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At least 19 recordsLinked to original sources

PubMed1992

Characterization of functional receptors for gastrointestinal hormones on human colon cancer cells.

Studies demonstrate that some colon cancers possess receptors for various gastrointestinal hormones or neurotransmitters, the occupation of which can affect growth. These results are limited because frequently only a small number of tumors are studied, only 1 or 2 receptors are sought, and the effect on cell function is not investigated. In the present study, 10 recently characterized human colon cancer cell lines were studied to determine whether they possess receptors for any of 12 different gastrointestinal hormones or neurotransmitters and to determine whether these receptors mediate changes in cellular function. Each of the cell lines exhibited receptors for at least one radioligand. Receptors for vasoactive intestinal peptide (VIP) and muscarinic cholinergic agents occurred on 60%, bombesin and gastrin on 30%, beta-adrenergic agents and gastrin-releasing peptide (GRP) on 20%, and somatostatin, opiates, neuromedin B, and substance P on 10%. Analysis of [3H]N-methylscopolamine binding revealed a Kd of 0.2 nM for N-methylscopolamine with a binding capacity of 2500 sites/cell. With the agonist carbamylcholine, the receptor exhibited 2 classes of binding sites: one of high affinity (Kd 55 microM) representing 75% of the binding sites and one of low affinity (Kd 0.3 mM) representing 25% of the binding sites. Analysis of 125I-[Tyr4]bombesin binding revealed a receptor of high affinity (Kd 2.1 microM) with a binding capacity of 3300 sites/cell. Inhibition of binding by agonists revealed relative potencies of 125I-[Tyr4]bombesin greater than GRP much greater than neuromedin B, and two recently described antagonists were similar in potency to GRP. Analysis of 125I-VIP binding revealed a receptor having 2 classes of binding sites: one of high affinity (Kd 3.6 nM) and one of low affinity (Kd 1.7 microM) which represented the majority of the 5.5 x 10(6) binding sites/cell. The relative potencies of agonists were VIP greater than helodermin greater than peptide histidine methionine greater than secretin. Evaluation of biological activity mediated by the muscarinic cholinergic and bombesin receptors revealed an increase of intracellular calcium and of inositol triphosphate by specific receptor agonists. The presence or absence of receptors detected by binding correlated closely with the ability of selective receptor agonists to alter cell function. These results demonstrate the presence of several different receptors for gastrointestinal hormones or neurotransmitters, some described for the first time, on human colon cancer cell lines, including bombesin-related peptides, VIP, somatostatin, substance P, beta-adrenergic agents, calcitonin gene-related peptide, gastrin, muscarinic cholinergic agents, and opiates.(ABSTRACT TRUNCATED AT 400 WORDS)

Bombesin↗

PubMed1992

Characterization of colonic circular smooth muscle cells in culture.

The receptor-binding properties of isolated rabbit colonic circular smooth muscle cells in primary culture have been investigated. In intact smooth muscle, acetylcholine, acting through M2 muscarinic receptors, and vasoactive intestinal polypeptide (VIP), acting through VIP receptors, are two of the principal neurotransmitters mediating contraction and relaxation, respectively. The muscarinic receptor was present in very high levels (600,000 receptors/cell) on freshly isolated colonic smooth muscle cells as shown by binding of the muscarinic receptor antagonist N-methylscopolamine (NMS). However, NMS binding sites decreased rapidly when the cells were placed in primary culture. After 21 h in culture, specific binding of [3H]NMS decreased to 20%, and after 48 h to less than 10% that of preculture values. This loss was not associated with a change in receptor affinity, since Kd was unchanged for the receptors still present. In contrast, high-affinity VIP receptors were expressed on cultured smooth muscle cells but could not be detected on freshly isolated cells. Cultured cells responded to VIP with an increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP), indicating that the VIP receptors were functionally coupled to adenylate cyclase. Cultured cells also responded to calcitonin gene-related peptide (CGRP) and forskolin with increased production of intracellular cAMP. In contrast, neither VIP nor CGRP elicited an increase in intracellular cAMP when added to freshly isolated cells. Furthermore, freshly isolated cells had a greatly diminished response to forskolin, suggesting that the isolation procedure not only destroyed cell surface receptors for VIP and CGRP, but also damaged the cells sufficiently to decrease cellular adenylate cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

C14-aminosteroid LNF 209, an agent with positive inotropic and antimuscarinic activity.

LNF 209 is a cardioactive steroid containing at position C14 a protonized amino group. It was tested whether LNF 209 has antimuscarinic properties besides its cardiotonic effect, which was also quantified. In guinea pig left atria (3 Hz, 0.9 mM Ca2+, 5.4 mM K+), LNF 209 increased the force of contraction at concentrations > or = 1 microM, thus having a tenfold lower potency than ouabain. Correspondingly, cardiac glycoside binding sites in guinea pig cardiac membranes labeled with [3H]ouabain were occupied by LNF 209 (Ki = 1.7 microM) at concentrations tenfold higher compared with ouabain (Kd = 0.14 microM). The negative inotropic effect of the muscarinic agonist oxotremorine was antagonized by LNF 209, but not by ouabain; in the Schild plot, the data for LNF 209 could be connected by a line with a slope of unity and a pA2 = 7.5. The binding of [3H]N-methylscopolamine ([3H]NMS) to the M2-cholinoceptors in guinea pig cardiac membranes was inhibited by LNF 209 with a Ki = 0.5 microM; LNF reduced the affinity of [3H]NMS binding without affecting the number of receptor sites. In isolated segments of guinea pig ileum, LNF 209 antagonized the effect of oxotremorine with a pA2 = 7.4. It is concluded that LNF 209 is a competitive antagonist at M-cholinoceptors without preference for the cardiac M2-cholinoceptors. Whether the antimuscarinic property is of significance in species highly sensitive to digitalis remains to be established.

Androstanes↗

PubMed1992

Heparin, dextran and trypan blue allosterically modulate M2 muscarinic receptor binding properties and interfere with receptor-mediated inhibition of adenylate cyclase.

The influences of heparin, dextran and trypan blue on muscarinic receptor binding properties and inhibition of adenylate cyclase were investigated in homogenates of the rat heart. These compounds caused a concentration-dependent enhancement in the specific binding of the muscarinic antagonist [3H]N-methylscopolamine ([3H]NMS) when measured at a radioligand concentration of approximately 0.5 nM in magnesium-containing, low ionic strength buffer. The maximal enhancements of [3H]NMS binding were 2.89-, 1.68- and 1.43-fold increases for heparin, dextran and trypan blue, respectively; the EC50 values for this effect were 0.12, 0.033 and 4.6 microM, respectively. The effects of heparin, dextran and trypan blue on [3H]NMS binding were attributed mainly to an increase in the overall affinity of muscarinic receptors for [3H]NMS, and were greatly attenuated by 100 mM NaCl. These effects were qualitatively similar to those produced by GTP. Heparin, dextran and trypan blue also affected the binding of the muscarinic agonist oxotremorine-M in a manner similar to that of GTP; that is, in the presence of these compounds, agonist affinity was decreased. Our experiments also showed that heparin and dextran attenuate the inhibition of adenylate cyclase activity caused by oxotremorine-M in myocardial homogenates without influencing basal adenylate cyclase activity. We conclude that heparin and dextran interfere with the muscarinic receptor-G protein coupling in the rat heart.

Adenylyl Cyclase Inhibitors↗

PubMed1992

Selectivity of class I antiarrhythmic agents, disopyramide, pirmenol, and pentisomide for peripheral muscarinic M2 and M3 receptors.

The interactions of the class I antiarrhythmic agents, disopyramide, pirmenol, and pentisomide with peripheral muscarinic receptors were investigated by binding assay with [3H]N-methylscopolamine ([3H]NMS) as a ligand. All the agents inhibited the specific [3H]NMS binding to membrane preparations obtained from guinea pig submandibular gland (SG) and urinary bladder (UB) smooth muscle. The competition curves of these agents for [3H]NMS binding to SG membranes were monophasic, indicating competition with [3H]NMS at a single site. Comparison of results with those of our previous binding experiments using guinea pig left atrial (LA) membranes, showed that pirmenol had sevenfold lower affinity for glandular-type muscarinic receptors (M3) than for cardiac-type muscarinic receptors (M2). On the other hand, the dissociation constants (Ki) for disopyramide and pentisomide in SG were comparable to the high-affinity Ki values for these agents at M2 receptors. The competition curves of the three agents for [3H]NMS binding to UB membranes were biphasic and showed high- and low-affinity states of binding. The high- and low-affinity Ki values for pirmenol in UB were similar to its Ki values at M2 and M3 receptors obtained in LA and SG, respectively. The high-affinity Ki values for disopyramide and pentisomide were consistent with the respective Ki values determined in SG, whereas the low-affinity binding sites for these agents were presumably the result of their allosteric interactions with the receptors. All agents at higher concentrations slowed the dissociation of [3H]NMS elicited by an excess of atropine in both UB and SG, thus indicating allosteric interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

A novel m2-selective muscarinic antagonist: binding characteristics and autoradiographic distribution in rat brain.

Although several m2-selective muscarinic antagonists have been described, they are not particularly potent. Thus, the development of potent m2-selective compounds remains an important goal. We now report that a bio-isoster of AQ-RA 741 is both one order of magnitude more potent and slightly more selective than previously described compounds. DIBA, a di-benzo derivative of AQ-RA 741, in which the pyridine of the tricycle is replaced with a benzene ring, had Ki values of 4, 0.3, 11 and 2 nM at m1 through m4 receptors, respectively. These values were determined in competition studies with [3H]N-methylscopolamine ([3H]NMS) in membranes from transfected A9 L cells (m1 and m3), rat heart (m2) and NG108-15 cells (m4). AQ-RA 741 had Ki values of 34, 4, 86 and 15 nM at each of these receptors. The autoradiographic distribution of DIBA binding sites was determined by competition studies of [3H]NMS in rat brain. At low concentration, DIBA reduced [3H]NMS binding most significantly from superior colliculi, thalamus, hypothalamus, pontine nucleus, and interpeduncular nucleus, and not appreciably from caudate nucleus, cerebral cortical regions, or hippocampus, consistent with its binding to m2 receptors. These data indicate that DIBA is the most potent, m2-selective muscarinic antagonist yet described. DIBA should therefore become a useful probe in future studies of muscarinic function.

Animals↗

PubMed1992

Activation of T84 cell chloride channels by carbachol involves a phosphoinositide-coupled muscarinic M3 receptor.

Muscarinic agonists stimulate Cl- secretion across monolayers of the colon tumor epithelial cell line, T84. The muscarinic receptor has been characterized in T84 cell homogenates by radioligand binding using [3H]N-methylscopolamine ([3H]NMS). [3H]NMS bound to a single population of sites at 25 degrees C in 100 mM NaCl, 20 mM HEPES, 10 mM MgCl2, pH 7.4 buffer, with calculated Kd = 278 (+/- 44) pM and Bmax = 40 (+/- 6) fmol/mg protein (n = 4). Binding was reversible (diss. t1/2 = 18 +/- 3 min) and stereoselective (dexetimide Ki = 0.3 nM) much greater than levetimide (Ki = 8300 nM). Antagonists exhibited the following rank order of potencies and Ki values (nM): atropine (0.54) greater than 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) (0.84) greater than dicyclomine (14) = hexahydrosiladifenidol (18) greater than pirenzepine (136) greater than AF-DX 116 (3610). The same sequence was observed for inhibition of carbachol-induced 125I efflux from T84 monolayers. This is indicative of an M3 'glandular' muscarinic receptor. Coupling to second messenger systems was examined by labelling monolayers with [14C]arachidonic acid (AA) or [3H]inositol. Carbachol (0.3 mM) did not release [14C]AA from labelled lipids, but ionomycin produced a dose-dependent increase in media [14C]AA. Carbachol (0.3 mM) elevated inositol monophosphate 14-fold. The results suggest that muscarinic agonists stimulate Cl- secretion by interacting with an M3 receptor coupled to inositide lipid hydrolysis.

Arachidonic Acid↗

PubMed1992

Distribution of beta-adrenergic receptor subtypes in human post-mortem brain: alterations in limbic regions of schizophrenics.

The distribution of the beta 1 (beta 1) and beta 2 (beta 2) subtypes of the beta-adrenergic receptor was examined in rat and nondiseased control human tissue. The distribution of the beta 1 and beta 2 receptors was also examined in schizophrenic cases, with additional studies in schizophrenic suicide and nonschizophrenic suicide cases. Scatchard analysis of the binding of [125I]iodopindolol (IPIN) to cortical membranes showed a similar Kd in human (177 pM) and rat (161 pM), but a lower maximum binding site (Bmax) in the human tissue (18.7 fmol/mg protein and 55.6 fmol/mg protein). For the autoradiographic studies [125I]IPIN was used to visualize both subtypes (total) or was displaced with the selective beta 1-receptor antagonist ICI-89,406 to visualize beta 2 sites, or with the selective beta 2-receptor antagonist ICI-118,551 to visualize beta 1 sites. Important differences in the regional distribution of the two subtypes of the beta-adrenergic receptors were noted between rat and human. In the nucleus accumbens and ventral putamen (ventral striatum), a patchy distribution of beta 1 receptors was observed that was not evident in the rat. These patches were aligned with markers of the matrix compartment of the striatum. The schizophrenic cases showed significant increases in the labeling of the beta 1-receptor patches with [125I]IPIN. In contrast to the frontal cortex of the nondisease controls, the parietal and temporal cortex showed a high ratio of beta 1 to beta 2 receptors and a highly laminar organization of the subtypes. [125I]IPIN binding to beta 1 receptors was highest in the external laminae with the reverse gradient for the beta 2 subtype. The medial temporal cortex displayed an alteration in the ratio of the 2 subtypes of the beta-adrenergic receptor, with the parahippocampus and hippocampus of the human, in contrast to the rat brain, predominantly expressing the beta 2 receptor. Moreover, there were consistently higher densities of beta 2 receptors in the hippocampus of the right hemisphere than the left hemisphere of the nondisease controls. There was not a left and right hemispheric asymmetry of beta 2 receptors in the hippocampus of elderly schizophrenics or in young schizophrenics who committed suicide. The asymmetry was evident in nonschizophrenic suicides, suggesting that the lack of asymmetry in the hippocampus of schizophrenics is evident early in the disease process. Thus limbic structures show alterations in the patterning of beta 1 and beta 2 receptors in the schizophrenic cases.

Adult↗

PubMed1992

Impairment of contractile response to carbachol and muscarinic receptor coupling in gastric antral smooth muscle cells isolated from diabetic streptozotocin-treated rats and db/db mice.

This work explored the role of the cholinergic pathway, assessed at a post-synaptic level by the use of isolated smooth muscle cells, in the impairment of antral motility associated with diabetic gastroparesis. Contractile response to carbachol--but not to erythromycin, a motilin receptor agonist--was abolished in antral smooth muscle cells isolated from (i) rats previously rendered diabetic by a single i.v. dose of streptozotocin (STZ, 60 mg/kg) and (ii) db/db spontaneously diabetic mice. Insulin treatment of STZ-rats was able to prevent the impairment of the carbachol contractile response, but not to reverse it once established. In STZ-rats, impairment of contractile response was not associated with a change in density of [3H]-N-methyl-scopolamine ([3H]-NMS) binding sites (approximately 1.5 fmol/mg protein). Displacement curve of the [3H]-NMS binding by carbachol was shifted to the right in diabetic rats as compared to controls. The addition of GTP-gamma-S induced a shift to the right of the displacement curve in control but not in diabetic animals. These results strongly suggest that diabetes is associated with an early and specific alteration of the muscarinic control of contraction of antral smooth muscles at a post-synaptic level, associated with an alteration of the GTP-binding proteins coupled to muscarinic receptors.

Animals↗

PubMed1992

Blockade of conditioned taste aversion by scopolamine and N-methyl scopolamine: associative conditioning, not amnesia.

The anticholinergic, scopolamine, consistently disrupts one-trial passive avoidance conditioning but the effects of such drugs on one-trial conditioned taste aversion (CTA) are variable and contradictory. In the present study, treatment of rats with scopolamine impaired the suppression of sucrose intake by post-ingestion administration of lithium chloride (LiCl) in a two-bottle choice test. A similar effect was obtained by using N-methyl scopolamine which penetrates the brain only to a limited degree on acute administration. The blockade of CTA could be prevented in three ways: (i) by exposing the rats to sucrose only on the training day, (ii) by pre-exposing the rats to both sucrose and scopolamine, and (iii) by using a less palatable sucrose/ascorbate mixture. The results demonstrate that the effect of scopolamine on taste aversion is not mediated by the central nervous system, and can be modified by altering the novelty and relative salience of the taste conditioned stimulus. These experiments suggest that conditioned associations between taste and LiCl, and scopolamine and LiCl may underlie the blockade of CTA by scopolamine.

Amnesia↗

PubMed1992

Increased muscarinic cholinergic receptor density on CD4+ lymphocytes in progressive multiple sclerosis.

We measured the density and affinity of muscarinic cholinergic receptors (MR) in 29 chronic progressive and ten stable multiple sclerosis (MS) patients and 27 control subjects using [3H]N-methyl-scopolamine. The density of MR on CD4+ lymphocytes was significantly higher in chronic progressive MS (CPMS) than in controls (7.9 +/- 0.7 vs. 4.5 +/- 0.4 fmol/10(6) cells, p less than 0.001). Stable patients did not differ significantly from control subjects. Receptors of the M1 subtype were measured on CD4+ lymphocytes of nine patients and seven controls with the selective antagonist [3H]methylpirenzepine: M1/total receptor ratio was 64.1% in CPMS and 81.2% in controls, suggesting a selective increase of M2-type MR in CPMS. The findings may relate to parasympathetic denervation hypersensitivity of lymphocytes or to lymphocyte activation which is known to be associated with increased MR number.

Adult↗

PubMed1992

Differential regulation of muscarinic and nicotinic cholinergic receptors and their mRNAs in cultured sympathetic neurons.

Mechanisms regulating expression of neuronal muscarinic and nicotinic receptors were examined in cultures of neonatal rat sympathetic neurons. Two factors known to stimulate cholinergic transmitter development in sympathetic neurons were examined for their effects on cholinergic receptor expression. A membrane associated factor (MANS46) and a diffusible factor produced by cultured rat fibroblasts (RFCM) each decreased muscarinic receptor number. By contrast, neither treatment altered levels of nicotinic receptors. Levels of muscarinic (m2) receptor mRNA were decreased by MANS but not by RFCM, indicating that effects of the two treatments were mediated by different mechanisms. Neither MANS nor RFCM altered levels of nicotinic alpha 3 or beta 2 mRNAs, consistent with the lack of change in numbers of nicotinic receptors. These observations indicate that receptor phenotype in developing neurons is subject to regulation by multiple epigenetic factors. Further, the same signals which regulate transmitter development may also regulate receptor expression in sympathetic neurons.

Animals↗

PubMed1992

Carbamazepine-induced neurotoxicity and its prevention by NMDA in cultured cerebellar granule cells.

Long-term neurotoxicity of carbamazepine was studied in cultured cerebellar granule cells. Treatment of cells with carbamazepine for 3 days induced a dose-dependent neurotoxicity detected by a loss of [3H]ouabain binding to Na+,K(+)-ATPase, and [3H]N-methyl scopolamine binding to muscarinic cholinergic receptors as well as by direct morphologic examination. NMDA protected against carbamazepine-induced toxicity and this protection was blocked by 2-amino-5-phosphono-valerate (APV). The neurotoxicity induced by carbamazepine may be involved in the teratogenic and adverse effects of overdose associated with the treatment of manic-depressive illness and seizures.

2-Amino-5-phosphonovalerate↗

PubMed1992

Lack of intrinsic activity and significant subtype selectivity of SR 95639A at muscarinic receptors.

We assessed the intrinsic activity of the purported selective muscarinic M1 receptor agonist SR 95639A (morpholinoethylamino-3-benzocyclohepta-(5,6-c)-pyridazine) in inducing several receptor-mediated signals. Our results indicate that SR 95639A lacks the ability to activate phosphoinositide hydrolysis in rat cerebral cortex or in Chinese hamster ovary cells transfected with the genes of the muscarinic m1 and m3 receptors. Similarly, this compound did not exhibit intrinsic activity in stimulating muscarinic receptors which inhibit cyclic AMP synthesis and did not suppress acetylcholine release in rat striatum. In addition, SR 95639A did not show a marked selectivity at the level of the ligand recognition site at the muscarinic M1, M2 and M3 receptors, since it bound to these receptor subtypes with equilibrium dissociation constants of 4, 6 and 11 microM, respectively.

Acetylcholine↗

PubMed1992

The effects of p-chlorophenylalanine-induced serotonin synthesis inhibition and muscarinic blockade on the performance of rats in a 5-choice serial reaction time task.

The effects of serotonergic dysfunction induced by treatment with p-chlorophenylalanine (PCPA), an inhibitor of serotonin synthesis, and cholinergic dysfunction induced by scopolamine on the performance of adult rats in the 5-choice serial reaction time task measuring selective attention were studied. Food-deprived rats were trained to detect and respond to brief flashes of light presented randomly in one of five locations, until they reached a stable level of performance (about 4 months). Scopolamine 0.2 mg/kg produced a marked variation in the performance but did not, however, induce any consistent impairment in the discriminative accuracy. Other doses of scopolamine (0.05 and 0.1 mg/kg) or N-methyl-scopolamine 0.2 mg/kg, a peripheral muscarinic receptor antagonist, did not affect discriminative accuracy. Furthermore, scopolamine as well as N-methyl-scopolamine produced a number of other performance deficits, such as significantly decreased overall probability of responding and significantly increased response latencies. PCPA treatment induced an almost total depletion (> 99%) of frontal cortical serotonin and its major metabolite 5-HIAA and reduced the frontal cortical concentrations of noradrenaline (-30%) and dopamine (-42%). During baseline testing conditions, there was a trend for the discriminative accuracy to be decreased by PCPA, although this effect failed to reach significance (P = 0.07). Presenting the stimuli at unpredictable intervals or reducing the intensity of the visual stimulus impaired discriminative accuracy in both PCPA-treated and control rats. The decrease in discriminative accuracy induced by PCPA reached statistical significance when the stimuli were presented faster than normally or the intensity of the visual stimulus was reduced. PCPA treatment did not make the rats more susceptible to the effects of scopolamine on discriminative accuracy. However, PCPA treatment also induced a number of other performance deficits, resulting in a decreased overall tendency to respond. In summary, there is a statistically non-significant trend for the discriminative accuracy to be decreased by PCPA treatment under normal testing conditions, and as the discrimination task is made more difficult (stimulus intensity reduction, presentation of the stimuli at faster than normal rates), the deficit in discriminative accuracy produced by PCPA treatment is revealed. The results suggest a role for brain serotonin in the general organization of behavior.

Animals↗

PubMedDate not supplied

Chronotropic effects of atropine sulfate and methscopolamine bromide in normal subjects and patients undergoing cardiac catheterization.

The increase in heart rate caused by intravenous injections of atropine sulfate (AS) and methscopolamine bromide (MSB) were compared in 12 healthy volunteers. AS, 200 micrograms, or MSB, 100 micrograms, were injected every 3 min for five doses on successive days. Mean heart rate from the second and fifth injections of MSB was significantly greater than after corresponding injections of atropine. The ratio of equivalent doses of MSB and AS was found to be 1:3. Three subjects with AS and four subjects with MSB exhibited reduced heart rates after the first injection. Excellent correlation of plasma atropine concentrations and heart rate increase were obtained in two volunteers (correlation coefficient, r = 0.84). In the second study, AS, 180 micrograms, and MSB, 60 micrograms, were compared in 20 patients undergoing cardiac catheterization and coronary arteriography. Both drugs produced equivalent increments in heart rate. MSB produced a more predictable dose response than AS. Side effects were similar with both drugs when equivalent doses were used. This study confirms previous investigations that MSB can be titrated by intravenous injections to increase heart rate to desired levels. Absence of central nervous system stimulation by MSB suggests that it may be more appropriate for use in patients with myocardial infarction.

Adolescent↗

PubMed1992

Lack of specific binding of bradykinin and D-Arg [Hyp3, Thi5, D-Tic, Oic8] bradykinin in membranes from rat heart.

Recent data demonstrated effects of bradykinin (BK) in the isolated perfused rat heart which could be blocked by BK antagonists. However, so far BK receptors in heart tissue have not been characterized. To search for BK receptors in rat hearts iodinated D-Arg[Hyp3,Thi5,D-Tic,Oic8]BK (Hoe 140) was used as a ligand for binding due to its high affinity and to its resistance to degradation. The labeled antagonist bound well to membranes prepared from rat ileum and could be displaced by the unlabeled antagonist as well as by BK and T-kinin in a concentration dependent manner. However, there was no specific binding detectable when membranes from rat left cardiac ventricle were used. To assure integrity of at least one other receptor in these membranes, binding of 3H-methylscopolamine and its displacement by the respective cold ligand was demonstrated. To rule out an occupation of the BK receptors by endogenous formed BK, the tissue was treated with an acidic buffer with high ionic strength to remove surface bound BK. However, this treatment did not unmask any specific binding for the BK antagonist. In view of the possibility that the structure of the labeled antagonist prevented its binding, experiments were carried out using tritiated BK itself. These experiments also failed to demonstrate specific binding sites which indicate that the structural aspects of Hoe 140 are probably not interfering in binding of the antagonist, especially since it binds to ileum. It is concluded, that there are too few binding sites for BK in the rat heart homogenate for ordinary binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Muscarinic receptor subtypes in human neuroblastoma cell lines SH-SY5Y and IMR-32 as determined by receptor binding, Ca++ mobilization and northern blotting.

Muscarinic receptor subtypes in neuroblastoma cell lines IMR-32 and SH-SY5Y were determined with receptor binding, Ca++ mobilization and Northern blotting. Displacement of [3H]NMS with pirenzepine in IMR-32 cells revealed apparent binding sites with Kd values of 5 (41%) and 237 nM (59%). With 4-diphenylacetoxy-N-metylpiperidine metiodid, a similar proportion of apparent high- and low-affinity binding was obtained: 36 (Kd = 0.26 nM) and 64% (Kd = 6.3 nM), respectively. In SH-SY5Y cells, two different affinities with apparent Kd of 40 (24%) and 460 nM (76%) could be distinguished with pirenzepine, even though the Kd of the apparent high-affinity site varied markedly (variation = 8.7-96.8 nM). Inhibition of carbachol-induced Ca++ mobilization displayed high sensitivity to 4-diphenylacetoxy-N-methylpiperidine metiodid in both cell lines. IMR-32 cells displayed high sensitivity to pirenzepine, whereas the sensitivity varied between different batches of SH-SY5Y cells. DNA fragments (approximately 1000 base pairs) from SH-SY5Y DNA amplified with polymerase chain reaction were used as probes for muscarinic receptor mRNA. Northern blotting with the Hm1-specific probe gave a stronger signal for SH-SY5Y than for IMR-32, whereas the result obtained with the Hm2-probe was the opposite. Also, the Hm3 mRNA was detected in SH-SY5Y cells. The Hm4 and Hm5 transcripts were not detected in either of these cell lines.

Blotting, Northern↗