Autoradiographic Localization of Angiotensin II Receptor Binding Sites on Noradrenergic Neurons of the Locus Coeruleus of the Rat

The locus coeruleus (LC) of the rat was lesioned by microinjection of selective neurotoxins into the brainstem. 6-Hydroxydopamine (6-OHDA), 3 micrograms/microliter, given unilaterally at two sites 0.6 mm apart on the rostro-caudal axis of the LC, was used to lesion catecholamine-containing neuronal elements. Ibotenic acid, 2.5 micrograms/0.5 microliters, administered similarly was used to lesion nerve cell bodies. Two weeks after administration of the neurotoxin, lesion efficacy was determined based on the norepinephrine content of the cerebral cortex ipsi- and contralateral to the lesion. 6-OHDA lesions of the LC caused a 46% reduction in ipsilateral cortical norepinephrine and a 60% reduction in specific 125I-[Sar1, Ile8]-angiotensin II (125I-SIAII) binding in the LC. Ibotenic acid lesions of the LC caused a 73% reduction in ipsilateral cortical norepinephrine and a 81% reduction in specific 125I-SIAII binding in the LC. These results indicate that AII receptor binding sites in the LC are localized on noradrenergic nerve cell bodies or their dendritic and axonal ramifications within the LC.     

Diethylstilbestrol potentiates and testosterone antagonizes the action of 3-methylcholanthrene on benzo(a) pyrene metabolism in hep G2 cells

We have used the human hepatoma cell line, Hep G2, to examine the ability of hormones and xenobiotics to modulate the hepatic induction of benzo(a)pyrene hydroxylase and epoxide hydrolase. Hep G2 cells were cultured in Eagle's Minimum Essential Medium supplemented with 10% fetal calf serum. 3-Methylcholanthrene, diethylstilbestrol, testosterone propionate, and combinations of 3-meth-ylcholanthrene, and each of the hormones were added directly to the culture media. We subsequently studied the metabolism of benzo(a)pyrene using cell lysates of the Hep G2 cells. Metabolites were quantitated by high-performance liquid chromatography (HPLC) using fluorodetection. Exposure to 3-methyl-cholanthrene alone resulted in an eightfold increase in total benzo(a)pyrene metabolites with a change of the predominant metabolite from the 3-hydroxy-benzo(a)pyrene to the carcinogenic pathway of the benzo(a)pyrene-7,8-diol. Diethylstilbestrol and testosterone propionate resulted in small, but significant, decreases in metabolism of benzo(a)pyrene. When exposed in combination with 3-methyl-cholanthrene, testosterone propionate antagonized and diethylstilbestrol potentiated the metabolism of benzo(a)pyrene. 3-Methylcholanthrene, diethylstilbestrol, and combinations of 3-methylcholanthrene and diethylstilbestrol or testosterone propionate resulted in increased epoxide hydrolase activity as compared to controls. These results, carried out in a human hepatoma cell line, lend support to a concern for potentiated toxicity and carcinogenicity following exposure to complex chemical mixtures.     

G Protein-mediated Activation of a Nonspecific Cation Current in Cultured Rat Retinal Pigment Epithelial Cells

We used whole-cell patch-clamp recording techniques to investigate G protein-activated currents in cultured rat retinal pigment epithelial (RPE) cells. Using 140 mm KCl intracellular and 130 mm NaCl extracellular solutions, rat RPE cells possessed both inward and outward K⁺ currents. Upon addition of the nonhydrolyzable guanine triphosphate analogue, guanosine-5′-O-(3-thiophosphate) (GTPγS, 0.1 mm), to the recording electrode, a nonspecific cation (NSC) current was elicited. The NSC current had a mean reversal potential of +5.7 mV in 130 mm extracellular NaCl with Cs⁺-aspartate in the pipette, and was not affected by alterations in the extracellular Ca²⁺ or Cl⁻ concentration. The GTPγS-activated current was found to be permeable to several monovalent cations (K⁺, Na⁺, choline, TRIS, and NMDG). Addition of fluoroaluminate, an activator of large molecular weight heterotrimeric GTP-binding proteins (G proteins), to the intracellular recording solution activated the NSC current. The G protein involved was pertussis toxin (PTX)-sensitive, since GTPγS failed to activate the NSC current in cells pretreated with PTX. Further investigation of second messenger molecules suggested that activation of the NSC current was not affected by alterations in intracellular Ca²⁺ or ATP. From these results, we conclude that a G protein-regulated NSC current is present in rat RPE cells. Activation of the NSC current may sufficiently depolarize RPE cells to activate outward K⁺ currents. This would provide a mechanism by which these cells could rid themselves of accumulated K⁺. Received: 25 January 1996/Revised: 24 April 1996     

A Ca2+-activated Whole-Cell Cl− Conductance In Human Placental Cytotrophoblast Cells Activated Via a G Protein

Whole-cell patch clamp experiments were performed on cultured human cytotrophoblast cells incubated for 24–48 hr after their isolation from term placentas. Cl⁻-selective currents were examined using K⁺-free solutions. Under nonstimulated conditions, most cells initially expressed only small background leak currents. However, inclusion of 0.2 mm GTPγS in the electrode solution caused activation of an outwardly rectifying conductance which showed marked time-dependent activation at depolarized potentials above +20 mV. Stimulation of this conductance by GTPγS was found to be Ca²⁺-dependent since GTPγS failed to activate currents when included in a Ca²⁺-free electrode solution. In addition, similar currents could be activated by increasing the [Ca²⁺] of the pipette solution to 500 nm. The Ca²⁺-activated conductance was judged to be Cl⁻-selective, since reversal potentials were predicted by Nernst equilibrium potentials for Cl⁻. This conductance could also be reversibly inhibited by addition of the anion channel blocker DIDS to the bath solution at a dose of 100 μm. Preliminary experiments indicated the presence of a second whole-cell anion conductance in human cytotrophoblast cells, which may be activated by cell swelling. Possible roles for the Ca²⁺-activated Cl⁻ conductance in human placental trophoblast are discussed. Received: 9 November 1995/Revised: 18 January 1996     

Nucleotide sequence and presumed secondary structure of the 28S rRNA of pea aphid implication for diversification of insect rRNA

Determination of the entire nucleotide sequence of the aphid 28S ribosomal RNA gene (28S rDNA) revealed that it is 4,147 by in length with a G + C content of 60.3%. Based on the nucleotide sequence, we constructed a presumed secondary-structure model of the aphid 28S rRNA which indicated that the aphid 28S rRNA is characterized by the length and high G + C content of its variable regions. The G + C content of the aphid's variable regions was much higher than that of the entire sequence of the 28S rRNA, which formed a striking contrast to those ofDrosophila with the G + C content much lower than the entire 28S molecule. In this respect, the aphid 28S rRNA somewhat resembled those of vertebrates. This is the third report of a complete large-subunit rRNA sequence from an arthropod, and the first 28S rRNA sequence for a nondipterous insect. Correspondence to: H. Ishikawa     

The C-Terminal Domain of the mGluR1 Metabotropic Glutamate Receptor Affects Sensitivity to Agonists

Abstract: The metabotropic glutamate receptor (mGluR) subtype 1 exists as at least three variants (−1a, −1b, and −1c) generated by alternative splicing at the C-terminal domain. Fluorometric Ca²⁺ measurements were used to compare the concentration dependency of agonist-induced rises in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in human embryonic HEK 293 cells transiently expressing rat mGluR1a, mGluR1b, or mGluR1c. The rank order of agonist potencies was quisqualate ≫ (2 S, 1' S, 2' S )-2-(carboxycyclopropyl)glycine (L-CCG-I) > (1 S, 3 R )-1-aminocyclopentane-1,3-dicarboxylic acid [(1 S, 3 R )-ACPD] and did not differ among the splice variants. However, agonists were consistently more potent at mGluR1a than at mGluR1c and mGluR1b. In the same system, we characterized the agonist pharmacology of two chimeric rat mGluR3/1 receptors where the first and/or the second intracellular loop(s) and the C-terminal domain were exchanged with the corresponding mGluR1a or mGluR1c sequences and that were previously shown to mediate elevations in [Ca²⁺]_i in response to agonists. The potency of agonists was higher at the chimera having the C-terminus of mGluR1a as compared with those having the mGluR1c C-terminus. Both chimeric mGluR3/1 receptors had the same rank order of agonist potencies: L-CCG-I ≫ (1 S, 3 R )-ACPD ∼ quisqualate. These data support the hypothesis that the C-terminal domain of mGluRs plays a role in determining the potency of agonists for inducing mGluR-mediated functional responses.     

The fate of immunoglobulin G fed to larvae of Ostrinia nubilalis

Since plants can be transformed genetically to produce functional antibodies, an immunological approach may be developed for controlling their arthropod pests. Specific antibodies would protect plants from arthropods if they could gain access to the pest antigen in sufficient amounts such that the normal function of the antigen is disrupted. In order to study the fate of ingested antibodies in the body of the European corn borer (ECB), Ostrinia nubilalis (Hübner), (Lepidoptera: Pyralidae), we fed the larvae on serum-containing diet. When larvae were fed on the serum-containing diet for various lengths of time between 12 and 96 h, no significant differences were noted in the immunoglobulin G (IgG) concentration in their body. Immediately after the larvae stopped feeding, the concentrations of the IgG in their midgut was about one half that of the diet itself, but it decreased significantly after 6 h and again after 18 h (about 3 and 10 fold, respectively). Immediately after the larvae stopped feeding, the concentration of the IgG in their hemolymph was about 1/500 that in the diet. The concentration of IgG in the hemolymph of ECB larvae was influenced directly by the titer of antibodies in their diet. During the first 6 h after the larvae stop feeding the concentration of IgG in their hemolymph did not decrease significantly; however, it did so after 18 h (about 6 fold). The possibility that specific antibodies will gain access to antigens in the ECB body is discussed.     

Long-Term Effect of Forskolin on the Activation of Adenylyl Cyclase in Astrocytes

Abstract: Long-term (48-h) forskolin treatment of rat astroglial cells led to a slight decrease (30–40%) in the response to isoproterenol, vasoactive-intestinal peptide, guanyl 5'-(βγ-imido)diphosphate, guanosine 5'- O -(3-thiotriphosphate) [GTP(S)], and AIF₄⁻ in crude membrane fractions. In contrast, the acute stimulatory effect of forskolin was increased by 1.25–1.5-fold. These two opposite effects of forskolin were mediated by a cyclic AMP-dependent mechanism. No changes in G_sα, G_iα, or Gβ protein levels could be determined by immunoblotting using specific antisera. No significant differences were observed in the ability of G proteins extracted from control and forskolin-treated cells to reconstitute a full adenylyl cyclase activity in membranes from S49 cyc⁻ cells, lacking G_sα protein. G_sα proteins were detected in two pools of membranes, one in the heavy sucrose fractions and the other in light sucrose fractions. Forskolin treatment of the cells shifted G_sα protein toward the light-density membranes. We did not find any significant change in the distribution of adenylyl cyclase. In contrast to the decreased stimulation of adenylyl cyclase activity by agonists acting via G_sα, observed in the crude membrane fraction, the responses of adenylyl cyclase to forskolin as well as to GTP(S) were increased in the purified plasma membrane fractions. These results may indicate that sensitization of the catalyst appears to be the dominant component in the astroglial cell response to long-term treatment by forskolin.