Endogenous c-N-Ras provides a steady-state anti-apoptotic signal

We report that c-N-Ras possesses an isoform-specific, functional role in cell survival under steady-state conditions. This function includes protection from programmed cell death by serum deprivation or upon treatment with apoptosis-inducing agents. The data demonstrate that c-N-Ras may play a functional role in the regulation of steady-state phosphorylated Akt and serine 136-phosphorylated Bad (Ser(136)-pBad). Immortalized N-Ras knockout fibroblasts possess nearly undetectable levels of steady-state Ser(136)-pBad. In contrast, wild-type control cells and the N-Ras knockout cells ectopically expressing c-N-Ras at control levels maintained easily detectable levels of Ser(136)-pBad both at steady-state and following treatment with tumor necrosis factor alpha. Similar results were seen with Ser(112)-pBad. These differences did not arise from differences in total Bad protein levels. These data correlate with the observation that the N-Ras knockout cells exhibit a heightened susceptibility to the induction of apoptosis. Ectopic expression of c-N-Ras in the N-Ras knockout cells at endogenous levels, compared with control cells, significantly rescues the apoptotically sensitive phenotype. Elevated expression of either c-Kirsten A-Ras or c-Kirsten B-Ras did not reverse the apoptotic sensitivity of the N-Ras knockout cells or result in increased levels of either phospho-Akt or phospho-Bad. Our results indicate that, at steady state, c-N-Ras possesses an isoform-specific, functional role in cell survival.     

Simultaneous disruption of mouse ASIC1a, ASIC2 and ASIC3 genes enhances cutaneous mechanosensitivity

Three observations have suggested that acid-sensing ion channels (ASICs) might be mammalian cutaneous mechanoreceptors; they are structurally related to Caenorhabditis elegans mechanoreceptors, they are localized in specialized cutaneous mechanosensory structures, and mechanical displacement generates an ASIC-dependent depolarization in some neurons. However, previous studies of mice bearing a single disrupted ASIC gene showed only subtle or no alterations in cutaneous mechanosensitivity. Because functional redundancy of ASIC subunits might explain limited phenotypic alterations, we hypothesized that disrupting multiple ASIC genes would markedly impair cutaneous mechanosensation. We found the opposite. In behavioral studies, mice with simultaneous disruptions of ASIC1a, -2 and -3 genes (triple-knockouts, TKOs) showed increased paw withdrawal frequencies when mechanically stimulated with von Frey filaments. Moreover, in single-fiber nerve recordings of cutaneous afferents, mechanical stimulation generated enhanced activity in A-mechanonociceptors of ASIC TKOs compared to wild-type mice. Responses of all other fiber types did not differ between the two genotypes. These data indicate that ASIC subunits influence cutaneous mechanosensitivity. However, it is unlikely that ASICs directly transduce mechanical stimuli. We speculate that physical and/or functional association of ASICs with other components of the mechanosensory transduction apparatus contributes to normal cutaneous mechanosensation.     

Contraceptive steroids alter the steady-state kinetics of bile acids

Contraceptive steroids increase the ratio of cholic acid to chenodeoxycholic acid in bile. This alteration may contribute to the development of cholesterol gallstones. The objective of this study was to measure the effects of contraceptive steroids on bile acid kinetics and to relate them to changes in cholesterol metabolism. Steady-state kinetics of bile acids were measured in 15 healthy women, on and off contraceptive steroids. Cholic acid synthesis increased 30.3% (P less than 0.025) and its pool increased by 37.4% (P less than 0.025). Chenodeoxycholic acid synthesis decreased 6.4% (P = 0.08) and its pool decreased by 11.8% (P less than 0.05) during use of contraceptive steroids. The fractional turnover rates of both primary bile acids did not change. The changes in kinetics of the primary bile acids were related to alterations in biliary lipid and cholesterol metabolism, separately reported. (J. Lipid Res. 1987. 28: 828-839). During use of contraceptive steroids, total bile acid pool and total bile acid synthesis correlated directly with cholesterol synthesis, assayed in mononuclear leukocytes (r = 0.50 and r = 0.54, respectively) but not with the plasma clearance of chylomicron remnants, measured with retinyl palmitate. The data indicate that contraceptive steroids directly alter the hepatic synthesis of bile acids and suggest that newly synthesized cholesterol may be a preferred substrate for bile acid synthesis during use of contraceptive steroids.     

A Method for Activation of Endogenous Acid-sensing Ion Channel 1a (ASIC1a) in the Nervous System with High Spatial and Temporal Precision

Protons activate acid-sensing ion channel 1a (ASIC1a) in the central nervous system (CNS) although the impact of such activation on brain outputs remains elusive. Progress elucidating the functional roles of ASIC1a in the CNS has been hindered by technical difficulties of achieving acidification with spatial and temporal precision. We have implemented a method to control optically the opening of ASIC1a in brain slices and also in awake animals. The light-driven H⁺ pump ArchT was expressed in astrocytes of mouse cortex by injection of adenoviral vectors containing a strong and astrocyte-specific promoter. Illumination with amber light acidified the surrounding interstitium and led to activation of endogenous ASIC1a channels and firing of action potentials in neurons localized in close proximity to ArchT-expressing astrocytes. We conclude that this optogenetic method offers a minimally invasive approach that enables examining the biological consequences of ASIC1a currents in any structure of the CNS and in the modulation of animal behaviors.     

Potentiation and Block of ASIC1a by Memantine

Acid-sensing ion channels (ASICs) are modulated by various classes of ligands, including the recently described hydrophobic monoamines, which inhibit and potentiate ASICs in a subunit-specific manner. In particular, memantine inhibits ASIC1a and potentiates ASIC2a homomers. The aim of the present work was to characterize action mechanism of memantine on recombinant ASIC1a expressed in CHO (Chinese hamster ovary) cells. We have demonstrated that effect of memantine on ASIC1a strongly depends on membrane voltage, conditioning pH value and application protocol. When applied simultaneously with activating acidification at hyperpolarized voltages, memantine caused the strongest inhibition. Surprisingly, application of memantine between ASIC1a activations at zero voltage caused significant potentiation. Analysis of the data suggests that memantine produces two separate effects, voltage-dependent open-channel block and shift of steady-state desensitization curve to more acidic values. Putative binding sites are discussed based on the computer docking of memantine to the acidic pocket and the pore region.     

Endogenous hormones subtly alter women's response to heat stress

The thermoregulatory responses of menstruant women to exercise in dry heat (dry-bulb temperature/wet-bulb temperature = 48/25 degrees C) were evaluated at three times during the menstrual cycle: menstrual flow (MF), 3-5 days during midcycle including ovulation (OV), and in the middle of the luteal phase (LU). Serum concentrations of estradiol-17 beta (E2), progesterone (Pg), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were measured by radioimmunoassay, and these values were used to determine the dates of OV (peak LH and FSH) and LU (peak postovulatory Pg). After heat acclimation, subjects received heat stress tests (HST) consisting of a 2-h cycle-ergometer exercise at 30% of maximal O2 consumption in the heat. Rectal (Tre) and mean skin (Tsk) temperatures, heart rate (HR), and sweat rate on the chest and thigh were recorded continuously. Total sweat loss (Msw), as indicated by weight loss, was recorded every 20 min, and equivalent water replacement was given. Steady-state exercise metabolic rate (M) was measured at 45 and 110 min. Seven of eight subjects had ovulatory cycles during experimental months. At rest, Tre was lowest at OV and significantly higher at LU. During steady-state exercise both Tre and Tsk were lowest at OV and significantly higher at LU. There were no differences between phases in Msw, sweat rate on the chest and thigh or M. Despite higher Tre and Tsk at LU, all subjects were able to complete the 2-h of exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational Changes Associated with Proton-dependent Gating of ASIC1a

Acid-sensing ion channels are proton-gated Na⁺ channels expressed predominantly in neurons. How channel structure translates an environmental stimulus into changes in pore permeability remains largely undefined. The pore of ASIC1 is defined by residues in the second transmembrane domain (TM2), although a segment of the outer vestibule is formed by residues of TM1. We used the voltage clamp fluorometry technique to define the role of the region preceding TM2 (pre-TM2) in activation and desensitization of mouse ASIC1a. Oocytes expressing E425C channels labeled with Alexa Fluor 488 C5-maleimide showed a change in the emission of the fluorescent probe in response to extracellular acidification. The time course of the change in fluorescence correlated with activation but not desensitization of E425C channels. The fluorescence emission did not change following extracellular acidification in oocytes carrying an inactivating mutation (W287G/E425C), although these channels were labeled and expressed at the plasma membrane. Our data indicate that pore opening occurs in conjunction with a conformational rearrangement of the pre-TM2. We observed a change in the emission of the fluorescent probe when labeled E425C channels transition from the desensitized to the resting state. The substituted-cysteine-accessibility method was used to determine whether the pre-TM2 has different conformations in the resting and desensitized states. State-dependent changes in accessibility to 2-[(trimethylammonium)ethyl]methanethiosulfonate bromide modification were observed in oocytes expressing K421C, K422C, Y424C, and E425C channels. Our results suggest that the pre-TM2 of ASIC1a undergoes dynamic conformational rearrangements during proton-dependent gating.     

Systemic injections of gastro-intestinal peptides alter behavior in rats

Twenty-four male albino rats were given daily intraperitoneal injections of vasoactive intestinal polypeptide (VIP), motilin, human gastrin I (1–17) or the diluent control vehicle at a dose of 100 μg/kg for four consecutive days and food intake, water intake, body weight, and running wheel activity were determined every 24 hours. Animals injected with motilin or human gastrin I (1–17) exhibited decreased food intake relative to those injected with VIP or diluent, which did not differ from each other, although food intake increased reliably over days. The mean water consumption followed the same pattern as that of food intake. As expected from the above results, VIP produced weight gains as compared with rats injected with motilin or gastrin but not reliably more than after diluent. A reliable effect of trials for weight gain was the greatest on day three. Running wheel activity was not affected by injections of human gastrin I (1–17), motilin, or diluent but was reliably decreased by VIP. No significant differences existed across days. Although the results indicate that GI peptides may affect behavior when injected systemically and that like other peptides they have multiple effects, caution is urged in the interpretation of behavioral results at this time.     

Systemic injections of gastro-intestinal peptides alter behavior in rats

Twenty-four male albino rats were given daily intraperitoneal injections of vasoactive intestinal polypeptide (VIP), motilin, human gastrin I (1–17) or the diluent control vehicle at a dose of 100 μg/kg for four consecutive days and food intake, water intake, body weight, and running wheel activity were determined every 24 hours. Animals injected with motilin or human gastrin I (1–17) exhibited decreased food intake relative to those injected with VIP or diluent, which did not differ from each other, although food intake increased reliably over days. The mean water consumption followed the same pattern as that of food intake. As expected from the above results, VIP produced weight gains as compared with rats injected with motilin or gastrin but not reliably more than after diluent. A reliable effect of trials for weight gain was the greatest on day three. Running wheel activity was not affected by injections of human gastrin I (1–17), motilin, or diluent but was reliably decreased by VIP. No significant differences existed across days. Although the results indicate that GI peptides may affect behavior when injected systemically and that like other peptides they have multiple effects, caution is urged in the interpretation of behavioral results at this time.     

Gating Transitions in the Palm Domain of ASIC1a

Acid-sensing ion channels (ASICs) are trimeric cation-selective proton-gated ion channels expressed in the central and peripheral nervous systems. The pore-forming transmembrane helices in these channels are linked by short loops to the palm domain in the extracellular region. Here, we explore the contribution to proton gating and desensitization of Glu-79 and Glu-416 in the palm domain of ASIC1a. Engineered Cys, Lys, and Gln substitutions at these positions shifted apparent proton affinity toward more acidic values. Double mutant cycle analysis indicated that Glu-79 and Glu-416 cooperatively facilitated pore opening in response to extracellular acidification. Channels bearing Cys at position 79 or 416 were irreversibly modified by thiol-reactive reagents in a state-dependent manner. Glu-79 and Glu-416 are located in β-strands 1 and 12, respectively. The covalent modification by (2-(trimethylammonium)ethyl) methanethiosulfonate bromide of Cys at position 79 impacted conformational changes associated with pore closing during desensitization, whereas the modification of Cys at position 416 affected conformational changes associated with proton gating. These results suggest that β-strands 1 and 12 contribute antagonistically to activation and desensitization of ASIC1a. Site-directed mutagenesis experiments indicated that the lower palm domain contracts in response to extracellular acidification. Taken together, our studies suggest that the lower palm domain mediates conformational movements that drive pore opening and closing events.     

Endogenous somatostatin-like peptides of rat basophilic leukemia cells

The tetradecapeptide somatostatin (SOM 14) and a 28-amino acid biosynthetic precursor (SOM 28) are constituents of diverse neuroendocrine tissues that are released by noxious stimuli from a subset of sensory nerve endings, and substantially modify the functions of basophils and mast cells. SOM-like factors were detected initially in the fluid phase of suspensions of immunologically challenged rat basophilic leukemia cells (RBL), and were purified from ethanol/0.2 M acetic acid (3/1, v/v) extracts of replicate portions of 3 X 10(9) RBL. Sephadex G-25 columns resolved factors of over 10,000, 2000 to 4000, and 300 to 1200 daltons that are antigenically related to SOM 14, as assessed by a radioimmunoassay specific for SOM 14. Only the two larger factors were detected by a radioimmunoassay for SOM 28(1-14), which binds to prepro-SOM and SOM 28 but not SOM 14. Reverse-phase high performance liquid chromatography distinguished the two smaller SOM peptides of RBL from SOM 28 and SOM 14, respectively. Amino acid analyses showed major differences in composition between the 2000 to 4000 dalton SOM of RBL and SOM 28. Picomolar to nanomolar concentrations of both of the smaller SOM peptides of RBL inhibited the IgE-dependent release of histamine from basophils to the same extent as SOM 14. The finding of 3 to 5 ng of structurally unique SOM-like peptides per 10(8) RBL suggests that endogenous mechanisms analogous to those of specialized sensory neurons may regulate the expression of hypersensitivity.     

A kinase-anchoring protein 150 and calcineurin are involved in regulation of acid-sensing ion channels ASIC1a and ASIC2a

Acid-sensing ion channel (ASIC) 1a and ASIC2a are acid-sensing ion channels in central and peripheral neurons. ASIC1a has been implicated in long-term potentiation of synaptic transmission and ischemic brain injury, whereas ASIC2a is involved in mechanosensation. Although the biological role and distribution of ASIC1a and ASIC2a subunits in brain have been well characterized, little is known about the intracellular regulation of these ion channels that modulates their function. Using pulldown assays and mass spectrometry, we have identified A kinase-anchoring protein (AKAP)150 and the protein phosphatase calcineurin as binding proteins to ASIC2a. Extended pulldown and co-immunoprecipitation assays showed that these regulatory proteins also interact with ASIC1a. Transfection of rat cortical neurons with constructs encoding green fluorescent protein- or hemagglutinin-tagged channels showed expression of ASIC1a and ASIC2a in punctate and clustering patterns in dendrites that co-localized with AKAP150. Inhibition of protein kinase A binding to AKAPs by Ht-31 peptide reduces ASIC currents in cortical neurons and Chinese hamster ovary cells, suggesting a role of AKAP150 in association with protein kinase A in ASIC function. We also demonstrated a regulatory function of calcineurin in ASIC1a and ASIC2a activity. Cyclosporin A, an inhibitor of calcineurin, increased ASIC currents in Chinese hamster ovary cells and in cortical neurons, suggesting that activity of ASICs is inhibited by calcineurin-dependent dephosphorylation. These data imply that ASIC down-regulation by calcineurin could play an important role under pathological conditions accompanying intracellular Ca(2+) overload and tissue acidosis to circumvent harmful activities mediated by these channels.     

Synthetic "interface" peptides alter dimeric assembly of the HIV 1 and 2 proteases.

Retroviral proteases are obligate homodimers and play an essential role in the viral life cycle. Dissociation of dimers or prevention of their assembly may inactivate these enzymes and prevent viral maturation. A salient structural feature of these enzymes is an extended interface composed of interdigitating N- and C-terminal residues of both monomers, which form a four-stranded beta-sheet. Peptides mimicking one beta-strand (residues 95-99), or two beta-strands (residues 1-5 plus 95-99 or 95-99 plus 95-99) from the human immunodeficiency virus 1 (HIV1) interface were shown to inhibit the HIV1 and 2 proteases (PRs) with IC50's in the low micromolar range. These interface peptides show cognate enzyme preference and do not inhibit pepsin, renin, or the Rous sarcoma virus PR, indicating a degree of specificity for the HIV PRs. A tethered HIV1 PR dimer was not inhibited to the same extent as the wild-type enzymes by any of the interface peptides, suggesting that these peptides can only interact effectively with the interface of the two-subunit HIV PR. Measurements of relative dissociation constants by limit dilution of the enzyme show that the one-strand peptide causes a shift in the observed Kd for the HIV1 PR. Both one- and two-strand peptides alter the monomer/dimer equilibrium of both HIV1 and HIV2 PRs. This was shown by the reduced cross-linking of the HIV2 PR by disuccinimidyl suberate in the presence of the interface peptides. Refolding of the HIV1 and HIV2 PRs with the interface peptides shows that only the two-strand peptides prevent the assembly of active PR dimers. Although both one- and two-strand peptides seem to affect dimer dissociation, only the two-strand peptides appear to block assembly. The latter may prove to be more effective backbones for the design of inhibitors directed toward retroviral PR dimerization in vivo.     

Intracellular ASIC1a regulates mitochondrial permeability transition-dependent neuronal death

Acid-sensing ion channel 1a (ASIC1a) is the key proton receptor in nervous systems, mediating acidosis-induced neuronal injury in many neurological disorders, such as ischemic stroke. Up to now, functional ASIC1a has been found exclusively on the plasma membrane. Here, we show that ASIC1a proteins are also present in mitochondria of mouse cortical neurons where they are physically associated with adenine nucleotide translocase. Moreover, purified mitochondria from ASIC1a^−/− mice exhibit significantly enhanced Ca²⁺ retention capacity and accelerated Ca²⁺ uptake rate. When challenged with hydrogen peroxide (H₂O₂), ASIC1a^−/− neurons are resistant to cytochrome c release and inner mitochondrial membrane depolarization, suggesting an impairment of mitochondrial permeability transition (MPT) due to ASIC1a deletion. Consistently, H₂O₂-induced neuronal death, which is MPT dependent, is reduced in ASIC1a^−/− neurons. Additionally, significant increases in mitochondrial size and oxidative stress levels are detected in ASIC1a^−/− mouse brain, which also displays marked changes (>2-fold) in the expression of mitochondrial proteins closely related to reactive oxygen species signal pathways, as revealed by two-dimensional difference gel electrophoresis followed by mass spectrometry analysis. Our data suggest that mitochondrial ASIC1a may serve as an important regulator of MPT pores, which contributes to oxidative neuronal cell death.     

A gating mutation in the internal pore of ASIC1a

Using a substituted cysteine accessibility scan, we have investigated the structures that form the internal pore of the acid-sensing ion channel 1a. We have identified the amino acid residues Ala-22, Ile-33, and Phe-34 in the amino terminus and Arg-43 in the first transmembrane helix, which when mutated into cysteine, were modified by intracellular application of MTSET, resulting in channel inhibition. The inhibition of the R43C mutant by internal MTSET requires opening of the channel. In addition, binding of Cd2+ ions to R43C slows the channel inactivation. This indicates that the first transmembrane helix undergoes conformational changes during channel inactivation. The effect of Cd2+ on R43C can be obtained with Cd2+ applied at either the extracellular or the intracellular side, indicating that R43C is located in the channel pore. The block of the A22C, I33C, and F34C mutants by MTSET suggests that these residues in the amino terminus of the channel also participate to the internal pore.     

Differential effects of N-glycans on surface expression suggest structural differences between the acid-sensing ion channel (ASIC) 1a and ASIC1b

ASICs (acid-sensing ion channels) are H(+)-gated Na(+) channels with a widespread expression pattern in the central and the peripheral nervous system. ASICs have a simple topology with two transmembrane domains, cytoplasmic termini and a large ectodomain between the transmembrane domains; this topology has been confirmed by the crystal structure of chicken ASIC1. ASIC1a and ASIC1b are two variants encoded by the asic1 gene. The variable part of the protein includes the cytoplasmic N-terminus, the first transmembrane domain and approximately the first third of the ectodomain. Both variants contain two consensus sequences for N-linked glycosylation in the common, distal part of the ectodomain. In contrast with ASIC1a, ASIC1b contains two additional consensus sequences in the variable, proximal part of the ectodomain. Here we show that all the extracellular asparagine residues within the putative consensus sequences for N-glycosylation carry glycans. The two common distal glycans increase surface expression of the channels, but are no absolute requirement for channel activity. In sharp contrast, the presence of at least one of the two proximal glycans, which are specific to ASIC1b, is an absolute requirement for surface expression of ASIC1b. This result suggests substantial differences in the structure of the proximal ectodomain between the two ASIC1 variants.     

VIP and related peptides induce rapid homologous desensitization in the human lymphoma SUP T1 cell line

Incubation of human SUP T1 lymphoblasts with VIP, helodermin and related peptides induced homologous desensitization within 5 min as indicated by: 1) a secondary decrease in cellular cyclic AMP levels, even in the presence of phosphodiesterase inhibitors, 2) a reduced capacity of cells to bind [125I]helodermin, 3) decreased helodermin stimulation of adenylate cyclase activity in membranes, and 4) unaffected NaF- and Gpp[NH]p-stimulated adenylate cyclase activities. The desensitizing ability of all peptides correlated with their efficacy to occupy cell receptors, except for [D-Phe2]VIP, a partial VIP agonist with low intrinsic activity, that did not desensitize.     

FAD-linked mutations in presenilin 1 alter the length of Abeta peptides derived from betaAPP transmembrane domain mutants

gamma-Secretase is an enzymatic activity responsible for the final cleavage of the amyloid precursor protein leading to the production of the amyloid beta-peptide (Abeta). gamma-Secretase is likely an aspartyl protease, since its activity can be inhibited by both pepstatin and active-site directed aspartyl protease inhibitors. Recent work has indicated that presenilins 1 and 2 may actually be the gamma-secretase enzymes. Presenilin (PS) mutations, which lead to an increase in the production of a longer form of Abeta, are also the most common cause of familial Alzheimer's disease (FAD). Therefore, in an attempt to better characterize the substrate preferences of gamma-secretase, we performed experiments to determine how FAD-linked mutations in PS1 would affect the generation of Abeta peptides from full length precursor substrates that we have previously demonstrated to be proteolytically cleaved at alternative sites and/or by enzymatic activities that are pharmacologically distinct. Presenilin mutations increased the production of Abeta peptides from sites distal to the primary cleavage site ('longer' peptides) and in several cases also decreased production of 'shorter' peptides. These results support a model in which the FAD-linked mutants subtly alter the conformation of the gamma-secretase complex to favor the production of long Abeta.     

FMRFamide-gated sodium channel and ASIC channels: a new class of ionotropic receptors for FMRFamide and related peptides

FMRFamide and related peptides typically exert their action through G-protein coupled receptors. However, two ionotropic receptors for these peptides have recently been identified. They are both members of the epithelial amiloride-sensitive Na+ channel and degenerin (ENaC/DEG) family of ion channels. The invertebrate FMRFamide-gated Na+ channel (FaNaC) is a neuronal Na+-selective channel which is directly gated by micromolar concentrations of FMRFamide and related tetrapeptides. Its response is fast and partially desensitizing, and FaNaC has been proposed to participate in peptidergic neurotransmission. On the other hand, mammalian acid-sensing ion channels (ASICs) are not gated but are directly modulated by FMRFamide and related mammalian peptides like NPFF and NPSF. ASICs are activated by external protons and are therefore extracellular pH sensors. They are expressed both in the central and peripheral nervous system and appear to be involved in many physiological and pathophysiological processes such as hippocampal long-term potentiation and defects in learning and memory, acquired fear-related behavior, retinal function, brain ischemia, pain sensation in ischemia and inflammation, taste perception, hearing functions, and mechanoperception. The potentiation of ASIC activity by endogenous RFamide neuropeptides probably participates in the response to noxious acidosis in sensory and central neurons. Available data also raises the possibility of the existence of still unknown FMRFamide related endogenous peptides acting as direct agonists for ASICs.