INTRODUCTION

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CALCIUM IS A critical intracellular second messenger that regulates a myriad of cell functions, including hormone secretion, cell motility, muscle contraction, and gene transcription (2). In addition to the role of Ca²⁺ as a regulator of normal physiological processes, Ca²⁺ is an important modulator of pathological processes, including inflammation (11) (via its effects on cytokine production and lymphocyte activation) and programmed cell death (4), i.e., apoptosis. Ca²⁺ homeostasis in an intact organ is more likely to reflect the actual in vivo state, because conditions of cell culture and isolation of cells cause large differences in cell Ca²⁺ handling (29). Also, the effects of cell-to-cell interaction via gap junctions and paracrine factors are present in intact organs but absent in isolated cells. Furthermore, studies indicate that the amplitude and frequency of Ca²⁺ oscillations encode important information in the single cell and likely in the whole organ as well (8, 15, 21). Recent advances in fluorescent microscopy have made it possible to examine intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and Ca²⁺ signaling in the intact perfused organ. Currently, only two studies by Robb-Gaspers and Thomas (21) and Nathanson et al. (15) have been performed in the perfused liver and only a few [Ca²⁺]_i agonists were examined.

The purpose of this study was to examine Ca²⁺ signaling in the intact liver using a variety of drugs that act at distinct mechanistic sites. We used laser scanning confocal microscopy to examine the effect of drugs that mobilize Ca²⁺ either directly, by a receptor-dependent mechanism, or indirectly, by inhibiting reuptake of Ca²⁺ into the endoplasmic reticulum. Arginine vasopressin (AVP) working through the V₁ receptor activates phospholipase C, inducing inositol 1,4,5-triphosphate (IP₃) production that diffuses to the IP₃ receptor on the endoplasmic reticulum and releases the IP₃-sensitive Ca²⁺ pool (15, 21, 22). In addition to the IP₃-sensitive Ca²⁺ store, a ryanodine-sensitive Ca²⁺ store is also present in many cell types, including hepatocytes (1). Although ryanodine binding and a ryanodine-sensitive Ca²⁺ pool have been demonstrated in hepatocytes, the effects of ryanodine have not been investigated in the intact liver. Recently, ATP has been shown to increase [Ca²⁺]_i in isolated hepatocytes (23). ATP acts to mobilize Ca²⁺ via a G protein-coupled P₂ purinoceptor. Evidence suggests that ATP and other nucleotides may be secreted into the extracellular space and provide a novel paracrine signaling pathway, but its effects in the intact liver are unknown (23). We also examined the effect of drugs that increase [Ca²⁺]_i by inhibiting reuptake of Ca²⁺ into the endoplasmic reticulum. Thapsigargin, a tumor-promoting sesquiterpene lactone, increases [Ca²⁺]_i by a receptor-independent mechanism (28). Thapsigargin irreversibly inhibits the endoplasmic reticulum Ca²⁺-ATPase pump, leading to depletion of intracellular Ca²⁺ stores. Cyclopiazonic acid (CPA) is a tetramic acid metabolite of Aspergillus and Penicillium that binds stoichiometrically to sarcoplasmic and endoplasmic reticulum Ca²⁺-ATPase, causing inhibition of the pump (4). Previously no studies on the effect of CPA on hepatocytes have been conducted. In contrast to the Ca²⁺ agonists, dantrolene reduces [Ca²⁺]_i by inhibiting release of Ca²⁺ from the sarcoplasmic and endoplasmic reticulum (5). Although the exact mechanism of action of dantrolene is not known, dantrolene is reported to cause a dose-dependent decrease in binding of ryanodine to hepatic microsomes. Novel observations on the effects of these drugs on Ca²⁺ signaling (Ca²⁺ waves and Ca²⁺ oscillations) in the intact liver and insight into Ca²⁺ regulation are the subjects of this report.

	EXPERIMENTAL PROCEDURES

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Isolated perfused rat liver technique. Male Sprague-Dawley rats (175-300 g; Harlan, Indianapolis, IN) were anesthetized with halothane, and the liver was perfused in situ via the hepatic portal vein with Earle's balanced salt solution (E-6132; Sigma, St. Louis, MO) according to the methods of Robb-Gaspers and Thomas (21). In brief, the median lobe of the liver was studied and other lobes were tied off and excised. Perfusion flow rate was 4 ml · min¹ · g¹ wet wt. Additional compounds added to Earle's perfusion media were (in mM) 1.0 lactate, 0.1 pyruvate, and 120 µM sulfobromophthalein (BSP), a competitive anion transport inhibitor that inhibits cellular extrusion of the fluorescent Ca²⁺ indicator. BSP does not affect liver viability or alter Ca²⁺ oscillations when added to isolated hepatocytes (21). Media were filtered with a 0.22-µm filter, and 5% adult bovine serum (Hyclone, Logan, UT) was added. Initially, the liver was perfused in a nonrecirculating mode for 10 min before it was changed to a recirculating system for an additional 15 min of recovery after surgery.

Loading of the liver with indo 1-AM. One milligram of the ratiometric fluorescent dye indo 1 acetoxymethyl ester (indo 1-AM; Molecular Probes, Eugene, OR) was dissolved in 25 µl of a 20% solution of Pluronic F-127 in DMSO (Molecular Probes). We diluted this 25-µl solution with an additional 50 µl of DMSO, and we slowly infused the resultant indo 1-AM solution into 250 ml of Earle's balanced salt solution while stirring. The liver then was perfused with the fresh solution containing indo 1-AM in a recirculating mode for 45 min. After confirmation of successful loading of the liver with the Ca²⁺ indicator via fluorescence microscopy, liver perfusion was changed to a nonrecirculating mode using buffer that did not contain indo 1-AM to wash out excess indo 1-AM. Temperature of the liver was maintained at 34°C, and media were oxygenated with 95% oxygen-5% carbon dioxide and perfused via a peristaltic pump (Cole-Palmer, Vernon Hills, IL).

Fluorescence imaging of [Ca²⁺]_i. Images were obtained using the Nikon RCM 8000 laser scanning confocal microscope system developed by Tsien (31) and as described previously (11). For measurement of [Ca²⁺]_i, the indo 1-loaded liver was excited at 351 nM with an argon ion laser (Coherent, Palo Alto, CA). Indo 1 is a dual-emission dye, and when it is excited at 351 nM, two wavelengths are emitted, i.e., a wavelength at 405 mM corresponding to indo 1 bound to Ca²⁺ and a wavelength at 480 mM corresponding to free indo 1. The emission bands at 400-440 nM and those >440 nM are separated by dichroic mirrors and long-path filters and detected by parallel photomultipliers. The analog signals are corrected for background and shading, digitized, ratioed, and displayed in color as high-resolution spatial concentration images. Images were stored on a Panasonic opticomagnetic disc recorder. For calibration of the indo 1 [Ca²⁺]_i determinations, the intensities of known test solutions of Ca²⁺ (Molecular Probes) varying from 0 to 1 mM were determined using the free acid indicator pentapotassium indo 1 (5 µM; Molecular Probes). These Ca²⁺ standards also contained K⁺ and phosphates to reflect the intracellular milieu. The ratiometric calculations were done using the method described by Grynkiewiez et al. (10).

Autofluoresence. An important point that was critical in accurately determining [Ca²⁺]_i was use of proper acquisition parameters (laser power, neutral density filters, pinhole size, photomultiplier gain) to minimize cell autofluorescence. Before loading with indo 1-AM, a background image of each liver was obtained. Using the lowest power settings on the argon laser and with the addition of neutral density filters, the background image of the hepatocytes exhibited minimal autofluorescence (Fig. 1, top). Small regions of bright fluorescence (~0.2-3 µm in diameter) were seen in cells abutting the hepatic plates. (Fig. 1, top). Suematsu et al. (26, 27) noted similar findings in liver undergoing ultraviolet illumination. These investigators reported that these areas were due to vitamin A autofluoresence located in the Ito cells (27). After obtaining background and shading images, we held constant all acquisition parameters affecting fluorescent image intensity (i.e., laser power, neutral density filters, photomultiplier tube gain, and pinhole size), and all observed changes in fluorescent intensity were likely to reflect changes due to the Ca²⁺ indicator indo 1-AM (Fig. 1, middle). The liver was observed frequently during loading in each experiment, and the pattern of autofluorescence did not change during the course of the experiment and was consistent throughout the liver. To ensure that the autofluorescent pattern of the liver had not changed during the course of the experiments, 100 µM MnCl₂ in Ca²⁺-free Hanks' balanced salt solution was infused at the end of selected experiments (n = 8; Fig. 1, bottom). Mn²⁺ rapidly enters the cell, presumably by Ca²⁺ channels, and binds to indo 1 with a 20-fold greater affinity than that of Ca²⁺, causing complete quenching of the fluorescence of indo 1 (24). Significantly, no evidence of phototoxicity or photobleaching was observed during the experiment and images could be obtained almost continuously for 3-5 min without any changes in indo 1 fluorescence.

View larger version (56K): [in this window] [in a new window]   Fig. 1.   Top: background image of liver (×200) obtained before loading with indo 1 acetoxymethyl ester (indo 1-AM), demonstrating minimal autofluorescence. Middle: image of liver (×200) obtained after loading of indo 1-AM. Identical acquisition parameters were used to obtain background image. Note marked increase in fluorescence in indo 1-AM-loaded liver. Intracellular free Ca2+ concentration ([Ca2+]i) in nanomoles is determined by reference to color scale bar at right. Same color scale bar is used for all subsequent images. Bottom: effect of MnCl2 (100 µM) to quench fluorescence of indo 1. Ten minutes after beginning of infusion of MnCl2, fluorescent image (middle) had markedly diminished and only background autofluorescence (top) remained.

Pharmacologic protocols. After using fluorescent microscopy to observe that the liver had been loaded with the Ca²⁺ indicator indo 1-AM, we infused Ca²⁺-active drugs via a separate syringe pump into a 20-ml mixing chamber located 10 cm upstream of the liver. This solution entered the liver at 4 ml · min¹ · g¹ wet wt via the portal vein, as stated previously. To avoid potential confounding effects of drugs on intracellular Ca²⁺ stores, we administered each drug to naive livers, i.e., livers that had not been treated with any other Ca²⁺ agonist. In addition, the effect of pretreatment of livers with ryanodine, dantrolene, thapsigargin, or CPA on Ca²⁺ immobilization due to AVP administration was determined.

Statistical analysis. Values expressed are means ± SE. Statistical significance was accepted at the 95% confidence limit.

	RESULTS

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Basal [Ca²⁺]_i in perfused livers. In addition to obtaining background and shading images on every liver before loading indo 1-AM, we also obtained images of indo 1-loaded livers before drug infusion. After successful loading of indo 1-AM, three to six images (i.e., baseline [Ca²⁺]_i) of different regions of the liver were obtained before evaluation of drug effects. There were no regional differences in the baseline [Ca²⁺]_i in the livers. We collected and stored [Ca²⁺]_i images of the liver ~30 s before the Ca²⁺ agonist drugs reached the liver. Images were obtained and stored every 4-5 s. In the series of images presented, the baseline image of [Ca²⁺]_i immediately before drug effect is included.

AVP-induced Ca²⁺ waves and oscillations. The dose-response effect of AVP on [Ca²⁺]_i and Ca²⁺ mobilization was investigated in the perfused liver. Infusion of a high dose of AVP (20 nM) caused a rapid increase in [Ca²⁺]_i starting at the central vein and moving out centrifugally through the entire field toward the periportal area (Fig. 2). The averaged baseline [Ca²⁺]_i over an entire field of hepatocytes was 153.3 ± 3.1 nM (n = 16 livers) before AVP and increased to a maximal value of 419.5 ± 22.8 nM (n = 16 livers) after 30 s to 2 min of infusion of the drug. The maximum amplitude of the [Ca²⁺]_i increase was consistent for all hepatocytes in the lobule; Kupffer cells did not respond to AVP. After stopping of AVP and washing out of the residual drug, [Ca²⁺]_i slowly decreased; at 11 min the decrease was ~60%, and at 20 min [Ca²⁺]_i was back to pre-AVP levels (Fig. 2). Specifically, [Ca²⁺]_i in hepatocytes farthest from the central hepatic vein returned to normal first, whereas [Ca²⁺]_i in hepatocytes located around the central vein remained elevated for the longest time (Fig. 2). Although Ca²⁺ wave propagation was usually uniform and homogeneous throughout the lobule, in 3 of the 16 livers treated with AVP, multiple initiation sites of the wave were observed (Fig. 3). The speed of propagation of the Ca²⁺ wave across the hepatic lobule in 16 livers was variable and ranged between 8 and 40 µm/s.

View larger version (113K): [in this window] [in a new window]   Fig. 2.   [Ca2+]i wave due to arginine vasopressin (AVP). Infusion of 20 nM AVP for ~1-2 min caused a rapid increase in [Ca2+]i from ~150 to ~400 nM; see [Ca2+]i scale bar in Fig. 1. Increase in [Ca2+]i began in central vein and spread centrifugally. After AVP was stopped, [Ca2+]i slowly returned to baseline.

View larger version (110K): [in this window] [in a new window]   Fig. 3.   [Ca2+]i wave due to AVP. Infusion of 20 nM AVP for ~1-2 min caused a [Ca2+]i wave that originated at multiple sites in the lobule. Central vein is present at bottom right of each image. This is an example of multiple foci of Ca2+ spikes.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   [Ca2+]i oscillations due to AVP. [Ca2+]i oscillations for 2 of the regions in liver perfused with 2.0 nM AVP. Oscillatory frequency is similar for the 2 regions, but oscillatory phases are slightly shifted. Note steady increase in baseline [Ca2+]i occurring before oscillatory spike.

ATP. ATP was infused for 3 min for each concentration, starting at 1.4 µM, followed by 7 µM, and ending at 17.5 µM (n = 6 livers). At 1.4 and 7 µM, ATP caused an increase in [Ca²⁺]_i in ~5-10% of hepatocytes scattered in a random fashion throughout the hepatic lobule (Fig. 5A). The increase in Ca²⁺ was not sustained and lasted <30 s of the 3-min infusion of ATP. Infusion of 17.5 µM ATP resulted in recruitment of additional numbers of hepatocytes responding with increased [Ca²⁺]_i (Fig. 5B). Note that the hepatocyte response was not a graded response; i.e., the hepatocytes that did respond all exhibited a maximal rise in [Ca²⁺]_i. In contrast to AVP, there was no preferential response of hepatocytes in the pericentral vein region. Also in contrast to AVP, the increase in [Ca²⁺]_i was always brief, with [Ca²⁺]_i returning to baseline in ~30 s despite continuation of the ATP infusion. Only on rare instances did ATP cause oscillations in [Ca²⁺]_i. A bolus injection of ATP (~150 µM) caused the entire hepatic lobule to increase [Ca²⁺]_i to >300 nM, and [Ca²⁺]_i returned quickly to baseline in <1 min.

View larger version (102K): [in this window] [in a new window]   Fig. 5.   Effect of ATP. A: 7.5 µM ATP caused a brief, rapid, localized increase in [Ca2+]i that did not propagate across hepatic lobule. Each image (×200 magnification) was acquired 3 s apart. B: 17.5 µM ATP caused a similar brief rapid increase in [Ca2+]i but occurred in more hepatocytes.

Thapsigargin. In four livers, 1 µM thapsigargin was infused over a 10-min time period immediately after loading of hepatocytes with the fluorescent Ca²⁺ indicator. Baseline [Ca²⁺]i was 151.7 ± 11.4 nM (n = 4). In contrast to the action of AVP, in which the Ca²⁺ wave began near the central vein, thapsigargin caused a steady progressive increase in [Ca²⁺]_i in all hepatocytes at the same time but no Ca²⁺ oscillations were observed (Fig. 6). The maximal increase in hepatocyte [Ca²⁺]_i due to thapsigargin was >1,896 nM. After washout of thapsigargin, [Ca²⁺]_i decreased slowly and progressively over the next 15-20 min. In some regions of the hepatic lobule, [Ca²⁺]_i returned to normal, and in others, [Ca²⁺]_i remained elevated in selected hepatocytes located around the central vein.

View larger version (167K): [in this window] [in a new window]   Fig. 6.   Effect of thapsigargin on [Ca2+]i. Thapsigargin (1 µM) did not cause a Ca2+ wave, but rather a steady progressive increase in [Ca2+]i occurred almost uniformly in entire lobule. Central vein is located at bottom right of each image.

CPA. CPA was infused at 2.1 µM, and [Ca²⁺]_i increased from 146.9 ± 12.9 to 250.4 ± 17.7 nM (n = 7). The increase in [Ca²⁺]_i occurred slowly and progressively over the entire ~7-min infusion period. Similar to thapsigargin but in contrast to AVP, the increase in [Ca²⁺]_i occurred uniformly throughout the entire hepatic lobule and was not more intense in the pericentral vein region. After infusion of CPA was stopped, [Ca²⁺]_i returned to baseline. Prior treatment with CPA did not blunt the hepatocyte [Ca²⁺]_i response to AVP. No oscillations in [Ca²⁺]_i were observed in any livers treated with CPA.

Ryanodine. In six livers, 14 µM ryanodine was infused for a 10-min time period after loading with indo 1-AM. Average baseline value of [Ca²⁺]_i was 136.8 ± 8.8 nM. Ryanodine had only a minimal effect on hepatocyte [Ca²⁺]_i. The effect of ryanodine consisted of a nonsustained Ca²⁺ spike in single cells, averaging approximately one to three cells per microscopic field. Ryanodine did not cause Ca²⁺ waves or oscillations. When AVP (450 nM) was infused after washout of ryanodine, a rapid sustained maximal Ca²⁺ response to AVP was demonstrated and pretreatment with ryanodine failed to blunt this effect.

Dantrolene. Ten micromolar dantrolene, which inhibits release of Ca²⁺ from sarcoplasmic and endoplasmic reticulum, was effective in causing a significant, rapid decrease in elevated [Ca²⁺]_i that occurred in hepatocytes after treatment with high-dose AVP (450 nM; Fig. 7). The decrease in [Ca²⁺]_i occurred almost immediately after the beginning of perfusion with dantrolene. However, pretreatment with 10 µM dantrolene did not prevent the maximal increase in [Ca²⁺]_i that occurred during administration of high-dose AVP (450 nM). In one of two livers, dantrolene also was effective in decreasing the elevated [Ca²⁺]_i that occurred after treatment with 1 µM thapsigargin. The decrease in [Ca²⁺]_i that occurred when dantrolene was infused into thapsigargin-treated hepatocytes was not uniform; i.e., only some hepatocytes responded by decreasing [Ca²⁺]_i, whereas other cells had no decrease.

View larger version (83K): [in this window] [in a new window]   Fig. 7.   Effect of dantrolene on [Ca2+]i. Elevated [Ca2+]i due to AVP was decreased by infusion of 10 µM dantrolene. [Ca2+]i decreased almost immediately after dantrolene reached liver. [Ca2+]i (nM) illustrated on scale bar at right.

Evaluation of intracellular compartmentation of indo 1 and liver autofluorescence with MnCl₂. Mn²⁺ rapidly enters the cell, presumably via Ca²⁺ channels (24). Mn²⁺ binds to indo 1 with a 20-fold greater affinity than that of Ca²⁺ and thereby causes complete quenching of the fluorescence of indo 1 free acid. Mn²⁺ has no effect on the Ca²⁺-insensitive fluorescence of the nondeesterified indo 1-AM (12, 24). MnCl₂ has been used in Ca²⁺ experiments for several purposes, including 1) detection of cell autofluorescence, 2) evaluation of fluorescence from incompletely hydrolyzed indo 1-AM, and 3) determination of the contribution of indo 1 fluorescence arising from the noncytosol, i.e., intracellular organelles (21, 24). Although Mn²⁺ will cause rapid quenching of cytosolic indo 1, Mn²⁺ may also cause quenching of indo 1 that is located in intracellular organelles, i.e., mitochondria, nucleus, and endoplasmic reticulum.

	DISCUSSION

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The present study evaluating the effect of various Ca²⁺ agonists on Ca²⁺ signaling in the intact liver provides insight into the regulation of intracellular Ca²⁺ in the whole organ. One of the most interesting and intriguing findings was the unique and specific effect of the individual drugs on Ca²⁺ mobilization. For example, both AVP and ATP mobilize Ca²⁺ from IP₃-sensitive Ca²⁺ stores (14, 21, 22). AVP acts on V_1a receptors to activate phospholipase C, thereby inducing IP₃ production. ATP mobilizes Ca²⁺ via a G protein-coupled P₂ purinoceptor, which also increases IP₃ (23). Despite both drugs causing increases in IP₃, the [Ca²⁺]_i response in the liver differed. AVP but not ATP caused a preferential increase in [Ca²⁺]_i in pericentrally located hepatocytes. A 2.0 nM concentration of AVP caused oscillations in [Ca²⁺]_i, whereas oscillations were infrequently observed with ATP. An organized Ca²⁺ wave that spread across the entire lobule was observed routinely with AVP, whereas ATP did not cause a Ca²⁺ wave. ATP did cause focal regions of hepatocytes to increase [Ca²⁺]_i, but the area did not involve the entire lobule. Also, the increase in [Ca²⁺]_i due to AVP was of much longer duration (min) than that due to ATP (s). Why ATP, which also generates IP₃, did not cause a Ca²⁺ wave is also not known.

In liver, hepatocytes are tightly coupled by gap junctions (22, 23). Tordjmann and associates (30) found that second messengers and [Ca²⁺]_i elevation in one hepatocyte cannot trigger Ca²⁺ responses in abutting cells, suggesting that diffusion across gap junctions (although required for coordination) is not sufficient by itself for the propagation of intercellular Ca²⁺ waves and the presence of hormone at the cell surface of each hepatocyte is required for cell-to-cell Ca²⁺ signal propagation (30). They also reported that functional differences between adjacent connected hepatocytes may be the basic mechanism for intercellular propagation of Ca²⁺ waves (30).

Studies by Nathanson et al. (15) have shown an increased number of V_1a receptors in hepatocytes located in the pericentral vein region. Therefore, this unique receptor distribution is the most likely rationale for the preferential [Ca²⁺]_i response to AVP of hepatocytes in the pericentral region (15). Robb-Gaspers and Thomas (21), however, noted that perfusion with low doses of vasopressin induced oscillations of hepatocyte Ca²⁺ that were coordinated across entire lobules of the liver by propagation of Ca²⁺ waves along the hepatic plates. At the subcellular level, these periodic Ca²⁺ waves initiated from the sinusoidal domain of cells within the periportal region and spread to the pericentral region. At higher vasopressin doses, a single Ca²⁺ wave was observed and the direction of Ca²⁺ wave propagation was reversed, being initiated in the pericentral region and spreading to the periportal region (21).

Although hepatocytes express ATP receptors (P₂) that on stimulation can evoke [Ca²⁺]_i signals, the distribution of ATP receptors within the lobule has not been determined (23). ATP is secreted from hepatocytes into the extracellular space, and it is believed that ATP serves as a novel paracrine signaling pathway (23). Our study in the intact liver confirms the ability of extracellularly administered ATP to cause increased [Ca²⁺]_i in neighboring hepatocytes located throughout the lobule. The increase in [Ca²⁺]_i due to AVP was of much longer duration than that due to ATP (s). This difference in the duration of the [Ca²⁺]_i spike with ATP versus AVP may have important cellular consequences. Because studies indicate that both the amplitude and duration of the agonist-induced increase in [Ca²⁺]_i are important in directing the particular cellular response, differences in the [Ca²⁺]_i signal due to AVP and ATP are partly responsible for the unique effects of the two hormones. Further substantiating the important role of Ca²⁺ oscillations is work by De Koninck and Schulman (8) demonstrating that the frequency of Ca²⁺ oscillations directly encoded the activity of the Ca²⁺- and calmodulin-dependent protein kinase II.

An interesting observation noted with AVP but not as readily apparent with ATP was a small increase in basal [Ca²⁺]_i (~20-40 mM) occurring 30-90 s after addition of AVP (Fig. 4). This increase in [Ca²⁺]_i immediately preceded the initiation of the Ca²⁺ wave and appeared to be present uniformly in all hepatocytes in the lobule. It may be that this increase in [Ca²⁺]_i is a triggering event for initiation of the Ca²⁺ wave, i.e., a Ca²⁺-induced Ca²⁺ release phenomenon (2, 3, 7). This finding has not been previously noted in other studies of the perfused liver (15, 21). Although hepatocytes did not exhibit a basal increase with ATP, they did demonstrate an "all or none" response to the drug. The larger dose of ATP caused recruitment of additional hepatocytes, all of which had a maximal Ca²⁺ response (Fig. 5). There was no increase in [Ca²⁺]_i in the majority of hepatocytes. Therefore, this Ca²⁺ response is similar to neuronal firing in that it is an all or none response.

In the present report, the order of administration of AVP was important in determining the Ca²⁺ response. If high-dose AVP (20 nM) was administered first, the entire hepatic lobule had a brisk sustained rise in [Ca²⁺]_i. However, if 20 nM AVP was administered after the 2 and 5 nM AVP doses, the increase in [Ca²⁺]_i was less robust and the Ca²⁺ wave progressed more slowly and on occasion was not sustained despite continued infusion of AVP. One possibility for the differential response is that intracellular Ca²⁺ stores had been partially depleted by the initial dose of AVP, resulting in a diminished Ca²⁺ response with subsequent doses of AVP (6). Interestingly, in experiments in which the -agonist isoproterenol was administered either before or after AVP, the increase in [Ca²⁺]_i was greater when the drug was given after AVP (unpublished data), suggesting that intracellular Ca²⁺ stores may not have been depleted by AVP. Another possible explanation for the decreased response to AVP could be internalization of the V₁ receptors producing receptor desensitization (16).

Lower concentrations of AVP (2.0 mM) caused oscillations in hepatocyte [Ca²⁺]_i (Fig. 4), as reported by Robb-Gaspers and Thomas (21). Both the amplitude (~200 mM) and frequency (~1 cycle/60 s) of the Ca²⁺ oscillations reported herein are in close agreement with those reported previously (21). Also consistent with their findings, the individual frequency of the oscillations is similar for individual hepatocytes located at different sites in the hepatic lobule, but the various frequencies are phase shifted, i.e., occur at different time points (Fig. 4) (21).

Ca²⁺ agonists: Thapsigargin and CPA. Thapsigargin and CPA, which increase [Ca²⁺]_i via a receptor and an IP₃-independent mechanism, did not cause either Ca²⁺ oscillations or a Ca²⁺ wave, and the increase in [Ca²⁺]_i occurred uniformly and simultaneously throughout the lobule. The absence of the Ca²⁺ wave with thapsigargin and CPA versus the presence of the Ca²⁺ wave with AVP may be due to the fact that neither thapsigargin nor CPA generate IP₃, whereas AVP does. Although the exact mechanism of the Ca²⁺ wave is not known, evidence suggests that diffusion of IP₃ through gap junctions may be involved (2, 6). Although thapsigargin and CPA failed to induce Ca²⁺ waves, several other findings should be noted. The magnitude of the increase in [Ca²⁺]_i with thapsigargin was greater than that with either CPA or AVP, which may be due to a dose-response effect; i.e., higher doses of CPA or AVP may cause an equivalent elevation in [Ca²⁺]_i. Alternatively, thapsigargin may be more efficient either in emptying Ca²⁺ stores or in emptying additional non-AVP-responsive Ca²⁺ stores. Thapsigargin almost completely blocked the ability of AVP to increase [Ca²⁺]_i. The different effect of both drugs may be due to the fact that thapsigargin is an irreversible inhibitor, whereas CPA is reversible. Other mechanisms for the increased [Ca²⁺]_i may be involved. Because all three drugs cause depletion of endoplasmic reticulum Ca²⁺ stores, activation of plasma membrane Ca²⁺ channels and increased influx of extracellular Ca²⁺ may result (19, 20).

Ryanodine. Several groups have identified separate ryanodine and IP₃ binding sites in hepatic microsomes (13). Studies also show that ryanodine is capable of mobilizing Ca²⁺ in the hepatocyte from microsomal stores that are distinct from those that can be regulated by IP₃ (1, 13, 16). In our study, 10 µM ryanodine had minimal effects in the isolated liver, and in only a few isolated hepatocytes (2-3 per high-powered field) was there a nonsustained increase in [Ca²⁺]_i. The dose of ryanodine in the present study (10 µM) was chosen because a dose-response curve with ryanodine on hepatocyte microsomal preparations demonstrated that 10 µM ryanodine caused 80% of the maximum release of Ca²⁺ (13). It is possible that higher concentrations of ryanodine might have produced a greater effect on [Ca²⁺]_i. Bazotte et al. (1), using 200 µM ryanodine in isolated hepatocytes, found a 24% increase in [Ca²⁺]_i. Nathanson et al. (16) reported that 50 µM ryanodine caused a 15 ± 5 nM increase in hepatocyte [Ca²⁺]_i from 113 ± 19 to 128 ± 19 nM. In contrast to thapsigargin, ryanodine failed to prevent the Ca²⁺ response to AVP in the perfused liver. Studies by Nathanson et al. (16) in isolated hepatocytes also demonstrated that 50 µM ryanodine did not inhibit the increase in [Ca²⁺]_i induced by AVP.

Dantrolene. Although the molecular mechanism of action of dantrolene is unknown, it has been shown to strongly inhibit ryanodine but not IP₃ binding to hepatic microsomal preparations. In addition to inhibition of ryanodine binding, there is one report that dantrolene and its analogs block dihydropyridine receptors. If confirmed, this may be another possible mechanism for its Ca²⁺ antagonist effect (9). Dantrolene also inhibits Ca²⁺-induced Ca²⁺ release in diverse types of cells, including neurons and smooth and skeletal muscle cells (5, 18). Pretreatment with dantrolene failed to prevent the hepatocyte Ca²⁺ response to our dose of 20 nM AVP, and it did not affect either the speed of the wave or the peak [Ca²⁺]_i rise. Nathanson and associates (16) noted that 10 µM dantrolene reduced the peak cytosolic Ca²⁺ response to 40 nM AVP and ANG II by 50% in isolated hepatocytes. A possible explanation for the different findings in the present study versus the report of Nathanson et al. (16) is the dose of AVP that was used in the two studies. Furthermore, in the current study, dantrolene was used as a pretreatment only and was not present in solution when the AVP was added. However, dantrolene was effective in decreasing the increased [Ca²⁺]_i that occurred with AVP. Within 1-2 min after beginning dantrolene, the increased [Ca²⁺]_i that resulted from AVP had decreased significantly.

Indo 1-AM intracellular compartmentation. Another potential problem of Ca²⁺ indicators is their transport from the cytosol into intracellular organelles such as the nucleus, endoplasmic reticulum, and mitochondria. If the Ca²⁺ indicators are sequestered in these organelles, an inaccurate assessment of cytosolic free Ca²⁺ concentration, i.e., [Ca²⁺]_i, will result (12, 24). Ca²⁺ indicators may give a falsely high value for [Ca²⁺]_i if localized in endoplasmic reticula or mitochondria. Although it may be difficult to determine if the Ca²⁺ indicator has been taken up by organelles, a heterogeneous appearance of the cell on microscopic examination may be one clue (16). No heterogeneous appearance occurred in any of the livers in the current study. In three livers, nuclear labeling with indo 1 appeared to be present, although this finding may have represented a layer of cytoplasm overlying the nucleus. Three-dimensional reconstruction could be used to determine if the nuclei did in fact load indo 1.

Perspectives