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Cardiovascular Research 2000 48(3):393-401; doi:10.1016/S0008-6363(00)00193-0
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Copyright © 2000, European Society of Cardiology

The interrelationship between chloride ions and endothelium on {alpha}1-adrenoceptor-mediated contractions in aortic rings from Dahl normotensive and hypertensive rats

Reza Tabrizchi* and Jennifer A Duggan

Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland St. John's, St. John's NF, Canada

* Corresponding author. Tel.: +1-709-737-6864; fax: +1-709-737-7010 rtabrizc{at}morgan.ucs.mun.ca

Received 11 May 2000; accepted 7 July 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: The effects of chloride-free buffer in the absence or presence of the nitric oxide synthase inhibitor, N{omega}-nitro-L-arginine methyl ester (L-NAME), or chloride channel antagonist, indanyloxyacetic acid 94 (IAA-94) on {alpha}1-adrenoceptor mediated contractions were investigated in aortic rings from Dahl salt-resistant normotensive (SRN) and salt-sensitive hypertensive (SSH) rats on a 4% salt diet. Methods: Systolic and diastolic blood pressure were measured via intra-arterial catheters under halothane anesthesia. Subsequently, the aorta was removed and cirazoline-induced contractions were recorded in normal Krebs and chloride-free buffer using ring preparation. Guanosine 3',5'-cyclic monophosphate (cyclic GMP) content of aortic rings was also determined by scintillation proximity assay kits. Results: Systolic and diastolic blood pressure of SSH (180/130±1/1, mean±S.E.; n = 14) were significantly higher than those of SRN (101/76±1/1, mean±S.E.; n = 14) 7 weeks after they were placed on a salt diet. While the presence of L-NAME failed to have any impact on cirazoline-induced contractions in aortic rings from SSH rats, it significantly accentuated the effects of cirazoline in tissues from SRN rats. On the other hand, IAA-94 was able to inhibit cirazoline-mediated contractions in aortic rings from both SRN and SSH rats. The removal of chloride ions potentiated contractions produced by cirazoline in tissues from SRN but not SSH rats. Moreover, cirazoline-evoked responses in tissues from SRN were not further accentuated by the inclusion of L-NAME in chloride-free buffer. Cirazoline-mediated contractions in tissues from SSH rats were not influenced by absence of chloride ions, and the presence of L-NAME. It was also apparent that the inclusion of IAA-94 in absence of chloride ions did not prevent the potentiation of responses to cirazoline. Removal of chloride ions did not significantly decrease basal cyclic GMP levels in aortic rings from either strain. Conclusions: Basal release of nitric oxide seems to make a greater contribution in the suppression of cirazoline-evoked contractions in vessels from SRN as opposed to SSH rats. Chloride channels appear to contribute to cirazoline-evoked contractions in normal Krebs but not in chloride-free buffer.

KEYWORDS Arteries; Hypertension; Nitric oxide; Cl-channel; Vasoactive agents

This article is referred to in the Editorial by M.Y. Alexander (pages 365–366) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
There is both physiological and pharmacological evidence in support of the view that calcium-activated chloride currents play a pivotal role in influencing changes in membrane potential in vascular smooth muscle [1]. Evidently, agonist-induced stimulation of calcium-activated chloride currents via the release of intracellular calcium accounts for membrane depolarization in vascular smooth muscle [2,3]. It would be prudent to suggest that one cellular mechanism by which agonists produce depolarization in blood vessels is by stimulation of calcium-activated chloride conductance which leads to the opening of voltage-gated calcium channels [2,4,5]. Therefore, the opening of voltage-dependent calcium channels and the subsequent calcium influx is a key factor in the process involving contractions in blood vessels [6–8].

A number of studies have revealed that selective antagonism of calcium-activated chloride channels results in inhibition of {alpha}-adrenoceptor mediated contraction in functioning blood vessels [6,7]. Evidence from our laboratory also suggests that chloride ions play an important role in {alpha}1-adrenoceptor-mediated vasoconstriction and contraction [8,9]. However, of special interest is a study that purports to the existence of an important link between the functional role of endothelium and chloride ions in functional blood vessels [10]. Essentially, it has been demonstrated that nitric oxide plays a critical role in modulating the contribution of chloride ions on noradrenaline-induced contractions in blood vessels [10]. Therefore, based on this view, chloride ion handling and endothelial regulation of chloride ions may have a pathophysiological significance in conditions where endothelial cell dysfunction has been found.

Certainly, there is extensive evidence in the literature which supports the view that alterations in endothelial cell function, in part, account for changes in the functional behavior of blood vessels in the developed state of hypertension [11]. For example, endothelium-dependent relaxation has been reported to be depressed in blood vessels from Dahl salt-sensitive hypertensive rats [12,13]. Moreover, findings that the addition of L-arginine can induce vascular dilation in blood vessels of normotensive Dahl salt-sensitive but not hypertensive Dahl salt-sensitive rats would support the view that the L-arginine-nitric oxide pathways could be altered subsequent to the development of hypertension in this strain of animal [14]. In addition, it has been suggested that vascular hypercontractile responses to noradrenaline in blood vessels of Dahl salt-sensitive rats are, in part, a consequence of impaired endothelial nitric oxide production [15]. Collectively, current evidence in the literature seems to support the idea that in Dahl salt-sensitive hypertensive rats, alterations in the functional behavior of blood vessels, in part, can be attributed to changes in the contribution made by the endothelial cells during excitation–contraction coupling processes.

Based on the idea that nitric oxide has an impact on chloride regulation and functional responses in blood vessels, and that this process may become dysfunctional in hypertension, here, in the present investigation our objectives were to determine: (a) the impact of chloride ions on {alpha}1-adrenoceptor mediated contractions, (b) the influence of chloride ions and nitric oxide synthase inhibitor, N{omega}-nitro-L-arginine methyl ester (L-NAME), on {alpha}1-adrenoceptor-mediated contractions, (c) the interaction between chloride ions and the chloride channel antagonist, indanyloxyacetic acid 94 (IAA-94), on contractions, and (d) the impact of chloride ions on basal levels of guanosine 3',5'-cyclic monophosphate (cyclic GMP) in blood vessels from Dahl salt-resistant normotensive and salt-sensitive hypertensive rats on a salt diet for 7 weeks.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996).

2.1 Surgical procedures
Female RAPP Dahl rats (salt-resistant and salt-sensitive) were purchased from Harlan (Indianapolis, IN, USA) at 4–5 weeks of age. Animals were housed (two per cage) with 12 h light/dark cycles and given free access to normal food (Purina rat chow) and tap water ad libitum. At 6–7 weeks of age, animals were placed on a diet containing 4% salt.

Following 7 weeks on a 4% salt diet, each rat was anaesthetised with halothane (5% in 100% oxygen for induction and 1.25% in 100% oxygen for maintenance), and a catheter (polyethylene tubing I.D. 0.58 mm, O.D. 0.965 mm) was inserted into the left femoral artery for the measurement of blood pressure. The catheter was filled with heparinized saline (25 iuml–1 in 0.9% NaCl), and blood pressure and heart rate were measured for 30 min continuously. Arterial blood pressure was recorded with a pressure transducer (Gould Statham, USA; Model PD23B) connected to a Gould Universal amplifier and recorder (Gould, France, Model 8188-2202-00). Heart rate was calculated from the blood pressure tracing. Subsequently, each animal was sacrificed and the thoracic aorta, heart and kidneys were removed. The weight of the left and right kidneys, right ventricle and left ventricle+septum were recorded.

2.2 Tissue isolation
The thoracic aorta was removed from the animals and dissected free of connective tissue at room temperature in Krebs buffer of the following composition (in mM): NaCl, 120; KCl, 4.6; glucose, 11; MgCl2, 1.2; CaCl2, 2.5; KH2PO4, 1.2; NaHCO3, 25.3. All experiments were carried out either in normal Krebs or chloride-free buffer of the following composition (in mM): C2H2COONa 120, C2H2COOK 3.5, glucose 11, MgSO4 1.2, Ca(C6H11O7)2 2.5, KH2PO4 1.2, and NaHCO3 25. The pH of the buffers following saturation with a 95% O2–5% CO2 gas mixture was 7.4.

2.3 Experimental protocol
Aortic rings (~2 mm in length) were mounted in 20-ml organ baths at 37°C under a force of 29.4 mN (the baseline tension employed in these studies was determined in a series of preliminary studies involving length tension curves with a single concentration of cirazoline (1.0 µM), and using tissues from both normotensive and hypertensive rats), and gassed continuously with a mixture of 95% O2–5% CO2. The tissues were equilibrated for 60 min and isometric tension was measured using force displacement transducers (Model FT03, Grass Instruments, MA, USA) connected to a polygraph (Model 7PCPB, Grass Instruments, MA, USA). Tissues were initially contracted twice with a single concentration of cirazoline (1.0 µM) over a 120-min period after the equilibration. Following the addition of the single concentration of cirazoline, each tissue was washed with fresh normal Krebs solution, and allowed to equilibrated for 60 min (washed once at 30 min with regular Krebs). The first cumulative concentration–response curve to cirazoline (1 nM–3 µM) was constructed after the initial contact with a single concentration of cirazoline. The tissues were then washed with regular Krebs and allowed to equilibrate for 30 min. After this period, each tissue was either washed with regular Krebs or chloride-free buffer and a second concentration–response curve to cirazoline was constructed in the presence of twice distilled water (20 µl), L-NAME (10 µM), or IAA-94 (30 µM). Twice distilled water, L-NAME or IAA-94 was left in contact with each tissues for 25 min prior to and throughout the construction of the second concentration–response curve to cirazoline (1 nM–10 µM).

In two additional groups of experiments, tissues (from Dahl normotensive and hypertensive rats) were allowed to equilibrated for 60 min. Each tissue was then contracted twice with a single concentration of cirazoline (1.0 µM) over a 120-min period. Each time the tissues were washed with fresh normal Krebs solution and each tissue was allowed to equilibrate for 60 min (washed once at 30 min with regular Krebs). Subsequently, each tissue was exposed to high K+ (80 mM). The aortic rings were then washed with normal Krebs solution and allowed to equilibrate for 30 min. After this period, each tissue was washed again with normal Krebs. Ten min after this wash, IAA-94 (30 µM) was added to each bath and 20 min later, tissues were again exposed to high K+ solutions in the presence of IAA-94. In experiments involving high K+, the appropriate quantities of K+ replaced Na+.

2.4 Cyclonucleotide studies
Aortic rings (~4 mm in length) were incubated in 125 ml of normal Krebs solution for 90 min and gassed continuously with a 95% O2–5% CO2 gas mixture kept at 37°C. The buffer was replaced with fresh buffer every 30 min. Each tissue was then transferred into a scintillation vial containing 10 ml of regular Krebs solution and allowed to equilibrate for 30 min at 37°C while gassed continuously with 95% O2–5% CO2. The physiological salt solution was then replaced with 10 ml of either normal Krebs or chloride-free buffer and allowed to equilibrate for 30 min. Subsequently, each tissue was rapidly frozen in liquid nitrogen and stored at –80°C until it was assayed for cyclic nucleotides.

Cyclic nucleotides in aortic rings were measured by the technique described by Delpy and associates [16]. Briefly, frozen aortic rings were homogenized in 0.4 ml of cold trichloroacetic acid (6%) using an electric tissue homogenizer (Dyna-mix Model 43, Fisher Scientific, ON, Canada). The homogenate was centrifuged using an eppendorf centrifuge (Model 5415C, Brinkman Instruments, NY, USA) at 5000 g for approximately 1 min and the supernatant was decanted without disturbing the protein pellet. Subsequently, the supernatant was washed four times with six volumes of water-saturated diethyl ether to remove the trichloroacetic acid. The upper ether layer was discarded after each wash. Following the final extraction, any remaining ether was evaporated under air stream. Throughout these procedures, the samples were consistently stored at approximately 4°C in an ice-water bath. The aqueous phase was then evaporated to dryness with a Speed-vac system (Model SVC100H, Savant Instruments, London, UK) for approximately 1.5 h and the dried extract dissolved in a suitable volume of Tris–EDTA assay buffer prior to analysis. Cyclic GMP content was determined by scintillation proximity assay kits (Amersham, ON, Canada). Protein concentration in the pellet was measured using Bradford's [17] method (Bio-Rad Life Research Product, ON, Canada).

2.5 Data and statistical analysis
Results from the contraction studies were calculated as a percentage of the maximum contraction induced by the agonist following the construction of the first concentration–response curve to cirazoline. Percent maximum, Hill coefficient (nH) and EC50 values were calculated for individual curves using a program executed on an IBM compatible microcomputer [18]. These parameters were determined by fitting the percent contractile response at increasing concentrations of agonist ([A]) by non-linear least squares to the relation Y = a+bX, where Y = response and X = [A]n/([A]n+[EC50]n) with n(nH) fixed at ‘floating’ integral values to obtain the best fit. Cyclic GMP was calculated as pmoles of cyclic nucleotide per mg of protein. Analysis of variance was used for comparisons of percent maximum, nH, pD2, as well as, cyclic nucleotide levels. For multiple comparisons, Duncan's multiple range test was used to compare between means. Un-paired student's t-test was used for comparison between other values (body weight, blood pressure, heart rate, ratio of right ventricle to left ventricle+septum and kidney weights). For all cases, a probability of error of less than 0.05 was selected as the criterion for statistical significance.

2.6 Chemicals
Stock solutions of all drugs, except indanyloxyacetic acid 94 (R(+)-IAA-94), were made in twice distilled water. A stock solution of 0.1 M IAA-94 was made in 0.1 M NaOH and subsequently diluted in twice distilled water. Cirazoline, L-NAME, and IAA-94 were purchased from Research Biochemical International (MA, USA).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
Both the systolic and diastolic blood pressures of Dahl salt-sensitive rats were significantly higher than those of Dahl salt-resistant rats 7 weeks after they were placed on a 4% salt diet. While the heart rate of Dahl salt-sensitive rats was slightly higher than those of salt-resistant rats on salt diet, this was found to be not significant (Table 1). However, the ratio of right ventricle to left ventricle plus septum was significantly smaller in Dahl salt-sensitive rats when compared to salt-resistant rats. Moreover, the kidney weight corrected for body weight was found to be greater in Dahl salt-sensitive than salt-resistant rats on salt diet (Table 1). It was observed that the Dahl salt-sensitive rats had a slightly, but significantly, higher body weight than Dahl salt-resistant rats 7 weeks after they were placed on the salt diet (Table 1).


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  		Table 1 Systolic and diastolic blood pressure (BP), heart rate (HR), ratio of right to left ventricle+septum (RV/LV+S), right kidney (RK) and left kidney (LK) weight and body weight (BW) of Dahl salt-resistant (SR) and salt-sensitive (SS) rats after 7 weeks on a 4% salt dieta

 
3.1 Effects of L-NAME and IAA-94 on cirazoline-induced contractions in normal Krebs solution
There were no significant differences between the control pD2 and nH values of the concentration–response curves to cirazoline in aortic rings among the various groups of animals within each strain (i.e. salt-resistant and salt-sensitive). Mechanical activity (contractions) produced by cirazoline in aortic rings obtained from salt-resistant rats were accentuated in the presence of L-NAME (10 µM), and inhibited by the presence of IAA-94 (30 µM) in normal Krebs solution (Fig. 1A and B). In experiments conducted using aortic rings from salt-resistant rats, inclusion of L-NAME in the Krebs solution resulted in a significant increase in the pD2 and an increase in the nH and maximal response of the concentration–response curve to cirazoline when compared to respective time-controlled experiments (Table 2). Experiments conducted in the presence of IAA-94 resulted in significant reductions in the maximal response without any significant effects on either the pD2 or nH of the concentration response curve to cirazoline in comparison to respective time-controlled experiments (Table 2).


Figure 1
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  		Fig. 1 Concentration–response curves to cirazoline in normal Krebs (A) in the absence (closed circles) or presence (opened circles) of L-NAME (10 µM), (B) in the absence (closed circles) or presence (opened circles) of IAA-94 (30 µM) in aortic rings from Dahl salt-resistant normotensive rats. Each point represents the mean of six experiments±S.E.

 

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  		Table 2 pD2, Hill coefficient (nH) and percent maximum response values obtained from individual concentration response curves in aortic ring preparations from Dahl salt-resistant rats on a 4% salt diet for 7 weeksa

 
We observed that, in experiments where aortic rings from salt-sensitive rats were used, cirazoline-induced contractions were not substantially affected by the presence of L-NAME when compared to respective time-controlled experiments (Fig. 2A; Table 3). This is clearly in contrast to our observations with the effects of L-NAME on cirazoline-induced contractions in aortic rings from salt-resistant rats (Fig. 1A; Table 2).


Figure 2
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  		Fig. 2 Concentration–response curves to cirazoline in normal Krebs (A) in the absence (closed circles) or presence (opened circles) of L-NAME (10 µM), (B) in the absence (closed circles) or presence (opened circles) of IAA-94 (30 µM) in aortic rings from Dahl salt-sensitive hypertensive rats. Each point represents the mean of six experiments±S.E.

 

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  		Table 3 pD2, Hill coefficient (nH) and percent maximum response values obtained from individual concentration–response curves in aortic ring preparations from Dahl salt-sensitive rats on a 4% salt diet for 7 weeksa

 
However, IAA-94 did attenuate mechanical responses to cirazoline in aortic rings from salt-sensitive rats (Fig. 2B). The presence of IAA-94 resulted in significant reduction in the pD2 and a significant decrease in the maximal response of the concentration–response curve to cirazoline without altering the nH (Table 3). The presence of IAA-94 (30 µM) did not significantly affect high K+ (80 mM) induced contractions in aortic rings from salt-resistant (–7%±3% n = 6) and salt-sensitive (–8%±3%; n = 6) rats.

3.2 Effects of L-NAME and IAA-94 on cirazoline-induced contractions in chloride-free buffer
The replacement of chloride ions with propionate ions potentiated mechanical contractions induced by cirazoline in aortic rings obtained from salt-resistant but not salt-sensitive rats (Fig. 3A and B). It was apparent that removal of chloride ions resulted in a significant increase in the pD2 and a significant increase in the maximal response to cirazoline when compared to respective time-controlled experiments in aortic rings from salt-resistant rats (Table 2). In contrast, in chloride-free buffer, cirazoline-induced responses were attenuated in aortic rings from salt-sensitive rats. The resultant effect was a significant reduction in the pD2 value when compared to respective time-controlled experiments, without any significant change to the maximal response or nH (Table 3).


Figure 3
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  		Fig. 3 Concentration–response curves to cirazoline (A) in normal Krebs (closed circles) or chloride-free buffer (opened circles) in aortic rings from Dahl salt-resistant normotensive rats, (B) in normal Krebs (closed circles) or chloride-free buffer (opened circles) in aortic rings from Dahl salt-sensitive hypertensive rats. Each point represents the mean of six experiments±S.E.

 
In experiments conducted using aortic rings from salt-resistant rats, inclusion of L-NAME in chloride-free buffer did not produce a significant additive effect on cirazoline-induced contractions when compared to either the effects of cirazoline in the presence of L-NAME in normal Krebs or under the condition where chloride ions were absent from the buffer (Table 2; Fig. 2A, 3A and 4AGoGo). Furthermore, the inclusion of L-NAME in chloride-free buffer did not have any substantive impact on cirazoline-mediated contractions in aortic rings from salt-sensitive rats when compared to effects of cirazoline in chloride-free buffer (Table 3; Fig. 3B and 5AGo).


Figure 4
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  		Fig. 4 Concentration–response curves to cirazoline (A) in normal Krebs in the absence (closed circles) or chloride-free buffer in the presence (opened circles) of L-NAME (10 µM), (B) in normal Krebs in the absence (closed circles) or chloride-free buffer in the presence (opened circles) of IAA-94 (30 µM) in aortic rings from Dahl salt-resistant normotensive rats. Each point represents the mean of six experiments±S.E.

 

Figure 5
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  		Fig. 5 Concentration–response curves to cirazoline (A) in normal Krebs in the absence (closed circles) or in chloride-free buffer in the presence (opened circles) of L-NAME (10 µM), (B) in normal Krebs in the absence (closed circles) or in chloride-free buffer in the presence (opened circles) of IAA-94 (30 µM) in aortic rings from Dahl salt-sensitive hypertensive rats. Each point represents the mean of six experiments±S.E.

 
The presence of IAA-94 in chloride-free buffer did not prevent the potentiation of responses to cirazoline in aortic rings from salt-resistant rats (Fig. 4B; Table 2). Moreover, the inclusion of IAA-94 in chloride-free buffer did not produce substantive additive inhibition of cirazoline-induced contractions in tissues from salt-sensitive rats when compared to the respective effects of cirazoline in the presence of IAA-94 in normal Krebs (Fig. 5B; Table 3).

3.3 Effects of chloride-free buffer on basal cyclic GMP levels
It is apparent that there are significant differences between basal cyclic GMP levels in tissues from Dahl salt-resistant in comparison to salt-sensitive rats (Table 4). While removal of chloride ions did slightly decrease basal cyclic GMP levels in aortic rings from both strains of animals, this was not found to be significant (Table 4).


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  		Table 4 Basal cyclic GMP levels (pmol/mg of protein) in aortic rings from Dahl salt-resistant and salt-sensitive rat on 4% salt diet for 7 weeks either in normal Krebs solution or chloride-free buffera

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The fact that the replacement of chloride ions with propionate ions in the physiological salt solution resulted in accentuation of cirazoline-mediated contractions in aortic rings from salt-resistant normotensive rats underlines the presence of an inherited inhibitory process that appears to be in place continuously in this conduit vessel. It has been recognized for some time that factor(s) released from endothelial cells tonically influences {alpha}-adrenoceptor mediated contractions in isolated blood vessels [19–21]. Previous observations had indicated that the removal of endothelium in blood vessels resulted in an increase in potency and/or efficacy of {alpha}-adrenoceptor agonists [18,20,22]. Moreover, in the presence of the nitric oxide inhibitor, L-NAME, noradrenaline-evoked contractions were found to be potentiated in rat isolated aortic rings [23].

Our current findings are consistent with previous reports where it was demonstrated that removal of chloride ions potentiated noradrenaline-evoked contractions in aortic rings from normotensive rats [10,24]. In fact, Lamb and Barna [10] had implied that the tonic release of nitric oxide, in part, controlled noradrenaline-induced activation of chloride currents. We find that while the presence of the nitric oxide synthase inhibitor, L-NAME, potentiated cirazoline-mediated contractions in aortic rings from salt-resistant normotensive rats, the combination of chloride-free buffer and L-NAME did not result in substantive additive effects. Moreover, based on our present observation, chloride-free buffer also did not significantly reduce basal levels of cyclic GMP. It is evident that the impact of the removal of chloride ions on cirazoline-induced mechanical activity was independent of basal cyclic GMP levels. However, while our findings indicate that an increase in responsiveness to cirazoline in chloride-free buffer was unlikely to be the result of reduced basal levels of cyclic GMP, they do not rule out the possibility that the basal release of nitric oxide may have been reduced in chloride-free buffer. Therefore, the idea that tonic release of nitric oxide may influence vascular contractions independent of cyclic GMP production needs to be considered. Nitric oxide has been found to produce relaxation in smooth muscle, in part, as a result of direct activation of calcium-activated potassium channels [25]. Certainly, electrophysiological evidence using whole-cell and single channel currents in single myocytes from guinea-pig proximal colon have also revealed that nitric oxide can directly activate calcium-activated potassium channels [26]. In addition, nitric oxide was found to directly induce activation of large conductance calcium-dependent potassium channels in mesenteric artery smooth muscle cells [27]. Taken together, the evidence in the literature supports the idea that nitric oxide affects smooth muscle function by direct actions on ion channels. Thus, the lack of additive effects by L-NAME in chloride-free buffer may point to an interaction between chloride ions and nitric oxide which is independent of processes associated with basal cyclic GMP production.

In the present investigation, we also found that the removal of chloride ions did not potentiate the responses to cirazoline in aortic rings from salt-sensitive hypertensive rats. It is evident that replacement of chloride ions with propionate resulted in inhibition of responses produced by cirazoline. In addition, L-NAME was not able to accentuate contractions induced by cirazoline in salt-sensitive hypertensive rats in normal Krebs. It is evident from the present investigation that blood vessels from salt-resistant normotensive rats had higher basal levels of cyclic GMP when compared to salt-sensitive hypertensive rats. However, it has been demonstrated that L-NAME is equally capable of potentiating cirazoline-mediated vasoconstriction in isolated perfused mesenteric blood vessels from normotensive and spontaneously hypertensive rats [28].

It is apparent that differences exist between the behaviors of blood vessels from salt-resistant normotensive and salt-sensitive hypertensive rats with respect to the impact that chloride ions have on mechanical activity. The paradoxical effects of chloride-free buffer on contractions produced by cirazoline in blood vessels from salt-resistant normotensive versus salt-sensitive hypertensive rats support a close link between chloride handling and endothelial cell function in these blood vessels. However, evidence from our laboratory had shown that removal of chloride ions from buffer results in attenuation of cirazoline-mediated vasoconstriction in perfused mesenteric blood vessels from two-kidney one-clip hypertensive rats as well as normotensive rats [9]. This may point to differences that exist between chloride handling by blood vessels in different regions of the body. These paradoxical observations with chloride-free buffer and L-NAME on cirazoline-mediated vasoconstriction in rat isolated mesenteric blood vessels from normotensive animals argue against a uniform link between chloride ions and tonic release of nitric oxide in blood vessels.

The putative chloride channel antagonist, IAA-94, inhibited cirazoline-evoked contractions in blood vessels from salt-resistant normotensive rats and salt-sensitive-hypertensive rats in regular Krebs solution. It is evident that, in the absence of chloride ions, the inhibitory effects of IAA-94 in blood vessels from salt-sensitive hypertensive rats were reduced. Moreover, IAA-94 was not able to impair the potentiating actions of chloride-free buffer on cirazoline-mediated contractions in blood vessels from salt-resistant normotensive rats. Our observation with IAA-94 in normal Krebs is consistent with the view that chloride channel antagonists are capable of inhibiting calcium-activated chloride currents thus impairing depolarization and the subsequent opening of voltage-gated calcium channels [1]. Evidently, IAA-94 is a very selective blocker of chloride channels in the epithelial cells [29]. Moreover, IAA-94 (same concentration as the present study) has been reported to be capable of inhibiting endothelin-evoked depolarization in cultured vascular smooth muscle cells [30]. In addition, endothelin-evoked vasoconstriction has also been found to be impaired by IAA-94 [30]. Carmines [31] has also reported that the vasoconstriction actions of angiotensin II on renal afferent arterioles were attenuated by IAA-94 while potassium-evoked responses were not affected. However, like other putative chloride channel antagonists, such as ethacrynic acid and anthracene-9-carboxylic acid, IAA-94 has been noted to activate potassium channels [32]. Thus, it can be argued that this may have been one mechanism by which IAA-94, in part, inhibited cirazoline-mediated contractions. This, perhaps, is unlikely to be the mechanism by which IAA-94 inhibited cirazoline-mediated contractions. It is evident from our present findings that removal of chloride ions was able to nullify the inhibitory effects of IAA-94 in aortic rings from salt-sensitive hypertensive rats. This would not have occurred if IAA-94 was primarily inhibiting cirazoline-mediated contractions by opening potassium channels. Moreover, the potentiation of cirazoline-mediated responses in chloride-free buffer was not impaired by the simultaneous presence of IAA-94 in aortic rings from salt-resistant normotensive rats. This may also point to the fact that the inhibitory effects of IAA-94 cannot be primarily attributed to its properties as a potassium channel opener. In addition, in the present investigation, IAA-94 was shown not to inhibit high K+ induced contractions. This would indicate that IAA-94 did not directly inhibit voltage-gated calcium channels. Collectively, our present findings support the view that the inhibitory effects of IAA-94 was most likely attributed to inhibition of calcium-activated chloride channels.

Time for primary review 22 days.


   Acknowledgements
 
This work was supported by a grant from Natural Sciences and Engineering Research Council of Canada. We would like to thank Ms. Deanne Ryan for her excellent technical assistance.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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