Ca2+ sensitization and the regulation of contractility in rat anococcygeus and retractor penis muscle

Stimulation of the RhoA/Rho-kinase (ROK) signaling represents a key step in the maintenance of agonist-induced contraction of smooth muscle. We aimed to demonstrate Ca(2+) sensitization in rat anococcygeus and retractor penis muscles and to identify the molecular expression of major components of this pathway. Both anococcygeus and retractor penis showed a similar expression of RhoA, ROKalpha, and ROKbeta at the protein level as well as the mRNA for RhoGEFs. Cumulative addition of the ROK inhibitors H-1152 (0.001-3 microM), Y-27632 (0.01-30 microM) or HA-1077 (0.01-30 microM) caused sustained relaxations of precontracted smooth muscle strips. Ca(2+) sensitization induced by phenylephrine, norepinephrine and carbachol was markedly antagonized by all three ROK inhibitors. In addition, the contractile response to KCl-induced depolarization was highly sensitive to these ROK inhibitors. H-1152 was approximately 8-20 more potent than Y-27632 and HA-1077 to inhibit contraction. Electrical field stimulation (EFS, 1-32 Hz) caused transient contractions in both anococcygeus and retractor penis muscle, which were blocked by tetrodotoxin (1 microM), phentolamine (1 microM) or bretylium tosylate (30 microM). Similarly, H-1152 (0.1-1 microM), Y-27632 (1-10 microM) or HA-1077 (1-10 microM) significantly reduced EFS-evoked contractions in a concentration-dependent manner. The results indicate that the RhoA/ROK-mediated Ca(2+) sensitization pathway is expressed in anococcygeus and retractor penis muscles and enhances contractions produced by receptor-dependent and independent mechanisms.     

AT_1受体在脑胆碱能刺激引起的孤束核TH-IR变化中的作用

本工作选用清醒SD雄性大鼠,利用免疫组织化学方法,观察大鼠侧脑室注射胆碱能激动剂氨甲酰胆碱后孤束核内儿茶酚胺能神经元活性的变化以及阻断脑AT1受体对上述变化的影响。免疫组织化学结果显示:侧脑室注射氨甲酰胆碱(.5μg)后40 min,孤束核内酪氨酸羟化酶(TH)免疫反应阳性神经元数目明显增多(P<0.05),免疫染色强度明显增强(P<0.05)。losartan(20μg)预处理后孤束核内TH免疫反应活性明显下降(P<0.05)。上述结果提示,侧脑室注射胆碱能激动剂氨甲酰胆碱对孤束核内儿茶酚胺能神经元具有兴奋作用,阻断脑血管紧张素能AT1受体可部分抑制氨甲酰胆碱在孤束核诱导的上述变化。     

The Effects of Carbachol on the Proximal and Distal Parts of Frog Motor Nerve Endings

Cholinomimetics not only activate postsynaptic cholinoreceptors in neuromuscular synapses, but also alter the process of acetylcholine secretion from nerve endings. However, the mechanism of action of cholinomimetics on the secretory process remains unidentified. We approached the question of the mechanism of the presynaptic action of cholinomimetics in the present study by investigating the effects of the n,m-cholinomimetic carbachol on nerve ending currents and postsynaptic membrane currents. Carbachol induced decreases in the postsynaptic response, without affecting the duration and amplitude of the nerve ending current in both the central and distal part of the nerve ending. However, carbachol increased the time between the arrival of the presynaptic action potential and the start of transmitter secretion. This effect on synaptic delay was more marked in the distal parts of the ending. The action of another potential modulator, extracellular potassium, was accompanied by decreases in presynaptic currents and also by increases in synaptic delay. These data provide evidence for the suppressive effect of carbachol on acetylcholine secretion acting via presynaptic metabotropic cholinoreceptors which control the level and time course of secretion of neurotransmitter quanta.     

WAY-131256 is an orally active,efficacious, and in vivo functionally selective M1 agonist

Computer modeling of carbachol docked in the human m₁ receptor binding pocket has been used to discover a series of carbamate and thiocarbamate chiral, conformationally restricted analogues of carbachol based on azabicyclo[2.2.1]heptan-3-ol. These molecules have been evaluated for affinity and efficacy at human muscarinic receptors (m₁–m₅) transfected into a CHO cell line. None of these compounds was selective in binding. Thiocarbamate analogues had greater affinity for the m₁ receptor subtype, but lower efficacy based on comparison of their ability to induce phosphoinositide (PI) turnover. Carbamate analogues had lower affinity for m₁ receptors than thiocarbamates and varied in efficacy from 10% to 100% of the carbachol response in phosphoinositide (PI) turnover. One of these analogues 3S,4R-azabicyclo[2.2.1]heptan-3-methylcarbamate, WAY-131256, (VI) has been characterized as an m₁/m₂ agonist in vitro. (VI) was equi-efficacious to the standard m₁ agonist, xanomeline (Phase III) in vivo in a scopolamine-impaired radial arm maze paradigm (MED 1 mg/kg, 5.88 mmol/kg for VI and MED 1 mg/kg, 3.55 mmoles/kg for xanomeline) and was approximately equal to xanomeline in an AF64A-impaired radial arm maze paradigm. Despite its lack of m₁ selectivity in vitro, in vivo experiments on (VI) indicated no significant effect on blood pressure or heart rate at 10 mg/kg (58.78 mmol/kg) (i.p.), and no peripheral side effects attributed to stimulation of either the m₂ or m₃ receptors (salivation, lacrimation, and chromodacryorrhea) up to doses of 30 mg/kg, 176.2 mmol/kg. These results may be explained by different receptor densities in various brain regions not accounted for in a transfected cell line or by metabolism of (VI) to a m₁ selective agonist in vivo. Drug Dev. Res. 40:185–192, 1997. © 1997 Wiley-Liss, Inc.     

Interactions between limbic, thalamo-striatal-cortical, and central autonomic pathways during epileptic seizure progression.

We used immunocytochemistry to determine the regional and temporal distribution of Fos protein expression in awake and unrestrained rats after a unilateral stereotaxic microinjection of a cholinergic agonist, carbachol, in the thalamic ventroposterolateral and reticular nuclei, previously shown to cause limbic and generalized convulsive seizures. The microinjection of carbachol elicits behavioral alterations including immobilization, staring, facial and jaw clonus, rearing, and falling, followed by recurrent generalized convulsive seizures, and a pattern of c-fos expression throughout the brain. In addition to the hypothalamic paraventricular and supraoptic nuclei, the initial induction of c-fos expression was observed as early as 15 minutes after the carbachol microinjection, in the piriform and entorhinal cortices, the thalamic paraventricular, the supramammilary, the lateral parabrachial nuclei, and the central gray. From 30 minutes to 2 hours, corresponding to the occurrence of motor expression of limbic and recurrent generalized convulsive seizures, Fos immunoreactivity was seen in a number of functionally related brain regions including the hippocampus, the amygdala, and the anterior thalamic nucleus (limbic system); the thalamus, the basal ganglia, and the cortex (thalamo-striatal-cortical system); and the hypothalamus, the central nucleus of the amygdala, the pons, and the medulla (central autonomic system). On the basis of the present results showing regional and temporal c-fos expression and well known neuroanatomical connections, we have constructed a neural network relating the limbic, thalamo-striatal-cortical, and central autonomic systems. This analysis provides, for the first time, neuronal circuits and pathways relating epilepsy-elicited behavioral expression of convulsive seizures and adaptive homeostatic responses and could serve as a basis for studying central autonomic regulation during epileptic disorders.     

Endoplasmic reticulum dynamics in hippocampal dendritic spines induced by agonists of type I metabotropic glutamate but not by muscarinic acetylcholine receptors

Neurons in the hippocampus exhibit subpopulations of dendritic spines that contain endoplasmic reticulum (ER). ER in spines is important for synaptic activity and its associated Ca(2+) signaling. The dynamic distribution of ER to spines is regulated by diacylglycerol and partly mediated by protein kinase C, metalloproteinases and γ-secretase. In this study, we explored whether pharmacological activation of type I metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors (mAChRs) known to activate phospholipase C would have any effect on spine ER content. We found that DHPG (100 μM) but not carbachol (10 μM) caused a reduction in the number of spines with ER. We further found that ER Ca(2+) depletion triggered by thapsigargin (200 nM) had no effect on ER localization in spines.     

Mechanisms underlying the antidiarrheal,antispasmodic and bronchodilator activities of Fumaria parviflora and involvement of tissue and species specificity

Ethnopharmacological relevance: In the Greco-Arab (Unani) traditional medicine, Fumaria parviflora Linn. is widely used in hypreractive gut and respiratory disorders including diarrhea, abdominal cramps, indigestion and asthma but scientific studies to provide rational for these medicinal uses are sparse. This study was therefore undertaken to provide ethnopharmacological basis for its medicinal use in diarrhea, abdominal cramps and asthma.     

Carbachol阻断亚硒酸钠对离体皮质神经元的致凋亡作用(英文)

在早期工作发现小剂量亚硒酸钠 (Sodiumselenite)对离体皮质神经元的致凋亡作用的基础上 ,观察了在亚硒酸钠致凋亡模型中加入M型胆碱能受体激动剂Carbachol(Carb)能否阻止凋亡过程和相伴随的有关基因表达的改变。结果显示 :①用不同剂量 (0 .0 0 4 ,0 .0 2 ,0 .1,0 .5,2 .5和 12 .5mmol/L)的Carb和 0 .5μmol/L亚硒酸钠共同处理培养的新生小鼠皮质神经元时 ,用琼脂糖凝胶电泳DNA梯形检测和流式细胞仪亚二倍体峰检测 ,都证明Carb表现出对亚硒酸钠致凋亡作用的剂量依赖性保护效应 ;②用RT PCR技术证明 ,在与Carb共同孵育的实验中 ,不再出现或减弱了亚硒酸钠单独处理时出现的bcl 2基因表达的下调和bax ,p53,c fos以及AChE基因表达的上调。上述结果表明 ,亚硒酸钠对皮质神经元的致凋亡作用和与之相伴随的基因表达的改变 ,可被一定浓度的Carb所阻断 ,提示亚硒酸钠致凋亡作用可能同本室发现的 β 淀粉样蛋白的C末端 5肽片段的致凋亡作用具有类似的途径 ,以类似的基因表达改变为基础 ,并对Carb的抗凋亡机制进行了讨论。     

Induction of c-fos and TIS genes in cultured rat astrocytes by neurotransmitters

The interaction of neurotransmitters with their specific receptors initiates a cascade of intracellular biochemical events which lead to induction of specific genes. Included in this cascade is the rapid and transient induction of a family of primary early response genes we term TIS genes (Lim et al.: Oncogene 1: 263-270, 1987). Expression of six TIS gene, including c-fos, was examined in secondary cultures of rat neocortical astrocytes exposed to muscarinic and adrenergic agonists and antagonists to study the early genomic responses which accompany neurotransmitter-induced alteration of glial morphology and physiology. Carbachol induced accumulation of mRNA for c-fos and the other TIS genes. Carbachol-mediated induction of TIS mRNA expression was sensitive to atropine blockade and was potentiated by lithium. Norepinephrine (NE), isoproterenol, or phenylephrine also induced TIS mRNA accumulation. In order to determine which second-messenger pathways mediate NE induction of TIS gene expression, the influences of the beta(B) antagonist propranolol (PR), the alpha I(AI) antagonist prazosin (PZ), and the alpha 2(A2) antagonist yohimbine (YB) were examined. The induction of TIS1 mRNA by NE was partially blocked by PR or PZ alone, and completely abolished by both antagonists in combination. YB had no effect on TIS1 mRNA expression. These results suggest that NE induces TIS1 mRNA through both B- and A 1-adrenergic, but not A2, pathways. The lack of effect of inhibitors of phospholipase A2 and cyclooxygenase suggests that the A1 component is mediated through a protein kinase C pathway. The induction of transient gene expression by neurotransmitters may mediate the secondary genomic responses and phenotypic changes occurring in astrocytes in response to alterations in neuronal neurotransmitter release.     

Modulation of acid secretion in common bile duct ligation-related gastropathy in Wistar rats

BACKGROUND: Portal hypertensive gastropathy is associated with fundic gland atrophy, resulting in a decrease in chief and parietal cells, and diminished acid secretion. METHODS: Acid secretion by isolated parietal cells was measured (acridine orange retention), along with the levels of various second messengers (intracellular Ca(2+), cyclic adenosine monophosphate and protein kinase C) in the common bile duct, ligated portal hypertensive rats and compared with sham-operated controls. RESULTS: There was a significant decrease in the response of isolated parietal cells to the secretagogues histamine and carbachol. This resulted in the blunted acid secretion in the common bile duct ligated group. In addition, all the second messengers studied were significantly decreased as compared with the sham-operated controls. CONCLUSION: These results suggest that the blunted acid secretory response in the portal hypertensive rat is caused by an alteration in the intracellular signal transduction mechanism.