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Am J Physiol Heart Circ Physiol 281: H2233-H2240, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 5, H2233-H2240, November 2001

Ca2+ influx mediates enhanced alpha 2-adrenergic contraction in aortas from rats treated with NOS inhibitor

Harshini Mukundan and Nancy L. Kanagy

Vascular Physiology Group, Department of Cell Biology and Physiology, Health Sciences Center, University of New Mexico, Albuquerque, New Mexico 87131-5218


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previously, we reported that aortic segments from rats made hypertensive with the nitric oxide synthase inhibitor Nomega -nitro-L-arginine (L-NNA) exhibit enhanced contractile sensitivity to both alpha 2-adrenergic receptor (alpha 2-AR) stimulation and to KCl-induced depolarization. We hypothesized that increased contractile responses to these agents was due to a change in the common effector L-type voltage-dependent calcium channel (VDCC). In aortic segments from control and L-NNA-treated rats, contraction to the alpha 2-AR agonist UK-14304 stimulated Ca2+ influx but released intracellular Ca2+ only in control arteries. UK-14304-induced contraction was blocked by the VDCC antagonist nifedipine in both control and L-NNA aortas but contraction of aortas from L-NNA-treated rats was blocked by lower concentrations. Calcium imaging studies in fura 2-loaded freshly isolated aortic vascular smooth muscle cells also demonstrated UK-14304-stimulated Ca2+ influx sensitive to nifedipine only in cells from L-NNA-treated rats. We conclude that alpha 2-AR contraction in the rat aorta is mediated primarily by Ca2+ influx and that L-NNA-induced hypertension increases the dependence of this contraction on VDCCs.

Nomega -nitro-L-arginine; alpha 2-adrenergic receptor; UK-14304; vascular smooth muscle cells; L-type voltage-dependent calcium channels; nifedipine; hypertension


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELIUM-DERIVED nitric oxide (NO) plays an important role in maintaining blood pressure. This is evidenced by the observation that mice deficient in endothelial NO synthase (NOS) exhibit hypertension. Clinical studies (reviewed in Refs. 4 and 6) have also demonstrated that a majority of human cases of hypertension are associated with impaired NO-mediated vasodilation and that this reduced endothelial NO production in humans appears to contribute to the pathology of hypertension (reviewed in Refs. 21 and 25). The relevance of the NOS inhibition model of hypertension in addressing these pathological changes has been established in many animal studies demonstrating that regardless of whether the loss of NO is the cause (reviewed in Ref. 31) or consequence of hypertension, it contributes to the vascular pathology of the disease.

We have previously reported (10) that rats made hypertensive with a NOS inhibitor (NOS-I) have an enhanced vascular contraction to alpha 2-adrenoceptor (alpha 2-AR) agonists. However, there is no change in the sensitivity to alpha 1-adrenergic receptor (alpha 1-AR) agonists. The mechanism for the vascular change leading to enhanced alpha 2-AR contraction is unclear. Furthermore, the contractile sensitivity to KCl is also enhanced in arteries from these animals (10), suggesting that augmented alpha 2-AR contractility may be mediated by a change in activation of voltage-dependent calcium channels (VDCCs), a common effector of KCl and alpha 2-AR contraction.

alpha -ARs are pharmacologically characterized as one of two subtypes, alpha 1 and alpha 2 (24). alpha 2-ARs are localized both in presynaptic nerve endings, where they mediate feedback inhibition of norepinephrine (NE) release, and at postsynaptic sites, where they mediate multiple effects including vasoconstriction (9).

A rise in myoplasmic free Ca2+ triggers alpha 2-AR-mediated constriction of vascular smooth muscle cells (VSMCs) (8). This increase may be mediated either by release of Ca2+ from the sarcoplasmic reticulum or by increased influx of Ca2+ through membrane channels. Calcium channels on VSMCs are predominantly voltage dependent and play an important role in both myogenic and agonist-stimulated tone (16). Two subtypes of VDCCs, L-type and T-type, have been identified on VSMCs (23). However, influx through L-type VDCCs appears to be the major pathway for Ca2+ entry into VSMCs and in the control of vascular tone (16, 26).

The relative contributions of Ca2+ release and influx to alpha 2-AR contraction are not precisely known. Several investigators have shown that alpha 2-AR contraction depends on extracellular Ca2+ to a greater extent than alpha 1-AR-mediated contraction (12, 26). Xiao and Pang (29) reported that pressor responses to the alpha 2-AR agonist UK-14304 in pithed rats were inhibited by diltiazem but that this VDCC blocker did not affect phenylephrine (PE)-mediated pressor responses. These observations suggest that, in vivo and in vitro, alpha 2-AR stimulates VDCC-mediated Ca2+-influx to a greater extent than alpha 1-AR.

In contrast, Morgan (18) has shown that stimulation of alpha 1-AR but not alpha 2-AR caused membrane depolarization in feline submucosal arterioles, suggesting that alpha 2-ARs do not activate VDCCs in this vascular bed. In addition, Motulsky et al. (19) demonstrated that verapamil blocked alpha 1-AR and alpha 2-AR contraction equally in multiple tissues.

Although these studies suggest that Ca2+ influx is important to alpha 2-AR contraction, the relative contributions of intracellular and extracellular stores are not known. Also, it is not clear whether there are differences in Ca2+-mobilization sources for alpha 1- and alpha 2-ARs. Finally, it has not been determined whether the increased vascular responsiveness to alpha 2-AR stimulation in arteries from rats made hypertensive by a NOS-I is mediated by altered contribution of one or both of these sources.

The main purpose of this study, therefore, was to define the relative contribution of intracellular and extracellular Ca2+ to alpha 2-AR contraction and to investigate the alteration in Ca2+ sources during NOS-I hypertension. We hypothesized that enhanced alpha 2- AR contraction in arteries from Nomega -nitro-L-arginine (L-NNA)-treated rats is mediated by increased Ca2+ influx through L-type VDCCs and that these channels are the primary source of activator Ca2+ for alpha 2-AR-mediated contraction.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of New Mexico. Male Sprague-Dawley rats (250-300 g) were used for all experiments. For 14 days, L-NNA-treated rats drank water containing L-NNA (0.5 g/l), and control rats drank tap water. Blood pressure was monitored using the indirect tail-cuff method (plethysmographic detection; IITC, Woodland Hills, CA). Pentobarbital sodium (50 mg/kg) was used to anesthetize animals on day 15. The animals were exsanguinated and the thoracic aorta was removed and placed in ice-cold physiological saline solution (PSS) consisting of (in mol/l) 1.3 × 10-1 NaCl, 4.7 × 10-3 KCl, 1.18 × 10-3 H2PO4, 1.17 × 10-3 MgSO4 · H2O, 14.9 × 10-3 NaHCO3, 5.5 × 10-3 dextrose, 0.026 × 10-3 EDTA, and 1.6 × 10-3 CaCl2.

Contractile Studies

Thoracic aorta segments from L-NNA-treated and control animals were used for all experiments. The aorta was cleaned of superficial fat and cut in 4-mm segments. The endothelium was removed by gently rubbing the lumen of the segment with a closed pair of fine-tipped forceps. The tissue was suspended in a bath of PSS warmed to 37°C and bubbled with 95% O2-5% CO2 using metal hooks attached to Grass FT03 force transducers connected to a Gould chart recorder. The segment was stretched to 2,000 mg of passive tension to facilitate maximal detection of active tension and allowed to equilibrate 60 min. During the last 30 min, indomethacin (10-6 mol/l) was added to the bath to inhibit the formation of cyclooxygenase metabolites. After the initial equilibration, viability of the tissue was confirmed by adding PE (10-7 mol/l). The removal of endothelium was confirmed by the absence of relaxation to ACh (10-6 mol/l) in contracted segments. The tissue was then washed for 60 min with PSS to remove PE and ACh before the start of the following experimental protocols.

Calcium influx and release studies. The relative contributions of Ca2+ influx and release from intracellular stores to alpha 1- and alpha 2-AR contraction were assessed using a modification of a procedure previously described (5, 7, 13). After initial PE contraction, tissues were washed repeatedly with PSS containing Ca2+ until the contraction returned to baseline (60 min). The aortic segments were subsequently washed with Ca2+-free PSS containing EGTA for 2 min to remove extracellular Ca2+ while keeping the intracellular stores intact.

Tissue was then challenged with either the alpha 1-AR agonist PE (10-6 mol/l) or the alpha 2-AR agonist UK-14304 (10-6 mol/l) to assess the contribution of intracellular stores alone to the contractile response [sarcoplasmic reticulum (SR) response]. Segments were then washed with Ca2+-free PSS to prevent reloading of intracellular stores. Ca2+-containing PSS was then added for 1 min and the tissue was again challenged with UK-14304 or PE (10-6 mol/l) to evaluate the response in the presence of extracellular Ca2+. After a final 60-min wash with PSS containing Ca2+, tissues were stimulated with the same agonist to assess the response in the presence of both intracellular and extracellular Ca2+ (see Fig. 2A).

Parallel sets of rings were stimulated with the same agonists but always in the presence of Ca2+ to evaluate the consistency of contraction to repeated agonist exposure.

In a separate set of experiments, the status of intracellular Ca2+ stores after the first UK-14304 or PE stimulation was assessed by adding NE (10-6 mol/l) to the bath to release agonist-sensitive intracellular stores. This was done to assure equivalent emptying of agonist-activated stores in both the PE- and UK-14304-treated tissues.

A third set of experiments was performed to evaluate the effect of completely emptying intracellular stores before assessing the response in the presence of extracellular Ca2+ alone. In these experiments, aortic segments were stimulated with UK-14304 or PE (10-6 mol/l) in the presence of both intracellular and extracellular Ca2+ to establish a baseline response. The segments were then stimulated with NE (10-6 mol/l) in Ca2+-free PSS containing EGTA (10-3 mol/l) to deplete agonist-sensitive intracellular stores. This was followed by 30 min of incubation with thapsigargin (10-6 mol/l) (11) to inhibit sarcoplasmic Ca2+-ATPase and prevent intracellular stores from refilling. After incubation, segments were stimulated with UK-14304 or PE (10-6 mol/l) in Ca2+-free PSS containing EGTA (10-3 mol/l). CaCl2 (1.6 × 10-3 mol/l) was then added to the bath in the continued presence of the agonist to stimulate contraction mediated entirely by Ca2+ influx.

BAY K 8644 concentration-response curves. Cumulative concentration-response curves were generated with the L-type Ca2+ channel agonist BAY K 8644 (Sigma, 10-9-10-6.5 mol/l). After maximal stimulation with BAY K 8644, tissues were washed for 60 min and stimulated with PE (10-6 mol/l) to assess viability. Only responses from rings that responded to PE were used.

Effect of nifedipine on alpha 2-AR contraction. Multiple concentration-response curves to UK-14304 (10-9-10-5.5 mol/l) in the presence of increasing concentrations of nifedipine (10-7-10-5 mol/l) were used to evaluate the role of L-type calcium channels in alpha 2-AR contraction. Simultaneous UK-14304-response curves in the absence of nifedipine were generated to assess attenuation of the UK-14304 response over time. Responses to vehicles (DMSO for UK-14304; ethanol for nifedipine and BAY K 8644) were also determined on separate vessels in parallel experiments.

Calcium-Imaging Studies

Isolation of rat thoracic aorta VSMCs. Cells were isolated from the thoracic aortae of control and L-NNA-treated rats under sterile conditions using presterilized solutions and media by a modification of the collagenase dissociation method of Khalil and Morgan (14). Thoracic aortas were cleaned, adventitia was removed, and the remaining medial layers were cut into 1 × 1-mm segments and transferred to a dissociation solution consisting of (mol/l) 1 NaCl, 10-1 KCl, 10-1 MgCl2, 1.0 HEPES, 10-1 CaCl2, and 10-2 glucose and 0.5 mg/ml BSA (pH 7.35). After a 10-min incubation period at room temperature, the solution was removed and an equivalent volume of dissociation solution containing 0.5 mg/ml dithiothreitol and 1.5 mg/ml papain was added. Tissue was incubated an additional 20 min at 37°C in this solution. The solution was then changed to a dissociation solution containing 1.9 mg/ml type II collagenase, 1 mg/ml pancreatic elastase, and 1 mg/ml trypsin inhibitor. The tissue was incubated in this solution at 37°C until digestion was complete (15-30 min). After digestion, cells were centrifuged at 900 g for 10 min at 4°C. Cells were suspended in 2 ml of VSMC media consisting of 10% fetal bovine serum, 30 µg fungizone, 1% penicillin-streptomycin, 0.5% L-glutamine in DMEM (all ingredients from GIBCO Life Technologies), warmed to 37°C, and then plated on 25-mm glass coverslips.

Calcium imaging. All experiments were conducted on VSMCs 48-72 h after isolation and plating. Cells were incubated with fura 2-AM (2 × 10-6 mol/l) for 40 min at room temperature in the dark. Coverslips were then inserted into an open microincubator (PDMI-2, Medical Systems) attached to the stage of an inverted microscope (Diaphot 300, Nikon; Tokyo, Japan). The microincubator was maintained at 37°C by means of a bipolar temperature controller (TC-202, Medical Systems). Images were collected using a digital charge-coupled device camera (SenSys 1400, Photometrics; Tuscon, AZ) and processed with Metafluor imaging software (Universal Imaging). Data are expressed as a ratio of emitted fluorescence at 510 nm in cells excited at 340 and 380 nm. Responses to UK-14304 (10-7 mol/l), nifedipine (10-7 mol/l), and ionomycin (10-6 mol/l) or their respective vehicles (DMSO, ethanol) were analyzed by directly adding the agonist/antagonists to the bath. Changes in the 340/380 nm emission ratios were recorded. For the UK-14304 stimulations, the maximum increase in the 340/380 nm ratio was collected during the plateau phase of the excitation response.

Data analysis and statistics. Data are reported as means ± SE. Nifedipine inhibition of UK-14304 contraction was analyzed by one-way ANOVA followed by the Student-Newman-Keuls post hoc test. For determining concentration effects within each group, one-way repeated-measures ANOVA with Tukey's post hoc test was used. Calcium imaging data was also evaluated by one-way ANOVA. Two-way ANOVA was used when applicable to compare between groups and treatments. The n value is the number of animals. A P value <=  0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

alpha 2-Adrenergic Contraction is Augmented in Aortas From L-NNA-Treated Rats

Several investigators have analyzed contractile responsiveness of tissue to adrenergic agonists during hypertension (10, 15). We confirmed our previous observation that aortic segments from L-NNA-treated rats exhibit a greater sensitivity and responsiveness to UK-14304 compared with controls. A greater concentration of UK-14304 was required to contract the control tissue (Fig. 1). We previously demonstrated that the sensitivity and responsiveness to the alpha 1-AR agonist PE is not altered in this preparation (10).


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  		Fig. 1.   Concentration-dependent contraction of aortic segments from Nomega -nitro-L-arginine (L-NNA) and control rats by UK-14304 (n = 12). Segments from L-NNA-treated rats have increased contractile sensitivity to UK-14304. Results are means ± SE. *Significant difference from control. PE, phenylephrine.

alpha 2-Adrenergic Contraction is Dependent on Extracellular Ca2+

To determine the relative contribution of extracellular and intracellular Ca2+ to UK-14304 contraction, aortic segments were stimulated with either UK-14304 or PE in the presence or absence of extracellular Ca2+. Stimulation with UK-14304 (10-6 mol/l) did not cause contraction in the absence of extracellular Ca2+ in segments from L-NNA-treated animals but did cause a small contraction in segments from control rats. Subsequent stimulation in the presence of extracellular Ca2+ elicited a large contractile response in both groups (Fig. 2). Therefore, the dependence of UK-14304 contraction on extracellular Ca2+ was greater in arteries from L-NNA-treated rats than in arteries from control rats. PE (10-6 mol/l) contracted segments in both the presence and absence of extracellular Ca2+. Additionally, aortas were stimulated with NE (10-6 mol/l) after the initial UK-14304 stimulation (in the absence of extracellular Ca2+) to deplete agonist-sensitive intracellular Ca2+ stores. This did not alter the magnitude of the subsequent response to UK-14304 in the presence of extracellular Ca2+ in tissues from the L-NNA-treated rats, suggesting that this response was totally mediated by Ca2+ influx. However, depletion of agonist-sensitive stores with NE in aortic segments from control rats caused a significant decrease in the subsequent contractile response (Fig. 3).


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  		Fig. 2.   A: typical tracing from a single UK-14304 experiment showing responses generated during each manipulation in an aortic segment from a L-NNA-treated animal. SR, sarcoplasmic reticulum release. B: UK-14304 (10-6 mol/l) responses in aortic segments from control and L-NNA-treated rats (n = 6). C: PE (10-6 mol/l) responses in aortic segments from control and L-NNA-treated rats (n = 6). Results are means ± SE. *Significant difference from control.



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  		Fig. 3.   UK-14304 (10-6 mol/l) responses in aortic segments from control and L-NNA-treated rats (n = 3). Intracellular stores were depleted with norepinephrine (NE, 10-6 mol/l) in the absence of extracellular Ca2+. Responses to PE were similar to those in Fig. 2C (data not shown). Results are means ± SE. *Significant differences from control. dagger Significant difference from initial response.

Similar experiments were carried out using the SR Ca2+-ATPase inhibitor thapsigargin to completely deplete Ca2+ stores before the influx-only experiment (Fig. 4). These data also demonstrate that contraction to UK-14304 in L-NNA-treated tissue requires only extracellular Ca2+. However, depletion of thapsigargin-sensitive stores in aortic segments from control rats caused a significant decrease in the subsequent contractile response, similar to that seen in the NE depletion studies. These studies indicate that the source of activator Ca2+ for UK-14304 contraction is altered in aortic segments from L-NNA-treated rats.


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  		Fig. 4.   UK-14304 (10-6 mol/l) responses in aortic segments from control and L-NNA-treated rats (n = 5). Intracellular Ca2+ stores were depleted by stimulating tissues with NE (10-6 mol/l) in the absence of extracellular Ca2+. Refilling of intracellular stores was inhibited by treatment with thapsigargin (10-6 mol/l, 30 min). Extracellular Ca2+ was depleted by washing tissues in Ca2+-free physiological saline solution containing EGTA (10-3 mol/l). Responses to PE were not different from baseline for any of the conditions (data not shown). Results are means ± SE. *Significant differences from control. dagger Significant difference from initial response.

Nifedipine inhibits UK-14304 contraction. To determine whether VDCCs mediate the increased Ca2+ influx during alpha 2-AR contraction, concentration-response curves to the alpha 2-AR agonist UK-14304 were generated in the presence of increasing concentrations of the dihydropyridine nifedipine. We observed that incubation with nifedipine (10-6 mol/l) significantly attenuated UK-14304 contraction in aortic segments from L-NNA-treated rats, whereas higher concentrations completely blocked contraction. Inhibition of contraction in tissue from control rats required a greater concentration of nifedipine, and even the highest concentration used (10-5 mol/l) did not completely abolish the response. These data suggest that UK-14304 contraction is mediated differently in arteries from L-NNA-treated and control rats (Fig. 5). Nifedipine vehicle (0.01% ethanol) did not affect contraction or viability.


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  		Fig. 5.   A: inhibition of UK-14304 contraction in control segments requires a greater concentration of nifedipine (Nif; n = 12). B: Nif causes concentration-dependent inhibition of UK-14304 contraction in aortic segments from L-NNA-treated rats (n = 8). Results are expressed as means ± SE. *Significant difference from vehicle.

Aortas From L-NNA-Treated Rats Exhibit Greater Contractility to BAY K 8644

The difference in the response of arteries from control and L-NNA-treated rats to the VDCC blocker nifedipine suggested there might be a difference in the expression or activity of VDCCs. This was evaluated using the VDCC agonist BAY K 8644. Segments from L-NNA-treated rats developed more tension and had a lower contractile threshold to BAY K 8644 than those from control rats (Fig. 6).


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  		Fig. 6.   Enhanced contraction of aortic segments from L-NNA-treated rats in response to BAY K 8644 compared with aortic segments from control rats (n = 8). Results are expressed as means ± SE. *Significant difference from control.

Calcium Influx in VSMCs From L-NNA-Treated Rats Was More Sensitive to Nifedipine

Calcium imaging studies on freshly isolated aortic smooth muscle cells revealed that individual cells from L-NNA-treated rats were more sensitive to nifedipine compared with control cells. UK-14304 (10-7 mol/l) generated a significant increase in the 340/380 nm excitation ratio in both control and L-NNA-treated cells. This increase was inhibited by nifedipine (10-7 mol/l) in L-NNA-treated cells but was not affected in control cells (Fig. 7). The viability of the cells was confirmed by adding the Ca2+ ionophore ionomycin (10-6 mol/l). Only data from cells that responded to ionomycin were used. Adding DMSO or ethanol to the bath did not effect the excitation ratio.


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  		Fig. 7.   A: typical tracing from a single experiment showing responses of an individual aortic vascular smooth muscle cell (VSMC) from an L-NNA-treated rat to UK-14304 (UK, 10-7 mol/l), Nif (10-7 mol/l), and ionomycin (Iono, 10-3 mol/l). Only data from cells that respond to Iono were used. B: summary of responses from VSMCs freshly isolated from control and L-NNA-treated rat aortas. Nif reverses the UK-14304-induced Ca2+ increase in VSMCs from L-NNA-treated rats but not in those from control (n = 8 animals in each group). Results are expressed as means ± SE. *Significant difference from control. dagger Significant difference from baseline intensity. #Significant difference from UK-14304 stimulation (P <=  0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies from our laboratory demonstrated an augmented contractile sensitivity to alpha 2-AR stimulation in mesenteric arteries and aortas from L-NNA-treated animals compared with controls (10). However, the mechanisms of this increased sensitivity were unclear. Several investigators have shown that L-type VDCCs in VSMCs play an important role in the regulation of vascular tone (12) and, specifically, in mediating alpha 2-AR contraction (20). Also, modifications in channel properties have been implicated in the pathology of different forms of hypertension (17). Patch-clamp studies (22) in VSMCs isolated from spontaneously hypertensive rats show an increased L-type Ca2+ current compared with controls, whereas Ohya et al. (20) demonstrated impaired activation of ATP-sensitive potassium channels in VSMCs from spontaneously hypertensive rats. These studies suggest that alterations in function or density of ion channels contribute to the vascular changes in hypertension.

These observations led us to hypothesize that increased arterial sensitivity to alpha 2-AR agonists in arteries from rats made hypertensive with L-NNA is due to increased Ca2+ influx through L-type VDCCs. Contractility studies and calcium-imaging studies were used to investigate the proposed hypothesis and to determine whether alpha 2-AR contraction is mediated by VDCC activation.

There were four major new findings from this study. First, augmented alpha 2-AR contraction of aortic rings from L-NNA-treated rats to UK-14304 depends on Ca2+ influx rather than release from intracellular stores (Figs. 2-4). Second, UK-14304 contraction in aortic rings from L-NNA-treated rats is mediated to a greater extent by Ca2+ influx through L-type VDCCs than is contraction in rings from control rats (Fig. 5). Third, aortic rings from L-NNA-treated rats demonstrated increased sensitivity and maximal contraction to the VDCC agonist BAY K 8644 compared with aortic rings from control rats (Fig. 6). Finally, nifedipine (10-7 mol/l) blocks Ca2+ increases to UK-14304 stimulation in isolated aortic VSMC from L-NNA-treated animals but does not affect UK-14304 Ca2+ signaling in VSMCs from control rats (Fig. 7).

The two main mechanisms that mediate increased intracellular Ca2+ concentration in VSMCs are Ca2+ influx through L-type VDCCs and release from intracellular stores. Results from these studies demonstrate that contraction to alpha 2-adrenergic agonists in aortic segments from L-NNA-treated rats is mediated by Ca2+ influx but not by release from intracellular stores. This is not true of aortic rings from control rats. Therefore, alpha 2-AR postreceptor signaling or VDCC regulation is altered in VSMC from L-NNA-treated rats. Our studies with the VDCC agonist BAY K 8644 suggest that the alteration is in part at the level of the channel, because there is augmented contraction to both UK-14304 and BAY K 8644.

Several investigators have demonstrated that in agonist-mediated contractions, Ca2+ entering VSMCs stimulates further release of Ca2+ from the intracellular stores (reviewed in Refs. 2 and 3). In our experiments, NE was used to deplete agonist-sensitive intracellular stores. In addition, studies were conducted using the Ca2+-ATPase inhibitor thapsigargin to prevent store refilling (11). Results from these experiments are consistent with the hypothesis that contraction to alpha 2-adrenergic agonists in aortic segments from L-NNA-treated rats is mediated by Ca2+ influx alone and not by release from intracellular stores. UK-14304-mediated contraction did not stimulate any Ca2+-mediated Ca2+ release in our experiments.

An interesting observation was that in aortic segments from control rats, contraction to UK-14304 after NE store depletion was diminished compared with baseline contraction (Fig. 3). However, this difference was not observed in the absence of NE stimulation (Fig. 2) or with inhibition of Ca2+-ATPases by thapsigargin (Fig. 4). This suggests that UK-14304 activates different signal transduction pathways under different conditions. It appears that UK-14304 contraction requires intact intracellular Ca2+ stores in control tissues but not in those from rats made hypertensive by L-NNA. The reason for this difference in source of activator Ca2+ was not directly tested but could be caused by either a difference in the subtype of receptor mediating the contraction or by a change in the second messenger cascade activated by the receptor. It is intriguing to speculate that NOS-I hypertension may cause a change in the expression of one or more components of the alpha 2-AR signaling pathway.

The L-type VDCC is the primary Ca2+ channel type in VSMCs and plays an important role in the regulation of vascular tone. Xiao and Rand (30) have shown that the Ca2+ channel blocker diltiazem significantly attenuated the pressor response to UK-14304 in pithed rats suggesting that Ca2+ influx through VDCCs mediates in vivo vasoconstriction to alpha 2-AR stimulation. Kannan and Seip (12) demonstrated that in rat mesenteric arteries, the VDCC blocker nitrendipine totally blocked alpha 2-AR-mediated contraction, whereas it only attenuated alpha 1-AR contraction. We report here that increasing concentrations of nifedipine inhibited the UK-14304 contraction in both control and L-NNA-treated tissue. However, a higher concentration of nifedipine was required to inhibit the contraction in the control tissue compared with the L-NNA-treated group. These data demonstrate that L-type VDCCs are the main portals of Ca2+ entry into aortic VSMCs from both control and L-NNA-treated animals stimulated with alpha 2-AR agonists. However, VSMCs from L-NNA-treated rats are more dependent on Ca2+ influx than are VSMCs from control rats.

Therefore, these observations consistently demonstrate that alpha 2-ARs depend more on VDCCs for contraction than do alpha 1-ARs and provide strong support for the proposal that these two receptors stimulate vasoconstriction through different intracellular signaling pathways. It is intriguing that in vascular segments from hypertensive rats, contraction to low concentrations of NE, the endogenous ligand for all ARs, is more sensitive to both alpha 2-AR antagonists (10) and to VDCC inhibitors (12) than contraction in segments from normotensive animals. Endogenous NE-mediated contraction may, therefore, rely on alpha 2-AR during low levels of sympathetic activity and on alpha 1-AR stimulation with increased release of NE. Increased sensitivity of the alpha 2-AR to NE stimulation during hypertension may, therefore, be an important contributor to increased sympathetic vasoconstriction. Results from the calcium imaging studies also support these observations.

These studies together demonstrate there are differences in the way intracellular Ca2+ is regulated by alpha 2-AR in L-NNA and control VSMCs. However, the mechanisms underlying these differences can only be postulated. One possible explanation could be open probability (Po) of L-type VDCCs in the VSMCs from L-NNA-treated rats. Po of L-type VDCCs is governed primarily by the membrane potential (Em), because depolarization activates the channel and hyperpolarization inactivates it. Martens and Gelband (17) have shown that VSMCs from spontaneously hypertensive rats and deoxycorticosterone acetate-hypertensive sensitive rats were ~20 mV more depolarized than controls. Depolarized resting Em in VSMCs from L-NNA-treated rats could, therefore, explain the increased Ca2+ influx and contractile sensitivity to UK-14304 in L-NNA tissue. Recent studies from our laboratory have indeed found that mesenteric artery VSMCs from L-NNA-treated rats have significantly depolarized Em compared with cells from controls (6a).

Our results with the dihydropyridine analog BAY K 8644 also support this hypothesis. BAY K 8644 is a L-type VDCC that more effectively activates the channel when the membrane is depolarized (28). Consistent with our hypothesis, BAY K 8644 contracted aortic segments from L-NNA-treated rats but elicited little or no contraction in segments from control rats. These data also suggest that L-NNA-treated tissue may have a relatively depolarized Em compared with vascular smooth muscle from controls.

Alternatively, calcium sensitivity of contraction could be augmented in arteries from L-NNA-treated rats. Under this scenario, similar levels of calcium entering through VDCCs would elicit a greater response due to enhanced myosin light chain phosphorylation. There is evidence in the literature (27) that arteries from hypertensive rats have enhanced calcium sensitivity, but this was not addressed in the current study.

In conclusion, our results indicate that increased vascular contractile sensitivity to alpha 2-AR contraction after in vivo NOS inhibition is mediated by increased Ca2+ influx through L-type VDCCs. In addition, we observed that UK-14304 contraction of control arteries requires intact intracellular Ca2+ stores, whereas store depletion does not affect UK-14304 contraction in arteries from hypertensive rats. We speculate that NOS-I hypertension alters the expression or activity of a component of the alpha 2-AR signal transduction pathway. These findings increase our understanding of the mechanisms of alpha 2-AR vasoconstriction and suggest that augmented alpha 2-AR contraction contributes to increased vascular resistance in forms of hypertension associated with impaired endothelial production of NO. Insights into the signal transduction mechanisms altered during NOS-I will lead to new therapeutic strategies in the treatment of a number of vascular disorders, including hypertension, atherosclerosis, restenosis, and thrombosis.


    ACKNOWLEDGEMENTS

We thank Pam Allgood for technical assistance and B. Walker and Rebecca Carter for reviewing the manuscript.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-03852 (to N. L. Kanagy). We acknowledge the University of New Mexico Research Project and Travel Grant (to H. Mukundan) for generous support.

Address for reprint requests and other correspondence: H. Mukundan, Vascular Physiology Group, Dept. of Cell Biology and Physiology, Univ. of New Mexico Health Sciences Center, 915 Camino de Salud NE, Albuquerque, NM 87131-5218 (E-mail: hmukundan{at}salud.unm.edu).

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.

Received 15 March 2001; accepted in final form 19 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 281(5):H2233-H2240
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


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