AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 292: H2973-H2981, 2007. First published February 9, 2007; doi:10.1152/ajpheart.00419.2006
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/6/H2973    most recent
00419.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhao, H.
Right arrow Articles by Smith, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhao, H.
Right arrow Articles by Smith, C. J.

Differential effects of phosphodiesterase PDE-3/PDE-4-specific inhibitors on vasoconstriction and cAMP-dependent vasorelaxation following balloon angioplasty

Hong Zhao, John Quilley, David C. Montrose, Swarna Rajagopalan, Qizhi Guan, and Carolyn J. Smith

Department of Pharmacology, New York Medical College, Valhalla, New York

Submitted 26 April 2006 ; accepted in final form 9 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
It is known that cAMP and cGMP are important for vasorelaxation, and cyclic nucleotide phosphodiesterases (PDEs) regulate their levels. Balloon angioplasty (BAL) is associated with reduced cAMP and cGMP levels, and inhibition of PDE-3 reduces restenosis. In this study, we found that BAL increased PDE-3 activity, which affected vasoreactivity of rat aortic rings 24-h post-BAL; these were compared with intact (INT) and ex vivo endothelium-denuded rings (RUB) from sham rats. In BAL and RUB rings, vasorelaxant responses to ACh were abolished. The EC50 for phenylephrine (PE) was 1.8-fold less in RUB than in INT or BAL, whereas the maximal contractile effect of PE was greater in BAL than in INT or RUB. PDE-3 inhibitors reduced the maximal response to PE by >65% in BAL compared with 10–30% in INT and RUB; the reduction of the maximal response to U-46619 was 37% in BAL compared with 8% in INT with no reduction in RUB. PDE-4 inhibitors reduced PE-induced tone by <30% in an endothelium-dependent manner. Vasorelaxant responses to agonists that utilize cAMP were greatly impaired in BAL and RUB rings, and inhibition of PDE-3 enhanced the vasorelaxant responses in BAL or RUB. Inhibition of PDE-4 increased vasorelaxant responses to isoproterenol (ISO) to a much lesser degree. Thus PDE-3 and PDE-4 inhibitors exhibited differential effects on PE-induced tone and vasorelaxant responses to ISO. Inhibition of PDE-3 also produced a greater increase in cAMP in BAL than INT or RUB rings. These results suggest that increased PDE-3 activity after BAL may promote a vasospastic state and that the reduction in cAMP may, possibly, influence vessel remodeling.

rat aorta; cyclic adenosine 3', 5'-cyclic monophosphate

BALLOON ANGIOPLASTY (BAL) is widely used to treat obstructive coronary artery disease, yet restenosis following BAL limits the efficacy of this procedure, which can be improved with a drug-eluting stent (3). In preclinical studies, it has been shown that enhanced smooth muscle cell (SMC) proliferation and migration account for the neointimal thickening that follows BAL (6, 21). Cyclic AMP inhibits proliferation of SMC (20) and other cells in vitro (4, 11, 44), and reduced cAMP levels were found in rat aorta following BAL (40). Thus increasing cAMP levels after BAL should help reduce neointimal thickening. Moreover, cAMP is an important second messenger for vasorelaxation (41), and reduced cAMP levels in BAL should affect vasoreactivity. Recent studies on {alpha}1-adrenoceptor-mediated vasoconstriction after BAL have yielded conflicting results, where both hyperreactivity and hyporeactivity were reported (1, 17, 29). The effects of BAL on responses to several vasorelaxant agents have also been studied, but only one addressed the cAMP pathway using a beta-adrenergic agonist (16). In that study, the authors showed an initially decreased beta-agonist-induced relaxation followed by an increase during neointimal formation.

Phosphodiesteases (PDEs) play an integral role in regulation of cyclic nucleotide levels. There are 11 PDE families in mammals, and differential splicing of certain families leads to at least 50 isoenzyme variants (for review, see Ref. 35). PDE-1, PDE-3, PDE-4, and PDE-5 are present in vascular SMC; PDE-1, PDE-2, PDE-3, PDE-4, and PDE-5 are also present in the endothelial cells (46, 47, 60). The contribution of PDE-3 and PDE-4 to the regulation of VSMC proliferation/migration has been well studied. One PDE-3 inhibitor, cilostazol, has been shown to downregulate the transcription factor E2F (22) and upregulate anti-oncogenes (cell cycle arrest) p53 and p21 (39). The cilostazol for restenosis trial indicates that cilostazol may be useful for countering restenosis after angioplasty (10, 59). However, to our knowledge, there are no reports concerning the effects of BAL-induced alterations in PDE activity as it relates to vasoactivity.

We hypothesized that BAL increases PDE activities and thus reduces cAMP and cGMP levels, which, in turn, modifies vascular responsiveness. In the present study, we examined the effect of BAL injury on vascular reactivity to an {alpha}1-adrenoceptor agonist and to several cAMP-dependent vasorelaxants. We also examined the effects of PDE-3 or PDE-4 inhibitors on these responses, as these agents affect vasorelaxation in the intact (INT) rat aorta (24, 25, 27, 28, 42, 50), canine coronary arteries (14), and human mesenteric vessels (26). We compared vasoreactivity of BAL aortic rings to sham INT and endothelium-denuded (RUB) rat aortic rings to further investigate the effect of injury following BAL, beyond the effect of denudation of the endothelium and the acute loss of endothelial-derived nitric oxide (NO).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
Angioplasty. Male Sprague-Dawley rats (326–350 g) were purchased from Charles River Laboratories (Wilmington, MA) and housed for at least 3 days before experiments. Animal use was approved by the New York Medical College Institutional Animal Care and Use Committee. Rats were anesthetized by intraperitoneal injection of 70 mg/kg nembutal sodium, and a 2-Fr Fogarty embolectomy balloon catheter (Edwards Life Sciences, Irvine, CA) was introduced through the left femoral artery to the aortic arch and pulled back 1 cm. The balloon was then inflated to 750–850 mmHg, and the catheter was partially withdrawn to the branch of the femoral artery. This process was repeated three times. The catheter was removed, the femoral artery sutured, and the incision clipped. For sham rats, the uninflated catheter was inserted only once into the left femoral artery without entering the aorta.

Preparation of isolated aortic rings. Preliminary studies from this laboratory showed that PDE-3 and PDE-4 expression and activity started to increase as early as 24 h after BAL, and the increase lasted 4–7 days; thus the 24-h time point was chosen for the experiments to examine the early effects of PDEs on the blood vessel. Following laparotomy and thoracotomy, the aorta from the heart to just above the kidneys was excised, flushed with Krebs solution (118 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4·7H2O, 2.5 mM CaCl2·2H2O, 10 mM dextrose, 25 mM sodium bicarbonate) to remove blood, and dissected free of periadventitial tissue. For contractility studies, the aorta was cut into 2.5-mm rings and kept in ice-cold Krebs until mounted onto hooks in a tissue bath chamber. For ex vivo removal of endothelium from sham rings (RUB), a toothpick was inserted into the ring and rolled gently for two revolutions. Hematoxylin and eosin staining confirmed that endothelium was removed in both BAL and RUB rings (data not shown).

Vascular contractility studies. Aortic vascular reactivity was measured using the Myobath System, and data from six to eight channels (6–8 rings) were collected using the MP100 Data Acquisition System (both from World Precision Instruments, Sarasota, FL). Rings stretched to 2 g of tension (baseline) were equilibrated for 60 min in 10-ml oxygenated Krebs buffer at 37°C. Experiments were initiated by measuring contractions to KCl (10–100 mM or 80 mM). Then contractile responses to the {alpha}1-agonist phenylephrine (PE) (1 nM–3 µM) were examined. After a maximal PE contraction was established, rings were relaxed with acetylcholine (ACh) (3 nM–3 µM). Subsequently, relaxant responses to one of the following drugs were evaluated in rings precontracted with PE (3 µM): isoproterenol (ISO), a nonselective beta-adrenoceptor agonist; carbacyclin [prostacyclin (PGI2) analog]; forskolin (FSK) to directly activate adenylate cyclase; or PDE-3 inhibitor OPC3911. Next, effects of PDE-3 inhibitors [OPC3911, cilostazol (OPC13013), and milrinone] or PDE-4 inhibitors (Ro20-1724 and cilomilast) were studied; rings were incubated with inhibitors for 10 min before eliciting a maximal contraction to PE. Contractile responses to U-46619 (thromboxane A2 receptor agonist, 100 nM) in rings incubated with OPC3911 (1 µM) for 25 min were also studied. PDE inhibitors cannot be easily washed out, and, therefore, only one inhibitor could be tested on each ring. In addition, maximum PE contractions were different in the three types of rings. Consequently, relaxant responses to receptor agonists were expressed in two ways: 1) as the percentage of the initial PE-induced contraction (%PE contraction) to compare ISO sensitivity, and 2) as absolute changes in tension. Since FSK cannot be easily removed after application, only one dose response could be obtained for each ring. Therefore, rings incubated with OPC3911 were compared with untreated control rings. Finally, the response to KCl (80 mM) was tested again at the end of the experiment to ensure that there was no temporal change in reactivity. Aortic rings were washed at least three times with Krebs solution, and tension was allowed to return to baseline after each set of treatments in two or three replicate rings, and the rings reequilibrated for at least 20 min before the next drug addition. Initial experiments showed no differences in the contractile responses of the proximal and distal parts of the aorta, and in subsequent experiments rings were randomized.

All of the PDE-3 and PDE-4 inhibitors compete for the active site of the enzyme. The reported IC50 values for selective inhibition of vascular PDE-3 activity are as follows: OPC3911 0.03 µM; milrinone 0.49 µM; and cilostazol 0.2 µM (27, 51). The reported IC50 for cilomilast inhibition of PDE-4 in U937 cells is 30 nM (19) and 0.4–0.7 µM for Ro20-1724 for recombinant human PDE-4B/4D (19, 56). Our studies typically used 0.1 µM OPC3911 (3.3 times IC50) and 0.5 µM cilostazol (2.5 times IC50). Since the effects of PDE-4 inhibitors were not as pronounced in preliminary studies, relatively higher concentrations were used vs. the PDE-3-selective drugs: 0.5 µM cilomilast (16.7 times IC50) and 10 µM Ro20-1724 (14.3 to 25 times IC50).

Medial SMC preparation and PDE activity assay. Medial SMC were prepared as described (37). Briefly, aortas were rapidly excised from the animal, rinsed in medium 199 with Hanks’ balanced salt solution, and incubated for 25 min in the same medium plus 1% collagenase, 0.25% elastase, and 1% soy bean trypsin inhibitor. After incubation, the adventitia and endothelium were rapidly stripped away from the media. Medial SMC were snap-frozen in liquid nitrogen and stored at –70°C. Tissues were then powdered and homogenized on ice with homogenate buffer (290 mM sucrose, 10 mM MOPS, 1.0 mM EGTA, 3.0 mM NaN3, 1.0 mM DTT, 3.0 mM benzamidine, 10 µg/ml pepstatin, 10 µg/ml leupeptin, 10 µg/ml antipain, 0.8 mM PMSF) and polytroned for two 10-s periods. The samples were then subjected to differential centrifugation at 1,000 g for 10 min, 7,500 g for 10 min followed by 40,000 g for 60 min at 4°C. The 40,000 g supernatants, the cytosolic fractions, were use for PDE assay.

For the cAMP PDE activity assay, 3H-cAMP was added at 100,000 dpm to buffer containing 50 mM HEPES, 0.1 mM EGTA, 8.3 mM MgCl2, and 0.1 µM cAMP in 200-µl volumes. Various concentrations of the test protein sample in 100 µl were added to start the reaction, and the mixture was incubated at 30°C for 15 min. The reaction was stopped by adding 100 µl stop mix (7.5 mM cAMP/3.33 mM 5'-AMP in 0.17 N HCl). Samples were neutralized with 100 µl buffer containing 250 mM Tris and 250 mM NaOH, pH 8.0. Then 100 µl venom mix (0.1875% Crotalus atrox in 100 mM Tris, pH 8.0) was added, and samples were incubated at 30°C for 20 min. The sample was then applied to DEAE-Sephadex A-25 column, and the eluate was collected in scintillation vials with 10 ml Beckman Readi-Gel. The column was washed with 3.6 ml H2O, and the eluate was also collected in the same vial. The radioactivity was measured in a liquid scintillation counter. PDE-3 or PDE-4 activities were determined as the activities inhibited by OPC3911 (1.5 µM) or Ro20-1724 (15 µM), respectively, added to the assay mix.

Western blot analysis of protein kinase A phosphorylation. For Western blot analysis, aortic rings were frozen in liquid nitrogen, homogenized in homogenate buffer, and centrifuged at 16,000 g for 20 min. Forty microliters of the supernatants were mixed with 10 µl of 5x sample buffer (312.5 mM Tris·HCl, pH 6.8, 10% SDS, 25% glycerol, 0.05% bromophenol blue), incubated at 95°C for 5 min, and then subjected to Western blot analysis with Bio-Rad (Hercules, CA) system and a protein kinase A (PKA) substrate antibody from Cell Signaling (no. 9621, Beverly, MA) at 1:3,000 dilution.

Determination of cAMP and cGMP levels in aorta. Aortic rings (3.5–4 mm) were incubated in Krebs buffer at 37°C for 10 min and then incubated with different drugs for another 10 min. The rings were then frozen in liquid nitrogen, homogenized in 5% TCA, and centrifuged at 16,000 g for 15 min. The supernatant was then subjected to water-saturated ether extraction for three times and heated at 70°C to remove trace amounts of ether. The cAMP and cGMP levels were then measured with Cayman (Ann Arbor, MI) cAMP or cGMP enzyme immunoassay kits. The dried TCA pellet was weighed, and the cAMP and cGMP levels were normalized to the weight.

Statistical analysis. The results shown are means ± SE. Data were analyzed by the Student's t-test with Excel (single point) or two-way ANOVA with SigmaStat 2.0 (whole curve). P < 0.05 was considered as significant. Curve fitting with SlideWrite V6 (1-Site Ligand model, log scale of drug concentration) was used to estimate EC50 values. The drug maximum reaction (Rmax) was the value for either contraction above baseline, or the relaxation of PE-induced contraction, measured at the highest drug concentration tested.

Drugs and chemicals. NaCl, MgSO4, CaCl2·2H2O, dextrose, and L-ascorbic acid were obtained from Fisher Scientific (Suwanee, GA), and carbacyclin from Biomol (Plymouth Meeting, PA). OPC3911 and cilostazol were generous gifts from Dr. Junichi Kambayashi (Otsuka, Rockville, MD). Cilomilast (SKB 207499) was generously provided by Dr. Theodore J. Torphy (formerly affiliated with SmithKline Beecham). All others were obtained from Sigma (St. Louis, MO).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANT
 REFERENCES
 
Differential effects of BAL and ex vivo endothelium denudation on contractile response to PE. To compare the effects of BAL and ex vivo removal of the endothelium, rings from BAL rats and two sets of rings from sham rats (INT and RUB) were studied. Contractile responses to PE differed between the INT and the other two groups (Fig. 1A). The EC50 for PE was 1.8-fold less in RUB (52 nM) vs. INT (95 nM) (P < 0.001), while the Rmax contraction at 3 µM PE was unchanged by acute removal of endothelium (Fig. 1A). In contrast, the EC50 for PE in BAL (92 nM) was similar to that for INT, but the Rmax was greater in BAL (*P < 0.005 vs. INT). Contractile responses to KCl were similar in INT, RUB, and BAL rings (Fig. 1B). The responses to 80 mM KCl were not different among the three groups of rings at the beginning and end of the contractile studies and were unaffected by the inhibitors, although the contractile response to KCl was increased by 12–13% in all three groups of rings at the end of the study (data not shown). It should be noted that, when the contractile response to PE was expressed as a percentage of the response to KCl (80 mM), the Rmax in RUB rings was greater than that in INT (P < 0.05) but was not different from that in BAL. ACh-induced relaxation of PE contracted rings was markedly impaired in both BAL and RUB rings compared with INT rings (data not shown).


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 1. Responses to vasoconstrictors in intact (INT) and deendothelialized (RUB) aortic rings and those from rats subjected to balloon angioplasty (BAL). A: dose-response curves to phenylephrine (PE) (INT: {square}, n = 16; RUB: {triangleup}, n = 14; BAL: bullet, n = 13; *P < 0.05, BAL vs. RUB or INT). B: dose-response curves to KCl (n = 5 for each group).

 
Differential effects of PDE-3 and PDE-4 inhibitors on PE-induced contraction. To determine the effect of PDE-3 and PDE-4 inhibitors on PE vasoconstriction, aortic rings were incubated with PDE-3 or PDE-4 inhibitors and then contracted with PE (3 µM). The resulting contraction (change in tension, dTinhibitor) was compared with a prior PE contraction of the same ring without the PDE inhibitor (dTwithout). As shown in Fig. 2A, in INT rings, two of the three PDE-3 inhibitors and both PDE-4 inhibitors significantly reduced PE-induced contraction by 20% (OPC3911, milrinone, Ro20-1724) to 30% (cilomilast) (*P < 0.05 vs. absence of inhibitors). In RUB rings, inhibitory effects of PDE-3 inhibitors were similar to INT (*P < 0.05 vs. absence of inhibitors), except that cilostazol also produced a significant 10% reduction in PE tone (*P < 0.001 vs. absence of cilostazol). In contrast to the INT rings, the PDE-4 inhibitors did not significantly affect the PE-induced contraction in RUB or BAL. In BAL rings, all three PDE-3 inhibitors produced a pronounced reduction (65 to 85%) in PE-induced contraction (*P < 0.0001 vs. absence of inhibitors), which was three to four times greater than the effects seen in the other groups (xP < 0.01 BAL vs. INT or RUB). A time control for a second PE concentration curve without any inhibitor showed no difference in INT and RUB rings. BAL showed a slight increase of 12–13% compared with the first contraction at maximum PE concentration (3 µM) (data not shown).


Figure 2
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 2. Effects of inhibitors of phosphodiesterase (PDE)-3 and PDE-4 on PE-induced contractions in INT and RUB aortic rings and those from rats subjected to BAL. A: PE (3 µM) contractions after incubation of PDE-3 and PDE-4 inhibitors normalized to PE contractions without inhibitors (INT, open bars; RUB, shaded bars; BAL, solid bars). dTinhibitor, change in tension with inhibitor; dTwithout, change in tension without inhibitor. For OPC3911 (0.1 µM), n = 8–12 per group; for milrinone (0.3 µM), n = 4–5 per group; for cilostazol (0.5 µM), Ro20-1724 (10 µM), and cilomilast (0.5 µM), n = 4 per group. *P < 0.05, with vs. absence of inhibitors; xP < 0.01, BAL vs. INT or RUB. B: vasorelaxant effects of the PDE-3 inhibitor OPC3911 in PE-contracted (3 µM) aortic rings (INT, {square}; RUB, {triangleup}; BAL, bullet; n = 5 for each group). *P < 0.05, BAL vs. INT.

 
To further clarify the effect of PDE-3 inhibitors on vasoconstriction, rings from the three groups were contracted with PE (3 µM), and then cumulative concentration-response curves were generated for OPC3911. As shown in Fig. 2B, the curve for OPC3911-induced vasorelaxation was shifted to the left in BAL rings compared with INT rings (*P < 0.05, INT vs. BAL). The concentration response in RUB rings was similar to that in INT.

Effect of PDE-3 inhibitors, OPC3911 and cilostazol, on ISO-induced relaxation. BAL, RUB, and INT rings were contracted with PE (3 µM), and then cumulative concentration-response curves were generated for ISO. The rings were then washed, incubated with the PDE-3 inhibitor, OPC3911 (0.1 µM) or cilostazol (0.5 µM), contracted with PE, and relaxed with ISO again as above. As shown in Fig. 3, ISO-induced relaxation was impaired in both BAL and RUB rings compared with INT rings (*P < 0.001, BAL or RUB vs. INT, whole curve). When normalized to corresponding PE contractions, after treatment with OPC3911, sensitivity to ISO in both BAL and RUB rings was indistinguishable from that of INT rings (Fig. 3A). Rmax with ISO (3 µM) was greatly improved in both BAL and RUB; in BAL, with OPC3911 (0.1 µM), only 8.96% PE contraction remained, compared with 61.9% without OPC3911, and, in RUB, it was 4.54 vs. 64.3% (Fig. 3A). In terms of change in tension, as shown in Fig. 2, PE-induced contraction was greatly reduced in BAL by OPC3911 (Fig. 3B) such that a minimal effect of ISO was required to produce maximum relaxation. In contrast, PE-induced contraction in INT and RUB was only slightly affected by OPC3911, but the dose-response curve to ISO in RUB was markedly shifted to the left (Fig. 3B). Another PDE-3 inhibitor, cilostazol, was tested for the effects on the ISO dose-response curve. Cilostazol (0.5 µM, Fig. 3C) showed similar leftward shifting of the ISO curve as OPC3911 (0.1 µM). Cilostazol improved the ISO sensitivity (*P < 0.001, BAL or RUB vs. INT without inhibitors, whole curve) in both BAL and RUB rings to near that of INT rings, and Rmax for ISO (3 µM) was greatly enhanced in both BAL and RUB. With cilostazol (0.5 µM) in BAL, only 13.0% PE contraction remained, compared with 59.0% without cilostazol, and, in RUB, the values were 19.8 vs. 63.7%. Another PDE-3 inhibitor, milrinone, showed similar results as cilostazol (data not shown).


Figure 3
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 3. Effect of the PDE-3 inhibitors, OPC3911 (A and B; OPC, 0.1 µM) and cilostazol (C; Cil, 0.5 µM), on isoproterenol (ISO)-induced relaxation of INT (squares; n = 4–10) and RUB (triangles; n = 4–8) aortic rings and those from rats after BAL (circles; n = 5–9). A: normalized to PE contraction with OPC3911. B: absolute change in tension with OPC3911. C: normalized to PE contraction with cilostazol. Rings were precontracted with PE (3 µM). Dotted lines and open symbols, untreated rings; solid lines and symbols, with inhibitors. *P < 0.001, BAL or RUB vs. INT.

 
Effect of PDE-3 inhibitor, OPC3911, on carbacyclin- and FSK-induced relaxation. BAL, RUB, and INT rings were contracted with PE (3 µM), and then cumulative concentration-response curves were generated for carbacyclin (Fig. 4A) and FSK (Fig. 4B). For carbacyclin, the rings were then washed, incubated with PDE-3 inhibitor OPC3911 (0.1 µM), contracted with PE, and relaxed with carbacyclin again. Since FSK cannot be washed from the rings after application, a separate set of rings was incubated with OPC3911 and compared with their untreated controls. Carbacyclin- and FSK-induced relaxation was impaired in both BAL and RUB rings compared with INT rings (*P < 0.001, BAL or RUB vs. INT, whole curve). OPC3911 (0.1 µM) improved ISO sensitivity in both BAL and RUB rings to near that of INT rings for both carbacyclin and FSK. Rmax for carbacyclin (3 µM) was affected as follows: in BAL treated with OPC3911 (0.1 µM), 31.3% PE contraction remained compared with 90.2% without OPC3911; in RUB, the values were 31.7 vs. 98.7%. FSK fully relaxed the rings with or without OPC3911, so no change of Rmax for FSK (3 µM) was observed.


Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 4. Effect of the PDE-3 inhibitor, OPC3911 (OPC, 0.1 µM), on relaxant responses to carbacyclin (A) and forskolin (FSK; B) in INT (squares) and RUB (triangles) aortic rings and those from rats following BAL (circles). For carbacyclin, n = 4 per group. For FSK, n = 4–7 for INT and RUB; n = 6–10 for BAL. Rings were precontracted with PE (3 µM). Dotted lines and open symbols, untreated rings; solid lines and symbols, with inhibitors. *P < 0.001, BAL or RUB vs. INT.

 
Effect of PDE-3 inhibitor, OPC3911, on U-46619-induced contraction. The effect of PDE-3 inhibitor, OPC3911, on vasoconstriction induced by U-46619 was studied. Again, the contraction in the presence of OPC3911 (1 µM; dTinhibitor) was compared with a prior U-46619 contraction of the same ring without the inhibitor (dTwithout). As shown in Fig. 5, incubation with OPC3911 reduced the tension differentially in the three types of rings (*P < 0.05, BAL vs. INT or RUB). The tension was reduced by 37% in BAL rings, whereas it was reduced by only 8% in INT rings with no reduction in RUB rings. However, in contrast to PE responses and OPC3911, for an effect on U-46619-induced contractile responses, a higher concentration of OPC3911 and a longer period of incubation were required, consistent with a report that PDE-3 inhibitors are more potent in relaxing rings contracted with norepinephrine than those contracted with U-46619 (43).


Figure 5
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 5. Effects of PDE-3 inhibitor OPC3911 (1 µM) on U-46619-induced contractions in INT and RUB aortic rings and those from rats subjected to BAL. U-46619 (100 nM) contractions after incubation of OPC3911 were normalized to U-46619 contractions without OPC3911 (INT, open bar; RUB, shaded bar; BAL, solid bar; n = 3 for each group; *P < 0.05, BAL vs. INT or RUB).

 
Effect of PDE-4 inhibitors, Ro20-1724 and cilomilast, on ISO-induced relaxation. The PDE-4 inhibitors, Ro20-1724 and cilomilast, were tested for their effects on responses to ISO in all three types of aortic rings (*P < 0.001, BAL or RUB vs. INT, whole curve). Both inhibitors increased the sensitivity to ISO. However, even with 10 µM of Ro20-1724 (Fig. 6A) or 0.5 µM of cilomilast (Fig. 6B), the ISO response in both BAL and RUB rings was less sensitive compared with that in INT rings (xP < 0.001, BAL or RUB vs. INT for both inhibitors, whole curve). Ro20-1724 (10 µM) moderately increased Rmax for ISO (3 µM) in BAL and RUB: in BAL, 40.5% PE contraction remained compared with 51.0% without Ro20-1724; in RUB rings, the values were 36.5 vs. 69.1%. Similar results were obtained for cilomilast (0.5 µM): in BAL with and without cilomilast, the respective values for the remaining PE contraction were 46.1 vs. 67.6% and in RUB, 46.5 vs. 73.4%.


Figure 6
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 6. Effects of Ro20-1724 (Ro, 10 µM; A) (n = 4 per group) and cilomilast (Cilom, 0.5 µM; B) (n = 3–5 per group) to inhibit PDE-4 on relaxant responses to ISO in INT (squares) and RUB (triangles) aortic rings and those from rats subjected to BAL (circles). Rings were precontracted with PE (3 µM). Dotted lines and open symbols, untreated rings; solid lines and symbols, with inhibitors. *P < 0.001, BAL or RUB vs. INT; xP < 0.001, BAL or RUB in the presence of PDE-4 inhibitor vs. INT in the presence of PDE-4 inhibitor.

 
PDE-3 and PDE-4 activity and cAMP-cGMP level in BAL and control aorta. PDE-3 and PDE-4 activity was measured in the cytosolic fraction of aortic smooth muscle from rats 24 h after BAL or sham operation. As shown in Fig. 7A, PDE-3 activity was increased 65.8% (*P < 0.005, BAL vs. sham), and PDE-4 activity was increased 52.1%, although not significantly (P = 0.076, BAL vs. sham).


Figure 7
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 7. PDE activities and cAMP-cGMP levels in aortas from rats subjected to BAL and sham-operated control rats (CON). A: activity of PDE-3 and PDE-4 in the cytosol of aortic medial smooth muscle cells from BAL (shaded bar, n = 3) and CON rats (open bar, n = 3). B: cAMP levels in BAL and INT and RUB aortic rings under basal conditions (n = 4–8 for each group) and after incubation with PE (n = 4 for each group); or after incubation with the PDE-3 inhibitor OPC3911 (OPC; n = 4–8 for each group); or with dual inhibitors of PDE-3 (OPC3911) and PDE-4 (Ro20-1724) (OPC + Ro; n = 4 for each group); or with dual inhibitors plus FSK (OPC + Ro + FSK; n = 4–8 for each group). C: cGMP levels in BAL and INT and RUB aortic rings under basal conditions (n = 4–8 for each group) and after incubation with PE (n = 4 for each group); or after incubation with OPC3911 (OPC; n = 4–8 for each group); or with dual inhibitors of OPC3911 and Ro20-1724 (OPC + Ro; n = 4 for each group); or with dual inhibitors plus FSK (OPC + Ro + FSK; n = 4–8 for each group). *P < 0.05, different treatments vs. basal level; {dagger}P < 0.05, BAL or RUB vs. INT; {ddagger}P < 0.05, BAL vs. RUB.

 
Increased PDE-3 activity should affect cAMP and, perhaps, cGMP levels. We thus measured these levels in BAL and INT and RUB aorta rings under basal conditions, or after incubation with PE, or after incubation with different combinations of PDE-3 and PDE-4 inhibitors and FSK (Fig. 7, B and C). We did not observe a difference in basal cAMP levels. In the presence of PE, cAMP levels tended to be lower in BAL than in INT, but the difference did not reach significance (P = 0.083), although the difference was significant between RUB and INT ({dagger}P < 0.05). When the PDE-3 inhibitor OPC3911 was added, there was a significant increase in cAMP in BAL (*P < 0.05, drug treatment vs. basal level), which was higher than in INT or RUB ({dagger}P < 0.05, BAL vs. INT; and {ddagger}P < 0.05, BAL vs. RUB). Dual inhibition of PDE-3 (OPC3911) and PDE-4 (Ro20-1724) further increased cAMP levels in all groups of rings (*P < 0.01, drug treatment vs. basal level), with BAL higher than RUB but not INT ({ddagger}P < 0.05, BAL vs. RUB). RUB showed lower cAMP levels than INT ({dagger}P < 0.05). When FSK was added with the dual inhibitors, cAMP levels increased in all groups of rings (*P < 0.05, drug treatment vs. basal level), but greater cAMP levels were seen in BAL compared with INT or RUB rings ({dagger}P < 0.05, BAL vs. INT; and {ddagger}P < 0.05, BAL vs. RUB). cGMP levels were lower in BAL compared with RUB ({ddagger}P < 0.05) but not with INT. In the presence of PE, cGMP levels were lower in BAL and RUB compared with INT ({dagger}P < 0.05), but the effect of the PDE inhibitors and FSK on the cGMP levels was less clear. Only OPC3911 alone but not dual inhibitors with or without FSK increased cGMP levels in INT and BAL (*P < 0.05, drug treatment vs. basal level). With OPC3911, RUB but not BAL showed lower cGMP level than INT ({dagger}P < 0.05). cGMP levels were lower in BAL and RUB compared with INT when dual PDE-3 and PDE-4 inhibitors, OPC3911 and Ro20-1724, were present ({dagger}P < 0.05, BAL vs. INT). When FSK was added with the inhibitors, no differences were observed between BAL and INT or RUB, but cGMP levels in RUB were lower than in INT ({dagger}P < 0.05).

PKA phosphorylation in BAL and CON aortic rings. Western blot analysis of PKA phosphorylation was carried out with an antibody raised against the PKA serine phosphorylation site. The signal strength of the bands between 50 and 150 kDa was quantified. Quantification was also carried out for Ponceau S staining for the similar region, and the ratio was used to estimate PKA phosphorylation levels. As shown in Fig. 8, A and B, the two bands around the 85-kDa marker contributed most to the quantitation, and the phosphorylation was weaker in BAL than in CON. The phosphorylation level in BAL is ~68% of that in CON (Fig. 8C, *P < 0.05).


Figure 8
View larger version (39K):
[in this window]
[in a new window]

  		Fig. 8. Decreased PKA phosphorylation in homogenates of rat aorta 24 h after BAL compared with sham-operated CON rats. A: Western blots showing PKA phosphorylation levels in BAL or CON aorta. B: Ponceau S staining of the blot showing protein loading. C: relative PKA phosphorylation level expressed as the ratio of PKA blot intensity to Ponceau S intensity in BAL (shaded bar, n = 3) and CON (open bar, n = 3) normalized to CON. The signal strengths of similar regions between 50 and 150 kDa for PKA blot or Ponceau S blot were quantified. *P < 0.05, BAL vs. CON.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANT
 REFERENCES
 
We have shown that PDE-3 activity is increased following BAL and that PDE-4 activity tended to increase but did not reach significance. We thus hypothesized that changes in activity would modify vascular responsiveness by influencing the levels of cyclic nucleotides. We showed that BAL modifies vascular responsiveness via mechanisms in addition to removal of the endothelium, which include increased activity of PDE-3.

We compared aortic rings from rats subjected to BAL to rings from sham rats. Because BAL results in the removal of the endothelium, we also used rings from sham rats where the endothelium was removed by rubbing. The differences between BAL and RUB rings are readily apparent when considering PE- or U-46619-induced contraction in the presence of PDE-3 inhibitors. Inhibition of PDE-3 reduced the contractile response to PE or U-46619 to a similar degree in INT and RUB rings but produced a much greater effect in BAL rings, whereby the maximal contractile response to PE or U-46619 was markedly reduced. This is consistent with increased PDE-3 activity in BAL rings, where inhibition of PDE-3 in BAL rings would result in a relatively greater increase in cAMP to counter the contractile effects of PE or U-46619. As responses to PE or U-46619 in RUB and INT rings were much less affected by inhibitors of PDE, this is consistent with the interpretation that PDE is at basal levels and, therefore, inhibition produces a smaller increase in cAMP. This idea was further supported by the observations that the PDE-3 inhibitor OPC3911, alone or with a PDE-4 inhibitor and FSK, induced a greater increase in cAMP levels in BAL vs. INT or RUB. We did not observe a difference in the basal cAMP level between BAL and INT or RUB aortic rings despite increased cytosolic medial smooth muscle PDE-3 activity in BAL. It is possible that the change in cAMP is subtle and/or is localized or that the sensitivity of the cAMP assay is not enough to elucidate the difference. We thus examined PKA phosphorylation by Western blot with an antibody raised against PKA serine phosphorylation site. The PKA phosphorylation level was lower in BAL than INT rings. The cGMP levels tended to be lower in BAL than control, but the difference did not achieve significance. Furthermore, the inhibitors of PDE and FSK had a limited effect on cGMP levels, and we suggest that the effects of PDE-3 inhibitors on contractile responses to PE were primarily via effects on cAMP levels.

The effect of PDE-3 on vasoreactivity after BAL is specific for this PDE family, as the effect of inhibition of PDE-4 was much less pronounced, suggesting that this isoform plays a minor role in hydrolyzing cyclic nucleotides that are increased in response to contraction. In addition, in INT rings, although inhibitors of both PDE-3 and PDE-4 moderately reduced PE-induced contraction, the effects of PDE-4 inhibitors are endothelium dependent, while those of PDE-3 inhibitors are not. This is consistent with previous studies showing that relaxation of PE-contracted rat aortic rings by PDE-3 inhibitors was endothelium independent (27), whereas relaxation induced by PDE-4 inhibitors was endothelium dependent (24). These different effects of PDE-3 and PDE-4 inhibitors may relate to different cellular locations for PDE-3 and PDE-4 (38). The hydrophobic NH2-terminal regions of PDE-3 are important for its membrane localization (53), while the distribution of PDE-4 is affected by interaction with B-arrestins (45) or A kinase anchoring proteins (2).

The different PE responses in BAL, INT, and RUB rings were agonist selective, since KCl-induced contractions were comparable in all groups before and after the inhibitors, whereas the PDE-3 inhibitors moderately reduced the response to PE in INT and RUB rings but produced a marked reduction in BAL rings. The maximal response to PE was greater in BAL rings than that in INT or RUB rings without PDE-3 inhibitors, but the difference between BAL and RUB was not apparent when the responses were expressed as a percentage of the response to KCl. We speculate that any increase in the maximal response to PE in BAL rings may result from increased activity of PDE, with a resultant reduction of cyclic nucleotides that may moderate contractile mechanisms. Surprisingly, the 50% effective dose for PE-induced contraction in RUB rings was approximately one-half of that for INT rings and was similar for BAL and INT rings. These findings do not agree with those of Lippolis et al. (29), who reported reduced maximal contractile responses to PE and KCl in BAL rat carotid artery. However, Heijenbrok et al. (17) found increased sensitivity to PE in the rat carotid artery immediately after injury, whereas contractile responses to KCl were unchanged, and Accorsi-Mendonca et al. (1) reported that the maximal response to PE was reduced in the injured rat carotid artery but enhanced in the contralateral vessel 4–7 days after BAL. Possible explanations for these variable results include different sources of vascular tissue (57), the degree of damage to the vessel (36), or different times of testing after injury.

We also studied the effects of PDE-3 inhibitors on cAMP-dependent vasorelaxant responses to ISO, carbacyclin, and FSK. cAMP-dependent vasorelaxant responses were greatly impaired in both BAL and RUB rings contracted with PE, which may relate to removal of endothelial NO and a reduction in cGMP, which inhibits PDE-3 activity (for reviews, see Refs. 9, 34). Therefore, removal of the endothelium and NO could be expected to increase PDE-3 activity with a resultant decrease in cAMP levels in response to cAMP-dependent agonists. This was supported by the results that, in the presence of PE, cAMP and cGMP levels were lower in RUB and also tended to be lower in BAL than that in INT, but the levels were similar in RUB and BAL. It may also explain why PE contractions in both BAL and RUB were different from those in INT. When normalized to maximum PE-induced contractions, responses to cAMP-dependent vasorelaxants were enhanced similarly in BAL and RUB rings by PDE-3 inhibitors to that observed in INT rings. However, as mentioned above, PDE-3 inhibitors greatly reduced PE-induced tension in BAL, and, therefore, the additional decrease in tension to produce maximal relaxation in response to an agonist was relatively small. This is consistent with the notion that only a small further increase in cAMP in response to an agonist is required for maximal relaxation in BAL. In contrast, PE-induced contraction in INT and RUB rings was much less affected by inhibition of PDE-3, and the leftward shift in the dose-response curve to agonists was much less in INT. In RUB rings, the effects of vasorelaxant agonists were markedly enhanced in the presence of PDE-3 inhibitors, such that the dose-response curves were similar to those of INT rings. Thus there is a synergistic interaction in RUB that is clearly different from BAL. Inhibitors of PDE-4 also enhanced the impaired vasorelaxant activity of ISO in RUB and BAL rings, but the effect was much less marked than that for inhibition of PDE-3, again suggesting a minor role for this isoform in vasorelaxation.

The ability of PDE-3 inhibitors to shift to the left the dose-response curves for all vasorelaxant agents including FSK, which directly activates adenylate cyclase, in RUB rings suggests that a combination of PDE-3 inhibitors with cAMP-dependent agonists could result in a better vasorelaxation. This could be useful in pathological conditions such as artherosclerosis and hypertension. Indeed, previous studies showed that milrinone restored the impaired ISO-response curve in hypoxia-induced pulmonary hypertension, whereas the PDE-4 inhibitor rolipram was much less effective (55). Furthermore, low doses of PDE inhibitors amplified the pulmonary vasodilatory effects of inhaled prostacyclin in rabbits (48, 49) and humans (13) with pulmonary hypertension.

Increased PDE-3 activity can account for our observations of altered vasoreactivity in BAL rings, and we found that PDE-3 activity was significantly increased following BAL. PDE-3 can be activated as a result of phosphorylation by PKA or Akt (PKB) (23, 33, 54, 58), and Akt can be activated by PDGF through phosphatidylinositol 3-kinase (5, 12), a pathway that is involved in regulating the vascular SMC phenotype (15). In rat carotid artery, PDGF was upregulated 6 h after BAL injury, and the PDGF receptors were upregulated 1–2 wk after BAL injury (32). We postulate that upregulation of PDGF could activate Akt, which, in turn, activates PDE-3. PDE-4 activity was increased after BAL, albeit insignificantly, and PDE-4 can be activated by PKA phosphorylation or ERK-mediated phosphorylation (8, 18). Chronic regulation of PDE-3 and PDE-4 expression by a cAMP-dependent feedback stimulus also has been demonstrated (7, 30, 31, 33, 52), and the mechanism underlying upregulation of PDEs by BAL remains to be determined.

In summary, increased PDE-3 activity after BAL modifies vascular responsiveness, which may have important functional implications for angioplasty.


   GRANT
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
REFERENCES
 
This study is supported by National Heart, Lung, and Blood Institute Grant 1R01HL069061 and a grant from the American Diabetes Association.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Zhao, Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595 (e-mail: hong_zhao{at}nymc.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 

  1. Accorsi-Mendonca D, Correa FM, Paiva TB, de Souza HP, Laurindo FR, de Oliveira AM. The balloon catheter induces an increase in contralateral carotid artery reactivity to angiotensin II and phenylephrine. Br J Pharmacol 142: 79–88, 2004.[CrossRef][ISI][Medline]
  2. Asirvatham AL, Galligan SG, Schillace RV, Davey MP, Vasta V, Beavo JA, Carr DW. A-kinase anchoring proteins interact with phosphodiesterases in T lymphocyte cell lines. J Immunol 173: 4806–4814, 2004.[Abstract/Free Full Text]
  3. Bhargava B, Karthikeyan G, Abizaid AS, Mehran R. New approaches to preventing restenosis. BMJ 327: 274–279, 2003.[Free Full Text]
  4. Blomhoff HK, Blomhoff R, Stokke T, deLange DC, Brevik K, Smeland EB, Funderud S, Godal T. cAMP-mediated growth inhibition of a B-lymphoid precursor cell line Reh is associated with an early transient delay in G2/M, followed by an accumulation of cells in G1. J Cell Physiol 137: 583–587, 1988.[CrossRef][ISI][Medline]
  5. Burgering BM, Coffer PJ. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376: 599–602, 1995.[CrossRef][Medline]
  6. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest 49: 327–333, 1983.[ISI][Medline]
  7. Conti M, Jin SL. The molecular biology of cyclic nucleotide phosphodiesterases. Prog Nucleic Acid Res Mol Biol 63: 1–38, 1999.[ISI][Medline]
  8. Conti M, Richter W, Mehats C, Livera G, Park JY, Jin C. Cyclic AMP-specific PDE4 phosphodiesterases as critical components of cyclic AMP signaling. J Biol Chem 278: 5493–5496, 2003.[Free Full Text]
  9. Degerman E, Belfrage P, Manganiello VC. Structure, localization, and regulation of cGMP-inhibited phosphodiesterase (PDE3). J Biol Chem 272: 6823–6826, 1997.[Free Full Text]
  10. Douglas JS Jr, Holmes DR Jr, Kereiakes DJ, Grines CL, Block E, Ghazzal ZM, Morris DC, Liberman H, Parker K, Jurkovitz C, Murrah N, Foster J, Hyde P, Mancini GB, Weintraub WS. Coronary stent restenosis in patients treated with cilostazol. Circulation 112: 2826–2832, 2005.[Abstract/Free Full Text]
  11. Dugan LL, Kim JS, Zhang Y, Bart RD, Sun Y, Holtzman DM, Gutmann DH. Differential effects of cAMP in neurons and astrocytes. Role of B-raf. J Biol Chem 274: 25842–25848, 1999.[Abstract/Free Full Text]
  12. Franke TF, Yang SI, Chan TO, Datta K, Kazlauskas A, Morrison DK, Kaplan DR, Tsichlis PN. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81: 727–736, 1995.[CrossRef][ISI][Medline]
  13. Haraldsson SA, Kieler-Jensen N, Ricksten SE. The additive pulmonary vasodilatory effects of inhaled prostacyclin and inhaled milrinone in postcardiac surgical patients with pulmonary hypertension. Anesth Analg 93: 1439–1445, 2001.[Abstract/Free Full Text]
  14. Harris AL, Grant AM, Silver PJ, Evans DB, Alousi AA. Differential vasorelaxant effects of milrinone and amrinone on contractile responses of canine coronary, cerebral, and renal arteries. J Cardiovasc Pharmacol 13: 238–244, 1989.[ISI][Medline]
  15. Hayashi K, Takahashi M, Kimura K, Nishida W, Saga H, Sobue K. Changes in the balance of phosphoinositide 3-kinase/protein kinase B (Akt) and the mitogen-activated protein kinases (ERK/p38MAPK) determine a phenotype of visceral and vascular smooth muscle cells. J Cell Biol 145: 727–740, 1999.[Abstract/Free Full Text]
  16. Heijenbrok FJ, Mathy MJ, Pfaffendorf M, van Zwieten PA. Beta-adrenergic responses are significantly enhanced in rat carotid artery with intimal hyperplasia. Naunyn Schmiedebergs Arch Pharmacol 362: 276–283, 2000.[CrossRef][ISI][Medline]
  17. Heijenbrok FJ, van der Wal AC, Pfaffendorf M, van Zwieten PA. Morphologic and functional consequences of intimal hyperplasia in the rat carotid artery. Naunyn Schmiedebergs Arch Pharmacol 357: 133–142, 1998.[CrossRef][ISI][Medline]
  18. Houslay MD, Adams DR. PDE4 cAMP phosphodiesterases: modular enzymes that orchestrate signalling cross-talk, desensitization and compartmentalization. Biochem J 370: 1–18, 2003.[CrossRef][ISI][Medline]
  19. Huang Z, Ducharme Y, Macdonald D, Robichaud A. The next generation of PDE4 inhibitors. Curr Opin Chem Biol 5: 432–438, 2001.[CrossRef][ISI][Medline]
  20. Indolfi C, Avvedimento EV, Di Lorenzo E, Esposito G, Rapacciuolo A, Giuliano P, Grieco D, Cavuto L, Stingone AM, Ciullo I, Condorelli G, Chiariello M. Activation of cAMP-PKA signaling in vivo inhibits smooth muscle cell proliferation induced by vascular injury. Nat Med 3: 775–779, 1997.[CrossRef][ISI][Medline]
  21. Jackson CL, Schwartz SM. Pharmacology of smooth muscle cell replication. Hypertension 20: 713–736, 1992.[Abstract/Free Full Text]
  22. Kim MJ, Park KG, Lee KM, Kim HS, Kim SY, Kim CS, Lee SL, Chang YC, Park JY, Lee KU, Lee IK. Cilostazol inhibits vascular smooth muscle cell growth by downregulation of the transcription factor E2F. Hypertension 45: 552–556, 2005.[Abstract/Free Full Text]
  23. Kitamura T, Kitamura Y, Kuroda S, Hino Y, Ando M, Kotani K, Konishi H, Matsuzaki H, Kikkawa U, Ogawa W, Kasuga M. Insulin-induced phosphorylation and activation of cyclic nucleotide phosphodiesterase 3B by the serine-threonine kinase Akt. Mol Cell Biol 19: 6286–6296, 1999.[Abstract/Free Full Text]
  24. Komas N, Lugnier C, Stoclet JC. Endothelium-dependent and independent relaxation of the rat aorta by cyclic nucleotide phosphodiesterase inhibitors. Br J Pharmacol 104: 495–503, 1991.[ISI][Medline]
  25. Lindgren S, Andersson KE. Comparison of the effects of milrinone and OPC 3911 with those of isoprenaline, forskolin and dibutyryl-cAMP in rat aorta. Gen Pharmacol 22: 617–624, 1991.[ISI][Medline]
  26. Lindgren S, Andersson KE, Belfrage P, Degerman E, Manganiello VC. Relaxant effects of the selective phosphodiesterase inhibitors milrinone and OPC 3911 on isolated human mesenteric vessels. Pharmacol Toxicol 64: 440–445, 1989.[ISI][Medline]
  27. Lindgren S, Rascon A, Andersson KE, Manganiello V, Degerman E. Selective inhibition of cGMP-inhibited and cGMP-noninhibited cyclic nucleotide phosphodiesterases and relaxation of rat aorta. Biochem Pharmacol 42: 545–552, 1991.[CrossRef][ISI][Medline]
  28. Lindgren SH, Andersson TL, Vinge E, Andersson KE. Effects of isozyme-selective phosphodiesterase inhibitors on rat aorta and human platelets: smooth muscle tone, platelet aggregation and cAMP levels. Acta Physiol Scand 140: 209–219, 1990.[ISI][Medline]
  29. Lippolis L, Sorrentino R, Popolo A, Maffia P, Nasti C, d. d'Emmanuele V, Marzocco S, Autore G, Pinto A. Time course of vascular reactivity to contracting and relaxing agents after endothelial denudation by balloon angioplasty in rat carotid artery. Atherosclerosis 171: 171–179, 2003.[CrossRef][ISI][Medline]
  30. Liu H, Maurice DH. Expression of cyclic GMP-inhibited phosphodiesterases 3A and 3B (PDE3A and PDE3B) in rat tissues: differential subcellular localization and regulated expression by cyclic AMP. Br J Pharmacol 125: 1501–1510, 1998.[CrossRef][ISI][Medline]
  31. Ma D, Wu P, Egan RW, Billah MM, Wang P. Phosphodiesterase 4B gene transcription is activated by lipopolysaccharide and inhibited by interleukin-10 in human monocytes. Mol Pharmacol 55: 50–57, 1999.[Abstract/Free Full Text]
  32. Majesky MW, Reidy MA, Bowen-Pope DF, Hart CE, Wilcox JN, Schwartz SM. PDGF ligand and receptor gene expression during repair of arterial injury. J Cell Biol 111: 2149–2158, 1990.[Abstract/Free Full Text]
  33. Manganiello VC, Degerman E. Cyclic nucleotide phosphodiesterases (PDEs): diverse regulators of cyclic nucleotide signals and inviting molecular targets for novel therapeutic agents. Thromb Haemost 82: 407–411, 1999.[ISI][Medline]
  34. Maurice DH. Cyclic nucleotide phosphodiesterase-mediated integration of cGMP and cAMP signaling in cells of the cardiovascular system. Front Biosci 10: 1221–1228, 2005.[CrossRef][ISI][Medline]
  35. Maurice DH, Palmer D, Tilley DG, Dunkerley HA, Netherton SJ, Raymond DR, Elbatarny HS, Jimmo SL. Cyclic nucleotide phosphodiesterase activity, expression, and targeting in cells of the cardiovascular system. Mol Pharmacol 64: 533–546, 2003.[Abstract/Free Full Text]
  36. Merhi Y, Guidoin R, Provost P, Leung TK, Lam JY. Increase of neutrophil adhesion and vasoconstriction with platelet deposition after deep arterial injury by angioplasty. Am Heart J 129: 445–451, 1995.[CrossRef][ISI][Medline]
  37. Miano JM, Tota RR, Vlasic N, Danishefsky KJ, Stemerman MB. Early proto-oncogene expression in rat aortic smooth muscle cells following endothelial removal. Am J Pathol 137: 761–765, 1990.[Abstract]
  38. Mongillo M, McSorley T, Evellin S, Sood A, Lissandron V, Terrin A, Huston E, Hannawacker A, Lohse MJ, Pozzan T, Houslay MD, Zaccolo M. Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases. Circ Res 95: 67–75, 2004.[Abstract/Free Full Text]
  39. Morishita R. A scientific rationale for the CREST trial results: evidence for the mechanism of action of cilostazol in restenosis. Atheroscler Suppl 6: 41–46, 2005.[ISI][Medline]
  40. Murakami H, Yayama K, Miao RQ, Wang C, Chao L, Chao J. Kallikrein gene delivery inhibits vascular smooth muscle cell growth and neointima formation in the rat artery after balloon angioplasty. Hypertension 34: 164–170, 1999.[Abstract/Free Full Text]
  41. Murray KJ. Cyclic AMP and mechanisms of vasodilation. Pharmacol Ther 47: 329–345, 1990.[CrossRef][ISI][Medline]
  42. Nakamura T, Houchi H, Minami A, Sakamoto S, Tsuchiya K, Niwa Y, Minakuchi K, Nakaya Y. Endothelium-dependent relaxation by cilostazol, a phosphodiesteras III inhibitor, on rat thoracic aorta. Life Sci 69: 1709–1715, 2001.[CrossRef][ISI][Medline]
  43. Onomoto M, Tsuneyoshi I, Yonetani A, Suehiro S, Matsumoto K, Sakata R, Kanmura Y. Differential pharmacologic sensitivities of phosphodiesterase-3 inhibitors among human isolated gastroepiploic, internal mammary, and radial arteries. Anesth Analg 101: 950–956, 2005.[Abstract/Free Full Text]
  44. Pastan IH, Johnson GS, Anderson WB. Role of cyclic nucleotides in growth control. Annu Rev Biochem 44: 491–522, 1975.[CrossRef][ISI][Medline]
  45. Perry SJ, Baillie GS, Kohout TA, McPhee I, Magiera MM, Ang KL, Miller WE, McLean AJ, Conti M, Houslay MD, Lefkowitz RJ. Targeting of cyclic AMP degradation to beta 2-adrenergic receptors by beta-arrestins. Science 298: 834–836, 2002.[Abstract/Free Full Text]
  46. Polson JB, Strada SJ. Cyclic nucleotide phosphodiesterases and vascular smooth muscle. Annu Rev Pharmacol Toxicol 36: 403–427, 1996.[CrossRef][ISI][Medline]
  47. Sadhu K, Hensley K, Florio VA, Wolda SL. Differential expression of the cyclic GMP-stimulated phosphodiesterase PDE2A in human venous and capillary endothelial cells. J Histochem Cytochem 47: 895–906, 1999.[Abstract/Free Full Text]
  48. Schermuly RT, Ghofrani HA, Enke B, Weissmann N, Grimminger F, Seeger W, Schudt C, Walmrath D. Low-dose systemic phosphodiesterase inhibitors amplify the pulmonary vasodilatory response to inhaled prostacyclin in experimental pulmonary hypertension. Am J Respir Crit Care Med 160: 1500–1506, 1999.[Abstract/Free Full Text]
  49. Schermuly RT, Roehl A, Weissmann N, Ghofrani HA, Schudt C, Tenor H, Grimminger F, Seeger W, Walmrath D. Subthreshold doses of specific phosphodiesterase type 3 and 4 inhibitors enhance the pulmonary vasodilatory response to nebulized prostacyclin with improvement in gas exchange. J Pharmacol Exp Ther 292: 512–520, 2000.[Abstract/Free Full Text]
  50. Schoeffter P, Lugnier C, Demesy-Waeldele F, Stoclet JC. Role of cyclic AMP- and cyclic GMP-phosphodiesterases in the control of cyclic nucleotide levels and smooth muscle tone in rat isolated aorta. A study with selective inhibitors. Biochem Pharmacol 36: 3965–3972, 1987.[CrossRef][ISI][Medline]
  51. Schror K. The pharmacology of cilostazol. Diabetes Obes Metab 4, Suppl 2: S14–S19, 2002.[CrossRef][ISI][Medline]
  52. Shakur Y, Holst LS, Landstrom TR, Movsesian M, Degerman E, Manganiello V. Regulation and function of the cyclic nucleotide phosphodiesterase (PDE3) gene family. Prog Nucleic Acid Res Mol Biol 66: 241–277, 2001.[ISI][Medline]
  53. Shakur Y, Takeda K, Kenan Y, Yu ZX, Rena G, Brandt D, Houslay MD, Degerman E, Ferrans VJ, Manganiello VC. Membrane localization of cyclic nucleotide phosphodiesterase 3 (PDE3). Two N-terminal domains are required for the efficient targeting to, and association of, PDE3 with endoplasmic reticulum. J Biol Chem 275: 38749–38761, 2000.[Abstract/Free Full Text]
  54. Smith CJ, Vasta V, Degerman E, Belfrage P, Manganiello VC. Hormone-sensitive cyclic GMP-inhibited cyclic AMP phosphodiesterase in rat adipocytes. Regulation of ins. J Biol Chem 266: 13385–13390, 1991.[Abstract/Free Full Text]
  55. Wagner RS, Smith CJ, Taylor AM, Rhoades RA. Phosphodiesterase inhibition improves agonist-induced relaxation of hypertensive pulmonary arteries. J Pharmacol Exp Ther 282: 1650–1657, 1997.[Abstract/Free Full Text]
  56. Wang P, Myers JG, Wu P, Cheewatrakoolpong B, Egan RW, Billah MM. Expression, purification, and characterization of human cAMP-specific phosphodiesterase (PDE4) subtypes A, B, C, and D. Biochem Biophys Res Commun 234: 320–324, 1997.[CrossRef][ISI][Medline]
  57. Ward MR, Kanellakis P, Ramsey D, Jennings GL, Bobik A. Response to balloon injury is vascular bed specific: a consequence of de novo vessel structure? Atherosclerosis 151: 407–414, 2000.[CrossRef][ISI][Medline]
  58. Wijkander J, Landstrom TR, Manganiello V, Belfrage P, Degerman E. Insulin-induced phosphorylation and activation of phosphodiesterase 3B in rat adipocytes: possible role for protein kinase B but not mitogen-activated protein kinase or p70 S6 kinase. Endocrinology 139: 219–227, 1998.[Abstract/Free Full Text]
  59. Zhang Z, Foster JK, Kolm P, Jurkovitz CT, Parker KM, Murrah NV, Anderson GT, Douglas JS Jr, Weintraub WS. Reduced 6-month resource use and costs associated with cilostazol in patients after successful coronary stent implantation: results from the Cilostazol for RESTenosis (CREST) trial. Am Heart J 152: 770–776, 2006.[CrossRef][ISI][Medline]
  60. Zhu B, Strada S, Stevens T. Cyclic GMP-specific phosphodiesterase 5 regulates growth and apoptosis in pulmonary endothelial cells. Am J Physiol Lung Cell Mol Physiol 289: L196–L206, 2005.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/6/H2973    most recent
00419.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhao, H.
Right arrow Articles by Smith, C. J.
Right arrow Search for Related Content
PubMed
Right arrow