Differing significance of Na(+)-Ca2+ exchange in the regulation of cytosolic Ca2+ in rat exocrine gland acini and cardiac myocytes.

In order to compare the importance of Na(+)-Ca2+ exchange in the regulation of cytosolic Ca2+ concentration (Ca2+i), acini obtained from rat pancreas and submandibular glands as well as cardiac myocytes were loaded with Na+ by inhibition of Na(+)-K+ ATPase activity then loaded with fura-2. In the exocrine tissues, incubation in K(+)-free buffer or with ouabain had no substantial effect on resting Ca2+i or on the changes in Ca2+i following exposure to carbachol as compared with acini incubated under control conditions. In contrast, rat cardiac myocytes, treated identically, showed marked changes in Ca2+i under resting and stimulated conditions as compared with controls. We conclude that the Na(+)-Ca2+ exchange systems of rat pancreatic and submandibular gland acini contribute little to the overall regulation of Ca2+i at rest during cholinergic stimulation.     

Regulation of the epithelial Na+ channel by cytosolic ATP

The epithelial Na+ channel (ENaC), composed of three subunits (alphabetagamma), is expressed in various Na(+)-absorbing epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. By using patch clamp techniques, we have examined the effect of cytosolic ATP on the activity of the rat alphabetagammaENaC (rENaC) stably expressed in NIH-3T3 cells and in Madin-Darby canine kidney epithelial cells. The inward whole-cell current attributable to rENaC activity ran down when these cells were dialyzed with an ATP-free pipette solution in the conventional whole-cell voltage-clamping technique. This run down was prevented by 2 mM ATP (but not by AMP or ADP) in the pipette solution or by the poorly or non-hydrolyzable analogues of ATP (adenosine 5'-O-(thiotriphosphate) and adenosine 5'-(beta,gamma-imino)triphosphate) in both cell lines, suggesting that protection from run down was mediated through non-hydrolytic nucleotide binding. Accordingly, we demonstrate binding of ATP (but not AMP) to alpharENaC expressed in Madin-Darby canine kidney cells, which was inhibited upon mutation of the two putative nucleotide-binding motifs of alpharENaC. Single channel analyses indicated that the run down of currents observed in the whole-cell recording was attributable to run down of channel activity, defined as NPo (the product of the number of channels and open probability). We propose that this novel ATP regulation of ENaC may be, at least in part, involved in the fine-tuning of ENaC activity under physiologic and pathophysiologic conditions.     

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.     

Measurement of cytosolic free Ca2+ in individual pancreatic acini

The kinetics of changes in cytosolic free Ca2+ ([Ca2+]i) were determined in individual rat pancreatic acini by microfluorimetry. Three major findings are reported. First, at maximal stimulatory concentrations for amylase release, both caerulein and bombesin induced an initial rise in [Ca2+]i followed by prolonged secondary oscillations of smaller amplitude. The latter effect was not observed with supramaximal doses of caerulein. Second, these cyclic changes were dependent, at least in part, on extracellular Ca2+. Finally, comparison of the threshold doses for [Ca2+]i mobilization and enzyme discharge demonstrated that pathways independent of an elevation of [Ca2+]i control the secretory activity of pancreatic acini at low, picomolar agonist concentrations.     

Elevation of cytosolic [Ca2+] due to intracellular Ca2+ release retards carbachol stimulation of divalent cation entry in rat parotid gland acinar cells

This study examines the activation of divalent cation entry into rat parotid gland acinar cells by using Mn2+ as a Ca2+ surrogate cation. Following muscarinic-cholinergic stimulation of dispersed parotid acini with carbachol (10 microM), the onset of internal Ca2+ release (cytosolic [Ca2+], [Ca2+]i, increase) and the stimulation of Mn2+ entry (increase in fura2 quenching) are not simultaneously detected. [Ca2+]i elevation, due to intracellular release, is detected almost immediately following carbachol addition and peak [Ca2+]i increase occurs at 6.0 +/- 0.8 sec. However, there is an interval (apparent lag) between carbachol addition and the detection of stimulated Mn2+ entry. This apparent lag is decreased from 26 +/- 3.1 sec to 9.2 +/- 1.5 sec when external Mn2+ ([Mn2+]0) is increased from 12.5 to 500 microM. It is not decreased further with increase in [Mn2+]0 from 500 microM to 1 mM (9.8 +/- 2.1 sec), although both intracellular free Mn2+ and [Mn2+-fura2]/[fura2] increase. Thus, at [Mn2+]0 < 500 microM, the observed lag time is partially due to a limitation in the magnitude of Mn2+ entry. Furthermore, neither peak [Ca2+]i nor the time required to reach peak [Ca2+]i is significantly altered by [Mn2+]0 (12.5 microM to 1 mM). At every [Mn2+]0 tested (i.e., 12.5 microM-1 mM), the apparent lag is significantly greater than the time required to reach peak [Ca2+]i. However, when carbachol stimulation of the [Ca2+]i increase is attenuated by loading the acini with the Ca2+ chelator, 2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA), there is no detectable lag in carbachol stimulation of Mn2+ entry (with 1 mM [Mn2+]0). Importantly, in BAPTA-loaded acini, carbachol stimulates Mn2+ entry via depletion of the internal Ca2+ pool and not via direct activation of other divalent cation entry mechanisms. Based on these results, we suggest that the apparent lag in the detection of carbachol stimulation of Mn2+ entry into parotid acinar cells is due to a retardation of Mn2+ entry by the initial increase in [Ca2+]i, due to internal release, which most likely occurs proximate to the site of divalent cation entry.     

Video imaging of cytosolic Ca2+ in pancreatic beta-cells stimulated by glucose, carbachol, and ATP.

In order to define the differences in the distribution of cytosolic free Ca2+ ([Ca2+]i) in pancreatic beta-cells stimulated with the fuel secretagogue glucose or the Ca(2+)-mobilizing agents carbachol and ATP, we applied digital video imaging to beta-cells loaded with fura-2.83% of the cells responded to glucose with an increase in [Ca2+]i after a latency of 117 +/- 24 s (mean +/- S.E., 85 cells). Of these cells, 16% showed slow wave oscillations (frequency 0.35/min). In order to assess the relationship between membrane potential and the distribution of the [Ca2+]i rise, digital image analysis and perforated patch-clamp methods were applied simultaneously. The system used allowed sufficient temporal resolution to visualize a subplasmalemmal Ca2+ transient due to a single glucose-induced action potential. Glucose could also elicit a slow depolarization which did not cause Ca2+ influx until the appearance of the first of a train of action potentials. [Ca2+]i rose progressively during spike firing. Inhibition of Ca2+ influx by EGTA abolished the glucose-induced rise in [Ca2+]i. In contrast, the peak amplitude of the [Ca2+]i response to carbachol was not significantly different in normal or in Ca(2+)-deprived medium. Occasionally, the increase of the [Ca2+]i rise was polarized to one area of the cell different from the subplasmalemmal rise caused by glucose. The amplitude of the response and the number of responding cells were significantly increased when carbachol was applied after the addition of high glucose (11.2 mM). ATP also raised [Ca2+]i and promoted both Ca2+ mobilization and Ca2+ influx. The intracellular distribution of [Ca2+]i was homogeneous during the onset of the response. A polarity in the [Ca2+]i distribution could be detected either in the descending phase of the peak or in subsequent peaks during [Ca2+]i oscillations caused by ATP. In the absence of extracellular Ca2+, the sequential application of ATP and carbachol revealed that carbachol was still able to raise [Ca2+]i after exhaustion of the ATP response. This may be due to desensitization to the former agonist, since the response occurred in the same area of the cell. These results reveal subtle differences in [Ca2+]i distribution following membrane depolarization with glucose or the application of Ca(2+)-mobilizing agonists.     

Regulation of Na+/Ca2+ exchange activity by cytosolic Ca2+ in transfected Chinese hamster ovary cells

Transfected Chinese hamster ovary cells stably expressing thebovine cardiacNa⁺/Ca²⁺exchanger (CK1.4 cells) were used to determine the range of cytosolic Ca²⁺ concentrations([Ca²⁺]_i)that activateNa⁺/Ca²⁺exchange activity. Ba²⁺ influx wasmeasured in fura 2-loaded, ionomycin-treated cells under conditions inwhich the intracellular Na⁺concentration was clamped with gramicidin at ~20 mM.[Ca²⁺]_iwas varied by preincubating ionomycin-treated cells with either theacetoxymethyl ester of EGTA or medium containing 0-1 mM added CaCl₂. The rate ofBa²⁺ influx increased in asaturable manner with[Ca²⁺]_i,with the half-maximal activation value of 44 nM and a Hill coefficientof 1.6. When identical experiments were carried out with cellsexpressing a Ca²⁺-insensitivemutant of the exchanger, Ba²⁺influx did not vary with[Ca²⁺]_i.The concentration for activation of exchange activity was similar tothat reported for whole cardiac myocytes but approximately an order ofmagnitude lower than that reported for excised, giant patches. Thereason for the difference in Ca²⁺regulation between whole cells and membrane patches is unknown.

     

Correlation between Ca2+-stimulated adenosine triphosphatase activity and Ca2+ uptake in microsomal fraction of rat submandibular gland

Both the Ca2+-ATPase activity and the Ca2+ uptake in a microsomal fraction of rat submandibular gland were inhibited by the addition of indomethacin in vitro. The decrease of both the Ca2+-ATPase activity and the Ca2+ uptake caused by the drug closely paralleled each other (r = 0.97). The inhibitory manner of indomethacin on Ca2+-ATPase and Ca2+ uptake was noncompetitive for Ca2+. These results suggest that the Ca2+-ATPase in the microsomal fraction of rat submandibular gland is a Ca2+ pump in this tissue.     

Role of extracellular Ca2+ in acetylcholine-induced repetitive Ca2+ release in submandibular gland acinar cells of the rat

Acetylcholine (ACh) caused repetitive transient Cl⁻ currents activated by intracellular Ca²⁺ in single rat submandibular grand acinar cells. As the concentration of ACh increased the amplitude and the frequency of the transient Cl⁻ currents increased. These responses occurred also in the absence of extracellular Ca²⁺ but disappeared after several minutes. Repetitive transient Cl⁻ currents were restored by readmission of Ca²⁺ to the extracellular solution. The higher the concentration of extracellular Ca²⁺ readmitted, the larger the amplitude of the transient Cl⁻ currents. Ca²⁺ entry through a store-coupled pathway was detected by application of Ca²⁺ to the extracellular solution during a brief cessation of stimulation with ACh. In these experiments too, the higher the concentration of Ca²⁺, the larger the transient Cl⁻ currents activated by Ca²⁺ released from the stores. The time course of decrease in total charge movements of repetitive transient responses to ACh with removal of extracellular Ca²⁺ depended on a decrease in charge movements of each transient event rather than a decrease in frequency of the repetitive events. The decrease of charge movements of each transient event was due to a decrease in its amplitude rather than its duration. The results suggest that in this cell type an amplitude-modulated mechanism is involved in repetitive Ca²⁺ release and that Ca²⁺ entry is essential to maintain the repetitive release of Ca²⁺. The results further suggest that the magnitude of Ca²⁺ entry determines the number of unitary stores filled with Ca²⁺ which can synchronously respond to ACh. © 1996 Wiley-Liss, Inc.     

Activation of ouabain-sensitive p-nitrophenylphosphatase by carbachol and cGMP in rat submandibular gland

Na+,K+-ATPase activity was monitored by measuring ouabain-sensitive K+-dependent p-nitrophenylphosphatase (p-NPPase) activity in rat submandibular gland slices. Carbachol (carbamylcholine chloride) stimulated the p-NPPase activity in the presence of calcium but not in its absence. Carbachol activation of the enzyme was totally ouabain sensitive and could be blocked by atropine. A minimal requirement of sodium ion extracellularly was required for this carbachol stimulation. cGMP and its dibutyryl analogue was also effective in stimulating the enzyme activity, whereas, cAMP was ineffective. Calcium, however, was not required for cGMP activation of the p-NPPase activity. The result indicates that calcium is the second messenger and cGMP is the tertiary connection between cholinergic stimulation and Na+,K+-ATPase activation in these glands. Activation of Na+,K+-ATPase is postulated to be responsible for primary fluid formation.     

The secretory response in dissociated acini from the rat submandibular gland

Rat submandibular gland was dissociated by enzymatic digestion with collagenase and hyaluronidase, followed by mild mechanical shearing and filtration through a nylon mesh. The dissociated cell populations contained predominantly groups of acinar cells which maintained their acinar arrangement. The morphological and functional viability of the cells was confirmed by electron microscopic examination and a normal secretory response to β-adrenergic or cholinergic stimulation was observed. Both isoproterenol (IPR) and carbachol caused the fusion of secretory granules into large vacuoles which were also continuous with the lumen, and into which the secretory product was released. Secretion was assessed quantitatively from the incorporation of ¹⁴C-glucosamine into the acinar cells and its subsequent release into the culture medium as labelled glycoprotein. IPR stimulated secretion to 125% of untreated controls in the concentration range 5 × 10^?5 to 5 × 10^?7 M, and to 110% of controls at 5 × 10^?8 M, after 40 min incubation. Carbachol stimulated secretion to 131% of controls at 5 × 10^?5 M and to 115% at 5 × 10^?6 M but had no effect at 5 × 10^?7 or 5 × 10^?8 M. The secretory response was blocked by the respective β-adrenergic and cholinergic antagonists, propranolol and atropine. These findings show that dissociated rat submandibular acinar cells provide a useful in vitro model for the study of mucus synthesis and secretion.     

Swelling-induced Ca2+ release from intracellular calcium stores in rat submandibular gland acinar cells

The effects of osmotically-induced cell swelling on cytoplasmic free Ca2+ concentration ([Ca2+]i) were studied in acinar cells from rat submandibular gland using microspectrofluorimetry. Video-imaging techniques were also used to measure cell volume. Hypotonic stress (78% control tonicity) caused rapid cell swelling reaching a maximum relative volume of 1.78 +/- 0.05 (n = 5) compared to control. This swelling was followed by regulatory volume decrease, since relative cell volume decreased significantly to 1.61 +/- 0.08 (n = 5) after 10 min exposure to hypotonic medium. Osmotically induced cell swelling evoked by medium of either 78% or 66% tonicity caused a biphasic increase of [Ca2+]i. The rapid phase of this increase in [Ca2+]i was due to release of Ca2 + from intracellular stores, since it was also observed in cells bathed in Ca2+-free solution. The peak increase of [Ca2+]i induced by cell swelling was 3.40 +/- 0.49 (Fura-2 F340/F380 fluorescence ratio, n = 11) and 3.17 +/- 0.43 (n = 17) in the presence and the absence of extracellular Ca2+, respectively, corresponding to an absolute [Ca2+]i of around 1 microm. We found that around two-thirds of cells tested still showed some swelling-induced Ca2+ release (SICR) even after maximal concentrations (10(-5) M - 10(-4) M) of carbachol had been applied to empty agonist-sensitive intracellular Ca2+ stores. This result was confirmed and extended using thapsigargin to deplete intracellular Ca2+ pools. Hypotonic shock still raised [Ca2+]i in cells pretreated with thapsigargin, confirming that at least some SICR occurred from agonist-insensitive stores. Furthermore, SICR was largely inhibited by pretreatment of cells with carbonyl cyanide m-cholorophenyl hydrazone (CCCP) or ruthenium red, inhibitors of mitochondrial Ca2+ uptake. Our results suggest that the increase in [Ca2+]i, which underlies regulatory volume decrease in submandibular acinar cells, results from release of Ca2+ from both agonist-sensitive and mitochondrial Ca2+ stores.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

Regulation of free cytosolic Ca2+ in the peptic and parietal cells of the rabbit gastric gland

Quin 2-loaded isolated rabbit gastric glands and purified peptic cells were used to measure free cytosolic Ca2+ ([Ca2+]i) during hormone stimulation. Rabbit gastric glands are composed of peptic and parietal cells with less than 1% endocrine cells. Although both cell types responded to the same hormones, they may be distinguished in terms of the source of Ca2+ bringing about the change in [Ca2+]i. Experiments were designed to assign changes in [Ca2+]i to either the peptic or parietal cells and to attempt to maintain these distinctions in the mixed cell population of gastric glands. It was shown that the peptide cholecystokinin octapeptide induced a rapid and transient increase in [Ca2+]i of isolated peptic cells. This signal was independent of medium Ca2+ and insensitive to the Ca2+ channel blockers La3+ and nifedipine. In gastric glands, the Ca2+ outdependent increase in (Ca2+)i (the secondary transient) was slower and dose dependently blocked by La3+ and nifedipine. This allowed [Ca2+]i levels in the physiologically more intact rabbit gastric glands to be dissected and correlated with fluorescence changes of quin 2 in either cell type. The transient increase in [Ca2+]i coincided with a burst of pepsin but not acid secretion. A subsequent slower phase of pepsin secretion took place while the cells restored near resting [Ca2+]i. Using a combination of the Ca2+ ionophore A23187 and the protein kinase C activating phorbol ester 12-O-tetra-decanoylphorbol 13-acetate, the hormone response pattern of pepsin secretion could be mimicked. The intracellular Ca2+ stores of the peptic cells in the gastric gland remained depleted of Ca2+ until specific antagonists were added. The reloading of intracellular stores required medium Ca2+ although [Ca2+]i was maintained at resting level during the entire reloading period. Hence, a specialized pathway of Ca2+ reloading is postulated.     

Dose-dependent morphological changes of intercellular canaliculi during stimulation with carbachol and isoproterenol in the isolated rat submandibular gland

Intercellular canaliculi (IC) form a primary mixing reservoir for transcellularly and paracellularly secreted saliva whose composition depends on the degree of elevation of cytosolic Ca2+ and of cytosolic cyclic AMP concentrations caused by the secretagogues employed. In perfused rat submandibular gland (SMG), appearance of exocytosis on IC reflected the quantity of secreted mucin. Morphological observations were carried out by HR-SEM using a modified osmium maceration method on specimens treated with CCh and/or ISP. Mild secretory stimulation revealed that exocytosis did not occur simultaneously, even along the same intercellular canaliculus. Higher doses did not alter the spatial distribution of exocytosis along intercellular canaliculi but increased its temporal frequency, dose dependently. These findings lead us to conclude that, under low levels of secretory stimulation, exocytosis does not show a dose-dependent change, but that its spatial and temporal frequency changes in a dose-dependent manner.