Molecular pharmacology of the calcitonin receptor.

The peptide hormone calcitonin is widely used therapeutically in the treatment of bone disorders such as Paget's disease, osteoporosis, and the hypercalcemia of some malignancies. However, emerging evidence suggests the actions of calcitonin via its G protein-coupled receptor, the calcitonin receptor, may not be limited to bone. Calcitonin receptors have also been identified in the central nervous system, testes, skeletal muscle, lymphocytes, and the placenta. We are now becoming aware that the complexity of the calcitonin response mediated by the calcitonin receptor can be influenced by accessory proteins, receptor isoforms, genetic polymorphisms, developmental and/or transcriptional regulation, feedback inhibition, and the specific cellular or tissue background. This article discusses what is known about the molecular and pharmacological actions of the calcitonin receptor and highlights areas of current research.

Attenuation of cocaine-induced genomic and functional responses in prenatal cocaine-exposed rabbits.

The effects of in utero cocaine exposure on cocaine-induced genomic and functional responses in postnatal life were examined. Pregnant Dutch Belted rabbits were injected intravenously, twice daily, with cocaine hydrochloride (4 mg/kg) or saline from day 8 through day 29 of pregnancy. Prenatally exposed kits were challenged with cocaine on postnatal day 20. In prenatal saline-exposed kits, cocaine induced time- and dose-dependent c-fos gene expression in both frontal cortex and striatum. Prenatal cocaine exposure reduced cocaine-induced c-fos responses by 35-58% in the frontal cortex and 37-41% in the striatum. Cocaine-induced functional responses that included head bobbing, seizure, and locomotor activity were also attenuated in prenatal cocaine-exposed kits. Cocaine-induced c-fos expression and functional responses were blocked by the D(1) dopamine receptor antagonist, SCH23390, or by the serotonin receptor antagonist, methysergide, but not by the D(2) dopamine receptor antagonist, L-sulpride. The results indicate that in utero cocaine exposure leads to diminished responses to cocaine challenge in the offspring, which may be mediated by prenatal cocaine-induced alterations in one or more components of the D(1) dopamine and/or serotonin receptor signaling systems during early postnatal life.

Receptor activity modifying proteins.

Our understanding of G protein-coupled receptor (GPCR) function has recently expanded to encompass novel protein interactions that underlie both cell-surface receptor expression and the exhibited phenotype. The most notable examples are those involving receptor activity modifying proteins (RAMPs). RAMP association with the calcitonin (CT) receptor-like receptor (CRLR) traffics this receptor to the cell surface where individual RAMPs dictate the expression of unique phenotypes. A similar function has been ascribed to RAMP interaction with the CT receptor (CTR) gene product. This review examines our current state of knowledge of the mechanisms underlying RAMP function.

Amylin receptor phenotypes derived from human calcitonin receptor/RAMP coexpression exhibit pharmacological differences dependent on receptor isoform and host cell environment.

Receptor activity modifying proteins (RAMPs) constitute a group of three proteins, designated as RAMP1, 2, and 3, which are able to effect functional changes in some members of the G protein-coupled receptor family. Thus, RAMP1 or RAMP3 can modify the calcitonin receptor (CTR) to also function as a high-affinity amylin receptor-like phenotype. To examine the RAMP/CTR interaction, individual RAMPs were coexpressed with either of the two human CTR (hCTR) isoforms, the insert negative (hCTR(I1-)) or the insert positive (hCTR(I1+)), in Chinese hamster ovary (CHO-P) or African monkey kidney (COS-7) cells. CHO-P cells provide an environment conducive to a low, but significant, level of amylin binding with either hCTR isoform alone, unlike in COS-7, where RAMP coexpression is imperative for amylin binding. Also, in CHO-P, hCTR(I1-) induced amylin binding with all three RAMPs, in contrast to COS-7, where only RAMP1 or RAMP3 generate an amylin receptor phenotype. hCTR(I1+) induced high-affinity amylin binding with any RAMP in either cell line. In COS-7 cells, hCTR(I1+)/RAMP-generated receptor displayed high- and low-affinity states, in contrast with the single-state binding seen with hCTR(I1-)/RAMP-generated receptor, whereas in CHO-P cells a two-affinity state receptor phenotype was evident with both hCTR isoforms. Endogenous RAMP expression is low and similar between cell lines. The results suggest that CTR/RAMP interaction in these cells is complex with other cellular factors such as the levels of different G proteins and/or receptor/RAMP stoichiometry following heterologous coexpression contributing to the ultimate receptor phenotype.

Multiple ramp domains are required for generation of amylin receptor phenotype from the calcitonin receptor gene product.

Calcitonin (CT), calcitonin gene-related peptide (CGRP), amylin, and adrenomedullin constitute a family of structurally related peptides that signal via either the calcitonin receptor-like receptor or the CT receptor, with receptor phenotype determined by coexpression of one of the three receptor activity-modifying proteins (RAMPs). The nature of the interaction between the receptor and RAMP was investigated using chimeras between RAMP1 and RAMP2 where the amino-terminal domain of RAMP1 was attached to the transmembrane domain and carboxy terminus of RAMP2 and called RAMP1/2, and vice versa for RAMP2/1. Cotransfection of wild-type or chimeric RAMPs with the insert-negative isoform of the human CT receptor (hCTR(I1-)) into COS-7 cells resulted in the expression of (125)I-rat amylin binding sites. Highest specific binding was observed when either RAMP1 or RAMP2/1 were cotransfected, indicating the importance of the RAMP transmembrane domain and/or carboxy terminus for the degree to which amylin receptors are expressed. In contrast, the phenotype generated was primarily determined by the amino terminus, with similar RAMP1- and RAMP1/2-induced receptor phenotypes that had higher affinity for human CGRPalpha and lower affinity for human calcitonin than the RAMP2- and RAMP2/1-induced receptors.

Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product.

Receptor activity-modifying proteins (RAMPs) are single-transmembrane proteins that transport the calcitonin receptor-like receptor (CRLR) to the cell surface. RAMP 1-transported CRLR is a calcitonin gene-related peptide (CGRP) receptor. RAMP 2- or RAMP 3-transported CRLR is an adrenomedullin receptor. The role of RAMPs beyond their interaction with CRLR, a class II G protein-coupled receptor, is unclear. In this study, we have examined the role of RAMPs in generating amylin receptor phenotypes from the calcitonin (CT) receptor gene product. Cotransfection of RAMP 1 or RAMP 3 with the human CT receptor lacking the 16-amino acid insert in intracellular domain 1 (hCTRI1-) into COS-7 cells induced specific 125I-labeled rat amylin binding. RAMP 2 or vector cotransfection did not cause significant increases in specific amylin binding. Competition-binding characterization of the RAMP-induced amylin receptors revealed two distinct phenotypes. The RAMP 1-derived amylin receptor demonstrated the highest affinity for salmon CT (IC50, 3.01 +/- 1.44 x 10(-10) M), a high to moderate affinity for rat amylin (IC50, 7.86 +/- 4.49 x 10(-9) M) and human CGRPalpha (IC50, 2.09 +/- 1.63 x 10(-8) M), and a low affinity for human CT (IC50, 4.47 +/- 0.78 x 10(-7) M). In contrast, whereas affinities for amylin and the CTs were similar for the RAMP 3-derived receptor, the efficacy of human CGRPalpha was markedly reduced (IC50, 1.12 +/- 0.45 x 10(-7) M; P <.05 versus RAMP 1). Functional cyclic AMP responses in COS-7 cells cotransfected with individual RAMPs and hCTRI1- were reflective of the phenotypes seen in competition for amylin binding. Confocal microscopic localization of c-myc-tagged RAMP 1 indicated that, when transfected alone, RAMP 1 almost exclusively was located intracellularly. Cotransfection with calcitonin receptor (CTR)I1- induced cell surface expression of RAMP 1. The results of experiments cross-linking 125I-labeled amylin to RAMP 1/hCTR-transfected cells with bis succidimidyl suberate were suggestive of a cell-surface association of RAMP 1 and the receptors. Our data suggest that in the CT family of receptors, and potentially in other class II G protein-coupled receptors, the cellular phenotype is likely to be dynamic in regard to the level and combination of both the receptor and the RAMP proteins.

Genomic responses to 5-HT1A or 5-HT2A/2C receptor activation is differentially regulated in four regions of rat brain.

The functional profiles of brain 5-HT1A and 5-HT2A/C receptors were assessed by quantitating changes in the immediate early genes -c-fos, ngf1c and tis1, following receptor activation with either 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin) or DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane). Stimulation of either class of 5-HT receptor elicited an induction of all three immediate early genes to varying extents in cortex, hippocampus and cerebellum, but not in striatum. The responses to 8-OH-DPAT peaked earlier than those to DOI. WAY 100135 (N-tertiobutyl-3-[4-(2-methoxyphenyl)-piperazinyl]-2-phenylpropana mide), the putative 5-HT1A receptor antagonist blocked the 8-OH-DPAT effect but not the responses to DOI. WAY 100135 by itself also elicited a relatively smaller genomic response. Ketanserin completely abolished the DOI-induced genomic responses. The results support the earlier findings that 5-HT1A receptor sites are abundant in frontal cortex and hippocampus. In addition, the robust genomic responses to 8-OH-DPAT as well as Northern hybridization with a cDNA probe for 5-HT1A mRNA in the cerebellum clearly implicate the functional expression of 5-HT1A receptors in this brain region. The responses to the 5-HT2 receptor agonist, DOI support a greater abundance of these receptors in the cortex, and relatively lower levels in hippocampus and cerebellum. The results suggest a differential induction pattern among the three immediate-early genes depending on the brain region and the 5-HT receptor subtype involved.

The expression of alpha 1 adrenoceptor subtypes changes with age in the rat aorta.

Previous studies showed that alpha 1 adrenoceptor-mediated contractile responses change with age in the rat aorta, becoming more sensitive to Ca++ channel blockers and less sensitive to chlorethylclonidine (CEC), suggesting a change in the alpha 1 adrenoceptor subtypes that are present. In this study, alpha 1 adrenoceptor density and alpha 1 adrenoceptor subtypes were measured in the Fischer 344 rat aorta during aging. Aortic alpha 1 adrenoceptor densities, determined by saturation binding of 2-[beta-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl] tetralone ([125I]-HEAT), were 47, 41 and 45 fmol/mg protein in 1-,6- and 24-month-old rats, respectively. The noncompetitive antagonist CEC completely blocked [125I]-HEAT binding in aortas from 1-month-old rats but inhibited binding only partially in aortas from older rats. Two binding sites were detected for norepinephrine and for WB4101 in all ages. The low-affinity constants for WB4101 (31-51 nM) were consistent with those for the alpha 1b adrenoceptor subtype, and this binding site decreased with age. The high-affinity constant for WB4101 (1.4 nM) in 1-month-old aorta was consistent with that for alpha 1d adrenoceptor subtype, whereas the high-affinity constants (0.03 nM) in 6- and 24-month-old aortas were consistent with those for the alpha 1a adrenoceptor subtype. At least three alpha 1 adrenoceptor subtypes appear to be colocalized in the rat aorta, so the binding affinities may reflect binding to more than one subtype. This makes it difficult to identify denfinitively the subtypes based on their radioligand binding characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic fluoxetine or desmethylimipramine treatment alters 5-HT2 receptor mediated c-fos gene expression.

These studies examined the effects of a 21-day treatment regime with either the tricyclic antidepressant, desmethylimipramine (DMI), or the selective 5-HT uptake inhibitor, fluoxetine, on 5-HT2 receptors in rat brain, as assessed by selective agonist-mediated c-fos gene expression. Chronic, but not acute, treatment with fluoxetine (10 mg/kg, i.p. for 21 days) resulted in supersensitization of the response to an acute challenge (4 mg/kg, i.p.) with the selective 5-HT2 agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI), both in frontal cortex and in hippocampus. Chronic treatment with DMI (10 mg/kg, i.p. for 21 days) resulted in a significant desensitization of the response to DOI. These findings are discussed in relation to the possible modes of action of these two clinically useful agents.

Cloning of cDNA for the glutamate-binding subunit of an NMDA receptor complex.

The amino acids L-glutamic and L-aspartic acids form the most widespread excitatory transmitter network in mammalian brain. The excitation produced by L-glutamic acid is important in the early development of the nervous system, synaptic plasticity and memory formation, seizures and neuronal degeneration. The receptors activated by L-glutamic acid are a target for therapeutic intervention in neurodegenerative diseases, brain ischaemia and epilepsy. There are two types of receptors for the excitatory amino acids, those that lead to the opening of cation-selective channels and those that activate phospholipase C (ref. 11). The receptors activating ion channels are NMDA (N-methyl-D-aspartate) and kainate/AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-sensitive receptors. The complementary DNAs for the kainate/AMPA receptor and for the metabotropic receptor have been cloned. We report here on the isolation and characterization of a protein complex of four major proteins that represents an intact complex of the NMDA receptor ion channel and on the cloning of the cDNA for one of the subunits of this receptor complex, the glutamate-binding protein.

Nucleotide sequence and transcript mapping of the HindIII F region of the Autographa californica nuclear polyhedrosis virus genome.

The organization of genes in the 2.95 kb EcoRI-SalI fragment (0.7 to 3.0 map units) located within the HindIII F region of the Autographa californica nuclear polyhedrosis virus genome was studied by a combination of DNA sequencing, Northern blot analysis, S1 mapping and primer extension analysis. In addition to the two divergent overlapping transcripts [leftward early (ES1) and rightward late (ES2)] previously reported, a third transcript which is present at 18 h p.i. and which also runs leftward, overlapping ES1 by 1600 nucleotides (nt) at the 3' end, was mapped to this region. The DNA sequence revealed the presence of three open reading frames (ORFs) of significant length. ORF-1 and ORF-2 correspond to the leftward transcripts, and code for potential polypeptides of 151 and 329 amino acids, respectively. ORF-3 which codes for a potential polypeptide of 167 amino acids is located on the opposite strand in a region for which no transcript mapping data are available. However, the conserved late gene promoter/cap site sequence (ATAAG) is present 23 nt upstream of the start of ORF-3.

Characterization of a cDNA encoding ricin E, a hybrid ricin-Ricinus communis agglutinin gene from the castor plant Ricinus communis.

Two classes of ricin cDNA clones have been identified and sequenced. The cDNA clone pBL-1 closely matches in nucleotide sequence the ricin genomic clone pAKG previously described by Halling et al., 1985 (Nucl. Acids Res. 13:8019). A second group of cDNA clones, represented by pBL-3, encode a hybrid protein (ricin E), having a ricin-like A chain and N-terminal half of the B chain and an RCA (Ricinus communis agglutinin)-like C-terminal half of the B chain.

Growth and carcass characteristics of cattle and buffalo breeds reared on a dry zone pasture in Sri Lanka (Ceylon).

Growth and carcass characteristics were measured on calves from three cattle breeds-the Sinhala, Red Sindhi and Friesian-and two buffalo breeds-the local buffalo and Murrah. The growth study was carried out on Brachiaria brizantha pasture over a period of 48 weeks. The two buffalo breeds grew fastest followed by the two Bos indicus breeds. The Friesian cattle grew at the slowest rate. The Sinhala and local buffalo had normal haematocrit values while the values for all other breeds were lower. The Sinhala and local buffalo had the highest carcass dressing percentages and the highest muscle; bone ratios. Tenderness measured by Warner-Bratzler shear force values and palatability scores by a taste panel were similar for meat from cattle and buffalo.