The G protein G _o is highly expressed in neurons and mediates effects of a group of rhodopsin-like receptors that includes the opioid, α ₂ -adrenergic, M2 muscarinic, and somatostatin receptors. In vitro , G _o is also activated by growth cone-associated protein of M _r 43,000 (GAP43) and the Alzheimer amyloid precursor protein, but it is not known whether this occurs in intact cells. To learn about the roles that G _o may play in intact cells and whole body homeostasis, we disrupted the gene encoding the α subunits of G _o in embryonic stem cells and derived G _o -deficient mice. Mice with a disrupted α _o gene (α _o −/− mice) lived but had an average half-life of only about 7 weeks. No G _o α was detectable in homogenates of α _o −/− mice by ADP-ribosylation with pertussis toxin. At the cellular level, inhibition of cardiac adenylyl cyclase by carbachol (50–55% at saturation) was unaffected, but inhibition of Ca ²⁺ channel currents by opioid receptor agonist in dorsal root ganglion cells was decreased by 30%, and in 25% of the α _o −/− cells examined, the Ca ²⁺ channel was activated at voltages that were 13.3 ± 1.7 mV lower than in their counterparts. Loss of α _o was not accompanied by appearance of significant amounts of active free βγ dimers (prepulse test). At the level of the living animal, G _o -deficient mice are hyperalgesic (hot-plate test) and display a severe motor control impairment (falling from rotarods and 1-inch wide beams). In spite of this deficiency, α _o −/− mice are hyperactive and exhibit a turning behavior that has them running in circles for hours on end, both in cages and in open-field tests. Except for one, all α _o −/− mice turned only counterclockwise. These findings indicate that G _o plays a major role in motor control, in motor behavior, and in pain perception and also predict involvement of G _o in Ca ²⁺ channel regulation by an unknown mechanism.

Pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 are neuropeptides of the vasoactive intestinal peptide/secretin/glucagon family. We previously described alternative splicing of the region encoding the third intracellular loop of the PACAP receptor generating six isoforms with differential signal transduction properties (Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H., and Journot, L. (1993) Nature 365, 170-175). In addition, we demonstrated that the potencies of the two forms of PACAP are similar for adenylate cyclase stimulation, whereas PACAP-38 is more potent than PACAP-27 in phospholipase C activation. In the present work, we document the existence of a new splice variant of the PACAP receptor that was characterized by a 21-amino-acid deletion in the N-terminal extracellular domain. We demonstrate that this domain modulates receptor selectivity with respect to PACAP-27 and -38 binding and controls the relative potencies of the two agonists in phospholipase C stimulation.

Cisplatin (CDDP) is a highly effective chemotherapeutic agent but with significant ototoxic side effects. Apoptosis is an important mechanism of cochlear hair cell loss following exposure to an ototoxic level of CDDP. This study examines intracellular pathways involved in hair cell death induced by CDDP exposure in vivo to develop effective therapeutic strategies to protect the auditory receptor from CDDP-initiated hearing loss. Guinea pigs were treated with systemic administration of CDDP. Cochlear hair cells from CDDP-treated animals exhibited classic apoptotic alterations in their morphology. Several important signaling events that regulate the death of CDDP-injured cochlear hair cells were identified. CDDP treatment induced the activation and redistribution of cytosolic Bax and the release of cytochrome c from injured mitochondria. Activation of caspase-9 and caspase-3, but not caspase-8, was detected after treatment with CDDP, and the cleavage of fodrin by activated caspase-3 was observed within damaged hair cells. Intracochlear perfusions with caspase-3 inhibitor (z-DEVD-fmk) and caspase-9 inhibitor (z-LEHD-fmk) prevent hearing loss and loss of sensory cells, but caspase-8 inhibitor (z-IETD-fmk) and cathepsin B inhibitor (z-FA-fmk) do not. Although the stress-activated protein kinase/c-Jun NH₂-terminal kinase (JNK) signaling pathway is activated in response to CDDP toxicity, intracochlear perfusion of d-JNKI-1, a JNK inhibitor, did not protect against CDDP ototoxicity but instead potentiated the ototoxic effects of CDDP. The results of the present study show that blocking a critical step in apoptosis may be a useful strategy to prevent harmful side effects of CDDP ototoxicity in patients having to undergo chemotherapy.

Pituitary adenylate cyclase–activating polypeptide (PACAP) is a ubiquitous neuropeptide of the vasoactive intestinal peptide (VIP) family that potentiates glucose-stimulated insulin secretion. Pancreatic β cells express two PACAP receptor subtypes, a PACAP-preferring (PAC1) and a VIP-shared (VPAC2) receptor. We have applied a gene targeting approach to create a mouse lacking the PAC1 receptor (PAC1^–/–). These mice were viable and normoglycemic, but exhibited a slight feeding hyperinsulinemia. In vitro, in the isolated perfused pancreas, the insulin secretory response to PACAP was reduced by 50% in PAC1^–/– mice, whereas the response to VIP was unaffected. In vivo, the insulinotropic action of PACAP was also acutely reduced, and the peptide induced impairment of glucose tolerance after an intravenous glucose injection. This demonstrates that PAC1 receptor is involved in the insulinotropic action of the peptide. Moreover, PAC1^–/– mice exhibited reduced glucose-stimulated insulin secretion in vitro and in vivo, showing that the PAC1 receptor is required to maintain normal insulin secretory responsiveness to glucose. The defective insulinotropic action of glucose was associated with marked glucose intolerance after both intravenous and gastric glucose administration. Thus, these results are consistent with a physiological role for the PAC1 receptor in glucose homeostasis, notably during food intake.