INTRODUCTION

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POSTSYNAPTIC ADRENERGIC RECEPTORS are present in a variety of tissues and regulate such diverse functions as adipose tissue lipolysis, platelet aggregation, vasoconstriction, and intestinal electrolyte secretion (27). Norepinephrine-stimulated vasoconstriction in vascular smooth muscle (VSM) is mediated by ₁- and ₂-adrenoreceptors (₁- and ₂-ARs, respectively). Although ₁-ARs are responsible for ~80% of norepinephrine vasoconstriction in arteries from normotensive rats (8), the contribution of _2i-ARs increases with hypertension (18). ₂-ARs are G_i protein-coupled receptors, and vasoconstriction is sensitive to genistein, erbstatin, and methyl-2,5-dihydroxycinnamate (16, 17). However, the specific tyrosine kinases involved and the role of tyrosine kinase activation in ₂-AR contraction are unknown.

Several tyrosine kinases linked to smooth muscle vasoconstriction can be activated by ₂-AR stimulation. These include Src (26), the epidermal growth factor (EGF) receptor kinase (24), and a dual serine/tyrosine kinase that is responsible for activating extracellular signal-regulated kinase (ERK) 1/2 called mitogen-activated protein kinase ERK kinase (MEK) (26, 29). These tyrosine kinases participate in vasoconstriction in a variety of ways. Src family tyrosine kinases can contribute to vasoconstriction by stimulating intracellular Ca²⁺ release and activating L-type Ca²⁺ channels (13, 35). Once transactivated, the EGF receptor can subsequently activate many signaling molecules that participate in vasoconstriction (6, 10). ERK 1/2, which is activated by MEK, can phosphorylate caldesmon and myosin light-chain kinase and result in enhanced Ca²⁺ sensitivity (2, 7). Thus tyrosine kinase activation can contract VSM by two potentially separate mechanisms: increased Ca²⁺ concentration ([Ca²⁺]) and increased Ca²⁺ sensitivity.

Vascular reactivity and tyrosine kinase activity are increased in several forms of hypertension (28, 30). We have previously demonstrated that vasoreactivity to ₂-AR stimulation is enhanced in rats with chronic nitric oxide synthase (NOS) inhibition-induced hypertension (LHR) compared with normotensive control rats (NR), whereas ₁-AR vasoreactivity remains unchanged (18). It is unknown whether tyrosine kinases contribute to the enhanced ₂-AR vasoreactivity that is associated with NOS inhibition hypertension. The goals of this study were to determine whether tyrosine kinases (specifically Src, the EGF receptor kinase, and ERK 1/2) contribute to ₂-AR contraction and to evaluate differences in tyrosine kinase signaling in arteries from NR and LHR. Previous work suggests that ₂-AR contraction is dependent on extracellular Ca²⁺ influx (1, 19, 20) and the activation of one or more tyrosine kinases (16). This combined with the observation that these tyrosine kinases can increase intracellular [Ca²⁺] led us to hypothesize that ₂-ARs activate Src, the EGF receptor kinase, and ERK 1/2 to contract rat aorta by increasing [Ca²⁺] and not by increasing Ca²⁺ sensitivity. In addition, we hypothesized that an increase in one or more of these pathways augments ₂-AR contraction in arteries from LHR. We tested these hypotheses in endothelium-denuded thoracic aortic rings from male Sprague-Dawley NR and LHR using the ₂-AR agonist UK-14304 and specific tyrosine kinase inhibitors. Additionally, the Ca²⁺ ionophore ionomycin was used to determine whether UK-14304 increases contraction in permeabilized arteries and whether tyrosine kinase inhibition attenuates that augmentation.

Together, the data presented here suggest that ₂-ARs stimulate contraction through two separate pathways: one is apparently enhanced with NOS inhibition hypertension, mediates activation of Ca²⁺ sensitivity, and is independent of tyrosine kinase activation; and the other is tyrosine kinase dependent and regulates intracellular [Ca²⁺].

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Hypertension induction and tissue collection. Male Sprague-Dawley rats (body wt 250-300 g) were given tap water containing 0.5 g/l N-nitro-L-arginine (LHR) or vehicle (NR) to drink for 14 days. Blood pressure values (measured by tail cuff; IITC; Woodland Hills, CA) and animal body weights were measured on days 0, 7, and 14. After the 14-day treatment period, animals were anesthetized with intraperitoneal pentobarbital sodium. Thoracic aortas were removed and placed in ice-cold physiological saline solution [PSS, which contained (in mM) 130 NaCl, 4.7 KCl, 1.18 KH₂PO₄, 1.17 MgSO₄ · 7H₂O, 14.9 NaHCO₃, 5.5 dextrose, 0.026 CaNa₂-EDTA, and 1.6 CaCl₂, pH 7.3]. Vessels were cleaned of visible fat, cut into 4-mm rings, and denuded of endothelium using the tip of closed, sharp forceps.

Contractile studies. Rings were attached to a force transducer (Grass) with stainless steel hooks, and generated tension was recorded on a polygraph (Gould). Two rings (one NR and one LHR) were placed in a water-jacketed tissue bath that contained 37°C PSS bubbled with 95% O₂-5% CO₂. Rings were stretched to 2,500 mg passive tension (determined by length-tension curve to allow maximum active-tension generation; data not shown) and allowed to equilibrate for 60 min. Indomethacin (1 µM) was added during the final 30 min of equilibration. To determine viability, rings were exposed to a single concentration of phenylephrine (0.1 µM). Lack of relaxation to ACh (1 µM) in phenylephrine-contracted rings was used to verify endothelium removal. Rings generating at least 1,000 mg tension to phenylephrine with <5% relaxation to ACh were washed until no active tension remained (30-60 min). Rings were then exposed to cumulative concentrations of the ₂-AR agonist UK-14304 in the presence or absence of specific tyrosine kinase inhibitors. Previous studies have shown that ₂-AR vasoconstriction is sensitive to genistein, erbstatin, and methyl-2,5-dihydroxycinnamate (16, 17). Tyrphostins are reported to be specific tyrosine kinase inhibitors with an inactive analog, although some nonspecific effects at concentrations of 100 µM have been reported (31, 37, 38). Inhibitor concentrations were chosen based on concentrations effective in similar preparations (3, 5, 23, 41). Time-control studies were performed with each experiment, and data were not reported if differences were apparent in time controls.

Western blot analysis. Rings were hung in water-jacketed tissue baths as for contractile experiments and equilibrated for 60 min. After equilibration, tissues were treated with the MEK inhibitor PD-98059 (10 µM) or vehicle for 30 min and then exposed to the ₂-AR agonist UK-14304 (10 µM) or EGF (1 µM). Exactly 5 min after agonist stimulation (appropriate activation time was determined by time course; data not shown), rings were removed from baths and placed into Dounce homogenizers that contained 150 µl of ice-cold lysis buffer [Tris-buffered saline (TBS) that contained 1 mM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml antipain, and 1 mM phenylmethylsulfonyl fluoride]. Tissues were rapidly homogenized on ice and sonicated 10 times with 1-s pulses. Tissue homogenates were centrifuged at 13,000 g for 3 min at 4°C. Protein concentration of the supernatant was determined using the Bradford method (Bio-Rad).

Statistics. Data were analyzed with one-way or two-way ANOVA as appropriate. Post hoc tests were performed using Student-Newman-Keuls test. P values 0.05 are considered significant. Contractile studies are expressed as the percent maximum tension to UK-14304. Percentages were arcsin transformed before statistical analysis to ensure normality.

Materials. Inhibitors {tyrphostin 23, tyrphostin 1, 4-amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), AG-1478, and PD-98059} were purchased from BioMol. Antibodies were purchased from Transduction Labs. Chemiluminescent reagents were purchased from Amersham.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Inhibition of ₂-AR contraction by tyrphostin 23. To confirm that tyrosine kinases are required for ₂-AR contraction, denuded thoracic aortic rings were treated for 30 min with vehicle (DMSO), tyrphostin 23 (15-50 µM), or the inactive analog of tyrphostin 23, tyrphostin 1 (50 µM). Tyrphostin 23 significantly and concentration dependently attenuated contraction to UK-14304 (Fig. 1, A and B), whereas the inactive analog was without effect (Fig. 1, C and D). The highest concentration of tyrphostin used, 50 µM, completely prevented the UK-14304 contraction.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Tyrphostin 23 (Tyr 23) concentration-dependently attenuates contraction to the 2-adrenoreceptor (2-AR) agonist UK-14304. Thoracic aortic rings from endothelium-denuded normotensive rats (NR, A and C) and rats made hypertensive with chronic nitric oxide synthase (NOS) inhibition (LHR, B and D) were used to generate consecutive cumulative concentration-response curves to UK-14304 (109 to 105 M) in the presence and absence of increasing concentrations of Tyr 23 or its inactive analog Tyr/Tyr 1. Highest concentration of Tyr 23 (50 µM) completely eliminated the contraction, whereas the same concentration of Tyr 1 was without effect. *Statistically significant difference between vehicle and treatment; n, no. of animals.

Involvement of specific tyrosine kinases. Although ₂-AR contraction requires a tyrosine kinase, the specific kinase involved is unknown. Src family kinases or the EGF receptor kinase can be activated by ₂-AR (24, 26). However, the current contractile studies suggest that neither Src nor the EGF receptor kinase participate in ₂-AR contraction in rat aorta. The Src family kinase inhibitor PP1 (0.1 and 1 µM) did not reduce UK-14304 contraction in rings from NR or LHR (Fig. 2, A and B). However, PP1 (0.1 and 1 µM) attenuated serotonin contraction in rings from both groups, which demonstrates that these concentrations of the inhibitor are effective against an agonist that activates Src (Ref. 3; Fig. 2, C and D). Additionally, AG-1478 (0.03 and 0.3 µM), a specific inhibitor of the EGF receptor kinase, did not affect UK-14304 contraction in either group at concentrations that have been shown to inhibit EGF receptor kinase in VSM (Refs. 5, 23; Fig. 3).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   4-Amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), the Src family kinase inhibitor, did not attenuate 2-AR contraction. PP1 (1 or 10 µM) did not affect UK-14304 contraction in endothelium-denuded thoracic aortic rings from either NR (A) or LHR (B). However, the same concentrations of PP1 reduced serotonin (109 to 105 M) contraction in both NR (C) and LHR (D) groups. This indicates that Src family tyrosine kinases do not participate in 2-AR contraction in rat aorta. *Statistically significant difference between vehicle and treatment; n, no. of animals.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Epidermal growth factor (EGF) receptor inhibitor AG-1478 does not affect UK-14304 contraction. Treatment of endothelium-denuded aortic rings from NR (A) and LHR (B) with AG-1478 did not affect contraction to the 2-AR agonist UK-14304 (109 to 105 M), which suggests that the EGF receptor kinase does not contribute to 2-AR contraction. n, No. of animals.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   The mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD-98059 attenuates contraction to the 2-AR agonist UK-14304 (109 to 105 M). Treatment with PD-98059 significantly reduces UK-14304 contraction in endothelium-denuded aortic rings from NR (A) and LHR (B), which suggests that ERK 1/2 contributes to 2-AR contraction. *Statistically significant difference between vehicle and treatment; n, no. of animals.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Densitometry data from Western blot analysis of ERK 1/2 activation after stimulation of aortic rings with the 2-AR agonist UK-14304 (UK; 10 µM) in the presence and absence of the MEK inhibitor PD-98059 (PD; 10 µM). UK-14304 activates p42 ERK (A) and p44 ERK (B) in endothelium-denuded aortic rings from NR and LHR. Representative blots of phosphorylated ERK 1/2 in aortas from NR (C) and LHR (D) are shown. Activation of ERK 1/2 is eliminated by pretreatment with PD-98059. *Statistically significant difference between vehicle (Veh) and treatment; n, no. of animals.

NOS-inhibition hypertension enhances basal Ca²+ sensitivity. Ionomycin was used to analyze Ca²⁺ sensitivity in the absence of agonists in denuded aortic rings from NR and LHR. Rings were permeabilized with ionomycin (1.5 µM) and exposed to extracellular Ca²⁺ (0.8 or 1.6 mM). We have observed that this concentration of ionomycin produces similar increases in intracellular [Ca²⁺] in aortic strips from both NR and LHR as measured by fura 2 Ca²⁺ imaging in aortic strips (unpublished observations). Aortic rings from LHR contracted more than rings from NR at each concentration of Ca²⁺ used (Fig. 6A). A representative trace is shown in Fig. 6B. In some aortas from NR, the contraction to Ca²⁺ decreased slightly over time. Results reported are the maximal tensions achieved.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Ionomycin-permeabilized aortic rings from LHR contract more to CaCl2 and UK-14304 than rings from NR (A). Endothelium-denuded thoracic aortic rings from NR and LHR were permeabilized with the Ca2+ ionophore ionomycin (1.5 µM) in a Ca2+-free physiological saline solution. Ca2+ added to the bath stimulates a greater contraction in rings from LHR than NR (hatched and shaded portion of bars). 2-AR agonist UK-14304 added on top of the Ca2+ contraction augments CaCl2 tension more in aortas from LHR than NR (open bars). A representative trace of the experiment at 0.8 mM CaCl2 is shown (B). This suggests that NOS inhibition hypertension may enhance basal and 2-AR activated Ca2+ sensitivity. *Statistically significant difference between vehicle and treatment; n, no. of animals.

Tyrosine kinases regulate intracellular [Ca²+] in UK-14304 contraction. Tyrosine kinases can affect both Ca²⁺ sensitivity and intracellular [Ca²⁺] in VSM contraction. To address the role of tyrosine kinases in ₂-AR contraction, in the presence of tyrphostin 23 (30 µM) or vehicle, denuded thoracic aortic rings from NR and LHR were permeabilized with ionomycin (1.5 µM) and contraction was elicited with CaCl₂ (0.8 or 1.6 mM). After the contraction plateaued, UK-14304 (10 µM) was added. At both concentrations of Ca²⁺, UK-14304 augmented CaCl₂ contraction (Fig. 6). At the lower concentration of CaCl₂ (0.8 mM), UK-14304 stimulated a greater augmentation of contraction in aortas from LHR than from NR. Although tyrphostin 23 (30 µM) decreased UK-14304 contraction in nonpermeabilized aortas from NR and LHR (see Fig. 1, A and B), the same concentration of tyrphostin did not attenuate augmented contraction to UK-14304 (10 µM) in ionomycin-permeabilized rings (Fig. 7). Tyrphostin 23 treatment increased UK-14304 augmentation of contraction in aortas from LHR, possibly owing to a slight attenuation of tension in 1.6 mM Ca²⁺ after tyrphostin administration.

View larger version (11K): [in this window] [in a new window]   Fig. 7.   Augmentation of Ca2+ contraction by the 2-AR agonist UK-14304 (10 µM) is unaffected by Tyr 23. Endothelium-denuded aortic rings from NR and LHR were treated with vehicle or Tyr 23. Treated rings were permeabilized with the Ca2+ ionophore ionomycin (1.5 µM) in the continued presence of the antagonist and exposed to 1.6 mM CaCl2. UK-14304 was added on top of the Ca2+ contraction. Data shown as milligrams of tension change from Ca2+ contraction. n, No. of animals.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This study examined the role of tyrosine kinase activation in ₂-AR arterial contraction and analyzed changes in arterial Ca²⁺ sensitivity after NOS inhibition hypertension. We observed that tyrosine kinases play a critical role in ₂-AR contraction. However, none of the specific kinases tested (Src, EGF receptor, and MEK) appear to be the primary tyrosine kinase activated. We also observed an apparent increase in Ca²⁺ sensitivity associated with ₂-AR activation that is insensitive to tyrosine kinase inhibition. This suggests that ₂-AR contraction may rely on a tyrosine kinase for increases in intracellular [Ca²⁺] and activate a second tyrosine kinase-independent pathway that increases Ca²⁺ sensitivity. Finally, we noted an apparent increase in basal Ca²⁺ sensitivity in aortas from LHR that may contribute to the previously noted increase in vasoreactivity to ₂-ARs and other agonists.

Tyrosine kinase involvement in vasoconstriction is not unique to ₂-ARs. Tyrosine kinases participate in vasoconstriction stimulated by many agonists including serotonin, angiotensin, and phenylephrine (3, 11, 34). However, the signaling pathways involved appear to be relayed through different specific tyrosine kinases and may be quite different for each of these contractile agonists. It has been hypothesized that ₂-AR contraction relies on Src (16), and recently Roberts (26) reported an Src component to ₂-AR contraction in porcine palmar lateral veins. However, the data presented here indicate that Src is not involved in ₂-AR contraction in rat aortas, which suggests that differences between species and vessels may exist. The inability of tyrphostin 23 to attenuate UK-14304 contraction in ionomycin-permeabilized rings suggests that the tyrosine kinase component of the ₂-AR contraction may control intracellular [Ca²⁺]. However, most studies demonstrating the influence of tyrosine kinase on intracellular [Ca²⁺] have focused on Src or ERK 1/2 (13, 33, 35). The results presented here indicate that neither of these are the primary target for tyrphostin 23 inhibition. Therefore, a unique tyrosine kinase appears to regulate intracellular [Ca²⁺] in ₂-AR contraction of the rat aorta.

The role of Src as a regulator of the L-type Ca²⁺ channel has been established in human VSM, rat ear artery, and rat mesenteric artery cells (13, 35, 37, 38, 40). Contraction of the rat aorta by 5-HT, serotonin, and angiotensin (11, 34) is sensitive to Src inhibition and relies in part on extracellular Ca²⁺ entry through L-type Ca²⁺ channels, which suggests that Src may regulate Ca²⁺ entry in the rat aorta. However, to our knowledge, this hypothesis has not been directly tested. This study provides novel evidence that in rat aortas, the ₂-AR contraction relies on a tyrosine kinase other than Src to regulate Ca²⁺ entry though L-type voltage channels. It is possible that the tyrosine kinase is actually regulating another component upstream of the Ca²⁺ channel. However, direct testing of this hypothesis would require additional experiments including concurrent measurement of intracellular Ca²⁺ and tension in the rat aorta in the presence and absence of tyrosine kinase inhibitors. Thus current data suggest that a tyrosine kinase regulates Ca²⁺ influx rather than Ca²⁺ sensitivity.

It is interesting to note that AT₁ and ₁-AR (both G_q receptors) activate Src with subsequent intracellular Ca²⁺ release and Ca²⁺ entry to cause contraction (11, 34). The ₂-AR on the other hand [a G_i protein-coupled receptor] does not rely on intracellular Ca²⁺ release (1, 19, 21). This is evident from our previous observation that in the presence of thapsigargin and after depletion of intracellular stores, ₂-AR contraction in the NR aorta is nearly 100% and contraction in the LHR aorta is 100% of responses in Ca²⁺-replete arteries (21).

Additionally, Src, ERK 1/2, and the EGF receptor participate in AT₁ and ₁-AR contractions (9, 11, 15, 34). These kinases can be activated by ₂-ARs in cell types other than VSM and may participate in the signaling cascade that leads to other vascular functions of the ₂-ARs such as migration or cell growth (25). However, the data presented here indicate that these tyrosine kinases do not play a primary role in ₂-AR VSM contraction. Therefore, although participation of tyrosine kinases in contraction is common, there appear to be at least two separate groups of receptors that use significantly different signaling pathways to produce tyrosine kinase-dependent vasoconstriction. In addition, the primary tyrosine kinases required for ₂-AR contraction remain unknown. G_i protein-coupled receptors can transactivate other growth factor receptors (including PDGF), which could potentially mediate vascular contraction (12). It is also possible that another cytosolic tyrosine kinase, such as PYK2, FAK, or paxillin, leads to ₂-AR contraction.

Although we demonstrate a reliance on tyrosine kinases for ₂-AR contraction in the rat aorta, no differences were noted in the ability of tyrphostin 23 to inhibit contraction between NR and LHR. Augmented tyrosine kinase-mediated vasoconstriction has been reported (28, 30) in spontaneously hypertensive rats (SHR). In SHR, increased expression of ERKs has also been observed (37). However, no association has been made between NOS inhibition hypertension and increased basal or agonist-stimulated tyrosine kinase activity. The data presented here do not directly evaluate kinase activity in NOS inhibition hypertension but do suggest that augmented tyrosine kinase activation does not cause enhanced ₂-AR contraction. Further studies, including kinase assays and concurrent intracellular Ca²⁺-tension measurements, are warranted to determine whether tyrosine kinases indeed play a larger role in other vasoconstriction pathways in arteries from LHR than in NR.

It has been well established that most models of hypertension have increased vasoreactivity. Although many have hypothesized that enhanced vasoreactivity is due to increases in intracellular [Ca²⁺] in hypertensive arteries (20, 32), there have been recent suggestions that Ca²⁺ sensitivity in the contractile apparatus is increased with hypertension (36). Increased Ca²⁺ sensitivity has been attributed to activation of protein kinase C (5, 14) and more recently to Rho-activated kinase (ROK; Refs. 22, 36). These kinases inhibit myosin light-chain phosphatase to augment force generation (4, 22). Additionally, the ROK inhibitor Y-27632 significantly reduces blood pressure in SHR compared with Wistar-Kyoto rats, which suggests that increased Ca²⁺ sensitivity may contribute to hypertension (36).

The data presented here are the first to associate NOS inhibition hypertension with increased Ca²⁺ sensitivity. Previous work from this laboratory demonstrated an increase in vascular reactivity to KCl and to ₂-AR agonists but not to ₁-AR agonists (18). The increase in vasoreactivity may be due to the same apparent enhancement of basal and ₂-AR-activated Ca²⁺ sensitivity indicated here, although concurrent measurements of Ca²⁺ and active tension are needed to confirm this observation.

In conclusion, this study presents evidence that in the rat aorta, ₂-AR contraction proceeds through two distinct pathways: a tyrosine kinase-independent pathway mediates ₂-AR activated Ca²⁺ sensitivity, and a tyrosine kinase-dependent pathway that does not include Src or EGF receptor kinase that regulates intracellular [Ca²⁺]. Increased CaCl₂ contraction and UK-14304 augmentation of contraction in permeabilized aortas from LHR suggest that NOS inhibition hypertension enhances basal and ₂-AR-activated Ca²⁺ sensitivity. Furthermore, because no change in tyrosine kinase sensitivity was apparent in aortas from LHR, it is possible that enhanced tyrosine kinase-independent Ca²⁺ sensitivity is responsible for increased vasoreactivity in aortas from LHR.