Expression of muscarinic acetylcholine and dopamine receptor mRNAs in rat basal ganglia.

Within the basal ganglia, acetylcholine and dopamine play a central role in the extrapyramidal control of motor function. The physiologic effects of these neurotransmitters are mediated by a diversity of receptor subtypes, several of which have now been cloned. Muscarinic acetylcholine receptors are encoded by five genes (m1-m5), and of the two known dopamine receptor subtypes (D1 and D2) the D2 receptor gene has been characterized. To gain insight into the physiological roles of each of these receptor subtypes, we prepared oligodeoxynucleotide probes to localize receptor subtype mRNAs within the rat striatum and substantia nigra by in situ hybridization histochemistry. Within the striatum, three muscarinic (m1, m2, m4) receptor mRNAs and the D2 receptor mRNA were detected. The m1 mRNA was expressed in most neurons (greater than 80%); the m2 mRNA, in neurons which were both very large and rare; and the m4 and D2 mRNAs, in 40-50% of the neurons, one-third of which express both mRNAs. Within the substantia nigra, pars compacta, only the m5 and D2 mRNAs were detected, and most neurons expressed both mRNAs. These data provide anatomical evidence for the identity of the receptor subtypes which mediate the diverse effects of muscarinic and dopaminergic drugs on basal ganglia function.     

D1 and D2 dopamine receptor mRNA in rat brain.

Physiological and pharmacological criteria have divided dopamine receptors into D1 and D2 subtypes, and genes encoding these subtypes have recently been cloned. Based on the sequences of the cloned receptors, we prepared oligodeoxynucleotide probes to map the cellular expression of the corresponding mRNAs in rat brain by in situ hybridization histochemistry. These mRNAs showed largely overlapping yet distinct patterns of expression. The highest levels of expression for both mRNAs were observed in the caudate-putamen, nucleus accumbens, and olfactory tubercle. Within the caudate-putamen, 47 +/- 6% and 46 +/- 5% of the medium-sized neurons (10-15 microns) expressed the D1 and D2 mRNAs, respectively, and only the D2 mRNA was observed in the larger neurons (greater than 20 microns). The D1 and D2 mRNAs were expressed in most cortical regions, with the highest levels in the prefrontal and entorhinal cortices. Within neocortex, D1 mRNA was observed primarily in layer 6 and D2 mRNA in layers 4-5. Within the amygdala, D1 mRNA was observed in the intercalated nuclei, and D2 mRNA in the central nucleus. Within the hypothalamus, D1 mRNA was observed in the suprachiasmatic nucleus and D2 mRNA in many of the dopaminergic cell groups. Within the septum, globus pallidus, superior and inferior colliculi, mammillary bodies, and substantia nigra only D2 mRNA was detected. These data provide insight into the neuroanatomical basis of the differential effects of drugs that act on D1 or D2 receptors.     

Axonal arborizations of a magnocellular basal nucleus input and their relation to the neurons in the thalamic reticular nucleus of rats.

A dense axonal plexus, arising in a portion of the magnocellular basal nucleus, was identified in the thalamic reticular nucleus in adult rats. The details of these axonal arbors as well as their relation to the neurons of the reticular nucleus were investigated by using Phaseolus vulgaris leucoagglutinin injections into the basal nucleus and intracellular injections of Lucifer yellow into reticular nucleus neurons. Axons arising in the caudal basal nucleus at the medial margin of the globus pallidus do not enter the dorsal thalamus but are confined to the reticular nucleus, where they arborize widely and densely. Neurons in the reticular nucleus are large, with sparsely spined and beaded dendrites, which radiate within the plane of the nucleus. Bouton-like swellings along basal nucleus axons are often found apposed to the somata of reticular nucleus neurons, although many are also apposed to dendrites. These morphological observations suggest a second potentially significant route, in addition to its well-known direct cortical projection, through which the magnocellular basal nucleus could influence cortical function: it may, by strategically modulating the excitability of reticular nucleus neurons, alter the general state of the thalamus and hence affect the initial transmission of information to the cortex.     

Immunohistochemical localization of the androgen receptor in rat and human tissues.

Immunohistochemical localization of the androgen receptor (AR) was performed in reproductive tissues, submaxillary gland, pituitary, and brain of the rat and in human prostate. AR was visualized using either of two polyclonal antibodies raised against peptides with sequences derived from rat and human AR. Tissue sections of 6-8 microns, frozen in isopentane and fixed in paraformaldehyde, were stained using immunoglobulin G fractions of immune, preimmune, and peptide-adsorbed immune sera in the avidin-biotin peroxidase procedure. AR was prominent in nuclei of acinar epithelial cells of epididymis, ventral prostate, seminal vesicle, and ductus deferens from the intact rat. Androgen withdrawal, 3 days after castration, resulted in the loss of receptor immunostaining, which was restored within 15 min of androgen administration. Stromal cell staining was absent or weak in the ventral prostate of intact rats, but was more evident in the epididymis. AR was confined to nuclei of cells within and bordering the interstitial compartment of the testis, including Sertoli cells, peritubular myoid cells, and interstitial cells, and was undetectable in germ cells. Submaxillary gland epithelial cells and a population of rat anterior pituitary cells showed strong nuclear staining of AR. In rat brain, AR was present in the medial preoptic, arcurate, and ventromedial nuclei of the hypothalamus, the medial nucleus of the amygdala, the CA-1 hippocampus, and the cortex. AR was prominent in acinar epithelial cells in human benign prostatic hyperplasia and was also present in stroma of fibromuscular benign hyperplasia. Heterogeneous staining was observed in stromal and epithelial cells of prostatic adenocarcinoma. The results of these studies indicate that AR can be detected immunohistochemically in a variety of tissues and cell types using antipeptide polyclonal antibodies. The presence of AR in tissues correlated with their known androgen responsiveness.     

Identification and localization of glucagon-like peptide-1 and its receptor in rat brain

The existence and distribution of glucagon-like peptide-1 (GLP-1) and its receptor in rat brain in relation to that of glucagon were examined. The concentration of GLP-1 immunoreactivity (GLP-1-IR), measured by a specific and sensitive RIA established in this study with anti GLP-1 serum (LMT-01), was found to be highest in the thalamus-hypothalamus, followed by the medulla oblongata. The distribution of glucagon-like immunoreactivity was similar to that of GLP-1-IR. However, appreciable glucagon immunoreactivity was detected only in the thalamus-hypothalamus. Gel filtration analysis showed the presence of GLP-1-IR of various molecular weights in the extract of thalamus-hypothalamus including that eluted at the same position as synthetic GLP-1 (1-37); moreover, HPLC analysis also confirmed the presence of GLP-1-IR, eluted at the exact position as synthetic GLP-1 (1-37). The distribution of receptors for GLP-1 corresponded with that of GLP-1-IR in the rat brain, except in the pituitary gland. The distribution of these receptors was also similar to that of glucagon receptors. The thalamus-hypothalamus, pituitary gland, and medulla oblongata were rich in GLP-1 and glucagon-binding sites. The binding affinities of GLP-1 and glucagon were in the nanomolar range [disocciation constant Kd approximately equal to 4 nM]. The presence of specific, high affinity receptors for GLP-1 was confirmed by demonstrating that GLP-1 stimulated cAMP formation in the thalamus-hypothalamus and the pituitary gland. The concentration of GLP-1 required for half-maximal stimulation of cAMP formation in these regions was about 1 nM. These results suggest that GLP-1 may be synthesized in certain parts of the brain and play a role as a neurosignal transmitter.     

Identification, characterization, and localization of the dopamine D3 receptor in rat brain using 7-[3H]hydroxy-N,N-di-n-propyl-2-aminotetralin.

We have identified 7-[3H]hydroxy-N,N-di-n-propyl-2-aminotetralin ([3H]7-OH-DPAT) as a selective probe for the recently cloned dopamine D3 receptor and used it to assess the presence of this receptor and establish its distribution and properties in brain. In transfected Chinese hamster ovary (CHO) cells, it binds to D3 receptors with subnanomolar affinity, whereas its affinity is approximately 100-, 1000-, and 10,000-fold lower at D2, D4, and D1 receptors, respectively. Specific [3H]7-OH-DPAT binding sites, with a Kd of 0.8 nM and a pharmacology similar to those at reference D3 receptors of CHO cells, were identified in rat brain. D3 receptors differ from D2 receptors in brain by their lower abundance (2 orders of magnitude) and distribution, restricted to a few mainly phylogenetically ancient areas--e.g., paleostriatum and archicerebellum--as evidenced by membrane binding are autoradiography studies. Native D3 receptors in brain are characterized by an unusually high nanomolar affinity for dopamine and a low modulatory influence of guanyl nucleotides on agonist binding. These various features suggest that D3 receptors are involved in a peculiar mode of neurotransmission in a restricted subpopulation of dopamine neurons.     

Immunohistochemical localization of cholecystokinin- and gastrin-like peptides in the brain and hypophysis of the rat

The distribution of gastrin-cholecystokinin-like peptide(s) is reported in brain and hypophysis of the rat. The unlabeled peroxidase-antiperoxidase complex immunohistochemical technique was used. Controls of specificity for various peptides were studied with solid-phase absorption. Colchicine treatment was necessary to obtain positivity in many neuronal cell bodies. In addition to their already known distribution, gastrin-cholecystokinins containing neural cell bodies and fibers were present in olfactory structures, in various preoptic and hypothalamic nuclei (except in mamillary bodies), in mesencephalic nucleus linearis rostralis, and in A-10, A-9, and A-8 regions of Dahlström and Fuxe, which include substantia nigra. From previous investigations and the present distribution study, it can be inferred that, although most of the brain material consists of cholecystokinin, gastrins may also be present in hypothalamo-posthypophyseal magnocellular cells, in nucleus tractus solitarii, and in the dorsal horn of the spinal cord. The distribution of positive cell bodies in the peripheral part of the paraventricular nucleus and in the dorsal part of the supraoptic nuclei in the hypothalamus is similar to that of oxytocin neurons. The localization of positive cell bodies in A-10, A-9, and A-8 regions of Dahlström and Fuxe is similar to that of dopaminergic neurons. The mesencephalic concentration of cell bodies and the wide distribution of fibers in striatal, hypothalamic, septal, and other hemispheric structures together with thick positive fibers in the medial forebrain bundle is consistent with the existence of ascending mesencephalic pathways, including the nigrostriate pathway.     

Antibodies to the beta-adrenergic receptor: attenuation of catecholamine-sensitive adenylate cyclase and demonstration of postsynaptic receptor localization in brain.

Antibodies to the beta 2-adrenergic receptor of frog erythrocytes have been raised in rabbits by immunization with purified receptor preparations. Binding of the antibodies to the receptors was demonstrated by immunoprecipitation and by the altered mobility of the antibody-bound receptors on steric-exclusion HPLC columns. As assessed by a radioimmunoassay developed with the antibody, beta 2-adrenergic receptors from several sources showed various degrees of immunological crossreactivity whereas several beta 1-adrenergic receptors did not crossreact. The antibody appeared to not bind at the ligand binding site of the receptor and did not perturb antagonist radioligand binding to the receptor. Nonetheless, the antibodies selectively attenuated catecholamine-stimulated adenylate cyclase. This suggests that the antibodies recognize and bind to domains of the receptor other than the binding site and that may be involved in coupling to other components of the adenylate cyclase system. Immunocytochemical techniques were used with the antibodies to delineate a postsynaptic localization of beta-adrenergic receptors in rat and frog brain. Thus, these anti-beta-adrenergic receptor antibodies provide a useful reagent for probing beta-adrenergic receptor structure, function, and localization.     

Expression and localization of human dopamine D2 and D4 receptor mRNA in the adrenal gland, aldosterone-producing adenoma, and pheochromocytoma

Aldosterone secretion is evidently regulated by a dopaminergic inhibitory mechanism. Pharmacological characterization and autoradiographic studies revealed D2-like receptors in the adrenal cortex, especially in the zona glomerulosa. However, the subtype of the dopamine receptors involving this regulation has not been elucidated. To investigate which subtype of receptors expresses in the adrenal cortex, we examined the messages of D2-like receptors, D2, D3, and D4, by RT-PCR and in situ hybridization of adrenal glands and adrenal neoplasm. Both D2 and D4 receptors were expressed in normal adrenal glands, pheochromocytoma, and aldosterone-producing adenoma. However, the D2 receptors were not universally expressed, in contrast with the D4 receptors that were detected in all cases of aldosterone-producing adenoma and adrenal remnant. No D3 receptor message was detected by RT-PCR in any adrenal sample. Both D2 and D4 receptors were expressed in significant amounts in the adrenal medulla and pheochromocytoma. In the adrenal cortex, the expression of the D2 receptors was in the zona glomerulosa and zona reticularis, with no different signal intensities between the two zones. D4 receptors were mainly localized in the zona glomerulosa and, to a lesser extent, in the zona reticularis. Both receptors were expressed at low levels in the zona fasciculata. In aldosterone-producing adenoma, the expression of D2 and D4 was especially found in nonzona fasciculata-like cells. To elucidate which dopamine receptor regulates aldosterone secretion, the effects of specific D2 and D4 antagonists, raclopride and clozapine, respectively, were examined in cultured NCI-H295 cells. Dopamine further increased angiotensin II-induced aldosterone secretion by 20%. In the presence of 1 microM dopamine and angiotensin II, 10(-5)-10(-7) M clozapine decreased aldosterone levels by 40-55%. The decrease in aldosterone secretion by clozapine was completely reversed when raclopride was added simultaneously. These data suggest that dopamine exerts dual effects on aldosterone secretion in NCI-H295 cells. Activation of D4 receptors can increase aldosterone secretion, whereas an inhibitory effect is mediated via D2 receptors. In summary, we demonstrated the existence of both D2 and D4 receptors in the human adrenal gland and adrenal neoplasm. Both receptors play significant roles in the modulation of aldosterone secretion, but in opposite directions.     

Localization of the mRNA for the dopamine D2 receptor in the rat brain by in situ hybridization histochemistry.

32P-labeled oligonucleotides derived from the coding region of rat dopamine D2 receptor cDNA were used as probes to localize cells in the rat brain that contain the mRNA coding for this receptor by using in situ hybridization histochemistry. The highest level of hybridization was found in the intermediate lobe of the pituitary gland. High mRNA content was observed in the anterior lobe of the pituitary gland, the nuclei caudate-putamen and accumbens, and the olfactory tubercle. Lower levels were seen in the substantia nigra pars compacta and the ventral tegmental area, as well as in the lateral mammillary body. In these areas the distribution was comparable to that of the dopamine D2 receptor binding sites as visualized by autoradiography using [3H]SDZ 205-502 as a ligand. However, in some areas such as the olfactory bulb, neocortex, hippocampus, superior colliculus, and cerebellum, D2 receptors have been visualized but no significant hybridization signal could be detected. The mRNA coding for these receptors in these areas could be contained in cells outside those brain regions, be different from the one recognized by our probes, or be present at levels below the detection limits of our procedure. The possibility of visualizing and quantifying the mRNA coding for dopamine D2 receptor at the microscopic level will yield more information about the in vivo regulation of the synthesis of these receptors and their alteration following selective lesions or drug treatments.     

Distinct roles of dopamine D2L and D2S receptor isoforms in the regulation of protein phosphorylation at presynaptic and postsynaptic sites

Dopamine D2 receptors are highly expressed in the dorsal striatum where they participate in the regulation of (i) tyrosine hydroxylase (TH), in nigrostriatal nerve terminals, and (ii) the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), in medium spiny neurons. Two isoforms of the D2 receptor are generated by differential splicing of the same gene and are referred to as short (D2S) and long (D2L) dopamine receptors. Here we have used wild-type mice, dopamine D2 receptor knockout mice (D2 KO mice; lacking both D2S and D2L receptors) and D2L receptor-selective knockout mice (D2L KO mice) to evaluate the involvement of each isoform in the regulation of the phosphorylation of TH and DARPP-32. Incubation of striatal slices from wild-type mice with quinpirole, a dopamine D2 receptor agonist, decreased the state of phosphorylation of TH at Ser-40 and its enzymatic activity. Both effects were abolished in D2 KO mice but were still present in D2L KO mice. In wild-type mice, quinpirole inhibits the increase in DARPP-32 phosphorylation at Thr-34 induced by SKF81297, a dopamine D1 receptor agonist. This effect is absent in D2 KO as well as D2L KO mice. The inability of quinpirole to regulate DARPP-32 phosphorylation in D2L KO mice cannot be attributed to decreased coupling of D2S receptors to G proteins, because quinpirole produces a similar stimulation of [(35)S]GTPgammaS binding in wild-type and D2L KO mice. These results demonstrate that D2S and D2L receptors participate in presynaptic and postsynaptic dopaminergic transmission, respectively.     

Cloning, molecular characterization, and chromosomal assignment of a gene encoding a second D1 dopamine receptor subtype: differential expression pattern in rat brain compared with the D1A receptor.

Multiple D1 dopaminergic receptor subtypes have been postulated on the basis of pharmacological, biochemical, and genetic studies. We describe the isolation and characterization of a rat gene encoding a dopamine receptor that is structurally and functionally similar to the D1 dopamine receptor. The coding region, which is intronless, encodes a protein of 475 amino acids (Mr 52,834) with structural features that are consistent with receptors coupled to guanine nucleotide-binding regulatory proteins. The expressed protein binds dopaminergic ligands and mediates stimulation of adenylyl cyclase with pharmacological properties similar to those of the D1 dopamine receptor. The gene encoding the human homologue of this receptor subtype is located to the short arm of chromosome 4 (4p16.3), the same region as the Huntington disease gene. In striking contrast to the previously cloned D1 receptor, little or no mRNA for the receptor described here was observed in striatum, nucleus accumbens, olfactory tubercle, and frontal cortex. High levels of mRNA for this receptor were found in distinct layers of the hippocampus, the mammillary nuclei, and the anterior pretectal nuclei, brain regions that have been shown to exhibit little or no D1 dopamine receptor binding. On the basis of its properties we propose that this dopamine receptor subtype be called D1B.     

胶质细胞源性神经营养因子及受体在Alzheimer病模型大鼠脑组织中的表达

目的　探讨胶质细胞源性神经营养因子 (GDNF)在 Alzheimer病 (AD)的发病机制中的作用。方法　应用免疫组化方法检测神经营养因子 (GDNF)及其受体 GFRα- 1、Ret在正常及 AD模型大鼠脑组织中的表达。结果　实验组 (手术侧 )海马 CA1区神经细胞数目较对侧 (非手术侧 )及正常对照组明显减少 (P<0 .0 5) ,非手术侧与对照组则无明显差别。实验组 (手术侧、非手术侧 )及对照组大鼠的大脑皮层、海马均有 GDNF及受体 GFRα- 1、Ret的阳性表达 ;阳性免疫反应产物定位于神经细胞浆内 ;阳性反应程度对照组、实验组 (非手术侧 )、实验组 (手术侧 )依次减弱 ,但差别无统计学意义。结论　在单侧隔 -穹窿海马伞切断制成的 AD大鼠脑组织中 ,手术损伤侧海马 CA1区神经元数目减少 ,但减少的神经元的种类不明 ,机制亦不明。在该模型大鼠脑组织中没有确切的 GDNF合成减少及 /或受体 (GFRα- 1、Ret)表达障碍 ,不能确定 GDNF在 AD发病机制中的作用以及在 AD治疗上的应用价值。     

Effects of tiodazosin, praxosin, trimazosin and phentolamine on blood pressure, heart rate and on pre- and postsynaptic alpha-adrenergic receptors in the rat

Subcutaneous administration of tiodazosin (0.1-3 mg/kg), prazosin (0.01-1 mg/kg), trimazosin (10-30 mg/kg) and phentolamine (0.1-3 mg/kg) to conscious spontaneously hypertensive rats (SHR) produced graded decreases in blood pressure with the order of potency being prazosin > tiodazosin > phentolamine > trimazosin. Heart rate was elevated predominantly only by phentolamine and this was consistent with the activity of this agent for both pre- and postsynaptic alpha-adrenergic receptors. In contrast, tiodazosin, prazosin and trimazosin showed selectivity only for postsynaptic alpha-adrenergic receptors. Acute oral administration of tiodazosin and prazosin indicated tiodazosin to be about 1/2 as potent as prazosin. However, chronic administration of equivalent doses of the two compounds for 25 and 52 days via the drinking water indicated approxiately equivalent, sustained reductions in blood pressure. Furthermore, at the end of the 52-day chronic dosing period tiodazosin caused appreciably less alpha-adrenergic receptor antagonist activity than prazosin as assessed by the norepinephrine dose-pressor response profiles. These results indicate that following chronic dosing with tiodazosin in the rat other mechanisms besides alpha-adrenergic receptor antagonist activity are probably contributing to the antihypertensive effect in the rat.     

Serotonin and dopamine receptors in the rat pituitary gland: autoradiographic identification, characterization, and localization

S2 serotonin and D2 dopamine receptors were identified, characterized, and localized in rat pituitary gland by quantitative light microscopic autoradiography. [3H]Spiperone was used to localize S2 serotonin and D2 dopamine receptors. A high concentration of D2 dopamine receptors [1 microM 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN)- or sulpiride-displaceable [3H]spiperone binding] was found in the rat intermediate lobe with much lower concentrations present in the anterior and posterior lobes. Significant densities of cinanserin-displaceable [3H]spiperone binding sites (i.e. S2 serotonin receptors) were present in all three lobes of the pituitary gland. [125I]Lysergic acid ([125I]LSD) was used to characterize further and selectively visualize S2 serotonin receptors in the rat pituitary. Data analysis by densitometry showed that [125I]LSD binding the rat intermediate pituitary was saturable and of high affinity with an apparent dissociation constant (Kd) of 1.2 nM. Data from competition studies using a variety of compounds showed a S2 serotonin receptor profile at this [125I]LSD binding site in rat pituitary. The highest concentration of [125I]LSD binding sites was found in the intermediate lobe with progressively lower concentrations present in the posterior and anterior lobes, respectively. There is a uniform pattern of distribution of S2 serotonin and D2 dopamine receptors within each lobe of the rat pituitary gland. The results of the present study provide the first identification of S2 serotonin receptors in the pituitary and confirm the heterogeneous distribution of D2 dopamine receptors within the rat pituitary. These data provide further evidence for the importance of dopamine in regulating pituitary function and suggest a physiological role for serotonin in regulating pituitary hormone secretion.     

The basal ganglia, the deep prepyriform cortex, and seizure spread: bicuculline is anticonvulsant in the rat striatum.

The gamma-aminobutyric acid antagonist, bicuculline methiodide (BMI), induces myoclonic seizures in rats when injected into the deep prepyriform cortex at concentrations lower than those that induce convulsions from the amygdala, hippocampus, or neocortex. This observation prompted the suggestion that the deep prepyriform cortex was responsible for seizure generation regardless of the neurotransmitter and neuronal circuits involved. Bilateral intrastriatal application of BMI protects rats against seizures induced by (i) local application of BMI into the deep prepyriform cortex and (ii) systemic application of bicuculline, pilocarpine (a cholinergic agonist), or kainic acid (a glutamate receptor agonist). The region of the striatum sensitive to the previously unknown anticonvulsant action of BMI is located in the immediate vicinity of the deep prepyriform cortex and is 100-150 times more sensitive to the anticonvulsant action relative to the sensitivity of the deep prepyriform cortex to the convulsant action of BMI. These data suggest a powerful gamma-aminobutyric acid-dependent gating role of the basal ganglia in determining the seizure threshold in the forebrain. This argues against the suggestion that the deep prepyriform cortex plays a crucial role in the generation of seizures following systemic administration of convulsants. The discovery of an anticonvulsant action of BMI in the rat striatum contradicts the gamma-aminobutyric acid theory of epilepsy, which implies that deficits in the gamma-aminobutyric acid-mediated inhibition in the central nervous system lead to the emergence of seizures.     

Immunocytochemical localization of the glucocorticoid receptor in rat brain, pituitary, liver, and thymus with two new polyclonal antipeptide antibodies.

The intracellular localization of the glucocorticoid receptor (GR) was studied in male rat brain, pituitary, liver, and thymus. Two new polyclonal anti-GR antibodies, GR 57 and GR 59, raised against two synthetic peptides (346-357 and 245-259) that correspond to unique regions of the amino-terminus of human GR were used. Vibratome sections (30-50 microns) of perfused brain and frozen sections (6-8 microns) of pituitary, liver, and thymus fixed in paraformaldehyde were incubated in preimmune serum, immunoserum, epitope-purified immunoserum, or peptide-absorbed immunoserum of either GR 57 or GR 59 and immunostained by the avidin-biotin peroxidase method. GR immunoreactivity (GR-ir) was primarily nuclear in brain, pituitary, liver, and thymus sections from intact rats. Adrenalectomy caused nuclear GR-ir to decrease and cytoplasmic GR-ir to increase. When adrenalectomized rats were treated with corticosterone (100 micrograms and 1 mg) or dexamethasone (1 microgram, 100 micrograms, and 1 mg), GR-ir was again predominantly nuclear. One microgram of corticosterone failed to cause nuclear GR-ir when administered to adrenalectomized rats. Immunoreactive neurons and glial cells were extensively distributed, with varied intensity, throughout the rat forebrain. The areas include cortex, septum, hippocampus, amygdala, thalamus, and hypothalamus. Cells with the strongest GR-ir were located in the caudate putamen, paraventricular, arcuate, and central amygdala nuclei, areas CA1-CA2 of the hippocampus, and laminae 4 and 5 of the cortex. In the pituitary, cells of the anterior and posterior lobes were GR immunoreactive, while those in the intermediate lobe were not. Hepatocytes of the liver and thymocytes and reticuloepithelial cells of the thymus were GR immunoreactive. The results show that GR can be localized immunocytochemically in numerous rat tissues using antipeptide polyclonal antibodies and correlated with the results of biochemical and ligand receptor studies.     

Calcium antagonist flunarizine hydrochloride affects striatal D2 dopamine receptors in the young adult and aged rat brain

The calcium (Ca) antagonist flunarizine hydrochloride (FNZ) has been reported to induce parkinsonism, especially in the elderly. The effects of FNZ on dopamine receptors in rat striatal membranes, especially in aged rats, were studied using radiolabeled receptor assay. Similar displacing potencies in [(3)H]spiperone bindings were exhibited for FNZ and the Ca antagonists verapamil and nicardipine. FNZ was found to directly and competitively effect D2 receptors (D2-Rs) as an antagonist, without effecting D1 receptors. Furthermore, the washing of preoccupied membranes revealed that FNZ has a long-acting potent effect on D2-Rs. The comparative study of FNZ and sulpiride in young-adult and aged rats showed that the effect of FNZ on D2-Rs was more marked in aged rats. These results might be related to FNZ-induced parkinsonism and its high incidence in the elderly.     

Effects of vitamin D and its metabolites on calcium transport in the diabetic rat.

We studied diabetic rats, 5 days after streptozotocin injection, and matched controls to determine whether depressed duodenal calcium absorption associated with uncontrolled diabetes in the rat would respond to vitamin D or its metabolites. At the appropriate time following the intravenous injection of 0.25 mug of either vitamin D3, 25-hydroxycholecalciferol (25-OHD3), 1,25-dihydroxycholecalciferol (1,25-OH)2D3), or 1alpha-hydroxycholecalciferol (1alpha-OHD3) to half of each diabetic and control group, calcium transport was evaluated using everted duodenal sacs with 0.4 mM40Ca and tracer 45Ca on both mucosal and serosal surfaces. All agents stimulated duodenal calcium absorption in controls. Diabetics responded only to 1,25-(OH)2D3, the metabolite that acts directly on the duodenum, and to its synthetic analog, 1alpha-OHD3. 1alpha-OHD3 is activated to 1,25-(OH)2D3 by 25-hydroxylation in the liver; 25-OHD3 must be 1alpha-hydroxylated in the kidney to be active. The stimulation of duodenal calcium absorption in diabetic rats by 1alpha-OHD3, but not by either vitamin D3 or 25-OHD3, is most consistent with a defect in vitamin D metabolism at the 1alpha-hydroxylation step in the kidney.