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Karl Kunzelmann

Publications and source records attributed to Karl Kunzelmann.

35 records · Page 2Linked to original sources
PubMed2004

The kappa-opioid receptor agonist asimadoline inhibits epithelial transport in mouse trachea and colon.

The potent kappa-opioid receptor agonist n-methyl-N-[(1S)-1-phenyl-2-((3S)-3-hydroxypyrrolidin-1-yl)-ethyl]-2,2-diphenyl-acetamide hydrochloride (asimadoline, EMD 61753) was initially developed for the treatment of chronic pain. Because opioids are well known to reduce secretion and to cause constipation, we investigated the effects on epithelial transport in murine trachea and colon. In Ussing chamber experiments, asimadoline (100 microM) decreased short-circuit currents in airways and colon epithelium. The inhibition of I(SC) was not blocked by naloxone (10 microM) or nor-binaltorphimine (10 microM), suggesting that the response was not mediated by kappa-opioid receptors. The effect of asimadoline on I(SC) was concentration-dependent with half-maximal inhibition of I SC at 23.7 (9.5-49.3) microM and was sensitive to the K+ channel blocker charybdotoxin (10 nM). The amiloride-sensitive Na+ current was reduced by asimadoline, but not in cAMP stimulated tissues. Asimadoline strongly inhibited transient Ca2+-dependent Cl- secretion, activated by the muscarinic receptor agonist carbachol (100 microM) or the purinergic agonist ATP (100 microM). Thus, asimadoline inhibits epithelial transport independent of kappa-opioid receptors, by inhibition of basolateral Ca2+-activated and charybdotoxin-sensitive K+ channels.

Acetamides↗
PubMed2004

Acute effects of parainfluenza virus on epithelial electrolyte transport.

Parainfluenza viruses are important causes of respiratory disease in both children and adults. In particular, they are the major cause of the serious childhood illness croup (laryngotracheobronchitis). The infections produced by parainfluenza viruses are associated with the accumulation of ions and fluid in the respiratory tract. It is not known, however, whether this accumulation is because of a direct effect of the viruses on ion and fluid transport by the respiratory epithelium. Here we show that a model parainfluenza virus (the Sendai virus), in concentrations observed during respiratory infections, activates Cl- secretion and inhibits Na+ absorption across the tracheal epithelium. It does so by binding to a neuraminidase-insensitive glycolipid, possibly asialo-GM1, triggering the release of ATP, which then acts in an autocrine fashion on apical P2Y receptors to produce the observed changes in ion transport. These findings indicate that fluid accumulation in the respiratory tract associated with parainfluenza virus infection is attributable, at least in part, to direct effects of the virus on ion transport by the respiratory epithelium.

Adenosine Triphosphate↗
PubMed2004

Effects of dietary lectins on ion transport in epithelia.

Phytohemagglutinins are widely distributed in common food items. They constitute a heterogeneous group of proteins, which are often resistant to proteolysis in the gastrointestinal tract. Upon binding to the luminal membrane of intestinal cells, they can interfere with digestive, protective or secretory functions of the intestine. Phytohemagglutinins present in red kidney beans and jackbeans have been shown to induce diarrhea and hypersecretion in human airways, but the underlying mechanisms remain obscure. We examined how agglutinins from wheat germ (WGA), soy bean (SBA), red kidney beans (Pha-E, Pha-L), and jackbeans (Con-A) affect ion transport in mouse airways and large intestine using Ussing chamber techniques. We found that Pha-E, Pha-L, and Con-A but not WGA and SBA inhibit electrogenic Na(+) absorption dose dependently in both colon and trachea. The inhibitory effects of Con-A on Na(+) absorption were suppressed by the sugar mannose, by inhibition of phospholipase C (PLC) and protein kinase C (PKC). Thus, nutritional phytohemagglutinins block salt absorption in a PLC- and PKC-dependent manner, probably by inhibition of the epithelial Na(+) channel (ENaC). This effect may be therapeutically useful in patients suffering from cystic fibrosis.

Animals↗
PubMed2004

Assessment of CFTR function in rectal biopsies for the diagnosis of cystic fibrosis.

The Ussing chamber technique has contributed significantly to our understanding of the role of ion transport in the pathogenesis of human diseases like cystic fibrosis (CF). Here, we summarize protocols developed to study the Cl- channel function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in rectal biopsies from normal individuals and CF patients. These protocols can be applied to study the function and pharmacological modulation of wild-type and mutant CFTR in the context of the native epithelium. Together with sweat testing and genetic analyses, these functional measurements may aid in establishing a diagnosis of CF.

Biopsy↗
PubMed2004

The DeltaF508 mutation results in loss of CFTR function and mature protein in native human colon.

BACKGROUND AND AIMS: Deletion of the codon for phenylalanine at position 508 (DeltaF508) is the most frequent disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In heterologous cells, defective processing of the DeltaF508 protein results in endoplasmic reticulum retention, proteolytic degradation, and absence of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent plasma membrane Cl(-) conductance. However, data with respect to the processing block of DeltaF508 protein in native epithelia are limited and conflicting. METHODS: To characterize both the fate and function of DeltaF508 protein in a native epithelium, we measured CFTR-mediated Cl(-) secretion, localization of the CFTR protein, and CFTR maturation in rectal biopsy specimens from normal individuals and DeltaF508 homozygous patients with cystic fibrosis (CF). RESULTS: Ussing chamber studies showed that cAMP-dependent and cholinergic Cl(-) secretion was absent from rectal tissues freshly excised from DeltaF508 homozygous patients with CF. By immunohistochemistry, we detected wild-type but not DeltaF508 CFTR at the luminal membrane of crypt colonocytes. By sequential immunoprecipitation and immunoblotting analyses, mature CFTR protein was detected in normal but not in DeltaF508 homozygous tissues. CONCLUSIONS: Collectively, these data show that there is insufficient maturation and transport of DeltaF508 CFTR from the endoplasmic reticulum to the apical membrane to support CFTR-mediated Cl(-) secretion in the CF colon.

Adolescent↗
PubMed2004

CFTR Cl- channel function in native human colon correlates with the genotype and phenotype in cystic fibrosis.

BACKGROUND & AIMS: Cystic fibrosis (CF) is caused by over 1000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and presents with a widely variable phenotype. Genotype-phenotype studies identified CFTR mutations that were associated with pancreatic sufficiency (PS). Residual Cl- channel function was shown for selected PS mutations in heterologous cells. However, the functional consequences of most CFTR mutations in native epithelia are not well established. METHODS: To elucidate the relationships between epithelial CFTR function, CFTR genotype, and patient phenotype, we measured cyclic adenosine monophosphate (cAMP)-mediated Cl- secretion in rectal biopsy specimens from 45 CF patients who had at least 1 non-DeltaF508 mutation carrying a wide spectrum of CFTR mutations. We compared CFTR genotypes and clinical manifestations of CF patients who expressed residual CFTR-mediated Cl- secretion with patients in whom Cl- secretion was absent. RESULTS: Residual anion secretion was detected in 40% of CF patients, and was associated with later disease onset (P < 0.0001), higher frequency of PS (P < 0.0001), and less severe lung disease (P < 0.05). Clinical outcomes correlated with the magnitude of residual CFTR activity, which was in the range of approximately 12%-54% of controls. CONCLUSIONS: Specific CFTR mutations confer residual CFTR function to rectal epithelia, which is related closely to a mild disease phenotype. Quantification of rectal CFTR-mediated Cl- secretion may be a sensitive test to predict the prognosis of CF disease and identify CF patients who would benefit from therapeutic strategies that would increase residual CFTR activity.

Adolescent↗
PubMed2004

First encounter: how pathogens compromise epithelial transport.

Pathogenic organisms trigger numerous signaling pathways that ultimately lead to drastic changes in physiological functions. Apart from altering structure and function of the epithelial tight junction barrier and activating inflammatory cascades, they induce changes in fluid and electrolyte transport. Pathogens do so by activating or by inhibiting ion channels and transporters, and the result might be to their benefit or to their disadvantage.

Animals↗
PubMed2003

Modulation of Ca2+-activated Cl- secretion by basolateral K+ channels in human normal and cystic fibrosis airway epithelia.

Human airway epithelia express Ca2+-activated Cl- channels (CaCC) that are activated by extracellular nucleotides (ATP and UTP). CaCC is preserved and seems to be up-regulated in the airways of cystic fibrosis (CF) patients. In the present study, we examined the role of basolateral K+ channels in CaCC-mediated Cl- secretion in native nasal tissues from normal individuals and CF patients by measuring ion transport in perfused micro Ussing chambers. In the presence of amiloride, UTP-mediated peak secretory responses were increased in CF compared with normal nasal tissues. Activation of the cAMP pathway further increased CaCC-mediated secretion in CF but not in normal nasal mucosa. CaCC-dependent ion transport was inhibited by the chromanol 293B, an inhibitor of cAMP-activated hKvLQT1 K+ channels, and by clotrimazole, an inhibitor of Ca2+-activated hSK4 K+ channels. The K+ channel opener 1-ethyl-2-benzimidazolinone further increased CaCC-mediated Cl- secretion in normal and CF tissues. Expression of hSK4 as well as hCACC-2 and hCACC-3 but not hCACC-1 was demonstrated by reverse transcriptase PCR on native nasal tissues. We conclude that Ca2+-activated Cl- secretion in native human airway epithelia requires activation of Ca2+-dependent basolateral K+ channels (hSK4). Co-activation of hKvLQT1 improves CaCC-mediated Cl- secretion in native CF airway epithelia, and may have a therapeutic effect in the treatment of CF lung disease.

Adolescent↗
PubMed2003

Pharmacotherapy of the ion transport defect in cystic fibrosis: role of purinergic receptor agonists and other potential therapeutics.

Cystic fibrosis (CF), is an autosomal recessive disease frequently seen in the Caucasian population. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF is characterized by enhanced airway Na(+) absorption, mediated by epithelial Na(+) channels (ENaC), and deficient Cl(-) transport. In addition, other mechanisms may contribute to the pathophysiological changes in the CF lung, such as defective regulation of HCO(3)(-) secretion. In other epithelial tissues, epithelial Na(+) conductance is either increased (intestine) or decreased (sweat duct) in CF. CFTR is a cyclic AMP-regulated epithelial Cl(-) channel, and appears to control the activity of several other transport proteins. Accordingly, defective epithelial ion transport in CF is likely to be a combination of defective Cl(-) channel function and impaired regulator function of CFTR, which in turn is linked to impaired mucociliary clearance and development of chronic lung disease. As the clinical course of CF is determined primarily by progressive lung disease, novel pharmacological strategies for the treatment of CF focus on correction of the ion transport defect in the airways. In recent years, it has been demonstrated that activation of purinergic receptors in airway epithelia by extracellular nucleotides (adenosine triphosphate/uridine triphosphate) has beneficial effects on mucus clearance in CF. Activation of the dominant class of metabotropic purinergic receptors, P2Y(2) receptors, appears to have a 2-fold benefit on ion transport in CF airways; excessive Na(+) absorption is attenuated, most likely by inhibition of the ENaC and, simultaneously, an alternative Ca(2+)-dependent Cl(-) channel is activated that may compensate for the CFTR Cl(-) channel defect. Thus activation of P2Y(2) receptors is expected to lead to improved hydration of the airway surface liquid in CF. Furthermore, purinergic activation has been shown to promote other components of mucociliary clearance such as ciliary beat frequency and mucus secretion. Clinical trials are under way to test the effect of synthetic purinergic compounds, such as the P2Y(2) receptor agonist INS37217, on the progression of lung disease in patients with CF. Administration of these compounds alone, or in combination with other drugs that inhibit accelerated Na(+) transport and help recover or increase residual activity of mutant CFTR, is most promising as successful therapy to counteract the ion transport defect in the airways of CF patients.

Airway Resistance↗
PubMed2002

ENaC is inhibited by an increase in the intracellular Cl(-) concentration mediated through activation of Cl(-) channels.

Activation of the CFTR Cl(-) channel inhibits epithelial Na(+) absorption, according to studies on native epithelia derived from airways, colon and kidney, and can also be demonstrated in overexpressing cells. However, Na(+) absorption is not inhibited by CFTR in the native sweat duct epithelium. The mechanism for the inhibition of epithelial sodium channels (ENaC) has been examined in most detail in Xenopus oocytes coexpressing CFTR and ENaC. It was shown that ENaC is inhibited during stimulation of CFTR in Xenopus oocytes, independent of the experimental setup and the magnitude of the whole-cell current. However, a minimal Cl(-) conductance is required for inhibition of ENaC, and inhibition is augmented at higher CFTR-to-ENaC currents ratios. Low-CFTR-to-ENaC conductance ratios may be the reason for the absence of ENaC inhibition, as described recently. Similar to CFTR, ClC-0 Cl(-) currents also inhibit ENaC, as well as high extracellular Na(+) and Cl(-) in partially permeabilized oocytes. Thus, inhibition of ENaC is not specific to CFTR and could be mediated by Cl(-) flow and/or changes in the intracellular Cl(-) concentration. These results are reminiscent of the Cl(-) feedback regulation observed in mouse mandibular duct cells. Current results obtained with ENaC mutants examined in Xenopus oocytes suggest a charge interaction of Cl(-) ions with the epithelial sodium channel.

Animals↗
PubMed2002

Gene complementation of airway epithelium in the cystic fibrosis mouse is necessary and sufficient to correct the pathogen clearance and inflammatory abnormalities.

Increasingly, cystic fibrosis (CF) is regarded as an inflammatory disorder where the response of the lung to Pseudomonas aeruginosa is exaggerated as a consequence of processes mediated by the product of the CF gene, CFTR. Of importance to any gene-replacement strategy for treatment of CF is the identification of the cell type(s) within the lung milieu that need to be corrected and an indication whether this is sufficient to restore a normal inflammatory response and bacterial clearance. We generated G551D CF mice transgenically expressing the human CFTR gene in two tissue compartments previously demonstrated to mediate a CFTR-dependent inflammatory response: lung epithelium and alveolar macrophages. Following chronic pulmonary infection with P. aeruginosa, CF mice with epithelial-expressed but not macrophage-specific CFTR showed an improvement in pathogen clearance and inflammatory markers compared with control CF animals. Additionally, these data indicate the general role for epithelial cell-mediated events in the response of the lung to bacterial pathogens and the importance of CFTR in mediating these processes.

1-Methyl-3-isobutylxanthine↗
PubMed2002

Mechanisms for the inhibition of amiloride-sensitive Na+ absorption by extracellular nucleotides in mouse trachea.

Purinergic stimulation of airway epithelial cells induces Cl- secretion and modulates Na+ absorption by an unknown mechanism. To gain insight into this mechanism, we used a perfused micro-Ussing chamber to assess transepithelial voltage (V(te)) and amiloride-sensitive short-circuit current (I(sc-Amil)) in mouse trachea. Exposure to apical ATP or UTP (each 100 micromol/l) caused a large initial increase in lumen negative V(te) and I(sc), corresponding to a transient Cl- secretion, while basolateral application of ATP/UTP induced only a small secretory response. Luminal, but not basolateral, application of nucleotides was followed by a sustained and reversible inhibition of I(sc-Amil) that was independent of extracellular Ca2+ or activation of protein kinase C and was not induced by carbachol (100 micromol/l) or the Ca2+ ionophore ionomycin (1 micromol/l). Removal of extracellular Cl- or exposure to 200 microM DIDS reduced UTP-mediated inhibition of I(sc-Amil) substantially. The phospholipase inhibitor U73122 (10 micromol/l) and pertussis toxin (PTX; 200 ng/ml) both attenuated UTP-induced Cl- secretion and inhibition of I(sc-Amil). Taken together, these data imply a contribution of Cl- conductance and PTX-sensitive G proteins to nucleotide-dependent inhibition of the amiloride-sensitive Na+ current in the mouse trachea.

Absorption↗
PubMed2002

Cloning and expression of the mouse glomerular podoplanin homologue gp38P.

BACKGROUND: Puromycin aminonucleoside nephrosis (PAN) is a rat model for human minimal change nephropathy. During PAN, severe proteinuria is induced that is paralleled by a reduced expression of a rat podocyte protein, named podoplanin. The protein probably plays a role in maintaining the unique shape of podocytes. Recently, attenuated amino acid transport has been observed in cultured mouse glomerular epithelial cells treated with puromycin aminonucleoside (PA). In the present study, gp38P, a protein homologous to rat podoplanin was cloned from mouse glomerular epithelial cells and was found to be down-regulated by PA. A role for gp38P in membrane transport in mouse podocytes has been suggested. METHODS: Based on homology to rat podoplanin, the protein gp38P was cloned from mouse glomerular epithelial cells by RT-PCR. Mouse glomerular epithelial cells, mouse cortical collecting duct cells, and Xenopus oocytes were treated with PA and the expression of gp38P was examined by RT-PCR and western blot analysis. Expression of gp38P in other mouse tissues was demonstrated by RT-PCR. The possible impact of gp38P on amino acid transport and folic acid uptake was examined in Xenopus oocytes. RESULTS: gp38P cloned from mouse glomerular epithelial cells showed strong homologies to rat podoplanin and gp38, a protein expressed in the thymus and other tissues. RT-PCR analysis demonstrated ubiquitous expression of gp38P in epithelial and non-epithelial tissues. Quantitative RT-PCR and western blot analysis indicated down-regulation of gp38P in PA-treated glomerular epithelial cells along with loss of cell shape and cell lysis, which was not observed in other cell types. When expressed in Xenopus oocytes, gp38P had no impact on folic acid uptake or transport activity of the amino acid co-transporters CAT1, EAAC1, and rBAT. CONCLUSION: Cultured mouse glomerular epithelial cells express the podoplanin homologue gp38P, which is down-regulated by PAs. gp38P is ubiquitously expressed and is likely to control specifically the unique shape of podocytes.

Amino Acid Sequence↗
PubMed2002

Activation of ion secretion via proteinase-activated receptor-2 in human colon.

Proteinase-activated receptor (PAR) type 2 (PAR-2) has been shown to mediate ion secretion in cultured epithelial cells and rat jejunum. With the use of a microUssing chamber, we demonstrate the role of PAR-2 for ion transport in native human colonic mucosa obtained from 30 normal individuals and 11 cystic fibrosis (CF) patients. Trypsin induced Cl(-) secretion when added to the basolateral but not luminal side of normal epithelia. Activation of Cl(-) secretion by trypsin was inhibited by indomethacin and was further increased by cAMP in normal tissues but was not present in CF colon, indicating the requirement of luminal CF transmembrane conductance regulator. Effects of trypsin were largely reduced by low Cl(-), by basolateral bumetanide, and in the presence of barium or clotrimazole, but not by tetrodotoxin. Furthermore, trypsin-induced secretion was inhibited by the Ca(2+)-ATPase inhibitor cyclopiazonic acid and in low-Ca(2+) buffer. The effects of trypsin were almost abolished by trypsin inhibitor. Thrombin, an activator of PAR types 1, 3, and 4, had no effects on equivalent short-circuit currents. The presence of PAR-2 in human colon epithelium was confirmed by RT-PCR and additional experiments with PAR-2-activating peptide. PAR-2-mediated intestinal electrolyte secretion by release of mast cell tryptase and potentiation of PAR-2 expression by tumor necrosis factor-alpha may contribute to the hypersecretion observed in inflammatory processes such as chronic inflammatory bowel disease.

Adult↗
PubMed2002

Electrolyte transport in the mammalian colon: mechanisms and implications for disease.

The colonic epithelium has both absorptive and secretory functions. The transport is characterized by a net absorption of NaCl, short-chain fatty acids (SCFA), and water, allowing extrusion of a feces with very little water and salt content. In addition, the epithelium does secret mucus, bicarbonate, and KCl. Polarized distribution of transport proteins in both luminal and basolateral membranes enables efficient salt transport in both directions, probably even within an individual cell. Meanwhile, most of the participating transport proteins have been identified, and their function has been studied in detail. Absorption of NaCl is a rather steady process that is controlled by steroid hormones regulating the expression of epithelial Na(+) channels (ENaC), the Na(+)-K(+)-ATPase, and additional modulating factors such as the serum- and glucocorticoid-regulated kinase SGK. Acute regulation of absorption may occur by a Na(+) feedback mechanism and the cystic fibrosis transmembrane conductance regulator (CFTR). Cl(-) secretion in the adult colon relies on luminal CFTR, which is a cAMP-regulated Cl(-) channel and a regulator of other transport proteins. As a consequence, mutations in CFTR result in both impaired Cl(-) secretion and enhanced Na(+) absorption in the colon of cystic fibrosis (CF) patients. Ca(2+)- and cAMP-activated basolateral K(+) channels support both secretion and absorption of electrolytes and work in concert with additional regulatory proteins, which determine their functional and pharmacological profile. Knowledge of the mechanisms of electrolyte transport in the colon enables the development of new strategies for the treatment of CF and secretory diarrhea. It will also lead to a better understanding of the pathophysiological events during inflammatory bowel disease and development of colonic carcinoma.

Animals↗
PubMed2002

Ion transport induced by proteinase-activated receptors (PAR2) in colon and airways.

Protease-activated receptors type 2 (PAR2) are activated by serine proteases like trypsin and mast cell tryptase. The function and physiological significance of PAR2 receptors is poorly understood, but recent studies suggest a role during inflammatory processes in both airways and intestine. PAR2 receptors are also likely to participate in the control of ion transport in these tissues. We demonstrate that stimulation of PAR2 in airways and intestine significantly enhanced ion transport. Trypsin induced Cl- secretion in both airways and intestine when added to the basolateral but not to the luminal side of these tissues. In both airways and intestine, stimulation of ion transport was largely dependent on the increase in intracellular Ca2+. Effects of trypsin were largely reduced by basolateral bumetanide and barium and by trypsin inhibitor. Thrombin, an activator of proteinase-activated receptors types 1, 3, and 4 had no effects on equivalent short-circuit current in either airways or intestine. Expression of PAR2 in colon and airways was further confirmed by reverse transcription-polymerase chain reaction. We postulate that these receptors play a significant role in the regulation of electrolyte transport, which might be important during inflammatory diseases of airways and intestine.

Animals↗