(8,9). In addition to its role as sensor of [Ca 2ϩ ] o , the CaR is also stimulated by aromatic amino acids (10) that, like [Ca 2ϩ ] o , induce striking and lasting CaR-mediated [Ca 2ϩ ] i oscillations (9, 11). However, the patterns of [Ca 2ϩ ] i oscillations induced by these agonists are different. Aromatic amino acid stimulation of the CaR induces repetitive, low frequency [Ca 2ϩ ] i spikes that return to the base-line level, a pattern known as transient oscillations. In contrast, [Ca 2ϩ ] o -elicited CaR activation produces high frequency sinusoidal oscillations upon a raised plateau level of [Ca 2ϩ ] i (9, 11). The amplitude, frequency, and duration of [Ca 2ϩ ] i oscillations are increasingly recognized as encoding important information for a variety of biological processes, and, consequently, there is intense interest in understanding the underlying mechanisms (12).Our previous results produced several lines of evidence indicating that PKCs negatively regulate the frequency of [Ca 2ϩ ] i oscillations induced by activation of the CaR by increases in [Ca 2ϩ ] o (11). We hypothesized that periodic phosphorylation of the CaR by PKCs provides the negative feedback needed to cause [Ca 2ϩ ] o -induced sinusoidal [Ca 2ϩ ] i oscillations. Intriguingly, the transient [Ca 2ϩ ] i oscillations produced by the CaR in response to amino acid stimulation appear to be mediated by a different pathway, but the mechanism(s) involved remained poorly understood.In the present study, we examined whether sinusoidal and transient [Ca 2ϩ ] i oscillations produced by the CaR in response to Ca 2ϩ or L-phenylalanine are mediated by different pathways. Using real time imaging of changes in phosphatidylinositol 4,5-biphosphate hydrolysis and generation of Ins(1,4,5)P 3 in single cells, we found that [Ca 2ϩ ] o -induced CaR activation

The protein kinase D (PKD) family consists of three serine/threonine kinases: PKC/PKD, PKD2, and PKC/PKD3. Whereas PKD has been the focus of most studies, virtually nothing is known about the effect of G protein-coupled receptor agonists (GPCR) on the regulatory properties and intracellular distribution of PKD3. Consequently, we examined the mechanism that mediates its activation and intracellular distribution. GPCR agonists induced a rapid activation of PKD3 by a protein kinase C (PKC)-dependent pathway that leads to the phosphorylation of the activation loop of PKD3. Comparison of the steady-state distribution of endogenous or tagged PKD3 versus PKD and PKD2 in unstimulated cells indicated that whereas PKD and PKD2 are predominantly cytoplasmic, PKD3 is present both in the nucleus and cytoplasm. This distribution of PKD3 results from its continuous shuttling between both compartments by a mechanism that requires a nuclear import receptor and a competent CRM1-nuclear export pathway. Cell stimulation with the GPCR agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD3 that is PKCdependent. Interestingly, the nuclear accumulation of PKD3 can be dramatically enhanced in response to its activation. Thus, this study demonstrates that the intracellular distribution of PKD isoenzymes are distinct, and suggests that their signaling properties are regulated by differential localization.

BACKGROUND & AIMS Opening of the mitochondrial permeability transition pore (MPTP) causes loss of the mitochondrial membrane potential (ΔΨm) and, ultimately, adenosine triphosphate depletion and necrosis. Cells deficient in cyclophilin D (CypD), a component of the MPTP, are resistant to MPTP opening, loss of ΔΨm, and necrosis. Alcohol abuse is a major risk factor for pancreatitis and is believed to sensitize the pancreas to stressors, by poorly understood mechanisms. We investigated the effects of ethanol on the pancreatic MPTP, the mechanisms of these effects, and their role in pancreatitis. METHODS We measured ΔΨm in mouse pancreatic acinar cells incubated with ethanol alone and in combination with physiologic and pathologic concentrations of cholecystokinin-8 (CCK). To examine the role of MPTP, we used ex vivo and in vivo models of pancreatitis, induced in wild-type and CypD−/− mice by a combination of ethanol and CCK. RESULTS Ethanol reduced basal ΔΨm and converted a transient depolarization, induced by physiologic concentrations of CCK, into a sustained decrease in ΔΨm, resulting in reduced cellular adenosine triphosphate and increased necrosis. The effects of ethanol and CCK were mediated by MPTP because they were not observed in CypD−/− acinar cells. Ethanol and CCK activated MPTP through different mechanisms— ethanol by reducing the ratio of oxidized nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide, as a result of oxidative metabolism, and CCK by increasing cytosolic Ca2+. CypD−/− mice developed a less-severe form of pancreatitis after administration of ethanol and CCK. CONCLUSIONS Oxidative metabolism of ethanol sensitizes pancreatic mitochondria to activate MPTP, leading to mitochondrial failure; this makes the pancreas susceptible to necrotizing pancreatitis.