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Grass carp somatolactin: II. Pharmacological study on postreceptor signaling mechanisms for PACAP-induced somatolactin- and -β gene expression

Endocrinology Division, School of Biological Sciences, University of Hong Kong, Hong Kong, China

Submitted 22 April 2008 ; accepted in final form 29 May 2008

	ABSTRACT

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Somatolactin (SL), the latest member of the growth hormone/prolactin family, is a novel pituitary hormone with diverse functions. However, the signal transduction mechanisms responsible for SL expression are still largely unknown. Using grass carp as an animal model, we examined the direct effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on SL gene expression at the pituitary level. In primary cultures of grass carp pituitary cells, SL and SLβ mRNA levels could be elevated by PACAP via activation of PAC-I receptors. With the use of a pharmacological approach, the AC/cAMP/PKA and PLC/inositol 1,4,5-trisphosphate (IP₃)/PKC pathways and subsequent activation of the Ca²⁺/calmodulin (CaM)/CaMK-II cascades were shown to be involved in PACAP-induced SL mRNA expression. Apparently, the downstream Ca²⁺/CaM-dependent cascades were triggered by extracellular Ca²⁺ ([Ca²⁺]_e) entry via L-type voltage-sensitive Ca²⁺ channels (VSCC) and Ca²⁺ release from IP₃-sensitive intracellular Ca²⁺ stores. In addition, the VSCC component could be activated by cAMP/PKA- and PLC/PKC-dependent mechanisms. Similar postreceptor signaling cascades were also observed for PACAP-induced SLβ mRNA expression, except that [Ca²⁺]_e entry through VSCC, PKC coupling to PLC, and subsequent activation of CaMK-II were not involved. These findings, taken together, provide evidence for the first time that PACAP can induce SL and SLβ gene expression in fish model via PAC-I receptors through differential coupling to overlapping and yet distinct signaling pathways.

pituitary adenylate cyclase-activating polypeptide; grass carp pituitary cells

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with diverse functions, is widely expressed in various tissues in mammals (5). The protein structure of PACAP is highly homologous to vasoactive intestinal polypeptide (VIP; Ref. 38), and the biological functions of the two peptides are mediated by VPAC receptors, which can bind PACAP and VIP with similar affinity. PACAP can also exert physiological actions independent of VIP via activation of PAC-I receptors, which are specific for PACAP with little/low binding affinity for VIP (10). In mammals, PACAP neurons can be located in the hypothalamus with nerve fibers extending into the median eminence (36). In addition, PACAP immunoreactivity can be detected in hypophysial portal blood (4) and PACAP treatment is known to stimulate pituitary hormone secretion, both in vivo and in vitro (32). The role of PACAP as a hypophysiotropic factor is well conserved in vertebrates, including the fish (42). Although modern-day bony fish do not have a hypophysial portal blood system, their pituitary is directly innervated by the hypothalamus (9) and nerve fibers with PACAP immunoreactivity have been identified in the pituitary of goldfish (41) and eel (23). In representative fish species, PACAP treatment can also induce GH [e.g., common carp (44) and salmon (29)] and LH secretion [e.g., goldfish (2)] through direct actions at the pituitary level.

In fish models, PACAP may also play a role in SL regulation. In stargazer, PACAP nerve fibers are located in the vicinity of SL cells in the pars intermedia (20). With the use of a cell immunoblot assay, the blotting area for SL immunoreactivity detected on the culture membrane with goldfish pituitary cells attached can be increased by PACAP treatment, implying that PACAP may induce SL release at the pituitary level (21). This idea is confirmed by our recent studies (12) in grass carp pituitary cells, in which SL release, SL content, and total SL production could be elevated by PACAP stimulation. In the same study, the increase in SL production also occurred with parallel rises in SL and SLβ mRNA levels and these stimulatory actions could be mimicked by the PAC-I receptor agonist Maxadilan but blocked by the PAC-I antagonist M65. These findings suggest that PACAP can not only induce SL secretion but also stimulate SL synthesis by upregulation of SL and SLβ gene expression via PAC-I receptors. To further examine the mechanisms for PACAP induction of SL gene expression, grass carp pituitary cells were used as a model to study the postreceptor signaling events involved in PACAP-induced SL and SLβ mRNA expression. By direct measurement of second messengers and pharmacological perturbation of signaling cascades, we demonstrate for the first time that PACAP can trigger SL and SLβ gene expression at the pituitary level by differential coupling of the Ca²⁺/calmodulin (CaM)-dependent pathway with the AC/cAMP/PKA and PLC/inositol 1,4,5-trisphosphate (IP₃)/PKC signaling mechanisms.

	MATERIALS AND METHODS

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Animals. One-year-old grass carps (Ctenopharyngodon idellus) with a body weight ranging from 1.5 to 2.0 kg were obtained from local markets. After acclimation in the laboratory, the fish were anesthetized in 0.05% MS222 (Sigma, St. Louis, MO) and killed for pituitary cell preparation according to the guidelines for animal use at the University of Hong Kong.

Test substances. Ovine PACAP₃₈ and human VIP were purchased from Bachem Fine Chemicals (La Jolla, CA), dissolved in double-distilled deionized water, and stored frozen at –80°C as 0.1 mM stocks in small aliquots. Forskolin, H89, IBMX, 1,2-dioctanoylglycerol (DiC8), 8-(4-chloro-phenylthio)-cAMP (CPT-cAMP), A23187 [GenBank] , Bay K8644, KN62, nifedipine, thapsigargin, 2-aminoethoxydiphenyl borate (2-APB), U73122 [GenBank] , GF109203, MDL 12330A, calphostin C, and calmidazolium were obtained from Calbiochem (San Diego, CA). Similar to the peptide hormones, these pharmacological agents were prepared as 10-mM frozen stocks in DMSO. Stock solutions of test substances were diluted with prewarmed (28°C) culture medium to appropriate concentrations 15 min before drug treatment. The final dilutions of DMSO were <0.1% and had no effects on SL gene expression in grass carp pituitary cells.

Measurement of SL and SLβ mRNA expression. Primary cultures of grass carp pituitary cells were prepared by the trypsin/DNase digestion method as described previously (43). After that, pituitary cells were seeded in 24-well plates at a density of 2.5 x 10⁶ cells·ml^–1·well^–1 and cultured for 15–18 h at 28°C in carp MEM (MEM Eagle Medium supplemented with 26 mM NaHCO₃, 25 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml fungizone, pH 7.7) with 5% FBS for recovery. On the following day, culture medium was replaced with serum-free carp MEM with the appropriate levels of test substances and the cells were allowed to incubate at 28°C for another 48 h. After drug treatment, total RNA was extracted from individual wells using Trizol and subjected to slot blot assays (45) using the DIG-labeled cDNA probes for carp SL and SLβ, respectively. The cDNA probes were prepared by a PCR DIG probe synthesis kit (Roche) using primers franking position 109-392 of grass carp SL (GenBank Accession No. EF372075) and position 197-486 of grass carp SLβ (Accession No. EF372074), respectively. In these studies, parallel blotting of 18S rRNA was also conducted to serve as an internal control.

Measurement of cAMP production. The neurointermediate lobe (NIL) of individual pituitaries was isolated by manual dissection under a stereomicroscope, and cell dispersion was conducted using trypsin/DNase digestion method (43). The cells prepared (refer to as "NIL cells") were seeded at a density of 2 x 10⁶ cells·2 ml^–1·dish^–1 in poly-D-lysine precoated 35-mm dishes and cultured overnight at 28°C in carp MEM with 5% FBS. On the following day, culture medium was replaced with 0.9 ml HEPES-buffered HBSS (39) with 0.1% BSA and 0.1 mM IBMX. IBMX, the inhibitor for phosphodiesterases, was included to prevent cAMP degradation in NIL cells. Drug treatment was initiated by the addition of 0.1 ml 10x stocks of PACAP at the appropriate concentrations, and the cells were allowed to incubate at 28°C for 15 min. After that, culture medium was harvested for the measurement of cAMP release whereas cellular cAMP content was extracted from NIL cells with 1 ml of ice-cold absolute ethanol. These cAMP samples were freeze-dried and stored at –20°C until their cAMP contents were quantified by a BioTrak [¹²⁵I]cAMP kit (Amersham, Piscataway, NJ).

Measurement of intracellular Ca²⁺ levels. Grass carp NIL cells were cultured on poly-D-lysine precoated coverslips (5 x 10⁵ cells·ml^–1·coverslip^–1) for single-cell Ca²⁺ imaging as described previously (35). Briefly, NIL cells were preloaded with the Ca²⁺-sensitive dye fura-2/AM (5 µM; Molecular Probes) for 45 min at room temperature. After that, ratiometric measurement of the intracellular Ca²⁺ level ([Ca²⁺]_i) was conducted using a PTI Epifluorescence Ca²⁺ Imaging System (Photon Technology International, Birmingham, NJ) with excitation wavelengths at 340 and 380 nm and emission wavelength at 510 nm. Given that variations in fura-2 loading were noted between different batches of NIL cells, Ca²⁺ calibration was not performed and the Ca²⁺ data were simply expressed as a ratio of fluorescence signals with excitation at 340 and 380 nm, respectively (as "F340/380 Ratio").

Western blot of CaM expression. Grass carp NIL cells (2.5 x 10⁶ cells·ml^–1·well^–1) were incubated at 28°C for 48 h in carp MEM containing increasing concentrations of PACAP. After that, Western blot was performed according to the standard procedures in our laboratory (11). Briefly, NIL cells were rinsed with PBS (pH 7.4) and cell lysate was prepared by three cycles of freezing and thawing in RIPA buffer (50 mM Tris·HCl, 150 mM NaCl, 1% NP-40, 1 mM EDTA, and 0.25% Na deoxycholate) with 1 mM PMSF and complete protease inhibitor cocktail (Roche). The lysate was resolved in a 10% gel by SDS-PAGE and transblotted onto a PVDF membrane at 65 V for 2.5 h. The membrane was then blocked by 2% nonfat dried milk and incubated overnight at 4°C with an antiserum raised against human CaM (1:1,000; Upstate, Milford, MA). On the following day, HRP-conjugated anti-mouse IgG (1:15,000; Bio-Rad, Hercules, CA) was added and SuperSignal WestPico (Pierce, Rockford, IL) was used as a substrate for signal development. In these studies, Western blot of β-actin using an actin Ab-1 kit (Oncogene, Boston, MA) was used as an internal control. Similar procedures were also used in this study for Western blot of PKC, PKCβ, and PKC using a PKC sampler kit (Transduction Laboratories, Lexingon, KY) according to the antibody dilutions recommended by the manufacturer.

Data transformation and statistics. For slot blot assays, SL and SLβ mRNA levels were quantified in terms of arbitrary density units and normalized against the amount of 18S RNA expressed in the same sample. Since no significant changes were observed for 18S RNA expression in these experiments, normalized data were simply transformed as a percentage of the mean value in the control group for statistical analysis (referred to as "%Ctrl"). For cAMP measurement, the data for cAMP release and cAMP content were expressed as picomoles cAMP detected per million cells. Data presented, expressed as means ± SE, are the results pooled from separate experiments and were analyzed using Student's t-test or two-way ANOVA followed by Fisher's least significant difference test. Differences were considered significant at P < 0.05.

	RESULTS

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PACAP induction of SL and SLβ mRNA expression. In our recent studies (12), grass carp PACAP was shown to increase SL and SLβ gene expression in the carp pituitary. Since grass carp PACAP is of limited supply, ovine PACAP was used in the present study. To ensure that ovine PACAP behaves similarly in terms of receptor specificity compared with fish PACAP, static incubation experiments were conducted in grass carp pituitary cells with PACAP and VIP of mammalian origin (Fig. 1). In this case, increasing concentrations of ovine PACAP (0.1–100 nM) could upregulate SL and SLβ mRNA levels in a dose-dependent manner. The minimal effective doses for PACAP induction of SL and SLβ gene expression were found to be at 0.1 nM level with maximal responses observed in the 10- to 100-nM dose range. The ED₅₀s for the stimulatory effects of PACAP on SL and SLβ mRNA levels were estimated to be 0.9 and 1.2 nM, respectively. In parallel experiments with VIP treatment, increasing doses of human VIP (0.1–100 nM) were not very effective in triggering SL gene expression, and significant rises in SL and SLβ mRNA levels could be noted only at a 100-nM dose. Apparently, the transcript expression of SL and SLβ at the pituitary level are more sensitive to PACAP stimulation compared with the corresponding responses caused by VIP and these pharmacological profiles are consistent with that reported for mammalian PAC-I receptors (10).

View larger version (32K): [in this window] [in a new window]   Fig. 1. Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on somatolactin (SL) and SLβ gene expression in grass carp pituitary cells. Pituitary cells were incubated for 48 h with increasing doses (0.1–1,000 nM) of ovine PACAP or human VIP. After drug treatment, total RNA was isolated and slot blots for SL (top) and SLβ mRNA (bottom) were conducted as described in MATERIALS AND METHODS. In these studies, parallel blotting of 18S RNA was used as an internal control. Data presented are expressed as means ± SE and are the pooled results from 4 separate experiments. Experimental groups denoted by the same letter represent a similar level of transcript expression [P > 0.05, ANOVA followed by Fisher's least significant difference (LSD) test]. Representative results of slot blots are also included for reference.

View larger version (46K): [in this window] [in a new window]   Fig. 2. Effects of activating cAMP-dependent cascades on SL and SLβ gene expression in grass carp pituitary cells. Pituitary cells were treated for 48 h with increasing doses of the AC activator forskolin (A; 0.001–10 µM) or the membrane permeant cAMP analog CPT-cAMP (B; 0.1–1,000 µM). After drug treatment, total RNA was extracted for SL and SLβ mRNA measurement and parallel blotting of 18S RNA was used as an internal control. Data presented, expressed as means ± SE, are pooled results from 3 separate experiments. Treatment groups denoted by the same letter represent a similar level of transcript expression (P > 0.05, ANOVA followed by Fisher's LSD test).

View larger version (18K): [in this window] [in a new window]   Fig. 3. Functional role of cAMP-dependent mechanisms in PACAP-induced SL and SLβ gene expression. A: effects of increasing concentrations of PACAP (0.1–1,000 nM) on cAMP release and cAMP content in neurointermediate lobe (NIL) cells prepared from the grass carp pituitary. *Significant difference compared with the respective control (P < 0.05, Student's t-test). B: effects of MDL12330A and H89 on PACAP-induced SL and SLβ mRNA expression. Pituitary cells were incubated for 48 h with ovine PACAP (10 nM) in the presence of absence of the AC inhibitor MDL12330A (MDL; 20 µM) or PKA inhibitor H89 (20 µM). After drug treatment, total RNA was extracted for SL and SLβ mRNA measurement. In this study, parallel blotting of 18S RNA was used as an internal control. Data presented, expressed as means ± SE, are pooled results from 4 separate experiments. Experimental groups denoted by the same letter represent a similar level of transcript expression (P > 0.05, ANOVA followed by Fisher's LSD test).

View larger version (38K): [in this window] [in a new window]   Fig. 4. Functional role of PLC/PKC-dependent mechanisms in SL and SLβ gene expression. A: effects of U73122 [GenBank] on PACAP-induced SL and SLβ gene expression. Pituitary cells were exposed to ovine PACAP (10 nM) for 48 h with or without simultaneous treatment of the PLC inhibitor U73122 [GenBank] (20 µM). B: effects of increasing doses of the PKC activator TPA (0.1–100 nM) on SL and SLβ mRNA expression in carp pituitary cells. The duration of TPA treatment was reduced to 6 h to avoid the potential problem of PKC desensitization. In this study, parallel treatment with the synthetic diacylglycerol analog DiC8 (10 µM) was also performed (inset). C: Western blot of PKC isoforms expressed in grass carp pituitary cells. Immunoreactivities of conventional PKCs, including PKC, PKCβ, and PKC, expressed in the carp pituitary were detected with the antibodies raised against the corresponding isoforms in rat (right). In parallel studies, Western blot for PKCβ and PKC was performed in carp pituitary cells with 6-h pretreatment with TPA (100 nM, left). In these experiments, protein lysate prepared from the rat brain was used as a positive control. Data presented are pooled results from 3 experiments. Treatment groups denoted by the same letter represent a similar level of transcript expression (P > 0.05, ANOVA followed by Fisher's LSD test).

View larger version (43K): [in this window] [in a new window]   Fig. 5. Effects of PKC inhibition on PACAP-induced SL and SLβ gene expression. Grass carp pituitary cells were incubated for 48 h with ovine PACAP (10 nM) in the presence or absence of the PKC inhibitors calphostin C (A; 20 µM) and GF10923 (B; 20 µM). After drug treatment, total RNA was extracted for SL and SLβ mRNA measurement and parallel blotting of 18S RNA was used as an internal control. Data presented are pooled results from 4 experiments, and treatment groups denoted by the same letter represent a similar level of transcript expression (P > 0.05, ANOVA followed by Fisher's LSD test).

View larger version (48K): [in this window] [in a new window]   Fig. 6. PACAP activation of Ca2+/calmodulin (CaM)-dependent cascades and function role of Ca2+ entry in SL and SLβ gene expression. A: effects of PACAP on intracellular Ca2+ levels in NIL cells prepared from the carp pituitary. NIL cells were preloaded with the Ca2+-sensitive dye fura-2 and treated with PACAP (10 nM) in normal culture medium (with 2.5 mM CaCl2) or in a Ca2+-free medium supplemented with 0.1 mM EGTA. B: effects of PACAP on CaM expression in grass carp NIL cells. NIL cells were incubated for 48 h with increasing doses of PACAP (0.1–100 nM). After drug treatment, cell lysate was prepared for Western blot using an antibody raised against human CaM. Western blot for β-actin was also conducted to serve as an internal control. C: effects of A23187 [GenBank] and Bay K8644 on SL and SLβ gene expression in carp pituitary cells. Pituitary cells were treated for 48 h with increasing levels of the Ca2+ ionophore A23187 [GenBank] (1–50 nM) or L-type voltage-sensitive Ca2+ channels (VSCC) activator Bay K8644 (10–1,000 nM). After that, total RNA was extracted for SL and SLβ mRNA measurement and parallel blotting of 18S RNA was used as an internal control. Data presented are pooled results from 4 separate experiments and groups denoted by the same letter represent a similar level of transcript expression (P > 0.05; ANOVA followed by Fisher's LSD test).

View larger version (42K): [in this window] [in a new window]   Fig. 7. Entry of extracellular Ca2+ in PACAP-induced SL and SLβ gene expression. Grass carp pituitary cells were treated for 48 h with ovine PACAP (10 nM) in A Ca2+-free medium with 0.1 mM EGTA or in B in the presence of the L-type VSCC blocker nifedipine (5 µM). After that, total RNA was extracted for SL and SLβ mRNA measurement and parallel blotting of 18S RNA was used as an internal control. Data presented are pooled results from 4 separate experiments, and treatment groups denoted by the same letter represent a similar level of transcript expression (P > 0.05, ANOVA followed by Fisher's LSD test).

View larger version (40K): [in this window] [in a new window]   Fig. 8. Intracellular Ca2+ mobilization in PACAP-induced SL and SLβ gene expression. Before PACAP stimulation, pituitary cells were pretreated with the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor thapsigargin (A; 50 nM) to deplete intracellular Ca2+ stores or the inositol 1,4,5-trisphosphate (IP3) receptor inhibitor 2-aminoethoxydiphenyl borate (2-APB; B; 100 µM) to block Ca2+ mobilization from IP3-sensitive Ca2+ stores. After that, the cells were challenged with ovine PACAP (10 nM) for 48 h before RNA extraction for SL and SLβ mRNA measurement. Parallel blotting of 18S RNA was used as the internal control. Data presented are pooled results from 4 experiments, and treatment groups denoted by the same letter represent a similar level of transcript expression (P > 0.05, ANOVA followed by Fisher's LSD test).

View larger version (42K): [in this window] [in a new window]   Fig. 9. Functional role of CaM and CaMK-II in PACAP-induced SL and SLβ gene expression. Pituitary cells were incubated for 48 h with PACAP (10 nM) with or without the simultaneous treatment of the CaM antagonist calmidazolium (A; 1 µM) or the CaMK-II inhibitor KN62 (B; 5 µM). After drug treatment, total RNA was extracted for SL and SLβ mRNA measurement and parallel blotting of 18S RNA was used as an internal control. Data presented are pooled results from 4 separate experiments, and treatment groups denoted by the same letter represent a similar level of transcript expression (P > 0.05; ANOVA followed by Fisher's LSD test).

View larger version (43K): [in this window] [in a new window]   Fig. 10. Functional role of Ca2+/CaM-dependent cascades in SL and SLβ gene expression induced by CPT-cAMP. Pituitary cells were incubated for 48 h with CPT-cAMP (100 µM) in Ca2+-free culture medium with 0.1 mM EGTA (A) or with cotreatment of the VSCC blocker nifedipine (B; 5 µM) or CaMK-II inhibitor KN62 (C; 5 µM). After drug treatment, RNA samples were isolated for SL and SLβ mRNA measurement and parallel blotting of 18S RNA was used as an internal control. Data presented are pooled results from 4 separate experiments, and treatment groups denoted by the same letter represent a similar level of transcript expression (P > 0.05, ANOVA followed by Fisher's LSD test).

View larger version (41K): [in this window] [in a new window]   Fig. 11. Functional role of Ca2+/CaM-dependent cascades in SL and SLβ gene expression induced by PKC activation. Pituitary cells were incubated for 6 h with TPA (100 nM) in Ca2+-free culture medium with 0.1 mM EGTA (A) or with cotreatment of the VSCC blocker nifedipine (B; 5 µM) or CaMK-II inhibitor KN62 (C; 5 µM). After that, total RNA samples were isolated for SL and SLβ mRNA measurement and parallel blotting of 18S RNA was used as an internal control. Data presented are pooled results from 4 separate experiments, and treatment groups denoted by the same letter represent a similar level of transcript expression (P > 0.05, ANOVA followed by Fisher's LSD test).

	DISCUSSION

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In mammalian cell models, PACAP not only can trigger cAMP synthesis via G_s coupling with AC (32) but also induce Ca²⁺ mobilization (1) through G_q/11 coupling with PLC (27). In porcine somaototrophs, PACAP-induced GH release is mediated by cAMP/PKA-, PLC/PKC-, and Ca²⁺-dependent cascades (19). In grass carp pituitary cells, PACAP can activate GH gene transcription via PAC-I receptors and these stimulatory effects are mediated by the AC/cAMP/PKA pathway coupled with [Ca²⁺]_e entry via L-type VSCC and subsequent activation of CaM/CaMK-II cascades (41). These previous findings have prompted us to speculate that other signaling pathways may be involved in PACAP-stimulated SL gene expression. In this study, PACAP treatment consistently triggered a transient rise in [Ca²⁺]_i and CaM expression in grass carp NIL cells, suggesting that the Ca²⁺/CaM-dependent mechanisms might have been activated. Since the Ca²⁺ responses could still be noted with reduced magnitude by removing [Ca²⁺]_e, it would be logical to assume that the Ca²⁺ responses triggered by PACAP may have two functional components, namely [Ca²⁺]_e entry from extracellular source and [Ca²⁺]_i mobilization of intracellular Ca²⁺ stores. In grass carp pituitary cells, [Ca²⁺]_e entry induced by A23187 [GenBank] or VSCC activation by Bay K8644 could elevate SL and SLβ gene expression. In contrast, SL mRNA expression caused by PACAP was inhibited by removing [Ca²⁺]_e using a Ca²⁺-free medium, blocking L-type VSCC by nifedipine, depleting [Ca²⁺]_i stores with thapsigargin, antagonizing endogenous CaM using calmidazolium, and inactivating CaMK-II by KN62. These results, as a whole, indicate that [Ca²⁺]_i mobilization and [Ca²⁺]_e entry through VSCC followed by activation of CaM/CaMK-II cascades are involved in PACAP induction of SL gene expression. In the case of SLβ expression, the Ca²⁺ dependence was found to be similar and yet different from the corresponding responses in SL gene. Although PACAP-stimulated SLβ mRNA expression could be negated by [Ca²⁺]_i depletion and CaM antagonism, it was not sensitive to the blockade by [Ca²⁺]_e removal, VSCC inhibition, or CaMK-II inactivation. Apparently, the effects of PACAP on SLβ gene expression are mediated by [Ca²⁺]_i mobilization and CaM activation but not by [Ca²⁺]_e entry through VSCC. Furthermore, CaMK-II is not involved in the downstream signaling after CaM activation. Given that multiple forms of CaMK have been reported and some of them (e.g., CaMK-IV) are known to be expressed in a tissue-specific manner (24), we do not exclude the possibility that other CaMKs may also be involved in PACAP-stimulated SLβ gene expression.

Apart from Ca²⁺-dependent mechanisms, differential coupling with the PLC/IP₃/PKC pathway was also observed for SL and SLβ gene expression. In grass carp pituitary cells, PKC activation by TPA and DiC8 was found to increase SL and SLβ mRNA levels, whereas inhibiting PLC using U73122 [GenBank] , blocking the IP₃ receptor with 2-APB, and PKC inactivation by calphostin C and GF10923 suppressed basal as well as PACAP-simulated SL mRNA expression. These results indicate that the PLC/IP₃/PKC pathway is involved in basal maintenance and PACAP induction of SL gene expression. Despite the fact that the rise in SLβ mRNA levels caused by PACAP could be negated by inhibiting PLC and IP₃ receptors, this stimulatory action was not affected by PKC blockade, suggesting that the PKC component may be uncoupled from PLC-dependent mechanisms leading to SLβ gene expression. Since PKC isoforms are expressed in the goldfish pituitary in a cell-type specific manner (17) and the atypical isoforms, e.g., PKC and PKC, are known to be insensitive to stimulation by phorbol ester and diacylglycerol (25), it is tempting to speculate that the lack of PKC involvement in PACAP-induced SLβ gene expression may be caused by different complements of PKC isoforms expressed in NIL cells with SL and SLβ expression. Since IP₃ receptors are Ca²⁺ channels responsible for [Ca²⁺]_i release from IP₃-sensitive Ca²⁺ stores (6), IP₃ produced after PLC activation in grass carp pituitary cells may act as a second messenger to trigger [Ca²⁺]_i mobilization and contribute to PACAP-induced SL gene expression through Ca²⁺/CaM-dependent mechanisms. In mammalian cell lines, the functionality of VSCC can be modulated by PKA and PKC via protein phosphorylation of ₁- and β_2a-subunits in the Ca²⁺ channel (14). These findings raise the possibility that the Ca²⁺/CaM-dependent cascades for SL gene expression may be secondarily coupled with the PKA- and PKC-dependent mechanisms. At the pituitary level, SL mRNA expression caused by activating PKA with CPT-cAMP or stimulating PKC with TPA were sensitive to the inhibition by Ca²⁺-free medium, VSCC blockade, and CaMK-II inactivation. Although similar inhibitions were also effective in blocking TPA-induced SLβ mRNA expression, the corresponding stimulation by CPT-cAMP was not affected. These results imply that [Ca²⁺]_e entry through VSCC and subsequent CaMK-II activation are acting downstream after PKC activation. However, the functional coupling of Ca²⁺/CaM-dependent mechanisms with the cAMP/PKA cascades is observed only in SL but not SLβ gene expression.

In summary, using grass carp pituitary cells as a model, we have confirmed that PACAP can induce SL and SLβ gene expression via activation of pituitary PAC-I receptors. Our results also demonstrate for the first time that PACAP-induced SL and SLβ gene expression are mediated by overlapping and yet distinct postreceptor signaling mechanisms (Fig. 12). At the pituitary level, PAC-I receptor activation can trigger SL gene expression via the AC/cAMP/PKA and PLC/IP₃/PKC pathways and subsequent activation of Ca²⁺/CaM/CaMK-II cascades. Apparently, the Ca²⁺/CaM-dependent mechanisms can be initiated by two functional components, namely [Ca²⁺]_e entry through VSCC and [Ca²⁺]_i mobilization of IP₃-sensitive Ca²⁺ stores. These two Ca²⁺ components in turn can be induced by PLC activation via PKC stimulation of L-type VSCC and IP₃ binding to IP₃ receptors in [Ca²⁺]_i stores. In pituitary cells with SL expression, VSCC can also be activated by the cAMP/PKA pathway, presumably via PKA activation. In the case of SLβ gene expression, similar involvement of the AC/cAMP/PKA and PLC/IP₃ cascades and a subsequent rise in [Ca²⁺]_i and CaM activation can be observed after PACAP stimulation. In contrast to SL gene expression, [Ca²⁺]_e influx via VSCC and subsequent CaMK-II activation play no functional role in PACAP action. Although PKC activation of VSCC can still occur in pituitary cells with SLβ expression, this PKC component is uncoupled from PLC and the downstream VSCC cannot be activated by the cAMP/PKA pathway. These findings, taken together, provide evidence that activation of PAC-I receptors expressed in two distinct populations of grass carp SL cells can stimulate SL and SLβ gene expression via differential coupling to different postreceptor signaling cascades. Given that PAC-I receptors with hip/hop inserts in the third intracellular loop (33) and amino acid substitutions in the transmembrane domains (3) are known to differentially activate cAMP-, PLC-, and Ca²⁺-dependent mechanisms, further investigations on the role of PAC-I receptor isoforms in PACAP-induced SL gene expression are clearly warranted.

View larger version (20K): [in this window] [in a new window]   Fig. 12. Working models for PACAP induction of SL and SLβ gene expression at the pituitary level (see DISCUSSION for details). [Ca2+]i, intracellular Ca2+; [Ca2+]e, extracellular Ca2+; CaM K-II, Ca2+/CaM-dependent protein kinase II.

	GRANTS

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	ACKNOWLEDGMENTS

 
We thank Dr. K. M. Chan (Chinese University of Hong Kong, Hong Kong) for support in this research project.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. O. L. Wong, Endocrinology Division, School of Biological Sciences, 4S-12 Kadoorie Biological Sciences Bldg., Univ. of Hong Kong, Pokfulam Road, Hong Kong SAR, P.R. China (e-mail: olwong{at}hkucc.hku.hk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

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