Sulphur-containing excitatory amino acid-evoked Ca(2+)-independent release of D-[3H]aspartate from cultured cerebellar granule cells: the role of glutamate receptor activation coupled to reversal of the acidic amino acid plasma membrane carrier. Sulphur-containing excitatory amino acid transmitter candidates (500 microM) stimulated the Ca(2+)-independent efflux of exogenously-supplied D-[3H]aspartate from primary cultures of cerebellar granule cells superfused continuously with HEPES-buffered saline containing CoCl2 (1 mM) in place of CaCl2. The stimulated release of D-[3H]aspartate was markedly attenuated by 200 microM 6,7-dinitroquinoxalinedione, a concentration at which the antagonist inhibits both non-N-methyl-D-aspartate and N-methyl-D-aspartate ionotropic excitatory amino acid receptors. The Ca(2+)-independent component of evoked release was also markedly attenuated and, in some cases, abolished by removing NaCl from the superfusion medium. Furthermore, when 700 microM dihydrokainate (demonstrated herein as a mixed/non-competitive inhibitor of the high-affinity dicarboxylic amino acid transporter in cultured granule cells) was included in the superfusion medium, stimulated efflux of D-[3H]aspartate was reduced by between 15-78% of the control response; the extent of inhibition varying with the agonist employed. In constrast, agents which act as competitive inhibitors of the plasma membrane carrier in granule cells, e.g. beta-methylene-D,L-aspartate, potentiated the release of D-[3H]aspartate in a synergistic manner. Taken together, these findings are consistent with a mechanism for the Ca(2+)-independent release of D-[3H]aspartate that is mediated predominantly by activation of excitatory amino acid receptors resulting in a reversal of the high-affinity dicarboxylic amino acid transport system. Although the physiological relevance of such non-vesicular release from the cytosol remains obscure and is still a matter of some debate, this mode of release may be of pathological significance.


A functional alpha-bungarotoxin receptor is present in chick cerebellum: purification and characterization. It has recently been demonstrated that alpha-bungarotoxin receptors, which behave as functional nicotinic receptors, are present in chick CNS. In this paper, we report the purification and characterization of a functional alpha-bungarotoxin receptor from chick cerebellum, a nervous tissue in which a clear inhibition of induced nicotine effects has been reported in vivo. This receptor contains at least three subunits of apparent mol. wt 52,000, 57,000 and 67,000. The use of monoclonal antibodies specific for the alpha 7 subunit demonstrated that 75% of the molecules present in our purified preparation belong to the alpha 7 subtype and that this antibody labels the 57,000 band in western blot, thus indicating that this is the toxin binding subunit. Reconstruction experiments in planar lipid bilayers show that this alpha-bungarotoxin receptor forms a cation selective channel whose opening is blocked by d-tubocurarine. Binding experiments on immobilized receptors over an alpha-bungarotoxin-Sepharose affinity column show that the ligand binding subunit is present in vivo in two copies per receptor. Immunological, pharmacological and functional experiments show that this purified receptor is very similar, but not identical, to the previously characterized chick optic lobe receptor, thus indicating the heterogeneity of these alpha-bungarotoxin receptors in the CNS.


Dopaminergic drugs reverse the impairment of radial-arm maze performance caused by lesions involving the cholinergic medial pathway. Pharmacological studies have shown that both cholinergic and dopaminergic transmitter systems are crucial for optimal choice accuracy in the radial-arm maze and that these systems interact in a complex fashion. Lesion studies have provided evidence that the basal nuclear complex of the forebrain, the origin of cholinergic projections to the cerebral mantle, may be critical for the cholinergic modulation of learning and memory. We have shown that knife-cut lesions of the medial cholinergic pathway significantly impair radial-arm maze choice accuracy performance. The current study examined the effectiveness of D1 and D2 ligands in counteracting this lesion-induced deficit. The adverse effects of medial cholinergic pathway lesions were diminished or reversed by daily treatment with a D1 agonist (SKF 38393), a D2 agonist (LY 171555) or a D1 antagonist (SCH 23390), but were not affected by treatment with a D2 antagonist (raclopride). The three beneficial treatments have previously been found to attenuate the adverse effects of nictonic or muscarinic blockade on choice accuracy performance in the radial-arm maze. The finding that these dopaminergic drugs ameliorate the memory deficit caused by lesions involving the cholinergic medial pathway suggests the importance of interactions between cholinergic and dopaminergic systems in radial-arm maze performance. These results may provide leads for the development of novel therapeutic approaches for treating human disorders thought to result from cholinergic hypofunction.


Repeated stimulation of D1 dopamine receptors causes time-dependent alterations in the sensitivity of both D1 and D2 dopamine receptors within the rat striatum. Recent evidence suggests that repeated stimulation of D1 dopamine receptors within the rat striatum leads to an enhancement of both D1 and D2 dopamine receptor-mediated responses. The present study used both behavioral observations and extracellular single unit recording techniques to investigate this phenomenon following repeated administration of selective D1 dopamine receptor agonists. Groups of rats received twice daily administration of either saline or the partial D1 dopamine receptor agonist SKF 38393 (8 mg/kg, s.c.) for three weeks. Rats were tolerant to the ability of SKF 38393 to enhance grooming behavior when tested immediately following the last of the 42 treatment injections. However, the ability of this last SKF 38393 injection to potentiate oral stereotyped behavior following administration of the D2 DA agonist quinpirole was still evident. Following a one-day withdrawal, grooming responses to SKF 38393 had returned to normal. At this time, administration of quinpirole, without concomitant SKF 38393, failed to significantly promote oral stereotypies, as is typical of normal rats. Following a one-week withdrawal period, SKF 38393-induced grooming behavior was significantly enhanced and quinpirole, administered without SKF 38393, produced pronounced oral stereotyped behavior in 10 of 12 rats tested. Following a one-month withdrawal, these sensitized responses were no longer evident. Single-cell recordings from rat lateral striatal neurons revealed similar time-dependent alterations in the effects of iontophoretically administered SKF 38393 and quinpirole. Current-response curves revealed that, without a withdrawal period, striatal neurons were subsensitive to the inhibitory effects of SKF 38393 but not quinpirole. The decreased inhibitory responses of striatal neurons to SKF 38393 returned to normal levels after a one-day withdrawal. Following a one-week withdrawal, the effects of both agonists were significantly greater than that in saline-treated controls. Normosensitivity was evident following a one-month withdrawal. Repeated administration of the full D1 DA agonist SKF 81297 (0.5 mg/kg, s.c., twice daily) also resulted in sensitized responses of striatal neurons following a one-week withdrawal, demonstrating that the sensitization to SKF 38393 was not due to its partial agonist character. The present findings provide both behavioral and electrophysiological evidence that repeated stimulation of D1 dopamine receptors results in a brief subsensitivity, followed by transient sensitization of the D1 receptors. The enhanced effects of D2 dopamine agonists might be due to an enhanced synergism (enabling) produced by endogenous dopamine stimulating supersensitive D1 receptors.(ABSTRACT TRUNCATED AT 400 WORDS)


Analysis of the prion protein gene in thalamic dementia. Thalamic degenerations or dementias are poorly understood conditions. The familial forms are (1) selective thalamic degenerations and (2) thalamic degenerations associated with multiple system atrophy. Selective thalamic degenerations share clinical and pathologic features with fatal familial insomnia, an autosomal dominant disease linked to a mutation at codon 178 of the prion protein (PrP) gene that causes the substitution of asparagine for aspartic acid (178Asn mutation). We amplified the carboxyl terminal coding region of the PrP gene from subjects with selective thalamic dementia or thalamic dementia associated with multiple system atrophy. Three of the four kindreds with selective thalamic dementia and none of the three kindreds with thalamic dementia associated with multiple system atrophy had the PrP 178Asn mutation. Thus, analysis of the PrP gene may be useful in diagnosing the subtypes of thalamic dementia. Moreover, since selective thalamic dementia with the PrP 178Asn mutation and fatal familial insomnia share clinical and histopathologic features, we propose that they are the same disease.


A PrP gene codon 178 base substitution and a 24-bp interstitial deletion in familial Creutzfeldt-Jakob disease. Several mutations in the prion protein (PrP) gene are associated with familial Creutzfeldt-Jakob disease (FCJD). We describe a family in which five members in three generations have had FCJD. The proband and some descendants of the affected members carried an abnormal PrP gene allele. This allele contained a 24-bp deletion from the tandem repeat region of the open reading frame and a codon 178 missense substitution. Observations suggest that the codon 178 mutation is involved in the pathogenesis of FCJD in the family described here. The 24-bp deletion may be an uncommon polymorphism.


Aromatic L-amino acid decarboxylase deficiency: clinical features, diagnosis, and treatment of a new inborn error of neurotransmitter amine synthesis. We report the clinical features, biochemical details, and treatment of the first detected cases of an inborn error of aromatic L-amino acid decarboxylase. Male monozygotic twins presented with extreme hypotonia and oculogyric crises. Concentrations of biogenic amines and their metabolites were reduced considerably both centrally and peripherally. Pterin and phenylalanine metabolism were normal. Activity of aromatic L-amino acid decarboxylase was virtually absent in a liver biopsy sample and greatly reduced in plasma. Concentrations of L-dopa, 3-methoxytyrosine, and 5-hydroxytryptophan were elevated in CSF, plasma, and urine. CSF S-adenosylmethionine concentrations were reduced. Pyridoxine treatment had no clinical effect but led to a fall in CSF L-dopa and 3-methoxytyrosine and a rise in S-adenosylmethionine. Treatment with either bromocriptine or tranylcypromine stopped the abnormal eye movements; tranylcypromine treatment also improved muscle tone and led to a rise in plasma norepinephrine and whole blood serotonin. Combined treatment with pyridoxine, bromocriptine, and tranylcypromine produced sustained improvement in tone and voluntary movements. The twins' parents were asymptomatic but had reduced plasma aromatic L-amino acid decarboxylase activity, consistent with heterozygosity. We monitored a subsequent pregnancy through biochemical analyses of a fetal liver biopsy sample and of amniotic fluid. We predicted an unaffected fetus, which was confirmed clinically and biochemically after birth.


Hyperammonemic alterations in the metabolism of glutamate and aspartate in rat cerebellar astrocytes. Pathophysiological concentrations of ammonia, both in vivo and in vitro, suppressed the production of 14CO2 from 14C-labelled glutamate and aspartate in astrocytes isolated from the rat cerebellum. Suppression of 14CO2 production with (aminooxy)acetic acid but not with glutamic acid diethyl ester indicated that transamination plays a major role in the oxidation of glutamate carbons. Activities of the enzymes, aspartate amino-transferase, alanine aminotransferase and glutaminase were decreased while those of glutamate dehydrogenase and glutamine synthetase were enhanced in the cerebellar astrocytes during hyperammonemic states. These results suggest an impairment of astrocytic glutamate metabolism during hyperammonemia.


The selective A2 adenosine receptor agonist CGS 21680 enhances excitatory transmitter amino acid release from the ischemic rat cerebral cortex. The effects of the selective adenosine A2 receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine hydrochloride (CGS 21680) on aspartate and glutamate release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (20 min) was elicited by four vessel occlusion. Pretreatment with CGS 21680 failed to alter basal excitatory amino acid levels, however, CGS 21680 at 10(-6) M significantly enhanced the ischemia-evoked release. Thus, aspartate and glutamate release during ischemia can be stimulated via the activation of A2 receptors, in addition to the suppression of excitatory amino acid release mediated by selective A1 receptor agonists.


Mossy fiber long-term potentiation shows specificity but no apparent cooperativity. Specificity in long-term potentiation (LTP) means that synapses onto a postsynaptic cell can potentiate independently of one another. Cooperativity refers to a requirement that some threshold number of afferents be co-activated to evoke LTP with a high-frequency stimulus. The induction of long-term potentiation (LTP) at the associational/commissural synapses onto hippocampal CA3 pyramidal cells shows clear cooperativity. LTP of mossy fiber inputs to these cells does not. Mossy fiber LTP does show synapse specificity. These results bear on the cellular mechanisms and the functions of mossy fiber LTP.


Effects of pretreatment with sublethal ischemia on the extracellular glutamate concentrations during secondary ischemia in the gerbil hippocampus evaluated with intracerebral microdialysis. To elucidate the role of glutamate in the pathogenesis of altered neuronal vulnerability following sublethal ischemia, we measured the extracellular concentrations of glutamate in the gerbil hippocampus using a microdialysis technique. During and immediately after 3-min forebrain ischemia, the extracellular glutamate level showed a striking increase. However, pretreatment with 2-min ischemia 1 h or 4 days before secondary 3-min ischemia did not alter the amount of glutamate released. The result indicates that cumulative damage and tolerance, which take place after pretreatment with sublethal ischemia, are not induced through modification of the amount of glutamate released during secondary ischemia.


Changes in hippocampal acetylcholine and glutamate extracellular levels during soman-induced seizures: influence of septal cholinoceptive cells. The changes in extracellular acetylcholine and glutamate levels were determined, during the course of seizures induced by soman, an irreversible inhibitor of acetylcholinesterase, in the CA1 hippocampal area of rats previously injected with atropine or normal saline into septum. The marked increases observed in soman-treated animals were abolished in rats receiving atropine. These data strongly suggest that, during soman intoxication, septal cholinoceptive cells play a key role in controlling the release of acetylcholine and glutamate in hippocampus. The mechanisms underlying this phenomenon are discussed.


Comparative neurotoxic potential of glutamate, endothelins, and platelet-activating factor in cerebral cortical cultures. The excitatory amino acid glutamate, the peptides endothelin-1 and -3, and the phospholipid platelet-activating factor have been implicated in ischemic injury to the nervous system. To determine if, like glutamate, endothelins and platelet-activating factor are directly toxic to neurons, we examined their effects on lactate dehydrogenase release and trypan blue dye exclusion in rat cerebral cortical cultures. Glutamate (1 mM) increased lactate dehydrogenase release by approximately 75% and reduced the number of cells excluding trypan blue dye by approximately 50%. In contrast, endothelins (0.5 and 100 nM) and platelet-activating factor (0.1 and 10 microM) had no effect on these indices of cell injury. Endothelins and platelet-activating factor appear more likely to act on blood vessels than on neurons or glia as mediators of ischemic injury.


Production of seizures and brain damage in rats by alpha-dendrotoxin, a selective K+ channel blocker. alpha-Dendrotoxin (Dtx), a snake polypeptide, increases neuronal excitability by blocking certain fast-activating, voltage-dependent K+ channels. Thus, the behavioural, electrocortical (ECoG) and neuropathological effects of Dtx, injected into rat brain areas, were studied. A unilateral injection of 35 pmol of Dtx into the CA1 hippocampal area or the dendate gyrus (DG; upper blade) immediately produced motor and ECoG seizures, followed at 24 h by multi-focal brain damage and significant neuronal loss. Whilst brain damage was seen bilaterally, significant neuronal loss occurred only in regions (CA1, CA3, CA4 and DG) ipsilateral to the site of injection. A lower dose (3.5 pmol) of toxin elicited motor and ECoG seizures but failed to produce brain damage. Seizures were observed 50 min after injecting Dtx (35 pmol) into the amygdala, though significant neuronal loss was not evident. 4-Aminopyridine (100 nmol), given into the CA1 area elicited a similar motor and ECoG pattern to that of Dtx except no brain damage could be seen at 24 h. Systemic pretreatment with antagonists of N-methyl-D-aspartate receptors (MK-801 or CGP 37849) did not protect against the effects typically evoked by injecting Dtx into the CA1 area.


Activation of quisqualate metabotropic receptors reduces glutamate and GABA-mediated synaptic potentials in the rat striatum. The role of quisqualate (QUIS) metabotropic receptors in the synaptic transmission in the striatum was investigated using the cortico-striatal slice preparation. Low concentrations (1-30 microM) of trans-1-amino-cyclopentyl-1,3- dicarboxylic acid (t-ACPD), a selective agonist of QUIS metabotropic receptors, decreased glutamate-mediated synaptic potentials (EPSPs) evoked in the striatum by the stimulation of cortico-striatal fibers. This agonist decreased also GABA-mediated depolarizing synaptic potentials evoked by intrastriatal stimulation in the presence of 6-cyano-7-nitro-quinoxaline-2,3- dione (CNQX); this effect was less potent than the action of t-ACPD on glutamate-mediated potentials. Low concentrations of t-ACPD did not affect the intrinsic membrane properties of striatal neurons and their postsynaptic responses to exogenous glutamate and GABA. Higher concentrations (50-100 microM) to t-ACPD caused membrane depolarizations and inward currents in several neurons. Our data suggest that low concentrations of t-ACPD selectively reduce synaptic transmission while higher concentrations of this agonist may cause a direct excitatory action on striatal neurons.


Alpha 2-adrenoceptor modulation of 5-HT biosynthesis in the rat brain. The purpose of the present study is to clarify the modulation of the biosynthesis of serotonin (5-HT) via the alpha 2-adrenoceptors in the brain. For this purpose, 5-hydroxytryptophan (5-HTP) accumulation was determined using an HPLC-ECD system in the presence of the inhibition of aromatic L-amino acid decarboxylase. Administration of alpha 2-adrenoceptor agonist, clonidine, produced a reduction of the in vivo 5-HTP accumulation in both the rat hippocampus and dorsal raphe nucleus. In addition, alpha 2-adrenoceptor antagonist, idazoxan, increased the 5-HTP accumulation in both the hippocampus and the dorsal raphe nucleus. In rats with catecholaminergic neurons denervated by pretreatment with 6-hydroxydopamine, clonidine failed to produce a reduction of 5-HTP accumulation in the dorsal raphe nucleus. On the other hand, hippocampal 5-HTP accumulation was decreased significantly. Brain tryptophan levels were unaffected by either clonidine or idazoxan. These results suggest that alpha 2-adrenoceptors might modulate serotonin biosynthesis and this modulation might be related to the neuroanatomical differences in the rat brain.


Evidence for the involvement of the mu but not delta opioid receptor subtype in the synergistic interaction between opioid and alpha 2 adrenergic antinociception in the rat spinal cord. The interaction between the spinal antinociceptive effects of selective mu or delta opioid agonists morphine and DSTBULET (Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr), respectively, and the selective alpha 2 adrenergic agonist dexmedetomidine was examined on convergent dorsal horn neuronal responses in the intact anaesthetized rat. The coadministration of intrathecal morphine (0.5 microgram, 2.5 micrograms) and dexmedetomidine (0.5 microgram) produced a greater than additive inhibition of C fibre-evoked responses. Inhibitions were reversed by either the opioid antagonist naloxone or the alpha 2 adrenergic antagonist atipamezole. The coadministration of intrathecal DSTBULET (1 microgram, 2.5 micrograms) and dexmedetomidine did not result in a supra-additive inhibition of C fibre-evoked responses. The results suggest that mu rather than delta opioid receptors are involved in the synergism of spinal opioid and alpha 2 adrenergic antinociception.


Excitatory effect of N-methyl-D-aspartate and kainate receptor on the 2-deoxyglucose uptake in the rat suprachiasmatic nucleus in vitro. The suprachiasmatic nuclei (SCN) have been identified as a pacemaker for many circadian rhythms in mammals. The previous findings indicate the excitatory amino acid (EAA) receptors play an important role in the transmission of light information from the retina to the circadian clocks. The 2-deoxyglucose (2DG) uptake shows a robust circadian change; high uptake during subjective day and low uptake subjective night. To determine whether EAA agonists regulate 2DG uptake in the SCN, we have measured 2DG uptake in the rat SCN in vitro. We report that, during the subjective night, glutamate, N-methyl-D-aspartate (NMDA) and kainic acid (KA) cause an increase in 2DG uptake and that NMDA- or KA-induced increase of 2DG uptake is blocked by co-treatments with competitive and non-competitive NMDA or KA receptor antagonists. EAA receptor agonist-induced increase in 2DG uptake occurs only during subjective night, at circadian times when photic phase shifting of activity occurs. Taken together, those data suggest that EAA may be an important transmitter of light information from retina to the SCN.


Trans-ACPD and L-APB presynaptically inhibit excitatory glutamatergic transmission in the basolateral amygdala (BLA). Intracellular recordings were obtained from neurones of the basolateral nucleus of the amygdala (BLA) and glutamate-mediated EPSPs evoked by stimulation of the stria terminalis (ST). The conformationally restricted analogue of glutamate trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) caused a dose-dependent reduction in EPSP amplitude, EC50 approximately 50 microM. This effect was mimicked by the glutamate autoreceptor agonist, L-aminophosphonobutyric acid (L-APB, 50 microM). Furthermore, the effects of submaximal concentrations (50 microM) of trans-ACPD and L-APB were additive. The reduction in EPSP amplitude is observed with concentrations of both drugs that have no effect on either the resting membrane potential or the input resistance of BLA neurones. In addition, these compounds can reduce EPSP amplitude but not the response to exogenous application of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA) suggesting activation of presynaptic receptors. These findings suggest that both trans-ACPD and L-APB act at presynaptic glutamate receptors on glutamatergic afferents to reduce excitatory transmission in the BLA.


Dynorphin, a preferential ligand for kappa-opioid receptors, is present in nerve fibers and immune cells within inflamed tissue of the rat. Exogenous kappa-opioid agonists have been shown to produce peripheral antinociceptive effects in inflamed tissue. This study sought to determine whether endogenous kappa-receptor ligands are present at the site of inflammation. In Freund's adjuvant-induced hindpaw inflammation in the rat, we show, by immunohistochemistry, that dynorphin is detectable within inflammatory cells and in the cutaneous nerves in a similar distribution as calcitonin gene-related peptide, a specific marker for sensory neurons. These findings extend our previous observations in that not only beta-endorphin and Met-enkephalin (mu- and delta-receptor ligands), but also a preferential kappa-ligand is present within inflamed subcutaneous tissue.


Dopamine D1 and D2 receptor interactions in the MPTP-treated marmoset. In a modified MPTP model of Parkinson's disease in the marmoset, both L-DOPA and the dopamine D2 agonist quinpirole were found to exhibit anti-bradykinetic activity. Both the dopamine D1 agonist SKF38393 and the D1 antagonist SCH23390 reduced the anti-bradykinetic action of L-DOPA and quinpirole. These results are discussed with respect to partial agonist activity of SKF38393 and the possibility that other dopamine receptors may be required for anti-Parkinsonian drug activity.


Loss of high-affinity alpha 2-adrenoceptors in Alzheimer's disease: an autoradiographic study in frontal cortex and hippocampus. We assessed, by quantitative autoradiography, the density of high-affinity alpha 2-adrenoceptors in hippocampus and frontal cortex sections from 18 patients dying with Alzheimer's disease (AD) in comparison with a control group of 13 matched cases. The full agonist [3H]bromoxidine (UK-14304) was used as a ligand. In AD brains, the specific binding of [3H]bromoxidine was markedly decreased both in frontal cortex, the reduction ranging from 55% on layer I (P less than 0.0005) to 40% loss on layers IV-VI (P less than 0.01), and in the hippocampus where the mean of alpha 2-receptor loss was 53% both for the CA1 (P less than 0.0005) and the dentate gyrus (P less than 0.005). This dramatic decrease in the density of functional, high-affinity alpha 2-adrenoceptors very probably reflects the neuronal loss described in locus coeruleus of AD brains. The important implications of these findings for the pharmacological treatment of AD are discussed.


Coactivation of D1 and D2 dopamine receptors is required for long-term synaptic depression in the striatum. Long-term changes of synaptic transmission following brief trains of high-frequency stimulation of excitatory pathways in the brain have attracted attention as a possible correlate of memory. In the cerebellum, concurrent activation of parallel fibers and climbing fibers leads to a long-term depression (LTD) of synaptic transmission, which may be the cellular substrate of motor learning in this structure. We report here for the first time that high-frequency stimulation of corticostriatal glutamatergic fibers in the striatum, another brain structure strongly involved in motor control, also induces LTD of synaptic transmission. Induction of striatal LTD is blocked either by SCH 23390, a D1 dopamine (DA) receptor antagonist or by L-sulpiride, a D2 DA receptor antagonist. The lesion of the nigrostriatal DAergic pathway abolishes LTD. After DA depletion, LTD can be restored by the application of exogenous DA. LTD can also be restored by coadministration of D1 and D2 DA receptor agonists, but not by the application of a single class of DA agonists alone. Our data show that coactivation of D1 and D2 DA receptors is required for LTD in the striatum. D1/D2 receptor cooperation in the induction of LTD may play a crucial role in the behavioural function of DA and in the therapeutic effects of DA agonists in Parkinson's disease.


Anterograde amnesia linked to benzodiazepines. Benzodiazepines, shown to affect memory, can produce anterograde amnesia (i.e., a loss of memory for events occurring forward in time). Following the ingestion of a benzodiazepine, short-term memory is not affected, but long-term memory is impaired. The memory loss may occur because events are not transferred from short-term memory to long-term memory and thus not consolidated into memory storage. Information stored prior to the ingestion of a benzodiazepine is not affected. Memory impairment is more likely in benzodiazepines that have a high benzodiazepine-receptor affinity, that accumulate in the body, that are given in high doses or intravenously, or that are eliminated slowly. Individuals taking benzodiazepines are often unaware of their memory impairment unless it is pointed out to them. Elderly clients experiencing memory impairment may be embarrassed to mention the problem. Alternatives to prescribing benzodiazepines include antidepressant medications, exercise or psychotherapy. When prescribing a benzodiazepine, it is important to fully inform patients of the drug's potential side effects and to maintain the lowest effective dose for the shortest period of time.


Intercellular adhesion molecule-1 (ICAM-1) and HLA-DR antigens in herpes keratitis. Intercellular adhesion molecule-1 (ICAM-1) is a cell surface glycoprotein that binds leukocyte function antigen-1 receptor on leukocytes, thereby regulating leukocyte trafficking and function at sites of inflammation. Recently, the authors demonstrated ICAM-1 in human corneas exposed to proinflammatory cytokines, but ICAM-1 has not been reported in corneal disease. In this study, the presence of ICAM-1 in human disciform herpes simplex virus (HSV) keratitis is investigated. Immunohistochemistry was performed for ICAM-1 on 4 keratoplasty specimens from patients with corneal scarring due to disciform HSV keratitis and 1 corneoscleral biopsy of a patient with active HSV keratoscleritis using specific, characterized monoclonal antibody to ICAM-1. Negative immunohistochemical controls included monoclonal antibodies to other vascular endothelial adhesion molecules or mouse serum. All 5 specimens demonstrated intense ICAM-1 immunoreactivity of keratinocytes, stromal keratocytes, and endothelial cells, predominantly in regions of leukocytic infiltration. Diffuse, intense HLA-DR positivity was detected throughout the corneas. The specimens failed to react with control antibodies. These results are the first to demonstrate ICAM-1 in human corneal disease and suggest important roles for ICAM-1 and HLA-DR co-expression in generating immune responses in HSV keratitis. Increased ICAM-1 expression in regions of leukocytic infiltration may regulate leukocyte-corneal cell binding, thereby promoting immune responses and damage by activated leukocytes.


Ocular jellyfish stings.

Ocular jellyfish stings. Corneal stings from the sea nettle (Chrysaora quinquecirrha) indigenous to the Chesapeake Bay are usually painful but self-limited injuries, with resolution in 24 to 48 hours. Five patients who developed unusually severe and prolonged iritis and intraocular pressure elevation after receiving corneal sea nettle stings were followed for 2 to 4 years. Decreased visual acuity, iritis, and increased intraocular pressure (32 to 48 mmHg) were noted in all cases. Iritis responded to topical corticosteroids and resolved within 8 weeks. Elevated intraocular pressure responded to topical beta blockers and oral carbonic anhydrase inhibitors. Mydriasis (4 of 5 cases), decreased accommodation (2 of 5 cases), peripheral anterior synechiae (2 of 5 cases), and iris transillumination defects (3 of 5 cases) also were noted. Mydriasis and decreased accommodation persisted for 5 months in 1 case and for more than 2 years in another. One patient has chronic unilateral glaucoma. Visual acuity returned to normal in all cases. The precise relationship between sea nettle venom and the observed clinical responses is not known. Corneal jellyfish stings usually produce a brief and self-limited reaction, but they do have the potential for long-term sequelae.


Neurotransmission in the auditory system. Neurotransmitters and neuromodulators thought to be active on neurons in the cochlea, CN, and SOC have been reviewed. The variety of neurotransmitters and neuromodulators present and likely colocalized in these neurons are the chemical substrates that link morphologically and physiologically diverse neurons to process sound information. The impact of the limited number of neurotransmitters and neuromodulators in the auditory system is magnified by their interaction with structurally diverse receptors; thus great functional diversity is possible. Moreover, the effects of neurotransmitters and neuromodulators are not limited to synaptic transmission but serve as trophic agents for the establishment of neuronal circuitry during development and the rearrangement of synapses as a result of sensory experience or injury. An understanding of the neurochemical aspects of sensory processing at these diverse synapses then is of fundamental importance in understanding the organization of the auditory system.


In situ hybridization for the localization of gene products in the auditory system. In this article, an overview of the technique and examples of our data demonstrate that our protocols produce excellent, reliable in situ hybridization of mRNAs and immunohistochemical localization of mRNA translation products in the temporal bone and brain stem. This in situ hybridization protocol has been used to localize eight different mRNAs, including four types of nicotinic acetylcholine receptor subunit mRNAs, actin mRNA, CGRP mRNA, and two mRNAs coding for gap junction proteins, in both paraffin-embedded and sectioned (6 microns) and cryostat sectioned (15 microns) temporal bones (unpublished data). In conclusion, in situ hybridization of mRNAs with single-stranded RNA probes allows screening of brain stem and temporal bone sections for the expression of specific genes, with cellular resolution, and this protocol gives reliable results for all mRNAs studied. Of great potential significance is the ability to assess variations in gene expression by in situ hybridization of specific mRNAs. It is expected that such variations accompany changes in the physiologic state of an organism, such as developmental, pathologic or experimentally induced, and that these variations in gene expression are important in understanding basic auditory processes at the cellular level.


Systemic injections of alpha-1 adrenergic agonists produce antinociception in the formalin test. The role of alpha 1 receptors in antinociception was investigated in the formalin test, a well established test of tonic pain. The effect of systemic injections of selective alpha 1-adrenergic agonists (phenylephrine and methoxamine), a mixed alpha agonist selective for alpha 2 receptors (ST-91), and 2 adrenergic antagonists (prazosin and idazoxan) was measured in groups of Long-Evans rats. All agonists tested produced significant antinociception in this test. Dose-response curves for each agonist were statistically parallel and equally efficacious (100% antinociception). Prior injection of 0.15 mg/kg prazosin (an alpha 1 antagonist) completely antagonized the antinociception produced by either an ED50 or a maximally effective dose of each agonist tested. Idazoxan (0.5 mg/kg), an alpha 2 antagonist, was without effect on the antinociception produced by phenylephrine or methoxamine. ST-91 produced significant antinociception in the presence of idazoxan although the response was different from that obtained with ST-91 alone. The observed antinociception in the formalin test was not due to drug-induced changes in peripheral inflammation as measured using plethysmometry. Moreover, none of the drugs tested produced significant changes in coordinated motor behavior (accelerated rotarod test) at doses that produced significant analgesia (ED50). We conclude that alpha 1 receptors contribute significantly to adrenergic analgesia in the formalin test by an undefined action on sensory processing mechanisms.


Reduction in adherence of Actinomyces viscosus after exposure to low-frequency acoustic energy. The ability of low-frequency (200 Hz) acoustic energy to reduce the adherence of Actinomyces viscosus T14V to saliva-treated hydroxyapatite (SHA) disks was studied. An acoustic pressure range between 0 and 65 kPa and exposure durations between 0 and 8 min were used to study the levels necessary to significantly alter adherence. The effects of acoustic exposure on both bacteria in liquid and bacteria already adhering to SHA disks were studied. A modified enzyme-linked immunosorbent assay was used to assess bacterial adherence. For bacterial suspensions exposed prior to addition to SHA disks, it was found that reductions in adherence were greater for lower bacterial concentrations. Exposure of bacteria already adhering to SHA disks resulted in a decrease in adherence that was independent of the bacterial concentration and linearly related to the logarithm of the exposure duration. In addition to affecting adherence, acoustic energy also dispersed bacterial aggregates. Our results support the concept that low-frequency sonic energy applied orally may be of therapeutic value in reducing adherence and colonization of teeth by plaque bacteria.


Characterization of the beta-adrenergic receptor in isolated human fetal lung type II cells. Functioning of the beta-adrenergic response system is important for successful transition of the neonate from fetal life to breathing air. We characterized the beta-adrenergic receptors on human fetal lung type II cells, the cell type responsible for many pulmonary responses sensitive to beta-adrenergic stimulation. Type II cells were isolated from human fetal lung explants, and membrane particulates prepared from these cells were used for radioligand binding studies. 125I-iodocyanopindolol, a specific beta-adrenergic antagonist, bound to a single class of saturable, high-affinity binding sites on type II cell membranes with a receptor concentration of 78 +/- 9 fmol receptor/mg membrane protein, a kd of 79 +/- 18 nM, and 958 +/- 120 receptors per cell. Binding was stereoselective with l-propranolol binding with higher affinity than the inactive d-isomer. The binding site had the characteristics of a beta 2-adrenergic receptor. The order of potency of beta-adrenergic agonists was isoproterenol greater than epinephrine much greater than norepinephrine. The beta 2-selective antagonist ICI 118,551 competed for a single class of high-affinity sites. Agonist binding affinity was reduced in the presence of guanyl nucleotides, consistent with receptors coupled to guanine nucleotide binding proteins. beta-Adrenergic agonists also stimulated adenylyl cyclase in these membrane preparations. 125I-iodocyanopindolol binding to membranes prepared from human fetal lung fibroblasts indicated fewer receptors (404 +/- 68) than were present on type II cells. Work by others has suggested a difference in lung function and lung beta-adrenergic receptor concentration between males and females.(ABSTRACT TRUNCATED AT 250 WORDS)


[Insulin sensitivity during use of adrenoblockers and adrenomimetics]. The effect of adrenergic blocking agents and adrenomimetics on sensitivity to insulin was studied in rat experiments. The sensitivity was judged from the duration of hypoglycemic coma development after intravenous infusion of 40 U/kg insulin. Sensitivity to insulin increased with the beta-adrenergic blocking agent obsidan and the alpha-adrenomimetic mesaton (phenylephrine hydrochloride) but diminished after infusion of the beta-adrenomimetic partusisten and the alpha-adrenergic blocking agent butyroxane. The obtained data are discussed from the standpoint of insulin-resistance control.


Dopamine inhibits Na/K-ATPase in single tubules and cultured cells from distal nephron. Dopamine decreases tubular sodium reabsorption, attributed in part to Na/K-ATPase inhibition in the proximal convoluted tubule (PCT). Because the final regulation of sodium excretion occurs in the collecting duct, where we have demonstrated specific dopamine DA1 binding sites, we examined the effects of dopamine, and of DA1 and DA2 receptor agonists on the Na/K pump in the microdissected rat cortical collecting duct (CCD) and in Madin-Darby canine kidney (MDCK) cells, a line derived from the dog distal nephron. Dopamine inhibited pump activity in CCD by approximately 40%-50%, an effect proportionally larger than in the PCT. Unlike in the latter, the effect of dopamine was reproduced by the DA1 agonist fenoldopam, which inhibited the CCD pump in dose-dependent manner (maximum, 10 microM). The DA2 agonist quinpirole was without effect, either alone or in combination with fenoldopam. These actions on Na/K-ATPase paralleled in reciprocal fashion effects on adenylate cyclase: dopamine or fenoldopam, but not quinpirole, produced a significant increase in cAMP content, and the stimulation by dopamine was blocked by SCH 23390. Inhibitors of cAMP phosphodiesterase (3-isobutyl-1-methyl-xanthine and theophylline), as well as forskolin and dibutyryl-cAMP, mimicked the effect of dopamine on the pump, underscoring the role of increased cAMP in this phenomenon. Both dopamine and fenoldopam inhibited Na/K-ATPase activity in MDCK cells. The results indicate that besides the PCT dopamine inhibits Na/K-ATPase activity in cells of the distal nephron, where its effect on the pump appears to be more pronounced and is mediated by activation of the DA1 receptor. The natriuretic effect of dopamine is probably exerted at both proximal and distal nephron sites.


Alpha 1- and beta-adrenergic interactions on L-type calcium current in cardiac myocytes. We investigated the mechanism by which alpha 1-adrenergic activation regulates basal and stimulated whole cell L-type Ca current (ICa) in rat ventricular myocytes using the physiological neurotransmitter, norepinephrine (NE, 10 microM). Stimulation of alpha 1-adrenoceptors, achieved by NE + 10 microM esmolol (a beta-receptor antagonist), had no significant effect on basal ICa. alpha 1-adrenergic activation had a marked inhibitory effect on ICa elevated by beta activation (NE + 1 microM) prazosin, an alpha 1-receptor antagonist) or activation of adenylyl cyclase by forskolin (25 microM); the inhibitory effect was reversible upon washout. However, alpha 1-adrenergic stimulation had no significant effect on ICa previously increased by intracellular application of cAMP (25 microM). The inhibitory effect seen on ICa elevated by NE showed no significant shift of either I-V or inactivation curves. It is unlikely that the inhibitory effect of alpha 1-adrenergic stimulation on NE or forskolin-elevated ICa is mediated through activation of Ca-dependent protein kinase C or changes in intracellular free Ca (pCa = 8.5, EGTA 5 mM) or cAMP-dependent phosphodiesterase. We conclude that alpha 1-adrenergic inhibition of beta-adrenergic stimulated-ICa is probably mediated through an as yet unknown G-protein. This inhibitory effect could serve as a regulatory feedback mechanism in physiological and pathophysiological settings.


Transplantation of umbilical cord blood in neuroblastoma. It has been shown that umbilical cord blood contains concentrations of hematopoietic progenitor cells equal to those of normal adult bone marrow. We successfully performed transplantation of cord blood combined with a hematopoietic growth factor (rGM-CSF) in a seven year old child suffering from neuroblastoma. Cord blood cells were collected from an HLA identical sibling at the time of delivery and stored in liquid nitrogen. The patient was conditioned with busulfan 600 mg/m2 and cyclophosphamide 200 mg/kg before receiving the thawed cells. There were no immediate side effects. Hematological reconstitution occurred promptly (granulocytes greater than 0.5 x 10(9)/L on day 13, platelets greater than 30 x 10(9)/L on day 40), although at day 10 the child experienced acute grade II cortico-sensitive GvHD. Mixed hematopoietic chimerism was observed 2 months later and complete remission lasted for 8 months.


Differential DNA binding properties of three human homeodomain proteins. The products of three human homeobox containing (HOX) genes, 2C, 3C and 4B, were produced in insect cells using the Baculovirus expression system and purified to near homogeneity. In this system we observed that the DNA binding forms of the three proteins are not glycosylated. HOX 3C and 4B are phosphorylated in insect cells, while HOX 2C is not. The three HOX proteins bind to a DNA sequence known to be a target site for Antennapedia protein with a very similar affinity (Kd = 1-2 x 10(-9) M). We then measured their binding properties to four human sequences present in the HOX 3D, 4C, 1C and 4B promoters. Two of these sequences have been reported to be binding sites for HOX proteins. HOX 2C, 3C and 4B behaved quite differently, showing low affinity for promoters of genes located upstream from their own gene in the HOX clusters and a higher affinity for regulatory sequences of their own gene and downstream HOX genes.


Participation of glutamic acid 23 of T4 endonuclease V in the beta-elimination reaction of an abasic site in a synthetic duplex DNA. T4 endonuclease V catalyzes the hydrolysis of the glycosyl bond of a thymine dimer in a DNA duplex and the cleavage of the 3'-phosphate by beta-elimination. We have previously identified a catalytic site for the first reaction (pyrimidine dimer-glycosylase activity) by systematic mutagenesis (Doi et al. Proc. Natl. Acad. Sci. USA 1992 in press) and by x-ray crystallography (Morikawa et al. Science, 256: 523-526, 1992). The results showed that replacement of Glu23 with either glutamine or aspartic acid completely abolished the glycosylase activity. We describe the investigation of the second reaction (apurinic/apyrimidinic endonuclease activity), using twenty two mutants of T4 endonuclease V plus a DNA mini duplex containing an abasic site. Replacement of Glu23 by glutamine abolished the second reaction, but replacement with aspartic acid did not. The pH optima of the mutant (23 Asp) and the wild type were found to be 5.0 and 5.5, respectively. We conclude that the carboxylate anion in position 23 may act as a general base in the beta-elimination reaction of the endonuclease.


A novel POU homeodomain gene specifically expressed in cells of the developing mammalian nervous system. We report the isolation of a novel human POU domain encoding gene named RDC-1. The POU domain of the RDC-1 encoded protein is highly related to the POU domain potentially encoded by the rat brain-3 sequence and to that of the Drosophila I-POU protein; outside of the POU region, RDC-1 is unrelated to any previously characterized protein. The RDC-1 gene is expressed almost exclusively in normal tissues and transformed cells of neural origin. In the developing mouse and human fetus, RDC-1 is expressed in a spatially and temporally restricted pattern that suggests a critical role in the differentiation of neuronal tissues. In addition, RDC-1 is expressed in a unique subset of tumors of the peripheral nervous system including neuroepitheliomas and Ewing's sarcomas but not neuroblastomas. Based on its unique structural characteristics and expression pattern, we discuss potential functions for the RDC-1 protein.


Mentally abnormal offenders. Evaluation and management of violence. Over the past two decades, much has been learned about the evaluation and management of violent patients. This has been translated into clinical guidelines for the evaluation and management of violent persons with psychiatric disorders. New research on neurotransmitters and the use of technology that can explore neurophysiologic and chemical activity in the brain promises continued advances in this area of psychiatry.


Tourette's syndrome. Current approaches to recognition and management. Once thought to be a rare, disabling disorder of psychogenic origin, Tourette's syndrome is now recognized as a chronic neurologic condition that is manageable in most patients. Individualized therapy is the key to helping patients achieve a productive and socially integrated life-style. This article discusses diagnostic criteria, effective management strategies, and promising new drug therapies.


Regional distribution of pyroglutamyl peptidase II in rabbit brain, spinal cord, and organs. Pyroglutamyl peptidase II (PPII) is a narrow specificity ectoenzyme that degrades thyrotropin-releasing hormone (TRH). We detected the enzyme in the brain of various mammals, with highest specific activity in rabbit brain. In this species, activity was heterogeneously distributed in the central nervous system. There was a 28-fold difference between regions of highest and lowest PPII activity. Enzyme activity was highest in the olfactory bulb and posterior cortex. In the spinal cord, activity was low but unevenly distributed, with highest values detected in the thoracic (T) region. Segments T1 and T2 activities were particularly high. Other organs contained low or undetectable levels of activity. The levels of TRH-like immunoreactivity (TRH-LI) in spinal cord segments were greatest in T3-T4 and lumbar L2-L6. Low concentrations were found in T1 and T9-T12. There was a partial correlation between the distribution of PPII activity and TRH receptors but not with TRH-LI levels. These results demonstrate that PPII is predominantly a central nervous system enzyme, and they support the hypothesis that PPII is responsible for degrading TRH released into the synaptic cleft.


D-Ala2,F5Phe4-dynorphin amide, an opiate with analgesic and toxic properties. A novel analog of dynorphin (1-13), D-Ala2,F5Phe4-dynorphin amide, was prepared and its pharmacological spectrum of activity was investigated. In a hot plate test on Swiss Webster and C57Bl mice, a 20 micrograms intracerebroventricular (icv) dose of the analog produced analgesia, which was greater in potency and duration than the parent dynorphin. This action of D-Ala2,F5Phe4-dynorphin amide was antagonized by the opiate receptor antagonist naloxone (2 mg/kg ip), administered either before or after the peptide. In addition to its analgesic action in mice, D-Ala2,F5Phe4-dynorphin amide produced a Straub tail and a catatonic-like state, both of which were also attenuated by naloxone. On the electrically-stimulated mouse vas deferens preparation, in vitro, D-Ala2,F5Phe4-dynorphin amide inhibited contractile activity and had an IC50 of 108.2 +/- 34.7 nM (SEM), about 4-fold weaker than that of dynorphin. This action was also attenuated by naloxone. An icv dose of 150 micrograms of D-Ala2,F5Phe4-dynorphin amide in mice, and a cumulative series of icv doses up to 2600 micrograms in anesthetized rats, failed to produce a lethal effect. No pathological changes were observed in mouse liver and kidney at 24 h after a 50 mg/kg dose of the peptide analog. In rats anesthetized with diallylbarbital (70 mg/kg ip) and urethane (280 mg/kg ip), D-Ala2,F5Phe4-dynorphin amide did not modify blood pressure, heart rate and respiratory rate. However, when mice were injected peripherally with single doses of D-Ala2,F5Phe4-dynorphin amide, convulsive episodes were produced, and lethal effects were observed with an LD50 of 60.0 mg/kg (95% confidence limits: 49.7-70.2 mg/kg) at 48 h. This action of D-Ala2,F5Phe4-dynorphin amide was not attenuated by naloxone (2.0 mg/kg, ip). Although analgesic and behavioral effects of D-Ala2,F5Phe4-dynorphin amide (e.g. Straub tail and catatonic-like state) are opiate-like, the lethal effect may be the consequence of actions of the peptide on non-opiate systems, Thus, the novel fluorinated dynorphin analog, D-Ala2,F5Phe4-dynorphin amide, may be a useful chemical tool for the study of opiate systems and their occasionally unanticipated biological or toxic actions.


The treatment of stable angina pectoris. A review of the available therapeutic measures for the management of stable angina pectoris is presented. The subject is critically examined by focusing on 5 main categories: general measures, drug therapy, coronary artery by-pass surgery, percutaneous transluminal coronary angioplasty and the handling of associated illnesses or disorders. General measures provide attention to the control or removal of aggravating conditions and to the institution of alterations in habits and life-style that will benefit the patient. Drug therapy depends upon the use of nitrates, beta adrenergic blockers and calcium entry blockers either alone or in combination. The mechanism of action of different drugs, its indications, as well as the dosage and frequency of use are reviewed. The indications for coronary artery by-pass surgery are presented as well as those for percutaneous transluminal angioplasty. An orientation in the selection of specific drugs when important concomitant diseases, such as hypertension and diabetes, coexist with stable angina pectoris is offered. The rationale behind the different alternatives is presented.


Anatomical evidence for interactions between somatostatin neurites in lamina II of the rat spinal cord. Light microscopic analysis of adult and 10-14-day-old rat spinal cords suggested that somatostatin-immunoreactive (SOM-I) fibers apposed SOM-I cell bodies in lamina (L) II. Electron microscopic analysis of these relationships at both ages showed the presence of direct appositions between SOM-I fibers and SOM-I cells. However, synapse formation between SOM-I fibers and cells was observed only in the young rat. Similarly, synapses between SOM-I fibers and SOM negative cell bodies were only found in the young animal. Adjacent SOM-I perikarya directly contacted each other, but, again, membrane specializations were evident only in the young rat. Within L I of the adult dorsal horn, a SOM-I fiber directly apposed an unlabeled cell body. Despite analysis of serial sections through the apposition, no synaptic contacts were observed.


Involvement of dopamine autoreceptors in the hypoactivity induced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) in mice. The effect of 8-OH-DPAT, a 5-HT1A receptor agonist, on the locomotor activity was analyzed in Albino Swiss mice. The studied drug (0.5-5 mg/kg) inhibited the spontaneous locomotor activity in mice. The hypoactivity induced by 8-OH-DPAT (1.5 mg/kg) was abolished by the dopamine (D1 and D2) receptor antagonist-haloperidol (0.00125 and 0.0025 mg/kg, but not in higher doses) and by the D2 antagonist with affinity for 5-HT1A and 5-HT2 receptors-spiperone (0.0025 and 0.005 mg/kg, but not in higher doses). The effect of 8-OH-DPAT was slightly reduced by the alpha 2-adrenoceptor antagonists: idazoxan (4 mg/kg), yohimbine (2 and 4 mg/kg) and rauwolscine (4 mg/kg). On the other hand, the non-selective 5-HT antagonist metergoline (0.5-4 mg/kg), the 5-HT1A antagonist NAN-190 (0.5-2 mg/kg), the beta-adrenoceptor blockers with high affinity for 5-HT1A and 5-HT1B receptors: pindolol and SDZ 21009 (2-8 mg/kg) and the agonist/antagonist of 5-HT1A receptors ipsapirone (2.5 and 5 mg/kg) did not affect the 8-OH-DPAT-induced hypoactivity. The obtained results suggest that the reduction of the spontaneous locomotor activity induced by 8-OH-DPAT results from a stimulation of dopamine autoreceptors, but not 5-HT receptors. Involvement of an alpha 2-adrenergic mechanism cannot be excluded.


Inhibition of nicotine-induced relaxation of the bovine retractor penis muscle by beta-adrenoceptor antagonists. The relative potency in inhibiting nicotine-induced relaxation of the bovine retractor penis muscle (BRP) was estimated for the racemates of seven beta-adrenoceptor antagonists, both of the optical isomers of propranolol, and lidocaine. The order of potency of the drugs studied was (+)-propranolol greater than (-)-propranolol greater than propranolol greater than alprenolol greater than metoprolol greater than lidocaine greater than acebutolol greater than pindolol greater than sotalol greater than atenolol. It is concluded that the inhibition of the relaxation was not due to blockade of beta-adrenoceptors but to the nonspecific effects of the beta-adrenoceptor antagonists. It is also concluded that the neurotransmitter(s) which was (were) released from the non-adrenergic non-cholinergic inhibitory nerves in the BRP did not relax the muscle by activating the beta-adrenoceptors. It is suggested that the beta-adrenoceptor antagonists inhibited the release of the inhibitory neurotransmitter(s) by a mechanism which is significantly correlated to their lipophilicity.


Modulation of the antigenic phenotype of human melanoma cells by differentiation-inducing and growth-suppressing agents. Tumor cells often display alterations in their normal program of cellular differentiation. A promising approach for the treatment of cancer involves the induction of terminal differentiation and a loss of proliferative capacity in cancer cells. In human melanoma cells, the combination of mezerein (MEZ) and fibroblast interferon (IFN-beta), results in a rapid and irreversible suppression of cell growth with a concomitant increase in the synthesis of melanin. The induction of terminal differentiation is associated with alterations in the expression of several cellular genes, including fibronectin, ISG-15 and ISG-54, and changes in the expression of specific cell surface antigens, including intercellular adhesion molecule-1 (ICAM-1) and HLA Class I antigens. In the HO-1 human melanoma cell line, induction of terminal differentiation by MEZ plus IFN-beta results in an induction and/or increased expression of ICAM-1, HLA Class I antigens and HLA Class II antigens. IFN-beta and MEZ alone can modulate expression of these antigens to a lower extent than does the combination of compounds. Induction of terminal differentiation and the irreversible suppression of cell growth is not a prerequisite for antigenic modulation in HO-1 cells. This is indicated by the inability of immune interferon (IFN-gamma), a strong inducer of ICAM-1, HLA Class I antigens and HLA Class II antigens synthesis, or the combination of IFN-beta plus IFN-gamma which synergistically but reversibly suppresses HO-1 growth, to induce melanin synthesis or terminal differentiation in HO-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)


[Signals regulating arterial vasomotricity and vasotrophicity]. Second messengers regulating the level of free intracellular calcium and the level of phosphorylation (kinase activities) transduce within the cells the mechanical and biochemical (endocrine, paracrine and autocrine) signals detected by the arterial wall. In arterial smooth muscle cells two signalling pathways produce opposite functional and structural effects: the breakdown of phosphoinositol leading to an increase in intracellular calcium level, and the cyclic guanosine monophosphate (GMP) pathway resulting in a decrease in calcium level. The arterial cyclic GMP pathway is predominantly under control of the endothelial function to secrete no rather than influenced by the atrial natriuretic factor. These two intracellular second messenger pathways are involved not only in the regulation of motricity, but also in the control of arterial wall trophicity, including hypertrophy, hyperplasia and collagen hypersecretion. Inactivation of the cyclic GMP pathway and activation of phosphoinositol breakdown predominate in vascular adaptation to the ageing process.


Comparison of Giardia isolates from different laboratories by isoenzyme analysis and recombinant DNA probes. A total of 13 new Giardia isolates were established in axenic culture. All of the new isolates were obtained by excystation of Giardia cysts from the feces of patients in Dutch hospitals. These isolates were subjected to isoenzyme and DNA analysis together with isolates from Poland, Belgium, and various other parts of the world. Isoenzyme analysis revealed that nearly all of the newly established isolates exhibited unique zymodemes. Isolates obtained from individuals from Belgium and Poland, on the other hand, displayed single zymodemes. Genomic DNA libraries were constructed from isolates belonging to the latter two zymodemes; specific and common recombinant DNA clones were selected from these libraries. Differential screening revealed that the two isolates had only 80% of the clones in common. Restriction-fragment-length polymorphism analysis using three different probes together with two synthetic probes that are complementary to Giardia structural protein genes led to the separation of all isolates into two major groups; within these groups, a further division could be made by application of other techniques or probes. The results of DNA analysis and zymodeme classification were in general agreement; in the present report they are compared with the data in the literature and discussed.


Overproduction, preparation of monoclonal antibodies and purification of E. coli asparagine synthetase A. In order to explore the structure--function relationship of the Escherichia coli asparagine synthetase A it was necessary to devise a system for overexpression of the gene and purification of the gene product. The E. coli asparagine synthetase A structural gene was fused to the 3' end of the human carbonic anhydrase II structural gene and overexpressed in E. coli. The gene product, a 66 kDa fusion protein, which exhibited asparagine synthetase activity, was purified in a single step by affinity chromatography and used as the antigen for the production of monoclonal antibodies. The monoclonal antibodies were screened by ELISA. Colonies were chosen which were positive for purified fusion protein and negative for purified human carbonic anhydrase II. The E. coli asparagine synthetase A gene was then overexpressed and the gene product was used without purification for the final screen. The antibodies selected were used for immunoaffinity chromatography to purify the recombinant overexpressed E. coli asparagine synthetase A. Thus, a procedure is now available so that asparagine synthetase A can be purified to homogeneity in a single step.


Alkaline phosphatase-somatostatin hybrid proteins as probes for somatostatin-14 receptors. By inserting appropriate peptide ligands into surface loops on globular proteins, we expect to develop probes for the location, accessibility, and steric and electrostatic environment of these ligand-binding sites on their membrane-bound receptors. Three residues in a loop on the surface of E. coli alkaline phosphatase were substituted by an 18-residue peptide containing the receptor-binding segment of somatostatin-14 without significantly affecting the catalytic properties of the enzyme. This hybrid protein was then used to investigate the ligand-binding site of somatostatin receptors. Tryptic cleavage of the hybrid protein within the inserted sequence, and binding of the hybrid protein to antisomatostatin antibodies demonstrated the surface accessibility of the guest peptide. Both the wild-type enzyme and the hormone-enzyme hybrid displaced 125I-labeled somatostatin from rat brain membrane receptors only at high concentrations. However, chemical cationization of the hybrid protein, which again did not disturb the phosphatase activity, enhanced its receptor-binding potency to a level only 23 times lower than that of somatostatin itself and 280 times higher than that of the cationized wild-type protein. This alkaline phosphatase/somatostatin hybrid protein appears, therefore, to be a suitable starting point for the development of probes for the steric and electrostatic environment of the ligand-binding site of somatostatin receptors.


Specific modulation of dopamine expression in neuronal hybrid cells by primary cells from different brain regions. MN9D is an immortalized dopamine-containing neuronal hybrid cell line. When MN9D cells were coaggregated with primary embryonic cells of optic tectum, a brain region that does not receive a dopaminergic innervation, there was a marked reduction in their dopamine content, tyrosine hydroxylase immunoreactivity, and tyrosine hydroxylase mRNA. Similar reductions in dopamine content were produced by coaggregation with cells from embryonic thalamus, another brain region devoid of dopaminergic innervation. Coaggregation of MN9D cells with dopaminoceptive cells from the corpus striatum or the cortex did not have a demonstrable stimulatory effect on the dopamine content of MN9D cells. The decrease in MN9D dopamine content produced by optic tectum cells was not reversed by addition of corpus striatum cells. Thus, the MN9D hybrid cells are able to respond to an inhibitory factor(s) from cells derived from brain areas that are not targets for dopaminergic neurons. Catecholamine-producing PC12 cells did not respond in a similar manner, suggesting that the response of MN9D cells is a function of their mesencephalic origin. Given the selective response of MN9D cells to different brain cell populations, this hybrid cell line should facilitate investigations of cell-cell interactions in the central nervous system that may be involved in the expression of neurotransmitter phenotype and establishment of specific neuronal connections.


Differential expression of arachidonate 5-lipoxygenase transcripts in human brain tumors: evidence for the expression of a multitranscript family. In addition to the important role of leukotrienes as mediators in allergy and inflammation, these compounds are also linked to pathophysiological events in the brain including cerebral ischemia, brain edema, and increased permeability of the blood-brain barrier in brain tumors. Although brain tumors have been shown to secrete leukotrienes, no studies to date have provided evidence for the tumor expression of genes encoding enzymes involved in leukotriene production. Therefore, the present study determined the abundance of the mRNA for arachidonate 5-lipoxygenase (5-LO; arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34), which is the rate-limiting enzyme in leukotriene synthesis, in a series of human brain tumors. Macrophage/monocyte infiltration of the tumor was estimated by measuring the abundance of the transcript for the 91-kDa glycoprotein phagocyte-specific oxidase (gp91-phox), which is the phagocyte-specific cytochrome b heavy chain. The present study shows that (i) the 5-LO transcript is expressed in normal bovine brain and in human brain tumors; (ii) the 5-LO gene in human brain tumors and in the dimethyl sulfoxide-induced promyelocytic human leukemic HL-60 cells is expressed as a multitranscript family (2.7, 3.1, 4.8, 6.4, 8.6 kilobases); and (iii) the abundance of 5-LO transcripts, the expression of the larger transcripts, and the 5-LO/gp91-phox ratio correlate with the tumor malignancy. Overall, the present study supports the hypothesis that the 5-LO gene product may play a role in human tumor-induced brain edemas and provides evidence for tumor-associated expression of high molecular weight 5-LO transcripts in human brain tumors.


Leukotriene A4 hydrolase: abrogation of the peptidase activity by mutation of glutamic acid-296. The metal-binding motif in the sequence of leukotriene A4 (LTA4) (EC 3.3.2.6), a bifunctional zinc metalloenzyme, contains a glutamic acid that is conserved in several zinc hydrolases. To study its role for the two catalytic activities, Glu-296 in mouse leukotriene A4 hydrolase was replaced by a glutamine or alanine residue by site-directed mutagenesis. Wild-type and mutated cDNAs were expressed four or five times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. With respect to their epoxide hydrolase activities--i.e., the conversion of LTA4 into leukotriene B4--the mutated enzymes [Gln296]LTA4 hydrolase and [Ala296]LTA4 hydrolase exhibited specific activities of 1070 +/- 160 and 90 +/- 30 nmol of LTB4 per mg of protein per min (mean +/- SD; n = 4 or 5), respectively, corresponding to 150% and 15% of unmutated enzyme. In contrast, when the mutated proteins were assayed for peptidase activity toward alanine-4-nitroanilide, they were found to be virtually inactive (less than or equal to 0.2% of unmutated enzyme). To serve as a positive control, we also replaced Ser-298 with an alanine residue, which resulted in a protein ([Ala298]LTA4 hydrolase) with catalytic properties almost indistinguishable from the wild-type enzyme. Substitution of Glu-296 by glutamine or alanine was also carried out with human LTA4 hydrolase, and the mutated human enzymes displayed specific activities similar to the corresponding mouse proteins. Zinc analyses of the purified mouse and human proteins confirmed that the mutations did not significantly influence their zinc content. In conclusion, the results of the present study indicate a direct catalytic role for Glu-296 in the peptidase reaction of LTA4 hydrolase, where it presumably acts as a base to polarize water, whereas its function, if any, is apparently not essential in the epoxide hydrolase reaction.


Activation of a D2 receptor increases electrical coupling between retinal horizontal cells by inhibiting dopamine release. In the fish retina, interplexiform cells release dopamine onto cone-driven horizontal cells. Dopamine decreases the electrical coupling between horizontal cells by activating adenylate cyclase through dopamine D1 receptors. Using intracellular recording, we have studied the effect of dopamine D2 receptor activation on horizontal cell electrical coupling in the intact goldfish retina. Superfusion of the D2 agonist LY171555 (quinpirole; 0.2-10 microM) increased horizontal cell coupling, as indicated by a decrease in responses to centered spots or slits of light. The length constant of the horizontal cell network increased an average of 31%. Although dopamine (0.5-20 microM) uncoupled horizontal cells, lower concentrations (e.g., 0.2 microM) initially uncoupled and then subsequently increased coupling beyond initial control levels. The coupling effect of LY171555 (10 microM) was blocked completely by prior application of the D1 agonist SKF 38393 at saturating (20 microM) or nonsaturating (2.5-5.0 microM) doses. Prior treatment of the retinas with 6-hydroxydopamine, which destroyed dopaminergic neurons, eliminated the coupling effect of LY171555 but not the uncoupling effect of SKF 38393. These results suggest that goldfish horizontal cells contain D1, but not D2, receptors and that dopamine activation of D2 autoreceptors on interplexiform cells inhibits dopamine release onto horizontal cells so that the electrical coupling between horizontal cells increases.


Point mutations in the tyrosine aminotransferase gene in tyrosinemia type II. Tyrosinemia type II (Richner-Hanhart syndrome, RHS) is a disease of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5), a 454-amino acid protein encoded by a gene with 12 exons. To identify the causative mutations in five TAT alleles cloned from three RHS patients, chimeric genes constructed from normal and mutant TAT alleles were tested in directing TAT activity in a transient expression assay. DNA sequence analysis of the regions identified as nonfunctional revealed six different point mutations. Three RHS alleles have nonsense mutations at codons 57, 223, and 417, respectively. One "complex" RHS allele carries a GT----GG splice donor mutation in intron 8 together with a Gly----Val substitution at amino acid 362. A new splice acceptor site in intron 2 of the fifth RHS allele leads to a shift in reading frame.


Prion protein preamyloid and amyloid deposits in Gerstmann-Sträussler-Scheinker disease, Indiana kindred. Gerstmann-Sträussler-Scheinker disease (GSS) is a familial neurological disorder pathologically characterized by amyloid deposition in the cerebrum and cerebellum. In GSS, the amyloid is immunoreactive to antisera raised against the prion protein (PrP) 27-30, a proteinase K-resistant peptide of 27-30 kDa that is derived by limited proteolysis from an abnormal isoform of a neuronal sialoglycoprotein of 33-35 kDa designated PrPSc. Polyclonal antibodies raised against synthetic peptides homologous to residues 15-40 (P2), 90-102 (P1), and 220-232 (P3) of the amino acid sequence deduced from hamster PrP cDNA were used to investigate immunohistochemically the distribution of PrP and PrP fragments in the brains of two patients from the Indiana kindred of GSS. Two types of anti-PrP-immunoreactive deposits were found: (i) amyloid deposits, which were exclusively labeled by anti-P1 antiserum to residues 90-102 of PrP, and (ii) preamyloid deposits, which were labeled by all anti-PrP antisera but did not exhibit the tinctorial and optical properties of amyloid. The latter appeared as diffuse immunostaining of the neuropil that targeted to areas in which amyloid deposits were most abundant. They were partially resistant to proteinase K digestion and consisted ultrastructurally of amorphous, flaky, electron-dense material. These findings substantiate our previous observation that the major amyloid component in the GSS Indiana kindred is an internal fragment of PrP and indicate that full-length abnormal isoforms of PrP and/or large PrP fragments accumulate in brain regions most affected by amyloid deposition. These findings support the view that in the GSS Indiana kindred a stepwise degradation of PrP occurs in situ in the process of amyloid fibril formation.


Instability of assembled T-cell receptor complex that is associated with rapid degradation of zeta chains in immature CD4+CD8+ thymocytes. The intracellular fate of newly synthesized T-cell receptor (TCR) chains was compared in CD4+CD8+ (double positive; DP) thymocytes and in CD4+CD8- or CD4-CD8+ (single positive; SP) thymocytes. Purified DP and SP thymocytes from normal adult mice were analyzed by pulse-chase metabolic labeling and immunoprecipitation with specific anti-TCR antibodies. Biosynthesis of invariant chains (CD3 gamma, -delta, -epsilon, and zeta) was comparable between DP and SP thymocytes, whereas DP thymocytes synthesized TCR alpha and TCR beta chains at lower and higher levels than SP thymocytes, respectively. These newly synthesized TCR chains were degraded at different rates in SP thymocytes based on their sensitivities for degradation as previously reported: TCR alpha, TCR beta, CD3 gamma, and CD3 delta chains were rapidly degraded and CD3 epsilon and zeta chains were stable. Although the degradation rates of clonotypic and invariant CD3 chains were similar in DP and SP thymocytes, the zeta subunit was rapidly degraded in DP thymocytes (t1/2, approximately 1.5 hr). Degradation of zeta was inhibited by NH4Cl, implicating lysosomes as the site of degradation. Comparison of TCR subunit assembly in DP and SP thymocytes demonstrated that, despite the same relative rate of formation of TCR complexes in a pulse period (30 min), complete complexes were unstable and degraded during the subsequent 6 hr of chase in DP thymocytes. This contrasted with the stability and a progressive increase in the levels of completely assembled complexes in SP thymocytes. Thus, these results demonstrate that a unique posttranslational regulation operates in the formation of TCR complexes in DP thymocytes and that lack of stability of complete TCR complexes is a crucial mechanism that may account for the limited surface TCR expression on this thymocyte subset.


Regulation of recombinant rat tyrosine hydroxylase by dopamine. Recombinant rat PC12 tyrosine hydroxylase, also called tyrosine 3-monooxygenase [L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], purified from Escherichia coli is in an activated form with a low Km for the tetrahydrobiopterin cofactor and a pH optimum of 6.5. Pretreatment with low levels of the derived product, dopamine, inhibits catalytic activity, increases the Km for the cofactor, and shifts the pH curve towards a more acidic pH optimum. Labeled dopamine binds to tyrosine hydroxylase with high affinity (Kd = 1 microM) but low stoichiometry (r = 0.08 mol/mol of enzyme subunit). The binding of dopamine results in the appearance of a blue-green chromophore with lambda max at approximately 660 nm, which is consistent with the formation of a catecholamine-iron complex. In the absence of dopamine, the recombinant enzyme cannot be further activated by phosphorylation with cAMP-dependent protein kinase, although as much as 1 mol of phosphate is incorporated per mol of subunit. In contrast, the enzyme pretreated with dopamine is activated by phosphorylation in the same fashion and to the same extent as the native hydroxylase. The results suggest that the high-affinity binding of catecholamine products is a pivotal post-translational modification that determines the state of enzyme activation and the response to phosphorylation.


Somatostatin receptors are expressed by immature cerebellar granule cells: evidence for a direct inhibitory effect of somatostatin on neuroblast activity. Somatostatin and somatostatin receptors are transiently expressed in the immature rat cerebellar cortex but virtually undetectable in the cerebellum of adults. Although somatostatin binding sites have been visualized during the postnatal period in the external granule cell layer, the type of cell that expresses somatostatin receptors has never been identified; thus, the potential function of somatostatin in the developing cerebellum remains unknown. In the present study, we have taken advantage of the possibility of obtaining a culture preparation that is greatly enriched in immature cerebellar granule cells to investigate the presence of somatostatin receptors and the effect of somatostatin on intracellular messengers on cerebellar neuroblasts in primary culture. Autoradiographic labeling revealed the occurrence of a high density of binding sites for radioiodinated Tyr-[D-Trp8]somatostatin-(1-14) on 1-day-old cultured immature granule cells. Saturation and competition studies showed the existence of a single class of high-affinity binding sites (Kd = 0.133 +/- 0.013 nM, Bmax = 3038 +/- 217 sites per cell). Somatostatin induced a dose-dependent inhibition of forskolin-evoked cAMP formation (ED50 = 10 nM), and this effect was prevented by preincubation of cultured immature granule cells with pertussis toxin. Somatostatin also caused a marked reduction of intracellular calcium concentration. These results show the presence of functionally active somatostatin receptors on immature granule cells. Our data suggest the possible involvement of somatostatin in the regulation of proliferation and/or migration of neuroblasts during the development of the cerebellar cortex.


Transfer and expression of the human multiple drug resistance gene into live mice. The human multiple drug resistance (MDR) gene has been used as a selectable marker to increase the proportion of bone marrow cells that contain and express this gene by drug selection. By constructing retroviral vectors containing and expressing the MDR gene and a nonselectable gene such as the beta-globin gene, enrichment for cells containing both of these genes can be achieved. A retroviral construct containing MDR cDNA in a Harvey virus-based vector has been used to transfect our ecotropic 3T3 retroviral packaging line GP+E86. Clones have been isolated by exposure of the retrovirally transfected cells (MDR producer cells) to colchicine (60 ng/ml), a selective agent that kills MDR-negative cells. Flow cytometry analysis (fluorescence-activated cell sorting) with an antibody to MDR demonstrates expression of human MDR protein on the surface of these colchicine-resistant producer clones. Untransfected GP+E86 cells are negative. Colchicine-resistant clones were titered using clone supernatants and the highest titer clone (4 x 10(4) viral particles per ml) was cocultured with 10(6) donor mouse bone marrow cells for 24-48 hr. The donor cells were then injected into congenic irradiated mice, and the presence of the MDR gene was assayed by the polymerase chain reaction (PCR) analysis using MDR-specific primers. In one experiment eight of nine transduced mice were positive for MDR by PCR of peripheral blood 14 and 50 days posttransplantation; after 240 days three of nine transduced mice were positive. Bone marrow obtained from one of these positive animals was stained with the MDR monoclonal antibody and the granulocyte population was analyzed by FACS. Approximately 14% of the total granulocyte pool contain increased levels of MDR protein. In addition, the bone marrow cells of several mice initially positive for MDR gene by PCR, and subsequently negative, were exposed to taxol, a drug whose detoxification depends on MDR gene expression; a positive signal was obtained in all of these mice, indicating drug selection of MDR-positive marrow cells. Cell sorting studies of these mice also show an increased number of high-MDR-expressing marrow cells, selected after exposure to taxol. Thus, in this live animal model MDR transduction is effective in selecting a human MDR-expressing population of marrow cells resistant to taxol chemotherapy. This strategy may, thus, be useful in humans to prevent the marrow toxicity induced by anticancer agents such as taxol and as a selectable marker to enrich for cells simultaneously transduced with a nonselectable gene.


Homology of a vesicular amine transporter to a gene conferring resistance to 1-methyl-4-phenylpyridinium. The vesicular amine transporter (VAT) catalyzes transport and storage of catechol and indolamines into subcellular organelles in a wide variety of cells. It plays a central role in neurotransmission and is the primary target for several pharmacological agents. One of the drugs, reserpine, binds very tightly to the transporter and remains bound even after solubilization, a finding that has proven useful for purification of the transporter from bovine adrenal medulla in a fully functional state. The sequences of 26 N-terminal amino acids and of an additional 7-amino acid internal peptide are presented. Antibodies against a synthetic peptide based on the above sequences immunoprecipitate the transporter, confirming the conclusion that the peptide sequence is derived from bovine VAT. To our knowledge, documentation of sequences of vesicular neurotransmitter transporters has not been presented previously. In addition, the sequences obtained are highly homologous to the predicted sequence of a protein from PC12 cells that confers to Chinese hamster ovary cells resistance to 1-methyl-4-phenylpyridinium (MPP+), an agent that causes parkinsonism in model systems, confirming the hypothesis that the protein conferring resistance to MPP+ is a VAT.


Cloning and expression of chicken erythrocyte transglutaminase. We report the sequences of cDNAs encoding chicken erythrocyte transglutaminase (EC 2.3.2.13). The complete mRNA consists of 3345/3349 nucleotides and predicts a single open reading frame. Nine peptide sequences derived from partial digests of the isolated protein agreed with the corresponding translation of the open reading frame. Approximately 60% identities between the avian protein and three related mammalian enzymes were found. Chicken erythrocyte transglutaminase mRNA is most abundant in red blood cells and kidney, and it accumulates during erythroid cell differentiation.


Cloning, genetic mapping, and expression analysis of an Arabidopsis thaliana gene that encodes 1-aminocyclopropane-1-carboxylate synthase. A genomic clone of one member of the Arabidopsis thaliana (L.) Heynh. 1-aminocyclopropane-1-carboxylate (ACC) synthase (S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) gene family (AT-ACC1) was isolated and sequenced. A region of homology was found in the 5'-untranslated region with the promoter of a zucchini and a tomato ACC synthase gene. Comparison of its primary structure with other ACC synthases revealed conservation of seven peptide regions as well as similarity with 11 amino acids of the catalytic site of aminotransferases. Genomic DNA gel blotting suggested the existence of an ACC synthase multigene family in Arabidopsis, possibly with three other members, none of which is very closely related to AT-ACC1. The existence of at least one other gene was confirmed by the isolation of a cDNA (AT-ACC2) from a flower-specific cDNA library. The AT-ACC1 gene was mapped on the Arabidopsis restriction fragment length polymorphism map and is located on the top of chromosome 1. This position does not correspond to any known mutation on the genetic map. Expression of the AT-ACC1 gene was studied by reverse transcription-PCR on total RNA. Messenger accumulation was strong in young leaves and flowers. The gene was not induced by wounding of young leaves or in seedlings in the presence of auxin. Ethylene exposure of mature plants led to an induction of AT-ACC1 gene expression. It is suggested that AT-ACC1 protein has a role in developmental control of ethylene synthesis.


Effects of methamphetamine and ethanol on learning and brain neurotransmitters in rats. The interactions of methamphetamine (MAMP) and ethanol (EtOH) on multiple active/passive avoidance performance and neurotransmitters in different brain regions were examined. After the acquisition schedules, rats were retrained under the influence of MAMP (2 mg/kg/day, IP), EtOH (2 g/kg/day, IP), and in combination over 20 days in rats (n = 6 per group). As a function of progress of drug treatment, MAMP-EtOH mixtures disrupt the learned avoidance performance and produced severe impairment of discriminative behavior caused by enhancement of excitability induced by MAMP when compared with MAMP only. At withdrawal, MAMP-EtOH-induced impairments of performance significantly persisted, whereas MAMP-only-induced impairments slightly recovered. At the eleventh day drug withdrawal, MAMP-only-induced alterations of neurotransmitter levels at different regions were alleviated by EtOH, but these did not return to normal levels. These data provide support for the direct antagonistic and indirect additive interactions following constant daily treatment with a combination of MAMP and EtOH. EtOH may be an important factor in MAMP abuse to MAMP-induced psychosis or neurotoxicity.


Beta-blocker effects on 24-h activity in normotensive and renovascular hypertensive baboons. Spontaneous motor activity of normotensive and renovascular hypertensive baboons was measured during oral dosing with the beta-adrenergic antagonists atenolol HCl (2.6 mg/kg/day) and d,l-propranolol HCl (6.8 mg/kg twice daily) in separate studies. Each study administered active drug for 21 consecutive days. Piezoelectric monitors sensitive to movement were worn continuously by the baboons. Propranolol decreased overall 24-h average activity during the third week of dosing in normotensive baboons but not in renovascular hypertensive baboons. The greatest reductions in activity averaged 20% at those times of day corresponding to the second daily drug dose both in normotensive baboons and, at this time of day only, in the majority of hypertensive baboons. Activity decreases reversed to baseline levels when propranolol was discontinued. For atenolol, most normotensive but no hypertensive baboons showed decreases in activity at the time of day corresponding to the daily drug dose.


Assessment of the stimulus properties of anxiolytic drugs by means of the conditioned taste aversion procedure. The conditioned taste aversion (CTA) procedure has recently been described as a more rapid alternative to two-lever operant procedures in drug discrimination research. We trained different groups of rats to discriminate the benzodiazepine chlordiazepoxide (CDP, 20 mg/kg) or the 5-hydroxytryptamine1A (5-HT1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (0.4 mg/kg) from saline by means of the CTA procedure. The results were in agreement with findings from two-lever operant drug discrimination procedures. However, discrimination training took 40 sessions in the case of CDP and 72 sessions for 8-OH-DPAT, which is comparable to results obtained with two-lever operant procedures. Dose-response curves were determined and generalization tests were performed for different benzodiazepine and nonbenzodiazepine anxiolytics. Baseline behavior deteriorated in the course of generalization and substitution testing, thus preventing further generalization testing. Our experience is that the use of the CTA procedure in drug discrimination research does not have sufficient advantages over traditionally used procedures to replace the latter.


Attenuation of alcohol consumption by MDMA (ecstasy) in two strains of alcohol-preferring rats. Alcohol preference and manifestation of alcoholism are thought by many to be associated with serotonin (5-HT) dysfunction in the brain. Thus, experiments were performed to determine the effect of acute and subchronic administration of (+/-) 3,4-methylenedioxymethamphetamine (MDMA), an amphetamine analog that stimulates 5-HT release, on alcohol preference in two strains of alcohol-preferring rats, the Fawn-Hooded (FH) and alcohol-preferring (P) rats. Rats were individually housed and provided free access to a solution of 10% ethanol, food, and water. Ethanol, food, and water intakes were measured daily. After establishing a stable baseline for ethanol and water intake, each rat was injected SC with a dose of 5.0 mg/kg MDMA or an equal volume of saline for 1 or 3 consecutive days. Body temperature was recorded immediately before and 120, 240, and 360 min after MDMA treatment. Ethanol, food, and water intake were measured for the preceding 24 h. Further, to determine the effect of MDMA on alcohol metabolism rats were injected with 5.0 mg/kg MDMA or saline and 15 min later with 2.5 g/kg alcohol. Then, blood alcohol levels were determined at 1, 3, and 5 h after alcohol administration. Our results show that a single administration of 5.0 mg/kg MDMA significantly decreased ethanol intake in both FH and P rats and increased water intake. Subchronic administration of 5.0 mg/kg MDMA for 3 consecutive days significantly attenuated alcohol intake in both strains but only increased water intake in P rats. Administration of MDMA induced hyper- and hypothermia in FH and P rats, respectively. This drug failed to exert any significant effect on the pharmacokinetics of alcohol, indicating a central effect.(ABSTRACT TRUNCATED AT 250 WORDS)


NMDA enhances the central depressant properties of ethanol in mice. Male Swiss-Webster mice were used to examine the effect of NMDA on the ethanol-induced loss of the righting reflex (LORR). The LORR was used as a measure of CNS depression. Immediately after animals regained the righting reflex following ethanol injection (4.0 g/kg, IP) mice received an ICV injection of saline or NMDA (10, 50, 100, or 500 nmol/kg) in a volume of 5 microliters. Upon ICV injection of NMDA, mice again lost the righting reflex and this effect of NMDA in the presence of ethanol occurred rapidly and in a dose-dependent manner. In another experiment DL-2-amino-5-phosphonovaleric acid (APV), a competitive antagonist of NMDA, was given ICV with NMDA (50 nmol/kg) in the presence of ethanol. APV (10 and 100 nmol/kg, ICV) significantly attenuated the response of NMDA to enhance the depressant action of ethanol. When bicuculline methiodide, an antagonist of GABA, was given ICV with NMDA (50 nmol/kg), bicuculline methiodide reduced the effect of NMDA to produce a second loss of the righting reflex (return to the LORR) in the presence of ethanol. When NMDA (100 nmol/kg, ICV) was injected in the absence of ethanol into mice, NMDA by itself did not produce a loss of the righting reflex. In this investigation, the results suggest that NMDA can augment ethanol-induced depression possibly through an interaction between glutamatergic and GABAergeric systems in the CNS.


Microinjections of dopamine agonists in the nucleus accumbens increase ethanol-reinforced responding. Long-Evans rats (N = 3) were trained to lever press on a fixed-ratio 4 (FR 4) schedule with ethanol (10% v/v) presented as the reinforcer. Each rat received a total of six bilateral nucleus accumbens microinjections, one per week. They were tested with one physiological saline control, three 20.0-microgram/brain d-amphetamine, and two 6.0-microgram/brain quinpirole injections given 10 min prior to operant sessions. Ethanol-reinforced responding terminated after approximately 10 min during control sessions. Microinjections of the D2 agonist quinpirole and the nonspecific dopamine (DA) agonist d-amphetamine increased total responding but produced slowed response rates that continued for 45-60 min. The slowed response rate produced by d-amphetamine resulted in a peak increase in interresponse times (IRTs) between 8-10 s, whereas quinpirole increased IRTs in the 14- to 16-s range, indicating that nonspecific DA activation resulted in higher rates of ethanol-reinforced responding than specific D2 activation although both drugs decreased local response rates. These data indicate that the amount and temporal extent of ethanol-reinforced responding are increased by microinjections of DA agonists in the nucleus accumbens and support the hypothesis that DA activity in this region is involved in the regulation of ethanol-reinforced responding.


Evidence for noradrenergic involvement in mediating the FG 7142 discriminative stimulus. Rats were trained to discriminate the stimulus properties of the benzodiazepine receptor partial inverse agonist beta-carboline-3-carboxylate acid methyl amide (FG 7142) (5.0 mg/kg) or the alpha 2-adrenergic receptor antagonist 17 alpha-hydroxyyohimban-16 alpha-carboxylic acid methyl ester (yohimbine) (3.0 mg/kg) from vehicle in a two-lever, food-motivated operant task. These compounds have in common a beta-carboline structure and anxiogenic behavioral profiles. The yohimbine discriminative stimulus was mimicked by the alpha 2-adrenergic receptor antagonist idazoxan and antagonized by the alpha 2-adrenergic receptor agonist clonidine, indicating that the yohimbine stimulus was mediated through the alpha 2-adrenergic receptor. The anxiogenic beta-carbolines FG 7142, 1,2,3,4-tetrahydro-beta-carboline (THBC), and norharmane, the anxiogenic/convulsant agent pentylenetetrazole (PTZ), and two physiological stressors failed to mimic the yohimbine discriminative stimulus. In contrast, both yohimbine and idazoxan dose responsively mimicked the anxiogenic FG 7142 stimulus. The present results demonstrate that an asymmetrical generalization exists between the discriminative stimuli produced by yohimbine and FG 7142. Furthermore, these data suggest that yohimbine can produce a multicomponent discriminative stimulus, part of which may be anxiogenic in nature. The ability of alpha 2-adrenergic receptor antagonists to mimic the FG 7142 cue suggests that activation of the noradrenergic system may underlie cues produced by benzodiazepine receptor inverse agonists.


Structure-activity relationship of new 10-13 membered oxygen-nitrogen heterocyclic systems containing two aromatic rings. The synthesis and biological activity of new 10-13 membered oxygen-nitrogen heterocyclic systems condensed with two aromatic rings is reported. The structure-activity relationship of these new compounds in X-ray investigation has been studied. Pharmacological investigations have shown that the compounds exhibit weak neuroleptic activity.


Effects of adrenoceptor agonists and antagonists on cardiovascular functional parameters in rats. The effects of intravenous administration of adrenoceptor agonists and antagonists on electrocardiographic or blood pressure (BP) functional parameters were assessed in urethane-anesthetized rats. The responses of cardiovascular functional parameters produced by these drugs included: (1) isoproterenol decreased the duration of a whole BP cycle (Wd), duration of the diastolic wave (Dd), peak amplitude of the systolic wave (SYa), amplitude of the diastolic notch (DNa), amplitude of the diastolic wave (DWa), pulse pressure (dp) and mean arterial pressure (mp) but increased the heart rate (HR) accompanied by prolonged R-R (RR) or P-P interval (PP) (2) propranolol decreased SYa, DNa, dp, mp, HR, the amplitude of the P wave (Pa) and amplitude of the S wave (Sa) but increased the duration of the QRS wave, P-R interval, duration of the R wave (Rd) and duration of the P wave (Pd); (3) adrenaline decreased HR (accompanied by prolonged RR and PP), Rd, Pa and amplitude of the T wave (Ta) but increased Pd, Wd, Dd, DNA, the time interval between aortic valve opening and closure (Dw), dp, mp, amplitude of the Q wave and amplitude of the R wave (Ra); (4) noradrenaline decreased HR (accompanied by prolonged RR and PP) and Pa but increased Wd, Pd, SYa, DNa, Dw, dp, mp, Ra and Ta; (5) phenylephrine decreased HR (accompanied by prolonged RR and PP) and Pa but increased Wd, Dd, DNa, mp and Ra; (6) phentolamine decreased SYa, DNa, DWa, Dw, dp and mp. This study illustrates the utility of the automated electrocardiogram (ECG) and BP analysis system for investigation of adrenoceptor agonists and antagonists. The use of this methodology not only reproduced most of cardiovascular functional parameter effects produced by these drugs using the conventional methodology but also realizes some new information about the drug-induced ECG or BP waveform effects.


Treatment of generalized anxiety disorder: comparison of a new beta-blocking drug (CGP 361 A), low-dose neuroleptic (flupenthixol), and placebo. In an attempt to evaluate an alternative drug treatment to benzodiazepines in generalized anxiety disorders, a placebo controlled trial was carried out with a new beta-adrenergic blocker (CPG 361 A). A low-dosage neuroleptic (flupenthixol) was included as a reference drug. Depending on the clinical assessment scales the placebo treatment resulted in moderate to excellent improvement in 36% to 56% of the patients after four weeks of treatment. The active drugs generally had a higher improvement range (from 31% to 80%). The global improvement scale was found to be better than the other scales in discriminating between placebo (50% improvement) and the active drugs (CGP 361 A brought about 78% improvement and flupenthixol brought about 80% improvement). However, only for flupenthixol was the difference of statistical significance.