Dose-dependency in spatial dynamics of [Ca2+]c in pancreatic acinar cells

Spatial dynamics of cytosolic concentration of Ca2+, [Ca2+]c, in stimulus-secretion coupling of rat pancreatic acinar cell was monitored by a digital image analysing technique using Fura-2. When freshly isolated acini were stimulated with lower concentrations of CCK-8 (5-30 pM), [Ca2+]c increase began at the region beneath the basolateral membrane and the [Ca2+]c increase depended on the presence of extracellular Ca2+ ([Ca2+]o). CCK-8 at higher concentrations (100 pM and 1 nM), however, caused [Ca2+]c increase even in the absence of [Ca2+]o. Low concentrations of G-protein activator, NaF (10 mM or lower), caused [Ca2+]o-dependent increase in [Ca2+]c, whereas higher concentrations of NaF (15 mM or higher) increased [Ca2+]c in the absence of [Ca2+]o. These results are compatible with the view that G-protein activated by a physiological concentration of secretagogue accelerates Ca2+ entry. This process is in contrast to the process of Ca2+ release from intracellular stores, which can be predominant when pharmacological or toxic concentration of the secretagogue was applied.     

High [Na+]i in cardiomyocytes from rainbow trout

Intracellular Na(+)-concentration, [Na(+)](i) modulates excitation-contraction coupling of cardiac myocytes via the Na(+)/Ca(2+) exchanger (NCX). In cardiomyocytes from rainbow trout (Oncorhyncus mykiss), whole cell patch-clamp studies have shown that Ca(2+) influx via reverse-mode NCX contributes significantly to contraction when [Na(+)](i) is 16 mM but not 10 mM. However, physiological [Na(+)](i) has never been measured. We recorded [Na(+)](i) using the fluorescent indicator sodium-binding benzofuran isophthalate in freshly isolated atrial and ventricular myocytes from rainbow trout. We examined [Na(+)](i) at rest and during increases in contraction frequency across three temperatures that span those trout experience in nature (7, 14, and 21 degrees C). Surprisingly, we found that [Na(+)](i) was not different between atrial and ventricular cells. Furthermore, acute temperature changes did not affect [Na(+)](i) in resting cells. Thus, we report a resting in vivo [Na(+)](i) of 13.4 mM for rainbow trout cardiomyocytes. [Na(+)](i) increased from rest with increases in contraction frequency by 3.2, 4.7, and 6.5% at 0.2, 0.5, and 0.8 Hz, respectively. This corresponds to an increase of 0.4, 0.6, and 0.9 mM at 0.2, 0.5, and 0.8 Hz, respectively. Acute temperature change did not significantly affect the contraction-induced increase in [Na(+)](i). Our results provide the first measurement of [Na(+)](i) in rainbow trout cardiomyocytes. This surprisingly high [Na(+)](i) is likely to result in physiologically significant Ca(2+) influx via reverse-mode NCX during excitation-contraction coupling. We calculate that this Ca(2+)-source will decrease with the action potential duration as temperature and contraction frequency increases.     

Endothelin increases [Ca2+]i in rat pancreatic acinar cells by intracellular release but fails to increase amylase secretion.

In individual fura-2 loaded cells of rat pancreatic acini endothelin-1 (ET-1) (10-50 nM) induced sustained oscillations in [Ca2+]i. At higher concentrations a larger, but transient increase in [Ca2+]i was observed, which was largely unaffected by removal of extracellular Ca2+. ET-1 induced the release of Ca2+i from the same store as cholecystokinin (CCK), but with less potency. At concentrations of endothelin which transiently increased Ca2+, ET-1 increased the accumulation of inositol phosphates. Specific binding sites for 125I-endothelin were demonstrated on rat pancreatic acini. A single class of binding sites was identified with an apparent Kd 108 +/- 12 pM and Bmax of 171 +/- 17 fmol/mg for ET-1. The relative potency order for displacing [125I]ET was ET-1 greater than ET-2 greater than ET-3. In contrast to CCK and the non-phorbol ester tumour promoter Thapsigargin (TG) which induce both transient and sustained components of [Ca2+]i elevation, ET-1 failed to increase amylase release over the range 100 pM-1 microM.     

Cardiotonic steroids differentially affect intracellular Na+ and [Na+]i/[K+]i-independent signaling in C7-MDCK cells

Recently, we reported that ouabain kills renal epithelial and vascular endothelial cells independently of elevation of the [Na(+)](i)/[K(+)](i) ratio. These observations raised the possibility of finding cardiotonic steroids (CTS) that inhibit the Na(+),K(+) pump without attenuating cell survival and vice versa. To test this hypothesis, we compared CTS action on Na(+),K(+) pump, [Na(+)](i) content, and survival of Madin-Darby canine kidney cells. At a concentration of 1 microM, ouabain and other tested cardenolides, as well as bufadienolides such as bufalin, cinobufagin, cinobufotalin, and telobufotoxin, led to approximately 10-fold inhibition of the Na(+),K(+) pump, a 2-3-fold decrease in staining with dimethylthiazol-diphenyltetrazolium (MTT), and massive death indicated by detachment of approximately 80% of cells and caspase-3 activation. In contrast, Na(+),K(+) pump inhibition and elevation of [Na(+)](i) seen in the presence of 3 microM marinobufagenin (MBG) and marinobufotoxin did not affect MTT staining and cell survival. Inhibition of the Na(+),Rb(+) pump in K(+)-free medium was not accompanied by a decline of MTT staining and cell detachment but increased sensitivity to CTS. In K(+)-free medium, half-maximal inhibition of (86)Rb influx was observed in the presence of 0.04 microM ouabain and 0.1 microM MBG, whereas half-maximal detachment and decline of MTT staining were detected at 0.03 and 0.004 microM of ouabain versus 10 and 3 microM of MBG, respectively. Both ouabain binding and ouabain-induced [Na(+)](i),[K(+)](i)-independent signaling were suppressed in the presence of MBG. Thus, our results show that CTS exhibit distinctly different potency in Na(+),K(+) pump inhibition and triggering of [Na(+)](i)/[K(+)](i)-independent signaling, including cell death.     

Epidermal growth factor inhibits both cholecystokinin octapeptide-induced inositol 1,4,5-trisphosphate production and [CA2+]i increase in rat pancreatic acinar cells

We have studied the effects of epidermal growth factor (EGF) on both cholecystokinin octapeptide (CCK-OP)-induced inositol-1,4,5 trisphosphate (IP3) production and on cytosolic free calcium concentrations [Ca2+]i by fluorescence measurements in fura-2-loaded pancreatic acini. Our data show that EGF inhibits CCK-OP induced IP3 production by 40 +/- 9% and decreases CCK-OP induced rise in cytosolic Ca2+ by 41 +/- 9%. These data indicate that activation of EGF receptors leads to inhibition of CCK-OP induced stimulation of phospholipase C (PLC).     

Modulation of [Ca2+]i signaling dynamics and metabolism by perinuclear mitochondria in mouse parotid acinar cells

Parotid acinar cells exhibit rapid cytosolic calcium signals ([Ca2+]i) that initiate in the apical region but rapidly become global in nature. These characteristic [Ca2+]i signals are important for effective fluid secretion, which critically depends on a synchronized activation of spatially separated ion fluxes. Apically restricted [Ca2+]i signals were never observed in parotid acinar cells. This is in marked contrast to the related pancreatic acinar cells, where the distribution of mitochondria has been suggested to contribute to restricting [Ca2+]i signals to the apical region. Therefore, the aim of this study was to determine the mitochondrial distribution and the role of mitochondrial Ca2+ uptake in shaping the spatial and temporal properties of [Ca2+]i signaling in parotid acinar cells. Confocal imaging of cells stained with MitoTracker dyes (MitoTracker Green FM or MitoTracker CMXRos) and SYTO dyes (SYTO-16 and SYTO-61) revealed that a majority of mitochondria is localized around the nucleus. Carbachol (CCh) and caged inositol 1,4,5-trisphosphate-evoked [Ca2+]i signals were delayed as they propagated through the nucleus. This delay in the CCh-evoked nuclear [Ca2+]i signal was abolished by inhibition of mitochondrial Ca2+ uptake with ruthenium red and Ru360. Likewise, simultaneous measurement of [Ca2+]i with mitochondrial [Ca2+] ([Ca2+]m), using fura-2 and rhod-FF, respectively, revealed that mitochondrial Ca2+ uptake was also inhibited by ruthenium red and Ru360. Finally, at concentrations of agonist that evoke[Ca2+]i oscillations, mitochondrial Ca2+ uptake, and a nuclear [Ca2+] delay, CCh also evoked a substantial increase in NADH autofluorescence. This autofluorescence exhibited a predominant perinuclear localization that was also sensitive to mitochondrial inhibitors. These data provide evidence that perinuclear mitochondria and mitochondrial Ca2+ uptake may differentially shape nuclear [Ca2+] signals but more importantly drive mitochondrial metabolism to generate ATP close to the nucleus. These effects may profoundly affect a variety of nuclear processes in parotid acinar cells while facilitating efficient fluid secretion.     

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.     

An insulin-sensitive cation channel controls [Na+]i via [Ca2+]o-regulated Na+ and Ca2+ entry.

The insulin-stimulated cation channel previously identified in patch-clamped muscle preparations is here shown to be responsible for bulk Na+ entry into the cell. The mainly Na+ current of the channel was shown to be accompanied by an inhibitory Ca2+ component responsible for oscillations. Here, using quantitative fluorescence imaging of Fura-2- and SBFI-loaded soleus muscle, we measure changes in [Na+]i and [Ca2+]i related to channel function. Insulin increased [Na+]i and [Ca+]i in a transient spike of < 1-min duration. There was a momentary dip in [Na+]i related to inhibition of the channel by the Ca2+ spike, and changes in external Ca2+ were shown to alter [Na+]i via the cation channel, all effects being blocked by the specific channel inhibitor mu-conotoxin, but not by tetrodotoxin. The [Ca2+]i spike could also be induced by 8-bromo cyclic-guanosine 5'-monophosphate, an analogue of the channel-activator cyclic-guanosine 5'-monophosphate (cGMP). In addition it was noted that insulin reduced the [Ca2+]i rise upon subsequent muscle depolarization by a factor of 3.5. Insulin could be substituted with phorbol ester for the same effect and HA1004, a protein kinase inhibitor, blocked the reduction.     

Inhibition of Na+,K+-ATPase by ouabain triggers epithelial cell death independently of inversion of the [Na+]i/[K+]i ratio

Treatment with ouabain led to massive death of principal cells from collecting ducts (C7-MDCK), indicated by cell swelling, loss of mitochondrial function, an irregular pattern of DNA degradation, and insensitivity to pan-caspase inhibitor. Equimolar substitution of extracellular Na(+) by K(+) or choline(+) sharply attenuated the effect of ouabain on intracellular Na(+) and K(+) content but did not protect the cells from death in the presence of ouabain. In contrast to ouabain, inhibition of the Na(+)/K(+) pump in K(+)-free medium increased Na(+)(i) content but did not affect cell survival. In control and K(+)-free medium, ouabain triggered half-maximal cell death at concentrations of approximately 0.5 and 0.05 microM, respectively, which was consistent with elevation of Na(+)/K(+) pump sensitivity to ouabain in K(+)-depleted medium. Our results show for the first time that the death of ouabain-treated renal epithelial cells is independent of the inhibition of Na(+)/K(+) pump-mediated ion fluxes and the [Na(+)](i)]/[K(+)](i) ratio.     

Stimulant-evoked depolarization and increase in [Ca2+] i in insulin-secreting cells is dependent on external Na+

Summary The patch-clamp technique and measurements of single cell [Ca²⁺] _i have been used to investigate the importance of extracellular Na⁺ for carbohydrate-induced stimulation of RINm5F insulin-secreting cells. Using patch-clamp whole-cell (current-clamp) recordings the average cellular transmembrane potential was estimated to be –60±1 mV (n=83) and the average basal [Ca²⁺] _i 102±6nm (n=37). When challenged with either glucose (2.5–10mm) ord-glyceraldehyde (10mm) the cells depolarized, which led to the initiation of Ca²⁺ spike potentials and a sharp rise in [Ca²⁺] _i . Similar effects were also observed with the sulphonylurea compound tolbutamide (0.01–0.1mm). Both the generation of the spike potentials and the increase in [Ca²⁺] _i were abolished when Ca²⁺ was removed from the bathing media. When all external Na⁺ was replaced with N-methyl-d-glucamine, in the continued presence of either glucose,d-glyceraldehyde or tolbutamide, a membrane repolarization resulted, which terminated Ca²⁺ spike potentials and attenuated the rise in [Ca²⁺] _i . Tetrodotoxin (TTX) (1–2 m) was also found to both repolarize the membrane and abolish secretagogue-induced rises in [Ca²⁺] _i .     

Elevated [Cl-]i, and [Na+]i inhibit Na+, K+, Cl- cotransport by different mechanisms in squid giant axons

Bumetanide-sensitive (BS) unidirectional fluxes of (36)Cl- or (22)Na+ were measured in internally dialyzed squid giant axons while varying the intra- or extracellular concentrations of Na+ and/or Cl-. Raising either [Cl-]i or [Na+]i resulted in a concentration-dependent reduction of the BS influx of both (36)Cl- and (22)Na+. Raising [Cl-]i above 200 mM completely blocked BS influxes. However, raising [Na+]i to 290 mM resulted in saturable but incomplete inhibition of both BS Na+ influx and BS Cl- influx. The consequences of varying intracellular Cl- on cotransporter effluxes were complex. At lower [Cl-]i values (below 100 mM) intracellular Cl- activated cotransporter effluxes. Surprisingly, however, raising [Cl-]i levels > 125 mM resulted in a [Cl-]i-dependent inhibition of BS effluxes of both Na+ and Cl-. On the other hand, raising [Na+]i resulted only in the activation of the BS Na+ efflux; intracellular Na+ did not inhibit BS efflux even at 290 mM. The inhibitory effects of intracellular Na+ on cotransporter-mediated influxes, and lack of inhibitory effects on BS effluxes, are consistent with the trans-side inhibition expected for an ordered binding/release model of cotransporter operation. However, the inhibitory effects of intracellular Cl- on both influxes and effluxes are not explained by such a model. These data suggest that Cl may interact with an intracellular site (or sites), which does not mediate Cl transport, but does modulate the transport activity of the Na+, K+, Cl- cotransporter.     

Ethanol augments elevated-[Ca2+]C induced trypsin activation in pancreatic acinar zymogen granules

It has been long recognized that significant percentage of patients with acute pancreatitis often presents with a history of excessive alcohol consumption; however, the patho-physiological effect of ethanol on acute pancreatitis remains poorly understood. Abnormally elevated cytosolic Ca²⁺ ([Ca²⁺]_C) has been found to be a shared phenomenon in acute pancreatitis that could induce trypsin premature activation. Here, we present the effects of ethanol to sensitize zymogen granules (ZGs) of pancreas acinar cells to elevated [Ca²⁺]_C leading to zymogen premature activation that could result in acute pancreatitis. The pH fluctuations ([pH]_G), Ca²⁺ concentration ([Ca²⁺]_G), and premature trypsin activation inside the ZGs were monitored directly with specific fluorescence indicators. Our results showed that ethanol could act directly on ZGs and cause ZGs more receptive to elevated [Ca²⁺]_C that could induce premature activation of zymogen (trypsin). This alcohol-induced effect is concentration dependent and strongly influenced by the surrounding [Ca²⁺]_C. The K⁺ channels on ZGs membranes are required in the sensitization process. Our observations provide a mechanistic understanding of the role of ethanol in the initiation phase of alcoholic pancreatitis.     

Mechanisms of nordihydroguaiaretic acid-induced [Ca2+]i increases in MDCK cells

The effect of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells has been investigated. NDGA (10-100 microM) increased [Ca2+]i concentration-dependently. The [Ca2+]i increase comprised an initial slow rise and a plateau over a time period of 5 min. Ca2+ removal partly inhibited the Ca2+ signals induced by 25-100 microM NDGA and abolished that induced by 10 microM NDGA. In Ca(2+)-free medium, pretreatment with 0.1 mM NDGA for 12 min abolished the [Ca2+]i increase induced by the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) and the endoplasmic reticulum (ER) Ca2+ pump inhibitor thapsigargin (1 microM). However, 0.1 mM NDGA still increased [Ca2+]i after Ca2+ stores had been depleted by pretreating with 2 microM CCCP, 1 microM thapsigargin and 0.1 mM cyclopiazonic acid. NDGA (50 microM) activated Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength, which was almost abolished by 50 microM La3+. This implies NDGA induced Ca2+ influx mainly via a La(3+)-sensitive pathway. Consistently, 50 microM La3+ pretreatment inhibited 0.1 mM NDGA-induced [Ca2+]i increase. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 0.1 mM NDGA in Ca(2+)-free medium, suggesting NDGA activated capacitative Ca2+ entry. Pretreatment with 0.1 mM NDGA for 200 s prior to Ca2+ did not alter 1 microM thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 reduced 0.1 mM NDGA-induced Ca2+ release by 65%, but inhibiting phospholipase C with 2 microM U73122 had little effect. This suggests NDGA-induced Ca2+ release was independent of inositol 1,4,5-trisphosphate (IP3), but was modulated by phospholipase A2.