19 results on '"Mollard, P."'
Search Results
2. A Secreted Fluorescent Reporter Targeted to Pituitary Growth Hormone Cells in Transgenic Mice
- Author
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Magoulas, C, McGuinness, L, Balthasar, N, Carmignac, D F, Sesay, A K, Mathers, K E, Christian, H, Candeil, L, Bonnefont, X, Mollard, P, and Robinson, I C. A. F
- Published
- 2000
3. TSH Pulses Finely Tune Thyroid Hormone Release and TSH Receptor Transduction
- Author
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Guillou, Anne, Kemkem, Yasmine, Lafont, Chrystel, Fontanaud, Pierre, Calebiro, Davide, Campos, Pauline, Bonnefont, Xavier, Fiordelisio-Coll, Tatiana, Wang, Ying, Brûlé, Emilie, Bernard, Daniel J, Le Tissier, Paul, Steyn, Frederik, and Mollard, Patrice
- Abstract
Detection of circulating TSH is a first-line test of thyroid dysfunction, a major health problem (affecting about 5% of the population) that, if untreated, can lead to a significant deterioration of quality of life and adverse effects on multiple organ systems. Human TSH levels display both pulsatile and (nonpulsatile) basal TSH secretion patterns; however, the importance of these in regulating thyroid function and their decoding by the thyroid is unknown. Here, we developed a novel ultra-sensitive ELISA that allows precise detection of TSH secretion patterns with minute resolution in mouse models of health and disease. We characterized the patterns of ultradian TSH pulses in healthy, freely behaving mice over the day-night cycle. Challenge of the thyroid axis with primary hypothyroidism because of iodine deficiency, a major cause of thyroid dysfunction worldwide, results in alterations of TSH pulsatility. Induction in mouse models of sequential TSH pulses that mimic ultradian TSH profiles in periods of minutes were more efficient than sustained rises in basal TSH levels at increasing both thyroid follicle cAMP levels, as monitored with a genetically encoded cAMP sensor, and circulating thyroid hormone. Hence, this mouse TSH assay provides a powerful tool to decipher how ultradian TSH pulses encode thyroid outcomes and to uncover hidden parameters in the TSH-thyroid hormone set-point in health and disease.
- Published
- 2024
- Full Text
- View/download PDF
4. Imaging and Manipulating Pituitary Function in the Awake Mouse.
- Author
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Hoa, Ombeline, Lafont, Chrystel, Fontanaud, Pierre, Guillou, Anne, Kemkem, Yasmine, Kineman, Rhonda D, Luque, Raul M, Fiordelisio Coll, Tatiana, Le Tissier, Paul, and Mollard, Patrice
- Abstract
Extensive efforts have been made to explore how the activities of multiple brain cells combine to alter physiology through imaging and cell-specific manipulation in different animal models. However, the temporal regulation of peripheral organs by the neuroendocrine factors released by the brain is poorly understood. We have established a suite of adaptable methodologies to interrogate in vivo the relationship of hypothalamic regulation with the secretory output of the pituitary gland, which has complex functional networks of multiple cell types intermingled with the vasculature. These allow imaging and optogenetic manipulation of cell activities in the pituitary gland in awake mouse models, in which both neuronal regulatory activity and hormonal output are preserved. These methodologies are now readily applicable for longitudinal studies of short-lived events (e.g., calcium signals controlling hormone exocytosis) and slowly evolving processes such as tissue remodeling in health and disease over a period of days to weeks.
- Published
- 2019
- Full Text
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5. The Processes of Anterior Pituitary Hormone Pulse Generation.
- Author
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Le Tissier, Paul, Fiordelisio Coll, Tatiana, and Mollard, Patrice
- Abstract
More than 60 years ago, Geoffrey Harris described his "neurohumoral theory," in which the regulation of pituitary hormone secretion was a "simple" hierarchal relationship, with the hypothalamus as the controller. In models based on this theory, the electrical activity of hypothalamic neurons determines the release of hypophysiotropic hormones into the portal circulation, and the pituitary simply responds with secretion of a pulse of hormone into the bloodstream. The development of methodologies allowing the monitoring of the activities of members of the hypothalamic-vascular-pituitary unit is increasingly allowing dissection of the mechanisms generating hypothalamic and pituitary pulses. These have revealed that whereas hypothalamic input is required, its role as a driver of pulsatile pituitary hormone secretion varies between pituitary axes. The organization of pituitary cells has a key role in the modification of their response to hypophysiotropic factors that can lead to a memory of previous demand and enhanced function. Feedback can lead to oscillatory hormone output that is independent of pulses of hypophysiotropic factors and instead, results from the temporal relationship between pituitary output and target organ response. Thus, the mechanisms underlying the generation of pulses cannot be generalized, and the circularity of feedforward and feedback interactions must be considered to understand both normal physiological function and pathology. We describe some examples of the clinical implications of recognizing the importance of the pituitary and target organs in pulse generation and suggest avenues for future research in both the short and long term.
- Published
- 2018
- Full Text
- View/download PDF
6. A New Perspective on Regulation of Pituitary Plasticity: The Network of SOX2-Positive Cells May Coordinate Responses to Challenge
- Author
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Le Tissier, Paul R, Murray, Joanne F, and Mollard, Patrice
- Abstract
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.
- Published
- 2022
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7. Tamoxifen Reduces Calcium Currents in a Clonal Pituitary Cell Line*
- Author
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SARTOR, P., primary, VACHER, P., additional, MOLLARD, P., additional, and DUFY, B., additional
- Published
- 1988
- Full Text
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8. Bisphenol A Effects on Gonadotroph Function: Disruption of Pituitary Cell-Cell Communication?
- Author
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Le Tissier, Paul R. and Mollard, Patrice
- Published
- 2016
- Full Text
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9. TSH Pulses Finely Tune Thyroid Hormone Release and TSH Receptor Transduction.
- Author
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Guillou A, Kemkem Y, Lafont C, Fontanaud P, Calebiro D, Campos P, Bonnefont X, Fiordelisio-Coll T, Wang Y, Brûlé E, Bernard DJ, Le Tissier P, Steyn F, and Mollard P
- Subjects
- Mice, Humans, Animals, Receptors, Thyrotropin, Thyrotropin, Thyroxine, Quality of Life, Thyroid Hormones pharmacology, Hypothyroidism, Thyroid Diseases
- Abstract
Detection of circulating TSH is a first-line test of thyroid dysfunction, a major health problem (affecting about 5% of the population) that, if untreated, can lead to a significant deterioration of quality of life and adverse effects on multiple organ systems. Human TSH levels display both pulsatile and (nonpulsatile) basal TSH secretion patterns; however, the importance of these in regulating thyroid function and their decoding by the thyroid is unknown. Here, we developed a novel ultra-sensitive ELISA that allows precise detection of TSH secretion patterns with minute resolution in mouse models of health and disease. We characterized the patterns of ultradian TSH pulses in healthy, freely behaving mice over the day-night cycle. Challenge of the thyroid axis with primary hypothyroidism because of iodine deficiency, a major cause of thyroid dysfunction worldwide, results in alterations of TSH pulsatility. Induction in mouse models of sequential TSH pulses that mimic ultradian TSH profiles in periods of minutes were more efficient than sustained rises in basal TSH levels at increasing both thyroid follicle cAMP levels, as monitored with a genetically encoded cAMP sensor, and circulating thyroid hormone. Hence, this mouse TSH assay provides a powerful tool to decipher how ultradian TSH pulses encode thyroid outcomes and to uncover hidden parameters in the TSH-thyroid hormone set-point in health and disease., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)
- Published
- 2023
- Full Text
- View/download PDF
10. Assessment of lactotroph axis functionality in mice: longitudinal monitoring of PRL secretion by ultrasensitive-ELISA.
- Author
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Guillou A, Romanò N, Steyn F, Abitbol K, Le Tissier P, Bonnefont X, Chen C, Mollard P, and Martin AO
- Subjects
- Animals, Dopamine D2 Receptor Antagonists pharmacology, Dopaminergic Neurons metabolism, Enzyme-Linked Immunosorbent Assay, Female, Hypothalamus cytology, Lactotrophs drug effects, Longitudinal Studies, Male, Mice, Mice, Inbred C57BL, Circadian Rhythm, Dopamine metabolism, Lactation, Lactotrophs metabolism, Prolactin metabolism
- Abstract
The pattern of prolactin (PRL) secretion depends on the physiological state. Due to insufficient detection sensitivity of existing assays, the precise description of these patterns in mice is lacking. We described an ultrasensitive ELISA assay that can detect mouse PRL in small fractions of whole blood, allowing longitudinal studies of PRL secretion profiles in freely moving mice. Over a 24-hour period, males displayed no oscillation in PRL levels, whereas virgin and lactating females showed large pulses. Peaks of PRL secretion reached 30-40 ng/mL in lactating female mice and rarely exceeded 10 ng/mL in virgin females. These pulses of PRL in lactating females were associated with suckling. The return of pups after an experimental 12-hour weaning induced a pulse of PRL release, reaching 100 ng/mL. This approach also enabled us to assess the inhibitory tone from hypothalamic dopamine neurons on PRL secretion. We used a dopamine D2 receptor antagonist to relieve pituitary lactotrophs from the tuberoinfundibular dopaminergic inhibitory tone and demonstrate a D2-induced PRL rise that can be used to evaluate both the secretory capacity of lactotrophs and the magnitude of the inhibitory tone on pituitary PRL release. We demonstrate that, although lactotroph function is altered to enhance chronic PRL output, their secretory response to acute stimulus is not modified during lactation and that chronic hyperprolactinemia is linked to a lower inhibitory tone. The combination of a sensitive PRL ELISA and administration of D2 receptor antagonist provide a unique opportunity to investigate the function and plasticity of the lactotroph axis in freely moving mice.
- Published
- 2015
- Full Text
- View/download PDF
11. Sustained alterations of hypothalamic tanycytes during posttraumatic hypopituitarism in male mice.
- Author
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Osterstock G, El Yandouzi T, Romanò N, Carmignac D, Langlet F, Coutry N, Guillou A, Schaeffer M, Chauvet N, Vanacker C, Galibert E, Dehouck B, Robinson IC, Prévot V, Mollard P, Plesnila N, and Méry PF
- Subjects
- Animals, Arcuate Nucleus of Hypothalamus immunology, Arcuate Nucleus of Hypothalamus metabolism, Arcuate Nucleus of Hypothalamus pathology, Biomarkers metabolism, Ependymoglial Cells immunology, Ependymoglial Cells metabolism, Gene Expression Regulation, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Growth Hormone-Releasing Hormone genetics, Growth Hormone-Releasing Hormone metabolism, Hypopituitarism immunology, Hypopituitarism metabolism, Hypopituitarism pathology, Hypothalamus immunology, Hypothalamus metabolism, Immunoglobulin G metabolism, Male, Median Eminence immunology, Median Eminence metabolism, Median Eminence pathology, Mice, Mice, Transgenic, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, Neurons immunology, Neurons metabolism, Permeability, Recombinant Fusion Proteins metabolism, Third Ventricle immunology, Third Ventricle metabolism, Third Ventricle pathology, Tight Junctions immunology, Tight Junctions metabolism, Brain Injuries physiopathology, Disease Models, Animal, Ependymoglial Cells pathology, Hypopituitarism etiology, Hypothalamus pathology, Neurons pathology, Tight Junctions pathology
- Abstract
Traumatic brain injury is a leading cause of hypopituitarism, which compromises patients' recovery, quality of life, and life span. To date, there are no means other than standardized animal studies to provide insights into the mechanisms of posttraumatic hypopituitarism. We have found that GH levels were impaired after inducing a controlled cortical impact (CCI) in mice. Furthermore, GHRH stimulation enhanced GH to lower level in injured than in control or sham mice. Because many characteristics were unchanged in the pituitary glands of CCI mice, we looked for changes at the hypothalamic level. Hypertrophied astrocytes were seen both within the arcuate nucleus and the median eminence, two pivotal structures of the GH axis, spatially remote to the injury site. In the arcuate nucleus, GHRH neurons were unaltered. In the median eminence, injured mice exhibited unexpected alterations. First, the distributions of claudin-1 and zonula occludens-1 between tanycytes were disorganized, suggesting tight junction disruptions. Second, endogenous IgG was increased in the vicinity of the third ventricle, suggesting abnormal barrier properties after CCI. Third, intracerebroventricular injection of a fluorescent-dextran derivative highly stained the hypothalamic parenchyma only after CCI, demonstrating an increased permeability of the third ventricle edges. This alteration of the third ventricle might jeopardize the communication between the hypothalamus and the pituitary gland. In conclusion, the phenotype of CCI mice had similarities to the posttraumatic hypopituitarism seen in humans with intact pituitary gland and pituitary stalk. It is the first report of a pathological status in which tanycyte dysfunctions appear as a major acquired syndrome.
- Published
- 2014
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12. Influence of estrogens on GH-cell network dynamics in females: a live in situ imaging approach.
- Author
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Schaeffer M, Hodson DJ, Meunier AC, Lafont C, Birkenstock J, Carmignac D, Murray JF, Gavois E, Robinson IC, Le Tissier P, and Mollard P
- Subjects
- Animals, Female, Green Fluorescent Proteins, Male, Mice, Mice, Transgenic, Sex Factors, Cell Movement, Estrogens pharmacology, Growth Hormone analysis, Pituitary Gland cytology, Time-Lapse Imaging
- Abstract
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.
- Published
- 2011
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13. Modulation of the tyrosine kinase receptor Ret/glial cell-derived neurotrophic factor (GDNF) signaling: a new player in reproduction induced anterior pituitary plasticity?
- Author
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Guillou A, Romanò N, Bonnefont X, Le Tissier P, Mollard P, and Martin AO
- Subjects
- Animals, Cell Proliferation, Cells, Cultured, Female, Glial Cell Line-Derived Neurotrophic Factor genetics, Immunohistochemistry, Lactation genetics, Lactation metabolism, Lactotrophs metabolism, Mice, Microscopy, Electron, Pituitary Gland cytology, Pituitary Gland ultrastructure, Polymerase Chain Reaction, Pregnancy genetics, Pregnancy metabolism, Proto-Oncogene Proteins c-ret genetics, Reproduction genetics, Signal Transduction genetics, Signal Transduction physiology, Somatotrophs metabolism, Weaning, Glial Cell Line-Derived Neurotrophic Factor metabolism, Pituitary Gland metabolism, Proto-Oncogene Proteins c-ret metabolism, Reproduction physiology
- Abstract
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.
- Published
- 2011
- Full Text
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14. Specific involvement of gonadal hormones in the functional maturation of growth hormone releasing hormone (GHRH) neurons.
- Author
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Gouty-Colomer LA, Méry PF, Storme E, Gavois E, Robinson IC, Guérineau NC, Mollard P, and Desarménien MG
- Subjects
- Action Potentials, Animals, Brain growth & development, Ethinyl Estradiol pharmacology, Female, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Male, Mice, Neurons cytology, Orchiectomy, Ovariectomy, Sex Characteristics, Sexual Maturation physiology, Synaptic Potentials physiology, Testosterone Propionate pharmacology, gamma-Aminobutyric Acid metabolism, Growth Hormone-Releasing Hormone metabolism, Neurons metabolism
- Abstract
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.
- Published
- 2010
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15. Different degrees of somatotroph ablation compromise pituitary growth hormone cell network structure and other pituitary endocrine cell types.
- Author
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Waite E, Lafont C, Carmignac D, Chauvet N, Coutry N, Christian H, Robinson I, Mollard P, and Le Tissier P
- Subjects
- Animals, Cell Communication physiology, Cell Count, Dwarfism, Pituitary etiology, Dwarfism, Pituitary genetics, Endocrine Cells metabolism, Female, Gene Dosage physiology, Genes, Transgenic, Suicide physiology, Human Growth Hormone genetics, Male, Mice, Mice, Inbred C57BL, Mice, Inbred CBA, Mice, Transgenic, Organ Size genetics, Organ Specificity genetics, Pituitary Gland metabolism, Somatotrophs metabolism, Viral Matrix Proteins genetics, Cell Communication genetics, Endocrine Cells cytology, Pituitary Gland cytology, Somatotrophs cytology
- Abstract
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.
- Published
- 2010
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16. Rhythmic bursts of calcium transients in acute anterior pituitary slices.
- Author
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Bonnefont X, Fiekers J, Creff A, and Mollard P
- Subjects
- Animals, Calcium metabolism, Electrophysiology, Female, Fluorescent Antibody Technique, Indirect, Fluorescent Dyes, Fura-2, In Vitro Techniques, Male, Microscopy, Confocal, Pituitary Gland, Anterior cytology, Pituitary Gland, Anterior drug effects, Rats, Rats, Wistar, Thyrotropin-Releasing Hormone pharmacology, Calcium Signaling physiology, Pituitary Gland, Anterior physiology
- Abstract
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.
- Published
- 2000
- Full Text
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17. Spontaneous and corticotropin-releasing factor-induced cytosolic calcium transients in corticotrophs.
- Author
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Guérineau N, Corcuff JB, Tabarin A, and Mollard P
- Subjects
- Action Potentials drug effects, Adenoma, Animals, Calcium Channel Blockers pharmacology, Calcium Channels physiology, Cytosol drug effects, Egtazic Acid pharmacology, Female, Humans, Isradipine, Membrane Potentials drug effects, Oxadiazoles pharmacology, Pituitary Gland drug effects, Pituitary Gland ultrastructure, Pituitary Neoplasms, Rats, Rats, Inbred Strains, Spectrometry, Fluorescence, Tetrodotoxin pharmacology, Tumor Cells, Cultured, Adrenocorticotropic Hormone metabolism, Calcium metabolism, Corticotropin-Releasing Hormone pharmacology, Cytosol metabolism, Pituitary Gland metabolism
- Abstract
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)
- Published
- 1991
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18. Electrical properties of cultured human adrenocorticotropin-secreting adenoma cells: effects of high K+, corticotropin-releasing factor, and angiotensin II.
- Author
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Mollard P, Vacher P, Guerin J, Rogawski MA, and Dufy B
- Subjects
- Action Potentials drug effects, Calcium pharmacology, Electric Conductivity, Electrophysiology, Humans, Lithium pharmacology, Membrane Potentials, Adenoma physiopathology, Adrenocorticotropic Hormone metabolism, Angiotensin II pharmacology, Corticotropin-Releasing Hormone pharmacology, Pituitary Neoplasms physiopathology, Potassium pharmacology
- Abstract
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.
- Published
- 1987
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19. Somatostatin blocks Ca2+ action potential activity in prolactin-secreting pituitary tumor cells through coordinate actions on K+ and Ca2+ conductances.
- Author
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Mollard P, Vacher P, Dufy B, and Barker JL
- Subjects
- Action Potentials drug effects, Animals, Calcium pharmacology, Electric Conductivity, Humans, Ion Channels drug effects, Ion Channels physiology, Kinetics, Membrane Potentials, Pertussis Toxin, Rats, Tumor Cells, Cultured, Virulence Factors, Bordetella pharmacology, Adenoma physiopathology, Calcium metabolism, Pituitary Neoplasms physiopathology, Potassium metabolism, Prolactin metabolism, Somatostatin pharmacology
- Abstract
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)
- Published
- 1988
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