Characterization of oscillations in cytosolic free Ca2+ concentration and measurement of cytosolic Na+ concentration changes evoked by angiotensin II and vasopressin in individual rat aortic smooth muscle cells. Use of microfluorometry and digital imaging

Dual wavelength microfluorometry was used to characterize the changes in cytosolic free Ca2+ concentration [( Ca2+]i) in individual cultured rat aortic vascular smooth muscle cells (VSMC). Angiotensin II (ANG II) at 10(-8) M induced a transient rise in [Ca2+]i from 43 +/- 2 to 245 +/- 23 nM, lasting for approximately 60 s (n = 42). In half of the population, discrete oscillations in [Ca2+]i of smaller amplitude occurred after the initial [Ca2+]i peak, with a period of 58 +/- 8 s and a maximum height of 132 +/- 24 nM. A similar oscillatory pattern was observed with arginine vasopressin (AVP). The oscillations depended upon the presence of extracellular Ca2+. Cytosolic free Na+ concentration ([Na+]i) in VSMC was also measured using the fluorescent Na+ probe sodium-binding benzofuran isophthalate. ANG II induced a gradual and sustained elevation of [Na+]i, from 24.0 +/- 6.2 to 36 +/- 9.7 mM. In response to AVP, [Na+]i rose to 41.0 +/- 11.6 mM. Video imaging of individual VSMC, with on-line ratio calibration of [Ca2+]i, revealed an inhomogeneous distribution of Ca2+ within the cell. [Ca2+] in the nucleus was invariably lower than in the cytoplasm in resting cells. In the cytoplasm, there were small regions in which [Ca2+] was elevated, or "hot spots." In Ca(2+)-containing medium, the initial rise in [Ca2+]i triggered by ANG II and AVP appeared to emanate from the hot spots and to spread evenly throughout the cytoplasm. Between [Ca2+]i oscillations, Ca2+ retreated back to the original hot spots. This study demonstrates the cellular and subcellular heterogeneity of [Ca2+]i both in resting VSMC and during stimulation by ANG II and AVP and reports the direct measurement of [Na+]i in VSMC. The results suggest an action of Ca2+ in both the initial and sustained phases of the response in VSMC and a link between changes in [Ca2+]i and [Na+]i.     

Kinetics and stoichiometry of coupled Na efflux and Ca influx (Na/Ca exchange) in barnacle muscle cells

Coupled Na+ exit/Ca2+ entry (Na/Ca exchange operating in the Ca2+ influx mode) was studied in giant barnacle muscle cells by measuring 22Na+ efflux and 45Ca2+ influx in internally perfused, ATP-fueled cells in which the Na+ pump was poisoned by 0.1 mM ouabain. Internal free Ca2+, [Ca2+]i, was controlled with a Ca-EGTA buffering system containing 8 mM EGTA and varying amounts of Ca2+. Ca2+ sequestration in internal stores was inhibited with caffeine and a mitochondrial uncoupler (FCCP). To maximize conditions for Ca2+ influx mode Na/Ca exchange, and to eliminate tracer Na/Na exchange, all of the external Na+ in the standard Na+ sea water (NaSW) was replaced by Tris or Li+ (Tris-SW or LiSW, respectively). In both Na-free solutions an external Ca2+ (Cao)-dependent Na+ efflux was observed when [Ca2+]i was increased above 10(-8) M; this efflux was half-maximally activated by [Ca2+]i = 0.3 microM (LiSW) to 0.7 microM (Tris-SW). The Cao-dependent Na+ efflux was half-maximally activated by [Ca2+]o = 2.0 mM in LiSW and 7.2 mM in Tris-SW; at saturating [Ca2+]o, [Ca2+]i, and [Na+]i the maximal (calculated) Cao-dependent Na+ efflux was approximately 75 pmol#cm2.s. This efflux was inhibited by external Na+ and La3+ with IC50's of approximately 125 and 0.4 mM, respectively. A Nai-dependent Ca2+ influx was also observed in Tris-SW. This Ca2+ influx also required [Ca2+]i greater than 10(-8) M. Internal Ca2+ activated a Nai-independent Ca2+ influx from LiSW (tracer Ca/Ca exchange), but in Tris-SW virtually all of the Cai-activated Ca2+ influx was Nai-dependent (Na/Ca exchange). Half-maximal activation was observed with [Na+]i = 30 mM. The fact that internal Ca2+ activates both a Cao-dependent Na+ efflux and a Nai-dependent Ca2+ influx in Tris-SW implies that these two fluxes are coupled; the activating (intracellular) Ca2+ does not appear to be transported by the exchanger. The maximal (calculated) Nai-dependent Ca2+ influx was -25 pmol/cm2.s. At various [Na+]i between 6 and 106 mM, the ratio of the Cao-dependent Na+ efflux to the Nai-dependent Ca2+ influx was 2.8-3.2:1 (mean = 3.1:1); this directly demonstrates that the stoichiometry (coupling ratio) of the Na/Ca exchange is 3:1. These observations on the coupling ratio and kinetics of the Na/Ca exchanger imply that in resting cells the exchanger turns over at a low rate because of the low [Ca2+]i; much of the Ca2+ extrusion at rest (approximately 1 pmol/cm2.s) is thus mediated by an ATP-driven Ca2+ pump.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMR studies of intracellular sodium ions in mammalian cardiac myocytes

The unambiguous measurement of intracellular sodium ion [Na+]i by the noninvasive NMR technique offers a new opportunity to monitor precisely the maintenance and fluctuations of [Na+]i levels in intact cells and tissues. The anionic frequency shift reagent, dysprosium (III) tripolyphosphate, which does not permeate intact cells, when added to suspensions of intact adult rat cardiac myocytes, alters the NMR frequency of extracellular sodium ions, [Na+]o, leaving that of intracellular ions, [Na+]i, unaffected. Using 23Na NMR in conjunction with this shift reagent, we have determined NMR-visible intracellular Na+ ion concentration in a suspension of isolated cardiac myocytes under standard conditions with insulin and Ca2+ in the extracellular medium to be 8.8 +/- 1.2 mmol/liter of cells (n = 4). This value is comparable to that measured by intracellular ion-selective microelectrodes in heart tissue. Cardiac myocytes incubated for several hours in insulin-deficient, Ca2+-containing medium prior to NMR measurement exhibited a somewhat lower [Na+]i value of 6.9 +/- 0.5 mmol/liter of cells (n = 3). Reversible Na+ loading of the cells by manipulation of extracellular calcium levels is readily measured by the NMR technique. Incubation of myocytes in a Ca2+-free, insulin-containing medium causes a 3-fold increase in [Na+]i to a level of 22.8 +/- 2.6 mmol/liter of cells (n = 10). In contrast to cells with insulin, insulin-deficient myocytes exhibit a markedly lower level of [Na+]i of only 14.6 +/- 2.0 mmol/liter of cells (n = 4) in Ca2+-free medium. These observations suggest that insulin may stimulate a pathway for Na+ influx in heart cells.     

Chick heart cells with high intracellular calcium concentration have a higher affinity for cardiac glycosides than those with low intracellular calcium concentration, as revealed by affinity labelling with a digoxigenin derivative.

Digital-imaging fluorescence microscopy with fura-2 allows the determination of intracellular calcium concentration ([Ca2+]i) in single cells. At a cell density of 10(5) cells/petri dish 44% of the chick embryo heart cells had a high [Ca2+]i of 99.4 +/- 7.1 nM and 56% of the cells a low [Ca2+]i of 27.8 +/- 4.4 nM (mean +/- SE). This laboratory previously reported that high-[Ca2+]i and low-[Ca2+]i cells from chick embryo hearts differ in their sensitivity to cardiac glycosides, as shown by measuring the increase in [Ca2+]i to reach a new steady state [Ahlemeyer, B., Weintraut, H., Seibold, G. & Schoner, W. (1991) in The sodium pump: recent developments (Kaplan, J. H. & De Weer, P., eds) pp. 653-656, Rockefeller University Press, New York]. This time we used N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate (HDMA) which binds irreversibly to amino groups of the Na+/K(+)-ATPase, and sheep anti-digoxigenin Fab fragments coupled with fluorescein isothiocyanate to identify different cardiac glycoside-binding sites. Half-maximal labelling of high-[Ca2+]i cells was obtained at 0.36 nM HDMA, and at 12.0 nM with the low-[Ca2+]i cells. Specific labelling of the cells by HDMA was 91% and 80% in high-[Ca2+]i and low-[Ca2+]i cells, respectively, as revealed by competition experiments with a 1000-fold excess of ouabain. HDMA half-maximally elevated the [Ca2+]i of high-[Ca2+]i cells at a concentration of 50 pM and that of low-[Ca2+]i cells at 8.0 nM. Concentrations higher than 0.1 microM produced signs of intoxication. When the labelled cells were subjected to a SDS/PAGE, a 100-kDa band was found to contain HDMA. The electrophoretic mobility of a protein labelled at 10 nM HDMA was slightly higher than that of a protein labelled at 1.0 microM. The data suggest that different isoforms of the alpha-subunit of Na+/K(+)-ATPase may exist in low-[Ca2+]i and high-[Ca2+]i cells of chick embryo heart.     

Deprivation of Na+, Ca2+ and Mg2+ from the extracellular solution increases cytosolic Ca2+ and stimulates catecholamine secretion from cultured bovine adrenal chromaffin cells

We report here that exposing cultured chromaffin cells to a low ionic strength medium (with sucrose in place of NaCl to maintain osmolarity) can induce a marked elevation in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine (CA) release. To determine the underlying mechanism, we first studied the effects of low [Na+]o on single cell [Ca2+]i (using fluo-3 as Ca2+ indicator) and CA release from many cells. In a Mg2+ and Ca2+-deficient medium, lowering the external concentration of Na2+ ([Na+]o) evoked CA secretion preceded by a transitory [Ca2+]i rise, the amplitude of which was inversely related to [Na+]o. By contrast, in the presence of either [Ca2+]o (2 mM) and [Mg2+]o (1.4 mM) or [Mg2+]o alone (3.4 mM), lowering the ionic strength was without effect. Furthermore, in a physiologic [Na+]o, [Ca2+]o and [Mg2+]o medium, two or three consecutive applications of the cholinergic agonist oxotremorine-M (oxo-M) consistently evoked a substantial [Ca2+]i rise. By contrast, consecutive applications of oxo-M in a Ca2+-deficient medium failed to evoke a rise in [Ca2+]i after the first exposure to the agonist. To clarify the underlying mechanism, we measured and compared the effects of low [Na+]o and the cholinergic agonists nicotine and oxo-M on changes in [Ca2+]i; we studied the effects of these agonists on both membrane potential, Vm (under current clamp conditions), and [Ca2+]i by single cell microfluorimetry (indo-1 as Ca2+ indicator). We observed that, in the presence of [Ca2+]o and [Mg2+]o, lowering [Na+]o had no effect on Vm. In a Ca2+-deficient medium, lowering [Na+]o depolarized the membrane from ca. –60 to –10 mV. As expected, we found that nicotine (10 M) depolarized the membrane (from ca. –60 to –20 mV) and simultaneously evoked a substantial [Ca2+]i rise that was [Ca2+]o-dependent. However, contrary to our expectations, we found that the muscarinic agonist oxo-M (50 M) also depolarized the membrane and induced an elevation in [Ca2+]i. Furthermore, both signals were blocked by D-tubocurarine, insinuating the nicotinic character of oxo-M in adrenal chromaffin cells from bovine. These results suggest that both nicotine and oxo-M stimulate Ca2+ entry, probably through voltage-gated Ca2+-channels. We also show here that oxo-M (and not low [Na+]o) stimulates phosphoinositide turnover.     

Augmentation of vasopressin-induced cAMP production by high potassium in rat renal papillary collecting tubule cells in culture.

The effect of high potassium, 60 mM KCl, on the cellular action of arginine vasopressin (AVP) was studied in rat renal papillary collecting tubule cells in culture. In the presence of 0.5 mM 3-isobutyl-1-methylxanthine AVP-induced cAMP production was enhanced by pretreatment of the cells with 60 mM KCl. Such an enhancement was not found in cells pretreated with Ca(2+)-free medium containing 1 mM EGTA or in Na(+)-free medium, which rather reduced AVP-induced cAMP production. Similar results were obtained with the blockers of cellular Ca2+ uptake, 1 x 10(-4) M verapamil and 1 x 10(-5) M nifedipine. The 60 mM KCl elevated the cellular sodium concentration ([Na+]i) from 15.1 to 18.8 mM, cellular pH (pHi) from 7.18 to 7.32, and basal cellular free calcium concentration ([Ca2+]i). These results indicate that high potassium promptly augments AVP-induced cAMP production in renal papillary collecting tubule cells. This effect is based on the alkalinized pHi and the increased [Ca2+]i.     

Effect of vasopressin on Na+ kinetics in cultured rat vascular smooth muscle cells

The effect of arginine vasopressin (AVP) on Na+ kinetics was examined in cultured rat vascular smooth muscle cells (VSMC) and rat renal papillary collecting tubule cells (RPCT) by the direct measurement of intracellular sodium concentration [(Na+]i) using fluorescence dye; SBFI. AVP increased [Na+]i in a dose-dependent manner at a concentration of 10(-9) M or higher in rat VSMC but did not affect [Na+]i in rat RPCT. The calcium (Ca2+)-free solution completely blocked the increasing effect of AVP on [Na+]i in rat VSMC. A Ca2+ ionophore, ionomycin (1-2 x 10(-6) M) increased [Na+]i both in rat VSMC and RPCT. The Ca2(+)-free solution abolished the ionomycin-increased [Na+]i both in rat VSMC and RPCT. These results therefore indicate that after binding the V1 receptor AVP increases [Na+]i mediated through an increase in cellular Ca2+ uptake in VSMC.     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na+ is known to increase the intracellular Ca2+ concentration ([Ca2+]i) in cardiac muscle, the exact mechanisms of low Na+ -induced increases in [Ca2+]i are not completely defined. To gain information in this regard, we examined the effects of low Na+ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca2+]i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na+ -induced increase in [Ca2+], was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na+ did not alter the ATP-induced increase in [Ca2+]i in the cardiomyocytes. This increase in [Ca2+]i due to low Na+ and elevated KCl was dependent on the extracellular concentration of Ca2+ (0.25-2.0 mM). The L-type Ca2+ -channel blockers, verapamil and diltiazem, at low concentrations (1 microM) depressed the low Na+, KCl-induced increase in [Ca2+]i without significantly affecting the response to low Na+ alone. The low Na+, high KCl-induced increase in [Ca2+]i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 microM), and diltiazem (5 and 10 microM) as well as with amiloride (5-20 microM), nickel (1.25-5.0 mM), cyclopiazonic acid (25 and 50 microM) and thapsigargin (10 and 20 microM). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 microM). These data suggest that in addition to the sarcolemmal Na+ - Ca2+ exchanger, both sarcolemmal Na+ - K+ ATPase, as well as the sarcoplasmic reticulum Ca2+ -pump play prominent roles in the low Na+ -induced increase in [Ca2+]i.     

Glucagon-like peptide-1 induces a cAMP-dependent increase of [Na+]i associated with insulin secretion in pancreatic beta-cells

Glucagon-like peptide-1 (GLP-1) elevates the intracellular free calcium concentration ([Ca2+]i) and insulin secretion in a Na+-dependent manner. To investigate a possible role of Na ion in the action of GLP-1 on pancreatic islet cells, we measured the glucose-and GLP-1-induced intracellular Na+ concentration ([Na+]i), [Ca2+]i, and insulin secretion in hamster islet cells in various concentrations of Na+. The [Na+]i and [Ca2+]i were monitored in islet cells loaded with sodium-binding benzofuran isophthalate and fura 2, respectively. In the presence of 135 mM Na+ and 8 mM glucose, GLP-1 (10 nM) strongly increased the [Na+]i, [Ca2+]i, and insulin secretion. In the presence of 13.5 mM Na+, both glucose and GLP-1 increased neither the [Na+]i nor the [Ca2+]i. In a Na+-free medium, GLP-1 and glucose did not increase the [Na+]i. SQ-22536, an inhibitor of adenylate cyclase, and H-89, an inhibitor of PKA, incompletely inhibited the response. In the presence of both 8 mM glucose and H-89, 8-pCPT-2'-O-Me-cAMP, a PKA-independent cAMP analog, increased the insulin secretion and the [Na+]i. Therefore, we conclude that GLP-1 increases the cAMP level via activation of adenylate cyclase, which augments the membrane Na+ permeability through PKA-dependent and PKA-independent mechanisms, thereby increasing the [Ca2+]i and promoting insulin secretion from hamster islet cells.     

A single cell model of myocardial reperfusion injury: Changes in intracellular Na+ and Ca2+ concentrations in guinea pig ventricular myocytes

To investigate the contribution of the changes in intracellular Na+ and Ca2+ concentrations ([Na+]i and [Ca2+]i) to myocardial reperfusion injury, we made an ischemia/reperfusion model in intact guinea pig myocytes. Myocardial ischemia was simulated by the perfusion of metabolic inhibitors (3.3 mM amobarbital and 5 M carbonyl cyanide m-chlorophenylhydrazone) with pH 6.6 and reperfusion was achieved by the washout of them with pH 7.4. [Na+]i increased from 7.9 ± 2.0 to 14.0 ± 3.4 mM (means ± S.E., p < 0.01) during 7.5 min of simulated ischemia (SI) and increased further to 18.8 ± 3.0 mM at 7.5 min after reperfusion. [Ca2+]i, expressed as the ratio of fluo 3 fluorescence intensity, increased to 133 ± 8% (p < 0.01) during SI and gradually returned to the control level after reperfusion. Intracellular pH decreased from 7.53 ± 0.04 to 6.31 ± 0.04 (p < 0.01) and recovered quickly after reperfusion. Reperfusion with the acidic solution or the continuous perfusion of hexamethylene amiloride (2 M) prevented the reperfusion-induced increase in [Na+]i. When the duration of SI was prolonged to 15 min, the cell response after reperfusion varied, 16 of 37 cells kept quiescent, 21 cells showed spontaneous Ca2+ waves, and 4 cells out of these 21 cells became hypercontracted. In quiescent cells, both [Na+]i and [Ca2+]i decreased immediately after reperfusion. In cells with Ca2+ waves, [Na+]i transiently increased further at the early phase of reperfusion, while [Ca+]i declined. In hypercontracted cells, [Na+]i increased as much as in Ca2+ wave cells, but [Ca2+]i increased extensively and both ion concentrations continued to increase. Reperfusion with the Ca2+-free solution prevented both the [Ca2+]i increase and morphological change. In the presence of ryanodine (10 M), the increase in [Ca2+]i after reperfusion was augmented and some cells became hypercontracted. We concluded that (1) Na+/H+ exchange is active both during SI and reperfusion, resulting in the additional [Na+]i elevation on reperfusion, (2) the [Na+]i level after reperfusion and the following Ca2+ influx via Na+/Ca2+ exchange are crucial for reperfusion cell injury, and (3) the Ca2+ buffering capacity of sarcoplasmic reticulum would also contribute to the Ca2+ regulation and cell injury after reperfusion.     

Trypanosoma cruzi: mechanisms of intracellular calcium homeostasis.

Regulation of intracellular Ca2+ homeostasis was characterized in epimastigote forms of Trypanosoma cruzi using the fluorescence probe Fura-2. Despite an increase in extracellular Ca2+, [Ca2+]o, from 0 to 2 mM, cytosolic Ca2+, [Ca2+]i, increased only from 85 +/- 9 to 185 +/- 21 nM, indicating the presence of highly efficient mechanisms for maintaining [Ca2+]i. Exposure to monovalent Na+ (monensin)-, K+ (valinomycin, nigericin)-, and divalent Ca2+ (ionomycin)-specific ionophores, uncouplers of mitochondrial respiration (oligomycin), inhibitors of Na+/K(+)-ATPase (ouabain), and Ca(2+)-sensitive ATPase (orthovanadate) in 0 or 1 mM [Ca2+]o resulted in perturbations of [Ca2+]i, the patterns of which suggested both sequestration and extrusion mechanisms. Following equilibration in 1 mM [Ca2+]o, incubation with orthovanadate markedly increased [Ca2+]i, results which are compatible with an active uptake of [Ca2+]i by endoplasmic reticulum. In contrast, equilibration in 0 or 1 mM [Ca2+]o did not influence the relatively smaller increase in [Ca2+]i following incubation with oligomycin, suggesting a minor role for the mitochondrial compartment. In cells previously equilibrated in 1 mM [Ca2+]o, exposure to monensin or ouabain, conditions known to decrease the [Na+]o/[Na+]i gradient, upon which the Na+/Ca2+ exchange pathways are dependent, markedly increased [Ca2+]i. In a complementary manner, decreasing the extracellular Na+ gradient with Li+ increased [Ca2+]i in a dose-dependent manner. Finally, the calcium channel blockers verapamil and isradipine inhibited the uptake of Ca2+ by greater than 50%, whereas diltiazem, nifedipine, and nicardipine were ineffective. The results suggest that epimastigote forms of T. cruzi maintain [Ca2+]i by uptake, sequestration, and extrusion mechanisms, with properties common to eukaryotic organisms.     

Cells from human bone giant cell tumors show a [Ca2+]o-sensing.

Giant cells from a human giant cell tumor of bone, showing several osteoclast features were tested for their capability of detecting the [Ca2+]o by a receptor like [Ca2+]o sensing. We found that cultured cells responded to elevation of [Ca2+]o, obtained adding 4 mM Ca2+ to the 2 mM Ca2+ containing buffer, by a transient increase of [Ca2+]i. Proliferative cells induced to differentiate by treatment with 10(-8) M 1,25 dihydroxyvitamin D3, were upregulated in their capability of responding to elevated [Ca2+]o. In fact, in this circumstance, the peak of [Ca2+]o-induced [Ca2+]i rise was increased compared to untreated cells. This suggests that 1,25 dihydroxyvitamin D3 induces a more efficient regulation of osteoclast activity.     

Ryanodine Inhibits Caffeine-Evoked Ca2+ Mobilization and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells

The effects of ryanodine, a selective inhibitor of the Ca(2+)-induced Ca2+ release mechanism, on caffeine-evoked changes in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine secretion were investigated using cultured bovine adrenal chromaffin cells. Caffeine (5-40 mM) caused a concentration-dependent transient rise in [Ca2+]i and catecholamine secretion in Ca2+/Mg(2+)-free medium containing 0.2 mM EGTA. Ryanodine (5 x 10(-5) M) alone had no effect on either [Ca2+]i or catecholamine secretion. Although the application of ryanodine plus caffeine caused the same increase in both [Ca2+]i and catecholamine secretion as those induced by caffeine alone, ryanodine (4 x 10(-7) - 5 x 10(-5) M) irreversibly prevented the increase in both [Ca2+]i and catecholamine secretion resulting from subsequent caffeine application over a range of concentrations. The secretory response to caffeine was markedly enhanced by replacement of Na+ with sucrose in Ca2+/Mg(2+)-free medium, and this enhanced response was also blocked by ryanodine. Caffeine was found to decrease the susceptibility of the secretory apparatus to Ca2+ in digitonin-permeabilized cells. These results indicate that caffeine mobilizes Ca2+ from intracellular stores, the function of which is irreversibly blocked by ryanodine, resulting in the increase in catecholamine secretion in the bovine adrenal chromaffin cell.     

The contributions of plasma membrane Na+-Ca2+-exchange and the Ca2+-ATPase to the regulation of cytosolic calcium ([Ca2+]i) in a clonal pituitary cell line (AtT-20) of mouse corticotropes

Single cell calcium microfluorimetry was used to examine the regulation of [Ca2+]i homeostasis in a clonal cell line of corticotropes (AtT-20 cells). Single cells, loaded with fura-2/AM, were exposed briefly to elevated potassium chloride (KCI, 40 mM, 5 sec). The time constant of decay of the [Ca2+]i signal was used as an index of [Ca2+]i extrusion and/or sequestration. Substitution of extracellular sodium with lithium, N-methyl-D-glucamine (NMDG), or Tris, increased resting levels of [Ca2+]i and significantly increased the time constant of [Ca2+]i decay by 40% compared to control indicating the participation of Na+-Ca2+-exchange. Prior exposure of single cells to thapsigargin (1 microM) or BuBHQ (10 microM). inhibitors of the SERCA Ca2+-ATPases, and/or the mitochondrial uncoupler FCCP (1 microM) did not significantly change the time constant of [Ca2+]i decay following KCl. Lanthanum ions (La3+), applied during the decay of the KCI-induced increase in [Ca2+]i, significantly increased the time constant of the return of [Ca2+]i to resting levels by 70% compared to control. Brief exposure of cells to sodium orthovanadate, an inhibitor of ATP-dependent pump activity, slowed and longer exposures prevented, the return of [Ca2+]i to resting levels. We conclude that neither intracellular SERCA pumps nor mitochondrial uptake contribute significantly to [Ca2+]i sequestration following a [Ca2+]i load and that the plasma membrane Ca2+-ATPase contributes to a greater extent than the Na+-Ca2+-exchanger to the return of [Ca2+]i to resting levels following a [Ca2+]i load under these experimental conditions.     

Agonist-specific regulation of [Na+]i in pancreatic acinar cells

In a companion paper (Zhao, H., and S. Muallem. 1995), we describe the relationship between the major Na+,K+, and Cl- transporters in resting pancreatic acinar cells. The present study evaluated the role of the different transporters in regulating [Na+]i and electrolyte secretion during agonist stimulation. Cell stimulation increased [Na+]i and 86Rb influx in an agonist-specific manner. Ca(2+)-mobilizing agonists, such as carbachol and cholecystokinin, activated Na+ influx by a tetraethylammonium-sensitive channel and the Na+/H+ exchanger to rapidly increase [Na+]i from approximately 11.7 mM to between 34 and 39 mM. As a consequence, the NaK2Cl cotransporter was largely inhibited and the activity of the Na+ pump increased to mediate most of the 86Rb(K+) uptake into the cells. Secretin, which increases cAMP, activated the NaK2Cl cotransporter and the Na+/H+ exchanger to slowly increase [Na+]i from approximately 11.7 mM to an average of 24.6 mM. Accordingly, secretin increased total 86Rb uptake more than the Ca(2+)- mobilizing agonists and the apparent coupling between the NaK2Cl cotransport and the Na+ pump. All the effects of secretin could be attributed to an increase in cAMP, since forskolin affected [Na+]i and 86Rb fluxes similar to secretin. The signaling pathways mediating the effects of the Ca(2+)-mobilizing agonists were less clear. Although an increase in [Ca2+]i was required, it was not sufficient to account for the effect of the agonists. Activation of protein kinase C stimulated the NaK2Cl cotransporter to increase [Na+]i and 86Rb fluxes without preventing the inhibition of the cotransporter by Ca(2+)-mobilizing agonists. The effects of the agonists were not mediated by changes in cell volume, since cell swelling and shrinkage did not reproduce the effect of the agonists on [Na+]i and 86Rb fluxes. The overall findings of the relationships between the various Na+,K+, and Cl- transporters in resting and stimulated pancreatic acinar cells are discussed in terms of possible models of fluid and electrolyte secretion by these cells.     

Energy requirements for the Na+ gradient in the oxygenated isolated heart: effect of changing the free energy of ATP hydrolysis

This study tests the hypothesis that a decrease of the free energy of ATP hydrolysis (Delta GATP) below a threshold value will inhibit Na+-K+-ATPase (Na+ pump) activity and result in an increase of intracellular Na+ concentration ([Na+]i) in the heart. Conditions were designed in which hearts were solely dependent on ATP derived from oxidative phosphorylation. The only substrate supplied was the fatty acid butyrate (Bu) at either low, 0.1 mM (LowBu), or high, 4 mM (HighBu), concentrations. Escalating work demand reduced the Delta GATP of the LowBu hearts. 31P, 23Na, and 87Rb NMR spectroscopy measured high-energy phosphate metabolites, [Na+]i, and Rb+ uptake. Rb+ uptake was used to estimate Na+ pump activity. To measure [Na+]i using a shift reagent for cations, extracellular Ca2+ was reduced to 0.85 mM, which eliminated work demand Delta GATP reductions. Increasing extracellular Na+ (Nae+) to 200 mM restored work demand Delta GATP reductions. In response to higher [Na+]e, [Na+]i increased equally in LowBu and HighBu hearts to approximately 8.6 mM, but Delta GATP decreased only in LowBu hearts. At lowest work demand the LowBu heart Delta GATP was -53 kJ/mol, Rb+ uptake was similar to that of HighBu hearts, and [Na+]i was constant. At highest work demand the LowBu heart Delta GATP decreased to -48 kJ/mol, the [Na+]i increased to 25 mM, and Rb+ uptake was 56% of that in HighBu hearts. At the highest work demand the HighBu heart Delta GATP was -54 kJ/mol and [Na+]i increased only approximately 10%. We conclude that a Delta GATP below -50 kJ/mol limits the Na+ pump and prevents maintenance of [Na+]i homeostasis.     

Contribution of sarcolemmal sodium-calcium exchange and intracellular calcium release to force development in isolated canine ventricular muscle

The aim of this work was to determine the relationship between peak twitch amplitude and sarcoplasmic reticulum (SR) Ca2+ content during changes of stimulation frequency in isolated canine ventricle, and to estimate the extent to which these changes were dependent upon sarcolemmal Na(+)-Ca2+ exchange. In physiological [Na+]o, increased stimulation frequency in the 0.2-2-Hz range resulted in a positive inotropic effect characterized by an increase in peak twitch amplitude and a decrease in the duration of contraction, measured as changes in isometric force development or unloaded cell shortening in intact muscle and isolated single cells, respectively. Action potentials recorded from single cells indicated that the inotropic effect was associated with a progressive decrease of action potential duration and a marked reduction in average time spent by the cell near the resting potential during the stimulus train. The frequency-dependent increase of peak twitch force was correlated with an increase of Ca2+ uptake into and release from the SR. This was estimated indirectly using the phasic contractile response to rapid (less than 1 s) lowering of perfusate temperature from 37 degrees C to 0-2 degrees C and changes of twitch amplitude resulting from perturbations in the pattern of electrical stimulation. Lowering [Na+]o from 140 to 70 mM resulted in an increase of contractile strength, which was accompanied by a similar increase of apparent SR Ca2+ content, both of which could be abolished by exposure to ryanodine (1 x 10(-8) M), caffeine (3 x 10(-3) M), or nifedipine (2 x 10(-6) M). Increased stimulation frequency in 70 mM [Na+]o resulted in a negative contractile staircase, characterized by a graded decrease of peak isometric force development or unloaded cell shortening. SR Ca2+ content estimated under identical conditions remained unaltered. Rate constants derived from mechanical restitution studies implied that the depressant effect of increased stimulation frequency in 70 mM [Na+]o was not a consequence of a decreased rate of refilling of a releasable pool of Ca2+ within the cell. These results demonstrate that frequency-dependent changes of contractile strength and intracellular Ca2+ loading in 140 mM [Na+]o require the presence of a functional sarcolemmal Na(+)-Ca2+ exchange process. The possibility that the negative staircase in 70 mM [Na+]o is related to inhibition of Ca(2+)-induced release of Ca2+ from the SR by various cellular mechanisms is discussed.     

Clearance of large Ca2+ loads in a single smooth muscle cell: examination of the role of mitochondrial Ca2+ uptake and intracellular pH

Redistribution of cytosolic free Ca2+ following Ca2+ influx into the cytoplasm was studied in single smooth muscle cells isolated from guinea-pig urinary bladder. Voltage-clamped cells were loaded with a low-affinity fluorophore Indo-1FF. A decay of free intracellular Ca2+ ([Ca2+]i) after the termination of the depolarizing pulse (1 s from -50 mV to +20 mV) was fitted with a single exponential and the effect of various substances on the time constant was compared. At a holding potential of +80 mV the [Ca2+]i decay was 1.56 times slower compared to that at -50 mV suggesting the presence of a voltage-dependent process redistributing Ca2+. In the presence of cyclopiazonic acid (CPA, 10 microM), an inhibitor of sarco(endo)plasmatic Ca2+ pump (SERCa), the [Ca2+]i decay was 3.93 times slower than that in the absence of the inhibitor. Introduction of a polycation Ruthenium Red (RR) (20 microM), an inhibitor of the mitochondrial Ca2+ uniporter, into a cell or collapsing a transmitochondrial H+ gradient with the protonophore CCCP (2 microM) slowed down the [Ca2+]i decay 6.05-fold and 9.78-fold, respectively. The apparent amplitude of [Ca2+]i increments was also increased by CCCP. Increasing H+ buffering power in the intracellular solution from 10 mM to 40 mM of HEPES greatly reduced the effect of CCCP on [Ca2+]i decay. A further increase in HEPES concentration to 100 mM eliminated the effects of CCCP both on the time course of [Ca2+]i decay and on the amplitude of [Ca2+]i increment. Perfusion of RR together with 100 mM HEPES into the cytoplasm was without effect on the decay time course of [Ca2+]i. The effect of CPA on [Ca2+]i decay was also reduced in cells loaded with 100 mM HEPES; the time constant in the presence of CPA was slowed down by a factor of 2.18. Application of 10 mM Na(+)-butyrate to the cells loaded with 10 mM HEPES resulted in a slowing down of [Ca2+]i decay: the time constant was increased by a factor of 5.84. Measurement of intracellular pH with SNARF-1 confirmed cytoplasmic acidification during application of Na(+)-butyrate and CCCP. It is concluded that the contribution of mitochondrial Ca2+ uptake to the rapid [Ca2+]i decay is much less than could be extrapolated from action of protonophores in these smooth muscle cells. The results also demonstrate the importance of intracellular pH for Ca2+ handling in the cytoplasm of smooth muscle cells.     

Ethanol-induced increases in [Ca2+]i and inositol (1,4,5) triphosphate in rat hepatocytes

Rat hepatocytes were studied for [Ca2+]i with Fura-2 at the single cell level using a microfluorometer-imaging system which showed that both the number of cells elevating [Ca2+]i and the magnitude of [Ca2+]i increase were directly dependent upon ethanol concentration between 50 mM and 1 M. Peak [Ca2+]i increases ranged from 27 nM with 50 mM ethanol to 57 nM after 1 M ethanol. Ethanol appeared to initiate calcium release from intracellular stores and caused a dose dependent production of inositol(1,4,5) triphosphate (Ins(1,4,5)P3) in hepatocytes. Low concentrations of ethanol (50-100 mM) did not significantly raise Ins(1,4,5)P3 although 300 mM-1 M increased Ins(1,4,5)P3 comparable to that found with vasopressin (5 nM). In summary, physiologic amounts of ethanol raise [Ca2+]i in rat hepatocytes, although at lower levels (50-100 mM) the changes may or may not be related to an Ins(1,4,5)P3 pathway.     

Unusual sensitivity of cytosolic free Ca2+ to changes in extracellular Ca2+ in rat C-cells

An essential function of C-cells is to monitor extracellular Ca2+ concentration ([Ca2+]e) and to respond to changes in [Ca2+]e by regulating hormone secretion. Using the calcitonin-secreting rat C-cell line rMTC 44-2, we have investigated a possible tight linkage between [Ca2+]e and cytosolic free Ca2+ ([Ca/+]i). We have demonstrated, using the Ca2+ indicator Quin 2, that the [Ca2+]i is particularly sensitive to changes in [Ca2+]e. Sequential increases in [Ca2+]e as small as 0.1 mM evoke clear elevations in [Ca2+]i. In contrast, other cell types tested did not alter their [Ca2+]i in response to increasing [Ca2+]e even to levels as high as 4.0 mM. Sequential 1.0 mM increments in [Ca2+]e caused the [Ca2+]i to rise from a base line of 357 +/- 20 nM Ca2+i at 1.0 mM Ca2+e to a maximum of 1066 +/- 149 nM Ca2+i at 5.0 mM Ca2+e. [Ca2+]e above 2.0 mM produced a biphasic response in [Ca2+]i consisting of an immediate (less than 5 s) spike followed by a decay to a new plateau. Treatment of rMTC 44-2 cells with either 50 mM K+ or 100 nM ionomycin at 1.0 mM Ca2+e caused an immediate spike in [Ca2+]i to micromolar levels. Pretreatment with EGTA or verapamil inhibited completely the increase in [Ca2+]i induced by 50 mM K+. However, pretreatment with EGTA only slightly attenuated the spike phase in [Ca2+]i produced by ionomycin, demonstrating that ionomycin released intracellular stores of calcium. We conclude that rMTC 44-2 cells regulate [Ca2+]i by monitoring small physiological changes in [Ca2+]e, the primary secretagogue for C-cells.