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Originally published In Press as doi:10.1074/jbc.M707115200 on September 24, 2007

J. Biol. Chem., Vol. 282, Issue 47, 34542-34554, November 23, 2007
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Atrial Natriuretic Peptide Attenuates Elevations in Ca2+ and Protects Hepatocytes by Stimulating Net Plasma Membrane Ca2+ Efflux*

Anne K. Green{ddagger}1, Rebecca C. Stratton{ddagger}2, Paul E. Squires{ddagger}, and Alec W. M. Simpson§

From the {ddagger}Department of Biological Sciences, The University of Warwick, Gibbet Hill Road, Coventry CV4 7AL and the §Department of Human Anatomy and Cell Biology, The University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool L69 3GE, United Kingdom

Received for publication, August 24, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Elevations in intracellular Ca2+ concentration and calpain activity are common early events in cellular injury, including that of hepatocytes. Atrial natriuretic peptide is a circulating hormone that has been shown to be hepatoprotective. The aim of this study was to examine the effects of atrial natriuretic peptide on potentially harmful elevations in cytosolic free Ca2+ and calpain activity induced by extracellular ATP in rat hepatocytes. We show that atrial natriuretic peptide, through protein kinase G, attenuated both the amplitude and duration of ATP-induced cytosolic Ca2+ rises in single hepatocytes. Atrial natriuretic peptide also prevented stimulation of calpain activity by ATP, taurolithocholate, or Ca2+ mobilization by thapsigargin and ionomycin. We therefore investigated the cellular Ca2+ handling mechanisms through which ANP attenuates this sustained elevation in cytosolic Ca2+. We show that atrial natriuretic peptide does not modulate the release from or re-uptake of Ca2+ into intracellular stores but, through protein kinase G, both stimulates plasma membrane Ca2+ efflux from and inhibits ATP-stimulated Ca2+ influx into hepatocytes. These findings suggest that stimulation of net plasma membrane Ca2+ efflux (to which both Ca2+ efflux stimulation and Ca2+ influx inhibition contribute) is the key process through which atrial natriuretic peptide attenuates elevations in cytosolic Ca2+ and calpain activity. Moreover we propose that plasma membrane Ca2+ efflux is a valuable, previously undiscovered, mechanism through which atrial natriuretic peptide protects rat hepatocytes, and perhaps other cell types, against Ca2+-dependent injury.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A rise in intracellular Ca2+ concentration ([Ca2+]i) is a common early event in cellular injury (1-6). Harmful increases in [Ca2+]i can result from increased Ca2+ influx, sustained Ca2+ release from intracellular stores, or a reduced rate of Ca2+ extrusion across the plasma membrane (1, 7-8).

In hepatocytes, increases in [Ca2+]i are associated with cytotoxicity (9) arising from diverse agents such as carbon tetrachloride (10, 11), paracetamol (12-14), extracellular ATP (15-19), and hydrophobic bile acids (20). Elevated [Ca2+]i also injures hepatocytes in several pathophysiological liver conditions, including ischemia-reperfusion injury (6, 21), endotoxaemia (22), and hemorrhagic shock (23).

The [Ca2+]i rise activates Ca2+-dependent enzymes including calpain, a neutral cysteine protease (24) that plays a key role in initiation of cellular injury and subsequent necrosis or apoptosis (6, 25-30). In hepatocytes, calpain promotes hepatocyte plasma membrane blebbing (31), induces the mitochondrial permeability transition in hepatocyte necrosis (32), and contributes to hepatocyte necrosis in anoxia (33). Calpain has been implicated in hepatotoxicity in ischemia-reperfusion injury (34, 35), hemorrhagic shock (36), and bile acid toxicity (20).

In the search for cytoprotective agents and therapeutic interventions, those which suppress injurious [Ca2+]i rises are considered highly promising (2-6, 30). Natriuretic peptides attenuate elevations in cytosolic free Ca2+ concentration ([Ca2+]c) in many cell types (37-50), including rat hepatocytes (51). Atrial natriuretic peptide (ANP)3 is a circulating hormone released mainly by atrial myocytes (52) in response to stress conditions such as volume expansion or cardiac hypoxia. In addition to its hypotensive, vasodilatory, and natriuretic effects in the cardiovascular and renal systems (53), increasing evidence supports a more widespread role for ANP in several other organs (54), including the liver. ANP elevates cGMP in rat hepatocytes (51, 55-56) through the guanylyl cyclase A receptor (57), and is cytoprotective in both isolated hepatocytes (51, 55, 58) and perfused liver (57, 59-62). An early preliminary study showed that ANP attenuated both oxidant-induced cell injury and the accompanying [Ca2+]i rise (51), suggesting that the hepatoprotective effects of ANP may be mediated by mechanisms involved in Ca2+ homeostasis (51, 55, 57). We have since examined the effects of ANP on physiological alterations in [Ca2+]c; we showed that ANP, through protein kinase G (PKG), attenuates [Ca2+]c oscillations, dramatically increases the basal rate of plasma membrane Ca2+ efflux and modestly inhibits basal Ca2+ influx in rat hepatocytes (56).

Although it has been demonstrated that ANP prevents oxidant-induced rises in hepatocyte [Ca2+]i (51), how ANP regulates pathophysiological elevations in hepatocyte [Ca2+]c has been very little studied. Cellular Ca2+ homeostasis is maintained through a complex interaction of Ca2+ fluxes between the cytosol, the intracellular stores of the endoplasmic reticulum and mitochondria and the extracellular milieu. A single early study based on 45Ca2+ flux measurements suggested the involvement of a decrease in Ca2+ influx in the hepatoprotective effect of ANP (55). However, this was not a true uni-directional measurement of Ca2+ influx and possible effects of ANP on other Ca2+ handling mechanisms were not investigated. Further investigation of the effects of ANP on pathophysiological [Ca2+]c and Ca2+ fluxes is therefore warranted.

Here we examine the effects of ANP on sustained rises in both cytosolic Ca2+ concentration ([Ca2+]c) and calpain activity and reduction in cell viability induced by extracellular ATP in single rat hepatocytes. ATP is released from cells, including hepatocytes, in response to stress, anoxia, or injury (15, 63). At low, close-to-physiological, concentrations, extracellular ATP serves as a signaling molecule, inducing oscillations in cytosolic concentration of free Ca2+ ([Ca2+]c) (56, 64, 65) and modulating physiological and metabolic processes in hepatocytes (66). In contrast, ATP at higher concentrations induces sustained elevations in [Ca2+]i (16-18, 64) that can lead to hepatocyte injury (18, 19) and death by necrosis and apoptosis (15-17). Ca2+-mediated injury to hepatocytes by extracellular ATP has been revealed by calpain activation (18) and plasma membrane blebbing (19), both early events in the initiation of cell injury (6). Rosser et al. (18) have shown that ATP increases hepatocyte calpain activity concomitantly with an increase in [Ca2+]i, suggesting that ATP stimulates calpain activity through elevation of [Ca2+]i.

We show here that ANP attenuates ATP-induced sustained rises in [Ca2+]c. We also show that ANP protects hepatocytes against elevations in calpain activity and decreases in cell viability mediated by both ATP, and the hydrophobic bile acid, taurolithocholate (TLC). We therefore investigate the cellular Ca2+ handling mechanisms through which ANP attenuates the sustained elevation in [Ca2+]c. We show that ANP does not modulate the release from or re-uptake of Ca2+ into intracellular stores but, through PKG, stimulates plasma membrane Ca2+ efflux from and inhibits ATP-stimulated Ca2+ influx into hepatocytes. We therefore propose that stimulation of net plasma membrane Ca2+ efflux is the key process through which ANP protects hepatocytes against harmful rises in [Ca2+]c.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Collagenase was from Roche Applied Sciences. Williams' medium E (WME) was from Invitrogen. ATP, [Arg8]vasopressin (AVP), ANP (rat atrial natriuretic factor 1-28), ionomycin, propidium iodide, sodium taurolithocholate (TLC), digitonin, BAPTA-AM, sodium nitroprusside (SNP), and 8-bromo-cGMP were from Sigma. Rp-8-pCPT-cGMPS (TEA salt), thapsigargin, and MDL 28170 were from CN Biosciences. FMC Sea-Plaque-agarose and FMC SeaPrep-agarose were from Flowgen Instruments Ltd., Sittingbourne, UK. Fura-2 pentapotassium salt, fura-2 dextran (10 kDa), t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin (Boc-Leu-Met-CMAC) and Alexa Fluor 594-conjugated donkey anti-goat IgG (H+L; 2 mg/ml) were from Molecular Probes. Caloxin 1b1 peptide was prepared by custom synthesis by Dalton Pharma Services, Toronto, ON, Canada. Primary anti-PKG antibodies were from Santa Cruz Biotechnology, Inc. Citifluor was from Agar Scientific Ltd., Essex, UK. All other chemicals (VWR International Ltd., Lutterworth, UK) were of the purest grade available. For preparation of the efflux medium, ultrapure water was prepared using the Milli-Q RG system (Millipore).

Hepatocytes
Single hepatocytes were isolated from 150 to 250-g male Wistar rats by collagenase digestion as described previously (67). For all experiments on populations of hepatocytes, cells were counted using a hemocytometer then stored in suspension in WME on ice.

Measurement of cGMP
Isolated rat hepatocytes, at a cell density of 106/ml, were incubated in suspension in extracellular buffer (ECB) of composition: 150 mM NaCl, 10 mM KCl, 1 mM MgCl2, 10 mM HEPES, 1.8 mM CaCl2, 11 mM glucose, pH 7.2, in a shaking water bath at 37 °C. ATP and ANP were added for the periods indicated. Cyclic GMP levels were measured using the acetylation method in a cGMP enzyme immunoassay system (Biotrak assays, GE Healthcare).

Measurement of [Ca2+]c in Single Hepatocytes
Preparation of Single Cells for Microinjection—After harvesting, the hepatocytes were incubated at 37 °C at low density (~103 cells/ml) in 1.7% SeaPrep-agarose in WME. Single hepatocytes were transferred to 0.1-mm path length microslides containing 1.2% FMC SeaPlaque-agarose in WME, which was subsequently gelled at 4 °C for 2 min. The cells were then held at 37 °C under a layer of liquid paraffin.

Fura-2 Dextran Preparation—Fura-2 dextran (10 kDa) was dissolved in (in mM): KCl 150, PIPES 1, pH 7.2, to makea5mM stock solution that was stored at -70 °C. For microinjection, a small aliquot of 5 mM fura-2 dextran was held as a droplet under liquid paraffin.

Microinjection and Data Acquisition—Freshly pulled pipettes were filled with fura-2 dextran by dipping the tip for a few seconds in the fura-2 dextran droplet. Individual hepatocytes were injected to ~0.5% of the cell volume as described previously (68). The fura-2 dextran, thus localized in the cytoplasmic compartment, reports [Ca2+]c only. Injected cells of healthy appearance were transferred within microslides to a heated (37 °C) stainless steel perfusion chamber on the stage of a Nikon Diaphot inverted epifluorescence microscope. Cells were superfused with ECB to which ATP, ANP, Rp-8-pCPT-cGMPS, and AVP were added as indicated. Cells were alternately excited at 340- and 380-nm wavelengths by means of a PTI D101 dual excitation light source. The 510-nm emission was detected by an ICCD camera (Photonic Science). [Ca2+]c was calculated by calibrating the fura-2 signal in situ as described previously (69).

Measurement of Calpain Activity in Hepatocytes
Calpain activity of intact rat hepatocytes in suspension was measured by the rate of generation of the fluorescent cleavage product of the calpain substrate Boc-Leu-Met-CMAC according to a method modified from those of Rosser et al. (18) and Han et al. (70). Boc-Leu-Met-CMAC is a non-fluorescent, membrane-permeant compound that diffuses readily into hepatocytes, where it is conjugated to glutathione. Intracellular calpain activity cleaves the bond between the methionine and the MAC-thiol groups, liberating and unquenching the highly fluorescent, membrane-impermeant MAC-thiol moiety within the cell. The slope of the fluorescent change with respect to time represents the intracellular calpain activity (18, 70). Before each assay, ~105 cells were washed 3 times in ECB. The cells were then resuspended in 1 ml of ECB at 37 °C, to which Boc-Leu-Met-CMAC (20 µM final concentration) was added. Fluorescence was measured, at 37 °C with constant stirring, using a PTI DeltaRam spectrofluorimeter, exciting at 380 nm and collecting emission at 460 nm.


Figure 1
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  		FIGURE 1.
ANP stimulates cGMP production in rat hepatocytes. Isolated rat hepatocytes were incubated at 37 °C in the presence of no treatment ({blacksquare}), 25 µM ATP ({diamondsuit}), 200 nM ANP ({blacktriangleup}), or 25 µM ATP + 200 nM ANP (bullet) for the periods indicated. Total cGMP levels were measured by enzyme immunoassay. *, significantly different from both control (no treatment) and ATP-treated hepatocytes at each corresponding time point.

 
Cell Viability
Cell viability was determined by propidium iodide fluorescence according to a method modified from that of Gores et al. (71). Before each assay, ~105 cells were washed 3 times in ECB. The cells were then incubated with 1 µM propidium iodide in ECB containing 0.2% bovine serum albumin for 10 min at 37 °C. 1 ml of the cell suspension was transferred to a fluorimeter cuvette and fluorescence was measured continuously (at 37 °C with constant stirring) using a PTI DeltaRam spectrofluorimeter, exciting at 515 nm and collecting emission at 610 nm. Agonists were added after 5 min (F0) and fluorescence was recorded for a further 15 min (F15). At the end of each experiment, digitonin (375 µM final concentration) was added to allow propidium iodide to enter cells and, after 20 min, produce maximum fluorescence (Fmax). The % non-viable cells after 15 min agonist exposure was calculated using equation: % nonviable cells = 100 x (F15 - F0)/(Fmax - F0).

Measurement of Ca2+ Efflux from Hepatocytes
Before each assay, ~106 cells were washed 3 times in ice-cold efflux medium, of composition: 150 mM NaCl, 10 mM KCl, 1 mM MgCl2, 10 mM HEPES, 11 mM glucose, pH 7.2. Ca2+ efflux from the intact rat hepatocytes was monitored by measuring the concentration of Ca2+ in the extracellular medium ([Ca2+]o) using fura-2 as extracellular Ca2+ reporter. Thus the cells were resuspended in 1 ml of efflux medium at 37 °C, to which the membrane-impermeant pentapotassium salt of fura-2 (10 µM final concentration) was added. Fluorescence was measured at 37 °C with constant stirring, using a PTI DeltaRam spectrofluorimeter, exciting alternately at 340 and 380 nm and collecting emission at 510 nm. [Ca2+]o was calculated by calibrating the photometric 340/380 nm ratio signal as described previously (56). [Ca2+]o never rose above 1.5 µM, ensuring a true, unidirectional, Ca2+ efflux measurement.

Measurement of Mn2+ Influx into Single Hepatocytes
Single hepatocytes, microinjected with fura-2 dextran (10 kDa), were superfused with 50 µM MnCl2 in ECB to which ATP, ANP, and Rp-8-pCPT-cGMPS were added as indicated. The fura-2 dextran, thus localized in the cytoplasmic compartment, reports Mn2+ influx into the cytosol only. Cells were alternately excited at 340 and 359 nm wavelengths by means of a PTI D101 dual excitation light source. The 510-nm emission was detected by an ICCD camera (Photonic Science).

Immunocytochemistry
Primary Antibodies—The following primary anti-PKG antibodies (Santa Cruz) were used: cGKI{alpha} (N-16), goat polyclonal, targeted against a 16-amino acid sequence near the N terminus of PKG I{alpha}; cGKIbeta (L-16), goat polyclonal, targeted against a 16-amino acid sequence near the N terminus of PKG Ibeta; cGKI{alpha}/beta (T-19), goat polyclonal, targeted against a 19-amino acid sequence near the C terminus of PKG I{alpha} and PKG Ibeta; cGKII (T-18), goat polyclonal, targeted against a 18-amino acid sequence near the N terminus of PKG II.

Immunolabeling—Hepatocytes were allowed to attach to glass coverslips for 6 and 24 h in WME at 37 °C and then fixed in 4% paraformaldehyde. Nonspecific binding was prevented by blocking for 1 h at 25 °C in phosphate-buffered saline (PBS) + 0.01% Triton X-100 containing 10% normal rabbit serum. Following three 5-min washes with PBS, cells were incubated at 4 °C overnight with the corresponding primary antibody diluted (1:100) in PBS + 0.01% Triton X-100. Following antibody incubation, cells were washed with PBS and then incubated in Alexa Fluor 594-conjugated donkey anti-goat IgG (H+L) for 2 h at 25 °C. The secondary antibody was diluted 1:1000 in PBS + 0.01% Triton X-100. Cells were then washed 3 times for 10 min in PBS and coverslips were mounted in citifluor (glycerol/PBS solution) on glass slides. Cells were examined under oil immersion (x63) using a Leica DMRE upright laser scanning confocal microscope with a TCS SP2 scan head.

Statistics
Differences between means were compared by Student's t test using a level of significance of p < 0.05. Data were expressed as mean ± S.E. All experiments are from at least three independent hepatocyte preparations.


Figure 2
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  		FIGURE 2.
ANP attenuates ATP-induced [Ca2+]c rises in single rat hepatocytes. Single, fura-2 dextran-injected rat hepatocytes responding to 25 µM ATP (A) with a rise in [Ca2+]c were co-supplied with 200 nM ANP (B) or 25 µM Rp-8-pCPT-cGMPS (Rp) and 200 nM ANP (C) for the periods indicated by the bars. The traces shown in A-C are representative of several experiments. D and E show, respectively, the calculated mean increase in amplitude (nM) above resting [Ca2+]c and the duration (s) of the [Ca2+]c rise above resting [Ca2+]c for each treatment. Calculated values are shown above each corresponding column. *, significantly different from control.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
ANP Stimulates cGMP Production in ATP-treated Rat Hepatocytes—Fig. 1 shows that 25 µM ATP does not elevate hepatocyte cGMP levels above those detected in control, untreated rat hepatocytes. ANP (200 nM) stimulates cGMP production in isolated rat hepatocytes both in the presence and absence of ATP.

ANP, through PKG, Attenuates ATP-induced Elevations in [Ca2+]c in Single Rat Hepatocytes—A single early study showed that ANP prevented oxidant-induced elevation of [Ca2+]i in populations of isolated rat hepatocytes (51). Here we examine the effect of ANP on harmful elevations in [Ca2+]c in single rat hepatocytes. Single rat hepatocytes responded to 25 µM ATP by the elevation of [Ca2+]c (Fig. 2A, representative of 7/7 hepatocytes; Fig. 2, D and E, mean data). Co-application of 200 nM ANP caused a decrease in both the amplitude and duration of the ATP-induced [Ca2+]c rise (Fig. 2B, representative of 6/6 hepatocytes; Fig. 2, D and E, mean data). In the presence of the membrane-permeant PKG inhibitor, Rp-8-pCPT-cGMPS, however, ANP did not significantly attenuate either the amplitude or duration of the ATP-induced rise in [Ca2+]c (Fig. 2C, representative of 6/6 hepatocytes; Fig. 2, D and E, mean data). ANP applied alone did not alter resting [Ca2+]c levels (5/5 hepatocytes; results not shown).

ANP, through PKG, Prevents ATP- and TLC-induced Stimulation of Calpain Activity in Rat Hepatocytes—Rosser et al. (18) have shown that extracellular ATP, through a rise in [Ca2+]i, stimulates a concomitant rise in calpain activity in rat hepatocytes. Here we examine whether ANP modulates the ATP- and Ca2+-dependent rise in calpain activity. Calpain activity of intact rat hepatocytes in suspension was measured by the accumulation of the fluorescent proteolytic product of the calpain substrate Boc-Leu-Met-CMAC. Fig. 3A shows a typical spectrofluorimetric recording of basal calpain activity following the addition of Boc-Leu-Met-CMAC to hepatocytes in suspension. Following the addition of Boc-Leu-Met-CMAC, there is an initial lag of ~60 s duration before a linear increase in fluorescence intensity is recorded. This observation is consistent with previous studies (18, 70). Following this initial lag, the rate of increase in fluorescent intensity is a measure of intracellular calpain activity (18, 70).

Basal calpain activity, in the absence of any Ca2+-elevating agent (Fig. 3A), was found to be consistent with previous estimates (18, 70). Fig. 3B shows that addition of ATP (25 µM) stimulated a rapid, mean 1.6-fold (Fig. 3E) elevation in calpain activity. Co-application of ANP (200 nM) prevented any rise in calpain activity above basal levels (Fig. 3C). We also show that the hydrophobic bile acid TLC stimulated a similar elevation in calpain activity (Fig. 3E), which was prevented upon co-application of ANP (Fig. 3E). TLC elevates hepatocyte Ca2+ (72-74) through release from intracellular stores by directly increasing store Ca2+ permeability (73).


Figure 3
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  		FIGURE 3.
ANP attenuates the ATP-induced elevation in calpain activity in rat hepatocytes. Calpain activity in populations of intact rat hepatocytes was measured at 37 °C, using the fluorigenic calpain substrate Boc-Leu-Met-CMAC. The traces shown in A-D are representative of several experiments. 20 µM Boc-Leu-Met-CMAC (BOC), 25 µM ATP, 200 nM ANP, or 25 µM Rp-8-pCPT-cGMPS (Rp) were added where indicated by the arrows. E shows calculated mean rates of increase in fluorescence (fluorescence units/s/105 cells) from several experiments. In the presence of 20 µM Boc-Leu-Met-CMAC, populations of intact rat hepatocytes were exposed to 25 µM ATP, ATP + 200 nM ANP, ATP + ANP + 25 µM Rp-8-pCPT-cGMPS (Rp), Rp alone, ANP alone, 200 µM TLC, TLC + ANP, TLC + ANP + Rp, 1 µM thapsigargin (Tg), Tg + ANP, 2 µM ionomycin (iono), iono + ANP; 20 µM BAPTA (30 min preincubation), BAPTA + ATP, BAPTA + Tg, BAPTA + TLC, 20 µM calpain inhibitor MDL 28170 (MDL), MDL + ATP, MDL + Tg, and MDL + TLC during continuous spectrofluorimetry. *, significantly different from control; **, significantly different from both control and ATP + ANP; *{dagger}, significantly different from both control and TLC + ANP; *{ddagger}, significantly different from both control and Tg + ANP; *§, significantly different from both control and iono + ANP.

 
In the presence of the membrane-permeant PKG inhibitor, Rp-8-pCPT-cGMPS (25 µM), however, ANP was without significant effect on either the ATP- (Fig. 3D) or TLC-stimulated elevation in calpain activity (Fig. 3E). Neither ANP nor Rp-8-pCPT-cGMPS applied alone altered basal calpain activity (Fig. 3E).

Fig. 3E shows that preincubation (30 min) of hepatocytes with the intracellular Ca2+ chelator BAPTA-AM (20 µM) inhibited calpain activity to ~35% of basal and prevented an increase in calpain activity in the presence of either ATP or TLC. (Preincubation of hepatocytes for 30 min with the Me2SO vehicle did not alter basal calpain activity; results not shown.) The endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (1 µM) and the Ca2+ ionophore ionomycin (2 µM), well characterized agents that elevate [Ca2+]c, both increased calpain activity in hepatocytes (Fig. 3E). The magnitude and the time scale of the ATP-, thapsigargin-, and ionomycin-induced rises in hepatocyte calpain activity observed here are very similar to the findings of Rosser et al. (18). These data, taken together, suggest that both the basal calpain proteolytic activity and the elevated proteolysis in the presence of ATP or TLC are dependent on [Ca2+]c.

Fig. 3E also shows that, in the added presence of ANP, neither thapsigargin nor ionomycin elevated calpain activity above basal levels. This finding lends further support to our hypothesis that ANP prevents the ATP-induced elevation in calpain activity by attenuating the rise in [Ca2+]c.

The specificity of the assay for calpain activity was assessed by measuring the effect of the membrane-permeant calpain inhibitor MDL 28170, which inhibits calpain activity in many cell types including hepatocytes (75). Fig. 3E shows that MDL 28170 (20 µM) inhibited basal generation of the fluorescent proteolysis product to ~20% of basal. MDL 28170 also prevented increases in proteolytic activity by ATP, TLC, and thapsigargin (Fig. 3E).


Figure 4
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  		FIGURE 4.
ANP protects hepatocytes from ATP- and TLC-induced cell death. Cell viability of populations of rat hepatocytes was measured by propidium iodide fluorimetry. In the presence of 1 µM propidium iodide, populations of rat hepatocytes were exposed to 25 µM ATP, ATP + 200 nM ANP, ATP + ANP + 25 µM Rp-8-pCPT-cGMPS (Rp), 200 µM TLC, TLC + ANP, TLC + ANP + Rp, Rp alone, and ANP alone for 15 min at 37 °C during continuous fluorimetry. The figure shows calculated mean % changes in number of non-viable cells (calculated from changes in propidium iodide fluorescence) from several experiments. The mean values are shown above each corresponding column. *, significantly different from control; **, significantly different from both control and ATP + ANP; *{dagger}, significantly different from both control and TLC + ANP.

 
ANP, through PKG, Prevents the ATP- and TLC-induced Decline in Hepatocyte Viability—Fig. 4 shows that exposure of hepatocytes to either ATP (25 µM) or TLC (200 µM) for 15 min significantly increased the percentage of non-viable cells in the population. Co-addition of ANP protected hepatocytes; in the presence of ANP, neither ATP nor TLC elevated the % of non-viable hepatocytes above control levels. The PKG inhibitor, Rp-8-pCPT-cGMPS, prevented hepatoprotection by ANP; in the presence of Rp-8-pCPT-cGMPS (25 µM), ANP was without significant effect on either the ATP- or TLC-induced hepatocyte death. Neither ANP nor Rp-8-pCPT-cGMPS alone significantly altered hepatocyte viability compared with control levels. These data suggest that the hepatoprotective effect of ANP is mediated by PKG.

ANP, through PKG, Stimulates Ca2+ Efflux from ATP-treated Rat Hepatocytes—As shown in Fig. 5, A and G, a basal rate of Ca2+ efflux was recorded from intact rat hepatocytes in the absence of agonist. This observation is consistent with the findings of our previous studies (56, 76). ATP (25 µM) did not alter the basal rate of Ca2+ efflux (Fig. 5, B and G). Fig. 5, C and G, show that ANP (200 nM) stimulated a large, approximate 2.7-fold, increase in the rate of Ca2+ efflux from ATP-treated hepatocytes. Stimulation of Ca2+ efflux by ANP was prevented by the PKG inhibitor, Rp-8-pCPT-cGMPS; in populations of hepatocytes to which Rp-8-pCPT-cGMPS (25 µM) had previously been added, addition of ANP and ATP did not significantly increase Ca2+ efflux above the basal rate (Fig. 5, D and G). Fig. 5G also confirms that Rp-8-pCPT-cGMPS did not, itself, alter the basal rate of Ca2+ efflux from hepatocytes.

In contrast, but consistent with our previous findings (56), co-addition of ATP and SNP (100 µM), an NO donor that activates soluble guanylyl cyclase and elevates hepatocyte cGMP (56), did not significantly alter the basal rate of Ca2+ efflux (Fig. 5G). In further agreement with our previous observations, a moderate stimulation of Ca2+ efflux was achieved by co-application of ATP and 8-bromo-cGMP (100 µM), a membrane permeant analogue of cGMP (Fig. 5G).

Fig. 5, E and G, confirm that the basal Ca2+ efflux recorded here is predominantly due to plasma membrane Ca2+-ATPase (PMCA) activity; caloxin 1b1 peptide, a specific PMCA inhibitor (77), inhibited basal Ca2+ efflux by ~65%. This observation is consistent with our previous demonstration, using a similar assay, of basal hepatocyte Ca2+ efflux inhibition by the PMCA inhibitors carboxyeosin and mini-glucagon (76). Moreover, neither ATP (Fig. 5G), nor ATP and ANP (Fig. 5, F and G) stimulated Ca2+ efflux in the presence of caloxin 1b1 peptide, indicating that the ANP-stimulated Ca2+ efflux is PMCA-mediated.

ANP, through PKG, Inhibits ATP-induced Cation Influx into Single Rat Hepatocytes—The quench by Mn2+ of cytoplasmic fura-2 fluorescence was used to estimate relative rates of Ca2+ influx into single hepatocytes. Fig. 6, A and E, show that addition of 50 µM Mn2+ to single unstimulated hepatocytes promptly caused a quenching of the fura-2 signal at both 340 nm and the Ca2+-insensitive wavelength, 359 nm. This observation of continuous basal Mn2+ influx into unstimulated hepatocytes is consistent with previous studies of both our laboratory (56, 69) and others (78, 79).

Fig. 6, B and E, show that addition of ATP (25 µM) increased the rate of Mn2+ influx into single hepatocytes, a finding consistent with previous reports (80). Although we have previously shown that ANP partially inhibits basal Mn2+ influx into hepatocytes (56), its effect on ATP-stimulated Mn2+ influx, which occurs through a distinct channel (79, 81), has not previously been examined. Co-addition of ANP (200 nM) completely prevented the stimulation of Mn2+ influx by ATP (Fig. 6, C and E). In the presence of the PKG inhibitor Rp-8-pCPT-cGMPS, however, ANP was without effect; Fig. 6, D and E, show that Rp-8-pCPT-cGMPS (25 µM) prevented any inhibition of ATP-stimulated Mn2+ influx by ANP.

ANP Does Not Modulate the Release from or Re-uptake of Ca2+ into Intracellular Stores—We have shown here that ANP attenuates [Ca2+]c rises, inhibits ATP-stimulated Ca2+ influx, and stimulates Ca2+ efflux in the presence of ATP. However, it is not known whether the attenuation of the [Ca2+]c rise by ANP is entirely attributable to its observed effects on plasma membrane Ca2+ fluxes. It is conceivable that ANP may also modulate intracellular Ca2+ handling by altering the release from and/or re-uptake of Ca2+ into intracellular stores. The aim of this set of experiments was to determine whether, in the absence of plasma membrane Ca2+ fluxes, ANP is still effective in modulating the agonist-evoked [Ca2+]c rise.

ATP could not be used as the Ca2+ mobilizing agonist in this set of experiments, because it forms a precipitate with La3+. Instead, hepatocytes were stimulated with 50 nM AVP. AVP and ATP have similar effects on hepatocyte [Ca2+]i; they release the same pool of intracellular Ca2+ (82), the [Ca2+]c response to AVP, like that to ATP, is attenuated by ANP (results not shown), they stimulate Ca2+ influx through a common channel (81) and 50 nM AVP, like 25 µM ATP, does not alter basal Ca2+ efflux (results not shown). In normal Ca2+-containing ECB in the absence of La3+, the [Ca2+]c rise induced in single fura dextran-injected hepatocytes by 50 nM AVP was similar, in terms of both mean increase in [Ca2+]c (592 ± 36 nM; n = 3) and duration (548 ± 66 s; n = 3), to that evoked by 25 µM ATP (Fig. 2, A, D, and E). A typical recording is shown in Fig. 7A.


Figure 5
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  		FIGURE 5.
Effects of ANP on the rate of Ca2+ efflux from ATP-treated rat hepatocytes. Ca2+ efflux from populations of intact rat hepatocytes was measured at 37 °C, using fura-2 in the extracellular medium. The traces shown in A-F are representative of several experiments. 25 µM ATP, 200 nM ANP, 25 µM Rp-8-pCPT-cGMPS (Rp), or 300 µM caloxin 1b1 (caloxin) were added where indicated by the arrows. Calculated rates of Ca2+ efflux are shown in G; the mean Ca2+ efflux values are shown above each corresponding column. *, significantly different from control.

 
Agonists were applied to single fura dextran-injected hepatocytes in the presence of 0.5 mM La3+ in nominally Ca2+-free ECB to eliminate both Ca2+ influx (79, 81) and Ca2+ efflux (82-84). Fig. 7B shows that stimulation of single fura dextran-injected rat hepatocytes with AVP under these conditions evoked a rise in [Ca2+]c, but that the amplitude of AVP-induced [Ca2+]c rise was considerably decreased compared with that seen in normal Ca2+-containing ECB in the absence of La3+ (Fig. 7A). This observation is consistent with several previous studies that have shown that the amplitude of the hepatocyte [Ca2+]c rise evoked by several agonists, including AVP and ATP, is significantly reduced when Ca2+ influx is prevented (85-88). Under these conditions (0.5 mM La3+ in nominally Ca2+-free ECB), however, i.e. in the absence of plasma membrane Ca2+ fluxes, any effect of ANP on the rates of rise or decay of the AVP-induced [Ca2+]c elevations will be attributable to modulation of stored Ca2+ release or re-uptake, respectively (89). Fig. 7, B-G, show that neither the rates of rise (Fig. 7D) or decay (Fig. 7E) nor the mean amplitude (Fig. 7F) or duration (Fig. 7G) of the AVP-induced [Ca2+]c response was altered by the co-addition of ANP (Fig. 7C).

PKG Isoforms I{alpha} and Ibeta Are Expressed in Rat Hepatocytes—Two mammalian isotypes of PKG have been described: type I, consisting of an {alpha} and a beta isoform, splice variants of a single gene, and type II (90).

Immunoreactivity, using the commercially available antibodies cGKI{alpha} (N-16) and cGKIbeta (L-16), demonstrated expression of PKG isoform I{alpha} and PKG isoform Ibeta (visualized using Alexa 594, red secondary) in isolated rat hepatocytes seeded onto coverglass for 6 (results not shown) or 24 h (Fig. 8). No staining was observed using the Alexa 594 secondary antibody alone. Both isoform I{alpha} and isoform Ibeta appear localized to hot spots at the plasma membrane and at a region adjacent to the nucleus, with lesser diffuse staining throughout the cytosol (Fig. 8). Similar immunostaining was also achieved using antibody cGKI{alpha}/beta (T-19), targeted against a 19-amino acid sequence near the C terminus of PKG I{alpha} and PKG Ibeta. We have therefore demonstrated positive immunostaining for PKG isotype I using antibodies raised against both the N and C termini. In contrast, an absence of staining using antibody cGKII (T-18) suggested a lack of PKG isoform II expression (results not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Extracellular ATP induces potentially harmful [Ca2+]i elevations in rat hepatocytes. We have shown here that ANP, through PKG, attenuates both the amplitude and duration of the ATP-induced rise in [Ca2+]c, the accompanying Ca2+-dependent elevation in calpain activity and the decline in cell viability. Our study has then characterized the mechanism underlying the hepatoprotective effect of ANP against Ca2+-dependent injury (51). We have shown that ANP both stimulates Ca2+ efflux from and inhibits Ca2+ influx into ATP-treated hepatocytes. Under experimental conditions where plasma membrane Ca2+ fluxes are eliminated, ANP is without effect on [Ca2+]c rises, suggesting that it does not modulate the release from or re-uptake of Ca2+ into intracellular stores. The data thus suggest that stimulation of net plasma membrane Ca2+ efflux (to which both Ca2+ efflux stimulation and Ca2+ influx inhibition contribute) is the key process through which ANP attenuates [Ca2+]c rises and consequent hepatocyte injury.


Figure 6
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  		FIGURE 6.
ANP, through PKG, attenuates ATP-stimulated Mn2+ influx into single rat hepatocytes. Single, fura-2 dextran-injected, rat hepatocytes were superfused with 50 µM Mn2+ (A) and 25 µM ATP (B), 25 µM ATP + 200 nM ANP (C), or 25 µM ATP + 200 nM ANP + 25 µM Rp-8-pCPT-cGMPS (Rp; D) for the periods indicated by the bars. Changes in fluorescence were recorded at the Ca2+-insensitive wavelength, 359 nm. Fluorescence was normalized to the maximum fluorescence units. The traces shown in A-D are representative of several experiments. Calculated changes in Mn2+ influx are shown in E. For each cell, the rate of Mn2+ quench following addition of agonist(s) was expressed as a percentage of its basal rate of Mn2+ quench; the mean percentage values from several cells were then calculated and are shown above each corresponding column. *, significantly different from control; **, significantly different from both control and ATP + ANP.

 
It has been estimated that basal calpain activity is present when hepatocyte [Ca2+]i is 100-200 nM and increases significantly at [Ca2+]i between 400 and 1000 nM (18). Our data agree well with this estimation; we show here that ATP elevated [Ca2+]c from ~150-200 to ~800 nM (Fig. 2A) and stimulated a 1.6-fold increase in calpain activity (Fig. 3, B and E). Furthermore, we demonstrate here, for the first time, that ANP prevents the ATP-induced elevation in calpain activity. This effect is entirely consistent with our observation that in the added presence of ANP, ATP elevated [Ca2+]c to only ~400 nM (Fig. 2B). Together with our demonstration that ANP also prevents stimulation of calpain activity by either thapsigargin or ionomycin, this suggests that ANP prevents the ATP-induced elevation in calpain activity by attenuating the rise in [Ca2+]c.

We also show that ANP prevents elevation of calpain activity and diminished cell viability induced by the hydrophobic bile acid TLC. Hydrophobic bile acids are classic hepatotoxicants in vivo, causing cholestatic liver injury. Numerous studies have demonstrated that hydrophobic bile acids, including TLC, elevate hepatocyte Ca2+ (72-74, 91-93) and calpain activity (20), which play a key role in bile acid hepatotoxicity (20, 72-74, 91-93).

Our data suggest that the observed effects of ANP on hepatocyte Ca2+ homeostasis, calpain activity, and viability are mediated by cGMP and PKG. We have shown that ANP, at the concentration used throughout this study, stimulates cGMP production in rat hepatocytes in either the presence or absence of ATP. The PKG inhibitor, Rp-8-pCPT-cGMPS, prevents attenuation by ANP of the ATP-induced rise in both hepatocyte [Ca2+]c and calpain activity and also its hepatoprotective effect. Rp-8-pCPT-cGMPS also prevents the ANP-mediated modulation of both Ca2+ efflux and Ca2+ influx recorded in the presence of ATP.

Several previous studies, in diverse cell types, have demonstrated PKG-mediated inhibition of Ca2+ influx by natriuretic peptides (38, 89, 94-97). In contrast, considerably fewer studies have demonstrated plasma membrane Ca2+ efflux stimulation by natriuretic peptides: ANP has been shown to stimulate Na+-dependent Ca2+ efflux from rat cardiomyocytes (98). ANP and 8-bromo-cGMP stimulate Na+-independent Ca2+ efflux from vascular smooth muscle cells (99). We have previously shown that ANP and cGMP, through PKG, stimulate basal Ca2+ efflux from rat hepatocytes (56), and we show here that ANP also dramatically stimulates Ca2+ efflux from ATP-treated hepatocytes. Consistent with our observations, however, are several reports of Ca2+ efflux stimulation by cGMP. cGMP, through PKG, stimulates Ca2+ efflux from endothelial cells (100) and platelets (101). Furthermore, cGMP regulates [Ca2+]c in smooth muscle cells through stimulation of the PMCA (102); a similar role for cGMP in pancreatic acinar cells has also been suggested (103). It is possible, therefore, that future studies may subsequently reveal the involvement of Ca2+ efflux stimulation in the ability of natriuretic peptides to attenuate elevations in [Ca2+]c in other cell types, as we have demonstrated here in rat hepatocytes.

Ca2+ efflux from rat hepatocytes is achieved by the PMCA; the predominant PMCA isoform in adult rat liver and isolated hepatocytes is PMCA1, whereas PMCA2 is also present at much lower levels (104). Na+/Ca2+ exchange does not play a role in the regulation of [Ca2+]c in this cell type (105). Whereas we have demonstrated previously (76), and confirm here, that basal Ca2+ efflux from hepatocytes is predominantly PMCA-mediated, we now also show that ANP-stimulated hepatocyte Ca2+ efflux is mediated by the PMCA. Stimulation of PMCA by PKG has been shown in intact cells (99, 101), crude membrane preparations (102, 106), and partially purified enzyme preparations (107, 108). In both smooth muscle preparations (102) and intact platelets (101), PKG increased the Vmax of the PMCA without affecting its Km. PKG stimulated smooth muscle PMCA activity by up to 4-fold; this stimulation was independent of, and additive to, the approximate 2-fold stimulation by calmodulin (102). Intriguingly, however, several studies have demonstrated that PMCA itself is not a substrate for PKG (107-109). Of the many PMCA isoforms identified, only one, PMCA1b, contains the consensus sequence for phosphorylation by cyclic nucleotide-dependent protein kinases (110). However, it has been demonstrated that PMCA1b is not phosphorylated by PKG, but that direct phosphorylation of PMCA is not required for its stimulation by PKG (108). Indeed, rather than PMCA stimulation by direct PKG phosphorylation, two alternative mechanisms for PKG-mediated PMCA stimulation were proposed. First, it was suggested that PKG may stimulate the PMCA indirectly via phosphorylation of a phosphatidylinositol kinase, thus generating phosphatidylinositol 4-phosphate that binds and activates the PMCA (107). Alternatively, it was postulated that PKG phosphorylates an intermediate 240-kDa protein, later identified as the type I inositol, 1,4,5-trisphosphate receptor (111), which is responsible for PMCA activation (108). However, the same group has since demonstrated that whereas PKG does indeed stimulate the PMCA without phosphorylating it, PKG-dependent phosphorylation of the 240-kDa protein/inositol 1,4,5-trisphosphate receptor is not an essential step (112).


Figure 7
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  		FIGURE 7.
In the presence of La3+, ANP does not alter the agonist-induced rise in hepatocyte [Ca2+]c. Single fura-2 dextran-injected rat hepatocytes were supplied with 50 nM AVP (A), 0.5 mM La3+ and 50 nM AVP (B), or 0.5 mM La3+ and AVP + 200 nM ANP (C) for the periods indicated by the bars. The dashed lines indicate the time at which the superfusion medium was changed to nominally Ca2+-free ECB. The traces shown in A-C are representative of several experiments. D-G show, respectively, the calculated mean rate of rise (nM [Ca2+]c/s), rate of fall (nM [Ca2+]c/s), increase in amplitude (nM) above resting [Ca2+]c and duration (s) of the [Ca2+]c rise above resting [Ca2+]c for each treatment. Calculated values are shown above each corresponding column.

 
We have demonstrated here, by immunocytochemistry, the presence of PKG isoforms I{alpha} and Ibeta in primary rat hepatocytes. High levels of PKG type I are found in vascular smooth muscle cells, endothelial cells, and platelets, where it is thought to attenuate elevations in [Ca2+]c (90). It has been shown that PKG I{alpha}, but not PKG Ibeta, stimulates the partially purified porcine aorta PMCA without any detectable PMCA phosphorylation (112). We therefore propose that PKG I{alpha} mediates ANP-induced stimulation of the PMCA in rat hepatocytes.

Moreover, it has been demonstrated that ANP causes PKG isoform I to translocate to the plasma membrane, whereas SNP, an NO donor that activates soluble guanylyl cyclase, does not (113). This is entirely consistent with our previous finding, confirmed here, that SNP, unlike ANP, does not stimulate hepatocyte Ca2+ efflux. We previously proposed that their differing abilities to stimulate Ca2+ efflux may be related to the differing cellular location of their respective target guanylyl cyclases (56). Thus, ANP may, through plasma membrane guanylyl cyclase A receptor-mediated localized cGMP elevation, activate PKG I{alpha}, causing it to stimulate the PMCA. Elevation of cGMP in the bulk cytosol by SNP may therefore, in contrast, be ineffective in stimulating the PMCA. Indeed, since we first demonstrated differential effects of soluble and particulate guanylyl cyclase activation on Ca2+ signaling (56, 89), others have reported similar effects in several cell types that have likewise been attributed to cGMP compartmentation (43, 115, 116).


Figure 8
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  		FIGURE 8.
PKG isoforms I{alpha} and Ibeta are expressed in rat hepatocytes. Isolated rat hepatocytes were immunostained with either anti-PKG1{alpha} (cGKI{alpha} (N-16), B) or anti-PKG1beta (cGKIbeta (L-16), D). Alexa 594 anti-goat (red) was used to visualize immunoreactivity. Corresponding phase images demonstrate general cell morphology (A and C, respectively). The scale bar represents 10 µm and data are representative of three separate experiments.

 
The ability of ANP to attenuate pathophysiological elevations in [Ca2+]c by stimulation of net Ca2+ efflux may be particularly valuable in cytoprotection of hepatocytes, because we also show here that ATP does not, itself, stimulate Ca2+ efflux from hepatocytes. This finding is consistent with our previous observation that the rate of basal Ca2+ efflux from hepatocytes is not altered by Ca2+-mobilizing agonists at lower concentrations that stimulate [Ca2+]c oscillations (56). This is in contrast to the ability of Ca2+-mobilizing agonists to stimulate Ca2+ efflux from several other cell types (89, 117). This interesting observation deserves separate further investigation, but may be related to reports that the PMCA in rat hepatocytes, unlike other cell types, is not subject to dynamic regulation by calmodulin (110, 117). The classical model for calmodulin regulation of the PMCA involves calmodulin binding to the PMCA in a Ca2+-dependent manner, stimulating its Vmax and, more dramatically, decreasing its Km (Ca2+) by 1 order of magnitude from 10-20 mM to ~0.5 µM (110). In rat hepatocyte plasma membranes, however, the PMCA already exhibits high Ca2+ affinity (Km ~ 0.5 µM) in the absence of exogenously added calmodulin (118). It has therefore been proposed that the rat hepatocyte PMCA may be permanently associated with calmodulin, which may thus maintain the PMCA in the high Ca2+ affinity form (110, 117). Although Ca2+ itself stimulates PMCA activity (110, 118), it is possible that, in rat hepatocytes, the PMCA is already close to Ca2+ saturation even in unstimulated cells, and thus may be insensitive to agonist-evoked [Ca2+]c elevations. Indeed, it has been shown that subplasmalemmal [Ca2+]is higher than bulk [Ca2+]c even in unstimulated cells (119). ANP in contrast, is effective in stimulating hepatocyte Ca2+ efflux because PKG-mediated PMCA stimulation increases Vmax (101) and is, as discussed above, independent of, and additive to, stimulation by calmodulin (102).

Most attempts to avert or alleviate Ca2+-dependent hepatocyte injury have involved prevention of plasma membrane Ca2+ influx, and have been met with some success (13, 120-122). However, inhibition of Ca2+ influx (123) or removal of extracellular Ca2+ (124) does not always prevent hepatocyte injury (123-125). In contrast, possible hepatoprotective effects of Ca2+ efflux stimulation alone have not been investigated. However, intracellular chelation of excess cytosolic Ca2+ has been shown to be highly effective in preventing and reversing hepatocyte injury (19, 126). It is possible that removal of excess Ca2+ from the cytosol could be more effective than prevention of Ca2+ influx, particularly when the injurious [Ca2+]i rises originate from intracellular stores. It is probable that the modulatory effect of ANP on elevated [Ca2+]c is determined by the net flux of Ca2+ across the plasma membrane, to which both increased efflux and decreased influx contribute. However, we propose that stimulation of Ca2+ efflux is a key valuable, previously undiscovered, mechanism in ANP-mediated hepatoprotection, rendering the hepatocyte able to actively clear Ca2+ overload, rather than simply preventing further [Ca2+]i increases.

Through this and our previous study we have thus shown that ANP attenuates both physiological (56) and pathophysiological Ca2+ signals in hepatocytes. ANP may therefore serve an in vivo role as a circulating modulator of both physiological and pathophysiological elevations in hepatocyte [Ca2+]c. Moreover, mRNA coding for natriuretic peptides has been demonstrated in human liver tissue (127) suggesting that, in addition to circulating ANP, liver may be exposed to locally generated natriuretic peptides.

Ca2+- and calpain-dependent injury is not unique to hepatocytes. Indeed, a rise in [Ca2+]c is a common pivotal event in cellular injury and has been implicated in numerous disease processes, for example, pancreatitis (3), cardiovascular disease (4), and neurotoxicity (5). Similarly, calpain activation plays a key role in cellular injury in diverse pathologies, including oxidant-induced pancreatic acinar cell injury (27), myocardial ischemia-reperfusion injury (128), neurotoxicity (129), spinal cord injury (28), and endothelial dysfunction in type 2 diabetes (29). Because natriuretic peptides act on several organs (54) and attenuate elevations in [Ca2+]c in many cell types (37-50), it is unlikely that their cytoprotective effects are restricted to liver. Indeed, natriuretic peptides may have an in vivo role as circulating inhibitors of pathophysiological elevations in [Ca2+]c and calpain activity.

Natriuretic peptides and their receptors may therefore be promising therapeutic targets to protect cells, including hepatocytes, against Ca2+- and calpain-mediated injury. Natriuretic peptide therapy is already in development for the management of cardiovascular disorders (53, 130-132); clinical intervention with natriuretic peptides has been approved for treatment of heart failure (133). Of particular interest is a report that ANP infusion decreases portal pressure and increases hepatic blood flow in cirrhotic patients (134). Moreover, a recent study has demonstrated that inhibition of ANP degradation decreases intrahepatic vascular resistance, with little effects on systemic hemodynamics in cirrhotic rats (114). We predict that such treatment will also protect hepatocytes against Ca2+- and calpain-dependent injury. Natriuretic peptide therapy may prove to have more widespread clinical utility for the prevention or alleviation of Ca2+- and calpain-dependent cellular injury in several organs.

In conclusion, our data show that ANP attenuates ATP-induced elevations in [Ca2+]c and calpain activity in rat hepatocytes by modulating plasma membrane Ca2+ fluxes. We demonstrate that stimulation of net plasma membrane Ca2+ efflux is the key process through which ANP protects hepatocytes. Moreover we propose that Ca2+ efflux stimulation is a valuable, previously undiscovered, mechanism in the protection of hepatocytes, and perhaps other cell types, against Ca2+-dependent injury.


   FOOTNOTES
 
* This work was supported in part by Wellcome Trust Grant 065845/Z/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

2 Biotechnology and Biological Sciences Research Council funded Ph.D. student. Back

1 To whom correspondence should be addressed. Tel.: 44-2476-574184; Fax: 44-2476-523701; E-mail: A.K.Green{at}warwick.ac.uk.

3 The abbreviations used are: ANP, atrial natriuretic peptide; [Ca2+]c, cytosolic concentration of free Ca2+; PKG, protein kinase G; TLC, taurolithocholate; SNP, sodium nitroprusside; WME, Williams' medium E; AVP, [Arg8]vasopressin; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester; Rp-8-pCPT-cGMPS, 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate; Boc-Leu-Met-CMAC, t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin; ECB, extracellular buffer; PBS, phosphate-buffered saline; PMCA, plasma membrane Ca2+-ATPase; PIPES, 1,4-piperazinediethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We are grateful for expert advice on caloxin peptides from Dr. A. K. Grover, Department of Medicine, McMaster University, Ontario, Canada.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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