Cyclic GMP induces oscillatory calcium signals in rat hepatocytes.

The ability of guanosine-3′,5′-cyclic monophosphate (cGMP) to induce increases in the intracellular free calcium ion concentration ([Ca2+]i) was studied at the single cell level in fura-2-loaded rat hepatocytes. Both 8-bromo-cGMP (Br-cGMP) and dibutyryl cGMP (db-cGMP) produced oscillatory [Ca2+]i increases in hepatocytes. In addition, Br-cGMP increased the frequency of agonist-induced spiking or converted [Ca2+]i oscillations into sustained nonoscillatory [Ca2+]i responses. Addition of the nitric oxide donor sodium nitroprusside also produced oscillatory [Ca2+]i increases similar to those generated by cGMP analogues. In the absence of extracellular Ca2+, cGMP-induced [Ca2+]i responses were significantly reduced and mainly appeared as single transient [Ca2+]i increases. The effects of cGMP analogues do not appear to be mediated by a secondary increase in cAMP or activation of cAMP-dependent protein kinase (PKA), since [Ca2+]i responses to cGMP analogues were inhibited by the G-kinase inhibitor 8-bromoguanosine-3′,5′-cyclic monophosphorothioate (Rp-Br-cGMP[S]). Both Br-cGMP and db-cGMP also increased [Ca2+]i in the presence of the PKA inhibitor 8-bromoadenosine-3′,5′-cyclic monophosphorothioate (Rp-Br-cAMP[S]) and when the cGMP-inhibitable cAMP phosphodiesterase activity was inhibited by pretreatment with siguazodan. Br-cGMP stimulated the Mn2+-induced quench of compartmentalized fura-2 in intact hepatocytes, indicating a site of action at the level of the Ca2+ stores. This locus was further supported by the finding that pretreatment of hepatocytes with Br-cGMP potentiated submaximal inositol 1,4,5-trisphosphate (InsP3)-induced Mn2+ quench in subsequently permeabilized hepatocytes. db-cGMP also decreased PKA-mediated back phosphorylation of the hepatic type-1 InsP3 receptor, indicating that G-kinase phosphorylates the InsP3 receptor at sites targeted by PKA. These data indicate that phosphorylation of the hepatic InsP3 receptor by G-kinase increases the sensitivity to InsP3 for [Ca2+]i release and is associated with the production of [Ca2+]i oscillations in single rat hepatocytes.

naling 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) [Ca 2ϩ ] i responses to submaximal concentrations of inositol phospholipid-linked agonists originate as repetitive oscillations in [Ca 2ϩ ] i that are spatially organized in the form of Ca 2ϩ waves (5). These [Ca 2ϩ ] i responses are mediated, at least in part, by an increased formation of the second messenger inositol-1,4,5-trisphosphate (InsP 3 ) 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 InsP 3 receptor regulates the sensitivity to both InsP 3 and Ca 2ϩ (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 InsP 3 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 [Ca 2ϩ ] i responses in a number of different cell types (12). For example, cGMP decreases [Ca 2ϩ ] 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 [Ca 2ϩ ] i signaling including activation of plasma membrane Ca 2ϩ pumps (17,18), stimulation of sarcoplasmic reticulum Ca 2ϩ pumps by phosphorylation of phospholamban (19 -21), inhibition of L-type voltage-gated calcium channels (14), and inhibition of InsP 3 -induced Ca 2ϩ release by phosphorylation of the InsP 3 receptor (22). In addition, Br-cGMP inhibits thrombin-induced InsP 3 formation and [Ca 2ϩ ] i responses by phosphorylating a pertussis toxin-sensitive Gprotein in Chinese hamster ovary cells transfected with Gkinase (23). Growth factor-mediated inositol phospholipid-specific phospholipase C activation and [Ca 2ϩ ] 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 [Ca 2ϩ ] i in sea urchin eggs by stimulating the synthesis of cyclic ADP ribose and the release of Ca 2ϩ from ryanodine-sensitive intracellular stores (25). A role for cGMP in the regulation of Ca 2ϩ entry has emerged from research in pancreatic acinar cells where intracellular perfusion of cGMP emulates the inward Ca 2ϩ entry current observed upon depletion of intracellular Ca 2ϩ stores (26). Further studies have revealed that depletion of intracellular Ca 2ϩ stores activates NOS to generate cGMP which, depending on the final concen-tration, can either stimulate or inhibit agonist-induced Ca 2ϩ entry in acinar cells (27). NO and cGMP have also been shown to potentiate Ca 2ϩ 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 [Ca 2ϩ ] 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 Ca 2ϩ 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 Ca 2ϩ homeostasis in rat hepatocytes. Our data show that cGMP phosphorylates the InsP 3 receptor in intact hepatocytes and increases the sensitivity to InsP 3 for [Ca 2ϩ ] i release. This results in the generation of [Ca 2ϩ ] i oscillations and to the enhancement of agonist-induced Ca 2ϩ responses.

EXPERIMENTAL PROCEDURES
Cell Isolation-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).

Measurement of InsP 3 -induced Mn 2ϩ
Quench of Compartmentalized Fura-2 in Permeabilized Hepatocytes-Intact hepatocytes (4 mg of protein/ml) were loaded with fura-2/AM (5 M) 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 Ca 2ϩ -free HEPES buffer. Before use the cells were washed once in Ca 2ϩ -free HEPES buffer containing 100 M EGTA. The cells were then permeabilized in buffer containing 120 mM KCl, 1 mM KH 2 PO 4 , 2 mM MgCl 2 , 10 mM NaCl, 5 g/ml oligomycin, 1 g/ml rotenone, 5 M carbonyl chlorophenylhydrazone, 2 mM ATP⅐Mg, 10 mM creatine phosphate, 1 unit/ml creatine kinase, 30 -50 g/ml digitonin, 2 M thapsigargin, and 1 g/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 M MnCl 2 . 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.
Fluorescence Imaging in Single Hepatocytes-Hepatocytes (5 ϫ 10 5 cells/Petri dish) were plated on glass coverslips coated with poly-Dlysine (5 g/cm 3 ) in 2 ml of insulin-free Williams E medium supplemented with 10% fetal calf serum, 10 units/ml penicillin, 10 g/ml streptomycin, 0.05 mg/ml gentamycin, 4 g/ml dexamethasone, and 2 mM glutamine. The cells were incubated for 30 min at 37°C under an atmosphere of CO 2 /air (5:95%) and then washed to remove unattached and nonviable cells. Measurements of [Ca 2ϩ ] i were obtained from cells loaded with fura-2 by incubation with fura-2/AM (5 M in 0.03% pluronic F-127) for 30 min at 37°C in HEPES buffer with 2% bovine serum albumin and 200 M sulfinpyrazone. This protocol resulted in Ͼ70% cytosolic loading of fura-2. For Mn 2ϩ 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 ϫ oil immersion objective, and chamber were thermostatically regulated at 37°C. A liquid N 2 -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 [Ca 2ϩ ] 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. [Ca 2ϩ ] i was calculated from the fluorescence measurements using the ratio method, after subtraction of fluorescence signals derived from compartmentalized dye, as described previously (36). Mn 2ϩ quenching of compartmentalized fura-2 was measured at 360-nm excitation.
Back Phosphorylation of the InsP 3 Receptor-The assay for back phosphorylation of the InsP 3 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 InsP 3 receptor antibody (raised against the C terminus of the type-1 InsP 3 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 MgCl 2 , 0.5 mM phenylmethylsulfonyl fluoride, and 10 g/ml each of aprotinin, soybean trypsin inhibitor, and leupeptin. Immunoprecipitated InsP 3 receptor was then incubated in 0.2 ml of phosphorylation buffer containing 100 units/ml of the catalytic subunit of PKA and 3-5 Ci of [␥-32 P]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 32 P-labeled InsP 3 receptor was quantitated from the autoradiographs with a laser densitometer. The localization of the InsP 3 receptor on the nitrocellulose was verified by immunoblotting.
Materials-[␥-32 P]ATP was obtained from DuPont NEN, Ins(1, 4, 5)P 3 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.

Cyclic GMP Increases [Ca 2ϩ ] i in Single Hepatocytes-
The effects of cGMP analogues on [Ca 2ϩ ] i were examined at the single cell level in fura-2-loaded freshly isolated hepatocytes. Fig. 1 shows typical [Ca 2ϩ ] i responses in single hepatocytes exposed to Br-cGMP (200 M) or db-cGMP (200 M). In the present study we have used Br-cGMP and db-cGMP interchangeably since the [Ca 2ϩ ] i responses to both compounds are essentially equivalent and observed over similar concentration ranges. 2 [Ca 2ϩ ] i responses produced by cGMP appeared as a series of base-line-separated [Ca 2ϩ ] 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 [Ca 2ϩ ] i responses frequently displayed secondary spiking activity during the falling phase of each [Ca 2ϩ ] 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 [Ca 2ϩ ] i responses at higher doses (data not shown). The [Ca 2ϩ ] 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 [Ca 2ϩ ] 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 M) (Fig. 2). The inability to fully suppress [Ca 2ϩ ] 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 [Ca 2ϩ ] 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- Addition of the NO donor SNP also generated [Ca 2ϩ ] i increases in hepatocytes. This is shown in Fig. 4 where addition of SNP produced oscillatory [Ca 2ϩ ] i increases, comparable with those elicited by cGMP. The SNP-induced [Ca 2ϩ ] i oscillations appeared after a well-defined latent period. Most cells responded with [Ca 2ϩ ] i oscillations in the range of 1-10 M SNP. Increasing doses of SNP increased the proportion of cells responding but did not produce a clear enhancement of [Ca 2ϩ ] i oscillation frequency in individual hepatocytes already responding to SNP. We have observed similar [Ca 2ϩ ] i responses with SIN-1 in hepatocytes (data not shown).
Role of Extracellular Ca 2ϩ -Previous studies in liver, and other cell types, have shown that [Ca 2ϩ ] i oscillations persist in the absence of extracellular Ca 2ϩ , although they tend to have a reduced frequency followed by eventual run down and stopping (36,41). To assess the contribution of extracellular Ca 2ϩ influx to the initiation and maintenance of cGMP-induced [Ca 2ϩ ] i oscillations, the ability of Br-cGMP to elicit [Ca 2ϩ ] i responses in the absence of extracellular Ca 2ϩ was examined. As shown in Fig. 5, removal of extracellular Ca 2ϩ did not significantly affect the amplitude (368 Ϯ 26 nM in the presence and 345 Ϯ 23 nM in the absence of extracellular Ca 2ϩ ) of Br-cGMP-induced [Ca 2ϩ ] i responses but significantly reduced their frequency or more often converted them to single transient [Ca 2ϩ ] i increases. Removal of extracellular Ca 2ϩ also had no effect on the time to peak Ca 2ϩ . We have observed similar effects of Ca 2ϩ depletion on vasopressin-induced [Ca 2ϩ ] i responses in primary cultured hepatocytes (36). This indicates that Ca 2ϩ influx is not required for the generation of Br-cGMP-induced [Ca 2ϩ ] i responses but plays an important role in sustaining the [Ca 2ϩ ] i oscillations by replenishing intracellular Ca 2ϩ stores.
Cyclic GMP Potentiates Agonist-induced [Ca 2ϩ ] i Responses-The data described above demonstrate that cGMP-induced [Ca 2ϩ ] i responses have a similar temporal organization and Ca 2ϩ 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 [Ca 2ϩ ] i response to phenylephrine in all cells, but the extent of this enhancement was determined by the magnitude of the phenylephrine-induced [Ca 2ϩ ] i increase (Fig. 6). Addition of Br- cGMP to cells responding to phenylephrine with one or two [Ca 2ϩ ] i oscillations resulted in a rapid increase in oscillation frequency (Fig. 6B), whereas cells displaying multiple [Ca 2ϩ ] i spikes were converted to sustained [Ca 2ϩ ] i responses (Fig. 6C). Br-cGMP was also able to generate [Ca 2ϩ ] i oscillations in cells that failed to respond to phenylephrine (Fig. 6A). In cells where phenylephrine caused a sustained [Ca 2ϩ ] i response Br-cGMP was unable to further increase [Ca 2ϩ ] i (data not shown). The amplitude of the oscillatory and sustained [Ca 2ϩ ] i responses produced by addition of Br-cGMP were similar to those generated by phenylephrine alone. A similar potentiation of agonistinduced [Ca 2ϩ ] i responses was observed upon addition of SNP to hepatocytes pre-exposed to phenylephrine (data not shown).
Cyclic GMP Stimulates Mn 2ϩ -induced Quenching of Compartmentalized Fura-2 in Intact Hepatocytes-Recent studies in intact and permeabilized cells have shown that Mn 2ϩ can be used as a Ca 2ϩ surrogate to monitor the divalent cation permeability of the InsP 3 -sensitive Ca 2ϩ channels associated with intracellular stores (42)(43)(44)(45). These experiments exploit the ability of Mn 2ϩ to quench the fluorescence of fura-2 entrapped within intracellular compartments and provide a direct measure of the gating properties of InsP 3 -sensitive Ca 2ϩ channels. We have utilized this technique to further resolve the contribution of intracellular Ca 2ϩ stores to the [Ca 2ϩ ] i responses generated by cGMP in hepatocytes. Changes in fura-2 fluorescence were monitored at 360 nm excitation (Ca 2ϩ -insensitive wavelength) and in Ca 2ϩ -free medium to facilitate Mn 2ϩ entry and eliminate contributions from Ca 2ϩ influx. Fig. 7 demonstrates that Mn 2ϩ 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 [Ca 2ϩ ] 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 Ca 2ϩ channels within intracellular stores. 3 Although it is not possible to monitor [Ca 2ϩ ] i changes simultaneously with the Mn 2ϩ 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 [Ca 2ϩ ] i oscillations observed under the same conditions.
Br-cGMP also potentiated the ability of phenylephrine to stimulate Mn 2ϩ quench of compartmentalized fura-2 in a manner that paralleled its effects on agonist-stimulated [Ca 2ϩ ] i signals. This is shown in Fig. 8 where the capacity of Br-cGMP to enhance phenylephrine-induced Mn 2ϩ quench was limited by the extent of the agonist response. In fact Br-cGMP had no affect in cells where the phenylephrine-induced Mn 2ϩ quench had already gone to completion (Fig. 8A). These results indicate that Br-cGMP mimics the effects of hormones that give [Ca 2ϩ ] i oscillations, by stimulating the sequential opening and closing of the intracellular Ca 2ϩ channels whose permeability to Mn 2ϩ is registered by the periodic quench of compartmentalized 3 Under these conditions Mn 2ϩ quench does not reflect activity of plasma membrane channels since cytosolic dye is already fully quenched with Mn 2ϩ , and previous studies have shown that these steps continue after removal of extracellular Mn 2ϩ (44,45). fura-2. In addition, Br-cGMP and phenylephrine appear to stimulate Mn 2ϩ entry into the same intracellular compartment, and this constitutes almost the entire ionomycin-sensitive Ca 2ϩ store.
Cyclic GMP Phosphorylates the Type-1 InsP 3 Receptor at a PKA-sensitive Site in Intact Hepatocytes-The type-1 InsP 3 receptor has been shown to be phosphorylated by PKA in intact rat hepatocytes (8). This phosphorylation leads to an increased sensitivity of the hepatic InsP 3 receptor to both Ca 2ϩ and InsP 3 (8,46,47) and has been suggested to underlie the stimulatory effects of cAMP on [Ca 2ϩ ] i signals in hepatocytes (47,48). Since the [Ca 2ϩ ] i increases generated by cGMP in this study appear to be mediated by G-kinase activation, and the type-1 InsP 3 receptor is known to be phosphorylated by G-kinase on a site also phosphorylated by PKA (22), we investigated whether cGMP phosphorylated the InsP 3 receptor in intact hepatocytes. The phosphorylation of the type-1 InsP 3 receptor was examined using a back phosphorylation assay, whereby solubilized extracts from control and cGMP-treated hepatocytes were immunoprecipitated with a type-1 InsP 3 receptor antibody before being phosphorylated in vitro by incubation with [ 32 P]ATP and the catalytic subunit of PKA. Accordingly, an increased phosphorylation of the InsP 3 receptor in intact hepatocytes should be reflected by a decreased incorporation of 32 P during the in vitro phosphorylation assay. Fig. 9 demonstrates that both db-cAMP and db-cGMP treatment of intact hepatocytes decreased 32 P incorporation into the immunoprecipitated InsP 3 receptor. The results summarized in Table I demonstrate that exposure to db-cGMP reduced 32 P incorporation by about 55% of that produced by db-cAMP. However, when both agents were added in combination their effects on 32 P incorporation were completely nonadditive. This suggests that db-cAMP and db-cGMP phosphorylate common sites on the InsP 3 receptor.
Cyclic GMP Potentiates InsP 3 -induced Mn 2ϩ Quench in Permeabilized Hepatocytes-As noted above, previous studies have reported that phosphorylation of the InsP 3 receptor by PKA potentiates InsP 3 -induced [Ca 2ϩ ] i release in hepatocytes (46 -48). To ascertain whether cGMP-mediated InsP 3 receptor phosphorylation elicited a similar sensitizing action, the effect of Br-cGMP on InsP 3 -induced Mn 2ϩ 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 Ca 2ϩ -free HEPES buffer before permeabilization in cytosolic buffer containing thapsigargin. The addition of thapsigargin depletes the intracellular Ca 2ϩ stores and enables the effects of cGMP on the Mn 2ϩ permeability of the InsP 3 receptor/channel to be monitored independent of changes in luminal Ca 2ϩ . 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 Mn 2ϩ -induced quench elicited by a submaximal dose of InsP 3 (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 Mn 2ϩ quench produced by a maximal dose of InsP 3 (5 M). This indicates that pretreatment with cGMP increases the number of InsP 3 -sensitive Ca 2ϩ channels that can be activated in the presence of submaximal concentrations of InsP 3 without altering the size of the InsP 3 -sensitive Ca 2ϩ pool, as measured by the Mn 2ϩ quench response to a maximal dose of InsP 3 .

DISCUSSION
In the present study we have shown that cGMP increases quench protocol to directly monitor the permeability of intracellular Ca 2ϩ channels in intact hepatocytes. These experiments utilize Mn 2ϩ to permeate intracellular Ca 2ϩ channels and quench the fluorescence of compartmentalized fura-2. The high affinity of fura-2 for Mn 2ϩ permits the rate and magnitude of Mn 2ϩ 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 Mn 2ϩ quench of compartmentalized fura-2 in intact hepatocytes by initiating a series of quench steps that comprise essentially all of the ionomycin-sensitive intracellular Ca 2ϩ store. Similar steps of Mn 2ϩ 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 [Ca 2ϩ ] i oscillations (44).
The ability of both cGMP and receptor agonists to promote the Mn 2ϩ quench of the entire ionomycin-sensitive store suggests that both agents stimulate [Ca 2ϩ ] i release from intracel-lular 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 Mn 2ϩ quench of compartmentalized dye could result from 1) both stimuli activating identical intracellular Ca 2ϩ channels, or 2) be due to activation of distinct Ca 2ϩ channels that have access to luminally connected intracellular compartments. For inositol phospholipid-specific phospholipase C-linked agonists such as phenylephrine, [Ca 2ϩ ] i mobilization has clearly been shown to be mediated by the binding of InsP 3 to an intracellular receptor that functions as a release channel for luminal Ca 2ϩ (6). The effects of cGMP on InsP 3 -induced Mn 2ϩ quench in permeabilized hepatocytes, and the similar kinetics and amplitude of cGMP-induced [Ca 2ϩ ] i responses to those of hormones in intact hepatocytes, suggest that cGMP and phenylephrine release [Ca 2ϩ ] i through common intracellular Ca 2ϩ channels.
The data obtained with the back phosphorylation assay further indicate that cGMP targets the InsP 3 receptor Ca 2ϩ channel by phosphorylating PKA-sensitive sites on the type-1 InsP 3 receptor. It is known that PKA phosphorylates two sites on the type-1 InsP 3 receptor at serine 1755 and 1589 (49) and that  InsP 3 receptor with PKA Isolated hepatocytes were incubated with drug for 10 min. Samples were centrifuged, solubilized, and extracts immunoprecipitated with type-1 InsP 3 receptor antibody. Immunoprecipitates were back-phosphorylated with [ 32 P]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 32 P-labeled InsP 3 receptor was quantitated from the autoradiographs with a laser densitometer and is expressed as the percentage of the label incorporated into the InsP 3 receptor immunoprecipitated from control cells. The data are the mean Ϯ S.E. of three experiments in which back phosphorylation assays for each condition were performed in duplicate. G-kinase only catalyzes the phosphorylation of one site at serine 1755 (22). The reduction in 32 P incorporation into the immunoprecipitated InsP 3 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 32 P incorporation into the purified cerebellar InsP 3 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 InsP 3 receptor is not fully phosphorylated in the presence of db-cAMP and that the combined effects of cGMP and cAMP on InsP 3 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 InsP 3 receptor phosphorylation in intact hepatocytes are consistent with the observed composite action of G-kinase and PKA on in vitro InsP 3 receptor phosphorylation (22).
The functional effects of InsP 3 receptor phosphorylation on InsP 3 -induced [Ca 2ϩ ] 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 InsP 3 -induced Ca 2ϩ release in microsomal membranes. By contrast, other reports have established that PKA phosphorylation of a homotetrameric type-1 cerebellar InsP 3 receptor in lipid vesicles increases InsP 3 -induced Ca 2ϩ release (52). Treatment of permeabilized hepatocytes with PKA catalytic subunit also potentiates InsP 3 -induced [Ca 2ϩ ] i release (46,47), and PKA activation in intact hepatocytes phosphorylates the type-1 InsP 3 receptor and increases its sensitivity to Ca 2ϩ and InsP 3 measured subsequent to permeabilization (8). In addition, activation of protein kinase C in isolated liver nuclei also appears to stimulate InsP 3 -induced [Ca 2ϩ ] i release (53). Our data indicate that phosphorylation of PKA-sensitive sites on the InsP 3 receptor in intact hepatocytes by cGMP-mediated G-kinase activation potentiates submaximal InsP 3 -induced [Ca 2ϩ ] i release in subsequently permeabilized hepatocytes. We propose that this sensitizing action underlies the generation of [Ca 2ϩ ] i oscillations by cGMP in single hepatocytes and its ability to potentiate [Ca 2ϩ ] 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 [Ca 2ϩ ] 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 [Ca 2ϩ ] i increase in this study, but neither SNP nor cGMP analogues had any effect on agonist-induced [Ca 2ϩ ] 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 InsP 3 -sensitive Ca 2ϩ 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 InsP 3 receptor by G-kinase (22) is associated with an inhibition of InsP 3 -induced Ca 2ϩ 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 InsP 3 receptor phosphorylation or by the distribution of different isoforms of the InsP 3 receptor. The contribution of membrane environment to the functional effects of InsP 3 receptor phosphorylation has been documented in hepatocytes, where the ability of PKA to stimulate InsP 3 receptor binding in permeabilized cells is lost when the InsP 3 receptor is detergentsolubilized (8). The expression of different receptor subtypes could also influence the degree to which cyclic nucleotides can phosphorylate the InsP 3 receptor since the PKA phosphorylation sites present on the type-1 InsP 3 receptor are not conserved in the type-2 or type-3 InsP 3 receptors (55). This diversity in InsP 3 receptor function could be extended further by the propensity of different InsP 3 receptor subunits to form homo-or heterotetrameric assemblies (56,57).
An additional finding of the present study was that cGMPinduced [Ca 2ϩ ] i responses in hepatocytes are not dependent on stimulated Ca 2ϩ entry. This excludes the possibility that [Ca 2ϩ ] i responses to cGMP are mediated by a direct action of cGMP on plasma membrane Ca 2ϩ channels. This is also supported by the finding that cGMP still potentiates agonist-induced [Ca 2ϩ ] i responses in the absence of extracellular Ca 2ϩ (data not shown). Our data also indicate that Ca 2ϩ entry does not contribute significantly to the elevation of [Ca 2ϩ ] i as the amplitude and time to peak of cGMP-induced [Ca 2ϩ ] i responses are unaffected by removal of extracellular Ca 2ϩ .
In conclusion, the data presented here demonstrate that the effects of cGMP on cellular Ca 2ϩ homeostasis in rat hepatocytes are mediated by activation of G-kinase. Phosphorylation of the InsP 3 receptor by cGMP increases the sensitivity to InsP 3 for [Ca 2ϩ ] i release and results in the production of [Ca 2ϩ ] 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 Ca 2ϩ signaling in liver by regulating InsP 3 receptor function.

TABLE II
Effect of cGMP on InsP 3 -Induced Mn 2ϩ 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 Ca 2ϩ -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 M Mn 2ϩ . The initial rates were calculated by linear regression over the linear portion of the quench response obtained after addition of 150 nM InsP 3 or 5 M InsP 3 . The size of the InsP 3 -sensitive pool is expressed as a percentage of the total ionomycin-quenchable fluorescence. The data represent the mean Ϯ S.E. of values obtained in 15-21 individual runs from 4 to 5 different hepatocyte preparations. Mn 2ϩ quench of fura-2 fluorescence was monitored with excitation at 360 nm and emission at 510 nm.