Search results
Found 12804 matches for
Autosomal dominant growth hormone deficiency disrupts secretory vesicles in vitro and in vivo in transgenic mice.
Autosomal dominant GH deficiency type II (IGHDII) is often associated with mutations in the human GH gene (GH1) that give rise to products lacking exon-3 ((Deltaexon3)hGH). In the heterozygous state, these act as dominant negative mutations that prevent the release of human pituitary GH (hGH). To determine the mechanisms of these dominant negative effects, we used a combination of transgenic and morphological approaches in both in vitro and in vivo models. Rat GC cell lines were generated expressing either wild-type GH1 (WT-hGH-GC) or a genomic GH1 sequence containing a G->A transition at the donor splice site of IVS3 ((Deltaexon3)hGH-GC). WT-hGH-GC cells grew normally and produced equivalent amounts of human and rGH packaged in dense-cored secretory vesicles (SVs). In contrast, (Deltaexon3)hGH-GC cells showed few SVs but accumulated secretory product in amorphous cytoplasmic aggregates. They produced much less rGH and grew more slowly than WT-hGH-GC cells. When cotransfected with an enhanced green fluorescent protein construct (GH-eGFP), which copackages with GH in SVs, WT-hGH-GC cells showed normal electron microscopy morphology and SV movements, tracked with total internal reflectance fluorescence microscopy. In contrast, coexpression of (Deltaexon3)hGH with GH-eGFP abolished the vesicular targeting of GH-eGFP, which instead accumulated in static aggregates. Transgenic mice expressing (Deltaexon3)hGH in somatotrophs showed an IGHD-II phenotype with mild to severe pituitary hypoplasia and dwarfism, evident at weaning in the most severely affected lines. Hypothalamic GHRH expression was up-regulated and somatostatin expression reduced in (Deltaexon3)hGH transgenic mice, consistent with their profound GHD. Few SVs were detectable in the residual pituitary somatotrophs in (Deltaexon3)hGH transgenic mice, and these cells showed grossly abnormal morphology. A low copy number transgenic line showed a mild effect relatively specific for GH, whereas two severely affected lines with higher transgene copy numbers showed early onset, widespread pituitary damage, macrophage invasion, and multiple hormone deficiencies. These new in vitro and in vivo models shed new light on the cellular mechanisms involved in IGHDII, as well as its phenotypic consequences in vivo.
Neutrophil interaction with inflamed postcapillary venule endothelium alters annexin 1 expression.
Annexin 1 (ANX-A1) exerts antimigratory actions in several models of acute and chronic inflammation. This is related to its ability to mimic the effect of endogenous ANX-A1 that is externalized on neutrophil adhesion to the postcapillary endothelium. In the present study we monitored ANX-A1 expression and localization in intravascular and emigrated neutrophils, using a classical model of rat peritonitis. For this purpose, a pair of antibodies raised against the ANX-A1 N-terminus (ie, able to recognize intact ANX-A1) or the whole protein (ie, able to interact with all ANX-A1 isoforms) was used by immunofluorescence and immunocytochemistry analyses. The majority ( approximately 50%) of ANX-A1 on the plasma membrane of intravascular neutrophils was intact. Extravasation into the subendothelial matrix caused loss of this pool of intact protein (to approximately 6%), concomitant with an increase in total amount of the protein; only approximately 25% of the total protein was now recognized by the antibody raised against the N-terminus (ie, it was intact). In the cytoplasm of these cells, ANX-A1 was predominantly associated with large vacuoles, possibly endosomes. In situ hybridization confirmed de novo synthesis of ANX-A1 in the extravasated cells. In conclusion, biochemical pathways leading to the externalization, proteolysis, and synthesis of ANX-A1 are activated during the process of neutrophil extravasation.
Presynaptic NMDARs in the hippocampus facilitate transmitter release at theta frequency.
A rise in [Ca(2+)](i) provides the trigger for neurotransmitter release at neuronal boutons. We have used confocal microscopy and Ca(2+) sensitive dyes to directly measure the action potential-evoked [Ca(2+)](i) in the boutons of Schaffer collaterals. This reveals that the trial-by-trial amplitude of the evoked Ca(2+) transient is bimodally distributed. We demonstrate that "large" Ca(2+) transients occur when presynaptic NMDA receptors are activated following transmitter release. Presynaptic NMDA receptor activation proves critical in producing facilitation of transmission at theta frequencies. Because large Ca(2+) transients "report" transmitter release, their frequency on a trial-by-trial basis can be used to estimate the probability of release, p(r). We use this novel estimator to show that p(r) increases following the induction of long-term potentiation.
Cellular in vivo imaging reveals coordinated regulation of pituitary microcirculation and GH cell network function.
Growth hormone (GH) exerts its actions via coordinated pulsatile secretion from a GH cell network into the bloodstream. Practically nothing is known about how the network receives its inputs in vivo and releases hormones into pituitary capillaries to shape GH pulses. Here we have developed in vivo approaches to measure local blood flow, oxygen partial pressure, and cell activity at single-cell resolution in mouse pituitary glands in situ. When secretagogue (GHRH) distribution was modeled with fluorescent markers injected into either the bloodstream or the nearby intercapillary space, a restricted distribution gradient evolved within the pituitary parenchyma. Injection of GHRH led to stimulation of both GH cell network activities and GH secretion, which was temporally associated with increases in blood flow rates and oxygen supply by capillaries, as well as oxygen consumption. Moreover, we observed a time-limiting step for hormone output at the perivascular level; macromolecules injected into the extracellular parenchyma moved rapidly to the perivascular space, but were then cleared more slowly in a size-dependent manner into capillary blood. Our findings suggest that GH pulse generation is not simply a GH cell network response, but is shaped by a tissue microenvironment context involving a functional association between the GH cell network activity and fluid microcirculation.
Rapid actions of 17beta-oestradiol on a subset of lactotrophs in the rat pituitary.
Increasingly the role of rapid mechanisms of steroid action in physiological regulation are being recognised. We have investigated rapid effects of 17beta-oestradiol (E) on prolactin (PRL) release in vitro. Pituitary segments from male rats were incubated for 5, 10 or 20 min in Earle's balanced salt solution containing 1.2 mM tannic acid (to enable visualisation of exocytosed secretory granules by electron microscopy) either alone (control) or containing 10(-10)-10(-8) M E conjugated to bovine serum albumin (E-BSA). PRL and leuteinising hormone (LH) release from pituitary segments were also determined in response to E and E-BSA by radioimmunoassay. Within 10 min E-BSA and E (10(-12)-10(-6) M) stimulated a significant (P < 0.05) concentration-dependent release of PRL but not LH. After exposure to experimental media for 5 min, only occasional exocytosis from type I lactotrophs (characterised by large polymorphic secretory granules) was observed in either control or E-BSA treated tissue. In contrast, E-BSA (10(-10)-10(-8) M) induced a significant (P < 0.05) increase in the number of exocytotic profiles from type II lactotrophs (characterized by smaller, spherical granules). This effect was not inhibited by removal of extracellular calcium, or by pre-treatment of cells with the RNA synthesis inhibitor actinomycin-D (0.5 microg ml(-1)), the protein synthesis inhibitor cycloheximide (1 microg ml(-1)) or the anti-oestrogen ICI 182,780 (1 microM). FACS analysis demonstrated binding of E-BSA-fluorescein isothiocyanate (FITC) (10(-10)-10(-7) M) to a subpopulation of anterior pituitary cells. The E-BSA-FITC binding sites assumed a patchy distribution across the cell surface. In conclusion, we report for the first time a rapid, non-genomic effect of E on PRL secretion in normal pituitary tissue.
Insulin and IGF-I inhibit GH synthesis and release in vitro and in vivo by separate mechanisms.
IGF-I is considered a primary inhibitor of GH secretion. Insulin may also play an important role in regulating GH levels because insulin, like IGF-I, can suppress GH synthesis and release in primary pituitary cell cultures and insulin is negatively correlated with GH levels in vivo. However, understanding the relative contribution insulin and IGF-I exert on controlling GH secretion has been hampered by the fact that circulating insulin and IGF-I are regulated in parallel and insulin (INSR) and IGF-I (IGFIR) receptors are structurally/functionally related and ubiquitously expressed. To evaluate the separate roles of insulin and IGF-I in directly regulating GH secretion, we used the Cre/loxP system to knock down the INSR and IGFIR in primary mouse pituitary cell cultures and found insulin-mediated suppression of GH is independent of the IGFIR. In addition, pharmacological blockade of intracellular signals in both mouse and baboon cultures revealed insulin requires different pathways from IGF-I to exert a maximal inhibitory effect on GH expression/release. In vivo, somatotrope-specific knockout of INSR (SIRKO) or IGFIR (SIGFRKO) increased GH levels. However, comparison of the pattern of GH release, GH expression, somatotrope morphometry, and pituitary explant sensitivity to acute GHRH challenge in lean SIRKO and SIGFRKO mice strongly suggests the primary role of insulin in vivo is to suppress GH release, whereas IGF-I serves to regulate GH synthesis. Finally, SIRKO and/or SIGFRKO could not prevent high-fat, diet-induced suppression of pituitary GH expression, indicating other factors/tissues are involved in the decline of GH observed with weight gain.
An investigation into pituitary gonadotrophic hormone synthesis, secretion, subunit gene expression and cell structure in normal and mutant male mice.
To investigate brain-pituitary-gonadal inter-relationships, we have compared the effects of mutations that perturb the hypothalamic-pituitary-gonadal axis in male mice. Specifically, serum and pituitary gonadotrophin concentrations, gonadotrophin gene expression, and gonadotroph structure and number were measured. Follicle-stimulating hormone (FSH)β knockout (FSHβKO), FSH receptor knockout (FSHRKO), luteinising hormone (LH) receptor knockout (LuRKO), hypogonadal (hpg), testicular feminised (tfm) and gonadectomised mice were compared with control wild-type mice or heterozygotes. Serum levels of LH were similar in FSHβKO, FSHRKO and heterozygote males despite decreased androgen production in KO males. As expected, there was no detectable FSH in the serum or pituitary and an absence of expression of the FSHβ subunit gene in FSHβKO mice. However, there was a significant increase in expression of the common α and LHβ subunit genes in FSHRKO males. The morphology of FSHβKO and FSHRKO gonadotrophs was not significantly different from controls, except that the subpopulation of granules consisting of an electron-dense core and electron-lucent 'halo' was not observed in FSHβKO gonadotrophs and the granules were smaller in diameter. In the gonadotrophin-releasing hormone deficient hpg mouse, gonadotrophin mRNA and hormone levels were significantly lower compared to control mice and gonadotrophs were correspondingly smaller, with less abundant endoplasmic reticulum and reduced secretory granules. In LuRKO, tfm and gonadectomised mice, hyperstimulation of LHβ and FSHβ mRNA and serum protein concentrations was reflected by subcellular changes in gonadotroph morphology, including more dilated rough endoplasmic reticulum and more secretory granules distributed adjacent to the plasma membrane. In summary, major differences in pituitary content and serum concentrations of the gonadotrophins LH and FSH have been found between normal and mutant male mice. These changes are associated with changes in transcriptional activity of the gonadotrophin subunit genes and are reflected by changes in the cellular structure and secretory granule architecture within the gonadotroph cells.
Existence of long-lasting experience-dependent plasticity in endocrine cell networks
Experience-dependent plasticity of cell and tissue function is critical for survival by allowing organisms to dynamically adjust physiological processes in response to changing or harsh environmental conditions. Despite the conferred evolutionary advantage, it remains unknown whether emergent experience-dependent properties are present in cell populations organized as networks within endocrine tissues involved in regulating body-wide homeostasis. Here we show, using lactation to repeatedly activate a specific endocrine cell network in situ in the mammalian pituitary, that templates of prior demand are permanently stored through stimulus-evoked alterations to the extent and strength of cell-cell connectivity. Strikingly, following repeat stimulation, evolved population behaviour leads to improved tissue output. As such, long-lasting experience-dependent plasticity is an important feature of endocrine cell networks and underlies functional adaptation of hormone release. © 2012 Macmillan Publishers Limited. All rights reserved.
Existence of long-lasting experience-dependent plasticity in endocrine cell networks.
Experience-dependent plasticity of cell and tissue function is critical for survival by allowing organisms to dynamically adjust physiological processes in response to changing or harsh environmental conditions. Despite the conferred evolutionary advantage, it remains unknown whether emergent experience-dependent properties are present in cell populations organized as networks within endocrine tissues involved in regulating body-wide homeostasis. Here we show, using lactation to repeatedly activate a specific endocrine cell network in situ in the mammalian pituitary, that templates of prior demand are permanently stored through stimulus-evoked alterations to the extent and strength of cell-cell connectivity. Strikingly, following repeat stimulation, evolved population behaviour leads to improved tissue output. As such, long-lasting experience-dependent plasticity is an important feature of endocrine cell networks and underlies functional adaptation of hormone release.
3D functional scaffolds for cardiovascular tissue engineering
© 2018 Elsevier Ltd. All rights reserved. The heart is responsible for providing blood flow to enable perfusion of all tissues and organs with oxygen and nutrients, and is, due to its high oxygen demand and continuous action, particularly susceptible to hypoxia and perfusion deficits. Since the heart is unable to cope with tissue damage and no curative treatment is available, researchers are actively investigating novel therapeutic strategies for cardiac regeneration. Cardiac cell therapy and tissue engineering have attracted considerable attention and clinical trials are ongoing to assess clinical efficacy and accommodate translation to the clinic. In addition, cardiac toxicity as a result of drug therapy is one of the main causes of compound attrition during drug development and post-marketing withdrawal. Moreover, although some cardiotoxicity assays have become standard in drug development, these assays require further improvement as they are prone to false-positive and false-negative outcomes. Stem cell and tissue engineering technologies have been proposed as potential tools for addressing these limitations. In this chapter, we will discuss current developments within these fields and make recommendations for future work.
Ambiguity in the Presentation of Decellularized Tissue Composition: The Need for Standardized Approaches.
Decellularization offers great potential to the field of tissue engineering, as this method gives rise to scaffold material with the native organ architecture by removing all cellular material and leaving much of the extracellular matrix (ECM) intact. However, many parameters may affect decellularization efficacy and ECM retention and, therefore, decellularization protocols need to be optimized for specific needs. This requires robust methods for comparison of decellularized tissue composition. Various representation methods are used in literature to express tissue composition (DNA, glycosaminoglycans, collagen, other ECM proteins, and growth factors). Here, we present and compare the various methods used and demonstrate that normalization to either dry or wet decellularized weight might be misleading and may overestimate true component retention. Moreover, the magnitude of the confounding effect is likely to be decellularization treatment dependent. As a result, we propose alternative comparison strategies: normalization to whole organ or to a unit of whole initial organ weight. We believe proper assessment of decellularized tissue composition is paramount for the successful comparison of different decellularization protocols and clinical translation.
Stem Cell Therapy for the Heart: Blind Alley or Magic Bullet?
When stressed by ageing or disease, the adult human heart is unable to regenerate, leading to scarring and hypertrophy and eventually heart failure. As a result, stem cell therapy has been proposed as an ultimate therapeutic strategy, as stem cells could limit adverse remodelling and give rise to new cardiomyocytes and vasculature. Unfortunately, the results from clinical trials to date have been largely disappointing. In this review, we discuss the current status of the field and describe various limitations and how future work may attempt to resolve these to make way to successful clinical translation.
Temporal accumulation and localization of isoflurane in the C57BL/6 mouse and assessment of its potential contamination in 19 F MRI with perfluoro-crown-ether-labeled cardiac progenitor cells at 9.4 Tesla.
PURPOSE: To assess the uptake, accumulation, temporal stability, and spatial localization of isoflurane (ISO) in the C57BL/6 mouse, and to identify its potential interference with the detection of labeled cardiac progenitor cells using 19 F MRI/MR spectroscopy (MRS). MATERIALS AND METHODS: Objectives are demonstrated using (a) in vitro ISO tests, (b) in vivo temporal accumulation/spatial localization C57BL/6 studies (n = 3), and (c) through injections of perfluoro-crown-ether (PFCE) labeled cardiac progenitor cells into femoral muscle areas of the murine hindlimb post-mortem (n = 1) using 1 H/19 F MRI/MRS at 9.4 Tesla. Data were acquired using double-gated spoiled gradient echo images and pulse-acquire spectra. For the in vivo study, the temporal stability of ISO resonances was quantified using coefficient of variability (CV) (5 min) estimates. RESULTS: Two ISO resonances were observed in vivo that correspond to the -CF3 and -OCHF2 moieties. CV values ranged between 3.2 and 6.4% (-CF3 ) and 6.4 and 11.2% (-OCHF2 ). Reductions of the ISO dose (2.0 to 1.7%) at 80 min postinduction had insignificant effects on ISO signals (P = 0.23; P = 0.71). PFCE-labeled cells exhibited a resonance at -16.25 ppm in vitro that did not overlap with the ISO resonances, a finding that is confirmed with MRS post-mortem using injected, labeled cells. Based on 19 F MRI, similar in vivo/post-mortem ISO compartmentalization was also confirmed in peripheral and thoracic skeletal muscles. CONCLUSION: Significant ISO accumulation was observed by 19 F MRS in vivo with temporally stable signals over 90 min postinduction. ISO effects on PFCE labels are anticipated to be minimal but may be more prominent for perfluoropolyether or perfluorooctyl bromide labels. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 1 J. MAGN. RESON. IMAGING 2017;45:1659-1667.