Large Intracellular Pool Research Articles

Modulation of the phosphate-starvation response in Escherichia coli by genetic manipulation of the polyphosphate pathways

The effect of intracellular polyphosphate on the phosphate-starvation response in Escherichia coli was studied by genetically manipulating the intracellular polyphosphate levels and by performing phosphate shifts on the genetically engineered strains. Strains that produced large quantities of polyphosphate and were able to degrade it induced the phosphate-starvation response to a lesser extent than wild-type strains, whereas strains that were unable to degrade a large intracellular polyphosphate pool induced the phosphate-starvation response to a greater extent than wild-type strains. These results have important implications for expression of heterologous genes under control of the phoA promoter. (c) 1996 John Wiley & Sons, Inc.

Diverging pathways for lipopolysaccharide and CD14 in human monocytes

CD14 is considered to be the major endotoxin (lipopolysaccharide [LPS]) binding molecule on human monocytes. It initiates cellular response, but its role in the clearance of LPS is not well understood. Under conditions that ensure totally CD14-dependent LPS binding on human monocytes, the internalization mechanisms of LPS and CD14 were studied. The uptake and intracellular distribution of fluorescein isothiocyanate (FITC)-LPS and CD14 was determined by flow cytometry, trypan blue quenching, and confocal fluorescence microscopy. Incubation of surface-biotinylated cells with LPS at 37 degrees C or 4 degrees C and subsequent subfractionation was used to further characterize CD14 internalization. The amount of the intracellular CD14 was estimated by CD14 enzyme-linked immunosorbent assay (ELISA). The internalization rate of 10 ng/ml FITC-LPS with 1% human serum was 1% of bound endotoxin per minute, whereas CD14 expression did not decrease at the same time surface. We proved the presence of an intracellular CD14 pool (2.68 x 10(6) molecules per unstimulated monocyte) and could show that internalized FITC-LPS molecules can be found in different intracellular compartments than CD14. Subfractionation of LPS-treated biotinylated monocytes showed no change in biotinylated CD14 in the membrane fraction independently of the incubation temperature (37 degrees C or at 4 degrees C) used, indicating that these CD14 molecules were not taken up by an active process. These data indicate the presence of a large intracellular CD14 pool in monocytes with a yet unknown function, and suggest that LPS and CD14 molecules can be internalized independently after association on the cell surface.

Carbon monoxide induces vasodilation and nitric oxide release but suppresses endothelial NOS.

The vascular effects of carbon monoxide (CO) resemble those of nitric oxide (NO), but it is unknown whether the two messengers converge or exhibit reciprocal feedback regulation. These questions were examined in microdissected perfused renal resistance arteries (RRA) studied using NO-sensitive microelectrodes. Perfusion of RRA with buffers containing increasing concentrations of CO resulted in a biphasic release of NO. The NO response peaked at 100 nM CO and then declined to virtually zero at 10 microM. When a series of 50-s pulses of 100 nM CO were applied repeatedly (150-s interval), the amplitude of consecutive NO responses was diminished. NO release from RRA showed dependence on L-arginine but not D-arginine, and the responses to CO were inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthases (NOS). CO (100 nM) also suppressed NO release induced by 100 microM carbachol, a potent agonist for endothelial NOS (eNOS). RRA from rats in which endogenous CO production from inducible HO was elevated (cobalt chloride 12 h prior to study) also showed suppressed responses to carbachol. Furthermore, responses consistent with these findings were obtained in juxtamedullary afferent arterioles perfused in vitro, where the vasodilatory response to CO was biphasic and the response to acetylcholine was blunted. Collectively, these data suggest that the CO-induced NO release could be attributed to either stimulation of eNOS or to NO displacement from a cellular storage pool. To address this, direct in vitro measurements with an NO-selective electrode of NO production by recombinant eNOS revealed that CO dose-dependently inhibits NO synthesis. Together, the above data demonstrate that, whereas high levels of CO inhibit NOS activity and NO generation, lower concentrations of CO induce release of NO from a large intracellular pool and, therefore, may mimic the vascular effects of NO.

Distribution of AMPA receptor subunits GluR1‐4 in the dorsal vagal complex of the rat: a light and electron microscope immunocytochemical study

The dorsal vagal complex, localized in the dorsomedial medulla, includes the nucleus tractus solitarii (NTS), the dorsal motor nucleus of the vagus nerve (DMN) and the area postrema (AP). The distribution of AMPA-preferring glutamate receptors (AMPA receptors) within this region was investigated using immunohistochemistry and antibodies recognizing either one (GluR1 or GluR4) or two (GluR2 and GluR3) AMPA receptors subunits. The distribution of GluR1 immunoreactivity showed high contrast of staining between strongly and lightly labeled areas. Labeling was intense in the AP and weak in the NTS, except for its medial and dorsalmost parts which exhibited moderate staining. Almost no GluR1 immunoreactivity was found in the DMN. GluR2/3 immunolabeling was present in the entire dorsal vagal complex. This labeling was strong in the AP, the DMN and the medial half of the NTS and moderate in the lateral half of the NTS, except for the interstitial subdivision which exhibited intense staining. Labeling induced by the GluR4 antibody was very weak throughout the dorsal vagal complex. Ultrastructural examination showed that GluR1 and GluR2/3 immunoreactivity was localized in neuronal cell bodies and dendrites. No labeled axon terminal or glial cell body was found. Immunoperoxidase staining in labeled cell bodies and dendrites was associated with intracellular organelles (microtubules, mitochondria, cisternae of the endoplasmic reticulum, . . .) and/or parts of the plasma membrane. Plasma membrane labeling was often associated with asymmetrical synaptic differentiations. No labeled symmetrical synapse was found using either GluR1 or GluR2/3 antibody. The present results show that AMPA receptors have a widespread distribution in neuronal perikarya and dendrites of the rat dorsal vagal complex. They suggest differences in subunit composition between AMPA receptors localized in the NTS, the DMN and the AP. Ultrastructural data are consistent with the fact that AMPA receptors associated with the plasma membrane are mostly synaptic receptors. However, they also suggest the existence of a large intracellular pool of receptor subunits in neuronal soma and dendrites. Synapse 34:55–67, 1999. © 1999 Wiley-Liss, Inc.

Distribution of AMPA receptor subunits GluR1-4 in the dorsal vagal complex of the rat: a light and electron microscope immunocytochemical study.

The dorsal vagal complex, localized in the dorsomedial medulla, includes the nucleus tractus solitarii (NTS), the dorsal motor nucleus of the vagus nerve (DMN) and the area postrema (AP). The distribution of AMPA-preferring glutamate receptors (AMPA receptors) within this region was investigated using immunohistochemistry and antibodies recognizing either one (GluR1 or GluR4) or two (GluR2 and GluR3) AMPA receptors subunits. The distribution of GluR1 immunoreactivity showed high contrast of staining between strongly and lightly labeled areas. Labeling was intense in the AP and weak in the NTS, except for its medial and dorsalmost parts which exhibited moderate staining. Almost no GluR1 immunoreactivity was found in the DMN. GluR2/3 immunolabeling was present in the entire dorsal vagal complex. This labeling was strong in the AP, the DMN and the medial half of the NTS and moderate in the lateral half of the NTS, except for the interstitial subdivision which exhibited intense staining. Labeling induced by the GluR4 antibody was very weak throughout the dorsal vagal complex. Ultrastructural examination showed that GluR1 and GluR2/3 immunoreactivity was localized in neuronal cell bodies and dendrites. No labeled axon terminal or glial cell body was found. Immunoperoxidase staining in labeled cell bodies and dendrites was associated with intracellular organelles (microtubules, mitochondria, cisternae of the endoplasmic reticulum,.) and/or parts of the plasma membrane. Plasma membrane labeling was often associated with asymmetrical synaptic differentiations. No labeled symmetrical synapse was found using either GluR1 or GluR2/3 antibody. The present results show that AMPA receptors have a widespread distribution in neuronal perikarya and dendrites of the rat dorsal vagal complex. They suggest differences in subunit composition between AMPA receptors localized in the NTS, the DMN and the AP. Ultrastructural data are consistent with the fact that AMPA receptors associated with the plasma membrane are mostly synaptic receptors. However, they also suggest the existence of a large intracellular pool of receptor subunits in neuronal soma and dendrites.

Immunolocalization of CD1d in human intestinal epithelial cells and identification of a beta2-microglobulin-associated form.

In order to better understand the role of intestinal CD1d, we sought to define the cellular localization and further characterize the biochemical structure of CD1d in human intestinal epithelial cells (IEC). Using a CD1d-specific rabbit anti-gst-CD1d antibody, immunoprecipitation of radiolabeled cell surface proteins detected a previously identified 37 kDa protein as well as a 48-50 kDa protein which were confirmed by Western blotting with a CD1d-specific mAb, D5. Immunoprecipitation of protein lysates with the CD1d-specific mAb, D5 and 51.1.3, and the beta2-microglobulin (beta2m)-specific mAb, BBM.1, followed by N-glycanase digestion and Western blotting with the D5 mAb showed that the 48-50 kDa protein was a beta2m-associated, CD1d glycoprotein. CD1d was immunolocalized to the apical and lateral regions of native small and large intestinal IEC as defined by confocal laser microscopy using the D5 mAb and the rabbit anti-gst-CD1d antibody. In addition, a large apical intracellular pool of CD1d was identified. Identical observations were made with polarized T84 cells. Selective biotin labeling of apical and basolateral cell surfaces followed by immunoprecipitation with the D5 mAb, N-glycanase digestion and avidin blotting confirmed the presence of glycosylated CD1d on both cell surfaces and immunolocalization of the 37 kDa non-glycosylated form of CD1d to the apical cell surface. These studies show that CD1d is located in an ideal position for luminal antigen sampling and presentation to subjacent intraepithelial lymphocytes.

Cholesterol Distribution in Living Cells: Fluorescence Imaging Using Dehydroergosterol as a Fluorescent Cholesterol Analog

Cholesterol is an important constituent of most mammalian cell membranes and its concentration in various cellular membranes is tightly regulated. Although there is much information about cholesterol distribution and trafficking in cells, it is primarily derived from indirect measurements, and the results obtained using different approaches are often conflicting. A cholesterol analog that faithfully mimics the properties of cholesterol and can be followed in living cells would thus be very useful. In this study, we report the fluorescence imaging of such an analog, dehydroergosterol (DHE), in living cells. DHE differs from cholesterol in having three additional double bonds and an extra methyl group. In model systems, DHE closely mimics the behavior of native cholesterol. Using triple-labeling studies, we show that DHE colocalizes extensively with endocytosed transferrin, an endocytic recycling compartment marker, and with a marker for the trans-Golgi network, Tac-TGN38. This distribution of DHE is qualitatively similar to that observed when cells are labeled with the fluorescent cholesterol-binding polyene antibiotic, filipin, although there are differences in apparent proportions of DHE and filipin that are localized at the plasma membrane. Another cholesterol derivative, 25-NBD-cholesterol, has a structure that is compromised by the presence of a bulky NBD group and does not distribute to the same organelles as DHE or filipin. In addition, we show in this manuscript that kinetic processes can be followed in living cells by monitoring recovery of DHE fluorescence in a photobleached region over time. Our observations provide evidence for the presence of a large intracellular cholesterol pool in the endocytic recycling compartment and the trans-Golgi network that might play important roles in the trafficking of lipids, lipid-anchored proteins, and transmembrane proteins that preferentially partition into cholesterol-enriched membrane domains. In addition, this intracellular cholesterol pool might be involved in the maintenance of cellular cholesterol homeostasis.

Presence of a large intracellular pool of C3a receptor (C3aR) in human T lymphocytes

Presence of a large intracellular pool of C3a receptor (C3aR) in human T lymphocytes

Efficient secretion of the model antigen M6-gp41E in Lactobacillus plantarum NCIMB 8826.

Four Lactobacillus strains (Lb. plantarum NCIMB 8826, Lb. paracasei LbTGS1.4, Lb. casei ATCC 393 and Lb. fermentum KLD) were tested for their ability to produce and secrete heterologous proteins. These strains were first screened with an alpha-amylase reporter under the control of a set of expression or expression/secretion signals from various lactic acid bacteria. With most of the constructions tested, the level of extracellular production was highest in Lb. plantarum NCIMB 8826, and lowest in Lb. paracasei LbTGS1.4. These two strains were next assayed using a model antigen consisting of the N-terminal part of the M6 protein from Streptococcus pyogenes fused to the linear epitope ELDKWAS from human immunodeficiency virus gp41 protein. Secretion of this heterologous protein was inefficient in Lb. paracasei LbTGS1.4, which accumulated a large intracellular pool of the unprocessed precursor, whereas Lb. plantarum NCIMB 8826 was able to secrete the antigen to a level as high as 10 mg l-1.

Expression of the IL-2 receptor gamma subunit in resting human CD4 T lymphocytes: mRNA is constitutively transcribed and the protein stored as an intracellular component.

IL-2 receptor (IL-2R) is composed of three subunits named IL-2R alpha, IL-2R gamma. Here, we study the expression of the IL-2R gamma in highly purified, resting peripheral human CD4 T lymphocytes. We show by FACS analysis that the IL-2R gamma subunit is not detectable at the cell surface of peripheral CD4 T lymphocytes. This result has been verified after acid treatment of the cell surface and analysis with three specific anti-IL-2R gamma mAb. Using RT-PCR and intracellular FACS analysis, we demonstrate that IL-2R gamma is constitutively expressed at the mRNA level and the protein is stored as an intracellular component in resting CD4 T lymphocytes. IL-2R alpha and beta subunits are not detectable by these methods. In addition, we show that CD4 T cell remain insensitive to a variety of cytokines that share IL-2R gamma as a common subunit of their receptors (e.g. IL-2, IL-4, IL-7 and IL-15). The kinetics of cell surface expression of IL-2R gamma have been studied after activation of CD4 T lymphocytes and compared with induction of IL-2R alpha and IL-2R beta. Maximum expression of IL-2R gamma is observed after 2 days of stimulation, and remains constant and comparable to IL-2R beta up to day 5. We conclude from these studies that IL-2R gamma is translocated to the membrane only after T cell activation and induction of the IL-2R alpha and IL-2R beta genes. We hypothesize that in CD4 T cells a large intracellular pool of IL-2R gamma is present but that its cell surface translocation depends on the expression of alpha and/or beta chains specific for IL-2, IL-4, IL-7, IL-9 or IL-15.

Effect of phytoplankton cell size on transient-state nitrate and ammonium uptake kinetics.

SUMMARYThe uptake of nitrate or ammonium (at a concentration of 10 mol I-1) by marine phytoplankton was studied in relation to cell size. Initial specific nitrate uptake rates by small (35000 m3) and large (130000 m3) cells of the diatom Ditylum brightwellii did not differ significantly. However, the larger cells maintained a high uptake rate for a longer time. Therefore, they accumulated nitrate in a higher biomass-specific pool than the smaller cells. In the dark, this effect was even more pronounced. Two smaller diatom species, Lauderia borealis (7474 m3) and Thalassiosira pseudonana (98 (m3), had lower initial specific nitrate uptake rates and lower intracellular pools. Transient-state ammonium uptake did not result in accumulation of large intracellular pools of ammonium. Theoretically, and on the basis of the presented results, we stress the dualistic functional role of the vacuole. A large vacuole is an effective way for larger algal species to possess a minimum cell nutrient quota/cell surface ratio which is in the range of smaller species. Furthermore, by functioning as a storage reservoir it reduces inhibition of the uptake rate by cytoplasmic accumulated nutrients. The effect of the latter mechanism is that larger algal species are better at nitrate uptake under fluctuating conditions. These results imply that, in nitrogen-controlled marine systems, resource competition under fluctuating nutrient concentrations can only lead to a shift towards larger phytoplankton species if nitrate rather than ammonium is the main nitrogen source. From theoretical considerations it is argued that the maximum growth rate of algae is determined by nutrient assimilation properties rather than by photosynthetic capacity.

Characterization of integrin expression and regulation on SW-480 human colon adenocarcinoma cells and the effect of rhodostomin on basal and upregulated tumor cell adhesion

Integrins are a superfamily of cell surface glycoproteins that mediate cell-extracellular matrix (ECM) and cell-cell adhesion. Immunofluorescence microscopy and flow cytometric analysis using anti-antigrin mAbs as the primary binding ligands demonstrated that the platelet integrin receptor α IIb β 3 , as well as α v β 3,α 5β 1 and α 6β 1 , are present on the surface of SW-480 human colon adenocarcinoma cells. Monoclonal antibodies (mAbs) against α IIb β 3 inhibited unstimulated basal adhesion to fibronectin by approximately 30% and 40%, respectively. The surface immunoreactivity of tumor cells for α IIb β 3 was enhanced by pretreatment (5 min) with a phorbol ester ( 12-O- tetradecanoylphorbol-13- acetate (TPA)) or a lipoxygenase metabolite of arachidonic acid, 12-hdyroxyeicosatetraenoic acid (12-HETE) in a dose- and time-dependent manner. SW-480 cells possess a large intracellular pool of α IIb β 3 , from which the receptor complex translocates to the cell surface following pretreatment with TPA or 12(S)- HETE . This pretreatment enhances adhesion to fibronectin, which is exclusively by α IIb β 3 integrins. Staurosporine was found to block α IIb β 3 up-regulation and enhanced-adhesion. TPA and 12-(S)_- HETE also facilitated the redisitribution of α IIbβ 3 during the enhanced-spreading process. Rhodostomin, an Arg-Gly-Asp-(RGD) containing antiplatelet snake venom peptide, was about 400-times more patent than RGDS at inhibiting control, TPA- or 12-(S)- HETE -enhanced adhesion of SW-480 cells tp fibronectin. The binding of mAbs against α IIb β 3, α v β 3 and α 5 1 was inhibited by pretreatment with rhodostomin, suggesting that rhodostomin binds via its RGD sequence to multiple integrain receptors (i.e., α IIb β 3 , α v β 3 , α 5β 1 ) expressed on the SW-480 cell surface, inhibiting cell adhesion to ECM.

Effects of perphenazine on the metabolism of inositol phospholipids in SK-N-BE(2) human neuroblastoma cells

Administration of myo-[ 3H]inositol to SK-N-BE(2) human neuroblastoma cells for 24 hr resulted in equilibrium labelling of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP 2), as well as in retention of a large intracellular pool of free myo-[ 3H]inositol. Equilibrium labelling was no longer observed when cells were treated for 2 hr with 20 μM perphenazine (PPZ) in label-free medium; under these conditions, myo-[ 3H]inositol from the retained intracellular pool was incorporated into PI and PIP but not into PIP 2. Analysis of water-soluble myo-[ 3H]inositol derivatives and inositol 1,4,5-trisphosphate mass determination indicated that PPZ did not stimulate phosphoinositide hydrolysis by phospholipase C. These results indicate that PPZ raises PI and PIP levels, whereas it is ineffective in expanding the PIP 2 pool. The latter effect is not due to a concomitant synthesis and hydrolysis of this lipid.

Regulation of CD2-Mediated Heterotypic Interactions of Murine T Lymphocytes

In this study we have investigated the expression and function of the murine CD2 receptor in T cells. The surface level of CD2 increased between 300 and 400% when T cells were activated, which also induced a rapid increase of nearly 40-fold in the steady-state levels of CD2 mRNA. Although the activated lymphocytes were found to internalize and degrade nearly 50% of membrane-bound CD2, in addition to shedding the CD2 receptor from the cell surface, this does not account for the discrepancy between the increase in mRNA and antigen levels during activation. These findings suggest that the expression of CD2 is also regulated by post-transcriptional processes which control the translational efficiency of the CD2 message. Activation of T cells enhanced their interaction with mesenchymal cell targets (fibroblasts) via a CD2-dependent adhesion pathway which was not inhibited by the anti-mouse CD48 (sgp-60) mAb OX78, the only counter-receptor thus far identified on other cells as a ligand for the mouse CD2 receptor. Moreover, since murine fibroblasts were found not to express CD48, our results implicate a novel ligand for CD2, possibly a homologue of the human LFA-3. This new pathway of heterotypic T cell interaction would be facilitated by utilization of the large intracellular pool of CD2 transcripts to up-regulate CD2 expression and, as this receptor is a major signal-transducing molecule, would further enhance T cell activation and increase CD2-mediated adhesion.

Quantitation of blood‐brain barrier ultrastructure

This paper describes a quantitative approach to evaluating the ultrastructural features of brain capillaries that relate to the low non-specific permeability of the blood-brain barrier (BBB). Critical features in this approach include examination of large numbers of tissue samples and consistent, objective means of measuring features of interest. Junctional clefts, i.e., continuous channels between tight junctional regions correlate well with the know vascular permeability, being low in normal adult blood-brain barrier, high in fetal brain, and high in tumours, both human and rat. Endothelial vesicles do not always correlate with vascular permeability. They have a low density in normal adult BBB, but are also low in fetal BBB and low in some intracranial tumour vessels. However, they have a high density in muscle capillaries, and others have shown that they increase in BBB vessels damaged by hypertension. Fenestrations are consistently high in leaky vessels, but not all leaky vessels have fenestrations. The density of mitochondria in endothelial cells is high in BBB vessels of some species but not in others. Glut-1, the glucose transporter of the BBB is asymmetrically distributed between the luminal and abluminal membranes of BBB capillaries, being almost four times as numerous on the abluminal face. A large intracellular pool of glucose transporter may provide a means for rapid upregulation of the surface transporters.

Energy metabolism in Leishmania.

Alanine plays a key role in the response of promastigotes to osmotic stress and to hypoxia. It is rapidly released in response to hypo-osmolality, is consumed from its large intracellular pool under iso-osmotic conditions even in the presence of glucose, and is synthesized under hyperosmotic conditions even in the absence of glucose. Its rate of oxidation, in the presence or absence of any of ten other amino acids tested, is strongly inhibited by hyperosmolality. Glucose oxidation is also inhibited by hyperosmolality, but to a lesser extent than that of alanine, and is inhibited by alanine, glutamate, and aspartate. Hyperosmolality also inhibits the incorporation of label from [2-14C]acetate into the putative storage carbohydrate, mannan, which occurs via the glyoxylate bypass and the as yet unexplored "mannoneogenic" pathway. The rates of glycolysis and of oxidation of several amino acids decrease with increasing culture age, but the capacity to oxidize fatty acids increases, and in cells from 3-day stationary phase cultures hyperosmolality enhances rather than inhibits alanine oxidation.

Regulation of mannitol biosynthesis and degradation by Cryptococcus neoformans.

Cryptococcus neoformans, an encapsulated yeast that is an opportunistic pathogen of AIDS patients, produced and secreted mannitol when incubated with an appropriate carbon source. Glucose, fructose, and mannose were good growth substrates and were converted to mannitol. Maltose and xylose were good growth substrates but were not converted to mannitol. Cells of C. neoformans that were grown on a non-mannitol-generating carbon source, such as peptone or xylose, were able to convert glucose to mannitol only after a prolonged lag period in the presence of glucose. It was concluded that the enzymes of the mannitol biosynthetic pathway were not constitutively expressed but were induced in response to glucose or to a glucose metabolite. Enzymes required to catabolize mannitol, however, were constitutively expressed. The production of mannitol was inhibited by anaerobiosis, by the respiratory poison rotenone, and by polyethylenesulfonate, a specific inhibitor of fungal NADP-dependent dehydrogenases. When cells were incubated with deuterated glucose, the deuterium content of the mannitol produced was much lower than that of the glucose precursor, indicating that the glucose was diluted by an intracellular pool of an intermediate. We had previously shown that C. neoformans contains a large intracellular pool of glucose 6-phosphate, and we now conclude that this pool of glucose 6-phosphate is metabolically active.

ω-conotoxin and Cd 2+ stimulate the recruitment to the plasmamembrane of an intracellular pool of voltage-operated Ca 2+ channels

125I-ω-conotoxin binding to neuroblastoma cells at 37°C continously increased, reaching a plateau after 6–8 hr; this was up to 6 times higher than that observed at lower temperatures. The same effect was induced by short pulses with w-conotoxin followed by a chase period at 37°C in control medium. Cd 2+ also induced up-regulation of surface 125I-ω-conotoxin-binding sites. Fura-2 and patch-clamp experiments showed that the recruited binding sites corresponded to functional voltage-operated Ca 2+ channels. Permeabilization experiments revealed a large intracellular pool of 125I-ω-conotoxin-binding sites, whose recruitment to the plasmamembrane was prevented by brefeldin A and nocodazole. These data suggest that specific stimuli might induce voltage-operated Ca 2+ channel translocation to plasmamembrane and, in this way, modulate presynaptic events.

Complex subcellular distribution of sodium-dependent amino acid transport systems in kidney cortex and LLC-PK1/Cl4 cells

To characterize the amino acid transport system in basolateral membranes and to test for possible intracellular loci of amino acid transport activity, we surveyed the distribution of L-alanine transport activity in rabbit proximal tubular cells and LLC-PK1/Cl4 cells. A three-dimensional separation procedure based on differential sedimentation, density gradient centrifugation, and counter-current distribution resolved 21 physically and biochemically distinct membrane populations from rabbit cortex. Inhibition of L-alanine transport by phenylalanine and N-(methylamino)isobutyric acid was used to delineate parallel amino acid transport pathways. Population n was identified as brush border membranes by virtue of its 16-fold maltase enrichment; 94% of its Na(+)-dependent alanine transport activity was mediated by systems previously shown to be characteristic of brush border membranes. Two populations, c' and c", which accounted for 25% of the total Na,K-ATPase activity, were identified as basalateral membranes on the basis of Na,K-ATPase cumulative enrichment factors of 15 and 21; 82% of the total alanine transport in these populations was mediated by a Na(+)-independent system similar to the classical system L. Na,K-ATPase, Na(+)-independent and Na(+)-dependent alanine transport activities were associated with intracellular membrane populations as well as with the plasma membranes. The major intracellular locus of Na,K-ATPase activity, population i accounted for roughly 31% of the Na,K-ATPase, maximally enriched ninefold; it contained 29% of the total system L transport activity. Population l, which was identified as endoplasmic reticulum because it was the major locus of membrane-bound NADPH cytochrome c reductase activity, contained 44% of the total system A transport. Three distinct Golgi-derived populations, m', m", and o, accounted for 39% of the total system A transport. A survey of the amino acid transport systems in LLC-PK1/Cl4 cells showed that the majority of system A-mediated amino acid transport was present in membranes of intracellular and possibly apical origin. The presence of large intracellular pools of amino acid transport activities might reflect newly synthesized transport proteins, ongoing membrane recycling or, perhaps, intracellular reserves available for rapid recruitment to the plasma membrane.

Sperm, Inositol Trisphosphate, and Thimerosal-Induced Intracellular Ca 2+ Elevations in Rabbit Eggs

Fertilization-induced calcium (Ca 2+) changes were examined in in vivo fertilized rabbit eggs, using the fluorescent Ca 2+ indicator fura-2 dextran. Twenty-four of 48 fertilized eggs exhibited repetitive Ca 2+ rises with intervals or 13 ± 1 min (means ± SEM) during the recording period of 45 min. None of the unfertilized eggs showed Ca 2+ rises (6/6). The mean peak Ca 2+ concentration ([Ca 2+] i) was 466 ± 30 n M and the average duration was 100 ± 3 sec. The amplitude of the Ca 2+ rises decreased and the duration increased as the stage of fertilization progressed from recently fertilized to pronuclear apposition ( P < 0.05). In unfertilized eggs, Ca 2+ release was elicited by injection of inositol 1,4,5 trisphosphate (InsP3; 5 μ M in the injection pipette) with a mean peak [Ca 2+] i of 764 ± 88 n M and a duration of 28 ± 1 sec ( n = 9). Injection of InsP3S3 (500 μ M), a nonmetabolizable analogue of InsP3, induced repetitive Ca 2+ rises different from sperm-induced rises in periodicity and duration. Exposure to 400 μ M thimerosal caused spontaneous Ca 2+ rises (1.4 ± 0.1 Ca 2+ rises in 45 min of measurements) with an amplitude of 1200 ± 54 n M and duration of 114 ± 8 sec. Heparin injection (100 mg/ml), an InsP3 receptor antagonist, blocked both InsP3 and thimerosal-induced spontaneous Ca 2+ rises. Successive application of InsP3 and thimerosal in Ca 2+-free medium showed that either InsP3 or thimerosal produced smaller Ca 2+ rise(s) when preceded by Ca 2+ rise(s) induced by the other agonist. The results of this study indicate that rabbit eggs, like other mammalian eggs, exhibit repetitive Ca 2+ rises during fertilization. InsP3 and thimerosal stimulate intracellular Ca 2+ release most likely from a common large intracellular pool by activating the InsP3 receptor.