Search

It has long been recognized that islet cell function is rapidly altered in vitro, but can be maintained, at least in part, when cells are layered on defined extracellular matrices. The present work addresses the influence of short-term cell-matrix interactions on islet beta-cell function and provides first insight into the molecular basis of these interactions. When primary rat beta-cells were allowed to attach to a matrix produced by a rat carcinoma cell line (804G), there was an increased insulin secretory response to secretagogues. This change was the result of an increase in the proportion of actively secreting beta-cells and in the amount of insulin secreted per active cell, as shown using the reverse hemolytic plaque assay. In turn, the spreading or flattening of beta-cells on this matrix was enhanced by secretagogues, and flattened cells secreted more insulin than rounded cells. Using indirect immunofluorescence, it was found that 1)alpha6beta1 integrins are present at the surface of islet cells in situ, 2) alpha6beta1 expression is heterogeneous among purified beta-cells and is upregulated by insulin secretagogues, 3) alpha6beta1 expression is higher in spreading cells, and 4) anti-alpha6beta1-specific antibodies decrease spreading. These observations demonstrate that islet cell-matrix interactions can improve the sensitivity of insulin cells to glucose and are mediated, at least in part, by alpha6beta1 integrins, suggesting that outside-in signaling through alpha6beta1 integrin plays a major role in the regulation of beta-cell function.

The cystic fibrosis (CF) gene encodes a cAMP-gated Cl- channel (cystic fibrosis transmembrane conductance regulator [CFTR]) that mediates fluid transport across the luminal surfaces of a variety of epithelial cells. We have previously shown that gap junctional communication and Cl- secretion were concurrently regulated by cAMP in cells expressing CFTR. To determine whether intercellular communication and CFTR-dependent secretion are related, we have compared gap junctional coupling in a human pancreatic cell line harboring the DeltaF508 mutation in CFTR and in the same cell line in which the defect was corrected by transfection with wild-type CFTR. Both cell lines expressed connexin45 (Cx45), as evidenced by RT-PCR, immunocytochemistry, and dual patch-clamp recording. Exposure to agents that elevate intracellular cAMP or specifically activate protein kinase A evoked Cl- currents and markedly increased junctional conductance of CFTR-expressing pairs, but not in the parental cells. The latter effect, which was caused by an increase in single-channel activity but not in unitary conductance of Cx45 channels, was not prevented by exposing CFTR-expressing cells to a Cl- channel blocker. We conclude that expression of functional CFTR restored the cAMP-dependent regulation of junctional conductance in CF cells. Direct intercellular communication coordinates multicellular activity in tissues that are major targets of CF manifestations. Consequently, defective regulation of gap junction channels may contribute to the altered functions of tissues affected in CF.

CD4, a member of the immunoglobulin superfamily, is not only expressed in T4 helper lymphocytes but also in myeloid cells. Receptor-mediated endocytosis plays a crucial role in the regulation of surface expression of adhesion molecules such as CD4. In T lymphocytes p56lck, a CD4-associated tyrosine kinase, prevents CD4 internalization, but in myeloid cells p56lck is not expressed and CD4 is constitutively internalized. In this study, we have investigated the role of cyclic AMP (cAMP) in the regulation of CD4 endocytosis in the myeloid cell line HL-60. Elevations of cellular cAMP were elicited by 1) cholera toxin, 2) pertussis toxin, 3) forskolin and IBMX, 4) NaF, or 5) the physiological receptor agonist prostaglandin E1. All five interventions led to an inhibition of CD4 internalization. Increased cAMP levels did not inhibit endocytosis per se, because internalization of insulin receptors and transferrin receptors and fluid phase endocytosis were either unchanged or slightly enhanced. The mechanism of cAMP inhibition was further analyzed at the ultrastructural level. CD4 internalization, followed either by quantitative electron microscopy autoradiography or by immunogold labeling, showed a rapid and temperature-dependent association of CD4 with clathrin-coated pits in control cells. This association was markedly inhibited in cells with elevated cAMP levels. Thus these findings suggest a second-messenger regulation of CD4 internalization through an inhibition of CD4 association with clathrin-coated pits in p56lck-negative cells.

Ex vivo labeling of islets with superparamagnetic iron oxide (SPIO) nanoparticles allows posttransplant MRI imaging of the graft. In the present study, we compare two clinical grade SPIOs (ferucarbotran and ferumoxide) in terms of toxicity, islet cellular uptake, and MRI imaging. Human islets (80-90% purity) were incubated for 24 h with various concentrations of SPIOs (14-280 mug/ml of iron). Static incubations were performed, comparing insulin response to basal (2.8 mM) or high glucose stimulation (16.7 mM), with or without cAMP stimulation. Insulin and Perl's (assessment of iron content) staining were performed. Electronic microscopy analysis was performed. Labeled islets were used for in vitro or in vivo imaging in MRI 1.5T. Liver section after organ removal was performed in the same plane as MRI imaging to get a correlation between histology and radiology. Postlabeling islet viability (80 +/- 10%) and function (in vitro static incubation and in vivo engraftment of human islets in nude mice) were similar in both groups. Iron uptake assessed by electron microscopy showed iron inclusions within the islets with ferucarbotran, but not with ferumoxide. MRI imaging (1.5T) of phantoms and of human islets transplanted in rats, demonstrated a strong signal with ferucarbotran, but only a weak signal with ferumoxide. Signal persisted for >8 weeks in the absence of rejection. An excellent correlation was observed between radiologic images and histology. The hepatic clearance of intraportally injected ferucarbotran was faster than that of ferumoxide, generating less background. A rapid signal decrease was observed in rejecting xenogeneic islets. According to the present data, ferucarbotran is the most appropriate of available clinical grade SPIOs for human islet imaging.

Gap junction channels provide a pathway for coordinating multicellular activity. To evaluate the contribution of cell-to-cell communication in the function of epithelial cells, we studied the strength of gap junctional coupling in pancreatic acinar and duct cells exposed to agents known to elevate the intracellular concentration of Ca(2+) or cAMP. In acinar cells, we observed that maximal concentrations of acetylcholine evoked a biphasic increase in cytosolic Ca(2+) mobilization. The second sustained phase, which depends on Ca(2+) influx into the cell, was associated with the rapid closure of gap junction channels. In duct cells, stimulation of CFTR-dependent Cl(-) currents with cAMP analogs markedly increased gap junctional conductance in pairs of cells. Interestingly, cAMP had no effect on intercellular communication between cells harboring the DeltaF508 mutation of CFTR. An abnormal pattern of gap junctional coupling may contribute to the altered functions of tissues affected in cystic fibrosis.

Methods to detect functional cystic fibrosis transmembrane conductance regulator (CFTR) are needed for the assessment of new therapies in cystic fibrosis (CF). We have combined patch-clamp and fluorimetric techniques to investigate whether the fluorescent voltage-sensitive probe bis-(1,3-diethylthiobarbituic anid) trimethine oxonol (Di5BAC2(3)) discriminates between changes of membrane potential (Vm evoked by cAMP in CF and CYFT-corrected epithelial cells. About 60% of the (FTR-correrced cells increased their membrane conductance and depolarized in response to cAMP, as compared to about 20% of CF cells. CFTR was found to contribute only to a fraction of the cAMP-induced responses, as indicated by the differential effects of Cl- channel blockers. Simultaneous reocording of fluorescence (AF) and membrane potential revealed that AF detected Vm changes as small as 10 mV . The relationship between deltaF and deltaVm however, was not proportional. When a large number of cells were analyzed by digital imaging, an increase in deltaF in response to cAMP was detected in the majority of CFTR-corrected cells, but only in a small proportion of CF cells. The results indicate that the DiSBAC2(3) approach is a valid tool to compare cell populations with different proportions of cells responding to CFTR arcivation by cAMP. It cannot be used, however, for quantitative assessment of functional CFTR in individual CF cells.

Superoxide anion (O2-.) production from human neutrophils stimulated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, 1 microM) was inhibited by preparations of the inhibitor of cAMP-dependent protein kinase, Rp-cyclic adenosine 3',5'-phosphorothioate (Rp-cAMPS, 100 microM). This effect of Rp-cAMPS was reversed by xanthine amine congener (0.1 microM), an adenosine receptor antagonist, and by low concentrations of adenosine desaminase (0.02 mg/ml). HPLC analysis shows that these preparations of Rp-cAMPS contained concentrations of adenosine which could produce significant inhibition of fMLP-induced O2-. production. These results suggest that Rp-cAMPS should be used with caution in cells or tissues containing adenosine receptors, and that preparations of Rp-cAMPS should be treated with adenosine desaminase before use to avoid activation of adenosine receptors.

Utilizing cocultures of mouse renal juxtaglomerular cells with bovine microvascular endothelial cells, we have examined whether endothelial cells exert direct influence on renin secretion from renal juxtaglomerular cells. In the presence of endothelial cells both spontaneous and forskolin (10 microM) or isoproterenol (10 microM) stimulated renin release were markedly attenuated. The stimulatory effect of the calmodulin antagonist calmidazolium (10 microM) on renin secretion was not altered by endothelial cells, whereas the stimulatory effect of ethylisopropylamiloride (50 microM) an inhibitor of sodium-proton exchange was enhanced in the presence of endothelial cells. Indomethacin (10 microM) and NG-monomethyl-l-arginine (NMMA) (1 mM) used to inhibit cyclooxygenase activity and production of endothelium-derived relaxing factor (EDRF) decreased spontaneous renin release in the presence of endothelial cells only, but had no effect on forskolin stimulated renin secretion. Endothelin (1 microM) inhibited cAMP stimulated renin release both in the absence and in the presence of endothelial cells. ATP (10 microM) which acts on both endothelial and juxtaglomerular cells via purinergic P2 receptors inhibited cAMP stimulated renin release only in the absence but not in the presence of endothelial cells. This modulatory effect of endothelial cells was no altered by indomethacin nor by NMMA. Taken together, our findings provide first evidence for a local control function of the endothelium on cAMP stimulated renin secretion from renal juxtaglomerular cells, which could in part be mediated by endothelin.

An image processing system was programmed to automatically track and digitize the movement of amebae under phase-contrast microscopy. The amebae moved in a novel chemotaxis chamber designed to provide stable linear attractant gradients in a thin agarose gel. The gradients were established by pumping attractant and buffer solutions through semipermeable hollow fibers embedded in the agarose gel. Gradients were established within 30 min and shown to be stable for at least a further 90 min. By using this system it is possible to collect detailed data on the movement of large numbers of individual amebae in defined attractant gradients. We used the system to study motility and chemotaxis by a score of Dictyostelium discoideum wild-type and mutant strains, including "streamer" mutants which are generally regarded as being altered in chemotaxis. None of the mutants were altered in chemotaxis in the optimal cAMP gradient of 25 nM/mm, with a midpoint of 25 nM. The dependence of chemotaxis on cAMP concentration, gradient steepness, and temporal changes in the gradient were investigated. We also analyzed the relationship between turning behavior and the direction of travel during chemotaxis in stable gradients. The results suggest that during chemotaxis D. discoideum amebae spatially integrate information about local increases in cAMP concentration at various points on the cell surface.

The role of Ca2+ on insulin release has been studied by the use of ionophore A23187. The ionophore complexes divalent cations and permits Ca2+ entry into cells by acting as a carrier in the plasma membranes. Cultured cells obtained by enzymatic digestion of pancreases from newborn rats were studied on the 3rd day of culture. With Ca2+ in the incubation medium the ionophore induced sustained insulin release even in the absence of glucose. Optimal effects of the ionophore were observed at 3 and 10 mug per ml in the presence of 0.3 to 1.0 mM Ca-2+. Under these conditions the insulin release was greater than that caused by 16.7 mM glucose. A graded response was observed to changes in Ca-2+ concentration from 0.1 to 1.0 mM Ca-2+. Higher Ca-2+ concentrations caused a large amount of insulin to be released promptly, but the release was not sustained. Mg-2+ and Sr-2+ were not found to substitute for Ca-2+. Ba-2+ at 0.3 mM stimulated insulin release even in the absence of ionophore. Cyclic adenosine 3':5'-monophosphate was able to increase ionophore-induced insulin release. The alpha-adrenergic effect of epinephrine to inhibit insulin release was not observed in the presence of Ca-2+ and the ionophore, and a stimulatory effect of epinephrine was seen. This unusual stimulatory effect of epinephrine was blocked by propranolol indicating a beta-adrenergic mechanism for epinephrine. It is concluded that Ca-2+, which plays an essential role in the stimulus-secretion coupling, can alone initiate and cause sustained insulin release.

It has become clear over the last few years that malignant transformation for several tumors results from failure of embryonic tissue to properly differentiate and activation of a mechanism which triggers uncontrolled cellular proliferation. Retinoblastoma fits this pattern, in that early inactivation of regulatory genes leads to proliferation of cells that are arrested in a primitive stage of development. Cultured Y-79 human retinoblastoma cells, however, can be induced to differentiate to a more "normal" stage in development through the use of several naturally occurring agents, such as butyrate, cyclic AMP, and retinoic acid. These agents cause reversible growth inhibition (e.g., retinoic acid) or result in cell death (e.g., butyrate). Proteins translated from mRNAs isolated from Y-79 cells are dramatically altered by all the above substances. Thus, alteration in transcriptional activity and changes in macromolecular synthesis can lead to the transition of rapidly growing, non-differentiated retinoblastoma cells to a non-proliferating, more differentiated state. The changes of the mRNA species induced with the various agents we have utilized, supports the hypothesis that differentiation and growth inhibition in Y-79 cells are programmed genetic events which can be controlled at least in vitro. Screening and testing of differentiating agents could be clinically useful for the treatment of retinoblastoma as well as for better understanding the biological control mechanisms involved in tumor growth. Anticancer Research