Lack of vascular connexin 40 is associated with hypertension and irregular arteriolar vasomotion1

Cor de Wit, Frederik Roos, Steffen-Sebastian Bolz and Ulrich Pohl

Physiologisches Institut, Ludwig-Maximilians-Universität München, 80336 Munich, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
Gap-junctional communication coordinates the behavior of individual cells in arterioles. Gap junctions are formed by connexins 40 (Cx40), Cx43, Cx37, and Cx45 in the vasculature. Previously, we have shown that lack of Cx40 impairs conduction of dilatory signals along arterioles. Herein, we examined whether hypertension is present in conscious animals and whether this is a direct effect or due to secondary mechanisms. Mean arterial pressure was elevated by 20–25 mmHg in conscious Cx40-deficient mice (Cx40-/-) compared with wild-type controls in both sexes. Differences in heart rate were not observed. Blockade of NO synthase increased pressure equally in both genotypes. Conversely, the angiotensin AT1-receptor antagonist, candesartan, decreased pressure to similar extents in Cx40-/- and wild-type mice. Acetylcholine and sodium nitroprusside (0.05–15 nmol) were equally potent and effective in decreasing pressure and inducing dilatory responses in the microcirculation. However, in contrast to wild type, Cx40-/- arterioles exhibited spontaneous, irregular vasomotion leading temporarily to complete vessel closure. We conclude that loss of Cx40 is associated with hypertension independent of the action of angiotensin II. It is also not related to an altered efficacy of NO or other endothelial dilators. However, the observed irregular vasomotion suggests that peripheral vascular resistance is affected.

gap junctions; signal transmission; cell communication; endothelium-dependent dilation; nitric oxide


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
COMMUNICATION BETWEEN CELLS in an intact organ is important to coordinate the behavior of individual cells and for proper organ function. Such a coordination is achieved in many organs by the direct transfer of current or small molecules across the membranes of adjacent cells. This is possible due to cell coupling via gap junctions which interconnect their cytoplasms (13, 29). Communication via gap junctions has also recently been demonstrated to be an important mechanism in regional coordination within the vascular system (5, 21, 22). Signals such as changes of membrane potential spread along endothelial and/or vascular smooth muscle cells, thereby enabling the arteriole to react as a single unit leading to constriction or dilation over a long distance (911, 23, 30). Such a mechanism is presumably involved in the control of functional hyperemia, as dilation of upstream arterioles in response to contraction of skeletal muscle fibers was attenuated after blockade of gap junctions (6).

Gap junctions are clusters of intercellular channels formed by connexin proteins. These connexins consist of a large family of different members, which are distinguished by their molecular weight (25). In vascular cells, four connexins have been found, namely Cx43, Cx40, Cx37, and Cx45 (8). Of these, Cx43 is the most abundant within the vascular smooth muscle, whereas Cx40 is mainly found in the endothelium (1, 12, 24, 27, 31).

The preferential location of the different connexins within the vascular system suggests that they may serve specialized functions. The specific role for a certain connexin is highlighted by our previous finding that the loss of Cx40 leads to a defect in cellular communication in vascular cells, most likely within the endothelial layer (7). Additionally, the loss of Cx40 was associated with hypertension in anesthetized male animals. To further delineate the role of connexins in hypertension, we now measured blood pressure in conscious animals of both sexes at different ages. Furthermore, we sought to determine whether hypertension is a direct effect or due to secondary mechanisms. To analyze the potential role of an alteration of endothelial NO production, which is known to result in hypertension, e.g., endothelial NO synthase deficiency (16), we studied the effect of an NO synthase inhibitor on blood pressure in both Cx40-deficient and wild-type mice. Furthermore, responses upon injection of an NO donor and the endothelium-dependent agonist, acetylcholine (ACh), were assessed. Recently, it has been shown that Cx40 is abundantly expressed in the kidney in the modified smooth muscle cells of the juxtaglomerular apparatus (14, 24), suggesting that the loss of Cx40 may lead to an alteration of renin release and subsequently enhanced levels of angiotensin II. To examine whether the hypertension observed in Cx40-/- animals is angiotensin II dependent, we injected an AT1-receptor antagonist. Furthermore, dilator responses to ACh were determined by direct visualization of the microcirculation.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
Animal preparation and experimental setup.
The care of the animals and the conduct of the experiments were in accordance with the rules of the German animal protection law. In this study, Cx40-/- mice and their wild-type littermates, described previously (17), were investigated. Cx40-/- mice with a mixed 129/Sv-C57BL/6 background were backcrossed five times into a C57BL/6 background, and their heterozygous offsprings were interbred to generate animals used in these experiments. For surgical procedures, all mice were anesthetized with droperidol (20 mg/kg), fentanyl (0.1 mg/kg), and midazolam (2 mg/kg ip). The right carotid or femoral artery was exposed and subsequently cannulated by a polyethylene tube for later measurement of arterial pressure in conscious animals. Likewise, a catheter was inserted into the right jugular vein. These catheters were tunneled subcutaneously and guided to the outside through a small skin incision at the back of the animal. The arterial catheter was connected to a pressure transducer located at the same hydrostatic level as the mouse (Statham, Costa Mesa, CA) via a swivel device (Instech Laboratories, Plymouth Meeting, PA). The pressure signal was processed with a computer-based monitoring system (XmAD, http://www.ibiblio.org/pub/Linux/science/lab) at a sampling rate of 1,000 Hz. Heart rate was calculated by analyzing software (XmANA) from the amplitude of the pressure signal.

Experimental protocols.
To obtain pressure values in conscious mice, the animals were allowed to recover for 12–16 h from anesthesia before the actual pressure measurements were started. After a 1-h period of measurements, the specific inhibitor of NO synthase, N{omega}-nitro-L-arginine (L-NA), was injected intravenously as a single bolus within 10 min (100 mg/kg in a volume of 20 ml/kg). To study the effect of endothelium-dependent and -independent vasodilators on blood pressure, ACh or sodium nitroprusside (SNP) was applied as a single bolus into the carotid artery at increasing dosages (from 0.05 to 15 nmol) in a volume of 50 µl to Cx40-/- mice and their wild-type littermates. Pressure was allowed to return to control values before the next bolus was applied, which normally occurred within 5–10 min depending on the dosage used. This experimental protocol was repeated after inhibition of NO synthase (see above). In a different experimental series, the AT1-receptor blocker candesartan (CV11974) was applied intravenously as a single bolus at increasing concentrations (0.01 to 10 mg/kg) to Cx40-/- mice and respective control animals. After each injected bolus, pressure was allowed to reach a new steady-state level for 30 min before the next concentration was given. Thus an additive concentration-response curve to candesartan was obtained. All mice were killed by an overdose of anesthesia at the end of the protocol. Hearts were carefully excised, and wet weight was determined.

Microcirculatory studies.
Mice were anesthetized with droperidol (20 mg/kg), fentanyl (0.1 mg/kg), and midazolam (2 mg/kg ip), followed by continuous infusion (jugular vein). The cremaster muscle was prepared as described (7) and superfused with bicarbonate-buffered salt solution (in mmol/l: 143 Na+, 6 K+, 2.5 Ca2+, 1.2 Mg2+, 128 Cl-, 25 HCO3-, 1.2 SO42-, and 1.2 H2PO4-). In each animal, 12 arterioles were studied and observed using a microscope (Metallux, Leitz) equipped with a video camera. Images were displayed on a video monitor and recorded on videotape for measurement of luminal diameters. Arteriolar diameters were measured shortly before and during the local superfusion of the endothelium-independent NO donor SNP (1 µmol/l) or the endothelial stimulator ACh (1, 10 µmol/l). Increasing concentrations of vasoactive drugs were applied consecutively, with a recovery period of 5 min between washout and application of the next concentration or drug. During this recovery period, the arterioles regained their baseline diameter. The same protocol was then repeated in the presence of the NO synthase inhibitor L-NA (30 µmol/l) and the cyclooxygenase inhibitor indomethacin (3 µmol/l). These inhibitors were added to the superfusion 30 min before the protocol was repeated. Some arterioles were monitored continuously to study vasomotion.

Solutions and drugs.
All substances, except for candesartan, which was a gift of Astra-Zeneca (Wedel, Germany), were purchased from Sigma (Deisenhofen, Germany). SNP was dissolved in 1 mmol/l sodium acetate at the day of experiment and stored in the dark. Candesartan (10 mg/kg) was dissolved in Na2CO3 (1 mol/l) and 0.9% NaCl (1:20 vol/vol). L-NA (5 mg/ml) was dissolved in isotonic NaCl at 70°C by vigorous stirring. All further dilutions and solutions were prepared in Ringer solution containing 147 mmol/l Na+, 4 mmol/l K+, 2.5 mmol/l Ca+, and 156 mmol/l Cl- or in the superfusion buffer.

Statistics and calculations.
Comparisons within groups were performed using paired t-tests and, for multiple comparisons, P values were corrected according to Bonferroni. Data between groups were compared by analysis of variance followed by post hoc analysis of the means. Differences were considered significant at a corrected error probability of P < 0.05. All data are means ± SE.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
Hemodynamics in conscious Cx40-deficient mice.
Arterial pressure was measured in conscious mice at different ages in both sexes. Values were obtained after the animals recovered from anesthesia, i.e., 12–16 h after catheterization of the artery. Mean arterial blood pressure was 92 ± 4 mmHg in male wild-type (Cx40+/+) mice at an age of 117 ± 13 days (n = 8 animals). Heart rate, which was obtained from the oscillations of the pressure signal, was 490 ± 30 beats/min. In conscious male Cx40-deficient (Cx40-/-) mice, arterial pressure was significantly elevated by ~25 mmHg (n = 8) without differences in heart rate (Fig. 1). In female mice, the femoral artery was cannulated instead of the carotid vessel (n = 8 each group). Pressure in female Cx40+/+ mice at an age of 247 ± 13 days was similar to that found in male animals (P = 0.25), although heart rate was slightly lower (female, 424 ± 14; male, 489 ± 25; P < 0.05). As was found in male animals, mean arterial pressure was significantly elevated by 20–25 mmHg in female Cx40-/- mice. This was not associated with changes in heart rate (Fig. 1). The same differences were found in old, female mice (468 ± 15 days).



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Fig. 1. Arterial pressure (top) and heart rate (bottom) in connexin 40 (Cx40)-deficient and wild-type mice. Pressure values were sampled at 1,000 Hz in conscious animals for a period of 1 h at 1 day after cannulating the carotid (male mice) or femoral artery (female mice). All values were summarized over the 1-h measuring period. Male animals were 114 ± 9, young females were 247 ± 13, and old females were 468 ± 15 days old. Mean arterial pressure was elevated in male and female Cx40-/- mice irrespective of the age of the animal. Heart rate was similar between genotypes; however, heart rates were higher in old vs. young mice. *P < 0.05 vs. Cx40+/+ mice; n = 8 animals of each genotype in each group.

 
Heart weights in Cx40-deficient mice.
To check whether the observed hypertension was associated with cardiac hypertrophy, heart weights were measured. In male animals of a similar age (121 ± 13 vs. 104 ± 9 days, P = 0.26), body weight of Cx40-/- mice was lower (25 ± 1 vs. 28 ± 1 g, P < 0.05) and heart weight was higher (195 ± 15 vs. 150 ± 6 mg, P < 0.05). Thus relative heart weight was significantly higher in Cx40-/- mice (7.8 ± 0.6 mg/kg body wt) compared with wild-type animals (5.3 ± 0.2 mg/kg body wt). Likewise, the relative heart weights of female Cx40-/- animals were significantly increased compared with Cx40+/+ mice. This was found in both young (at 247 ± 13 days of age: Cx40+/+, 5.3 ± 0.2; Cx40-/-, 6.1 ± 0.3 mg/kg body wt; P < 0.05) and old mice (age of 468 ± 15 days: Cx40+/+, 5.2 ± 0.4; Cx40-/-, 6.5 ± 0.4 mg/kg body wt; P < 0.05). These differences were also due to lower body weights as well as higher heart weights in Cx40-/- animals.

Effect of inhibition of NO synthase.
The systemic blockade of NO synthase by intravenous infusion of L-NA (100 mg/kg within 10 min) increased blood pressure in both genotypes significantly (Table 1). The increase in pressure was similar in both groups (Cx40+/+, 17 ± 4%; Cx40-/-, 12 ± 3%; P = 0.4, n = 6 each group) and was accompanied by a sharp decrease in heart rate (Cx40+/+, -34 ± 7%; Cx40-/-, -31 ± 4%; P = 0.7). Interestingly, mean arterial pressure was elevated in Cx40-/- animals before and after NO synthase inhibition (Table 1).


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Table 1. Effect of NO synthase inhibition on circulatory parameters in Cx40-deficient mice

 
Responses to endothelium-dependent and -independent vasodilators.
To assess a potential role of Cx40-mediated cell coupling on resistance vessel reactivity, responses to intra-arterial bolus injection of ACh or SNP were measured in Cx40-/- mice. In both genotypes, ACh induced a concentration-dependent decrease in mean arterial pressure. Pressure declined immediately after injection and reached a plateau within 15 s, which was stable for the next 45–55 s. Pressure gradually returned to control level, which was re-attained depending on the ACh concentration within 1–4 min after injection (Table 2). Minimum values during this period are depicted in Fig. 2. Heart rate remained unchanged at low ACh dosages and decreased by 79 ± 32 and 90 ± 37 beats/min with the highest amounts of ACh used (5 and 15 nmol, respectively). The effective ACh dosages and the duration of the responses were similar in both genotypes. Pressure decreases in Cx40-/- mice tended to be higher in amplitude, compared with controls, resulting in the similar minimal pressure levels observed (Fig. 2). In both genotypes, blockade of NO synthase with L-NA (100 mg/kg iv) reduced the duration of the ACh responses (Table 1) but not the maximal pressure drop (Fig. 2). Similarly, the injection of the NO donor, SNP, induced concentration-dependent decreases of arterial pressure in both genotypes. However, similar to the results with ACh, pressure drops observed upon SNP injection tended to be higher in Cx40-/- mice. Thus the same pressure level resulted after the highest dosage of SNP, despite a higher initial control level in Cx40-/- animals (Fig. 3). L-NA did not affect SNP-induced responses in Cx40+/+ or in Cx40-/- mice (data not shown).


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Table 2. Time to peak of ACh-induced pressure drop and its duration before and after L-NA

 


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Fig. 2. Changes of blood pressure upon ACh application in Cx40-deficient mice. ACh was injected as a bolus into the carotid artery before (left) and after inhibition of NO synthase (right) in young, male mice. The injection of ACh as a bolus induced a decrease of arterial pressure within 10 s, reaching a minimum between 20 and 70 s. The pressure then gradually returned to control level within 180 s. Data are minimal values observed (black symbols), and, for reference, values obtained immediately before bolus application are also plotted (gray symbols) for each injection and genotype separately. In both genotypes (open symbols, Cx40+/+; filled symbols, Cx40-/-), ACh induced concentration-dependent pressure drops, and the amount of ACh necessary to induce a significant change was similar in both types of mice. Note that at the highest concentration of ACh, arterial pressure was no longer different between the genotypes, indicating that there appears to be a minimal level that can be reached by ACh injection. Right: responses obtained after blockade of NO synthase by L-NA (100 mg/kg iv). However, maximal pressure drops remained unaffected in both genotypes by L-NA. *P < 0.05 vs. before ACh injection.

 


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Fig. 3. Effect of sodium nitroprusside (SNP) injection on blood pressure in Cx40-deficient mice. SNP was injected as a bolus into the carotid artery in young male mice. Data are minimal mean pressure values obtained upon bolus injection of SNP (black symbols). For reference, control pressure for each injection and genotype, as obtained immediately before bolus application, is also plotted (gray symbols). In both Cx40+/+ and Cx40-/- genotypes, SNP induced concentration-dependent pressure drops, and the minimum amount of SNP necessary to induce a significant drop was similar in both genotypes. As was found with ACh, pressure values of both genotypes converged with increasing concentrations of SNP. *P < 0.05 vs. before SNP injection.

 
Responses within the microcirculation.
In additional experiments, dilator responses were assessed in the microcirculation (n = 4 animals of each genotype). Resting tone of the observed arterioles was 66 ± 3% in Cx40+/+ (n = 48) and 63 ± 4% of the maximal diameter in Cx40-/- mice (n = 47). Dilations upon superfusion of ACh or SNP were similar in Cx40+/+ and Cx40-/- mice. Indomethacin and L-NA reduced dilations at low ACh concentrations but were without effect at 10 µmol/l in both genotypes (Fig. 4). However, although dilatory responses were comparable, some Cx40-/- arterioles exhibited an irregular vasomotion pattern that was never observed in wild-type arterioles. The observed vasomotion pattern was characterized by a constriction over a short distance, which spread to downstream locations similar to a propagating, propulsive wave. Eventually, these constrictions led to a complete, but temporary, flow stop [Figs. 5 and 6; the pictures are stills from a movie demonstrating this phenomenon, which may be downloaded from our server (http://wpl70n.physiol.med.uni-muenchen.de/cx40/cx40.avi) and is also available as Supplementary Material at the Physiological Genomics web site].2 When observed in an arteriole, these constrictions could be abrogated by the superfusion of ACh or SNP but returned upon removal of the dilator substance. In contrast to this irregular behavior, normal vasomotion is characterized by simultaneous diameter changes along the entire vessel length and can be observed in wild-type and Cx40-deficient mice (Fig. 6D).



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Fig. 4. Arteriolar dilations upon ACh and SNP administration in wild-type and Cx40-deficient mice. Arterioles were visualized by intravital microscopy in the cremaster muscle. The dilation upon ACh was attenuated in the presence of L-NA (30 µmol/l) and indomethacin (Indo, 3 µmol/l) at 1 µmol/l ACh, but not at 10 µmol/l. Responses to SNP remained unaffected. However, dilatory responses to ACh or SNP were similar in both Cx40+/+ and Cx40-/- genotypes, regardless of the presence of L-NA and indomethacin. *P < 0.05 vs. control responses; n = 48 arterioles in 4 animals in each genotype.

 


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Fig. 5. Irregular vasomotion in Cx40-deficient arterioles. The arteriole was visualized by intravital microscopy in the cremaster muscle; blood flow is from bottom to top. In Cx40-deficient mice, but never in wild-type or endothelial NO synthase-deficient mice, which are also hypertensive, an irregular vasomotion was observed. A constriction occurred spontaneously within an arteriole (arrow in B), which traveled to downstream sites (arrows in CE) leading to a transient flow stop (D). This constriction released spontaneously (F) and started again at an upstream site. A movie demonstrating this phenomenon can be downloaded from our server (http://wpl70n.physiol.med.uni-muenchen.de/cx40/cx40.avi) and is also available as Supplementary Material at the Physiological Genomics web site. The pictures shown are stills from this movie taken within an interval of 5 s (see time stamp). Calibration bar is 50 µm.

 


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Fig. 6. Temporal and spatial analysis of the irregular vasomotion in Cx40-deficient arterioles. The arteriole was visualized over a length of 196 µm and flow direction was from bottom to top (arrow in A). B: diameters at different positions along the vessel (as marked from a to g in A) over time. A localized constriction appears first at the bottom, upstream part (a) and travels quickly to downstream positions (b and c). Even further downstream, the constriction travels slower, and thus the arteriole is constricted at certain positions for longer periods (d to g). C: diameter of this vessel over one cycle (7 s) along the entire observed arteriolar length. Note that the diameter axis is reversed for easier visualization of constrictions, represented by peaks. Initially, the constriction travels quickly, but it slows down before it releases. D: for comparison, normal vasomotion in a different arteriole characterized by simultaneous diameter changes along the entire vessel length. This can be observed in wild-type and Cx40-deficient mice.

 
Effect of AT1-receptor blockade in Cx40-/- mice.
It has been reported that Cx40 is abundantly expressed in the juxtaglomerular apparatus in the kidney (14, 24), and Cx40-dependent cell coupling may be associated with the release of renin. To determine acute constrictor effects of angiotensin, the AT1-receptor blocker, candesartan, was applied systemically. At the lowest concentrations (0.01 and 0.1 mg/kg) candesartan decreased mean arterial pressure in Cx40+/+ mice (n = 7), but higher concentrations (1.0 and 10.0 mg/kg) had no additional effect. This indicates a complete blockade of the pressor effects of angiotensin II via AT1 receptors. In Cx40-/- animals, candesartan decreased arterial pressure to a similar degree (Fig. 7, n = 7), and the highest dosage (10 mg/kg) was without additional effect. However, pressure was still significantly elevated in Cx40-/- mice compared with controls (Fig. 7). Heart rate was not different at any concentration between genotypes, although slight decreases were observed in Cx40-/- mice upon injection of 0.1 and 1.0 mg/kg candesartan.



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Fig. 7. Effect of AT1-receptor blockade on hemodynamics in Cx40-/--deficient mice. Graphs show blood pressure (left) and heart rate (right) in response to additive concentrations of the AT1-receptor blocker, candesartan, applied systemically to conscious animals. In Cx40+/+ (open symbols) as well as in Cx40-/- mice (solid symbols) the blockade of AT1 receptors led to a significant dose-dependent decrease of blood pressure (left). However, at higher concentrations a further effect on blood pressure was no longer observed, indicating a complete blockade of the pressor effect of angiotensin II. Despite the fact that the angiotensin II-mediated pressor effects were completely abrogated, arterial pressure was still elevated in Cx40-/- mice compared with controls. Significant differences in heart rate between genotypes were not detected (right). *P < 0.05 vs. wild-type animals. #P < 0.05 vs. previous lower candesartan concentration applied; n = 7 animals in each group.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
This study demonstrates that the loss of Cx40 is associated with arterial hypertension in both sexes at different ages in conscious mice. The present observations extend our previous study, in which we found elevated pressure in anesthetized Cx40-deficient animals. An impaired action or release of endothelial NO or alterations in the efficacy of other endothelial dilators, often implicated in hypertension, were not responsible for Cx40-/--associated hypertension, as judged by pressure drops and arteriolar dilations upon ACh or SNP. Moreover, it seems that the hypertension is not related to alterations in the renin-angiotensin system or to increased sympathetic outflow. Thus, although it is still unknown how a defect in cell coupling due to the loss of Cx40 elevates arterial pressure, the data suggest that Cx40 serves an important function within the vascular wall and that coupling via Cx40 contributes to the control of peripheral vascular resistance. The latter assumption is highlighted by the finding of an irregular vasomotion leading eventually to complete arteriolar flow stop.

Hypertension in Cx40-deficient mice was of a similar magnitude in both sexes at different ages. In all groups studied, this was not accompanied by increased heart rate. This suggests that the hypertension was not caused by an activation of the sympathetic system. Although we did not measure cardiac output, we speculate that an increase in cardiac output is not the cause of the hypertension, because expression of Cx40 in the heart is mainly restricted to atrial myocytes and the conduction system (28), where heart rate was not increased. On the other hand, alterations within the vascular system have been shown previously in these mice (7, 18). Moreover, irregular vasomotion patterns were observed (Fig. 5, 6). Taken together, it is more likely that peripheral vascular resistance is affected by Cx40 deficiency. Young Cx40-deficient mice (~3 mo of age) were already hypertensive and remained so up to 15 mo age. Such a chronically elevated blood pressure should result in cardiac hypertrophy, which was indeed the case. In all groups studied, the relative heart weight was found to be higher in Cx40-deficient mice. In contrast to Cx40 deficiency, targeted endothelial deletion of Cx43 was without effect on arterial pressure (26) or was associated with hypotension and bradycardia (19). Why alterations of cellular coupling by either the ubiquitous deletion of Cx40 or endothelial deletion of Cx43 lead to such different effects is interesting, but remains unexplained.

Several mechanisms which may lead to elevated pressure were excluded to be involved in the hypertension associated with Cx40 deficiency. First, the elevation of blood pressure is not caused by impaired action or release of endothelial NO because the blockade of NO synthase did not offset the difference between genotypes. Moreover, the efficacy of NO as well as ACh in reducing pressure were similar in both wild-type and Cx40-/- mice, suggesting that the dilator potency of enhanced levels of NO is also not affected by the loss of Cx40. Recently, it has been proposed that dilations induced by ACh in mice involve a mechanism that is distinct from NO and prostaglandins (2). Also in the present study, systemic blockade of NO synthase did not affect the maximal amplitude, but only the duration of ACh-induced pressure drops, suggesting that other endothelial dilator mechanisms, namely endothelium-derived hyperpolarizing factor (EDHF), are important. Interestingly, arterioles in the cremaster dilated upon ACh after inhibition of NO synthase and cyclooxygenase in wild-type and Cx40-/- mice, and this NO- and prostaglandin-independent dilation is often attributed to EDHF. Reportedly, EDHF requires gap junctional communication to induce dilation (2). Our data show that Cx40-dependent cell coupling is not crucial for this dilation. This is in line with the recent observation showing that different peptides targeted to specific connexins attenuated responses attributed to EDHF only if all connexin blocking peptides were applied together (4). However, EDHF may not be a single entity in different vascular beds in mice, as others have reported that EDHF is a P-450 metabolite (15) or hydrogen peroxide (20), and both may act independently of gap junctional coupling.

Cx40 is strongly expressed within the kidney in the juxtaglomerular apparatus (14, 24). Therefore, cell coupling via Cx40 may play a role in renin release. To test whether a potentially enhanced release of renin is causing the observed hypertension, we blocked the vasoconstriction induced by angiotensin II (3). Systemic application of an AT1 receptor antagonist significantly reduced arterial pressure in both genotypes, indicating a continuous pressor effect of angiotensin II. A sufficient blockade of these receptors was achieved, as increasing the dosage of the blocker did not further reduce arterial pressure. However, Cx40-/- mice still exhibited a significantly higher blood pressure compared with controls. This suggests that acute pressor effects of angiotensin II are not responsible for the observed hypertension. As already mentioned, heart rate was not altered in Cx40-/- mice. This argues against an activation of the sympathetic system as a cause of the hypertension. The fact that inhibition of NO synthase led to a sharp decrease in heart rate verifies the intactness of the baroreflex and sympathetic regulation.

Having thus exhausted the most important mechanisms resulting in hypertension, we conclude that the elevated arterial pressure is related to a new mechanism involving gap junctional communication via Cx40. This could be caused by a reduction of overall junctional conductance, altered gating properties, or channel selectivity. The latter possibilities may result from a different composition of heteromeric channels in Cx40-deficient vessels. Alternatively, an overexpression of other connexins as shown to be the case for Cx37 (18) may be causal. However, the mechanism by which a defect in cell coupling causes an elevation of pressure is still unknown. The fact that Cx40-deficient mice displayed an irregular vasomotion, which led temporarily to a complete flow stop, suggests that Cx40-dependent cellular coupling, presumably within the endothelial cell layer, is involved in the regulation of vascular resistance. If these irregular patterns are also found in other vascular beds, then this could be a cause for enhanced peripheral vascular resistance.

In summary, we showed that the loss of cell coupling via Cx40 is associated with hypertension in conscious animals in both sexes. However, this does not alter the efficacy of the endothelial dilator NO or other dilators released upon ACh application. Additionally, the hypertension is unlikely to be due to an activation of the renin-angiotensin system or to an activation of the sympathetic nervous system. The data show that the specific functions of Cx40 cannot be fully compensated by another member of the connexin family expressed in vascular cells despite endothelial upregulation and redistribution of Cx37 in Cx40-deficient mice (18). We propose that Cx40-dependent cell coupling has an important role in the control of peripheral vascular resistance and that defects in cell coupling within the vascular tree should be regarded as a possible cause of hypertension in humans.


    ACKNOWLEDGMENTS
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 553, project B2, to U. Pohl) and the Friedrich-Baur Stiftung (to C. de Wit).


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: C. de Wit, Physiologisches Institut, Ludwig-Maximilians-Universität, Schillerstr. 44, 80336 Munich, Germany (E-mail: dewit{at}lmu.de).

10.1152/physiolgenomics.00169.2002.

1 This article was submitted for review in response to a Special Call for Papers on "Comparative Genomics." Back

2 The Supplementary Material (a movie file) for this article is available online at http://physiolgenomics.physiology.org/cgi/content/full/13/2/169/DC1. Back


    References
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 

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