Receptor Localization Studies Research Articles

Addition of a Carboxy-Terminal Green Fluorescent Protein Does Not Alter the Binding and Signaling Properties of Relaxin Family Peptide Receptor 3

The relaxin family peptide receptor 3 (RXFP3) is the cognate receptor for the neuropeptide relaxin-3. RXFP3 was tagged at the carboxy-terminus with a variant of the green fluorescent protein (GFP(2)) for use in receptor localization studies. RXFP3-GFP(2) was examined to ensure it retained binding and signaling properties similar to untagged RXFP3. Competition for [(125)I]INSL5/H3 relaxin chimera binding to RXFP3 and RXFP3-GFP(2) indicated that the carboxy-terminal tag did not affect receptor binding or receptor internalization. RXFP3-GFP(2) activated ERK1/2 with a similar potency to RXFP3 when transiently expressed in CHO-K1 or HEK293T cells, suggesting that the GFP(2) tag did not affect receptor function. This study demonstrated that addition of a carboxy-terminal fusion protein to RXFP3 did not alter the binding or signaling properties of RXFP3, making RXFP3-GFP(2) a useful tool for future receptor localization and trafficking studies.

Peripherally restricted opioid drugs: advances and retreats

The aim of this article is to familiarize anesthesiologists with recent research investigating peripheral opioid analgesia. The review focuses on studies of peripheral receptor distribution in humans and clinical data that support the hypothesis of peripheral analgesia that have been published over the past 2 years. Recent anatomical studies using human tissue have detected the presence of mu-opioid receptors in epidermal and dermal layers of normal skin, although expression was not altered in certain dermal clinical conditions. Nerve fibers of human dental pulp also contained detectable mu-opioid receptors. A survey of the clinical literature on peripherally mediated mu analgesia suggests that under conditions of inflammation, the onset of analgesia is typically delayed, but once initiated, is of long duration. A peripherally selective kappa agonist was efficacious in relieving the pain associated with chronic pancreatitis. Peripheral analgesia mediated through the mu-opioid receptor has been demonstrated in a variety of clinical settings, with the preponderance of data generated with arthroscopic procedures. Receptor localization studies using human tissue have corroborated the presence of the mu-opioid receptor in the periphery. Future research is needed to investigate the clinical utility of kappa and delta-opioid receptor agonists in the peripheral setting.

Topically administered CB 2-receptor agonist, JWH-133, does not decrease intraocular pressure (IOP) in normotensive rabbits

Recent IOP and receptor localization studies suggest that the IOP effects of cannabinoids are mediated via ocular CB 1 receptors. However, it is not yet known whether CB 2 receptor agonists decrease IOP. In this study, the IOP-lowering effects of the CB 2 receptor agonist JWH-133 were studied in normotensive rabbits, and compared with CP55,940. JWH-133 and CP55,940 were dissolved in aqueous hydroxypropyl-β-cyclodextrin (HP-β-CD) solutions and propylene glycol. The eye drops (25 μl) were administered unilaterally to the rabbit eye, and IOPs were measured at fixed time intervals. JWH-133, dissolved in either HP-β-CD (doses = 10 μg and 25 μg) or propylene glycol (dose = 62.5 μg), did not have any effect on IOP when compared to vehicle treatments. In contrast, CP55,940 formulated in HP-β-CD (doses = 25 μg and 62.5 μg) or propylene glycol (dose = 62.5 μg) reduced IOP significantly compared to vehicle treatments. The results suggest that topically administered CB 2 receptor agonist, JWH-133, does not decrease IOP in normotensive rabbits at the doses and formulations used, and thus, CB 2 receptor agonists may not be useful as cannabinoid-based IOP-lowering therapeutics.

Modulation of lateral perforant path excitatory responses by metabotropic glutamate 8 (mGlu8) receptors

The contribution of metabotropic glutamate 8 (mGlu8) receptors to modulation of medial and lateral perforant path (MPP and LPP) inputs to the dentate gyrus was investigated using electrophysiological recording of field excitatory postsynaptic potentials (fEPSPs) from hippocampal slices taken from wild-type and mGlu8 receptor knockout animals. Application of the selective group III mGlu receptor agonist, L-AP4 (1–100 μM), reduced fEPSPs evoked by LPP, but not MPP stimulation in wild-type slices in a concentration-dependent manner (EC 50=4.7 μM). The selective mGlu8 receptor agonist, DCPG (1–30 μM) also suppressed LPP fEPSPs with an EC 50 value of 3.1 μM. The L-AP4-induced reduction in LPP fEPSPs could be blocked by the group III antagonist, MSOP (100 μM) in wild-type slices and was eliminated in mGlu8 receptor-deficient slices. Additional experiments showed that MPP fEPSPs were suppressed by the group II agonist, LY379268 (0.01–3 μM) in control slices (EC 50=153.1 nM); an effect that was not altered in mGlu8 receptor knockout slices (EC 50=153.8 nM) . In addition, LY379268 had little effect on fEPSPs evoked by LPP stimulation in mGlu8 receptor-deficient slices. In conjunction with recent receptor localization studies, these results suggest that the mGlu8 receptors serve as autoreceptors on LPP afferents to the dentate gyrus.

Characterization of Central Inhibitory Muscarinic Autoreceptors by the Use of Muscarinic Acetylcholine Receptor Knock-Out Mice

Forebrain muscarinic acetylcholine (ACh) receptors (mAChRs; M1-M5) are predicted to play important roles in many fundamental central functions, including higher cognitive processes and modulation of extrapyramidal motor activity. Synaptic ACh levels are known to be regulated by the activity of presynaptic muscarinic autoreceptors mediating inhibition of ACh release. Primarily because of the use of ligands with limited receptor subtype selectivity, classical pharmacological studies have led to conflicting results regarding the identity of the mAChR subtypes mediating this activity in different areas of the brain. To investigate the molecular identity of hippocampal, cortical, and striatal inhibitory muscarinic autoreceptors in a more direct manner, we used genetically altered mice lacking functional M2 and/or M4 mAChRs [knock-out (KO) mice]. After labeling of cellular ACh pools with [3H]choline, potassium-stimulated [3H]ACh release was measured in superfused brain slices, either in the absence or the presence of muscarinic drugs. The nonsubtype-selective muscarinic agonist, oxotremorine (0.1-10 microm), inhibited potassium-stimulated [3H]ACh release in hippocampal, cortical, and striatal slices prepared from wild-type mice by up to 80%. This activity was totally abolished in tissues prepared from M2-M4 receptor double KO mice. Strikingly, release studies with brain slices from M2 and M4 receptor single KO mice indicated that autoinhibition of ACh release is mediated primarily by the M2 receptor in hippocampus and cerebral cortex, but predominantly by the M4 receptor in the striatum. These results, together with additional receptor localization studies, support the novel concept that autoinhibition of ACh release involves different mAChRs in different regions of the brain.

Localization of tachykinin receptors and Fos-like immunoreactivity induced by substance P in guinea-pig brain.

1. In the present study, a comparison was made between the distribution of tachykinin NK1 and NK3 receptor immunoreactivity and the distribution of Fos-like immunoreactivity induced by the tachykinin agonist substance P (SP) in the guinea-pig brain. 2. In agreement with results from previous studies in rat brain, NK1 receptor-immunoreactive neurons were found to be widely distributed throughout the brain in the striatum and in diencephalic and mesencephalic structures, while NK3 receptor-immunoreactive neurons were mainly in telencephalic structures. Considerable overlap was observed between NK1 and NK3 receptor distributions. 3. Substance P induced Fos-like immunoreactivity (Fos-LI) in extensive areas of the guinea-pig brain. The induction of Fos-LI was markedly inhibited in many areas by pretreatment with the NK1 receptor antagonist SR 140333. The NK3 receptor antagonist SR 142801 reduced Fos-LI staining in fewer areas, although a reduction was observed in the cortex, striatum and hypothalamus. 4. In general, tachykinin receptors were located at sites corresponding to areas of functional activation by SP, as shown by Fos-LI. These results extend previous studies by adding a functional dimension to tachykinin receptor localization studies.

Distribution of Fos-like immunoreactivity in guinea-pig brain following administration of the neurokinin-1 receptor agonist, [Sar 9,Met(O 2) 11]substance P

The tachykinins are a family of peptides with putative neurotransmitter roles in the nervous system. They mediate their effects via neurokinin-1, neurokinin-2 and neurokinin-3 receptors. There has been increasing interest in the therapeutic application of the tachykinin neurokinin-1 receptor antagonists in the treatment of pain and emesis, and more recently in depression. However, the central role of neurokinin-1 receptors is not well understood. The aims of the present study were to determine the behavioural responses of guinea-pigs, and the distribution of Fos-like immunoreactivity in the guinea-pig brain, following intracerebroventricular administration of the neurokinin-1 receptor-selective agonist, [Sar 9,Met(O 2) 11]substance P. The effects of pretreatment with the neurokinin-1 receptor antagonist, SR 140333, were also investigated. Administration of [Sar 9,Met(O 2) 11]substance P induced increased locomotor activity, as well as face washing, grooming and wet-dog shake behaviours, all of which were inhibited by the neurokinin-1 receptor antagonist, SR 140333, indicating the involvement of neurokinin-1 receptors. In order to localize the brain regions activated by [Sar 9,Met(O 2) 11]substance P, the distribution of neurons expressing Fos-like immunoreactivity was examined. [Sar 9,Met(O 2) 11]substance P induced increased Fos-like immunoreactivity in widespread areas, including the frontal cortex, hippocampus, amygdala, thalamus, hypothalamus, periaqueductal gray, area postrema and nucleus of the solitary tract. SR 140333 reduced Fos-like immunoreactivity induced by [Sar 9,Met(O 2) 11]substance P in most areas. Thus, brain regions associated with emotion, sensation, learning and memory, autonomic regulation and emesis were activated by stimulation of neurokinin-1 receptors. The present data have added a functional domain to previous neurokinin-1 receptor localization studies by describing the extensive regions of the CNS that may be activated by stimulation of these receptors, and the potential of neurokinin-1 receptor antagonists to inhibit activation of these regions.

Comparison of Antibodies Directed Against Receptor Segments of NPY-Receptors

The Y1-, Y2-, Y4- and Y5-receptor, which belong to the rhodopsin-like G-protein coupled, 7 transmembrane helix spanning receptors, bind the 36-mer neuromodulator NPY (neuropeptide Y) with nanomolar affinity. Synthetic fragments of the second (E2) and third (E3) extracellular loop were used to generate subtype selective anti-receptor antibodies against the Y-receptors. As investigated on intact receptors by ELISA and on solubilized receptors by SDS-PAGE and subsequent Western blotting, subtype selectivity was only partly achieved. Nevertheless, selectivity can be obtained by using several antisera in combination. These antibodies represent tools for molecular mass determination, receptor purification by affinity chromatography with antibody-columns and receptor localization studies.

A three-receptor model for the interaction of the K88 fimbrial adhesin variants of Escherichia coli with porcine intestinal epithelial cells.

Four phenotypes of pigs distinguished by the variant(s) of K88 fimbrial adhesin (K88ab, K88ac, K88ad) that bind to their intestinal epithelial cells (I-none of the variants, II-K88ad, III-K88ab and K88ac, and IV-all three variants) have been identified. We hypothesize that the differences between the phenotypes are defined by the presence or absence of K88 adhesin receptors. We propose a three-receptor model to account for the observed phenotypes: 1) Receptor bed which binds all three variants and is found in phenotype IV, 2) Receptor be which binds K88ab and K88ac and is found in phenotype III and IV, and 3) Receptor d which binds K88ad and is found in phenotype II. We have identified the be receptor activity as a pair of mucin-type sialoglycoproteins (210 and 240 kDa). Although neither the bcd nor d receptor has been identified biochemically, their presence has been established using both blocking and receptor localization studies. Blocking studies using phenotype IV brush borders demonstrated that K88ab and K88ac fimbriae block the binding of E. coli expressing any of the K88 variants, but K88ad fimbriae block only K88ad E. coli binding. These results indicate that two receptors (bcd and bc) exist in the phenotype IV animals. Receptor localization studies on intestinal sections from phenotype IV animals showed that K88ab and K88ac adhesin binding is continuous from the crypt to the tip of the villus. The binding of the K88ad adhesin binding is multifocal in phenotype IV pigs, but continuous from crypt to tip of the villus in sections of phenotype II pigs. These studies verify the presence of two receptors (bcd and bc) in phenotype IV animals, and indicate that the K88ad receptor in phenotype IV animals (bcd) is different than in phenotype II animals (d).

Labeling of human platelet plasma membrane thromboxane A 2/prostaglandin H 2 receptors using SQB, a novel biotinylated receptor probe

This study reports the synthesis, biological evaluation, and application of a new biotinylated derivative 1-[[1 S-[1α,2α( Z),3α,4α]]-7-[3-[[[[(1-oxocyclohexylpropyl)amino]acetyl]amino]methyl]-7-oxabi-cyclo [2.2.1]hept-2-yl]-5-heptenoyl]-2-[hexahydro-2′-oxo-1 H-thieno[3′,4′ d][imidazole-4′-pentanoyl]hydrazine (SQB) of the thromboxane A 2/prostaglandin H 2 (TXA 2/PGH 2) receptor antagonist [1 S-[1α,2α( Z),3α, 4α]]-7-[3-[[[[(1-oxocyclohexylpropyl)amino]acetyl]amino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ31,491). SQB was synthesized by reacting SQ31.491 with biotin hydrazide, and the product was purified by flash chromatography. It was found that SQB specifically inhibited platelet aggregation in response to U46619 with an ic 50 of 275 nM. On the other hand, SQB did not inhibit adenosine diphosphate or A23187-induced aggregation. Competition binding studies revealed that SQB produced a concentration-dependent inhibition of [ 3H]-[1S-[1α,2β(5Z),3β,4α]]-7-[3-[[2[( phenylamino)carbonyl]hydrazino]methyl]-7- oxabicyclo[2.2.1] hept-2- yl]-5- heptenoic acid ([ 3H]SQ29,548) specific binding in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-solubilized platelet membranes, with a K i of 220 nM. The shape of the SQB inhibition binding curve was indistinguishable from that produced by the TXA 2/PGH 2 receptor antagonist BM13.177. Finally, incubation of gel-filtered platelets or platelet-rich plasma with SQB and fluorescein isothiocyanate (FITC)-avidin demonstrated fluorescent labeling of platelet plasma membrane TXA 2/PGH 2 receptors. Furthermore, this SQB-FITC fluorescent labeling was reduced significantly by co-incubation of the platelets with the TXA 2/PGH 2 antagonist SQ29,548. Based on the ability of SQB-FITC-avidin to label intact platelets, it can be concluded: (1) that a pool of platelet TXA 2/PGH 2 receptors resides in the plasma membrane; and (2) that the binding domains for these receptors are oriented at or near the external membrane surface. Collectively, these data demonstrate that SQB is a highly specific probe for TXA 2/PGH 2 receptors, which should be of significant value for receptor localization studies in platelets and other tissues.

The area postrema: a cardiovascular control centre at the blood-brain interface?

The area postrema (AP) is one of the circumventricular organs of the brain and as such it is highly vascular and lacks the normal blood-brain barrier. Anatomical tracing studies have demonstrated afferent projections to AP originating from the paraventricular nucleus, lateral parabrachial nucleus (l-PBN), nucleus tractus solitarius (NTS), as well as the vagus nerve. AP neurons have been shown to project primarily to l-PBN, and NTS. Receptor localization studies have reported dense aggregations of many specific peptide receptors in AP including those for angiotensin II (ANG), atrial natriuretic peptide (ANP), and endothelin (ET). Electrical stimulation studies have shown that activation of AP neurons at low frequencies (less than 15 Hz) results in decreases in blood pressure and heart rate, while higher frequency (greater than 20 Hz) stimulation causes increases in blood pressure. These low frequency effects on blood pressure and heart rate appear to result from activation of separate components of the autonomic nervous system. Extracellular single unit recordings have identified two functionally separate populations of AP neurons: one responsive to circulating ANG and a second apparently responsive to changes in blood pressure. In addition, AP neurons are activated by increases in circulating ET. Afferent inputs to AP neurons from 1-PBN have separate excitatory (12% of AP neurons) or inhibitory (12% of AP neurons) effects on a relatively small proportion of AP neurons. In contrast, preliminary evidence suggests a much more broadly distributed excitatory input to approximately 70% of tested AP neurons originating from the aortic depressor nerve. These studies provide considerable evidence implicating the AP as a significant neural structure regulating the cardiovascular system.

Light microscopic localization of brain opiate receptors: a general autoradiographic method which preserves tissue quality

A general technique is described for using slide-mounted unfixed tissue sections to characterize and visualize drug and neurotransmitter receptors in brain or other tissues. The preparation of material, from fresh frozen, unfixed brain to dried sections securely attached to slides, is described in detail. The tissue can be kept intact during incubation at varying temperatures in solutions containing radiolabeled ligand, ions, buffers, and allosteric effectors. Strategies are described for determining optimal stereospecific binding with highest signal-to-noise ratios and for determining that a "meaningful" receptor is being studied. Dry formaldehyde fixation by vapors from heated paraformaldehyde preserves the tissue quality and traps the ligand near its site on the receptor, permitting subsequent histological processing through alcohols, solvents, and aqueous media, including liquid nuclear track emulsion. Visualization of [3H]naloxone- or [3H]enkephalin-labeled opiate receptor distributions in rat and human brains is achieved by tritium-sensitive film or by classical "wet" emulsion autoradiography. The advantages of the film include its ease of use and the ability to quantify receptor density by densitometry which can be computer-assisted. The advantage of the emulsion is the greater resolution and the concomitant appearance of morphology in cell-stained sections. Examples of correlations of opiate receptor distributions which underlying cytoarchitecture illustrate the potential for receptor localization studies.