Astrocyte sodium signaling and neuro-metabolic coupling in the brain.

At tripartite synapses, astrocytes undergo calcium signaling in response to release of neurotransmitters and this calcium signaling has been proposed to play a critical role in neuron-glia interaction. Recent work has now firmly established that, in addition, neuronal activity also evokes sodium transients in astrocytes, which can be local or global depending on the number of activated synapses and the duration of activity. Furthermore, astrocyte sodium signals can be transmitted to adjacent cells through gap junctions and following release of gliotransmitters. A main pathway for activity-related sodium influx into astrocytes is via high-affinity sodium-dependent glutamate transporters. Astrocyte sodium signals differ in many respects from the well-described glial calcium signals both in terms of their temporal as well as spatial distribution. There are no known buffering systems for sodium ions, nor is there store-mediated release of sodium. Sodium signals thus seem to represent rather direct and unbiased indicators of the site and strength of neuronal inputs. As such they have an immediate influence on the activity of sodium-dependent transporters which may even reverse in response to sodium signaling, as has been shown for GABA transporters for example. Furthermore, recovery from sodium transients through Na(+)/K(+)-ATPase requires a measurable amount of ATP, resulting in an activation of glial metabolism. In this review, we present basic principles of sodium regulation and the current state of knowledge concerning the occurrence and properties of activity-related sodium transients in astrocytes. We then discuss different aspects of the relationship between sodium changes in astrocytes and neuro-metabolic coupling, putting forward the idea that indeed sodium might serve as a new type of intracellular ion signal playing an important role in neuron-glia interaction and neuro-metabolic coupling in the healthy and diseased brain.

Role of the JNK pathway in NMDA-mediated excitotoxicity of cortical neurons.

Excitotoxic insults induce c-Jun N-terminal kinase (JNK) activation, which leads to neuronal death and contributes to many neurological conditions such as cerebral ischemia and neurodegenerative disorders. The action of JNK can be inhibited by the D-retro-inverso form of JNK inhibitor peptide (D-JNKI1), which totally prevents death induced by N-methyl-D-aspartate (NMDA) in vitro and strongly protects against different in vivo paradigms of excitotoxicity. To obtain optimal neuroprotection, it is imperative to elucidate the prosurvival action of D-JNKI1 and the death pathways that it inhibits. In cortical neuronal cultures, we first investigate the pathways by which NMDA induces JNK activation and show a rapid and selective phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7), whereas the only other known JNK activator, mitogen-activated protein kinase kinase 4 (MKK4), was unaffected. We then analyze the action of D-JNKI1 on four JNK targets containing a JNK-binding domain: MAPK-activating death domain-containing protein/differentially expressed in normal and neoplastic cells (MADD/DENN), MKK7, MKK4 and JNK-interacting protein-1 (IB1/JIP-1).

Glucose and lactate are equally effective in energizing activity-dependent synaptic vesicle turnover in purified cortical neurons.

This study examines the role of glucose and lactate as energy substrates to sustain synaptic vesicle cycling. Synaptic vesicle turnover was assessed in a quantitative manner by fluorescence microscopy in primary cultures of mouse cortical neurons. An electrode-equipped perfusion chamber was used to stimulate cells both by electrical field and potassium depolarization during image acquisition. An image analysis procedure was elaborated to select in an unbiased manner synaptic boutons loaded with the fluorescent dye N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43). Whereas a minority of the sites fully released their dye content following electrical stimulation, others needed subsequent K(+) depolarization to achieve full release. This functional heterogeneity was not significantly altered by the nature of metabolic substrates. Repetitive stimulation sequences of FM1-43 uptake and release were then performed in the absence of any metabolic substrate and showed that the number of active sites dramatically decreased after the first cycle of loading/unloading. The presence of 1 mM glucose or lactate was sufficient to sustain synaptic vesicle cycling under these conditions. Moreover, both substrates were equivalent for recovery of function after a phase of decreased metabolic substrate availability. Thus, lactate appears to be equivalent to glucose for sustaining synaptic vesicle turnover in cultured cortical neurons during activity.

Relationship between L-glutamate-regulated intracellular Na+ dynamics and ATP hydrolysis in astrocytes.

Glutamate uptake into astrocytes and the resulting increase in intracellular Na+ (Na+(i)) have been identified as a key signal coupling excitatory neuronal activity to increased glucose utilization. Arguments based mostly on mathematical modeling led to the conclusion that physiological concentrations of glutamate more than double astrocytic Na+/K+-ATPase activity, which should proportionally increase its ATP hydrolysis rate. This hypothesis was tested in the present study by fluorescence monitoring of free Mg2+ (Mg2+(i)), a parameter that inversely correlates with ATP levels. Glutamate application measurably increased Mg2+(i) (i.e. decreased ATP), which was reversible after glutamate washout. Na+(i) and ATP changes were then directly compared by simultaneous Na+(i) and Mg2+ imaging. Glutamate increased both parameters with different rates and blocking the Na+/K+-ATPase during the glutamate-evoked Na+(i) response, resulted in a drop of Mg2+(i) levels (i.e. increased ATP). Taken together, this study demonstrates the tight correlation between glutamate transport, Na+ homeostasis and ATP levels in astrocytes.

Early acquisition of typical metabolic features upon differentiation of mouse neural stem cells into astrocytes.

Specific metabolic features, such as glutamate reuptake, have been associated with normal functions of mature astrocytes. In this study, we examined whether these characteristics are acquired together with classical phenotypic markers of differentiated astrocytes. Differentiation of E14 mouse neurospheres into astrocytes was induced by the addition of fetal bovine serum (FBS). Degree of differentiation was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence for both GFAP and nestin. Neural stem cells expressed nestin but not GFAP, while differentiated astrocytes were immunopositive for GFAP but displayed low levels of nestin expression. A strong increase in the expression of the glutamate transporter GLAST and the monocarboxylate transporter MCT1 accompanied phenotypic changes. In addition, active glutamate transport appeared in differentiated astrocytes, as well as their capacity to increase aerobic glycolysis in response to glutamate. Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor, but not interleukin-6, triggered the expression of phenotypic and morphological characteristics of astrocytes. In addition, exposure to LIF led to the appearance of metabolic features typically associated with astrocytes. Altogether, our results show that acquisition of some specific metabolic features by astrocytes occurs early in their differentiation process and that LIF represents a candidate signal to induce their expression.

Insights into the mechanisms of ifosfamide encephalopathy: drug metabolites have agonistic effects on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors and induce cellular acidification in mouse cortical neurons.

Therapeutic value of the alkylating agent ifosfamide has been limited by major side effects including encephalopathy. Although the underlying biochemical processes of the neurotoxic side effects are still unclear, they could be attributed to metabolites rather than to ifosfamide itself. In the present study, the effects of selected ifosfamide metabolites on indices of neuronal activity have been investigated, in particular for S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA). Because of structural similarities of SCMC with glutamate, the Ca(2+)(i) response of single mouse cortical neurons to SCMC and TDGA was investigated. SCMC, but not TDGA, evoked a robust increase in Ca(2+)(i) concentration that could be abolished by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but only partly diminished by the N-methyl-D-aspartate receptor antagonist 10,11-dihydro-5-methyl-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK=801). Cyclothiazide (CYZ), used to prevent AMPA/kainate receptor desensitization, potentiated the response to SCMC. Because activation of AMPA/kainate receptors is known to induce proton influx, the intracellular pH (pH(i)) response to SCMC was investigated. SCMC caused a concentration-dependent acidification that was amplified by CYZ. Since H(+)/monocarboxylate transporter (MCT) activity leads to similar cellular acidification, we tested its potential involvement in the pH(i) response. Application of the lactate transport inhibitor quercetin diminished the pH(i) response to SCMC and TDGA by 43 and 51%, respectively, indicating that these compounds may be substrates of MCTs. Taken together, this study indicates that hitherto apparently inert ifosfamide metabolites, in particular SCMC, activate AMPA/kainate receptors and induce cellular acidification. Both processes could provide the biochemical basis of the observed ifosfamide-associated encephalopathy.

Identification of a mammalian H(+)-myo-inositol symporter expressed predominantly in the brain.

Inositol and its phosphorylated derivatives play a major role in brain function, either as osmolytes, second messengers or regulators of vesicle endo- and exocytosis. Here we describe the identification and functional characterization of a novel H(+)-myo- inositol co-transporter, HMIT, expressed predominantly in the brain. HMIT cDNA encodes a 618 amino acid polypeptide with 12 predicted transmembrane domains. Functional expression of HMIT in Xenopus oocytes showed that transport activity was specific for myo-inositol and related stereoisomers with a Michaelis-Menten constant of approximately 100 microM, and that transport activity was strongly stimulated by decreasing pH. Electrophysiological measurements revealed that transport was electrogenic with a maximal transport activity reached at pH 5.0. In rat brain membrane preparations, HMIT appeared as a 75-90 kDa protein that could be converted to a 67 kDa band upon enzymatic deglycosylation. Immunofluorescence microscopy analysis showed HMIT expression in glial cells and some neurons. These data provide the first characterization of a mammalian H(+)-coupled myo- inositol transporter. Predominant central expression of HMIT suggests that it has a key role in the control of myo-inositol brain metabolism.

Effects of glial glutamate transporter inhibitors on intracellular Na+ in mouse astrocytes.

The effects of inhibitors of the glial Na+/glutamate co-transporter on the intracellular Na+ concentration ([Na+](i)) were investigated in mouse cortical astrocytes. [Na+](i) was monitored by fluorescence microscopy on single astrocytes using the Na+-sensitive probe sodium-binding benzofuran isophtalate. Application of the competitive inhibitors threo-beta-hydroxyaspartate (THA) and trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC) resulted in robust and reversible increases in [Na+](i) that were comparable in shape to the response to glutamate but about twice lower in amplitude. As previously observed with glutamate, the amplitude of the [Na+](i) response to these compounds was concentration-dependent with EC(50) values of 11.1 microM (THA) and 7.6 microM (t-PDC), as was the initial rate of [Na+](i) rise (EC(50) values of 14.8 microM for THA and 11.5 microM for t-PDC). Both compounds diminished the response to subsequent glutamate applications, possibly because of an inhibitory effect of the intracellularly-accumulated compounds. In comparison, the newly-developed compound threo-beta-benzyloxyaspartate (TBOA) alone did not cause any significant alteration of [Na+](i) up to a concentration of 500 microM . TBOA inhibited the [Na+](i) response evoked by 200 microM glutamate in a concentration-dependent manner with IC(50) values of 114 and 63 microM, as measured on the amplitude and the initial rate, respectively. The maximum inhibition of glutamate-evoked [Na+](i) increase by TBOA was approximately 70%. The residual response persisted in the presence of a non-NMDA receptor antagonist or the inhibitor of the GLT-1 glutamate transporters, dihydrokainate (DHK). In view of the complete reversibility of its effects, TBOA represents a very useful pharmacological tool for studies of glutamate transporters.

A quantitative analysis of L-glutamate-regulated Na+ dynamics in mouse cortical astrocytes: implications for cellular bioenergetics.

The mode of Na+ entry and the dynamics of intracellular Na+ concentration ([Na+]i) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. An elevation of [Na+]i was evoked by glutamate, whose amplitude and initial rate were concentration dependent. The glutamate-evoked Na+ increase was primarily due to Na+-glutamate cotransport, as inhibition of non-NMDA ionotropic receptors by 6-cyano-7-nitroquinoxiline-2,3-dione (CNQX) only weakly diminished the response and D-aspartate, a substrate of the glutamate transporter, produced [Na+]i elevations similar to those evoked by glutamate. Non-NMDA receptor activation could nevertheless be demonstrated by preventing receptor desensitization using cyclothiazide. Thus, in normal conditions non-NMDA receptors do not contribute significantly to the glutamate-evoked Na+ response. The rate of Na+ influx decreased during glutamate application, with kinetics that correlate well with the increase in [Na+]i and which depend on the extracellular concentration of glutamate. A tight coupling between Na+ entry and Na+/K+ ATPase activity was revealed by the massive [Na+]i increase evoked by glutamate when pump activity was inhibited by ouabain. During prolonged glutamate application, [Na+]i remains elevated at a new steady-state where Na+ influx through the transporter matches Na+ extrusion through the Na+/K+ ATPase. A mathematical model of the dynamics of [Na+]i homeostasis is presented which precisely defines the critical role of Na+ influx kinetics in the establishment of the elevated steady state and its consequences on the cellular bioenergetics. Indeed, extracellular glutamate concentrations of 10 microM already markedly increase the energetic demands of the astrocytes.

Roles of Na(+)-Ca2+ exchange and of mitochondria in the regulation of presynaptic Ca2+ and spontaneous glutamate release.

The release of neurotransmitter from presynaptic terminals depends on an increase in the intracellular Ca2+ concentration ([Ca2+]i). In addition to the opening of presynaptic Ca2+ channels during excitation, other Ca2+ transport systems may be involved in changes in [Ca2+]i. We have studied the regulation of [Ca2+]i in nerve terminals of hippocampal cells in culture by the Na(+)-Ca2+ exchanger and by mitochondria. In addition, we have measured changes in the frequency of spontaneous excitatory postsynaptic currents (sEPSC) before and after the inhibition of the exchanger and of mitochondrial metabolism. We found rather heterogeneous [Ca2+]i responses of individual presynaptic terminals after inhibition of Na(+)-Ca2+ exchange. The increase in [Ca2+]i became more uniform and much larger after additional treatment of the cells with mitochondrial inhibitors. Correspondingly, sEPSC frequencies changed very little when only Na(+)-Ca2+ exchange was inhibited, but increased dramatically after additional inhibition of mitochondria. Our results provide evidence for prominent roles of Na(+)-Ca2+ exchange and mitochondria in presynaptic Ca2+ regulation and spontaneous glutamate release.

Acute application of the tricyclic antidepressant desipramine presynaptically stimulates the exocytosis of glutamate in the hippocampus.

Tricyclic antidepressants (e.g., imipramine, desipramine) are currently used in the treatment of mood disorders such as depression. At the cellular level they inhibit the re-uptake of the exocytosed monoamines serotonin and noradrenaline. However, they also stimulate phospholipase C activity and the production of the second messenger inositol 1,4,5-trisphosphate. Since phospholipase C activation can also lead to the production of the protein kinase C activator diacylglycerol, we have undertaken experiments to see whether acutely applied desipramine could change the synaptic strength of neurons in a protein kinase C-dependent manner. Experiments performed with cultured hippocampal neurons dissociated from neonatal rats revealed that desipramine rapidly enhanced the spontaneous vesicular release of glutamate. This was observed by measuring the frequency of tetrodotoxin-resistant spontaneous excitatory postsynaptic currents. Analysis of amplitude distribution histograms indicated a presynaptic site of action. The protein kinase inhibitor staurosporine and down-regulation of protein kinase C activity greatly reduced the desipramine-dependent enhancement of the frequency of tetrodotoxin-resistant spontaneous excitatory postsynaptic currents. This presynaptic modulation requires SNARE proteins because cleavage of SNAP-25 with the botulinum neurotoxin A strongly reduced the desipramine-induced glutamate release. Thus, acute applications of desipramine stimulated the ongoing neurotransmitter release pathway, probably by activating protein kinase C. Our data indicate that tricyclic antidepressant drugs not only act on serotoninergic and/or noradrenergic cells but can also modify the activity of glutamatergic neurons.

Permissive role of cAMP in the oscillatory Ca2+ response to inositol 1,4,5-trisphosphate in rat hepatocytes.

Rat hepatocytes respond to alpha-adrenergic stimulation by intracellular production of myo-inositol 1,4,5-trisphosphate (IP3) which stimulates the periodic release and reuptake of intracellular store (IS) Ca2+. The generation of these Ca2+ oscillations was investigated by simultaneously monitoring Ca2+ changes in the cytosol and IS by combined fluorescence microscopy and whole-cell patch clamp. Intracellular IP3 perfusion (1-50 microM in the pipette) produced three types of Ca2+ response: understimulation, oscillations and overstimulation, i.e. with Ca2+ levels not returning to baseline. In a total of 57 experiments, only three displayed oscillations during continuous IP3 infusion, in a narrow range of IP3 concentration centred around 5-8 microM in the pipette. In oscillating cells, cytosolic Ca2+ spikes were synchronized with transient Ca2+ depletions of the IS, consistent with a direct exchange of Ca2+ between the two compartments. Application of 8-Br-cAMP to cells infused with IP3 increased the probability of eliciting Ca2+ oscillations by a factor of 4-5 for IP3 concentrations in the range 1-10 microM, whereas IP3 concentrations above 10 microM always resulted in overstimulation. IP3 photorelease experiments and measurements of IS Ca2+ content indicated that 8-Br-cAMP enhanced the affinity of the IP3 receptor and increased the pool of releasable Ca2+. We propose that cAMP has a permissive role in the generation of IP3-induced Ca2+ oscillations by extending the window of IP3 concentrations able to elicit oscillations.

Involvement of calmodulin in the activation of store-operated Ca2+ entry in rat hepatocytes.

The possible participation of calmodulin in the activation of store-operated Ca2+ entry (SOC) in single rat hepatocytes was investigated microspectrofluorimetrically. SOC was triggered after discharging intracellular Ca2+ stores using the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin in the absence of external Ca2+. Re-admission of bath Ca2+ caused a rapid and pronounced Ca2+ entry. The calmodulin antagonists calmidazolium or CGS 9343B applied before the thapsigargin treatment inhibited SOC, whereas they were ineffective when added after the thapsigargin-induced Ca2+ transient. This study suggests that activation of calmodulin after the elevation of cytosolic Ca2+ associated with the emptying of Ca2+ stores is involved in the triggering of SOC in hepatocytes.

Perturbation of myo-inositol-1,4,5-trisphosphate levels during agonist-induced Ca2+ oscillations.

Agonist-induced Ca2+ oscillations in rat hepatocytes involve the production of myo-inositol-1,4,5-trisphosphate (IP3), which stimulates the release of Ca2+ from intracellular stores. The oscillatory frequency is conditioned by the agonist concentration. This study investigated the role of IP3 concentration in the modulation of oscillatory frequency by using microinjected photolabile IP3 analogs. Photorelease of IP3 during hormone-induced oscillations evoked a Ca2+ spike, after which oscillations resumed with a delay corresponding to the period set by the agonists. IP3 photorelease had no influence on the frequency of oscillations. After photorelease of 1-(alpha-glycerophosphoryl)-D-myo-inositol-4,5-diphosphate (GPIP2), a slowly metabolized IP3 analog, the frequency of oscillations initially increased by 34% and declined to its original level within approximately 6 min. Both IP3 and GPIP2 effects can be explained by their rate of degradation: the half-life of IP3, which is a few seconds, can account for the lack of influence of IP3 photorelease on the frequency, whereas the slower metabolism of GPIP2 allowed a transient acceleration of the oscillations. The phase shift introduced by IP3 is likely the result of the brief elevation of Ca2+ during spiking that resets the IP3 receptor to a state of maximum inactivation. A mathematical model of Ca2+ oscillations is in satisfactory agreement with the observed results.

Modulation of hormone-induced calcium oscillations by intracellular pH in rat hepatocytes.

Single isolated rat hepatocytes were used to investigate the influence of intracellular pH (pHi) on hormone-induced cytosolic Ca2+ oscillations, using videofluorescence microscopy. Although pHi did not vary after alpha-adrenergic stimulation, manipulations of pHi induced pronounced alterations in the frequency of oscillations. Increasing the resting pHi with ammonium chloride (5-20 mM), trimethylammonium (2-10 mM), or triethylammonium (1.2-8 mM) reduced the frequency of oscillations. A change in pHi of > 0.25 was sufficient to reversibly inhibit oscillations. This effect could be overcome by increasing the agonist concentration or by adding 8-bromoadenosine 3',5'-cyclic monophosphate, an agent known to potentiate the alpha-adrenergic response. Cellular acidification, obtained by the ammonium prepulse method as well as by application of acetate or the ionophore nigericin, in the continuous presence of agonist was accompanied by a modest frequency increase of the oscillations, leading in some cases to an overstimulated state. This study indicates that pHi, within a range of values expected to occur in vivo (0.1-0.2 pH units), exerts a chronotropic effect on phenylephrine-induced Ca2+ oscillations. In contrast, oscillations induced by ADP or vasopressin were pHi invariant.

Agonist-specific behaviour of the intracellular Ca2+ response in rat hepatocytes.

A variety of agonists stimulate in hepatocytes a response that takes the shape of repetitive cytosolic free Ca2+ transients called Ca2+ oscillations. The shape of spikes and the pattern of oscillations in a given cell differ depending on the agonist of the phosphoinositide pathway that is applied. In this study, the response of individual rat hepatocytes to maximal stimulation by arginine vasopressin (AVP), phenylephrine and ADP was investigated by fluorescence microscopy and flash photolysis. Hepatocytes loaded with Ca2+-sensitive probes were stimulated with a first agonist to evoke a maximal response, and then a second agonist was added. When phenylephrine or ADP was used as the first agonist, AVP applied subsequently could elicit an additional response, which did not happen when AVP was first applied and phenylephrine or ADP was applied later. Cells microinjected with caged myo-inositol 1,4,5-trisphosphate (IP3) were challenged with the different agonists and, when a maximal response was obtained, photorelease of IP3 was triggered. Cells maximally stimulated with AVP did not respond to IP3 photorelease, whereas those stimulated with phenylephrine or ADP responded with a fast Ca2+ spike above the elevated steady-state level, which was followed by an undershoot. In contrast, with all three agonists, IP3 photorelease triggered at the top of an oscillatory Ca2+ transient was able to mobilize additional Ca2+. These experiments indicate that the differential response of cells to agonists is found not only during Ca2+ oscillations but also during maximal agonist stimulation and that potency and efficacy differences exist among agonists.

A critical evaluation of in situ measurement of mitochondrial electrical potentials in single hepatocytes.

The range of applicability and the critical parameters involved in the assessment of mitochondrial electrical potential (delta psi mit) using epifluorescence microscopy were evaluated based on both theoretical and experimental analysis. Rat hepatocytes loaded with the potential-dependent fluorescent dye rhodamine 123 exhibited the expected heterogeneity of fluorescence distribution with dark regions corresponding to the nucleus and bright regions corresponding to the mitochondria-rich cytosol. Calibration of the signal was performed by permeabilizing the cell membrane for monovalent cations using nystatin and gramicidin, and equilibrating the cell with a K(+)-free bath solution. A voltage-clamp at defined delta psi mit was then achieved after addition of valinomycin in the presence of different K+ concentrations in the bath. Theoretical analysis indicated that the ratio of fluorescence intensity measured in mitochondria-rich and mitochondria-poor regions of cell was related with delta psi mit and yielded quantitative estimates of electrical potential with an accuracy of 10-20 mV. The ratio tended to plateau at potentials more negative than-140 mV, showing a limitation of the technique. Manoeuvres such as imposing a heavy ATP demand or interfering with the mitochondrial respiration depolarized mitochondria, while delta psi mit was not altered in a measurable manner during Ca2+ oscillations consecutive to alpha 1-agonist stimulation.

Determination of the Na permeability of the tight junctions of MDCK cells by fluorescence microscopy.

The kinetics of Na movement across the tight junctions of MDCK cells, grown on coverslips and perfused with HEPES or bicarbonate Ringer at 37 degrees C, were investigated after filling the lateral intercellular spaces (LIS) of the epithelium with SBFO, an Na-sensitive fluorescent dye. Dilution and bi-ionic potential measurements showed that MDCK cell tight junctions, although cation-selective, were poorly permeable to N-methyl-D-glucamine Cl (NMDG) but freely permeable to Li. In previous experiments in which Na was replaced by NMDG, a very slow decrease in LIS Na concentration (time constant = 4.8 min) resulted. In the present study, reduction of perfusate Na from 142 to 14 or 24 mM with Na replaced by Li caused LIS Na concentration to decrease with a time constant of 0.43 min. The time constant for Na increase of the LIS was 0.28 min, significantly shorter than that for Na decrease because of the additional component of transcellular Na influx. Ouabain eliminated the transcellular component and equalized the time constants for Na influx and efflux. These results were incorporated into a mathematical model which enabled calculation of the transcellular and paracellular Na fluxes during fluid reabsorption. Regulation of the Na permeability of individual tight junctions by protein kinase A (PKA) was evaluated by treating the monolayers with the Sp-cAMPS, a cAMP substitute, or Rp-cAMPS, a specific inhibitor of PKA. Stimulation of PKA strikingly increased tight junctional permeability while PKA inhibition diminished junctional Na permeability.

Simultaneous measurements of Ca2+ in the intracellular stores and the cytosol of hepatocytes during hormone-induced Ca2+ oscillations.

Simultaneous Ca2+ measurements in the cytosol and intracellular stores (IS) of rat hepatocytes were performed using two Ca(2+)-sensitive probes (Fluo-3 and Mag-fura-2), and combined whole-cell patch clamp and fluorescence microscopy. A steady-state Ca2+ concentration of approximately 630 microM was estimated in the IS. alpha 1-Adrenergic stimulation induced periodic elevations of cytosolic Ca2+ and parallel synchronized transient declines in the IS. Subsequent application of the intracellular Ca(2+)-pump inhibitor thapsigargin resulted in a release of Ca2+ from the IS to reach a level of Ca2+ depletion much lower than the lowest transient decline observed during the oscillations.