Pheochromocytoma cell lines from heterozygous neurofibromatosis knockout mice.

Transplantable tumors and cell lines have been developed from pheochromocytomas arising in mice with a heterozygous knockout mutation of the neurofibromatosis gene, Nf1. Nf1 encodes a ras-GTPase-activating protein, neurofibromin, and mouse pheochromocytoma (MPC) cells in primary cultures typically show extensive spontaneous neuronal differentiation that may result from the loss of the remaining wild-type allele and defective regulation of ras signaling. However, all MPC cell lines express neurofibromin, suggesting that preservation of the wild-type allele may be required to permit the propagation of MPC cells in vitro. MPC lines differ from PC12 cells in that they express both endogenous phenylethanolamine N-methyltransferase (PNMT) and full-length PNMT reporter constructs. PNMT expression is increased by dexamethasone and by cell-cell contact in suspension cultures. Mouse pheochromocytomas are a new tool for studying genes and signaling pathways that regulate cell growth and differentiation in adrenal medullary neoplasms and are a unique model for studying the regulation of PNMT expression.

Incorporation of urea and ammonia nitrogen into ileal and fecal microbial proteins and plasma free amino acids in normal men and ileostomates.

BACKGROUND: The importance of urea nitrogen reutilization in the amino acid economy of the host remains to be clarified. OBJECTIVE: The objective was to explore the transfer of (15)N from orally administered [(15)N(2)]urea or (15)NH(4)Cl to plasma free and intestinal microbial amino acids. DESIGN: Six men received an L-amino acid diet (167 mg N*kg(-)(1)*d(-)(1); 186 kJ*kg(-)(1)*d(-)(1)) for 11 d each on 2 different occasions. For the last 6 d they ingested [(15)N(2)]urea or, in random order, (15)NH(4)Cl (3.45 mg (15)N*kg(-)(1)*d(-)(1)). On day 10, a 24-h tracer protocol (12 h fasted/12 h fed) was conducted with subjects receiving the (15)N tracer hourly. In a similar experiment, (15)NH(4)Cl (3.9 mg (15)N*kg(-)(1)*d(-)(1)) was given to 7 ileostomates. (15)N Enrichments of urinary urea and plasma free and fecal or ileal microbial protein amino acids were analyzed. RESULTS: (15)N Retention was significantly higher with (15)NH(4)Cl (47.7%; P < 0.01) than with [(15)N(2)]urea (29.6%). Plasma dispensable amino acids after the (15)NH(4)Cl tracer were enriched up to 20 times (0. 2-0.6 (15)N atom% excess) that achieved with [(15)N(2)]urea. The (15)N-labeling pattern of plasma, ileal, and fecal microbial amino acids (0.05-0.45 (15)N atom% excess) was similar. Appearance of microbial threonine in plasma was similar for normal subjects (0.14) and ileostomates (0.17). CONCLUSION: The fate of (15)N from urea and NH(4)Cl differs in terms of endogenous amino acid metabolism, but is similar in relation to microbial protein metabolism. Microbial threonine of normal and ileostomy subjects appears in the blood plasma but the net contribution to the body threonine economy cannot be estimated reliably from the present data.

Availability of intestinal microbial lysine for whole body lysine homeostasis in human subjects.

We have investigated whether there is a net contribution of lysine synthesized de novo by the gastrointestinal microflora to lysine homeostasis in six adults. On two separate occasions an adequate diet was given for a total of 11 days, and a 24-h (12-h fast, 12-h fed) tracer protocol was performed on the last day, in which lysine turnover, oxidation, and splanchnic uptake were measured on the basis of intravenous and oral administration of L-[1-(13)C]lysine and L-[6,6-(2)H(2)]lysine, respectively. [(15)N(2)]urea or (15)NH(4)Cl was ingested daily over the last 6 days to label microbial protein. In addition, seven ileostomates were studied with (15)NH(4)Cl. [(15)N]lysine enrichment in fecal and ileal microbial protein, as precursor for microbial lysine absorption, and in plasma free lysine was measured by gas chromatography-combustion-isotope ratio mass spectrometry. Differences in plasma [(13)C]- and [(2)H(2)]lysine enrichments during the 12-h fed period were observed between the two (15)N tracer studies, although the reason is unclear, and possibly unrelated to the tracer form per se. In the normal adults, after (15)NH(4)Cl and [(15)N(2)]urea intake, respectively, lysine derived from fecal microbial protein accounted for 5 and 9% of the appearance rate of plasma lysine. With ileal microbial lysine enrichment, the contribution of microbial lysine to plasma lysine appearance was 44%. This amounts to a gross microbial lysine contribution to whole body plasma lysine turnover of between 11 and 130 mg. kg(-1). day(-1), depending on the [(15)N]lysine precursor used. However, insofar as microbial amino acid synthesis is accompanied by microbial breakdown of endogenous amino acids or their oxidation by intestinal tissues, this may not reflect a net increase in lysine absorption. Thus we cannot reliably estimate the quantitative contribution of microbial lysine to host lysine homeostasis with the present paradigm. However, the results confirm the significant presence of lysine of microbial origin in the plasma free lysine pool.