Single calcium-dependent cation channels in mouse pancreatic acinar cells

Summary The Ca²⁺-activated nonselective cation channel in mouse pancreatic acini has been studied with the help of patch-clamp single-channel current recording in both the cell-attached conformation and in excised inside-out membrane patches. In intact resting mouse pancreatic acinar cells no unitary activity was observed. Adding saponin to the bath solution to disrupt the plasma membrane (apart from the isolated patch membrane from which current recording was made) evoked unitary inward current steps when the free ionized Ca²⁺ concentration in the bath ([Ca²⁺] _i ) was 5×10^–8 m or above. When an electrically isolated patch membrane was excised and the internal aspects of the plasma membrane were exposed to the bath solution, channel activation could be obtained when [Ca²⁺] _i was 10^–7 m or above. However, with the passage of time the total inward current declined and about 1 min after excision no unitary current steps could be observed. At this stage Ca²⁺ in micromolar concentration was needed to open the channels and several hundred micromoles of Ca²⁺ per liter were required for maximal channel activation. Our results indicate that the Ca²⁺-activated nonselective cation channel is more sensitive to internal Ca²⁺ than hitherto understood and that it may therefore play a role under physiological conditions in intact cells.     

Extracellular ATP elevates intracellular free calcium in rat parotid acinar cells

The effect of extracellular ATP on intracellular free Ca2+ was characterized in quin2-loaded parotid acinar cells. ATP specifically increased the intracellular Ca2+ concentration six-fold above a basal level of 180 nM. Of other purine nucleotides tested, only adenylylthiodiphosphate (ATP gamma S) had significant activity. ATP and the muscarinic agonist carbachol increased intracellular Ca2+ even in the absence of extracellular Ca2+. Both agonists stimulated K+ release, which was followed by reuptake of K+, even in the continued presence of agonist. In the absence of Mg2+, ATP was much more potent but no more efficacious in elevating intracellular Ca2+, suggesting that ATP4- is the active species. The effect of ATP was reversed by removal with hexokinase, arguing against a role for an active contaminant of ATP and against a non-specific permeabilizing effect of extracellular ATP. Lactate dehydrogenase release was unaffected by a maximally effective concentration of ATP. These observations are consistent with a possible neurotransmitter role for ATP in the rat parotid gland.     

Calcium influx factor directly activates store-operated cation channels in vascular smooth muscle cells

Recently, we described a novel 3-pS Ca(2+)-conducting channel that is activated by BAPTA and thapsigargin-induced passive depletion of intracellular Ca(2+) stores and likely to be a native store-operated channel in vascular smooth muscle cells (SMC). Neither Ca(2+) nor inositol 1,4,5-trisphosphate or other second messengers tested activated this channel in membrane patches excised from resting SMC. Here we report that these 3-pS channels are activated in inside-out membrane patches from SMC immediately upon application of Ca(2+) influx factor (CIF) extracted from mutant yeast, which has been previously shown to activate Ca(2+) influx in Xenopus oocytes and Ca(2+) release-activated Ca(2+) current in Jurkat cells. In bioassay experiments depletion of Ca(2+) stores in permeabilized human platelets resulted in the release of endogenous factor, which activated 3-pS channels in isolated inside-out membrane patches excised from SMC and exposed to permeabilized platelets. The same 3-pS channels in excised membrane patches were also activated by acid extracts of CIF derived from human platelets with depleted Ca(2+) stores, which also stimulated Ca(2+) influx upon injection into Xenopus oocytes. Specific high pressure liquid chromatography fractions of platelet extracts were found to have CIF activity when injected into oocytes and activate 3-pS channels in excised membrane patches. These data show for the first time that CIF produced by mammalian cells and yeast with depleted Ca(2+) stores directly activates native 3-pS cation channels, which in intact SMC are activated by Ca(2+) store depletion.     

Activation of nonselective cation channels by physiological cholecystokinin concentrations in mouse pancreatic acinar cells.

The activation of the nonselective cation channels in mouse pancreatic acinar cells has been assessed at low agonist concentrations using patch-clamp whole cell, cell-attached patch, and isolated inside-out patch recordings. Application of acetylcholine (ACh) (25-1,000 nM) and cholecystokinin (CCK) (2-10 pM) evoked oscillatory responses in both cation and chloride currents measured in whole cell experiments. In cell-attached patch experiments we demonstrate CCK and ACh evoked opening of single 25-pS cation channels in the basolateral membrane. Therefore, at least a component of the whole cell cation current is due to activation of cation channels in the basolateral acinar cell membrane. To further investigate the reported sensitivity of the cation channel to intracellular ATP and calcium we used excised inside-out patches. Micromolar Ca2+ concentrations were required for significant channel activation. Application of ATP and ADP to the intracellular surface of the patch blocked channel opening at concentrations between 0.2 and 4 mM. The nonmetabolizable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP, 0.2-2 mM), also effectively blocked channel opening. The subsequent removal of ATP caused a transient increase in channel activity not seen with the removal of ADP or AMP-PNP. Patches isolated into solutions containing 2 mM ATP showed channel activation at micromolar Ca2+ concentrations. Our results show that ATP has two separate effects. The continuous presence of the nucleotide is required for operation of the cation channels and this action seems to depend on ATP hydrolysis. ATP can also close the channel and this effect can be demonstrated in excised inside-out patches when ATP is added to the bath after a period of exposure to an ATP-free solution. This action does not require ATP hydrolysis. Under physiological conditions hormonal stimulation can open the nonselective cation channels and this can be explained by the rise in the intracellular free Ca2+ concentration.     

Distinct characteristics of receptor-operated Ca2+ influx and refilling in pancreatic acinar cells

Ca2+ influx from the extracellular space in nonexcitable cells occurs via receptor-operated and refilling processes. However, they showed different characteristics with respect to the Mn2+ permeability, depletion of intracellular Ca2+ stores, and sensitivity to the K+ ionophore valinomycin in rat pancreatic acinar cells. While Mn2+ did not enter into the cells during the refilling phase, the opposite was true in receptor-operated Ca2+ influx (ROCI) evoked by carbachol (CCh). ROCI occurred in the absence of intracellular Ca2+ release from the stores. Valinomycin abolished the second response of Ca2+ elicited by CCh, whereas it had no effect on ROCI. These observations suggest that receptor-operated Ca2+ channels (ROCCs) and refilling channels may be different in rat pancreatic acinar cells.     

Extracellular ATP stimulates inositol phospholipid turnover and calcium influx in C6 glioma cells

Extracellular ATP caused a dose-dependent accumulation of inositol phosphates and a rise in cytosolic free Ca²⁺ ([Ca²⁺]_i) in C₆ glioma cells with an EC₅₀ of 60±4 and 10±5 M, respectively. The threshold concentration of ATP (3 M) for increasing [Ca²⁺]_i was approximately 10-fold less than that for stimulating phosphoinositide (PI) turnover. The PI response showed a preference for ATP; ADP was about 3-fold less potent than ATP but had a comparable maximal stimulation (11-fold of the control). AMP and adenosine were without effect at concentrations up to 1 mM. ATP-stimulated PI metabolism was found to be partially dependent on extracellular Ca²⁺ and Na⁺ but was resistant to tetrodotoxin, saxitoxin, amiloride, ouabain, and inorganic blockers of Ca²⁺ channels (Co²⁺, Mn²⁺, La³⁺, or Cd²⁺). In Ca²⁺-free medium, ATP caused only a transient increase in [Ca²⁺]_i as opposed to a sustained [Ca²⁺]_i increase in normal medium. The ATP-induced elevation of [Ca²⁺]_i was resistant to Na⁺ depletion and treatment with saxitoxin, verapamil and nisoldipine, but was attentuated by La³⁺. The differences in the characteristics of ATP-caused P₁ hydrolysis and [Ca²⁺]_i rise suggest that ATP receptors are independently coupled to phospholipase C and receptor-gated Ca²⁺ channels. Because of the robust effect of ATP in stimulating PI turnover and the apparent absence of P₁-purinergic receptors, the C₆ glioma cell line provides a useful model for investigating the transmembrane signalling pathway induced by extracellular ATP. The mechanisms underlying the unexpected finding of [Na⁺]_o dependency for ATP-induced PI turnover require further investigation.Abbreviations PI phosphoinositide - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - PDBu phorbol 12, 13-dibutyrate - PSS physiological saline solution - IP inositol phosphates - IP₁ inositol monophosphate - IP₂ inositol bisphosphate - IP₃ inositol trisphosphate - IP₄ inositol (1,3,4,5) tetrakisphosphate - PLC phospholipase C     

Extracellular ATP activates Ca2(+)-dependent K+ conductance via Ca2+ influx in mouse macrophages

1. Using the perforated patch recording, the effects of ATP on membrane current were investigated in mouse peritoneal macrophages. 2. Extracellularly applied ATP induced a biphasic current consisting of a initial inward current [Ii(ATP)] followed by an outward current [Io(ATP)]. These currents were associated with a marked increase in conductance at their peaks. 3. Ii(ATP) reversed close to 0 mV and was attenuated by removal of external Na+. 4. Io(ATP) reversed near -80 mV and was increased by decreasing the external concentration of K+. 5. Io(ATP) was completely abolished by removal of external Ca2+, treatment with an intracellular Ca2+ chelator, the acetoxymethyl ester of 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid (BAPTA-AM) and bath applied quinidine but not tetraethylammonium (TEA) or apamin. 6. These results suggest that Ii(ATP) and Io(ATP) are due to an activation of nonspecific cationic and Ca2(+)-dependent K+ conductances, respectively, and raise the possibility that the putative ATP receptor may be important in regulating macrophage functions, motility, phagocytosis and cytokines secretion.     

Intracellular pH regulation in the mouse lacrimal gland acinar cells

Summary Intracellular pH (pH _i ) of the acinar cells of the isolated, superfused mouse lacrimal gland has been measured using pH-sensitive microelectrodes. Under nonstimulated condition pH _i was 7.25, which was about 0.5 unit higher than the equilibrium pH. Alterations of the external pH by ±0.4 unit shifted pH _i only by ±0.08 unit. The intracellular buffering value determined by applications of 25mm NH ₄ ⁺ and bicarbonate buffer solution gassed with 5% CO₂/95% O₂ was 26 and 46mm/pH, respectively Stimulation with 1 m acetylcholine (ACh) caused a transient, small decrease and then a sustained increase in pH _i . In the presence of amiloride (0.1mm) or the absence of Na⁺, application of ACh caused a significant decrease in pH _i and removal of amiloride or replacement with Na⁺-containing saline, respectively, rapidly increased the pH _i . Pretreatment with DIDS (0.2mm) did not change the pH _i of the nonstimulated conditions; however, it significantly enhanced the increase in pH _i induced by ACh. The present results showed that (i) there is an active acid extrusion mechanism that is stimulated by ACh; (ii) stimulation with ACh enhances the rate of acid production in the acinar cells; and (iii) the acid extrusion mechanism is inhibited by amiloride addition to and Na⁺ removal from the bath solution. We suggest that both Na⁺/H⁺ and HCO ₃ ^– /Cl^– exchange transport mechanisms are taking roles in the intracellular pH regulation in the lacrimal gland acinar cells.     

Extracellular ATP increases free cytosolic calcium in rat parotid acinar cells. Differences from phospholipase C-linked receptor agonists.

The effects of extracellular ATP on intracellular free calcium concentration [( Ca2+]i), phosphatidylinositol (PtdIns) turnover, amylase release and Ca2+-activated membrane currents were examined in isolated rat parotid acinar cells and contrasted with the effects of receptor agonists known to activate phospholipase C. ATP was more effective than muscarinic and alpha-adrenergic agonists and substance P as a stimulus for elevating [Ca2+]i (as measured with quin2). The ATP effect was selectively antagonized by pretreating parotid cells with the impermeant anion-exchange blocker 4,4'-di-isothiocyano-2,2'-stilbenedisulphonate (DIDS), which also inhibited binding of [alpha-32P]ATP to parotid cells. By elevating [Ca2+]i, ATP and the muscarinic agonist carbachol both activated Ca2+-sensitive membrane currents, which were measured by whole-cell and cell-attached patch-clamp recordings. However, there were marked contrasts between the effects of ATP and the receptor agonists linked to phospholipase C, as follows. (1) Although the combination of maximally effective concentrations of carbachol, substance P and phenylephrine had no greater effect on [Ca2+]i than did carbachol alone, there was some additivity between maximal ATP and carbachol effects. (2) Intracellular dialysis with guanosine 5'-[beta-thio]diphosphate did not block activation of ion channels by ATP, but did block channel activation by the muscarinic agonist carbachol. This suggests that a G-protein is involved in the muscarinic response, but not in the response to ATP. (3) Despite its pronounced effect on [Ca2+]i, ATP had little effect on PtdIns turnover in these cells, in contrast with the effects of carbachol and other Ca2+-mobilizing agents. (4) Although ATP was able to stimulate amylase release from parotid acinar cells, the stimulation was only 33 +/- 9% of that obtained with phospholipase C-linked receptor agonists. These differences suggest that ATP increases [Ca2+]i through specific activation of a pathway which is distinct from that shared by the classical phospholipase C-linked receptor agonists.     

Receptor-mediated metabolism of the phosphoinositides and phosphatidic acid in rat lacrimal acinar cells.

The metabolism of the inositol lipids and phosphatidic acid in rat lacrimal acinar cells was investigated. The muscarinic cholinergic agonist methacholine caused a rapid loss of 15% of [32P]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and a rapid increase in [32P]phosphatidic acid (PtdA). Chemical measurements indicated that the changes in 32P labelling of these lipids closely resembled changes in their total cellular content. Chelation of extracellular Ca2+ with excess EGTA caused a significant decrease in the PtdA labelling and an apparent loss of PtdIns(4,5)P2 breakdown. The calcium ionophores A23187 and ionomycin provoked a substantial breakdown of [32P]PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P); however, a decrease in [32P]PtdA was also observed. Increases in inositol phosphate, inositol bisphosphate and inositol trisphosphate were observed in methacholine-stimulated cells, and this increase was greatly amplified in the presence of 10 mM-LiCl; alpha-adrenergic stimulation also caused a substantial increase in inositol phosphates. A23187 provoked a much smaller increase in the formation of inositol phosphates than did either methacholine or adrenaline. Experiments with excess extracellular EGTA and with a protocol that eliminates intracellular Ca2+ release indicated that the labelling of inositol phosphates was partially dependent on the presence of extracellular Ca2+ and independent of intracellular Ca2+ mobilization. Thus, in the rat lacrimal gland, there appears to be a rapid phospholipase C-mediated breakdown of PtdIns(4,5)P2 and a synthesis of PtdA, in response to activation of receptors that bring about an increase in intracellular Ca2+. The results are consistent with a role for these lipids early in the stimulus-response pathway of the lacrimal acinar cell.     

Extracellular calcium and the organization of tight junctions in pancreatic acinar cells

In pancreatic lobules incubated in Ca²⁺-free Krebs-Ringer bicarbonate solution +0.5 mM EGTA tight junctions are first disarrayed and then break up into fasciae occludentes and small fibrillar fragments, which move laterally in the plane of the plasmalemma and often wind up around the gap junctions. The interruption of the continuity of tight junctions results in the disappearance of the difference in intramembranous particle density between the lateral and luminal regions of the plasmalemma. These results are consistent with the interpretation of tight junctions as dynamic structures, probably resulting from a specific polymerization of intramembranous particles and confirm that tight junctions might have a role in establishing and maintaining the regional differences of the plasmalemma.     

Functional homogeneity of the non-mitochondrial Ca2+ pool in intact mouse lacrimal acinar cells.

In the absence of extracellular Ca2+, treatment of mouse lacrimal acinar cells with maximal concentrations of methacholine released Ca2+ from intracellular stores. No additional Ca2+ was mobilized by subsequent application of the intracellular Ca(2+)-ATPase inhibitor, thapsigargin, the stable inositol 1,4,5-trisphosphate ((1,4,5)IP3) analog, inositol 2,4,5-trisphosphate ((2,4,5)IP3) (by microinjection), or the Ca2+ ionophore, ionomycin. However, following prolonged activation of cells by methacholine in the presence of extracellular Ca2+, Ca2+ accumulated into a pool which was released by ionomycin but not by thapsigargin. This latter accumulation was blocked by prior microinjection of ruthenium red, indicating that it represents mitochondrial uptake. In saponin-permeabilized lacrimal cells, two Ca(2+)-sequestering pools were detected: (i) a ruthenium red-sensitive, thapsigargin-insensitive pool, presumed to be the mitochondria; and (ii) a ruthenium red-insensitive, thapsigargin-sensitive pool. Only the thapsigargin-sensitive pool accumulated Ca2+ at concentrations similar to those in unstimulated cells. The thapsigargin-sensitive Ca2+ pool was sensitive to (1,4,5)IP3; however, in contrast to findings in intact cells, only 44% of this pool was releasable by (1,4,5)IP3 or (2,4,5)IP3. These data indicate that, in intact lacrimal acinar cells, all exchangeable (ionomycin-sensitive) Ca2+ residues in a pool which responds homogeneously to agonists, (1,4,5)IP3, and thapsigargin. Prolonged elevation of [Ca2+]i results in Ca2+ accumulation into a second, ruthenium red-sensitive pool, presumably mitochondria. Finally, permeabilization of the cells fragments the non-mitochondrial pool, resulting in two pools, one sensitive and one insensitive to (1,4,5)IP3.     

Thapsigargin activates a calcium influx pathway in the unfertilized mouse egg and suppresses repetitive calcium transients in the fertilized egg.

At fertilization, the sperm initiates development of the mouse egg by inducing a large transient increase in the intracellular Ca2+ concentration ([Ca2+]i), which is followed by repetitive transient increases in [Ca2+]i. To determine how the repetitive Ca2+ transients are produced, thapsigargin, an inhibitor of the endoplasmic reticulum Ca-ATPase, was used to deplete intracellular Ca2+ stores within the egg. In the unfertilized egg, thapsigargin (1-50 microM) caused a slowly rising and falling transient increase in [Ca2+]i with or without extracellular Ca2+. An influx pathway for Ca2+ is activated by thapsigargin, since an immediate increase in [Ca2+]i occurred when Ca2+ was added to eggs after thapsigargin treatment in a Ca2+, Mg(2+)-free medium. This suggests that Ca2+ entry in the mouse egg may be coupled to the emptying of an intracellular store. The magnitude of the first Ca2+ transient at fertilization was reduced by as much as 84% in eggs pretreated with thapsigargin. Reduction of extracellular Ca2+, by addition of a Ca2+ chelator, suppressed the repetitive Ca2+ transients following fertilization. The Ca2+ transients also require filling of an intracellular store; they were suppressed when thapsigargin was added before or after fertilization. These results support the hypothesis that the first sperm-induced Ca2+ transient at fertilization depletes an intracellular Ca2+ store, triggering an increase in plasma membrane Ca2+ permeability, and that the enhanced Ca2+ influx causes repetitive Ca2+ transients due to the periodic filling and emptying of an intracellular Ca2+ store.     

Activation of rat basophilic leukemia cells. Temporal identification of the signal calcium influx mediated by the receptor-operated channel pathway

Antigen-induced 45calcium influx into rat basophilic leukemia (RBL) cells was examined with emphasis on the early time domain under conditions that exclude loss of the cation during the subsequent washing step. Such preparations demonstrate a distinct, temporally separate influx peaking at 3 min, followed by a substantial efflux. This internalized 45Ca2+ approaches millimolar total intracellular concentration and is therefore either sequestered or becomes bound to intracellular components (proteins). The amplitude of this influx is linearly proportional to IgE-receptor occupancy at low receptor occupancies, and is sensitive to the anti-allergic drug cromolyn. Furthermore, the timing of both the maximal uptake and the maximal susceptibility to cromolyn correlates with the Quin-2 signal in these cells, and the initial degranulation pattern bears some resemblance to trends in the 45Ca2+ uptake curve. These qualities suggest that the early peak at 2-3 min, rather than any later 45Ca2+ uptake, comprises the initial signalling Ca2+ pool. Maximal apparent inhibition by cromolyn for 45Ca2+ uptake was about 65% and required a 10-15-min preincubation with the cells. The inhibitory effect was limited to the peak at 3 min, suggesting that tracer incorporation beyond 5-6 min largely involves other pools or pathways, triggered by receptor aggregation, yet only indirectly related to channel activity or to the signal proper. A quantitative similarity was found between the early peak measured on intact cells and the single channel conductance measured on reconstituted planar bilayers containing the purified receptor for IgE and the purified cromolyn-binding protein [Corcia, A. et al. (1986) EM BO J. 5, 849-854]. This, as well as the effects of cromolyn, support the assumption that cromolyn-binding protein is a major constituent involved in this early influx, or that influx is a principal pathway for the signaling calcium mass.     

Monitoring of the activation of receptor-operated calcium channels in human platelets

Activation of receptor-operated calcium channels has been monitored by measurements of the quenching of the fluorescence of intracellularly trapped fura-2 by Mn entering from the extracellular medium. Release of calcium from intracellular stores was followed simultaneously by measurements of the ratio of the fluorescence excited at 340 and 380 nm. Thrombin, ADP, platelet-activating-factor (PAF) and collagen, all produced both release of calcium from the intracellular stores and uptake of Mn from the extracellular medium. The uptake of Mn, but not the increase of (Ca2+)i, was blocked by nickel. These results suggest the existence of plasma membrane calcium channels which can be activated by the different agonists tested here. The activation of calcium channels was very fast and transient with ADP and PAF, fast and maintained with thrombin, and delayed with collagen.     

Extracellular ATP4- promotes cation fluxes in the J774 mouse macrophage cell line

Extracellular ATP stimulates transmembrane ion fluxes in the mouse macrophage cell line J774. In the presence of Mg2+, nonhydrolyzable ATP analogs and other purine and pyrimidine nucleotides do not elicit this response, suggesting the presence of a specific receptor for ATP on the macrophage plasma membrane. One candidate for such a receptor is the ecto-ATPase expressed on these cells. We, therefore, investigated the role of this enzyme in ATP-induced 86Rb+ efflux in J774 cells. The ecto-ATPase had a broad nucleotide specificity and did not hydrolyze extracellular ATP in the absence of divalent cations. 86Rb+ efflux was not blocked by inhibition of the ecto-ATPase and did not require Ca2+ or Mg2+. In fact, ATP-stimulated 86Rb+ efflux was inhibited by Mg2+ and correlated with the availability of ATP4- in the medium. In the absence of divalent cations, the slowly hydrolyzable ATP analogs adenosine 5'-(beta, gamma-imido)triphosphate (AMP-PNP) and adenosine 5'-O-(3-thio)triphosphate (ATP-gamma-S) also stimulated 86Rb+ efflux, albeit at higher concentrations than that required for ATP4-. Exposure of J774 cells to 10 mM ATP for 45 min caused death of 95% of cells. By this means we selected variant J774 cells that did not exhibit 86Rb+ efflux in the presence of extracellular ATP but retained ecto-ATPase activity. These results show that the ecto-ATPase of J774 cells does not mediate the effects of ATP on these cells; that ATP4- and not MgATP2- promotes 86Rb+ efflux from these cells; and that hydrolysis of ATP is not required to effect this change in membrane permeability. These findings suggest that J774 cells possess a plasma membrane receptor which binds ATP4-, AMP-PNP, and ATP-gamma-S, and that the ecto-ATPase limits the effects of ATP on these cells by hydrolyzing Mg-ATP2-.     

Vasoactive intestinal peptide activates Ca2(+)-dependent K+ channels through a cAMP pathway in mouse lacrimal cells

The action of vasoactive intestinal peptide (VIP) on Ca2(+)-dependent K+ currents, in dissociated mouse lacrimal cells, was investigated using patch clamp techniques. In whole cell recordings, VIP (10-100 pM) increased the magnitude of the Ca2(+)-dependent K+ current. In single channel recordings, VIP increased the fraction of time the large charybdotoxin-sensitive Ca2(+)-activated K+ channel spent in the open state. The activity of this channel was also increased by adding forskolin or 8-bromo cAMP to the bath. Additionally, application of either cAMP or catalytic subunit of cAMP-dependent protein kinase directly to the cytoplasmic surface of excised inside out patches reversibly lengthened the time Ca2(+)-activated K+ channels spent in the open state. These data suggest that VIP stimulates Ca2(+)-activated K+ channels by a cAMP-dependent pathway in mouse lacrimal acinar cells.     

Acetylcholine-Induced Na+ influx in the mouse lacrimal gland acinar cells: Demonstration of multiple Na+ transport mechanisms by intracellular Na+ activity measurements

Summary In the isolated, superfused mouse lacrimal gland, intracellular Na⁺ activities (aNa _i ) of the acinar cells were directly measured with double-barreled Na⁺-selective microelectrodes. In the nonstimulated conditionaNa _i was 6.5±0.5 mM and membrane potential (V _m ) was –38.9±0.4 mV. Addition of 1 mM ouabain or superfusion with a K⁺-free solution slightly depolarized the membrane and caused a gradual increase inaNa _i . Stimulation with acetylcholine (ACh, 1 M) caused a membrane hyperpolarization by about 20 mV and an increase inaNa _i by about 9 mM in 5 min. The presence of amiloride (0.1 mM) reduced the ACh-induced increase inaNa _i by approximately 50%, without affectingV _m and input resistance in both nonstimulated and ACh-stimulated conditions. Acid loading the acinar cells by an addition/withdrawal of 20 mM NH₄Cl or by replacement of Tris⁺-buffer saline solution with HCO ₃ ^– /CO₂-buffered solution increasedaNa _i by a few mM. Superfusion with a Cl^–-free NO ₃ ^– solution or 1 mM furosemide or 0.5 mM bumetanide-containing solution had little effect on the restingaNa _i levels, however, it reduced the ACh-induced increase inaNa _i by about 30%. Elimination of metabolite anions (glutamate, fumarate and pyruvate) from the superfusate reduced both the restingaNa _i and the ACh-induced increase inaNa _i .The present results suggest the presence of multiple Na⁺ entry mechanisms activated by ACh, namely, Na⁺/H⁺ exchange, Na-K-Cl cotransport and organic substrate-coupled Na⁺ transport mechanisms.