Demonstration of functional neuronal beta3-adrenoceptors within the enteric nervous system

BACKGROUND & AIMS: Although the beta(3)-adrenoceptor (AR) has been suggested to be involved in regulation of gut motility and visceral algesia, the precise mechanisms have been unknown. beta(3)-AR has been postulated to have a nonneuronal expression, being initially characterized in adipocytes and subsequently in the smooth muscle. We aimed to investigate the expression of beta(3)-AR in human enteric nervous system and its role in motility and visceral algesia. METHODS: The expression of beta(3)-AR in human colon myenteric and submucosal plexus was investigated using immunohistochemistry. The effects of a beta(3)-AR agonist on nerve-evoked and carbachol-induced contractions as well as somatostatin release were investigated in strips of human colon. The effect of an agonist on diarrhea and visceral pain was investigated in vivo in rat models. RESULTS: beta(3)-AR is expressed in cholinergic neurons in the myenteric plexus and submucosal plexus of human colon. Activation of beta(3)-AR causes the release of somatostatin from human isolated colon. In a rat model of visceral pain, beta(3)-AR agonist elicits somatostatin-dependent visceral analgesia. beta(3)-AR agonists inhibit cholinergically mediated muscle contraction of the human colon, as well as chemically induced diarrhea in vivo in a rat model. CONCLUSIONS: This is the first demonstration of expression of beta(3)-AR in the enteric nervous system. Activation of these receptors results in inhibition of cholinergic contractions and enhanced release of somatostatin, which may lead to visceral analgesia and inhibition of diarrhea. Therefore, beta(3)-AR could be a novel therapeutic target for functional gastrointestinal disorders.     

Ca2+-dependent differential development of carbachol-induced desensitization to receptor agonists and high K+ in guinea-pig taenia caeci

1. Carbachol (CCh)-induced desensitization to CCh was interrupted by a transient resensitization during its early stage, with concomitant changes at the muscarinic receptor/G-protein level in smooth muscle of guinea-pig taenia caeci. To assess whether such a peculiar desensitizing process may heterologously regulate smooth muscle contraction, we examined the developmental processes of CCh-induced desensitization to histamine and high K(+) and compared it with that to CCh. 2. Under Ca(2+)-containing physiological conditions, treatment with 10(-4) mol/L CCh for 30 min induced heterologous desensitization to histamine and high K(+). The development of desensitization to histamine was interrupted by a transient resensitization at 1 min in a manner similar to that to CCh. In contrast, CCh-induced desensitization to high K(+) reached a peak at 1 min and was followed by a gradual resensitization up to a partial restoration at 30 min. 3. Under Ca(2+)-free conditions containing 0.2 mmol/L EGTA, treatment with 10(-4) mol/L CCh for 30 min failed to induce heterologous desensitization to either histamine or high K(+), whereas the CCh treatment developed homologous desensitization to CCh in a simple time-dependent manner without a resensitization phase. 4. These results suggest that cellular responsiveness to receptor agonists and non-receptor-mediated depolarizing stimulation is differentially regulated by Ca(2+)-dependent heterologous desensitization in smooth muscle.     

Genetic disruption of G proteins,Gi2α or Goα, does not abolish inotropic and chronotropic effects of stimulating muscarinic cholinoceptors in atrium

Background and purpose:

Classically, stimulation of muscarinic cholinoceptors exerts negative inotropic and chronotropic effects in the atrium of mammalian hearts. These effects are crucial to the vagal regulation of the heart beat. This effect is assumed to be mediated via GTP binding (G) proteins, because they can be abolished by Pertussis toxin. However, it is unknown which G proteins are involved.

Experimental approach:

We studied contractility in isolated left or right atrium from genetically manipulated mice with deletion of one of two G proteins, either of the α subunit of G_i2 protein (G_i2α) or of the α subunit of G_o protein (G_oα). Preparations were stimulated with carbachol alone or after pretreatment with the β-adrenoceptor agonist isoprenaline. For comparison, the effects of carbachol on L-type Ca²⁺-channels in isolated ventricular cardiomyocytes were studied.

Key results:

The negative inotropic and chronotropic effects of carbachol alone or in the presence of isoprenaline were identical in atria from knockout or wild-type mice. However, the effect of carbachol on isoprenaline-activated L-type Ca²⁺-channel in isolated ventricular cardiomyocytes was greatly attenuated in both types of knockout mice studied.

Conclusions and implications:

These data imply that there is either redundancy of G proteins for signal transduction or that Pertussis toxin-sensitive proteins other than G_i2α and G_oα mediate the vagal stimulation in the atrium. Moreover, different G proteins mediate the effect of carbachol in ventricle compared with atrium.     

In vivo and in vitro pharmacological characterization of SVT-40776, a novel M3 muscarinic receptor antagonist,for the treatment of overactive bladder

Background and purpose:

Highly selective M₃ muscarinic receptor antagonists may represent a better treatment for overactive bladder syndrome, diminishing side effects. Cardiac side effects of non-selective antimuscarinics have been associated with activity at M₂ receptors as these receptors are mainly responsible for muscarinic receptor-dependent bradycardia. We have investigated a novel antimuscarinic, SVT-40776, highly selective for M₃ over M₂ receptors (Ki = 0.19 nmol·L⁻¹ for M₃ receptor affinity). This study reports the functional activity of SVT-40776 in the bladder, relative to its activity in atria.

Experimental approach:

In vitro and ex vivo (oral dosing) inhibition of mouse detrusor and atrial contractile responses to carbachol were used to study the functional activity of SVT-40776. The in vivo efficacy of SVT-40776 was characterized by suppression of isovolumetric spontaneous bladder contractions in anaesthetized guinea pigs after intravenous administration.

Key results:

SVT-40776 was the most potent in inhibiting carbachol-induced bladder contractions of the anti-cholinergic agents tested, without affecting atrial contractions over the same range of concentrations. SVT-40776 exhibited the highest urinary versus cardiac selectivity (199-fold). In the guinea pig in vivo model, SVT-40776 inhibited 25% of spontaneous bladder contractions at a very low dose (6.97 µg·kg⁻¹ i.v), without affecting arterial blood pressure.

Conclusions and implications:

SVT-40776 is a potent inhibitor of M₃ receptor-related detrusor contractile activity. The absence of effects on isolated atria preparations represents an interesting characteristic and suggests that SVT-40776 may lack unwanted cardiac effects; a feature especially relevant in a compound intended to treat mainly elderly patients.British Journal of Pharmacology (2009) doi:10.1111/j.1476-5381.2008.00082.x     

Nonlinear dynamical analysis of carbachol induced hippocampal oscillations in mice

Aim： Hippocampal neuronal network and synaptic impairment underlie learning and memory deficit in Alzheimer＇s disease （AD） patients and animal models. In this paper, we analyzed the dynamics and complexity of hippocampal neuronal network synchronization induced by acute exposure to carbachol, a nicotinic and muscarinic receptor co-agonist, using the nonlinear dynamical model based on the Lempel-Ziv estimator. We compared the dynamics of hippocampal oscillations between wild-type （WT） and triple-transgenic （3xTg） mice, as an AD animal model. We also compared these dynamic alterations between different age groups （5 and 10 months）. We hypothesize that there is an impairment of complexity of CCh- induced hippocampal oscillations in 3xTg AD mice compared to WT mice, and that this impairment is age-dependent. Methods： To test this hypothesis, we used electrophysiological recordings （field potential） in hippocampal slices. Results： Acute exposure to 100 μmol/L CCh induced field potential oscillations in hippocampal CA1 region, which exhibited three distinct patterns： （1） continuous neural firing, （2） repeated burst neural firing and （3） the mixed （continuous and burst） pattern in both WT and 3xTg AD mice. Based on Lempel-Ziv estimator, pattern （2） was significantly lower than patterns （1） and （3） in 3xTg AD mice compared to WT mice （P〈0.001）, and also in 10-month old WT mice compared to those in 5-month old WT mice （P〈0.01）. Conclusion： These results suggest that the burst pattern （theta oscillation） of hippocampal network is selectively impaired in 3xTg AD mouse model, which may reflect a learning and memory deficit in the AD patients.     

阿托品对人视网膜色素上皮细胞D407株 表达及分泌TGF β2的调控

目的：通过观察不同浓度阿托品联合10-5 mol/L卡巴胆碱干预下D407细胞表达及分泌TGF β2的变化,探讨阿托品对RPE细胞表达及分泌TGF β2的调控作用。方法：常规培养D407细胞,药物干预前换用无血清培养基培养, 分为4组。（1）实验组（A组）：A1~A5组依次加入10-4～10-8 mol/L阿托品,孵育30 min后每组加入10-5 mol/L卡巴胆碱;（2）阴性对照组（B组）：B1~B5组依次加入10-4～10-8 mol/L阿托品;（3）阳性对照组（C组）：加入10-5 mol/L卡巴胆碱;（4）空白对照组（D组）：不加药物。干预24 h后采用RT PCR,Western印迹及ELISA法检测细胞胞浆中TGF β2 mRNA及蛋白质的表达水平及上清液中TGF β2的含量。统计学方法采用单因素方差分析。结果：实验组D407细胞胞浆TGF β2 mRNA和蛋白质表达水平及上清中TGF β2蛋白质含量均较阳性对照组低,10-4 mol/L阿托品可完全阻断10-5 mol/L卡巴胆碱上调TGF β2表达及分泌的作用,其效应具有浓度依赖性（F=1 056.897, 1 320.170, 475.657;P＜0.001）。阴性对照组D407细胞胞浆TGF β2 mRNA和蛋白质表达水平及上清中TGF β2蛋白质含量与空白对照组比较差异无统计学意义（P＞0.05）。结论：阿托品可有效抑制卡巴胆碱促进人RPE细胞表达及分泌TGF β2的功能,提示M受体参与介导此过程。     

Disparate cholinergic currents in rat principal trigeminal sensory nucleus neurons mediated by M1 and M2 receptors: a possible mechanism for selective gating of afferent sensory neurotransmission

Neurons situated in the principal sensory trigeminal nucleus (PSTN) convey orofacial sensory inputs to thalamic relay regions and higher brain centres, and the excitability of these ascending tract cells is modulated across sleep/wakefulness states and during pain conditions. Moreover, acetylcholine release changes profoundly across sleep/wakefulness states and ascending sensory neurotransmission is altered by cholinergic agonists. An intriguing possibility is, therefore, that cholinergic mechanisms mediate such state-dependent modulation of PSTN tract neurons. We tested the hypotheses that cholinergic agonists can modulate PSTN cell excitability and that such effects are mediated by muscarinic receptor subtypes, using patch-clamp methods in rat and mouse. In all examined cells, carbachol elicited an electrophysiological response that was independent of action potential generation as it persisted in the presence of tetrodotoxin. Responses were of three types: depolarization, hyperpolarization or a biphasic response consisting of hyperpolarization followed by depolarization. In voltage-clamp mode, carbachol evoked corresponding inward, outward or biphasic currents. Moreover, immunostaining for the vesicle-associated choline transporter showed cholinergic innervation of the PSTN. Using muscarinic receptor antagonists, we found that carbachol-elicited PSTN neuron hyperpolarization was mediated by M2 receptors and depolarization, in large part, by M1 receptors. These data suggest that acetylcholine acting on M1 and M2 receptors may contribute to selective excitability enhancement or depression in individual, rostrally projecting sensory neurons. Such selective gating effects via cholinergic input may play a functional role in modulation of ascending sensory transmission, including across behavioral states typified by distinct cholinergic tone, e.g. sleep/wakefulness arousal levels or neuropathic pain conditions.     

Role of gap junctions in endothelium-derived hyperpolarizing factor-mediated vasodilatation in rat renal artery

AIM: To investigate the effects of gap junction inhibitors on endothelium-derived but nitric oxide (NO)- and prostacyclin (PGI2)-independent vasodilatations induced by carbachol in the rat isolated renal artery. METHODS: Isolated renal arteries were mounted on a wire myograph apparatus were tissues treated with the nitric oxide synthase inhibitor N^ω-nitro-L-arginine methyl ester hydrochloride (NAME; 100 μmol/L) and indomethacin (10 μmol/L) and precontracted with phenylephrine (0.1 μmol/L). NAME and indomethacin treated Carbachol (0.01-10 μmol/L)- or sodium nitroprusside (SNP; 1-300 nmol/L)-induced mediated relaxations were observed in the presence of gap junction inhibitors. RESULTS: Carbachol produced a concentration-dependent relaxation in tissues treated with NAME (100 μmol/L) and indomethacin (10 μmol/L). This relaxation was not affected by hemoglobin (3 μmol/L), but was inhibited by charybdotoxin (200 nmol/L) and ouabain (30 μmol/L). The putative gap junction inhibitors, GAP 27 peptides with sequence homology to connexins 40 and 43 respectively reduced carbachol- but not SNP-induced relaxations mediated by endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations. The inhibition by the connexin 43 inhibitor was greater than that of the connexin 40 inhibitor. CONCLUSION: The results indicate the presence of gap junctions sensitive to 43^GAP 27 and 43^GAP 27 in the rat renal artery and each of these different types of gap junctions plays a role in the NO- and PGI2-independent relaxations induced by carbachol in this blood vessel. However, connexin 43 appears to play a more predominant role in mediating gap junction communications in the rat renal artery.     

Inhibition of calcium/calmodulin-dependent protein kinase II normalizes diabetes-induced abnormal vascular reactivity in the rat perfused mesenteric vascular bed

1. Calcium/calmodulin-dependent protein kinase II (CaMKII) has an important function in mediating insulin release but its role in the development of diabetes-induced cardiovascular complications is not known. 2. We investigated the ability of a chronic administration of KN-93 (5 mg kg(-1) alt diem for 4 weeks), an inhibitor of CaMKII, to modulate the altered vasoreactivity of the perfused mesenteric bed to common vasoconstrictors and vasodilators in streptozotocin (STZ)-induced diabetes. 3. The vasoconstrictor responses induced by noradrenaline (NE), endothelin-1 (ET-1), and angiotensin II (Ang II), were significantly increased whereas, vasodilator responses to carbachol and histamine were significantly reduced in the perfused mesenteric bed of the STZ-diabetic rats as compared with non-diabetic controls. 4. Inhibition of CaMKII by KN-93 treatment did not affect blood glucose levels but produced a significant normalization of the altered agonist-induced vasoconstrictor and vasodilator responses. KN-93 did not affect agonist-induced responses in control animals. In addition, KN-93 significantly reduced weight loss in diabetic rats. 5. The present data suggest that CaMKII is an essential mediator in the development of diabetic vascular dysfunction and may also play an important role in signalling pathways leading to weight loss during diabetes.