Regulatory Peptides, 37 (1992) 195-203
195
© 1992 Elsevier Science Publishers B.V. All rights reserved. 0167-0115/92/$05.00 REGPEP 01136
Stimulatory effect of vasoactive intestinal peptide (VIP) on cyclic AMP production in rat peritoneal macrophages J . J . Segura, J . M . Guerrero, R. G o b e r n a and J . R . Calvo Departamento de Bioquimica Mddica y Biologia Molecular, Facultad de Medicina, Universidad de Sevilla, Sevilla (Spain)
(Received 10 June 1991; revisedversion received26 September 1991; accepted 22 October 199I) K e y words." VIP; Rat peritoneal macrophage; Cyclic AMP; Neuropeptide;
Neuroimmunoregulation
Summary Vasoactive intestinal peptide (VIP) stimulated cyclic AMP production in rat peritoneal macrophages. The stimulatory effect of VIP was dependent on time, temperature and cell concentration, and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX). At 15°C, the response occurred in the 0.1-1000 nM range of VIP concentrations. Half maximal stimulation of cellular cyclic AMP (EDso) was obtained at 1.2 + 0.5 nM VIP, and maximal stimulation (about 3-fold basal level) was obtained between 100-1000 nM. The cyclic AMP system of rat peritoneal macrophages showed a high specificity for VIP. The order of potency observed in inducing cyclic AMP production was VIP > rGRF > h G R F > PHI > secretin. Glucagon, insulin, pancreastatin and octapeptide of cholecystokinin did not modify cyclic AMP levels at concentrations as high as 1/~M. The fl-adrenergic agonist isoproterenol increased the cyclic AMP production and show additive effect with VIP. Somatostatin inhibits the accumulation of cyclic AMP in the presence of both vasoactive intestinal peptide and isoproterenol. The finding of a VIP-stimulated cyclic AMP system in rat peritoneal macrophages, together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, strongly suggest that VIP may be involved in the regulation of macrophage function.
Correspondence: J.R. Calvo, Departamento de Bioquimica Mrdica y BiologiaMolecular, Facultad de Medicina, Universidad de Sevilla, Avda. Sanchez Pizjuan, 4. 41009-Sevilla, Spain.
196 Introduction
Numerous reports have suggested an interaction between the neuroendocrine and immune systems [1-3] and, particularly, several studies have implicated vasoactive intestinal peptide (VIP) as an immunoregulatory peptide [4-6]. Specific receptors for VIP have been demonstrated on different types of lymphoid cells in several species and in human monocytes [7-12]. In this context, we recently characterized specific and functional receptors for VIP in rat peritoneal macrophages [ 13]. It is now clearly established that the biological activity of VIP is mediated by stimulation of plasma membrane adenylate cyclase of target cells. VIP is a potent activator of cyclic AMP production system in different cells [ 14-17]. The increased cyclic AMP level subsequently triggers the activation of a cyclic AMP-dependent protein-kinase [ 18,19]. In the present paper, we have investigated the capacity of VIP to stimulate cyclic AMP production in rat peritoneal macrophages as another prerequisite to assess a physiological role of this neuropeptide in the regulation of macrophage functions.
Materials and Methods
Chemicals Synthetic rat VIP, porcine peptide histidine isoleucine amide (PHI), rat and human growth hormone releasing factors (rGRF and hGRF, respectively), secretin, octapeptide of cholecystokinin (CCK 26-38), porcine pancreastatin and somatostatin, were purchased from Peninsula Laboratories Europe (Merseyside, U.K.); porcine glucagon and porcine monocomponent insulin from Novo (Copenhagen, Denmark); bacitracin, bovine serum albumin (fraction V), 3-isobutyl-1-methylxanthine (IBMX), propranolol and isoproterenol from Sigma (St. Louis, MO, U.S.A.); cyclic AMP assay kit was obtained from Radiochemical Center (Amersham, U.K.). All other chemicals were reagent grade.
Preparation of peritoneal macrophages Peritoneal macrophages were isolated from Wistar rats by the method of Tsunawaky and Nathan [20]. As previously descrived [ 13 ], approx. 959/0 cells were viable by trypan blue exclusion.
Cyclic AMP production Cyclic AMP production was determined with minor modifications as previously [ 15]. In a standard assay, rat peritoneal macrophages (106 cells/ml) were incubated in 0.5 ml of 35 mM Tris-HC1 buffer (pH 7.5) containing 50 mM NaCI, 1.4~o (w/v) bovine serum albumin, 1 mg/ml bacitracin, 0.2 mM 3-isobutyl-l-methylxanthine (IBMX) in the absence or presence of increasing concentrations of unlabelled VIP (0.01-1000 nM). After 45 min incubation at 15 °C, reaction was stopped by the addition of 2.5 ml methanol. The precipitate was removed by centrifugation, aliquots of the supernatant were evaporated and cyclic AMP was measured by a kit cyclic AMP assay system.
197
Statistical analysis Each experiment was carried out at least three times. All experiments were performed in triplicate. Statistical significance was determined by Student's unpaired t-test. A value of P < 0.05 (one-tailed) was considered statistically significant.
Results
Effect of time, temperature, phosphodiesterase inhibitor, cell concentration and pH on cyclic AMP production stimulated by vasoactive intestinal peptide The stimulation of cyclic A M P production by VIP was observed under all conditions tested. In the presence of 0.2 m M 3-isobutyl-l-methylxanthine as a phosphodiesterase inhibitor, VIP-stimulated cyclic A M P production was dependent on time and temperature (Fig. 1). At 30 °C, a maximally effective dose of VIP (100 nM) elicited a rapid increase of cyclic A M P levels that became maximal at 30 min (about 3-fold increase above basal) and decreased thereafter. Reducing the incubation temperature from 30 to 15 °C caused a small decrease in both rate and extent of cyclic A M P production but an apparent steady state could be observed at about 30-45 min and remained constant until 120 min. Maximum increase above cyclic A M P basal levels was induced by VIP in the presence of I B M X 0.2 mM. Addition of phosphodiesterase inhibitor at higher concentrations did not result in further increase of the stimulatory effect of VIP on cyclic A M P production (Fig. 2). However, in the absence of I B M X the increase above cyclic A M P basal levels produced by VIP was reduced by about 90%. A 45 min incubation at 15 °C
6
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I 90
I 120
5 o
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3
a.
u ._ I
u
1 0
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I 30
I 60 Time (min)
Fig. 1. Time-courseof VIP-induced cyclicAMP accumulation by rat peritoneal macrophages as a function of temperature. Cells (106/ml) were incubated in the presence of 0.2 mM 3-isobutyl-l-methylxanthinewith (open symbols)or without (closedsymbols)10- 7 M VIP at 15 °C (r"l, l ) or 30 °C (O, O), and the reaction was stopped at the indicated time. Each point is the mean of triplicates. Two other separate experiments gave similar results.
198
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195
© 1992 Elsevier Science Publishers B.V. All rights reserved. 0167-0115/92/$05.00 REGPEP 01136
Stimulatory effect of vasoactive intestinal peptide (VIP) on cyclic AMP production in rat peritoneal macrophages J . J . Segura, J . M . Guerrero, R. G o b e r n a and J . R . Calvo Departamento de Bioquimica Mddica y Biologia Molecular, Facultad de Medicina, Universidad de Sevilla, Sevilla (Spain)
(Received 10 June 1991; revisedversion received26 September 1991; accepted 22 October 199I) K e y words." VIP; Rat peritoneal macrophage; Cyclic AMP; Neuropeptide;
Neuroimmunoregulation
Summary Vasoactive intestinal peptide (VIP) stimulated cyclic AMP production in rat peritoneal macrophages. The stimulatory effect of VIP was dependent on time, temperature and cell concentration, and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX). At 15°C, the response occurred in the 0.1-1000 nM range of VIP concentrations. Half maximal stimulation of cellular cyclic AMP (EDso) was obtained at 1.2 + 0.5 nM VIP, and maximal stimulation (about 3-fold basal level) was obtained between 100-1000 nM. The cyclic AMP system of rat peritoneal macrophages showed a high specificity for VIP. The order of potency observed in inducing cyclic AMP production was VIP > rGRF > h G R F > PHI > secretin. Glucagon, insulin, pancreastatin and octapeptide of cholecystokinin did not modify cyclic AMP levels at concentrations as high as 1/~M. The fl-adrenergic agonist isoproterenol increased the cyclic AMP production and show additive effect with VIP. Somatostatin inhibits the accumulation of cyclic AMP in the presence of both vasoactive intestinal peptide and isoproterenol. The finding of a VIP-stimulated cyclic AMP system in rat peritoneal macrophages, together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, strongly suggest that VIP may be involved in the regulation of macrophage function.
Correspondence: J.R. Calvo, Departamento de Bioquimica Mrdica y BiologiaMolecular, Facultad de Medicina, Universidad de Sevilla, Avda. Sanchez Pizjuan, 4. 41009-Sevilla, Spain.
196 Introduction
Numerous reports have suggested an interaction between the neuroendocrine and immune systems [1-3] and, particularly, several studies have implicated vasoactive intestinal peptide (VIP) as an immunoregulatory peptide [4-6]. Specific receptors for VIP have been demonstrated on different types of lymphoid cells in several species and in human monocytes [7-12]. In this context, we recently characterized specific and functional receptors for VIP in rat peritoneal macrophages [ 13]. It is now clearly established that the biological activity of VIP is mediated by stimulation of plasma membrane adenylate cyclase of target cells. VIP is a potent activator of cyclic AMP production system in different cells [ 14-17]. The increased cyclic AMP level subsequently triggers the activation of a cyclic AMP-dependent protein-kinase [ 18,19]. In the present paper, we have investigated the capacity of VIP to stimulate cyclic AMP production in rat peritoneal macrophages as another prerequisite to assess a physiological role of this neuropeptide in the regulation of macrophage functions.
Materials and Methods
Chemicals Synthetic rat VIP, porcine peptide histidine isoleucine amide (PHI), rat and human growth hormone releasing factors (rGRF and hGRF, respectively), secretin, octapeptide of cholecystokinin (CCK 26-38), porcine pancreastatin and somatostatin, were purchased from Peninsula Laboratories Europe (Merseyside, U.K.); porcine glucagon and porcine monocomponent insulin from Novo (Copenhagen, Denmark); bacitracin, bovine serum albumin (fraction V), 3-isobutyl-1-methylxanthine (IBMX), propranolol and isoproterenol from Sigma (St. Louis, MO, U.S.A.); cyclic AMP assay kit was obtained from Radiochemical Center (Amersham, U.K.). All other chemicals were reagent grade.
Preparation of peritoneal macrophages Peritoneal macrophages were isolated from Wistar rats by the method of Tsunawaky and Nathan [20]. As previously descrived [ 13 ], approx. 959/0 cells were viable by trypan blue exclusion.
Cyclic AMP production Cyclic AMP production was determined with minor modifications as previously [ 15]. In a standard assay, rat peritoneal macrophages (106 cells/ml) were incubated in 0.5 ml of 35 mM Tris-HC1 buffer (pH 7.5) containing 50 mM NaCI, 1.4~o (w/v) bovine serum albumin, 1 mg/ml bacitracin, 0.2 mM 3-isobutyl-l-methylxanthine (IBMX) in the absence or presence of increasing concentrations of unlabelled VIP (0.01-1000 nM). After 45 min incubation at 15 °C, reaction was stopped by the addition of 2.5 ml methanol. The precipitate was removed by centrifugation, aliquots of the supernatant were evaporated and cyclic AMP was measured by a kit cyclic AMP assay system.
197
Statistical analysis Each experiment was carried out at least three times. All experiments were performed in triplicate. Statistical significance was determined by Student's unpaired t-test. A value of P < 0.05 (one-tailed) was considered statistically significant.
Results
Effect of time, temperature, phosphodiesterase inhibitor, cell concentration and pH on cyclic AMP production stimulated by vasoactive intestinal peptide The stimulation of cyclic A M P production by VIP was observed under all conditions tested. In the presence of 0.2 m M 3-isobutyl-l-methylxanthine as a phosphodiesterase inhibitor, VIP-stimulated cyclic A M P production was dependent on time and temperature (Fig. 1). At 30 °C, a maximally effective dose of VIP (100 nM) elicited a rapid increase of cyclic A M P levels that became maximal at 30 min (about 3-fold increase above basal) and decreased thereafter. Reducing the incubation temperature from 30 to 15 °C caused a small decrease in both rate and extent of cyclic A M P production but an apparent steady state could be observed at about 30-45 min and remained constant until 120 min. Maximum increase above cyclic A M P basal levels was induced by VIP in the presence of I B M X 0.2 mM. Addition of phosphodiesterase inhibitor at higher concentrations did not result in further increase of the stimulatory effect of VIP on cyclic A M P production (Fig. 2). However, in the absence of I B M X the increase above cyclic A M P basal levels produced by VIP was reduced by about 90%. A 45 min incubation at 15 °C
6
u
O
o
0
I 90
I 120
5 o
-6
4
E Q. ..~
3
a.
u ._ I
u
1 0
0
I 30
I 60 Time (min)
Fig. 1. Time-courseof VIP-induced cyclicAMP accumulation by rat peritoneal macrophages as a function of temperature. Cells (106/ml) were incubated in the presence of 0.2 mM 3-isobutyl-l-methylxanthinewith (open symbols)or without (closedsymbols)10- 7 M VIP at 15 °C (r"l, l ) or 30 °C (O, O), and the reaction was stopped at the indicated time. Each point is the mean of triplicates. Two other separate experiments gave similar results.
198
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