Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.
Amino Acids ; Binding Sites ; Calcium ; Cytosol ; Glutamic Acid ; Myocytes, Smooth Muscle ; Permeability ; Polyamines ; Receptors, Muscarinic ; Spermine ; Transient Receptor Potential Channels

BACKGROUND: Efficacy and safety of tiotropium bromide, a muscarinic receptor antagonist, in treatment of asthma have been reported. However, its effect on airway remodeling in chronic asthma of the elderly has not been clearly verified. The objective of this study was to investigate the effect of tiotropium and expression of muscarinic receptors as its related mechanism in an aged mouse model of chronic asthma with airway remodeling. METHODS: BALB/c female mice age 6 weeks, 9 and 15 months were sensitized and challenged with ovalbumin (OVA) for three months. Tiotropium bromide was administered during the challenge period. Airway hyperresponsiveness (AHR) and pulmonary inflammation were measured. Parameters of airway remodeling, and expression levels of M2 and M3 receptors were examined. RESULTS: Total cell with eosinophils, increased in the OVA groups by age, was decreased significantly after treatment with tiotropium bromide, particularly in the age group of 15 months. AHR and levels of interleukin (IL)-4, IL-5, and IL-13 were decreased, after tiotropium administration. In old aged group of 9- and 15-months-treated groups, hydroxyproline contents and levels of α-smooth muscle actin were attenuated. Tiotropium enhanced the expression of M2 but decreased expression of M3 in all aged groups of OVA. CONCLUSION: Tiotropium bromide had anti-inflammatory and anti-remodeling effects in an aged mouse model of chronic asthma. Its effects seemed to be partly mediated by modulating expression M3 and M2 muscarinic receptors. Tiotropium may be a beneficial treatment option for the elderly with airway remodeling of chronic asthma.
Actins ; Aged ; Airway Remodeling ; Animals ; Asthma* ; Eosinophils ; Female ; Humans ; Hydroxyproline ; Interleukin-13 ; Interleukin-5 ; Interleukins ; Mice* ; Ovalbumin ; Ovum ; Pneumonia ; Receptors, Muscarinic* ; Tiotropium Bromide*

Most diabetic patients experience diabetic mellitus (DM) urinary bladder dysfunction. A number of studies evaluate bladder smooth muscle contraction in DM. In this study, we evaluated the change of bladder smooth muscle contraction between normal rats and DM rats. Furthermore, we used pharmacological inhibitors to determine the differences in the signaling pathways between normal and DM rats. Rats in the DM group received an intraperitoneal injection of 65 mg/kg streptozotocin and measured blood glucose level after 14 days to confirm DM. Bladder smooth muscle contraction was induced using acetylcholine (ACh, 10⁻⁴ M). The materials such as, atropine (a muscarinic receptor antagonist), U73122 (a phospholipase C inhibitor), DPCPX (an adenosine A1 receptor antagonist), udenafil (a PDE5 inhibitor), prazosin (an α₁-receptor antagonist), papaverine (a smooth muscle relaxant), verapamil (a calcium channel blocker), and chelerythrine (a protein kinase C inhibitor) were pre-treated in bladder smooth muscle. We found that the DM rats had lower bladder smooth muscle contractility than normal rats. When prazosin, udenafil, verapamil, and U73122 were pre-treated, there were significant differences between normal and DM rats. Taken together, it was concluded that the change of intracellular Ca²⁺ release mediated by PLC/IP3 and PDE5 activity were responsible for decreased bladder smooth muscle contractility in DM rats.
Acetylcholine ; Animals ; Atropine ; Blood Glucose ; Calcium Channels ; Humans ; Injections, Intraperitoneal ; Muscle, Smooth* ; Papaverine ; Prazosin ; Protein Kinase C ; Rats* ; Receptor, Adenosine A1 ; Receptors, Muscarinic ; Streptozocin ; Type C Phospholipases ; Urinary Bladder* ; Verapamil

Cardiovascular diseases are life-threatening illnesses with high morbidity and mortality. Suppressed vagal (parasympathetic) activity and increased sympathetic activity are involved in these diseases. Currently, pharmacological interventions primarily aim to inhibit over-excitation of sympathetic nerves, while vagal modulation has been largely neglected. Many studies have demonstrated that increased vagal activity reduces cardiovascular risk factors in both animal models and human patients. Therefore, the improvement of vagal activity may be an alternate approach for the treatment of cardiovascular diseases. However, drugs used for vagus nerve activation in cardiovascular diseases are limited in the clinic. In this review, we provide an overview of the potential drug targets for modulating vagal nerve activation, including muscarinic, and β-adrenergic receptors. In addition, vagomimetic drugs (such as choline, acetylcholine, and pyridostigmine) and the mechanism underlying their cardiovascular protective effects are also discussed.
Acetylcholine ; pharmacology ; Animals ; Cardiovascular Diseases ; drug therapy ; Cholinergic Agents ; therapeutic use ; Humans ; Receptors, Muscarinic ; drug effects ; Sympathetic Nervous System ; drug effects ; physiopathology ; Vagus Nerve ; drug effects ; physiopathology

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.
Calcium ; Carbachol ; Humans ; Hydrogen-Ion Concentration ; Ion Channels ; Kidney ; Mass Screening ; Mutagenesis, Site-Directed ; Mutant Proteins ; Protein Kinase C ; Receptors, Muscarinic

It is known that top-down associative inputs terminate on distal apical dendrites in layer 1 while bottom-up sensory inputs terminate on perisomatic dendrites of layer 2/3 pyramidal neurons (L2/3 PyNs) in primary sensory cortex. Since studies on synaptic transmission in layer 1 are sparse, we investigated the basic properties and cholinergic modulation of synaptic transmission in layer 1 and compared them to those in perisomatic dendrites of L2/3 PyNs of rat primary visual cortex. Using extracellular stimulations of layer 1 and layer 4, we evoked excitatory postsynaptic current/potential in synapses in distal apical dendrites (L1-EPSC/L1-EPSP) and those in perisomatic dendrites (L4-EPSC/L4-EPSP), respectively. Kinetics of L1-EPSC was slower than that of L4-EPSC. L1-EPSC showed presynaptic depression while L4-EPSC was facilitating. In contrast, inhibitory postsynaptic currents showed similar paired-pulse ratio between layer 1 and layer 4 stimulations with depression only at 100 Hz. Cholinergic stimulation induced presynaptic depression by activating muscarinic receptors in excitatory and inhibitory synapses to similar extents in both inputs. However, nicotinic stimulation enhanced excitatory synaptic transmission by ~20% in L4-EPSC. Rectification index of AMPA receptors and AMPA/NMDA ratio were similar between synapses in distal apical and perisomatic dendrites. These results provide basic properties and cholinergic modulation of synaptic transmission between distal apical and perisomatic dendrites in L2/3 PyNs of the visual cortex, which might be important for controlling information processing balance depending on attentional state.
Animals ; Automatic Data Processing ; Dendrites ; Depression ; Inhibitory Postsynaptic Potentials ; Kinetics ; Pyramidal Cells ; Rats ; Receptors, AMPA ; Receptors, Muscarinic ; Synapses ; Synaptic Transmission ; Visual Cortex

Xylitol is well-known to have an anti-caries effect by inhibiting the replication of cariogenic bacteria. In addition, xylitol enhances saliva secretion. However, the precise molecular mechanism of xylitol on saliva secretion is yet to be elucidated. Thus, in this study, we aimed to investigate the stimulatory effect of xylitol on saliva secretion and to further evaluate the involvement of xylitol in muscarinic type 3 receptor (M3R) signaling. For determining these effects, we measured the saliva flow rate following xylitol treatment in healthy individuals and patients with dry mouth. We further tested the effects of xylitol on M3R signaling in human salivary gland (HSG) cells using real-time quantitative reverse-transcriptase polymerase chain reaction, immunoblotting, and immunostaining. Xylitol candy significantly increased the salivary flow rate and intracellular calcium release in HSG cells via the M3R signaling pathway. In addition, the expressions of M3R and aquaporin 5 were induced by xylitol treatment. Lastly, we investigated the distribution of M3R and aquaporin 5 in HSG cells. Xylitol was found to activate M3R, thereby inducing increases in Ca²⁺ concentration. Stimulation of the muscarinic receptor induced by xylitol activated the internalization of M3R and subsequent trafficking of aquaporin 5. Taken together, these findings suggest a molecular mechanism for secretory effects of xylitol on salivary epithelial cells.
Aquaporin 5 ; Bacteria ; Calcium ; Calcium Signaling ; Candy ; Epithelial Cells ; Humans ; Immunoblotting ; Mouth ; Polymerase Chain Reaction ; Receptors, Muscarinic ; Saliva ; Salivary Glands ; Xylitol

Muscarinic acetylcholine receptors (mAChRs), including M1-M5 subtypes, are classic receptors in regulating water, ion, and solute transport in salivary gland. Our work focuses on the studies on the expression pattern and function of mAChR in the submandibular gland (SMG), and the underlying mechanism involved in the mAChR-regulated secretion, together with the effect of parasympathectomy on the salivary secretion. Microvascular autotransplantation of SMG into the temporal fossa provides a continuous and endogenous source of fluids, and is currently an effective method for treating severe keratoconjunctivitis sicca. By using RT-PCR, Western blotting, and immunofluorescence, our data demonstrated that the expression of M1 and M3 subtypes were decreased in latent period in rabbit SMG autotransplantation model, whereas carbachol stimulation promoted the salivary secretion, as well as M1 and M3 expressions. By contrast, mAChRs were hypersensitive in epiphora SMGs, whereas atropine gel and botulinum toxin A application significantly inhibited the hypersecretion in both animal models and patients. Furthermore, the possible intracellular signal molecules involved in the mAChR-modulated salivary secretion were explored. Activation of mAChR upregulated the expression of aquaporin 5 (AQP5), the main transporter that mediated water secretion through transcellular pathway, and led to AQP5 trafficking from lipid rafts to non-lipid microdomain. Extracellular signal-regulated kinase 1/2 (ERK1/2) was involved in the mAChR-regulated AQP5 content. mAChR activation also modulated the expression, distribution, and function of tight junction proteins, and increased paracellular permeability. ERK1/2/β-arrestin2/clathrin/ubiquitin signaling pathway was responsible for the mAChR-regulated downregulation of tight junction molecule claudin-4. Cytoskeleton filamentous actin (F-actin) was also involved in the distribution and barrier function of epithelial tight junctions. Besides, endothelial tight junctions were opened by mAChR agonist-evoked salivation in the mice. Furthermore, parasympathetic denervation increased resting salivary secretion in the long terminrats and minipigs. Taken together, our work demonstrated that mAChR regulated saliva secretion via transcellular and paracellular pathways in SMG epithelium as well as tight junction opening in SMG endothelium. Modulation of mAChR might be a promising strategy to ameliorate SMG dysfunction.
Animals ; Aquaporin 5 ; Carbachol ; Humans ; Mice ; Rabbits ; Receptors, Muscarinic ; Salivation ; Submandibular Gland

PURPOSE: Many patients with benign prostatic hyperplasia need treatment for remaining storage symptoms after surgery. Therefore, we evaluated the effect of the phytotherapeutic agent WSY-1075 on persistent detrusor overactivity (DO) after the relief of bladder outlet obstruction (BOO). MATERIALS AND METHODS: Rats were assigned to 3 groups: control (n=6), persistent DO (n=6), and persistent DO treated with the phytotherapeutic agent WSY-1075 (n=6). Persistent DO after relief of partial BOO was generated in the rat model, and 6 of the rats with this condition were orally administered WSY-1075. After 4 weeks of administration, cystometry was performed. Additionally, 8-hydroxy-2-deoxyguanosine and superoxide dismutase were measured to evaluate oxidative stress in the bladder. Pro-inflammatory cytokines, such as interleukin-8 and tumor necrosis factor-α, were analyzed, as were the M2 and M3 muscarinic receptors of the bladder. RESULTS: Significantly increased contraction pressure and a decreased contraction interval were observed in the persistent DO group after relief of BOO. Moreover, oxidative stress, pro-inflammatory cytokines, and M3 muscarinic receptors were significantly increased. After treatment with WSY-1075, significantly reduced DO was observed by cystometry in comparison with the persistent DO group. Additionally, significantly decreased levels of oxidative stress, pro-inflammatory cytokines, and M3 muscarinic receptors in the bladder were observed after treatment with WSY-1075. CONCLUSIONS: Treatment with WSY-1075 improved persistent DO after the relief of BOO mediated by antioxidative and anti-inflammatory effects. Further studies are necessary to identify the exact mechanism of the treatment effect of WSY-1075.
Animals ; Cytokines ; Humans ; Interleukin-8 ; Lower Urinary Tract Symptoms ; Models, Animal ; Necrosis ; Oxidative Stress ; Phytotherapy ; Prostatic Hyperplasia ; Rats ; Receptors, Muscarinic ; Superoxide Dismutase ; Urinary Bladder Neck Obstruction* ; Urinary Bladder* ; Urinary Bladder, Overactive

BACKGROUND/OBJECTIVES: Fermented Laminaria japonica (FL), a type sea tangle used as a functional food ingredient, has been reported to possess cognitive improving properties that may aid in the treatment of common neurodegenerative disorders, such as dementia. MATERIALS/METHODS: We examined the effects of FL on scopolamine (Sco)- and ethanol (EtOH)-induced hippocampus-dependent memory impairment, using the Passive avoidance (PA) and Morris water maze (MWM) tests. To examine the underlying mechanisms associated with neuroprotective effects, we analyzed acetylcholine (ACh) and acetylcholinesterase (AChE) activity, brain tissue expression of muscarinic acetylcholine receptor (mAChR), cAMP response element binding protein (CREB) and extracellular signal-regulated kinases 1/2 (ERK1/2), and immunohistochemical analysis, in the hippocampus of mice, compared to current drug therapy intervention. Biochemical blood analysis was carried out to determine the effects of FL on alanine transaminase (ALT), aspartate transaminase (AST), and triglyceride (TG) and total cholesterol (TC) levels. 7 groups (n = 10) consisted of a control (CON), 3 Sco-induced dementia and 3 EtOH-induced dementia groups, with both dementia group types containing an untreated group (Sco and EtOH); a positive control, orally administered donepezil (Dpz) (4mg/kg) (Sco + Dpz and EtOH + Dpz); and an FL (50 mg/kg) treatment group (Sco + FL50 and EtOH + FL50), orally administered over the 4-week experimental period. RESULTS: FL50 significantly reduced EtOH-induced increase in AST and ALT levels. FL50 treatment reduced EtOH-impaired step-through latency time in the PA test, and Sco- and EtOH-induced dementia escape latency times in the MWM test. Moreover, anticholinergic effects of Sco and EtOH on the brain were reversed by FL50, through the attenuation of AChE activity and elevation of ACh concentration. FL50 elevated ERK1/2 protein expression and increased p-CREB (ser133) in hippocampus brain tissue, according to Western blot and immunohistochemistry analysis, respectively. CONCLUSION: Overall, these results suggest that FL may be considered an efficacious intervention for Sco- and EtOH-induced dementia, in terms of reversing cognitive impairment and neuroplastic dysfunction.
Acetylcholine ; Acetylcholinesterase ; Alanine Transaminase ; Animals ; Aspartate Aminotransferases ; Blotting, Western ; Brain ; Cholesterol ; Cognition Disorders* ; Cyclic AMP Response Element-Binding Protein ; Dementia* ; Drug Therapy ; Ethanol ; Extracellular Signal-Regulated MAP Kinases ; Functional Food ; Hippocampus ; Immunohistochemistry ; Laminaria* ; Memory ; Mice* ; Neurodegenerative Diseases ; Neuronal Plasticity* ; Neuroprotective Agents ; Receptors, Muscarinic ; Scopolamine Hydrobromide ; Triglycerides ; United Nations ; Water