Medium viscosity regulates the activity of membrane-bound and soluble phospholipase A2

Medium viscosity is a regulator of very low density lipoprotein production by cultured hepatocytes; their secretion and synthesis are inversely proportional to the extracellular fluid viscosity. The possibility that the mechanism of this extracellular effect on cell function involves modulation of cell membrane component(s) was considered. Along with this assumption, we studied the effect of medium viscosity on the activity of phospholipase A2 (PLA2), an enzyme present in the cell surface membrane, and the activity has been correlated with cellular secretion. We have found that culture medium viscosity inhibits the activity of PLA2 in the plasma membrane of cultured liver cells, concomitantly with the inhibition of lysosomal enzyme and lipoprotein secretion. It was also found that the degradation of liposomal phosphatidylcholine by soluble snake venom PLA2 is inversely proportional to the solvent viscosity. The possibility that the effect of medium viscosity on the enzymatic reaction involves the modulation of dynamic properties of membrane phospholipids was then considered. This hypothesis was examined by monitoring the fluorescence depolarization of fluorophores incorporated into phospholipid vesicles. No significant effect of the solvent viscosity on the phospholipid bilayer was observed. It is proposed that the regulation of cellular secretion by extracellular fluid viscosity involves modulation of the cell membrane PLA2 activity.     

Inhibitory effects of the porcine follicular fluid on estradiol and progesterone secretion by cultured rat granulosa cells

The effect of porcine follicular fluid on estradiol and progesterone secretion was examined using a rat granulosa cell culture with FSH and testosterone in the medium. Follicular fluids from small (less than 5 mm) and large (greater than 6 mm) follicles (SFFI, LFF1) were treated with charcoal, and then fractionated by filtration through an Amicon XM-50 and an PM-10 membrane. The addition of 25% SFF1 and LFF1 into the culture system significantly inhibited estradiol and progesterone secretion (P less than 0.005). These inhibitory activities were observed in PM-10 retentates (10,000-50,000 MW) and filtrates (less than 10,000 MW) of SFF1 and LFF1. The addition of XM-50 filtrates (less than 50,000 MW) of SFF1 and LFF1 caused a dose-dependent inhibition of estradiol and progesterone secretion. The dose-response relationship between the filtrates and estradiol secretion was linear with a significant correlation coefficient. The addition of the filtrates exerted no inhibitory effect on the growth of the cells cultured. XM-50 filtrate of LFF1 from a batch with a low ratio of small/large follicles showed a lower inhibitory activity on estradiol secretion than that of LFF1, while the inhibitory activities in both filtrates on progesterone secretion were almost equivalent. These results suggest that the follicular fluid of small porcine follicle contains nonsteroidal regulators capable of inhibiting estradiol and progesterone secretion by cultured rat granulosa cells, and that the estradiol secretion inhibitor activity decreases in the fluid of large follicle while the progesterone secretion inhibitor activity does not decrease in it.     

Morphological changes in the clasper gland of the Atlantic stingray, Dasyatis sabina, associated with the seasonal reproductive cycle

The clasper gland of the Atlantic stingray, Dasyatis sabina, was examined over a 1-year period, covering an entire reproductive cycle. Changes in clasper gland tissue architecture, fluid production, and cell proliferation were assessed. No changes in tissue architecture were observed. Evidence of cell proliferation in the gland epithelium was not detected using immunocytochemistry for proliferating cell nuclear antigen, a cellular marker of mitosis. Epithelial cells were not observed to undergo mitosis, and cell membranes remained intact. The lack of structural changes and epithelial cell proliferation supports the proposed merocrinal mode of fluid secretion. Rays captured in nonbreeding months had clasper glands that exhibited tubules with reduced lumens. In contrast, rays caught during the breeding season had clasper gland tubules with enlarged lumens. Clasper gland fluid production was quantified through measurements of the fluid area and tubule area calculated from digital images. Clasper gland fluid production was significantly higher during the mating period than during months not associated with copulatory activity. These data support the notion that the clasper gland is involved in stingray copulatory activity. This study adds to the limited amount of literature focused on this poorly understood component of reproduction in skates and rays.     

Secretion by human fibroblasts of monocyte chemoattractant protein-1, the product of gene JE

We recently purified human monocyte chemoattractant protein-1 (MCP-1) from culture fluids of either human glioma cell lines or mitogen-stimulated human peripheral blood mononuclear leukocytes. It has now been shown that MCP-1 is the product of the gene JE, which was first recognized by its expression in fibroblasts stimulated with platelet-derived growth factor (PDGF). We therefore studied secretion of MCP-1 by three human fibroblast cell lines. Monocyte chemotactic activity was found in culture fluids of all three lines after growth to confluence in DMEM-10% FCS, and the amounts secreted per cell were comparable for the three lines. The MRC-5 line was chosen for further study. Monocyte chemotactic activity secretion by confluent MRC-5 cultures continued after a switch to serum-free medium and was not inhibited by anti-PDGF antibody, indicating that secretion may not have been caused by autocrine release of PDGF. When concentrated serum-free MRC-5 culture fluid was injected into an HPLC gel filtration column, only one chemotactic activity peak was observed, which was in the same location as glioma-derived MCP-1. The activity was completely absorbed out by an anti-MCP-1 affinity column, which indicates that all the chemotactic activity in MRC-5 culture fluid was accounted for by MCP-1. PDGF caused a marked increase in chemotactic activity over that found in serum-free culture fluid of MRC-5 or 501T cells. Immunoprecipitation by anti-human MCP-1 showed two bands, corresponding to the two forms of MCP-1 previously described (MCP-1 alpha and beta); and the amounts increased in response to PDGF stimulation. Thus, the reported increase in human fibroblast JE mRNA in response to PDGF-containing serum stimulation is reflected in increased secretion of the MCP-1 gene product.     

Tolvaptan inhibits ERK-dependent cell proliferation, Cl⁻ secretion, and in vitro cyst growth of human ADPKD cells stimulated by vasopressin

In autosomal dominant polycystic kidney disease (ADPKD), arginine vasopressin (AVP) accelerates cyst growth by stimulating cAMP-dependent ERK activity and epithelial cell proliferation and by promoting Cl(-)-dependent fluid secretion. Tolvaptan, a V2 receptor antagonist, inhibits the renal effects of AVP and slows cyst growth in PKD animals. Here, we determined the effect of graded concentrations of tolvaptan on intracellular cAMP, ERK activity, cell proliferation, and transcellular Cl(-) secretion using human ADPKD cyst epithelial cells. Incubation of ADPKD cells with 10(-9) M AVP increased intracellular cAMP and stimulated ERK and cell proliferation. Tolvaptan caused a concentration-dependent inhibition of AVP-induced cAMP production with an apparent IC(50) of ～10(-10) M. Correspondingly, tolvaptan inhibited AVP-induced ERK signaling and cell proliferation. Basolateral application of AVP to ADPKD cell monolayers grown on permeable supports caused a sustained increase in short-circuit current that was completely blocked by the Cl(-) channel blocker CFTR(inh-172), consistent with AVP-induced transepithelial Cl(-) secretion. Tolvaptan inhibited AVP-induced Cl(-) secretion and decreased in vitro cyst growth of ADPKD cells cultured within a three-dimensional collagen matrix. These data demonstrate that relatively low concentrations of tolvaptan inhibit AVP-stimulated cell proliferation and Cl(-)-dependent fluid secretion by human ADPKD cystic cells.     

A model for fluid secretion in the exocrine pancreas

Fluid secretion by the isolated rabbit pancreas is strongly dependent on the presence of Na+ in the bathing medium. Substitution of Na+ by another cation such as Li+ or K+ causes an inhibition of fluid secretion rate and a change in the composition of the secreted fluid which is dependent on the nature of the substituent cation. Stimulation of the pancreas by CCK-8 or carbachol increases paracellular ion permeability and, in some cases, also fluid secretion rate. We present a simple, quantitative model for ion and water secretion which accounts for the effects observed upon Na+ substitution and stimulation. The main features are active, Na+-dependent transcellular HCO3- transport and passive, paracellular cation and anion permeation. The activity of the HCO3- pump is dependent on the energy status of the cell and on the Na+ concentration in the bathing medium, and is competitively inhibited by K+. The paracellular ion permeabilities can be modulated by stimulatory agonists. We examine the extent to which, according to the model, fluid secretion is controlled by the various system parameters such as ion permeabilities and ion pump activity, and by external parameters such as the ion concentrations in the bathing medium. In addition, calculation of the effects of changes in these parameters are carried out in order to gain more insight in the mechanisms of secretion.     

A stimulatory effect of the fluid from preimplantation rabbit blastocysts upon luteinization of monkey granulosa cell cultures.

Blastocyst fluid was aspirated from Day 6 1/2--7 rabbit blastocysts and was added to cultures of granulosa cells obtained from preovulatory follicles of untreated rhesus monkeys or from follicles of monkeys or from follicles of monkeys treated with PMSG. The stimulation of progesterone secretion was measured and equated with that produced by hCG. The hCG-like activity was also measured in a radioreceptor assay using 125I-labelled hCG and porcine granulosa cells. In 8 out of 10 experiments with cultured cells from untreated monkeys, addition of 20% blastocyst fluid from Days 6--9 of culture stimulated progesterone secretion by 2- to 6-fold. Similar findings were obtained in 5 experiments with cultures from PMSG-treated monkeys except that the blastocyst fluid was added from Days 0 to 6 of culture. The granulosa cells in such cultures underwent morphological luteinization. Compared to a standard of purified hCG the blastocyst fluid contained about 0.76--2.5 ng hCG-like activity/ml which was non-dialysable. The radioreceptor assay indicated the presence of 0.5--2.5 ng hCG-like material/ml.     

A pyruvate cycling pathway involving cytosolic NADP-dependent isocitrate dehydrogenase regulates glucose-stimulated insulin secretion

Glucose-stimulated insulin secretion (GSIS) from pancreatic islet beta-cells is central to control of mammalian fuel homeostasis. Glucose metabolism mediates GSIS in part via ATP-regulated K+ (KATP) channels, but multiple lines of evidence suggest participation of other signals. Here we investigated the role of cytosolic NADP-dependent isocitrate dehydrogenase (ICDc) in control of GSIS in beta-cells. Delivery of small interfering RNAs specific for ICDc caused impairment of GSIS in two independent robustly glucose-responsive rat insulinoma (INS-1-derived) cell lines and in primary rat islets. Suppression of ICDc also attenuated the glucose-induced increments in pyruvate cycling activity and in NADPH levels, a predicted by-product of pyruvate cycling pathways, as well as the total cellular NADP(H) content. Metabolic profiling of eight organic acids in cell extracts revealed that suppression of ICDc caused increases in lactate production in both INS-1-derived cell lines and primary islets, consistent with the attenuation of pyruvate cycling, with no significant changes in other intermediates. Based on these studies, we propose that a pyruvate cycling pathway involving ICDc plays an important role in control of GSIS.     

An antidiuretic factor in the forest ant: purification and physiological effects on the Malpighian tubules

Formica polyctena antidiuretic factor (FopADF) was purified from a 15% trifluoroacetic acid (TFA) extract of the abdomens of 150,000 worker ants. After solid phase extraction of the crude extract and reversed-phase HPLC on two C(18) columns, an antidiuretic factor was isolated. Tested at a concentration of 1.0 ant-equivalents/μl (ant-eq/μl), the factor reversibly inhibited fluid secretion of isolated Malpighian tubules to 29+/-5% (mean+/-SE, n=24) of the control value. The same concentration of FopADF reversibly depolarized both the basolateral membrane potential (V(bl)), from -21+/-2 mV to -3+/-1 mV (n=5), and the apical membrane potential (V(ap)), from -65+/-5 mV to -20+/-5 mV (n=5). Similar effects on fluid secretion and V(ap) were caused by a TFA extract of the haemolymph of ants with non-secreting tubules. Unfortunately, further purification of FopADF on a C(4) column led to a loss of activity in the fluid secretion assay. This is the first time an endogenous antidiuretic factor acting directly on Malpighian tubules has been partially purified and shown to depolarize the tubule cell membranes.     

Local regulation of granulosa cell maturation

Fluid from small antral follicles inhibits several functions of porcine granulosa cells from 3-10-mm follicles in vitro, whereas fluid from large follicles stimulates cells from small follicles. Local factors may be needed in vivo to enable granulosa cells to fully respond to gonadotrophins. Only those follicles containing local stimulators may develop while those containing inhibitors may become arrested in development or become atretic. We have compared the actions of GnRH analogs and chondroitin sulfate (CS) on porcine granulosa cell steroidogenesis with actions of follicular fluids. GnRH agonist mimicked follicular fluid inhibition of progesterone secretion but GnRH antagonist did not antagonize follicular fluid's inhibitory actions. GnRH antagonist mimicked follicular fluid enhancement of basal and LH-stimulated progesterone secretion, but did not mimic follicular fluid enhancement of FSH action or stimulation of estrogen secretion. GnRH agonist blocked the enhancement of LH-stimulated progesterone secretion by both GnRH antagonist and stimulatory follicular fluid. CS inhibited basal and LH-stimulated progesterone secretion but did not inhibit pregnenolone utilization, aromatase activity or estrogen secretion. GnRH-like molecules and CS may be partially responsible for follicular fluid actions on granulosa cells. The actions of other molecules are needed to explain the total effects of follicular fluids on granulosa cells.     

A soluble form of the interleukin 4 receptor in biological fluids

Murine biological fluids and murine cell culture supernatants were analyzed for the presence of soluble murine interleukin 4 receptor (sIL4R) with the use of two monoclonal antibodies directed against the receptor. Mouse urine, serum, ascitic fluid, and cell culture supernatants contained varying levels of immunoreactive protein. All of the immunoreactive protein possessed interleukin 4 (IL 4) binding activity. Following partial purification of ascitic fluid a protein was isolated that binds IL 4 with high affinity. This data is consistent with the fact that murine biological fluids contain a soluble version of the murine IL 4 receptor that arises via secretion of the soluble receptor and/or via shedding of the extracellular portion of the full-length receptor from the cell surface.     

Stimulation of protein secretion in the initial segment of the rat epididymis by fluid from the ram rete testis

Zone 1A of the ductus epididymidis was perfused with ovine rete testis fluid (nRTF) and modifications of it, and a synthetic medium (sRTF) based on the inorganic composition of nRTF. There was little fluid transport by the duct mucosa and nRTF stimulated protein secretion. The secretagogue activity was not extracted by charcoal, was sensitive to protease digestion and was present in a portion of nRTF with a molecular weight of greater than 10,000. The addition of bovine serum albumin to the sRTF stimulated protein secretion, but not to the same extent as equal amounts of protein in nRTF. Polyacrylamide gel electrophoresis of the perfusates showed that proteins with molecular weights of 19,000 (all rats studied), and 22,000, 30,000 and 60,000 (at least half the rats studied) were secreted into the perfusion fluids as well as some blood proteins, but the pattern of secretion was not affected by the composition of the perfusion fluid.     

Localization of proteolytic enzymes in cells of Bacillus intermedius

The activities of proteinases in the culture fluid and cellular fractions of Bacillus intermedius 3-19 grown under various conditions were studied. Thiol-dependent serine proteinase was the prevalent enzyme in the total pool of extracellular proteinases (70%); its catalytically active form was also detected in the cell membrane and, during active enzyme production, in the cell wall. Another enzyme, glutamyl endopeptidase (10% of the total pool), was detected in the cell membrane; it was also found in the cell wall and cytoplasm during active enzyme secretion into the growth medium. Production of these enzymes was maximal on medium containing inorganic phosphate and gelatin and decreased 2- to 4-fold on medium with glucose and lactate. The level of activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins. The addition of CoCl2 (2 mM) into the medium caused an essential increase in extracellular glutamyl endopeptidase activity and promoted the release of the membrane-bound enzyme into the culture fluid. Proteolytic activity towards casein was also detected in the cytoplasm. The proteinases localized in the cytoplasm were shown to differ in their properties from those secreted.     

Anion secretion by the isolated rabbit pancreas

The isolated rabbit pancreas secretes a fluid containing chloride and bicarbonate in about equal concentrations. Replacement of bicarbonate by acetate, phosphate or isethionate, replacement of Na⁺ by Li⁺ and addition of ouabain to the bathing medium of the pancreas inhibit the secretion of fluid, chloride and bicarbonate in a similar fashion and by maximally 100%. Replacement of chloride by isethionate inhibits fluid secretion by maximally 50%, chloride secretion by 90% and bicarbonate secretion by 20%. It is concluded that fluid secretion is based on a Na⁺-gradient-dependent bicarbonate influx or proton efflux in the ductular cell, and that the secretion of chloride is secondary to that of bicarbonate.     

Peroxidase Activity in the Leaf Elongation Zone of Tall Fescue : II. Spatial Distribution of Apoplastic Peroxidase Activity in Genotypes Differing in Length of the Elongation Zone

Previous work suggested that cell wall peroxidase activity increased as cells were displaced through the elongation zone in leaf blades of tall fescue (Festuca arundinacea Schreb.). In this study, two genotypes that differ in length of the elongation zone were used to examine the relationship between peroxidase activity in apoplastic fluid of intact leaf blade segments and the spatial distribution of leaf growth. Apoplastic fluid was extracted by vacuum infiltration and centrifugation, and peroxidase activity was assayed spectrophotometrically. Isoelectric focusing was used to characterize the isoforms of apoplastic peroxidase within the region of elongation and in the region of secondary cell wall deposition, which is distal to the elongation zone. A striking correlation was found in each genotype between both the location and timing of increase in apoplastic peroxidase activity and the onset of growth deceleration. Only cationic isoforms of apoplastic peroxidase could be identified in the elongation zone, whereas additional anionic isoforms appeared in the region of secondary cell wall deposition. We conclude that cessation of elongation growth in tall fescue leaf blades is likely to be related to the secretion of cationic isoforms of peroxidase into the cell wall.     

Fine structure of the spermatheca and of the accessory glands in Orchesella villosa (Collembola, Hexapoda)

The spermatheca and the accessory glands of the collembolan Orchesella villosa are described for the first time. Both organs exhibit ultrastructural differences, according to the time of the intermolt in which the specimens were observed. A thick cuticular layer lines the epithelial cells of the accessory glands. In the reproductive phase, they are involved in secretory activity; a moderately dense secretion found in the apical cell region opens into the gland lumen. Cells with an extracellular cistern are intermingled with the secretory cells. These cells could be involved in fluid secretion, with the secretory product opening into the cistern which is filled with an electron-transparent material. After the reproductive phase, the gland lumen becomes filled with a dense secretion. The accessory gland secretion may play a protective role towards the eggs. The spermatheca is located between the accessory glands; its epithelium is lined by a thin cuticle forming spine-like projections into the lumen and consists of cells provided with an extracellular cistern. Secretory cells, similar to those seen in the accessory glands, are missing. Cells with a cistern could be involved in the production of a fluid secretion determining sperm unrolling and sperm motility.     

Regulation of protein secretion in Chinese hamster ovary cells by cell cycle position and cell density : Plasminogen activator, procollagen and fibronectin

To study the relationship between cell growth control, cell contact, and protein secretion, we examined the production of plasminogen activator, procollagen, and fibronectin by Chinese hamster ovary (CHO) fibroblasts, both as a function of position in the cell cycle and as a function of cell density. CHO fibroblasts that were synchronized at hourly intervals throughout the cell cycle by mitotic selection in an automated roller bottle apparatus secreted plasminogen activator only during the G2 and M phases of the cell cycle (10–14 h after mitotic selection). Cell-associated plasminogen activator activity was variable during G1 and S, but was greatly reduced during G2 and M. In contrast, secretion of the connective tissue matrix proteins, procollagen and fibronectin, was controlled by cell density rather than by cell cycle position. Type III procollagen and fibronectin were secreted throughout the cell cycle with no pronounced variations. Type I procollagen was not secreted by cycling cells and was observed in confluent cultures only after 24–48 h. To correlate these changes in protein secretion patterns with cell shape and contact, we used scanning electron microscopy (SEM) to study the appearance of CHO cells after mitotic selection. Actively dividing cells retained a high proportion of rounded, ruffled, and blebbed cells during all phases of the cell cycle. Only with increased cell density in contact-inhibited confluent cultures did most cells begin to flatten and spread. Thus, secretion of and attachment to extracellular matrix did not occur in rapidly dividing cells, but appeared to require the increased cell-cell contact and spreading that accompanies contact inhibition of growth. On the other hand, increased secretion of plasminogen activator was directly related to cell division and may be part of a sequence of events that allows cells growing in culture to loosen extracellular attachments in preparation for rounding and cytokinesis.     

Stimulatory and inhibitory effects of endogenous factors in head extracts of Formica polyctena (Hymenoptera) on the fluid secretion of Malpighian tubules

This study was designed to investigate the regulation of fluid secretion by the Malpighian tubules of the worker ant Formica polyctena (Hymenoptera). Different solvent systems were used to make crude head extracts and to determine the solubility of the diuretic factors. Surprisingly, when distilled water, acid acetone, methanol and 15% trifluoroacetic acid (TFA) were used as solvents, two consecutive significant stimulations of fluid secretion were obtained: the first, when adding the extract to the tubule and the second, when washing it out. Extract obtained with a fifth solvent, Ringer solution, gave an almost complete but reversible inhibition of fluid secretion. Extracts were prepurified by means of a disposable C₁₈ column by elution with 20, 40, 60 and 80% acetonitrile. When the fractions were kept apart the 40% acetonitrile fraction caused an inhibition of fluid secretion. The 20, 60 and 80% acetonitrile fractions on the other hand resulted in two consecutive stimulations as described above. The dose-response curve for 15% TFA extract was bell-shaped with a threshold concentration of 1 × 10⁻³ heads/μl Ringer. A maximum response (stimulation of fluid secretion by a factor of 3.3 ± 0.72, n = 10) was observed with a concentration of 5 × 10⁻² heads/μl Ringer. Higher concentrations resulted in small increases of fluid secretion rates and in the appearance of the second stimulation when the extract was washed out. The activity present in the heads of Formica was not destroyed by boiling or by proteolytic enzymes (trypsin, chymotrypsin, pronase E and proteinase K). Only immobilized aminopeptidase M, which destroys the activity of peptides with a free N-terminus, had a significant effect on the activity of a 15% TFA head extract. Various biogenic amines were tested for their ability to mimic the effect of the head extracts. Only octopamine and dopamine evoked a small and transient increase in secretion rate. Thus biogenic amines probably do not contribute to a large extent to the response of Formica tubules to the crude head extract. The possibility that both diuretic and antidiuretic factors are present in the extract is discussed.     

SecA: the ubiquitous component of preprotein translocase in prokaryotes

SecA is an obligatory component of the complex hetero-septameric translocase of prokaryotes. It is unique in that it exists as two forms within the holoenzyme; first, as a structural component of the preprotein channel and second, as an ATP-dependent membrane cycling factor facilitating the translocation of a broad class of proteins across the cytoplasmic membrane. While the translocase activity of SecA appears to be functionally conserved, it is not clear whether the mechanisms of regulation of the secA gene are similarly maintained. The recent characterization of an ATP-dependent RNA helicase activity of SecA offers a unique mechanism for SecA to communicate the secretion status of the cell to the appropriate regulatory circuits simply by the unwinding of an appropriate RNA target. Resolution of these two activities through combined biochemical, genetic, and biophysical studies should lead to a better understanding of the role of SecA in bacterial secretion.     

The influence of starvation and natural refeeding on the rate of triacylglycerol/fatty acid substrate cycling in brown adipose tissue and different white adipose sites of the ratin vivo. The role of insulin and the sympathetic nervous system

Triacylglycerol/fatty acid substrate cycling was measuredin vivo in brown adipose tissue (BAT) and white adipose tissue (WAT) of fed, starved and refed rats. Starvation (24 h) significantly decreased the rate of cycling in BAT, and refeeding chow diet led to a rapid, 6-fold increase in cycling. Cycling rate in WAT was much lower than in BAT, and was not influenced by fasting or refeeding. Similar rates of cycling were found in epididymal, mesenteric, subcutaneous, and scapular WAT depots. Sympathetic denervation of interscapular BAT abolished the response of the tissue to refeeding, as did acute suppression of insulin secretion. Similarly, rats fasted for 3 days showed no acute increase in the activity of the cycle following refeeding.