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R Eckert

Publications and source records attributed to R Eckert.

At least 37 records · Page 2Linked to original sources
PubMed1998

Spatial differences in gap junction gating in the lens are a consequence of connexin cleavage.

Gap junctions in the vertebrate lens exhibit spatial differences in pH gating: those in the cortical fibre cells close upon tissue acidification while those in the core region do not. It has been speculated that this difference in channel gating is a consequence of the cleavage of the connexins (Cx) that form the gap junction channels. We report the construction of a truncation mutant of ovine Cx50 which mimicks the cleavage in the intact lens. The construct when expressed in Xenopus oocytes results in the formation of functional channels. Comparison with full-length Cx50 revealed a significant reduction in the pH-sensitivity of the truncated form. This is the first evidence linking the non-uniform gating of gap junction channels in the lens with connexin cleavage. It also reveals how fibre cells in the core region remain connected despite the acidic environment caused by elevated lactate levels.

Animals↗
PubMed1998

Characterization of the gap junction protein connexin37 in murine endothelium, respiratory epithelium, and after transfection in human HeLa cells.

Affinity-purified antibodies to oligopeptides derived from two different regions of the carboxyterminus and cytoplasmic loop or to the last 103 C-terminal amino acids of mouse connexin37 (Cx37) were used to characterize expression of this gap junctional protein in endothelium of several murine tissues. Cx37 was expressed in endothelium of large blood vessels in brain, liver, kidney, spleen, heart, and lung, but not in capillaries. In addition, weak Cx37 immuno-signals were observed in lung respiratory epithelium of small bronchi and in alveolar epithelial cells of bronchioli. The ratios of Cx37 protein to Cx37 mRNA in adult and embryonic kidney as well as skin were 29-303-fold larger than in lung, suggesting that Cx37 mRNA was translated at different efficiencies in kidney and skin versus lung. Cx37 protein was more abundant in embryonic kidney and lung than in the corresponding adult tissues. After differential centrifugation of plasma membrane fractions in sucrose gradients, we found that Cx37-containing gap junctions in lung were much smaller than Cx32 and Cx26 aggregates from liver. HeLa cells were transfected with mouse Cx37 cDNA. In these cells, mouse Cx37 protein was phosphorylated mainly at serine, less at tyrosine, and very little at threonine residues. Three conductance states were resolved at 110, 240, and 315 pS.

Animals↗
PubMed1998

A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes.

Gap junctions provide direct intercellular communication by linking adjacent cells with aqueous pores permeable to molecules up to 1 kDa in molecular mass and 8-14 A in diameter. The identification of over a dozen connexins in the mammalian gap junction family has stimulated interest in the functional significance of this diversity, including the possibility of selectivity for permeants as seen in other channel classes. Here we present a quantitative comparison of channel permeabilities of different connexins expressed in both HeLa transfectants (rat Cx26, rat Cx32 and mouse Cx45) and Xenopus oocytes (rat Cx26 and rat Cx32). In HeLa cells, we examined permeability to two fluorescent molecules: Lucifer Yellow (LY: anionic, MW 457) and 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI, cationic, MW 350). A comparison of the kinetics of fluorescent dye transfer showed Cx32, Cx26 and Cx45 to have progressively decreasing permeabilities to LY, but increasing permeabilities to DAPI. This pattern was inconsistent with selection based on physical size of the probe, nor could it be accounted for by the differences between clones in the electrical conductance of the monolayers. In Xenopus oocytes, where electrical and dye coupling could be assessed in the same cells, Cx32 coupled oocytes showed an estimated 6-fold greater permeability to LY than those coupled by Cx26, a comparable result to that seen in HeLa cells, where an approximately 9-fold difference was seen. The oocyte system also allowed an examination of Cx32/Cx26 heterotypic gap junction that proved to have a permeability intermediate between the two homotypic forms. Thus, independent of the expression system, it appears that connexins show differential permeabilities that cannot be predicted based on size considerations, but must depend on other features of the probe, such as charge.

Animals↗
PubMed1998

A distinct membrane current in rat lens fiber cells isolated under calcium-free conditions.

PURPOSE: To investigate whether lens fiber cells isolated and maintained under calcium-free conditions exhibit distinct membrane currents. METHODS: Fiber cells were isolated from the cortical portion of neonate rat lenses using a trypsin digestion protocol and were maintained in EDTA-buffered Ringer's solutions. Membrane currents were recorded from fiber bundles using the whole-cell patch-clamp technique. RESULTS: Cortical fiber cells of up to 600-microm length were viable and amenable to whole-cell patch-clamp recording. The major current recorded under these conditions was a slowly activating, voltage-dependent current that was markedly increased on membrane depolarization. This current appeared to be fiber cell specific and had similar properties to currents elicited by gap junction hemichannels previously recorded by others in Xenopus oocytes. CONCLUSIONS: This is the first report of whole-cell patch-clamp recordings from intact elongated fiber cells. Fiber cells kept in calcium-free bath medium appear to be electrically "leaky" and exhibit a distinct membrane current that has not been described previously for lens cells. This current is unlikely to be active in the normal lens but may play a role in the depolarized cataractogenic lens.

Animals↗
PubMed1998

Expression profiles of P2-receptor isoforms P2Y1 and P2Y2 in the rat lens.

PURPOSE: To verify at the molecular level that P2 receptors are expressed in the lens and to determine their expression profiles. METHODS: The reverse transcription-polymerase chain reaction (RT-PCR) was used to screen rat lens epithelial and fiber cells for the expression of the ionotropic P2X2 receptor and the G-protein-linked receptor isoforms P2Y1 and P2Y2. Northern blot analysis was used to confirm the level of expression of P2Y1 and P2Y2. The profile of P2-receptor isoform expression in the lens was identified using an indirect RT-PCR in situ hybridization procedure on paraffin sections of whole rat eyes. RESULTS: P2X2-receptor transcripts could not be detected in lens epithelial or fiber cells. P2Y1- and P2Y2-receptor transcripts were detected in lens fiber cells but not in the epithelial cells. Their expression profiles were maximal in the lens cortex and mostly overlapped each other, except that transcripts for the P2Y2-receptor isoform appeared earlier in the not yet fully elongated fiber cells in the lens bow region. CONCLUSIONS: The molecular data support physiological evidence previously reported by others that P2Y receptors are expressed in the lens and identifies cortical fiber cells as the principal site of expression.

Animals↗
PubMed1997

Understanding intercellular interactions and cell adhesion: lessons from studies on protein-metal interactions.

To understand cell-cell interactions and the interactions of cells to non-biological materials, studies on binding forces between cellular proteins and between proteins and non-biological material such as metal surfaces are essential. The adsorption of proteins to solid-water interfaces is a multifactorial and a multistep process. First steps are determined by long-range interactions where surface properties such as hydrophobicity, distribution of charged groups, ion concentrations and pH play important roles. In later steps structural rearrangements in the protein molecule and dehydration effects become more important making the adsorption process often irreversible. In the following we demonstrate that protein A and tubulin have a specific type of interaction to metal surfaces probably as an intermediate step in the adsorption process. The proteins were attached to the tip of a microfabricated cantilever in such a way that only one molecule interacts with the surface. By recording force-distance curves with an atomic force microscope the adhesion forces of single molecules binding to gold, titanium and indium-tinoxid surfaces were measured.

Animals↗
PubMed1997

Gap junction channels: new roles in disease.

The importance of intercellular communication to complex cellular processes such as development, differentiation, growth, propagation of electrical impulses and diffusional feeding has long been appreciated. The realization that intercellular communication is mediated by gap junction channels, which are in turn comprised of a diverse family of proteins called the connexins, has provided new tools and avenues for studying the role of intercellular communication in these important cellular processes. The identification of different connexin isoforms has not only enabled the development of specific reagents to study connexin expression patterns, but has also allowed the functional properties of the different connexin isoforms and how they interact with each other, to be explored. Increasingly, the knowledge gained from studying connexin diversity is being used to investigate the role played by gap junction channels in a number of diseases. In this article we highlight selected cases where gap junction channels have been shown or are believed to be directly involved in the disease process.

Animals↗
PubMed1996

Retinoid X receptor-specific retinoids inhibit the ability of retinoic acid receptor-specific retinoids to increase the level of insulin-like growth factor binding protein-3 in human ectocervical epithelial cells.

The hormones derived from vitamin A and related synthetic ligands (retinoids) are important regulators of differentiation and development and have been shown to be therapeutically useful in the treatment of cervical cancer. All-trans-retinoic acid exerts its effects by activation of retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers. These heterodimers bind to the retinoic acid response elements of target genes to regulate gene expression. RXR ligands act through RXR homodimers to regulate gene expression. In the present study, we describe the effects of RAR- and RXR-specific ligands on the regulation of insulin-like growth factor binding protein-3 (IGFBP-3) production and cell proliferation in human ectocervical epithelial (ECE) cell lines. Treatment of ECE16-1 cells with a RAR-specific ligand (TTNPB) or a ligand that interacts with both RAR and RXR receptors (9-cis-retinoic acid) increases IGFBP-3 levels and suppresses cell proliferation. In contrast, RXR-specific ligands (AGN191701, SR11217, and SR11237) do not regulate proliferation and slightly suppress the IGFBP-3 level. Cotreatment with increasing concentrations (0.01-1000nm) of RXR-specific ligand antagonizes the growth suppressive and IGFB-3-increasing effects of 1000 nM TTNPB. Similar results are observed in two other ECE cell lines, ECE16-D1 and ECE16-D2. These results indicate that RXR-specific ligands can antagonize RAR responses in these cell lines and suggest that a RAR-specific retinoid may be superior to one with mixed RAR/RXR binding activity for inhibiting cervical cancer cell proliferation. Moreover, the antagonism of RAR-dependent responses by RXR-specific ligands is consistent with a squelching model in which the RXR-specific ligand drives formation of RXR/RXR homodimers at the expense of the more active RAR/RXR heterodimers.

Cell Division↗
PubMed1995

Ryanodine-sensitive intracellular Ca2+ stores in isolated rabbit penile erectile tissue.

Norepinephrine release from adrenergic nerve terminals leads to a rise in intracellular Ca2+, which promotes penile smooth muscle contraction and detumescence. Ca2+ sources are the extracellular space and sarcoplasmic Ca2+ stores. To elucidate the role of intracellular stores strips from rabbit erectile tissue were investigated in an organ bath study. Contractions were elicited by phenylephrine (PE) and electrical stimulation. Incubation in Ca(2+)-free solution as well as exposure to nifedipine did not abolish electrical or PE-induced contraction. Ryanodine (10(-5) mol/l), a functional blocker of sarcoplasmic Ca2+ channels, significantly reduced PE response. In the presence of caffeine (10(-3) mol/l) the effect was significantly enhanced. Addition of nifedipine nearly abolished the contraction. These results provide evidence for intracellular Ca2+ pools in cavernosal tissue and indicate that the alpha 1-adrenoceptor-induced contraction requires the opening of voltage-gated Ca2+ channels and the release of Ca2+ from intracellular stores.

Animals↗
PubMed1995

Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells.

DNAs coding for seven murine connexins (Cx) (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43, and Cx45) are functionally expressed in human HeLa cells that were deficient in gap junctional communication. We compare the permeabilities of gap junctions comprised of different connexins to iontophoretically injected tracer molecules. Our results show that Lucifer yellow can pass through all connexin channels analyzed. On the other hand, propidium iodide and ethidium bromide penetrate very poorly or not at all through Cx31 and Cx32 channels, respectively, but pass through channels of other connexins. 4,6 Diamidino-2-phenylindole (DAPI) dihydrochloride shows less transfer among Cx31 or Cx43 transfectants. Neurobiotin is weakly transferred among Cx31 transfectants. Total junctional conductance in Cx31 or Cx45 transfected cells is only about half as high as in other connexin transfectants analyzed and does not correlate exactly with any of the tracer permeabilities. Permeability through different connexin channels appears to be dependent on the molecular structure of each tracer, i.e. size, charge and possibly rigidity. This supports the hypothesis that different connexin channels show different permeabilities to second messenger molecules as well as metabolites and may fulfill in this way their specific role in growth control and differentiation of cell types. In addition, we have investigated the function of heterotypic gap junctions after co-cultivation of two different connexin transfectants, one of which had been prelabeled with fluorescent dextran beads. Analysis of Lucifer yellow transfer reveals that HeLa cells expressing Cx31 (beta-type connexin) do not communicate with any other connexin transfectant tested but only with themselves. Two other beta-type connexin transfectants, HeLa-Cx26 and -Cx32, do not transmit Lucifer yellow to any of the alpha-type connexins analyzed. Among alpha-type connexins, Cx40 does not communicate with Cx43. Thus, connexins differ in their ability to form functional heterotypic gap junctions among mammalian cells.

Animals↗
PubMed1995

Penile sympathic skin response in erectile dysfunction.

Penile sympathic skin response (PSSR) was measured in 20 normal patients and in 46 patients with erectile dysfunction (ED). PSSR could be registered in 80% of normal patients. The latencies were 1,100-1,600 ms. In patients with ED, latencies were 1,240-3,640 ms. A reproducible normal latency of PSSR (< 1,600 ms) indicates a normal sympathic innervation of the penis. Long latencies (> 1,800 ms) indicate neuropathy of unmyelinated nerval fibers. The loss of a reproducible answer however allows no exact diagnostic information. PSSR is a useful diagnostic test for detecting neuropathy of unmyelinated nerval fibers as a possible cause of ED.

Adult↗
PubMed1995

Inhibition of L-type calcium currents in guinea pig ventricular myocytes by the kappa-opioid agonist U50488H does not involve binding to opiate receptors.

In guinea pig ventricular myocytes the kappa-opioid agonist trans(+/-)-3,3-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide methanesulfonate salt (U50488H) inhibited L-type calcium currents (ICa) as measured by the patch clamp technique in a dose-dependent fashion. The basal, as well as the isoprenaline and 8-Br-cAMP-stimulated, ICa were found to be reduced by U50488H. The inhibition was reversible and was not prevented on preincubation with pertussis toxin or the receptor antagonists naloxone and naltrexone. Naltrexone alone also caused an inhibition of ICa. Leucine enkephaline, a peptide opiate agonist, had no effect on ICa. U50488H did not alter the current/voltage relationship of the calcium current. The inhibition was independent of the cytosolic calcium concentration because it was also observed in the presence of 10 mM BAPTA in the pipette. If the compound was applied intracellularly via a perfused patch pipette there was no inhibition of ICa. The calcium current stimulated by the dihydropyridine calcium agonist Bay K 8644 was also reduced by U50488H. Conversely the inhibition of ICa by U50488H could be antagonized by Bay K 8644. In conclusion, these results demonstrate that binding to specific membrane receptors is not involved in the inhibition of L-type calcium current by U50488H and other nonpeptide opioid agonists. A direct interaction with the channel molecule at the exterior of the cell is probably involved.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1994

Muscular cavernous single cell analysis in patients with venoocclusive dysfunction.

Enzymatically isolated smooth muscle cells of the corpora cavernosa obtained from open biopsies of 15 patients, clinically nonresponding to papaverine/phentolamine and prostaglandin E1 (PGE1) and classified by cavernosometry, were examined using the patch-clamp technique in the whole-cell configuration mode simultaneously monitoring the intracellular calcium concentration by means of the Ca(2+)-sensitive fluorescence dye FURA-II. It could be demonstrated that extracellularly applied PGE1 induces smooth muscle relaxation by inhibition of voltage-dependent L-type Ca2+ currents (58 +/- 8%). Compared to intact cavenous tissue (n = 5), the smooth muscle cells of 14/15 PGE1 nonresponders had no evidence of functional disturbance. Due to intact smooth muscle cells in most cases, etiology of venoocclusive dysfunction remains unclear.

Alprostadil↗
PubMed1994

Immunochemical and electrophysiological characterization of murine connexin40 and -43 in mouse tissues and transfected human cells.

Human HeLa or SkHep1 cells, defective in intercellular communication through gap junctions, were transfected with coding sequences of murine connexin40 (Cx40) and -43. The transfected cells were restored in gap junctional coupling as shown by 100-fold increased electrical conductance. When studied by the double whole-cell patch-clamp technique, Cx40 HeLa transfectants exhibited single channel conductances of gamma = 121 +/- 7 pS and gamma = 153 +/- 5 pS. They were voltage gated with an equivalent gating charge of z = 4.0 +/- 0.5 for a voltage of half-maximal inactivation U0 = 44 +/- 7 mV. The corresponding values of connexin43 (Cx43) HeLa transfectants are: gamma = 60 +/- 4 pS and gamma = 40 +/- 2 pS as well as z = 3.7 +/- 0.8 and U0 = 73 +/- 7 mV. Transfer of the dye Lucifer Yellow was always considerably lower in Cx40- than in Cx43-transfectants though their total junctional conductance was similar or even higher than for Cx43-transfectants. In order to characterize cell and tissue distribution as well as phosphorylation of connexin40 and -43 proteins, antibodies to C-terminal oligopeptides of these proteins were prepared and used for immunoblotting, immunoprecipitation, and immunofluorescence analysis of transfected cells where they exhibited the punctate pattern characteristic of gap junctions on contacting membranes. Phosphorylation of connexin40 was shown by immunoprecipitation from 32P-labeled, transfected SkHep1 cells. Analyses of protein distribution in tissues revealed that the amount of connexin40 detected in heart was higher than in lung which is the inverse of the level of connexin40 mRNA in these tissues, suggesting posttranscriptional control of expression. Connexin40 protein in adult mouse heart and skin is about 20-fold more abundant than in the corresponding embryonic tissue. Connexin43 in adult mouse heart appears to be more highly phosphorylated than in embryonic heart or in transfected human cells.

Adenocarcinoma↗
PubMed1993

Role of intracellular Ca(2+)-stores in the 5-hydroxytryptamine-induced Ca(2+)-current inhibition in NG108-15 hybrid cells.

Mouse neuroblastoma x rat glioma hybrid cells (N x G, NG108-15) were used to study the mechanism of Ca(2+)-current (ICa) inhibition by 5-hydroxytryptamine (5-HT). 5-HT caused a dose-dependent decrease of ICa which was abolished by ICS 205-930 (10)(-8) M) while 2-methyl-5-HT was an agonist. Intracellular infusion of GDP beta S (50 microM) prevented the 5-HT-induced inhibition of ICa whereas pertussis toxin (PTX) pretreatment did not alter the 5-HT response. The 5-HT-induced inhibition depended on the free Ca(2+)-concentration in the pipette solution. Pretreating N x G cells with low molecular weight (LMW) heparin (160 micrograms/ml), 200 microM ryanodine or 2-10 mM caffeine attenuated the 5-HT-induced inhibition of ICa. From these results we suggest that the 5-HT-induced ICa inhibition requires release of Ca2+ from intracellular stores.

Animals↗
PubMed1993

Biophysical characterization of gap-junction channels in HeLa cells.

HeLa cells seem not to be junctionally coupled when probed with techniques such as Lucifer yellow spreading and/or ionic coupling measured with three inserted microelectrodes. When investigated with double whole-cell patch-clamp measurements, HeLa cells in monolayer cultures were electrically coupled in 39% of the cases with very low transjunctional conductances (average one to five open channels). These gap-junction channels had a single-channel conductance gamma = 26 +/- 6 pS and were voltage-gated with an equivalent gating charge z = 3.1 +/- 1.5 for a voltage of half-maximal inactivation Uo = 49 +/- 10 mV. The voltage-dependent component represents only 31 +/- 8% of the total junctional conductance. The voltage-insensitive conductance is characterized by a residual open probability po (infinity) = 0.34 +/- 0.12, which corresponds to a ratio Gmin/Gmax = 0.50 +/- 0.12. Dissociation of monolayer cells into cell pairs yielded about 58% coupled cell pairs with no notably altered single-channel properties.

Connexins↗
PubMed1993

Involvement of intracellular Ca2+ release mechanism in adenosine-induced cardiac Ca2+ current inhibition.

BACKGROUND: Myocardial contractility is regulated by changes in the free intracellular Ca2+ concentration [Ca2+]i, which is determined by concerted interactions of Ca2+ influx through voltage-dependent Ca2+ channels, release of Ca2+ from intracellular pools, and Ca2+ extrusion systems. Although adenosine has been reported to attenuate postischemic stunning, the mechanism is unknown. METHODS: The patch clamp technique and the Ca(2+)-sensitive fluorescence dye FURA II were used in isolated ventricular cardiomyocytes to study the effects of adenosine on the L-type Ca2+ channel current (ICa) and on [Ca2+]i. RESULTS: Adenosine decreased the basal ICa by 76% +/- 4% (100 nmol/L, n = 48) and simultaneously elevated [Ca2+]i up to 1.8 mumol/L (n = 37) by adenosine-A1-receptor activation. The adenosine-evoked responses were not sensitive to pertussis toxin (n = 12), but intracellular application of guanosine diphosphate-beta sulfate (10 mumol/L) reduced the effects of adenosine by 86.7% +/- 7% (n = 9). Block of intracellular Ca2+ release by ryanodine and thapsigargin attenuated, whereas intracellularly applied inositol 1,4,5-trisphosphate (n = 18) mimicked the adenosine-evoked responses. CONCLUSIONS: These findings suggest that coupling of adenosine to A1-receptors induced G protein-mediated activation of phospholipase C followed by generation of inositol 1,4,5-trisphosphate, which releases Ca2+ from intracellular Ca2+ stores. The inhibition of ICa by depletion of intracellular Ca2+ stores could play a major role in preventing ischemia-induced Ca2+ overload.

Adenosine↗