P. De Neef

VIP activation of rat anterior pituitary adenylate cyclase

The brain gut octacosapeptide VIP (vasoactive intestinal peptide) is found in discrete areas in the cerebral cortex, the hypothalamus and the posterior pituitary gland. Specific VIP binding sites are coupled to an adenylate cyclase system in synaptic membranes from guinea pig brain. Besides, the concentration of VIP in the hypothalamo-hypophysial portal vessels is much higher than in the systemic blood. The peptide has however no established function in the hypophysis. These data document the presence in the rat pituitary of functional VIP receptors existing in the form of a VIP-stimulated adenylate cyclase system and suggest that VIP might be a major peptidic activator of rat adenopituitary membrane adenylate cyclase.

Development of selective agonists and antagonists for the human vasoactive intestinal polypeptide VPAC2 receptor

Ro 25-1553 is a cyclic VIP derivative with a high affinity for the VPAC(2) receptor subtype. Our goal was to identify the modifications that support its selectivity for VPAC(2) receptors, and to develop a VIP or Ro 25-1553 analog behaving as a high affinity, VPAC(2) selective antagonist. The selectivity of Ro 25-1553 for the human receptor was supported mainly by the acetylation of the amino-terminus, by the introduction of a lysine residue in position 12, and by the carboxyl-terminal extension. The lactam bridge created between positions 21 and 25 contributed to the affinity of the compound for the VIP receptors but participated only marginally to its selectivity. Deletion of the first five aminoacid residues led to a low affinity antagonist with a low selectivity. Introduction of a D-Phe residue in position 2 reduced the affinity, the selectivity and the intrinsic activity, the compound being a partial agonist. Myristoylation of the amino-terminus of [K(12)]VIP(1-26) extended carboxyl-terminally with the -K-K-G-G-T sequence of Ro 25-1553 led to a high affinity, selective VPAC(2) receptor antagonist. This molecule represents the first selective human VPAC(2) receptor antagonist described to date.

Characterization of Secretin and Vasoactive Intestinal Peptide Receptors in Rat Pancreatic Plasma Membranes Using the Native Peptides, Secretin-(7–27) and Five Secretin Analogues

A comparison has been made of the ability of vasoactive intestinal peptide (VIP), secretin, secretin analogues, and secretin-(7-27) to stimulate adenylate cyclase in rat pancreatic plasma membranes. A parallel study of the capacity of peptides of the VIP-secretin family to compete with 125I-VIP for binding to the same plasma membranes was conducted. This allowed a classification of VIP-secretin receptors into three subtypes: (1) VIP-preferring receptors; (2) high-affinity secretin receptors, and (3) low-affinity secretin receptors. The properties of secretin at high-affinity secretin receptors were likely to reflect a contribution of membranes from centroacinar and duct cells.

Effects of HIS1 modifications on the ability of vasoactive intestinal peptide to stimulate adenylate cyclase from rat and human tissues

The importance of the N-terminal His residue of VIP for stimulating adenylate cyclase was appreciated by estimating the intrinsic activity and EC50 of four VIP analogues on membranes from rat lung, liver, brain, anterior pituitary, and pancreas, and on human heart membranes. In all tissue preparations tested except one, the order of efficacy (and often potency) was: VIP greater than (Ac-His1)VIP greater than (Phe1)VIP = (3-Me-His1)VIP greater than (D-His1)VIP. In rat heart membranes, the order of efficacy was somewhat different: VIP greater than (Ac-His1)VIP = (Phe1)VIP greater than (D-His1)VIP greater than (3-Me-His1)VIP. These data demonstrated the key role of His1 in VIP in activating adenylate cyclase. They suggest that a given VIP analogue might act as full agonist in tightly coupled adenylate cyclase systems (such as those of rat lung and liver membranes) whereas the same analogue could not promote full activity in poorly coupled systems (such as that present in rat brain synaptic membranes).

Vasoactive Intestinal Peptide (VIP) and peptide having N-terminal histidine and C-terminal isoleucine amide (PHI) stimulate adenylate cyclase activity in human heart membranes

The presence of receptors, recognized by Vasoactive Intestinal Peptide (VIP) and Peptide having N-terminal Histidine and C-terminal Isoleucine amide (PHI), was documented in membranes from human right auricle and left ventricular cardiac muscle by the ability of these peptides to stimulate adenylate cyclase. The capacity of VIP and PHI to activate the enzyme was comparable, in auricle as well as ventricle membranes, the affinity of the system being moderately higher for VIP than for PHI. In auricles, dose-effect curves appeared compatible with the coexistence of high-affinity and low-affinity VIP receptors. PHI could not, however, discriminate these subclasses of VIP receptors.

Expression of pituitary adenylate cyclase activating polypeptide (PACAP) receptors in human glial cell tumors

Twenty-three human gliomas were analyzed: 13 astroglial neoplasms including three grade II, four grade III, and six grade IV tumors; seven ependymomas; and three oligodendrogliomas. A crude membrane fraction was prepared within 30 min after surgical removal of the tumors and was immediately tested for the presence of pituitary adenylate cyclase activating polypeptide (PACAP) receptors. PACAP stimulated adenylate cyclase activity in 23 tumors, but a specific binding of [125I-acetyl-His1]PACAP-27 was detected in only 16 tumors. In all cases, PACAP-27 and -38 were equipotent (Kd or Kact of 1-3 nM) and were 100- to 1000-fold more potent than VIP. PACAP stimulated threefold the adenylate cyclase activity in the presence of GTP. The results were compatible with an interaction of PACAP with a highly selective type I PACAP receptor and not with a high-affinity VIP/PACAP type II receptor. The presence of PACAP receptors on glial neoplasic opens the possibility of a control of the tumor growth by this family of peptides.

A comparison between muscarinic receptor occupancy, adenylate cyclase inhibition, and inotropic response in human heart.

SummaryBinding to muscarinic receptors was compared with adenylate cyclase inhibition in membranes derived from human heart auricles, and with inhibition of the contraction of auricular muscle fibers.In the absence of GTP, agonists recognized two classes of receptors both of which bound antagonists with the same affinity. In the presence of GTP, both classes of receptors for agonists were converted into a single low affinity state.Carbachol and oxotremorine inhibited adenylate cyclase activity by 43%, pilocarpine being less efficient (−28%). The 3 agonists exerted similar inhibitory effects on the inotropic response, in 7 out of 9 preparations of electrically- and norepinephrine-stimulated fibers. Dose-effect curves suggested that spareness (or an amplification mechanism) was implicated in the occupancy of low affinity binding sites by carbachol and oxotremorine (but not by the partial agonist pilocarpine) and the resulting inhibition of both adenylate cyclase activity and contractile force.

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.

Amino acid sequence of VIP, PHI and secretin from the rabbit small intestine

VIP, PHI and secretin were purified from rabbit small intestine throughout a maximum of 6 chromatographic steps. After elution on a reverse phase C18 column, the 3 peptides were separated on a Fractogel column using specific radioimmunoassays for detection. After cation exchange chromatography on Mono S, the final steps were performed using a reverse phase RP8-e column. For these steps, radioreceptor assays were utilized to detect VIP and PHI. We confirmed that the VIP sequence of rabbit was identical to that of porcine VIP. The PHI sequence was also found identical to that of porcine PHI. By contrast, rabbit secretin was highly original, differing from porcine secretin in having Leu, Arg and Leu-NH2 residues instead of Phe, Ser and Val-NH2 in, respectively, position 6, 16 and 27.

Inhibitory effects of quinidine on rat heart muscarinic receptors

Quinidine inhibited binding of the labelled agonist [3H]oxotremorine M [( 3H]Oxo-M) and the labelled antagonist [3H]N-methylscopolamine [( 3H]NMS) to rat heart muscarinic receptors. Kinetic studies demonstrated that quinidine decreased the association rates (I50: 4 and 7.5 microM) and dissociation rates (I50: 100 and 68 microM) of [3H]Oxo-M and [3H]NMS, with different potencies. These cooperative effects explained the low Hill coefficients and apparent selectivity of quinidine competition curves.