mouse trpv4  (Novus Biologicals)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa

doi: 10.1007/s00018-024-05341-w

Figure Lengend Snippet: Phylogeny of the TRPV4 in vertebrates and expression of different Trpv4 splice variants in seabream testis and spermatozoa. a Bayesian majority rule consensus tree (1 million MCMC generations, aamodel = mixed) of a ClustalX amino acid alignment of Trpv4 protein sequences rooted with the ghost shark Trpv4. The GenBank accession numbers of the sequences are listed in Supplementary Table . Bayesian posterior probabilities are shown at each node with the scale bar indicating expected substitution rates per site. b Schematic representation of the gilthead seabream trpv4 exons, and the polypeptides of wild-type Trpv4 (Trpv4_v1) and splice variants Trpv4_v2 and _v10 (Ensembl accession numbers ENSSAUT00010061485.1, ENSSAUT00010061488.1 and ENSSAUT00010061519.1, respectively). The proline-rich domain (PRD) and ankyrin repeat domain (ARD) are shown for each protein. c Representative RT-PCR detection of seabream trpv4_v1, _v2 and _v10 mRNAs in testis and ejaculated spermatozoa (SPZ). The N line is the negative control (absence of RT during cDNA synthesis). The arrows indicate the specific amplified transcripts, and the sizes (kb) of molecular markers are indicated on the left. The positions on the trpv4 genomic sequence of the different oligonucleotide primers employed are indicated in b

Article Snippet: Antibodies against human or mouse TRPV4 were purchased from Novus Biologicals (# NBP2-41262) and Invitrogen (# OSR00136W), respectively.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa

doi: 10.1007/s00018-024-05341-w

Figure Lengend Snippet: Both Trpv4 and Aqp4a are expressed in seabream spermatozoa. a Immunostaining (left panels, bright field [BF]; right panels, epifluorescence images) of Trpv4 in ejaculated spermatozoa using two commercial rabbit antibodies against mammalian TRPV4 (α-TRPV4-1 [Novus Biologicals # NBP2-41262] and α-TRPV4-2 [Invitrogen # OSR00136W]), showing that the Trpv4 immunoreaction (red color) was localized along the flagellum (arrows). The nucleus is counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bars, 5 μm. b Immunoblots of total membrane protein extracts from X. laevis oocytes uninjected (Uninj.), oocytes injected with cRNA encoding seabream Trpv4_v1, and sperm flagella (Sp), using the two TRPV4 antibodies. Spermatozoa extracts were treated with or without PNGase F (plus or minus) prior to electrophoresis. Arrows indicate monomers, while the asterisks indicate glycosylated forms. The arrowheads indicate other potential post-translational modifications of the ion channel. Molecular mass markers (kDa) are on the left. c Immunolocalization of seabream Aqp4a (green) along the tail of the spermatozoa (arrows) using a paralog-specific chicken antibody (α-Aqp4a). The negative controls (right panels) were incubated with the primary antibody preadsorbed with the antigenic peptide. Labels and scale bars as in A. d Schematic diagram of the Aqp4a topology depicting the cytoplasmatic N- and C-termini (NT and CT, respectively), the six transmembrane α-helices (1–6), and the five loops ( A-E ). The two translation initiating methionines (M1 and M43) in the NT are shown. e, f Immunoblot of X. laevis oocytes uninjected (Uninj) or expressing the Aqp4a-M1 or Aqp4a-M43 isoforms ( e ), and of sperm flagella showing the expression of both isoforms (arrows) ( f ). For the latter immunoblot, the α-Aqp4a was preadsorbed with the antigenic peptide before immunoblotting to verify the specificity of the reactive bands. Labels as in b . g Double immunostaining of Trpv4 and Aqp4a in seabream spermatozoa using the TRPV4-1 and Aqp4a antibodies showing co-localization of both channels (arrows). Labels and scale bars as in a . h Co-immunoprecipitation of Trpv4 and Aqp4a in ejaculated spermatozoa using either of the two commercial TRPV4 antibodies or immunoglobulin G (IgG) as control. The immunoprecipitates were immunoblotted with the TRPV4 or Aqp4a antibodies as indicated. Labels as in b . IgG-HC, IgG heavy chain

Article Snippet: Antibodies against human or mouse TRPV4 were purchased from Novus Biologicals (# NBP2-41262) and Invitrogen (# OSR00136W), respectively.

Techniques: Immunostaining, Western Blot, Membrane, Injection, Electrophoresis, Incubation, Expressing, Double Immunostaining, Immunoprecipitation, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa

doi: 10.1007/s00018-024-05341-w

Figure Lengend Snippet: Functional characterization of seabream Trpv4 variants in X. laevis oocytes. a Representative double immunostaining of uninjected oocytes and oocytes expressing Aqp4a-M43 together with human influenza hemagglutinin (HA)-tagged Trpv4_v1, _v2 or _v10 using a seabream Aqp4a-specific antiserum and anti-HA antibodies. The oocyte plasma membrane was counterstained with wheat germ agglutinin (WGA). Scale bars, 10 μm. b-c Representative volume ( b ) and current ( c ) traces obtained from oocytes voltage-clamped at Vm = − 20 mV and challenged with a hypo- or hyperosmotic gradient (− 100 mOsm: blue bars and + 100 mOsm: red bars, respectively). The traces were recorded with a 200-ms step protocol (as indicated by numbers 1–4) from an uninjected oocyte and oocytes expressing either Aqp4a, Trpv4_v1, _v2 or _v10 alone, or Aqp4a together with Trpv4_v1, _v2 or _v10. d-f Summarized I/V curves from oocytes expressing Aqp4a plus Tpv4_v1, _v2 or _v10 in control solution (white) or during application of a hyposmotic (blue) or hyperosmotic solution (red). g-i Trpv4_v1, _v2 or _v10-mediated current activity at -85 mV obtained after exposure to -100 mOsm (blue) or + 100 mOsm (red) normalized to that obtained in control conditions. In d-i panels, values ( n = 5–9 oocytes; white dots in g-i ) are presented as mean ± SEM. The paired normalized data in g-i were statistically analyzed by one sample t -test (***, p < 0.001, with respect to the same oocytes at 220 mOsm prior to osmotic challenge; ns, not statistically significant). The controls (uninjected oocytes and oocytes expressing Aqp4a or Tpv4_v1, Trpv4_v2 or Trpv4_v10 alone) and exposed to an isosmotic solution or during application of a hyposmotic or hyperosmotic solution are shown in Supplementary Fig.

Article Snippet: Antibodies against human or mouse TRPV4 were purchased from Novus Biologicals (# NBP2-41262) and Invitrogen (# OSR00136W), respectively.

Techniques: Functional Assay, Double Immunostaining, Expressing, Membrane, Control, Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa

doi: 10.1007/s00018-024-05341-w

Figure Lengend Snippet: Inhibition of Trpv4_v1 and _v10 with different TRPV4 blockers in X. laevis oocytes. a-l Summarized I/V curves from oocytes expressing either Tpv4_v1 ( a-f ) or _v10 ( g-l ) alone in control solution (white dots) or after 5 min exposure to different TRPV4 blockers at two concentrations, RN-1734 (10 and 100 µM, blue and red dots, respectively), ruthenium red (RR, 1 and 10 µM, blue and red dots, respectively) and HC-067047 (1 and 10 µM, blue and red dots, respectively). m-r The Trpv4_v1- ( m-o ) or _v10- ( p-r ) mediated current activity at -85 mV obtained after exposure to the different TRPV4 blockers was normalized to that obtained in control conditions. In all panels, data are presented as mean ± SEM ( n = 5–6 oocytes; white dots in m-r ). The paired normalized values were statistically analyzed by one sample t -test (*, p < 0.05; **, p < 0.01; ***, p < 0.001, with respect to the same oocytes before treatment with the inhibitors; ns, not statistically significant)

Article Snippet: Antibodies against human or mouse TRPV4 were purchased from Novus Biologicals (# NBP2-41262) and Invitrogen (# OSR00136W), respectively.

Techniques: Inhibition, Expressing, Control, Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa

doi: 10.1007/s00018-024-05341-w

Figure Lengend Snippet: Inhibition of Trpv4 or Aqp4a impairs seabream sperm motility. a-c Inhibition of the percentage of motility and progressivity (% MOT and % PROG, respectively) and curvilinear velocity (VCL) at 5, 30 and 60 s post-activation induced by increasing doses of the TRPV4 antagonist RN-1734 or the Aqp4a antibody (α-Aqp4a), or by 20 µM of TGN-020. Control spermatozoa were treated with 0.5% DMSO (vehicle, a and c ) or 200 nM IgY ( b ). In all panels, the data are the mean ± SEM ( n = 5–8 males, one ejaculate per male; white dots). Statistical differences in a and b within each time point were measured by one-way ANOVA, or Kruskal-Wallis test, followed by Dunn’s multiple comparisons test ( a and b ), or by an unpaired Student t -test ( c ). *, p < 0.05; **, p < 0.01; ***, p < 0.001, with respect to non-treated sperm. The brightfield and immunostaining images in panel b confirm the specific binding of α-Aqp4a to Aqp4a in the spermatozoon flagellum through the labelling of either IgY- or α-Aqp4a in vitro-treated spermatozoa with anti-chicken secondary antibodies. The nucleus of the spermatozoa was counterstained with DAPI (blue). Scale bars, 5 μm

Article Snippet: Antibodies against human or mouse TRPV4 were purchased from Novus Biologicals (# NBP2-41262) and Invitrogen (# OSR00136W), respectively.

Techniques: Inhibition, Activation Assay, Control, Immunostaining, Binding Assay, In Vitro

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa

doi: 10.1007/s00018-024-05341-w

Figure Lengend Snippet: Model for the RVI mechanism in post-activated seabream spermatozoa. Upon release into the hyperosmotic seawater a rapid water efflux mediated by Aqp1aa results in cell shrinkage and subsequent [Ca 2+ ] i increase, both triggering flagellar motility. The rapid spermatozoon shrinkage could induce the activation of NKCC1 and Na + , K + , Cl − influx, which would then drive water uptake through Aqp4a, and perhaps also by NKCC1 supported water fluxes. Water influx into the spermatozoon could induce local swelling of the flagellum, triggering the activation of the Trpv4 and a further local Ca 2+ influx, which may activate signaling events for the stimulation of L-type Ca 2+ channels, and perhaps of other Ca 2+ channels present in the spermatozoa, thereby allowing a massive Ca 2+ influx. The increased accumulation of Ca 2+ and other ions would facilitate further Aqp4a-mediated water uptake in the spermatozoon, and feasibly also through Aqp1ab1 and/or -10bb present in the anterior region of the flagellum, to promote a fast volume recovery and to maintain motility

Article Snippet: Antibodies against human or mouse TRPV4 were purchased from Novus Biologicals (# NBP2-41262) and Invitrogen (# OSR00136W), respectively.

Techniques: Activation Assay

Journal: Fluids and Barriers of the CNS

Article Title: Lysophosphatidic acid as a CSF lipid in posthemorrhagic hydrocephalus that drives CSF accumulation via TRPV4-induced hyperactivation of NKCC1

doi: 10.1186/s12987-022-00361-9

Figure Lengend Snippet: LPA is a novel endogenous agonist of luminal membraneous TRPV4. A Representative current traces obtained from uninjected or TRPV4-expressing oocytes during a 200 ms step protocol (upper panel, control solution; lower panel, LPA-containing solution). B Averaged I/V curves from TRPV4–expressing oocytes in control solution, n = 9 (black), during application of LPA without, n = 8 (orange) or with TRPV-4 inhibition, n = 8 (+RN, purple). Uninjected oocytes treated with LPA are shown in grey, n = 12. C TRPV4-mediated current activity (at V m = − 85 mV) summarized in control solution (black) and after exposure to LPA without (orange) or with TRPV4 inhibition (purple). D Transcript abundance (in TPM) of members of the transient receptor potential vanilloid family obtained from RNA sequencing of rat choroid plexus; TRPV1, 0.4; TRPV2, 0.5; TRPV4, 58.5; TRPV6, 0.06. E Transcript abundance of all filtered ion channels. F Representative immunolabeling of TRPV4 in rat choroid plexus. Nuclei staining by DAPI. Scale bar = 5 μm. Statistical evaluation with one-way ANOVA with Dunnett’s post hoc test **P < 0.01; ***P < 0.001

Article Snippet: Blockage was done with 10% swine serum diluted in PBS + 1%Tween-20 (PBST), and the sections were immunolabeled with primary rabbit anti-NKCC1 (Abcam #AB59791, 1:400) and mouse anti-TRPV4 (BD BioSciences #AB53079, 1:500) overnight at 4 °C, and Alexa Fluor TM 488 and Alexa Fluor TM 647 (ThermoFisher Scientific 1:500) for 2 h at room temperature.

Techniques: Expressing, Inhibition, Activity Assay, RNA Sequencing Assay, Immunolabeling, Staining

Journal: Fluids and Barriers of the CNS

Article Title: Lysophosphatidic acid as a CSF lipid in posthemorrhagic hydrocephalus that drives CSF accumulation via TRPV4-induced hyperactivation of NKCC1

doi: 10.1186/s12987-022-00361-9

Figure Lengend Snippet: TRPV4 activity modulates ICP and CSF production in vivo. A Schematic illustrating the cranial window (shown as a dotted circle) into which the ICP probe is positioned, LV; lateral ventricle. B ICP as a function of time upon infusion of control solution, n = 5 (black) or TRPV4 inhibitor-containing solution, n = 5 (RN; purple) shown as 5 min average values normalized to the baseline. Inset, initial ICP, n = 10. C Summarized changes in ICP with control solution (black) and TRPV4 inhibitor-containing solution after 1.5 h. D Schematic of the VCP method used to determine the CSF production rate. E Representative time course of the dextran ratio (outflow/inflow) with a mock solution change with control solution (indicated with a grey bar). Inset, average CSF production rate, n = 7. F CSF production as a function of time. Data normalized to the last four samples before solution change to either the control solution, n = 7 (black), the TRPV4 activator GSK, n = 6 (green), or the TRPV4 inhibitor RN, n = 6 (purple). G Summarized CSF production rates in % of control after exposure to vehicle (black), TRPV4 activation (green) and TRPV4 inhibition (purple). H Correctly targeted dye delivery in mid-saggital sections of a rat brain. I A representative image of a rat after injection of IRDye 800CW carboxylate dye (superimposed pseudo-color). The square placed in line with lambda indicates the area of dye content quantification. J Representative images obtained at t = 0.5 (t 0.5 ) and t = 5 (t 5 ) min in control solution (left) or upon TRPV4 inhibition (RN; right). K The dye intensity normalized to that obtained in the first image and plotted as a function of time representing flow rate for control, n = 5 (black), TRPV4 activation, n = 6 (GSK, green) and TRPV4 inhibition, n = 6 (RN, purple). L Quantification of the dye intensity (flow rate) determined from linear regression in K over the 5 min. time window from control (black), TRPV4 activation (green) and TRPV4 inhibition (purple). Statistical evaluation with Student’s t- test ( C ) or one-way ANOVA with Dunnett’s post hoc test ( F and K ) *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: Blockage was done with 10% swine serum diluted in PBS + 1%Tween-20 (PBST), and the sections were immunolabeled with primary rabbit anti-NKCC1 (Abcam #AB59791, 1:400) and mouse anti-TRPV4 (BD BioSciences #AB53079, 1:500) overnight at 4 °C, and Alexa Fluor TM 488 and Alexa Fluor TM 647 (ThermoFisher Scientific 1:500) for 2 h at room temperature.

Techniques: Activity Assay, In Vivo, Activation Assay, Inhibition, Injection

Journal: Fluids and Barriers of the CNS

Article Title: Lysophosphatidic acid as a CSF lipid in posthemorrhagic hydrocephalus that drives CSF accumulation via TRPV4-induced hyperactivation of NKCC1

doi: 10.1186/s12987-022-00361-9

Figure Lengend Snippet: TRPV4 co-localizes with—and regulates—NKCC1. A Representative immunolabeling in rat choroid plexus of TRPV4 (red) and NKCC1 (green). DAPI is used for nuclei staining. Scale bar = 10 μm. B Proximity ligation assayed interaction complex between NKCC1 and TRPV4 shown as red speckles. Inset: Absence of primary antibodies against TRPV4 and NKCC1. Scale bar = 10 μm. C Efflux of 86 Rb + from choroid plexus (inset) in control settings without, n = 5 (black) or with NKCC1 inhibition by bumetanide (BUM), n = 5 (grey), or upon TRPV4 activation by GSK without, n = 5 (green) or with BUM, n = 5 (light green). GSK-mediated 86 Rb + efflux obtained with inclusion of the TRPV4 inhibitor RN, n = 5 (dotted, purple line). Y-axis is the natural logarithm of the amount of left in the choroid plexus at time t (A t ) divided by the amount at time 0 (A 0 ). D NKCC1-mediated efflux rate constants for 86 Rb + in control, n = 5 (black) or upon TRPV4 activation, n = 5 (GSK; green). Statistical evaluation with Student’s t- test. ***P < 0.001

Article Snippet: Blockage was done with 10% swine serum diluted in PBS + 1%Tween-20 (PBST), and the sections were immunolabeled with primary rabbit anti-NKCC1 (Abcam #AB59791, 1:400) and mouse anti-TRPV4 (BD BioSciences #AB53079, 1:500) overnight at 4 °C, and Alexa Fluor TM 488 and Alexa Fluor TM 647 (ThermoFisher Scientific 1:500) for 2 h at room temperature.

Techniques: Immunolabeling, Staining, Ligation, Inhibition, Activation Assay

Journal: Fluids and Barriers of the CNS

Article Title: Lysophosphatidic acid as a CSF lipid in posthemorrhagic hydrocephalus that drives CSF accumulation via TRPV4-induced hyperactivation of NKCC1

doi: 10.1186/s12987-022-00361-9

Figure Lengend Snippet: TRPV4-mediated Ca 2+ fluctuations activate NKCC1 in a WNK-SPAK-dependent manner. A Efflux of 86 Rb + from choroid plexus in control solution, n = 4 (black), in Ca 2+ -free solution, n = 4 (grey), upon TRPV4-activation by GSK with, n = 4 (green) or without Ca 2+ , n = 4 (light green). B GSK-sensitive efflux rate constants for 86 Rb + with (green) or without (light green) Ca 2+ present. C Schematic of the hypothesized LPA-induced TRPV4-mediated Ca 2+ influx regulating WNK-SPAK-mediated phosphorylation of NKKC1. D Transcript abundance of filtered kinases in rat choroid plexus with rank of SPAK and WNK kinases depicted. E Network analysis depicting published protein-protein associations amongst those illustrated in Panel A. F 86 Rb + efflux from choroid plexus in control settings without, n = 6 (black) or with SPAK inhibition, n = 6 (grey), or upon TRPV4 activation by GSK without, n = 6 (green) or with, n = 6 (light green) SPAK inhibition. Y-axis is the natural logarithm of the amount of left in the choroid plexus at time t (A t ) divided by the amount at time 0 (A 0 ). G GSK-mediated efflux rate constants for 86 Rb + without (green) or with SPAK-inhibition (light green). H 86 Rb + efflux in control settings without, n = 5 (black) or with, n = 5 (grey) WNK inhibition, or upon TRPV4 activation by GSK without, n = 5 (green) or with, n = 5 (light green) WNK inhibition. I GSK-mediated efflux rate constants for 86 Rb + without (green) or with (light green) WNK-inhibition. J 86 Rb + efflux in control settings without, n = 5 (black) or with, n = 5 (grey) NKCC1 inhibition by bumetanide (BUM), or upon application of LPA without, n = 5 (orange) or with, n = 5 (black/orange) NKCC1 inhibition (BUM). K BUM-sensitive efflux rate constants for 86 Rb + in control setting (black) or upon LPA application (orange). Statistical evaluation with Student’s t- test. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: Blockage was done with 10% swine serum diluted in PBS + 1%Tween-20 (PBST), and the sections were immunolabeled with primary rabbit anti-NKCC1 (Abcam #AB59791, 1:400) and mouse anti-TRPV4 (BD BioSciences #AB53079, 1:500) overnight at 4 °C, and Alexa Fluor TM 488 and Alexa Fluor TM 647 (ThermoFisher Scientific 1:500) for 2 h at room temperature.

Techniques: Activation Assay, Inhibition