Development of endothelium-dependent relaxation in canine coronary collateral arteries

BACKGROUND: Little information exists regarding development of vasomotor control mechanisms during coronary collateral artery maturation. Therefore, we studied endothelium-dependent relaxation of canine collateral arteries isolated 2, 4, and 9 months after placement of an ameroid occluder around the proximal left circumflex coronary artery. RESULTS: Collateral arteries isolated after 2 months exhibited markedly reduced endothelium-dependent relaxation in response to acetylcholine (ACh; 10(-10) to 10(-4) mol/L) and bradykinin (BK; 10(-11) to 10(-6) mol/L) compared with relaxation of noncollateral coronary arteries (P<0.01). In contrast, endothelium-independent relaxation of collateral arteries to nitroprusside was only slightly reduced compared with relaxation of noncollateral arteries (P<0.05). Endothelium-dependent relaxation of collateral arteries isolated after 4 and 9 months was increased significantly, to the extent that relaxation to ACh and BK was not significantly different between collateral and noncollateral arteries at these periods. Inhibition of nitric oxide synthesis with NT-nitro-L-arginine methyl ester (L-NAME; 100 micromol/L) markedly inhibited ACh-induced relaxation in all noncollateral arteries and in collateral arteries isolated after 9 months. However, neither L-NAME nor indomethacin (5 micromol/L) alone inhibited ACh-mediated relaxation of collateral arteries isolated after 4 months. ACh-induced relaxation of these collateral arteries was only inhibited when arteries were preconstricted with 30 mmol/L K+ and pretreated with L-NAME and indomethacin (ie, when synthesis/effects of nitric oxide, prostaglandins, and endothelium-derived hyperpolarizing factor were inhibited). CONCLUSIONS: Development of endothelium-dependent relaxation in canine coronary collateral arteries is not complete after 2 months. After 4 months, endothelium-dependent relaxation of collateral arteries is similar to relaxation of noncollateral arteries, but the relaxation exhibits decreased dependence on synthesis of nitric oxide and increased involvement of prostaglandins and endothelium-derived hyperpolarizing factor(s). After 9 months of development, collateral arteries exhibit normal nitric oxide-dependent relaxation, similar to noncollateral arteries.     

Hypomagnesemia inhibits nitric oxide release from coronary endothelium: protective role of magnesium infusion after cardiac operations

BACKGROUND: Postoperative hypomagnesemia is common in patients who have undergone cardiac operations and is associated with clinically significant morbidity resulting from atrial and ventricular dysrhythmias. Magnesium supplementation may increase the cardiac index in the early postoperative period. METHODS: The action of the magnesium cation on coronary vascular reactivity was studied. Segments of canine epicardial coronary artery were suspended in organ chambers to measure isometric force (95% O2/5% CO2, 37 degrees C). RESULTS: In coronary segments constricted with prostaglandin F2alpha (2 x 10[-6] mol/L), acetylcholine and adenosine diphosphate (10[-9] to 10[-4] mol/L) induced vasodilation in arteries with endothelium (n=10, each group; p < 0.05). Acetylcholine-mediated vasodilation was blocked by NG-monomethyl-L-arginine (10[-4] mol/L) and NG-nitro-L-arginine (10[-4] mol/L), two inhibitors of nitric oxide synthesis from L-arginine (n=10, p < 0.05). The removal of magnesium from the organ chamber solution impaired vasodilation in response to acetylcholine and adenosine diphosphate. However, normal endothelium-dependent vasodilation could be restored by return of magnesium to the bathing solution. Vascular relaxation in response to bradykinin (10[-9] to 10[-6] mol/L), which was found to induce endothelium-dependent vasodilation independent of nitric oxide production, was unaffected by magnesium removal (n=10). CONCLUSIONS: Hypomagnesemia selectively impaired the release of nitric oxide from the coronary endothelium. Because nitric oxide is a potent endogenous nitro-vasodilator and inhibitor of platelet aggregation and adhesion, hypomagnesemia could promote vasoconstriction and coronary thrombosis in the early postoperative period.     

Vasodilator responses of coronary resistance arteries of exercise-trained pigs

BACKGROUND: The purpose of this study was to test the hypothesis that vasodilator responses of porcine coronary resistance arteries are increased by exercise training. METHODS AND RESULTS: Yucatan miniature swine were randomly divided into groups of exercise-trained (ET) and sedentary (SED) control pigs. ET pigs were placed on a progressive treadmill training program lasting 16 to 20 weeks, and SED pigs remained inactive during the same time period. Coronary resistance arteries 64 to 157 microns in diameter were isolated for in vitro evaluation of relaxation responses to the endothelium-independent dilators sodium nitroprusside (1 x 10(-10) to 1 x 10(-4) mol/L) and adenosine (1 x 10(-10) to 1 x 10(-5) mol/L) and to bradykinin (1 x 10(-13) to 3 x 10(-7) mol/L), an endothelium-dependent agent. Relaxation responses to adenosine and sodium nitroprusside were not altered by exercise training. Endothelium-dependent relaxation to bradykinin was enhanced in coronary resistance arteries from ET pigs (IC50: ET, 0.07 +/- 0.02 nmol/L; SED, 1.59 +/- 0.09 nmol/L). To determine whether prostanoids and/or the nitric oxide synthase pathway were involved in the ET-induced changes in bradykinin-induced vasodilation, responses to bradykinin were examined in coronary resistance arteries from both ET and SED pigs in the presence of indomethacin and in the presence of nitro-monomethyl L-arginine (L-NMMA). Both indomethacin and L-NMMA produced significant inhibition of the bradykinin-induced relaxation in vessels from both groups. Despite decreased bradykinin-induced relaxation after indomethacin, bradykinin-induced vasodilation was still enhanced in vessels from the ET group. L-NMMA caused greater inhibition of the bradykinin-induced relaxation in coronary resistance arteries from ET pigs relative to arteries from SED pigs and eliminated the training-induced enhancement of the bradykinin responses. CONCLUSIONS: These results suggest that exercise training enhances bradykinin-induced vasodilation through increased endothelium-derived relaxing factor/nitric oxide production by the L-arginine/nitric oxide synthase pathway.     

Swelling in the isolated perfused cornea induced by 12(R)hydroxyeicosatetraenoic acid

PURPOSE: To evaluate the effect of 12(R)hydroxyeicosatetraenoic acid (12(R)HETE) on corneal swelling when directly perfused to human and rabbit corneal endothelium. METHOD: Excised rabbit and human corneas were mounted in the in vitro specular microscope and the endothelium was perfused with 12(R)HETE at 10(-5), 10(-6), and 10(-7) mol/l. Both 12(R)HETE and 12(S)HETE were compared at equal molar (10(-6) mol/l) concentrations. The reversal of 12(R)HETE and ouabain corneal swelling was also compared. Endothelial permeability to carboxyfluorescein was measured after 12(R)HETE perfusion. High-performance liquid chromatographic analysis confirmed that 12(R)HETE remained in the perfusion media. RESULTS: 12(R)HETE caused a dose-dependent corneal swelling of 25 +/- 2, 24 +/- 1, and 14 +/- 0.5 microns/hr at 10(-5), 10(-6), and 10(-7) mol/l, respectively. Equal molar concentrations (10(-6) mol/l) of 12(S)HETE did not cause corneal swelling. Removal of the 12(R)HETE from the perfusion media resulted in reversal of corneal swelling whereas corneal swelling induced by ouabain did not reverse after ouabain removal. 12(R)HETE (10(-6) mol/l) perfused to the human corneal endothelium inhibited temperature reversal corneal thinning when compared to the paired corneal endothelium perfused with BSS Plus (Alcon Laboratories, Inc., Fort Worth, TX). Na/K adenosine triphosphatase activity was inhibited by 10(-6) mol/l ouabain by 35%, 10(-6) mol/l 12(R)HETE by 54%, and 10(-6) mol/l 12(S)HETE by 0.5%. Endothelial permeability to carboxyfluorescein was unaffected by 12(R)HETE. CONCLUSION: 12(R)HETE causes corneal swelling by inhibiting endothelial pump function. This inhibition of transport appears to be at least partly mediated by inhibition of endothelial Na/K adenosine triphosphatase.     

Linoleic acid induces relaxation and hyperpolarization of the pig coronary artery

Linoleic acid, a polyunsaturated C18 fatty acid, is one of the major fatty acids in the coronary arterial wall. Although diets rich in linoleic acid reduce blood pressure and prevent coronary artery disease in both humans and animals, very little is known about its mechanism of action. We believed that its beneficial effects might be mediated by changes in vascular tone. We investigated whether linoleic acid induces relaxation of porcine coronary artery rings and the mechanism involved in this process. Linoleic acid and two of its metabolites, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-hydroperoxyoctadecadienoic acid (13-HPODE), induced dose-dependent relaxation of prostaglandin (PG) F2alpha-precontracted rings that was not affected by indomethacin (10[-5] mol/L), a cyclooxygenase inhibitor, or cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC; 10[-5] mol/L), a lipoxygenase inhibitor. Removal of endothelial cells had no effect on vasorelaxation, suggesting a direct effect on the vascular smooth muscle cells (VSMC). When rings were contracted with KCl, linoleic acid failed to induce relaxation. Although tetrabutylammonium (5 x 10[-3] mol/L), a nonselective K+ channel blocker, slightly inhibited the relaxation caused by linoleic acid, glibenclamide (10[-6] mol/L), an ATP-sensitive K+ channel blocker, and charybdotoxin (7.5x10[-8] mol/L) or tetraethylammonium (5x10[-3] mol/L), two different Ca2+-activated K+ channel blockers, had no effect. However, relaxation was completely blocked by ouabain (5x10[-7] mol/L), a Na+/K+-ATPase inhibitor, or by a K+-free solution. In addition, linoleic acid (10[-6] mol/L) caused sustained hyperpolarization of porcine coronary VSMC (from -49.5+/-2.0 to -60.7+/-4.2 mV), which was also abolished by ouabain. We concluded that linoleic acid induces relaxation and hyperpolarization of porcine coronary VSMC via a mechanism that involves activation of the Na+/K+-ATPase pump.     

Endothelium-dependent relaxation in response to ethanol in the porcine isolated pulmonary artery

Many drugs cannot be dissolved in distilled water and so other solvents such as ethanol, dimethylsulphoxide and methanol are used. Because very little is known about the direct effects of these three solvents on the cardiovascular system, we have examined their effects on isolated pulmonary and coronary arteries from the pig. Increasing concentrations of ethanol, dimethylsulphoxide and methanol induced relaxation in porcine pulmonary (at 1.2% v/v, 59.9+/-9.0% (n =9), 55.9+/-9.0% (n =6) and 12.3+/-6.4% (n = 8), respectively, of U46619-induced tone) and coronary arteries (at 1.2% v/v, 69.9+/-7.1% (n = 10), 78.9+/-6.1% (n = 7) and 12.9+/-8.2% (n = 6) respectively, of U46619-induced tone). In the pulmonary arteries the relaxation in response to ethanol was found to be endothelium-dependent whereas the responses to dimethylsulphoxide and methanol were unaffected by removal of the endothelium. In the coronary arteries the relaxation to all three solvents was independent of the presence of the endothelium. Comparison of the sensitivity of the tissues to the solvents showed that ethanol and dimethylsulphoxide produced comparative responses in both the pulmonary and coronary arteries, whereas methanol was much less potent. The endothelium-dependent response to ethanol in the porcine pulmonary artery (maximum response, Emax, 67.1+/-9.3% of U46619-induced tone, n = 7) was attenuated by the cyclooxygenase inhibitor, flurbiprofen (Emax 31.9 +/- 12.0%, n=7), the nitric oxide synthase inhibitor, L-NAME (NG-nitro-L-arginine methyl ester; Emax 23.5+/-10.2%, n = 7)) and the combination of both inhibitors (Emax 18.3+/-7.8%, n = 7). The residual relaxatory response to ethanol was abolished, and converted into a contractile response, both by removal of the endothelium (at 1.7% v/v ethanol 27.3+/-11.5% of U46619-induced tone, n=7) and by the addition of a low concentration of KC1 (49.9-/+10.3%, n=6), suggesting the release of a non-prostanoid, non-nitric oxide factor from the endothelium. This response, however, was not attenuated by the cannabinoid receptor-antagonist SR141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide HCL; 52.5-/+4.3% relaxation, n =8), suggesting that the factor released in this preparation by ethanol is not a cannabinoid. The results of this study indicate that many solvents commonly used in pharmacological experiments have pronounced vasoactive properties. Methanol might be the vehicle of choice, because it was the least active solvent, whereas high concentrations of ethanol might influence vascular function at both the level of the smooth muscle and the endothelium, with the action on the endothelium involving the release of endothelium-derived relaxing factors.     

Nitric oxide regulation of corticotropin-releasing hormone release from the human perfused placenta in vitro

We have investigated the regulatory role of nitric oxide (NO) in corticotropin-releasing hormone (CRH) release from the human perfused placental lobule in vitro. The effects of the NO donor sodium nitroprusside, the NO synthase inhibitor N omega-nitro-L-arginine, and the NO substrate L-arginine on human (h) placental CRH secretion have been studied. Single lobules of term placentae were bilaterally perfused with Krebs solution (5 mL/min; 95% O2-5% CO2; 37 C; pH 7.3). Fetal and maternal perfusates were collected at 4 C every 30 min for 3 h. CRH immunoreactivity (CRH-IR) in perfusates was measured by RIA using the 41-residue synthetic CRH as standard, 125I-labeled Tyr-hCRH as tracer, and a rabbit anti-CRH antibody Y2BO. The sensitivity of the assay was 0.13 pmol/L. Under basal conditions, human perfused placentae in vitro continuously secreted CRH-IR, which diluted in parallel to a synthetic hCRH-(1-41) standard curve. Size-exclusion chromatography of placental perfusates using a Sephadex G-50 column indicated that placental CRH-IR predominately coeluted with hCRH-(1-41) standard. Basal maternal perfusate CRH-IR levels (27 +/- 4 pmol/L) released from perfused placental lobules were nearly 10-fold greater than fetal perfusate CRH-IR levels (3.4 +/- 0.7 pmol/L; P < 0.05). Infusion of sodium nitroprusside (30-100 mumol/L) into the maternal and fetal placental circulations inhibited CRH-IR release into maternal perfusate in a concentration-dependent manner, but did not inhibit CRH-IR release into the fetal perfusate. N omega-nitro-L-arginine (100 mumol/L) increased placental CRH-IR secretion into fetal perfusate, and this effect was reversed by the infusion of L-arginine (100 mumol/L), which also reduced release below basal levels. In contrast, maternal perfusate CRH-IR levels were not affected by N omega-nitro-L-arginine or L-arginine. These results indicate that the human perfused placenta in vitro releases a substance of similar mol wt and hCRH-IR. Moreover, modulators of the NO signaling pathway differentially affect placental secretion of CRH-IR into the maternal and fetal perfusates. These data are consistent with the involvement of NO in the regulation of placental CRH release during pregnancy.     

The fetoplacental pressor effects of low-dose acetylsalicylic acid and angiotensin II in the ex vivo cotyledon model

OBJECTIVE: Our purpose was to investigate perfusion pressure changes ex vivo induced by angiotensin II on fetoplacental vasculature pretreated with low-dose acetylsalicylic acid. STUDY DESIGN: Two cotyledons from each of 12 placentas were perfused. The intervillous space of one cotyledon was infused with acetylsalicylic acid (5 x 10(-5) mol/L) similar to the serum concentration of women receiving daily low-dose aspirin therapy (60 to 81 mg). The control cotyledon was infused with an equivalent amount of normal saline solution. Two doses of angiotensin II, 1 x 10(-11.5) and 1 x 10(-10) moles, were injected as boluses into the chorionic arteries of each cotyledon. A 3 x 10(-7) mole dose of angiotensin II was also injected into the intervillous space. Statistical analysis was performed with analysis of variance, and results are expressed as mean pressure change in millimeters of mercury +/- SEM. RESULTS: Perfusion pressure response did not vary between cotyledons pretreated with acetylsalicylic acid and control cotyledons when 3 x 10(-7) moles of angiotensin II was injected into the intervillous space (8.0 +/- 1.9 mm Hg vs 9.8 +/- 1.6 mm Hg, p = 0.59). There were no differences between cotyledons in pressure response to 1 x 10(-11.5) moles of angiotensin II injected into the fetal circuit (5.9 +/- 0.8 mm Hg vs 6.7 +/- 0.9 mm Hg, p = 0.51). However, in the cotyledons pretreated with acetylsalicylic acid there was a decrease in the pressor response to 1 x 10(-10) moles of angiotensin II (14.1 +/- 1.4 mm Hg vs 21.5 +/- 3.3 mm Hg, p = 0.05). CONCLUSIONS: Low-dose aspirin infused into the intervillous space decreases vasoconstriction elicited by angiotensin II in the fetoplacental compartment. This suggests that maternal low-dose aspirin therapy has effects in the fetoplacental circulation in addition to its effects in the maternal circulation.     

Role of K+ channel opening and stimulation of cyclic GMP in the vasorelaxant effects of nicorandil in isolated piglet pulmonary and mesenteric arteries: relative efficacy and interactions between both pathways

1. The effects of the K+ channel opener levcromakalim, the guanylate cyclase stimulant nitroprusside and the dual drug nicorandil (K+ channel opener and guanylate cyclase stimulant) were analysed in piglet isolated endothelium-denuded pulmonary (PA) and mesenteric (MA) arteries stimulated by noradrenaline (NA) or by the thromboxane A2 mimetic U46619. 2. Nicorandil, levcromakalim and verapamil were less potent in PA than in MA, the efficacy of levcromakalim was also reduced in PA. The effects of nicorandil and levcromakalim were similar in arteries pre-contracted by NA and U46619, whereas verapamil was more potent in arteries pre-contracted by NA. Nitroprusside was equipotent in MA pre-contracted by either NA or U46619 and in PA pre-contracted by NA whereas in PA pre-contracted by U46619, nitroprusside showed lower potency and efficacy. 3. The relaxant effects of levcromakalim and nitroprusside were inhibited by 10(-5) M glibenclamide and 10(-6) M ODQ, respectively. Nicorandil-induced relaxation was inhibited by ODQ in all experimental conditions, whereas glibenclamide had inhibitory effects in PA and MA pre-contracted by U46619, had no effect in PA pre-contracted by NA and in MA pre-contracted by NA it was only inhibitory in the presence of ODQ. 4. No apparent interactions were found between nitroprusside and levcromakalim as indicated by the lack of effects of pretreatment with one of them (producing 20-35% relaxation) on the potency of the relaxant response to the other. However, in PA pre-contracted by U46619, where nitroprusside or levcromakalim induced only partial relaxation, the combination of both mechanisms (either by combining nitroprusside plus levcromakalim or by nicorandil) was able to induce full vasodilatation. 5. In conclusion, K+ channel opening and guanylate cyclase stimulation are independent pathways that induce additive vasorelaxation in piglet PA and MA. The mechanism of action of nicorandil is dependent on the artery and on the nature of the agonist employed to precontract the artery. The relative efficacy of K+ channel opening vs guanylate cyclase stimulation may partially explain the preferential contribution of each mechanism to the relaxant effects of nicorandil.     

Dopamine-induced relaxation in isolated intrarenal arteries from adult stroke-prone spontaneously hypertensive rats

1. The relaxant effects of dopamine (DA) on the intrarenal arteries obtained from 6 month old stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wistar-Kyoto (WKY) rats were pharmacologically investigated in vitro. 2. DA (10(-7)-3 x 10(-5) mol/L) produced endothelium-independent relaxation on the arterial rings which had been incubated with phenoxybenzamine (2 x 10(-6) mol/L) and precontracted with KCl. 3. DA-induced relaxation was greater in the arterial rings from SHRSP than in those from WKY. SKF 38393 (10(-8)-10(-6) mol/L) partially mimicked DA-produced relaxation in both groups. SCH 23390 dose-dependently inhibited DA-induced relaxation with pD'2 value of 9.33 for SHRSP and of 9.26 for WKY. 4. There were no significant differences between SHRSP and WKY in the relaxation caused by forskolin, dibutyryl cyclic AMP, or 3-isobutyl-1-methylxanthine. 5. These results suggested that DA1 receptor-mediated relaxation was increased in the intrarenal arteries from SHRSP, and this increase might not be associated with altered vasodilation mediated by cyclic AMP.     

Role of nitric oxide in cyclosporine A-induced hypertension

Cyclosporine A (CsA) is an immunosuppressive agent that also causes hypertension. The effect of CsA on vascular responses was determined in Sprague-Dawley rats and isolated rat aortic rings. Male rats weighing 250 to 300 g were given either CsA (25 mg. kg-1. d-1) in olive oil or vehicle by intraperitoneal injection for 7 days. CsA administration produced a 42% increase (P<0.001) in mean arterial pressure (MAP) that reached a plateau after 3 days. Conversely, the levels of both nitrate/nitrite, metabolites of nitric oxide (NO), and cGMP, which mediates NO action, decreased by 50% (P<0.001) and 35% (P<0.001), respectively, in the urine. Thoracic aortic rings from rats treated with CsA and precontracted with endothelin (10(-9) mol/L) showed a 35% increase (P<0.001) in tension, whereas endothelium-dependent relaxation induced by acetylcholine (ACh, 10(-9) mol/L) was inhibited 65% (P<0.001) compared with that in untreated rats. This response was similar to that of endothelium-denuded aortic rings from untreated rats in which ACh-induced relaxation was completely abolished (P<0.001), but relaxation induced by S-nitroso-N-acetylpenicillamine (SNAP, 10(-8) mol/L) was unaffected (P<0.001). ACh-induced formation of both nitrate/nitrite and cGMP by both denuded and CsA-treated aortic rings was inhibited 95% (P<0.001) and 65% (P<0.001), respectively, compared with intact aortic rings. The effects of CsA were reversed both in vivo and in vitro by pretreatment with L-arginine (10 mg. kg-1. d-1 IP), the precursor of NO. There were no changes in MAP and tension in rats treated with L-arginine alone. In summary, CsA inhibits endothelial NO activity, with resulting increases in MAP and tension, and this inhibition can be overcome by parenteral administration of L-arginine.     

Autacoid interactions in the regulation of blood flow in the human placenta

1. Interactions between autacoids may play important roles in the regulation of blood flow in the foetal placenta. In order to investigate this aspect of placental haemodynamics, human normal-term placentae were perfused in vitro and the responses of the foetal vessels to various combinations of vasoactive agents were determined. 2. Vasoconstriction responses to 5-hydroxytryptamine (5-HT) were potentiated in the presence of endothelin-1 (ET-1), the thromboxane A2-mimetic U46619 and a nitric oxide synthase inhibitor, N-nitro-L-arginine (NOLA), but not in the presence of angiotensin II. 3. N-Nitro-L-arginine caused vasoconstriction of the perfused placenta and indomethacin attenuated this effect and blocked the potentiation of the 5-HT response by NOLA. 4. Indomethacin did not affect ET-1-induced pressure increases and infusion of U46619 had no effect on release of ET-like immunoreactivity into the foetal placental circulation. 5. The present study provides evidence of interactions between several autacoids in human perfused placentae in vitro. These interactions may play important roles in foetal placental haemodynamics in normal or pathological situations.     

The effects of combined antifolates on inhibition of growth of murine leukemia cells cultured in vitro

PURPOSE: Long-term patency of cryopreserved vascular grafts is determined by maintained cellular and tissue viability, which implies preservation of various biochemical, smooth muscle, and endothelial functions. Therefore, it was investigated whether the presence of fetal calf serum (FCS) in the cryomedium improves the postthaw contractile and endothelial function of human arteries. METHODS: Rings from human mesenteric (HMA) and left circumflex coronary arteries (HCA) obtained from organ donors were randomized into three groups and studied either unfrozen or after storage for 3 to 6 weeks at -196 degrees C while suspended in Krebs-Henseleit solution without or with 20% FCS as the vehicles and 1.8 mol/L dimethyl sulfoxide and 0.1 mol/L sucrose as cryoprotecting agents. The samples were slowly frozen to -70 degrees C and then stored in liquid nitrogen. Before use, the tissues were thawed within 3 minutes in a 40 degrees C water bath. RESULTS: After thawing the sensitivity to various agonists and maximal responses to the endothelium-independent relaxing agent sodium nitroprusside were unchanged. However, after cryopreservation of HMA was performed without and with FCS, maximal contractile responses to noradrenaline were significantly reduced to 10.1 +/- 0.7 gm and 9.9 +/- 0.9 gm compared with 13.3 +/- 0.6 gm in unfrozen HMA (mean +/- SEM, n = 15). After cryopreservation of HCA was performed without and with FCS, maximal contractile responses to prostaglandin F2 alpha (6.9 +/- 0.4 gm in unfrozen HCA) were significantly reduced to 4.3 +/- 0.3 gm and 3.8 +/- 0.2 gm (mean +/- SEM, n = 6). In both types of arteries cryopreservation also attenuated significantly the endothelium-dependent relaxant responses to bradykinin during U46619 (10 nmol/L)-induced tone. In HMA the maximal bradykinin-induced relaxation (85% +/- 4%) was significantly diminished to 29% +/- 7% and 38% +/- 9% after cryopreservation without and with FCS (mean +/- SEM, n = 6). In HCA maximal bradykinin-induced relaxation (88% +/- 4%) was significantly diminished to 26% +/- 10% and 36% +/- 11% after cryopreservation without and with FCS (mean +/- SEM, n = 6). This result was reflected by a marked endothelial denudation in all groups of cryopreserved arteries. Neither functional nor morphologic preservation of the endothelial cell lining was significantly improved by FCS supplementation of the cryomedium. CONCLUSIONS: Cryopreservation diminished contractile and endothelium-dependent relaxant responses of human arteries. The presence of FCS in the cryomedium did not modify these changes.     

Ouabain amplifies contractile responses to phenylephrine in rat tail arteries in hypertension

Ouabain, a cardiac glycoside, binds to the alpha-subunits of Na+, K(+)-ATPase and inhibits Na+ pump activity. It has been proposed that endogenous ouabain, by inhibiting vascular Na+, K(+)-ATPase, can increase vascular resistance and thus may contribute to hypertension. One of the consequences of inhibition of the membrane Na+ pump is enhanced responsiveness of vascular smooth muscle to vasopressor substances. The purpose of the present study was to determine whether ouabain can enhance the responsiveness of the vasculature in hypertension. In the present study 100 microM ouabain enhanced the contractile response elicited by phenylephrine in isolated, perfused tail arteries from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. The enhanced contractile response was more pronounced in the arteries of the SHR. We demonstrated that this concentration of ouabain inhibits the Na+ pump activity, measured as ouabain-sensitive 86Rb uptake, by about 65%, in isolated tail arteries. We conclude that ouabain can sensitize the vascular smooth muscle to the effects of vasopressor substances and this effect is more pronounced in genetically hypertensive rats. Endogenous ouabain may contribute to the pathophysiology of hypertension by enhancing vascular tone.     

Altered neuropeptide Y Y1 responses in mesenteric arteries in rats with congestive heart failure

The aim of the present study was to elucidate if the potentiating effect of neuropeptide Y on various vasoactive agents in vitro is (1) altered in mesenteric arteries from rats with congestive heart failure and (2) mediated by the neuropeptide Y Y1 receptor. The direct vascular effects of neuropeptide Y and its modulating effects on the contractions induced by endothelin-1-, noradrenaline-, 5-hydroxytryptamine (5-HT)-, U46619-(9,11-dideoxy-11alpha, 9alpha-epoxymethano-prostaglandin F2alpha) and ATP, and acetylcholine-induced dilatations were studied in the presence and absence of the neuropeptide Y Y1 antagonist, BIBP3226 (BIBP3226?(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl ]-D-arginine-amide?). Neuropeptide Y, per se, had no vasoactive effect in the arteries. The potency of endothelin-1 was significantly decreased in congestive heart failure rats. Neuropeptide Y and neuropeptide Y-(13-36) potentiated the endothelin-1-induced contraction in congestive heart failure mesenteric arteries. In 20% of the congestive heart failure rats, sarafotoxin 6c induced a contraction of 31+/-4%. Neuropeptide Y also potentiated U46619- and noradrenaline-induced contractions but not 5-HT-induced contractions in congestive heart failure arteries. In sham-operated animals neuropeptide Y potentiated noradrenaline- and 5-HT-induced contractions. These potentiations were inhibited by BIBP3226. Acetylcholine induced an equipotent relaxation in both groups which was unaffected by neuropeptide Y. In conclusion, neuropeptide Y responses are altered in congestive heart failure rats. The potentiating effect differs between vasoactive substances. Neuropeptide Y Y1 and non-neuropeptide Y1 receptors are involved.     

Inhibition of cAMP mediated relaxation in rat coronary vessels by block of Ca++ activated K+ channels

The hypothesis for this study is that block of calcium activated potassium (KCa) channels inhibits cAMP induced relaxation in pressurized rat coronary resistance arteries. Pressure-diameter experiments with septal arteries (200-270 microns internal diameter at 60 mmHg and maximum dilation) showed significant basal tone over a range of pressure from 40-120 mmHg. The level of tone was increased with the thromboxane A2 analogue 9,11-dideoxy-11 alpha, 9 alpha-epoxy-methanoprostaglandin F2 alpha (U46619) in all experiments. Receptor activation of the cAMP pathway was done with adenosine (ADO) and isoproterenol (ISO). Tetraethylammonium ion (TEA+), 1mM, significantly inhibited relaxation to ADO (10(-6)-10(-3)M) with a maximal inhibition of 75 +/- 7% (as a % of maximum diameter change with the vasodilator alone) at 10(-3)M ADO. TEA+ inhibited ISO (10(-6)M) relaxation by 63 +/- 9%. Direct activation of the cAMP pathway was done with forskolin and 8-bromo-cAMP. TEA+ significantly inhibited forskolin (10(-6)-10(-4)M) induced relaxation with a maximal inhibition of 81.3 +/- 1.2% at 10(-4)M forskolin. TEA+ and iberiotoxin (10(-7)M) significantly inhibited 8- bromo-cAMP (10(-3)M) induced relaxation by 72 +/- 5% and 56 +/- 3% respectively. The effect of TEA+ on relaxation induced by nitroprusside (a cGMP dependent vasodilator) was not significant. The results show that rat coronary resistance arteries possess significant myogenic tone and modulation of Kca channels plays a major role in cAMP mediated relaxation.     

Inhibition of alpha1-adrenoceptor-mediated contractions in isolated tail arteries and aorta of the rat by alpha2-adrenoceptor agonists

The effects of alpha2-adrenoceptor agonists, clonidine, tizanidine and UK-14304 on alpha1-adrenoceptor-mediated contractile responses were studied in isolated tail arteries and thoracic aorta of the rat. When applied during sustained contractile responses to almost maximum concentration (10 microM) of phenylephrine, clonidine (0.3 microM to 100 microM) produced concentration-dependent relaxations in both tissues. The maximum relaxation was smaller in tail arteries than in thoracic aorta. Clonidine up to 100 microM failed to relax both tissues precontracted with KCl (60 microM) or U-46619 (1 microM), a thromboxane mimetic. The clonidine-induced relaxation in tail arteries, was reversed by alpha2-adrenoceptor antagonists, yohimbine and idazoxane. Effects of the alpha2-adrenoceptor antagonists were concentration-dependent (0.1 microM to 1 microM), but not in a competitive manner. On the other hand, the relaxation in thoracic aorta was not significantly antagonized by these alpha2-adrenoceptor antagonists. Tizanidine and UK-14304 also relaxed both tail arteries and thoracic aorta precontracted with phenylephrine. The characteristics of the relaxation and their antagonism by yohimbine in both arteries were similar to those induce by clonidine. In tail arteries, NG-nitro-L-arginine, a nitric oxide synthase inhibitor, at a concentration that completely inhibited acetylcholine-induced relaxations did not significantly affect the relaxation induced by clonidine. In contrast, the relaxation of thoracic aorta in response to clonidine was partly reduced in the presence of NG-nitro-L-arginine. These results indicate that the alpha2-adrenoceptor agonists selectively inhibit the contractions induced by phenylephrine in both tissues. Regional differences in the modes of the inhibition by the alpha2-adrenoceptor agonists exist.     

Innervation and nitric oxide modulation of mesenteric arteries of the golden hamster

Immunohistochemical and pharmacological techniques were used to examine perivascular nerves, endothelium and the effects of inhibition of nitric oxide synthesis on responses in mesenteric arteries/perfused mesenteric arterial beds of the Golden hamster. Frequency-dependent vasoconstrictions to electrical field stimulation and dose-dependent vasoconstrictions to noradrenaline were significantly augmented by NG-nitro-L-arginine methyl ester (10(-5) M), an inhibitor of nitric oxide synthase. In preparations with tone raised with methoxamine (10 microM) dose-dependent relaxations to ATP, but not to acetylcholine, were blocked by NG-nitro-L-arginine methyl ester. In the presence of guanethidine (5 microM) to block sympathetic neurotransmission there was no neurogenic relaxation to electrical field stimulation. Furthermore, the sensory neurotoxin capsaicin (0.05-5 nmol) did not elicit relaxation. Immunohistochemical studies demonstrated dense plexuses of fibres immunoreactive for tyrosine hydroxylase and neuropeptide Y, a plexus of moderate density for calcitionin gene-related peptide and an absence of fibres immunoreactive for substance P and vasoactive intestinal polypeptide. Of particular interest is the finding that whereas sympathetic perivascular nerves and nitric oxide regulate the function of hamster mesenteric arteries, there is no apparent motor function of calcitonin gene-related peptide-containing sensory nerves.     

Alpha-2 adrenoceptor subtype causing nitric oxide-mediated vascular relaxation in rats

The alpha-2 adrenoceptor subtype and its signal transduction pathway mediating vascular relaxation in rats were studied in vitro using rings of superior mesenteric arteries. Removal of endothelium or incubation with NG-nitro-L-arginine completely blocked relaxant responses to UK14,304, suggesting endothelium-derived nitric oxide mediates relaxation. The order of potency for full (F) or partial (P) agonists causing relaxation was guanabenz (P) > UK14,304 (F) > clonidine (P) > epinephrine (F) > norepinephrine (F). Affinities (Ka) of alpha-2 adrenoceptor subtype-selective drugs for blocking relaxation were obtained in side-by-side experiments comparing rat mesenteric arteries with pig coronary arteries. Relaxation of pig coronary arteries is known to be mediated by the alpha-2A adrenoceptor subtype. Ka values in nM for rauwolscine (19), WB-4101 (265), SKF-104078 (197), spiroxatrine (128), and prazosin (1531) for blocking relaxation in rat arteries were consistent with their affinities for binding at the alpha-2D adrenoceptor subtype. Ka values for rauwolscine and WB-4101, drugs distinguishing the alpha-2D from the alpha-2A adrenoceptor subtype, were significantly higher in blocking relaxation of rat arteries compared with pig arteries, suggesting the alpha-2D adrenoceptor subtype mediates NO-induced relaxation in rat arteries. We used forskolin to oppose alpha-2 adrenoceptor-mediated inhibition of cAMP formation by directly stimulating cAMP formation in endothelium. Forskolin did not affect the relaxant response to UK14,304, suggesting that cAMP is not involved in the coupling of alpha-2 adrenoceptors to nitric oxide-induced vascular relaxation.     

Involvement of calcitonin gene-related peptide (CGRP) receptors in insulin-induced vasodilatation in mesenteric resistance blood vessels of rats

1. The vascular effect of insulin in the mesenteric resistance blood vessel and the role of calcitonin generelated peptide (CGRP)-receptor in insulin-induced vascular responsiveness were investigated in rats. 2. The mesenteric vascular beds isolated from Wistar rats were perfused with Krebs solution, and perfusion pressure was measured with a pressure transducer. In preparations contracted by perfusion with Krebs solution containing methoxamine in the presence of guanethidine, the perfusion of insulin (from 0.1 to 3000 nM) caused a concentration-dependent decrease in perfusion pressure due to vasodilatation. The pD2 value and maximum relaxation (%) were 6.94+/-0.22 and 43.9+/-5.2, respectively. 3. This vasodilator response to insulin was unaffected by 100 nM propranolol (beta-adrenoceptor antagonist) plus 100 nM atropine (muscarinic cholinoceptor antagonist), 100 microM L-NG-nitroarginine (nitric oxide synthase inhibitor), 1 microM ouabain (Na+-K+ ATPase inhibitor), or 1 microM glibenclamide (ATP sensitive K+-channel inhibitor). 4. In preparations without endothelium, perfusion of insulin produced a marked vasodilatation. The pD2 value and maximum relaxation (%) were 7.62+/-0.21 and 81.0+/-4.6, respectively, significantly greater than in preparations with intact endothelium. 5. The vasodilator responses to insulin in the preparations without endothelium were significantly inhibited by CGRP[8 37], a CGRP receptor antagonist, whereas pretreatment with capsaisin, a toxin for CGRP-containing nerves, did not affect insulin-induced vasodilatation. 6. These results suggest that insulin induces non-adrenergic, non-cholinergic and endothelium-independent vasodilatation, which is partially mediated by CGRP receptors.