Blockade of alpha-adrenergic receptors by analogues of phosphatidylcholine

The experimental evidence reviewed in this article suggests that the kidneys may have an additional function in regulating blood pressure besides their role in controlling both blood volume by urine formation and the relative state of vasoconstriction by the renin-angiotensin system. That is, the kidneys may have an additional influence upon the vasculature of a hormonal vasodilating system. The interstitial cells of the renal medulla appear to be mediating this activity and lipid compounds have been extracted from the renal medulla which display depressor activity. One such compound, the antihypertensive polar renomedullary lipid (APRL), has been demonstrated to consist of specific alkyl ether analogues of phosphatidylcholine. The vascular responses to these compounds include vasodilation of both arterioles and venules, rapid lowering of arterial blood pressure with little or no tachycardia, increased depressor activity in hypertensive animals, and blockade of vascular smooth muscle alpha 1-adrenergic receptors. Most recently, APRL and a synthetic analogue, 1-0-octadecyl-2-acetyl-sn-glycero-3-phosphorylcholine, have been used to demonstrate alpha-adrenergic receptor blockade on a smooth muscle cell line (DDT1) by radioligand assays. This action may be due to the insertion of these compounds into cell membranes causing subsequent steric interactions and blockade of the alpha-adrenergic receptor.     

Increased agonist affinity is induced in tetranitromethane-modified muscarinic receptors

Tetranitromethane (TNM) modifies the muscarinic receptors from rat cerebral cortex. The modified receptor possesses an increased binding affinity (6-9-fold) toward several agonists such as acetylcholine, carbamoylcholine, arecoline, etc. The binding of antagonists (Bmax and Kd) is only slightly altered. The effects of TNM treatment can be prevented by atropine, thus indicating that TNM modifies residue(s) at the binding site. We carried out a series of successive chemical modifications which indicated that the modified residue(s) is (are) most probably a tyrosyl and not a cysteinyl residue. This conclusion gains support from the pH profile of agonist binding, which suggests the involvement of a residue with an apparent pK comparable to that of the phenolic hydroxyl of a nitrotyrosyl residue. The binding properties of the modified receptor, when compared to those of the native one, clearly indicate that the response to TNM modification with respect to the binding of agonists such as acetylcholine and carbamoylcholine is different from that when oxotremorine and its analogue are employed. This is interpreted as being the result of different binding modes exhibited by the various agonists. Nitration of the receptors can be prevented by the presence of an antagonist but not by an agonist. We propose that this differential response is due to the formation of ligand-receptor complexes that differ with respect to the microenvironment of the modified tyrosyl residue.     

Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics

The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-[3H]piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. In contrast, the major differences between the kinetic binding parameters of agonists and antagonists to the low affinity agonist binding sites are in the association rate constants, which were 2-5 orders of magnitude lower for agonists. This demonstrates that there are basic differences in the interactions of agonists with the low and high affinity sites. Our findings also suggest that isomerization of the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.     

Modulation of pancreatic muscarinic receptors by weaning

We have investigated the effects of early and delayed weaning on the development of the rat pancreatic muscarinic receptors. Weaning after 12, 14 and 16 complete days resulted in significantly increased concentrations of muscarinic receptors. Milk feeding, as the exclusive source of food, from day 12 to 23, 25 or 28, was associated with slight non significant decreases in receptor concentration. In both instances, early or delayed weaning, the apparent KDs of (3H-QNB binding were not affected. It is suggested that early solid food intake can modulate rapidly the pancreatic muscarinic receptor population while exclusive milk feeding does not seem to influence the dynamic of the muscarinic receptor population.     

Modulation of neuritogenesis by astrocyte muscarinic receptors

Astrocytes have been shown to release factors that have promoting or inhibiting effects on neuronal development. However, mechanisms controlling the release of such factors from astrocytes are not well established. Astrocytes express muscarinic receptors whose activation stimulates a robust intracellular signaling, although the role of these receptors in glial cells is not well understood. Acetylcholine and acetylcholine receptors are present in the brain before synaptogenesis occurs and are believed to be involved in neuronal maturation. The present study was undertaken to investigate whether stimulation of muscarinic receptors in astrocytes would modulate neurite outgrowth in hippocampal neurons. Rat hippocampal neurons, co-cultured with rat cortical astrocytes previously exposed to the cholinergic agonist carbachol, displayed longer neurites. The effect of carbachol in astrocytes was due to the activation of M3 muscarinic receptors. Exposure of astrocytes to carbachol increased the expression of the extracellular matrix proteins fibronectin and laminin-1 in these cells. This effect was mediated in part by an increase in laminin-1 and fibronectin mRNA levels and in part by the up-regulation of the production and release of plasminogen activator inhibitor-1, an inhibitor of the proteolytic degradation of the extracellular matrix. The inhibition of fibronectin activity strongly reduced the effect of carbachol on the elongation of all the neurites, whereas inhibition of laminin-1 activity reduced the elongation of minor neurites only. Plasminogen activator inhibitor-1 also induced neurite elongation through a direct effect on neurons. Taken together, these results demonstrate that cholinergic muscarinic stimulation of astrocytes induces the release of permissive factors that accelerate neuronal development.     

Autoantibodies enhance agonist action and binding to cardiac muscarinic receptors in chronic Chagas' disease

Chronic Chagasic patient immunoglobulins (CChP-IgGs) recognize an acidic amino acid cluster at the second extracellular loop (el2) of cardiac M(2)-muscarinic acetylcholine receptors (M(2)AChRs). These residues correspond to a common binding site for various allosteric agents. We characterized the nature of the M(2)AChR/CChP-IgG interaction in functional and radioligand binding experiments applying the same mainstream strategies previously used for the characterization of other allosteric agents. Dose-response curves of acetylcholine effect on heart rate were constructed with data from isolated heart experiments in the presence of CChP or normal blood donor (NBD) sera. In these experiments, CChP sera but not NBD sera increased the efficacy of agonist action by augmenting the onset of bradyarrhythmias and inducing a Hill slope of 2.5. This effect was blocked by gallamine, an M(2)AChR allosteric antagonist. Correspondingly, CChP-IgGs increased acetylcholine affinity twofold and showed negative cooperativity for [(3)H]-N-methyl scopolamine ([(3)H]-NMS) in allosterism binding assays. A peptide corresponding to the M(2)AChR-el2 blocked this effect. Furthermore, dissociation assays showed that the effect of gallamine on the [(3)H]-NMS off-rate was reverted by CChP-IgGs. Finally, concentration-effect curves for the allosteric delay of W84 on [(3)H]-NMS dissociation right shifted from an IC(50) of 33 nmol/L to 78 nmol/L, 992 nmol/L, and 1670 nmol/L in the presence of 6.7 x 10(- 8), 1.33 x 10(- 7), and 2.0 x 10(- 7) mol/L of anti-el2 affinity-purified CChP-IgGs. Taken together, these findings confirmed a competitive interplay of these ligands at the common allosteric site and revealed the novel allosteric nature of the interaction of CChP-IgGs at the M(2)AChRs as a positive cooperativity effect on acetylcholine action.     

Blockade of GABAA receptor channels by niflumic acid prevents agonist dissociation

The modulation by the nonsteroidal anti-inflammatory drug niflumic acid (NFA) of the GABAA receptor-mediated currents was studied in acutely isolated cerebellar Purkinje cells using the whole-cell recording and fast drug application system. At concentrations of 3–300 μM NFA potentiated GABA (2 μM)-activated currents, and at concentrations of 1–3 mM NFA blocked these responses. The NFA-induced block was strongly voltage-dependent. Analysis of the voltage dependence of the block suggests that the blocking action of NFA is a result of NFA binding at the site located within GABAA channel pore. The termination of GABA and NFA application was followed by a transient increase of the inward current — “tail” current. These data suggest that NFA acts as a sequential open channel blocker, which prevents dissociation of agonist while the channel is blocked. The tail current develops because, prior to dissociation of agonist, the channels that are in the blocked state must return to the close state via the open state. The tail currents were compared in the presence and absence of gabazine, a competitive antagonist that also allosterically inhibits GABA_A receptors. Application of gabazine only during development of tail current did not change neither amplitude nor time course of this current, while noncompetitive antagonists picrotoxin and penicillin blocked it. Protection of tail current from gabazine block indicates that GABA cannot dissociate from the open-blocked state and the agonist was trapped on the receptor while the channel was open. Trapping was specific for the agonist, because the positive allosteric modulator zolpidem (benzodiazepine site agonist) was able to potentiate the tail current in the absence of GABA in the external solution. Our observations provide a model-independent functional support of the hypothesis that open channel block of GABAA channels by NFA prevents an escape of the agonist from its binding sites.     

Purification of muscarinic acetylcholine receptors by affinity chromatography

Calf forebrain homogenates contain 2.8 pM muscarinic acetylcholine receptors per mg of protein. [3H]Antagonist saturation binding experiments under equilibrium conditions revealed a single class of sites with equilibrium dissociation constants of 0.82 nM for [3H]dexetimide and 0.095 nM for [3H]quinuclidinyl benzilate. Displacement binding studies with agonists revealed the presence of low and high affinity sites. Here we describe the solubilization of muscarinic acetylcholine receptors with digitonin and their purification by affinity chromatography using an affinity gel which consisted of dexetimide coupled to Affi-Gel 10 (i.e., carboxy N-hydroxysuccinimide esters linked via a 1 nm spacer arm to agarose beads). Purified proteins were obtained by specific elution with muscarinic drugs, i.e., the antagonist atropine and the irreversible ligand propylbenzilylcholine mustard. SDS-polyacrylamide gel electrophoresis of the radioiodinated purified preparations revealed a major 70-K protein.     

Activation of solubilized G-proteins by muscarinic acetylcholine receptors

Binding of GTP and its analogue, guanosine 5′-O-[γ-thio]triphosphate (GTP[S]) to G-proteins, and release of GTP[S] from G-proteins are stimulated by muscarinic acetylcholine (mACh) receptors in intact cardiac membranes. Upon solubilization of receptors and G-proteins by membrane extraction with the detergent, 3-[(cholamidopropyl)dimethylammonio]-1-propanesulphonate, followed by sucrose density gradient centrifugation, agonist-liganded mACh receptors stimulated binding of GTP[S] and hydrolysis of GTP by G-proteins with similar requirements as in intact membranes. One soluble agonist-activated mACh receptor induced binding of GTP[S] to several (about seven) soluble G-proteins. In contrast to intact membranes, however, agonist activation of mACh receptors did not induce release of GTP[S] from solubilized G-proteins. The data presented indicate that mACh receptors can interact with and efficiently activate G-proteins even in solution, whereas the possible interaction of receptors with GTP[S]-liganded G-proteins observed in intact membranes is lost upon solubilization of these components.     

Maturation of muscarinic agonist receptors in rat developing pancreas and its relation to maximal enzyme secretion

The true K_Ds of [³H] (?) QNB binding to muscarinic receptors were found to be 4.13 and 6.43 × 10^?11 M in pancreas of 21 day fetal and adult rats. The competition curves of specific [³H] (?) QNB bound by two antagonists have shown that the affinity of these drugs did not change with age with Hill coefficients near unity. However, with the agonist carbamylcholine as the competitive drug, a more flat curve was obtained with a Hill coefficient below unity. At least two populations of carbamylcholine binding sites were found with different K_Ds: a K_H around 7 × 10^?7 M and a K_L around 3 × 10^?5 M. These two populations were present during all the developmental periods studied. The ED₅₀ of bethanechol stimulated amylase secretion did not change within the age limit studied (from 11 to 365 days). The high affinity sites for carbamylcholine would seem to be the receptor implicated in the physiological response of the pancreas.     

Constitutively active muscarinic receptors

Mutations that increase constitutive activity and alter ligand binding have been used to investigate the structure and mechanism of activation of muscarinic receptors. These data are reviewed with reference to the recently published three-dimensional structure of rhodopsin. Residues in TM3 and TM6 where amino acid substitutions increased constitutive activity align with residues within the core of the receptor. A nucleus of these residues is located immediately below the predicted binding site of acetylcholine. The i2 loop where mutations also increase constitutive activity was found to loop away from the i3 loop, which has been found to modulate G-protein coupling specificity.     

Striatal muscarinic receptors: Regulation by dopaminergic agonists

The effects of apomorphine on the binding properties of striatal muscarinic receptors were investigated using the specific muscarinic antagonist, [³H](?)3-quinuclidinyl benzilate ([³H](?)QNB). When binding measurements were made in 50 mM sodium/HEPES buffer, pH 7.4, containing Mg⁺², the binding of [³H](?)QNB was consistent with the presence of two binding sites; 57% of the sites had a high affinity dissociation constant of 0.030 nM whereas the remaining sites had a low affinity dissociation constant of 0.64 nM. Apomorphine (1.0 μM) enhanced the binding of [³H](?)QNB by an apparent conversion of low to high affinity sites. A variety of other agents were screened for their ability to enhance [³H](?)QNB binding, and a pattern generally consistent with a dopaminergic effect was observed although some evidence for a β-adrenergic effect was demonstrable. The potent neuroleptics haloperidol, spiperone and sulpiride failed to antagonize the apomorphine enhancement of [³H](?)QNB binding as well as some adrenergic antagonists. However, the potent inhibitors of the dopamine-sensitive adenylate cyclase, α-flupenthixol and fluphenazine, specifically blocked the apomorphine enhancement of [³H](?)QNB binding with K_i values of approximately 0.1 μM.     

Sphingosine kinase-mediated calcium signaling by muscarinic acetylcholine receptors

Based on the finding that G protein-coupled receptors (GPCRs) can induce Ca2+ mobilization, apparently independent of the phospholipase C (PLC)/inositol-1,4,5-trisphosphate (IP3) pathway, we investigated whether sphingosine kinase, which generates sphingosine-1-phosphate (SPP), is involved in calcium signaling by mAChR and other GPCRs. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,/N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by M2 and M3 mAChRs in HEK-293 cells without affecting PLC activation. Activation of M2 and M3 mAChR rapidly and transiently stimulated production of SPP. Furthermore, microinjection of SPP into HEK-293 cells induced rapid and transient Ca2+ mobilization. Pretreatment of HEK-293 cells with the calcium chelator BAPTA/AM fully blocked mAChR-induced SPP production. On the other hand, incubation of HEK-293 cells with calcium ionophores activated SPP production. Similar findings were obtained for formyl peptide and P2Y2 purinergic receptors in HL-60 cells. On the basis of these studies we propose, that following initial IP3 production by receptor-mediated PLC activation, a local discrete increase in [Ca2+]i induces sphingosine kinase stimulation, which ultimately leads to full calcium mobilization. Thus, sphingosine kinase activation most likely represents an amplification system for calcium signaling by mAChRs and other GPCRs.     

Phosphorylation of the cardiac muscarinic receptor in intact chick heart and its regulation by a muscarinic agonist

We have tested the possibility that regulation of cardiac muscarinic receptor function may involve receptor phosphorylation. Chick heart muscarinic receptors were purified from relatively small amounts of tissue to near homogeneity using a three-step chromatographic procedure that utilized the affinity chromatography procedure of Haga and Haga (Haga, K., and Haga, T. (1983) J. Biol. Chem. 258, 13575-13579). The purified preparations contained a single major peptide which migrated on sodium dodecyl sulfate gels with an apparent Mr of 79,000. When receptors were purified from 32P-bathed hearts, a single major phosphopeptide eluted from the affinity column and comigrated on sodium dodecyl sulfate gels with the band of stained receptor. Treatment of hearts with the agonist carbachol led to marked increases (10-12-fold) in the phosphorylation of the receptor. The results show that the muscarinic receptor is a phosphoprotein in cardiac tissue and that treatment with a receptor agonist regulates its phosphorylation in the intact cell.     

G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na+ channel interaction

Our previous experiments in membranes prepared from rat heart and brain led us to suggest that the binding of agonists to the muscarinic receptors and to the Na+ channels is a coupled event mediated by guanine nucleotide binding protein(s) [G-protein(s)]. These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of [3H]acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of [3H]batrachotoxin to Na+ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced 22Na+ uptake in the presence and absence of tetrodotoxin, which blocks Na+ channels. Our findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na+ channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components--receptor, G-protein, and Na+ channel--is such that at resting potential the muscarinic receptor induces opening of Na+ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.     

Quinuclidinone O-Alkynyloximes with muscarinic agonist activity.

A series of quinuclidinone O-alkynyloximes (14-19) were synthesized and evaluated in radioligand displacement assays for binding affinities to M1-M3 muscarinic receptors. Radioligand displacement assays were carried out using [3H] oxotremorine-M and [3H] pirenzepine on rat cortical tissue and [3H] N-methylscopolamine on rat heart and submandibulary glands. Two alkynyloximes 15 and 18 had pirenzepine/oxotremorine M ratios which were indicative of muscarinic agonist and partial agonist activity, respectively. They were tested for their mnemonic effects in mice using the swimming escape task and found to attenuate scopolamine induced impairment of the task in mice at 2mg/kg. The results show that the O-alkynyloxime moiety linked to azacycles of appropriate size and rigidity (for example quinuclidine and tropane) is a potentially useful muscarinic pharmacophore that can be exploited for the design of muscarinic agonists.     

Blockade of immune receptors of mouse lymphocytes by several lectins

It has been shown that incubation of mouse spleen lymphocytes with lectins PHA, ConA, LSG decreased the number of luminous cells in comparison with the control numbers. The luminous rabbit sera against mouse immunoglobulins was used in the experiments. The more was the concentration of lectins the less was the luminescence of the lymphocytes. The maximum effect was shown for PHA. The researchers came to the conclusion that the lectins used had a pronounced non-specific capacity to block the immune receptors of mouse lymphocytes.     

An alkylating derivative of oxotremorine interacts irreversibly with the muscarinic receptor

A 2-chloroethylamine derivative of oxotremorine was studied in pharmacological experiments and muscarinic receptor binding assays. The compound, N-[4-(2-chloroethylmethylamino)-2-butynyl]-2-pyrrolidone (BM 123), forms an aziridinium ion in aqueous solution at neutral pH that stimulates contractions of the guinea pig ileum with a potency similar to that of oxotremorine. Following the initial stimulation, there is a long lasting period of lack of sensitivity of the guinea pig ileum to muscarinic agonists. BM 123 also produces muscarinic effects in vivo. When homogenates of the rat cerebral cortex were incubated with BM 123 and assayed subsequently in muscarinic receptor binding assays, a loss of binding capacity for the muscarinic antagonist, [3H]N-methylscopolamine ( [3H]NMS), was noted without a change in affinity. Similar observations were made in [3H]1-3-quinuclidinyl benzilate ( [3H]1-QNB) binding assays on the forebrains of mice that had been injected with BM 123 24 hr earlier. The loss in receptor capacity for both [3H]NMS and [3H]1-QNB was prevented by atropine treatment. Kinetic studies of the interaction of BM 123 with homogenates of the rat cerebral cortex in vitro showed that the half-time for the loss of [3H]1-QNB binding sites increased from 10 to 45 min as the concentration of BM 123 decreased from 10 to 1 microM. In contrast to the aziridinium ion, the parent 2-chloroethylamine compound and the alcoholic hydrolysis product were largely devoid of pharmacological and binding activity.