Disordered differentiation of mouse embryonic lungs in organ cultures under the action of nitrosomethylurea

We have studied the effect of nitrosomethylurea (NMU) on differentiation of early rudiments of mouse embryonic lungs (12th day of embryogenesis) explanted into an organ culture. We have demonstrated that nontoxic doses of NMU are capable of accelerating normal lung differentiation both at the early (increase in the number of epithelial buds) and at the late (increase in the number of explants with regions of well-developed alveoli) stages of cultivation. However, NMU induces disturbances of differentiation, which appear as polycystic structures and hyperplastic nodules generally absent in the control.     

Effect of nitrosourea in small doses on the frequency of mutation in Salmonella typhimurium

The effect of N-nitroso-N-methylurea (NMU), N-nitroso-N,N'-dimethylurea (NDMU) and N-nitroso-N-ethylurea (NEU) at doses less than 100 mkg/ml on mutability of Salmonella typhimurium strains of Ames' system (G-46, TA-1950, TA-1535, TA-100, TA-1538) has been studied. NMU and NEU at doses of 5-10 mkg/ml have been found to increase the survival and decrease the number of reversions from auxotrophity in histidine to prototrophity. The effect of given doses of NMU and NEU on bacteria repair activity has been shown. The role of pk M101 plasmide in this process is being discussed. NDMU in contrast to NMU and NEU induces read frome shift mutations and exhibits high mutagenous activity at all doses examined.     

Distribution of neuromedin U receptor subtype 2 mRNA in the rat brain

Neuromedin U (NMU) is a family of peptides found in the gut and the central nervous system [Neuroscience 25 (1988) 797; Biochem. Biophys. Res. Commun. 130 (1985) 1078]. While several peripheral activities such as uterus stimulating and hypertensive effects have been described for NMU [Biochem. Biophys. Res. Commun. 130 (1985) 1078], its role in the CNS remains poorly understood. Recently, we reported the identification of two receptors for NMU (NMU1R and NMU2R), and demonstrated that NMU may play a role in regulating feeding behavior. The central effect of NMU is likely mediated primarily via NMU2R, since NMU1R is detectable only in the periphery, but not in the brain [Nature 406 (2000) 70]. In this report, we describe detailed mapping of NMU2R mRNA expression in the rat brain by in situ hybridization. The most intense signals were observed in the ependymal cell layer along the wall of the third ventricle in the hypothalamus, CA1 region of the hippocampus, indusium griseum and septohippocampal nucleus. Moderate expression was detected in the hypothalamic paraventricular nucleus, dorsal raphe nucleus as well as a number of other brain structures. The presence of NMU2R in the hypothalamus is consistent with its role in energy balance. Significant levels of expression of NMU2R elsewhere in the brain may suggest additional physiological functions for this neuropeptide.     

Neuromedin U type 1 receptor stimulation of A-type K+ current requires the βγ subunits of Go protein, protein kinase A, and extracellular signal-regulated kinase 1/2 (ERK1/2) in sensory neurons

Although neuromedin U (NMU) has been implicated in analgesia, the detailed mechanisms still remain unclear. In this study, we identify a novel functional role of NMU type 1 receptor (NMUR1) in regulating the transient outward K(+) currents (I(A)) in small dorsal root ganglion (DRG) neurons. We found that NMU reversibly increased I(A) in a dose-dependent manner, instead the sustained delayed rectifier K(+) current (I(DR)) was not affected. This NMU-induced I(A) increase was pertussis toxin-sensitive and was totally reversed by NMUR1 knockdown. Intracellular application of GDPβS (guanosine 5'-O-(2-thiodiphosphate)), QEHA peptide, or a selective antibody raised against the Gα(o) or Gβ blocked the stimulatory effects of NMU. Pretreatment of the cells with the protein kinase A (PKA) inhibitor or ERK inhibitor abolished the NMU-induced I(A) response, whereas inhibition of phosphatidylinositol 3-kinase or PKC had no such effects. Exposure of DRG neurons to NMU markedly induced the phosphorylation of ERK (p-ERK), whereas p-JNK or p-p38 was not affected. Moreover, the NMU-induced p-ERK increase was attenuated by PKA inhibition and activation of PKA by foskolin would mimic the NMU-induced I(A) increase. Functionally, we observed a significant decrease of the firing rate of neuronal action potential induced by NMU and pretreatment of DRG neurons with 4-AP could abolish this effect. In summary, these results suggested that NMU increases I(A) via activation of NMUR1 that couples sequentially to the downstream activities of Gβγ of the G(o) protein, PKA, and ERK, which could contribute to its physiological functions including neuronal hypoexcitability in DRG neurons.     

Mutagenesis in cultured mammalian cells. II. Induction of gene mutations in Chinese hamster cells

Mutations controlling the resistance to 6-mercaptopurine (6-M) and the ability to multiply in a medium with a low concentration of glucose (“glucose-independent” mutants) were induced in cultured Chinese hamster cells by N-nitrosomethylurea (NMU), 5-bromodeoxyuridine (BUdR), UV and X-rays. The chemical agents were found to be very active in induction of mutations to 6-M resistance (NMU and BUdR) and mutations of “glucose independence” (NMU). These agents increase the yield of mutations as compared to the spontaneous mutation rate by about two orders of magnitude. The induced rate of 6-M-resistant mutations by X-rays was 2.0 ? 10⁻⁷ per viable cell per roentgen. BUdR approximately equally increases the cell's sensitivity to both inactivating and mutagenic action of X-rays. The maximum induction of mutations to 6-M resistance by UV was observed at 100 erg/mm². This dose leads to 1 16-fold increase of the mutation frequency as compared to the spontaneous rate. Further increase of the UV dose up to 200 erg/mm² resulted in a lower yield of mutations per dose unit. The highest yield of mutations to 6-M resistance induced by NMU, BUdR and X-rays was observed if cells were plated in selective medium several generations after the mutagenic treatment. The maximum yield of mutations to 6-M resistance induced by UV and of glucose-independence induced by NMU was recorded if cells were transferred to selective media immediately after treatment. The kinetics of expression of mutations and the decline of their number observed after prolonged incubation of treated cells in nonselective conditions are discussed.     

Cardiovascular actions of central neuromedin U in conscious rats

Neuromedin U (NMU) is a brain-gut peptide, which peripherally stimulates smooth muscle, increases of blood pressure, alters ion transport in the gut, controls local blood flow, and regulates adrenocortical function. Although intracerebroventricular (i.c.v.) administration of NMU is known to decrease food intake and body weight, little is known about its effect on other physiological functions. We examined the effects of i.c.v. administration of NMU on mean arterial pressure (MAP), heart rate (HR), and plasma norepinephrine in conscious rats. Neuromedin U (0.05 and 0.5 nmol) provoked an increase in MAP (93.8 +/- 0.5 to 123.5 +/- 1.7 and 94.7 +/- 0.8 to 132.7 +/- 3.0 mm Hg, respectively) and HR (334.9 +/- 6.0 to 494.1 +/- 6.9 and 346.3 +/- 3.3 to 475.1 +/- 8.9 beats/min, respectively). In contrast, plasma norepinephrine increased only with a high dose of neuromedin U. Intravenously administered NMU (0.5 nmol) elicited a small and short lasting increase in MAP, compared to that by i.c.v. NMU. These results indicate that central neuromedin U regulates sympathetic nervous system activity and affects cardiovascular function.     

Design,synthesis and biological activity of flavonoid derivatives as selective agonists for neuromedin U 2 receptor

Central neuromedin U 2 receptor (NMU2R) plays important roles in the regulation of food intake and body weight. Identification of NMU2R agonists may lead to the development of pharmaceutical agents to treat obesity. Based on the structure of rutin, a typical flavonoid and one of the NMU2R agonists we previously identified from an in-house made natural product library, 30 flavonoid derivatives have been synthesized and screened on a cell-based reporter gene assay. A number of compounds were found to be selective and highly potent to NMU2R. For example, the EC₅₀ value of compound NRA 4 is very close to that of NMU, the endogenous peptide ligand of NMU2R. Structure–activity relationship analysis revealed that a 3-hydroxyl group in ring C and a 2′-fluoride group in ring B were essential for this class of compounds to be active against NMU2R.     

Effects of neuromedin U on the pulsatile LH secretion in ovariectomized rats in association with feeding conditions

We examined the effects of intracerebroventricular injection of neuromedin U (NMU), at a dose that is reported to induce satiety in rats, on the pulsatile luteinizing hormone (LH) secretion in adult ovariectomized (OVX) rats under a normal feeding or a 48-h fasted condition. In OVX rats under the normal feeding condition, injection of NMU (1 nmol/3 microl) significantly decreased the mean LH concentration without affecting the frequency or amplitude of LH pulses, but under the 48-h fasted condition, it significantly decreased the mean LH concentration and the frequency of LH pulses without affecting the amplitude. The interpulse interval was significantly lengthened by NMU injection under the normal and the 48-h fasted condition, but the effect under the 48-h fasted condition was greater than under the normal feeding condition. We also confirmed that the 48-h fasted condition per se did not affect the pulsatile LH secretion in OVX rats. We suggest that NMU and fasting synergistically inhibit the pulsatile LH secretion, even though NMU has been said to act as a satiety factor.     

Neuromedin U causes biphasic cardiovascular effects and impairs baroreflex function in rostral ventrolateral medulla of spontaneously hypertensive rat

Neuromedin U (NMU) causes biphasic cardiovascular and sympathetic responses and attenuates adaptive reflexes in the rostral ventrolateral medulla (RVLM) and spinal cord in normotensive animal. However, the role of NMU in the pathogenesis of hypertension is unknown. The effect of NMU on baseline cardiorespiratory variables in the RVLM and spinal cord were investigated in urethane-anaesthetized, vagotomized and artificially ventilated male spontaneously hypertensive rats (SHR) and Wistar–Kyoto rats (WKY). Experiments were also conducted to determine the effects of NMU on somatosympathetic and baroreceptor reflexes in the RVLM of SHR and WKY. NMU injected into the RVLM and spinal cord elicited biphasic response, a brief pressor and sympathoexcitatory response followed by a prolonged depressor and sympathoinhibitory response in both hypertensive and normotensive rat models. The pressor, sympathoexcitatory and sympathoinhibitory responses evoked by NMU were exaggerated in SHR. Phrenic nerve amplitude was also increased following intrathecal or microinjection of NMU into the RVLM of both strains. NMU injection into the RVLM attenuated the somatosympathetic reflex in both SHR and WKY. Baroreflex sensitivity was impaired in SHR at baseline and further impaired following NMU injection into the RVLM. NMU did not affect baroreflex activity in WKY. The present study provides functional evidence that NMU can have an important effect on the cardiovascular and reflex responses that are integrated in the RVLM and spinal cord. A role for NMU in the development and maintenance of essential hypertension remains to be determined.     

Quantitating the frequency of initiation and cH-ras mutation in in situ N-methyl-N-nitrosourea-exposed rat mammary gland

Mammary carcinogenesis is a multistep process consisting minimally of initiation and promotion/progression stages. The rate-limiting stage in the carcinogenesis process is undetermined but can in part be addressed by estimating the frequency of initiation, a heritable early event. Here, we use an in vivo limiting dilution transplantation assay to estimate initiation frequency in a rat mammary epithelial stem-like cell population that was exposed in situ to 50 mg/kg N-methyl-N-nitrosourea (NMU) administered i.v. We estimate that this dose resulted in the killing of 65% of exposed mammary cells. Known numbers of cells surviving NMU exposure were grafted into fat-pads of recipient rats in which the cells grew and differentiated into structurally and functionally normal mammary glands. Recipient rats were hormonally manipulated to provide maximal promotion of initiated cells. Mammary carcinomas developing at graft sites were quantitated over a 2-year period. Based on these results, we estimate that at least 1 surviving NMU-exposed mammary cell in 7,200 was initiated. Seventeen % of these graft site carcinomas had an activated H-ras oncogene with a G to A mutation in codon 12. This suggests that at least 1 mammary cell in 43,000 was mutated in this fashion by in situ exposure to NMU. These data suggest that cH-ras represents approximately 1 of 5 of the initiation events produced by NMU exposure of rat mammary glands.     

Novel role of the anorexigenic peptide neuromedin U in the control of LH secretion and its regulation by gonadal hormones and photoperiod

Neuromedin U (NMU) is a widely spread neuropeptide, with predominant expression at the gastrointestinal tract and brain, putatively involved in the regulation of a diversity of biological functions, including food intake, energy balance and circadian rhythms; all closely related to reproduction. Yet, the implication of NMU in the control of the gonadotropic axis remains scarcely studied. We report herein analyses on the hypothalamic expression and function of NMU in different physiological and experimental states of the rat reproductive system. Expression of NMU mRNA at the hypothalamus was persistently detected along female postnatal development, with maximum levels in adulthood that fluctuated across the cycle and were modulated by ovarian steroids. Acute central administration of NMU evoked increases of serum LH levels in pubertal female rats, while repeated injection of NMU tended to advance vaginal opening. Likewise, central injection of NMU increased serum LH concentrations in cycling female rats, with peak responses in estrus. In contrast, NMU significantly inhibited preelevated LH secretion in gonadectomized and kisspeptin-treated rats. Finally, in noncycling females due to photoperiodic manipulation (constant light), hypothalamic NMU mRNA levels were markedly depressed, but relative LH responses to exogenous NMU were significantly augmented. All together, our present data support a predominant stimulatory role of NMU in the control of the female gonadotropic axis, which appears under the influence of developmental, hormonal, and photoperiodic cues, and might contribute to the joint regulation of energy balance, biological rhythms, and reproduction.     

Heat shock modification of nitrosomethylurea action on sunflower seedlings

Separate and combined action of nitrosomethylurea (NMU) and heat shock (HS) on germination of seeds of the inbred line 3629 and derived nuclear and plastome mutant lines, n-chlorina-1 and en-chlorina-5, respectively, were studied. NMU at a concentration of 0.015%, as well as HS (40 degrees C), had no effect on the spontaneous level of chromosome aberrations in root meristem of sunflower seedlings. However, at a concentration of 0.03%, the mutagen considerably increased the frequency of chromosome rearrangements. Pretreatment with heat for 30 min enhanced the cytogenetic effect of NMU (0.03%) on line 3629 seedlings. In the nuclear mutant, the reaction of root meristematic cells did not depend on the additional heat treatment. The combined action of HS and NMU at early germination stages had no effect on the survival of line 3629 plants and plastome mutant en-chlorina-5; the survival of n-chlorina-1 plants decreased. The highest frequency of M1 plants with chlorophyllic abnormalities (green revertants, lethals, and variegated forms) was observed in n-chlorina-1 line.     

Regulation of gonadotropin secretion and puberty onset by neuromedin U

Neuromedin U (NMU), an anorexigenic peptide, was originally isolated from porcine spinal cord in 1985. As NMU is abundant in the anterior pituitary gland, we investigated the effects of NMU on gonadotropin secretion. Both NMU and its receptors, NMUR1 and NMUR2, were expressed in the pituitary gland. NMU suppressed LH and FSH releases from rat anterior pituitary cells. Moreover, NMU-deficient mice exhibit an early onset of vaginal opening. The LHbeta/FSHbeta ratio, which is an index of puberty onset, is high in young NMU-deficient mice. These results indicate that NMU suppresses gonadotropin secretion and regulates the onset of puberty.     

Effect of N-nitroso-N-methylurea on viability and mutagenic response of repair-deficient strains of Escherichia coli

Various E. coli mutants, deficient in DNA repair, differed in their response to increasing concentrations of N-nitroso-N-methylurea (NMU).Loss of viability due to exposure to NMU was greatest in those strains with a reduced capacity for repair of single-strand breaks. Viability of wild-type and uvrA^? strains was not affected by NMU concentrations up to 3.0 mM. Some loss of viability occurred, at the higher NMU concentrations, in both strains carrying exrA^? while strains carrying uvrA^?polA^? or recA^? were the most sensitive. The results support the hypothesis that the lethal effect of NMU on repair-deficient E. coli was due to its ability to induce single-strand breaks.Induction of mutations by NMU was observed in all the strains used and the results suggested that NMU damage per se was the major mutational event. The dose response curve for induction of revertants by NMU was, however, influenced by the repair system(s) present. The number of revertants scored at the higher NMU concentrations was greater in those strains lacking the recA and polA dependent repair functions than in the wild-type strain. However, at NMU concentrations below 2.0 mM the numbers of revertants induced in exrA^? carrying strains, prossessing accurate rec-dependent repair, were lower than the comparable wild-type values. The evidence suggests that the uvrA gene product also acts on some, possibly non-mutagenic, types of NMU damage and that error-prone repair of these lesions increases the number of potential revertants.     

Serum oxytocinase activity is related to tumor growth parameters in N-methyl nitrosourea induced rat breast cancer

Oxytocinase has been reported to hydrolyse the peptide hormone oxytocin (OT). We have previously described changes in oxytocinase activity in human breast cancer, where a highly significant increase occurred in tumoral tissue. In the present work, we analysed oxytocinase activity in serum of rats with breast cancer induced by N-methyl-nitrosourea (NMU). We also correlated these data with the number and size of tumors and the body weight of the animals to evaluate the putative value of this activity as a biological marker of the disease. Our results confirm the involvement of OT in carcinogenesis and suggest a mayor role for oxytocinase activity in the development of breast cancer.     

Modification of the Effect of Nitrosomethylurea on Sunflower Seedlings by Heat Shock

Separate and combined action of nitrosomethylurea (NMU) and heat shock (HS) on germination of seeds of the inbred line 3629 and derived nuclear and plastome mutant lines, n-chlorina-1 and en-chlorina-5, respectively, were studied. NMU at a concentration of 0.015%, as well as HS (40°C), had no effect on the spontaneous level of chromosome aberrations in root meristem of sunflower seedlings. However, at a concentration of 0.03%, the mutagen considerably increased the frequency of chromosome rearrangements. Pretreatment with heat for 30 min enhanced the cytogenetic effect of NMU (0.03%) on line 3629 seedlings. In the nuclear mutant, the reaction of root meristematic cells did not depend on the additional heat treatment. The combined action of HS and NMU at early germination stages had no effect on the survival of line 3629 plants and plastome mutant en-chlorina-5; the survival of n-chlorina-1 plants decreased. The highest frequency of M₁plants with chlorophyllic abnormalities (green revertants, lethals, and variegated forms) was observed in n-chlorina-1 line.     

PEGylation of Neuromedin U yields a promising candidate for the treatment of obesity and diabetes

Neuromedin U (NMU) is an endogenous peptide, whose role in the regulation of feeding and energy homeostasis is well documented. Two NMU receptors have been identified: NMUR1, expressed primarily in the periphery, and NMUR2, expressed predominantly in the brain. We recently demonstrated that acute peripheral administration of NMU exerts potent but acute anorectic activity and can improve glucose homeostasis, with both actions mediated by NMUR1. Here, we describe the development of a metabolically stable analog of NMU, based on derivatization of the native peptide with high molecular weight poly(ethylene) glycol (PEG) ('PEGylation'). PEG size, site of attachment, and conjugation chemistry were optimized, to yield an analog which displays robust and long-lasting anorectic activity and significant glucose-lowering activity in vivo. Studies in NMU receptor-deficient mice showed that PEG-NMU displays an expanded pharmacological profile, with the ability to engage NMUR2 in addition to NMUR1. In light of these data, PEGylated derivatives of NMU represent promising candidates for the treatment of obesity and diabetes.     

Neuromedin U suppresses prolactin secretion via dopamine neurons of the arcuate nucleus

Neuromedin U (NMU) has a precursor that contains one additional peptide consisting of 33 or 36 amino acid residues. Recently, we identified this second peptide from rat brain and designated it neuromedin U precursor-related peptide (NURP), showing it to stimulate prolactin release from the pituitary when injected via the intracerebroventricular (icv) route. Here, we examined whether NMU, like NURP, also stimulates prolactin release. Unlike NURP, icv injection of NMU significantly decreased the secretion of prolactin from the pituitary. This suppression of prolactin release by NMU was observed in hyper-prolactin states such as lactation, stress, pseudopregnancy, domperidone (dopamine antagonist) administration, and icv injection of NURP. Immunohistochemical analysis revealed that icv injection of NMU induced cFos expression in dopaminergic neurons of the arcuate nucleus, but not the substantia nigra. Mice with double knockout of NMU and neuromedin S (NMS), the latter also binding to NMU receptors, showed a significant increase of the plasma prolactin level after domperidone treatment relative to wild-type mice. These results suggest that NMU and NURP may play important reciprocal roles in physiological prolactin secretion.