The Role of Potassium Channels in Relaxant Effect of Levosimendan in Rat Small Mesenteric Arteries

Summary We investigated both the effect of levosimendan and the role of various potassium channels in KCl-precontracted rat small mesenteric arteries. Levosimendan (10⁻⁶−10⁻³ M) or cromakalim (CRO, 10⁻⁷−10⁻⁴ M) produced concentration-dependent relaxation responses in small mesenteric arteries precontracted by 30 mM KCl. The relaxant responses to levosimendan in KCl-precontracted arteries did not differ significantly between endothelium-intact and endothelium-denuded preparations. Incubation of rat small mesenteric arterial segments with ATP-dependent potassium channel (KATP) blocker glibenclamide (GLI, 10⁻⁶ M) for 30 min significantly inhibited the relaxant responses to both levosimendan and CRO. Neither the Ca²⁺-activated potassium channel (KCa) blocker iberiotoxin (10⁻⁷ M) nor the voltage-dependent potassium channel (KV) blocker 4-aminopyridine (5 mM) incubation for 30 min caused significant alterations in relaxant responses to levosimendan in KCl-precontracted small mesenteric arteries. These findings suggested that levosimendan-induced relaxation responses in isolated rat small mesenteric arteries were neither depended on endothelium nor inhibited by the blockers of KV or KCa but, they rather seem to depend on the activation of KATP.     

The Gender Differences in the Relaxation to Levosimendan of Human Internal Mammary Artery

Purpose The mechanism of the vasorelaxation to levosimendan varies depending on the vascular bed and species studied. Here, we examined the vasorelaxation to levosimendan as well as its modification by various potassium channel antagonists in human internal mammary artery (IMA) obtained from male and female patients. Methods IMA grafts were supplied from 27 male and 19 age-matched female patients undergoing coronary bypass operation. The contraction to noradrenaline and relaxation to levosimendan were studied in IMA rings obtained from both gender. The relaxations to levosimendan were also assessed in the presence of glibenclamide (10 μM), an adenosine triphosphate-sensitive potassium channel (K_ATP) blocker, or charybdotoxin (100 nM), a calcium-activated potassium channel (K_Ca) blocker, or 4-aminopyridine(1 mM), a voltage-sensitive potassium channel (K_v) inhibitor. Results Concentration–response curves to noradrenaline were not different in IMA rings from either gender. Pretreatment with levosimendan (3 × 10⁻⁷ M) slightly modified the contractions to noradrenaline in both gender. Levosimendan (10⁻⁹–10⁻⁵ M) produced concentration-dependent relaxation in IMA rings, contracted by noradrenaline (5 × 10⁻⁶ M), from males and females. The vasodilatory effects of levosimendan were more pronounced in the arteries from males (83%) than females (69%), in term of the maximal relaxation (E _max). Charybdotoxin and glibenclamide significantly inhibited the relaxation to levosimendan in the arteries from males but not in those of females. Conclusions The vasodilating efficacy of levosimendan and its relaxation mechanism differs between the arteries from males and females, which may have clinical consequences in the treatment of heart failure.     

Relaxant effects of forskolin on guinea pig tracheal smooth muscle

We investigated the relaxant effects of forskolin, a diterpene derivative isolated from the roots ofColeus forskohlii, on guinea pig airway smooth muscle by measuring the isometric tension of tracheal smooth muscle in vitro and transcutaneous Po₂ during the histamine inhalation test (HIT) in vivo. Forskolin (10⁻⁹–10⁻⁵ M) caused dose-dependent relaxant effects on resting tone and on leukotriene C₄ (10⁻⁷ M)-, leukotriene D₄ (10⁻⁷ M)-, and carbachol (3 × 10⁻⁶ M)-induced contraction of tracheal smooth muscle. Moreover, with propranolol pretreatment the relaxant effect of forskolin on tracheal smooth muscle did not change, whereas with the same pretreatment the relaxant effect of isoproterenol diminished. Forskolin (10⁻⁸–10⁻⁶ M) raised tissue cyclic AMP levels dose-dependently in tracheal smooth muscle (6.7–359.9 pmol/mg protein). Forskolin (1 mg/kg) administered subcutaneously raised the respiratory threshold of (RT-histamine in the HIT. The determination of the RT-histamine by measuring tcPo₂ was possible without anesthesia. These results suggest that forskolin relaxes airway smooth muscle in guinea pigs in vitro and in vivo by raising tissue cyclic AMP levels and that its actions are independent ofβ-adrenoceptors.     

Budesonide Down-Regulates Eosinophil Locomotion But Has No Effects on ECP Release or on H2O2 Production

Treatment of allergic asthma with inhaled corticosteroids results in local down-regulation of proinflammatory cytokine synthesis and in marked decrease in tissue eosinophilia. Blood concentrations of inhaled corticosteroids, although significantly lower than those measured in the lung, may still have antiinflammatory effects on circulating eosinophils, reducing their ability to migrate. The aim of our study was to evaluate in vitro the activity of budesonide on blood eosinophils by measuring their chemotactic response, eosinophil cationic protein (ECP) release, and hydrogen peroxide (H₂O₂) production in the presence of different drug concentrations similar to those obtained at airway level (10⁻⁸ and 10⁻⁷ M) and at blood level (10⁻¹⁰ and 10⁻⁹ M). Partially purified blood eosinophils, isolated from 23 asthmatic subjects, were used to evaluate the activity of budesonide on: (1) chemotaxis toward the activated fifth component of complement (C5a, 0.1 μg/ml) or recombinant human (rh) interleukin (IL)-5 (200 pg/ml), (2) ECP release by cells stimulated with tetradecanoylphorbol acetate (TPA) and (3) H₂O₂ production by TPA-activated cells. The chemotactic response to C5a was down-regulated significantly by budesonide only by the highest concentrations tested (10⁻⁸ and 10⁻⁷ M); differently, budesonide was effective in inhibiting eosinophil migration toward rhIL-5, at all concentrations tested (p < 0.01, each comparison). By contrast, no drug-induced modifications were observed in ECP release or in H₂O₂ production (p > 0.05, each comparison). We conclude that concentrations of budesonide similar to those obtained in vivo are effective in inhibiting eosinophil locomotion but not in down-regulating the release of reactive oxygen species and granule-associated proteins. Accepted for publication: 11 February 1999     

The Highly Expressed and Inducible Endogenous NAD(P)H:quinone Oxidoreductase 1 in Cardiovascular Cells Acts as a Potential Superoxide Scavenger

It has recently been demonstrated that purified NAD(P)H:quinone oxidoreductase 1 (NQO1) is able to scavenge superoxide (O₂^•−) though the rate of reaction of O₂^•− with NQO1 is much lower than the rate of enzymatic dismutation catalyzed by superoxide dismutase (SOD). This study was undertaken to determine if the endogenously expressed NQO1 in cardiovascular cells could scavenge O₂^•−. We observed that NQO1 was highly expressed in cardiovascular cells, including rat aortic smooth muscle A10 and cardiac H9c2 cells, as well as normal human aortic smooth muscle and endothelial cells. NQO1, but not SOD in the cardiovascular cells was highly inducible by 3H-1,2-dithiole-3-thione (D3T). Cytosols from H9c2 and human aortic smooth muscle cells (HASMCs) were isolated to determine the O₂^•− scavenging ability of the endogenously expressed NQO1 by using pyrogallol autooxidation assay. We showed that cytosols from the above cells inhibited pyrogallol autooxidation in an NADPH or NADH-dependent manner. The NADH/NADPH-dependent inhibition of pyrogallol autooxidation by the cytosols was completely abolished by the NQO1-specific inhibitor, ES936, suggesting that the endogenously expressed NQO1 could scavenge O₂^•−. In the presence of NADH/NADPH, cytosols from D3T-treated cells showed increased ability to scavenge O₂^•− as compared to cytosols from untreated cells. This increased ability to scavenge O₂^•− was also completely reversed by ES936. 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin-trapping experiments using potassium superoxide as a O₂^•− generator further confirmed the ability of NQO1 from HASMCs to scavenge O₂^•−. The spin-trapping experiments also showed that induction of NQO1 by D3T in HASMCs augmented the O₂^•− scavenging ability. Taken together, these results demonstrate that the highly expressed and inducible endogenous NQO1 in cardiovascular cells may act as a potential O₂^•− scavenger.     

Mechanism of leukotriene-induced contraction of isolated guinea pig tracheal smooth muscle

The mechanical response of guinea pig tracheal smooth muscle to leukotriene (LT) B₄, C₄, D₄ and E₄ was investigated, and the effects of several agents on LT-induced contraction were determined. Agents used in the present study were: verapamil, a Ca-channel blocker; dibutyryl cyclic-AMP (DBcAMP); aminophylline; N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), a calmodulin antagonist; N-(6-aminohexyl)-1-naphthalene-sulfonamide hydrochloride (W-5), resembling W-7 in structure but without calmodulin antagonism. The results were as follows: 1) LTB₄ had a contractile activity, dependent on the [Ca]o concentration; it was suppressed by a [Mg]o increase to 1.2 mM and over. Little effect was observed from other drugs. 2) Contractile activities of LTC₄, D₄ and E₄ were dose-dependent, depending upon [Ca]o as well as LTB₄. 3) Verapamil (5×10⁻⁶−10⁻³ M), DBcAMP (3×10⁻⁶−3×10⁻³ M) and aminophylline (3×10⁻⁸−3×10⁻³ M) suppressed LTs (C₄, D₄, and E₄, 10⁻⁸ M)-induced contraction dose-dependently. However, W-7 and W-5 did not suppress LT-induced contraction. These results suggest that the LTC₄-, D₄-and E₄-induced responses could be explained by the effect of LTC₄, D₄ and E₄ on increasing Ca influx through the plasma membrane.     

Effects of aging on ATP sensitive K+ channels and on prostanoid production in the rat mesenteric bed.

The aim of the present work was to evaluate, in the rat isolated mesenteric bed, whether increasing age is associated with alterations in the ATP sensitive K⁺ channels functionality. Moreover, studies were performed in order to evaluate the effects of aging on the synthesis of vascular prostanoids as well as on its possible contribution to the pressor responses of this vascular bed. Male Wistar rats of 3 month (adults) and 24 month (aged) were used. Although no differences were found among adult and aged rats in pressor responses to 2–30 nmol noradrenaline and to 40–160 nmol KCl, the relaxant responses to the K⁺ channel opener, 10⁻⁶ M cromakalim, were significantly diminished in the aged group compared to the adults. On the other hand, whereas PGF₂α and 6-keto PGF₁α production was not modified with age, the thromboxane B₂ and prostaglandin E₂ production in the mesenteric bed from 24 month old rats was significantly increased compared to the adult group. Furthermore, the cyclooxigenase synthesis inhibitor, 10⁻⁵ M indomethacin reduced the pressor responses induced by noradrenaline in the mesenteric beds from adults but not from aged rats. It is concluded that there is an age related reduction in the functionality of the ATP sensitive K⁺ channels in the rat mesenteric bed. In addition, aging produces an increase in the release of vasoconstrictor as well as of vasodilator prostanoids, whose contribution to noradrenaline induced pressor responses appears to be less relevant in the older animals.     

Hydrogen peroxide-induced vascular relaxation in porcine coronary arteries is mediated by Ca2+-activated K+ channels

Summary Hydrogen peroxide (H₂O₂) elicited concentration-dependent relaxation of endothelium-denuded rings of porcine coronary arteries. The relaxation induced by the H₂O₂ was markedly attenuated by 10μM 1H-[1,2,4]oxadiazolo [4,3,a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase, or by 100nM charybdotoxin, an inhibitor of large-conductance Ca²⁺-activated K⁺ (K_Ca) channels. A combination of the ODQ and charybdotoxin abolished the H₂O₂-induced relaxation. Pretreatment with 25 μM of an Rp stereoisomer of adenosine-3′,5′-cyclic monophosphothioate (Rp-cAMPS), 20μM glibenclamide, or 1mM 4-aminopyridine did not affect the vascular response to H₂O₂. The presence of catalase at 1000U/ml significantly attenuated the H₂O₂-induced relaxation. Exposure of cultured smooth muscle cells to H₂O₂ activated K_Ca channels in a concentration-dependent manner in cell-attached patches. Pretreatment with catalase significantly inhibited the activation of K_Ca channels. Rp-cAMPS did not inhibit the H₂O₂-induced activation of K_Ca channels. The activation of K_Ca channels by H₂O₂ was markedly decreased in the presence of ODQ. However, even in the presence of ODQ, H₂O₂ activated K_Ca channels in a concentration-dependent manner. In inside-out patches, H₂O₂ significantly activated K_Ca channels through a process independent of cyclic guanosine 3′,5′-monophosphate (cGMP). In conclusion, H₂O₂ elicits vascular relaxation due to activation of K_Ca channels, which is mediated partly by a direct action on the channel and partly by activation of soluble guanylate cyclase, resulting in the generation of cGMP.     

Melatonin inhibits vasopastic action of hydrogen peroxide in human umbilical artery

ABSTRACT: We evaluated the antioxidant property of melatonin as it relates to the vasospastic effect of hydrogen peroxide (H₂O₂) on the human umbilical artery. Helical sections of umbilical arteries were obtained from healthy pregnant women who were delivered between weeks 37 and 39 of gestation. Changes in maximal potassium chloride (KCl)-induced tension were measured in arterial segments with intact endothelium. Segments were treated with H₂O₂ alone, or were pretreated either with an H₂O₂scavenger (catalase, 2000 IU), a hydroxyl radical scavenger (mannitol, 10^?2M), a nitric oxide-synthesis inhibitor (L-N^G-monomethyl arginine, LNMA, 2 × 10^?4M), or melatonin (10^?6M to 10^?4M). The effect of H₂O₂(10^?4M) on the relaxation induced by the calcium ionophore A23187 was also determined in arterial segments, with or without pretreatment with melatonin (10^?6M, 10^?4M). H₂O₂ (10^?6 M to 10^?4 M) potentiated vascular tension in a concentration-dependent manner (P < 0.0001). Pretreatment with LNMA significantly suppressed the vasospastic effect of H₂O₂ (P < 0.0001). Pretreatment with either catalase or mannitol significantly reduced the vasospastic effect of H₂O₂ (P < 0.005, P < 0.002, respectively). Melatonin also significantly reduced the vasospastic effect of H₂O₂ in a concentration-dependent manner (H₂O₂ 10^?6 M, P < 0.0001: H₂O₂ 10^?5 M, P<0.0001: H₂O₂ 10^?4M, P < 0.00001). Pretreatment with H₂O₂ significantly inhibited the relaxation induced by the calcium ionophore A23187 (P < 0.005). Treatment with melatonin prior to exposure to H₂O₂ significantly restored the relaxation induced by A23187 (P < 0.005). Results suggest that H₂O₂ potentiates vascular tension in the human umbilical artery, perhaps by suppressing the endothelial synthesis of nitric oxide. Melatonin significantly suppressed the vasospastic effect of H₂O₂, possibly due to its ability to scavenge the hydroxyl radical.     

Melatonin suppresses vasospastic effect of hydrogen peroxide in human umbilical artery: Relation to calcium influx

ABSTRACT: We evaluated the hydroxyl radical scavenging effect of melatonin on the vasospastic action induced by hydrogen peroxide in human umbilical artery. Helical sections were made of umbilical arteries obtained from healthy pregnant women who were delivered between 37 and 39 weeks of gestation. Changes in maximal potassium chloride (KC1, 10^?2 M)-induced tension were measured in umbilical artery segments with intact endothelium. Segments were treated with H₂O₂ (10^?9 M to 10^?7 M) only, or were pretreated with an H₂O₂ scavenger (catalase, 2,000 IU), a hydroxyl radical scavenger (mannitol, 10^?2 M), or melatonin (10^?8 M to 10^?6 M). The effect of H₂O₂ on the response of the segments of umbilical artery to external calcium was determined. Changes in KCl-induced contraction were also determined in segments pretreated with an inhibition of intracellular calcium release (ryanodine, 10^?4 M) prior to exposure to H₂O₂. Pretreating the segments of umbilical arteries with H₂O₂ (10^?8 M, 10^?7 M) significantly potentiated the maximal contraction induced by KC1 (P < 0.0001, P < 0.03, respectively). Pretreatment with either catalase or mannitol significantly reduced the vasospastic effect of H₂O₂ (10^?8 M) (P < 0.0001, P < 0.0001, respectively). Melatonin also significantly reduced the vasospastic effect of H₂O₂ (10^?8 M), in a concentration-dependent manner (P<0.0001). H₂O₂ (10^?8 M) significantly increased the contractile response to external calcium. Melatonin pretreatment significantly suppressed the contractile response to external calcium. Treatment with ryanodine prior to exposure to H₂O₂ did not affect KCl-induced contraction. Results suggest that H₂O₂ potentiates the KCl-induced maximal contraction of the human umbilical artery, perhaps by increasing calcium influx via activation of the voltage-dependent calcium channel. Melatonin significantly suppresses the vasospastic effect of H₂O₂, probably due to its scavenging of the hydroxyl radical.     

Growth hormone-releasing peptide-2 (GHRP-2) does not act via the human growth hormone-releasing factor receptor in GC cells

Effect of growth hormone-releasing peptide-2 (GHRP-2) on ovine somatotrophs is abolished by a growth hormone-releasing factor (GRF) receptor antagonist, which raises the possibility that GHRP-2 may act on GRF receptors. In the present study, we used rat pituitary GC cells with or without stable transfection of cDNA coding for the human GRF receptor (GC/R⁺ or GC/R⁻) to determine whether or not GHRP-2 acts via the GRF receptor. Northern blot analysis indicated that GRF receptor mRNA was undetectable in GC/R⁻ cells, whereas a high level of expression occurred in GC/R⁺ cells that were transfected by GRF receptor cDNA. In GC/R⁻ cells, incubation with up to 10⁻⁷ M of either hGRF or GHRP-2 did not alter the intracellular cAMP, [Ca²⁺]i, or GH secretion. In GC/R⁺ cells, hGRF (10⁻¹¹–10⁻⁷ M) increased cAMP levels in a concentration-dependent manner up to 20-fold. This increase in cAMP levels was blocked by a GRF receptor antagonist, [Ac-Tyr¹, d-Arg²]-GRF 1–29, but not by a Ca²⁺ channel blocker, NiCl₂ (0.5 mM). GH secretion and [Ca²⁺]i were, however, not increased by hGRF. Incubation of the transfected cells with 10⁻¹¹–10⁻⁸ M GHRP-2 did not modify intracellular cAMP levels. This result suggests that GHRP-2 does not act through the GRF receptor.     

Epigallocatechin-3-Gallate Protects Na<Superscript>+</Superscript> Channels in Rat Ventricular Myocytes Against Sulfite

Sulfite (bisulfite/sulfite) can affect voltage-gated sodium (Na⁺) channels (VGSC) in a concentration-dependent manner in isolated rat ventricular myocytes. In this study, the effect of epigallocatechin-3-gallate (EGCG) on VGSC in isolated ventricular myocytes was studied. Ventricular myocytes were exposed to 10 μM bisulfite/sulfite for 10 min, and EGCG was then administered in different concentrations (10, 30, 50 μg ml⁻¹). Decreased activity of superoxide dismutase, catalase (CAT) and glutathione peroxidase (GPx) was observed after bisulfite/sulfite exposure, with significant increase in Na⁺ currents (I _Na) and alterations in half-activation voltage and half-inactivation voltage. Intracellular reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂), hydroxyl (OH^·), and superoxide anion (O₂^·−) were increased. After EGCG treatment, activity of the aforementioned enzymes increased while the ROS level decreased. The effects progressed with increasing amounts of EGCG, up to a level similar to blank control at the dose of 50 μg ml⁻¹ EGCG, EGCG also reduced the I _Na and reversed the alterations in half-activation voltage and half-inactivation voltage. In conclusion, EGCG could protect Na⁺ channels in rat ventricular myocytes against the oxidative damage induced by sulfite as a scavenger of the ROS.     

Involvement of Nitric Oxide and Potassium Channels in the Reduction of Basal Tone Produced by Blockade of Thromboxane A2/Prostaglandin H2/ Receptors in Aortic Rings Of Hypertensive Rats

This study was designed to investigate involvement of potassium channels in the action of nitric oxide facilitating reduction of basal tone by thromboxane A₂/prostaglandin H₂receptor blockade with ifetroban in rings of thoracic aorta taken from rats with aortic coarctation-induced hypertension. Ifetroban-induced reduction of basal tone in aortic rings without drug pretreatment was attenuated (P<0.05) in rings pretreated with the nitric oxide synthesis inhibitor Nω-nitro-L-arginine methyl ester (L-NAME; 3 × 10<^-4mol/L; 0.55±0.09 g versus 0.23±0.07 g). The vasorelaxing effect of ifetroban also was decreased (P<0.05) in preparations pretreated with a potassium channel blocker, either tetraethylammonium (TEA; 10^-2mol/L) or 4-aminopyridine (4-AP; 3 × 10^-3mol/L). Ifetroban-induced reduction of basal tone was not attenuated in preparations pretreated first with L-NAME and then with sodium nitroprusside (SNP; 6±1 nmol/L) to compensate for the loss of endogenous nitric oxide. However, the facilitatory effect of SNP on ifetroban-induced relaxation of aortic rings pretreated with L-NAME alone was not demonstrable in rings pretreated with L-NAME plus TEA or 4-AP. These observations suggest that a mechanism involving nitric oxide and potassium channels facilitates the reduction in basal tone produced by ifetroban in aortic rings of rats with aortic coarctation-induced hypertension     

Effects of Recycled Paper Dust Extracts on Isolated Guinea Pig Trachea

The effect of paper dust collected at two different locations in a paper recycling plant (PD1 and PD2) on isolated nonsensitized guinea pig tracheal smooth muscle was studied in vitro. Dust extracts were prepared as a 1:10 w/v aqueous solution. Dose-related contractions of guinea pig tracheal rings were elicited with both PD1 and PD2. Pharmacologic studies were performed with atropine (10⁻⁶ M), indometacin (10⁻⁶ M), pyrilamine (10⁻⁶ M), LY171883 (10⁻⁵ M), nordihydroguaiaretic acid (10⁻⁵ M), and TMB8 (10⁻⁵ M). The possible role of endogenous neuropeptides in this constrictor process was studied by depleting neural mediators with capsaicin (5 × 10⁻⁶ M) before challenge with dust extracts. Constrictor effects were partially inhibited by a wide variety of the mediator blocking agents. The effects of both extracts were almost totally inhibited by the anticholinergic agent atropine, suggesting that a principal pathway mediating this response may involve the parasympathetic nervous system. The intracellular calcium-blocking agent TMB8 also induced a reduction of the contractile responses to PD1 and PD2 concsistent with the well established role of intracellular calcium in smooth muscle constriction. Pretreatment with capsaicin significantly increased the contractile activity of paper dust extracts but only at the higher doses of these extracts. This suggests that the effect of paper dust is not initiated by the release of mediators stored in sensory nerves but that the prerelease of these mediators may enhance the constrictor effects of these dusts. We suggest that paper dust extracts cause dose-related airway smooth muscle constriction possibly associated with the release of cholinergic as well as other mediators. The constrictor effect does not require tissue presensitization or the release of neuropeptides from sensory nerves. Accepted for publication: 21 March 1997     

Relaxant properties mediated by nitroglycerin in aortic coarctation hypertensive rats

 Nitroglycerin-mediated vasorelaxation is chiefly attributed to the cyclic guanosine monophosphate (cGMP)-dependent pathway, and partly to the cGMP-independent pathway via calcium-activated K⁺ channels (K_Ca). To investigate whether chronic hypertension alters responses of vascular smooth muscle to vasoactive agonists, we determined nitroglycerin-mediated relaxation of aortic rings from coarctation hypertensive rats. Banding the abdominal aorta above the renal arteries for 4 weeks elevated blood pressure and caused cardiac hypertrophy by 49%. In response to nitroglycerin, the relaxation of aortic rings precontracted with 10⁻⁷ M norepinephrine was lower in the banded group than in the sham-operated group. Methylene blue, a guanylate cyclase inhibitor, suppressed a greater part of nitroglycerin-mediated relaxation and reached similar levels of relaxation in the two groups. Charybdotoxin, a specific K_Ca channel blocker, also suppressed the relaxation by about 40% in the aortic rings from sham-operated animals, but not in those from the banded group. The response to charybdotoxin was markedly diminished or virtually eliminated in the banded group in the presence or absence of methylene blue. The combination of charybdotoxin and methylene blue nearly abolished nitroglycerin-mediated relaxation in the sham-operated group, whereas nitroglycerin-mediated relaxation was seen to remain in the banded group. These results indicate that the involvement of cGMP-independent K_Ca channels in nitroglycerin-mediated relaxation disappeared after the development of hypertension produced by aortic coarctation. Received: May 20, 2002 / Accepted: July 27, 2002 Correspondence to K. Okumura     

Atypical Adrenoceptor-mediated Relaxation of Canine Pulmonary Artery Through a Cyclic Adenosine Monophosphate–dependent Pathway

To determine whether functional atypical β-adrenoceptors (β₃-adrenoceptors) are present in pulmonary vascular smooth muscle, we studied isolated canine pulmonary arterial rings under isometric conditions in vitro. Addition of β-adrenoceptor agonists produced a concentration-dependent relaxation of noradrenaline-precontracted tissues, a rank order potency being isoproterenol (1) > salbutamol (0.95) > selective β₃-adrenoceptor agonists, CL 316243 (0.85), and BRL 37344 (0.83). A marked desensitization to salbutamol occurred by pretreatment with salbutamol but not with CL 316243. When β₁-adrenoceptors had been blocked, the relaxant responses to salbutamol were competitively antagonized by the β₂-adrenoceptor antagonist ICI 118551 with a pA₂ value of 7.67 ± 0.21 (mean ± S.E.), but the response to CL 316243 was weekly antagonized by ICI 118551 only at a high concentration of 10⁻⁵ M, where an apparent pA₂ value was 5.24. In contrast, cyanopindolol, a nonselective β-adrenoceptor antagonist, antagonized CL 316243–induced relaxation in a competitive manner with a pA₂ of 6.10 ± 0.11. This pA₂ value was lower than that when salbutamol was used as an agonist (6.69 ± 0.14, p < 0.01). Intracellular 3′,5′-cyclic adenosine monophosphate (cAMP) levels were increased by CL 316243 in a concentration-dependent fashion, an effect that was not altered by ICI 118551. These results suggest that β₃-adrenoceptors may exist in canine pulmonary artery smooth muscle and that stimulation of this atypical receptor causes vasodilation through a cAMP-dependent pathway. Accepted for publication: 17 June 1999     

Antioxidant Function of Ambroxol in Mononuclear and Polymorphonuclear Cells in Vitro

This study quantifies the antioxidant function of ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine) in vitro. Polymorphonuclear cells (PMN) and mononuclear cells were isolated from the blood of healthy volunteers (n= 46) to determine reactive oxygen species (ROS) by luminol-enhanced chemiluminescence. Ambroxol or the controls N-acetylcysteine (NAC), nacystelyn (NAL), glutathione (GSH), superoxide dismutase (SOD), catalase, and the combination of SOD/catalase were incubated for 1 or 2 h with zymosan-activated cells in vitro using concentrations ranging from 10⁻⁶ to 10⁻³ mol/liter. Reduction of ROS-mediated luminescence was similar within the cell types. Ambroxol (10⁻⁴ mol/liter) reduced ROS about 75% (1-h incubation) and 98% (2-h incubation), respectively (p < 0.001). SOD and SOD/catalase, but not the H₂O₂-catalyzing substances (NAC, NAL, GSH, and catalase), reduced cellular ROS. This indicates that inflammatory cells predominantly generate O⁻ ₂, which can be scavenged by ambroxol. The antioxidant function of ambroxol with increasing incubation time suggests additional cellular antiinflammatory properties of this substance. Our results indicate that good antioxidant function of ambroxol is related mainly to direct scavenger function of reactive oxygen metabolites such as O⁻ ₂. However, an antioxidative effect of ambroxol may also be associated with the reduction of prooxidative metabolism in inflammatory cells. Concluding from this observation, and because of the well known high affinity of ambroxol for lung tissue, ambroxol may be an alternative in antioxidant augmentation therapy, particularly in pulmonary diseases characterized by an overburden of toxic oxygen metabolites. Accepted for publication: 5 December 1996     

Involvement of cyclo-oxygenase-generated vasodilating eicosanoid(s) in addition to nitric oxide in endothelin-1-induced endothelium-dependent vasorelaxation in guinea pig aorta

Summary This study investigates the vasodilatory effects of endothelin-1 (ET-1) in isolated guinea pig aortic rings in vitro. Cumulative dose-response curves to ET-1 were constructed and ET-1 actions on prostaglandin F₂ (PGF₂)-precontraction were studied in both endothelium-intact and endothelium-denuded preparations, in the presence or absence of a cyclooxygenase inhibitor (indomethacin) and/or nitric oxide inhibitors (N^G-nitro-L-arginine methyl ester and hemoglobin). In endothelium-intact preparations, pretreatment with indomethacin (10^–5M, 30 min), alone or in combination with N^G-nitro-L-arginine methyl ester (L-NAME, 10^–4M), significantly augmented the constrictive responses to ET-1, whereas indomethacin, L-NAME, and hemoglobin (10^–5M) had no significant effects in the endothelium-denuded preparations. Furthermore, in PGF₂-precontracted, endothelium-intact preparations, ET-1, at a dose of 10^–9M, induced initial relaxation followed by subsequent contraction, while it only contracted the endothelium-denuded preparations. The initial relaxation was abolished by indomethacin, but not by L-NAME or hemoglobin. In addition, this relaxation was not inhibited by a specific ET_A receptor antagonist, BQ-123 (6 × 10^–6M). In addition to the involvement of nitric oxide, these results show the involvement of cyclo-oxygenase-generated vasodilating eicosanoid(s) derived from endothelium in ET-1-induced vasorelaxation in guinea pig aorta in vitro. The results also indicate that this vasorelaxation is mediated by ET_B receptor activation.This study was financially supported by Toyobo Biotechnology Foundation, and by Grants-in-Aid for scientific research from the Ministry of Education, Science and Culture of Japan (project numbers: 3770533 and 04670571) and the Ministry of Health and Welfare of Japan.     

Effects of nitric oxide donors on vascular smooth muscles depend on a type of vascular smooth-muscle preactivation

The abilities of such therapeutic nitrovasodilators as sodium nitroprusside (SNP) and glyceryl trinitrate (GTN) to dilate vascular smooth muscles (VSM) and affect intracellular calcium concentration level ([Ca²⁺]_i) in a rat tail artery were tested under different types of preactivation. To shed light on mechanisms underlying possible differences in the action of these two nitric oxide (NO) donors, simultaneous measurements of [Ca²⁺]_i and contractile force were done. All vascular rings were precontracted either using a high-K⁺-Krebs solution or phenylephrine (PE). It was shown that the effect of both NO donors strongly depended on a type of VSM preactivation. The EC₅₀ for GTN under K⁺ stimulation of VSM comprised (2.48±1.6)×10⁻⁵ M, whereas the mean EC₅₀ under PE stimulation was (3.05±2.3)×10⁻⁴ M (p<0.05, n=9). The EC₅₀ for SNP under K⁺ stimulation of VSM comprised (1.09±0.47)×10⁻⁷ M, whereas the EC₅₀ under PE stimulation was (8.01±2.4)×10⁻⁶ M (p<0.05, n=9). GTN demonstrated a significant discrepancy in the magnitude of changes in [Ca²⁺]_i and related VSM relaxant responses, indicating the ability of GTN to relax VSM in the absence of a proportional decrease in [Ca²⁺]_i. The main peculiarity of SNP action under K⁺ stimulation as compared to PE stimulation was the transient decrease in [Ca²⁺]_i while relaxation was sustained. Therefore, both NO donors demonstrated their ability to produce vasorelaxation as a result of an alteration in myofilament calcium sensitivity. These data clearly indicate that the sensitivity of VSM to NO donors is higher under K⁺ depolarization than that seen under PE stimulation, indicating that Ca²⁺ entry through voltage-operated calcium channels is more sensitive to NO as compared to calcium mobilization by means of Ca²⁺ entry through receptor-operated calcium channels or intracellular Ca²⁺ release, or both.     

Effects of carbamylcholine chloride on human antral gastrin mRNA levels

The effects of the muscarinic receptor agonist, carbamylcholine chloride (carbachol), on gastrin release and gastrin mRNA levels in human antral mucosa (n=15) were determined. During a-2-h incubation period, carbachol (10⁻⁶−10⁻⁴M) decreased gastrin mRNA levels to 71±8% (10⁻⁶M), 40±8% (10⁻⁵M), and 33±5% (10⁻⁴M) of control levels. Carbachol (10⁻⁵M) decreased intracellular gastrin (from 1634±103 to 1272±126 pg/mg tissue protein), while it increased gastrin release into the medium (from 609±48 to 918±68 pg/ml per mg tissue protein). After 6-and 9-h culture, carbachol gradually increased gastrin mRNA levels, by 96±12% and 126±23%, respectively. Atropine sulfate (10⁻⁵ M) completely inhibited the carbachol-induced changes. Cycloheximide markedly decreased tissue gastrin concentration, but increased gastrin mRNA levels, whereas it had no effects on gastrin release. These findings suggested that carbachol may have a time-related biphasic action on human antral gastrin biosynthesis.