Carbachol-stimulated calcium entry in SH-SY5Y human neuroblastoma cells: which route?

M3 muscarinic receptors expressed on SH-SY5Y human neuroblastoma cells are linked to phosphoinositide turnover and rises in [Ca2+]i. The rise in [Ca2+]i is biphasic with the peak phase being due to release from an intracellular Ins(1,4,5)P3-sensitive site and the plateau phase being due to Ca2+ entry across the plasma membrane. Ca2+ entry does not appear to involve voltage sensitive Ca2+ channels, a pertussis toxin sensitive G-protein-operated Ca2+ channel or Ins(1,4,5)P3/Ins(1,3,4,5)P4-operated Ca2+ channel. We suggest that carbachol-stimulated Ca2+ entry in SH-SY5Y human neuroblastoma cells occurs via receptor operated Ca2+ channels and through capacitive refilling.     

Regulation of Muscarinic Agonist-Induced Activation of Phosphoinositidase C in Electrically Permeabilized SH-SY5Y Human Neuroblastoma Cells by Guanine Nucleotides

myo-[3H]Inositol-labelled SH-SY5Y cells were permeabilized with electrical discharges. 3H-Inositol phosphate formation in cells shown to be fully permeable was stimulated by the muscarinic agonist carbachol, by guanosine 5'-(gamma-thio)triphosphate [GTP(S)], and by guanosine 5'-(beta gamma-imido)diphosphate (GppNHp). Synergism was observed on coincubation of these GTP analogues with carbachol. GTP was also stimulatory and guanosine 5'-(beta-thio)diphosphate was inhibitory in the presence of agonist. Atropine blocked the effects of carbachol. Stimulation by GTP(S) (0.1 mM) occurred after a 1-2-min lag, whereas Ca2+ (0.5 mM), carbachol (1 mM), and carbachol plus GTP(S) stimulated without delay. The effects of carbachol plus GTP(S) but not those of Ca2+ were inhibited by spermine (4 mM). Accumulation of 3H-inositol phosphates was enhanced by Li+ (4 mM) only in intact cells. In intact or permeabilized cells, the "partial" agonist arecoline was maximally 40-50% as efficacious as carbachol. In permeabilized cells, the maximal effects of carbachol and arecoline were enhanced 2.8- and 5.3-fold, respectively, by 0.1 mM GTP(S), but only the EC50 for carbachol was substantially reduced. The binding affinity of carbachol but not that of arecoline in permeabilized cells was significantly reduced by 0.1 mM GppNHp. These data indicate that a guanine nucleotide-binding regulatory protein is involved in coupling muscarinic receptors to phosphoinositidase C in SH-SY5Y cells and that the activity of this protein influences the relationship between receptor occupation and phosphoinositide response.     

12-O-Tetradecanoylphorbol 13-Acetate and Forskolin Modify Muscarinic Receptor-Linked Ca2+ Mobilization in SH-SY5Y Neuroblastoma Cells Through Different Mechanisms

The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which causes differentiation of SH-SY5Y neuroblastoma cells, reduces carbachol binding and carbachol-stimulated Ca2+ mobilization in these cells. The decrease in responsiveness to carbachol is due partially to a reduction in the amount of Ca2+ released by the cells and partially to a decrease in the sensitivity of the cells to carbachol. These effects probably can be attributed to a reduction in muscarinic receptor number and a decrease in receptor affinity, respectively. Forskolin, an alkaloid known to cause an increase in cellular cyclic AMP, enhances Ca2+ influx into the cells without affecting the cytosolic free Ca2+ concentration. The alkaloid causes an apparent restoration of the reduced Ca2+ release, caused by TPA, but does not affect the sensitivity of the cells to carbachol. Forskolin increases the decay of carbachol-induced increase in cytosolic Ca2+. The effects of TPA appear to be linked directly to receptor function, whereas those of forskolin are due to the effect of cyclic AMP on cellular Ca2+ metabolism.     

(-)-Indolactam V activates protein kinase C and induces changes in muscarinic receptor functions in SH-SY5Y human neuroblastoma cells

The effects of a synthetic protein kinase C (PKC) activator, (-)-indolactam V (ILV), were studied in SH-SY5Y human neuroblastoma cells. (-)-ILV induced a translocation of PKC from cytosol to plasma membrane and displaced 3H-phorbol dibutyrate binding in the micromolar range. In addition, (-)-ILV caused a decreased sensitivity of cells to muscarinic agonist-induced Ca2+ mobilization measured with quin-2 and induced a down-regulation of cell surface muscarinic receptors. All the changes induced by (-)-ILV were similar in magnitude to those seen with the phorbol ester tetradecanoyl phorbol acetate (TPA). The results suggest that (-)-ILV is a full activator of PKC and a promising alternative to phorbol esters in studies on mechanism of actions of PKC.     

Differentiation-associated decrease in muscarinic receptor sensitivity in human neuroblastoma cells

Muscarinic receptor-linked increases in intracellular free Ca2+ as measured with quin-2 and Ca2+ release from monolayers of cells have been measured in the human neuroblastoma cell line SH-SY5Y. Induction of differentiation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to a decrease in the sensitivity of the cells to low concentrations of agonists with respect to the induced increase in cytosolic free Ca2+ and stimulation of Ca2+ efflux. No decrease in agonist binding affinity was observed when the displacement of a labelled antagonist, 3H-NMS, by a non-labelled agonist was studied.     

Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C.

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SY5Y human neuroblastoma cells has been investigated. The dose-response relations of carbachol-induced cAMP synthesis and carbachol-induced rise in intracellular free Ca2+ were similar. The stimulated cAMP synthesis was inhibited by about 50% when cells were entrapped with the Ca2+ chelator BAPTA or in the presence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca2+ ionophore, ionomycin and by TPA, an activator of PKC. When added together TPA and ionomycin had a synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE1 or PGE1 + pertussis toxin carbachol stimulated cAMP production to the same extent as in control cells. Ca2+ and protein kinase C thus seem to be the mediators of muscarinic-receptor linked cAMP synthesis by a direct action on adenylate cyclase.     

The Epithelial Phenotype of Human Neuroblastoma Cells Expresses Bradykinin, Endothelin, and Angiotensin II Receptors That Stimulate Phosphoinositide Hydrolysis

The neuroblastoma line SK-N-SH consists of distinct and interconverting cell types, which include a neuroblast phenotype (SH-SY5Y), an epithelial phenotype (SH-EP), and an intermediate cell type (SH-IN). In SH-SY5Y cells, only muscarinic receptor activation produced stimulation of phosphoinositide turnover, whereas in SH-EP cells, where muscarinic receptors are not present, the peptides bradykinin, endothelin, and angiotensin II stimulated phosphoinositide hydrolysis with EC50 values of 16, 6, and 0.7 nM, respectively, and a rank order of maximal effects of bradykinin greater than endothelin greater than angiotensin II. Fetal calf serum at concentrations between 1 and 10% was also a potent stimulator of phosphoinositide hydrolysis in SH-EP cells but not in SH-SY5Y cells. In the intermediate cell clone, SH-IN, phosphoinositide hydrolysis was stimulated not only by muscarinic receptors, but also by endothelin, bradykinin, and serum, an indication that this cell type harbors all the kinds of receptors that are differentially expressed in the other two cell types. The effects of the three peptides--bradykinin, endothelin, and angiotensin II--on phosphoinositide hydrolysis in SH-EP cells were additive, a result suggesting that the three kinds of receptors may activate distinct transducer proteins and/or phospholipase C subtypes. Pretreatment of intact SH-EP cells with pertussis toxin under conditions sufficient to ADP-ribosylate 90-95% of the endogenous guanine nucleotide regulatory protein substrates did not impair the ability of any of the receptors to stimulate phosphoinositide hydrolysis in any of the cell types. In contrast, short-term exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (1 microM) abolished the stimulation of phosphoinositide hydrolysis mediated by peptide receptors in SH-EP cells and partially inhibited that by muscarinic receptors in SH-SY5Y cells. Prolonged incubation of SH-EP cells with phorbol ester resulted in a recovery of receptor responsiveness, the extent and rate of which were different for each receptor type. In contrast, there was no recovery of responsiveness for muscarinic receptors in SH-SY5Y cells. The pattern of phorbol ester-mediated effects depended on the cell rather than on the receptor type. In fact, muscarinic receptor responsiveness in SH-IN, the intermediate cell type, was desensitized by and recovered from treatment with phorbol esters in a manner more similar to peptide receptors in SH-EP than to muscarinic receptors in SH-SY5Y. These data suggest that the transduction mechanisms by which distinct receptor types are coupled to phosphoinositide hydrolysis in the three cell phenotypes differ in sensitivity to feedback regulation by protein kinase C.     

Apparent noncompetitive antagonism of muscarinic receptor mediated Ca2+ mobilization by some muscarinic antagonists.

Ca2+ mobilizations in SH-SY5Y and IMR-32 human neuroblastoma cell lines were measured using the fluorescent Ca2+ indicator fura-2. A variety of antagonists (atropine, pirenzepine, 4-DAMP and N-methyl-scopolamine) inhibited carbamyl choline-induced transient Ca2+ mobilization both in a competitive and a noncompetitive manner. The apparent noncompetitive inhibition constants were lower in IMR-32 than in SH-SY5Y cells even when the competitive inhibition constants were similar. This may relate to the previously reported differential expression of muscarinic receptor subtypes in these cell lines.     

Receptor-Coupled Phosphoinositide Hydrolysis in Human Retinal Pigment Epithelium

Carbachol and histamine stimulated phosphoinositide (PPI) hydrolysis in cultured human retinal pigment epithelium (RPE), as reflected by an accumulation of 3H-inositol phosphates in the presence of 10 mM Li+. Carbachol increased PPI hydrolysis to greater than 600% of basal with an EC50 of 60 microM; stimulation was linear up to 60 min. This activation likely occurred via the M3 muscarinic cholinergic receptor based on the IC50 values for 4-diphenylacetoxy-N-methylpiperidine methiodide (0.47 nM), pirenzepine (280 nM), and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]-acetyl]-5,11- dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one (1.4 microM). Carbachol-mediated PPI hydrolysis was decreased by 80% in the absence of extracellular Ca2+. Histamine stimulated PPI turnover in a linear manner by 180% with an EC50 of 20 microM by the H1 histaminergic receptor. Serotonin, glutamate, norepinephrine, and dopamine were inactive. In human RPE, the resting cytoplasmic Ca2+ concentration, as determined by fura-2 fluorescence, was 138 +/- 24 nM. On the addition of carbachol, there was a 180% increase in peak intracellular Ca2+; addition of histamine increased intracellular Ca2+ by 187%. These results suggest receptor-mediated, inositol lipid hydrolysis is coupled to intracellular Ca2+ flux in human RPE.     

Inositol 1,4,5-Trisphosphate Receptor Immunoreactivity in SH-SY5Y Human Neuroblastoma Cells Is Reduced by Chronic Muscarinic Receptor Activation

Inositol 1,4,5-trisphosphate (InsP3) receptor immunoreactivity in SH-SY5Y human neuroblastoma cells was monitored with a monoclonal antibody raised against the mouse cerebellar InsP3 receptor. Recognition of a protein corresponding to the InsP3 receptor (molecular mass, approximately 275 kDa) was inhibited markedly following exposure of cells to 0.1 mM carbachol. This effect was half-maximal and maximal at approximately 2 and approximately 6 h, respectively; was blocked by atropine; but was not mimicked by thapsigargin, K+, or phorbol 12-myristate 13-acetate. However, the decrease in immunoreactivity following exposure of cells to carbachol for 5 h was blocked if the extracellular Ca2+ concentration was reduced from 1.3 mM to 200 nM. This manipulation also reduced markedly carbachol-induced increases in InsP3 concentration at 5 h. These data indicate that chronic muscarinic stimulation of phosphoinositide hydrolysis reduces InsP3 receptor concentration in SH-SY5Y cells, perhaps via a mechanism that involves prolonged elevation of InsP3 levels.     

Inhibition of polyphosphoinositide turnover in rat cerebral cortex by clonidine

In the rat brain, a number of receptors are linked to phospholipase C which catalyzes the hydrolysis of membrane inositol phospholipids; stimulation of alpha 1-adrenergic receptors, for example, increases polyphosphoinositide turnover, but stimulation of alpha 2-receptors does not. The hydrolysis of inositol phospholipids in rat cortical slices was investigated using a direct assay involving prelabeling these lipids with 3H-inositol and then measuring the formation of 3H-inositol phosphates in the presence of lithium ions. As expected, clonidine, an alpha 2-agonist, did not stimulate the formation of 3H-inositol phosphates; however, clonidine antagonized the ability of noradrenaline to stimulate 3H-inositol phosphate formation. This effect was not blocked by antagonists of alpha 2, 5HT2, H2, or muscarinic receptors. Clonidine did not affect carbachol-stimulated 3H-inositol phosphate formation.     

Muscarinic Receptor-Stimulated Phosphoinositide Turnover in Human SK-N-SH Neuroblastoma Cells: Differential Inhibition by Agents that Elevate Cyclic AMP

The possibility that an increased intracellular concentration of cyclic AMP (cAMP) can regulate the extent of muscarinic receptor-stimulated phosphoinositide (PPI) turnover in the human neuroblastoma cell line SK-N-SH was examined. Addition of either forskolin (or its water-soluble analog, L-85,8051), theophylline, isobutylmethylxanthine, or cholera toxin, agents that interact with either the catalytic unit of adenylate cyclase, cAMP phosphodiesterase, or the guanine nucleotide binding protein linked to adenylate cyclase activation, resulted in a 45-181% increase in cAMP concentration and a 27-70% inhibition of carbachol-stimulated inositol phosphate release. Through the use of digitonin-permeabilized cells, the site of inhibition was localized to a step at, or distal to, the guanine nucleotide binding protein that regulates phospholipase C activity. In contrast, when intact SK-N-SH cells were exposed to prostaglandin E1, the ensuing increases in cAMP were not accompanied by an inhibition of stimulated PPI turnover. These differential effects of increased cAMP concentrations on stimulated PPI turnover may reflect the compartmentation of cAMP within SK-N-SH cells.     

Muscarinic Receptor Sequestration in SH-SY5Y Neuroblastoma Cells Is Inhibited when Clathrin Distribution Is Perturbed

Abstract: The possibility that clathrin plays a role in the agonist-mediated sequestration of muscarinic cholinergic receptors in human SH-SY5Y neuroblastoma cells has been investigated by the application of experimental paradigms previously established to perturb clathrin distribution and receptor cycling events. Preincubation of SH-SY5Y cells under hypertonic conditions resulted in a pronounced inhibition of agonist-induced muscarinic receptor sequestration (70–80% at 550 mOsm), which was reversed when cells were returned to isotonic medium. Depletion of intracellular K⁺ or acidification of the cytosol also resulted in >80% inhibition of muscarinic receptor sequestration. Under conditions of hypertonicity, depletion of intracellular K⁺, or acidification of cytosol, muscarinic receptor-stimulated phosphoinositide hydrolysis and Ca²⁺ signaling events were either unaffected or markedly less inhibited than receptor sequestration. That these same experimental conditions did perturb clathrin distribution was verified by immunofluorescence studies. Hypertonicity and depletion of intracellular K⁺ resulted in a pronounced accumulation of clathrin in the perinuclear region, whereas acidification of the cytosol resulted in the appearance of microaggregates of clathrin throughout the cytoplasm and at the plasma membrane. The results are consistent with the possibility that muscarinic receptors in SH-SY5Y cells are endocytosed via a clathrin-dependent mechanism.     

应用两种方法诱导SH-SY5Y细胞分化的研究

目的：观察全反式维甲酸（ATRA）处理和ATRA与十四烷酰佛波醇乙酸酯（TPA）序贯处理（ATRA/TPA）对人类神经母细胞瘤细胞系SH-SY5Y细胞增殖抑制和形态分化的影响。方法：应用10μM ATRA处理6天和10μM ATRA处理3天继以80 nMTPA处理3天这两种方法使SH-SY5Y细胞分化;用倒置光学显微镜动态观察SH-SY5Y细胞形态学变化;并用MTT比色法比较两种分化方法对SH-SY5Y细胞的体外抗增殖作用。结果：ATRA处理和ATRA与TPA序贯处理对SH-SY5Y细胞都有抗增值和诱导细胞分化作用,细胞形态发生明显的变化,分化成神经元表型,前者主要表现为两端带有长突起的纺锤体样细胞形态,而后者主要是由细胞体延伸出多个突起的多边形的细胞。ATRA分化6天的细胞的存活率下降为78.7%±2.0%。当去除ATRA后,继续培养1天的细胞存活率上升为89%±0.2%,而继续培养2天的细胞存活率为86.3%±1.4%;ATRA与TPA序贯分化6天细胞存活率下降为75.9±0.4%。当去除TPA后,继续培养一天的细胞存活率为75.5±0.7%,继续培养2天的细胞存活率为74.9±1.0%。结论：维甲酸（ATRA）处理和ATRA与十四烷酰佛波醇乙酸酯（TPA）序贯处理（ATRA/TPA）均能明显诱导SH-SY5Y细胞分化。这两种分化细胞为神经科学的研究提供了优良的体外培养模型细胞,尤其是ATRA与TPA序贯处理能获得分化完全而稳定的神经元样细胞。     

Mitogenic response of human SH-SY5Y neuroblastoma cells to insulin-like growth factor I and II is dependent on the stage of differentiation

Human insulin-like growth factor I and II (IGF-I and IGF-II) in concentrations of 1-30 ng/ml, were shown to stimulate ornithine decarboxylase activity and [3H]thymidine incorporation in human SH-SY5Y neuroblastoma cells. Proliferation of these cells was also stimulated by IGF-I and II when added to RPMI 1640 medium, fortified with selenium, hydrocortisone, transferrin, and beta-estradiol. Labeled IGF-I and II bound to SH-SY5Y cells. The cross-reaction pattern of IGF-I, IGF-II, and insulin in competing with the binding of labeled IGF-I and IGF-II, respectively, indicated that SH-SY5Y cells express both type I and type II IGF receptors. Treatment of SH-SY5Y cells for 4 d with 12-O-tetradecanoylphorbol-13-acetate (TPA), which resulted in morphological and functional differentiation and growth inhibition, abolished the mitogenic response to both IGF-I and II. Concomitantly, the binding of IGF-II disappeared almost totally, which offers a possible explanation for the reduced biological response to IGF-II after TPA treatment. In contrast, the IGF-I binding in TPA-treated cells was only reduced to approximately 70% of the binding to control cells. It is therefore not excluded that the IGF-I receptor could be uncoupled by TPA, with persistent binding capacity for IGF-I.     

Protein kinase C activation and down-regulation in relation to phorbol ester-induced differentiation of SH-SY5Y human neuroblastoma cells

The role of protein kinase C activation in changes in muscarinic receptor functions and in the appearance of biochemical properties characteristic of neuronal cells was studied in SH-SY5Y human neuroblastoma cells induced to differentiate with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). A decrease in muscarinic receptor sensitivity with respect to agonist induced Ca2+ mobilization and receptor number parallelled the increase in membrane-associated protein kinase C (PK-C) activity. These changes occurred during the first 6 h of culture, and they were associated with rounding-up of cells. A subsequent decrease in particulate PK-C activity was followed by an increase in noradrenaline content, the appearance of an electrically excitable membrane, and an increase in the level of neuron-specific enolase. These changes were accompanied by a pronounced neurite outgrowth. 1-(5-Isoquinolinesulphonyl)-2-methylpiperazine (H-7), an inhibitor of PK-C and cyclic nucleotide-dependent protein kinases, enhanced the morphological differentiation induced by TPA, whereas N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA-1004), which primarily inhibits cyclic nucleotide-dependent protein kinases, had no effect on the TPA-induced phenotypic differentiation. H-7 inhibited the decrease in muscarinic receptor sensitivity and receptor number, but had no effect on the appearance of the electrically excitable membrane or on the increase in the neuron-specific enolase level. Both H-7 and HA-1004 inhibited the TPA-induced increase in noradrenaline content.     

Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc mRNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC8) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA and DiC8 it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.     

Muscarinic-receptor-mediated changes in intracellular Ca2+ and inositol 1,4,5-trisphosphate mass in a human neuroblastoma cell line, SH-SY5Y.

This study reports increased intracellular Ca2+ and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to muscarinic-cholinergic stimulation of human neuroblastoma (SH-SY5Y) cells. Carbachol stimulation leads to a rapid increase in intracellular Ca2+ and Ins(1,4,5)P3 mass, both reaching a peak at around 10 s and then declining to a new maintained phase significantly above basal. Dose-response analysis of peak and plateau phases of intracellular Ca2+ shows different agonist potencies for both phases, carbachol being more potent for the plateau phase. The plateau-phase intracellular Ca2+ was dependent on extracellular Ca2+, which is admitted to the cell through a non-voltage-sensitive Ni2(+)-blockable Ca2+ channel. Using a Mn2+ quench protocol, we have shown that Ca2+ entry occurs early during the discharge of the internal stores. The plateau phase (Ca2(+)-channel opening) is dependent on the continued presence of agonist, since addition of atropine closes the Ca2+ channel and intracellular Ca2+ declines rapidly back to basal. We also failed to detect a refilling transient when we added back Ca2+ after intracellular Ca2+ had reached a peak and then declined in Ca2(+)-free conditions. These data strongly suggest that muscarinic stimulation of SH-SY5Y cells leads to a rapid release of Ca2+ from an Ins(1,4,5)P3-sensitive internal store and a parallel early entry of Ca2+ across the plasma membrane.     

Inositol 1,4,5-trisphosphate-independent calcium signalling by platelet-derived growth factor in the human SH-SY5Y neuroblastoma cell.

In adherent SH-SY5Y human neuroblastoma cells, activation of G-protein-coupled muscarinic M3 receptors evoked a biphasic elevation of both intracellular [Ca(2+)] ([Ca(2+)]i) and inositol-1,4,5-trisphosphate (D-Ins(1,4,5)P3) mass. In both cases, temporal profiles consisted of rapid transient elevations followed by a decline to a lower, yet sustained level. In contrast, platelet-derived growth factor (PDGF), a receptor tyrosine kinase agonist acting via PDGF receptor b chains in these cells, elicited a slow and transient elevation of [Ca(2+)]i that returned to basal levels within 5 to 10 min with no evidence of inositol phosphate generation. Full responses for either receptor type required intracellular and extracellular Ca(2+) and mobilization of a shared thapsigargin-sensitive intracellular Ca(2+) store. Strategies that affected the ability of D-Ins(1,4,5)P3 to interact with the Ins(1,4,5)P3-receptor demonstrated an Ins(1,4,5)P3-dependency of the muscarinic receptor-mediated elevation of [Ca(2+)]i but showed that PDGF-mediated elevations of [Ca(2+)]i are Ins(1,4,5)P3-independent in these cells.     

The Intact Human Neuroblastoma Cell (SH-SY5Y) Exhibits High-Affinity [3H]Pirenzepine Binding Associated with Hydrolysis of Phosphatidylinositols

The binding of [3H]pirenzepine to a human neuroblastoma cell line (SH-SY5Y) and its correlation with hydrolysis of phosphatidylinositols were characterized. Specific [3H]pirenzepine binding to intact cells was rapid, reversible, saturable, and of high affinity. Kinetic studies yielded association (k+1) and dissociation (k-1) rate constants of 5.2 +/- 1.4 X 10(6) M-1 min-1 and 1.1 +/- 0.06 X 10(-1) min-1, respectively. Saturation experiments revealed a single class of binding sites (nH = 1.1) for the radioligand with a total binding capacity of 160 +/- 33 fmol/mg protein and an apparent dissociation constant of 13 nM. The specific [3H]pirenzepine binding was inhibited by the presence of selected muscarinic drugs. The order of antagonist potency was atropine sulfate greater than pirenzepine greater than AF-DX 116, with K0.5 of 0.53 nM, 2.2 nM, and 190 nM, respectively. The binding properties of [3H](-)-quinuclidinyl benzilate and its quaternary derivative [3H](-)-methylquinuclidinyl benzilate were also investigated. The muscarinic agonist carbachol stimulated formation of inositol phosphates which could be inhibited by muscarinic antagonists. The inhibition constants of pirenzepine and AF-DX 116 were 11 nM and 190 nM, respectively. In conclusion, we show that the nonclassical muscarinic receptor antagonist [3H]pirenzepine identifies a high-affinity population of muscarinic sites which is associated with hydrolysis of phosphatidylinositols in this human neuroblastoma cell line.