Growth hormone responses to melatonin in man.

Oral administration of 1 gram of melatonin caused rapid and significant elevations of serum human growth hormone in eight out of nine normal male subjects. The mean (+/- standard error of the mean) of the peak response by growth hormone was 22.9 +/- 4.6 microunits per milliliter. The increased secretion of growth hormone may be due to interaction of melatonin with hypothalamic serotoninergic receptors.

Suppression of human growth hormone secretion by melatonin and cyproheptadine.

Pituitary growth hormone (GH) release in the rat is stimulated via serotoninergic pathways and can be inhibited by treatment with compounds that act as serotonin antagonists, such as cyproheptadine or the pineal gland hormone, melatonin. To investigate a possible role for serotonin in the control of human GH release, the effects of cyproheptadine and melatonin administration on the GH responses of normal male subjects were examined. The oral administration of cyproheptadine (8-12 mg daily for 5 days) to normal subjects reduced their GH responses to both insulin-induced hypoglycemia and physical exercise to a highly significant extent. Similarly, the mean GH responses of 10 subjects to insulin-induced hypoglycemia were significantly reduced after the prior oral administration of melatonin (1 g). The data presented show that serotonin antagonism has a similar effect on GH secretion in man to that observed in the rat and provides further evidence for serotoninergic, and possibly pineal, involvement in the control of human GH secretion.

Membrane permeability of redox active metal chelators: an important element in reducing hydroxyl radical induced NAD+ depletion in neuronal cells.

There is substantial evidence implicating increased production of the hydroxyl radical and oxidative stress in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD). Significant amounts of hydroxyl radicals will be produced in the presence of hydrogen peroxide and redox active iron via Fenton chemistry. Increased iron levels within the cytoplasm of vulnerable neurons suggest that this may also be an important site of oxidative activity. We investigated the likelihood that intracellular, rather than extracellular chelation of ferrous or ferric iron may be more effective in reducing hydroxyl radical induced cell damage and preserving NAD(+) levels and cell viability. Using intracellular NAD(H) measurements as an indicator of cell viability we found that membrane permeable ferrous chelators were most efficient in preserving cellular NAD(+) levels. Hydrophilic, ferrous or ferric chelators and lipophilic ferric chelators were essentially ineffective in preventing cellular NAD(+) depletion when added at physiological concentrations. We propose that lipophilic ferrous chelators, due to their actions inside the cell, are effective agents for moderating neuronal damage in conditions such as AD where intracellular oxidative stress plays a significant role in disease pathology.

Tolbutamide increases hypothalamic serotonin activity in the rat.

Sulfonylureas are potent hypoglycemic agents; however, their mechanism of action remains incompletely understood. Recent data indicate that hypothalamic norepinephrine (NE) plays a major role in mediating the central neural regulation of blood glucose. We therefore examined whether the sulfonylurea tolbutamide might lower serum glucose via an effect on hypothalamic NE neuronal activity, and compared the effects with those of 2-deoxyglucose-induced neuroglycopenia and of chronic insulin administration. Serum glucose levels fell and serum insulin levels rose 10, 20, and 30 min after acute tolbutamide injection. Serum glucose concentrations were reduced after chronic tolbutamide administered in drinking water, but serum insulin did not change. Hypothalamic NE neuronal activity was increased 10 min after tolbutamide administration, but not at the later times, nor during chronic tolbutamide administration. However, consistent with a rise in serotonin (5-HT) neuronal activity, hypothalamic 5-hydroxyindoleacetic acid (5-HIAA) and the 5-HIAA/5-HT ratio rose 30 min after acute tolbutamide and during chronic tolbutamide administration. This rise was not due to neuroglycopenia per se, since hypothalamic NE neuronal activity was increased and hypothalamic 5-HT neuronal activity was reduced after 2-deoxyglucose-induced neuroglycopenia. Furthermore, the effect of chronic tolbutamide contrasted with that of chronic insulin administration where hypothalamic NE neuronal activity was increased, while hypothalamic 5-HT neuronal activity was unchanged. We conclude that tolbutamide does not lower serum glucose via a direct effect on hypothalamic NE neuronal activity; however, we note that tolbutamide specifically increases hypothalamic 5-HT neuronal activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of d-fenfluramine on basal glucose turnover and fat-feeding-induced insulin resistance in rats.

There is evidence that fenfluramine improves insulin action independently of its anorectic and weight-loss-inducing properties. Chronic d-fenfluramine also reduces hypothalamic noradrenergic tone, which correlates highly with hepatic glucose output. We report that chronic d-fenfluramine (5 mg.kg-1.day-1) ameliorates insulin resistance induced by high-fat feeding. Insulin action was assessed in adult male rats at basal insulin levels and at hyperinsulinemia (approximately 140 mU/L with the euglycemic clamp technique). Hepatic glucose production, peripheral glucose disposal, and individual tissue glucose metabolism were determined from bolus injections of [3H]-2-deoxyglucose and [14C]glucose. Food intake was matched between groups. Basal glucose turnover was reduced 28% (P less than .05) in fat-fed rats receiving d-fenfluramine (fat + fen). The glucose infusion rate to maintain euglycemia was 22.0 +/- 1.1 mg.kg-1.min-1 in the high-carbohydrate-fed rats, 8.2 +/- 1.0 in fat-fed rats, and 15.1 +/- 0.5 in the fat + fen group. Peripheral glucose disposal, reflecting measured skeletal muscle changes, was reduced by fat feeding (from 23.5 +/- 1.0 to 13.8 +/- 0.6 mg.kg-1.min-1) but was improved by d-fenfluramine (16.9 +/- 0.5, P less than .05 vs. fat fed). Impaired suppression of hepatic glucose output by insulin, caused by fat feeding, was totally reversed by d-fenfluramine. Thus, d-fenfluramine counteracted diet-induced insulin resistance, with the predominant effect on the liver. We hypothesize that d-fenfluramine improves insulin action by reducing hypothalamic noradrenergic tone, which in turn reduces the neural drive to hepatic glucose output and improves the hepatic response to insulin.

Steroid estrogens in ocean sediments.

This paper gives results from a study measuring the abundance of steroid hormones in ocean sediments in the proximity of a deep ocean sewage outfall. The outfall is discharge point for an enhanced primary sewage treatment plant and sediment samples were taken adjacent and 7 km from the outfall. All samples contained steroid estrogens at nanogram per gram levels with higher concentrations at the 7 km sampling site. The concentration of estrone ranged from (0.16-1.17 ng/g), 17beta-estradiol (0.22-2.48 ng/g) and the synthetic 17alpha-ethinylestradiol (<0.05-0.5 ng/g). The values detected correspond with estimates based on the proportion of estrogens sorbed to particles in the effluent and the expected proportion of particles originating from sewage in the ocean sediments. The results suggest that estrogens associated with the particulate fraction aggregate on contact with high ionic strength seawater and settle to the seafloor after discharge through deep ocean outfalls.

Steroid estrogens in primary and tertiary wastewater treatment plants.

The concentrations of two natural estrogens (estrone (E1) and Estradiol (E2)) and one synthetic progestin (Ethinylestradiol (EE2)) were measured for different unit operations in an advanced sewage treatment plant and in a large coastal enhanced primary sewage treatment plant. The average influent concentration to both plants was similar: 55 and 53 ng/L for E1 and 22 and 12 ng/L for E2 for the advanced and enhanced primary STPs, respectively. The activated sludge process at the advanced STP removed up to 85% and 96% of E1 and E2, respectively. The enhanced primary sewage treatment plant was mostly ineffective at removing the steroids with only 14% of E1 and 5% of E2 being removed during the treatment process. EE2 was not been detected during the study period in the influent or effluent of either STP. The difference in the observed removal between the two plants is primarily linked to plant performance but the extent to which removal of steroid estrogens is due to bacterial metabolism (i.e. the advanced STP) rather than adsorption to the bacterial biomass remains unclear. The poor removal observed for the coastal enhanced primary STP may have implications for the receiving environment in terms of a greater potential for abnormal reproductive systems in marine animals, particularly if discharges are into large bays or harbours where flushing is limited.

Hypothalamic monoamine control of stress-induced adrenocorticotropin release in the rat.

Despite evidence that ACTH release after stress is under excitatory hypothalamic control, a stimulatory role for any of the monoamine neurotransmitters is yet to be clearly demonstrated. In the present investigation computerized gas chromatography/mass spectrometry was used to assess the neuronal activities of hypothalamic dopamine, norepinephrine (NE), and serotonin (5-HT) in rats after stress-induced ACTH release. Medial basal hypothalamic NE neuronal activity as assessed by the ratio of 3,4-dihydroxyphenylethyleneglycol (DHPG) to NE. (DHPG/NE) was elevated (P less than 0.0005) within 2 min after a 3-min cold water swim stress. Ether stress also caused a marked elevation in NE activity (P less than 0.0025). A highly significant positive correlation between the ratio of hypothalamic DHPG/NE and serum corticosterone was found over a large population of normal and stressed rats. Consistent with this relationship between hypothalamic NE neuronal activity and ACTH release being a causal one were the findings that 1) adrenalectomized rats exhibited markedly elevated hypothalamic DHPG/NE ratio and serum ACTH (both P less than 0.0005) together with serum corticosterone levels reduced to about 3% of control levels (P less than 0.0005), and 2) the administration to rats of the alpha-blocker yohimbine or the antianxiety agent diazepam resulted in significant changes in hypothalamic NE activity, together with parallel changes in ACTH secretion. In hypothyroid rats, which have elevated hypothalamic 5-HT activity, and in normal gentled rats, stress caused a significant reduction in hypothalamic 5-HT activity. High hypothalamic activity of dopamine or 5-HT in hypothyroid rats did not significantly affect basal ACTH levels nor prevent the responses to either cold water swim or ether stress, and both stresses resulted in elevated hypothalamic DHPG/NE, serum ACTH, and serum corticosterone (all P less than 0.005) in these animals. From these data it is concluded that NE is an excitatory hypothalamic monoamine for ACTH release in stress and that hypothalamic 5-HT activity is reduced after stress.

Hyperinsulinemia suppresses glucose utilization in specific brain regions: in vivo studies using the euglycemic clamp in the rat.

It has been suggested that insulin acts centrally by altering brain glucose uptake. Previous studies of the effect of insulin on brain glucose metabolism have been difficult to interpret due to lack of steady state conditions for glucose and/or insulin. To determine whether insulin per se alters brain glucose metabolism, we measured glucose utilization rates, using the deoxyglucose method, in the medial basal hypothalamus, locus coeruleus, and motor cortex of conscious, unrestrained rats undergoing 2-h euglycemic clamps (blood glucose, 4.1 +/- 0.1 mmol/liter) at increasing insulin infusion rates. Plateau insulin levels were 29 +/- 5 mU/liter (controls) and 48 +/- 4, 146 +/- 8, 670 +/- 40, and 7560 +/- 410 mU/liter (clamped). Glucose utilization rates in the medial basal hypothalamus fell significantly from 60 +/- 4 mumol/100 g X min (controls) to 46 +/- 3, 39 +/- 2, 35 +/- 2, and 39 +/- 3 mumol/100 g X min in respective insulin-infused animals (P less than 0.01 vs. controls). Similar falls of up to 39% and 48% were seen in the locus coeruleus and motor cortex, respectively. A significant inverse correlation was found between the glucose utilization rate in each brain region and the log plasma insulin level. The reduction in glucose utilization rate was associated with marked increases in serum corticosterone levels (995 +/- 157 vs. 91 +/- 31 nmol/liter in controls, P less than 0.001). Serum potassium was significantly lower in clamped animals (5.2 +/- 0.3 to 5.9 +/- 0.3 mmol/liter) than in controls (7.0 +/- 0.4 mmol/liter, P less than 0.01). However, the inverse correlation between regional brain glucose utilization and log plasma insulin was independent of changes in serum potassium, while there was no independent correlation with serum potassium. The data reveal reduced glucose utilization in specific brain regions in the presence of insulin levels both equal to and above those found in the postabsorptive state and support a direct effect of insulin in suppressing regional brain glucose utilization.

Rapid bidirectional effects of insulin on hypothalamic noradrenergic and serotoninergic neuronal activity in the rat: role in glucose homeostasis.

Glucose release from the liver is mediated by hypothalamic norepinephrine (NE) neuronal activity, but glucose itself (or a metabolite of it) exerts negative feedback effects on central NE activity. The aim of the present study was to investigate a possible role for insulin in these central glucose homeostatic mechanisms. Computerized gas chromatography-mass spectrometry was used to assess the neuronal activities of hypothalamic NE and serotonin [5-hydroxytryptamine (5-HT)] in rats after acute insulin (2 U/kg) administration. Medial basal hypothalamic NE neuronal activity was assessed by the ratio of 3,4-dihydroxyphenylethyleneglycol to NE. The ratio was suppressed (P less than 0.005) 10 min after insulin administration but rose again to be significantly higher (P less than 0.05) than in saline controls by 30 min and at 45 min post insulin was highly significantly elevated. Hypothalamic 5-HT neuronal activity was assessed by the ratio of 5-hydroxyindoleacetic acid to 5-HT and showed effects opposite to those on NE and was elevated (P less than 0.0005) 10 min post insulin. Significant changes in serum corticosterone and GH levels also occurred after insulin administration, and the changes in these two hormones were positively associated with the changes in hypothalamic neuronal activities of NE and 5-HT, respectively. Serum glucagon levels were found to be significantly elevated in association with the secondary rise in hypothalamic NE activity but did not fall in the 10 min postinsulin phase thus indicating stimulation of pancreatic glucagon release by central NE neuronal pathways. The hypothalamic NE and 5-HT neural responses to a bolus dose of insulin were unaffected by feeding or fasting. These results and evidence that brain glucose utilization is reduced in the immediate postinsulin period suggest that the rapid effects of insulin on hypothalamic NE and 5-HT neuronal activities is a direct one not mediated by stimulation of brain glucose uptake.

The stimulation of human prolactin secretion by 3-Iodo-L-tyrosine.

Oral administration of a single 1 g dose of MIT to 10 normal male and female subjects resulted in a rise in serum prolactin in each subject. The mean peak level of serum prolactin attained by the 10 subjects was 36.3 plus or minus 7.9 ng/ml which was highly significantly elevated (P smaller than 0.0005) above the mean basal level of 5.3 plus or minus 1.0 ng/ml. While there was no significant difference between the basal serum prolactin levels of male and female subjects, the mean peak level attained by male subjects following MIT (18.8 plus or minus 3.3 ng/ml) was significantly less (P smaller than 0.0025) than that recorded for the female group (62.5 plus or minus 9.1). Serum levels of growth hormone (GH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), triiodothyronine (T3), thyroxine (T4) and cortisol were not significantly altered following MIT administration. The complete absence of side effects due to MIT make it a suitable drug for the acute clinical assessment of pituitary prolactin reserve.

Dopamine infusion studies in patients with pathological hyperprolactinemia: evidence of normal prolactin suppressibility but abnormal dopamine metabolism.

The cause of pathological hyperprolactinemia (PHP) is not known, although it has been postulated that lactotroph refractoriness to dopamine (DA) is an important factor. We, therefore, studied PRL suppressibility in response to iv DA administered in a dose designed to achieve peripheral plasma DA concentrations in the range reported in rat pituitary portal plasma. Nine normal subjects and 14 patients (8 with probable microadenomas and 6 with macroadenomas) with PHP were studied during a 4-h infusion of 0.5 micrograms/kg X min DA. Serum PRL levels decreased in normal subjects to 18 +/- 3% (mean +/- SEM) of initial basal levels, not significantly different from the fall to 22 +/- 2% of basal levels found in patients with PHP. Maximal PRL suppression in patients with probable microadenomas (21 +/- 2%) was not significantly different from that in those with macroadenomas (22.8 +/- 2.0%). Upon cessation of DA infusion, there was a rapid rebound in PRL release, which was significantly greater (P less than 0.05) in patients (155 +/- 15%) than in normal subjects (118 +/- 13%). Plasma DA levels were measured by gas chromatography/mass spectroscopy and were significantly higher (P less than 0.05) in patients than in normal subjects during the infusion. The MCR for DA in normal subjects (12.5 +/- 2.8 liters/kg X h) was significantly greater (P less than 0.005) than that in patients (6.2 +/- 0.5 liters/kg X h). The data show that the release of PRL in patients with PHP is not refractory to physiological concentrations of DA, but that there is an abnormality of peripheral DA metabolism in these patients. We conclude that the development of PHP is unlikely to be due to insensitivity of lactotrophs to endogenous DA and that patients with PHP have a systemic disturbance of DA metabolism.

Measurements of protein carbonyls, ortho- and meta-tyrosine and oxidative phosphorylation complex activity in mitochondria from young and old rats.

Mitochondrial bioenergetic function is often reported to decline with age and the accumulation of oxidative damage is thought to contribute. However, there are considerable uncertainties about the amount and significance of mitochondrial oxidative damage in aging. We hypothesized that, as radical production in mitochondria is greater than the rest of the cell, protein oxidative damage should accumulate more in mitochondria than the cytoplasm, and that this relative accumulation should increase with age. To test these hypotheses we measured the accumulation of three markers of protein oxidative damage in liver, brain, and heart from young and old rats. Ortho- and meta-tyrosine levels in protein hydrolysates were measured by a gas chromatography/mass spectrometry assay, and protein carbonyl content was determined by ELISA. Using these assays we found no evidence for increased protein oxidative damage in mitochondria relative to the cytosol. Most increases found in protein oxidative damage on aging were modest for all three tissues and there was no consistent pattern of increased oxidative damage in mitochondrial proteins on aging. Mitochondrial oxidative phosphorylation complex activities were also assessed revealing 39-42% decreases in F0F1--ATP synthase activity in liver and heart on aging, but not in other oxidative phosphorylation complexes. These findings have implications for the contribution of mitochondrial oxidative damage and dysfunction to aging.

IFN-beta1b induces kynurenine pathway metabolism in human macrophages: potential implications for multiple sclerosis treatment.

Interferon-beta(1b) (IFN-beta(1b)) has limited efficacy in the treatment of relapsing-remitting multiple sclerosis (RRMS). The kynurenine pathway (KP) is chiefly activated by IFN-gamma and IFN-alpha, leading to the production of a variety of neurotoxins. We sought to determine whether IFN-beta(1b) induces the KP in human monocyte-derived macrophages, as one explanation for its limited efficacy. Serial dilutions of IFN-beta(1b) (at concentrations comparable to those found in the sera of IFN-beta(1b)-treated patients) were added to human macrophage cultures. Supernatants were collected at various time points and assayed for the KP end product, quinolinic acid (QUIN). The effect of IFN-beta(1b) on the KP enzymes indoleamine 2,3-dioxygenase (IDO), 3-hydroxyanthranilate dioxygenase (3HAO), and quinolinate phosphoribosyltransferase (QPRTase) mRNA expression was assessed by semiquantitative RT-PCR. IFN-beta(1b) (> or =10 IU/ml) led to increased mRNA expression of both IDO and QUIN production (7901 +/- 715 nM) after 72 h at 50 IU/ml IFN-beta(1b) (p < 0.0001). This study demonstrates that IFN-beta(1b), in pharmacologically relevant concentrations, induces KP metabolism in human macrophages and may be a limiting factor in its efficacy in the treatment of MS. Inhibitors of the KP may be able to augment the efficacy of IFN-beta in MS.

Selective inhibition of nitric oxide production during cardiac allograft rejection causes a small increase in graft survival.

Nitric oxide production is increased in allograft rejection and may have both beneficial and deleterious effects on graft function and survival. In animal models, conventional immunosuppressive agents have been shown to decrease nitric oxide production. The aim of our study was to determine what effect augmentation and selective inhibition of nitric oxide production may have on graft survival by using the model of heterotopic cardiac transplantation in the rat. L-Arginine, the naturally occurring substrate for nitric oxide production, was administered subcutaneously at 200 mg/kg/day. L-NG-monomethyl-L-arginine (L-NMMA) is a selective inhibitor of nitric oxide synthase and was administered at 500 mg/kg/day to allograft recipients from the day of operation. Endogenous nitric oxide production was quantified by analysis of urinary nitrate excretion, and time to rejection was determined by graft palpation. L-Arginine did not significantly alter urinary nitrate excretion by iso- or allografts, suggesting that nitric oxide production is not a substrate-limited process in this model. Graft survival in this group was unchanged. L-NMMA produced a small increase in graft survival from 5.1 +/- 0.1 to 6.3 +/- 0.3 days compared with control allografts (P = 0.001) and abolished the rise in urinary nitrate excretion seen with control allografts. Lower doses of L-NMMA produced dose-related decrements in urinary nitrate excretion, but did not alter graft survival. We found that allograft rejection can proceed to graft loss despite complete inhibition of the increase in nitric oxide production that occurs during untreated rejection. The small increase in graft survival suggests that nitric oxide plays a minor role as a cytotoxic effector molecule in this model of acute rejection.

Urinary nitrate excretion is a noninvasive indicator of acute cardiac allograft rejection and nitric oxide production in the rat.

Cytokine induction of calcium-independent nitric oxide synthase is associated with production of large amounts of nitric oxide (NO). NO is a free radical that is rapidly degraded to nitrite and nitrate. Measurement of plasma and urinary nitrate is an indirect marker of NO production and previous studies have demonstrated that plasma nitrate rises with allograft rejection. The purpose of this study was to examine the temporal relationship between the rise in urinary nitrate excretion and the onset of graft rejection, and to determine the effect of conventional immunosuppression on nitrate excretion. The heterotropic model of cardiac transplantation in the rat was used, with Brown-Norway to Lewis allografts and Lewis to Lewis isograft controls. Twenty-four-hour urine specimens were collected before and after transplantation. Urinary nitrate excretion was measured by gas chromatography/mass spectrometry. Each group was treated with (1) no immunosuppression, (2) dexamethasone (3 mg/kg), or (3) CsA (10 mg/kg) on days 0, 1, and 2. Time to rejection for untreated allografts was 5.1 +/- 0.1 days, extending to 8.4 +/- 0.5 and 9.6 +/- 0.4 days with dexamethasone and CsA treatment, respectively. There was a significant rise in nitrate excretion on days 4, 7, and 9 for control, dexamethasone-treated, and CsA-treated allografts, respectively, preceding evidence of rejection. Untreated allograft rejection was associated with a peak in nitrate excretion 8 times that of basal excretion by isografts. Treatment of the allografts with dexamethasone and CsA significantly attenuated peak nitrate excretion compared with untreated allografts with a only a 2- to 3-fold rise preceding rejection. Results indicate that allograft rejection is associated with a dramatic increase in peak urinary nitrate excretion that is attenuated by standard immunosuppressive therapy. An increase in nitrate excretion precedes evidence of graft rejection, and may serve as a noninvasive marker of graft rejection.

Direct pituitary inhibition of prolactin secretion by dopamine and noradrenaline in sheep.

The effects of dopamine, noradrenaline and 3,4-dihydroxyphenylacetic acid (DOPAC) on the release of prolactin were examined in ovariectomized ewes. Infusion of dopamine (0.5 or 1 microgram/kg per min for 2 h i.v.) reduced plasma prolactin concentrations in a dose-dependent manner, whereas DOPAC (5 or 10 micrograms/kg per min for 2 h i.v.) had no effect. In a further series of experiments, ovariectomized hypothalamopituitary disconnected ewes were given dopamine or noradrenaline (each at 0.5 or 1 microgram/kg per min for 2 h i.v.), and both amines reduced mean plasma concentrations of prolactin with similar potency in a dose-dependent manner. These effects were blocked by treatment with pimozide and prazosin respectively. During the infusion of dopamine, the peripheral plasma concentrations of DOPAC and 3,4-dihydroxyphenylethyleneglycol (DHPG) were increased (DOPAC, 22 +/- 7 (S.E.M.) to 131 +/- 11 nmol/l; DHPG, 2.9 +/- 0.3 to 6.4 +/- 0.2 nmol/l), but plasma concentrations of dopamine and noradrenaline did not change. Finally, administration of domperidone, a specific dopamine receptor antagonist that does not cross the blood-brain barrier, resulted in a sustained increase in plasma prolactin concentrations in ovariectomized ewes. We conclude that the secretion of prolactin from the pituitary gland is under dual inhibitory regulation by both dopamine and noradrenaline in the sheep.

Concentrations of dopamine and noradrenaline in hypophysial portal blood in the sheep and the rat.

The concentrations of dopamine, noradrenaline and their respective primary neuronal metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethyleneglycol (DHPG) were measured in the hypophysial portal and peripheral plasma of sheep and rats by combined gas chromatography-mass spectrometry. Hypophysial portal and jugular blood samples were taken at 5- to 10-min intervals for 3-7 h from six conscious ovariectomized ewes. Blood was also collected for 30 min under urethane anaesthesia from the cut pituitary stalk from 16 pro-oestrous female and five intact male rats. In ovariectomized ewes, noradrenaline concentrations were higher in hypophysial portal plasma than in peripheral plasma (6.6 +/- 0.8 vs 2.2 +/- 0.4 nmol/l). In contrast, dopamine was undetectable (less than 1 nmol/l) in the portal and peripheral plasma of all ewes. Plasma levels of DOPAC and DHPG in portal and jugular samples were similar. In all pro-oestrous female rats, plasma concentrations of dopamine were higher in portal blood than in jugular blood (8.0 +/- 1.4 vs 4.8 +/- 0.6 nmol/l). Detectable concentrations of dopamine were measured in the portal plasma of two out of five male rats. Noradrenaline concentrations were higher in portal plasma than in peripheral plasma of both female (8.3 +/- 1.7 vs 3.7 +/- 0.6 nmol/l) and male (14.8 +/- 2.7 vs 6.1 +/- 1.2 nmol/l) rats. These data show that noradrenaline, but not dopamine, is secreted into the long portal vessels in sheep. The results suggest that there are species differences in the secretion of hypothalamic dopamine into hypophysial portal blood.

Quantification of 3-nitrotyrosine in biological tissues and fluids: generating valid results by eliminating artifactual formation.

Reactive nitrogen species such as peroxynitrite can nitrate specific amino acids, whether free or protein bound, and 3-nitrotyrosine is believed to be one marker of this reaction. To examine the significance of this pathway in biological systems we have developed an accurate, sensitive, and specific assay for 3-nitrotyrosine based on combined liquid chromatography tandem mass spectrometry. Our approach allowed simultaneous analysis of both tyrosine and 3-nitrotyrosine and employs isotopomer standards (i.e., [15N1, 13C9]-tyrosine and [13C6]-3nitrotyrosine). Calibration curves were linear (r2 = 0.999) across the range 0.5-100 pg/microL (i.e., 2.2-442 fmol/microL), and the detection limit for standard samples was 0.5 pg/microL (2.2 fmol/microL, or 10 fmol on column; S/N = 5) or 1 pg/microL (4.4 fmol/microL) for extracted (biological) samples. As a component of this study we have undertaken an extensive investigation of artifactual formation of 3-nitrotyrosine under conditions that exist during sample extraction and derivatization. Our studies show that under appropriate conditions (low pH, elevated temperatures, and in the presence of a vast excess of the two substrates, tyrosine and the nitrate anion), 3-nitrotyrosine can readily be formed as an artifact.

Growth hormone and cortisol secretion after oral clonidine in healthy adults.

The purpose of this study was to evaluate oral clonidine for testing growth hormone (GH) responsiveness in healthy adults. Oral clonidine (0.15 mg) produced a satisfactory GH response (greater than 4 ng/ml from basal) in eight out of 10 subjects, which is similar to rates reported after an equivalent intravenous dose. Elevated GH levels at baseline occurred in four out of five female subjects; this did not affect the clonidine-induced GH release. There were no significant differences at any time point in plasma prolactin or cortisol levels following clonidine, compared to placebo controls. Adequate plasma clonidine levels (greater than 0.4 ng/ml) were achieved in all subjects, with corresponding reductions in mean arterial blood pressure, but with only minimal adverse effects. Results from this study indicate that oral clonidine is a reliable method for testing GH responsiveness in adult subjects.