AJP - Heart AJP: Regulatory, Integrative and Comparative Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 287: H216-H224, 2004. First published March 11, 2004; doi:10.1152/ajpheart.00915.2003
0363-6135/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/1/H216    most recent
00915.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (29)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kansui, Y.
Right arrow Articles by Iida, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kansui, Y.
Right arrow Articles by Iida, M.

Angiotensin II receptor blockade corrects altered expression of gap junctions in vascular endothelial cells from hypertensive rats

Yasuo Kansui,1 Koji Fujii,1 Keiichiro Nakamura,2 Kenichi Goto,1 Hideyuki Oniki,1 Isao Abe,1 Yosaburo Shibata,2 and Mitsuo Iida1

1Department of Medicine and Clinical Science, and 2Department of Developmental Molecular Anatomy, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan

Submitted 29 September 2003 ; accepted in final form 8 March 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Blockade of the renin-angiotensin system improves the impaired endothelium-dependent relaxations associated with hypertension and aging, partly through amelioration of endothelium-derived hyperpolarizing factor (EDHF)-mediated responses. Although the nature of EDHF is still controversial, recent studies have suggested the involvement of gap junctions in EDHF-mediated responses. Gap junctions consist of connexins (Cx), and we therefore tested whether the expression of Cx in vascular endothelial cells would be altered by hypertension and antihypertensive treatment. Spontaneously hypertensive rats (SHR) were treated with either the angiotensin II type 1 receptor antagonist candesartan or the combination of hydralazine and hydrochlorothiazide for 3 mo from 5 to 8 mo of age. Confocal laser scanning microscopy after immunofluorescent labeling with antibodies against Cx37, Cx40, and Cx43 revealed that the expression of Cx37 and Cx40 in endothelial cells of the mesenteric artery was significantly lower in SHR than in WKY. Treatment with candesartan, but not the combination of hydralazine and hydrochlorothiazide, significantly increased the expression of Cx37 and Cx40, although blood pressure decreased similarly. On the other hand, the expression of Cx43, though scarce and heterogeneous, was increased in SHR compared with WKY, and candesartan treatment lowered the expression of Cx43. These findings suggest that renin-angiotensin system blockade corrects the decreased expression of Cx37 and Cx40 in arterial endothelial cells of hypertensive rats, partly independently of blood pressure, whereas the expression of Cx43 changed in the opposite direction. It remains to be clarified whether these changes in Cx37 and Cx40 are related to endothelial function, particularly that attributable to EDHF.

connexin; hypertension; treatment; renin-angiotensin system; mesenteric artery

ENDOTHELIAL CELLS play an important role in the regulation of vascular tone by the release of vasoactive factors, such as nitric oxide (15, 33), prostaglandins, and endothelium-derived hyperpolarizing factor (EDHF) (5, 6). EDHF relaxes underlying smooth muscle cells through membrane hyperpolarization (5, 14). Although the nature of EDHF is still debated (1–3, 9, 12, 29, 47, 48), several recent studies (10, 17, 37) have suggested the involvement of gap junctions in the EDHF-type response, which may permit the electrical transmission of hyperpolarization from endothelial cells to smooth muscle cells.

The gap junction channel is composed of two hemichannels, named connexons; each connexon comprises six subunit proteins called connexins (Cx). Three proteins, Cx37, Cx40, and Cx43, have been to be expressed mainly in vascular endothelial cells (20, 35, 49). These connexins form channels that are suggested to possess different biophysical properties, e.g., conductances, selectivities of passage of small molecular weight substances, and current-carrying ions (41, 45).

We have previously shown that in the rat mesenteric artery, EDHF-mediated hyperpolarization and relaxation are impaired by hypertension as well as with aging (13, 14), and that antihypertensive treatments, particularly those using inhibitors of the renin-angiotensin system, improve impaired EDHF-mediated responses (18, 19, 24, 32). Furthermore, our previous study (17) and other studies (38, 43) have demonstrated that gap junctions play a critical role in the action of EDHF in this arterial bed. In this context, it is tempting to speculate that connexins, composing gap junctions, would be altered by hypertension and drug treatments in the rat mesenteric artery, which might be associated with alterations of the EDHF-mediated responses.

The present study tested whether the expression of connexins in rat vascular endothelial cells would be altered by hypertension and antihypertensive treatment, especially treatment involving inhibitors of the renin-angiotensin system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation of arteries. This study was approved by the Committee on the Ethics of Animal Experimentation of Kyushu University. Male spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto rats (WKY) were fed a standard rat chow and had free access to tap water. At the age of 5 mo, SHR were assigned to one control (SHR-CON) and to one of two treatment groups. The latter two groups were treated for 3 mo with either the angiotensin type 1 (AT1) receptor antagonist candesartan cilexetil (SHR-CAN; 5 mg·kg–1·day–1) or the combination of hydralazine and hydrochlorothiazide (SHR-HH; 25 and 7.5 mg·kg–1·day–1, respectively) from 5 to 8 mo of age. All drugs were given in the drinking water. Water intake was checked three times a week, and drug concentrations were adjusted to achieve the above daily doses. Age-matched, untreated WKY (WKY-CON) served as controls (n = 4–6 in each group). In addition, in some studies, 15-wk-old, young SHR (SHR-Y) and WKY (WKY-Y) also served as young controls (n = 5 in each group).

Systolic blood pressure was measured in conscious rats before and at the end of the treatment period. The drugs were withdrawn 2 days before the experiments. The rats were deeply anesthetized with ether and killed by decapitation. The main trunk (elastic arteries) and the second or third branches (muscular arteries) of the mesenteric artery were excised, and heparin (600 units in 300 µl) in PBS was injected into the mesenteric artery to prevent clotting. The artery was cut into 2-mm-long rings in PBS for use in the confocal laser scanning microscopy experiment with immunofluorescence.

Immunohistochemistry. The arteries were cut along the longitudinal axis for whole mount preparation. Whole mount samples were fixed in acetone at –20°C for 1 h. After being washed briefly with PBS, the samples were incubated in a blocking solution (PBS containing 1% nonfat dry milk and 0.5% Triton X-100) for 1 h at room temperature. Samples were exposed to Cx37 (affinity-purified rabbit anti-Cx37, Alpha Diagnostic International, dilution 1:1,000), Cx40 (affinity-purified rabbit anti-Cx40, Chemicon International, dilution 1:1,000), or Cx43 antibodies (affinity-purified rabbit anti-Cx43, Zymed Laboratories, dilution 1:1,000) at 4°C overnight (20, 30, 31). After being washed in PBS for 1 h, the samples were incubated with secondary antibodies (Alexa488-conjugated goat anti-rabbit IgG antibodies, Molecular Probes; dilution 1:500) for 2 h. For negative control, primary antibodies were omitted. After being washed in PBS, the samples were mounted with the endothelial layer up in the mounting medium Vectashield containing propidium iodide (Vector Laboratories) for counterstaining nuclei. The samples were observed with the use of a confocal laser-scanning microscope (model LSM310, Zeiss). The number of immunolabeled connexin plaques per endothelial cell was determined from 10 cells in each animal.

Some samples in the form of double-stained cryosections were also estimated. Samples were quickly embedded in Tissue-Tek OCT compound and were frozen in liquid nitrogen. Cross-sectional cryosections (4 µm thick) were mounted on poly-L-lysine-coated glass slides. The sections were air dried and fixed in acetone at –20°C for 20 min. After being briefly washed with PBS, the mounted sections were incubated in a blocking solution (PBS containing 5% nonfat dry milk) for 1 h, and then with primary antibodies for 2 h at room temperature. For double stainings, pairs of a rabbit polyclonal antibody [anti-Cx37 (dilution 1:500), anti-Cx40 (dilution 1:1,000), or anti-Cx43 (1:1,000) antibodies] and a mouse monoclonal antibody [anti-{alpha}-smooth muscle actin antibodies (mouse anti-{alpha}-smooth muscle actin, Sigma; dilution 1:1,000)] were chosen. After being washed with PBS, the samples were incubated with secondary antibodies (Alexa488-conjugated goat anti-rabbit IgG and Alexa594-conjugated goat anti-mouse IgG) for 1 h at a dilution of 1:1,000. Samples were then washed in PBS and observed via the confocal laser-scanning microscope.

Statistical analysis. Results are given as means ± SE. Data were analyzed by one-way ANOVA, followed by Scheffé's test for multiple comparisons or paired Student's t-test, if appropriate. A level of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Systolic blood pressure, heart rate, and body weight. The systolic blood pressure, heart rates, and body weights of WKY and SHR before and at the end of the treatment period are shown in Table 1. Chronic treatment with candesartan cilexetil and the combination of hydralazine and hydrochlorothiazide lowered the blood pressure to comparable extents.


View this table:
[in this window]
[in a new window]
  		Table 1. Systolic blood pressure, heart rate, and body weight before and after 3 mo of treatment in the six study groups

 
Connexin expression. First, the distribution of Cxs was investigated in cross-sectional sections of the elastic and muscular arteries from the rat mesenteric arterial bed. In the elastic arteries of normotensive WKY rats, Cx37 was found in both the endothelium and the media. Cx40 was expressed in the endothelium, but not in the media. A small expression of Cx43 was observed in the media only. In the muscular arteries of WKY, both Cx37 and Cx40 existed abundantly in the endothelium. Cx43 was not observed in these arteries. Because quantitative analysis of Cxs was difficult in cross-sectional preparations, we focused on the expression of Cxs on endothelial cells using whole mount preparations in the following experiments.

In investigating the expression of these Cxs on the endothelial cells with whole mount preparations, the side opposite branching was chosen for the experiment regarding the main trunk of the mesenteric arteries because this side provides a continuous population of endothelial cells probably due to the minimal effect of flow stress in this region. In the branching side of the arteries, endothelial cells showed irregular and changed shapes especially near the branching region.

In the main trunk of the mesenteric artery, the number of Cx37 and Cx40 plaques per endothelial cell was significantly decreased in SHR-CON compared with WKY-CON (Figs. 1, 3A, and 3C). Antihypertensive treatment with candesartan cilexetil significantly increased the number of Cx37 and Cx40 in SHR. Treatment with a combination of hydralazine and hydrochlorothiazide tended to increase the number of Cxs, but the difference did not reach statistical significance (Cx37: WKY-CON, 76 ± 2; SHR-CON, 66 ± 2; SHR-HH, 72 ± 2; and SHR-CAN, 81 ± 3/endothelial cell vs. Cx40: WKY-CON, 89 ± 2; SHR-CON, 75 ± 2; SHR-HH, 78 ± 3; and SHR-CAN, 87 ± 2/endothelial cell) (Figs. 1, 3A, and 3C for P values). The expression of Cx43 was very scarce and heterogeneous and was therefore difficult to evaluate; however, Cx43 appeared to be increased in SHR compared with WKY, and candesartan treatment, but not the combination of hydralazine and hydrochlorothiazide, decreased the expression of Cx43 (WKY-CON, 11 ± 2; SHR-CON, 32 ± 4; SHR-HH, 33 ± 4; and SHR-CAN, 15 ± 3/endothelial cell) (Figs. 1 and 3E).



View larger version (84K):
[in this window]
[in a new window]
  		Fig. 1. Expression of connexins (Cx) 37, 40, and 43 (green) in endothelial cells of the rat elastic arteries of mesentery in Wistar-Kyoto-Control (WKY-CON), spontaneously hypertensive rats (SHR)-CON (SHR-CON), SHR-hydralazine and hydrochloride (SHR-HH), and SHR-candesartan (SHR-CAN). Nuclei of endothelial cells were counterstained with propidium iodide (red). Bar = 20 µm.

 


View larger version (33K):
[in this window]
[in a new window]
  		Fig. 3. Bar graph showing the expression of Cx37, 40, and 43 per endothelial cell in the mesenteric arteries of WKY-CON, SHR-CON, SHR-HH, and SHR-CAN. Expression of Cx37 (A), Cx40 (C), and Cx43 (D) in the elastic artery; Cx37 (B), Cx40 (D), and Cx43 (E) in the muscular artery. *P < 0.05 vs. SHR-CON by one-way ANOVA; {dagger}P < 0.05 vs. the elastic artery of the same group.

 
Second or third branches of the mesenteric arteries have also been evaluated. The expression of Cx37 and Cx40 per endothelial cell was significantly higher in these small arteries than in the main trunk of the mesenteric arteries. On the other hand, the expression of Cx43 was less frequent in the second and third branches compared with the main trunk of the mesenteric arteries.

The expression of Cx37 and Cx40 was significantly smaller in SHR-CON than in WKY-CON as was the case with the main trunk. Antihypertensive treatment with candesartan, but not the combination of hydralazine and hydrochlorothiazide, significantly increased the expression of Cx37 and Cx40 [Cx37: WKY-CON, 90 ± 1; SHR-CON, 84 ± 2; SHR-HH, 87 ± 4; and SHR-CAN, 95 ± 4/endothelial cell (see Fig. 2, 3B, and 3D for P values). The expression of Cx43 was significantly higher in SHR-CON than in WKY-CON, and markedly decreased in the two treatment groups [Cx43: WKY-CON, 1 ± 0; SHR-CON, 37 ± 4; SHR-HH, 4 ± 1; and SHR-CAN, 1 ± 1 (see Figs. 2 and 3F for P values).



View larger version (88K):
[in this window]
[in a new window]
  		Fig. 2. Expression of Cx37, 40, and 43 (green) in endothelial cells of the muscular arteries of mesentery in WKY-CON, SHR-CON, SHR-HH, WKY-Y, and SHR-Y. Nuclei of endothelial cells were counterstained with propidium iodide (red). Bar = 20 µm.

 
Age-related changes in connexin expression were also investigated by comparing 8-mo-old WKY and SHR with WKY-Y and SHR-Y. Expression of Cx37, but not Cx40, was significantly lower in WKY-CON compared with WKY-Y (Cx37: WKY-CON, 76 ± 2; WKY-Y, 84±2/endothelial cell vs. Cx40: WKY-CON, 89 ± 2; WKY-Y, 92 ± 4/endothelial cell) (see Figs. 1, 4A, and 4C for P values). In SHR, Cx37 and Cx40 expression was significantly decreased in SHR-CON compared with SHR-Y (Cx37: SHR-CON, 66 ± 2; SHR-Y, 79 ± 2/endothelial cell vs. Cx40: SHR-CON, 75 ± 2; SHR-Y 88 ± 2/endothelial cell), and the expression of Cx37 and Cx40 tended to be lower in SHR-Y compared with WKY-Y (see Figs. 1, 4A, and 4C for P values). On the other hand, Cx43 expression was increased in SHR-CON and WKY-CON compared with SHR-Y and WKY-Y, respectively (WKY-CON, 11 ± 2; WKY-Y, 6 ± 2/endothelial cell; SHR-CON, 32 ± 4; SHR-Y 14 ± 3), and the expression of Cx43 was significantly increased in SHR-Y compared with WKY-Y (see Figs. 1 and 4E for P values).



View larger version (38K):
[in this window]
[in a new window]
  		Fig. 4. Bar graph showing the expression of Cx37, 40, and 43 per endothelial cell in the mesenteric arteries of WKY-CON, WKY-Y, SHR-CON, and SHR-Y. Expression of Cx37 (A), Cx40 (C), and Cx43 (D) in the elastic artery; Cx37 (B), Cx40 (D), and Cx43 (E) in the muscular artery. *P < 0.05 vs. SHR-CON; {dagger}P < 0.05 vs. the elastic artery of the same group; {ddagger}P < 0.05 vs. SHR-Y; and ¶P < 0.05 vs. WKY-Y.

 
In the second or third branches of the mesenteric arteries, Cx37 and Cx40 expression was similar between WKY-CON and WKY-Y (Cx37: WKY-CON, 90 ± 1; WKY-Y, 93 ± 2/endothelial cell vs. Cx40: WKY-CON, 102 ± 3; WKY-Y, 100 ± 2/endothelial cell) (Figs. 2, 4B, and 4D). In SHR, Cx40, but not Cx37, was significantly decreased in SHR-CON compared with SHR-Y (Cx37: SHR-CON, 84 ± 2; SHR-Y, 89 ± 1/endothelial cell; Cx40: SHR-CON, 84 ± 3; SHR-Y, 104 ± 3/endothelial cell). The expression of Cx37 and Cx40 was similar between WKY-Y and SHR-Y (see Figs. 2, 4B, and 4D for P values). On the other hand, expression of Cx43 tended to be increased in SHR-CON compared with SHR-Y (WKY-CON, 1 ± 0; WKY-Y, 0 ± 0/endothelial cell vs. SHR-CON 37 ± 4; SHR-Y 8 ± 5). Furthermore, expression of Cx43 was significantly increased in SHR-Y compared with WKY-Y (see Figs. 2 and 4F for P values).

No Cx was found when the primary antibodies were omitted for a negative control.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrated that the expression of Cx37 and Cx40 was significantly decreased in vascular endothelial cells from SHR compared with WKY. Chronic treatment with the AT1 receptor antagonist candesartan, but not the traditional combination of hydralazine and hydrochlorothiazide, led to a significant increase in the expression of Cx37 and Cx40 in endothelial cells, although blood pressure was reduced similarly by both treatments. The expression of Cx43 was relatively difficult to evaluate because of scarce and heterogeneous distribution; however, the expression of Cx43 appeared to be increased in SHR compared with WKY, and treatment with candesartan lowered Cx43 expression. These findings suggest that the inhibition of the renin-angiotensin system restores the decreased expression of Cx37 and Cx40 in the vascular endothelial cells of hypertensive rats, whereas Cx43 appears to exhibit changes in the opposite direction.

In the endothelial cells of the rat large and small mesenteric arteries, Cx40 was most abundantly expressed, followed by Cx37. Cx43 was only scarcely observed in the endothelial cells of normotensive rats. In the media, small expression of Cx37 and Cx43, but not of Cx40, was observed. Although Cx45 has also been identified in certain vascular tissues (26, 27), we could not detect expression of Cx45 in the endothelial cells of the rat mesenteric artery (data not shown), which is consistent with some other studies (25, 36). The specificity of antibodies against Cx37, Cx40, and Cx43 used in this study has been confirmed in the previous studies from our and other laboratories (20, 30, 31). With the use of the same antibodies as used in this study, Gustafsson et al. (20) have confirmed specific stainings for Cx37, Cx40, and Cx43 for corresponding antibodies in the rat mesenteric vascular bed (20).

Several recent studies, including those on the rat mesenteric arteries, suggest that gap junctions play an important role in EDHF-mediated responses (17, 37, 38, 43). It is assumed that hyperpolarization or small molecules generated in endothelial cells may spread to underlying smooth muscle cells via the myoendothelial gap junction (4, 37, 38, 44, 47). The relative importance of EDHF to relaxation and the spatial distribution of gap junctions appear to coincide; i.e., EDHF has been shown to play a more important role in small arteries than in large arteries, and the incidence of myoendothelial gap junctions, determined by electron microscopy, is higher in small mesenteric arteries than in large mesenteric arteries, the findings being suggestive of the role of gap junctions in EDHF-mediated responses (37). In this study, the expression of Cx37 and Cx40 was also higher in small mesenteric arteries (muscular arteries) than in large mesenteric arterial trunks (elastic arteries), implying the greater involvement of gap junctional communication in smaller arteries, although such Cxs expression may represent mainly those between endothelial cells.

We have previously demonstrated that EDHF-mediated hyperpolarization and relaxation are impaired by hypertension and with aging (13, 14). We also found that antihypertensive treatment improves the impairment of EDHF-mediated responses associated with hypertension and aging (18, 19, 24, 32). The direction of changes of the expression of Cx37 and Cx40 in the present study, i.e., a decrease in hypertension and with aging and an increase with antihypertensive treatment, especially with that including the AT1 receptor antagonist, appeared to coincide with changes in the EDHF-mediated responses in our previous studies. Furthermore, differences in the expression of Cx37 and Cx40 between SHR and WKY appeared to be greater in older rats than in younger rats, suggesting that the expression of these connexins is downregulated during the course of hypertension. In addition, the expression of Cx37 tended to decrease with aging even in normotensive rats. On the other hand, the expression of Cx43 increased in hypertension and with aging and decreased with the AT1 receptor antagonist, changes opposite to those in EDHF-mediated responses.

It is at present unclear which connexins are involved mainly in the EDHF-type responses. Furthermore, three types of heterocellular couplings via gap junctions exist and each might contribute to EDHF-mediated responses in blood vessels, namely those between endothelial cells, smooth muscle cells, and endothelial and smooth muscle cells. In fact, the combination of gap junction inhibitors targeted to each of these connexins, Cx37, Cx40, and Cx43, was required to abolish the EDHF-mediated relaxation in the rat hepatic artery (4). It has also been demonstrated that electrical signals in the vessel wall may travel quite effectively in the longitudinal direction primarily through endothelial cells via gap junctions, indicating the importance of the gap junction between endothelial cells (11, 40, 48). Theoretically, the impairment of EDHF responses could be accounted for by a defect in any of these three gap junction pathways. The expression of connexins observed in this study may represent mainly those between endothelial cells. Providing that homocellular, as well as heterocellular, gap junctions play a role in EDHF-mediated responses in the rat mesenteric arteries, it is possible to speculate that a decrease in the expression of Cx37 and Cx40 between endothelial cells might lead to an impaired conduction between endothelial cells, resulting in an overall reduction in the EDHF-type response. Indeed, conduction of vasodilation along arterioles was severely attenuated in mice deficient of Cx40 (7). Alternatively, some of the expression of Cx37 and Cx40 observed in the present study might represent those of myoendothelial gap junctions. Although we have previously demonstrated that gap junction inhibitors, such as carbenoxolone, greatly inhibit EDHF-type responses in the rat mesenteric arteries, suggesting a critical importance of gap junctions in these responses (17), further studies are needed to test whether changes in the expression of connexins observed in this study would actually be related to the alteration of EDHF-mediated responses in hypertension. Studies using connexin-mimetic peptides homologous to the extracellular loops of connexins to inhibit gap junctional communication would help to elucidate these issues. Furthermore, the present findings obtained with immunological staining should be confirmed using other measures of gap junction function and expression, such as intercellular dye transfer and Western blotting and mRNA expression of the connexins in the endothelial cell layer.

The present findings of decreased expression of Cx37 and Cx40 in hypertension are consistent with those of Rummery et al. (35) obtained in the rat caudal arteries and extend their study by showing for the first time that the inhibitor of the renin-angiotensin system by the angiotensin II receptor antagonist candesartan corrects the decreased expression of these Cxs. Such an effect of candesartan is likely to be in part due to the inhibition of the renin-angiotensin system per se rather that to its blood pressure lowering effect alone, because the traditional combination of hydralazine and hydrochlorothiazide only partially increased the expression of Cx37 and Cx40 despite achieving a similar reduction in blood pressure. This is also consistent with our previous study concerning the effect of drug treatment on EDHF-mediated response; i.e., a more marked improvement of EDHF-mediated responses was observed in association with the inhibitors of renin-angiotensin system than with other classes of antihypertensive agents. It remains to be determined whether the renin-angiotensin system and its inhibition directly influence the expression of Cx37 and Cx40 in vascular walls or indirectly through correction of vascular remodelings (39).

In the present study, Cx43 increased by hypertension and decreased by antihypertensive treatment, especially with candesartan. In fact, the regulation of Cx43 appeared to differ from those of Cx37 and Cx40 (8, 16, 22, 34); e.g., in cardiac cells, it has been demonstrated that the expression of Cx43 and Cx40 is differentially regulated (8). Gabriels et al. (16) suggested that Cx43, unlike Cx37 and Cx40, localized to regions that experience turbulent shear stress from disturbed blood flow in endothelial cells of the rat aorta. It is, therefore, possible to speculate that endothelial Cx43 expression might increase due to turbulent shear stress and higher shear stress in hypertension and decrease by antihypertensive treatment. Consistent with our findings, Cx43 expression was increased in tissue homogenized from aortas of mineralocorticoid-salt hypertension and renal artery clip hypertension rats (21, 46). On the other hand, in the study by Rummery et al. (35), Cx43 was decreased in endothelial cells from SHR compared with WKY, although to a lesser extent compared with Cx37 and Cx40. A decrease in Cx43 m RNA expression was also reported in the aorta of rats treated with NG-nitro-L-arginine methyl ester (23).

It appears, therefore, that the expression of Cx43 in the vascular wall may differ depending on the type of hypertension, vascular beds, and/or cell types (i.e., endothelial cells or smooth muscle cells). Interestingly, endothelial cell-specific knockout of Cx43 has been shown to cause hypotension in mice (28), although overall role of Cx43 in the regulation of vascular tone requires further investigation.

There are some reports suggesting a possible link between AT1 receptor activation and Cx43 expression. In cultured rat cardiac cells, angiotensin II has been shown to increase Cx43 but not Cx40 expression through AT1 receptors, which involves the signal transduction pathways, including extracellular signal-regulated kinase 1/2 and p38 (34). It has also been shown that cyclical mechanical stretch increases angiotensin II production and Cx43 gene expression in cultured rat cardiomyocytes, partially through AT1 receptors (42). These in vitro findings obtained in cardiac cells suggest possible association among mechanical stress, angiotensin II, and Cx43 expression. It is unknown whether angiotensin II exhibits similar effects on Cx43 expression in vascular endothelial cells. In any case, the present study for the first time revealed the possibility that chronic in vivo blockade of the renin-angiotensin system could modulate endothelial cell connexin expression, either positively (in the case of Cx37 and Cx40) or negatively (Cx43).

In conclusion, the present study has demonstrated that the expression of Cx37 and Cx40 in endothelial cells from the mesenteric arteries was decreased in hypertensive rats, and the treatment with AT1 receptor antagonist increased the expression of Cx37 and Cx40 in hypertensive rats, likely in part through blockade of the renin-angiotensin system per se. On the other hand, the expression of Cx43 in endothelial cells changed in the opposite direction. It remains to be determined whether the alteration in the expression of connexins and its modulation by angiotensin II receptor blockade are related to changes in EDHF-mediated responses in hypertension.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by Grant-in-Aid for Scientific Research 13670722 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Tokyo). A portion of this study was conducted at the Kyushu University Station for Collaborative Research.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Fujii, Dept. of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu Univ., Maidashi 3-1-1, Higashi-ku, Fukuoka, 812-8582, Japan (E-mail: fujii{at}intmed2.med.kyushu-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Beny JL and Pacicca C. Bidirectional electrical communication between smooth muscle and endothelial cells in the pig coronary artery. Am J Physiol Heart Circ Physiol 266: H1465–H1472, 1994.[Abstract/Free Full Text]
  2. Campbell WB, Gebremedhin D, Pratt PF, and Harder DR. Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res 78: 415–423, 1996.[Abstract/Free Full Text]
  3. Campbell WB and Harder DR. Endothelium-derived hyperpolarizing factors and vascular cytochrome P450 metabolites of arachidonic acid in the regulation of tone. Circ Res 84: 484–488, 1999.[Free Full Text]
  4. Chaytor AT, Taylor HJ, and Griffith TM. Gap junction-dependent and -independent EDHF-type relaxations may involve smooth muscle cAMP accumulation. Am J Physiol Heart Circ Physiol 282: H1548–H1555, 2002.[Abstract/Free Full Text]
  5. Chen G, Suzuki H, and Weston AH. Acetylcholine releases endothelium-derived hyperpolarizing factor and EDRF from rat blood vessels. Br J Pharmacol 95: 1165–1174, 1988.[Web of Science][Medline]
  6. Cohen RA and Vanhoutte PM. Endothelium-dependent hyperpolarization. Beyond nitric oxide and cyclic GMP. Circulation 92: 3337–3349, 1995.[Free Full Text]
  7. De Wit C, Roos F, Bolz SS, Kirchhoff S, Kruger O, Willecke K, and Pohl U. Impaired conduction of vasodilation along arterioles in connexin40-deficient mice. Circ Res 86: 649–655, 2000.[Abstract/Free Full Text]
  8. Dhein S, Polontchouk L, Salameh A, and Haefliger JA. Pharmacological modulation and differential regulation of the cardiac gap junction proteins connexin 43 and connexin 40. Biol Cell 94: 402–422, 2002.
  9. Edwards G, Dora KA, Gardener MJ, Garland CJ, and Weston AH. K+ is an endothelium-derived hyperpolarizing factor in rat arteries. Nature 396: 269–272, 1998.[CrossRef][Medline]
  10. Edwards G, Feletou M, Gardener MJ, Thollon C, Vanhoutte PM, and Weston AH. Role of gap junctions in the responses to EDHF in rat and guinea-pig small arteries. Br J Pharmacol 128: 1788–1794, 1999.[CrossRef][Web of Science][Medline]
  11. Emerson GG and Segal SS. Electrical activation of endothelium evokes vasodilation and hyperpolarization along hamster feed arteries. Am J Physiol Heart Circ Physiol 280: H160–H167, 2001.[Abstract/Free Full Text]
  12. Fisslthaler B, Popp R, Kiss L, Potente M, Harder DR, Fleming I, and Busse R. Cytochrome P450 2C is an EDHF synthase in coronary arteries. Nature 401: 493–497, 1999.[CrossRef][Medline]
  13. Fujii K, Ohmori S, Tominaga M, Abe I, Takata Y, Ohya Y, Kobayashi K, and Fujishima M. Age-related changes in endothelium-dependent hyperpolarization in the rat mesenteric artery. Am J Physiol Heart Circ Physiol 265: H509–H516, 1993.[Abstract/Free Full Text]
  14. Fujii K, Tominaga M, Ohmori S, Kobayashi K, Koga T, Takata Y, and Fujishima M. Decreased endothelium-dependent hyperpolarization to acetylcholine in smooth muscle of the mesenteric artery of spontaneously hypertensive rats. Circ Res 70: 660–669, 1992.[Abstract/Free Full Text]
  15. Furchgott RF and Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: 373–376, 1980.[CrossRef][Medline]
  16. Gabriels JE and Paul DL. Connexin43 is highly localized to sites of disturbed flow in rat aortic endothelium but connexin37 and connexin40 are more uniformly distributed. Circ Res 83: 636–643, 1998.[Abstract/Free Full Text]
  17. Goto K, Fujii K, Kansui Y, Abe I, and Iida M. Critical role of gap junctions in endothelium-dependent hyperpolarization in rat mesenteric arteries. Clin Exp Pharmacol Physiol 29: 595–602, 2002.[CrossRef][Web of Science][Medline]
  18. Goto K, Fujii K, Onaka U, Abe I, and Fujishima M. Renin-angiotensin system blockade improved endothelial dysfunction in hypertension. Hypertension 36: 575–580, 2000.[Abstract/Free Full Text]
  19. Goto K, Fujii K, Onaka U, Abe I, and Fujishima M. Angiotensin-converting enzyme inhibitor prevents age-related endothelial dysfunction. Hypertension 36: 581–587, 2000.[Abstract/Free Full Text]
  20. Gustafsson F, Mikkelsen HB, Arensbak B, Thuneberg L, Neve S, Jensen LJ, and Holstein-Rathlou NH. Expression of connexin 37, 40 and 43 in rat mesenteric arterioles and resistance arteries. Histochem Cell Biol 119: 139–148, 2003.[Web of Science][Medline]
  21. Haefliger JA, Castillo E, Waeber G, Bergonzelli GE, Aubert JF, Sutter E, Nicod P, Waeber B, and Meda P. Hypertension increases connexin43 in a tissue-specific manner. Circulation 95: 1007–1014, 1997.[Abstract/Free Full Text]
  22. Haefliger JA, Demotz S, Braissant O, Suter E, Waeber B, Nicod P, and Meda P. Connexin 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension. Kidney Int 60: 190–201, 2001.[CrossRef][Web of Science][Medline]
  23. Haefliger JA, Meda P, Formenton A, Wiesel P, Zanchi A, Brunner HR, Nicod P, and Hayoz D. Aortic connexin43 is decreased during hypertension induced by inhibition of nitric oxide synthase. Arterioscler Thromb Vasc Biol 19: 1615–1622, 1999.[Abstract/Free Full Text]
  24. Kansui Y, Fujii K, Goto K, Abe I, and Iida M. Angiotensin II receptor antagonist improves age-related endothelial dysfunction. J Hypertens 20: 439–446, 2002.[CrossRef][Web of Science][Medline]
  25. Ko YS, Coppen SR, Dupont E, Rothery S, and Severs NJ. Regional differentiation of desmin, connexin43, and connexin45 expression patterns in rat aortic smooth muscle. Arterioscler Thromb Vasc Biol 21: 355–364, 2001.[Abstract/Free Full Text]
  26. Kumai M, Nishii K, Nakamura K, Takeda N, Suzuki M, and Shibata Y. Loss of connexin45 causes a cushion defect in early cardiogensis. Development 127: 3501–3512, 2000.[Abstract]
  27. Kruger O, Plum A, Kim JS, Winterhager E, Maxeiner S, Hallas G, Kirchhoff S, Traub O, Lamers WH, and Willecke K. Defective vascular development in connexin 45-deficient mice. Development 127: 4179–4193, 2000.[Abstract]
  28. Liao Y, Day KH, Damon DN, and Duling BR. Endothelial cell-specific knockout of connexin 43 causes hypotension and bradycardia in mice. Proc Natl Acad Sci USA 98: 9989–9994, 2001.[Abstract/Free Full Text]
  29. Matoba T, Shimokawa H, Nakashima M, Hirakawa Y, Mukai Y, Hirano K, Kanaide H, and Takeshita A. Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice. J Clin Invest 106: 1521–1530, 2000.[Web of Science][Medline]
  30. Nakamura K and Shibata Y. Connexin43 expression in network-forming cells at the submucosal-muscular border of guinea pig and dog colon. Cells Tiss Org 165: 16–21, 1995.
  31. Nakamura K, Inai T, Nakamura K, and Shibata Y. Distribution of gap junction protein connexin 37 in smooth muscle cells of the rat trachea and pulomonary artery. Arch Histol Cytol 62: 27–37, 1999.[CrossRef][Web of Science][Medline]
  32. Onaka U, Fujii K, Abe I, and Fujishima M. Antihypertensive treatment improves endothelium-dependent hyperpolarization in the mesenteric artery of spontaneously hypertensive rats. Circulation 98: 175–182, 1998.[Abstract/Free Full Text]
  33. Palmer RM, Ferrige AG, and Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327: 524–526, 1987.[CrossRef][Medline]
  34. Polontchouk L, Ebelt B, Jackels M, and Dhein S. Chronic effects of endothein 1 and angiotensin II on gap junctions and intercellular communication in cardiac cells. FASEB J 16: 87–89, 2002.[Abstract/Free Full Text]
  35. Rummery NM, McKenzie KU, Whitworth JA, and Hill CE. Decreased endothelial size and connexin expression in rat caudal arteries during hypertension. J Hypertens 20: 247–253, 2002.[CrossRef][Web of Science][Medline]
  36. Rummery NM, Hickey H, McGurk G, and Hill CE. Connexin37 is the major connexin expressed in the media of caudal artery. Arterioscler Thromb Vasc Biol 22: 1427–1432, 2002.[Abstract/Free Full Text]
  37. Sandow SL and Hill CE. Incidence of myoendothelial gap junctions in the proximal and distal mesenteric arteries of the rat is suggestive of a role in endothelium-derived hyperpolarizing factor-mediated responses. Circ Res 86: 341–346, 2000.[Abstract/Free Full Text]
  38. Sandow SL, Tare M, Coleman HA, Hill CE, and Parkington HC. Involvement of myoendothelial gap junctions in the actions of endothelium-derived hyperpolarizing factor. Circ Res 90: 1108–1113, 2002.[Abstract/Free Full Text]
  39. Schiffrin EL, Park JB, and Pu Q. Effect of crossing over hypertensive patients from a beta-blocker to an angiotensin receptor antagonist on resistance artery structure and on endothelial function. J Hypertens 20: 71–78, 2002.[CrossRef][Web of Science][Medline]
  40. Segal SS, Welsh DG, and Kurjiaka DT. Spread of vasodilation and vasoconstriction along feed arteries and arterioles of hamster skeletal muscle. J Physiol 516: 283–291, 1999.[Abstract/Free Full Text]
  41. Shibata Y, Kumai M, Nishii K, and Nakamura K. Diversity and molecular anatomy of gap junctions. Med Electron Microsc 34: 153–159, 2001.[CrossRef][Medline]
  42. Shyu KG, Chen CC, Wang BW, and Kuan P. Angiotensin II receptor antagonist blocks the expression of connexin43 induced by cyclical mechanical stretch in cultured neonatal rat cardiac myocytes. J Mol Cell Cardiol 33: 691–698, 2001.[CrossRef][Web of Science][Medline]
  43. Tare M, Coleman HA, and Parkington HC. Glycyrrhetinic derivatives inhibit hyperpolarization in endothelial cells of guinea pig and rat arteries. Am J Physiol Heart Circ Physiol 282: H335–H341, 2002.[Abstract/Free Full Text]
  44. Taylor HJ, Chaytor AT, Edwards DH, and Griffith TM. Gap junction-dependent increases in smooth muscle cAMP underpin the EDHF phenomenon in rabbit arteries. Biochem Biophys Res Commun 283: 583–589, 2001.[CrossRef][Web of Science][Medline]
  45. Valiunas V, Beyer EC, and Brink PR. Cardiac gap junction channels show quantitative differences in selectivity. Circ Res 91: 104–111, 2002.[Abstract/Free Full Text]
  46. Watts SW and Webb RC. Vascular gap junctional communication is increased in mineralocorticoid-salt hypertension. Hypertension 28: 888–893, 1996.[Abstract/Free Full Text]
  47. Yamamoto Y, Imaeda K, and Suzuki H. Endothelium-dependent hyperpolarization and intercellular electrical coupling in guinea-pig mesenteric arterioles. J Physiol 514: 505–513, 1999.[Abstract/Free Full Text]
  48. Yamamoto Y, Klemm MF, Edwards FR, and Suzuki H. Intercellular electrical communication among smooth muscle and endothelial cells in guinea-pig mesenteric arterioles. J Physiol 535: 181–195, 2001.[Abstract/Free Full Text]
  49. Yeh HI, Rothery S, Dupont E, Coppen SR, and Severs NJ. Individual gap junction plaques contain multiple connexins in arterial endothelium. Circ Res 83: 1248–1263, 1998.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
A. Ellis, K. Goto, D. J. Chaston, T. D. Brackenbury, K. R. Meaney, J. R. Falck, R. J. H. Wojcikiewicz, and C. E. Hill
Enalapril Treatment Alters the Contribution of Epoxyeicosatrienoic Acids but Not Gap Junctions to Endothelium-Derived Hyperpolarizing Factor Activity in Mesenteric Arteries of Spontaneously Hypertensive Rats
J. Pharmacol. Exp. Ther., August 1, 2009; 330(2): 413 - 422.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
A. Just, L. Kurtz, C. de Wit, C. Wagner, A. Kurtz, and W. J. Arendshorst
Connexin 40 Mediates the Tubuloglomerular Feedback Contribution to Renal Blood Flow Autoregulation
J. Am. Soc. Nephrol., July 1, 2009; 20(7): 1577 - 1585.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
S. Dal-Ros, C. Bronner, C. Schott, M. O. Kane, M. Chataigneau, V. B. Schini-Kerth, and T. Chataigneau
Angiotensin II-Induced Hypertension Is Associated with a Selective Inhibition of Endothelium-Derived Hyperpolarizing Factor-Mediated Responses in the Rat Mesenteric Artery
J. Pharmacol. Exp. Ther., February 1, 2009; 328(2): 478 - 486.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
A. Makino, O. Platoshyn, J. Suarez, J. X.-J. Yuan, and W. H. Dillmann
Downregulation of connexin40 is associated with coronary endothelial cell dysfunction in streptozotocin-induced diabetic mice
Am J Physiol Cell Physiol, July 1, 2008; 295(1): C221 - C230.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
K. A. Dora, N. T. Gallagher, A. McNeish, and C. J. Garland
Modulation of Endothelial Cell KCa3.1 Channels During Endothelium-Derived Hyperpolarizing Factor Signaling in Mesenteric Resistance Arteries
Circ. Res., May 23, 2008; 102(10): 1247 - 1255.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
P. Winter and K. A. Dora
Spreading dilatation to luminal perfusion of ATP and UTP in rat isolated small mesenteric arteries
J. Physiol., July 1, 2007; 582(1): 335 - 347.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
A. Virdis, R. Colucci, M. Fornai, E. Duranti, C. Giannarelli, N. Bernardini, C. Segnani, C. Ippolito, L. Antonioli, C. Blandizzi, et al.
Cyclooxygenase-1 Is Involved in Endothelial Dysfunction of Mesenteric Small Arteries From Angiotensin II-Infused Mice
Hypertension, March 1, 2007; 49(3): 679 - 686.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
X. F. Figueroa, B. E. Isakson, and B. R. Duling
Vascular Gap Junctions in Hypertension
Hypertension, November 1, 2006; 48(5): 804 - 811.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. Feletou and P. M. Vanhoutte
Endothelial dysfunction: a multifaceted disorder (The Wiggers Award Lecture)
Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H985 - H1002.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
V. V. Matchkov, A. Rahman, L. M. Bakker, T. M. Griffith, H. Nilsson, and C. Aalkjaer
Analysis of effects of connexin-mimetic peptides in rat mesenteric small arteries
Am J Physiol Heart Circ Physiol, July 1, 2006; 291(1): H357 - H367.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
S. Mather, K. A. Dora, S. L. Sandow, P. Winter, and C. J. Garland
Rapid Endothelial Cell-Selective Loading of Connexin 40 Antibody Blocks Endothelium-Derived Hyperpolarizing Factor Dilation in Rat Small Mesenteric Arteries
Circ. Res., August 19, 2005; 97(4): 399 - 407.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. Earley, T. C. Resta, and B. R. Walker
Disruption of smooth muscle gap junctions attenuates myogenic vasoconstriction of mesenteric resistance arteries
Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2677 - H2686.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/1/H216    most recent
00915.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (29)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kansui, Y.
Right arrow Articles by Iida, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kansui, Y.
Right arrow Articles by Iida, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.