The vasodilator effect of thiamylal in dog mesenteric artery: contribution of intracellular action.

The effects of thiamylal on contractions induced by various mechanisms were investigated in mesenteric arteries isolated from dogs. Thiamylal (10(-4) to 10(-3) M) significantly inhibited contractions induced by KCl (20 mM) in normal media, and those induced by norepinephrine (10(-5) M) in normal and Ca(2+)-free media. Caffeine-induced contraction was significantly inhibited by thiamylal in the concentrations greater than 3 x 10(-5) M in intact fibers and 10(-5) M in chemically skinned fibers. Chemically skinned fibers that were precontracted with Ca2+ were relaxed by thiamylal in concentrations lower than those required to relax intact fibers that were precontracted with KCl (20 mM); the ED50 was 1.52 x 10(-5) M in skinned fibers and 5.50 x 10(-4) M in intact fibers. These results suggest that intracellular mechanisms are involved in thiamylal-induced vasodilatation of dog mesenteric artery.     

Guanylyl cyclase mediates ANP-induced vasoconstriction of murine splenic vessels

We have previously shown that ANP causes differential constriction of the splenic vasculature of the rat (veins greater than arteries), which may be inhibited by blocking the production of cGMP with A7195. In this paper, we report experiments done on vessels derived from guanylyl cyclase (GC)-A knockout mice. Small splenic arteries ( approximately 150-microm diameter) and veins ( approximately 250-microm diameter) were dissected from male GC-A-deficient 129sv mice or age-matched wild-type controls and mounted in a wire myograph. In the wild-type mice, ANP exhibited higher potency in the veins than in the arteries (EC(50) values wild-type mice: artery, 8 +/- 3 x 10(-9) M, n = 5 vs. vein, 6 +/- 4 x 10(-10) M, n = 5; P < 0.05). The concentration-response curve for ANP-induced vasoconstriction was also shifted leftward in denuded compared with intact arteries (EC(50) values: denuded artery: 5 +/- 3 x 10(-10) M, n = 5 vs. intact artery, 8 +/- 3 x 10(-9) M, n = 5; P < 0.05), i.e., the denuded vessels were more reactive. By contrast, ANP caused no significant change in tension from baseline in intact splenic arteries, intact splenic veins, or denuded splenic arteries derived from the GC-A-deficient mice, although these vessels did show normal concentration-dependent increases in tension to phenylephrine. We conclude that ANP causes vasoconstriction in the splenic vasculature by an endothelium-independent mechanism, mediated via guanylyl cyclase.     

Mesenteric arterial relaxation to calcitonin gene-related peptide is increased during pregnancy and by sex steroid hormones

The present study investigated whether pregnancy and circulatory ovarian hormones increase the sensitivity of the mesenteric artery to calcitonin gene-related peptide (CGRP)-induced relaxation and possible mechanisms involved in this process. Mesenteric arteries from young adult male rats or female rats (during estrous cycle, after ovariectomy, at Day 20 of gestation, or Postpartum Day 2) were isolated, and the responsiveness of the vessels to CGRP was examined with a small vessel myograph. The CGRP (10(-10) to 10(-7) M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in mesenteric arteries of all groups. Arterial relaxation sensitivity to CGRP was significantly (P < 0.05) greater in female rats compared with male rats. Pregnancy increased the sensitivity to CGRP significantly (P < 0.05) compared to ovariectomized and Postpartum Day 2 rats. In pregnant rats, CGRP-receptor antagonist, CGRP(8-37), inhibited the relaxation responses produced by CGRP. The CGRP-induced relaxation was not affected by N(G)-nitro-l-arginine methyl ester (nitric oxide inhibitor, 10(-4) M) but was significantly (P < 0.05) attenuated by an inhibitor of guanylate cyclase (1H-[1 , 2 , 4 ]oxadizaolo[4 , 3 -a]quinoxalin-1-one, 10(-5) M). Relaxation responses of CGRP on mesenteric arteries were blocked (P < 0.05) by a cAMP-dependent protein kinase A inhibitor, Rp-cAMPs (10(-5) M). The CGRP-induced vasorelaxation was significantly (P < 0.05) attenuated by calcium-dependent (tetraethylammonium, 10(-3) M), but not ATP-sensitive (glybenclamide, 10(-5) M), potassium channel blocker. Therefore, the results of the present study suggest that mesenteric vascular sensitivity to CGRP is higher during pregnancy and that cAMP, cGMP, and calcium-dependent potassium channels appear to be involved. Therefore, we propose that CGRP-mediated vasodilation may be important to maintain vascular adaptations during pregnancy.     

BK(Ca) channels compensate for loss of NOS-dependent coronary artery relaxation in cardiomyopathy

Previously, we showed that development of myocardial necrotic lesions is associated with impaired endothelium-dependent coronary artery relaxation in young cardiomyopathic hamsters. Since active necrosis declines with aging, this study was designed to determine whether coronary artery endothelium-dependent relaxation to ACh is restored and to identify the mechanisms mediating this effect. Intraluminal diameter was recorded in coronary arteries (150-250 micrometer) from control (C, 297 +/- 5 days old) and cardiomyopathic (M, 296 +/- 4 days old) hamsters. Relaxation to ACh (10(-9)-3 x 10(-5) M) was similar in vessels from C and M hamsters. However, mechanisms mediating relaxation to ACh were altered. Inhibition of nitric oxide synthase (NOS) activity with N-nitro-L-arginine (1 mM) had a greater inhibitory effect in vessels from C hamsters, indicating a reduction in NOS-dependent relaxation in vessels from M hamsters. Conversely, inhibition of large Ca(2+)-dependent K(+) (BK(Ca)) channels with charybdotoxin (CTX, 0.1 microM) had a greater inhibitory effect in vessels from M hamsters. In the presence of both N-nitro-L-arginine and CTX, relaxation to ACh was abolished in both groups. CTX (0.1 micrometer) produced a 50 +/- 4 and 30 +/- 3% contraction of vessels from M and C hamsters, respectively, indicating an enhanced role for BK(Ca) channels in regulation of coronary artery tone in M hamsters. Finally, vasodilatory cyclooxygenase products contributed to ACh-induced relaxation in vessels from M, but not C, hamsters. In conclusion, NOS-dependent relaxation of coronary small arteries is reduced in the late stage of cardiomyopathy. An increase in relaxation mediated by BK(Ca) channels and vasodilatory cyclooxygenase products compensates for this effect.     

Biphasic effects of exogenous phospholipase D on vessel tone

There exists functional significance for agonists stimulated phospholipase D (PLD) and exogenous PLD in different systems, but the functional importance of PLD on vessel tone is not clear. We studied the functional importance of PLD in whole animals and in isolated vessel preparation. In in vivo study, we demonstrated that intravenous injection of PLD at 10, 30, and 100 units to male Sprague-Dawley rats did not significantly alter the mean arterial blood pressure and heart rate in 30 minutes. However, in a denuded vessel preparation, PLD from 10(-3) units/ml to 10 units/ml induced vasoconstriction from 2.8 +/- 1.7 % to 30.5 +/- 1.7 % of maximal KCI contraction in calcium enriched physiological salt solution (PSS). This vasoconstrictive effects of PLD were significantly inhibited by omission of extracellular calcium from PSS or by pretreatment the vessels with nifedipine (10(-6) M). Pretreated the denuded vessels with a protein kinase C inhibitor, chelerythrine (10(-6) M), did not significantly alter the vasoconstrictive effects of PLD. These results indicate that calcium channel rather than protein kinase C activation is involved in PLD-induced vasoconstriction. In endothelium-intact vessels, application of PLD from 10(-4) units/ml to 10 units/ml induced endothelium dependent relaxation in vessels precontracted with phenylephrine (10(-6) M). This relaxation effects of PLD were inhibited by pretreatment of vessels with indomethacin (10(-5) M) or with N-nitro-L-arginine (L-NAME, 10(-4) M), suggesting prostaglandin and nitric oxide released by PLD stimulation. The biphasic effects of PLD on vessel tone are mediated by extracellular calcium and by endothelium-derived nitric oxide and prostaglandin.     

Apelin effects in human splanchnic arteries. Role of nitric oxide and prostanoids

Apelin effects were examined in human splanchnic arteries from liver donors (normal arteries) and from liver recipients. Segments 3 mm long were obtained from mesenteric arteries taken from liver donors (normal arteries), and from hepatic arteries taken from cirrhotic patients undergoing liver transplantation (liver recipients), and the segments were mounted in organ baths for isometric tension recording. In arteries under resting conditions, apelin (10(-10)-10(-6) M) caused no effect in any of the arteries tested. In arteries precontracted with the thromboxane A(2) analogue U46619 (10(-7)-10(-6) M), apelin (10(-10)-10(-6) M) produced concentration-dependent relaxation that was lower in hepatic than in mesenteric arteries, whereas sodium nitroprusside (10(-8)-10(-4) M) produced a similar relaxation in both types of arteries. The inhibitor of nitric oxide synthesis N(w)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) diminished the relaxation to apelin in mesenteric but not in hepatic arteries. The inhibitor of cyclooxygenase meclofenamate (10(-5) M) did not affect the relaxation provoked by apelin in both types of arteries. Therefore, apelin may produce relaxation in normal human splanchnic arteries, and this relaxation may be mediated in part by nitric oxide without involvement of prostanoids. This relaxation as well as the role of nitric oxide may be decreased in splanchnic arteries from cirrhotic patients.     

Hydrogen sulfide as an endogenous regulator of vascular smooth muscle tone in trout

Hydrogen sulfide (H(2)S) is an endogenous vasodilator in mammals, but its presence and function in other vertebrates is unknown. We generated H(2)S from NaHS and examined the effects on isolated efferent branchial arteries from steelhead (stEBA) or rainbow (rtEBA) trout. H(2)S concentration was measured colorimetrically (CM) and with ion-selective electrodes (ISE) in rainbow trout plasma. NaHS produced a triphasic response consisting of a relaxation (phase 1), constriction (phase 2), and relaxation (phase 3) in both unstimulated vessels and in stEBA precontracted with carbachol (Carb). Phase 1 and phase 3 in stEBA were decreased and phase 2 increased in unstimulated vessels by K(+)(ATP) channel inhibition (glibenclamide), or a cocktail of inhibitors of cyclooxygenase, lipoxygenase, and cytochrome P-450 (indomethacin, esculetin, and clotrimazole). Inhibition of soluble guanylate cyclase with ODQ o NS-2028 inhibited phase 3 in stEBA, although NaHS decreased cGMP production by tEBA. stEBA phase 2 contractions were partially inhibited by the myosin light chain kinase inhibitor, ML-9, but unaffected by L-type calcium channel inhibition (methoxyverapamil), whereas contraction in tEBA was partially inhibited by nifedipine or removal of extracellular calcium. Phase 3 relaxations were more pronounced in stEBA precontracted with Carb and no epinephrine (NE) than those cont acted by KCl or K(2)SO(4). stEBA phase 2 and phase 3 responses were dose dependent (EC(50) = 1.1 +/- 1.2 x 10(-3) M and 6.7 +/- 0.9 x 10(-5) M, respectively; n = 7). NaHS was also vasoactive in steelhead bulbus arteriosus, celiac mesenteric arteries, and anterior cardinal veins. Rainbow trout plasma sulfide concentration was 4.0 +/- 0.3 x 10(-5) M, n = 4 (CM) and 3.8 +/- 0.4 x 10(-5) M, n = 9 (ISE); similar to phase 3 EC(50). Because NaHS has substantial vasoactive effects at physiological plasma concentrations, we propose that its soluble derivative, H(2)S, is a tonically active endogenous vasoregulator in trout.     

Inhibition of acetylcholine-induced EDHF response by elevated glucose in rat mesenteric artery

The effects of high glucose on endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations of isolated rat mesenteric artery and the possible involvement of reactive oxygen species in these responses were investigated. After precontraction with phenylephrine (3 x 10(-8)-10(-7) M), acetylcholine (10(-8)-3 x 10(-6) M) and A 23187 (10(-8)-3 x 10(-6) M), a calcium ionophore, induced concentration-dependent relaxations in the presence of N(W)-nitro-l-arginine methyl ester (L-NAME) (10(-4) M) and indomethacin (10(-5) M). These relaxations were abolished in the presence of charybdotoxin (2 x 10(-7) M) plus apamin (10(-7) M) and were assumed to be mediated by EDHF. Effects of elevated glucose were examined by incubating the arterial rings for 6 h in Krebs-Henseleit solution containing 22.2 mM glucose. Under these conditions relaxation to acetylcholine was significantly attenuated but was unchanged when the tissues were incubated for 6 h in solution containing 11.1 mM mannitol used as hyperosmotic control. Addition of superoxide dismutase (SOD) (75 U/ml) and combination of SOD with catalase (200 U/ml) during incubation with high glucose significantly preserved the impairment of EDHF-mediated relaxations to acetylcholine. A 23187-induced endothelium-dependent relaxation was not affected by high glucose. Similarly, relaxations to pinacidil (10(-10)-10(-5) M) and to sodium nitroprusside (SNP) (10(-10)-3 x 10(-7) M) were also unchanged in the rings exposed to high glucose. These results suggest that in rat mesenteric arteries exposed to elevated glucose receptor-dependent EDHF-mediated relaxations (acetylcholine-induced) are impaired whereas receptor-independent ones (A 23187-induced) and responses to smooth muscle relaxants that exert their effects through mechanisms independent of endothelium are unaffected. Our findings lead us to propose that reactive oxygen species like superoxide ((.)O(2)(-)) and hydrogen peroxide (H(2)O(2)) do seem to play a role in the impairment of EDHF-mediated relaxations in the presence of elevated glucose.     

ACh-induced relaxations of rabbit small mesenteric arteries: role of arachidonic acid metabolites and K+

ACh-induced endothelium-dependent relaxation in rabbit small mesenteric arteries is resistant to N-nitro-L-arginine (L-NA) and indomethacin but sensitive to high K+, indicating the relaxations are mediated by endothelium-derived hyperpolarizing factors (EDHFs). The identity of the EDHFs in this vascular bed remains undefined. Small mesenteric arteries pretreated with L-NA and indomethacin were contracted with phenylephrine. ACh (10(-10) to 10(-6) M) caused concentration-dependent relaxations that were shifted to the right by lipoxygenase inhibition and the Ca(2+)-activated K+ channel inhibitors apamin (100 nM) or charybdotoxin (100 nM) and eliminated by the combination of apamin plus charybdotoxin. Relaxations to ACh were also blocked by a combination of barium (200 microM) and apamin but not barium plus charybdotoxin. Addition of K+ (10.9 mM final concentration) to the preconstricted arteries elicited small relaxations. K+ addition before ACh restored the charybdotoxin-sensitive component of relaxations to ACh. K+ (10.9 mM) also relaxed endothelium-denuded arteries, and the relaxations were inhibited by barium but not by charybdotoxin and apamin. With the use of whole cell patch-clamp analysis, ACh (10(-7) M) stimulated voltage-dependent outward K+ current from endothelial cells, which was inhibited by charybdotoxin, indicating K+ efflux. Arachidonic acid (10(-7) to 10(-4) M) induced concentration-related relaxations that were inhibited by apamin but not by charybdotoxin and barium. Addition of arachidonic acid after K+ (10.9 mM) resulted in more potent relaxations to arachidonic acid compared with control without K+ (5.9 mM). These findings suggest that, in rabbit mesenteric arteries, ACh-induced, L-NA- and indomethacin-resistant relaxation is mediated by endothelial cell K+ efflux and arachidonic acid metabolites, and a synergism exists between these two separate mechanisms.     

Role of arachidonic acid lipoxygenase metabolites in acetylcholine-induced relaxations of mouse arteries

Arachidonic acid (AA) metabolites function as EDHFs in arteries of many species. They mediate cyclooxygenase (COX)- and nitric oxide (NO)-independent relaxations to acetylcholine (ACh). However, the role of AA metabolites as relaxing factors in mouse arteries remains incompletely defined. ACh caused concentration-dependent relaxations of the mouse thoracic and abdominal aorta and carotid, femoral, and mesentery arteries (maximal relaxation: 57 ± 4%, 72 ± 4%, 82 ± 3%, 80 ± 3%, and 85 ± 3%, respectively). The NO synthase inhibitor nitro-L-arginine (L-NA; 30 μM) blocked relaxations in the thoracic aorta, and L-NA plus the COX inhibitor indomethacin (10 μM) inhibited relaxations in the abdominal aorta and carotid, femoral, and mesenteric arteries (maximal relaxation: 31 ± 10%, 33 ± 5%, 41 ± 8%, and 73 ± 3%, respectively). In mesenteric arteries, NO- and COX-independent relaxations to ACh were inhibited by the lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA; 10 μM) and BW-755C (200 μM), the K(+) channel inhibitor apamin (1 μM), and 60 mM KCl and eliminated by endothelium removal. They were not altered by the cytochrome P-450 inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (20 μM) or the epoxyeicosatrienoic acid antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 μM). AA relaxations were attenuated by NDGA or apamin and eliminated by 60 mM KCl. Reverse-phase HPLC analysis revealed arterial [(14)C]AA metabolites that comigrated with prostaglandins, trihydroxyeicosatrienoic acids (THETAs), hydroxyepoxyeicosatrienoic acids (HEETAs), and hydroxyeicosatetraenoic acids (HETEs). Epoxyeicosatrienoic acids were not observed. Mass spectrometry confirmed the identity of 6-keto-PGF(1α), PGE(2), 12-HETE, 15-HETE, HEETAs, 11,12,15-THETA, and 11,14,15-THETA. AA metabolism was blocked by NDGA and endothelium removal. 11(R),12(S),15(S)-THETA relaxations (maximal relaxation: 73 ± 3%) were endothelium independent and blocked by 60 mM KCl. Western immunoblot analysis and RT-PCR of the aorta and mesenteric arteries demonstrated protein and mRNA expression of leukocyte-type 12/15-LO. Thus, in mouse resistance arteries, 12/15-LO AA metabolites mediate endothelium-dependent relaxations to ACh and AA.     

Nitric oxide-mediated nonadrenergic noncholinergic relaxation of piglet pulmonary arteries decreases with postnatal age.

Nonadrenergic noncholinergic (NANC) vasodilator mechanisms may contribute to the maintenance of adult pulmonary and systemic vascular tone. However, their actions in the neonatal circulation have not been studied. We aimed to investigate NANC vasorelaxation in neonatal and 2-week-old piglet pulmonary and mesenteric arteries and to examine the potential role of nitric oxide (NO) in this phenomenon. Responses to electric field stimulation (EFS, 50V, 0.25-32 Hz) were investigated in pulmonary and mesenteric artery rings (external diameter 150-200 microm) precontracted with the thromboxane A2 mimetic U46619, in the presence of guanethidine (10 microM) and atropine (10 microM). Under these conditions, EFS resulted in a frequency dependent relaxation of newborn pulmonary (maximal relaxation of 53+/-9.1%), mesenteric (68.8.2+/-7.1%) and 2-wk-old mesenteric (46 6.3%) arteries but this relaxation was significantly reduced (4.5+/-2.2%) in 2-week-old pulmonary arteries. In neonatal pulmonary arteries, the neurotoxin tetrodotoxin (0.3 muM), the NO synthase inhibitor L-NAME (0.1 mM), and the guanylyl cyclase inhibitor ODQ (10 microM) abolished EFS-induced relaxations, suggesting that NANC relaxation of porcine neonatal pulmonary arteries is mediated by NO, which is probably neuronal in origin. However, The expression in pulmonary arteries of the neuronal NO synthase (nNOS), as determined by Western-blot analysis, increased with postnatal age whereas the expression of the endothelial NOS (eNOS) did not change. In conclusion, NANC relaxation is present in neonatal pulmonary and mesenteric arteries and it is, at least partially, mediated through NO. NANC relaxation of porcine pulmonary and mesenteric arteries decreases with postnatal maturation.     

Effect of small changes in extracellular magnesium concentration on the tone of feline mesenteric arteries: involvement of endothelium.

The aim of this study was to investigate the effect of small alterations in extracellular magnesium concentration on the tone of feline mesenteric arteries and to examine the role of endothelium in these responses. We measured isometrical tension of isolated arterial rings, placed between two stainless steel wires in a tissue chamber containing Krebs-Henseleit solution, aerated with a gas mixture containing 95% O2 and 5% CO2 at 37 degrees C. After precontraction with noradrenaline, a decrease of extracellular magnesium concentration from 1.2 mM to 1.0 and 0.8 mM resulted in sustained relaxations, whereas the elevation of extracellular magnesium from 0.8 mM to 1.2 mM caused an increase in vascular tone when endothelium was intact. The magnesium-withdrawal related dilations were absent in endothelium-denuded vessels and were inhibited by oxyhemoglobin (5 x 10(-6) M) and methylene blue (10(-5) M), suggesting the involvement of endothelium-derived relaxing factor in this vascular response. Nifedipine (5 x 10(-7) M) or dichlorobenzamil (3 x 10(-5) M), however, did not affect the magnesium-deficiency related relaxations. Therefore, in this vascular action of magnesium, nifedipine-sensitive calcium channels or the sodium- calcium antiport system are not involved. We conclude that small alterations in extracellular magnesium concentration, possibly within the physiological range, are able to modify the basal formation and release of EDRF, and thus alter arterial smooth muscle tone in this vascular bed. This endothelium- and magnesium-dependent system appears to be more sensitive than the direct smooth muscle actions of magnesium. The possible physiological and pathophysiological consequences of these observations are discussed.     

Disruption of endothelial caveolae is associated with impairment of both NO- as well as EDHF in acetylcholine-induced relaxation depending on their relative contribution in different vascular beds

Caveolae represent an important structural element involved in endothelial signal-transduction. The present study was designed to investigate the role of caveolae in endothelium-dependent relaxation of different vascular beds. Caveolae were disrupted by cholesterol depletion with filipin (4x10(-6) g L(-1)) or methyl-beta-cyclodextrin (MCD; 1x10(-3) mol L(-1)) and the effect on endothelium-dependent relaxation was studied in rat aorta, small renal arteries and mesenteric arteries in the absence and presence of L-NMMA. The contribution of NO and EDHF, respectively, to total relaxation in response to acetylcholine (ACh) gradually changed from aorta (71.2+/-6.1% and 28.8+/-6.1%), to renal arteries (48.6+/-6.4% and 51.4+/-6.4%) and to mesenteric arteries (9.1+/-4.0% and 90.9+/-4.1%). Electron microscopy confirmed filipin to decrease the number of endothelial caveolae in all vessels studied. Incubation with filipin inhibited endothelium-dependent relaxation induced by cumulative doses of ACh (3x10(-9)-10(-4) mol L(-1)) in all three vascular beds. In aorta, treatment with either filipin or MCD only inhibited the NO component, whereas in renal artery both NO and EDHF formation were affected. In contrast, in mesenteric arteries, filipin treatment only reduced EDHF formation. Disruption of endothelial caveolae is associated with the impairment of both NO and EDHF in acetylcholine-induced relaxation.     

Epoxyeicosatrienoic acid-induced relaxation is impaired in insulin resistance

We assessed the effect of epoxyeicosatrienoic acids (EETs) in intact mesenteric arteries and Ca(2+)-activated K(+) (BK(Ca)) channels of isolated vascular smooth muscle cells from control and insulin-resistant (IR) rats. The response to 11,12-EET and 14,15-EET was assessed in small mesenteric arteries from control and IR rats in vitro. Mechanistic studies were performed in endothelium intact or denuded arteries and in the presence of pharmacological inhibitors. Moreover, EET-induced activation of the BK(Ca) channel was assessed in myocytes in both the cell-attached and the inside-out (I/O) patch-clamp configurations. In control arteries, both EET isomers induced relaxation. Relaxation was impaired by endothelium denudation, N(omega)-nitro-L-arginine, or iberiotoxin (IBTX), whereas it was abolished by IBTX + apamin or charybdotoxin + apamin. In contrast, the EETs did not relax IR arteries. In control myocytes, the EETs increased BK(Ca) activity in both configurations. Conversely, in the cell-attached mode, EETs had no effect on BK(Ca) channel activity in IR myocytes, whereas in the I/O configuration, BK(Ca) channel activity was enhanced. EETs induce relaxation in small mesenteric arteries from control rats through K(Ca) channels. In contrast, arteries from IR rats do not relax to the EETs. Patch-clamp studies suggest impaired relaxation is due to altered regulatory mechanisms of the BK(Ca) channel.     

β-ADRENOCEPTOR-MEDIATED RELAXATION INHIBITED BY TETRAPENTYLAMMONIUM IONS IN RAT MESENTERIC ARTERY

The aim of this study was to examine the contribution of K⁺ channel activation to β-adrenoceptor-mediated relaxation in rat mesenteric arteries. Isoprenaline and fenoterol concentration-dependently relaxed the phenylephrine-preconstricted endothelium-intact arteries of the rat with EC₅₀ values of 0.26 ± 0.03 μM and 0.87 ± 0.12 μM, respectively. β-Adrenoceptor-mediated relaxation was significantly attenuated upon removal of endothelium. Tetrapentylammonium ions (TPA⁺) at low concentrations (1–5 μM) inhibited relaxations induced by β-adrenoceptor agonists in arteries with and without endothelium, while glibenclamide (3 μM) had no effect. TPA⁺ (5 μM) inhibited isoprenaline-induced relaxation in the presence of either iberiotoxin (100 nM) or glibenclamide (3 μM). TPA⁺ did not alter forskolin-induced relaxation. In the presence of 60 mM extracellular K⁺, the relaxations induced by two agonists were reduced in endothelium-intact arteries and abolished in endothelium-denuded arteries. The present results suggest that the activation of TPA⁺-sensitive K⁺ channels contributes toward the relaxations mediated through β- and β₂-adrenoceptor stimulation in rat mesenteric arteries. © 1998 Elsevier Science Inc.     

Parathyroid hormone-related protein-mediated responses in pulmonary arteries and veins of newborn lambs

PTHrP has important roles in lung development and function. Here we determined the vasomotor responses of isolated pulmonary arteries and veins of newborn and adult sheep to PTHrP. In vessels constricted with endothelin-1, PTHrP (PTHrP 1-34) caused greater relaxation of veins than of arteries. In both vessel types, relaxation to the peptide was less in adult than in newborn vessels. In newborn lambs, PTHrP-induced relaxation was not affected by endothelium removal, inhibition of eNOS, or inhibition of adenylyl cyclases by SQ-22536. However, relaxation was attenuated by 4-aminopyridine, inhibitor of voltage-dependent potassium channels, in both arteries and veins, and by charybdotoxin, inhibitor of calcium-activated potassium channels, in veins. When vessels were saturated with 8-BrcAMP (3 x 10(-4) M), to eliminate relaxation mediated by endogenous cAMP, PTHrP-induced relaxation was partially attenuated. In vessels treated with 8-BrcAMP (3 x 10(-4) M), 4-aminopyridine but not charybdotoxin inhibited relaxation induced by PTHrP 1-34 in both arteries and veins. Radioimmunoassay showed that, in the presence of a general phosphodiesterase inhibitor, PTHrP caused a concentration-dependent increase in intracellular cAMP content in arteries and veins, which was largely abolished by SQ-22536. Our results demonstrate that PTHrP is a potent vasodilator of pulmonary vessels, with a greater effect in veins than in arteries. Relaxation induced by the peptide contains both cAMP-dependent and -independent components. In both arteries and veins, voltage-dependent potassium channels mediate the response to PTHrP, at least in part, in a cAMP-independent fashion; and in veins, calcium-activated potassium channels may be stimulated by elevated cAMP levels.     

Effects of prostaglandins and arachidonic acid on baboon cerebral and mesenteric arteries

Effects of prostaglandins (PGs) E1, E2, F2 alpha and I2 in a wide range of concentration were examined in mesenteric and cerebral arteries isolated from mature baboons. PGs E1, E2 and F2 alpha at low concentrations (10(-10) to 10(-7) M) elicited relaxation in helically cut strips of cerebral arteries precontracted with phenylephrine. In contrast, the PGs did not cause relaxation in the mesenteric artery. PGI2 (10(-9) to 10(-6) M) produced marked relaxation in both arteries. The EC25 for PGI2 in the mesenteric artery was significantly lower than that in the cerebral artery. During baseline conditions, cerebral arteries contracted in response to high concentrations (greater than 10(-7) M) of PGs E1, E2 and F2 alpha. In mesenteric arteries, a large contraction was induced by PGs F2 alpha and E2 but not by PGE1. Arachidonic acid (10(-6) M) produced an aspirin-inhibitable relaxation in both arteries to a similar extent, so that the vasodilator PG(s) formed in the two different arterial walls appear to exert a similar relaxant action. Thus, the baboon mesenteric artery was more sensitive to PGI2 for the relaxant effect than was the cerebral artery, while PGs F2 alpha, E1 and E2 caused only a contraction in the mesenteric artery but both relaxation and contraction in the cerebral artery.     

Endothelium modulates anion channel-dependent aortic contractions to iodide

Anion currents contribute to vascular smooth muscle (VSM) membrane potential. The substitution of extracellular chloride (Cl) with iodide (I) or bromide (Br) initially inhibited and then potentiated isometric contractile responses of rat aortic rings to norepinephrine. Anion substitution alone produced a small relaxation, which occurred despite a lack of active tone and minimal subsequent contraction of endothelium-intact rings (4.2 +/- 1.2% of the response to 90 mM KCl). Endothelium-denuded rings underwent a similar initial relaxation but then contracted vigorously (I > Br). Responses to 130 mM I (93.7 +/- 1.9% of 90 mM KCl) were inhibited by nifedipine (10(-6) M), niflumic acid (10(-5) M), tamoxifen (10(-5) M), DIDS (10(-4) M), and HCO(-)(3)-free buffer (HEPES 10 mM) but not by bumetanide (10(-5) M). Intact rings treated with N(omega)-nitro-L-arginine (10(-4) M) responded weakly to I (15.5 +/- 2.1% of 90 mM KCl), whereas hemoglobin (10(-5) M), indomethacin (10(-6) M), 17-octadecynoic acid (10(-5) M), and 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one (10(-6) M) all failed to augment the response of intact rings to I. We hypothesize that VSM takes up I primarily via an anion exchanger. Subsequent I efflux through anion channels having a selectivity of I > Br > Cl produces depolarization. In endothelium-denuded or agonist-stimulated vessels, this current is sufficient to activate voltage-dependent calcium channels and cause contraction. Neither nitric oxide nor prostaglandins are the primary endothelial modulator of these anion channels. If they are regulated by an endothelium-dependent hyperpolarizing factor it is not a cytochrome P-450 metabolite.     

Calcium Uptake and Associated Adenosine Triphosphatase Activity of Isolated Platelet Membranes

A platelet subcellular fraction, sedimenting between 14,000 and 40,000 g and consisting primarily of membrane vesicles, accumulates up to 200–400 nmoles calcium/mg protein in the presence of ATP and oxalate. Steady-state levels of calcium accumulation are attained in 40–60 min. Calcium uptake requires adenosine triphosphate (ATP), is enhanced by oxalate, and is accompanied by the release of inorganic phosphate. Calcium accumulation and phosphate release require magnesium and are inhibited by Salyrgan (10 µM) and adenosine diphosphate (ADP) (1 mM), but not by ouabain (0.1 mM). The ATPase activity is stimulated by low concentrations of calcium (5–10 µM) and is inhibited by 2 mM EGTA. Electron microscopic histochemistry using lead nitrate to precipitate released phosphate results in lead precipitates localized primarily at the inner surface of membrane vesicles. These results provide evidence for a membrane ATPase that is stimulated by low concentrations of calcium and may be involved in the transport of calcium across the membrane. It is postulated that the observed calcium uptake activity is an in vitro manifestation of a calcium extrusion pump in the intact platelet.     

Cyclooxygenase- and lipoxygenase-dependent relaxation to arachidonic acid in rabbit small mesenteric arteries

We recently reported that the lipoxygenase product 11,12,15-trihydroxyeicosatrienoic acid (THETA) mediates arachidonic acid (AA)-induced relaxation in the rabbit aorta. This study was designed to determine whether this lipoxygenase metabolite is involved in relaxation responses to AA in rabbit small mesenteric arteries. AA (10(-9)-10(-4) M) produced potent relaxations in isolated phenylephrine-preconstricted arteries, with a maximal relaxation of 99 +/- 0.5% and EC(50) of 50 nM. The cyclooxygenase (COX) inhibitors indomethacin (10 microM), NS-398 (10 microM, selective for COX-2), and SC-560 (100 nM, selective for COX-1) caused a marked rightward shift of concentration responses to AA. With the use of immunohistochemical analysis, both COX-1 and COX-2 were detected in endothelium and smooth muscle of small mesenteric arteries. Indomethacin-resistant relaxations were further reduced by the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC; 1 muM), nordihydroguaiaretic acid (NDGA; 1 microM), and ebselen (1 microM). HPLC analysis showed that [(14)C]AA was metabolized by mesenteric arteries to PGI(2), PGE(2), THETAs, hydroxyepoxyeicosatrienoic acids (HEETAs), and 15-hydroxyeicosatetraenoic acid (15-HETE). The production of PGI(2) and PGE(2) was blocked by indomethacin, and the production of THETAs, HEETAs, and 15-HETE was inhibited by CDC and NDGA. Column fractions corresponding to THETAs were further purified, analyzed by gas chromatography/mass spectrometry, and identified as 11,12,15- and 11,14,15-THETA. PGI(2), PGE(2), and purified THETA fractions relaxed mesenteric arteries precontracted with phenylephrine. The AA- and THETA-induced relaxations were blocked by high K(+) (60 mM). These findings provide functional and biochemical evidence that AA-induced relaxation in rabbit small mesenteric arteries is mediated through both COX and lipoxygenase pathways.