The influence of shrinkage-cracking on the drying behaviour of White Portland cement using Single-Point Imaging (SPI)

We examined membrane fluidity of bovine adrenal chromaffin cells and chromaffin granules using cationic trimethylammonium derivative of diphenylhexatriene (TMA-DPH) as a fluorescence probe. After adding TMA-DPH to the suspension of chromaffin cells and that of granules, it first bound to the outer layer of the plasma membrane of the cells and that of the granule membrane, then gradually penetrated the inner layer of each membrane and distributed to both leaflets of the respective membranes. Accompanying increases in the ratio of incorporated probe on the cytoplasmic side of the chromaffin cell membrane, its fluorescence anisotropy gradually decreased. However, in chromaffin granules, the fluorescence anisotropy gradually increased with increases in the ratio of incorporated probe. These findings suggest that the inner layer of the plasma membrane and outer layer of the granular membrane are more fluid than the corresponding side of each membrane, which is suitable for the fusion between both membranes. We also examined the effect of trichosporin-B-VIa, a fungal ion channel forming alpha-aminoisobutyric acid-containing peptide, on the fluidity of chromaffin cells using TMA-DPH. The peptide decreased the fluorescence anisotropy and increased the fluorescence intensity in the concentration range that induced Ca2+ dependent catecholamine secretion, suggesting that a change in lipid dynamics of the lipid bilayer of the plasma membrane was induced by this peptide.     

Ca2+-triggered peptide secretion in single cells imaged with green fluorescent protein and evanescent-wave microscopy

Green fluorescent protein fused to human chromogranin B or neuropeptide Y was expressed in PC12 cells and caused bright, punctate fluorescence. The fluorescent points colocalized with the endogenous secretory granule marker dopamine beta-hydroxylase. Stimulation of live PC12 cells with elevated [K+], or of permeabilized PC12 cells with Ca2+, led to Ca2+-dependent loss of fluorescence from neurites. Ca2+ stimulated secretion of both fusion proteins equally well. In living cells, single fluorescent granules were imaged by evanescent-wave fluorescence microscopy. Granules were seen to migrate; to stop, as if trapped by plasmalemmal docking sites; and then to disappear abruptly, as if through exocytosis. Evidently, GFP fused to secreted peptides is a fluorescent marker for dense-core secretory granules and may be used for time-resolved microscopy of single granules.     

Evaluation of the electrostatic field strength at the site of exocytosis in adrenal chromaffin cells

Exocytosis in secretory cells consists of release from intracellular storage granules directly into the extracellular space via fusion of the granule membrane with the plasma membrane of the cell. It is considered here as comprising two distinct processes. One is the close apposition of granule and plasma membranes. The other arises from interactions between the two membranes during the process of apposition, leading to the formation of a fusion pore. In the following it is shown for the case of the adrenal medullary chromaffin cell that the fusion pore can be ascribed to electroporation of the granule membrane, triggered by the strong electric field existing at the site of exocytosis. Based on an electric surface charge model of the cytoplasmic side of the plasma membrane, resulting from the negatively charged phosphatidylserine groups, it is found that the electrostatic field strength at the site of exocytosis reaches values on the order of 10(8) V/m at small intermembrane distances of 3 nm and lower. The field strength increases with the size of the disc-shaped plasma membrane region generating the electric field, reaching an approximate limit for a radius of 10 nm, at a surface charge density of 5.4 x 10(-2) C/m2. According to previous experimental evaluations of threshold field strength, this field is sufficiently strong to cause membrane electroporation. This step is a precondition for the subsequent membrane fusion during the ongoing process of apposition, leading to secretion.     

The last few milliseconds in the life of a secretory granule. Docking, dynamics and fusion visualized by total internal reflection fluorescence microscopy (TIRFM)

We have monitored single vesicles (granules) in bovine adrenal chromaffin cells using an optical sectioning technique, total internal reflection fluorescence microscopy (TIRFM). With TIR, fluorescence excitation is limited to an optical slice near a glass/water interface. In cells located at the interface, granules loaded with fluorescent dye can be visualized near to or docked at the plasma membrane. Here we give evidence that (1) TIRFM resolves single vesicles and (2) the fluorescence signal originates from vesicles of roughly 350 nm diameter, presumably large dense core vesicles (LDCVs). (3) Diffusional spread of released vesicle contents can be resolved and serves as a convenient criterion for a fusion event. (4) We give details on vesicle properties in resting cells, such as lateral mobility of chromaffin granules, number density, and frequency of spontaneous fusion or withdrawal into the cytoplasm. (5) Upon stimulation with high extracellular potassium, TIRFM reports depletion of the 'visible pool' of vesicles closest to the plasma membrane within hundreds of milliseconds, consistent with previous concepts of a release-ready pool. We conclude that TIRFM constitutes an independent assay for pool depletion. TIRFM will allow us to study aspects of secretion that have previously been inaccessible in living cells, in particular the spatial relations and dynamics of vesicles prior to and during exocytosis and re-supply of the near-membrane pool of vesicles.     

Neuronal Ca2+ sensor 1, the mammalian homologue of frequenin, is expressed in chromaffin and PC12 cells and regulates neurosecretion from dense-core granules

Neuronal Ca2+ sensor 1 (NCS-1) is the mammalian homologue of the Ca2+-binding protein frequenin previously implicated in regulation of neurotransmission in Drosophila (Pongs, O., Lindemeier, J., Zhu, X. R., Theil, T., Endelkamp, D., Krah-Jentgens, I., Lambrecht, H.-G., Koch, K. W., Schwemer, J., Rivosecchi, R., Mallart, A., Galceran, J. , Canal, I., Barbas, J. A., and Ferrus, A. (1993) Neuron 11, 15-28). NCS-1 has been considered to be expressed only in neurons, but we show that NCS-1 expression can be detected in bovine adrenal chromaffin and PC12 cells, two widely studied model neuroendocrine cells. NCS-1 was present in both cytosolic and membrane fractions including purified chromaffin granules, and in immunofluorescence, its distribution overlapped with peripheral punctate staining seen with the synaptic-like microvesicle marker synaptophysin in PC12 cells. The possible functional role of NCS-1 in exocytosis of dense-core granules was tested using transient transfection in PC12 cells and assay of co-transfected growth hormone (GH) release. Overexpression of NCS-1 increased evoked GH release in intact cells in response to ATP. No effect of overexpression was seen on GH release because of Ca2+ in permeabilized cells suggesting that NCS-1 may have a regulatory but not direct role in neurosecretion.     

Degranulation of chicken heterophil leucocytes during phagocytosis, studied by phase contrast and interference microscopy

The dynamic aspects of degranulation of chicken heterophil leucocytes during phagocytosis have been studied by phase contrast and interference microscopy. Both standard (8 fps) and high-speed (400 fps) cine-photomicrographic recordings of this process under phase contrast are presented. Lysis of individual granules is usually completed in less than 60 milliseconds. During lysis of each granule a rounded phase dense body is ejected into the phagocytic vacuole. Measurements made by interference microscopy show that there is usually a substantial fall from a protein concentration of c. 100 per cent. w/v for intact granules to a concentration of 16 per cent. w/v for the vacuole resulting from their lysis; this can only be explained by a rapid intake of water into the granule matrix following membrane fusion. The intake of water that accompanies granule lysis causes swelling of the granule matrix, and is thought to explain the mechanism of ejection of the phase dense body. Granule lysis is not dependent on the intake of water, since occasional vacuoles have been observed which showed no fall in protein concentration relative to the intact granules. The membrane around the intact granule effectively excludes the entry of water into the concentrated hygroscopic granule matrix, but once membrane fusion occurs this barrier to the entry of water is usually lost.     

Brefeldin A inhibits the constitutive-like secretion of a sulfated protein in pancreatic acinar cells

Sulfation is a common posttranslational modification of secretory proteins and serves as a valuable marker of constitutive and regulated secretory pathways. We investigated the cellular localization and the secretory behavior of sulfated macromolecules in the mouse pancreatic acinar cell. The major sulfated proteins of the cell were present in isolated zymogen granules, as determined by metabolic labeling with [35S]sulfate and subcellular fractionation. The sulfated proteins fell into three groups: gp300 is not secreted and is a component of the zymogen granule membrane; pancreatic lipase (56 kDa) and a 40 kDa protein are soluble and exhibit regulated secretion kinetics; and p82 is initially granule membrane associated, but is released from the cell with constitutive-like kinetics as a 75 kDa protein (p75). Secretion of p75 could be stimulated for up to 4 h after pulse labeling, presumably from immature secretory granules, but not after 6 h of chase. Treatment of cells with brefeldin A (BFA) at the start of the [35S]sulfate pulse resulted in almost total inhibition of sulfation. Addition of BFA during the chase (0-2 h) allowed normal basal and stimulated secretion of regulated secretory proteins, but reversibly inhibited the constitutive-like secretion of p75. In this case, the behavior of p75 was maintained as that of a regulated secretory protein for up to 6 h of chase. In untreated cells, immunofluorescence of p82/p75 was along the acinar lumen, and in small punctate structures in the apical cytoplasm. In BFA-treated cells, immunolabeling of p82/p75 was lost from the acinar lumen, and cytoplasmic labeling was finer and appeared to be associated with the secretory granule membranes. These data suggest a role for brefeldin A-sensitive coat formation in maturation of secretory granules after they bud from the TGN.     

Lack of the response to nicotine in 21-day-old rat adrenal chromaffin cells in vivo

Response to nicotine of adrenal chromaffin cells was studied in suckling and young adult male rats in vivo. When 5 mg/kg of nicotine was injected subcutaneously to 8-week-old rats, the content of adrenaline and noradrenaline in the chromaffin granule fraction decreased about by 36 and 45%, respectively, 2 min after the administration. In electron microscopy, the number of chromaffin granules in the perinuclear region of adrenaline-storing cells decreased markedly. The number of vacuoles, probably produced by membrane recycling resulting from exocytosis, increased significantly in adrenaline- and noradrenaline-storing cells. Omega-shaped profiles (exocytosis) were frequently observed both in adrenaline- and noradrenaline-storing cells. On the other hand, nicotine injection did not significantly alter the catecholamine content in the 21-day-old rat chromaffin granule fraction, although severe convulsion was evoked. In electron microscopy, the changes indicative of exocytosis mentioned above were scarcely observed. Cholinergic nerve fibers of mature appearance were observed in the adrenal medulla of 21-day-old rats. These results indicate that the responsiveness of the chromaffin cells to nicotine in 21-day-old rats differs from that in 8-week-old rats.     

Estimation of mean exocytic vesicle capacitance in mouse adrenal chromaffin cells

Whole-cell membrane capacitance measurements are frequently used to monitor neuronal and nonneuronal secretory activity. However, unless individual fusion events can be resolved, the type of the fusing vesicles cannot be identified in these experiments. Here we apply statistical analysis of trial-to-trial variations between depolarization-induced capacitance increases of mouse adrenal chromaffin cells and obtain estimates for the capacitance contribution of individual exocytic vesicles between 0.6 and 2 fF. For comparison, measurements of membrane capacitance were combined with amperometric recordings of catecholamine release during intracellular perfusion of chromaffin cells with high [Ca2+]. Crosscorrelation of both signals yielded a mean capacitance contribution of individual catecholaminergic vesicles of 1.3 fF. We suggest that depolarization-induced capacitance increases in mouse adrenal chromaffin cells mainly represent fusion of chromaffin granules.     

Oxidoreductase activities of chromaffin granule ghosts isolated from the bovine adrenal medulla

1. Based on estimated s-values of subpopulations of bovine adrenal chromaffin granules (B?dtker-Naess, V., Slinde, E., Terland, O. and Flatmark, T. (1978) Biochim. Biophys. Acta 541, 124--134) a new large-scale procedure is described for the isolation of the total population of chromaffin granules by differential centrifugation in 0.25 M sucrose. 2. Using the total population of chromaffin granules obtained by differential centrifugation, final purification was achieved by density-gradient centrifugation in either sucrose or Percoll-sucrose. In either case, the isolated granule fractions were contaminated with mitochondria to about the same degree. 3. Chromaffin granule ghosts, obtained by hypoosmotic lysis of granules isolated by sucrose density-gradient, centrifugation, were subjected to centrifugation on a discontinuous density gradient (buffer/0.9 M sucrose). By this procedure a substantial purification of the ghosts was achieved as determined from measurements of protein and various marker enzymes. 4. In contrast to preparations of chromaffin granule ghosts prepared by previous standard procedures, those purified by gradient centrifugation (on 0.9 M sucrose) did not reveal any NADH-linked cytochrome b-561 reductase activity. However, experimental evidence is presented for the existence of an intrinsic NADH-oxidizing enzyme system in the granule membrane. 5. No significant difference was observed in the specific content of cytochrome b.561 and NADH:(acceptor) oxidoreductase activities between ghost preparations obtained from populations of heavy and light chromaffin granules. 6. The functional significance of cytochrome b-561 and the NADH:(acceptor) oxidoreductase activities of the granule membrane remains to be determined.     

Identification and characterization of a novel 9.2-kDa membrane sector-associated protein of vacuolar proton-ATPase from chromaffin granules

Vacuolar proton-translocating ATPase (holoATPase and free membrane sector) was isolated from bovine chromaffin granules by blue native polyacrylamide gel electrophoresis. A 5-fold excess of membrane sector over holoenzyme was determined in isolated chromaffin granule membranes. M9.2, a novel extremely hydrophobic 9.2-kDa protein comprising 80 amino acids, was detected in the membrane sector. It shows sequence and structural similarity to Vma21p, a yeast protein required for assembly of vacuolar ATPase. A second membrane sector-associated protein (M8-9) was identified and characterized by amino-terminal protein sequencing.     

Quantitative analysis of the movements of cytoplasmic granules in polarized fibroblasts

Movements of cytoplasmic organelles were analyzed in Vero fibroblasts. In the cells polarized at the edge of an experimental wound, cytoplasmic granules moved randomly (Brownian motions) and by separate jumps (saltatory movements). The displacement of granules by the Brownian motions exceeded by more than an order of magnitude that of the mitochondria similar by weight. Lipid droplets moved predominantly by saltations, whereas mitochondria and lysosomes moved much less often. In a front part of the polarized cells, the main directions of saltatory movements were from the nucleus to the leading edge of a cell and back, whereas the tangential movements (across the long axis of a cell) were less than 1%. 90% of saltatory movements occurred in the area starting 10-12 microm from the nucleus and ending 10-12 microm from the leading edge of a cell. The average rate of saltatory movements of the granules (2.38 microm/s) was identical in both directions. The average length of the track was 7.49 microm; the maximum track length reached 30 microm. An increase in the granule diameter from 0.3 to 1.4 microm resulted in a minor (statistically insignificant) decrease in the average rate of the movements. The average rate of saltatory movements of mitochondria was 1.00 microm/s, and the average track length was 6.04 microm. Therefore, mitochondria, in contrast to lipid droplets, are rigidly fixed in the cytoplasm, and the force holding mitochondria is equal to the force produced by the microtubule-associated motors. Taking into account the characteristic of the centrifugal saltations, we suggest that they are mediated by an unusual dynein.     

Cortical granule exocytosis is triggered by different thresholds of calcium during fertilisation in sea urchin eggs

In sea urchin eggs, fertilisation is followed by a calcium wave, cortical granule exocytosis and fertilisation envelope elevation. Both the calcium wave and cortical granule exocytosis sweep across the egg in a wave initiated at the point of sperm entry. Using differential interference contrast (DIC) microscopy combined with laser scanning confocal microscopy, populations of cortical granules undergoing calcium-induced exocytosis were observed in living urchin eggs. Calcium imaging using the indicator Calcium Green-dextran was combined with an image subtraction technique for visual isolation of individual exocytotic events. Relative fluorescence levels of the calcium indicator during the fertilisation wave were compared with cortical fusion events. In localised regions of the egg, there is a 6s delay between the detection of calcium release and fusion of cortical granules. The rate of calcium accumulation was altered experimentally to ask whether this delay was necessary to achieve a threshold concentration of calcium to trigger fusion, or was a time-dependent activation of the cortical granule fusion apparatus after the 'triggering' event. Calcium release rate was attenuated by blocking inositol 1,4,5-triphospate (InsP3)-gated channels with heparin. Heparin extended the time necessary to achieve a minimum concentration of calcium at the sites of cortical granule exocytosis. The data are consistent with the conclusion that much of the delay observed normally is necessary to reach threshold concentration of calcium. Cortical granules then fuse with the plasma membrane. Further, once the minimum threshold calcium concentration is reached, cortical granule fusion with the plasma membrane occurs in a pattern suggesting that cortical granules are non-uniform in their calcium sensitivity threshold.     

Identification of a potential effector pathway for the trimeric Go protein associated with secretory granules. Go stimulates a granule-bound phosphatidylinositol 4-kinase by activating RhoA in chromaffin cells

Besides having a role in signal transduction, heterotrimeric G proteins may be involved in membrane trafficking events. In chromaffin cells, Go is associated with secretory organelles, and its activation inhibits the ATP-dependent priming of exocytosis. By using permeabilized cells, we previously described that the control exerted by the granule-bound Go on exocytosis may be related to effects on the cortical actin network through a sequence possibly involving Rho. To provide further insight into the function of Rho in exocytosis, we focus here on its intracellular localization in chromaffin cells. By immunoreplica analysis, immunoprecipitation, and confocal immunofluorescence, we found that RhoA is specifically associated with the membrane of secretory chromaffin granules. Parallel subcellular fractionation experiments revealed the occurrence of a mastoparan-stimulated phosphatidylinositol 4-kinase activity in purified chromaffin granule membranes. This stimulatory effect of mastoparan was mimicked by GAP-43, an activator of the granule-associated Go, and specifically inhibited by antibodies against Galphao. In addition, Clostridium botulinum C3 exoenzyme completely blocked the activation of phosphatidylinositol 4-kinase by mastoparan. We propose that the control exerted by Go on peripheral actin and exocytosis is related to the activation of a downstream RhoA-dependent phosphatidylinositol 4-kinase associated with the membrane of secretory granules.     

Structure of the human sensorimotor system. I: Morphology and cytoarchitecture of the central sulcus

Chromaffin cells grafted to the brain of animals with experimental parkinsonism and patients with Parkinson's disease can restore nigrostriatal functions. Mechanisms underlying these beneficial effects are unknown, but may include growth factors rather than the minute amounts of dopamine (DA) liberated from chromaffin cells. We now report that protein from chromaffin granules, which release their contents by exocytosis, promotes survival and uptake of 3H-DA of mesencephalic DAergic neurons in vitro and protect against N-methylpyridinium ion toxicity. This neurotrophic effect is accompanied by cell proliferation and mediated by astroglial cells induced in these cultures. Inhibition of cell proliferation and concomitant astrogliosis by 5-fluorodeoxyuridine and alpha-aminoadipic acid abolishes the trophic effect. Two highly specific inhibitors of the epidermal growth factor receptor (EGFR) signal transduction pathway, 4,5-dianilinophthalimide (10 microM) and tyrphostin B56 (10 microM), selectively block the neurotrophic capacity of chromaffin granule protein. As expected, they also block the mitogenic effects of EGF and TGF-alpha. However, these two mitogens do not mimic the pronounced mitogenic and trophic actions of chromaffin granule protein. Culture medium conditioned by mesencephalic cells pretreated with chromaffin granule protein promotes survival of DAergic neurons without increasing numbers of astroglial cells. The effective molecule is unlikely to be glial cell line-derived neurotrophic factor, whose mRNA is not detectable in cultures treated with chromaffin granule protein. We conclude that chromaffin granules contain a putatively novel growth factor, which signals through the EGFR and may be responsible for the known protective and restorative actions of chromaffin cell grafts to the lesioned nigrostriatal system.     

Exocytotic competence and intergranular fusion in cord blood-derived eosinophils during differentiation

We studied degranulation of single cord blood-derived mononuclear cells differentiating to eosinophils in cultures containing recombinant human interleukin-5 (rhIL-5) and rhIL-3 by whole-cell patch-clamp capacitance measurements. As in mature cells, degranulation can be stimulated by intracellular application of guanosine-5'-O-(3-thiotriphosphate) (GTP) gamma S after 10 days in culture, simultaneously with the first morphological appearance of granules. These results demonstrate that the fusion machineries for exocytotic fusion are present and functional as soon as the granules are formed, presumably at the myeloblast stage. In the third week, the total amount of granules exocytosed upon stimulation is similar to that in mature eosinophils from peripheral blood. The capacitance step size distributions in promyelocytes and myelocytes confirm that mature large specific granules are formed by homotypic fusion of unit granules with similar size. Homotypic fusion is facilitated during early stages of differentiation associated with granulogenesis. Between day 10 and day 35 in culture the plasma membrane area of resting cells decreases from approximately 700 microns2 to approximately 400 microns2, approaching the value of mature cells from peripheral blood. The most prominent decrease occurs between day 25 and day 35 and is accompanied by the appearance of an exocytotic component due to small vesicles. This suggests that a class of small secretory vesicles is formed by endocytosis during a late phase in maturation.     

Segregation of nitric oxide synthase expression and calcium response to nitric oxide in adrenergic and noradrenergic bovine chromaffin cells

Previous work has demonstrated that nitric oxide can be an important intracellular messenger in the regulation of neurosecretion in chromaffin cells. Since standard chromaffin cell cultures are mixed populations of noradrenaline and adrenaline producing cells, it would seem important to understand the functional differences between these individual components. The use of fluorescence imaging techniques for the recording of cytosolic calcium from single chromaffin cells together with the immunoidentification of individual cells with specific antibodies against tyrosine hydroxylase, N-phenyl ethanolamine methyl transferase and nitric oxide synthase, has allowed us to measure single-cell calcium responses in identified adrenergic, noradrenergic and nitrergic chromaffin cells, thus helping us to clarify the differential role of nitric oxide in the function of these chromaffin cell types. 53 +/- 2% of chromaffin cells were able to synthesize nitric oxide (nitric oxidesynthase-positive cells), these cells being mainly noradrenergic (82 +/-2%). Results indicate that nitric oxide donors such as sodium nitroprusside, molsidomine and isosorbide dinitrate evoke [Ca2+]i increases in a 62 +/- 4% of chromaffin cells, the response to nitric oxide donors being between 30 and 50% of that of 20 microM nicotine. Cells responding to nitric oxide donors were mainly adrenergic (68 +/- 5%) although 45 +/- 9% of noradrenergic cells also gave [Ca2+]i increasing responses. The distribution of nitric oxide responding cells between nitric oxide synthase-positive and negative was very similar in the whole population (63 +/- 5 and 60 +/- 7%, respectively), but these differences were more prominent when considering the distribution of nitric oxide response between noradrenergic and adrenergic nitric oxide synthase-positive cells; while 73 6% of adrenergic nitric oxide synthase-positive cells evoke [Ca2+]i increases by nitric oxide stimulation, only 35 +/- 11% of noradrenergic nitric oxide synthase-positive cells respond. Taken together these results seem to indicate that (i) nitric oxide could act within adrenal medulla as both an intracellular and intercellular messenger; and (ii) noradrenergic cells seem to be specialized in nitric oxide synthesis while adrenergic cells with an endocrine function could mainly act as a target of neurosecretory action of this second messenger.     

Fusion of azurophil granules with phagosomes and activation of the tyrosine kinase Hck are specifically inhibited during phagocytosis of mycobacteria by human neutrophils

Pathogenic mycobacteria parasitize macrophages and reside within phagosomes, which do not fuse with lysosomal granules. Mycobacteria are also internalized by neutrophils, which possess at least two types of granules, specific and azurophil granules, the latter being specialized lysosomes. Here, we investigated the ability of mycobacteria to inhibit the fusion of these granules with their phagosomes in human neutrophils. It was found that when pathogenic (Mycobacterium kansasii and Mycobacterium avium) or nonpathogenic (Mycobacterium smegmatis and Mycobacterium phlei) mycobacteria were internalized by neutrophils, they induced the inhibition of azurophil granule fusion with phagosomes even when they were serum opsonized. In contrast, secretion of specific granule content and production of O2-, both of which contribute to the neutrophil bactericidal response, were triggered. Hck is a Src family tyrosine kinase associated with azurophil granules. During internalization of zymosan, azurophil granules fused with phagosomes and Hck was activated and translocated to the phagosomal membrane, whereas in neutrophils engulfing mycobacteria, Hck did not translocate and remained unactivated. The activation of the tyrosine kinase Fgr was not affected. These results indicate that 1) pathogenic and nonpathogenic mycobacteria trigger similar bactericidal responses in neutrophils, 2) phagocytosis and fusion of azurophil granules can be uncoupled by mycobacteria, and 3) Hck could be one of the key elements of the azurophil secretory pathway that are altered during phagocytosis of mycobacteria.     

Kinetic and allosteric cooperativity in L-adenosine transport in chromaffin cells. A mnemonical transporter

In cultured chromaffin cells and plasma membrane vesicles from chromaffin tissue, the transport of D-[3H]adenosine followed Michaelis-Menten saturation kinetics, with Km values of 1.5 +/- 0.3 microM and 1.9 +/- 0.2 microM, respectively. The transport of the isomer, L-[3H]adenosine, showed sigmoidal kinetics in both preparations. In plasma membrane vesicles the S0.5 was 2.5 +/- 0.2 microM with a Hill coefficient of 2.8 and the Vmax value of 0.26 +/- 0.01 pmol s-1 (mg of protein)-1. In cultured chromaffin cells the kinetic parameters for L-[3H]adenosine were S0.5 = 6.2 +/- 0.2 microM and a Vmax 19.7 +/- 0.5 pmol/min per 10(6) cells, with a pronounced positive cooperativity. The Hill coefficient was 4.9. The transport of the L-isomer in cultured cells followed Michaelis-Menten kinetics at the lowest concentrations employed, below 2 microM. On the basis of these results, we propose a kinetic model whereby the adenosine transporter functions mnemonically.     

Reticulospinal actions on primary afferent depolarization of cutaneous and muscle afferents in the isolated frog neuraxis

The disposition and orientation of mouse ductin (the subunit c of the vacuolar H(+)-ATPase) in gap junctions has been examined. Like the Nephrops norvegicus (arthropod) form, mouse ductin in the intact junctional structure is resistant to high levels of nonspecific proteinase, suggesting that it is for the most part buried in the bilayer. Antisera to an octapeptide near the N-terminus cross-react with ductins in gap junction preparations from four different mouse tissues, from chicken and Xenopus laevis liver, and from N. norvegicus hepatopancreas. The antisera and antibodies, affinity purified against the octapeptide, agglutinate isolated gap junctions, suggesting that the N-terminus is located on the exposed surface, equivalent to the cytoplasmic face of an intercellular gap junction. The antibodies also block dye coupling when injected into cells in culture, confirming the cytoplasmic location of the epitope. The lipophylic reagent dicylohexyl carbodiimide (DCCD), which targets carboxyl groups within the membrane and selectively reacts with ductin in N. norvegicus gap junction preparations, rapidly inhibits junctional communication. Bafilomycin A1, which inhibits V-ATPase and stops vacuolar acidification, does not affect dye coupling, showing that the inhibition seen with antibodies and DCCD is not an indirect consequence of their action on the ductin of V-ATPase. Consistent with this interpretation the anti-peptide antibodies do not bind to intact chromaffin granules or inhibit their V-ATPase activity, but do bind to osmotically disrupted granule membrane. This suggests that ductin has an orientation (N-terminus pointing away from the cytoplasm) in the vacuolar membrane opposite to that in the gap junction membrane.