The uptake of calcium by isolated chromaffin granules of the adrenal medulla

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) catalyses the interconversion of biologically active cortisol to inactive cortisone in man, and corticosterone to 11-dehydrocorticosterone in rodents. As such, this enzyme has been shown to confer aldosterone-selectivity on the mineralocorticoid receptor and to modulate cortisol/corticosterone access to the glucocorticoid receptor (GR). Two kinetically distinct isoforms of this enzyme have been characterized in both rodents and man; a low-affinity NADP(H)-dependent enzyme (11 beta-HSD1) which predominantly acts as an oxoreductase and, more recently, a high-affinity NAD-dependent uni-directional dehydrogenase (11 beta-HSD2). In this study we have analysed the expression of both 11 beta-HSD1 and 11 beta-HSD2 isoforms in rat adrenal cortex and medulla and have investigated their possible roles with respect to glucocorticoid-regulated enzymes mediating catecholamine biosynthesis in adrenal medullary chromaffin cells. Using a rat 11 beta-HSD1 probe and a recently cloned in-house mouse 11 beta-HSD2 cDNA probe, Northern blot analyses revealed expression of mRNA species encoding both 11 beta-HSD1 (1.4 kb) and 11 beta-HSD2 (1.9 kb) in the whole adrenal. Consistent with this, 11 beta-dehydrogenase activity (pmol 11-dehydrocorticosterone formed/mg protein per h, mean +/- S.E.M.) in adrenal homogenates, when incubated with 50 nM corticosterone in the presence of 200 microM NAD, was 97.0 +/- 9.0 and with 500 nM corticosterone in the presence of 200 microM NADP, was 98.0 +/- 1.4. 11-Oxoreductase activity (pmol corticosterone formed/mg protein per h) with 500 nM 11-dehydrocorticosterone in the presence of 200 microM NADPH, was 187.7 +/- 31.2. In situ hybridization studies of rat adrenal cortex and medulla using 35 S-labelled antisense 11 beta-HSD1 cRNA probe revealed specific localization of 11 beta-HSD1 mRNA expression predominantly to cells at the corticomedullary junction, most likely within the inner cortex. In contrast, 11 beta-HSD2 mRNA was more abundant in cortex versus medulla, and was more uniformly distributed over the adrenal gland. Negligible staining was detected using control sense probes. Ingestion of the 11 beta-HSD inhibitor, glycyrrhizic acid (> 100 mg/kg body weight per day for 4 days) resulted in significant inhibition of adrenal NADP-dependent (98.0 +/- 1.4 vs 42.5 +/- 0.4) and NAD-dependent (97.0 +/- 9.0 vs 73.2 +/- 6.7) 11 beta-dehydrogenase activity and 11-oxoreductase activity (187.7 +/- 31.2 vs 67.7 +/- 15.3). However, while levels of 11 beta-HSD1 mRNA were similarly reduced (0.85 +/- 0.07 vs 0.50 +/- 0.05 arbitrary units), those for 11 beta-HSD2 remained unchanged (0.44 +/- 0.03 vs 0.38 +/- 0.01). Levels of mRNA encoding the glucocorticoid-dependent enzyme phenylethanolamine N-methyltransferase which catalyses the conversion of noradrenaline to adrenaline, were also significantly reduced in those rats given glycyrrhizic acid (1.12 +/- 0.04 vs 0.78 +/- 0.04), while those for the glucocorticoid-independent enzyme tyrosine hydroxylase (1.9 kb), which catalyses the conversion of tyrosine to DOPA, were unchanged (0.64 +/- 0.04 vs 0.61 +/- 0.04). In conclusion, the rat adrenal gland expresses both 11 beta-HSD1 and 11 beta-HSD2 isoforms. 11 beta-HSD1 gene expression is localized to the adrenal cortico-medullary junction, where it is ideally placed to regulate the supply of cortex-derived corticosterone to the medullary chromaffin cells. This, together with our in vivo studies, suggests that 11 beta-HSD1 may play an important role with respect to adrenocorticosteroid regulation of adrenaline biosynthesis. The role of 11 beta-HSD2 in the adrenal remains to be elucidated.     

The effect of thrombin on the uptake and transformation of arachidonic acid by human platelets

Washed human platelets take up arachidonic acid from plasma and incorporate the fatty acid into the major classes of complex lipids. Thrombin impairs net incorporation. It activates endogenous phospholipases which liberate arachidonic acid from phospholipids. As a consequence of thrombin induced aggregation platelets release arachidonic acid intermediates formed by the action of platelet fatty acid cyclooxygenase and by platelet fatty acid lipoxygenase. Cyclooxygenase, but not lipoxygenase, is inhibited by aspirin and indomethicin. Analysis of the pathways of arachidonic acid metabolism may furnish new insight into platelet function and into disorders of primary hemostasis.     

Selective uptake of intact parathyroid hormone by the liver: differences between hepatic and renal uptake

Hepatic and renal extraction of immunoreactive parathyroid hormone (i-PTH) was studied in awake dogs with explanted kidneys and chronic indwelling hepatic vein catheters. After a single injection of bovine PTH 1-84 (b-PTH 1-84), hepatic arteriovenous (A-V) differences for immunoreactive PTH (i-PTH) was 39% at 2 min after injection but decreased to 0% by 25 min, despite high levels of i-PTH in the arterial circulation. Gel filtration of arterila and hepatic venous samples obtained when hepatic A-V differences for i-PTH were demonstrable revealed hepatic uptake of the intact hormone and addition of a smaller COOH-terminal fragment, eluting just after the intact hormone, to the hepatic venous blood. Gel filtration of samples obtained 20-30 min after injection of b-PTH was demonstrable) revealed no detectable intact hormone in the circulation. Levels of COOH-terminal fragments of the hormone at the time were identical in arterial and hepatic venous samples. In additional experiemtns no hepatic A-V difference was observed after the injection of the synthetic bovine PTH 1-34 (syn b-PTH 1-34). By comparison there was a demonstrable A-V difference of 20% across the kidney for both intact PTH and COOH-terminal fragments that persisted until i-PTH disappeared from the circulation. The kidney also demonstrated an A-V difference of 22% after injection of syn b-PTH 1-34. These studies demonstrate selective extraction of intact PTH but not of its fragments by the liver. The kidney, on the other hand, extracted the intact hormone and both COOH and NH2 terminal fragments. The studies demonstrate that the kidney was the only organ of those examined that detectably removed the fragments of PTH from the circulation.     

Encoding of three-dimensional structure-from-motion by primate area MT neurons

We see the world as three-dimensional, but because the retinal image is flat, we must derive the third dimension, depth, from two-dimensional cues. Image movement provides one of the most potent cues for depth. For example, the shadow of a contorted wire appears flat when the wire is stationary, but rotating the wire causes motion in the shadow, which suddenly appears three-dimensional. The neural mechanism of this effect, known as 'structure-from-motion', has not been discovered. Here we study cortical area MT, a primate region that is involved in visual motion perception. Two rhesus monkeys were trained to fixate their gaze while viewing two-dimensional projections of transparent, revolving cylinders. These stimuli appear to be three-dimensional, but the surface order perceived (front as opposed to back) tends to reverse spontaneously. These reversals occur because the stimulus does not specify which surface is in front or at the back. Monkeys reported which surface order they perceived after viewing the stimulus. In many of the neurons tested, there was a reproducible change in activity that coincided with reversals of the perceived surface order, even though the stimulus remained identical. This suggests that area MT has a basic role in structure-from-motion perception.     

Selective representation of relevant information by neurons in the primate prefrontal cortex

The severe limitation of the capacity of working memory, the ability to store temporarily and manipulate information, necessitates mechanisms that restrict access to it. Here we report tests to discover whether the activity of neurons in the prefrontal (PF) cortex, the putative neural correlate of working memory, might reflect these mechanisms and preferentially represent behaviourally relevant information. Monkeys performed a 'delayed-matching-to-sample' task with an array of three objects. Only one of the objects in the array was relevant for task performance and the monkeys needed to find that object (the target) and remember its location. For many PF neurons, activity to physically identical arrays varied with the target location; the location of the non-target objects had little or no influence on activity. Information about the target location was present in activity as early as 140ms after array onset. Also, information about which object was the target was reflected in the sustained activity of many PF neurons. These results suggest that the prefrontal cortex is involved in selecting and maintaining behaviourally relevant information.     

Assessment of glucosylifosfamide mustard biodistribution in rats with prostate adenocarcinomas by means of in vivo 31P NMR and in vitro uptake experiments

A combined in vitro/in vivo study was performed to evaluate the possible application of phosphorus (31P) NMR spectroscopy for therapy monitoring and to investigate glucosylifosfamide mustard (Glc-IPM) transport and biodistribution by radiotracer techniques. Dynamic in vivo 31P NMR measurements were performed in rats with prostate adenocarcinoma after i.v. injection of 1 mmol/kg body weight (bw) of ifosfamide (IFO) (n = 4) and 1 mmol/kg bw (n = 4) or 2.15 mmol/kg bw (n = 9) of Glc-IPM. In a biodistribution study with 14C-labeled Glc-IPM and a final dose of 0.8 mmol Glc-IPM/kg bw, the animals were killed 5, 30, 60, and 120 min after drug administration, an ethanol extraction was performed from several tissues, and the dose per g tissue was calculated. The same tumor cell line was used in saturation and competition experiments to further elucidate the transport mechanism. The 31P NMR signals of IFO and Glc-IPM showed no overlap with the endogenous phosphorus peaks. A rapid washout with a half-life between 25.9 +/- 5.6 min for the lower dose and 34.3 +/- 4.2 min for the higher dose of Glc-IPM was observed in the tumor. No statistically significant change of the pH value was observed during the examination period. The beta-nucleoside 5'-triphosphate (NTP)/inorganic phosphate (Pi) signal intensity ratio showed a tendency to decrease but without statistical significance. A rapid elimination was demonstrated by both the noninvasive NMR technique and the biodistribution study. No saturation was found in vitro for the Glc-IPM uptake, even at the concentration of 5 mM. Furthermore, the Glc-IPM uptake was not inhibited by the presence of 2-deoxyglucose and vice versa. The data show that the pharmacokinetics of Glc-IPM in the tumor can be followed in vivo by 31P NMR. The results presented are evidence for diffusion as the transport mechanism for Glc-IPM in this tumor model. However, the better visualization of Glc-IPM as compared to ifosfamide may be due to metabolic trapping of a negatively charged metabolite after deglycosylation.     

Transport of intact porphyrin by HpuAB, the hemoglobin-haptoglobin utilization system of Neisseria meningitidis

The meningococcal hemA gene was cloned and used to construct a porphyrin biosynthesis mutant. An analysis of the hemA mutant indicated that meningococci can transport intact porphyrin from heme (Hm), hemoglobin (Hb), and Hb-haptoglobin (Hp). By constructing a HemA- HpuAB- double mutant, we demonstrated that HpuAB is required for the transport of porphyrin from Hb and Hb-Hp.     

The influence of prostate volume on the ratio of free to total prostate specific antigen in serum of patients with prostate carcinoma and benign prostate hyperplasia

BACKGROUND: Determining the ratio of free to total prostate specific antigen (f-PSA to t-PSA, calculated as the percentage of f-PSA [f-PSA%]) in serum allow for a clearer distinction between patients with prostate carcinoma (PCa) and patients with benign prostate hyperplasia (BPH) than determining the level of t-PSA alone. To find influencing factors on f-PSA%, the authors investigated prostate volume, TNM classification, and tumor stage. METHODS: The authors measured f-PSA and t-PSA in 36 men with untreated PCa (tumor classification: T1, 2, 3pNO, MO), 44 patients with BPH, and 54 healthy controls. Prostate volume was determined by transrectal ultrasound. RESULTS: The median values of t-PSA and f-PSA% were 7.8 micrograms/L and 10.5% in PCa patients, 4.3 micrograms/L and 20.8% in patients with BPH, and 1.4 micrograms/L and 23.6% in the control group. Patients with PCa had a significantly lower proportion of f-PSA than BPH patients and healthy men. There was no correlation of f-PSA% to TNM stage or tumor grade. In PCa patients a significant positive correlation (correlation coefficient [r] = 0.51, P < 0.001) was found between f-PSA% and prostate volume, whereas there was no significant correlation in BPH patients (r = -0.27, P > 0.05). There was a significant difference in f-PSA% between PCa and BPH patients with prostate volumes smaller than 40 cm3 (9.0% vs. 21.6%, P < 0.01) but not between patients in these 2 groups with prostate volumes exceeding 40 cm3 (15.1% vs. 18.2%, P = 0.11). CONCLUSIONS: Determining the ratio of f-PSA to t-PSA to discriminate between PCa and BPH patients yields significant results only in men with a prostate volume of less than 40 cm3.     

Differential uptake of estramustine phosphate metabolites and its correlation with the levels of estramustine binding protein in prostate tumor tissue

Estracyt (EMP) has been used for the treatment of hormone refractory prostate cancer for many years. Recently, new data from combination studies have given rise to new interest in this old drug. Explanations for the synergy found in the clinic are many, but one major factor may be the previous indication that the drug accumulates in the prostate tumor. We have, therefore, examined the level of the four metabolites, estromustine (EoM), estramustine (EaM), estrone, and estradiol in the tumor and serum of 14 patients with T2 and T3 prostate cancer receiving a single i.v. dose of 600 mg of EMP, about 12 h before radical prostatectomy. Because it has been suggested that the uptake into the prostate tumor is due to binding to the estramustine binding protein (EMBP), we have in addition measured the level of EMBP in the prostate tumor tissue. The main serum and tissue metabolite in all patients was EoM followed by EaM, estrone, and estradiol. The levels for EoM ranged from 63.8-162.8 ng/ml in the serum and from 64.8-1209 ng/ml in the prostate tumor, resulting in a mean ratio for serum to tumor of 1:5. The levels for EaM ranged from 8.3-51.4 ng/ml in the serum and 73.9-563.4 ng/ml in the tumor, giving a mean ratio for serum to tumor of 1:13. The levels of EMBP were higher in T3 tumors than in T2 tumors, 54.1 and 40.7 ng/g tissue, respectively. A significant correlation was found between the levels of EaM (r = 0.60) and the levels of EMBP in the tumor. These data demonstrate that 12 h after a single i.v. dose of 600 mg of EMP the levels of the cytotoxic metabolites EoM and EaM are substantially higher in the tumor than in the serum of the same patient and that a correlation exists between the levels of EaM in the tumor and the levels of EMBP. Thus, this supports the hypothesis that the EMBP is responsible for the retention of EoM and EaM in the prostate tumor.     

Ossification within the primate larynx

Ossification of the laryngeal cartilages is a random occurrence possibly related to age and sex. A wide variety of primate larynges have been examined radiologically, by macroscopic section or after whole organ serial section with the aim of determining presence of ossification. The results of this investigation will be presented in an attempt to establish possible correlation between structure, function and degree of ossification within each specific larynx. This is a unique enquiry which may assist in the understanding of ossification within the human larynx.     

The effect of thalamic lesions on primate taste preference

Taste preferences and aversions were examined before and after the placement of bilateral, electrophysiologically guided electrolytic lesions of the thalamic taste area in five Old World monkeys. The only consistent changes shown by all lesioned monkeys on the 30-min, single-bottle intake test were a significantly attenuated consumption of 0.003 M quinine hydrochloride and a significantly elevated intake of 0.1 M sodium chloride. These specific taste deficits are contrary to the global hypogeusia predicted on the basis of previous research. A revised definition of the boundaries of the gustatory thalamus is suggested and the merits of the thalamic monkey preparation are compared with those of the decerebrate rat model.     

The prostate anesthetic block for outpatient prostate surgery

Four patients with systemic autoimmune disorders, 3 of a serious nature, presented to 1 cardiologist over a 20-month span. In 3 of these cases, an HMG-CoA reductase inhibitor was presumably etiologic, while in the fourth case, the HMG-CoA reductase inhibitor might have unmasked the disorder. It would be useful to determine the true frequency of this complication, particularly in older patients not included in most of the statin trials to date. It is well established that autoimmune phenomena and particularly the development of autoantibodies increase with age. The data presented in this report that the group of HMG-CoA reductase inhibitors could be a heretofore poorly recognized etiologic agent. This issue might be addressed by a case-control study looking at the prevalence of statin use in elderly patients with systemic autoimmune disorders and in controls. Until then, the authors advise caution in the use of this class of medications in patient subgroups for whom no clear-cut clinical benefit has yet been proven.     

Energy-dependent uptake of calcium by the yeast Schizosaccharomyces pombe

1. In resting cells of the fission yeast Schizosaccharomyces pombe, the uptake of calcium is stimulated by the addition of 90 mM glucose in the presence as in the absence of respiration and inhibited by Antimycin A in the absence of exogenous carbon source. This uptake therefore requires fermentative or respiratory metabolic energy. 2. The calcium uptake by S. pombe exhibits saturation kinetics and high affinity for calcium. At external pH 4.5, the apparent Km is 45 muM ca2+ 400 muM of other divalent cations exert competitive inhibitions of calcium uptake in the following order of affinities: Sr2+ greater than Mn2+ greater than Co2+ greater than Mg2+. Inhibition by KCl is also observed but is of non-competitive type and requires high concentrations of the order of 40 mM. 3. At 30 degrees C, the uptake rate of calcium is about 10-times higher at pH 8925 than at pH 4.0. An extrusion of 45Ca2+, the rate of which is estimated to be lower than one-fifth of the uptake, is observed in the presence of glucose when the external pH is acid. 4. At external pH 4.5, low concentrations of lanthanum chloride, ruthenium red and hexamine cobaltichloride are inhibitory for the uptake of calcium by the yeast cells. 5. In presence of Antimycin A, the uncouplers: NaN3, dinitrophenol, and concentrations of crobonylcyanide m-chlorophenylhydrazone higher than 80 muM inhibit the calcium uptake by glycolysing cells. In the presence of glucose, the K+ ionophore Dio-9 dnhances severalfold the uptake of calcium even at 2 degrees C. 6. It is concluded that S. pombe possess an active transport system for low concentrations of calcium. This transport seems to be dependent on an electric potential (negative inside) across the cellular membrane.     

Microsphere uptake by the intestine of White Leghorn chickens

A study was conducted to determine the effective size for latex microsphere uptake in the intestine of white leghorn chickens. Three trials were conducted in which ligated intestinal segments of anesthetized 8-wk-old chickens were injected with 0.2-, 0.5-, 2-, 6-, 10-, or 20-mu diameter fluoresceinated latex microspheres. Microspheres were counted in brush border, epithelium, and lamina propria of each intestinal segment, liver, and spleen. After 1 hr, the 0.2-, 0.5-, and 2-mu microspheres were oriented along the brush border of epithelial cells and microsphere uptake into the epithelium and lamina propria was observed in the duodenum, ileum, cecum, cecal tonsil, and colon. Uptake of microspheres of 6, 10, and 20 mu diameter into epithelium and lamina propria was not observed in any intestinal segment. Also, no microspheres of any diameter were observed in sections of liver and spleen to suggest that there was no appreciable entry of microspheres into the bloodstream within 1 hr after administration. The results indicated that uptake of microspheres by the chicken intestine is a size-dependent process with microspheres < or = 2 mu being taken up to an equal extent by most segments of intestine.     

Biphasic L-arginine uptake by the isolated guinea-pig heart

L-Arginine is the physiological substrate for the formation of nitric oxide (NO) and accounts for the biological activity of endothelium-derived relaxing factor. We have studied L-arginine transport in the heart using a rapid dual-isotope dilution technique. The time course of L-[3H]arginine uptake (extraction) by the isolated perfused guinea-pig heart was found to occur in two phases. The first phase reached a plateau in 6.6 +/- 0.6 s and lasted 8.8 +/- 0.7 s, whereas the second phase developed a plateau after 16.3 +/- 0.8 s. The first phase of maximal uptake (Umax,1) accounted for 13.4 +/- 1.4% of the total uptake and the second (Umax,2) for 32.3 +/- 1.8%. The two phases of uptake were inhibited by unlabelled L-arginine in a dose-dependent manner, which suggests that both phases are carrier mediated. The degree of inhibition of Umax,1 and Umax,2 by unlabelled L-arginine was not significantly different. Studies of the kinetics of uptake of these processes revealed an apparent Km,1 of 183 +/- 10 microM with a Vmax,1 of 50 +/- 10 nmol min-1 g-1 for the first phase and Km,2 of 167 +/- 14 microM with a Vmax,2 of 93 +/- 13 nmol min-1 g-1 for the second phase of uptake. These results suggest a similar affinity for the receptors of both transport systems, but with different values for Vmax (P < 0.05). In contrast, 1 mM unlabelled D-arginine had no effect on either the first or second phase of uptake of L-[3H]arginine by the heart, which suggests that these processes are stereospecific. In the presence of the L-stereoisomer of nitro-arginine-mono-methyl ester (L-NAME), a potent inhibitor of NO synthesis, the Umax,1 was inhibited by about 60% while Umax,2 was inhibited by only 20%, which suggests that there is a difference in the effect of L-NAME on the two phases of L-arginine uptake. The first phase most probably represents uptake into the capillary wall, i.e. endothelium and smooth muscle, while the second phase represents entry into the extra-endothelial compartment, i.e. the cardiac myocytes and fibroblasts.     

PET 2-fluoro-2-deoxyglucose uptake in rat prostate adenocarcinoma during chemotherapy with gemcitabine

This study was performed to investigate the effect of the new chemotherapeutic agent gemcitabine on glucose transport and metabolism in prostate carcinoma in vitro and in vivo. METHODS: After transplantation of rat prostate adenocarcinoma cells, dynamic PET measurements with fluorine-18-labeled 2-fluoro-2-deoxy-D-glucose (18FDG) were performed in 15 animals before and 1 day after therapy with 90 mg/kg of body weight (n = 8) and 180 mg/kg of body weight (n = 7) gemcitabine. In the second examination, the animals received a simultaneous injection of 18FDG and [3H]thymidine. Quantitative evaluation of the PET data was done using the standardized uptake value (SUV) as well as a three-compartment pharmacokinetic model. Furthermore, the incorporation of [3H]thymidine into the DNA was determined. In vitro measurements of the FDG, 3-O-methylglucose and thymidine uptake were performed immediately and 4 hr after a 24-hr incubation period with different doses of gemcitabine. RESULTS: FDG-SUV and the metabolic rate of FD 3 utilization did not change significantly after therapy. However, the values for the transport rate constants K1 and K2 increased significantly. The incorporation of thymidine into the DNA of treated tumors showed an 80% decline as compared with a control group. In the cell culture experiments, a dose-dependent increase of FDG (up to 178%) and 3-O-methylglucose uptake (up to 305%) was demonstrated. The thymidine uptake showed a 96% decline in the nucleic acid fraction and an increase of up to 337% in the cytoplasmic fraction. CONCLUSION: The more global measures of FDG metabolism as SUV and metabolic rate of FDG utilization were unchanged after therapy, while DNA synthesis and cell viability declined. However, in vitro and in vivo evidence of an enhancement of glucose transport is presented, indicating that quantification by modelling may be superior for the evaluation of metabolic effects during chemotherapy.