Localization of angiotensin-converting enzyme, angiotensin II, angiotensin II receptor subtypes, and vasopressin in the mouse hypothalamus

The hypothalamic angiotensin II (Ang II) system plays an important role in pituitary hormone release. Little is known about this system in the mouse brain. We studied the distribution of angiotensin-converting-enzyme (ACE), Ang II, Ang II receptor subtypes, and vasopressin in the hypothalamus of adult male mice. Autoradiography of binding of the ACE inhibitor [¹²⁵I]351A revealed low levels of ACE throughout the hypothalamus. Ang II- and vasopressin-immunoreactive neurons and fibers were detected in the paraventricular, accessory magnocellulary, and supraoptic nuclei, in the retrochiasmatic part of the supraoptic nucleus and in the median eminence. Autoradiography of Ang II receptors was performed using [¹²⁵I]Sar¹–Ang II binding. Ang II receptors were present in the paraventricular, suprachiasmatic, arcuate and dorsomedial nuclei, and in the median eminence. In all areas [¹²⁵I]Sar¹–Ang II binding was displaced by the AT₁ receptor antagonist losartan, indicating the presence of AT₁ receptors. In the paraventricular nucleus [¹²⁵I]Sar¹–Ang II binding was displaced by Ang II (K_i=7.6×10⁻⁹) and losartan (K_i=1.4×10⁻⁷) but also by the AT₂ receptor ligand PD 123319 (K_i=5.0×10⁻⁷). In addition, a low amount of AT₂ receptor binding was detected in the paraventricular nucleus using [¹²⁵I]CGP 42112 as radioligand, and the binding was displaced by Ang II (K_i=2.4×10⁻⁹), CGP 42112 (K_i=7.9×10⁻¹⁰), and PD 123319 (K_i=2.2×10⁻⁷). ACE, Ang II, and AT₁ as well as AT₂ receptor subtypes are present in the mouse hypothalamus. Our data are the basis for further studies on the mouse brain Ang II system.     

Discrete regional distributions of thyrotropin releasing hormone (TRH) receptor binding in monkey central nervous system

Thyrotropin releasing hormone (TRH) directly influences central nervous system (CNS) function, independent of its pituitary action. Although these CNS effects have been behaviorally characterized, information is not yet available on the precise regional distribution of its receptor. TRH receptor binding was examined in the monkey CNS by the radioreceptor assay for clarifying the site of TRH action. TRH was bound to brain tissue membranes via high-affinity (K_d = 5.9 × 10⁻⁹M) and low-affinity (K_d = 11.2 × 10⁻⁸M) components. TRH receptor binding varied dramatically throughout the monkey brain, with more than a 40-fold variation. The limbic system contained the greatest amount of binding. The next highest areas were the cerebral cortex, hypothalamus, interpeduncular nucleus and periaqueductal gray matter of the midbrain. Receptor binding was very low or not detectable in the medial thalamus, cerebellum, brain stem, spinal cord and white matter. These data suggest that TRH has an effect on the CNS via limbic system, cerebral cortex and midbrain.     

Temperature-dependent effect of zolpidem on the GABAA receptor-mediated response at recombinant human GABAA receptor subtypes

The effects of zolpidem on the two forms of recombinant human GABA_A receptors (α1β2γ2s and α3β2γ2s) at different temperatures were functionally investigated, using the whole-cell patch recording configuration. In both forms, zolpidem potentiated the response to GABA in a concentration-dependent manner. At 16°C, the apparent dissociation constant (K_D) values for the α1β2γ2s and α3β2γ2s forms were 3.7×10⁻⁸ and 5.6×10⁻⁷ M, respectively. When the temperature was increased to 36°C, the K_D values for the α1β2γ2s and α3β2γ2s forms were 2.1×10⁻⁷ and 1.5×10⁻⁶ M, respectively. Although the affinity ratio was reduced from 15.1 to 7.1-fold the selectivity of zolpidem for the α1β2γ2s still remained at 36°C.     

Analysis of regional variations in the affinities of muscarinic agonists in the rat brain

The brain stem of the rat has a higher affinity toward muscarinic agonists than does the forebrain. Receptor occupancy curves of both regions of the brain deviate from simple mass-action binding. The characteristics of the binding in each region are compatible with the existence of two non-interacting binding sites, and are not attributable to desensitization or to negatively cooperative binding within a small oligomer; however, the possibility of large oligomers remains to be excluded. The agonist binding data were analyzed by a linear transformation of Scatchard-like inhibition curves of the binding of the antagonist [³H]quinuclidinyl benzilate (QNB). Such analysis, based on a model of two subpopulations of receptors in each area, shows the subpopulations of the brain stem and the forebrain to be distinct. Brain stem: 44% of receptors possess high affinity with dissociation constant for carbachol,K_H = 2.8 × 10⁻⁸M, dissociation constant of low-affinity receptor,K_L = 2.3 × 10⁻⁶M; forebrain: 41% high affinity,K_H = 2.1 × 10⁻⁷M, K_L = 1.7 × 10⁻⁵M. The data suggest that whole brain contains at least three major muscarinic receptors, which can be distinguished on the basis of their affinities for agonists.     

α-Bungarotoxin-acetylcholine receptors in the chick ciliary ganglion: Effects of deafferentation and axotomy

α-Bungarotoxin (α-BuTX) binds in a saturable and practically irreversible fashion to membrane-associated receptors in the ciliary ganglion of the adult chick. The binding of toxin to receptors is competitively inhibited by nicotinic cholinergic ligands, and for these properties the receptors are regarded as acetylcholine receptors of the nicotinic type (α-BuTX-AChRs). The rate constant of association (K₁) and dissociation (K₋₁) of the toxin-receptor reaction has been estimated to be K₁ = 7.4 × 10⁴ M⁻¹sec⁻¹ and K₋₁ = 9.6 × 10⁻⁶sec⁻¹, respectively. Light autoradiography shows that most, if not all, the receptors are related to surface membrane, probably to synaptic areas of both choroid and ciliary neurons. The choroid neurons contain more receptors that the ciliary ones. A single chick ciliary ganglion binds specically 47 fmole of α-BuTX in situ corresponding to 2.83 × 10¹⁰ α-BuTX-AChRs/ganglion.No changes in number and distribution of the toxin receptors occur following preganglionic denervation. Conversely, postganglionic axotomy causes a rapid disappearance of the receptors in situ. Since binding experiments in vitro revealed a partial, instead of a total, loss of the receptors, it is suggested that the disappearance of the receptors in situ includes both a partial loss of the original receptors and the masking of the residual ones.     

Subtypes of β-adrenergic receptors in rat cerebral microvessels

The¹²⁵I-labeled iodohydroxybenzylpindolol (IHYP) binding to β-receptors on brain microvessels is inhibited by isoproterenol, epinephrine and norepinephrine, with K_i values of 2 × 10⁻⁷M, 2.5 × 10⁻⁶M and 1.2 × 10⁻⁵M, respectively. A modified Scatchard analysis of the inhibitory effects of practolol, metroprolol and zinterol on IHYP binding has shown that the proportion of β₂-receptors in our preparation is about 80% of the total β-adrenergic receptor population. Our data indicate that the β-adrenergic receptors located on cerebral microvessels are of both β₁ and β₂ types, with a predominance of the β₂ type.     

Androgen receptor in the rat brain — assays and properties

The existence and relevance of an androgen receptor in the developing brain has been a matter of controversy. We here describe both sucrose density gradient and hydroxylapatite assays which clearly define a distincy androgen receptor in the 24-day-old rat hypothalamus, amygdala and preoptic region, but not in the cortex. This receptor has considerable affinity for estradiol-17ß, thus perhaps accounting for some uncertainty about its nature, but none for diethylstilbestrol or other estrogens, antiestrogens or glucocorticoids. Its K_d for both dihydrotestosterone and testosterone is about 1 × 10⁻⁹ M and for estradiol about 2 × 10⁻⁸ M. Its properties are generally consistent with those of androgen receptor reported for other tissues.     

Characterization of insulin binding to skeletal muscle sarcolemma during the development of diabetes

The aim of the present study was to characterize insulin binding to isolated skeletal muscle sarcolemma during the development of experimental diabetes. Experimental diabetes with a persistent hyperglycemia of 363 ± 23 mg glucose/dl was induced in Sprague-Dawley rats by a single intravenous injection of streptozotocin (65 mg/kg body weight). Insulin binding to skeletal muscle sarcolemma was measured in control, 6-day, and 18-day diabetic rats, the latter group of which demonstrated signs of peripheral diabetic neuropathy and muscle wasting. Analysis of the binding data in an insulin concentration range of 7.5 × 10⁻¹²to 10⁻⁸m yielded curvilinear Scatchard plots. After 18 days, insulin binding increased in diabetic sarcolemma from 0.025 to 0.048 pmol/mg with no change in the dissociation constant (K_D = 1.69 × 10⁻¹⁰m) of the high-affinity segment of the Scatchard plots. Insulin binding was antagonized by insulin analogs in proportion to their biological potency (proinsulin > desoctapeptide glucagon).     

Binding of angiotensins to rat brain tissue: Structure activity relationship

The binding of ³H-angiotensin II to a synaptosome-enriched fraction of the subcortical part of rat brain was studied. In this fraction specific high-affinity binding sites for angiotensin II were demonstrated. The binding sites were saturated at a ligand concentration of 2×10⁻⁹ M. Scatchard analysis revealed a single class of binding sites with an apparent maximal binding capacity of 14 fmoles/mg of protein and an equilibrium dissociation constant, K_d, of 0.9 × 10⁻⁹ M. The specific binding at the K_d concentration amounted to 59% of the total binding and was reversible. The association and dissociation rate constants (k₁ and k₋₁) were 0.0212 nM⁻¹ min⁻¹ and 0.0196 min⁻¹, respectively. Binding was dependent on both incubation time and tissue concentration in the incubation mixture. Angiotensins with biological activity in the brain, i.e., angiotensins I, II, III, and the fragments (3–8) and (4–8) competed with ³H-angiotensin II for the binding sites with IC₅₀'s of 9×10⁻⁸ M, 2×10⁻⁹ M, 4× 10⁻⁹ M, 4× 10⁻⁷ M and 4×10⁻⁶, respectively. In the presence of 1 mM of the converting enzyme inhibitor SQ 14,225 the IC₅₀ for angiotensin I was 2 × 10⁻⁷ M. Competition by the biologically active fragment angiotensin (5–8) could not be demonstrated. The latter peptide, however, was highly metabolized during the incubation under the assay conditions used. The binding potency of the various angiotensins paralleled their dipsogenic and presser potency. The present data indicate the possible physiological involvement of these binding sites as specific receptors in the actions of angiotensins in the brain.     

Inhibition of brain adenylate cyclase by A1 adenosine receptors: Pharmacological characteristics and locations

When tested under conditions reducing the endogenous production of adenosine (presence of adenosine deaminase (ADA) 1.6 IU/ml; and deoxyadenosine triphosphate (d-ATP)), and in the presence of both NaCl and GTP, the ADA-resistant analog phenylisopropyladenosine (PIA) inhibited the adenylate cyclase of several tissues. These tissues included:(1) 5 brain areas of adult rats (frontal and parietal cortex, cerebellum cortex, hippocampus and striatum) — hypothalamus and mid-brain adenylate cyclases were not inhibited by PIA; (2) astrocytes in primary cultures prepared from cerebral cortex of newborn mice; and (3) neurons in primary cultures prepared from striata of 15-day-old mouse embryos.The specificity profile of the adenosine receptor involved in the inhibition was determined in astrocytes. It was typical of an A₁ adenosine receptor (high affinity of PIA;K_aapp: 9 ± 5 × 10⁻⁹M(n= 4) compared to the affinity of 5′-N-ethylcar☐amide adenosine (NECA);K_aapp: 1.3 ± 0.6 × 10⁻⁷M (n= 3). There was an excellent correlation between the affinities of several adenosine agonists and antagonists for A₁ receptors coupled with an adenylate cyclase in astrocytes and for the receptors labeled with N⁶-cyclohexyl-[³H]adenosine in brain cortex.In adult rat striatum as well as in astrocytes and striatal neurons in culture the adenylate cyclase was inhibited by low PIA concentrations through A₁ receptors and stimulated by higher concentrations through A₂ receptors. In contrast, A₂ receptors were not detected in adult rat cerebral cortex.In adult rat striatum, A₁ and dopamine receptors coupled with an adenylate cyclase seemed to be located on different cell populations. In contrast, in astrocytes A₁ and β-adrenergic receptors coupled with adenylate cyclase were apparently located on the same cells.     

Kinetic analysis ofl-leucine transport across the blood-brain barrier

Unidirectionall-leucine influx across cerebral capillaries was measured at different concentrations with an in situ rat brain perfusion technique, which has been several advantages over presently-used methods such as the Brain Uptake Index (BUI) technique. The maximal influx rate (V_max) and half-saturation concentration (K_m) equaled1.07 ± 0.02 × 10^3- μmol·s⁻·⁻¹and0.026 *+- 0.002 μmol·ml⁻¹, respectively, for the saturable component, and the constant for non-saturable equaled6.8 ± 1.4 × 10⁻⁵s⁻¹. These values differ by 3–4-fold from respective values obtained with the BUI technique.     

Peptide displacement of [H]5-hydroxytryptamine binding to bovine cortical membranes

Chemical studies have demonstrated that peptides such as the encephalitogenic (EAE) peptide of myelin basic protein (MBP) and luteinizing hormone-releasing hormone (LHRH) can bind serotonin (5-hydroxytryptamine, 5-HT) in vitro. The present research was undertaken to determine whether such binding interferes with 5-HT binding to its 5-HT₁ receptors on bovine cerebral cortical membranes. EAE peptide and LHRH displaced [³H]5-HT with IC₅₀s of 4.0 × 10⁻⁴ and 1.8 × 10⁻³M respectively. MBP itself also showed apparent displacing ability with an IC₅₀ of 6.0 × 10⁻⁵M, though it also caused aggregation of cortical membranes that might have interfered with normal receptor binding. These results support previous suggestions that the tryptophan peptide region of MBP may act as a 5-HT receptor in the neural system. We also tested the effects of muramyl dipeptide (N-acetyl-muramyl-L-Ala-D-isoGln, MD), a bacterial cell-wall breakdown product that acts as a slow-wave sleep promoter, binds to LHRH and EAE peptide, and competes for 5-HT binding sites on macrophages. It showed no significant displacement of 5-HT binding to cortical membranes (IC₅₀ > 10^t-1 M), but its D-Ala analogue did (IC₅₀ = 1.7 × 10⁻³M). Thus, it seems likely that the 5-HT-related effects of naturally occurring muramyl peptides are physiologically limited by receptor types.     

Production of interleukin-1 by microglia in response to substance P: role for a non-classical NK-1 receptor

Substance P (SP) is a central and peripheral neurotransmitter which has been found in multiple sclerosis plaques. SP stimulates peripheral immune cells and may play a role in some chronic inflammatory diseases. Human peripheral monocyte/macrophages have been shown to produce the inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor α (TNFα) in response to SP. Therefore, in this study we examined rat brain microglia for the presence of SP receptors and production of IL-1 and TNFα in response to SP. Microglia had 4900±950(mean ± SE) receptors per cell fitting a two-site model. Four percent of these were high-affinity receptors with a K_d of 8.2 × 10⁻⁸ M ± 3.6 × 10⁻⁸ M (mean ± SE), and 96% of them were low-affinity receptors with a K_d of 2.1 × 10⁻⁶ M ± 5.2 × 10⁻⁷ M (mean ± SE). Competitive studies with CP 96,345 and other SP analogs demonstrate these to be non-classical NK-1 receptors. SP alone did not stimulate IL-1 or TNFα production. However, SP in synergy with lipopolysaccharide (LPS) quadrupled IL-1 production compared to LPS alone, but did not affect TNFα production. These results have implications for certain inflammatory conditions in the central nervous system.     

Prostaglandin E1 modifies porcine cerebral arterial tone through cyclooxygenase related eicosanoid(s) release

The effects of prostaglandin E₁ (PGE₁) on porcine cerebral arteries were studied in the absence and presence of cyclooxygenase inhibitors, 5×10⁻⁶ M indomethacin or 10⁻⁴ M aspirin. In preparations placed at a resting tension, 3×10⁻⁹ to 10⁻⁶ M PGE₁ caused dose-dependent contractions. In prostaglandin F_2α (PGF_2α)- contracted preparations, low concentrations of 10⁻⁹ and 3 × 10⁻⁹ M PGE₁ did not modify arterial tone and higher concentrations of 10⁻⁸ to 10⁻⁶ M PGE₁ further increased the arterial tone in all preparations tested. In contrast, in the presence of the cyclooxygenase inhibitors, low concentration of 10⁻⁹ and 3×10⁻⁹ M PGE₁ markedly decreased the arterial tone induced by PGF_2α and higher dose of 10⁻⁸ to 10⁻⁶ M PGE₁ increased the arterial tone in 15 of 18 preparations (83%). These findings suggest that PGE₁ modifies porcine cerebral vascular tone at least partly through cyclooxygenase-related eicosanoid(s) production.     

Binding of []insulin to specific receptors and stimulation of nucleotide incorporation in cells cultured from rat brain

The occurrence of insuling receptors and biological responses to insulin has been investigated in trypsin-dissociated fetal rat brain cells maintained in culture for 8 days. Binding of [¹²⁵]insulin to brain cells in culture was time- and pH-dependent and 85–90% specific. Porcine insulin competed for [¹²⁵]insulin binding in a dose-dependent manner. Unrelated polypeptides, including angiotensin II, glucagon, bovine growth hormone, and bovine prolactin did not compete for [¹²⁵]insulin binding. The half-life of [¹²⁵]insulin dissociation from receptors at 24°C was 15 min and a plot of ln[B/Bo] vs time suggested two dissociation rate constants of2.7 × 10⁻⁴ sec⁻¹ and5.0 × 10⁻⁵ sec⁻¹. Scatchard analysis of the binding data gave a curvelinear plot which may indicate negative cooperativity or the occurrence of both high affinity(K_a = 2 × 10¹¹M⁻¹) and low affinity(K_a = 4 × 10¹⁰M⁻¹) sites. Of the estimated total of 4.9 × 10⁴ binding sites per cell, 28–30% appear to be high affinity sites.Incubation of cultures with insuling caused a time- and dose-dependent stimulation of [³H]thymidine and [³H]uridine incorporation into TCA-precipitable material. Maximum stimulation of thymidine incorporation (2–5-fold) occured 11 h after incubation with 167 nM insulin. The same concentration of insulin caused a 2.2-fold increase in [³H]uridine incorporation in 2 h. These results indicate that cells cultured from rat brain contain specific insulin receptors capable of mediating effects of insulin on macromolecular synthesis in the central nervous system.     

Effects of the bile salt sodium deoxycholate, protamine, and inflammatory mediators on the potassium permeability of the frog nerve perineurium

An electrophysiological method was used to measure the potassium permeability (P_K) of the perineurium of the sciatic nerve of frogs Rana temporaria and R. pipiens. Isolated but intact nerves were mounted in a grease-gap chamber, and compound action potential and DC potential monitored. Change in the DC potential (ΔDC) in response to challenge with 100 mM [K⁺] Ringer was used to assess the K⁺ permeability of the perineurium, since change in DC potential under these conditions reflected changes in the axonal resting potential. The permeability of the perineurium was calculated from the published calibration curve relating ΔDC to bathing [K⁺] in desheathed nerves of Abbott et al. (1997). In the control condition, P_K was <1.1×10⁻⁶ cm·s⁻¹. The bile salt sodium deoxycholate (DOC, 1–4 mM) caused a dose-dependent increase in P_K, which reached a maximum of 1.7×10⁻⁵ cm·s⁻¹ after 2-min exposure to 4 mM DOC, but access of K⁺ to the endoneurial compartment was more restricted after DOC than after desheathing. Protamine phosphate (1 mM) and protamine sulphate (0.1–5 mg/ml equals 0.125–6.25 mM) had no effect on P_K. Neither histamine (0.4–40 mg/ml), bradykinin (0.1–5 mg/ml) nor serotonin (5-hydroxytryptamine, 0.1–5 mg/ml) affected P_K. The frog nerve perineurium appears to be relatively insensitive to chemical agents and inflammatory mediators, in contrast to the endothelial cells forming the endoneurial blood–nerve barrier and the blood–brain barrier.     

Localization and characterization of epidermal growth-factor receptors in the developing rat medial septal area in culture

The presence and binding properties of epidermal growth-factor receptors (EGF-Rs) in different cell types purified from the rat medial septal area in culture were investigated. We report that astrocytes, oligodendrocytes and neurons from this area possess EGF-Rs while microglia do not. EGF-binding sites are detectable on astrocytes derived from the medial septum of both embryonic and neonatal rats. Scatchard analysis of the data for astrocytes from the fetal rats show that EGF specifically binds to both high- (K_d = 7.21 × 10⁻¹⁰ M, B_max = 3602 receptors/cell) and low-affinity (K_d = 3.99 × 10⁻⁸ 10⁻⁸ M, B_max = 6,265 receptors/cell) receptors on these cells. On the other hand, astrocytes purified from neonatal tissue possess a greater number of high-affinity receptors (B_max = 10,938 receptors/cell) when compared with the embryonic astroglia. With time in culture, the number of both types of receptors on neonatal astrocytes decreases. Oligodendrocytes also possess high- and low-affinity EGF-Rs with dissociation constants of 3.25 × 10⁻¹⁰ M and 3.85 × 10⁻⁸ M, respectively. The number of receptors on oligodendrocytes is significantly lower than those on neonatal astrocytes (B_max = 1185 and 25,081 receptors/cell for high- and low-affinity binding sites, respectively). Finally, neurons from this area also exhibit two different EGF-R types with dissociation constants similar to those described for astrocytes. As the number of receptors/neuron (B_max = 136 and 1159 receptors/cell for high- and low-affinity binding sites, respectively) appears to be extremely low, it is possible that EGF specifically binds only to a subpopulation of neurons from this area. These studies demonstrate which cell types in the developing medial sepal area posses EGF-Rs and provide a detailed characterization of these binding sites. These EGF-R-bearing cells may be potential targets for this growth factor or for transforming growth factor α in this brain area.     

Virol A, a toxic trans-polyacetylenic alcohol of Cicuta virosa, selectively inhibits the GABA-induced Cl current in acutely dissociated rat hippocampal CA1 neurons

The effects of virol A (VA), a toxic component of Cicuta virosa (water hemlock), on the GABA-induced Cl⁻ current (I_GABA) in acutely dissociated rat hippocampal CA1 neurons were investigated using whole-cell patch-clamp techniques. VA reversibly reduced I_GABA and the muscimol (Mus)-induced current (I_Mus) in a concentration-dependent manner. The IC₅₀ values for VA against I_GABA and I_Mus were 9.6×10⁻⁷ and 9.8×10⁻⁷ M, respectively. VA shifted the EC₅₀ value of I_GABA from 6.5×10⁻⁶ to 2.1×10⁻⁵ M, whereas it had no effect on the maximum response, thereby suggesting that VA inhibited I_GABA in a competitive manner. VA had no apparent effect on current–voltage relationships for I_GABA, thus indicating the lack of voltage-dependency. On the other hand, application of VA (10⁻⁶ M) did not additionally reduce the I_GABA suppressed by >10⁻⁵ M picrotoxin. VA but not bicuculline accelerated the decay phase of I_GABA, as was seen with picrotoxin. Moreover, pre-application of 10⁻⁵ M VA reduced I_GABA. VA did not inhibit that induced by glycine (10⁻⁴ M). These results indicate that VA inhibits I_GABA by acting both on the GABA agonist site and on the Cl⁻ channel of the GABA_A receptor–channel complex. VA is a structurally novel type of compound that selectively inhibits the GABA_A receptor–Cl⁻ channel complexes in mammalian central nervous system neurons.     

Modulation of glycine-induced currents by zinc and other metal cations in neurons acutely dissociated from the dorsal motor nucleus of the vagus of the rat

Effects of Zn²⁺ and other polyvalent cations on glycine-induced currents in the freshly dissociated rat dorsal motor nucleus of the vagus neurons were investigated under voltage-clamp conditions by the use of the nystatin-perforated patch recording configuration. Glycine evoked a Cl⁻ current in a concentration-dependent manner with a half-maximum effective concentration (EC₅₀) of 3.3×10⁻⁵ M. Strychnine inhibited the 3×10⁻⁵ M glycine-induced current in a concentration-dependent manner with a half-maximal inhibitory concentration (IC₅₀) of 6.8×10⁻⁷ M. At low concentrations (3×10⁻⁸ M–3×10⁻⁶ M), Zn²⁺ potentiated the current elicited by 3×10⁻⁵ M glycine. On the other hand, at concentrations higher than 10⁻⁵ M, Zn²⁺ inhibited the glycine response. The biphasic action of Zn²⁺ was mimicked by Ni²⁺, but La³⁺ and Co²⁺ had only potentiating effect. Zn²⁺ shifted the concentration–response curve for the glycine-induced current without changing the maximum response, and the EC₅₀ values for the glycine response in the absence and presence of 10⁻⁶ M and 10⁻⁴ M Zn²⁺ were 3.3×10⁻⁵ M, 1.3×10⁻⁵ M and 1.3×10⁻⁴ M, respectively. These results suggest that the biphasic modulation of glycine response by Zn²⁺ results from changes in apparent glycine affinity.     

A single (−)-nicotine injection causes change with a time delay in the affinity of striatal D2 receptors for antagonist, but not for agonist, nor in the D2 receptor mRNA levels in the rat substantia nigra

The in vitro and in vivo effects of (−)-nicotine on dopamine D₂ receptors in the rat neostriatum have been studied using biochemical binding, in situ hybridization and immunocytochemistry. A single i.p. injection (1 mg/kg) of (−)-nicotine resulted in a reduction of theK_D value of the D₂ antagonist [³H]raclopride binding sites in rat neostriatal membrane preparations at 12 h without any significant change in theB_max value. This action of (−)-nicotine was counteracted by pretreatment 15 min earlier with the nicotine antagonist mecamylamine (1 mg/kg, i.p.). However, theK_D and theB_max values of the D₂ agonist [³H]NPA binding sites in the rat neostriatal membrane preparations were not significantly affected 0.5–48 h after a single i.p. injection with 1 mg/kg of (−)-nicotine. No significant change in neostriatal D₂ receptor mRNA levels was observed at any time interval after the (−)-nicotine injection. No significant change was observed in tyrosine hydroxylase (TH) immunoreactivity in either the substantia nigra or the neostriatum, nor in nigral TH mRNA levels during the time interval studied (4–24 h posttreatment). Furthermore, addition of low (10 nM) or high (1 μM) concentrations of (−)-nicotine in vitro to rat neostriatal membranes did not alter the characteristics of [³H]raclopride or [³H]NPA binding. These results indicate that a single (−)-nicotine injection can produce a selective and delayed increase in the affinity of D₂ receptors for the antagonist, but not for the agonist without modifying the levels of D₂ receptor mRNA, probably via the activation of central nicotinic receptors.