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Endocrinology Vol. 140, No. 12 5691-5697
Copyright © 1999 by The Endocrine Society

ARTICLES

Ceramide Enhances Growth Hormone (GH)-Releasing Hormone-Stimulated Cyclic Adenosine 3',5'-Monophosphate Accumulation but Inhibits GH Release in Rat Anterior Pituitary Cells1

Tetsuo Negishi, Constance L. Chik and Anthony K. Ho

Departments of Physiology and Medicine (C.L.C.), Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Address all correspondence and requests for reprints to: Dr. C. L. Chik, Department of Medicine, 7–26 Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7. E-mail: cchik{at}ualberta.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, the effect of ceramide on GH-releasing hormone (GHRH)-stimulated cAMP accumulation and GH release in rat anterior pituitary cells was investigated. C2-, C6-, and C8-ceramide were found to enhance GHRH-stimulated cAMP accumulation. In contrast, their effects on GHRH-stimulated GH release were inhibitory. Treatment with a glucosylceramide synthase inhibitor produced a similar enhancing effect on cAMP accumulation and an inhibitory effect on GH release. To identify the pathway through which ceramide mediated its effect, it was found that ceramide inhibited GH release stimulated by KCl, BayK 8644, and a GH-releasing peptide, but not that stimulated by ionomycin or an activator of protein kinase C. Direct measurement of intracellular Ca2+ revealed that C2-ceramide inhibited GHRH- and KCl-mediated increases in intracellular Ca2+, suggesting that ceramide probably inhibits GH release through inhibition of the L-type Ca2+ channels. As for its mechanism on cAMP accumulation, the enhancing effect of ceramide on GHRH-stimulated cAMP accumulation was abolished in the presence of a phosphodiesterase inhibitor, isobutylmethylxanthine, suggesting that ceramide enhances the cAMP response through inhibition of its metabolism. Taken together, our results suggest that ceramide plays an important role in the regulation of GHRH-stimulated responses in somatotrophs. By reducing GH secretion while enhancing cAMP accumulation, ceramide may promote the synthesis and storage of GH in rat anterior pituitary cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE RAT anterior pituitary gland, the release of GH is primarily under the regulation of two neuropeptides, GH-releasing hormone (GHRH) and somatostatin, the former stimulating and the latter inhibiting the release of GH (1). The mechanisms by which GHRH stimulates and somatostatin inhibits GH release involve the adenylyl cyclase/cAMP pathway and changes in intracellular Ca2+ (1, 2, 3). Increased cAMP levels lead to the opening of a Na+ permeable ion channel, depolarization of the cell membrane and influx of Ca2+ through the L-type Ca2+ channels (4, 5). This increase in intracellular Ca2+ concentration ([Ca2+]i) promotes GH release through the process of exocytosis (6). The production of cAMP and GH release is modulated by additional signaling mechanisms, including protein kinase C, tyrosine kinase, diacylglycerol, and phospholipases (7, 8, 9). There is also evidence that GHRH induces the transcription of GH gene through a cAMP-dependent process (10).

Another signaling mechanism that could potentially modulate cAMP production and GH release in the rat anterior pituitary gland is the sphingomyelin pathway. This pathway mediates the action of cytokines such as interleukin-1ß, interferon-{gamma}, and tumor necrosis factor-{alpha} (11, 12, 13). Ceramide is produced after sphingomyelin hydrolysis by activation of a sphingomyelinase (12, 13). Sphingolipid metabolites, including ceramide, sphingosine, and sphingosine-1-phosphate, are emerging as a new class of second messengers that are involved in cellular proliferation, differentiation, and apoptosis (12, 14). Ceramide is also involved in the regulation of Ca2+ homeostasis and intracellular enzymes such as protein kinase C (14, 15, 16, 17, 18), known mechanisms that can modulate cAMP production and GH release. Taken together, these observations suggest that ceramide may play an important modulatory role on the responses of somatotrophs to GHRH stimulation. In this study, we investigated whether ceramide had an effect on GHRH-stimulated cAMP accumulation and GH release and, if ceramide modulated GH release, whether this modulation was related to its effect on cAMP accumulation or changes in [Ca2+]i.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Synthetic rat GHRH was obtained from Peninsula Laboratories, Inc. (San Carlos, CA). Isobutylmethylxanthine (IBMX), forskolin, and (Bu)2cAMP were purchased from Sigma Chemical Co. (St. Louis, MO). Ala-His-D-ßNal-Ala-Trp-D-Phe-Lys-NH2 (GHRP-1) was a gift from Dr. C. Y. Bowers (Tulane University, New Orleans, LA). Ceramides, ionomycin, and 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP) were obtained from Calbiochem (La Jolla, CA). Fura-2, the acetoxymethyl esters of fura-2, and EGTA were purchased from Molecular Probes, Inc. (Eugene, OR). DMEM was purchased from Biofluids (Rockville, MD). [125I]cAMP and [125I]GH were obtained from ICN (Costa Mesa, CA). All other chemicals were of the purest grade available. Antibody for the RIA of cAMP was a gift from Dr. A. Baukal (NICHHD, NIH, Bethesda, MD). The GH assay kit was obtained from the Pituitary Hormone Distribution Agency of the NIDDK (Baltimore, MD).

Preparation and treatment of rat anterior pituitary cells
All procedures were reviewed and approved by the health sciences animal and welfare committee of the University of Alberta (Edmonton, Canada). Male Sprague Dawley rats (180–200 g) were decapitated, and the pituitary glands were collected in ice-cold PBS. The pars nervosa-intermedia were discarded, and the anterior pituitaries were minced into small fragments and dissociated by enzymatic digestion (18, 19). Cell yield was approximately 106 cells/gland with greater than 90% viability. Cells were suspended in DMEM with FCS (10%, vol/vol) and plated onto multiwelled dishes at a density of 120,000 cells/well. They were then incubated under a humidified atmosphere of 95% air-5% CO2 at 37 C. After 48 h, the cells were washed twice with DMEM containing 1% BSA and equilibrated for 30 min before performing the experiments.

Experimental design
The plated cells were washed a third time, and the medium bathing the cells was replaced by medium in which the drugs were dissolved. Drugs were dissolved in at least a 200-fold concentrated solution in H2O or dimethylsulfoxide and diluted to the final concentration in DMEM (pH 7.4). The concentration of dimethylsulfoxide never exceeded 0.5%. After 15 min, the medium was removed and assayed for GH release. The attached cells were lysed by alternate freezing and thawing in 5 mM acetic acid, and intracellular accumulation of cAMP was measured.

Cyclic nucleotide and GH assays
For cAMP measurements, the cell lysate was boiled for 5 min and assayed using a RIA procedure in which samples were acetylated before analysis (19, 20, 21). Intra- and interassay coefficients of variation were less than 5%. The results were expressed as femtomoles per 120,000 cells. Medium GH was assayed in duplicate using a double antibody RIA, and the results were expressed as picograms per 120,000 cells. Intra- and interassay variations for GH were less than 10%.

Determination of intracellular Ca2+
Intracellular Ca2+ was determined using a fluorescent Ca2+ indicator, fura-2 (19, 20, 22). Cells (1 x 106) were pelleted and resuspended in culture medium (DMEM with HEPES, pH 7.2). The cells were loaded by incubation with 5 µM fura-2 AM for 45 min at 37 C. The cells were then pelleted, washed twice, and resuspended in a fresh buffered salt solution that contained 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.2 mM MgCl2, 1.2 mM KH2PO4, 25 mM HEPES, and 6 mM glucose, pH 7.2. Aliquots of this suspension (2.0 ml) were transferred to a cuvette for the fluorescence signal determination, using a SLM Amico DMX 1000 fluorescence spectrophotometer with a thermostatically controlled cell holder fitted with a magnetic stirrer. [Ca2+]i of pituitary cells was determined by monitoring the ratio of the fluorescence emission signal at 510 nm (8-nm slit width), with the excitation wavelength set at 380 and 340 nm (8-nm slit width). The temperature was maintained at 37 C. The free Ca2+ concentration was calculated according to the equation established by Poenie et al. (22): intracellular Ca2+ = Kd x Fo/Fs x (R - Ro)/(Rs - R), where Kd is the dissociation constant of fura-2-Ca2+ complex (225 nM), Fo and Fs are the fluorescence intensities at 380 nm for free (o) and Ca2+-saturated (s) dye, and R, Ro, and Rs are the ratio of the dye fluorescence intensities at 340 and 380 nm for unknown, free, and Ca2+-saturated dye, respectively. Both Fs and Rs were determined by lysing the cells with Triton X-100 (0.1%), whereas Fo and Ro were determined by addition of 10 mM EGTA to the lysed cell suspension.

Statistical analysis
Data are presented as the mean ± SEM of the amount of cAMP or GH obtained from three independent experiments, each performed in quadruplicate. Data were analyzed by ANOVA and Duncan’s multiple range test. The paired t test was used for the analysis of [Ca2+]i measurement. Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of ceramides on GHRH-stimulated cAMP accumulation and GH release
Treatment of rat anterior pituitary cells with GHRH (10 nM) for 15 min caused a significant increase in cAMP accumulation and GH release (Fig. 1Go). Although C6-ceramide (30 µM) had no effect on basal cAMP accumulation or GH release, pretreatment with C6-ceramide for 5 min significantly enhanced GHRH-stimulated cAMP accumulation in a concentration-dependent manner (Fig. 1Go). C6-ceramide (3 µM) caused a 30% increase in GHRH-stimulated cAMP accumulation and a 60% increase was observed with 30 µM (Fig. 1Go). In contrast, C6-ceramide inhibited GHRH-stimulated GH release in a concentration-dependent manner (Fig. 1Go). C6-ceramide (10 µM) caused a 30% inhibition of the GHRH-stimulated GH release, whereas a 45% inhibition was observed with 30 µM (Fig. 1Go).



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  		Figure 1. Effect of C6-ceramide on GHRH-stimulated GH release and cAMP accumulation. Rat anterior pituitary cells were incubated in DMEM and pretreated with C6-ceramide (C6; 3–30 µM) for 5 min. The cells were then stimulated with GHRH (10 nM) for an additional 15 min in the absence or presence of C6. Each value represents the mean ± SEM of determinations performed in quadruplicate from three independent experiments. *, P < 0.05; **, P < 0.01 (compared with the corresponding treatment with GHRH).

 
Similar results were obtained with 30 µM C2- and C8-ceramide. Neither C2- nor C8-ceramide had an effect on basal cAMP accumulation or GH release (data not shown). Pretreatment with C2- or C8-ceramide for 5 min enhanced GHRH-stimulated cAMP accumulation while inhibiting GHRH-stimulated GH release (Fig. 2Go). In contrast, C2-dihydroceramide (30 µM), an inactive analog, had no effect on basal or GHRH-stimulated cAMP accumulation or GH release (Fig. 2Go). Of the three active ceramides, the enhancing effects of C6- and C8-ceramide (30 µM) on GHRH-stimulated cAMP were greater than those of C2-ceramide (30 µM), whereas the inhibitory effects of the three ceramides (30 µM) on GHRH-stimulated GH release were similar (Fig. 2Go).



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  		Figure 2. Effects of ceramides on the GHRH-stimulated cAMP accumulation and GH release. Rat anterior pituitary cells were incubated in DMEM and pretreated with 30 µM of C2-ceramide (C2), C6-ceramide (C6), C8-ceramide (C8), or C2-dihydroceramide (dhC2) for 5 min. The cells were then stimulated with GHRH (10 nM) for an additional 15 min in the presence of different ceramides. Each value represents the mean ± SEM of determinations performed in quadruplicate from three independent experiments. *, P < 0.05; **, P < 0.01 (compared with the corresponding treatment with GHRH).

 
Effect of PPMP on GHRH-stimulated GH release and cAMP accumulation
Treatment with a glucosylceramide synthase inhibitor, PPMP, has been shown to induce an increase in cellular ceramide levels (23, 24). Pretreatment with PPMP (10 µM) for 15 min, like treatment with C2-, C6, and C8-ceramides, was effective in enhancing GHRH-stimulated cAMP accumulation (Table 1Go). The inhibitory effect of PPMP (10 µM) on GHRH-stimulated GH release was also similar to that observed with the active ceramides (Table 1Go).


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  		Table 1. Effect of PPMP on GHRH-stimulated cAMP accumulation and GH release

 
Effect of C6-ceramide on agonist-stimulated GH release
To identify the mechanism through which ceramide inhibits GH release, the effect of ceramide on agonists that have been shown to induce GH release by different mechanisms was determined. Treatment with PMA (100 nM), an activator of protein kinase C, caused a 4.4-fold increase in GH release (Fig. 3Go). Pretreatment with C6-ceramide (30 µM) for 5 min had no effect on the PMA-induced GH release (Fig. 3Go). In contrast, C6-ceramide significantly inhibited GH release stimulated by a membrane-permeable analog of cAMP, (Bu)2cAMP (1 mM; Fig. 3Go). C6-ceramide was also effective in inhibiting GH release stimulated by a GH-releasing peptide, GHRP-1 (100 nM; Fig. 3Go).



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  		Figure 3. Effects of C6-ceramide on GH release stimulated by different agonists. Rat anterior pituitary cells were incubated in DMEM and pretreated with C6-ceramide (C6; 30 µM) for 5 min. Top panel, The cells were then stimulated with PMA (100 nM), (Bu)2cAMP (db-cA; 1 mM), or GHRP-1 (100 nM) for an additional 15 min in the presence or absence of C6 (30 µM). Bottom panel, The cells were then stimulated with KCl (30 mM), BayK 8644 (BayK; 10 µM), or ionomycin (ION; 10 µM) for an additional 15 min in the presence or absence of C6 (30 µM). Each value represents the mean ± SEM of determinations performed in quadruplicate from three independent experiments. *, P < 0.05; **, P < 0.01 (compared with the corresponding treatment without ceramide).

 
GH release can also be stimulated by agents that elevate [Ca2+]i. Treatment with a depolarizing concentration of K+, BayK 8644 (an agonist of the L-type Ca2+ channel) or ionomycin (a Ca2+ ionophore) was effective in inducing GH release in rat anterior pituitary cells (Fig. 3Go). Pretreatment with C6-ceramide for 5 min selectively reduced GH release stimulated by KCl (30 mM) and BayK 8644 (10 µM). In contrast, C6-ceramide had no effect on GH release induced by ionomycin (10 µM; Fig. 3Go).

The inhibitory effects of C6-ceramide on KCl- and BayK 8644-stimulated GH release were also observed with 30 µM of C2- and C8-ceramide (Fig. 4Go). Furthermore, C6-ceramide (30 µM) caused an increase in the EC50 value of the KCl-stimulated GH release (Fig. 5Go). In contrast, C6-ceramide had no effect on the maximal GH release stimulated by KCl (60 mM; Fig. 5Go).



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  		Figure 4. Effects of ceramides on KCl- or BayK 8644-stimulated GH release. Rat anterior pituitary cells were incubated in DMEM and pretreated with 30 µM C2-ceramide (C2), C6-ceramide (C6), C8-ceramide (C8), or C2-dihydroceramide (dhC2) for 5 min. The cells were then stimulated with KCl (30 mM) or BayK 8644 (BayK; 10 µM) for an additional 15 min in the presence of different ceramides. Each value represents the mean ± SEM of determinations performed in quadruplicate from three independent experiments. *, P < 0.05; **, P < 0.01 (compared with the corresponding treatment without ceramide).

 


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  		Figure 5. Effect of C6-ceramide on KCl-stimulated GH release. Rat anterior pituitary cells were incubated in DMEM and pretreated with C6-ceramide (C6; 30 µM) for 5 min. The cells were then stimulated with varying concentrations of KCl (10–60 mM) for an additional 15 min in the absence or presence of C6. Each value represents the mean ± SEM of determinations performed in quadruplicate from three independent experiments. *, P < 0.05 (compared with the corresponding treatment without ceramide).

 
Effect of C6-ceramide on GHRH- and KCl-mediated increases in [Ca2+]i
The above observations suggest that ceramide probably inhibits GHRH-stimulated GH release by reducing [Ca2+]i. This was tested directly by measuring [Ca2+]i using a fluorescent Ca2+ indicator, fura-2. In fura-2-loaded anterior pituitary cells, the resting [Ca2+] was 135 ± 3.3 nM as in previous studies (19, 20) (Fig. 6Go and Table 2Go). Treatment with GHRH (100 nM) or KCl caused increases in [Ca2+]i to 185 ± 5.5 and 195 ± 6.4 nM, respectively. C2-ceramide and C2-dihydroceramide caused a small increase in the fluorescent signal (Fig. 6Go and Table 2Go) that was caused by autofluorescence as similar increases were observed in the cell-free system (data not shown). Treatment with C2-ceramide reduced the GHRH (100 nM)- and KCl (30 mM)-mediated increases in [Ca2+]i (Fig. 6Go and Table 2Go). In contrast, C2-dihydroceramide, the inactive analog, was ineffective (Fig. 6Go and Table 2Go).



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  		Figure 6. Effects of ceramide on GHRH-stimulated increases in [Ca2+]i. Rat anterior pituitary cells loaded with fura-2 were treated with A) GHRH (100 nM); B) C2-ceramide (C2; 30 µM) and GHRH (100 nM); C) C2-dihydroceramide (dhC2; 30 µM) and GHRH (100 nM); D) KCl (30 mM); E) C2 (30 µM) and KCl (30 mM); and F) dhC2 (30 µM) and KCl (30 mM). The ratio of the fluorescence emission signal at 510 nm with the excitation wavelengths set at 340 nm and 380 nm was continually recorded and calibrated as described. The tracing is representative of at least three independent experiments.

 

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  		Table 2. Effect of ceramides on GHRH- and KCl-mediated increases in intracellular Ca2+

 
Effect of C6-ceramide on GHRH- and forskolin-stimulated cAMP accumulation and GH release in the presence of IBMX
To determine whether the enhancing effect of ceramide on cAMP accumulation was due to inhibition of metabolism of cAMP, the effect of C6-ceramide on GHRH-stimulated cAMP accumulation was determined in the presence of a phosphodiesterase inhibitor, IBMX. In the presence of IBMX (1 mM), the enhancing effect of C6-ceramide (30 µM) on GHRH (10 nM)-stimulated cAMP accumulation was abolished (Fig. 7Go). However, C6-ceramide remained effective in reducing GHRH-stimulated GH release in IBMX-treated cells (Fig. 7Go).



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  		Figure 7. Effects of C6-ceramide on GHRH- or forskolin-stimulated cAMP accumulation and GH release in the absence or presence of IBMX. Rat anterior pituitary cells were incubated for 5 min with C6-ceramide (C6; 30 µM). The cells were then stimulated with GHRH (10 nM) or forskolin (FORSK; 3 µM) for an additional 15 min in the absence or presence of IBMX (1 mM). Each value represents the mean ± SEM of determinations performed in quadruplicate from three independent experiments. *, P < 0.05; **, P < 0.01 (compared with the corresponding treatment without ceramide).

 
When forskolin was used to stimulate cAMP accumulation and GH release, C6-ceramide (30 µM) also enhanced the forskolin (3 µM)-stimulated cAMP accumulation while inhibiting GH release (Fig. 4Go). The enhancing effect of C6-ceramide on forskolin-stimulated cAMP accumulation was abolished in the presence of IBMX (1 mM) (Fig. 7Go). However, the inhibitory effect of C6-ceramide on forskolin-stimulated GH release persisted in the presence of IBMX (Fig. 7Go). These observations suggest that phosphodiesterase is a probable site of action for the ceramide effect on cAMP accumulation.

Effect of C6-ceramide on GHRH-stimulated cAMP accumulation in the presence of ionomycin
To determine whether the effect of ceramide on GHRH-stimulated cAMP accumulation was related to Ca2+ entry, the effect of ceramide was tested in the presence of ionomycin. In the presence of ionomycin (10 µM), ceramide (30 µM) remained effective in enhancing the GHRH (10 nM)-stimulated cAMP accumulation (Table 3Go), suggesting that the effect of ceramide on cAMP accumulation is independent of changes in [Ca2+]i.


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  		Table 3. Effect of ceramide on GHRH-stimulated cAMP accumulation in the presence of ionomycin

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Signaling through the sphingomyelin pathway, which is present in most mammalian cells, has generally been accepted as an important mechanism in regulating cellular processes such as growth, differentiation, apoptosis, and cell cycle arrest (14). Activation of this pathway involves generation of ceramide through hydrolysis of sphingomyelin by sphingomyelinase. The ceramide produced, which acts as a second messenger, has been shown to modulate the activities of different kinases and phosphatases in mediating its biological responses (14, 17). The production of cAMP and GH release in the rat anterior pituitary gland is modulated by several signaling mechanisms, including protein kinase C, tyrosine kinase, diacylglycerol, and phospholipases (7, 8, 9). Our results indicate that although ceramide on its own does not have a direct effect on GH release or cyclic nucleotide synthesis, ceramide has two distinct effects on the GHRH-mediated signaling mechanisms. Whereas ceramide enhances GHRH-stimulated cAMP accumulation, its effect on GH release is inhibitory.

In this study, we found that C2-, C6-, and C8-ceramide, three active ceramides, have similar effects on GHRH-stimulated cAMP accumulation and GH release. In contrast, C2-dihydroceramide, an inactive analog of ceramide, has no effect on GHRH-stimulated cAMP accumulation and GH release, suggesting that the effects of ceramide on the GHRH-stimulated responses are unlikely to be due to nonspecific effects of the drugs used. In support of this is the observation that treatment with PPMP, which inhibits the metabolism of ceramide (23, 24), has the same effect as C2-, C6-, or C8-ceramide on GHRH-stimulated cAMP accumulation and GH release.

Even though ceramide has an enhancing effect on GHRH-stimulated cAMP accumulation, several observations indicate that the inhibitory effect of ceramide on GH release is independent of its enhancing effect on cAMP accumulation. First, C6-ceramide is effective in inhibiting GH release stimulated by GHRP-1, KCl, or BayK 8644, three treatments that have no effect on cAMP accumulation (19, 20). Second, the inhibitory effect of C6-ceramide on GH release is observed when cells are stimulated by the membrane-permeable cAMP analog, (Bu)2cAMP. Third, the observation that ceramide remains effective in inhibiting GHRH-stimulated GH release in the presence of IBMX also argues against ceramide acting at the cAMP level.

Our results indicate that ceramide probably inhibits GHRH-stimulated GH release through a Ca2+-dependent mechanism. This is based on the observations that ceramide is effective in inhibiting GH release stimulated by the releasing peptide, GHRP-1. GHRP-1, which has no effect on cAMP accumulation, is known to induce a biphasic increase in [Ca2+]i involving acute mobilization of intracellular Ca2+ followed by a persistent plateau due to Ca2+ entry through the L-type Ca2+ channels (20, 25). Although protein kinase C is involved in the action of GHRP-1 (26), ceramide is unlikely to act on protein kinase C, as it has no effect on the PMA-mediated GH release. Furthermore, GHRH does not activate protein kinase C in rat somatotrophs (27).

Additional studies using a depolarizing concentration of K+ or BayK 8644 (an L-type Ca2+ channel agonist) to increase GH release support an inhibitory effect of ceramide on the L-type Ca2+ channels. Consistent with this conclusion is the observation that ceramide has no effect on GH release stimulated by ionomycin, the Ca2+ ionophore. Furthermore, by measuring [Ca2+]i directly, ceramide is effective in inhibiting GHRH- and KCl-induced increases in [Ca2+]i. It is of interest to note that ceramide has been shown to inhibit these channels in rat ventricular myocytes (28). Although the inhibitory effect of ceramide on GH release appears to be mediated through the L-type Ca2+ channels, this effect can be reversed by using a higher concentration of KCl to depolarize the cells. Therefore, ceramide appears to reduce the sensitivity of these channels to changes in membrane potential.

In contrast to its effect on GH secretion, our results show that ceramide enhances cAMP accumulation by inhibiting its metabolism. This is suggested by the study in which IBMX was used to inhibit phosphodiesterase activities. In the presence of IBMX, ceramide has no effect on GHRH- or forskolin-stimulated cAMP accumulation. As ceramide reduces GHRH-mediated increases in [Ca2+]i, one possible explanation is that the enhancing effect on cAMP accumulation could be explained by a reduced calmodulin-dependent cyclic nucleotide phosphodiesterase activity. However, this is an unlikely mechanism, because ceramide remains effective in enhancing GHRH-stimulated cAMP accumulation when [Ca2+]i is elevated by ionomycin, a Ca2+ ionophore. Therefore, unlike its effect on GH release, the effect of ceramide on cAMP accumulation appears to be independent of changes in [Ca2+]i. In support of this, the enhancing effect of ceramide on cAMP accumulation was also abolished in the presence of a type IV cAMP-specific phosphodiesterase inhibitor, rolipram (our unpublished observation). It should be mentioned that ceramide has previously been shown to increase intracellular cAMP accumulation in rat pinealocytes by inhibiting its synthesis (29). In contrast, the increase in cAMP production by ceramide in airway smooth muscle cells is through activation of adenylyl cyclase (30). Therefore, the mechanism through which ceramide increases cAMP production appears to be tissue specific.

The use of exogenous ceramides and PPMP suggests that the observed effects on GH release and cAMP accumulation are probably related to activation of the sphingomyelin cycle. As ceramide is rapidly converted to sphingosine and sphingosine-1-phosphate, it is possible that the observed effects of ceramide and PPMP could be mediated by these lipid metabolites. In our preliminary study, only sphingosine-1-phosphate, but not sphingosine, was found to have a small effect on GHRH-stimulated cAMP accumulation (our unpublished observation). However, it is of interest to note that the L-type Ca2+ channels are inhibited by sphingosine in GH4C1 cells (31, 32).

Whereas the inhibitory effect of ceramide on GHRH-stimulated GH release appears to be mediated through its effect on the L-type Ca2+ channels, the enhancing effect of ceramide on GHRH-stimulated cAMP accumulation appears to be through its action on phosphodiesterases. This dual effect of ceramide suggests that an immediate consequence of activation of the sphigomyelin cycle is inhibition of GH release due to a reduced Ca2+ influx through the L-type Ca2+ channels. However, activation of this cycle also results in enhanced cAMP accumulation, and this could lead to activation of GH gene transcription (10).

Our results suggest that ceramide, through the two distinct effects on cAMP metabolism and L-type Ca2+ channels, appears to convert the GHRH signal from one that enhances both secretion and synthesis to one that favors synthesis over secretion. This observation is probably of physiological importance and of relevance to the role of cytokine on the regulation of GH release. Cytokines, including interleukin-1ß, interferon-{gamma}, and tumor necrosis factor-{alpha}, which activate the sphingomyelin cycle in other cells (11, 12, 13), have previously been shown to modulate the release of pituitary hormones, including GH (33, 34, 35). Endotoxin, which has a short term inhibitory effect on GH secretion, has been shown to increase the amplitude of GH pulses after 24 h (36). It will be of interest to determine whether the endotoxin-mediated changes in GH release are secondary to the effects of ceramide observed in our study.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada. Back

Received May 14, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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