Volume-sensitive Cl- current in bovine adrenocortical cells: comparison with the ACTH-induced Cl- current.

In a previous study performed on zona fasciculata (ZF) cells isolated from calf adrenal glands, we identified an ACTH-induced Cl- current involved in cell membrane depolarization. In the present work, we describe a volume-sensitive Cl- current and compare it with the ACTH-activated Cl- current. Experiments were performed using the whole-cell patch-clamp recording method, video microscopy and cortisol-secretion measurements. In current-clamp experiments, hypotonic solutions induced a membrane depolarization to -22 mV. This depolarization, correlated with an increase in the membrane conductance, was sensitive to different Cl- channel inhibitors. In voltage-clamp experiments, hypotonic solution induced a membrane current that slowly decayed and reversed at -21 mV. This ionic current displayed no time dependence and showed a slight outward rectification. It was blocked to variable extent by different conventional Cl- channel inhibitors. Under hypotonic conditions, membrane depolarizations were preceded by an increase in cell volume that was not detected under ACTH stimulation. It was concluded that hypotonic solution induced cell swelling, which activated a Cl- current involved in membrane depolarization. Although cell volume change was not observed in the presence of ACTH, biophysical properties and pharmacological profile of the volume-sensitive Cl- current present obvious similarities with the ACTH-activated Cl- current. As compared to ACTH, hypotonic solutions failed to trigger cortisol production that was weakly stimulated in the presence of high-K+ solution. This shows that in ZF cells, membrane depolarization is not a sufficient condition to fully activate secretory activities.

Characterization of a Na+-Ca2+ exchanger NCX1 isoform in bovine fasciculata cells of adrenal gland.

The Na+-Ca2+ exchanger (NCX) is well known to regulate intracellular Ca2+ concentration in many excitable cells. To date, three members of the NCX exchanger gene family have been identified: NCX1, NCX2 and NCX3. In zona fasciculata of the bovine adrenal gland, a Na+-Ca2+ exchanger has been recently found to display biochemical similarities with the NCX1 exchanger of renal basolateral cells. Although a Na+-Ca2+ exchanger has already been characterized in zona medulla of adrenal gland, such an exchanger has never been studied in the adrenal cortex. Thus, we have used a reverse transcriptase-polymerase chain reaction (RT-PCR) assay with specific NCX primers on fasciculata cell cultures. The sequencing of the amplified fragment revealed, for the first time, the presence of a NACA3 isoform of the NCX1 exchanger in the adrenal cortex, similar to the one found in adrenal chromaffin cells and in renal cortex. This isoform is 92 and 94% identical in the portion compared with kidney Na+-Ca2+ exchanger NACA3 of rat and pig, respectively.

Characterization of Na/Ca exchange in plasmalemmal vesicles from zona fasciculata cells of the bovine adrenal gland.

The presence of an Na/Ca exchange system in fasciculata cells of the bovine adrenal gland was tested using isolated plasmalemmal vesicles. In the presence of an outwardly Na(+) gradient, Ca(2+) uptake was about 2-fold higher than in K(+) condition. Li(+) did not substitute for Na(+) and 5 mM Ni(2+) inhibited Ca(2+) uptake. Ca(2+) efflux from Ca(2+)-loaded vesicles was Na(+)-stimulated and Ni(2+)-inhibited. The saturable part of Na(+)-dependent Ca(2+) uptake displayed Michaelis-Menten kinetics. The relationship of Na(+)-dependent Ca(2+) uptake versus intravesicular Na(+) concentration was sigmoid (apparent K(0.5) approximately 24 mM; Hill number approximately 3) and Na(+) acted on V(max) without significant effect on K(m). Na(+)-stimulated Ca(2+) uptake was temperature-dependent (apparent Q(10) approximately 2.2). The inhibition properties of several divalent cations (Cd(2+), Sr(2+), Ni(2+), Ba(2+), Mn(2+), Mg(2+)) were tested and were similar to those observed in kidney basolateral membrane. The above results indicate the presence of an Na/Ca exchanger located on plasma membrane of zona fasciculata cells of bovine adrenal gland. This exchanger displays similarities with that of renal basolateral cell membrane.

Two types of pharmacologically distinct Ca(2+) currents with voltage-dependent similarities in zona fasciculata cells isolated from bovine adrenal gland.

Voltage-activated Ca(2+) currents, in zona fasciculata cells isolated from calf adrenal gland, were characterized using perforated patch-clamp recording. In control solution (Ca(2+): 2.5 mm) a transient inward current was followed, in 40% of the cells, by a sustained one. In 20 mm Ba(2+), 61% of the cells displayed an inward current, which consisted of transient and sustained components. The other cells produced either a sustained or a transient inward current. These different patterns were dependent upon time in culture. Current-voltage relationships show that both the transient and sustained components activated, peaked and reversed at similar potentials: -40, 0 and +60 mV, respectively. The two components, fully inactivated at -10 mV, were separated by double-pulse protocols from different holding potentials where the transient component could be inactivated or reactivated. The decaying phase of the sustained component was fitted by a double exponential (time constants: 1.9 and 20 sec at +10 mV); that of the transient component was fitted by a single exponential (time constant: 19 msec at +10 mV). Steady-state activation and inactivation curves of the two components were superimposed. Their half activation and inactivation potentials were similar, about -15 and -34 mV, respectively. The sustained component was larger in Ba(2+) than in Sr(2+) and Ca(2+). Ni(2+) (20 microm) selectively blocked the transient component while Cd(2+) (10 microm) selectively blocked the sustained one. (+/-)Bay K 8644 (0.5 microm) increased the sustained component and nitrendipine (0.5-1 microm) blocked it selectively. The sustained component was inhibited by calciseptine (1 microm). Both components were unaffected by omega-conotoxin GVIA and MVIIC (0.5 microm). These results show that two distinct populations of Ca(2+) channels coexist in this cell type. Although the voltage dependence of their activation and inactivation are comparable, these two components of the inward current are similar to T- and L-type currents described in other cells.

Activation by angiotensin II of Ca(2+)-dependent K+ and Cl- currents in zona fasciculata cells of bovine adrenal gland.

The effects of angiotensin II (100 nM) on the electrical membrane properties of zona fasciculata cells isolated from calf adrenal gland were studied using the whole cell patch recording method. In current-clamp condition, angiotension II induced a biphasic membrane response which began by a transient hyperpolarization followed by a depolarization more positive than the control resting potential. These effects were abolished by Losartan (10(-5) M), an antagonist of angiotensin receptors of type 1. The angiotensin II-induced transient hyperpolarization was characterized in voltage-clamp condition from a holding potential of -10 mV. Using either the perforated or the standard recording method, a transient outward current accompanied by an increase of the membrane conductance was observed in response to the hormonal stimulation. This outward current consisted of an initial fast peak followed by an oscillating or a slowly decaying plateau current. In Cl(-)-free solution, the outward current reversed at -78.5 mV, a value close to EK. It was blocked by external TEA (20 mM) and by apamin (50 nM). In K(+)-free solution, the transient outward current, sensitive to Cl- channel blocker DPC (400 microM), reversed at -52 mV, a more positive potential than ECl. Its magnitude changed in the same direction as the driving force for Cl-. The hormone-induced transient outward current was never observed when EGTA (5 mM) was added to the pipette solution. The plateau current was suppressed in nominally CA(2+)-free solution (47% of cells) and was reversibly blocked by Cd2+ (300 microM) but not by nisoldipine (0.5-1 microM) which inhibited voltage-gated Ca2+ currents identified in this cell type. The present experiments show that the transient hyperpolarization induced by angiotensin II is due to Ca(2+)-dependent K+ and Cl- currents. These two membrane currents are co-activated in response to an internal increase of [Ca2+]i originating from intra- and extracellular stores.

The transient outward current of isolated bovine adrenal zona fasciculata cells: comparison between standard and perforated patch recording methods.

Voltage-clamp experiments were performed on single bovine adrenal fasciculata cells in short-term primary culture using either standard (broken membrane) or perforated whole-cell patch clamp recording. The membrane current measured with the perforated method was dominated by a very stable transient outward current. By contrast, the transient outward current recorded using the standard method was unstable. The reversal potential of the transient outward current varied linearly with the logarithm of [K+]e with a slope of 47 mV per decade. The onset of activation was sigmoidal and was fitted with a power function where n = 4. Time constants ranged from 1 to 4 msec with a maximum at -25 mV. The steady-state activation curve spanned the voltage range -50 +80 mV without reaching a clear maximum. During a pulse, the current decayed in a biexponential manner. Time constants tau 1 and tau 2 were voltage-dependent and ranged from 50 to 200 msec respectively for a voltage step at +50 mV. The steady-state inactivation was dependent on the conditioning pulse duration. Using short conditioning pulses (1.2 sec), the curve which spanned the voltage range -40 to -20 mV, was 15 mV more positive than that obtained with longer conditioning pulses (60 sec). Time constants of this "very slow inactivation" process (tau vs) determined for voltage steps at -60 and -50 mV were 15 and 10 sec respectively. A "facilitation process" of the peak current was observed when the duration or the amplitude of conditioning pulses were increased in the voltage range -100 to -50 mV. Recovery from inactivation followed a biexponential time course which seemed a mixture of both inactivation processes. In some experimental conditions, isolated cells were able to produce overshooting action potentials. These results are discussed in relation with the membrane electrogenesis of this cell type.

The Desheathed Periphery of Aplysia Giant Neuron. Fine Structure and Measurement of [Ca2+]o Fluctuations with Calcium-selective Microelectrodes.

The visceral ganglion of Aplysia was mechanically desheathed after protease softening of the connective tissue to permit the positioning of ion-selective electrodes in the vicinity of the neuronal membrane. The effects of this treatment on satellite glia and neuronal cytology were observed by electron microscopy. The intracellular alterations were not suggestive of serious membrane damage but the cohesion between glial and neuronal membranes was affected-the glial processes appeared to retract from the trophospongium and in some cases the neuronal membrane was completely naked. The external calcium activity [Ca2+]o at the surface of identified giant neuron, R2, was measured using double-barrelled calcium-selective microelectrodes. A decrease of approximately 1 mM in [Ca2+]o could be recorded only during trains of action potentials induced by intracellular depolarizing current injection, and when the electrode was pushed firmly against the neuron surface. A recovery from this decrease in [Ca2+]o could sometimes, but not always, be observed during the phase of induced neuronal activity.

Membrane currents that govern smooth muscle contraction in a ctenophore.

Ctenophores are transparent marine organisms that swim by means of beating cilia; they are the simplest animals with individual muscle fibres. Predatory species, such as Beroe ovata, have particularly well-developed muscles and are capable of an elaborate feeding response. When Beroe contacts its prey, the mouth opens, the body shortens, the pharynx expands, the prey is engulfed and the lips then close tightly. How this sequence, which lasts 1 s, is accomplished is unclear. The muscles concerned are structurally uniform and are innervated at each end by a neuronal nerve net with no centre for coordination. Isolated muscle cells studied under voltage-clamp provide a solution to this puzzle. We find that different groups of muscle cells have different time-dependent membrane currents. Because muscle contraction depends upon calcium entry during each action potential, these different currents produce different patterns of contraction. We conclude that in a simple animal such as a ctenophore, a sophisticated set of membrane conductances can compensate for the absence of an elaborate system of effectors.

Isolation of functional giant smooth muscle cells from an invertebrate: structural features of relaxed and contracted fibers.

The giant smooth muscle fibers of a ctenophore were isolated by enzymatic digestion. These fibers are multinucleated cells, up to 50 micrometers in diameter and 2 cm in length. Their ultrastructure and membrane electrical properties are similar to those of in situ fibers. Relaxed, coiled (partially contracted), and fully shortened states were distinguished in isolated cells and studied by scanning and transmission electron microscopy. Calcium-containing mitochondrial granules were found in the coiled cells but not in either the relaxed or the fully shortened cells. The relaxed cell is characterized in cross section by the density of myosin filaments (457 +/- 15 per micrometer2) and the thin-to-thick filament ratio (5.2 +/- 0.2). In the coiled cell, the muscle lattice does not expand uniformly, as shown by the variability of myosin spacing, and the thin-to-thick filament ratio decreases. Both clockwise and counterclockwise coiling occur along the same fiber. The implications of these findings with respect to the structure of the contractile apparatus are discussed.

Cell junctions in the excitable epithelium of bioluminescent scales on a polynoid worm: a freeze-fracture and electrophysiological study.

The bioluminescent scales of the polynoid worm Acholoe are covered by a dorsal and ventral monolayer of epithelium. The luminous activity is intracellular and arises from the ventral epithelial cells, which are modified as photocytes. Photogenic and non-photogenic epithelial cells have been examined with regard to intercellular junctions and electrophysiological properties. Desmosomes, septate and gap junctions are described between all the epithelial cells. Lanthanum impregnation and freeze-fracture reveal that the septate junctions belong to the pleated-type found in molluscs, arthropods and other annelid tissues. Freeze-fractured gap junctions show polygonal arrays of membrane particles on the P face and complementary pits on the E face. Gap junctions are of the P type as reported in vertebrate, mollusc and some annelid tissues. Intracellular current passage also induces propagated non-overshooting action potentials in all the epithelial cells; in photocytes, an increase of injected current elicits another response which is a propagated 2-component overshooting action potential correlated with luminous activity. This study shows the coexistence of septate and gap junctions in a conducting and excitable invertebrate epithelium. The results are discussed in relation to the functional roles of intercellular junctions in invertebrate epithelia. It is concluded that the gap junctions found in this excitable epithelium represent the structural sites of the cell-to-cell propagation of action potentials.