Effects of potassium deficiency on potassium, polyamines and amino acids in mouse tissues.

Sexual dimorphism in potassium content was found in plasma, kidney, heart and skeletal muscle of CD1 mice. We observed that feeding mice with a K(+)-deficient diet had an uneven and gender-dependent effect on organ weight and tissue potassium concentrations. Treatment produced a marked decrease in plasma, pancreas and skeletal muscle K(+) levels in both sexes, and a reduction in kidney, liver and heart potassium concentrations in females. Moreover, K(+) deficiency produced a 2-3-fold increase in the concentrations of cationic amino acids, such as arginine and lysine in both heart and skeletal muscle of the two sexes, a slight increase ( approximately 37%) in renal arginine in the male mice. The concentrations of these amino acids in plasma and other tissues in both sexes remained unaltered. Polyamine levels in heart, liver, skeletal muscle and pancreas from male and female mice were not affected by K(+) deficiency. However, in the male kidney potassium deficiency was accompanied by an increase of putrescine and spermidine concentration, and a reduction of putrescine excretion into the urine, even though renal K(+) concentration was not significantly affected and ornithine decarboxylase activity was dramatically decreased. The general lack of correlation between tissue potassium decrease and the increase in organic cations suggests that it is unlikely that the changes observed could be related with an attempt of the tissues to compensate for the reduction in cellular positive charge produced by the fall in K(+) content. The mechanisms by which these changes are produced are discussed, but their physiological implications remain to be determined.

Long-term doxycycline-controlled expression of human tyrosine hydroxylase after direct adenovirus-mediated gene transfer to a rat model of Parkinson's disease.

Developments of technologies for delivery of foreign genes to the central nervous system are opening the field to promising treatments for human neurodegenerative diseases. Gene delivery vectors need to fulfill several criteria of efficacy and safety before being applied to humans. The ability to drive expression of a therapeutic gene in an adequate number of cells, to maintain long-term expression, and to allow exogenous control over the transgene product are essential requirements for clinical application. We describe the use of an adenovirus vector encoding human tyrosine hydroxylase (TH) 1 under the negative control of the tetracycline-sensitive gene regulatory system for direct injection into the dopamine-depleted striatum of a rat model of Parkinson's disease. This vector mediated synthesis of TH in numerous striatal cells and transgene expression was observed in a large proportion of them for at least 17 weeks. Furthermore, doxycyline, a tetracycline analog, allowed efficient and reversible control of transgene expression. Thus, the insertion of a tetracycline-sensitive regulatory cassette into a single adenovirus vector provides a promising system for the development of successful and safe therapies for human neurological diseases. Our results also confirm that future effective gene replacement approaches to Parkinson's disease will have to consider the concomitant transfer of TH and GTP-cyclohydrolase transgenes because the synthesis of the TH cofactor tetrahydrobiopterin may be crucial for restoration of the dopaminergic deficit.

Adenovirus-mediated over-expression of TGFbeta1 in the striatum decreases dopaminergic cell survival in embryonic nigral grafts.

Neurotrophic factors may be valuable for improving the survival and the functional efficacy of fetal nigral grafts to treat Parkinson's disease (PD). However, further characterization of their effects is required. New methods of protein delivery also need to be explored to supply sustained and regulated levels of these molecules. Gene transfer via adenoviral vectors is a promising strategy for this purpose. We show herein the effect of adenovirus-mediated transforming growth factor beta1 (TGFbeta1) gene transfer on fetal nigral grafts in a rat model of PD. Direct injection of AdTGFbeta1 into the dopamine-depleted striatum decreased the survival of the transplanted tyrosine hydroxylase-positive (TH+) neurons and impaired the functional efficacy of grafts. Viral toxicity to the graft was avoided by separating the site of viral infection from the transplant by a distance that allowed TGFbeta1 effect on the graft. This infection protocol may be useful for delivering secreted molecules with neurotrophic effects to dopaminergic grafts.

Postnatal exposure to androgens alters renal ornithine decarboxylase ontogeny and abolishes renal sexual dimorphism in mice.

The mouse kidney presents marked sexual dimorphism, manifested not only in renal size but also in the subcellular structure and enzyme activity. Ornithine decarboxylase (ODC), a key enzyme in the biosynthetic pathway of polyamines, is induced in the kidney by androgens, and its activity is higher in the kidney of male mice. The renal differences between male and female mice are not manifested during the first weeks of life and start to be expressed after weaning, simultaneously with the increase in plasma testosterone concentration. Treatment of newborn mice before postnatal day 21 with a single dose of testosterone propionate (TP, 200 microg/animal) did not increase renal ODC activity or renal size. From day 21 the same treatment elicited significant increases in renal ODC, especially in females where the basal activity of control animals was much lower than in males. The repeated injection of TP during the first 10 days of life (200 microg/animal, days 1, 4, 7 and 10) promoted an early increase in renal ODC activity but abolished the physiological rise observed in male mice at puberty and adulthood. This treatment dramatically reduced the secretion of the sexual hormones, testosterone, estradiol and progesterone, by the gonads, and diminished renal size as well as ODC and beta-glucuronidase activities in male mice. Stanozolol produced effects similar to those of TP, while the nonsteroidal antiandrogen, flutamide, did not apparently affect the normal development of the male or female kidney. The results indicate that: (a) kidney sexual dimorphism is not congenital; (b) neonatal androgens are not required to induce the sexual dimorphism of the mouse kidney; (c) the neonatal kidney is unresponsive to testosterone; (d) the premature and repeated exposure to supraphysiological levels of testosterone may accelerate the ontogeny of renal ODC but can abolish later testosterone secretion and hence alter the sexual characteristics of the male kidney, and (e) the postnatal treatment with androgens does not affect the response of the adult kidney to exogenous androgens. One can conclude that the postnatal manipulation of androgens may accelerate the development of the mechanisms of androgen responsiveness in some tissues but it may alter neural structures, probably the GnRH pulse generator, that control testosterone secretion.

Antiandrogenic effect of RU-486 in the mouse kidney.

RU-486 (mifepristone) is a synthetic steroid with potent antiprogesterone and antiglucocorticoid activity, that is currently used as a contraceptive agent. In the present work we have evaluated the antiandrogenic effect of this compound on mouse kidney, a very well known extragenital model of androgen action by studying the effect of RU-486 on renal parameters that depend on androgens, such as renal ornithine decarboxylase (ODC) activity and kidney hypertrophy, as well as the inhibitory action of mifepristone on the induction of renal ODC and kidney hypertrophy elicited by testosterone treatment in female mice and in castrated male. The results showed that: (1) 48 hr after treatment of male mice with of RU-486 (50 mg/kg, four injections) renal ODC activity decreased from 3.381 +/- 490 nmol CO2/h.g to 605 +/- 163 (SD, n = 5); (2) in female mice or orchidectomized male mice, RU-486 also inhibited the renal ODC induction elicited by exogenous administration of testosterone propionate (TP), the magnitude of the inhibition was dependent on the doses of TP and RU-486 used. While RU-486 at a dose of 25 mg/kg inhibited more than 80% ODC induction produced by treatment with 5 mg/kg TP, the same dose did not significantly affect ODC when the dose of TP was increased up to 100 mg/kg. Higher concentration of RU-486 (200 mg/kg) clearly inhibited the increase in ODC produced by treatment with TP 100 mg/kg; (3) RU-486 was more effective in blocking the anabolic effects produced by stanozolol, a steroidal anabolizing agent, than those produced by testosterone; and (4) RU-486 was less effective than the nonsteroidal antiandrogen flutamide in inhibiting renal ODC activity in male mice. Our results clearly indicate that RU-486 possesses moderate antiandrogenic activity in mouse kidney. The possibility that RU-486 may have similar effects in man should be considered when using this drug.

Hypokalemia decreases testosterone production in male mice by altering luteinizing hormone secretion.

Potassium deficiency produced by feeding mice a low potassium diet caused a marked decrease in plasma and testicular testosterone concentrations and a concomitant fall in the weight of seminal vesicles and in renal ornithine decarboxylase activity. All of these parameters were rapidly restored when potassium supply was normalized. Immunocytochemical analysis of gonadotropes and plasma LH values suggested that the pulsatile liberation of LH by the pituitary was impaired in the potassium-deficient male mice. Because the synthesis of testosterone in the potassium-deficient mice was stimulated by exogenous LH, hCG, or GnRH, one can conclude that alteration of the transcellular potassium gradient could affect the regulation of the hypothalamo-hypophyseal-testicular axis by affecting the pulsatile release of GnRH. Our results showing that the stimulation of LH secretion after castration was similar in control and potassium-deficient male mice suggest that a testicular factor(s) different from testosterone could be implicated in the abnormal regulation of LH secretion in potassium-deficient mice. We conclude that plasma potassium concentration is an important factor in the regulation of gonadotropin secretion and testicular functions.

Neuronal regulation of ornithine decarboxylase induced by androgens in the mouse kidney.

1. Neuronal, but not circulating catecholamines, regulate the induction of ornithine decarboxylase (ODC) by testosterone in the mouse kidney. 2. Central and peripheral catecholamine-depleting agents, such as reserpine or alpha-methyl-p-tyrosine, exerted a more pronounced effect on renal ODC than the selective agents tetrabenazine or guanethidine. 3. Benserazide and haloperidol decreased the induction of renal ODC produced by testosterone in female mice. 4. Renal denervation produced a partial inhibition of renal ODC in male mice and decreased the induction of ODC elicited by testosterone in female mice. 5. These results suggest that both peripheral sympathetic neurons as well as central related factors can modulate the effect of androgens on renal ODC activity.

An evaluation of the role of polyamines in different models of kidney hypertrophy in mice.

The role of ornithine decarboxylase (ODC) and polyamines in kidney hypertrophy is controversial. Since part of this controversy could be related to differences in the model system used by the different authors, we studied the changes in renal ODC and polyamines in six different models of kidney hypertrophy in mice, including compensatory renal hypertrophy produced by unilateral nephrectomy, experimental diabetes, potassium depletion and treatment with hormones such as testosterone, thyroxine and fluorocortisone. Only in the case of renal hypertrophy produced by testosterone administration was there a significant increase in ODC activity and putrescine content in the kidneys. However, the concomitant treatment with difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, as a 2% solution in the drinking water completely abolished the increase of renal ODC, but the kidney weights increased and other androgenic effects, such as the induction of renal beta-glucuronidase, were not affected. Moreover, DFMO-treatment did not prevent the kidney enlargement produced in other types of hypertrophy, even in the cases associated with hyperplasia. The present results support the premise that, at least in mice, the increase in ODC activity and polyamine biosynthesis is not required for kidney growth, and also that in most cases renal enlargement is not accompanied by any increase in the polyamine content.

Involvement of polyamines in the contragestational effect of hyperthermia.

Although hyperthermia produces teratogenic effects in a great variety of animal species, the molecular mechanisms by which hyperthermia exerts its action remain unknown. We have studied the implications of polyamines in contragestational hyperthermia in rats. Our results show that the contragestational action of hyperthermia when applied in consecutive periods during days 8, 9 and 10 of rat pregnancy could be completely prevented by the previous administration of polyamines (putrescine and spermidine, 0.6 mmoles/kg and 0.03 mmoles/kg respectively) in combination with the diamine oxidase inhibitor aminoguanidine or by this inhibitor alone (0.12 mmoles/kg). The administration of polyamines alone partially prevented the fetotoxic effect of hyperthermia but produced a marked mortality (50%) in the pregnant rats. These findings support a major and complex role of polyamines in the mechanisms of hyperthermia-mediated teratogenesis, and suggest that the oxidative catabolism of polyamines could be in part responsible of the deleterious effect produced by hyperthermia in rat pregnancy.

Postnatal development of ornithine decarboxylase and polyamines in the mouse kidney: influence of testosterone.

The role of ornithine decarboxylase (ODC) and polyamines in kidney growth during the postnatal development of mice and the influence of testosterone were investigated. A marked sexual dimorphism in renal size was evident after the 3rd week of life, corresponding with the rise in circulating testosterone and the increase in renal ODC and urinary excretion of putrescine in male mice. Renal putrescine and spermidine gradually decreased during the first 3 weeks of life and did not correlate with ODC activity. Treatments with alpha-difluoromethylornithine, and ODC-specific inhibitor, and the antiandrogen flutamide during weeks 4 and 5 showed that both compounds decreased renal ODC activity but only flutamide impaired kidney growth, suggesting that renal growth in mice is regulated by androgens but is independent of the induction of ODC activity.

Potassium regulates plasma testosterone and renal ornithine decarboxylase in mice.

Potassium deficiency produced different effects in the kidney of male or female mice. While in female, potassium deficiency caused a marked renal hypertrophy with no significant changes in testosterone-regulated enzymes, such as ornithine decarboxylase and beta-glucuronidase, in the male the same treatment provoked a marked fall of these enzymes owing to a dramatic decrease in plasma testosterone. Potassium replenishment restored plasma testosterone and renal enzymatic activities. These results show for the first time, that potassium modulates circulating testosterone and suggest that this cation could exert an important regulatory role in controlling androgen actions.