Structural Requirements of Maxadilan,a Non-Mammalian Potent Vasodilatory Peptide,for Interaction with the PAC-1 Receptor from Rat Brain Using a Synthetic Mini-Library

Although there is no amino acid sequence similarity between maxadilan (Maxa) and pituitary adenylate cyclase activating polypeptide (PACAP), our synthetic Maxa was found to bind PACAP specific receptors (PAC-1 receptors) with a high affinity, but low potency for the accumulation of cAMP in PC12 cells. Competitive binding studies of ¹²⁵I-PACAP-27 to rat cortical membranes allowed exploration of the structural requirements for this interaction using mini-libraries constructed by solid-phase peptided synthesis, that include disulfide isomers, N-, C- and middle segment deleted peptides and analogs. Maxa as well as PACAP38 inhibited the specific binding of ¹²⁵I-PACAP-27 with IC₅₀ values of 3.89 and 4.90 nM, respectively. The most potent derivative of our synthetic Maxa-analogs with an IC₅₀ value of 1.99 nM was Maxa[1–23 + 43–61, S–S^14–51 Ala^1,5] which consists of N- (position 1–23) and C- (position 43-61) terminal linear fragments cross-linked by a disulfide bridge between positions 14 and 51. This peptide did not increase intracellular cAMP, at a concentration of 100 nM, but inhibited cAMP accumulation induced by 1 nM PACAP-27 in PC12 cells, whereas wild Maxa increased intracellular cAMP although it was weaker than PACAP-27. Our data suggest deletion of the middle segment between residues 24–42 affords some derivatives that behave as low affinity antagonists. This paper is dedicated to the memory of Professor Bruce Merrifield, a pioneer and one of the most important contributors to solid-phase synthesis.     

Receptors for the vasodilator maxadilan are expressed on selected neural crest and smooth muscle-derived cells

Maxadilan is a potent vasodilator peptide isolated from salivary glands of the blood feeding sand fly Lutzomyia longipalpis. The peptide relaxes rabbit aortic rings in an endothelium independent manner while elevating levels of cAMP and has been found to bind to membrane homogenates from brain. These studies on tissues have now been expanded with an examination of binding and signaling of maxadilan to a number of established cell lines and primary cultures. The data reveal that maxadilan binds to and stimulates the accumulation of cAMP in the rat pheochromocytoma line PC12 and the human neuroblastoma line NBfl. Accumulation of cAMP occurred in a transformed mouse pancreatic smooth muscle line (MILE) and primary rabbit aorta smooth muscle cells. The peptide did not bind to or induce cAMP formation in the rat thoracic aorta line L6. Scatchard analysis of binding to the PC12 and NBfl lines indicates that maxadilan binds to a single class of high-affinity receptors. Similar pharmacologic actions and possible structural homologies between maxadilan and calcitonin generelated peptide (CGRP) suggested the possibility that they shared receptors. However, competition studies and comparative second messenger analysis reveal that maxadilan does not interact with receptors for CGRP, amylin or adrenomedullin and suggest that this peptide may bind to a novel receptor whose endogenous ligand remains unknown.     

重组Maxadilan的制备与鉴定

为利用基因工程技术获得重组Maxadilan(RMMAX), 根据Maxadilan的氨基酸序列, 设计并人工合成了在原核表达的基因。克隆到表达载体pKYB, 重组质粒pKYB-MAX转化表达宿主菌Escherichia coli strain ER2566, 构建表达工程菌。用诱导剂IPTG诱导由目的多肽、内含肽和几丁质结合域(Chitin binding domain, CBD)组成的“三元”融合蛋白表达; 用几丁质珠亲和层析纯化了裂解液中的融合蛋白, 用b-巯基乙醇切割融合蛋白, 获得目的蛋白。所得的多肽经激光飞行质谱测定分子量结果与理论值相符, 生物活性分析表明, 重组Maxadilan有显著的提升血糖的作用。     

Maxadilan Activates PAC1 Receptors Expressed in Xenopus laevis Melanophores

Functional interactions between ligands and their cognate receptors can be investigated using the ability of melanophores from Xenopus laevis to disperse or aggregate their pigment granules in response to alterations in the intracellular levels of second messengers. We have examined the response of long‐term lines of cultured melanophores from X. laevis to pituitary adenylate cyclase activating peptide (PACAP), a neuropeptide with vasodilatory activity, and maxadilan, a vasodilatory peptide present in the salivary gland extracts of the blood feeding sand fly. Pituitary adenylate cyclase activating peptide increased the intracellular levels of cyclic adenosine monophosphate (cAMP) and induced pigment dispersion in the pigment cells, confirming that melanophores express an endogenous PACAP receptor. Maxadilan did not induce a response in non‐transfected melanophores. When the melanophores were transfected with complementary DNA (cDNA) from the three different members of the PACAP receptor family, maxadilan induced pigment dispersion specifically and cAMP accumulation in melanophores transfected with the cDNA for PAC1 receptors but not VPAC1 or VPAC2 receptors. A melanophore line was generated that stably expresses the PAC1 receptor.     

Long-term administration of maxadilan improves glucose tolerance and insulin sensitivity in mice

Maxadilan and its truncated variant, M65, are agonist and antagonist specific, respectively, for the PAC1 receptor. PAC1 is the specific receptor for the pituitary adenylate cyclase-activating peptide (PACAP), which is not shared by vasoactive intestinal peptide (VIP). PACAP is a ubiquitous peptide of the glucagon superfamily that is involved in glucose homeostasis and regulation of insulin secretion. This study employed the recombinant maxadilan and M65 to evaluate the PAC1 receptor-mediated effects on energy metabolism using NIH mice. First, the acute effect of maxadilan-induced hyperglycemia was blocked by M65. In long-term studies, NIH mice were given daily intraperitoneal injections with maxadilan, M65, or vehicle for 21 days. Maxadilan suppressed feeding and enhanced water intake significantly for the first several days. After that period, maxadilan treatment continued to promote food and water intake. Long-term administration of maxadilan led to an increase in body weight (P<0.01), decrease in body fat (P<0.01), down-regulation of basal plasma glucose (P<0.01), upregulation of basal plasma insulin (P<0.01) and improved glucose tolerance (P<0.01) and insulin sensitivity (P<0.01). An elevation in plasma LDL (P<0.01) was also observed in the maxadilan group. However, M65 displayed no significant adverse effects on the aforementioned parameters except basal plasma glucose (P<0.05). The significant changes induced by maxadilan indicate that the PAC1 receptor plays multiple key roles in carbohydrate metabolism, lipid metabolism and energy homeostasis in mice.