nitroarginine and iberiotoxin

To observe the effect of β₃adrenoceptors (β₃-AR) activation on rat thoracic aorta smooth muscle contractility and the possible related mechanism.. The endothelium removed thoracic aorta was pre-contracted with 30 mmol/L KCl physiological saline solution (PSS). Then the tension of the thoracic aorta was recorded in presence of BRL37344 (BRL) to determine the action of β₃-AR. The tension of the thoracic aorta was also recorded in the presence of Propranolol (PRA), SR59230A (SR), L-NNA, H-89 and Iberiotoxin (IBTX) respectively to reveal the underling mechanism of β₃-AR activation on rat vascular smooth muscle. Immunohistochemistry was adopted to confirm the existence and the distribution of β₃-AR in rat thoracic aorta.. The results showed that: (1) The thoracic aorta was relaxed by β₃-AR activation, with a relaxation percentage of (10.59 ± 0.79). (2) β₃-AR was expressed in both endothelial and smooth muscle layer in thoracic aorta sections of rats. (3) PRA did not block the effect of BRL on the thoracic aorta. The relaxation actions of BRL could be antagonized by pre-incubating the thoracic aorta with SR. (4) L-NNA (a NOS inhibitor) and H-89 (a PKA inhibitor) reversed the relaxation effect of BRL on vascular smooth muscle. (5) The effect of BRL was decreased after application of Ibriotoxin (IBTX), a large conductance calcium dependent potassium channel blocker.. The results confirmed that activation of β₃-AR led to relaxation of thoracic aorta smooth muscle. The relaxation action of β₃-AR on smooth muscle of rat thoracic aorta was related to activation of NOS and PKA signaling pathway. Large conductance Ca²⁺-K⁺ channels were involved in the relaxation action of β₃-AR activation on rat thoracic aorta smooth muscle.

Topics: Animals; Aorta, Thoracic; In Vitro Techniques; Isoquinolines; Large-Conductance Calcium-Activated Potassium Channels; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroarginine; Peptides; Propanolamines; Propranolol; Rats; Receptors, Adrenergic, beta-3; Signal Transduction; Sulfonamides

To determine whether palmitic acid methyl ester (PAME) or methyl palmitate is the retina-derived relaxing factor (RRF).. A superfusion bioassay cascade technique was used with rat isolated retina as donor tissue and rat aortic ring as detector tissue. The superfusate was analyzed with gas chromatography/mass spectrometry (GC/MS). The biochemical and pharmacologic characteristics of RRF and PAME were compared.. The authors demonstrated that the retina on superfusion with Krebs solution spontaneously released RRF (indicated by aortic ring relaxation) and PAME (measured by GC/MS). The release of RRF and PAME was calcium dependent because the release was abolished when the retinas were superfused with calcium-free Krebs solution. Furthermore, aortic relaxations induced by RRF and PAME were not affected after heating their solutions at 70 degrees C for 1 hour, suggesting that both are heat stable. Exogenous PAME concentration dependently induced aortic relaxation with EC50 of 0.82+/-0.75 pM. The aortic relaxations induced by RRF and exogenous PAME were inhibited by 4-aminopyridine (2 mM) and tetraethylammonium (TEA, 10 mM) but were not affected by TEA at 1 mM or 3 mM, glibenclamide (3 microM), or iberiotoxin (100 nM). The vasodilator activity of Krebs solution containing RRF or exogenous PAME was greatly attenuated after hexane extraction.. RRF and PAME share similar biochemical properties and react similarly to all pharmacologic inhibitors examined. Both act primarily on the voltage-dependent K+ (Kv) channel of aortic smooth muscle cells, causing aortic relaxation. These results suggest that PAME is the hydrophobic RRF.

Topics: 4-Aminopyridine; Animals; Aorta; Biological Assay; Calcium; Enzyme Inhibitors; Hexanes; Isotonic Solutions; Male; Miconazole; Nitroarginine; Palmitates; Peptides; Potassium Channel Blockers; Proadifen; Rats; Rats, Sprague-Dawley; Retina; Retinal Vessels; Solvents; Tetraethylammonium; Vasodilation

In addition to nitric oxide (NO), it is thought that an endothelium-derived hyperpolarizing factor (EDHF) plays an important role in the relaxation of penile arteries. Recently, it has been shown that C-type natriuretic peptide (CNP) shows the characteristics of EDHF in systemic small arteries.. To investigate the mechanism involved in CNP-evoked vasodilatation and to address whether CNP is an EDHF in human penile resistance arteries.. Erectile tissue was obtained in connection with transsexual operations. Intracavernous penile resistance arteries were isolated and mounted in microvascular myographs for recording of isometric tension. Membrane potential was recorded by the use of a small glass electrode inserted in the smooth muscle layer.. In vitro evidence for hyperpolarization and vasorelaxation induced by CNP.. Acetylcholine (ACh) and CNP hyperpolarized smooth muscle membrane potential in resting penile resistance arteries. In penile small arteries incubated with inhibitors of NO synthase and cyclooxygenase and contracted with phenylephrine, ACh and CNP evoked concentration-dependent relaxations with maximum of 56 +/- 6% and 71 +/- 6%, respectively. Addition of a combination of blockers of small- and intermediate-conductance calcium-activated K(+) channels, apamin plus charybdotoxin, respectively, and a combination thought to block the smooth muscle response of EDHF-type relaxation, barium plus ouabain, markedly reduced ACh- and CNP-evoked relaxation. Iberiotoxin, a blocker of big-conductance calcium-activated K(+) channels inhibited the vasorelaxant responses evoked by ACh and CNP. A selective natriuretic peptide receptor type C (NPR-C) agonist, C-atrial natriuretic factor(4-23) (cANF(4-23)), induced relaxations with less maximum response compared to CNP.. The present findings suggest that CNP possesses the characteristics of an EDHF in human penile resistance arteries. By activation of natriuretic peptide receptor type B and NPR-C receptors, CNP causes relaxation by activation, respectively, of large-conductance calcium-activated K(+) channels and Na(+)/K(+)-adenosine triphosphatase (ATPase), and barium-sensitive inward rectifier K(+) channels. Modulation of the CNP pathway opens for new treatment modalities of erectile dysfunction.

Topics: Acetylcholine; Adolescent; Adult; Apamin; Arteries; Barium Compounds; Charybdotoxin; Chlorides; Cyclooxygenase Inhibitors; Enzyme Inhibitors; Humans; Indomethacin; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Male; Membrane Potentials; Middle Aged; Muscle, Smooth, Vascular; Natriuretic Agents; Natriuretic Peptide, C-Type; Neurotoxins; Nitroarginine; Ouabain; Penis; Peptides; Vascular Resistance; Vasodilator Agents

Conducted vasodilatation (CVD) reflects the initiation and rapid (>mm s(-1)) spread of hyperpolarization along the endothelium and into smooth muscle. The ion channels that initiate CVD remain unclear as do signalling pathways that may complement electromechanical relaxation. Using isolated pressurized (75 mmHg; 37 degrees C) feed arteries (n=63; diameter: rest: 53 +/- 2 microm, maximal: 98 +/- 2 microm) from hamster retractor skeletal muscle, we investigated the contribution of calcium-activated potassium channels (KCa) and endothelium-derived autacoids to CVD. Local delivery (1 microm micropipette tip; 500-2000 ms pulse) of acetylcholine (ACh) at the downstream end initiated a local increase in endothelial cell [Ca2+]i (Fura-PE3; Deltaratio 340/380 nm = 0.215 +/- 0.032) that preceded CVD along the entire vessel. During local perifusion with KCa antagonists, iberiotoxin (5 microm) had no effect, but charybdotoxin (CTX, 5 microm) + apamin (APA, 10 microm) abolished CVD reversibly. Remarkably, this local inhibition of KCa unmasked a 'slow-conducted vasodilatation' (SCVD) that spread >1200 microm at approximately 21 microm s(-1) (n=27). Recorded 500 microm upstream from the ACh stimulus, a rise in endothelial cell [Ca2+]i (Deltaratio 340/380 nm) = 0.146 +/- 0.017; P<0.05) preceded SCVD (Deltadiameter = 14 +/- 3 microm) by approximately 10 s. Before KCa inhibition, antagonism of nitric oxide synthase (Nomega-nitro-L-arginine, 250 microm; l-NNA) and cyclooxygenase (indomethacin, 5 microm; INDO) had no effect on the amplitude of CVD yet response duration decreased by one-third (P<0.05). During local CTX + APA perifusion, L-NNA + INDO abolished SCVD while conducted [Ca2+]i responses remained intact. Thus, ACh triggers electromechanical relaxation of smooth muscle cells along the vessel initiated by local KCa, and the ensuing 'wave' of Ca2+ along the endothelium releases autacoids to promote pharmacomechanical relaxation.

Topics: Acetylcholine; Animals; Arteries; Calcium; Charybdotoxin; Cricetinae; Cyclooxygenase Inhibitors; Endothelium, Vascular; Enzyme Inhibitors; Indomethacin; Male; Mechanotransduction, Cellular; Mesocricetus; Microscopy, Video; Muscle, Skeletal; Muscle, Smooth, Vascular; Neurotoxins; Nitroarginine; Peptides; Potassium Channels, Calcium-Activated; Vasodilation; Vasodilator Agents

In this study, we examined the effects of Salvia miltiorrhiza (Danshen) crude extract, some of its lipid-soluble components (tanshinone I, tanshinone II(A), cryptotanshinone, dihydroisotanshinone I) and the water-soluble compounds (danshensu and salvianolic acid B) on the K(+) channels such as the iberiotoxin-sensitive Ca(2+)-activated K(+) (BK(Ca)) channels and the glibenclamide-sensitive ATP-dependent K(+) (IK(ATP)) channels of the porcine left anterior descending coronary artery smooth muscle cells. Cumulative application of salvianolic acid B (30-300 microM) caused a l-NNA (100 microM)-insensitive, potentiation of the outward BK(Ca) current amplitude with no apparent effect on the IK(ATP) channels opening. Salvianolic acid B (300 microM) caused an ODQ (10 microM, a guanylate cyclase inhibitor)-sensitive enhancement of the outward BK(Ca) current amplitude. In contrast, none of the other isolated chemical constituents of S. miltiorrhiza modified the openings of the two types of K(+) channels studied. In conclusion, our results suggest that salvianolic acid B, a major hydrophilic constituent found in Radix S. miltiorrhiza, activated the opening of the BK(Ca) channels of the porcine coronary artery smooth muscle cells through the activation of guanylate cyclase without the involvement of the nitric oxide synthase activation.

Topics: Animals; Benzofurans; Coronary Vessels; Cromakalim; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Enzyme Activation; Enzyme Inhibitors; Glyburide; Guanylate Cyclase; In Vitro Techniques; Ion Channel Gating; Membrane Potentials; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitroarginine; Oxadiazoles; Patch-Clamp Techniques; Peptides; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Calcium-Activated; Quinoxalines; Receptors, Drug; Salvia miltiorrhiza; Swine

During cerebral ischemia, both hypoxia and hypercapnia appear to produce marked dilatation of the cerebral arteries. Hypercapnia and hypoxia may be accompanied by extracellular and intracellular acidosis, which is another potent dilator of cerebral arteries. However, the precise mechanism by which acidosis produces dilatation of the cerebral arteries is not fully understood. The objective of the present study was to examine the mechanisms by which intracellular acidosis produces dilatation of the basilar artery in vivo.. Using a cranial window in anesthetized rats, we examined responses of the basilar artery to sodium propionate, which was used to cause intracellular acidosis specifically. Expression of subunits of potassium channels was determined by reverse transcription and polymerase chain reaction (RT-PCR).. Topical application of propionate increased diameter of the basilar artery in a concentration-related manner. Propionate-induced dilatation of the artery was attenuated by glibenclamide, an inhibitor of ATP-sensitive potassium channels. However, inhibitors of nitric oxide synthase (N(G)-nitro-L-arginine), large-conductance calcium-activated potassium channels (iberiotoxin), and cyclooxygenase (indomethacin) did not affect the vasodilatation. Expression of mRNA for SUR2B and Kir6.1 was detected, with the use of RT-PCR, in the cultured basilar arterial muscle cells.. The findings suggest that intracellular acidification may produce dilatation of the basilar artery through activation of ATP-sensitive potassium channels in vivo. Kir6.1/SUR2B may be the major potassium channels that mediate propionate-induced dilatation of the artery.

Topics: Amiloride; Animals; ATP-Binding Cassette Transporters; Basilar Artery; Cells, Cultured; Cyclooxygenase Inhibitors; Enzyme Inhibitors; Glyburide; Hydrogen-Ion Concentration; Indomethacin; Intracellular Fluid; Ion Transport; Macromolecular Substances; Male; Muscle, Smooth, Vascular; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Organ Specificity; Pancreas; Peptides; Potassium; Potassium Channels; Potassium Channels, Calcium-Activated; Potassium Channels, Inwardly Rectifying; Propionates; Rats; Rats, Sprague-Dawley; Receptors, Drug; Sodium-Hydrogen Exchangers; Sulfonylurea Receptors; Vasodilation

Vasomotor reactions upon focal stimulation of arterioles have been shown to be conducted along the vascular wall. Such a conduction, which is assumed to reflect the spread of electrical signals, may contribute to coordination of responses within a vascular segment. We aimed to identify which endothelial autacoid(s) act as mediators of the local and conducted dilator responses, respectively. To this end, arterioles in the hamster cremaster microcirculation were locally stimulated with endothelium-dependent [acetylcholine (ACh)] or endothelium-independent dilators [sodium nitroprusside (SNP)], and the resulting changes in diameter were measured using a videomicroscopy technique at the site of application and up to 1.4 mm upstream at distant sites. Experiments were also performed after blockade of nitric oxide (NO) synthase, cyclooxygenase, P-450 monooxygenase, or K(+) channels. Dilations upon ACh (71 +/- 3%) were conducted rapidly (<1 s) to upstream sites (at 1.4 mm: 37 +/- 5%). Although the NO donor SNP induced a similar local dilation (71 +/- 7%), this response was not conducted. Maximal amplitudes of ACh-induced dilations were not attenuated after inhibition of NO synthase and cyclooxygenase at the local and remote sites. However, additional treatment with a P-450 monooxygenase blocker (sulfaphenazole) strongly attenuated the local response (from 62 +/- 9 to 17 +/- 5%) and abrogated dilations at distant sites (at 0.67 mm: from 23 +/- 4% to 4 +/- 3%). Likewise, 17-octadecynoic acid strongly attenuated local and remote responses. Blockers of Ca(2+)-dependent K(+) channels (charybdotoxin or iberiotoxin) attenuated dilations at the local and remote sites after focal application at the ACh stimulation site. In marked contrast, treatment of the upstream site with these blockers was without any effect. We conclude that upon local stimulation with ACh, a cytochrome P-450 monooxygenase product is generated that induces local dilation via the activation of Ca(2+)-dependent K(+) channels and initiates conduction of the dilation. In contrast to the local site, neither activation of these K(+) channels nor the synthesis of NO or prostaglandins is necessary to dilate the arterioles at remote, distant sites. This suggests that endothelium-derived hyperpolarizing factor serves as an important mediator to initiate conducted dilations and, by doing so, may act as a key player in the coordination of arteriolar behavior in the microcirculatory network.

Topics: Acetylcholine; Animals; Arterioles; Autacoids; Biological Factors; Charybdotoxin; Cricetinae; Endothelium, Vascular; Enzyme Inhibitors; Male; Mesocricetus; Muscle, Skeletal; Nitric Oxide; Nitroarginine; Nitroprusside; Peptides; Prostaglandins; Sulfaphenazole; Vasodilation; Vasodilator Agents

1. The direct and endothelium-dependent effects of lipopolysaccharide (LPS) were investigated on resistance and conductance arteries from normotensive Wistar (NWR) and spontaneously hypertensive (SHR) rats. 2. In both NWR and SHR, LPS induced dose-dependent relaxations of the mesenteric vascular bed, which were inhibited by L-NNA in SHR but not in NWR. Iberiotoxin (IBTX) inhibited the responses to LPS in both groups, indicating the participation of high conductance Ca(2+)-dependent K(+) channels. 3. In mesenteric artery rings, the resting membrane potentials and the hyperpolarizing responses of NWR to LPS did not differ in endothelized and denuded preparations but L-NNA inhibited the responses only in endothelized rings. These responses were reduced by bosentan, suggesting that endothelin release may mask a possible hyperpolarizing response to LPS. The hyperpolarizing responses to LPS were blocked by IBTX in both endothelized and de-endothelized NWR rings. In the SHR only intact rings showed hyperpolarization to LPS, which was inhibited by IBTX and byL-NNA. 4. In SHR aortic endothelized or denuded rings, LPS induced hyperpolarizing responses which, in endothelized rings, were partially blocked by L-NNA, by IBTX or by glibenclamide, but totally abolished by IBTX plus glibenclamide. No response to LPS was observed in NWR aortic rings. 5. Our results indicate that LPS activates large conductance Ca(2+)-sensitive K(+) channels located in the smooth muscle cell membrane both directly and indirectly, through NO release from the endothelium in NWR, whereas NO is the major mediator of the LPS responses in SHR resistance vessels.

Topics: Animals; Aorta, Thoracic; Arteries; Dose-Response Relationship, Drug; Hypertension; In Vitro Techniques; Lipopolysaccharides; Male; Membrane Potentials; Mesenteric Arteries; Nitric Oxide; Nitroarginine; Peptides; Potassium Channels; Rats; Rats, Inbred SHR; Rats, Wistar; Vasodilation

Nitric oxide (NO) synthase (NOS) inhibition with N(omega)-nitro-L-arginine (L-NNA) produces L-NNA hypertensive rats (LHR), which exhibit increased sensitivity to voltage-dependent Ca(2+) channel-mediated vasoconstriction. We hypothesized that enhanced contractile responsiveness after NOS inhibition is mediated by depolarization of membrane potential (E(m)) through attenuated K(+) channel conductance. E(m) measurements demonstrated that LHR vascular smooth muscle cells (VSMCs) are depolarized in open, nonpressurized (-44.5 +/- 1.0 mV in control vs. -36.8 +/- 0.8 mV in LHR) and pressurized mesenteric artery segments (-41.8 +/- 1.0 mV in control vs. -32.6 +/- 1.4 mV in LHR). Endothelium removal or exogenous L-NNA depolarized control VSMCs but not LHR VSMCs. Superfused L-arginine hyperpolarized VSMCs from both the control and LHR groups and reversed L-NNA-induced depolarization (-44.5 +/- 1.0 vs. -45.8 +/- 2.1 mV). A Ca(2+)-activated K(+) channel agonist, NS-1619 (10 microM), hyperpolarized both groups of arteries to a similar extent (from -50.8 +/- 1.0 to -62.5 +/- 1.2 mV in control and from -43.7 +/- 1.1 to -55.6 +/- 1.2 mV in LHR), although E(m) was still different in the presence of NS-1619. In addition, superfused iberiotoxin (50 nM) depolarized both groups similarly. Increasing the extracellular K(+) concentration from 1.2 to 45 mM depolarized E(m), as predicted by the Goldman-Hodgkin-Katz equation. These data support the hypothesis that loss of NO activation of K(+) channels contributes to VSMC depolarization in L-NNA-induced hypertension without a change in the number of functional large conductance Ca(2+)-activated K(+) channels.

Topics: Animals; Arginine; Benzimidazoles; Calcium; Cell Membrane; Endothelium, Vascular; Enzyme Inhibitors; Hypertension; Male; Membrane Potentials; Mesenteric Arteries; Muscle, Smooth, Vascular; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroarginine; Penicillamine; Peptides; Potassium; Potassium Channel Blockers; Potassium Channels; Rats; Rats, Sprague-Dawley

Angiotensin II (Ang II) is one of the most potent vasoconstrictor substances, yet paradoxically, Ang II may dilate certain vascular beds via an undefined mechanism. Ang II-induced vasoconstriction is mediated by the AT(1) receptor, whereas the relative expression and functional importance of the AT(2) receptor in regulating vascular resistance and blood pressure are unknown. We now report that Ang II induces relaxation of mesenteric microvessels and that this vasodilatory response was unaffected by losartan, an AT(1) receptor antagonist, but was inhibited by PD123,319, a selective antagonist of AT(2) receptors. In addition, reverse transcriptase-polymerase chain reaction studies revealed high amounts of AT(2) receptor mRNA in smooth muscle from these same microvessels. Ang II-induced relaxation was inhibited by either tetraethylammonium or iberiotoxin, suggesting involvement of the large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel. Subsequent whole-cell and single-channel patch-clamp studies on single myocytes demonstrated that Ang II increases the activity of BK(Ca) channels. As in our tissue studies, the effect of Ang II on BK(Ca) channels was inhibited by PD123,319, but not by losartan. In light of these consistent findings from tissue physiology, molecular studies, and cellular/molecular physiology, we conclude that Ang II relaxes microvessels via stimulation of the AT(2) receptor with subsequent opening of BK(Ca) channels, leading to membrane repolarization and vasodilation. These findings provide evidence for a novel endothelium-independent vasodilatory effect of Ang II.

Topics: Angiotensin II; Animals; Calcium; Cells, Cultured; Dose-Response Relationship, Drug; Endothelin-1; Imidazoles; In Vitro Techniques; Losartan; Male; Membrane Potentials; Mesenteric Arteries; Muscle, Smooth, Vascular; Nitroarginine; Patch-Clamp Techniques; Peptides; Potassium Channels; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Angiotensin; RNA, Messenger; Tetraethylammonium; Vasodilation

Carbon monoxide (CO) is an endogenous dilator in the newborn cerebral microcirculation. Other dilators include prostanoids and nitric oxide (NO), and interactions among the systems are likely. Experiments on anesthetized piglets with cranial windows address the hypothesis that CO-induced dilation of pial arterioles involves interaction with the prostanoid and NO systems. Topical application of CO or the heme oxygenase substrate heme-L-lysinate (HLL) produced dilation. Indomethacin, N(omega)-nitro-L-arginine (L-NNA), and either iberiotoxin or tetraethylammonium chloride (TEA) were used to inhibit prostanoids, NO, and Ca(2+)-activated K(+) (K(Ca)) channels, respectively. Indomethacin, L-NNA, iberiotoxin, or TEA blocked cerebral vasodilation to CO and HLL. Vasodilations to both CO and HLL were returned to indomethacin-treated piglets by topical application of iloprost. Vasodilations to both CO and HLL were returned to L-NNA-treated piglets by sodium nitroprusside but not iloprost. In iberiotoxin- or TEA-treated piglets, dilations to CO and HLL could not be restored by either iloprost or sodium nitroprusside. The dilator actions of CO involve prostacyclin and NO as permissive enablers. The permissive actions of prostacyclin and NO may alter the K(Ca) channel response to CO because neither iloprost nor sodium nitroprusside could restore dilation to CO when these channels were blocked.

Topics: Animals; Animals, Newborn; Arterioles; Carbon Monoxide; Heme; Indomethacin; Kinetics; Lysine; Microcirculation; Muscle, Smooth, Vascular; Nitric Oxide; Nitroarginine; Peptides; Pia Mater; Potassium Channels; Scorpion Venoms; Swine; Tetraethylammonium; Vasodilation; Vasodilator Agents

Dilatation of cerebral arterioles in response to arachidonic acid is dependent on activity of cyclooxygenase. In this study, we examined mechanisms that mediate dilatation of the basilar artery in response to arachidonate. Diameter of the basilar artery (baseline diameter = 216 +/- 7 micrometer) (means +/- SE) was measured using a cranial window in anesthetized rats. Arachidonic acid (10 and 100 microM) produced concentration-dependent vasodilatation that was not inhibited by indomethacin (10 mg/kg iv) or N(G)-nitro-L-arginine (100 microM) but was inhibited markedly by baicalein (10 micrometerM) or nordihydroguaiaretic acid (NDGA; 10 microM), inhibitors of the lipoxygenase pathway. Dilatation of the basilar artery was also inhibited markedly by tetraethylammonium ion (TEA; 1 mM) or iberiotoxin (50 nM), inhibitors of calcium-dependent potassium channels. For example, 10 microM arachidonate dilated the basilar artery by 19 +/- 7 and 1 +/- 1% in the absence and presence of iberiotoxin, respectively. Measurements of membrane potential indicated that arachidonate produced hyperpolarization of the basilar artery that was blocked completely by TEA. Incubation with [(3)H]arachidonic acid followed by reverse-phase and chiral HPLC indicated that the basilar artery produces relatively small quantities of prostanoids but large quantities of 12(S)-hydroxyeicosatetraenoic acid (12-S-HETE), a lipoxygenase product. Moreover, the production of 12-HETE was inhibited by baicalein or NDGA. These findings suggest that dilatation of the basilar artery in response to arachidonate is mediated by a product(s) of the lipoxygenase pathway, with activation of calcium-dependent potassium channels and hyperpolarization of vascular muscle.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Basilar Artery; Cyclooxygenase Inhibitors; Enzyme Inhibitors; Flavanones; Flavonoids; Indomethacin; Lipoxygenase; Male; Membrane Potentials; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitroarginine; Peptides; Potassium Channels; Rats; Rats, Sprague-Dawley; Tetraethylammonium; Tritium; Vasodilation

In porcine coronary arteries, smooth muscle hyperpolarizations produced by the nitric oxide donor, NOR-1, and the prostacyclin analogue, iloprost, were compared with those induced by substance P and bradykinin and attributed to the endothelium-derived hyperpolarizing factor (EDHF). In the presence of 300 microM L-nitroarginine and 10 microM indomethacin, iloprost-induced hyperpolarizations were partially inhibited by 10 microM glibenclamide whereas those to NOR-1, substance P and bradykinin were unaffected. Hyperpolarizations produced by maximally-effective concentrations of NOR-1 and NS1619 were identical (to -65 mV). They were significantly less than those generated by either substance P or bradykinin (to approximately -80 mV) and were abolished by iberiotoxin 100 nM, a concentration which had essentially no effect on responses to substance P or bradykinin. Incubation of segments of intact arteries for 16 - 22 h in bicarbonate-buffered Krebs solution had little effect on EDHF responses to substance P or bradykinin. In contrast, after incubation for this period of time in HEPES-buffered Tyrode solution or Krebs containing 10 mM HEPES the EDHF response to substance P was abolished and that to bradykinin was markedly reduced. The residual bradykinin-induced hyperpolarization following incubation in Tyrode solution was inhibited by iberiotoxin and by 10 microM 17-octadecynoic acid. We conclude that substance P activates only the EDHF pathway in the presence of nitric oxide synthase and cyclo-oxygenase inhibitors. Incubation in HEPES-buffered Tyrode solution abolishes the EDHF responses to substance P and bradykinin to reveal an additional hyperpolarizing mechanism, associated with the opening of K(+) channels, activated only by bradykinin.

Topics: Animals; Bicarbonates; Biological Factors; Bradykinin; Buffers; Coronary Vessels; Cromakalim; Endothelium, Vascular; Epoprostenol; Glyburide; HEPES; In Vitro Techniques; Indomethacin; Isotonic Solutions; Membrane Potentials; Muscle, Smooth, Vascular; Nitric Oxide Donors; Nitroarginine; Peptides; Sodium Chloride; Substance P; Swine; Time Factors; Vasodilator Agents

1. To elucidate the mechanism of the relaxation mediated by endothelium-derived hyperpolarizing factors (EDHFs), the effect of brefeldin A, a membrane transport blocker, on cytosolic Ca(2+) concentration ([Ca(2+)]i) and tension was determined in the porcine coronary arterial strips. We also examined the effect of brefeldin A on [Ca(2+)]i in the endothelial cells of the porcine aortic valve. 2. In the presence of 10 microM indomethacin and 30 microM N(G)-nitro-L-arginine (L-NOARG), both bradykinin and substance P induced a transient decrease in [Ca(2+)]i and tension in arterial strips contracted with 100 nM U46619 (thromboxane A2 analogue). A 6 h pre-treatment with 20 microg ml(-1) brefeldin A abolished the bradykinin-induced relaxation, while it had no effect on the substance P-induced relaxation. 3. In the absence of indomethacin and L-NOARG, brefeldin A had no effect on the bradykinin-induced relaxation during the contraction induced by U46619 or 118 mM K(+). 4. The indomethacin/L-NOARG-resistant relaxation induced by bradykinin was completely inhibited by 3 mM tetrabutylammonium (non-specific Ca(2+)-activated K(+) channel blocker), while that induced by substance P was not inhibited by 3 mM tetrabutylammonium or 1 mM 4-aminopyridine (voltage-dependent K(+) channels blocker) alone, but completely inhibited by their combination. 5. Brefeldin A had no effect on the [Ca(2+)]i elevation in endothelial cells induced by bradykinin or substance P. 6. In conclusion, bradykinin produce EDHF in a brefeldin A-sensitive mechanism in the porcine coronary artery. However, this mechanism is not active in a substance P-induced production of EDHF, which thus suggests EDHF to be more than a single entity.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 4-Aminopyridine; Animals; Apamin; Biological Factors; Bradykinin; Brefeldin A; Calcium; Coronary Vessels; Dose-Response Relationship, Drug; Endothelium, Vascular; Female; In Vitro Techniques; Indomethacin; Male; Nitroarginine; Peptides; Potassium; Potassium Channel Blockers; Quaternary Ammonium Compounds; Substance P; Swine; Vasoconstriction; Vasoconstrictor Agents; Vasodilation

We examined pial arteriolar reactivity to a partially endothelial nitric oxide synthase (eNOS)-dependent vasodilator ADP as a function of chronic estrogen status. The eNOS-dependent portion of the ADP response was ascertained by comparing ADP-induced pial arteriolar dilations before and after suffusion of a NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA; 1 mM) in intact, ovariectomized (Ovx), and 17beta-estradiol (E2)-treated Ovx females. We also examined whether ovariectomy altered the participation of other factors in the ADP response. Those factors were the following: 1) the prostanoid indomethacin (Indo); 2) the Ca2+-dependent K+ (K(Ca)) channel, iberiotoxin (IbTX); 3) the ATP-regulated K+ (K(ATP)) channel glibenclamide (Glib); 4) the K(Ca)-regulating epoxygenase pathway miconazole (Mic); and 5) the adenosine receptor 8-sulfophenyltheophylline (8-SPT). In intact females, the eNOS-dependent (L-NNA sensitive) portion of the ADP response represented approximately 50% of the total. The ADP response was retained in the Ovx rats but L-NNA sensitivity disappeared. On E2 replacement, the initial pattern was restored. ADP reactivity was unaffected by Indo, Glib, Mic, and 8-SPT. IbTX was associated with 50-80% reductions in the response to ADP in the intact group that was nonadditive with L-NNA, and 60-100% reductions in the Ovx group. The present findings suggest that estrogen influences the mechanisms responsible for ADP-induced vasodilation. The continued sensitivity to IbTX in Ovx rats, despite the loss of a NO contribution, is suggestive of a conversion to a hyperpolarizing factor dependency in the absence of E2.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Antifungal Agents; Arterioles; Calcium; Cardiovascular Agents; Endothelium, Vascular; Estrogens; Female; Glyburide; Hypoglycemic Agents; Indomethacin; Miconazole; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroarginine; Ovariectomy; Peptides; Pia Mater; Potassium Channels; Rats; Rats, Sprague-Dawley; Theophylline; Vasodilation

1. The contribution of an endothelium-derived hyperpolarizing factor (EDHF) was investigated in saphenous and mesenteric arteries from endothelial nitric oxide synthase (eNOS) (-/-) and (+/+) mice. 2. Acetylcholine-induced endothelium-dependent relaxation of saphenous arteries of eNOS(-/-) was resistant to N(omega)-nitro-L-arginine (L-NNA) and indomethacin, as well as the guanylyl cyclase inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a) quinoxalin-1-one(ODQ). 3. Potassium (K(+)) induced a dose-dependent vasorelaxation which was endothelium-independent and unaffected by either L-NNA or indomethacin in both saphenous and mesenteric arteries from eNOS(-/-) or (+/+) mice. 4. Thirty microM barium (Ba(2+)) and 10 microM ouabain partially blocked potassium-induced, but had no effect on acetylcholine-induced vasorelaxation in saphenous arteries. 5. Acetylcholine-induced relaxation was blocked by a combination of charybdotoxin (ChTX) and apamin which had no effect on K(+)-induced relaxation, however, iberiotoxin (IbTX) was ineffective against either acetylcholine- or K(+)-induced relaxation. 6. Thirty microM Ba(2+) partially blocked both K(+)- and acetylcholine-induced relaxation of mesenteric arteries, and K(+), but not acetylcholine-induced relaxation was totally blocked by the combination of Ba(2+) and ouabain. 7. These data indicate that acetylcholine-induced relaxation cannot be mimicked by elevating extracellular K(+) in saphenous arteries from either eNOS(-/-) or (+/+) mice, but K(+) may contribute to EDHF-mediated relaxation of mesenteric arteries.

Topics: Acetylcholine; Animals; Apamin; Arteries; Barium; Biological Factors; Charybdotoxin; Enzyme Inhibitors; In Vitro Techniques; Mesenteric Veins; Mice; Mice, Knockout; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroarginine; Ouabain; Peptides; Potassium; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Inwardly Rectifying; Sodium-Potassium-Exchanging ATPase

Pulmonary venous constriction leads to significant pulmonary hypertension and increased edema formation in several models using newborns. Although alkalosis is widely used in treating neonatal and pediatric pulmonary hypertension, its effects on pulmonary venous tone have not previously been directly measured. This study sought to determine whether alkalosis caused pulmonary venous relaxation and, if so, to identify the mediator(s) involved. Pulmonary venous rings (500-microm external diameter) were isolated from 1-wk-old piglets and precontracted with the thromboxane mimetic U-46619. Responses to hypocapnic alkalosis were then measured under control conditions after inhibition of endothelium-derived modulator activity or K(+) channels. In control rings, alkalosis caused a 34.4 +/- 4.8% decrease in the U-46619-induced contraction. This relaxation was significantly blunted in rings without functional endothelium and in rings treated with nitric oxide synthase or guanylate cyclase inhibitors. However, neither cyclooxygenase inhibition nor voltage-dependent, calcium-dependent, or ATP-dependent K(+)-channel inhibitors altered alkalosis-induced relaxation. These data suggest that alkalosis caused significant dilation of piglet pulmonary veins that was mediated by the nitric oxide-cGMP pathway.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 4-Aminopyridine; Alkalosis; Animals; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Glyburide; Hypoglycemic Agents; In Vitro Techniques; Nitric Oxide; Nitroarginine; Oxadiazoles; Peptides; Potassium Channels; Pulmonary Veins; Quinoxalines; Swine; Vasoconstrictor Agents; Vasodilation

The role of L-type Ca(2+) channels in the relaxation to nitric oxide (NO)-mediated MaxiK(Ca) channel activation was examined in guinea pig aorta. Acetylcholine (ACh) produced an endothelium-dependent relaxation of guinea pig aorta precontracted with noradrenaline (NA), which was abolished by an NO synthase inhibitor, N(G)-nitro-L-arginine (L-NNA). Both endothelium-dependent relaxation by ACh and endothelium-independent relaxation by an NO donor, (+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR3), were strongly suppressed by a soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ), suggesting that increased intracellular cGMP plays the key role in both responses. ACh- and NOR3-induced relaxations were significantly suppressed by iberiotoxin (IbTX), a selective blocker of MaxiK(Ca) channels. ACh- and NOR3-induced relaxations were greatly attenuated when arteries were precontracted with high KCl instead of NA, supporting the idea that K(+) channel activation mediates the relaxant responses. (6) NOR3-induced relaxations were not affected by a L-type Ca(2+) channel blocker, diltiazem. Furthermore, endothelium-independent relaxation by a K(ATP) channel opener, (+)-7,8-dihydro-6, 6-dimethyl-7-hydroxy-8-(2-oxo-1-piperidinyl)-6H-pyrano[2,3-f] benz-2,1, 3-oxadiazole (NIP-121) was not affected by diltiazem and nicardipine. These findings suggest that blockade of L-type Ca(2+) channels is not a major mechanism responsible for the vascular relaxation due to NO-mediated MaxiK(Ca) channel activation in guinea pig aorta.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Calcium Channel Blockers; Calcium Channels, L-Type; Drug Interactions; Female; Guinea Pigs; In Vitro Techniques; Large-Conductance Calcium-Activated Potassium Channels; Male; Nitric Oxide; Nitric Oxide Donors; Nitro Compounds; Nitroarginine; Norepinephrine; Oxadiazoles; Peptides; Piperidines; Potassium Channels; Potassium Channels, Calcium-Activated; Potassium Chloride; Quinoxalines; Vasodilation

To date, the release of the endothelium-derived hyperpolarizing factor (EDHF) has been demonstrated only in response to receptor-dependent Ca2+-elevating agonists. Since endothelial cells in situ are continuously subjected to rhythmic distension, we investigated the effect of rhythmic stretch on the release of EDHF from isolated porcine coronary arteries. In the combined presence of diclofenac and N(G)-nitro-L-arginine (L-NNA), sinusoidal pressure oscillations (from 40 to 50 mm Hg, 4 minutes, 1.5 Hz) led to simultaneous oscillations in the external diameter of coronary artery segments, the amplitude of which were decreased by iberiotoxin and apamin and also by endothelial denudation. In order to directly demonstrate the release of EDHF, the intraluminal solution from endothelium-intact coronary segments exposed to pulsatile stretch was applied to detector rat aortic smooth muscle cells, the membrane potential of which was continuously measured using the patch-clamp technique. The hyperpolarization of detector cells induced by the intraluminal solution was proportional to the amplitude of the pressure oscillations applied to the donor artery and was attenuated by either preincubation of donor arteries with 17-octadecynoic acid or application of either tetrabutylammonium or iberiotoxin to detector cells. In contrast to the bradykinin-induced release of EDHF, the EDHF synthesized in response to pulsatile stretch did not exhibit any tachyphylaxis. These findings demonstrate for the first time that the synthesis of EDHF in coronary arteries can be mechanically stimulated by rhythmic vessel wall distension and suggest that the continuous release of EDHF may contribute to the adjustment of an adequate vascular compliance and to the control of coronary blood flow.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Apamin; Biological Factors; Cells, Cultured; Coronary Vessels; Diclofenac; Electric Stimulation; Endothelium, Vascular; Fatty Acids, Unsaturated; In Vitro Techniques; Muscle, Smooth, Vascular; Nitroarginine; Oscillometry; Patch-Clamp Techniques; Peptides; Rats; Stress, Mechanical; Swine; Vasodilation

The first goal of this study was to examine the hypothesis that dilatation of the basilar artery in response to activation of ATP-sensitive K+ channels is mediated by nitric oxide (NO). Diameter of the basilar artery (209 +/- 5 microns, mean +/- SE) was measured using a cranial window in anesthetized rats. Aprikalim (a direct activator of ATP-sensitive K+ channels) dilated the basilar artery under control conditions. Inhibition of endogenous NO production with NG-nitro-L-arginine (L-NNA, 10(-4) M) did not alter responses to aprikalim. The second goal was to determine whether vasodilatation in response to NO is dependent on activation of calcium-activated K+ channels. Tetraethylammonium (TEA, 10(-3) M), an inhibitor of calcium-activated K+ channels, did not affect dilator responses to sodium nitroprusside (an NO donor) under control conditions. Responses to nitroprusside (10(-8) and 10(-7) M) were augmented more than twofold during application of L-NNA. In the presence of L-NNA, the augmented portion of the response to nitroprusside was inhibited by TEA and iberiotoxin (5 x 10(-8) M, a highly selective inhibitor of calcium-activated K+ channels), but it was not inhibited by glibenclamide (10(-6) M), an inhibitor of ATP-sensitive K+ channels. These findings suggest that dilator responses of the basilar artery to an activator of ATP-sensitive potassium channels are not mediated by NO. Calcium-activated K+ channels may not normally contribute to dilator responses of the basilar artery to nitroprusside. The effects of TEA and iberiotoxin suggest that when endogenous production of NO is inhibited, sodium nitroprusside causes the opening of calcium-activated K+ channels, contributing to an augmented vasodilator response.

Topics: Animals; Basilar Artery; Enzyme Inhibitors; Glyburide; Male; Nitric Oxide; Nitroarginine; Nitroprusside; Papaverine; Peptides; Potassium Channel Blockers; Potassium Channels; Rats; Rats, Sprague-Dawley; Tetraethylammonium; Tetraethylammonium Compounds; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

The relaxation of rat mesenteric resistance arteries to acetylcholine was studied in vessels (normalised internal diameter 230-330 microns) mounted in an isometric myograph and contracted with noradrenaline (5 microM). Removal of the endothelium abolished acetylcholine-induced vasorelaxation, whereas pretreatment with NG-nitro-L-arginine (500 microM) only inhibited the response partly. The relaxation was, however, completely inhibited by NG-nitro-L-arginine when the arteries were contracted with 80 mM K+. Acetylcholine-induced vasorelaxation was also attenuated by pretreatment with the K+ channel blocker, iberiotoxin (100 nM), and the combined pretreatment with iberiotoxin+NG-nitro-L-arginine completely blocked vasorelaxation to acetylcholine. Further, vasorelaxation to acetylcholine was attenuated by tetraethylammonium (5 mM), 4-aminopyridine (1 mM), and BaCl2 (100 microM), respectively, whereas glibenclamide (1 microM) and indomethacin (10 microM) were devoid of effect. Vasorelaxation to the nitric oxide donor sodium nitroprusside was not influenced by iberiotoxin. We conclude that in rat mesenteric resistance arteries, there is a significant nitric oxide-independent component of acetylcholine-induced vasorelaxation, which is mediated by activation of several types of K+ channels, in particular large conductance Ca(2+)-dependent K+ channels.

Topics: Acetylcholine; Animals; In Vitro Techniques; Male; Mesenteric Arteries; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitroarginine; Norepinephrine; Peptides; Potassium; Potassium Channel Blockers; Potassium Channels; Rats; Vascular Resistance

Nonadrenergic, noncholinergic (NANC) relaxations were elicited by field stimulation (1-16 Hz, 1 msec, 12 V for 15 sec) of guinea pig trachea desensitized with capsaicin (3 microM); pretreated with atropine (1 microM), propranolol (1 microM), indomethacin (3 microM) and alpha-chymotrypsin (2 U/ml) and contracted with 3 microM histamine. The nitric oxide (NO) synthase inhibitor L-nitro-N-arginine (L-NNA) significantly inhibited these responses, which is indicative of NO involvement. The ability of the large conductance Ca(++)-activated K+ channel antagonists iberiotoxin (IbTx) and charybdotoxin (ChTx) and the small conductance Ca(++)-activated K+ channel antagonist apamin to modify relaxations to NANC nerve stimulation and to the NO donor 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1) was studied. Both IbTx (100 nM) and ChTx (100 nM) were found to inhibit the L-NNA-sensitive relaxations elicited by field stimulation and to inhibit the relaxations to SIN-1. In contrast, apamin did not inhibit the relaxations to either field stimulation or SIN-1. These results suggest that in the guinea pig trachea, responses to endogenous or exogenously added NO are at least in part mediated by the large conductance Ca(++)-activated K+ channel.

Topics: Animals; Arginine; Charybdotoxin; Dose-Response Relationship, Drug; Electric Stimulation; Guinea Pigs; In Vitro Techniques; Male; Molsidomine; Muscle Relaxation; Nitric Oxide; Nitroarginine; Peptides; Potassium Channels; Scorpion Venoms; Trachea