Altered expression and localization of the tight junction protein ZO-1 in primary and metastatic pancreatic cancer.

INTRODUCTION: ZO-1 is a tight junction membrane protein that plays a critical role in cell-cell interaction, proliferation, and differ entiation. AIM: To localize and evaluate the expression of ZO-1 in the normal human pancreas, in pancreatic ductal adenocarcinoma (PDAC), and in chronic pancreatitis (CP). METHODOLOGY AND RESULTS: Northern and Western blot analysis revealed ZO-1 expression in all six tested pancreatic cancer cell lines. Expression of ZO-1 mRNA was increased sixfold in PDAC samples in comparison with normal samples (p = 0.04). Confocal microscopy revealed the presence of ZO-1 in the apical and apicolateral areas of ductular cells in the normal pancreas. Similarly, in CP, ZO-1 was localized at apical and apicolateral areas of small proliferating ductular cells and large metaplastic ducts. In PDAC, however, ZO-1 expression was observed irrespective of whether the cancer cells formed duct-like structures or exhibited a diffuse infiltrating pattern. Metastatic pancreatic cancer cells within lymph nodes displayed variable staining patterns, ranging from apical and apicolateral to a diffuse membranous staining. CONCLUSION: These observations suggest that ZO-1 is overexpressed in PDAC and raise the possibility that this overexpression may confer a metastatic advantage to pancreatic cancer cells.

Glucagon-like peptide 1 elevates cytosolic calcium in pancreatic beta-cells independently of protein kinase A.

Glucagon-like peptide 1 (7-36)amide (GLP-1) is an insulinotropic intestinal peptide hormone with a potential role as antidiabetogenic therapeutic agent. It mediates a potentiation of glucose-induced insulin secretion, by activation of adenylate cyclase and subsequent elevation of cytosolic free calcium, [Ca2+]cyt. We investigated the role of protein kinase A (PKA) in GLP-1 signal transduction, using isolated mouse islets as well as the differentiated beta-cell line INS-1. Two specific inhibitors of PKA, (Rp)-adenosine cyclic 3',5'-phosporothioate (Rp-cAMPS, up to 3 mM) and KT5720 (up to 10 microM), did not inhibit the GLP-1-induced [Ca2+]cyt elevation. Another PKA inhibitor, H-89, reduced the [Ca2+]cyt elevation only when applied at high concentrations (10-40 microM), higher than sufficient for PKA inhibition in many cell types. Furthermore, at these concentrations, H-89 also inhibited presumably PKA-independent processes such as glucose-induced [Ca2+]cyt elevations and intracellular calcium storage. This suggests a PKA-independent action of H-89. Similarly to H-89, the potent but unselective protein kinase inhibitor staurosporine inhibited the GLP-1-induced [Ca2+]cyt elevation only at high concentrations, at which it also inhibited glucose-induced [Ca2+]cyt elevations. The same observations as with GLP-1 were made when adenylate cyclase was stimulated with forskolin, for selective examination of signal transduction downstream of receptor and G protein. Our results suggest that the GLP-1-induced [Ca2+]cyt elevation is mediated independently of PKA and thus belongs to the yet-little-characterized ensemble of effects that are mediated by binding of cAMP to other target proteins.

Transformation-dependent calcium influx by voltage-operated calcium channels in stellate cells of rat liver.

BACKGROUND/AIMS: The transformation of hepatic stellate cells into myofibroblasts is a key step in the pathogenesis of fibrotic liver diseases. The intracellular signaling associated with hepatic stellate cell transformation becomes a point of interest, especially the role of cytosolic free calcium concentration ([Ca2+]i). The aim of the study was to investigate possible differences between various transformation phenotypes of hepatic stellate cells with regard to the calcium influx mediated by L-type voltage-operated calcium channels (L-type VOC). METHODS: Hepatic stellate cells were isolated from rat liver by pronase-collagenase reperfusion and cultured under standard conditions. The transformation of hepatic stellate cells was stimulated by treatment with transforming growth factor-beta (TGF-beta) or inhibited with interferon-gamma (IFN-gamma) and characterized by immunocytochemistry for smooth muscle alpha-actin and determination of hyaluronan in the culture media with a ligand binding assay. [Ca2+]i was measured in individual cells with fluorescence microscopy using fura-2. VOCs were activated by the standard procedure of extracellular potassium elevation, to achieve depolarization, and identified by various controls. RESULTS: In transformed myofibroblasts the activation of VOCs by potassium elevation from 5.4 mmol/l to 50.4 mmol/l led to a 19% increase in [Ca2+]i in contrast to 0.2% in hepatic stellate cells cultured for 3 days. In 7-day old hepatic stellate cells, after stimulation of cell transformation with TGF-beta-1, an enhanced [Ca2+]i response to potassium elevation was detected, while inhibition of transformation with IFN-gamma for the same time caused a decreased calcium signal compared with untreated control cultures. Short-term treatment with the cytokines (1 day) did not influence depolarization-dependent calcium signals. CONCLUSION: The results show the [Ca2+]i increase via L-type VOCs to be dependent on the transformation level of hepatic stellate cells into myofibroblasts which can be influenced by the long-term treatment of hepatic stellate cells with TGF-beta or IFN-gamma. In contrast, there is no evidence for direct regulation of VOC activity by TGF-beta or IFN-gamma after short-term exposure.

A nutrient-regulated cytosolic calcium oscillator in endocrine pancreatic glucagon-secreting cells.

We investigated the influence of nutrients on spontaneous cytosolic calcium oscillations in InR1-G9 glucagonoma cells, a model for pancreatic alpha-cells. The oscillations depended on calcium release from stores and on calcium influx, partly through voltage-dependent calcium channels. Oscillations required the presence of at least 1 mM glucose, 50 microM alanine, or 50 microM glutamine, but were terminated by higher nutrient concentrations (40 mM glucose, or above 2 mM alanine or glutamine). The effects depended on the metabolism of the nutrients. Glutamine and alanine hyperpolarized the cells. This effect was inhibited (glutamine) or attenuated (alanine) by 1 mM ouabain. Our findings suggest that [Ca2+]i regulation in alpha-cells is dominated by slow oscillations induced by a lack of metabolic energy, resulting in decreased calcium export and storage, as well as increased calcium influx, partly due to depolarization caused by reduced sodium pump activity. These processes, leading to an elevated cytosolic calcium concentration, may mediate oscillations by calcium-induced calcium release from intracellular stores.

Measurement of calcium influx in surface-fixed single sperm cells: efficiency of different immobilization methods.

Calcium fluxes across the plasma membrane of spermatozoa are part of the signal transduction pathway during sperm capacitation. To identify the topography, the time sequence and frequency of calcium fluxes in motile human spermatozoa, individual spermatozoa have to be locally immobilized in a measurement chamber to allow the quantification of ionized calcium by use of a microspectro-photometric method (FURA-2AM). In this study, we compared different immobilization methods using agarose-, gelatin-, laminin- and poly-L-lysine-coated glass slides. Optimal results were obtained with poly-L-lysine coating, which permitted the adhesion of motile spermatozoa that could be analysed microspectro-photometrically. The loss of adherent spermatozoa during the washing procedures was below 10%. However, a major disadvantage of poly-L-lysine coating is that this polymer itself induces a low calcium flux in spermatozoa. Comparing all tested variations, laminin offered the best adhesion result without any detectable effects on calcium fluxes. Our method allowed the rapid change of incubation fluid and calcium concentrations around individual motile spermatozoa and the reproducible quantification of calcium fluxes in single motile spermatozoa.

Leptin: a potent inhibitor of insulin secretion.

The hormone leptin is expressed and secreted by the adipose tissue and impacts on the central nervous system. Leptin is involved in the regulation of energy balance, satiety, and body composition. The lack of active leptin results in obesity, high food intake, hyperglycemia, and hyperinsulinemia. We present data supporting effects of leptin on the endocrine pancreas. We found the leptin receptor to be expressed in insulin- and glucagon-secretin cells derived from mouse, hamster, and rat pancreas. In the isolated perfused rat pancreas leptin is a potent inhibitor of basal and glucose-induced insulin secretion, especially during the first phase of the insulin response. At isolated mouse islets and insulin-secreting INS-1 cells leptin reduced promptly and persistently the intracellular Ca2+ levels. Cytoplasmic Ca2+ oscillation amplitude was decreased and the oscillation frequency increased. These findings suggest functional active receptors for leptin on insulin-secreting B-cells. Therefore, leptin is a metabolic hormone and not only a signal to the brain indicating filled fat stores. Our data suggest that leptin is also a signal back to the endocrine pancreas that no more insulin is required to replenish fat stores. Thus, an "adipo-insular axis" operating with two arms exists: insulin and glucagon are signals to the adipocyte. This releases leptin, which could be the mediator of the respective feedback to the pancreas. A defective leptin suppression of insulin secretion could contribute to hyperinsulinemia and disturbances of glucose metabolism.

Interaction of GLP-I and leptin at rat pancreatic B-cells: effects on insulin secretion and signal transduction.

The incretin effect is reduced in NIDDM, although a corresponding attenuation of incretin hormone secretion does not occur. We characterized the direct interaction of GLP-I, an important incretin hormone, and leptin on insulin secretion and signal transduction in B-cells. Leptin inhibited GLP-I stimulated insulin release from the isolated perfused rat pancreas. Both phases of the biphasic insulin secretory response were inhibited. GLP-I receptor binding and GLP-I induced cAMP generation remained unchanged. Leptin reduced the GLP-I mediated increase of cytosolic Ca2+ concentration. It had similar effects on calcium elevations induced by forskolin. The effect was more pronounced during the plateau phase than during the initial peak. These effects could help to explain leptin's inhibitory effects on insulin secretion. The inhibition of GLP-I's insulinotropic effects by leptin may be an interesting aspect in the pathophysiology of NIDDM. The existence of an "adipo-insular axis" is suggested, in which leptin represents a negative feed-back signal from the adipose tissue to the endocrine pancreas.

The positive charge of the imidazole side chain of histidine7 is crucial for GLP-1 action.

Glucagon-like peptide-1(7-36)amide/(7-37) (GLP-1) is an incretin hormone which plays an important role in postprandial glucose homeostasis. Since GLP-1 potentiates glucose-induced insulin secretion, stimulates insulin biosynthesis and inhibits glucagon release, it is a potential tool for the treatment of diabetes mellitus. For this, an exact understanding of the structural/functional moieties of the peptide is mandatory. The present study investigates the importance of structural features of histidine7 at the N-terminus for GLP-1 action. Based upon binding and activity data obtained from ten different GLP-1 analogues we show that not the positive charge of the free alpha-amino group but the positive charge of the imidazole side chain of histidine is crucial for GLP-1 action. The presence of a ring structure and a basic function as well as the correct positioning of both seems to be decisive.

Agonist-releasable intracellular calcium stores and the phenomenon of store-dependent calcium entry. A novel hypothesis based on calcium stores in organelles of the endo- and exocytotic apparatus.

Store-dependent calcium entry represents a little characterized calcium permeation pathway that is present in a variety of cell types. It is activated in an unknown way by depletion of intracellular calcium stores, for example in the course of phospholipase C stimulation. Current hypotheses propose that depleted calcium stores signal their filling state to this permeation pathway either by direct, protein-mediated interaction or by release of a small, diffusible messenger. The further characterization of store-dependent calcium entry will benefit from progress in the identification of the intracellular calcium storing compartments. Recent findings reviewed here suggest that these compartments include parts of the organelle system that is involved in endo- and exocytosis. This commentary describes a novel model of store-dependent calcium entry based on calcium stores belonging to the endo- and exocytotic organelle system. Such calcium stores could establish a tubule-like connection with the extracellular space, in analogy to the cellular compartments that contain the insulin-sensitive glucose transporter or the gastric proton pump. This connection will provide a pathway for store-dependent calcium entry. Under store depletion, extracellular calcium will permeate through the tubule-like connection into the store lumen and from there into the cytosol. The consequences of this model for the development of drugs modulating store-dependent calcium entry are discussed.

Molecular cloning, functional expression, and signal transduction of the GIP-receptor cloned from a human insulinoma.

Glucose-dependent insulinotropic polypeptide (GIP) plays an important role in the regulation of postprandial insulin secretion and proinsulin gene expression of pancreatic beta-cells. This study demonstrates the molecular cloning of a cDNA for the GIP-receptor from a human insulinoma lambda gt11 cDNA library. The cloned cDNA encoded a seven transmembrane domain protein of 466 amino acids which showed high homology (41%) to the human glucagon-like peptide 1 (GLP-1) receptor. Homology to the GIP receptor from rat or hamster was 79% and 81%, respectively. When transfected stably into fibroblast CHL-cells a high affinity receptor was expressed which coupled to the adenylate cyclase with normal basal cAMP and increasing intracellular cAMP levels under stimulation with human GIP-1-42 (EC50 = 1.29 x 10(-13) M). The receptor accepted only human GIP 1-42 (Kd = 1.93 +/- 0.2 x 10(-8) M) and porcine truncated GIP 1-30 (Kd = 1.13 +/- 0.1 x 10(-8) M) as high affinity ligands. At 1 microM, exendin-4 and (9-39)amide weakly reduced GIP-binding (25%) whereas secretin, glucagon, glucagon-like peptide-1, vasoactive intestinal polypeptide, peptide histidine-isoleucine, and pituitary adenylyl cyclase activating peptide were without effect. In transfected CHL cells, GIP-1-42 did not increase intracellular calcium. Northern analysis revealed one transcript of human GIP receptor mRNA with an apparent size of 5.5 kb. The exact understanding of GIP receptor regulation and signal transduction will aid in the understanding of the incretin hormone's failure to exert its biological action at the pancreatic B-cell in type II diabetes mellitus.

Iron sequestration by the yeast vacuole. A study with vacuolar mutants of Saccharomyces cerevisiae.

To investigate iron uptake by the yeast vacuole, we examined iron accumulation and subcellular distribution in two Saccharomyces cerevisiae mutant strains with distinct alterations in vacuolar morpholgy and functions, the vacuolar protein targeting mutants vpt13 and vpt16. The mutant vpt13 is deficient in vacuolar acidification but possesses a morphologically normal vacuole, whereas vpt16 is devoid of any vacuole-like structure. The vacuole-lacking mutant vpt16 displayed reduced iron accumulation at high and low extracellular iron concentrations. Iron accumulation by vpt13 was not reduced at low extracellular iron concentrations, and only slightly reduced at high external iron. Subcellular fractionation of vpt13 after iron loading confirmed the vacuolar localization of the accumulated iron. Vacuolar iron sequestration thus is not dependent on vacuolar acidification, in contrast to vacuolar uptake of other metal cations such as calcium, strontium, manganese, and zinc. Conclusively, the vacuolar iron-uptake mechanism is different from the cation-proton exchangers mediating uptake of these other metal cations. Regarding the lacking dependence on vacuolar acidification as well as the high amounts of iron accumulated by the vacuole, vacuolar iron uptake could be mediated by an iron pump similar to the known ATP-dependent mechanisms of active transport.

Spontaneous calcium oscillations in clonal endocrine pancreatic glucagon-secreting cells.

Measurements of the cytosolic calcium concentration in single cells of the clonal endocrine pancreatic glucagon-secreting cell line INR1 G9 revealed the existence of spontaneous calcium oscillations in 20-70% of these cells. Inhibition of these spontaneous oscillations by thapsigargin as well as the phospholipase C inhibitor U 73122 demonstrated involvement of calcium release from intracellular stores, probably mediated by a high basal activity of phospholipase C. Removal of extracellular calcium but not the L-type calcium channel antagonists verapamil or nifedipine terminated the spontaneous oscillations, suggesting that calcium influx by a pathway distinct from L-type channels contributed to the oscillations. Similar spontaneous calcium oscillations could be the pacemaker of pulsatile glucagon release in endocrine pancreatic A-cells.

Signal transduction of the GLP-1-receptor cloned from a human insulinoma.

GLP-1 (glucagon-like peptide 1 (7-36) amide) plays an important role in the regulation of insulin secretion and proinsulin gene expression of pancreatic beta-cells. Patients with insulinoma tumors show uncontrolled insulin hypersecretion. This study demonstrates the molecular cloning of a cDNA for the GLP-1 receptor from a human insulinoma employing a lambda-gt11 cDNA library. The cloned cDNA encoded a seven transmembrane domain protein of 463 amino acids which showed high homology to the GLP-1 receptor in normal human pancreas. Four amino acid exchanges were found in comparison to a receptor sequence obtained from regular pancreatic islets. When transfected transiently into COS-7 or stably into fibroblast CHL cells a high affinity receptor was expressed which coupled to the adenylate cyclase with normal basal cAMP and increasing intracellular cAMP levels under GLP-1 stimulation. The receptor accepted GLP-1 and the non-mammalian agonist exendin-4 as high affinity ligands. In transfected COS-7 cells, GLP-1 did not influence intracellular calcium, whereas in the stably transfected fibroblasts GLP-1 transiently increased intracellular calcium to a small extent. The understanding of GLP-1 receptor regulation and signal transduction will aid in the discovery of compounds that act as agonists of the GLP-1 receptor for potential use in the treatment of diabetes and will facilitate the understanding of its expression under normal and pathophysiological conditions.

An investigation on the role of vacuolar-type proton pumps and luminal acidity in calcium sequestration by nonmitochondrial and inositol-1,4,5-trisphosphate-sensitive intracellular calcium stores in clonal insulin-secreting cells.

To test whether in RINm5F rat insulinoma cells luminal acidity and the activity of a vacuolar-type proton pump are involved in calcium sequestration by intracellular calcium stores sensitive to inositol 1,4,5-trisphosphate (InsP3) we examined the effects of various proton-conducting ionophores and ammonium chloride, and of bafilomycin, a specific inhibitor of vacuolar proton pumps, on this parameter. Bafilomycin in concentrations up to 1 microM did not affect calcium sequestration by nonmitochondrial, InsP3-sensitive stores at all; 50 microM carbonylcyanide m-chlorophenylhydrazone, 50 microM monensin and 30 mM NH4Cl, which are diverse ways to dissipate transmembrane pH gradients, did not inhibit calcium sequestration. This argues against signficant involvement of internal acidity and vacuolar proton pumps in calcium sequestration by InsP3-sensitive stores in RINm5F cells. The proton-potassium-exchanging ionophore nigericin (20-100 microM), however, inhibited calcium sequestration by nonmitochondrial and InsP3-sensitive stores. This effect was dependent on the presence of potassium and could be reversed by inclusion of carbonylcyanide m-chlorophenylhydrazone or acetate in the incubation medium. Thus, the inhibitory effect of nigericin appears to be based on proton extrusion coupled to potassium influx across the membrane of calcium stores in RINm5F cells, creating an internal alkalinization of these stores. The effect of nigericin implies the continuous maintenance of an outside-to-inside potassium concentration gradient by nonmitochondrial calcium stores in RINm5F cells. This feature will be of potential interest in the identification of InsP3-sensitive calcium-storing organelles.

Protein kinase C activates capacitative calcium entry in the insulin secreting cell line RINm5F.

This study examines the calcium store-regulated (capacitative) calcium influx pathway in the endocrine pancreatic cell line RINm5F, utilizing thapsigargin. After preincubation of the cells with the phorbol ester TPA, thapsigargin induced a sustained elevation of cytosolic calcium as well as a sustained stimulation of manganese entry, the latter being used to assess calcium influx. Thapsigargin given alone provoked a smaller and only transient elevation of cytosolic calcium and stimulation of manganese entry. The protein kinase C inhibitor staurosporine antagonized the effect of the phorbol ester. Verapamil, nifedipine, or measures to hyperpolarize the cells exerted no inhibitory action against this effect, which excludes an involvement of voltage-dependent calcium channels. In conclusion, our data shows for the first time that protein kinase C stimulation activates the capacitative calcium influx pathway of endocrine pancreatic insulin-producing cells.

A novel membrane associated carbonyl reducing enzyme is present in smooth endoplasmic reticulum of mouse liver.

Evidence supporting the existence of two distinct carbonyl (metyrapone) reducing enzymes which differ in subcellular localization and immunological homology has been provided. A soluble enzyme, designated as carbonyl reductase (EC 1.1.1.184) is located in the cytosol. The distribution of the second, membrane associated, MPON-reductase shows an excellent linear correlation to NADPH-cytochrome c reductase and, on the other hand, is reciprocal to the RNA/protein ratio of submicrosomal preparations. This indicates that the membrane associated MPON-reductase is exclusively located in the smooth endoplasmic reticulum. Using antibodies against the purified membrane associated enzyme the extent of immunological crossreaction corresponds well to the specific activities of MPON-reductase in the granular fractions, thus further confirming the localization of this enzyme within this organelle. The absence of antigenic crossreaction to cytosolic MPON-reductase indicates differences also in terms of the immunological relationship between the two enzymes.

Vanadium uptake by yeast cells.

During incubation with vanadyl, Saccharomyces cerevisiae yeast cells were able to accumulate millimolar concentrations of this divalent cation within an intracellular compartment. The intracellular vanadyl ions were bound to low molecular weight substances. This was indicated by the isotropic nature of the electron paramagnetic resonance (EPR) spectra of the respective samples. Accumulation of intracellular vanadyl was dependent on presence of glucose during incubation. It could be inhibited by various di- and trivalent metal cations. Of these cations lanthanum displayed the strongest inhibitory action. If yeast cells were exposed to more than 50 microM vanadyl sulfate at a pH higher than 4.0, a potassium loss into the medium was detected. The magnitude of this potassium loss suggests a damage of the plasma membrane caused by vanadyl. Upon addition of vanadate to yeast cells surface-bound vanadyl was detectable after several minutes by EPR. This could be the consequence of extracellular reduction of vanadate to vanadyl. The reduction was followed by a slow accumulation of intracellular vanadium, which could be inhibited by lanthanum or phosphate. Therefore, permeation of vanadyl into the cells can be assumed as one mechanism of vanadium accumulation by yeast during incubation with vanadate.