Receptor-mediated adenylate cyclase-coupled mechanism for PGE₂ inhibition of insulin secretion in HIT cells

Prostaglandin E₂ (PGE₂) inhibits glucose-induced insulin secretion, and inhibitors of PGE₂ synthesis augment this event. However, there has been confusion regarding prostaglandin regulation of insulin secretion, partly because no mechanism has been demonstrated for the inhibitory action of PGE₂ on β-cell function. These studies were performed with a clonal cell line of glucose-responsive β-cells (HIT cells) to determine whether PGE₂ effects on insulin secretion are receptor mediated and, if so, whether the postreceptor effects are mediated by inhibitory regulatory components (N(i)) of adenylate cyclase. Saturable [³H]PGE₂ binding to HIT cells was demonstrated. This binding was dissociable and specific for prostaglandins of the E series. Scatchard analyses of binding data indicated a single class of sites with a K(d) of ~1 x 10^-9 M. Guinea pigs islets were also demonstrated to have a single class of binding sites with a similar K(d) but only 22% as many binding sites (0.060 vs. 0.013 pmol/mg protein, HIT cells vs. guinea pig islet). HIT cells were demonstrated to synthesize PGE₂ and this synthesis was inhibitable by acetylsalicylic acid. Accumulation of cAMP by HIT cells was inhibited in a concentration-dependent manner by PGE₂ with an IC₅₀ of ~1 x 10^-9 M. Insulin secretion by HIT cells during static incubations with 11.1 mM glucose was also inhibited by PGE₂ in a concentration-dependent manner with an IC₅₀ of 1 x 10^-9 M. PGE₂ was more potent than epinephrine but less potent than somatostatin in this regard. Maximum inhibition of glucose-induced insulin secretion was 26, 37, and 29% of control values for somatostatin, PGE₂, and epinephrine, respectively. Incubation of HIT cell membranes with pertussis toxin and [α-³²P]NAD with subsequent electrophoresis via sodium dodecyl sulfate-polyacrylamide gels revealed ADP-ribosylation of a component consistent with the N(i) component of adenylate cylcase. Pretreatment of intact HIT cells with pertussus toxin prevented subsequent labeling of this component when membranes were incubated with pertussis toxin and [³²P]NAD. Pertussis toxin partially prevented the inhibitory effects of PGE₂ but not NiCl₂, on glucose-induced insulin secretion. These findings uniquely demonstrate inhibition of PGE₂ of glucose-induced insulin secretion from HIT cells. The mechanism of PGE₂ action appears to involve specific PGE receptors that activate the N(i) component of adenylate cyclase, resulting in diminished cAMP.