Transport of l-leucine hydroxy analogue and l-lactate in rabbit small-intestinal brush-border membrane vesicles

Substitution of the -amino group of amino acids by hydroxyl groups yields hydroxy analogues (HA), which have been ascribed beneficial effects in nitrogensparing diets for uremic patients. In this study, intestinal uptake of l-leucine HA (l-LeuHA) and l-lactate into rabbit jejunal brush-border membrane vesicles was investigated. An inward-directed H⁺ or Na⁺ gradient stimulated uptake of both labelled substrates in a voltageclamped assay. The H⁺ gradient was the major driving force of uptake as compared with the Na⁺ gradient, and it led to a transient accumulation of both l-LeuHA and l-lactate. The proton ionophore carbonylcyanide p trifluoromethoxyphenylhydrazone (FCCP) reduced the initial H⁺-gradient-driven uptake rates of both substrates, but was without effect on Na⁺-gradient-driven uptakes. The H⁺-gradient-driven l-LeuHA uptake was saturable (apparent K_t = 15.4 mM). -HA of l-leucine, l-isoleucine, l-valine, d-leucine, d-valine or l-lactate inhibited the H⁺-gradient-driven l-LeuHA or l-lactate uptakes whereas free branched-chain amino acids had no effect. Preloading the vesicles with one of the l-or d-HA of branched-chain amino acids or with l-lactate stimulated tracer l-LeuHA and also tracer l-lactate uptakes in the presence of a H⁺ gradient. It is concluded that H⁺-gradient-driven transport of l- and d-stereoisomeric HA of branched-chain amino acids as well as of l-lactate across rabbit intestinal brush-border membranes is mediated by the same carrier. Furthermore, there exists a Na⁺gradient-driven l-lactate transport system in the rabbit intestinal brush-border membrane.     

Immunologic roles of hyaluronan

Hyaluronan (HA), a large glycosaminoglycan composed of d-N-acetylglucosamine and d-glucuronic acid, is expressed in virtually all tissues and has long been considered to serve as a structural component or filling material in the tissue interstitium (Filler Theory). This idea was revised with the discovery of HA-binding proteins that introduced the concept that HA may also serve as an adhesive substrate for cellular trafficking (Adhesion Theory). Most recently, it has been shown that HA fragments can deliver maturational signals to dendritic cells (DCs) and high molecular weight HA polymers can deliver costimulatory signals to T-cells (Signaling Theory). Thus, HA may represent an important component of the immune system. Recently, we have evaluated the impact of HA on Langerhans cell (LC) maturation and migration using a novel peptide inhibitor of HA function, termedPep-1 (GAHWQFNALTVR). As skin-specific members of the DC family, LCs are crucial for the initiation of cutaneous immune responses. Local injections of Pep1 prevented hapten-induced LC migration from, the epidermis, providing the first experimental evidence that HA facilitates their emigration. Moreover, Pep-1 also significantly inhibited the hapteninduced maturation of LCs in vivo as assessed by cell morphology, costimulatory molecule expression, and their ability to induce proliferation of allogeneic T-cells. HA therefore has dual functionality to facilitate LC migration and maturation, the two critical events for the initiation of adaptive immune responses. Finally, we have observed that DC-dependent, antigen-specific T-cell proliferation and cytokine secretion is blocked by Pep-1. These results have revealed a previously unrecognized role for HA in antigen presentation. Thus, far from an inert structural biopolymer, HA represents a multifunctional carbohydrate mediator of immune processes.     

Effects of gamma-aminobutyric acid receptor agonists and antagonist on LHRH-synthesizing neurons as detected by immunocytochemistry and in situ hybridization

Summary Gamma-aminobutyric acid (GABA) is thought to play an important role in the regulation of luteinizing hormone-releasing hormone (LHRH) release but its role in the regulation of LHRH gene expression and LHRH synthesis is not known. We hypothesized that since GABA appears to have primarily inhibitory effects on LHRH cells (at the level of the cell body), GABA may act to decrease LHRH gene expression and peptide synthesis. This hypothesis was tested by examining the effect of GABA receptor activation and GABA receptor blockade on LHRH mRNA and peptide levels employing in situ hybridization and immunocytochemistry. Cells in the preoptic area (POA) of ovariectomized (ovx) rats were selectively exposed in vivo to specific GABA-ergic receptor agonists or an antagonist for up to 24 h. THIP, a specific GABA _a receptor agonist, did not have a significant effect on either the intensity of LHRH immunoreactivity, or the number of LHRH-ir cells, observed as compared to controls. Baclofen, a GABA _b receptor agonist appeared to decrease the number of cells with the greatest intensity of LHRH immunoreactivity, compared to controls. In situ hybridization, with either a tritiated RNA probe or a ³²P-labelled oligonucleotide, complementary to LHRH mRNA, revealed that THIP either had no effect on the labelling intensity (³²P-labelled oligonucleotide) or (contrary to our hypothesis) a slight excitatory effect on the level of LHRH mRNA detected per cell (tritiated RNA probe). Bicuculline (a specific GABA _a receptor antagonist) decreased both the number of labelled cells observed per section through the POA, and the intensity of labelling observed in sections hybridized with the ³²P-labelled oligonucleotide. These results suggest that in the POA GABA _a receptors do not exert an inhibitory effect on LHRH gene expresssion, but rather could affect LH perhaps by electrically inhibiting LHRH neurons. In contrast, baclofen appeared to exert an inhibitory effect on LHRH gene expression, since the number of grains per labelled cell in the POA of baclofen treated rats was lower than the grains per labelled cell of control rats. Also, similar to the results obtained with immunocytochemistry, in situ hybridization following baclofen treatment suggested that activation of GABA _b receptors is able to reduce the number of neurons with the highest levels of LHRH mRNA. Thus, in the POA, GABA acting through GABA _b receptors could be effective through changes in mRNA or peptide synthesis.     

Endogenous protease-dependent replication of human influenza viruses in two MDCK cell lines

Summary.  Multi-cycle replication and plaque formation of influenza A and B viruses and cleavage activation of their hemagglutinin (HA) by an endogenous protease(s) were examined in two MDCK cell lines, MDCK(−) and MDCK(+). No exogenous trypsin was required for multi-cycle replication and plaque formation of all the influenza A viruses tested in the MDCK(+) cell, while those of the viruses in the MDCK(−) cell were completely trypsin-dependent. In both cell lines, on the other hand, influenza B viruses grew well in the absence of trypsin. The capability of multiple replication and plaque formation of the influenza viruses correlated with cleavage of the HA precursor (HA₀) to HA₁ and HA₂, indicating that both cell lines express an HA activating endoprotease(s); that of the MDCK(+) cell activates the HA of influenza A and B viruses, and that of the MDCK(−) cell does only the HA of influenza B virus. Furthermore, the protease of the MDCK(+) cell was strongly suggested to be present on the cell surface and a serine protease. The MDCK(+) cell would be useful for isolation of influenza viruses from clinical specimens and for screening of protease inhibitors for anti-influenza virus drugs. Received April 9, 1998. Accepted May 22, 1998     

Inhibitory mechanisms in epileptiform activity induced by low magnesium

In rat hippocampal slices epileptiform activity was induced by superfusion with Mg²⁺-free artificial cerebrospinal fluid (ACSF). Paroxysmal depolarization shifts (PDS) were evoked by electrical stimulation of Schaffer collaterals. To investigate the afterpotentials that follow PDS, intracellular recordings were made from CA1 pyramidal cells. The experiments revealed that several components are engaged in the generation of PDS afterpotentials in Mg²⁺-free ACSF. A long lasting component which determined the overall duration of the PDS afterhyperpolarization was blocked by intracellular application of ethylenebis(oxonitrilo)-tetraacetate (EGTA); concomitantly, the afterhyperpolarizations following depolarizing current injections were blocked. This indicated that the long lasting component was due to a slow Ca²⁺-activated K⁺ current. The block of Ca²⁺-activated K⁺ current uncovered a depolarizing PDS afterpotential with an N-shaped voltage dependence, suggesting that this depolarizing afterpotential component may be due to an N-methyl d-aspartate (NMDA) conductance. Intracellular injection of Cl^– revealed that the PDS were followed by Cl^– currents lasting about 500 ms. This component could be blocked by application of bicuculline suggesting that it is due to a synaptically GABA-mediated (i.e. -aminobutyric acid) Cl^– current. A comparison of PDS afterpotentials in Mg²⁺-free ACSF and those in other models of epileptiform activity suggests that similar sequences of inhibitory components are activated in spite of different pharmacological alterations of membrane conductances which induce the epileptiform discharges.     

Identification of an immunoreactive non-proteinaleous component in Borrelia burgdorferi

Investigations of immunoblots using Borrelia burgdorferi antigen demonstrated that a band, migrating faster than the bromophenol blue front in sodium dodecyl sulfate-gel electrophoresis, reacted strongly with sera containing anti-Borrelia burgdorferi antibodies preferentially of the IgG class. Extraction of this antigenic component and chemical analyses showed that the substance was composed mainly of fatty acids and carbohydrates. Typical structures of classical lipooolysaccharides such as 3-deoxy-d-manno-2-octulosonic acid, hydroxy fatty acids or lipid A could not be detected.     

Effect of feeding a high protein diet on amino acid uptake into rat intestinal brush border membrane vesicles

Uptake of the neutral amino acidl-leucine into isolated rat intestinal brush border membrane (=BBM) vesicles and into a jejunal mucosa preparation as affected by the protein content of the diet was investigated. Adult rats fed either a high carbohydrate (HC) diet (11% protein) or a high protein (HP) diet (77% protein) for several weeks were used for the experiments.The time course ofl-leucine uptake into BBM vesicles prepared from the small intestine of HC-or HP-rats was studied under conditions of an inwardly directed Na⁺-gradient and under Na⁺-equilibrium conditions. Furthermore, in one series of experiments the Na⁺-equilibrium was replaced by a K⁺-equilibrium. l-leucine uptake under Na⁺-gradient conditions displayed the overshoot phenomenon typically associated with Na⁺-gradient-dependent active transport processes in BBM vesicles and the overshoot in group HP exceeded that in group HC significantly.Under both Na⁺-and K⁺-equilibrium conditionsl-leucine uptake into the BBM-vesicles also was faster in group HP.Finallyl-leucine uptake into jejunal mucosa in group HP exceeded that in group HC, too.The results therefore indicate that Na⁺-dependent and Na⁺-independent transport of neutral amino acids across the intestinal brush border membrane adapts to the dietary protein level.Some of the results were reported in a preliminary form [16]     

Sugar transport in isolated rat kidney papillary collecting duct cells

d-Glucose is an important substrate of energy metabolism and osmolyte synthesis in the renal papillary collecting duct. In order to characterize the cellular entry ofd-glucose in this tubular segment, collecting duct cells were isolated from rat kidney papilla and the rate ofd-glucose uptake was measured indirectly by monitoring thed-glucose-dependent O₂ uptake in the presence of the uncoupler CCCP.d-Glucose uptake was found to be sodium-independent and not sensitive to phlorizin even at a concentration of 10^–3 M. Uptake was, however, completely inhibited by 10^–5 M cytochalasin B and 10^–4 M phloretin. The apparentK _i for cytochalasin B was 1.5×10^–6 M and for phloretin 2.0×10^–5 M. Studies on the substrate specificity revealed that at 1 mMd-mannose is taken up and metabolized to the same extent asd-glucose. A 50-fold higher concentration of 2-deoxy-d-glucose and 2-amino-2-deoxy-d-glucose inhibitedd-glucose uptake completely whereas -methyl-d-glucoside,d-allose, andd-galactose were without effect. Under conditions whered-glucose utilization was maximally stimulated an apparentK _m of 1.2 mM and aV _max of 1 mmold-glucose/g protein hour was found ford-glucose uptake.These results indicate that thed-glucose uptake into papillary collecting duct cells is probably mediated by a transport system similar to the one found in basal-lateral membranes of pelarized renal, intestinal, and liver cells as well as in nonpolarized fat cells and erythrocytes.Supported by grant DFG Gr 916/1-1     

Isolation and preliminary characterization of a highly cytolytic influenza B virus variant with an aberrant NS gene

By repeated backcrosses of influenza virus A/Aichi/2/68 (H3N2) with B/Yamagata/1/73 in MDCK cells, a virus clone with HA of B serotype (clone B/610B5B/201, or clone 201) was obtained, which formed sharp plaques in MDCK cells and induced a severe cell lysis early after infection. Its structural proteins were indistinguishable from those of B/Yamagata. Electrophoresis of the RNA of the clone also showed an identical pattern to that of B/Yamagata except RNA segment 8 (NS gene), which migrated faster than the corresponding segment of B/Yamagata in a 2.8% polyacrylamide gel. Within the clone 201-infected MDCK cells, only one species of nonstructural (NS) polypeptide was demonstrable, which had the same electrophoretic mobility as NS2 of B/Yamagata, and any band which might be taken as the counterpart of NS1 of B/Yamagata was not detectable on the gel. Peptide mapping revealed that NS of clone 201 was structurally different from either NS1 or NS2 of wild-type B/Yamagata. NS gene and its function of clone 201 was successfully transferred to B/Lee/40 by genetic reassortment.     

Ultrastructural study of plasma membrane GM1 in neuroectodermal cells using cholera-peroxidase

Summary Cholera toxin was coupled to peroxidase to yield a highly specific ultrastructural marker for G _m1 gangliosides. Study of embryonic brain cells in culture revealed intense binding of choleraperoxidase by plasma membranes of both neurons and glial cells. In contrast, long-term monolayer glioblastoma cultures, including one producing C-type virus, revealed virtually no labelling of their plasma membranes. Such cells were shown to be capable of incorporating exogenously applied G_{m 1} into their plasma membranes. Studies with fixed brain and synaptosomal fractions were in accord with results on embryonic brain cells in culture, and autoradiographic findings with¹²⁵I cholera supported observations made utilizing cholera—peroxidase. From our studies there is some indication that long-term propagation in vitro alters the plasma membrane G _m1.     

Sodium dependence of the amino acid transport in the proximal convolution of the rat kidney

Summary With the technique of stop flow microperfusion with simultaneous capillary microperfusion the zero net flux transtubular concentration differences (c) of labelled amino acids which are equivalent to their active transport rates were measured. Alll-amino acids tested (phenylalanine, histidine, aminobicycloheptane-carboxylic acid, aminoisobutyric acid; lysine, ornithine, arginine; aspartic acid; proline and glycine) showed a considerable c, i.e. active transport rate. When, however, the ambient sodium was replaced by choline the c values dropped to zero. An analysis of the Na⁺ dependence of the ornithine transport revealed that the sodium-dependence is of the mixed type, i.e. thatK _m decreased andV _max increased with increasing Na⁺ concentration to the same extent.In contrast to other biological systems no mutual interaction between the Na⁺-dependentd-glucose andl-histidine transport could be observed.Incidental to these studies it was observed that the active transport rate ofd-histidine was in the range of 40% of that of thel-isomer while ford-phenylalanine it was only in the range of 10% of the active transport of thel-isomer. Furthermore it was found that thel-aspartic acid transport was already saturated at a luminall-aspartic acid concentration of 0.05 mmol/l while that ofl-phenylalanine was not saturated even at a luminal concentration of 9 mmol/l.     

Cell transformation induces a cytoplasmic Ca2+ oscillator in Madin-Darby canine kidney cells

Alkaline stress transforms Madin-Darby canine kidney (MDCK) cells as indicated by loss of epithelial structure, multilayer cell growth and formation of foci. In the present study we report that transformed MDCK cells (MDCK-F cells) exhibit spontaneous and lasting oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i), which are absent in non-transformed cells. Oscillations, as revealed by Fura-2 video imaging, were due to the activity of an inositol 1,4,5-trisphosphate-(InsP ₃)-sensitive Ca²⁺ store since their frequency was dependent on bradykinin concentration and they were abolished by the phosphoinositidase C inhibitor U73122. Moreover, blockers of the cytoplasmic Ca²⁺-ATPase, thapsigargin and 2,5-di-(tetr-butyl)-1,4-benzohydroquinone inhibited oscillatory activity. In contrast, neither injection of ruthenium red, ryanodine nor caffeine had any effect on oscillations. Analysis of the spatial distribution of [Ca²⁺]_i showed that Ca²⁺ transients originated from an initiation site constant for a given cell and spread through the cell as an advancing Ca²⁺ wave. Oscillations started in a random manner from single cells and spread over neighbouring cells, suggesting a kind of intercellular communication. We conclude that MDCK-F cells have acquired the ability for endogenous Ca²⁺ release through transformation. Oscillations are primarily due to the activity of an InsP ₃-sensitive cytosolic Ca²⁺ oscillator.     

Changes of plasma membrane permeability in neutrophils treated with polycations

The effects of the polycations poly-l-arginine, poly-l-lysine, and polyethyleneimine on rabbit neutrophil membrane permeability were compared. LDH release, quin2 release from quin2-loaded cells, and increase of indol fluorescence were considered as measures for changes in membrane permeability. All polycations cause abundant LDH release. Quin2 release occurs more rapidly than LDH release, and the increase of indol fluorescence is even faster. Apparently polycation-induced permeability changes occur gradually, allowing the influx (or efflux) of small molecules more rapidly than larger ones. A number of divalent and trivalent cations inhibit polycation-induced LDH and quin2 release in a way that resembles the inhibition of other cytotoxic agents described in literature. In the absence of extracellular Ca²⁺, the polycations induce little lysozyme release. In the presence of extracellular Ca²⁺, there is abundant lysozyme release, indicating that the influx of Ca²⁺ causes exocytosis. Exocytosis still occurs when Ca²⁺ is added some time after poly cation addition, indicating that polycation treatment leaves the cells largely intact. All polycations tested have in common that they cause gradual changes in the permeability of the plasma membrane only, which opens the possibility to use them as membrane-permeabilizing agents for the study of Ca²⁺-induced exocytosis.     

Global,synchronous oscillations in cytosolic calcium and adherence in bradykinin‐stimulated Madin‐Darby canine kidney cells

Aims and Methods: Intercellular Ca²⁺ oscillations are a universal mode of signalling in both excitable and non‐excitable cells. Here, we study the relationship between Ca²⁺ signalling and coherent changes in adhesion properties by measuring the transepithelial impedance across bradykinin‐stimulated Madin‐Darby canine kidney (MDCK) cell layers grown on a microelectrode. During hormone stimulation, the impedance is found to oscillate, reflecting that the cells undergo morphological/adhesive alterations with high spatio‐temporal organization. The experiments are supplemented with parallel, digital imaging fluorescence microscopy of bradykinin‐induced single‐cell Ca²⁺ oscillations. Results: In agreement with previous experiments, MDCK cells are found to elicit synchronous, multicellular Ca²⁺ oscillations in response to hormone stimulus. The periods of the Ca²⁺ oscillations and the electrical fluctuations are found to coincide. Further, blocking of gap junctions by 18α‐glycyrrhetinic acid causes a loss of synchrony in Ca²⁺ signals and inhibition of impedance oscillations, emphasizing the importance of gap junctions in the signal transduction process. Conclusion: Based on these observations it is concluded that the co‐ordinated adhesive changes in MDCK cells are a direct consequence of synchronized Ca²⁺ oscillations. Calcium signalling represents an efficient way of organizing physiological responses in a tissue. A possible functional implication of the structural changes might be to modulate transportation of various substances across the cell sheet.     

Impact of the variations in potential glycosylation sites of the hemagglutinin of H9N2 influenza virus

Variations in the potential glycosylation sites were observed in hemagglutinin (HA) sequences of H9N2 avian influenza virus isolated in China, deposited in the Influenza Virus Resource of NCBI before 2017, which showed a deleted glycosylation site at amino acid residue 218, and an introduced glycosylation site at amino acid residue 313. Based on the variations in the glycosylation sites at these amino acids, H9N2 avian influenza viruses could be divided into three categories. Firstly, most of the H9N2 influenza viruses were 218G⁺ viruses; less 313G⁺ viruses were isolated between 1997 and 2004. Secondly, the occurrence of the 218G⁺/313G⁺ viruses increased, while the 218G⁺/313G^? viruses decreased from 2005 to 2012. Thirdly, from 2013 to 2016, the 218G^?/313G⁺ viruses were predominant compared to the 218G⁺/313G⁺ viruses. Here, based on an F/98 virus backbone, a 218G⁺/313G^? virus, two reassortment viruses were generated, and named rF/HA218G⁺/313G⁺ and rF/HA 218G⁺/313G^?, respectively. HA protein migration demonstrated that the glycosylation sites at amino acid residues 313 and 218 were both functional. The absence of the glycosylation site at amino acid residue 218 and the presence of the glycosylation site at amino acid residue 313 increased antibody binding and moderately prevented the virus from escaping neutralization with homologous antisera. Additionally, compared to the F/98 virus (218G⁺/313G^?), the viruses rF/HA218G⁺/313G⁺ or rF/HA218G^?/313G⁺ showed significantly increased infectivity of MDCK cells, chicken embryo eggs, and trachea and lung tissue of SPF chickens, but did not display differences in airborne spread in chickens or infectivity of mice compared with its parental virus F/98.

     

GLIOMA INVASION IN VITRO IS MEDIATED BY CD44–HYALURONAN INTERACTIONS

Invasion is a clinically important problem contributing to mortality and morbidity in patients with gliomas, but the mechanism(s) by which glioma cells invade surrounding brain structures is poorly understood. Various experimental models have been used in attempts to elucidate the process of glioma invasion. An in vitro model which is increasingly being employed involves measurement of the rate of invasion of tumour cells through Matrigel^®—a complex mixture of extracellular matrix components derived from the Engelbroth–Holm–Swarm (EHS) sarcoma. This model has been used to examine the possibility that extracellular hyaluronan (HA) might facilitate the invasive behaviour of human glioma cells. The major component of Matrigel^® is laminin, with smaller amounts of collagen IV, heparan sulphate proteoglycans, entactin, and nidogen, but it lacks HA. In our experiments, we have incorporated HA into Matrigel^® and have measured its effect on the rate of invasion of human glioma cells in a modified Boyden chamber assay system. The incorporation of HA (50–800 mg/cm²) resulted in a dose-dependent increase in invasion. Invasion was enhanced by up to 70 per cent in comparison with HA-free Matrigel^®. Since CD44 is a major HA receptor expressed on gliomas, it might have a role in the HA-mediated facilitation of invasion. This was tested by blocking CD44 with specific antibody, which resulted in a 43 per cent reduction in invasion rate. We conclude that in an in vitro model system, HA enhances invasion of glioma cells and that the mechanism involves a CD44–HA interaction. © 1997 by John Wiley & Sons, Ltd.     

Cytoplasmic and nuclear input virus RNPs in influenza virus-infected cells

Chicken fibroblasts and MDCK cells were infected with influenza virus labelled with either 3H-uridine or 14C-amino acids, and the location in infected cells and properties of input virus-labelled structures were studied. Input virus RNA and protein were found in the cytoplasm of nuclei 1 h p.i. A part of the intranuclear parental structures was associated with chromatin while the other part could be extracted from nucleoplasm by 0.16 M-NaCl and represented free ribonucleoprotein (RNP) particles. These RNPs sedimented in glycerol velocity gradients at 40 to 70S, very similar to cytoplasmic RNPs, but differed distinctly from them in buoyant density. The bulk of cytoplasmic RNPs after fixation with formaldehyde banded in CsCl at 1.34 g/ml while nucleoplasmic RNPs banded at 1.39 or 1.41 g/ml. RNPs isolated from virions and infected cells contained the NP polypeptide which was revealed by SDS-PAGE analysis as a double band. The ratio of the two bands varied in cytoplasmic and nucleoplasmic RNPs, the lower band being dominant in cytoplasmic but not in nucleoplasmic RNPs. In addition, cytoplasmic RNPs were phosphorylated. The possible significance of intracellular RNP modifications for virus replication is discussed.     

Variables of the rubella hemagglutination test employing freeze-dried erythrocytes

Summary The variables which affect the interaction between freeze-dried one-day-old chick erythrocytes and rubella hemagglutinin prepared from rubella-infected porcine kidney cells were defined and evaluated. The sensitivity of the hemagglutination (HA) reaction is much greater at pH 6.0 to 6.2 than at pH 7.0 to 7.5. HEPES (N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid) diluent with added Ca²⁺ or Mg²⁺ ion gave four- to eightfold higher HA titers than one without divalent cations.The development of agglutinated and non-agglutinated erythrocyte patterns depended much upon the concentrations of gelatin and albumin in the HEPES diluent. Gelatin especially was essential to obtain stable and clearly distinguishable patterns.Optimal conditions for the agglutination of freeze-dried erythrocytes by rubella hemagglutinin were provided when a HEPES-buffered saline at pH 6.2, containing 10^–3 m CaCl₂, 0.2 per cent bovine serum albumin, and 0.0025 per cent gelatin was employed throughout as a diluent for serum, hemagglutinin, and freeze-dried erythrocyte suspension. This diluent gave maximally sensitive and reproducible results in rubella HA and hemagglutination-inhibition (HI) tests employing freeze-dried erythrocytes.With 1 Figure     

Cell signalling-mediating insulin increase of mRNA expression for cationic amino acid transporters-1 and -2 and membrane hyperpolarization in human umbilical vein endothelial cells

Insulin induces vasodilatation in human subjects and increases l-arginine transport and NO synthesis in human umbilical vein endothelial cells (HUVEC). Cell signalling events associated with insulin effects on activity and mRNA expression of the human cationic amino acid transporters 1 (hCAT-1) and 2B (hCAT-2B) are unknown. l-Arginine transport and eNOS activity were determined in HUVEC exposed to insulin. mRNA levels for hCAT-1, hCAT-2B and eNOS were quantitated by real time RT-PCR and endothelial NO synthase (eNOS) protein was identified by Western blot analysis. Intracellular Ca²⁺, l-arginine and l-citrulline levels, l-[³H]citrulline formation from l-[³H]arginine, cGMP formation, nitrite level, ATP release and membrane potential were determined. Insulin increased l-arginine transport and the mRNA levels for hCAT-1 and hCAT-2B and eNOS expression and activity. Insulin also induced membrane hyperpolarization and increased intracellular Ca²⁺, l-[³H]citrulline, cGMP and nitrite formation. Insulin-mediated stimulation of the l-arginine/NO pathway is thus associated with increased hCAT-1 and hCAT-2B mRNA, and eNOS expression, via mechanisms involving membrane hyperpolarization, mitogen-activated protein kinases p42 and p44, phosphatidylinositol 3-kinase, NO and protein kinase C. We have characterized a cell signalling pathway by which hyperinsulinaemia could lead to vasodilatation in human subjects, and which could have implications in patients in whom plasma insulin levels are altered, such as in diabetes mellitus.