Differential influence of cAMP on the expression of the three subtypes (ATA1, ATA2, and ATA3) of the amino acid transport system A. (17/351)

Treatment of HepG2 cells with forskolin led to 60-100% stimulation of system A activity, measured as the Na+-dependent uptake of alpha-(methylamino)isobutyric acid. The stimulation was reproducible with cholera toxin and dibutyryl cAMP, and inhibitable by H7, a non-specific protein kinase inhibitor. The stimulatory effect was eliminated by cycloheximide and actinomycin D. The forskolin effect was associated with an increase in the maximal velocity of the transport system, with no change in substrate affinity. These cells express three different subtypes of system A (ATA1, ATA2, and ATA3). Treatment with forskolin increased the steady-state levels of ATA1 and ATA2 mRNAs, but decreased that of ATA3 mRNA.  (+info)

Parathyroid hormone (PTH)-(1-14) and -(1-11) analogs conformationally constrained by alpha-aminoisobutyric acid mediate full agonist responses via the juxtamembrane region of the PTH-1 receptor. (18/351)

The N-terminal portion of parathyroid hormone is critical for PTH-1 receptor (P1R) activation and has been postulated to be alpha-helical when bound to the receptor. We investigated whether substitution of the sterically hindered and helix-promoting amino acid alpha-aminoisobutyric acid (Aib) in N-terminal PTH oligopeptides would improve the capacity of the peptide to activate the P1R. Analysis of the effects of individual Aib substitutions at each position in [Ala(3,12),Gln(10),Har(11),Trp(14)]PTH(1-14)NH(2) ([M]PTH(1-14)) on cAMP-stimulating potency in HKRK-B28 cells revealed that Aib at most positions diminished potency; however, Aib at positions 1 and 3 enhanced potency. Thus [Aib(1,3),M]PTH(1-14) was approximately 100-fold more potent than [M]PTH(1-14) (EC(50) = 1.1 +/- 0.1 and 100 +/- 20 nm, respectively), approximately 100,000-fold more potent than native PTH(1-14), and 2-fold more potent than PTH(1-34). The shorter peptide, [Aib(1,3),M]PTH(1-11), was also fully efficacious and 1,000-fold more potent than [M]PTH(1-11) (EC(50) 4 +/- 1 nm versus 3 +/- 1 microm). In cAMP stimulation assays performed in COS-7 cells expressing P1R-delNt, a receptor that lacks most of the N-terminal extracellular domain, [Aib(1,3),M]PTH(1-14) was 50-fold more potent than [M]PTH(1-14) (EC(50) = 0.7 +/- 0.2 versus 40 +/- 2 nm) and 1,000-fold more potent than PTH(1-34) (EC(50) = 700 nm). [Aib(1,3),M]PTH(1-14), but not PTH(1-34), inhibited the binding of (125)I-[Aib(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH(1-21) NH(2) to hP1R-delNt (IC(50) = 1,600 +/- 200 nm). The Aib(1,3) substitutions in otherwise unmodified PTH(1-34) enhanced potency and binding affinity on hP1R-delNt, but they had no effect for this peptide on hP1R-WT. Circular dichroism spectroscopy demonstrated that the Aib-1,3 substitutions increased helicity in all peptides tested, including PTH(1-34). The overall data thus suggest that the N-terminal residues of PTH are intrinsically disordered but become conformationally constrained, possibly as an alpha-helix, upon interaction with the activation domain of the PTH-1 receptor.  (+info)

Effects of mild hypothermia on blood-brain barrier disruption during isoflurane or pentobarbital anesthesia. (19/351)

BACKGROUND: This study was performed to determine whether mild hypothermia (32 degrees C) could attenuate the degree of blood-brain barrier (BBB) disruption caused by a hyperosmolar solution and whether the degree of disruption would vary depending on anesthetic agents. METHODS: Rats were assigned to one of four groups: normothermic isoflurane, normothermic pentobarbital, hypothermic isoflurane, and hypothermic pentobarbital. During isoflurane (1.4%; normothermic or hypothermic) or pentobarbital (50 mg/kg administered intraperitoneally; normothermic or hypothermic) anesthesia, the external carotid artery and the femoral artery and vein were catheterized. Body temperature was maintained at 37 and 32 degrees C for the normothermic and hypothermic groups, respectively. To open the BBB, 25% mannitol was infused through the right carotid artery at the rate of 0.25 ml x kg(-1) x s(-1) for 30 s. The transfer coefficient of 14C-alpha-aminoisobutyric acid was determined. RESULTS: Blood pressure was similar among the four groups of animals. The degree of the BBB disruption by hyperosmolar mannitol was less with isoflurane than pentobarbital anesthesia in the normothermic groups (transfer coefficient: 29.9 +/- 17.1 and 50.4 +/- 17.5 microl x g(-1) x min(-1) for normothermic isoflurane and pentobarbital, respectively; P < 0.05). Mild hypothermia decreased the BBB disruption during anesthesia with both anesthetic agents (hypothermic isoflurane: 9.8 +/- 8.3 microl x g(-1) x min(-1), P < 0.05 vs. normothermic isoflurane; hypothermic pentobarbital: 30.2 +/- 13.9 microl x g(-1) x min(-1), P < 0.05 vs. normothermic pentobarbital), but the disruption was less during isoflurane anesthesia (hypothermic isoflurane vs. hypothermic pentobarbital, P < 0.005). In the contralateral cortex, there were no significant differences among these four experimental groups. CONCLUSIONS: The data demonstrated that hypothermia was effective in attenuating BBB disruption by hyperosmolar mannitol during isoflurane as well as pentobarbital anesthesia. The degree of disruption appeared smaller during isoflurane than during pentobarbital anesthesia in both the normothermic as well as the hypothermic groups.  (+info)

One-way fluxes of alpha-aminoisobutyric acid in Ehrlich ascites tumor cells. Trans effects and effects of sodium and potassium. (20/351)

One-way fluxes in the steady state and one-way influxes at zero intracellular concentrations were measured for alpha-aminoisobutyric acid (AIB) in Ehrlich ascites tumor cells at 32 degrees C. The one-way fulxes show trans effects in the concentration of AIB and are dependent on sodium levels. The one-way fluxes for initial influx and for the steady state were fitted with the equations derived for the frequently used two-state carrier model. Estimates of the parameters of these equations were obtained with use of nonlinear least squares. These gave relatively good fits of the flux data and the data on steady-state distribution ratios. The two-state carrier model predicted a trans inhibition of one-way influx and a trans stimulation of one-way efflux. The former phenomenon has been demonstrated for AIB transport in Ehrlich ascites cells and there is evidence, through less firm, for the latter.  (+info)

Side chain effect on ion channel characters of Aib rich peptides. (21/351)

As models of ion channel proteins and naturally occurring pore-forming peptides, we designed a series of Aib rich peptides [Ac-(Aib-Xxx-Aib-Ala)(5)-NH(2) (Xxx = Lys, Glu, Ser, and Gly: BXBA-20)] to investigate the effects of the side chains of the amino acid residues Lys, Glu, Ser, and Gly on the conformation and electrophysiological properties of ion channels. The conformation of peptides and their affinity for phospholipid membranes were evaluated by CD spectroscopy. Patch-clamp experiments revealed that all BXBA-20 peptides form ion channels in DPhPC bilayers exhibiting clearly resolved transitions between the open and closed states. The channel forming frequency was in the order BKBA-20>BEBA-20>BSBA-20>BGBA-20. In the case of BKBA-20 and BEBA-20, the self-assembled conductive oligomers expressed homogeneous and voltage-independent single channel conductances. In contrast, heterogeneous conductance was observed in BSBA-20 and BGBA-20 ion channels under similar experimental conditions. From these results, we conclude that peptides with a high degree of helical conformation, high amphipathicity, high affinity for lipid membranes, and self-associating characters in vesicles are most suitable for inducing ion channels with a high frequency of occurrence. Moreover, BEBA-20, BSBA-20, and BGBA-20 channels were cation-selective, whereas the BKBA-20 channel was non-selective.  (+info)

Localization and functional relevance of system a neutral amino acid transporters in cultured hippocampal neurons. (22/351)

Glutamine and alanine are important precursors for the synthesis of glutamate. Provided to neurons by neighboring astrocytes, these amino acids are internalized by classical system A amino acid carriers. In particular, System A transporter (SAT1) is a highly efficient glutamine transporter, whereas SAT2 exhibits broad specificity for neutral amino acids with a preference for alanine. We investigated the localization and the functional relevance of SAT1 and SAT2 in primary cultures of hippocampal neurons. Both carriers have been expressed since early developmental stages and are uniformly distributed throughout all neuronal processes. However, whereas SAT1 is present in axonal growth cones and can be detected at later developmental stages at the sites of synaptic contacts, SAT2 does not appear to be significantly expressed in these compartments. The non-metabolizable amino acid analogue alpha-(methylamino)-isobutyric acid, a competitive inhibitor of system A carriers, significantly reduced miniature excitatory postsynaptic current amplitude in neurons growing on top of astrocytes, being ineffective in pure neuronal cultures. alpha-(Methylamino)-isobutyric acid did not alter neuronal responsitivity to glutamate, thus excluding a postsynaptic effect. These data indicate that system A carriers are expressed with a different subcellular distribution in hippocampal neurons and play a crucial role in controlling the astrocyte-mediated supply of glutamatergic neurons with neurotransmitter precursors.  (+info)

Characterization of neutral amino acid transport in a marine pseudomonad. (23/351)

The transport of neutral amino acids in marine pseudomonad B-16 (ATCC 19855) has been investigated. From patterns of competitive inhibition, mutant analysis, and kinetic data, two active transport systems with overlapping substrate specificities were distinguished and characterized. One system (DAG) served glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid (AIB) and, to a lesser extent, L-alanine and possibly other related neutral D- and L-amino acids. The other system (LIV) showed high stereospecificity for neutral amino acids with the L configuration and served primarily to transport L-leucine, L-isoleucine, L-valine, and L-alanine. This system exhibited low affinity for alpha-aminoisobutyric acid. Neither system was able to recognize structural analogues with modified alpha-amino or alpha-carboxyl groups. The kinetic parameters for L-alanine transport by the DAG and LIV systems were determined with appropriate mutants defective in either system. For L-alanine, Kt values of 4.6 X 10(-5) and 1.9 X 10(-4) M and Vmax values of 6.9 and 20.8 nmol/min per mg of cell dry weight were obtained for transport via the DAG and LIV systems respectively. alpha-Aminoisobutyric acid transport heterogeneity was also resolved with the mutants, and Kt values of 2.8 X 10(-5) and 1.4 X 10(-3) M AIB were obtained for transport via the DAG and LIV systems, respectively. Both systems required Na+ for activity (0.3 M Na+ optimal) and in this regard are distinguished from systems of similar substrate specificity reported in nonmarine bacteria.  (+info)

Stimulation of protein synthesis in cultured heart muscle cells by glucose. (24/351)

Glucose stimulated the rate of incorporation of [3H]leucine into HCLO4-insoluble fraction of cultured rat heart muscle cells under both aerobic and anaerobic conditions. In the aerobic system the incorporation proceeded at a constant rate during 3h of incubation with and without glucose whereas in the anaeorbic system the incorporation ceased after approx. 60 min and could be renewed only by the addition of glucose. No correlation was found to exist between the above effect of glucose on protein synthesis and glucose-dependent changes in the intracellular ATP concentration. The extent of the stimulation of protein synthesis was related to the concentration of glucose. The effect of glucose was suppressed by cycloheximide but was not affected by actinomycin D. Glucose had no effect on the rate of transport of alpha-aminoisobutyric acid. Mannose also stimulated [3H]leucine incorporation. Substances that did not produce lactate were ineffective. Iodoacetate inhibited the stimulatory effect of glucose, but pyruvate, which by itself had no apprecialbe stimulatory action, relieved the inhibition induced by iodoacetate. There was no concomitant change in the concentration of ATP when iodoacetate inhibition was reversed by pyruvate. L-Lactate or other intermediates of energy metabolism could not relieve the inhibitory effect of iodoacetate.  (+info)