trpm4 inhibitors  (Eppendorf AG)

Journal: Bioactive Materials

Article Title: Mechanically sensitized hydrogel microspheres trigger membrane receptor switch for cartilage repair

doi: 10.1016/j.bioactmat.2026.03.017

Figure Lengend Snippet: OBNC microspheres activate integrin receptors and mechanosensitive calcium channels. A) Mechanistic diagram of integrin activation verified using fluorophores. B-C) Fluorescence microscopy images of MSCs loaded on HAMA or OBNC hydrogel. D) Fluorescence intensity of single cell in each group was quantified. E) Fluorescence microscopy of MSCs loaded on OBNC hydrogel after different treatments. F) Fluorescence intensity in the whole field of view for each group. G) Fluorescence intensity in the single cell for each group. H) Schematic representation of patch clamp experiments. I) Electrical signals generated by MSCs in response to mechanical stimulation. J) Statistical analysis of poking currents (n = 6). K) The concentration of calcium ions in stem cells of different groups as detected by flow cytometry (siRNA1: targeting the TRPM4 gene, siRNA2: targeting the PIEZO1 gene). L) Quantitative analysis of flow cytometric results (∗ symbol represents comparison with HAMA group, # symbol represents comparison with OBNC group). (ns: non-significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ## P < 0.01, ### P < 0.001).

Article Snippet: TRPC1 inhibitor (0.3 nM, Pico145, CAS No. 1628287-16-0), TRPM7 inhibitor (1.0 μM, VPC4, CAS No. 945604-76-2), TRPV2 inhibitor (5.0 μM, compound IV2-1, CAS No. 2242724-49-6), TRPM4 inhibitor (1.5 μM, CBA, CAS No. 351424-20-9), PIEZO1 inhibitor (2.5 μM, GsMTx4, CAS No. 1209500-46-8), integrin αvβ5 inhibitor (8.0 nM, Compound 12, CAS No.: 2615912-33-7), integrin αvβ1 inhibitor (0.3 nM, Compound C8, CAS No. 1689540-62-2), integrin α5β1 inhibitor (10 μM, ATN-161, 904763-27-5), and CDK5 inhibitor (5 nM, CDK5-IN-1, 2,639,540-19-3) were purchased from MCE Biotechnology Co., LTD. After the MSCs were treated, the cRGD solution was added at a concentration of 1:200 and incubated in the dark for 15 min, and the results were observed by fluorescence microscopy.

Techniques: Activation Assay, Fluorescence, Microscopy, Single Cell, Patch Clamp, Generated, Concentration Assay, Flow Cytometry, Comparison

Journal: eNeuro

Article Title: Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits

doi: 10.1523/ENEURO.0332-17.2018

Figure Lengend Snippet: Immunolabeling of TRPM4 and TRPC3 channels in pre-BötC neurons and motoneurons. A , B , Confocal fluorescence microscopy images of coronal sections from neonatal mouse medulla at the level of the pre-BötC, showing widely distributed neuronal labeling by TRPM4 ( A1 , low magnification; A2 , higher magnification of dashed box in A1 ) and TRPC3 ( B ) channel antibodies (red) within the pre-BötC region and motoneurons within the semicompact division of nucleus ambiguus (NAsc). Note extensive antibody labeling also outside of these regions ( A1 ). C , D , Confocal images of the pre-BötC region from adult rats, showing neuronal labeling by TRPM4 ( C ) and TRPC3 ( D ) channel antibodies within the pre-BötC and labeling of NAsc motoneurons. E , F , Confocal images of the hypoglossal (XII) motor nucleus on one side of the medulla at the level containing the pre-BötC from neonatal rat ( E ) and mouse ( F ), showing TRPM4 ( E ) and TRPC3 ( F ) channel antibody labeling (red) of XII motoneurons identified by ChAT immunolabeling (green). Merged images (right in E and F ) show antibody colabeling of XII motoneurons. A–D have the same dorso-medial anatomic orientation. d, dorsal; m, medial; V4, 4th ventricle.

Article Snippet: We analyzed the time course of perturbations of the inspiratory burst frequency, amplitude, and duration of integrated XII inspiratory motor output after application to the slice bathing solution of the putative selective TRPM4 channel inhibitor (9-phenanthrol, Millipore, 10–50 μ m ; ), the selective TRPC3 channel inhibitor (Pyrazole compound-3: Pyr3, Millipore, 10–50 μ m ; ), and for comparison, the putative I CAN blocker flufenamic acid (FFA, Sigma-Aldrich, 20–75 μ m ; ; ).

Techniques: Immunolabeling, Fluorescence, Microscopy, Labeling, Antibody Labeling

Journal: eNeuro

Article Title: Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits

doi: 10.1523/ENEURO.0332-17.2018

Figure Lengend Snippet: Expression of TRPM4 and TRPC3 channel mRNA in glutamatergic and glycinergic/GABAergic pre-BötC inspiratory neurons. A , Overview of experimental in vitro neonatal rat rhythmic slice preparation showing whole-cell patch-clamp recording from the pre-BötC inspiratory neurons and suction electrode recordings from hypoglossal (XII) nerves to monitor inspiratory activity. NAsc, semicompact division of nucleus ambiguus; V4, fourth ventricle. B , Two-photon single optical plane images of pre-BötC inspiratory neuron (arrow) targeted for whole-cell recording and subsequent harvesting of cytoplasm, showing Ca 2+ -sensitive dye (OGB) labeling ( B1 ) and Dodt structural image ( B2 ). B3 , Identification of inspiratory neuron by verifying that the Ca 2+ fluorescence signals in real time are synchronized with integrated inspiratory XII nerve activity (∫ XII). C1 , Current-clamp recording (upper traces) from excitatory pre-BötC inspiratory neuron in B illustrates inspiratory bursts synchronized with ∫ XII. Under voltage-clamp (lower traces), the same neuron exhibited rhythmic inward synaptic currents synchronized with ∫ XII. This neuron was shown to be excitatory (VgluT2-expressing) by post hoc single-cell RT-PCR (below). C2 , Current-clamp recording (upper traces) and voltage-clamp recording (lower traces) from inhibitory pre-BötC inspiratory neuron illustrating inspiratory bursts and rhythmic inward synaptic currents synchronized with ∫ XII. This neuron was shown to be inhibitory (co-expression of GlyT2 and GAD67 mRNA) by post hoc single-cell RT-PCR (see below). D , Representative electrophoresis gel generated by single-cell multiplex RT-PCR from mRNA in cytoplasm harvested during whole-cell recording from two electrophysiologically identified pre-BötC inspiratory neurons ( C1 and C2 ) in neonatal rat slices. In addition to cDNA probes for TRPM4 and TRPC3 channel mRNA, probes for vesicular glutamate transporter type 2 (VgluT2), glycine transporter type 2 (GlyT2), and glutamatic acid decarboxylase 67 (GAD67) mRNA were used to identify excitatory or inhibitory neuronal phenotypes, examples of which are shown. Expected numbers of base pairs (bp) for reaction products are indicated. Assays for both of these neurons had clean negative controls from “mock harvests” in the slice and appropriate positive controls from 100 pg total rat brain RNA run as RT template (not shown, see Materials and Methods).

Article Snippet: We analyzed the time course of perturbations of the inspiratory burst frequency, amplitude, and duration of integrated XII inspiratory motor output after application to the slice bathing solution of the putative selective TRPM4 channel inhibitor (9-phenanthrol, Millipore, 10–50 μ m ; ), the selective TRPC3 channel inhibitor (Pyrazole compound-3: Pyr3, Millipore, 10–50 μ m ; ), and for comparison, the putative I CAN blocker flufenamic acid (FFA, Sigma-Aldrich, 20–75 μ m ; ; ).

Techniques: Expressing, In Vitro, Slice Preparation, Patch Clamp, Activity Assay, Labeling, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Generated, Multiplex Assay

Journal: eNeuro

Article Title: Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits

doi: 10.1523/ENEURO.0332-17.2018

Figure Lengend Snippet: Pharmacological inhibition of TRPM4, TRPC3, and I CAN does not affect variability of inspiratory rhythm in vitro . A , B , Poincaré maps ( A ) from a representative in vitro rat slice experiment with inhibition of TRPM4 and TRPC3 channels, respectively, by 9-phenanthrol (9-Phen in B ) and Pyr3, illustrating geometric fits of XII inspiratory period data and representations of short-term (SD1) and long-term (SD2) variability measures (see Methods for definitions), and summary data ( B ) for the analyzed group ( n = 6 each). C , D , Equivalent sets of data from in vitro mouse slice experiments. Variability measures normalized to control values were not statistically significant in all cases in B and D . T TOT , respiratory period.

Article Snippet: We analyzed the time course of perturbations of the inspiratory burst frequency, amplitude, and duration of integrated XII inspiratory motor output after application to the slice bathing solution of the putative selective TRPM4 channel inhibitor (9-phenanthrol, Millipore, 10–50 μ m ; ), the selective TRPC3 channel inhibitor (Pyrazole compound-3: Pyr3, Millipore, 10–50 μ m ; ), and for comparison, the putative I CAN blocker flufenamic acid (FFA, Sigma-Aldrich, 20–75 μ m ; ; ).

Techniques: Inhibition, In Vitro, Control

Journal: eNeuro

Article Title: Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits

doi: 10.1523/ENEURO.0332-17.2018

Figure Lengend Snippet: Perturbations of imaged pre-BötC inspiratory Ca 2+ activity and electrophysiologically recorded hypoglossal motor output in the VgluT2-GCaMP6f transgenic mouse in vitro slice during application of TRPM4 channel inhibitor. A , Example of two-photon single optical plane image showing Cre-dependent GCaMP6f expression in pre-BötC glutamatergic neurons. B , Background subtracted ( F – F 0 ) Δ F image showing increased GCaMP6f fluorescence of individual neurons for the optical plane shown in A . C , Integrated inspiratory hypoglossal activity (∫ XII) and the spatially averaged field GCaMP6f fluorescence transients (Δ F ), quantified as F – F 0 , of the optical plane shown in A during control time, and 10 and 15 min after bath-application of 9-phenanthrol (50 µ m ). D , Inspiratory burst-wise correlations of the field fluorescence Δ F and ∫ XII amplitudes (XII Amp; colored dots), and their grouped averages (circles with error cross bars: mean values ± SEM), normalized to their control values, for time windows 5, 10, 15, and 20 min after 9-phenanthrol application. Note that the 15- and 20-min point clusters are nearly superimposed, indicating quasi–steady state of the perturbations was achieved by 20 min. The identity line (dashed) and linear regression line (solid; Pearson correlation coefficient r = 0.74) indicate significant correlation between peak field Δ F and XII Amp.

Article Snippet: We analyzed the time course of perturbations of the inspiratory burst frequency, amplitude, and duration of integrated XII inspiratory motor output after application to the slice bathing solution of the putative selective TRPM4 channel inhibitor (9-phenanthrol, Millipore, 10–50 μ m ; ), the selective TRPC3 channel inhibitor (Pyrazole compound-3: Pyr3, Millipore, 10–50 μ m ; ), and for comparison, the putative I CAN blocker flufenamic acid (FFA, Sigma-Aldrich, 20–75 μ m ; ; ).

Techniques: Activity Assay, Transgenic Assay, In Vitro, Expressing, Fluorescence, Control

Journal: eNeuro

Article Title: Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits

doi: 10.1523/ENEURO.0332-17.2018

Figure Lengend Snippet: Single-neuron GCaMP6f fluorescence signal tracking during TRPM4 channel inhibition. A , Single optical plane image of the pre-BötC region with cells of interest (1–8) in a rhythmically active neonatal medullary in vitro slice preparation from the VgluT2-GCaMP6f transgenic mouse. Regions of interest detected algorithmically (see Methods) for quantifying somal fluorescence transients are outlined in cyan. Color scale of pixels immediately surrounding some of the cells (2, 3, 7, 8) was adjusted to more clearly delineate neuron soma in this image. B , Examples of time series of single-neuron GCaMPF6f fluorescence transients synchronous with integrated inspiratory hypoglossal activity (∫ XII, red) used for single-neuron Δ F analysis during control period, and 10 and 20 min after bath application of 50 µ m 9-phenanthrol.

Article Snippet: We analyzed the time course of perturbations of the inspiratory burst frequency, amplitude, and duration of integrated XII inspiratory motor output after application to the slice bathing solution of the putative selective TRPM4 channel inhibitor (9-phenanthrol, Millipore, 10–50 μ m ; ), the selective TRPC3 channel inhibitor (Pyrazole compound-3: Pyr3, Millipore, 10–50 μ m ; ), and for comparison, the putative I CAN blocker flufenamic acid (FFA, Sigma-Aldrich, 20–75 μ m ; ; ).

Techniques: Fluorescence, Inhibition, In Vitro, Slice Preparation, Transgenic Assay, Activity Assay, Control

Journal: eNeuro

Article Title: Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits

doi: 10.1523/ENEURO.0332-17.2018

Figure Lengend Snippet: Effects of TRPM4, TRPC3, and I CAN channel inhibitors on inspiratory Ca 2+ activity of the pre-BötC field and glutamatergic neurons expressing GCaMP6f. A , Example of relationship between normalized pre-BötC peak field GCaMP6f ΔF and normalized individual inspiratory cell ΔF, 10 and 20 min after bath-applied 50 µ m 9-phenanthrol. Eight neurons (shown in ) were tracked through time (connected green dots) within the two-photon optical section using automated ROI detection. Group mean values ± SEM of the normalized fluorescence transients are plotted (green filled circles with error bars) and the identity line (dashed) is indicated. Note that two of the neurons showed augmented fluorescence transients in this example, but the mean group cellular Δ F nevertheless followed the field Δ F . B , Group summary of effects of TRPM4, TRPC3, and I CAN inhibitors on the inspiratory pre-BötC field Δ F and cellular Δ F . Left: mean values of cellular ΔF (red, n = 6 neurons; green, n = 8, same as A ) ± SEM during control period, 10 min, and 20 min after bath-applied 9-phenanthrol from two slices are indicated (diamonds and error crosses). Inspiratory neurons with unaffected or increased ΔF amplitude included in the group statistics are plotted individually at the top. Middle: three-experiment summary for Pyr3 (red, n = 6 neurons; green, n = 13; blue, n = 4). Right: two-experiment summary for FFA (red, n = 12 neurons; green, n = 6). Identity line (dashed) is indicated.

Article Snippet: We analyzed the time course of perturbations of the inspiratory burst frequency, amplitude, and duration of integrated XII inspiratory motor output after application to the slice bathing solution of the putative selective TRPM4 channel inhibitor (9-phenanthrol, Millipore, 10–50 μ m ; ), the selective TRPC3 channel inhibitor (Pyrazole compound-3: Pyr3, Millipore, 10–50 μ m ; ), and for comparison, the putative I CAN blocker flufenamic acid (FFA, Sigma-Aldrich, 20–75 μ m ; ; ).

Techniques: Activity Assay, Expressing, Fluorescence, Control

Journal: eNeuro

Article Title: Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits

doi: 10.1523/ENEURO.0332-17.2018

Figure Lengend Snippet: Time courses of perturbations of respiratory neural activities by TRPM4 channel inhibitor in mature rat and mouse arterially perfused in situ brainstem–spinal cord preparations. A–C , Time courses of integrated burst amplitudes (normalized to mean control amplitudes, pink mountain plots) of inspiratory pre-BötC neural population activity (pre-BötC) obtained by extracellular recordings, vagus nerve (VN) inspiratory (Insp) and postinspiratory (post-I) activity (solid black line in middle panel in A ), phrenic nerve (PN) inspiratory activity, and respiratory frequency ( f R ) in perfused preparation from mature (4-wk-old) rat. TRPM4 inhibitor 9-phenanthrol (50 µ m ) was added to the perfusate at the vertical dashed line. B, Cycle-triggered overplots of the three neurograms (pre-BötC, VN, and PN) digitally triggered at the onset of PN activity (vertical solid line) before (red, corresponding to time points marked by arrow a in C ) and after the inhibitor (black traces, at arrow b in C ). C , Dynamic raster plots of cycle-triggered PN inspiratory (red) and VN including post-I (cyan, right side) activities. After 9-phenanthrol, T I (red) was prolonged, PN and VN inspiratory amplitude declined (darkened red), and f R increased (see A ) as post-I activity amplitude declined. D–F , Same type of data sets and analysis for an adult (4-mo-old) mouse preparation showing perturbations of pre-BötC, VN, and PN activity, including loss of VN post-I activity and associated increase of f R , following administration of 9-phenanthrol (20 µ m ).

Article Snippet: We analyzed the time course of perturbations of the inspiratory burst frequency, amplitude, and duration of integrated XII inspiratory motor output after application to the slice bathing solution of the putative selective TRPM4 channel inhibitor (9-phenanthrol, Millipore, 10–50 μ m ; ), the selective TRPC3 channel inhibitor (Pyrazole compound-3: Pyr3, Millipore, 10–50 μ m ; ), and for comparison, the putative I CAN blocker flufenamic acid (FFA, Sigma-Aldrich, 20–75 μ m ; ; ).

Techniques: In Situ, Control, Activity Assay

Journal: eNeuro

Article Title: Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits

doi: 10.1523/ENEURO.0332-17.2018

Figure Lengend Snippet: Summary of effects of TRPM4 and TRPC3 channel inhibitors on respiratory activities in arterially perfused brainstem–spinal cord preparation in situ from mature rats and adult mice. A , B , Summary time courses (solid lines: mean values; lighter color bands: ± SEM) of the amplitudes of integrated inspiratory pre-BötC neural population activity, VN post-inspiratory (post-I) activity, and PN inspiratory activity from rat ( A , 9-phenanthrol, n = 6; Pyr3, n = 8) and mouse ( B , 9-phenanthrol, n = 7; Pyr3, n = 6) preparations, showing significant reduction of all amplitudes (normalized to mean control values) by both TRPM4 (9-phenanthrol, red) and TRPC3 (Pyr3, blue) channel inhibitors. Bottom panels show group summaries for inspiratory frequency ( f R ) and inspiratory activity time ( T I ). Time of drug administration (9-phenanthrol, 20–50 µ m ; Pyr3, 50 µ m ) is indicated by vertical dashed lines.

Article Snippet: We analyzed the time course of perturbations of the inspiratory burst frequency, amplitude, and duration of integrated XII inspiratory motor output after application to the slice bathing solution of the putative selective TRPM4 channel inhibitor (9-phenanthrol, Millipore, 10–50 μ m ; ), the selective TRPC3 channel inhibitor (Pyrazole compound-3: Pyr3, Millipore, 10–50 μ m ; ), and for comparison, the putative I CAN blocker flufenamic acid (FFA, Sigma-Aldrich, 20–75 μ m ; ; ).

Techniques: In Situ, Activity Assay, Control

Journal: eNeuro

Article Title: Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits

doi: 10.1523/ENEURO.0332-17.2018

Figure Lengend Snippet: Time-dependent changes in the amplitude of integrated XII inspiratory burst activities (XII Amp) and the field GCaMP6f fluorescence transients (Δ F ) of the pre-BötC glutamatergic population after TRPM4, TRPC3, and I CAN inhibitors. A , Control experiments ( n = 5 mice) to test for possible photobleaching and time-dependent changes in population activity, in which calcium imaging was performed without any drug application with exactly the same protocol of image acquisition as the pharmacological experiments. The results (mean normalized values ± SEM) show that there were no significant changes in the pre-BötC field Δ F amplitude, XII Amp (normalized to control values), and normalized inspiratory burst frequency ( f R ). B , Group summary data (mean normalized values ± SEM) for 9-phenanthrol, Pyr3, and FFA ( n = 5, 5, and 4, respectively) shows reduction in both XII Amp and the pre-BötC field Δ F amplitude, while f R changed nonsignificantly after applying channel inhibitors in all cases. C , Time-dependent reductions of XII Amp and field Δ F amplitudes after drug application were positively correlated (solid lines: linear regression; Pearson linear correlation coefficient for 9-phenanthrol, Pyr3, and FFA: r = 0.864, 0.845, and 0.749, respectively). The linear regression on mean amplitude reduction between XII Amp and field Δ F for 9-phenanthrol, Pyr3, and FFA yielded corresponding linear models with slopes m = 0.859, 0.463, and 0.676, and intercepts b = 0.103, 0.499, and 0.277, respectively. Dashed lines represent the identity line.

Article Snippet: We analyzed the time course of perturbations of the inspiratory burst frequency, amplitude, and duration of integrated XII inspiratory motor output after application to the slice bathing solution of the putative selective TRPM4 channel inhibitor (9-phenanthrol, Millipore, 10–50 μ m ; ), the selective TRPC3 channel inhibitor (Pyrazole compound-3: Pyr3, Millipore, 10–50 μ m ; ), and for comparison, the putative I CAN blocker flufenamic acid (FFA, Sigma-Aldrich, 20–75 μ m ; ; ).

Techniques: Fluorescence, Control, Activity Assay, Imaging