Characterization of alpha-adrenoceptors involved in central thermoregulation in rabbits

Intracerebroventricular (icv) injection of methyldopa induced body temperature changes in the rabbits. The dose of 100 micrograms/kg did not produce any significant change on body temperature whereas 250 micrograms/kg of the drug induced hyperthermia. Higher dose of 500 micrograms/kg produced initial hypothermia which was followed by hyperthermia. On further increase of the dose to 1 mg/kg, consistent hypothermia was evident. Prazosin, a specific post-synaptic alpha 1 adrenoceptor blocker, induced hypothermia whereas piperoxan (presynaptic alpha 2 antagonist) produced hyperthermia. The pretreatment with prazosin, blocked the hyperthermic response of methyldopa. The initial hypothermia by 500 micrograms/kg of methyldopa was also potentiated. The pretreatment with piperoxan completely blocked the hypothermia but had no effect on hyperthermic response of methyldopa. Pretreatment of rabbits with both prazosin and piperoxan completely blocked the hypothermia as well as hyperthermic response of methyldopa. Thus it appeared that both presynaptic alpha 2 and postsynaptic alpha 1 adrenoceptors are involved in central thermoregulation in rabbits.     

Characterization of pre- and postsynaptic dopamine receptors in Lymnaea

1. The effects of dopamine and several synthetic agonists and antagonists were studied using two identified neurons of the snail Lymnaea stagnalis. 2. In both the buccal-2 (B-2) neurons and the pedal giant (RPeD1) neuron dopamine elicited a hyperpolarizing response at least partly due to potassium efflux. RPeD1 is itself dopaminergic, implicating autoreceptors in its response to dopamine. 3. The following agents were tested: agonists--LY171555, pergolide, SKF38393, (-)-3-PPP, R(-)NPA and dopamine; antagonists--SCH23390, sulpiride, and metaclopramide. Dibutyryl cAMP was applied to determine whether the response is cAMP-mediated. 4. Results indicate that the pharmacological profiles of dopamine receptors on these neurons are inconsistent with those of either D-1, D-2 or autoreceptors in mammals.     

Renal dopamine receptors are involved in the development of cardiac hypertrophy

The present study examined the effect of fenoldopam, a known dopamine-1 receptor (DA1) agonist in order to understand its involvement in the cardiac hypertrophic process. Male Sprague-Dawley rats underwent abdominal aortic constriction (AB) with placement of a suprarenal ligature while sham operated animals served as controls. The AB groupsshowed an increase in their heart wt, left ventricular (LV) wt, heart wt/body wt and LV wt/body wt ratio. Furthermore, the length of these hearts, as measured from the auriculoventricular border to the apex, LV wall and interventricular (IV) septal thickness were increased from control levels. Treatment with SCH 23390, a DA1 antagonist, on the other hand, was able to partially regress the cardiac hypertrophic changes. All these parameters were also increased in control animals treated with fenoldopam (F). Such changes were more striking in the F+AB group which showed a significant acceleration of the cardiac hypertrophic process on super-imposing the two treatments. Plasma dopamine and renin activity were increased in all the groups as compared to control. These results indicate that dopamine receptors are implicated in the development of cardiac hypertrophy.     

Dorsal hippocampal dopamine receptors are involved in mediating ethanol state-dependent memory

In the present study, the effects of bilateral injections of dopaminergic agents into the hippocampal CA1 regions (intra-CA1) on ethanol (EtOH) state-dependent memory were examined in mice. A single-trial step-down passive avoidance task was used for the assessment of memory retention in adult male NMRI mice. Pre-training intra-peritoneal (i.p.) administration of EtOH (0.25, 0.5 and 1 g/kg) dose dependently induced impairment of memory retention. Pre-test administration of EtOH (0.5 g/kg)-induced state-dependent retrieval of the memory acquired under pre-training EtOH (0.5 g/kg) influence. Intra-CA1 administration of the dopamine D(1) receptor agonist, SKF 38393 (0.5, 1 and 2 g/mouse) or the dopamine D(2) receptor agonist, quinpirole (0.25, 0.5 and 1 microg/mouse) alone cannot affect memory retention. While, pre-test intra-CA1 injection of SKF 38393 (2 microg/mouse, intra-CA1) or quinpirole (0.25, 0.5 and 1 microg/mouse, intra-CA1) improved pre-training EtOH (0.5 g/kg)-induced retrieval impairment. Moreover, pre-test administration of SKF 38393 (0.5, 1 and 2 microg/mouse, intra-CA1) or quinpirole (0.5 and 1 microg/mouse, intra-CA1) with an ineffective dose of EtOH (0.25 g/kg) significantly restored the retrieval and induced EtOH state-dependent memory. Furthermore, pre-training injection of the dopamine D(1) receptor antagonist, SCH 23390 (4 microg/mouse), but not the dopamine D(2) receptor antagonist, sulpiride, into the CA1 regions suppressed the learning of a single-trial passive avoidance task. Pre-test intra-CA1 injection of SCH 23390 (2 and 4 microg/mouse, intra-CA1) or sulpiride (2.5 and 5 microg/mouse, intra-CA1) 5 min before the administration of EtOH (0.5 g/kg, i.p.) dose dependently inhibited EtOH state-dependent memory. These findings implicate the involvement of a dorsal hippocampal dopaminergic mechanism in EtOH state-dependent memory and also it can be concluded that there may be a cross-state dependency between EtOH and dopamine.     

Characterization of D2 receptors and dopamine levels in the thalamus of the rat.

We kinetically characterized D2 receptors in thalami pooled from a group of Sprague-Dawley rats and then determined thalamic levels of dopamine (DA), homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), and norepinephrine (NE) in relation to a measure of thalamic DA D2 receptor densities in another group of rats. The equilibrium dissociation constant (kd) was estimated as 0.1 nM by three independent methods, while the Bmax for thalamic D2 receptors was found to be 6.4 fmol/mg p using 3H-spiperone as ligand and ketanserin to occlude 5HT2 binding. Kinetic constants were in agreement with previously reported kinetic data from rodent caudate-putamen. This suggests that thalamic D2 receptors are similar to D2 receptors from other brain areas. Mean thalamic levels of DA (22.6 ng/mg p), DOPAC (1.19 ng/mg p) and HVA (0.31 ng/mg p) concur with previous reports of a sparse distribution of thalamic DA neurons. D2 receptor densities were positively correlated with DA metabolites DOPAC (P less than .05; r = 0.423) and HVA (P less than .05; r = 0.368), but not DA or NE. These results establish fundamental characteristics of thalamic DA neurotransmission to assist in the investigation of behavioral pharmacology of this area.     

Characterization of central melatonin receptors using 125I-melatonin

The binding of 125I-melatonin, a potent analog of melatonin, to rat brain synaptosomal preparations was investigated. 125I-melatonin bound with high affinity (Kd = 38 nM) to a single class of sites (Bmax = 81 fmol/mg protein). Kinetic studies indicated that binding was time-dependent and reversible. Specific 125I-melatonin binding was inhibited by melatonin, and was unaffected by other structurally related compounds including serotonin. Binding of 125I-melatonin was greatly reduced if the synaptosomal preparations were pretreated by heat or trypsin but was unaffected by freeze-thawing. These results suggest that 125I-melatonin may serve as a valuable probe for studying melatonin receptors.     

Characterization of brain D2 dopamine receptors

In order to characterize and partially purify solubilized dopamine receptors, canine brain striatum microsomes were solubilized with 1% digitonin, and enriched by either gel permeation chromatography, preparative vertical column isoelectric focusing, or sucrose gradient ultracentrifugation. Chromatography on Sephacryl S-300 in buffer (contaning 0.05% Triton X-100) yielded a Stokes radius of 5.8 nm. Isoelectric focusing of the solubilized, radiolabelled receptor produced peaks of [³H]spiperone radioactivity corresponding to isoelectric values of 5.0 and 7.8, of which the former has been shown elsewhere to be the intact D₂ dopamine receptor. Sucrose density gradient ultracentrifugation, again in buffer containing 0.05% Triton X-100, indicated a hydrodynamic mol. wt of 136,000 Daltons, which corresponds closely to the value of 123,000 Daltons estimated using radiation inactivation. Such molecular characterization will aid in the distinction of dopamine receptor subtypes.     

Characterization and mapping of bovine dopamine receptors 1 and 5

A cDNA encoding the bovine dopamine receptor 1 (DRD1) was isolated from a bovine cDNA library, cloned and completely sequenced. The coding region showed 93 and 91% sequence identity on DNA level and 96 and 94% on protein level with its respective porcine and human orthologs. The bovine DRD1 and dopamine receptor 5 (DRD5) were mapped, respectively, to BTA10 and 6 by radiation hybrid mapping. One SNP was found in DRD1 and four in DRD5. Using polymerase chain reaction-restriction fragment length polymorphism, 11 different European cattle breeds were screened for the presence of the DRD1 and DRD5 substitutions. Allele frequencies for DRD1 and DRD5 alleles were very similar across all the breeds examined. Allele frequency discrepancies were found between Belgian Blue beef breed and the other breeds.     

Characterization of enzymes involved in the central metabolism of Gluconobacter oxydans

Gluconobacter oxydans is an industrially important bacterium that lacks a complete Embden–Meyerhof pathway (glycolysis). The organism instead uses the pentose phosphate pathway to oxidize sugars and their phosphorylated intermediates. However, the lack of glycolysis limits the amount of NADH as electron donor for electron transport phosphorylation. It has been suggested that the pentose phosphate pathway contributes to NADH production. Six enzymes predicted to play central roles in intracellular glucose and gluconate flux were heterologously overproduced in Escherichia coli and characterized to investigate the intracellular flow of glucose and gluconates into the pentose phosphate pathway and to explore the contribution of the pentose phosphate pathway to NADH generation. The key pentose phosphate enzymes glucose 6-phosphate dehydrogenase (Gox0145) and 6-phosphogluconate dehydrogenase (Gox1705) had dual cofactor specificities but were physiologically NADP- and NAD-dependent, respectively. Putative glucose dehydrogenase (Gox2015) was NADP-dependent and exhibited a preference for mannose over glucose, whereas a 2-ketogluconate reductase (Gox0417) displayed dual cofactor specificity for NAD(P)H. Furthermore, a putative gluconokinase and a putative glucokinase were identified. The gluconokinase displayed high activities with gluconate and is thought to shuttle intracellular gluconate into the pentose phosphate pathway. A model for the trafficking of glucose and gluconates into the pentose phosphate pathway and its role in NADH generation is presented. The role of NADPH in chemiosmotic energy conservation is also discussed.     

Multiple classes of dopamine receptors in mammalian central nervous system: the involvement of dopamine-sensitive adenylyl cyclase.

Two classes of dopamine receptor mechanism are defined according to their association with, or independence from, a dopamine-sensitive adenylyl cyclase. Dopamine receptors unrelated to adenylyl cyclase are designated type alpha. Dopamine receptors linked to adenylyl cyclase are designated type beta. Drugs discriminate between the two receptor mechanisms. The dopaminergic ergots (lisuride, lergotrile and CB-154) and their antagonists (such as metoclopramide) are relatively specific for the alpha-dopaminergic receptor in the anterior pituitary. Other agonists (e.g. apomorphine and dopamine) and antagonists (e.g. antipsychotic phenothiazines and butyrophenones) affect both classes of receptor.     

The central effect of noradrenaline on thermoregulation in the neonatal guinea pig.

Noradrenaline (10 microgram) injected into the lateral cerebral ventricle of guinea pigs aged 2 to 12 days produced a rapid increase in oxygen consumption and in colonic temperature at an ambient temperature of 30 degrees C. The increase was most pronounced in the youngest animals and decreased with advancing age, but was still significant at 12 days of age. Species differences and the role of ambient temperature in the responses are discussed.     

Characterization of central cholecystokinin receptors using a radioiodinated octapeptide probe

We have developed a binding assay for 125I-Bolton-Hunter-labeled cholecystokinin octapeptide (125I-(BH)CCK8) using mouse cerebral cortex membrane preparations. This ligand interacts with cortical membrane preparations in a saturable, high-affinity manner, satisfying the requirements for specific cholecystokinin receptor labeling. Salt is required for maximal binding and BSA is specifically inhibitory with cerebral cortical but not with pancreatic sites. Cholecystokinin peptides as small as CCK30-33 displace binding at low nanomolar concentrations. Dissociation of 125I-(BH)CCK8 is biphasic in both mouse and guinea pig cortex. Pretreatment of membranes at 37 degrees C results in a marked loss of recognition sites, suggesting that the sites may be rapidly metabolized in vivo. After 37 degrees C pretreatment, the loss of CCK recognition sites corresponds to a selective loss of the slow component of dissociation curves. This selective elimination of one dissociation population, as well as the biphasic dissociation kinetics, suggests that at least two distinct CCK receptor subtypes exist in the brain.     

Two types of dopamine receptors in behavioral regulation.

Evidence has been presented indicating the existence of distinct dopamine receptors in the brain. The experimental proof that two types of dopamine receptors--the excitation-mediating (DAe) receptors and the inhibition-mediating (DAi) receptors--exist in the snail brain is put forward. The functioning of DAi and DAe receptors in locomotor activity and stereotyped behavior is discussed along with implications of dopaminergic involvement in a gratification system.     

Iodobenzamide for in vivo exploration of central dopamine receptors: evaluation in animal models of supersensitivity

Iodobenzamide is a promising agent to investigate D2 receptors by SPECT in living human brain. In this work, we have evaluated this radiolabeled compound in two animal models of D2 receptors supersensitivity. In the first model, rats were treated chronically with haloperidol during three weeks (S.C. injection of 0.5 mg/kg/day). One week after the last day of treatment, they were I.V. injected with 125I-IBZM. In vivo specific binding study showed a 45 percent increase of 125I-IBZM fixation in the striatum of treated rats. In a second step of experiments, animals were unilaterally lesioned by a stereotaxic injection of 6-OHDA in the substantia nigra, 23 days before receiving 125I-IBZM. Autoradiographic analysis of coronal brain sections showed a 38 percent enhancement of 125I-IBZM in vivo binding in the striatum on the lesioned side as compared to the contralateral intact side; this increase occurred in striatal lateral area. These data demonstrate that 125I-IBZM is convenient to detect alterations of dopamine D2 receptors in vivo in the rat. Thus IBZM labelled with 123I can be a very useful imaging agent for the exploration of D2 receptors in pathological situations.     

Neuroglial relationship in experimental central demyelination in rabbits.

Experimental allergic encephalomyelitis (EAE) induced in the rabbit, initially showed inflammatory lesions, followed by demyelination. The EAE lesions also exhibited an ascendency in their appearance, i.e. they involved the spinal cord, brain stem and cerebellum in that order. During the inflammatory stage of EAE, the astrocytes became hypertrophied and the oligodendrocytes were seen to be degenerating. The inflammatory cells included lymphocytes, plasma cells, and gitter cells. When demyelination had set in, there appeared a paucity of oligodendrocytes and a marked astrocytosis in and around the lesions. The relationship of the oligodendrocytes with the maintenance of myelin in the central nervous system is discussed.