Application Of Acetylcholine Research Articles

Cholinergic regulation of cardiac pacemaker activity by L-type Cav1.3 channels

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondation Recherche Médicale Introduction The cholinergic regulation of heart rate (HR) is mediated by acetylcholine (ACh)-dependent activation of M2-receptors (M2R). Activated M2R promote release of the βγ-subunit of G-proteins to directly gate GIRK1/4 channels (underlying the cardiac IKACh current), while αi-subunits inhibit adenylate cyclase (AC) activity. AC inhibition reduces the intracellular concentration of cAMP, decreasing the activity of ion channels involved in pacemaking, including "funny" f-(HCN4) and L-type Cav1.3 calcium channels. Purpose To determine the role of L-type Cav1.3 channels in cholinergic regulation of heart rate. Methods We recorded the frequency of activation and position of pacemaker leading site in ex vivo sinus nodes and the HR of isolated Langendorff perfused hearts of mice at baseline or during ACh perfusion. We used control wild type (WT) mice, and five genetically modified mouse models: Cav1.3 knockout (KO, ablated Cav1.3-mediated L-type current), GIRK4KO (ablated IKACh current), HCN4-CNBD (selective deletion of cAMP-dependent regulation of HCN4), GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO. We performed in vivo telemetric recordings of heart rate (HR) in WT and GIRK4KO/Cav1.3KO animals. Results Data from optical mapping experiments showed that, under basal conditions, perfusion of 3 μM ACh significantly reduced the frequency of action potentials in WT (44%), HCN4-CNBD (38%), Cav1.3KO (65%) and GIRK4KO (8%) isolated mouse sinus node tissues. ACh application did not significantly affect the frequency of action potentials recorded in tissue from GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO animals. Furthermore, in all sinus nodes tested, regardless of genotype, ACh shifted the pacemaker leading site from its normal position by at least 0.7 mm. Upon stimulation of the β-adrenergic pathway by Isoproterenol, to reproduce conditions of accentuated antagonism, 3µM ACh reduced HR in isolated hearts from WT (43.8%), HCN4-CNBD (38.7%), Cav1.3KO (25,4%), GIRK4KO (16.9%) and GIRK4KO/HCN4-CNBD (16.4%) mice. No significant HR reduction was recorded in hearts from GIRK4KO/Cav1.3KO animals. In vivo data indicate that HR reduction induced by combined injection of Hexamethonium ( a Nicotinic acetylcholine receptor blocker) with Carbamoylcholine (CCH, M2 receptor agonist) or with 2-Chloro-N6-Cyclopentyladenosine (CCPA, A1 receptor agonist) is higher in WT than in GIRK4KO/Cav1.3KO animals (68% vs 48% CCH, and 79% vs 62% CCPA, respectively). Conclusion Our data indicate that L-type Cav1.3 channels are involved in cholinergic regulation of heart rate in mice. In addition, when the intracellular concentration of cAMP is elevated (i.e. under conditions of accentuated antagonism), cholinergic regulation of sinus node pacemaking is reliant on Cav1.3 and KACh channels.

Functional and Molecular Properties of DYT-SGCE Myoclonus-Dystonia Patient-Derived Striatal Medium Spiny Neurons.

Myoclonus-dystonia (DYT-SGCE, formerly DYT11) is characterized by alcohol-sensitive, myoclonic-like appearance of fast dystonic movements. It is caused by mutations in the SGCE gene encoding ε-sarcoglycan leading to a dysfunction of this transmembrane protein, alterations in the cerebello-thalamic pathway and impaired striatal plasticity. To elucidate underlying pathogenic mechanisms, we investigated induced pluripotent stem cell (iPSC)-derived striatal medium spiny neurons (MSNs) from two myoclonus-dystonia patients carrying a heterozygous mutation in the SGCE gene (c.298T>G and c.304C>T with protein changes W100G and R102X) in comparison to two matched healthy control lines. Calcium imaging showed significantly elevated basal intracellular Ca2+ content and lower frequency of spontaneous Ca2+ signals in SGCE MSNs. Blocking of voltage-gated Ca2+ channels by verapamil was less efficient in suppressing KCl-induced Ca2+ peaks of SGCE MSNs. Ca2+ amplitudes upon glycine and acetylcholine applications were increased in SGCE MSNs, but not after GABA or glutamate applications. Expression of voltage-gated Ca2+ channels and most ionotropic receptor subunits was not altered. SGCE MSNs showed significantly reduced GABAergic synaptic density. Whole-cell patch-clamp recordings displayed elevated amplitudes of miniature postsynaptic currents and action potentials in SGCE MSNs. Our data contribute to a better understanding of the pathophysiology and the development of novel therapeutic strategies for myoclonus-dystonia.

Influence of the cumulative effect of zoledronic acid on periodontal microcirculation in rats.

To evaluate the effect of repeated administration of zoledronic acid (ZA) on the development and severity of osteonecrosis of the jaws. In the experiment, 36 rats were used, which were divided into 4 groups: group 1 was injected with saline for 6 weeks, group 2 was injected with ZA once, group 3 was injected zoledronic acid for 3 weeks once a week, group 4 was injected with ZA for 6 weeks once a week. While taking medications, the tooth was removed. The volumetric blood flow rate was studied using laser and high-frequency ultrasound Doppler in the area of the periodontium of an extracted tooth in rats with the application of acetylcholine. Bone tissue was examined out using CBCT. In group 2, there was a violation of blood flow in the mucous membrane, bone microcirculation, but no reliable data was obtained in the bone defect in comparison with group 1. In groups 3 and 4, there was significantly disrupted blood flow. This led to an increase of the osteonecrosis (maximum at the 4th group), which was confirmed by data obtained using CBCT. An inverse relationship was observed between the blood flow of bone tissue and the size of the defect after tooth extraction. The introduction of ZA in a dose-dependent fashion resulted in impaired endothelial vasodilation and impaired blood flow to extraction sockets. These findings might explain the development of osteonecrosis of the mandible following removal of a first molar.

Functional and Pathological Influence of Hypothermia on Spike Activity of Cortical Neurons

In sensorimotor cortical slices of guinea pig using local iontophoretic application of glutamate to the soma and dendrites it was found that a decrease of temperature of incubating fluid from 34 to 21°C - 22°C changes the somatic responses to the local injection of glutamate to the dendritic loci, while the responses to iontophoretic application of glutamate to the soma remain unchanged. Hypothermic changes in reactivity to dendritic stimulation start below 30°C and coincide with changes in the spontaneous activity of neurons, both in the direction of increasing and decreasing the frequency of firing in different nerve cells. On hypothermic decrease of spontaneous activity, the latencies of evoked dendritic responses on the soma became more longer, while on hypothermic increase of firing level, somatic spike responses to iontophoretic application of glutamate to dendritic loci appeared with shorter latencies. Hypothermic changes in the physiological parameters of neurons were accompanied by a drop in spike amplitude at the same temperature and with its further decrease. At the same time, there was a decrease of spike reaction to iontophoretic application of acetylcholine below 30°. It is proposed that the reason for hypothermic changes of neuronal activity is decreasing rate of M-cholinergic process at 27°C - 29°C which leads to opening K+ channels of neuronal membranes and hence to attenuation of conductive function of dendrites and to imbalance of K+ ion homeostasis. Peculiarities of hypothermic regulation of neuronal spike activity depend on individual functional properties of cortical neurons.

Regulation of Vascular Smooth Muscle Contractions in the Model of Metabolic Syndrome.

Reduced glucose tolerance, hyperglycemia, and imbalance in lipid levels were found in rats with metabolic syndrome induced by a high-fat, high-carbohydrate diet. The contractile responses of intact and endothelium-denuded aortic smooth muscle segments from rats with metabolic syndrome to application of acetylcholine, phenylephrine, sodium nitroprusside, and forskolin were studied by mechanographic method. It was found that endothelial dysfunction develops against the background of metabolic and hemodynamic disorders in metabolic syndrome. It was shown that the regulation of vasoconstrictor reactions of vascular smooth muscles in metabolic syndrome is due to a decrease in Ca2+ entry, mainly voltage-independent, as well as changes in the function of cGMP- and cAMP-activated K+-channels.

Comparative transcriptome analysis reveals the regulatory effects of acetylcholine on salt tolerance of Nicotiana benthamiana

Salinity is a major cause of crop losses worldwide. Acetylcholine (ACh) can ameliorate the adverse effects of abiotic stresses on plant growth, including salinity stress; however, the underlying molecular mechanisms of this process are unclear. Here, seedlings of Nicotiana benthamiana grown under normal conditions or exposed to 150 mmol L−1 NaCl salinity stress were then treated with a root application of 10 μM ACh. Exogenous ACh application resulted in the downregulation of the activity of the antioxidant enzymes, ascorbate peroxidase, and catalase. ACh-treated plants had lower levels of reactive oxygen species, including the superoxide anion radical and hydrogen peroxide. Transcriptome analysis indicated that ACh treatment under salt stress promoted the differential expression of 658 genes in leaves of N. benthamiana (527 were upregulated and 131 were downregulated). Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that exogenous ACh application was associated with a substantial increase in the transcripts of genes related to cell wall peroxidases, xyloglucan endotransglucosylases or hydrolases, and expansins, indicating that ACh activates cell wall biosynthesis in salt-stressed plants. ACh also enhanced the expression of genes associated with the auxin, gibberellin, brassinosteroid, and salicylic acid signalling pathways, indicating that ACh induces the activation of these pathways under salt stress. Collectively, these findings indicate that ACh-induced salt tolerance in N. benthamiana seedlings is mediated by the inhibition of antioxidant enzymes, activation of cell wall biosynthesis, and hormone signalling pathways. Stress-induced genes involved in osmotic regulation and oxidation resistance were induced by ACh under salt stress. The genes whose transcript levels were elevated by ACh treatment in salt-stressed N. benthamiana could be used as molecular markers of the physiological status of plants under salt stress.

Effects of exenatide on microvascular reactivity in patients with type 2 diabetes and coronary artery disease: A randomized controlled study.

We studied the effect of the GLP-1RA exenatide on skin microvascular function in patients with T2DM and CAD. Thirty-five patients with T2DM, CAD, and HbA1C 42-86mmol/mol were randomized to treatment with exenatide or conventional non-GLP-1-based therapy for 12weeks. Skin microvascular function was examined in the forearm by LDF and iontophoretic application of acetyl choline and SNP, and by PORH at baseline and after 12weeks. Blood samples for fasting plasma glucose, HbA1C, and lipid profile were collected. At 12weeks, patients on exenatide showed reductions in HbA1C (from 63.5±13 to 60.7±14mmol/mol, p=.065), body weight (from 92.6±16 to 89±16kg, p<.001), and systolic blood pressure (from 141±13 to 134±16mmHg, p<.05) as compared to the conventionally treated group. There were no significant changes in skin microvascular function between or within the two groups at follow-up. Three months' daily treatment with the GLP-1RA exenatide in T2DM patients with CAD showed no significant effects on skin microvascular function or blood glucose control, while this study confirms a reduction in body weight and blood pressure by exenatide, as compared to conventional antidiabetic drug treatment.

Gamma-aminobutyric acid (GABA) and acetylcholine (ACh) alleviate water deficit effects in soybean: From gene expression up to growth performance

Bioregulators act as priming agents under abiotic stress conditions. Bioregulators such as gamma-aminobutyric acid (GABA) and acetylcholine (ACh) affect the efficiency of the antioxidant system and the regulation of the stomatal aperture, respectively. The aim of this study was to verify a possible synergistic effect among these bioregulators as attenuators of the effects of water deficiency in Glycine max. We combined the application of GABA and ACh at 2.0 mM in soybean plants under different water regimes. The factors studied were: 1) application of Gaba and ACh in seeds (S); in leaves (L); in seeds and leaves (SL); control without application (C); and (2) water regimes at 100 % field capacity (FC) and water deficit (WD). When the application of bioregulators were removed from the process, a severe decrease in photosynthesis capacity (93 %) was observed on the sixth day (after withholding water), as well as a higher expression of the genes known to be induced by water deficit. The combination of GABA and ACh applied to seeds and leaves under water deficit resulted in a lower decline in photosynthesis, as well as better water-use efficiency and biomass production. Soybean plants subjected to this treatment also showed lower expression of GmABA2, GmLEA3 and GmP5CS genes, lower proline content and increased activity of SOD, CAT and APX compared to the control treatment. The results indicate that the combined exogenous application of GABA and ACh in soybean plants acted to promote increased tolerance to water deficit, showing their potential for use on agricultural areas which are prone to droughts.

Cholinergic regulation of cardiac pacemaker activity by l-type cav1.3 channels

Abstract Introduction The cholinergic regulation of heart rate (HR) is mediated by acetylcholine (ACh)-dependent activation of M2-receptors (M2R). Activated M2R promote release of the βγ-subunit of G-proteins to directly gate GIRK1/4 channels (underlying the cardiac IKACh current), while αi-subunits inhibit adenylate cyclase (AC) activity. AC inhibition reduces the intracellular concentration of cAMP, decreasing the activity of ion channels involved in pacemaking, including “funny” f-(HCN4) and L-type Cav1.3 calcium channels. Purpose To determine the importance of L-type Cav1.3 channels in the cholinergic regulation of heart rate. Methods We recorded the frequency and the position of the pacemaker leading site in ex vivo sinus nodes and the HR of isolated Langendorff perfused hearts of mice in control or during ACh perfusion. We used control wild type (WT) mice, and five genetically modified mouse models: Cav1.3 knockout (KO, ablated Cav1.3-mediated L-type current), GIRK4KO (ablated IKACh current), HCN4-CNBD (selective deletion of cAMP-dependent regulation of HCN4), GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO. Results Data from optical mapping experiments showed that, under basal conditions, perfusion of 3 μM ACh significantly reduced the frequency of action potentials in WT (44%), HCN4-CNBD (38%), Cav1.3KO (65%) and GIRK4KO (8%) isolated mouse sinus node tissues. ACh application did not significantly affect the frequency of action potentials recorded in tissue from GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO animals. Furthermore, in all the sinus node tissues tested, regardless of the genotypes, ACh shifted the pacemaker leading site from its normal position by at least 0.7 mm. Upon stimulation of the β-adrenergic pathway by Isoproterenol, to reproduce conditions of accentuated antagonism, 3μM ACh reduced HR in isolated hearts from WT (43.8%), HCN4-CNBD (38.7%), Cav1.3KO (25,4%), GIRK4KO (16.9%) and GIRK4KO/HCN4-CNBD (16.4%) mice. No significant HR reduction was recorded in hearts from GIRK4KO/Cav1.3KO animals. Conclusion Our data indicate that L-type Cav1.3 channels are involved in cholinergic regulation of heart rate in mice. In addition, when the intracellular concentration of cAMP is elevated (i.e. under conditions of accentuated antagonism), the cholinergic regulation of sinus node pacemaking is predominantly ensured by Cav1.3 and KACh channels. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): “Fondation pour la recherche medicale” FRM

Nicotinic acetylcholine receptors in the synganglion of the tick Ixodes ricinus: Functional characterization using membrane microtransplantation

Nicotinic acetylcholine receptors are an important class of excitatory receptors in the central nervous system of arthropods. In the ticks Ixodes ricinus, the functional and pharmacological properties of nicotinic receptors located in their neurons are still unknown. The objective of this study was to characterize the pharmacological properties of tick nicotinic receptors using membrane microtransplantation in Xenopus laevis oocytes and two-electrodes voltage clamp method. The membranes microtransplanted were extracted from the tick synganglion. We found that oocytes microtransplanted with tick synganglion membranes expressed nicotinic acetylcholine receptor subtypes which were activated by acetylcholine (1 mM) and nicotine (1 mM). Currents induced by pressure application of acetylcholine and nicotine were diminished by 10 nM α-bungarotoxin and methyllycaconitine, suggesting that they expressed two subtypes of nicotinic receptors, α-bungarotoxin-sensitive and -insensitive, respectively. In addition, we found that nicotine receptors expressed in the synganglion membranes were poorly sensitive to the neonicotinoid insecticides clothianidin (CLT), imidacloprid (IMI), acetamiprid (ACE) and thiamethoxam (TMX), in agreement with their lack of activity as acaricides. Interestingly, current amplitudes were strongly potentialized in the presence of 1 μM PNU-120596. CLT was more active as an agonist than IMI, TMX and ACE. Finally, we demonstrated that microtransplantation of purified membrane from the tick synganglion can be a valuable tool for the development and screening of compounds targeting tick nicotinic acetylcholine receptor subtypes.

Nicotine Self-Administration Induces Plastic Changes to Nicotinic Receptors in Medial Habenula.

Chronic nicotine upregulates nicotinic acetylcholine receptors (nAChRs) throughout the brain, and reducing their activity may promote somatic and affective states that lead to nicotine seeking. nAChRs are functionally upregulated in animal models using passive nicotine administration, but whether/how it occurs in response to volitional nicotine intake is unknown. The distinction is critical, as drug self-administration (SA) can induce neurotransmission and cellular excitability changes that passive drug administration does not. In this study, we probed the question of whether medial habenula (MHb) nAChRs are functionally augmented by nicotine SA. Male rats were implanted with an indwelling jugular catheter and trained to nose poke for nicotine infusions. A saline SA group controlled for non-specific responding and nicotine-associated visual cues. Using patch-clamp whole-cell recordings and local application of acetylcholine, we observed robust functional enhancement of nAChRs in MHb neurons from rats with a history of nicotine SA. To determine whether upregulated receptors are generally enhanced or directed to specific cellular compartments, we imaged neurons during recordings using two-photon laser scanning microscopy (2PLSM). nAChR activity at the cell soma and on proximal and distal dendrites was examined by local nicotine uncaging using a photoactivatable nicotine (PA-Nic) probe and focal laser flash photolysis. Results from this experiment revealed strong nAChR enhancement at all examined cellular locations. Our study demonstrates nAChR functional enhancement by nicotine SA, confirming that volitional nicotine intake sensitizes cholinergic systems in the brain. This may be a critical plasticity change supporting nicotine addiction.

The Effect of Plantain Active Ingredient Aucubin on Isolated Rat Smooth Muscle Tissue and Primary Cell Lines

This study aims to assess the pharmacodynamics of aucubin, the active ingredient in plantain (Plantago), on in vitro rat bladder and trachea tissues, and explore its cell protective effects on primary lung and kidney cell lines. The study was carried out via repeated applications of acetylcholine, atropine, verapamil and oxybutynin, alongside Ca++ in a calcium-free environment, on urinary bladder tissue, and repeated applications of acetylcholine, atropine, carbachol and mecamylamine on trachea tissue. At the same time, cell viability and catalase and superoxide dismutase activity was measured on primary cell lines. The results indicate that aucubin has a relaxant effect on urinary bladder and trachea tissues. It is conceived that aucubin acts as a cholinergic antagonist through different subreceptors (muscarinic-3 receptors). The results also indicate that aucubin has a dose-dependent effect on primary lung and kidney cell lines, with increased cell viability, reduced cytotoxicity and antioxidant effects. In conclusion, it is suggested that plantain, which is used in folk medicine, and its active ingredient aucubin might have beneficial effects on bronchoconstriction and other respiratory conditions, as well as on pulmonary and renal diseases, urinary incontinence, etc.

P1161CUTANEOUS MICROCIRCULATORY DYSFUNCTION IN PERITONEAL DIALYSIS PATIENTS

Abstract Background and Aims Microvascular impairment is an early step in cardiovascular disease (CVD), the major cause of morbidity and mortality in patients with CKD. Changes in skin microvascular reactivity have been shown to reflect more widespread changes in the systemic and coronary microcirculation. Microvascular function is attenuated by advancing age and conditions that commonly coexist with CKD such as diabetes and hypertension. We investigated whether skin microvascular reactivity is impaired in peritoneal dialysis (PD) patients compared with healthy controls and whether this impairment is independent of comorbidity. Method Forearm skin vasculature was examined in 27 healthy controls, 27 patients on PD and 27 controls matched to the PD patients for age, gender, diabetes and previous CV events. Microvascular function was assessed using laser Doppler flowmetry in combination with post-occlusive reactive hyperaemia (a test of generalised microvascular function) and iontophoretic application of acetylcholine (ACh) and sodium nitroprusside (SNP) to investigate endothelium dependant and non-endothelium dependant vasodilation respectively. Results Peak post-occlusive flow was lower in the PD patients than in both the healthy controls and the co-morbidity matched group; 90.45 AU (60.9-128.35) in healthy controls, 73AU (58.8-134.4) in matched controls and 56.95AU (45.1-89.8) in PD patients (p=0.0239 healthy controls versus patients, p=0.0373 matched controls versus patients). SNP-mediated vasodilatation was statistically lower in the PD group compared with healthy controls (p= 0.016), ACh response was lower but did not reach statistical significance (p= 0.076). ACh and SNP-mediated vasodilatation trended towards being lower in PD patients than matched controls but did not reach statistical significance. Conclusion The PD patients were characterised by a generalised dysfunction of the skin microcirculation compared with healthy controls and controls matched for factors known to affect the microcirculation. This appears to be the result of impaired endothelium dependant and independent vasodilatory mechanisms.

Acetylcholine ameliorates the adverse effects of cadmium stress through mediating growth, photosynthetic activity and subcellular distribution of cadmium in tobacco (Nicotiana benthamiana).

Acetylcholine (ACh), a well-known major neurotransmitter, plays a potential role in response to abiotic stresses. However, the mechanism of ACh-mediated cadmium (Cd) toxicity in tobacco seedlings is largely uncharacterized. In this study, a hydroponics experiment was conducted under 100μM Cd stress in the presence or absence of ACh (50μM) to investigate the potential effects of ACh on Cd toxicity. The results revealed that ACh application effectively alleviated Cd-induced reductions in plant growth, photosynthetic pigments and gas exchange attributes and improved the photosystem II activity. Ultrastructural observation indicated that Cd exposure ruptured the internal structure of chloroplasts, and even caused the accumulation of osmiophilic granules in chloroplasts, whereas these phenomena were alleviated by the addition of ACh. Cd stress also caused a marked increase in oxidative stress, as evidenced by the accumulation of O2- and H2O2, which were efficiently minimized after ACh application by up-regulating antioxidant enzyme activities (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX; glutathione reductase, GR). Besides, Cd stress considerably increased the levels of glutathione (GSH), Non-protein thiols (NPTs) and phytochelatins (PCs), whereas ACh application to Cd-stressed seedlings further increased those contents, thereby enhancing the tolerance of Cd-stressed plants. Moreover, exogenously applied ACh declined the accumulation of Cd and minimized the damage from Cd toxicity by modulating the distribution of Cd in the vacuole and cell wall. Therefore, these results provide insights into the ameliorative effects of ACh on Cd-induced a series of physiological reactions.

Mechanism of gamete shedding in starfish: Involvement of acetylcholine in extracellular Ca2+-dependent contraction of gonadal walls

Starfish are suitable animals for the study of hormonal regulatory mechanism of oocyte maturation and ovulation. Although contraction of the gonadal walls is essential for the shedding gametes, little was known about the mechanism. When ovaries of starfish Patiria pectinifera were incubated in Ca2+-free seawater in the presence of 1-methyladenine (1-MeAde), the germinal vesicles in oocytes broke down, but no ovulation occurred. Verapamil, a potent inhibitor of voltage-dependent Ca2+ channels, inhibited 1-MeAde-induced ovulation. These results suggest that extracellular Ca2+ and its influx are indispensable for gamete shedding. Furthermore, acetylcholine (ACh) was involved in extracellular Ca2+-dependent contractions of gonadal walls. Although 1-MeAde failed to induce contraction of the gonadal walls in normal seawater containing L-glutamic acid, application of ACh or carbachol, an agonist for ACh receptor, could bring about shedding of mature oocytes. Atropine, a competitive antagonist of the muscarinic ACh receptor, inhibited 1-MeAde-induced ovulation, but a nicotinic ACh receptor antagonist mecamylamine had no effect. Furthermore, ACh was detected in the ovaries and testes in P. pectinifera. These findings suggest that ACh acts on muscarinic ACh receptors in gonadal walls to induce peristaltic contractions caused by Ca2+ influx via Ca2+ channels in the gonadal wall muscle for gamete shedding. The present study also provides new insight into the regulatory mechanism that 1-MeAde acts on secretion of ACh in ovaries and testes.

Роль NO и H 2 S в регуляции тонуса церебральных сосудов при хронической болезни почек

Chronic kidney disease (CKD) is a widely spread condition that is a risk factor for the development of cerebrovascular diseases and neurological complications. Currently the mechanisms by which CKD increases the risk of brain damage are poorly understood. In our study, NO- and H2S-mediated changes in the cellular and molecular mechanisms of cerebral artery tone regulation were investigated in male Wistar rats in vivo with a CKD model. CKD was modeled by removal of 5/6 renal tissue at 4 months of age. After 4 months, part of the parietal bone and the dura mater were removed, and the diameter of the arteries was measured under the action of blockers and signaling pathway inhibitors using a microscope. In nephrectomized rats, the reactions of cerebral arteries to the application of acetylcholine (AH) manifested mainly in the form of constriction, while vasodilation predominated in the control group. Sodium nitroprusside led to a dilatation of the arteries, the amplitude of which in rats with CKD was significantly lower compared to the control group. Methylene blue and glibenclamide in rats with CKD led to a slight weakening of reactions to ACh, while in control group rats, the dilatation of arteries was significantly reduced. The degree of dilatation of cerebral arteries on H2S in rats with CKD was significantly less compared to rats in the control group. Propargylglycine caused only slight changes in artery diameter of CKD rats, while in the control group the reaction to propargylglycines was statistically significant. We conclude that the CKD attenuates endothelium-dependent and endothelium-independent NO- and H2S-mediated dilator reactions in cerebral arteries.

NO-dependent mechanism of vasodilation in pial arteries of nefrectomizedrats

THE AIM: to study changes in the NO-mediated dilatation mechanism in the pial arteries of the brain of nephrectomized rats.MATERIALS AND METHODS: The study was conducted on Wistar-Kyoto rats. At 4 months of age, a two-stage nephrectomy was performed. After 3 months, the reaction of the arteries of the pial membrane of the brain to agonists and antagonists was investigated by in vivo microscopy (the diameter of the arteries was measured against the action of acetylcholine, sodium nitroprusside and methylene blue). Besides, the perfusion of the brain tissue was measured for subsequent calculations of the values of the components of the vascular tone.RESULTS: It was shown that the application of acetylcholine to the pial membrane led to a change in the diameter of the arteries. In nephrectomized rats under the action of acetylcholine, a significantly larger number of arteries in the constriction state was registered compared to the control group. The effect of sodium nitroprusside in the control group was accompanied by a dilatation of 100 % of the pial arteries; in the nephrectomized rat group, dilatation was detected in 83.2 ± 4.7 % of the arteries. When methylene blue was used in a group of nephrectomized rats, a smaller number of arteries in the constriction state was detected as compared to the control group.CONCLUSION: In nephrectomized rats, pronounced disorders of the NO-mediated mechanism of the pial arteries of the brain were found, leading to an increase in the endothelial component of the vascular tone. The endothelium of the pial arteries of nephrectomized rats produces less NO both spontaneously and when stimulated with acetylcholine. In nephrectomized rats, abnormalities in the signal cascade of NO →sGC → cGMP in the smooth muscle cells of the pial arteries were revealed, which is confirmed by their lesser ability to dilate to the use of exogenous NO.

Propofol But Not Desflurane Maintains Rat Cerebral Arteriolar Responses to Acetylcholine During Acute Hyperglycemia.

Acute hyperglycemia causes vascular endothelial dysfunction in various organs including the cerebral vessels. It is associated with increased mortality and morbidity in the perioperative period. The impact of anesthetic agents on cerebral vasodilatory responses during hyperglycemia remains unclear. We investigated endothelial function in rat cerebral arterioles during acute hyperglycemia, under propofol or desflurane anesthesia. A closed cranial window preparation was used to measure changes in pial arteriole diameter induced by topical application of acetylcholine (ACh), an endothelium-dependent vasodilator, in rats anesthetized with propofol or desflurane. Pial arteriole responses to ACh were measured during normoglycemia and hyperglycemia. We then investigated whether the response of cerebral arterioles to acute hyperglycemia under propofol anesthesia were related to propofol or its vehicle, intralipid. ACh resulted in a dose-dependent dilation of cerebral arterioles during propofol and desflurane anesthesia under normoglycemic conditions. The vasodilatory effects of ACh were also maintained under hyperglycemic conditions during propofol anesthesia, but the vasodilator response to ACh was significantly impaired during hyperglycemia compared with normoglycemia with desflurane anesthesia. The vasodilatory effects of ACh were maintained during normoglycemia and hyperglycemia in rats receiving propofol or intralipid. Rat pial arteriole responses to ACh are maintained during conditions of acute hyperglycemia with propofol anesthesia but suppressed compared with normoglycemia with desflurane anesthesia.

NITRIC OXIDE SYNTHASE DYSFUNCTION UNDERLIES CEREBROVASCULAR DEFICITS IN MOUSE MODELS OF TAUOPATHY

Our laboratory recently reported the accumulation of pathogenic soluble aggregated tau (tau oligomers) in the cerebral microvasculature of human patients with tauopathies, including Alzheimer's disease (AD). The functional consequences of cerebrovascular tau accumulation, however, are not yet understood. The aim of the present study was to determine whether brain microvascular tau accumulation leads to microvascular dysfunction. To this end, we assessed the coupling between increased neural activity and increased cerebral blood flow, a highly regulated processes called neurovascular coupling (NVC), in several different models of tauopathy. Laser Doppler flowmetry was used to determine changes in cerebral blood flow evoked by whisker stimulation, a measure of NVC, in JNPL3, PS19, and hTau mice expressing the P301L mutation, P301S mutation, and wild-type human tau respectively, compared to appropriate controls for each strain. Inhibitors of nitric oxide synthases (NOS) were topically applied on the stimulated barrel cortex during NVC to elucidate the contribution of neuronal NOS (nNOS) and other NOS (endothelial NOS, eNOS) to NVC in each strain. In a separate group of animals, we measured vascular reactivity as endothelium-dependent vasodilation following topical application of acetylcholine (ACh). To assess the impact of pathogenic tau on NOS function, we treated primary neurons or brain vascular endothelial cells with soluble tau aggregates and measured activity of nNOS and eNOS as relative phosphorylated and total enzyme levels in neurons and endothelial cells respectively. Our data indicate that isolated overexpression of WT or mutant tau in models of tauopathy impairs NVC responses, and that this deficit is mediated by a reduction in nNOS activity in vivo. Further, our studies show that tauopathy also impairs endothelium-dependent vasoreactivity. We found that soluble tau aggregates inhibit the phosphorylation of NOS in primary cultured cells. Thus, pathogenic tau aggregates may impair NOS activation, leading to microvascular dysfunction in tauopathies. Tauopathies are associated with cerebrovascular dysfunction. The inhibition of NOS phosphorylation by pathogenic soluble tau aggregates may underlie microvascular dysfunction in tauopathies. Thus, therapeutic modulation of pathogenic tau may mitigate brain microvascular deficits, which occur prior to clinical onset in Alzheimer's disease and potentially other tauopathies.

Probing spatial inhomogeneity of cholinergic changes in cortical state in rat

Acetylcholine (ACh) plays an essential role in cortical information processing. Cholinergic changes in cortical state can fundamentally change how the neurons encode sensory input and motor output. Traditionally, ACh distribution in cortex and associated changes in cortical state have been assumed to be spatially diffuse. However, recent studies demonstrate a more spatially inhomogeneous structure of cholinergic projections to cortex. Moreover, many experimental manipulations of ACh have been done at a single spatial location, which inevitably results in spatially non-uniform ACh distribution. Such non-uniform application of ACh across the spatial extent of a cortical microcircuit could have important impacts on how the firing of groups of neurons is coordinated, but this remains largely unknown. Here we describe a method for applying ACh at different spatial locations within a single cortical circuit and measuring the resulting differences in population neural activity. We use two microdialysis probes implanted at opposite ends of a microelectrode array in barrel cortex of anesthetized rats. As a demonstration of the method, we applied ACh or neostigmine in different spatial locations via the microdialysis probes while we concomitantly recorded neural activity at 32 locations with the microelectrode array. First, we show that cholinergic changes in cortical state can vary dramatically depending on where the ACh was applied. Second, we show that cholinergic changes in cortical state can vary dramatically depending on where the state-change is measured. These results suggests that previous work with single-site recordings or single-site ACh application should be interpreted with some caution, since the results could change for different spatial locations.