Gap junctional coupling between progenitor cells of regenerating retina in the adult newt

Gap junctional coupling between progenitor cells of regenerating retina in the adult newt was examined by a slice-patch technique. Retinal slices at the early regeneration stage comprised one to two layers of cells with mitotic activity, progenitor cells. These cells were initially voltage-clamped at a holding potential of -80 mV, near their resting potentials, and stepped to either hyperpolarizing or depolarizing test potentials under suppression of voltage-gated membrane currents. About half the cells showed passively flowing currents that reversed polarity around their resting potentials. The currents often exhibited a voltage- and time-dependent decline. As the difference between the test potential and resting potential increased, the time until the current decreased to the steady-state level became shorter and the amount of steady-state current decreased. Thus, the overall current profile was almost symmetrical about the current at the resting potential. Input resistance estimated from the initial peak of the currents was significantly smaller than that expected in isolated progenitor cells. In a high-K(+) solution, which decreased the resting potential to around 0 mV, the symmetrical current profile was also obtained, but only when the membrane potential was held at 0 mV before the voltage steps. These observations suggest that the current was driven and modulated by the junctional potential difference between the clamping cell and its neighbors. In addition, we examined effects of uncoupling agents on the currents. A gap junction channel blocker, halothane, suppressed the currents almost completely, indicating that the currents are predominantly gap junctional currents. Furthermore, injection of biocytin into the current-recorded cells revealed tracer coupling. These results demonstrate that progenitor cells of regenerating retina couple with each other via gap junctions, and suggest the presence of their cytoplasmic communication during early retinal regeneration.     

Extracellular calcium affects the membrane currents of cultured human keratinocytes.

Electrophysiologic properties of cultured human keratinocytes were studied using the patch voltage-clamp technique. Undifferentiated, proliferative keratinocytes grown in low Ca2+ medium had an average resting membrane potential of -24 mV. Voltage-clamp experiments showed that these cells had two membrane ionic currents: a large voltage-independent leak conductance, and a smaller voltage-dependent Cl- current that activated with depolarization. Increasing the extracellular Ca2+ concentration from 0.15 to 2 mM resulted in a doubling of the magnitude of the voltage-gated current and a shift in current activation to more negative potentials. Since levels of extracellular Ca2+ can alter the morphology and differentiation state of keratinocytes, the finding of a Ca2(+)-activated Cl- current in these cells suggests a role for this conductance in the initiation of differentiation.     

Human cord blood CD34+ progenitor cells acquire functional cardiac properties through a cell fusion process

The efficacy of cardiac repair by stem cell administration relies on a successful functional integration of injected cells into the host myocardium. Safety concerns have been raised about the possibility that stem cells may induce foci of arrhythmia in the ischemic myocardium. In a previous work (36), we showed that human cord blood CD34(+) cells, when cocultured on neonatal mouse cardiomyocytes, exhibit excitation-contraction coupling features similar to those of cardiomyocytes, even though no human genes were upregulated. The aims of the present work are to investigate whether human CD34(+) cells, isolated after 1 wk of coculture with neonatal ventricular myocytes, possess molecular and functional properties of cardiomyocytes and to discriminate, using a reporter gene system, whether cardiac differentiation derives from a (trans)differentiation or a cell fusion process. Umbilical cord blood CD34(+) cells were isolated by a magnetic cell sorting method, transduced with a lentiviral vector carrying the enhanced green fluorescent protein (EGFP) gene, and seeded onto primary cultures of spontaneously beating rat neonatal cardiomyocytes. Cocultured EGFP(+)/CD34(+)-derived cells were analyzed for their electrophysiological features at different time points. After 1 wk in coculture, EGFP(+) cells, in contact with cardiomyocytes, were spontaneously contracting and had a maximum diastolic potential (MDP) of -53.1 mV, while those that remained isolated from the surrounding myocytes did not contract and had a depolarized resting potential of -11.4 mV. Cells were then resuspended and cultured at low density to identify EGFP(+) progenitor cell derivatives. Under these conditions, we observed single EGFP(+) beating cells that had acquired an hyperpolarization-activated current typical of neonatal cardiomyocytes (EGFP(+) cells, -2.24 ± 0.89 pA/pF; myocytes, -1.99 ± 0.63 pA/pF, at -125 mV). To discriminate between cell autonomous differentiation and fusion, EGFP(+)/CD34(+) cells were cocultured with cardiac myocytes infected with a red fluorescence protein-lentiviral vector; under these conditions we found that 100% of EGFP(+) cells were also red fluorescent protein positive, suggesting cell fusion as the mechanism by which cardiac functional features are acquired.     

A mathematical model of electrotonic interactions between ventricular myocytes and fibroblasts

Functional intercellular coupling has been demonstrated among networks of cardiac fibroblasts, as well as between fibroblasts and atrial or ventricular myocytes. In this study, the consequences of these interactions were examined by implementing the ten Tusscher model of the human ventricular action potential, and coupling it to our electrophysiological models for mammalian ventricular fibroblasts. Our simulations reveal significant electrophysiological consequences of coupling between 1 and 4 fibroblasts to a single ventricular myocyte. These include alterations in plateau height and/or action potential duration (APD) and changes in underlying ionic currents. Two series of simulations were carried out. First, fibroblasts were modeled as a spherical cell with a capacitance of 6.3 pF and an ohmic membrane resistance of 10.7 G Omega. When these "passive" fibroblasts were coupled to a myocyte, they caused slight prolongation of APD with no changes in the plateau, threshold for firing, or rate of initial depolarization. In contrast, when the same myocyte-fibroblast complexes were modeled after addition of the time- and voltage-gated K(+) currents that are expressed in fibroblasts, much more pronounced effects were observed: the plateau height of the action potential was reduced and the APD shortened significantly. In addition, each fibroblast exhibited significant electrotonic depolarizations in response to each myocyte action potential and the resting potential of the fibroblasts closely approximated the resting potential of the coupled ventricular myocyte.     

Modulation of potassium current characteristics in human myometrial smooth muscle by 17beta-estradiol and progesterone

The K(+) channel currents are important modulators of smooth muscle membrane potential and excitability. We assessed whether voltage-gated K(+) currents from human myometrium are regulated by placental steroid hormones during pregnancy and labor. Pregnant human myometrial cells were isolated from samples obtained at cesarean section. Primary cultured cells were treated with 100 nM 17beta-estradiol, 1 microM progesterone, or both hormones in combination for 24 h. Acute effects of the two hormones were also determined. The K(+) currents were recorded using the standard whole-cell, patch-clamp technique. Primary cultures possessed both delayed rectifier (I(KV)) and A-like (I(KA)) voltage-gated K(+) currents. The 24-h 17beta-estradiol treatment caused a hyperpolarizing shift in the steady-state inactivation of both I(KV) and I(KA). Progesterone treatment also shifted the inactivation of I(KA) and increased I(KV) amplitude by 60%-110%. Conversely, the combined treatment had no effect on these currents. Neither 17beta-estradiol (0.1-1 microM) nor progesterone (1-5 microM) had any effect on the K(+) current when applied acutely. These results show that 17beta-estradiol should inhibit myometrial K(+) channel activity, whereas progesterone is likely to have the opposite effect. These results are consistent with the respective procontractile and proquiescence roles for 17beta-estradiol and progesterone in human uterus during pregnancy.     

Hyperpolarization-activated current (I(h)) in ganglion-cell photoreceptors

Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and serve as the primary retinal drivers of non-image-forming visual functions such as circadian photoentrainment, the pupillary light reflex, and suppression of melatonin production in the pineal. Past electrophysiological studies of these cells have focused on their intrinsic photosensitivity and synaptic inputs. Much less is known about their voltage-gated channels and how these might shape their output to non-image-forming visual centers. Here, we show that rat ipRGCs retrolabeled from the suprachiasmatic nucleus (SCN) express a hyperpolarization-activated inwardly-rectifying current (I(h)). This current is blocked by the known I(h) blockers ZD7288 and extracellular cesium. As in other systems, including other retinal ganglion cells, I(h) in ipRGCs is characterized by slow kinetics and a slightly greater permeability for K(+) than for Na(+). Unlike in other systems, however, I(h) in ipRGCs apparently does not actively contribute to resting membrane potential. We also explore non-specific effects of the common I(h) blocker ZD7288 on rebound depolarization and evoked spiking and discuss possible functional roles of I(h) in non-image-forming vision. This study is the first to characterize I(h) in a well-defined population of retinal ganglion cells, namely SCN-projecting ipRGCs.     

白介素1β抑制培养的大鼠皮层神经元钠电流峰值和动作电位幅度

白介素1β(interleukin-1β,IL-1β)是重要的促炎细胞因子,在中枢神经系统的生理学和病理学过程中发挥关键作用.电压门控钠通道是可兴奋细胞电学活动的基础,控制神经元的兴奋性和动作电位.最近的研究又显示了IL-1β与电压门控通道之间的相互作用.为考察中枢神经元中IL-1β与电压门控钠通道之间的相互作用,本研...     

Role of hyperpolarization-activated currents for the intrinsic dynamics of isolated retinal neurons

The intrinsic dynamics of bipolar cells and rod photoreceptors isolated from tiger salamanders were studied by a patch-clamp technique combined with estimation of effective impulse responses across a range of mean membrane voltages. An increase in external K(+) reduces the gain and speeds the response in bipolar cells near and below resting potential. High external K(+) enhances the inward rectification of membrane potential, an effect mediated by a fast, hyperpolarization-activated, inwardly rectifying potassium current (K(IR)). External Cs(+) suppresses the inward-rectifying effect of external K(+). The reversal potential of the current, estimated by a novel method from a family of impulse responses below resting potential, indicates a channel that is permeable predominantly to K(+). Its permeability to Na(+), estimated from Goldman-Hodgkin-Katz voltage equation, was negligible. Whereas the activation of the delayed-rectifier K(+) current causes bandpass behavior (i.e., undershoots in the impulse responses) in bipolar cells, activation of the K(IR) current does not. In contrast, a slow hyperpolarization-activated current (I(h)) in rod photoreceptors leads to pronounced, slow undershoots near resting potential. Differences in the kinetics and ion selectivity of hyperpolarization-activated currents in bipolar cells (K(IR)) and in rod photoreceptors (I(h)) confer different dynamical behavior onto the two types of neurons.     

Expression and high glucose-mediated regulation of K+ channel interacting protein 3 (KChIP3) and KV4 channels in retinal Müller glial cells

Normal vision depends on the correct function of retinal neurons and glia and it is impaired in the course of diabetic retinopathy. Müller cells, the main glial cells of the retina, suffer morphological and functional alterations during diabetes participating in the pathological retinal dysfunction. Recently, we showed that Müller cells express the pleiotropic protein potassium channel interacting protein 3 (KChIP3), an integral component of the voltage-gated K(+) channels K(V)4. Here, we sought to analyze the role of KChIP3 in the molecular mechanisms underlying hyperglycemia-induced phenotypic changes in the glial elements of the retina. The expression and function of KChIp3 was analyzed in vitro in rat Müller primary cultures grown under control (5.6 mM) or high glucose (25 mM) (diabetic-like) conditions. We show the up-regulation of KChIP3 expression in Müller cell cultures under high glucose conditions and demonstrate a previously unknown interaction between the K(V)4 channel and KChIP3 in Müller cells. We show evidence for the expression of a 4-AP-sensitive transient outward voltage-gated K(+) current and an alteration in the inactivation of the macroscopic outward K(+) currents expressed in high glucose-cultured Müller cells. Our data support the notion that induction of KChIP3 and functional changes of K(V)4 channels in Müller cells could exert a physiological role in the onset of diabetic retinopathy.     

Membrane ionic currents in Rhodobacter capsulatus. Evidence for electrophoretic transport of K+, Rb+ and NH4+

1. The cytoplasmic membrane ionic current of cells of Rhodobacter capsulatus, washed to lower the endogenous K+ concentration, had a non-linear dependence on the membrane potential measured during photosynthetic illumination. Treatment of the cells with venturicidin, an inhibitor of the H(+)-ATP synthase, increased the membrane potential and decreased the membrane ionic current at values of membrane potential below a threshold. 2. The addition of K+ or Rb+, but not of Na+, led to an increase in the membrane ionic current and a decrease in the membrane potential in either the presence or absence of venturicidin. Approximately 0.4 mM K+ or 2.0 mM Rb+ led to a half-maximal response. At saturating concentrations of K+ and Rb+, the membrane ionic currents were similar. The membrane ionic currents due to K+ and Rb+ were not additive. The K(+)-dependent and Rb(+)-dependent ionic currents had a non-linear relationship with membrane potential: the alkali cations only increased the ionic current when the membrane potential lay above a threshold value. The presence of 1 mM Cs+ did not lead to an increase in the membrane ionic current but it had the effect of inhibiting the membrane ionic current due to either K+ or Rb+. 3. Photosynthetic illumination in the presence of either K+ or Rb+, and weak acids such as acetate, led to a decrease in light-scattering by the cells. This was attributed to the uptake of potassium or rubidium acetate and a corresponding increase in osmotic strength in the cytoplasm. 4. The addition of NH4+ also led to an increase in membrane ionic current and to a decrease in membrane potential (half-maximal at 2.0 mM NH4+). The relationship between the NH4(+)-dependent ionic currents and the membrane potential was similar to that for K+. The NH4(+)-dependent and K(+)-dependent ionic current were not additive. However, illumination in the presence of NH4+ and acetate did not lead to significant light-scattering changes. The NH4(+)-dependent membrane ionic current was inhibited by 1 mM Cs+ but not by 50 microM methylamine. 5. It is proposed that the K(+)-dependent membrane ionic current is catalysed by a low-affinity K(+)-transport system such as that described in Rb. capsulatus [Jasper, P. (1978) J. Bacteriol. 133, 1314-1322]. The possibility is considered that, as well as Rb+, this transport system can also operate with NH4+. However, in our experimental conditions NH4+ uptake is followed by NH3 efflux.(ABSTRACT TRUNCATED AT 400 WORDS)     

TREK-1 K+ channels couple angiotensin II receptors to membrane depolarization and aldosterone secretion in bovine adrenal glomerulosa cells

Bovine adrenal glomerulosa (AZG) cells were shown to express bTREK-1 background K(+) channels that set the resting membrane potential and couple angiotensin II (ANG II) receptor activation to membrane depolarization and aldosterone secretion. Northern blot and in situ hybridization studies demonstrated that bTREK-1 mRNA is uniformly distributed in the bovine adrenal cortex, including zona fasciculata and zona glomerulosa, but is absent from the medulla. TASK-3 mRNA, which codes for the predominant background K(+) channel in rat AZG cells, is undetectable in the bovine adrenal cortex. In whole cell voltage clamp recordings, bovine AZG cells express a rapidly inactivating voltage-gated K(+) current and a noninactivating background K(+) current with properties that collectively identify it as bTREK-1. The outwardly rectifying K(+) current was activated by intracellular acidification, ATP, and superfusion of bTREK-1 openers, including arachidonic acid (AA) and cinnamyl 1-3,4-dihydroxy-alpha-cyanocinnamate (CDC). Bovine chromaffin cells did not express this current. In voltage and current clamp recordings, ANG II (10 nM) selectively inhibited the noninactivating K(+) current by 82.1 +/- 6.1% and depolarized AZG cells by 31.6 +/- 2.3 mV. CDC and AA overwhelmed ANG II-mediated inhibition of bTREK-1 and restored the resting membrane potential to its control value even in the continued presence of ANG II. Vasopressin (50 nM), which also physiologically stimulates aldosterone secretion, inhibited the background K(+) current by 73.8 +/- 9.4%. In contrast to its potent inhibition of bTREK-1, ANG II failed to alter the T-type Ca(2+) current measured over a wide range of test potentials by using pipette solutions of identical nucleotide and Ca(2+)-buffering compositions. ANG II also failed to alter the voltage dependence of T channel activation under these same conditions. Overall, these results identify bTREK-1 K(+) channels as a pivotal control point where ANG II receptor activation is transduced to depolarization-dependent Ca(2+) entry and aldosterone secretion.     

Effect of capsaicin on voltage-gated currents of trigeminal neurones in cell culture and slice preparations

Effects of capsaicin on voltage-gated currents were examined in vitro by whole-cell patch-clamp recordings from small neurones of rat trigeminal ganglia either in slice preparations or in different cell cultures. Cells were classified as sensitive to capsaicin if they responded with inward current and/or conductance change to the agent in nanomolar concentration. Capsaicin (150 to 330 nM) in sensitive cells reduced the mixed inward current evoked by depolarizing step or ramp commands in all preparations. In cultured cells, the inward current was depressed to 32.78 +/- 26.42% (n = 27) of the control. Both the tetrodotoxin-sensitive and -resistant inward currents were affected. The data support the concept that capsaicin besides acting on VR-1 receptors inhibits also some voltage gated channels. In 34 cultured cells, capsaicin increased the slope conductance to 170.5 +/- 68%. Percentage of capsaicin sensitive cells observed in nerve growth factor-treated cultured cell populations was higher (77.8%) than in the two other preparations (14.3 or 38.8%). It is concluded that 1) depression of the voltage-gated currents may play an important role in the functional desensitization of the sensory receptors and in the analgesic effect induced by the agent and 2) cell body of sensory neurones under native condition seems less sensitive to capsaicin then that of cells cultured in the presence of nerve growth factor.     

Patch-clamp study of neurons and glial cells in isolated myenteric ganglia

Most of the physiological information on the enteric nervous system has been obtained from studies on preparations of the myenteric ganglia attached to the longitudinal muscle layer. This preparation has a number of disadvantages, e.g., the inability to make patch-clamp recordings and the occurrence of muscle movements. To overcome these limitations we used isolated myenteric ganglia from the guinea pig small intestine. In this preparation movement was eliminated because muscle was completely absent, gigaseals were obtained, and whole cell recordings were made from neurons and glial cells. The morphological identity of cells was verified by injecting a fluorescent dye by micropipette. Neurons displayed voltage-gated inactivating inward Na(+) and Ca(2+) currents as well as delayed-rectifier K(+) currents. Immunohistochemical staining confirmed that most neurons have Na(+) channels. Neurons responded to GABA, indicating that membrane receptors were retained. Glial cells displayed hyperpolarization-induced K(+) inward currents and depolarization-induced K(+) outward currents. Glia showed large "passive" currents that were suppressed by octanol, consistent with coupling by gap junctions among these cells. These results demonstrate the advantages of isolated ganglia for studying myenteric neurons and glial cells.     

Calcitonin gene-related peptide suppresses pacemaker currents by nitric oxide/cGMP-dependent activation of ATP-sensitive K(+) channels in cultured interstitial cells of Cajal from the mouse small intestine

The effects of calcitonin gene-related peptide (CGRP) on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine were investigated using the whole-cell patch clamp technique at 30 degrees . Under voltage clamping at a holding potential of -70 mV, CGRP decreased the amplitude and frequency of pacemaker currents and activated outward resting currents. These effects were blocked by intracellular GDPbetaS, a G-protein inhibitor and glibenclamide, a specific ATP-sensitive K(+) channels blocker. During current clamping, CGRP hyperpolarized the membrane and this effect was antagonized by glibenclamide. Pretreatment with SQ-22536 (an adenylate cyclase inhibitor) or naproxen (a cyclooxygenase inhibitor) did not block the CGRP-induced effects, whereas pretreatment with ODQ (a guanylate cyclase inhibitor) or L-NAME (an inhibitor of nitric oxide synthase) did. In conclusion, CGRP inhibits pacemaker currents in ICC by generating nitric oxide via G-protein activation and so activating ATP-sensitive K(+) channels. Nitric oxide- and guanylate cyclase- dependent pathways are involved in these effects.     

Somatostatin decreases voltage-gated Ca2+ currents in GH3 cells through activation of somatostatin receptor 2

The secretion of growth hormone (GH) is inhibited by hypothalamic somatostatin (SRIF) in somatotropes through five subtypes of the somatostatin receptor (SSTR1-SSTR5). We aimed to characterize the subtype(s) of SSTRs involved in the Ca2+ current reduction in GH3 somatotrope cells using specific SSTR subtype agonists. We used nystatin-perforated patch clamp to record voltage-gated Ca2+ currents, using a holding potential of -80 mV in the presence of K+ and Na+ channel blockers. We first established the presence of T-, L-, N-, and P/Q-type Ca2+ currents in GH3 cells using a variety of channel blockers (Ni+, nifedipine, omega-conotoxin GVIA, and omega-agatoxin IVA). SRIF (200 nM) reduced L- and N-type but not T- or P/Q-type currents in GH3 cells. A range of concentrations of each specific SSTR agonist was tested on Ca2+ currents to find the maximal effective concentration. Activation of SSTR2 with 10(-7) and 10(-8) M L-797,976 decreased the voltage-gated Ca2+ current and abolished any further decrease by SRIF. SSTR1, SSTR3, SSTR4, and SSTR5 agonists at 10(-7) M did not modify the voltage-gated Ca2+ current and did not affect the Ca2+ current response to SRIF. These results indicate that SSTR2 is involved mainly in regulating voltage-gated Ca2+ currents by SRIF, which contributes to the decrease in intracellular Ca2+ concentration and GH secretion by SRIF.     

Inwardly rectifying K(+) channels in esophageal smooth muscle

The whole cell patch-clamp technique was used to investigate whether there were inwardly rectifying K(+) (K(ir)) channels in the longitudinal muscle of cat esophagus. Inward currents were observable on membrane hyperpolarization negative to the K(+) equilibrium potential (E(k)) in freshly isolated esophageal longitudinal muscle cells. The current-voltage relationship exhibited strong inward rectification with a reversal potential (E(rev)) of -76.5 mV. Elevation of external K(+) increased the inward current amplitude and positively shifted its E(rev) after the E(k), suggesting that potassium ions carry this current. External Ba(2+) and Cs(+) inhibited this inward current, with hyperpolarization remarkably increasing the inhibition. The IC(50) for Ba(2+) and Cs(+) at -60 mV was 2.9 and 1.6 mM, respectively. Furthermore, external Ba(2+) of 10 microM moderately depolarized the resting membrane potential of the longitudinal muscle cells by 6.3 mV while inhibiting the inward rectification. We conclude that K(ir) channels are present in the longitudinal muscle of cat esophagus, where they contribute to its resting membrane potential.     

Effect of superoxide derived from lucifer yellow CH on voltage-gated currents of mouse taste bud cells

Lucifer yellow CH (LY), a fluorescent membrane-impermeable cell marker dye, has been routinely loaded into cells through recording electrodes to visualize these cells after electrophysiological investigation, without considering its pharmacological effect. Recently, we showed that the exposure of cells loaded with LY to light for microscopy produced unidentified radical species that retarded the inactivation of voltage-gated Na+ currents irreversibly (Higure Y et al. 2003). Here, we show that superoxide dismutase, an enzyme that decomposes superoxide, reverses the retardation effect, which assures that superoxide is the unidentified radical species. The estimated mean lifetime of superoxide in recording electrodes (in the absence of cytoplasm) is approximately 6 min, and hence, the Na+ currents are retarded even in the dark, when LY is exposed to light before being loaded into the cell. Superoxide has no effect on voltage-gated Cl- currents. These results show that superoxide action on ion channels is rather selective. The breakdown of superoxide inside cells and the effect of endogenous superoxide on the superoxide-susceptible channels are discussed.     

Voltage-gated inward currents of morphologically identified cells of the frog taste disc

We used the patch clamp technique to record from taste cells in vertical slices of the bullfrog (Rana catesbeiana) taste disc. Cell types were identified by staining with Lucifer yellow in a pipette after recording their electrophysiological properties. Cells could be divided into the following three groups: type Ib (wing) cells with sheet-like apical processes, type II (rod) cells with single thick rod-like apical processes and type III (rod) cells with thin rod-like apical processes. No dye-coupling was seen either between cells of the same type or between cells of different types. We focused on the voltage-gated inward currents of the three types of cells. Type Ib and type II cells exhibited tetrodotoxin (TTX)-sensitive voltage-gated Na+ currents. Surprisingly, type III cells showed TTX-resistant voltage-gated Na+ currents and exhibited a lack of TTX-sensitive Na+ currents. TTX-resistant voltage-gated Na+ currents in taste cells are reported for the first time here. The time constant for the inactivating portion of the voltage-gated inward Na+ currents of type III cells was much larger than that of type Ib and type II cells. Therefore, slow inactivation of inward Na+ currents characterizes type III cells. Amplitudes of the maximum peak inward currents of type III cells were smaller than those of type Ib and type II cells. However, the density (pA/pF) of the maximum peak inward currents of type III cells was much higher than that of type Ib cells and close to that of type II cells. No evidence of the presence of voltage-gated Ca2+ channels in frog taste cells has been presented up to now. In this study, voltage-gated Ba2+ currents were observed in type III cells but not in type Ib and type II cells when the bath solution was a standard Ba2+ solution containing 25 mM Ba2+. Voltage-gated Ba2+ currents were blocked by addition of 2 mM CoCl2 to the standard Ba2+ solution, suggesting that type III cells possess the voltage-gated Ca2+ channels and they do classical (calcium-influx) synaptic transmission. It appears that type III cells are taste receptor cells.     

Plasmalemmal voltage-activated K(+) currents in protoplasts from tobacco BY-2 cells: possible regulation by actin microfilaments?

Plasmalemmal ionic currents from enzymatically isolated protoplasts of suspension-cultured tobacco 'Bright Yellow-2' cells were investigated by whole-cell patch-clamp techniques. In all protoplasts, delayed rectifier outward K(+) currents having sigmoidal activation kinetics, no inactivation, and very slow deactivation kinetics were activated by step depolarization. Tail current reversal potentials were close to equilibrium potential E(K) when external [K(+)] was either 6 or 60 mM. Several channel blockers, including external Ba(2+), niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, inhibited this outward K(+) current. Among the monovalent cations tested (NH(4)(+), Rb(+), Li(+), Na(+)), only Rb(+) had appreciable permeation (P(Rb)/P(K) (=) 0.7). In addition, in 60 mM K(+) solutions, a hyperpolarization-activated, time-dependent, inwardly rectifying K(+) current was observed in most protoplasts. This inward current activated very slowly, did not inactivate, and deactivated quickly upon repolarization. The tail current reversal potential was very close to E(K), and other monovalent cations (NH(4)(+), Rb(+), Li(+), Na(+)) were not permeant. The inward current was blocked by external Ba(2+) and niflumic acid. External Cs(+) reversibly blocked the inward current without affecting the outward current. The amplitude of the inward rectifier K(+) current was generally small compared to the amplitude of the outward K(+) current in the same cell, although this was highly variable. Similar amplitudes for both currents occurred in only 4% of the protoplasts in control conditions. Microfilament-depolymerizing drugs shifted this proportion to about 12%, suggesting that microfilaments participate in the regulation of K(+) currents in tobacco 'Bright Yellow-2' cells.     

Multipotent progenitor cells derived from adult peripheral blood of swine have high neurogenic potential in vitro

Peripheral blood-derived multipotent adult progenitor cells (PBD-MAPCs) are a novel population of stem cells, isolated from venous blood of green fluorescent protein transgenic swine, which proliferate as multicellular non-adherent spheroids. Using a simple differentiation protocol, a large proportion of these cells developed one of five distinct neural cell phenotypes, indicating that these primordial cells have high neurogenic potential. Cells exhibiting neural morphologies developed within 48 h of exposure to differentiation conditions, increased in percentage over 2 weeks, and stably maintained the neural phenotype for three additional weeks in the absence of neurogenic signaling molecules. Cells exhibited dynamic neural-like behaviors including extension and retraction of processes with growth cone-like structures rich in filamentous actin, cell migration following a leading process, and various cell-cell interactions. Differentiated cells expressed neural markers, NeuN, β-tubulin III and synaptic proteins, and progenitor cells expressed the stem cell markers nestin and NANOG. Neurally differentiated PBD-MAPCs exhibited voltage-dependent inward and outward currents and expressed voltage-gated sodium and potassium channels, suggestive of neural-like membrane properties. PBD-MAPCs expressed early neural markers and developed neural phenotypes when provided with an extracellular matrix of laminin without the addition of cytokines or growth factors, suggesting that these multipotent cells may be primed for neural differentiation. PBD-MAPCs provide a model for understanding the mechanisms of neural differentiation from non-neural sources of adult stem cells. A similar population of cells, from humans or xenogeneic sources, may offer the potential of an accessible, renewable and non-tumorigenic source of stem cells for treating neural disorders.