Cloning and characterization of a zebrafish homologue of human AQP1: a bifunctional water and gas channel

Chen LM, Zhao J, Musa-Aziz R, Pelletier MF, Drummond IA, Boron WF. Cloning and characterization of a zebrafish homologue of human AQP1: a bifunctional water and gas channel. Am J Physiol Regul Integr Comp Physiol 299: R1163-R1174, 2010. First published August 25, 2010; doi:10.1152/ajpregu.00319.2010.-The mammalian aquaporins AQP1, AQP4, and AQP5 have been shown to function not only as water channels but also as gas channels. Zebrafish have two genes encoding an AQP1 homologue, aqp1a and aqp1b. In the present study, we cloned the cDNA that encodes the zebrafish protein Aqp1a from the 72-h postfertilization (hpf) embryo of Danio rerio, as well as from the swim bladder of the adult. The deduced amino-acid sequence of aqp1a consists of 260 amino acids and is 59% identical to human AQP1. By analyzing the genomic DNA sequence, we identified four exons in the aqp1a gene. By in situ hybridization, aqp1a is expressed transiently in the developing vasculature and in erythrocytes from 16 to 48 h of development. Later, at 72 hpf, aqp1a is expressed in dermal ionocytes and in the swim bladder. Western blot analysis of adult tissues reveals that Aqp1a is most highly expressed in the eye and swim bladder. Xenopus oocytes expressing aqp1a have a channel-dependent (*) osmotic water permeability (P(f)*) that is indistinguishable from that of human AQP1. On the basis of the magnitude of the transient change in surface pH (Delta pHS) that were recorded as the oocytes were exposed to either CO(2) or NH(3), we conclude that zebrafish Aqp1a is permeable to both CO(2) and NH(3). The ratio (Delta pHS*)CO2/P(f)* is about half that of human AQP1, and the ratio (Delta pHS*)NH3/P(f)* is about one-quarter that of human AQP1. Thus, compared with human AQP1, zebrafish Aqp1a has about twice the selectivity for CO(2) over NH(3).

Anesthetic Activation of Central Respiratory Chemoreceptor Neurons Involves Inhibition of a THIK-1-Like Background K(+) Current

At surgical depths of anesthesia, inhalational anesthetics cause a loss of motor response to painful stimuli (i.e., immobilization) that is characterized by profound inhibition of spinal motor circuits. Yet, although clearly depressed, the respiratory motor system continues to provide adequate ventilation under these same conditions. Here, we show that isoflurane causes robust activation of CO(2)/pH-sensitive, Phox2b-expressing neurons located in the retrotrapezoid nucleus (RTN) of the rodent brainstem, in vitro and in vivo. In brainstem slices from Phox2b-eGFP mice, the firing of pH-sensitive RTN neurons was strongly increased by isoflurane, independent of prevailing pH conditions. At least two ionic mechanisms contributed to anesthetic activation of RTN neurons: activation of an Na(+)-dependent cationic current and inhibition of a background K(+) current. Single-cell reverse transcription-PCR analysis of dissociated green fluorescent protein-labeled RTN neurons revealed expression of THIK-1 (TWIK-related halothane-inhibited K(+) channel, K(2P)13.1), a channel that shares key properties with the native RTN current (i.e., suppression by inhalational anesthetics, weak rectification, inhibition by extracellular Na(+), and pH-insensitivity). Isoflurane also increased firing rate of RTN chemosensitive neurons in urethane-anesthetized rats, again independent of CO(2) levels. In these animals, isoflurane transiently enhanced activity of the respiratory system, an effect that was most prominent at low levels of respiratory drive and mediated primarily by an increase in respiratory frequency. These data indicate that inhalational anesthetics cause activation of RTN neurons, which serve an important integrative role in respiratory control; the increased drive provided by enhanced RTN neuronal activity may contribute, in part, to maintaining respiratory motor activity under immobilizing anesthetic conditions.

Analysis of peptides in prohormone convertase 1/3 null mouse brain using quantitative peptidomics

P>Neuropeptides are produced from larger precursors by limited proteolysis, first by endopeptidases and then by carboxypeptidases. Major endopeptidases required for these cleavages include prohormone convertase (PC) 1/3 and PC2. In this study, quantitative peptidomics analysis was used to characterize the specific role PC1/3 plays in this process. Peptides isolated from hypothalamus, amygdala, and striatum of PC1/3 null mice were compared with those from heterozygous and wild-type mice. Extracts were labeled with stable isotopic tags and fractionated by HPLC, after which relative peptide levels were determined using tandem mass spectrometry. In total, 92 peptides were found, of which 35 were known neuropeptides or related peptides derived from 15 distinct secretory pathway proteins: 7B2, chromogranin A and B, cocaine- and amphetamine-regulated transcript, procholecystokinin, proenkephalin, promelanin concentrating hormone, proneurotensin, propituitary adenylate cyclase-activating peptide, proSAAS, prosomatosatin, provasoactive intestinal peptide, provasopressin, secretogranin III, and VGF. Among the peptides derived from these proteins, similar to 1/3 were decreased in the PC1/3 null mice relative to wild-type mice, similar to 1/3 showed no change, and similar to 1/3 increased in PC1/3 null. Cleavage sites were analyzed in peptides that showed no change or that decreased in PC1/3 mice, and these results were compared with peptides that showed no change or decreased in previous peptidomic studies with PC2 null mice. Analysis of these sites showed that while PC1/3 and PC2 have overlapping substrate preferences, there are particular cleavage site residues that distinguish peptides preferred by each PC.

CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice

Purkinje cell degeneration (pcd) mice have a mutation within the gene encoding cytosolic carboxypeptidase 1 (CCP1/Nna1), which has homology to metallocarboxypeptidases. To assess the function of CCP1/Nna1, quantitative proteomics and peptidomics approaches were used to compare proteins and peptides in mutant and wild-type mice. Hundreds of peptides derived from cytosolic and mitochondrial proteins are greatly elevated in pcd mouse hypothalamus, amygdala, cortex, prefrontal cortex, and striatum. However, the major proteins detected on 2-D gel electrophoresis were present in mutant and wild-type mouse cortex and hypothalamus at comparable levels, and proteasome activity is normal in these brain regions of pcd mice, suggesting that the increase in cellular peptide levels in the pcd mice is due to reduced degradation of the peptides downstream of the proteasome. Both nondegenerating and degenerating regions of pcd mouse brain, but not wild-type mouse brain, show elevated autophagy, which can be triggered by a decrease in amino acid levels. Taken together with previous studies on CCP1/Nna1, these data suggest that CCP1/Nna1 plays a role in protein turnover by cleaving proteasome-generated peptides into amino acids and that decreased peptide turnover in the pcd mice leads to cell death.-Berezniuk, I., Sironi, J., Callaway, M. B., Castro, L. M., Hirata, I. Y., Ferro, E. S., Fricker, L. D. CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice. FASEB J. 24, 1813-1823 (2010). www.fasebj.org

Hemopressins and other hemoglobin-derived peptides in mouse brain: comparison between brain, blood, and heart peptidome and regulation in Cpefat/fat mice

P>Many hemoglobin-derived peptides are present in mouse brain, and several of these have bioactive properties including the hemopressins, a related series of peptides that bind to cannabinoid CB1 receptors. Although hemoglobin is a major component of red blood cells, it is also present in neurons and glia. To examine whether the hemoglobin-derived peptides in brain are similar to those present in blood and heart, we used a peptidomics approach involving mass spectrometry. Many hemoglobin-derived peptides are found only in brain and not in blood, whereas all hemoglobin-derived peptides found in heart were also seen in blood. Thus, it is likely that the majority of the hemoglobin-derived peptides detected in brain are produced from brain hemoglobin and not erythrocytes. We also examined if the hemopressins and other major hemoglobin-derived peptides were regulated in the Cpefat/fat mouse; previously these mice were reported to have elevated levels of several hemoglobin-derived peptides. Many, but not all of the hemoglobin-derived peptides were elevated in several brain regions of the Cpefat/fat mouse. Taken together, these findings suggest that the post-translational processing of alpha and beta hemoglobin into the hemopressins, as well as other peptides, is up-regulated in some but not all Cpefat/fat mouse brain regions.

Novel endogenous peptide agonists of cannabinoid receptors

Hemopressin (Hp), a 9-residue alpha-hemoglobin-derived peptide, was previously reported to function as a CB(1) cannabinoid receptor antagonist (1). In this study, we report that mass spectrometry (MS) data from peptidomics analyses of mouse brain extracts identified N-terminally extended forms of Hp containing either three (RVD-Hp alpha) or two (VD-Hp alpha) additional amino acids, as well as a beta-hemoglobinderived peptide with sequence similarity to that of hemopressin (VD-Hp beta). Characterization of the alpha-hemoglobin-derived peptides using binding and functional assays shows that in contrast to Hp, which functions as a CB(1) cannabinoid receptor antagonist, both RVD-Hp alpha and VD-Hp alpha function as agonists. Studies examining the increase in the phosphorylation of ERK1/2 levels or release of intracellular Ca(2+) indicate that these peptides activate a signal transduction pathway distinct from that activated by the endo-cannabinoid, 2-arachidonoylglycerol, or the classic CB(1) agonist, Hu-210. This finding suggests an additional mode of regulation of endogenous cannabinoid receptor activity. Taken together, these results suggest that the CB(1) receptor is involved in the integration of signals from both lipid-and peptide-derived signaling molecules.-Gomes, I., Grushko, J. S., Golebiewska, U., Hoogendoorn, S., Gupta, A., Heimann, A. S., Ferro, E. S., Scarlata, S., Fricker, L. D., Devi, L. A. Novel endogenous peptide agonists of cannabinoid receptors. FASEB J. 23, 3020-3029 (2009). www.fasebj.org

Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium-dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca(2+), with an estimated K(d) value of 0.52 mu m. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187-stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187-stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells.

Effects of Exercise Training on Hepatic Microsomal Triglyceride Transfer Protein Content in Rats

Microsomal triglyceride transfer protein (MTP) is a protein that exerts a central regulatory role in very-low-density lipoprotein (VLDL) assembly and secretion. The purpose of the study was to investigate the effects of all exercise-training program oil hepatic content of MTP and its relation to hepatic VLDL-triglyceride (VLDL-TG) production in response to lipid infusion. Female rats either fed a standard (SD) or all obesity-induced high-fat (HF; 43% as energy) diet for 8 weeks were Subdivided into sedentary (Sed) and trained (Tr) groups. Exercise training consisted Of Continuous running on a motor-driven rodent treadmill 5 times/week for 8 weeks. At the end of this period, all rats in the fasted state were intravenously infused with a 20% Solution of intralipid for 3 h followed by all injection of Triton WR1339 to block lipoprotein lipase. An additional control grout) consisting of Sed rats fed the SD diet was infused with saline (0.9% NaCl). Plasma TG accumulation was thereafter measured during 90 min to estimate VLDL-TG production. Under HF diet, hepatic MTP content and plasma TG accumulation after Triton blockade (thus reflecting VLDL-TG synthesis and secretion) were not changed in Sed rats, whereas liver TG content was highly increased (similar to 90%; p<0.01). Oil the other hand, training reduced liver MTP protein content in both SD(-18%) and HF(-23%) fed rats(p<0.05). Plasma VLDL-TG accumulation was also lower (p<0.05) in Tr than in Sed rats fed the HF diet. This effect was not observed in SD fed rats. Furthermore, the exercise training-induced decrease in VLDL-TG production in HF rats was associated with a decrease in liver TG levels. It is Concluded that in addition to a reduction in liver TG content, exercise training reduces VLDL synthesis and/or secretion in HF fed rats probably via MTP regulation.

Peptidomic Analysis of Human Cell Lines

Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEIC293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the PI position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells.

O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway

Aims Glycosylation with beta-N-acetylglucosamine (O-GlcNAcylation) is one of the most complex post-translational modifications. The cycling of O-GlcNAc is controlled by two enzymes: UDP-NAc transferase (OGT) and O-GlcNAcase (OGA). We recently reported that endothelin-1 (ET-1) augments vascular levels of O-GlcNAcylated proteins. Here we tested the hypothesis that O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway. Methods and results Incubation of vascular smooth muscle cells (VSMCs) with ET-1 (0.1 mu M) produces a time-dependent increase in O-GlcNAc levels. ET-1-induced O-GlcNAcylation is not observed when VSMCs are previously transfected with OGT siRNA, treated with ST045849 (OGT inhibitor) or atrasentan (ET(A) antagonist). ET-1 as well as PugNAc (OGA inhibitor) augmented contractions to phenylephrine in endothelium-denuded rat aortas, an effect that was abolished by the Rho kinase inhibitor Y-27632. Incubation of VSMCs with ET-1 increased expression of the phosphorylated forms of myosin phosphatase target subunit 1 (MYPT-1), protein kinase C-potentiated protein phosphatase 1 inhibitor protein (protein kinase C-potentiated phosphatase inhibitor-17), and myosin light chain (MLC) and RhoA expression and activity, and this effect was abolished by both OGT siRNA transfection or OGT inhibition and atrasentan. ET-1 also augmented expression of PDZ-Rho GEF (guanine nucleotide exchange factor) and p115-Rho GEF in VSMCs and this was prevented by OGT siRNA, ST045849, and atrasentan. Conclusion We suggest that ET-1 augments O-GlcNAcylation and this modification contributes to increased vascular contractile responses via activation of the RhoA/Rho-kinase pathway.

Local and cardiorenal effects of periodontitis in nitric oxide-deficient hypertensive rats

Objective: In this study we have assessed the renal and cardiac consequences of ligature-induced periodontitis in both normotensive and nitric oxide (NO)-deficient (L-NAME-treated) hypertensive rats. Materials and methods: Oral L-NAME (or water) treatment was started two weeks prior to induction of periodontitis. Rats were sacrificed 3, 7 or 14 days after ligature placement, and alveolar bone loss was evaluated radiographically. Thiobarbituric reactive species (TBARS; a lipid peroxidation index), protein nitrotyrosine (NT; a marker of protein nitration) and myeloperoxidase activity (MPO; a neutrophil marker) were determined in the heart and kidney. Results: In NO-deficient hypertensive rats, periodontitis-induced alveolar bone loss was significantly diminished. In addition, periodontitis-induced cardiac NT elevation was completely prevented by L-NAME treatment. On the other hand L-NAME treatment enhanced MPO production in both heart and kidneys of rats with periodontitis. No changes due to periodontitis were observed in cardiac or renal TBARS content. Conclusions: In addition to mediating alveolar bone loss, NO contributes to systemic effects of periodontitis in the heart and kidney. (C) 2010 Elsevier Ltd. All rights reserved.

Glucocorticoids Exacerbate Lipopolysaccharide-Induced Signaling in the Frontal Cortex and Hippocampus in a Dose-Dependent Manner

Although the anti-inflammatory actions of glucocorticoids (GCs) are well established, evidence has accumulated showing that proinflammatory GC effects can occur in the brain, in a poorly understood manner. Using electrophoretic mobility shift assay, real-time PCR, and immunoblotting, we investigated the ability of varying concentrations of corticosterone (CORT, the GC of rats) to modulate lipopolysaccharide (LPS)-induced activation of NF-kappa B (nuclear factor kappa B), expression of anti- and proinflammatory factors and of the MAP (mitogen-activated protein) kinase family [ERK (extracellular signal-regulated kinase), p38, and JNK/ SAPK (c-Jun N-terminal protein kinase/ stress-activated protein kinase)], and AKT. In the frontal cortex, elevated CORT levels were proinflammatory, exacerbating LPS effects on NF-kappa B, MAP kinases, and proinflammatory gene expression. Milder proinflammatory GCs effects occurred in the hippocampus. In the absence of LPS, elevated CORT levels increased basal activation of ERK1/ 2, p38, SAPK/ JNK, and AKT in both regions. These findings suggest that GCs do not uniformly suppress neuroinflammation and can even enhance it at multiple levels in the pathway linking LPS exposure to inflammation.

Nox2 B-loop peptide, Nox2ds, specifically inhibits the NADPH oxidase Nox2

In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1, Nox2, and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibition of these isoforms is an area of intense current investigation. In this study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B-loop of Nox2, would inhibit ROS production by the Nox2-. but not the Noxl- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2ds was assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1- or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibited superoxide (O(2)(center dot-)) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance confirmed that Nox2ds inhibits O(2)(center dot-) production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production by either Nox1- or Nox4-oxidase. These findings demonstrate that Nox2ds is a selective inhibitor of Nox2-oxidase and support its utility to elucidate the role of Nox2 in organ pathophysiology and its potential as a therapeutic agent. (C) 2011 Elsevier Inc. All rights reserved.

Matrix metalloproteinases cleave the beta(2)-adrenergic receptor in spontaneously hypertensive rats

Rodrigues SF, Tran ED, Fortes ZB, Schmid-Schonbein GW. Matrix metalloproteinases cleave the beta(2)-adrenergic receptor in spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 299: H25-H35, 2010. First published April 9, 2010; doi:10.1152/ajpheart.00620.2009.-We recently observed the enhanced serine and matrix metalloproteinase (MMP) activity in the spontaneously hypertensive rat (SHR) compared with its normotensive Wistar-Kyoto (WKY) rat and the cleavage of membrane receptors in the SHR by MMPs. We demonstrate in vivo that MMP-7 and MMP-9 injection leads to a vasoconstrictor response in microvessels of rats that is blocked by a specific MMP inhibitor (GM-6001, 1 mu M). Multiple pathways may be responsible. Since the beta(2)-adrenergic receptor (beta(2)-AR) is susceptible to the action of endogenous MMPs, we hypothesize that MMPs in the plasma of SHRs are able to cleave the extracellular domain of the beta(2)-AR. SHR arterioles respond in an attenuated fashion to beta(2)-AR agonists and antagonists. Aorta and heart muscle of control Wistar rats were exposed for 24 h (37 C) to fresh plasma of male Wistar and WKY rats and SHRs with and without doxycycline (30 mu M) and EDTA (10 mM) to reduce MMP activity. The density of extracellular and intracellular domains of beta(2)-AR was determined by immunohistochemistry. The density of the extracellular domain of beta(2)-AR is reduced in aortic endothelial cells and cardiac microvessels of SHRs compared with that of WKY or Wistar rats. Treatment of the aorta and the heart of control Wistar rats with plasma from SHRs, but not from WKY rats, reduced the number of extracellular domains, but not intracellular domains, of beta(2)-AR in aortic endothelial cells and cardiac microvessels. MMP inhibitors (EDTA and doxycycline) prevented the cleavage of the extracellular domain. Thus MMPs may contribute to the reduced density of the extracellular domain of beta(2)-AR in blood vessels and to the increased arteriolar tone of SHRs compared with normotensive rats.

Changes in extracellular levels of glutamate in the nucleus accumbens after ethanol-induced behavioral sensitization in adolescent and adult mice

Repeated administration of low doses of ethanol gradually increases locomotor responses to ethanol in adult Swiss mice. This phenomenon is known as behavioral sensitization. However, we have shown that adolescent Swiss mice show either behavioral tolerance or no sensitization after repeated ethanol injections. Although the mesolimbic dopamine system has been extensively implicated in behavioral sensitization, several studies have demonstrated an important role of glutamatergic transmission in this phenomenon. In addition, relatively few studies have examined the role of developmental factors in behavioral sensitization to ethanol. To examine the relationship between age differences in behavioral sensitization to ethanol and the neurochemical adaptations related to glutamate within nucleus accumbens (NAc), in vivo microdialysis was conducted in adolescent and adult Swiss mice treated with ethanol (1.8 g/kg) or saline for 15 days and subsequently challenged with an acute dose (1.8 g/kg) of ethanol 6 days later. Consistent with previous findings, only adult mice demonstrated evidence of behavioral sensitization. However, ethanol-treated adolescent mice demonstrated a 196.1 +/- 40.0% peak increase in extracellular levels of glutamate in the NAc after ethanol challenge in comparison with the basal values, whereas ethanol-treated adult mice demonstrated a 52.2 +/- 6.2% reduction in extracellular levels of glutamate in the NAc after ethanol challenge. These observations suggest an age-dependent inverse relationship between behavioral and glutamatergic responses to repeated ethanol exposure. (C) 2011 Elsevier Inc. All rights reserved.