Vasoactive intestinal peptide receptor regulation of cAMP accumulation and glycogen hydrolysis in the human Ewing's sarcoma cell line WE-68

Abstract

This study describes functional characteristics of receptors for vasoactive intestinal peptide (VIP) on human Ewing's sarcoma WE-68 cells. These characteristics include 125I-VIP binding capacity cellular cAMP generation, glycogen hydrolysis, and pharmacological specificity. Binding studies with 125I-VIP showed specific, saturable, binding sites for VIP in WE-68 cells. Scatchard analysis revealed the presence of a single class of high-affinity binding sites that exhibited a dissociation constant ( K d ) of 90 pM and a maximal binding capacity ( B max) of 24 fmol/mg of protein. VIP and VIP-related peptides competed for 125I-VIP binding in the following order of potency: human (h) VIP > human peptide with N-terminal histidine and C-terminal methionine (PHM) > chicken secretion > > porcine secretin. Glucagon and the C-terminal fragments VIP[10-28] and VIP[16-28] and the VIP analogue ( d-Phe 2)VIP did not inhibit 125I-VIP binding. Addition of hVIP to WE-68 cells provoked marked stimulation of cAMP accumulation. h VIP stimulated increases in cAMP content were rapid, concentration-dependent, and potentiated by 3-isobutyl-1-methylxanthine (IBMX). Half-maximal stimulation ( ec 50) occured at 150 nM hVIP. The ability of hVIP and analogues to stimulate cAMP generation paralleled their potencies in displacing 125I-VIP binding. ( d-Phe 2)VIP, VIP[10-28], VIP[16-28], and ( p- Cl- d - Phe 6 , Leu 17)VIP, a putative receptor antagonist, affetected neither basal cAMP levels nor hVIP-induced cAMP accumulation. WE-68 cell responses to hVIP were desentisized by prior exposure to hVIP. Desensitization to hVIP did not modify the cAMP response to β-adrenergic stimulation, and β-adrenergic agonist desensitization did not modify responses to hVIP. hVIP also induced a time-a and concentration-dependent hydrolysis of 3H-glycogen newly formed from 3H-glucose in WE-68 cultures. hVIP maximally decreased 3H-glycogen content by 36% with an ec 50 value of about 8 nM. The order of potency of structurally related peptides of hVIP for stimulation of glycogenolysis correlated with their order of potency for inhibition of 125I-VIP binding. IBMX potentiated the glycogenolytic action of hVIP and PHM. The simultaneous presence of the calcium channel antagonist verapamil or the calcium ionophore A 23187 did not influence the glycogenolytic and cAMP stimulatory effects of hVIP. Collectively, these data indicate that Ewing's sarcoma (WE-68) cells are endowed with genuine VIP receptors which are coupled to the formation of cAMP that probabaly serves a second messenger role in stimulating glycogen hydrolysis in these cells in response to VIP. The data also indicate that (a) the entire sequence of VIP is necessary for full potency, and (b) the N-terminal portion is crucial for the binding to receptors and the biological activity of VIP.