PubMed Health⌕ Search

Biomedical subjects

Martin Diener

Publications and source records attributed to Martin Diener.

13 recordsLinked to original sources
PubMed2006

Characterization of inositol 1,4,5-trisphosphate (IP3) receptor subtypes at rat colonic epithelium.

The aim of the present study was the characterization of the subtypes of inositol 1,4,5-trisphosphate receptors (IP3R) in rat colonic epithelium. A monoclonal antibody against IP3R1 did not stain the colonic epithelial cells. In contrast, IP3R2 and IP3R3 were found within the epithelium; however, with a distinct intracellular localization and differences in their distribution along the crypt axis. IP3R2 immunoreactivity was found within the nuclei of the epithelial cells. The signal was distributed all over the nucleus and not restricted to the nuclear envelope as demonstrated by counterstaining with lamin B1 and electron microscopical examination after immunogold labelling. In contrast, an antibody against IP3R3 stained the epithelial cells mostly in their apical half in accordance with the typical localization of IP3R in organelles such as the endoplasmic reticulum. In addition, there was a gradient from the surface region towards the crypt fundus, where the IP3R3 signal could not be detected. Despite the strong IP3R3-gradient, in saponin-permeabilized colonic crypts exogenously administered IP3 or adenophostin A evoked a similar depletion of mag-fura-2-loaded intracellular Ca2+ stores in crypt and surface cells suggesting a contribution of the nuclear IP3R2 to the Ca2+ release. This conclusion was confirmed by experiments with isolated nuclei from colonic epithelium, at which IP3 was able to induce changes in the Ca2+ concentration, which were inhibited by 2-aminoethoxy-diphenylborate (2-APB), a blocker of IP3 receptors. These results demonstrate that the colonic epithelial cells undergo changes in IP3R subtype expression during differentiation.

Animals↗
PubMed2006

Histamine-induced ion secretion across rat distal colon: involvement of histamine H1 and H2 receptors.

The aim of the present study was to investigate the effect of histamine, a product of e.g. mast cells, on short-circuit current (I(sc)) across rat distal colon. Histamine concentration-dependently stimulated an increase in I(sc), which often was preceded by a transient negative current. Neither a release of neurotransmitters nor a release of prostaglandins contributed to the histamine response. The histamine-induced increase in I(sc) was blocked by the histamine H(1) antagonist, pyrilamine, but was resistant against the histamine H(2) antagonist, cimetidine. Conversely, the histamine H(1) agonist, TMPH (2-(3-trifluoromethylphenyl)histamine), exclusively evoked an increase in I(sc), whereas the histamine H(2) agonist, amthamine, evoked only a decrease in I(sc) suggesting that stimulation of different types of histamine receptors is responsible for the two phases of the response evoked by native histamine. Histamine induces the opening of glibenclamide-sensitive Cl(-) channels and of charybdotoxin-sensitive K(+) channels in the apical membrane as demonstrated by experiments at basolaterally depolarized epithelia. A further action site is the basolateral membrane, because histamine stimulates a charybdotoxin- and tetrapentylammonium-sensitive K(+) conductance in this membrane as observed in tissues, in which the apical membrane was permeabilized with an ionophore, nystatin. The increase in I(sc) evoked by histamine was blocked after depletion of intracellular Ca(2+) stores with cyclopiazonic acid and after blockade of inositol 1,4,5-trisphosphate (IP(3)) receptors, suggesting a release of stored Ca(2+). This was confirmed by the observation that the histamine H(1) agonist TMPH induced an increase in the fura-2 ratio signal of epithelial cells within isolated colonic crypts. Consequently, the mediator histamine seems to stimulate both histamine H(1) and H(2) receptors, from which the former seems to be prominently involved in the induction of epithelial chloride secretion.

Animals↗
PubMed2006

Epithelial muscarinic M1 receptors contribute to carbachol-induced ion secretion in mouse colon.

Cholinergically induced intestinal anion secretion is generally believed to be caused by stimulation of epithelial muscarinic M3 receptors, whereas muscarinic M1 receptors are thought to be localized primarily on enteric neurons. In order to test this assumption, carbachol-stimulated Cl- secretion across distal colon, measured as increase in short-circuit current (I(sc)), was compared between M1-knockout (M1R-KO) and M3-knockout (M3R-KO) mice. Surprisingly, the maximal increase in I(sc) evoked by carbachol was more than twice as large in M3R-KO compared to M1R-KO mice. This difference was not due to a reduced secretory capacity of the epithelium from M3R-KO animals, as forskolin stimulated a similar maximal I(sc) in both types of animals. The neurotoxin tetrodotoxin diminished, but did not abolish the secretory response evoked by carbachol in M3R-KO distal colon, suggesting the existence of epithelial muscarinic receptors other than the type M3. Furthermore, in muscarinic receptor wild-type animals, the muscarinic M1 receptor antagonist pirenzepine inhibited the carbachol-stimulated I(sc) by more than 70% suggesting the presence of epithelial muscarinic M1 receptors; a conclusion, which was confirmed by the identification of mRNA for muscarinic M1 receptors in isolated crypts from wild-type colon. Consequently, epithelial muscarinic receptors from the type M1 contribute to cholinergically induced ion secretion in mouse colon.

Animals↗
PubMed2005

The epidermal growth factor-pathway is not involved in down-regulation of Ca(2+)-induced Cl- secretion in rat distal colon.

Ca(2+)-dependent secretagogues such as carbachol induce a transient Cl- secretion followed by long-lasting inhibition (run-down) of secretion. In the colonic tumour cell line, T84, epidermal growth factor (EGF) inhibits Ca(2+)-dependent secretion, whereas antagonists of the EGF-signalling pathway slow down its run-down. The aim of the present study was to investigate whether a similar mechanism underlies the down-regulation of carbachol-induced Cl- secretion measured as change in short-circuit current (I(sc)) in a native intestinal epithelium, i.e. rat distal colon. In contrast to the colonic tumour cell line, EGF (1-100 microg/l) induced a transient secretory I(sc) and did not interfere with a subsequent administration of carbachol. Pretreatment with inhibitors of enzymes involved in the signalling cascade induced by EGF, i.e. tyrphostin AG1478, an inhibitor of the EGF receptor protein tyrosine kinase, PD 98059, an inhibitor of MAP kinase, and wortmannin, a blocker of the phosphatidylinositol-3-kinase, did also not affect the action of carbachol on transepithelial I(sc). In order to investigate potential effects of these inhibitors on apical Cl- channels, the basolateral membrane was depolarized and a Cl- current across the apical membrane was driven by a Cl- gradient. Under these conditions, carbachol evoked a transient increase in I(sc), caused by the stimulation of Ca(2+)-dependent Cl- channels, followed by a long-lasting down-regulation of apical Cl- conductance leading to a decrease in I(sc). All blockers of the EGF-signalling pathway tested did not interfere with the action of carbachol at the apical membrane. Consequently, the EGF-pathway seems not to be involved in the down-regulation of Ca(2+)-dependent Cl- secretion across rat colon.

Animals↗
PubMed2005

Upregulation of cyclooxygenase-2 and thromboxane A2 production mediate the action of tumor necrosis factor-alpha in isolated rat myenteric ganglia.

Intact myenteric ganglia from 4- to 10-day-old rats were isolated from the small intestine. The preparations were cultured overnight, and drugs were applied within this time frame (20 h). Whole cell patch-clamp technique was used to measure basal membrane potential and carbachol-induced depolarization at neurons within these ganglia. Pretreatment with TNF-alpha (100 ng/ml) hyperpolarized the membrane (from -31.0 +/- 2.7 mV under control conditions to -61.2 +/- 3.2 mV in the presence of the cytokine) and potentiated the depolarization induced by carbachol (from 5.2 +/- 0.7 mV under control conditions to 27.5 +/- 2.0 mV in the presence of the cytokine). These effects were mimicked by carbocyclic thromboxane A2 (10(-6) mol/l), a stable thromboxane A2 agonist. The TNF-alpha action was inhibited by 1-benzylimidazole (2 x 10(-4) mol/l), a thromboxane synthase inhibitor, and BAY U 3405 (5 x 10(-4) mol/l), an inhibitor of thromboxane receptors. Measurements of thromboxane production in the supernatant of the culture revealed an increased concentration of thromboxane B2, the stable metabolite of thromboxane A2, after exposure to TNF-alpha. Immuncytochemical staining for cyclooxygenase-2 (COX-2) and the neuronal marker microtubule-associating protein-2 revealed an upregulation of COX-2 in myenteric neurons after exposure to the cytokine. These results demonstrate the involvement of COX-2 and the subsequent production of thromboxane A2 in the presence of TNF-alpha.

Animals↗
PubMed2004

Stimulation of colonic anion secretion by monochloramine: action sites.

During inflammatory bowel disease, reactive oxygen metabolites are released by phagocytes reacting with intraluminal NH3 to produce monochloramine (NH2Cl). NH2Cl is assumed to play role in the pathogenesis of inflammation-associated diarrhoea, as it is able to induce intestinal secretion. The aim of the present study was to determine the action sites of NH2Cl in rat colonic epithelium with Ussing chamber and fura-2 experiments. In intact mucosa, NH2Cl (5.10(-6)-10(-4) mol.l(-1)) evoked a concentration-dependent increase in short-circuit current (Isc), consistent with the induction of anion secretion, as demonstrated by anion substitution and transport blocker experiments. When the apical membrane was permeabilised by the ionophore nystatin, two basolateral action sites of NH2Cl (5.10(-5) mol.l(-1)) could be identified, i.e. an increase in the K+ conductance and a stimulation of the Na+-K+ pump. When tissues were basolaterally depolarised by a high K+ concentration, the stimulation of an apical Cl- conductance by NH2Cl was observed. In isolated colonic crypts loaded with the Ca2+-sensitive fluorescent dye fura-2, NH2Cl (5.10(-5) mol.l(-1)) evoked an increase in the intracellular Ca2+ concentration. This increase was independent from the presence of Ca2+ in the extracellular medium, but was inhibited by blockade of intracellular sarcoplasmatic, endoplasmatic Ca2+-ATPases with cyclopiazonic acid (10(-5) mol.l(-1)). The NH2Cl-evoked Ca2+ release was sensitive against inhibition of ryanodine receptors with ruthenium red (5.10(-5) mol.l(-1)) and against inhibition of inositol-1,4,5-trisphosphate (IP3) receptors with 2-aminoethoxydiphenylborate (10(-4) mol.l(-1)). Both blockers also inhibited the NH2Cl-induced increase in Isc. These results indicate that an intracellular Ca2+ release via ryanodine and/or IP3 receptors is involved in oxidant stimulation of anion secretion in rat colon.

Ammonium Chloride↗
PubMed2004

Effects of dopamine on ion transport across the rat distal colon.

Dopamine (5.10(-6)-5.10(-4) M) induced a concentration-dependent decrease in short-circuit current (I(sc)) across the rat distal colon. This response was preceded by a transient and inconsistent increase in I(sc). The alpha-adrenoceptor blocker phentolamine and the inhibitors of dopamine-2-like (D(2)-like) receptors L-741,626 and L-745,870 inhibited the dopamine response, suggesting a contribution of adrenergic and dopaminergic receptors. The decrease in I(sc) evoked by dopamine was inhibited by bumetanide, an inhibitor of the basolateral Na(+)-K(+)-2 Cl(-) cotransporter responsible for the uptake of K(+), and by quinine, a blocker of apical K(+) channels, indicating that stimulation of K(+) secretion contributes to the measured change in I(sc). In patch-clamp experiments dopamine hyperpolarized the membrane and increased cellular K(+) current. This response was not concomitant with a change in the intracellular [Ca(2+)] as demonstrated in parallel fura-2 experiments. These results demonstrate that dopamine, like other catecholamines, stimulates colonic K(+) secretion.

Adrenergic Antagonists↗
PubMed2002

Ryanodine receptors and the mediation of Ca2+-dependent anion secretion across rat colon.

The properties of the Na(+)-Ca(2+) exchanger in isolated crypts from rat colon were studied using the Fura-2 imaging technique. The transport mode of the exchanger was reversed by replacing extracellular Na(+) by the impermeable cation, N-methyl-D-glucamine (NMDG(+)), so that the transporter mediated a Ca(2+) influx into the cells. Depletion of intracellular Ca(2+) stores by inhibitors of sarcoplasmatic endoplasmatic calcium ATPases (SERCA), i.e., cyclopiazonic acid (10(-5) mol l(-1)) or thapsigargin (10(-6) mol l(-1)), reduced the increase in [Ca(2+)](i) evoked by superfusion with NMDG(+), suggesting a cross-talk between the exchanger and the Ca(2+) stores. However, measurement of Ca(2+) influx with the Mn(2+) quench technique revealed that the activity of the exchanger was independent of the filling state of the stores. Instead, the obvious inhibition of the [Ca(2+)](i)response by SERCA blockers was due to a reduction of Ca(2+)-induced Ca(2+) release after inhibition of store-refilling. The functional presence of ryanodine receptors was demonstrated by the increase in [Ca(2+)](i)evoked by ryanodine (10(-7) to 3x10(-4) mol l(-1)) in a concentration-dependent manner. This effect was mimicked by cADP ribose (10(-5) mol l(-1)) in crypts permeabilized with saponin (10 mg l(-1)). Ruthenium red (5x10(-5) mol l(-1)) or high concentrations of ryanodine (3x10(-4) mol l(-1)) inhibited this response. In Ussing chamber experiments ruthenium red (5x10(-4) mol l(-1)) or a high concentration of ryanodine (10(-3) mol l(-1)) inhibited the increase in short-circuit current evoked by the cholinergic agonist, carbachol (5x10(-5) mol l(-1)). Consequently, Ca(2+)-induced Ca(2+) release may act as kind of amplifier during Ca(2+)-dependent Cl(-) secretion in order to maintain a long-lasting increase in the intracellular Ca(2+) concentration.

Animals↗
PubMed2002

Ca(2+)-dependent and -independent Cl(-) secretion stimulated by the nitric oxide donor, GEA 3162, in rat colonic epithelium.

The lipophilic nitric oxide-liberating drug, 1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162), concentration-dependently induced a Cl(-) secretion in rat colon. At a low concentration (5 x 10(-5) M), the action was Ca(2+)-dependent, whereas at a high concentration (5 x 10(-4) M), the response was independent from extracellular Ca(2+). Fura-2 experiments at isolated colonic crypts revealed that GEA 3162 induced an increase of the cytoplasmic Ca(2+) concentration due to an influx of extracellular Ca(2+), probably mediated by an activation of a nonselective cation conductance as demonstrated by whole-cell patch-clamp studies. After depolarization of the basolateral membrane, GEA 3162 (5 x 10(-4) M) stimulated a current, which was suppressed by glibenclamide but was resistant against blockade of protein kinases by staurosporine, suggesting an activation of apical Cl(-) channels directly by the nitric oxide (NO) donor. After permeabilizing the apical membrane with the ionophore, nystatin, GEA 3162 (5 x 10(-4) M) activated basolateral K(+) conductances and the Na(+)-K(+)-ATPase. Thus, the lipophilic NO donor GEA 3162 stimulates a Cl(-) secretion in a Ca(2+)-dependent and -independent manner.

Animals↗
PubMed2002

Methods for the study of ionic currents and Ca2+-signals in isolated colonic crypts.

Isolated epithelial cells from intestinal mucosae are a suitable object for the study of the regulation of ion transport in the gut. This regulation possesses a great importance for human and veterinary medicine, as diarrheal diseases, which often are caused by an inadequate activation of intestinal anion secretion, are one of the major lethal diseases of children or young animals. The aim of this paper is to describe a method for the isolation of intact colonic crypts, e.g. for the subsequent investigation of the regulation of anion secretion by the intracellular second messenger, Ca(2+) using electrophysiological and imaging techniques.

Journal Article↗
PubMed2002

Action of palytoxin on apical H+/K+-ATPase in rat colon.

Palytoxin stimulated a cation-dependent short-circuit current (Isc) in rat distal and proximal colon in a concentration-dependent fashion when applied to the mucosal surface of the tissue. The distal colon exhibited a higher sensitivity to the toxin. The palytoxin-induced Isc was blocked by vanadate but was resistant to ouabain or scilliroside, suggesting the conversion of a vanadate-sensitive H+/K+-ATPase into an electrogenic cation transporter. Cation substitution experiments with basolaterally depolarized tissues suggested an apparent permeability of the palytoxin-induced conductance of Na+>K+>Li+. Immunohistochemical control experiments confirmed the absence of the Na+/K+-ATPase in the apical membrane. Consequently, the pore-forming action of palytoxin is not restricted to Na+/K+-ATPase but is also observed with the colonic H+/K+-ATPase.

Acrylamides↗
PubMedDate not supplied

Listeria monocytogenes mediated CFTR transgene transfer to mammalian cells.

BACKGROUND: Several approaches for gene therapy of cystic fibrosis using viral and non-viral vectors are currently being undertaken. Nevertheless, the present data suggest that vectors currently being used will either have to be further modified or, alternatively, novel vector systems need to be developed. Recently, bacteria have been proven as suitable vehicles for DNA transfer to a wide variety of eukaryotic cells. In this study, we assessed the ability of the facultative intracellular pathogen Listeria monocytogenes to deliver a cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR) to CHO-K1 cells, since these cells have been extensively used for heterologous CFTR expression. METHODS: An established in vitro gene transfer system based on antibiotic-mediated lysis of intracellular L. monocytogenes was exploited to transfer eukaryotic expression plasmids. Transient as well as stable CFTR transgene expression was analyzed by microscopical and biochemical methods; functionality was tested by whole-cell patch-clamp recordings. RESULTS: L. monocytogenes mediated gene transfer to CHO-K1 cells was facilitated by an improved transfection protocol. In addition, the use of the isogenic mutant L. monocytogenes hlyW491A, engineered to produce a hemolysin variant with low toxigenic activity, greatly enhanced the efficiency of gene transfer. This strain allowed the transfer of functional CFTR to CHO-K1 cells. CONCLUSIONS: This is the first demonstration of L. monoyctogenes mediated CFTR transgene transfer. The successful in vitro transfer suggests that L. monocytogenes might be a potential vector for cystic fibrosis gene therapy or alternative applications and deserves further investigation in vitro as well as in vivo.

Animals↗