Alpha-1-adrenoceptor subtype selective regulation of connexin 43 expression in rat cardiomyocytes

Diana Marcela Rojas Gomez, Jan Sebastian Schulte, Friedrich Wilhelm Mohr, Stefan Dhein

Resultado de la investigación: Article

19 Citas (Scopus)

Resumen

Connexin43 (Cx43) is the predominant intercellular gap junction protein in the heart providing intercellular communication for the cell-to-cell transfer of electrical activation. In a previous study, we could show that α-adrenoceptor stimulation can affect Cx43 expression and function. We now wanted to elucidate which α1-adrenoceptor subtype might be involved. Cultured neonatal rat cardiomyocytes were exposed to various concentrations of phenylephrine (0.1-1,000 nM) for 24 h (n=6). Thereafter, cells were harvested, and after lysis, Cx43 content was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Results were normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Finally, we determined the effect of this treatment on intercellular gap junction conductivity using dual whole-cell voltage clamp. Similarly, we tested the effect of an additional treatment with either 10 nM prazosin or, to assess the subtypes, 10 nM of the α1A-antagonist RS17053 (n=6), 500 nM of the α1B-antagonist AH1111OA (n=6), or 50 nM of the α1D-antagonist BMY7378 (n=6). Moreover, we incubated the cells for 24 h with the α1A-adrenoceptor agonist A61603 (10 nM). Phenylephrine led to enhanced Cx43 expression with a pEC50 8.00±0.06. The other cardiac connexins, Cx40 and Cx45, as well as GAPDH were not affected. This increase in Cx43 expression resulted in enhanced gap-junction conductance (44±4 nS vs 26±4 nS). As expected, the increased Cx43 expression could be antagonized by prazosin. Moreover, it was nearly completely inhibited by BMY7378 but was not significantly affected by RS17053. AH1111OA led to a moderate but incomplete inhibition. In contrast, β-actin expression was also up-regulated by phenylephrine but was inhibited by prazosin or RS17053, while it was not affected by BMY7378 or AH1111OA. About 24 h exposure to the α1A-adrenoceptor agonist A61603 led to a twofold increase in β-actin but did not affect Cx43. The low pEC50 value of about 1 nM for noradrenaline reported in our earlier study fits well to the hypothesis of an effect mediated predominantly via α1D-adrenoceptors, which is further supported by the finding of a nearly complete antagonisation of the phenylephrine effect by BMY7378. Thus, we conclude that cardiac Cx43 expression seems to be regulated via α1-adrenoceptors predominantly by subtype α1D-adrenoceptors, while other proteins like β-actin seem to be regulated via α1A-adrenoceptors.

Idioma originalEnglish
Páginas (desde-hasta)77-85
Número de páginas9
PublicaciónNaunyn-Schmiedeberg's Archives of Pharmacology
Volumen377
N.º1
DOI
EstadoPublished - 1 mar 2008

Huella dactilar

Connexin 43
Cardiac Myocytes
Adrenergic Receptors
Phenylephrine
A 61603
Prazosin
Actins
Glyceraldehyde-3-Phosphate Dehydrogenases
Intercellular Junctions
Gap Junctions
Connexins
Cell Communication
Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis
Norepinephrine
Western Blotting

ASJC Scopus subject areas

  • Pharmacology

Citar esto

Rojas Gomez, Diana Marcela ; Schulte, Jan Sebastian ; Mohr, Friedrich Wilhelm ; Dhein, Stefan. / Alpha-1-adrenoceptor subtype selective regulation of connexin 43 expression in rat cardiomyocytes. En: Naunyn-Schmiedeberg's Archives of Pharmacology. 2008 ; Vol. 377, N.º 1. pp. 77-85.
@article{3852e779dd614144b3aaf2ae56b79966,
title = "Alpha-1-adrenoceptor subtype selective regulation of connexin 43 expression in rat cardiomyocytes",
abstract = "Connexin43 (Cx43) is the predominant intercellular gap junction protein in the heart providing intercellular communication for the cell-to-cell transfer of electrical activation. In a previous study, we could show that α-adrenoceptor stimulation can affect Cx43 expression and function. We now wanted to elucidate which α1-adrenoceptor subtype might be involved. Cultured neonatal rat cardiomyocytes were exposed to various concentrations of phenylephrine (0.1-1,000 nM) for 24 h (n=6). Thereafter, cells were harvested, and after lysis, Cx43 content was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Results were normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Finally, we determined the effect of this treatment on intercellular gap junction conductivity using dual whole-cell voltage clamp. Similarly, we tested the effect of an additional treatment with either 10 nM prazosin or, to assess the subtypes, 10 nM of the α1A-antagonist RS17053 (n=6), 500 nM of the α1B-antagonist AH1111OA (n=6), or 50 nM of the α1D-antagonist BMY7378 (n=6). Moreover, we incubated the cells for 24 h with the α1A-adrenoceptor agonist A61603 (10 nM). Phenylephrine led to enhanced Cx43 expression with a pEC50 8.00±0.06. The other cardiac connexins, Cx40 and Cx45, as well as GAPDH were not affected. This increase in Cx43 expression resulted in enhanced gap-junction conductance (44±4 nS vs 26±4 nS). As expected, the increased Cx43 expression could be antagonized by prazosin. Moreover, it was nearly completely inhibited by BMY7378 but was not significantly affected by RS17053. AH1111OA led to a moderate but incomplete inhibition. In contrast, β-actin expression was also up-regulated by phenylephrine but was inhibited by prazosin or RS17053, while it was not affected by BMY7378 or AH1111OA. About 24 h exposure to the α1A-adrenoceptor agonist A61603 led to a twofold increase in β-actin but did not affect Cx43. The low pEC50 value of about 1 nM for noradrenaline reported in our earlier study fits well to the hypothesis of an effect mediated predominantly via α1D-adrenoceptors, which is further supported by the finding of a nearly complete antagonisation of the phenylephrine effect by BMY7378. Thus, we conclude that cardiac Cx43 expression seems to be regulated via α1-adrenoceptors predominantly by subtype α1D-adrenoceptors, while other proteins like β-actin seem to be regulated via α1A-adrenoceptors.",
keywords = "Adrenoceptor, Connexin, Gap junction, Pharmacology, Phenylephrine",
author = "{Rojas Gomez}, {Diana Marcela} and Schulte, {Jan Sebastian} and Mohr, {Friedrich Wilhelm} and Stefan Dhein",
year = "2008",
month = "3",
day = "1",
doi = "10.1007/s00210-007-0244-9",
language = "English",
volume = "377",
pages = "77--85",
journal = "Naunyn-Schmiedeberg's Archives of Pharmacology",
issn = "0028-1298",
publisher = "Springer Verlag",
number = "1",

}

Alpha-1-adrenoceptor subtype selective regulation of connexin 43 expression in rat cardiomyocytes. / Rojas Gomez, Diana Marcela; Schulte, Jan Sebastian; Mohr, Friedrich Wilhelm; Dhein, Stefan.

En: Naunyn-Schmiedeberg's Archives of Pharmacology, Vol. 377, N.º 1, 01.03.2008, p. 77-85.

Resultado de la investigación: Article

TY - JOUR

T1 - Alpha-1-adrenoceptor subtype selective regulation of connexin 43 expression in rat cardiomyocytes

AU - Rojas Gomez, Diana Marcela

AU - Schulte, Jan Sebastian

AU - Mohr, Friedrich Wilhelm

AU - Dhein, Stefan

PY - 2008/3/1

Y1 - 2008/3/1

N2 - Connexin43 (Cx43) is the predominant intercellular gap junction protein in the heart providing intercellular communication for the cell-to-cell transfer of electrical activation. In a previous study, we could show that α-adrenoceptor stimulation can affect Cx43 expression and function. We now wanted to elucidate which α1-adrenoceptor subtype might be involved. Cultured neonatal rat cardiomyocytes were exposed to various concentrations of phenylephrine (0.1-1,000 nM) for 24 h (n=6). Thereafter, cells were harvested, and after lysis, Cx43 content was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Results were normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Finally, we determined the effect of this treatment on intercellular gap junction conductivity using dual whole-cell voltage clamp. Similarly, we tested the effect of an additional treatment with either 10 nM prazosin or, to assess the subtypes, 10 nM of the α1A-antagonist RS17053 (n=6), 500 nM of the α1B-antagonist AH1111OA (n=6), or 50 nM of the α1D-antagonist BMY7378 (n=6). Moreover, we incubated the cells for 24 h with the α1A-adrenoceptor agonist A61603 (10 nM). Phenylephrine led to enhanced Cx43 expression with a pEC50 8.00±0.06. The other cardiac connexins, Cx40 and Cx45, as well as GAPDH were not affected. This increase in Cx43 expression resulted in enhanced gap-junction conductance (44±4 nS vs 26±4 nS). As expected, the increased Cx43 expression could be antagonized by prazosin. Moreover, it was nearly completely inhibited by BMY7378 but was not significantly affected by RS17053. AH1111OA led to a moderate but incomplete inhibition. In contrast, β-actin expression was also up-regulated by phenylephrine but was inhibited by prazosin or RS17053, while it was not affected by BMY7378 or AH1111OA. About 24 h exposure to the α1A-adrenoceptor agonist A61603 led to a twofold increase in β-actin but did not affect Cx43. The low pEC50 value of about 1 nM for noradrenaline reported in our earlier study fits well to the hypothesis of an effect mediated predominantly via α1D-adrenoceptors, which is further supported by the finding of a nearly complete antagonisation of the phenylephrine effect by BMY7378. Thus, we conclude that cardiac Cx43 expression seems to be regulated via α1-adrenoceptors predominantly by subtype α1D-adrenoceptors, while other proteins like β-actin seem to be regulated via α1A-adrenoceptors.

AB - Connexin43 (Cx43) is the predominant intercellular gap junction protein in the heart providing intercellular communication for the cell-to-cell transfer of electrical activation. In a previous study, we could show that α-adrenoceptor stimulation can affect Cx43 expression and function. We now wanted to elucidate which α1-adrenoceptor subtype might be involved. Cultured neonatal rat cardiomyocytes were exposed to various concentrations of phenylephrine (0.1-1,000 nM) for 24 h (n=6). Thereafter, cells were harvested, and after lysis, Cx43 content was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Results were normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Finally, we determined the effect of this treatment on intercellular gap junction conductivity using dual whole-cell voltage clamp. Similarly, we tested the effect of an additional treatment with either 10 nM prazosin or, to assess the subtypes, 10 nM of the α1A-antagonist RS17053 (n=6), 500 nM of the α1B-antagonist AH1111OA (n=6), or 50 nM of the α1D-antagonist BMY7378 (n=6). Moreover, we incubated the cells for 24 h with the α1A-adrenoceptor agonist A61603 (10 nM). Phenylephrine led to enhanced Cx43 expression with a pEC50 8.00±0.06. The other cardiac connexins, Cx40 and Cx45, as well as GAPDH were not affected. This increase in Cx43 expression resulted in enhanced gap-junction conductance (44±4 nS vs 26±4 nS). As expected, the increased Cx43 expression could be antagonized by prazosin. Moreover, it was nearly completely inhibited by BMY7378 but was not significantly affected by RS17053. AH1111OA led to a moderate but incomplete inhibition. In contrast, β-actin expression was also up-regulated by phenylephrine but was inhibited by prazosin or RS17053, while it was not affected by BMY7378 or AH1111OA. About 24 h exposure to the α1A-adrenoceptor agonist A61603 led to a twofold increase in β-actin but did not affect Cx43. The low pEC50 value of about 1 nM for noradrenaline reported in our earlier study fits well to the hypothesis of an effect mediated predominantly via α1D-adrenoceptors, which is further supported by the finding of a nearly complete antagonisation of the phenylephrine effect by BMY7378. Thus, we conclude that cardiac Cx43 expression seems to be regulated via α1-adrenoceptors predominantly by subtype α1D-adrenoceptors, while other proteins like β-actin seem to be regulated via α1A-adrenoceptors.

KW - Adrenoceptor

KW - Connexin

KW - Gap junction

KW - Pharmacology

KW - Phenylephrine

UR - http://www.scopus.com/inward/record.url?scp=39149131662&partnerID=8YFLogxK

U2 - 10.1007/s00210-007-0244-9

DO - 10.1007/s00210-007-0244-9

M3 - Article

C2 - 18193202

AN - SCOPUS:39149131662

VL - 377

SP - 77

EP - 85

JO - Naunyn-Schmiedeberg's Archives of Pharmacology

JF - Naunyn-Schmiedeberg's Archives of Pharmacology

SN - 0028-1298

IS - 1

ER -