Calcium SignalingLawrence D. Gaspers, Nicola Pierobon, Andrew P. Thomas

Calcium Signaling

Lawrence D. Gaspers, Nicola Pierobon, Andrew P. Thomas

18

18.1

Ca

Signaling in Liver

Ca

is a ubiquitous and versatile signaling molecule controlling the activity of a broad spectrum of bio- logical events [2–4, 13, 74]. Agonist-evoked changes in cytosolic free Ca

([Ca

]

) can be spatially local- ized or global in nature, with information encoded in the frequency of Ca

spikes or the magnitude of the Ca

increase. The ability to control Ca

events in both space and time permits the transmission of a diverse set of intracellular messages to downstream Ca

-sensitive targets. Calcium ions can exert con- trol by either binding directly to the target protein, as is the case for intramitochondrial dehydrogenas- es [41], or indirectly through Ca

-binding proteins, such as calmodulin or calcineurin [40, 42] and even at the genetic level through transcriptional repres- sors (i.e. DREAM) [9].

Ca

signaling is likely to be of critical impor- tance for hepatocyte function within the intact liver.

Hepatocytes are large polarized epithelia cells with different Ca

-sensitive metabolic activities occur- ring within the basolateral and apical membrane domains. One pertinent example is the actin- and myosin-containing microfilaments surrounding the apical membrane, which has been implicated in Ca

-dependent contraction of the bile canaliculi [7, 33, 78, 79, 83]. Moreover, the functional capac- ity of the hepatic lobule displays an asymmetrical distribution between hepatocytes; ATP-utilizing pathways, such as gluconeogenesis and urea syn- thesis, predominate in the periportal zone, whereas glutamine production and glycolytic activity are higher in pericentral hepatocytes [31, 32]. Each dis- tinct metabolic zone presumably contains it own unique repertoire of hormone receptors, intracellu- lar Ca

requirements and Ca

sensors. Thus, inter- cellular Ca

movements between lobular zones may be required for the liver to function in a coordinated fashion under high metabolic demand.

18.2

Phosphoinositide Signaling Pathway

Glycogenolytic hormones, such as α

-adrenergic ag- onists, vasopressin and ATP, evoke [Ca

]

increases by stimulating the production of the Ca

-mobiliz- ing second messenger, inositol-1,4,5-trisphosphate (IP

). This is achieved through receptor-associated heterotrimeric G proteins, which activate phos- phohypherationinositide-specific phospholipase C (PLC) [2, 13, 53]. Under basal conditions, heterot- rimeric G proteins consisting of α, β, and γ subunits are associated together in an inactive GDP-bound state. Agonist binding to cell surface receptors stim- ulates the exchange of GDP for GTP on the G α subu- nit, promoting its dissociation from the βγ dimer.

The G γ subunits are modified by protein prenyla- tion, which promotes the association of the βγ dimer with membranes. The activated G α subunits also re- main attached to the plasma membrane through a covalently attached palmitate moiety in the COOH terminal. All α subunits of the G

subfamily interact with the C2 domain of PLC- β mediating the trans- location of the enzyme to the plasma membrane.

The G βγ dimer has also been proposed to enhance

membrane localization of PLC- β by binding to the

pleckstrin homology (PH) domain in the NH

ter-

minal [53]. Membrane localization increases the hy-

drolysis of a minor membrane phospholipid, phos-

phatidylinositol-4,5-bisphosphate (PIP

) resulting

in the co-release of two second messengers: IP

and

diacylglycerol (DAG). The lipophilic DAG recruits

protein kinase C (PKC) to the plasma membrane

activating one branch of the phosphoinositide-de-

pendent signaling pathway. At the same time, IP

is

released into the cytosol where it mobilizes inter-

nal Ca

stores in the endoplasmic reticulum upon

binding to the IP

receptors (IP

R) [2, 48]. The de-

pletion of luminal Ca

stores stimulates the influx

of external Ca

to sustain the agonist signal and

refill the internal stores [51]. The signaling pathway

is turned off by intrinsic GTPase activity of the G α

subunit slowly hydrolyzing GTP back to GDP, which

allows reassembly with the G βγ dimer into an inac- tive complex.

Epidermal growth factor and hepatocyte growth factor are important agonists regulating liver growth and regeneration. These growth factors also acti- vate bifurcated signaling pathways, including Ca

mobilization in isolated hepatocytes. In this case, growth factor challenge evokes receptor dimeriza- tion resulting in autophosphorylation of the receptor on specific tyrosine residues. These phosphorylated protein domains are high affinity docking sites for SH2-containing proteins, like PLC- γ. After binding to the SH2-domain, PLC- γ is activated by receptor- mediated phosphorylation. Moreover, binding to growth factor receptors tethers the enzyme close to its substrate, PIP

, in the plasma membrane [2, 13, 53]. The end result of these events is an increase in IP

and DAG formation.

18.3

IP3-Dependent [Ca

]

Oscillations in Liver

At the single cell level, receptor-mediated activa- tion of the phosphoinositide signaling pathway of- ten generates complex spatiotemporal intracellular [Ca

]

responses. Peter Cobbold and coworkers were the first group to demonstrate that hormones coupled to PLC activation elicit a series of discrete, baseline-separated [Ca

]

spikes or oscillations in aequorin-injected hepatocytes [81, 82]. We have used Ca

-sensitive fluorescent indicators in com- bination with digital fluorescence imaging mi- croscopy and laser scanning confocal microscopy techniques to confirm these initial observations in isolated hepatocytes [59, 60, 71] and have adapted these approaches to extend the study of Ca

signal- ing into the intact liver [21, 49, 55, 56, 72–74].

In agreement with our previous studies con- ducted in primary cultured hepatocytes, challeng- ing the liver with a continuous perfusion of sub- maximal agonist concentrations evokes periodic [Ca

]

spikes in the individual hepatocytes of the perfused liver (Fig. 18.1). In both cultured cells and hepatocytes within the intact liver, the agonist con- centration determines the frequency of the [Ca

]

oscillations, whereas the amplitude and kinetics of the individual [Ca

]

oscillation are independent of agonist concentration [55, 59, 72]. This phenome- non has been termed frequency-modulation [1] and is thought to regulate Ca

-sensitive targets with greater fidelity than other types of [Ca

]

signals (e.g. amplitude-modulation), especially at low levels of agonist stimulation [71, 74]. Frequency encoded Ca

signals have been shown to be more effec- tive in controlling mitochondrial metabolism [24]

initiating gene expression [39] and capable of dif- ferentially activating transcription factors [17]. To use the information encoded in the incoming Ca

signals necessitates that downstream Ca

-sensitive proteins can decode or count the [Ca

]

spikes. The rates of activation and inactivation of PKC [46] or Ca

/calmodulin-dependent protein kinase II [16]

in response to oscillatory Ca

signals suggest that these enzymes are capable of discriminating be- tween Ca

signals that differ in spike frequency, converting the digitized Ca

response into discrete levels of kinase activity [16]. Moreover, we have shown that mitochondria can decode the frequency of Ca

signals into different time-averaged levels of NAD(P)H production; the result of a complex in- terplay between Ca

-activated intramitochondrial dehydrogenases and Ca

-dependent stimulation of the respiratory chain [24, 57].

The traces in Fig. 18.1 (a, b) illustrate two exam- ples of the agonist concentration exerting control over [Ca

]

signals in hepatocytes of the perfused liver. At low vasopressin concentrations, individual

Fig. 18.1 Vasopressin-evoked [Ca²⁺]_i signals in the intact per- fused liver. a, b Fura-2/AM-loaded livers were challenged with the indicated hormone concentrations. c [Ca²⁺]_i oscillations in

two hepatocytes displaced 70 µm along the hepatic plate. All traces are typical, single-cell [Ca²⁺]_i responses.

[Ca

]

spikes are separated by relatively long peri- ods during which [Ca

]

is maintained close to ba- sal values. Increasing the hormone concentration from 50 to 200 pM vasopressin evoked either an increase in the number of Ca

spikes per minute (Fig. 18.1a) or resulted in a sustained increase in [Ca

]

(Fig. 18.1b). Note that there is a difference in hormone sensitivity between the two hepatocytes in response to the initial agonist challenge.

The [Ca

]

spikes in individual hepatocytes of the perfused liver are also spatially organized as regenerative intracellular Ca

waves, with similar properties to those described for isolated hepato- cytes. In both cases, the rate of [Ca

]

rise was simi- lar throughout the cell, but the Ca

responses were offset in direct proportion to the distance from the site of Ca

wave initiation. Intracellular Ca

waves commenced predominately close to the basolateral membrane or sometimes occurred more diffusely throughout the cell (Fig. 18.4). The latter observa- tion may reflect Ca

waves propagating in the Z-di- rection or from out of the focal plane. Once initiated, intracellular Ca

waves propagate throughout the cell at a constant velocity and are unaffected by ago- nist type or concentration. The calculated rates of Ca

wave propagation were 15–25 µm/s in both iso- lated hepatocytes [60] and individual hepatocytes of the perfused liver [55]. Depleting extracellular Ca

for short periods prior to agonist challenge did not alter the rate of intracellular Ca

waves (unpub- lished observations) suggesting that Ca

influx is not mandatory. However, it should be noted the ex- tracellular Ca

omission did slow the rate of inter- cellular Ca

waves (see below).

18.4

Intercellular Ca

Waves

Ca

signals are not limited to the boundaries of the one cell, but rather the [Ca

]

increase can be com- municated to neighboring cells giving rise to inter- cellular Ca

waves. Ca

waves passing through multiple cells and up to 1 mm in distance have been reported in a variety of cell preparations, which preserved the functional integrity of the intact tis- sue [64] and in perfused organs [43, 49, 55, 84, 85].

Intercellular Ca

waves have been evoked by local or global application of Ca

-mobilizing hormones [55], mechanical stimulation of a single cell [63] or microinjection/photo release of Ca

-mobilizing second messengers [10, 63]. Intercellular Ca

waves can display remarkable multicellular organiza- tion, propagating as radial [55], circuitous or even spiral Ca

waves [26] at speeds up to 100 µm/s. A

potentially interesting property of some Ca

waves is their ability to pass between different cell types.

Such “heterotypic” calcium signaling has been dem- onstrated to occur between photocytes and their supporting cells, oocytes and follicular cells, mixed glia and in astrocyte/endothelial cell co-cultures [19, 38, 44, 62]. Intercellular Ca

waves in the as- trocyte syncitium have been reported to pass into neighboring neurons and modulate their excitabil- ity [45]. Conversely, electrical stimulation of neural circuits can trigger Ca

waves in glial cell popula- tions in both hippocampal slices [15] and the neu- romuscular junction [30, 52].

The mechanism whereby Ca

waves pass be- tween cells is the subject of some debate: both intra- cellular and extracellular pathways have been pro- posed. Calcium waves may propagate by diffusion of a second messenger through gap junctions or by the secretion of a Ca

-mobilizing agonist, poten- tially ATP. Evidence for a role of gap junctions has been provided by studies demonstrating blockage of intercellular Ca

waves by gap junction inhibitors [6, 75] or electroporation of antibodies to connexins [5], a gap junction constituent. Indeed, gap junctions are permeable to both Ca

and the second messen- ger, IP

[61]. Moreover, the extent of intercellular Ca

waves has been correlated with the expression levels of connexin proteins. Thus, intercellular Ca

waves are not observed in cell lines expressing low levels of connexins but occur in cultures transfected with connexins [12]. Heterogeneity in gap junction permeability between cells may provide an elegant means for “routing” Ca

waves through the tissue, while the level of connexin expression or subtype composition may regulate the rate of intercellular Ca

waves.

In other cell preparations, Ca

waves propagate

across cell-free boundaries and the path of these

Ca

waves can be influenced by the direction of

extracellular perfusate flow [28, 47]. Such studies

suggest that upon stimulation certain cell types se-

crete an extracellular message; most likely ATP that

induces Ca

increases in neighboring cells [23, 28,

47]. The field is further complicated by the observa-

tion that overexpressing connexins in gap-junction-

deficient cell lines enhances ATP release during

mechanical stimulation [14]. This discrepancy may

be explained by the fact that unpaired connexins or

hemichannels in the plasma membrane appear to

form Ca

-regulated ATP-secreting channels [22,

69]. Indeed, photorelease of caged IP

and the con-

sequential rise in Ca

has been shown to stimulate

ATP release, presumably mediated through con-

nexin hemichannels [8]. Moreover, focal mechani-

cal stimulation of single cultured hepatocytes has

been reported to release ATP evoking a [Ca

]

rise

in neighboring hepatocytes and bile ductal cells through the activation of metabotrophic purinergic receptors [65]. Finally, there is evidence that hor- monal stimulation can increase cell volume in the perfused liver [29, 76], which is a potent stimulus for ATP efflux in rat and human hepatocytes [18, 20]. Thus, it is possible that both gap junctions and ATP release are involved in propagating the agonist- evoked intercellular Ca

wave. The relative contri- butions of these different pathways in mediating the propagation of intercellular Ca

waves will most likely vary considerably depending upon the tissue and agonist type.

18.5

Intercellular Ca

Waves in the Perfused Liver

Remarkably, IP

-dependent Ca

signals are or- ganized at the multicellular level in the perfused

liver and do not occur asynchronously. The traces in Fig. 18.1c depict vasopressin-evoked [Ca

]

re- sponses in two hepatocytes separated by 70 µm along the same hepatic plate. The [Ca

]

spikes in the two cells have the same periodicity, but are phase-shifted in time. The mechanism underlying this observation is the coordinated propagation of intercellular Ca

waves from cell to cell along the hepatic plate (Figs. 18.2, 18.4). Thus, the rising phase of each [Ca

]

spike passes sequentially into neighboring hepatocytes.

Fig. 18.2 shows a series of confocal images of fluo-3 fluorescence in two adjacent hepatic lobules.

The periportal (PP) and pericentral (PC) zones were identified by infusing fluorescein-labeled bovine serum albumin (F-BSA) into the portal circulation and recording the flow pattern of the dye. F-BSA does not readily cross the hepatocyte plasma mem- brane or access the bile canaliculi, and thus is an excellent marker for the sinusoidal space. The PP zones are defined as those regions displaying the first increase in fluorescence during F-BSA infusion

Fig. 18.2 Fluo-3/AM-loaded liver was sequentially challenged with 1–10 nM glucagon (top) followed by 80 pM vasopressin (bottom). The images (597×406 µm) were acquired with laser

scanning confocal microscopy. Ca²⁺ increases are depicted in white and time(s) after hormone infusion is shown bottom right.

(Fig. 18.3). The white overlay superimposed onto the grayscale images in Figs. 18.2 and 18.4 depicts the sites of fluorescence increase at the indicated time points during hormone challenge.

Each vasopressin-evoked [Ca

]

spike initiates from a small number of hepatocytes located just outside the portal tract, and then propagates in a ra- dial fashion into the portal vein and outward to the pericentral region (Fig. 18.2, bottom panels). The initiator cells act like pacemakers entraining the fre- quency and spatial pattern of [Ca

]

oscillations for the entire lobule. These intercellular [Ca

]

waves are not self-propagating and require the continuous presence of the hormone to pass through the entire lobule. The direction of the translobular Ca

wave is periportal to pericentral during low hormone challenge. This orientation is also observed during retrograde perfusion via the hepatic vein [55], but is reversed at supramaximal vasopressin doses that generate sustained [Ca

]

increases. Thus, the path of Ca

wave propagation is independent of the di- rection of perfusion flow.

Coordinated intralobular [Ca

]

waves were routinely observed when livers were stimulated with vasopressin or α

-adrenergic agonists, but not for all agonists reported to elevate [Ca

]

in isolated hepa- tocytes. Glucagon is a key gluconeogenic and glycog- enolytic hormone that regulates glucose output from the liver. Several groups have previously shown that physiological levels of glucagon can evoke [Ca

]

increases in hepatocyte suspensions [11, 68], which may be mediated by a rise in either IP

or cAMP [68, 77]. Challenging the perfused liver with 1–10 nM glucagon elicited asynchronous [Ca

]

oscilla- tions in a limited number of hepatocytes (Fig. 18.2, top panels) that were randomly distributed in both periportal and pericentral regions. These [Ca

]

signals propagated along the hepatic plate for rela- tively short distances (several cells) before dissipat- ing. Moreover, these asynchronous Ca

responses were similar to the Ca

signals obtained with sub- threshold vasopressin doses (>10 pM). Supraphysi- ological levels of glucagon (100 nM) did evoke a sustained increase in [Ca

]

in all of the cells in the hepatic lobule. However, these were non-oscillatory Ca

signals and intercellular Ca

waves followed the progression of the hormone infusion. These data suggest that physiological doses of glucagon do not evoke an increase in IP

production sufficient to fully mobilize internal Ca

stores.

Stimulating the nerve tracts surrounding the portal vein enhances glucose and lactate release from the perfused liver, while inducing an overflow of norepinephrine into the hepatic vein [27, 67].

These data are consistent with hepatic sympathetic nerves controlling glycogen breakdown in the liver.

Most postganglionic sympathetic neurons store and co-release ATP along with norepinephrine [80].

Norepinephrine binds to α

-adrenergic receptors on the hepatocytes stimulating IP

-dependent [Ca

]

signals with a consequent increase in phosphorylase a activity. On the other hand, little is known about the role of ATP in controlling glucose metabolism.

In our hands, low levels of ATP (1–10 µM) evoked [Ca

]

oscillations in hepatocytes mainly near the portal tract and these Ca

waves propagated across a third to a half of the lobule before termi- nating (unpublished observations). The pattern of Ca

responses was not due to an uneven distribu- tion of purinergic receptors; maximal ATP (100 µM) challenge evoked a sustained [Ca

]

increase in all hepatocytes in the lobule [49]. It is possible that during low ATP challenge the ligand is degraded by ecto-ATPase before reaching the pericentral hepa-

Fig. 18.3 Periportal (PP) and pericentral (PC) zones of the he- patic lobule are determined by infusing fluorescein-conjugated bovine serum albumin (F-BSA) into the portal vein. Time(s) rela-

tive to F-BSA infusion is shown top right. The sites of initial fluo- rescence increase are indicated (arrows).

tocytes. Since intercellular Ca

waves require the continuous presence of agonist to propagate across the entire lobule, ATP hydrolysis could explain the early termination of the Ca

wave. The functional consequences of these “truncated” Ca

waves are currently unknown, but may be important in main- taining some of the distinct metabolic zones in the hepatic lobule.

When examined with sufficient spatial and temporal resolution, intracellular and intercellu- lar [Ca

]

waves were easily observed propagating from cell to cell along the hepatic plates (Fig. 18.4).

Intercellular [Ca

]

waves show a clear delay at each cell–cell boundary, which presumably reflects the period required to regenerate a propagating Ca

- mobilizing second messenger in the adjacent hepa- tocyte. The propagation of intercellular Ca

waves, in both isolated hepatocyte triplets [75] and the perfused liver (unpublished observations), are sen- sitive to gap junction inhibitors and experimental maneuvers designed to disrupt cell-to cell-contacts.

Moreover, vasopressin-evoked intercellular Ca

waves are observed after prior desensitization of purinergic receptors with supramaximal ATP con- centrations [49]. Taken together, these data suggest that intercellular Ca

waves in liver are predomi- nately mediated through gap junctions. The second messenger involved in propagating the Ca

signal between hepatocytes has yet to be determined; how- ever, hepatic gap junctions are permeable to both Ca

and IP

[61]. Although the rate of propaga- tion of intracellular Ca

waves was independent of hormone concentration, the time delay for the Ca

signal to pass between cells was inversely related to the agonist dose. Thus, the strength of the extracel-

lular stimuli, in the intact liver, can be encoded in both the frequency of [Ca

]

spiking and the rate at which these Ca

signals propagate across the lob- ule. Frequency-modulated Ca

signals will most likely be the determining factor regulating the in- tensity of the cellular response in individual hepa- tocytes, whereas the rate of intercellular Ca

wave propagation may determine the degree of coordina- tion between the lobular zones.

As mentioned previously, [Ca

]

oscillations oc- cur asynchronously throughout the lobule at sub- threshold vasopressin concentrations, the result of limited intercellular Ca

waves. Challenging the liver with bradykinin, to evoke Ca

mobilization in hepatic sinusoidal endothelial cells, markedly increased the frequency of vasopressin-induced [Ca

]

oscillations plus the rate and extent to which the intercellular Ca

waves propagated across the lobule. The effects of bradykinin were mimicked by nitric oxide donors and blocked by inhibitors of nitric oxide formation [49]. These data suggest that crosstalk between nitric oxide and Ca

signaling pathways may be another important mechanism in fine-tuning liver function.

18.6

Ca

and Mitochondrial Metabolism

A rise in [Ca

]

stimulates contractile, secretory or metabolic pathways augmenting ATP demand in the cytosol. To maintain cellular energy homeos- tasis and cell viability, the rate of ATP production must match utilization. This requires coordinating

Fig. 18.4 Confocal images (143×105 µm) illustrating the cell-to-cell propagation of [Ca²⁺]_i oscillations. Dots show the initiating hepatocytes and arrows depict the ap- parent direction of the Ca²⁺

waves. Ca²⁺ increases are depicted in white and time(s) after vasopressin (100 nM) infusion is shown top left.

the activation of mitochondrial oxidative phosphor- ylation with the flux rate through cytosolic ATP-re- quiring reactions. In many cell types, mitochondria are strategically localized close to Ca

release sites, such that [Ca

]

increases from either internal Ca

stores or Ca

influx across the plasma membrane can be rapidly transported into the mitochondrial matrix [24, 37, 41, 54, 57, 58]. The consequent eleva- tion in mitochondrial Ca

([Ca

]

) stimulates the Ca

-sensitive intramitochondrial dehydrogenases, resulting in elevation of NAD(P)H [24, 41, 57, 58]. The preferential coupling between increases of [Ca

]

and [Ca

]

is one proposed mechanism to coor- dinate mitochondrial ATP production with cellular energy demand [41, 58]. However, efficient transfer of Ca

from the cytosol to the mitochondria only occurs during the rising phase of a Ca

spike, when a gradient of high [Ca

]

that is sufficient to activate mitochondrial Ca

uptake develops close to the re- lease channel [24, 37, 54, 57, 58]. Consequently, mi- tochondrial metabolism is maximally stimulated by periodic [Ca

]

oscillations at frequencies above 0.5 spikes per minute. In contrast, sustained increases in [Ca

]

evoked by maximal hormone doses can only transiently elevate [Ca

]

, and consequently do not sustain the activity of Ca

-sensitive intrami- tochondrial dehydrogenases for a prolonged period [24, 57, 58].

Agonist-evoked [Ca

]

responses can also regu- late the activity of the aspartate/glutamate carrier in the mitochondrial inner membrane [36]. This transporter is an integral component of the malate/

aspartate shuttle, which transfers cytosolic NADH into the mitochondrial matrix for oxidation. Thus, Ca

-dependent stimulation of intramitochondrial dehydrogenases and the transport of cytosolic re- ducing equivalents work in a concerted fashion to increase supply of substrates for oxidative me- tabolism. In isolated hepatocytes, Ca

-mobilizing hormones stimulate an increase in mitochondrial membrane potential ( ∆Ψ

), which is the potential energy used to drive ATP production [57, 58]. This appears to occur through a Ca

-dependent inhibi- tion of mitochondrial pyrophosphatases leading to K

entry into the mitochondrial matrix, which stim- ulates volume increases and electron flux through the respiratory chain [25]. Finally, Ca

can directly stimulate F

F

-ATPase synthetic activity and thus, a higher rate of ATP production [70]. Taken together, these data suggest that Ca

ions can coordinate the entire ATP synthesis pathway from substrate supply to the finished product.

18.7

Hormone-Evoked Redox Changes in the Perfused Liver

We have previously shown that the activation of Ca

-sensitive intramitochondrial dehydrogenases can be monitored in real time by changes in the relative redox state of mitochondrial pyridine and flavin nucleotides [21, 24, 57]. Ca

-mobilizing hor- mones stimulate mitochondrial NADH production resulting in an increase in cellular autofluorescence at 360 nm. The reduced flavin has a lower fluores- cent signal at 470 nm than the oxidized form, thus an opposite signal change occurs for flavoproteins during hormone challenge (Fig. 18.5).

Cellular flavoprotein fluorescence originates predominately from the mitochondrial matrix, since the flavin fluorescence is quenched in most cytosolic proteins [66]. It has been estimated that lipoamide dehydrogenases and electron transfer fla- voproteins constitute 75% of total flavoprotein fluo- rescence in isolated mitochondria [34, 35]. Lipoam- ide dehydrogenase is an integral component of the Ca

-sensitive intramitochondrial pyruvate and 2-oxoglutarate dehydrogenases [50] and their fla- vin cofactors are in direct equilibrium with the mi- tochondrial NAD

/NADH pool. On the other hand, NADH fluorescence can derive from both cytosolic and mitochondrial compartments. We have utilized 2-photon confocal microscopy to evaluate the rela- tive contribution of the cytosolic and mitochondrial pools to the overall NADH fluorescence signal. The image in Fig. 18.5a was acquired with multi-photon excitation and shows the endogenous pyridine nu- cleotide fluorescence in a fluo4/AM-loaded intact liver. The NAD(P)H signal originates primarily from the mitochondria, the bright punctuate structures within the two hepatocytes. The infrared absorp- tion for fluo4 and pyridine nucleotide are spectrally distinct, therefore fluo4 does not interfere with py- ridine nucleotide measurements. Hence, it should be possible to monitor hormone-evoked NADH increases with multi-photon excitation simultane- ously with Ca

responses using fluo4 and a 488 nm argon laser line.

Challenging perfused livers with vasopressin

concentrations that evoke oscillatory Ca

respons-

es resulted in a sustained increase in NAD(P)H pro-

duction, which was paralleled by a reduction in mi-

tochondrial flavoproteins (Fig. 18.5b). At the end of

the experiment, ketone bodies were infused into the

portal vein to evaluate the relative change in mito-

chondrial redox couples (Fig. 18.5b). We have show

previously that the mitochondrial NAD

/NADH

ratio can be manipulated experimentally with ac- etoacetate and β-hydroxybutyrate [24, 57]. Ketone bodies are interconverted by the mitochondrial ma- trix enzyme, β-hydroxybutyrate dehydrogenase, which utilizes mitochondrial NADH and NAD

, re- spectively. In this experiment, vasopressin evoked an approximately 50% reduction in mitochondrial NADH and FAD redox couples in equilibrium with β-hydroxybutyrate dehydrogenase.

The vasopressin-evoked rise in NAD(P)H fluo- rescence propagated across the lobule as a redox wave. In agreement with our isolated hepatocyte studies, hormone-dependent increases in NAD(P)H decayed more slowly than the [Ca

]

spike, result- ing in a sustained mitochondrial response at low frequency of Ca

spiking (not shown). Stimula- tion with sub-threshold agonist doses generates asynchronous [Ca

]

oscillations, which do not propagate as coordinated intercellular Ca

waves across the lobule. Under these conditions, the mi- tochondrial NAD(P)H responses are not sustained and limited to a few cells surrounding the portal tract. Increasing the agonist dose above the thresh- old (50–150 pM vasopressin) initiated a coordinated intercellular Ca

wave and a concomitant NAD(P)H wave across the lobule. These studies strongly sug- gest that intercellular Ca

waves allow the entire lobule to respond to agonist stimulation in a coor- dinated fashion. Moreover, these studies raise the possibility that other Ca

-dependent processes in the liver are also regulated in a similar manner.

18.8 Summary

The strength of the extracellular agonist signal is thought to be encoded primarily by the frequency of [Ca

]

spikes (temporal aspect), whereas the in- tracellular [Ca

]

wave serves to propagate the Ca

signal, thus ensuring that the entire cell is exposed to the full strength of the Ca

signal (spatial as- pect). In the intact liver, agonist strength may also be conveyed by the rate of intercellular Ca

wave propagation. Moreover, the translobular movement of Ca

provides a mechanism to coordinate the dif- ferent metabolic zones in the liver. The ensemble of Ca

signals is recognized by downstream Ca

-sen- sitive pathways, such as mitochondrial NADH pro- duction, and converted into steady-state changes in metabolic output.

Selected Reading

Rhee SG. Regulation of phosphoinositide-specific phospholi- pase C. Annu Rev Biochem 2001;70:281–312. (A concise and readable review highlighting the enzymes involved in phos- phoinositide signaling pathways.)

Berridge MJ. Inositol trisphosphate and calcium signalling. Na- ture 1993;361:315–325. (A short, but detailed primer devot- ed to IP3-dependent Ca²⁺ signaling.)

Duchen MR. Contributions of mitochondria to animal physiol- ogy: from homeostatic sensor to calcium signalling and cell death. J Physiol (Lond) 1999;516:1–17. (A general review de- scribing mitochondrial Ca²⁺ homeostasis.)

Hajnóczky G, Robb-Gaspers LD, Seitz MB, Thomas AP. Decoding of cytosolic calcium oscillations in the mitochondria. Cell 1995;82:415–424.

Fig. 18.5 a Two-photon image of pyridine nucleotide fluores- cence in the intact liver. b Simultaneous measurement of flavin (blue) and pyridine (gray) nucleotide fluorescence changes dur-

ing vasopressin challenge. Acetoacetate (AcAc) and β-hydroxy- butyrate (β-HB) are added to assess the size of the mitochondrial redox pool.

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