Role of Trpc channels, Stim1 and Orai1 in PGF(2α)-induced calcium signaling in NRK fibroblasts.

Normal rat kidney (NRK) fibroblasts exhibit growth-dependent changes in electrophysiological properties and intracellular calcium dynamics. The transition from a quiescent state to a density-arrested state results in altered calcium entry characteristics. This coincides with modulation of the expression of the genes encoding the calcium channels Trpc1, Trpc6 and Orai1, and of the intracellular calcium sensor Stim1. In the present study we have used gene selective short hairpin (sh) RNAs against these various genes to investigate their role in (a) capacitative store-operated calcium entry (SOCE); (b) non-capacitative OAG-induced receptor-operated calcium entry (ROCE); and (c) prostaglandin F(2α) (PGF(2α))-induced Ca(2+)-oscillations in NRK fibroblasts. Intracellular calcium measurements revealed that knockdown of the genes encoding Trpc1, Orai1 and Stim1 each caused a significant reduction of SOCE in NRK cells, whereas knockdown of the gene encoding Trpc6 reduced only the OAG-induced ROCE. Furthermore, our data show that knockdown of the genes encoding Trpc1, Orai1 and Stim1, but not Trpc6, substantially reduced the frequency (up to 60%) of PGF(2α)-induced Ca(2+) oscillations in NRK cells. These results indicate that in NRK cells distinct calcium channels control the processes of SOCE, ROCE and PGF(2α)-induced Ca(2+) oscillations.

Different roles of inositol 1,4,5-trisphosphate receptor subtypes in prostaglandin F(2alpha)-induced calcium oscillations and pacemaking activity of NRK fibroblasts.

We investigated the role of inositol 1,4,5-trisphosphate (IP(3))-receptor isoforms in the prostaglandin F(2alpha) (PGF(2alpha))-induced calcium oscillations and pacemaking activity of normal rat kidney (NRK) fibroblasts. Reverse transcription polymerase chain reaction (RT-PCR) studies revealed that NRK fibroblasts express only the IP(3)-receptor subtypes IP(3)R1 and IP(3)R3. Quantitative RT-PCR analysis demonstrated that their expression levels varied as a function of the growth status of NRK cells; NRK cells made quiescent (Q) by serum deprivation expressed significantly higher levels of subtypes 1 and 3 than cells grown to density-arrest (DA). Using Ca(2+)-imaging techniques, we show that the frequency of PGF(2alpha)-induced calcium oscillations in DA-cells is lower than in Q-cells. To study whether these differences in the frequency of calcium oscillations relate to the relative amounts of IP(3)-receptor subtypes expressed by the cells, we knocked down the genes for either IP(3)-receptor subtype by using an shRNA approach. Knockdown of the IP(3)R1 gene significantly decreased the frequency of the PGF(2alpha)-induced calcium oscillations in both Q- and DA-cells. It also reduced the frequency of the repetitive firing of calcium action potentials by DA-cells. In contrast, knockdown of the IP(3)R3 gene caused an increase in the frequency of both processes, suggesting a role for this receptor subtype as an anti-Ca(2+)-oscillatory unit in NRK fibroblasts. Our findings indicate that the reduction in the frequency of PGF(2alpha)-induced calcium oscillations in DA-cells compared with Q-cells results from the reduced expression ratio of IP(3)R1 versus IP(3)R3 receptors in DA-cells. Moreover, these data provide direct evidence that the frequency of IP(3)-dependent calcium oscillations determines the periodicity of action potential firing by density-arrested NRK fibroblasts.

Growth-dependent modulation of capacitative calcium entry in normal rat kidney fibroblasts.

Normal rat kidney (NRK) fibroblasts have electrophysiological properties and intracellular calcium dynamics that are dependent upon their growth stage. In the present study we show that this differential behavior coincides with a differential calcium entry that can be either capacitative or non-capacitative. Confluent cells made quiescent by serum deprivation, which have a stable membrane potential near -70 mV and do not show spontaneous intracellular calcium oscillations, primarily exhibit the capacitative mechanism for calcium entry, also called store-operated calcium entry (SOCE). When the quiescent cells are grown to density-arrest in the presence of EGF as the sole polypeptide growth factor, these cells characteristically fire spontaneously repetitive calcium action potentials, which propagate throughout the whole monolayer and are accompanied by intracellular calcium transients. These density-arrested cells appear to exhibit in addition to SOCE also receptor-operated calcium entry (ROCE) as a mechanism for calcium entry. Furthermore we show that, in contrast to earlier studies, the employed SOCs and ROCs are permeable for both calcium and strontium ions. We examined the expression of the canonical transient receptor potential channels (Trpcs) that may be involved in SOCE and ROCE. We show that NRK fibroblasts express the genes encoding Trpc1, Trpc5 and Trpc6, and that the levels of their expression are dependent upon the growth stage of the cells. In addition we examined the growth stage dependent expression of the genes encoding Orai1 and Stim1, two proteins that have recently been shown to be involved in SOCE. Our results suggest that the differential expression of Trpc5, Trpc6, Orai1 and Stim1 in quiescent and density-arrested NRK fibroblasts is responsible for the difference in regulation of calcium entry between these cells. Finally, we show that inhibition or potentiation of SOCE and ROCE by pharmacological agents has profound effects on calcium dynamics in NRK fibroblasts.

Role of the prostanoid FP receptor in action potential generation and phenotypic transformation of NRK fibroblasts.

By using an shRNA approach to knockdown the expression of the prostaglandin (PG)-F(2alpha) receptor (FP-R), the role of PGF(2alpha) in the process of phenotypic transformation of normal rat kidney (NRK) fibroblasts has been studied. Our data show that PGF(2alpha) up-regulates Cox-2 expression both at the mRNA and protein level, indicating that activation of FP-R in NRK fibroblasts induces a positive feedback loop in the production PGF(2alpha). Knockdown of FP-R expression fully impaired the ability of PGF(2alpha) to induce a calcium response and subsequent depolarization in NRK cells. However, these cells could still undergo phenotypic transformation when treated with a combination of EGF and retinoic acid, but in contrast to the wild-type cells, this process was not accompanied by a membrane depolarization to -20 mV. Knockdown of FP-R expression also impaired the spontaneous firing of calcium action potentials by density-arrested NRK cells. These data show that a membrane depolarization is not a prerequisite for the acquisition of a transformed phenotype. Furthermore, our data provide the first direct evidence that activity of PGF(2alpha) by putative pacemaker cells underlies the generation of calcium action potentials in NRK monolayers.

Local induction of pacemaking activity in a monolayer of electrically coupled quiescent NRK fibroblasts.

Cultures of normal rat kidney (NRK) fibroblasts may display spontaneous calcium action potentials which propagate throughout the cellular monolayer. Pacemaking activity of NRK cells was studied by patch clamp electrophysiology and vital calcium imaging, using a new experimental approach in which a ring was placed on the monolayer in order to physically separate pacemakers within or under the ring and follower cells outside the ring. Stimulation of cells inside the ring with IP(3)-generating hormones such as prostaglandin F(2alpha) (PGF(2alpha)) resulted in the induction of periodic action potentials outside the ring, which were abolished when the L-type calcium channel blocker nifedipine was added outside the ring, but not inside the ring. PGF(2alpha)-treated cells displayed asynchronous IP(3)-mediated calcium oscillations of variable frequency, while follower cells outside the ring showed synchronous calcium transients which coincided with the propagating action potential. Mathematical modelling indicated that addition of PGF(2alpha) inside the ring induced both a membrane potential gradient and an intracellular IP(3) gradient, both of which are essential for the induction of pacemaking activity under the ring. These data show that intercellular coupling between PGF(2alpha)-treated and non-treated cells is essential for the generation of a functional pacemaker area whereby synchronization of calcium oscillations occurs by activation of L-type calcium channels.

Fast calcium wave propagation mediated by electrically conducted excitation and boosted by CICR.

We have investigated synchronization and propagation of calcium oscillations, mediated by gap junctional excitation transmission. For that purpose we used an experimentally based model of normal rat kidney (NRK) cells, electrically coupled in a one-dimensional configuration (linear strand). Fibroblasts such as NRK cells can form an excitable syncytium and generate spontaneous inositol 1,4,5-trisphosphate (IP(3))-mediated intracellular calcium waves, which may spread over a monolayer culture in a coordinated fashion. An intracellular calcium oscillation in a pacemaker cell causes a membrane depolarization from within that cell via calcium-activated chloride channels, leading to an L-type calcium channel-based action potential (AP) in that cell. This AP is then transmitted to the electrically connected neighbor cell, and the calcium inflow during that transmitted AP triggers a calcium wave in that neighbor cell by opening of IP(3) receptor channels, causing calcium-induced calcium release (CICR). In this way the calcium wave of the pacemaker cell is rapidly propagated by the electrically transmitted AP. Propagation of APs in a strand of cells depends on the number of terminal pacemaker cells, the L-type calcium conductance of the cells, and the electrical coupling between the cells. Our results show that the coupling between IP(3)-mediated calcium oscillations and AP firing provides a robust mechanism for fast propagation of activity across a network of cells, which is representative for many other cell types such as gastrointestinal cells, urethral cells, and pacemaker cells in the heart.

Hysteresis and bistability in a realistic cell model for calcium oscillations and action potential firing.

Many cells reveal oscillatory behavior. Some cells reveal action-potential firing resulting from Hodgkin-Huxley (HH) type dynamics of ion channels in the cell membrane. Another type of oscillation relates to periodic inositol triphospate (IP3)-mediated calcium transients in the cytosol. In this study we present a bifurcation analysis of a cell with an excitable membrane and an IP3-mediated intracellular calcium oscillator. With IP3 concentration as a control parameter the model reveals a complex, rich spectrum of both stable and unstable solutions with hysteresis corresponding to experimental data. Our results reveal the emergence of complex behavior due to interactions between subcomponents with a relatively simple dynamical behavior.

Stabilizing role of calcium store-dependent plasma membrane calcium channels in action-potential firing and intracellular calcium oscillations.

In many biological systems, cells display spontaneous calcium oscillations (CaOs) and repetitive action-potential firing. These phenomena have been described separately by models for intracellular inositol trisphosphate (IP3)-mediated CaOs and for plasma membrane excitability. In this study, we present an integrated model that combines an excitable membrane with an IP3-mediated intracellular calcium oscillator. The IP3 receptor is described as an endoplasmic reticulum (ER) calcium channel with open and close probabilities that depend on the cytoplasmic concentration of IP3 and Ca2+. We show that simply combining this ER model for intracellular CaOs with a model for membrane excitability of normal rat kidney (NRK) fibroblasts leads to instability of intracellular calcium dynamics. To ensure stable long-term periodic firing of action potentials and CaOs, it is essential to incorporate calcium transporters controlled by feedback of the ER store filling, for example, store-operated calcium channels in the plasma membrane. For low IP3 concentrations, our integrated NRK cell model is at rest at -70 mV. For higher IP3 concentrations, the CaOs become activated and trigger repetitive firing of action potentials. At high IP3 concentrations, the basal intracellular calcium concentration becomes elevated and the cell is depolarized near -20 mV. These predictions are in agreement with the different proliferative states of cultures of NRK fibroblasts. We postulate that the stabilizing role of calcium channels and/or other calcium transporters controlled by feedback from the ER store is essential for any cell in which calcium signaling by intracellular CaOs involves both ER and plasma membrane calcium fluxes.

Autocrine production of prostaglandin F2alpha enhances phenotypic transformation of normal rat kidney fibroblasts.

We have used normal rat kidney (NRK) fibroblasts as an in vitro model system to study cell transformation. These cells obtain a transformed phenotype upon stimulation with growth-modulating factors such as retinoic acid (RA) or transforming growth factor-beta (TGF-beta). Patch-clamp experiments showed that transformation is paralleled by a profound membrane depolarization from around -70 to -20 mV. This depolarization is caused by a compound in the medium conditioned by transformed NRK cells, which enhances intracellular Ca2+ levels and thereby activates Ca2+-dependent Cl- channels. This compound was identified as prostaglandin F2alpha (PGF2alpha) using electrospray ionization mass spectrometry. The active concentration in the medium conditioned by transformed NRK cells as determined using an enzyme immunoassay was 19.7 +/- 2.5 nM (n = 6), compared with 1.5 +/- 0.1 nM (n = 3) conditioned by nontransformed NRK cells. Externally added PGF2alpha was able to trigger NRK cells that had grown to density arrest to restart their proliferation. This proliferation was inhibited when the FP receptor (i.e., natural receptor for PGF2alpha) was blocked by AL-8810. RA-induced phenotypic transformation of NRK cells was partially (approximately 25%) suppressed by AL-8810. Our results demonstrate that PGF2alpha acts as an autocrine enhancer and paracrine inducer of cell transformation and suggest that it may play a crucial role in carcinogenesis in general.

Modeling action potential generation and propagation in NRK fibroblasts.

Normal rat kidney (NRK) fibroblasts change their excitability properties through the various stages of cell proliferation. The present mathematical model has been developed to explain excitability of quiescent (serum deprived) NRK cells. It includes as cell membrane components, on the basis of patch-clamp experiments, an inwardly rectifying potassium conductance (G(Kir)), an L-type calcium conductance (G(CaL)), a leak conductance (G(leak)), an intracellular calcium-activated chloride conductance [G(Cl(Ca))], and a gap junctional conductance (G(gj)), coupling neighboring cells in a hexagonal pattern. This membrane model has been extended with simple intracellular calcium dynamics resulting from calcium entry via G(CaL) channels, intracellular buffering, and calcium extrusion. It reproduces excitability of single NRK cells and cell clusters and intercellular action potential (AP) propagation in NRK cell monolayers. Excitation can be evoked by electrical stimulation, external potassium-induced depolarization, or hormone-induced intracellular calcium release. Analysis shows the roles of the various ion channels in the ultralong ( approximately 30 s) NRK cell AP and reveals the particular role of intracellular calcium dynamics in this AP. We support our earlier conclusion that AP generation and propagation may act as a rapid mechanism for the propagation of intracellular calcium waves, thus contributing to fast intercellular calcium signaling. The present model serves as a starting point to further analyze excitability changes during contact inhibition and cell transformation.

Ionic basis for excitability of normal rat kidney (NRK) fibroblasts.

Ionic membrane conductances of normal rat kidney (NRK) fibroblasts were characterized by whole-cell voltage-clamp experiments on single cells and small cell clusters and their role in action potential firing in these cells and in monolayers was studied in current-clamp experiments. Activation of an L-type calcium conductance (GCaL) is responsible for the initiation of an action potential, a calcium-activated chloride conductance (GCl(Ca)) determines the plateau phase of the action potential, and an inwardly rectifying potassium conductance (GKir) is important for the generation of a resting potential of approximately -70 mV and contributes to action potential depolarization and repolarization. The unique property of the excitability mechanism is that it not only includes voltage-activated conductances (GCaL, GKir) but that the intracellular calcium dynamics is also an essential part of it (via GCl(Ca)). Excitability was found to be an intrinsic property of a fraction (approximately 25%) of the individual cells, and not necessarily dependent on gap junctional coupling of the cells in a monolayer. Electrical coupling of a patched cell to neighbor cells in a small cluster improved the excitability because all small clusters were excitable. Furthermore, cells coupled in a confluent monolayer produced broader action potentials. Thus, electrical coupling in NRK cells does not merely serve passive conduction of stereotyped action potentials, but also seems to play a role in shaping the action potential.

Fenamates: a novel class of reversible gap junction blockers.

The effect of fenamates on gap junctional intercellular communication was investigated in monolayers of normal rat kidney (NRK) fibroblasts and of SKHep1 cells overexpressing the gap junction protein connexin43 (Cx43). Using two different methods to study gap junctional intercellular communication, single electrode voltage-clamp step response measurements and dye microinjection, we show that fenamates are reversible blockers of Cx43-mediated intercellular communication. After adding fenamates to a confluent monolayer of electrically coupled NRK fibroblasts, the voltage step-induced capacitive current transient changed from a transient characteristic for charging multiple coupled cell capacitances to one characteristic for a single cell in isolation. The capacitance of completely uncoupled cells was 19.7 +/- 1.0 pF (mean +/- S.E.M.; n = 11). Junctional conductance between the patched cell and the surrounding cells in the monolayer changed from >140.7 +/- 9.6 nS (mean +/- S.E.M.; n = 14) to <1.4 +/- 0.4 nS (mean +/- S.E.M.; n = 11) after uncoupling. Electrical coupling could be restored to >51.8 +/- 4.2 nS (mean +/- S.E.M.; n = 11) by washout of the fenamates. Voltage-clamp step response measurements showed that the potency of fenamates in inhibiting electrical coupling decreases in the order meclofenamic acid > niflumic acid > flufenamic acid. The half-maximal concentration determined by dye-coupling experiments was 25 and 40 microM for meclofenamic acid and flufenamic acid, respectively. Inhibition of gap junctional communication by fenamates did not involve changes in intracellular calcium or pH, and was unrelated to protein kinase C activity or an inhibition of cyclooxygenase activity. Voltage-clamp step response measurements in confluent monolayers of SKHep1 cells that had been stably transfected with Cx43 revealed that fenamates are potent blockers of Cx43-mediated intercellular communication. In conclusion, fenamates represent a novel class of reversible gap junction blockers that can be used to study the role of Cx43-mediated gap junctional intercellular communication in biological processes.

Voltage-dependent reversal of anodic galvanotaxis in Nyctotherus ovalis.

Aerobic and anaerobic ciliates swim towards the cathode when they are exposed to a constant DC field. Nyctotherus ovalis from the intestinal tract of cockroaches exhibits a different galvanotactic response: at low strength of the DC field the ciliates orient towards the anode whereas DC fields above 2-4 V/cm cause cathodic swimming. This reversal of the galvanotactic response is not due to backward swimming. Rather the ciliates turn around and orient to the cathode with their anterior pole. Exposure to various cations, chelators, and Ca(2+)-channel inhibitors suggests that Ca(2+)-channels similar to the "long lasting" Ca(2+)-channels of vertebrates are involved in the voltage-dependent anodic galvanotaxis. Evidence is presented that host-dependent epigenetic factors can influence the voltage-threshold for the switch from anodic to cathodic swimming.

Phenotypic transformation of normal rat kidney fibroblasts by endothelin-1. Different mode of action from lysophosphatidic acid, bradykinin, and prostaglandin f2alpha.

In the present study, we compared the effects of endothelin (ET)-1 on cell proliferation and second messenger induction in normal rat kidney (NRK) fibroblasts, with those of other activators of G-protein-coupled receptors such as prostaglandin (PG)-F2alpha, bradykinin (BK), and lysophosphatidic acid (LPA). LPA is mitogenic by itself, while the other factors require the presence of EGF. In density-arrested NRK cells, ET-1 and LPA induce phenotypic transformation rapidly, with similar kinetics as retinoic acid (RA) and transforming growth factor (TGF)-beta, while BK and PGF2alpha only do so with delayed kinetics. ET-1 and PGF2alpha are strong inducers of anchorage-independent growth, with a similar level of induction as TGFbeta, in contrast to LPA and BK. When investigating the second messenger generation, we found that ET-1 is the strongest activator of arachidonic acid release and phosphatidylinositol diphosphate hydrolysis. Only in the case of ET-1 the cell depolarization is not reversible upon removal of the factor. Similarly, only the ET-1-induced transient enhancement of intracellular calcium concentration is paralleled by both homologous and heterologous desensitization. In conclusion, these data show that ET-1 is a potent inducer of second messengers and phenotypic transformation in NRK cells, with characteristics that clearly differ from those of other activators of G-protein-coupled receptors, most likely as a result of prolonged receptor activation.

Electrically excitable normal rat kidney fibroblasts: A new model system for cell-semiconductor hybrids.

In testing various designs of cell-semiconductor hybrids, the choice of a suitable type of electrically excitable cell is crucial. Here normal rat kidney (NRK) fibroblasts are presented as a cell line, easily maintained in culture, that may substitute for heart or nerve cells in many experiments. Like heart muscle cells, NRK fibroblasts form electrically coupled confluent cell layers, in which propagating action potentials are spontaneously generated. These, however, are not associated with mechanical disturbances. Here we compare heart muscle cells and NRK fibroblasts with respect to action potential waveform, morphology, and substrate adhesion profile, using the whole-cell variant of the patch-clamp technique, atomic force microscopy (AFM), and reflection interference contrast microscopy (RICM), respectively. Our results clearly demonstrate that NRK fibroblasts should provide a highly suitable test system for investigating the signal transfer between electrically excitable cells and extracellular detectors, available at a minimum cost and effort for the experimenters.

Synchronized calcium spiking resulting from spontaneous calcium action potentials in monolayers of NRK fibroblasts.

The correlation between the intracellular Ca2+ concentration ([Ca2+]i) and membrane potential in monolayers of density-arrested normal rat kidney (NRK) fibroblasts was investigated. Using the fluorescent probe Fura-2, spontaneous repetitive spike-like increases in [Ca2+]i (Ca2+ spikes) were observed that were synchronised throughout the entire monolayer. Ca2+ spikes disappeared in Ca(2+)-free solutions and could be blocked by the L-type Ca2+ channel antagonist felodipine. Simultaneous measurements of [Ca2+]i and membrane potential showed that these Ca2+ spikes were paralleled by depolarisations of the plasma membrane. Using patch clamp measurements, action potential-like depolarisations consisting of a fast spike depolarisation followed by a plateau phase were seen with similar kinetics as the Ca2+ spikes. The action potentials could be blocked by L-type Ca2+ channel blockers and were dependent on extracellular Ca2+. The plateau phase was predominantly determined by a Cl- conductance and was dependent on intracellular Ca2+. The presence of voltage-dependent L-type Ca2+ channels in NRK cells was confirmed by patch clamp measurements in single cells. It is concluded that monolayers of density-arrested NRK fibroblasts exhibit spontaneous Ca2+ action potentials leading to synchronised Ca2+ spiking. This excitability of monolayers of fibroblasts may represent a novel Ca2+ signaling pathway in electrically coupled fibroblasts, cells that were hitherto considered to be inexcitable.

Membrane depolarization in NRK fibroblasts by bradykinin is mediated by a calcium-dependent chloride conductance.

The effects of the phosphoinositide-mobilizing agonist bradykinin (BK) on membrane potential and intracellular calcium in monolayers of normal rat kidney (NRK) fibroblasts were investigated. BK induced a rapid transient depolarization in these cells, which was mimicked by other phosphoinositide-mobilizing factors such as prostaglandin F2alpha (PGF2alpha), lysophosphatidic acid (LPA), platelet-derived growth factor (PDGF-BB), and serum. Depolarization by BK was independent of extracellular Ca2+ or Na+. It was shown using extracellular Cl- substitutions that the depolarization was caused by an increased Cl- conductance. Depolarization was inhibited by 5-nitro-2-3-phenylpropyl(amino)benzoic acid (NPPB), niflumic acid, and flufenamic acid, inhibitors of calcium-dependent chloride channels. The depolarization provoked by BK could be mimicked by raising intracellular calcium with ionomycin or thapsigargin and could be blocked with geneticin, a blocker of phospholipase C. When intracellular calcium was buffered by loading the cells with 1,2-bis(2-aminophenoxy)ethane-NNN'N'-tetra-acetic acid (BAPTA), depolarization was prevented. We conclude that in NRK fibroblasts extracellular stimuli that increase intracellular calcium, depolarize the cells via the activation of a calcium-dependent chloride conductance. In addition to an increase in intracellular calcium, depolarization may be an important effector pathway in response to extracellular stimuli in fibroblasts. It is hypothesized that, in electrically coupled cells such as NRK fibroblasts, intercellular transmission of these depolarizations may represent a mechanism to coordinate uniform multicellular responses to Ca2+-mobilizing agonists.

Synchronized Ca2+ signaling by intercellular propagation of Ca2+ action potentials in NRK fibroblasts.

The intercellular propagation of Ca2+ waves by diffusion of inositol trisphosphate has been shown to be a general mechanism by which nonexcitable cells communicate. Here, we show that monolayers of normal rat kidney (NRK) fibroblasts behave like a typical excitable tissue. In confluent monolayers of these cells, Ca2+ action potentials can be generated by local depolarization of the monolayer on treatment with either bradykinin or an elevation of the extracellular K+ concentration. These electronically propagating action potentials travel intercellularly over long distances in an all-or-none fashion at a speed of approximately 6.1 mm/s and can be blocked by L-type Ca2+ channel blockers. The action potentials are generated by depolarizations beyond the threshold value for L-type Ca2+ channels of about -15 mV. The result of these locally induced, propagating Ca2+ action potentials is an almost synchronous, transient increase in the intracellular Ca2+ concentration in large numbers of cells. These data show that electrically coupled fibroblasts can form an excitable syncytium, and they elucidate a novel mechanism of intercellular Ca2+ signaling in these cells that may coordinate synchronized multicellular responses to local stimuli.

Determination of gap junctional intercellular communication by capacitance measurements.

Electrical coupling between cells is usually measured using the double-patch-clamp technique with cell pairs. Here, a single patch-clamp technique that is not limited to cell pairs is described to determine electrical coupling between cells. Capacitance measurements in clusters of normal rat kidney (NRK) fibroblasts were used to study intercellular communication. In the whole-cell patch-clamp configuration capacitive transients were evoked by applying small voltage pulses. Total membrane capacitance was calculated from these capacitive transients after determination of access resistance, membrane conductance, and the decay constant of the transients, or alternatively by integrating the current transient. We found that in clusters of one to ten cells, membrane capacitance increased linearly with cell number, showing that the cells are electrically coupled. Membrane conductance of the cluster of cells also increased, as expected for cells that are well coupled. In subconfluent and confluent cultures, high membrane conductances together with large capacitive transients were observed, indicative of electrical coupling. Capacitance could only be determined qualitatively under these conditions, due to space clamp problems. In the presence of the gap junctional inhibitors halothane, heptanol or octanol, capacitance of all clusters of cells fell to single-cell levels, showing a complete uncoupling of the cells. The tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also uncoupled the cells completely, with 10 min. We conclude that capacitance measurements can provide a useful tool to study changes in intercellular communication in clusters of cells.

Bradykinin-induced growth inhibition of normal rat kidney (NRK) cells is paralleled by a decrease in epidermal-growth-factor receptor expression.

Normal rat kidney fibroblasts, grown to density arrest in the presence of epidermal growth factor (EGF), can be induced to undergo phenotypic transformation by treatment with transforming growth factor beta or retinoic acid. Here we show that bradykinin blocks this growth-stimulus-induced loss of density-dependent growth arrest by a specific receptor-mediated mechanism. The effects of bradykinin are specific, and are not mimicked by other phosphoinositide-mobilizing agents such as prostaglandin F2 alpha. Northern-blot analysis and receptor-binding studies demonstrate that bradykinin also inhibits the retinoic acid-induced increase in EGF receptor levels in these cells. These studies provide additional evidence that EGF receptor levels modulate EGF-induced expression of the transformed phenotype in these cells.