bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutrophil Activation and Mediators of Inflammation in Chronic Venous Insufficiency
Authors: Smith Philip D. Coleridge
Published: April 2003

The effect of venous hypertension on the state of activation of leucocytes has been investigated in patients with venous disease and control subjects. Leucocytes become ‘trapped’ in the circulation of the leg during periods of venous hypertension produced by sitting or standing. This is greater in the limbs of patients with chronic venous disease than controls. Studies of the plasma levels of neutrophil granule enzymes show that these are increased during periods of venous hypertension, suggesting that this causes activation of the neutrophils. Investigation of the leucocyte surface ligand CD11b shows that the more activated neutrophils and monocytes are sequestered during venous hypertension. Measurement of plasma levels of the soluble parts of the vascular (VCAM), intercellular (ICAM) and endothelial leucocyte (ELAM) adhesion molecules show that these are all elevated in patients with chronic venous disease compared to controls. Following 30 min of venous hypertension produced by standing, these levels are further increased. These data suggest that venous hypertension causes neutrophil and monocyte activation, which in turn causes injury to the endothelium. I believe that this may be the mechanism that initiates the pathological processes which lead to venous ulceration. It has recently been shown that the venotonic drug Daflon 500 mg (450 mg diosmin, 50 mg hesperidin, Servier, France) influences these processes. Surface expression of CD62L is reduced in neutrophils and monocytes, and plasma levels of soluble endothelial adhesion molecules are reduced. These observations may explain the anti-inflammatory effects of Daflon 500 mg.
Dual Effect of Adenosine on Vascular Smooth Muscle [3H]-Thymidine DNA Labeling: Receptor-Mediated Modulation of DNA Synthesis and Inhibition of Thymidine Uptake
Authors: Thorin-Trescases Nathalie , Ono Yasuhiro , Tremblay Johanne , Hamet Pavel , Orlov Sergei N.
Published: April 2003

This study examined the contribution of cAMP signaling to the modulation of vascular smooth muscle cell (VSMC) proliferation by adenosine. At a concentration of 1 mM, adenosine inhibited [3H]-thymidine uptake, measured as the initial rate of isotope influx, by 10-fold. Diminution of [3H]-thymidine uptake by adenosine was independent of the presence of A1- and A2-receptor antagonists, indicating that adenosine competes with thymidine for plasma membrane transporter-binding sites. Considering these results, in order to estimate [3H]-thymidine DNA labeling, VSMCs were preincubated with adenosine for 48 h, and adenosine was then omitted during the subsequent 2 h of incubation in [3H]-thymidine-containing medium. In serum-depleted VSMCs, preincubation with 100 µM or 1,000 µM adenosine augmented DNA synthesis by approximately 6- and 3-fold, respectively, whereas the increment of DNA synthesis triggered by serum was decreased in the presence of adenosine by 20–30%. Both cAMP production and inhibition of DNA synthesis by adenosine in serum-supplied cells were independent of the presence of the A1-antagonist 1,2-dipropyl-8-cyclopentylxanthine (DPCPX), but were abolished by the A2-antagonist 1,3-dimethyl-7-propylxanthine (DMPX). In contrast, the activation of DNA synthesis in serum-depleted cells by adenosine was decreased in the presence of DPCPX and DMPX by approximately 30 and 40%, respectively. Both in serum-supplied and -depleted VSMCs, dose-dependent elevation of cAMP production with an adenylate cyclase activator, forskolin, reduced DNA synthesis by up to 40–60%. Thus, our results show that in addition to suppressing thymidine uptake, adenosine depresses the DNA synthesis triggered by serum-derived growth factors and stimulates DNA synthesis in serum-depleted cells. These data also suggest that the inhibition of DNA synthesis is mediated by cAMP production where the activation of DNA synthesis is independent of cAMP signaling.
Isolated Systolic Hypertension from a Vascular Point of View
Authors: Muntinga J.H.J. , Heesen W.F. , Smit A.J. , Visser K.R. , May J.F.
Published: April 2003

Due to the results of antihypertensive intervention studies, isolated systolic hypertension (ISH) has gained new interest lately. Yet, apart from increased aortic stiffness, the specific pathophysiological features of ISH have remained largely undetermined. Therefore, we investigated the elastic properties of the vascular bed of an upper arm segment in uncomplicated ISH patients and matched normotensive controls using an electrical bioimpedance technique. Compared with the controls, the compliance of the arterial bed as a whole at normotensive blood pressure level was on the average 108.0% higher (p < 0.005) in the hypertensive patients. The blood volume of the arterial bed as a whole at operating blood pressure level and that of the larger arteries were significantly higher (40.5%, p < 0.05, and 40.5%, p < 0.01, respectively). The same held true for the venous blood volume (64.4%, p < 0.05), and for the width of the arterial compliance-pressure relation (34.6%, p < 0.01). We concluded that ISH is a separate pathophysiological entity in which all parts of a peripheral vascular bed are changed and the decreased buffering function of the aorta and large arteries is partly compensated for by an increase in small artery compliance.
Functional Assessment of Coronary Arteries by Poststenotic Intravascular Doppler Ultrasound
Authors: Porenta Gerold , Binder Thomas , Moertl Deddo , Zehetgruber Manfred , Graf Senta , Maurer Gerald , Probst Peter
Published: April 2003

This study sought to delineate the impact of the rate pressure product on intraluminal Doppler velocity measurements and to determine the relation between poststenotic vasodilator reserve and percent luminal obstruction in coronary vessels. Twenty patients with single-vessel coronary disease were studied prior to coronary angioplasty and at follow-up 6 months later. Intracoronary velocity reserve after administration of adenosine was measured distal to the stenosis with a Doppler-tipped guide wire and was compared to quantitative coronary angiography and adenosine myocardial perfusion scintigraphy. The rate pressure product was confirmed as significant covariate (ANCOVA, p < 0.005) of intracoronary Doppler reserve. When normalized to rate pressure product, poststenotic Doppler velocity reserve in stenosed arteries was significantly lower than in patent arteries as classified by quantitative coronary angiography (1.7 ± 0.6 vs. 2.9 ± 0.5, p < 0.001) and perfusion scintigraphy (1.5 ± 0.4 vs. 2.8 ± 0.5, p < 0.001). Normalized Doppler velocity reserve showed a nonlinear but highly significant relation to percent area stenosis [y = 3.0·(1 – exp[0.081 (x – 100)]), p < 0.001]. When normalized Doppler velocity reserve was less than 2.0, coronary disease was identified with 95% specificity and 94% sensitivity in comparison to perfusion scintigraphy. Thus, in coronary arteries poststenotic Doppler reserve and percent area stenosis show a significant nonlinear relation. Doppler velocity reserve when normalized to rate pressure product can be used to characterize the hemodynamic impact of coronary obstructions.
Prostacyclin Inhibits the Production of MMP-9 Induced by Phorbol Ester through Protein Kinase A Activation, but Does Not Affect the Production of MMP-2 in Human Cultured Mesangial Cells
Authors: Kitahara Masashi , Ichikawa Motoki , Kinoshita Tatsuya , Shiozawa Satoshi , Shigematsu Hidekazu , Komiyama Atsushi
Published: April 2003

Background/Aims: The imbalance between degradation and synthesis of the glomerular extracellular matrix (ECM) causes glomerular sclerosis in various types of glomerulonephritis. We investigated the effect of prostacyclin, which is an inflammatory mediator, on the production of matrix metalloproteinase (MMP)-9 and MMP-2 in human cultured mesangial cells. The synthesis of Jun proteins and Ets-1 proteins, which are related with MMP-9 gene, was also studied. Methods: The production of MMP-9 and MMP-2 was investigated by gelatin zymography. Western blotting was undertaken to analyze the protein synthesis of Jun and Ets-1. Results: Prostacyclin inhibited the production of MMP-9 induced by phorbol ester. The inhibitory effect by prostacyclin was reversed in part by the pretreatment with an inhibitor of protein kinase A, such as H-89. Forskolin also inhibited the production of MMP-9. The production of MMP-2 was constitutionally seen and was not influenced by prostacyclin and forskolin. The synthesis of Jun protein augmented by phorbol ester was suppressed by prostacyclin. Ets-1 protein was constitutionally synthesized and was not affected by phorbol ester and prostacyclin. Conclusion: Prostacyclin plays an important role in inflammatory glomerular disorders by regulating the metabolism of ECM. The production of MMP-9 and MMP-2 may be under the different control pathways.
Cardiac Hypertrophy in the Prague- Hypertensive Rat Is Associated with Enhanced JNK2 but not ERK Tissue Activity
Authors: Vogel Volker , Bokemeyer Dirk , Heller Jiri , Kramer Herbert J.
Published: April 2003

Mitogen-activated protein (MAP) kinases are important intracellular mediators for proliferation and hypertrophy and therefore may also regulate cardiomyoblast growth in hypertensive heart disease. Thus, the aim of the present study was to examine the activities of MAP kinases, namely extracellular signal-regulated kinase (ERK)1,2, c-Jun NH2-terminal kinases (JNK)1,2 and p38 MAP kinase, in myocardial tissue of 12-week-old Prague normotensive (PNR) and hypertensive rats (PHR), a model of genetic hypertension with marked cardiac hypertrophy. Systolic blood pressure was 121 ± 5 in PNR and 208 ± 15 mm Hg in PHR (p < 0.01). Total heart weight was 247 ± 4 in PNR vs. 316 ± 4 mg/100 g body weight in PHR (p < 0.01). Left and right ventricular weights were 121 ± 5 and 53 ± 3 in PNR vs. 168 ± 4 (p < 0.01) and 57 ± 2 mg/100 g body weight (n.s.) in PHR. Using anti-ERK2 Western blot analysis as well as immunocomplex ERK activity assay, we found no activation of ERK2 in left or right ventricular tissue of PHR and PNR. Similary, p38 MAP kinase phosphorylation and activity were not detectable. In contrast, Western blot analysis using antiphospho-JNK antibodies revealed in myocardial tissue of right and left ventricles significantly greater phosphorylation of JNK2 in PHR than in PNR. This finding was confirmed by immunocomplex JNK activity assay using ATF-2 as substrate, which demonstrated a significant increase in JNK activity in the left ventricle of PHR as compared to PNR (6.4 ± 1.5 vs. 2.5 ± 0.5 OD; each n = 5; p < 0.05). In conclusion, cardiac JNK2 seems to be regulated differently from ERK2 in this rat model. In PHR, as compared to PNR, we found enhanced activity of JNK2 in the left and right ventricles suggesting that JNK2 is involved in hypertensive cardiac disease. The rise in JNK in both ventricles may result indirectly from humoral stimuli, e.g., endothelin-1 and/or angiotensin II, and may contribute to ventricular hypertrophy in this model of spontaneous hypertension.
Absence of Cytokine Response to Bacterial Challenge in Human Tubuloepithelial Cells
Authors: Leeker Antje , Ernst Martin , Lankenau Petra , Krüger Sabine , Kreft Burkhard
Published: April 2003

In acute bacterial renal infections, which are most frequently caused by Escherichia coli, tubuloepithelial cells are involved with respect to bacterial adherence, invasion and cytotoxicity. In addition, cytokines expressed by tubuloepithelial cells may be relevant for the recruitment of inflammatory cells and tissue damage in bacterial interstitial nephritis. We asked which inflammatory cytokines are produced by primary human tubuloepithelial cells following in vitro exposure to E. coli and found no release of IL-6, IL-8 and TNF-α by tubular cells challenged by bacteria. Purified virulence factors (fimbriae, lipopolysaccharide) from E. coli were also without effects on cytokine release by tubular cells. Since lymphocytic infiltration is a characteristic feature in the chronic form of interstitial nephritis, MHC class II expression by tubular cells in response to bacterial coincubation was analyzed. Exposure to both IFN-γ and E. coli enhanced MHC class II expression on tubuloepithelial cells. In conclusion, tubuloepithelial cells may play an active role in the local defense against bacteria, e.g. by expressing MHC class II molecules. However, in vitro inflammatory cytokines are not induced by E. coli in this cell population.
Lack of Association between Gly460Trp Polymorphism of Alpha-Adducin Gene and Salt Sensitivity of Blood Pressure in Polish Hypertensives
Authors: Ciechanowicz Andrzej , Widecka Krystyna , Drozd Radosław , Adler Grażyna , Cyryłowski Lech , Czekalski Stanisław
Published: April 2003

Background: Previous studies have suggested that α-adducin (α-ADD) polymorphism may identify patients with a salt-sensitive form of hypertension. Aim: To investigate the association between Gly460Trp polymorphism of α-ADD and the pattern of blood pressure response to subacute (1 week) salt loading and depletion in young adult thin Polish hypertensives. Methods: The study group consisted of 44 subjects with salt-sensitive hypertension (SS) and 24 subjects with non-salt-sensitive hypertension (SR). Genomic DNA isolated from peripheral blood leukocytes was amplified by PCR method with primers flanking the polymorphic region. The mismatch near to 3′-end of the upstream primer was introduced to create a Nla III restriction site in Trp 460 allele. In addition, excreted fraction of filtered sodium (FENa), plasma renin activity (PRA) and plasma concentrations of aldosterone (ALDO) were determined on normal, low and high salt diets. Results: FENa on normal or high salt diets were significantly lower in the SS hypertensives as compared with the SR patients. PRA in SS group was also significantly lower as compared with results in SR group, but only on high salt diet. No significant difference was detected in frequencies of genotypes and alleles of α-ADD gene between SS and SR subjects. An additional analysis with regard to genotype (Gly/Gly vs. Gly/Trp+Trp/Trp) showed no significant difference in changes of blood pressure as well as in results of laboratory investigations. Conclusion: Our results suggest lack of association between Gly460Trp polymorphism of α-adducin gene and salt sensitivity of blood pressure in Polish hypertensives.
Vertebrobasilar Strokes in Children
Authors: Al Tawari A. , Ouvrier R.A. , Steinberg A.W. , Grattan-Smith P. , Antony A.
Published: April 2003

Objectives: This study describes the rare disorder of vertebrobasilar strokes in children and provides an assessment strategy to alert the clinician towards the possibility of this disorder. Method: A retrospective study of vertebrobasilar strokes over 20 years was conducted in the Neurology Department at the New Children’s Hospital, Sydney, Australia. Results: There were 6 males and 3 females between the ages of 18 months and 15 years. Frequently presenting features included headache, hemiparesis, ataxia and cranial nerve palsies. Aphasia occurred in 2 and afebrile seizures in 1 patient. Five patients had depressed consciousness extending to coma in 3. Etiological factors included trauma to the head or neck in 5, rheumatic endocarditis with artificial valve embolism in 1, brain stem encephalitis in 1 and undetermined etiology in 2. Diagnostic neuroimaging studies including MRI showed infarction in the territories of the occluded arteries. MRA showed occlusion of the vertebral artery and/or its branches in 5 patients, while the other 4 patients had involvement of the basilar artery and/or its branches. No abnormalities of blood coagulation were detected. Five patients were treated with anticoagulants. Acute hydrocephalus secondary to cerebellar infarction developed in 3 patients. Recurrent vertebrobasilar strokes were reported in 2 males, each of whom was treated surgically. There was 1 late death. Residual neurological deficits were seen in 5 patients and 3 made a complete recovery. Conclusion: Although this disorder is rare in pediatric patients, it is important to consider it in those children with appropriate neurological symptoms and signs.
Carcinoma Originating from the Ectopic Mammary Gland of the Axilla
Authors: Hamid H.L.A. , Farghaly M.M. , Baker H.I. , Al-Manee M.S.
Published: April 2003

Carcinoma originating from ectopic breast tissue is a rare condition comprising only 0.3% of all breast cancers. The case presented here is the first of its kind to be reported in Kuwait. A review of the literature is provided, and a rationale for the treatment of this condition.
Cyclical Expression of GnRH and GnRH Receptor mRNA in Lymphoid Organs
Authors: Jacobson Jill D. , Crofford Leslie J. , Sun Lihua , Wilder Ronald L.
Published: April 2003

Gonadotropin-releasing hormone (GnRH) is known to possess dirct immunomodulatory effects. We have previously demonstrated that the administration of GnRH analogues modulates the expression of murine lupus independently of effects on gonadal steroids. We speculate that GnRH exerts direct actions at the level of the immune cells. GnRH receptors have been identified on lymphocytes. Because GnRH and GnRH receptor (GnRH-R) expression varies with the estrous cycle at the levels of the hypothalamus and pituitary, we speculated that similar cyclicity might be demonstrable in lymphoid tissue. In this report, we used competitive reverse transcription PCR to quantitate the expression of GnRH and GnRH-R mRNA in lymphoid organs throughout the estrous cycle in mice. We demonstrate that the pattern of expression of GnRH-R mRNA in spleen agrees closely with the pattern in the pituitaries of the same mice and the pattern previously reported in the rat pituitary, with significantly increased levels of expression seen on the afternoon of proestrus compared to the morning of metestrus. A similar pattern is seen with GnRH-R mRNA expression in the thymus. Furthermore, we show that the expression of GnRH mRNA in both thymus and spleen agrees closely with the pattern of expression of its receptor, with significantly increased levels of expression seen on the afternoon of proestrus compared to the morning of metestrus. Additional in vitro studies demonstrate that both GnRH and estradiol significantly increase the expression of GnRH-R mRNA in immune cells. These findings support an active role for GnRH in the immune system.
Dopamine Produced by the Stomach May Act as a Paracrine/ Autocrine Hormone in the Rat
Authors: Mezey Éva , Eisenhofer Graeme , Hansson Stefan , Hunyady Béla , Hoffman Beth J.
Published: April 2003

Dopamine (DA) has been suggested to be a protective factor in the gastrointestinal tract but neither a source of DA nor its exact targets of action have been identified. In this study, we demonstrate high levels of DA (and DOPA) which persist after chemical sympathectomy in the gastric juice of rats. Immunostaining and in situ hybridization histochemistry reveal the presence of tyrosine hydroxylase (TH), DA transporter and vesicular monamine transporters in the acid-producing parietal cells. Like DA, TH enzyme activity remains after chemical sympathectomy. We also demonstrate active reuptake and storage of DA that indicates a regulated release of this neurohormone from parietal cells. DA D1b receptor mRNA is the most abundant DA receptor subtype in gastric and duodenal epithelium. Therefore, we suggest that selective DA D1b receptor agonists may be useful adjuncts in the treatment of duodenal and gastric ulcers. Gastric epithelia possess the hallmarks of functional DA neuroendocrine cells, suggesting that DA has an important role in self-protective mechanisms of the gastrointestinal tract. These findings should allow elucidation of DA role in normal and disease states in the stomach and duodenum.
Estrogen Receptor Expression in Brainstem Noradrenergic Neurons of the Sheep
Authors: Simonian Sharon X. , Delaleu Bernadette , Caraty Alain , Herbison Allan E.
Published: April 2003

Noradrenergic neurons are implicated in the estrogen-dependent neural regulation of luteinizing hormone secretion in a variety of mammalian species. The current study has used immunocytochemical methods to determine whether estrogen receptors (ER) are expressed within the brainstem of the ewe and to establish their relationship to noradrenergic neurons. Using a monoclonal mouse antiserum directed against the N-terminal of ERα, four distinct populations of ERα-immunoreactive cells were identified in ovine medulla and pons. The largest population was found in the superficial laminae of the spinal nucleus of the trigeminal nerve, followed by the nucleus tractus solitarius, lateral area postrema, and ventrolateral medulla. Double-labelling immunocytochemistry using antisera directed against the ERα and dopamine-β-hydroxylase revealed that noradrenergic neurons expressing ER immunoreactivity were only found in ventrolateral medulla (A1 cell group) and nucleus tractus solitarius (A2 cell group). No double-labelled cells were identified in the A5, A6, or A7 noradrenergic cell groups. ERs were expressed with a clear rostrocaudal topography within the A1 and A2 populations, with 80–90% of noradrenergic neurons expressing ERα in the caudalmost medulla as compared with less than 5% rostral to the obex. Our findings demonstrate that, as in the rat, the ovine A1 and A2 neurons express ERs in a defined topographical manner, while, dissimilar to the rat, ERα is not synthesized by noradrenergic neurons in the other cell groups. These observations indicate that A1 and A2 noradrenergic neurons in the ovine brainstem are likely to be influenced by circulating estrogens and lay the neuroanatomical foundations for investigating the functional role of these cell populations within the gonadotropin-releasing hormone neuron network of the sheep.
Pharmacological Evidence that Neurotensin Mediates Prolactin- Induced Activation of Tuberoinfundibular Dopamine Neurons
Authors: Hentschel Kenneth , Cheung Sun , Moore Kenneth E. , Lookingland Keith J.
Published: April 2003

The purpose of this study was to investigate the role of neurotensin (NT) receptors in mediating the stimulatory effects of prolactin on the activity of tuberoinfundibular dopamine (TIDA) neurons in male and female rats. TIDA neuronal activity was estimated by measuring concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) in terminals of these neurons in the median eminence (ME). Haloperidol activates TIDA neurons indirectly by blocking D2 receptors on pituitary lactotropes, thereby increasing secretion of prolactin. Twelve hours after administration of haloperidol (1 mg/kg, s.c.), DOPAC concentrations in the ME were increased. Blockade of NT receptors with the selective antagonist SR-48692 had no effect per se on basal DOPAC concentrations in the ME but produced a dose-related (10–1,000 µg/kg, i.p.; 1 h) reversal of haloperidol-induced increases in ME DOPAC concentrations. In contrast, SR-48692 had no effect on either basal or haloperidol-induced increases in plasma prolactin. SR-48692 also blocked the stimulatory effects of prolactin (10 µg/rat, i.c.v.; 12 h) on ME DOPAC concentrations. SR-48692 was equally effective in blocking the stimulatory effects of haloperidol and prolactin on TIDA neurons in male and female rats. These results suggest that NT mediates the induced stimulatory effect of hyperprolactinemia on the activity of TIDA neurons in both males and females, whereas the tonic regulation of these neurons by prolactin in females occurs via an NT-independent mechanism.
Ovarian Steroids Modulate Responsiveness to Dopamine and Expression of G-Proteins in Lactotropes
Authors: Livingstone John D. , Lerant Anna , Freeman Marc E.
Published: April 2003

The effects of chronic ovarian steroid treatment on the secretory activity of individual lactotropes and the mechanisms modulating their responsiveness to dopamine (DA) were studied. Female rats were ovariectomized (OVX) and implanted with Silastic capsules containing progesterone (P4), 17β), 17β-estradiol (E2) or both E2 (E2+P4). Ten days after surgery, anterior pituitaries were enzymatically dispersed and the reverse hemolytic plaque assay (RHPA) was performed to assess the release of prolactin (PRL) from individual lactotropes. RHPA was combined with immunocytochemistry (ICC) for PRL, Gαs or Giα3/Gαo proteins. E2 treatment alone or in combination with P4 increased the percentage of immunoreactive lactotropes among anterior pituitary cells. Incidence of active (plaque-forming) lactotropes, however, was increased both in P4-, and E2-treated rats and E2+P4 treatment increased it even further. While P4 treatment did not affect the frequency distribution of lactotropes, both E2 and E2+P4 treatments increased the large plaque-forming lactotrope population. This increase was reflected by the significantly greater mean plaque areas of lactotropes from E2- and E2+P4-treated rats compared to OVX or P4-treated animals. The responsiveness of lactotropes to DA from P4-treated rats did not differ from that of OVX rats: thus challenge with 1 µM DA inhibited the release of PRL, while 100 pM DA had no effect. E2 and E2+P4 treatments, however, profoundly changed the lactotrope’s responsiveness: challenge with 1 µM DA had no effect and 100 pM DA resulted in moderate stimulation of PRL release in E2+P4 rats. Double-label ICC revealed that ovarian steroid treatments did not affect the expression of Gαs in lactotropes. The incidence of Giα3/Gαo-immunoreactive lactotropes, however, decreased after E2 treatment, alone or in combination with P4. Although expression of Gαs was similar in all plaque-forming cells regardless of plaque size, lactotropes expressing Giα3/Gαo were more likely to form small plaques in all treatment groups. These data suggest: (1) ovarian steroid treatment recruits quiescent lactotropes to release PRL; (2) E2 treatment alone or in combination with P4 increases the amount of PRL rleased by individual lactotropes; (3) E2-induced alterations in the frequency distribution and lactotrope responsiveness to DA may be due in part to a decreased expression of Giα3/Gαo.
Large Somal Size Is Associated with the Expression of Galanin but Not with Neuronal Birthdate in the Sexually Dimorphic Male Nucleus of Ferret’s Preoptic Area/Anterior Hypothalamus
Authors: Park Joong-Jean , Patel Jayen H. , Dad Luqman K. , Tobet Stuart A. , Baum Michael J.
Published: April 2003

Using Nissl and Golgi stains, a sexually dimorphic male nucleus (MN) comprised of a cluster of large cells with large dendritic arbors has been identified in the dorsal preoptic area/anterior hypothalamus (POA/AH) of male ferrets. The MN-POA/AH is formed only in males by the action of estradiol derived from the neural aromatization of testosterone during the last quarter of a 41-day gestation. The ferret’s dorsal POA/AH is also characterized by a sex difference in the expression of the neuropeptide galanin which first arises in males around embryonic day (e) 34. We asked whether the male-typical phenotype of large somal size is related to birthdate and/or the capacity of dorsal POA/AH neurons to express galanin. In experiment 1 we labeled cohorts of cells born on E20, E24, or E28 by injecting the amniotic sacs of individual fetuses with the thymidine analogue bromodeoxyuridine (BrdU). On postnatal day 20, BrdU-immunoreactive cells were visualized immunohistochemically, counterstained with cresyl violet, and their somal sizes were measured. BrdU-immunoreactive cells were significantly larger in the males’ MN-POA/AH than in a comparable region of females, regardless of when they were born between E20 and E28. In experiment 2 galanin-immunoreactive cells in the dorsal POA/AH of adult ferrets were visualized immunohistochemically, and their somal sizes were measured. Somal areas of galanin-immunoreactive cells were significantly larger in the MN-POA/AH of intact, breeding, or castrated and testosterone-treated males than in the corresponding area of females. Our results suggest that cells in the males’ MN-POA/AH are more likely to be larger than cells in females’ corresponding region, regardless of birthdate. Finally, in adulthood the male-typical phenotype of large Nissl-stained somal areas of MN-POA/AH cells may, in part, reflect their increased galanin expression.
Neuronal Labeling in the Rat Brain and Spinal Cord from the Ovary Using Viral Transneuronal Tracing Technique
Authors: Gerendai Ida , Tóth Ida E. , Boldogkői Zsolt , Medveczky István , Halász Béla
Published: April 2003

In the present investigations the viral transneuronal labeling method, which is able to reveal hierarchial chains of central nervous system (CNS) neurons, was applied to identify sites in the CNS connected with the ovary and presumably involved in the control of ovarian functions. Pseudorabies virus was injected into the ovaries of rats and a few days later (at various times after the injection) the spinal cord and brain were examined for virus-infected neurons from the ovary. The virus-labeled nerve cells were identified by immunocytochemistry using polyclonal antiviral antibody. Virus-labeled neurons were detected both in the spinal cord and the brain. In the spinal cord such elements were observed in the intermediolateral cell column, in the dorsal horn close to the marginal zone and in the central autonomic nucleus. In the medulla oblongata and pons, neurons of several nuclei and cell groups (area postrema, nucleus of the solitary tract, dorsal vagal complex, nucleus ambiguus, paragigantocellular nucleus, parapyramidal nucleus, A1, A5 and A7 cell groups, caudal raphe nuclei, locus ceruleus, subceruleus nucleus, Barrington’s nucleus, Kölliker-Fuse nucleus) were found to be transneuronally labeled. In the mesencephalon, the ventrolateral part of the periaqueductal gray matter contained virus-labeled neurons. In the diencephalon, a very intensive cell body labeling was observed in the hypothalamic paraventricular nucleus and a few virus-infected neurons could be detected in the lateral and dorsal hypothalamus, in the arcuate nucleus, zona incerta, perifornical area and in the anterior hypothalamus. Concerning the telencephalic structures, virus-labeled cells were found in the bed nucleus of the stria terminalis and in the central amygdala nucleus. These findings provide the first neuromorphological evidence for the existence of a multisynaptic neuronal pathway between the ovary and the CNS, and give a detailed account of the structures involved in this pathway.
Diurnal Rhythmicity of Gonadotropin-Releasing Hormone Gene Expression in the Rat
Authors: Gore Andrea C.
Published: April 2003

The release of the decapeptide gonadotropin-releasing hormone (GnRH) from neuroterminals in the median eminence varies depending upon the stage of development, time of day, reproductive season, and day of the ovarian cycle. With respect to the biosynthetic events responsible for such alterations in GnRH release, we have reported that increases in GnRH mRNA occur concomitantly with or precede the proestrous GnRH/LH surge in female rats, and the pubertal increase in GnRH. Because a hallmark of puberty is the development of nocturnal increases in gonadotropin/GnRH release, in the present study, we examined whether a rhythm in GnRH gene expression occurs that may be responsible for this rhythm of neurosecretion. Additionally, we extended the previous finding of diurnal rhythms of GnRH gene expression in cycling females to adult male rats. The mechanism for these changes, i.e. GnRH gene transcription, RNA processing or a posttranscriptional mechanisms was also elucidated. In experiment I, GnRH gene expression was measured in female rats at different stages of puberty [P25 (juvenile); ∼P37 (day of vaginal opening); and P45 (postpubertal)] at 10.00, 14.00, 18.00, 22.00, 2.00 and 6.00 h. GnRH mRNA levels increased significantly during pubertal development, independent of any changes in GnRH gene transcription or RNA processing, indicating a posttranscriptional mechanism. GnRH mRNA levels did not vary with time of day in these pubertal rats, indicating that the diurnal rhythm of GnRH release that develops during puberty is probably a posttranslational event. In experiment II, GnRH gene expression was measured in adult male rats at the same 4-hour intervals. GnRH mRNA levels varied significantly with time of day: they increased from 10.00 to 14.00 h, remained elevated through 22.00 h, then decreased significantly to the nadir at 10.00 h. The mechanism for these changes appears to involve processing or stability of the GnRH mRNA in the nucleus. These results indicate that GnRH mRNA levels are regulated differentially with time of day depending upon age and gender.
Delta Opiate Receptors Account for the Castration-Induced Unmasking of Gonadotropin-Releasing Hormone Binding Sites in the Rat Pituitary
Authors: Leblanc Pierre , L’Héritier Andrée , Kordon Claude
Published: April 2003

Under control incubation conditions, gonadotropin-releasing hormone (GnRH) binds only a fraction of its receptors in rat-cultivated pituitary cells. Unmasking of the remaining receptors, which have been termed ‘cryptic’, requires drug- or peptide-induced protein kinase activation. Spontaneous masking however is not observed on pituitary cells sampled from castrated male rats, suggesting the presence of an intrinsic unmasking factor. Many endogenous factors could theoretically account for the effect. Here we attempted to identify the factor involved by taking advantage of their differential dependency upon second messengers and transduction cascades. Spontaneous unmasking of GnRH binding was found reversed by pertussis toxin (PTX), an inhibitor of αi and αo subunits of heterotrimeric G proteins, and by U73122, a phospholipase C (PLC) inhibitor. In contrast, desensitization of protein kinase C (PKC) or inhibition of tyrosine kinase by herbimycin were ineffective. Among endogenous pituitary factors able to unmask GnRH receptors in pituitary cells from normal male rats, as EGF, NPY or opiate peptides, only the latter were found to correspond to this transduction profile. In an attempt to characterize the pharmacology of opiate effects, naloxone (10 µM), a poorly selective opiate antagonist, restored masking of GnRH binding in cells from castrates. Only the δ antagonist naltrindole (1 µM) was able to mimick the action of naloxone. Conversely, when tested on cells from intact animals, morphine (10 µM), as well as dslet (1 µM) and met-ENK (10 nM), preferential δ agonists, but not dago and β-endorphin or U50488 H and dynorphin, respectively µ and ĸ agonists, were able to suppress masking. Among opioid peptides endogenous to the pituitary, only met-ENK was able to unmask cryptic receptors, an effect antagonized by naltrindole. We conclude that an opiate δ receptor subtype is endogenously activated in the pituitary of castrated male rats to prevent masking of GnRH binding.
Ultrastructural Immunolocalization of Leptin Receptor in Mouse Brain
Authors: De Matteis Rita , Cinti Saverio
Published: April 2003

Antibodies directed to amino acids 877–894 (M-18) and 32–51 (K-20) were used to localize leptin receptor by immunocytochemistry in mouse brain. Both antibodies stained several hypothalamic nuclei (paraventricular nucleus, supraoptic nucleus, supraoptic retrochiasmatic nucleus, suprachiasmatic nucleus, preoptic area, ventromedial nucleus, dorsomedial nucleus, lateral hypothalamus, arcuate nucleus, ventral and dorsal premammillary nuclei), the thalamic and amygdaloid nuclei, neurons of the neocortex and archicortex and the epithelial cells of the choroid plexus. While M-18 staining was concentrated in the Golgi area, with K-20 it was dispersed in the cytoplasm. Glial cells were stained only by K-20. These results suggest that the trans-membrane forms of the receptor are concentrated at the membrane level of the Golgi complex of neurons and in epithelial cells of the choroid plexus while the soluble form is dispersed in their cytoplasm. Glial cells express only the soluble form.
Thyroid Hormone-Dependent Regulation of Galanin Synthesis in Neurons and Glial Cells after Colchicine Administration
Authors: Calzà Laura , Giardino Luciana , Hökfelt Tomas
Published: April 2003

Galanin and galanin message-associated peptide (GMAP) synthesis is up-regulated in neurons and glial cells in the adult rat brain by different experimental manipulations, including intracerebroventricular colchicine. We have previously reported that the galanin expression is severely attenuated in some neurons in adult hypothyroidism. In order to further investigate the role of thyroid hormone for the in vivo regulation of galanin gene expression, we have studied the effect of intraventricular administration of colchicine on prepro-galanin (ppGAL) mRNA expression in the brain of normal and hypothyroid, adult male rats. While ppGAL mRNA levels were markedly elevated in a great number of glial cells in the white and gray matter in normal rats, this effect was almost completely abolished in hypothyroid rats. In contrast, colchicine-induced up-regulation of galanin/GMAP expression occurs in the hypothalamic paraventricular nucleus both of euthyroid and hypothyroid rats, although with a slightly different time course.
Gonadal and Age-Related Influences on NMDA-Induced Growth Hormone Secretion in Male Rats
Authors: Pinilla Leonor , Gonzalez Lucas , Tena-Sempere Manuel , Dieguez Carlos , Aguilar Enrique
Published: April 2003

Activation of N-methyl-D,L-aspartic acid (NMDA) receptors stimulates growth hormone (GH) secretion. The mechanisms involved in this action are still a matter of debate. Present experiments were carried out to assess specifically: (1) the age-related changes in NMDA effects; (2) the physiological role of NMDA in pulsatile GH secretion; (3) the hypothalamic and/or pituitary actions of NMDA, and (4) the influence of gonadal function on NMDA-induced GH release. NMDA (15 mg/kg i.p.) stimulated GH secretion in neonatal, prepubertal and adult males, this effect being blocked by MK-801, a selective antagonist of NMDA receptors. In adult males, pulsatile GH secretion was abolished after administration of MK-801 and AP-5, antagonists of NMDA receptors. The stimulatory effect of NMDA on GH release was exerted at the hypothalamic level, since in vitro GH secretion was slightly inhibited in the presence of NMDA (0.5 mM). The increase in GH release after NMDA treatment cannot be explained through an increase in GHRH release, as the NMDA effect persisted in animals pretreated with GHRH antiserum and in those neonatally injected with mono- sodium glutamate, a drug that destroys GHRH neurons. In addition, NMDA-induced GH secretion was independent of testicular function since it remained after orchidectomy, testosterone replacement as well as after permanent damage of testicular function by neonatal administration of estrogens (500 µg on day 1 of life). We conclude that NMDA receptors play a physiological role stimulating GH secretion through a hypothalamic mechanism that is, at least partially, not GHRH-dependent, and is not modulated by testicular secretion.
Effects of Centrally Administered Pituitary Adenylate Cyclase-Activating Polypeptide on c-fos Gene Expression and Heteronuclear RNA for Vasopressin in Rat Paraventricular and Supraoptic Nuclei
Authors: Nomura Masayoshi , Ueta Yoichi , Serino Ryota , Yamamoto Yukiyo , Shibuya Izumi , Yamashita Hiroshi
Published: April 2003

The effects of intracerebroventricular (i.c.v.) administration of pituitary adenylate cyclase-activating polypeptide (PACAP) on the expression of c-fos gene as well as heteronuclear (hn) RNA for arginine vasopressin (AVP) in paraventricular (PVN) and supraoptic nuclei (SON) of rats were investigated by immunohistochemistry for c-fos protein (Fos) and in situ hybridization histochemistry for c-fos mRNA and AVP hnRNA. The i.c.v. administration of PACAP (200 pmol/rat) caused a marked induction of Fos-like immunoreactivity (LI) in PVN and SON. The nuclear Fos-LI existed in AVP-LI containing cells in the PVN and SON. The expression of the c-fos gene in the PVN and SON was increased in a dose-related manner 30 min after i.c.v. administration of PACAP. PACAP-induced expression of the c-fos gene was significantly reduced by pretreatment with a PACAP receptor antagonist, PACAP-(6-38)-NH2. In addition, Fos-LI and the expression of the c-fos gene were also observed in the periventricular region of the third ventricle after i.c.v. administration of PACAP. The induction of c-fos gene expression in the PVN and SON reached a maximum 30 min after PACAP administration. The expression of c-fos gene in the PVN and SON induced by i.c.v. administration of vasoactive intestinal peptide (200 pmol/rat) was weaker than that induced by PACAP. The hnRNA for AVP in the PVN and SON was significantly increased 30 min after i.c.v. administration of PACAP (200 pmol/rat). Our results suggest that PACAP activates PVN and SON neurons via PACAP receptors and, in parallel, transcription of the AVP gene in the PVN and SON.
Potentiation of Prolactin Secretion following Lactotrope Escape from Dopamine Action
Authors: del Mar Hernández María , García Ferreiro Rafael E. , García David E. , Hernández María Elena , Clapp Carmen , Martínez de la Escalera Gonzalo
Published: April 2003

Hypothalamic dopamine (DA) tonically inhibits prolactin (PRL) release from the anterior pituitary gland. Transient escapes from this DA tone elicit a pronounced potentiation of the PRL-releasing action of secretagogues such as thyrotropin-releasing hormone (TRH). Previous evidence has suggested that modulation of Ca2+ channels can be involved in this potentiation. With a lactotropic cell line (GH4C1) expressing human D2-DA receptors, we tested the hypothesis that a brief escape from the tonic inhibitory action of DA triggers a facilitation of Ca2+ influx through Ca2+ channels. We initially found that in these cells, DA effectively and reversibly inhibited PRL secretion, and reversibly enhanced an inwardly rectifying K+ current. The effects of DA administration and withdrawal on Ca2+ currents were examined using the patch-clamp technique in the whole-cell configuration and Ba2+ as a divalent charge carrier through Ca2+ channels. Macroscopic Ba2+ currents were significantly decreased by short term (1–10 min) applications of DA (500 nM), which further declined following 24 h of constant exposure to DA. After DA removal, a biphasic facilitation of the density of Ba2+ currents was observed. An initial 2-fold enhancement of conductance was detected between 10 and 40 min, followed by a second facilitation of the same magnitude observed 24 h after DA withdrawal. The present results directly demonstrate that dissociation of DA from D2-receptors expressed in GH4C1 lactotrope cells causes an increase of high-voltage-activated Ca2+ channel function, which may play an important role in the cross-talking amplification of endocrine cascades such as that involved in the TRH-induced PRL-release potentiating action of DA withdrawal.
Potentiation of Prolactin Secretion following Lactotrope Escape from Dopamine Action
Authors: Hernández María Elena , del Mar Hernández María , Díaz-Muñoz Mauricio , Clapp Carmen , de la Escalera Gonzalo Martínez
Published: April 2003

Modulation of Ca2+ channels has been shown to alter cellular functions. It can play an important role in the amplification of signals in the endocrine system, including the pleiotropically regulated pituitary lactotropes. Prolactin (PRL) secretion is tonically inhibited by dopamine (DA), the escape from which triggers acute episodes of hormone secretion. The magnitude of these episodes is explained by a potentiation of the PRL-releasing action of secretagogues such as thyrotropin-releasing hormone (TRH). While the mechanisms of this potentiation are not fully understood, it is known to be mimicked by the dihydropyridine, L-type Ca2+ channel agonist Bay K 8644 and blocked by nifedipine and methoxyverapamil. The potentiation is also blocked by inhibitors of cyclic AMP-dependent protein kinase and protein kinase C. We recently described that the escape from tonic actions of DA results in increased macroscopic Ca2+ currents in GH4C1 lactotropic clonal cells transfected with a cDNA encoding the long form of the human D2-DA receptor. Here we show that the withdrawal from DA potentiates the PRL-releasing action of TRH in GH4C1/D2-DAR cells to the same extent as in enriched lactotropes in primary culture. In both experimental models a low density of dihydropyridine receptors was shown by (+)-[3H]PN200-110 binding. Photoaffinity labelling with the dihydropyridine [3H]azidopine revealed a protein consistent with the α1 subunit of L-type Ca2+ channels of 165–170 kDa. In both experimental models, the facilitation of TRH action triggered by the escape from DA was correlated with an enhanced rate of phosphorylation of this putative α1 subunit, the nature of which was further supported by immunoprecipitation with selective antibodies directed against the α1C and α1D subunit of voltage-gated calcium channels. We propose that PKA- and PKC-dependent phosphorylation of the α1 subunit of high voltage activated, L-type Ca2+ channels is responsible for the facilitation of Ca2+ currents in lactotropes, and hence for the potentiation of secretagogue-mediated PRL secretion. Thus, phosphorylation of Ca2+ channels in endocrine cells may be a mechanism for the regulation of various functions including amplification of hormone secretion.
Modulation of Prohormone Convertase 2 in Spinal Cord during Gestation and Hormone-Simulated Pregnancy
Authors: Varshney Charu , Rivera Mildred , Gintzler Alan R.
Published: April 2003

Gestation as well as its hormonal simulation (HSP) is characterized by an enhanced spinal dynorphin/ĸ-opioid antinociception. This antinociception is accompanied by decreased content of dynorphin precursor intermediates and increased content of mature dynorphin peptides (1–17 and 1–8) in the lumbar spinal region. This suggests that augmented processing of spinal dynorphin precursor intermediates is an adaptive mechanism used by dynorphin neurons to meet increased synthetic demands necessitated by increased dynorphin neurotransmission. Prohormone convertase (PC) 1 and 2 represent major secretory granule proteolytic processing activities capable of converting neuroendocrine and neurotransmitter peptide (dynorphin) precursor intermediates to their mature, biologically active products. Accordingly, the current investigation was undertaken to assess their potential relevance to peptidergic (dynorphin) neuronal functional plasticity in vivo. In order to evaluate a molecular biological parameter of PC2 synthesis, a solution hybridization assay was developed with which to quantify changes in the spinal lumbar content of its mRNA. This study demonstrates that during gestation and HSP, lumbar PC2 protein content, but not that of PC1, is augmented. The increase in lumbar PC2 during HSP indicates that the pregnancy blood concentration profile of 17β-estradiol and progesterone is a predominant facet of the pregnant condition responsible for its modulation during this condition. In contrast to the elevated content of lumbar PC2 protein, levels of PC2 mRNA in the lumbar cord of pregnant or HSP rats were essentially unchanged. This indicates that increased transcriptional activity is not, necessarily, a prerequisite for increased PC2 protein content to be manifest. These observations suggest positive modulation of PC2 to be a critical component of the mechanism(s) by which spinal dynorphin neurons adapt to the demand-induced increased production of mature dynorphin peptides.