Neurotensin potentiates the endogenous dopamine release from striatal synaptosomes of rat

The effect of neurotensin (NT) on the release of endogenous dopamine (DA) of rat striatal synaptosomes was studied. In the basic medium with Ca++ (5mM K+ and 1.2 mM Ca++), spontaneous release of DA was determined to be 12.03 +/- 1.12 pmol/mg protein, while in the Ca++-free basic medium containing EGTA (2.0 mM), the amount of DA released was still up to 11.2 +/- 1.06 pmol/mg protein. NT in 10(-4)-10(-6) M range tested potentiated both the spontaneous and K+-induced release of DA in Ca++-free medium. In addition, NT in 10(-4) M, but not in lower concentrations tested, potentiated the spontaneous, Ca++-dependent release of DA. It is suggested that the effect of NT on DA release is mediated by the specific NT receptors at the DA axonal terminals. The possibility, however, that NT has some influence on the carrier-mediated process of the membrane might not be ruled out.     

Distribution and subtype determination ofβ-receptors in the spinal cord of the adult rat

1. We determined the number of beta-receptors in the whole spinal cord of the adult rat and in the cervical, thoracal, and lumbal/sacral parts. 2. The undivided spinal cord contains 47 +/- 10 fmol/mg beta-receptors (KD = 2066 +/- 982 pmol/liter), and the cervical part of the spinal cord contains 53 +/- 8 fmol/mg protein (KD = 3224 +/- 1775 pmol/liter). The thoracal part shows 40 +/- 1 fmol/mg protein (KD = 3229 +/- 104 pmol/liter), and the lumbal/sacral spinal cord contains 48 +/- 8 fmol/mg protein (KD = 3610 +/- 1610 pmol/liter). 3. Competitive inhibition studies with l-practolol, dl-atenolol, and ICI 118,551 were performed and we calculated by a computer program in the whole spinal cord the following ratio of beta-receptor subtypes: 80 +/- 5% Beta 1-receptors and 20 +/- 5% beta 2-receptors. 4. The basal and (-)-isoproterenol- and NaF-stimulated activity of adenylate cyclase was highest in the cervical part of the spinal cord and equally distributed between the thoracal and the lumbal/sacral parts. 5. The whole synaptosomal protein of the cervical part of the spinal cord contained 132 +/- 20 fmol, the thoracal part 117 +/- 3 fmol, and the lumbal/sacral part 133 +/- 22 fmol.     

Neurotensin elevates hepatic bile acid secretion in chickens by a mechanism requiring an intact enterohepatic circulation

Neurotensin (NT), given intravenously at 10-50 pmol/kg per min to anesthetized female chickens equipped with a bile duct fistula, dose-dependently elevated hepatic bile flow and bile acid output but only when the enterohepatic circulation was maintained by returning the bile to the intestinal lumen. Infusion of NT at 10 and 50 pmol/kg per min increased the average hepatic bile acid output over a 30-min period to 138 +/- 11 and 188 +/- 13% of control, respectively. During infusion of NT, plasma levels of immunoreactive NT (iNT) increased in time from the basal level (14 +/- 1.3 pM) to reach steady state at 30 min. There was a near linear relationship between the dose of NT infused and the increment in plasma iNT. In addition, infusion of NT at 40 pmol/kg min gave a plasma level of iNT (approximately/= 88 pM) which was within the range of those observed during duodenal perfusion with lipid (54-300 pM) and near to that measured in hepatic portal blood from fed animals (52 +/- 5 pM). Perfusion of duodenum with lipid released endogenous NT and increased the rate of hepatic bile flow. When NT antagonist SR48692 was given, bile flow rate decreased to the basal level. These results suggest that intestinal NT, released by lipid, may participate in the regulation of hepatic bile acid output by a mechanism requiring an intact enterohepatic circulation.     

Effect of neurotensin and neurotensin fragments on gastric acid secretion in man

The effect of intravenous infusion of neurotensin (NT) and NT-fragments on pentagastrin stimulated gastric acid secretion was investigated in healthy subjects. Neurotensin was infused in three doses (72, 144 and 288 pmol/kg per h). An N-terminal fragment (NT 1-8), a C-terminal fragment (NT 8-13) and an NT-analogue, substituted at the C-terminal tyrosine residue (Phe11-NT) were infused in two doses (72 and 144 pmol/kg per h). Concentrations of the infused peptides were measured in peripheral venous blood by radioimmunoassay. Plasma levels of NT 1-13, NT 1-8 and Phe11-NT increased in a dose-dependent manner; NT 1-13 to 50 (34-69), 78 (54-113) and 143 (112-242) pmol/l (medians and range) at 72, 144 and 288 pmol/kg per h, NT 1-8 to 405 (340-465) and 1215 (915-1300) pmol/l, and Phe11-NT to 200 (110-245) and 390 (250-410) pmol/l at 72 and 144 pmol/kg per h, respectively. Increases in plasma levels of NT 8-13 could not be detected during the infusion, suggesting that the fragment is rapidly metabolized in man. Neurotensin 1-13 inhibited gastric acid secretion in a dose-dependent manner and the decrease in gastric acid secretion was linearly related to plasma levels of NT 1-13. Neurotensin 1-8 and NT 8-13 inhibited gastric acid secretion only at 144 pmol/kg per h, while the analogue Phe11-NT had no effect. The results showed that the inhibition of gastric acid secretion produced by NT was dose-dependent and linearly related to circulating levels of NT, and that under physiological conditions this effect presumably is elicited by the C-terminal part of the peptide.     

Liberation of cyclic AMP and catecholamine from the heart during left stellate stimulation in the anesthetized dog

In open-chest pentothal-chloralose anesthetized dogs, plasma catecholamine and cyclic AMP levels were evaluated in the aortic and coronary sinus blood, during stimulations of the left ansa subclavia (1, 2, and 4 Hz). Basal aortic and coronary sinus catecholamine levels were respectively 0.373 +/- 0.090 and 0.259 +/- 0.048 ng/mL and cyclic AMP levels averaged 21.4 +/- 1.4 and 20.9 +/- 1.6 pmol/mL. Statistically significant increases in cyclic AMP levels were induced by sympathetic stimulations at 1 Hz (2.0 +/- 0.6 pmol/mL, 2 Hz (2.5 +/- 1.2 pmol/mL) and 4 Hz (6.5 +/- 1.5 pmol/mL), concomitantly with elevations of coronary sinus catecholamine levels. Sotalol (5 mg/kg) abolished the increases in coronary sinus cyclic AMP levels induced in coronary sinus cyclic AMP output averaged 282 +/- 30 pmol/min (1 Hz), 662 +/- 160 pmol/min (2 Hz), and 1679 +/- 242 pmol/min (4 Hz). Sympathetically induced cyclic AMP output (4Hz) was blunted by sotalol (-81 +/- 14 pmol/min). Aortic cyclic AMP levels were not significantly influenced by stellate stimulation. Intense correlations were found between increased in coronary sinus plasma catecholamines and cyclic AMP concentration levels (r = 0.81, slope - 1.45, ordinate = -1.42, n = 15) as well as between delta cyclic AMP output versus delta catecholamine output values in the coronary sinus (r = 0.93. slope output levels. Intracoronary infusion of phenylephrine (10 micrograms/min) or nitroprusside (200 micrograms/min) had no influence on cyclic AMP plasma levels whereas aortic and coronary sinus levels were respectively increased 5.5 +/- 1.9 and 7.3 +/- 1.4 pmol/mL during the administration of isoproterenol (5 micrograms/min). These data suggested that plasma cyclic AMP constitutes a sensitive index of cardiac beta-adrenergic activity elicited by the release of endogenous catecholamine during stellate stimulations.     

Sex-hormone-binding globulin and albumin concentrations in human cerebrospinal fluid

Polyacrylamide gel electrophoresis of plasma and concentrated cerebrospinal fluid (CSF) preincubated with tritium labelled 5 alpha-dihydrotestosterone (DHT) showed identical migration of the radioactivity, indicating the presence of sex-hormone-binding globulin (SHBG) in human CSF. The concentrations of SHBG (measured as the binding capacity) and albumin were measured in concentrated CSF (12 women and 1 man) and samples of plasma of some patients (9 women). SHBG could not be detected in 6 of the CSF samples, and the mean value of the determinable samples was 42.3 +/- 13.4 pmol/l. The mean +/- SE of the SHBG concentration in plasma was 90.8 +/- 8.9 nmol/l and the mean albumin concentrations in CSF and plasma were 3.4 +/- 0.6 mumol/l and 670 +/- 107 mumol/l respectively. The distribution ratio for SHBG over the blood-CSF barrier was 10 times higher than for albumin. It was concluded that the SHBG-binding in the CSF is negligible but that the albumin-binding may contribute to the CSF concentrations of testosterone and estradiol, which are 10-25% above the plasma unbound concentrations.     

Adenosine transporters in chromaffin cells: subcellular distribution and characterization

Adenosine transporters in freshly isolated and cultured chromaffin cells were quantified by the [3H]dipyridamole binding technique, showing a maximal bound capacity of 0.4 +/- 0.05 pmol/10(6) cells (240,000 +/- 20,000 transporters by cell). Scatchard analysis showed a similar affinity for [3H]dipyridamole in isolated cells and subcellular fractions (Kd = 5 +/- 0.6 nM). For enriched plasma membrane preparations and chromaffin granule membranes, the maximal binding capacities were also very similar, 2.3 +/- 0.3 and 1.8 +/- 0.4 pmol/mg protein, respectively. When [3H]nitrobenzylthioinosine was employed as a radioligand, the maximal bound capacity in cultured chromaffin cells was 0.053 +/- 0.004 pmol/10(6) cells (32,000 +/- 3000 transporters per cell) with a high affinity constant (Kd = 0.25 +/- 0.03 nM); similar values were obtained in all subcellular fractions (Kd = 0.1 +/- 0.01). Also, plasma and chromaffin granule membranes showed similar maximal binding values (0.4 +/- 0.06 pmol/mg protein). Photoincorporation studies with [3H]nitrobenzylthioinosine into plasma membrane polypeptides showed the presence of three molecular species of 115 +/- 10; 58 +/- 6 and 42 +/- 5 kDa. Chromaffin granule membranes showed only the 105 +/- 9 and 51 +/- 4 molecular species.     

Pharmacokinetics of 19-nortestosterone esters in normal men

A reliable method for the isolation of 19-nortestosterone (NT), testosterone (T) and dihydrotestosterone (DHT) by high-performance liquid chromatography (HPLC) and quantitation of the individual steroids by radioimmunoassays is described. The method was used to measure serum concentrations of NT, T and DHT in a pharmacokinetic study and in a clinical trial for male fertility control. Following intramuscular injection of either 50 mg 19-nortestosterone-3-(p-hexoxyphenyl)-propionate (NP) or 50 mg 19-nortestosterone-decanoate (ND) serum NT increased rapidly to maximal concentrations of 4.6 +/- 3.2 and 2.0 +/- 1.3 nmol/l (+/-SD), respectively, in the 6 volunteers. The half-life time was 8 days for ND and 21 days for NP. Based on these findings a clinical trial with NP was performed. NP was given to 5 healthy men in doses of 100 mg/week for the first 3 weeks followed by 200 mg/week for 10 further weeks. Serum NT levels increased gradually and maximal concentrations were reached in the 13th treatment week (20.2 +/- 3.4 nmol/l). Measurable amounts of NT were detectable for 19 weeks after the last injection. The study shows that NT accumulates under this treatment regime and wider spacing of the injection intervals may be possible in future trials.     

Measurement of kynurenic acid in mammalian brain extracts and cerebrospinal fluid by high-performance liquid chromatography with fluorometric and coulometric electrode array detection

Kynurenic acid is a broad-spectrum excitatory amino acid (EAA) receptor antagonist which is present in the mammalian central nervous system. We describe a method for the measurement of kynurenic acid using isocratic reverse-phase high-performance liquid chromatography (HPLC) with fluorometric detection enhanced by Zn2+ as a postcolumn reagent. The method requires no prior sample preparation procedures other than extraction with 0.1 M HClO4. The reliability of the primary fluorometric method was verified by comparing measurements of tissue concentrations of kynurenic acid in human cerebral cortex and putamen using three different methods of separation with fluorometric detection, as well as four methods utilizing HPLC with coulometric electrode array system (CEAS) detection. All seven methods produced comparable results. The concentration of kynurenic acid in human cerebral cortex was 2.07 +/- 0.61 pmol/mg protein, and in human putamen, 3.38 +/- 0.81 pmol/mg protein. Kynurenic acid was also found to be present in human cerebrospinal fluid (CSF) at a concentration of 5.09 +/- 1.04 nM. The regional distribution of kynurenic acid in the rat brain was examined. Kynurenic acid concentrations were highest in brainstem (149.6 fmol/mg protein) and olfactory bulb (103.9 fmol/mg protein) and lowest in thalamus (26.0 fmol/mg protein). There were no significant postmortem changes in kynurenic acid concentrations in cerebral cortex, hippocampus, and striatum at intervals ranging from 0 to 24 h. Perfusion of the cerebral vasculature with normal saline prior to sacrifice did not significantly alter kynurenic acid content in rat hippocampus, cerebral cortex, or striatum. The analytical methods described are the most sensitive (10-30 fmol injection-1) and specific (utilizing both excitation and emissions properties and electrochemical reaction potentials, respectively) methods for determining kynurenic acid in brain tissue extracts and CSF. These methods should prove useful in examining whether kynurenic acid modulates EAA-mediated neurotransmission under physiologic conditions, as well as in determining the role of kynurenic acid in excitotoxic neuronal death.     

Brainstem deficiency of the 14-3-3 regulator of serotonin synthesis: a proteomics analysis in the sudden infant death syndrome

Impaired brainstem responses to homeostatic challenges during sleep may result in the sudden infant death syndrome (SIDS). Previously we reported a deficiency of serotonin (5-HT) and its key biosynthetic enzyme, tryptophan hydroxylase (TPH2), in SIDS infants in the medullary 5-HT system that modulates homeostatic responses during sleep. Yet, the underlying basis of the TPH2 and 5-HT deficiency is unknown. In this study, we tested the hypothesis that proteomics would uncover previously unrecognized abnormal levels of proteins related to TPH2 and 5-HT regulation in SIDS cases compared with controls, which could provide novel insight into the basis of their deficiency. We first performed a discovery proteomic analysis of the gigantocellularis of the medullary 5-HT system in the same data set with deficiencies of TPH2 and 5-HT levels. Analysis in 6 SIDS cases and 4 controls revealed a 42-75% reduction in abundance in 5 of the 6 isoforms identified of the 14-3-3 signal transduction family, which is known to influence TPH2 activity (p < 0.07). These findings were corroborated in an additional SIDS and control sample using an orthogonal MS(E)-based quantitative proteomic strategy. To confirm these proteomics results in a larger data set (38 SIDS, 11 controls), we applied Western blot analysis in the gigantocellularis and found that 4/7 14-3-3 isoforms identified were significantly reduced in SIDS cases (p ≤ 0.02), with a 43% reduction in all 14-3-3 isoforms combined (p < 0.001). Abnormalities in 5-HT and TPH2 levels and 5-HT(1A) receptor binding were associated with the 14-3-3 deficits in the same SIDS cases. These data suggest a potential molecular defect in SIDS related to TPH2 regulation, as 14-3-3 is critical in this process.     

Uptake and Metabolism of Serotonin by Ependymal Primary Cultures

Serotonin uptake and metabolism was studied in ependymal primary cultures. Serotonin uptake was facilitated by two different systems, one of which was the neuronal serotonin transporter SERT, exhibiting a Vmax value of 3.8+/-0.1 pmol x min(-1) x (mg protein)(-1) and an apparent Michaelis-Menten constant of 0.41+/-0.03 microM. The main product of metabolism was 5-hydroxyindole acetic acid, which resulted from the action of monoamine oxidase A. This enzyme showed a maximal rate of 0.85+/-0.02 nmol x min(-1) x (mg protein)(-1) and a Michaelis-Menten constant of 78+/-5 microM. Ependymal cells were able to dispose of extracellular serotonin with initial rates of approximately 600 pmol x min(-1) x (mg protein)(-1) and of 4 pmol x min(-1) x (mg protein)(-1) when challenged with 500 microM and 1 microM extracellular serotonin, respectively. Ependymal cells are concluded to facilitate the "sink" action of the CSF by removing waste compounds upon passing of the fluid from the parenchymal extracellular space into the ventricular system.     

Cyclic nucleotide function in trachealis muscle of dogs with and without airway hyperresponsiveness

We examined basal adenosine 3',5'-cyclic monophosphate (cAMP) levels, isoproterenol (ISO)-stimulated cAMP responses, basal cAMP, and guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase (PDE) activities and protein-kinase (PK) activities in trachealis muscle from five Basenji-greyhound (BG) and four greyhound dogs to determine whether the inverse relationship between in vivo and in vitro airway responsiveness could be due to altered cyclic nucleotide metabolism. Basal cAMP levels were not significantly different (PNS) in muscle from BG (11.6 +/- 0.53 pmol/mg protein) and greyhound dogs (10.30 +/- 1.60 pmol/mg protein). The cAMP responses to stimulation with ISO were enhanced in BG compared with greyhound dogs. The low Michaelis constant (1) for Km-cAMP PDE activity (Km = 0.63 microM) was significantly less (P less than 0.005) in BG dogs (1.54 +/- 0.28 pmol.min-1.mg protein-1) than greyhounds (11.76 +/- 2.48). Endogenously active PK activity was significantly greater (P less than 0.005) in BG (54.74 +/- 5.39 pmol.min-1.mg protein-1) than in greyhound dogs (15.50 +/0 2.20). Increases in PK activity with 5 microM cAMP added were not significantly different between BG (14.79 +/- 6.00) and greyhound dogs (7.04 +/- 2.14). Approximately 90% of both endogenous PK activity and cAMP-activated PK activity in BG and greyhound dogs was inhibited by a cAMP-dependent PK inhibitor (PKI'). These data suggest that decreased cyclic nucleotide degradation due to decreased cyclic nucleotide PDE activity with increased PK could account for the in vitro hyporesponsiveness of airway smooth muscle in BG dogs as a protective adaptive mechanism.     

Presence and Measurement of Imidazoleacetic Acid, a γ-Aminobutyric Acid Agonist, in Rat Brain and Human Cerebrospinal Fluid

Imidazoleacetic acid (IAA) was unequivocally demonstrated in rat brain, human CSF, and human plasma by a gas chromatographic-mass spectrometric method that can reliably quantify as little as 8 pmol, i.e., 1 ng. Owing to tautomerism of the imidazole ring, IAA and [15N, 15N]IAA, the internal standard, each formed two chromatographically distinct isomers after derivatization of the ring nitrogens with either ethyl chloroformate or methyl chloroformate. The isomers of n-butyl(N-ethoxycarbonyl)imidazole acetate and n-butyl(N-methoxycarbonyl)imidazole acetate were identified by analysis with methane chemical ionization and electron impact ionization of molecular and fragment ions. The levels (mean +/- SEM) of free IAA were 140 +/- 14 pmol/g and 2.7 +/- 0.2 pmol/ml in brains of untreated rats and human lumbar CSF, respectively. Mean levels of IAA in brains of anesthetized rats, perfused free of blood, did not differ significantly from mean levels of anesthetized, nonperfused controls or from untreated rats. The source or sources of IAA in brain and CSF are unknown. Because IAA is a potent agonist at gamma-aminobutyrate receptors, it merits examination as a regulator in brain.     

VIP in cerebrospinal fluid of patients with multiple sclerosis

The concentration of VIP was measured radioimmunochemically in cerebrospinal fluid (CSF) from 14 healthy volunteers and from 22 patients with multiple sclerosis. Significantly lower levels of VIP was obtained in the patients (18 +/- 3 pmol/l) than in controls (37 +/- 4 pmol/l). There was no correlation between the level of VIP in CSF and other CSF parameters such as albumin. IgG or cell content; nor between VIP concentration and the physical handicap or neuropsychiatric symptoms. There was a trend towards lower values of VIP in patients with steadily progressing rather than intermittent course of the disease but the difference between the groups was not significant.     

Effects of iron deficiency and exercise on myoglobin in rats

The effects of iron deficiency and endurance training on muscle myoglobin (Mb), body weights, and blood lactic acid concentration were studied in rats. Fifty animals were divided into four groups: anemic trained (AT), normal trained (NT), anemic sedentary (AS), and normal sedentary (NS). Following 5 weeks of dietary control, the mean hemoglobin values for the AT and AS rats were 0.013 +/- 0.002 mmol X l-1 (8.7 +/- 1.4 g X dl-1) and 0.014 +/- 0.003 mmol X l-1 (9.2 +/- 1.7 g X dl-1) respectively, and did not significantly change throughout the study. AT and NT rats were run on a motor driven treadmill 4 days/week for 6 weeks up to a pre-established time of 90 min. Following the training, body weights of the AT (157 +/- 13 g) and NT (153 +/- 13 g) rats were lower than their respective sedentary groups AS (172 +/- 9 g) and NS (176 +/- 15 g). Resting blood lactic acid concentration following training was lower in both trained groups, AT (3.3 +/- 2.0 mM) and NT (2.3 +/- 1.9 mM) compared to AS (8.2 +/- 2.6 mM) and NS (3.8 +/- 1.6 mM). Training increased Mb concentration in hearts of both the anemic and normal trained groups (AT, 0.66 +/- 0.13 mg X g-1; NT, 0.95 +/- 0.08 mg X g-1) compared to the sedentary groups (AS, 0.44 +/- 0.08 mg X g-1; NS, 0.70 +/- 0.13 mg X g-1). Only the AT rats showed an increase in skeletal muscle Mb. This study provides evidence that myoglobin may limit aerobic metabolism.     

PM-induced cardiac oxidative stress and dysfunction are mediated by autonomic stimulation

Epidemiological studies show that increases in particulate air pollution (PM) are associated with increases in cardiopulmonary morbidity and mortality. However, the mechanism(s) underlying the cardiac effects of PM remain unknown. We used pharmacological strategies to determine whether oxidants are implicated in PM-dependent cardiac dysfunction and whether PM-induced increase in autonomic stimulation on the heart mediates cardiac oxidative stress and toxicity. Adult Sprague-Dawley rats were exposed to either intratracheal instillation of urban air particles (UAP 750 microg) or to inhalation of concentrated ambient particles (CAPs mass concentration 700+/-180 microg/m3) for 5 h. Oxidative stress and cardiac function were evaluated 30 min after UAP instillation or immediately after exposure to CAPs. Instillation of UAP led to significant increases in heart oxidants measured as organ chemiluminescence (UAP: 38+/-5 cps/cm2, sham: 10+/-1 cps/cm2) or thiobarbituric acid reactive substances (TBARS, UAP: 76+/-10, Sham 30+/-6 pmol/mg protein). Heart rate increased immediately after exposure (UAP: 390+/-20 bpm, sham: 350+/-10 bpm) and returned to basal levels over the next 30 min. Heart rate variability (SDNN) was unchanged immediately after exposure, but significantly increased during the recovery phase (UAP: 3.4+/-0.2, Sham: 2.4+/-0.3). To determine the role of ROS in the development of cardiac malfunction, rats were treated with 50 mg/kg N-acetylcysteine (NAC) 1 h prior to UAP instillation or CAPs inhalation. NAC prevented changes in heart rate and SDNN in UAP-exposed rats (340+/-8 and 2.9+/-0.3, respectively). To investigate the role of the autonomic nervous system in PM-induced oxidative stress, rats were given 5 mg/kg atenolol (beta-1 receptor antagonist), 0.30 mg/kg glycopyrrolate (muscarinic receptor antagonist) or saline immediately before exposure to CAPs aerosols. Both atenolol and glycopyrrolate effectively prevented CAPs-induced cardiac oxidative stress (CL(ATEN): 11+/-1 cps/cm2, CL(GLYCO): 10+/-1 cps/cm2, TBARS(ATEN): 40+/-6 pmol/mg protein, TBARS(GLYCO): 38+/-6 pmol/mg protein). These data indicate that PM exposure increases cardiac oxidants via autonomic signals and the resulting oxidative stress is associated with significant functional alterations in the heart.     

Relationship of CSF pH, O2, and CO2 responses in metabolic acidosis and alkalosis in humans

The effect of induced metabolic acidosis (48 h of NH4Cl ingestion, BE - 10.6 +/- 1.1) and alkalosis (43 h of NaHCO3- ingestion BE 8.8 +/- 1.6) on arterial and lumber CSF pH, Pco2, and HCO3- and ventilatory responses to CO2 and to hypoxia was assessed in five healthy men. In acidosis lumbar CSF pH rose 0.033 +/- 0.02 (P less than 0.05). In alkalosis CSF pH was unchanged. Ventilatory response lines to CO2 at high O2 were displaced to the left in acidosis (9.0 +/- 1.4 Torr) and to the right in alkalosis (4.5 +/- 1.5 Torr) with no change in slope. The ventilatory response to hypoxia (delta V40) was increased in acidosis (P less than 0.05) and it was decreased in four subjects in alkalosis (P, not significant). We conclude that the altered ventilatory drives of steady-state metabolic imbalance are mediated by peripheral chemoreceptors, and in acidosis the medullary respiratory chemoreceptor drive is decreased.     

Effects of ketoconazole on rat testicular steroidogenic enzymatic activities

Ketoconazole (K) is an antifungal imidazole derivative which has been shown to be a potent inhibitor of testosterone (T) biosynthesis in rodents and humans. To study the effect of K on rat testicular steroidogenesis we measured the activities of five testicular microsomal steroidogenic enzymes in K-treated rats and controls. Thirty male adult rats were given either 2 mg K or water every 12 hours by mouth during 5 days. Mean testicular weight was similar in both groups of animals. The K-treated group had a T serum concentration of 83 +/- 14 ng/dL whereas it was 94 +/- 16 ng/dL in the control group (NS). The K-treated animals had decreased activities of the 3 beta-hydroxysteroid dehydrogenase (830 +/- 48 vs 2,245 +/- 109 pmol/mg protein/min, P less than 0.001), 17-hydroxylase (243 +/- 5 vs 676 +/- 17 pmol/mg protein/min, P less than 0.001), 17-ketosteroid reductase (31 +/- 2 vs 169 +/- 7 pmol/mg protein/min, P less than 0.001), and aromatase enzymes (92 +/- 6 vs 123 +/- 7 pmol/mg protein/min, P less than 0.01). The 17,20-desmolase activity was similar in both groups of animals (210 +/- 4 vs 171 +/- 18 pmol/mg protein/min). We conclude that K given orally to rats inhibits the activity of several testicular steroidogenic enzymes.     

A slab gel electrophoresis technique for measurement of plasma retinol-binding protein, cellular retinol-binding and retinoic-acid-binding proteins in human skin

At least four different proteins that bind retinoids could be present in a vitamin A target tissue like the skin. In order to separate cellular retinoid-binding proteins (CRBP and CRABP) from serum retinol-binding protein (RBP) and albumin, a one-step procedure was devised. The technique is based on slab polyacrylamide gel electrophoresis (PAGE) of the extracted proteins incubated with tritiated retinoids. The procedure was used to study binding proteins in the skin. The results show that epidermal extracts (the epithelial part of the skin) contain no RBP activities whereas dermal extracts (the mesenchymal part of the skin) contain 1.6 +/- 0.81 pmol/mg protein of RBP. This technique further showed higher levels of CRABP in both epidermal (9.05 +/- 1.16 pmol/mg protein) and dermal (1.5 +/- 0.54 pmol/mg protein) extracts than those previously determined by other less specific techniques. On the other hand CRBP levels were found to be lower in the two tissues (epidermis 0.2 +/- 0.1 pmol/mg and dermis 0.12 +/- 0.05 pmol/mg protein). New conditions to measure specifically CRABP with the charcoal/dextran technique could be developed and analyzed by the PAGE technique; a dissociation constant of 13.7 nM was then calculated for epidermal CRABP. This PAGE technique appears to be the most appropriate method for the study of retinoid-binding proteins including RBP in human skin.     

Steroid sulfatase activity in leukocytes: a comparative study in 45,X; 46,Xi(Xq) and carriers of steroid sulfatase deficiency

The enzyme steroid sulfatase (STS) hydrolyses 3-beta-hydroxysteroid sulfates. The female-male STS activity ratio is 1.04-1.7:1 in several cell lines in adults and reaches 2:1 in prepubertal subjects. In fibroblasts, STS values in X-chromosome abnormalities show a partial positive correlation according to the number of X-chromosomes. X-linked ichthyosis (XLI) carriers, with only one copy of the STS gene, present lower STS levels than normal controls. This study analyzes the STS activity in leukocytes of 46,Xi(Xq); 45,X; XLI carriers and normal controls using 7-[3H]-dehydroepiandrosterone sulfate as substrate. X-monosomy (1.07 +/- 0.18 pmol/mg protein/h), Xq isochromosome (1.02 +/- 0.12 pmol/mg protein/h) and normal females (1.03 +/- 0.11 pmol/mg protein/h) had similar STS values (p > 0.05). XLI-carriers and males showed the lowest STS levels (0.34 +/- 0.04 pmol/mg protein/h, p < 0.001 and 0.82 +/- 0.14 pmol/mg protein/h, p < 0.05, respectively). Female-male STS activity ratio in leukocytes was 1.3:1. These data indicate that a complex mechanism regulates the STS expression depending on each type of cell line.