INTRODUCTION

An increase in intracellular Ca²⁺ ([Ca²⁺]_i)¹ is the key signaling event employed by a diverse range of extracellular stimuli to coordinate cell function (1). In both isolated single hepatocytes (2, 3) and intact liver (4) [Ca²⁺]_i responses to submaximal concentrations of inositol phospholipid-linked agonists originate as repetitive oscillations in [Ca²⁺]_i that are spatially organized in the form of Ca²⁺ waves (5). These [Ca²⁺]_i responses are mediated, at least in part, by an increased formation of the second messenger inositol-1,4,5-trisphosphate (InsP₃) and its ability to specifically interact with an intracellular receptor that functions as a release channel for luminal calcium (6, 7). The phosphorylation state of the InsP₃ receptor regulates the sensitivity to both InsP₃ and Ca²⁺ (8) and has potential for control by a number of different protein kinases, including PKA, protein kinase C, calcium-calmodulin-dependent protein kinase, and G-kinase (9). The functional effects of InsP₃ receptor phosphorylation can be inhibitory or stimulatory depending on cell type (10) and are counterbalanced by protein phosphatases (8, 11).

Cyclic GMP has been shown to influence [Ca²⁺]_i responses in a number of different cell types (12). For example, cGMP decreases [Ca²⁺]_i in vascular smooth muscle (13), cardiac myocytes (14), platelets (15), and cerebellar neurons (16) by a mechanism involving activation of G-kinase. Several intracellular targets have been proposed to mediate the inhibitory action of G-kinase on [Ca²⁺]_i signaling including activation of plasma membrane Ca²⁺ pumps (17, 18), stimulation of sarcoplasmic reticulum Ca²⁺ pumps by phosphorylation of phospholamban (19, 20, 21), inhibition of L-type voltage-gated calcium channels (14), and inhibition of InsP₃-induced Ca²⁺ release by phosphorylation of the InsP₃ receptor (22). In addition, Br-cGMP inhibits thrombin-induced InsP₃ formation and [Ca²⁺]_i responses by phosphorylating a pertussis toxin-sensitive G-protein in Chinese hamster ovary cells transfected with G-kinase (23). Growth factor-mediated inositol phospholipid-specific phospholipase C activation and [Ca²⁺]_i signals are also inhibited by pretreatment with cGMP and NO donors in NIH-3T3 fibroblasts and tumor epithelial cells (24). By contrast, cGMP increases [Ca²⁺]_i in sea urchin eggs by stimulating the synthesis of cyclic ADP ribose and the release of Ca²⁺ from ryanodine-sensitive intracellular stores (25). A role for cGMP in the regulation of Ca²⁺ entry has emerged from research in pancreatic acinar cells where intracellular perfusion of cGMP emulates the inward Ca²⁺ entry current observed upon depletion of intracellular Ca²⁺ stores (26). Further studies have revealed that depletion of intracellular Ca²⁺ stores activates NOS to generate cGMP which, depending on the final concentration, can either stimulate or inhibit agonist-induced Ca²⁺ entry in acinar cells (27). NO and cGMP have also been shown to potentiate Ca²⁺ entry in epidermal growth factor- and platelet-derived growth factor-stimulated NIH-3T3 cells (28).

In liver relatively little is known about the functional role of cGMP and its ability to interfere with [Ca²⁺]_i signaling (29). Hepatocytes are known to contain soluble guanylate cyclase and produce large amounts of cGMP in vitro when stimulated with a combination of lipopolysaccharide and cytokines (30). This is mediated by an activation of guanylate cyclase resulting from the induction of NOS and generation of NO (30). Hepatocytes do not appear to express the constitutive form of NOS (31), but inducible NOS has been cloned from human hepatocytes and appears to be a distinct isoform with a partial dependence on Ca²⁺ and calmodulin (32). Recent studies have also shown that NO can interfere with canalicular contraction in coupled rat hepatocytes (33, 34). In the present study we have examined the effects of cGMP on cellular Ca²⁺ homeostasis in rat hepatocytes. Our data show that cGMP phosphorylates the InsP₃ receptor in intact hepatocytes and increases the sensitivity to InsP₃ for [Ca²⁺]_i release. This results in the generation of [Ca²⁺]_i oscillations and to the enhancement of agonist-induced Ca²⁺ responses.

EXPERIMENTAL PROCEDURES

All the experiments in this study were performed on freshly isolated hepatocytes that were prepared by collagenase perfusion of livers obtained from male Sprague-Dawley rats (250-300 g), as described previously (35).

Intact hepatocytes (4 mg of protein/ml) were loaded with fura-2/AM (5 µM) for 35 min at 37 °C in HEPES buffer containing 2% bovine serum albumin. After loading, the cells were washed and stored at 4 °C in Ca²⁺-free HEPES buffer. Before use the cells were washed once in Ca²⁺-free HEPES buffer containing 100 µM EGTA. The cells were then permeabilized in buffer containing 120 mM KCl, 1 mM KH₂PO₄, 2 mM MgCl₂, 10 mM NaCl, 5 µg/ml oligomycin, 1 µg/ml rotenone, 5 µM carbonyl chlorophenylhydrazone, 2 mM ATP·Mg, 10 mM creatine phosphate, 1 unit/ml creatine kinase, 30-50 µg/ml digitonin, 2 µM thapsigargin, and 1 µg/ml each of the protease inhibitors pepstatin, antipain, and leupeptin with pH adjusted to 7.2 using Tris base. The quenching of compartmentalized fura-2 was monitored after addition of 40 µM MnCl₂. Incubations were performed in the cuvette of a fluorimeter (Photo Technology Deltascan) maintained at 37 °C with continuous stirring. The excitation wavelength was 360 nm with emission at 510 nm.

Hepatocytes (5 × 10⁵ cells/Petri dish) were plated on glass coverslips coated with poly-D-lysine (5 µg/cm³) in 2 ml of insulin-free Williams E medium supplemented with 10% fetal calf serum, 10 units/ml penicillin, 10 µg/ml streptomycin, 0.05 mg/ml gentamycin, 4 µg/ml dexamethasone, and 2 mM glutamine. The cells were incubated for 30 min at 37 °C under an atmosphere of CO₂/air (5:95%) and then washed to remove unattached and nonviable cells. Measurements of [Ca²⁺]_i were obtained from cells loaded with fura-2 by incubation with fura-2/AM (5 µM in 0.03% pluronic F-127) for 30 min at 37 °C in HEPES buffer with 2% bovine serum albumin and 200 µM sulfinpyrazone. This protocol resulted in >70% cytosolic loading of fura-2. For Mn²⁺ quenching of compartmentalized fura-2 in intact hepatocytes, the cells were loaded with fura-2/AM for 45-60 min to increase the amount of compartmentalized dye.

Fluorescence images were obtained as described previously (36, 37). Coverslips with attached fura-2-loaded hepatocytes were transferred to a chamber with 1 ml of HEPES buffer and mounted on the stage of a Zeiss IM-35 inverted microscope. The stage, 20 × oil immersion objective, and chamber were thermostatically regulated at 37 °C. A liquid N₂-cooled charged coupled device camera (Photometrics Ltd.) was used as the imaging device. Images were digitized at 12-bit resolution and stored and analyzed with a Heurikon HK68/M10 computer. For [Ca²⁺]_i measurements fluorescent images were collected alternately at excitation wavelengths of 340 and 380 nm (10 nm bandwidth) with an emission wavelength of 460-600 nm. [Ca²⁺]_i was calculated from the fluorescence measurements using the ratio method, after subtraction of fluorescence signals derived from compartmentalized dye, as described previously (36). Mn²⁺ quenching of compartmentalized fura-2 was measured at 360-nm excitation.

The assay for back phosphorylation of the InsP₃ receptor was performed as described previously (8). Hepatocytes were preincubated in Dulbecco's modified Eagle's medium for 10 min at 37 °C and then incubated with drug for 10 min. The cells were centrifuged, solubilized in hepatocyte solubilization buffer, and the extracts immunoprecipitated with an InsP₃ receptor antibody (raised against the C terminus of the type-1 InsP₃ receptor). The immunoprecipitates were washed in a phosphate buffer containing 120 mM KCl, 50 mM Tris-HCl (pH 7.2), 0.1% Triton X-100 (w/v), 0.3 mM MgCl₂, 0.5 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of aprotinin, soybean trypsin inhibitor, and leupeptin. Immunoprecipitated InsP₃ receptor was then incubated in 0.2 ml of phosphorylation buffer containing 100 units/ml of the catalytic subunit of PKA and 3-5 µCi of [-³²P]ATP (3000 Ci/mmol). Immunoprecipitates were phosphorylated for 15 min at 30 °C, and the reaction was terminated by washing the protein A-Sepharose beads with phosphorylation buffer containing unlabeled MgATP (1 mM). The phosphorylated proteins in the immunoprecipitates were separated on 5% SDS-polyacrylamide gels and transferred to nitrocellulose, which was then autoradiographed. The amount of ³²P-labeled InsP₃ receptor was quantitated from the autoradiographs with a laser densitometer. The localization of the InsP₃ receptor on the nitrocellulose was verified by immunoblotting.

[-³²P]ATP was obtained from DuPont NEN, Ins(1, 4, 5)P₃ from LC Services, and ionomycin from Calbiochem. Fura-2 acetoxymethyl ester (Fura2/AM) and pluronic F-127 were from Molecular Probes Inc. Williams E medium was from Life Technologies, Inc., Dulbecco's modified Eagle's medium was from Life Technologies, and collagenase was from Worthington. Rp-Br-cAMP[S] and Rp-Br-cGMP[S] were from Biolog Life Science Institute. Siguazodan (SKF94836) was a gift from Dr. K. Murray (SmithKline Beecham Pharmaceuticals, U. K.). All other chemicals were obtained from Sigma or Fisher.

RESULTS

The effects of cGMP analogues on [Ca²⁺]_i were examined at the single cell level in fura-2-loaded freshly isolated hepatocytes. Fig. 1 shows typical [Ca²⁺]_i responses in single hepatocytes exposed to Br-cGMP (200 µM) or db-cGMP (200 µM). In the present study we have used Br-cGMP and db-cGMP interchangeably since the [Ca²⁺]_i responses to both compounds are essentially equivalent and observed over similar concentration ranges.² [Ca²⁺]_i responses produced by cGMP appeared as a series of base-line-separated [Ca²⁺]_i spikes with similar kinetics and amplitude to those previously obtained with inositol phospholipid-linked agonists in this preparation (36). The only significant differences were that cGMP-induced [Ca²⁺]_i responses frequently displayed secondary spiking activity during the falling phase of each [Ca²⁺]_i oscillation. As shown previously with receptor-linked agonists (36), individual hepatocytes displayed significant variation in the sensitivity to cGMP analogues, with cells already responding to low doses of cGMP producing sustained [Ca²⁺]_i responses at higher doses (data not shown). The [Ca²⁺]_i responses induced by cGMP could be mediated by activation of G-kinase or be due to activation of PKA and/or increases in cAMP resulting from an inhibition of cAMP PDE activity. However, in all cases [Ca²⁺]_i responses to submaximal concentrations of Br-cGMP were significantly, or completely, inhibited by addition of the G-kinase inhibitor Rp-Br-cGMP[S] (200 µM) (Fig. 2). The inability to fully suppress [Ca²⁺]_i responses in some cells is probably explained by the fact that Rp-Br-cGMP[S] is a competitive inhibitor of G-kinase (38). This indicates that cGMP-induced [Ca²⁺]_i increases are mediated by activation of G-kinase and argue against indirect effects of cGMP due to increases in cAMP and/or activation of PKA, as shown in some other cell types (39, 40). This is further demonstrated by the finding that inhibition of PDE III (cGMP-inhibitable cAMP phosphodiesterase) with siguazodan (30 µM) failed to increase [Ca²⁺]_i and did not inhibit cGMP-induced [Ca²⁺]_i signals (Fig. 3). Inhibition of PKA by pretreatment with Rp-Br-cAMP[S] (200 µM) also failed to prevent [Ca²⁺]_i responses to cGMP in hepatocytes (data not shown).

Addition of the NO donor SNP also generated [Ca²⁺]_i increases in hepatocytes. This is shown in Fig. 4 where addition of SNP produced oscillatory [Ca²⁺]_i increases, comparable with those elicited by cGMP. The SNP-induced [Ca²⁺]_i oscillations appeared after a well-defined latent period. Most cells responded with [Ca²⁺]_i oscillations in the range of 1-10 µM SNP. Increasing doses of SNP increased the proportion of cells responding but did not produce a clear enhancement of [Ca²⁺]_i oscillation frequency in individual hepatocytes already responding to SNP. We have observed similar [Ca²⁺]_i responses with SIN-1 in hepatocytes (data not shown).

Previous studies in liver, and other cell types, have shown that [Ca²⁺]_i oscillations persist in the absence of extracellular Ca²⁺, although they tend to have a reduced frequency followed by eventual run down and stopping (36, 41). To assess the contribution of extracellular Ca²⁺ influx to the initiation and maintenance of cGMP-induced [Ca²⁺]_i oscillations, the ability of Br-cGMP to elicit [Ca²⁺]_i responses in the absence of extracellular Ca²⁺ was examined. As shown in Fig. 5, removal of extracellular Ca²⁺ did not significantly affect the amplitude (368 ± 26 nM in the presence and 345 ± 23 nM in the absence of extracellular Ca²⁺) of Br-cGMP-induced [Ca²⁺]_i responses but significantly reduced their frequency or more often converted them to single transient [Ca²⁺]_i increases. Removal of extracellular Ca²⁺ also had no effect on the time to peak Ca²⁺. We have observed similar effects of Ca²⁺ depletion on vasopressin-induced [Ca²⁺]_i responses in primary cultured hepatocytes (36). This indicates that Ca²⁺ influx is not required for the generation of Br-cGMP-induced [Ca²⁺]_i responses but plays an important role in sustaining the [Ca²⁺]_i oscillations by replenishing intracellular Ca²⁺ stores.

The data described above demonstrate that cGMP-induced [Ca²⁺]_i responses have a similar temporal organization and Ca²⁺ dependence to those generated by receptor agonists. It was therefore of interest to establish the nature of any interaction between these two stimuli. For these experiments Br-cGMP was added to single hepatocytes during continuous exposure to phenylephrine. Br-cGMP potentiated the [Ca²⁺]_i response to phenylephrine in all cells, but the extent of this enhancement was determined by the magnitude of the phenylephrine-induced [Ca²⁺]_i increase (Fig. 6). Addition of Br-cGMP to cells responding to phenylephrine with one or two [Ca²⁺]_i oscillations resulted in a rapid increase in oscillation frequency (Fig. 6B), whereas cells displaying multiple [Ca²⁺]_i spikes were converted to sustained [Ca²⁺]_i responses (Fig. 6C). Br-cGMP was also able to generate [Ca²⁺]_i oscillations in cells that failed to respond to phenylephrine (Fig. 6A). In cells where phenylephrine caused a sustained [Ca²⁺]_i response Br-cGMP was unable to further increase [Ca²⁺]_i (data not shown). The amplitude of the oscillatory and sustained [Ca²⁺]_i responses produced by addition of Br-cGMP were similar to those generated by phenylephrine alone. A similar potentiation of agonist-induced [Ca²⁺]_i responses was observed upon addition of SNP to hepatocytes pre-exposed to phenylephrine (data not shown).

Recent studies in intact and permeabilized cells have shown that Mn²⁺ can be used as a Ca²⁺ surrogate to monitor the divalent cation permeability of the InsP₃-sensitive Ca²⁺ channels associated with intracellular stores (42, 43, 44, 45). These experiments exploit the ability of Mn²⁺ to quench the fluorescence of fura-2 entrapped within intracellular compartments and provide a direct measure of the gating properties of InsP₃-sensitive Ca²⁺ channels. We have utilized this technique to further resolve the contribution of intracellular Ca²⁺ stores to the [Ca²⁺]_i responses generated by cGMP in hepatocytes. Changes in fura-2 fluorescence were monitored at 360 nm excitation (Ca²⁺-insensitive wavelength) and in Ca²⁺-free medium to facilitate Mn²⁺ entry and eliminate contributions from Ca²⁺ influx. Fig. 7 demonstrates that Mn²⁺ quench of fura-2 fluorescence in intact hepatocytes is biphasic and consists of an initial rapid quench of the cytosolic dye component followed by a slow quench of the compartmentalized dye, as described previously (44). Addition of Br-cGMP after the cytosolic dye was completely quenched resulted in a significant increase in the rate of quench of the residual compartmentalized dye. As reported previously, under conditions of vasopressin-induced [Ca²⁺]_i oscillations (44), the quench of fura-2 in the intracellular stores was composed of a series of individual steps, and this encompassed almost all of the ionomycin-sensitive intracellular stores. This stepwise quench of the compartmentalized component of fura-2 fluorescence reflects the periodic opening and closing of the Ca²⁺ channels within intracellular stores.³ Although it is not possible to monitor [Ca²⁺]_i changes simultaneously with the Mn²⁺ quench of compartmentalized fura-2, the latency to the first quench step, the duration of the rapid phase of each step, and the frequency of the steps were all consistent with the temporal pattern of [Ca²⁺]_i oscillations observed under the same conditions.

Br-cGMP also potentiated the ability of phenylephrine to stimulate Mn²⁺ quench of compartmentalized fura-2 in a manner that paralleled its effects on agonist-stimulated [Ca²⁺]_i signals. This is shown in Fig. 8 where the capacity of Br-cGMP to enhance phenylephrine-induced Mn²⁺ quench was limited by the extent of the agonist response. In fact Br-cGMP had no affect in cells where the phenylephrine-induced Mn²⁺ quench had already gone to completion (Fig. 8A). These results indicate that Br-cGMP mimics the effects of hormones that give [Ca²⁺]_i oscillations, by stimulating the sequential opening and closing of the intracellular Ca²⁺ channels whose permeability to Mn²⁺ is registered by the periodic quench of compartmentalized fura-2. In addition, Br-cGMP and phenylephrine appear to stimulate Mn²⁺ entry into the same intracellular compartment, and this constitutes almost the entire ionomycin-sensitive Ca²⁺ store.

The type-1 InsP₃ receptor has been shown to be phosphorylated by PKA in intact rat hepatocytes (8). This phosphorylation leads to an increased sensitivity of the hepatic InsP₃ receptor to both Ca²⁺ and InsP₃ (8, 46, 47) and has been suggested to underlie the stimulatory effects of cAMP on [Ca²⁺]_i signals in hepatocytes (47, 48). Since the [Ca²⁺]_i increases generated by cGMP in this study appear to be mediated by G-kinase activation, and the type-1 InsP₃ receptor is known to be phosphorylated by G-kinase on a site also phosphorylated by PKA (22), we investigated whether cGMP phosphorylated the InsP₃ receptor in intact hepatocytes. The phosphorylation of the type-1 InsP₃ receptor was examined using a back phosphorylation assay, whereby solubilized extracts from control and cGMP-treated hepatocytes were immunoprecipitated with a type-1 InsP₃ receptor antibody before being phosphorylated in vitro by incubation with [³²P]ATP and the catalytic subunit of PKA. Accordingly, an increased phosphorylation of the InsP₃ receptor in intact hepatocytes should be reflected by a decreased incorporation of ³²P during the in vitro phosphorylation assay. Fig. 9 demonstrates that both db-cAMP and db-cGMP treatment of intact hepatocytes decreased ³²P incorporation into the immunoprecipitated InsP₃ receptor. The results summarized in Table I demonstrate that exposure to db-cGMP reduced ³²P incorporation by about 55% of that produced by db-cAMP. However, when both agents were added in combination their effects on ³²P incorporation were completely nonadditive. This suggests that db-cAMP and db-cGMP phosphorylate common sites on the InsP₃ receptor.

Table I. Effect of cyclic nucleotides on back phosphorylation of hepatic type-1 InsP3 receptor with PKA Isolated hepatocytes were incubated with drug for 10 min. Samples were centrifuged, solubilized, and extracts immunoprecipitated with type-1 InsP3 receptor antibody. Immunoprecipitates were back-phosphorylated with [32P]ATP and the catalytic subunit of PKA. The phosphorylated immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and then autoradiographed. The amount of 32P-labeled InsP3 receptor was quantitated from the autoradiographs with a laser densitometer and is expressed as the percentage of the label incorporated into the InsP3 receptor immunoprecipitated from control cells. The data are the mean ± S.E. of three experiments in which back phosphorylation assays for each condition were performed in duplicate. Additions 32P incorporation % of control None 100 db-cAMP (0.1 mM) 34.4  ± 15.0 db-cGMP (0.1 mM) 61.1  ± 11.0 db-cAMP (0.1 mM) + db-cGMP (0.1 mM) 33.0  ± 16.0

As noted above, previous studies have reported that phosphorylation of the InsP₃ receptor by PKA potentiates InsP₃-induced [Ca²⁺]_i release in hepatocytes (46, 47, 48). To ascertain whether cGMP-mediated InsP₃ receptor phosphorylation elicited a similar sensitizing action, the effect of Br-cGMP on InsP₃-induced Mn²⁺ quench of compartmentalized fura-2 was examined in permeabilized hepatocytes, as described previously (42, 44, 46). For these experiments fura-2-loaded intact hepatocytes were preincubated at 37 °C for 5 min with Br-cGMP in Ca²⁺-free HEPES buffer before permeabilization in cytosolic buffer containing thapsigargin. The addition of thapsigargin depletes the intracellular Ca²⁺ stores and enables the effects of cGMP on the Mn²⁺ permeability of the InsP₃ receptor/channel to be monitored independent of changes in luminal Ca²⁺. Pretreatment of intact hepatocytes with Br-cGMP (1 mM) resulted in a 2-fold increase in the rate and a 26% increase in the magnitude of the Mn²⁺-induced quench elicited by a submaximal dose of InsP₃ (150 nM) in permeabilized cells (Fig. 10 and Table II). Under identical experimental conditions Br-cGMP did not significantly affect the rate or magnitude of the Mn²⁺ quench produced by a maximal dose of InsP₃ (5 µM). This indicates that pretreatment with cGMP increases the number of InsP₃-sensitive Ca²⁺ channels that can be activated in the presence of submaximal concentrations of InsP₃ without altering the size of the InsP₃-sensitive Ca²⁺ pool, as measured by the Mn²⁺ quench response to a maximal dose of InsP₃.

Table II. Effect of cGMP on InsP3-Induced Mn2+ quench of compartmentalized fura-2 in permeabilized hepatocytes Intact hepatocytes were incubated in the presence or absence of 1 mM Br-cGMP for 5 min in Ca2+-free HEPES buffer before permeabilization in cytosolic-like buffer containing thapsigargin and digitonin. The quenching of compartmentalized dye was monitored in the presence of 40 µM Mn2+. The initial rates were calculated by linear regression over the linear portion of the quench response obtained after addition of 150 nM InsP3 or 5 µM InsP3. The size of the InsP3-sensitive pool is expressed as a percentage of the total ionomycin-quenchable fluorescence. The data represent the mean ± S.E. of values obtained in 15-21 individual runs from 4 to 5 different hepatocyte preparations. Mn2+ quench of fura-2 fluorescence was monitored with excitation at 360 nm and emission at 510 nm. Additions Initial rate Pool size fluorescence units/s % ionomycin 150 nM InsP3 560  ± 63 31.1  ± 1.4 150 nM InsP3 + 1 mM Br-cGMP 1037  ± 130a 39.2  ± 1.5a 5 µM InsP3 5095  ± 524 50.9  ± 0.9 5 µM InsP3 + 1 mM Br-cGMP 6012  ± 451 51.9  ± 0.8 a  Statistically different from Mn2+ quench responses to 150 nM InsP3 obtained in the absence of Br-cGMP, calculated by Student's t test (p < 0.05).

DISCUSSION

In the present study we have shown that cGMP increases [Ca²⁺]_i and potentiates the Ca²⁺-mobilizing action of receptor agonists in rat hepatocytes. The effects of cGMP on hepatocyte Ca²⁺ homeostasis are mediated by activation of G-kinase and mimicked by the spontaneous release of NO through treatment with SNP. The [Ca²⁺]_i responses induced by cGMP display a similar temporal organization to those obtained with phenylephrine and other InsP₃-linked agonists and are observed in the absence of extracellular Ca²⁺. These findings suggest that alterations in plasma membrane Ca²⁺ fluxes are not integral to the basic mechanism of cGMP-induced [Ca²⁺]_i increases and indicate a site of action at the level of intracellular Ca²⁺ stores. This is further supported by results obtained with the Mn²⁺ quench protocol to directly monitor the permeability of intracellular Ca²⁺ channels in intact hepatocytes. These experiments utilize Mn²⁺ to permeate intracellular Ca²⁺ channels and quench the fluorescence of compartmentalized fura-2. The high affinity of fura-2 for Mn²⁺ permits the rate and magnitude of Mn²⁺ quenching to be used as indices of net channel permeability and the size of the accessible intracellular stores, respectively (42, 44). The data obtained with this experimental paradigm show that cGMP increases the rate of Mn²⁺ quench of compartmentalized fura-2 in intact hepatocytes by initiating a series of quench steps that comprise essentially all of the ionomycin-sensitive intracellular Ca²⁺ store. Similar steps of Mn²⁺ quench have been described previously with receptor agonists and have been interpreted to reflect the sequential opening and closing of the intracellular channels responsible for the generation of [Ca²⁺]_i oscillations (44).

The ability of both cGMP and receptor agonists to promote the Mn²⁺ quench of the entire ionomycin-sensitive store suggests that both agents stimulate [Ca²⁺]_i release from intracellular stores that are luminally continuous (44). This is also supported by the finding that cGMP fails to produce additional quench when the response to phenylephrine has gone to completion. The effects of cGMP and phenylephrine on the Mn²⁺ quench of compartmentalized dye could result from 1) both stimuli activating identical intracellular Ca²⁺ channels, or 2) be due to activation of distinct Ca²⁺ channels that have access to luminally connected intracellular compartments. For inositol phospholipid-specific phospholipase C-linked agonists such as phenylephrine, [Ca²⁺]_i mobilization has clearly been shown to be mediated by the binding of InsP₃ to an intracellular receptor that functions as a release channel for luminal Ca²⁺ (6). The effects of cGMP on InsP₃-induced Mn²⁺ quench in permeabilized hepatocytes, and the similar kinetics and amplitude of cGMP-induced [Ca²⁺]_i responses to those of hormones in intact hepatocytes, suggest that cGMP and phenylephrine release [Ca²⁺]_i through common intracellular Ca²⁺ channels.

The data obtained with the back phosphorylation assay further indicate that cGMP targets the InsP₃ receptor Ca²⁺ channel by phosphorylating PKA-sensitive sites on the type-1 InsP₃ receptor. It is known that PKA phosphorylates two sites on the type-1 InsP₃ receptor at serine 1755 and 1589 (49) and that G-kinase only catalyzes the phosphorylation of one site at serine 1755 (22). The reduction in ³²P incorporation into the immunoprecipitated InsP₃ receptor by PKA after treatment of intact hepatocytes with cGMP was about 55% of that obtained with cAMP. This is similar to the results of a previous study where maximal ³²P incorporation into the purified cerebellar InsP₃ receptor (exclusively type-1) in the presence of G-kinase was shown to be 49-65% of that generated by PKA (22). Our finding that the InsP₃ receptor is not fully phosphorylated in the presence of db-cAMP and that the combined effects of cGMP and cAMP on InsP₃ receptor phosphorylation are nonadditive is consistent with the fact that serine 1755 is preferentially phosphorylated by PKA (49). Thus, extensive phosphorylation of this residue by PKA could preclude further phosphorylation in the presence of activators of both PKA and G-kinase. Overall, the combined effects of cGMP and cAMP on InsP₃ receptor phosphorylation in intact hepatocytes are consistent with the observed composite action of G-kinase and PKA on in vitro InsP₃ receptor phosphorylation (22).

The functional effects of InsP₃ receptor phosphorylation on InsP₃-induced [Ca²⁺]_i release varies between different cell types and have even been shown to be inconsistent in studies on the same cell type (7). For example, phosphorylation by PKA in cerebellum (50) and platelets (51) inhibits InsP₃-induced Ca²⁺ release in microsomal membranes. By contrast, other reports have established that PKA phosphorylation of a homotetrameric type-1 cerebellar InsP₃ receptor in lipid vesicles increases InsP₃-induced Ca²⁺ release (52). Treatment of permeabilized hepatocytes with PKA catalytic subunit also potentiates InsP₃-induced [Ca²⁺]_i release (46, 47), and PKA activation in intact hepatocytes phosphorylates the type-1 InsP₃ receptor and increases its sensitivity to Ca²⁺ and InsP₃ measured subsequent to permeabilization (8). In addition, activation of protein kinase C in isolated liver nuclei also appears to stimulate InsP₃-induced [Ca²⁺]_i release (53). Our data indicate that phosphorylation of PKA-sensitive sites on the InsP₃ receptor in intact hepatocytes by cGMP-mediated G-kinase activation potentiates submaximal InsP₃-induced [Ca²⁺]_i release in subsequently permeabilized hepatocytes. We propose that this sensitizing action underlies the generation of [Ca²⁺]_i oscillations by cGMP in single hepatocytes and its ability to potentiate [Ca²⁺]_i responses to receptor agonists.

Our data differ from results recently obtained with NO donors and cGMP analogues in hepatocyte couplets (33, 34). Thus, Dufour et al. (33) have shown that pretreatment with SIN-1 and cGMP inhibits agonist-induced [Ca²⁺]_i increases and bile canalicular contraction in rat hepatocyte doublets. By contrast, Burgstahler and Nathanson (34) have shown that pretreatment with SNP potentiates, and cGMP analogues have no effect on the rate of vasopressin-induced canalicular contraction in rat hepatocyte couplets. SNP produced a small [Ca²⁺]_i increase in this study, but neither SNP nor cGMP analogues had any effect on agonist-induced [Ca²⁺]_i responses. The reasons for the contradictory findings of these two reports and the differences between the results of the present study are unknown. It is possible that an elevation in the levels of constitutive PKA phosphorylation may have precluded or altered the effects of cGMP in the earlier studies. It is also possible that the InsP₃-sensitive Ca²⁺ stores could have been depleted during the 15-min cGMP pretreatment protocols used by Dufour et al. (33). In addition, Burgstahler and Nathanson (34) have shown that high concentrations of SIN-1, in the absence of superoxide dismutase, can be toxic to hepatocytes due to peroxynitrite formation.

The results of the present study also differ from those obtained in smooth muscle where phosphorylation of the InsP₃ receptor by G-kinase (22) is associated with an inhibition of InsP₃-induced Ca²⁺ release (54). The reason for these differences between cell types is unclear, although they could reflect the presence or absence of regulatory factors that mediate InsP₃ receptor phosphorylation or by the distribution of different isoforms of the InsP₃ receptor. The contribution of membrane environment to the functional effects of InsP₃ receptor phosphorylation has been documented in hepatocytes, where the ability of PKA to stimulate InsP₃ receptor binding in permeabilized cells is lost when the InsP₃ receptor is detergent-solubilized (8). The expression of different receptor subtypes could also influence the degree to which cyclic nucleotides can phosphorylate the InsP₃ receptor since the PKA phosphorylation sites present on the type-1 InsP₃ receptor are not conserved in the type-2 or type-3 InsP₃ receptors (55). This diversity in InsP₃ receptor function could be extended further by the propensity of different InsP₃ receptor subunits to form homo- or heterotetrameric assemblies (56, 57).

An additional finding of the present study was that cGMP-induced [Ca²⁺]_i responses in hepatocytes are not dependent on stimulated Ca²⁺ entry. This excludes the possibility that [Ca²⁺]_i responses to cGMP are mediated by a direct action of cGMP on plasma membrane Ca²⁺ channels. This is also supported by the finding that cGMP still potentiates agonist-induced [Ca²⁺]_i responses in the absence of extracellular Ca²⁺ (data not shown). Our data also indicate that Ca²⁺ entry does not contribute significantly to the elevation of [Ca²⁺]_i as the amplitude and time to peak of cGMP-induced [Ca²⁺]_i responses are unaffected by removal of extracellular Ca²⁺.

In conclusion, the data presented here demonstrate that the effects of cGMP on cellular Ca²⁺ homeostasis in rat hepatocytes are mediated by activation of G-kinase. Phosphorylation of the InsP₃ receptor by cGMP increases the sensitivity to InsP₃ for [Ca²⁺]_i release and results in the production of [Ca²⁺]_i oscillations at the single cell level. These findings describe a new functional role for NO and cGMP in hepatocytes and reveal an additional regulatory mechanism whereby these intracellular messengers modulate Ca²⁺ signaling in liver by regulating InsP₃ receptor function.