Calcitonin gene-related peptide and its receptor in the thymus

Calcitonin gene-related peptide (CGRP), a 37-amino acid residue neuropeptide, was immunostained in rat thymus at two sites: a subpopulation of thymic epithelial cells, namely subcapsular/perivascular cells, were heavily stained besides some nerve fibers surrounding arteries and arterioles. The administration of nanomolar concentrations of rat -CGRP dose-dependently raised intracellular cyclic adenosine monophosphate (cAMP) levels in isolated rat thymocytes (half-maximum stimulation 1 nM) but not in cultured rat thymic epithelial cells. Peptides structurally related to CGRP (i.e., rat calcitonin or amylin) had no effect. CGRP(8–37), an N-terminally truncated form, acted as an antagonist. Peripheral blood lymphocytes did not respond to CGRP, suggesting that receptors are present only on a subpopulation of thymocytes but not on mature T cells. This was substantiated by visualization of CGRP receptors on single cells by use of CGRP-gold and -biotin conjugates of established biological activity: only a small proportion of isolated thymocytes was surface labeled. In situ, the CGRP conjugates labeled receptors on large thymocytes residing in the outer cortical region of rat thymus pseudolobules. Thus, immunoreactive CGRP is found in subcapsular/perivascular thymic epithelial cells and acts via specific CGRP receptors on thymocytes by raising their intracellular cAMP level. It is suggested that CGRP is a paracrine thymic mediator that might influence the differentiation, maturation, and proliferation of thymocytes.     

巴氏碳球C_(60)对体外培养HeLa细胞的光敏杀伤效应

采用MTT比色分析法,观察了不同C_(60)浓度和不同光照强度下C_(60)对体外培养的HeLa细胞的光敏杀伤效应。结果表明,C_(60)在30μg/ml,光强4000Lux的条件下即可杀伤大部分细胞。受伤细胞圆缩、脱壁,里面颗粒增多,失去表面微绒毛状结构。当光强增大时,细胞表面甚至出现破损。     

Sodium currents in toad cardiac pacemaker cells

Cells in the pacemaker region of toad (Bufo marinus) sinus venosus had spontaneous rhythmic action potentials. The rate of firing of action potentials, the rate of diastolic depolarization and the maximum rate of rise of action potentials were reduced by TTX (10 nm to 1 m). Currents were recorded with the whole cell, tight seal technique from cells enzymatically dissociated from this region. Cells studied were identified as pacemaker cells by their characteristic morphology, spontaneous rhythmic action potential activity that could be blocked by cobalt but not by TTX and lack of inward rectification. When calcium, potassium and nonselective cation currents (I_f) activated by hyperpolarization were blocked, depolarization was seen to generate transient and persistent inward currents. Both were sodium currents: they were abolished by tetrodotoxin (10 to 100 nm), their reversal potential was close to the sodium equilibrium potential and their amplitude and reversal potential were influenced as expected for sodium currents when extracellular sodium ions were replaced with choline ions. The transient sodium current was activated at potentials more positive than –40 mV while the persistent sodium current was obvious at more negative potentials. It was concluded that, in toad pacemaker cells, TTX-sensitive sodium currents contributing both to the upstroke of action potentials and to diastolic depolarization may play an important role in setting heart rate.We thank the Australian National Heart Foundation for their support. D.A.S. is an NHMRC Senior Research Officer.     

Sodium channels in cultured neuroblastoma cells grown in high glucose or l-Fucose

Patch clamp techniques were used to record whole cell and single channel Na⁺ currents from NB41A3 neuroblastoma cells grown in culture. Cells were grown for two weeks in control medium or medium supplemented with 30 mm d-glucose of 30 mm l-fucose.Cells exposed to glucose or l-fucose had smaller whole cell Na⁺ currents than cells grown in unsupplemented medium, consistent with earlier studies (Yorek, Stefani & Wachtel, 1994). Whole cell macroscopic currents showed no change in activation or inactivation kinetics. Single channel current properties and opening probability were also unchanged.The number of [³H]saxitoxin binding sites, and therefore the total number of Na⁺ channels, was not reduced in cells grown in glucose or l-fucose (Yorek et al., 1994). Therefore, we conclude that some of the channels must have been rendered nonfunctional by the conditioning media. The finding that single channel properties are not altered suggests that channels become nonfunctional in an all-or-none manner.This work was supported by Merit Review Awards to M.A. Yorek and R.E. Wachtel from the Department of Veterans Affairs and by National Institutes of Health grant DK45453 to M.A. Yorek.     

Repetitive DNA sequences located in the central region of the human mdr1 (multidrug resistance) gene may account for a gene fusion event during its evolution

The mdr1 gene, first member of the human multidrug-resistance gene family, is a major gene involved in cellular resistance to several drugs used in anticancer chemotherapy. Its product, the drug-excreting P-glycoprotein, shows a bipartite structure formed by two similar adjacent halves. According to one hypothesis, the fusion of two related ancestral genes during evolution could have resulted in this structure. The DNA sequence analysis of the introns located in the region connecting the two halves of the human mdr1 gene revealed a highly conserved poly(CA) · poly (TG) sequence in intron 15 and repeated sequences of the Alu family in introns 14 and 17. These repeated sequences most likely represent molecular fossils of ancient DNA elements which were involved in such a recombination event. Correspondence to: M. Pauly     

Pertussis Toxin-Sensitive G Protein α-Subunits: Production of Monoclonal Antibodies and Detection of Differential Increases on Differentiation of PC12 and LA-N-5 Cells

Abstract: Monoclonal antibodies were produced that are specific for the three major pertussis toxin-sensitive G protein α-subunits present in mammalian brain—αo, αi1, and αi2—using purified bovine brain G proteins, purified rat brain G proteins, and purified recombinant αi2, respectively. These monoclonal antibodies were used to monitor changes in the concentrations of the three G protein α-subunits during differentiation of PC12 cells and human neuroblastoma LA-N-5 cells. In PC12 cells, levels of αi1 but not αi2 increased during nerve growth factor-induced differentiation. In contrast, αi2 but not αi1 increased when LA-N-5 cells were differentiated with retinoic acid. The concentration of αo increased in both cell lines during differentiation. Electrophoretic resolution of αo subtypes revealed that although αo2 was the major subtype in undifferentiated cells, only the concentration of αo1 increased during differentiation of both PC12 and LA-N-5 cells. The level of 43-kDa growth-associated protein, a protein known to associate with αo, increased similarly to that of αo1. ADP-ribosylation of αo, αi1, and αi2 with pertussis toxin did not alter the reactivities of the monoclonal antibodies, but toxin treatment of cells reduced the concentrations of each protein after 24 h. There was no change in the concentration of αq, which is not ADP-ribosylated by pertussis toxin. Thus, these new monoclonal antibodies enabled the detection of differential increases in subtypes of αi and αo associated with neuronal differentiation.     

The Distribution and Biochemical Properties of a Cdc2-Related Kinase, KKIALRE, in Normal and Alzheimer Brains

Abstract: The biochemical properties and distribution of a Cdc2-related kinase, KKIALRE, were studied in brain tissues and cultured cells with antibodies to a subregion of KKIALRE protein deduced from cDNA. In adult human brain, the KKIALRE-immunoreactive protein consisted of four or five isoforms having a molecular size of 40–52 kDa, whereas in fetal brain, there was one protein of ∼48 kDa. Cultured astrocytes, neuroblastoma cells, and mouse brains contained the fetal form of KKIALRE protein. KKIALRE-immunoreactive proteins were capable of phosphorylating histone and synthetic peptides with the X-Ser-Pro-X motif, indicating that these proteins belong to the proline-directed Ser/Thr protein kinase family. The KKIALRE immunoreactivity was detected primarily in fibrous astrocytes in white matter and perivascular and subpial spaces, as well as in Bergmann glia in the cerebellum. In fetal brains radial glia were weakly immunoreactive. Reactive astrocytes were more intensely labeled than other glia. Neurons in normal brains and brains with Alzheimer's disease (AD) displayed no KKIALRE immunoreactivity. KKIALRE immunoreactivity was similar in neurons with and without neurofibrillary tangles. The results indicate that in CNS, the KKIALRE protein is mainly a glial protein that is up-regulated in gliosis and that it probably plays no role in the hyperphosphorylation of τ in AD brains.     

Regulation of Phenylethanolamine N-Methyltransferase Gene Expression by Imidazoline Receptors in Adrenal Chromaffin Cells

Abstract: As adrenal medullary chromaffin cells express imidazoline binding sites in the absence of α₂-adrenergic receptors, these cells provide an ideal system in which to determine whether imidazolines can influence catecholamine gene expression through nonadrenergic receptors. This study evaluates the ability of clonidine and related drugs to regulate expression of the gene for the epinephrine-synthesizing enzyme phenylethanolamine N -methyltransferase (PNMT) in the rat adrenal gland and in bovine adrenal chromaffin cell cultures. In vivo, PNMT and tyrosine hydroxylase (TH) mRNA levels increase in rat adrenal medulla after a single injection of clonidine. Clonidine also dose-dependently stimulates PNMT mRNA expression in vitro in primary cultures of bovine chromaffin cells, with a threshold dose of 0.1 μ M . Other putative imidazoline receptor agonists, including cimetidine, rilmenidine, and imidazole-4-acetic acid, likewise enhance PNMT mRNA production showing relative potencies that correlate with their binding affinities at chromaffin cell I₁-imidazoline binding sites. The effects of clonidine on PNMT mRNA appear to be distinct from and additive with those exerted by nicotine. Moreover, neither nicotinic antagonists nor calcium channel blockers, which attenuate nicotine's influence on PNMT mRNA production, diminish clonidine's effects on PNMT mRNA. Although 100 μ M clonidine diminishes nicotine-stimulated release of epinephrine and norepinephrine in chromaffin cells, this effect appears unrelated to stimulation of imidazoline receptor subtypes. This is the first report to link imidazoline receptors to neurotransmitter gene expression.     

Differential Calcitonin Gene-Related Peptide (CGRP) and Amylin Binding Sites in Nucleus Accumbens and Lung: Potential Models for Studying CGRP/Amylin Receptor Subtypes

Abstract: Calcitonin gene-related peptide (CGRP), a 37-amino-acid peptide, is a member of a small family of peptides including amylin or islet amyloid polypeptide and salmon calcitonin. These related peptides have been shown to display similar effects on in vitro and in vivo carbohydrate metabolism. The present study was initiated to identify and characterize the binding sites for these peptides in lung and nucleus accumbens membranes prepared from pig and guinea pig. Both tissues in either species displayed high-affinity (2-[¹²⁵I]iodohistidyl¹⁰)humanCGRPα ([¹²⁵I]hCGRPα) binding (IC₅₀ = 0.4–7.7 nM), which was displaced by hCGRP_8–37α with equally high affinity (IC₅₀ = 0.4–7.3 nM). High-affinity binding for [¹²⁵I]Bolton-Hunter human amylin ([¹²⁵I]BH-h-amylin) was also observed in these tissues (IC₅₀ = 0.2–6.0 nM). In membranes from the nucleus accumbens of both species, salmon calcitonin competed for amylin binding sites with high affinity (IC₅₀ = 0.1 nM) but was poor in competing for amylin binding in lung membranes. Rat amylin_8–37 competed for [¹²⁵I]hCGRPα binding with higher affinity (IC₅₀ = 5.4 nM) compared with [¹²⁵I]BH-h-amylin binding (IC₅₀ = 200 nM) in porcine nucleus accumbens, whereas in guinea pig nucleus accumbens, the IC₅₀ values for rat amylin_8–37 were 117 and 12 nM against [¹²⁵I]hCGRPα and [¹²⁵I]BH-h-amylin, respectively. Also, functional studies evaluating the activation of adenylate cyclase and generation of cyclic AMP in response to these agonists indicated that hCGRPα (EC₅₀ = 0.3 nM), h-amylin (EC₅₀ = 150 nM), and salmon calcitonin (EC₅₀ = 1,000 nM) activated adenylate cyclase, resulting in increased cyclic AMP production in porcine lung membranes that was antagonized by hCGRP_8–37α. The affinity of hCGRP_8–37α was similar for all three peptides. The cyclic AMP responses to amylin and salmon calcitonin were significantly (p < 0.05) lower than that of hCGRPα and not additive, suggesting that they are acting as partial agonists at the same CGRP1-type receptor in porcine lung membranes. Similar observations were made for guinea pig lung membranes. However, human amylin and salmon calcitonin were weaker than hCGRPα in activating lung adenylate cyclase. None of these peptides activated adenylate cyclase in membranes prepared from the nucleus accumbens of both species. The data from these studies demonstrate both species and tissue differences in the existence of distinct CGRP and amylin binding sites and present a potential opportunity to study further CGRP and amylin receptor subtypes.     

Dopamine D4-Like Receptor Elevation in Schizophrenia: Cloned D2 and D4 Receptors Cannot Be Discriminated by Raclopride Competition Against [3H]Nemonapride

Abstract: Three independent studies have found that the density of dopamine D4-like receptors is elevated in postmortem brain striata in schizophrenia. This elevation has been questioned by a fourth study that used a different method and failed to detect a biphasic component when raclopride was used to compete against the binding of 1 n M [³H]nemonapride to schizophrenia tissue. To test whether this competition method could distinguish between dopamine D2 and D4 receptors, the present study used mixtures of only these two cloned receptors, free of all other receptors. Using combinations of cloned dopamine D2 and D4 receptors, this competition method could not resolve these components up to a level of 48% D4 receptors. Thus, the objections raised by the findings of the fourth study, mentioned above, do not appear valid. Furthermore, the present results indicate that the data using such a competition method actually mask a manyfold marked elevation in the density of dopamine D4-like receptors in schizophrenia.