Desensitizing GABAC receptors on carp retinal bipolar cells

Bicuculline- and baclofen-insensitive GABA receptors (GABAC receptors) on bipolar cells acutely dissociated from carp retina were investigated with using the whole-cell patch-clamp recording technique. The currents of these cells mediated by GABAC receptors showed striking desensitization, even at low concentrations of GABA. Both the time constant tau of the GABAC current decay and the extent of desensitization were significantly different from that of GABAC receptors previously observed in other retinas and elsewhere in the CNS, suggesting that the GABAC receptors of carp bipolar cells might be distinct in intracellular mechanisms and subunit composition.     

Contributions of GABAA receptors and GABAC receptors to acetylcholine release and directional selectivity in the rabbit retina

GABA is a major inhibitory neurotransmitter in the mammalian retina and it acts at many different sites via a variety of postsynaptic receptors. These include GABAA receptors and bicuculline-resistant GABAC receptors. The release of acetylcholine (ACh) is inhibited by GABA and strongly potentiated by GABA antagonists. In addition, GABA appears to mediate the null inhibition which is responsible for the mechanism of directional selectivity in certain ganglion cells. We have used these two well-known examples of GABA inhibition to compare three GABA antagonists and assess the contributions of GABAA and GABAC receptors. All three GABA antagonists stimulated ACh release by as much as ten-fold. By this measure, the ED50s for SR-95531, bicuculline, and picrotoxin were 0.8, 7.0, and 14 microM, respectively. Muscimol, a potent GABAA agonist, blocked the effects of SR-95531 and bicuculline, but not picrotoxin. This indicates that SR-95531 and bicuculline are competitive antagonists at the GABAA receptor, while picrotoxin blocks GABAA responses by acting at a different, nonreceptor site such as the chloride channel. In the presence of a saturating dose of SR-95531 to completely block GABAA receptors, picrotoxin caused a further increase in the release of ACh. This indicates that picrotoxin potentiates ACh release by a mechanism in addition to the block of GABAA responses, possibly by also blocking GABAC receptors, which have been associated with bipolar cells. All three GABA antagonists abolished directionally selective responses from ON/OFF directional-selective (DS) ganglion cells. In this system, the ED50S for SR-95531, bicuculline, and picrotoxin were 0.7 microM, 8 microM, and 94.6 microM, respectively. The results with SR-95531 and bicuculline indicate that GABAA receptors mediate the inhibition responsible for directional selectivity. The addition of picrotoxin to a high dose of SR-95531 caused no further increase in firing rate. The comparatively high dose required for picrotoxin also suggests that GABAC receptors do not contribute to directional selectivity. This in turn suggests that feedforward GABAA inhibition, as opposed to feedback at bipolar terminals, is responsible for the null inhibition underlying directional selectivity.     

Molecular studies on kinin receptors

We describe the results of functional studies on DNA clones encoding functional bradykinin receptors derived from human, rat, and mouse sources and including both genomic and complementary DNA clones. In both the Xenopus oocyte and the COS cell expression systems, the receptors from human and rat showed the pharmacological properties of B2 receptors, but receptors from mouse displayed both B1- and B2-like pharmacological properties. We further investigated the molecular relationship between the B1 and B2 receptor subtypes expressed by a human fibroblast cell line, and we demonstrate that these two receptor subtypes are encoded by distinct mRNA species.     

Molecular medicine of chemokine receptors]

Chemokines belong to a family of small secreted proteins that play essential roles in the recruitment and activation of leukocytes at the sites of inflammation. Thirteen chemokine receptors have already been cloned and shown to be organized with a structure of seven-transmembrane-domain receptors, a structure typical of classical G-protein coupled receptors. Most of chemokine receptors display overlapping specificities and most of chemokines bind several different receptors. Engagement of receptors results in the elevation of cytosolic calcium and activation of PKC via inositol trisphosphate and diacylglycerol, respectively. Furthermore, involvement of protein tyrosine kinases, MAP kinase and P13 kinase in the signaling pathway is demonstrated.     

Molecular chaperones and subcellular trafficking of steroid receptors

Unliganded steroid receptors exist as heteromeric complexes comprised of heat shock and immunophilin proteins that associate either directly or indirectly with receptor carboxyl-terminal ligand-binding domains. Molecular chaperons, and other proteins associated with steroid receptors, play an important role in the maturation of receptors to a hormone-binding competent state. Steroid receptor-associated 90 and 70 kDa heat shock proteins, hsp90 and hsp70, respectively, have well established roles in protein folding in addition to participating in numerous subcellular trafficking pathways. In this review, we discuss the possible roles that molecular chaperons, such as hsp90, hsp70 and DnaJ proteins, have in steroid receptor trafficking within two distinct subcellular compartments, i.e. the cytoplasm and nucleus.     

Molecular biology of opioid receptors: recent advances

Endogenous opioid peptides and opiates like morphine produce their pharmacological effects through the membrane bound opioid receptors. These receptors belong to a superfamily of G-protein-coupled receptors, all of which possess seven membrane-spanning regions. Structure-activity relationship studies of opioids opened up new avenues for the pharmacological characterization of the opioid receptors. As a further advancement in this direction, molecular cloning has led to the identification of three different types of opioid receptors -- OP1 (delta), OP2 (kappa) and OP3 (mu) -- thereby supporting the results of earlier pharmacological studies which postulated their existence. The three opioid receptors are highly homologous. Consequent to the development of highly specific and selective agonists and antagonists, it was proposed that the three types of opioid receptors could be further categorized into different subtypes. However, the molecular biology data generated so far do not support the presence of the various subtypes of the three well-characterized opioid receptors. Recent strides towards the advancement of our knowledge relating to the molecular biology of these receptors have been reviewed in this article.     

Molecular characteristics of insect vitellogenins and vitellogenin receptors

The recent cloning and sequencing of several insect vitellogenins (Vg), the major yolk protein precursor of most oviparous animals, and the mosquito Vg receptor (VgR) has brought the study of insect vitellogenesis to a new plane. Insect Vgs are homologous to nematode and vertebrate Vgs. All but one of the insect Vgs for which we know the primary structure are cleaved into two subunits at a site [(R/K)X(R/K)R or RXXR with an adjacent beta-turn] recognized by subtilisin-like proprotein convertases. In four of the Vgs, the cleavage site is near the N-terminus, but in one insect species, it is near the C-terminus of the Vg precursor. Multiple alignments of these Vg sequences indicate that the variation in cleavage location has not arisen through exon shuffling, but through local modifications of the amino acid sequences. A wasp Vg precursor is not cleaved, apparently because the sequence at the presumed ancestral cleavage site has been mutated from RXRR to LYRR and is no longer recognized by convertases. Some insect Vgs contain polyserine domains which are reminiscent of, but not homologous to, the phosvitin domain in vertebrate Vgs. The sequence of the mosquito VgR revealed that it is a member of the low-density lipoprotein receptor (LDLR) family. Though resembling chicken and frog VgRs, which are also members of the LDLR family, it is twice as big, carrying two clusters of cysteine-rich complement-type (Class A) repeats (implicated in ligand-binding) instead of one like vertebrate VgRs and LDLRs. It is very similar in sequence and domain arrangement to the Drosophila yolk protein receptor (YPR), despite a non-vitellogenin ligand for the latter. Though vertebrate VgRs, insect VgR/YPRs, and LDLR-related proteins/megalins all accommodate one cluster of eight Class A repeats, fingerprint analysis of the repeats in these clusters indicate they are not directly homologous with one another, but have undergone differing histories of duplications, deletions, and exon shuffling so that their apparent similarity is superficial. The so-called epidermal growth factor precursor region contains two types of motifs (cysteine-rich Class B repeats and YWXD repeats) which occur independently of one another in diverse proteins, and are often involved in protein-protein interactions, suggesting that they potentially are involved in dimerization of VgRs and other LDLR-family proteins. Like the LDLR, but unlike vertebrate VgRs and the Drosophila YPR, the mosquito VgR contains a putative O-linked sugar region on the extra-cellular side of the transmembrane domain. Its function is unclear, but may protect the receptor from membrane-bound proteases. The cytoplasmic tail of insect VgR/YPRs contains a di-leucine (or leucine-isoleucine) internalization signal, unlike the tight-turn tyrosine motif of other LDLR-family proteins. The importance of understanding the details of yolk protein uptake by oocytes lies in its potential for exploitation in novel insect control strategies, and the molecular characterization of the proteins involved has made the development of such strategies a realistic possibility.     

Molecular biology of microglia cytokine and chemokine receptors and microglial activation

Activation of brain microglial cells can be subdivided into a number of stages. Early stages likely are proliferation and migration to sites of cell damage. These two stages have been studied exemplarily on the IL-3 receptor beta-subunit and on the CC-chemokine receptor 5 using molecular biological methods. First, IL-3 receptor beta-subunit cDNA has been cloned in full length from rat microglia. Since cultured microglia are already activated to some extent, mRNA of this subunit has been detected in the isolated cells, but was absent in normal rat brain. Lipopolysaccharide (LPS) increased this mRNA in the cultured cells and LPS injected into the circulation of rats induced the mRNA specifically in brain microglia as revealed by in situ hybridizations. Next, we obtained partial cDNAs of receptor-coupled protein tyrosine kinases JAK 1 and JAK 2. These mRNAs were present both in cultured microglia and in rat brain, but were not influenced by LPS. Finally, a full-length cDNA of the rat chemokine receptor 5 has been obtained by PCR methodology. Its mRNA was increased by administration of LPS both in cultured microglia and in vivo. It is expected, that further investigations on these receptors could help to develop improved strategies to combat chronic inflammatory events in the brain.     

Molecular and pharmacological characterization of GABAA receptors in the rat pituitary

Levels of mRNA for the major subunits of the GABAA receptor were assayed in the rat pituitary anterior and neurointermediate lobes by ribonuclease protection assay. alpha 1, beta 1, beta 2, beta 3, and gamma 2s were found to be the predominant subunits in the anterior lobe, whereas alpha 2, alpha 3, beta 1, beta 3, gamma 2s, and gamma 1 were the predominant subunits expressed in the neurointermediate lobe. alpha 5, alpha 6, and delta subunits were not detectable. Hill and Scatchard analysis of [3H] muscimol binding to anterior and neurointermediate lobe membranes showed high-affinity binding sites with dissociation constants of 5.6 and 4.5 nM, respectively, and Hill coefficients near 1. Muscimol sites were present at a maximum of 126 fmol/mg in the anterior lobe and 138 fmol/mg in the neurointermediate lobe. The central-type benzodiazepine antagonist [3H]Ro 15-1788 bound to a high-affinity site with a dissociation constant of 1.5 nM in both tissues, at a maximum of 60 fmol/mg in anterior pituitary and 72 fmol/mg in neurointermediate lobe. A Hill coefficient of 1 was measured for this site in both tissues. Assays of CL 218,872 displacement of Ro 15-1788 were consistent with a pure type I benzodiazepine site in the anterior lobe and a pure type II site in the intermediate lobe. These results are consistent with both tissue-specific expression of particular GABAA receptor subunits and receptor heterogeneity within individual cells in the pituitary.     

Androgen receptors and skeletal muscle composition in trotters treated with nandrolone laureate

To study the effects of nandrolone laureate (19-nortestosterone) on muscle hypertrophy and concentration of androgen receptors (AR), biopsy specimens were taken from the middle gluteal muscle of 6 Finnhorse trotters (geldings and mares) undergoing training before, immediately after, and 13 weeks after a 14-week treatment with nandrolone. Another 6 similarly trained horses served as controls. An additional 10 mares and 10 geldings were used to study annual variation in muscle concentration of AR. AR was immunohistochemically localized in the nuclei. AR concentration remained constant during the first 14 weeks of the study, but increased significantly during the 13-week follow-up period in both groups. This finding can be explained by the annual variation in AR. In the anabolic steroid (AS)-treated horses, but not in the controls (C), the cross-sectional area of the type I fibres increased significantly during the treatment period, and the percentage of type IIA fibres correlated positively with AR concentration at the end of nandrolone treatment. In the AS group, the concentration of DNA decreased during the 13-week follow-up period, and the percentage of H-chains in the isoenzymes of LDH increased. Protein concentration increased in both groups during the follow-up period. Glycogen content and the activity of citrate synthase in muscle during the study remained unchanged. It can thus be concluded that AS produce differing effects on type I and type II fibres, and the AR concentration in equine muscle may contribute to the change observed in the middle gluteal muscle.     

Molecular interaction of Agouti protein and Agouti-related protein with human melanocortin receptors

Agouti protein and the Agouti-related protein (AGRP) are antagonists of the melanocortin-3 receptor and melanocortin-4 receptor. Both proteins contain 10 cysteines in the C-terminal domain arranged in five disulfide bonds. One possible arrangement of the disulfide bonds predicts an octapeptide loop, and the chemical properties of four residues within this loop (residues 111-114 in human AGRP) bear striking resemblance to those of several melanocortin peptides, including alpha-MSH, MT-II, and SHU-9119. We showed that cyclic synthetic octapeptides based on the sequence of this loop from Agouti protein or human AGRP are functional antagonists of the human melanocortin-4 receptor. All peptides had a lower affinity for the melanocortin-3 receptor than for the melanocortin-4 receptor. Substitution of serines for cysteines resulted in linear peptides which had reduced binding affinities for both receptors. Mutational analysis of human AGRP indicated that its C-terminal domain is functionally equivalent to the intact human AGRP. The RFF111-113 triplet appears to be the most critical portion of AGRP in determining the binding affinity for both melanocortin-3 and melanocortin-4 receptors. These data strongly suggest that the loop defined by Cys-110 and Cys-117 is critical in determining the antagonist activity of human AGRP. Our data provide indirect evidence for the suggestion that the Cys-110 to Cys-117 octapeptide loop of human AGRP mimics the conformation of alpha-MSH, MT-II, and SHU-9119.     

Molecular pharmacology of homologues of ibotenic acid at cloned metabotropic glutamic acid receptors

We have studied the effects of the enantiomers of 2-amino-3-(3-hydroxyisoxazol-5-yl)propionic acid (homoibotenic acid, HIBO) and analogues substituted with a methyl, bromo or butyl group in the four position of the ring at cloned metabotropic glutamate (mGlu) receptors expressed in Chinese hamster ovary (CHO) cells. In contrast to the parent compound ibotenic acid, which is a potent group I and II agonist, the (S)-forms of homoibotenic acid and its analogues are selective and potent group I antagonists whereas the (R)-forms are inactive both as agonists and antagonists at group I, II, and III mGlu receptors. Interestingly, (S)-homoibotenic acid and the analogues display equal potency at both mGlu1alpha and mGlu5a with Ki values in the range of 97 to 490 microM, (S)-homoibotenic acid and (S)-2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid [(S)-4-butylhomoibotenic acid] displaying the lowest and highest potency, respectively. The homoibotenic acid analogues thereby differ from mGlu receptor antagonists derived from phenylglycine such as (S)-4-carboxyphenylglycine which only antagonizes mGlu1alpha (Ki = 18 microM) showing no effect at mGlu5a (Ki > 300 microM).     

Molecular mechanisms for the regulation of the expression and function of muscarinic acetylcholine receptors

The regulation of muscarinic acetylcholine receptor expression and function was investigated in cultured cells and in knockout mice. Muscarinic agonist exposure causes m2 receptor desensitization and sequestration and decreases the expression of cardiac potassium channels. The expression of m2 receptors in chick retina is regulated by a developmentally regulated secreted factor. Mice lacking the m1 receptor exhibit a loss of muscarinic regulation of M-current potassium channel activity and pilocarpine-induced seizures.     

Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3

Galanin is a 29- or 30-amino acid peptide with wide-ranging effects on hormone release, feeding behavior, smooth muscle contractility, and somatosensory neuronal function. Three distinct galanin receptor (GALR) subtypes, designated GALR1, 2, and 3, have been cloned from the rat. We report here the cloning of the human GALR2 and GALR3 genes, an initial characterization of their pharmacology with respect to radioligand binding and signal transduction pathways, and a profile of their expression in brain and peripheral tissues. Human GALR2 and GALR3 show, respectively, 92 and 89% amino acid sequence identity with their rat homologues. Radioligand binding studies with 125I-galanin show that recombinant human GALR2 binds with high affinity to human galanin (K(D) = 0.3 nM). Human GALR3 binds galanin with less affinity (IC50 of 12 nM for porcine galanin and 75 nM for human galanin). Human GALR2 was shown to couple to phospholipase C and elevation of intracellular calcium levels as assessed by aequorin luminescence in HEK-293 cells and by Xenopus melanophore pigment aggregation and dispersion assays, in contrast to human GALR1 and human GALR3, which signal predominantly through inhibition of adenylate cyclase. GALR2 mRNA shows a wide distribution in the brain (mammillary nuclei, dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic, and arcuate nuclei), and restricted peripheral tissue distribution with highest mRNA levels detected in human small intestine. In comparison, whereas GALR3 mRNA was expressed in many areas of the rat brain, there was abundant expression in the primary olfactory cortex, olfactory tubercle, the islands of Calleja, the hippocampal CA regions of Ammon's horn, and the dentate gyrus. GALR3 mRNA was highly expressed in human testis and was detectable in adrenal gland and pancreas. The genes for human GALR2 and 3 were localized to chromosomes 17q25 and 22q12.2-13.1, respectively.     

Molecular evolution of the C-terminal cytoplasmic domain of a superfamily of bacterial receptors involved in taxis

Twenty-nine proteins from 16 different species of prokaryotes revealed an extensive sequence homology with the cytoplasmic domain of the Escherichia coli aspartate receptor. The high percentage of identity indicated that they constitute a superfamily of proteins. A consensus secondary structure consisting mostly of alpha-helices was predicted. The occurrence of a seven-residue repeat (a-b-c-d-e-f-g), in which both the a and d residues were hydrophobic with few exceptions, provided additional evidence for a conserved alpha-helical conformation. Sequence alignments, together with the predicted secondary structure, led to identification of the boundaries for the functional units constituting the cytoplasmic domain. Putative methylation sites were assigned for all the members of this superfamily. These proteins could be grouped into three classes based on the presence of 14-residue insertion/deletion regions found within both the signalling and the methylation functional units of the cytoplasmic domain. The gene coding for the C-terminal cytoplasmic domain of these proteins apparently evolved through gene duplication from a common ancestor in which the four original 14-residue insertion/deletion regions were deleted two by two during evolution.