Bradykinin receptors in isolated rat duodenum

Pharmacological properties of the bradykinin receptors in the isolated rat duodenum were investigated by examining the relaxant and contractile responses to bradykinin and [des-Arg9]-bradykinin, an agonist of B1 receptors. A specific desensitization and de novo formation for B1 receptors were observed. Changes in medium pH caused a decrease in the responses to bradykinin and [des-Arg9]-bradykinin of rat duodenum. Urea incubation in test tube inhibited the responses to bradykinin and [des-Arg9]-bradykinin of rat duodenum, while urea in bathing medium was ineffective. These findings strongly suggested that (a) ionic bonds are important in the interaction between bradykinin and its receptors, and (b) B2 receptors in rat duodenum are different from those in guinea pig ileum.     

Rotavirus nonstructural protein NSP2 self-assembles into octamers that undergo ligand-induced conformational changes

The nonstructural protein NSP2 is a component of the rotavirus replication machinery and binds single-stranded RNA cooperatively, with high affinity, and independent of sequence. Recently, NSP2 has been shown to form multimers and to possess an NTPase activity, but its precise function remains unclear. In the present study, we have characterized the solution structure of recombinant NSP2 by velocity and equilibrium ultracentrifugation, dynamic light scattering, and circular dichroism spectroscopy. We found that NSP2 exists as an octamer, which is functional in the binding of RNA and ADP. In the presence of magnesium, a partial dissociation of the octamer into smaller oligomers was observed. This was reversed by binding of ADP and RNA. We observed an increased sedimentation rate in the presence of ADP and a nonhydrolyzable ATP analogue, which suggests a change toward a significantly more compact octameric conformation. The secondary structure of NSP2 showed a high fraction of beta-sheet, with small changes induced by magnesium that were reversed in the presence of RNA. That NSP2 can exist in different conformations lends support to the previously proposed hypothesis (Taraporewala, Z., Chen, D., and Patton, J. T. (1999) J. Virol. 73, 9934-9943) of its function as a molecular motor involved in the packaging of viral mRNA.     

Single cell observation of ligand-induced desensitization of B-cell membrane immunoglobulin-mediated calcium signals.

Using a digital imaging fluorescence microscope we have observed the membrane immunoglobulin (mIg)-induced desensitization of calcium signals in individual BAL17 B lymphoma cells which express two kinds of antigen receptors, mIgM and mIgD. The mIgD-mediated desensitization was partly abrogated by pretreating the cells with phorbol 12-myristate 13-acetate (PMA) for 24 h, however, the mIgM-mediated one was not affected by the pretreatment. This supports the idea that at least two mechanisms are operative for mIg-induced desensitization in B cells.     

Internalization and desensitization of adenosine receptors

Until now, more than 800 distinct G protein-coupled receptors (GPCRs) have been identified in the human genome. The four subtypes of the adenosine receptor (A(1), A(2A), A(2B) and A(3) receptor) belong to this large family of GPCRs that represent the most widely targeted pharmacological protein class. Since adenosine receptors are widespread throughout the body and involved in a variety of physiological processes and diseases, there is great interest in understanding how the different subtypes are regulated, as a basis for designing therapeutic drugs that either avoid or make use of this regulation. The major GPCR regulatory pathway involves phosphorylation of activated receptors by G protein-coupled receptor kinases (GRKs), a process that is followed by binding of arrestin proteins. This prevents receptors from activating downstream heterotrimeric G protein pathways, but at the same time allows activation of arrestin-dependent signalling pathways. Upon agonist treatment, adenosine receptor subtypes are differently regulated. For instance, the A(1)Rs are not (readily) phosphorylated and internalize slowly, showing a typical half-life of several hours, whereas the A(2A)R and A(2B)R undergo much faster downregulation, usually shorter than 1 h. The A(3)R is subject to even faster downregulation, often a matter of minutes. The fast desensitization of the A(3)R after agonist exposure may be therapeutically equivalent to antagonist occupancy of the receptor. This review describes the process of desensitization and internalization of the different adenosine subtypes in cell systems, tissues and in vivo studies. In addition, molecular mechanisms involved in adenosine receptor desensitization are discussed.     

Intracellular calcium and desensitization of acetylcholine receptors

Acetylcholine (ACh) was applied iontophoretically to voltage-clamped endplates in frog muscle. The current induced by prolonged application of ACh decreases progressively as the membrane becomes desensitized. Desensitization was sharply localized, and at a distance of 15 micrometer or less the ACh sensitivity of the membrane remained normal. Desensitization still occurred in muscles exposed to Ca2+-free media for several hours. In these conditions the rate of desensitization was not greatly affected by altering the membrane potential. In normal Ringer (1.8 mM Ca2+) desensitization was more pronounced and ACh application was frequently accompanied by localized contraction of the muscle fibre. Both the desensitization and the contraction were reduced after intracellular injection of EGTA, probably because this opposes the rise in internal Ca2+ normally caused by ACh action.     

Using ligand-induced conformational change to screen for compounds targeting G-protein-coupled receptors

The authors describe a novel drug strategy designed as a primary screen to discover either antagonist or agonist compounds targeting G-protein-coupled receptors (GPCRs). The incorporation of a nuclear localization sequence (NLS, a 5 amino acid substitution), in a location in helix 8 of the GPCR structure, resulted in ligand-independent receptor translocation from the cell surface to the nucleus. Blockade of the GPCR-NLS translocation from the cell surface was achieved by either antagonist or agonist treatments, each achieving their result in a sensitive concentration-dependent manner. GPCR-NLS translocation and blockade occurred regardless of the identity of the G-protein-coupling, and thus this assay is also ideally suited for identification of compounds targeting orphan GPCRs. The GPCR-NLS trafficking was visualized by fusion to fluorescent detectable proteins. Quantification of this effect was measured by determining the density of cell surface receptors, using enzyme fragment complementation in a manner suitable for high-throughput screening. Thus, the authors have developed a cellular assay for GPCRs suitable for compound screening without requiring prior identification of an agonist or knowledge of G-protein-coupling.     

Carbachol induces desensitization of cardiac beta-adrenergic receptors through muscarinic M1 receptors

Incubation of enzymatically dissociated cardiac myocytes with carbachol leads to a time- and concentration-dependent loss of beta-receptors assayable with [3H]CGP-12177. This loss is due to a redistribution of beta-receptors from the plasma membrane to a cytosol-derived vesicular fraction, consistent with an internalization process. The carbachol effects are not influenced by gallamine or oxotremorine which interact with the high-affinity (M2) muscarinic receptors. These results suggest that carbachol-induced desensitization is secondary to activation of protein kinase C by diacylglycerols generated through M1 receptor-linked phosphoinositide hydrolysis.     

AMPA receptors undergo channel arrest in the anoxic turtle cortex

Without oxygen, all mammals suffer neuronal injury and excitotoxic cell death mediated by overactivation of the glutamatergic N-methyl-D-aspartate receptor (NMDAR). The western painted turtle can survive anoxia for months, and downregulation of NMDAR activity is thought to be neuroprotective during anoxia. NMDAR activity is related to the activity of another glutamate receptor, the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR). AMPAR blockade is neuroprotective against anoxic insult in mammals, but the role of AMPARs in the turtle's anoxia tolerance has not been investigated. To determine whether AMPAR activity changes during hypoxia or anoxia in the turtle cortex, whole cell AMPAR currents, AMPAR-mediated excitatory postsynaptic potentials (EPSPs), and excitatory postsynaptic currents (EPSCs) were measured. The effect of AMPAR blockade on normoxic and anoxic NMDAR currents was also examined. During 60 min of normoxia, evoked peak AMPAR currents and the frequencies and amplitudes of EPSPs and EPSCs did not change. During anoxic perfusion, evoked AMPAR peak currents decreased 59.2 +/- 5.5 and 60.2 +/- 3.5% at 20 and 40 min, respectively. EPSP frequency (EPSP(f)) and amplitude decreased 28.7 +/- 6.4% and 13.2 +/- 1.7%, respectively, and EPSC(f) and amplitude decreased 50.7 +/- 5.1% and 51.3 +/- 4.7%, respectively. In contrast, hypoxic (Po(2) = 5%) AMPAR peak currents were potentiated 56.6 +/- 20.5 and 54.6 +/- 15.8% at 20 and 40 min, respectively. All changes were reversed by reoxygenation. AMPAR currents and EPSPs were abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In neurons pretreated with CNQX, anoxic NMDAR currents were reversibly depressed by 49.8 +/- 7.9%. These data suggest that AMPARs may undergo channel arrest in the anoxic turtle cortex.     

Untangling ligand induced activation and desensitization of G-protein-coupled receptors

Long-term treatment with a drug to a G-protein-coupled receptor (GPCR) often leads to receptor-mediated desensitization, limiting the therapeutic lifetime of the drug. To better understand how this therapeutic window might be controlled, we created a mechanistic Monte Carlo model of the early steps in GPCR signaling and desensitization. Using this model we found that the rates of G-protein activation and receptor phosphorylation can be partially decoupled by varying the drug-receptor dissociation rate constant, k(off), and the drug's efficacy, alpha. The maximum ratio of G-protein activation to receptor phosphorylation (GARP) was found for drugs with an intermediate k(off) value and small alpha-value. Changes to the cellular environment, such as changes in the diffusivity of membrane molecules and the G-protein inactivation rate constant, affected the GARP value of a drug but did not change the characteristic shape of the GARP curve. These model results are examined in light of experimental data for a number of GPCRs and are found to be in good agreement, lending support to the idea that the desensitization properties of a drug might be tailored to suit a specific application.     

Tachykinin peptide-induced activation and desensitization of neurokinin 1 receptors

The actions of four tachykinins on inhibition and desensitization of the M-current of bullfrog sympathetic neurons have been characterized. Radioligand binding parameters of the tachykinins were determined at a neurokinin receptor in a heterologous expression system. The correlation between binding, signaling and receptor regulation was investigated. A correlation between receptor binding and signaling was found between the peptides; however, their ability to produce desensitization was not correlated with binding and signaling. These results show that the ability of a tachykinin peptide to induce signal activation is not indicative of its ability to induce receptor regulation.     

Adenosine deaminase affects ligand-induced signalling by interacting with cell surface adenosine receptors

Adenosine deaminase (ADA) is not only a cytosolic enzyme but can be found as an ecto-enzyme. At the plasma membrane, an adenosine deaminase binding protein (CD26, also known as dipeptidylpeptidase IV) has been identified but the functional role of this ADA/CD26 complex is unclear. Here by confocal microscopy, affinity chromatography and coprecipitation experiments we show that A₁ adenosine receptor (A₁R) is a second ecto-ADA binding protein. Binding of ADA to A₁R increased its affinity for the ligand thus suggesting that ADA was needed for an effective coupling between A₁R and heterotrimeric G proteins. This was confirmed by the fact that ASA, independently of its catalytic behaviour, enhanced the ligand-induced second messenger production via A₁R. These findings demonstrate that, apart from the cleavage of adenosine, a further role of ecto-adenosine deaminase on the cell surface is to facilitate the signal transduction via A₁R.     

Interactions between opioid and chemokine receptors: heterologous desensitization

The opioid and chemokine receptors are both members of the seven transmembrane G protein-coupled receptor (GPCR) superfamily. Desensitization is believed to be a major element of the regulation of the function of these receptors, and recent findings suggest that both agonist-dependent (homologous) desensitization and heterologous desensitization can control receptor activity. The cross-desensitization between opioid and chemokine receptors has significant implications for our understanding of both the regulation of leukocyte trafficking, as well as the regulation of chemokine receptor function in inflammatory disease states. We also review findings which suggest that pro-inflammatory chemokine receptor-induced heterologous desensitization of opioid receptors has important implications for the regulation of opioid receptor function in the nervous system.     

Hepatic receptor for asialo-glycoproteins. Ultrastructural demonstration of ligand-induced microaggregation of receptors

Endocytosis of asialo-glycoproteins in hepatocytes is mediated by a lectin-like receptor with specificity for D-galactose. Early events of receptor-ligand interactions have been studied by ultrastructural analysis. Hepatocytes were isolated from the rat liver by collagenase perfusion and incubated with a galactosylated electron dense marker (gold-Gal-BSA, glactosylated bovine serum albumin adsorbed onto colloidal gold particles). Initial binding of gold-Gal-BSA particles occurs to receptors diffusely distributed at hepatic microvilli of the former space of Disé. No lectin activity was found in membrane areas that had formed in situ the region of hepatic cell contact or bile canaliculi. Microaggregation of receptor-ligand complexes is seen as an early consequence of particle binding. Microaggregates contain 2-5 particles and are located outside coated pits. After prolonged incubation larger clusters are formed, these are found associated with coated membrane areas. It is concluded that at least three steps precede the uptake of galactosylated proteins by hepatocytes. These are: (i) binding of ligand at diffusely distributed binding sites; (ii) local microaggregation of receptor-ligand complexes; (iii) formation of larger clusters and association with coated pits.     

Bradykinin B2 receptors mediate pulmonary sympathetic afferents induced reflexes in rabbits

Endogenous bradykinin (BK) is an established mediator of pulmonary inflammation, yet its role in lung disease is unclear. In the rabbit, injecting BK into the lung parenchyma elicits reflex hyperpnea, tachypnea, hypotension, and bradycardia by stimulating pulmonary sympathetic afferents. To further explore bradykinin effects, breathing pattern (phrenic nerve and abdominal muscle activities) and hemodynamics (blood pressure and heart rate) were examined in anesthetized, open-chest, and mechanically ventilated rabbits. Three receptor agonists [bradykinin, selective B(1) (des-Arg(9)-BK), and selective B(2) (Tyr(8)-BK)], as well as three B(2) receptor antagonists, B6029 (N alpha-Adamantaneacetyl)-Bradykinin, B(1)650 (D-Arg-[Hyp(3), Thi(5,8), D-Phe(7)]-Bradykinin, or Hoe-140 (D-Arg-[Hyp(3), Thi(5), D-Tic(7), Oic(8)] bradykinin), were used to identify the responsible receptor subtype. In both intact and vagotomized rabbits, injecting BK or a selective B(2) agonist into the lung elicited similar cardiopulmonary responses. These reflex responses were greatly attenuated or blocked by pre-injecting B(2) antagonists into the right atrium or into the lung parenchyma. In contrast, the B(1) agonist elicited fewer cardiopulmonary effects in intact rabbits and had no effect in vagotomized rabbits. We conclude that BK stimulates pulmonary sympathetic afferents [Soukhova, G., Wang, Y., Ahmed, M., Walker, J., Yu, J., 2003. Bradykinin stimulates respiratory drive by activating pulmonary sympathetic afferents in the rabbit. J. Appl. Physiol. 95, 241-249.; Wang, Y., Soukhova, G., Proctor, M., Walker, J., Yu, J., 2003. Bradykinin causes hypotension by activating pulmonary sympathetic afferents in the rabbit. J. Appl. Physiol. 95, 233-240.], eliciting a characteristic cardiopulmonary reflex via B(2) receptors.     

Short-term disappearance of muscarinic cell surface receptors in carbachol-induced desensitization

N6-Phenylisopropyladenosine was employed in the absence of endogenous adenosine to explore the influence exerted by the R-site over the antagonistic interaction of insulin and catecholamines on several parameters of fat cell metabolism. When no hormones were present, N6-phenylisopropyladenosine had little or no effect; however, the nucleoside potentiated insulin inhibition of catecholamine-stimulated events, such as lipolysis, and, conversely, diminished or blocked catecholamine inhibition of insulin-stimulated processes, such as 2-deoxyglucose uptake, glucose oxidation and esterification, even under conditions where N6-phenylisopropyladenosine, alone, was ineffective in reversing catecholamine actions.     

Calcium-dependent,slow desensitization distinguishes different types of glutamate receptors

1. L-Glutamate, the most likely transmitter of rapid excitatory synaptic interactions in the brain and spinal cord, is a potent neurotoxin. Mechanisms that terminate the action of glutamate are, therefore, likely to be important for maintaining the integrity of glutaminoceptive neurons. In this study, we show that glutamate currents evoked in voltage-clamped chick motoneurons fade during prolonged or repeated application of glutamate by pressure ejection from nearby pipettes. 2. The magnitude of the decline depends on the Ca2+/Mg2+ ratio in the extracellular medium. With Ca2+ = 10.0 mM and no added Mg, the steady-state glutamate current amounted to 50% of the initial value. 3. Single-channel measurements indicate that the fade is due to receptor desensitization rather than to agonist-induced channel blockade, as the mean channel open time within bursts is independent of the agonist concentration. 4. Application of more selective agonists showed that Ca2+-dependent slow desensitization involved only G1 (NMDA) receptors. G2 responses (activated by kainate and quisqualate) did not exhibit this slow phase of desensitization under the same conditions.     

Bradykinin receptors in intestinal smooth muscles and their post-receptor events related to calcium

The effects of trifluoperazine and verapamil on bradykinin- and des-Arg(9)-bradykinin induced responses of isolated rat duodenum and guinea-pig ileum were investigated to elucidate post-bradykinin receptor events. Verapamil and trifluoperazine inhibited bradykinin induced relaxations and contractions and des-Arg(9)- bradykinin induced contractions in rat duodenum. Bradykinin induced contractions of ileum were also inhibited by trifluoperazine and. verapamil. Since non-competitive affinity constants of trifluoperazine and verapamil for the relaxant responses to bradykinin in duodenum and for the contractile responses to bradykinin in ileum are different, post-bradykinin receptor events related to calcium may be different in ileum and duodenum. In addition, affinity constants of bradykinin in guinea-pig ileum and rat duodenum are also disparate suggesting the presence of different types of bradykinin B(2) receptors in these two organs.     

Differential desensitization and internalization of three different bullfrog gonadotropin-releasing hormone receptors

We previously demonstrated the presence of three distinct types of the gonadotropin-releasing hormone receptor (GnRHR) in a bullfrog (denoted bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). The bfGnRHRs exhibited differential tissue distribution and ligand selectivity. In the present study, we demonstrated the desensitization and internalization kinetics of these receptors in both transiently-transfected HEK293 cells and retrovirus-mediated stable cells. The time-course accumulation of the inositol phosphate in response to GnRH revealed that bfGnRHR-1 and -2 were rapidly desensitized, whereas bfGnRHR-3 was slowly desensitized. A comparison of the internalization kinetics revealed the most rapid rate and highest extent of internalization of bfGnRHR-2 among the three receptors. Interestingly, the mechanisms that underlie the receptor internalization appear to differ from each other. Internalization of bfGnRHR-1 was dependent on both dynamin and beta-arrestin, whereas those of bfGnRHR-2 and -3 were dependent on dynamin, but not on arrestin. These results, therefore, suggest that differential regulatory mechanisms for desensitization and internalization of the GnRHR are involved in diverse cellular and physiological responses to GnRH stimulation.