Peri-conceptional obesogenic exposure induces sex-specific programming of disease susceptibilities in adult mouse offspring.

Vulnerability of the fetus upon maternal obesity can potentially occur during all developmental phases. We aimed at elaborating longer-term health outcomes of fetal overnutrition during the earliest stages of development. We utilized Naval Medical Research Institute (NMRI) mice to induce pre-conceptional and gestational obesity and followed offspring outcomes in the absence of any postnatal obesogenic influences. Male adult offspring developed overweight, insulin resistance, hyperleptinemia, hyperuricemia and hepatic steatosis; all these features were not observed in females. Instead, they showed impaired fasting glucose and a reduced fat mass and adipocyte size. Influences of the interaction of maternal diet∗sex concerned offspring genes involved in fatty liver disease, lipid droplet size regulation and fat mass expansion. These data suggest that a peri-conceptional obesogenic exposure is sufficient to shape offspring gene expression patterns and health outcomes in a sex- and organ-specific manner, indicating varying developmental vulnerabilities between sexes towards metabolic disease in response to maternal overnutrition.

Genome-wide, large-scale production of mutant mice by ENU mutagenesis.

In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html).

Dissection of biochemical borderline phenotypes in carriers and genetic variants of medium-chain acyl-CoA dehyrogenase deficiency: implications for newborn screening [corrected].

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) represents a potentially fatal fatty acid beta-oxidation disorder. Newborn screening (NBS) by tandem mass spectrometry (MS/MS) has been implemented worldwide, but is associated with unresolved questions regarding population heterogeneity, burden on healthy carriers, cut-off policies, false-positive and negative rates. In a retrospective case-control study, 333 NBS samples showing borderline acylcarnitine patterns but not reaching recall criteria were genotyped for the two most common mutations (c.985A>G/c.199C>T) and compared with genotypes and acylcarnitines of 333 controls, 68 false-positives, and 34 patients. c.985A>G was more frequently identified in the study group and false-positives compared to controls (1:4.3/1:2.3 vs. 1:42), whereas c.199C>T was found more frequently only within the false-positives (1:23). Biochemical criteria were devised to differentiate homozygous (c.985A>G), compound heterozygous (c.985A>G/c.199C>T), and heterozygous individuals. Four false-negatives were identified because our initial algorithm required an elevation of octanoylcarnitine (C(8)) and three secondary markers in the initial and follow-up sample. The new approach allowed a reduction of false-positives (by defining high cut-offs: 1.4 micromol/l for C(8); 7 for C(8)/C(12)) and false-negatives (by sequencing the ACADM gene of few suspicious samples). Our validation strategy is able to differentiate healthy carriers from patients doubling the positive predictive value (42-->88%) and to target NBS to MCADD-subsets with potentially higher risk of adverse outcome. It remains controversial, if NBS programs should aim at identifying all subsets of all diseases included. Because the natural course of milder variants cannot be assessed by observational studies, our strategy could serve as a general model for evaluation of MS/MS-based NBS.

Autoregulation of bradykinin receptors and bradykinin-induced prostacyclin formation in human fibroblasts.

The interaction of bradykinin (BK) with its specific receptors on intact cultured human fibroblasts results in production of prostaglandins, including prostacyclin (PGI2), and accumulation of cyclic AMP. Incubation of cells with 1 microM BK for 5 min at 37 degrees C led to a marked reduction (75-90%) in BK-induced PGI2 release and in total number of [3H]BK-binding sites with no change in dissociation constant (6.1 and 7.6 nM for control and BK-treated cells, respectively). The decrease in receptor number did not result from BK transferred from the first incubation into the binding assay. BK-induced receptor loss was temperature dependent; exposure of cells to BK at 4 degrees C had little or no effect on receptor number. After incubation with BK for approximately equal to 15 min, further incubation in the absence of BK for 30 min at 37 degrees C almost completely restored both receptor number and BK-induced PGI2 release. With more prolonged exposure to BK (greater than 1 h), restoration of receptors was inversely related to the length of exposure and the concentration of BK. Recovery was unaffected by cycloheximide. During prolonged incubation without removal of BK, cells began to recover receptors by 5 h; greater than 99% of the bradykinin initially present disappeared by 3 h. Bacitracin greatly retarded BK disappearance and totally prevented recovery. These observations provide direct evidence that the number of BK receptors on cultured human fibroblasts can be regulated by BK itself. In addition, it appears that BK-degrading systems, by influencing local concentrations of the peptide, may play an important role in the autoregulation of BK receptors. The presence of highly active degradation systems might serve to protect target tissues from developing chronic insensitivity to BK and, perhaps, similar peptides.

Studies on the role of specific cell surface receptors in the removal of lipoprotein (a) in man.

The binding of 125I-lipoprotein (a) [Lp(a)] to cell surface receptors was studied on cultured human fibroblasts. The results were compared with corresponding data obtained with 125I-low density lipoproteins (LDL). Equilibrium binding studies showed that Lp(a) is bound with high affinity by the cell surface receptors. The maximum binding capacity for Lp(a) was 37% lower than for LDL. For Lp(a) and LDL, the Scatchard plots displayed linearity, indicating a single category of binding sites. Half-maximal saturation occurred at a concentration of 9.52 +/- 1.04 nM for Lp(a) and 7.76 +/- 1.29 nM for LDL. Competition binding experiments revealed that Lp(a) and LDL are nearly equally potent in competing each other for the binding sites. Binding of Lp(a) and LDL were followed by suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Cyclohexanedione treatment of Lp(a) and LDL completely abolished receptor binding. Neither Lp(a) nor LDL were specifically bound by fibroblasts obtained from a patient with homozygous familial hypercholesterolemia (FH). The removal mechanisms for Lp(a) and LDL were further compared by in vivo studies. Radioiodinated Lp(a) and LDL were injected intravenously into 12 normolipemic individuals to measure kinetic parameters of these two lipoproteins simultaneously in each subject. Mean fractional catabolic rate (FCR) of Lp(a) was 0.260 +/- 0.060 and mean FCR of LDL was 0.377 +/- 0.077 (mean +/- SD). In each subject, FCR of Lp(a) was lower than the FCR of LDL; the mean difference was 31%. The absolute synthetic rate of Lp(a) was significantly lower than the corresponding value of LDL. In each individual, the percentage of total Lp(a) that was contained in the intravascular space was higher than the corresponding value of LDL; the mean difference was 19%. A highly significant positive correlation was found between FCR of LDL and FCR of Lp(a) (r = 0.853, P less than 0.01). No relationship was found between the serum concentration of LDL-apolipoprotein B and Lp(a). The serum level of Lp(a) was positively related to the absolute rate of Lp(a) synthesis (r = 0.979, P less than 0.01). The serum level of LDL-apolipoprotein B was inversely related to FCR of LDL (r = 0.613, P less than 0.05). In a patient with homozygous FH, FCR of LDL was 0.205 and FCR of Lp(a) was 0.210. The results of these studies show that Lp(a) is specifically bound with high affinity to the same receptors of human fibroblasts as LDL. The affinity and maximum binding capacity are slightly lower for Lp(a) than for LDL. The results of the turnover studies are consistent with the assumption that Lp(a) is removed from the plasma by similar mechanisms as LDL.

Receptors for bradykinin in intact cultured human fibroblasts. Identification and characterization by direct binding study.

Bradykinin receptors on cultured human fibroblasts were characterized using [2,3-prolyl-3,4-3H(N)]bradykinin as radioligand. During incubation with intact fibroblasts, intact [3H]bradykinin was lost much more rapidly at 37 degrees than at 4 degrees C as determined by bioassay, high-performance liquid chromatography, and ion-exchange chromatography, and is likely to be degraded. At 4 degrees, but not at 37 degrees C, bradykinin remained intact in the presence of 2 mM bacitracin, but not in the presence of soybean trypsin inhibitor or SQ-20881, an inhibitor of kininase II. Specific binding at 4 degrees C was saturable with a maximum number of binding sites of 230 +/- 18 fmol/mg protein (mean +/- SE, n = 4) and a dissociation constant of 4.6 +/- 0.5 nM (mean +/- SE, n = 4). Linear Scatchard plots, Hill coefficients close to unity (0.95-1.06), and the failure of excess bradykinin to influence dissociation kinetics are consistent with a single component binding system with no significant cooperativity. Na+ at physiological concentrations and Ca++ or Mg++ at 3-10 mM reduced binding by 25%. The relative potencies of bradykinin analogues and unrelated peptides in competing for [3H]bradykinin binding indicated a specificity of the binding sites consistent with that of a B2 type receptor. Potencies of the peptides in displacing [3H]bradykinin correlated with their abilities to release prostacyclin, determined as its metabolite 6-keto-PGF1 alpha. This system, the first in which bradykinin receptors on human cells have been characterized, should prove useful for investigation of the regulation of bradykinin-influenced biological processes.

Influence of plasmalogen deficiency on membrane fluidity of human skin fibroblasts: a fluorescence anisotropy study.

The influence of plasmalogen deficiency on membrane lipid mobility was determined by measuring fluorescence anisotropy of trimethylammoniumdiphenylhexatriene (TMA-DPH) and diphenylhexatrienylpropanoylhydrazylstachyose (glyco-DPH) inserted in the plasma membranes of human skin fibroblasts deficient in plasmalogens. The cells used were from patients affected with cerebrohepatorenal (Zellweger) syndrome (CHRS) or rhizomelic chondrodysplasia punctata. Their plasmalogen content (0-5% of total phospholipid) is significantly reduced compared with that of control cells from healthy donors (13-15% of total phospholipid) or of CHRS fibroblasts supplemented with the plasmalogen precursor, hexadecylglycerol. Plasmalogen-deficient cells consistently showed lower fluorescence anisotropies of membrane-bound DPH fluorophores corresponding to higher membrane lipid mobilities as compared to controls. However, very similar lipid mobilities were found for sonicated aqueous dispersions of phospholipids extracted either from CHRS or control cells. Therefore, the differences observed with living cells are not due to differences in the overall physical properties of the membrane lipid constituents. Other phenomena such as lipid asymmetry and/or plasmalogen-protein interactions may be responsible for the effects observed in the biomembranes.

B2 bradykinin receptors in cultured neonatal rat cardiomyocytes mediate a negative chronotropic and negative inotropic response.

Receptors for bradykinin (BK) were characterized in primary cultures of beating neonatal rat cardiomyocytes using [3H]BK was radioligand. Degradation studies demonstrated that [3H]BK was stable for at least 2 h when incubated with cardiomyocytes at 2 and 37 degrees C in the presence of bacitracin in combination with captopril or ramiprilat. Without these inhibitors, > 80% of the [3H]BK was degraded within 2 h at 37 degrees C. This indicates that angiotensin-converting enzyme (ACE) is responsible for the main BK-degrading activity in cardiomyocytes. Scatchard plots were linear and gave a Kd of 1.5 +/- 0.8 nmol/l (mean +/- SD, n = 4) and a maximum binding capacity of 55-125 fmol/mg protein. Association and dissociation studies showed that binding of [3H]BK was saturable and reversible. Binding of [3H]BK at 37 degrees C led to internalization of the ligand. Competition studies with B1 and B2 agonists and antagonists were consistent with a B2 subtype of receptor. Addition of BK to beating cardiomyocytes (> 1 nmol/l) at 37 degrees C gave a strong but transient negative chronotropic effect. This response was paralleled by changes in the pulsation amplitude, which indicated a simultaneous negative inotropic effect of BK. These results provide a basis for the hypothesis that ACE inhibition exerts its cardioprotective effect at the level of a population of cardiomyocytes by virtue of kinin receptor-mediated mechanisms.

Evaluation of saliva as a specimen for monitoring zidovudine therapy in HIV-infected patients.

To facilitate studies of the pharmacokinetic properties of zidovudine, the relationship between plasma and salivary concentrations of the drug was studied, after oral dosage, in 10 HIV-infected patients. Zidovudine concentrations were determined in plasma, unstimulated mixed saliva and citric-acid-stimulated mixed saliva over a period of 3 1/2 hours by high-performance liquid chromatography. Correlation coefficients were r = 0.97 (P less than 0.0001) for stimulated saliva compared with plasma and r = 0.89 (P less than 0.0001) for unstimulated saliva, with average values in unstimulated saliva being 113.8 +/- 44.6% in plasma and 67.8 +/- 25.4% in stimulated saliva. Stimulated saliva values found to be 70% of the total reflected the concentration of the unbound drug in plasma. Except for a shorter half-life time (t1/2) in saliva, pharmacokinetic parameters showed a good correlation in the three types of specimen. These findings and the convenience of sample collection suggest that citric-acid-stimulated saliva might be an appropriate specimen for monitoring zidovudine therapy.

Demonstration of receptor binding of two apo-B containing lipoproteins by differential labelling with colloidal gold.

The interaction of two types of apo-B containing lipoproteins, human low density lipoprotein (LDL) and lipoprotein-a (Lp(a], with cultured human skin fibroblasts was studied by differential colloidal gold labelling in conjunction with thin sectioning and surface replication techniques. After separate exposure of the fibroblasts to either gold labelled LDL or Lp(a) for 15 to 30 min at 37 degrees C, labelled lipoproteins were predominantly found in coated pit areas. Excess of unlabelled LDL or Lp(a) completely displaced the gold labelled lipoproteins, indicating specific binding by the LDL-receptor. Simultaneous exposure of fibroblasts to LDL-16 nm gold and Lp(a)-40 nm gold conjugates revealed that both LDL and Lp(a) are bound in the same coated pit and internalized into the same endosome. In contrast to native lipoproteins, gold labelled acetylated lipoproteins were found diffusely distributed on membrane surface areas predominantly representing fibronectin-containing fibrils.

Predicting phenotype in steroid 21-hydroxylase deficiency? Comprehensive genotyping in 155 unrelated, well defined patients from southern Germany.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders. CAH is most often caused by deficiency of steroid 21-hydroxylase. The frequency of CYP21-inactivating mutations and the genotype-phenotype relationship were characterized in 155 well defined unrelated CAH patients. We were able to elucidate 306 of 310 disease-causing alleles (diagnostic sensitivity, 98.7%). The most frequent mutation was the intron 2 splice site mutation (30.3%), followed by gene deletions (20.3%), the I172N mutation (19.7%) and large gene conversions (7.1%). Five point mutations were detected that have not been described in other CAH cohorts. Genotypes were categorized in 4 mutation groups (null, A, B, and C) according to their predicted functional consequences and compared to the clinical phenotype. The positive predictive value for null mutations (ppv(null)) was 100%, as all patients with these mutations had a salt-wasting phenotype. In mutation group A (intron 2 splice site mutation in homozygous or heterozygous form with a null mutation), the ppv(A) to manifest with salt-wasting CAH was 90%. In group B predicted to result in simple virilizing CAH (I172N in homozygous or compound heterozygous form with a more severe mutation), ppv(B) was 74%. In group C (P30L, V281L, P453S in homozygous or compound heterozygous form with a more severe mutation), ppv(C) was 64.7% to exhibit the nonclassical form of CAH, but 90% when excluding the P30L mutation. Thus, in general, a good genotype-phenotype relationship is shown in patients with either the severest or the mildest mutations. A considerable degree of divergence is observed within mutation groups of intermediate severity. As yet undefined factors modifying 21-hydroxylase gene expression and steroid hormone action are likely to account for these differences in phenotypic expression.

Thromboxane A2 analogue U 46619 enhances tumour cell proliferation in HeLa cells via specific receptors which are apparently distinct from TXA2 receptors on human platelets.

In this study, we have demonstrated for the first time by using U 46619, a stable analogue of thromboxane A2 (TXA2), that TXA2 exerts a cell proliferative effect on HeLa cells which is mediated by specific TXA2 receptors, inasmuch as the cell proliferation could be dose-dependently suppressed by TXA2 receptor antagonist BM 13177. The investigation of the phospholipase C pathway by U 46619 and prostaglandin H2 (PGH2) in the presence and absence of BM 13177 in cells with or without pertussis toxin pretreatment, as well as radioligand receptor binding studies, revealed that, in contrast to TXA2 receptors on human platelets, where TXA2 and PGH2 share the same receptor binding sites, HeLa cells possess distinct receptors for TXA2 and PGH2.

[Hyp3]-bradykinin and [Hyp3]-Lys-bradykinin interact with B2-bradykinin receptors and stimulate inositol phosphate production in cultured human fibroblasts.

The recently isolated, naturally occurring peptide hormones [Hyp3]-bradykinin and [Hyp3]-Lys-bradykinin were investigated for their agonist activity on solubilized binding sites from human fibroblasts. Both ligands competed with [3H]bradykinin binding in a dose-dependent fashion with potencies similar to bradykinin (BK) and Lys-BK. Biological activity was assessed by determination of inositol phosphate accumulation and cyclic 3',5'-adenosine monophosphate synthesis in intact cultured cells. Stimulation by the hydroxylated peptides resulted in a pronounced accumulation of both parameters with similar effectiveness as BK and Lys-BK. These results indicate that [Hyp3]-BK and [Hyp3]-Lys-BK are agonists at the bradykinin receptor system with properties comparable to their non-hydroxylated analogues. This suggests that hydroxylation of kinins does not alter receptor interaction or signal transduction in cultured human fibroblasts.

Genomic organization and chromosomal localization of the human peroxisomal membrane protein-1-like protein (PXMP1-L) gene encoding a peroxisomal ABC transporter.

The cDNA of the peroxisomal membrane protein-1-like protein (PXMP1-L, synonyms: PMP69, P70R), a novel peroxisomal ATP binding cassette transporter of yet unknown function, has recently been cloned. The best known peroxisomal member of this protein family is the adrenoleukodystrophy protein, defects of which are the underlying cause of X-linked adrenoleukodystrophy (X-ALD). Here we describe the complete exon-intron structure (19 exons and 18 introns covering 16.0 kb) of the human PXMP1-L gene, transcript variants, the localization on chromosome 14q24 by cytogenetic analysis and sequencing of the putative promoter region. PXMP1-L has been proposed to play a role as a modifier in determining the phenotypic variations observed in X-ALD. The data presented will enable sequence analysis of the PXMP1-L gene in X-ALD patients and facilitate the analysis of PXMP1-L function.

Cloning and characterization of the gene encoding the human peroxisomal assembly protein Pex3p.

Proteins essential for the assembly of functional peroxisomes are designated peroxins and are encoded by PEX genes. In yeast, Pex3p was previously identified as a peroxisomal integral membrane protein indispensable for peroxisome biogenesis and integrity. Here we report the cloning of the orthologous human PEX3 gene. It encodes a polypeptide of 373 amino acids (42 kDa) and is expressed in all tissues examined. As shown by transfection of epitope tagged constructs and immunofluorescence analysis, human Pex3p is localized at the peroxisome. The N-terminal 40 amino acids were revealed to be sufficient to target a GFP reporter protein to the peroxisome. A positively charged five amino acid sequence within this N-terminal region is highly conserved from yeast to human Pex3p. Overexpression of human Pex3p leads to proliferation of ER membranes in COS7 cells. Since disruption of human peroxins has been shown to result in peroxisomal biogenesis disorders, PEX3 is another candidate gene being involved in this disease group.

The mouse gene encoding the peroxisomal membrane protein 1-like protein (PXMP1-L): cDNA cloning, genomic organization and comparative expression studies.

PXMPI-L (synonyms: PMP69, P70R) is a peroxisomal protein that belongs to the ABC-transporter superfamily. Its closest homolog is the peroxisomal membrane protein 1 (PMP70). We have cloned the mouse PXMP1-L gene. It encodes a 606 amino acid protein. In contrast to the human and the rat, mouse PXMP1-L is predominantly expressed in the liver. The mouse PXMP1-L gene consists of 19 exons and spans 21 kb of genomic sequence. No obvious peroxisome proliferator response element has been found in 1.1 kb of the putative promoter region. No coordination of constitutive or fenofibrate-induced expression of PXMP1-L with other peroxisomal ABC transporters was observed so that an obligate exclusive heterodimer formation is not likely to occur. The data presented will be particularly useful for the generation of a mouse model defective in PXMP1-L in order to elucidate the yet unknown function of this protein.

Expression and functional properties of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator fused to glutathione-S-transferase.

CFTR-NBF-2 was expressed in Escherichia coli in fusion with glutathione-S-transferase, the soluble portion was purified and identified as a structured protein by its CD spectrum. Association reactions of the recombinant NBF-2 with adenine nucleotides were monitored qualitatively by demonstrating its ability to bind specifically to ATP-, ADP- and AMP-affinity agarose and quantitatively by recording the fluorescence enhancement of excited trinitrophenol (TNP)-labelled adenine nucleotides occurring as a result of binding to NBF-2. Best-fit monophasic binding curves to the fluorescence data indicated Kd values of 22 microM for TNP-ATP, 39 microM for TNP- ADP and 2.1 microM for TNP-AMP. The corrected Kd values for unlabelled adenine nucleotides competing with the fluorophores were determined to be 37 microM for ATP, 92 microM for ADP and 12 microM for AMP. The recombinant NBF-2 did not show any hydrolytic activity on ATP (detection limit 0.001 s-1). Our findings support the concept of a central role of NBF-2 in CFTR activity regulation acting as an allosteric switch between channel opening and closing and give the first experimental evidence that the channel inhibitor AMP could act via NBF-2.

A recombinant polypeptide model of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator is a GTP-binding protein.

Association reactions of a recombinant CFTR-NBF-2 polypeptide fused to glutathione S-transferase with guanine nucleotides were monitored quantitatively by recording the fluorescence enhancement of excited trinitrophenol (TNP)-labelled GTP after binding to NBF-2. Binding of TNP-GTP to the recombinant NBF-2 polypeptide was characterized by a Kd value of 3.9 microM. The corrected Kd values for unlabelled guanine nucleotides were determined to be 33 microM for GTP, 92 microM for GDP and 217 microM for GMP. TNP-ATP bound to NBF-2 was competitively displaced by GTP indicating a common binding site for both nucleotides. The recombinant NBF-2 did not show an intrinsic GTPase activity above a detection limit of 0.007 min(-1). Our findings provide the first experimental evidence that NBF-2 can act as a GTP-binding subunit that would favor the release of GDP after GTP hydrolysis.

Fish oil supplementation improves visual evoked potentials in children with phenylketonuria.

Visual evoked potentials (VEP) were measured in 36 patients with early-treated phenylketonuria (PKU; aged 1 to 11 years) and good metabolic control before and after supplementation with omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) from fish oil. Patients with PKU had significantly longer P100 latencies than 22 age-matched control subjects. After 3 months of LC-PUFA supplementation, VEP latencies improved significantly in PKU patients but did not change in 12 untreated healthy children. The authors conclude that omega-3 LC-PUFA are essential substrates for nervous system function even beyond infancy.

Synthesis of kininogen and degradation of bradykinin by PC12 cells.

1. In this study, the abilities of PC12 cells to synthesize and degrade kinins were investigated. Kinin formation was assessed as kinin and kininogen content of cells and supernatants in serum-free incubations by use of a bradykinin-specific radioimmunoassay. Expression of kininogen mRNA was demonstrated by reverse-transcriptase PCR. Kinin degradation pathways of intact PC12 cells were characterized by identification of the kinin fragments generated from tritiated bradykinin either in the absence or presence of the angiotensin I-converting enzyme inhibitor ramiprilat. 2. Kinin immunoreactivity in the supernatant of PC12 cell cultures accumulated in a time-dependent fashion during incubations in serum-free media. This effect was solely due to de novo synthesis and release of kininogen (35 pg bradykinin h-1 mg-1 protein) since it could be suppressed by cycloheximide. Continuous synthesis of kininogen was a specific property of PC12 cells, as it was not observed in cultured macro- or microvascular endothelial cells. PC12 cells contained only minor amounts of stored kininogen. The rate of kininogen synthesis was not affected by ramiprilat, bacterial lipopolysaccharide, nerve growth factor or dexamethasone, but was stimulated 1.4 fold when cells were pretreated for 1 day with 1 microM desoxycorticosterone. 3. By use of cDNA probes specific for kininogen subtype mRNAs, expression of low-molecular-weight kininogen and T-kininogen in PC12 cells was confirmed. Expression of high molecular weight kininogen mRNA was also shown, though only at the lowest limit of detection of the assay. 4. Degradation of tritiated bradykinin by PC12 cells occurred with a half-life of 48 min resulting in the main fragments [1-7]- and [1-5]-bradykinin. The degradation rate of bradykinin decreased to 15% in the presence of ramiprilat (250 nM). Apart from angiotensin I-converting enzyme direct cleavage of bradykinin to [1-7]- and [1-5]-bradykinin still occurred under this condition as a result of additional kininase activities. 5. Along with previous findings of B2-receptor-mediated catecholamine release, these results now confirm the hypothesis that a cellular kinin system is expressed in PC12 cells. The presence of such a system may reflect a role of kinins as local neuromodulatory mediators in the peripheral sympathetic system.