Ca2+ release and Ca2+ influx in Chinese hamster ovary cells expressing the cloned mouse B2 bradykinin receptor: tyrosine kinase inhibitor-sensitive and -insensitive processes

A cDNA encoding a mouse B₂ bradykinin (BK) receptor was stably transfected in Chinese hamster ovary (CHO) cells. In two resulting transformants, mouse B₂ BK receptor was found to induce a twofold elevation in the inositol-1,4,5-trisphosphate level. In a pertussis toxin-insensitive manner, BK also produced a biphasic increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). The initial elevation in [Ca²⁺]_i was abolished by thapsigargin pretreatment in Ca²⁺-free medium. The second phase was dependent on external Ca²⁺. The BK/inositol trisphosphate- and thapsigargin-sensitive Ca²⁺ stores required extracellular Ca²⁺ for refilling. Ca²⁺ influx induced by BK and thapsigargin was confirmed by Mn²⁺ entry through Ca²⁺ influx pathways producing Mn²⁺ quenching. Genistein, a tyrosine kinase inhibitor, partially decreased the BK-induced [Ca²⁺]_i increase during the sustained phase and the rate of Mn²⁺ entry. BK had essentially no effect on the intracellular cyclic AMP level. The results suggest that the mouse B₂ BK receptor couples to phospholipase C in CHO cells and that its activation results in biphasic [Ca²⁺]_i increases, by mobilization of intracellular Ca²⁺ and store-depletion-mediated Ca²⁺ influx, the latter of which is tyrosine phosphorylation-dependent.     

Tetanus toxin treatment in vitro inhibits the release of GABA from rat cerebral cortex slices evoked by high K+ and Na+-free media

The influence of tetanus toxin in vitro on the release of exogenous [³H]GABA was studied with rat cerebral cortex slices. The influx, long-term accumulation and spontaneous efflux of GABA were not modified by the toxin. The release induced by high K⁺ (50 mM) medium from the superfused slices pretreated with the toxin was significantly inhibited in a time- and dose-dependent fashion. This release was attenuated during superfusion with Ca²⁺-free medium and the toxin no longer affected the remaining Ca²⁺-independent release. The release induced by Na⁺-free media did not require extracellular Ca²⁺ ions, and the toxin inhibited the release both with and without Ca²⁺. The toxin treatment had no marked influence on the ouabain (20 μM) or veratrine (25–50 μM)-induced release of GABA. The toxin treatment in vitro appears to modify some step(s) in the stimulated release of GABA without affecting its unstimulated membrane transport. Tetanus toxin may thus prove a valuable tool in studying the mechanisms of the release of GABA and possibly other inhibitory transmitters in synapses of the central nervous system.     

Reduced external calcium or sodium stimulates calcium influx in Pelvetia eggs

The effect of external calcium and sodium ion concentrations on the calcium fluxes on the Pelvetia fastigiata De Toni egg was measured. Decreasing external [Ca²⁺] greatly increased the permeability of the eggs to Ca²⁺; at 1 mM external Ca²⁺ this permeability was 60 times as great as it was at the normal [Ca²⁺] of 10 mM. Lowering the external [Na⁺] also increased Ca²⁺ influx; at 2 mM Na⁺, the Ca²⁺ influx was 2–3 times as great as it was at the normal [Na⁺] if choline was used as a Na⁺ substitute. Lithium was less effective as a Na⁺ substitute in increasing Ca²⁺ influx. The extra Ca²⁺ influx in low [Na⁺] seemed to be dependent on internal [Na⁺]. The Ca²⁺ efflux increased transiently and then declined in low Na⁺ media.     

Ca2+ influx mechanisms in caveolae vesicles of pulmonary smooth muscle plasma membrane under inhibition of α2β1 isozyme of Na+/K+-ATPase by ouabain

AimsWe sought to determine the mechanisms of an increase in Ca²⁺ level in caveolae vesicles in pulmonary smooth muscle plasma membrane during Na⁺/K⁺-ATPase inhibition by ouabain.Main methodsThe caveolae vesicles isolated by density gradient centrifugation were characterized by electron microscopic and immunologic studies and determined ouabain induced increase in Na⁺ and Ca²⁺ levels in the vesicles with fluorescent probes, SBFI-AM and Fura2-AM, respectively.Key findingsWe identified the α₂β₁ and α₁β₁ isozymes of Na⁺/K⁺-ATPase in caveolae vesicles, and only the α₁β₁ isozyme in noncaveolae fraction of the plasma membrane. The α₂-isoform contributes solely to the enzyme inhibition in the caveolae vesicles at 40 nM ouabain. Methylisobutylamiloride (Na⁺/H⁺-exchange inhibitor) and tetrodotoxin (voltage-gated Na⁺-channel inhibitor) pretreatment prevented ouabain induced increase in Na⁺ and Ca²⁺ levels. Ouabain induced increase in Ca²⁺ level was markedly, but not completely, inhibited by KB-R7943 (reverse-mode Na⁺/Ca²⁺-exchange inhibitor) and verapamil (L-type Ca²⁺-channel inhibitor). However, pretreatment with tetrodotoxin in conjunction with KB-R7943 and verapamil blunted ouabain induced increase in Ca²⁺ level in the caveolae vesicles, indicating that apart from Na⁺/Ca⁺-exchanger and L-type Ca²⁺-channels, “slip-mode conductance” of Na⁺ channels could also be involved in this scenario.SignificanceInhibition of α₂ isoform of Na⁺/K⁺-ATPase by ouabain plays a crucial role in modulating the Ca²⁺ influx regulatory components in the caveolae microdomain for marked increase in (Ca²⁺)_i in the smooth muscle, which could be important for the manifestation of pulmonary hypertension.     

Sodium-calcium exchange in renal epithelial cells: Dependence on cell sodium and competitive inhibition by magnesium

Summary Kinetic properties of Na⁺–Ca²⁺ exchange in a renal epithelial cell line (LLC-MK₂) were assessed by measuring cytosolic free Ca²⁺ with fura-2 and⁴⁵Ca²⁺ influx. Replacing external Na⁺ with K⁺ produced relatively small increases in free Ca²⁺ and⁴⁵Ca²⁺ uptake unless the cells were incubated with ouabain. Ouabain markedly increased cell Na⁺ and strongly potentiated the effect of replacing external Na⁺ with K⁺ on free Ca²⁺ and⁴⁵Ca²⁺ uptake.⁴⁵Ca²⁺ influx in 140mm K⁺ or N-methyl-d-glucamine minus influx in 140mm Na⁺ was used to quantify Na⁺–Ca²⁺ exchange activity of Na⁺-loaded cells. The dependence of exchange on cell Na⁺ was sigmoidal; theK _0.5 was 26±3 mmol/liter cell water space, and the Hill coefficient was 3.1±0.2. The kinetic features of the dependence of exchange on cell Na⁺ partly account for the small increase in Ca²⁺ influx when all external Na⁺ is replaced by K⁺. Besides raising cell Na⁺ ouabain appears to activate the exchanger. Magnesium competitively inhibited exchange activity. The potency of Mg²⁺ was 8.2-fold lower with potassium instead of N-methyl-d-glucamine or choline as the replacement for external Na⁺. Potassium also increased theV _max of exchange by 86% and had no effect on theK _m for Ca²⁺. The exchanger does not cause detectable²²Na⁺–Mg²⁺ exchange and does not appear to require K⁺ or transport⁸⁶Rb⁺. Although exchange activity was plentiful in the epithelial cells from monkey kidney, others from amphibian, canine, opossum, and porcine kidney had no detectable exchange activity. All of the measured kinetic properties of Na⁺–Ca²⁺ exchange in the renal epithelial cells are very similar to those of the exchanger in rat aortic myocytes.     

Ouabain-induced perturbations in intracellular ionic homeostasis regulate death receptor-mediated apoptosis

Apoptosis is defined by specific morphological and biochemical characteristics including cell shrinkage (termed apoptotic volume decrease), a process that results from the regulation of ion channels and plasma membrane transporter activity. The Na⁺–K⁺-ATPase is the predominant pump that controls cell volume and plasma membrane potential in cells and alterations in its function have been suggested to be associated with apoptosis. We report here that the Na⁺–K⁺-ATPase inhibitor ouabain, potentiates apoptosis in the human lymphoma Jurkat cells exposed to Fas ligand (FasL) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not other apoptotic agents such as H₂O₂, thapsigargin or UV-C implicating a role for the Na⁺–K⁺-ATPase in death receptor-induced apoptosis. Interestingly, ouabain also potentiated perturbations in cell Ca²⁺ homeostasis only in conjunction with the apoptotic inducer FasL but not TRAIL. Ouabain did not affect alterations in the intracellular Ca²⁺ levels in response to H₂O₂, thapsigargin or UV-C. FasL-induced alterations in Ca²⁺ were not abolished in Ca²⁺-free medium but incubation of cells with BAPTA-AM inhibited both Ca²⁺ perturbations and the ouabain-induced potentiation of FasL-induced apoptosis. Our data suggest that the impairment of the Na⁺–K⁺-ATPase activity during apoptosis is linked to perturbations in cell Ca²⁺ homeostasis that modulate apoptosis induced by the activation of Fas by FasL.     

Sarcolemmal Na+-Ca2+ exchange and Ca2+-pump activities in cardiomyopathies due to intracellular Ca2+-overload

In order to identify defects in Na⁺-Ca²⁺ exchange and Ca²⁺-pump systems in cardiomyopathic hearts, the activities of sarcolemmal Na⁺-dependent Ca²⁺ uptake, Na⁺-induced Ca²⁺ release, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase were examined by employing cardiomyopathic hamsters (UM-X7.1) and catecholamine-induced cardiomyopathy produced by injecting isoproterenol into rats. The rates of Na⁺-dependent Ca²⁺ uptake, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities of sarcolemmal vesicles from genetically-linked cardiomyopathic as well as catecholamine-induced cardiomyopathic hearts were decreased without any changes in Na⁺-induced Ca²⁺-release. Similar results were obtained in Ca²⁺-paradox when isolated rat hearts were perfused for 5 min with a medium containing 1.25 mM Ca²⁺ following a 5 min perfusion with Ca²⁺-free medium. Although a 2 min reperfusion of the Ca²⁺-free perfused hearts depressed sarcolemmal Ca²⁺-pump activities without any changes in Na⁺-induced Ca²⁺-release, Na⁺-dependent Ca²⁺ uptake was increased. These results indicate that alterations in the sarcolemmal Ca²⁺-efflux mechanisms may play an important role in cardiomyopathies associated with the development of intracellular Ca²⁺ overload.     

Calcium-salinity interactions affect ion transport in Chara corallina

Detached internodes of Chara corallina survived in solutions containing 100 mol m^?3 NaCl when the external concentration of Ca²⁺ was greater than 1 mol m^?3. Na⁺ influx was roughly proportional to external Na⁺ up to 100 mol m^?3 NaCl. Na⁺ influx involved two components: a Ca²⁺-insensitive influx which allowed the passage of Na⁺ independently of external Ca²⁺; and a Ca²⁺-inhibitable mechanism where Na⁺ influx was inversely proportional to external Ca²⁺. The Ca²⁺-inhibitable Na⁺ influx was similar to the Ca²⁺-inhibitable K⁺ influx. Mg²⁺ and Ba²⁺ were able to substitute for Ca²⁺ in partially inhibiting Na⁺ influx in the absence of external Ca²⁺. The effect of Ca²⁺ appears specific to Na⁺ and K⁺ influx since the effects of a Ca²⁺-free solution on the influx of some other cations, anions and neutral compounds is small. It is suggested that Na⁺ influx via the Ca²⁺-inhibitable mechanism represents Na⁺ leakage through K⁺ channels and that cell death at high salinity occurs due to a cytotoxic Na⁺ influx via this mechanism.     

Mechanistic distinction between activation and inhibition of (Na+K)-ATPase-mediated Ca influx in cardiomyocytes

(Na⁺+K⁺)-ATPase (NKA) mediates positive inotropy in the heart. Extensive studies have demonstrated that the reverse-mode Na⁺/Ca²⁺-exchanger (NCX) plays a critical role in increasing intracellular Ca²⁺ concentration through the inhibition of NKA-induced positive inotropy by cardiac glycosides. Little is known about the nature of the NCX functional mode in the activation of NKA-induced positive inotropy. Here, we examined the effect of an NKA activator SSA412 antibody on ⁴⁵Ca influx in isolated rat myocytes and found that KB-R7943, a NCX reverse-mode inhibitor, fails to inhibit the activation of NKA-induced ⁴⁵Ca influx, suggesting that the Ca²⁺ influx via the reverse-mode NCX does not mediate this process. Nifedipine, an L-type Ca²⁺ channel (LTCC) inhibitor, completely blocks the activation of NKA-induced ⁴⁵Ca influx, suggesting that the LTCC is responsible for the moderate increase in intracellular Ca²⁺. In contrast, the inhibition of NKA by ouabain induces 4.7-fold ⁴⁵Ca influx compared with the condition of activation of NKA. Moreover, approximately 70% of ouabain-induced ⁴⁵Ca influx was obstructed by KB-R7943 and only 30% was impeded by nifedipine, indicating that both the LTCC and the NCX contribute to the rise in intracellular Ca²⁺ and that the NCX reverse-mode is the major source for the ⁴⁵Ca influx induced by the inhibition of NKA. This study provides direct evidence to demonstrate that the activation of NKA-induced Ca²⁺ increase is independent of the reverse-mode NCX and pinpoints a mechanistic distinction between the activation and inhibition of the NKA-mediated Ca²⁺ influx path ways in cardiomyocytes.     

Controls on na influx in corn roots

We have investigated the effects of hyperpolarization and depolarization, and the presence of K⁺ and/or Ca²⁺, on ²²Na⁺ influx into corn (Zea mays L.) root segments. In freshly excised root tissue which is injured, Na⁺ influx is unaffected by hyperpolarization with fusicoccin, or depolarization with uncoupler (protonophore), or by addition of K⁺. However, added Ca²⁺ suppresses Na⁺ influx by 60%. In washed tissue which has recovered, Na⁺ influx is doubled over that of freshly excised tissue, and the influx is increased by fusicoccin and suppressed by uncoupler. This energy-linked component of Na⁺ influx is completely eliminated by low concentrations of K⁺, leaving the same level and kind of Na⁺ influx seen in freshly excised roots. The K⁺-sensitive energy linkage appears to be by the carrier for active K⁺ influx. Calcium is equally inhibitory to Na⁺ influx in washed as in fresh tissue. Other divalent cations are only slightly less effective. Net Na⁺ uptake was about 25% of ²²Na⁺ influx, but proportionately the response to K⁺ and Ca²⁺ was about the same.

The constancy of K⁺-insensitive Na⁺ influx under conditions known to hyperpolarize and depolarize suggests that if Na⁺ transport is by means of a voltage-sensitive channel, the rise or fall of channel resistance must be proportional to the rise or fall in potential difference. The alternative is a passive electroneutral exchange of ²²Na⁺ for endogenous Na⁺. The data suggest that an inwardly directed Na⁺ current is largely offset by an efflux current, giving both a small net uptake and isotopic exchange.

     

Ca2+ dependency of Na+ transport by rabbit renal brush border membrane

Summary Intracellular Ca²⁺ has been suggested to play an important role in the regulation of epithelial Na⁺ transport. Previous studies showed that preincubation of toad urinary bladder, a tight epithelium, in Ca²⁺-free medium enhanced Na⁺ uptake by the subsequently isolated apical membrane vesicles, suggesting the downregulation of Na⁺ entry across the apical membrane by intracellular Ca²⁺. In the present study, we have examined the effect of Ca²⁺-free preincubation on apical membrane Na⁺ transport in a leaky epithelium, i.e., brush border membrane (BBM) of rabbit renal proximal tubule. In contrast to toad urinary bladder, it was found that BBM vesicles derived from proximal tubules incubated in 1mm Ca²⁺ medium exhibited higher Na⁺ uptake than those derived from proximal tubules incubated in Ca²⁺-free EGTA medium. Such effect of Ca²⁺ in the preincubation medium was temperature dependent and could not be replaced by another divalent cation, Ba²⁺ (1mm). Ca²⁺ in the preincubation medium did not affect Na⁺-dependent BBM glucose uptake, and its effect on BBM Na⁺ uptake was pH gradient dependent and amiloride (10^–3 m) sensitive, suggesting the involvement of Na⁺/H⁺ antiport system. Addition of verapamil (10^–4 m) to 1mm Ca²⁺ preincubation medium abolished while ionomycin (10^–6 m) potentiated the effect of Ca²⁺ to increase BBM Na⁺ uptake, suggesting that the effect of Ca²⁺ in the preincubation medium is likely to be mediated by Ca²⁺-dependent cellular pathways and not due to a direct effect of extracellular Ca²⁺ on BBM. Neither the proximal tubule content of cAMP nor the inhibitory effect of 8, bromo-cAMP (0.1mm) on BBM Na⁺ uptake was affected by the presence of Ca²⁺ in the preincubation medium, suggesting that Ca²⁺ in the preincubation medium did not increase BBM Na⁺ uptake by removing the inhibitory effect of cAMP. Addition of calmodulin inhibitor, trifluoperazine (10^–4 m) to 1mm Ca²⁺ preincubation medium did not prevent the increase in BBM Na⁺ uptake. The effect of Ca²⁺ was, however, abolished when protein kinase C in the proximal tubule was downregulated by prolonged (24 hr) incubation with phorbol 12-myristate 13-acetate (10^–6 m). In summary, these results show the Ca²⁺ dependency of Na⁺ transport by renal BBM, possibly through stimulation of Na⁺/H⁺ exchanger by protein kinase C.     

Effects of the calcium ionophore A-23187 on the regulation of sugar transport in muscle

The distribution of (¹⁴C)-3-0-methyl-D-glucose and of (⁴⁵Ca) was followed in perifused left atria and intact hemidiaphragms of the rat. The carboxylic calcium ionophore A-23187 affected sugar and Ca²⁺ influx in parallel, with low concentrations inhibiting and higher ones stimulating influx under basal conditions. The stimulation of sugar transport by insulin, high concentrations of adrenaline or ouabain, or by K⁺-free medium was antagonized by the calcium ionophore. Likewise, A-23187 counteracted the depression of sugar transport caused by low concentrations of ouabain or adrenaline. These results support a role of Ca²⁺ in the regulation of sugar transport in muscle. However, increased influx of Ca²⁺ cannot explain all the effects of A-23187. It is suggested that the ionophore may also act by releasing Ca²⁺ from intracellular storage and binding sites.     

Characterization of Low pH-induced Catecholamine Secretion in the Rat Adrenal Medulla

Abstract: Catecholamine (CA) secretion was evoked when the isolated rat adrenal gland was perfused with HEPES-buffered Krebs solution acidified by the addition of HCI or by gassing with 95% O₂/5% CO₂. The secretion was detectable at pH 7.0 and increased with decreasing pH until at ～6.4. The low pH-induced CA secretion consisted of two phases, an initial transient response followed by a sustained phase. An intracellular Ca²⁺ antagonist, 3,4,5-trimethoxybenzoic acid 8-(N,N-diethylamino)octyl ester, selectively inhibited the initial phase of secretion. Both of the responses were resistant to nifedipine, a blocker of voltage-gated Ca²⁺ channel, but were completely inhibited in Ca²⁺-free (1 mM EGTA containing) solution. Adrenaline was an exclusive component in CAs released by low pH. The time course and extent of intracellular acidification caused either by low pH in the external medium or by the offset of a transitory NH₄CI application had no correlation with those of the secretory responses in the corresponding period. These results suggest that extracellular acidification preferentially activates adrenaline secretive cells to evoke CA secretion and that this low pH-induced CA secretion may be mediated by dihydropyridine-insensitive Ca²⁺ influx. Furthermore, the initial transient phase of the low pH-induced CA secretion might be caused by a Ca²⁺ release from intracellular stores, which is also induced by the Ca²⁺ influx.     

Control of sodium influx by calcium and turgor in two charophytes differing in salinity tolerance

The effects of Ca²⁺ and cell turgor on Na⁺ influx were examined in two charophytes, lamprothamnium papulo-SUM (salt-tolerant) and Chara corallina (salt-sensitive), to try to identify causes of salinity toxicity. Mortality was associated with Na+ influx, with the two species showing similar sensitivities to high Na⁺ influx. In Lamprothamnium, toxic influxes of Na⁺ occurred at much higher external Na⁺ concentrations than in Chara. The differences in Na⁺ influx at the same Na⁺ concentration were not due to different responses to external Ca²⁺. Lamprothamnium adjusts its turgor in response to increasing NaCl whereas Chara cannot. In solutions of KC1 up to at least 200 mol m_-3, however, Chara regulated turgor, and when KC1 was subsequently replaced with NaCl, Na+ influx was low and similar to that in Lamprothamnium at the same Na* concentration. Chara cells which were not turgor-adjusted in KCI had Na⁺ influxes 2-5-fold higher than the turgid cells. Thus, it appears that turgor is a major determinant of Na⁺ influx, and therefore of cell survival. We found no evidence that the mechanism of Na⁺ influx in Chara is different from that in Lamprothamnium. Higher susceptibility of Chara to NaCl seems to result from inability to regulate turgor, in turn leading to toxic Na⁺ influx.     

Na+-dependent Ca2+ uptake in isolated opercular epithelium of Fundulus heteroclitus

It is concluded that Ca²⁺ transport across the basolateral membranes of the ionocytes in killifish skin is mediated for the major part by a Na⁺/Ca²⁺-exchange mechanism that is driven by the (transmembrane) Na⁺ gradient established by Na⁺/K⁺-ATPase. The conclusion is based, firstly, on the biochemical evidence for the presence of a Na⁺/Ca²⁺-exchanger next to the Ca²⁺-ATPase in the basolateral membranes of killifish gill cells. Secondly, the transcellular Ca²⁺ uptake measured in an Ussing chamber setup was 85% and 80% reduced in freshwater (FW) and SW (SW) opercular membranes, respectively, as the Na⁺ gradient across the basolateral membrane was directly or indirectly (by ouabain) reduced. Thapsigargin or dibutyryl-cAMP/IBMX in SW opercular membranes reduced Ca²⁺ influx to 46%, comparable to the effects seen in FW membranes [reduction to 56%; Marshall et al. 1995a]. Basal Ca²⁺ influx across the opercular membrane was 48% lower in membranes from fish adapted to SW than in membranes from fish adaptated to FW. Branchial Na⁺/K⁺-ATPase activity was two times higher in SW adapted fish. Accepted: 29 October 1996     

Multiple effects of Na+ removal on pancreatic secretion in vitro

Summary The effects of removing Na⁺ from the incubation medium on basal and secretagogue induced zymogen release by pancreatic fragments and isolated pancreatic acini were studied by both morphological evaluation and measurement of amylase release. In both fragments and isolated acini, removal of Na⁺ led to an increased basal secretion of zymogen granule contents from acinar cells via exocytosis; secretory material, however, accumulated in acinar and ductular lumina as a result of the lack of fluid secretion necessary to wash out the enzymes. In studies with fragments, after Na⁺ removal there was no significant increase in amylase release into the medium; isolated acini, in contrast, showed an increased amylase release consistent with the shorter distance from the acinar lumen to the bathing medium. Stimulation with either bethanechol or caerulein led to a further depletion of zymogen granules in both preparations; in the absence of Na⁺ secretory product accumulated in intracellular lakes as well as in duct lumens. The hypothesis that Na⁺ influx is important in stimulus-secretion coupling to release intracellular Ca²⁺ was directly tested by measuring ⁴⁵Ca²⁺ efflux. No effect of removing Na⁺ on ⁴⁵Ca²⁺ efflux was seen. It was concluded, therefore, that while Na⁺ is essential for pancreatic fluid secretion, it is not necessary for the secretion of zymogen granule contents into acinar lumina.Supported by NIH grant GM-19998 from the United States Public Health Service     

Acceleration of Sodium-Calcium Exchange Activity during ATP-induced Calcium Release in Transfected Chinese Hamster Ovary Cells

The P_2U purinergic agonist ATP (0.3 mM) elicited an increase in [Ca²⁺]_i due to Ca²⁺ release from intracellular stores in transfected Chinese hamster ovary cells that express the bovine cardiac Na⁺/Ca²⁺ exchanger (CK1.4 cells). The following observations indicate that ATP-evoked Ca²⁺ release was accompanied by a Ca²⁺- dependent regulatory activation of Na⁺/Ca²⁺ exchange activity: Addition of extracellular Ca²⁺ (0.7 mM) 0–1 min after ATP evoked a dramatic rise in [Ca²⁺]_i in Na⁺-free media (Li⁺ substitution) compared to Na⁺-containing media; no differences between Na⁺- and Li⁺-based media were observed with vector-transfected cells. In the presence of physiological concentrations of extracellular Na⁺ and Ca²⁺, the ATP-evoked rise in [Ca²⁺]_i declined more rapidly in CK1.4 cells compared to control cells, but then attained a long-lived plateau of elevated [Ca²⁺]_i which eventually came to exceed the declining [Ca²⁺]_i values in control cells. ATP elicited a transient acceleration of exchange-mediated Ba²⁺ influx, consistent with regulatory activation of the Na⁺/Ca²⁺ exchanger. The acceleration of Ba²⁺ influx was not observed in vector-transfected control cells, or in CK1.4 cells in the absence of intracellular Na⁺ or when the Ca²⁺ content of the intracellular stores had been reduced by prior treatment with ionomycin. The protein kinase C activator phorbol 12-myristate 13-acetate attenuated the exchange-mediated rise in [Ca²⁺]_i under Na⁺-free conditions, but did not inhibit the ATP-evoked stimulation of Ba²⁺ influx. The effects of PMA are therefore not due to inhibition of exchange activity, but probably reflect the influence of protein kinase C on other Ca²⁺ homeostatic mechanisms. We conclude that exchange activity is accelerated during ATP-evoked Ca²⁺ release from intracellular stores through regulatory activation by increased [Ca²⁺]_i. In the absence of extracellular Ca²⁺, the stimulation of exchange activity is short-lived and follows the time course of the [Ca²⁺]_i transient; in the presence of extracellular Ca²⁺, we suggest that the exchanger remains activated for a longer period of time, thereby stabilizing and prolonging the plateau phase of store-dependent Ca²⁺ entry.     

Extracellular Mg regulates the intracellular Na concentration in rat sublingual acini

The intracellular free Na⁺ concentration ([Na⁺]_i) increases during muscarinic stimulation in salivary acinar cells. The present study examined in rat sublingual acini the role of extracellular Mg²⁺ in the regulation of the stimulated [Na⁺]_i increase using the fluorescent sodium indicator benzofuran isophthalate (SBFI). The muscarinic induced rise in [Na⁺]_i was approximately 4-fold greater in the absence of extracellular Mg²⁺. When Na⁺ efflux was blocked by the Na⁺,K⁺-ATPase inhibitor ouabain, the stimulated [Na⁺]_i increase was comparable to that seen in an Mg²⁺-free medium. Moreover, ouabain did not add further to the stimulated [Na⁺]_i increase in an Mg²⁺-free medium suggesting that removal of extracellular Mg²⁺ may inhibit the Na⁺ pump. In agreement with this assumption, ouabain-sensitive Na⁺ efflux and rubidium uptake were reduced by extracellular Mg²⁺ depletion. Our results suggest that extracellular Mg²⁺ may regulate [Na⁺]_i in sublingual salivary acinar cells by modulating Na⁺ pump activity.