Macroscopic Ca2+ -Na+ and K+ currents in single heart and aortic cells

Summary The whole-cell voltage clamp technique was used to study the slow inward currents and K⁺ outward currents in single heart cells of embryonic chick and in rabbit aortic cells. In single heart cells of 3-day-old chick embryo three types of slow inward Na⁺ currents were found. The kinetics and the pharmacology of the slow I_Na, were different from those of the slow Ica in older embryos. Two types of slow inward currents were found in aortic single cells of rabbit; angiotensin 11 increased the sustained type and d-cAMP and d-cGMP decreased the slow transient component. Two types of outward K⁺ currents were found in both aortic and heart cells. Single channel analysis demonstrated the presence of a high single K⁺ channel conductance in aortic cells. In cardiac and vascular smooth muscles, slow inward currents do share some pharmacological properties, although the regulation of these channels by cyclic nucleotides and several drugs seems to be different.     

Macroscopic Ca2+ -Na+ and K+ currents in single heart and aortic cells

The whole-cell voltage clamp technique was used to study the slow inward currents and K+ outward currents in single heart cells of embryonic chick and in rabbit aortic cells. In single heart cells of 3-day-old chick embryo three types of slow inward Na+ currents were found. The kinetics and the pharmacology of the slow INa were different from those of the slow ICa in older embryos. Two types of slow inward currents were found in aortic single cells of rabbit; angiotensin II increased the sustained type and d-cAMP and d-cGMP decreased the slow transient component. Two types of outward K+ currents were found in both aortic and heart cells. Single channel analysis demonstrated the presence of a high single K+ channel conductance in aortic cells. In cardiac and vascular smooth muscles, slow inward currents do share some pharmacological properties, although the regulation of these channels by cyclic nucleotides and several drugs seems to be different.     

Voltage-activated Ca2+ currents in insulin-secreting cells

Membrane voltage and voltage-clamped membrane currents have been investigated with the whole-cell patch clamp method in the insulin-secreting cell line RINm5F. The mean resting membrane potential of RINm5F cells was found to be -52 mV. Overshooting spike potentials could be evoked by depolarising voltage steps in the absence of a secretagogue. Inward membrane currents evoked by depolarising voltage steps were dependent upon extracellular Ca2+ and blocked by Co2+, nifedipine and verapamil. Outward membrane currents which were evoked by depolarising voltage steps to positive membrane potentials were reduced when Ca2+ entry was prevented. It is concluded that the voltage-activated Ca2+ currents underlie the voltage-activated spike potentials recorded from insulin-secreting cells.     

Modulation of cytosolic and nuclear Ca2+ and Na+ transport by taurine in heart cells

Brain membranes contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. We have previously postulated: (a) that this kind of tubulin is a peripheral membrane protein that arises from microtubules that in vivo interact with membranes and (b) that the hydrophobic behaviour is due to the interaction of tubulin with a membrane component. Here we report the in vitro conversion of hydrophilic into hydrophobic tubulin by incubating microtubule associated proteins (MAPs) free taxol-stabilized microtubules with Triton X-100 solubilized membranes. After incubation, the microtubules were sedimented, depolymerized and subjected to partition into Triton X-114. Part of the tubulin was isolated in the detergent phase and contained, as observed in native membranes, a high proportion of the acetylated isotype. Because of the high proportion of acetylated tubulin the in vitro conversion resembles the in vivo interaction. Electrophoretic analysis of the detergent phase shows, besides tubulin, two major protein bands of 29 and 100 kDa molecular mass. The ability of the solubilized membranes to convert hydrophilic into hydrophobic tubulin is greatly diminished if the solubilized membrane preparation is preincubated in the presence of trypsin or heated at 90°C for 5 min, indicating that the membrane component that confers the hydrophobic behaviour to tubulin is of proteinaceous nature.     

Voltage-gated Ca2+ currents in rat gastric enterochromaffin-like cells

Enterochromaffin-like (ECL) cells are histamine-containingendocrine cells in the gastric mucosa that maintain a negative membranepotential of about 50 mV, largely due to voltage-gated K⁺ currents [D. F. Loo, G. Sachs, and C. Prinz. Am. J. Physiol. 270 (Gastrointest Liver Physiol. 33):G739-G745, 1996]. The current study investigated thepresence of voltage-gated Ca²⁺channels in single ECL cells. ECL cells were isolated from rat fundicmucosa by elutriation, density gradient centrifugation, and primaryculture to a purity >90%. Voltage-gatedCa²⁺ currents were measured insingle ECL cells using the whole cell configuration of the patch-clamptechnique. Depolarization-activated currents were recorded in thepresence of Na⁺ orK⁺ blocking solutions and additionof 20 mM extracellular Ca²⁺. ECLcells showed inward currents in response to voltage steps that wereactivated at a test potential of around 20 mV with maximalinward currents observed at +20 mV and 20 mM extracellular Ca²⁺. The inactivation rate of thecurrent decreased with increasingly negative holding potentials and wastotally abolished at a holding potential of 30 mV. Addition ofextracellular 20 mM Ba²⁺ insteadof 20 mM Ca²⁺ increased thedepolarization-induced current and decreased the inactivation rate. Theinward current was fully inhibited by the specific L-typeCa²⁺ channel inhibitor verapamil(0.2 mM) and was augmented by the L-typeCa²⁺ channel activator BAY K 8644 (0.07 mM). We conclude that depolarization activateshigh-voltage-activated Ca²⁺channels in ECL cells. Activation characteristics,Ba²⁺ effects, and pharmacologicalresults imply the presence of L-type Ca²⁺ channels, whereasinactivation kinetics suggest the presence of additional N-typechannels in rat gastric ECL cells.

     

The co-presence of Na+/Ca2+-K+ exchanger and Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

We have previously shown that there is high Na(+)/Ca(2+) exchange (NCX) activity in bovine adrenal chromaffin cells. In this study, by monitoring the [Ca(2+)](i) change in single cells and in a population of chromaffin cells, when the reverse mode of exchanger activity has been initiated, we have shown that the NCX activity is enhanced by K(+). The K(+)-enhanced activity accounted for a significant proportion of the Na(+)-dependent Ca(2+) uptake activity in the chromaffin cells. The results support the hypothesis that both NCX and Na(+)/Ca(2+)-K(+) exchanger (NCKX) are co-present in chromaffin cells. The expression of NCKX in chromaffin cells was further confirmed using PCR and northern blotting. In addition to the plasma membrane, the exchanger activity, measured by Na(+)-dependent (45)Ca(2+) uptake, was also present in membrane isolated from the chromaffin granules enriched fraction and the mitochondria enriched fraction. The results support that both NCX and NCKX are present in bovine chromaffin cells and that the regulation of [Ca(2+)](i) is probably more efficient with the participation of NCKX.     

Ca2+ currents in cerebral artery smooth muscle cells of rat at physiological Ca2+ concentrations

Single Ca2+ channel and whole cell currents were measured in smooth muscle cells dissociated from resistance-sized (100-microns diameter) rat cerebral arteries. We sought to quantify the magnitude of Ca2+ channel currents and activity under the putative physiological conditions of these cells: 2 mM [Ca2+]o, steady depolarizations to potentials between -50 and -20 mV, and (where possible) without extrinsic channel agonists. Single Ca2+ channel conductance was measured over a broad range of Ca2+ concentrations (0.5-80 mM). The saturating conductance ranged from 1.5 pS at 0.5 mM to 7.8 pS at 80 mM, with a value of 3.5 pS at 2 mM Ca (unitary currents of 0.18 pA at -40 mV). Both single channel and whole cell Ca2+ currents were measured during pulses and at steady holding potentials. Ca2+ channel open probability and the lower limit for the total number of channels per cell were estimated by dividing the whole-cell Ca2+ currents by the single channel current. We estimate that an average cell has at least 5,000 functional channels with open probabilities of 3.4 x 10(-4) and 2 x 10(-3) at -40 and -20 mV, respectively. An average of 1-10 (-40 mV and -20 mV, respectively) Ca2+ channels are thus open at physiological potentials, carrying approximately 0.5 pA steady Ca2+ current at -30 mV. We also observed a very slow reduction in open probability during steady test potentials when compared with peak pulse responses. This 4- 10-fold reduction in activity could not be accounted for by the channel's normal inactivation at our recording potentials between -50 and -20 mV, implying that an additional slow inactivation process may be important in regulating Ca2+ channel activity during steady depolarization.     

Inhibitory effect of n-3 fish oil fatty acids on cardiac Na+/Ca2+ exchange currents in HEK293t cells

Abnormal activity of the cardiac Na+/Ca2+ exchanger (NCX1) can affect intracellular Ca2+ homeostasis and cause arrhythmias. The n-3 polyunsaturated fatty acids (PUFAs), however, may prevent arrhythmias. To test the effect of PUFAs on the cardiac NCX1 current (I(NCX1)), the canine NCX1 cDNA was expressed in human embryonic kidney (HEK293t) cells. The average density of I(NCX1) was 10.9+/-2.6 pA/pF (n=44) in NCX1-transfected cells and eicosapentaenoic acid (EPA, C20:5n-3) significantly inhibited I(NCX1) The suppression of I(NCX1) by EPA was concentration-dependent with an IC50 of 0.82+/-0.27 microM. EPA had a similar effect on outward or inward I(NCX1). Docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (AA, C20:4n-6) also significantly inhibited I(NCX1), whereas the saturated fatty acid, stearic acid (SA, C18:0), did not. Our data demonstrate that the n-3 PUFAs significantly suppress cardiac I(NCX1), which is probably one of their protective effects against lethal arrhythmias.     

Na+/Ca2+ Exchange and Cellular Ca2+ Homeostasis

The Na⁺/Ca²⁺ exchange system is the primary Ca²⁺ efflux mechanism in cardiac myocytes, and plays an important role in controlling the force of cardiac contraction. The exchanger protein contains 11 transmembrane segments plus a large hydrophilic domain between the 5th and 6th transmembrane segments; the transmembrane regions are reponsible for mediating ion translocation while the hydrophilic domain is responsible for regulation of activity. Exchange activity is regulated in vitro by interconversions between an active state and either of two inactive states. High concentrations of cytosolic Na⁺ or the absence of cytosolic Ca²⁺ promote the formation of the inactive states; phosphatidylinositol-(4,5)bisphosphate (or other negatively charged phospholipids) and cytosolic Ca²⁺ counteract the inactivation process. The importance of these mechanisms in regulating exchange activity under normal physiological conditions is uncertain. Exchanger function is also dependent upon cytoskeletal interactions, and the exchanger's location with respect to intracellular Ca²⁺-sequestering organelles. An understanding of the exchanger's function in normal cell physiology will require more detailed information on the proximity of the exchanger and other Ca²⁺-transporting proteins, their interactions with the cytoskeleton, and local concentrations of anionic phospholipids and transported ions.     

The oppositely directed Ca2+ and Na+ transmembrane transport in algal cells

Summary The countertransport of Ca²⁺ and Na⁺ across the membranes of the unicellular fresh-water algaChlamydomonas reinhardtii CW-15 and twoDunaliella species differing in salt tolerance was studied. All algae used are devoid of cell walls. The calcium uptake by twoDunaliella species depended markedly on the intracellular sodium concentration. This calcium uptake was accompanied by Na⁺ release. For 15 and 30 s after artificial gradient formation (Na_int ⁺ greater than Na_ext ⁺) the ratio of released Na⁺ to absorbed Ca²⁺ was 31 and 41, respectively. For the extremely halotolerantD. salina, the apparent Michaelis constant of the Ca²⁺ uptake was 33 M, and for the marine halotolerant algaD. maritima, it was equal to 400 M, presuming more efficient Na⁺-for-Ca²⁺ exchange inD. salina cells. Ouabain, an inhibitor of Na⁺/K⁺-ATPase, suppressed Na⁺ transfer by 25%, whereas the agents blocking Ca²⁺-channels did not affect the transport of Ca²⁺ and Na⁺. The oppositely directed transmembrane Ca²⁺ and Na⁺ transfer was shown to depend on the external concentrations of Na⁺ and H⁺. In the fresh-water algaC. reinhardtii CW-15 (Na_ext ⁺ greater than Na_int ⁺), the direction of Ca²⁺ and Na⁺ fluxes across the plasma membrane was opposite to those described for Dunaliella cells. The results obtained point to the ability of the Na⁺-Ca²⁺ exchanger function in plasma membranes of algal cells.