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RECEPTORS AND SIGNAL TRANSDUCTION
1Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; and 2Department of Ophthalmology, Tokyo Women's Medical School, Tokyo, Japan
Submitted 10 January 2007 ; accepted in final form 5 August 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1-adrenergic agonist), or epidermal growth factor (EGF). The latter three agonists are known to activate p44/p42 MAPK. p44/p42 MAPK activity and protein secretion were measured. As measured by Western blot analysis, DBcAMP inhibited both basal and agonist-stimulated p44/p42 MAPK activity. Cellular cAMP levels were increased by 1) using two different cell-permeant cAMP analogs, 2) activating adenylyl cyclase (L-858051), or 3) activation of Gs-coupled receptors (VIP). The cell-permeant cAMP analogs, L-858051, and VIP inhibited basal p44/p42 MAPK activity by 50, 40, and 40%, respectively. DBcAMP and VIP inhibited carbachol- and EGF-stimulated MAPK activity. cAMP, but not VIP, inhibited phenylephrine-stimulated MAPK activity. Potentiation of secretion was detected when carbachol, phenylephrine, or EGF was simultaneously added with DBcAMP. We conclude that increasing cellular cAMP levels inhibits p44/p42 MAPK activity and that this could account for potentiation of secretion obtained when cAMP was elevated and Ca2+ and PKC were increased by agonists. adenosine 3',5'-cyclic monophosphate; dibutyryl adenosine 3',5'-cyclic monophosphate
The lacrimal gland is a polarized secretory tissue that secretes proteins, water, and electrolytes. This secretion is necessary to maintain the health of the ocular surface and is regulated by parasympathetic and sympathetic nerves. Binding of the parasympathetic neurotransmitter vasoactive intestinal peptide (VIP) to its receptor induces activation of a cAMP-dependent pathway (14) through the Gs G protein subunit, which in turn stimulates adenylyl cyclase (AC) (7). Activating AC with VIP or the AC activator forskolin or preventing cAMP breakdown by inhibiting the cyclic nucleotide phosphodiesterase (PDE) causes an increase in intracellular cAMP levels, which in turn activates PKA (10, 17). That activation of PKA mediates VIP-induced protein secretion was demonstrated using two PKA inhibitors, protein kinase inhibitor (PKI) and H-89. VIP also increases the intracellular Ca2+ concentration ([Ca2+]i) in the lacrimal gland (12).
Another parasympathetic neurotransmitter, acetylcholine, activates M3 muscarinic receptors located on the basolateral membranes of acinar cells (15, 20). The activated M3 receptor then interacts with a Gq/11 G protein subtype (18) to activate phospholipase C-β (PLC-β) to break down phosphatidylinositol bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (1,4,5-IP3) and diacylglycerol (DAG). The 1,4,5-IP3 binds to IP3 receptors located on intracellular Ca2+ stores in the endoplasmic reticulum and increases the [Ca2+]i. DAG activates a family of enzymes known as protein kinase C (PKC). The lacrimal gland contains PKC-, -
, -
, and -
and protein kinase D (PKD). Both increasing the [Ca2+]i and activation of PKC-, -, and - play integral roles in protein secretion stimulated by cholinergic agonists. Activation of M3 receptors also stimulates the non-receptor tyrosine kinases Pyk2 and Src that subsequently stimulate the Ras/Raf, mitogen-activated protein kinase kinase (MEK), and p44/p42 MAPK pathway. Interestingly, investigators in our laboratory (24) found that activation of the p44/p42 MAPK pathway attenuates cholinergic agonist-stimulated protein secretion in the lacrimal gland.
Norepinephrine, a sympathetic neurotransmitter, activates both - and β-adrenergic receptors. Activation of β-adrenergic receptors by isoproterenol stimulates the cAMP signaling pathway to induce protein secretion (19). Activation of 1D-adrenergic receptors in the lacrimal gland by phenylephrine stimulates production of nitric oxide (NO). NO activates guanylate cyclase to increase guanosine 3',5'-cyclic monophosphate (cGMP) to induce secretion (13). 1D-Adrenergic receptors also activate the p44/p42 MAPK pathway by increasing epidermal growth factor (EGF) ectodomain shedding to transactivate the EGF receptor (EGFR) to stimulate p44/p42 MAPK activity. Similarly to its effect on cholinergic agonist-induced secretion, activation of MAPK inhibits protein secretion stimulated by 1D-adrenergic agonists (4, 22).
Another stimulus of protein secretion in the lacrimal gland is EGF (20). EGF binds to the EGFR to recruit adapter molecules that activate the three major downstream signaling pathways: MAPK, phosphatidylinositol-3 kinase (PI3K), or PLC-
. To stimulate the p44/p42 MAPK pathway, the adapter proteins Shc and Grb2 are recruited to the phosphorylated EGFR, leading to activation of Ras, Raf, MEK, and MAPK (24–26). EGF simulates lacrimal gland protein secretion by stimulating PLC- to increase the [Ca2+]i and activate PKC but not PI3K or p44/p42 MAPK (24).
In 1984, investigators in our laboratory (8) found that if a Ca2+- or PKC-dependent agonist, such as carbachol or phenylephrine, was combined with a cAMP-dependent agonist, such as VIP, synergism or potentiation of secretion occurred. In other words, the response to the two agonists was greater than the sum of the individual responses. The potentiation also occurred if cAMP was increased independently of receptor activation. Potentiation of protein secretion also occurred when 1-adrenergic agonists and β-adrenergic agonists were used together (9, 18).
Several possible mechanisms by which potentiation of protein secretion could occur include potentiation of the increase in the [Ca2+]i, potentiation of the activation of PKC, and potentiation of the cellular cAMP concentration. However, all of these possibilities have been disproved (7, 9) . We now hypothesize that an inhibition of p44/p42 MAPK activity causes potentiation of protein secretion by relieving its attenuating effects on agonist-stimulated secretion.
In the present study, we used freshly isolated, nontransfected lacrimal gland acinar cells and found that potentiation of secretion and inhibition of p44/p42 MAPK occurred when cAMP analogs and Ca2+/PKC-dependent agonists were added simultaneously. Increasing cellular cAMP levels inhibited both basal and agonist-induced p44/p42 MAPK activity. Agonists known to increase cAMP in lacrimal gland cells also inhibited p44/p42 MAPK activity. We conclude that inhibition of p44/p42 MAPK by cAMP may be responsible for the potentiation of secretion that occurs when cAMP is elevated in conjunction with Ca2+/PKC-dependent agonists.
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MATERIALS AND METHODS |
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-butyryl]-, dihydrochloride (L-858051) and VIP were obtained from Calbiochem (San Diego, CA). Carbachol and phenylephrine were acquired from Sigma (St. Louis, MO) and EGF from Pepro Tech (Rocky Hill, NJ). For Western blot analysis, antibodies against phosphorylated p44/p42 MAPK and total p42 MAPK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and monoclonal antibodies against total cAMP response element binding protein (CREB) and phosphorylated CREB were obtained from Affinity BioReagents (Golden, CO). For measurement of peroxidase secretion, the fluorescent molecule Amplex red (Molecular Probes, Eugene, OR) was acquired from Molecular Probes. Preparation of lacrimal gland acini. All experiments were approved by the Schepens Eye Research Institute Animal Care and Use Committee. Both exorbital lacrimal glands were removed from male Sprague-Dawley rats that had been anesthetized with CO2 for 1 min and then decapitated. Lacrimal glands were trimmed of fatty and connective tissue and fragmented into small pieces 2–3 mm in diameter. The pieces were then washed at 37°C in Krebs-Ringer bicarbonate (KRB) buffer (in mM: 119 NaCl, 4.8 KCl, 1 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 25 NaHCO3) supplemented with 10 mM HEPES, 5.5 mM glucose, and 0.5% BSA (KRB-HEPES), pH 7.4. Lacrimal gland acini were prepared by incubating tissue pieces with collagenase (CLSIII; 150 U/ml) in 10 ml of KRB-HEPES buffer for 40 min at 37°C under a stream of 95% O2-5% CO2. Lacrimal gland lobules were subjected to gentle pipetting 10 times at regular time intervals through tips of decreasing diameter. The preparation was then filtered through nylon mesh, and the acini were pelleted with a 3-min centrifugation at 5,000 rpm. The pellet was washed twice by centrifugation (5,000 rpm, 3 min) through a 4% BSA solution made in KRB-HEPES buffer. The dispersed acini were allowed to recover for 60 min in 5 ml of fresh KRB-HEPES buffer containing 0.5% BSA.
Detection of MAPK and CREB activation by Western blotting. Lacrimal gland acini were incubated for the indicated time period with agonists at the indicated concentrations. To terminate the reaction, ice-cold KRB-HEPES buffer without BSA was added. The acini were centrifuged, and 100 µl of ice-cold RIPA buffer (10 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 100 µg/ml PMSF, 30 µl/ml aprotinin, and 1 mM Na3VO3) were added. The pellet was then sonicated. After a 10,000 rpm centrifugation for 30 min at 4°C, proteins in the supernatant were removed and separated by SDS-PAGE (10% acrylamide gels) and then transferred to nitrocellulose membranes. Activated p44/p42 MAPK and CREB were detected with antibodies that specifically recognize the phosphorylated (activated) pools of enzymes. A secondary antibody conjugated to horseradish peroxidase was used and detected with the enhanced chemiluminescence method. Films were scanned and analyzed using NIH Image software. Values for phosphorylated enzymes (amounts for p42 and p44 MAPK were added together) were normalized to the amount of total enzyme by using antibodies to total enzyme (phosphorylated and nonphosphorylated) and were compared with the control value that was set at 1.
Measurement of peroxidase secretion. Lacrimal gland acini were incubated in 0.5% BSA-KRB-HEPES buffer in duplicate for 20 min with agonists or no additions or with simultaneous addition of two compounds. After a brief centrifugation, supernatant was collected and peroxidase activity was measured in duplicate in both the supernatant and the pellet fractions using Amplex red. Briefly, 100 µl of sample were incubated with 4 mM hydrogen peroxide and 0.4 mM Amplex red in 50 mM Tris·HCl, pH. 8.0. Oxidation of Amplex red by peroxidase in the presence of hydrogen peroxide produces a highly fluorescent molecule, resorufin. The amount produced was read using a fluorescence microplate reader (model FL600; Bio-Tek, Winooski, VT) with an excitation wavelength of 530 nm and an emission wavelength of 590 nm.
Data presentation and statistic analysis. Results are means ± SE. Data were analyzed using Student's t-test. P < 0.05 was considered statistically significantly different.
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RESULTS |
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Effect of activating AC and inhibiting phosphodiesterase on p44/p42 MAPK activity. Another method for increasing intracellular cAMP concentrations is to prevent its degradation by inhibiting PDE or to stimulate its synthesis by activating AC. To determine the effects of inhibiting PDE or activating AC on p44/p42 MAPK activity, we increased cAMP levels by inhibiting PDE with IBMX or activating AC with the forskolin analog L-858051. As shown in Fig. 4, a 30-min incubation with DBcAMP at 10–3 M and L-858051 at 10–4 M significantly inhibited basal p44/p42 MAPK activity by 22 ± 5.0 and 39 ± 3.0% compared with no addition. The decrease in basal p42/p44 MAPK activity was not significant with IBMX at 10–3 M. Simultaneous addition of IBMX (10–3 M) and L-858051 (10–4 M) or all three compounds [DBcAMP (10–3 M), IBMX (10–3 M), and L-858051 (10–4 M)] to acini significantly decreased basal p44/p42 MAPK activity by 68 ± 6.0 or 66 ± 4.0%, respectively. As a positive control, carbachol (10–4 M) increased p44/p42 MAPK activity 2.0 ± 0.4-fold over basal level (Fig. 4B). Thus increasing the cAMP concentration by activating AC decreases p44/p42 MAPK activity. Simultaneous inhibition of PDE and activation of AC results in a greater inhibition than the use of permeable cAMP analogs, AC activators, or PDE inhibitors alone.
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Effect of increasing cAMP levels on agonist-stimulated p44/p42 MAPK activity. We next determined the effect of increasing cAMP levels on agonist-induced p44/p42 MAPK activity. In Fig. 6, A and B, the membrane-permeant cAMP analog DBcAMP (10–3 M) significantly inhibited basal p44/p42 MAPK activity by 30 ± 4.0% when incubated for 5 min. To determine whether agonist-induced p44/p42 MAPK activity is also inhibited by cAMP, lacrimal gland acini were incubated simultaneously with DBcAMP (10–3 M) and carbachol (10–4 M), phenylephrine (10–4 M), or EGF (10–7 M) for 5 min. Our laboratory (22) previously showed that these three compounds each activate p44/p42 MAPK. Carbachol significantly increased p44/p42 MAPK activity by 2.2 ± 0.5-fold. Addition of DBcAMP (10–3 M) significantly inhibited this increase by 56%. Phenylephrine significantly increased p44/p42 MAPK activity by 1.3 ± 0.1-fold. Addition of DBcAMP completely inhibited this response. EGF significantly increased p44/p42 MAPK activity by 1.8 ± 0.3-fold. Addition of DBcAMP also completely inhibited the EGF-stimulated increase in p44/p42 MAPK activity.
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Effect of VIP on agonist-stimulated p44/p42 MAPK activity. To determine whether VIP had an effect on agonist-induced p44/p42 MAPK activity, lacrimal gland acini were incubated simultaneously with VIP (10–8 M) and carbachol (10–4 M), phenylephrine (10–4 M), or EGF (10–7 M) for 5 min. VIP alone significantly inhibited basal p44/p42 MAPK activity (Fig. 7, A–C). Carbachol increased p44/p42 MAPK activity to 2.5 ± 0.3-fold above basal level, which was significantly decreased by 85 ± 13% by the simultaneous addition of VIP (Fig. 7A). Phenylephrine increased p42/p44 MAPK activity to 1.8 ± 0.6-fold above basal level. Interestingly, VIP did not have any effect on phenylephrine-induced increase in p44/p42 MAPK activity (n = 3, Fig. 7B). EGF increased p42/p44 MAPK activity to 2.1 ± 0.3-fold above basal, which was completely inhibited by the simultaneous addition of VIP (Fig. 7A). These data show that increasing cellular cAMP levels with VIP significantly reduced basal and carbachol- and EGF-induced p44/p42 MAPK activation.
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1D-adrenergic agonists. To test whether cAMP also potentiates peroxidase secretion by affecting the muscarinic pathway, the 1D-adrenergic pathway, or the EGF pathway, DBcAMP (10–3 M) was added alone or with carbachol (10–5 and 10–4 M), phenylephrine (10–5 and 10–4 M), or EGF (10–8 and 10–7 M) for 20 min. Theoretical additivity was calculated as the experimental value of DBcAMP-stimulated protein secretion plus the experimental value of each agonist-stimulated protein secretion value minus the experimental basal value. As shown in Fig. 8A, DBcAMP at 10–3 M and carbachol at 10–4 and 10–5 M significantly increased peroxidase secretion compared with the basal value. Simultaneous addition of DBcAMP with carbachol at both concentrations potentiated secretion by 181% at 10–4 M and 208% at 10–5 M carbachol compared with the theoretical additivity values. In Fig. 8B, phenylephrine at 10–4 and 10–5 M significantly increased secretion compared with the basal value. When DBcAMP 10–3 M was added simultaneously with phenylephrine, secretion was significantly potentiated by 240% at 10–4 M and 287% at 10–5 M phenylephrine compared with the theoretical additivity values. EGF at 10–8 M significantly increased peroxidase secretion. Simultaneous addition of DBcAMP 10–3 M with EGF significantly potentiated secretion by 380% at 10–7 M and 323% at 10–8 M EGF compared with the theoretical additivity values (n = 7) (Fig. 8C).
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DISCUSSION |
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1D-adrenergic agonists known to increase the [Ca2+]i and to activate PKC- and MAPK, even though the cellular mechanisms of action for these agonists differ. We now show that potentiation of secretion also can occur with a cAMP-dependent stimulus and the growth factor EGF, which increases protein secretion by increasing the [Ca2+]i and activating PKC. The potentiation of protein secretion under all these conditions did not appear to occur 1) at the receptor level, since either activating AC or using membrane-permeable cAMP analogs to replace cAMP-dependent agonists could increase potentiation of secretion; 2) at the level of the increase in [Ca2+]i, since the [Ca2+]i was not potentiated; or 3) at the cAMP level, since the cAMP level was not potentiated (9). In the present study, we identified a possible mechanism that could account for potentiation of secretion, that of inhibition of basal and agonist-activated p44/p42 MAPK activity through an increase in cellular cAMP concentration. Since increasing p44/p42 MAPK activity attenuates agonist-stimulated protein secretion (24), decreasing p44/p42 MAPK activity would increase protein secretion. This mechanism of potentiation is supported by previous findings in our laboratory (22, 24) that inhibition of p44/p42 MAPK with the MEK inhibitor U-0126 increases cholinergic and 1-adrenergic agonist as well as EGF-induced-protein secretion. In the present study, simultaneous addition of a membrane-permeable cAMP analog with a cholinergic agonist, an 1D-adrenergic agonist, or EGF potentiated protein secretion and inhibited agonist-induced p44/p42 MAPK activity. Evidence that cAMP can mediate an inhibitory response and alter another signaling pathway also was shown by Burgering et al. (2) in NIH-3T3 cells. They demonstrated that elevation of the intracellular cAMP levels results in the inhibition of growth factor-induced mitogenesis. In these cells, cAMP regulation of the MAPK cascade provides important cross talk between hormone and growth factor signaling, as occurs in the lacrimal gland. The effect of cAMP on inhibition of p44/p42 MAPK was robust. It occurred when intracellular cAMP levels were increased by diverse mechanisms. These included increasing the receptor-mediated activity with VIP, using three different cell-permeable cAMP analogs (DBcAMP, Sp-5,6-DCl-cBiMPS, and 8-BrcAMP), and activating AC with a forskolin analog. Furthermore, if cAMP levels were increased with the use of a membrane-permeant cAMP analog, activation of AC, and inhibition of cAMP PDE simultaneously, basal p44/p42 MAPK activity was inhibited to a greater extent than compared with the effect of the membrane-permeant analog alone. It is not clear why IBMX alone did not have a significant effect on basal p44/p42 MAPK activity. It is possible that the lack of effect could be due to the fact that multiple isoforms of PDEs exist, some of which are insensitive to IBMX (21). It is not know which types of PDEs are present in the lacrimal gland. Regardless, these data indicate that increasing cAMP levels by multiple different mechanisms inhibits basal p44/p42 MAPK activity.
The inhibition of basal p44/p42 MAPK activity is functional, since prevention of cAMP breakdown by inhibiting cAMP PDE on its own stimulates protein secretion, as does activation of AC and the use of the membrane-permeant analog (7). The stimulation of secretion by increasing cAMP levels could be due to an effect of cAMP directly in the secretory process, on MAPK, or both. Unfortunately, we cannot use U-0126, which inhibits MEK, to test this hypothesis, since both U-0126 and cAMP inhibit p44/p42 MAPK. Therefore, inhibition of MAPK by cAMP cannot be reversed by inhibition of MEK with U-0126. Thus MEK inhibitors cannot be used to demonstrate that cAMP-induced secretion or cAMP-induced potentiation of secretion occurs via p44/p42 MAPK.
To ensure that the membrane-permeant cAMP analog DBcAMP increased intracellular cAMP levels in rat lacrimal gland acinar cells, we measured CREB activity. It is well known that CREB is stimulated by protein kinases such as PKA (36). Although CREB can be activated by a number of different kinases and [Ca2+]i (16), only PKA should be activated in unstimulated acinar cells incubated with cAMP analogs. The increase, then, is consistent with an increase in intracellular cAMP.
In the present study, VIP inhibited p44/p42 MAPK. In addition, simultaneous addition of VIP with cholinergic agonists and EGF inhibited the increase in MAPK activity normally seen with these stimuli. This finding supports the hypothesis that MAPK is responsible for potentiation of secretion under these conditions. However, simultaneous addition of VIP and 1-adrenergic agonists had no effect on 1D-adrenergic agonist-stimulated MAPK activity despite the fact that VIP potentiated 1D-adrenergic agonist-stimulated protein secretion. It is well established in the lacrimal gland that cholinergic and 1D-adrenergic agonists stimulate protein secretion by activating different signaling pathways. Cholinergic agonists increase [Ca2+]i and activate PKC- and -, whereas 1D-adrenergic agonists increase both the amount of NO/cGMP and the activity of PKC- (13, 27). It also is known that VIP increases the amount of intracellular cAMP concentration in the cell and causes a small increase in [Ca2+]i (14). The increase in [Ca2+]i could activate enzymes in other pathways in addition to the cAMP pathway. It is possible, then, that the VIP and 1D-adrenergic agonists pathways interact at another step in the secretory process to cause the potentiation of secretion seen with these two agonists.
Potentiation of secretion occurred when a membrane-permeable cAMP analog was used simultaneously with a cholinergic agonist, an 1D-adrenergic agonist, and EGF, agonists known to activate p44/p42 MAPK. This suggests that potentiation of secretion occurs not only in neurally stimulated secretion but also when growth factors were used. Because the pathways used by these agonists and EGF are different, it is not surprising that the effect of exogenous cAMP analog was slightly different for each agonist. For secretion, greater potentiation occurred with cAMP and either EGF or the 1D-adrenergic agonist than with cAMP and the cholinergic agonist. This is similar to the inhibition of p44/p42 MAPK that occurred with cAMP and either EGF or the 1D-adrenergic agonist, which was greater than that with cAMP and the cholinergic agonist. EGF and 1D-adrenergic agonists both activate the EGFR, but EGF causes a greater activation of p44/p42 MAPK than the 1D-adrenergic agonist. Interestingly, cholinergic agonists caused a greater activation of p44/p42 MAPK than EGF or the 1D-adrenergic agonist but caused less potentiation of secretion. This could be explained by cholinergic agonists not activating EGFR, as do the other two agonists, indicating that there may be a second mechanism, a non-cAMP pathway, used to cause potentiation of secretion. The existence of a second mechanism for cholinergic agonists is consistent with DBcAMP only partially inhibiting cholinergic agonist activation of MAPK.
Potentiation of secretion occurred when either maximal or submaximal concentrations of agonists were used with the membrane-permeant cAMP analog. The use of a membrane-permeant cAMP analog and an activator of AC is in contrast to previous findings in which VIP was used to increase cellular cAMP levels (8). When VIP was used with a maximal concentration of a cholinergic or 1D-adrenergic agonist, potentiation of secretion did not occur, although it occurred at submaximal concentrations of these latter two agonists. A possible explanation is that receptor-mediated interactions are limited by saturation of the agonist-receptor interaction. Another possible explanation is that there is another overlap, in addition to MAPK, between the VIP-dependent and cholinergic agonist, 1D-adrenergic agonist, and EGF signaling pathways. This overlap could be the increase in [Ca2+]i given that stimulation of all four pathways increases [Ca2+]i whereas the use of endogenous cAMP analogs does not. This is consistent with previously published work from our laboratory showing that increasing cAMP by activating AC with forskolin potentiated secretion when used with both maximal and submaximal concentrations of cholinergic agonists.
We conclude that increasing intracellular cAMP levels by multiple mechanisms inhibits p44/p42 MAPK activity. This could alleviate the inhibitory effect of p44/p42 MAPK on agonist-stimulated lacrimal gland secretion and account for the potentiation of secretion that occurs when cellular cAMP levels are elevated by a receptor-mediated activation, activation of AC, or the use of permeable cAMP analogs along with simultaneous stimulation with a Ca2+/PKC-dependent agonist.
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FOOTNOTES |
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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.
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REFERENCES |
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