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Keywords:

  • Vasoactive intestinal polypeptide;
  • Primary chromaffin cells;
  • Pituitary adenylate cyclase-ac-tivating polypeptide;
  • Calcium influx;
  • Calcineurin activation

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Radioimmunoassay for VIP
  5. Northern analysis of VIP mRNA
  6. RESULTS AND DISCUSSION
  7. REFERENCES

Abstract : A > 15-fold increase in vasoactive intestinal polypeptide (VIP) mRNA and VIP peptide levels occurred in primary chromaffin cells following exposure to the neurotrophic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP)-27 with an EC50 of ~2 nM. PACAP induction of VIP expression was blocked by methoxyverapamil or by a combination of nimodipine and ω-conotoxin MVIIC, indicating a requirement for PACAP-initiated calcium entry through voltage-dependent calcium channels for regulation of VIP biosynthesis. Ascomycin, which inhibits calcineurin through formation of an ascomycin/FKBP12/calcineurin ternary complex, abolished the PACAP-evoked increase in VIP expression, whereas rapamycin, which also binds to FKBP12 but does not cause inhibition of calcineurin, did not. Cyclosporin A, which inhibits calcineurin through formation of a cyclosporin A/cyclophilin/calcineurin complex, also abolished PACAP-evoked VIP biosynthesis. These data indicate that PACAP regulates the expression of VIP via a signaling pathway that requires calcium influx and activation of calcineurin.

The neuropeptide PACAP (for ppituitary adenylate cyclase-activating polypeptide) was originally isolated from the hypothalamus (Miyata et al., 1989). PACAP is a potent secretagogue for prolactin, acetylcholine, and other hormones and neurotransmitters throughout the neuroendocrine axis and in the nervous system (Arimura and Shioda, 1995). It is also a developmentally important neurotrophic factor and is neuroprotective after ischemic injury in the brain (Uchida et al., 1996 ; Sheward et al., 1998 ; Waschek et al., 1998). PACAP is released from the splanchnic nerve in vivo (Wakade, 1986 ; Przywara et al., 1996). It evokes catecholamine secretion from chromaffin cells through opening of voltage-dependent calcium channels and regulates catecholamine biosynthesis in the adrenal medulla via the tyrosine hydroxylase gene (Rius et al., 1994 ; Haycock, 1996). PACAP evokes secretion and biosynthesis of the adrenomedullary neuropeptides Met-enkephalin and atrial and brain natriuretic peptides from chromaffin cells (Babinski et al., 1996 ; Hahm et al., 1998) and of chromogranin A in PC12 cells (Taupenot et al., 1998). The type I PACAP receptor is abundant in adrenal medulla, and the hop variant of this receptor is the dominant PACAP receptor in bovine chromaffin cells (Spengler et al., 1993 ; Tanaka et al., 1998). PACAP acting through the type I PACAP receptor elicits a rise in cyclic AMP levels and increases cytosolic calcium concentrations through extracellular calcium influx and inositol trisphosphate (IP3)-mediated calcium mobilization from intracellular stores in chromaffin cells (Babinski et al., 1996 ; Tanaka et al., 1998). Elevation of intracellular cyclic AMP levels, increased cytosolic calcium concentrations, or activation of protein kinase C can each independently lead to up-regulation of neuropeptide gene expression in chromaffin cells (MacArthur and Eiden, 1996). PACAP regulation of biosynthesis of vasoactive intestinal polypeptide (VIP) is of particular interest because VIP is itself a neurotrophic neuropeptide and with PACAP may constitute a two-component neuropeptide self-regulatory network involved in neuroendocrine cell proliferation and differentiation (Muller et al., 1995 ; Lelievere et al., 1996). Here, we show that PACAP is an important regulator of VIP biosynthesis via PACAP-initiated calcium influx and activation of the serine/threonine phosphatase calcineurin.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Radioimmunoassay for VIP
  5. Northern analysis of VIP mRNA
  6. RESULTS AND DISCUSSION
  7. REFERENCES

Chromaffin cell culture

Chromaffin cells were obtained from steer adrenal medullae (Hahm et al., 1998). At 24 h after plating into 24-well dishes (500,000 cells/ml per well), cells were preincubated with appropriate agents [D600, ascomycin, cyclosporin A (CsA), or rapamycin] by addition of 100 μl of a 10× stock solution to each well. Thirty minutes later, medium was removed and replaced with complete medium without nystatin, containing vehicle or PACAP-27, with or without the agents added during the preincubation period. For measurement of cell VIP content, medium was removed by aspiration, and cells were extracted for VIP peptide assay after addition of 500 μl of 0.1 M HCl to each well (Eiden et al., 1984). For quantification of VIP mRNA, cells were harvested 18-24 h after drug additions into sodium dodecyl sulfate-EDTA-Tris buffer containing 100 μg/ml proteinase K (SETpK), as described previously (Hahm et al., 1998). The drugs were initially dissolved in ethanol, water, or complete medium so that the final concentrations of vehicle were <0.1% (vol/vol) in the final incubation medium.

Radioimmunoassay for VIP

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Radioimmunoassay for VIP
  5. Northern analysis of VIP mRNA
  6. RESULTS AND DISCUSSION
  7. REFERENCES

VIP was assayed directly in aliquots of culture medium and in lyophilized 0.1 M HCl extracts of chromaffin cells, as previously described (Pruss et al., 1985).

Northern analysis of VIP mRNA

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Radioimmunoassay for VIP
  5. Northern analysis of VIP mRNA
  6. RESULTS AND DISCUSSION
  7. REFERENCES

Northern blot analysis of VIP mRNA was performed with riboprobe transcribed in vitro from human VIP cDNA. Uniformity of total chromaffin cell RNA per lane was verified by staining of ribosomal (18S and 28S) RNA with ethidium bromide following electrophoresis. Quantitation of VIP mRNA was performed by densitometric scanning of autoradiograms of each northern blot.

Materials

PACAP-27 was purchased from Phoenix Pharmaceuticals (Mountain View, CA, U.S.A.) ; ascomycin, methoxyverapamil (D600), rapamycin, and CsA from Sigma (Kansas City, MO, U.S.A.) ; and nimodipine and ω-conotoxin MVIIC from Calbiochem (San Diego, CA, U.S.A.).

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Radioimmunoassay for VIP
  5. Northern analysis of VIP mRNA
  6. RESULTS AND DISCUSSION
  7. REFERENCES

PACAP potently increased the biosynthesis of VIP from chromaffin cells (Fig. 1). Enhanced synthesis of VIP by PACAP likely involves activation of the VIP gene, because there was a parallel increase in both VIP mRNA and peptide levels following exposure to 0.1-10 mM PACAP (Fig. 1). Ionotropic effects of PACAP receptor occupancy are responsible for PACAP-27-induced VIP up-regulation, because both peptide and mRNA inductions were blocked by the voltage-dependent calcium channel blocker D600 (Fig. 2). As D600 has been reported to block nicotine-stimulated sodium as well as calcium influx into chromaffin cells (Corcoran and Kirshner, 1983), we also examined the effects of specific blockade of L, P/Q, and N voltage-dependent calcium channels, with nimodipine and ω-conotoxin MVIIC in combination. This treatment also blocked PACAP induction of VIP biosynthesis (Table 1). The immunosuppressant ascomycin, which binds to FKBP12 and inhibits calcineurin activity (Liu et al., 1991 ; Kay, 1996), abolished both PACAP-evoked VIP biosynthesis and mRNA up-regulation, at 100 nM (Fig. 2). CsA binds the immunophilin protein cyclophilin rather than FKBP12, but also inhibits calcineurin through formation of an immunosuppressant/cyclophilin/calcineurin complex (Liu et al., 1991 ; Steiner et al., 1997). CsA abolished PACAP-evoked VIP biosynthesis at concentrations as low as 100 nM, whereas rapamycin, which binds FKBP12 but unlike ascomycin does not form a calcineurin-inhibiting complex (Dumont et al., 1990 ; Yakel, 1997 ; Snyder et al., 1998), did not abolish PACAP-stimulated VIP biosynthesis at concentrations up to 1,000 nM (Fig. 3).

image

Figure 1. Up-regulation of VIP in chromaffin cells following stimulation with PACAP-27. Following a complete change of medium on day 2 in vitro, fresh medium containing 0.1-10 nM PACAP-27 or medium alone was added to each well. Seventy-two hours later, chromaffin cells were harvested for radioimmunoassay of VIP. Data are mean ± SEM (bars) values of triplicate determinations from a single experiment, which was repeated with similar results. Inset : Up-regulation of VIP mRNA by PACAP-27. Cells were harvested for quantification of VIP mRNA by northern blot analysis 18-24 h after treatment with 0.1-10 nM PACAP-27 or vehicle alone. Data are representative of triplicate experiments.

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image

Figure 2. Blockade of PACAP-evoked upregulation of VIP and VIP mRNA by ascomycin (Asco) and D600. Chromaffin cells were pretreated with 0.1 μM Asco, 30 μM D600, or vehicle for 30 min, and then medium was replaced with the same concentration of Asco, D600, or vehicle alone with or without 10 nM PACAP-27. A : At 72 h after addition of PACAP or vehicle, cellular VIP content was measured as described in Fig. 1. Data are mean ± SEM (bars) values of triplicate determinations from a single experiment, which was repeated with similar results. B : At 18 h after addition of PACAP or vehicle, cells were harvested for quantification of VIP mRNA by northern blot analysis. Data are mean ± SEM (bars) values of triplicate determinations, one of which is illustrated in the inset, and are expressed in arbitrary densitometric units (ADU).

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Table 1. PACAP-27 stimulation of VIP biosynthesis is blocked by pretreatment with nimodipine plus ω-conotoxin MVIICData are mean ± SEM values for triplicate wells from a single chromaffin cell dispersion. Cells were cultured as described in Materials and Methods. Medium was removed and replaced with fresh medium or medium containing ω-conotoxin MVIIC plus nimodipine. Thirty minutes later, medium was again replaced with medium alone or medium containing PACAP-27 and/or ω-conotoxin MVIIC plus nimodipine at the concentrations shown. Seventy-two hours later, cells were harvested for VIP radioimmunoassay.
ConditionVIP (pg/well)
Medium alone15.4 ± 1.6
10 nM PACAP3,362 ± 185
1 μMω-conotoxin MVIIC/10 μM nimodipine11.7 ± 2.4
10 nM PACAP + 1 μMω-conotoxin MVIIC/10 μM nimodipine187 ± 16
image

Figure 3. Inhibition of PACAP-evoked up-regulation of VIP biosynthesis by ascomycin (Asco) and CsA but not by rapamycin (Rapa). Chromaffin cells were pretreated with 1-100 nM Asco, 100 and 1,000 nM CsA, 100 and 1,000 nM Rapa, or vehicle for 30 min, and then medium was replaced with the same concentration of Asco, CsA, Rapa, or vehicle, with or without 10 nM PACAP-27. Seventy-two hours later, chromaffin cell VIP content was measured as described in Fig. 1. Asco, CsA, or Rapa alone at each concentration had no effect on basal concentration of VIP. Data are mean ± SEM (bars) values of triplicate determinations from a single experiment, which was repeated with similar results.

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The data presented above indicate that PACAP-evoked up-regulation of VIP biosynthesis requires calcium entry through voltage-dependent calcium channels and activation of calcineurin. The simplest interpretation of these data is that calcium influx, initiated by PACAP receptor stimulation, triggers calcium/calmodulin-dependent activation of calcineurin (Klee et al., 1988). PACAP receptor occupancy results in increased levels of several second messengers in chromaffin cells, including cyclic AMP, IP3, and calcium (via influx as well as mobilization from intracellular stores) (Tanaka et al., 1998, and references therein). Therefore, additional signaling pathways besides those initiated by calcium, and indeed other calciumdependent signaling pathways besides those involving calcineurin, could be involved in PACAP signaling to the VIP gene. Calcineurin may be a primary component of PACAP signaling in chromaffin cells or may play a permissive role, perhaps by allowing relief from inhibition via dephosphorylation of negative regulatory sites of trans-acting factors such as CREB (Sun et al., 1994), which are responsive to PACAP signaling in other cell systems (Muller et al., 1998).

The requirement for calcium influx in PACAP regulation of VIP biosynthesis may have physiological relevance to PACAP synergism with spontaneous electrical activity and the actions of other neurotrophins/growth factors to regulate the biosynthesis of specific neuropeptides in neuroendocrine cells. For example, calcium influx is not required for up-regulation of enkephalin biosynthesis stimulated by PACAP in chromaffin cells (Hahm et al., 1998). The distinct calcium requirements for PACAP regulation of two important neuropeptide genes may provide a mechanism for differential expression of enkephalin and VIP in peripheral and central neurons that respond to PACAP and other neurotrophins during development.

The involvement of the threonine/serine phosphatase calcineurin in the regulation of the VIP gene by PACAP reveals a previously unremarked signal transduction pathway leading to activation of VIP gene transcription. There are several known possibilities for calcineurin involvement in calcium-initiated signal transduction. These include activation of nitric oxide synthase, intracellular calcium mobilization via modulation of ryanodine and IP3 receptors, and activation and nuclear translocation of the trans-acting factor NFAT (Schreiber and Crabtree, 1992 ; Snyder et al., 1998, and references therein). Although nitric oxide synthase activation is implicated in neurotransmitter release and neurotoxicity, there is no evidence as yet for its involvement in neuropeptide biosynthesis. Modulation of ryanodine and IP3 receptor function by FK506 augments, rather than inhibits, intracellular calcium mobilization. Activation of NFAT, on the other hand, is a potentially likely locus for calcineurin action in the context of VIP gene activation, because an NFAT response element on the VIP gene sensitive to regulation by calcium has been recently identified (Symes et al., 1998). By the criterion of inhibition by ascomycin or CsA, calcineurin is also implicated in the regulation of both the glucagon (Schwaninger et al., 1993) and the δ-opioid receptor (Buzas et al., 1998) genes by depolarization. Calcineurin may be an essential signaling component for transsynaptic regulation of hormone secretion and biosynthesis in mature neuroendocrine cells and neurons and for the mutual interactions between signaling by PACAP and VIP postulated to occur during nervous system development and neuroendocrine tumor progression (Muller et al., 1995 ; Waschek et al., 1998).

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Radioimmunoassay for VIP
  5. Northern analysis of VIP mRNA
  6. RESULTS AND DISCUSSION
  7. REFERENCES
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