Biomedical subjects

M G Currie

Publications and source records attributed to M G Currie.

At least 73 records · Page 4Linked to original sources

PubMed1992

Human guanylin: cDNA isolation, structure, and activity.

Guanylin is a mammalian peptide homologue of heat-stable enterotoxins that acts on intestinal guanylate cyclase to elicit an increase in cyclic GMP. We have isolated a cDNA encoding an apparent precursor of guanylin from a human intestinal cDNA library. The mRNA is expressed at high levels in human ileum and colon. Human guanylin stimulated increases in T84 cell cyclic GMP levels, displaced 125I-labelled heat-stable enterotoxin (STa) binding to this cell line, and stimulated increases in short-circuit current (Isc) of isolated rat proximal colonic mucosa. This peptide may play a role in regulating fluid and electrolyte absorption in human intestines.

Amino Acid Sequence↗

PubMed1992

Rat guanylin cDNA: characterization of the precursor of an endogenous activator of intestinal guanylate cyclase.

Guanylin is a recently discovered endogenous activator of intestinal guanylate cyclase that was purified from intestinal tissue. Clones have been isolated which demonstrate that the guanylin peptide is contained within a 115 amino acid apparent preprohormone encoded by a 600 base messenger RNA in rat jejunum. The messenger RNA is found predominantly in intestinal tissues, showing a striking gradient of expression ranging from undetectable in esophagus and stomach to abundant in colon. Guanylin may serve a paracrine function to regulate intestinal guanylate cyclase activity, cyclic GMP levels, and thereby, fluid and electrolyte absorption. We hypothesize that the heat stable enterotoxins mimic the endogenously produced guanylin to cause diarrhea.

Amino Acid Sequence↗

PubMed1992

Suppression of atrial natriuretic peptide (ANP) receptor recovery from homologous down-regulation by 8-bromo-cGMP in endothelial cells.

In a previous study we reported that cyclic GMP (cGMP) selectively down-regulates the atrial natriuretic peptide clearance receptor (C-ANP receptor) in the cultured bovine pulmonary artery endothelial (CPAE) cell line. Our efforts in the current study are directed towards determining the effects of cGMP on C-ANP receptor recycling and de novo synthesis following homologous down-regulation by atriopeptin III (APIII, rat ANP 103-126). [125I]APIII binding to CPAE cells was decreased to 45.3 +/- 1.0% of control following the pretreatment with 100 nM APIII for 24 h. After the removal of APIII, the decreased [125I]APIII binding gradually recovered up to 68.8 +/- 1.8% of control for 24 h: a 'long-term recovery'. When CPAE cells were pretreated with 1 nM APIII for 30 min, [125I]APIII binding was also decreased to 62.6 +/- 2.6% of control. Following the removal of APIII, the decrease in [125I]APIII binding quickly recovered to 87.7 +/- 2.7% of control for 1 h: a 'short-term recovery'. 8-bromo-cGMP suppressed the long-term recovery of ANP receptor in a dose-dependent manner, while it had no effect on the short-term recovery. Both actinomycin D (1 ng/ml) and cycloheximide (10 ng/ml) significantly (P less than 0.01) suppressed the long-term recovery, but failed to affect the short-term recovery, whereas, the short-term recovery was significantly (P less than 0.01) inhibited by either 10 mM NH4Cl or 0.2 mM chloroquine which inhibits the recycling of internalized ANP receptor. These findings suggest that new ANP receptor synthesis is necessary for long-term but not for short-term recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Guanylin: an endogenous activator of intestinal guanylate cyclase.

Intestinal guanylate cyclase mediates the action of the heat-stable enterotoxin to cause a decrease in intestinal fluid absorption and to increase chloride secretion, ultimately causing diarrhea. An endogenous ligand that acts on this guanylate cyclase has not previously been found. To search for a potential endogenous ligand, we utilized T84 cells, a human colon carcinoma-derived cell line, in culture as a bioassay. This cell line selectively responds to the toxin in a very sensitive manner with an increase in intracellular cyclic GMP. In the present study, we describe the purification and structure of a peptide from rat jejunum that activates this enzyme. This peptide, which we have termed guanylin, is composed of 15 amino acids and has the following amino acid sequence, PNTCEICAYAACTGC, as determined by automated Edman degradation sequence analysis and electrospray mass spectrometry. Analysis of the amino acid sequence of this peptide reveals a high degree of homology with heat-stable enterotoxins. Solid-phase synthesis of this peptide confirmed that it stimulates increases in T84 cyclic GMP levels. Guanylin required oxidation for expression of bioactivity and subsequent reduction of the oxidized peptide eliminated the effect on cyclic GMP, indicating a requirement for cysteine disulfide bond formation. Synthetic guanylin also displaces heat-stable enterotoxin binding to cultured T84 cells. Based on these data, we propose that guanylin is an activator of intestinal guanylate cyclase and that it stimulates this enzyme through the same receptor binding region as the heat-stable enterotoxins.

Amino Acid Sequence↗

PubMed1992

Inhibition of endothelial cell clearance of atrial natriuretic peptide by cyclic GMP treatment.

In a previous study, we reported that cyclic GMP (cGMP) selectively down-regulates the clearance receptor (C-receptor) for atrial natriuretic peptide (ANP) in the cultured bovine pulmonary artery endothelial (CPAE) cell line. The present study was undertaken in order to examine the effect of cGMP on the internalization of the ANP-receptor complex in CPAE cells. Maximum binding of [125I]APIII to the cells significantly decreased following the treatment with 1 mM 8-bromo-cGMP for 48 or 72 h. Scatchard analysis of the binding assay data from the treated cells showed a decrease in Bmax (616 to 411 fmol/mg protein) without a significant change in Kd. Removal of cell surface-bound APIII by acetic acid revealed that not only the surface binding, but also the internalization of APIII significantly decreased in 8-bromo-cGMP-treated cells, indicating a decrease in receptor-mediated uptake of ANP into the cells. These results suggest that cGMP regulates the clearance of ANP by vascular endothelial cells.

Analysis of Variance↗

PubMed1992

Difference in effect of atrial natriuretic peptide on cGMP in aortic and coronary smooth muscle cells.

Blood vessels show a heterogeneous response to the atrial natriuretic peptide (ANP). In our experiments thoracic aorta from the guinea pig relaxed in response to atriopeptin III (AP; rat ANP-103-126) and to sodium nitroprusside (SNP). In contrast, in perfused guinea pig hearts, AP III produced no change in coronary flow, while SNP increased flow. In smooth muscle cells cultured from the coronary system (CASM) and from the thoracic aorta (TASM), we compared receptor binding and the effects on guanosine 3',5'-cyclic monophosphate (cGMP) production of AP III. AP III bound specifically with equal affinity and with equivalent numbers of binding sites in both cell types. AP III produced a dose-dependent increase in cGMP in TASM (50% effective concentration approximately 3 nM) with a maximum 11-fold increase over basal at 1 microM AP III. In contrast, in CASM, AP III failed to increase cGMP. Nitroprusside increased cGMP in both cell types. Autoradiograms of 125I-labeled AP III linked to cell membranes showed bands at 70 kDa (ANP-C receptor) in both cell types. A second band at 140 kDa (ANP-B receptor) was only seen in TASM. These results suggest that smooth muscle cells of coronary resistance vessels of the guinea pig do not express the particulate guanylyl cyclase that is activated by ANP.

Animals↗

PubMed1991

Cyclic GMP down-regulates atrial natriuretic peptide receptors on cultured vascular endothelial cells.

Down-regulation of atrial natriuretic peptide (ANP) receptors was investigated using a cultured bovine pulmonary artery endothelial (CPAE) cell line. Endothelial cells have been shown to possess two subtypes of ANP receptors, a guanylate cyclase-coupled receptor (B-receptor) and a clearance receptor (C-receptor). The treatment with APIII, rat ANP (103-126), at concentrations of 10(-8) to 10(-6) M for 24 h, resulted in a significantly (p less than 0.01) greater decrease in maximum 125I-APIII binding to CPAE cells than the identical concentration of API, rat ANP (103-123). APIII at concentrations of 10(-8) to 10(-6) M stimulated cyclic GMP (cGMP) production 3.3-17.5-fold greater than similar concentrations of API. From these findings, we hypothesized that cGMP produced following ANP binding to the B-receptor participates in ANP receptor regulation. M&B 22948, a selective inhibitor of cGMP-specific phosphodiesterase, significantly (p less than 0.01) potentiated the effect of both API and APIII on 125I-APIII binding, while M&B 22948 itself had no significant effect on 125I-APIII binding. Treatment of the cells with 1 mM 8-bromo-cGMP also significantly (p less than 0.01) decreased 125I-APIII binding to the cells, and a potentiation of this effect was observed by M&B 22948. Scatchard analysis of binding data from 8-bromo-cGMP-treated cells showed a significant decrease in Bmax (1.79 +/- 0.15 to 1.20 +/- 0.07 fmol/mg protein, p less than 0.05) without a significant change in Kd. Affinity cross-linking of 125I-APIII to 8-bromo-cGMP-treated cells showed a decrease in the labeling of 60- and 70-kDa bands corresponding to the C-receptor. In addition, the APIII-stimulated cGMP response remained unchanged in the 8-bromo-cGMP-treated cells, indicating that the B-receptor was not down-regulated. We conclude that cGMP regulates ANP-binding sites on the endothelial cell and that the evidence indicates that the C-receptor may preferentially be down-regulated by cGMP in CPAE cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Atrial natriuretic peptide inhibits oxidant-induced increases in endothelial permeability.

Chemically and enzymatically generated oxidants alter endothelial cell shape, increase macromolecular permeability across endothelial cell monolayers, and increase lung microvascular permeability. We examined the effect of ANP (atrial natriuretic peptide) on oxidant-induced injuries to bovine aortic endothelial cell monolayers and to isolated, perfused rabbit lungs. Treatment of cultured endothelial monolayers with glucose oxidase (1.4 U/ml) caused changes in cell shape characterized by a retraction of cells and the formation of numerous intercellular gaps. Glucose oxidase treatment also caused a reduction in F-actin stress fibers visualized by rhodamine-phalloidin fluorescence. Pretreatment (5 min) of the endothelial monolayers with ANP (10(-7) M) attenuated the oxidant-induced changes in cell shape and reduction in F-actin staining. In addition, ANP significantly (P less than 0.05) reduced increases in endothelial monolayer permeability to albumin resulting from glucose oxidase treatment. Oxidant-induced injury of isolated, perfused rabbit lungs produced pulmonary edema measured as an increase in lung weight. This increase in weight was significantly (P less than 0.05) inhibited by pretreatment of lungs with ANP (10(-7) M). Collectively, these results suggest that ANP may act to preserve endothelial barrier function and reduce edema formation caused by oxidant injury.

Animals↗

PubMed1991

Atriopeptin stimulates chloride secretion in cultured shark rectal gland cells.

Monolayer cultures of shark rectal gland (SRG) epithelial cells were treated with atriopeptin (AP), and the effects on Cl- secretion and intracellular guanosine 3',5'-cyclic monophosphate (cGMP) accumulation were examined. Basolateral or apical exposure to 10(-7) M AP markedly stimulated (8-fold) Cl(-)-dependent, bumetanide-sensitive, short-circuit current (Isc). The AP-stimulated Isc exhibited transient oscillations before reaching a steady state. This behavior is not observed when Isc is activated by other secretagogues such as vasoactive intestinal peptide, 2-chloroadenosine, forskolin, or ionomycin. Intracellular cGMP was concomitantly elevated (10-fold) by 10(-7) M AP. Both Isc stimulation and cGMP accumulation responses exhibited a similar dose dependency beginning at an AP concentration of 1 nM. The bilateral response to AP suggests the presence of receptors on both apical and basolateral plasma membranes. These results are the first demonstration of a direct effect of AP on Cl(-)-secreting epithelial cells. These data also suggest a role for cGMP in mediating Cl- secretion in these cells.

2-Chloroadenosine↗

PubMed1991

Glucocorticoid regulation of atrial natriuretic peptide receptors on cultured endothelial cells.

Cultured pulmonary artery endothelial (CPAE) cells possess specific high affinity receptors for atrial natriuretic peptide (ANP). CPAE cells were used to investigate the regulation of ANP receptors by glucocorticoids. Treatment of CPAE cells with dexamethasone (1 microM) produced a 69.3 +/- 23% (P less than 0.01) increase in the maximum binding of [125I]ANP to CPAE cells without affecting the affinity of binding. The EC50 for the dexamethasone effect was approximately or equal to 0.5 nM, with a maximum effect at 10 nM. The effect was time dependent and developed over 16-72 h, and it could be inhibited by cycloheximide (0.075 micrograms/ml), indicating a requirement for de novo protein synthesis. The glucocorticoid receptor antagonist RU 486 completely inhibited the dexamethasone effect. In affinity cross-linking experiments, dexamethasone increased the labelling of the ANP-R2, or clearance, receptor, whereas labeling of the ANP-R1 receptor could not be detected. Despite the increase in maximum binding, dexamethasone treatment produced a significant (P less than 0.01) decrease in the ANP-stimulated cyclic GMP response of CPAE cells and no change in the affinity of binding for the truncated ANP analog, atriopeptin I. These results indicate that the dexamethasone effect is mediated through glucocorticoid receptors and may be selective for the nonguanylate cyclase-coupled, or ANP-R2, receptor subtype. This study provides direct evidence for regulation of ANP receptors by steroid hormones.

Animals↗

PubMed1990

Atrial natriuretic peptide regulation of endothelial permeability is mediated by cGMP.

Previous studies in our laboratory showed that ANP inhibits increases in endothelial monolayer permeability to macromolecules induced by thrombin. In this present study, we investigated the second messenger system involved in the influence of ANP on monolayer permeability. In bovine aortic endothelial cells (BAEC), ANP (100 nM) caused increased cGMP levels which were measurable at 30 sec and maximal at 3 min. Addition of 8-bromo cGMP (1 mM) to BAEC monolayers mimicked the actions of ANP by inhibiting thrombin- mediated increases in permeability to [125I]-labeled bovine serum albumin. Inhibition of increases in permeability by lower concentrations of ANP was enhanced by the cGMP-selective phosphodiesterase inhibitor, M&B 22948 (100 microM). The use of ANP structural analogs which stimulate cGMP production (AP III or BNP) prevented thrombin-induced increases in monolayer permeability, whereas AP-I, which does not increase cGMP levels, was ineffective.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1990

Effect of glucocorticoids on the binding of atrial natriuretic peptide to endothelial cells.

Cultured bovine pulmonary artery endothelial cells were found to possess specific, high affinity receptors for atrial natriuretic peptide (ANP). When cells which had been maintained in medium containing 10% fetal bovine serum were placed in serum-free medium for 24 h, there was a 50% decrease in ANP receptor density. Addition of hydrocortisone (20 microM) or dexamethasone (1 microM) to the serum-free medium for 48 h resulted in an increase in ANP binding sites with no change in the affinity of the sites for ANP. The glucocorticoid antagonist RU 486 (1 microM) significantly (P less than 0.05) decreased the binding of ANP to cultured pulmonary artery endothelial cells in the presence of 1% fetal bovine serum. These results suggest a role for glucocorticoids in the regulation of ANP receptors on vascular endothelial cells.

Animals↗

PubMed1990

A rapid and economical method of preparing radioiodinated cyclic nucleotide derivatives for use in radioimmunoassays.

2'-O-succinyladenosine 3':5'-cyclic monophosphate tyrosyl methyl ester (ScAMP-TME) and 2'-O-succinylguanosine 3':5'-cyclic monophosphate tyrosyl methyl ester (ScGMP-TME) were radioiodinated using chloramine T and Na125I. The resulting radiolabeled cyclic nucleotide derivatives, ScAMP-125I-TME and ScGMP-125I-TME, were subsequently purified by reverse-phase chromatography on Sep-Pak C18 cartridges (Waters Associates, Milford, MA) and tested as tracers in sensitive radioimmunoassays for cAMP and cGMP, respectively. Purified ScAMP-125I-TME and ScGMP-125I-TME functioned in the respective radioimmunoassays for up to 12 weeks when suspended in a 1:1 (v:v) mixture of n-propanol and 20 mM sodium acetate, pH 6.0. Thus, this purification method enables rapid and economical preparation of tracers for cyclic nucleotide radioimmunoassays. Furthermore, our findings suggest that reverse-phase chromatography may be applicable to the purification of other small polar molecules to which tyrosyl groups have been added for the purpose of radioiodination.

Cyclic AMP↗

PubMed1989

Atrial and brain natriuretic peptides share binding sites in the kidney and heart.

We examined the distribution of binding sites for atrial natriuretic peptide (ANP) and the recently discovered brain natriuretic peptide (BNP) in rat kidney and heart by receptor autoradiography. In frozen kidney sections, both 125I-ANP and 125I-BNP exhibited localized binding to cortical glomeruli. The binding of each radiolabeled peptide was abolished by inclusion of either excess (1 microM) unlabeled ANP or excess unlabeled BNP, suggesting that ANP and BNP share cortical glomerular binding sites. In frozen cardiac sections, ANP and BNP binding sites were localized to the endothelium of the endomural channels and endocardium. As was the case for kidney, binding of each peptide at these sites was abolished by the presence of excess unlabeled ANP or BNP, suggesting that these natriuretic peptides share binding sites in the heart as well. To explore further the possibility that ANP and BNP utilize the same receptor(s), we performed competitive binding experiments using cultured pulmonary artery endothelial (CPAE) cells. ANP and BNP competitively displaced one another with equivalent IC50 values from CPAE cell binding sites. Furthermore, both ANP and BNP elevated the levels of cGMP, a putative second messenger for ANP, in these cells. These data are consistent with the observation that BNP, like ANP, causes a natriuresis and diuresis in rats, and suggest that BNP may possess other biological activities known for ANP.

Animals↗

PubMed1989

Atrial natriuretic peptide binding sites in the mammalian heart: localization to endomural vessels.

The distribution of binding sites for atrial natriuretic peptide in cardiac ventricles of several mammalian species, including rat and human, was determined by in vitro autoradiography. The results revealed a unique anatomic localization of atrial natriuretic peptide binding sites to endomural vessels (Thebesian vessels), which communicate directly with the ventricular chambers. Digital image analysis indicated that these vascular channels possessed binding site densities comparable to those of the renal glomeruli, a major target site for circulating atrial natriuretic peptide. In contrast, no specific labeling of branches of the coronary arteries and veins was detected. The discrete localization of atrial natriuretic peptide binding sites to this "primitive" cardiac circulatory system allows speculation as to the role of this hormone in the regulation of endocardial circulation during cardiac development, normal ventricular function, and in coronary insufficiency.

Adult↗

PubMed1989

Enhanced activity of the cardiac endocrine system during right ventricular hypertrophy.

In a model of pulmonary hypertension induced by a single injection of monocrotaline (MCT), we observed a time-dependent right ventricular hypertrophy, which became apparent in treated rats 21 days after administration of MCT and progressed through day 45. Associated with this right ventricular hypertrophy were time-dependent increases in ventricular levels of immunoreactive atrial natriuretic peptide (iANP). Forty-five days after MCT treatment, treated rats exhibited a 72-fold increase in right ventricular iANP levels and a 7-fold increase in left ventricular iANP levels. Hybridization analysis of total RNA extracted from cardiac tissue indicated that both atrial and ventricular ANP mRNA levels were elevated in treated rats. These data suggest that during pulmonary hypertension and cardiac hypertrophy the endocrine activity of the heart expands to include ventricular tissue. ANP binding site autoradiography revealed decreased binding site density in the kidney and hearts of treated rats at 49 days, consistent with the occurrence of desensitization/down-regulation. Enhanced ventricular ANP production may serve as a compensatory response to sustained elevation of pulmonary arterial pressure or may function as an autocrine/paracrine system regulating cardiac function. In either case, the effects of augmented ANP production may be subject to modulation by the status of ANP receptors in target organs and cells.

Animals↗

PubMed1989

Atriopeptin inhibition of thrombin-mediated changes in the morphology and permeability of endothelial monolayers.

To determine the role of endothelial atriopeptin (AP) receptors, we examined the effect of AP-III on the morphology and macromolecular permeability of monolayer cultures of bovine aortic endothelial cells. AP-III alone (10(-9)-10(-6) M) had no observable effect on the morphology of the monolayers or their permeability to 125I-labeled albumin. However, incubation of the endothelial monolayers with AP-III (10(-8)-10(-6) M) antagonized thrombin-induced (1 unit/ml) cell-shape change and the formation of intercellular gaps. AP-III also opposed the effect of thrombin on the distribution of actin filaments in the endothelial cytoskeleton. Further, thrombin caused a 2-fold increase in monolayer permeability to 125I-labeled albumin, which was abolished by 10(-8)-10(-6) M AP-III pretreatment. Taken together with the findings that AP-III exhibited specific and saturable binding in these cells, these data suggest that AP regulates endothelial permeability through a receptor-mediated process.

Actins↗

PubMed1989

Co-localization of a kallikrein-like serine protease (arginine esterase A) and atrial natriuretic peptide in rat atrium.

Atrial natriuretic peptide (ANP) is stored in atrial granules primarily as a larger molecular weight precursor (pro-ANP), which is believed to be rapidly converted to an active peptide of 28 amino acids during or shortly after secretion. A tissue kallikrein-like serine protease has been suggested as a potential processing enzyme. In the present immunocytochemical study, using specific monoclonal antibodies, we found that esterase A, a kallikrein-like serine protease, was demonstrable in rat atrial myocytes and in ventricular myocytes, and was capable of cleaving pro-ANP to yield a low molecular weight product. Using colloidal gold immunocytochemistry at the electron microscopic level, we have found esterase A in atrial myocytes, both in granules and in another subcellular site that corresponds to sarcoplasmic reticulum. Double-label electron microscopic immunocytochemical results indicated that esterase A can co-localize with ANP in granules of atrial myocytes.

Animals↗