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.

Generation of nanostructures of mica supported lysozyme and lysozyme-nanogold conjugates by diving tip nanowriting.

Nanostructures of lysozyme molecules and lysozyme-nanogold conjugates were generated by atomic force microscopy in contact-, tapping- and force-distance- mode on mica in aqueous solution. In contact mode at high ionic strength, adjusted lysozyme concentration and lower loading force a monolayer of defined structure and orientation of lysozyme can be formed by the scan process of the tip. A lateral resolution of the monolayer of about 80 nm could be achieved. At larger loading forces besides a lysozyme monolayer also 3D- aggregates could be generated in parallel. In force-distance mode the volume of 3D-aggregates was studied as function of lysozyme concentration, loading force and number or frequency of up- and down-movement of the tip. Also in tapping mode 3D-aggregates were generated at the selected incubation conditions. Application of the linescan mode for solutions of nanogold or lysozyme-nanogold conjugates allowed the formation of monlayers of linear shape with lateral resolution of about 35 nm on mica. Nanogold line-structures could be connected to macroscopic gold contacts. It is postulated that adjustment of electrostatic interaction between lysozyme and substrate and the applied loading force is critical for monolayer formation. Different to the underlying mechanism of the well-established dip-pen nanolithography (DPN) (Piner et al., 1999) for the presented method of diving tip nanowriting (DTN) adsorption of the molecules from the aqueous bulk phase to the tip and thereafter the flow to the mica surface is discussed. DTN could be used to either contact proteins electrically or to form preaggregates for protein crystallization (Wiechmann et al.

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.

Analysis of protein crystal growth at molecular resolution by atomic force microscopy.

High-resolution atomic force microscopy (AFM) studies have been performed to analyze the molecularity of growth steps of the (1 1 0) face of tetragonal lysozyme crystals. Besides a major population of step heights of about 5.5 nm also step heights of about half this size were observed. The latter steps always appeared pairwise. Both surfaces the 1 1 0) face and the (1 0 1) face could be imaged at molecular level. Comparison of the height pattern of the corresponding surface structure indicates that the (1 1 0) face is relatively smooth of less than 0.2 nm compared to the (1 0 1) face of about 1.5 nm. AFM linescan images of the (1 0 1) face indicate the insertion of lysozyme aggregates in solution to the crystal surface rather than lysozyme monomers. This study suggests that insertion of lysozyme aggregates in the solution yields growth steps of the (1 1 0) face of monomolecular as well as of bimolecular unit height.

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.

Effects of tetrabutylhydroperoxide on hepatocyte ion channels.

As shown previously, tetrabutylhydroperoxide (t-BOOH) shrinks hepatocytes by release of cellular K(+). The hyperpolarization, the increase of K(+) selectivity, and the Ba(2+) sensitivity of K(+) release have been taken as evidence of activation of K(+) channels. To further define the ion channels involved, patch-clamp studies have been performed on rat hepatocytes. The data show that 0.1 mmol/l t-BOOH inhibits an inwardly rectifying K(+) channel and simultaneously activates a 35-pS K(+) channel. It is suggested that the activation of the latter channel population contributes to the observed K(+) release of hepatocytes following exposure to t-BOOH.

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.

Manipulation and molecular resolution of a phosphatidylcholine-supported planar bilayer by atomic force microscopy.

The morphology of supported planar bilayers has been investigated below phase transition temperature by atomic force microscopy in contact and tapping mode. The bilayers were formed by the vesicle-spreading technique. In contact mode at low scanning forces of about 1 nN true molecular resolution could be achieved for supported phosphatidylcholine bilayers. The resolution was confirmed by experiments that captured the location, average area of individual lipid headgroups and the manipulation of the bilayer surface. Repeated scanning in contact mode shifted the random topology of the surface consecutively to a striped pattern. Height profiles of defect-containing bilayers were analyzed. The shape of the defects became smooth by repeated scanning. The height profiles allowed the estimation of the indentation of the tip into the surface-adsorbed membrane. In tapping mode a disordered pattern of headgroups became visible. Our morphological data at molecular resolution suggest that the native arrangement of the choline head-groups is disordered, free of large packing defects and becomes ordered in Schallamach waves by scanning in contact mode.

The phosphatase inhibitor okadaic acid blocks KCl-depolarization-induced rise of cytosolic calcium of rat insulinoma cells (RINm5F).

It has been shown that okadaic acid (OA) diminishes insulin secretion of rat pancreatic islets in response to glucose, glyceraldehyde and KCl. Glucose, glyceraldehyde and KCl cause release of insulin by depolarization and subsequent opening of L-type calcium channels. Calcium entry into cells is thought to be related to protein phosphorylation. To evaluate whether or not OA mediated inhibition of insulin secretion in response to depolarization might be due to an interference with calcium uptake, we studied its effect on KCl (30 mM)-induced increases of cytosolic calcium and discharge of insulin in the insulin secreting clonal tumor cell line RINm5F. OA inhibited KCl-stimulated insulin release in concentrations > or = 1 microM. In intact RINm5F cells similar concentrations of OA decreased the activity of protein phosphates PP-1/PP-2A and inhibited the depolarization-induced rise of cytosolic calcium ([Ca2+]i). The latter action could also be achieved with the protein phosphatase inhibitor calyculin A, whereas the OA analogue 1-nor-okadaone, which is without effect on phosphatases, did not affect [Ca2+]i or insulin release. It is concluded that depression of depolarization-induced insulin secretion by OA is due to inhibition of calcium entry along voltage dependent calcium channels. The data also suggest that in RINm5F cells protein phosphatases PP-1/PP-2A are related to the function of voltage-dependent calcium channels.

Modulation of Neisseria porin (PorB) by cytosolic ATP/GTP of target cells: parallels between pathogen accommodation and mitochondrial endosymbiosis.

PorB of the pathogenic Neisseria species belongs to the large family of pore-forming proteins (porins) produced by gram-negative bacteria. PorB is exceptional in that it is capable of translocating vectorially into membranes of infected target cells and functions in the infection process. Here we report on an unexpected similarity between Neisserial PorB and mitochondrial porins. Both porin classes interact with purine nucleoside triphosphates, which down-regulate pore size and cause a shift in voltage dependence and ion selectivity. Patch-clamp analyses indicate that PorB channel activity is tightly regulated in intact epithelial cells. In light of recent findings on the pivotal role of PorB in virulence and the prevention of phagosome lysosome fusion, these data provide important mechanistic clues on the intracellular pathogen accommodation reminiscent of mitochondrial endosymbiosis.

Hodgkin-Huxley analysis of a GCAC1 anion channel in the plasma membrane of guard cells.

A quantitative analysis of the time- and voltage-dependent kinetics of the Guard Cell Anion Channel (GCAC1) current in guard cell protoplasts from Vicia faba was analyzed using the whole-cell patch clamp technique. The voltage-dependent steady-state activation of GCAC1 current followed a Boltzmann distribution. For the corresponding steady-state value of the activation variable a power of two was derived which yielded suitable fits of the time course of voltage-dependent current activation. The GCAC1 mediated chloride current could successfully be described in terms of the Hodgkin-Huxley equations commonly evoked for the Na channel in nerve. After step depolarizations from a potential in the range of the resting potential to potentials above the equilibrium potential for chloride an activation and also an inactivation could be described. The gating of both processes exhibited an inverse relationship on the polarity of the applied step potentials in the order of milliseconds. Deactivating tail currents decline exponentially. The presented analysis contributes to the understanding of the rising phase of the observed action potentials in guard cells of V. faba. Evidence is presented that the voltage-dependent kinetic properties of the GCAC1 current are different from those properties described for the excitable anion currents in the plasmalemma of Chara corallina (Beilby & Coster, 1979a).

Electrophysiological effects of progesterone on hepatocytes.

The addition of progesterone (1-100 mumol/l) to the extracellular fluid bathing rat hepatocytes led to a rapid and fully reversible depolarization of the cell membrane. The progesterone-induced depolarization was paralleled by a decrease of potassium selectivity and an increase of cell membrane resistance and was abolished in the presence of the potassium channel blocker barium. Accordingly, in whole cell recordings, progesterone led to a decrease of the cell membrane conductance. 17 alpha-Hydroxyprogesterone and beta-estradiol were less effective by a factor of 10, whereas cholesterol, corticosterone and hydrocortisone did not significantly alter the potential difference across the cell membrane. In conclusion, acute administration of progesterone depolarized rat hepatocytes by decreasing the potassium conductance of the cell membrane.

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.

Morphological alterations and cytoskeletal reorganization in opossum kidney (OK) cells during osmotic swelling and volume regulation.

Cells from a variety of tissues regulate their volume when exposed to anisotonic conditions. After exposure of cells to hypotonic conditions, the rapid phase of cell swelling is followed by a slower phase of cell shrinkage towards the initial volume. The present study investigates morphological alterations of adherent and fully spread cells after exposure to hypotonic conditions and the reorganization of cytoskeletal components such as F-actin, actin-binding proteins, microtubules and intermediate-sized filaments. We used cells of a continuous epithelial cell line from the opossum kidney (OK cells), which were exposed to hypotonic conditions for a period of 60 min at 25 degrees C. The osmolarity was reduced by 40% from 320 mosmol/l (isotonic conditions) to 192 mosmol/l (hypotonic conditions). The initial swelling after exposure of OK cells to hypotonic conditions caused enhanced ruffling membrane activity, formation of lamellipodia and an extended space between adjacent cells which was caused by a more rounded cell shape. Moreover, the height of cells located in the centre of cell clusters increased by 32 +/- 8% (mean value +/- SEM) as checked by morphometric analysis of the vertical distance between the apical and basolateral F-actin domain. Although the fluorescence intensity and organization of F-actin in a horizontal direction remained unaltered during cell swelling, we observed a loss of periodicity and irregular distribution of myosin aggregates and a partial rearrangement of vimentin filaments in the form of short fragments. In all experiments the organization of microtubles was observed to be unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)

Atomic force microscopy of peritoneal macrophages after particle phagocytosis.

The atomic force microscope was used to image peritoneal macrophages after phagocytosis of latex beads with 0.45 microns in diameter and of zymosan particles. The rigidity of the phagocytosed material allowed to image the live membrane at forces below 2 nN. Repeated scanning of the membrane unavoidably caused the protrusion of the beads and increased their virtual height. The influence of fixation by glutaraldehyde on the image and the corresponding force vs. distance curves were analyzed and compared. Short treatment with Triton X-100 enabled us to identify intracellular components, such as embedded latex beads, cell nucleus and cytoskeletal strands. The data demonstrate that it is possible to image living cells if they are bolstered by stiff material.

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.