Prolactin selectively stimulates ornithine decarboxylase in the lateral lobe of the rat prostate

In androgenized-hypophysectomized rats, ovine prolactin stimulated the activity of the ornithine decarboxylase (ODC) of the lateral lobes, but not the ventral and dorsal lobes of the prostate glands in a time- and dose-dependent fashion. High degrees of enzyme stimulation were associated with significant elevations in the endogenous levels of its product, putrescine. The relative response to prolactin over basal activities was relatively unaffected by indomethacin but decreased with cycloheximide, suggesting that prostaglandins do not mediate the effects of the hormone, but that a high rate of protein synthesis is a prerequisite for its expression. Indomethacin alone significantly increased the basal activity of the enzyme above control levels, suggesting that prostaglandins may normally exert a degree of inhibition on the ODC. The selective activation of the lateral lobe ODC supports previous reports of a differential response of the various prostatic lobes to prolactin, and also provides a convenient biochemical response for examining details of prolactin action on this organ.     

Effect of a tyrosine kinase inhibitor, genistein, on the actions of prolactin in cultured mouse mammary tissues.

Genistein, an inhibitor of tyrosine kinase, was used to determine the possible role of tyrosine kinase in the prolactin (PRL) stimulation of milk product formation and ornithine decarboxylase (ODC) activation in cultured mouse mammary gland tissue. Genistein (10-200 microM) inhibited in a dose-response fashion the PRL stimulation of casein, lipid and lactose synthesis as well as ODC activation. Genistein, however, did not inhibit the phospholipase C, phorbol myristate acetate or cAMP effects on ODC activation. These results suggest the possible involvement of tyrosine kinase in the mechanism by which PRL expresses its effects in mammary gland tissues.     

Studies of hypertension-induced vascular hypertrophy in cultured smooth muscle cells from spontaneously hypertensive rats

Mechanisms of vascular hypertrophy induced by hypertension were studied in cultured aortic smooth muscle cells from spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) and compared with those from normotensive Wistar-Kyoto (WKY) rats. Fetal calf serum-stimulated ornithine decarboxylase (ODC) activity of cultured smooth muscle cells was greater in SHR and SHRSP than in WKY. Beta- but not alpha-adrenergic agonist stimulated ODC activity acutely in cultured smooth muscle cells from WKY, and isoprenaline-induced activation was blocked by the beta-blocker, propranolol, and enhanced by the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine. These results indicate that cultured vascular smooth muscle cells from SHR and SHRSP are more prone to increase the protein synthesis than those from WKY through the trophic induction of ODC activity and that the regulation of ODC activity by catecholamines is mediated through beta-agonistic effect in cultured smooth muscle cells.     

Induction of a protein inhibitor to ornithine decarboxylase by the end products of its reaction.

Putrescine, the end-product of ornithine decarboxylase (ODC: L-ornithine carboxylyase, EC; 4.1.1.17) action, induces the synthesis of a protein(s), in L1210, neuroblastoma, and H-35 cells as well as in rat liver, which inhibits ODC activity. Spermidine and spermine, distal products of ODC activity, also induce the synthesis of a similar protein in H-35 cells. These ODC-inhibitors are heat-labile, trypsin-sensitive, and their induction is dependent upon protein synthesis. They have short half-lives which range from 18 to 66 min; these half-lives are similar to those of the ODC derived from the same source. They are noncompetitive inhibitors of ODC activity with an apparent molecular weight of 26,500. Each inhibitor crossreacts with the ODC's of the other cells and forms an enzyme-inhibitor complex which is stable during Sephadex chromatography; however, after treatment with ammonium sulfate, enzyme and inhibitor activities can be dissociated and recovered intact from the same column. We propose the name antizyme for proteins whose synthesis is induced by the proximal or distal products of the enzyme they inhibit.     

Regulation of thyroid ornithine ornithine decarboxylase (ODC) by thyrotropin. I. The rat.

We studied the effects of TSH on rat thyroid ornithine decarboxylase (ODC) activity. After 1 day of goitrogen treatment, there was an abrupt fall in serum triiodothyronine (T3) a rise in circulating TSH, and a dramatic increase in thyroid ODC activity. Despite the continued rise in TSH and progressive increase in thyroid gland size with further treatment, thyroid ODC activity declined on the third day and remained at submaximal levels. Thyroid ODC activity was also stimulated in a dose-related manner by administration of exogenous TSH. Little TSH effect was noted before 3 h. Maximal ODC activity occurred between 4 and 5 h. The TSH stimulation of ODC could be inhibited by pretreatment with actinomycin D or cycloheximide, suggesting that the increase in ODC activity requires new RNA and protein synthesis. Although pretreatment with agents that alter microtubule structure (e.g., colchicine and vinblastine) prevent stimulation of ODC activity by TSH, additional data suggest, but do not confirm, that hrmone secretion and ODC activation may be dissociable. Further studies were undertaken to determine whether cyclic AMP (cAMP) or prostaglandins played any role in the regulation of thyroidal ODC activity. Dibutyryl cAMP, alone, or together with aminophylline, did not stimulate thyroidal ODC activity in dosages which concomitantly stimulated adrenal enzyme activity. Likewise, prostaglandin E2 (PGE2) did not stimulate thyroidal ODC activity, but did stimulate adrenal enzyme activity in a dose-related manner. However, pre-treatment of rats with inhibitors of prostaglandin synthesis prevented the activation of thyroidal ODC BY TSH. One inhibitor, indomethacin, attenuated the TSH stimulation of enzyme activity in a dose-related manner. Indomethacin pretreatment also resulted in approximately a 10-fold decrease in thyroidal prostaglandin levels. Exogenous PGE9, in dosages as high as 500 pg, did not overcome the inhibitory effect of indomethacin on ODC activation. Although the precise role for endogenous prostaglandins remains to be defined, it does appear that a reduction in thyroidal prostaglandins prevents activation of the enzyme by TSH.     

Ornithine decarboxylase activity and polyamines in the anterior pituitary gland during the rat oestrous cycle

The biosynthesis of polyamines, an ubiquitous group of amines shown to be essential for normal cellular growth and differentiation, was studied in the rat anterior pituitary gland during the different stages of the oestrous cycle. The activity of ornithine decarboxylase (ODC), which catalyses the rate-limiting step in the biosynthesis of polyamines, was low during oestrus, metoestrus and dioestrus. However, a marked transitory rise in ODC activity was found in the pituitary gland on the evening of pro-oestrus. The rise in ODC activity was accompanied by an increase in the pituitary content of the polyamines putrescine and spermidine. Ovariectomy did not significantly change the basal ODC activity in the pituitary gland. Oestrogen treatment of ovariectomized rats resulted in a marked stimulation of pituitary polyamine biosynthesis. The largest effects were observed when oestrogen was given as two injections 72 h apart, which gave rise to levels of ODC activity comparable to those observed on the evening of pro-oestrus. The increase in polyamine synthesis in the anterior pituitary gland during pro-oestrus appeared not to be related to the preovulatory secretion of LH or prolactin, since neither LH-releasing hormone nor thyrotrophin-releasing hormone (which induces a secretion of prolactin) affected pituitary ODC activity. The observed biosynthesis of polyamines may be associated with the cellular proliferation which occurs in the anterior pituitary gland at oestrus.     

Stimulation of ornithine decarboxylase activity by arginine vasopressin in the rat medullary thick ascending limb of Henle's loop

The effect of arginine vasopressin (AVP) on rat renal ornithine decarboxylase (ODC) activity was investigated by a cytochemical technique optimized for use in the medullary thick ascending limb of Henle's loop (mTAL). Stimulation of ODC activity by AVP was confined to the mTAL. Peaks in enzyme activity in cultured rat renal segments occurred after tissue had been exposed to AVP for 3 or 8 min and these times of maximal stimulation did not change with the concentration of AVP. There was a dose-dependent response in ODC activity over the AVP concentration range 0.01 10 fmol/l. The ODC response to AVP was totally blocked by specific antiserum to AVP and reduced by 70% with the specific inhibitor to ODC, difluoromethyl ornithine.     

Regulation of casein synthesis by polyamines in mammary gland explants of mice.

Studies were carried out to determine whether the actions of prolactin on the metabolism of the mammary gland may involve polyamines. In mouse mammary gland explants that were preincubated for 2 days with insulin plus hydrocortisone, the rate of [3H]leucine incorporation into casein was enhanced in a prolactin-like manner during a further incubation with spermidine plus cyclic GMP or phospholipase A. Putrescine (0.5 mM) plus PGF2alpha, cyclic GMP or arachidonic acid also enhanced the rate of casein synthesis: but PGF2alpha plus 0.5 mM arginine, ornithine or spermine had no effect. Methyl GAG, an inhibitor of the enzyme S-adenosyl-L-methionine decarboxylase (which is required for the conversion of putrescine to spermidine), abolished the putrescine plus PGF2alpha stimulation of casein synthesis. Since this drug did not affect the action of spermidine plus PGF2alpha on casein synthesis, the specific action of spermidine on casein synthesis is suggested. Neither arginine, ornithine nor the polyamines, by themselves, affected the rate of [3H]uridine incorporation into RNA or the rate of [3H]leucine incorporation into casein. Spermidine levels were elevated within 4 h after adding prolactin to explants which were preincubated for 2 days with insulin plus hydrocortisone; this effect was apparent during incubation periods of up to 48 h with prolactin. Arginase and ornithine decarboxylase activities were also elevated in response to prolactin. Arginase activity was only elevated, however, during long incubation periods with prolactin, i.e., during incubation periods of longer than 2 days. In contrast, ornithine decarboxylase activity was elevated by prolactin within a 30 min incubation period; this effect was maximal after 2 h and persisted during exposure periods of up to 24 h.     

Influence of ovarian ornithine decarboxylase in folliculogenesis and luteinization

LH plays a relevant role in folliculogenesis, ovulation, and luteinization. Although ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is a target of LH in the ovary, the functional significance of ODC induction has remained elusive. Our study reveals that the blockade of the induction of ovarian ODC by means of the specific inhibitor alpha-difluoromethylornithine (DFMO) affects folliculogenesis and luteinization. In immature female mice, DFMO was found to inhibit ovarian growth, the formation of Graafian follicles, and the secretion of progesterone and estradiol. In adult cycling females, the administration of DFMO on the evening/night of proestrus markedly decreased plasma progesterone levels at diestrus, which was associated to the decrease in the expression of steroidogenic factor 1, cytochrome cholesterol side chain cleavage enzyme, and steroidogenic acute regulatory protein in the ovary and to a reduced vascularization of the corpora lutea. These effects were not reverted by the administration of gonadotropins or prolactin. ODC immunoreactivity was also stimulated by LH in theca and granulosa cells of antral follicles but not in preantral follicles. Overall, these experiments demonstrate that elevated ODC values found in the ovary of immature and adult mice play a relevant function in ovarian physiology and that ODC/polyamines must be considered as important mediators of some of the effects of LH on follicular development and luteinization.     

Stimulation of hepatic and renal ornithine decarboxylase activity by selected amino acids

The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, increases after a protein meal. The effect of amino acid mixtures on hepatic and renal ODC activity and polyamine content was studied in postabsorptive and 72-hour fasted rats. Fasting decreased ODC activity in liver and in kidney by approximately 50%. Hepatic ODC activity increased tenfold 4 hours after intraperitoneal injection of either 1 g/kg of a synthetic mixture of 17 amino acids or of casein hydrolysate to fed rats and about 20-fold in fasted rats. Renal ODC activity increased four- and tenfold respectively. A mixture of glutamate, aspartate, and alanine at concentrations given in the hydrolysate reproduced the full amino acid effect. No amino acid was effective when given alone, nor were mixtures of the other amino acid constituents of the hydrolysate. Glutamate + alanine was ineffective as were glucose or various combinations of arginine, ornithine, aspartate and NH₃. Ornithine + glutamate or aspartate + glutamate were active but stimulated less than aspartate + glutamate + alanine. Hepatic and renal putrescine content increased in parallel with ODC activity. The data suggest that specific amino acids possess the full ODC-stimulating capability of a high quality protein and that polyamine synthesis is linked to urea cycle activity.     

Effects of gastrin and difluoromethylornithine on growth of human colon cancer

The effect of difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase activity, was evaluatedin vivo andin vitro on the growth of a gastrin-sensitive human colon carcinoma (WiDr).In vivo, mice bearing the tumor treated with pentagastrin had larger tumors with higher ornithine decarboxylase activity and polyamine content (P<0.05) than mice not treated with pentagastrin. Difluoromethylornithine treatment significantly decreased ornithine decarboxylase in both the pentagastrin-treated and the untreated animals; however, DFMO had no effect on tumor volume, weight, protein, or DNA content. In cell culture, gastrin treatment increased WiDr cell number and [³H]thymidine incorporation in the presence or absence of serum. In serum-free conditions, however, gastrin stimulated cell growth without concomitantly increasing ODC activity. DFMO, on the other hand, decreased both ODC activity and growth. These studies suggest that the trophic effect of gastrin on WiDr human colon cancer is independent of ODC activity. Since gastrin treatment increased ODC activityin vivo, gastrin may interactin vitro with other factors present in serum that can alter ODC activity.Supported by NIH First CA50303 and American College of Physicians Research and Teaching Award to Dr. Smith.     

Cyclosporine inhibits prolactin induction of ornithine decarboxylase in rat tissues

Cyclosporine (CyA), formerly cyclosporin A, significantly inhibited the ability of prolactin (PRL) to elevate ornithine decarboxylase (ODC) activity in a variety of rat tissues. Administration of PRL to hypophysectomized rats also resulted in an induction of ODC activity which was inhibited markedly in all tissues studied in the presence of CyA. Transglutaminase ( TGase ) activity was not affected in any significant manner by PRL or CyA in most tissues studied. However, it was elevated in the adrenal by 10(-8) M PRL. Bromocryptine, which selectively antagonizes pituitary PRL release, decreased the kidney ODC basal levels to 30% of vehicle control and serum PRL level to 4.3 +/- 1.4 compared to 28 +/- 10 in controls, suggestive of PRL maintenance of steady-state ODC activity in the kidney. CyA administration did not affect the action of glucagon, a known cyclic AMP-mediated hormone, or 8-bromo-cyclic AMP on kidney ODC activity. The elevation of rat kidney ODC activity by dexamethasone and triiodothyronine (T3), compounds which elevated serum prolactin levels in all cases, was also blocked by administration of CyA. Epidermal growth factor (EGF), which did not induce rat kidney ODC activity by itself, was capable of producing a small increment in ODC activity in the presence of CyA. The marked effect of CyA to selectively block ODC induction by PRL may be due to the ability of CyA to interact with receptor-required phospholipids in membranes and thus to antagonize hormone-receptor interaction.     

Decreased ornithine decarboxylase activity in the kidneys of Dahl salt-sensitive rats.

To assess the roles of polyamines (putrescine, spermidine, and spermine) and ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, in the development of salt-sensitive hypertension, we evaluated activity and expression of ODC, urinary polyamine excretion, and antizyme (endogenous ODC inhibitor protein) expression in Dahl salt-sensitive (SS) and salt-resistant (SR) rats after they were fed on a low (0.3%) or high (4%) salt diet for 4 weeks. We also examined the effects of spermidine and difluoromethylornithine (DFMO: a specific inhibitor of ODC) on the systolic blood pressure and ODC protein expression in SS rats fed a high salt diet. Renal ODC activity and urinary polyamine excretion in SS rats were lower than those in SR rats after 4 weeks treatment with a low or high salt diet. The renal ODC protein expression of SS rats was paradoxically increased as compared to the SR group. A high salt diet did not alter ODC activity but increased ODC protein only in SS rats. ODC mRNA and antizyme protein expressions were not significantly different among the four groups. Spermidine supplementation attenuated and DFMO exaggerated hypertension in SS rats fed a high salt diet. Spermidine down-regulated and DFMO up-regulated renal ODC protein in SS rats on a high salt diet. ODC activity was decreased but protein was paradoxically increased in kidneys of SS rats. ODC protein was suggested to increase in compensation for the inhibition of its activity. Impaired ODC activity and polyamine production in the kidney may exaggerate salt-sensitive hypertension in SS rats.     

Polyamines and growth regulation of cultured human breast cancer cells by 17 beta-oestradiol

The growth of ZR-75-1 cells, a line of human breast cancer cells in culture, is stimulated by oestradiol and inhibited by anti-oestrogens. Changes in growth rate caused by these agents are accompanied by changes in activity of ornithine decarboxylase, a rate-limiting enzyme for polyamine synthesis. Furthermore, the growth inhibition caused by tamoxifen, an anti-oestrogen, can be reversed by the addition of spermine, spermidine or putrescine to the cells. Insulin can also stimulate ZR-75-1 cell growth and this is again accompanied by an increase in ODC activity. The reduced cell growth rate observed when the cells become confluent is associated with a marked decrease in ornithine decarboxylase activity. Experiments performed with DFMO, a specific and irreversible inhibitor of ODC, show that this compound can prevent the stimulation of growth by oestradiol and that this may be overcome by the addition of putrescine to the cells. It would appear that increased ODC activity and polyamine synthesis are necessary components of the stimulation of breast cancer cell growth by oestradiol but that other growth regulatory stimuli also may act via this enzyme.     

Protein kinase C alpha, epsilon and AP-1 mediate prolactin regulation of mitochondrial aspartate aminotransferase expression in the rat lateral prostate

Citrate accumulation and secretion are physiological functions of the prostate gland that are regulated by testosterone and prolactin. The metabolic pathway for citrate production in the prostate involves the activity of mitochondrial aspartate aminotransferase (mAAT). The expression of mAAT in the prostate is regulated by prolactin through a signal transduction pathway mediated by protein kinase C (PKC). In this report we determined which PKC isoforms are expressed in rat lateral prostate epithelial cells and their activation by prolactin. Eight PKC isoforms are expressed in the ventral and lateral prostate lobes. Although all eight isoforms are expressed, only PKC and PKC were stimulated by prolactin and only in the lateral prostate lobe. Activator protein-1 (AP-1) appears to be the target of prolactin-PKC signaling because prolactin stimulated nuclear protein binding to an AP-1 consensus oligodeoxynucleotide. Moreover, the nuclear binding protein stimulated by prolactin also bound an mAAT oligodeoxynucleotide that contained an AP-1 consensus sequence and which competed for binding with the consensus AP-1 oligodeoxynucleotide. A PKC antisense oligodeoxynucleotide blocked expression of mAAT mRNA. Thus, we conclude that PKC is a specific PKC isoform that mediates via AP-1 the signal for prolactin regulation of mAAT gene expression in rat lateral prostate epithelial cells.     

Thyroid-stimulating hormone regulation of ornithine decarboxylase activity in the thyroid.

TSH (1.0 U im) caused a 22-fold increase in thyroidal ornithine decarboxylase activity (ODC) 6 hours after administration in intact rats. Hypophysectomized rats treated with 1 U TSH showed a 5-fold increase in thyroid ODC activity. This stimulation appeared to be specific for TSH since hormones known to induce ODC activity in other target tissues, such as ACTH or LH, showed no significant stimulation. DIBUTYRYL CYCLIC AMP and aminopylline caused a 12-fold increase in ODC activity 5 hours after administration. Prostaglandins have also been implicated in the TSH-induced stimulation of cyclic AMP. Indomethacin (1.0 mg/100 g body wt, ip), an inhibitor of prostaglandin synthesis, was administered 3 hours before TSH with a resulting 30% diminution (P less than .001) in ODC activity compared with the administration of TSH alone. To rule out the possibility that the increase in ODC activity with TSH might be due to increased thyroid hormone secretion, ODC activity was evaluated 6 hours after triiodothyronine administration (60 mug/100 g body wt), and no significant increase in thyroid ODC activity was found. Stimulation of ODC activity was 90% inhibited by the intraperitoneal administration of actinomycin D (80 mug/100 g body wt) or cycloheximide (400 mug/100 g body wt) given simultaneously with TSH. These results indicated that TSH specifically stimulated thyroid ODC activity, which may be important for the growth-promoting action of the hormone on the thyroid gland. This action may be mediated by cAMP and prostaglandins and may require new protein synthesis.     

Epidermal growth factor regulation of DNA synthesis in human colonic lamina propria lymphocytes

Epidermal growth factor (EGF) is a potent growth factor for many tissues including the gastrointestinal tract. EGF is present in the gut lumen and is absorbed through the mucosa in the developing animals. In addition, EGF has been found to alter the immune system. In this study, we investigated thein vitro effect of EGF on normal colonic lamina propria lymphocyte DNA synthesis and ornithine decarboxylase activity. Human colonic lamina propria lymphocytes were isolated by collagenase-EDTA digestion. The effect of EGF on Con A-stimulated lymphocyte thymidine incorporation was tested. We observed that EGF suppressed DNA synthesis and ornithine decarboxylase (ODC) activity in lamina propria lymphocytes. EGF did not alter the time course of thymidine incorporation into LPL stimulated by the combination of phorbol 12,13-dibutyrate (PDB) and ionomycin. Our data suggest that (1) EGF suppresses DNA synthesis in human colonic lamina propria lymphocytes as well as ODC activity and (2) this inhibition may be mediated through protein kinase C or calcium flux. We postulate that EGF may have a role in modulating the human gut immune system.This work was supported in part by grant CA43280 from the National Institutes of Health.