Using nanoparticle optics assay for direct observation of the function of antimicrobial agents in single live bacterial cells

Multidrug resistance (MDR) has been reported in both prokaryotes and eukaryotes, underscoring the challenge of design and screening of more efficacious new drugs. For instance, the efflux pump of Pseudomonas aeruginosa (gram-negative bacteria) can extrude a variety of structurally and functionally diverse substrates, which leads to MDR. In this study, we present a new platform that studies modes of action of antibiotics in living bacterial cells (P. aeruginosa), in real-time, at nanometer scale and single-cell resolution using nanoparticle optics and single living cell imaging. The color index of silver (Ag) nanoparticles (violet, blue, green, and red) is used as the sized index (30 +/- 10, 50 +/- 10, 70 +/- 10, and 90 +/- 10 nm) for real-time measurement of sized transformation of the cell wall and membrane permeability at the nanometer scale. We have demonstrated that the number of Ag nanoparticles accumulated in cells increases as the aztreonam (AZT) concentration increases and as incubation time increases, showing that AZT induces the sized transformation of membrane permeability and the disruption of the cell wall. The results demonstrate that nanoparticle optics assay can be used as a new powerful tool for real-time characterization of modes of action of antimicrobial agents in living cells at the nanometer scale. Furthermore, studies of mutants of WT bacteria (nalB-1 and DeltaABM), suggest that an efflux pump (MexA-MexB-OprM) effectively extrudes substrates (nanoparticles) out of the cells, indicating that the MDR mechanism involves the induction of changes in membrane permeability and the intrinsic pump machinery.     

Fluorescence study of lectinlike receptors involved in the flocculation of the yeast Saccharomyces cerevisiae.

Flocculation of some yeasts involves lectinlike receptors with two different patterns of inhibition by sugars: mannose sensitive (MS) and glucose-mannose sensitive (GMS). The visualization and quantification of these receptors were performed using neoglycoproteins fluorescent probes. Fluorescence microscopy showed a homogeneous distribution of surface receptors for the strain belonging to the MS group and a polar distribution for cells belonging to the GMS group. Affinity constants, estimated by fluorimetry, were shown to have different values (MS, 2.6 +/- 0.7 x 10(5) M-1; GMS, 2 +/- 1 x 10(6) M-1), but the number of sites was estimated to be smaller for strain NCYC 1195 which belongs to the GMS group than for strain NCYC 869 from the MS group (MS, 2.4 +/- 0.2 x 10(7) sites/cell; GMS, 3.9 +/- 0.8 x 10(6) sites/cell).     

Biologically synthesized fluorescent CdS NPs encapsulated by PHB

Here an attempt was made to biologically synthesize fluorescent cadmium sulfide nanoparticles and to immobilize the synthesized nanoparticles in PHB nanoparticles. The present study uses Brevibacterium casei SRKP2 as a potential producer for the green synthesis of CdS nanoparticles. Biologically synthesized nanoparticles were characterized and confirmed using electron microscopy and XRD. The size distribution of the nanoparticles was found to be 10-30 nm followed by which the consequence of time, growth of the organism, pH, concentration of CdCl(2) and Na(2)S on the synthesis of nanoparticles were checked. Enhanced synthesis and fluorescence emission of CdS nanoparticles were achieved at pH 9. The synthesized CdS NPs were immobilized with PHB and were characterized. The fluorescent intensity of the CdS nanoparticles remained unaffected even after immobilization within PHB nanoparticles.     

Intrinsically fluorescent luteinizing hormone receptor demonstrates hormone-driven aggregation

The possibility that LH receptors exist as isolated molecules when unbound and aggregate upon binding gonadotropins has previously been untestable in viable cells for want of a suitable nonhormone probe. We have now expressed in CHO cells an intrinsically-fluorescent LH receptor involving enhanced green fluorescent protein (GFP) fused to the C-terminus of the rat LH receptor (rLHR-GFP). More than half of these receptors (54 +/- 4%) are located on the plasma membrane and are functional: cAMP levels increase 3-5 fold in response to 10 nM LH or hCG. In fluorescence photobleaching recovery studies at 37 degrees C, 54 +/- 13% of unoccupied rLHR-GFP were laterally mobile with a diffusion coefficient D of 16 +/- 3.5 x 10(-10)cm2sec-1. Introduction of 10 nM LH for 1 h slowed receptor lateral diffusion to 6.6 +/- 1.3 x 10(-10)cm2sec-1 and reduced fluorescence recovery after photobleaching to 27 +/- 1%. Following treatment with 1 nM hCG, rLHR-GFP were laterally immobile and were distributed into small fluorescent patches over the cell surface. Thus, unoccupied rLHR-GFP receptors apparently exist as dispersed plasma membrane proteins with comparatively fast lateral diffusion. Interaction of receptors with LH or hCG caused clustering of rLHR-GFP receptors, significantly restricting lateral diffusion.     

Developmental peculiarities of short-term nongenomic effect of aldosterone on intracellular sodium concentration in rat distal nephron

Intracellular sodium concentration in CCD fragments micro-dissected from the kidneys of 10-day and adult rats using fluorescent dye Na+ Green, was studied. The steady state level of intracellular sodium was lower in fragments from amiloride kidney (24.50 +/- 1.3 and 35.3 +/- 4.9 mM, n = 8, respectively). Amiloride reduced this parameter but, in the epithelia cells from immature kidney, the effect of imaloride was less pronounced. Fast nongenomic action of aldosterone on intracellular sodium concentration was found in both groups. Aldosterone significantly raised the steady state sodium level in epithelial cells in conditions of low sodium concentration (14 mM) in outer medium in both type of CCD fragments: from 10-day pups and adult animals (4.0 +/- 1, 5.5 +/- 0.5 and 5.1 +/- 0.2, 7.9 +/- 0.2 mM, n = 8). We suggest that, along with the well-known mineral-corticoid effect, aldosterone takes part in regulation of cell volume in CCD rat kidney from the earliest stages of postnatal ontogenesis.     

Alteration of deposition pattern and pulmonary response as a result of improved dispersion of aspirated single-walled carbon nanotubes in a mouse model

Nanoparticles have a fundamental dimension of <100 nm. However, on suspension in media, agglomerates of nanoparticles are the more common structure. This is particularly evident in prior intratracheal instillation or aspiration studies of single-walled carbon nanotubes (SWCNT), in which granulomatous lesions encased by epithelioid macrophages were produced by large agglomerates. In this study, we tested the hypothesis of whether exposure to more dispersed SWCNT structures would alter pulmonary distribution and response. A dispersed preparation of single-walled carbon nanotubes (DSWCNT) with a mean diameter of 0.69 microm was given by pharyngeal aspiration to C57BL/6 mice. Electron microscopy demonstrated a highly dispersed, interstitial distribution of DSWCNT deposits by 1 day postexposure. Deposits were generally <1 microm. Macrophage phagocytosis of DSWCNT was rarely observed at any time point. Lung responses were studied by lavage and morphometry at 1 h, 1 day, 7 day, and 1 mo after a single DSWCNT exposure of 10 microg/mouse. Lung sections and lavage cells demonstrated an early, transient neutrophilic and inflammatory phase that rapidly resolved and was similar to that observed with large agglomerates. No granulomatous lesions or epithelioid macrophages were detected. Morphometric measurement of Sirius red staining was used to assess the connective tissue response. The average thickness of connective tissue in alveolar regions was 0.10 +/- 0.02, 0.09 +/- 0.02, 0.10 +/- 0.01, 0.48 +/- 0.04, and 0.88 +/- 0.19 microm for PBS and 1-h, 1-day, 7-day, and 1-mo postexposure groups, respectively. The results demonstrate that dispersed SWCNT are rapidly incorporated into the alveolar interstitium and that they produce an increase in collagen deposition.     

Single-particle tracking of oriC-GFP fluorescent spots during chromosome segregation in Escherichia coli

DNA regions close to the origin of replication were visualized by the green fluorescent protein (GFP)-Lac repressor/lac operator system. The number of oriC-GFP fluorescent spots per cell and per nucleoid in batch-cultured cells corresponded to the theoretical DNA replication pattern. A similar pattern was observed in cells growing on microscope slides used for time-lapse experiments. The trajectories of 124 oriC-GFP spots were monitored by time-lapse microscopy of 31 cells at time intervals of 1, 2, and 3 min. Spot positions were determined along the short and long axis of cells. The lengthwise movement of spots was corrected for cell elongation. The step sizes of the spots showed a Gaussian distribution with a standard deviation of approximately 110 nm. Plots of the mean square displacement versus time indicated a free diffusion regime for spot movement along the long axis of the cell, with a diffusion coefficient of 4.3+/-2.6x10(-5) microm2/s. Spot movement along the short axis showed confinement in a region of the diameter of the nucleoid ( approximately 800 nm) with an effective diffusion coefficient of 2.9+/-1.7x10(-5) microm2/s. Confidence levels for the mean square displacement analysis were obtained from numerical simulations. We conclude from the analysis that within the experimental accuracy--the limits of which are indicated and discussed--there is no evidence that spot segregation requires any other mechanism than that of cell (length) growth.     

The osmotic characteristics of human fetal liver-derived hematopoietic stem cell candidates

Hematopoietic stem cells derived from fetal liver have promising therapeutic potential for allotransplantation but require a specific protocol to minimize the damage produced by cryopreservation procedures. In this study, a fundamental approach was applied for designing a cell preservation protocol. To this end, the biophysical characteristics that describe the osmotic reaction of CD34(+)CD38(-) human fetal liver stem cell candidates were studied using fluorescent microscopy. The osmotically inactive volume of the stem cell candidates was determined as 48% of the isotonic volume. The permeability coefficients for water and Me(2)SO were determined at T = +22 degree C: L(p) = 0.27 +/- 0.03 microm x min(-1)atm(-1), P(Me(2)SO)) = 2.09 +/- 0.85 x 10 (-4) cm x min(-1), sigma (Me(2)SO)) = 0.63 +/- 0.03 and at T = +12 degree C: L(p) = 0.15 +/-0.02 microm x min(-1)atm(-1), P(Me(2)SO)) = 6.44 +/-1.42 x 10 (-5) cm x min(-1), sigma (Me(2)SO)) = 0.46 +/- 0.05. The results obtained suggest that post-hypertonic and hypotonic stress are the possible reasons for damage to a CD34(+)CD38(-) cell during the cryopreservation procedure.     

Use of a trapped fluorescent indicator to demonstrate effects of thyroliberin and dopamine on cytoplasmic calcium concentrations in bovine anterior pituitary cells

The fluorescent calcium indicator 'quin2' was used to demonstrate changes in cytoplasmic calcium concentrations in bovine anterior pituitary cells. The basal calcium concentration was 0.21 +/- 0.02 microM (mean of 4 cell preparations). Thyroliberin (TRH) (10(-10) - 10(-6) M) rapidly and at the higher concentrations transiently increased the concentration. Dopamine (10(-10) - 10(-7) M) decreased the concentration transiently and more slowly. At 10(-5) M, dopamine prevented the increase in calcium concentration caused by 10(-9) M TRH, and partially inhibited the increase caused by higher concentrations of the peptide. The data support the hypothesis that calcium is the second messenger for TRH, and suggest that dopamine inhibits TRH-induced prolactin secretion by preventing the calcium concentration from exceeding the level necessary to increase secretion.     

Inhaled corticosteroid effects both eosinophilic and non-eosinophilic inflammation in asthmatic patients

AIM: To determine induced sputum cell counts and interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha) and leukotriene B4 (LTB4) levels as markers of neutrophilic inflammation in moderate persistent asthma, and to evaluate the response to inhaled steroid therapy. METHODS: Forty-five moderate asthmatic patients and 10 non-smoker controls were included in this study. All patients received inhaled corticosteroid (800 microg of budesonide) for 12 weeks. Before and after treatment pulmonary function tests were performed, and symptom scores were determined. Blood was drawn for analysis of serum inflammatory markers, and sputum was induced. RESULTS: Induced sputum cell counts and inflammatory markers were significantly higher in patients with asthma than in the control group. The induced sputum eosinophil counts of 12 patients (26%) were found to be less than 5%, the non-eosinophilic group, and sputum neutrophil counts, IL-8 and TNF-alpha levels were significantly higher than the eosinophilic group (neutrophil, 50+/-14% versus 19+/-10%, p<0.01). In both groups, there was a significant decrease in sputum total cell counts and serum and sputum IL-8, TNF-alpha and LTB4 levels after the treatment. There was no change in sputum neutrophil counts. Although the sputum eosinophil count decreased only in the eosinophilic subjects, there was no significant difference in inflammatory markers between the groups. The symptom scores were significantly improved after treatment, while the improvement did not reach statistical significance on pulmonary function test parameters. CONCLUSION: Notably, in chronic asthma there is a subgroup of patients whose predominant inflammatory cells are not eosinophils. Sputum neutrophil counts and neutrophilic inflammatory markers are significantly higher in these patients. In the non-eosinophilic group, inhaled steroid caused an important decrease in inflammatory markers; however, there was no change in the sputum eosinophil and neutrophil counts.     

Cilostazol suppresses adhesion of human neutrophils to HUVECs stimulated by FMLP and its mechanisms

The interaction between neutrophils and endothelial cells (ECs) is of great importance in many physiological and pathological progresses. Although cilostazol (CLZ), a novel selective phosphodiesterase (PDE) type 3 inhibitor, has been proved to be useful in vasodilatation and inhibition of platelet aggregation, its effect on adhesion is not clearly known. In this study, we examined the effects and investigated the mechanisms of cilostazol on neutrophil adhesion to human umbilical endothelial cells (HUVECs) triggered by N-formyl-methionyl-leucyl-phenylal-anine (FMLP), a chemotactic peptide. The soluble vascular cell adhesive molecule-1 (sVCAM-1) release from FMLP (10 microM)-stimulated HUVECs was determined by ELISA kits. Fluo-2, a fluorescent indicator, was used to investigate intracellular free calcium concentration ([Ca2+]i) in HUVECs. HL-60 cells were induced to be neutrophilic by DMSO and loaded with Fluo-3, another fluorescent indicator, to detect [Ca2+]i, and CLA was used as a chemiluminescent indicator to determine superoxide production in neutrophilic cells. The result showed that CLZ (1-100 microM) significantly inhibited neutrophil adhesion to FMLP-stimulated HUVECs. In HUVECs, CLZ obviously downregulated sVCAM-1 level, while it had no meaningful influence [Ca2)]i. But in neutrophils, FMLP-activated superoxide generation and [Ca2+]i increase were found being inhibited by exposure to CLZ . Furthermore, we also demonstrated that Ca2+ increase was preceded to the superoxide generation in neutrophils. The results suggest that CLZ involves in adhesion reactions between neutrophil and ECs, partly via VCAM-1 expression in ECs, and decreasing [Ca2+]i induced activation of neutrophils, which means a lot to prevent atherosclerosis and other cardiovascular diseases.     

Oral gene delivery: design of polymeric carrier systems shielding toward intestinal enzymatic attack

The gastrointestinal tract poses a variety of morphological and physiological barriers to the expression of target genes. The aim of this study was to evaluate the stability of cationic polymer/pDNA nanoparticles toward salts and enzymes of the intestinal fluid. Within this study, a chitosan-enzyme inhibitor conjugate has been generated and characterized. Based on this conjugate, nanoparticles with pDNA were generated to enhance transfection rate in oral gene delivery. The enzyme inhibitor aurintricarboxylic acid (ATA) was covalently bound to chitosan to improve the enzymatic stability of nanoparticles formed with this polymer and pDNA. Chitosan-ATA/pDNA nanoparticles showed a size of 98.5 +/- 26 nm and a zeta potential of -13.26 +/- 0.24 mV (n = 3-4). Stability studies with salt solution, lysozyme, DNase, and freshly collected porcine intestinal fluid showed that chitosan-ATA/pDNA nanoparticles are significantly (p < 0.05) more stable than unmodified chitosan/pDNA nanoparticles. Apart from improved stability, chitosan-ATA/pDNA nanoparticles showed a 2.6-fold higher transfection rate than chitosan/pDNA nanoparticles in the Caco-2 cell line, thus creating a promising carrier for orally administered therapeutic genes.     

Performance of fluorescent europium(III) nanoparticles and colloidal gold reporters in lateral flow bioaffinity assay

Lateral flow (LF) immunoassays (i.e., immunochromatographic assays) have traditionally been applied to analytes that do not require very high analytical sensitivity or quantitative results. The selection of potential analytes is often limited by the performance characteristics of the assay technology. Analytes with more demanding sensitivity requirements call for reporter systems enabling high analytical sensitivity. In this study, we systematically compared the performance of fluorescent europium(III) [Eu(III)] chelate dyed polystyrene nanoparticles and colloidal gold particles in lateral flow assays. The effect of time-resolved measurement mode was also studied. Because binder molecules used in immunoassays might not behave similarly when conjugated to different reporter particles, two model assays were constructed to provide reliable technical comparison of the two reporter systems. The comparative experiment demonstrated that the fluorescent nanoparticles yielded 7- and 300-fold better sensitivity compared with colloidal gold in the two test systems, respectively. Although the two reporter particles may induce variable effects using individual binders, overall the high specific activity of Eu(III) nanoparticles has superior potential over colloidal gold particles for the development of robust high-sensitivity bioaffinity assays.     

Formation of a fluorescent glucocorticoid receptor-steroid complex in HTC cell cytosol

An intensely fluorescent rhodamine derivative of dexamethasone (i.e. Dex-C2-Rho) was synthesized. Dex-C2-Rho possessed high affinity for HTC cell glucocorticoid receptors in cell-free systems. Whole cell activity and receptor affinity of Dex-C2-Rho were both much lower, apparently due to problems with cell permeability and/or metabolism. A specific, fluorescent receptor-steroid complex at concentrations as low as 1 X 10(-9) M could readily be observed with crude HTC cell receptors after removal of the free Dex-C2-Rho. This appears to be the first report of a fluorescent glucocorticoid receptor-steroid complex.     

Measurement of diffusion within the cell wall in living roots of Arabidopsis thaliana

To quantify the diffusion constant of small molecules in the plant cell wall, fluorescence from carboxyfluorescein (CF) in the intact roots of Arabidopsis thaliana was recorded. Roots were immersed in a solution of the fluorescent dye and viewed through a confocal fluorescence microscope. These roots are sufficiently transparent that much of the apoplast can be imaged. The diffusion coefficient, D(cw), of CF in the cell wall was probed using two protocols: fluorescence recovery after photobleaching and fluorescence loss following perfusion with dye-free solution. Diffusion coefficients were obtained from the kinetics of the fluorescent transients and modelling apoplast geometry. Apoplastic diffusion constants varied spatially in the root. In the elongation zone and mature cortex, D(cw)=(3.2+/-1.4)x10(-11) m(2) s(-1), whereas in mature epidermis, D(cw)=(2.5+/-0.7)x10(-12) m(2) s(-1), at least an order of magnitude lower. Relative to the diffusion coefficient of CF in water, these represent reductions by approximately 1/15 and 1/195, respectively. The low value for mature epidermis is correlated with a suberin-like permeability barrier that was detected with either autofluorescence or berberine staining. This study provides a quantitative estimate of the permeability of plant cell walls to small organic acids-a class of compounds that includes auxin and other plant hormones. These measurements constrain models of solute transport, and are important for quantitative models of hormone signalling during plant growth and development.     

A method for determining the individual optical characteristics of posttranslational fluorescent forms of fluorescent proteins with the use of nonlinear laser fluorimetry

A method for determining the individual optical characteristics (fluorescence quantum yield, the rate constant and quantum yield of singlet-triplet conversion, excitation of fluorescence cross-section, extinction coefficient) and concentration correlations between the fluorescent forms of fluorescent proteins arising in the reaction of posttranslational chromophore formation has been developed, which is based on combined application of absorption spectroscopy and classical and nonlinear laser fluorimretry. The method allows one to determine the share of fluorescent forms in the mixture of chromoproteins. The individual optical characteristics of the red form of the fluorescent protein mRFP1 has been determined: the fluorescence quantum yield eta = 0.24 +/- 0.03; the extinction coefficient in the maximum of absorbance band (584 nm) epsilon = 213 +/- 40 mM(-1) cm(-1) (the cross-section of absorbance sigma = (8.2 +/- 1.5).10(-16) cm2); the constant of singlet-triplet conversion rate K32 = (0 +/- 0.6)-10970 s(-1). The part of the red form in the mixture of chromoproteins is 26 +/- 6%.     

Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion.

The green fluorescent protein (GFP) was used as a noninvasive probe to quantify the rheological properties of cell cytoplasm. GFP mutant S65T was purified from recombinant bacteria for solution studies, and expressed in CHO cell cytoplasm. GFP-S65T was brightly fluorescent in solution (lambda ex 492 nm, lambda em 509 nm) with a lifetime of 2.9 ns and a rotational correlation time (tc) of 20 ns. Recovery of GFP fluorescence after photobleaching was complete with a half-time (t1/2) in aqueous saline of 30 +/- 2 ms (5-micron diameter spot), giving a diffusion coefficient of 8.7 x 10(-7) cm2/s. The t1/2 was proportional to solution viscosity and was dependent on spot diameter. In contrast to fluorescein. GFP photobleaching efficiency was not affected by solution O2 content, triplet state quenchers, singlet oxygen scavengers, and general radical quenchers. In solutions of higher viscosity, an additional, rapid GFP recovery process was detected and ascribed to reversible photobleaching. The t1/2 for reversible photobleaching was 1.5-5.5 ms (relative viscosity 5-250), was independent of spot diameter, and was unaffected by O2 or quenchers. In cell cytoplasm, time-resolved microfluorimetry indicated a GFP lifetime of 2.6 ns and a tc of 36 +/- 3 ns, giving a relative viscosity (cytoplasm versus water) of 1.5. Photobleaching recovery of GFP in cytoplasm was 82 +/- 2% complete with a t1/2 of 83 +/- 6 ms, giving a relative viscosity of 3.2. GFP translational diffusion increased 4.7-fold as cells swelled from a relative volume of 0.5 to 2. Taken together with measurements of GFP translation and rotation in aqueous dextran solutions, the data in cytoplasm support the view that the primary barrier to GFP diffusion is collisional interactions between GFP and macromolecular solutes.     

Imaging of the GI tract by QDs loaded heparin-deoxycholic acid (DOCA) nanoparticles

This study presents an approach to deliver non invasive, near-IR imaging agent using oral delivery system. Low molecular weight heparin (LMWH)-deoxycholic acid (DOCA)/(LHD) nanoparticles formed by a self-assembly method was prepared to evaluate their physicochemical properties and oral absorption in vitro and in vivo. Near-IR QDs were prepared and loaded into LHD nanoparticles for imaging of the gastro-intestinal (GI) tract absorption. Q-LHD nanoparticles were almost spherical in shape with diameters of 194-217nm. The size and fluorescent intensity of the Q-LHD nanoparticles were stable in 10% FBS solution and retained their fluorescent even after 5 days of incubation. Cell viability of Q-LHD nanoparticles maintained in the range of 80-95% for 24h incubation. No damage was found in tissues or organs during animal experiments. The in vivo oral absorption of Q-LHD was observed in SKH1 mice for 3h under different doses. From the results, we confirmed that Q-LHD was absorbed mostly into the ileum of small intestine containing intestinal bile acid transporter as observed in TEM and molecular imaging system. Our designed nanoparticles could be administered orally for bio-imaging and studying the bio-distribution of drug.     

Agonist-induced cytoplasmic volume changes in cultured rabbit parietal cells

Concomitant Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange activation occurs during stimulation of acid secretion in cultured rabbit parietal cells, possibly related to a necessity for volume regulation during the secretory process. We investigated whether cytoplasmic volume changes occur during secretagogue stimulation of cultured rabbit parietal cells. Cells were loaded with the fluorescent dye calcein, and the calcein concentration within a defined cytoplasmic volume was recorded by confocal microscopy. Forskolin at 10(-5) M, carbachol at 10(-4) M, and hyperosmolarity (400 mosmol) resulted in a rapid increase in the cytoplasmic dye concentration by 21 +/- 6, 9 +/- 4, and 23 +/- 5%, respectively, indicative of cell shrinkage, followed by recovery to baseline within several minutes, indicative of regulatory volume increase (RVI). Depolarization by 5 mM barium resulted in a decrease of the cytoplasmic dye concentration by 10 +/- 2%, indicative of cell swelling, with recovery within 15 min, and completely prevented forskolin- or carbachol-induced cytoplasmic shrinkage. Na(+)/H(+) exchange inhibitors slightly reduced the initial cell shrinkage and significantly slowed the RVI, whereas 100 microM bumetanide had no significant effect on either parameter. We conclude that acid secretagoguges induce a rapid loss of parietal cell cytoplasmic volume, followed by RVI, which is predominantly mediated by Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange.     

The murine IgA-secreting plasmacytoma MOPC-315 expresses somatostatin receptors

We have previously shown that some neuropeptides had a profound effect on in vitro Ig synthesis (especially IgA) and mitogen-driven murine lymphocyte proliferation. MOPC-315, an IgA-secreting plasmacytoma line, has been extensively used in studies of the regulation of IgA synthesis. In this report we show that the neuropeptide somatostatin (SOM) inhibits proliferation ([3H]thymidine uptake) of MOPC-315 and also inhibits IgA synthesis in vitro. MOPC-315 cells bind both fluorescent SOM and [125I]SOM specifically. On cytofluorimetric analysis, 68 +/- 6.8% (mean +/- SE, n = 7) of MOPC 315 cells labeled with fluorescent SOM and this staining was compatible by incubation with an excess of unlabeled peptide. Specific [125I]SOM binding increased linearly with cell concentration, was rapid and achieved equilibrium after 20 min at 4 degrees C. It was temperature-dependent, readily reversible, and under equilibrium conditions demonstrated a dissociation constant of 1.6 +/- 0.7 nM (mean +/- SE, n = 5). Scatchard analysis showed that MOPC-315 cells had 40,733 +/- 16,050 (mean +/- SE) binding sites for SOM per cell. The characteristics of the interactions of SOM with MOPC-315 cells suggest a specific receptor-mediated mechanism whereby this neuropeptide may modulate lymphocyte function.