Acetylcholine Concentration-response Curve Research Articles

Hydroxyurea does not reverse functional alterations of the nitric oxide-cGMP pathway associated with priapism phenotype in corpus cavernosum from sickle cell mouse.

Sickle cell disease (SCD) is a genetic disorder that has been associated with priapism. The role of hydroxyurea, a common SCD therapy, in influencing the nitric oxide (NO)-cGMP pathway and its effect on priapism is unclear. To investigate the effect of hydroxyurea treatment on smooth muscle relaxation of corpus cavernosum induced by stimulation of the NO-cGMP pathway in SCD transgenic mice and endothelial NO synthase gene-deficient (eNOS-/-) mice, which are used as model of priapism associated with the low bioavailability of endothelial NO. Four-month-old wild-type (WT, C57BL/6), SCD transgenic, and eNOS-/- male mice were treated with hydroxyurea (100 mg/Kg/day) or its vehicle (saline) daily for three weeks via intraperitoneal injections. Concentration-response curves for acetylcholine (ACh), sodium nitroprusside (SNP), and electrical field stimulation (EFS) were generated using strips of mice corpus cavernosum. The SCD mice demonstrated an amplified CC relaxation response triggered by ACh, EFS, and SNP. The corpus cavernosum relaxation responses to SNP and EFS were found to be heightened in the eNOS-/- group. However, the hydroxyurea treatment did not alter these escalated relaxation responses to ACh, EFS, and SNP in the corpus cavernosum of the SCD group, nor the relaxation responses to EFS and SNP in the eNOS-/- group. In conclusion, hydroxyurea is not effective in treating priapism associated with SCD. It is likely that excess plasma hemoglobin and reactive oxygen species, which are reported in SCD, are reacting with NO before it binds to GCs in the smooth muscle of the corpus cavernosum, thus preventing the restoration of baseline NO/cGMP levels. Furthermore, the downregulation of eNOS in the penis may impair the pharmacological action of hydroxyurea at the endothelial level in SCD mice. This study emphasize the urgency for exploring alternative therapeutic avenues for priapism in SCD that are not hindered by high plasma hemoglobin and ROS levels.

Pharmacologycal activity of peperina (Minthostachys verticillata) on gastrointestinal tract

Ethnopharmacological relevanceMinthostachys verticillata (Griseb.) Epling (Lamiaceae), known as Peperina is a medicinal native plant, with a traditional use as a digestive, antispasmodic and antidiarrheic. Aim Of The StudyDespite its folkloric use, no scientific evaluation of this plant related to the gastrointestinal inflammatory process has been carried out to date. The present study aims to assess the effects of M. verticillata on gastrointestinal system in experimental models. Materials and methodsM. verticillata (250 and 500 mg/kg) was orally tested in a colitis model induced by acetic acid. Colon weight/length ratio, oxidative stress (oxidized and reduced glutathione), histological changes using Alcian blue and hematoxylin & eosin staining and expression of IL1β, TNFα, iNOS, COX-2 were evaluated. The effect of the extract in three additional in vivo models were studied: intestinal motility and diarrhea induced by ricin oil, and visceral pain induced by intracolonic administration of capsaicin. Finally, the activity on concentration response curves of acetylcholine, calcium chloride, potassium and serotonin were achieved in isolated rat jejunum. ResultsIn the colitis model, M. verticillata induced a significant reduction in the colon weight/length ratio, oxidative stress and expression levels of IL-1β, iNOS and COX-2. Also, the extract diminished the severity of microscopic tissue damage and showed protective effect on goblet cells. Intestinal motility, diarrhea, visceral pain-related behaviors and referred hyperalgesia were significantly reduced when the animals were treated with the extract. Furthermore, in isolated jejunum, M. verticillata significantly reduced the contraction induced by serotonin and acetylcholine. Likewise, the extract non-competitively inhibited the response-concentration induced by CaCl2 and inhibited both low and high K+-induced contractions. ConclusionsThis is the first study to validate traditional use of M. verticillata for digestive disorders and demonstrated that its aqueous extract could represent a promising strategy in targeting the multifactorial pathophysiology of inflammatory bowel disease.

High concentration of uric acid failed to affect endothelial function of small mesenteric arteries, femoral arteries and aortas from aged Wistar-Kyoto rats.

It is known that a high level of uric acid (UA) in plasma, hyperuricemia (HU), is associated with the increased risk of cardiovascular diseases (CVDs). Endothelial damage has been suggested as a potential mechanism involved in HU-induced CVDs, especially in patients with the accumulation of other cardiovascular risk factors. However, the role of UA in the pathogenesis of endothelial dysfunction is still a matter of debate. It is unclear whether UA is a causative risk factor in endothelial dysfunction, an inert marker or an endothelium-protective molecule with respect to its antioxidant properties. Of note, only a few studies have been conducted to investigate the effect of UA on vascular endothelium-dependent relaxation. Therefore, we have studied the acute in vitro effects of high UA concentrations on the endothelial function of arteries isolated from aged rats. Experiments were performed in small mesenteric arteries (SMAs), femoral arteries and thoracic aortas isolated from 68-week-old and 57-week-old male Wistar-Kyoto rats. Vascular reactivity was investigated in isometric conditions using the wire myograph and organ chamber. Acetylcholine (ACh) was used to investigate endothelium-dependent vasorelaxation. Then, UA was added to the myograph or organ chamber at 600 μmol/l (arteries from 68-week-old rats) or 1200 μmol/l (arteries from 57-week-old rats) and incubated for 1 h, and this was followed by determining the ACh concentration-response curve. UA had no significant effect on ACh-induced vasorelaxation and pD2 values in all investigated groups. Likewise, no significant differences in noradrenaline- (SMAs), serotonin- (femoral arteries) and phenylephrine-induced (aortas) vasoconstriction were observed after UA pre-incubation. In conclusion, high concentrations of UA administered acutely failed to affect endothelial function and did not provoke endothelial dysfunction in resistant mesenteric arteries, medium-sized and large arteries from aged rats.

Effect of Dietary Nicotinamide Mononucleotide (NMN) on Function and Mechanics of Skeletal Muscle Arteries from Aged Mice

Ageing is associated with a decline in vascular function and increased risk of cardiovascular disease. Dietary NMN, an NAD+ precursor, increases muscle vascularity, blood flow and exercise performance in aged mice 1. This study determined the effects of dietary NMN on endothelium‐dependent dilation (EDD) and vascular stiffness in skeletal muscle arteries isolated from aged mice. Male and female C57BL/6J mice received NMN (400 mg/kg) in drinking water for 8–10 weeks; mice were ~97 weeks old at the end of the treatment period. Segments of the saphenous and gracilis muscle arteries (SA and GMA respectively) were removed. Functional responses were examined using pressure myography. SA and some GMA were pre‐constricted with phenylephrine. Pressure‐induced myogenic tone was enhanced in GMA from NMN‐treated male mice. NMN treatment enhanced EDD in the GMA of both male and female mice as measured by a leftward shift in the pEC50 of acetylcholine concentration‐response curves. EDD in the SA from either sex was not altered by NMN treatment. NMN treatment also did not affect responses to the endothelium‐independent vasodilator sodium nitroprusside in either artery, from either sex. The nitric oxide synthase (NOS) inhibitor L‐NAME (100 μM) strongly inhibited EDD in the SA but caused only minor inhibition of EDD in the GMA, from both sexes. In the GMA from both sexes, EDD was significantly inhibited by blockers of Ca2+‐sensitive K+ channels (KCa). Analysis of relative EC50‐shift suggested intermediate‐conductance‐KCa (IKCa)‐mediated vasodilation was enhanced in the GMA from NMN‐treated mice. Stress‐strain relationships were obtained from arteries under passive conditions, over the pressure‐range 5–120 mmHg. Analysis of these relationships showed NMN treatment increased compliance of GMA, but not SA, from both sexes. SA from female mice were less compliant than SA from male mice, regardless of NMN treatment. These studies suggest NMN treatment can enhance EDD, decrease arterial stiffness and improve blood flow autoregulation in small resistance (GMA) arteries, but not larger conduit (SA) arteries in skeletal muscle from aged mice. The increased EDD may be due to increased IKCa‐mediated vasodilation, but not increased nitric oxide‐mediated effects. These changes may contribute to the enhanced muscle blood flow and exercise performance observed in aged NMN‐treated mice.

Abstract 125: Low-Grade Chronic Infection Induces Vascular Dysfunction and Remodeling in Salt Sensitive Hypertension

According to the new AHA guidelines, over half of Americans have hypertension. Multiple factors contribute to hypertension including genetics, diet, stress, and infection. N-formyl peptides (NFPs) are a potent chemoattractant that bind formyl peptide receptors (FPR), an innate immune receptor. Interestingly, the only source of NFPs are bacteria and mitochondria, which evolved from bacteria. We have observed that FPR is expressed in arteries and its activation with bacterial (fMLPs) or mitochondrial (FMIT) NFPs induces vascular remodeling via actin polymerization. Given that increased gut permeability is present in hypertension, we questioned if bacterial NFPs derived from a leaky gut also play a role in hypertension. We hypothesized that bacterial NFPs activate FPR leading to vascular dysfunction and remodeling. We used male (6 weeks) Dahl Resistant (DR) and Sensitive (DS) rats on a low (0.3 %) or high salt (2 %) diet for 9 weeks. After 6 weeks some animals on high salt received water + antibiotic [neomycin (NEO), 0.5 g/L or amoxicillin (AMO), 50 mg/kg/day) for 3 weeks to decrease bacterial levels. Statistics: t-test *p<0.05. We observed that FPR mRNA expression was increased in aorta from DS (low-salt) when compared to DR (low-salt) (DR: 0.96 ± 0.7 vs. DS: 1.3 ± 1.7*), suggesting that FPR is activated prior to the onset of hypertension. High-salt diet increased FPR mRNA expression in aorta from DR, but did not exacerbate it in DS compared to DS (low-salt). Phenylephrine and acetylcholine concentration-response curves (1nM – 1μM) were performed in mesenteric resistance arteries using wire myography. Due to increased arterial stiffness from severe hypertension in DS (high salt), these rats showed a decrease in contraction compared to DR. DS treated with NEO or AMO ameliorated this response (tension (mN): Emax: DS 11 ± 4 vs. DS+ NEO 17 ± 2*, S + AMO 17 ± 0.9*). No changes were observed in the relaxation curve. Using pressure myograph, it was observed that NEO and AMO reverted vascular remodeling [wall thickness (μm): DS 55 ± 4 vs. DS + NEO 38 ± 9* and DS + AMO 31± 9*; cross sectional area (μm 2 ): DS 45,624 ± 1370 vs. DS + NEO 28,700 ± 8370* and DS + AMO 23,276 ± 9775*]. We concluded that low-grade chronic infection plays a role in vascular dysfunction and remodeling observed in hypertension.

Sustained exposure to prostaglandin D2 augments the contraction induced by acetylcholine via a DP1 receptor-mediated activation of p38 in bronchial smooth muscle of naive mice.

Prostaglandin D2 (PGD2), one of the key lipid mediators of allergic airway inflammation, is increased in the airways of asthmatics. However, the role of PGD2 in the pathogenesis of asthma is not fully understood. In the present study, effects of PGD2 on smooth muscle contractility of the airways were determined to elucidate its role in the development of airway hyperresponsiveness (AHR). In a murine model of allergic asthma, antigen challenge to the sensitized animals caused a sustained increase in PGD2 levels in bronchoalveolar lavage (BAL) fluids, indicating that smooth muscle cells of the airways are continually exposed to PGD2 after the antigen exposure. In bronchial smooth muscles (BSMs) isolated from naive mice, a prolonged incubation with PGD2 (10−5 M, for 24 h) induced an augmentation of contraction induced by acetylcholine (ACh): the ACh concentration-response curve was significantly shifted upward by the 24-h incubation with PGD2. Application of PGD2 caused phosphorylation of ERK1/2 and p38 in cultured BSM cells: both of the PGD2-induced events were abolished by laropiprant (a DP1 receptor antagonist) but not by fevipiprant (a DP2 receptor antagonist). In addition, the BSM hyperresponsiveness to ACh induced by the 24-h incubation with PGD2 was significantly inhibited by co-incubation with SB203580 (a p38 inhibitor), whereas U0126 (a ERK1/2 inhibitor) had no effect on it. These findings suggest that prolonged exposure to PGD2 causes the BSM hyperresponsiveness via the DP1 receptor-mediated activation of p38. A sustained increase in PGD2 in the airways might be a cause of the AHR in allergic asthmatics.

Relaxant effects of Azadirachta indica A. Juss var. siamensis Valeton flower extract on isolated rat ileum contractions and the mechanisms of action

Azadirachta indica A. Juss var. siamensis Valeton or commonly known as Siamese neem is one of the most well-known plant in traditional Ayurvedic medicine. The aim of the present study was to investigate the relaxant effects of A. indica on isolated rat ileum contractions and its potential underlying mechanisms involved. The isometric contractions of ileum segments were investigated in organ baths for spontaneous activity and response to aqueous extract of Siamese neem flower (SNF). The spasmolytic action of the extract was also assessed on contraction induced by acetylcholine and high potassium. Our findings indicate that cumulative concentrations of SNF aqueous extract induced relaxant effect on spontaneous rat ileum contractions. The extract has also suppressed the cumulative concentration response curve for acetylcholine and pottasium ions-induced contraction. The presence and absence of propranol (antagonist of β-adrenergic receptor) and l-Name (antagonist of nitric oxide synthase) in SNF aqeous extract co-treatment demonstrated no significant different in term of contraction activity when compared to SNF extract treatment alone. The treatment of SNF extract caused a significant inhibition in tissue contraction stimulated by accumulation of calcium ions. Our results showed the relaxant effect of SNF aqueous extract on the isolated rat ileum. In short, the SNF aqueous extract exhibited an inhibitory effect on the spontaneous ileum contactions particularly on the contraction stimulated by acetylcholine and high potassium. The observed effect might acted through the modulation of calcium channels. This findings provide a pharmacological basis for the traditional use of SNF for the treatment of gastrointestinal spasms.

Endothelial dysfunction of internal thoracic artery graft in patients with chronic kidney disease

ObjectivesThe present study was designed to evaluate the association between chronic kidney disease and the endothelial function of internal thoracic artery (ITA) grafts in patients undergoing coronary bypass surgery. An isometric tension study was performed in ITA strips obtained during surgery. Concentration-response curves for acetylcholine (ACh) and sodium nitroprusside were constructed in ITA strips partially precontracted with phenylephrine under the inhibition of cyclooxygenase. The integrity of the endothelium was verified histologically by en-face staining of the luminal surface with the use of silver nitrate solution. ResultsIn endothelium-intact ITA strips, ACh produced a concentration-dependent relaxation in patients with glomerular filtration rate (GFR, mL/min/1.73 m2) > 60. A concentration-dependent relaxation response also was observed in patients with GFR 30 to 60, but it was reduced significantly compared with those with GFR > 60. In both groups, removal of endothelium or treatment with nitric oxide (NO) synthase inhibitors almost abolished the ACh-induced relaxation. On the other hand, in patients with GFR < 30, mild contraction rather than relaxation was induced at a high concentration of ACh, which was modified neither by treatment with NO synthase inhibitors nor by removal of the endothelium. Vasodilator responses to sodium nitroprusside were comparable among the 3 groups. The relaxation of endothelium-intact strips to a peak ACh concentration correlated positively with GFR. This relationship held true in a multiple linear regression model, and interaction terms between GFR and other risk factors were not statistically significant. ConclusionsEndothelial function of ITA grafts to release NO is impaired at the time of surgery in patients with chronic kidney disease.

The Prokinetic, Laxative, and Antidiarrheal Effects of Morus nigra: Possible Muscarinic, Ca(2+) Channel Blocking, and Antimuscarinic Mechanisms.

Morus nigra Linn. (black mulberry) is used in gastrointestinal ailments. This study demonstrates gut modulatory properties of M. nigra. The prokinetic, laxative, and antidiarrheal activities of M. nigra were assessed in mice, while isolated rabbit jejunum and guinea-pig ileum were used to explore insight into mechanism(s). At 30 and 70 mg/kg, the crude extract of M. nigra (Mn.Cr) exhibited atropine-sensitive prokinetic and laxative effects, similar to carbachol (CCh). While at higher doses (100, 300, and 500 mg/kg), Mn.Cr offered protection against castor oil-induced diarrhea. In rabbit jejunum, Mn.Cr and its chloroform fraction inhibited CCh-induced contractions more potently compared with high K(+) (80 mm). Conversely, petroleum fraction was more potent against high-K(+) -induced contractions. At 0.01 mg/mL, Mn.Cr caused a parallel shift in acetylcholine concentration-response curves (CRCs) followed by a non-parallel shift at 0.03 mg/mL, similar to dicyclomine. At further tested concentrations, Mn.Cr (0.1 and 0.3 mg/mL) and petroleum fraction suppressed Ca(2+) CRCs, similar to verapamil. In guinea-pig ileum, Mn.Cr, its aqueous and ethyl acetate fractions exhibited atropine-sensitive gut stimulant activity along with additional uncharacterized excitatory response in the aqueous fraction only. These results suggest that black mulberry possesses prokinetic, laxative, and antidiarrheal effects, putatively mediated through cholinomimetic, antimuscarinic, and Ca(2+) antagonist mechanisms, respectively. Copyright © 2016 John Wiley & Sons, Ltd.

Differential α4(+)/(−)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms

Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(−)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(−)α4 site with lower agonist affinity than the α4(+)/(−)β2 sites. However, the relative roles of the conserved α4(+)/(−)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (−)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with 125I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(−)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(−)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.

Lisinopril alters contribution of nitric oxide and K(Ca) channels to vasodilatation in small mesenteric arteries of spontaneously hypertensive rats.

To investigate lisinopril effect on the contribution of nitric oxide (NO) and K(Ca) channels to acetylcholine (ACh)-induced relaxation in isolated mesenteric arteries of spontaneously hypertensive rats (SHRs). Third branch mesenteric arteries isolated from lisinopril treated SHR rats (20 mg/kg/day for ten weeks, SHR-T) or untreated (SHR-UT) or normotensive WKY rats were mounted on tension myograph and ACh concentration-response curves were obtained. Westernblotting of eNOS and K(Ca) channels was performed. ACh-induced relaxations were similar in all groups while L-NMMA and indomethacin caused significant rightward shift only in SHR-T group. Apamin and TRAM-34 (SK(Ca) and IK(Ca) channels blockers, respectively) significantly attenuated ACh-induced maximal relaxation by similar magnitude in vessels from all three groups. In the presence of L-NMMA, indomethacin, apamin and TRAM-34 further attenuated ACh-induced relaxation only in SHR-T. Furthermore, lisinopril treatment increased expression of eNOS, SK(Ca) and BK(Ca) proteins. Lisinopril treatment increased expression of eNOS, SK(Ca), BK(Ca) channel proteins and increased the contribution of NO to ACh-mediated relaxation. This increased role of NO was apparent only when EDHF component was blocked by inhibiting SK(Ca) and IK(Ca) channels. Such may suggest that in mesenteric arteries, non-EDHF component functions act as a reserve system to provide compensatory vasodilatation if (and when) hyperpolarization that is mediated by SK(Ca) and IK(Ca) channels is reduced.

Decreased endothelial nitric oxide, systemic oxidative stress, and increased sympathetic modulation contribute to hypertension in obese rats

We investigated the involvement of nitric oxide (NO) and reactive oxygen species (ROS) on autonomic cardiovascular parameters, vascular reactivity, and endothelial cells isolated from aorta of monosodium glutamate (MSG) obese rats. Obesity was induced by administration of 4 mg/g body wt of MSG or equimolar saline [control (CTR)] to newborn rats. At the 60th day, the treatment was started with N(G)-nitro-L-arginine methyl ester (L-NAME, 20 mg/kg) or 0.9% saline. At the 90th day, after artery catheterization, mean arterial pressure (MAP) and heart rate were recorded. Plasma was collected to assess lipid peroxidation. Endothelial cells isolated from aorta were evaluated by flow cytometry and fluorescence intensity (FI) emitted by NO-sensitive dye [4,5-diaminofluoresceindiacetate (DAF-2DA)] and by ROS-sensitive dye [dihydroethidium (DHE)]. Vascular reactivity was made by concentration-response curves of acetylcholine. MSG showed hypertension compared with CTR. Treatment with L-NAME increased MAP only in CTR. The MSG induced an increase in the low-frequency (LF) band and a decrease in the high-frequency band of pulse interval. L-NAME treatment increased the LF band of systolic arterial pressure only in CTR without changes in MSG. Lipid peroxidation levels were higher in MSG and were attenuated after L-NAME. In endothelial cells, basal FI to DAF was higher in CTR than in MSG. In both groups, acetylcholine increased FI for DAF from basal. The FI baseline to DHE was higher in MSG than in CTR. Acetylcholine increased FI to DHE in the CTR group, but decreased in MSG animals. We suggest that reduced NO production and increased production of ROS may contribute to hypertension in obese MSG animals.

Abstract 290: Endothelial Dysfunction in a Rat Model of Asphyxia-Induced Cardiac Arrest

Background: Altered vasoreactivity and elevated markers of endothelial activation are found following cardiac arrest (CA) and resuscitation. However, it is unknown whether the described altered vasoreactivity reflects influence from humoral factors, affection of the underlying smooth muscle or a change in the endothelium itself. Furthermore, it is unknown how the inflammatory markers correlate to endothelial function. The aim of the present study was to assess endothelial function and the relation to plasma markers of inflammation and adhesion, CA and resuscitation. Methods: Male Sprague Dawley rats underwent 5 min. of asphyxia-induced CA and subsequent resuscitation. After return of spontaneous circulation (ROSC) animals were euthanized after 30 min. or 2 hrs. Sham operated animals were used as controls. At the end of the experiment blood was drawn and analyzed for IL-1β, IL-6, IL-10, TNF-α, ICAM-1, VCAM-1, VEGF-α, vWF and sP-Selectin. Segments of the Left coronary artery (LCA) and mesenteric resistance arteries were mounted in microvascular myographs. Concentration-response curves for acetylcholine (ACH), NS309, and sodium nitroprusside where obtained. Results: LCA-segments exhibited an impaired vasodilation to ACH after 30 min. of ROSC as CA animals dilated to a max of 66 ± 2 vs. 94 ± 8% in the sham group (Mean ± SE; P=0.0014). After 2 hrs. of ROSC vasoreactivity was comparable to sham groups for all drugs tested. The mesenteric arteries exhibited a larger response to NS309 after 2 hrs. of ROSC compared to 2 hr. sham (logEC50 ± SE: 0.43 ± 0.05 vs. 1.85 ± 0.23 µM; P&lt;0,001). Levels of sP-selectin, vWF and sVCAM-1 were elevated in both CA groups vs. sham groups, with no correlation to vasoreactivity. Conclusion: Endothelium-dependent ACH-induced nitric oxide (NO)-mediated vasodilation was blunted in LCA segments in the early post-resuscitation phase, NS309-induced endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation was enhanced in mesenteric resistance arteries in the intermediate post-resuscitation phase. There was no relation of vasoreactivity to plasma markers of adhesion and inflammation. The altered vascular reactivity may explain impaired coronary flow and hypotension in the restitution phase after cardiac arrest.

Characterization of 5-hydroxytryptamine-induced contraction and acetylcholine-induced relaxation in isolated chicken basilar artery

The aim of the present study was to clarify the responsiveness of the chicken basilar artery to 5-hydroxytryptamine (5-HT) and acetylcholine (ACh) and to characterize the related receptor subtypes in vitro. Basilar arteries were obtained from freshly slaughtered broiler chickens. The 5-HT induced concentration-dependent contraction of the arteries. The concentration-response curves for 5-HT were shifted 30-fold to the right by methiothepin (a 5-HT(1) and 5-HT(2) receptor antagonist) and 3-fold to the right by ketanserin (a 5-HT(2) receptor antagonist). In the presence of ketanserin, the concentration-response curve for 5-HT was shifted 10-fold to the right by methiothepin. The pA(2) value for methiothepin was 8.26. The ACh induced concentration-dependent relaxation under conditions of precontraction by 5-HT. The concentration-response curve for ACh was shifted to the right by atropine [a nonselective muscarinic (M) receptor antagonist] and hexahydro-sila-difenidol hydrochloride, a p-fluoroanalog (pFHHSiD, an M(3) receptor antagonist), but not by pirenzepine (an M(1) receptor antagonist) or methoctramine (an M(2) receptor antagonist). The pA(2) value for pFHHSiD was 7.55. Nω-Nitro-l-arginine (a nitric oxide synthase inhibitor) inhibited ACh-induced relaxation by approximately 50%. These results suggest that 5-HT induces contraction via activation of 5-HT(1) and 5-HT(2) receptors and that ACh induces relaxation via activation of the M(3) receptor. The 5-HT(1) receptor might play a dominant role in 5-HT-induced contraction. One of the factors involved in ACh-induced relaxation is probably nitric oxide released from endothelial cells.

Development of an ex vivo model for pharmacological experimentation on isolated tissue preparation

Pharmacology as a subject depends largely on experiments conducted in laboratory animals. Experimental animals like rat, guinea pig, rabbit, etc. are used for the biological assay. For the teaching purposes to use isolated strip preparations from various organs, the laboratory animal species has to be sacrificed just for a piece of tissue. The present study was aimed to develop ex vivo model for pharmacological experimentation, which will mimic the actual laboratory condition without sacrificing the experimental animals. Dose response curve of acetylcholine alone and in presence of different concentrations of atropine was plotted using isolated chicken ileum, chicken duodenum, rat ileum, and rat duodenum and their EC50 values were compared. The effect of atropine in terms of its type of antagonism was predicted based on Schild plot and pA2 values were obtained. The chicken ileum and duodenum were also evaluated for four- and three-point bioassay, respectively. The results suggested that acetylcholine produced a dose-dependent increase in contraction in both chicken and rat ileum and duodenum preparation. The concentration response curve of acetylcholine in chicken ileum shifted toward left side of rat ileum with a higher EC50 value. Atropine shifted the concentration response curve of acetylcholine toward right with a change in EC50 value. Schild plots indicated that antagonism produced by atropine was found to be competitive in nature. The pA2 values of atropine were found significantly high with isolated chicken ileum as compared to rat ileum preparation. It is concluded that isolated chicken ileum and duodenum preparation can be employed for routine experiments of pharmacology subject and the use of these isolated preparations is a novel approach for managing pharmacological experiments and importantly, without sacrificing the experimental animals.

Impairment of Endothelium-dependent Relaxation and Changes in Levels of cyclic GMP in Carotid Arteries from Stroke-prone Spontaneously Hypertensive Rats

Endothelium-dependent relaxation of carotid arteries and changes in levels of cyclic (c)GMP between stroke-prone spontaneously hypertensive (SHRSP) and Wistar-Kyoto (WKY) rats have been compared. The concentration-response curve for acetylcholine (ACh)-induced relaxation was shifted to the right in carotid arteries from SHRSP. Relaxation responses produced by calcimycin (A 23187) and melittin, both endothelium-dependent agents, were depressed in carotid arteries from SHRSP. Relaxation responses produced by sodium nitroprusside and 8-Br-cGMP were similar to those in strips from WKY. ACh-induced production of cGMP was significantly decreased in carotid arteries from SHRSP when compared with the level for similarly treated strips from WKY. These results suggest that functional changes in endothelium, but not guanylate cyclase activity or cGMP sensitivity in the carotid arteries, may occur in hypertension. Thus, impaired endothelium-dependent relaxation in SHRSP may play an important role in hypertensive vascular diseases such as stroke.

M1 and M3 Muscarinic Receptors Mediate Relaxation and Contraction in Canine Nasal Veins

Acetylcholine (ACh) has been shown to induce nasal congestion via vasorelaxation of intranasal posterior collecting veins (PCV) coupled with vasocontraction of extranasal outflow veins (dorsal nasal vein [DNV] and sphenopalatine vein [SPV]). The aim of this study was to characterize the muscarinic receptor subtype(s) involved in ACh-induced relaxation and contraction in canine nasal veins. PCV, DNV, and SPV were isolated from the canine nose. In vitro isometric tension of segments from these veins was monitored to reflect vascular reactivity. ACh concentration-response curve was studied in the presence of muscarinic receptor subtype inhibitors. Immunohistochemical localization of M(1)-M(5) receptor subtypes in the veins was performed. ACh-induced relaxation in PVC was inhibited by pertussis toxin (PTX; inhibitor of G-protein that couples M(2)/M(4) receptors), methoctramine (selective M(2) muscarinic receptor inhibitor), muscarinic toxin 7 (MT-7; selective M(1) muscarinic receptor inhibitor), and 4-diphenylacetoxy-methylpiperidine methiodide (4-DAMP; selective M(3) muscarinic receptor inhibitor). ACh-induced contraction in SPV and DNV was potentiated by PTX and methoctramine but was inhibited by MT-7 and 4-DAMP. Immunohistochemistry confirmed the presence of five muscarinic receptor subtypes in the endothelium of nasal veins, with staining of M(3) > M(1) > M(5) > M(2) = M(4) in PVC but M(2) = M(4) > M(3) > M(1) > M(5) in outflow veins. M(1) and M(3) receptor subtypes were localized in the smooth muscles of both types of veins. The results show that ACh relaxes intranasal veins and contracts extranasal veins primarily via M(1) and M(3) muscarinic receptor subtypes, implying the therapeutic value of M(1)/M(3)-specific or highly selective anticholinergics on nasal congestion.

Sphingosine-1-phosphate augments agonist-mediated contraction in the bronchial smooth muscles of mice

The effects of sphingosine-1-phosphate (S1P) on bronchial smooth muscle (BSM) contractility were investigated in naive mice. S1P had no effect on the basal tone of the isolated BSM tissues. However, in the presence of S1P(10-6 M), the BSM contractions induced by acetylcholine (ACh) and endothelin-1 (ET-1) were significantly augmented: both the ACh and ET-1 concentration-response curves were significantly shifted to the left. In contrast, the pretreatment with S1P had no effect on the contractions induced by high K+ depolarization. It is thus possible that S1P augments BSM contraction induced by the activation of G protein-coupled receptors.

Chronic Treatment With N-acetylcysteine Improves Cardiac Function but Does Not Prevent Progression of Cardiomyopathy in Syrian Cardiomyopathic Hamsters

Oxidative stress has been postulated to contribute to the onset and development of heart failure (HF). The efficacy of antioxidant therapy in HF, however, remains controversial. This study evaluates the effect of the antioxidant N-acetylcysteine (NAC, 1 g/kg per day) on cardiovascular function in 2- and 6-month-old Bio-TO2 Syrian cardiomyopathic hamsters (SCH) after treatment for 1 month and 5 months with this drug. Endothelial function, systolic blood pressure (SBP), and echocardiographic parameters were evaluated. Age-matched F1-B golden hamsters were used as controls. One month of NAC administration significantly decreased SBP in 2-month-old SCH (n = 5, P < 0.001) without modifying echocardiographic values. Five-month treatment of cardiomyopathic animals with the antioxidant improved the acetylcholine-induced relaxation in aortic rings by 24% (E( Max) value from 45.8% ± 4% to 55.3% ± 2% n = 7, P < .05) but did not modify EC(50) values for the acetylcholine concentration-response curve. In addition, 5-month administration of NAC to SCH increased ejection fraction from 39% ± 4% to 57% ± 4% (n = 11, P < .001) and decreased left ventricular end-diastolic and end-systolic volumes (from 0.38 ± 0.04 mL/100 g body weight (BW) and 0.22 ± 0.03 mL/100 g BW, before, to 0.24 ± 0.04 mL/100 g BW and 0.12 ± 0.03 mL/100 g BW after treatment, P < .01). Cardiac output index also improved after 5 months of treatment, although it did not reach statistical significance. These results suggest that antioxidant therapy alone decreases ventricular dilatation and improves cardiovascular function in this animal model of dilated cardiomyopathy, but it does not prevent the appearance of HF.

Abattoir-sourced Isolated Ileum from Gallus gallus domesticus as an Experimental Tool

A study was undertaken to determine the longevity of active muscarinic receptors on abattoir-sourced isolated ileum preparations from Gallus gallus domesticus, with a view to using the tissue as an experimental tool for functional response assays in laboratory experiments. A concentration-response curve for acetylcholine (1-256 μM) was plotted, in the presence and absence of 1, 3 and 6 nM atropine. In a second experiment, unknown concentrations of acetylcholine samples were determined by using an interpolation method. In this experiment, four sample concentrations were used and the calculated values were found to be almost equal to the actual values. Finally, an experiment was carried out to elucidate the effects of post-sacrifice time on the contractile response of the tissue. The results showed that the tissue maintained considerable contractile response at the 6-hour post-sacrifice time-point. Competitive antagonistic activity was observed between acetylcholine and atropine on the chicken ileum, and the pA2 value was calculated to be 9.21 by using an Arunlakshana-Schild plot. The results suggest that isolated ileum preparations of Gallus gallus domesticus, obtained from a meat abattoir, can be used as a basic experimental tool for bioassays in routine laboratory experiments. However, its potential as a research tool still needs to be confirmed.