Exogenous nitric oxide inhibits apoptosis in guinea pig gastric mucous cells.

BACKGROUND: Increased nitric oxide (NO) synthase activity and enhanced apoptosis are features of gastric mucosa infected with Helicobacter pylori and a causative relation has been suggested. However, although NO can promote apoptosis, its actions vary with cell type. AIMS: To determine whether exogenous NO, derived from an NO donor, might promote or counteract apoptosis in gastric mucous epithelial cells. METHODS: Primary cultures of guinea pig gastric mucosal cells were exposed to the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) for 24 hours. Apoptosis was detected from nuclear staining with Hoechst 33258, in situ nick end labelling of DNA, and the presence of DNA "ladders" in cell extracts. Cyclic GMP content and caspase activity were determined by immunoassay and fluorimetric assay respectively. RESULTS: SNAP 1 mM did not alter the small proportion of cells on the culture plate (3-6%) which exhibited features of apoptosis. However, SNAP produced an inhibition of apoptosis, and of caspase 3 like activity, when enhanced by 25 microM N-hexanoyl-D-sphingosine (C(6)-ceramide), or by detachment of cells from the culture plate. The guanylate cyclase inhibitor, 1H-1, 2, 4-oxadiazole-4, 3-a-quinoxaline-1-one (ODQ), prevented the stimulation of cyclic GMP by SNAP, but not the anti- apoptotic effects of the NO donor. The cyclic GMP analogues 8-bromo-cyclic GMP and 8-(4-chlorophenylthio) guanosine-3',5'- cyclic monophosphate did not significantly inhibit apoptosis in the mucosal cells. CONCLUSIONS: Exogenous NO inhibited apoptosis in guinea pig gastric mucous cells by a mechanism which did not involve elevation of cyclic GMP. NO, if produced from NO synthase during infection with H pylori, may therefore counter the proapoptotic effects of this pathogen.

Effects of four trihalomethanes on DNA strand breaks, renal hyaline droplet formation and serum testosterone in male F-344 rats.

All four possible trihalomethanes (THMs) containing bromine and chlorine, as well as perchloroethylene (PCE), were evaluated for their ability to produce DNA strand breaks, alpha 2u-globulin rich renal deposits, and testosterone changes in male F-344 rats. Rats received daily equimolar doses (0.75 or 1.5 mmol/kg) of THMs or PCE (1000 mg/kg) in 4% Emulphor vehicle by oral gavage for 7 days. No significant DNA strand breaks were produced by any THM or PCE treatment. PCE treatment produced increased hyaline droplet formation in renal tubules. However, all THM treatments reduced or eliminated the appearance of renal hyaline droplets. All four THM treatments also produced a decrease in serum testosterone concentrations on day 7, which might account for decreased hyaline droplet formation. No significant increase in cell proliferation, measured by [3H]thymidine incorporation in vivo, appeared in this 1-week study.

Identification of a novel thrombin receptor sequence required for activation-dependent responses.

Thrombin receptor (TR) activation by alpha-thrombin requires proteolytic cleavage, although synthetic peptides modeled after the new N-terminus directly effect receptor activation without cleavage, presumably by interacting with an unidentified region of the receptor. To further define critical residues responsible for receptor activation, we performed epitope mapping of anti-TR1-160, a previously described polyclonal antibody that inhibits peptide ligand-induced receptor activation in various cell types expressing a functional TR. An enzyme-linked immunosorbent assay (ELISA) using overlapping decapeptides derived from the TR extracellular domains identified four immunodominant peaks within the long N-terminal extension centered between amino acids 34-44, 48-67, 65-79, and 87-94. Soluble peptides derived from regions 83-94, but not those from other regions of the receptor, neutralized the ability of anti-TR1-160 to inhibit peptide ligand-induced platelet aggregation, suggesting that antibodies directed against this region of the TR are important in ligand-mediated activation. Thrombin receptor mutants lacking discrete regions of the TR were subsequently evaluated using microinjected Xenopus oocytes. Whereas a TR mutant lacking amino acid residues Thr67-Lys82 (TR delta 67-82) showed normal to exaggerated responses to either alpha-thrombin or synthetic peptide ligands, only TR mutants with limited deletions spanning the residues Gln83-Ser93 exhibited dysfunctional responses to either agonist (200 nmol/L alpha-thrombin or 200 mumol/L TR42-47). These data provide a model for receptor activation that implicates a discrete and previously uncharacterized sequence within the TR N-terminal extension that is necessary for initiation of signal transduction events independent of the initiating agonist.

The VLA-2 (alpha 2 beta 1) I domain functions as a ligand-specific recognition sequence for endothelial cell attachment and spreading: molecular and functional characterization.

The integrin VLA-2 (alpha 2 beta 1), generally considered to represent the specific collagen receptor on human endothelial cells, contains an alpha 2-subunit inserted I domain with structural similarity to the type A domains found within the recently described superfamily of receptor-ligand recognition proteins. This region of the cDNA has now been isolated and used for molecular and functional characterization of this heterodimeric receptor complex. Comparative sequence analysis with the porcine homologue revealed 93% amino acid sequence identity, suggestive of a developmentally conserved function. To complete structure/function studies, this region of the human cDNA was expressed as a chimeric protein in Escherichia coli, and a rabbit polyclonal antibody (anti-I domain) was used to study determinants of endothelial cell attachment and spreading in vitro. Quantifiable and visual disruption of endothelial cell attachment to gelatin, type I collagen, and laminin was evident using the specific anti-I domain antibody, with minimal inhibitory effects demonstrable using fibronectin or fibrinogen matrices. Therefore, these data would suggest that the alpha 2 beta 1 I domain confers ligand-binding specificity for both known alpha 2 beta 1 substrates (laminin and collagen), and that this region subserves a regulatory function in the molecular processes controlling endothelial cell attachment and spreading in vitro.

Chromosomal assignment of the human thrombin receptor gene: localization to region q13 of chromosome 5.

A functional thrombin receptor (TR) structurally related to other members of the seven-transmembrane receptor family has been isolated from diverse cellular types intimately involved in the regulation of the thrombotic response. This receptor recapitulates many of the previously identified sequelae of thrombin-mediated cell activation phenomenon, and requires proteolytic cleavage for downstream effector-response coupling events. Using two complementary approaches, we have now completed the chromosomal assignment of the human thrombin receptor gene. Discordancy analysis of polymerase chain reaction products from a human-rodent hybrid cell mapping panel assigned the sequence to human chromosome 5 with no observed discordancies. Cytogenetic localization using fluorescence in situ hybridization on human metaphase chromosomes specifically localized the human TR gene to region q13 of chromosome 5, confirming its presence as a single-locus gene in the human genome. The chromosomal localization of the human TR gene is at or contiguous with the proximal breakpoint site identified in the majority of patients with the 5q- syndrome (dysmegakaryocytopoiesis and refractory anemia).

The thrombin receptor extracellular domain contains sites crucial for peptide ligand-induced activation.

A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize alpha-thrombin- and peptide ligand-induced activation responses using two different antibodies: anti-TR34-52 directed against a 20-amino acid peptide spanning the thrombin cleavage site, and anti-TR1-160 generated against the NH2-terminal 160 amino acids of the TR expressed as a chimeric protein in Escherichia coli. Activation-dependent responses to both alpha-thrombin (10 nM) and peptide ligand (20 microM) were studied using fura 2-loaded cells and microspectrofluorimetry. Whereas preincubation of CHO-TR with anti-TR34-52 abolished only alpha-thrombin-induced [Ca2+]i transients, preincubation with anti-TR1-160 abrogated both alpha-thrombin- and peptide ligand-induced responses. This latter inhibitory effect was dose dependent and similar for both agonists, with an EC50 of approximately 90 micrograms/ml. Anti-TR1-160 similarly abolished peptide ligand-induced [Ca2+]i transients in platelets and HUVECs, whereas qualitatively different responses characterized by delayed but sustained elevations in [Ca2+]i transients were evident using alpha-thrombin. Platelet aggregation to low concentrations of both ligands was nearly abolished by anti-TR1-160, although some shape change remained; anti-TR34-52 only inhibited alpha-thrombin-induced aggregation. These data establish that a critical recognition sequence for peptide ligand-mediated receptor activation is contained on the NH2-terminal portion of the receptor, upstream from the first transmembrane domain. Furthermore, alpha-thrombin-induced activation of HUVECs and platelets may be partially mediated by an alternative mechanism(s) or receptor(s).

Quantitative image cytometry of hepatocytes expressing gamma-glutamyl transpeptidase and glutathione S-transferase in diethylnitrosamine-initiated rats treated with phenobarbital and/or phthalate esters.

Image cytometry was used to quantify the volume of liver expressing two histochemical markers associated with neoplasia, gamma-glutamyl transpeptidase (GGT) and the placental isozyme of glutathione S-transferase (GST-P). Rats were treated with diethylnitrosamine (DENA) followed by phenobarbital (PB), di(2-ethylhexyl)phthalate (DEHP), or di-n-octyl-phthalate (DOP) for 26 weeks. In one series, PB-treated rats were given 2.0%, 0.5%, or 0.1% DEHP in the feed. GGT expression was detected diffusely throughout the liver parenchyma in several treatment groups so that any enhanced expression in altered foci (AF) and nodules (N) was not apparent. GST-P was detected only in AF and N. GST-P may represent a second genetic alteration, as GST-P+ AF and N also expressed GGT but not the reverse. The peroxisome proliferator DEHP inhibited expression of GGT or GST-P in livers of either DENA-treated or DENA+PB-treated rats. With GST-P the reduction was correlated to a reduced number of AF and N. In contrast, DEHP's stereoisomer, DOP, was as effective as PB in promoting expression of both markers. We conclude that image cytometry of hepatocytes expressing GST-P can be used in the bioassay of the carcinogenic potential of chemicals that affect liver proliferation.

The role of hyperplastic nodules in dichloroacetic acid-induced hepatocarcinogenesis in B6C3F1 male mice.

Dichloroacetic acid (DCA) has recently been shown to increase significantly the incidence of hepatic adenomas (HAs) and hepatocarcinomas (HCs) in male B6C3F1 mice. Although little is known about the mechanism of DCA carcinogenesis, chronic ingestion of the compound in drinking water induces primarily hyperplastic nodules (HNs) prior to the appearance of HAs and HCs. Given the putative preneoplastic potential of the HNs, we undertook this study to determine the role of the HNs in the progression of DCA-induced hepatocarcinogenesis. This role was assessed by detecting the expression of five different tumor markers: p21 ras, p39 c-jun, phosphotyrosine, tumor-associated aldehyde dehydrogenase and alpha-fetoprotein, all known from previous studies to be expressed more often in neoplastic liver lesions than in normal liver. Tumor marker expression was detected by immunohistochemical methods using formalin-fixed, paraffin-embedded sections of normal B6C3F1 mouse liver, and DCA-induced HNs, HAs and HCs. The results demonstrated that, except for the c-jun marker, HNs expressed the markers significantly less often than either HAs or HCs. Equal expression of c-jun occurred in any of the three lesion types. Although these results could be used to argue that no relationship existed between HNs and later-appearing HAs and HCs, those HNs that were marker positive contained small nests of marker-positive hepatocytes among a field of normally appearing unstained hepatocytes. No similar nests of marker-positive cells were detected in any area of normal liver outside the HNs. Also very few altered hepatic foci (AF) were detected with these markers or with hematoxylin and eosin, or with histochemical stains for ATPase or glucose-6-phosphatase deficiencies. These results suggested that these nests within some HNs were areas of transformed, or neoplastic hepatocytes. Phenotypic heterogeneity analysis, in which the number of tumor markers co-expressed by any given lesion was examined, confirmed a significantly greater percentage of HAs and HCs expressing multiple markers than HNs. Those HNs that expressed multiple markers, however, expressed at the same frequency as HAs and HCs and the expression was confined to the same nests of cells. Taken together, these data suggest that these nests of marker-positive cells within the HNs were neoplastic and could develop into later-appearing HAs and/or HCs. The absence of marker expression in normal liver and limited expression in the few AF indicates that the HNs may be the only significant preneoplastic lesion in DCA-induced hepatocarcinogenesis.

Aldosterone production and hormone responsiveness in adrenal glomerulosa cells from cows of different ages.

Aldosterone production by suspensions of adrenal glomerulosa cells obtained from young and old cows was measured. Basal steroidogenesis was lower in cells from old cows, as were the responses to angiotensin II (AII), potassium, ACTH and dibutyryl cyclic AMP. Receptors for AII and aldosterone production from added progesterone were the same in old and young cells. Synthesis of pregnenolone from endogenous precursor, the 'early pathway' of aldosteronogenesis, was lower in old cells than in young. AII-stimulated incorporation of 32P into phosphatidylinositol and the change in 45Ca2+ flux induced by AII were diminished in old cells. Overall protein synthesis, measured by 3H leucine incorporation, was lower in old cells than young, but was not affected by AII in either. Diminished responsiveness of adrenal glomerulosa cells from old animals results from a change in postreceptor events that affect the early pathway of aldosteronogenesis.

Thyroid status and thermogenesis in rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Several key aspects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity resemble the effects of hypothyroidism, while in other ways the toxic responses are characteristics of hyperthyroidism. Whether thyroid dysfunction plays a role in TCDD toxicity remained unknown, however. We therefore determined the dose-related effects of TCDD treatment on plasma concentrations of L-thyroxine (T4), 3,5,3'-triiodo-L-thyronine (T3), and thyroid-stimulating hormone (TSH), and compared these changes with signs of TCDD toxicity. We also determined whether indices of functional thyroid status (and thermogenesis) were altered in response to TCDD treatment. Young adult male Sprague-Dawley rats were given single oral doses of TCDD (6.25-100 micrograms/kg) and evaluated 1 week later. Toxicity, measured by decreases in feed intake and body weight, ranged from minimal to severe. Plasma concentrations of T4 were greatly reduced at all doses tested, while T3 was increased in a dose-related fashion (up to 35%). TSH was elevated but was inversely proportional to dose. Thyroid histology was unremarkable, and TCCD treatment had little effect on the ability of rats to raise serum T4, T3, and TSH concentrations in response to acute cold stress. TCDD treatment caused a slight (8%) decrease in basal metabolic rate, yet comparable decreases were seen in pair-fed control animals. Thermogenesis, as measured by O2 consumption and colonic temperatures in rats exposed to various ambient temperatures, was only marginally affected. In summary, although thyroid hormone concentrations were markedly altered, rats given doses of TCDD sufficient to cause overt toxicity appeared to be essentially euthyroid. These results do not support proposals by other researchers that altered thyroid status is a major contributor to TCDD toxicity and/or a key response to TCDD exposure.

Relationship of alterations in energy metabolism to hypophagia in rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Efficiency of energy utilization was evaluated temporally in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 50 micrograms/kg)-treated male their pair-fed counterparts, and a group with ad libitum access to ground feed. TCDD-treated rats exhibited a progressive reduction in feed intake and body weight. The weight loss of vehicle-treated rats, pair-fed to the TCDD-treated group, was comparable to that found in rats receiving TCDD. Following treatment, rats administered TCDD were as efficient in absorbing feed energy from the gut as control rats. This was evidenced by similar relative relative digestible energy values in TCDD-treated rats, their pair-fed partners, and a group with ad libitum access to feed. Equivalent decreases in oxygen consumption and carbon dioxide production in TCDD-treated rats and their pair-fed counterparts, relative to rats with ad libitum access to feed, suggested that the decrease in both of these parameters in TCDD-treated rats was secondary to hypophagia and/or weight loss. Decline of respiratory quotient (RQ) to almost 0.7 in both TCDD-treated rats and their pair-fed counterparts is indicative of fat combustion. By Day 17 post-treatment, RQ increased significantly in the TCDD-treated and pair-fed groups possibly due to a limitation in the availability of lipid stores. Also, TCDD-treated rats and their pair-fed partners diminished their water intake to a similar extent without reducing urine output. Likewise, urinary excretion of both energy and urea was decreased to the same extent in rats treated with TCDD as it was in their pair-fed counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of two hexachlorobiphenyl isomers on ornithine decarboxylase induction: inhibitory effects correlate with aryl hydrocarbon hydroxylase activity.

The effects of 2,2',4,4',5,5'-hexachlorobiphenyl (2,4,5-HCB) or 3,3',4,4',5,5'-hexachlorobiphenyl (3,4,5-HCB) on hepatic ornithine decarboxylase (ODC) induction by dexamethasone were investigated. At one week after a single i.p. dose of corn oil or 2,4,5,-HCB and 4 h after administration of dexamethasone, rats exhibited 50- to 60-fold increases of ODC activity. However, rats that had received 3,4,5-HCB in place of 2,4,5-HCB exhibited only a 8-fold increase in ODC activity in response to dexamethasone administration. 2,4,5-HCB administration resulted in increased hepatic aryl hydrocarbon hydroxylase (AHH) activity. Administration of 3,4,5-HCB produced increased AHH activity and decreased N-demethylase activity. It is suggested that the ODC-inhibitory effects may have resulted from Ah-receptor-mediated events.

Androgenic deficiency in male rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the male reproductive system were investigated. Sexually mature (290 g) Sprague-Dawley rats were given single oral doses of TCDD sufficient to cause varying degrees of hypophagia and impaired body weight gain. The largest doses decreased plasma testosterone and dihydrotestosterone concentrations by 90 and 75%, respectively, from ad libitum-fed control values, while decreasing seminal vesicle and ventral prostate weights by 68 and 48%. On Day 7, the approximate ED50 for these responses was 15 micrograms TCDD/kg, a nonlethal dose. Reductions in caput epididymis and testis weights were also observed. The androgenic deficiency was seen as early as 2 days after dosing and persisted for at least 12 days. Based on data from pair-fed control rats, only about half the decreases in accessory sex organ weights and in plasma androgen concentrations could be accounted for by TCDD-induced hypophagia or body weight loss. These signs of androgenic deficiency were not the result of stress (based in part on plasma corticosterone assays), nor could they be accounted for by the known effects of TCDD on steroid metabolism. While the TCDD-induced depression in plasma testosterone concentrations appears to be the primary event observed, the mechanism by which testosterone concentrations were decreased remains unknown. The androgenic deficiency may account for the male reproductive pathology and dysfunction in animals treated with overtly toxic doses of TCDD.

Hypothyroxinemia and hypothermia in rats in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin administration.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment on body temperature and serum and tissue levels of thyroid hormones, glucose, glucagon, insulin, and somatostatin were investigated. Within 7 days following TCDD administration (45 micrograms/kg), rats exhibited hypothyroidism compared to pair-fed controls and rats fed ad libitum. Body temperature was maintained in the pair-fed and ad libitum-fed controls but was significantly decreased in TCDD-treated rats at 2 days. Within 2 weeks of the administration of 90 micrograms TCDD/kg, body temperature was below 35 degrees C with the lowest mean value of 34.5 degrees C recorded on Day 16. Mean body temperatures for control rats ranged from 36.8 to 37.5 degrees C. One week after TCDD administration (45 micrograms/kg), serum thyroxine (T4) declined to 46% of pair-fed controls. The decreased free-thyroxine index indicated that the measured decrease in thyroxine reflected decreased hormone concentrations as opposed to altered protein binding. Hypoglycemia occurred in TCDD-treated rats subsequent to hypothyroxinemia and hypothermia, but it did not develop in the pair-fed controls. At 1 week after administration of 45 micrograms TCDD/kg, serum and pancreatic insulin levels were reduced to 25 and 76% of ad libitum-fed controls, respectively. Hypophagia was determined to be responsible for the decreased growth rate and hypoinsulinemia but did not account for hypothyroxinemia, hypothermia, and hypoglycemia following the administration of TCDD. No significant alterations were detected in serum glucagon or in pancreatic, hepatic, or serum somatostatin levels. Decreased somatostatin in the gastric antrum coincided with a 29% increase in stomach dry weight. The delayed toxicity of TCDD may result, in part, from these hormonal alterations.

Inhibition of ornithine decarboxylase activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the activity of rat liver ornithine decarboxylase (ODC) were investigated. Sixteen hours after partial hepatectomy, rats that had been pretreated with TCDD for 1 week exhibited a 3- to 4-fold increase in ODC activity, while vehicle controls exhibited to 8- to 10-fold increase. This inhibition of ODC induction by TCDD was time dependent since TCDD administration at the time of partial hepatectomy did not produce inhibitory effects on the subsequent ODC induction. ODC induction after either aminophylline or dexamethasone administration, agents which act via cAMP-mediated and direct nuclear events, respectively, also was inhibited by pretreatment with TCDD. It was concluded that TCDD pretreatment decreased the ability of the liver to respond to hormonal stimulation as reflected in the attenuation of ODC induction. RNA polymerase I activity, which positively correlates with ODC activity in growth and development, decreased concomitantly with decreased induction of ODC. In unstimulated liver RNA polymerase I activity, as well as protein, DNA, and RNA levels, remained unchanged 1 week after TCDD. However, TCDD administration resulted in decreased liver concentrations of putrescine and spermidine, but not spermine. This suggests that TCDD pretreatment results in a time-dependent decrease in hormone responsivity.