Epidermal growth factor stimulates Ca2+ uptake of human erythrocytes

To examine the functional significance of epidermal growth factor (EGF) binding sites present on the human erythrocyte membrane [Engelmann et al. (1992) Am J Hematol 39:239–241], the effect of EGF on ⁴⁵Ca²⁺ uptake and on ²²Na⁺ efflux from these cells has been studied. In all cases media contained 1.25 mM Ca²⁺, whereas Na⁺ and K⁺ were varied. In 140 mM Na⁺/5 mM K⁺ medium EGF (250 ng/ml) stimulated ⁴⁵Ca²⁺ uptake by 50%–90% in quin-2-loaded cells, and by up to threefold in untreated cells. Increasing extracellular K⁺ up to 75 mM at the expense of extracellular Na²⁺ stimulated the EGF-induced ⁴⁵Ca²⁺ uptake by about twofold compared to 145 mM Na⁺ medium both in quin-2-loaded and in untreated cells. In 145 mM K⁺ medium, however, no EGF-induced ⁴⁵Ca²⁺ uptake was detectable in quin-2-loaded cells, while in untreated cells Ca²⁺ entry was stimulated twofold by EGF. After increasing intracellular Na⁺ from 6 mmol/l cells to 18 mmol/l cells in untreated cells suspended in 145 mM K⁺ medium, ⁴⁵Ca²⁺ uptake induced by EGF gradually increased. In contrast, in 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, ⁴⁵Ca²⁺ uptake accelerated by EGF was largely unaffected by a modified red cell Na⁺ content. When ²²Na-loaded untreated red cells were suspended in 145 mM K⁺ medium EGF stimulated red cell ²²Na⁺ efflux by more than threefold. In 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, no ²²Na⁺ efflux induced by the growth factor was evident. The results are consistent with the idea that EGF stimulates (at least) two components of ⁴⁵Ca²⁺ uptake in human erythrocytes. One of the two is unmasked in 145 mM K⁺ medium, inhibited by quin-2 loading, accelerated by intracellular Na⁺ and appears to involve reversed Na⁺/Ca²⁺ exchange.     

Na+ currents in cultured mouse pancreatic B-cells

Pancreatic B-cells, kept in culture for 1–4 days, were studied in the whole-cell, cell-attached and outside-out modes of the patch clamp technique. B-cells were identified by the appearance of electrical activity in the cell-attached mode when the bath glucose was raised from 3 to 20 mM. In whole-cell, 80% of these cells showed a transient inward Na⁺ current, when depolarizing pulses were preceded by holding potentials, or prepulses to potentials more negative than –80 mV. The midpoint (E _h) of the inactivation curve (h ) was at –109 mV in 2.6 mM Ca²⁺, 1.2 mM Mg²⁺ and –120 mV in 0.2 mM Ca²⁺, 3.6 mM Mg²⁺. In 2.6 mM Ca²⁺, inactivation was fully removed atE<–150 mV. Na⁺ currents activated atE>–60 mV and were largest at around –10 mV (120 mM Na⁺). The kinetic parameters of activation (t _p) and inactivation ()_h were similar to those of other mammalian Na⁺ channels. Unitary currents with an amplitude of approximately 1 pA at –30 mV (140 mM Na⁺) with a similar voltage-dependence and time-course of mean current were recorded from outside-out patches. The results show that B-cells have a voltage-dependent Na⁺ current which, owing to the voltage-dependence of inactivation, is unlikely to play a major role in glucose-induced electrical activity.     

Effects of magnesium on45Ca uptake and release at different sites in rabbit aortic smooth muscle

Effects of 1.5 mM Mg²⁺ on muscle tension and on⁴⁵Ca uptake and release at different sites in the rabbit aortic media-intimal layer were investigated. The sustained contraction induced by either 10^?6 M norepinephrine (NE) or 60 mM K⁺ was not affected by 1.5 mM Mg²⁺ in the presence of 1.5 mM Ca²⁺. However, the contractions elicited with NE or K⁺ in 0.03 mM Ca²⁺-containing solution were inhibited by 1.5 mM Mg²⁺ by 67% and 27%, respectively. Total⁴⁵Ca uptake measured in the presence of either 1.5 mM or 0.03 mM Ca²⁺ was not affected by 1.5 mM Mg²⁺. The rate of residual⁴⁵Ca uptake (⁴⁵Ca uptake followed by a wash in La³⁺-containing solution at low temperature) measured in the presence of 1.5 mM Ca²⁺ was slightly lower in the presence of 1.5 mM Mg²⁺. However, the increase in rate of residual⁴⁵Ca uptake induced by NE or the net increase in the residual⁴⁵Ca uptake induced by K⁺ was not decreased by 1.5 mM Mg²⁺. The residual⁴⁵Ca uptake measured in the presence of 0.03 mM Ca²⁺ was reduced to 64% and 24% of controls by addition of 1.5 mM Mg²⁺ or Sr²⁺, respectively. A part of the residual⁴⁵Ca was released by NE. Uptake of⁴⁵Ca at this NE-affected Ca²⁺ site did not take place in the presence of 1.5 mM Mg²⁺ when the Ca²⁺ concentration of the medium was 0.03 mM. However, this⁴⁵Ca uptake component was only partially inhibited when the Ca²⁺ concentration of the medium was 1.5 mM. The NE-induced increase in⁴⁵Ca efflux was not inhibited by 1.5 mM Mg²⁺. From these results, Mg²⁺ appears to be a weak antagonist for both Ca²⁺ entry into the vascular smooth muscle cell and Ca²⁺ binding at a high affinity intracellular site.     

ATP suppresses activity of Ca2+ -activated K+ channels by Ca2+ chelation

Ca²⁺-activated maxi K⁺ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pflügers Arch 417:517, 1990), channel activity (NP _o) was stimulated by 3 M [Ca²⁺]_c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na₂ATP. The following results suggest that suppression of NP _o by Na₂ATP is due to Ca²⁺ chelation and hence reduction of [Ca²⁺]_c and reduced Ca²⁺ activation of the channel. The effect was absent when Mg ATP was used instead of Na₂ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca²⁺]_c was buffered with 10 mM HDTA (apparent pK _Ca 5.58) instead of EGTA (pK _Ca 6.8). A Ca²⁺-sensitive electrode system indicated that 1 mM Na₂ATP reduced [Ca²⁺]_c in 1 mM Ca-EGTA from 3 M to 1.4 M. Na₂ATP, Na₂GTP, Li₄AMP-PNP and NaADP reduced measured [Ca²⁺]_c in parallel with their suppression of NP _o. After the Na₂ATP-induced reduction of [Ca²⁺]_c was re-adjusted by adding either CaCl₂ or MgCl₂, the effect of Na₂ATP on NP _o disappeared. In vivo, intracellular [Mg²⁺] exceeds free [ATP^4–], hence ATP modulation of maxi K⁺ channels due to Ca²⁺ chelation is without biological relevance.     

A Na+-activated K+ current (I K,Na) is present in guinea-pig but not rat ventricular myocytes

 The effects of removing extracellular Ca²⁺ and Mg²⁺ on the membrane potential, membrane current and intracellular Na⁺ activity (a ⁱ _Na) were investigated in guinea-pig and rat ventricular myocytes. Membrane potential was recorded with a patch pipette and whole-cell membrane currents using a single-electrode voltage clamp. Both guinea-pig and rat cells depolarize when the bathing Ca²⁺ and Mg²⁺ are removed and the steady-state a ⁱ _Na increases rapidly from a resting value of 6.4± 0.6 mM to 33±3.8 mM in guinea-pig (n=9) and from 8.9±0.8 mM to 29.3±3.0 mM (n=5) in rat ventricular myocytes. Guinea-pig myocytes partially repolarized when, in addition to removal of the bathing Ca²⁺ and Mg²⁺, K⁺ was also removed, however rat cells remained depolarized. A large diltiazem-sensitive inward current was recorded in guinea-pig and rat myocytes, voltage-clamped at –20 mV, when the bathing divalent cations were removed. When the bathing K⁺ was removed after Ca²⁺ and Mg²⁺ depletion, a large outward K⁺ current developed in guinea-pig, but not in rat myocytes. This current had a reversal potential of –80±0.7 mV and was not inhibited by high Mg²⁺ or glybenclamide indicating that it is not due to activation of non-selective cation or adenosine triphosphate (ATP)-sensitive K channels. The current was not activated when Li⁺ replaced the bathing Na⁺ and was blocked by R-56865, suggesting that it was due to the activation of K_Na channels. Received: 15 October 1998 / Received after revision: 22 January 1999 / Accepted: 5 February 1999     

Contraction of epithelial (MDCK) cells in response to low extracellular calcium is dependent on extracellular sodium

Like other cells of epithelial origin, MDCK cells respond with a reversible structural transformation to a diminution in the concentration of extracellular Ca²⁺. Upon deprivation of Ca²⁺ in the medium the cells undergo an active contraction mediated by the actin-myosin cytoskeleton, in parallel to detachment of the intercellular contacts and appearance of free spaces in the epithelium or monolayer (Castillo et al., 1998). We now present results indicating that the decrease of external Ca²⁺ plays an indirect and non-specific role in activating contraction, probably by allowing an influx of Na⁺. The omission of external Ca²⁺ had no effect when it was replaced by Mg²⁺, Ba²⁺ or Hg²⁺, and the addition of any of these divalent cations induced relaxation of cells previously contracted by exposure to low Ca²⁺. A null or weak response was observed also when Ca²⁺ was lowered in a solution where Na⁺ was replaced by choline or in the presence of amiloride (30 M), which reduces the permeability of the plasma membrane to Na⁺. Restitution of Na⁺ or removal of amiloride were followed by contraction in the same cultures. Li⁺ proved an able substitute of Na⁺ as requisite for cell contraction in response to Ca²⁺ depletion. Monensin (0.1 mM) –an ionophore selective for Na⁺– and to a lesser extent ouabain (0.1 mM) –an inhibitor of Na⁺ extrusion across the plasma membrane– , both stimulated contraction in the presence of the normal level of external Ca²⁺. Decreasing by half the normal concentration of external K⁺ facilitated cell contraction, but typical responses were observed when K⁺ was increased to 40 mM by partial substitution for Na⁺. These findings attest that cell contraction in response to low Ca²⁺ is likely due to an increase in the permeability of the plasma membrane to Na⁺, though not to membrane depolarization as such. Evidences from other motile systems suggest that Na⁺ influx might in turn cause an elevation of cytoplasmic Ca²⁺, which activates the actin-myosin cytoskeleton.     

The effect of high K+ depolarization and verapamil on45Ca2+ fluxes in the canine myocardium

The effects of high K⁺ depolarization and verapamil on Ca²⁺ uptake and the total intracellular Ca²⁺ content of canine ventricular muscle strips (0.5 mm thick) were investigated. High K⁺ (96 mM) increased Ca²⁺ uptake above control and maintained this enhanced uptake throughout a 90 second measuring period. Verapamil (5×10^–6M) significantly (p<0.05) inhibited this high K⁺ stimulated uptake. However, verapamil (5×10^–6M) also had a direct effect on Ca²⁺ fluxes, causing a significant increase in both Ca²⁺ uptake (p<0.001) and total intracellular Ca²⁺ content (p<0.001) in the resting tissue. Therefore, verapamil's apparent inhibition of high K⁺ stimulated Ca²⁺ uptake may have resulted from some mechanism other than Ca²⁺ channel blockade.     

Contraluminal para-aminohippurate (PAH) transport in the proximal tubule of the rat kidney

In order to study the characteristics of contraluminal para-aminohippurate transport into proximal tubular cells the stopped flow capillary perfusion method was applied. The disappearance of³H-paraaminohippurate from the capillary perfusate at different concentrations and contact times was measured and saturation type behaviour was found with aK _m of 0.08±0.01 (SE) mmol/l,J _max of 1.1±0.1 pmol·s^–1·cm^–1 andr, the final extracellular/intracellular distribution ratio of 0.93±0.03. Omission of Na⁺ from the capillary test perfusate caused a small reduction of contraluminal PAH uptake at small transport rates (0.1 mmol/l PAH in the test perfusate) but not at high transport rates (1.0 mmol/l PAH in the test perfusate). Change of K⁺ between 0 and 40 mmol/l and pH between 6.0 and 8.0 did not influence contraluminal PAH uptake. Isotonic replacement of chloride by gluconate, nitrate, sulfate, phosphate, methanesulfonate or increase in bicarbonate to 50 mmol/l did not influence PAH uptake at small transport rates. But isotonic sulfate and phosphate, as well as 50 mmol/l HCO ₃ ^– and 25 mmol/l Hepes in isotonic solutions reduced PAH uptake at high transport rates. Addition of 5 mmol/l Ca²⁺, Mg²⁺, Mn²⁺, Ba²⁺, Cd²⁺ to isotonic Na⁺-gluconate solution did not influence PAH uptake except for Mg²⁺ and Mn²⁺ which inhibited uptake at small transport rates only. Preperfusion of the peritubular capillaries with rat serum, Na⁺ gluconate (Ca²⁺-+Mg²⁺-free), Na⁺ gluconate (Ca²⁺-+Mg²⁺-free) plus 10 mmol/l lactate or pyruvate or 0.1 mmol/l 2-oxoglutarate did not influence PAH uptake at small PAH transport rates, but inhibited at high transport rates. Preperfusion of the capillaries for 10 s with Na⁺-, Ca²⁺- and Mg²⁺-free solutions reduced PAH uptake in the presence of Na⁺ at both transport rates. A second 10 s preperfusion — after the first 10 s Na⁺-, Ca²⁺-, Mg²⁺-free preperfusion — with serum or solutions which contained Na⁺ and Ca²⁺ or Mg²⁺ restored the PAH fluxes to control values. The data are compatible with the hypothesis that contraluminal PAH uptake occurs by a saturable transport mechanism in exchange for other intracellular anions rather than in cotransport with Na⁺ ions. It was, however, not possible to identify the type of counteranions involved. The large effect of cation replacement on para-aminohippurate transport, which was reported in many previous studies with kidney slices, is not a direct effect on the para-aminohippurate transporter, but is rather caused indirectly via cell metabolism and/or changed ion gradients.     

Modulation of phospholipase A2 activity in human synovial fluid by cations

Cell-free, Ca²⁺ dependent phospholipase A₂ activity (PLA₂) was measured in human synovial fluid of patients with various kinds of arthritis using [1–¹⁴C] oleate-labelled autoclaved Escherichia coli as substrate. PLA₂ activity at pH 7.0 and with 5 mM added Ca²⁺ was stimulated and then inhibited in a dose-dependent fashion by NaCl; maximal stimulation of 8.8 fold was found at 150 mM Na⁺. Similar effects were obtained with K⁺ Li⁺ and Ru⁺. In the absence of added Na⁺, PLA₂ activity was maximal with 25 mM Ca²⁺ (145 nmols/hr/mg), but in the presence of 150 mM Na⁺, activity was maximal with 4 mM Ca²⁺ (415 nmols/hr/mg). PLA₂ activity was optimal between pH 6.5–8.0 in presence of 150 mM Na⁺¹ and 4 mM Ca²⁺. There was no significant difference between PLA₂ activity in synovial fluids from rhematoid and other types of arthritis. Neutral active, Ca²⁺-dependent PLA₂ activity in acid extracts of human platelets, plasma, polymorphonuclear leukocytes and synovial fluid varied in response to added Na⁺. In presence of 150 mM added Na⁺ and 5 mM PLA₂ activity in human synovial fluid was inhibited by all multivalent cations tested. In the absence of Na⁺, Cu²⁺ and Mg²⁺ stimulated PLA₂ activity in a dose dependent fashion; whereas, Fe²⁺, Fe³⁺ and Al³⁺ were inhibitory. The extent of stimulation by Mg²⁺ was inversely related to the concentration of added Ca²⁺.     

Effects of glucagon on Na+, Cl−, K+, Mg2+ and Ca2+ transports in cortical and medullary thick ascending limbs of mouse kidney

The effects of glucagon on transepithelial Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ net fluxes were investigated in isolated perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. Transepithelial ion net fluxes (J _Na ⁺,J _Cl ^–,J _K ⁺,J _Ca ²⁺,J _Mg ²⁺) were determined by electron probe analysis of the collected tubular fluid. Simultaneously the transepithelial voltage (PD_te) and the transepithelial resistance (R _te) were recorded. In cTAL-segments (n=8), glucagon (1.2×10^–8 mol · l^–1) stimulated significantly the reabsorption of Na⁺, Cl^–, Ca²⁺ and Mg²⁺J _Na ⁺ increased from 204±20 to 228±23 pmol · min^–1 · mm^–1,J _Cl ^– from 203±18 to 234±21 pmol · min^–1 · mm^–1,J _Ca ²⁺ from 0.52±0.13 to 1.34±0.30 pmol · min^–1 · mm^–1 andJ _Mg ²⁺ from 0.51±0.08 to 0.84±0.08 pmol · min^–1 · mm^–1.J _K+ remained unchanged: 3.2±1.3 versus 4.0±1.9 pmol · min^–1 · mm^–1. Neither PD_te (16.3±1.5 versus 15.9±1.4 mV) norR _te (22.5±3.0 versus 20.3±2.6 cm²) were changed significantly by glucagon. However, in the post-experimental periods a significant decrease in PD_te and increase inR _te were noted. In mTAL-segments (n=9), Mg²⁺ and Ca²⁺ transports were close to zero and glucagon elicited no significant effect. The reabsorptions of Na⁺ and Cl^–, however, were strongly stimulated:J _Na ⁺ increased from 153±17 to 226±30 pmol · min^–1 · mm^–1 andJ _Cl ^– from 151±23 to 243±30 pmol · min^–1 · mm^–1. The rise in NaCl transport was accompanied by an increase in PD_te from 10.3±1.1 to 12.3±1.2 mV and a decrease inR _te from 19.1±2.7 to 17.8±2.0 cm². No net K⁺ movement was detectable either in the absence or in the presence of glucagon. A micropuncture study carried out in hormone-deprived rats indicated that glucagon stimulates Na⁺, Cl^–, K⁺, Mg²⁺ and Ca²⁺ reabsorptions in the loop of Henle. In conclusion our data demonstrate that glucagon stimulates NaCl reabsorption in the mTAL segment and to a lesser extent in the cTAL segment whereas it stimulates Ca²⁺ and Mg²⁺ reabsorptions only in the cortical part of the thick ascending limb of the mouse nephron. These data are in good agreement with, and extend, those obtained in vivo on the rat with the hormone-deprived model.This study was supported by the Commission des Communautés Européennes, Grant no. ST 23, 00951F (CD) and by Wissenschaftsausschuß der Nato über den DAAD     

Role of Na+/Ca2+ exchange in transcellular Ca2+ transport across primary cultures of rabbit kidney collecting system

Cells from connecting tubule and cortical collecting duct of rabbit kidney were isolated by immunodissection with mAb R2G9 and cultured on permeable filters. Confluent monolayers developed an amiloride-sensitive transepithelial potential difference of –50±1 mV (lumen negative) and a transepithelial resistance of 507±18 cm². Transepithelial Ca²⁺ transport increased dose-dependently with apical [Ca²⁺] and, in solutions containing 1 mM Ca²⁺, the active transcellular Ca²⁺ transport rate was 92±2 nmol h^–1 cm^–2. Transcellular Ca²⁺ transport was dependent on basolateral Na⁺ (Na _b ⁺ ). Isoosmotic substitution of Na _b ⁺ for N-methylglucamine resulted in a concentration-dependent decrease in Ca²⁺ absorption, with maximal inhibition of 67±5%. A Hill plot of the Na⁺-dependence yielded a coefficient of 1.9±0.4, indicating more than one Na⁺ site on a Na⁺-dependent Ca²⁺ transport system. In addition, the absence of Ca _b ²⁺ resulted in a significant increase in Ca²⁺ transport both in the presence and absence of Na _b ⁺ . Added basolaterally, ouabain (0.1 mM) inhibited Ca²⁺ transport to the same extent as did Na⁺-free solutions, while bepridil (0.1 mM), an inhibitor of Na⁺/Ca²⁺ exchange, reduced Ca²⁺ transport by 32±6%. Methoxyverapamil, felodipine, flunarizine and diltiazem (10 M) were without effect. Depolarisation of the basolateral membrane, by raising [K⁺]_b to 60 mM, significantly decreased transcellular Ca²⁺ transport, which is indicative of electrogenic Na⁺/Ca²⁺ exchange. In conclusion, active Ca²⁺ transport in the collecting system of rabbit kidney is largely driven by basolateral Na⁺/Ca²⁺ exchange. However, a residual Ca²⁺ absorption of about 30% was always observed, suggesting that other Ca²⁺ transport mechanisms, presumably a Ca²⁺-ATPase, participate as well.     

ATP-sensitive K+ channels in rat ventricular myocytes are blocked and inactivated by internal divalent cations

K⁺ currents were recorded from ATP-sensitive channels in inside-out patches from isolated rat ventricular myocytes. In the absence of internal divalent cations the current voltage relationship could be described by constant-field assumptions with a permeability of 1.25×10^–13 cm²/s; outward currents saturated under a high driving force for K⁺ movement. Internal 0.1–5.0 mM Mg²⁺, 0.1 M Ca²⁺ and 10 mM Na⁺ each depressed the flux of K⁺ ions moving outwards through open channels. Internal 0.1–5.0 mM Mg²⁺, 0.1–1.0 M Ca²⁺ and 1–10 M Ba²⁺ and Sr²⁺ blocked K⁺ channel activity in a dose-and voltage-dependent manner. Run-down channels could be reactivated by Mg-ATP, but not by AMP-PNP, ATPS or Mg-free ATP which suggested that phosphorylation of the channels was involved in their activity. Ca²⁺ (>=1 M) and Sr²⁺ (1 mM) markedly inactivated K⁺ ATP channels, millimolar Ba²⁺ or Mg²⁺ were less effective. This suggested that the run down of the channels was a Ca²⁺-dependent dephosphorylation of the K⁺ channel protein.     

Occurrence and properties of a (Na+−K+)-activated ATPase in the mucosa of the rat intestine

Summary The existence of an ouabain-sensitive (Na⁺–K⁺)-activated ATPase system has been demonstrated in the total intestine of the rat. The (Na⁺–K⁺)-ATPase activity was about 10–15% of the total ATPase in 4 equal parts of the small intestine; in the colon about 35% of the total ATPase was (Na⁺–K⁺)-activated ATPase. The highest (Na⁺–K⁺)-ATPase activity has been observed in the first and second part of the small intestine, while in the colon the activity was 2–4 times higher than in the ileum.The (Na⁺–K⁺)-ATPase of rat colon required both Na⁺ (K _m=8.3 mmoles/l) and K⁺ (K _m=0.6 mmoles/l). Maximal activation of the (Na⁺–K⁺)-ATPase system required 2 mM Mg²⁺ at an ATP concentration of 2 mM. The pH optimum for (Na⁺–K⁺)-ATPase of rat colon was 7.5, while the Mg²⁺-activated ATPase activity had a pH optimum of 8.6. The (Na⁺–K⁺)-ATPase was inhibited by ouabain (pI ₅₀=3.6).The relation between the differences in (Na⁺–K⁺)-ATPase activity and Na⁺-absorption on different parts of the intestine is discussed.     

Ca2+, Mg2+ and K+ transport in the cortical and medullary thick ascending limb of the rat nephron: influence of transepithelial voltage

Isolated segments of rat cortical (cTAL) and medullary (mTAL) thick ascending limbs were microperfused and the transepithelial net fluxes (J_x) were determined by measuring the composition of the collected fluid with an electron microprobe. When perfused with symmetrical solutions both segments showed similar J_Na and j_Cl and lumen-positive transepithelial voltage (V _te=7–8 mV). J_Mg, J_Ca and J_K were not significantly different from zero. When perfused with asymmetrical solutions (lumen 50 mM, bath 150 mM NaCl), the mean V_te were 23 mV and 17 mV in the cTAL and mTAL respectively; this rise was accompanied by significant increases in J_Mg and J_Ca in the cTAL, but not in the mTAL, and a marked increase in J_K in both segments. It is concluded that, in the rat, divalent cations can be reabsorbed in the cTAL, and K⁺ can be reabsorbed in the cTAL and mTAL. The transport is voltage-dependent. The mTAL can reabsorb neither Mg²⁺ nor Ca²⁺, whatever V_te.     

(Na+K+)-activated ATPase in human cornea

Summary Distribution and principal characteristics of (Na⁺K⁺)-activated ATPase in human cornea were investigated.(Na⁺K⁺)-ATPase was present in both epithelium and endothelium, whereas the corneal stroma did not exhibit significant enzyme activity.In homogenates specific activity of the (Na⁺K⁺)-ATPase was 2.3-fold higher in endothelium than in epithelium. Calculation of total enzyme activity revealed a 6.1-fold higher content of (Na⁺K⁺)-ATPase in the epithelium.In the epithelium a 7-fold enrichment of (Na⁺K⁺)-ATPase compared to the homogenate was obtained in the 150–1500×g _av fraction. Maximum enrichment in the endothelium was 3.5-fold and was achieved in the 1500–2500×g _av fraction. Both fractions showed, however, the same specific activity.The pH-optimum of (Na⁺K⁺)-ATPase in the 150–1500×g _av fraction ranged from 8.0–8.2 in both epithelium and endothelium.In the epithelial 150–1500×g _av fraction the apparentK _m-values were 4.0 mM for Na⁺, 2.8 mM for K⁺ and 0.12 mM for Mg²⁺ · ATP in equimolar concentrations.The inhibition constant of epithelial (Na⁺K⁺)-ATPase for ouabain was determined asK _i=3.3×10^–7 M.The present data support the view that control of corneal hydration in man is a function of both endothelium and epithelium.     

Effects of parathyroid hormone and calcitonin on Na+, Cl−, K+, Mg2+ and Ca2+ transport in cortical and medullary thick ascending limbs of mouse kidney

The effect of parathyroid hormone (PTH) on transepithelial Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ transport was investigated in isolated perfused cortical thick ascending limbs (cTAL) and that of human calcitonin (hCT) was tested in both cortical and medullary thick ascending limbs (mTAL) of the mouse nephron. The transepithelial ion net fluxes (J _x) were determined by electron probe analysis of the perfused and collected fluids. Simultaneously, the transepithelial voltage (PD_te) and resistance (R _te) were recorded. In cTAL segments, PTH and hCT significantly stimulated the reabsorption of Na⁺, Cl^–, Ca²⁺ and Mg²⁺. hCT generated a net K⁺ secretion towards the lumen and PTH tended to exert the same effect. Neither PD_te nor R _te were significantly altered by either PTH or hCT. However, in the post-experimental period a significant decrease in PD_te was noted. Time control experiments carried out under similar conditions revealed a significant decrease in PD_te with time, which could have masked the hormonal response. In mTAL segments, Mg²⁺ and Ca²⁺ transport was close to zero. hCT did not exert any detectable effect on either PD_te or J _Cl ^–, J _Na ⁺ J _K ⁺, J _Mg ²⁺ and J _Ca ²⁺ in these segments. In conclusion, our data demonstrate that PTH and hCT stimulate NaCl reabsorption as well as Mg²⁺ and Ca²⁺ reabsorption in the cTAL segment of the mouse. These data are in agreement with and extend data obtained in vivo in the rat.     

Role of voltage- and Ca2+-dependent K+ channels in the control of glucose-induced electrical activity in pancreatic B-cells

Low concentrations of tetraethylammonium chloride (TEA), which inhibit voltage- and Ca²⁺-sensitive K⁺ channels (K⁺-VCa channels), were used to investigate whether these channels play a role in the control of glucose-induced electrical activity (slow waves with spikes) in mouse pancreatic B-cells. Addition of 2 mM TEA to a medium containing 0, 3 or 6 mM glucose had no effect on the membrane potential of B-cells or on ⁸⁶Rb⁺ efflux and insulin release from isolated islets. In 10 mM glucose, 0.5–2 mM TEA produced a concentration-dependent increase in spike amplitude without modifying slow-wave duration or frequency. Insulin release was only slightly increased under these conditions. In conclusion, K⁺-VCa channels are not operative when the B-cell membrane is not depolarized (in low glucose). They appear to play a role in the repolarization of the spikes but not in that of the slow waves. In contrast to ATP-sensitive K⁺ channels, K⁺-VCa channels are not a target on which glucose acts to regulate electrical activity in B-cells and, hence, insulin release.     

Effects of Mg2+ and Ca2+ on Noradrenaline Release and Uptake in Adrenergic Nerve Granules in Different Media

Euler , U. S. V. and F. Lishajko . Effects of Mg²⁺ and Ca²⁺ on noradrenaline release and uptake in adrenergic nerve granules in different media. Acta physiol. scand. 1973. 89. 415–422. Uptake and release of noradrenaline (NA) from a suspension of storage granules from bovine splenic nerves was measured after incubation in various media and effects of added Mg²⁺ and Ca²⁺ observed. No significant differences in NA release rate were observed in different media when NA uptake was prevented by addition of potassium ferricyanide to the medium. Uptake was facilitated by phosphate ions 5–30 mM but counteracted by monovalent cations. In sucrose-phosphate media uptake of NA almost balanced spontaneous release. Mg²⁺ and Ca²⁺ had only a small action on NA release in the absence of NA uptake. In sucrose phosphate media Mg²⁺ facilitated uptake, whereas Ca²⁺ enhanced NA release. This action was associated with the formation of a calcium phosphate precipitate. No release or uptake of NA was observed during incubation at ice-water temperature, nor did Mg²⁺ or Ca²⁺ have any effect. The results tend to show that release and uptake of NA in storage granules are separate, temperature-dependent processes, influenced by mono- and divalent cations and phosphate ions.     

Caffeine-induced depolarization in amphibian skeletal muscle fibres: role of Na+/Ca2+ exchange and K+ release

Caffeine (4 mM ) produces a depolarization of about 10 mV in frog muscle fibres (Leptodactylus ocellatus). The aim of this work was to study the mechanisms of this effect. An approximately threefold rise in membrane resistance [Cl⁻-free (SO₄^2–) medium] substantially increased, and both Na⁺-free medium and Ni²⁺ (5 mM ) reduced, the caffeine-induced depolarization. In voltage-clamped (–60 mV) short fibres from lumbricalis muscle of the toad (Buffo arenarum), caffeine generated an inward current of 4.13 ± 0.48 μA cm^–2. This caffeine-induced current was reduced by 60% in Na⁺-free medium, 44% in the presence of 5 mM amiloride and 48% by 5 mM Ni²⁺, suggesting that the activation of the Na⁺–Ca²⁺ exchanger in its forward mode may play a role in the observed electrical effects of the drug. Caffeine also produced a marked release of K⁺. Net K⁺ efflux increased from 3.5 ± 0.2 (control) to 22.1 ± 2.3 pmol s^–1 cm^–2 (caffeine). It is shown that in the presence of the drug, [K⁺] in the lumen of the T tubules may well increase to levels which could produce, in part, both the observed depolarization and the caffeine-induced current under voltage clamp conditions. The caffeine-induced K⁺ efflux was not reduced by 5 mM Ni²⁺. At a holding potential of 30 mV the caffeine-induced current was reversed (outward) and roughly halved by 5 mM Ni²⁺. The Ni²⁺-sensitive fraction of the caffeine-induced current, assumed to represent the Na⁺–Ca²⁺ exchanger current, had an estimated reversal potential close to 12 mV ([Na⁺]_o=115 mM ; [Ca²⁺]_o=1 mM ). In conclusion, the depolarizing effect of caffeine described here would be produced by two mechanisms: (a) an inward current generated by the activation of the Na⁺–Ca²⁺ exchanger in its forward mode, and (b) the rise of the external [K⁺] in restricted spaces like the T tubules.     

A rapidly inactivating Ca2+-dependent K+ current in pheochromocytoma cells (PC12) of the rat

The membrane electrical properties of undifferentiated pheochromocytoma cells of the rat (PC12) were studied using both current-and voltage-clamp techniques with the use of low-resistance blunt-tipped micropipettes (patch electrodes). In the presence of tetrodotoxin (TTX, 2–3 M), a spike-like wave form with a prominent after-hyperpolarization (AHP) was recorded following brief (< 10 ms) depolarizing current pulses. The inorganic divalent cations, Cd²⁺ (0.5 mM), Mn²⁺ (4mM), and 0 mM Ca²⁺/4 mM Mg²⁺ solution prolonged the duration, attenuated the AHP, slowed the rate of repolarization, and slightly enhanced the amplitude of this wave form. A rapidly inactivating outward current was recorded in over 70% of the cells under voltage-clamp conditions. This transient current was elicited at about ±30 mV, and was blocked by tetraethylammonium (5 mM), inorganic divalent cations (Cd²⁺, 0.5 mM; Mn²⁺, 4 mM; Ba²⁺, 3 mM), and removal of Ca²⁺ (0 mM Ca²⁺/4 mM Mg²⁺) from the local perfusion medium. In addition, 4-aminopyridine (5 mM), which blocks the transient outward K⁺ current I_A in a variety of excitable cells, did not have any appreciable effect on this rapidly inactivating current. Moreover, it was possible to elicit the current at a holding potential of ±40 mV. The reversal potential of this current was ±90 mV, and shifted positively when extracellular K⁺ concentrations were elevated. It is concluded that PC12 cells have a rapidly inactivating Ca²⁺ -dependent K⁺ current. A possible explanation for the transient nature of this current may be the presence of an effective intracellular Ca²⁺ buffering (uptake or extrusion) system.