A subchronic application period of glucocorticoids leads to rat cognitive dysfunction whereas physostigmine induces a mild neuroprotection.

The cholinergic neurotransmitter system and prolonged glucocorticoid-induced stress can affect cognitive functions in opposite ways. While pharmacological enhancement of cholinergic neurotransmission is known to induce neuroprotective effects, chronic glucocorticoids impair cognitive functions. Up to now, there is no consensus as to whether a subchronic stress period of several days would affect cognitive function. The goal of this study was to investigate whether or not repeated applications of physostigmine over 3 days lead to protective effects on rat spatial cognitive abilities in contrast to the deteriorating effect on rat cognitive function after corticosterone treatment. Furthermore, we wanted to investigate in what extent this cognition-modulating effect is associated with rat cerebral acetylcholinergic system. Male adult rats (n = 40) were randomly divided into four groups with n = 10 per group: (I) placebo-, (II) corticosterone- (15 mg/day), (III) physostigmine- (0.014 mg/day), and (IV) physostigmine + corticosterone-treated rats. Body mass and plasma corticosterone concentrations were measured. Psychometric investigations were conducted using a Morris water maze before and after a subchronic treatment. In cerebral tissue, ACh and acetylcholinesterase (AChE) content and ACh receptor density were determined. Tissue corticosterone concentration was measured in cerebral cortex, hippocampus, and adrenal glands. In corticosterone-treated rats, reduced spatial cognitive abilities were associated with a significant increase in plasma (+25%) and cerebral corticosterone levels (+350%) parallelled by a significant reduction in adrenal gland concentrations (-84%) as compared to placebo. Repeated physostigmine injections improved rats' spatial memory and increased cerebral ACh and AChE content (p < 0.05). When physostigmine was administered at the same time as corticosterone (group IV), it was not able to reverse the corticosterone effect. A significant correlation was detected between cerebral AChE and corticosterone concentrations as well as between AChE and psychometric parameters. We conclude that subchronic exogenous corticosterone administration induces memory dysfunction whereas physostigmine exerts cognitive-enhancing effects if given for 3 days. An apparently existing interaction between glucocorticoid excess and ACh metabolism is discussed.

Negative association between plasma cortisol levels and aggression in a high-risk community sample of adolescents.

In this study, the association of aggressive behavior and personality traits with plasma cortisol levels was investigated in a high-risk community sample of adolescents. Plasma cortisol levels were collected in 245 fifteen-year-olds (118 males, 127 females) from an epidemiological cohort study of children at risk for psychopathology. Additionally, measures of reactive and proactive aggression, externalizing behavior and callous-unemotional together with impulsive personality features were assessed. Both subtypes of aggression as well as delinquent behavior and impulsive personality traits showed significant negative correlations with plasma cortisol levels. This association was observed in males, but not in females. In both gender groups, callous-unemotional traits were unrelated to plasma cortisol levels. This result suggests that the association between cortisol levels and aggression in adolescents is mediated rather by impulsivity than by unemotional or psychopathic traits.

Cortisol metabolism in depressed patients and healthy controls.

BACKGROUND: Chronic stress as well as major depressive disorders are associated with hypercortisolemia and impaired hypothalamic-pituitary-adrenocortical axis functioning. The aim of this study was to determine whether in major depression changes in the activity patterns of local modulators of glucocorticoid action might contribute to an increase in cortisol bioavailability and if they change during antidepressant treatment and clinical response. METHODS: Concentrations of urinary total cortisol (UFF), urinary total cortisone (UFE), tetrahydrocortisone (THE), tetrahydrocortisol (THF) and allo-THF (5alpha-THF) were measured in 10-hour nocturnal urine samples of 19 depressed patients and 15 healthy controls. The activity of 11beta-hydroxysteroid dehydrogenases (11beta-HSD) as well as 5alpha- and 5beta-reductases was assessed by calculating the ratios of glucocorticoid metabolites. Patients were treated for 28 days with either mirtazapine or venlafaxine. Enzyme activity was observed during the course of treatment and compared to healthy controls. Responders to treatment were selected for this analysis. RESULTS: Depressed patients showed reduced 5alpha-reductase activity manifested as a significantly lower amount of 5alpha-THF (102.8 +/- 167.2 vs. 194.6 +/- 165.8 microg, p = 0.019). The increase in the UFF/UFE ratio (0.73 +/- 0.32 vs. 0.29 +/- 0.13, p < 0.0001) indicates reduced activity of renal 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2). During pharmacological treatment, 5alpha-reductase activity in patients returned to the level of the control group, while the decrease in 11beta-HSD2 activity persisted until day 28. CONCLUSIONS: Our results show changes in activity of intracellular modulators of steroid action in major depressive disorders, particularly a reduced activity of the intracellular cortisol-deactivating enzymes 5alpha-reductase and 11beta-HSD2. These changes suggest an increase in cortisol bioavailability within tissues.

Reduction in rat phosphatidylethanolamine binding protein-1 (PEBP1) after chronic corticosterone treatment may be paralleled by cognitive impairment: a first study.

Chronic stress is associated with hippocampal atrophy and cognitive dysfunction. This study investigates how long-lasting administration of corticosterone as a mimic of experimentally induced stress affects psychometric performance and the expression of the phosphatidylethanolamine binding protein (PEBP1) in the adult hippocampus of one-year-old male rats. Psychometric investigations were conducted in rats before and after corticosterone treatment using a holeboard test system. Rats were randomly attributed to 2 groups (n = 7) for daily subcutaneous injection of either 26.8 mg/kg body weight corticosterone or sesame oil (vehicle control). Treatment was continued for 60 days, followed by cognitive retesting in the holeboard system. For protein analysis, the hippocampal proteome was separated by 2D electrophoresis (2DE) followed by image processing, statistical analysis, protein identification via peptide mass fingerprinting and gel matching and subsequent functional network mapping and molecular pathway analysis. Differential expression of PEBP1 was additionally quantified by Western blot analysis. Results show that chronic corticosterone significantly decreased rat hippocampal PEBP1 expression and induced a working and reference memory dysfunction. From this, we derive the preliminary hypothesis that PEBP1 may be a novel molecular mediator influencing cognitive integrity during chronic corticosterone exposure in rat hippocampus.

Thyroid cysts: a new extra-adrenal site of aldosterone synthase expression and increased aldosterone content.

BACKGROUND: The rapid re-accumulation of fluid following aspiration of thyroid cystic lesions suggests that active transport of sodium and water may be involved in volume regulation of these lesions. In this study we address the possibility that aldosterone may take part in this process. SUBJECTS AND METHODS: Thirty-one patients (29 women and two men), with a mean age of 52.7 +/- 13.2 years (range: 27-77 years) underwent evaluation for thyroid nodules that had a sonographic cystic component. Cystic fluid obtained by FNA biopsy was sent for cytological examination and biochemical measurements. In 10 patients, material was collected for RNA extraction and determination of aldosterone synthase expression by RT-PCR amplification. RESULTS: All lesions were benign, cystic, colloid nodules. Cyst fluid aldosterone levels as measured by routine radioimmunoassay (RIA) were elevated above the normal plasma levels in all but five patients. Mean aldosterone levels were 27.1 +/- 22.9 ng/dl (SD) (range: 5.9-117.5 ng/dl). In contrast, cyst cortisol values were in the low, low normal serum range (6.2 +/- 2.9 microg/dl, range: 0.2-10.2 microg/dl). Sodium, chloride and potassium levels were 137 +/- 4.7 mEq/l, 98 +/- 5 mEq/l and 4.9 +/- 1.4 mEq/l, respectively. Plasma aldosterone levels were normal in all patients tested. To confirm these results, 12 samples were assayed after extraction and chromatography using a highly specific antibody. Cyst aldosterone levels in this group were elevated above the normal serum range in all but one patient (mean concentration: 24.5 +/- 14.6 ng/dl, range: 8.72-40.1 ng/dl). In this group, 18(OH)B levels were within the normal plasma range (12-55 ng/dl) in all but one patient (34.9 +/- 17 ng/dl). Furthermore, aldosterone synthase mRNA expression was found in aspirates of four of 10 patients. CONCLUSIONS: The increased aldosterone concentration and the presence of aldosterone synthase expression suggest that aldosterone may be locally produced and secreted in thyroid tissue. The pathophysiological implications of this finding remain to be established.

Chronic corticosterone-induced deterioration in rat behaviour is not paralleled by changes in hippocampal NF-kappaB-activation.

We investigated whether long-lasting stress induced by chronic glucocorticoid (GC) exposure affects activation of brain NF-kappaB and whether these changes are related to functional deterioration and structural changes in the rat hippocampus. Psychometric investigations were conducted using a holeboard test system in 28 one-year-old male Wistar rats. Thereafter, rats were divided into three groups for daily administration of 10 mg corticosterone (treatment) or sesame oil (placebo = sham control for effects of the vehicle) for 60 days. Additional control rats did not receive any treatment or handling until the end of the experiment. Behavioural and cognitive changes were tested again in the holeboard system. Rat body weights and corticosterone concentrations in plasma, hippocampus and urine were determined and adrenal glands were investigated histopathologically. Hippocampal concentrations of corticosterone, NF-kappaB and I-kappaBalpha were determined using RIA, EMSA and Western blotting techniques, respectively. Structural changes in rat hippocampus were measured using magnetic resonance imaging techniques. High peripheral corticosterone concentrations after chronic treatment led to significant reductions in rat body weight. Significant atrophy of both adrenal glands with marked histological deterioration was detected. Furthermore, an increase in hippocampal corticosterone concentrations was observed after chronic administration. Chronic corticosterone treatment also significantly altered behaviour and working and reference memory capacity without changing hippocampal structure. Daily injections of sesame oil in the placebo group, however, were also sufficient to reduce the pellet-finding time. However, neither in the corticosterone group nor in the placebo group were behavioural changes paralleled by significant changes in brain NF-kappaB activation and I-kappaBalpha expression. Thus, cognitive alterations in rats seen after chronic corticosterone exposure are not paralleled by hippocampal NF-kappaB modulation.

Kallidin-like peptide mediates the cardioprotective effect of the ACE inhibitor captopril against ischaemic reperfusion injury of rat heart.

1. The potential cardioprotective effect of ACE inhibitors has been attributed to the inhibition of bradykinin degradation. Recent data in rats documented a kallidin-like peptide, which mimics the cardioprotective effect of ischaemic preconditioning. This study investigates in isolated Langendorff rat heart the effect of the ACE inhibitor captopril, the role of bradykinin, kallidin-like peptide, and nitric oxide (NO). 2. The bradykinin level in the effluent of the control group was 14.6 pg ml(-1) and was not affected by captopril in the presence or absence of kinin B2-receptor antagonist, HOE140. 3. The kallidin-like peptide levels were approximately six-fold higher (89.8 pg ml(-1)) and increased significantly by treatment with captopril (144 pg ml(-1)), and simultaneous treatment with captopril and HOE140 (197 pg ml(-1)). 4. Following 30 min ischaemia in the control group, the creatine kinase activity increased from 0.4 to 53.4 U l(-1). In the captopril group and in the captopril+L-NAME group, the creatine kinase activity was significantly lower (18.5 and 22.8 U l(-1)). This beneficial effect of captopril was completely abolished by the kinin B2-receptor antagonist, HOE140, as well as by the kallidin antiserum. 5. Perfusion of the hearts with kallidin before the 30 min ischaemia, but not with bradykinin, yielded an approximately 50% reduction in creatine kinase activity after reperfusion. 6. Pretreatment with L-NAME alone and simultaneously with captopril, and with kallidin, respectively, suggests a kinin-independent action of NO before the 30 min ischaemia on coronary flow and a kinin-dependent action after ischaemia. 7. These data show that captopril increases kallidin-like peptide in the effluent. Kallidin-like peptide via kinin B2 receptor seems to be the physiological mediator of cardioprotective actions of captopril against ischaemic reperfusion injury. HOE140 as well as the kallidin antiserum abolished the cardioprotective effects of captopril.

Rat tissue kallikrein releases a kallidin-like peptide from rat low-molecular-weight kininogen.

The kallikrein-kinin system is subdivided into the plasma and tissue-kallikrein-kinin system, with bradykinin (BK) and kallidin (KAL) (Lys(0)-bradykinin) as functional peptides. This occurs in both humans and other mammals. Both peptides are released by plasma and tissue-kallikrein. BK, but not KAL, has been detected in rats until now. One can explain this observation by the structural differences found in the sequence of rat high- and low-molecular kininogen containing an Arg-residue instead of a Lys-residue in front of the N-terminus of the BK sequence. Nevertheless, we were able to measure a kallidin-like peptide (KLP), in rat plasma and urine, using a specific KAL antiserum. In order to confirm our data, we isolated low-molecular-weight kininogen from rat plasma and incubated it with purified rat glandular kallikrein. The generated peptide was retained on a high-pressure liquid chromatography column and displaced by an excess of angiotensin I. The KLP-containing fraction was identified with the KLP radioimmunoassay. A specific ion signal with a mass to charge ratio (m/z) of 1216.73 was detected with matrix-assisted laser desorption/ionization mass spectrometry. As proposed earlier, the structure of this peptide is Arg(1)-KAL, instead of Lys(1)-KAL. The structural similarity between the Lys- and the Arg-residue explains the high crossreactivity (80%) of KLP with the specific KAL antibody. The incubation of KLP with angiotensin-converting enzyme yields two molecules with masses of 913.4 and 729.3 containing the sequence H-Arg-Arg-Pro-Pro-Gly-Phe-Ser-Pro-OH and H-Arg-Arg-Pro-Pro-Gly-Phe-OH. The enzymatic cleavage could be inhibited by captopril. The data suggest that in rats, as in other mammals, the tissue kallikrein-kinin system mediates its physiological effects via a kallidin-like peptide, which is Arg(1)-kallidin (Arg(0)-bradykinin).

A kallidin-like peptide is a protective cardiac kinin, released by ischaemic preconditioning of rat heart.

Bradykinin is thought to play a major role among the endogenous cardioprotective candidates of ischaemic preconditioning (IPC). Little attention has been paid to the fact that in the tissue kallidin (KAL), rather than bradykinin might be the physiological mediator of the kallikrein-kinin system. In order to evaluate the importance of one or the other peptide the release and effect of both kinins has been investigated in isolated rat hearts following IPC. Bradykinin- and a KAL-like peptide were measured in the effluent of the rat isolated Langendorff heart with two different specific radioimmunoassays. The creatine kinase activity in the effluent was judged as degree of cardiac injury caused by ischaemia. During IPC, which consists of three 5 min no-flow and 5 min reperfusion cycles prior to the 30 min ischaemia, the bradykinin level in the effluent did not change significantly (15.4-19.4 pg ml(-1)). In the control group the bradykinin levels were 15.9-16.6 pg ml(-1). During IPC KAL-like peptide (Arg(1)-, instead of Lys(1)-KAL), which has recently been verified by mass spectrometry, displays 5.8-fold higher levels in the effluent and significantly increases in the same time interval from 90.4 to 189 pg ml(-1). After 30 min ischaemia the bradykinin levels in the IPC group were not significantly different to those of the control group (18.7 vs 14.4 pg ml(-1)). The KAL-like peptide levels in the IPC group vs the control group were 105 vs 86.1 pg ml(-1). By the 30 min ischaemia the creatine kinase activity in the IPC group increased from 0.367 to 8.93 U l(-1) (before and 10-30 min after ischaemia). In the control group during the same time period the creatine kinase levels increased from 0.277 to 34.9 U l(-1). The low increase in creatine kinase activity following IPC was taken as equivalent of the cardioprotective action. A KAL antibody or HOE140 (kinin B(2)-receptor antagonist) completely abolished this beneficial effect of IPC (36.6 and 53.0 U l(-1)) when added to the perfusion medium during the reperfusion cycles of IPC prior to the 30 min ischaemia. Our data suggest that in rat hearts KAL-like peptide rather than bradykinin is the physiological compound activated by IPC and acting via the cardiac kinin B(2)-receptor. Thus, endogenously generated KAL-like peptide seems to play a major role in the cardioprotection of IPC.

A particular phenotype in a girl with aldosterone synthase deficiency.

Aldosterone synthase deficiency (ASD) usually presents in infancy as a life-threatening electrolyte imbalance. A 4-wk-old child of unrelated parents was examined for failure to thrive and salt-wasting. Notable laboratory findings were hyperkalemia, high plasma renin, and low-normal aldosterone levels. Urinary metabolite ratios of corticosterone/18-hydroxycorticosterone and 18-hydroxycorticosterone/aldosterone were intermediate between ASD type I and type II. Sequence analysis of CYP11B2, the gene encoding aldosterone synthase (P450c11AS), revealed that the patient was a compound heterozygote carrying a previously described mutation located in exon 4 causing a premature stop codon (E255X) and a further, novel mutation in exon 5 that also causes a premature stop codon (Q272X). The patient's unaffected father was a heterozygous carrier of the E255X mutation, whereas the unaffected mother was a heterozygous carrier of the Q272X mutation. Therefore, the patient's CYP11B2 encodes two truncated forms of aldosterone synthase predicted to be inactive because they lack critical active site residues as well as the heme-binding site. This case of ASD is of particular interest because despite the apparent lack of aldosterone synthase activity, the patient displays low-normal aldosterone levels, thus raising the question of its source.

Endocrine disorders of sodium regulation. Role of adrenal steroids in genetic defects causing sodium loss or sodium retention.

Salt and water homoeostasis is tightly regulated by a variety of control mechanisms with the adrenal steroid hormone aldosterone playing a central role. Defects or disturbances in these systems lead to either salt loss, which is life threatening in the neonatal period, or sodium retention causing hypertension. Rapid and accurate diagnosis is required to avoid severe complications. During the last few years molecular genetic advances have been identified as the basic genetic defects for a number of clinical syndromes. This knowledge has considerably increased our understanding of the basic pathways involved in sodium and water homoeostasis and of the pathophysiology of these syndromes, particularly the hypertension. In this review we have summarized the biochemical, physiological and genetic basis for clinical syndromes presenting with salt loss and failure to thrive as well as the rare but important genetic syndromes causing sodium retention and hypertension. Early diagnosis and identification will help to prevent severe complications, but it has to be emphasized that the complicated cascade of aldosterone action is still relatively poorly understood. Further syndromes may exist which once identified will help to better understand the basic physiology of aldosterone action.

Association of testosterone and dihydrotestosterone with externalizing behavior in adolescent boys and girls.

BACKGROUND: While an association between androgens and different types of aggression has been well documented in male offenders, the influence of androgens on externalizing behavior in adolescents at risk for antisocial behavior has not been investigated so far. METHODS: Plasma levels of the main androgen metabolites testosterone (T) and 5alpha-dihydrotestosterone (DHT) were measured in N = 87 fourteen-year-old (36 boys, 51 girls) from a prospective longitudinal study of children at risk. Externalizing behavior at age 8, 11 and 14 was assessed using the Achenbach Child Behavior Checklist (CBCL) and Teacher Report Form (TRF). RESULTS: Significant higher androgen levels (T, DHT) were found in male, but not in female adolescents with elevated scores of externalizing behavior. Moreover, boys with persistent externalizing behavior exhibited the highest levels of plasma androgens. CONCLUSIONS: There is a link between T, DHT and externalizing behavior in male adolescents at risk for psychopathology. Due to the findings of highest androgen levels in boys with persistent externalizing behavior, a role of androgens in the development of disruptive or later antisocial disorders can be hypothesized.

Urinary tetrahydroaldosterone as a screening method for primary aldosteronism: a comparative study.

BACKGROUND: The major aldosterone metabolite 3 alpha,5 beta tetrahydroaldosterone reflects up to 45% of the aldosterone secretion. Its 24-h urinary excretion is likely to provide an accurate index of the daily aldosterone production and to be an indicator for primary aldosteronism (PA). METHODS: In a prospective study, the validity of tetrahydroaldosterone as a screening test for PA was evaluated in comparison to serum potassium, plasma aldosterone, plasma renin activity, plasma aldosterone/renin activity ratio (PARR), as well as 24-h urinary aldosterone-18-glucuronide and free aldosterone. A total of 111 normotensive individuals, 412 PA patients and 1453 essential hypertensive patients, were studied. The effect of blood sampling technique on potassium level was also investigated. RESULTS: Tetrahydroaldosterone differentiated PA from essential hypertension with a sensitivity of 96% and a specificity of 95%. The sensitivity was 89% for plasma aldosterone, 87% for free aldosterone, 85% for PARR, 71% for aldosterone-18-glucuronide and 51% for renin activity. Specificities varied between 91% and 85%. The combined use of the parameters plasma aldosterone > or =9.0 ng/dL and PARR > or =25 resulted in a sensitivity of 82% and specificity of 95%. Forearm exercise proved to be a source of erroneous elevations of potassium sufficient to obscure the suspicion of PA. CONCLUSION: The data suggest that tetrahydroaldosterone is the most reliable screening test for PA. Tetrahydroaldosterone determination in combination with aldosterone-18-glucuronide and free aldosterone increases diagnostic specificity for PA. Potassium, renin, plasma aldosterone, and basal PARR are inadequate screening procedures because they are subject to high rates of false-positive and false-negative results.

Cortisol and cortisone in human follicular fluid and serum and the outcome of IVF treatment.

BACKGROUND: The glucocorticoid status of ovarian follicular fluid has been linked to oocyte quality. The aim of this study was to examine whether the concentrations of cortisone and cortisol and their calculated ratios in the follicular fluid and serum samples are predictive of IVF outcome. METHODS: In the prospective study of 387 patients (420 treatment cycles) undergoing IVF treatment the concentrations of cortisone and cortisol were measured with specific assays, and their calculated ratios in the follicular fluid and serum samples obtained after ovarian stimulation and induced ovulation were determined. RESULTS: In 75 patients, treatment resulted in clinical pregnancy and was associated with significantly lower follicular cortisone (24+/-12 versus 29+/-16 nmol/l, P<0.002) and higher cortisol/cortisone ratio (7.24+/-2.22 versus 6.45+/-2.17 nmol/l, P<0.007). In addition, the ratios of serum cortisone and cortisol to follicular cortisone and cortisol were significantly higher in those women who became pregnant. CONCLUSIONS: We propose that the follicular fluid glucocorticoid concentration resulting from the conditions in the circulation and the course of the intrafollicular cortisol-cortisone interconversion appear to play a role in the outcome of IVF.

Regulatory effects of salt diet on renal renin-angiotensin-aldosterone, and kallikrein-kinin systems.

The interaction of the renin-angiotensin-aldosterone system (RAAS) and the kallikrein-kinin system (KKS) was investigated in rats fed on a low, normal, and high-salt diet for 2 weeks. At the beginning of the second week, either a B2-receptor antagonist (icatibant), or an AT1-receptor antagonist (losartan), or an aldosterone receptor antagonist (spironolactone) was applied via osmotic pump delivering a constant amount of drug for 7 days. The urinary bradykinin (BK) levels corresponded with increasing NaCl diet and the activity of urinary kallikrein. However, in agreement with other investigators we found a down-regulation of the renal kallikrein gene expression in response to an increasing NaCl diet. Renal kinins are able to stimulate the renal kallikrein expression as well as the renal excretion of active kallikrein via the B2-receptor. The release of renal kallikrein is also mediated by angiotensin II (AngII). After high-salt diet the blood pressure was significantly increased. Losartan and spironolactone were not effective in reducing this increase, as AngII and aldosterone should be low during high-salt diet. However, low-salt diet also yielded an increase in blood pressure, which, however, could be abolished following losartan infusion. The data suggest that the expression of renal kallikrein mRNA is mainly regulated by dietary salt intake. However, kinins are able to stimulate the kallikrein gene expression, as well as the renal kallikrein release. Angll mediates only a stimulatory effect on the urinary kallikrein release. In contrast to the general belief, our data support the opinion that low-salt diet is able to mediate an increase in blood pressure, as the RAAS is stimulated in response to a marked salt deficiency.