Selenium distribution and metabolic profile in relation to nutritional selenium status in rats.

The disposition of selenium (Se) was investigated in Wistar rats of various Se status after an intravenous injection of 82Se-selenite. Various fractions of plasma, urine, and cytosols from liver and kidney were separated by high performance liquid chromatography (HPLC), coupled with an inductively coupled argon plasma-mass spectrometry (ICP-MS). The technique allowed simultaneous differentiation of the fate of injected and endogenous Se, and if it was influenced by the previous Se burden in the tissues. A broad Se-peak from plasma was resolved in two fractions by assessing the m/z 82/78 ratios. Urinary profiles indicated that the metabolism of Se was dose-dependent; monomethylselenol being the primary metabolite of Se in untreated animals, whereas noticeable amount of trimethylselenonium ion was detected after the injection of 82Se. Liver and kidney cytosols contained complex Se-enriched fractions, a positive identification of which was not done in this study. In most cases, the enrichment of tissue fractions with the stable isotope was altered by the dietary Se levels, the isotope nevertheless was exchanged with the endogenous Se in various macromolecules to a varying degree.

Systemic dispositions of molybdenum and copper after tetrathiomolybdate injection in LEC rats.

Mutant Long-Evans rats with a cinnamon coat-color (LEC rats) have been established as an animal model for Wilson disease, a genetic disorder of copper (Cu) metabolism. Systemic disposition of molybdenum (Mo) and altered distributions of Cu were compared in eight organs between LEC rats and Wistar rats (normal) at different times after a single intraperitoneal injection of tetrathiomolybdate (TTM) for chelation therapy. Excretion through urine and feces was also examined. Hepatic disposition of Mo was dramatically increased in LEC rats, suggesting that the interaction of TTM with Cu results in enhanced uptake of Mo. Concentrations of Mo and Cu decreased in the liver of LEC rats over time, whereas those in the spleen increased. Although the concentration of Mo taken up by the kidney decreased over time after an initial increase in both rats, Cu concentration increased over time. Cu was not redistributed to the brain. Excretion of Mo through urine was decreased and that into feces was increased in LEC rats compared with those in Wistar rats. These results indicate that TTM is taken up by the liver depending on the Cu content, and the Cu and Mo removed from the liver are mostly excreted through feces. Redistribution of Cu was observed in the spleen and kidneys, but not in the brain.

Molybdenum and copper kinetics after tetrathiomolybdate injection in LEC rats: specific role of serum albumin.

Chelation therapy with tetrathiomolybdate (TTM) was applied to Long-Evans rats with a cinnamon coat-color (LEC rats), an animal model for Wilson disease, to remove copper (Cu) accumulated in the liver in a form bound to metallothionein (MT). Changes in molybdenum (Mo) and Cu concentrations and their biological forms in serum of LEC rats determined at different times after a single intraperitoneal injection were compared with those of Wistar (normal) rats. The change in Mo concentration in serum of normal rats was mono-phasic, whereas in LEC rats it was bi-phasic. The phase in normal rats and the first phase in LEC rats appeared to reflect the process of uptake and disappearance of TTM in the livers of Wistar and LEC rats. On the other hand, the second phase in LEC rats paralleled the changes of Cu and appeared to reflect the complex formation (Cu/thiomolybdate complex) between Mo and Cu accumulated in the liver. The complex was specifically bound to albumin as determined by high performance liquid chromatography with inductively coupled plasma-mass spectrometry (HPLC/ICP-MS). The results suggested that the changes in the Mo concentration in serum reflected the amount of Cu in the liver.

Enriched stable isotopes of elements used as tracers: methods of presenting high-performance liquid chromatographic-inductively coupled argon plasma mass spectrometric data.

High-performance liquid chromatography (HPLC) of biological fluids and tissue cytosolic preparations was employed in conjunction with argon-induced inductively coupled plasma mass spectrometry (ICP-MS) to investigate the distribution of stable isotopes as tracers. The common way of presenting the data from the ICP-MS is by plotting the count rates versus the retention time of HPLC fractions. Additional information can be derived, e.g., the composite peaks can be further resolved, and the level of enrichment in various biological components can be expressed by alternative ways of presenting these data. The two additional approaches described here involve presenting the ratios of enriched tracer with a suitable naturally abundant mass number of the same element, and by expressing the extent of enrichment by the tracer isotope in a given fraction to that of the same mass number in the fraction derived from an untreated source. Each method of presentation has different merits and drawbacks. The data therefore may be best presented in more than one way to emphasize the conclusions from a given experiment. Observations are presented after simultaneously injecting stable isotopes of three essential elements, copper, selenium and zinc, into mice. Plasma and liver cytosolic fractions were analysed and data represented in different ways as indicated above.

Detection of selenium-containing biological constituents by high-performance liquid chromatography-plasma source mass spectrometry.

Mass spectrometry with inductively coupled argon plasma excitation (ICP-MS) was used as a multi-element specific detection method for HPLC for the speciation of selenium (Se) in biological samples. Se-containing biological constituents were separated on a size-exclusion column and were detected Se-specifically at m/z 78 and 82 for natural abundance 78Se and 82Se, respectively. Se peaks not identical with known authentic samples were detected. Diets with different Se contents induced changes in the distributions Se-containing constituents more in urine, kidney and liver samples than in plasma and red blood cells samples. The results indicate that HPLC-ICP-MS is a specific and sensitive means for the speciation of Se-containing biological constituents.

Prolactin stimulates [3H]dopamine release from dispersed rat tubero-infundibular dopaminergic neurons and dopamine decreases gonadotropin-releasing hormone release induced by calcium ionophore.

In order to investigate the involvement of prolactin-dopamine and dopamine-gonadotropin interactions in the hypothalamo-pituitary axis of hyperprolactinemia, in vitro studies were performed using primary cultures of dispersed rat hypothalamic heterogeneous cells containing tubero-infundibular dopaminergic neurons or gonadotropin-releasing hormone (GnRH) neurons. We observed that prolactin caused dose-dependent stimulation of [3H]dopamine release after a 16-h incubation. Staurosporin (10 nmol/l), an inhibitor of protein kinase C, significantly reduced the [3H]dopamine release induced by prolactin (1 mg/l). Incubation of tubero-infundibular dopaminergic neurons with prolactin (1 mg/l) had no effect on intracellular cyclic adenosine monophosphate accumulation. Dopamine (1 mumol/l) significantly (p < 0.01) reduced the release of GnRH induced by 50 mumol/l calcium ionophore from dispersed hypothalamic cells from the preoptic area, while prolactin had no effect on GnRH release. These data support the hypothesis that the antigonadotropic effect of prolactin on the hypothalamus is mediated by an inhibitory effect of dopamine on GnRH release.

Atrial and brain natriuretic peptides enhance dopamine accumulation in cultured rat hypothalamic cells including dopaminergic neurons.

Dopamine accumulation in hypothalamic cells by atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was analyzed using newborn rat hypothalamic cells in culture. Both ANP and BNP caused a dose-dependent increase in [3H]-dopamine accumulation in the cells. ANP increased [3H]-dopamine accumulation significantly within 20 min. The effects of ANP and BNP on dopamine accumulation paralleled an increase in intracellular cGMP concentration. (Bu)2-cGMP and sodium nitroprusside, a stimulator of the soluble form of guanylate cyclase, also enhanced [3H]-dopamine accumulation. ANP had no effect on efflux of [3H] radioactivity after [3H]-dopamine uptake. These results suggest that a change in cGMP is one of the intermediate steps in dopamine accumulation in hypothalamic cells by ANP and BNP.

Vasoactive intestinal peptide enhances dopamine accumulation in primary cell culture of rat hypothalamus.

The effect of vasoactive intestinal peptide (VIP) and PHI-27 on dopamine accumulation in cultured rat hypothalamic cells was investigated. VIP enhanced [3H]dopamine accumulation dose dependently. This effect was significant at 10(-8)-10(-5) M VIP with a concomitant increase in intracellular cyclic AMP (cAMP), and reached its plateau level at 10(-6) M VIP. VIP increased [3H]dopamine accumulation significantly within 15 min. PHI-27 and dibutyryl cAMP ((Bu)2-cAMP) also enhanced [3H]dopamine accumulation. These results suggest that VIP enhances dopamine accumulation in hypothalamic cells by increasing intracellular cAMP.

Effect of prolactin on the secretion of hypothalamic GnRH and pituitary gonadotropins.

In order to clarify the mechanism by which excess PRL inhibits gonadotropin release, in vivo and in vitro studies were performed with adult female rats. First, we examined the effect of hyperprolactinemia, produced by implantation of anterior pituitary glands under the kidney capsule, on catecholamine turnover in the medial basal hypothalamus (MBH) and on GnRH concentrations in MBH and hypophyseal portal blood. Rats bearing pituitary transplants exhibited increased turnovers of dopamine (DA) in the MBH, decreased concentrations of GnRH in the MBH and in plasma of hypophyseal portal blood and impaired gonadotropin release from the pituitary gland. Second, we examined the effects of PRL on DA release and of DA on GnRH release from rat hypothalamic cells. We observed that PRL stimulated [3H] DA release, and DA inhibited ionophore-induced GnRH release from dispersed hypothalamic cells. Third, we examined the effect of PRL on estrogen-induced LH release using the in vitro perfusion system. We found that administration of PRL suppressed estrogen-induced LH release by suppressing GnRH release from the hypothalamus. These findings suggest that chronic hyperprolactinemia may increase dopaminergic tone in the MBH that may inhibit GnRH secretion from the MBH and LH release from the pituitary and that these processes may be responsible for disturbances of cyclic hypothalamic pituitary-ovarian activity.

Adenosine 3',5'-cyclic monophosphate enhances dopamine accumulation in rat hypothalamic cell culture containing dopaminergic neurons.

The regulation of dopamine accumulation in cultured rat hypothalamic cells by adenosine 3',5'-cyclic monophosphate (cAMP) was investigated in cultures of newborn rat hypothalamic cells. Both dibutyryl cAMP, (Bu)2-cAMP, and forskolin enhanced [3H]dopamine accumulation in a dose-dependent manner. cAMP also enhanced [3H]dopamine accumulation, but to a lesser degree. Neither n-butyrate nor adenosine alone enhanced [3H]dopamine accumulation. (Bu)2-cAMP had no effect on basal efflux of [3H]radioactivity. The effect of (Bu)2-cAMP appeared on day 5 of culture, reached a maximum on day 6, and then rapidly decreased. These results suggest that dopamine uptake by cultured rat hypothalamic cells is regulated by intracellular cAMP.

Involvement of extracellular calcium and arachidonate in [3H] dopamine release from rat tuberoinfundibular neurons.

The mechanism of [3H]dopamine [( 3H]DA) release was investigated using primary cultures of dispersed cells from the rat tuberoinfundibular region, which contains tyrosine hydroxylase (TH)-like immunoreactive neurons. The calcium ionophore A23187 at 10 nM and above caused a significant and dose-dependent increase in [3H]DA release. In the presence of 50 microM A23187, [3H]DA release was detectable within 30 s and reached a plateau in 15 min. The induction of [3H]DA release by 50 microM A23187 was abolished by lowering the extracellular calcium concentration with 2 mM EDTA. Maitotoxin, another calcium-channel activator, also increased [3H]DA release at a concentration of 50 ng/ml. Exogenous additions of 100 mIU/ml phospholipase A2 and 10 microM arachidonate caused significant release of [3H]DA. Furthermore, A23187 stimulated [3H]arachidonate release from tuberoinfundibular dopaminergic (TIDA) neurons in a dose- and time-dependent manner. These results suggest that extracellular calcium and arachidonate are involved in the process of [3H]DA release from rat TIDA neurons.