GABAB antagonists: Resolution,absolute stereochemistry,and pharmacology of (R)- and (S)-phaclofen

Phaclofen, which is the phosphonic acid analogue of the GABA_B agonist (RS)-3-(4-chlorophenyl)-4-aminobutyric acid (baclofen), is a GABA_B antagonist. As part of our studies on the structural requirements for activation and blockade of GABA_B receptors, we have resolved phaclofen using chiral chromatographic techniques. The absolute stereochemistry of (?)-(R)-phaclofen was established by X-ray crystallographic analysis. (?)-(R)-Phaclofen was shown to inhibit the binding of [³H]-(R)-baclofen to GABA_B receptor sites on rat cerebellar membranes (IC₅₀ = 76 ± 13 μM), whereas (+)-(S)-phaclofen was inactive in this binding assay (IC₅₀ > 1000 μM). (?)-(R)-Phaclofen (200 μM) was equipotent with (RS)-phaclofen (400 μM) in antagonizing the action of baclofen in rat cerebral cortical slices, while (+)-(S)-phaclofen (200 μM) was inactive. The structural similarity of the agonist (R)-baclofen and the antagonist (?)-(R)-phaclofen suggests that these ligands interact with the GABA_B receptor sites in a similar manner. Thus, it may be concluded that the different pharmacological effects of these compounds essentially result from the different spatial and proteolytic properties of their acid groups. © 1994 Wiley-Liss, Inc.     

Alterations in the Intrinsic Burst Activity of Purkinje Neurons in Offspring Maternally Exposed to the CB1 Cannabinoid Agonist WIN 55212-2

Burst firing plays an important role in normal neuronal function and dysfunction. In Purkinje neurons, where the firing rate and discharge pattern encode the timing signals necessary for motor function, any alteration in firing properties, including burst activity, may affect the motor output. Therefore, we examined whether maternal exposure to the cannabinoid receptor agonist WIN 55212-2 (WIN) may affect the burst firing properties of cerebellar Purkinje cells in offspring. Whole-cell somatic patch-clamp recordings were made from cerebellar slices of adult male rats that were exposed to WIN prenatally. WIN exposure during pregnancy induced long-term alterations in the burst firing behavior of Purkinje neurons in rat offspring as evidenced by a significant increase in the mean number of spikes per burst (p < 0.05) and the prolongation of burst firing activity (p < 0.01). The postburst afterhyperpolarization potential (p < 0.001), the mean intraburst interspike intervals (p < 0.001) and the mean intraburst firing frequency (p < 0.001) were also significantly increased in the WIN-treated group. Prenatal exposure to WIN enhanced the firing irregularity as reflected by a significant decrease in the coefficient of variation of the intraburst interspike interval (p < 0.05). Furthermore, whole-cell voltage-clamp recordings revealed that prenatal WIN exposure significantly enhanced Ca²⁺ channel current amplitude in offspring Purkinje neurons compared to control cells. Overall, the data presented here strongly suggest that maternal exposure to cannabinoids can induce long-term changes in complex spike burst activity, which in turn may lead to alterations in neuronal output.     

The Sodium-Potassium Pump Controls the Intrinsic Firing of the Cerebellar Purkinje Neuron

In vitro, cerebellar Purkinje cells can intrinsically fire action potentials in a repeating trimodal or bimodal pattern. The trimodal pattern consists of tonic spiking, bursting, and quiescence. The bimodal pattern consists of tonic spiking and quiescence. It is unclear how these firing patterns are generated and what determines which firing pattern is selected. We have constructed a realistic biophysical Purkinje cell model that can replicate these patterns. In this model, Na⁺/K⁺ pump activity sets the Purkinje cell''s operating mode. From rat cerebellar slices we present Purkinje whole cell recordings in the presence of ouabain, which irreversibly blocks the Na⁺/K⁺ pump. The model can replicate these recordings. We propose that Na⁺/K⁺ pump activity controls the intrinsic firing mode of cerbellar Purkinje cells.     

The accumulation of cyclic AMP and cyclic GMP in guinea pig brain slices: Effect of calcium ions,norepinephrine and adenosine

The effect of Ca²⁺ and putative neurotransmitters on formation of cyclic AMP and cyclic GMP has been studied in incubated slices of brain tissue. Cyclic AMP levels in cerebellar slices after about 90 min of incubation ranged from 10 pmol/mg protein in rabbit, to 25 in guinea pig, to 50 in mouse and 200 in rat. Cyclic GMP levels in the same four species showed no correlation with cyclic AMP levels and were, respectively, 1.3, 20, 5 and 30 pmol/mg protein. The absence of calcium during the prolonged incubation of cerebellar slices had little effect on final levels of cyclic AMP, while markedly decreasing final levels of cyclic GMP. Reintroduction of Ca²⁺ resulted in a rapid increase in cerebellar levels of cyclic GMP which was most pronounced for guinea pig where levels increased nearly 7-fold within 5 min. Prolonged incubation of guinea pig cerebral cortical slices in calcium-free medium greatly elevated cyclic AMP levels apparently through enhanced formation of adenosine, while having little effect on final levels of cyclic GMP. Norepinephrine and adenosine elicited accumulations of cyclic AMP and cyclic GMP in both guinea pig cerebral cortical and cerebellar slices. Glutamate, γ-aminobutyrate, glycine, carbachol, and phenylephrine at concentrations of 1 mM or less had little or noe effect on cyclic nucleotide levels in guinea pig cerebellar slices. Prostaglandin E₁ and histamine slightly increased cerebellar levels of cyclic AMP. Isoproterenol increased both cyclic AMP and cyclic GMP. The accumulation of cyclic AMP and cyclic GMP elicited by norepinephrine in cerebellar slices appeared, baed on dose vs. response curves, agonist-antaganonist relationships and calcium dependency, to involve in both cases activation of a similar set of ß-adrenergic receptors. In cerebellar slices accumulations of cyclic AMP and cyclic GMP elicted by norepinephrine and by a depolarizing agent, veratridine, were strongly dependent on the presence of calcium. The stimulatory effects of adenosine on cyclic AMP and cyclic GMP formation were antagonized by theophylline. The lack of correlations between levels of cyclic AMP and cyclic GMP under the various conditions suggested independent activation of cyclic AMP- and cyclic GMP-generating systems in guinea pig cerebellar slices by interactions with Ca²⁺, norephinephrine and adenosine.     

The binding of fluorocatecholamines to adrenergic and dopaminergic receptors in rat brain membranes

2-Fluoronorepinephrine (IC₅₀ ≈0.7 μM) is a relatively selective ligand for displacement of radioactive dihydroalprenolol from β₁-adrenergic receptors in membrane preparations from rat cerebral cortex. It is less potent (IC₅₀ ≈10 μM) in displacing dihydroalprenolol from β₂-adrenergic receptors in rat cerebellar membranes and in displacing clonidine from α₂-adrenergic receptors in rat cerebral cortical membranes. It is much less potent (IC₅₀ > 100 μM) in displacing WB-4101 from α₁-adrenergic receptors in rat cerebral cortical membranes. In contrast, 6-fluoronorepinephrine is relatively selective for α-adrenergic receptors, being at least 50–200 times more potent at such receptors than at β-adrenergic receptors. 5-Fluoronorepinephrine like norepinephrine does not exhibit remarkable selectivity towards α- and β-adrenergic receptors. The 2-, 5- and 6-fluorodopamines are more potent ligands at α₁-adrenergic receptors than at α₂- and β-adrenergic receptors but the specificity is not markedly affected by the position of the fluorine substituent. The results suggest that the specificity exhibited by the 2- and 6-fluoronorepinephrine at adrenergic receptors is not primarily due to fluorine-induced changes in the physicochemical properties of the aromatic ring, but instead to stereoselective interactions of the ethanolamine side chain of norepinephrine with fluorine at either the 2- or 6-ring positron. The fluorodopamines like dopamine itself are more potent at dopaminergic than at α- or β-adrenergic receptors. The 2-, 5- and 6-fluorodopamines are all nearly equipotent with dopamine in the displacement of radioactive spiroperidol from dopaminergic receptors in membrane preparations from rat striatum, while the 2- and 6-fluorodopamine are somewhat less potent than dopamine or 5-fluorodopamine in displacement of radioactive apomorphine in striatal membranes.     

AMPA receptor agonists: Resolution,configurational assignment,and pharmacology of (+)-(S)- and (-)-(R)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-isoxazol-4-yl]-propionic acid (2-Py-AMPA)

We have previously shown that whereas (RS)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid (APPA) shows the characteristics of a partial agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, (S)-APPA is a full AMPA receptor agonist and (R)-APPA a weak competitive AMPA receptor antagonist. This observation led us to introduce the new pharmacological concept, functional partial agonism. Recently we have shown that the 2-pyridyl analogue of APPA, (RS)-2-amino-3-[3-hydroxy-5-(2-pyridyl)isoxazol-4-yl]propionic acid (2-Py-AMPA), is a potent and apparently full AMPA receptor agonist, and this compound has now been resolved into (+)- and (-)-2-Py-AMPA (ee ≥ 99.0%) by chiral HPLC using a Chirobiotic T column. The absolute stereochemistry of the enantiomers of APPA has previously been established by X-ray analysis, and on the basis of comparative studies of the circular dichroism spectra of the enantiomers of APPA and 2-Py-AMPA, (+)- and (-)-2-Py-AMPA were assigned the (S)- and (R)-configuration, respectively. In a series of receptor binding studies, neither enantiomer of 2-Py-AMPA showed detectable affinity for kainic acid receptor sites or different sites at the N-methyl-D-aspartic acid (NMDA) receptor complex. (+)-(S)-2-Py-AMPA was an effective inhibitor of [³H]AMPA binding (IC⁵⁰ = 0.19 ± 0.06 μM) and a potent AMPA receptor agonist in the rat cortical wedge preparation (EC₅₀ = 4.5 ± 0.3 μM) comparable with AMPA (IC₅₀ = 0.040 ± 0.01 μM; EC₅₀ = 3.5 ± 0.2 μM), but much more potent than (+)-(S)-APPA (IC₅₀ = 5.5 ± 2.2 μM; EC₅₀ = 230 ± 12 μM). Like (-)-(R)-APPA (IC₅₀ > 100 μM), (-)-(R)-2-Py-AMPA (IC₅₀ > 100 μM) did not significantly affect [³H]AMPA binding, and both compounds were week AMPA receptor antagonists (K_i = 270 ± 50 and 290 ± 20 μM, respectively). Chirality 9:274–280, 1997. © 1997 Wiley-Liss, Inc.     

Activation of corticotropin-releasing factor 2 receptor inhibits Purkinje neuron P-type calcium currents via Goα-dependent PKC epsilon pathway

Corticotropin-releasing factor (CRF) receptors have been demonstrated to be widely expressed in the central nervous system and in many peripheral tissues of mammalians. However, it is still unknown whether CRF receptors will function in cerebellar Purkinje neurons. In the present study, we investigated the expression profile of CRF receptors in rat cerebellum and identified a novel functional role of CRFR2 in modulating Purkinje neuron P-type Ca²⁺ currents (P-currents). We found that CRFR2α mRNA, but not CRFR1 and CRFR2β, was endogenously expressed in rat cerebellum. Activation of CRFR2 by UCN2 inhibited P-currents in a concentration-dependent manner (IC₅₀ ~ 0.07 µM). This inhibitory effect was abolished by astressin2B, a CRFR2 antagonist, and was blocked by GDP-β-S, pertussis toxin, or a selective antibody raised against the G_oα. Inhibition of phospholipase C (PLC) blocked the inhibitory action of UCN2. The application of diacylglycerol (DAG) antagonist, 1-hexadecyl-2-acetyl-sn-glycerol, as well as inhibition of either protein kinase C or its epsilon isoform (PKCε) abolished the UCN2 effect while 1-oleoyl-2-acetyl-sn-glycerol (EI-150), a membrane-permeable DAG analogue, occluded UCN2-mediated inhibition. In addition, UCN2 significantly increases spontaneous firing frequency of Purkinje neurons in cerebellar slices. In summary, activation of CRFR2 inhibits P-currents in Purkinje neurons via G_oα-dependent PLC/PKCε pathway, which might contribute to its physiological functions in the cerebellum.     

Formation of a functional adrenergic input to intraocular cerebellar grafts: Ingrowth of inhibitory sympathetic fibers

The intraocular transplantation technique was used to study the ingrowth of peripheral sympathetic adrenergic nerves from the iris into transplants of fetal rat cerebellum, and the possible function of these nerves. The transplants, grown in oculo for one-half to eight months, were analyzed by fluorescence histochemistry and electrophysiological techniques. Peripheral sympathetic adrenergic fibers from the iris were able to grow into the cerebellar transplants and arborize in a pattern similar to that in situ, appearing in all three cortical layers and the noncortical areas of the transplants. The density of visible nerves without pretreatment and after preincubation in 10⁻⁶ or 10⁻⁵ M α-methylnorepinephrine was comparable to mature rat cerebellum. The spontaneous discharge of the Purkinje cells in oculo was inhibited by microiontophoresis of norepinephrine (NE) and amphetamine in sympathetically innervated, as well as sympathectomized transplants denervated by ganglionectomy. The NE response was blocked by the adrenergic β-receptor blocker MJ-1999. GABA also inhibited the Purkinje cell activity while glutamate accelerated the discharge. Parenteral amphetamine inhibited Purkinje cell activity in sympathetically innervated transplants, but was ineffective in denervated transplants. The Purkinje cell spontaneous activity was inhibited by electrical stimulation of the NE fiber input through the cervical sympathetic trunk. This inhibition could be antagonized by parenteral reserpine or the β-adrenergic antagonist propranolol. The responses of the Purkinje cells within the transplants to drugs and transmitters mimic those of the adult rat in situ. In view of the fluorescence histochemical evidence for an ingrowth of peripheral sympathetic adrenergic fibers into the cerebellar transplants, and the results of stimulating the sympathetic trunk, it is suggested that peripheral adrenergic fibers may be able to establish functional connections with the Purkinje cells similar to the cerebellar adrenergic synapses normally formed in situ by fibers from the locus coeruleus.     

Characteristics of the catecholamine and histamine receptor sites mediating accumulation of cyclic adenosine 3',5'-monophosphate in guinea pig brain

—Five areas of guinea pig brain were examined to determine the properties of the receptor sites mediating increases in [³H]adenosine 3′,5′-monophosphate (cyclic AMP). Both epinephrine and histamine were effective in causing increases in cyclic AMP in slices derived from cerebral cortex, hippocampus or amygdala, but not in diencephalon or brainstem. Stimulation of slices of cerebral cortex by either epinephrine or histamine resulted in a small, but reproducible, decrease in specific radioactivity of the [³H]-cyclic AMP produced, as did stimulation of the hippocampus by epinephrine. The catecholamine receptor was an α-adrenergic receptor in all three areas where epinephrine was effective; α-adrenergic stimulation, but not β-adrenergic stimulation, increased levels of [³H]-cyclic AMP. Furthermore, α-, but not β-adrenergic blocking agents, prevented the epinephrine- induced increase of both [³H]- and total cyclic AMP in cerebral cortex and hippocampus. Only antihistaminic agents were capable of antagonizing the histamine-induced increase of both [³H]- and total cyclic AMP in these two brain areas. The catecholamine receptor in the amygdala also appeared to be an α-adrenergic receptor. The effects of histamine and epinephrine together were far greater than the sum of effects of either hormone alone in both cerebral cortex and hippocampus.     

Independent and Coordinate Regulation of β1- and β2-Adrenergic Receptors in Rat C6 Glioma Cells

Abstract

Rat C6 glioma cells have both β₁- and β₂-adrenergic receptors in ～ 7:3 ratio. When the cells were exposed to the β-adrenergic agonist isoproterenol, there was a rapid sequestration of up to 50% of the surface receptor population over a 30-min period as measured by the loss of binding of the hydrophilic ligand [³H] CGP-12177 to intact cells. Using the β₂-selective antagonist CGP 20712A to quantify the proportion of the two subtypes, it was found that although both β₁ and β₂ receptors were sequestered, the latter were sequestered initially twice as fast as the former. More prolonged agonist exposure led to a down-regulation of ～ 90% of the total receptor population by 6 h as measured by the loss of binding of the more hydrophobic ligand [¹²⁵I] iodocyanopindolol to cell lysates. The two subtypes, however, underwent down-regulation with similar kinetics. Treatment of the cells with agents that raise cyclic AMP levels such as cholera toxin and forskolin resulted in a slower, but still coordinated down-regulation of both subtypes. Thus, there appears to be both independent and coordinate regulation of endogenous β₁-and β₂-adrenergic receptors in the same cell line.     

II. Beta-adrenergic receptors and catecholamine sensitive adenylate cyclase in the developing rat lung

β-Adrenergic receptors were identified in membrane fractions of fetal and postnatal rat lung with the β-adrenergic antagonist (?)?[³H] dihydroalprenolol, (?)?[³H] DHA. β-Receptor number (Bmax) increased 11-fold from day 18 of gestation to day 28 of postnatal life, 46±7 to 491±69 femtomole·mg^?1 protein. Neither the K_D, approximately 0.8nM for [³H]DHA, nor the β-adrenergic subtype changed with age. Classical agonists competed for the β-receptor with properties characteristic of β₂-adrenergic binding. Analysis of the inhibition of receptor binding by selective β-adrenergic agents demonstrated approximately 75% β₂ and 25% β₁ β-adrenergic subtypes in fetal rat lung membranes. The increase in β-adrenergic receptor during development was associated with adenylate cyclase activity which was sensitive to catecholamines at all ages studied, supporting the possible role of the β-adrenergic receptor system in the postnatal regulation of pulmonary function.     

Intracellular Ca regulates spike encoding at cortical GABAergic neurons and cerebellar Purkinje cells differently

Spike encoding at GABAergic neurons plays an important role in maintaining the homeostasis of brain functions for well-organized behaviors. The rise of intracellular Ca²⁺ in GABAergic neurons causes synaptic plasticity. It is not clear how intracellular Ca²⁺ influences their spike encoding. We have investigated this issue at GFP-labeled GABAergic cortical neurons and cerebellar Purkinje cells by whole-cell recording in mouse brain slices. Our results show that an elevation of intracellular Ca²⁺ by infusing adenophostin-A lowers spike encoding at GABAergic cortical neurons and enhances encoding ability at cerebellar Purkinje cells. These differential effects of cytoplasmic Ca²⁺ on spike encoding are mechanistically associated with Ca²⁺-induced changes in the refractory periods and threshold potentials of sequential spikes, as well as with various expression ratios of CaM-KII to calcineurin in GABAergic cortical neurons and cerebellar Purkinje cells.     

Accumulation of inositol phosphates and cyclic AMP in guinea-pig cerebral cortical preparations: Effects of norepinephrine,histamine, carbamylcholine and 2-chloroadenosine

Norepinephrine and serotonin augment by about 2-fold the accumulation of cyclic [3H]AMP elicited by 2-chloroadenosine in [³H]adenine-labeled guinea-pig cerebral cortical slices. Histamine causes a 3-fold augmentation. The first two agents have no effect on cyclic AMP alone, while histamine has only a small effect alone. The augmentation of the 2-chloroadenosine response appears to be mediated by α₁-adrenergic, 5HT₂-serotonergic and H₂-histaminergic receptors. VIP-elicited accumulations of cyclic AMP are also augmented through stimulation of α₁-adrenergic, 5HT₂-serotonergic and H₁-histaminergic receptors. Activation of these amine receptors also increases the turnover of phosphatidylinositols in [³H]inositol-labeled guinea pig cerebral cortical slices. Norepinephrine causes a 5-fold, serotonin a 1.2-fold, and histamine a 2.5-fold increase in accumulations of [³H]inositol phosphates. 2-Chloroadenosine, vasoactive intestinal peptide, baclofen, and somatostatin have no effect on phosphatidylinositol turnover, nor do the last two agents augment accumulations of cyclic AMP elicited by 2-chloroadenosine. The data suggest a possible relationship between turnover of phosphatidylinositol and the augmentations of the cyclic AMP accumulations elicited by biogenic amines in brain slices.     

Characteristics of β-adrenergic-agonist binding to rat adipocyte membranes: Evidence that (±)-[3H]hydroxybenzylisoproterenol interacts selectively with the adipocyte β-adrenergic receptors

The binding characteristics of the β-adrenergic agonist $\text{(±)-[}{}^3\text{H]hydroxybenzylisoproterenol}$ to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37°C (second order rate constant k₁=1.37·10⁷·M^?1·min^?1). Dissociation of specific binding by 0.5 mM (?)-isoproterenol suggested dissociation from two different sites with respective dissociation rate constants k₂ of 0.106·min^?1 and 0.011·min^?1.[³H]Hydroxybenzylisoproterenol binding was saturable (B_max=690±107 fmol/mg protein), yielding curvilinear Scatchard plots. Computer modeling of these data were consistent with the existence of two classes of [³H]hydroxybenzylisoproterenol binding sites, one having high affinity (K_D=3.5±0.7 nM) but low binding capacity (10% of the total sites) and one haveing low affinity (K_D=101±20 nM) but high binding capacity (90% of the sites). Adrenergic ligands competed with [³H]hydroxybenzylisoproterenol binding with the following order of potency=(?)-propranolol>(?)-isoproterenol>(?)-norepinephrine≈ (?)-epinephrine>>(+)-isoproterenol=(+)-propranolo, which is consistent with binding to β₁-adrenergic receptors. Competition curves of [³H]hydroxybenzylisoproterenol binding by the β-agonist (?)-isoproterenol were shallow and modeled to two affinity states of binding, whereas, competition curves by β-antagonist (?)-propranolol were steeper with Hill number near to one. Gpp[NH]p severely reduced [³H]hydroxybenzyl-isoproterenol binding, an effect which apparently resulted from the reduction of the number of both the high and low affinity sites. In membranes which had been previously exposed to (?)-isoproterenol, then number of [³H]hydroxybenzylisoproterenol binding sites was reduced by 50%, an effect which apparently resulted from the loss of part of both the high and low affinity state binding sites. Finally, the ability of (?)-isoproterenol to stimulate adenylate cyclase correlate closely with the ability of (?)-isoproterenol to displace [³H]hydroxybenzylisoproterenol binding. Comparison of these findings with the binding characteristics of the β-antagonist [³H]dihydroalprenolol to rat adipocyte membranes, led to conclude that [³H]hydroxybenzylisoproterenol can be successfully used to label the β-adrenergic receptors of rat fat cells and suggests that it might be a better ligand than [³H]dihydroalprenolol in these cells.     

Antagonism of alpha- and beta-adrenergic-mediated accumulations of cyclic AMP in rat cerebral cortical slices by the beta-antagonist (-)alprenolol.

(?)Alprenolol, a compound reported to bind with a high degree of specificity and stereoselectivity to β-adrenergic receptors from rat cerebral cortex completely inhibited the accumulations of cyclic AMP elicited by maximally effective concentrations of norepinephrine and epinephrine at antagonist concentrations as low as 10^?5M. Other β-adrenergic antagonists such as (?)propranolol, (±)sotalol, and (+)alprenolol only partially antagonized accumulations of cyclic AMP elicited by these catecholamines even at 10-fold higher concentrations. α-Adrenergic antagonists such as phentolamine, phenoxybenzamine and clonidine only partially antagonized inhibited the accumulation of cyclic AMP elicited by methoxamine, a compound shown to stimulate the accumulation of cyclic AMP by interaction with α-adrenergic receptors. The results indicate that in brain tissue containing a mixed population of α- and β- adrenergic linked cyclic AMP generating systems, (?)alprenolol does not exhibit absolute specificity for β-receptors.     

Facilitation of norepinephrine release from cerebral cortex is mediated by β2-adrenergic receptors

Facilitatory effects of prenalterol and albuterol (β1- and β2-selective adrenergic agonists, respectively) in the absence and presence of propranolol (a nonselective β-adrenergic antagonist), ICI 89,406 or ICI 118,551 (β1- and β2-selective adrenergic antagonists, respectively) on electrical stimulation-evoked release of ³H-NE from rat cerebral cortical slices were assessed. Albuterol (0.1 –100 nM) increased evoked release of ³H-NE from the cerebral cortical slices with greater potency than prenalterol (1 – 100 nM). The β2-adrenergic antagonist ICI 118,551 (1 nM) and propranolol (50 nM) abolished the facilitatory effects of albuterol (0.1 and 10 nM). In contrast, the βl-adrenergic antagonist ICI 89,406 (1 nM) did not alter the release-enhancing effect of albuterol. Prenalterol (10 and 100 nM)-induced facilitation of evoked release of ³H-NE was abolished by ICI 118,551; propranolol reduced the effect of 10 nM prenalterol and abolished that of 100 nM prenalterol. ICI 89,406 inhibited the effect of 100 nM prenalterol without altering that of 10 nM prenalterol. Basal release of ³H-NE was not altered by the drugs used in this study. These results suggest that facilitation of ³H-NE release induced by β-adrenergic agonists is mediated primarily by β2-adrenergic receptors.     

Accumulation of adenosine 3',5'-monophosphate in rat brain slices: effects of prostaglandins.

Prostaglandin E₁ and E₂ and 15(S)-15-methyl PGE₂ methyl ester stimulate the accumulation of radioactive cyclic AMP in brain slices from Sprague-Dawley rats, labelled during a prior incubation with [¹⁴C] adenine. Prostaglandins A₁ and B₁ have marginal effects and prostaglandin F_1α has no effect. Relatively high concentrations of about 80 μM PGE₁, PGE₂ and 15(S)-15-methyl PGE₂ are required to elicit a maximal 2–5 fold increase in accumulation of cyclic AMP in slices from cerebrum, but significant increases are elicited by 3.5 μM prostaglandin. Similar increases are elicited in slices from neocortex, striatum or midbrain-thalamus-hypothalamus, while lesser increases pertain in slices from cerebellum, medulla-pons or hippocampus. The accumulation of cyclic AMP elicited by PGE₁ in slices from cerebrum was not blocked by naloxone, propranololphentolamine, tetracaine, theophylline, or by nearly equimolar concentrations of either of two prostaglandin antagonists, 7-oxa-13-prostynoic acid and the dibenzoxazepine hydrazide, SC 19220. Morphine potentiated the effects of PGE₁. The combination of 85 μM PGE₁ with either isoproterenol, norepinephrine, adenosine or veratridin did not increase the accumulation of cycli AMP significantly above those elicited by the isoproterenol, norepinephrine, adenosine or veratridine alone. The combined effect of PGE₁ and norepinephrine in the presence of a β-adrenergic antagonist, sotalol, was, however, additive. The results indicate that PGE₁ stimulates cyclic AMP formation in rat brain slices, but that it either has antagonist activity with respect to accumulations of cyclic AMP-elicited by other agents or has no detectable agonist activity when cyclases are maximally stimulated by other agents.     

Ischemia deteriorates the spike encoding of rat cerebellar Purkinje cells by raising intracellular Ca2+

Ischemia-induced excitotoxicity at cerebellar Purkinje cells is presumably due to a persistent glutamate action. To the fact that they are more vulnerable to ischemia than other glutamate-innervated neurons, we studied whether additional mechanisms are present and whether cytoplasm Ca²⁺ plays a key role in their ischemic excitotoxicity. Ischemic changes in the excitability of Purkinje cells were measured by whole-cell recording in cerebellar slices of rats with less glutamate action. The role of cytoplasm Ca²⁺ was examined by two-photon cellular imaging and BAPTA infusion in Purkinje cells. Lowering perfusion rate to cerebellar slices deteriorated spike timing and raised spike capacity of Purkinje cells. These changes were associated with the reduction of spike refractory periods and threshold potentials, as well as the loss of their control to spike encoding. Ischemia-induced functional deterioration at Purkinje neurons was accompanied by cytoplasm Ca²⁺ rise and prevented by BAPTA infusion. Therefore, the ischemia destabilizes the spike encoding of Purkinje cells via raising cytoplasm Ca²⁺ without a need for glutamate, which subsequently causes their excitotoxic death.     

Alpha-adrenergic interaction with stimulators of cyclic AMP concentrations in canine thyroid slices.

Thyroid stimulating hormone (TSH) increased cyclic AMP levels approximately 10–20 fold in canine thyroid slices after 30 min incubation. Thereafter the cyclic AMP level declined reaching about 50% of the maximal by 90 min even in the presence of 10 mM theophylline. When phentolamine, an α-adrenergic blocker, was added with TSH to the incubation medium, the decline of cyclic AMP levels that followed the peak was markedly diminished. The maximal effect of phentolamine was observed at a concentration of 10^?6M. A similar decline of the cyclic AMP levels after the peak was observed when the tissues was stimulated by prostaglandin E₁ or cholera toxin and the decline was again prevented by phentolamine. Phentolamine alone had no significant effect on the basal cyclic AMP levels. Phenylephrine, an α-adrenergic agonist, diminished the rise of cyclic AMP levels induced by TSH.Norephinephrine, a physiologic adrenergic stimulator, caused a marked inhibition of the elevation of cyclic AMP levels induced by prostaglandin E₁ or cholera toxin as was the case by TSH (Life Sciences 21, 607, 1977). The norepinephrine effect was abolished by phentolamine, but not by propranolol, a β-adrenergic blocker.These results indicate that α-adrenergic actions may be involved in the counter-regulation of cyclic AMP levels in canine thyroid glands.     

ADENOSINE 3'',5''-MONOPHOSPHATE IN GUINEA PIG CEREBRAL CORTICAL SLICES: EFFECTS OF α- AND β-ADRENERGIC AGENTS, HISTAMINE, SEROTONIN AND ADENOSINE

—Norepinephrine and epinephrine, in combination with either adenosine or histamine, enhanced the accumulation of cyclic AMP in guinea pig cerebral cortical slices. Isoproterenol had only marginal effects under the same conditions. Studies with d- and l-norepinephrine and with the α- and β-adrenergic blocking agents, phenoxybenzamine, phentolamine, dihydroergokryptamine, propranolol and sotalol, indicated that the effect of catecholamines on cyclic AMP levels in this tissue was stereo-specific and was mediated primarily via interaction with a classical α-adrenergic receptor. Studies with the antihistaminics, diphenhydramine and pheniramine, and the antiserotonin agent, methysergide, indicated that guinea pig cerebral cortical slices contain receptors for histamine and serotonin, whose activation also stimulates an enhanced accumulation of cyclic AMP in the presence of adenosine.