Binding Ability Of Receptors Research Articles

Comparative In-Silico Molecular Docking of Silymarin for SARS-CoV-2 Receptor

The COVID-19 pandemic has spread worldwide in over 185 countries, with millions of infections and hundreds of thousands of deaths. The current pandemic has made the situation worse, forcing the development of better treatment. In this work, the binding ability of COVID-19 receptors with silymarin has been analyzed using AutoDock 1.4.6. Further, it is compared with the standard drug remdesivir. Silymarin, a potential phytochemical compound obtained from the seeds of the Silybum marianum (milk thistle) plant, has been documented as an antiviral agent against several viruses. So silymarin can also be an effective compound in the treatment of COVID-19. This study aims to determine the binding ability of COVID-19 receptors towards silymarin and further comparative analysis by remdesivir. Drug Discovery Studio version 2021 software was used to analyze ligands and targets. AutoDock 1.4.6 software was used to perform the docking study. Among the various receptors, 5N11 (Human beta1-coronavirus (β1CoV) OC43), 7MJP (SARS-CoV-2 receptor binding domain in complex with neutralizing antibody COVA2-39), 7JMO (SARS-CoV-2 receptor-binding domain in complex with neutralizing antibody COVA2-04) receptors showed the highest binding ability of -8.09, -7.23, -6.96 kcal/mol towards silymarin compared to the standard remdesivir having the docking score of -5.21, -3.76, -2.97 kcal/mol, respectively. By the comparative analysis, silymarin has a better and highest binding ability.

Exploring the mechanism of Buxue Yimu Pill on hemorrhagic anemia through molecular docking, network pharmacology and experimental validation.

Buxue Yimu Pill (BYP) is a classic gynecological medicine in China, which is composed of Angelica sinensis (Oliv.) Diels, Leonurus japonicus Houtt, Astragalus membranaceus (Fisch.) Bunge, Colla corii asini and Citrus reticulata Blanco. It has been widely used in clinical therapy with the function of enriching Blood, nourishing Qi, and removing blood stasis. The current study was designed to determine the bioactive molecules and therapeutic mechanism of BYP against hemorrhagic anemia. Herein, GC-MS and UPLC/Q-TOF-MS/MS were employed to identify the chemical compounds from BYP. The genecards database (https: //www.genecards.org/) was used to obtain the potential target proteins related to hemorrhagic anemia. Autodock/Vina was adopted to evaluate the binding ability of protein receptors and chemical ligands. Gene ontology and KEGG pathway enrichment analysis were conducted using the ClusterProfiler. As a result, a total of 62 candidate molecules were identified and 152 targets related to hemorrhagic anemia were obtained. Furthermore, 34 active molecules and 140 targets were obtained through the virtual screening experiment. The data of molecular-target (M-T), target-pathway (T-P), and molecular-target-pathway (M-T-P) network suggested that 32 active molecules enhanced hematopoiesis and activated the immune system by regulating 57 important targets. Pharmacological experiments showed that BYP significantly increased the counts of RBC, HGB, and HCT, and significantly down-regulated the expression of EPO, IL-6, CSF3, NOS2, VEGFA, PDGFRB, and TGFB1. The results also showed that leonurine, leonuriside B, leosibiricin, ononin, rutin, astragaloside I, riligustilide and levistolide A, were the active molecules closely related to enriching Blood. In conclusion, based on molecular docking, network pharmacology and validation experiment results, the enriching blood effect of BYP on hemorrhagic anemia may be associated with hematopoiesis, anti-inflammation, and immunity enhancement.

The ligand binding ability of dopamine D1 receptors synthesized using a wheat germ cell-free protein synthesis system with liposomes

G-protein coupled receptors (GPCRs) share a common seven-transmembrane topology and mediate cellular responses to a variety of extracellular signals. However, structural and functional approaches to GPCRs have often been limited by the difficulty of producing a sufficient amount of receptor protein using conventional expression systems. We synthesized human dopamine D1 receptors using a wheat cell-free protein synthesis system with liposomes and then analyzed their receptor binding ability. We determined the specific binding of [3H]SCH23390 to the synthesized receptors generated from a cell-free protein synthesis system or rat striatal membranes. From Scatchard plot analysis, the dissociation constant (Kd) and the maximum density (Bmax) of the synthesized receptors were 6.61±0.06nM and 1.85±0.05pmol/mg protein, respectively. The same analysis for rat striatal membrane gave a Kd of 2.67±0.05nM and Bmax of 0.70±0.10pmol/mg protein. Using a competition binding assay, Ki values of antagonists, SCH23390, LE300 and SKF83566, for the synthetic receptors were the same as those for rat striatal membranes, but Ki values of agonists, A68930, SKF38393 and dopamine, were 5–17 fold higher than those for rat striatal membranes. These results suggest that the dopamine D1 receptors synthesized in liposomes have a functional binding capacity. The different patterns of binding of antagonists and agonists to the synthetic receptors and rat striatal membranes indicate that G proteins are involved in agonist binding to dopamine D1 receptors. The cell-free protein synthesis method with liposomes will be invaluable for the functional analysis of GPCRs.

Synthesis and anion binding properties of new dihomooxacalix[4]arene diurea and dithiourea receptors

Functionalization of the lower rim of p-tert-butyldihomooxacalix[4]arene with two (thio)ureido moieties provided new diurea (n-propyl 5a, tert-butyl 5b and phenyl 5c) and dithiourea (phenyl 5d) derivatives, all in the cone conformation, as shown by NMR studies. The X-ray crystal structure of 5c is reported. The binding ability of these neutral receptors towards a large variety of anions was assessed by 1H NMR titrations. The structures and complexation energies of some complexes were also studied using DFT methods. The data showed that, in general, the association constants decrease with decrease of anion basicity and they are strongly dependent on the nature of the substituent at the urea moiety. Phenyl-(thio)urea derivatives 5c and 5d are the best anion receptors, showing the strongest complexation for F− (log Kass=2.70 and 2.75, respectively) and also for the oxoanions AcO−, BzO− and H2PO4−. These results were corroborated by DFT calculations.

Chromogenic anion molecular probes based on β,β’-disubstituted calix[4]pyrroles

The reaction of octamethylcalix[4]pyrrole with 3-(dimethylamino)prop-2-enal and POCl3 affords a mixture of isomeric calix[4]pyrroles bearing two formylethenyl groups at the β-pyrrolic positions. These dialdehydes were used in Knoevenagel reactions with malononitrile and indene-1,3-dione leading to new chromogenic anion molecular probes. The binding ability of the new receptors was investigated by UV–vis spectroscopy and NMR, indicating the formation of supramolecular 1:2 host:guest complexes with high affinity constants.

Bidentate Urea Derivatives of p-tert-Butyldihomooxacalix[4]arene: Neutral Receptors for Anion Complexation

Three new bidentate ureidodihomooxacalix[4]arene derivatives (phenyl 5a, n-propyl 5b, and tert-butyl 5c) were synthesized in four steps from the parent compound p-tert-butyldihomooxacalix[4]arene and obtained in the cone conformation, as shown by NMR studies. The binding ability of these neutral receptors toward spherical, linear, trigonal planar, and tetrahedrical anions was assessed by (1)H NMR and UV-vis titrations. The structures and complexation energies of some complexes were also studied by DFT methods. The data showed that the association constants are strongly dependent on the nature of the substituent (aryl/alkyl) at the urea moiety. In general, for all the receptors, the association constants decrease with decrease of anion basicity. Ph-urea 5a is the best anion receptor, showing the strongest complexation for F(-) (log K(assoc) = 3.10 in CDCl3) and also high binding affinity for the carboxylates AcO(-) and BzO(-). Similar results were obtained by UV-vis studies and were also corroborated by DFT calculations.

Comparative study of colorimetric sensors based on newly synthesized Schiff bases

New sensitive cation receptors 2a and 2b containing oligoether linked Schiff bases were prepared and their ability to calorimetrically sense transition metal ions was investigated. Addition of metal ions to a methanol solution of 2a, 2b can result in an obvious colour change (from colourless to yellowish-brown for Cu2+ and colourless to yellow for Co2+, Ni2+, Cd2+, Zn2+) and UV–vis absorption spectral change in a short time. The colour change and binding ability of receptors with Cu2+ ion are more compare to other ions, the detection limit is allowable to 1.0×10−5 and 1.0×10−6M level of Cu2+, 1.0×10−4 and 1.0×10−5M level of Co2+, Ni2+, Cd2+, Zn2+ according to visual colour change and UV–vis spectral change, respectively. This indicated that these two receptors are highly selective for those metal ions over other ions like Ca2+, Mg2+, Mn2+, Hg2+, Cr3+, Fe2+, Pb2+.

The tautomerization between keto- to phenol-hydrazone induced by anions in the solution

Abstract Two simple anion receptors, 2-[(2-hydroxy-5-nitrophenyl)methylene]hydrazone ( 1 ) and 2-[(3,5-dibromo-2-hydroxyphenyl)methylene]hydrazone ( 2 ) with –OH binding sites, were synthesized and characterized. The anion binding ability of receptors 1 and 2 with halide anions (F − , Cl − , Br − and I − ), AcO − and H 2 PO 4 - was investigated using visual (naked-eye), UV–vis titration experiments in dry DMSO together with DFT theoretical calculation. The addition of F − , AcO − and H 2 PO 4 - to the host solution resulted in a red shift of the charge-transfer absorbance band accompanied by a color change from yellow to orange in the naked-eye experiments. Receptor 1 containing a nitro group at the para position and receptor 2 containing two bromine groups at the ortho and para positions both showed strong binding ability for H 2 PO 4 - ion in the form of phenol-hydrazone. Moreover, receptor 1 , induced by anion species in the solution, converted to the form of phenol-hydrazone from keto-hydrazone.

Pyrrolic tripodal receptors for carbohydrates. Role of functional groups and binding geometry on carbohydrate recognition

The contribution from several H-bonding groups and the impact of geometric requirements on the binding ability of benzene-based tripodal receptors toward carbohydrates have been investigated by measuring the affinity of a set of structures toward octyl β-D-glucopyranoside, selected as a representative monosaccharide. The results reported in the present study demonstrate that a judicious choice of correct geometry and appropriate functional groups is critical to achieve the complementary hydrogen bonding interactions required for an effective carbohydrate recognition.

Exploring the Binding Ability of Phenanthroline-Based Polyammonium Receptors for Anions: Hints for Design of Selective Chemosensors for Nucleotides

The synthesis of receptor 2,6,10,14,18-pentaaza[20]-21,34-phenanthrolinophane (L1), containing a pentaamine chain linking the 2,9 positions of a phenanthroline unit, is reported. The protonation features of L1 and of receptor 2,6,10,14,18,22-hexaaza[23]-24,37-phenanthrolinophane (L2) have been studied by means of potentiometric, (1)H NMR, and spectrofluorimetric measurements; this study points out that the fluorescent emission of both receptors depends on the protonation state of the polyamine chain. In fact, the receptors are emissive only at neutral or acidic pH values, where all the aliphatic amine groups are protonated. Potentiometric titrations show that L2 is able to bind selectively ATP over TTP, CTP, and GTP. This selectivity is lost in the case of L1. (1)H and (31)P NMR measurements and molecular mechanics calculations show that the phosphate chains of nucleotides give strong electrostatic and hydrogen-bonding interactions with the ammonium groups of the protonated receptors, while the nucleobases interact either via pi-stacking with phenanthroline or via hydrogen bonding with the ammonium groups. Of note, MM calculations suggest that all nucleotides interact in an inclusive fashion. In fact, in all adducts the phosphate chain is enclosed within the receptor cavities. This structural feature is confirmed by the crystal structure of the [(H(6)L2)(2)(TTP)(2)(H(2)O)(2)](4+) adduct. Fluorescence emission measurements at different pH values show that L2 is also able to ratiometrically sense ATP in a narrow pH range, thanks to emission quenching due to a photoinduced electron transfer (PET) process from an amine group of the receptor to the excited phenanthroline.

Binding of Ionic Species: A General Approach To Measuring Binding Constants and Assessing Affinities

Bound together: The association of receptors with ionic species cannot be assimilated to the binding of neutral guests. When dealing with salts, both ion pairing and binding to the free and the ion-paired ionic guest determine the actual association pattern (see figure). The general issue of measuring association constants and assessing affinities for ions is addressed and validated in two cases of anion binding.A general approach to the largely underestimated issue of measuring binding constants and assessing affinities in the binding of ionic species is described. The approach is based on a rigorous, nongraphical determination of binding constants in multiequilibrium systems by nonlinear regression of chemical shift data from NMR titrations and on the use of the BC(50) descriptor for assessing affinities and ranking the binding ability of receptors on a common scale. The approach has been validated with two tripodal anion-binding receptors, namely, a ureidic (1) and a pyrrolic (2) receptor, binding to tetramethylammonium chloride in CDCl(3)/CD(3)CN (80:20). A set of five and six formation constants could be measured for 1 and 2, respectively, including, in addition to the ion pair, complexes of the free and the ion-paired anion. The BC(50) values calculated from the measured constants allowed a quantitative assessment of each receptor's binding affinity towards the chloride anion, the pyrrolic receptor showing a 15-fold larger affinity over the ureidic receptor, a figure that quantifies the improvement obtained by replacing the amido-pyrrolic for ureidic binding groups on the tripodal scaffold of the receptor. The results have shown that, in contrast to common practice, neither of the two systems could be appropriately described by a 1:1 association with the anion only, but required the ion-pairing and ion-pair binding equilibria to be taken into account because these contribute substantially to the complexation process. The BC(50) descriptor has also been shown to be a useful and general tool for the assessment of affinities of systems involving ionic species. The required extension of the BC(50) binding descriptor to include the treatment of ion-binding has been described in detail.

An amphioxus orthologue of the estrogen receptor that does not bind estradiol: Insights into estrogen receptor evolution

BackgroundThe origin of nuclear receptors (NRs) and the question whether the ancestral NR was a liganded or an unliganded transcription factor has been recently debated. To obtain insight into the evolution of the ligand binding ability of estrogen receptors (ER), we comparatively characterized the ER from the protochordate amphioxus (Branchiostoma floridae), and the ER from lamprey (Petromyzon marinus), a basal vertebrate.ResultsExtensive phylogenetic studies as well as signature analysis allowed us to confirm that the amphioxus ER (amphiER) and the lamprey ER (lampER) belong to the ER group. LampER behaves as a "classical" vertebrate ER, as it binds to specific DNA Estrogen Responsive Elements (EREs), and is activated by estradiol (E2), the classical ER natural ligand. In contrast, we found that although amphiER binds EREs, it is unable to bind E2 and to activate transcription in response to E2. Among the 7 natural and synthetic ER ligands tested as well as a large repertoire of 14 cholesterol derivatives, only Bisphenol A (an endocrine disruptor with estrogenic activity) bound to amphiER, suggesting that a ligand binding pocket exists within the receptor. Parsimony analysis considering all available ER sequences suggest that the ancestral ER was not able to bind E2 and that this ability evolved specifically in the vertebrate lineage. This result does not support a previous analysis based on ancestral sequence reconstruction that proposed the ancestral steroid receptor to bind estradiol. We show that biased taxonomic sampling can alter the calculation of ancestral sequence and that the previous result might stem from a high proportion of vertebrate ERs in the dataset used to compute the ancestral sequence.ConclusionTaken together, our results highlight the importance of comparative experimental approaches vs ancestral reconstructions for the evolutionary study of endocrine systems: comparative analysis of extant ERs suggests that the ancestral ER did not bind estradiol and that it gained the ability to be regulated by estradiol specifically in the vertebrate lineage, before lamprey split.

Anion Binding versus Intramolecular Hydrogen Bonding in Neutral Macrocyclic Amides

Although amide groups are important hydrogen-bond donors in natural and synthetic anion receptors, studies on structure-affinity relationships of amide-based macrocyclic receptors are still very limited. Therefore, we synthesized a series of macrocyclic tetraamides 5-8 derived from 1,3-benzenedicarboxylic (isophthalic) acid and aliphatic alpha,omega-diamines of different lengths. (1)H NMR titrations in DMSO solution show that the anion affinity of these receptors decreases with increasing size of the macrocycle irrespective of the anion, and this suggests a minor role of geometric complementarity. Comparison with their previously studied pyridine congeners reveals that the isophthalic acid based macrocycles are less potent, in contrast to what was found for simple model diamides. Combined theoretical and experimental structural studies were carried out to determine the reasons behind this behaviour. The results show that the unexpectedly low anion binding ability of the isophthalic acid-based receptors is due to the self-complementary nature of the isophthalic bis-amide fragments: when two such moieties are present within a sufficiently flexible macrocycle, they adopt syn-anti conformations and bind each other by two strong intramolecular hydrogen bonds that close the macrocyclic cavity. Nevertheless, anion binding is able to break these hydrogen bonds and switch a macrocycle into a convergent all-syn conformation. Despite the ill-preorganized conformation, 20-membered receptor 6 is better than either its open-chain analogue (macrocyclic effect) and/or its isomer having differently placed carbonyl groups. The crystal structures of four anion complexes of the macrocyclic receptors are reported. X-ray studies and solution NMR data confirmed the inclusive nature of the complexes and pointed to strong involvement of aromatic CH hydrogen atoms in anion binding.

Synthesis and binding ability of bile acid-based receptors for recognition of flavin analogues

Novel cholaphanes 6a, b, based on lithocholic and deoxycholic acids, were synthesised through 3a, b by a sequence of reactions involving Cs-salt methodology of macrocyclisation. Cholaphanes 6a, b and acyclic steroidal receptors 3a, b bind flavin analogues via three hydrogen bonds in CHCl 3.

Photoinduced control of cation binding ability of non-conjugated bichromophoric receptors

Several receptors based on nonconjugated bichromophores displaying phototunable cation binding properties have been designed and studied. Some of these systems undergo a reversible photocyclisation to produce a crown ether or a cryptand; the photoswitching induces significant changes on a long time scale (hours to months after illumination) of the complexing ability of the material. Other bitopic receptors display a sudden and transitory change of cation concentration only during the singlet excited state lifetime.

Enhanced Carboxylate Binding Using Urea and Amide-Based Receptors with Internal Lewis Acid Coordination: A Cooperative Polarization Effect

A structural design strategy is described that greatly improves the acetate binding ability of neutral urea and amide-based receptors. The enhanced binding is due to a cooperative polarization effect which is induced by intramolecular coordination of the urea or amide carbonyl to a Lewis acidic boronate group. A series of boronate-ureas, 3, and a related bis(boronate-amide), 23, were prepared in two steps from 2-(aminophenyl)boronic acid and their structures elucidated using X-ray crystallography and other spectrometric methods. The abilities of the receptors to associate with tetrabutylammonium acetate in dimethyl sulfoxide solution were determined by (1)H NMR titration experiments. Association constants were calculated using nonlinear curve-fitting methods. The boronate-ureas 3 strongly bind to acetate in dimethyl sulfoxide solution with association constants as high as 6 x 10(4) M(-)(1). This is more than 150 times greater than the association constants for control urea receptors that lacked an appropriate boron substituent. Thermodynamic studies indicate that the enhanced association is due to a favorable enthalpic change. Additional NMR studies eliminated the possibility of proton transfer to the acetate during complex formation. Molecular modeling indicates that the boronate-ureas exhibit improved acetate binding because the intramolecular coordination (i) induces a larger host dipole moment which strengthens the guest/host ion-dipole interaction, and (ii) increases the positive surface potential at the urea NH residues which strengthens short range Coulombic interactions with the anionic acetate. The observed association constants correlate better with calculated host dipole moments, suggesting that for the boronate-ureas described here this is the more influential factor controlling association.

High Affinity Carboxylate Binding Using Neutral Urea-Based Receptors with Internal Lewis Acid Coordination.

Uncharged receptors for anions and cations have many potential applications ranging from membrane transport carriers for ion-selective electrodes to reaction catalysts.1 As a consequence, there is a need to design and synthesize neutral receptors with high binding affinities and/ or high binding selectivities. Unlike cation receptors, there are essentially no examples of biotic, low molecular weight hosts for anions.2 Synthetic anion receptors have to be designed de novo using the principles of molecular recognition. The neutral anion-receptors reported to date have employed either Lewis acid-base,3 hydrogen bonding,4 and/or ion-dipole interactions.5 Most of the hydrogen bonding systems have used urea groups as the recognition motif. Urea-based hosts have been shown to associate with carboxylates, phosphates, and sulfonates to produce bidentate hydrogen-bonded complexes such as 1.4 In this paper we describe a structural design strategy that greatly improves the anion binding ability of neutral urea-based receptors. It is likely that this strategy can be incorporated into the designs of other amide-based molecular recognition systems. The formation of supramolecular complex 1 is driven primarily by hydrogen bonding and ion-dipole interactions.6,7 These bonding interactions can be strengthened by cooperative polarization of the urea group, which is accomplished by coordinating the urea carbonyl to a Lewis acid.8 A major design challenge is to ensure that the Lewis acid is held in the correct spatial orientation with a high effective molarity. One possible solution is the Lewis acid-urea conjugate 2. In line with our current interest in organoboron receptors,9 we designed boronate-urea 3 as a first-generation example of this receptor class. The valence bond structure for receptor 3 can be represented by two limiting forms, 3a or 3b. Prior to this study there was literature precedent to suggest that 3b would be the major resonance contributor.10

VEGF121, a Vascular Endothelial Growth Factor (VEGF) Isoform Lacking Heparin Binding Ability, Requires Cell-surface Heparan Sulfates for Efficient Binding to the VEGF Receptors of Human Melanoma Cells

Four vascular endothelial growth factor (VEGF) splice variants containing 121, 165, 189, and 206 amino acids are produced from a single human gene as a result of alternative splicing. VEGF121 is not a heparin-binding protein, while the other VEGF species possess heparin binding ability. YU-ZAZ6 human melanoma cells expressed the mRNA encoding the VEGF receptor flt-1, but not the mRNA encoding the VEGF receptor KDR/flk-1. Both VEGF121 and VEGF165 bound to the VEGF receptors of these cells. Unexpectedly, heparin inhibited the binding of VEGF121 as well as the binding of VEGF165 to the VEGF receptors of the melanoma cells. Digestion of the cells with heparinase also inhibited the binding of both VEGF variants. The VEGF165 binding ability of heparinase-digested cells could be partially restored by the addition of exogenous heparin to the binding reaction. In contrast, the addition of heparin to heparinase-digested cells did not restore VEGF121 binding. These results suggest that cell-surface heparan sulfates may regulate the binding ability of the VEGF receptors of the melanoma cells. They also indicate that heparin is not able to fully substitute for cell surface-associated heparan sulfates since VEGF121 binding to the VEGF receptors of heparinase-treated cells is not restored by heparin. These data suggest that changes in the composition of cell-surface heparin-like molecules may differentially affect the interaction of various VEGF isoforms with VEGF receptors.

CHOLECYSTOKININ RECEPTOR BINIDIING AFTER LONG-TERM ETHANOL TREATMENT IN RATS

Brain cholecystokinin (CCK) receptors have been implicated in anxiety disorders and suicidal behaviour. We have examined the radioligand binding ability of CCK and benzodiazepine receptors in rat brain after long-term intermittent voluntary vs voluntary and forced low-dose ethanol exposure. During 58 weeks, one group of rats had a choice between ethanol and water as the drinking fluid for 24 hr each week. Another group of rats had the same weekly choice between ethanol and water, but at the end of each 24 hr choice period, ethanol (2.0 g/kg) was injected. During the second period of ethanol treatment, lasting for 32 weeks, both ethanol-treated groups had continuous free access to ethanol and water. These two treatments have previously been shown to induce partially different neurochemical alterations. In the present investigation, benzodiazepine receptor binding in the frontal cortex, hippocampus and striatum was similar in both ethanol treatment groups compared to controls. CCK receptor binding in the hippocampus and striatum did not differ between the three groups; however, in the frontal cortex, there was an increase in the apparent number of CCK binding sites in the group of rats submitted to voluntary plus forced ethanol exposure as compared to the control group or the voluntary intake group. These results suggest that long-term ethanol treatment may lead to alterations in brain CCK-ergic neurotransmission, but that the changes are specific to the treatment schedule.

Novel regulation of muscarinic receptors and their coupling with G proteins in smooth muscle: transient resensitization during desensitizing process.

1. Muscarinic stimulation of the smooth muscle of guinea-pig taenia caeci was produced with 10(-4) M carbachol for 15 s, 30 s, 1 min, 2 min and 30 min, and the time course of developing desensitization was studied by measuring the muscle contractility and the binding characteristics of muscarinic receptors. 2. The contractile response to carbachol was analyzed using dose-response curves. The response to 10(-7) M carbachol was reduced by treatment for 15 s with 10(-4) M carbachol (fast desensitization), but recovered partially after 30 s treatment and completely after 1 min treatment (resensitization). Contractility was reduced again after 2 min and 30 min treatment (re-desensitization). 3. The affinity of carbachol for muscarinic receptors was changed by the carbachol treatment in a manner similar to the contractility. Thus, the affinity was reduced at 15 s, restored slightly at 30 s and completely at 1 and 2 min, and was reduced again at 30 min. 4. 5'-Guanylylimidodiphosphate (GppNHp), a non-hydrolysable analogue of guanosine triphosphate (GTP) reduced the affinity of muscarinic receptors for carbachol via guanine nucleotide-binding regulatory proteins (G proteins). A similar effect was observed in tissues desensitized by 15 s carbachol treatment. This effect disappeared after 30 s, recovered completely after 1 and 2 min, and disappeared again after 30 min carbachol treatment. 5. Neither the dissociation constant (Kd value) nor the maximal binding (Bmax) of [3H]-quinuclidinyl benzilate ([3H]-QNB) to muscarinic receptors were changed by the carbachol treatment. 6. These results indicate that the whole process of desensitization, resensitization and re-desensitization are related to changes in the binding ability of muscarinic receptors, in their coupling with G proteins and in the post-receptor steps of the signal transduction. We emphasize that the desensitizing process involves an early transient phase of resensitization that could be caused by restoration of both the affinity of carbachol for muscarinic receptors and their coupling with G proteins. This novel resensitization mechanism may have some physiological significance for cellular homoeostasis by modulating cellular responsiveness transiently or even in an oscillatory manner during the process of desensitization.