Organ Bath Research Articles

In vitro inhibition of uterine contractions using electrospun nanofibers loaded with nifedipine and ML7.

To formulate a nanofiber-based controlled drug delivery system that could be effective in preventing uterine contractions and can be used for the treatment of preterm labor. We utilized uterine tissue samples obtained from ten pregnant women who underwent cesarean section at term to investigate the effect of nanofibers on spontaneous and induced myometrial contractions. We prepared nifedipine and ML7-loaded nanofibers using the electrospinning method with Poly(D,L-lactide-co-glycolide) (PLGA) polymer, resulted in seven groups of nanofibers, including a control group. Group I served as the control, Group II was non-drug loaded nanofiber, Group III was nifedipine (10-5 M) loaded nanofiber, Group IV was ML7 (3x10-5 M) loaded nanofiber, Group V was ML7 (3x10-5 M) and nifedipine (10-5 M) nanofiber, Group VI was ML7 (3x10-5 M) and nifedipine (3x10-5 M) nanofiber, and Group VII was ML7 (3x10-5 M) and nifedipine (10-4 M) nanofiber. To evaluate the contractile response, the nanofibers loaded with different doses of ML7 and nifedipine were applied onto the tissue strips, and in vitro organ bath experiments were performed. Full-thickness uterine samples were cleared of the serosa and surrounding tissues, and eight strips (3x10 mm) were prepared from each sample. The seven different nanofiber formulations were gently placed and sutured onto the strips, with one strip always kept as the time control. We recorded spontaneous, KCl-induced, and stimulated cumulative oxytocin-induced contractions from all samples in all groups. After completing all experiments, the viability of the strips was checked, and weight measurement was recorded. The administration of drug-loaded polymers resulted in a significant decrease in both the frequency and intensity of spontaneous and induced contractions in all groups (p<0.01). No significant difference was observed between the control group and the non-drug-loaded nanofiber group in post hoc analysis (p=0.704). In terms of amplitude and frequency of contractions, the most significant decrease was observed in group VII at cumulative oxytocin doses compared to the control and non-drug-loaded nanofiber groups (p<0.05). Moreover, group VI also showed a significant decrease in contraction intensity and frequency compared to the control and non-drug-loaded nanofiber groups (p<0.05). While the use of nifedipine and/or ML7-loaded nanofibers decreased both intensity and frequency of contraction, this attenuation was not significant compared to the control and empty polymer groups. However, a more significant inhibition was observed when ML7 was used with nifedipine at doses of 3x10-5 M and 10-4 M. The results indicate that human uterine contractions can be inhibited using calcium channel blocker (nifedipine) and myosin light chain kinase inhibitor (ML7) loaded nanofibers in uterine tissue strips. These results strongly suggested the potential for the development of locally effective and safe controlled drug release systems to prevent premature birth.

Combined Uterorelaxant Effect of Magnesium Sulfate and Terbutaline: Studies on Late Pregnant Rat Uteri In Vitro and In Vivo

(Acta Obstet Gynecol Scand. 2023;102:457–464) Preterm birth is a common and impactful complication of pregnancy and is defined as the delivery of a fetus between 20 and 37 weeks of gestation. It can lead to further complications and even developmental problems for the child that can be long term. Standard treatment for symptoms of preterm labor involves tocolytic drugs to stop contractions and delay delivery long enough for interventions to occur, minimizing effects on the infant. Although many different drugs are used, the efficacy and side effects of many are not well characterized or understood. This study was designed to assess the effectiveness and side effects of a combination of drugs: β2-agonist terbutaline and magnesium sulfate (MgSO4). This was done by examining the combined administration of the drugs in an isolated organ bath and performing in vivo smooth muscle electromyographic studies in pregnant rats.

Contractions Induced in Human Pulmonary Arteries by a H2S Donor, GYY 4137, Are Inhibited by Low-Frequency (20 kHz) Ultrasound.

The present study aimed to investigate the effect of a H2S donor, GYY 4137, on human pulmonary arteries and whether low-frequency ultrasound (20 kHz, 4 W/cm2) inhibits GYY 4137 contractions. Functional studies were conducted on human and rat pulmonary arteries mounted on microvascular myographs. We placed an ultrasonic gadget in the tissue organ bath to insonate the arteries with low-frequency ultrasound. To measure the effect of the low-frequency ultrasound on the entrance of extracellular Ca2+, the preparations were placed in a Ca2+-free solution, and the thromboxane agonist, U46619, and extracellular calcium were added in the presence of insonation. In isolated human pulmonary arteries, GYY 4137 induced contractions, which were most pronounced in the arteries contracted with the thromboxane analogue, U46619. The transient GYY4137 contractions were reversed by low-frequency ultrasound, a blocker of KV7 channels, XE-991 (10 µM), and glibenclamide (1 μM), a blocker of ATP-sensitive channels. Low-frequency ultrasound also inhibited the contractions induced by the smooth muscle entrance of increasing extracellular calcium concentrations. The present findings show that GYY 4137 can cause a transient contraction of pulmonary arteries in human arteries. GYY 4137 alone does not cause significant vascular contraction in rat lung arteries, but it contracts rat lung arteries precontracted with U46619. The transient contractions induced by GYY 4137 can be inhibited by low-frequency ultrasound, probably by counteracting the influx of external Ca2+. The effect of low-frequency ultrasound counteracts contraction in pulmonary arteries; therefore, a possibility could be to develop a larger device allowing treatment of patients with pulmonary hypertension.

Mechanisms involved in the muscle relaxing effects of STW 5 in guinea pig stomach.

The herbal preparation STW 5 ameliorates functional dyspepsia partly by relaxing smooth muscle of the proximal stomach, thus improving gastric accommodation. We explored the unknown pathways responsible for this effect by testing targets known to modulate gastric smooth muscle relaxation. STW 5-induced relaxation of smooth muscle strips from guinea pig gastric corpus before and after pharmacological interventions were recorded with force transducers in an organ bath. ORAI1 mRNA expression was tested in the proximal stomach. Blockade of Ca2+-activated K+ and Cl- channels, voltage-gated L- or T-type Ca2+ channels, TRPA1-, TRPV1-, adenosine or 5-HT4 receptors, antagonizing ryanodine receptors, inhibiting cyclooxygenase or sarcoplasmic reticulum calcium ATPase did not affect STW 5-evoked relaxation. Likewise, protein-kinase A or G were not involved. However, the relaxation evoked by STW 5 was significantly reduced by phorbol-12-myristat-13-acetat, an activator of protein-kinase C, by 2- aminoethyldiphenylborinate, an inhibitor of the IP3 receptor-mediated Ca2+ release from the sarcoplasmic reticulum or by SKF-96365, a nonselective store-operated calcium entry (SOCE) blocker. Furthermore, the mixed TRPC3/SOCE inhibitor Pyr3, but not the selective TRPC3 blocker Pyr10, reduced the effect of STW 5. Finally, BTP2, a potent blocker of ORAI-coupled SOCE, almost abolished STW 5-evoked relaxation. Expression of ORAI1 could be demonstrated in the corpus/fundus. STW 5 inhibited SOCE, most likely ORAI channels, which are modulated by IP3- and PKC-dependent mechanisms. Our findings impact on the design of drugs to induce muscle relaxation and help identify phytochemicals with similar modes of actions to treat gastrointestinal disturbances.

Inhibition of PDE-4 isoenzyme attenuates frequency and overall contractility of agonist-evoked ureteral phasic contractions.

The aim of this study was to investigate the functional role of phosphodiesterase enzymes (PDE) in the isolated porcine ureter. Distal ureteral strips were mounted in organ baths and pre-contracted with 5-HT (100 μM). Upon generation of stable phasic contractions, PDE-4 and PDE-5 inhibitors were added cumulatively to separate tissues. PDE-4 inhibitors, such as rolipram (10 nM and greater) and roflumilast (100 nM and greater), resulted in significant attenuation of ureteral contractile responses, while a higher concentration of piclamilast (1 μM and greater) was required to induce a significant depressant effect. The attenuation effect by rolipram was abolished by SQ22536 (100 μM). PDE-5 inhibitors, such as sildenafil and tadalafil, were not nearly as effective and were only able to suppress the 5-HT-induced contractions at higher concentrations of 1 μM. Rolipram significantly enhanced the depressant effect of forskolin, while sodium nitroprusside-induced attenuation of contractile responses remained unchanged in the presence of tadalafil. In summary, our study demonstrates that PDE-4 inhibitors are effective in attenuating 5-HT-induced contractility in porcine distal ureteral tissues, while PDE-5 inhibitors are less effective. These findings suggest that PDE-4 inhibitors, such as rolipram, may hold promise as potential therapeutic agents for the treatment of ureteral disorders attributable to increased intra-ureteral pressure.

Functional Analysis of TRPA1, TRPM3, and TRPV1 Channels in Human Dermal Arteries and Their Role in Vascular Modulation.

Transient receptor potential (TRP) channels are pivotal in modulating vascular functions. In fact, topical application of cinnamaldehyde or capsaicin (TRPA1 and TRPV1 channel agonists, respectively) induces "local" changes in blood flow by releasing vasodilator neuropeptides. We investigated TRP channels' contributions and the pharmacological mechanisms driving vasodilation in human isolated dermal arteries. Ex vivo studies assessed the vascular function of artery segments and analyzed the effects of different compounds. Concentration-response curves to cinnamaldehyde, pregnenolone sulfate (PregS, TRPM3 agonist), and capsaicin were constructed to evaluate the effect of the antagonists HC030031 (TRPA1); isosakuranetin (TRPM3); and capsazepine (TRPV1). Additionally, the antagonists/inhibitors olcegepant (CGRP receptor); L-NAME (nitric oxide synthase); indomethacin (cyclooxygenase); TRAM-34 plus apamin (K+ channels); and MK-801 (NMDA receptors, only for PregS) were used. Moreover, CGRP release was assessed in the organ bath fluid post-agonist-exposure. In dermal arteries, cinnamaldehyde- and capsaicin-induced relaxation remained unchanged after the aforementioned antagonists, while PregS-induced relaxation was significantly inhibited by isosakuranetin, L-NAME and MK-801. Furthermore, there was a significant increase in CGRP levels post-agonist-exposure. In our experimental model, TRPA1 and TRPV1 channels seem not to be involved in cinnamaldehyde- or capsaicin-induced relaxation, respectively, whereas TRPM3 channels contribute to PregS-induced relaxation, possibly via CGRP-independent mechanisms.

Heme-induced corpus cavernosum relaxation and its implications for priapism in sickle cell disease: a mechanistic insight.

Patients with sickle cell disease (SCD) experience intravascular hemolysis, leading to elevated plasma heme levels. This phenomenon has been associated with increased priapism in men with SCD. The heme group can be metabolized by heme oxygenase (HO), generating carbon monoxide (CO), which is known to promote smooth muscle relaxation via soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate (cGMP). However, the effects of heme on the relaxation responses of corpus cavernosum (CC) have not been investigated. To evaluate the functional and biochemical effects of the heme group on mouse CC smooth muscle in vitro. Male C57BL/6 mice were used. CC tissues were mounted in organ baths. Measurement of cGMP in mice CC was evaluated. The cumulative addition of heme concentrations promoted the relaxation of CC. HO inhibitor (1J, 100μM) or sGC inhibitor (ODQ, 10μM) blocked the relaxing effect of the heme group. Pre-incubation of CC with heme (100μM) enhanced relaxation induced by acetylcholine, sodium nitroprusside, and nitrergic relaxation (electrical field stimulation), which was abolished by 1J or ODQ. The heme group increased the cGMP production in CC, which was abolished by 1J or ODQ. cGMP levels were significantly higher in CC treated with heme, and pre-incubation with compound 1J or ODQ abolished the effect of heme in raising cGMP levels. The HO-CO-sGC-cGMP pathway appears to play a crucial role in promoting CC relaxation. Our study provides novel insight into the role of group heme in CC relaxation and its potential contribution to priapism in SCD. Heme may serve as a pharmacological target for new therapies to prevent priapism.

Zinc-aspirin preconditioning reduces endothelial damage of arterial grafts in a rodent model of revascularization.

Coronary artery bypass grafting (CABG) is the most common cardiac surgical procedure. The prognosis of revascularization via CABG is determined by the patency of the used grafts, for which an intact endothelium is essential. The degree of ischemia-reperfusion injury (IRI), which occurs during the harvest and implantation of the grafts, is an important determinant of graft patency. Preconditioning with aspirin, a nonsteroidal anti-inflammatory drug has been shown to reduce the functional and molecular damage of arterial grafts in a rodent model. Studies have found that the zinc-aspirin complex may be able to exert an even better protective effect in pathological cardiovascular conditions. Thus, our aim was to characterize the protective effect of zinc-aspirin complex on free arterial grafts in a rodent model of revascularization. Donor Lewis rats were treated with either zinc-aspirin, aspirin, or placebo (n = 8) for 5 days, then the aortic arches were harvested and stored in cold preservation solution and implanted heterotopically in the abdominal cavity of the recipient rats, followed by 2 h of reperfusion. There was also a non-ischemia-reperfusion control group (n = 8). Functional measurements using organ bath and histomorphological changes using immunohistochemistry were analyzed. The endothelium dependent maximal vasorelaxation was improved (non-transplanted control group: 82% ± 3%, transplanted control group: 14% ± 2%, aspirin group: 31% ± 4%, zinc-aspirin group: 52% ± 4%), the nitro-oxidative stress and cell apoptosis decreased, and significant endothelial protection was shown in the groups preconditioned with aspirin or zinc-aspirin. However, zinc-aspirin proved to be more effective in the reduction of IRI, than aspirin alone. Preconditioning with zinc-aspirin could be a promising way to protect the function and structural integrity of free arterial grafts, thus improving the outcomes of CABG.

Optogenetic Activation of Cholinergic Enteric Neurons Reduces Inflammation in Experimental Colitis

Background & AimsIntestinal inflammation is associated with loss of enteric cholinergic neurons. Given the systemic anti-inflammatory role of cholinergic innervation, we hypothesized that enteric cholinergic neurons similarly possess anti-inflammatory properties and may represent a novel target to treat inflammatory bowel disease (IBD). MethodsMice were fed 2.5% dextran sodium sulfate (DSS) for seven days to induce colitis. Cholinergic enteric neurons, which express choline acetyltransferase (ChAT), were focally ablated in the mid-colon of ChAT::Cre;R26-iDTR (ChAT-iDTR) mice by local injection of diphtheria toxin prior to colitis induction. Activation of enteric cholinergic neurons was achieved using ChAT::Cre;R26-ChR2 (ChAT-ChR2) mice, in which ChAT+ neurons express channelrhodopsin-2 (ChR2), with daily blue light stimulation (BLS) delivered via an intracolonic probe during the 7 days of DSS treatment. Colitis severity, ENS structure, and smooth muscle contractility were assessed by histology, immunohistochemistry, qPCR, organ bath, and electromyography. In vitro studies assessed the anti-inflammatory role of enteric cholinergic neurons on cultured muscularis macrophages. ResultsAblation of ChAT+ neurons in DSS-treated mice exacerbated colitis, as measured by weight loss, colon shortening, histologic inflammation, and CD45+ cell infiltration, and led to colonic dysmotility. Conversely, optogenetic activation of enteric cholinergic neurons improved colitis, preserved smooth muscle contractility, protected against loss of cholinergic neurons, and reduced proinflammatory cytokine production. Both ACh and optogenetic cholinergic neuron activation in vitro reduced proinflammatory cytokine expression in LPS-stimulated muscularis macrophages. ConclusionsThese findings demonstrate that enteric cholinergic neurons have an anti-inflammatory role in the colon and should be explored as a potential IBD treatment.

Time-dependent progress of lower urinary tract dysfunction in streptozotocin-induced diabetic rats with or without low-dose insulin treatment.

Objectives: To examine time-dependent functional and structural changes of the lower urinary tract in streptozotocin-induced diabetic rats with or without low-dose insulin treatment and explore the pathophysiological characteristics of insulin therapy on lower urinary tract dysfunction (LUTD) caused by diabetes mellitus (DM). Methods: Female Sprague-Dawley rats were divided into five groups: normal control (NC) group, 4 weeks insulin-treated DM (4-DI) group, 4 weeks DM (4-DM) group, 8 weeks insulin-treated DM (8-DI) group and 8 weeks DM (8-DM) group. DM was initially induced by i.p. injection of streptozotocin (65 mg/kg), and then the DI groups received subcutaneous implantation of insulin pellets under the mid dorsal skin. Voiding behavior was evaluated in metabolic cages. The function of bladder and urethra in vivo were evaluated by simultaneous recordings of the cystometrogram and urethral perfusion pressure (UPP) under urethane anesthesia. The function of bladder and urethra in vitro were tested by organ bath techniques. The morphologic changes of the bladder and urethra were investigated using Hematoxylin-Eosin and Masson's staining. Results: Both 4-and 8-weeks diabetic rats have altered micturition patterns, including increased 12-h urine volume, urinary frequency/12 hours and voided volume. In-vivo urodynamics showed the EUS bursting activity duration is longer in 4-DM group and shorter in 8-DM group compared to NC group. UPP change in 8-DM were significantly lower than NC group. While none of these changes were found between DI and NC groups. Organ bath showed the response to Carbachol and EFS in bladder smooth muscle per tissue weights was decreased significantly in 4- and 8-weeks DM groups compared with insulin-treated DM or NC groups. In contrast, the contraction of urethral muscle and maximum urethral muscle contraction per gram of the tissue to EFS stimulation were significantly increased in 4- and 8-weeks DM groups. The thickness of bladder smooth muscle was time-dependently increased, but the thickness of the urethral muscle had no difference. Conclusions: DM-induced LUTD is characterized by time-dependent functional and structural remodeling in the bladder and urethra, which shows the hypertrophy of the bladder smooth muscle, reduced urethral smooth muscle relaxation and EUS dysfunction. Low-dose insulin can protect against diuresis-induced bladder over-distention, preserve urethral relaxation and protect EUS bursting activity, which would be helpful to study the slow-onset, time-dependent progress of DM-induced LUTD.

Impact of intravascular hemolysis on functional and molecular alterations in the urinary bladder: implications for an overactive bladder in sickle cell disease.

Patients with sickle cell disease (SCD) display an overactive bladder (OAB). Intravascular hemolysis in SCD is associated with various severe SCD complications. However, no experimental studies have evaluated the effect of intravascular hemolysis on bladder function. This study aimed to assess the effects of intravascular hemolysis on the micturition process and the contractile mechanisms of the detrusor smooth muscle (DSM) in a mouse model with phenylhydrazine (PHZ)-induced hemolysis; furthermore, it aimed to investigate the role of intravascular hemolysis in the dysfunction of nitric oxide (NO) signaling and in increasing oxidative stress in the bladder. Mice underwent a void spot assay, and DSM contractions were evaluated in organ baths. The PHZ group exhibited increased urinary frequency and increased void volumes. DSM contractile responses to carbachol, KCl, α-β-methylene-ATP, and EFS were increased in the PHZ group. Protein expression of phosphorylated endothelial NO synthase (eNOS) (Ser-1177), phosphorylated neuronal NO synthase (nNOS) (Ser-1417), and phosphorylated vasodilator-stimulated phosphoprotein (VASP) (Ser-239) decreased in the bladder of the PHZ group. Protein expression of oxidative stress markers, NOX-2, 3-NT, and 4-HNE, increased in the bladder of the PHZ group. Our study shows that intravascular hemolysis promotes voiding dysfunction correlated with alterations in the NO signaling pathway in the bladder, as evidenced by reduced levels of p-eNOS (Ser-1177), nNOS (Ser-1417), and p-VASP (Ser-239). The study also showed that intravascular hemolysis increases oxidative stress in the bladder. Our study indicates that intravascular hemolysis promotes an OAB phenotype similar to those observed in patients and mice with SCD.

Mefenamic Acid Reduces the Contractility of Isolated Uterine Smooth Muscle of Rat

Objective: Mefenamic acid is a nonsteroidal anti-inflammatory drug widely used for the treatment of pain and inflammation. The drug is indicated in patients with mild to moderate pain and inflammatory diseases, usually toothache, painful menstruation, muscle or joint pains and postpartum pain. There are not enough studies showing the effectiveness of this drug, which is significantly effective in gynecological pains, on the uterine contraction and relaxation mechanism. This study was carried out to investigate the effects of mefenamic acid on the uterine contraction and relaxation mechanism. Materials and Methods: In the study, seven female intact Sprague-Dawley rats in the diestrus period were used. Longitudinal myometrium sections of 1.2 cm length, 2 mm width and 1 mm thickness from animals were suspended in an isolated organ bath containing crebs solution. After the regulation period, mefenamic acid was administered at a dose of 300 μM. Contractile changes were monitored using an isometric transducer. Before and after the application, the area under the curve (AUC), frequency and peak to peak (p-p) values were normalized as % change. Statistical analyzes of the data were performed with the Paired Sample T test in SPSS 22.0 program. Results: Mefenamic acid caused a statistically significant decrease in the p-p, AUC and frequency values of spontaneous uterine contractions at 300 μM dose (p&lt;0.001). Conclusion: Mefenamic acid has an inhibitory effect on uterine contractions. This drug, which is widely used in the clinic for menstrual pain caused by abnormal uterine contractions, especially in young women, may show its analgesic effect by inhibiting uterine contractions.

Smooth muscle electromyography for detecting major alterations in the estrus cycle in rats.

Determining the female animal cycle is crucial in preclinical studies and animal husbandry. Changes in hormone levels during the cycle affect physiological responses, including altered contractility of the visceral smooth muscle. The study aimed to identify estrus and anestrus using smooth muscle electromyographic (SMEMG) measurements, in vivo fluorescent imaging (IVIS) and in vitro organ contractility of the uterus and cecum. The study involved sexually mature female Sprague-Dawley rats, aged 10-12 weeks. The rats received a daily injection of cetrorelix acetate solution for 7 days, while another group served as the control. The animals were subjected to gastrointestinal and myometrial SMEMG. The change in αvβ3 integrin activity was measured with IVIS in the abdominal cavity. Contractility studies were performed in isolated organ baths using dissected uterus and cecum samples. Plasma samples were collected for hormone level measurements. A 3-fold increase in spontaneous contraction activity was detected in SMEMG measurements, while a significant decrease in αvβ3 integrin was measured in the IVIS imaging procedure. Cetrorelix reduced the level of LH and the progesterone / estradiol ratio, increased the spontaneous activity of the cecum rings, and enhanced KCl-evoked contractions in the uterus. We found a significant change in the rate of SMEMG signals, indicating simultaneous increases in the contraction of the cecum and the non-pregnant uterus, as evidenced by isolated organ bath results. Fluorescence imaging showed high levels of uterine αvβ3 integrin during the proestrus-estrus phase, but inhibiting the sexual cycle reduced fluorescence activity. Based on the results, the SMEMG and IVIS imaging methods are suitable for detecting estrus phase alterations in rats.

Inhibitory Effects of Mefenamic Acid on Rat Urinary Bladder Contractions In Vitro ( Effects of Mefenamic Acid on Urinary Bladder Contractions )

Objective: In this study, the effect of mefenamic acid, a nonsteroidal anti-inflammatory drug, on the unconstrained contractile movement of the bladder muscle of female Spraque Dawley rats in diestrus was investigated. Since the effect of mefenamic acid on bladder smooth muscle is not known and its use for other conditions may cause possible side effects on the bladder, its effect was investigated for the possibility of using it as an active substance in the treatment of bladder problems. Materials and Methods: In the experimental study, 1.5 cm long smooth muscle strips from seven female Sprague Dawley rats were prepared and suspended in an isolated organ bath system containing Krebs solution under 1.5 g tension. The system was gassed continuously with oxygen/carbon dioxide mixture (95%:5%) and isometric contractions were recorded. At a dose of 300 µM, the contraction/relaxation effects of mefenamic acid concentration on bladder smooth muscle were investigated. The area under the curve (AUC) and amplitude values of bladder contractions were analyzed before and after mefenamic acid administration. With the data obtained from the analysis, the effect of mefenamic acid on bladder contractions was evaluated using paired t-tests in SPSS Statistical Software. Results: The decrease in area and amplitude values was statistically significant (P&lt;0.05). Mefenamic acid had an inhibitory effect on bladder contractions. Conclusion: The present findings demonstrate the relaxant effect of mefenamic acid on the rat bladder and if similar effects are observed in human studies, mefenamic acid may be effective in the treatment of voiding problems such as bladder incontinence.

6-Nitrodopamine is an endogenous mediator of the rabbit corpus cavernosum relaxation.

6-Nitrodopamine (6-ND) is a novel endogenous catecholamine that has a potent relaxant action on vascular smooth muscle in vitro. To evaluate the basal release of 6-ND and noradrenaline from rabbit-isolated corpus cavernosum (RbCC) and its relaxing action on this tissue. Rabbit corpus cavernosa were dissected and suspended in a 5-mL organ bath containing oxygenated Krebs-Henseleit's solution. 6-ND and noradrenaline release was quantified by liquid chromatography coupled to tandem mass spectrometry. The relaxant activity of 6-ND was assessed in RbCC strips pre-contracted with endothelin-1 (10nM). Rabbit corpus cavernosum presented basal release of both 6-ND (2.9±0.8ng/mL, n=12) and noradrenaline (1.7±1.3ng/mL, n=12). The 6-ND release was reduced by pre-treatment with Nω-nitro-l-arginine methyl ester (l-NAME) (100 µM), whereas that of noradrenaline was unaffected. Tetrodotoxin (TTX, 1 µM) abolished the noradrenaline release but had no effect on 6-ND release, indicating a non-neurogenic origin for 6-ND. 6-ND and the selective dopamine D2-agonist L-741,626 caused concentration-dependent RbCC relaxations (pEC50 of 11±0.15 and 11.15±0.28, respectively). Pre-treatment with either l-NAME or the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-on (ODQ) (100 µM) caused a rightward shift of the concentration-response curve to 6-ND, without affecting the L-741,626 responses. In TTX (100nM)-pre-treated preparations, neither l-NAME nor ODQ shifted the 6-ND concentration-response curve. Dopamine, noradrenaline, and adrenaline caused concentration-dependent RbCC contractions. Pre-incubation with 6-ND concentration-dependently inhibited the dopamine-induced contractions, without affecting those induced by either noradrenaline or adrenaline. 6-Nitrodopamine is the most potent endogenous relaxant agent in RbCC ever described and represents a novel mechanism by which NO causes corpus cavernosum smooth muscle relaxation. The finding that 6-ND acts as a truly selective dopamine D2-receptor antagonist indicates that the balance of dopamine and 6-ND release/synthesis may be the main mechanism that modulates corpus cavernosum smooth muscle tonus in vivo.

5-hydroxytryptamine-evoked contractile activity of large gut in neonatal albino rats

Objectives: Serotonin, also known as 5-hydroxytryptamine (5-HT), is a monoamine neurotransmitter. It is abundantly present in the gut of mammals. The extent to which it contributes to the contraction of the neonatal gut requires further investigation. This study aimed to assess the effect of 5-HT on the contractions of the large intestine in newborn albino rats. Materials and Methods: The colon and rectum samples were collected from neonatal and adult albino rats for analysis. Further, in an organ bath, isometric contractions of these isolated gut segments were recorded, in vitro, using a force transducer and a computerised chart recorder, with and without 5-HT in different groups. The 5-HT-induced contractions were also recorded in gut segments pre-treated with various antagonists. Results: 5-HT (0.01–10 μM) caused a significantly (P &lt; 0.05) greater contractile response (g/g wet tissue) in neonate rats as compared to adults. The response was greater in the rectum as compared to the colon in both neonates and adults. In neonate rats, ondansetron, a 5-HT3 antagonist, could not block the 5-HT-induced large gut contractions, while, in adult rats, it significantly blocked the 5-HT-evoked gut contractility. Methysergide, a 5-HT1/2/5-7 antagonist, blocked the response in both the adult and neonate rectum. Conclusion: The 5-HT-evoked response is mediated through 5-HT3 receptor subtypes in adults but not in neonate colon and rectum, indicating possible changes in the distribution of 5-HT receptors in the colon and rectum during development. Furthermore, atropine (a muscarinic cholinergic blocker) and hexamethonium (a ganglion blocker) could not affect the 5-HT-evoked responses in the neonate or adult rats’ colons or rectum. The effect of 5-HT did not appear to involve cholinergic or enteric ganglionic elements.

Mechanism of action of &lt;i&gt;Pterorhachis&lt;/i&gt; &lt;i&gt;zenkeri&lt;/i&gt; (Meliaceae), a sexual enhancer: effect on vas deferens contractility

Background: Pterorhachis. zenkeri commonly known as “Ayilalou” or “Démareur” is an aphrodisiac plant used by Cameroonian traditional healers to enhance libido and sperm production. This study was undertaken to investigate the aphrodisiac mechanism of P. zenkeri on the contractile activity of vas deferens with regard to its function on ejaculation. Materials and Methods: The proximal parts of rat vas deferens were mounted in a 20 mL organ bath containing Krebs solution at 37°C. Cumulative dose-response curves were recorded with KCl (35-280 mM), adrenaline (7-70 µM) and aqueous extract of P. zenkeri (3,55-21,3 mg/mL). Prazosin (type 1 alpha-adrenergic receptor antagonist, 10 µM), Nifedipine (a specific L-type calcium channel antagonist, 5 µM), 2-AminoethoxydiPhenyl Borate (2-APB, IP3 receptor antagonist, 200 μM) and calcium-free medium were further used to study the mechanism of action. Results: KCl, adrenaline and aqueous extract of P. zenkeri showed a concentration-dependent effect on vas deferens contractibility. Nifedipine and Pazosin partially inhibited the effects of P. zenkeri. In calcium-free medium containing Ethylene Glycol-bis (2-aminoethylether)-N,N,N’N’-tetraacetic acid (EGTA, 1µM), P. zenkeri triggered vas deferens contraction which was completely abolished in the presence of 2-APB. These results suggest that the aqueous extract of P. zenkeri possesses bioactive compounds capable of inducing vas deferens contractions through type 1 alpha adrenergic receptors and mobilization of extra and intracellular calcium. Conclusion: These results confirm the aphrodisiac properties of P. zenkeri through the activation of the emissive phase of ejaculation.

Comparative vascular effects of levetiracetam and valproate with hyperhomocysteinemia in rat models

Abstract Objectives Hyperhomocysteinemia (HHcy) a significant risk factor for vascular disease, often emerges in epilepsy with the use of antiepileptic drugs. In this relationship, our study investigates the combined effects of HHcy and antiepileptics on vascular function using a rat model. Methods Fourty two rats were included and divided into six groups as, 1-Control, 2-L-Met, 3-LEV injected, 4-LEV-injected + L-Met, 5-VAL-injected, 6-VAL injected + L-Met. L-Methionine (L-Met) was added to drinking water of rats for 1 month to develop HHcy. Simultaneously, intraperitoneal (ip) injections of sodium valproate (VAL) and levetiracetam (LEV) were administered. Effects were comparatively investigated, and noradrenaline (NA), followed by acetylcholine (ACh) and glyceryl trinitrate (GTN) were applied in organ bath system. Agonist doses were expressed as ten base logarithm (M) through 10−9, 10−8, 10−7, 10−6, 10−5, 10−4 mol/L in dose-response graph. Results NA contractions between LEV and LEV + L-Met groups showed statistical significance (LEV Emax=288.50 ± 46.54, LEV + L-Met Emax=480.40 ± 78.83) (p&lt;0.05) however, no significance was observed among the other groups. ACh relaxations between Control-L-Met (Control Inhmax=12.65 ± 2.09, L-Met Inhmax=50.05 ± 7.43) (p&lt;0.05), and Control-Val + L-Met (Control Emax=328.20 ± 52.83, VAL + L-Met Emax=452.60 ± 71.53) (p&lt;0.01), groups showed statistical significance. Between other groups, no significance was observed. In GTN relaxations, no statistical significance was observed. Conclusions This study highlights the adverse impact of HHcy on aortic relaxation. Further impairment was observed with VAL compared to other treatment and control groups. These findings underscore the importance of considering vascular side effects when selecting antiepileptic drugs. Ultimately, our study contributes valuable insights that may aid the choice of appropriate treatment strategies to mitigate potential vascular complications of HHcy.

Investigating the Effect of Serotonin II and III Receptor Inhibitors on the Chronotropic Changes of the Heart to Isoproterenol in Rat Models of Biliary Cirrhosis

Cirrhotic patients have hyperdynamic circulation and at-rest tachycardia, and agents that activate the sympathetic pathway, such as physical practice and pharmacologic stimulations, compared with the normal population, cannot cause enough increase in heartbeat, a condition known as cirrhotic cardiomyopathy. Concerning the presentation of 5-HT2 &amp; 5-HT3 receptors in rat hearts, we used Ketanserin as a 5-HT2 receptor inhibitor &amp; Tropisetron as a 5-HT3 receptor inhibitor to evaluate chronic therapeutic effects of 5-HT2 &amp; 5-HT3 antagonists on the cardiac chronotropic response of cirrhotic rats to adrenergics. Cirrhosis was induced by surgical ligation of the bile duct in Male Wistar rats, and another group remained sham. A week after bile duct ligation or sham surgery, the subjects were given an intraperitoneal injection of either saline or Tropisetron (2 mg/kg). In other BDL &amp; Sharm groups, the subjects were given an intraperitoneal injection of either saline or Ketanserin (6 mg/kg) every other 3 days in the last 3 weeks. Four weeks after bile duct ligation or sham surgery, the atria were isolated and chronotropic responsiveness to Isoproterenol was assessed using a standard organ bath. Our data showed that chronic treatment with Tropisetron (5-HT3 antagonist) in cirrhotic rats could decrease the cardiac chronotropic response. Chronic treatment with Tropisetron can cause a significant decrease in cardiac chronotropic response to Isoproterenol in healthy and cirrhotic rats, even lower than that in cirrhotic rats (without any special treatment). Chronic treatment with Ketanserin cannot change their impaired chronotropic response to Isoproterenol.&#x0D;

Nitric oxide and ion channels mediate L-cysteine-induced inhibition of colonic smooth muscle contraction.

Previous studies have suggested that L-cysteine regulates gut motility through hydrogen sulfide. However, the mechanisms involved in the L-cysteine-induced response have not been extensively studied. This study aimed to investigate the underlying mechanisms of action of L-cysteine on spontaneous contraction of rat colon. Longitudinal and circular muscle strips from rat middle colon were prepared to measure the spontaneous contractile activities of colon in an organ bath system. Whole-cell voltage-clamp techniques were applied to record the currents of L-type voltage-dependent Ca2+ channels (VDCCs) and voltage-gated K+ channels (Kv) in isolated smooth muscle cells (SMCs) from colon. L-cysteine inhibited the spontaneous contraction of longitudinal and circular muscle strips from the rat colon in a concentration-dependent manner. The inhibition induced by L-cysteine was significantly decreased by inhibitors of H2S synthesis (p < 0.05). Furthermore, the suppression induced by L-cysteine was partially attenuated by tetrodotoxin, L-NNA and glibenclamide (p < 0.05). Whole-cell voltage-clamp recordings showed that L-cysteine caused a remarkable reduction in the peak currents of VDCCs and significantly increased the membrane currents of Kv channels in isolated SMCs (p < 0.05). We concluded that L-cysteine inhibits the contractile activities of smooth muscle strips from the rat colon. The relaxation in response to L-cysteine may be in part mediated by a nitrergic pathway and by inhibiting the VDCCs in combination with a direct activation of the KV channels and KATP channels.