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【摘要】 Dilation of rat preglomerular microvessels (PGMV) by activation of adenosine A 2A receptors (A 2A R) is coupled to epoxyeicosatrienoic acid (EET) release. We have investigated the commonality of this signal transduction pathway, i.e., sequential inhibition of G s, adenylyl cyclase, PKA, and Ca 2+ -activated K + (K Ca ) channel activity, to the vasoactive responses to A 2A R activation by a selective A 2A agonist, CGS-21680, compared with those of 11,12-EET. Male Sprague-Dawley rats were anesthetized, and microdissected arcuate arteries (110-130 µm) were cannulated and pressurized to 80 mmHg. Vessels were superfused with Krebs solution containing N G -nitro-L-arginine methyl ester ( L -NAME) and indomethacin and preconstricted with phenylephrine. We assessed the effect of 3-aminobenzamide (10 µM), an inhibitor of mono-ADP-ribosyltranferases, on responses to 11,12-EET (3 nM) and CGS-21680 (10 µM) and found that both were inhibited by 70% ( P < 0.05), whereas the response to SNP (10 µM) was unaffected. Furthermore, 11,12-EET (100 nM), like cholera toxin (100 ng/ml), stimulated ADP-ribose formation in homogenates of arcuate arteries compared with control. SQ-22536 (10 µM), an inhibitor of adenylyl cyclase activity, and myristolated PKI (14-22) amide (5 µM), an inhibitor of PKA, decreased activity of 11,12-EET and CGS-21680. Incubation of 11,12-EET (3 nM-3 µM) with PGMV resulted in an increase in cAMP levels ( P < 0.05). The responses to both 11,12-EET and CGS-21680 were significantly reduced by superfusion of iberiotoxin (100 nM), an inhibitor of K Ca channel activity. Thus in rat PGMV activation of A 2A R is coupled to EET release upstream of adenylyl cyclase activation and EETs stimulate mono-ADP-ribosyltransferase, resulting in Gs protein activation.
【关键词】 cytochrome P rat arcuate artery
IN THE KIDNEY, ADENOSINE (A) participates in the regulation of vascular tone and tubular function ( 21 ). The physiological effects of adenosine are observed in nearly every tissue and organ and are expressed in preglomerular microvessels (PGMV) ( 12, 22 ). Adenosine participates in the regulation of renal vascular tone by stimulating primarily A 1 and A 2A receptors (R) in PGMV; stimulation of A 1 R constricts the renal vasculature ( 18 ), whereas stimulation of A 2A R increases renal blood flow ( 27 ), stimulates renin secretion ( 32 ), and decreases blood pressure ( 26 ). These receptors are members of the large family of seven-transmembrane-spanning heterotrimeric G protein-coupled receptors ( 35 ). Activation of the high-affinity A 1 R inhibits adenylyl cyclase via G i, whereas activating the low-affinity A 2A R stimulates adenylyl cyclase via G s ( 12 ). The PGMV, which occupy a key position in the regulation of the renal circulation ( 33 ), are endowed with high levels of cytochrome P -450 (CYP) isoforms, epoxygenases, and - and -1 hydroxylase, which metabolize arachidonic acid (AA) to four regioisomeric cis -epoxyeicosatrienoic acids (EETs), 5,6-, 8,9-, 11,12- and 14,15-EETs, and 19- and 20- HETE, respectively ( 5 ).
The vasodilator responses of EETs have been ascribed to activation of large-conductance Ca 2+ -activated K + channels (K Ca ); EETs have been reported to stimulate G s activity ( 1, 13 ) via ADP-ribosylation, in a manner similar to cholera toxin ( 28, 29 ). ADP-ribosyltranferases, which have prominent nicotinamide adenine dinucleotide (NAD)-glycohydrolase activity, ( 15 ), catalyze the transfer of ADP-ribose (ADPR), an intracellular signaling molecule, to G proteins ( 15 ). Endogenous ADP-ribosylation of G proteins and increased adenylyl cyclase activity occur in response to stimulation of adenosine receptors in adipocytes ( 10 ). In renal afferent arteries, Imig et al. ( 19, 20 ) have shown that the sulfonimide analog of 11,12-EET was the most potent dilator EET, which activates K Ca channel activity through a PKA-dependent mechanism. Activation of A 2A R results in stimulation of G s protein-mediated increases in adenylyl cyclase/PKA activity ( 12 ).
Activation of A 2A R by 2-[4-(2-carboxyethyl)phenethylamino]-5'- N -ethylcarboxamide-adenosine (CGS-21680), a selective A 2A R agonist ( 27 ), dilates PGMV ( 38 ) by acting on K + channels independently of a nitric oxide (NO) component ( 37 ), findings that are consistent with an EET acting as a second messenger. We have observed that activation of A 2A R with CGS-21680 dilates rat pressurized arcuate arteries via an EET-dependent mechanism ( 7 ). Furthermore, CGS-21680 increased EET levels without affecting HETE levels of isolated PGMV. CGS-21680-stimulated EET levels were abolished by preincubation with either an A 2A R antagonist, ZM-241385, or a selective epoxygenase inhibitor, methylsulfonyl-propargyloxyphenylhexanamide (MS-PPOH) ( 3 ), and were independent of NO and cyclooxygenase (COX) activity. The responses to 2-chloroadenosine, a nonspecific adenosine agonist, were also diminished by epoxygenase inhibition, indicating that EETs contribute to adenosine-induced dilation ( 7 ). In arcuate arteries, 5,6-EET and 11,12-EET were equipotent dilators and were more active than 8,9-EET, whereas 14,15-EET was inactive ( 7 ). However, the vasodilator response to 5,6-EET was abolished by inhibition of COX, whereas that to 11,12-EET was not, making 11,12-EET a more likely candidate for mediating arcuate arterial dilation in response to activating A 2A R.
The transduction mechanism by which EETs mediate the vasodilator effect of adenosine has not been addressed. As activation of A 2A R results in stimulation of G s protein-mediated increases in adenylyl cyclase/PKA activity ( 12 ) and addition of an 11,12-EET analog to renal afferent arterioles results in a PKA-dependent dilation ( 19 ), we investigated the commonality of this signal transduction pathway, i.e., sequential inhibition of G s, adenylyl cyclase, PKA, and K Ca channel activity, to the vasoactive responses to A 2A activation by a selective A 2A agonist, CGS-21680, compared with that of 11,12-EET. We have provided evidence that activation of A 2A R in pressurized arcuate arteries is coupled to EET release upstream of adenylyl cyclase activation and that EETs stimulate mono-ADP-ribosyltransferase, resulting in G s protein activation. The proposed signaling pathway for adenosine stimulation of EET formation via A 2A R and resulting dilation of rat PGMV are depicted in Fig. 1.
Fig. 1. Proposed signaling pathway for adenosine stimulation of EET formation, via adenosine A 2A receptors (A 2A R), and results in dilation of rat preglomerular microvessels (PGMV). K Ca, Ca 2+ -activated K + channel.
MATERIALS AND METHODS
Pressurized renal arcuate artery preparation. The experimental procedures used in this study were approved by the New York Medical College Institutional Animal Care and Use Committee and conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats (Charles River) were housed using a 12:12-h light-dark cycle (lights on 0600-1800) with access to water and standard laboratory rat chow (0.8% NaCl, Purina) available ad libitum. Renal arteries were isolated from 8- to 10-wk-old rats anesthetized with pentobarbital sodium (100 mg/kg body wt), as described elsewhere ( 17, 25 ). Segments of arcuate artery were microdissected from hemisected kidneys at 4°C and carefully cleared of adherent connective tissue and tubules and mounted on glass micropipettes, with monofilament silk, in a l-ml water-jacketed perfusion vessel chamber ( 7 ). Pressure was gradually increased to 80 mmHg with a pressure-servo unit (Living System Instrumentation) and maintained throughout all experiments. The microvessel chamber was placed on the stage of an inverted microscope (Nikon) with attached video camera (CCD). The change in internal diameters of the arcuate arteries was continuously measured with a video dimension analyzer (Living System Instrumentation) and recorded on a pen recorder. Isolated arteries were continuously superfused with Krebs solution, equilibrated with 95% O 2 -5% CO 2 at 37°C at the rate of 1 ml/min and equilibrated for 1 h. The composition of Krebs solution was (in mmol/l) 118 NaCl, 4.7 KCl, 1.19 KH 2 PO 4, 1.19 MgSO 4, 1.9 CaCl 2, 25 NaHCO 3, and 5.5 glucose, pH 7.4. We included N G -nitro-L-arginine methyl ester ( L -NAME; 200 µM), a NO synthesis inhibitor, and indomethacin (10 µM), a COX inhibitor, to avoid any potential interaction of either NO ( 36 ) or COX ( 8 ) with CYP-derived AA metabolite levels.
Vascular responses to CGS-21680, sodium nitroprusside (SNP), and 8-bromo-cAMP (8-BrcAMP; Sigma, St. Louis, MO), and 11,12-EET (Biomol Research Laboratories, Plymouth Meeting, PA) were recorded. Internal diameters were measured 1-2 min after administration of each agonist, with 10-min washout periods between addition of agonists and antagonists. After we obtained control responses, enzyme inhibitors were added to the superfusate for 15 min before the administration of agonists was repeated. We studied the effect of myristolated PKI (14-22) amide (mPKI; 5 µM; Biomol), an inhibitor of PKA activity; okadaic acid, an inhibitor of type 2A protein phosphatase (PP2A; 10 nM); SQ-22536 (10 µM; Cayman), an inhibitor of adenylyl cyclase activity; 3-aminobenzamide (3-AM; 10 µM; Sigma), an inhibitor of mono-ADP-ribosyltransferase, and MS-PPOH (12 µM), a selective epoxygenase inhibitor ( 3 ). Stock solutions were prepared (SQ-22536, 3-AM, okadaic acid, MS-PPOH, and 11,12-EET in ethanol and mPKI in Krebs solution) and diluted in Krebs solution, so that the ethanol concentration was <0.01% at the time of administration to arcuate arteries. Stock solutions of CGS-21680 were made up in Krebs solution and SNP in water.
Assay of NAD-glycohydrolase in arcuate artery homogenate. Rat arcuate/interlobular arteries were microdissected, pooled, and homogenized in ice-cold HEPES buffer containing (in mM) 25 Na-HEPES, 1 EDTA, 255 sucrose, and 0.1 phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 6,000 g for 5 min. at 4°C, and the supernate was stored at -80°C. To determine the activity of NAD-glycohydrolase, which converts NAD to ADPR, the homogenates (50 µg) were incubated for 60 min, at 37°C, with 1 mM NAD in buffer containing (in mM) 250 potassium gluconate, 250 N -methylglucamine, 20 HEPES, and 1 MgCl 2 (pH 7.2) ( 29 - 31 ). The arterial homogenates were preincubated with either 11,12-EET or cholera toxin (100 ng/ml), as a positive control, and then NAD was added and incubated for 60 min. The reactions were stopped by snap freezing in liquid N 2. Before HPLC analysis, samples were centrifuged at 4°C to remove proteins. Nucleotides were separated on a Supelcosil LC-18 (3 µm; 4.6 x 150 mm) column using an isocratic mobile phase of 150 mmol/l ammonium acetate (pH 5.5) at a flow rate of 1.0 ml/min. Quantitation was performed based on the elution profile of standards, cyclic (c) ADPR (cADPR), NAD, and ADPR, monitored by UV absorbance (254 nm), and conversion was calculated from the area under the curve.
Isolation of rat PGMV for measurement of EETs and cAMP. The isolation of PGMV, using the iron oxide technique, from the kidneys of anesthetized (pentobarbital sodium; 100 mg/kg body wt) male Sprague-Dawley rats (8-10 wk old; Charles River) has been described previously ( 6, 9 ). PGMV were washed three times in Tyrode's solution containing indomethacin (10 µM) and L -NAME (200 µM) and gassed with 95% O 2 -5% CO 2. The composition of Tyrode's solution was (in mM) 138.0 NaCl, 2.7 KCl, 1.8 CaCl 2, 1.0 MgCl 2, 11.9 NaHCO 3, 0.42 NaHPO 4, and 5.6 glucose, pH 7.4. Based on light microscopic examination, only preparations that had minimal proximal tubular contamination (<5%) were used for the experiments. Protein concentration was determined using the Bradford method ( 2 ) after vessels were suspended in 1 N NaOH for 2-3 days and homogenized.
Quantitation of EETs. Suspensions of PGMV ( 0.5 mg protein/ml) were incubated with NADPH (1 mM) in the presence or absence of 8-BrcAMP (1 mM) at 37°C for 15 min ( 7 ). MS-PPOH (12 µM) was added to the incubates for a 10- to 15-min preincubation at 4°C before the addition of NADPH. The PGMV and media were acidified to pH 4.0 with 9% formic acid. After addition of internal standards (2 ng of D 8 8,9-/11,12-EET, 1 ng of D 8 14,15-EET; Biomol), the samples were extracted twice with 2 x Vol ethyl acetate and evaporated to dryness. The samples were purified by reverse-phase (RP)-HPLC, and fractions containing EETs were derivatized and quantitated as described ( 7, 8 ). The endogenous EETs (ion m/z 319) were identified by comparison of GC retention times with authentic D 8 8, 9-, 11,12-, and 14,15-EET ( m/z 327) standards. The highly labile 5,6-EET was not measured.
Vascular cAMP determination. Isolated PGMV were incubated, as described above, in the presence and absence of 11,12-EET (3 nM-3 µM) and CGS-21680 (1-100 µM) for 5 min. The effect of MS-PPOH on CGS 21680-induced cAMP levels was determined by preincubating PGMV with MS-PPOH (12 µM) for 15 min and then washing the vessels in fresh buffer containing MS-PPOH before addition of CGS-21860. After incubation, the supernates were transferred to Eppendorfs containing 10 µl concentrated HCl and snap frozen in liquid nitrogen. To assay cAMP, samples and cAMP standards underwent acetylation and cAMP levels were measured by ELISA, according to the suggested procedure provided by Biomol.
Statistical analysis. Comparisons among several groups were made by analysis of variance followed by a modified t -test. Paired analyses were used when comparisons were made of data obtained from the same experimental preparation (i.e., basal and stimulated levels). Data are expressed as means ± SE, and a P value of <0.05 was considered significant.
RESULTS
A 2A R activation and 11,12-EET stimulate ADP-ribosylation of G proteins. The functional responses to CGS-21680 and 11,12-EET were evaluated on pressurized arcuate arteries [inner diameter (ID) 100-130 µm]. Arteries were superfused from the outset with indomethacin (10 µm) and L -NAME (200 µm), and only vessels that constricted to phenylephrine (20 nm) were studied. As activation of A 2A R with CGS-21680 stimulates G s ( 12 ), we hypothesized that 11,12-EET-induced dilation is mediated via G s. Therefore, we assessed the effect of 3-AM (10 µM; n = 6), an inhibitor of mono-ADP-ribosyltranferases, on responses to 11,12-EET (3 nM) and CGS-21680 (10 µM) and found that both were inhibited by 70% ( P < 0.05), whereas the response to SNP (10 µM) was unaffected ( Fig. 2 ).
Fig. 2. Arcuate renal arteries with internal diameter (ID) of 110-130 µm were pressurized (80 mmHg) and superfused with Krebs solution containing N G -nitro- L -arginine methyl ester ( L -NAME; 200 µM) and indomethacin (10 µM). The change in ID in response to addition of 11,12-EET, CGS-21860, or sodium nitroprusside (SNP) was measured in the presence and absence of 3-aminobenzamide. Values are means ± SE. * P < 0.05 vs. control; n = 6.
We next evaluated the effect of 11,12-EET on ADPR and cADPR formation in microdissected arcuate arteries. As shown in Fig. 3, 11,12-EET (100 nM), like cholera toxin (100 ng/ml), stimulated ADPR formation in homogenates of arcuate arteries compared with control.
Fig. 3. Top : reverse-phase (RP)-HPLC separation of standards of NAD, ADP-ribose (ADPR), and cyclic (c) ADPR (cADPR). Middle : RP-HPLC trace of incubates of microdissected arcuate artery homogenates showing the separation of NAD, ADPR, and cADPR. Bottom : stimulation of arcuate artery ADPR formation by 11,12-EET and cholera toxin. Values are means ± SE. AU, arbitrary units. * P < 0.05 vs. control; n = 4.
CGS-21680 and 11,12-EET vasodilate by activation of an adenylyl cyclase/PKA-dependent mechanism. As CGS-21680 is known to vasodilate by stimulating adenylyl cyclase/PKA activity, we used inhibitors of adenylyl cyclase/PKA to assess the contribution of this signaling pathway to the responses to both CGS-21680 and 11,12-EET in rat arcuate arteries. Superfusion of pressurized arcuate arteries with SQ-22536 (10 µM; n = 6), an inhibitor of adenylyl cyclase activity, decreased responses to CGS-21680 and 11,12-EET, reducing the ID from 22 ± 2 to 1 ± 1 µm ( P < 0.05) and from 24 ± 5 to 9 ± 5 µm ( P < 0.05), respectively ( Fig. 4 ). Myristolated PKI (14-22) amide (5 µM; n = 6), an inhibitor of PKA activity, diminished the dilator responses to CGS-21680 and 11,12-EET by 88 and 95%, respectively ( Fig. 5 ). The responses to SNP were not diminished by either SQ-22536 or mPKI ( Figs. 4 and 5 ).
Fig. 4. An adenylyl cyclase (AC) inhibitor, SQ-22536 (10 µM), significantly reduced the vasodilator response of 11,12-EET and abolished the effect of CGS-21680, whereas the dilator responses to 8-bromo-cAMP (8-BrcAMP) and SNP were unaffected. Values are means ± SE. * P < 0.05 vs. control; n = 6.
Fig. 5. Myristolated PKI (14-22) amide (mPKI; 5 µM), a PKA inhibitor, resulted in a significant reduction of the vasodilator response to 11,12-EET and CGS-21680 (CGS), respectively. The response to SNP was unaltered by mPKI. Values are means ± SE. * P < 0.05 vs. control; n = 6.
As these data suggest that activation of A 2A R is coupled to EET release upstream of adenylyl cyclase activation, we determined the effect of 5-min incubation with CGS-21680 and 11,12-EET on isolated PGMV cAMP levels. CGS-21680 (100 µM) stimulated cAMP release that was reduced by preincubation of PGMV with MS-PPOH, indicating that EETs are necessary for cAMP production. Incubation of PGMV with 11,12-EET (30 nM-3 µM) for 5 min resulted in a dose-dependent increase in cAMP levels ( Fig. 6 ). Further evidence that EET formation was upstream of adenylyl cyclase was obtained by incubating PGMV with 8-BrcAMP (1 mM), a permeable cAMP analog, and quantitating both EET formation and vasodilator activity. Incubation of PGMV with 8-BrcAMP did not stimulate EET release ( Fig. 7 ). Addition of 8-BrcAMP increased the ID of arcuate arteries by 36 ± 3 µm, but dilator responses to 8-BrcAMP were unaffected by either SQ-22536 ( Fig. 4 ) or by MSPPOH (12 µM; Fig. 7 ).
Fig. 6. Incubation of CGS-21680 with PGMV resulted in increased cAMP production compared with that of control. Stimulated cAMP levels, with CGS-21680, were reduced in the presence of methylsulfonyl-propargyloxyphenylhexanamide (MS-PPOH). Furthermore, incubation of PGMV with 11,12-EET resulted in a dose-dependent increase in cAMP levels. Values are means ± SE. * P < 0.05 vs. control; n = 4.
Fig. 7. Top : effect of 8-BrcAMP (1 mM) on the internal diameter of isolated, pressurized arcuate arteries measured in the presence and absence of an epoxygenase inhibitor (MS-PPOH; 12 µM). Values are means ± SE. * P < 0.05 vs. control; n = 6. Bottom : incubation of PGMV with 8-BrcAMP, in the presence and absence of MS-PPOH (12 µM), did not stimulate EET release. Values are means ± SE, n = 4.
CGS-21680 and 11,12-EET activates K Ca channel activity through a PP2A-dependent mechanism. We have investigated the contribution of PP2A activity as a signaling molecule involved in the renal vasodilator response to 11,12-EET and CGS-21680. Okadaic acid, an inhibitor of PP2A activity, reduced responses to both 11,12-EET and CGS-21680, whereas the responses to SNP were unaffected ( Fig. 8 ).
Fig. 8. Okadaic acid, an inhibitor of protein phosphatase 2A, reduced the vasodilator responses to 11,12-EET and CGS-21680. The response to SNP was unaffected. Values are means ± SE. * P < 0.05 vs. control; n = 6.
Although the role of K Ca channels in the mechanism of dilation of EETs has been elucidated, the effect of CGS-21680 on K Ca channel activity is unclear. As shown in Fig. 9, the responses to both 11,12-EET and CGS-21680 were significantly reduced by superfusion of iberiotoxin (100 nM), an inhibitor of K Ca channel activity.
Fig. 9. Iberiotoxin, a Ca 2+ -activated K + (K Ca ) channel blocker, inhibited the vasodilator responses to 11,12-EET and CGS-21860. Values are means ± SE. * P < 0.05 vs. control; n = 4.
DISCUSSION
This study provides further evidence that, in pressurized arcuate arteries, EETs are mediators of the dilator response to A 2A R agonists ( 7 ) and activation of A 2A R is coupled to EET release upstream of adenylyl cyclase activation. EETs mediate the vasodilation of A 2A R activation by stimulation of mono-ADP-ribosyltransferase ( Fig. 1 ).
Several investigators have reported that EETs stimulate G s activity ( 1, 13, 28, 29 ) in a manner similar to cholera toxin, an exogenous ADP-ribosyltransferase, which activates transfer of ADPR from NAD to G s with subsequent activation of adenylate cyclase ( 29 ). Endogenous ADP-ribosylation of G proteins and increased adenylyl cyclase activity occur in response to stimulation of adenosine receptors in adipocytes ( 10, 23 ). We showed that 11,12-EET, like cholera toxin, stimulated NAD-dependent ADPR formation by arcuate arterial homogenates and that 3-AM, an inhibitor of NAD-glycohydrolase, reduced the dilator response to 11,12-EET and CGS-21680, without affecting the responsiveness to SNP. In coronary vascular smooth muscle, EETs significantly increased ADPR production from NAD and increased K Ca channel activity, effects blocked by cibaron blue, an inhibitor of NAD-glycohydrolase activity ( 30 ). The 11,12-EET mediated intracellular signal transduction by transferring ADPR to a 52-kDa receptor protein, which is recognized by an antibody against G s ( 29 ). In coronary vessels, EETs increase production of ADPR, but not cADPR ( 29 ). However, in other tissues, e.g., pulmonary artery endothelial cells, hormonally stimulated increases in cADPR levels have been reported ( 1, 41 ). It has also been reported that rat arcuate arteries exhibit high cADPR formation ( 39 ); however, we did not observe a peak corresponding to cADPR and, therefore, it is possible that in our experiments, cADPR was converted to ADPR by cADPR hydrolase ( 39 ).
As adenosine binding to A 2A R triggers de novo synthesis of EETs ( 7 ), presumably A 2A R stimulation acts in a similar manner to that of bradykinin, which is linked to activation of phospholipases in various arterial preparations ( 14, 43 ). In rat cortical collecting ducts, adenosine inhibition of epithelial sodium channel activity is mediated by 11,12-EET via a phospholipase A 2 mechanism ( 42 ).
Both A 2A R and A 2B R are known to be coupled through G s to the activation of adenylyl cyclase and PKA activity ( 12 ) and K + channel activation ( 38, 40 ). We investigated the effect of sequential inhibition of this pathway and showed that the vasodilator responses to 11,12-EET and CGS-21680 were decreased in the presence of SQ-22536, an inhibitor of adenylyl cyclase activity. Furthermore, addition of 8-BrcAMP resulted in vasodilation, but responses were unaffected by either SQ-22536 or MS-PPOH. Moreover, vascular EET levels were not stimulated by 8-BrcAMP. That EETs were acting upstream of adenylyl cyclase was determined by comparing the activity of CGS-21680 and 11,12-EET on cAMP generation by PGMV. Incubation of PGMV with 11,12-EET resulted in an increase in cAMP levels, and CGS 21680-induced stimulation of cAMP was inhibited by preincubation of PGMV with MS-PPOH.
Imig et al. ( 19 ) have shown that inhibition of PKA activity significantly attenuated the afferent arteriole response to the 11,12 EET analog, and our data are in agreement with this study. Inhibition of PKA activity with mPKI inhibited responses to CGS-21680 and 11,12-EET and also reduced responses to 2-chloroadenosine, a nonselective agonist of all adenosine receptors (data not shown). From our study, we conclude that CGS-21680 and 11,12 EET dilate arcuate arteries in an adenylyl cyclase/PKA-dependent manner. However, in coronary arteries, neither 11,12-EET nor adenosine has been shown to stimulate adenylyl cyclase or PKA activity ( 16, 30 ). The apparent differences between the mechanism of 11,12 EET activity and the cAMP/PKA involvement in coronary vs. renal arteries, may reside in vessel size, as larger caliber vessels are less sensitive to the vasorelaxant effects of EETs. In coronary arteries ( 250-300 µm), 11,12-EET is 100-fold less active than afferent or arcuate arteries ( 20-100 µm) ( 7, 20, 30 ). Thus adenylyl cyclase/PKA may not be stimulated in larger coronary arteries or insufficient levels of cAMP formed for detection. In addition to bovine coronary artery studies, in human embryonic kidney 293 cells (HEK 293), 11,12-EET does not stimulate adenylyl cyclase or PKA activity. However, in HEK 293 cells, K Ca channel activity was increased by forskolin, an adenylyl cyclase activator, an effect blocked by KT-5720, a PKA inhibitor. The effect of 11,12 EET or cholera toxin on K Ca channel activity was unaffected by adenylyl cyclase inhibition with SQ-22536, thus providing evidence that 11,12-EET activity is independent of the adenylyl cyclase/PKA pathway ( 13 ). These discrepancies may relate to the degree of coupling of G s to adenylyl cyclase or to the quantity or isoforms of adenylyl cyclase and PKA in different cell types, vascular beds, or species. In bovine coronary arteries, G s has been proposed to directly increase K Ca channel activity ( 4, 28 ), whereas in HEK 293 cells the effect of cholera toxin on K Ca activity was unaffected by SQ-22536, suggesting poor coupling between G s and adenylyl cyclase in this cell line.
Although the vasodilator effect of EETs has been attributed to activation of K Ca channels, the mechanism by which A 2A R activation results in vasodilation is unclear. In rabbit arcuate arteries, the dilator response to CGS-21680 was inhibited by 40% with a mixture of K + channel blockers, although individual blockers were ineffective ( 38 ). In rat perfused hydronephrotic kidneys, adenosine-mediated vasodilation via A 2A R was blocked with glibenclamide, an inhibitor of K ATP channels ( 40 ). In our study, the vasodilator responses to 11,12-EET and CGS-21680 were diminished by iberiotoxin, an inhibitor of intermediary K + channels. Furthermore, we show that PKA-mediated phosphorylation of PP2A contributed to renal vasoactivity of 11,12-EET and CGS-21680, data in agreement with those reported for vasoactivity of 11,12-EET in microvascular myocytes ( 11 ). PP2A comprises a diverse family of phosphoserine- and phosphothreonine-specific enzymes and plays a prominent role in the regulation of specific signal transduction cascades and is often found in association with other phosphatases and kinases. PP2A interacts with a substantial number of other cellular proteins, which are PP2A substrates targeting PP2A to different subcellular compartments or affecting enzyme activity ( 24, 44 ).
In this study, we did not address an alternative transduction sequence that has been proposed for -adenoreceptor-induced dilation of rat pulmonary arteries ( 1 ), relaxation being partially due to a cAMP/PKA-dependent increase in cADPR synthesis and subsequent Ca 2+ release via ryanodine receptors, leading to activation of K Ca channels and membrane hyperpolarization.
In conclusion, we have addressed the commonality of the signal transduction pathway to the vasoactivity of A 2A R activation and 11,12-EET in pressurized arcuate arteries. Our findings support ADP-ribosylation of G proteins and generation of cAMP as key components in the signaling system activated by the A 2A R-EET pathway, producing renal vasodilation. The schema proposed in Fig. 1 indicates the signaling pathway by which adenosine stimulates EET formation, resulting in dilation of rat PGMV. As the components of the signaling pathway are present in both endothelial and vascular smooth muscle cells, this mechanism may have an autocrine and/or a paracrine function. As the A 2A receptors are located on endothelial cells of the renal vasculature ( 37 ), EETs may be released to activate ADPR of smooth muscle cells or, alternatively, downstream mediators such as cAMP may stimulate smooth muscle kinases.
The proposed signal transduction pathway for 11,12-EET-induced vasodilation may have broader implications as the antifibrinolytic properties of 11,12-EET have been linked to stimulation of G s /adenylyl cyclase/PKA activity ( 34 ), which may confer additional support to the notion of EETs acting as cardioprotective eicosanoids.
GRANTS
This research was supported in part by National Institutes of Health Grants HL-34300, HL-25394, and GM-31278.
ACKNOWLEDGMENTS
We thank Melody Steinberg for editorial assistance in preparing this manuscript.
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作者单位：1 Department of Pharmacology, New York Medical College, Valhalla, New York; and 2 Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas