p2x4 receptor  (Alomone Labs)


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    Structured Review

    Alomone Labs p2x4 receptor
    The negative chronotropic effect of ATP is mediated by activation of <t>P2X4.</t> The negative chronotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X7 receptor antagonist, A438079 (3 µM, A ), the P2X4 receptor antagonist, 5-BDBD (10 µM, B ) and the positive allosteric modulator of the P2X4 receptor, ivermectin (30 µM, C ). The negative chronotropic effect of CTP (1 mM, D ) either in the absence or in the presence of 5-BDBD (10 µM), is also shown for comparison. Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from baseline; horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p
    P2x4 Receptor, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy"

    Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2019.01103

    The negative chronotropic effect of ATP is mediated by activation of P2X4. The negative chronotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X7 receptor antagonist, A438079 (3 µM, A ), the P2X4 receptor antagonist, 5-BDBD (10 µM, B ) and the positive allosteric modulator of the P2X4 receptor, ivermectin (30 µM, C ). The negative chronotropic effect of CTP (1 mM, D ) either in the absence or in the presence of 5-BDBD (10 µM), is also shown for comparison. Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from baseline; horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p
    Figure Legend Snippet: The negative chronotropic effect of ATP is mediated by activation of P2X4. The negative chronotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X7 receptor antagonist, A438079 (3 µM, A ), the P2X4 receptor antagonist, 5-BDBD (10 µM, B ) and the positive allosteric modulator of the P2X4 receptor, ivermectin (30 µM, C ). The negative chronotropic effect of CTP (1 mM, D ) either in the absence or in the presence of 5-BDBD (10 µM), is also shown for comparison. Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from baseline; horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p

    Techniques Used: Activation Assay

    Selective blockage of P2X4 and of NCX transporter partially offsets the negative inotropic effect of ATP in paced rat ventricular strips. The negative inotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X4 receptor antagonist, 5-BDBD (10 µM, A and B ) and of the NCX inhibitor, KB-R7943 (3 µM, C and D ). Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from the baseline isometric tension of RV strips, measured as the active tension (mN/mg of wet tissue weight, panels A and C ) and the derivative of developed force over time (+dF/dt, mN/s, panels B and D ); horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p
    Figure Legend Snippet: Selective blockage of P2X4 and of NCX transporter partially offsets the negative inotropic effect of ATP in paced rat ventricular strips. The negative inotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X4 receptor antagonist, 5-BDBD (10 µM, A and B ) and of the NCX inhibitor, KB-R7943 (3 µM, C and D ). Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from the baseline isometric tension of RV strips, measured as the active tension (mN/mg of wet tissue weight, panels A and C ) and the derivative of developed force over time (+dF/dt, mN/s, panels B and D ); horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p

    Techniques Used:

    Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-002, C-terminus, Alomone) and NCX1 (Anx-011, Alomone) protein in the sinoatrial node (SAN), right atria (RA) and right ventricle (RV). The SAN was identified based on its low Cx43 (green) and high HCN4 (magenta) protein expression (left hand-side images). Images were taken from whole-mount heart preparations including the three analyzed regions, SAN, RA and RV. Dashed lines represent boundaries of the SAN. The pulmonary parenchyma was used as a structural support to facilitate immunostaining of myocardial sections and it is visible in the bottom right quadrant of each SAN image. White arrows indicate blood vessels including the SAN artery. Scale bar 30 µm. Images are representative of three different individuals.
    Figure Legend Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-002, C-terminus, Alomone) and NCX1 (Anx-011, Alomone) protein in the sinoatrial node (SAN), right atria (RA) and right ventricle (RV). The SAN was identified based on its low Cx43 (green) and high HCN4 (magenta) protein expression (left hand-side images). Images were taken from whole-mount heart preparations including the three analyzed regions, SAN, RA and RV. Dashed lines represent boundaries of the SAN. The pulmonary parenchyma was used as a structural support to facilitate immunostaining of myocardial sections and it is visible in the bottom right quadrant of each SAN image. White arrows indicate blood vessels including the SAN artery. Scale bar 30 µm. Images are representative of three different individuals.

    Techniques Used: Expressing, Immunostaining

    The mechanism underlying the dual P2X4 receptor-mediated effects on cardiac chronotropy and inotropy implicates downstream modulation of NCX activity (digitalis-like phenomenon). Besides ion fluxes carried by pacemaker HCN channels (not represented), the unstable resting membrane potential and the spontaneous firing of SAN cardiomyocytes are attributed mainly to the electrogenic NCX transport operating in the forward Ca 2+ -extrusion mode. Na + influx through the P2X4 receptor pore dissipates the electrochemical gradient of this ion across the plasma membrane leading to inhibition and/or reversion of the NCX pacemaker current. This may justify slowing down of SAN cells depolarizations and the negative chronotropic effect of ATP. Likewise, intracellular Ca 2+ accumulation due both (1) to Ca 2+ influx through the P2X4 receptor pore, and (2) to reversal of NCX activity may explain the positive inotropic effect of the P2X4 receptor in paced ventricular cardiomyocytes. Figure composition used elements from Servier Medical Art .
    Figure Legend Snippet: The mechanism underlying the dual P2X4 receptor-mediated effects on cardiac chronotropy and inotropy implicates downstream modulation of NCX activity (digitalis-like phenomenon). Besides ion fluxes carried by pacemaker HCN channels (not represented), the unstable resting membrane potential and the spontaneous firing of SAN cardiomyocytes are attributed mainly to the electrogenic NCX transport operating in the forward Ca 2+ -extrusion mode. Na + influx through the P2X4 receptor pore dissipates the electrochemical gradient of this ion across the plasma membrane leading to inhibition and/or reversion of the NCX pacemaker current. This may justify slowing down of SAN cells depolarizations and the negative chronotropic effect of ATP. Likewise, intracellular Ca 2+ accumulation due both (1) to Ca 2+ influx through the P2X4 receptor pore, and (2) to reversal of NCX activity may explain the positive inotropic effect of the P2X4 receptor in paced ventricular cardiomyocytes. Figure composition used elements from Servier Medical Art .

    Techniques Used: Activity Assay, Inhibition

    2) Product Images from "Single-Dose P2 X4R Single-Chain Fragment Variable Antibody Permanently Reverses Chronic Pain in Male Mice"

    Article Title: Single-Dose P2 X4R Single-Chain Fragment Variable Antibody Permanently Reverses Chronic Pain in Male Mice

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms222413612

    Evidence of P2X4R scFv 95 in brain and trigeminal ganglia, while P2X4R scFv 95 treatment prevents the P2X4R protein elevation in trigeminal ganglia of untreated FRICT-ION mice. ( A ). His-tag biomarker in the brain indicates brain penetrance of the P2X4R scFv 95 in amygdala, medulla, and trigeminal ganglia. ( B ). Bar graph of the scFv His-tag in the amygdala, brainstem medulla dorsal horn, and trigeminal ganglia (TG) in the Western blots. The His-tag remains at 7 weeks after the single i.p. dose was given in male mice with FRICT-ION. Almost no His-tag was detected in the medial prefrontal cortex (mPFC) in these comparisons with normalized intensity in arbitrary units. ( C ). The P2X4 protein increased in trigeminal ganglia of FRICT-ION mice with neuropathic pain (week 10) was not evident in mice treated with P2X4R scFv 95. n = 3, One-way ANOVA, * p
    Figure Legend Snippet: Evidence of P2X4R scFv 95 in brain and trigeminal ganglia, while P2X4R scFv 95 treatment prevents the P2X4R protein elevation in trigeminal ganglia of untreated FRICT-ION mice. ( A ). His-tag biomarker in the brain indicates brain penetrance of the P2X4R scFv 95 in amygdala, medulla, and trigeminal ganglia. ( B ). Bar graph of the scFv His-tag in the amygdala, brainstem medulla dorsal horn, and trigeminal ganglia (TG) in the Western blots. The His-tag remains at 7 weeks after the single i.p. dose was given in male mice with FRICT-ION. Almost no His-tag was detected in the medial prefrontal cortex (mPFC) in these comparisons with normalized intensity in arbitrary units. ( C ). The P2X4 protein increased in trigeminal ganglia of FRICT-ION mice with neuropathic pain (week 10) was not evident in mice treated with P2X4R scFv 95. n = 3, One-way ANOVA, * p

    Techniques Used: Mouse Assay, Biomarker Assay, Western Blot

    Effect of in vitro P2X4R scFv95 treatment on electrophysiological properties of TG neuron primary cultures from FRICT-ION mice. ( A ). Distribution of single vs. multiple action potential (AP) firing from untreated FRICT-ION and P2X4 scFv95 pre-treated FRICT-ION TG neurons. Prevalence of multiple firing was higher in untreated TG neurons from FRICT-ION mice than those with P2X4R scFv95 pre-treatment. ( B ). Prevalence of rebound firing in untreated FRICT-ION and P2X4R scFv95 pre-treated FRICT-ION TG neuronal cultures. Neurons with more rebound firing were observed more often in untreated cultures than in P2X4R-scFv-treated TG neurons from FRICT-ION mice. Effect of P2X4R-scFv treatment on electrophysiological properties of ( C ). large-diameter TG neurons ( > 25 microns) and ( D ). small- to medium-diameter TG-neurons (
    Figure Legend Snippet: Effect of in vitro P2X4R scFv95 treatment on electrophysiological properties of TG neuron primary cultures from FRICT-ION mice. ( A ). Distribution of single vs. multiple action potential (AP) firing from untreated FRICT-ION and P2X4 scFv95 pre-treated FRICT-ION TG neurons. Prevalence of multiple firing was higher in untreated TG neurons from FRICT-ION mice than those with P2X4R scFv95 pre-treatment. ( B ). Prevalence of rebound firing in untreated FRICT-ION and P2X4R scFv95 pre-treated FRICT-ION TG neuronal cultures. Neurons with more rebound firing were observed more often in untreated cultures than in P2X4R-scFv-treated TG neurons from FRICT-ION mice. Effect of P2X4R-scFv treatment on electrophysiological properties of ( C ). large-diameter TG neurons ( > 25 microns) and ( D ). small- to medium-diameter TG-neurons (

    Techniques Used: In Vitro, Mouse Assay

    3) Product Images from "Distinct purinergic signaling pathways in prepubescent mouse spermatogonia"

    Article Title: Distinct purinergic signaling pathways in prepubescent mouse spermatogonia

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201611636

    P2X7 constitutes the low-affinity spermatogonial ATP sensor. (A) Exemplary whole-cell voltage-clamp recordings of currents triggered by repetitive stimulation (1 s; ISI = 3 s; S 1 , S 6 ) with ATP (300 µM; left) or BzATP (300 µM; right), respectively. Traces from untransfected spermatogonia (black) and cells transfected with either nontarget negative control siRNA (gray) or a construct targeting P2X4 (green) are overlaid. (B) Quantification of results from repetitive stimulation experiments as shown in A. Peak current densities (means ± SEM) are color coded and plotted as a function of stimulus repetition. Cells were analyzed under control conditions ( n = 33/13 [ATP/BzATP]; black), after transfection with nontargeting siRNA ( n = 16/23 [ATP/BzATP]; gray), and after P2X4 knockdown ( n = 16/23 [ATP/BzATP]; green). Inset (left) shows the data delimited by the dashed rectangle at an increased scale. Asterisks (*) indicate statistical significance, P
    Figure Legend Snippet: P2X7 constitutes the low-affinity spermatogonial ATP sensor. (A) Exemplary whole-cell voltage-clamp recordings of currents triggered by repetitive stimulation (1 s; ISI = 3 s; S 1 , S 6 ) with ATP (300 µM; left) or BzATP (300 µM; right), respectively. Traces from untransfected spermatogonia (black) and cells transfected with either nontarget negative control siRNA (gray) or a construct targeting P2X4 (green) are overlaid. (B) Quantification of results from repetitive stimulation experiments as shown in A. Peak current densities (means ± SEM) are color coded and plotted as a function of stimulus repetition. Cells were analyzed under control conditions ( n = 33/13 [ATP/BzATP]; black), after transfection with nontargeting siRNA ( n = 16/23 [ATP/BzATP]; gray), and after P2X4 knockdown ( n = 16/23 [ATP/BzATP]; green). Inset (left) shows the data delimited by the dashed rectangle at an increased scale. Asterisks (*) indicate statistical significance, P

    Techniques Used: Transfection, Negative Control, Construct

    Posttranscriptional gene silencing confirms P2X4 as the high-affinity spermatogonial ATP sensor. (A) Bar chart quantifying siRNA-dependent selective knockdown of spermatogonial gene expression in vitro. When transfected with one of two siRNA constructs (green), knockdown of P2X4 expression was confirmed by quantitative real-time PCR. Transcript quantities (means ± SEM) are normalized to mRNA levels in untransfected spermatogonia and compared with cells treated with nontargeting siRNA controls (gray). Administration of both targeting siRNAs effectively reduced P2X4 transcription as compared with both untransfected spermatogonia and negative (nontargeting siRNA) controls. Asterisks (*) denote statistical significance, P
    Figure Legend Snippet: Posttranscriptional gene silencing confirms P2X4 as the high-affinity spermatogonial ATP sensor. (A) Bar chart quantifying siRNA-dependent selective knockdown of spermatogonial gene expression in vitro. When transfected with one of two siRNA constructs (green), knockdown of P2X4 expression was confirmed by quantitative real-time PCR. Transcript quantities (means ± SEM) are normalized to mRNA levels in untransfected spermatogonia and compared with cells treated with nontargeting siRNA controls (gray). Administration of both targeting siRNAs effectively reduced P2X4 transcription as compared with both untransfected spermatogonia and negative (nontargeting siRNA) controls. Asterisks (*) denote statistical significance, P

    Techniques Used: Expressing, In Vitro, Transfection, Construct, Real-time Polymerase Chain Reaction

    P2X4 is a candidate mediator of the high-affinity ATP response in spermatogonia. (A) Reverse transcription PCR reveals transcripts for P2rx2 , P2rx4 , and P2rx7 in both Sertoli–germ cell co-cultures and whole testis preparations at P7. A faint band for P2rx3 -encoding cDNA is only amplified from whole testis samples but not co-cultures. Primer pairs generate amplificates of 150–250 bp, respectively. cDNA from whole brain/spinal cord serves as positive control, whereas no template controls are devoid of detectable signals. (B–E) Exemplary whole-cell voltage-clamp recordings of ATP-induced currents (10 µM; 1 s; V hold = −80 mV; S 1 , S 6 ) under control conditions and during treatment with suramin (10 µM and 100 µM; B), extracellular Cu 2+ (100 µM; C), reduced pH (6.3; D), and ivermectin (3 µM; E). Altered conditions were established for at least 60 s (preincubation). (F) Bar diagram (means ± SEM) depicting the quantitative analysis of experiments exemplified in B–E. Note the discontinuous ordinate (//). Numbers of cells tested are indicated above bars. Asterisks (*) denote statistical significance, P ≤ 0.01 (paired t test).
    Figure Legend Snippet: P2X4 is a candidate mediator of the high-affinity ATP response in spermatogonia. (A) Reverse transcription PCR reveals transcripts for P2rx2 , P2rx4 , and P2rx7 in both Sertoli–germ cell co-cultures and whole testis preparations at P7. A faint band for P2rx3 -encoding cDNA is only amplified from whole testis samples but not co-cultures. Primer pairs generate amplificates of 150–250 bp, respectively. cDNA from whole brain/spinal cord serves as positive control, whereas no template controls are devoid of detectable signals. (B–E) Exemplary whole-cell voltage-clamp recordings of ATP-induced currents (10 µM; 1 s; V hold = −80 mV; S 1 , S 6 ) under control conditions and during treatment with suramin (10 µM and 100 µM; B), extracellular Cu 2+ (100 µM; C), reduced pH (6.3; D), and ivermectin (3 µM; E). Altered conditions were established for at least 60 s (preincubation). (F) Bar diagram (means ± SEM) depicting the quantitative analysis of experiments exemplified in B–E. Note the discontinuous ordinate (//). Numbers of cells tested are indicated above bars. Asterisks (*) denote statistical significance, P ≤ 0.01 (paired t test).

    Techniques Used: Polymerase Chain Reaction, Amplification, Positive Control

    4) Product Images from "Distinct purinergic signaling pathways in prepubescent mouse spermatogonia"

    Article Title: Distinct purinergic signaling pathways in prepubescent mouse spermatogonia

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201611636

    P2X7 constitutes the low-affinity spermatogonial ATP sensor. (A) Exemplary whole-cell voltage-clamp recordings of currents triggered by repetitive stimulation (1 s; ISI = 3 s; S 1 , S 6 ) with ATP (300 µM; left) or BzATP (300 µM; right), respectively. Traces from untransfected spermatogonia (black) and cells transfected with either nontarget negative control siRNA (gray) or a construct targeting P2X4 (green) are overlaid. (B) Quantification of results from repetitive stimulation experiments as shown in A. Peak current densities (means ± SEM) are color coded and plotted as a function of stimulus repetition. Cells were analyzed under control conditions ( n = 33/13 [ATP/BzATP]; black), after transfection with nontargeting siRNA ( n = 16/23 [ATP/BzATP]; gray), and after P2X4 knockdown ( n = 16/23 [ATP/BzATP]; green). Inset (left) shows the data delimited by the dashed rectangle at an increased scale. Asterisks (*) indicate statistical significance, P
    Figure Legend Snippet: P2X7 constitutes the low-affinity spermatogonial ATP sensor. (A) Exemplary whole-cell voltage-clamp recordings of currents triggered by repetitive stimulation (1 s; ISI = 3 s; S 1 , S 6 ) with ATP (300 µM; left) or BzATP (300 µM; right), respectively. Traces from untransfected spermatogonia (black) and cells transfected with either nontarget negative control siRNA (gray) or a construct targeting P2X4 (green) are overlaid. (B) Quantification of results from repetitive stimulation experiments as shown in A. Peak current densities (means ± SEM) are color coded and plotted as a function of stimulus repetition. Cells were analyzed under control conditions ( n = 33/13 [ATP/BzATP]; black), after transfection with nontargeting siRNA ( n = 16/23 [ATP/BzATP]; gray), and after P2X4 knockdown ( n = 16/23 [ATP/BzATP]; green). Inset (left) shows the data delimited by the dashed rectangle at an increased scale. Asterisks (*) indicate statistical significance, P

    Techniques Used: Transfection, Negative Control, Construct

    Posttranscriptional gene silencing confirms P2X4 as the high-affinity spermatogonial ATP sensor. (A) Bar chart quantifying siRNA-dependent selective knockdown of spermatogonial gene expression in vitro. When transfected with one of two siRNA constructs (green), knockdown of P2X4 expression was confirmed by quantitative real-time PCR. Transcript quantities (means ± SEM) are normalized to mRNA levels in untransfected spermatogonia and compared with cells treated with nontargeting siRNA controls (gray). Administration of both targeting siRNAs effectively reduced P2X4 transcription as compared with both untransfected spermatogonia and negative (nontargeting siRNA) controls. Asterisks (*) denote statistical significance, P
    Figure Legend Snippet: Posttranscriptional gene silencing confirms P2X4 as the high-affinity spermatogonial ATP sensor. (A) Bar chart quantifying siRNA-dependent selective knockdown of spermatogonial gene expression in vitro. When transfected with one of two siRNA constructs (green), knockdown of P2X4 expression was confirmed by quantitative real-time PCR. Transcript quantities (means ± SEM) are normalized to mRNA levels in untransfected spermatogonia and compared with cells treated with nontargeting siRNA controls (gray). Administration of both targeting siRNAs effectively reduced P2X4 transcription as compared with both untransfected spermatogonia and negative (nontargeting siRNA) controls. Asterisks (*) denote statistical significance, P

    Techniques Used: Expressing, In Vitro, Transfection, Construct, Real-time Polymerase Chain Reaction

    P2X4 is a candidate mediator of the high-affinity ATP response in spermatogonia. (A) Reverse transcription PCR reveals transcripts for P2rx2 , P2rx4 , and P2rx7 in both Sertoli–germ cell co-cultures and whole testis preparations at P7. A faint band for P2rx3 -encoding cDNA is only amplified from whole testis samples but not co-cultures. Primer pairs generate amplificates of 150–250 bp, respectively. cDNA from whole brain/spinal cord serves as positive control, whereas no template controls are devoid of detectable signals. (B–E) Exemplary whole-cell voltage-clamp recordings of ATP-induced currents (10 µM; 1 s; V hold = −80 mV; S 1 , S 6 ) under control conditions and during treatment with suramin (10 µM and 100 µM; B), extracellular Cu 2+ (100 µM; C), reduced pH (6.3; D), and ivermectin (3 µM; E). Altered conditions were established for at least 60 s (preincubation). (F) Bar diagram (means ± SEM) depicting the quantitative analysis of experiments exemplified in B–E. Note the discontinuous ordinate (//). Numbers of cells tested are indicated above bars. Asterisks (*) denote statistical significance, P ≤ 0.01 (paired t test).
    Figure Legend Snippet: P2X4 is a candidate mediator of the high-affinity ATP response in spermatogonia. (A) Reverse transcription PCR reveals transcripts for P2rx2 , P2rx4 , and P2rx7 in both Sertoli–germ cell co-cultures and whole testis preparations at P7. A faint band for P2rx3 -encoding cDNA is only amplified from whole testis samples but not co-cultures. Primer pairs generate amplificates of 150–250 bp, respectively. cDNA from whole brain/spinal cord serves as positive control, whereas no template controls are devoid of detectable signals. (B–E) Exemplary whole-cell voltage-clamp recordings of ATP-induced currents (10 µM; 1 s; V hold = −80 mV; S 1 , S 6 ) under control conditions and during treatment with suramin (10 µM and 100 µM; B), extracellular Cu 2+ (100 µM; C), reduced pH (6.3; D), and ivermectin (3 µM; E). Altered conditions were established for at least 60 s (preincubation). (F) Bar diagram (means ± SEM) depicting the quantitative analysis of experiments exemplified in B–E. Note the discontinuous ordinate (//). Numbers of cells tested are indicated above bars. Asterisks (*) denote statistical significance, P ≤ 0.01 (paired t test).

    Techniques Used: Polymerase Chain Reaction, Amplification, Positive Control

    5) Product Images from "The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy"

    Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2019.01103

    The negative chronotropic effect of ATP is mediated by activation of P2X4. The negative chronotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X7 receptor antagonist, A438079 (3 µM, A ), the P2X4 receptor antagonist, 5-BDBD (10 µM, B ) and the positive allosteric modulator of the P2X4 receptor, ivermectin (30 µM, C ). The negative chronotropic effect of CTP (1 mM, D ) either in the absence or in the presence of 5-BDBD (10 µM), is also shown for comparison. Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from baseline; horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p
    Figure Legend Snippet: The negative chronotropic effect of ATP is mediated by activation of P2X4. The negative chronotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X7 receptor antagonist, A438079 (3 µM, A ), the P2X4 receptor antagonist, 5-BDBD (10 µM, B ) and the positive allosteric modulator of the P2X4 receptor, ivermectin (30 µM, C ). The negative chronotropic effect of CTP (1 mM, D ) either in the absence or in the presence of 5-BDBD (10 µM), is also shown for comparison. Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from baseline; horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p

    Techniques Used: Activation Assay

    Selective blockage of P2X4 and of NCX transporter partially offsets the negative inotropic effect of ATP in paced rat ventricular strips. The negative inotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X4 receptor antagonist, 5-BDBD (10 µM, A and B ) and of the NCX inhibitor, KB-R7943 (3 µM, C and D ). Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from the baseline isometric tension of RV strips, measured as the active tension (mN/mg of wet tissue weight, panels A and C ) and the derivative of developed force over time (+dF/dt, mN/s, panels B and D ); horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p
    Figure Legend Snippet: Selective blockage of P2X4 and of NCX transporter partially offsets the negative inotropic effect of ATP in paced rat ventricular strips. The negative inotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X4 receptor antagonist, 5-BDBD (10 µM, A and B ) and of the NCX inhibitor, KB-R7943 (3 µM, C and D ). Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from the baseline isometric tension of RV strips, measured as the active tension (mN/mg of wet tissue weight, panels A and C ) and the derivative of developed force over time (+dF/dt, mN/s, panels B and D ); horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p

    Techniques Used:

    Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-002, C-terminus, Alomone) and NCX1 (Anx-011, Alomone) protein in the sinoatrial node (SAN), right atria (RA) and right ventricle (RV). The SAN was identified based on its low Cx43 (green) and high HCN4 (magenta) protein expression (left hand-side images). Images were taken from whole-mount heart preparations including the three analyzed regions, SAN, RA and RV. Dashed lines represent boundaries of the SAN. The pulmonary parenchyma was used as a structural support to facilitate immunostaining of myocardial sections and it is visible in the bottom right quadrant of each SAN image. White arrows indicate blood vessels including the SAN artery. Scale bar 30 µm. Images are representative of three different individuals.
    Figure Legend Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-002, C-terminus, Alomone) and NCX1 (Anx-011, Alomone) protein in the sinoatrial node (SAN), right atria (RA) and right ventricle (RV). The SAN was identified based on its low Cx43 (green) and high HCN4 (magenta) protein expression (left hand-side images). Images were taken from whole-mount heart preparations including the three analyzed regions, SAN, RA and RV. Dashed lines represent boundaries of the SAN. The pulmonary parenchyma was used as a structural support to facilitate immunostaining of myocardial sections and it is visible in the bottom right quadrant of each SAN image. White arrows indicate blood vessels including the SAN artery. Scale bar 30 µm. Images are representative of three different individuals.

    Techniques Used: Expressing, Immunostaining

    The mechanism underlying the dual P2X4 receptor-mediated effects on cardiac chronotropy and inotropy implicates downstream modulation of NCX activity (digitalis-like phenomenon). Besides ion fluxes carried by pacemaker HCN channels (not represented), the unstable resting membrane potential and the spontaneous firing of SAN cardiomyocytes are attributed mainly to the electrogenic NCX transport operating in the forward Ca 2+ -extrusion mode. Na + influx through the P2X4 receptor pore dissipates the electrochemical gradient of this ion across the plasma membrane leading to inhibition and/or reversion of the NCX pacemaker current. This may justify slowing down of SAN cells depolarizations and the negative chronotropic effect of ATP. Likewise, intracellular Ca 2+ accumulation due both (1) to Ca 2+ influx through the P2X4 receptor pore, and (2) to reversal of NCX activity may explain the positive inotropic effect of the P2X4 receptor in paced ventricular cardiomyocytes. Figure composition used elements from Servier Medical Art .
    Figure Legend Snippet: The mechanism underlying the dual P2X4 receptor-mediated effects on cardiac chronotropy and inotropy implicates downstream modulation of NCX activity (digitalis-like phenomenon). Besides ion fluxes carried by pacemaker HCN channels (not represented), the unstable resting membrane potential and the spontaneous firing of SAN cardiomyocytes are attributed mainly to the electrogenic NCX transport operating in the forward Ca 2+ -extrusion mode. Na + influx through the P2X4 receptor pore dissipates the electrochemical gradient of this ion across the plasma membrane leading to inhibition and/or reversion of the NCX pacemaker current. This may justify slowing down of SAN cells depolarizations and the negative chronotropic effect of ATP. Likewise, intracellular Ca 2+ accumulation due both (1) to Ca 2+ influx through the P2X4 receptor pore, and (2) to reversal of NCX activity may explain the positive inotropic effect of the P2X4 receptor in paced ventricular cardiomyocytes. Figure composition used elements from Servier Medical Art .

    Techniques Used: Activity Assay, Inhibition

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    Alomone Labs p2x4 receptor
    The negative chronotropic effect of ATP is mediated by activation of <t>P2X4.</t> The negative chronotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X7 receptor antagonist, A438079 (3 µM, A ), the P2X4 receptor antagonist, 5-BDBD (10 µM, B ) and the positive allosteric modulator of the P2X4 receptor, ivermectin (30 µM, C ). The negative chronotropic effect of CTP (1 mM, D ) either in the absence or in the presence of 5-BDBD (10 µM), is also shown for comparison. Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from baseline; horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p
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    The negative chronotropic effect of ATP is mediated by activation of P2X4. The negative chronotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X7 receptor antagonist, A438079 (3 µM, A ), the P2X4 receptor antagonist, 5-BDBD (10 µM, B ) and the positive allosteric modulator of the P2X4 receptor, ivermectin (30 µM, C ). The negative chronotropic effect of CTP (1 mM, D ) either in the absence or in the presence of 5-BDBD (10 µM), is also shown for comparison. Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from baseline; horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p

    Journal: Frontiers in Pharmacology

    Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

    doi: 10.3389/fphar.2019.01103

    Figure Lengend Snippet: The negative chronotropic effect of ATP is mediated by activation of P2X4. The negative chronotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X7 receptor antagonist, A438079 (3 µM, A ), the P2X4 receptor antagonist, 5-BDBD (10 µM, B ) and the positive allosteric modulator of the P2X4 receptor, ivermectin (30 µM, C ). The negative chronotropic effect of CTP (1 mM, D ) either in the absence or in the presence of 5-BDBD (10 µM), is also shown for comparison. Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from baseline; horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p

    Article Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-024, extracellular loop, Alomone) and of the HCN4 channel (Agp-004, Alomone) in the rat sinoatrial node (SAN); images obtained in right atria (RA) and right ventricle (RV) are also shown for comparison.

    Techniques: Activation Assay

    Selective blockage of P2X4 and of NCX transporter partially offsets the negative inotropic effect of ATP in paced rat ventricular strips. The negative inotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X4 receptor antagonist, 5-BDBD (10 µM, A and B ) and of the NCX inhibitor, KB-R7943 (3 µM, C and D ). Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from the baseline isometric tension of RV strips, measured as the active tension (mN/mg of wet tissue weight, panels A and C ) and the derivative of developed force over time (+dF/dt, mN/s, panels B and D ); horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p

    Journal: Frontiers in Pharmacology

    Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

    doi: 10.3389/fphar.2019.01103

    Figure Lengend Snippet: Selective blockage of P2X4 and of NCX transporter partially offsets the negative inotropic effect of ATP in paced rat ventricular strips. The negative inotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X4 receptor antagonist, 5-BDBD (10 µM, A and B ) and of the NCX inhibitor, KB-R7943 (3 µM, C and D ). Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from the baseline isometric tension of RV strips, measured as the active tension (mN/mg of wet tissue weight, panels A and C ) and the derivative of developed force over time (+dF/dt, mN/s, panels B and D ); horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p

    Article Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-024, extracellular loop, Alomone) and of the HCN4 channel (Agp-004, Alomone) in the rat sinoatrial node (SAN); images obtained in right atria (RA) and right ventricle (RV) are also shown for comparison.

    Techniques:

    Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-002, C-terminus, Alomone) and NCX1 (Anx-011, Alomone) protein in the sinoatrial node (SAN), right atria (RA) and right ventricle (RV). The SAN was identified based on its low Cx43 (green) and high HCN4 (magenta) protein expression (left hand-side images). Images were taken from whole-mount heart preparations including the three analyzed regions, SAN, RA and RV. Dashed lines represent boundaries of the SAN. The pulmonary parenchyma was used as a structural support to facilitate immunostaining of myocardial sections and it is visible in the bottom right quadrant of each SAN image. White arrows indicate blood vessels including the SAN artery. Scale bar 30 µm. Images are representative of three different individuals.

    Journal: Frontiers in Pharmacology

    Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

    doi: 10.3389/fphar.2019.01103

    Figure Lengend Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-002, C-terminus, Alomone) and NCX1 (Anx-011, Alomone) protein in the sinoatrial node (SAN), right atria (RA) and right ventricle (RV). The SAN was identified based on its low Cx43 (green) and high HCN4 (magenta) protein expression (left hand-side images). Images were taken from whole-mount heart preparations including the three analyzed regions, SAN, RA and RV. Dashed lines represent boundaries of the SAN. The pulmonary parenchyma was used as a structural support to facilitate immunostaining of myocardial sections and it is visible in the bottom right quadrant of each SAN image. White arrows indicate blood vessels including the SAN artery. Scale bar 30 µm. Images are representative of three different individuals.

    Article Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-024, extracellular loop, Alomone) and of the HCN4 channel (Agp-004, Alomone) in the rat sinoatrial node (SAN); images obtained in right atria (RA) and right ventricle (RV) are also shown for comparison.

    Techniques: Expressing, Immunostaining

    The mechanism underlying the dual P2X4 receptor-mediated effects on cardiac chronotropy and inotropy implicates downstream modulation of NCX activity (digitalis-like phenomenon). Besides ion fluxes carried by pacemaker HCN channels (not represented), the unstable resting membrane potential and the spontaneous firing of SAN cardiomyocytes are attributed mainly to the electrogenic NCX transport operating in the forward Ca 2+ -extrusion mode. Na + influx through the P2X4 receptor pore dissipates the electrochemical gradient of this ion across the plasma membrane leading to inhibition and/or reversion of the NCX pacemaker current. This may justify slowing down of SAN cells depolarizations and the negative chronotropic effect of ATP. Likewise, intracellular Ca 2+ accumulation due both (1) to Ca 2+ influx through the P2X4 receptor pore, and (2) to reversal of NCX activity may explain the positive inotropic effect of the P2X4 receptor in paced ventricular cardiomyocytes. Figure composition used elements from Servier Medical Art .

    Journal: Frontiers in Pharmacology

    Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

    doi: 10.3389/fphar.2019.01103

    Figure Lengend Snippet: The mechanism underlying the dual P2X4 receptor-mediated effects on cardiac chronotropy and inotropy implicates downstream modulation of NCX activity (digitalis-like phenomenon). Besides ion fluxes carried by pacemaker HCN channels (not represented), the unstable resting membrane potential and the spontaneous firing of SAN cardiomyocytes are attributed mainly to the electrogenic NCX transport operating in the forward Ca 2+ -extrusion mode. Na + influx through the P2X4 receptor pore dissipates the electrochemical gradient of this ion across the plasma membrane leading to inhibition and/or reversion of the NCX pacemaker current. This may justify slowing down of SAN cells depolarizations and the negative chronotropic effect of ATP. Likewise, intracellular Ca 2+ accumulation due both (1) to Ca 2+ influx through the P2X4 receptor pore, and (2) to reversal of NCX activity may explain the positive inotropic effect of the P2X4 receptor in paced ventricular cardiomyocytes. Figure composition used elements from Servier Medical Art .

    Article Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-024, extracellular loop, Alomone) and of the HCN4 channel (Agp-004, Alomone) in the rat sinoatrial node (SAN); images obtained in right atria (RA) and right ventricle (RV) are also shown for comparison.

    Techniques: Activity Assay, Inhibition

    Evidence of P2X4R scFv 95 in brain and trigeminal ganglia, while P2X4R scFv 95 treatment prevents the P2X4R protein elevation in trigeminal ganglia of untreated FRICT-ION mice. ( A ). His-tag biomarker in the brain indicates brain penetrance of the P2X4R scFv 95 in amygdala, medulla, and trigeminal ganglia. ( B ). Bar graph of the scFv His-tag in the amygdala, brainstem medulla dorsal horn, and trigeminal ganglia (TG) in the Western blots. The His-tag remains at 7 weeks after the single i.p. dose was given in male mice with FRICT-ION. Almost no His-tag was detected in the medial prefrontal cortex (mPFC) in these comparisons with normalized intensity in arbitrary units. ( C ). The P2X4 protein increased in trigeminal ganglia of FRICT-ION mice with neuropathic pain (week 10) was not evident in mice treated with P2X4R scFv 95. n = 3, One-way ANOVA, * p

    Journal: International Journal of Molecular Sciences

    Article Title: Single-Dose P2 X4R Single-Chain Fragment Variable Antibody Permanently Reverses Chronic Pain in Male Mice

    doi: 10.3390/ijms222413612

    Figure Lengend Snippet: Evidence of P2X4R scFv 95 in brain and trigeminal ganglia, while P2X4R scFv 95 treatment prevents the P2X4R protein elevation in trigeminal ganglia of untreated FRICT-ION mice. ( A ). His-tag biomarker in the brain indicates brain penetrance of the P2X4R scFv 95 in amygdala, medulla, and trigeminal ganglia. ( B ). Bar graph of the scFv His-tag in the amygdala, brainstem medulla dorsal horn, and trigeminal ganglia (TG) in the Western blots. The His-tag remains at 7 weeks after the single i.p. dose was given in male mice with FRICT-ION. Almost no His-tag was detected in the medial prefrontal cortex (mPFC) in these comparisons with normalized intensity in arbitrary units. ( C ). The P2X4 protein increased in trigeminal ganglia of FRICT-ION mice with neuropathic pain (week 10) was not evident in mice treated with P2X4R scFv 95. n = 3, One-way ANOVA, * p

    Article Snippet: Cells were fixed in 2% paraformaldehyde and blocked in 2% FBS/BSA blocking buffer (1 h) prior to overnight incubation in primary antibody (rabbit anti-P2X4R, 1:1000, APR-024, lot#APR024AN0225, Alomone Labs, Jerusalem, Israel).

    Techniques: Mouse Assay, Biomarker Assay, Western Blot

    Effect of in vitro P2X4R scFv95 treatment on electrophysiological properties of TG neuron primary cultures from FRICT-ION mice. ( A ). Distribution of single vs. multiple action potential (AP) firing from untreated FRICT-ION and P2X4 scFv95 pre-treated FRICT-ION TG neurons. Prevalence of multiple firing was higher in untreated TG neurons from FRICT-ION mice than those with P2X4R scFv95 pre-treatment. ( B ). Prevalence of rebound firing in untreated FRICT-ION and P2X4R scFv95 pre-treated FRICT-ION TG neuronal cultures. Neurons with more rebound firing were observed more often in untreated cultures than in P2X4R-scFv-treated TG neurons from FRICT-ION mice. Effect of P2X4R-scFv treatment on electrophysiological properties of ( C ). large-diameter TG neurons ( > 25 microns) and ( D ). small- to medium-diameter TG-neurons (

    Journal: International Journal of Molecular Sciences

    Article Title: Single-Dose P2 X4R Single-Chain Fragment Variable Antibody Permanently Reverses Chronic Pain in Male Mice

    doi: 10.3390/ijms222413612

    Figure Lengend Snippet: Effect of in vitro P2X4R scFv95 treatment on electrophysiological properties of TG neuron primary cultures from FRICT-ION mice. ( A ). Distribution of single vs. multiple action potential (AP) firing from untreated FRICT-ION and P2X4 scFv95 pre-treated FRICT-ION TG neurons. Prevalence of multiple firing was higher in untreated TG neurons from FRICT-ION mice than those with P2X4R scFv95 pre-treatment. ( B ). Prevalence of rebound firing in untreated FRICT-ION and P2X4R scFv95 pre-treated FRICT-ION TG neuronal cultures. Neurons with more rebound firing were observed more often in untreated cultures than in P2X4R-scFv-treated TG neurons from FRICT-ION mice. Effect of P2X4R-scFv treatment on electrophysiological properties of ( C ). large-diameter TG neurons ( > 25 microns) and ( D ). small- to medium-diameter TG-neurons (

    Article Snippet: Cells were fixed in 2% paraformaldehyde and blocked in 2% FBS/BSA blocking buffer (1 h) prior to overnight incubation in primary antibody (rabbit anti-P2X4R, 1:1000, APR-024, lot#APR024AN0225, Alomone Labs, Jerusalem, Israel).

    Techniques: In Vitro, Mouse Assay

    P2X7 constitutes the low-affinity spermatogonial ATP sensor. (A) Exemplary whole-cell voltage-clamp recordings of currents triggered by repetitive stimulation (1 s; ISI = 3 s; S 1 , S 6 ) with ATP (300 µM; left) or BzATP (300 µM; right), respectively. Traces from untransfected spermatogonia (black) and cells transfected with either nontarget negative control siRNA (gray) or a construct targeting P2X4 (green) are overlaid. (B) Quantification of results from repetitive stimulation experiments as shown in A. Peak current densities (means ± SEM) are color coded and plotted as a function of stimulus repetition. Cells were analyzed under control conditions ( n = 33/13 [ATP/BzATP]; black), after transfection with nontargeting siRNA ( n = 16/23 [ATP/BzATP]; gray), and after P2X4 knockdown ( n = 16/23 [ATP/BzATP]; green). Inset (left) shows the data delimited by the dashed rectangle at an increased scale. Asterisks (*) indicate statistical significance, P

    Journal: The Journal of General Physiology

    Article Title: Distinct purinergic signaling pathways in prepubescent mouse spermatogonia

    doi: 10.1085/jgp.201611636

    Figure Lengend Snippet: P2X7 constitutes the low-affinity spermatogonial ATP sensor. (A) Exemplary whole-cell voltage-clamp recordings of currents triggered by repetitive stimulation (1 s; ISI = 3 s; S 1 , S 6 ) with ATP (300 µM; left) or BzATP (300 µM; right), respectively. Traces from untransfected spermatogonia (black) and cells transfected with either nontarget negative control siRNA (gray) or a construct targeting P2X4 (green) are overlaid. (B) Quantification of results from repetitive stimulation experiments as shown in A. Peak current densities (means ± SEM) are color coded and plotted as a function of stimulus repetition. Cells were analyzed under control conditions ( n = 33/13 [ATP/BzATP]; black), after transfection with nontargeting siRNA ( n = 16/23 [ATP/BzATP]; gray), and after P2X4 knockdown ( n = 16/23 [ATP/BzATP]; green). Inset (left) shows the data delimited by the dashed rectangle at an increased scale. Asterisks (*) indicate statistical significance, P

    Article Snippet: Primary antibodies were anti-DAZL (1:500; Abcam), anti-Sloα1 (extracellular; 1:500; Alomone Labs), anti-P2X4 (extracellular; 1:100, Alomone Labs), and anti-P2X7 (extracellular; 1:500; Alomone Labs).

    Techniques: Transfection, Negative Control, Construct

    Posttranscriptional gene silencing confirms P2X4 as the high-affinity spermatogonial ATP sensor. (A) Bar chart quantifying siRNA-dependent selective knockdown of spermatogonial gene expression in vitro. When transfected with one of two siRNA constructs (green), knockdown of P2X4 expression was confirmed by quantitative real-time PCR. Transcript quantities (means ± SEM) are normalized to mRNA levels in untransfected spermatogonia and compared with cells treated with nontargeting siRNA controls (gray). Administration of both targeting siRNAs effectively reduced P2X4 transcription as compared with both untransfected spermatogonia and negative (nontargeting siRNA) controls. Asterisks (*) denote statistical significance, P

    Journal: The Journal of General Physiology

    Article Title: Distinct purinergic signaling pathways in prepubescent mouse spermatogonia

    doi: 10.1085/jgp.201611636

    Figure Lengend Snippet: Posttranscriptional gene silencing confirms P2X4 as the high-affinity spermatogonial ATP sensor. (A) Bar chart quantifying siRNA-dependent selective knockdown of spermatogonial gene expression in vitro. When transfected with one of two siRNA constructs (green), knockdown of P2X4 expression was confirmed by quantitative real-time PCR. Transcript quantities (means ± SEM) are normalized to mRNA levels in untransfected spermatogonia and compared with cells treated with nontargeting siRNA controls (gray). Administration of both targeting siRNAs effectively reduced P2X4 transcription as compared with both untransfected spermatogonia and negative (nontargeting siRNA) controls. Asterisks (*) denote statistical significance, P

    Article Snippet: Primary antibodies were anti-DAZL (1:500; Abcam), anti-Sloα1 (extracellular; 1:500; Alomone Labs), anti-P2X4 (extracellular; 1:100, Alomone Labs), and anti-P2X7 (extracellular; 1:500; Alomone Labs).

    Techniques: Expressing, In Vitro, Transfection, Construct, Real-time Polymerase Chain Reaction

    P2X4 is a candidate mediator of the high-affinity ATP response in spermatogonia. (A) Reverse transcription PCR reveals transcripts for P2rx2 , P2rx4 , and P2rx7 in both Sertoli–germ cell co-cultures and whole testis preparations at P7. A faint band for P2rx3 -encoding cDNA is only amplified from whole testis samples but not co-cultures. Primer pairs generate amplificates of 150–250 bp, respectively. cDNA from whole brain/spinal cord serves as positive control, whereas no template controls are devoid of detectable signals. (B–E) Exemplary whole-cell voltage-clamp recordings of ATP-induced currents (10 µM; 1 s; V hold = −80 mV; S 1 , S 6 ) under control conditions and during treatment with suramin (10 µM and 100 µM; B), extracellular Cu 2+ (100 µM; C), reduced pH (6.3; D), and ivermectin (3 µM; E). Altered conditions were established for at least 60 s (preincubation). (F) Bar diagram (means ± SEM) depicting the quantitative analysis of experiments exemplified in B–E. Note the discontinuous ordinate (//). Numbers of cells tested are indicated above bars. Asterisks (*) denote statistical significance, P ≤ 0.01 (paired t test).

    Journal: The Journal of General Physiology

    Article Title: Distinct purinergic signaling pathways in prepubescent mouse spermatogonia

    doi: 10.1085/jgp.201611636

    Figure Lengend Snippet: P2X4 is a candidate mediator of the high-affinity ATP response in spermatogonia. (A) Reverse transcription PCR reveals transcripts for P2rx2 , P2rx4 , and P2rx7 in both Sertoli–germ cell co-cultures and whole testis preparations at P7. A faint band for P2rx3 -encoding cDNA is only amplified from whole testis samples but not co-cultures. Primer pairs generate amplificates of 150–250 bp, respectively. cDNA from whole brain/spinal cord serves as positive control, whereas no template controls are devoid of detectable signals. (B–E) Exemplary whole-cell voltage-clamp recordings of ATP-induced currents (10 µM; 1 s; V hold = −80 mV; S 1 , S 6 ) under control conditions and during treatment with suramin (10 µM and 100 µM; B), extracellular Cu 2+ (100 µM; C), reduced pH (6.3; D), and ivermectin (3 µM; E). Altered conditions were established for at least 60 s (preincubation). (F) Bar diagram (means ± SEM) depicting the quantitative analysis of experiments exemplified in B–E. Note the discontinuous ordinate (//). Numbers of cells tested are indicated above bars. Asterisks (*) denote statistical significance, P ≤ 0.01 (paired t test).

    Article Snippet: Primary antibodies were anti-DAZL (1:500; Abcam), anti-Sloα1 (extracellular; 1:500; Alomone Labs), anti-P2X4 (extracellular; 1:100, Alomone Labs), and anti-P2X7 (extracellular; 1:500; Alomone Labs).

    Techniques: Polymerase Chain Reaction, Amplification, Positive Control