Gas chromatography/mass spectrometry in metabolic profiling of biological fluids.

One of the objectives of metabonomics is to identify subtle changes in metabolite profiles between biological systems of different physiological or pathological states. Gas chromatography mass spectrometry (GC/MS) is a widely used analytical tool for metabolic profiling in various biofluids, such as urine and blood due to its high sensitivity, peak resolution and reproducibility. The availability of the GC/MS electron impact (EI) spectral library further facilitates the identification of diagnostic biomarkers and aids the subsequent mechanistic elucidation of the biological or pathological variations. With the advent of new comprehensive two dimensional GC (GC x GC) coupled to time-of-flight mass spectrometry (TOFMS), it is possible to detect more than 1200 compounds in a single analytical run. In this review, we discuss the applications of GC/MS in the metabolic profiling of urine and blood, and discuss its advances in methodologies and technologies.

Identification of novel genes that are differentially expressed in human colorectal carcinoma.

By using mRNA differential display technology, we have identified three cDNA clones, myl 3, myl 4, and myl 6, to show significant changes in expression between human colorectal tumor and paired normal tissue. Northern blot analysis indicated that clone myl 3 expression was elevated in normal tissue, and clone myl 4 expression was elevated in tumor tissue. Nucleotide sequence analysis revealed that clones myl 3 and myl 4 have not been previously identified. However, clone myl 6 appears to be the human homolog of the 3' end region of tissue inhibitor of metalloproteinase 3 (TIMP-3). Northern blot analysis showed that a 2.5 kb TIMP-3 transcript was expressed at a much higher level in normal tissue than the colorectal tumor.

Modeling Caco-2 permeability of drugs using immobilized artificial membrane chromatography and physicochemical descriptors.

This study evaluates the potential of immobilized artificial membrane (IAM) chromatography, in combination with other physicochemical descriptors for high-throughput absorption profiling during lead optimization. An IAM chromatographic method was developed and validated. Absorption profiles of 32 structurally diverse compounds (acidic, basic, neutral and amphoteric) were then evaluated based on their IAM retention factor (log k'IAM), molecular weight (MW), calculated log P (C log P), polar surface area (PSA), hydrogen bonding capacity (HBD and HBA) and calculated Caco-2 permeability (QPCaco). Using regression and stepwise regression analysis, experimental Caco-2 permeability was correlated against log k'IAM and a combination of various physicochemical variables for quantitative structural-permeability relationship (QSPR) study. For the 32 structurally diverse compounds, log k'IAM correlated poorly with Caco-2 permeability values (R2 = 0.227). Stepwise regression analysis confirmed that Clog, PSA, HBD and HBA parameters are not statistically significant and can be eliminated. Correlation between Caco-2 cell uptake and log k'IAM was enhanced when molecular size factor (MW) was included (R2 = 0.555). The exclusion of 11 compounds (paracellularly and actively transported, Pgp substrates and blocker, and molecules with MW lesser than 200 and greater than 800) improved the correlation between Caco-2 permeability, IAM and MW factors to R2 value of 0.84. The results showed that IAM chromatography can only profile the passive absorption of drug molecules. Finally, it was confirmed in this study that the IAM model can accurately identify the Caco-2 permeability of nontransported Pgp substrates, such as verapamil and ketoconazole, through passive permeation because of their high permeability. IAM chromatography, combined with molecular size factor (MW), is useful for elucidating biopartitioning mechanism of drugs.

Immunoreactive methionine-enkephalin in cerebrospinal fluid and blood plasma during acute stress in conscious sheep.

The opioid peptide methionine-enkephalin (Met-enkephalin) was measured in plasma and cerebrospinal fluid (CSF) of sheep in which the cisterna magna, carotid artery, and jugular vein were chronically cannulated. Venous blood plasma and CSF were collected before and after stress treatment and in control studies in conscious animals. Plasma and CSF were extracted with octadecylsilica and oxidized, and Met-enkephalin was measured as its Met-sulfoxide derivative by specific RIA. The molecular form of immunoreactive Met-enkephalin was characterized by peptide size exclusion chromatography of an octadecylsilica extract of sheep plasma through Bio-Gel P2, followed by reverse phase liquid chromatography, and was identical to Met-enkephalin and Met-sulfoxide-enkephalin. Insulin-induced hypoglycemia produced an elevation of plasma cortisol and an increase in the plasma concentration of Met-enkephalin. Acute hemorrhage led to an earlier and greater rise in plasma cortisol than that associated with insulin-induced hypoglycemia, but did not increase the concentration of Met-enkephalin in plasma. Neither form of acute stress increased the concentration of Met-enkephalin in CSF. These studies confirm that secretion of Met-enkephalin into blood can be dissociated from stimulation of the pituitary-adrenocortical system. They also show that circulating Met-enkephalin is elevated in conscious sheep during acute hypoglycemic stress, but plasma Met-enkephalin is unlikely to exert effects on the opiate receptors of periaqueductal or spinal nociceptive neurons under these conditions, since it does not enter cerebrospinal fluid in significant amounts.

Acute hemorrhagic stress in conscious sheep elevates immunoreactive beta-endorphin in plasma but not in cerebrospinal fluid.

The effects of acute hemorrhagic stress on the concentrations of immunoreactive beta-endorphin (IR beta EP) in cerebrospinal fluid (CSF) and blood plasma were investigated in conscious sheep in which the cisterna magna, a carotid artery, and a jugular vein were chronically cannulated. Serial samples of CSF and jugular venous blood were collected before and after acute arterial hemorrhage and in control experiments. Basal concentrations of IR beta EP were higher in plasma than in CSF. Plasma concentrations of cortisol and IR beta EP increased within 45 min of the commencement of hemorrhage and returned to near baseline levels within 2.25 h. The concentrations of cortisol and IR beta EP in plasma observed after hemorrhage were significantly different from those observed in controls (analysis of variance). Neither the molar nor the relative changes from initial concentrations of IR beta EP in CSF were significantly different between hemorrhage-stressed and controls by analysis of variance. These results show that hemorrhagic stress in conscious sheep elevates concentrations of IR beta EP in plasma but not in CSF, indicating that pituitary beta EP secreted into blood does not enter CSF in significant amounts.

A corticotropin-releasing hormone type I receptor antagonist delays parturition in sheep.

In sheep, corticotropin-releasing hormone (CRH) can stimulate the fetal release of ACTH to produce a cortisol surge which leads to the onset of parturition. We tested the hypothesis that fetal CRH is a primary factor in the onset of parturition in sheep by using a Type I CRH receptor antagonist, antalarmin, to block the endogenous action of CRH. Pregnant ewes were cannulated at 130-135 days of gestation. Five catheters were placed into the amniotic sac, fetal femoral artery, fetal tarsal vein, maternal jugular vein and carotid artery. After 5 days' recovery, blood samples from maternal and fetal vessels were collected at the following times: a day before the start of infusion, at [-1, 0, 1, 2, 4, 8 and 24]h, on the first day of infusion, and thereafter daily throughout a 10-day infusion. Animals (n=6 per group) received infusions into a fetal vein of either a vehicle comprising 1:1 mixture of ethanol and polyethoxylated castor oil (Cremophor EL) or antalarmin (50 g/L) in the vehicle at a rate of 0.3 mL/h. The plasma samples were assayed for ACTH and cortisol using commercial RIA kits. Fetuses infused with vehicle delivered at a mean gestational age of 141.8 +/- 0.9 days compared with antalarmin-infused sheep at 148.8 +/- 1.6 days (P = 0.0036, unpaired Student's t-test). Fetal ACTH and cortisol did not change in the antalarmin-infused sheep after 3 days' infusion compared to significant increases in vehicle-infused sheep (P=0.004 and P = 0.016 respectively, ANOVA). These data show that CRH receptor antagonism in the fetus can delay the onset of parturition. It supports the hypothesis that hypothalamic CRH drives fetal production of ACTH and is essential for the onset of parturition triggered by a surge in fetal cortisol.

Beta-endrophin immunoreactivity during human pregnancy.

The human placenta has been implicated as a source of numerous peptide hormones during pregnancy. Since the immunoassay detection of the proopiomelanocortin derived peptide beta-endorphin (beta E) in placental extracts in 1978, it has remained uncertain whether placental beta E immunoreactivity (IR) is 1) secreted into the maternal circulation and 2) opiate receptor active during pregnancy. To elucidate the nature of beta E IR in the placenta, both beta E IR and N-alpha-acetylated beta E (Ac beta E) IR were simultaneously measured in extracts of human pituitaries, placentas, and plasma by two homologous RIAs. Pituitary extracts (n = 6) contained 38 +/- 7 nmol beta E IR per g wet wt tissue (mean +/- SEM), of which only 20 +/- 4 pmol/g were Ac beta E IR. Term placental extracts (n = 19) had 201 +/- 30 fmol/g wet wt total beta E IR and 30 +/- 3 fmol/g wet wt total Ac beta E IR, which comprised 15% of total beta E IR in placental extracts. Total plasma beta E IR rose from 28 weeks gestation (8.5 +/- 0.3 fmol/mL, n = 159) to peak at labor (50 +/- 4 fmol/mL, n = 98; P < 0.01) but total Ac beta E IR was found in only four 28-week (1.7 +/- 0.9 fmol/mL) and 42 labor plasma samples (0.9 +/- 0.1 fmol/mL). Gel filtration chromatography of placental and pituitary extracts showed that while less than 1% of the beta E31-size material was acetylated in the pituitary, up to 60% of the beta E31-size material in placental extracts was acetylated. In pooled third trimester plasma extracts, however, only 4% of the beta E31-size material was acetylated. Furthermore, the ratio of beta E31:beta-lipotropin in pituitary extracts (n = 3) was 0.5; pooled plasma-0.5, and placental extracts (n = 5)-1.2. These data indicate that 1) the placenta extensively N-alpha-acetylates beta E31 destroying its opiate bioactivity while the pituitary does not; 2) beta E IR in pregnant women's plasma is similar to pituitary beta E IR, being mostly nonacetylated and similar in size to beta-lipotropin. These findings are consistent with a pituitary source for the elevated plasma beta E IR found during late pregnancy which may, in turn, be a consequence of elevated plasma concentrations of placentally secreted plasma corticotropin-releasing factor IR present during the third trimester.

Nitric oxide inhibits corticotropin-releasing hormone exocytosis but not synthesis by cultured human trophoblasts.

Nitric oxide (NO) plays an important role in many cell-cell signaling systems, but its mechanism of action is variable. We have previously reported that NO reduces secretion of the peptide hormone, CRH, from cultured placental cells and the perfused placenta. Because placental CRH production seems linked to human parturition, we wished to explore the mechanism of action of NO in this setting in more detail. We report here that in the placenta, NO specifically inhibited CRH exocytosis, not synthesis, and that endogenous NO affects this process. Cytotrophoblasts were prepared from term human placentas and cultured as monolayers. CRH immunoreactivity in the cell supernatants and cell extracts were measured by RIA. CRH messenger RNA was determined by Northern blot analysis. Sodium nitroprusside (SNP; 1-100 mumol/L) and S-nitroso-N-acetyl-penicillamine (SNAP; 1-100 mumol/L), NO donors, significantly reduced basal CRH concentration in the media, while increasing the concentration of CRH in the cells (P < 0.01), suggesting that exocytosis of CRH was inhibited. These effects could be attenuated by the NO scavenger hemoglobin (20 micrograms/mL). KCl (45 mmol/L), which causes exocytosis by depolarizing the cell membrane, increased CRH release by 2- to 3-fold, and this was inhibited by SNP. Basal release of CRH was augmented by the NO synthase competitive inhibitor N omega-L-arginine methyl ester (1 mmol/L; P < 0.01) and the guanylate cyclase inhibitor, LY83583 (1 mumol/L; P < 0.01). The inhibitory effect of SNP was also blocked by LY83583. CRH messenger RNA content did not change when the placental cells were incubated with SNP, N omega-L-arginine methyl ester, and LY83583 for 6 and 24 h, and this was consistent with studies showing that total CRH immunoreactivity (cells plus media) did not change in the presence of SNP. These studies indicate that exogenous NO inhibits CRH exocytosis, rather than biosynthesis, by human trophoblasts and that endogenous NO has tonic inhibitory effects on CRH release by these cells. The inhibitory effect of NO on basal and stimulated CRH release by placental trophoblasts seems to be a guanylate cyclase-mediated inhibition of exocytosis.

Corticotropin-releasing hormone gene expression in primary placental cells is modulated by cyclic adenosine 3',5'-monophosphate.

CRH, the principal neuropeptide regulator of pituitary ACTH secretion, is also expressed in placenta. Placental CRH has been linked to the process of human parturition. However, the mechanisms regulating transcription of the CRH gene in placenta remain unclear. cAMP signaling pathways play important roles in regulating the expression of a diverse range of endocrine genes in the placenta. Therefore, we have explored the effect of cAMP on CRH promoter activity in primary cultures of human placental cells. Both forskolin and 8-bromo-cAMP, activators of protein kinase A, can increase CRH promoter activity 5-fold in transiently transfected human primary placental cells, in a manner that parallels the increase in endogenous CRH peptide. Maximal stimulation of CRH promoter activity occurs at 500 micromol/L 8-bromo-cAMP and 10 micromol/L forskolin. Electrophoretic mobility shift assay and mutation analysis combined with transient transfection demonstrate that in placental cells cAMP stimulates CRH gene expression through a cAMP regulatory element in the proximal CRH promoter region and involves a placental nuclear protein interacting specifically with the cAMP regulatory element.

Corticotropin-releasing hormone directly and preferentially stimulates dehydroepiandrosterone sulfate secretion by human fetal adrenal cortical cells.

Estrogens produced by the placenta play a pivotal role in the endocrine control of pregnancy and induce many of the key changes involved in parturition. The placentae of humans and higher primates use the C19 androgen dehydroepiandrosterone sulfate (DHEA-S) supplied by the fetal adrenals as the principal substrate for estrogen synthesis. Thus, secretion of androgens by the fetal adrenals may be central to the process of primate parturition. The timing of human parturition also is correlated with placental CRH concentrations in the maternal circulation. Because the mechanisms that regulate DHEA-S production by the fetal adrenals are incompletely understood, we examined whether there is a functional relationship between CRH and steroid production by human fetal adrenal cortical cells. Using Northern blot analysis, we detected messenger RNA transcripts (2.7 kb) encoding the type-1 CRH receptor in total RNA extracted from midgestation human fetal adrenals, suggesting that the fetal adrenal cortex may be directly responsive to CRH. To test this, primary cultures of human fetal adrenal cortical cells were exposed to human CRH. Human CRH increased DHEA-S production by cultured human fetal adrenal cortical cells in a dose-dependent fashion, with an ED50 of 10-100 pmol/L. Human CRH was as effective as ACTH at stimulating DHEA-S production; however, it was 70% less potent than ACTH at stimulating cortisol production, indicating that its actions were preferentially directed toward increasing DHEA-S synthesis. Consistent with this thesis, we found that CRH increased abundance of messenger RNA encoding cytochrome P450 cholesterol side-chain cleavage and 17alpha-hydroxylase/17,20 lyase but not 3beta-hydroxysteroid dehydrogenase in adrenal cells. CRH did not alter cell number, indicating that it is not mitogenic for fetal adrenal cortical cells. These data demonstrate a direct functional interaction between CRH and the fetal adrenal. Therefore, placental CRH production, which rises exponentially during human pregnancy, may play a key role in promoting DHEA-S production by the fetal adrenals, which could lead to increasing placental estrogen synthesis and contribute to the process of parturition in humans.

Glucocorticoid stimulation of corticotropin-releasing hormone gene expression requires a cyclic adenosine 3',5'-monophosphate regulatory element in human primary placental cytotrophoblast cells.

Production of placental CRH, which is identical to the peptide synthesized and secreted in the hypothalamus, has been linked to human parturition. Glucocorticoids stimulate placental CRH secretion and messenger ribonucleic acid expression, in contrast to their inhibition of CRH synthesis in the hypothalamus. A positive feedforward loop involving glucocorticoid-CRH-ACTH-glucocorticoid is thought to drive the exponential increase in placental CRH leading to delivery. Tissue-specific effects of glucocorticoids on CRH expression are therefore of interest. Using human primary placental cells, we investigated the mechanism by which glucocorticoids stimulate placental CRH gene expression. Nuclear run-on transcription shows that in human placental cells glucocorticoids up-regulate transcription of human CRH (hCRH). Using transient transfection assays we demonstrate that dexamethasone up-regulates both basal and cAMP-stimulated hCRH promoter activity, correlating well with the increase in endogenous CRH peptide levels. Through mutagenesis and deletion analyses we show that dexamethasone stimulation of hCRH gene transcription requires a functional cAMP regulatory element (CRE); this CRE is adequate to confer dexamethasone stimulation upon a heterologous promoter, and electrophoretic mobility shift assay studies show that a placental nuclear protein specifically binds to the hCRH CRE.

Corticotropin-releasing hormone in baboon pregnancy.

During human pregnancy, plasma CRH immunoreactivity (CRH-IR) rises progressively, peaking during labor and falling after delivery. Among animal species, only higher primates have elevated CRH-IR during pregnancy. This study examines whether changes in plasma CRH-IR in the baboon (Papio hamadryas) are similar to those in the human. CRH-IR was determined by RIA in 16 baboons at different stages of gestation (44 samples) and in 3 males. Assays were performed on Vycor extracts of plasma and CRH-IR diluted in parallel to synthetic human (h) CRH-41 standard. Reverse phase high pressure liquid chromatography and size-exclusion chromatography with Sephadex G-50 showed that baboon CRH-IR eluted in a position similar to that of hCRH-41. Regression analysis revealed a cubic association between plasma CRH-IR and gestational age, with peak concentrations occurring at 60 days gestation (term = 182 days). Although greatly elevated concentrations persisted throughout pregnancy, concentrations in the first half (1-91 days) were significantly higher (mean +/- SEM, 1.9 +/- 0.3 nM/L; n = 27) than in the second half (92-182 days; 1.0 +/- 0.2 nM/L; n = 11; P < 0.003 by t test). CRH-IR fell to low levels by day 1 postpartum. The concentration of total cortisol in nonpregnant animals was 1370.9 +/- 134.9 nM/L (n = 5), which was similar to pregnancy levels (1346.3 +/- 356.1 nM/L; n = 28); there was no gestational age-related pattern evident. Plasma corticosteroid-binding globulin was estimated by RIA, and plasma free cortisol was calculated to be 73 +/- 14 nM/L in pregnant animals and showed no gestational age-related changes. The mean progesterone concentration in the pregnant baboon was 12.5 +/- 2.2 nM/L (7-169 days; n = 27). There was no significant change in progesterone levels during the period of gestation studied; however, they were higher than nonpregnant levels. Baboon and human plasma (0.1 mL each) were incubated with [125I]Tyr-hCRH in Tris-HCl buffer (pH 7.5) and chromatographed with Sephadex G-75, using the same buffer. The radioactivity of fractions was determined, and no CRH-binding protein was identified in baboon plasma. This study indicates that gestational changes in CRH-IR in the baboon are different from those observed in humans. There is a dissociation between maternal plasma CRH and cortisol. The apparent lack of bioactivity of baboon plasma CRH is not due to a circulating binding protein, which is absent in this species.

Vacuolating toxin from Helicobacter pylori activates cellular signaling and pepsinogen secretion in human gastric adenocarcinoma cells.

We investigated cellular signaling and pepsinogen secretion in the human gastric adenocarcinoma cell line AGS which was pretreated with the purified vacuolating cytotoxin from Helicobacter pylori. Results indicated that vacuolating toxin increased the levels of inositol phosphates, cytosolic free calcium concentration, adenosine 3',5'-cyclic monophosphate, and phosphorylation of 31 kDa and 22 kDa proteins in the host cells. Moreover, pepsinogen secretion from AGS cells was stimulated with increasing concentrations of cytotoxin. We conclude that besides the H. pylori cytotoxin-induced cellular vacuoles, cytotoxin-stimulated signaling mediators and pepsinogen release are important factors involved in the etiology of chronic active gastritis and peptic ulcer disease.

Identification of neutral and acidic sphingomyelinases in Helicobacter pylori.

We demonstrated for the first time the presence of sphingomyelinase (SMase) in Helicobacter pylori. Activation of SMase has been implicated as the cause of elevation of cellular ceramide levels and consequently of apoptosis. The data indicate that there are two classes of SMase, defined by their optimal pHs and cellular locations, existing in H. pylori. One is an Mg(2+)-dependent membrane-bound enzyme with an optimal activity at pH 7, and the other is an Mg(2+)-independent cytosolic enzyme with an optimal activity at pH 5. Bisalumin, a bismuth salt, was found to inhibit the activities of both forms of SMase regardless of the presence of Mg2+. By Western blot analysis, the membrane-bound SMases of H. pylori and Bacillus cereus were shown to be antigenically related and to have a similar denatured molecular mass of 28 kDa.

Preparation and characterization of immunogens for antibody production against metanephrine and normetanephrine.

A two-step zero-length cross-linking procedure using active esters was successfully adopted for conjugating metanephrine (MN) and normetanephrine (NM) to bovine serum albumin (BSA). The protein was activated with water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in the presence of N-hydroxysulfosuccimide (sulfo-NHS), leading to the formation of active N-succinimidyl esters of some glutamic and aspartic acid carboxyls. The pertinence of this reaction for the coupling of these haptens to carboxylate groups was confirmed via reaction with a model compound, 2-hydroxybenzoic acid, and subsequent characterization using atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used for the quantitative assessment of the hapten/protein ratios of these conjugates. This technique of conjugate characterization demonstrated greater resolution in molecular weight determination compared to nondenaturing polyacrylamide gel electrophoresis (native PAGE). Preliminary results from enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA procedures using test antisera confirmed that the synthesized immunogens were highly antigenic and elicited specific antibody responses in BALB/c mice against the haptens.

Application of an ELISA-elution assay as a screening tool for dissociation of yolk antibody-antigen complexes.

A modified enzyme-linked immunosorbent assay termed ELISA-elution assay was used as a screening tool to compare the efficiency of eluents for the dissociation of hen yolk immunoglobulin IgY bovine IgG complexes. The potential denaturing effects of the eluents were also monitored. Different buffers (pH 2.3-7.5), containing various types and concentrations of salts (NaCl, (NH4)2SO4 and MgCl2) as well as polyols (ethylene glycol (EG) and glycerol) were compared to the commonly reported glycine x HCl (pH 2.8) buffer and to a commercially available eluent, Actisep. Acidic pH buffers, Actisep and MgCl2 (3.5 M with EG or 4 M without EG) all successfully dissociated IgY from immobilized IgG. However, some denaturation was apparent using MgCl, and, to a lesser extent, Actisep. Furthermore, these same eluents demonstrated a diminished ability for liberating IgG from immobilized IgY(IgG). Information on eluent efficacy obtained by the ELISA-elution assays was applied to selectively isolate lower affinity antibodies for immunoaffinity column chromatography.

What should men know about prostate-specific antigen screening before giving informed consent?

PURPOSE: Since prostate-specific antigen (PSA) screening is controversial, some authorities recommend that patients give informed consent before testing. We identified and compared what facts experts and patients thought men should know. SUBJECTS AND METHODS: We recruited a Delphi panel of national experts (6 urologists and 6 non-urologists) and conducted 6 focus groups of couples (48 subjects) with 24 screened and unscreened men from a university hospital. We ranked key facts that experts and couples thought men ought to know before consenting to PSA screening and conducted a multidisciplinary focus group to help interpret the findings. RESULTS: All participants would disclose that false positive and false negative results can occur and that it is not known whether PSA screening reduces prostate cancer mortality. The 12 experts would disclose the uncertain benefits of treating early, localized prostate cancer. All 24 couples would disclose that the PSA test is a blood test and that patients may worry about results. The 6 urologists would disclose that prostate cancer is often incurable when symptoms appear; the 6 non-urologists, that it can be asymptomatic. The 12 couples with screened men would disclose that the PSA test can detect cancer sooner than the digital rectal examination; the 12 couples with unscreened men, that PSA testing is controversial. CONCLUSIONS: Physicians and patients agree upon some facts that men should know about PSA screening before giving informed consent. However, physicians fail to emphasize other facts that patients find important. Physicians may differ by expertise; patients, by experience. Our findings provide content for informed consent for PSA screening, and our method may be useful for other controversial tests.

Enhanced role for the opening of potassium channels in relaxant responses to acetylcholine after myocardial ischaemia and reperfusion in dog coronary arteries.

1. Anaesthetized dogs were subjected to 1 h occlusion of the left circumflex coronary artery followed by 2 h of reperfusion. Relaxant responses were examined in coronary artery rings removed proximal (nonischaemic) or distal (ischaemic) to the site of occlusion. 2. Relaxant responses to acetylcholine (ACh) were similar in nonischaemic and ischaemic artery rings. In addition ACh-induced relaxation of nonischaemic and ischaemic artery rings was equally susceptible to inhibition of nitric oxide (NO) synthase using L-N(G)-nitroarginine (L-NOARG, 10(-4) M), or to inhibition of soluble guanylate cyclase using 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10(-5) M). 3. In nonischaemic arteries, the relaxation to ACh was unaffected by high K+ (67 mM) but in ischaemic arteries, the maximum relaxation to ACh was significantly reduced from 113+/-6 to 60+/-2% (ANOVA, P<0.05). Tetraethylammonium (TEA, 10(-3) M), an inhibitor of large conductance calcium activated potassium (BK(Ca)) channels did not inhibit the response to ACh in nonischaemic arteries but in ischaemic arteries TEA significantly shifted the concentration response curve to ACh to the right (pEC(50); nonischaemic, 7.07+/-0.25; ischaemic, 6.54+/-0.21, P<0.01, ANOVA) without decreasing the maximum relaxation. TEA did not affect the responses to sodium nitroprusside in either nonischaemic or ischaemic arteries. 4. In conclusion, ischaemia/reperfusion did not change the sensitivity of endothelium-dependent relaxation to L-NOARG or ODQ indicating that ischaemia did not affect the contribution of NO or cyclic GMP to ACh-induced relaxation. However, in ischaemic arteries the opening of the BK(Ca) channels contributed to relaxation caused by ACh whereas TEA had no effect in nonischaemic arteries. The factor responsible for the opening of this potassium channel was a factor other than NO and may be endothelium derived hyperpolarizing factor (EDHF).

Detection of mRNA of sodium iodide symporter in benign and malignant human thyroid tissues.

Human sodium iodide symporter (hNIS) is an intrinsic membrane protein with 12 transmembrane regions, which shows homology to other sodium-dependent transporters. There is controversy as to the amount of hNIS expression in different kinds of human thyroid cancer tissues and cell lines. In this study, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect mRNA of hNIS in various fresh normal, benign tissues and malignant human thyroid tissues. The forward primer was nested hNIS-5' primer containing the sequences: ACCTGGAAATGCGCTTCAGC. The reverse primer was nested hNIS-3' primer containing the sequences: AAGCATGACACCGCGTGCCA. The results revealed three of three normal tissues, six of eight nodular hyperplasia, two of two hyperthyroidism, one of three follicular adenomas, five of ten papillary thyroid carcinomas, one of one follicular carcinoma and zero of one metastatic follicular tissues demonstrated positive results for hNIS in thyroid epithelial cells. A higher percentage of positive results of the symporter mRNA were found in normal benign thyroid tissues and the thyroid tissues of hyperthyroidism, and nodular hyperplasia (84.6%); however follicular adenoma, papillary and follicular thyroid carcinomas demonstrated a lower percentage of expression in the RT-PCR studies (46.7%). Serum thyrotropin levels and the degree of differentiated components presented in cancer tissues have been mentioned as important factors for hNIS expression in the cancer tissues. The discrepancies of the expression of hNIS in in vivo and in vitro studies need further investigation. In conclusion, hNIS was found in higher ratios in normal and benign thyroid tissues than in the malignant tissues. In addition, the RT-PCR technique hNIS did not detect the transporter in most papillary thyroid cancer tissues.

Secretion of beta-endorphin into the maternal circulation by uteroplacental tissues in response to hypoglycaemic stress.

The placenta has been shown to contain ACTH and beta-endorphin but the roles of these peptides are unknown. To investigate whether they are released into the maternal circulation from the placenta in response to physiological stimuli the effects of hypoglycaemic stress were investigated. Plasma samples were collected from the femoral artery (FA) and uterovarian (UV) vein of nine pregnant sheep before and during hypoglycaemia induced by intravenous insulin (100U). Plasma concentrations of ovine beta-endorphin (o beta-EP) were measured by radioimmunoassay. Concentrations of o beta-EP rose in both vessels by 60 min after insulin. The peak concentrations of o beta-EP (pmol/l) were 122 +/- 29 (mean +/- SEM, n = 8) in the UV and 96 +/- 24 (n = 9) fmol/ml in the FA 60 min after insulin injection. There was no difference between the concentrations of o beta-EP in the vessels before insulin injection but at 60 and 120 min after insulin the concentrations of o beta-EP were significantly higher in the UV than FA (P less than 0.02, analysis of variance). This indicates that the pregnant uterus or placenta can respond to hypoglycaemia by secreting beta-EP into the maternal circulation. It is therefore possible that placental pro-opiomelanocortin (POMC) peptides may have a role in maternal endocrinology and metabolism.