VIP antagonist demonstrates differences in VIP- and PHI-mediated stimulation and inhibition of ACTH and corticosterone secretion in rats

Regulatory Peptides 59 (1995) 321-333

ELSEVIER

VIP antagonist demonstrates differences in VIP- and PHI-mediated stimulation and inhibition of ACTH and corticosterone secretion in rats Larry D. Alexander, Linda D. Sander

*

Department of Physiology, Meharry Medical College, School of Graduate Studies and Research, 1005 Dr. D.B. Todd Jr. Boulevard, Nashville, TN 37208, USA

Received 16 August 1994; revised 14 June 1995; accepted 16 June 1995

Abstract Previous studies in our laboratory have demonstrated that PVN administration of equimolar doses of VIP and PHI induce similar increases in plasma ACTH and CORT concentrations via the release of CRF and vasopressin in fasted, freely moving rats studied during the early light cycle. The purpose of these investigations was to determine whether VIP and PHI act via the same receptor and/or mechanism. Individual studies involving the PVN administration of either VIP or PHI in doses ranging from 0.3 to 31).0 nmol/rat demonstrated that VIP increases both ACTH and CORT secretion throughout the administered range. In contrast, PHI was an effective stimulant in doses up to 15 nmol/rat but had no effect on either ACTH or CORT at a dose of 30 nmol/rat thus yielding a bell-shaped dose-response curve. When increasing doses of PHI (0.15-3.0 nmol/rat) were administered against a background of VIP (3.0 nmol/rat) predictably additive responses were observed; however, when increasing doses of VIP (0.15-3.0 nmol/rat) were administered with PHI (3.0 nmol/rat) only the higher doses of VIP facilitated the PHI-induced secretion while the lower doses of VIP actually reduced the PHi-induced ACTH secretion. Fina]ly, pretreatment with [Lys 1, Pro2'5,Arg3'4,Tyr6]-VIP, anVIP (1.5 nmol/rat) totally suppressed VIP-induced ACH-I secretion but had no effect on PHI-induced secretion. These studies collectively suggest that VIP and PHI utilize different receptors/mechanisms to regulate HPA secretion. Furthermore, when a range of doses of anVIP (1.5-30.0 nmol/rat) was tested against VIP (3.0 nmol/rat), ACTH secretion was totally suppressed at all doses of the antagonist. However, the maximal reduction of CORT secretion occurred at the lowest dose of anVIP and increasing doses were less and less effective, suggesting that not only PHI but VIP may also both stimulate and inhibit HPA secretion. While both the stimulatory artd the inhibitory actions of PHI appear to involve ACTH, only the stimulatory action of VIP is ACTH-dependent. Keywords: Pituitary hormone; Hypothalamus; Neuropeptide; Vasoactive intestinal peptide; Peptide histidine isoleucine; VIP-antagonist;

Paraventricularnucleus

1. Introduction

* Correspondingauthor. Fax: + 1 (615) 3276655.

Vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI), structurally related mem-

0167-0115/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0167-0115(95)00087-9

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L.D. Alexander, L.D. Sander/Regulatory Peptides 59 (1995) 321-333

bers of the secretin/glucagon family of neuropeptides, have both been shown to stimulate ACTH and corticosterone (CORT) secretion in rats [1-3]. Previous studies in our laboratory have demonstrated that both peptides are more effective in stimulating ACTH secretion when administered into the paraventricular nucleus (PVN) of the hypothalamus than when given intravenously (i.v.) [4,5] and that stimulation by both peptides is mediated via both corticotropin releasing factor (CRF) and vasopressin (AVP) [6]. The observed similarities in the actions of VIP and PHI [4-7] coupled with the facts that the two peptides are frequently co-localized in neuronal tissues, including parvocellular neurons of the PVN [8-11] and co-released in response to stimulation [9,12] caused us to question whether both peptides might, in fact, be acting at the same receptor and/or through the same mechanism. The purpose of the present study was fourfold: (1) to determine the maximally effective doses of VIP and PHI; (2) to determine whether the co-administration of VIP and PHI elicits an additive response; (3) to determine whether the VIP receptor-antagonist [Lys 1, Pro 2'5, Arg3'4,Tyr6]-vasoactive intestinal peptide (anVIP) inhibits VIP-stimulated ACTH and CORT secretion, and (4) to determine if PHI-stimulated ACTH and C ORT secretion involves PHI interacting as an agonist with the same VIP receptors.

histidine isoleucine (PHI), rat, Mr: 3011.8; and the selective VIP-receptor antagonist [Lys 1, Pro 2'5, Arg3'a,Tyr6]-vasoactive intestinal peptide (anVIP), Mr: 3467.1. All compounds were obtained from Bachem California, Torrance, CA, USA. Saline served as the solvent and control solution. 2.3. P V N cannulation

In all experiments, rats were fitted with a chronic PVN cannula 7 days prior to the experiment. Animals were anesthetized with ketamine:acepromazine (87:4 m g / k g BW, i.p.) and placed on a stereotaxic instrument; a single guide cannula was lowered into the brain. PVN cannulae were constructed in our laboratory using 21 gauge stainless-steel hypodermic tubing [4,5]. The guide cannula with a removable inner stylette was stereotaxically implanted towards the PVN. Implantation coordinates, taken from the atlas of Paxinos and Watson [13], were 6.8 mm anterior to the interaural line, 0.4 mm lateral to the midline and 6.0 mm ventral to the skull surface. The incisor bar was set at 3.3 mm below the interaural line. The cannula was fixed onto the skull with anchor screws and dental cement. The animals were given a minimum recovery period of 7 days. During this period, the animals were handled and mock-injected daily in order to adapt them to the test procedures.

2. Materials and methods 2.4. Intravenous catheterization 2.1. Animals

The experiments were performed on fasted, freely moving male, Sprague-Dawley rats (Harlan Sprague-Dawley Co., Inc., Indianapolis, IN, USA) weighing 250-300 g at the time of the experiments. The animals were housed in individual hanging wire mesh-bottomed cages in a temperature-controlled room at 22 + 2°C. They were maintained on laboratory rat chow and water ad libitum. The animals were kept on a 12/12 h light/dark cycle with lights on at 0700h. 2.2. Drugs

The compounds used were vasoactive intestinal peptide (VIP), porcine/human, Mr: 3326.2; peptide

Twenty-four hours before the study, some animals were prepared with an indwelling jugular catheter prepared from a 20 cm length of Silastic tubing (0.025 in ID; 0.04 in OD; Dow Coming, Midland, MI, USA). The tubing was inserted under ketamine : acepromazine anesthesia (87 : 4 mg/kg, BW, i.p.) into the jugular vein and advanced into the right atrium. The catheter was then secured. To prevent clot formation at its tip inside the vein, the catheter was filled with a solution of 40% polyvinylpyrrolidone (PVP, M r 45,000) in saline ( w / v ) containing 50 units of heparin per ml. The catheter was mn under the skin of the back, externalized at the mid-back region and sealed with a stainless-steel stylette. This procedure afforded the rats

L.D. Alexander,L.D. Sander/RegulatoryPeptides59 (1995) 321-333 easy access to rat chow and water and allowed normal sleeping posture.

2.5. Experimental procedures The experiments were carried out between 0900h and 1100h in overnight fasted, freely moving male rats. On the day of tile experiment, the rats were left undisturbed in individual plastic cages in a quiet room for an adaptation period of at least 60 min before beginning the study. In some studies, blood samples were obtained by decapitation and collected into chilled tubes containing 0.5 ml of 7.5 g% EDTA. In animals prepared with i.v. catheters, blood samples (0.5 ml) for plasma ACTH and CORT determinations were drawn from the catheter immediately before as well as at various time points after PVN administration of the drugs or saline. The blood volume was immediately replaced with a physiological saline-heparin solution. Blood samples were collected into pre-chilled tubes containing 0.05 ml of 7.5 g% EDTA. Afte~r centrifuging the blood for 15 min at 4°C, plasma was separated and stored at - 2 0 ° C until the assays were run. Before storage, aprotinin (500 K I U / m l plasma) was added to all samples to be used for ACTH determinations.

2.6. Experiment 1: effect of VIP and PHI on ACTH and CORT secretion: dose-response relationship In two separate studies, VIP (0.30-30.0 nmol) or PHI (0.30-30.0 nmol) was administered into the PVN and blood samples were obtained by decapitation 15 min later. VIP and PHI were solubilized in 0.9% saline and administered in a total volume of 1.0 /xl. The peptide was gradually injected into the PVN over a period of 5-10 s using a 5-/xl Hamilton syringe with polyethylene tubing connecting the syringe to the injector. The injector (26 gauge) extended 2.4 mm beyond the guide cannula. Saline served as the control The rats were randomly assigned to each treatment group on an alternate day basis until each animal was tested with a single dose of drug. The time course of the experiment was chosen on the bases of previous experiments [4,5] so that blood samples were obtained at the time point when ACTH and CORT responses to VIP or PHI were maximal. Although the lower doses of VIP and

323

PHI in this study were used in the previous studies, this work was repeated with additional higher doses of peptides in order to determine the maximally effective doses of each peptide.

2.7. Experiment 2: effect of co-administration of VIP and PHI on ACTH and CORT secretion VIP (3.0 nmol), PHI (3.0 nmol) or co-administration of a fixed dose of VIP (3.0 nmol) with varying doses of PHI (0.15-3.0 nmol) or vice versa, was administered into the PVN. The various drugs and dose combinations were studied in random order and each animal was used only once. Peptides and peptide combinations were solubilized in 0.9% saline which served as the control solution. At the beginning of the study, a 0.5 ml blood sample was collected via the i.v. catheter. The saline or peptides alone or_in various dose combinations were then injected into the PVN at 0 min. All drugs were infused in a total volume of 0.5/zl. Additional blood samples were collected at 15, 30 and 60 min after drug administration.

2.8. Experiment 3: effect of VIP receptor-antagonist (anVIP) on VIP-stimulated ACTH and CORT secretion: dose-response study The VIP receptor-antagonist [Lys 1, Pro 2'5, Arg3'4,Tyr6]-VIP (1.5-30.0 nmol/rat) was injected into the PVN 5 min prior to the injection of VIP (3.0 nmol). Saline served as the control for both VIP and VIP antagonist. Fifteen minutes after the second injection, blood samples were collected by decapitation. All drugs were solubilized in 0.9% saline and administered in a total volume of 1.0 /zl. Rats were randomly assigned to groups on an alternate day basis until each animal was tested with a single dose of drug. All animals were tested only once.

2.9. Experiment 4: effect of VIP receptor-antagonist (anVIP) on VIP-stimulated ACTH and CORT secretion: time course study At the beginning of the study, a blood sample was collected via the i.v. catheter. The VIP receptor antagonist [Lys a, Pro 2'5, Arg3'4,Tyr 6]-VIP (1.5 nmol) or saline was then injected into the PVN 5 min prior

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L.D. Alexander, L.D. Sander/ Regulatory Peptides59 (1995) 321-333

to the injection of VIP (3.0 nmol). One group of control animals received anVIP followed by saline 5 min later; a second control group received 2 saline injections. Additional blood samples were collected at 5, 15, 30 and 60 min after the second injection. All drugs and saline were administered in a total volume of 1.0/zl. Each animal was tested only once. The choice of the dose of antagonist was based on experiment 3. 2.10. Experiment 5: effect of VIP antagonist (anVIP) on VIP- and PHI-induced A C T H and CORT secretion To determine whether PHI cross-reacts with VIP in stimulating A C T H and CORT release, we examined the effect of the VIP antagonist [Lys 1, Pro 2'5, Arg3'n,Tyr6]-VIP on PHI-induced A C T H and CORT secretion. The antagonist (1.5 nmol) was injected into the PVN 5 min prior to the injection of 3.0 nmol of either VIP or PHI. Control animals were injected with the antagonist or with saline alone. Fifteen minutes after the second injection, blood samples were collected by decapitation. 2.11. Histology At the completion of experiments all rats were injected with methylene blue dye (1 /zl) through the A

same injectors. The animals in experiments 1, 3 and 5 were decapitated and the brains were removed promptly and stored in 10% buffered formalin solution. Animals in experiments 2 and 4 were given a large dose of ketamine:acepromazine anesthesia (130 : 6 m g / k g BW, i.p.) and perfused transcardially with buffered 10% formalin. The rats were then decapitated and the brains removed and stored in 10% formalin solution for a minimum of 5 days. Using a vibratome, serial coronal sections of the brain, 100 /zm thick were cut, mounted on slides, stained using 0.5% cresyl violet, and subjected to histological examination of the PVN cannula placement site. All cannula placements were in the region of the PVN. 2.12. Radioimmunoassays Plasma A C T H concentrations were assayed using the A C T H RIA kit available from Diagnostic System Laboratories, Inc., Webster, TX, USA; intra-assay variability of 4.5-8.0% and inter-assay variability of 8.0-12.0%. Specificity, as determined by the manufacturer, showed 100% cross-reactivity between human and rat A C T H (1-39) and human A C T H (1-24), 0% cross-reactivity to A C T H (1-10) and less than 0.1% cross-reactivity with /3-endorphin, /3-1ipotropin, Met and Leu-enkephalin, prolactin and several other peptides tested. Plasma corticosterone con50-

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Fig. 1. The ACTH (A) and CORT (B) responses to VIP (0.30-30.0 nmol) injected into the PVN of fasted, freely moving male rats. The animals were decapitated at + 15 rain. The results represent the mean + S.E.M. of 5 animals. Statistical significance: *P < 0.05; * *P < 0.01; * * *P < 0.001; #P < 0.0001 vs. saline-treated group, ap < 0.05; bp < 0.01; Cp < 0.001 vs. graded dose of anVIP.

L.D. Alexander, L.D. Sander~Regulatory Peptides 59 (1995) 321-333

centrations were determined using antibody and label available from ICN Biochemicals, Costa Mesa, CA, USA; intra-assay variability 6.4-7.7% and inter-assay variability of 7.0-13.0%.

2.13. Statistical ana,~sis Results are prese:ated as mean + S.E.M. and represent groups of 5 - 8 animals per treatment. The data were evaluated by one- or two-way analysis of variance or a two-way analysis of variance corrected for repeat measures followed by Newman Keul's post hoc test for multiple comparisons. The limit of significance was a P-value of < 0.05.

3. Results 3.1. Experiment 1: effect of VIP and PHI on ACTH and CORT secretion: dose-response relationship VIP (0.30-30.0 nmol/rat) injected into the PVN caused significant increases in ACTH secretion. Plasma ACTH concentration increased from a basal level of 49.0 + 7.8 p g / m l to a VIP-stimulated maximal value of approximately 650.0 _ 101.3 p g / m l , a 13-fold increase, which was obtained with the highest dose of VIP (30 nmol/rat; P < 0.001). The EDs0

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was estimated to be 1.0 nmol. Doses of 3.0 and 15.0 nmol of VIP caused 8-and 10-fold increases in the plasma concentration of ACTH ( 4 2 5 . 8 _ 118.2 p g / m l , P < 0.05, and 512.5 __+114.0 p g / m l , P < 0.01), respectively, compared to the saline-control group. The lowest dose of VIP (0.30 nmol/rat) had no effect on plasma ACTH ( 6 2 . 0 _ 5.9 p g / m l ) secretion (Fig. 1A). Comparing the effects of sequentially increasing doses of VIP on plasma ACTH levels, a statistically significant difference was observed only between the responses to 0.30 and 3.0 nmol VIP ( P < 0.01). Thus, the ACTH responses to higher doses of VIP (15.0 and 30.0 nmol) were not significantly greater than that seen with the dose of 3.0 nmol. VIP also caused dose-dependent increases in plasma CORT concentration: saline < 0.30 nmol ( P < 0.0001) < 3.0 nmol ( P < 0.01) < 15 nmol ( P < 0.05) = 30.0 nmol. Plasma CORT increased from a basal level of 3.5 + 1.6 /zg% to a maximal value of approximately 43.6 + 2.3/xg%, a 12-fold increase in response to the highest dose of VIP (30 nmol, P < 0.0001). Doses of 0.3, 3.0 and 15.0 nmol caused 7-, 9- and l 1-fold increases in plasma CORT ( 2 6 . 4 _ 2.1, 33.2 + 1.6 and 39.8 + 1.4 /xg%, P < 0.0001) levels, respectively, compared to the saline-treated controls (Fig. 1B). The ED50 was approximately 0.36 nmol.

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Fig. 2. The ACTH (A) alad CORT (B) responses to PHI (0.30-30.0 nmol) injected into the PVN of fasted, freely moving male rats. The animals were decapitated at +15 min. The results represent the mean + S.E.M. of five animals. Statistical significance: *P < 0.05; * *P < 0.01; * * *P < 0.(p01; #P < 0.0001 vs. saline-treated group, ap < 0.05; bp < 0.01; Cp < 0.001 vs. graded dose of anVIP.

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L.D. Alexander, L.D. Sander/Regulatory Peptides 59 (1995) 321-333

PHI (0.30-30.0 nmol/rat) injected into the PVN also had a significant effect on plasma A C T H (Fig. 2A) and CORT (Fig. 2B) secretion. Both plasma A C T H and CORT were maximally stimulated by 15.0 nmol PHI (588.0 + 5.9 vs. control 4 3 . 4 _ 5.8 p g / m l , P < 0.0001 and 3 1 . 6 _ 5.5 vs. 6.5 _ 1 . 9 /xg%, P < 0.001, respectively). An additional increase of PHI concentration (30.0 nmol) caused reductions in the stimulatory responses for both A C T H (197.4 _ 25.8 p g / m l ) and CORT (17.1 _ 4.1 g%) which were not significantly different from salinetreated controls rather than stimulation as seen with VIP. A comparison of the effects of sequentially increasing doses of PHI on plasma A C T H levels indicated that the responses to 3.0 and 15.0 nmol of PHI did not differ. A dose of 30.0 nmol PHI led to a significant reduction in plasma A C T H compared to 15.0 nmol ( P < 0.0001). With regard to CORT levels, the response to 15.0 nmol was significantly greater than that to 3.0 nmol ( P < 0.05); 30.0 nmol PHI resulted in a significant reduction ( P < 0.01 vs. 15.0 nmol) The EDs0 values for plasma A C T H and CORT were approximately 1.0 and 0.70 nmol, respectively.

3.2. Experiment 2: effect of co-administration of VIP and PHI on ACTH and CORT secretion Figs. 3 and 4 show the A C T H and CORT responses to PVN injections of varying doses (0.15-3.0 nmol) of VIP and PHI in fasted, freely moving male rats, respectively. The peak A C T H and CORT responses to all doses of VIP or PHI occurred within 15 min of administration. When the two peptides were administered alone in a dose of 3.0 nmol, both peptides caused peak A C T H (VIP: 1 8 9 . 2 + 6.0 p g / m l , P < 0.01; PHI: 164.7 + 7.6 p g / m l , P < 0.01) levels which were significantly elevated at 15 min compared to the time-matched saline control (41.3 + 8.2 p g / m l ) group (Fig. 3A and Fig. 4A, respectively). In comparison, the VIP (3.0 nmol) was a slightly stronger stimulant of basal A C T H secretion at 15 min than an equimolar dose of PHI ( P < 0.01). When VIP and PHI were given simultaneously as a fixed dose of VIP (3.0 nmol) and increasing doses of PHI (0.15-3.0 nmol), the two peptides increased plasma A C T H levels at 15 rain above that observed

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Fig. 3. Plasma ACrH (A) and CORT (B) responses following microinjection of a fixed dose of VIP (3 nmol/rat) with co-administration of increasing doses of PHI (0.15-3.0 nmol) into the hypothalamic paraventricular nucleus (PVN) of fasted, freely moving male rats. There were six animals in each experimental group. Each point represents mean _+S.E.M. All animals, peptides and doses were part of a single study. Significance: * *P < 0.01, VIP vs. time-matched control; +P < 0.01, co-administration of varying doses of PHI vs. time-matched VIP (3 nmol).

with VIP alone: VIP + 0.15 nmol PHI (210 +_ 1.8 p g / m l ) , VIP + 0.30 nmol PHI (239.7 _+ 10.6 p g / m l ) , VIP + 1.5 nmol PHI (292.5 _ 28.2 p g / m l ) and VIP + PHI 3.0 nmol ( 3 4 8 . 2 _ 25.0 p g / m l ) ( P < 0.01, Fig. 3A). Plasma A C T H response to the addition of 0.30 nmol PHI was significantly elevated

L.D. Alexander, L.D. Sander~Regulatory Peptides 59 (1995) 321-333

up to 15 min, whereas, the addition of the higher two doses (1.5 and 3.0 nmol) of PHI elevated the plasma A C T H responses for up to 60 min. The A C T H

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Time ( mln ) Fig. 4. Plasma ACTH (A) and CORT (B) responses following microinjection of a fixed dose of PHI (3.0 nmol/rat) with co-administration of increasing doses of VIP (0.15-3.0 nmol) into the hypothalamicparaventricular nucleus (PVN) in fasted, freely moving male rats. There were 6 animals in each experimental group. Each point represents mean+S.E.M. All animals, peptides and doses were part of a single study. For purposes of comparison. The ACTH and CORT responses to saline and to 3.0 nmoi VIP + 3.0 nmol PHI are repeated from Fig. 3. Significance: *P < 0.05, * *P < 0.01, PHI vs. time-matched control; +P < 0.01, co-administration of varying doses of VIP vs. time-matched PHI (3 nmol).

327

response to the two peptides given in combination at the highest dose (3.0 nmol): 384.2-l-25.0 p g / m l was slightly but not significantly greater than the sum of the A C T H responses of the two peptides given individually: VIP (3.0 nmol), 189.2 + 6.0 + PHI (3.0 nmol) 164.7 _ 7.6 = 353.9 + 13.6 p g / m l . In contrast, when VIP and PHI were given simultaneously as a fixed dose of PHI (3 nmol/rat) with increasing doses of VIP (0.15-3.0 nmol), the addition of small amounts of VIP (0.15 or 0.30 nmol) resulted in peak A C T H responses (92.5 ___19.9 or 116.7 + 28.4 p g / m l , respectively) which were significantly lower ( P < 0.01) than that of PHI alone. The addition of larger amounts of VIP (1.5 or 3.0 nmol) increased the effectiveness of PHI stimulation of plasma A C T H (213.7___ 19.6 or 384.2 + 25.0 p g / m l , P < 0.01) secretion (Fig. 4A). The addition of the highest dose of VIP significantly elevated plasma A C T H levels for up to 60 rain. PVN injections of either 3.0 nmol of VIP or PHI alone significantly increased plasma CORT (33.8 ___ 2.7/zg%, P < 0.01 and 26.1 + 5.2 /.tg%, P < 0.05, respectively) levels at 15 min compared to the timematched saline control (11.3 ___4 . 5 / z g % ) group (Fig. 3B and Fig. 4B, respectively). There was no significant difference between peak CORT released by the VIP and PHI. With a fixed dose of VIP (3 nmol/rat), the addition of all doses of PHI (0.15-3.0 nmol) increased the effectiveness of VIP stimulation o f plasma CORT secretion; however, only the higher two doses of PHI (1.5 and 3.0 nmol) resulted in peak CORT values (61.8 ___6.6 and 72.5 ___6.9/xg%, P < 0.01, respectively) that were significantly different from VIP alone. The addition of the highest dose of PHI significantly elevated plasma CORT levels for up to 60 min. As with ACTH, the peak CORT response to the co-administration of VIP + PHI was not significantly greater than the sum of the peak responses to the two peptides given individually (Fig. 3B). CORT release induced by the background injection of PHI was not influenced by the two lower doses of VIP; however, the two higher doses (1.5 and 3.0 nmol) resulted in a significant increase in the PHI-stimulated peak response: PHI + 1.5 nmol VIP 36.4 __. 5.3 and PHI + 3.0 nmol VIP 72.5 + 6.9/zg%, P < 0.01, respectively (Fig. 4B). The addition of the higher two doses of VIP significantly elevated plasma A C T H levels for up to 60 min.

328

L.D. Alexander, L.D. Sander~Regulatory Peptides 59 (1995) 321-333

3.3. Experiment 3: effect of VII) receptor-antagonist (anVIP) on VIP-stimulated ACTH and CORT secretion." dose-response study PVN-injection of VIP (3 nmol/rat) significantly increased plasma ACTH (473.6 + 51.0 pg/ml, P < 0.0001) and CORT (35.5 + 3.2 /xg%, P < 0.0001) by approximately 214 and 400% as compared to saline-treated controls (150.7 + 12.0 p g / m l and 7.1 + 1.1 /~g%), respectively. Why the control ACTH levels were higher in this study than in other experiments is unknown. Nonetheless, pretreatment with the VIP receptor antagonist [Lys ~, Pro 2'5, Arg3'4,Tyr6]-VIP, anVIP, (1.5-30.0 nmol/rat) inhibited the VIP-stimulated ACTH and CORT secretion. The lowest administered dose of anVIP totally suppressed VIP-stimulated ACTH secretion (195.0 + 46.6 pg/ml, P < 0.0001; Fig. 5A) and increasing the dose of anVIP had no additional effect; none of the ACTH responses to VIP + anVIP were significantly different from the saline control or from each other. In contrast, not only did the lowest dose of anVIP (1.5 nmol) have the maximal inhibitory effect on VIP-stimulated CORT secretion, but increasing the dose of antagonist resulted in less and less suppression: VIP + 1.5 nmol anVIP, 11.7 + 1.2; VIP + 3.0 nmol anVIP, 17.9 + 2.0; VIP + 6.0 nmol anVIP, 23.0 + 1.1 and VIP + 30.0 nmol anVIP, 23.1 + 1.1 /xg%, P < 0.001; reductions of 84, 62, 44% and 44%, respectively. The CORT response to 1.5 nmol anVIP + VIP was not significantly greater than saline control; however, the 3.0-30.0 nmol anVIP + VIP responses were ( P < 0.001). A significant difference was also observed between sequentially increasing doses of anVIP: saline = 1.5 nmol anVIP + VIP < 3.0 nmol anVIP + VIP ( P < 0.05) < 6.0 nmol anVIP + VIP ( P < 0.05) = 30.0 nmol anVIP + VIP.

antagonist (1.5 nmol), by itself, had no significant effect on plasma ACTH levels. However, pretreatment with the VIP antagonist inhibited the 15 min VIP-induced ACTH peak response (80.0 + 7.75 pg/ml, P < 0.0001) by 94% and totally abolished the 30 and 60 min responses. Also, no significant

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3.4. Experiment 4: effect of VIP receptor-antagonist (anVIP) on VIP-stimulated ACTH and CORT secretion: time course study PVN injection of VIP (3 nmol/rat) increased plasma ACTH significantly. Consistent with previous studies, plasma ACTH peaked at 15 min (249 + 27.7 p g / m l vs. saline control 73.0 _+ 3.3 pg/ml; P < 0.0001) and decreased thereafter, returning near basal levels within 60 min. The VIP receptor-

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Fig. 5. The effect of pretreatment with the competitive antagonist of VIP, [Lysl,Pro 2,s, Arg3,4,Tyr6]-VIP, anVIP, on VIP-induced plasma ACI'H (A) and CORT (B) secretion in fasted, freely moving male rats. [Lysl,Pro 2,5, Arg3,4,Tyr6 ]-VIP (1.5-30.0 nmol) was injected into the PVN at - 5 min and VIP (3.0 nmol) or saline (Sal) was injected into the PVN at time 0. The animals were decapitated at 15 rain. The results represent the mean + S.E.M. of 7-8 animals. Statistical significance: #P < 0.0001 Sal vs. VIP, *P<0.0001 VIP vs. anVIP, ap<0.001; bp<0.0001 Sai vs. anVIP + VIP, + P < 0.05 vs. graded dose of anVIP.

L.D. Alexander, L.D. Sander~Regulatory Peptides 59 (1995) 321-333

differences in plasma ACTH levels were observed among the saline only, the anVIP + saline or the anVIP + VIP treated animals at any time point (Fig. 6A). Similarly to previious results, VIP (3.0 nmol/rat) stimulated plasma CORT significantly. The time response was similar to that seen in the VIP-induced ACTH response, with the peak CORT response occurring at 15 min (50.6 _ 1.2 /zg%, P < 0.0001). In contrast to plasma ACTH, VIP-induced CORT levels remained elevated for the duration of the 60 min. Pretreatment with the VIP receptor-antagonist (1.5 nmol/rat) inhibited the 15 and 30 min VIP-stimulated CORT respone;es (14.9 _ 0.70 and 12.0 _ 1.2 /zg%, P < 0.0001)by approximately 76 and 73%, respectively, and abolished the 60 min VIP-stimulated CORT response (4.8 _ 0.71/xg%, P < 0.0001). The VIP antagonis~I, by itself, had no effect on plasma CORT concentrations (Fig. 6B). In comparison, the VIP-induced ACTH was totally blocked at all time points, whereas the CORT response was not. These results agree with those in experiment 3 suggesting that the VIP-induced plasma CORT secretion seen in both studies :may involve a non-ACTH-mediated effect on the adrenal.

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L.D. Alexander, L.D. Sander/Regulatory Peptides 59 (1995) 321-333

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the VIP receptor-antagonist had no effect on PHI-induced CORT secretion (Fig. 7B). anVIP alone had no effect on either ACTH or CORT secretion. Nor were there differences in ACTH levels between anVIP + VIP and anVIP alone or saline. In contrast, CORT levels in the anVIP + VIP treated animals were significantly greater than both saline controls and anVIP alone demonstrating once again the persistence, of CORT secretion despite complete suppression of ACTH.

4. Discussion Although the brain-gut peptides VIP and PHI are structurally similar, are often co-localized and co-released and both act via CRF and vasopressin to stimulate ACTH and CORT secretion when administered into the PVN of the rat hypothalamus, the current studies collectively suggest that these peptides utilize different receptors a n d / o r mechanisms to elicit the same responses. Firstly, as demonstrated in experiment one, although both peptides initiate

very similar stimulatory responses when administered at doses of or below 15 nmol/rat, at higher levels of peptide administration, VIP continues to stimulate ACTH and thus CORT secretion, whereas PHI has no apparent effect on the HPA axis, resulting in the development of a bell-shaped dose-response curve. Secondly, when increasing doses of PHI are added to a background stimulation by VIP (experiment 2), both ACTH and CORT secretion increase in a predictably additive fashion. In contrast, when increasing doses of VIP are added to a background stimulation by PHI, only the higher two doses of VIP supplement the PHI-induced secretion of ACTH and CORT while the lower doses of VIP actually suppress the PHI-induced stimulation of ACTH secretion (while having no effect on CORT stimulation). And, thirdly, anVIP, an established receptor-antagonist of VIP, totally blocks VIP-induced stimulation of ACTH and substantially reduces CORT secretion while having no effect on PHI-induced ACTH and CORT secretion (experiment 5). Each of these findings is consistent with the involvement of two or more receptors and or mechanisms in

L.D. Alexander, L.D. Sander~Regulatory Peptides 59 (1995) 321-333

mediating the effects of VIP and PHI on the HPA axis. Both pharmacological and molecular studies have confirmed the existence of two or more VIP receptors with differing affinities for the various members of the secretin/glucagon family of neuropeptides and differing peripheral and central distributions [14-16]. VIP 1 receptors are found in the periphery (e.g. lung and liver) and brain (e.g. hippocampus, cortex and hypotha]Lamus) tissue, whereas the VIP2 receptors are distinc, t for the central nervous system (CNS) (e.g. suprachiasmatic nucleus and hippocampus). These VIP-receptors also recognize other VIPrelated peptides suc,h as secretin and PHI to some extent [17,18] which may help explain some of the similarities in actions among these peptides. CNS-receptors specific for PHI have not yet been described. Since [Lys 1, Pro:"'5,Arg3'4,Tyr6]-VIP, the receptor antagonist used in this study, is thought to bind with equal affinities to all known VIP receptors [19], its lack of effect on PHI-stimulated ACTH and CORT secretion favors the existence of an unrelated PHI receptor. Using the same antagonist, Murase et al. [20] recently demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP), another VIP-related peptide, also acts via non-VIP receptors to stimulate AVP release in the rat. Whether PHI and PACAP use the sar~Lereceptor can not be determined at this time. The current studies also suggest that VIP influences HPA secretion via at least two mechanisms, one of which appears to be independent of ACTH. In both the co-administration study (experiment 2) involving increasing VIP in the presence of background PHI and all three of the anVIP studies (experiments 3-5), divergent changes in ACTH and CORT secretion following manipulation of VIP suggest that VIP may alter CORT secretion without the involvement of ACTH. In the co-administration study, low doses of VIP administered concomitantly with PHI resulted in significantly lower levels of ACTH than observed with PHI alone, while CORT levels appeared to be unaffected. Higher doses of VIP increased both PHI-induced ACTH and CORT. Although the small magnitude of these differences is such that they could be explained on the bases of sampling times and differences in hormone half-lives, the differences in the

331

ACTH and CORT responses to anVIP are not so easily dismissed. In all three of the anVIP studies conducted (which involved differing groups of animals), anVIP, at a dose of 1.5 nmol/rat, totally suppressed VIP-induced ACTH secretion to levels which did not differ statistically from the saline control levels or anVIP alone. In contrast, in all three studies, CORT secretion in the presence of VIP + anVIP while substantially reduced, was not totally suppressed. Furthermore, as the level of anVIP was increased (experiment 3) the ACTH concentration remained suppressed while that of CORT exhibited less and less suppression. The mechanism of this dis-inhibition is presently unknown. The possibility that anVIP might possess weak agonistic properties would not explain the divergent responses of ACTH and CORT. However, these findings would be consistent with an ACTH-mediated stimulatory pathway for VIP and an inhibitory pathway which acts on CORT secretion independently of ACTH. The data further suggest that the stimulatory pathway is more sensitive (high affinity) to anVIP than the inhibitory pathway (low affinity?). Numerous studies support the contention that VIP influences CORT secretion via peripheral mechanisms which do not involve pituitary ACTH, such as a direct action on the adrenal gland [2], changes in adrenal blood flow [21] a n d / o r central nervous system changes resulting in peripheral splanchnic nerve release of VIP [22]. However, since the VIP and anVIP used in this study were administered into the PVN (previous studies have shown that PVN administration is much more sensitive to VIP than systemic administration), it is unlikely that changes in peripherally acting VIP are responsible for the A C T H CORT divergence. On the other hand, it is also possible that VIP-invoked CORT secretion may involve a central VIP pathway mediated via the splanchnic nerves [23]. Interestingly, immunohistochemical studies have identified VIP-containing neurons and nerve fibers in sympathetic ganglia [21,24] and VIP has been shown to stimulate adrenal sympathetic (splanchnic) nerves [23]. Furthermore, nerve fibers of the PVN have been shown to project directly to sympathetic ganglia in the spinal cord [25], although the presence of VIP within these fibers has yet to be established.

332

L.D. Alexander, L.D. Sander~Regulatory Peptides 59 (1995) 321-333

Even though the current status of a hypothalamosympathetic-adrenal pathway involving VIP is theoretical, the fact that large quantities of VIP are found in neurons and nerve fibers of the hypothalamus [26], the existence of a direct hypothalamo-sympathetic pathway [25] and evidence that sympathetic ganglia neurons have peptidergic receptors for VIP [22,24], suggest that PVN VIP may make efferent inputs to sympathetic neurons which eventually influence the adrenocortical function. Although splanchnic nerve stimulation has been shown to increase adrenocortical activity [27-29], recent studies by Jasper et al. [30] have suggested that the low level of CORT secretion seen during the daily trough of the circadian rhythm may be due to splanchnic nerve mediated inhibition of CORT secretion. Since the current studies were performed during the early hours of the light cycle, such splanchnic nerve mediated inhibition, if it exists, would have been operational. Thus, it would appear that VIP-induced (CRF/AVP-mediated) ACTH secretion at this time overcomes such inhibition. On the other hand, the concomitant administration of anVIP, by blocking the presumably high-affinity ACTH-linked sites may, through dis-inhibition, reveal the existence of a lower-affinity inhibitory pathway. In conclusion, these observations strongly suggest the existence of multiple receptors and/or mechanisms mediating VIP- and PHI-induced regulation of plasma ACTH and CORT secretion. PHI may act through two receptors/mechanisms, one stimulatory (low doses) and one inhibitory (high doses) to regulate both ACTH and CORT, whereas VIP-mediated HPA activity most likely involves an ACTH-dependent stimulatory and an ACTH-independent inhibitory pathway.

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

Acknowledgements [13]

This research was supported by funds from NIH 3SO6GM8037 and NSF RIMI HRD-9106096.

[14]

References

[15]

[1] Itoh, S., Hirota, R. and Katsuura, G., Effect of cholecystokinin octapeptide and vasoactive intestinal polypeptide on

adrenocortical secretion in the rat, Jpn. J. Physiol., 32 (1982) 553-560. Cunningham, L.A. and Holzwarth, M.A., Vasoactive intestinal peptide stimulates adrenal aldosteronc and corticosterone secretion, Endocrinology, 122 (1988) 2090-2097. Tilders, F., Tatemoto, IC and Berkenbosch, F., The intestinal peptide PHI-27 potentiates the action of corticotropin-releasing factor on ACTH release from rat pituitary fragments in vitro, Endocrinology, 115 (1984) 1633-1635. Alexander, L.D. and Sander, LD., Vasoactive intestinal peptide stimulates ACTH and corticosterone release after injection into the PVN, Regul. Pept., 51 (1994) 221-227. Alexander, L.D., Sander, L.D., Hooper, T. and Washington, V., Peptide histidine isoleucine induced elevations in ACTH and corticosterone in the rat, Peptides, 15 (1994) 1021-1025. Alexander, L.D. and Sander, LD., Involvement of vasopressin and corticotropin-releasing hormone in VIP-and PHI-induced secretion of ACTH and corticosterone, Neuropeptides, 28 (1995) 167-173. Ohta, H., Kato, Y., Tojo, K., Shimatsu, A., Inoue, T., Kabayama, Y. and Imura, H., Further evidence that peptide histidine isoleucine (PHI) may function as a prolactin releasing factor in rats, Peptides, 6 (1985) 709-712. Ceccatelli, S., Eriksson, M. and H6kfelt, T., Distribution and coexistence of corticotropin -releasing factor -, neurotensin -, enkephalin-, cholecystokinin-, galanin-and vasoactive intestinal polypeptide/peptide histidine isoleucine-like peptides in the parvocellular part of the paraventricular nucleus, Neuroendocrinology, 49 (1989) 309-323. Fahrenkrug, J., Co-existence and co-secretion of the structurally related peptides VIP and PHI, Scand. J. Clin. Lab. Invest., 47 (Suppl. 186) (1987) 43-50. H~ikfelt, T., Fahrenkrug, J., Ju, G., Ceccatelli, S., Tsuruo, Y., Meister, B., Mutt, V., Rundgren, M., Brodin, E., Terenius, L., Hulting, A.-L., Werner, S., Bj6rklund, H. and Vale, W., Analysis of peptide histidine-isoleucine/vasoactive intestinal polypeptide-immunoreactive neurons in the central nervous system with special reference to their relation to corticotropin releasing factor-and enkephalin-like immunoreactivities in the paraventricular hypothalamic nucleus, Neuroscience, 23 (1987) 827-857. H6kfelt, T., Fahrenkrug, J., Tatemoto, K., Mutt, V. and Werner, S., PHI, a VIP-like peptide is present in the rat median eminence, ACTA Physiol. Scand., 116 (1982) 469. Ohta, T., Ito, S. and Ohga, A., Co-release of PHI and VIP in dog stomach by peripheral and central vagal stimulation, Br. J. Pharmacol., 100 (1990) 231-236. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1986. Ishihara, T., Shigemoto, R., Mori, K., Takahashi, K. and Nagata, S., Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide, Neuron, 8 (1992) 811-819. Lutz, E.M., Sheward, W.J., West, K.M., Morrow, J.A., Fink, G. and Harmar, A.J., The VIP 2 receptor: molecular characterization of a cDNA encoding a novel receptor for vasoactive intestinal peptide, FEBS Lett., 334 (1993) 3-8.

L.D. Alexander, L.D. Sander/Regulatory Peptides 59 (1995) 321-333 [16] Taylor, D.P. and Pert, C.B., Vasoactive intestinal polypeptide: specific binding to rat brain membranes, Proc. Natl. Acad. Sci., USA, 76 (1979) 660-664. [17] Couvineau, A., Voisin, T., Guijarro, L. and Laburthe, M., Purification of vascactive intestinal peptide receptor from porcine liver by a newly designed one-step affinity chromatography, J. Biol. Chem., 265 (1990) 13386-13390. [18] Robberecht, P., Cauvin, A., Gourlet, P. and Christophe, J., Heterogeneity of VIP receptors, Arch. Int. Pharmacodyn., 303 (1990) 51-66. [19] Gozes, I. and Brenn~man, D.E., VIP: molecular biology and neurobiological function, Mol. Neurobiol., 3 (1989) 201-236. [20] Murase, T., Kondo, K., Otake, K. and Oiso, Y., Pituitary adenylate cyclase-activating polypeptide stimulates arginine vasopressin release in conscious rats, Neuroendocrinology, 57 (1993) 1092-1096. [21] Lundberg, J.M., Fal~trenkrug, J., H6kfelt, T., Martling, C.R., Larrson, O., Tatemc,to, K. and Anggard, A., Coexistence of peptide HI (PHI) and VIP in nerves regulating blood flow and bronchial smooth muscle tone in various mammals including man, Peptidcs, 5 (1984) 593-606. [22] Giachetti, A., Said, S.I., Reynolds, R.C. and Koniges, F.C., Vasoactive intestinal polypeptide in brain: localization in and release from isolated nerve terminals, Proc. Natl. Acad. Sci., USA, 74 (1977) 34714-3428. [23] Somiya, H. and Tonoue, T., Neuropeptides as central integrators of autonomic nerve activity: effects of TRH, SRIF, VIP and bombesin on gastric and adrenal nerves, Regul. Pept., 9 (1984) 47-52.

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[24] H6kfelt, T., Elfvin, L.-G., Schultzberg, M., Fuxe, K., Said, S.I., Mutt, V. and Goldstein, M., Immunohistochemical evidence of vasoactive intestinal peptide-containing neurons and nerve fibers in sympathetic ganglia, Neuroscience, 2 (1977) 885-896. [25] Buijs, R.M., Kalsbeek, A., Van der Woude, T.P., Van Heerikhuize, J.J. and Shinn, S., Suprachiasmatic nucleus lesion increases corticosterone secretion, Am. J. Physiol., 264 (1993) R1186-R1192. [26] Larsson, L.L, Ultrastructural localization of a new neuronal peptide (VIP), Histochemistry, 54 (1977) 173-176. [27] Edwards, A.V. and Jones, C.T., The effect of splanchnic nerve stimulation on adrenocortical activity in conscious calves, J. Physiol., 382 (1987) 385-396. [28] Bonstein, S.R., Ehrhart-Bornstein, M., Scherbaum, W.A., Pfeiffer, E.F. and Hoist, J.J., Effects of splanchnic nerve stimulation on the adrenal cortex may be mediated by chromaffin cells in a paracrine manner, Endocrinology, 127 (1990) 900-906. [29] Engeland, W.C. and Gann, D.S., Splanchnic nerve stimulation modulates steroid secretion in hypophysectomized dogs, Neuroendocrinology, 50 (1989) 124-131. [30] Jasper, M.S. and Engeland, W.C., Splanchnic neural activity modulates ultradian and circadian rhythms in adrenocortical secretion in awake rats, Neuroendocrinology, 59 (1994) 9 7 109.