Jason I. E. Bruce

Molecular and Functional Identification of a Ca2+ (Polyvalent Cation)-sensing Receptor in Rat Pancreas

The balance between the concentrations of free ionized Ca2+ and bicarbonate in pancreatic juice is of critical importance in preventing the formation of calcium carbonate stones. How the pancreas regulates the ionic composition and the level of Ca2+ saturation in an alkaline environment such as the pancreatic juice is not known. Because of the tight cause-effect relationship between Ca2+ concentration and lithogenicity, and because hypercalcemia is proposed as an etiologic factor for several pancreatic diseases, we have investigated whether pancreatic tissues express a Ca2+-sensing receptor (CaR) similar to that recently identified in parathyroid tissue. Using reverse transcriptase-polymerase chain reaction and immunofluorescence microscopy, we demonstrate the presence of a CaR-like molecule in rat pancreatic acinar cells, pancreatic ducts, and islets of Langerhans. Functional studies, in which intracellular free Ca2+concentration was measured in isolated acinar cells and interlobular ducts, show that both cell types are responsive to the CaR agonist gadolinium (Gd3+) and to changes in extracellular Ca2+ concentration. We also assessed the effects of CaR stimulation on physiological HCO3 −secretion from ducts by making measurements of intracellular pH. Luminal Gd3+ is a potent stimulus for HCO3 − secretion, being equally as effective as raising intracellular cAMP with forskolin. These results suggest that the CaR in the exocrine pancreas monitors the Ca2+ concentration in the pancreatic juice, and might therefore be involved in regulating the level of Ca2+ in the lumen, both under basal conditions and during hormonal stimulation. The failure of this mechanism might lead to pancreatic stone formation and even to pancreatitis.

Cytosolic Ca2+ and Ca2+‐activated Cl− current dynamics: insights from two functionally distinct mouse exocrine cells

The dynamics of Ca2+ release and Ca2+‐activated Cl− currents in two related, but functionally distinct exocrine cells, were studied to gain insight into how the molecular specialization of Ca2+ signalling machinery are utilized to produce different physiological endpoints: in this case, fluid or exocytotic secretion. Digital imaging and patch‐clamp methods were used to monitor the temporal and spatial properties of changes in cytosolic Ca2+ concentration ([Ca2+]c) and Cl− currents following the controlled photolytic release of caged‐InsP3 or caged‐Ca2+. In parotid and pancreatic acinar cells, changes in [Ca2+]c and activation of a Ca2+‐activated Cl− current occurred with close temporal coincidence. In parotid, a rapid global Ca2+ signal was invariably induced, even with low‐level photolytic release of threshold amounts of InsP3. In pancreas, threshold stimulation generated an apically delimited [Ca2+]c signal, while a stronger stimulus induced a global [Ca2+]c signal which exhibited characteristics of a propagating wave. InsP3 was more effective in parotid, where [Ca2+]c signals initiated with shorter latency and exhibited a faster time‐to‐peak than in pancreas. The increased potency of InsP3 in parotid probably results from a four‐fold higher number of InsP3 receptors as measured by radiolabelled InsP3 binding and western blot analysis. The Ca2+ sensitivity of the Cl− channels in parotid and pancreas was determined from the [Ca2+]‐current relationship measured during a dynamic ‘Ca2+ ramp’ produced by the continuous, low‐level photolysis of caged‐Ca2+. In addition to a greater number of InsP3 receptors, the Cl− current density of parotid acinar cells was more than four‐fold greater than that of pancreatic cells. Whereas activation of the current was tightly coupled to increases in Ca2+ in both cell types, local Ca2+ clearance was found to contribute substantially to the deactivation of the current in parotid. These data reveal specializations of common modules of Ca2+‐release machinery and subsequent effector activation that are specifically suited to the distinct functional roles of these two related cell types.

Modulation of [Ca2+]i Signaling Dynamics and Metabolism by Perinuclear Mitochondria in Mouse Parotid Acinar Cells

Parotid acinar cells exhibit rapid cytosolic calcium signals ([Ca2+]i) that initiate in the apical region but rapidly become global in nature. These characteristic [Ca2+]i signals are important for effective fluid secretion, which critically depends on a synchronized activation of spatially separated ion fluxes. Apically restricted [Ca2+]i signals were never observed in parotid acinar cells. This is in marked contrast to the related pancreatic acinar cells, where the distribution of mitochondria has been suggested to contribute to restricting [Ca2+]i signals to the apical region. Therefore, the aim of this study was to determine the mitochondrial distribution and the role of mitochondrial Ca2+ uptake in shaping the spatial and temporal properties of [Ca2+]i signaling in parotid acinar cells. Confocal imaging of cells stained with MitoTracker dyes (MitoTracker Green FM or MitoTracker CMXRos) and SYTO dyes (SYTO-16 and SYTO-61) revealed that a majority of mitochondria is localized around the nucleus. Carbachol (CCh) and caged inositol 1,4,5-trisphosphate-evoked [Ca2+]i signals were delayed as they propagated through the nucleus. This delay in the CCh-evoked nuclear [Ca2+]i signal was abolished by inhibition of mitochondrial Ca2+ uptake with ruthenium red and Ru360. Likewise, simultaneous measurement of [Ca2+]i with mitochondrial [Ca2+] ([Ca2+]m), using fura-2 and rhod-FF, respectively, revealed that mitochondrial Ca2+ uptake was also inhibited by ruthenium red and Ru360. Finally, at concentrations of agonist that evoke [Ca2+]i oscillations, mitochondrial Ca2+ uptake, and a nuclear [Ca2+] delay, CCh also evoked a substantial increase in NADH autofluorescence. This autofluorescence exhibited a predominant perinuclear localization that was also sensitive to mitochondrial inhibitors. These data provide evidence that perinuclear mitochondria and mitochondrial Ca2+ uptake may differentially shape nuclear [Ca2+] signals but more importantly drive mitochondrial metabolism to generate ATP close to the nucleus. These effects may profoundly affect a variety of nuclear processes in parotid acinar cells while facilitating efficient fluid secretion.

A model of calcium waves in pancreatic and parotid acinar cells

We construct a mathematical model of Ca(2+) wave propagation in pancreatic and parotid acinar cells. Ca(2+) release is via inositol trisphosphate receptors and ryanodine receptors that are distributed heterogeneously through the cell. The apical and basal regions are separated by a region containing the mitochondria. In response to a whole-cell, homogeneous application of inositol trisphosphate (IP(3)), the model predicts that 1), at lower concentrations of IP(3), the intracellular waves in pancreatic cells begin in the apical region and are actively propagated across the basal region by Ca(2+) release through ryanodine receptors; 2), at higher [IP(3)], the waves in pancreatic and parotid cells are not true waves but rather apparent waves, formed as the result of sequential activation of inositol trisphosphate receptors in the apical and basal regions; 3), the differences in wave propagation in pancreatic and parotid cells can be explained in part by differences in inositol trisphosphate receptor density; 4), in pancreatic cells, increased Ca(2+) uptake by the mitochondria is capable of restricting Ca(2+) responses to the apical region, but that this happens only for a relatively narrow range of [IP(3)]; and 5), at higher [IP(3)], the apical and basal regions of the cell act as coupled Ca(2+) oscillators, with the basal region partially entrained to the apical region.

A Role for Phosphorylation of Inositol 1,4,5-Trisphosphate Receptors in Defining Calcium Signals Induced by Peptide Agonists in Pancreatic Acinar Cells

Stimulation of pancreatic acinar cells with acetylcholine (ACh) and cholecystokinin (CCK) results in an elevation of cytosolic calcium ([Ca2+] c ) through activation of inositol 1,4,5-trisphosphate receptors (InsP3R). The global temporal pattern of the [Ca2+] c changes produced by ACh or CCK stimulation differs significantly. The hypothesis was tested that CCK stimulation results in a protein kinase A (PKA)-mediated phosphorylation of InsP3R and this event contributes to the generation of agonist-specific [Ca2+] c signals. Physiological concentrations of CCK evoked phosphorylation of the type III InsP3R, which was blocked by pharmacological inhibition of PKA. Imaging of fura-2-loaded acinar cells revealed that the rate of [Ca2+] c rise during CCK-evoked oscillations slows with each subsequent oscillation, consistent with a developing modulation of release, whereas the kinetics of ACh-evoked oscillations remain constant. Stimulation of cells with ACh following activation of PKA resulted in a slowing of the ACh-evoked [Ca2+] c rise, which now resembled a time-matched CCK response. PKA activation also resulted in a slowing of [Ca2+] c increases elicited by photolysis of caged InsP3. Targeted, PKA-mediated phosphorylation of type III InsP3R is involved in a physiological CCK response, as disruption of the targeting of PKA with the peptide HT31 resulted in marked changes in the CCK-evoked [Ca2+] c signal but had no effect on ACh-evoked responses. Stimulation of cells with bombesin, which evokes [Ca2+] c oscillations indistinguishable from those produced by CCK, also results in PKA-mediated phosphorylation of type III InsP3R. Thus, we conclude that PKA-mediated phosphorylation of type III InsP3R is a general mechanism by which the patterns of [Ca2+] c oscillations are shaped in pancreatic acinar cells.

Ion channel diversity, channel expression and function in the choroid plexuses

Knowledge of the diversity of ion channel form and function has increased enormously over the last 25 years. The initial impetus in channel discovery came with the introduction of the patch clamp method in 1981. Functional data from patch clamp experiments have subsequently been augmented by molecular studies which have determined channel structures. Thus the introduction of patch clamp methods to study ion channel expression in the choroid plexus represents an important step forward in our knowledge understanding of the process of CSF secretion.Two K+ conductances have been identified in the choroid plexus: Kv1 channel subunits mediate outward currents at depolarising potentials; Kir 7.1 carries an inward-rectifying conductance at hyperpolarising potentials. Both K+ channels are localised at the apical membrane where they may contribute to maintenance of the membrane potential while allowing the recycling of K+ pumped in by Na+-K+ ATPase. Two anion conductances have been identified in choroid plexus. Both have significant HCO3- permeability, and may play a role in CSF secretion. One conductance exhibits inward-rectification and is regulated by cyclic AMP. The other is carried by an outward-rectifying channel, which is activated by increases in cell volume. The molecular identity of the anion channels is not known, nor is it clear whether they are expressed in the apical or basolateral membrane. Recent molecular evidence indicates that choroid plexus also expresses the non-selective cation channels such as transient receptor potential channels (TRPV4 and TRPM3) and purinoceptor type 2 (P2X) receptor operated channels. In conclusion, good progress has been made in identifying the channels expressed in the choroid plexus, but determining the precise roles of these channels in CSF secretion remains a challenge for the future.

Calcium-activated K + channels increase cell proliferation independent of K + conductance

The intermediate-conductance calcium-activated potassium channel (IK1) promotes cell proliferation of numerous cell types including endothelial cells, T lymphocytes, and several cancer cell lines. The mechanism underlying IK1-mediated cell proliferation was examined in human embryonic kidney 293 (HEK293) cells expressing recombinant human IK1 (hIK1) channels. Inhibition of hIK1 with TRAM-34 reduced cell proliferation, while expression of hIK1 in HEK293 cells increased proliferation. When HEK293 cells were transfected with a mutant (GYG/AAA) hIK1 channel, which neither conducts K(+) ions nor promotes Ca(2+) entry, proliferation was increased relative to mock-transfected cells. Furthermore, when HEK293 cells were transfected with a trafficking mutant (L18A/L25A) hIK1 channel, proliferation was also increased relative to control cells. The lack of functional activity of hIK1 mutants at the cell membrane was confirmed by a combination of whole cell patch-clamp electrophysiology and fura-2 imaging to assess store-operated Ca(2+) entry and cell surface immunoprecipitation assays. Moreover, in cells expressing hIK1, inhibition of ERK1/2 and JNK kinases, but not of p38 MAP kinase, reduced cell proliferation. We conclude that functional K(+) efflux at the plasma membrane and the consequent hyperpolarization and enhanced Ca(2+) entry are not necessary for hIK1-induced HEK293 cell proliferation. Rather, our data suggest that hIK1-induced proliferation occurs by a direct interaction with ERK1/2 and JNK signaling pathways.

Differential regulation of the apical plasma membrane Ca 2+-ATPase by protein kinase A in parotid acinar cells

Cross-talk between intracellular calcium ([Ca2+]i) signaling and cAMP defines the specificity of stimulus-response coupling in a variety of cells. Previous studies showed that protein kinase A (PKA) potentiates and phosphorylates the plasma membrane Ca2+-ATPase (PMCA) in a Ca2+-dependent manner in parotid acinar cells (Bruce, J. I. E., Yule, D. I., and Shuttleworth, T. J. (2002) J. Biol. Chem. 277, 48172–48181). The aim of this study was to further investigate the spatial regulation of [Ca2+]i clearance in parotid acinar cells. Par-C10 cells were used to functionally isolate the apical and basolateral PMCA activity by applying La3+ to the opposite side to inhibit the PMCA. Activation of PKA (using forskolin) differentially potentiated apical [Ca2+]i clearance in mouse parotid acinar cells and apical PMCA activity in Par-C10 cells. Immunofluorescence of parotid tissue slices revealed that PMCA1 was distributed throughout the plasma membrane, PMCA2 was localized to the basolateral membrane, and PMCA4 was localized to the apical membrane of parotid acinar cells. However, in situ phosphorylation assays demonstrated that PMCA1 was the only isoform phosphorylated by PKA following stimulation. Similarly, immunofluorescence of acutely isolated parotid acinar cells showed that the regulatory subunit of PKA (RIIβ) translocated to the apical region following stimulation. These data suggest that PKA-mediated phosphorylation of PMCA1 differentially regulates [Ca2+]i clearance in the apical region of parotid acinar cells because of a dynamic translocation of PKA. Such tight spatial regulation of Ca2+ efflux is likely important for the fine-tuning of Ca2+-dependent effectors close to the apical membrane important for the regulation of fluid secretion and exocytosis.

Insulin Protects Pancreatic Acinar Cells from Palmitoleic Acid-induced Cellular Injury

Background: Palmitoleic acid is a major pancreatitis-inducing agent. Results: Insulin protected cells from palmitoleic acid (POA)-induced ATP depletion, inhibition of the plasma membrane calcium pump (PMCA), cytotoxic Ca2+ overload and necrosis. Conclusion: Insulin protects against acinar cell injury induced by pancreatitis-inducing agents. Significance: This provides an important therapeutic strategy for treating pancreatitis with insulin therapy. Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired mitochondrial metabolism, cytosolic Ca2+ ([Ca2+]i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca2+]i are linked critically by the ATP-driven plasma membrane Ca2+-ATPase (PMCA) important for maintaining low resting [Ca2+]i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca2+]i was measured by fura-2 imaging. An in situ [Ca2+]i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess cellular ATP depletion. Ethanol (100 mm) and POAEE (100 μm) induced a small but irreversible Ca2+ overload response but had no significant effect on PMCA activity. POA (50–100 μm) induced a robust Ca2+ overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca2+ overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced Ca2+ overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002. These data provide the first evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis.

Insulin Protects Pancreatic Acinar Cells from Cytosolic Calcium Overload and Inhibition of Plasma Membrane Calcium Pump

Background: Impaired metabolism and cytosolic Ca2+ overload in pancreatic acinar cells can trigger pancreatitis. Results: Insulin protected cells from oxidant-induced Ca2+ overload, inhibition of the plasma membrane calcium pump (PMCA), and ATP depletion. Conclusion: Insulin switches metabolism toward glycolysis and fuels the PMCA even when mitochondria are impaired. Significance: This mechanism may provide an important therapeutic strategy for pancreatitis. Acute pancreatitis is a serious and sometimes fatal inflammatory disease of the pancreas without any reliable treatment or imminent cure. In recent years, impaired metabolism and cytosolic Ca2+ ([Ca2+]i) overload in pancreatic acinar cells have been implicated as the cardinal pathological events common to most forms of pancreatitis, regardless of the precise causative factor. Therefore, restoration of metabolism and protection against cytosolic Ca2+ overload likely represent key therapeutic untapped strategies for the treatment of this disease. The plasma membrane Ca2+-ATPase (PMCA) provides a final common path for cells to “defend” [Ca2+]i during cellular injury. In this paper, we use fluorescence imaging to show for the first time that insulin treatment, which is protective in animal models and clinical studies of human pancreatitis, directly protects pancreatic acinar cells from oxidant-induced cytosolic Ca2+ overload and inhibition of the PMCA. This protection was independent of oxidative stress or mitochondrial membrane potential but appeared to involve the activation of Akt and an acute metabolic switch from mitochondrial to predominantly glycolytic metabolism. This switch to glycolysis appeared to be sufficient to maintain cellular ATP and thus PMCA activity, thereby preventing Ca2+ overload, even in the face of impaired mitochondrial function.