Evaluation of 2 celecoxib derivatives： analgesic effect and selectivity to cyclooxygenase-2/1

AIM: To evaluate the analgesic effects of 2 celecoxib derivatives and their inhibitory effects on cyclooxygenase (COX). METHODS: Four antinociceptive assays were used: the acetic acid-induced writhing test, hot plate test, hot tail-flick test and formalin test. Three doses were used in the analgesic assays and ED50 values were calculated. For the selectivity assay, macrophages were incubated with test compounds at various concentrations and then stimulated with calcimycin or lipopolysaccharide (LPS). The amounts of 6-keto-prostaglandin F1alpha(6-keto-PGF1alpha) and prostaglandin E2 (PGE2) in the supernatant were examined by radioimmunoassay (RIA). The selectivity of the test compounds was expressed as the IC(50,COX-1)/IC(50,COX-2) value. RESULTS: Celecoxib and its 2 derivatives had a significant analgesic effect. The ED50 values of celecoxib, PC-406 and PC-407 were 94.2, 67.9, and 63.3 mg/kg, respectively, for the acetic acid-induced writhing test; 104.7, 89.1, and 30.0 mg/kg, respectively, for the hot tail-flick response test; 60.7, 56.7, and 86.2 mg/kg, respectively, for the hot plate response test; 67.1, 55.8, and 68.8 mg/kg, respectively, for the formalin-induced response. That is, the ED50 of PC-406 was the lowest for the formalin and hot plate tests, which focus on changes above the spinal cord level; however, the ED50 of PC-407 was lowest for the tail-flick and writhing tests, which focus on changes at the spinal cord level. Celecoxib and PC-407 inhibited COX-1 with IC50 values of 39.8 and 27.5 nmol/L, respectively. PC-406 inhibited COX-1 with an IC50 value of more than 1000 nmol/L. The IC50 values for the effect of celecoxib, PC-406 and PC-407 on COX-2 were 4.8, 8.9, and 1.9 nmol/L respectively. The IC(50, COX-1)/IC(50,COX-2) ratios for celecoxib and PC-407 were 8.3 and 14.4, respec-tively. For PC-406, the ratio was greater than 112.2. CONCLUSION: Derivatives of celecoxib via substitution with an isopropyl or naphthyl group at the 5 position in the pyrazole ring still have analgesic effects and the ability to selectively inhibit COX-2. Substitution with a naphthyl group may have more effect on the peripheral pain pathway, whereas substitution with an isopropyl group may have more effect on the central pain pathway. This phenomenon occurs partly because substitution with an isopropyl group is more beneficial for COX-2 selectivity than is substitution with a naphthyl group.     

Synthesis and SAR of Benzisothiazole- and Indolizine-β-d-glucopyranoside Inhibitors of SGLT2

d-glucopyranoside inhibitors of human SGLT2 are described. The synthesis of the C-linked heterocyclic glucosides took advantage of a palladium-catalyzed cross-coupling reaction between a glucal boronate and the corresponding bromo heterocycle. The compounds have been evaluated for their human SGLT2 inhibition potential using cell-based functional transporter assays, and their structure−activity relationships have been described. Benzisothiazole-C-glucoside 16d was found to be an inhibitor of SGLT2 with an IC₅₀ of 10 nM.     

Occurrence, pharmacology and function of presynaptic α2-autoreceptors in α2 A/D-adrenoceptor-deficient mice

The occurrence, pharmacological properties and function of alpha2-autoreceptors were studied in hippocampal slices, occipito-parietal cortex slices, segments of heart atria and segments of the vas deferens of wildtype (WT) mice and mice in which the alpha2 A/D-adrenoceptor gene had been disrupted (alpha2 A/D(KO)). Tissues were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically. Stimulation periods for brain slices consisted either of 1 pulse or of 2-64 pulses delivered at 1-s intervals; stimulation periods for peripheral tissues consisted either of 1 POP (pseudo-one-pulse; brief burst of 20 pulses/50 Hz) or of 2-4 POPs delivered at 1-s intervals. Single pulses or POPs were used to study the effect of medetomidine and its interaction with antagonists. One or more pulses or POPs per stimulation period were used to study alpha2-autoinhibition. Medetomidine decreased the evoked overflow of tritium in WT tissues. In alpha2 A/D(KO) tissues, the inhibition was slightly (peripheral tissues) or greatly (brain slices) attenuated but not abolished. Phentolamine, rauwolscine, spiroxatrine, 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), tolazoline and prazosin antagonized the effect of medetomidine in all tissues. Their pKd values against medetomidine were compared with pKd values at prototypical alpha2 binding sites by means of a correlation analysis. For WT brain and atrial autoreceptors, the correlations indicated an alpha2D pharmacology, whereas for WT vas deferens autoreceptors they favoured an alpha2B pharmacology. In the KO tissues, any correlation with alpha2D was lost, and the non-alpha2 A/D-autoreceptors displayed alpha2B or alpha2C pharmacology. When 2 or more pulses or POPs were applied to WT tissues per stimulation period, the pulse number-overflow curve (POP number-overflow curve) was flat, indicating that overflow elicited by p pulses (POPs) was much smaller than p times the overflow elicited by a single pulse (POP); moreover, rauwolscine caused a pulse (POP) number-dependent and, at high pulse (POP) numbers, large increase in evoked tritium overflow. In alpha2 A/D(KO) tissues, the pulse (POP) number-overflow curve was much steeper, indicating that overflow elicited by p pulses (POPs) was closer to p times the overflow elicited by a single pulse (POP); moreover, rauwolscine caused no (atria) or only a small increase in overflow, and did so in brain slices only at high pulse numbers (16 and 64). In conclusion, the predominant alpha2D pharmacology of the autoreceptors in WT tissues supports the idea that the main mammalian presynaptic alpha2-autoreceptors belong to the alpha2 A/D subtype. However, alpha2 A/D-deficient animals also possess autoreceptors. As expected, these non-alpha2 A/D-autoreceptors display alpha2B or alpha2C pharmacology. In WT animals, alpha2B- or alpha2C-autoreceptors or both may coexist with alpha2 A/D-autoreceptors, at least in peripheral tissues. Little autoinhibition by released noradrenaline in trains of pulses remains when the alpha2 A/D-adrenoceptor is lacking, again in accord with a predominance of alpha2 A/D-autoreceptors.     

The effects of PGE2 and PGF2α on rhythmic contractions of sphincter of Oddi

• 1.1. Spontaneous rhythmic activity observed in some of the guinea-pig sphincter of Oddi preparations was completely abolished by PGE₂ (10⁻⁸ M) but not altered by PGF_2α (10⁻⁶ M).
• 2.2. Indomethacin (10⁻⁵ M), a cyclooxygenase inhibitor, elicited long-lasting rhythmic contractions in 50% of the preparations tested, which did not show any spontaneous activity. PGE₂ (10⁻⁸ M) completely inhibited, however PGF_2α (10⁻⁸-10⁻⁶ M) did not change the rhythmic contractions induced by indomethacin.
• 3.3. Both initial phasic contraction and the frequency and amplitude of peristaltic waves induced by ACh (10⁻³ M) were increased by indomethacin (10⁻⁵ M), decreased by PGE₂ (10⁻⁷ M) and not altered by PGF_2α (10⁻⁷ M).

     

Characterization and in vitro dissolution behaviour of ketoconazole/β- and 2-hydroxypropyl-β-cyclodextrin inclusion compounds

The effect of β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin on the solubility of ketoconazole in different media were studied. A type A_L solubility diagram was obtained for ketoconazole and the two cyclodextrins in buffer solution, pH 5 and pH 6. The stability constants between ketoconazole and the two cyclodextrins were calculated from the phase solubility diagrams. Increased ionization of the imidazole derivative decreased the values of the stability constants. The formation of solid inclusion complexes were experimentally prepared by the kneading and spray-drying techniques. In order to confirm solid complex formation, X-ray diffractometry and differential scanning calorimetry were used. It was found that the spray-drying technique could be used to prepare the amorphous state of drug inclusion complexes. The dissolution rates of ketoconazole from the inclusion complex made by spray-drying were faster than the pure drug, kneading systems and the physical mixtures of drug and cyclodextrins. The enhanced dissolution rate of spray-dried products might be attributed to the decreased particle size, the high-energetic amorphous state and inclusion complex formation.     

Downregulation of NO and PGE2 in LPS-stimulated BV2 microglial cells by trans-isoferulic acid via suppression of PI3K/Akt-dependent NF-κB and activation of Nrf2-mediated HO-1

Little is known about whether trans-isoferulic acid (TIA) regulates the production of lipopolysaccharide (LPS)-induced proinflammatory mediators. Therefore, we examined the effect of TIA isolated from Clematis mandshurica on LPS-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in BV2 microglial cells. We found that TIA inhibited the production of LPS-induced NO and PGE₂ without accompanying cytotoxicity in BV2 microglial cells. TIA also downregulated the expression levels of specific regulatory genes such as inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) by suppressing LPS-induced NF-κB activity via dephosphorylation of PI3K/Akt. In addition, we demonstrated that a specific NF-κB inhibitor PDTC and a selective PI3K/Akt inhibitor, LY294002 effectively attenuated the expression of LPS-stimulated iNOS and COX-2 mRNA, while LY294002 suppressed LPS-induced NF-κB activity, suggesting that TIA attenuates the expression of these proinflammatory genes by suppressing PI3K/Akt-mediated NF-κB activity. Our results showed that TIA suppressed NO and PGE₂ production through the induction of nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent heme oxygenase-1 (HO-1). Taken together, our data indicate that TIA suppresses the production of proinflammatory mediators such as NO and PGE₂, as well as their regulatory genes, in LPS-stimulated BV2 microglial cells, by inhibiting PI3K/Akt-dependent NF-κB activity and enhancing Nrf2-mediated HO-1 expression.     

Effect of catecholamic acid on detoxication and distribution of NiCl_2 in mice and rats

目的：研究双酚胺酸（ＣＢＭＩＤＡ）对氯化镍的解毒作用．方法：ＮｉＣｌ２中毒后，立即给予ＣＢＭＩＤＡ，记录动物存活数；小鼠ｉｖ６３ＮｉＣｌ２后给药，测定２４ｈ组织中６３镍；用整体放射自显影术，显示小鼠体内６３镍分布．结果：ｓｃＣＢＭＩＤＡ０５－１５ｇ·ｋｇ－１对ｉｐＮｉＣｌ２５００ｍｇ·ｋｇ－１有解毒作用；小鼠ｉｐＮｉＣｌ２ＬＤ５０为８２８ｍｇ·ｋｇ－１，给药１５或２５ｇ·ｋｇ－１，ＬＤ５０分别为７８９和８２０ｍｇ·ｋｇ－１；大鼠ｉｍＣＢＭＩＤＡ５００ｍｇ·ｋｇ－１使ＮｉＣｌ２的ＬＤ５０提高８倍；组织中６３镍测定和定位显示，ＣＢＭＩＤＡ减少肺和血液中６３镍，增加了骨中６３镍，２４ｈ尿、粪６３镍排出与对照组无明显差异．结论：ＣＢＭＩＤＡ有效地解除镍毒性，提高动物存活率，降低镍在肺部的滞留．     

Cellular pharmacology of multi- and duplex drugsconsisting of ethynylcytidine and 5-fluoro-2′-deoxyuridine

Prodrugs can have the advantage over parent drugs in increased activation and cellular uptake. The multidrug ETC-L-FdUrd and the duplex drug ETC-FdUrd are composed of two different monophosphate-nucleosides, 5-fluoro-2′deoxyuridine (FdUrd) and ethynylcytidine (ETC), coupled via a glycerolipid or phosphodiester, respectively. The aim of the study was to determine cytotoxicity levels and mode of drug cleavage. Moreover, we determined whether a liposomal formulation of ETC-L-FdUrd would improve cytotoxic activity and/or cleavage. Drug effects/cleavage were studied with standard radioactivity assays, HPLC and LC-MS/MS in FM3A/0 mammary cancer cells and their FdUrd resistant variants FM3A/TK⁻. ETC-FdUrd was active (IC₅₀ of 2.2 and 79 nM) in FM3A/0 and TK⁻ cells, respectively. ETC-L-FdUrd was less active (IC₅₀: 7 nM in FM3A/0 vs 4500 nM in FM3A/TK⁻). Although the liposomal formulation was less active than ETC-L-FdUrd in FM3A/0 cells (IC₅₀:19.3 nM), resistance due to thymidine kinase (TK) deficiency was greatly reduced. The prodrugs inhibited thymidylate synthase (TS) in FM3A/0 cells (80–90%), but to a lower extent in FM3A/TK⁻ (10–50%). FdUMP was hardly detected in FM3A/TK⁻ cells. Inhibition of the transporters and nucleotidases/phosphatases resulted in a reduction of cytotoxicity of ETC-FdUrd, indicating that this drug was cleaved outside the cells to the monophosphates, which was verified by the presence of FdUrd and ETC in the medium. ETC-L-FdUrd and the liposomal formulation were neither affected by transporter nor nucleotidase/phosphatase inhibition, indicating circumvention of active transporters. In vivo, ETC-FdUrd and ETC-L-FdURd were orally active. ETC nucleotides accumulated in both tumor and liver tissues. These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation.     

Restoration of defective L-type Ca2+ current in cardiac myocytes of type 2 diabetic db/db mice by Akt and PKC-ι

Diabetes is associated with an increased risk of heart failure and the development of a cardiomyopathy whose etiology is only partially understood. Ca entry through the voltage-dependent L-type Ca channel CaV1.2 initiates the contractile cycle in cardiac myocytes. Decreased cardiac contractility and depressed CaV1.2 function have been reported in obese type 2 diabetic db/db mice. Here, we demonstrate that a reduction in phosphoinositide 3-kinase (PI3K) signaling is a major contributor to the altered function of CaV1.2 in db/db cardiac myocytes. Using the whole-cell patch clamp technique, we determined that intracellular infusion of cardiac myocytes from db/db mice with phosphatidylinositol 3,4,5-trisphosphate (PIP3), the second messenger produced by PI3K, increased the L-type Ca current (ICa,L) density nearly to the level seen in wild-type cells. PIP3 also reversed the positive shift in the voltage dependence of the steady-state current activation observed in db/db myocytes. Infusion of protein kinases that act downstream of PI3K also affected ICa,L. Akt1 and Akt2 were as effective as PIP3 in restoring the ICa,L density in db/db myocytes but did not affect the voltage dependence of current activation. The infusion of atypical PKC-ι (the human homolog of mouse PKC-λ) caused a small but significant increase in the ICa,L density and completely reversed the shift in voltage dependence of steady-state current activation. These results indicate that a defect in PI3K/PIP3/Akt/PKC-λ signaling is mainly responsible for the depressed CaV1.2 function in the hearts of db/db mice with type 2 diabetes.     

Single-dose tolerance and pharmacokinetics of 2-n-propyl-2(E)-pentenoate (Δ2(E)-valproate) in healthy male volunteers

2-n-Propyl-2(E)-pentenoic acid (^2(E)-valproate) was administered to healthy volunteers in oral doses of 50–800 mg. The drug was tolerated well and no significant adverse effects were observed. Pharmacokinetic parameters were determined. Valproate and 3-keto-valproate were detected as metabolites.     

Effects of β2-agonist- and dexamethasone-treatment on relaxation and regulation of β-adrenoceptors in human bronchi and lung tissue

1. Long-term treatment with β₂-adrenoceptor agonists can lead to a decreased therapeutic efficacy of bronchodilatation in patients with obstructive pulmonary disease. In order to examine whether or not this is due to β-adrenoceptor desensitization, human bronchial muscle relaxation was studied in isolated bronchial rings after pretreatment with β₂-adrenoceptor agonists. Additionally, the influence of pretreatment with dexamethasone on desensitization was studied.
2. The effect of β₂-agonist incubation alone and after coincubation with dexamethasone on density and affinity of β-adrenoceptors was investigated by radioligand binding experiments.
3. In human isolated bronchi, isoprenaline induces a time- and concentration-dependent β-adrenoceptor desensitization as judged from maximal reduction in potency by a factor of 7 and reduction of 73±4% in efficacy of isoprenaline to relax human bronchial smooth muscle.
4. After an incubation period of 60 min with 100 μmol l⁻¹ terbutaline, a significant decline in its relaxing efficacy (81±8%) and potency (by a factor 5.5) occurred.
5. Incubation with 30 μmol l⁻¹ isoprenaline for 60 min did not impair the maximal effect of a subsequent aminophylline response but led to an increase in potency (factor 4.4).
6. Coincubation of dexamethasone with isoprenaline (120 min; 30 μmol l⁻¹) preserved the effect of isoprenaline on relaxation (129±15%).
7. In radioligand binding experiments, pretreatment of lung tissue for 60 min with isoprenaline (30 μmol l⁻¹) resulted in a decrease in β-adrenoceptor binding sites (B_max) to 64±1.6% (P<0.05), while the antagonist affinity (K_D) for [³H]-CGP-12177 remained unchanged.
8. In contrast, radioligand binding studies on lung tissue pretreated with either dexamethasone (30 μmol l⁻¹) or isoprenaline (30 μmol l⁻¹) plus dexamethasone (30 μmol l⁻¹) for 120 min did not lead to a significant change of B_max (160±22.1% vs 142.3±28.7%) or K_D (5.0 nmol l⁻¹ vs 3.5 nmol l⁻¹) compared to the controls.
9. In conclusion, pretreatment of human bronchi with β-adrenoceptor agonists leads to functional desensitization and, in lung tissue, to down-regulation of β-adrenoceptors. This effect can be counteracted by additional administration of dexamethasone. Our model of desensitization has proved useful for the identification of mechanisms of β-adrenoceptor desensitization and could be relevant for the evaluation of therapeutic strategies to counteract undesirable effects of long-term β-adrenoceptor stimulation.

     

Determination of tramadol and metabolites by HPLC-FL and HPLC–MS/MS in urine of dogs

Tramadol is a centrally acting analgesic drug used in veterinary and human clinical practice. Its metabolism has been largely characterized in human being but is still long to be comprehended in several animal species, especially in the dog. The aim of the present study was to develop and validate a new analytical procedure to investigate HPLC the metabolization/elimination process tramadol in urine of dogs by HPLC-FL or HPLC–MS/MS. A single oral dose of tramadol (4 mg/kg) was administered to 4 male Beagle dogs and the urine was naturally collected. This matrix either hydrolyzed than un-hydrolyzed was extracted with different blends of solvents to detect the total or free form of the analytes, respectively.     

β1 and β2 Stimulatory effects of prenalterol

Summary Prenalterol, previously characterized as a functionally cardioselective partial -adrenoceptor agonist, was shown to relax K⁺-elicited contractures in the uterine muscle from progesterone pretreated rats (pD ₂ 7.7) and to increase beating rate in the rat right atrium (pD ₂ 8.0) at about the same concentrations with maximal effects corresponding to 94 and 82% respectively of those of isoproterenol. Terbutaline, with equal maximal effects as isoproterenol, was 50 times more potent in the uterus (pD ₂ 7.8) than in the right atrium (pD ₂ 6.1). Both tissues displayed a high sensitivity to isoproterenol (pD ₂ 9.1 in both tisues) indicating large receptor reserves for the full agonist.The maximal relaxing effect of prenalterol in the uterus was obtained at about a three-fold increase of the cyclic AMP content, which is similar to that obtained with isoproterenol at a corresponding relaxation.The effects in the uterine muscle of all three agonists were mediated through ₂-adrenoceptors since ₂-adrenoceptor blockers (ICI 118,551 and IPS 339) antagonized the effects in concentrations which had only marginal effects on the atrial responses of the agonists. The ₁-antagonists pafenolol and pamatolol in concentrations higher than those, which blocked the effects of the agonists on beating rate, were devoid of inhibitory effects in the uterus.These results indicate that prenalterol possesses the ability to elicit a functional response by stimulation of either ₁- or adrenoceptors provided that the tissue has a large spare receptor reserve for full agonists.     

Sp-2-propylthio-ATP-α-B and Sp-2-propylthio-ATP-α-B,β-γ-dichloromethylene are novel potent and specific agonists of the human P2Y11 receptor

The human P2Y₁₁ nucleotide receptor mRNA was found in virtually all human tissues, and the receptor serves many physiological roles, such as immune response regulation. The Ala-87-Thr-P2Y₁₁ receptor single nucleotide polymorphism was linked to increased risk for acute myocardial infarction. To facilitate the development of new therapeutic applications involving cells expressing several P2 receptor subtypes, the availability of specific and potent agonists is mandatory. Here, we synthesized a series of novel adenine nucleotide derivatives, based upon the potent P2Y₁₁ receptor agonists AR-C67085. Features of the novel nucleotide derivatives are a propylthio substitution at C2-adenine and a Pα-borano or Pα-thio substitution of non-bridging oxygen atom. The latter substitutions introduce a chiral center at the α-phosphate. Sp-isomers of Pα-borano- and Rp-isomers of Pα-thio-substituted nucleotides are preferred by the P2Y₁₁ receptor. As recently reported by us, diastereoselectivity of the P2Y₁₁ receptor is opposite to that of the P2Y₁ receptor. Therefore, we exploit this characteristic to increase nucleotide selectivity. At the P2Y₁₁ receptor, the Sp-isomers of 2-propylthio-ATP-α-B (2B) and 2-propylthio-ATP-α-B,β-γ-dichloromethylene (4B) were the most potent of the novel nucleotide series, with EC₅₀ values of 0.03 μM for both, being ca. 80-fold more potent than 2-propylthio-ATP and ATP (EC₅₀ = 2.6 μM). We conclude that the borano-substitution at the α-phosphate of 2-propylthio-ATP enhances nucleotide potency at the P2Y₁₁ receptor. The combination with a Pβ-Pγ-dichloromethylene group in 4B results in a nucleotide, which shows higher selectivity for the P2Y₁₁ receptor over the P2Y₁ receptor than 2B making it the most promising of the novel P2Y₁₁ receptor agonists.     

Effects of Sr2+ and Mg2+ on the phospholipase A and the presynaptic neuromuscular blocking actions of β-bungarotoxin,crotoxin and taipoxin

1.beta-Bungarotoxin, crotoxin and taipoxin, presynaptic neurotoxins of snake venom origin, have about the same phospholipid-splitting activities as a much less toxic cobra phospholipase A2 in the presence of Ca2+ and deoxycholate. 2. Sr2+ was a much less effective activator of the enzymes than is Ca2+, the activation by Sr2+ being only 3-6% for beta-bungarotoxin and crotoxin and 12% for taipoxin. 3. Sr2+ also inhibited the Ca2+ -activated enzymes by 80% in the cases of beta-bungarotoxin and crotoxin, but only 16% in the case of taipoxin. 4. Mg2" had no significant effect on beta-bungarotoxin or crotoxin, but activated taipoxin in the presence or absence of Ca2". 5. In Sr2+ -Tyrode lacking Ca2+ all three toxins exhibited the same immediate depression followed by facilitation in the rat and mouse diaphragms, but the final blocking activity was only 3-10% with beta-bungarotoxin and crotoxin and was 30% with taipoxin. 6. In Sr2+ -Tyrode, increasing in the rate of nerve stimulation had less accelerating effect on the development of neuromuscular block than in Ca2+ -Tyrode for any of the toxins. 7. Removal of Mg2+ from Sr2+ -Tyrode did not diminish the potency of taipoxin in blocking neuromuscular transmission, suggesting that enzyme activity at the outer surface of the axolemma does not contribute to the neuromuscular blocking action. 8. All of the results indicate that there are close correlations between the presynaptic activities of these toxins and their phospholipid-splitting activities in the cationic environment prevailing in the axoplasm. Apparently the final blocking effect of these toxins is due to phospholipase A action within the nerve terminal.     

Role of the soluble guanylyl cyclase α1/α2 subunits in the relaxant effect of CO and CORM-2 in murine gastric fundus

Carbon monoxide (CO) has been shown to cause enteric smooth muscle relaxation by activating soluble guanylyl cyclase (sGC). In gastric fundus, the sGCα₁β₁ heterodimer is believed to be the most important isoform. The aim of our study was to investigate the role of the sGCα₁/α₂ subunits in the relaxant effect of CO and CORM-2 in murine gastric fundus using wild-type (WT) and sGCα₁ knock-out (KO) mice. In WT mice, CO (bolus)-induced relaxations were abolished by the sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), while CORM-2- and CO (infusion)-induced relaxations were only partially inhibited by ODQ. In sGCα₁ KO mice, relaxant responses to CO and CORM-2 were significantly reduced when compared with WT mice, but ODQ still had an inhibitory effect. The sGC sensitizer 1-benzyl-3-(5′-hydroxymethyl-2′-furyl-)-indazol (YC-1) was able to potentiate CO- and CORM-2-induced relaxations in WT mice but lost this potentiating effect in sGCα₁ KO mice. Both in WT and sGCα₁ KO mice, CO-evoked relaxations were associated with a significant cGMP increase; however, basal and CO-elicited cGMP levels were markedly lower in sGCα₁ KO mice. These data indicate that besides the predominant sGCα₁β₁ isoform, also the less abundantly expressed sGCα₂β₁ isoform plays an important role in the relaxant effect of CO in murine gastric fundus; however, the sGC stimulator YC-1 loses its potentiating effect towards CO in sGCα₁ KO mice. Prolonged administration of CO—either by the addition of CORM-2 or by continuous infusion of CO—mediates gastric fundus relaxation in both a sGC-dependent and sGC-independent manner.