The effect of ulinastatin on CFU-C colony formation in mouse myelocyte cultures

The mixture of 5'-guanylylimidodiphosphate (Gpp(NH)p)/EDTA/NaCl has been used to delineate low-affinity conditions for agonists binding to G-protein-linked receptors. The effects of this mixture on [3H]RX821002 (2-methoxyidazoxan) binding to alpha2-adrenoceptors were evaluated in different tissues. The density of alpha2-adrenoceptors in the presence of the mixture was 11, 78 and 60% higher in human cortex (predominant alpha2A), human caudate (alpha2A + alpha2C) and rat kidney (alpha2A + alpha2B), respectively, than in its absence. In rat kidney, masking of alpha2B-adrenoceptors by ARC239 (2-[2-[4-(o-methoxyphenyl)-piperazin-1-yl]-ethyl]-4,4-dimethyl-1,3 -(2H,4H)-isoquinolindione) (50 nM) or masking of alpha2A-adrenoceptors by BRL44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidaz ole) (100 nM) demonstrated that the increase was in the alpha2B-adrenoceptor but not in the alpha2A-adrenoceptor subtype.     

Pharmacological characterization of [3H]idazoxan, [3H]RX821002 and p-[125I]iodoclonidine binding to alpha 2-adrenoceptors in rat cerebral cortical membranes

Binding characteristics of alpha 2-adrenoceptors in rat cerebral cortical membranes were compared using the antagonist radioligands [3H]idazoxan, [3H]2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline ([3H]RX821002), and the partial agonist radioligand [125I]2-[2,6-(dichloro-4-iodophenyl)imino]imidazoline ([125I]iodoclonidine). With [3H]RX821002 and alpha 2-adrenoceptor subtype-selective competitors, both alpha 2A/D- and alpha 2C-adrenoceptor subtypes were detected, suggesting rat cortical membranes contain approximately 90% alpha 2A/D-adrenoceptor subtype and 10% alpha 2C-adrenoceptor subtype. Only alpha 2A/D-adrenoceptors were detected with [3H]idazoxan and [125I]iodoclonidine. All three radioligands bound to a single high affinity site (Kd = 0.3-1.6 nM). However, the densities of sites labeled by [3H]idazoxan and [125I]iodoclonidine were 50% greater than the density labeled by [3H]RX821002, likely representing non-adrenoceptor binding sites. The density of [125I]iodoclonidine binding sites in glycylglycine buffer was similar to that labeled by [3H]RX821002. These results suggest that: (1) alpha 2A/D-adrenoceptors are the predominant subtype in rat cerebral cortex, (2) demonstrate that the small number of alpha 2C-adrenoceptors in this tissue can be detected using prazosin to displace [3H]RX821002 binding, and (3) non-adrenoceptor binding with [125I]iodoclonidine can be minimized with the use of glycylglycine buffer.     

The development of an instrument to measure self-consistency

These studies examined which alpha 2-adrenoceptor subtype is expressed in the hypothalamus and preoptic area and the influence of estradiol administration on alpha 2-adrenoceptors in the hypothalamus of female rats. The alpha 2-adrenoceptor antagonist [3H] RX821002 bound to a single site in hypothalamus, preoptic area, and cortex membranes, with high affinity and low nonspecific binding, as determined by Scatchard and kinetic binding analyses. Competition for [3H]RX821002 binding in the hypothalamus and preoptic area by various noradrenergic agonists and antagonists revealed a unique pharmacological specificity with a high degree of similarity to that of the alpha 2D-adrenoceptor. Norepinephrine displacement of [3H]RX821002 binding in hypothalamic membranes from ovariectomized animals was monophasic and characterized by high affinity. In contrast, norepinephrine competition for [3H]RX821002 binding sites in the hypothalamus from rats exposed to estradiol for 48 hr was biphasic, and norepinephrine bound to both a high (18%) and a low (82%) affinity site in these membranes. Thus, the formation of agonist high affinity alpha 2D-adrenoceptor complexes was inhibited by prior exposure to estrogen. In both control and estradiol-exposed hypothalamic membranes, 100 microM 5'-guanylylimidodiphosphate [Gpp(NH)p] converted the norepinephrine competition curves to ones characterized by monophasic, low affinity binding. In addition, binding of the full alpha 2-adrenoceptor agonist [3H]UK-14,304 in the hypothalamus and preoptic area of female rats was concentration-dependently diminished by Gpp(NH)p treatment. Complete loss of [3H]UK-14,304 binding was effected by 100 microM Gpp(NH)p. This suggests that [3H]UK-14,304 may be useful in labeling the agonist high affinity state of alpha 2-adrenoceptors. Decreasing the incubation temperature in saturation studies from 25 degrees to 0 degrees increased [3H]UK-14,304 binding in hypothalamic membranes of control rats but not in membranes from estradiol-treated rats. Estradiol treatment for 48 hr decreased [3H]UK-14,304 binding in hypothalamic membranes by 34% (0 degrees) to 60% (25 degrees), without changing the Kd. These results suggest that the alpha 2D-adrenoceptor is the predominant subtype in the hypothalamus and preoptic area of female rats and that estradiol treatment markedly reduces the number of alpha 2D-adrenoceptors in the agonist high affinity state.     

Allosteric control of acetylcholinesterase activity by monoclonal antibodies

The aim of this study was to assess the effect of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)-induced alpha2-adrenoceptor inactivation on regulatory G proteins and the recovery of agonist and antagonist binding sites. EEDQ induced a rapid increase in the abundance of rat brain cortical Galphai1/2 proteins (30% at 6 h) which reached a maximum at 4 days (45%) and which then slowly returned (7-30 days) to control values. EEDQ did not alter significantly the levels of Galphai3 and Galphao proteins. By using the standard monoexponential model, the analysis of the recovery of alpha2-adrenoceptor density (6 h-30 days) with [3H]UK 14304 (bromoxidine) and [3H]RX 821002 (2-metoxy idazoxan) in the cerebral cortex did not reveal differences in receptor turnover parameters. However, the recovery of [3H]UK 14304 binding fitted best to a new biphasic recovery model, suggesting the existence of two distinct phases of recovery of agonist sites (r1 and r2 = 15.7 and 7.4 fmol mg protein(-1) day(-1); k1 and k2 = 0.51 and 0.25 day(-1); (t1/2)1 and (t1/2)2 = 1.4 and 2.7 days). In contrast, the recovery of [3H]RX 821002 antagonist sites did not fit to the biphasic model (r = 8.1, k = 0.14, t1/2 = 4.9). Because agonist binding requires coupling to G proteins, the present results suggest that the rapid over-expression of Galphai1/2 proteins induced by EEDQ is related to the biphasic recovery of [3H]UK 14304 binding. The possible implication of the faster recovery of alpha2-adrenoceptor function after EEDQ inactivation is discussed.     

Transition probability cell cycle model. Part I--Balanced growth

In this study, we have identified and characterized functional alpha2-adrenergic receptor (alpha2-AR) subtypes in human corpus cavernosum and in cultured human corpus cavernosum smooth muscle cells. Analysis of total RNA, isolated from whole corpus cavernosum tissue and smooth muscle cells, by RNase protection assays, demonstrated expression of mRNA for alpha2A, alpha2B, and alpha2C adrenergic receptor subtypes in whole tissue and alpha2A and alpha2C subtypes in cultured smooth muscle cells. Binding studies with [3H]RX821002 (a highly selective and specific ligand for alpha2-adrenergic receptor) in isolated membrane fractions of human corpus cavernosum smooth muscle cells, demonstrated specific alpha2-AR binding sites with high affinity (Kd = 0.63 nM) and limited capacity (25-30 fmol/mg protein). Binding of [3H]RX821002 was displaced with the nonselective alpha-AR antagonist, phentolamine, and with the alpha-AR agonist, norepinephrine, in a dose-dependent manner, but not by the selective alpha1-AR agonist, phenylephrine. Binding of [3H]rauwolscine was also displaced by phentolamine. UK 14,304, a selective alpha2-AR agonist, inhibited forskolin-induced cyclic adenosine monophosphate (cAMP) synthesis in cultured human corpus cavernosum smooth muscle cells and induced dose-dependent contractions of tissue strips in organ bath chambers. UK 14,304-induced contractions were inhibited with alpha2-AR selective antagonists, rauwolscine and delquamine (RS 15385-197). These observations suggest that in human corpus cavernosum, norepinephrine (NE) and epinephrine may activate postsynaptic alpha2-AR subtypes, in addition to activating alpha1-AR subtypes, on smooth muscle cells, contributing to local control of human corpus cavernosum smooth muscle tone, in vivo.     

Multiple alpha 2 adrenergic receptor subtypes. I. Comparison of [3H]RX821002-labeled rat R alpha-2A adrenergic receptors in cerebral cortex to human H alpha2A adrenergic receptor and other populations of alpha-2 adrenergic subtypes

In the present study, we examined the binding of the alpha-2 adrenergic receptor (AR) antagonist [3H]-(2-(2-methoxy-1,4-benzodioxan- 2yl)-2-imidazoline ([3H]RX821002) to alpha-2 AR in rat cerebral cortex (CC) and compared the properties of these sites to those of rat alpha-2A (R alpha-2A) AR in submaxillary gland (SMG), human alpha-2A (H alpha-2A) AR in human platelets and alpha-2B AR in neonatal rat lung. In the presence of guanidinium phosphate, [3H]RX821002 bound with high affinity to a large and homogeneous population of sites in CC (Kd = 0.30 +/- 0.03 nM and Bmax = 271 +/- 7 fmol/mg of protein), SMG (Kd = 0.7 and Bmax = 274), human platelets (Kd = 0.6 nM and Bmx = 189) and neonatal rat lung (kd = 0.9 and Bmax = 161). A total of 34 chemically diverse AR ligands monophasically inhibited the binding of [3H]RX821002 from each site with, for the CC, the most potent ligand being atipamezole (Ki = 0.2 nM). For all ligands, and at each site, Hill coefficients did not differ significantly from unity. Although the profiles of inhibition of [3H]RX821002 were virtually identical in rat CC and SMG, these populations revealed several marked differences to human platelets; the alkaloids, rauwolscine and yohimbine, as well as the benzodioxane, [2-(2,6- dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane] (WB 4101), displayed about 10-fold lower affinity for R alpha-2A as compared to H alpha-2A sites, whereas the benzopyrrolidines, fluparoxan and des-fluorofluparoxan, showed about 10-fold greater affinity for R alpha-2A sites. Further, whereas the calculation of potency ratios for selected pairs of ligands, as well as of correlation coefficients, revealed virtual identity between R alpha-2A AR in CC and SMG, these analyses revealed that each of these populations of R alpha-2A AR clearly differed to H alpha-2A AR in human platelets. In addition, both R alpha-2A AR in rat CC and SMG as well as H alpha-2A AR in human platelets markedly differed to alpha-2B AR in neonatal rat lung; thus, they showed 20-fold higher affinity for [2-(2H-(1-methyl-1,3-dihydroisoindole)methyl)-4,5- dihydroimidazoline] (BRL 44408), oxymetazoline, guanfacine and guanabenz yet 10- to 100-fold lower affinity for [2-(2-4-o- methoxyphenyl)piperazine-1-yl)-ethyl)-4,4-dimethyl-1,3-(2H,4H)- isoquinolinedione] (ARC 239) prazosin, chlorpromazine and corynanthine. Similar differences in R alpha-2A and H alpha-2A sites to alpha-2C sites were apparent upon analysis of literature data.(ABSTRACT TRUNCATED AT 400 WORDS)     

The visual instruction system

alpha 2-Adrenoceptors are remarkably regulated by developmental factors. In this study alpha 2-adrenoceptor subtypes have been characterised in neonatal and adult rat spinal cords. In saturation experiments, a 5% proportion of [3H]rauwolscine binding has a high affinity component, representing the alpha 2C-subtype in both tissues. Competition studies with [3H]RX821002 indicate that in both tissues the alpha 2A/D subtype is expressed similarly.     

Up-regulation of immunolabeled alpha2A-adrenoceptors, Gi coupling proteins, and regulatory receptor kinases in the prefrontal cortex of depressed suicides

Suicide and depression are associated with an increased density of alpha2-adrenoceptors (radioligand receptor binding) in specific regions of the human brain. The function of these inhibitory receptors involves various regulatory proteins (Gi coupling proteins and G protein-coupled receptor kinases, GRKs), which work in concert with the receptors. In this study we quantitated in parallel the levels of immunolabeled alpha2A-adrenoceptors and associated regulatory proteins in brains of suicide and depressed suicide victims. Specimens of the prefrontal cortex (Brodmann area 9) were collected from 51 suicide victims and 31 control subjects. Levels of alpha2A-adrenoceptors, Galphai1/2 proteins, and GRK 2/3 were assessed by immunoblotting techniques by using specific polyclonal antisera and the immunoreactive proteins were quantitated by densitometry. Increased levels of alpha2A-adrenoceptors (31-40%), Galphai1/2 proteins (42-63%), and membrane-associated GRK 2/3 (24-32%) were found in the prefrontal cortex of suicide victims and antidepressant-free depressed suicide victims. There were significant correlations between the levels of GRK 2/3 (dependent variable) and those of alpha2A-adrenoceptors and Galphai1/2 proteins (independent variables) in the same brain samples of suicide victims (r = 0.56, p = 0.008) and depressed suicide victims (r = 0.54, p = 0.041). Antemortem antidepressant treatment was associated with a significant reduction in the levels of Galphai1/2 proteins (32%), but with modest decreases in the levels of alpha2A-adrenoceptors (6%) and GRK 2/3 (18%) in brains of depressed suicide victims. The increased levels in concert of alpha2A-adrenoceptors, Galphai1/2 proteins, and GRK 2/3 in brains of depressed suicide victims support the existence of supersensitive alpha2A-adrenoceptors in subjects with major depression.     

Effects of imidazoline derivatives on cholinergic motility in guinea-pig ileum: involvement of presynaptic alpha2-adrenoceptors or imidazoline receptors?

The present study investigates the possibility that imidazoline receptors mediate modulation of cholinergic motor functions of the guinea-pig ileum. For this purpose, the effects of a series of compounds with known affinity for alpha2-adrenoceptors and/or imidazoline recognition sites were examined on the cholinergic twitch contractions evoked by electrical field stimulation (0.1 Hz) of longitudinal muscle-myenteric plexus preparations. Additional experiments were carried out on ileal strips preincubated with [3H]choline, superfused with physiological salt solution containing hemicholinium-3, and subjected to electrical field stimulation (1 Hz). The stimulation-induced outflow of radioactivity was taken as an index of endogenous acetylcholine release. Alpha-methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline and xylazine caused a concentration-dependent inhibition of twitch responses (IC50 from 0.13 to 1.05 microM; Emax from 85.9 to 92.5%). Rilmenidine and agmatine were less potent in reducing the twitch activity, and the latter compound acted also with low intrinsic activity (IC50=44.9 microM; Emax=35.5%). In interaction experiments, the inhibitory action of clonidine on twitch responses was competitively antagonized by RX 821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline), idazoxan, rauwolscine, yohimbine and BRL 44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)-methyl] -4,5-dihydroimidazoline), whereas prazosin (10 microM), ARC 239 (2-(2,4-(O-methoxy-phenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl- 1,3-(2H,4H)-isoquinolindione; 10 microM) and BRL 41992 (1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo[c,f]imidazol[1,5-a]a zepine; 10 microM) were without effect. Rauwolscine antagonized the inhibitory effects of various agonists on ileal twitch activity in a competitive manner and with similar potency. Agmatine and idazoxan did not significantly modify the twitch contractions when tested in the presence of alpha2-adrenoceptor blockade by rauwolscine (3 microM) or RX 821002 (1 microM). Linear regression analysis showed that the affinity values of antagonists correlated with their affinity at the alpha2A and alpha2D binding sites as well as at previously classified alpha2A/D adrenoceptor subtypes, whereas no significant correlation was obtained when comparing the potency estimates of agonists and antagonists with the affinity at I1 or I2 binding sites. When tested on the electrically induced outflow of tritium, alpha-methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline, xylazine and rilmenidine yielded inhibitory concentration-response curves which were shifted rightward to a similar extent in the presence of rauwolscine (3 microM). In the absence of further drugs, agmatine significantly reduced the evoked tritium outflow at the highest concentrations tested (10 and 100 microM), whereas idazoxan (up to 100 microM) was without effect. When RX 821002 (1 microM) was added to the superfusion medium, neither agmatine nor idazoxan modified the evoked outflow of radioactivity. The results argue against modulation by imidazoline receptors of acetylcholine release from myenteric plexus nerve terminals. They provide evidence that compounds endowed with imidazoline-like structures affect the cholinergic motor activity of the guinea-pig ileum by interacting with presynaptic alpha2-adrenoceptors belonging to the alpha2D subtype.     

Characterization of adrenoceptor types and subtypes in American bullfrogs acclimated to warm or cold temperature

While indirect evidence suggested that the responsiveness of frog adrenoceptors changes in response to temperature, direct measurement of adrenoceptor binding following acclimation to warm and cold temperatures had not been done. In the present study, the radioligands [3H]prazosin, [3H]RX821002, and [125I]cyanopindolol were used to label and quantify alpha 1-, alpha 2-, and beta-adrenoceptors in bullfrogs acclimated to warm or cold environments. The number of alpha 1-, alpha 2-, and beta-adrenoceptors in atrium, ventricle, and kidney membranes was not significantly different between warm- and cold-acclimated frogs. Characterization of receptor subtypes using pharmacological antagonists demonstrated that alpha 2-adrenoceptors in frog spinal cord and kidney were of the same pharmacological subtype, which is similar to the mammalian alpha 2A-subtype. The beta-adrenoceptor in frog ventricle, atrium, and kidney was the beta 2-subtype. These results suggest that while the alpha 1-, alpha 2-, and beta-adrenoceptor types have evolved in the frog, multiple subtypes of adrenoceptors are not necessary for physiological regulation in this species.     

Effects of the antiparkinsonian drug budipine on neurotransmitter release in central nervous system tissue in vitro

The effects of the antiparkinsonian drugs budipine and biperiden on spontaneous and electrically evoked release of dopamine (DA), acetylcholine (ACh), GABA or noradrenaline (NA) were studied in caudate nucleus or cortex slices, respectively, of the rabbit brain. Whereas both drugs (1-10 microM) strongly increased spontaneous [3H]outflow in caudate nucleus slices preincubated with [3H]DA, budipine inhibited but biperiden facilitated the evoked DA release. In the presence of the DA-reuptake inhibitor nomifensine, a significant part of the budipine-induced basal [3H] outflow consisted of unmetabolized DA. Synaptosomal high-affinity uptake of [3H]DA was only weakly affected by budipine and biperiden (IC50 values, 11 and 9 microM, respectively). Budipine enhanced also basal [3H]outflow from cortex slices prelabeled with [3H]NA, however this outflow consisted mainly of NA metabolites even in the presence of cocaine. The evoked release of [3H]ACh in rabbit caudate nucleus slices preincubated with [3H] choline was almost unaffected by budipine but enhanced by biperiden in the absence of further drugs. In the presence of nomifensine, however, budipine inhibited, but biperiden still enhanced, the evoked ACh release. Moreover, both drugs showed antimuscarinic properties in the presence of the ACh esterase inhibitor physostigmine, i.e., they facilitated the evoked ACh release, exhibiting pA2 values of about 6.9 (budipine) and 8.3 (biperiden). Addition of the D2 receptor antagonist domperidone diminished all inhibitory effects of budipine on the evoked ACh release. The evoked overflow of [3H] in caudate nucleus slices preincubated with [3H]GABA was reduced by both budipine and biperiden. It is concluded that both anticholinergic and indirect dopaminomimetic properties contribute to the antiparkinsonian effects of budipine, whereas biperiden exhibits mainly anticholinergic effects. Moreover, both drugs might disinhibit GABA controlled neurons in the central nervous system.     

Simultaneous quantification of serotonin, dopamine and noradrenaline levels in single frontal cortex dialysates of freely-moving rats reveals a complex pattern of reciprocal auto- and heteroreceptor-mediated control of release

In the present study, a novel and exceptionally sensitive method of high-performance liquid chromatography coupled to coulometric detection, together with concentric dialysis probes, was exploited for an examination of the role of autoreceptors and heteroceptors in the modulation of dopamine, noradrenaline and serotonin levels in single samples of the frontal cortex of freely-moving rats. The selective D3/D2 receptor agonist, CGS 15855A [(+/-)-trans-1,3,4,4a,5,10b-hexahydro-4-propyl-2H-[1]benzopyrano[3 ,4-b]-pyridin-9-ol], and antagonist, raclopride, respectively decreased (-50%) and increased (+60%) levels of dopamine without significantly modifying those of serotonin and noradrenaline. The selective alpha2-adrenergic receptor agonist, dexmedetomidine, markedly decreased noradrenaline levels (-100%) and likewise suppressed those of serotonin and dopamine by -55 and -45%, respectively. This effect was mimicked by the preferential alpha2-adrenergic receptor agonist, guanabenz (-100%, -60% and -50%). Furthermore, the alpha2-adrenergic receptor antagonist, RX 821,002 [2(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline], and the preferential alpha2A-adrenergic receptor antagonist, BRL 44408 [2-(2H-(1-methyl-1,3-dihydroisoindole)methyl)-4,5-dihydroimidaz ole], both evoked a pronounced elevation in levels of noradrenaline (+212%, +109%) and dopamine (+73%, +85%). In contrast, the preferential alpha(2B/2C)-adrenergic receptor antagonist, prazosin, did not modify noradrenaline and dopamine levels. RX 821,002 and BRL 44408 did not significantly modify levels of serotonin, whereas prazosin decreased these levels markedly (-55%), likely due to its alpha1-adrenergic receptor antagonist properties. The selective serotonin-1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), reduced serotonin levels (-65%) and increased those of dopamine and noradrenaline by +100%), and +175%, respectively. The selective serotonin-1A antagonist, WAY 100,635 [N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclo- hexanecarboxamide], which had little affect on monoamine levels alone, abolished the influence of 8-OH-DPAT upon serotonin and dopamine levels and significantly attenuated its influence upon noradrenaline levels. Finally, the selective serotonin-1B agonist, GR 46611 [3-[3-(2-dimethylaminoethyl)-1H-indol-5-yl]-N-(4-methoxybenzyl)acrylamid e], decreased serotonin levels (-49%) and the serotonin-1B antagonist, GR 127,935 [N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-2'-methyl-4'-(5-me thyl-1,2,4-oxadiazol-3-yl)-biphenyl-4-carboxamide], which did not significantly modify serotonin levels alone, abolished this action of GR 46611. Levels of dopamine and noradrenaline were not affected by GR 46611 or GR 127,935. In conclusion, there is a complex pattern of reciprocal autoreceptor and heteroceptor control of monoamine release in the frontal cortex. Most notably, activation of alpha2-adrenergic receptors inhibits the release of noradrenaline, dopamine and serotonin in each case, while stimulation of serotonin-1A receptors suppresses serotonin, yet facilitates noradrenaline and dopamine release. In addition, dopamine D2/D3 autoreceptors restrain dopamine release while (terminal-localized) serotonin-1B receptors reduce serotonin release. Control of serotonin release is expressed phasically and that of noradrenaline and dopamine release tonically.     

High affinity [3H]imipramine and [3H]paroxetine binding sites in suicide brains

Specific binding of [3H]imipramine and [3H]paroxetine was simultaneously examined in human brains (frontal cortex, temporal cortex, cingulate cortex, hypothalamus, hippocampus and amygdala) from 11 controls and 11 depressed suicide victims. A single saturable high affinity site was obtained for both radioligands. Age was not related to significant changes in [3H]imipramine and [3H]paroxetine binding parameters, which indicates the stability of the brain serotonergic system with increasing age. A major finding of the present study concerns the existence of a significant decrease in the maximum number (Bmax) of [3H]imipramine binding sites in hippocampus from depressed suicides as compared with the control group, without changes in the binding affinity (Kd). In contrast, when [3H]paroxetine was used as radioligand, no changes in either Bmax or Kd were detected in any of the brain regions studied. These findings suggest that [3H]imipramine may be a better marker than [3H]paroxetine when alterations in the presynaptic serotonergic uptake site are to be detected.     

Active immunization using dendritic cells mixed with tumor cells inhibits the growth of primary breast cancer

The potential role of alpha2-adrenoceptors in modulating the activity of adenylyl cyclase in the rat striatum was examined. The selective alpha2-adrenoceptor agonist, UK14,304, produced a concentration-dependent inhibition of forskolin-stimulated accumulation of cAMP in striatal slices. The effect of UK14,304 was reversed by pre-incubation of striatal slices with the selective alpha2-adrenoceptor antagonist, RX821002. To determine whether alpha2C-adrenoceptors contribute to the alpha2-adrenoceptor-induced inhibition of forskolin-stimulated cAMP accumulation, an antisense oligodeoxynucleotide directed against alpha2C-adrenoceptor mRNA (alpha(2C)AS) or a random sequence (RS) was infused directly into the striatum. The ability of alpha(2C)AS to reduce the expression of alpha2C-adrenoceptors has been previously demonstrated. Alpha2C(AS) infusions did not reduce the ability of UK14,304 to inhibit forskolin-stimulated cAMP accumulation. Instead, alpha(2C)AS significantly enhanced forskolin-stimulated cAMP accumulation on the infusion side compared to the contralateral striatum. In contrast to the effects of alpha(2C)AS, infusions of RS had no effects on forskolin-stimulated cAMP accumulation or on the ability of UK14,304 to inhibit this effect. Incubation of striatal slices from untreated rats with RX821002 could mimic the ability of alpha(2C)AS infusion to enhance forskolin-stimulated cAMP accumulation, and did so in a concentration-dependent manner. Alpha2-adrenoceptors are negatively coupled to adenylyl cyclase in the rat striatum and alpha2C-adrenoceptors appear to be under tonic activation by an endogenous ligand in striatal slices.     

Biotransformation of 17-alkylsteroids in the equine: gas chromatographic-mass spectral identification of ten intermediate metabolites of methyltestosterone

Parenterally administered domoic acid, a structural analog of the excitatory amino acids glutamic acid and kainic acid, has specific effects on brain histology in rats, as measured using different anatomic markers. Domoic acid-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex, and different anatomic markers can detect these neurotoxic effects to varying degrees. Here we report effects of domoic acid administration on quantitative indicators of brain metabolism and gliosis. Domoic acid, 2.25 mg/kg i.p., caused stereotyped behavior and convulsions in approximately 60% of rats which received it. Six to eight days after domoic acid or vehicle administration, the animals were processed to measure regional brain incorporation of the long-chain fatty acids [1-(14)C]arachidonic acid ([14C]AA) and [9,10-(3)H]palmitic acid ([3H]PA), or regional cerebral glucose utilization (rCMRglc) using 2-[1-(14)C]deoxy-D-glucose, by quantitative autoradiography. Others rats were processed to measure brain glial fibrillary acidic protein (GFAP) by enzyme-linked immunosorbent assay. Domoic acid increased GFAP in the anterior portion of cerebral cortex, the caudate putamen and thalamus compared with vehicle. However, in rats that convulsed after domoic acid GFAP was significantly increased throughout the cerebral cortex, as well as in the hippocampus, septum, caudate putamen, and thalamus. Domoic acid, in the absence of convulsions, decreased relative [14C]AA incorporation in the claustrum and pyramidal cell layer of the hippocampus compared with vehicle-injected controls. In the presence of convulsions, relative [14C]AA incorporation was decreased in hippocampus regions CA1 and CA2. Uptake of [3H]PA into brain was unaffected. Relative rCMRglc decreased in entorhinal cortex following domoic acid administration with or without convulsions. These results suggest that acute domoic acid exposure affects discrete brain circuits by inducing convulsions, and that domoic acid-induced convulsions cause chronic effects on brain function that are reflected in altered fatty acid metabolism and gliosis.     

Lymphocyte proliferative responses to recombinant bovine herpes virus type 1 (BHV-1) glycoprotein gD (gIV) in immune cattle: identification of a T cell epitope

Effects of the antipsychotics risperidone and clozapine on 5-HT2 and D2-dopamine receptor binding were examined using [3H]N-methylspiperone ([3H]NMSP) and in vitro receptor autoradiography on human whole hemisphere cryosections. The 5-HT2 receptor antagonist ketanserin and the D2-dopamine receptor antagonist raclopride were used as references. [3H]NMSP binding was observed in caudate nucleus, putamen, and cerebral cortex indicating binding to D2-dopamine and 5-HT2 receptors. Risperidone and clozapine counteracted the binding to both receptor types. This was in contrast to raclopride, which selectively blocked the D2-dopamine receptors in the basal ganglia, and ketanserin, which selectively blocked the 5-HT2 receptors in the cerebral cortex. Risperidone (100 nM and 10 microM) blocked up to 90% of [3H]NMSP binding to both receptor types, whereas the blocking capacity of clozapine (10 microM) was lower (approximately 60%). The lack of total blockade of D2-dopamine receptors is in line with results obtained in with [11C]raclopride and positron emission tomography studies of clozapine treated human subjects. However, autoradiographic studies of clozapine competition of [3H]raclopride binding show total displacement of the binding at high clozapine concentrations, thus contradicting the PET results with [11C]raclopride, as well as the autoradiographic results obtained with [3H]NMSP. In conclusion it can be stated that pharmacological concentrations of the two drugs clozapine and risperidone block a large proportion of D2-dopamine receptors and 5-HT2 receptors in the human brain. Moreover, the study shows the usefulness of human whole hemisphere autoradiography for the study of interaction of drugs with different central neurotransmitter receptors.     

Differential effects of aging on binding sites of the activated NMDA receptor complex in mice

The NMDA receptor site has been shown to be vulnerable to the effects of aging. Decreases in binding to the receptor site of up to 50% have been reported in aged animals. The present study was designed to quantitate and compare the effects of aging on multiple binding sites of the NMDA receptor complex in various brain regions. Autoradiography with [3H]glutamate, [3H]CPP, [3H]glycine, [3H]MK801 and [3H]TCP was performed on brain sections from 3, 10 and 28-30 month old C57B1/6 mice. The percent declines between 3 and 28-30 months of age in [3H]-glutamate (15-35% declines) and [3H]CPP (20-42% declines) binding were similar within most cortical regions and the caudate nucleus but [3H]glutamate binding showed less change (0-11% declines) than [3H]CPP (13-27% declines) in the occipital/temporal cortex and hippocampal regions. [3H]MK801 and [3H]TCP binding, stimulated by 10 microM glutamate, exhibited intermediate aging changes between the glycine and NMDA sites, both in percent decline (3-28% and 0-26%, respectively) and in the number of brain regions involved. [3H]Glycine binding, stimulated by 10 microM glutamate, showed no significant overall effect of age (declines ranged from 0-34%). [3H]CPP binding was significantly more affected than [3H]glycine binding in many regions. These results suggest that aging has heterogeneous effects on different sites on the NMDA receptor complex throughout the brain and on NMDA receptor agonist versus antagonist binding in selected brain regions.     

A postmortem study of frontal cortical dopamine D1 receptors in schizophrenics, psychiatric controls, and normal controls

We tested the hypothesis that aberrant dopaminergic innervation in frontal and cingulate cortices of schizophrenic patients might be revealed by examining dopamine D1 receptor density in these brain regions. A quantitative autoradiographic assay with [3H]-SCH 23390 was performed with samples from schizophrenic patients, normal controls, neuroleptic-treated controls, and suicides. There was a significant elevation in specific binding of [3H]-SCH 23390 in the intermediate layer of the prefrontal cortex from neuroleptic-treated controls (p = .05). Elevated [3H]-SCH 23390 binding in several layers from prefrontal and cingulate cortex was observed in schizophrenic subjects, although these results did not reach statistical significance. When data from subjects who had received neuroleptics (schizophrenics and neuroleptic controls) were compared to subjects who had not received neuroleptics (normal controls and suicides), there was a significant elevation in receptor density in both the prefrontal (p = .05) and cingulate cortices (p = .03). These data suggest that elevated [3H]-SCH 23390 binding in human prefrontal and cingulate cortices may occur with chronic neuroleptic treatment, although increased receptor density that may exist as a feature of psychotic illnesses cannot be excluded.     

Selective loss of pallidal dopamine D2 receptor density in hepatic encephalopathy

The binding parameters of [3H]SCH 23390 and [3H]spiperone (radioligands for dopamine D1 and D2 receptors, respectively) were investigated in autopsied frontal cortex, caudate nucleus and globus pallidus/putamen of cirrhotic patients who died in hepatic coma as well as in age- and sex-matched controls. Specific [3H]SCH 23390 binding site densities were unchanged in all regions; in contrast, specific [3H]spiperone binding site density was decreased (by 44%, P < 0.001) in the globus pallidus/putamen of patients with HE. Decreased densities of pallidal D2 binding sites could relate to the motor dysfunctions commonly encountered in human HE.