The secretion of fluid and electrolytes by salivary gland acinar cells requires the coordinated regulation of multiple water and ion transporter and channel proteins. Notably, all the key transporter and channel proteins in this process appear to be activated, or are up-regulated, by an increase in the intracellular Ca2+ concentration ([Ca2+]i). Consequently, salivation occurs in response to agonists that generate an increase in [Ca2+]i. The mechanisms that act to modulate these increases in [Ca2+]i obviously influence the secretion of salivary fluid. Such modulation may involve effects on mechanisms of both Ca2+ release and Ca2+ entry and the resulting spatial and temporal aspects of the [Ca2+]i signal, as well as interactions with other signaling pathways in the cells. The molecular cloning of many of the transporter and regulatory molecules involved in fluid and electrolyte secretion has yielded a better understanding of this process at the cellular level. The subsequent characterization of mice with null mutations in many of these genes has demonstrated the physiological roles of individual proteins. This review focuses on recent developments in determining the molecular identification of the proteins that regulate the fluid secretion process.

The literature suggests that the physiological functions for which mitochondria sequester Ca(2+) are (1). to stimulate and control the rate of oxidative phosphorylation, (2). to induce the mitochondrial permeability transition (MPT) and perhaps apoptotic cell death, and (3). to modify the shape of cytosolic Ca(2+) pulses or transients. There is strong evidence that intramitochondrial Ca(2+) controls both the rate of ATP production by oxidative phosphorylation and induction of the MPT. Since the results of these processes are so divergent, the signals inducing them must not be ambiguous. Furthermore, as pointed out by Balaban [J. Mol. Cell. Cardiol. 34 (2002 ) 11259-11271], for any repetitive physiological process dependent on intramitochondrial free Ca(2+) concentration ([Ca(2+)](m)), a kind of intramitochondrial homeostasis must exist so that Ca(2+) influx during the pulse is matched by Ca(2+) efflux during the period between pulses to avoid either Ca(2+) buildup or depletion. In addition, mitochondrial Ca(2+) transport modifies both spatial and temporal aspects of cytosolic Ca(2+) signaling. Here, we look at the amounts of Ca(2+) necessary to mediate the functions of mitochondrial Ca(2+) transport and at the mechanisms of transport themselves in order to set up a hypothesis about how the mechanisms carry out their roles. The emphasis here is on isolated mitochondria and on general mitochondrial properties in order to focus on how mitochondria alone may function to fulfill their physiological roles even though the interactions of mitochondria with other organelles, particularly with endoplasmic and sarcoplasmic reticulum [Sci. STKE re1 (2004) 1-9], may also influence this story.

The effects of acetylcholine (ACh), cholecystokinin (CCK), internally applied GTP‐gamma‐S, inositol trisphosphate [Ins (1,4,5) P3] or Ca2+ on the cytoplasmic free Ca2+ concentration [( Ca2+]i) were assessed by simultaneous microfluorimetry (fura‐2) and measurement of the Ca2(+)‐dependent Cl‐ current (patch‐clamp whole‐cell recording) in single internally perfused mouse pancreatic acinar cells. ACh (0.1‐0.2 microM) evoked an oscillating increase in [Ca2+]i measured in the cell as a whole (microfluorimetry) which was synchronous with oscillations in the Ca2(+)‐dependent Cl‐ current reporting [Ca2+]i close to the cell membrane. In the same cells a lower ACh concentration (0.05 microM) evoked shorter repetitive Cl‐ current pulses that were not accompanied by similar spikes in the microfluorimetric recording. When cells did not respond to 0.1 microM ACh, caffeine (1 mM) added on top of the sustained ACh stimulus resulted in [Ca2+]i oscillations seen synchronously in both types of recording. CCK (10 nM) also evoked [Ca2+]i oscillations, but with much longer intervals between slightly broader Ca2+ pulses. Internal perfusion with 100 microM GTP‐gamma‐S evoked [Ca2+]i oscillations with a similar pattern. Ins (1,4,5) P3 (10 microM) evoked repetitive shortlasting spikes in [Ca2+]i that were only seen in the Cl‐ current traces, except in one small cell where these spikes were also observed synchronously in the microfluorimetric recording. Caffeine (1 mM) broadened these Ca2+ pulses. [Ca2+]i was also directly changed, bypassing the normal signalling process, by infusion of a low or high Ca2+ solution into the pipette.(ABSTRACT TRUNCATED AT 250 WORDS)

Summary Phospholipase Cε (PLCε) is a multifunctional enzyme implicated in cardiovascular, pancreatic and inflammatory functions. Here we show that conditional deletion of PLCε in mouse cardiac myocytes protects from stress-induced pathological hypertrophy. PLCε siRNA in ventricular myocytes decreases endothelin-1 (ET-1)-dependent elevation of nuclear calcium and activation of nuclear protein kinase D (PKD). PLCε scaffolded to muscle-specific A kinase anchoring protein (mAKAP), along with PKCε and PKD, localizes these components at or near the nuclear envelope and this complex is required for nuclear PKD activation. Phosphatidylinositol 4-phosphate (PI4P) is identified as a perinuclear substrate in the Golgi apparatus for mAKAP-scaffolded PLCε. We conclude that perinuclear PLCε, scaffolded to mAKAP in cardiac myocytes, responds to hypertrophic stimuli to generate DAG from PI4P in the Golgi apparatus, in close proximity to the nuclear envelope, to regulate activation of nuclear PKD, and hypertrophic signaling pathways.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are tetrameric intracellular Ca2+-release channels with each subunit containing a binding site for IP3 in the N-terminus. We provide evidence that four IP3 molecules are required to activate the channel under diverse conditions. Comparing the concentration-response relationship for binding and Ca2+ release suggested that IP3Rs are maximally occupied by IP3 before substantial Ca2+ release occurs. We showed that ligand binding–deficient subunits acted in a dominant-negative manner when coexpressed with wild-type monomers in the chicken immune cell line DT40-3KO, which lacks all three genes encoding IP3R subunits, and confirmed the same effect in an IP3R-null human cell line (HEK-3KO) generated by CRISPR/Cas9 technology. Using dimeric and tetrameric concatenated IP3Rs with increasing numbers of binding-deficient subunits, we addressed the obligate ligand stoichiometry. The concatenated IP3Rs with four ligand-binding sites exhibited Ca2+ release and electrophysiological properties of native IP3Rs. However, IP3 failed to activate IP3Rs assembled from concatenated dimers consisting of one binding-competent and one binding-deficient mutant subunit. Similarly, IP3Rs containing two monomers of IP3R2short, an IP3 binding-deficient splice variant, were nonfunctional. Concatenated tetramers containing only three binding competent ligand-binding sites were nonfunctional under a wide range of activating conditions. These data provide definitive evidence that IP3-induced Ca2+ release only occurs when each IP3R monomer within the tetramer is occupied by IP3, thereby ensuring fidelity of Ca2+ release.