Potassium currents in cultured rabbit retinal pigment epithelial cells

Membrane potential and ionic currents were studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell patch clamp and perforated-patch recording techniques. RPE cells exhibited both outward and inward voltage-dependent currents and had a mean membrane capacitance of 26±12 pF (sd, n=92). The resting membrane potential averaged ?31±15 mV (n=37), but it was as high as ?60 mV in some cells. When K⁺ was the principal cation in the recording electrode, depolarization-activated outward currents were apparent in 91% of cells studied. Tail current analysis revealed that the outward currents were primarily K⁺ selective. The most frequently observed outward K⁺ current was a voltage- and time-dependent outward current (I _K) which resembled the delayed rectifier K⁺ current described in other cells. I _K was blocked by tetraethylammonium ions (TEA) and barium (Ba²⁺) and reduced by 4-aminopyridine (4-AP). In a few cells (3–4%), depolarization to ?50 mV or more negative potentials evoked an outwardly rectifying K⁺ current (I _Kt) which showed more rapid inactivation at depolarized potentials. Inwardly rectifying K⁺ current (I _KI) was also present in 41% of cells. I _KI was blocked by extracellular Ba²⁺ or Cs⁺ and exhibited time-dependent decay, due to Na⁺ blockade, at negative potentials. We conclude that cultured rabbit RPE cells exhibit at least three voltage-dependent K⁺ currents. The K⁺ conductances reported here may provide conductive pathways important in maintaining ion and fluid homeostasis in the subretinal space.     

α2-Adrenoceptor-Mediated Inhibition of Electrically Evoked [3H]Noradrenaline Release from Chick Sympathetic Neurons: Role of Cyclic AMP

Abstract: This study explores the role of cyclic AMP in electrically evoked [³H]noradrenaline release and in the α₂-adrenergic modulation of this release in chick sympathetic neurons. Along with an increase in stimulation-evoked tritium overflow, applications of forskolin enhanced the formation of intracellular cyclic AMP. Both effects of forskolin were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The forskolin-induced increase in overflow was abolished by the Rp-diastereomer of cyclic AMP-thioate, an antagonist at cyclic AMP-dependent protein kinases, and 1,9-dideoxy-forskolin, an inactive analogue at adenylyl cyclase, had no effect on the evoked overflow. A 24-h pretreatment with either cholera toxin or forskolin reduced the subsequent forskolin-induced accumulation of cyclic AMP and inhibited the stimulation-evoked release. Basal cyclic AMP production, however, remained unaltered after forskolin treatment and was enhanced after 24 h of cholera toxin exposure. The α₂-adrenergic agonist bromoxidine did not affect the formation of cyclic AMP stimulated by forskolin but reduced electrically evoked release. However, effects of bromoxidine on ³H overflow were attenuated by forskolin as well as by 8-bromo-cyclic AMP. Effects of bromoxidine on [³H]noradrenaline release were paralleled by an inhibition of voltage-activated Ca²⁺ currents, primarily through a delayed time course of current activation. This effect was abolished when either forskolin or 8-bromo-cyclic AMP was included in the pipette solution. Both substances, however, failed to affect Ca²⁺ currents in the absence of bromoxidine. These results suggest that the signaling cascade of the α₂-adrenergic inhibition of noradrenaline release involves voltage-activated Ca²⁺ channels but not cyclic AMP. Elevated levels of cyclic AMP, however, antagonize this α₂-adrenergic reduction, apparently through a disinhibition of Ca²⁺ channels.     

Depressant effects of prostacyclin in human atrial fibers and cardiomyocytes

The aim of this study was to characterize the electropharmacological effects of prostacyclin (PGI₂) in human atrial fibers and cardiomyocytes. Atrial tissues obtained from the hearts of 28 patients undergoing corrective cardiac surgery were used. Transmembrane action potentials were recorded using a conventional microelectrode technique, and twitch force by a transducer. Effects of PGI₂ (1 nM–10 µM) on action potential characteristics and contraction of atrial fibers were evaluated in normal [K]_o (4 mM) and high [K]_o (27 mM) in the absence and presence of cardiotonic agents. In addition, atrial and ventricular myocytes were isolated enzymatically from atrial tissues and hearts of 4 patients undergoing cardiac transplant. The effects of PGI₂ on Na- and Ca-dependent inward currents (I_Na and I_Ca) of cardiomyocytes were tested. In 9 human atrial fibers showing fast-response action potentials (mean dV/dt_max = 101 ± 15 Vs^–1) in 4 mM [K]_o, PGI₂ did not influence dV/dt_max of phase 0 depolarization even at 1 µM. However, at a concentration as low as 10 nM, PGI₂ depressed spontaneous rhythms or slow-response action potentials in high-K-depolarized fibers. PGI₂ also depressed delayed afterdepolarizations and aftercontractions induced by cardiotonic agents. In isolated cardiomyocytes, PGI₂ reduced I_Ca but not I_Na. The present findings show that, in human atrial fibers and cardiomyocytes, PGI₂ induces greater depressant effects on the slow-response action potential, I_Ca and triggered activity than on the fast-response action potential. It is suggested that PGI₂ may act through a selective reduction of transmembrane Ca influx.     

Hydrodynamics in early animal evolution

Choanoflagellates and sponges feed by filtering microscopic particles from water currents created by the flagella of microvillar collar complexes situated on the cell bodies of the solitary or colonial choanoflagellates and on the choanocytes in sponges. The filtering mechanism has been known for more than a century, but only recently has the filtering process been studied in detail and also modelled, so that a detailed picture of the water currents has been obtained. In the solitary and most of the colonial choanoflagellates, the water flows freely around the cells, but in some forms, the cells are arranged in an open meshwork through which the water can be pumped. In the sponges, the choanocytes are located in choanocyte chambers (or choanocyte areas) with separate incurrent and excurrent canals/pores located in a larger body, which enables a fixed pattern of water currents through the collar complexes. Previous theories for the origin of sponges show evolutionary stages with choanocyte chambers without any opening or with only one opening, which makes separation of incurrent and excurrent impossible, and such stages must have been unable to feed. Therefore a new theory is proposed, which shows a continuous evolutionary lineage in which all stages are able to feed by means of the collar complexes.     

High-Resolution Performance of Polycaprolactone Functionalized with Guanidinium Ionic Liquid for Gas Chromatography

This work firstly reported a new polycaprolactone based material functionalized with guanidinium ionic liquid (PCL-GIL) as the stationary phase with high resolution performance for capillary gas chromatography (GC). It is composed of polycaprolactone (PCL) and guanidinium ionic liquid (GIL) with amphiphilic conformation. The PCL-GIL capillary column coated by static method exhibited high column efficiency of 3942 plates/m and moderate polarity. As a result, the PCL-GIL column exhibited high-resolution capability. For a mixture of 27 analytes with a wide ranging polarity and outperformed the PCL-2OH and HP-35 columns, showing its advantageous separation capability for analytes of diverse types. Moreover, the PCL-GIL column showed high resolving capability for various positional isomers and cis-/trans-isomers, including alkylbenzenes, chlorobenzenes, naphthalenes, bromonitrobenzenes, chloronitrobenzenes, benzaldehydes, phenols, alcohols, respectively. In a word, PCL derivatized by GIL units as a new type of stationary phase has a promising future in GC separations.     

Distribution of Plasma Membrane H+-ATPase and Polar Current Patterns in Leaves and Stems of Elodea canadensis*

The proton pumping ATPase in the plasma membrane of Elodea canadensis is believed to play a major role in inorganic carbon acquisition. To investigate potentially different carbon uptake strategies within the same plant, plasma membrane H⁺-ATPase distribution and polar current patterns were investigated in Elodea leaves and stems. Specific activity of plasma membrane H⁺-ATPase in leaf microsomal fractions was tenfold higher than in stem derived microsomes. Probing western blots with a monoclonal antibody specific for plasma membrane H⁺-ATPase, yielded strongly visible double bands at 100 kDa in leaf microsome preparations, whereas little antigen was detected in analogous stem microsome preparations. Using the same plasma membrane H⁺-ATPase specific antibody on tissue sections, the enzyme was found almost exclusively localized at the border of cells at the lower leaf surface. A positive ion current leaving the lower leaf surface was measured, using a vibrating probe device. Part of this current entered the upper leaf surface and part of it the internodes of the stem. The experimental results support the view, that Elodea leaves have different means of inorganic carbon uptake than stem internodes.     

Sodium currents in toad cardiac pacemaker cells

Cells in the pacemaker region of toad (Bufo marinus) sinus venosus had spontaneous rhythmic action potentials. The rate of firing of action potentials, the rate of diastolic depolarization and the maximum rate of rise of action potentials were reduced by TTX (10 nm to 1 m). Currents were recorded with the whole cell, tight seal technique from cells enzymatically dissociated from this region. Cells studied were identified as pacemaker cells by their characteristic morphology, spontaneous rhythmic action potential activity that could be blocked by cobalt but not by TTX and lack of inward rectification. When calcium, potassium and nonselective cation currents (I_f) activated by hyperpolarization were blocked, depolarization was seen to generate transient and persistent inward currents. Both were sodium currents: they were abolished by tetrodotoxin (10 to 100 nm), their reversal potential was close to the sodium equilibrium potential and their amplitude and reversal potential were influenced as expected for sodium currents when extracellular sodium ions were replaced with choline ions. The transient sodium current was activated at potentials more positive than –40 mV while the persistent sodium current was obvious at more negative potentials. It was concluded that, in toad pacemaker cells, TTX-sensitive sodium currents contributing both to the upstroke of action potentials and to diastolic depolarization may play an important role in setting heart rate.We thank the Australian National Heart Foundation for their support. D.A.S. is an NHMRC Senior Research Officer.