Canine Saphenous Vein Research Articles

Cardiovascular responses produced by 5-hydroxytriptamine:a pharmacological update on the receptors/mechanisms involved and therapeutic implications

The complexity of cardiovascular responses produced by 5-hydroxytryptamine (5-HT, serotonin), including bradycardia or tachycardia, hypotension or hypertension, and vasodilatation or vasoconstriction, has been explained by the capability of this monoamine to interact with different receptors in the central nervous system (CNS), on the autonomic ganglia and postganglionic nerve endings, on vascular smooth muscle and endothelium, and on the cardiac tissue. Depending, among other factors, on the species, the vascular bed under study, and the experimental conditions, these responses are mainly mediated by 5-HT(1), 5-HT(2), 5-HT(3), 5-HT(4), 5-ht(5A/5B), and 5-HT(7) receptors as well as by a tyramine-like action or unidentified mechanisms. It is noteworthy that 5-HT(6) receptors do not seem to be involved in the cardiovascular responses to 5-HT. Regarding heart rate, intravenous (i.v.) administration of 5-HT usually lowers this variable by eliciting a von Bezold-Jarisch-like reflex via 5-HT(3) receptors located on sensory vagal nerve endings in the heart. Other bradycardic mechanisms include cardiac sympatho-inhibition by prejunctional 5-HT(1B/1D) receptors and, in the case of the rat, an additional 5-ht(5A/5B) receptor component. Moreover, i.v. 5-HT can increase heart rate in different species (after vagotomy) by a variety of mechanisms/receptors including activation of: (1) myocardial 5-HT(2A) (rat), 5-HT(3) (dog), 5-HT(4) (pig, human), and 5-HT(7) (cat) receptors; (2) adrenomedullary 5-HT(2) (dog) and prejunctional sympatho-excitatory 5-HT(3) (rabbit) receptors associated with a release of catecholamines; (3) a tyramine-like action mechanism (guinea pig); and (4) unidentified mechanisms (certain lamellibranch and gastropod species). Furthermore, central administration of 5-HT can cause, in general, bradycardia and/or tachycardia mediated by activation of, respectively, 5-HT(1A) and 5-HT(2) receptors. On the other hand, the blood pressure response to i.v. administration of 5-HT is usually triphasic and consists of an initial short-lasting vasodepressor response due to a reflex bradycardia (mediated by 5-HT(3) receptors located on vagal afferents, via the von Bezold-Jarisch-like reflex), a middle vasopressor phase, and a late, longer-lasting, vasodepressor response. The vasopressor response is a consequence of vasoconstriction mainly mediated by 5-HT(2A) receptors; however, vasoconstriction in the canine saphenous vein and external carotid bed as well as in the porcine cephalic arteries and arteriovenous anastomoses is due to activation of 5-HT(1B) receptors. The late vasodepressor response may involve three different mechanisms: (1) direct vasorelaxation by activation of 5-HT(7) receptors located on vascular smooth muscle; (2) inhibition of the vasopressor sympathetic outflow by sympatho-inhibitory 5-HT(1A/1B/1D) receptors; and (3) release of endothelium-derived relaxing factor (nitric oxide) by 5-HT(2B) and/or 5-HT(1B/1D) receptors. Furthermore, central administration of 5-HT can cause both hypotension (mainly mediated by 5-HT(1A) receptors) and hypertension (mainly mediated by 5-HT(2) receptors). The increasing availability of new compounds with high affinity and selectivity for the different 5-HT receptor subtypes makes it possible to develop drugs with potential therapeutic usefulness in the treatment of some cardiovascular illnesses including hypertension, migraine, some peripheral vascular diseases, and heart failure.

Mechanisms of vasoconstriction with direct skin cooling in humans

direct skin cooling causes a vasoconstriction partly dependent on α2-adrenergic function. In in vitro studies, this is a manifestation of reactive oxygen species stimulating Rho and Rho kinase to cause translocation of α2C-receptors from the Golgi to the plasma membrane. In this issue of American

β-Adrenoceptors and potassium channels

The existence of two subtypes of adrenergic receptors, the α-adrenoceptors (αAR) and the β-adrenoceptors (βAR), was first demonstrated by Ahlquist (1948), and since then they have been extensively studied and characterised. They are receptors for the endogenous catecholamines epinephrine and norepinephrine and have a very wide tissue distribution (Table 1). In the case of βAR, Lands et al. (1967) found evidence for the existence of two types of βAR β1 and β2—by measuring the relative potency of sympathomimetic amines in different tissues. Subsequently, Emorine et al. (1989) isolated and cloned a third βARsubtype—the β3 adrenoceptor. Pharmacological evidence suggested a fourth type of βAR (Kaumann 1997; Molenaar et al. 1997; Galitzky et al. 1998), but it has not been cloned. In fact, recent evidence suggests that the putative β4AR, identified in the heart, is probably a low-affinity state of the β1AR (Granneman 2001; Kaumann et al. 2001; Sarsero et al. 2003; Joseph et al. 2004a,b; Arch 2004). All βAR are G-protein-coupled receptors, the predominant signal transduction mechanism of which is coupling of a Gs protein to adenylyl cyclase (AC), which becomes activated and catalyses the conversion of adenosine triphosphate (ATP) to cyclic 3′,5′ adenosine monophosphate (cAMP). cAMP activates protein kinase A (PKA) by binding to its regulatory subunit, causing PKA to dissociate from the catalytic subunit, thereby rendering it active. PKA is a serine/threonine protein kinase that targets a number of intracellular proteins, thereby eliciting a series of specific cellular responses. Such is the well-known “classical” mechanism for βAR signal-transduction. In the last two decades, however, it has become clear that this is by no means the only mechanism by which βAR give rise to intracellular responses. For example, in the heart, β2AR can couple to both Gs and Gi proteins. Coupling to Gs activates the cAMP-PKA pathway, mediating positive inotropic effects in the same manner as β1AR; by contrast, coupling to the Gi protein modifies the outcome of Gs signalling through activation of the PI3 kinase-Akt pathway (Zheng et al. 2005). In rat aorta, β2AR give rise to stimulation of nitric oxide biosynthesis, partially through PKA signalling but partly also through PI3 kinase-Akt activation (Ferro et al. 2004). In skeletal and cardiac muscle cells, Gs can directly enhance the activation of plasmalemmal voltage-sensitive Ca channels (Brown and Birnbaumer 1988), and this represents another potential mechanism of βAR signalling which is Gs-mediated but cAMP-PKA-independent. Interestingly, the coupling to voltage-sensitive Ca channels may differ between β1AR and β2AR (Foerster et al. 2004) In more recent years, it has become evident that, in a wide variety of tissues, βAR can also cause activation of potassium channels to give rise to their cellular effects; and interestingly, the potassium channels involved appear to vary widely from one tissue to another, for no obvious reason. In conscious dogs, β2AR-mediated dilatation of resistance coronary vessels involves both the opening of KATP channels and nitric oxide formation, and inhibition of nitric oxide synthase was found to antagonise lemakalim responses (Ming et al. 1997). In isolated canine saphenous veins and rat mesenteric arteries, the βAR agonist Naunyn-Schmiedeberg’s Arch Pharmacol (2006) 373:183–185 DOI 10.1007/s00210-006-0065-2

Modulation of phosphatidylinositol 3-kinase signaling reduces intimal hyperplasia in aortocoronary saphenous vein grafts

Fifty percent of human aortocoronary saphenous vein grafts are occluded after 10 years. Intimal hyperplasia is an initial step in graft occlusion and consists of vascular smooth muscle cell proliferation. Phosphatidylinositol 3-kinase and its downstream regulator, the inositol 3-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), are important regulators of vascular smooth muscle cell proliferation, migration, and cell death. This study tests whether overexpression of PTEN in aortocoronary saphenous vein grafts can reduce intimal hyperplasia. Adult dogs underwent aortocoronary bypass grafting to the left anterior descending artery by using the autologous saphenous vein. Saphenous vein grafts were treated with phosphate-buffered saline (n = 9), empty adenovirus (n = 8), or adenovirus encoding for PTEN (n = 8). Arteriography at 30 and 90 days assessed saphenous vein graft patency. A subset received saphenous vein grafts treated with a marker transgene (beta-galactosidase, n = 3), empty adenovirus (n = 4), or adenovirus encoding for PTEN (n = 4) and were killed on postoperative day 3 to confirm expression. Vascular smooth muscle cells were isolated from canine saphenous vein infected with adenovirus encoding for PTEN, and immunoblotting and proliferation assays were performed. Saphenous vein graft transgene expression was confirmed by means of immunohistochemistry, immunoblotting, and polymerase chain reaction. Arteriograms revealed all saphenous vein grafts to be patent. Saphenous vein grafts treated with adenovirus encoding for PTEN demonstrated reduced intimal area compared with those treated with empty adenovirus and phosphate-buffered saline (1.39 +/- 0.11 vs 2.35 +/- 0.3 and 2.57 +/- 0.4 mm 2 , P < .05), and the intima/media ratio was lower in saphenous vein grafts treated with adenovirus encoding for PTEN (0.50 +/- 0.05 vs 1.43 +/- 0.18 and 1.11 +/- 0.14, P < .005). PTEN overexpression in vascular smooth muscle cells inhibited platelet-derived growth factor-induced phosphorylation of Akt, a downstream effector of phosphatidylinositol 3-kinase. PTEN-treated vascular smooth muscle cells demonstrated decreased basal, platelet-derived growth factor-stimulated, and serum-stimulated proliferation. This study demonstrates that PTEN overexpression in aortocoronary saphenous vein grafts reduces intimal hyperplasia. The mechanism of this antiproliferative effect in vascular smooth muscle cells is likely due to inhibition of phosphatidylinositol 3-kinase signaling through Akt, with resultant decreases in vascular smooth muscle cell growth and survival. Therefore modulation of the phosphatidylinositol 3-kinase pathway through PTEN overexpression might represent a novel therapy to prevent saphenous vein graft intimal hyperplasia after coronary artery bypass grafting.

Activation of mitogen-activated protein kinases during preparation of vein grafts and modulation by a synthetic inhibitor

ObjectiveLong-term durability of saphenous vein grafts used for coronary artery bypass grafting is limited by neointimal formation. Arterial vascular injury is known to activate intracellular mitogen-activated protein kinases, including extracellular signal-regulated kinases and c-jun N-terminal kinases, that affect cell differentiation, proliferation, migration, and apoptosis. This study tests the hypothesis that these mitogen-activated protein kinases are activated in saphenous veins during preparation for coronary artery bypass grafting. MethodsSaphenous veins were harvested from 10 patients undergoing coronary artery bypass grafting. A specimen from each vein was placed in ice-cold lysis buffer immediately after harvesting (t = 0). The remaining tissue was incubated at room temperature in normal saline, 0.1% dimethylsulfoxide (vehicle), or 50 mmol/L PD98059 (mitogen-activated protein kinase kinase-1/2 inhibitor) until the vein was grafted (mean 50 minutes). To study kinetics of intracellular signaling pathways, canine saphenous veins were harvested, and mitogen-activated protein kinases and PI-3 kinase pathways were studied after different incubation time intervals. Extracted proteins were analyzed by Western blotting or in vitro kinase assay. ResultsThe human saphenous veins showed elevated levels of active extracellular signal-regulated kinase after harvesting (t = 0) and prior to implant (t = 1). Incubation with PD98059 resulted in decreased activation of extracellular signal-regulated kinase. Kinetics of canine saphenous veins showed extracellular signal-regulated kinase and c-jun N-terminal kinase activation, in a time-dependent manner, along with activation of the growth factor-regulated PI3 kinase pathway. ConclusionsThis study characterizes activation of extracellular signal-regulated kinases and c-jun N-terminal kinases during vein graft preparation and demonstrates the ability to inhibit extracellular signal-regulated kinase activation by simple incubation with a specific inhibitor. Further studies are needed to evaluate the significance of these findings with respect to graft durability.

Gene transfer to coronary artery bypass conduits

Background. Gene therapy is a rational approach to prevention of stenosis in saphenous vein grafts used as conduits for coronary artery bypass grafting. To explore this possibility we developed methods for adenoviral-mediated gene transfer to canine saphenous veins. Methods. During a single procedure, autogenous canine saphenous vein segments were transduced ex vivo and used as coronary artery bypass grafts. The proximal end of each vein was ligated, adenovirus containing the Escherichia coli β-galactosidase gene (Ad.CMVLacZ) was delivered at titers of 2.5 × 10 9 or 5 × 10 9 plaque-forming units (pfu)/mL to the lumen through a distal heparin lock, and the segment was immersed in the viral solution for 1 hour at 37°C. Control segments were exposed to diluent alone in an identical manner. Aortocoronary anastomoses were made using cardiopulmonary bypass. Transgene expression was assessed qualitatively and quantitatively after 3 days. Results. β-Galactosidase levels showed a dose-dependent increase: 0.00 ± 0.00 ng/mg total protein for controls; 5.60 ± 2.27 ng/mg total protein for a viral titer of 2.5 × 10 9 pfu/mL and 11.97 ± 6.14 ng/mg for 5 × 10 9 pfu/mL. The two dosage groups differed significantly from each other ( p = 0.035) and from controls ( p = 0.003). X-gal staining demonstrated mostly endothelial and scattered adventitial transgene expression. Conclusions. Transgene expression after ex vivo gene transfer into saphenous vein grafts in a canine coronary artery bypass model indicates that this method may be useful for delivery of therapeutic genes to prevent or retard vein graft arteriosclerosis.

Low 4-aminopyridine concentration-induced contraction is mediated by neuronal noradrenaline in canine saphenous vein

4-Aminopyridine (4-AP), a known inhibitor of the voltage-dependent potassium channels, is able to increase the basal tone of different types of blood vessel preparations. In order to determine the efficiency of 4-AP in veins and to clarify its possible mechanism of action, the aim of the present study was to determine the basal tone and release of radiolabelled tissue noradrenaline (NA) after administration of low 4-AP concentrations. Experiments were performed in canine saphenous vein in the absence and presence of functional endothelium. 4-AP (0.012–5 μM) enhanced the basal tone of venous rings without and with endothelium (maximum tone at 5 μM 4-AP: 2.20±1.29 and 1.3±0.57 mN, respectively). NA stores of the venous tissue were loaded by adding 1 mM NA to the tissue for 10 min and then washed out. After loading the NA-stores of venous tissue, 4-AP-induced contractions were significantly increased both in the absence and presence of endothelium (maximum tone at 5 μM 4-AP after loading with NA: 10.51±3.64 and 10.52±4.69 mN, respectively). Following NA loading, chemical denervation of the endothelium denuded venous preparations by 0.5 mM 6-hydroxydopamine (6-OHDA) completely abolished the contractions evoked by 4-AP. After incubation of the saphenous preparations with 3H-NA, 5 μM 4-AP significantly increased tritium-efflux from the tissue. These results provide evidence for the efficiency of 4-AP on the basal tone of isolated canine saphenous vein when applied in low concentrations. Furthermore, it is suggested that this action of 4-AP may considerably depend on the release of NA from the perivascular nerve endings.

Regulation of Na+pump expression by vascular smooth muscle cells

The Na(+) pump and its regulation is important for maintaining membrane potential and transmembrane Na(+) gradient in all mammalian cells and thus is essential for cell survival and function. Vascular smooth muscle cells (VSMC) have a relatively low number of pump sites on their membrane compared with other cells. We wished to determine the mechanisms for regulating the number of pump sites in these cells. We used canine saphenous vein VSMC cultured in 10% serum and passaged one time. These cells were subcultured in 5% serum media with low K(+) (1 mM vs. control of 5 mM), and their pump expression was assessed. These VSMC upregulated their pump sites as early as 4 h after treatment (measured by [(3)H]ouabain binding). At this early time point, there was no detectable increase in protein expression of either alpha(1)- or beta(1)-subunits of the pump shown by Western blots. When the cells were treated with the phosphoinositide 3-kinase (PI-3-K) inhibitor LY-294002 (which is known to inhibit cytoplasmic transport processes) in low-K(+) media, the pump site upregulation was inhibited. These data suggest that the low-K(+)-induced upregulation of Na(+) pump number can occur by translocation of preformed pumps from intracellular stores.

Inhibitory effect of valves on endothelium-dependent relaxations to calcium ionophore in canine saphenous vein.

The present study was designed to evaluate endothelium-dependent relaxation to the calcium ionophore A-23187 in isolated canine saphenous veins. Isometric force recordings and cGMP measurements using isolated veins with and without valves were performed. During contractions to U-46619 (3 x 10(-7) M), endothelium-dependent relaxations to A-23187 (10(-9)-10(-6) M) were significantly reduced in rings with valves compared with rings without valves. Endothelial removal abolished A-23187-induced relaxation. Relaxations to forskolin (FK; 10(-8)-10(-5) M) and diethylaminodiazen-1-ium-1,2-dionate; DEA-NONOate, 10(-9)-10(-5) M) were identical in rings with and without valves. In rings without valves, a nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME; 3 x 10(-4) M), and a cyclooxygenase inhibitor, indomethacin (10(-5) M), partially reduced A-23187-induced relaxation. However, in rings with valves, L-NAME had no effect, whereas indomethacin abolished the relaxation to A-23187. A selective soluble guanylate cyclase inhibitor, 1H-[1,2,4]-oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 3x10(-6) M), had no effect on the relaxation to A-23187 in either group. In contrast, ODQ abolished the A-23187-induced increase in cGMP levels, suggesting that relaxation to nitric oxide released by A-23187 is independent of increases in cGMP. These results demonstrate that endothelium-dependent relaxation to A-23187 is reduced in regions of veins with valves compared with relaxation in the nonvalvular venous wall. Lower production of nitric oxide in endothelial cells of valvular segments appears to be a mechanism responsible for reduced reactivity to A-23187.

Adventitial versus intimal liposome-mediated ex vivo transfection of canine saphenous vein grafts with endothelial nitric oxide synthase gene

Purpose: Experiments were designed (1) to evaluate liposome-mediated endothelial constitutive nitric oxide synthase (ecNOS) transfection in vein grafts and (2) to compare intimal and adventitial routes of transfection. Methods: Male mongrel dogs (N = 36) underwent bilateral femoral artery bypass grafting with the lateral saphenous vein. In each animal one vein was transfected with plasmid (pVR1012) containing the ecNOS gene, and another vein was transfected with plasmid alone (control). Gene transfer was performed from either the intimal surface (Group I, n = 18) or the adventitial surface (Group II, n = 18). In each group there were three transfection subgroups (n = 6 each): (a ) 10 μg/mL naked plasmid DNA, (b ) 10 μg/mL plasmid DNA + liposome (LipofectAMINE PLUS), and (c ) 100 μg/mL plasmid DNA + LipofectAMINE PLUS. Grafts were harvested on the third postoperative day, and the transfection was assessed with molecular techniques and enzyme assay for activity of NOS by conversion of tritiated l-arginine to tritiated l-citrulline. Proliferating cells were quantified with a digital analysis of histologic sections after nuclear antigen Ki-67 (MIB1) immunohistochemistry. Results: Transgene was identified with polymerase chain reaction in all ecNOS-transfected grafts, regardless of transfection modality. However, significant transcription of the ecNOS transgene was observed only in Group IIc (mean ecNOS messenger RNA, 8.7 ± 1.7 vs 3.1 ± 0.7 × 10–2 attomole/μL, in transfected compared with control grafts, respectively, P =.01). NOS activity increased approximately twofold in this group (11.58 ± 2.1 and 6.3 ± 1.0 pmol tritiated l-citrulline per milligram protein per hour in transfected and control grafts, respectively, P =.05). Numbers of proliferating cells did not differ among ecNOS-transfected and control grafts in any transfection group. Conclusion: These results suggest that ecNOS transfection of vein grafts is feasible through intimal and adventitial routes with naked DNA or a liposomal vector. However, efficient transcription of the transgene is evident at postoperative day 3 only after adventitial transfection of 100 μg/mL of the gene. (J Vasc Surg 2000;32:1190-200.)

Short-term treatment with transdermal nicotine affects the function of canine saphenous veins

Short-term treatment with transdermal nicotine affects the function of canine saphenous veins

Short-term treatment with transdermal nicotine affects the function of canine saphenous veins.

Experiments were designed to determine the effects of nicotine treatment on the functions of saphenous veins used for coronary artery bypass grafts in dogs. Dogs received either no treatment or transdermal nicotine for 5 weeks at doses of 11 mg, 22 mg or 44 mg/day. Saphenous veins were removed and suspended for the measurement of isometric force in organ chambers. Endothelium was removed mechanically from some rings. N(G)-mono-methyl-L-arginine (L-NMMA; 10(-4) M) was used to inhibit the production of nitric oxide. Contractions to alpha2-adrenergic stimulation were decreased in veins from dogs treated with a 22-mg/day dose of transdermal nicotine. In addition, endothelium-dependent relaxations to adenosine-diphosphate (10(-8)-10(-4) M) and the calcium ionophore A23,187 (10(-8)-10(-6) M) were decreased in veins from dogs with a 22-mg/day dose and increased in veins from dogs treated with a 44-mg/day dose. These relaxations were inhibited by L-NMMA. Plasma concentrations of oxidized products of nitric oxide were decreased only in dogs treated with 22 mg/day of nicotine. The relaxation of rings without endothelium (direct response on the smooth muscle) to nitric oxide were not altered by nicotine treatment. These results suggest that the short-term treatment of dogs with intermediate (22 mg/day) but not low (11 mg/day) or high (44 mg/day) doses of transdermal nicotine decreases the endothelial function of veins used for coronary artery bypass grafts. Therefore, changes in plasma products of nitric oxide and endothelium-dependent relaxations mediated by nitric oxide are related to the dose of nicotine treatment.

Alterations in vascular reactivity in isolated vessel segments from dogs with naturally occurring heart failure

To investigate if functional vascular reactivity is altered in heart failure, the reactivity of isolated canine saphenous vein (SV) and femoral artery (FA) rings, from control dogs and dogs with naturally occurring heart failure was examined. In both vessels, relaxation responses to the endothelium-dependent vasodilator, acetylcholine were unaffected by heart failure. In the FA, in heart failure, there was a significant reduction in the potency of the agonist noradrenaline (pEC506·05±0·07 (N = 8) and 5·54 ± 0·13 (N = 7) for control and heart failure respectively). There was no significant alteration in potency in the SV. In addition, in the FA the maximum responses to both noradrenaline (control 3·64 ± 0·31 KPa, (N = 8); failure 5·11 ± 0·35 KPa, (N = 7) P = 0·004) and potassium chloride (control 2·18 ± 0·26 KPa, (N = 8); failure 4·46 ± 0·25 KPa, (N = 7) P = 0·001) were significantly increased in heart failure. It is suggested that enhanced agonist induced responses, in the femoral artery, in dogs with heart failure, may limit blood flow to exercising skeletal muscle and subsequently reduce exercise tolerance.

Recording of the saphenous vein compliance by an ultrasonic echo-tracking device in the dog: effects of S 18149.

1. Saphenous vein reactivity was recorded in the anaesthetized dog by use of an ultrasonic echo-tracking device to measure the internal diameter of the vein and to calculate the venous compliance. This method was used to investigate the effects of a new partial alpha1/alpha2-adrenoceptor agonist, S 18149, on the canine saphenous vein in vivo after intravenous (i.v.) or oral administration. 2. Venoconstrictions induced by i.v. or local administration of compounds were evaluated by continuous recording of the internal diameter of the saphenous vein with the echo-tracking method. Venous compliance was calculated in two ways: (1) as the slope of the diameter-pressure curve obtained by increasing the venous pressure with an inflatable cuff and (2) in veins in which pressure was higher than 12 mmHg, pulsatile variations in the venous diameter and venous pressure were detected and used to calculate the pulsatile compliance of the vein. 3. S 18149 administered i.v. at 0.5 microg kg(-1) min(-1) for 10 min induced a decrease in the saphenous vein diameter (-15+/-3%) and blood flow (-72+/-6%) associated with an increase in saphenous vein resistance; at the dose used, S 18149 did not modify venous pressure and caused only a weak increase in arterial pressure (+7+/-2 mmHg). 4. The pulsatile compliance of the saphenous vein averaged 8.65+/-1.37 mm2 x 100 mmHg(-1) in control dogs and was significantly decreased to 5.13+/-0.68 mm2 x 100 mmHg(-1) in the same animals after treatment with S 18149 at 100 microg kg(-1) per os (n=10). The saphenous vein compliance calculated with the increased external pressure method averaged 24.90+/-1.49 microm mmHg(-1) in control dogs and was significantly reduced in the same animals after treatment with S 18149 at 100 microg kg(-1) per os to 9.06+/-3.42 microm mmHg(-1) (n=5). When constrictions of the vein were induced with increasing doses of (-)-phenylephrine, injected locally at 1, 3 or 6 microg min(-1), only the responses obtained with the lower dose of (-)-phenylephrine were increased in dogs treated with S 18149 100 microg kg(-1) per os (-16+/-4% versus -4+/-3%, n=5). 5. These results show that the high resolution echo-tracking device previously used for arterial compliance measurements, allows the detection of pulsatile changes in the canine saphenous vein and thus permits calculation of both the pulsatile and the static compliance of superficial veins in vivo. Using this technique, we have demonstrated that the novel alpha-adrenoceptor agonist S 18149 constricts the canine saphenous vein in vivo and decreases the saphenous vein compliance after oral administration.

Hyperpolarization and Relaxation of Canine Vascular Smooth Muscle to Vasoactive Intestinal Polypeptide

This study was designed to determine the influence of the endothelium on the hyperpolarization induced by vasoactive intestinal polypeptide (VIP) in smooth muscle cells of canine blood vessels, and the potential contribution that these electrophysiologic changes may make to the relaxant effects of VIP. Membrane potential was measured in isolated canine coronary arteries and saphenous veins, using glass microelectrodes. Isometric force was recorded in a conventional organ chamber. All experiments were performed in the presence of indomethacin. VIP induced concentration-dependent and endothelium-independent hyperpolarization of the saphenous vein. This response was abolished by glibenclamide. VIP did not induce hyperpolarization of coronary arterial smooth muscle either in the presence or absence of the endothelium. VIP caused concentration-dependent and endothelium-independent relaxations of both arterial and venous rings. The relaxation of the saphenous vein to VIP was not influenced by glibenclamide. These data suggest that hyperpolarization of the cell membrane does not play a significant role in the relaxation of canine blood vessels to VIP.

Prejunctional alpha2A-autoreceptors in the canine saphenous vein.

The aim of the study was to determine the subtype of prejunctional alpha2-autoreceptors in the canine saphenous vein. Segments of the vein were incubated with 3H-noradrenaline and subsequently perifused with modified Krebs-Henseleit solution. Five periods of electrical stimulation were applied (S1-S5; each for 2 min, 1 Hz). Concentration-response curves for the inhibitory effect of the alpha2-adrenoceptor agonists oxymetazoline and UK-14,304 and for the facilitatory effect of nine antagonists were determined. Correlations between the pEC30%s for the antagonists obtained in the present study and the pKis for the same antagonists obtained in tissues expressing only alpha2A- (HT29 cells), alpha2B- (neonatal rat lung), alpha2C- (OK cells) or alpha2D-adrenoceptors (rat submaxillary gland) showed that the alpha2-autoreceptors in the canine saphenous vein resemble most closely the alpha2A-subtype. Furthermore, oxymetazoline was a highly potent agonist (pIC50% = 8.10) and prazosin was a weak antagonist (pEC30% = 6.46), confirming that the alpha2-adrenoceptors involved in the modulation of the response to electrical stimulation of the canine saphenous vein do not belong to either the alpha2B- or alpha2C-subtype. On the other hand, the EC30% ratios phentolamine/rauwolscine and idazoxan/rauwolscine were much closer to the ratios obtained at alpha2-autoreceptors of the rabbit- (alpha2A) than of the guinea-pig brain cortex (alpha2D). The results suggest that the sympathetic nerves of the canine saphenous vein are endowed with alpha2A-adrenoceptors.

Migratory abilities of different vascular cells from the tunica media of canine vessels.

In response to injury, vascular smooth muscle cells (VSMC) are thought to migrate toward the area of damage, where they participate in the reparative process. We have recently identified and isolated two distinct cell types (VSMC and type 2 cells) from the tunica media of canine carotid artery and saphenous vein. The purpose of these experiments was to determine whether both cell types were able to migrate in response to a variety of chemoattractants. A multiwell Boyden chamber and a wound migration assay were used to assess the migratory ability of these cells in vitro. The results indicated that VSMC did not exhibit directed migration in response to either 10% fetal bovine serum or platelet-derived growth factor (PDGF)-AB. In contrast, type 2 cells migrated to serum, PDGF-AB, and PDGF-BB but not to PDGF-AA, endothelin (ET)-1, or ET-3. No difference in migratory ability was detected between type 2 cells isolated from carotid arteries or saphenous veins. It is concluded that the migratory ability of cells within the tunica media of vessels from adult animals are not equal, suggesting that only selected cells may participate in vascular wall repair.

Evidence for the Presence of Regional Differences in the Subtype Specificity of Muscarinic Receptors in Rabbit Lower Urinary Tract

To elucidate the subtype specificity of muscarinic cholinergic receptors in mediating contractile responses in the lower urinary tract, we investigated contractile and biochemical properties of muscarinic receptors in bladder dome, bladder base and urethra of the rabbit. Isometric contractile response curves to increasing concentrations of carbachol were constructed in the absence and presence of various concentrations of subtype selective muscarinic antagonists. Bladder dome, bladder base, and urethra demonstrate different characteristics in terms of efficacy and potency with respect to carbachol-induced contractile responses. Emax values are significantly larger and ED50 values are significantly smaller in bladder dome and bladder base than in urethra. Calculation of the pA2 values, the negative logarithm of the antagonist affinity constant (KB), for a series of muscarinic antagonists, i.e., atropine (nonselective), pirenzepine (M1 selective), methoctramine (M2 selective), and 4-DAMP (M1/M3 selective) indicate that the carbachol-induced contractile response in bladder dome and bladder base is mediated through the M3 receptor subtype whereas the carbachol-induced contractile response in urethra is probably mediated through the M1 and/or M3 and possibly M2 subtypes. Muscarinic cholinergic antagonists inhibit [3H]quinulidinyl benzilate binding to bladder dome, bladder base and urethra with the following rank order of affinities: atropine > 4-DAMP > methoctramine > pirenzepine. The binding data indicate the predominance of the M2 receptor subtype in all three regions.

The effects of cryopreservation on the biaxial mechanical properties of canine saphenous veins.

The experimental and analytical methods presented in the companion paper [1] are used here to study the effects of cryopreservation on the in vitro biaxial random-elastic mechanical properties of canine saphenous veins. The properties of specimens tested in their physiological range of loadings immediately after thawing were not significantly different from their properties before cryopreservation. However, they stiffened significantly in the few hours following thawing. This effect was not observed for aging fresh specimens, nor for cyanide-poisoned specimens, indicating that the static tone of the venous smooth muscle may be affected by cryopreservation and thawing, but the elastin and collagen fibers are most likely unaffected.

Bradykinin-induced contractions of canine saphenous veins: mediation by B2 receptors and involvement of eicosanoids.

1. Experiments were designed to determine the subtype of kinin-receptors mediating the contraction of venous smooth muscle to bradykinin and to investigate the involvement of metabolites of arachidonic acid in this response. 2. Bradykinin (10(-9) to 10(-6) M) caused concentration-dependent contractions of the canine isolated saphenous vein without endothelium, which were potentiated by indomethacin (10(-5) M, an inhibitor of cyclo-oxygenase). The concentration-response curve was biphasic, reaching an asymptote at 10(-8) M and a secondary maximal response at 10(-6) M. 3. Bradykinin (10(-8) M to 3 x 10(-6) M) caused a three fold stimulation in the release of the vasodilator prostaglandin E2 (PGE2) and a two fold stimulation of that of the vasodilator prostacyclin, measured by the production of 6-keto-PGF1 alpha (its stable breakdown product). 4. Under control conditions, nordihydroguaiaretic acid (NDGA, 10(-5) M), an inhibitor of lipoxygenase, did not affect the response to bradykinin. In the presence of indomethacin (10(-5) M), NDGA reduced contractions to bradykinin, suggesting the involvement of lipoxygenase metabolites in the potentiation evoked by the inhibitor of cyclo-oxygenase. 5. The selective B1 receptor agonist [des-Arg9]-bradykinin, in the concentration-range 10(-6) to 10(-5) M, induced contractions, which were abolished by the B2 receptor antagonist D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]-bradykinin (Hoe 140; 10(-6) M). The selective B1 receptor antagonist [des-Arg9, Leu8]-bradykinin, (10(-7) to 10(-5) M) had no significant effect on bradykinin-induced contractions. 6. The B2 receptor antagonists Hoe 140 (10(-8) to 10(-6) M) and D-Arg [Hyp3, D-Phe7]-bradykinin (10(-7) to 10(-5) M) shifted the concentration-response curve to bradykinin to the right in a concentration-dependent manner. 7. These results indicate that, in the canine saphenous vein, bradykinin causes contraction by activating B2 receptors. This results in the production of metabolites of arachidonic acid, which play a key role in the contraction of canine saphenous venous smooth muscle.