A possible role of BDNF in prostate cancer detection.

Many studies have demonstrated that both normal and malignant prostate cells respond to a variety of growth factors, while several significant differences were found between normal and tumoural cells. The aim of this study was to focus on the localization and distribution of the immuno-reactivity for neurotrophins (NTs) and neurotrophin receptors (NTRs) in normal, hyperplastic and prostate cancer cells, obtained from 40 subjects. We studied samples obtained from 16 prostate cancer (PC, retropubic radical prostatectomy), 20 benign prostatic hyperplasia (BPH, supra-pubic prostatectomy) and normal peripheral prostate tissue from four fresh male cadavers. Samples were examined via immunohistochemical techniques in order to detect the expression of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and their own receptors TrkA, p75, TrkB and TrkC. We observed a high expression of BDNF and TrkB in PC and BPH, though no immuno-reactivity was found for p75. Low expression was reported by other NTs and NTRs in the normal peripheral prostate zone, BPH and PC. These data suggest a possible predictive role for NTs and NTRs, especially for BDNF and TrkB, in the diagnosis and/or management of prostate cancer. The absence of p75 expression confirms its supposed role in apoptotic phenomenon.

Immunohistochemical profile of some neurotransmitters and neurotrophins in the seminiferous tubules of rats treated by lonidamine.

Lonidamine (LND) or [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid] is an anticancer and antispermatogenic drug that exerts a large number of effects on tumor cells and germ cells. Sexually mature male Sprague-Dawley rats were housed at 22 degrees C on a 12-h light/12-h dark cycle 1 week before the experiments, with free access to food and water. LND was suspended in 0.5% methylcellulose at a concentration of 10 mg/mL and administered orally at the dose of 10 mL/kg (b.w.) as a single dose. Control rats received an equal amount of vehicle. Testes were removed, fixed for 24 h in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate (pH 7.2 at 22 degrees C), rinsed with the same buffer, and stored at room temperature. From each sample, a block of tissue was removed by sectioning through the organ. After dehydration in ethanol at increasing concentrations (70-100%), each block was embedded in paraffin and serial 5 mm thick sections were cut using a rotatory microtome. The immunoreactivity for NTs has been observed in spermatogonia of untreated rats, while the rats treated with LND showed an immunohistochemical localization in all the stages of germinal cells. The generally well-expressed immunoreactivity for the neurotrophins receptors in treated rats observed in our study is presumably attributable to alterations of the receptors' structure and/or expression leading to changes of the activity, affinity, localization or protein interactions that may depend on sensitization of ion channels (induced by LND). Neurotrophins (NTs) appear to be interesting proteins for the modulation of sperm maturation and motility with a prominent role for the nerve growth factor (NGF), that may exert an autocrine or paracrine role. We therefore investigated the location and distribution of immunoreactivity for some neurotransmitters (SP, VIP, CGRP, nNOS, Chat), neurotrophins (NGF, BDNF, NT-3) and their own receptors (TrKA, TrKB, TrKC, p75) in the seminiferous tubules of male rats treated by LND in the light of the literature on this topic.

Expression of neurotransmitters and neurotrophins in neurogenic inflammation of the rat retina.

Antidromic stimulation of the rat trigeminal ganglion triggers the release of substance P (SP) and calcitonin gene-related peptide (CGRP) from sensory nerve terminals of the capsaicin sensitive C-fibers. These pro-inflammatory neuropeptides produce a marked hyperemia in the anterior segment of the eye, accompanied by increased intraocular pressure, breakdown of the blood-aqueous barrier and myosis. To assess the effects of neurogenic inflammation on the retina, specifically on the immunostaining of neurotransmitters and neurotrophins, as well as on the expression of neurotrophin receptors in the retina. RT-PCR was also accomplished in control and stimulated animals to confirm the immunohistochemical results. In the electrically stimulated eyes, immunostaining for SP, CGRP, VIP and nNOS demonstrated a marked increase in the RPE/POS (Retinal Pigment Epithelium/Photoreceptor Outer Segments), in the inner and outer granular layers and in the ganglion cells in comparison to the control eyes. CGRP and SP were found increased in stimulated animals and this result has been confirmed by RT- PCR. Changes in neurotrophin immunostaining and in receptor expression were also observed after electric stimulation of trigeminal ganglia. Decrease of BDNF and NT4 in the outer and inner layers and in ganglion cells was particularly marked. In stimulated rat retinas immunostaining and RT-PCR showed a NGF expression increase. Neurotrophin receptors remained substantially unchanged. These studies demonstrated, for the first time, that antidromic stimulation of the trigeminal ganglion and subsequent neurogenic inflammation affect immunostaining of retinal cell neurotransmitter/neuropeptides and neurotrophins as well as the expression of neurotrophin receptors.

Expression of neurotransmitters and neurotrophins in human adenoid tissue.

Mucosae-associated lymphoid tissues are richly innervated and the mucosae contain peptidergic nerve endings associated with different types of cells and macrophages. The lymphatic tissue is known to interact with the nervous system and several organs, implicated in the host response to a wide range of stressors, and is also richly innervated. We focussed our attention on the immune organs with particular regard to the human adenoid lymphatic tissues in order to investigate the neuroimmune links and the possible existence of relationships among different neurotransmitters and lymphocytes, macrophages, epithelial cells and nerve fibers by testing the expression of certain neurotransmitters and neurotrophins (NTs) with their own receptors.

Enzyme histochemistry of glutamate dehydrogenase in ageing rat cerebellar cortex.

The influence of ageing on glutamate dehydrogenase activity was studied in the cerebellar cortex of 3-month-old (young), 12-month-old (adult) and 26-month-old (aged) male Sprague-Dawley rats by using an enzyme histochemical technique. In young rats the enzyme reactivity was observed in the neuropil of the molecular layer as well as in the perikarya of basket cells and of stellate cells; within the cytoplasm of Purkinje neurons and in synaptic glomeruli of the granular layer. Glutamate dehydrogenase activity was significantly increased in the cerebellar cortex of adult rats and decreased in old animals. The synaptic glomeruli of the granular layer were the structures of the cerebellar cortex more remarkably affected by age-related changes. The possibility that decreased glutamate catabolism occurring in the ageing cerebellar cortex may result in an excess of the amino acid and may contribute to the nerve cell loss occurring in the cerebellum of old rats is discussed.

Age-related changes of the metabolic profile of rat cerebellar cortex: enzyme histochemical study.

The influence of aging on the metabolic profile of cerebellar cortex was studied in young (3-month-old), adult (12-month-old) and aged (26-month-old) male Sprague-Dawley rats using enzyme histochemical techniques. The following enzymatic activities related to energy transduction were examined: lactate-(LDH) and succinate-(SDH) dehydrogenases; NADH2-tetrazolium reductase (NADHD) and alpha-glycerophosphate-dehydrogenase (GPDH). The intensity of enzymatic staining within the neuropil of molecular and granular layers as well as within the cytoplasm of Purkinje neurons of young, adult and aged animals was assessed microphotometrically. In the molecular layer LSH, SDH and NADHD levels were reduced in old rats; GPDH was decreased both in adult and old animals. In Purkinje neurons no age-related changes of the enzymatic activities under study were observed. In the granular layer LDH and GPDH showed an age-dependent loss; SDH and NADHD were unchanged. The possibility that age-related changes of the enzymatic activities under study may be due to impaired energy production mechanisms and/or represent the consequence of reduced energetic needs resulting from the documented age-dependent loss of synapses in the molecular or in the granular layers of cerebellar cortex is discussed.

Age-related changes in adrenaline content of rat splanchnic blood vessels.

The influence of ageing on the adrenaline content of the superior mesenteric artery and vein, renal artery and vein and portal vein was studied in 3-month- (young), 12-month- (adult) and 24-month-old (old) male Wistar rats using radioenzymatic assay for the measurement of catecholamine levels. Adrenaline concentrations were unchanged in the vascular wall of the blood vessels examined in adult rats, but were significantly decreased in the vascular wall of the superior mesenteric, renal and portal veins of old rats. In contrast, no age-dependent changes of adrenaline levels were found in the vascular wall of the superior mesenteric or renal arteries. The possibility that the loss of adrenaline concentrations in the venous vascular wall may be in some way related to the cardiovascular impairment occurring with age is discussed.

NADPH-diaphorase histochemistry in the rat cerebral cortex and hippocampus: effect of electrolytic lesions of the nucleus basalis magnocellularis.

Unilateral or bilateral electrolytic lesions of the nucleus basalis magnocellularis (NBM) increased NADPH-diaphorase in the fronto-parietal cortex and in the CA1-CA3 fields of the hippocampus. NBM is the cholinergic basal forebrain nucleus supplying the fronto-parietal cortex but not the hippocampus. This increase was more remarkable at 4 weeks than at 2 weeks after lesioning. Monolateral or bilateral lesioning of the NBM increased to a similar extent NADPH-diaphorase. The number of neurons expressing NADPH-diaphorase was not statistically different between sham-operated and NBM-lesioned rats. These results indicate that similarly as reported in experimental damage of several brain areas, lesions of the NBM induce NADPH-diaphorase. The induction of this marker for nitric oxide synthase occurs both in the target of projections arising from the NBM such as the frontal cortex and in an area not directly supplied by NBM such as the hippocampus. Lesion-induced NADPH-diaphorase increase may contribute to neurodegenerative changes caused by damage of the NBM area.

Influence of age on L-type Ca2+ channels in the pulmonary artery and vein of spontaneously hypertensive rats.

The influence of age on the density and localization of L-type Ca2+ channels was studied during development of hypertension in the pulmonary artery and vein of spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto (WKY) rats by radioligand binding assay and light microscope autoradiography. SHR were examined at 6 weeks (juvenile, pre-hypertensive stage), 12 weeks (young, developing hypertension) and 24 weeks (mature, established hypertension). The dihydropyridine-type Ca2+ antagonist [3H]nicardipine was used as a radioligand. It was bound specifically to sections of rat pulmonary artery and vein. Dissociation constant (Kd) values were similar in WKY rats and SHR, whereas maximum density of binding sites (Bmax) values increased in SHR in comparison with WKY rats. This increase was noticeable from the pre-hypertensive phase. The pharmacological profile of [3H]nicardipine binding was similar in different age groups of either normotensive and hypertensive rats. Quantitative analysis of autoradiographs from SHR revealed a progressive increase of silver grains in smooth muscle of tunica media and to a lesser extent in the adventitia of pulmonary artery but not of pulmonary vein from pre-hypertensive stage to developing hypertension. No further changes were observed in established hypertension. The above data indicate that the density of L-type Ca2+ channels of pulmonary arteries is increased in SHR. This augmentation after the pre-hypertensive phase suggests the occurrence of dysregulation of Ca2+ handling in the pulmonary vasculature of developing SHR.

Effect of nicardipine treatment upon cardiac hypertrophy in spontaneously hypertensive rats: a morphometric and ultrastructural study.

OBJECTIVE: The present study was designed to investigate the effect of nicardipine administration upon systolic blood pressure (SBP) and cardiac hypertrophy in spontaneously hypertensive rats (SHR). DESIGN: SBP, heart: and left ventricle: body weight ratios, the cross-sectional area of cardiocytes, and the ultrastructure of the left ventricle were evaluated. METHODS: Ten-week old male SHR and age-matched normotensive Wistar-Kyoto rats were studied for 12 weeks. One group of SHR was treated for 12 weeks with a daily oral dose of 1 mg/kg nicardipine and another group with 1 mg/kg hydralazine; Wistar-Kyoto rats were used as a normotensive control group. Light and electron microscope techniques associated with image analysis and morphometry were used. RESULTS: Nicardipine administration normalized SBP values and significantly reduced the heart: and left ventricle: body weight ratios. Moreover, administration reduced the cross-sectional area of cardiocytes by approximately 38% in subendocardium and by 24% in subepicardium. Hydralazine administration significantly reduced SBP values but had no effect upon heart: or left ventricle: body weight ratios or the cross-sectional area of cardiocytes. Electron microscopy showed that nicardipine treatment was able to reduce the hypertension-dependent changes in cardiac ultrastructure consisting of alternations to intercalated discs and line Z morphology as well as in the decrease of the mitochondria: myofibrils ratio. CONCLUSIONS: The above data indicate that nicardipine administration is able to reduce SBP and to counter the development of structural and ultrastructural changes in cardiac morphology which represent a common complication of arterial hypertension.

Labeling of dopamine D3 and D4 receptor subtypes in human peripheral blood lymphocytes with [3H]7-OH-DPAT: a combined radioligand binding assay and immunochemical study.

Molecular biology studies have demonstrated that human peripheral blood lymphocytes express dopamine D2-like receptors belonging to the D3 and D4 receptor subtypes, whereas the characterization of these receptors using radioligand binding assay techniques provided conflicting results. The preferential dopamine D3 receptor agonist [3H]7-hydroxy-N, N-di-n-propyl-2-aminotetralin ([3H]7-OH-DPAT) was used recently for labeling lymphocyte dopamine D3 receptor. However, the selectivity of this compound for the D3 receptor was questioned. In this study we have investigated human peripheral blood lymphocyte dopamine receptor subtypes labeled by [3H]7-OH-DPAT using a conventional radioligand binding assay technique and antibodies against dopamine D2-like receptor subtypes. [3H]7-OH-DPAT was specifically bound to intact human peripheral blood lymphocytes with a dissociation constant (Kd) value of 0.32 + 0.03 nM and a maximum density of binding sites (Bmax) of 18.2 + 0.8 fmol/2 x 10(6) cells. [3H]7-OH-DPAT binding was unaffected by antibodies against dopamine D2 and D2S receptors. Anti-dopamine D3 and D4 receptor antibodies reduced [3H]7-OH-DPAT binding by about 53% and 32% respectively. Combination of anti D3 and D4 receptor antibodies reduced remarkably [3H]7-OH-DPAT binding. The above results suggest that the dopamine receptor agonist [3H]7-OH-DPAT labels dopamine D3 and D4 receptor subtypes in human peripheral blood lymphocytes. The use of antibodies raised against dopamine receptor subtypes in combination with radioligand binding assay may contribute to define receptor subtypes expressed by human peripheral blood lymphocytes in health and disease.

Identification of dopamine plasma membrane and vesicular transporters in human peripheral blood lymphocytes.

Plasma membrane dopamine transporter (DAT), vesicular monoamine transporters (VMAT) type-1 and -2 and the expression of the dopaminergic markers dopamine and tyrosine hydroxylase were assessed in membranes and/or in cytospin centrifuged human peripheral blood lymphocytes. The radiolabeled DAT ligand [3H]GBR12935 was bound to peripheral lymphocytes in a manner consistent with the specific binding to a dopamine uptake system, with a dissociation constant similar to that found in striatum, but with a lower density of binding sites. On the other hand, no specific binding occurred in cerebellum used as a test tissue not expressing DAT. Western blot analysis using antibodies raised against amino or carboxy terminus of DAT or against VMAT-1 or VMAT-2 revealed labeling of single bands of approximately 76, 55 or 68 KDa, respectively, displaying similar migration characteristics in lymphocytes and test tissues used for comparison. Immunofluorescence revealed that anti-dopamine, anti-tyrosine hydroxylase, anti-DAT, anti-VMAT-1 and anti-VMAT-2 antibodies labeled the total population of cytospin-centrifuged lymphocytes mounted on microscope slides. Confocal laser microscopy demonstrated that dopamine and VMAT-2 immunoreactivity was developed mainly in cytoplasmic punctiform areas likely corresponding to vesicles and to a lower extent was associated to plasma membrane. Tyrosine hydroxylase immunoreactivity was diffused to cytoplasm and to plasma membrane of lymphocytes, whereas DAT and VMAT-1 immunoreactivity were located almost exclusively in lymphocyte plasma membrane and cytoplasm, respectively. Lymphocyte DAT characterized in this study has probably functional relevance as [3H]dopamine was taken up by intact lymphocytes and uptake was inhibited specifically by compounds known to affect dopamine transport. These findings indicate that human peripheral blood lymphocytes possess DAT plasma membrane and VMAT-1 and VMAT-2 transporters. Increasing evidence indicates that dopamine transporter changes may be related to neuronal injury. In view of this assessment of lymphocyte DAT and VMAT transporters can be considered for identifying pathologies characterized by impaired dopaminergic neurotransmission.

Age-dependent changes in the expression of dopamine receptor subtypes in human peripheral blood lymphocytes.

The pharmacological profile and the density of dopamine D3 and D5 receptor subtypes expressed by human peripheral blood lymphocytes of subjects of different ages (ranging from 20 to 75 years) were assessed using radioligand binding techniques. Dopamine D3 receptor was assayed with [3H]7-hydroxy-N,N-di-n-propyl-2-aminotetraline ([3H]7-OH-DPAT) as a ligand. Dopamine D5 receptor was assayed using [3HIR]-(+)-(-chloro-2,3,4,5, tetrahydro-5-phenyl-1H-3-benzazepin-al-hemimaleate) ([3H]SCH 23390) as a ligand. The affinity and the pharmacological profile of [3H]7-OH-DPAT and [3H]SCH 23390 at dopamine D3 and D5 receptor, respectively, were similar in subjects of different ages. The density of dopamine D3 receptor binding sites was slightly decreased in subjects of 30-39 years in comparison with younger individuals. A remarkable loss of dopamine D3 receptor was then found between 40 and 49 years of age in comparison with younger subjects. A further slight decrease was noticeable between 50 and 59 years of age. The number of [3H]7-OH-DPAT binding sites was then stabilized after 60 years of age. The density of dopamine D5 receptor binding sites did not show age-dependent changes. The above findings indicate the occurrence of a decline in the density of lymphocyte dopamine D3 but not D5 receptor between adult and mature subjects. The possibility that dopamine D3 receptor assay in peripheral blood lymphocytes may represent a tool for investigating dopamine receptor function in aging and age-related neurological disorders is discussed.

Muscarinic cholinergic receptor subtypes in the pigeon bursa of Fabricius: a radioligand binding and autoradiographic study.

The pharmacological profile and the anatomical localisation of muscarinic cholinergic receptor subtypes were studied in the pigeon bursa of Fabricius, using radioligand binding and autoradiographic techniques with [3H]quinuclidinyl benzilate (QNB) as a ligand. [3H]QNB was specifically bound to sections of bursa of Fabricius. The binding was time-, temperature- and concentration-dependent. The dissociation constant was 0.31 +/- 0.02 nM, and the maximum density of binding sites averaged 38 +/- 2.5 fmol/mg protein. The pharmacological profile of [3H]QNB binding to sections of pigeon bursa of Fabricius was consistent with the labelling of M2, M3 and M4 muscarinic receptor subtypes. Light microscope autoradiography showed the localisation of [3H]QNB binding sites in the medulla, in follicular septa, in the cortico-medullary border and in lesser amounts in the cortical layer. The functional significance of these receptors should be clarified in future studies.

Pharmacological characterization and anatomical localization of prejunctional beta-adrenoceptors in the rat kidney.

1. The subtype and anatomical localization of beta-adrenoceptors mediating facilitation of stimulus-induced overflow of noradrenaline ('prejunctional beta-adrenoceptors') are not conclusively known to date. The present study was undertaken to characterize these receptors by use of pharmacological methods as well as to define their localization (prejunctional or postjunctional) with radio-ligand binding and autoradiography techniques combined with surgical denervation of the sympathetic innervation to the rat kidney. 2. Exposure of the kidney to (-)-isoprenaline, the nonselective beta-adrenoceptor agonist, resulted in a dose-dependent facilitation of stimulus-induced neurotransmitter overflow. This response was inhibited by propranolol, the beta 1- and beta 2-adrenoceptor antagonist, with a pA2 of 9.20 suggesting that the prejunctional beta-adrenoceptors are not of the beta 3-subtype. 3. The rank order of potency and potency ratios of beta-adrenoceptor agonists at renal prejunctional beta-adrenoceptors (EC50 for agonist/EC50 for (-)-isoprenaline) were: (-)-isoprenaline (1) > procaterol (2) > salbutamol (3) > adrenaline (10) > (+)-isoprenaline (25). However, dobutamine, the beta 1-adrenoceptor agonist, failed to enhance stimulus-induced overflow of noradrenaline. These results are indicative of the presence of beta 2-adrenoceptors as prejunctional beta-adrenoceptors. 4. Facilitation elicited by (-)-isoprenaline and procaterol, the selective beta 2-adrenoceptor agonist, was inhibited by ICI 118,551, the selective beta 2-adrenoceptor antagonist, with pKb values of 9.20 and 9.35, respectively at renal prejunctional beta-adrenoceptors. Similarly, the pKb values of metoprolol, the selective beta 1-adrenoceptor antagonist, at renal prejunctional beta-adrenoceptors were determined to be 6.25 and 6.18 against (-)-isoprenaline and procaterol, respectively. These results suggest the presence of a homogeneous population of beta 2-adrenoceptors as prejunctional beta-adrenoceptors. 5. Radio-ligand binding analysis of renal beta-adrenoceptors revealed the prevalence of the beta 1-subtype as compared to the beta 2-subtype (63% vs 37%). However, surgical denervation of the rat kidney, resulting in more than 90% reduction in renal noradrenaline content, selectively reduced the beta 2-adrenoceptor population by 80%, implying the presence of beta 2-adrenoceptors on renal sympathetic nerve terminals. 6. Autoradiographic analysis demonstrated the presence of beta 1-adrenoceptors on cortical structures such as glomeruli and tubules. beta-Adrenoceptors were found to be present on tubules (minor population), collecting tubules in outer medulla and the adventitia and adventitial-medial border of intraparenchymal branches of the renal artery. Surgical denervation of the rat kidney resulted in the disappearance of Beta2-adrenoceptors associated with the intraparenchymal branches, without affecting the Beta-adrenoceptor populations at other sites. These results support the notion that the Beta2-subtype is present on renal sympathetic nerve terminals and demonstrate that these prejunctional Beta2-adrenoceptors are associated with the renal vasculature and not with renal tubules.7. The results of the present investigation demonstrate that renal prejunctional Beta-adrenoceptors are of the Beta2-subtype in nature. These receptors are present on sympathetic nerve terminals which are associated with the renal vasculature.

Pharmacological characterisation of Ca2+ channels of the L-type in human peripheral blood lymphocytes.

Ca2+ channels of the L-type were characterised in intact human peripheral blood lymphocytes using a radioligand binding technique and the dihydropyridine-type Ca2+ channel antagonist [3H](+)-PN 200-110 (isopropyl-4-(2,1,3-benzoxadiazol-4-yl)1,4-dihydro-5-methoxycarbon yl-2, 6-dimethyl-3-pyridine carboxylate) as a ligand. [3H](+)-PN 200-110 binding to human peripheral blood lymphocytes was time-, temperature-, concentration-dependent and of high affinity. The dissociation constant (Kd) value was 0.4 +/- 0.02 nM and the maximum binding capacity (Bmax) was 33.5 +/- 1.6 fmol/10(6) cells. Pharmacological analysis of [3H](+)-PN 200-110 binding to human peripheral blood lymphocytes was consistent with the labelling of a Ca2+ channel of the L-type. In fact, dihydropyridine derivatives were the most potent competitors of [3H](+)-PN 200-110 binding, whereas phenylalkylamine and benzothiazepine compounds or non-selective Ca2+ channel modulators were weak or ineffective displacers. These findings are the first observation that human peripheral blood lymphocytes express Ca2+ channels of the L-type. The possibility that Ca2+ channel antagonists may interfere with immune system function is discussed.

Effect of ipsilateral lesioning of the nucleus basalis magnocellularis and of L-alpha-glyceryl phosphorylcholine treatment on choline acetyltransferase and acetylcholinesterase in the rat fronto-parietal cortex.

The present study assesses the effect of unilateral lesions of the nucleus basalis magnocellularis (NBM) and of treatment with L-alpha-glyceryl phosphorylcholine (GFC, choline alfoscerate) on the acetylcholine-synthesizing (choline acetyltransferase (ChAT)), and acetylcholine-degradating (acetylcholinesterase (AChE)) enzymes in the rat fronto-parietal cortex ipsilateral to the lesion. Ibotenic acid injections in the right NBM area caused a significant decrease of both ChAT and AChE activities as well as of histochemically reactive stores of AChE in the right fronto-parietal cortex. Treatment with GFC restored in part the loss of ChAT and AChE activities. Moreover, AChE reactivity is restored in the fronto-parietal cortex of NBM-lesioned rats treated with GFC. GFC is a precursor in the biosynthesis of brain phospholipids which increases the bioavailability of acetylcholine in the nervous tissue. The possible relevance of the restoration of the marker enzymes of cholinergic neurotransmission by GFC in an animal model of cholinergic hypofunction is considered.

Dopamine D4 receptor in human peripheral blood lymphocytes: a radioligand binding assay study.

The expression of dopamine D4 receptor was investigated in human peripheral blood lymphocytes with a radioligand binding assay technique, using [3H]clozapine as radioligand. [3H]Clozapine was specifically bound to human peripheral blood lymphocytes. The binding was time-, temperature-, and concentration-dependent and of high affinity, with a dissociation constant (K(d)) value of 0.34 +/- 0.02 nM and a maximum density of binding sites (B(max)) value of 27 +/- 1.4 fmol/10(6) cells. The pharmacological profile of [3H]clozapine binding to human peripheral blood lymphocytes was similar to that found in Chinese hamster ovary (CHO) cells transfected with the D4 clone (D4.2 variant). The above results are consistent with molecular biology studies demonstrating the expression of a dopamine D4 receptor in immune cells and in human peripheral blood lymphocytes. The availability of a rapid and sensitive radioligand binding assay technique for the dopamine D4 receptor in human peripheral blood lymphocytes may contribute to better define the role of this dopamine receptor subtype in neurological and psychiatric disorders.

Muscarinic cholinergic receptor subtypes in human peripheral blood lymphocytes.

The subtypes of muscarinic cholinergic receptors were studied in human peripheral blood lymphocytes with radioligand binding techniques and the non-selective muscarinic cholinergic receptor antagonist [3H]quinuclidinyl benzylate (QNB) as a ligand. [3H]QNB was bound to human peripheral lymphocytes in a manner consistent with the labelling of muscarinic cholinergic receptors. The dissociation constant (Kd) value was 0.60 +/- 0.08 nM and the maximum density of binding sites (Bmax) was 2.33 +/- 0.03 fmol/2.2 x 10(6) cells. The binding was time-, temperature- and concentration-dependent, belonging to a single class of high affinity sites. Analysis of the pharmacological profile of [3H]QNB binding in the presence of compounds specific for the different muscarinic receptor subtypes suggests that human peripheral blood lymphocytes express mainly muscarinic cholinergic M2 and M3 receptor subtypes and to a lesser extent muscarinic M4 receptors. The characterization of the subtypes of muscarinic cholinergic recognition sites expressed by human peripheral blood lymphocytes may represent a tool for investigating the possible relationships between immune and cholinergic systems in normal and pathologic conditions.

Influence of splenectomy on the properties of erythrocytes with hemoglobin Volga disease.

Hemoglobin Volga is a rare unstable hemoglobin in which there is a replacement of an internal alanine residue, beta 27 (B9), by an aspartate. From a clinical point of view it is characterized by a moderately severe Heinz body hemolytic anemia. A possible clue to the cause of the hemolysis is the increased vulnerability to oxidation of Hb Volga, with increased free radical turnover and consequent damage to the red cell membrane. Splenectomy performed on carriers of Hb Volga may have a positive outcome leading to improvements in both the clinical condition of the patient and hematological variables such as hemoglobin concentration and bilirubin concentration. With the aim of defining a more complete biochemical picture of the beneficial effect of splenectomy in this disease, we have evaluated some enzymic activities of the red cells of a young patient with Hb Volga disease, before and after splenectomy. In particular, we have investigated the activity of superoxide dismutase (superoxide: superoxide oxidoreductase), glutathione peroxidase (GSH peroxidase, glutathione: hydrogen-peroxidase oxidoreductase) and catalase (hydrogen-peroxide: hydrogen-peroxide oxidoreductase) that catalyze reactions relevant to the steady-state concentration of potentially toxic oxygen derivatives such as O2- and H2O2. Besides, we have carried out experiments on the erythrocytic membrane to evaluate eventual changes on the chemical (i.e. peroxidation) and physico-chemical (i.e. fluidity) properties following surgery.