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Cholinergic innervation in the pancreas controls both the release of digestive enzymes to support the intestinal digestion and absorption, as well as insulin release to promote nutrient use in the cells of the body. The effects of muscarinic receptor stimulation are described in detail for endocrine beta cells and exocrine acinar cells separately. Here we describe morphological and functional criteria to separate these two cell types in situ in tissue slices and simultaneously measure their response to ACh stimulation on cytosolic Ca2+ oscillations [Ca2+]c in stimulatory glucose conditions. Our results show that both cell types respond to glucose directly in the concentration range compatible with the glucose transporters they express. The physiological ACh concentration increases the frequency of glucose stimulated [Ca2+]c oscillations in both cell types and synchronizes [Ca2+]c oscillations in acinar cells. The supraphysiological ACh concentration further increases the oscillation frequency on the level of individual beta cells, inhibits the synchronization between these cells, and abolishes oscillatory activity in acinar cells. We discuss possible mechanisms leading to the observed phenomena.

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

Opening of two-pore domain K

In β cells, stimulation by metabolic, hormonal, neuronal, and pharmacological factors is coupled to secretion of insulin through different intracellular signaling pathways. Our knowledge about the molecular machinery supporting these pathways and the patterns of signals it generates comes mostly from rodent models, especially the laboratory mouse. The increased availability of human islets for research during the last few decades has yielded new insights into the specifics in signaling pathways leading to insulin secretion in humans. In this review, we follow the most central triggering pathway to insulin secretion from its very beginning when glucose enters the β cell to the calcium oscillations it produces to trigger fusion of insulin containing granules with the plasma membrane. Along the way, we describe the crucial building blocks that contribute to the flow of information and focus on their functional role in mice and humans and on their translational implications.

Residing in the islets of Langerhans in the pancreas, β cells contribute to glucose homeostasis by managing the body's insulin supply. Although it has been acknowledged that healthy β cells engage in heavy cell-to-cell communication to perform their homeostatic function, the exact role and effects of such communication remain partly understood. We offer a novel, to our knowledge, perspective on the subject in the form of 1) a dynamical network model that faithfully mimics fast calcium oscillations in response to above-threshold glucose stimulation and 2) empirical data analysis that reveals a qualitative shift in the cross-correlation structure of measured signals below and above the threshold glucose concentration. Combined together, these results point to a glucose-induced transition in β-cell activity thanks to increasing coordination through gap-junctional signaling and paracrine interactions. Our data and the model further suggest how the conservation of entire cell-cell conductance, observed in coupled but not uncoupled β cells, emerges as a collective phenomenon. An overall implication is that improving the ability to monitor β-cell signaling should offer means to better understand the pathogenesis of diabetes mellitus.

NMDA receptors promote repolarization in pancreatic beta cells and thereby reduce glucose-stimulated insulin secretion. Therefore, NMDA receptors are a potential therapeutic target for diabetes. While the mechanism of NMDA receptor inhibition in beta cells is rather well understood at the molecular level, its possible effects on the collective cellular activity have not been addressed to date, even though proper insulin secretion patterns result from well-synchronized beta cell behavior. The latter is enabled by strong intercellular connectivity, which governs propagating calcium waves across the islets and makes the heterogeneous beta cell population work in synchrony. Since a disrupted collective activity is an important and possibly early contributor to impaired insulin secretion and glucose intolerance, it is of utmost importance to understand possible effects of NMDA receptor inhibition on beta cell functional connectivity. To address this issue, we combined confocal functional multicellular calcium imaging in mouse tissue slices with network science approaches. Our results revealed that NMDA receptor inhibition increases, synchronizes, and stabilizes beta cell activity without affecting the velocity or size of calcium waves. To explore intercellular interactions more precisely, we made use of the multilayer network formalism by regarding each calcium wave as an individual network layer, with weighted directed connections portraying the intercellular propagation. NMDA receptor inhibition stabilized both the role of wave initiators and the course of waves. The findings obtained with the experimental antagonist of NMDA receptors, MK-801, were additionally validated with dextrorphan, the active metabolite of the approved drug dextromethorphan, as well as with experiments on NMDA receptor KO mice. In sum, our results provide additional and new evidence for a possible role of NMDA receptor inhibition in treatment of type 2 diabetes and introduce the multilayer network paradigm as a general strategy to examine effects of drugs on connectivity in multicellular systems., Author summary Information processing in tissues is regulated by networks of interacting cells and through crosstalk with their environment. This is also true for islets of Langerhans in which several hundreds of beta cells are interconnected to ensure a proper secretion of insulin, a hormone crucial for the control of metabolic homeostasis. Since the loss of coordinated cellular activity is accompanied by a disruption of insulin secretion patterns, one of the main hallmarks of diabetes, it is of utmost importance to understand the underlying mechanisms that drive collective cellular activity in the islets and how these are affected in disease. Recently, NMDA receptor inhibitors were shown to boost beta cell activity and insulin secretion and were therefore suggested as potential antidiabetic drugs. However, their impact on the multicellular dynamics is not known. In the present study, we address this issue by combining high-frequency functional multicellular calcium imaging in pancreas tissue slices with network science approaches. Our findings reveal that NMDA receptor inhibition increases and synchronizes the activity of beta cell populations. Moreover, by considering individual intercellular calcium waves as network layers we show that NMDA receptor inhibition stabilizes the course of intercellular signals and confines the wave initiator regions.

Objective In adults, improper arterial function has been linked to cognitive impairment. The pulse wave velocity (PWV), augmentation index (AIx) and other vascular parameters are useful indicators of arterial health. In our study, we monitored arterial properties, body constitution, school success, and motor skills in young adolescents. We hypothesize that reduced cognitive and motor abilities have a vascular origin in children. Methods We analysed 81 healthy school children aged 11–16 years. Anthropometry central systolic arterial pressure, body mass index (BMI), standard deviation scores (SDS) BMI, general school performance grade, and eight motor tests were assessed. PWV, AIx, and central systolic arterial pressure (SBPao) were measured. Results AIx and SBPao correlated negatively with school performance grades. Extremely high AIx, PWV and SBPao values were observed in 5% of children and these children had average to low school performance. PWV correlated significantly with weight, height, and waist and hip circumference. AIx, PWV, school success, and BMI correlated strongly with certain motor functions. Conclusions Increased AIx and SBPao are associated with lower school and motor performance in children. PWV is influenced by the body’s constitution.

The intestinal epithelium is composed of diverse cell types, most abundant being the enterocytes. Among other functions, they maintain the intestinal barrier and play a critical role in the absorption of nutrients, drugs and toxins. This study describes the development and characterization of human intestinal epithelial cells (HUIEC), a spontaneously arising cell line established by selective trypsinization and cloning of the intestinal epithelium, resulting in a uniform population of highly epithelial cells with a strong growth potential.

Research on skeletal muscles suffers from a lack of appropriate human models to study muscle formation and regeneration on the regulatory level of single cells. This hampers both basic understanding and the development of new therapeutic approaches. The use of imaging multicolour flow cytometry and myogenic stem cells can help fill this void by allowing researchers to visualize and quantify the reaction of individual cultured cells to bioactives or other physiological impulses. As proof of concept, we subjected human CD56+ satellite cells to reference bioactives follistatin and Malva sylvestris extracts and then used imaging multicolor flow cytometry to visualize the stepwise activation of myogenic factors MyoD and myogenin in individual cells. This approach enabled us to evaluate the potency of these bioactives to stimulate muscle commitment. To validate this method, we used multi-photon confocal microscopy to confirm the potential of bioactives to stimulate muscle differentiation and expression of desmin. Imaging multicolor flow cytometry revealed statistically significant differences between treated and untreated groups of myogenic progenitors and we propose the utilization of this concept as an integral part of future muscle research strategies.

In this work we have compared the ultrastructural characteristics of major pancreatic endocrine cells, pituitary melanotrophs and adrenal chromaffin cells in the normal mouse strain (wild type, WT) and mice with a known secretory deficit, the Rab3a knockout strain (Rab3a KO). For this purpose, pancreata, pituitary glands and adrenal glands from the Rab3a KO and from the WT mice were analysed, using conventional transmission electron microscopy (TEM). In order to assess the significance of the presence of Rab3a proteins in the relevant cells, we focused primarily on their secretory vesicle morphology and distribution. Our results showed a comparable general morphology in Rab3a KO and WT in all assessed endocrine cell types. In all studied cell types, the distribution of secretory granules along the plasma membrane (number of docked and almost-docked vesicles) was comparable between Rab3a KO and WT mice. Specific differences were found in the diameters of their secretory vesicles, diameters of their electron-dense cores and the presence of autophagic structures in the cells of Rab3A KO mice only. Occasionally, individual electron-dense round vesicles were present inside autophagosome-like structures; these were possibly secretory vesicles or their remnants. The differences found in the diameters of the secretory vesicles confirm the key role of Rab3a proteins in controlling the balance between secretory vesicle biogenesis and degradation, and suggest that the ablation of this protein probably changes the nature of the reservoir of secretory vesicles available for regulated exocytosis.

Pancreatic beta cells act as glucose sensors, in which intracellular ATP ([ATP](i)) are altered with glucose concentration change. The characterization of voltage-gated sodium channels under different [ATP](i) remains unclear. Here, we demonstrated that increasing [ATP](i) within a certain range of concentrations (2-8 mM) significantly enhanced the voltage-gated sodium channel currents, compared with 2 mM cytosolic ATP. This enhancement was attenuated by even high intracellular ATP (12 mM). Furthermore, elevated ATP modulated the sodium channel kinetics in a dose-dependent manner. Increased [ATP](i) shifted both the current-voltage curve and the voltage-dependent inactivation curve of sodium channel to the right. Finally, the sodium channel recovery from inactivation was significantly faster when the intracellular ATP level was increased, especially in 8 mM [ATP](i), which is an attainable concentration by the high glucose stimulation. In summary, our data suggested that elevated cytosolic ATP enhanced the activity of Na(+) channels, which may play essential roles in modulating β cell excitability and insulin release when blood glucose concentration increases.

Corneal wound healing is often affected by TGF-β–mediated fibrosis and scar formation. Guided fibrosis with IGF-1 and antifibrotic substances might maintain corneal transparency. Primary human corneal keratocytes under serum-free conditions were used as a model of corneal stromal wounding, with markers of corneal fibrosis and opacity studied under TGF-β2 stimulation. Single-cell imaging flow cytometry was used to determine nuclearization of Smad3 and intracellular fluorescence intensity of Smad7 and the corneal crystallin aldehyde dehydrogenase 3A1. Extracellular matrix proteoglycans keratocan and biglycan were quantified using ELISAs. On the TGF-β2 background, the keratocytes were treated with IGF-1 and suberoylanilidehydroxamic acid (SAHA) or halofuginone ± IGF-1. IGF-1 alone decreased Smad3 nuclearization and increased aldehyde dehydrogenase 3A1 expression, with favorable extracellular matrix proteoglycan composition. SAHA induced higher Smad7 levels and inhibited translocation of Smad3 to the nucleus, also when combined with IGF-1. Immunofluorescence showed that myofibroblast transdifferentiation is attenuated and appearance of fibroblasts is favored by IGF-1 alone and in combination with the antifibrotic substances. The TGF-β/Smad pathway of fibrosis and opacity was inhibited by IGF-1 and further with SAHA in particular and with halofuginone. IGF-1 is thus a valid aid to antifibrotic treatment, with potential for effective and transparent corneal wound healing.

Ionotropic glutamate receptors (iGluRs) are expressed in islets and insulinoma cells and involved in insulin secretion. However, the exact roles that iGluRs play in β cells remain unclear. Here, we demonstrated that GluR2-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) were expressed in mouse β cells. Glutamate application increased both cytosolic calcium and the number of docked insulin-containing granules, which resulted in augmentation of depolarization-induced exocytosis and high-glucose-stimulated insulin release. While glutamate application directly depolarized β cells, it also induced an enormous depolarization when K(ATP) channels were available. Glutamate application reduced the conductance of K(ATP) channels and increased voltage oscillations. Moreover, actions of AMPARs were absent in Kir6.2 knock-out mice. The effects of AMPARs on K(ATP) channels were mediated by cytosolic cGMP. Taken together, our experiments uncovered a novel mechanism by which AMPARs participate in insulin release.

Inactivating mutations in the transcription factor hepatocyte nuclear factor (HNF) 1A cause HNF1A–maturity-onset diabetes of the young (HNF1A-MODY), the most common monogenic form of diabetes. To examine HNF1A-MODY-induced defects in gene expression, we performed a microarray analysis of the transcriptome of rat INS-1 cells inducibly expressing the common hot spot HNF1A frameshift mutation, Pro291fsinsC-HNF1A. Real-time quantitative PCR (qPCR), Western blotting, immunohistochemistry, reporter assays, and chromatin immunoprecipitation (ChIP) were used to validate alterations in gene expression and to explore biological activities of target genes. Twenty-four hours after induction of the mutant HNF1A protein, we identified a prominent down-regulation of the bone morphogenetic protein 3 gene (Bmp-3) mRNA expression. Reporter assays, qPCR, and Western blot analysis validated these results. In contrast, inducible expression of wild-type HNF1A led to a time-dependent increase in Bmp-3 mRNA and protein levels. Moreover, reduced protein levels of BMP-3 and insulin were detected in islets of transgenic HNF1A-MODY mice. Interestingly, treatment of naïve INS-1 cells or murine organotypic islet cultures with recombinant human BMP-3 potently increased their insulin levels and restored the decrease in SMAD2 phosphorylation and insulin gene expression induced by the HNF1A frameshift mutation. Our study suggests a critical link between HNF1A-MODY-induced alterations in Bmp-3 expression and insulin gene levels in INS-1 cells and indicates that the reduced expression of growth factors involved in tissue differentiation may play an important role in the pathophysiology of HNF1A-MODY.

Cyclic AMP regulates the late step of Ca²+-dependent exocytosis in many secretory cells through two major mechanisms: a protein kinase A-dependent and a cAMP-GEF/Epac-dependent pathway. We designed a protocol to characterize the role of these two cAMP-dependent pathways on the Ca²+ sensitivity and kinetics of regulated exocytosis in mouse pancreatic beta cells, using a whole-cell patch-clamp based capacitance measurements. A train of depolarizing pulses or slow photo-release of caged Ca²+ were stimuli for the exocytotic activity. In controls, due to exocytosis after slow photo-release, the C(m) change had typically two phases. We observed that the Ca²+-dependency of the rate of the first C(m) change follows saturation kinetics with high cooperativity and half-maximal rate at 2.9±0.2 μM. The intracellular depletion of cAMP did not change amp1, while rate1 and amp2 were strongly reduced. This manipulation pushed the Ca²+-dependency of the exocytotic burst to significantly lower [Ca²+](i). To address the question of which of the cAMP-dependent mechanisms regulates the observed shifts in Ca²+ dependency we included regulators of PKA and Epac2 activity in the pipette solution. PKA activation with 100 μM 6-Phe-cAMP or inhibition with 500 μM Rp-cAMPs in beta cells significantly shifted the EC(50) in the opposite directions. Specific activation of Epac2 did not change Ca²+ sensitivity. Our findings suggest that cAMP modulates Ca²+-dependent exocytosis in mouse beta cells mainly through a PKA-dependent mechanism by sensitizing the insulin releasing machinery to [Ca²+](i); Epac2 may contribute to enhance the rates of secretory vesicle fusion.

α-Neurexins constitute a family of neuronal cell surface molecules that are essential for efficient neurotransmission, because mice lacking two or all three α-neurexin genes show a severe reduction of synaptic release. Although analyses of α-neurexin knock-outs and transgenic rescue animals suggested an involvement of voltage-dependent Ca2+channels, it remained unclear whether α-neurexins have a general role in Ca2+-dependent exocytosis and how they may affect Ca2+channels. Here we show by membrane capacitance measurements from melanotrophs in acute pituitary gland slices that release from endocrine cells is diminished by >50% in adult α-neurexin double knock-out and newborn triple knock-out mice. There is a reduction of the cell volume in mutant melanotrophs; however, no ultrastructural changes in size or intracellular distribution of the secretory granules were observed. Recordings of Ca2+currents from melanotrophs, transfected human embryonic kidney cells, and brainstem neurons reveal that α-neurexins do not affect the activation or inactivation properties of Ca2+channels directly but may be responsible for coupling them to release-ready vesicles and metabotropic receptors. Our data support a general and essential role for α-neurexins in Ca2+-triggered exocytosis that is similarly important for secretion from neurons and endocrine cells.

Cyclic AMP regulates Ca2+-dependent exocytosis through a classical protein kinase A (PKA)-dependent and an alternative cAMP–guanine nucleotide exchange factor (GEF)/Epac-dependent pathway in many secretory cells. Although increased cAMP is believed to double secretory output in isolated pituitary cells, the direct target(s) for cAMP action and a detailed and high-time resolved analysis of the effect of intracellular cAMP levels on the secretory activity in melanotrophs are still lacking. We investigated the effect of 200 μm cAMP on the kinetics of secretory vesicle depletion in mouse melanotrophs from fresh pituitary tissue slices. The whole-cell patch-clamp technique was used to depolarize melanotrophs and increase the cytosolic Ca2+ concentration ([Ca2+]i). Exogenous cAMP elicited an about twofold increase in cumulative membrane capacitance change and ∼34% increase of high-voltage activated Ca2+ channel amplitude. cAMP-dependent mechanisms did not affect [Ca2+]i, since the application of forskolin failed to change [Ca2+]i in melanotrophs, a phenomenon readily observed in anterior lobe. Depolarization-induced secretion resulted in two distinct kinetic components: a linear and a threshold component, both stimulated by cAMP. The linear component (ATP-independent) probably represented the exocytosis of the release-ready vesicles, whereas the threshold component was assigned to the exocytosis of secretory vesicles that required ATP-dependent reaction(s) and > 800 nm[Ca2+]i. The linear component was modulated by 8-pCPT-2Me-cAMP (Epac agonist), while either H-89 (PKA inhibitor) or Rp-cAMPS (the competitive antagonist of cAMP binding to PKA) completely prevented the action of cAMP on the threshold component. In line with this, 6-Phe-cAMP, (PKA agonist), increased the threshold component. From our study, we suggest that the stimulation of cAMP production by application of oestrogen, as found in pregnant mice, increases the efficacy of the hormonal output through both PKA and cAMP–GEFII/Epac2-dependent mechanisms.

Cl− ions are known regulators of Ca2+-dependent secretory activity in many endocrine cells. The suggested mechanisms of Cl− action involve the modulation of GTP-binding proteins, voltage-activated calcium channels or maturation of secretory vesicles. We examined the role of cytosolic Cl− ([Cl−]i) and Cl− currents in the regulation of secretory activity in mouse melanotrophs from fresh pituitary tissue slices by using the whole-cell patch-clamp. We confirmed that elevated [Cl−]i augments Ca2−-dependent exocytosis and showed that Cl− acts on secretory vesicle maturation. The latter process was abolished by a V-type H−-ATPase blocker (bafilomycin), intracellular 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS), a Cl− channel blocker, and tolbutamide, a sulphonylurea implicated in secretory vesicle maturation. In a small subset of cells, block of plasmalemmal Cl− current by DIDS reversibly enhanced endocytosis. The direct activation of G-proteins by GTP-γ-S, a non-hydrolysable GTP analogue, did not restore the impaired secretion observed in low [Cl−]i conditions. The amplitude of voltage-activated calcium currents was unaffected by the [Cl−]i. Furthermore, two Cl−-permeable channels, calcium-activated Cl− channels and GABAA receptors, appeared as major regulators of intracellular Cl− homeostasis. In conclusion, the predominant underlying mechanism of Cl− action is mediated by intracellular Cl− fluxes during vesicle maturation, rather than activation of G-proteins or modulation of voltage-activated Ca2+ channels.

Enlargeosomes, a new type of widely expressed cytoplasmic vesicles, undergo tetanus toxin-insensitive exocytosis in response to cytosolic Ca2+concentration ([Ca2+]i) rises. Cell biology of enlargeosomes is still largely unknown. By combining immunocytochemistry (marker desmoyokin-Ahnak, d/A) to capacitance electrophysiology in the enlargeosome-rich, neurosecretion-defective clone PC12-27, we show that 1) the two responses, cell surface enlargement and d/A surface appearance, occur with similar kinetics and in the same low micromolar [Ca2+]irange, no matter whether induced by photolysis of the caged Ca2+compound o-nitrophenyl EGTA or by the Ca2+ionophore ionomycin. Thus, enlargeosomes seem to account, at least in large part, for the exocytic processes triggered by the two stimulations. 2. The enlargeosome membranes are resistant to nonionic detergents but distinct from other resistant membranes, rich in caveolin, Thy1, and/or flotillin1. 3. Cell cholesterol depletion, which affects many membrane fusions, neither disrupts enlargeosomes nor affects their regulated exocytosis. 4. The postexocytic cell surface decline is [Ca2+]idependent. 5. Exocytized d/A-rich membranes are endocytized and trafficked along an intracellular pathway by nonacidic organelles, distinct from classical endosomes and lysosomes. Our data define specific aspects of enlargeosomes and suggest their participation, in addition to cell differentiation and repair, for which evidence already exists, to other physiological and pathological processes.

Regeneration of skeletal muscle after injury is limited by scar formation, slow healing time and a high recurrence rate. A therapy based on platelet-rich plasma (PRP) has become a promising lead for tendon and ligament injuries in recent years, however concerns have been raised that PRP-derived TGF-β could contribute to fibrotic remodelling in skeletal muscle after injury. Due to the lack of scientific grounds for a PRP -based muscle regeneration therapy, we have designed a study using human myogenic progenitors and evaluated the potential of PRP alone and in combination with decorin (a TGF-β inhibitor), to alter myoblast proliferation, metabolic activity, cytokine profile and expression of myogenic regulatory factors (MRFs). Advanced imaging multicolor single-cell analysis enabled us to create a valuable picture on the ratio of quiescent, activated and terminally committed myoblasts in treated versus control cell populations. Finally high-resolution confocal microscopy validated the potential of PRP and decorin to stimulate the formation of polynucleated myotubules. PRP was shown to down-regulate fibrotic cytokines, increase cell viability and proliferation, enhance the expression of MRFs, and contribute to a significant myogenic shift during differentiation. When combined with decorin further synergistc effects were identified. These results suggest that PRP could not only prevent fibrosis but could also stimulate muscle commitment, especially when combined with a TGF-β inhibitor.

Gold nanoparticles (GNPs) are claimed as outstanding biomedical tools for cancer diagnostics and photo-thermal therapy, but without enough evidence on their potentially adverse immunological effects. Using a model of human dendritic cells (DCs), we showed that 10 nm- and 50 nm-sized GNPs (GNP10 and GNP50, respectively) were internalized predominantly via dynamin-dependent mechanisms, and they both impaired LPS-induced maturation and allostimulatory capacity of DCs, although the effect of GNP10 was more prominent. However, GNP10 inhibited LPS-induced production of IL-12p70 by DCs, and potentiated their Th2 polarization capacity, while GNP50 promoted Th17 polarization. Such effects of GNP10 correlated with a stronger inhibition of LPS-induced changes in Ca2+ oscillations, their higher number per DC, and more frequent extra-endosomal localization, as judged by live-cell imaging, proton, and electron microscopy, respectively. Even when released from heat-killed necrotic HEp-2 cells, GNP10 inhibited the necrotic tumor cell-induced maturation and functions of DCs, potentiated their Th2/Th17 polarization capacity, and thus, impaired the DCs' capacity to induce T cell-mediated anti-tumor cytotoxicity in vitro. Therefore, GNP10 could potentially induce more adverse DC-mediated immunological effects, compared to GNP50.

Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca2+-regulated exocytosis. Previous functional analysis in pituitary melanotrophs described Rab3a as a positive regulator of Ca2+-dependent exocytosis. However, the precise role of the Rab3a isoform on the kinetics and intracellular [Ca2+] sensitivity of regulated exocytosis, which may affect the availability of two major peptide hormones, α-melanocyte stimulating hormone (α- MSH) and β-endorphin in plasma, remain elusive. We employed Rab3a knock-out mice (Rab3a KO) to explore the secretory phenotype in melanotrophs from fresh pituitary tissue slices. High resolution capacitance measurements showed that Rab3a KO melanotrophs possessed impaired Ca2+-triggered secretory activity as compared to wild-type cells. The hampered secretion was associated with the absence of cAMP-guanine exchange factor II/ Epac2- dependent secretory component. This component has been attributed to high Ca2+-sensitive release-ready vesicles as determined by slow photo-release of caged Ca2+. Radioimmunoassay revealed that α-MSH, but not β-endorphin, was elevated in the plasma of Rab3a KO mice, indicating increased constitutive exocytosis of α-MSH. Increased constitutive secretion of α-MSH from incubated tissue slices was associated with reduced α-MSH cellular content in Rab3a-deficient pituitary cells. Viral re-expression of the Rab3a protein in vitro rescued the secretory phenotype of melanotrophs from Rab3a KO mice. In conclusion, we suggest that Rab3a deficiency promotes constitutive secretion and underlies selective impairment of Ca2+-dependent release of α-MSH. peerReviewed

In endocrine cells within islets of Langerhans calcium ions couple cell stimulation to hormone secretion. Since the advent of modern fluorimetry, numerous in vitro studies employing primarily isolated mouse islets have investigated the effects of various secretagogues on cytoplasmic calcium, predominantly in insulin-secreting beta cells. Due to technical limitations, insights of these studies are inherently limited to a rather small subpopulation of outermost cells. The results also seem to depend on various factors, like culture conditions and duration, and are not always easily reconcilable with findings in vivo. The main controversies regard the types of calcium oscillations, presence of calcium waves, and the level of synchronized activity. Here, we set out to combine the in situ acute mouse pancreas tissue slice preparation with noninvasive fluorescent calcium labeling and subsequent confocal laser scanning microscopy to shed new light on the existing controversies utilizing an innovative approach enabling the characterization of responses in many cells from all layers of islets. Our experiments reproducibly showed stable fast calcium oscillations on a sustained plateau rather than slow oscillations as the predominant type of response in acute tissue slices, and that calcium waves are the mechanistic substrate for synchronization of oscillations. We also found indirect evidence that even a large amplitude calcium signal was not sufficient and that metabolic activation was necessary to ensure cell synchronization upon stimulation with glucose. Our novel method helped resolve existing controversies and showed the potential to help answer important physiological questions, making it one of the methods of choice for the foreseeable future.

We have used the whole-cell patch-clamp technique to study changes in membrane conductance and membrane capacitance after osmotic swelling in rat hepatocytes. Hypoosmotic solutions induced an instantaneous increase in the volume of patch-clamped cells that was followed by a slow decline reminiscent of regulatory volume decrease as seen in intact cells. These morphological changes were associated with a transient increase in membrane conductance. The rise in conductance was not correlated with changes in capacitance, neither in time after the initiation of cell swelling nor in magnitude. Therefore we conclude that an osmotically induced increase in conductance is probably a result of the activation of existent channels in the plasmalemma and not a result of the fusion of vesicle membrane containing ionic channels.

Stimulus-secretion coupling at the cellular level is studied by measuring changes in membrane capacitance in a variety of secretory cells. Attempts to gain new insight into the control of exocytosis in adenohypophysial cells by the patch-clamp technique are briefly outlined.

Fast neurotransmission and slower hormone release share the same core fusion machinery consisting of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. In evoked neurotransmission, interactions between SNAREs and the Munc18-1 protein, a member of the Sec1/Munc18 (SM) protein family, are essential for exocytosis, whereas other SM proteins are dispensable. To address if the exclusivity of Munc18-1 demonstrated in neuroexocytosis also applied to fast insulin secretion, we characterized the presence and function of Munc18-1 and its closest homologue Munc18-2 in β-cell stimulus-secretion coupling. We show that pancreatic β-cells express both Munc18-1 and Munc18-2. The two Munc18 homologues exhibit different subcellular localization, and only Munc18-1 redistributes in response to glucose stimulation. However, both Munc18-1 and Munc18-2 augment glucose-stimulated hormone release. Ramp-like photorelease of caged Ca(2+) and high resolution whole-cell patch clamp recordings show that Munc18-1 and Munc18-2 overexpression shift the Ca(2+) sensitivity of the fastest phase of insulin exocytosis differently. In addition, we reveal that Ca(2+) sensitivity of exocytosis in β-cells depends on the phosphorylation status of the Munc18 proteins. Even though Munc18-1 emerges as the key SM-protein determining the Ca(2+) threshold for triggering secretory activity in a stimulated β-cell, Munc18-2 has the ability to increase Ca(2+) sensitivity and thus mediates the release of fusion-competent granules requiring a lower cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i)(.) Hence, Munc18-1 and Munc18-2 display distinct subcellular compartmentalization and can coordinate the insulin exocytotic process differently as a consequence of the actual [Ca(2+)](i).

Phosphatidylinositol-4, 5-bisphosphate [PI(4,5)P(2)] has been implicated in the priming of large dense-core vesicles in many secretory cells; however, its role in the Ca(2+)-dependent secretory activity in pituitary cells remains elusive. We assessed the effect of elevated intracellular PI(4,5)P(2) on the kinetics of Ca(2+)-dependent exocytosis, using a whole-cell patch-clamp technique in wild-type mouse melanotrophs from fresh pituitary tissue slices. We found that 1 micromol/L PI(4,5)P(2) significantly increased Ca(2+)-dependent exocytosis of vesicles that need to go through ATP-dependent reactions; however, the exocytosis of release-ready vesicles (ATP-independent release) and voltage-activated Ca(2+) currents remained unaffected. We suggest that PI(4,5)P(2) increases the size of the readily releasable vesicle pool by regulating the effectiveness of vesicular mobilization and fusion in an ATP-dependent manner.

The mechanisms governing the fast, regulated exocytosis of enlargeosomes have been unknown, except for the participation of annexin-2 in a pre-fusion step. We investigated whether any SNAREs are involved. In PC12-27 cells, which are enlargeosome-rich, the expressed SNAREs exhibited various distributions (trans-Golgi network, scattered puncta, plasma membrane); however, only VAMP4 was colocalized in discrete puncta with the enlargeosome marker desmoyokin. The exocytosis of the organelle, revealed by capacitance increases and by surface appearance of desmoyokin, was largely inhibited by microinjection of anti-VAMP4, anti-syntaxin-6 and anti-SNAP23 antibodies, by incubation with botulinum toxin E, and by transfection of VAMP4 and syntaxin-6 siRNAs. Microinjection of the antibodies anti-VAMP7, anti-VAMP8 and anti-syntaxin-4, and transfection with the VAMP8 siRNA were ineffective. Inhibition of enlargeosome exocytosis by VAMP4 siRNA also occurred in a cell type that was competent for neurosecretion, SH-SY5Y. Moreover, in cells expressing a VAMP4-GFP construct, enlargeosome exocytosis and surface appearance of fluorescence occurred concomitantly, and many ensuing surface patches were co-labelled by GFP and desmoyokin. VAMP4, an R-SNARE that has never been shown to participate in regulated exocytoses, therefore appears to be harboured in the membrane of enlargeosomes and to be a member of the machinery mediating their regulated exocytosis. Syntaxin-6 and SNAP23 appear also to be needed for the process to occur; however, the mechanism of their participation, whether direct or indirect, remains undefined.

Cyclin-dependent kinase 5 (Cdk5) is emerging as a multifunctional kinase involved in regulating numerous cellular processes. Lately, Cdk5 has also emerged as a key controller of regulated membrane fusion in secretory cells. The pancreatic β-cell is highly specialized to secrete insulin in response to elevated glucose concentrations in the blood. The final biochemical events leading to insulin release from the β-cell are governed by a secretion apparatus that is similar to the presynaptic machinery mediating synaptic transmission in neuronal networks. We now summarize recent developments in the field of Cdk5 and regulated exocytosis and also present some novel findings regarding Cdk5’s effect on insulin secretion.

Members of the Rab3 (A–D) subfamily of small GTPases are believed to play a key role in regulated exocytosis. These proteins share ∼80% identity at amino acid level. The question of whether isoforms of Rab3 are functionally redundant was the subject of this study. We used RT-PCR analysis, in situ hybridization histochemistry, and confocal microscope-based analysis of immunocytochemistry to show that rat melanotrophs contain about equal amounts of Rab3A and Rab3B transcripts as well as proteins. Therefore, these cells are a suitable model to study the subcellular distribution and the role of these paralogous isoforms in regulated exocytosis. Secretory activity of single cells was monitored with patch-clamp capacitance measurements, and the cytosol was dialyzed with a high-calcium-containing patch pipette solution. Preinjection of antisense oligodeoxyribonucleotides specific to Rab3A, but not to Rab3B, induced a specific blockage of calcium-dependent secretory responses, indicating an exclusive requirement for Rab3A in melanotroph cell-regulated secretion. Although the injection of purified Rab3B protein was ineffective, the injection of recombinant Rab3A proteins into rat melanotrophs revealed that regulated secretion was stimulated by a GTP-bound Rab3A with an intact COOH terminus and inhibited by Rab3AT36N, impaired in GTP binding. These results indicate that Rab3A, but not Rab3B, enhances secretory output from rat melanotrophs and that their function is not redundant.

Eukaryotic cells internalize extracellular macromolecules by en- docytosis and it was shown that Rab5 protein is required for this process. While it is clear that endocytosis consists of vesicle fission from the plasma mem- brane, the role of Rab5 protein in the plasma membrane surface area changes is still unclear. Here we studied whether Rab5 is required for membrane sur- face area changes in rat melanotrophs—cells deriving from the pituitary pars intermedia. The presence of this protein in melanotrophs was probed by immu- nocytochemistry and its putative role in membrane area dynamics was moni- tored electrophysiologically with membrane capacitance measurements as this parameter directly reflects changes in membrane surface area. We found that Rab5 protein exists in melanotrophs. At (Ca 2+ )i < 3 M, endocytosis-dominat- ed membrane capacitance decrease was found to be blocked by microinjection of specific Rab5 antibody. At high (Ca 2+ )i, Rab5 antibody did not affect the steady-state increase in membrane capacitance, while it elevated the rate of membrane capacitance increase, which is consistent with an inhibition of en- docytosis.

Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A(2) acts intracellularly. As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them. Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus. The transport of proteins into the nucleus proceeds via the cytosol of a cell, therefore, ammodytoxin passed the cytosol of the neuron on its way to the nucleus. Although it is not yet clear how ammodytoxin is translocated into the cytosol of the neuron, our results demonstrate that its stability in the cytosol is not in question, providing the evidence that the toxin can act in this cellular compartment.

Taking tissue slices of the embryonic and newborn pancreas is a novel approach for the study of the perinatal development of this gland. The aim of this study was to describe the morphology and physiology of in vivo and in vitro developing beta-cells. In addition, we wanted to lay a foundation for the functional analysis of other pancreatic cells, either alone or as part of an integrative pancreatic physiology approach. We used cytochemistry and light microscopy to detect specific markers and the whole-cell patch-clamp to assess the function of single beta-cells. The insulin signal in the embryonic beta-cells was condensed to a subcellular compartment and redistributed throughout the cytosol during the first 2 days after birth. The hormone distribution correlated well with the development of membrane excitability and hormone release competence in beta-cells. Endocrine cells survived in the organotypic tissue culture and maintained their physiological properties for weeks. We conclude that our preparation fulfills the criteria for a method of choice to characterize the function of developing pancreas in wild-type and genetically modified mice that die at birth. We suggest organotypic culture for in vitro studies of the development and regeneration of beta-cells.

Synaptotagmin I (Syt I), a low-affinity Ca2+-binding protein, is thought to serve as the Ca2+sensor in the release of neurotransmitter. However, functional studies on the calyx of Held synapse revealed that the rapid release of neurotransmitter requires only approximately micromolar [Ca2+], suggesting that Syt I may play a more complex role in determining the high-affinity Ca2+dependence of exocytosis. Here we tested this hypothesis by studying pituitary cells, which possess high- and low-affinity Ca2+-dependent exocytic pathways and express Syt I. Using patch-clamp capacitance measurements to monitor secretion and the acute antisense deletion of Syt I from differentiated cells, we have shown that the rapid and the most Ca2+-sensitive pathway of exocytosis in rat melanotrophs requires Syt I. Furthermore, stimulation of the Ca2+-dependent exocytosis by cytosol dialysis with solutions containing 1 μM [Ca2+] was completely abolished in the absence of Syt I. Similar results were obtained by the preinjection of antibodies against the CAPS (Ca2+-dependent activator protein for secretion) protein. These results indicate that synaptotagmin I and CAPS proteins increase the probability of vesicle fusion at low cytosolic [Ca2+].

We used the whole-cell patch-clamp technique to study the secretory activity of single cells by monitoring changes in membrane capacitance [Neher, E. and Marty, A. (1982) Proc. Natl. Acad. Sci. USA 79, 523–535] in anterior pituitary cells. Unexpectedly we have observed that increasing intracellular chloride ions stimulate Ca2+-induced exocytosis in a dose-dopendent fashion (Kd = 12 mM). These results demonstrate a role of cytosolic chloride ions in the regulation of exocytotic secretion in anterior pituitary cells. It is suggested that chloride channels, in addition to playing a part in regulating membrane electrical activity [Korn, S.J., Bolden, A. and Horn, A. (1991) J. Physiol. 439, 423–437; Penner, R., Matthews, G. and Horn, A. (1988) Nature 334, 499–504] and cytosolic pH [Kaila, K. and Voipio, J. (1987) Nature 330, 163–165], are also involved in the modulation of cytosolic chloride concentration and thus in the control of exocytosis.

Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca 2+ -dependent activator protein for secretion), a protein required for Ca 2+ -dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca 2+ elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca 2+ dependencies. A threshold of ≈10 μM Ca 2+ was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca 2+ activity < 10 μM. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca 2+ and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.

Besides having a role in signal transduction some trimeric G-proteins may be involved in a late stage of exocytosis. Using immunocytochemistry and confocal microscopy we found that Gi(3)-protein resides mainly in the plasma membrane, whereas Gi(1/2-)protein is preferentially associated with secretory granules. To study the function of trimeric Gi(3)- and Gi(1/2)-proteins, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. We report here that mastoparan, an activator of trimeric G-proteins, enhances calcium-induced secretory activity in rat melanotrophs. The introduction of synthetic peptides corresponding to the C-terminal domain of the (α)-subunit of Gi(3)- and Gi(1/2)-proteins indicated that Gi(3)peptide specifically blocked the mastoparan-stimulated secretory activity, which indicates an involvement of a trimeric Gi(3)-protein in mastoparan-stimulated secretory activity. Flash photolysis of caged Ca(2+)-elicited biphasic capacitance increases consisting of a fast and a slower component. Injection of anti-Gi(3) antibodies selectively inhibited the slow but not the fast component of secretory activity in rat melanotrophs. We propose that the plasma membrane-bound Gi(3)-protein may be involved in regulated secretion by specifically controlling the slower kinetic component of exocytosis.

The whole-cell patch-clamp membrane capacitance measurement was used to monitor secretory activity in rat melanotrophs, while rab3AL, putative effector domain peptides of Rab3 small GTPases (20-30 kDa), were introduced into cytosol. In melanotrophs dialyzed with calcium free solutions membrane capacitance tends to decrease slightly. This decrease is further potentiated with GDPbetaS (500 microM). We found that rab3AL (100 microM) stimulated secretory activity in the absence of calcium. The rab3AL response was qualitatively comparable to the response to mastoparan (1 microM), an activator of certain heterotrimeric GTP-binding proteins. Interestingly, inclusion of GDPbetaS (500 microM) resulted in a blockade of both rab3AL and mastoparan induced responses. We conclude that rab3AL and mastoparan induce calcium-independent stimulation of secretory activity in rat melanotrophs by activation of a downstream heterotrimeric GTP-binding protein.

Publisher Summary This chapter discusses how synthetic peptides to different regions of transmembrane receptors and GTP binding proteins have increased the understanding of the molecular mechanism of signal transduction and signaling pathways involved in the control of membrane traffic and fusion in secretory cells. A powerful approach has been to use both peptides and antibodies to different G proteins to define which ones are important in signal transduction and release. Carboxy-terminal peptides to G proteins have been shown to prevent coupling of G proteins to their respective receptors. In particular, residues 345–354 of G αi3 are found to specifically block a stimulation of release from mast cells. In addition to this, an antibody to G αi3 is found to cause a specific inhibition of GTPγS induced release in permeabilized mast cells. Smg's act as regulatory switches whose activity is controlled by cycling between the active GTP bound and inactive GDP states. Unlike G proteins, smg's have slow intrinsic rates of activation and inactivation and the hunt for regulatory proteins for both arms of the cycle began. Two classes of regulatory proteins exist—the guanine nucleotide exchange factors (GNEFs), which catalyze release of bound GDP, promoting its replacement by GTP, and the GTPase-activating proteins (GAPs), which speed up GTP hydrolysis as an irreversible step in the cycle. GNEFs can be either stimulatory or inhibitory.

We investigated the role of ADP-ribosylation factor (ARF) in regulated exocytosis in patch-clamped rat melanotrophs. Addition of brefeldin A (BFA) to inhibit activation of endogenous ARF protein was found to attenuate regulated secretory activity monitored as changes in membrane capacitance (Cm). A synthetic peptide to amino acids 46-61 of ARF (P-14) was also found to inhibit Ca(2+)-induced secretory activity in these cells. This inhibition was not apparent with a scrambled amino acid sequence of ARF-P14 peptide. This paper provides the first patch-clamp study to suggest that the small GTP-binding protein ARF is required to trigger release of secretory granules from rat pituitary melanotrophs.

Calcium ions play an important role in both intra- and intercellular signaling. In pancreatic acinar cells intracellular Ca(2+) regulates exocytotic secretion and fluid secretion. In this paper we study the typical experimental traces of Ca(2+) responses in pancreatic acinar cells obtained in response to the physiological agonist acetylcholine. To determine whether they are stochastic or deterministic in nature, we analyze the traces with methods of nonlinear time series analysis. In particular, by performing surrogate data tests and employing a determinism test for short time series, we show that the responses of pancreatic acinar cells to acetylcholine are stochastic with only faintly expressed deterministic features. Presented results thus corroborate the notion that mathematical models should take stochasticity explicitly into account when describing intra- and intercellular processes, and that indeed further efforts should be directed toward this subject.