8-bromocyclic-gmp and guanylin

Membrane guanylate cyclase C (GC-C) is the receptor for guanylin, uroguanylin, and heat-stable enterotoxin (STa) in the intestine. GC-C-deficient mice show resistance to STa in intestine but saluretic and diuretic effects of uroguanylin and STa are not disturbed. Here we describe the cellular effects of these peptides using immortalized human kidney epithelial (IHKE-1) cells with properties of the proximal tubule, analyzed with the slow-whole-cell patch clamp technique. Uroguanylin (10 or 100 nm) either hyperpolarized or depolarized membrane voltages (V(m)). Guanylin and STa (both 10 or 100 nm), as well as 8-Br-cGMP (100 microm), depolarized V(m). All peptide effects were absent in the presence of 1 mm Ba(2+). Uroguanylin and guanylin changed V(m) pH dependently. Pertussis toxin (1 microg/ml, 24 h) inhibited hyperpolarizations caused by uroguanylin. Depolarizations caused by guanylin and uroguanylin were blocked by the tyrosine kinase inhibitor, genistein (10 microm). All three peptides increased cellular cGMP. mRNA for GC-C was detected in IHKE-1 cells and in isolated human proximal tubules. In IHKE-1 cells GC-C was also detected by immunostaining. These findings suggest that GC-C is probably the receptor for guanylin and STa. For uroguanylin two distinct signaling pathways exist in IHKE-1 cells, one involves GC-C and cGMP as second messenger, the other is cGMP-independent and connected to a pertussis toxin-sensitive G protein.

Topics: Bacterial Toxins; Barium; Cells, Cultured; Cyclic GMP; Enterotoxins; Escherichia coli Proteins; Gastrointestinal Hormones; Genistein; Guanylate Cyclase; Humans; Hydrogen-Ion Concentration; Kidney Tubules, Proximal; Natriuretic Peptides; Peptides; Pertussis Toxin; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction; Virulence Factors, Bordetella

Guanylin, an activator of the guanylyl cyclase C receptor in the apical membrane of intestinal epithelium, modulates intestinal fluid and electrolyte transport. The bioactive 15-amino acid peptide originally isolated from rat intestine represents the C-terminal part of a longer, 115-residue prepropeptide. The aim of the present study was to characterize the direction and molecular form in which guanylin is secreted from the colonic mucosa, as well as the mechanisms that trigger its secretion. Isolated rat colonic mucosa was mounted in Ussing chambers, allowing the separate determination of apical and basolateral release. After HPLC purification, two different molecular forms of guanylin were identified in the apical incubation media by combining a bioassay for guanylyl cyclase C activation, a specific guanylin enzyme-linked immunosorbent assay and mass spectrometry, as well as sequence analysis: a bioactive form coeluting with synthetic 15-residue guanylin and the 94-residue propeptide, guanylin-22-115. The basal concentration of proguanylin at the apical side of epithelia was about 15-fold higher, compared with that of the small, bioactive peptide. In the basolateral incubation media, no proguanylin and only very low amounts of bioactive guanylin were detected. Incubation with carbachol led to a significant increase of about 7-fold in the release of proguanylin to both sides of the isolated epithelia. On the apical side, a concomitant increase of the small, bioactive peptide was observed; whereas, on the basolateral side, its concentration remained unchanged. Vasoactive intestinal peptide or the NO-donor S-nitroso-N-acetylpenicillamine did not affect guanylin secretion. Our results suggest that, in the intestine, guanylin is secreted mainly to the luminal side of the epithelium. The peptide is released as a 94-residue propeptide, which is then processed to a smaller, bioactive form (luminocrine secretion). Carbachol stimulates the release of proguanylin to both sides of the intestinal mucosa, but a parallel increase in the bioactive C-terminal derivative only occurs on the apical side. In vivo, the basolateral release could be a source of circulating proguanylin, which might be processed proteolytically to the active peptide in distant target tissues (endocrine secretion).

Topics: Amino Acid Sequence; Animals; Biological Assay; Carbachol; Colon; Cyclic GMP; Drug Stability; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Intestinal Mucosa; Molecular Sequence Data; Natriuretic Peptides; Peptides; Protein Precursors; Rats; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Sensitivity and Specificity; Sequence Analysis, Protein

1. Most segments of the gastrointestinal tract secrete HCO3-, but the molecular nature of the secretory mechanisms has not been identified. We had previously speculated that the regulator for intestinal electrogenic HCO3- secretion is the cystic fibrosis transmembrane regulator (CFTR) channel. To prove this hypothesis, we have now measured HCO3- secretion by pH-stat titration, and recorded the electrical parameters of in vitro duodenum, jejunum and ileum of mice deficient in the gene for the CFTR protein ('CF-mice') and their normal littermates. 2. Basal HCO3- secretory rates were reduced in all small intestinal segments of CF mice. Forskolin, PGE2, 8-bromo-cAMP and VIP (cAMP-dependent agonists), heat-stable enterotoxin of Escherichia coli (STa), guanylin and 8-bromo-cGMP (cGMP-dependent agonists) and carbachol (Ca2+ dependent) stimulated both the short-circuit current (Isc) and the HCO3- secretory rate (JHCO3-) in all intestinal segments in normal mice, whereas none of these agonists had any effect on JHCO3- in the intestine of CF mice. 3. To investigate whether Cl(-)-HCO3- exchangers, which have been implicated in mediating the response to some of these agonists in the intestine, were similarly active in the small intestine of normal and CF mice, we studied Cl- gradient-driven 36Cl- uptake into brush-border membrane (BBM) vesicles isolated from normal and CF mouse small intestine. Both the time course and the peak value for 4,4'-diisothiocyanostilbene-2',2-disulphonic acid (DIDS)-inhibited 36Cl- uptake was similar in normal and CF mice BBM vesicles. 4. In summary, the results demonstrate that the presence of the CFTR channel is necessary for agonist-induced stimulation of electrogenic HCO3- secretion in all segments of the small intestine, and all three intracellular signal transduction pathways stimulate HCO3- secretion exclusively via activation of the CFTR channel.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Bacterial Toxins; Bicarbonates; Calcium; Carbachol; Colforsin; Cyclic AMP; Cyclic GMP; Cystic Fibrosis Transmembrane Conductance Regulator; Dinoprostone; Duodenum; Enterotoxins; Escherichia coli Proteins; Gastrointestinal Hormones; Ileum; In Vitro Techniques; Intestinal Mucosa; Intestine, Small; Jejunum; Kinetics; Mice; Mice, Knockout; Microvilli; Natriuretic Peptides; Peptides; Vasoactive Intestinal Peptide

The human colon carcinoma cell lines Caco-2 and HT-29 take up taurine actively. Treatment of Caco-2 cells with Escherichia coli heat-stable enterotoxin (STa) or with guanylin inhibited taurine uptake by approximately 40%. In contrast, neither STa nor guanylin changed the uptake of taurine in HT-29 cells. The inhibition in Caco-2 cells was associated with a decrease in the maximal velocity as well as in the affinity of the transporter. STa caused a 21-fold increase in guanosine 3',5'-cyclic monophosphate (cGMP) levels in Caco-2 cells with no change in cAMP levels. Neither cGMP nor cAMP levels were affected by STa treatment in HT-29 cells. Experiments with protein kinase inhibitors suggested that protein kinase A may mediate the observed effects of STa on taurine uptake. In accordance with this suggestion, treatment of Caco-2 cells with cholera toxin, which elevated intracellular cAMP levels, was found to inhibit taurine uptake. The steady state levels of the taurine transporter mRNA transcripts were not altered as a result of STa treatment. Studies with Caco-2 cells grown on permeable filters revealed that STa acts from the apical side. The taurine uptake from the apical side was inhibited by STa, but the taurine uptake from the basolateral side remained unaffected. It is suggested that the activity of the intestinal taurine transporter may be regulated by protein kinase A at a posttranslational level and that the intestinal absorption of taurine may be impaired during infection with enterotoxigenic strains of E. coli.

Topics: Alkaloids; Bacterial Toxins; Biological Transport; Carrier Proteins; Cells, Cultured; Cholera Toxin; Cyclic AMP; Cyclic GMP; Enterotoxins; Escherichia coli; Escherichia coli Proteins; Gastrointestinal Hormones; Humans; Intestinal Mucosa; Membrane Glycoproteins; Membrane Transport Proteins; Natriuretic Peptides; Peptides; RNA, Messenger; Staurosporine; Taurine