Vasoactive intestinal polypeptide and glucagon: stimulation of adenylate cyclase activity via distinct receptors in liver and fat cell membranes

Vasoactive intestinal polypeptide (VIP), a peptide hormone that is chemically and biologically related to glucagon and secretin, stimulates the activity of adenylate cyclase in liver and fat cell membranes. Effects of combinations of VIP with glucagon and secretin at concentrations that maximally activate adenylate cyclase suggest that in adipose tissue, the three hormones act on the same enzyme, whereas in liver, VIP and secretin activate a common enzyme that is distinct from that responding to glucagon. Studies with radioiodinated derivatives of VIP and glucagon indicate that these hormones interact with separate receptors. Secretin, which gives a maximal stimulation of adenylate cyclase activity virtually identical to that elicited by VIP, inhibits the binding of the latter to its receptor. However, the apparent affinity of secretin for adenylate cyclase and for the VIP receptor is about two order of magnitude lower than that of VIP. It is suggested that VIP and secretin may activate adenylate cyclase via a common receptor.     

Do Secretin and Vasoactive Intestinal Peptide Have Independent Receptors on Striatal Neurons and Glial Cells in Primary Cultures?

Vasoactive intestinal peptide (VIP) and secretin are two related peptides that activate adenylate cyclase on membranes of striatal neurons and glial cells from embryonic mouse brain grown in primary culture. On the two cell types, the maximal activation that could be induced by secretin was only 40% above basal activity, which represented less than 15% of the maximal effect obtainable with VIP. From competition experiments performed on glial cells and the neuroblastoma X glioma hybrid, NG 108-15, a cell line known to possess both VIP and secretin sensitive-adenylate cyclase, we demonstrate that secretin does not activate VIP receptors. Furthermore, secretin has an apparent high affinity (EC50 10(-8) M) for its receptors on striatal neurons and NG 108-15 whereas an apparent low affinity (EC50 7 X 10(-6) M) was found on striatal glial cells. This suggests the existence of either two distinct secretin receptors or a desensitized form.     

Presence of vasoactive intestinal peptide receptors coupled to adenylate cyclase in rat lung membranes

(1) The binding of 125I-labelled vasoactive intestinal peptide (VIP) to a particulate fraction from rat lung was rapid, temperature dependent, saturable and specific. This process was also reversible and 125I-labelled VIP dissociation was accelerated by guanine triphosphate nucleotides. The curves describing the inhibition of tracer binding by peptides of the VIP-secretin family suggested the presence of at least two classes of VIP receptor: a "high-affinity' type with decreasing affinity for VIP in the order: VIP = [Val5]secretin greater than [Ala4, Val5]secretin; and a "low-affinity type' with decreasing affinity for VIP in the order: VIP greater than [Val5]secretin greater than [Ala4, Val5]secretin = secretin greater than [Ala4]secretin. (2) VIP and related peptides stimulated the adenylate cyclase activity of the same lung membrane preparation more efficiently than beta-adrenergic agonists and prostaglandins E1 and E2. The dose-effect curves of stimulation of adenylate cyclase by VIP and parent peptides were also compatible with the existence of two classes of VIP receptor, the relative peptide potencies being identical with their ability to compete with 125I-labelled VIP for binding.     

Pharmacology and molecular identification of secretin receptors in rat gastric glands

The structure of the secretin receptor in purified plasma membranes isolated from the antral and fundic parts of the rat gastric mucosa was probed, using the cross linking reagent dithiobis succinimidyl propionate (DSP) and HPLC-purified [125I] secretin. [125I] secretin binding sites were preferentially located in rat antrum and displayed the pharmacological properties expected for specific secretin receptors: secretin greater than helodermin greater than rhGRF greater than rPHI. SDS gel electrophoresis of the solubilized receptor allowed identification of two radiolabeled peptides of 62 and 33 KDa connected by disulfide bonds. According to the sensitivity of the 62 KDa component to low doses of secretin and to GTP, it constitutes the membrane domain involved in the physiological regulation of adenylate cyclase by secretin in rat gastric glands.     

Concentration-dependent inactivation of superoxide dismutase

1. Vasoactive intestinal peptide (VIP) receptors were identified in crude rat hepatic membranes by 125I-labelled VIP binding and by the ability of VIP to stimulate adenylate cyclase activity. The specificity of these receptors was evaluated by the capacity of secretin, synthetic secretin analogues, and secretin fragments to inhibit 125I-labelled VIP binding and to stimulate adenylate cyclase. 2. The results were compatible with the existence of two classes of VIP binding sites that could be distinguished according to their affinity for VIP and their specificity. High-affinity sites were more specific for VIP as secretin was 175 times less potent than VIP for recognition of these sites while being only 33 times less potent than VIP for recognition of low-affinity sites. 3. Secretin analogues, monosubstituted in position 2, 3, 4 or 6 were less potent than secretin for adenylate cyclase stimulation as well as for the recognition of the two classes of receptors. [Val5]secretin was more potent than secretin and appeared definitely more VIP-like than secretin; [Ala4, Val5] and [D-Ala4,Val5]secretin were equipotent to secretin. 4. The fragment secretin (7-27) was unable to recognize VIP receptors and to stimulate adenylate cyclase. The substituted fragment [Gln9,Asn15]secretin (5-27) recognized these receptors with weak potency but could not activate the enzyme.     

Secretin Stimulates Cyclic AMP Formation in the Rat Brain

The effects of secretin on cyclic AMP levels in the rat brain were determined. Incubation of rat brain frontal cortex slices with secretin or the structurally related peptides peptide histidine leucine (PHI) or vasoactive intestinal polypeptide (VIP) in the presence of 10 mM theophylline resulted in a dose-dependent increase in the cyclic AMP levels. The half-maximal increase in cyclic AMP occurred using a 1 microM dose of secretin or a 2 microM dose of PHI or VIP. Preincubation of slices with secretin-(5-27) produced a dose-dependent inhibition of the secretin but not VIP- or PHI-stimulated increase in the cyclic AMP content. Also, in receptor binding studies, secretin-(5-27) produced a dose-dependent inhibition (Ki = 400 nM) of 125I-secretin but not of 125I-VIP binding to rat brain membranes. Guanyl-5'-yl imidodiphosphate decreased the affinity of radiolabelled secretin binding as a result of an increased rate of dissociation of bound 125I-secretin. These data suggest that secretin receptors in the rat brain may be coupled to adenylate cyclase in a stimulatory manner and that secretin-(5-27) may function as a central secretin receptor antagonist.     

Binding of vasoactive intestinal peptide and its stimulation of adenylate cyclase through two classes of receptors in rat liver membranes effects of 12 secretin analogues and 2 secretin fragments

1. Vasoactive intestinal peptide (VIP) receptors were identified in crude rat hepatic membranes by ¹²⁵I-labelled VIP binding and by the ability of VIP to stimulate adenylate cyclase activity. The specificity of these receptors was evaluated by the capacity of secretin, synthetic secretin analogues, and secretin fragments to inhibit ¹²⁵I-labelled VIP binding and to stimulate adenylate cyclase. 2. The results were compatible with the existence of two classes of VIP binding sites that could be distinguised according to their affinity for VIP and their specificity. High-affinity sites were more specific for VIP as secretin was 175 times less potent than VIP for recognition of these sites while being only 33 times less potent than VIP for recognition of low-affinity sites. 3. Secretin analogues, monosubstituted in position 2, 3, 4, or 6 were less potent than secretin for adenylate cyclase stimulation as well as for the recognition of the two classes of receptors. [Val⁵]Secretin was more potent than secretin and appeared definitely more VIP-like than secretin; [Ala⁴, Val⁵]secretin were equipotent to secretin. 4. The fragment secretin (7–27) was unable to recognize VIP receptors and to stimulate adenylate cyclase. The substituted fragment [Gln[⁹,Asn¹⁵]secretin (5–27) recognized these receptors with weak potency but could not activate the enzyme.     

Specific labelling by [125I]helodermin of high-affinity VIP receptors in rat liver membranes

Helodermin, a newly isolated peptide from Gila Monster venom, is structurally related to VIP and secretin. When used as radioligand, [125I]helodermin bound rapidly and reversibly to crude rat liver membranes, the dissociation being accelerated by GTP. Competition binding curves of [125I]helodermin and [125I]VIP with unlabelled peptides showed the following order of decreasing affinity: VIP greater than helodermin greater than secretin greater than hpGRF(1-29)-NH2. The shape of binding curves and of concurrent adenylate cyclase activation is compatible with the specific labelling, by [125I]helodermin, of a class of high-affinity VIP receptors that is capable to stimulate adenylate cyclase.     

Evidence that helodermin, a newly extracted peptide from Gila monster venom, is a member of the secretin/VIP/PHI family of peptides with an original pattern of biological properties

Helodermin, a newly isolated peptide from the venom of Gila monster (Heloderma suspectum) was shown to stimulate the adenylate cyclase activity of rat pancreatic membranes as efficiently as secretin and VIP. It also increased cyclic AMP levels and inhibited [125I]VIP binding in rat pancreatic acini. Finally, helodermin activated adenylate cyclase in membranes from rat heart, rat brain, and human heart, showing properties analogous yet distinct from those of secretin, VIP and PHI.     

The interaction of secretin with pancreatic membranes.

1. 125I-labelled secretin bound rapidly and specifically to membranes from cat pancreas. Binding of labelled hormone was competitively inhibited by unlabelled secretin in the same range of concentrations that stimulated pancreatic adenylate cyclase in these membranes. The dissociation constant of the membrane binding sites for unlabelled secretin as evaluated by these displacement experiments was 4.1-10(-9) M and the number of binding sites 1.0 pmol per mg of membrane protein. 2. Studies using different concentrations of [125I]secretin (at a constant ratio of labelled to unlabelled hormone) revealed a similar value of 4-4-10(-9) M for the dissociation constant. 3. Both the association and dissociation rate constants of [125I]secretin binding were temperature sensitive; the dissociation rate constant increased more rapidly with increase in temperature. The ratio k-1/k+1 (at 22 degrees C) gave a dissociation constant of 3.7-10(-9)M which agrees closely with the figure obtained from equilibrium data. These data indicate that 125I-labelled secretin and unlabelled secretin bind to the same binding site on pancreatic membranes, with high affinity. 4. Unlabelled secretin stimulated pancreatic adenylate cyclase with an apparent Km of 8.4-10(-9) M, while [125I]secretin apparently did not stimulate the adenylate cyclase. Together with the binding data this might suggest that different portions of the secretin molecule are responsible for binding and adenylate cyclase activation. 5. Studies on the specificity of [125I]secretin binding carried out with various peptide hormones (glucagon, human gastrin, pancreozymin and caerulein) which are all inefficient in stimulating pancreatic fluid secretin, showed that these hormones have no influence on the binding of [125I]secretin. In contrast, vasoactive intestinal polypeptide, which stimulates pancreatic fluid and bicarbonate secretion, showed a competitive inhibition of secretin binding to the plasma membrane preparation.     

The role of phospholipids in TSH stimulation of adenylate cyclase in thyroid plasma membranes

Treatment of bovine thyroid plasma membranes with phospholipase A or C inhibited the stimulation of adenylate cyclase activity by thyroid-stimulating hormone (TSH). In general, basal and NaF-stimulated adenylate cyclase activity was not influenced by such treatment. When plasma membranes were incubated with 1–2 units/ml phospholipase A, subsequent addition of phosphatidylcholine or phosphatidylserine but not phosphatidylethanolamine partially restored TSH stimulation. Phosphatidylcholine was more effective than phosphatidylserine in that it caused greater restoration of the TSH response and smaller amounts of phosphatidylcholine were active. However, when the TSH effect was obliterated by treatment of plasma membranes with 10 units/ml phospholipase A, phospholipids were unable to restore any response to TSH. Lubrol PX, a nonionic detergent, inhibited basal, TSH- and NaF-stimulated adenylate cyclase activities in thyroid plasma membranes. Although phosphatidylcholine partially restored TSH stimulation of adenylate cyclase activity in the presence of Lubrol PX, it did not have a similar effect on the stimulation induced by NaF. These results indicate that phospholipids are probably essential components in the system by which TSH stimulates adenylate cyclase activity in thyroid plasma membranes. The effects do not seem to involve the catalytic activity of adenylate cyclase but the data do not permit a distinction between decreased binding of TSH to its receptor or impairment of the signal from the bound hormone to the enzyme activity.     

Pharmacological characterization of the novel helodermin/VIP receptor present in human SUP-T1 lymphoma cell membranes

[Acetyl-His1]VIP stimulated adenylate cyclase with higher potency than VIP in membranes from human SUP-T1 lymphoblasts and was used as an efficient radioiodinated ligand with low non-specific binding to evaluate the relationship between receptor occupancy and adenylate cyclase activation and the possible interference of peptide T (an epitope derived from HIV envelope protein gp120). Various peptides inhibited [125I-acetyl-His1]VIP binding and activated the enzyme, their order of potency being: helodermin greater than [acetyl-His1]VIP greater than VIP = PHI = [Phe1]VIP greater than [D-Phe2]VIP = [D-Ala4]VIP = [D-Phe4]PHI greater than or equal to [D-Phe4]VIP greater than [D-His1]VIP giving further support for the existence of a novel subtype of helodermin/VIP receptors. [D-Ala1]peptide T and VIP-(10-28) did not recognize the binding site and did not inhibit, even at high concentration, VIP - or VIP analogue - stimulated adenylate cyclase activities.     

Identification of high affinity receptors for vasoactive intestinal peptide on human lymphocytes of B cell lineage

Specific, high affinity receptors for vasoactive intestinal peptide (VIP) have been identified on a human pre-B cell line, Nalm 6, and on a human plasma cell line, Dakiki. The single class of high affinity sites exhibited a KD of 12.6 +/- 2.9 nM for VIP in Nalm 6 cells and 9.1 +/- 2.7 nM in Dakiki plasma cells. The homologous peptides, peptide histidine methionine (PHM), growth hormone releasing factor (GHRF), and secretin were all less effective than VIP in competitively inhibiting binding of 125I-VIP to Nalm 6 and Dakiki plasma membranes. The putative receptor was characterized as a 47-kDa protein using covalent cross-linking techniques and VIP stimulated adenylate cyclase in pre-B cells. Human lymphocytes of B cell lineage thus appear to express functional VIP receptors homologous to the receptor identified in T lymphoblasts, brain, pituitary, and intestine.     

Pharmacological characterization of VIP receptors in human lung membranes

The ability of VIP, PHI, secretin, helodermin, and seven N-terminally D-amino monosubstituted VIP and PHI analogs to occupy (125I)iodo-VIP labeled receptors and to activate adenylate cyclase was tested on human lung membranes purified by the method of Schachter et al. Best fitted Kd, Kact and % of max. values suggested the coexistence, in near equal proportions, of two classes of VIP-preferring binding sites coupled to adenylate cyclase that showed similar decreasing affinity for: VIP greater than (D-Ala4)-VIP greater than (D-Asp3)-VIP = (D-Ser2)-VIP greater than (D-His1)-VIP greater than PHI greater than (D-Phe2)-VIP greater than (D-Phe4)-VIP. (D-Arg2)-VIP was a non-selective agonist. A third receptor type, coupled to adenylate cyclase and showing high affinity for secretin and helodermin but not for VIP, was also detected.     

Pharmacology and molecular identification of vasoactive intestinal peptide (VIP) receptors in normal and cancerous gastric mucosa in man

In human antral membranes, VIP and its natural analogs inhibited the binding of HPLC-purified 125I-VIP, according to the following order of potency: VIP greater than rh GRF greater than helodermin greater than r PHI greater than PHM greater than p PHI greater than hp GRF greater than h, p secretin. No specific binding was detected in plasma membranes purified from the human fundus. In human antral membranes, Scatchard plots were compatible with the existence of two classes of VIP receptors, the first class with high affinity and low binding capacity (Kd = 0.1 nM, Bmax = 10 fmol/mg protein) and another class with a low affinity and higher binding capacity (Kd = 12) nM, Bmax = 1,000 fmol/mg protein). The structure of the VIP receptor in purified plasma membranes prepared from human antral glands and from the HGT-1 human gastric cancer cells was subsequently probed using the cross-linking reagent DSP and 125I-VIP. In agreement with the pharmacological study and the Scatchard analysis of the binding data, SDS gel electrophoresis of the solubilized receptor identified two radiolabeled peptides Mr 67,000 and 34,000 containing disulfide bonds. According to its sensitivity to low doses of VIP and to GTP, the Mr 67,000 binding site represents the membrane domains involved in the physiologial regulation of adenylate cyclase by VIP in normal and transformed human gastric epithelia.     

Vasoactive intestinal peptide: autocrine growth factor in neuroblastoma.

Neuroblastoma is the most common solid tumor of children less than 5 years of age; yet the biology of this tumor is poorly understood. Neuroblastoma tumors are derived from neural crest precursors; they synthesize both adrenergic and peptidergic neurotransmitters. This study determined VIP receptor expression in primary neuroblastoma tumors prior to chemotherapy. The VIP receptor was expressed in 12 of 15 neuroblastoma tumors as determined by direct binding studies (KD = 1.3-12.4 nM) and VIP-mediated stimulation of adenylate cyclase. The VIP stimulation index for adenylate cyclase in the primary tumor was inversely correlated with the VIP content of the tumor, suggesting that VIP regulates its own receptor expression. Similar observations were made in vitro by comparison of two human neuroblastoma cell lines, IMR32 and SKNSH. Both cell lines were demonstrated to express specific, high affinity VIP receptors (KD = 4 nM and 2.5 nM for IMR32 and SKNSH, respectively). IMR32 cells contained very low levels of VIP (0.6 pg VIP/10(6) cells). Exogenous VIP stimulated adenylate cyclase 22-fold over basal activity and VIP inhibited proliferation of IMR32 cells by 49% in 6-day cultures. On the other hand, SKNSH cells synthesized high levels of VIP (6.3 pg/10(6) cells), metabolized VIP rapidly and demonstrated a low level of VIP-mediated stimulation of adenylate cyclase; their proliferation rate was minimally inhibited by exogenous VIP. These observations help validate the hypothesis that VIP serves as an autocrine growth factor in neuroblastoma.     

Effects of PHI on vasoactive intestinal peptide receptors and adenylate cyclase activity in lung membranes. A comparison in man, rat, mouse and guinea pig

The presence of receptors, recognized by vasoactive intestinal peptide (VIP) as well as by PHI (a peptide with N-terminal histidine and C-terminal isoleucine amide), was documented in lung membranes from rat, mouse, guinea pig and man by the ability of these receptors, once occupied, to stimulate adenylate cyclase. In lung membranes from rat, mouse and guinea pig, the capacity of VIP, PHI and secretin to stimulate the enzyme and the potency of the same peptides to compete with 125I-VIP for binding to VIP receptors were similar, the affinity decreasing in the order: VIP greater than PHI greater than secretin. In addition, dose-effect curves were compatible with the coexistence of high-affinity and low-affinity VIP receptors, in the four animal species considered. If PHI was able to recognize all VIP receptors it could not, however, discriminate the subclasses of VIP receptors.     

Inhibition of neuroblastoma adenylate cyclase by cannabinoid and nantradol compounds

This study was undertaken to ascertain the effects of cannabinoid drugs on prostanoid-stimulated adenylate cyclase in neuroblastoma cells. This report demonstrates that Δ⁹-tetrahydrocannabinol (THC) and levonantradol could decrease initial rate cyclic AMP accumulation in response to prostacyclin in intact cells. Basal accumulation was also diminished. Prostanoid-stimulated adenylate cyclase in a membrane preparation from these cells was inhibited by cannabinoid and nantradol compounds. However, this inhibition was not competitive with prostaglandin E₁ or prostacyclin. Further, inhibition was also observed when the enzyme was stimulated by peptide hormones at the secretin receptor. In contrast, enzyme activated by NaF was not inhibited by cannabinoid compounds. Cyclic AMP phosphodiesterase activity in subcellular fractions was unaltered by these agents. These data demonstrate that cannabinoid and nantradol compounds decrease cyclic AMP accumulation in neuronally derived cells, and that this results from an inhibition of basal and hormone-stimulated adenylate cyclase activity.     

Adenylate cyclase stimulation by VIP in rat and human parotid membranes

Adenylate cyclase activity was stimulated by vasoactive intestinal peptide (VIP) in rat parotid membranes, in the presence of 100 microM guanosine triphosphate (GTP). The threshold concentration of VIP was 300 nM and the activity doubled at the maximal VIP concentration tested (30 microM). The relative potency of peptides of the VIP family was: VIP greater than peptide histidine isoleucinamide (PHI) greater than secretin. The beta-adrenergic agent isoproterenol was a more efficient activator of rat parotid adenylate cyclase and its stimulatory effect, like that of VIP, depended on the presence of GTP. The effects of VIP and isoproterenol were both potentiated by 10 microM forskolin. By comparison with rat parotid preparations, membranes from a human parotid gland responded similarly to the VIP family of peptides (VIP greater than PHI greater than secretin). In both rat and human parotid membranes, two proteins (Mr 44 kDa and 53 kDa) of the alpha-subunit of Ns (the guanyl nucleotide-binding stimulatory protein) were labelled by ADP-ribosylation, in the presence of cholera toxin. Taken together, these results indicate that VIP receptors, when coupled to Ns, were able to activate the adenylate cyclase system in rat and human parotid membranes.     

Importance of disulfide bonds in receptors for vasoactive intestinal peptide and secretin in rat pancreatic plasma membranes

(1) Vasoactive intestinal peptide (VIP), secretin, and C-terminal octapeptide of cholecystokinin (CCK-8) receptors were identified in rat pancreatic plasma membranes by the ability of these peptides to stimulate adenylate cyclase activity. The membrane preparation procedure was conducted through a series of steps including discontinuous sucrose density gradient fractionation. 5 mM β-mercaptoethanol was added stepwise. Membrane preparations obtained stepwise were preincubated for 10 min at 25°C in the presence of various concentrations of β-mercaptoethanol or dithiothreitol before assaying adenylate cyclase. The use of the reducing agents exerted no effect on p[NH]ppG-, NaF-, and CCK-8- stimulated activities. By contrast, stimulation of adenylate cyclase by low VIP concentrations was specifically altered when β-mercaptoethanol was used during tissue homogeneization at 5°C. (2) In addition, both VIP and secretin responses were highly sensitive towards a preincubation of 10 min at 25°C in the presence of dithiothreitol. (3) These results were likely to reflect alterations at the receptor level. ¹²⁵I-VIP binding was, indeed, reduced after dithiothreitol preincubation, low concentrations of the thiol reagent decreasing the apparent number of high-affinity VIP receptors and higher dithiothreitol concentrations reducing the affinity of VIP receptors.