Claudin-1, -3, -4 and -7 gene expression analyses in canine prostate carcinoma and mammary tissue derived cell lines.

Claudins (CLDNs) are transmembrane proteins localised in the cell membrane of epithelial cells composing a structural and functional component of the tight junction protein complexes. In canine tumors deregulations of the CLDN expression patterns were described immunohistochemically. Targeting of claudin proteins has further been evaluated to establish novel therapeutic approaches by directed claudin binding. Precondition for the development of claudin targeting approaches in canine cells is the possibility to characterise claudin expression specifically and the availability of claudin positive cell lines. Herein PCR/qPCR assays were established allowing a rapid qualitative and quantitative characterisation of CLDN-1, -3, -4 and -7 gene expression in canine cell lines and tissues. Further commercially available antibodies were used to verify CLDN gene expression on protein level by Western blots. The developed assays were used to analyse six canine cell lines derived from mammary and prostate tissue for their CLDN-1, -3, -4 and -7 expressions. The canine cell line DT08/40 (prostate transitional cell carcinoma) was used for the establishment of specific CLDNs -1, -3, -4 and -7PCR/qPCR. The designed assays were verified by amplicon cloning and sequencing. Gene expressions were verified on protein level by Western blot. Additionally further cell lines were analysed for their CLDN-1, -3, -4 and -7 expression on mRNA and protein level (mammary derived cell lines: MTH53A (non-neoplastic), ZMTH3 (adenoma), MTH52C (carcinoma); prostate derived cell lines: DT08/46 and CT1258 (both adenocarcinoma).The screened cell lines showed expression for the CLDNs as follows: DT08/46 and DT08/40: CLDN-1, -3, -4 and -7 positive; CT1258: CLDN-1, -3, -4 and -7 negative; ZMTH3 and MTH52C: CLDN-1 and -7 positive, CLDN-3 and -4 negative; MTH53A: CLDN-1, -3 and -4 negative, CLDN-7 positive. Western blot analyses reflect the detected CLDN-1, -3, -4 and -7 expressions in the analysed cell lines. The established CLDN-1, -3, -4 and -7 PCR/qPCR assays allow a qualitative and quantitative characterisation of canine CLDN gene expression. Characterisation of CLDN expression in six canine cell lines led to the identification of two canine prostate tissue derived CLDN expressing cell lines. These cell lines serve as candidates for further research on CLDN-based functional and therapeutic approaches.

Fs-laser-induced Ca2+ concentration change during membrane perforation for cell transfection.

Fs-laser based opto-perforation is a gentle method for gene transfer into sensitive cells such as stem cells or primary cells. The high selectivity and the low damage to the cell lead to a high efficiency of transfection. However, there are side effects which induce stress to the cell due to the exchange of intra- and extracellular media as well as the disintegration of the structure of biomolecules resulting from the laser exposure. Moreover, the mechanisms of the optical transfection are still unclear. In this paper, we present our study on calcium (Ca(2+)) homeostasis during cell surgery, especially during laser induced membrane perforation. We show that the manipulation of cells can induce an increase in the cytosolic Ca(2+) concentration. This increase was not observed if the manipulation of the cells was performed in absence of the extracellular calcium indicating the importance of the Ca(2+) uptake. We found, that the uptake of extracellular Ca(2+) strongly depends on the repetition rate and the irradiation time of the laser pulses. The exposure for several seconds to kHz pulses even induces Ca(2+) induced Ca(2+) release. Dependent on the location of perforation, probably in the vicinity of an intracellular Ca(2+) stock, an instantaneous intracellular Ca(2+) release can be induced. Since Ca(2+) could be involved in negative side effect by cell surgery, we propose an application of the optoperforation technique in nominal Ca(2+)-free external solution.

Dipyridamole increases gap junction coupling in bovine GM-7373 aortic endothelial cells by a cAMP-protein kinase A dependent pathway.

The scrape-loading/dye transfer technique was applied on the bovine aortic endothelial cell line GM-7373 to analyze the effects of the antithrombolytic drug dipyridamole on gap junction coupling in endothelial cells. We found that a cell treatment for 24 h with dipyridamole in therapeutically relevant concentrations (1-100 microM) increased gap junction coupling in a dose dependent manner. Similar to dipyridamole, forskolin as well as 8-Br-cAMP increased the gap junction coupling, while dibutyryl-cGMP (db-cGMP) did not affect the gap junction coupling of the GM-7373 endothelial cells. In parallel, a pharmacological inhibition of protein kinase A (PKA) with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89), antagonised the action of dipyridamole on gap junction coupling. We propose that the observed dipyridamole induced increase in gap junction coupling in endothelial cells is related to a cAMP-PKA dependent phosphorylation pathway. The report shows that gap junction coupling in endothelial cells is a suitable therapeutic target for treatment of cardiovascular diseases.

Quantified femtosecond laser based opto-perforation of living GFSHR-17 and MTH53 a cells.

Opto-perforation is an interesting alternative to conventional techniques for gene transfer into living cells. The cell membrane is perforated by femtosecond (fs) laser pulses, in order to induce an uptake of macromolecules e.g. DNA. In this study, we successfully transfected a canine cell line (MTH53a) with GFP vector or a vector coding for a GFP-HMGB1 fusion protein. The transfected cells were observed 48 hours after treatment and they were not showing any signs of apoptosis or necrosis. Based on simultaneously measured membrane potential changes during the perforation, we were able to calculate and experimentally verify that the relative volume exchanged is 0.4 times the total cell volume. Thus, for first time a quantitative predication of the amount of uptaken molecules and therefore a quantification of the transfection is possible. Additionally, this method offers new high efficient possibilities for critical transfection approaches involving special cell types, e.g. primary and stem cells.

Gap junctions and cochlear homeostasis.

Gap junctions play a critical role in hearing and mutations in connexin genes cause a high incidence of human deafness. Pathogenesis mainly occurs in the cochlea, where gap junctions form extensive networks between non-sensory cells that can be divided into two independent gap junction systems, the epithelial cell gap junction system and the connective tissue cell gap junction system. At least four different connexins have been reported to be present in the mammalian inner ear, and gap junctions are thought to provide a route for recycling potassium ions that pass through the sensory cells during the mechanosensory transduction process back to the endolymph. Here we review the cochlear gap junction networks and their hypothesized role in potassium ion recycling mechanism, pharmacological and physiological gating of cochlear connexins, animal models harboring connexin mutations and functional studies of mutant channels that cause human deafness. These studies elucidate gap junction functions in the cochlea and also provide insight for understanding the pathogenesis of this common hereditary deafness induced by connexin mutations.

Gap junction coupling and apoptosis in GFSHR-17 granulosa cells.

Recently, we found that intracellular washout of cGMP induces gap junction uncoupling and proposed a link between gap junction uncoupling and stimulation of apoptotic reactions in GFSHR-17 granulosa cells. In the present report we show that an inhibitor of guanylyl cyclase, ODQ, reduces gap junction coupling and promotes apoptotic reactions such as chromatin condensation and DNA strand breaks. To analyze whether gap junction uncoupling and induction of apoptotic reactions are related, the cells were treated with heptanol and 18 beta-GA, two known gap junction uncouplers. Gap junction coupling of GFSHR-17 cells could be restored if the incubation time with the gap junction uncouplers was less than 10 min. A prolonged incubation time irreversibly suppressed gap junction coupling and caused chromatin condensation as well as DNA degradation. The promotion of apoptotic reactions by heptanol or 18 beta-GA was not observed in cells with low gap junction coupling like HeLa cells, indicating that the observed genotoxic reactions are not caused by unspecific effects of gap junction uncouplers. Additionally, it was observed that heptanol or 18 beta-GA did not induce a sustained rise of [Ca(2+)](i). The effects of gap junction uncouplers could not be suppressed by the presence of 8-Br-cGMP. It is discussed that irreversible gap junction uncoupling can be mediated by cGMP-dependent as well as cGMP-independent pathways and in turn could lead to stimulation of apoptotic reactions in granulosa cells.

Structural calorimetry of main transition of supported DMPC bilayers by temperature-controlled AFM.

Atomic force microscopy at high temperature resolution (DeltaT < or approximately 0.1 K) provided a quantitative structural calorimetry of the transition from the fluid (Lalpha)- to the gel (Pbeta')-phase of supported dimyristoylphosphatidylcholine bilayers. Besides a determination of the main transition temperature (T0) and the van't Hoff transition enthalpy (DeltaHvH), the structural analysis in the nm-scale at T close to T0 of the ripple phase allowed an experimental estimation of the area of cooperative units from small lipid domains. Thereby, the corresponding transition enthalpy (DeltaH) of single molecules could be determined. The lipid organization and the corresponding parameters T0 and DeltaHvH (DeltaH) were modulated by heptanol or external Ca2+ and compared with physiological findings. The size of the cooperative unit was not significantly affected by the presence of 1 mM heptanol. The observed linear relationship of DeltaHvH and T0 was discussed in terms of a change in heat capacity.

Regulation of ion fluxes, cell volume and gap junctional coupling by cGMP in GFSHR-17 granulosa cells.

Gap junctional communication between granulosa cells seems to play a crucial role for follicular growth and atresia. Application of the double whole-cell patch-clamp- and ratiometric fura-2-techniques allowed a simultaneous measurement of gap junctional conductance ( G(j)) and cytoplasmic concentration of free Ca(2+) ([Ca(2+)](i)) in a rat granulosa cell line GFSHR-17. The voltage-dependent gating of G(j) varied for different cell pairs. One population exhibited a bell-shape dependence of G(j) on transjunctional voltage, which was strikingly similar to that of Cx43/Cx43 homotypic gap junction channels expressed in pairs of oocytes of Xenopus laevis. Within 15-20 min, gap junctional uncoupling occurred spontaneously, which was preceded by a sustained increase of [Ca(2+)](i) and accompanied by shrinkage of cellular volume. These responses to the whole-cell configuration were avoided by absence of extracellular Ca(2+), blockage of K(+) efflux, or addition of 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) to the pipette solution. Even in the absence of extracellular Ca(2+) or blockage of K(+) efflux, formation of whole-cell configuration generated a Ca(2+) spike that could be suppressed by the presence of 8-Br-cGMP. We propose that intracellular cGMP regulates Ca(2+) release from intracellular Ca(2+) stores, which activates sustained Ca(2+) influx, K(+) efflux and cellular shrinkage. We discuss whether gap junctional conductance is directly affected by cGMP or by cellular shrinkage and whether gap junctional coupling and/or cell shrinkage is involved in the regulation of apoptotic/necrotic processes in granulosa cells.

Hydrogen peroxide inhibits gap junctional coupling and modulates intracellular free calcium in cochlear Hensen cells.

The double whole-cell patch-clamp configuration was applied to analyze gap junctional conductance (Gj) of isolated pairs of cochlear supporting Hensen cells of guinea pig under control conditions and in the presence of hydrogen peroxide (H2O2). Under control conditions, the dependence of Gj on transjunctional voltage (Vj) appeared to vary between different cell pairs with a maximum value of about 40 nS at Vj close to 0 mV. The voltage dependence and the maximum amplitude of Gj stayed constant for at least 2 hr. Addition of H2O2 to the bath at concentrations above 0.08 mm caused a significant decrease of Gj, but the membrane potential of about -30 mV was not affected. In parallel, intracellular free calcium ([Ca2+]i) was followed using fura-2. At 0.8 mm H2O2, a sustained increase of [Ca2+]i was observed, while 0.08 mm H2O2 evoked an oscillating-like behavior of [Ca2+]i. We propose that the H2O2-evoked inhibition of gap junctional coupling of Hensen cells is closely related to pathophysiological conditions such as noise- induced hearing loss, aminoglycoside-related ototoxicity and presbycusis, which are known to be associated with production of free radicals.

Synaptic activity modulates the induction of bidirectional synaptic changes in adult mouse hippocampus.

Activity-dependent synaptic plasticity is critical for learning and memory. Considerable attention has been paid to mechanisms that increase or decrease synaptic efficacy, referred to as long-term potentiation (LTP) and long-term depression (LTD), respectively. It is becoming apparent that synaptic activity also modulates the ability to elicit subsequent synaptic changes. We provide direct experimental evidence that this modulation is attributable, at least in part, to variations in the level of postsynaptic depolarization required for inducing plasticity. In slices from adult hippocampal CA1, a brief pairing protocol known to produce LTP can also induce LTD. The voltage-response function for the induction of LTD and LTP in naive synapses exhibits three parts: at a postsynaptic membrane potential during pairing (V(m)) -20 mV, LTP is generated. This function varies with initial synaptic efficacy. In depressed synapses, Theta(-), the V(m) above which LTD is generated, is shifted toward more depolarized V(ms) and Theta(+), the LTD-LTP crossover point or, equivalently, the V(m) above which LTP is induced, toward more polarized V(ms). Conversely in potentiated synapses, Theta(-) is shifted toward more polarized V(ms). Therefore synaptic activity changes synaptic efficacy and accordingly adjusts the voltages for eliciting subsequent synaptic modifications. The concomitant shifts in the voltages for inducing LTD and LTP in opposite directions promote synaptic potentiation and inhibit synaptic depression in depressed synapses and vice versa in potentiated synapses.

Regulation of lens rCx46-formed hemichannels by activation of protein kinase C, external Ca(2+) and protons.

Rodent lens connexin46 (rCx46) formed active voltage-dependent hemichannels when expressed in Xenopus oocytes. Time-dependent macroscopic currents were evoked upon depolarization. The observed two activation time constants were weakly voltage-dependent and in the order of hundreds of milliseconds and seconds, respectively. Occasionally, the macroscopic steady-state current and the corresponding current-voltage curve showed inactivation at high depolarizing voltages (>+50 mV). To account for the fast recovery from inactivation (<2 msec) favored by hyperpolarization, a four-state kinetic model (C(1)(closed) <--> C(2)(closed) <--> O(open) <--> I(inactivated)) is proposed. In the absence of inactivation, the macroscopic conductance decreased and inactivation became visible at voltages positive of +50 mV when the rCx46-expressing oocytes were treated with the protein-kinase-C-activators OAG or TPA, high external concentrations of Ca(2+) or H(+). However, the underlying mechanisms of OAG, H(+) or Ca(2+) action were different. While OAG did not alter the voltage-dependent activation of the rCx46-hemichannels, an increase in the external Ca(2+) or H(+) level shifted the voltage threshold for activation to more positive voltages. In contrast to Ca(2+), protons were not effective in the physiological concentration range. We propose that under physiological conditions only external Ca(2+) and intracellular PKC-dependent processes regulate rCx46 in the lens.

Inhibition of gap junctional coupling in cochlear supporting cells by gentamicin.

Gap junctional coupling of cochlear supporting cells is thought to be responsible for spatial potassium buffering of the microenvironment of outer hair cells (OHC). OHC of the organ of Corti are considered as the target of aminoglycoside-induced ototoxicity. Due to the proposed functional relationship between OHC and cochlear supporting cells we investigated a possible involvement of the supporting Hensen cells in the ototoxic effect of the aminoglycoside gentamicin. Isolated Hensen cell pairs were superfused by gentamicin-containing bath solutions. Using the double whole-cell patch-clamp method gentamicin (10 microM) inhibited gap junctional conductance by about 90%, whereas the membrane potential of about -27 mV remained unchanged. Since the inhibitory effect was suppressed by the addition of catalase, the gentamicin mediated effect probably is due to production of free radicals. It is proposed that formation of free radicals in supporting cells inhibits gap junctional coupling whereby the spatial potassium buffer mechanism and, thus, the fine tuning of the cochlear OHC is impaired.

Inactivation of expressed and conducting rCx46 hemichannels by phosphorylation.

Hemichannels of rat connexin 46 (rCx46) were expressed in Xenopus laevis oocytes and analysed by two-electrode voltage-clamp experiments. It is established that rCx46 hemichannels can be activated at low external Ca2+ and positive membrane potentials. Upon larger depolarizations, the hemichannels of oocytes activate in a time-dependent manner, occasionally followed by a spontaneous inactivation. We found that, in the absence of inactivation, treatment of oocytes with 1-oleoyl-2-acetyl-sn-glycerol (OAG), an activator of protein kinase C (PKC), reversibly reduced the amplitude of the rCx46-mediated current and, after an incubation time of about 30 min, induced inactivation of the voltage-dependent current. After wash-out of OAG the corresponding membrane conductance increased and the inactivation behaviour disappeared. The OAG-induced inactivation, as well as the spontaneous inactivation, could be removed by application of the specific PKC inhibitor calphostin C and also by phloretin. The data provide evidence that the activation and inhibition of PKC affect the rCx46-mediated membrane conductance as well as the voltage-dependent current inactivation in an inverse manner.

Cholecystokinin-octapeptide affects the fluorescence signal of a single pancreatic acinar cell loaded with the acrylodan-labelled MARCKS peptide, a protein kinase C substrate.

We used a fluorescent derivative of the myristoylated, alanine-rich C kinase substrate (MARCKS) peptide as a probe for protein kinase C (PKC) activation by cholecystokinin-octapeptide (CCK-8) in isolated pancreatic acinar cell pairs. The diffusion of the acrylodan-labelled MARCKS-peptide into the cell interior could be monitored by the increase of fluorescence in the whole-cell patch-clamp configuration. Addition of 10 pM CCK-8 to the bath induced repetitive fluctuations of the fluorescent signal in the time range of 4-5 min. With 1 nM CCK-8 a sustained decrease of the signal was observed. Addition of polymyxin B, a specific inhibitor of PKC activation, to the pipette filling solution suppressed the CCK-8-induced change of fluorescence. The data indicate activation of PKC by CCK-8 in pancreatic acinar cells and could be compared with the previously studied CCK-8-induced gap junction uncoupling.

Regulation of gap junctional coupling in isolated pancreatic acinar cell pairs by cholecystokinin-octapeptide, vasoactive intestinal peptide (VIP) and a VIP-antagonist.

Cholecystokinin-octapeptide (CCK-OP) induces a time- and dose-dependent decrease of gap junctional conductance in isolated pairs of pancreatic acinar cells. In double whole-cell experiments, the time course could be described by the latency and the half-life time (t1/2) of cell-to-cell uncoupling. The latency shows a biphasic dependence on [CCK-OP] with a minimum of about 50 sec at 10(-9) M CCK-OP. In the presence of vasoactive intestinal peptide (VIP), the biphasic relationship is shifted to lower CCK-OP concentrations. The increase of latency at high concentrations of CCK-OP (> 10(-9) M) was blocked by addition of a VIP-antagonist. t1/2 decreases monophasically with increasing [CCK-OP]. Addition of GTP gamma S to the pipette solution suppresses the [CCK-OP] dependence of the latency and potentiates the uncoupling phase. The kinetic data are discussed in terms of CCK binding to receptors of high and low affinity. Evidence is presented that secretion and cell-to-cell coupling are not related by an all-or-none process, but that for physiological CCK-OP concentrations, gap junctional uncoupling follows secretion.

Gap junctional conductance tunes phase difference of cholecystokinin evoked calcium oscillations in pairs of pancreatic acinar cells.

Using the double whole-cell recording technique we measured the gap junctional conductance (gj), while simultaneously monitoring the cytoplasmic free calcium concentration ([Ca2+i]) in isolated acinar cell pairs from the pancreas of mouse. Physiological concentrations of the secretagogue cholecystokinin-octapeptide (CCK-OP) elicited [Ca2+i] oscillations and after a delay in the minute range reduced gj. The results provide the first evidence suggesting that gj regulates the phase difference (delta phi) of [Ca2+i] oscillations between neighbouring cells not in an all or none process, but with decreasing absolute value of gj delta phi monotonically increases. CCK-OP induced electrical uncoupling as well as the corresponding increase of delta phi could be blocked completely by the protein kinase C inhibitor polymyxin B. The data support the hypothesis that Ca2+ flow through gap junctions synchronizes [Ca2+i] oscillations between jap junctional coupled cells.

Gap junctional permeability is affected by cell volume changes and modulates volume regulation.

Isolated pancreatic acinar cell pairs became electrically uncoupled by exposure to a mild hypotonic shock. Reduction of bath osmolarity caused a delayed closure of gap junctional channels in the minute range. Dialysis of cell pairs by GTP[S] in the double whole-cell patch-clamp mode shortened the latency and shifted the hypotonically induced electrical uncoupling to lower osmolarity changes. Cellular treatment with cytochalasin B catalyzed electrical uncoupling by a hypotonic shock. In all cases, electrical uncoupling could be blocked completely by the protein kinase C (PKC) inhibitor polymyxin B. These results provide the first evidence suggesting that changes of cell volume and gap junctional permeability are correlated and that a G-protein dependent mechanism is involved. Evidence is presented that gap junctional coupling modulates volume regulation.