Characterization of the Glutamate Receptors Mediating Release of Somatostatin from Cultured Hippocampal Neurons

Abstract: l -Glutamate, NMDA, dl -α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin-like immunoreactivity (SRIF-LI) from primary cultures of rat hippocampal neurons. In Mg²⁺-containing medium, the maximal effects (reached at ∼100 µ M ) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 µ M (AMPA), 39 µ M (glutamate), 41 µ M (KA), and 70 µ M (NMDA). The metabotropic receptor agonist trans -1-aminocyclopentane-1,3-dicarboxylate did not affect SRIF-LI release. The release evoked by glutamate (100 µ M ) was abolished by 10 µ M dizocilpine (MK-801) plus 30 µ M 1-aminophenyl-4-methyl-7,8-methylenedioxy-5 H -2,3-benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA + AMPA. The release elicited by NMDA was sensitive to MK-801 but insensitive to GYKI 52466. The AMPA- and KA-evoked releases were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) or by GYKI 52466 but were insensitive to MK-801. The release of SRIF-LI elicited by all four agonists was Ca²⁺ dependent, whereas only the NMDA-evoked release was prevented by tetrodotoxin. Removal of Mg²⁺ caused increase of basal SRIF-LI release, an effect abolished by MK-801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved.     

Immunological Characterization and Transmembrane Topology of 5-Hydroxytryptamine3 Receptors by Functional Epitope Tagging

Abstract: 5-Hydroxytryptamine₃ (5-HT₃) receptors are the only known monoamine receptors mediating fast excitatory responses in mammalian neurons. Their primary structure as well as their electrophysiological and pharmacological properties show a phylogenetic relation to nicotinic acetylcholine, GABA_A, and glycine receptors. As a prototypical member of this gene superfamily, we investigated the membrane topology of functional homomeric 5-HT₃ receptors by using epitope tagging of the channel subunits expressed in heterologous systems. Visualization of 5-HT₃ receptors in transfected COS-7 cells, either in western blot (molecular mass 61.2 ± 0.8 kDa) or in situ, was performed with previously characterized antibodies recognizing artificial epitopes as well as with anti-fusion protein antibodies directed against a wild-type receptor intracellular domain. The extracellular location of the distal C-terminal tagged domain demonstrates the presence of a fourth transmembrane domain in 5-HT₃ serotonin-gated channels. In this region, the significant homology between members of this class of neurotransmitter-gated channels suggests strongly that they have a common transmembrane organization basically different from glutamate-gated and ATP-gated channels.     

Desensitization of the Neurokinin 1 Receptor Is Mediated by the Receptor Carboxy-Terminal Region, but Is Not Caused by Receptor Internalization

Abstract: The carboxy-terminal cytoplasmic regions of the rat neurokinin 1 (substance P) and neurokinin 2 (neurokinin A) receptors have been exchanged to determine if this region of the neurokinin 1 receptor is involved in its desensitization. When expressed at similar levels in stably transfected Chinese hamster ovary (CHO) cell lines, receptors containing the carboxy-terminal region of the neurokinin 1 receptor desensitized significantly more (as measured by reduction of the inositol 1,4,5-trisphosphate response) when preexposed for 1 min to 1 µ M neurokinin, indicating a role for the carboxy-terminal region of the neurokinin 1 receptor in its desensitization. Measurement of receptor internalization using radiolabeled neurokinins (0.3 n M ) indicated that ∼75–80% of the receptors were internalized in each cell line after 10 min at 37°C, with no observable correlation between neurokinin receptor desensitization and internalization. Measurement of loss of receptor surface sites for cell lines CHO NK1 and CHO NK1NK2 following exposure to 1 µ M substance P also indicated no obvious relationship between the percent desensitization and percent of receptors internalized. Also, two inhibitors of neurokinin 1 receptor internalization, phenylarsine oxide and hyperosmolar sucrose, did not inhibit neurokinin 1 receptor desensitization. The protein kinase inhibitors Ro 31-8220, staurosporine, and Zn²⁺ had no effect on neurokinin 1 receptor desensitization, indicating that the kinases affected by these agents are not rate-limiting in neurokinin 1 receptor desensitization in this system.     

Differential Inhibition of Secretagogue-Stimulated Sodium Uptake in Adrenal Chromaffin Cells by Activation of D4 and D5 Dopamine Receptors

Abstract: Recent studies have demonstrated that D1-selective and D2-selective dopamine receptor agonists inhibit catecholamine secretion and Ca²⁺ uptake into bovine adrenal chromaffin cells by receptor subtypes that we have identified by PCR as D5, a member of the D1-like dopamine receptor subfamily, and D4, a member of the D2-like dopamine receptor subfamily. The purpose of this study was to determine whether activation of D5 or D4 receptors inhibits influx of Na⁺, which could explain inhibition of secretion and Ca²⁺ uptake by dopamine agonists. D1-selective agonists preferentially inhibited both dimethylphenylpiperazinium- (DMPP) and veratridine-stimulated ²²Na⁺ influx into chromaffin cells. The D1-selective agonists chloro-APB hydrobromide (CI-APB; 100 µ M ) and SKF-38393 (100 µ M ) inhibited DMPP-stimulated Na⁺ uptake by 87.5 ± 2.3 and 59.7 ± 4.5%, respectively, whereas the D2-selective agonist bromocriptine (100 µ M ) inhibited Na⁺ uptake by only 22.9 ± 5.0%. Veratridine-stimulated Na⁺ uptake was inhibited 95.1 ± 3.2 and 25.7 ± 4.7% by 100 µ M CI-APB or bromocriptine, respectively. The effect of CI-APB was concentration dependent. A similar IC₅₀ (∼18 µ M ) for inhibition of both DMPP- and veratridine-stimulated Na⁺ uptake was obtained. The addition of 8-bromo-cyclic AMP (1 m M ) had no effect on either DMPP- or veratridine-stimulated Na⁺ uptake. These observations suggest that D1-selective agonists are inhibiting secretagogue-stimulated Na⁺ uptake in a cyclic AMP-independent manner.     

Nicotinic Autoreceptors Mediating Enhancement of Acetylcholine Release Become Operative in Conditions of "Impaired" Cholinergic Presynaptic Function

Abstract: The existence in the mammalian CNS of release-inhibiting muscarinic autoreceptors is well established. In contrast, few reports have focused on nicotinic autoreceptors mediating enhancement of acetylcholine (ACh) release. Moreover, it is unclear under what conditions the function of one type of autoreceptor prevails over that of the other. Rat cerebrocortex slices, prelabeled with [³H]choline, were stimulated electrically at 3 or 0.1 Hz. The release of [³H]ACh evoked at both frequencies was inhibited by oxotremorine, a muscarinic receptor agonist, and stimulated by atropine, a muscarinic antagonist. Nicotine, ineffective at 3 Hz, enhanced [³H]ACh release at 0.1 Hz; mecamylamine, a nicotinic antagonist, had no effect at 3 Hz but inhibited [³H]ACh release at 0.1 Hz. The cholinesterase inhibitor neostigmine decreased [³H]ACh release at 3 Hz but not at 0.1 Hz; in the presence of atropine, neostigmine potentiated [³H]ACh release, an effect blocked by mecamylamine. In synaptosomes depolarized with 15 mM KCI, ACh inhibited [³H]ACh release; this inhibition was reversed to an enhancement when the external [Ca²⁺] was lowered. The same occurred when, at 1.2 mM Ca²⁺, external [K⁺] was decreased. Oxotremorine still inhibited [³H]ACh release at 0.1 mM Ca²⁺. When muscarinic receptors were inactivated with atropine, the K⁺ (15 mM)-evoked release of [³H]ACh (at 0.1 mM Ca²⁺) was potently enhanced by ACh acting at nicotinic receptors (EC₅₀? 0.6 µM). In conclusion, synaptic ACh concentration does not seem to determine whether muscarinic or nicotinic autoreceptors are activated. Although muscarinic autoreceptors prevail under normal conditions, nicotinic autoreceptors appear to become responsive to endogenous ACh and to exogenous nicotinic agents under conditions mimicking impairment of ACh release. Our data may explain in part the reported efficacy of cholinesterase inhibitors (and nicotinic agonists) in Alzheimer's disease.     

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.     

Developmental and Regional Expression of NMDA Receptor Subtypes Containing the NR2D Subunit in Rat Brain

Abstract: The regional and developmental expression of NMDA receptors containing the NR2D subunit was analyzed on the level of the subunit mRNA and protein in rat brain. RNase protection experiments indicated that among two proposed splice variants of the NR2D subunit, only the NR2D-2 subunit is expressed. The regional distribution of the NR2D subunit protein was visualized with a newly developed NR2D-2 subunit-specific antiserum on brain sections using the histoblot technique. In adult brain, NR2D immunoreactivity was mainly restricted to diencephalic, mesencephalic, and brainstem structures. During postnatal development, the NR2D subunit was detected transiently in certain regions, such as the ventro-basal complex of the thalamus, hippocampus, inferior colliculus, and brainstem reticular formation, suggesting that NR2D subunit-containing receptors play a role in these brain areas only during development. The level of NR2D subunit mRNA and protein decreased during late postnatal development. However, significant levels of NR2D subunit mRNA and protein were present in adulthood, in particular, in the globus pallidus, thalamus, subthalamic nuclei, and superior colliculus. These results indicate a functional relevance for NMDA receptors containing the NR2D subunit in the developing and adult brain, although its expression in the adult brain is less prominent and restricted to a few brain areas.     

Opioid receptors: Some perspectives from early studies of their role in normal physiology,stress responsivity,and in specific addictive diseases

The early history of research on the possible existence of specific opioid receptors and on developing a new form of pharmacotherapy for the treatment of heroin addiction in New York City, from 1960–1973, along with the special relationships between two leading scientists conducting these research efforts, Dr. Eric Simon and Dr. Vincent P. Dole Jr., are presented in a historical perspective. The linkage of these early efforts and the subsequent identification and the elucidation of the effects of exogenous opiates acting at specific opiate receptors in human physiology, including some findings from perspective studies of heroin addicts at time of entry to and during methadone maintenance treatment, are presented in the context of the important clues which thereby were provided concerning the possible roles of the endogenous opioids in normal mammalian physiology. From many of these early clinical research findings and studies in animal models, the hypothesis that the endogenous opioids system may play an important role in stress responsivity was formulated along with the related hypothesis, first presented in the early 1970s, that an atypical responsivity to stress and stressors might be involved in the acquisition and persistence of, and relapse to specific addictive diseases, including heroin addiction, cocaine dependency and alcoholism. More recent studies of the possible involvement of the specific opioid receptors in these three addictive diseases—heroin addiction, cocaine addiction and alcoholism—from our laboratory are discussed in a historical perspective of the development of these ideas from the early research findings of not only Dr. Eric Simon, but his numerous colleagues in opioid research in the United States and throughout the world. Special issue dedicated to Dr. Eric J. Simon.