Calcium Ion Mobilization Research Articles

Gene Expression Analysis in HBV Transgenic Mouse Liver: A Model to Study Early Events Related to Hepatocarcinogenesis

Hepatitis B virus (HBV) is one of the major etiological factors responsible for the development of hepatocellular carcinoma (HCC). We used a transgenic mouse, containing HBV sequences, as a model system to unravel the molecular mechanisms of hepatocarcinogenesis induced by HBV. We chose this animal model because it consistently develops liver cancer after intermediate steps that mimic the natural history of HBV infection in humans. In this study, we focus our attention on the early events leading to liver cancer. We compared the gene expression profile of 3-month-old transgenic mice with that of 3-month-old wild-type (wt) animals. In the transgenic mouse, microarray data analysis showed a total of 45 significantly differentially expressed genes, 25 highly expressed (fold change > or =2; P = 0.0025), and 20 downregulated (fold change < or =0.5; P = 0.0025). These genes belong to several different functional categories such as the regulation of immunological response, transcription, intracellular calcium ion mobilization, regulation of cell cycle and proliferation, NF-kappab signal transduction cascades, and apoptosis. In particular, the upregulation of the antiapoptotic gene NuprI and the downregulation of the proapoptotic gene Bnip3 were found. This observation was supported by an in vitro apoptosis assay that showed downregulation of apoptosis in hepatocytes of HBV transgenic mouse compared with wt mice treated with staurosporine. In conclusion, our experimental approach allowed identification of new genes modulated by HBV and showed that the apoptotic process was deregulated in transgenic mouse hepatocytes. These data shed light on one possible mechanism by which HBV induces hepatocarcinogenesis.

Phyto- and Endogenous Estrogens Differently Activate Intracellular Calcium Ion Mobilization in Bovine Endometrial Cells

The main purpose of this study was to check whether phyto- and endogenous estrogens influence calcium ion mobilization [Ca(2+)](i) in bovine endometrial cells and whether this action is connected with biological effects i.e. prostaglandin (PG)F(2alpha) production. In our study we used two calcium measurement methods by comparing the microscopic method with widely used quantitative - spectrofluorometric method of [Ca(2+)](i) measurement. We also wanted to confirm whether visualization of calcium ion [Ca(2+)](i) in cells using microscopic method supported by micro image analysis (Micro Image Olympus system) reflects real, qualitative changes in the ion concentration. In both methods a cell-permeable form of fluorescent [Ca(2+)](i) indicator Fura-2 was used. Cultured bovine endometrial epithelial and stromal cells influenced by phorbol-2-myristate-13-acetate (PMA; positive control), estradiol 17-beta (E(2); endogenous estrogen) and active metabolites of phytoestrogens (environmental estrogens) were used as a model to study PGF(2alpha) secretion and [Ca(2+)](i) mobilization in the cells. Equol and para-ethyl-phenol in doses of 10(-8)-10(-6) M increased PGF(2alpha) concentration both in epithelial and stromal cells (P<0.05). In both methods, equol and para-ethyl-phenol did not cause intracellular [Ca(2+)](i) mobilization in epithelial and stromal cells (P>0.05). Both methods revealed that only E(2) and PMA induced intracellular [Ca(2+)](i) mobilization in epithelial and stromal cells (P<0.05). The results of both methods were highly correlated (P<0.001; r=0.82 for epithelial cells and r=0.89 for stromal cells). In conclusion, both methods gave approximately the same results and showed that phytoestrogens, in contrast to PMA and E(2), did not cause intracellular [Ca(2+)](i) mobilization in endometrial cells. The obtained results proved that the [Ca(2+)](i) visualization method supported by micro image analysis can produce similar results to the spectrofluorometric method.

Kisspeptin-10-Induced Signaling of GPR54 Negatively Regulates Chemotactic Responses Mediated by CXCR4: a Potential Mechanism for the Metastasis Suppressor Activity of Kisspeptins

The product of the KiSS-1 gene is absent or expressed at low level in metastatic melanoma and breast cancer compared with their nonmetastatic counterparts. A polypeptide derived from the KiSS-1 product, designated kisspeptin-10 (Kp-10), activates a receptor coupled to Galphaq subunits (GPR54 or KiSS-1R). To study the mechanism by which Kp-10 antagonizes metastatic spread, the effect on CXCR4-mediated signaling, which has been shown to direct organ-specific migration of tumor cells, was determined. Kp-10 blocked chemotaxis of tumor cells expressing CXCR4 in response to low and high concentrations of SDF-1/CXCL12 and inhibited mobilization of calcium ions induced by this ligand. Pretreatment with Kp-10 did not induce down-modulation of cell surface CXCR4 expression, reduce affinity for SDF-1/CXCL12, or alter Galphai subunit activation stimulated by this ligand. Although Kp-10 stimulated prolonged phosphorylation of extracellular signal-regulated kinase 1/2, it inhibited the phosphorylation of Akt induced by SDF-1. The ability of Kp-10 to inhibit signaling and chemotaxis induced by SDF-1 indicates that activation of GPR54 signaling may negatively regulate the role of CXCR4 in programming tumor metastasis.

Antiulcer Properties of the Ethanol Root Extract of Cissampelos mucronata.

ABSTRACTThe study was undertaken to investigate the antiulcer properties of the ethanol root extract of Cissampelos mucronata. A. Rich (Menispermaceae). Three models of experimental ulcer induction in rats were employed in the study. Oral median lethal dose (LD50) was determined in mice using Lorke's method, and the isolated tissue preparations were used to investigate the effect of the extract on calcium ion mobilization in the ileum and H2-receptor blocking property in nongravid rat uterine strip. Study of the acute toxicity test indicated an LD50 of 288.53 mg/kg. Antiulcer study showed that the extract exhibited dose-dependent antiulcer properties in the order indomethacin > histamine > stress-induced ulcers. The ulcer index recorded for the extract against ulcer induced by indomethacin and histamine were significant at p < 0.05. In vitro. pharmacological studies indicated that the extract possessed ability to block the voltage-gated calcium channels; nevertheless, it did not antagonize histamine-induced relaxation of the rat uterine smooth muscle. The results indicate that the ethanol root extract of C. mucronata. exhibited antiulcer property especially against indomethacin-induced ulcer. Cytoprotection and antispasmodic mechanisms of action may be responsible for the antiulcer properties.

Effect of Spermidine on Intracellular Calcium Ion Mobilization in Chicken Phagocytes Treated with Leukotriene B4 (LTB4)

The effect of spermidine, a type of polyamine present in exudates from inflamed tissues and bacteria-infected sites in humans, on calcium mobilization of leukotriene B4 (LTB4)-stimulated chicken abdominal macrophages and heterophils was examined in vitro. The intracellular calcium ion concentration [Ca2+]i was elevated immediately and reached a maximum a few seconds after the addition of LTB4. The elevated [Ca2+]i returned to the basal (resting) level approximately 2min after LTB4 addition. Pretreatment of cells with spermidine significantly prolonged the duration of [Ca2+]i elevation in LTB4-treated chicken phagocytes. However, no change was induced in the level of [Ca2+]i elevation by pretreating the LTB4-treated cells with spermidine. These results suggest that polyamines would enhance the activities of chicken macrophages and heterophils in inflammation sites and bacteria-infected sites through delaying the return to resting levels of stimulated cells, as in the case of human polymorphonuclear leukocytes.

Corticotropin-releasing hormone activates protein kinase C in an isoenzyme-specific manner

Protein kinase C (PKC) has recently emerged as mediator of corticotropin-releasing hormone (CRH) effects. Aim of the present study was to study the effects of CRH on each PKC isoenzyme. As a model we have used the PC12 rat pheochromocytoma cell line, expressing the CRH type 1 receptor (CRHR1). Our data were as follows: (a) CRH-induced rapid phosphorylation of conventional PKCα and PKCβ, accompanied by parallel increase of their concentration within nucleus. (b) CRH suppressed the phosphorylation of novel PKCδ and PKCθ, which remained in the cytosol. (c) CRH-induced transient phosphorylation of atypical PKCλ and had no effect on PKCμ. (d) The effect of CRH on each PKC isoenzyme was blocked by a CRHR1 antagonist. (e) Blockade of conventional PKC phosphorylation inhibited CRH-induced calcium ion mobilization from intracellular stores as well as the CRH-induced apoptosis and Fas ligand production. In conclusion, our findings suggest that CRH via its CRHR1 receptor differentially regulates PKC-isoenzyme phosphorylation, an apparently physiologically relevant effect since blockade of conventional PKC phosphorylation abolished the biological effect of CRH.

Roles of Protein Kinase A (PKA) and PKC on Corticotropin-Releasing Hormone (CRH)-Induced Elevation of Cytosolic Calcium from Extra- and Intra-cellular Sources

Corticotropin-releasing hormone (CRH) affects cytosolic calcium ion levels. The aim of the present work was to examine the role of protein kinase A (PKA)- and PKC-dependent signalling pathways in mediating the effect of CRH on calcium ion influx (from extra-cellular sources) and calcium ion mobilization (from intra-cellular stores). In this study, we employed a well-known model of neural crest-derived cells, the PC12 rat pheochromocytoma cell line. We found that CRH increased the concentration of cytosolic calcium ions in calcium-rich and in calcium-free media. In both conditions, an inhibitor of PKA phosphorylation abolished the effect of CRH. In contrast, the inhibitor of PKC phosphorylation blocked the effect of CRH only in calcium-free conditions. The phorbol ester PMA, activator of PKC, accelerated the steep of the curve of cytosolic calcium ion increase from intra-cellular stores. These data suggest that: (a) CRH induces calcium ion entrance into the cytoplasm from both extra-cellular sources (influx) and from intra-cellular stores (mobilization); (b) the PKA-dependent signalling pathway mediates both effects of CRH; and (c) the PKC-dependent signalling pathway mediates only the CRH-induced mobilization of calcium ions from intra-cellular stores. Thus, this is the first report demonstrating that distinct signalling pathways control the effects of CRH on calcium ion influx and on calcium ion mobilization from intra-cellular stores.

Promotion of B cell immune responses via an alum-induced myeloid cell population.

Exposure of naïve B cells to the cytokine interleukin-4 (IL-4) and/or antigen leads to a state of "priming," in which subsequent aggregation of major histocompatibility complex class II molecules induces the mobilization of calcium ions and cell proliferation. However, it is not clear how critical this priming is for immune responses or how it is normally induced in vivo. Injection of mice with the commonly used adjuvant alum led to priming of splenic B cells and to the accumulation in the spleen of a previously unknown population of IL-4-producing, Gr1+ cells. These cells and IL-4 were both required for in vivo priming and expansion of antigen-specific B cells, as well as for optimal production of antibody. These studies reveal a key role for a previously unknown accessory myeloid cell population in the generation of humoral immune responses.

Antispasmodic and hypotensive effects of Ferula asafoetida gum extract

The effects of Ferula asafoetida gum extract on the contractile responses of the isolated guinea-pig ileum induced by acetylcholine, histamine and KCl, and on the mean arterial blood pressure of rat were investigated. In the presence of extract (3 mg/ml), the average amplitude of spontaneous contractions of the isolated guinea-pig ileum was decreased to 54±7% of control. Exposure of the precontracted ileum by acetylcholine (10 μM) to Ferula asafoetida gum extract caused relaxation in a concentration-dependent manner. Similar relaxatory effect of the extract was observed on the precontracted ileum by histamine (10 μM) and KCl (28 mM). However, when the preparations were preincubated with indomethacin (100 nM) and different antagonists, such as propranolol (1 μM), atropine (100 nM), chlorpheniramine (25 nM) then were contracted with KCl, exposure to the extract (3 mg/ml) did not cause any relaxation. Furthermore, Ferula asafoetida gum extract (0.3–2.2 mg/100 g body weight) significantly reduced the mean arterial blood pressure in anaesthetised rats. It might be concluded that the relaxant compounds in Ferula asafoetida gum extract interfere with a variety of muscarinic, adrenergic and histaminic receptor activities or with the mobilisation of calcium ions required for smooth muscle contraction non-specificly.

Antispasmodic and hypotensive effects of Ferula asafoetida gum extract

The effects of Ferula asafoetida gum extract on the contractile responses of the isolated guinea-pig ileum induced by acetylcholine, histamine and KCl, and on the mean arterial blood pressure of rat were investigated. In the presence of extract (3 mg/ml), the average amplitude of spontaneous contractions of the isolated guinea-pig ileum was decreased to 54±7% of control. Exposure of the precontracted ileum by acetylcholine (10 μM) to Ferula asafoetida gum extract caused relaxation in a concentration-dependent manner. Similar relaxatory effect of the extract was observed on the precontracted ileum by histamine (10 μM) and KCl (28 mM). However, when the preparations were preincubated with indomethacin (100 nM) and different antagonists, such as propranolol (1 μM), atropine (100 nM), chlorpheniramine (25 nM) then were contracted with KCl, exposure to the extract (3 mg/ml) did not cause any relaxation. Furthermore, Ferula asafoetida gum extract (0.3–2.2 mg/100 g body weight) significantly reduced the mean arterial blood pressure in anaesthetised rats. It might be concluded that the relaxant compounds in Ferula asafoetida gum extract interfere with a variety of muscarinic, adrenergic and histaminic receptor activities or with the mobilisation of calcium ions required for smooth muscle contraction non-specificly.

Role of placental alkaline phosphatase in the internalization of trypomatigotes of Trypanosoma cruzi into HEp2 cells.

In vitro, Trypanosoma cruzi invades a wide variety of mammalian cells by an unique process that is still poorly understood. Trypomastigotes adhere to specific receptors on the outer membrane of host cells before intracellular invasion, causing calcium ion mobilization and rearrangement of host cell microfilaments. To test if placental alkaline phosphatase (PLAP), a trophoblast plasma membrane protein anchored by a glycosylphosphatidylinositol molecule, is involved in the transplacental transmission of this parasite. We cultured HEp2 cells with the parasite and studied PLAP and actin microfilaments. The results were correlated with invasion rate. Human HEp2 tumour cells express PLAP. HEp2 cells infected with trypomastigotes showed alteration in their alkaline phosphatase activity and a different pattern of actin organization, compared to control cells. Perturbation of PLAP from HEp2 cells before infection with T. cruzi trypomastigotes decreased the invasion rate. Placental alkaline phosphatase could be involved in the internalization of T. cruzi into HEp2 cells, via activation of tyrosine kinase and rearrangement of actin microfilaments.

Trypanosoma cruzi Infection Induces Microfilament Depletion in Human Placenta Syncytiotrophoblast

Congenital Chagas disease, endemic in Latin America, is associated with premature labour, miscarriage, and placentitis. Metacyclic trypomastigotes adhere to specific receptors on the outer membrane of host cells as a prelude to intracellular invasion, causing calcium ion mobilization, rearrangement of host cell microfilaments, recruitment of lysosomes and parasite internalization. The actin cytoskeleton plays an important role in many cellular processes including the parasite invasion into mammalian cells. In order to observe if placental cytoskeleton is altered in the process of parasite invasion into placental villi, actin microfilaments were studied. Using immunohistochemical techniques, it was observed that the presence of actin in the syncytiotrophoblast was intense throughout the brush border in control placentae belonging to non-chagasic women. But after culture with the trypomastigote, this labelling disappeared, indicating that the parasite induced disassembly of the cortical actin cytoskeleton when the placenta was infected. As a control, placentae from chagasic women were studied, and no actin was found. The same results were obtained by the electron microscope. We confirmed that cortical actin rearrangements may be an early step in the Trypanosoma cruzi invasion mechanism into placental cells, in order to allow lysosomes access to the plasma membrane, and formation of the parasitophorous vacuole. The recruitment of lysosomes occurs directly beneath the invasion site, and this process is required for parasite internalization.

Redirecting migration of T cells to chemokine secreted from tumors by genetic modification with CXCR2.

T-cell-based immunotherapies provide a promising means of cancer treatment although durable antitumor responses are infrequent. A potential reason for these shortcomings may lie in the observed lack of trafficking of specific T cells to tumor. Our increasing knowledge of the process of trafficking involving adhesion molecules and chemokines affords us the opportunity to intervene and correct deficiencies in this process. Chemokines can be expressed by a range of tumors and may serve as suitable targets for directing specific T cells toward tumor. We initially sought to identify which chemokines were produced by a range of human tumor cell lines, and which chemokines and chemokine receptors were expressed by cultured T cells. We identified two chemokines: Growth-Regulated Oncogene-alpha (Gro-alpha; CXCL1) and Regulated on Activation Normal T Cell-Expressed and Secreted (RANTES; CCL5), to be secreted by several human tumor cell lines. Expression was also detected in fine-needle aspirates of melanoma from patients. In addition, we determined the expression of several chemokine receptors on cultured human T cells including CCR1, CCR2, CCR4, CCR5, CXCR3, and CXCR4. Cultured, activated human T cells expressed the chemokines lymphotactin (XCL1), RANTES, macrophage inflammatory protein-1 alpha (MIP-1 alpha; CCL3) and MIP-1 beta (CCL4), but no appreciable Gro-alpha. In a strategy to direct T cells toward chemokines expressed by tumors we chose Gro-alpha as the target chemokine because it was produced by tumor and not by T cells themselves. However, T cells did not express the receptor for Gro-alpha, CXCR2, and therefore, T cells were transduced with a retroviral vector encoding CXCR2. Calcium ion mobilization, an important first step in chemokine receptor signaling, was subsequently demonstrated in transduced T cells in response to Gro-alpha. In addition, Gro-alpha was chemotactic for T cells expressing CXCR2 in vitro toward both recombinant protein and tumor-derived chemokine. Interestingly we demonstrate, for the first time, that Gro-alpha was able to induce interferon-gamma (IFN-gamma) secretion from transduced T cells, thereby extending our knowledge of other potential functions of CXCR2. This study demonstrates the feasibility of redirecting the migration properties of T cells toward chemokines secreted by tumors.

An investigation of the ionophoric characteristics of destruxin A

Destruxin A, a cyclohexadepsipeptide related to the enniatins and beauvericin, exhibits ionophoric properties. Calcium ion mobilization across liposomal membrane barriers, for example, has been demonstrated using the calcium ion-sensitive dyes Arsenazo III and Fura-2. Initial molecular mechanics/molecular dynamics calculations indicate the potential for destruxin A to form a coordination complex with calcium in which the divalent cation is bound at the center of a sandwich formed by two molecules of destruxin A. This novel calcium ion binding may help explain the diverse biological effects exhibited by the destruxins.

Outside-in Signaling Pathway Linked to CD146 Engagement in Human Endothelial Cells

CD146 (S-Endo 1 Ag or MUC18) is a transmembrane glycoprotein expressed on endothelial cells on the whole vascular tree. CD146 is located at the intercellular junction where it plays a role in the cohesion of the endothelial monolayer. CD146 engagement initiates an outside-in signaling pathway involving the protein tyrosine kinases FYN and FAK as well as paxillin. Here we report that CD146 engagement by its specific monoclonal antibody in human umbilical vein endothelial cells induces a Ca(2+) influx that is sensitive to thapsigargin and EGTA treatment, indicating that CD146 engagement initiates a store-operated calcium mobilization. In addition, biochemical and pharmacological analysis revealed that CD146 engagement initiates the tyrosine phosphorylation of phospholipase C-gamma, Pyk2, and p130(Cas). Pharmacological inhibition of Ca(2+) flux with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethyl ester and EGTA indicated that an increase in Ca(2+) is required for Pyk2 and p130(Cas) tyrosine phosphorylation. Moreover, a complex association was observed between Pyk2, p130(Cas), and paxillin. These results indicate that CD146 is coupled to a FYN-dependent pathway that triggers Ca(2+) flux via phospholipase C-gamma activation leading subsequently to the tyrosine phosphorylation of downstream targets such as Pyk2, p130(Cas), FAK, and paxillin. In addition to its role in cell-cell adhesion, CD146 is a signaling molecule involved in the dynamics of actin cytoskeleton rearrangement.

Cultured human mast cells derived from umbilical cord blood cells in the presence of stem cell factor and interleukin-6 cannot be a model of human skin mast cells: fluorescence microscopic analysis of intracellular calcium ion mobilization

To know whether cultured human mast cells raised from umbilical cord blood cells in the presence of stem cell factor (SCF) and interleukin-6 (IL-6) can be a model of human skin mast cells, the cells were stimulated, and intracellular calcium ion ([Ca 2+] i) mobilization was analyzed by fluorescence microscopic techniques in parallel with a measurement of histamine released from the cells. When IgE-sensitized mast cells were activated by anti-IgE, [Ca 2+] i elevation began at the periphery and subsequently proceeded toward the center of the cells. The increase in [Ca 2+] i in calcium ionophore A23187-stimulated mast cells began at the center and spread to the periphery of the cells. Significant histamine release was observed by each stimulation. However, either compound 48/80 or substance P failed to increase [Ca 2+] i with no appreciable histamine release. This study shows that there is heterogeneity of [Ca 2+] i mobilization in the activated human mast cells, and that cultured human mast cells derived from umbilical cord blood cells in the presence of SCF and IL-6 can not be a model of human skin mast cells.

Involvement of protein tyrosine kinases in activation of human eosinophils by platelet-activating factor.

Activation of human eosinophils by platelet-activating factor (PAF) involves multiple signal transduction pathways. Among these, protein kinase C has been demonstrated both to mediate respiratory burst and to suppress an alternative pathway of activation of respiratory burst and arachidonic acid metabolism in eosinophils. We utilized inhibitors of protein tyrosine kinases (PTK) to elucidate the role of PTK in PAF-induced activation of eosinophils. Eosinophils were isolated from peripheral blood of atopic donors and stimulated with PAF in the absence or presence of broad-spectrum PTK inhibitors-genistein or lavendustin A; an inhibitor of mitogen-activated protein (MAP) kinase activation-tyrphostin AG126; or an inhibitor of Janus kinase 2 (Jak2)-tyrphostin B42 (AG490). PAF induced superoxide anion (O2-*) generation, leukotriene C4 (LTC4) release, intracellular calcium ion mobilization and tyrosine phosphorylation of multiple eosinophil proteins in a concentration-dependent manner. All of these responses were concentration-dependently inhibited by genistein; lavendustin A also exhibited potent inhibition of PAF-induced LTC4 release. AG126 had no effect on either O2-* generation or LTC4 release, while AG490 inhibited both responses, albeit less effectively than genistein. We conclude that PAF activates PTK in human eosinophils and that this signalling pathway is involved in eliciting respiratory burst and leukotriene production. The specific PTK(s) involved are unknown but may include Jak2.

Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns.

Inositol 1,4,5-trisphosphate (IP3) is a second messenger that elicits complex spatiotemporal patterns of calcium ion (Ca2+) mobilization and has essential roles in the regulation of many cellular functions. In Madin-Darby canine kidney epithelial cells, green fluorescent protein-tagged pleckstrin homology domain translocated from the plasma membrane to the cytoplasm in response to increased concentration of IP3. The detection of translocation enabled monitoring of IP3 concentration changes within single cells and revealed spatiotemporal dynamics in the concentration of IP3 synchronous with Ca2+ oscillations and intracellular and intercellular IP3 waves that accompanied Ca2+ waves. Such changes in IP3 concentration may be fundamental to Ca2+ signaling.

NADH-Linked Substrate-Mediated Enhancement of Mitochondrial Calcium Accumulation and DNA Single-Strand Breakage Elicited bytert-Butylhydroperoxide: The Source of the Cation Is a Ryanodine-Sensitive Calcium Store

We previously found that the membrane-permeant NADH-linked substrates pyruvate and β-hydroxybutyrate enhance the formation of DNA single-strand breaks induced bytert-butylhydroperoxide (tB-OOH) in intact U937 cells. This effect is mediated by a process involving enforced mitochondrial calcium accumulation in the absence of discernible elevation in the cytosolic concentration of free calcium ions. We now show that the intracellular source of the cation is a ryanodine-sensitive calcium store. A high concentration of ryanodine, which suppressed the caffeine-mediated mobilization of calcium ions, also abolished the effects of the NADH-linked substrates on the mitochondrial accumulation of the cation as well as on the tB-OOH-induced genotoxic response. These data constitute a novel demonstration of a physiological mechanism with important pathological implications.

Influence of atropine on carbachol dual effect on Ca2+ mobilization in SH-SY5Y neuroblastoma cells.

The muscarinic receptor stimulated mobilisation of calcium ions in SH-SY5Y neuroblastoma cells was measured as a function on carbachol and atropine concentrations. The combined application of this pair of muscarinic agonist and antagonist yielded a set of bell-shaped dose-response curves. In the presence of atropine the cell responses were smaller and the up-going phase of these relationships was shifted towards higher agonist concentration, while the down-going phase of these curves was not influenced by the antagonist. These results pointed to a similar mechanism of the receptor inhibition at high carbachol (agonist) concentrations and by atropine (antagonist).