INDI is a similarity-based drug–drug interaction prediction method that can infer both pharmacokinetic and pharmacodynamic interactions, as well as their severity levels. Both known and predicted drug interactions are found to be prevalent in clinical practice.

The enhanced metabolism of phosphoinositides, which is associated with a wide variety of stimuli and physiological responses, has been studied intensively. Berridge and his collaborators demonstrated that the first measurable reaction following cell membrane receptor activation is a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and that the product of this reaction, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), could cause a release of non-mitochondrial calcium. These findings have been verified in other systems. Although the relationship between the hydrolysis of PtdIns(4,5)P2 and the mobilization of intracellular calcium was clearly demonstrated, the direct link between Ins(1,4,5)P3 production and the physiological response was only implied. We have investigated the possibility that the intracellular release of Ins(1,4,5)P3 mediates the muscarinic-cholinergic response is Xenopus oocytes, and we show here that intracellularly injected Ins(1,4,5)P3 mimics the muscarinic depolarizing chloride current in Xenopus oocytes. This is the first demonstration of a direct link between phosphoinositides metabolism and a neuro-transmitter-induced physiological response.

SUMMARY1. The membrane response to acetylcholine (ACh), inositol 1,4,5-trisphosphate (IP3) and intracellular Ca21 was studied in Xenopus laevis oocytes under voltageclamp conditions.2. Shallow, submembranal injections of IP3 in the animal hemisphere of the oocyte evoked a two-component response comprised of a rapid, transient component followed by a slow, sustained component.3. When the injection pipette was inserted further into the cell (to 300 ,um below the cell membrane), the fast component diminished and the slow component remained unchanged or even increased.4. The rapid component exhibited an apparent higher sensitivity to 1P3 compared to the slow component.5. The two components of the IP3 response were retained in a Ca2+-free environment.6. Injection of a single large dose (20-50 pmol) of CaCl2 into the oocyte evoked a typical two-component response, whereas repetitive threshold doses (0-1 pmol CaCl2) elicited large current fluctuations which developed into a small depolarization current.7. The delay in the peak of the slow component of the response to either IP3 or to CaC12 injections appeared too long to be accounted for by diffusion alone.8. Depletion of oocyte Ca2' by the divalent cation ionophore A23187 (>1 ,IM)inhibited the response to ACh and 'P3. Low concentrations of A23187 selectively inhibited the rapid component of the ACh response, though not the rapid component of the IP3 response. 9. Our data suggest that the two-component membrane response to ACh in Xenopus oocytes can be accounted for by ACh-induced elevation of IP3 and subsequent IP3-induced release of intracellular Ca2 .

We studied the activity of a truncated thyrotropin-releasing hormone receptor (TRH-R), which lacks the last 59 amino acids of the carboxyl tail, where Cys-335 was mutated to a stop codon (C335Stop) (Nussenzveig, D. R., Heinflink, M., and Gershengorn, M. C. (1993) J. Biol. Chem. 268, 2389-2392). In Xenopus laevis oocytes expressing C335Stop TRH-Rs, TRH binding was higher, whereas chloride current, 45Ca2+ efflux, and [Ca2+]i responses evoked by TRH were 23, 39, and 21%, respectively, of those in oocytes expressing wild type mouse pituitary TRH-Rs (WT TRH-Rs). In oocytes expressing C335Stop TRH-Rs, basal 45Ca2+ efflux and [Ca2+]i were twice those in oocytes expressing WT TRH-Rs; chelation of Ca2+ caused a rapid increase in holding current, which is consistent with basal activation; and coexpression with other receptors caused inhibition of the responses to the other cognate agonists. In AtT20 pituitary cells stably expressing C335Stop TRH-Rs, thyrotropin-releasing hormone (TRH)-independent inositol phosphate formation was 1.32 +/- 0.11-fold higher, basal [Ca2+]i was 1.8 +/- 0.2-fold higher, and the [Ca2+]i response to TRH was much lower than in cells expressing WT TRH-Rs. We conclude that a TRH-R mutant truncated at Cys-335 exhibits constitutive activity that results in desensitization of the response to TRH.

The involvement of cellular processes in the biphasic dynamics of heat acclimation was studied. Key steps in the cholinergic signal transduction pathway for water secretion were measured in the submaxillary gland of acclimating [2-day short-term heat acclimation (STHA) and 30-day long-term heat acclimation (LTHA) at 34 degrees C] or acute heat-stressed (2 h at 40 degrees C) rats in vitro. Both the carbamylcholine (CCh)-induced maximal fractional rate and the total 86Rb+ efflux, reflecting K+ efflux and water transport, transiently decreased in STHA (P < 0.001). In LTHA, the total K+ efflux increased (P < 0.001), whereas the maximal fractional rate of efflux increased only slightly. During STHA, the density of the high-affinity binding site of the muscarinic receptors (MRs) increased by 50% and their affinity for the muscarinic antagonist [3H]-N-methylscopolamine decreased transiently by 87%. Basal cytosolic Ca2+ concentration ([Ca2+]i) decreased (P < 0.05), but the peak CCh-induced [Ca2+]i increase resembled the control values. In LTHA, MR density continued to increase (100%; P < 0.05), whereas affinity resumed control values. Basal and CCh-induced [Ca2+]i increases returned to control levels. We conclude that glandular cellular processes follow a biphasic pattern with major apparent changes attributable to events distal to the [Ca2+]i rise. This was further validated by employing heat stress, which produced qualitatively different effects on the MR profile with a decrease in 86Rb+ efflux comparable to STHA. Hence, although heat-induced changes in the proximal components of the signal transduction pathway may contribute to altered regulatory span, the predominant apparent cellular effect is on the distal part of the pathway.