Transcriptomic analysis of polyamine-related genes and polyamine levels in placenta, yolk sac and fetus during the second half of mouse pregnancy.

In mammals, polyamines are essential for the maintenance of cell growth. Although early studies reported the highest values of mammalian ornithine decarboxylase (ODC) activity, a key enzyme in polyamine biosynthesis, in rodent placenta, the role of this enzyme in the second half of rodent pregnancy is still controversial. In order to get new insights on polyamine metabolism during this period of pregnancy, we studied polyamine levels, ODC expression and activity and transcript profile of different polyamine-related genes in mouse placenta, fetus and yolk sac. Results indicated that ODC activity and protein levels were higher in placenta than in fetus and yolk sac, especially in the labyrinth, although no correlation between ODC activity and polyamine levels were observed. The half-life of placental ODC ( approximately 190 min) was also higher than the fetal one ( approximately 24 min). Messenger RNAs of all biosynthetic and retroconversion enzymes of polyamine metabolism were present in the three gestational compartments analyzed, as well as those of antizymes 1 and 2 and antizyme inhibitor 1. However, no expression of antizyme 3 and antizyme inhibitor 2 was detected. The catabolic enzyme diamine oxidase was expressed only in the maternal part of placenta but not in the fetal part or in the fetus. The expansion of polyamine pools in the fetus was markedly higher than in placenta, in spite of its lower biosynthetic activity. Our results suggest that the elevated polyamine biosynthetic activity of mouse placenta is required to satisfy the high demand of polyamines required by the growing fetus, during the later period of pregnancy.

Influence of dietary arginine on the anabolic effects of androgens.

Feeding mice an arginine-deficient diet decreased plasma concentrations of arginine, citrulline and ornithine in the females and arginine in the males, abolishing the sexual dimorphic pattern of these amino acids found in mice fed the standard diet. In addition, the restriction of dietary arginine produced a marked decrease in body and renal weights as well as in the activity of renal ornithine decarboxylase, decreases that were gender dependent since they were observed exclusively in males. The fact that these changes were not associated with the decrease in the circulating levels of testosterone and that the dietary arginine restriction prevented the body weight gain induced by testosterone treatment of female mice fed the standard diet indicates that dietary arginine is required for the anabolic action of androgens. Moreover, under certain conditions that could compromise the renal synthesis of arginine, as in the compensatory renal hypertrophy that follows unilateral nephrectomy, the myotrophic effect of testosterone was transiently impaired. The results also revealed that arginine deficiency produced an opposite effect in the expression of IGF-I and IGF-binding protein 1 in the liver and kidney. Taken together, our results indicate that dietary arginine may be relevant to the anabolic action of testosterone, and suggest that this effect may be mediated by changes in the insulin-like growth factor system.

Gender-related differences in carnosine, anserine and lysine content of murine skeletal muscle.

The aminoacyl-imidazole dipeptides carnosine (beta-alanyl-L-histidine) and anserine (beta-alanyl-1-methyl-histidine) are present in relatively high concentrations in excitable tissues, such as muscle and nervous tissue. In the present study we describe the existence of a marked sexual dimorphism of carnosine and anserine in skeletal muscles of CD1 mice. In adult animals the concentrations of anserine were higher than those of carnosine in all skeletal muscles studied, and the content of aminoacyl-imidazole dipeptides was remarkably higher in males than in females. Postnatal ontogenic studies and hormonal manipulations indicated that carnosine synthesis was up-regulated by testosterone whereas anserine synthesis increased with age. Regional variations in the concentrations of the dipeptides were observed in both sexes, skeletal muscles from hind legs having higher amounts of carnosine and anserine than those present in fore legs or in the pectoral region. The concentration of L-lysine in skeletal muscles also showed regional variations and a sexual dimorphic pattern with females having higher levels than males in all muscles studied. The results suggest that these differences may be related with the anabolic action of androgens on skeletal muscle.

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.

Regulation of ornithine decarboxylase in B16 mouse melanoma cells: synergistic activation of melanogenesis by alphaMSH and ornithine decarboxylase inhibition.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the biosynthesis of polyamines, a family of cationic compounds required for optimal cell proliferation and differentiation. Within mammalian melanocytes, the expression of genes regulating cell growth and/or differentiation can be controlled by alpha-melanocyte-stimulating hormone (alphaMSH) and other melanogenesis modulating agents. In the B16 mouse melanoma model, alphaMSH stimulates melanogenesis by upmodulation of tyrosinase (tyr) activity, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibits melanin synthesis. Therefore, we analyzed the regulation of ODC by these agents, as related to changes in the melanogenic pathway. Treatment of B16 cells with TPA or alphaMSH rapidly stimulated ODC activity. The effect was stronger for TPA and appeared mainly posttranslational. Irreversible inhibition of ODC with the active site-directed inhibitor alpha-difluoromethylornithine (DFMO) did not block TPA-mediated inhibition of tyr. Conversely, prolonged treatment of B16 cells with DFMO stimulated tyr activity by a posttranslational mechanism, probably requiring polyamine depletion. Combination treatment with alphaMSH and DFMO synergistically activated tyr. Therefore, ODC induction is not involved in the melanogenic response of B16 cells to alphaMSH. Rather, increased intracellular concentrations of polyamines following ODC induction might constitute a feedback mechanism to limit melanogenesis activation by alphaMSH.

Preoperative values of CA 15-3 and CEA as prognostic factors in breast cancer: a multivariate analysis.

The role of circulating tumor markers in providing prognostic information has not been widely studied. In the current study, serum levels of the carbohydrate antigen 15-3 (CA 15-3) and carcinoembryonic antigen (CEA) were determined preoperatively in 364 breast cancer patients with no clinical signs of metastasis. The prognostic relevance of these markers for recurrence (175/364) and death of disease (104/175) was determined by Cox multivariate analysis, including the comparison with classical prognostic factors. High levels of both tumor markers were associated with aneuploid tumors with high S-phase fraction and high ornithine decarboxylase activity. CA 15-3 was highly associated with the number of positive lymph nodes and peritumoral lymphatic or blood vessel invasion. No significant associations were found between CEA or CA 15-3 levels and histologic grade, necrosis and steroid receptor status. In univariate analysis, preoperative values, using optimum cutoff values of CA 15-3 (40 U/ml) and CEA (6 ng/ml), were statistically significant for relapse-free survival and overall survival. In multivariate analysis, only node status, DNA ploidy and ornithine decarboxylase activity were independent predictors for relapse-free survival; the estrogen receptor status was a predictor of overall survival. In node-negative patients, ornithine decarboxylase activity was the only factor selected for relapse-free survival. In node-positive patients, the number of lymph nodes and DNA ploidy were the only variables selected for relapse-free survival or overall survival. Estrogen receptor and ornithine decarboxylase activity were excluded for relapse-free survival, but were significant prognostic factors for overall survival.

Aminoglutethimide, a steroidogenesis inhibitor, abolishes hormonal induction of ornithine decarboxylase in steroidogenic tissues: evidence for its role as cAMP-dependent protein kinase inhibitor.

Aminoglutethimide (AMG), a potent inhibitor of steroidogenesis used in the treatment of breast cancer and some adrenal pathologies, abolished the induction of ornithine decarboxylase (ODC) elicited by peptide hormones and by dibutyryl-cAMP in steroidogenic tissues. This effect seems to be related to an inhibition of cAMP-dependent protein kinase (IC50 = 287 microM) rather than blockade of the steroidogenic pathway. This inhibition may explain some of the effects observed in AMG treatment which cannot be ascribed to its direct effect on the cytochrome P450scc complex or aromatase. Taking into account that ODC, the rate-limiting enzyme in polyamine synthesis, is elevated in many types of cancer and that overexpression of this enzyme is associated with cell transformation, one may speculate that the inhibitory action of AMG on protein kinase A represents a positive colateral effect of this drug in cancer therapy.

Evolución del sector femoropoplíteo tras cirugía proximal / Evolution of infrainguinal disease after inflow procedures

Objetivos: Estudio descriptivo de las características clínicas, arteriográficas y hemodinámicas del sector femoropoplíteo de un grupo de pacientes operados del sector aortoiliaco por patología oclusiva y ver la evolución natural del sector infrainguinal durante 5 años de seguimiento desde la cirugía proximal. Métodos: Se seleccionaron 103 pacientes operados del sector proximal entre 1991-1994. Mediante una revisión retrospectiva, se siguió cada uno de estos pacientes, desde el momento de la cirugía proximal, así como en sus revisiones posteriores durante 5 años. Los 103 pacientes fueron clasificados en dos grupos,: GRUPO A, que supone el 31 por ciento, sólo con patología proximal, y el GRUPO B, el 69 por ciento, con patología simultánea de ambos sectores. Se define un subgrupo R con aquellos pacientes de ambos grupos que precisaron cirugía infrainguinal a lo largo de esos 5 años. En el GRUPO B la edad media era superior, existía mayor prevalencia de diabetes y los pacientes se encontraban en estadios clínicos más avanzados. Tanto en el GRUPO A, como en el GRUPO B, la cirugía proximal predominante fue el bypass aortobifemoral, existiendo una permeabilidad a los 5 años superior en el grupo A. En el GRUPO B, simultáneamente a la cirugía proximal, se realizaron más técnicas quirúrgicas asociadas entre las que se incluyen: la endarterectomía femoral, la ampliación de la anastomosis plastiando la profunda, etc. Resultados: La tasa de reintervenciones infrainguinales posteriores fue del 5 por ciento para el GRUPO A y del 36 por ciento para el GRUPO B. La mayor parte de los pacientes que precisaron reintervenciones posteriores presentaban afectación más extensa de la arteria femoral profunda y de ejes distales. Conclusiones: La cirugía infrainguinal se practicó mayoritariamente en aquellos enfermos con patología oclusiva o estenótica del sector femoropoplíteo en el momento de la cirugía proximal (es decir, grupo B). La afectación de ejes distales y de la arteria femoral profunda son las principales variables relacionadas con la probabilidad de cirugía infrainguinal simultánea y en dos tiempos (AU)

Prognostic value of ornithine decarboxylase and polyamines in human breast cancer: correlation with clinicopathologic parameters.

The polyamines putrescine, spermidine, and spermine and ornithine decarboxylase (ODC), the rate-limiting enzyme in their biosynthetic pathway, play an important role in cell proliferation, differentiation, and transformation. In the present study, we have analyzed polyamine concentrations and ODC activity in samples from benign breast diseases (n = 36), benign breast tissue adjacent to the primary carcinoma (n = 19), and breast carcinoma (n = 104). ODC activity in primary carcinoma was significantly higher (2.42 +/- 0.22 nmol CO2/h g; P < 0.001) than that found in benign breast (0.62 +/- 0.15 nmol CO2/h g) or in breast tissue adjacent to the primary carcinoma (0.52 +/- 0.16 nmol CO2/h g). The total polyamine content of breast cancer tissues was higher than in benign breast diseases (704.3 +/- 38.3 nmol/g wet weight versus 295.8 +/- 27.4 nmol/g wet weight) and correlated well with ODC activity (Pearson, r = 0.42; P < 0.001). ODC activity correlated with histological grade, peritumoral lymphatic or blood vessel invasion, S-phase fraction, and cathepsin D. Total polyamine concentration increased with S-phase fraction, cathepsin D, and aneuploidy. No significant correlation was found between ODC or polyamines and tumor size, lymph node involvement, or steroid receptor status. A major finding in our study was that ODC activity was an independent prognostic factor for recurrence and death. The results indicate that the estimation of ODC activity and polyamines in human breast carcinoma might be useful to determine tumor aggressiveness and suggest that ODC may have a potential value as both a prognostic factor and a chemoprevention target in human breast cancer.

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.

Hypokalemia alters sex hormone and gonadotropin levels: evidence that FSH may be required for luteinization.

Hypokalemia produced different effects on steroid sex hormone concentrations in plasma and ovary in the mouse. Estradiol levels were slightly increased, whereas circulating progesterone was markedly decreased in all estrous periods. The preovulatory surge of gonadotropins and the secondary surge of follicle-stimulating hormone (FSH) at estrus were also decreased, but basal levels of both gonadotropins were unaffected. Supplementation with luteinizing hormone (LH), FSH, or gonadotropin-releasing hormone (GnRH) at proestrus rapidly normalized plasma and ovarian progesterone levels at this stage of the estrous cycle. Plasma progesterone levels at diestrus were restored only by combined treatment, at the periovulatory stage, with LH and FSH or GnRH but not by LH or FSH alone. The results demonstrate a lack of steroidogenic activity in the corpus luteum of the potassium-deficient mice and, furthermore, that FSH plays an important role in luteinization in the hypokalemic mice. We conclude that alteration of the transcellular potassium gradient may affect the regulation of the periovulatory surge of gonadotropins and progesterone secretion, probably by altering the release of GnRH from the hypothalamus. In addition, the results suggest that FSH may play a certain role as a luteotropic hormone in mice.

The thyrotropin-releasing hormone-like peptides pGlu-Phe-Pro amide and pGlu-Glu-Pro amide increase plasma triiodothyronine levels in the mouse; the activity is sensitive to testosterone.

Three naturally occurring peptides, pGlu-Glu-Pro amide, pGlu-Phe-Pro amide and pGlu-Gln-Pro amide, with similar structures to thyrotropin releasing hormone (TRH) have recently been identified but no studies of their in vivo activities have been reported previously. We describe here the ability of pGlu-Phe-Pro amide and pGlu-Glu-Pro amide to influence thyroid status. Subcutaneous administration of these 'TRH-like' peptides in male and female CDI mice led to increased levels of triiodothyronine (T3) and to a lesser extent tetraiodothyronine (T4) in the circulation. pGlu-Phe-Pro amide was more potent than pGlu-Glu-Pro amide; it exhibited a similar potency to pGlu-His-Pro amide (TRH). pGlu-Phe-Pro amide, pGlu-Glu-Pro amide and TRH produced significantly greater effects in the female than in the male. Castration of male mice led to increased activities, with potencies comparable to those seen in the female; in contrast treatment of female mice with testosterone resulted in reduced activities, similar to those observed in the control male. The effects of potassium deprivation on the activities of the TRH-like peptides were also investigated. This diet, which results in decreased testosterone levels in the male, led to increased activities of the TRH-like peptides and TRH, approaching the potencies observed in the female. The results demonstrate that the TRH-like peptides pGlu-Phe-Pro amide and pGlu-Glu-Pro amide which occur naturally in the thyroid gland exhibit biological activity in influencing thyroid status in vivo. The activities are sensitive to testosterone.

Interference of the antihormone RU486 in the determination of testosterone and estradiol by enzyme-immunoassay.

The antihormone RU486 (mifepristone, 11beta-(4-dimethylaminophenyl)-17beta-hydroxy-17alpha-(prop- 1-ynyl)-estra-4,9-dien-3-one) is currently used in many endocrinological studies and in clinical practice as a contraceptive agent. The results presented here indicate that the synthetic steroid RU486 may interfere in determinations of testosterone and estradiol when using some commercial kits. Although the cross reactivity is low (0.30% for testosterone and 0.16% for estradiol), in animal experiments RU486 doses higher than 5 mg/kg may produce false positive results in the estimation of plasma testosterone (specially in castrated male or female mice) or estradiol concentrations, even in samples obtained 48 h after the administration of this antihormone.

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.

Effect of environmental temperature on tissue lead accumulation in mice repeatedly treated with lead acetate.

The effect of environmental temperature on lead accumulation in tissues of mice repeatedly treated with lead acetate (2 mg/kg per day and 5 mg/kg per day) for 3 or 6 weeks was studied. In blood, kidney and liver, the amount of lead accumulated after 3 weeks of treatment was markedly higher in animals exposed to 22 degrees C than those maintained at 35 degrees C. Conversely, when the treatment was extended to 6 weeks, lead concentrations in the liver and kidney were equal or higher respectively, in the mice exposed to 35 degrees C. In the brain, lead concentration was lower than that found in kidney and liver and it was independent of dose and ambient temperature of lead being higher at 35 degrees C than at 22 degrees C. These results demonstrate that environmental temperature influences the amount of lead accumulated in some rodent tissues, and that the duration of the treatment modifies the effect produced by temperature, suggesting that the changes elicited during the period of acclimation to the hot environment could be responsible for these findings.

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.

Diazepam potentiates the positive inotropic effect of isoprenaline in rat ventricle strips: role of cyclic AMP.

The responses of the electrically driven right ventricle strip of the rat heart to isoprenaline and other cyclic AMP-related inotropic agents were recorded in the absence and in the presence of diazepam. Isoprenaline, in concentrations ranging from 10 nM to 1 microM, significantly increased, in a concentration-dependent manner, the contractile force in this preparation. Diazepam (10 microM) produced a leftward shift in the isoprenaline concentration-response curve and significantly reduced its EC50. Higher concentrations of diazepam (100 microM) produced no further shift, but reduced the maximum of the concentration-response curve of isoprenaline. Forskolin (0.5-10 microM), which directly stimulates adenyl cyclase, also produced a concentration-dependent increase in cardiac contractility. Diazepam (10 microM) displaced to the left the concentration-response curve for forskolin and reduced its EC50. The cyclic AMP analogous dibutyryl cyclic AMP (0.1-1 mM) produced concentration-dependent positive inotropic effects which were not significantly modified in the presence of diazepam (10 microM). Diazepam (10 microM) significantly enhanced the cyclic AMP production induced by isoprenaline (0.1 microM) and forskolin (10 microM) by about 136% and 35% respectively. These results indicate that diazepam potentiates the positive inotropic effect induced by beta-adrenoceptor agonists, probably by increasing cyclic AMP production induced by these agents.

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.