Escitalopram prolonged fear induced by simulated public speaking and released hypothalamic-pituitary-adrenal axis activation

Simulated public speaking (SPS) test is sensitive to drugs that interfere with serotonin-mediated neurotransmission and is supposed to recruit neural systems involved in panic disorder. The study was aimed at evaluating the effects of escitalopram, the most selective serotonin-selective reuptake inhibitor available, in SPS. Healthy males received, in a double-blind, randomized design, placebo (n = 12), 10 (n = 17) or 20 (n = 14) mg of escitalopram 2 hours before the test. Behavioural, autonomic and neuroendocrine measures were assessed. Both doses of escitalopram did not produce any effect before or during the speech but prolonged the fear induced by SPS. The test itself did not significantly change cortisol and prolactin levels but under the higher dose of escitalopram, cortisol and prolactin increased immediately after SPS. This fear-enhancing effect of escitalopram agrees with previously reported results with less selective serotonin reuptake inhibitors and the receptor antagonist ritanserin, indicating that serotonin inhibits the fear of speaking in public.

Reduction of ICAM-1 expression by carbon monoxide via soluble guanylate cyclase activation accounts for modulation of neutrophil migration

Previously, it was demonstrated that the heme/heme oxygenase (HO)/carbon monoxide (CO) pathway inhibits neutrophil recruitment during the inflammatory response. Herein, we addressed whether the inhibitory effect of the HO pathway on neutrophil adhesion and migration involves the reduction of intracellular adhesion molecule type (ICAM)-1 and beta(2)-integrin expression. Mice pretreated with a specific inhibitor of inducible HO (HO-1), zinc protoporphyrin (ZnPP) IX, exhibit enhanced neutrophil adhesion and migration induced by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS). These findings are associated with an increase in ICAM-1 expression on mesentery venular endothelium. In accordance, HO-1 inhibition did not enhance LPS-induced neutrophil migration and adhesion in ICAM-1-deficient mice. Furthermore, the treatment with a CO donor (dimanganese decacarbonyl, DMDC) that inhibits adhesion and migration of the neutrophils, reduced LPS-induced ICAM-1 expression. Moreover, neither DMDC nor ZnPP IX treatments changed LPS-induced beta(2)-integrin expression on neutrophils. The effect of CO on ICAM-1 expression seems to be dependent on soluble guanylate cyclase (sGC) activation, since 1H-(1,2,4)oxadiazolo (4,3-a)quinoxalin-1-one (sGC inhibitor) prevented the observed CO effects. Finally, it was observed that the nitric oxide (NO) anti-inflammatory effects on ICAM-1 expression appear to be indirectly mediated by HO-1 activation, since the inhibition of HO-1 prevented the inhibitory effect of the NO donor (S-nitroso-N-acetylpenicillamine) on LPS-induced ICAM-1 expression. Taken together, these results suggest that CO inhibits ICAM-1 expression on endothelium by a mechanism dependent on sGC activation. Thus, our findings identify the HO-1/CO/guanosine 3`5`-cyclic monophosphate pathway as a potential target for the development of novel pharmacotherapy to control neutrophil migration in inflammatory diseases.

Is Suppression of Hypothalamic-Pituitary-Adrenal Axis Significant During Clinical Treatment of Phimosis?

Purpose: Corticoids have been an option for phimosis treatment since 1993. However, long-term use or repeated cycles pose a concern regarding drug absorption and consequent systemic effects. The aim of this study was to investigate the effect of topical corticoids used in treating phimosis on the hypothalamus-pituitary-adrenal axis in children. Materials and Methods: A total of 31 children were included in the study. Cortisol secretion was evaluated by the measurement of salivary cortisol in saliva samples collected at 9:00 a.m, before starting treatment and after 8 weeks of topical treatment with 0.05% clobetasol propionate. Salivary cortisol was determined by radioimmunoassay. To confirm that use of clobetasol propionate was not detected by the assay, the presence of cortisol circadian rhythm was checked by an extra saliva sample obtained at 11:00 p.m. from 10 children, and was observed to be maintained in all of them. Results: No significant difference in salivary cortisol levels was observed between samples obtained at 9:00 a.m. before starting treatment and after completing treatment when the entire group was analyzed. However, in 2 children the salivary cortisol levels after treatment were lower than the cutoff value (358 ng/dl) assumed to be suggestive of hypothalamus-pituitary-adrenal axis suppression. Conclusions: Topical clobetasol propionate used twice daily for clinical treatment of phimosis does not affect the hypothalamus-pituitary-adrenal axis in most patients. However, salivary cortisol level should be considered as a laboratory marker in long-term treatment or during repeated cycles to detect possible hypothalamus-pituitary-adrenal axis suppression.

Dorsal and ventral medullary catecholamine cell groups contribute differentially to systemic interleukin-1 beta-induced hypothalamic pituitary adrenal axis responses

Medial parvocellular paraventricular corticotropin-releasing hormone (mPVN CRH) cells are critical in generating hypothalamic-pituitary-adrenal (HPA) axis responses to systemic interleukin-1 beta (IL-1 beta). However, although it is understood that catecholamine inputs are important in initiating mPVN CRH cell responses to IL-1 beta, the contributions of distinct brainstem catecholamine cell groups are not known. We examined the role of nucleus tractus solitarius (NTS) and ventrolateral medulla (VLM) catecholamine cells in the activation of mPVN CRH, hypothalamic oxytocin (OT) and central amygdala cells in response to IL-1 beta (1 mug/kg, i.a.). Immunolabelling for the expression of c-fos was used as a marker of neuronal activation in combination with appropriate cytoplasmic phenotypic markers. First we confirmed that PVN 6-hydroxydopamine lesions, which selectively depleted catecholaminergic terminals, significantly reduced IL-1 beta -induced mPVN CRH cell activation. The contribution of VLM (A1/C1 cells) versus NTS (A2 cells) catecholamine cells to mPVN CRH cell responses was then examined by placing ibotenic acid lesions in either the VLM or NTS. The precise positioning of these lesions was guided by prior retrograde tracing studies in which we mapped the location of IL-1 beta -activated VLM and NTS cells that project to the mPVN. Both VLM and NTS lesions reduced the mPVN CRH and OT cell responses to IL-1 beta. Unlike VLM lesions, NTS lesions also suppressed the recruitment of central amygdala neurons. These studies provide novel evidence that both the NTS and VLM catecholamine cells have important, but differential, contributions to the generation of IL-1 beta -induced HPA axis responses. Copyright (C) 2001 S. Karger AG, Basel.

Role of circumventricular organs in pro-inflammatory cytokine-induced activation of the hypothalamic-pituitary-adrenal axis

1. The past 15 years has seen the emergence of a new field of neuroscience research based primarily on how the immune system and the central nervous system can interact. A notable example of this interaction occurs when peripheral inflammation, infection or tissue injury activates the hypothalamic- pituitary-adrenal axis (HPA). 2. During such assaults, immune cells release the pro- inflammatory cytokines interleukin (IL)-1, IL-6 and tumour necrosis factor-alpha into the general circulation. 3. These cytokines are believed to act as mediators for HPA axis activation. However, physical limitations of cytokines impede their movement across the blood-brain barrier and, consequently, it has been unclear as to precisely how and where IL-1beta signals cross into the brain to trigger HPA axis activation. 4. Evidence from recent anatomical and functional studies suggests two neuronal networks may be involved in triggering HPA axis activity in response to circulating cytokines. These are catecholamine cells of the medulla oblongata and the circumventricular organs (CVO). 5. The present paper examines the role of CVO in generating HPA axis responses to pro-inflammatory cytokines and culminates with a proposed model based on cytokine signalling primarily involving the area postrema and catecholamine cells in the ventrolateral and dorsal medulla.

No evidence of a role for activating CDK2 mutations in melanoma

Inactivation of p16(INK4a) and/or activation of cyclin-dependent kinase-4 (CDK4) are strongly associated with both susceptibility and progression in melanoma. Activating CDK4 mutations prevent the binding and inhibition of CDK4 by p16(INK4a). A second, more indirect role for CDK4 is in late G(1), where It may sequester the inhibitors p27(KIP1) or p21(CIP1) away from CDK2, and in doing so upregulate the CDK2 activity necessary for cells to proceed completely through G(1) into S phase. As the pivotal residues around the most predominant R24C activating CDK4 mutation are invariant between CDK2 and CDK4, we speculated that the pivotal arginine (position 22 in CDK2), or a nearby residue, may be mutated in some melanomas, resulting in the diminution of its binding and inhibition by p27(KIP1) or p21(CIP1). However, except for a silent polymorphism, we detected no variants within this region of the CDK2 gene in 60 melanoma cell lines. Thus, if CDK2 activity is dysregulated in melanoma it is likely to occur by a means other than mutations causing loss of direct inhibition. We also examined the expression of the CDK2 gene in melanoma cell lines, to assess its possible co-regulation with the gene for the melanocyte-lineage antigen pmel17, which maps less than 1 kb away in head to head orientation with CDK2 and may be transcribed off the same bidirectional promoter. However, expression of the genes is not co-regulated. (C) 2001 Lippincott Williams & Wilkins.

Rapid onset of pituitary apoplexy after goserelin implant for prostate cancer: need for heightened awareness

No Abstract

Adrenocorticotropin stimulation tests in patients with hypothalamic-pituitary disease: low dose, standard high dose and 8-h infusion tests

Objectives Low doses of ACTH [1-24] (0.1, 0.5 and 1.0 mug per 1.73 m(2)) may provide a more physiological level of adrenal stimulation than the standard 250 mug test, but not all studies have concluded that the 1.0 fig is a more sensitive screening test for central hypoadrenalism. Eight-hour infusions of high dose ACTH [1-24] have also been suggested as a means of assessing the adrenals' capacity for sustained cortisol secretion. In this study, we compared the diagnostic accuracy of three low dose ACTH tests (LDTs) and the 8-h infusion with the standard 250 jig test (HDT) and the insulin hypoglycaemia test (IHT) in patients with hypothalamic-pituitary disease. Subjects and design Three groups of subjects were studied. A healthy control group (group 1, n=9) and 33 patients with known hypothalamic or pituitary disease who were divided into group 2 (n=12, underwent IHT) and group 3 (n=21, IHT contraindicated). Six different tests were performed: a standard IHT (0.15 U/kg soluble insulin); a 60-minute 250 mug HDT; three different LDTs using 0.1 mug, 0.5 mug and 1.0 mug (all per 1.73 m(2)); and an 8-h infusion test (250 mug ACTH [1-24] at a constant rate over 8 h). Results Nine out of the 12 patients in group 2 failed the IHT. Three out of 12 patients from group 2 who clearly passed the IHT, also passed all the ACTH [1-24] stimulation tests. Seven of the 9 patients who failed the lHT, failed by a clear margin (peak cortisol

A cassette ligation strategy with thioether replacement of three Gly-Gly peptide bonds: Total chemical synthesis of the 101 residue protein early pregnancy factor [psi(CH2S)(28-29,56-57,76-77)]

The 101 residue protein early pregnancy factor (EPF), also known as human chaperonin 10, was synthesized from four functionalized, but unprotected, peptide segments by a sequential thioether ligation strategy. The approach exploits the differential reactivity of a peptide-NHCH2CH2SH thiolate with XCH2CO-peptides, where X = Cl or I/Br. Initial model studies with short functionalized (but unprotected) peptides showed a significantly faster reaction of a peptide-NHCH2CH2SH thiolate with a BrCH2CO-peptide than with a CICH2CO-peptide, where thiolate displacement of the halide leads to chemoselective formation of a thioether surrogate for the Gly-Gly peptide bond. This rate difference was used as the basis of a novel sequential ligation approach to the synthesis of large polypeptide chains. Thus, ligation of a model bifunctional N-alpha-chloroacetyl, C-terminal thiolated peptide with a second N-alpha-bromoacetyl peptide demonstrated chemoselective bromide displacement by the thiol group. Further investigations showed that the relatively unreactive N-alpha-chloroacetyl peptides could be activated by halide exchange using saturated KI solutions to yield the highly reactive No-iodoacetyl peptides. These findings were used to formulate a sequential thioether ligation strategy for the synthesis of EPF, a 101 amino acid protein containing three Gly-Gly sites approximately equidistantly spaced within the peptide chain. Four peptide segments or cassettes comprising the EPF protein sequence (BrAc-[EPF 78-101] 12, ClAc-[EPF 58-75]-[NHCH2CH2SH] 13, ClAc-[EPF 30-55]-[NHCH2CH2SH] 14, and Ac-[EPF 1-27]-[NHCH2CH2SH] 15) of EPF were synthesized in high yield and purity using Boc SPPS chemistry. In the stepwise sequential ligation strategy, reaction of peptides 12 and 13 was followed by conversion of the N-terminal chloroacetyl functional group to an iodoacetyl, thus activating the product peptide for further ligation with peptide 14. The process of ligation followed by iodoacetyl activation was repeated to yield an analogue of EPF (EPF psi(CH2S)(28-29,56-57,76-77)) 19 in 19% overall yield.

Distribution of two splice variants of the glutamate transporter GLT-1 in rat brain and pituitary

We have performed immunocytochemistry on rat brains using a highly specific antiserum directed against the originally described form of the glutamate transporter GLT-1 (referred to hereafter as GLT-1alpha), and another against a C-terminal splice variant of this protein, GLT-1B. Both forms of GLT-1 were abundant in rat brain, especially in regions such as the hippocampus and cerebral cortex, and macroscopic examination of sections suggested that both forms were generally regionally coexistent. However, disparities were evident; GLT-1alpha was present in the intermediate lobe of the pituitary gland, whereas GLT-1B was absent. Similar marked disparities were also noted in the external capsule, where GLT1A labeling was abundant but GLT-1B was only occasionally encountered. Conversely, GLT-1B was more extensively distributed, relative to GLT-1alpha, in areas such as the deep cerebellar nuclei. In most regions, such as the olfactory bulbs, both splice variants were present but differences were evident in their distribution. In cerebral cortex, patches were evident where GLT-1B was absent, whereas no such patches were evident for GLT-1alpha. At high resolution, other discrepancies were evident; double-labeling of areas such as hippocampus indicated that the. two splice variants may either be differentially expressed by closely apposed glial elements or that the two splice variants may be differentially targeted to distinct membrane domains of individual glial cells. (C) 2002 Wiley-Liss, Inc.

Localization of taurine transporters, taurine, and H-3 taurine accumulation in the rat retina, pituitary, and brain

The nervous system contains an abundance of taurine, a neuroactive sulfonic acid. Antibodies were generated against two cloned high-affinity taurine transporters, referred to in this study as TAUT-1 and TAUT-2. The distribution of such was compared with the distribution of taurine in the rat brain, pituitary, and retina. The cellular pattern of [H-3] taurine uptake in brain slices, pituitary slices, and retinas was examined by autoradiography. TAUT-2 was predominantly associated with glial cells, including the Bergmann glial cells of the cerebellum and astrocytes in brain areas such as hippocampus. Low-level labeling for TAUT-2 was also observed in some neurones such as CA1 pyramidal cells. TAUT-1 distribution was more limited; in the posterior pituitary TAUT-1 was associated with the pituicytes but was absent from glial cells in the intermediate and anterior lobes. Conversely, in the brain TAUT-1 was associated with cerebellar Purkinje cells and, in the retina, with photoreceptors and bipolar cells. Our data suggest that intracellular taurine levels in glial cells and neurons may be regulated in part by specific high-affinity taurine transporters. The heterogeneous distribution of taurine and its transporters in the brain does not reconcile well with the possibility that taurine acts solely as a ubiquitous osmolyte in nervous tissues. (C) 2002 Wiley-Liss, Inc.

Autosomal dominant hypocalcemia: A novel activating mutation (E604K) in the cysteine-rich domain of the calcium-sensing receptor

We report a novel activating mutation (E604K) of the calcium-sensing receptor in a family with autosomal dominant hypocalcemia. Whereas all affected individuals exhibited marked hypocalcemia, some cases with untreated hypocalcemia exhibited seizures in infancy, whereas others were largely asymptomatic from birth into adulthood. The missense mutation E604K (G2182A, GenBank accession no. U20759), which affects an amino acid residue in the C terminus of the cysteine-rich domain of the extracellular head, co-segregated with hypocalcemia in all seven individuals for whom DNA was available. Two unaffected, normocalcemic members of the family did not exhibit the mutation. The molecular impact of the mutation on two key components of the signaling response was assessed in HEK-293 cells transiently transfected with cDNA corresponding to either the wild-type calcium-sensing receptor or the E604K mutation derived by site-directed mutagenesis. There was a significant leftward shift in the concentration response curves for the effects of extracellular Ca2+ on both intracellular Ca2+ mobilization (determined by aequorin luminescence) and MAPK activity (determined by luciferase expression). The C terminus of the cysteine-rich domain of the extracellular head may normally act to suppress receptor activity in the presence of low extracellular Ca2+ concentrations.

A low concentration of ouabain (0.18 µg/kg) enhances hypertension in spontaneously hypertensive rats by inhibiting the Na+ pump and activating the renin-angiotensin system

We investigated the effects of low ouabain concentrations on systolic (SAP) and diastolic (DAP) arterial pressures and on pressor reactivity in 3-month-old male spontaneously hypertensive rats (SHR). Arterial blood pressure (BP) and pressor reactivity to phenylephrine (PHE) were investigated before and after 0.18 μg/kg ouabain administration (N = 6). The influence of hexamethonium (N = 6), canrenone (N = 6), enalapril (N = 6), and losartan (N = 6) on ouabain actions was evaluated. Ouabain increased BP (SAP: 137 ± 5.1 to 150 ± 4.7; DAP: 93.7 ± 7.7 to 116 ± 3.5 mmHg; P < 0.05) but did not change PHE pressor reactivity. Hexamethonium reduced basal BP in control but not in ouabain-treated rats. However, hexamethonium + ouabain increased DAP sensitivity to PHE. Canrenone did not affect basal BP but blocked ouabain effects on SAP. However, after canrenone + ouabain administration, DAP pressor reactivity to PHE still increased. Enalapril and losartan reduced BP and abolished SAP and DAP responses to ouabain. Enalapril + ouabain reduced DAP reactivity to PHE, while losartan + ouabain reduced SAP and DAP reactivity to PHE. In conclusion, a small dose of ouabain administered to SHR increased BP without altering PHE pressor reactivity. Although the renin-angiotensin system (RAS), Na+ pump and autonomic reflexes are involved in the effects of ouabain on PHE reactivity, central mechanisms might blunt the actions of ouabain on PHE pressor reactivity. The effect of ouabain on SAP seems to depend on the inhibition of both Na+ pump and RAS, whereas the effect on DAP seems to depend only on RAS.

Cholesterol 24S-Hydroxylase overexpression inhibits the liver X receptor (LXR) pathway by activating small guanosine triphosphate-binding proteins (sGTPases) in neuronal cells

The neuronal-specific cholesterol 24S-hydroxylase (CYP46A1) is important for brain cholesterol elimination. Cyp46a1 null mice exhibit severe deficiencies in learning and hippocampal long-term potentiation, suggested to be caused by a decrease in isoprenoid intermediates of the mevalonate pathway. Conversely, transgenic mice overexpressing CYP46A1 show an improved cognitive function. These results raised the question of whether CYP46A1 expression can modulate the activity of proteins that are crucial for neuronal function, namely of isoprenylated small guanosine triphosphate-binding proteins (sGTPases). Our results show that CYP46A1 overexpression in SH-SY5Y neuroblastoma cells and in primary cultures of rat cortical neurons leads to an increase in 3-hydroxy-3-methyl-glutaryl-CoA reductase activity and to an overall increase in membrane levels of RhoA, Rac1, Cdc42 and Rab8. This increase is accompanied by a specific increase in RhoA activation. Interestingly, treatment with lovastatin or a geranylgeranyltransferase-I inhibitor abolished the CYP46A1 effect. The CYP46A1-mediated increase in sGTPases membrane abundance was confirmed in vivo, in membrane fractions obtained from transgenic mice overexpressing this enzyme. Moreover, CYP46A1 overexpression leads to a decrease in the liver X receptor (LXR) transcriptional activity and in the mRNA levels of ATP-binding cassette transporter 1, sub-family A, member 1 and apolipoprotein E. This effect was abolished by inhibition of prenylation or by co-transfection of a RhoA dominant-negative mutant. Our results suggest a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons increase isoprenoid synthesis and sGTPase prenylation. This leads to a reduction in LXR activity, and consequently to a decrease in the expression of LXR target genes.

Structural stability of adenylate kinase from the sulfate-reducing bacteria Desulfovibrio gigas

Biophysical Chemistry 110 (2004) 83–92