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Intracellular spermine prevents acid-induced uncoupling of Cx43 gap junction channels.

Skatchkov SN, Bukauskas FF, Benedikt J, Inyushin M, Kucheryavykh YV - Neuroreport (2015)

Bottom Line: Our results showed (i) a higher increase in gap junctional communication at higher concentrations of cytoplasmic spermine, and (ii) that spermine prevented uncoupling of gap junctions at low intracellular pH.Taken together, we conclude that spermine enhances Cx43-mediated gap junctional communication and may preserve neuronal excitability during ischemia and trauma when pH in the brain acidifies.We, therefore, suggest a new role of spermine in the regulation of a Cx43-based network under (patho)physiological conditions.

View Article: PubMed Central - PubMed

Affiliation: Departments of aPhysiology bBiochemistry, Universidad Central del Caribe, School of Medicine, Bayamón, Puerto Rico cDominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, New York, New York, USA.

ABSTRACT
Polyamines (PAs), such as spermine and spermidine, modulate the activity of numerous receptors and channels in the central nervous system (CNS) and are stored in glial cells; however, little attention has been paid to their role in the regulation of connexin (Cx)-based gap junction channels. We have previously shown that PAs facilitate diffusion of Lucifer Yellow through astrocytic gap junctions in acute brain slices; therefore, we hypothesized that spermine can regulate Cx43-mediated (as the most abundant Cx in astrocytes) gap junctional communication. We used electrophysiological patch-clamp recording from paired Novikoff cells endogenously expressing Cx43 and HeLaCx43-EGFP transfectants to study pH-dependent modulation of cell-cell coupling in the presence or absence of PAs. Our results showed (i) a higher increase in gap junctional communication at higher concentrations of cytoplasmic spermine, and (ii) that spermine prevented uncoupling of gap junctions at low intracellular pH. Taken together, we conclude that spermine enhances Cx43-mediated gap junctional communication and may preserve neuronal excitability during ischemia and trauma when pH in the brain acidifies. We, therefore, suggest a new role of spermine in the regulation of a Cx43-based network under (patho)physiological conditions.

No MeSH data available.


Related in: MedlinePlus

Effect of spermine (SPM) on Cx43 gap junction channels. (a) Examples of junctional current (Ij) records from HeLaCx43-EGFP cell pairs in response to a repeated step-ramp-step protocol shown in (b). The presence of SPM in the pipette solution caused an increase, whereas the absence of SPM produced a decrease in Ij over time. (b) Voltage (Vj) protocol used to study Ij changes over time. (c) Summarized data after 10 min of recording in the presence or absence of 0.5 mM SPM in the pipette solution. Gap junctional conductance was calculated and normalized. Error bars represent SEM. *Significant difference (P<0.05, n=9 in each group). (d) Averaged junctional conductance (gj) measurements in Novikoff cell pairs using different intracellular concentrations of SPM: no SPM, 1 mM SPM, 5 mM SPM, and 10 mM SPM. (e) Summarized data of experiments shown in (d) at 10 min of the recordings. Error bars represent SEM. *Significant difference between groups (P<0.05, n=5 in each group).
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Figure 1: Effect of spermine (SPM) on Cx43 gap junction channels. (a) Examples of junctional current (Ij) records from HeLaCx43-EGFP cell pairs in response to a repeated step-ramp-step protocol shown in (b). The presence of SPM in the pipette solution caused an increase, whereas the absence of SPM produced a decrease in Ij over time. (b) Voltage (Vj) protocol used to study Ij changes over time. (c) Summarized data after 10 min of recording in the presence or absence of 0.5 mM SPM in the pipette solution. Gap junctional conductance was calculated and normalized. Error bars represent SEM. *Significant difference (P<0.05, n=9 in each group). (d) Averaged junctional conductance (gj) measurements in Novikoff cell pairs using different intracellular concentrations of SPM: no SPM, 1 mM SPM, 5 mM SPM, and 10 mM SPM. (e) Summarized data of experiments shown in (d) at 10 min of the recordings. Error bars represent SEM. *Significant difference between groups (P<0.05, n=5 in each group).

Mentions: Typically it takes about 2 min to dialyze and fully replenish glial cells with PAs through a patch pipette 16. We found enhanced gap junctional communication in Novikoff (Fig. 1d and e) and HeLaCx43-EGFP cells (Fig. 1a–c; by 214±25%, P<0.05, n=9) with the presence of spermine in both pipettes. In contrast, if patch pipettes did not contain spermine, we observed a run-down of Ij (Fig. 1a and d). The latter suggests that washout of endogenous PAs from cells (any proliferating cells contain endogenous PAs) triggers moderate uncoupling of the cells. While keeping the internal pHi stable at 7.2, we observed that the gap junctional communication increased with a rise in spermine concentration in the pipette solution from 1 to 5 and 10 mM by 164±20, 229±34, and 384±55%, respectively (P<0.05, n=5 in each group; Fig. 1d and e).


Intracellular spermine prevents acid-induced uncoupling of Cx43 gap junction channels.

Skatchkov SN, Bukauskas FF, Benedikt J, Inyushin M, Kucheryavykh YV - Neuroreport (2015)

Effect of spermine (SPM) on Cx43 gap junction channels. (a) Examples of junctional current (Ij) records from HeLaCx43-EGFP cell pairs in response to a repeated step-ramp-step protocol shown in (b). The presence of SPM in the pipette solution caused an increase, whereas the absence of SPM produced a decrease in Ij over time. (b) Voltage (Vj) protocol used to study Ij changes over time. (c) Summarized data after 10 min of recording in the presence or absence of 0.5 mM SPM in the pipette solution. Gap junctional conductance was calculated and normalized. Error bars represent SEM. *Significant difference (P<0.05, n=9 in each group). (d) Averaged junctional conductance (gj) measurements in Novikoff cell pairs using different intracellular concentrations of SPM: no SPM, 1 mM SPM, 5 mM SPM, and 10 mM SPM. (e) Summarized data of experiments shown in (d) at 10 min of the recordings. Error bars represent SEM. *Significant difference between groups (P<0.05, n=5 in each group).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493885&req=5

Figure 1: Effect of spermine (SPM) on Cx43 gap junction channels. (a) Examples of junctional current (Ij) records from HeLaCx43-EGFP cell pairs in response to a repeated step-ramp-step protocol shown in (b). The presence of SPM in the pipette solution caused an increase, whereas the absence of SPM produced a decrease in Ij over time. (b) Voltage (Vj) protocol used to study Ij changes over time. (c) Summarized data after 10 min of recording in the presence or absence of 0.5 mM SPM in the pipette solution. Gap junctional conductance was calculated and normalized. Error bars represent SEM. *Significant difference (P<0.05, n=9 in each group). (d) Averaged junctional conductance (gj) measurements in Novikoff cell pairs using different intracellular concentrations of SPM: no SPM, 1 mM SPM, 5 mM SPM, and 10 mM SPM. (e) Summarized data of experiments shown in (d) at 10 min of the recordings. Error bars represent SEM. *Significant difference between groups (P<0.05, n=5 in each group).
Mentions: Typically it takes about 2 min to dialyze and fully replenish glial cells with PAs through a patch pipette 16. We found enhanced gap junctional communication in Novikoff (Fig. 1d and e) and HeLaCx43-EGFP cells (Fig. 1a–c; by 214±25%, P<0.05, n=9) with the presence of spermine in both pipettes. In contrast, if patch pipettes did not contain spermine, we observed a run-down of Ij (Fig. 1a and d). The latter suggests that washout of endogenous PAs from cells (any proliferating cells contain endogenous PAs) triggers moderate uncoupling of the cells. While keeping the internal pHi stable at 7.2, we observed that the gap junctional communication increased with a rise in spermine concentration in the pipette solution from 1 to 5 and 10 mM by 164±20, 229±34, and 384±55%, respectively (P<0.05, n=5 in each group; Fig. 1d and e).

Bottom Line: Our results showed (i) a higher increase in gap junctional communication at higher concentrations of cytoplasmic spermine, and (ii) that spermine prevented uncoupling of gap junctions at low intracellular pH.Taken together, we conclude that spermine enhances Cx43-mediated gap junctional communication and may preserve neuronal excitability during ischemia and trauma when pH in the brain acidifies.We, therefore, suggest a new role of spermine in the regulation of a Cx43-based network under (patho)physiological conditions.

View Article: PubMed Central - PubMed

Affiliation: Departments of aPhysiology bBiochemistry, Universidad Central del Caribe, School of Medicine, Bayamón, Puerto Rico cDominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, New York, New York, USA.

ABSTRACT
Polyamines (PAs), such as spermine and spermidine, modulate the activity of numerous receptors and channels in the central nervous system (CNS) and are stored in glial cells; however, little attention has been paid to their role in the regulation of connexin (Cx)-based gap junction channels. We have previously shown that PAs facilitate diffusion of Lucifer Yellow through astrocytic gap junctions in acute brain slices; therefore, we hypothesized that spermine can regulate Cx43-mediated (as the most abundant Cx in astrocytes) gap junctional communication. We used electrophysiological patch-clamp recording from paired Novikoff cells endogenously expressing Cx43 and HeLaCx43-EGFP transfectants to study pH-dependent modulation of cell-cell coupling in the presence or absence of PAs. Our results showed (i) a higher increase in gap junctional communication at higher concentrations of cytoplasmic spermine, and (ii) that spermine prevented uncoupling of gap junctions at low intracellular pH. Taken together, we conclude that spermine enhances Cx43-mediated gap junctional communication and may preserve neuronal excitability during ischemia and trauma when pH in the brain acidifies. We, therefore, suggest a new role of spermine in the regulation of a Cx43-based network under (patho)physiological conditions.

No MeSH data available.


Related in: MedlinePlus