Developmental regulation of ornithine decarboxylase (ODC) in rat testis: comparison of changes in ODC activity with changes in ODC mRNA levels during testicular maturation.

These studies were undertaken to analyze the changes in testicular ornithine decarboxylase (ODC) mRNA levels and ODC activity in rats from birth to maturity. Levels of ODC mRNA were initially low in animals aged 10-17 days. Beginning at 21 days, ODC mRNA levels began to rise, reaching maximal levels by 40 days (p less than 0.01). The size of the 2.2- and 2.6-kb ODC mRNAs did not appear to change with age, as determined by Northern blot analysis. The increase in ODC mRNA that began at 21 days paralleled the increase in testis weight. This increase in ODC mRNA preceded the appearance of rat protamine-1 mRNA, a germ cell-specific mRNA found in round spermatids, which was first detected on Day 40. In contrast, levels of sulfated glycoprotein-2 mRNA, which, in the testis, is found exclusively in Sertoli cells, were highest at Day 17 and thereafter declined gradually with age. Unlike the increase in ODC mRNA levels, ODC activity was highest in 10-day-old animals and thereafter declined steadily with age, reaching minimal levels by 40 days (p less than 0.01). Thus, the increase in testicular ODC mRNA levels was in marked contrast to the decrease in testicular ODC activity. Incubation of cytosolic extract from 40-day-old animals with that from 10- or 17-day-old animals inhibited ODC activity approximately 50%, when compared to cytosols from 10- or 17-day-old animals. Dialysis of cytosol from 40-day-old animals prior to incubation with cytosol from 10-day-old animals relieved this inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative effects of TGFβ on proliferation of 7- and 14-day-old chick embryo fibroblasts and lack of involvement of the ODC/PA system in the TGFβ signaling pathway

The growth regulatory activity of transforming growth factor β (TGFβ) on chick embryo skin fibroblasts was compared in two developmental ages, days 7 and 14. The time course of ³H-thymidine incorporation, an S-phase marker of replication, was determined during 36 hr of TGFβ treatment. Seven-day-old cells showed a prereplicative phase of 6 hr, and 14-day-old cells showed a prereplicative phase of 12 hr. DNA synthesis peaked at 24 hr in 7-day-old fibroblasts and was 10 times higher than that in 14-day-old fibroblasts. Ornithine decarboxylase (ODC) activity and content of the natural polyamines spermine (Spm), spermidine (Spd), and putrescine (Put) differed during cell cycle. ODC activity peaked at 12 hr in 7-day-old cells and at 6 hr in 14-day-old cells. Its level was two times higher at day 7 and was associated with a greater content of ODC mRNA. The maximum of polyamine (PA) concentration was determined after 12 hr of treatment in 7-day-old cells and after 36 hr in 14-day-old cells. These findings indicate that the TGFβ proliferative response of embryo fibroblasts changes during development and is associated with activation of the ODC/PA system. Cotreatment with α-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ODC, did not reduced growth rate. Inhibition of ODC resulted in levels of Put and Spd comparable to that of quiescent fibroblasts, whereas Spm concentration remained higher. Because an altered ODC metabolism does not convey the effects of TGFβ on DNA synthesis, the ODC/PA system may not play a role in the pathway of TGFβ signaling. J. Cell. Physiol. 178:304–310, 1999. © 1999 Wiley-Liss, Inc.     

Cholinergic agents: antinociception without morphine type dependence in rats

We have confirmed the work of others showing that loss in body weight is a predictable and consistent sign of opiate withdrawal in rats. Rats that were treated chronically with either oxotremorine or physostigmine displayed no weight loss or other signs of opiate-like withdrawal when the drugs were withdrawn. Furthermore, there was no difference in weight loss between morphine dependent rats substituted with saline and those substituted with either cholinergic drug. However, we did observe an increased mortality among rats substituted with a cholinergic agent compared with saline. Rats infused with a mixture of morphine plus oxotremorine or morphine plus physostigmine showed less weight loss, but not fewer behavioral signs, after the end of the infusion than rats treated only with morphine. It is concluded that the cholinergic agents did not cause a morphine-like physical dependence themselves, but appeared to antagonize to some extent the development or manifestation of opiate dependence.     

Opiate Modulation of Striatal Dopamine and Hippocampal Norepinephrine Release Following Morphine Withdrawal

When opiates are abruptly withdrawn after chronic treatment, increases in hippocampal noradre-nergic function are observed which are accompanied by decreases in striatal dopamine release. The latter effects have to shown to persist for several weeks following the onset of opiate withdrawal. We examined the long-term effects of opiate withdrawal on 4-aminopyridine and potassium stimulated release of striatal dopamine and hippocampal norepinephrine. Tissue samples were obtained either from rats that had been exposed to opiate withdrawal following a seven day morphine infusion or sham treated control subjects. At 48 hours after the onset of withdrawal (cessation of morphine infusions), slices were loaded with [³H] neurotransmitter, washed extensively, and exposed to different drug treatments. 4-aminopyridine induced concentration related increases in striatal dopamine release, which was 36% calcium independent. Similar values for fractional release of striatal dopamine were obtained in morphine withdrawn and control subjects, for both potassium and 4-aminopyridine induced release. In addition, thresholds for 4-aminopyridine or potassium induced release of striatal dopamine did not differ between control and morphine withdrawn subjects. Treatment with 1.0 M morphine sulfate potentiated potassium evoked release of norepinephrine to an equal extent in both morphine withdrawn and sham treated hippocampal tissue. Exposure to a threshold concentration of potassium (8.0 mM), stimulated increased release of hippocampal norepinephrine in a significantly greater fraction of tissue samples obtained from morphine withdrawn animals. Although these results do not support changes in striatal dopamine release following opiate withdrawal, opiate mechanisms appear to be important determinants of in vitro hippocampal norepinephrine release.     

Ornithine Decarboxylase Activity During Development of Cerebellar Granule Neurons

Abstract: Ornithine decarboxylase (ODC), the key enzyme for polyamine biosynthesis, dramatically decreases in activity during normal cerebellar development, in parallel with the progressive differentiation of granule neurons. We have studied whether a similar pattern is displayed by cerebellar granule neurons during survival and differentiation in culture. We report that when granule cells were kept in vitro under trophic conditions (high K⁺ concentration), ODC activity progressively decreased in parallel with neuronal differentiation. Under nontrophic conditions (cultures kept in low K⁺ concentration), the enzymatic activity dropped quickly in parallel with an increased apoptotic elimination of cells. Cultures kept in high K⁺ but chronically exposed to 10 m M lithium showed both an increased rate of apoptotic cell death at 2 and 4 days in vitro and a quicker drop of ODC activity and immunocytochemical staining. A short chronic treatment of rat pups with lithium also resulted in transient decrease of cerebellar ODC activity and increased programmed cell death, as revealed by in situ detection of apoptotic granule neurons. The present data indicate that a sustained ODC activity is associated with the phase of survival and differentiation of granule neurons and that, conversely, conditions that favor their apoptotic elimination are accompanied by a down-regulation of the enzymatic activity.     

The effect of tripelennamine alone in combination with opiates to produce antinociception in mice

Antinociception (ANTI) was assessed in male CD-1 mice by a modification of Haffner's tail clamp procedure. Studies revealed that tripelennamine (Tp) alone produced antinociception (ANTI) in mice and also caused potentiation when combined with morphine (M) or nalbuphene (NB). Naloxone (Nx) only partially blocked the effect of Tp, but fully blocked M. Although atropine (At) had no intrinsic ANTI activity, it enhanced that of Tp but not M. Histamine (Hm) had no intrinsic ANTI activity, nor did it interact with either Tp or M. The partial abolition of Tp ANTI, in contrast to complete blockade of M effects with Nx, appears to indicate that Tp can stimulate the opiate receptor as well as another receptor for ANTI at a different locus. The combination of Tp with various opiates may have considerable abuse potential.     

Ornithine decarboxylase is a mediator of c-Myc-induced apoptosis.

c-Myc plays a central role in the regulation of cell cycle progression, differentiation, and apoptosis. However, the proteins which mediate c-Myc function(s) remain to be determined. Enforced c-myc expression rapidly induces apoptosis in interleukin-3 (IL-3)-dependent 32D.3 murine myeloid cells following IL-3 withdrawal, and this is associated with the constitutive, growth factor-independent expression of ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis. Here we have examined the role of ODC in c-Myc-induced apoptosis. Enforced expression of ODC, like c-myc, is sufficient to induce accelerated death following IL-3 withdrawal. ODC induced cell death in a dose-dependent fashion, and alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC enzyme activity, effectively blocked ODC-induced cell death. ODC-induced cell death was due to the induction of apoptosis. We also demonstrate that ODC is a mediator of c-Myc-induced apoptosis. 32D.3-derived c-myc clones have augmented levels of ODC enzyme activity, and their rates of death were also a function of their ODC enzyme levels. Importantly, the rates of death of c-myc clones were inhibited by treatment with DFMO. These findings demonstrate that ODC is an important mediator of c-Myc-induced apoptosis and suggest that ODC mediates other c-Myc functions.     

Regulation of G Protein-Coupled Receptor Kinase 2 in Brains of Opiate-Treated Rats and Human Opiate Addicts

Abstract: The effects of opiate drugs (heroin, morphine, and methadone) on the levels of G protein-coupled receptor kinase 2 (GRK2) were studied in rat and human brain frontal cortices. The density of brain GRK2 was measured by immunoblot assays in acute and chronic opiate-treated rats as well as in opiate-dependent rats after spontaneous or naloxone-precipitated withdrawal and in human opiate addicts who had died of an opiate overdose. In postmortem brains from human addicts, total GRK2 immunoreactivity was not changed significantly, but the level of the membrane-associated kinase was modestly but significantly increased (12%) compared with matched controls. In rats treated chronically with morphine or methadone modest increases of the enzyme levels (only significant after methadone) were observed. Acute treatments with morphine and methadone induced dose- and time-dependent increases (8–22%) in total GRK2 concentrations [higher increases were observed for the membrane-associated enzyme (46%)]. Spontaneous and naloxone-precipitated withdrawal after chronic morphine or methadone induced a marked up-regulation in the levels of total GRK2 in the rat frontal cortex (18–25%). These results suggest that GRK2 is involved in the short-term regulation of μ-opioid receptors in vivo and that the activity of this regulatory kinase in brain could have a relevant role in opiate tolerance, dependence, and withdrawal.     

Regulation of ODC activity in the thymus and liver of rats by adrenal hormones

The activity of L-ornithine decarboxylase (EC 4.1.1.17, ODC) has become a useful indicator of hormone responsiveness. Various regimens of dexamethasone, aldosterone and epinephrine, alone or in combination, were administered to adrenalectomized rats either in acute or chronic doses. In addition, adrenalectomized rats, which were chronically treated with aldosterone and epinephrine, were given a single injection of 50 micrograms dexamethasone and sacrificed at various time intervals after hormone treatment. Hepatic and thymic ODC activity was measured. The expected dexamethasone effect, an increase in hepatic and a decrease in thymic ODC, was observed. This study also revealed that aldosterone induced similar responses in these tissues. Epinephrine had the opposite effect since chronic administration of dexamethasone or aldosterone with epinephrine resulted in control levels of ODC. Furthermore, when aldosterone and epinephrine were chronically administered to adrenalectomized rats, to study the acute effects of dexamethasone on rat thymus and liver, the time course of the response in each tissue was found to be distinct. The influence of the adrenal gland on rat thymus and liver is not restricted only to glucocorticoids, but may also involve other hormones which it secretes.     

Androgen-dependence of ornithine decarboxylase in the rat epididymis

Ornithine decarboxylase (ODC) activity was measured in epididymides of 45-day-old rats. Higher ODC activity was detected in the corpus and cauda than in the caput epididymidis. Bilateral castration for 7 days caused epididymal ODC to fall to undetectable values, whereas testosterone restored activity to normal values. The effect of the androgen was significantly inhibited by cyproterone acetate. The caput was more sensitive to the action of testosterone than were the corpus and caudal segments. Unilateral castration for 4 or 8 days did not affect ODC on the control or castrated side, but the activity fell in epididymides of both sides after removal of the remaining testis. These results show that epididymal ODC activity is androgen-dependent.     

Epidermal ornithine decarboxylase and polyamines in mice exposed to 50 Hz magnetic fields and UV radiation

We studied the influence of magnetic fields (MFs) and simulated solar radiation (SSR) on ornithine decarboxylase (ODC) and polyamines in mouse epidermis. Chronic exposure to combined MF and SSR did not cause persistent effects on ODC activity or polyamines compared to the animals exposed only to UV, although the same MF treatment was previously found to accelerate skin tumor development. In an acute 24-h experiment, an elevation of putrescine and down-regulation of ODC activity was observed in the animals exposed to a 100-μT MF. No effect was seen 24 h after a single 2-MED (minimal erythemal dose) exposure to SSR. The results indicate that acute exposure to 50 Hz MF does exert distinctive biological effects on epidermal polyamine synthesis. Bioelectromagnetics 19:388–391, 1998. © 1998 Wiley-Liss, Inc.     

Calcium stimulates ornithine decarboxylase activity in cultured mammalian epithelial cells

Ornithine decarboxylase (ODC) activity usually rises to a peak a few hours after a trophic stimulus. The stimulation of ODC has been shown to depend on extracellular calcium in several in vitro eukaryotic systems. We have investigated the effect of calcium concentration on ODC activity and have found that ODC is stimulated when CaCl2 alone is added to calcium-deprived cells. Epithelial cells from calf esophagus were cultured and grown until stratified. Replacement of medium with fresh serum-free medium resulted in stimulation of ODC activity, which peaked at 4 hours and declined to basal level by 10 hours. Subsequent depletion of Ca2+ either by addition of ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or by replacement of medium with Ca2+-free medium, resulted in obliteration of ODC activity 4 hours later. Conversely, cultures in which medium was replaced with Ca2+-free medium and at 10 hours were repleted with Ca2+ (either by addition of CaCl2 or by replacement of medium with Ca2+-containing medium) exhibited a pronounced elevation of ODC activity 4 hours later. ODC activity peaked at 6 hours after the addition of CaCl2 and declined by 8 hours. The effect was elicited by a wide range of concentrations of added Ca2+ from 0.1 mM to 4.0 mM, but was maximal at 1.0 mM. ODC activity was totally abolished if either cycloheximide (10 micrograms/ml) or putrescine (10 mM) was added to cultures immediately prior to Ca2+ addition. Actinomycin D (2, 5, or 10 micrograms/ml) added 30 minutes before Ca2+ did not prevent the stimulation of ODC by added Ca2+. Stimulation by Ca2+ is dependent on (1) absence of Ca2+ during the initial 10-hour incubation and (2) duration of incubation in Ca2+-free medium prior to Ca2+ replenishment. The results indicate that Ca2+ can increase ODC in epithelial cells exposed to Ca2+-depleted medium and that the increase in ODC depends on protein synthesis but is not inhibited by actinomycin D.     

Effects of material methadone administration on ornithine decarboxylase in brain and heart of the offspring: Relationships of enzyme activity to dose and to growth impairment in the rat

Administration of 5 mg/kg of methadone daily to pregnant and nursing rats produced substantial retardation of body, brain and heart growth in the offspring; alterations in biochemical development also were present in the methadone-exposed pups, as evidenced by delays in the maturational declines of brain and heart ornithine decarboxylase (ODC) activity. Lowering the dose of methadone to levels which did not affect growth or brain ODC, still resulted in pro-found abnormalities in the developmental pattern of heart ODC. These studies indicate that downward adjustment of maternal methadone dosage to a point where birthweights and body and organ growth rates are normal, does not eliminate all of the biochemical alterations associated with the perinatal opiate syndrome.     

Regulated endocytosis of opioid receptors: cellular mechanisms and proposed roles in physiological adaptation to opiate drugs

Opiate drugs such as morphine and heroin are among the most effective analgesics known. Prolonged or repeated administration of opiates produces adaptive changes in the nervous system that lead to reduced drug potency or efficacy (tolerance), as well as physiological withdrawal symptoms and behavioral manifestations such as craving when drug use is terminated (dependence). These adaptations limit the therapeutic utility of opiate drugs, particularly in the treatment of chronically painful conditions, and are thought to contribute to the highly addictive nature of opiates. For many years it has been proposed that physiological tolerance to opiate drugs is associated with a modification of the number or functional activity of opioid receptors in specific neurons. We now understand certain mechanisms of opioid receptor desensitization and endocytosis in considerable detail. However, the functional roles that these mechanisms play in the complex physiological adaptation of the intact nervous system to opiates are only beginning to be explored.     

Hypoxic incubation blunts the development of thermogenesis in chicken embryos and hatchlings

We asked to what extent sustained hypoxia during embryonic growth might interfere with the normal development of thermogenesis. White Leghorn chicken eggs were incubated at 38 degrees C either in normoxia (Nx, 21% O2) or in hypoxia [Hx, 15% O2, from embryonic day 5 (E5) until hatching]. The Hx embryos had lower body weight (W) throughout incubation, and hatching was delayed by about 10 h. For both groups, all measurements were conducted in normoxia. At embryonic day E11, the static temperature-oxygen consumption (ambient T-Vo2) curve was typically ectothermic (Q10 = 1.92-1.94) and similar between Nx and Hx. Toward the end of incubation (E20), the Q10 averaged 1.41 +/- 0.06 in Nx and 1.79 +/- 0.08 in Hx (P < 0.005), indicating that the onset of the thermogenic response in Hx lagged behind Nx. In the 1-day-old hatchlings (H1), body weight did not significantly differ between Nx and Hx. At H1, the T-Vo2 curves were endothermic-type, and more so in the older (>8 h old) than in the newly hatched (<8 h old) chicks, whether examined statically or dynamically as a function of time. In either case, the thermogenic responses of Hx were lower than those of Nx. In a 43-31 degrees C thermocline, the preferred T of the Hx hatchlings was around 37.3 degrees C, and similar to Nx, suggesting a similar setpoint for thermoregulation. We conclude that hypoxic incubation blunted the development of thermogenesis. This could be interpreted as an example of epigenetic regulation, in which an environmental perturbation during early development alters the phenotypic expression of a regulatory system.