Agonist-receptor Binding Research Articles

Regional Variation of Bitter Taste and Aftertaste in Humans.

Despite widespread and persistent myths of a tongue map, all 5 prototypical taste qualities are sensed over the entire tongue. However, modern psychophysical data also suggest there may be more nuanced differences in suprathreshold intensity across oral loci, especially for bitterness. Here, we test whether bitter stimuli matched for whole-mouth intensity differ in perceived intensity across regions of the oral cavity in 2 experiments. Experiment 1 consisted of a whole-mouth sip and spit approach and Experiment 2 consisted of a spatial taste test using cotton swabs. In Experiment 1, participants (n = 63) rated overall intensity of 3 bitter solutions at 5 different loci (front, middle, back of tongue; roof of mouth; and lip). Temporal effects were explored using in-mouth and aftertaste ratings. In Experiment 2, participants (n = 48) rated the intensity of quinine and Tetralone solutions after solutions were painted on fungiform, circumvallate, and foliate papillae with a swab. After the spatial taste test, participants completed a questionnaire on self-reported beer intake. Analysis of variance results of both experiments show a significant locus by stimulus interaction, suggesting different bitterants were perceived differently across the various loci. This result was apparently driven by low-intensity ratings for Tetralone on the anterior tongue. Aftertaste ratings in Experiment 1 also revealed significant temporal effects: ratings on the anterior tongue decreased for all bitterants and ratings for quinine decreased at all loci. Reasons for these effects are not known but may suggest differential expression of bitter taste receptors or differences in bitter agonist-receptor binding affinity across tongue regions.

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein-coupled receptor (GPCR) activation. Agonist-receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape.

Molecular mechanisms of endothelial hyperpermeability in sepsis

There is endothelial hyperpermeability in sepsis,trauma,ischaemia-reperfusion injury,acute respiratory distress syndrome and thrombosis,while an important mechanism underlying this process is increased paracellular leakage of plasma fluid and protein because of cytoskeleton contration under the role of histamine, thrombin,vascular endothelial growth factor and activated neutrophil. Structural changes initiate with agonist-receptor binding,followed by activation of intracellular signalling molecules,then phosphorylate alter the hypermeability of cell-cell adhesion. Targeting key signalling molecules that mediate endothelial-junction-cytoskeleton dissociation demonstrates a therapeutic potential to improve vascular barrier function during inflammatory injury. Key words: Endothelial cell; Microvascular permeability; Signal transduction; Sepsis

Multiparametric Homogeneous Method for Identification of Ligand Binding to G Protein-Coupled Receptors: Receptor–Ligand Binding and β-Arrestin Assay

Two homogeneous assay systems have been combined to provide a new cell-based functional assay. The assay can be used to identify ligand binding to β(2)-adrenergic receptors, but also the downstream response can be determined in the same assay. Both the quenching resonance energy transfer (QRET) and the DiscoveRx PathHunter assay formats allow the use of intact cells. The homogeneous QRET technique is a single-label approach based on nonspecific quenching of the time-resolved luminescence, enabling agonist and antagonist receptor binding measurements. The commercial PathHunter assay is in turn based on enzyme fragment complementation, which can be detected on the basis of chemiluminescence signal. In the PathHunter technology the enzyme complementation is recorded immediately downstream of agonist-induced receptor activation. The new multiparametric detection technology combines these two assay methods enabling the identification of agonist, and antagonist binding to the receptor, and the agonist-induced response. Using the QRET and the PathHunter methods a panel of β(2)-adrenergic receptor ligands (epinephrine, terbutaline, metaproterenol, salmeterol, propranolol, alprenolol, bisoprolol, ICI 118,551, and bucindolol) was tested to prove the assay performance. The signal-to-background ratio for tested ligands ranged from 5 to 11 and from 6 to 18 with QRET and PathHunter, respectively. Combined homogeneous assay technique can provide an informative method for screening purposes and an efficient way to monitor receptor-ligand interaction, thus separating agonist from antagonist.

Two Neuronal Nicotinic Acetylcholine Receptors, α4β4 and α7, Show Differential Agonist Binding Modes

Nicotinic acetylcholine receptors (nAChRs) are pentameric, neurotransmitter-gated ion channels responsible for rapid excitatory neurotransmission in the central and peripheral nervous systems, resulting in skeletal muscle tone and various cognitive effects in the brain. These complex proteins are activated by the endogenous neurotransmitter ACh as well as by nicotine and structurally related agonists. Activation and modulation of nAChRs has been implicated in the pathology of multiple neurological disorders, and as such, these proteins are established therapeutic targets. Here we use unnatural amino acid mutagenesis to examine the ligand binding mechanisms of two homologous neuronal nAChRs: the α4β4 and α7 receptors. Despite sequence identity among the residues that form the core of the agonist-binding site, we find that the α4β4 and α7 nAChRs employ different agonist-receptor binding interactions in this region. The α4β4 receptor utilizes a strong cation-π interaction to a conserved tryptophan (TrpB) of the receptor for both ACh and nicotine, and nicotine participates in a strong hydrogen bond with a backbone carbonyl contributed by TrpB. Interestingly, we find that the α7 receptor also employs a cation-π interaction for ligand recognition, but the site has moved to a different aromatic amino acid of the agonist-binding site depending on the agonist. ACh participates in a cation-π interaction with TyrA, whereas epibatidine participates in a cation-π interaction with TyrC2.

Motoneuron Excitability and Muscle Spasms Are Regulated by 5-HT2Band 5-HT2CReceptor Activity

Immediately after spinal cord injury (SCI), a devastating paralysis results from the loss of brain stem and cortical innervation of spinal neurons that control movement, including a loss of serotonergic (5-HT) innervation of motoneurons. Over time, motoneurons recover from denervation and function autonomously, exhibiting large persistent calcium currents (Ca PICs) that both help with functional recovery and contribute to uncontrolled muscle spasms. Here we systematically evaluated which 5-HT receptor subtypes influence PICs and spasms after injury. Spasms were quantified by recording the long-lasting reflexes (LLRs) on ventral roots in response to dorsal root stimulation, in the chronic spinal rat, in vitro. Ca PICs were quantified by intracellular recording in synaptically isolated motoneurons. Application of agonists selective to 5-HT(2B) and 5-HT(2C) receptors (including BW723C86) significantly increased the LLRs and associated Ca PICs, whereas application of agonists to 5-HT(1), 5-HT(2A), 5-HT(3), or 5-HT(4/5/6/7) receptors (e.g., 8-OH-DPAT) did not. The 5-HT(2) receptor agonist-induced increases in LLRs were dose dependent, with doses for 50% effects (EC(50)) highly correlated with published doses for agonist receptor binding (K(i)) at 5-HT(2B) and 5-HT(2C) receptors. Application of selective antagonists to 5-HT(2B) (e.g., RS127445) and 5-HT(2C) (SB242084) receptors inhibited the agonist-induced increase in LLR. However, antagonists that are known to specifically be neutral antagonists at 5-HT(2B/C) receptors (e.g., RS127445) had no effect when given by themselves, indicating that these receptors were not activated by residual 5-HT in the spinal cord. In contrast, inverse agonists (such as SB206553) that block constitutive activity at 5-HT(2B) or 5-HT(2C) receptors markedly reduced the LLRs, indicating the presence of constitutive activity in these receptors. 5-HT(2B) or 5-HT(2C) receptors were confirmed to be on motoneurons by immunolabeling. In summary, 5-HT(2B) and 5-HT(2C) receptors on motoneurons become constitutively active after injury and ultimately contribute to recovery of motoneuron function and emergence of spasms.

A retro‐inverso α‐melanocyte stimulating hormone analog with MC1R‐binding selectivity

α-melanocyte stimulating hormone (α-MSH) is a tridecapeptide fragment of pro-opiomelanocortin (POMC) with broad effects on appetite, skin pigmentation, hormonal regulation, and potential roles in both inflammation and autoimmunity. The use of this peptide as an anti-inflammatory agent is limited by its low selectivity between the melanocortin receptors, susceptibility to proteolytic degradation, and rapid clearance from circulation. A retro-inverso (RI) sequence of α-MSH was characterized for receptor activity and resistance to protease. This peptide demonstrated surprisingly high selectivity for binding the melanocortin receptor 1 (MC1R). However, RI-α-MSH exhibited a diminished binding affinity for MC1R compared to α-MSH. Mapping of the residues critical for agonist activity, receptor binding, and selectivity by alanine scanning, identified the same critical core tetrapeptide required for the native peptide. Modest improvements in affinity were obtained by conservative changes employing non-natural amino acids and substitution of the C-terminal sequence with a portion of a MC1R ligand peptide previously identified by phage display. Recombination of these elements yielded a peptide with an identical K(i) as α-MSH at MC1R and a lower EC(50) in Mel-624 melanoma cells. A number of other structural modifications of the RI peptide were found to differ in effect from those reported for the L-form α-MSH, suggesting a significantly altered interaction with the MC1R.

The Role of Fluorine Atoms in a Fluorinated Prostaglandin Agonist

The role of covalently bound fluorine atoms in fluorinated prostaglandin agonist–receptor binding was studied using molecular dynamics and the fragment molecular orbital method. In agreement with experimental data, the simulated geometries of the fluorine and surrounding amino acid residues suggest that CF⋅⋅⋅H and F⋅⋅⋅CO interactions positively contribute to the receptor binding affinity of these ligands. The pair interaction energy profile of the fluorinated agonist confirmed these interactions. A similar profile obtained for the parent agonist suggested that the ω-chains play equivalent roles. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Modeling the dynamics of airway constriction: effects of agonist transport and binding

Recent advances have revealed that during exogenous airway challenge, airway diameters cannot be adequately predicted by their initial diameters. Furthermore, airway diameters can also vary greatly in time on scales shorter than a breath. To better understand these phenomena, we developed a multiscale model that allowed us to simulate aerosol challenge in the airways during ventilation. The model incorporates agonist-receptor binding kinetics to govern the temporal response of airway smooth muscle contraction on individual airway segments, which, together with airway wall mechanics, determines local airway caliber. Global agonist transport and deposition are coupled with pressure-driven flow, linking local airway constrictions with global flow dynamics. During the course of challenge, airway constriction alters the flow pattern, redistributing the agonist to less constricted regions. This results in a negative feedback that may be a protective property of the normal lung. As a consequence, repetitive challenge can cause spatial constriction patterns to evolve in time, resulting in a loss of predictability of airway diameters. Additionally, the model offers new insights into several phenomena including the intra- and interbreath dynamics of airway constriction throughout the tree structure.

Molecular mechanisms of endothelial hyperpermeability: implications in inflammation

Endothelial hyperpermeability is a significant problem in vascular inflammation associated with trauma, ischaemia-reperfusion injury, sepsis, adult respiratory distress syndrome, diabetes, thrombosis and cancer. An important mechanism underlying this process is increased paracellular leakage of plasma fluid and protein. Inflammatory stimuli such as histamine, thrombin, vascular endothelial growth factor and activated neutrophils can cause dissociation of cell-cell junctions between endothelial cells as well as cytoskeleton contraction, leading to a widened intercellular space that facilitates transendothelial flux. Such structural changes initiate with agonist-receptor binding, followed by activation of intracellular signalling molecules including calcium, protein kinase C, tyrosine kinases, myosin light chain kinase, and small Rho-GTPases; these kinases and GTPases then phosphorylate or alter the conformation of different subcellular components that control cell-cell adhesion, resulting in paracellular hypermeability. Targeting key signalling molecules that mediate endothelial-junction-cytoskeleton dissociation demonstrates a therapeutic potential to improve vascular barrier function during inflammatory injury.

Three dimensional modeling of N-terminal region of galanin and its interaction with the galanin receptor.

The neuropeptide galanin comes under the powerful and versatile modulators of classical neurotransmitters and is present in brain tissues, which are intimately involved in epileptogenesis. It acts as appealing targets for studying basic mechanisms of seizure initiation and arrest, and for the development of novel approaches for various neurodegenerative diseases. Galanin is widely distributed in the mammalian brain which controls various processes such as sensation of pain, learning, feeding, sexual behaviour, carcinogenesis, pathophysiology of neuroendocrine tumors and others. The function of galanin can be exploited through its interaction with three G-protein coupled receptors subtypes such as GalR1, GalR2 and GalR3. The N-terminal region of galanin comprises about highly conserved 15 amino acid residues, which act as the crucial region for agonist-receptor binding. We have constructed a theoretical structural model for the N-terminal region of galanin from Homo sapiens by homology modeling. The stereochemistry of the model was checked using PROCHECK. The functionally conserved regions were identified by surface mapping of phylogenetic information generated by online web algorithm ConSurf. The docking studies on the pharmacologically important galanin receptors with the theoretical model of N-terminal region of galanin predicted crucial residues for binding which would be useful in the development of novel leads for neurodegenerative disorders.

Effect of Dopamine on Vascular Reactivity in Near-Term Lamb Carotids: Role of the Endothelium

Many neonates are diagnosed with hypotension in the first 24 hr of life. Those with severe hypotension are often given high doses of dopamine at 10 to 20 microg/kg/min. This study examined the hypothesis that dopamine, a vasoactive drug commonly used in the neonatal intensive care unit, alters vascular reactivity. Vascular reactivity was measured by comparing 5HT dose-response characteristics in untreated near-term lamb common carotid arteries and arteries treated with 15 microg/kg/min of dopamine. The authors found that dopamine pretreatment for 60 min significantly potentiated 5HT-induced contractile tone by approximately 100% ( p < .05). This observed increase in tone was accompanied by a significant decrease in the affinity of 5HT to its receptor ( p < .05), suggesting an activation of a separate contractile pathway, or a mechanism downstream from agonist-receptor binding. Interestingly, an increase in contractility was observed only in endothelium-intact arteries. In arteries with denuded endothelium, dopamine pretreatment resulted in a small but significant decrease in tone compared to control arteries ( p < .05), suggesting a vasodilatory mechanism unmasked by endothelium removal. Although multiple mechanisms can increase vascular resistance, these data described the in vitro effects of high doses of dopamine on vascular tone as well as the role of the endothelium in dopaminemediated vasoconstriction.

Binding site prediction of galanin peptide using evolutionary trace method

Galanin is a neuropeptide with aminoacid length ranging from 29 to 31 is widely distributed in central and peripheral nervous system. Galanin controls various psychological processes such as sensation of pain, learning, feeding, and sexual behaviour. The N-terminal region of this neuropeptide has highly conserved 15 amino acids, which is triggered by galanin receptors. We performed evolutionary trace analysis for galanin sequences to gather information about functional residues. The consensus pattern given by the evolutionary trace (ET) analysis is supported by CLUSTALW and WEBLOGO results. Our observations strongly suggest the presence of functional residues in the N-terminal region of galanin for agonist-receptor binding.

Mechanisms of direct inhibitory action of propofol on uterine smooth muscle contraction in pregnant rats.

Although propofol directly inhibits uterine smooth muscle contraction, the mechanisms of this effect are still unknown. The current study aimed to clarify the mechanisms of the inhibitory effect of propofol on oxytocin-induced uterine smooth muscle contraction by measuring (1) the concentration of intracellular free Ca(2+) ([Ca(2+)](i)) simultaneously with muscle tension, (2) the amount of intracellular inositol 1,4,5-triphosphate ([IP(3)](i)), and (3) voltage-dependent Ca(2+) channel (VDCC) activity. Uterine smooth muscle tissues were obtained from pregnant rats (in late gestation). [Ca(2+)](i) with isometric tension was monitored by the 500-nm light emission ratio of preloaded Ca(2+) indicator fura-2. [IP(3)](i) and VDCC activity were measured by radioimmunoassay and patch clamp techniques, respectively. The uterine smooth muscle was stimulated by 20 nm oxytocin and exposed to propofol (10(-7) approximately 10(-4) m). Propofol had significant inhibitory effects on oxytocin-induced uterine smooth muscle contraction and increased [Ca(2+)](i) in pregnant rats in a dose-dependent manner, without affecting the agonist-receptor binding affinity. Propofol inhibited the increase in [IP(3)](i) induced by oxytocin. Propofol also inhibited VDCC activity in both activated and inactivated states. The solvent Intralipid had no effects on these parameters. Propofol inhibits oxytocin-induced uterine smooth muscle contraction, at least in part, by decreasing [Ca(2+)](i) without affecting agonist-receptor binding; the inhibitory effect of propofol on [Ca(2+)](i) might be mediated both by a decrease in [IP(3)](i) and by inhibition of VDCC activity.

The metabolic evidence of synergistic effect between ohmefentanyl and [D-Pen 2, D-Pen 5] enkephalin on differentiated SH-SY5Y cells in humans

Interactions between selective opioid agonists acting at μ- and δ-opioid receptors were evaluated by co-administering a low-effective dose of the selective μ-opioid receptor agonist ohmefentanyl (OMF) with sequentially increasing doses of the selective δ-opioid receptor agonist [D-Pen 2, D-Pen 5] enkephalin (DPDPE). Microphysiometer was used to measure the extracellular acidification rate (ECAR) of living cells in real-time, which reflected the functional activity after agonist-receptor binding. The synergy (i.e. a more than additive effect) was observed with combinations of these two opioid agonists on differentiated SH-SY5Y cells functionally expressing both μ- and δ-opioid receptors. The demonstration of the synergy suggests that the agonists of the subtypes of opioid receptors can interact at cellular level.

Effects of halothane and isoflurane on beta-adrenoceptor-mediated responses in the vascular smooth muscle of rat aorta.

Although previous studies have proposed that anesthetics may influence signal transduction systems, their effects on the beta-adrenoceptor-mediated system have not been fully characterized in vascular smooth muscle. The aim of this study was to determine how halothane and isoflurane affect beta-adrenoceptor-mediated vasodilation in rat aorta and what mechanisms were involved. Isometric tension and the intracellular calcium ion concentration ([Ca2+]i) were measured concomitantly in rat aortic strips from which the endothelium was removed. Strips precontracted with norepinephrine were dilated with the beta-adrenoceptor agonist, isoproterenol; the adenylyl cyclase activator, forskolin; or the membrane-permeable dibutyryl cyclic adenosine monophosphate (cAMP) with or without halothane or isoflurane. The effects of the anesthetics on each vasodilator were compared with the control responses. Beta-adrenoceptor binding characteristics and affinity for agonists were determined with [125I]-iodocyanopindolol with and without halothane or isoflurane. Furthermore, concentrations of cAMP induced by either isoproterenol or forskolin were measured with or without the anesthetics using an enzyme immunoassay procedure. Halothane and isoflurane attenuated vasodilation and [Ca2+]i decreases induced by isoproterenol, whereas both anesthetics only slightly affected vasodilation and [Ca2+]i decreases induced by forskolin and dibutyryl cAMP. Halothane and isoflurane had no effect on beta-adrenoceptor binding characteristics and affinity for agonists. Three percent halothane or 4% isoflurane significantly reduced cAMP levels induced by isoproterenol but not by forskolin. Halothane and isoflurane, at clinically relevant concentrations, can interfere with beta-adrenoceptor-mediated responses in the rat aorta at the steps after the agonist-receptor binding but before the adenylyl cyclase activation.

Regulation of Agonist-Receptor Binding by G Proteins and Divalent Cations in Spermatozoa Solubilized A1 Adenosine Receptors

Solubilized A1 adenosine receptor (A1AR) was used to investigate the effect of several cations on agonist-binding characteristics and GTP hydrolysis. It was shown by Western blot with Gβ-M14 that this preparation contains both G proteins and receptor. The role of the receptor molecule is to facilitate the activation of G proteins as α-GTP complex, and GTP hydrolysis has important consequences for the basic deactivation mechanism. Divalent cations, such as Mn2+, Ca2+, and Mg2+, potentiated the agonist-specific binding: Mn2+had the highest apparent affinity with half-maximal effect at 50 μM. Binding assays, performed in the presence of 100 μM Mn2+, showed an increase in the apparent affinity of the binding sites, whereas, in the presence of 1 mM Mg2+, significant alteration of the apparent affinity, but not of the number of sites, was detected. Concentrations of 1 mM Mg2+and 100 μM Mn2+enhanced GTPase activity, whereas 5 mM Ca2+resulted in the increase ofVmaxvalues without significant alterations ofKm. In the presence of A1-specific agonists, Mn2+and Mg2+caused a decrease ofVmaxvalues and an increase of GTP affinity. Other cations, such as Co2+, Cd2+, Cu2+, and Zn2+, inhibited the binding capacity but caused almost no changes in GTP hydrolysis kinetics.

Prostacyclin receptor desensitization is a reversible phenomenon in human platelets.

Long-term exposure of platelets to endogenous or exogenous prostacyclin or its analogues might result in desensitization of the platelet prostacyclin receptor in vitro and in vivo accompanied by a loss in receptor density on the platelet surface and a reduced sensitivity toward the inhibitory effects of prostacyclins. However, the reversibility of this process in platelets has not yet been investigated. Human platelets desensitized by the chemically stable prostacyclin analogue iloprost showed a significant reduction in [3H]-iloprost binding sites that was reversed by saponin permeabilization. This indicates functionally active internalized prostacyclin receptors. To assess whether the internalized prostacyclin receptors recycle to the cell surface after withdrawal of the agonist, iloprost sensitivity and prostacyclin receptor binding properties of iloprost (30 nmol/L, 2 hours) desensitized platelets incubated in iloprost-free autologous plasma were investigated. While desensitized platelets showed a significant increase in IC50 for iloprost inhibition of thrombin-induced platelet aggregation, serotonin release, and p-selectin expression and a reduced iloprost-stimulated cAMP formation, platelet iloprost sensitivity was restored 3 hours after iloprost withdrawal. In addition, the significant reduction in Bmax and the increase in K(D) of prostacyclin receptors in desensitized platelets as revealed by [3H]-iloprost binding studies also returned to the initial values. These results indicate that prostacyclin receptors internalized during short-term desensitization are not degraded but can be recycled rapidly to the platelet surface in a functionally active form after withdrawal of the agonist.

Structural definition of the C5a C terminus by two-dimensional nuclear magnetic resonance spectroscopy.

The serum glycoprotein C5a, which is derived from the proteolytic cleavage of complement protein C5, has been implicated in the pathogenesis of a number of inflammatory and allergic conditions. Because C5a induces an inflammatory response upon binding to a specific receptor, structural and mutagenesis studies were carried out to gain a better understanding of this binding interaction. These studies led to the first structural definition of the C terminus of recombinant human (rh)-C5a, determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Our results show that the C terminus adopts an alpha-helical conformation spanning residues 69 to 74, while the core domain exists as an antiparallel alpha-helical bundle. This C-terminal helix is connected to the core by a short loop that orients Arg 74 adjacent to Arg 62. Point mutation analysis had already revealed that residues 62 and 74 significantly contribute to agonist activity and receptor binding. Correlation of the C5a tertiary structure with mutational analyses clarifies the significance of the functional and binding properties of Arg 62 and suggests that both Arg 62 and Arg 74 interact at the same binding site on the receptor.

Direct inhibitory mechanisms of halothane on human platelet aggregation.

Although halothane directly inhibits platelet aggregation, the mechanisms of this effect are still unknown. The current study aimed to clarify the inhibitory mechanisms of halothane on thrombin-induced human platelet aggregation by measuring (1) platelet-surface glycoprotein Ib expression, (2) the concentration of intracellular free Ca2+ ([Ca2+]i) measured simultaneously with aggregation, (3) the concentration of intracellular inositol 1,4,5-triphosphate, and (4) the concentration of intracellular cyclic 3',5'-adenosine monophosphate ([cAMP]i). Washed platelet suspensions, obtained from healthy volunteers, were preincubated with halothane (0-2 mM) for 2 min and then exposed to 0.02 units/ml thrombin for 3 min. The glycoprotein Ib bound to fluorescein-labeled antibody was measured by fluorescence flow cytometry. [Ca2+]i was measured, simultaneously with aggregation, in Fura-2 (Ca2+ indicator)-loaded platelets by use of a fluorometer. Inositol 1,4,5-triphosphate and [cAMP]i were measured by radioimmunoassay. Halothane had no effect on glycoprotein Ib expression with or without thrombin. Halothane decreased the thrombin stimulated [Ca2+]i transient and inhibited platelet aggregation in a dose-dependent manner, both in the presence and in the absence of external Ca2+. Isoflurane had no apparent effect on either platelet aggregation or [Ca2+]i in the absence of external Ca2+. Halothane inhibited the increase in inositol 1,4,5-triphosphate induced by thrombin. Halothane moderately but significantly increased [cAMP]i, but the adenylate cyclase activator forskolin (which has the same inhibitory ability on aggregation as halothane) increased [cAMP]i to a much greater extent than did halothane. Halothane inhibits thrombin-induced human platelet aggregation by decreasing [Ca2+]i without inhibiting agonist-receptor binding; the inhibitory effect of halothane on [Ca2+]i might be mediated by a decrease in inositol 1,4,5 triphosphate and in part by an increase in [cAMP]i.