivation of spontaneously active pituitary cells has marked a new step in our knowledge of how endocrine cells secreting from LDCV interact with one another in the absence of any stimulus. Although domains of spontaneously coactive cells have been extensively described, namely in the brain (16, 17, 36, 37), synchronization between excitable endocrine cells has only been reported in secretagogue-stimulated cells, such as beta cells from pancreatic islets exposed to high glucose levels. The latter are electrically silent at rest (i.e. at low glucose concentrations), while they display synchronized bursts of Ca2+-driven action potentials only in response to their fuel secretagogue (38, 39).
In the brain, two distinct mechanisms support the spread of
coactivation between spontaneously excitable cells. Synaptic
transmission synchronizes neuronal activity in many brain areas (15),
whereas coupling through gap junctions underlies local coactivation in, e.g. neocortex neuronal domains (16). Our results strongly
suggest that gap junctions cause coupling of excitable pituitary cell subsets and thereby allow the synchronization of spontaneous
[Ca2+]i transients in these assemblies of cells.
Two observations concur with this proposal. Cell injection of small
tracers (LY or Neurobiotin) results in the selective labeling of
coactive cells and halothane, a gap junction blocker, markedly lowers
the appearance of dye coupling without affecting the time course of spontaneous [Ca2+]i transients. Since all these
studies have been done in slices, the relevance of these findings
in vivo is yet unknown. Nevertheless, we and others have
already observed LY transfer between unidentified pituitary cells in
both neonatal rat pituitary slices maintained in long term (1 month)
organotypic culture (9) and rat hemipituitaries (24). Ultrastructural
and immunohistofluorescent studies have also demonstrated the presence
of gap junction plaques and the expression of two connexin types (Cx26
and Cx43) in the anterior pituitary (22, 40, 41). Altogether, this
strongly suggests that cell-to-cell calcium signaling mediated by gap
junction communication is indeed present in the gland and is not a side effect of the acute slice preparation.
Line scanning mode experiments reveal that the speed of coactivation is higher than 1,000 µm/s. Although we do not rule out that Ca2+ or a metabolite (e.g. inositol 1,4,5-trisphosphate) can slowly diffuse through gap junctions in the cell assemblies (13, 14, 42), cell coactivation underlying synchronized [Ca2+]i transients is associated with a much faster and regenerative mechanism in the anterior pituitary. If single action potentials drive spontaneous [Ca2+]i transients, they should therefore act as a trigger for coactivation between gap junction-coupled cells. However, the occurrence of synchronized [Ca2+]i transients upon removing the blockade of Ca2+ entry suggests that maintenance of connectivity is not due to Ca2+ entry per se.
The mechanism driving the recruitment of coactive cells is tantalizing. What are the trigger cells? Given that external voltage stimulation causes a [Ca2+]i rise, which can propagate to adjoining cells, we suggest that electrical coupling mediates intercellular communication between excitable cells. However, the entrainment is not likely to be associated with the firing of fast-spiking cells which "chatter" slower cells (17), since the frequencies of [Ca2+]i transients are roughly similar in both synchronous and asynchronous cells. Interestingly, trigger cells can alternate with time within the coactive cell domain. The mechanisms that dictate the wide range of spiking patterns in the excitable pituitary cells remain hard to identify despite the fact that the voltage-gated channels that open during the action potential have been well characterized (35, 43). One would therefore assume that the stochastic occurrence of action potentials would continuously determine which cell triggers spikes in neighboring cells via gap junctions. Paracrine interactions may also play a significant role in the selection of trigger cells. Since single action potentials seem to be efficient enough to trigger fast exocytosis in gland (adrenal) slices (44), a minute fraction of fast-acting factors (e.g. ATP, dopamine copacked with hormones in LDCV) (19, 21) readily released upon single action potentials would quickly alter the firing frequency of any coactive cells (45) and thereby change the hierarchy of the propagation of electrical events. Finally, dye-coupled cells are not always within a single field at a single plane of focus suggesting that the cell phasing synchronization can be out of focus during optical recordings. A fine strategy for studying the organization of [Ca2+]i events within coactive cell domains would therefore consist in applying three-dimensional imaging in real time.
The significance of spontaneously coactive cell domains in the anterior pituitary can be viewed in terms of [Ca2+]i signal and hormone secretion. Cells containing GH in their LDCV prevail in coactive cell assemblies. Interestingly, coactive GH-containing cells often coexist with nearby asynchronous GH cells. Hence, the patterns of GH release should depend on the integration of the exocytotic activities of both synchronous and asynchronous cells. Morphological studies have also suggested a polarized phenotype for GH cells in situ since the latter are mainly arranged in palisades alongside fenestrated capillaries (10). Thus, the concurrent level of secretory efficiency within the capillaries depends on the topographical distribution of the two distinct GH subsets within the columns of pituitary cells (so-called cell cords), which are separated by basal laminae, connective tissue and blood vessels (10).
An association between GH-positive and GH-negative cells was also encountered, suggesting that the intricate pattern of spontaneous coactivation may encode the trigger for releasing factors other than hormones. In our view, neurotransmitter-like factors found in LDCV (e.g. dopamine in somatotrophs and lactotrophs) (19) could represent putative candidates since their fast and evanescent actions on pituitary cells (46) could provide a fine tuning of nearby cell activities.
Besides a possible role for local control of secretion, coactivation of excitable endocrine cells may serve a more integrated function in the entire gland. This can require a substantial number of connections between synchronized groups. Can a mechanism account for close synchrony despite the apparent wide range of distances (and the presence of connective tissue) between spontaneously coactive areas in vivo? The episodic releases of hypothalamic secretagogues (e.g. GH-releasing factor) (11) might periodically alter the number of coupled cells by acting on gap junctions (47, 48). This is certainly plausible, but these connections might not be dense enough to allow synchronization across very distant coactive areas. An alternative scenario is that other cells might pace collective rhythms within the anterior pituitary gland. Folliculostellate cells would be good candidates since they form a cell network, which extends throughout the anterior pituitary gland (49). These cells are coupled by gap junctions (24, 49, 50) and communicate with endocrine cells via paracrine interactions (20) and gap junctions (23, 24). Extensive analysis of these two putative mechanisms would be of particular interest, insofar as long distance synchrony may be an important determinant for shaping the patterns of hormone release in the systemic circulation.
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ACKNOWLEDGEMENTS |
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We are indebted to Drs. D. Debanne and O. Manzoni for critical reading, and M. Passama, A. Carrette, and R. Jaoul for their excellent technical assistance. We thank the National Hormone and Pituitary Program and the National Institutes of Health, NIDDK, for the reagents.
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FOOTNOTES |
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* This work was supported by grants from Institut National de la Santé et de la Recherche Médicale (U469), Région Languedoc-Roussillon, Association pour la Recherche sur le Cancer, Fondation pour la Recherche Médicale, and Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche (ACC-SV11).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 31-4-6714-2925;
Fax: 33-4-6754-2432; E-mail: guerinea{at}u469.montp.inserm.fr.
1 The abbreviations used are: LDCV, large dense core vesicles; [Ca2+]i, cytosolic free Ca2+ concentration; GH, growth hormone; fluo-3/AM, fluo-3 acetoxymethyl ester; LY, Lucifer yellow; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PIPES, piperazine-N,N'-bis-[2-ethanesulfonic acid]; TBS, Tris-buffered saline.
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REFERENCES |
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