Electrical coupling and uncoupling of exocrine acinar cells.

The electrical communication network in the mouse pancreatic acinar tissue has been investigated using simultaneous intracellular recording with two separate microelectrodes and direct microscopical control of the localizations of the microelectrode tips. All cells within one acinus were electrically coupled, and the coupling coefficient (the electrotonic potential change in a cell neighboring to the cell into which current is injected [V2] divided by the electrotonic potential change in the cell of current injection [V1]) between two cells near each other (less than 50 micron) was always close to 1. Cells farther apart (50-100 micron) were, in some cases, coupled; in other cases, there was no coupling at all. Coupling coefficients varied between 0 and 1. There was rarely electrical coupling over distances of more than 110 micron. Using microiontophoretic acetylcholine (ACh) application, it was possible to evoke almost complete electrical uncoupling of two previously coupled pancreatic or lacrimal acinar cells from different acini or within one acinus. The effects were fully and quickly reversible. While the ACh-evoked uncoupling in the pancreas was associated with membrane depolarization, ACh caused hyperpolarization in the lacrimal acinar cells. The uncoupling was associated with a very marked reduction in electrical time constant, indicating a reduction in input capacitance (effective surface cell membrane area). The concentrations of stimulants needed to evoke reduction in pancreatic cell-to-cell coupling were 1 micron for ACh, 0.14 nM for caerulein, and 3 nM for bombesin. These concentrations are smaller than those required to evoke maximal enzyme secretion.

Mitochondrial injury in pancreatitis.

Pancreatitis is an increasingly common disease that carries a significant mortality and which lacks specific therapy. Pathological calcium signalling is an important contributor to the initiating cell injury, caused by or acting through mitochondrial inhibition. A principal effect of disordered cell signalling and impaired mitochondrial function is cell death, either by apoptosis that is primarily protective, or by necrosis that is deleterious, both locally and systemically. Mitochondrial calcium overload is particularly important in necrotic injury, which may include damage mediated by the mitochondrial permeability transition pore. The role of reactive oxygen species remains controversial. Present understanding of the part played by disordered pancreatic acinar calcium signalling and mitochondrial inhibition offers several new potential therapeutic targets.

Calcium signalling and pancreatic cell death: apoptosis or necrosis?

Secretagogues, such as cholecystokinin and acetylcholine, utilise a variety of second messengers (inositol trisphosphate, cADPR and nicotinic acid adenine dinucleotide phosphate) to induce specific oscillatory patterns of calcium (Ca(2+)) signals in pancreatic acinar cells. These are tightly controlled in a spatiotemporal manner, and are coupled to mitochondrial metabolism necessary to fuel secretion. When Ca(2+) homeostasis is disrupted by known precipitants of acute pancreatitis, for example, hyperstimulation or non-oxidative ethanol metabolites, Ca(2+) stores (endoplasmic reticulum and acidic pool) become depleted and sustained cytosolic [Ca(2+)] elevations replace transient signals, leading to severe consequences. Sustained mitochondrial depolarisation, possibly via opening of the mitochondrial permeability transition pore (MPTP), elicits cellular ATP depletion that paralyses energy-dependent Ca(2+) pumps causing cytosolic Ca(2+) overload, while digestive enzymes are activated prematurely within the cell; Ca(2+)-dependent cellular necrosis ensues. However, when stress to the acinar cell is milder, for example, by application of the oxidant menadione, release of Ca(2+) from stores leads to oscillatory global waves, associated with partial mitochondrial depolarisation and transient MPTP opening; apoptotic cell death is promoted via the intrinsic pathway, when associated with generation of reactive oxygen species. Apoptosis, induced by menadione or bile acids, is potentiated by inhibition of an endogenous detoxifying enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1), suggesting its importance as a defence mechanism that may influence cell fate.

Attraction or repulsion by local Ca(2+) signals.

Recent studies have shown that cytosolic Ca(2+) signals, generated on one side of a nerve growth cone, can induce turning either towards or away from the side of the Ca(2+) signal, depending on the global Ca(2+) level. The results indicate that local Ca(2+) signals may provide important directional cues for axon guidance.

Calcium signalling: store-operated channel found at last.

The intestinal Ca(2+) transport protein CaT1 has recently been shown to have the biophysical characteristics of the Ca(2+)-release activated Ca(2+) channel that refills internal Ca(2+) stores following agonist-elicited release. This finding highlights a hitherto unrecognized link between epithelial Ca(2+) transport and Ca(2+) signalling.

Membrane repair: Ca(2+)-elicited lysosomal exocytosis.

Cells in exposed positions are subject to injury and therefore need membrane repair mechanisms. Ca(2+) entry inevitably follows membrane rupture and recent studies indicate that this elicits repair via Ca(2+)-activated exocytosis of lysosomes, regulated by lysosomal synaptotagmin VII.

Calcium uptake via endocytosis with rapid release from acidifying endosomes.

A number of specific cellular Ca2+ uptake pathways have been described in many different cell types [1] [2] [3]. The possibility that substantial quantities of Ca2+ could be imported via endocytosis has essentially been ignored, although it has been recognized that endosomes can store Ca2+ [4] [5]. Exocrine cells can release significant amounts of Ca2+ via exocytosis [6], so we have investigated the fate of Ca2+ taken up via endocytosis into endosomes. Ca2+-sensitive and H+-sensitive fluorescent probes were placed in the extracellular solution and subsequently taken up into fibroblasts by endocytosis. Confocal microscopy was used to assess the distribution of fluorescence intensity. Ca2+ taken up by endocytosis was lost from the endosomes within a few minutes, over the same period as endosomal acidification took place. The acidification was inhibited by reducing the extracellular Ca2+ concentration, and Ca2+ loss from the endosomes was blocked by bafilomycin (100 nM), a specific inhibitor of the vacuolar proton ATPase. Quantitative evaluation indicated that endocytosis causes substantial import of Ca2+ because of rapid loss from early endosomes.

Intracellular glucose switches between cyclic ADP-ribose and inositol trisphosphate triggering of cytosolic Ca2+ spiking.

Cyclic ADP-ribose (cADPR) is a potentially important intracellular Ca2+ releasing messenger [1-5]. In pancreatic acinar cells where intracellular infusion of both inositol trisphosphate (IP3) and cADPR evoke repetitive Ca2+ spiking [6], the cADPR antagonist 8-NH2-cADPR [7], which blocks cADPR-evoked but not IP3-evoked Ca2+ spiking, can abolish Ca2+ spiking induced by physiological levels of the peptide hormone cholecystokinin (CCK) [8]. We have tested the effect of intracellular glucose on the ability of IP3, cADPR and CCK to induce cytosolic Ca2+ spikes in pancreatic acinar cells. In order to gain access to the intracellular cytosol, we used the whole-cell configuration of the patch-clamp technique [9] and monitored cytosolic Ca2+ concentration changes by measuring the Ca(2+)-dependent ionic current [10-13]. Glucose (300 microM to 10 mM) in the patch pipette/intracellular solution prevented cADPR from evoking Ca2+ spiking. The same effect was observed with 2-deoxy-glucose, but not L-glucose. In contrast, glucose potentiated IP3-evoked Ca2+ spiking. CCK evoked Ca2+ spiking irrespective of the presence or absence of intracellular glucose, but the cADPR antagonist 8-NH2-cADPR blocked CCK-evoked Ca2+ spiking only in the absence of intracellular glucose. This suggests that the hormone can evoke Ca2+ spiking via either the IP3 or the cADPR pathway. The intracellular glucose level may control a switch between these two pathways.

In vitro action of bombesin on amylase secretion, membrane potential, and membrane resistance in rat and mouse pancreatic acinar cells. A comparison with other secretagogues.

Bombesin caused depolarization of rat or mouse pancreatic acinar cell membrane, reduction of membrane resistance, and a steep rise in amylase output from superfused pancreatic fragments. These effects were similar to those previously described for acetylcholine, cholecystokinin, and gastrin. The dose-response curves for these three effects of bombesin were very similar, with effects being detectable at concentrations of about 30 pM and maximal effects at about 10 nM. The equilibrium potential for the membrane action of bombesin, i.e., the membrane potential at which bombesin did not cause any change in membrane potential, was -16 mV. Similar values for equilibrium potential were obtained with acetylcholine, caerulein and pentagastrin. Bombesin in the higher dose range (10 nM) caused electrical uncoupling of acinar cells within an acinus, i.e., a marked increase in junctional membrane resistance. Similar uncoupling effects were observed after acetylcholine, caerulein, and pentagastrin stimulation. In conclusion, bombesin acts on the pancreatic acinar plasma membrane in exactly the same way as acetylcholine and cholecystokinin-pancreozymin. The electrical uncoupling caused by stimulation is evidence for an increase in cytosol free calcium ion concentration.

Can Ca2+ be released from secretory granules or synaptic vesicles?

Recent studies on single isolated secretory granules, as well as secretory granules in intact cells, show that Ca2+ is released from the granule store into the cytosol via a messenger-mediated pathway following agonist binding. This is important for generation of localized cytosolic Ca2+ signals which can initiate exocytosis.

Spatial dynamics of second messengers: IP3 and cAMP as long-range and associative messengers.

Recent imaging experiments have revealed the distinct spatial dynamics of second-messenger actions. In general, actions of Ca2+ tend to be local, whereas those of other messengers such as inositol 1,4,5-trisphosphate (IP3) and cAMP are long range. In pancreatic acinar cells, IP3 generated at the base can diffuse across the cell and evoke a spatially confined Ca2+ signal in the apical pole, triggering enzyme and fluid secretion. Similar mechanisms might also operate in other cell types. We propose that the distinct dynamics of messengers might be relevant to neuronal function: IP3 and cAMP could convey signals over long distances along neurites, and serve as mediators for association and co-operation, for example, during learning.

New Ca2+-releasing messengers: are they important in the nervous system?

In the nervous system, Ca2+ signalling is determined primarily by voltage-gated Ca2+-selective channels in the plasma membrane, but there is increasing evidence for involvement of intracellular Ca2+ stores in such signalling. It is generally assumed that neurotransmitter-elicited release of Ca2+ from internal stores is primarily mediated by Ins(1,4,5)P3, as originally discovered in pancreatic acinar cells. The more-recently discovered Ca2+-releasing messenger, cyclic ADP-ribose (cADPR), which activates ryanodine receptors, has so far only been implicated in a few cases, and the possible importance of another Ca2+-releasing molecule, nicotinic acid adenine dinucleotide phosphate (NAADP), has been ignored. Recent investigations of the action of the brain-gut peptide cholecystokinin on pancreatic acinar cells have indicated that NAADP and cADPR receptors are essential for Ca2+ release. Tools are available for testing the possible involvement of NAADP and cADPR in neurotransmitter-elicited intracellular Ca2+ release, and such studies could reveal complex mechanisms that control this release in the nervous system.

The endoplasmic reticulum: one continuous or several separate Ca(2+) stores?

The Ca2+ store and sink in the endoplasmic reticulum (ER) is important for Ca2+ signal integration and for conveyance of information in spatial and temporal domains. Textbooks regard the ER as one continuous network, but biochemical and biophysical studies revealed apparently discrete ER Ca2+ stores. Recent direct studies of ER lumenal Ca2+ movements show that this organelle system is one continuous Ca2+ store, which can function as a Ca2+ tunnel. The concept of a fully connected ER network is entirely compatible with evidence indicating that the distribution of Ca2+ -release channels in the ER membrane is discontinuous with clustering in certain localities.

Modulation of Ca2+- and voltage-activated K+ channels by internal Mg2+ in salivary acinar cells.

High-conductance K+ channels are known to be activated by internal Ca2+ and membrane depolarization. The effects of changes in internal Mg2+ concentration have now been investigated in patch-clamp single-channel current experiments on excised membrane fragments from mouse acinar cells. It is shown that Mg2+ in the concentration range 10(-6)-10(-3) M evokes a dose-dependent K+ channel activation at a constant Ca2+ concentration of 10(-8) M. The demonstration that changes in [Mg2+]i between 2.5 X 10(-4) and 1.13 X 10(-3) M has effects on the channel open-state probability indicates that fluctuations in [Mg2+]i in intact cells may influence the control of channel opening.

Voltage-activation of high-conductance K+ channel in the insulin-secreting cell line RINm5F is dependent on local extracellular Ca2+ concentration.

Patch-clamp single-channel current recording experiments have been carried out on intact insulin-secreting RINm5F cells. Voltage-activation of high-conductance K+ channels were studied by selectively depolarizing the electrically isolated patch membrane under conditions with normal Ca2+ concentration in the bath solution but with or without Ca2+ in the patch pipette solution. When Ca2+ was present in the pipette, 40 mV to 120 mV depolarizing pulses (100 ms) from the normal resting potential (-70 mV) regularly evoked tetraethylammonium-sensitive large outward single-channel currents and the average open state probability during the pulses varied from about 0.015 (40 mV pulses) to 0.1 (120 mV pulses). In the absence of Ca2+ in the pipette solution the same protocol resulted in fewer and shorter K+ channel openings and the open-state probability varied from about 0.0015 (40 mV pulses) to about 0.03 (120 mV pulses). It is concluded that Ca2+ entering voltage-gated channels raises [Ca2+]i locally and thereby markedly enhances the open-state probability of tetraethylammonium-sensitive voltage-gated high-conductance K+ channels.

Inhibition of Ca2+-activated K+ channels in pig pancreatic acinar cells by Ba2+, Ca2+, quinine and quinidine.

Patch-clamp whole-cell and single-channel current recordings were made from pig pancreatic acinar cells to test the effects of quinine, quinidine, Ba2+ and Ca2+. Voltage-clamp current recordings from single isolated cells showed that high external concentrations of Ba2+ or Ca2+ (88 mM) abolished the outward K+ currents normally associated with depolarizing voltage steps. Lower concentrations of Ca2+ only had small inhibitory effects whereas 11 mM Ba2+ almost blocked the K+ current. 5.5 mM Ba2+ reduced the outward K+ current to less than 30% of the control value. Both external quinine and quinidine (200-500 microM) markedly reduced whole-cell outward K+ currents. In single-channel current studies it was shown that external Ba2+ (1-5 mM) markedly reduced the probability of opening of high-conductance Ca2+ and voltage-activated K+ channels whereas internal Ba2+ (6 X 10(-6) to 3 X 10(-5) M) caused activation at negative membrane potentials and inhibition at positive potentials. Quinidine (200-400 microM) evoked rapid chopping of single K+ channel openings acting both from the outside and inside of the membrane and in this way markedly reduced the total current passing through the channels.

Two different types of electrogenic amino acid action on pancreatic acinar cells.

The electrogenic action of the basic amino acid, L-arginine, has been compared with the action of the neutral amino acids, L-alanine and glycine, in mouse pancreatic acinar cells. All three amino acids cause membrane depolarization, but while the reversal potential for the action of the neutral amino acids is close to the calculated value of the Na equilibrium potential (+30 mV) the reversal potential for the L-arginine effects is +7 mV. The neutral amino acids exhibit mutual inhibition, but L-arginine did not inhibit the L-alanine- or glycine-evoked depolarization nor did the neutral amino acids inhibit the action of L-arginine. While L-alanine markedly depressed acetylcholine-evoked depolarization, L-arginine had no such effect. It is concluded that there are at least two quite different types of electrogenic amino acid action in pancreatic acinar cells.

Calcium-activated cation channel in rat thyroid follicular cells.

Using the patch-clamp single-channel current recording technique, a cation channel in the contraluminal membrane of rat thyroid follicular cells has been characterized. The channel has a unit conductance of about 35 pS and is equally permeable to sodium and potassium. The pattern of channel opening and closing is independent of the membrane potential. The channel is only operational when the ionized calcium concentration in the fluid which is in contact with the inside of the membrane is at least 1 microM. This conductance pathway can be classified as a calcium dependent non-selective cation channel and could explain stimulant-evoked depolarizations in the thyroid follicular cells.

Thyrotropin controls cyclic nucleotide metabolism of thyroid follicular cells without affecting membrane potential or input resistance.

The action of thyrotropin on the rat thyroid follicular cell has been investigated using continuous transmembrane potential and input resistance recording from individual cells in in vitro preparations. The membrane potential in this study was -68.0 mV +/- 0.6 (mean+/-S.E.). Over a wide range of concentrations (1.5-50 mU/ml), thyrotropin failed to affect membrane potential or input resistance while 20 mU/ml thyrotropin was shown to elicit complex time-dependent changes in tissue levels of both cyclic AMP and cyclic GMP. The present results reveal that thyrotropin-receptor interaction does not affect plasma membrane permeability, but is characterized by complex changes in endogenous cyclic nucleotide metabolism.

Effects of alanine on insulin-secreting cells: patch-clamp and single cell intracellular Ca2+ measurements.

The effects of alanine, glucose and tolbutamide on insulin-secreting cells (RINm5F) have been investigated using patch-clamp and single cell intracellular Ca2+ measurements. When directly challenged with the amino acid L-alanine (2-10 mM) the cells underwent a sharp depolarization, which led to the generation of Ca2+ spike potentials and an increase in [Ca2+]i. The L-alanine-induced depolarization was associated with a net inward membrane current but no measurable change in the resistance of the cell. The latter effect was found to be in contrast to the actions of glucose (5-10 mM) and tolbutamide (100 microM), both of which depolarized cells and raised [Ca2+]i by an increase in the input resistance of the cell membrane, due to the closure of ATP-sensitive potassium channels. In the complete absence of external Na+ (by replacement with 140 mM NMDG+), L-alanine had no effects on either the membrane potential or [Ca2+]i. Similarly, replacing Na+ with NMDG+ in the continued presence of the amino acid resulted in a repolarization of the membrane and an attenuation of the L-alanine-induced rise in [Ca2+]i. The Na+ channel blocker TTX (1-2 microM) had no effects on the alanine-evoked electrical activity. Exchange of the L-form of the amino acid with the D-stereoisomer had similar actions to those of removing external Na+, since D-alanine had no effects on the membrane potential or [Ca2+]i. The actions of L-alanine were also found to be mimicked by the N-methylated amino acid analogue methylamino isobutyric acid (MeAIB) (2-10 mM), suggesting that the A-type electrogenic amino acid cotransport system operates in the RINm5F insulin-secreting cell line.