Characterization of SNF 9007, a novel cholecystokinin/opioid ligand in mouse ileum in vitro: Evidence for involvement of cholecystokinin(A) and cholecystokinin(B) receptors in regulation of ion transport

The effects of cholecystokinin (CCK) fragments and Asp-Tyr-D-Phe-Gly-Trp- [N-Me]Nle-Asp-Phe-NH₂ 1(SNF 9007), a synthetic CCK analog which binds with high affinity to CCK(B) and opioid delta receptors, were evaluated in isolated sheets of mouse ileum mounted in Ussing flux chambers. Serosal, but not mucosal, administration of cholecystokinin octapeptide-sulfated [CCK₈(s)] and cholecystokinin tetrapeptide (30-33) [CCK₄(30-33)] produced a brief, concentration-related increase in short circuit current (I(sc)) without changing tissue conductance. Serosal, but not mucosal, SNF 9007 produced a similar concentration-related increase in I(sc) which was followed by an immediate concentration-related and sustained decrease in I(sc); no decrease in I(sc) was observed for either CCK₈ or CCK₄(30-33). The increase and subsequent decrease in the SNF 9007 I(sc) response were respectively classified as phase I (i.e., CCK-like) and phase II (opioid-like) activity. CCK₈(s) and SNF 9007 (phase I) were active at low nanomolar concentrations, whereas CCK₄(30-33) was active only at high nanomolar concentrations: the rank order of potencies to increase I(sc) was CCK₈(s) > SNF 9007 > CCK₄(30- 33). Devazepide (L364,718), a selective antagonist of CCK(A) receptors, effectively blocked the action of CCK₈(s), but not that of CCK₄(30-33) or SNF 9007 (phase I). In contrast, 3R[+]-N-[2,3-dihydro-1-methyl-2-oxo-5- phenyl-1H-benzodiazepin-3-yl]-N'-[3-methyl-phenyl]urea (L365,260), a selective CCK(B) receptor antagonist, blocked the action of CCK₄(30-33) and SNF 9007 (phase I), and also antagonized CCK(B)(s), though to a lesser degree. The phase II response of SNF 9007 was antagonized by N,N-diallyl- Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864), a selective opioid delta receptor antagonist; this opioid antagonist did not influence the phase I response. Neither L364,718 or L365,260 influenced the SNF 9007 phase II response. Serosal pretreatment of tissues with tetrodotoxin, or the ganglionic blocker chlorisondamine, significantly blocked the actions of CCK₈(s) and CCK₄(30- 33), and both phase I and phase II responses to SNF 9007. Further, these peptides produced no significant response in mucosal preparations of ileum physically stripped of the enteric ganglia and muscularis externa. The data suggest that ileal ion-transport can be modulated by the activation of neural CCK(A) or CCK(B) receptors which are located partly preganglionically and that these receptors can be selectively activated by derivatives or analogs of CCK. CCK₈(s) appears to produce its effects predominately, but not exclusively, at the CCK(A) receptor, whereas SNF 9007 and CCK₄(30-33) selectively activate CCK(B) receptors in mouse ileum; SNF 9007 (phase I) is several-fold more potent than CCK₄(30-33) in influencing ion transport at the CCK(B) receptor. Finally, SNF 9007 has the unusual profile of acting at opioid delta receptors to produce a subsequent decrease in I(sc). These data demonstrate the importance of both CCK(A) and CCK(B), as well as opioid delta, receptors in the regulation of ion transport in the same intestinal segment.