Effects of changes in extracellular potassium,magnesium and calcium concentration on synaptic transmission in area CA1 and the dentate gyrus of rat hippocampal slices

The dependence of stimulus-induced synaptic potentials on changes of extracellular ionic concentrations of potassium ([K⁺]_o 3, 5, 8 mM), magnesium ([Mg²⁺]_o 2, 4, 8 mM) and calcium [Ca²⁺]_o (2 mM and continuous lowering by washing with Ca²⁺-free solutions) was investigated in area CA1 and dentate gyrus of rat hippocampal slices. Field potentials (fps), [K⁺]_o and [Ca²⁺]_o were measured with double-barreled ion selective/reference microelectrodes. Paired pulse stimulation (interval 50-ms) was applied either to the lateral perforant path or to the Schaffer collaterals. Elevation of [K⁺]_o from 5 to 8 mM and of [Mg²⁺]_o from 2 to 8 mM depressed the rise of excitatory postsynaptic potentials, as well as the amplitude of population spikes. With elevation of [K⁺]_o, the effect was stronger in the dentate gyrus, while with elevation of [Mg²⁺]_o, the reduction was more pronounced in area CA1. During washout of Ca²⁺, synaptic potentials became reduced and finally depressed. The [Ca²⁺]_o at which synaptic transmission was blocked increased with higher [Mg²⁺]_o and decreased with a change of [K⁺]_o from 3 to 5 mM, whereas with an elevation of [K⁺]_o from 5 to 8 mM, it rose in area CA1 but was reduced in dentate gyrus. All ionic changes also affected frequency habituation and potentiation in paired pulse experimentes. In dentate gyrus, frequency habituation was reversed to frequency potentiation with moderate lowering of [Ca²⁺]_o and with elevation of [Mg²⁺]_o and [K⁺]_o. In contrast, in area CA1 frequency potentiation was reduced upon elevation of [K⁺]_o.     

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

Paradox response of frog muscle membrane to changes in external potassium

Applying conventional microelectrode technique the anomalous behaviour of membrane potential in response to changes in [K⁺]_o was demonstrated in normal and cevadine-treated muscles bathed in Cl^–-free medium. Partial repolarization of the cevadine-depolarized membrane and reappearance of the slow membrane potential oscillation (SMPO) were induced by elevating [K⁺]_o from 2.5 mM to 10–20 mM. Both effects were reversed by return to 2.5 mM [K⁺]_o. The K-induced repolarization was markedly reduced by 20 mM Cs⁺, but not by 0.1 mM ouabain, 1 mM 4-aminopyridine, or 1 mM diethyl-pyrocarbonate. The elevation of [K⁺]_o failed to repolarize muscle fibers that had been depolarized only to a small extent. No K-induced repolarization has been observed in Cl^–-containing fluid. In cevadine-free experiments the omission of potassium from the extracellular space in Cl^–-free solution hyperpolarized some of the fibers, while depolarized others. Strong electrical stimuli applied in zero K-zero Cl solution turned all the fibers into depolarized state; on returning to 2.5 mM [K⁺]_o complete repolarization was achieved in most of the fibers. It has been concluded that the paradox response of the muscle membrane to changes in [K⁺]_o can be attributed to the K-dependent conductance changes of the inward rectifier K channel providing an explanation for the plateau-formation of SMPO and for the existence of two stable levels of membrane potential of the skeletal muscle bathed in Cl^–-free medium.     

Potassium-induced relaxation in isolated cerebral arteries contracted with prostaglandin F2α

Summary In helically cut strips of canine cerebral arteries exposed to 5.4 mM [K⁺]_o and contracted with prostaglandin F₂, the addition of K⁺ in concentrations ranging from 0.5–5 mM caused a dose-related relaxation. The relaxing effect of K⁺ was potentiated at reduced [K⁺]_o and suppressed at reduced [Na⁺]_o. Reduction of Cl^– from bathing media failed to alter the effect of K⁺. Removal of external Ca²⁺ markedly attenuated the K⁺-induced relaxation and increase in [Ca²⁺]_o also attenuated the relaxation. Similar relaxation was induced by K⁺ in cerebral arteries from other species including humans, puppies, cats and rabbits. The addition of K⁺ also elicited a relaxation in peripheral arteries, including coronary, femoral, mesenteric and renal, contracted with prostaglandin, but this relaxation was markedly less than in cerebral arteries. The content of Na⁺ in freshly excised cerebral arteries was significantly greater than that of peripheral arteries, while the content of K⁺ in these arteries was not significantly different.The present study provides further evidence to support the hypothesis that an electrogenic Na⁺ pump is involved in the genesis of K⁺-induced relaxation. The Na⁺ pump does not appear to be fully activated at normal [K⁺]_o of 5.4 mM in cerebral arteries.     

Stimulus-induced changes in extracellular Na+ and Cl− concentration in relation to changes in the size of the extracellular space

Summary Extracellular Na⁺- and Cl^–-concentrations ([Na⁺]_o, [Cl^–]_o) were recorded with ion-selective microelectrodes during repetitive stimulation and stimulus-induced self-sustained neuronal afterdischarges (SAD) in the sensorimotor cortex of cats. In all cortical layers [Na⁺]_o initially decreased by 4–7 mM. In depths of more than 600 m below the cortical surface such decreases usually turned into increases of 2–6 mM during the course of the SADs, whereas in superficial layers [Na⁺]_o never rose above its resting level. [Cl^–]_o always showed an increase in the course of the SADs often preceded by an initial small decrease. The average increase at a depth of 1,000 m was about 7 mM. [Cl^–]_o reached peak values at about the end of the ictal period, whereas [Na⁺]_o reached its maximum shortly after the end of the SAD, at times when [K⁺]_o was still elevated above the baseline concentration.These data indicate that the extracellular osmolarity can increase during SAD by up to 30 mM. Such an increase in osmolarity can be explained by an increase in the number of intracellular particles, caused by cleavage of larger molecules during enhanced metabolism. This could lead to cell-swelling due to passive water influx from the extracellular space (ES). However, the resulting reduction of the size of the ES is calculated to be less than 10% for an increase in intracellular osmolarity by 30 mOsm. This value is too small as compared to previously measured ES-reductions under similar conditions (i.e., 30% reduction at 1,000 m; Dietzel et al. 1980). Reductions of the size of the ES that accompany the observed changes in the ionic environment, are quantitatively explained on the basis of the extended glial buffering mechanism described in the preceding paper.Supported by the Deutsche Forschungsgemeinschaft, grant no. He 1128/2-2     

Membrane potential modulation of ionomycin-stimulated Ca<Superscript>2+</Superscript> entry via Ca<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange and SOC in rat submandibular acinar cells

Ionomycin (IM) at 5 μM mediates the Ca²⁺/H⁺ exchange, while IM at 1 μM activates the store-operated Ca²⁺ entry channels (SOCs). In this study, the effects of depolarization on both pathways were examined in rat submandibular acinar cells by increasing extracellular K⁺ concentration ([K⁺]_o). IM (5 μM, the Ca²⁺/H⁺ exchange) increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) to an extremely high value at 151 mM [K⁺]_o. However, with increasing [K⁺]_o, the rates of Ca²⁺ entry decreased in a linear relationship. The reversal potential (E _rev) for the Ca²⁺/H⁺ exchange was +93 mV, suggesting that IM (5 μM) exchanges 1 Ca²⁺ for 1 H⁺. Thus, depolarization decreases the Ca²⁺ influx via the Ca²⁺/H⁺ exchange because of its electrogenicity (1 Ca²⁺ for 1 H⁺). On the other hand, IM (1 μM, the SOCs) abolished an increase in [Ca²⁺]_i at 151 mM [K⁺]_o. With increasing [K⁺]_o, the rate of Ca²⁺ entry immediately decreased linearly. The E _rev for the SOC was +3.7 mV, suggesting that the SOCs are nonselective cation channels and less selective for Ca²⁺ over Na⁺ (P _Ca/P _Na = 8.2). Moreover, an increase in extracellular Ca²⁺ concentration (20 mM) enhanced the Ca²⁺ entry via the SOCs at 151 mM [K⁺]_o, suggesting depolarization does not inhibit the SOCs and decreases the driving force for the Ca²⁺ entry. This suggests that membrane potential changes induced by a secretory stimulation finely regulate the [Ca²⁺]_i via the SOCs in rat submandibular acinar cells. In conclusion, IM increases [Ca²⁺]_i via two pathways depending on its concentration, the exchange of 1 Ca²⁺ for 1 H⁺ at 5 μM and the SOCs at 1 μM.     

马桑内酯致痫大鼠海马神经细胞外Na+、K+活度的变化

目的和方法：应用Na⁺、K⁺选择性微电极检测马桑内酯致痫大鼠海马及海马脑片神经细胞外Na⁺、K⁺活度的改变。结果：海马内注射马桑内酯（5 μL,5×10^-4 mol/L）致痫大鼠30 s、1 min和2 min后,海马神经细胞外Na⁺活度分别低于对照组27.7 mmol/L、50.3 mmol/L和57.8 mmol/L,而K活度则分别高于对照组2.3 mmol/L、2.4 mmol/L和2.9 mmol/L（P<0.01）。3 min后,K⁺活度基本恢复至对照水平,而Na⁺活度仍持续低于对照水平（P<0.01）。海马脑片的实验结果与在体实验相似。结论：海马神经细胞处于癫痫状态时,存在Na⁺内流、K⁺外流现象。     

Effects of extracellular K+ and Ca2+ on membrane potential,contraction and86Rb+ efflux in guinea-pig mesotubarium

The effects of varying extracellular concentrations of K⁺ and Ca²⁺ [K⁺]_o and [Ca²⁺]_o on force development and membrane potential were investigated in the guinea-pig mesotubarium. At [K⁺]_o up to 40 mM, spontaneous action potentials were present, while higher [K⁺]_o gave sustained contractures at a stable membrane potential (−24 to −12 mV for [K⁺]_o from 60 to 120 mM). Tension decreased successively with increasing [K⁺]_o from 30 to 120 mM. The relaxing potency of the dihydropyridine Ca²⁺ antagonist, felodipine, increased as the membrane was depolarized with increasing [K⁺]_o and action potentials ceased. These results are compatible with the existence of Ca²⁺ channels showing voltage-dependent affinity with dihydrophyridines. Increasing [Ca²⁺]_o from 2.5 to 10 mM caused membrane hyperpolarization by about 11 mV and was accompanied by a lower frequency of spontaneous contractions and a longer duration of the relaxation between contractions.⁸⁶Rb⁺ efflux measurements in 60 mM K⁺ in the absence and presence of felodipine revealed a Ca²⁺-dependent component of the voltage-activated efflux. In normal solution (5.9 mM K⁺), efflux in the presence of felodipine was similar to the minimal value during normal spontaneous activity. The results indicate regulation of the permeability of K⁺ channels by the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and suggest participation of such channels in the generation of the regularly occurring bursts of action potentials characteristic of spontaneous activity in the mesotubarium.     

Activation of K+ and Cl− channels by Ca2+ and cyclic AMP in dissociated kidney epithelial (MDCK) cells

In dissociated MDCK cells, activators of the cyclic AMP system cause depolarization detectable by changes in fluorescence of the membrane potential sensitive dye bisoxonol. Addition of forskolin (60 M), vasopressin (2 M), 8-bromo-cyclic AMP (0.5 mM) or l-epinephrine (10 M) depolarized the cells substantially in low Cl^– (5 mM) but had little effect in high Cl^– (140 mM) solution. These results are consistent with cyclic AMP activation of Cl^– channels. The Ca²⁺-ionophore ionomycin (1 M) produced a rapid hyperpolarization in low and high Cl^– solutions, consistent with K⁺ channel opening. Using a clonal subline, MDCK-14, the magnitude of the ionomycin hyperpolarization was roughly proportional to the concomitant rise in [Ca²⁺]_i as measured with the intracellular Ca²⁺ probe indo-I. Both l-epinephrine and isoproterenol appeared to activate the Cl^– channels. However only l-epinephrine produced a [Ca²⁺]_i rise and a transient hyperpolarization (due to K⁺ channel opening), which preceeded the depolarization due to Cl^– channel opening. The l-epinephrine-induced [Ca²⁺]_i response of the heterogeneous MDCK cell population but not of the clonal subline MDCK-14 was inhibited by removal of extracellular Ca²⁺. In the latter only the slow secondary phase of the [Ca²⁺]_i rise was affected by Ca²⁺ removal. It is concluded that l-epinephrine activates K⁺ and Cl^– channels in a sequential manner in MDCK cells by Ca²⁺ and cAMP signals, presumably via - and -adrenergic receptors located on the same cell.Abbreviations MDCK cells Madin Darby Canine Kidney cells - [Ca²⁺]_i intracellular calcium concentration - [Cl^–]_i intracellular chloride concentration - [Cl^–]_o extracellular chloride concentration - [Na⁺+K⁺]_i intracellular concentration of Na⁺ and K⁺ - [Na⁺+K⁺]_o extracellular concentration of Na⁺ and K⁺ - E_M transmembrane potential - E_Cl chloride equilibrium potential - E_K potassium equilibrium potential - bis-oxonol [bis(1,3-diethylthio-barbiturate)] trimethine oxonol - DMSO dimethylsulfoxide - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis (-aminoethyl ether) N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid - NMG⁺ N-methylglucamine - RPMI medium Rosewell Park Memorial Institute medium     

Spontaneous Na+ and Ca2+ spike firing of cerebellar Purkinje neurons at high pressure

 The effects of high pressure (up to 10.1 MPa) on the spontaneous firing of Purkinje neurons in guinea-pig cerebellar slices were studied using the macropatch clamp technique. Pressure did not significantly alter the single somatic Na⁺ spike parameters or the frequency of regular Na⁺ spike firing. When Na⁺ currents were blocked by 0.5–1 μM tetrodotoxin (TTX), a pressure of 10.1 MPa slightly reduced the dendritic Ca²⁺ spike amplitude to 90.2±3.1% of its control value, and slowed its kinetics. The effects of pressure on the single Ca²⁺ spike were even less prominent when K⁺ currents were blocked by 5 mM 4-aminopyridine (4-AP). Pressure prolonged the active period of Ca²⁺ spike firing to 152.2±10.4% of the control value. Within the active period pressure increased the inter-spike interval to 164.9±8.7% and suppressed the typical firing of doublets. The latter changes were reversed by a high extracellular potassium concentration ([K⁺]_o) and 1 μM 4-AP, whereas in the presence of 5 mM 4-AP the pattern was insensitive to pressure. A high [Ca²⁺]_o reduced the firing frequency and suppressed doublet firing in a manner reminiscent of the pressure effect, but these changes could not be reversed by 4-AP. A low [Ca²⁺]_o slightly increased the firing of doublets. These results show that the single somatic Na⁺ spike is insensitive and the dendritic Ca²⁺ spike is only mildly sensitive to pressure. However, alterations in Ca²⁺ spike firing pattern suggest that modulation of dendritic K⁺ currents induce depression of dendritic excitability at pressure. Received: 19 May 1998 / Received after revision: 15 July 1998 / Accepted: 3 September 1998     

Changes in [Ca2+]o and [K+]o during repetitive electrical stimulation and during pentetrazol induced seizure activity in the sensorimotor cortex of cats

Changes in [Ca²⁺]_o and [K⁺]_o were measured in the sensorimotor cortex of cats during repetitive electrical stimulation and during pentetrazol induced epileptiform activity. Repetitive stimulation of the thalamic ventrobasal complex (VB) or of the cortical surface (CS) caused decreases in [Ca²⁺]_o by up to 0.45 mM and increases in [K⁺]_o by up to 7 mM. Maximum reductions of [Ca²⁺]_oΔ[Ca²⁺]_o were found in depths of 100 to 300 μm below cortical surface, while rises in [K⁺]_o were largest in depths of 600 to 1000 μm dependent on stimulation site. At depths below 700–900 μm increases in [K⁺]_o were often accompanied by rises in [Ca²⁺]_o of about 0.2 mM. Pentetrazol (PTZ) when injected at doses of 25 to 40 mg/kg body weight induced spontaneous seizure activity, which was in about 40% preceeded by a slight fall of baseline [Ca⁺]_o. Repetitive stimulation and spontaneous seizures resulted in Δ[Ca²⁺]_o of up to 0.6 mM, whereas rises in [K⁺]_o remained limited to a ‘ceiling level’ of about 10 mM. After PTZ application, peak Δ[Ca²⁺]_o were found at the same recording sites, but, in contrast to normal cortex, decreases in [Ca²⁺]_o were observed in all cortical layers. The enhanced Ca²⁺-signals after PTZ application and the observed reductions of [Ca²⁺]_o before seizure onset suggest that PTZ utilizes Ca²⁺-dependent mechanisms to initiate seizure activity.     

Cell-volume-dependent vascular smooth muscle contraction: role of Na+, K+, 2Cl− cotransport, intracellular Cl− and L-type Ca2+ channels

This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca²⁺ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca²⁺_o. Thirty minutes preincubation with bumetanide, a potent Na⁺,K⁺,Cl^– cotransport (NKCC) inhibitor, decreased Cl^–_i content, nifedipine-sensitive ⁴⁵Ca uptake and contractions triggered by modest depolarization ([K⁺]_o=36 mM). Elevation of [K⁺]_o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cl^–_i-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further.     

Effects of reduced electrochemical Na+ gradient on contractility in skeletal muscle: role of the Na+-K+ pump

 Continued excitation of skeletal muscle may induce a combination of a low extracellular Na⁺ concentration ([Na⁺]_o) and a high extracellular K⁺ concentration ([K⁺]_o) in the T-tubular lumen, which may contribute to fatigue. Here, we examine the role of the Na⁺-K⁺ pump in the maintenance of contractility in isolated rat soleus muscles when the Na⁺, K⁺ gradients have been altered. When [Na⁺]_o is lowered to 25 mM by substituting Na⁺ with choline, tetanic force is decreased to 30% of the control level after 60 min. Subsequent stimulation of the Na⁺-K⁺ pump with insulin or catecholamines induces a decrease in [Na⁺]_i and hyperpolarization. This is associated with a force recovery to 80–90% of the control level which can be abolished by ouabain. This force recovery depends on hyperpolarization and is correlated to the decrease in [Na⁺]_i (r = 0.93; P<0.001). The inhibitory effect of a low [Na⁺]_o on force development is considerably potentiated by increasing [K⁺]_o. Again, stimulation of the Na⁺-K⁺ pump leads to rapid force recovery. The Na⁺-K⁺ pump has a large potential for rapid compensation of the excitation-induced rundown of Na⁺, K⁺ gradients and contributes, via its electrogenic effect, to the membrane potential. We conclude that these actions of the Na⁺-K⁺ pump are essential for the maintenance of excitability and contractile force. Received: 19 December 1996 / Received after revision: 25 March 1997 / Accepted: 2 April 1997     

Intracellular Cl− concentration in striated intralobular ducts from rabbit mandibular salivary glands

Intralobular striated ducts have been isolated from rabbit mandibular salivary glands and maintained in primary culture for up to 2 days. Such ducts were loaded with the Cl^–-sensitive fluorescent dyeN-(ethoxycarbonylmethyl)-(6-methoxyquinolinium bromide) (MQAE) and intracellular Cl^– concentration ([Cl^–]_i monitored using a fluorescence microscope. Intracellular Cl^– could be rapidly and reversibly emptied from striated duct cells by replacing Cl^– in the superfusing solution with NO ₃ ^– . [Cl^–]_i could be lowered by removal of external Na⁺, exposure to 10 M amiloride or to 10 M 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS). Both amiloride and DIDS were able to inhibit the recovery of [Cl^–]_i after an initial exposure to Na⁺- or Cl^–-free solution. The amiloride derivatives, benzamil (2 M) and N-isobutyl-N-methylamiloride (MIBA), (10 M) also lowered [Cl^–]_i by similar amounts as 10 M amiloride. Varying external K⁺ concentration ([K⁺]_o) also affected [Cl^–]_i. Increasing [K⁺]_o increased [Cl^–]_i, but decreasing [K⁺]_o did not decrease [Cl^–]_i. Instead, [Cl^–]_i was also increased when [K⁺]_o was lowered below the control value. Bumetanide (0.1 mM) lowered [Cl^–]_i by only a small amount, while ouabain (1 mM) had no significant effect on [Cl^–]_i. These data are consistent with current models of electrolyte transport in salivary ducts which include Cl^– channels, Na⁺ channels, and Na⁺/H⁺ exchangers in the apical membrane. The effects of low [K⁺]_o can be interpreted in terms of a K⁺-dependent exit mechanism for Cl^–.     

Cytosolic free calcium in single microdissected rat cortical collecting tubules

Cytosolic free Ca²⁺ ([Ca²⁺]_i) was measured in single fragments of rat cortical collecting tubule (CCT) by using fura-2 and a tubule superfusion device. Under basal conditions, i.e. with 1 mM of external Ca²⁺ ([Ca²⁺]_o), the average steady state [Ca²⁺]_i was 179±16 nM (n=44 tubules). Random alterations of [Ca²⁺]_o between 0 mM and 4 mM led to corresponding variations in steady state [Ca²⁺]_i levels, which were linearly correlated with [Ca²⁺]_o (average slope 93±34 nM [Ca²⁺]_i per 1 mM [Ca²⁺]_o for six tubules). In contrast, [Ca²⁺]_i was little affected by decreasing external Na⁺ concentration. Cell membrane depolarization with 100 mM of external K⁺ induced a sustained drop in [Ca²⁺]_i (21% as an average). The data suggest that steady state [Ca²⁺]_i in CCT cells resulted from a non-saturable passive entry of calcium ions across cell membranes balanced with an active extrusion by calcium ATPase (pump and leak mechanism). The passive component cannot be accounted for either by Na⁺/Ca²⁺ exchangers nor by voltage-dependent calcium channels; it is best explained by the presence of voltage-independent calcium channels in cell membranes.     

Ionic mechanism of ouabain-induced swelling of leech Retzius neurons

By using electrophysiological and microfluorimetric methods, we found that leech Retzius neurons swell after inhibition of the Na⁺–K⁺ pump by the cardiac glycoside ouabain. To explore the mechanism of this swelling, we measured the effect of ouabain on [Na⁺]_i, [K⁺]_i, and [Cl⁻]_i, as well as on the membrane potential, by applying triple-barrelled ion-sensitive microelectrodes. As shown previously, ouabain induced a marked [Na⁺]_i increase, a [K⁺]_i decrease, and a membrane depolarization, and it also evoked an increase in [Cl⁻]_i. The analysis of the data revealed a net uptake of NaCl, which quantitatively explained the ouabain-induced cell swelling. In the absence of extracellular Na⁺ or Cl⁻, NaCl uptake was excluded, and the cell volume remained unaffected. Likewise, NaCl uptake and, hence, cell swelling did not occur when the Na⁺–K⁺ pump was inhibited by omitting bath K⁺. Also, in K⁺-free solution, [Na⁺]_i increased and [K⁺]_i dropped, but [Cl⁻]_i slightly decreased, and after an initial, small membrane depolarization, the cells hyperpolarized for a prolonged period. It is concluded that the ouabain-induced NaCl uptake is caused by the depolarization of the plasma membrane, which augments the inwardly directed electrochemical Cl⁻ gradient.     

Relation between extracellular [K+], membrane potential and contraction in rat soleus muscle: modulation by the Na+-K+ pump

An increased extracellular K⁺ concentration ([K⁺]₀) is thought to cause muscle fatigue. We studied the effects of increasing [K⁺]₀ from 4 mM to 8–14 mM on tetanic contractions in isolated bundles of fibres and whole soleus muscles from the rat. Whereas there was little depression of force at a [K⁺]₀ of 8–9 mM, a further small increase in [K⁺]₀ to 11–14 mM resulted in a large reduction of force. Tetanus depression at 11 mM [K⁺]_o was increased when using weaker stimulation pulses and decreased with stronger pulses. Whereas the tetanic force/resting membrane potential (E _M) relation showed only moderate force depression with depolarization from –74 to –62 mV, a large reduction of force occurred whenE _M fell to –53 mV. The implications of these relations to fatigue are discussed. Partial inhibition of the Na⁺-K⁺ pump with ouabain (10^–6 M) caused additional force loss at 11 mM [K⁺]₀. Salbutamol, insulin, or calcitonin gene-related peptide all stimulated the Na⁺-K⁺ pump in muscles exposed to 11 mM [K⁺ ₀] and induced an average 26–33% recovery of tetanic force. When using stimulation pulses of 0.1 ms, instead of the standard 1.0-ms pulses, force recovery with these agents was 41–44% which was significantly greater (P < 0.025). Only salbutamol caused any recovery ofE _M (1.3 mV). The observations suggest that the increased Na⁺ concentration difference across the sarcolemma, following Na⁺-K⁺ pump stimulation, has an important role in restoring excitability and force.     

Intracellular calcium in mammalian brain cells: fluorescence measurements with quin2

Summary Dispersed brain cells from 12–14 day old mouse embryos were loaded with the Ca²⁺-sensitive fluorescent probe, quin2 and shown to have a resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) of 158 nM (SE ± 5) in the presence of 1 mM [Ca²⁺]_o. When external [Ca²⁺] was raised from 0 to 1 mM there was an increase of [Ca²⁺]_i of 70 nM; with further additions of Ca to >10 mM [Ca²⁺]_o the level of [Ca²⁺]_i increased by <25 nM. Releasable intracellular Ca²⁺ stores, estimated from the increase in [Ca²⁺] produced by 4Br A23187 in the absence of extracellular Ca²⁺, were 24 fmol/10⁶ cells. A small increase in [Ca²⁺]_i could be produced by the mitochondrial inhibitor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). When extracellular K⁺ was raised by 10–20 mM, intracellular Ca²⁺ levels increased from 152 (SE ± 7) to 204 nM (SE ± 10). These K⁺-induced increases in [Ca²⁺]_i were blocked by verapamil, did not occur in the absence of extracellular Ca²⁺, and presumably reflect the activation of voltage-dependent Ca²⁺ channels. N-methyl-D-aspartic acid (NMDA) evoked an increase in [Ca²⁺]_i, while the kainate-like lathyrus sativus neurotoxin, L-3-oxalyl-amino-2aminopropionic acid (L-3,2-OAP) did not; this is consistent with previous observations of different and respectively Ca²⁺-dependent and -independent mechanisms of action of these excitatory amino acids.     

Osmolality- and Na+-dependent effects of hyperosmotic NaCl solution on contractile activity and Ca2+ cycling in rat ventricular myocytes

Hypertonic NaCl solutions have been used for small-volume resuscitation from hypovolemic shock. We sought to identify osmolality- and Na⁺-dependent components of the effects of the hyperosmotic NaCl solution (85 mOsm/kg increment) on contraction and cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in isolated rat ventricular myocytes. The biphasic change in contraction and Ca²⁺ transient amplitude (decrease followed by recovery) was accompanied by qualitatively similar changes in sarcoplasmic reticulum (SR) Ca²⁺ content and fractional release and was mimicked by isosmotic, equimolar increase in extracellular [Na⁺] ([Na⁺]_o). Raising osmolality with sucrose, however, augmented systolic [Ca²⁺]_i monotonically without change in SR parameters and markedly decreased contraction amplitude and diastolic cell length. Functional SR inhibition with thapsigargin abolished hyperosmolality effects on [Ca²⁺]_i. After 15-min perfusion, both hyperosmotic solutions slowed mechanical relaxation during twitches and [Ca²⁺]_i decline during caffeine-evoked transients, raised diastolic and systolic [Ca²⁺]_i, and depressed systolic contractile activity. These effects were greater with sucrose solution, and were not observed after isosmotic [Na⁺]_o increase. We conclude that under the present experimental conditions, transmembrane Na⁺ redistribution apparently plays an important role in determining changes in SR Ca²⁺ mobilization, which markedly affect contractile response to hyperosmotic NaCl solutions and attenuate the osmotically induced depression of contractile activity.     

Stimulation-induced calcium signalling and ion transport in rat pancreatic acini

In the present study we have characterized receptor-mediated Ca²⁺ signalling patterns as well as Ca²⁺-mediated ion transport mechanisms in collagenase isolated rat pancreatic acini. Measurements of the initial Ca²⁺ response to maximal carbachol stimulation revealed a rapid increase in [Ca²⁺]_i, which, in general, occurred synchronously throughout the cells. Less frequently, not all cells in the acinus responded to carbachol, but did respond to subsequent stimulation with bombesin, indicating that not all cells possess receptors for all the applied agonists. In view of the heterogeneity in the agonist-evoked Ca²⁺ responses, ionomycin was used to assess the role of Ca²⁺ in activating K⁺, Na⁺ and Cl^- transport mechanisms. Ionomycin induced a rise in [Ca²⁺]_i, thereby increasing Cl^- permeability as well as stimulating K⁺ efflux, probably through non-specific cation channels. However, the resting K⁺ efflux was insensitive to blockers of non-specific cation channels, indicating the existence of a selective resting K⁺ conductance. Ionomycin also stimulated influx of Na⁺, which in part was mediated by non-specific cation channels. The changes in ion fluxes measured in the present study revealed that when [Ca²⁺]_i is raised in rat pancreatic acini, they gain Na⁺ and Cl^- and lose K⁺, with non-specific cation channels being essential for this process.