Adenosine responses in experimental vein bypass grafts☆☆☆★★★

Article Outline

• Abstract
• MATERIALS AND METHODS
  • Membrane preparation and radioligand binding
  • Data and statistical analysis
• RESULTS
  • Adenosine receptors and their responses
  • Role of NO, prostaglandins, and membrane channels
  • Role of G-proteins
• DISCUSSION
• Acknowledgements
• References
• Copyright

Abstract 

Purpose: Veins develop unique endothelial and smooth muscle cell physiologic phenotypes after implantation as vein grafts in the arterial circulation. Receptor-mediated relaxation is reduced or absent in these grafts. This study examines the responses of vein grafts to adenosine, a known potent endogenous vasodilator, and compares these responses with the native veins and arteries. Methods: The presence of adenosine receptors (A₁ and A₂) by radioligand binding and the in vitro responses to the adenosine analogue N-ethyl-carboxyamido-adenosine were assessed in precontracted common carotid jugular vein bypass grafts placed in New Zealand white rabbits for 28 days. Results were compared with those obtained in precontracted jugular veins and carotid arteries. Both endothelialized and de-endothelialized vessels were studied. The contribution of nitric oxide (NO) and prostanoid production to relaxation was also determined by preincubation with the specific inhibitors <!--**Check here**-->l-monomethylarginine and indomethacin, respectively. Finally, the in vitro relaxation in response to the respective A₁ and A₂ adenosine receptor agonists R-phenyl-isopropyl-adenosine and CGS-21680 was also examined. Results: The results show that the adenosine-induced responses of the vein grafts differ from those of the jugular vein and carotid artery. First, in contrast to the carotid artery, vein graft adenosine-mediated relaxation is NO and prostanoid dependent, similar to the response of the jugular vein. Second, A₁ receptor activation in the vein graft produces an endothelium-dependent contractile response. Third, the A₂ receptor-mediated responses in the vein grafts appear to be independent of the endothelium. Fourth, radioligand studies show the presence of both receptor subtypes (A₁ and A₂) on the vein grafts with a ratio (A₁/A₂ = 1.4) closer to that of the jugular vein (A₁/A₂ = 1.8) than to that seen in the carotid artery (A₁/A₂ = 0.5). Conclusions: Vein graft adenosine responses appear to be unique in that they neither maintain a venous phenotype nor acquire an arterial phenotype. In particular, endothelial A₁ receptor-mediated responses change from relaxation to contraction, and receptor activated NO-mediated relaxation is preserved within the vein grafts probably via A₂ receptor signalling. (J Vasc Surg 1998;28:929-38.)

The use of veins as cardiovascular conduits remains important for cardiovascular surgeons. The universal response of veins after insertion into the arterial circulation is the development of intimal hyperplasia,¹ a proliferative smooth muscle cell and connective tissue lesion, which develops early after insertion of the vein into the arterial circulation. Clinically, this process appears to stabilize within the first 2 postoperative years in most vein grafts. The development of intimal hyperplasia has been associated with alterations in the functions of both endothelial and smooth muscle cells.² Experimental models and retrieved clinical material have shown alterations in the contractile and relaxant functions mediated by the endothelial and smooth muscle cells of vein grafts.¹ Receptor-activated endothelial-derived relaxation (mediated with nitric oxide [NO] or prostanoid) appears to be impaired compared with the native vein. Smooth muscle cell contractility and nonendothelial-mediated relaxation are also altered compared with native vein. These studies suggest changes in the endothelial cell and smooth muscle cell physiologic phenotypes in these vessels as compared with native vessels.¹, ² However, most of these studies on relaxation in vein grafts have focused on systems that involve cyclic guanosine monophosphate–mediated relaxation. The response of vein grafts to a cyclic adenosine-monophosphate activator adenosine is, at present, unknown. Adenosine is considered a major component of local hemostasis in all organ systems and can regulate a diverse series of functions, such as bronchoconstriction, smooth muscle cell tone, neurotransmitter release, platelet and white blood cell function, cardiac rate and contractility, and renal renin release and lipolysis.³ Endothelial and smooth muscle cells possess a series of specific adenosine receptors to produce relaxation under homeostatic conditions. These adenosine receptors are, for the most part, coupled to adenylate cyclase; the high-affinity A₁ receptor inhibits the enzyme, and the low-affinity A₂ receptor stimulates the enzyme. In addition, adenosine-induced relaxation has been shown in various organ systems to be mediated with NO, prostanoids, and adenosine triphosphate–dependent K⁺ channels. This study examines in vitro adenosine receptor–mediated responses and receptor expression in experimental vein grafts, jugular veins, and carotid arteries of the rabbit.

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MATERIALS AND METHODS 

Male New Zealand white rabbits weighing 2.0 to 2.5 kg were used in the study. The animals underwent a right carotid interposition bypass grafting with the ipsilateral external jugular vein.⁴ In this model, after 4 weeks, stable intimal hyperplasia developed, with a confluent endothelium and no evidence of an inflammatory infiltrate or residual platelet deposition. The animals were killed 4 weeks after surgery with pentobarbital sodium (Anthony Products Co, Arcadia, Calif). The vein graft, the contralateral jugular vein, and the common carotid artery were harvested for in vitro isometric tension studies. Mid portions of the vein graft were chosen because of the uniform characteristics of the intimal hyperplasia in these areas, which would minimize changes induced by vein artery mismatching, surgical variability, and different degrees of flow disturbance and intimal hyperplasia development. Animal care complied with the “Principles of Laboratory Animal Care,” as formulated by the National Society for Medical Research, and the “Guide for the Care and Use of Laboratory Animals,” issued by the National Institute of Health (US Department of Health and Human Services, NIH Publication No 86-23, revised 1985).

The vein graft, jugular vein, and carotid artery were isolated as described previously.⁴ The middle of each vessel was sectioned in situ into 4 5-mm segments and excised. Each vessel ring was suspended from 2 stainless steel hooks in 5 mL–capacity organ baths that contained oxygenated Krebs solution (122 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgCl₂, 2.5 mmol/L CaCl₂, 15.4 mmol/L NaHCO₃, 1.2 mmol/L KH₂PO₄ and 5.5 mmol/L glucose). The solution was maintained at 37°C and bubbled with a mixture of 95% O₂ and 5% CO₂. One hook was fixed to the bottom of the bath, and the other was connected to a force transducer (Myograph F-60, Narco Bio-Systems, Houston, Tex). The isometric responses of the tissue were recorded on a multichannel polygraph (Physiograph Mk111-S, Narco Bio-Systems). The tissues then were placed under 0.5g tension and allowed to equilibrate in physiologic Krebs solution for 1 hour. During the equilibration period, the Krebs solution was replaced every 15 minutes. After equilibration, the optimal resting tension for each ring was determined by the maximal response to a modified oxygenated Krebs solution that contained 60 mmol/L KCl, 66.7 mmol/L NaCl, 1.2 mmol/L MgCl₂, 2.5 mmol/L CaCl₂, 15.4 mmol/L NaHCO₃, 1.2 mmol/L KH₂PO₄, and 5.5 mmol/L glucose at resting tensions ranging from 0.5 μg to 2.5 μg. On the basis of these results, each ring was set at its optimal tension for subsequent dose response experiments. The potassium chloride [60 mmol/L] contractile responses were 150 ± 50 mg, 300 ± 30 mg, and 820 ± 40 mg for the jugular vein, vein graft, and carotid artery, respectively. All vessels (external jugular vein, common carotid artery, and vein graft) were precontracted with prostaglandin F_2a to achieve 70% of the potassium chloride contractile response. Both endothelialized and de-endothelialized vessels were examined. De-endothelialization of the rings was achieved by mechanical denudation of the rings with a balloon fogarty (Baxter Healthcare Corp, Irvine, Calif) embolectomy catheter before mounting and was confirmed by a lack of relaxation to acetylcholine as previously described.⁵

There were 3 series of experiments. In the first series, the responses to N-ethyl-carboxyamido-adenosine (NECA; 1010 to 104 mol/L), R-phenyl-isopropyl-adenosine (R-PIA; 1010 to 104 mol/L; selective A₁ agonist), and CSG-21680 (CSG; 1010 to 104 mol/L; selective A₂ agonist) were examined. In the second series, the effects of xanthine amine congener (XAC; 8-4-[2-amino-ethylamino]carbonyl-methyl-oxy-phenyl-1,3-dipropylxanthine; 105 mol/L for 30 minutes; specific A₁ blockade), N^G-monomethyl-L-arginine (L-NMMA; 105 mol/L for 30 minutes; NO synthesis inhibition), glybenclamide (105 mol/L for 30 minutes; K⁺ channel blockade), and indomethacin (105 mol/L for 30 minutes; cyclo-oxygenase inhibition) on the responses were tested. In the third series, the linkage of the responses to guanosine triphosphate (GTP) binding proteins was tested with incubation with pertussis toxin, an uncoupler of G_i/G_o (100 ng/mL for 60 minutes) that produces complete adenosine diphosphate ribosylation of G_i/G_o in these tissues, or cholera toxin, an uncoupler of G_S (200 ng/mL for 180 minutes) that promotes complete adenosine diphospate ribosylation of G_S in these tissues.⁶ All drugs were obtained from Sigma Chemical Co (St Louis, Mo).

Membrane preparation and radioligand binding 

Vein grafts, jugular veins, and carotid arteries were harvested at 28 days and cleaned of all adventitia before radioligand assay studies. The single layer of endothelial cells is unlikely to make a significant contribution to the data obtained. For [¹²⁵I]PAPA-APEC (2-[4-(2-{[4-aminophenyl]-methylcarbonyl}-ethyl)-phenyl]-ethylamino-5'-N-ethylcarboxamidoadenosine) and [¹²⁵I]APNEA (N⁵-2-[4-amino-3-iodophenyl]ethyladenosine) binding, membranes were prepared as previously described⁷ and resuspended in 50 mmol/L Tris-HCl, 1 mmol/L ethylene-diamine tetraacetic acid, pH 7.4, that contained 10 mmol/L MgCl₂ (50/10) and approximately 3 units/mL adenosine deaminase. Light vesicles were prepared as previously described.⁸ Before preparation of the vesicles, vessels first were treated with concanavalin A (0.25 mg/mL) for 30 minutes at 37°C to prevent sequestration of the A₁ adenosine receptor during the homogenization. [¹²⁵I]PAPA-APEC and [¹²⁵I]APNEA were synthesized and radiolabeled as described previously.⁷, ⁹

For A₁ adenosine receptor binding, membranes (20 to 40 μg/assay tube) were incubated for 1 hour at 37°C with 1 nmol/L [¹²⁵I]APNEA in a total volume of 250 μL of 50/10 buffer (50 mmol/L Tris-HCl, 10 mmol/L MgCl₂, 1 mmol/L ethylenediamine tetraacetic acid). This incubation time was optimal to ensure steady-state binding as determined from [¹²⁵I]APNEA association curves (data not shown). Theophylline (1 mmol/L) was used to define nonspecific binding, which normally averaged from 30% to 50% of the total binding. After incubation, membranes were rapidly filtered over 25 mm glass fiber filters (No 32; Schleicher and Schuell, Keene, NH) by vacuum and were washed 3 times with 3 mL of ice cold 50/10/1 buffer that contained 0.03% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propansulfonate). Filters were allowed to extract for at least 12 hours in toluene-based scintillation fluid before counting.

For A₂ adenosine receptor binding, membranes (60 to 100 μg of protein) were incubated for 1 hour with [¹²⁵I]PAPA-APEC (1 nmol/L) in a total volume of 250 μL of 50/10 buffer. Nonspecific binding was defined with 1 mmol/L theophylline and averaged about 50% of total binding at a concentration of the radioligand at the K_d. This concentration of theophylline was optimal and defined a similar level of nonspecific binding as that defined by NECA (100 μmol/L). Separation and counting of the bound radioligands was performed as described above. Results are expressed as a ratio of the binding of A₁ to that seen for A₂.

Data and statistical analysis 

The dose-response curves were analyzed, and the IC₅₀ value (median effective inhibitory concentration) for relaxation of each ring was calculated by logistic analysis and expressed as the pD₂ (–log₁₀[IC₅₀]).¹⁰ Where contraction occurs, the EC₅₀ value (median effective concentration) is reported. Data are presented as the arithmetric mean ± the standard error of the mean (SEM). Statistical differences between groups were tested with either one-way analysis of variance or with post hoc Tukey-Kramer comparison testing. A P value of less than .05 was regarded as significant.

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RESULTS 

Adenosine receptors and their responses 

Radioligand binding studies showed the presence of A₁ and A₂ receptors in the vein grafts with a ratio (A₁/A₂) of 1.4, in the jugular veins with a ratio of 1.8, and in the carotid arteries with a ratio of 0.5. To evaluate the effect of adenosine activation on vasorelaxation, we stimulated all 3 vessels with the adenosine agonist NECA. Vein grafts responded with a blunted maximal relaxation (up to 104 mol/L) compared with either the jugular vein or the carotid artery (Figs 1 through 3).

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• Fig. 1. 

  Adenosine-mediated relaxation in vein grafts. Cumulative dose response curves to NECA (○), CGS (▵) and R-PIA (□) in endothelialized (A) and de-endothelialized (B) vein grafts. NECA response with (●) and without (○) preincubation with XAC is shown in C. Values are the mean ± standard error of the mean of percent precontracted tension. Sensitivities are presented in Tables I and II.

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• Fig. 2. 

  Adenosine-mediated relaxation in jugular veins. Cumulative dose response curves to NECA (○), CGS (▵) and R-PIA (□) in endothelialized (A) and de-endothelialized (B) jugular veins. Values are the mean ± standard error of the mean of percent precontracted tension. Sensitivities are presented in Tables I and II.

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• Fig. 3. 

  Adenosine-mediated relaxation in carotid arteries. Cumulative dose response curves to NECA (○), CGS (▵ ) and R-PIA (□) in endothelialized (A) and de-endothelialized (B) carotid arteries. Values are the mean ± standard error of the mean of percent precontracted tension. Sensitivities are presented in Tables I and II.

Both the jugular veins and the carotid arteries relaxed in response to NECA (Figs 2 and 3). The lower doses of the NECA physiologically are more relevant and closer to what is experienced in the body given the rapid degradation of adenosine. Thus, the data at the lower concentrations as opposed to the higher concentrations are more physiologic. Because the effect of adenosine can be mediated through 2 distinct receptors A₁ and A₂, we tested the response of the vessels to the A₁ agonist, R-PIA, and the A₂ agonist, CGS. R-PIA induced vasoconstriction in the vein grafts (Fig 1) and CGS induced endothelium-independent relaxation equivalent to NECA. To determine whether the contractile response to R-PIA was caused by endothelial cell A₁ receptors or A₁ smooth muscle cell receptors, we evaluated the response of endothelial denuded vein graft segments to R-PIA. Endothelial denudation of the vein graft restored R-PIA–mediated relaxation (Fig 1) but at a much reduced level compared with that of jugular vein (Table I). To evaluate the contribution of A₁ receptors to the NECA response, we preincubated the vein grafts with the A₁ antagonist XAC (105 mol/L for 30 minutes). In the presence of XAC, the relaxation to NECA in the vein graft was enhanced, which further suggests that A₁ receptors are mediating contraction instead of relaxation in this vessel. The contractile response to R-PIA was abolished and that to CGS was unaffected (Fig 1; Table I).

Similarly, we tested the responses of the jugular veins and the carotid arteries to the A₁ agonist, R-PIA, and the A₂ agonist, CGS. Both R-PIA and CGS induced endothelium-independent relaxation in the jugular veins (Table I).

Table I. Receptor specific responses

*P = .89 for the comparison of jugular veins and carotid arteries.

Values are the pD₂ (–log₁₀[IC₅₀0) and are presented as the mean ± standard error of mean. Statstical analysis by analysis of variance with post hoc Tukey-Kramer comparison testing.

†P = .31 for the analysis of variance comparison of jugular veins and carotid arteries.

‡P < .05 compared with control vein graft.

§P = .47 for the analysis of variance comparison of control jugular veins, vein grafts, and carotid arteries.

P = .0002 for the analysis of variance comparison of denuded jugular veins, vein grafts, and carotid arteries.

¶P < .001 compared with control jugular vein.

#P < .01 compared with control carotid artery.

After pretreatment with XAC, the response of the jugular vein to NECA was significantly reduced (Table I) and the response to R-PIA was abolished; the response to CGS was unaffected by pretreatment with XAC. In the carotid arteries, CGS-induced relaxation displayed an endothelial-based component, but that to R-PIA was endothelium independent (Table II).

Table II. Responses to adenosine

Values are the pD₂ (–log₁₀[IC₅₀]) and are presented as the mean ± standard error of the mean. Statistical analysis by analysis of variance with post hoc Tukey-Kramer comparison testing.

*P = .45 for the analysis of variance comparison of control jugular veins, vein grafts, and carotid arteries.

†P = .024 for the analysis of variance comparison of denuded jugular veins, vein grafts, and carotid arteries (carotid artery vs vein graft, P < .05).

‡P < .05 compared with control vein graft.

§P < .01 compared with control vein graft.

P < .05 compared with control jugular vein.

¶P < .001 compared with control jugular vein.

#P < .01 compared with control carotid artery.

**P < .001 compared with control carotid artery.

Preincubation with XAC did produce a decrease in the response to NECA in the carotid artery, but this did not achieve significance (Table II). In the presence of XAC, the response to R-PIA was abolished and that to CGS was unaffected by pretreatment with XAC.

Role of NO, prostaglandins, and membrane channels 

To examine the contribution of NO production, prostanoid generation, or K⁺ channels activation—each of which are known to contribute to adenosine-mediated relaxation—the 3 vessels were preincubated with L-NMMA (a NO synthase inhibitor), indomethacin (a cyclo-oxygenase inhibitor), and glybenclamide (K⁺ channel blockade), respectively, before stimulation with NECA. Relaxation in vein grafts to NECA was sensitive to L-NMMA and indomethacin inhibition and independent of glybenclamide (Table II). The jugular veins relaxed to NECA with a response that could be partially inhibited by L-NMMA and indomethacin (Table II). Incubation with glybenclamide had no effect on the NECA response (Table II). The carotid arteries relaxed in response to NECA, and this relaxation was sensitive to the presence of indomethacin and glybenclamide but unaffected by L-NMMA (Table II). Given the striking differences in the responses to the respective A₁ and A₂ agonists, we performed a similar series of pretreatments before testing with R-PIA or CGS. In the vein graft, A₁-mediated contractile response was enhanced with preincubation with L-NMMA (9.05 ± 0.33, P < .05; mean ± SEM; EC₅₀) and indomethacin (10.5 ± 0.45, P < .05; EC₅₀) but was unaffected with preincubation with glybenclamide as compared with control (8.21 ± 0.25; EC₅₀). In the vein graft, A₂ response was unaffected by denudation (6.42 ± 0.14, mean ± SEM; IC₅₀) and was sensitive to L-NMMA (5.79 ± 0.14, P < .05; IC₅₀) and indomethacin (5.86 ± 0.15, P < .05; IC₅₀) but was independent of glybenclamide (5.64 ± 0.49; IC₅₀) as compared with control (6.18 ± 0.13; IC₅₀).

Role of G-proteins 

The linkage of the NECA responses to GTP-binding proteins was tested with incubation with pertussis toxin (uncoupler of G_i/G_o ) or cholera toxin (uncoupler of G_S ). Although cholera toxin abolished the response of the vein grafts to NECA, a component of this relaxation was also pertussis toxin sensitive (Table II). This suggests involvement of both types of alpha subunits (G_i/G_o and G_S), with G_s as the predominant GTP-binding protein linked to the receptor systems. Jugular veins responded to NECA with pertussis toxin–sensitive but cholera toxin–insensitive responses, which suggests signal transduction through G_i/G_o alpha subunits (Table II). Carotid arteries showed pertussis toxin–sensitive and cholera toxin–sensitive NECA-mediated responses, which suggests signal transduction through both G_i/G_o and G_S alpha subunits (Table II). Because of the different responses to the respective A₁ and A₂ agonists, we performed a similar series of pretreatments with pertussis and cholera toxins before testing with R-PIA or CGS. In the vein graft, A₁-mediated contractile response was enhanced by the presence of pertussis toxin (9.17 ± 0.31; EC₅₀) but was cholera toxin insensitive as compared with control (8.21 ± 0.25; EC₅₀). In contrast, the A₂ responses were cholera toxin sensitive (no response) and pertussis toxin insensitive (5.36 ± 0.60; IC₅₀).

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DISCUSSION 

The physiology of vein grafts after implantation into the arterial circulation is altered, with changes noted in the responses of the vein graft's smooth muscle cells to multiple contractile agonists. Although the pathologic implications of these responses remain to be defined, these changes do reflect a change in physiologic phenotype. In concert with these alterations in the smooth muscle cells, there are changes in the function of the endothelial cells. The most striking of these changes has been the reduction in receptor-activated NO-mediated relaxation and the apparent changes in the pattern of PGI₂ (decreased) and thromboxane A₂ (increased) production. These changes in important vessel wall modulators are considered to contribute to the pathologic processes that lead to the development of intimal hyperplasia in a vein graft. To date, the actions of adenosine in vein grafts has not been determined. This study shows that the responses of the vein grafts to this agonist in relation to the native vein and artery appear unique. First, in contrast to the carotid artery, vein graft adenosine-mediated relaxation is NO and prostanoid dependent, which is similar to the response of the jugular vein. Second, A₁-receptor activation in the vein graft produces an endothelium-dependent contractile response. Third, the A₂ receptor–mediated responses in the vein grafts appear independent of the endothelium. Fourth, radioligand studies confirm the presence of both receptor subtypes (A₁ and A₂) on the vein grafts, with a ratio closer to that of the jugular vein than to that seen in the carotid artery. And fifth, adenosine-receptor transduction by G-protein alpha subunits in the vein graft involves both α_i/α_o and α_S, which is more like the carotid artery.

Adenosine receptor–activated relaxation could be inhibited partly with preincubation with a nitric oxide synthase inhibitor, which suggests for the first time that receptor-mediated activation of this system is intact in vein grafts. The results would suggest that activation of the NOS is A₂ receptor mediated. These findings would further support the results obtained in this model and in retrieved human vein grafts that show the presence of constitutive NOS, which can be activated by nonreceptor-mediated endothelium-dependent agonists. The conversion of a receptor-mediated response from relaxation to contraction has been shown before in these vein grafts. In the jugular vein, serotonin-mediated endothelium-dependent relaxation is by activation of the 5-HT₁ receptor. After vein grafting, this response to serotonin changes from endothelium-dependent relaxation to endothelium-independent contraction and appears to be caused by the development of 5-HT₂ receptor subtypes on the smooth muscle cells of the intimal hyperplasia.¹¹ Similarly, others have reported that acetylcholine will relax the native vein and induce contraction in vein grafts, which suggests a change in the muscarinic receptors from activation of relaxation to the activation of contraction. The conclusions from these varied studies are that there is a defect in receptor activation or signal transduction in the endothelial cells of the vein graft that reduces the vessels ability to relax and modulate smooth muscle cells to increase contraction.

Changes in the hemodynamic environment of the vessel play a role in the functional changes in the adenosine responses shown for the graft endothelial cells. This response is composed of 2 components: an initial response to injury and a prolonged response to changes in the flow and shear stress across the endothelial cell monolayer. Altered endothelial cell signalling, whether by the acetylcholine receptor or by the adenosine A₁ receptor, may reflect the needs of the endothelium to modulate remodelling of the vessel wall and allow for adaptive functional responses to accommodate arterial levels of shear stress. The response to NECA is enhanced when the endothelium is removed. This suggests that activation of A₁ on endothelial cells produces a contractile response that is counterbalanced nearly completely by the A₂ response on the underlying smooth muscle cells. In relation to the acetylcholine receptor, it appears that receptor-induced NO-mediated endothelial-dependent relaxation is disturbed after insertion into the arterial circulation, with a loss of a response to the acetylcholine receptor but intact responses to calcium ionophore (receptor-independent NO-mediated endothelial-independent relaxation) and sodium nitroprusside (cGMP-mediated relaxation). We have shown, in the case of the acetylcholine receptor, that return of a vein graft to the venous circulation for 14 days after an initial exposure of the vein graft to the arterial circulation for 14 days will reverse the unresponsiveness of the acetylcholine receptor and allow receptor-induced NO-mediated endothelium-dependent relaxation. Whether modulation of the adenosine receptor would occur by manipulation of the hemodynamic environment is not presently known.

Adenosine-mediated relaxation in the jugular veins and carotid arteries was prostanoid dependent. Jugular veins also used NO as a secondary mediator, and carotid arteries used a K⁺-dependent channel. In the jugular veins and the carotid arteries, A₁-mediated relaxation (R-PIA) was endothelial independent, which suggests the presence of A₁ receptors on smooth muscle cells, and A₂ responses (CGS) showed a significant endothelial-dependent component, which suggests the presence of A₂ receptors on both the endothelial and smooth muscle cells. Relaxation in the endothelialized carotid arteries was mediated predominantly by A₂ receptors, which is similar to functional results reported by others for rabbit arterial tissue.¹² The radioligand studies in the carotid arteries confirm the presence of a significant A₂-receptor subtype as compared with the A₁ subtype.

A previous study by this laboratory showed that there was increased expression of α_i2 and α_S expression and de novo detectable expression of α_i3 in vein grafts as compared with jugular veins; these changes were associated with the development of pertussis toxin–sensitive contractile responses.⁶ These data suggest that intimal hyperplasia is associated with increased or novel expression of G-proteins in vivo coupled with a change in receptor-G protein coupling. Adenosine A₁ receptors interact with multiple G proteins subtypes (α_i3 > α_i2 > α_i1), and adenosine A₂ receptors interact with α_S G proteins.¹³, ¹⁴ The ability of pertussis toxin to inhibit a response suggests the presence of a receptor coupled to α_i or α_o, and the ability of cholera toxin to inhibit a response suggests the presence of a receptor coupled to α_S. Most reports have shown that pertussis toxin–sensitive G proteins mediate responses for the A₁ receptor in a variety of tissues. The involvement of G proteins in the response mediated by the A₂ response is limited. In human coronary arteries, A₁ and A₂ adenosine responses appear to be mediated by both pertussis toxin–sensitive and cholera toxin–sensitive receptors.¹⁵, ¹⁶, ¹⁷ R-PIA responses in bovine pulmonary artery endothelial cells have been shown to be pertussis toxin sensitive.¹⁸ In this study, we find that adenosine-mediated relaxation in the jugular vein was pertussis toxin–sensitive but cholera toxin–insensitive, and in the carotid artery, the responses were both pertussis toxin–sensitive and cholera toxin–sensitive, which suggests that α_i/α_o but not α_S participate in the adenosine responses in the jugular vein, but in the carotid artery, both α_i/α_o and α_S participate in the responses. The responses in the vein graft appear to be both pertussis toxin–dependent and cholera toxin–dependent, which suggests the participation of both α_i/α_o and α_S in the responses. Thus, there is a dichotomy existing in this study. Adenosine-mediated relaxation in the vein graft is NO-based and prostanoid-based like the jugular vein, but adenosine-receptor transduction in the vein graft involves both α_i/α_o and α_S, which is more like that seen in the carotid artery.

In summary, this study shows that there are changes in the adenosine-mediated vasoreactivity in vein grafts as compared with jugular veins because of a change in endothelial A₁ receptor-mediated response from relaxation to contraction. The adenosine responses of the vein grafts are also different from the carotid artery. The endothelial cells of the vein graft appear to have undergone a phenotypic change, which perhaps involved changes in signal transduction protein expression and linkages. If one considers that the changes in adenosine-receptor responses observed in this study are identifying an endothelial cell phenotype that is involved in the intimal hyperplastic process, then all variables that play a role in initiating intimal hyperplasia in the vein graft must be considered as possible initiators of the process. This study has characterized the differences in the adenosine responses between the 3 vessels, has shown that there are changes in adenosine responses, and has defined the receptor subtype involved, the type of G-proteins used, and the possible secondary mediators responsible. However, we have not as yet defined the cause of the change in endothelial cell phenotype, the subtype of A₂ receptors present, and the way in which the intracellular signalling pathways of the A₁ and A₂ receptors in the vein grafts are altered. Further studies will be required to define these pathways.

In conclusion, adenosine-mediated responses in vein grafts appear to be unique on both a functional and a signal transduction basis. The responses neither maintain a venous phenotype nor acquire an arterial phenotype in response to implantation.

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Acknowledgements 

We acknowledge the technical assistance of L. Barber. Microsutures were a gift of Ethicon Inc, Somerville, NJ.

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