Variation of cell and matrix morphologies in articular cartilage among locations in the adult human knee

OBJECTIVE: Understanding of articular cartilage physiology, remodelling mechanisms, and evaluation of tissue engineering repair methods requires reference information regarding normal structural organization. Our goals were to examine the variation of cartilage cell and matrix morphology in different topographical areas of the adult human knee joint. METHODS: Osteochondral explants were acquired from seven distinct anatomical locations of the knee joints of deceased persons aged 20-40 years and prepared for analysis of cell, matrix and tissue morphology using confocal microscopy and unbiased stereological methods. Differences between locations were identified by statistical analysis. RESULTS: Medial femoral condyle cartilage had relatively high cell surface area per unit tissue volume in the superficial zone. In the transitional zone, meniscus-covered lateral tibia cartilage showed elevated chondrocyte densities compared to the rest of the knee while lateral femoral condyle cartilage exhibited particularly large chondrocytes. Statistical analyses indicated highly uniform morphology throughout the radial zone (lower 80% of cartilage thickness) in the knee, and strong similarities in cell and matrix morphologies among cartilage from the femoral condyles and also in the mediocentral tibial plateau. Throughout the adult human knee, the mean matrix volume per chondron was remarkably constant at approximately 224,000 microm(3), corresponding to approximately 4.6 x 10(6) chondrons per cm(3). CONCLUSIONS: The uniformity of matrix volume per chondron throughout the adult human knee suggests that cell-scale biophysical and metabolic constraints may place limitations on cartilage thickness, mechanical properties, and remodelling mechanisms. Data may also aid the evaluation of cartilage tissue engineering treatments in a site-specific manner. Results indicate that joint locations which perform similar biomechanical functions have similar cell and matrix morphologies; findings may therefore also provide clues to understanding conditions under which focal lesions leading to osteoarthritis may occur.

Constitutive interaction of the P2Y2 receptor with the hematopoietic cell-specific G protein G(alpha16) and evidence for receptor oligomers

Hematopoietic cells uniquely express G(alpha16), a G protein alpha-subunit of the G(q)-type. G(alpha16) is obligatory for P2Y2 receptor-dependent Ca2+-mobilization in human erythroleukemia cells and induces hematopoietic cell differentiation. We tested whether P2Y2 receptors physically interact with G(alpha16). Receptor and G protein were fused to cyan (CFP) and yellow (YFP) variants of the green fluorescent protein (GFP), respectively. When expressed in K562 leukemia cells, the fusion proteins were capable of triggering a Ca2+-signal upon receptor stimulation, demonstrating their functional integrity. In fluorescence resonance energy transfer (FRET) measurements using confocal microscopy, a strong FRET signal from the plasma membrane region of fixed, resting cells was detected when the receptor was co-expressed with the G protein as the FRET acceptor, as well as when the CFP-tagged receptor was co-expressed with receptor fused to YFP. We conclude that, under resting conditions, G(alpha16) and P2Y2 receptors form constitutive complexes, and that the P2Y2 receptor is present as an oligomer.

Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy

BACKGROUND CONTEXT Proteolytic enzyme digestion of the intervertebral disc (IVD) offers a method to simulate a condition of disc degeneration for the study of cell-scaffold constructs in the degenerated disc. PURPOSE To characterize an in vitro disc degeneration model (DDM) of different severities of glycosaminoglycans (GAG) and water loss by using papain, and to determine the initial response of the human mesenchymal stem cells (MSCs) introduced into this DDM. STUDY DESIGN Disc degeneration model of a bovine disc explant with an end plate was induced by the injection of papain at various concentrations. Labeled MSCs were later introduced in this model. METHODS Phosphate-buffered saline (PBS control) or papain in various concentrations (3, 15, 30, 60, and 150 U/mL) were injected into the bovine caudal IVD explants. Ten days after the injection, GAG content of the discs was evaluated by dimethylmethylene blue assay and cell viability was determined by live/dead staining together with confocal microscopy. Overall matrix composition was evaluated by histology, and water content was visualized by magnetic resonance imaging. Compressive and torsional stiffness of the DDM were also recorded. In the second part, MSCs were labeled with a fluorescence cell membrane tracker and injected into the nucleus of the DDM or a PBS control. Mesenchymal stem cell viability and distribution were evaluated by confocal microscopy. RESULTS A large drop of GAG and water content of the bovine disc were obtained by injecting >30 U/mL papain. Magnetic resonance imaging showed Grade II, III, and IV disc degeneration by injecting 30, 60, and 150 U/mL papain. A cavity in the center of the disc could facilitate later injection of the nucleus pulposus tissue engineering construct while retaining an intact annulus fibrosus. The remaining disc cell viability was not affected. Mesenchymal stem cells injected into the protease-treated DDM disc showed significantly higher cell viability than when injected into the PBS-injected control disc. CONCLUSIONS By varying the concentration of papain for injection, an increasing amount of GAG and water loss could be induced to simulate the different severities of disc degeneration. MSC suspension introduced into the disc has a very low short-term survival. However, it should be clear that this bovine IVD DDM does not reflect a clinical situation but offers exciting possibilities to test novel tissue engineering protocols.

Size-Dependent Uptake of Particles by Pulmonary Antigen-Presenting Cell Populations and Trafficking to Regional Lymph Nodes

The respiratory tract is an attractive target organ for novel diagnostic and therapeutic applications with nano-sized carriers, but their immune effects and interactions with key resident antigen-presenting cells (APCs) such as dendritic cells (DCs) and alveolar macrophages (AMs) in different anatomical compartments remain poorly understood. Polystyrene particles ranging from 20 nm to 1,000 nm were instilled intranasally in BALB/c mice, and their interactions with APC populations in airways, lung parenchyma, and lung-draining lymph nodes (LDLNs) were examined after 2 and 24 hours by flow cytometry and confocal microscopy. In the main conducting airways and lung parenchyma, DC subpopulations preferentially captured 20-nm particles, compared with 1,000-nm particles that were transported to the LDLNs by migratory CD11blow DCs and that were observed in close proximity to CD3+ T cells. Generally, the uptake of particles increased the expression of CD40 and CD86 in all DC populations, independent of particle size, whereas 20-nm particles induced enhanced antigen presentation to CD4+ T cells in LDLNs in vivo. Despite measurable uptake by DCs, the majority of particles were taken up by AMs, irrespective of size. Confocal microscopy and FACS analysis showed few particles in the main conducting airways, but a homogeneous distribution of all particle sizes was evident in the lung parenchyma, mostly confined to AMs. Particulate size as a key parameter determining uptake and trafficking therefore determines the fate of inhaled particulates, and this may have important consequences in the development of novel carriers for pulmonary diagnostic or therapeutic applications.

Effect of zinc binding residues in growth hormone (GH) and altered intracellular zinc content on regulated GH secretion.

Endocrine cells store hormones in concentrated forms (aggregates) in dense-core secretory granules that are released upon appropriate stimulation. Zn(2+) binding to GH through amino acid residues His18, His21, and Glu174 are essential for GH dimerization and might mediate its aggregation and storage in secretory granules. To investigate whether GH-1 gene mutations at these positions interfere with this process, GH secretion and intracellular production were analyzed in GC cells (rat pituitary cell line) transiently expressing wt-GH and/or GH Zn mutant (GH-H18A-H21A-E174A) in forskolin-stimulated vs nonstimulated conditions. Reduced secretion of the mutant variant (alone or coexpressed with wt-GH) compared with wt-GH after forskolin stimulation was observed, whereas an increased intracellular accumulation of GH Zn mutant vs wt-GH correlates with its altered extracellular secretion. Depleting Zn(2+) from culture medium using N,N,N',N'-tetrakis(2-pyridylemethyl)ethylenediamine, a high-affinity Zn(2+) chelator, led to a significant reduction of the stimulated wt-GH secretion. Furthermore, externally added Zn(2+) to culture medium increased intracellular free Zn(2+) levels and recovered wt-GH secretion, suggesting its direct dependence on free Zn(2+) levels after forskolin stimulation. Confocal microscopy analysis of the intracellular secretory pathway of wt-GH and GH Zn mutant indicated that both variants pass through the regulated secretory pathway in a similar manner. Taken together, our data support the hypothesis that loss of affinity of GH to Zn(2+) as well as altering intracellular free Zn(2+) content may interfere with normal GH dimerization (aggregation) and storage of the mutant variant (alone or with wt-GH), which could possibly explain impaired GH secretion.

Th17 cells and tissue remodeling in atopic and contact dermatitis

BACKGROUND: Eczematous skin lesions of atopic dermatitis (AD) as well as allergic and irritant contact dermatitis (ACD, ICD) are characterized by the same typical clinical signs, although due to different causes. In both AD and ACD, the presence of T helper 17 cells which play an important role in host defense, has been reported. Furthermore, IL-17 is involved in tissue repair and remodeling. This study aimed to investigate IL-17 expression in acute eczematous skin lesions and correlate it with markers of remodeling in AD, ACD, and ICD. METHODS: Skin specimens were taken from positive patch test reactions to aeroallergens, contact allergens, and irritants at days 2, 3, and 4. Inflammatory cells as well as the expression of cytokines and extracellular matrix proteins were evaluated by immunofluorescence staining and confocal microscopy. RESULTS: Allergic contact dermatitis and ICD were characterized by IFN-γ expression, whereas in AD lesions, IL-13 expression and high numbers of eosinophils were the prominent phenotype. Expression of IL-17, but also IL-21 and IL-22, was observed in all eczema subtypes. The number of IL-22+ T cells correlated with the number of eosinophils. Markers of remodeling such as MMP-9, procollagen-3, and tenascin C were observed in all acute eczematous lesions, while a correlation of IL-17+ T cell numbers with tenascin C-expressing cells and MMP-9+ eosinophils was apparent. CONCLUSION: The expression of IL-17 and related cytokines, such as IL-22, was demonstrated in acute eczematous lesions independent of their pathogenesis. Our results suggest a potential role for IL-17 in remodeling of the skin.

Empirical chemosensitivity testing in a spheroid model of ovarian cancer using a microfluidics-based multiplex platform.

The use of biomarkers to infer drug response in patients is being actively pursued, yet significant challenges with this approach, including the complicated interconnection of pathways, have limited its application. Direct empirical testing of tumor sensitivity would arguably provide a more reliable predictive value, although it has garnered little attention largely due to the technical difficulties associated with this approach. We hypothesize that the application of recently developed microtechnologies, coupled to more complex 3-dimensional cell cultures, could provide a model to address some of these issues. As a proof of concept, we developed a microfluidic device where spheroids of the serous epithelial ovarian cancer cell line TOV112D are entrapped and assayed for their chemoresponse to carboplatin and paclitaxel, two therapeutic agents routinely used for the treatment of ovarian cancer. In order to index the chemoresponse, we analyzed the spatiotemporal evolution of the mortality fraction, as judged by vital dyes and confocal microscopy, within spheroids subjected to different drug concentrations and treatment durations inside the microfluidic device. To reflect microenvironment effects, we tested the effect of exogenous extracellular matrix and serum supplementation during spheroid formation on their chemotherapeutic response. Spheroids displayed augmented chemoresistance in comparison to monolayer culturing. This resistance was further increased by the simultaneous presence of both extracellular matrix and high serum concentration during spheroid formation. Following exposure to chemotherapeutics, cell death profiles were not uniform throughout the spheroid. The highest cell death fraction was found at the center of the spheroid and the lowest at the periphery. Collectively, the results demonstrate the validity of the approach, and provide the basis for further investigation of chemotherapeutic responses in ovarian cancer using microfluidics technology. In the future, such microdevices could provide the framework to assay drug sensitivity in a timeframe suitable for clinical decision making.

T-cadherin loss promotes experimental metastasis of squamous cell carcinoma

T-cadherin is gaining recognition as a determinant for the development of incipient invasive squamous cell carcinoma (SCC). However, effects of T-cadherin expression on the metastatic potential of SCC have not been studied. Here, using a murine model of experimental metastasis following tail vein injection of A431 SCC cells we report that loss of T-cadherin increased both the incidence and rate of appearance of lung metastases. T-cadherin-silenced SCC metastases were highly disordered with evidence of single cell dissemination away from main foci whereas SCC metastases overexpressing T-cadherin developed as compact, tightly organised sheets. SCC cell adhesion to vascular endothelial cells (EC) in culture was increased for T-cadherin-silenced SCC and decreased for T-cadherin-overexpressing SCC. Confocal microscopy showed that T-cadherin-silenced SCC adherent on EC display an elongated morphology with long thin extensions and a high degree of intercalation within the EC monolayer, whereas SCC overexpressing T-cadherin formed poorly-spread multicellular aggregates that remain on the outer surface of the EC monolayer. T-cadherin-deficient SCC or human keratinocyte cells exhibited increased transendothelial migration in vitro which could be attenuated in the presence of EGFR inhibitor gefitinib. Our data suggest that loss of T-cadherin can increase metastatic potential and aggressiveness of SCC, possibly due to facilitating arrest and extravasation through the vascular wall and/or more efficient establishment of metastases in the new microenvironment.

Quantitation of contacts among sensory, motor, and serotonergic neurons in the pedal ganglion of aplysia.

Present models of long-term sensitization in Aplysia californica indicate that the enhanced behavioral response is due, at least in part, to outgrowth of sensory neurons mediating defensive withdrawal reflexes. Presumably, this outgrowth strengthens pre-existing connections by formation of new synapses with follower neurons. However, the relationship between the number of sensorimotor contacts and the physiological strength of the connection has never been examined in intact ganglia. As a first step in addressing this issue, we used confocal microscopy to examine sites of contact between sensory and motor neurons in naive animals. Our results revealed relatively few contacts between physiologically connected cells. In addition, the number of contact sites was proportional to the amplitude of the EPSP elicited in the follower motor neuron by direct stimulation of the sensory neuron. This is the first time such a correlation has been observed in the central nervous system. Serotonin is the neurotransmitter most closely examined for its role in modulating synaptic strength at the sensorimotor synapse. However, the structural relationship of serotonergic processes and sensorimotor synapses has never been examined. Surprisingly, serotonergic processes usually made contact with sensory and motor neurons at sites located relatively distant from the sensorimotor synapse. This result implies that heterosynaptic regulation is due to nondirected release of serotonin into the neuropil.

THE EXPRESSION AND CELLULAR LOCALIZATION OF CC-CHEMOKINE RECEPTOR 5 (CCR5) AFTER TRAUMATIC BRAIN INJURY

Traumatic brain injury results from a primary insult and secondary events that together result in tissue injury. This primary injury occurs at the moment of impact and damage can include scalp laceration, skull fraction, cerebral contusions and lacerations as well as intracranial hemorrhage. Following the initial insult, a delayed response occurs and is characterized by hypoxia, ischemia, cerebral edema, and infection. During secondary brain injury, a series of neuroinflammatory events are triggered that can produce additional damage but may also help to protect nervous tissue from invading pathogens and help to repair the damaged tissue. Brain microglia and astrocytes become activated and migrate to the site of injury where these cells secrete immune mediators such as cytokines and chemokines. CC-chemokine receptor 5 (CCR5) is a member of the CC chemokine receptor family of seven transmembrane G protein coupled receptors. CCR5 is expressed in the immune system and is found in monocytes, leukoctyes, memory T cells, and immature dendritic cells. Upon binding to its ligands, CCR5 functions in the chemotaxis of these immune cells to the site of inflammation. In the CNS, CCR5 and its ligands are expressed in multiple cell types. In this study, I investigated whether CCR5 expression is altered in brain after traumatic brain injury. I examined the time course of CCR5 protein expression in cortex and hippocampus using quantitative western analysis of tissues from injured rat brain after mild impact injury. In addition, I also investigated the cellular localization of CCR5 before and after brain injury using confocal microscopy. I have observed that after brain injury CCR5 is upregulated in a time dependent manner in neurons of the parietal cortex and hippocampus. The absence of CCR5 expression in microglia and its delayed expression in neurons after injury suggests a role for CCR5 in neuronal survival after injury.

THE EXPRESSION AND CELLULAR LOCALIZATION OF CC-CHEMOKINE RECEPTOR 5 (CCR5) AFTER TRAUMATIC BRAIN INJURY

Traumatic brain injury results from a primary insult and secondary events that together result in tissue injury. This primary injury occurs at the moment of impact and damage can include scalp laceration, skull fraction, cerebral contusions and lacerations as well as intracranial hemorrhage. Following the initial insult, a delayed response occurs and is characterized by hypoxia, ischemia, cerebral edema, and infection. During secondary brain injury, a series of neuroinflammatory events are triggered that can produce additional damage but may also help to protect nervous tissue from invading pathogens and help to repair the damaged tissue. Brain microglia and astrocytes become activated and migrate to the site of injury where these cells secrete immune mediators such as cytokines and chemokines. CC-chemokine receptor 5 (CCR5) is a member of the CC chemokine receptor family of seven transmembrane G protein coupled receptors. CCR5 is expressed in the immune system and is found in monocytes, leukoctyes, memory T cells, and immature dendritic cells. Upon binding to its ligands, CCR5 functions in the chemotaxis of these immune cells to the site of inflammation. In the CNS, CCR5 and its ligands are expressed in multiple cell types. In this study, I investigated whether CCR5 expression is altered in brain after traumatic brain injury. I examined the time course of CCR5 protein expression in cortex and hippocampus using quantitative western analysis of tissues from injured rat brain after mild impact injury. In addition, I also investigated the cellular localization of CCR5 before and after brain injury using confocal microscopy. I have observed that after brain injury CCR5 is upregulated in a time dependent manner in neurons of the parietal cortex and hippocampus. The absence of CCR5 expression in microglia and its delayed expression in neurons after injury suggests a role for CCR5 in neuronal survival after injury.

DIRECT INPUTS TO OFF Α AND G9 GANGLION CELLS FROM AII AMACRINE CELLS IN RABBIT RETINA

In the mammalian retina, AII amacrine cells are essential in the rod pathway for dark-adapted vision. But they also have a “day job”, to provide inhibitory inputs to certain OFF ganglion cells in photopic conditions. This is known as crossover inhibition. Physiological evidence from several different labs implies that AII amacrine cells provide direct input to certain OFF ganglion cells. However, previous EM analysis of the rabbit retina suggests that the dominant output of the AII amacrine cell in sublamina a goes to OFF cone bipolar cells (Strettoi et al., 1992). Two OFF ganglion cell types in the rabbit retina, OFF α and G9, were identified by a combination of morphological criteria such as dendritic field size, dye coupling, mosaic properties and stratification depth. The AII amacrine cells (AIIs) were labeled with an antibody against calretinin and glycine receptors were marked with an antibody against the α1 subunit. This material was analyzed by triple-label confocal microscopy. We found the lobules of AIIs made close contacts at many points along the dendrites of individual OFF α and G9 ganglion cells. At these potential synaptic sites, we also found punctate labeling for the glycine receptor α1 subunit. The presence of a post-synaptic marker such as the α1 glycine receptor at contact points between AII lobules and OFF ganglion cells supports a direct inhibitory input from AIIs. This pathway provides for crossover inhibition in the rabbit retina whereby light onset provides an inhibitory signal to OFF α and G9 ganglion cells. Thus, these two OFF ganglion cell types receive a mixed excitatory and inhibitory drive in response to light stimulation.

A Cell Biological Determination of Integrator Subunit Localization

Uridine-rich small nuclear (U snRNAs), with the exception of the U6 snRNA, are RNA polymerase II (RNAPII) transcripts. The mechanism of 3’ cleavage of snRNAs has been unknown until recently. This area was greatly advanced when 12 of the Integrator complex subunits (IntS) were purified in 2005 through their interaction with the C-terminal domain (CTD) of the large subunit (RpbI) of RNAPII. Subsequently, our lab performed a genome-wide RNAi screen that identified two more members of the complex that we have termed IntS13 and IntS14. We have determined that IntS9 and 11 mediate the 3’ cleavage of snRNAs, but the exact function of the other subunits remains unknown. However, through the use of a U7 snRNA-GFP reporter and RNAi knockdown of the Integrator subunits in Drosophila S2 cells, we have shown that all subunits are required for the proper processing of snRNAs, albeit to differing degrees. Because snRNA transcription takes place in the nucleus of the cell, it is expected that all of the Integrator subunits would exhibit nuclear localization, but the knowledge of discrete subnuclear localization (i.e. to Cajal bodies) of any of the subunits could provide important clues to the function of that subunit. In this study, we used a cell biological approach to determine the localization of the 14 Integrator subunits. We hypothesized that the majority of the subunits would be nuclear, however, a few would display distinct localization to the Cajal bodies, as this is where snRNA genes are localized and transcribed. The specific aims and results are: 1. To determine the subcellular localization of the 14 Integrator subunits. To accomplish this, mCherry and GFP tagged clones were generated for each of the 14 Drosophila and human Integrator subunits. Confocal microscopy studies revealed that the majority of the subunits were diffuse in the nucleus, however, IntS3 formed discrete subnuclear foci. Surprisingly, two of the subunits, IntS2 and 7 were observed in cytoplasmic foci. 2. To further characterize Integrator subunits with unique subcellular localizations. Colocalization studies with endogenous IntS3 and Cajal body marker, coilin, showed that these two proteins overlap, and from this we concluded that IntS3 localized to Cajal bodies. Additionally, colocalization studies with mCherry-tagged IntS2 and 7 and the P body marker, Dcp1, revealed that these proteins colocalize as well. IntS7, however, is more stable in cytoplasmic foci than Dcp1. It was also shown through RNAi knockdown of Integrator subunits, that the cytoplasmic localization of IntS2 and 7 is dependent on the expression of IntS1 and 11 in S2 cells.

Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation.

INTRODUCTION Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. METHODS Bone marrow-derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4(+) T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. RESULTS The frequency of PS particle-positive CD11c(+)/CD11b(+) BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4(+) T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. CONCLUSION These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4(+) T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles.

Alteration of ZnT5-mediated zinc import into the early secretory pathway affects the secretion of growth hormone from rat pituitary cells

BACKGROUND Aggregation of growth hormone (GH) required for its proper storage in granules is facilitated by zinc (Zn(2+)) transported by specific zinc transporters in and out of the regulated secretory pathway. Slc30a5 (ZnT5) was reported to have the highest gene expression among all zinc transporters in primary mouse pituitary cells while ZnT5-null mice presented with abnormal bone development and impaired growth compared to wild-type counterparts. METHODS In vitro studies performed in GH3 cells, a rat pituitary cell line that endogenously produces rat GH (rGH), included analysis of: cytoplasmic Zn(2+) pool changes after altering rSlc30a5 expression (luciferase assay), rZnT5 association with different compartments of the regulated secretory pathway (confocal microscopy), and the rGH secretion after rSlc30a5 knock-down (Western blot). RESULTS Confocal microscopy demonstrated high co-localization of rZnT5 with ER and Golgi (early secretory pathway) while siRNA-mediated knock-down of rSlc30a5 gene expression led to a significant reduction in rGH secretion. Furthermore, altered expression of rSlc30a5 (knock-down/overexpression) evoked changes in the cytoplasmic Zn(2+) pool indicating its important role in mediating Zn(2+) influx into intracellular compartments of the regulated secretory pathway. CONCLUSION Taken together, these results suggest that ZnT5 might play an important role in regulated GH secretion that is much greater than previously anticipated.