Ultrastructural morphometry of parathyroid cells in rats of different ages

Summary Parathyroid glands of newborn to 1-year-old male Sprague-Dawley rats were fixed by perfusion or by immersion, and prepared for electron microscopy. Parathyroid glands fixed by immersion exhibited parenchymal cells with variable ultrastructure, indicating that these cells were in different stages of the proposed secretory cycle in parathyroid cells. In contrast, parathyroid cells of glands fixed by perfusion were uniform in ultrastructure, suggesting that all cells were in the same stage of secretory activity. Parathyroid glands of 3-, 4-, 6-, 8- and 10-week-old rats also were fixed by perfusion and analysed by electron-microscopic morphometry. These data demonstrated an increase in cell volume and in surface area of the membrane compartments concerned with parathyroid hormone secretion: these changes were not related to variations in serum calcium concentration. Both the qualitative observations and the quantitative data do not favour the idea of a secretory cycle in rat parathyroid cells.     

Development of a bioartificial pancreas: I. long-term propagation and basal and induced secretion from entrapped betaTC3 cell cultures

Bioartificial pancreatic constructs based on immunoisolated, insulin-secreting cells have the potential for providing effective, long-term treatment of type I (insulin-dependent) diabetes. Use of insulinoma cells, which can be amplified in culture, relaxes the tissue availability limitation that exists with normal pancreatic islet transplantations. We have adopted mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads as our model system for a bioartificial pancreas, and we have characterized the effects of long-term propagation and of glucose concentration step changes on the bioenergetic status and on the metabolic and secretory activities of the entrapped cells. Cell bioenergetics were evaluated nonivasively by phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy, and metabolic and secretory parameters by assaying cell culture medium. Data indicate that net cell growth occurred between days 3 and 10 of the experiment, resulting in an approximate doubling of the overall metabolic and secretory rates and of the intracellular metabolite levels. Concurrently, a reorganization of cell distribution within the beads was observed. Following this growth period, the measured metabolic and secretory parameters remained constant with time. During glucose step changes in the perfusion medium from a high concentration of 12 to 15 mM to 0 mM for 4.5 h to the same high glucose concentration, the oxygen consumption rate was not affected, whereas insulin secretion was always glucose-responsive. Intracellular nucleotide triphosphates did not change during 0 mM glucose episodes performed early in culture history, but they declined by 20% during episodes performed later in the experiment. It is concluded that the system of APA-entrapped betaTC3 cells exhibits several of the desirable characteristics of a bioartificial pancreas device, and that a correlation between ATP and the rate of insulin secretion from betaTC3 cells exists for only a domain of culture conditions. These findings have significant implications in tissue engineering a long-term functional bioartificial endocrine pancreas, in developing noninvasive methods for assessing construct function postimplantation, and in the biochemical processes associated with insulin secretion.     

Improved perfusion conditions for patch-clamp recordings on human erythrocytes

Various configurations of the patch-clamp method are powerful tools for examining the transport of charged solutes across biological membranes. Originally developed for the study of relatively large cells which adhere to solid surfaces under in vitro culture, these methods have been increasingly applied to small cells or organelles in suspension. Under these conditions, a number of significant technical problems may arise as a result of the smaller geometry. Here, we examined these problems using human erythrocytes infected with the malaria parasite, Plasmodium falciparum, a system where experimental differences and the technical difficulty of erythrocyte patch-clamp have hindered universal agreement on the properties of the induced ion channels. We found that patch-clamp recordings on infected erythrocytes are especially susceptible to artifacts from mechanical perturbations due to solution flow around the cell. To minimize these artifacts, we designed a new perfusion chamber whose geometry allows controlled solution flow around the fragile erythrocyte. Not only were recordings acquired in this chamber significantly less susceptible to perfusion artifacts, but the chamber permitted rapid and reversible application of known inhibitors with negligible mechanical agitation. Electrophysiological recordings then faithfully reproduced several findings made with more traditional methods. The new perfusion chamber should also be useful for patch-clamp recordings on blood cells, protoplasts, and organelles.     

Kinetics of albumin- and alpha-fetoprotein-production during rat liver development

Synthesis of most of the plasma proteins is one of the main functions of the hepatocytes. Albumin synthesis is quantitatively the most abundant. In the present study we investigated albumin- and alpha-fetoprotein-gene-expression, and the function of the secretory apparatus during rat liver development. To this purpose we used the method of radioactive biosynthetic labeling of newly synthesized albumin and alpha-fetoprotein (AFP) to monitor the secretory capacity of endodermal cells derived from ventral foregut region (embryonic day 10, E10), and of embryonic and fetal hepatoblasts. Synthesis and secretion of albumin and AFP were already detected in the low numbered ventral foregut endodermal cells; fibrinogen synthesis was detectable in the E12 hepatoblasts, which were in higher number. The whole secretory machinery was functional from the earliest stages of liver development, and the speed of secretion was comparable with that of the adult hepatocytes. There was almost 4-fold increase of hepatoblasts cell volume in fetal stage compared with embryonic stage. The model used suggests that the hepatocyte secretory apparatus is already functional before the emergence of the liver bud. This is the first comparative report to analyze the hepatocyte secretory function, cell proliferation and cell volume during liver development.     

Altered phospholipid secretion in type II pneumocytes isolated from streptozotocin-diabetic rats

To study the effect of diabetes on pulmonary surfactant secretion, type II pneumocytes from adult streptozotocin-induced diabetic rats were placed in short-term culture. As opposed to a linear secretory rate by control type II cells, the secretory rate of type II cells from diabetic animals was biphasic reaching a minimum at 1.5 h. When exogenous surfactant containing radioactive phosphatidylcholine was added to the incubation media for 1.5 h, the cells from diabetic animals incorporated more exogenous phosphatidylcholine into lamellar bodies than control cells. This suggests that in the type II cell from diabetic animals, the rate of reutilization is greater than the rate of secretion until 1.5 h, at which time the rate of secretion becomes greater. The altered secretory pattern was reversed by in vivo insulin treatment 30 min prior to killing but not by the addition of insulin to the incubation media. When challenged by isoproterenol, a beta-adrenergic agonist, the secretory pattern of cells from diabetic animals was biphasic as observed with basal secretion; however, secretion was stimulated 30% as opposed to 100% increase in control cells. These data suggest that basal and stimulated secretion are altered in the cultured type II cell from diabetic animals and restored by in vivo but not in vitro insulin treatment.     

Optical chamber system designed for microscopic observation of living cells under extremely high hydrostatic pressure

A novel pressure chamber system has been developed for the study of living cells under conditions of extremely high hydrostatic pressure up to 100 MPa (1 atm = 0.101325 MPa). The temperature in the chamber is thermostatically controlled in the range from 2 degrees to 80 degrees C. Two high-pressure pumps are employed for continuous perfusion of the chamber with culture medium and a chemical solution under high hydrostatic pressure conditions. The chamber has a 2-mm-thick glass window 2 mm in diameter, with a minimum working distance of 3.8 mm. The chamber system is designed to be adaptable to a variety of microscopic and imaging techniques. Using this chamber system, we successfully carried out real-time observations of elongated Escherichia coli and rounded HeLa cells under pressure.     

UNSTIMULATED SECRETION OF PROTEIN FROM RAT EXOCRINE PANCREAS CELLS

Our earlier work demonstrated that the rate of protein synthesis in the exocrine cells of the rat pancreas is constant in different physiological states, including prolonged fasting. In this study we have followed the fate of the protein in the pancreatic cells of the fasting animal in vivo as well as in vitro. The data were obtained by quantitative radioautography and by biochemical determinations. In nonanesthesized, fasting rats, without cannulated pancreatic duct, some 80% of the proteins synthesized at a given time leaves the cell within 12 hr by way of secretion, intracellular breakdown not being important. Two mechanisms of fasting secretion exist. The first, starting at a slow rate after 20 min, is inferred to result from fortuitous contacts of young secretory granules with the apical cell membrane. The rate of secretion is the same in vivo as in vitro, at least during the first 4 hr after pulse labeling. Within 7 hr about 20% of the total amount of newly synthesized protein has left the cell. The second mechanism consists of an orderly movement of the mass of secretory granules towards the apical cell membrane as caused by the continuous assembly of new granules. The granules that come into contact with the cell membrane are discharged. It takes about 7–12 hr for secretory protein transported in this way to reach the cell membrane. The addition of new secretory granules to those present is essential for the second mechanism, for the blockade of protein synthesis by cycloheximide decreases the rate of this phase of secretion without interfering with the secretory process proper. Atropin does not inhibit the fasting secretion in vitro, nor does extensive washing of the tissue slices, excluding possible secretagogues as important factors in fasting secretion.     

Flow in the well: computational fluid dynamics is essential in flow chamber construction

A perfusion system was developed to generate well defined flow conditions within a well of a standard multidish. Human vein endothelial cells were cultured under flow conditions and cell response was analyzed by microscopy. Endothelial cells became elongated and spindle shaped. As demonstrated by computational fluid dynamics (CFD), cells were cultured under well defined but time varying shear stress conditions. A damper system was introduced which reduced pulsatile flow when using volumetric pumps. The flow and the wall shear stress distribution were analyzed by CFD for the steady and unsteady flow field. Usage of the volumetric pump caused variations of the wall shear stresses despite the controlled fluid environment and introduction of a damper system. Therefore the use of CFD analysis and experimental validation is critical in developing flow chambers and studying cell response to shear stress. The system presented gives an effortless flow chamber setup within a 6-well standard multidish.     

Comparison of results using the gel microdrop cytokine secretion assay with ELISPOT and intracellular cytokine staining assay

The gel microdrop (GMD) secretion assay involves encapsulating single cells in a biotinylated agarose matrix, addition of a streptavidin bridge, diffusion of a biotinylated capture antibody, and detection of secreted molecules using a fluorescently labeled reporter antibody. Using flow cytometry, encapsulated cells can be analyzed or recovered based on cell type and secretory profile. Using murine Th2 cell line D10.G4.1 as a model, we recently demonstrated the feasibility of using the GMD cytokine secretion assay combined with flow cytometry to detect IL-4-producing cells after stimulation with the mitogen, Con A. In addition, subpopulations of encapsulated cells secreting IL-4 were simultaneously characterized by immunophenotyping. We found good correlation between results using the GMD cytokine secretion assay and results with the standard ELISPOT and intracellular cytokine (ICC) assays. The GMD cytokine secretion assay permits simultaneous detection of secreted cytokine and determination of cell surface phenotype on viable, single cells. Moreover, using fluorescence activated cell sorting (FACS), secreting cells of interest can be sorted, recovered, and cultured for further studies.     

Secretion of Carboxypeptidase E from Cultured Astrocytes and from AtT-20 Cells, a Neuroendocrine Cell Line: Implications for Neuropeptide Biosynthesis

Cultured astrocytes have recently been shown to produce certain neuropeptides, as well as neuropeptide processing enzymes. To characterize the secretory pathway in cultured astrocytes, we used the neuropeptide processing enzyme carboxypeptidase E (CPE) as a marker for neuropeptide secretion. Cultured astrocytes and AtT-20 cells, a mouse pituitary-derived neuroendocrine cell line, were labeled with [35S]Met for 15 min and then chased with unlabeled Met. CPE was isolated from either medium or cell extracts using a substrate affinity column. The time course of secretion of radiolabeled CPE was significantly different for cultured astrocytes as compared with AtT-20 cells. CPE was rapidly secreted from the astrocytes after a 30-min lag time, presumably reflecting transport through the endoplasmic reticulum and Golgi apparatus, followed by constitutive secretion. The secretion of radiolabeled CPE was essentially complete by 2 h. In contrast, only a portion of the radiolabeled CPE was secreted from AtT-20 cells over a 2-3-h period, indicating that the majority of newly synthesized CPE is stored, presumably in secretory granules within the AtT-20 cells. The regulation of CPE secretion from astrocytes was also examined. CPE secretion is stimulated two- to threefold by prolonged treatment (3-48 h) with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) but not by treatment with other secretagogues that stimulate CPE secretion from AtT-20 cells (forskolin, isoproterenol, A23187, and vasoactive intestinal peptide) or short (less than 3 h) exposure to TPA. Taken together, these results indicate that the secretory pathway for CPE, and presumably neuropeptides, is substantially different in astrocytes than the secretory pathway for CPE in neuroendocrine cells.     

Determination of erythrocyte transit times through micropores. I--Basic operational principles

To study the transit times of each red blood cell passing through cylindrical micropores and in order to evaluate sub-population of cells with regard to their deformability, we have developed a new system called the cell transit time analyser (CTTA). By using an AC voltage (100 KHz) across a special filter, we measure the electrical conductance change produced by the cells passing through the pores under a known driving pressure. This computer based device provides the distribution of transit times tau for 2000 cells in 1 minute and as a result the mean transit time [tau]. Experiments with red cells were designed to evaluate the flow behavior of both normal cells and cells whose mechanical properties were artificially altered. Cell volume was changed by use of non-isotonic media. Cell shape and cell volume were modified by varying the pH of the suspending buffer. Results of these experiments are: 1) a skew distribution of transit times towards high tau values for both control cells and artificially altered cells is observed: 2) [tau] is minimum for isotonic conditions and increases sharply for either hypotonic or hypertonic media: 3) [tau] is minimum at physiological pH and increases for either acid or alcaline changes of pH.     

Ultrasonic cell separator as a cell retaining device for high density cultures of plant cell

A system of ultrasonic filter device consisted of an ultrasonic generator, ultrasonic cell separation chamber (resonator) and a guide column, which was developed for suspension cultures of a plant cell. The key operation parameters affecting the efficiency of separation of cells from medium fluid were found to be the voltage of ultrasonic generator, the convective flow rate, and the distance between transducer and reflector. In the high density cultures ofAloe saponaria (>17 g DCW/L), the ultrasonic filter was so efficient that the cell holding time in the separation chamber was 10-fold higher than the case without ultrasonic wave at a convective flow rate of 0.24 cm/min. Furthermore, in perfusion type of high cell density cultures, cell aggregates were observed to be densely held in the ultrasonic chamber by ultrasonic force overcoming both gravitational and drag forces by pump. The accumulated cells were finally overflowed after the holding capacity of the chamber was reached. Back pressure was applied periodically to the resonator to flush cells back to bioreactor. The ultrasonic cell separator could operate over 75 min at a convective flow rate of 0.1 cm/min and at a cell concentration of 17 g DCW/L.     

The anal glands of Rhapidostreptus virgator (Diplopoda: Spirostreptidae)

Summary In Rhapidostreptus virgator exocrine gland complexes are found in the anal valves of both sexes. Every gland complex consists of about 200 secretory units, each of which is comprised of four cells: two secretory cells, an intermediary cell, and a canal cell. The amount of secretion produced by these glands varies during the intermoult cycle: it is very small in freshly moulted individuals (postmoult phase), at a medial level during the following intermoult phase, and very large in the premoult phase. The secretion may be used to form the excrement clumps and above all to build the moulting chamber.     

Molecular machinery and mechanism of cell secretion

Secretion occurs in all living cells and involves the delivery of intracellular products to the cell exterior. Secretory products are packaged and stored in membranous sacs or vesicles within the cell. When the cell needs to secrete these products, the secretory vesicles containing them dock and fuse at plasma membrane-associated supramolecular structures, called porosomes, to release their contents. Specialized cells for neurotransmission, enzyme secretion, or hormone release use a highly regulated secretory process. Similar to other fundamental cellular processes, cell secretion is precisely regulated. During secretion, swelling of secretory vesicles results in a build-up of intravesicular pressure, allowing expulsion of vesicular contents. The extent of vesicle swelling dictates the amount of vesicular contents expelled. The discovery of the porosome as the universal secretory machinery, its isolation, its structure and dynamics at nanometer resolution and in real time, and its biochemical composition and functional reconstitution into artificial lipid membrane have been determined. The molecular mechanism of secretory vesicle swelling and the fusion of opposing bilayers, that is, the fusion of secretory vesicle membrane at the base of the porosome membrane, have also been resolved. These findings reveal, for the first time, the universal molecular machinery and mechanism of secretion in cells.     

The vesicle-trafficking protein munc18b increases the secretory capacity of mammalian cells

Heterologous protein production in mammalian cells is often challenged by the bottleneck of the secretory machinery, which prevents producer cells from fully exploiting their physiologic capacity in the production of biopharmaceuticals. Recent advances in the understanding of the molecular mechanisms of vesicle trafficking have enabled the identification of key regulators that control the flow of recombinant proteins along the secretory pathway. Here, we report that transgenic expression of Munc18b, a Sec1/Munc18 (SM) protein regulating the fusion of secretory vesicles to the plasma membrane, enhances the secretory capacity of HeLa, HEK-293 and HT-1080 and so increases overall production of different secreted human glycoproteins as well as the titer of lentiviral particles produced in HEK-293-derived helper cells. Targeted interventions in secretory vesicle trafficking by Munc18b is a novel secretion engineering strategy, which harnesses the full secretory capacity of mammalian cells. Secretion engineering is the latest-generation metabolic engineering strategy, which could improve future therapies by increasing the production of biopharmaceuticals by boosting the secretion performance of cell implants in cell therapy initiatives and by raising the production titers of transgenic viral particles used for gene therapy applications.     

The fine structure of the ‘accessory salivary glands’ of the tettigoniid, Homorocoryphus nitidulus

A study has been carried out to determine the anatomy and fine structure of these apparently undescribed glands. Two major types of cell are present: secretory and epithelial. The secretory cells contain a much branched extracellular chamber into which secretion is discharged and from which it is carried to the median cuticle-lined duct by fine cuticular ductules.     

THE MECHANISM OF AQUEOUS HUMOR FORMATION INFERRED FROM CHEMICAL STUDIES ON BLOOD-AQUEOUS HUMOR DYNAMICS

The importance of considering the effect of a possible flow out of the anterior chamber before inferring any mechanism of aqueous humor formation from the relative concentration of a substance in the aqueous humor and plasma under equilibrium conditions has been stressed. Several processes to account for the chemical equilibria between aqueous humor and blood based on the ultrafiltration and secretion hypotheses with a possible simultaneous loss of aqueous humor by flow have been outlined. On the basis of these processes, equations were formulated which would relate the rates of transfer into and out of the anterior chamber to the ratio of concentration of a substance in the aqueous to that in the blood at various intervals after its introduction into the blood. The explanation of equilibrium ratios above and below one for aqueous constituents is made apparent from the mathematical formulations. For each substance tested a determination was made of the best fit when the concentration in the aqueous humor is plotted against time. This fit was obtained by plotting the rate of transfer in against the rate of transfer out of the anterior chamber for all of the experimentally found concentration ratios on the basis of both the ultrafiltration and secretory hypotheses. Two sets of values were obtained from these calculations, one set for each hypothesis. The substantial agreement of all the experimental data with an assumed rate of leakage out of the anterior chamber of approximately 4 c. mm. per minute was shown to be compatible only with the idea that all the monovalent electrolytes tested entered the anterior chamber as a result of secretory process. It could not be decided from these chemical studies whether the non-electrolytes and the one multivalent electrolyte tested enter the anterior chamber by ultrafiltration or secretion. Experimental findings from other sources were cited which would suggest that non-electrolytes enter the anterior chamber as a result of ultrafiltration. The implications of the mechanism outlined in the paper with respect to intraocular pressure have been discussed. Supplementary evidence from the literature has been given in support of the conclusions presented here.     

Development of a novel microperfusion chamber for determination of cell membrane transport properties.

A novel microperfusion chamber was developed to measure kinetic cell volume changes under various extracellular conditions and to quantitatively determine cell membrane transport properties. This device eliminates modeling ambiguities and limitations inherent in the use of the microdiffusion chamber and the micropipette perfusion technique, both of which have been previously validated and are closely related optical technologies using light microscopy and image analysis. The resultant simplicity should prove to be especially valuable for study of the coupled transport of water and permeating solutes through cell membranes. Using the microperfusion chamber, water and dimethylsulfoxide (DMSO) permeability coefficients of mouse oocytes as well as the water permeability coefficient of golden hamster pancreatic islet cells were determined. In these experiments, the individual cells were held in the chamber and perfused at 22 degrees C with hyperosmotic media, with or without DMSO (1.5 M). The cell volume change was videotaped and quantified by image analysis. Based on the experimental data and irreversible thermodynamics theory for the coupled mass transfer across the cell membrane, the water permeability coefficient of the oocytes was determined to be 0.47 micron. min-1. atm-1 in the absence of DMSO and 0.65 microns. min-1. atm-1 in the presence of DMSO. The DMSO permeability coefficient of the oocyte membrane and associated membrane reflection coefficient to DMSO were determined to be 0.23 and 0.85 micron/s, respectively. These values are consistent with those determined using the micropipette perfusion and microdiffusion chamber techniques. The water permeability coefficient of the golden hamster pancreatic islet cells was determined to be 0.27 microns. min-1. atm-1, which agrees well with a value previously determined using an electronic sizing (Coulter counter) technique. The use of the microperfusion chamber has the following major advantages: 1) This method allows the extracellular condition(s) to be readily changed by perfusing a single cell or group of cells with a prepared medium (cells can be reperfused with a different medium to study the response of the same cell to different osmotic conditions). 2) The short mixing time of cells and perfusion medium allows for accurate control of the extracellular osmolality and ensures accuracy of the corresponding mathematical formulation (modeling). 3) This technique has wide applicability in studying the cell osmotic response and in determining cell membrane transport properties.     

A new principle of cell cultivation: No air-liquid interface but gas exchange across a synthetic membrane

Summary A completely liquid-filled growth chamber for axenic cultures ofTetrahymena pyriformis is described; gas exchange is ensured across a synthetic membrane. The chamber may be incorporated into a continuous flow system with inoculation and removal of cell samples under sterile conditions. Initially, the generation time of the cells was slightly prolonged, about 10%, but after some cell doublings decreased to 5%. The capacity of the cells to form food vacuoles (endocytosis) was unaltered during growth in the chamber. The synthetic membrane was highly permeable to O₂ and CO₂; however, cells grown in the chamber contained small refractive granules. The culture chamber permits the culture volume to be varied and it may be used for other protozoa, bacteria, and even tissue culture cells.     

The regulated secretory pathway in CD4(+) T cells contributes to human immunodeficiency virus type-1 cell-to-cell spread at the virological synapse

Direct cell-cell spread of Human Immunodeficiency Virus type-1 (HIV-1) at the virological synapse (VS) is an efficient mode of dissemination between CD4(+) T cells but the mechanisms by which HIV-1 proteins are directed towards intercellular contacts is unclear. We have used confocal microscopy and electron tomography coupled with functional virology and cell biology of primary CD4(+) T cells from normal individuals and patients with Chediak-Higashi Syndrome and report that the HIV-1 VS displays a regulated secretion phenotype that shares features with polarized secretion at the T cell immunological synapse (IS). Cell-cell contact at the VS re-orientates the microtubule organizing center (MTOC) and organelles within the HIV-1-infected T cell towards the engaged target T cell, concomitant with polarization of viral proteins. Directed secretion of proteins at the T cell IS requires specialized organelles termed secretory lysosomes (SL) and we show that the HIV-1 envelope glycoprotein (Env) localizes with CTLA-4 and FasL in SL-related compartments and at the VS. Finally, CD4(+) T cells that are disabled for regulated secretion are less able to support productive cell-to-cell HIV-1 spread. We propose that HIV-1 hijacks the regulated secretory pathway of CD4(+) T cells to enhance its dissemination.