Effects of Hyperglycemia and Protein Kinase C on Connexin43 Expression in Cultured Rat Retinal Pigment Epithelial Cells

Previous results demonstrated that the intercellular communication mediated by gap junctions in retinal pigment epithelial (RPE) cells from the healthy Long Evans (LE) rat strain is higher than that from the dystrophic Royal College of Surgeons (RCS) rat strain. We examined connexin (Cx) expression in both cell types. At the mRNA level, a qualitatively similar expression pattern was found whereby Cx26, Cx32, Cx36, Cx43, Cx45 and Cx46 were all expressed. At the protein level, only Cx43 and Cx46 were detected. Expression of both isoforms was higher in LE-RPE as compared to RCS-RPE by a factor of 1.25 and 2 respectively. Phosphorylation of Cx43 was increased upon activation of protein kinase C (PKC) by 1 μM phorbol 12-myristate 13-acetate (PMA). The phosphorylation status was not changed in hyperglycemic conditions, but this treatment strongly decreased total Cx43 levels to about 75 and 40% (in LE-RPE and RCS-RPE cells respectively) of the control level in LE-RPE cells. This decrease could be overcome by PKC downregulation. These results demonstrate that PKC activation and hyperglycemic conditions have different effects on Cx43 and that PKC is involved in the metabolic pathway induced by hyperglycemic conditions. Received: 21 July 2000/Revised: 19 January 2001     

Expression of Cyclic Nucleotide-Gated Cation Channels in Airway Epithelial Cells

Using the whole-cell patch-clamp technique, the selectivity and pharmacology of 8-Br-cGMP-stimulated currents in the human alveolar cell line A549 was compared to 8-Br-cGMP-stimulated currents in HK293 cells transfected with hαCNC1. Whole cell currents stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 or A549 cells are carried by inward sodium and outward potassium with nearly the same selectivity. The whole-cell inward currents that are stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 are inhibited by l-cis-diltiazem with an IC₅₀ of 154 μm, by 2′,4′-dichlorobenzamil with an IC₅₀ of 50 μm and by amiloride with an IC₅₀ of 133 μm. The whole-cell inward currents in A549 cells that are stimulated by 8-Br-cGMP, are inhibited by l-cis-diltiazem with an IC₅₀ of 87 μm, by 2′4′-dichlorobenzamil with an IC₅₀ of 38 μm and by amiloride with an IC₅₀ of 32 μm suggesting that these airway cells contain cyclic nucleotide-gated cation channels. RT-PCR data suggest that mRNA of both αCNC1 and βCNC subunits are present in A549 cells and the presence of the βCNC subunit, may as previously reported, increase the affinity of these channel blockers compared to the hαCNC1 subunit alone. The mRNA of two other isoforms of this channel, CNC2 and CNC3, are also expressed in the A549 cell line. This study documents the IC₅₀ of externally applied channel blockers that can be used for in vitro or in vivo experiments to document sodium absorption via cyclic nucleotide-gated cation channels in airway cells. Received: 24 February/Revised: 28 May 1999     

Calcium-Sensitive Nonselective Cation Channel Identified in the Epithelial Cells Isolated from the Endolymphatic Sac of Guinea Pigs

We identified a Ca²⁺-sensitive cation channel in acutely dissociated epithelial cells from the endolymphatic sac (ES) of guinea pigs using the patch-clamp technique. Single-channel recordings showed that the cation channel had a conductance of 24.0 ± 1.3 pS (n= 8) in our standard solution. The relative ionic permeability of the channel was in the order K⁺= Na⁺ > Ca²⁺≫ Cl⁻. This channel was weakly voltage-dependent but was strongly activated by Ca²⁺ on the cytosolic side at a concentration of around 1 mm in inside-out excised patches. With cell-attached patches, however, the channel was activated by much lower Ca²⁺ concentrations. Treatment of the cells, under cell-attached configuration, with ionomycin (10 μm), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 μm), or ATP (1 mm), which increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), activated the channel at an estimated [Ca²⁺]_i from 0.6 μm to 10 μm. It is suggested that some activators of the channel were deteriorated or washed out during the formation of excised patches. Based on this Ca²⁺ sensitivity, we speculated that the channel contributes to the regulation of ionic balance and volume of the ES by absorbing Na⁺ under certain pathological conditions that will increase [Ca²⁺]_i. This is the first report of single-channel recordings in endolymphatic sac epithelial cells. Received: 24 October 2000/Revised: 10 April 2001     

Nonselective Cation and BK Channels in Apical Membrane of Outer Sulcus Epithelial Cells

The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K _Ca ) channel and (iii) a small K⁺ channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear current-voltage (I-V) relationship (27 pS) and was equally conductive for Na⁺ and K⁺, but not permeable to Cl⁻ or N-methyl-d-glucamine. Channel activity required the presence of Ca²⁺ at the cytosolic face, but was detected at Ca²⁺ concentrations as low as 10⁻⁷ m (open probability (P _o ) = 0.11 ± 0.03, n= 8). Gadolinium decreased P _o of the NSC channel from both the external and cytosolic side (IC₅₀∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K⁺ could be fitted to the I-V relationship in asymmetrical K⁺ and Na⁺ solutions. The channel was impermeable to Cl⁻ and N-methyl-d-glucamine. P _o of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca²⁺. TEA (20 mm), charybdotoxin (100 nm) and Ba²⁺ (1 mm) but not amiloride (1 mm) reduced P _o from the extracellular side. In contrast, external flufenamic acid (100 μm) increased P _o and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward current. We conclude that the NSC channel is Ca²⁺ activated, voltage-insensitive and involved in both constitutive K⁺ and Na⁺ reabsorption from endolymph while the BK channel might participate in the K⁺ pathway under stimulated conditions that produce an elevated intracellular Ca²⁺ or depolarized membrane potential. Received: 14 October 1999/Revised: 10 December 1999     

Role and Source of ATP for Activation of Nonselective Cation Channels by AlF Complex in Guinea Pig Chromaffin Cells

Intracellular dialysis with the solution containing the G protein activator, AlF complex, induced an inward nonselective cation current (I _NS) at −55 mV in chromaffin cells. Amplitudes of I _NS induced by dialysis with ATP-free AlF solutions progressively diminished as cells were pretreated with cyanide, a mitochondrial inhibitor. After a 10-min pretreatment, generation of I _NS by the AlF complex depended on exogenous ATP delivered from pipette solution. The relationship between amplitudes of I _NS and concentrations of MgATP was well expressed by a rectangular hyperbola with an EC₅₀ of 0.265 mm. This result suggests that the cyanide treatment almost depleted ATP near the plasma membrane. On the other hand, a similar cyanide treatment of adrenal medullary preparations did not induce a marked decrease in cellular ATP content. GTP, ITP, or UTP could not substitute for ATP in generation of I _NS by the AlF complex. Similarly, the substitution of ATP with non- or poorly hydrolyzable ATP analogues did not aid in generating I _NS. Bath application of the kinase inhibitor, H-7 (100 μm), suppressed AlF-induced I _NS in a manner depending on intracellular Mg²⁺. We conclude that ATP is a prerequisite for generation of I _NS as a phosphoryl donor and that mitochondria is the main source of ATP. Received: 17 April 1996/Revised: 26 July 1996     

Mechanism of Shrinkage Activation of Nonselective Cation Channels in M-1 Mouse Cortical Collecting Duct Cells

It has previously been shown that osmotic cell shrinkage activates a nonselective cation (NSC) channel in M-1 mouse cortical collecting duct cells [54] and in a variety of other cell types [20]. In the present study we further characterized the shrinkage-activated NSC channel in M-1 cells and its mechanism of activation using whole-cell current recordings. Osmotic cell shrinkage induced by addition of 100 mm sucrose to the bath solution caused a 20-fold increase in whole-cell inward currents from −10.8 ± 1.5 pA to −211 ± 10.2 pA (n= 103). A similar response was observed when cell shrinkage was elicited using a hypo-osmotic pipette solution. This indicates that cell shrinkage and not extracellular osmolarity per se is the signal for current activation. Cation substitution experiments revealed that the activated channels discriminate poorly between monovalent cations with a selectivity sequence NH₄ (1.2) ≥ Na⁺ (1) ≈ K⁺ (0.9) ≈ Li⁺ (0.9). In contrast there was no measurable permeability for Ca²⁺ or Ba²⁺ and the cation-to-anion permeability ratio was about 14. The DPC-derivatives flufenamic acid, 4-methyl-DPC and DCDPC were the most effective blockers followed by LOE 908, while amiloride and bumetanide were ineffective. The putative channel activator maitotoxin had no effect. Current activation was dependent upon the presence of intracellular ATP and Mg²⁺ and was inhibited by staurosporine (1 μm) and calphostin C (1 μm). Moreover, cytochalasin D (10 μm) and taxol (2 μm) reduced the current response to cell shrinkage. These findings suggest that the activation mechanism of the shrinkage-activated NSC channel involves protein kinase mediated phosphorylation steps and cytoskeletal elements. Received: 3 May 2000/Revised: 6 July 2000     

Lipofuscin Accumulation in Cultured Retinal Pigment Epithelial Cells Causes Enhanced Sensitivity to Blue Light Irradiation

Lipofuscin accumulates with age within secondary lysosomes of retinal pigment epithelial (RPE) cells of humans and many animals. The autofluorescent lipofuscin pigment has an excitation maximum within the range of visible blue light, while it is emitting in the yellow-orange area. This physico-chemical property of the pigment indicates that it may have a photo-oxidative capacity and, consequently, then should destabilize lysosomal membranes of blue-light exposed RPE. To test this hypothesis, being of relevance to the understanding of age-related macular degeneration, cultures of heavily lipofuscin-loaded RPE cells were blue-light–irradiated and compared with respect to lysosomal stability and cell viability to relevant controls. To rapidly convert primary cultures of RPE, obtained from neonatal rabbits, into aged, lipofuscin-loaded cells, they were allowed to phagocytize artificial lipofuscin that was prepared from outer segments of bovine rods and cones. Following blue-light irradiation, lysosomal membrane stability was measured by vital staining with the lysosomotropic weak base, and metachromatic fluorochrome, acridine orange (AO). Quantifying red (high AO concentration within intact lysosomes with preserved proton gradient over their membranes) and green fluorescence (low AO concentration in nuclei, damaged lysosomes with decreased or lost proton gradients, and in the cytosol) allowed an estimation of the lysosomal membrane stability after blue-light irradiation. Cellular viability was estimated with the delayed trypan blue dye exclusion test. Lipofuscin-loaded blue-light–exposed RPE cells showed a considerably enhanced loss of both lysosomal stability and viability when compared to control cells. It is concluded that the accumulation of lipofuscin within secondary lysosomes of RPE sensitizes these cells to blue light by inducing photo-oxidative alterations of their lysosomal membranes resulting in a presumed leakage of lysosomal contents to the cytosol with ensuing cellular degeneration of apoptotic type. The suggested mechanism may have bearings on the development of age-related macular degeneration. © 1997 Elsevier Science Inc.     

Apical Nonspecific Cation Channels in Everted Collecting Tubules of Potassium-Adapted Ambystoma

We observed intermediate conductance channels in approximately 20% of successful patch-clamp seals made on collecting tubules dissected from Ambystoma adapted to 50 mm potassium. These channels were rarely observed in collecting tubules taken from animals which were maintained in tap water. Potassium-adaptation either leads to an increase in the number of channels present or activates quiescent channels. In cell-attached patches the conductance averaged 30.3 ± 2.4 (9) pS. Since replacement of the chloride in the patch pipette with gluconate did not change the conductance, the channel carries cations, not anions. Notably, channel activity was observed at both positive and negative pipette voltages. When the pipette was voltage clamped at 0 mV or positive voltages, the current was directed inward, consistent with the movement of sodium into the cell. The pipette voltage at which the polarity of the current reversed (movement of potassium into the pipette) was −29.6 ± 6.5(9) mV. Open probability at 0 mV pipette voltage was 0.08 ± 0.03 and was unaffected when the apical membrane was exposed to either 2 × 10⁻⁶ or 2 × 10⁻⁵ m of amiloride. Exposure of the basolateral surface of the tubule to a saline containing 15 mm potassium caused a significant increase (P less than 0.001) in the open probability of these channels to 0.139 ± 0.002 without affecting the conductance of the apical channel. These data illustrate the presence of an intermediate conductance, poorly selective, amiloride-insensitive cation channel in native vertebrate collecting tubule. We postulate that, at least in amphibia, this channel may be used to secrete potassium. Received: 14 January 2000/Revised: 16 June 2000     

大鼠视网膜色素上皮细胞分离的改良及其MMP表达

目的探索和优化大鼠视网膜色素上皮(RPE)细胞分离培养的方法,评价RPE细胞的存活状态及细胞基质金属蛋白酶(MMP)的表达,为相关眼底疾病的研究提供细胞来源。方法采用改良的三步酶消化法分离大鼠RPE细胞,并进行细胞体外培养。倒置显微镜观察细胞形态,细胞生长曲线评价不同培养代数的RPE细胞的增殖活力。免疫荧光检测CRALBP和角蛋白表达鉴定RPE细胞,并观察不同培养代数RPE细胞中多种基质金属蛋白酶的表达。结果分离培养的RPE细胞可呈梭形、六角形,并维持RPE细胞特征性蛋白CRALBP和角蛋白表达,但细胞内色素成分随着细胞分裂和传代次数的增多逐渐减少。基质金属蛋白酶MMP2、MMP3、MMP9和MMP10在第1代和第3代RPE细胞中均表达阳性,且表达强度未见明显改变。结论应用改良的三步酶消化法可以成功的分离培养大鼠RPE细胞,并在第1代和第3代RPE细胞维持基质金属蛋白酶MMP2、MMP3、MMP9和MMP10的阳性表达。体外培养的大鼠RPE细胞为研究视网膜相关疾病提供了细胞模型。     

X-Ray Microanalysis and Phagocytotic Activity of Cultured Retinal Pigment Epithelial Cells in Hypoxia

Purpose: To investigate the influence of the functional and morphological changes induced in retinal pigment epithelial (RPE) cells by retinal ischemia, we evaluated the phagocytotic activity, the concentration of various elements, and ultrastructure in cultured RPE cells in hypoxia. Methods: The concentrations of oxygen in incubators were adjusted to 20, 10, and 1% by the addition of nitrogen for 72 hr. To observe phagocytotic activity and its relationship to actin filaments, the filaments of RPE cells incubated with fluoresbrite carboxylate YG microspheres were stained with rhodamine phalloidin. Some of the specimens were subjected to X-ray microanalysis by scanning electron microscope after being fixed, freeze-dried, and coated with carbon to investigate the cytoplasmic concentration of elements. A part of the latter specimens was also observed by transmission electron microscope after being embedded in epon and cut into ultrathin sections to see the ultra-structural changes inside cell. Results: Lowering oxygen concentrations from 20% to 1% swelled RPE cells and decreased the number of fluoresbrite carboxylate YG microspheres phagocytized by RPE cells. Phagocytosis of a large amount of latex beads (30 μl) for 24 hr in 1% oxygen caused a disruption of RPE cells. Na, S, and P were detected in RPE cells cultured in 20% oxygen. Reducing the oxygen concentration from 20 to 10 or 1% significantly decreased Na and increased S. Mitochondria were observed in RPE cells in 20 and 10% oxygen, but many vacuoles were observed in the cytoplasm in 1% oxygen. Conclusion: Hypoxia as low as 1% oxygen induced malfunction of phagocytosis and the fragility of RPE cells. We could speculate the imbalance of the electrolytes such as Na or a decrease of antioxidants such as glutathione containing S as a reason of disturbance of cell viability.     

Histamine Increases [Ca2+] in and Activates Ca-K and Nonselective Cation Currents in Cultured Human Capillary Endothelial Cells

We characterized the effects of histamine on intracellular Ca²⁺ and activation of ionic currents in human capillary endothelial cells. Histamine produced both a transient and sustained increase in intracellular Ca²⁺. The transient response was mediated largely through intracellular Ca²⁺ release and the sustained response was due to extracellular Ca²⁺ entry. The increase in intracellular Ca²⁺ by histamine was not affected by the H2 blocker cimetidine. But was entirely blocked by the H1 antagonist diphenhydramine showing that the histamine response in these cells is mediated through the H1 receptor. A transient ionic current is coactivated with the histamine-induced increase in intracellular Ca²⁺ and this current has several properties of a nonselective, Ca²⁺ permeable, cation channel (NSC). The magnitude of the NSC current does not strictly correlate with intracellular Ca²⁺ levels. A Ca²⁺-activated K⁺ current (BKCA) is activated by the increase in intracellular Ca²⁺ and this current is blocked by the selective BKCA blocker iberiotoxin. Received: 16 June 1999/Revised: 22 September 1999     

Voltage-Induced Activation of Mechanosensitive Cation Channels of Leech Neurons

The voltage dependence of stretch-activated cation channels in leech central neurons was studied in cell-free configurations of the patch-clamp technique. We established that stretch-activated channels excised from identified cell bodies of desheathed ganglia, as well as from neurons in culture, were slowly and reversibly activated by depolarizing membrane potentials. Negative pressure stimuli, applied to the patch pipette during a slow periodical modulation of membrane potential, enhanced channel activity, whereas positive pressures depressed it. Voltage-induced channel activation was observed, with soft glass pipettes, both in inside-out and outside-out membrane patches, at negative and positive reference potentials, respectively. The results presented in this study demonstrate that membrane depolarization induces slow activation of stretch-activated channels of leech central neurons. This phenomenon is similar to that found in Xenopus oocytes, however, some peculiar features of the voltage dependence in leech stretch-activated channels indicate that specific membrane-glass interactions might not necessarily be involved. Moreover, following depolarization, stretch-activated channels in membrane patches from neurons in culture exhibited significantly shorter delay to activation (sec) than their counterparts from neurons of freshly isolated ganglia (hundreds of sec).     

An L-type Calcium Channel in Renal Epithelial Cells

A voltage-activated Ca⁺⁺ channel has been identified in the apical membranes of cultured rabbit proximal tubule cells using the patch-clamp technique. With 105 mm CaCl₂ solution in the pipette and 180 NaAsp in the bath, the channel had a conductance of 10.4 ± 1.0 pS (n= 8) in on-cell patches, and 9.8 ± 1.1 pS (n= 8) in inside-out patches. In both on-cell and inside-out patches, the channel is active by membrane depolarization. For this channel, the permeation to Ba⁺⁺ and Ca⁺⁺ is highly selective over Na⁺ and K⁺ (P_Ca(Ba):P_Na(K) >200:1). The sensitivity to dihydropyridines is similar to that for L-type channels where the channel was blocked by nifedipine (10 μm), and activated by Bay K 8644 (5 μm). When activated by Bay K 8644, the channel showed subconductance levels. Treatment with forskolin (12.5 μm), phorbol ester (1 μm), or stretching (40 cm water) did not activate this channel. These results indicate that this Ca⁺⁺ channel is mostly regulated by membrane voltage, and appears to be an epithelial class of L-type Ca⁺⁺ channel. As such, it may participate in calcium reabsorption during periods of enhanced sodium reabsorption, or calcium signaling in volume regulation, where membrane depolarization occurs for prolonged periods. Received: 1 April 1996/Revised: 5 August 1996     

Sodium-dependent and -independent Choline Uptake by Type II Epithelial Cells from Rat Lung

The uptake of ³H-labeled choline by a suspension of isolated type II epithelial cells from rat lung has been studied in a Ringer medium. Uptake was linear for 4 min at both 0.1 μm and 5.0 μm medium choline; at 5 μm, only 10% of the label was recovered in a lipid fraction. Further experiments were conducted at the low concentration (0.1 μm), permitting characterization of the properties of high-affinity systems. Three fractions of choline uptake were detected: (i) a sodium-dependent system that was totally inhibited by hemicholinium-3 (HC-3); (ii) a sodium-independent uptake, when Na⁺ was replaced by Li⁺, K⁺ or Mg²⁺, inhibited by HC-3; (iii) a residual portion persisting in the absence of Na⁺ and unaffected by HC-3. Choline uptake was sigmoidally related to the medium Na⁺ concentration. Kinetic properties of the uptake of 0.1 μm ³H-choline in the presence and absence of medium Na⁺ were examined in two ways. (a) Inhibition by increasing concentrations of unlabeled choline (0.5–100 μm) was consistent with the presence of two Michaelis-Menten-type systems in the presence of Na⁺; a Na⁺-dependent portion (a mean of 0.52 of the total) had a K _m for choline of 1.5 μm while K _m in the absence of Na⁺ (Li⁺ substituting) was 18.6 μm. (b) Inhibition by HC-3 (0.3–300 μm) gave K_i values of 1.7 μm and 5.0 μm HC-3 for the Na⁺-dependent and -independent fractions. The apparent K _m of the Na⁺-dependent uptake is lower than that reported previously for lung-derived cells and is in the range of the K _m values reported for high-affinity, Na⁺-dependent choline uptake by neuronal cells. Received: 18 February 1997/Revised: 7 December 1997     

Calcium Channel Current in Rat Dental Pulp Cells

Voltage-gated Ca²⁺ currents in early-passage rat dental pulp cells were studied using whole-cell patch-clamp techniques. With Ba²⁺ as the charge carrier, two prominent inwardly-directed currents, I _f and I _s , were identified in these cells that could be distinguished on the basis of both kinetics and pharmacology. I _f was activated by membrane depolarizations more positive than −30 mV, and displayed fast inactivation kinetics, while I _s was activated by steeper depolarizations and inactivated more slowly. At peak current, time constants of inactivation for I _f and I _s were ∼17 vs.∼631 msec. Both I _f and I _s could be blocked by lanthanum. By contrast, only I _s was sensitive to either Bay-K or nifedipine, a specific agonist and antagonist, respectively, of L-type Ca²⁺ channels. I _s was also blocked by the peptide omega-Conotoxin GVIA. Taken together, results suggested that I _f was mediated by divalent cation flow through voltage-gated T-type Ca²⁺ channels, whereas I _s was mediated by L- and N-type Ca²⁺ channels in the pulp cell membrane. The expression of these prominent, voltage-gated Ca²⁺ channels in a presumptive mineral-inductive phenotype suggests a functional significance vis a vis differentiation of dental pulp cells for the expression and secretion of matrix proteins, and/or formation of reparative dentin itself. Received: 29 November 1999/Revised: 24 April 2000     

Activation of Divalent Cation Influx into S. cerevisiae Cells by Hypotonic Downshift

Subjecting Saccharomyces cerevisiae cells to a hypotonic downshift by transferring cells from YPD medium containing 0.8 m sorbitol to YPD medium without sorbitol induces a transient rapid influx of Ca²⁺ and other divalent cations into the cell. For cells grown in YPD at 37°C, this hypotonic downshift increases Ca²⁺ accumulation 6.7-fold. Hypotonic downshift-induced Ca²⁺ accumulation and steady-state Ca²⁺ accumulation in isotonic YPD medium are differentially affected by dodecylamine and Mg²⁺. The Ca²⁺-influx pathway responsible for hypotonic-induced Ca²⁺ influx may account for about 10–35% of Ca²⁺ accumulation by cells growing in YPD. Ca²⁺ influx is not required for cells to survive a hypotonic downshift. Hypotonic downshift greatly reduces the ability of S. cerevisiae cells to survive a 5-min exposure to 10 mm Cd²⁺ suggesting that mutants resistant to acute Cd²⁺ exposure may help identify genes required for hypotonic downshift-induced divalent cation influx. Received: 14 January 1997/Revised: 20 June 1997     

NaF Potentiates a K+-selective Ion Channel in G292 Osteoblastic Cells

Clinical studies have established that NaF increases mineral content in bone, although the cellular mechanisms underlying its osteoinductive effects remain unclear. Because metabolic effects of fluoride have been linked to ion flux and alterations in membrane potential, we used patch-clamp recording techniques to examine the electrophysiological response of osteoblastic cells to NaF. In these experiments, we show that NaF increased the amplitude and P _open of a 73 pS potassium-selective ion channel. The effect of NaF depended on extracellular Ca²⁺ and could be blocked by a combination of calcium-channel blocking agents, suggesting that potentiation of channel activity was dependent on external calcium. Because all patches were in the cell-attached configuration, the effect of NaF was presumably indirect. Although the underlying cellular mechanisms remain unclear, our findings suggest that activity of calcium and/or potassium-selective channels via second messenger cascades may mediate many of the early events involved in the response of bone cells to inorganic fluoride. Received: 30 March 1995/Revised: 13 October 1995     

Identification of Anion-selective Channels in the Basolateral Membrane of Mitochondria-rich Epithelial Cells

Epithelial cells of toad (Bufo bufo) skin were isolated by treatments of the epidermis with collagenase and trypsin. Cl^- channels in the basolateral membrane from soma or neck of mitochondria-rich cells were studied in cell-attached and excised inside-out configurations. Of a total of 87 sealed patches only 28 (32%) were electrically active, and in these we identified four different types of Cl^- channels. The two major populations constituted Ohmic Cl^- channels with limiting conductance (γ_125/125) of 10 pS and 30 pS, respectively. A much rarer 150 pS Ohmic Cl^- channel was also characterized. From i/V relationships of individual channels the following Goldman-Hodgkin-Katz permeabilities were calculated, 2.2 (±0.1) × 10^-14, 5.7 (±0.7) × 10^-14, and 32 (±2) × 10^-14 cm³/sec, for the 10, 30 and 150 pS Cl^- channels, respectively. The 30 pS channel was activated by hyperpolarization. The gating kinetics of the 150 pS channel was complex with burstlike closures within openings of long duration. The fourth type of Cl^- channel was studied in patches generating `noisy currents' with no discrete single-channel events, but with vanishing fluctuations at pipette potentials near E _Cl. Noise analysis revealed a power spectrum with cutoff frequencies of 1.2 and 13 Hz, indicating that resolution of kinetic steps was limited by small channel currents rather than fast channel gating. From the background noise level we estimated the channel conductance to be less than 1.7 pS. Despite the fact that the majority of patches did not contain electrically active Cl^- channels, patches being active, generally, contained more than a single active channel. Thus, for the above three types of resolvable channels, the mean number of active channels per patch amounted to 2.1, 1.4, and 2.0, respectively. This observation, like the finding of few patches with several unresolvable channels, indicates that electrically active Cl^- channels are organized in clusters. Received: 10 October 1996/Revised: 8 January 1997     

Embryonic Rat Hippocampal Neurons and GABAA Receptor Subunit-Transfected Non-neuronal Cells Release GABA Tonically

We used patch-clamp recording techniques to investigate the contribution of GABA to baseline membrane properties in cultured embryonic rat hippocampal neurons. Almost all of the neurons recorded with Cl⁻-filled pipettes and clamped at negative potentials exhibited baselines that were noticeably noisy, with microscopic fluctuations superimposed on the macroscopic holding current. A gentle steam of saline applied to the neuronal surface rapidly and reversibly reduced the baseline current and fluctuations, both of which were completely eliminated by bicuculline. Fluctuation analysis showed that the variance in the baseline current signal was exponentially distributed with estimated kinetics comparable to those activated by submicromolar concentrations of exogenous GABA. The kinetics of Cl⁻ channels activated by endogenous GABA displayed a potential sensitivity comparable to those activated by exogenous GABA. Non-neuronal cells stably transfected with α₁ and γ₂ GABA_A receptor subunits exhibited little baseline current variance when recorded with Cl⁻-filled pipettes. Addition of micromolar GABA to the extracellular saline or to the pipette solution induced a saline- and bicuculline-sensitive baseline current signal comparable to that recorded in hippocampal neurons. Thus, both intra- and extracellular sources of GABA could contribute to the baseline properties recorded in these cultured neurons. Received: 13 January 1997/Revised: 16 April, 1988