Free Krebs-Henseleit Solution Research Articles

Investigating the mechanisms underlying U46619-induced contraction on porcine lower esophageal sphincter.

Gastroesophageal reflux disease (GERD) is associated with an incompetent lower esophageal sphincter (LES), resulting in the reflux of gastric contents into the esophagus. U46619, a thromboxane A2 (TXA2) receptor agonist, induces contractions in various smooth muscles. Therefore, this study aimed to investigate the effects and mechanisms of action of U46619 on the porcine LES. To achieve this, contractions of the clasp and sling strips of the porcine LES, induced by U46619 were measured using isometric transducers. Furthermore, the contractile mechanism of U46619 in the porcine LES was investigated by pretreating the strips with atropine (a muscarinic receptor antagonist), tetrodotoxin (a neuronal sodium channel blocker), nifedipine (a calcium channel blocker), and Ca2+-free Krebs-Henseleit solution. Additionally, reverse transcription polymerase chain reaction and immunohistochemistry (IHC) were performed to determine the presence of the TXA2 receptor in porcine LES. The results of this study demonstrated that U46619 caused marked concentration-dependent contractions in both porcine sling and clasp strips. The mechanism of U46619-induced contraction of the porcine LES was found to be related to calcium channels. Furthermore, the reverse transcription PCR analysis and IHC revealed that the TXA2 receptor was expressed in the clasp and sling fibers of porcine LES. Consequently, this study suggests that U46619 mediates the contraction of porcine LES through calcium channels and has potential as a therapeutic approach for treating GERD. Significance Statement This study establishes that U46619 induces concentration-dependent contractions in porcine LES, primarily mediated by calcium channels. The presence of the TXA2 receptor in LES clasp and sling fibers is confirmed. These findings highlight U46619's potential as a GERD therapeutic by targeting calcium channels for LES contraction modulation.

Salvia miltiorrhiza Induces Tonic Contraction of the Lower Esophageal Sphincter in Rats via Activation of Extracellular Ca2+ Influx.

Up to 40% of patients with gastroesophageal reflux disease (GERD) suffer from proton pump inhibitor refractory GERD but clinically the medications to strengthen the lower esophageal sphincter (LES) to avoid irritating reflux are few in number. This study aimed to examine whether Salvia miltiorrhiza (SM) extracts induce tonic contraction of rat LES ex vivo and elucidate the underlying mechanisms. To investigate the mechanism underlying the SM extract-induced contractile effects, rats were pretreated with atropine (a muscarinic receptor antagonist), tetrodotoxin (a sodium channel blocker), nifedipine (a calcium channel blocker), and Ca2+-free Krebs-Henseleit solution with ethylene glycol tetraacetic acid (EGTA), followed by administration of cumulative dosages of SM extracts. SM extracts induced dose-related tonic contraction of the LES, which was unaffected by tetrodotoxin, atropine, or nifedipine. However, the SM extract-induced LES contraction was significantly inhibited by Ca2+-free Krebs-Henseleit solution with EGTA. Next, SM extracts significantly induce extracellular Ca2+ entry into primary LES cells in addition to intracellular Ca2+ release and in a dose-response manner. Confocal fluorescence microscopy showed that the SM extracts consistently induced significant extracellular Ca2+influx into primary LES cells in a time-dependent manner. In conclusion, SM extracts could induce tonic contraction of LES mainly through the extracellular Ca2+ influx pathway.

Vasorelaxant Effect of Osterici Radix Ethanol Extract on Rat Aortic Rings

The root of Ostericum koreanum Maximowicz has been used as a traditional medicine called “Kanghwal” in Korea (or “Qianghuo” in China). The purpose of this study was to investigate the vasorelaxant activity and mechanism of action of an ethanol extract of the O. koreanum root (EOK). We used isolated rat aortic rings to assess the effects of EOK on various vasorelaxant or vasoconstriction factors. EOK induced vasorelaxation in phenylephrine hydrochloride (PE) or KCl precontracted aortic rings in a concentration-dependent manner. However, the vasorelaxant effects of EOK on endothelium-intact aortic rings were reduced by pretreatment with L-NAME or methylene blue. In Ca2+-free Krebs-Henseleit solution, pretreatment with EOK (0.3 mg/mL) completely inhibited PE-induced constriction. In addition, EOK (0.3 mg/mL) also completely inhibited vasoconstriction induced by supplemental Ca2+ in aortic rings that were precontracted with PE or KCl. Furthermore, the EOK-induced vasorelaxation in PE-contracted aortic rings was inhibited by preincubation with nifedipine. These results indicate that the vasorelaxant effects of EOK are responsible for the induction of NO formation from L-Arg and NO-cGMP pathways, blockage of the extracellular Ca2+ entry via the receptor-operative Ca2+ channel and voltage-dependent calcium channel, and blockage of sarcoplasmic reticulum Ca2+ release via the inositol triphosphate pathway.

Zero flow global ischemia-induced injuries in rat heart are attenuated by natural honey.

In the present study, effects of preischemic administration of natural honey on cardiac arrhythmias and myocardial infarction size during zero flow global ischemia were investigated in isolated rat heart. The isolated hearts were subjected to 30 min zero flow global ischemia followed by 120 min reperfusion then perfused by a modified drug free Krebs-Henseleit solution throughout the experiment (control) or the solution containing 0.25, 0.5, 1 and 2% of natural honey for 15 min before induction of global ischemia (treated groups), respectively. Cardiac arrhythmias were determined based on the Lambeth conventions and the infarct size was measured by computerized planimetry. Myocardial infarction size was 55.8±7.8% in the control group, while preischemic perfusion of honey (0.25, 0.5, 1 and 2%) reduced it to 39.3±11, 30.6±5.5 (P<0.01), 17.9±5.6 (P<0.001) and 8.7±1.1% (P<0.001), respectively. A direct linear correlation between honey concentrations and infarction size reduction was observed (R(2)=0.9948). In addition, total number of ventricular ectopic beats were significantly decreased by all used concentrations of honey (P<0.05) during reperfusion time. Honey (0.25, 0.5 and 1 %) also lowered incidence of irreversible ventricular fibrillation (P<0.05). Moreover, number and duration of ventricular tachycardia were reduced in all honey treated groups. Preischemic administration of natural honey before zero flow global ischemia can protect isolated rat heart against ischemia/reperfusion injuries as reduction of infarction size and arrhythmias. Maybe, antioxidant and free radical scavenging activities of honey, reduction of necrotized tissue and providing energy sources may involve in these cardioprotective effects of honey.

Acute administration of natural honey protects isolated heart in normothermic ischemia.

This study intended to assess the efficacy of acute administration of natural honey on cardiac arrhythmias and infarct size when it is used during the normothermic ischemia in isolated rat heart. During 30 min of regional normothermic ischemia followed by 120 min of reperfusion, the isolated hearts were perfused by a modified drug free Krebs-Henseleit solution (control) or the solution containing 0.125, 0.25, 0.5 and 1% of freshly prepared natural honey (test groups), respectively. Cardiac arrhythmias were analyzed and determined through the recorded ECGs. The infarct size was measured using computerized planimetry package. At the ischemic phase, honey (0.25 and 0.5%) decreased the number and duration of ventricular tachycardia (VT), total number of ventricular ectopic beats (VEBs), duration and incidence of reversible ventricular fibrillation (VF) and total VF (p < 0.05 for all). During the reperfusion, concentrations of 0.125, 0.25 and 0.5% lowered the number of VT (p < 0.05), duration of reversible VF (p < 0.01) and total number of VEBs (p < 0.05). In addition, VT duration was reduced significantly with honey 0.125 and 0.25%. Moreover, the infarct size was 45.6 ± 3.4% in the control group, while the perfusion of honey (0.125, 0.25 and 0.5%) reduced it to 14.8 ± 5.1 (p < 0.001), 24.6 ± 7.3 (p < 0.01) and 31.4 ± 7.3% (p < 0.05), respectively. Regarding the results, it is concluded that the acute administration of natural honey in normothermic ischemia conditions can protect the rat heart as the reduction of infarct size and arrhythmias. Conceivably, the antioxidant and free radical scavenging activity, the reduction of necrotized tissue and the providence of rich energy source are more important mechanisms in cardioprotective effects of natural honey.

The Effect Of Ethanol On Tracheal Smooth Muscle Of Cat

Previously we have shown that ethanol induces contraction of pulmonary artery smooth muscle (PASM). However, the effect of ethanol on tracheal smooth muscle (TSM) and the mechanisms involved are not well understood. Therefore, we hypothesized that ethanol induces contractile responses of TSM and extra cellular Ca2+ is involved to this effect. In vitro force measurements were performed in TSM strips, excised from cats (n=29). Tissues were stabilized in Krebs‐Henseleit (KH) solution and treated with different concentrations of ethanol (46 – 276 mM). The role of extra cellular Ca2+ was studied by incubation of tissue in a Ca2+ free KH solution or using a Ca2+ channel blocker‐nifedipine. The role of nitric oxide (NO) was assessed by inhibition of NO synthase (NOS) using Nω‐nitro‐L‐arginine methyl ester. The ethanol induced concentration‐dependent contraction of TSM. Lack of Ca2+ in medium resulted with decrease of TSM tone likewise Ca2+ channel blocker. Inhibition of NOS, significantly increased contractile responses of TSM to the ethanol at lower ad middle concentrations, but reduced these responses at higher concentrations. In addition, inhibition of NOS significantly potentiated TSM tone to ACh.These data demonstrate that ethanol induces TSM contraction of cat and extra cellular Ca2+ is an important involved factor. NO release as a relaxant molecule counteracts the effect of ethanol and other agonists.

Selective antagonism activity of alkaloids from bulbs Fritillariae at muscarinic receptors: Functional studies

15 alkaloids were isolated from five Fritillariae species and 6 derivatives were synthesized. Alkaloids having anticholinergic effect on guinea-pig tracheal smooth muscle were screened out and their mechanism was further studied on the cAMP formation in Chinese hamster ovary cells stably expressing human muscarinic M2 receptor (CHO-hM2 cells) and intracellular calcium ([Ca 2+] i) transient in Chinese hamster ovary cells stably expressing human muscarinic M3 receptor (CHO-hM3 cells). In normal Krebs–Henseleit (KH) solution, imperialine ( 15), 3 β-acetylimperialine ( 16) and sinpeinine A ( 17) concentration-dependently relaxed 1 μM carbachol-induced contraction of guinea-pig tracheal rings with EC 50 of 4.19, 1.71 and 4.67 μM, respectively. In Ca 2+-free KH solution, 10 μM 3 β-acetylimperialine ( 16), imperialine ( 15) and sinpeinnine A ( 17) caused 97.42%, 5.45% and 6.55% inhibition, respectively, which indicated that the three components might inhibit muscarinic receptor in different mechanism. Results of muscarinic M2 receptor-inhibited cAMP formation in CHO-hM2 cells showed that imperialine ( 15) and sinpeinine A ( 17) could potently elevate the cAMP formation whereas 3 β-acetylimperialine ( 16) only had weak effect on antagonism of cAMP inhibition. Furthermore, the investigations of muscarinic M3 receptor-stimulated [Ca 2+] i transient in CHO-hM3 cells revealed that imperialine ( 15) and sinpeinine A ( 17) could not antagonize [Ca 2+] i transient, but 3 β-acetylimperialine ( 16) significantly inhibited [Ca 2+] i peak elevation with an IC 50 of 5.26 μM. The functional studies suggest that the mechanism of relaxant action of imperialine ( 15) and sinpeinine A ( 17) is due to their selective inhibitory effects on muscarinic M2 receptors and the mechanism of 3 β-acetylimperialine ( 16) originates from its selective muscarinic M3 receptors antagonism.

Contractile effects of angiotensin peptides in rat aorta are differentially dependent on tyrosine kinase activity.

It has been suggested that tyrosine kinase activity participates in the regulation of signal transduction associated with angiotensin II (Ang II)-induced pharmaco-mechanical coupling in rat aortic smooth muscle. We further tested the effects of genistein, a tyrosine-kinase inhibitor, and its inactive analogue, daidzein, on angiotensin I (Ang I), angiotensin III (Ang III) and angiotensin IV (Ang IV) contractions, as compared with those on Ang II. Genistein partially inhibited Ang II- and Ang I-induced contractions. The genistein-induced inhibition was more evident on Ang III and especially important on Ang IV contractile effects. Thus, Ang IV- and Ang III-induced contractions seem to be more dependent on tyrosine kinase activity than those evoked by Ang II or Ang I. Daidzein did not significantly affect the contractile effects of any of angiotensin peptides tested. These results clearly suggest that the inhibition of the action of angiotensin peptides actions by genistein is mediated by inhibition of endogenous tyrosine kinase activity. Furthermore, our data show that the type and/or intensity of tyrosine kinase activity is differentially associated with the contractile effects of different angiotensin peptides in rat aorta. Nifedipine, a blocker of membrane L-type Ca2+ channels, strongly inhibited Ang IV-induced contractions. At the same time, it significantly inhibited Ang III contractile effects as compared with Ang II and Ang I contractions. Meanwhile, we observed a close relationship between calcium influx and tyrosine kinase phosphorylation activity under the stimulatory effects of angiotensin peptides. Furthermore, genistein did not significantly influence the phasic contractions induced by angiotensin peptides in Ca2+-free Krebs-Henseleit solution. Thus, it appears that Ca2+ influx, rather than the release of Ca2+ from IP3-sensitive stores, may play a major role in the contractile effects of angiotensin peptides in rat aorta via tyrosine kinase activation. One argument against a direct action of genistein on the Ca2+ channel itself is that it did not markedly affect the K+-induced contraction (depolarisation) in rat aorta. At the same time, a potential role for tyrosine kinase activity in the process of calcium entry is suggested. An elevation of intracellular calcium via tyrosine kinase-mediated processes may mediate the actions of G-protein coupled receptor agonists in smooth muscle, including angiotensin peptides.

Purinoceptor-stimulated phosphoinositide hydrolysis in Madin-Darby canine kidney (MDCK) cells.

Extracellular nucleotides, acting through P2-purinoceptors, have been implicated in the regulation of ion transport in epithelia, including Madin-Darby canine kidney (MDCK) cells. In this study, experiments were conducted to characterize the P2-purinoceptor subtype on MDCK cells responsible for stimulating inositol phosphate (IP) accumulation using a range of nucleotide analogues. In Ca2+- and Mg2+-free Krebs-Henseleit solution (KHS), ATP, UTP, and ATPgammaS caused an increase in IP accumulation as a function of concentration with comparable kinetics. The order of potency for the nucleotide analogues was UTP = ATPgammaS > ATP = 2-chloro ATP (Cl-ATP) >> alpha,beta-methylene ATP (alpha,beta-MeATP) = 2-methylthio ATP (2MeSATP). Selective agonists for P1-, P2X- and P2Y-purinoceptors, such as N6-cyclopentyl adenosine, AMP, alpha,beta-MeATP, and 2MeSATP, had little effect. Stimulation of MDCK cells with maximally effective concentrations of ATP and UTP showed no additive effect and furthermore, ATP, UTP, and ATPgammaS induced cross-desensitization of the IP response, suggesting that ATP and UTP act upon a common nucleotide receptor, i.e. a P2U-purinoceptor. In Ca2+- and Mg2+-containing KHS, the concentration-response curves of ATP, UTP, and ATPgammaS were shifted to the right of those obtained in Ca2+- and Mg2+-free buffer, and asymptotic maxima were not reached, indicating that ATP4- and not MgATP2- or CaATP2- was the active agonist. Pretreatment of MDCK cells with pertussis toxin (PTX) inhibited ATP- and UTP-induced IP accumulation in a concentration-dependent fashion but did not completely abolish the IP accumulation, indicating that a PTX-sensitive G protein was partially involved in the IP response. In conclusion, ATP- and UTP-stimulated IP accumulation in MDCK cells appears to be mediated through the activation of P2U-purinoceptors coupled to a G protein that is partially sensitive to PTX. A form of nucleotide uncomplexed with divalent ions such as ATP4- seems to be the preferential agonist form for the purinoceptors on MDCK cells.

Presynaptic site of action underlying the ethanol-induced inhibition of norepinephrine release evoked by stimulation of N-methyl- d-aspartate (NMDA) receptors in rat cerebral cortex

Rat brain cortex synaptosomes pre-incubated with [ 3H]norepinephrine were used (1) to provide evidence that part of the NMDA receptors mediating stimulation of norepinephrine (NE) release are located on the noradrenergic varicosities themselves, (2) to characterize these receptors and (3) to examine whether ethanol specifically inhibits the NMDA-evoked NE release via a presynaptic site of action. In synaptosomes superfused with Mg 2+-free Krebs-Henseleit solution, NMDA (2-min exposur) stimulated tritium overflow in a concentration- and glycine-dependent manner. The stimulatory effect of NMDA was not altered by tetrodotoxin but was abolished by omission of Ca 2+ from the superfusion fluid and was considerably reduced in the presence of 1.2 mM Mg 2+. dl-(E)-2-Amino-4-methyl-5-phosphono-3-pentanoic acid (CGP 37849; a competitive NMDA receptor antagonist) produced a parallel shift of the concentration-response curve for NMDA to the right, whereas dizocilpine (MK-801; an antagonist at the phenyclidine, PCP, recognition site of the NMDA-gated ion channel) reduced the maximum effect of NMDA. Ethanol inhibited the NMDA-evoked tritium overflow in a concentration-dependent manner. In contrast, in synaptosomes superfused with Ca 2+-free Krebs-Henseleit solution containing 15 mM K + throughout, ethanol did not affect the tritium overflow evoked by 2 min introduction of 75 μM Ca 2+ into the superfusion fluid. This Ca 2+ -evoked overflow was also not altered by tetrodotoxin and dizocilpine, but was inhibited by the inorganic Ca 2+ channel antagonist Cd 2+. Three main conclusions can be drawn: (1) NMDA receptors mediating stimulation of NE release are also located on the cortical noradrenergic varicosities (and not only on so far unknown excitatory interneurones within the cortex); (2) these receptors exhibit the characteristic pharmacological features of the NMDA receptor system; (3) ethanol selectively inhibits the NE release evoked by stimulation of the presynaptic NMDA receptors, leaving the Ca 2+-evoked release promoted by high K + unaffected. This finding is compatible with the suggestions that the NMDA receptor system itself is a site of action of ethanol.

Effects of Na+, K(+)-pump inhibitors on acetylcholine-induced relaxation in the rabbit aorta.

The purpose of this study was to investigate the effects of inhibitors of the Na+, K(+)-pump and membrane depolarizing agents on endothelium-dependent acetylcholine-induced relaxation in the rabbit thoracic aorta. Aortic rings were prepared from the rabbit descending thoracic aorta and the contractility of the ring was measured in various conditions such as application of ouabain, exposure to K(+)-free Krebs-Henseleit solution and high K+. Ouabain or exposure to K(+)-free Krebs-Henseleit solution inhibited acetylcholine or sodium nitroprusside-induced relaxation. KCl also inhibited the acetylcholine or sodium nitroprusside-induced relaxation. These results suggest that the Na+, K(+)-pump may play a role in endothelium-dependent acetylcholine-induced relaxation.

Ethanol inhibits the N-methyl-D-aspartate (NMDA)-induced attenuation of the NMDA-evoked noradrenaline release in the rat brain cortex: interaction with NMDA-induced desensitization

The influence of ethanol, AP5 (DL-2-amino-5-phosphonopentanoic acid) and dizocilpine (MK-801) ((+)-5-methyl-10,11-dihydro-5H-dibenzo (a,b)-cyclo-hept-5,10-imine hydrogen maleate) on the NMDA-induced attenuation of the NMDA-evoked noradrenaline release was examined in rat brain cortex slices preincubated with 3H-noradrenaline. The slices were superfused with Mg(2+)-free Krebs-Henseleit solution. Tritium overflow (corresponding to 3H-noradrenaline release) was stimulated by 300 mumol/l NMDA for 2 min. Presence of 10-100 mumol/l NMDA from 20 to 2 min before stimulation concentration-dependently inhibited the NMDA (300 mumol/l)-evoked 3H overflow, suggesting an agonist-induced desensitization attributable to the modification of events at the NMDA receptor itself and/or distal to this receptor system. The desensitizing effect of 100 mumol/l NMDA was almost complete and was not diminished when the time of preexposure was decreased to 10 min, and when NMDA was removed from the superfusion fluid for up to 5 min before the stimulus; however, the desensitizing effect was reduced after further decrease in the duration of preexposure to NMDA or further prolongation of the interval between preexposure and stimulation. Ethanol inhibited the NMDA-induced 3H overflow (IC50 45 mmol/l); this effect was almost abolished when ethanol was omitted from the superfusion fluid from 2 min before stimulation onward. Ethanol, when simultaneously present with 100 mumol/l NMDA in the superfusion fluid from 20 to 2 min before the NMDA stimulus, concentration-dependently (IC50 112 mmol/l) decreased the inhibitory effect of NMDA.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of increased responses of the aorta to .ALPHA.-adrenoceptor agonists in streptozotocin-induced diabetic rats.

To investigate the influence of diabetes on the responsiveness of the cardiovascular system, we have examined the effects of various agents on the reactivity of the vascular smooth muscle of aortic strips obtained from age-matched control and diabetic rats. Norepinephrine (NE) contracted the aortic strips obtained from age-matched control and diabetic rats in a concentration-dependent manner, but maximal contraction of aortic strips in response to NE was enhanced in diabetic rats. The EC50 value for NE in the diabetic aortic strips was similar to that in the aorta from age-matched controls. Ca-induced contracture of the aortic strips which were depolarized with isotonic K+ (60 mM) was potentiated in aortic strips from diabetic rats, when compared with those from age-matched controls. Ca-induced contracture of aortic strips, preincubated with 10(-6) M NE and 10(-6) M nicardipine in Ca2+-free Krebs-Henseleit solution (KHS), was not significantly different in age-matched control and diabetic rats. Bay K 8644, an activator of calcium channels, produced an increase in the force of contraction of the slightly depolarized aorta from diabetic rats. Phasic contraction induced by phenylephrine (PE) in the presence of 10(-6) M nicardipine in Ca2+-free KHS was significantly enhanced in aortic strips obtained from diabetic rats. These results demonstrate that NE-induced contraction of the aortic strips obtained from diabetic rats was significantly enhanced, and that this increased contractile response of the aorta to NE may be due to an increased influx of extracellular calcium through the voltage-dependent Ca2+ channels, but not through the receptor-operated Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of calcium in arachidonic acid-induced contractions of guinea pig airways

We have previously shown that arachidonic acid (AA)-induced contractions of indomethacin-pretreated guinea pig trachea and parenchyma are due to the synthesis of leukotrienes C 4 and D 4. The present experiments were designed to investigate the role of calcium (Ca 2+) in the above. AA (66 μM)-induced contractions of trachea, but not parenchyma, were reduced in Ca 2+-free Krebs-Henseleit solution (KHS). However the contractions of both trachea and parenchyma were abolished in Ca 2+-free KHS with either lanthanum chloride (1 mM) or EDTA (300 μM). The Ca 2+ antagonists, verapamil (100 μM), nitrendipine (100 μM), and TMB-8 (100 μM), reduced AA-induced contractions of both trachea and parenchyma. Re-addition of Ca 2+ (2.2 mM) to trachea and parenchyma in Ca 2+-free KHS in the presence of lanthanum restored the AA-induced contractions. This effect of Ca 2+ was reduced by verapamil (100 μM) or nitrendipine (100 μM). LTC 4-induced contractions of trachea and parenchyma were unaffected by nitrendipine (100 μM), whereas tracheal contractions were reduced in Ca 2+-free KHS. Both tracheal and parenchymal contractions to LTC 4 were reduced in Ca 2+-free KHS in the presence of lanthanum chloride (1 mM). We conclude that superficially bound pools of Ca 2+ are important in AA-induced contractions of the airways. Furthermore, nitrendipine reduces AA-induced contractions by inhibiting AA metabolism and not by inhibiting airway smooth muscle contraction induced by released leukotrienes.

On aminophenazone metabolism in the isolated perfused rat liver

Isolated rat livers were perfused with recycling protein free Krebs-Henseleit solution for 80 min. Aminophenazone (0.87 mM, 1.7 mM), 4-methylaminoantipyrine (0.86 mM), 4-aminoantipyrine (1.6 mM), or 4-acetylaminoantipyrine (0.86 mM) was added to the perfusion medium.