Biomedical subjects

A Schwab

Publications and source records attributed to A Schwab.

At least 19 recordsLinked to original sources

PubMed2006

A novel function of capsaicin-sensitive TRPV1 channels: involvement in cell migration.

Cell migration relies on a tight temporal and spatial regulation of the intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i in turn depends on Ca2+ influx via channels in the plasma membrane whose molecular nature is still largely unknown for migrating cells. A mechanosensitive component of the Ca2+ influx pathway was suggested. We show here that the capsaicin-sensitive transient receptor potential channel TRPV1, that plays an important role in pain transduction, is one of the Ca2+ influx channels involved in cell migration. Activating TRPV1 channels with capsaicin leads to an acceleration of human hepatoblastoma (HepG2) cells pretreated with hepatocyte growth factor (HGF). The speed rises by up to 50% and the displacement is doubled. Patch clamp experiments revealed the presence of capsaicin and resiniferatoxin (RTX)-sensitive currents. In contrast, HepG2 cells kept in the absence of HGF are not accelerated by capsaicin and express no capsaicin- or RTX-sensitive current. The TRPV1 antagonist capsazepine prevents the stimulation of migration and inhibits capsaicin-sensitive currents. Finally, we compared the contribution of capsaicin-sensitive TRPV1 channels to cell migration with that of mechanosensitive TRPV4 channels that are also expressed in HepG2 cells. A specific TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate, does not increase the displacement. In summary, we assigned a novel role to capsaicin-sensitive TRPV1 channels. They are important Ca2+ influx channels required for cell migration.

Calcium↗

PubMed2005

Functional role of Na+-HCO3- cotransport in migration of transformed renal epithelial cells.

Cell migration is crucial for immune defence, wound healing or formation of tumour metastases. It has been shown that the activity of the Na(+)-H(+) exchanger (NHE1) plays an important role in cell migration. However, so far it is unknown whether Na(+)- HCO(3)(-) cotransport (NBC), which has similar functions in the regulation of intracellular pH (pH(i)) as NHE1, is also involved in cell migration. We therefore isolated NHE-deficient Madin-Darby canine kidney (MDCK-F) cells and tested whether NBC compensates for NHE in pH(i) and cell volume regulation as well as in migration. Intracellular pH was measured with the fluorescent pH indicator 2'7'-bis(carboxyethyl)-5-carboxyfluorescein (BCECF). The expression of NBC isoforms was determined with semiquantitative PCR. Migration was monitored with time-lapse video microscopy and quantified as the displacement of the cell centre. We found that MDCK-F cells express the isoform NBC1 (SLCA4A gene product) at a much higher level than the isoform kNBC3 (SLCA4A8 gene product). This difference is even more pronounced in NHE-deficient cells so that NBC1 is likely to be the major acid extruder in these cells and the major mediator of propionate-induced cell volume increase. NHE-deficient MDCK-F cells migrate more slowly than normal MDCK-F cells. NBC activity promotes migration during an acute intracellular acid load and increases migratory speed and displacement on a short timescale (< 30 min) whereas it has no effect on the long-term behaviour of migrating MDCK-F cells. Taken together, our results show that NBC actvity, despite many functional similarities, does not have the same importance for cell migration as NHE1 activity.

Animals↗

PubMed2003

Low-threshold heat receptor in chick sensory neurons is upregulated independently of nerve growth factor after nerve injury.

In mammals, the cloned low-threshold heat receptor, vanilloid receptor subtype 1 (VR1), is involved in the genesis of thermal hyperalgesia after inflammation. However, there is evidence that VR1 is not involved in the thermal hyperalgesia that occurs after nerve injury. In search for other heat receptors which might be involved in this phenomenon, we previously demonstrated that chick dorsal root ganglion neurons, which are insensitive to capsaicin, respond to low-threshold heat. Here, we investigated whether expression of the low-threshold noxious heat receptor in chicks is regulated by nerve growth factor (NGF), as VR1 is in mammals. Heat (44 degrees C) responsiveness of isolated dorsal root ganglion neurons of chicks was investigated (i) under culture conditions for up to 4 days with and without NGF and (ii) after a tight ligation of the sciatic nerve for up to 6 days, using cobalt-uptake method. In every case, a significant upregulation in the proportion of heat-responsive neurons was observed. On the molecular level, there was an increase of chick VR1 mRNA level in dorsal root ganglion cells cultured for 3 days in medium lacking NGF. In rat dorsal root ganglion neurons cultured for 1-4 days without NGF, patch-clamp experiments revealed that after 1 day almost all neurons responding to heat also responded to capsaicin, whereas after 3-4 days, more than one-half of the heat-responsive neurons did not respond to capsaicin. These data suggest the existence of low-threshold heat receptors in chick dorsal root ganglion neurons, the expression of which is regulated independently of NGF.

Animals↗

PubMed2002

Regulation of a calcium-sensitive K+ channel (cIK1) by protein kinase C.

Ca2+-sensitive K+ channels (IK1 channels) are required for many physiological functions such as cell proliferation, epithelial transport or cell migration. They are regulated by the intracellular Ca2+ concentration and by phosphorylation-dependent reactions. Here, we investigate by means of the patch-clamp technique mechanisms by which protein kinase C (PKC) regulates the canine isoform, cIK1, cloned from transformed renal epithelial (MDCK-F) cells. cIK1 elicits a K+-selective, inwardly rectifying, and Ca2+-dependent current when expressed in HEK293 or CHO cells. It is inhibited by charybdotoxin, clotrimazole, and activated by 1-ethyl-2-benzimidazolone. cIK1 is activated by intracellular application of ATP or ATP[gS]. ATP-dependent activation is reversed by PKC inhibitors (bisindolylmaleimide, calphostin C), while stimulation with ATP[gS] resists PKC inhibition. Stimulation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) leads to the acute activation of cIK1 currents, which are blocked by PKC inhibitors. In contrast, PKC depletion by overnight incubation with PMA prevents ATP-dependent cIK1 activation. Neither single mutations nor the simultaneous mutation of all PKC sites (T101, S178, T329) to alanine alter the acute regulation of cIK1 channels by PKC. However, current amplitudes of CIK1-T329A and the triple mutant are dramatically increased upon long-term treatment with PMA. These mutations thereby disclose an inhibitory effect on cIKl current of the PKC site at T329. Our results indicate that cIK1 channel activity is regulated in two ways. PKC-dependent activation of cIK1 channels occurs indirectly, while the inhibitory effect probably requires a direct interaction with the channel protein.

Adenosine Triphosphate↗

PubMed2001

Determination of free and conjugated indole-3-acetic acid, tryptophan, and tryptophan metabolites in grape must and wine.

Tryptophan (Trp) and its metabolites, especially indole-3-acetic acid (IAA), are considered to be potential precursors of 2-aminoacetophenone (AAP), an aroma compound that causes an "untypical aging off-flavor" (UTA) in Vitis vinifera wines. In this study, RP-HPLC with fluorescence detection was used for the qualitative and quantitative analysis of Trp and Trp metabolites in grape musts and wines to which different viticultural measures had been applied (time of harvest, soil treatment, leaf plucking, vine prune). An alkaline hydrolysis was developed to release bound IAA and Trp. A sensitive and selective determination of different Trp metabolites was achieved after solid phase extraction using a strong anion exchange material. In the examined grape musts, more than 95% of the total IAA was bound either as ester conjugate or as amide conjugate. Free IAA and other Trp metabolites were below the detection limit (<3 microg/L) or could be determined only in traces. Their amounts increased significantly during fermentation, whereas the amount of Trp decreased. It could be shown that the different viticultural measures applied (except the vine prune) as well as the climatic conditions of the vintage exhibited significant influences on the amounts of Trp and Trp metabolites in grape musts or wines.

Chromatography, High Pressure Liquid↗

PubMed2001

The neural correlates of person familiarity. A functional magnetic resonance imaging study with clinical implications.

Neural activity was measured in 10 healthy volunteers by functional MRI while they viewed familiar and unfamiliar faces and listened to familiar and unfamiliar voices. The familiar faces and voices were those of people personally known to the subjects; they were not people who are more widely famous in the media. Changes in neural activity associated with stimulus modality irrespective of familiarity were observed in modules previously demonstrated to be activated by faces (fusiform gyrus bilaterally) and voices (superior temporal gyrus bilaterally). Irrespective of stimulus modality, familiarity of faces and voices (relative to unfamiliar faces and voices) was associated with increased neural activity in the posterior cingulate cortex, including the retrosplenial cortex. Our results suggest that recognizing a person involves information flow from modality-specific modules in the temporal cortex to the retrosplenial cortex. The latter area has recently been implicated in episodic memory and emotional salience, and now seems to be a key area involved in assessing the familiarity of a person. We propose that disturbances in the information flow described may underlie neurological and psychiatric disorders of the recognition of familiar faces, voices and persons (prosopagnosia, phonagnosia and Capgras delusion, respectively).

Acoustic Stimulation↗

PubMed2001

Function and spatial distribution of ion channels and transporters in cell migration.

Cell migration plays a central role in many physiological and pathophysiological processes, such as embryogenesis, immune defense, wound healing, or the formation of tumor metastases. Detailed models have been developed that describe cytoskeletal mechanisms of cell migration. However, evidence is emerging that ion channels and transporters also play an important role in cell migration. The purpose of this review is to examine the function and subcellular distribution of ion channels and transporters in cell migration. Topics covered will be a brief overview of cytoskeletal mechanisms of migration, the role of ion channels and transporters involved in cell migration, and ways by which a polarized distribution of ion channels and transporters can be achieved in migrating cells. Moreover, a model is proposed that combines ion transport with cytoskeletal mechanisms of migration.

Animals↗

PubMed2001

Ion channels and transporters on the move.

Cell migration plays a crucial role in a variety of (patho)physiological processes such as immune defense, wound healing, and formation of tumor metastases. Detailed models have been developed to describe cytoskeletal mechanisms of migration. However, evidence is accumulating that the activity of ion channels and transporters is also required for optimal cell locomotion.

Animals↗

PubMed2000

Volume dynamics in migrating epithelial cells measured with atomic force microscopy.

Migration of transformed renal epithelial cells (transformed Madin-Darby canine kidney cells, MDCK-F cells) relies on the activity of a Ca(2+)-sensitive K+ channel (IK channel) that is more active at the rear end of these cells. We have postulated that intermittent IK channel activity induces local cell shrinkage at the rear end of migrating MDCK-F cells and thereby supports the cytoskeletal mechanisms of migration. However, due to the complex morphology of MDCK-F cells we have not yet been able to measure volume changes directly. The aim of the present study was to devise a new technique employing atomic force microscopy (AFM) to measure the volume of MDCK-F cells in their physiological environment and to demonstrate its dependence on IK channel activity. The spatial (x, y' and z) co-ordinates of each pixel of the three-dimensional image of MDCK-F cells allow calculation of the volume of the column "underneath" a given pixel. Thus, total cell volume is the sum of all pixel-defined columns. The mean volume of 17 MDCK-F cells was 2500+/-300 fl. Blockade of the IK channel with the specific inhibitor charybdotoxin (CTX) increased cell volume by 17+/-4%; activation of IK by elevating the intracellular [Ca2+] with the Ca2+ ionophore ionomycin decreased cell volume by 19+/-3%. Subtraction images (experimental minus control) reveal that swelling and shrinkage occur predominantly at the rear end of MDCK-F cells. In summary, our experiments show that AFM allows the measurement not only of total cell volume of living cells in their physiological environment but also the tracing of local effects induced by the polarized distribution of K+ channel activity.

Animals↗

PubMed2000

Polarization of Na(+)/H(+) and Cl(-)/HCO (3)(-) exchangers in migrating renal epithelial cells.

Cell migration is crucial for processes such as immune defense, wound healing, or the formation of tumor metastases. Typically, migrating cells are polarized within the plane of movement with lamellipodium and cell body representing the front and rear of the cell, respectively. Here, we address the question of whether this polarization also extends to the distribution of ion transporters such as Na(+)/H(+) exchanger (NHE) and anion exchanger in the plasma membrane of migrating cells. Both transporters are required for locomotion of renal epithelial (Madin-Darby canine kidney, MDCK-F) cells and human melanoma cells since their blockade reduces the rate of migration in a dose-dependent manner. Inhibition of migration of MDCK-F cells by NHE blockers is accompanied by a decrease of pH(i). However, when cells are acidified with weak organic acids, migration of MDCK-F cells is normal despite an even more pronounced decrease of pH(i). Under these conditions, NHE activity is increased so that cells are swelling due to the accumulation of organic anions and Na(+). When exclusively applied to the lamellipodium, blockers of NHE or anion exchange inhibit migration of MDCK-F cells as effectively as when applied to the entire cell surface. When they are directed to the cell body, migration is not affected. These data are confirmed immunocytochemically in that the anion exchanger AE2 is concentrated at the front of MDCK-F cells. Our findings show that NHE and anion exchanger are distributed in a polarized way in migrating cells. They are consistent with important contributions of both transporters to protrusion of the lamellipodium via solute uptake and consequent volume increase at the front of migrating cells.

Amiloride↗

PubMed1999

Determination of tryptophan and tryptophan metabolites in grape must and wine.

Tryptophan (Trp) and its metabolites, especially indole-3-acetic acid (IAA), are considered as potential precursors of 2-aminoacetophenone (AAP), an aroma compound which causes the "untypical aging off-flavor" (UTA) in Vitis vinifera white wines. In this study RP-HPLC with fluorescence detection was used for the qualitative and quantitative analysis of Trp and Trp-metabolites in 39 grapes, 22 grape musts and 16 wines, to which different viticultural conditions (ripeness, pruning, strip of leaves, soil condition) have been applied. A sensitive and selective determination was achieved after solid phase extraction using an anion exchange material. Only traces of Trp-metabolites could be determined in the examined grapes and grape musts, but their amounts increased significantly during fermentation, whereas the amount of Trp decreased. Different viticultural measures, besides the time of grape harvest, showed no significant influences on the amount of Trp and Trp-metabolites.

Acetophenones↗

PubMed1999

Migration of transformed renal epithelial cells is regulated by K+ channel modulation of actin cytoskeleton and cell volume.

Migration of transformed renal epithelial (MDCK-F) cells depends on the polarized activity of a Ca2+-sensitive K+ channel (IK channel; Pflügers Arch 432:R87-R93, 1996). This study was aimed at elucidating the functional link between the IK channel and the actin cytoskeleton which is required for cell locomotion. We monitored migration of MDCK-F cells with video microscopy, quantified filamentous actin with phalloidin binding, and measured the intracellular Ca2+ concentration ([Ca2+]i) with the fluorescent dye fura-2/AM. We compared the effects of IK channel activation or inhibition with those of hypotonic swelling or hypertonic shrinkage. IK channel inhibition with charybdotoxin (CTX) or cell swelling (omission of up to 50 mmol/l NaCl) as well as IK channel activation with 1-ethyl-2-benzimidazolinone (1-EBIO) or cell shrinkage (addition of up to 100 mmol/l mannitol) reduce the rate of migration dose-dependently by up to 80%, i.e., to the same extent as cytochalasin D. Inhibition of migration is accompanied either by actin depolymerization (CTX and cell swelling) or by actin polymerization (1-EBIO and cell shrinkage). Changes of migration and phalloidin binding induced by CTX and cell swelling or by 1-EBIO and cell shrinkage, respectively, are linearly correlated with each other. CTX and cell swelling elicit a rise of [Ca2+]i whereas 1-EBIO and cell shrinkage induce a slight decrease of [Ca2+]i in most MDCK-F cells. Taken together IK-channel-dependent perturbations of cell volume and anisotonicity elicit virtually identical effects on migration, actin filaments and [Ca2+]i. We therefore suggest that cell volume - possibly via [Ca2+]i - is the link between IK channel activity, actin filaments and migration. We propose a model for how temporal and local changes of cell volume can support the migration of MDCK-F cells.

Actins↗

PubMed1999

Molecular and functional characterization of the small Ca(2+)-regulated K+ channel (rSK4) of colonic crypts.

Colonic crypt cells possess basolateral Ca(2+)-regulated K+ channels which support Cl- secretion by providing the necessary driving force. The pharmacological characteristics of these channels were examined in Ussing chamber experiments of rat and rabbit colon mucosa by the use of blockers. The chromanol 293B, a blocker of KVLQT1 channels, and clotrimazole (CTZ), a blocker of small Ca(2+)-activated K+ channels, blocked stimulated Cl- secretion completely. Small-conductance Ca(2+)-activated K+ channels (SK) in excised basolateral patches of rat colonic crypts were inhibited concentration dependently by the imidazoles CTZ, NS004 and NS1619 and activated by 1-EBIO. These properties are similar to those of the known human SK channel (hSK4). hSK4-expressing Xenopus laevis oocytes showed ionomycin-activated and CTZ-inhibited K+ currents. When P2Y2 receptors were coexpressed these currents were also activated by ATP. The concentration/response curve was identical to that of rat SK channels. In human colonocytes (T84) exposed to hSK4 antisense probes, but not to sense probes, carbachol-induced K+ currents were attenuated. With RT-PCR an hSK4 could be demonstrated in human colon and in T84 colonocytes. By homology cloning the SK of the rat colon (rSK4) was identified. This protein has a high homology to hSK4 and mouse IK1. These data indicate that the Ca(2+)-activated and imidazole-inhibited basolateral K+ current in the colon is caused by SK4 channels.

Animals↗

PubMed1999

K(+) channel-dependent migration of fibroblasts and human melanoma cells.

Previously, we showed that migration of transformed renal epithelial cells (MDCK-F cells) is a K(+) channel-dependent process [J Clin Invest 1994;93:1631]. In order to determine whether K(+) channel activity is a general requirement for locomotion, we extended our observations to NIH3T3 fibroblasts and human melanoma cells. Migration of both cell types and its dependence on K(+) channel activity was measured at the single cell level by time lapse photography in the absence and presence of the specific K(+) channel blocker charybdotoxin (CTX). Locomotion of both cell types is inhibited by K(+) channel blockade. CTX slows down migration of fibroblasts and of melanoma cells dose-dependently by up to 61 +/- 11%. These findings suggest that K(+) channel activity is a general prerequisite for migration. To determine whether CTX-induced inhibition of migration of fibroblasts and melanoma cells involves quantitative changes of actin filaments, we indirectly measured filamentous actin by quantitating binding of fluorescently labeled phalloidin. Whereas CTX elicits a decrease of bound phalloidin in fibroblasts there is an increase in melanoma cells. Since migration of tumor cells is required for invading surrounding tissue, we developed an assay to test whether CTX-induced inhibition of migration also impairs invasion of melanoma cells. Melanoma cells were seeded on a layer of high resistance renal epithelial cells (MDCK cells clone C7; transepithelial resistance R(te) >3,000 Omegacm(2)) and R(te) was measured daily. R(te) starts to decrease 2 days after seeding of melanoma cells onto MDCK-C7 cells. By day 7, R(te) has dropped to 24 +/- 1.5% of control. K(+) channel blockade with CTX (10 nmol/l) cannot prevent or delay this drop of R(te). R(te) reaches the same level with or without CTX. These results indicate that the disruption of an epithelial layer, unlike migration of melanoma cells, cannot be modulated by K(+) channel blockade.

3T3 Cells↗

PubMed1998

Migrating transformed MDCK cells are able to structurally polarize a voltage-activated K+ channel.

Cell migration of transformed renal epithelial cells (MDCK-F) depends-in addition to cytoskeletal mechanisms-on the polarized activity of a Ca2+-sensitive K+ channel in the rear part of the cells. However, because of the lack of specific markers for this channel we are not able to determine whether a polarized distribution of the channel protein underlies its functional polarization. To determine whether the migrating MDCK-F cells have retained the ability to target K+ channels to distinct membrane areas we stably transfected the cells with the voltage-dependent K+ channel Kv1.4. Stable expression and insertion into the plasma membrane could be shown by reverse transcription-PCR, genomic PCR, Western blot, and patch-clamp techniques, respectively. The distribution of Kv1.4 was assessed with indirect immunofluorescence by using conventional and confocal microscopy. These experiments revealed that Kv1.4 is expressed only in transfected cells where it elicits the typical voltage-dependent, rapidly inactivating K+ current. The Kv1.4 protein is clustered at the leading edge of protruding lamellipodia of migrating MDCK-F cells. This characteristic distribution of Kv1.4 provides strong evidence that migrating MDCK-F cells are able to insert ion channels into the plasma membrane in an asymmetric way, which reflects the polarization of migrating cells in the plane of movement. These findings suggest that not only epithelial cells and nerve cells, but also migrating cells, can create functionally distinct plasma membrane areas.

Animals↗

PubMed1998

Measurement of human nasal potential difference to teach the theory of transepithelial fluid transport.

We describe a novel student course in membrane physiology in which students record their own nasal potential difference, i.e., the transepithelial potential difference of the respiratory mucosa in the nose. The nasal potential difference monitors directly, and in vivo, changes in the apical cell membrane potential of the respiratory mucosa induced by activators and inhibitors of ion channel activities. Basic principles of transepithelial fluid transport are taught by applying an appropriate perfusion protocol to the respiratory epithelium to either depolarize or hyperpolarize the membrane potential of the luminal cell side, thereby increasing or decreasing the nasal potential difference. This course was given at the Department of Physiology at the University of Würzburg in 1997, and responses of the students as reported on questionnaires were mainly positive.

Biological Transport↗

PubMed1998

MR imaging and localized proton MR spectroscopy in late infantile neuronal ceroid lipofuscinosis.

PURPOSE: Late juvenile neuronal ceroid lipofuscinosis (NCL) is a lysosomal neurodegenerative disorder caused by the accumulation of lipopigment in neurons. Our purpose was to characterize the MR imaging and spectroscopic findings in three children with late infantile NCL. METHODS: Three children with late infantile NCL and three age-matched control subjects were examined by MR imaging and by localized MR spectroscopy using echo times of 135 and 5. Normalized peak integral values were calculated for N-acetylaspartate (NAA), choline, creatine, myo-inositol, and glutamate/glutamine. RESULTS: MR imaging revealed volume loss of the CNS, most prominently in the cerebellum. The T2-weighted images showed a hypointense thalamus and hyperintense periventricular white matter. Proton MR spectra revealed progressive changes, with a reduction of NAA and an increase of myo-inositol and glutamate/glutamine. In long-standing late infantile NCL, myo-inositol became the most prominent resonance. Lactate was not detectable. CONCLUSION: MR imaging in combination with proton MR spectroscopy can facilitate the diagnosis of late infantile NCL and help to differentiate NCL from other neurometabolic disorders, such as mitochondrial or peroxisomal encephalopathies.

Aspartic Acid↗

PubMed1997

Life on biomembranes viewed with the atomic force microscope.

Since its invention in 1986, the atomic force microscope (AFM) has become one of the most widely used near-field microscopes. Surfaces of hard samples are imaged almost routinely with atomic resolution. Soft biological surfaces, however, are still challenging. In this brief review, the AFM technique is introduced to the experimental biologist. We discuss recent data on imaging molecular structures of biomembranes, and give detailed information on the application of the AFM with three representative examples. One is imaging plasma membrane turnover of transformed renal epithelial cells during migration in vivo, another is visualizing a cloned and isolated potassium channel usually located in kidney, and a third is imaging macromolecular pore complexes of the nuclear envelope of aldosterone-sensitive kidney cells and of Xenopus laevis oocytes. The review ends with the conclusion that nuclear pores can serve as birthday candles on a Guglhupf.

Animals↗