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SummaryThe ATP-sensitive K+channel (KATP) is a key regulator of hormone secretion from pancreatic islet endocrine cells. Using direct measurements of KATPchannel activity in pancreatic β cells and the lesser-studied α cells, from both humans and mice, we demonstrate that a glycolytic metabolon locally controls KATPchannels on the plasma membrane. The two ATP-consuming enzymes of upper glycolysis, glucokinase and phosphofructokinase, generate ADP that activates KATP. Substrate channeling of fructose 1,6-bisphosphate through the enzymes of lower glycolysis fuels pyruvate kinase, which directly consumes the ADP made by phosphofructokinase to raise ATP/ADP and close the channel. We further demonstrate the presence of a plasma membrane NAD+/NADH cycle, whereby lactate dehydrogenase is functionally coupled to glyceraldehyde-3-phosphate dehydrogenase. These studies provide direct electrophysiological evidence of a KATP-controlling glycolytic signaling complex and demonstrate its relevance to islet glucose sensing and excitability.Graphical abstractHoet al. demonstrate that the enzymes of glycolysis, as well as lactate dehydrogenase, form a plasma membrane-associated metabolon with intrinsic ATP/ADP and NAD+/NADH cycles. The subcellular location of this signaling complex allows both ATP-consuming and ATP-producing enzymes to locally control the activity of ATP-sensitive K+channels in pancreatic α and β cells from humans and mice.HighlightsKATPchannels are regulated by a glycolytic metabolon on the plasma membrane.Substrate channeling occurs between the consecutive enzymes of glycolysis.Upper glycolysis produces ADP that is used directly by lower glycolysis to make ATP.LDH and GADPH facilitate a plasma membrane-associated NAD+/NADH redox cycle.

β-cells use pyruvate kinase to sense nutrients and respond with appropriate insulin secretion. Here, we used β-cell deletion to identify the functions of constitutively active PKm1 and allosterically recruitable PKm2, and assess their regulation by mitochondrial PEP from PCK2. We demonstrate that both PKm isoforms are present in the KATP channel microcompartment. Compartmentation nullifies the disparities in PK isoform expression and activity, allowing the minor but recruitable PKm2 isoform to fully participate in KATP regulation. However, this shared control does not extend to β-cell response to amino acids, which requires PKm1 for the initiation of Ca2+ influx and insulin secretion and PKm2 for their termination. Mitochondrial PEP is required for this cycling between “on” and “off” states, as demonstrated by β-cell-specific deletion of PCK2. PCK2 was revealed to be an essential mechanism of β-cell cataplerosis, possessing metabolic control over cytosolic ATP/ADP generation in response to anaplerotic fuels. Further, PCK2 is required for amino acid-stimulated Ca2+ influx (via PKm1) and efflux (via PKm2) . These data provide strong genetic evidence for a revised oscillatory model of β-cell metabolism in which PKM1- and PKM2-driven PEP cycles play unique roles in the initiation and termination of nutrient-stimulated insulin secretion. Disclosure H.R.Foster: None. T.Ho: None. E.Potapenko: None. R.L.Cardone: n/a. R.Kibbey: Consultant; Agios, Inc. M.J.Merrins: None. Funding NIH/NIDDK (R01DK113103) HRSA (T32HP10010) NIH/NIA (T32AG000213)

SUMMARYPyruvate kinase (PK) and the phosphoenolpyruvate (PEP) cycle play key roles in nutrient-stimulated KATP channel closure and insulin secretion. To identify the PK isoforms involved, we generated mice lacking β-cell PKm1, PKm2, and mitochondrial PEP carboxykinase (PCK2) that generates mitochondrial PEP. Glucose metabolism generates both glycolytic and mitochondrially-derived PEP, which triggers KATP closure through local PKm1 and PKm2 signaling at the plasma membrane. Amino acids, which generate mitochondrial PEP without producing glycolytic fructose 1,6-bisphosphate to allosterically activate PKm2, signal through PKm1 to raise ATP/ADP, close KATP channels, and stimulate insulin secretion. Raising cytosolic ATP/ADP with amino acids is insufficient to close KATP channels in the absence of PK activity or PCK2, indicating that KATP channels are regulated by mitochondrially-derived PEP that provides ATP via plasma membrane-associated PK, but not via mitochondrially-derived ATP. Following membrane depolarization, the PEP cycle is also involved in an “off-switch” that facilitates KATP channel reopening and Ca2+ extrusion, as shown by PK activation experiments and β-cell PCK2 deletion that prolonged Ca2+ oscillations and increased insulin secretion. In conclusion, the differential response of PKm1 and PKm2 to the glycolytic and mitochondrial sources of PEP influences the β-cell nutrient response, and controls the oscillatory cycle regulating insulin secretion.

Pancreatic β-cells couple nutrient metabolism with appropriate insulin secretion. Here, we examined the requirements of pyruvate kinase (PK) isoforms in fuel sensing through the β-cell specific deletion of consititutively active PKM1 and allosterically recruitable PKM2 (PKM1- βKO, PKM2-βKO). β-cell deletion of PEP carboxykinase (PCK2-βKO) was used to block mitochondrial PEP production, leaving glycolytic PEP production intact. In vivo, only the PKM1-βKO mice showed glucose intolerance, and ex vivo insulin secretion was correspondingly reduced. Glucose-dependent Ca2+ oscillations were comparable in PKM1 βKO and PKM2-βKO islets, while the oscillatory frequency and duty cycle in PCK2-βKO islets increased relative to controls, suggesting redundancy in the PK response to glycolytic but not mitochondrial PEP. Using direct single-channel measurements of KATP in intact β-cells, amino acids were sufficient to stimulate KATP closure in control and PKM2-βKO cells, but failed to do so in the PCK2-βKO and PKM1-βKO models. PK activators (PKa) rescued KATP closure in PKM1- but not PCK2-deficient β-cells, arguing that PKM1 and PCK2 mediate membrane depolarization in response to mitochondrial fuels. The dependence of PKM2 on glucose was confirmed by the ability of PKa to rescue KATP closure in excised membrane patches from PKM1-βKO mice. Consistently, calcium influx stimulated by amino acids at low glucose was deficient in islets from PKM1-βKO and PCK2-βKO mice. PKa was sufficient to rescue the calcium defect in the PKM1-βKO islets but did not fully rescue the PCK2-βKO. Our findings provide strong genetic and electrophysiological evidence that the PCK2-PKM1 pathway is uniquely suited to direct the active (secretory) phase of calcium oscillations through direct effects on KATP, when FBP is low and PKM2 is inactive. While more limited in function, PKM2 can be therapeutically targeted, endowing it with the ability to respond to mitochondrial PEP and boost insulin secretion. Disclosure H. R. Foster: None. T. Ho: None. E. Potapenko: None. S. L. Lewandowski: None. S. Sdao: None. H. R. Vandeusen: None. R. L. Cardone: None. R. Kibbey: Research Support; Self; Agios, Inc. M. J. Merrins: None. Funding Health Resources and Services Administration (T32HP10010); National Institute on Aging (T32AG000213); National Institutes of Health (R01DK113103)

Acidocalcisomes are acidic calcium stores rich in polyphosphate (polyP) and are present in trypanosomes and also in a diverse range of other organisms. Ca2+ is released from these organelles through a channel, inositol 1,4,5-trisphosphate receptor (TbIP3R), which is essential for growth and infectivity of the parasite Trypanosoma brucei. However, the mechanism by which TbIP3R controls Ca2+ release is unclear. In this work, we expressed TbIP3R in a chicken B lymphocyte cell line in which the genes for all three vertebrate IP3Rs were stably ablated (DT40–3KO). We show that IP3-mediated Ca2+ release depends on Ca2+ but not on ATP concentration and is inhibited by heparin, caffeine, and 2-aminomethoxydiphenyl borate (2-APB). Excised patch clamp recordings from nuclear membranes of DT40 cells expressing only TbIP3R disclosed that luminal inorganic orthophosphate (Pi) or pyrophosphate (PPi), and neutral or alkaline pH can stimulate IP3-generated currents. In contrast, polyP or acidic pH did not induce these currents, and nuclear membranes obtained from cells expressing rat IP3R were unresponsive to polyP or its hydrolysis products. Our results are consistent with the notion that polyP hydrolysis products within acidocalcisomes or alkalinization of their luminal pH activate TbIP3R and Ca2+ release. We conclude that TbIP3R is well-adapted to its role as the major Ca2+ release channel of acidocalcisomes in T. brucei.

Acidocalcisomes of Trypanosoma brucei and the acidocalcisome-like vacuoles of Saccharomyces cerevisiae are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate–sodium symporter (TbPho91 and Pho91p in T. brucei and yeast, respectively), but the roles of these transporters in growth and orthophosphate (Pi) transport are unclear. We found here that Tbpho91−/− trypanosomes have a lower growth rate under phosphate starvation and contain larger acidocalcisomes that have increased Pi content. Heterologous expression of TbPHO91 in Xenopus oocytes followed by two-electrode voltage clamp recordings disclosed that myo-inositol polyphosphates stimulate both sodium-dependent depolarization of the oocyte membrane potential and Pi conductance. Deletion of the SPX domain in TbPho91 abolished this stimulation. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate generated outward currents in Na+/Pi-loaded giant vacuoles prepared from WT or from TbPHO91-expressing pho91Δ strains but not from the pho91Δ yeast strains or from the pho91Δ strains expressing PHO91 or TbPHO91 with mutated SPX domains. Our results indicate that TbPho91 and Pho91p are responsible for vacuolar Pi and Na+ efflux and that myo-inositol polyphosphates stimulate the Na+/Pi symporter activities through their SPX domains.

Pancreatic β-cells couple nutrient metabolism with appropriate insulin secretion. Here, we show that pyruvate kinase (PK), which converts ADP and phosphoenolpyruvate (PEP) into ATP and pyruvate, underlies β-cell sensing of both glycolytic and mitochondrial fuels. Plasma membrane-localized PK is sufficient to close K(ATP) channels and initiate calcium influx. Small-molecule PK activators increase the frequency of ATP/ADP and calcium oscillations and potently amplify insulin secretion. PK restricts respiration by cyclically depriving mitochondria of ADP, which accelerates PEP cycling until membrane depolarization restores ADP and oxidative phosphorylation. Our findings support a compartmentalized model of β-cell metabolism in which PK locally generates the ATP/ADP required for insulin secretion. Oscillatory PK activity allows mitochondria to perform synthetic and oxidative functions without any net impact on glucose oxidation. These findings suggest a potential therapeutic route for diabetes based on PK activation that would not be predicted by the current consensus single-state model of β-cell function.

Toxoplasma gondii , an obligate intracellular parasite, is capable of invading virtually any nucleated cell. Active invasion by the parasite is a precise process and intracellular parasites reside and replicate inside a parasitophorous vacuole (PV). The membrane of the PV functions as a molecular sieve allowing for its ionic milieu to be in equilibrium with the host cytosol. The parasite would thus be exposed to the ionic fluctuations of the host cytoplasm, such as increases in host cytosolic Ca2+ during Ca2+ signaling events. Ca2+ is a universal signaling molecule and both the host cell and T. gondii will utilize Ca2+ signaling to stimulate diverse cellular functions. Using T. gondii tachyzoites and host cells expressing genetically encoded Ca2+ indicators, we demonstrate that host Ca2+ signaling impacts intracellular Ca2+ levels of the parasite. We propose that Ca2+ influx derived from host Ca2+ signaling into the parasite is utilized to maintain the intracellular Ca2+ stores replenished as the parasite replicates within the low Ca2+ environment of the host cell. Intracellular Ca2+ store(s) release is needed to reach the initiation spike in Ca2+ that meets the threshold of Ca2+ needed to initiate the tightly coordinated process of egress. Post activation of the signal and subsequent microneme secretion, would cause breakdown of the host cell and allow for extracellular Ca2+ influx, causing a second, larger spike in Ca2+ and activation of the glideosome, the main motility machinery. To directly deliver precise concentrations of ions needed for egress, we used patch-clamped infected host cells and monitored parasite egress. In doing so, we discovered an alternative role for K+ in timing parasite egress. This is the first study using whole-cell patch clamp to study the role of ions such as K+ and Ca2+ in T. gondii egress.

SUMMARYPancreatic β-cells couple nutrient metabolism with appropriate insulin secretion. Here, we show that pyruvate kinase (PK), which converts ADP and phosphoenolpyruvate (PEP) into ATP and pyruvate, underlies β-cell sensing of both glycolytic and mitochondrial fuels. PK present at the plasma membrane is sufficient to close KATPchannels and initiate calcium influx. Small-molecule PK activators increase β-cell oscillation frequency and potently amplify insulin secretion. By cyclically depriving mitochondria of ADP, PK restricts oxidative phosphorylation in favor of the mitochondrial PEP cycle with no net impact on glucose oxidation. Our findings support a compartmentalized model of β-cell metabolism in which PK locally generates the ATP/ADP threshold required for insulin secretion, and identify a potential therapeutic route for diabetes based on PK activation that would not be predicted by the β-cell consensus model.GRAPHICAL ABSTRACTThe consensus model for β-cell glucose sensing supports a dominant role for OxPhos. This model doesn’t fully explain the observed metabolic and electrophysiologic oscillations associated with glucose-stimulated insulin secretion. Lewandowskiet al. challenge this model by mechanistically connecting the anaplerotic PEP cycle to the electrically silent triggering phase, and OxPhos to the electrically active secretory phase. Here, the allosteric recruitment of pyruvate kinase directs metabolic traffic between the two cycles and identifies potential therapeutic strategies for diabetes based on pharmacologic pyruvate kinase activation.HIGHLIGHTSCompartmentalized pyruvate kinase (PK) activity underlies β-cell fuel sensingMembrane-associated PK closes KATPchannels and controls calcium influxBy lowering ADP, PK toggles mitochondria between OxPhos and PEP biosynthesisPharmacologic PK activation increases oscillatory frequency and amplifies secretion

Inorganic pyrophosphate (PPi) is a by-product of biosynthetic reactions and has bioenergetic and regulatory roles in a variety of cells. Here we show that PPi and other pyrophosphate-containing compounds, including polyphosphate (polyP), can stimulate sodium-dependent depolarization of the membrane potential and Pi conductance in Xenopus oocytes expressing a Saccharomyces cerevisiae or Trypanosoma brucei Na+/Pi symporter. PPi is not taken up by Xenopus oocytes, and deletion of the TbPho91 SPX domain abolished its depolarizing effect. PPi generated outward currents in Na+/Pi-loaded giant vacuoles prepared from wild-type or pho91Δ yeast strains expressing TbPHO91 but not from the pho91Δ strains. Our results suggest that PPi, at physiological concentrations, can function as a signaling molecule releasing Pi from S. cerevisiae vacuoles and T. brucei acidocalcisomes. IMPORTANCE Acidocalcisomes, first described in trypanosomes and known to be present in a variety of cells, have similarities with S. cerevisiae vacuoles in their structure and composition. Both organelles share a Na+/Pi symporter involved in Pi release to the cytosol, where it is needed for biosynthetic reactions. Here we show that PPi, at physiological cytosolic concentrations, stimulates the symporter expressed in either Xenopus oocytes or yeast vacuoles via its SPX domain, revealing a signaling role of this molecule.

Acidocalcisomes are acidic calcium stores rich in polyphosphate (polyP) and are present in trypanosomes and also in a diverse range of other organisms. Ca(2+) is released from these organelles through a channel, inositol 1,4,5-trisphosphate receptor (TbIP(3)R), which is essential for growth and infectivity of the parasite Trypanosoma brucei. However, the mechanism by which TbIP(3)R controls Ca(2+) release is unclear. In this work, we expressed TbIP(3)R in a chicken B lymphocyte cell line in which the genes for all three vertebrate IP(3)Rs were stably ablated (DT40–3KO). We show that IP(3)-mediated Ca(2+) release depends on Ca(2+) but not on ATP concentration and is inhibited by heparin, caffeine, and 2-aminomethoxydiphenyl borate (2-APB). Excised patch clamp recordings from nuclear membranes of DT40 cells expressing only TbIP(3)R disclosed that luminal inorganic orthophosphate (P(i)) or pyrophosphate (PP(i)), and neutral or alkaline pH can stimulate IP(3)-generated currents. In contrast, polyP or acidic pH did not induce these currents, and nuclear membranes obtained from cells expressing rat IP(3)R were unresponsive to polyP or its hydrolysis products. Our results are consistent with the notion that polyP hydrolysis products within acidocalcisomes or alkalinization of their luminal pH activate TbIP(3)R and Ca(2+) release. We conclude that TbIP(3)R is well-adapted to its role as the major Ca(2+) release channel of acidocalcisomes in T. brucei.

Toxoplasma gondii is an obligate intracellular parasite and replicates inside a parasitophorous vacuole (PV) within the host cell. The membrane of the PV (PVM) contains pores that permits for equilibration of ions and small molecules between the host cytosol and the PV lumen. Ca(2+) signaling is universal and both T. gondii and its mammalian host cell utilize Ca(2+) signals to stimulate diverse cellular functions. Egress of T. gondii from host cells is an essential step for the infection cycle of T. gondii and a cytosolic Ca(2+) increase initiates a Ca(2+) signaling cascade that culminates in the stimulation of motility and egress. In this work, we demonstrate that intracellular T. gondii tachyzoites are able to take up Ca(2+) from the host cytoplasm during host signaling events. Both intracellular and extracellular Ca(2+) sources are important in reaching a threshold of cytosolic Ca(2+) needed for successful egress. Two peaks of Ca(2+) were observed in single parasites that egressed with the second peak resulting from Ca(2+) entry. We patched infected host cells to allow the delivery of precise concentrations of Ca(2+) for stimulation of motility and egress. Using this approach of patching infected host cells allowed to determine that increasing the host cytosolic Ca(2+) to a specific concentration can trigger egress, which is further accelerated by diminishing the concentration of potassium (K(+)).

Acidocalcisomes of Trypanosoma brucei and the acidocalcisome-like vacuoles of Saccharomyces cerevisiae are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate–sodium symporter (TbPho91 and Pho91p in T. brucei and yeast, respectively), but the roles of these transporters in growth and orthophosphate (P(i)) transport are unclear. We found here that Tbpho91(−/−) trypanosomes have a lower growth rate under phosphate starvation and contain larger acidocalcisomes that have increased P(i) content. Heterologous expression of TbPHO91 in Xenopus oocytes followed by two-electrode voltage clamp recordings disclosed that myo-inositol polyphosphates stimulate both sodium-dependent depolarization of the oocyte membrane potential and P(i) conductance. Deletion of the SPX domain in TbPho91 abolished this stimulation. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate generated outward currents in Na(+)/P(i)-loaded giant vacuoles prepared from WT or from TbPHO91-expressing pho91Δ strains but not from the pho91Δ yeast strains or from the pho91Δ strains expressing PHO91 or TbPHO91 with mutated SPX domains. Our results indicate that TbPho91 and Pho91p are responsible for vacuolar P(i) and Na(+) efflux and that myo-inositol polyphosphates stimulate the Na(+)/P(i) symporter activities through their SPX domains.

Trypanosoma cruzi is an important human and animal pathogen and the etiologic agent of Chagas disease. The parasite undergoes drastic changes in its metabolism during its life cycle. Amino acid consumption becomes important in the infective stages and leads to the production of ammonia (NH3), which needs to be detoxified. We report here the identification of an ammonium (NH4+) transporter that localizes to acidic compartments and is important for replication, differentiation, and resistance to starvation and osmotic stress., Trypanosoma cruzi, the etiologic agent of Chagas disease, undergoes drastic metabolic changes when it transits between a vector and mammalian hosts. Amino acid catabolism leads to the production of ammonium (NH4+), which needs to be detoxified. However, T. cruzi does not possess a urea cycle, and it is unknown how intracellular levels of ammonium are controlled. In this work, we identified an intracellular ammonium transporter of T. cruzi (TcAMT) that localizes to acidic compartments (reservosomes, lysosomes). TcAMT has 11 transmembrane domains and possesses all conserved and functionally important amino acid residues that form the pore in other ammonium transporters. Functional expression in Xenopus oocytes followed by a two-electrode voltage clamp showed an inward current that is NH4+ dependent at a resting membrane potential (Vh) lower than −120 mV and is not pH dependent, suggesting that TcAMT is not an NH4+/H+ cotransporter but an NH4+ or NH3/H+ transporter. Ablation of TcAMT by clustered regularly interspaced short palindromic repeat analysis with Cas9 (CRISPR-Cas9) resulted in significant defects in epimastigote and amastigote replication, differentiation, and resistance to starvation and osmotic stress. IMPORTANCE Trypanosoma cruzi is an important human and animal pathogen and the etiologic agent of Chagas disease. The parasite undergoes drastic changes in its metabolism during its life cycle. Amino acid consumption becomes important in the infective stages and leads to the production of ammonia (NH3), which needs to be detoxified. We report here the identification of an ammonium (NH4+) transporter that localizes to acidic compartments and is important for replication, differentiation, and resistance to starvation and osmotic stress.

Neurohumoral activation, which includes augmented plasma levels of the neurohormone vasopressin (VP), is a common finding in heart failure (HF) that contributes to morbidity and mortality in this disease. While an increased activation of magnocellular neurosecretory cells (MNCs) and enhanced glutamate function in HF is well documented, the precise underlying mechanisms remain to be elucidated. Here, we combined electrophysiology and protein measurements to determine whether altered glial glutamate transporter function and/or expression occurs in the hypothalamic supraoptic nucleus (SON) during HF. Patch-clamp recordings obtained from MNCs in brain slices show that pharmacological blockade of astrocyte glutamate transporter 1 (GLT1) function [500 μM dihydrokainate (DHK)], resulted in a persistent N-methyl-d-aspartate receptor (NMDAR)-mediated inward current (tonic INMDA) in sham rats, an effect that was significantly smaller in MNCs from HF rats. In addition, we found a diminished GLT1 protein content in plasma membrane (but not cytosolic) fractions of SON punches in HF rats. Conversely, astrocyte GLAST expression was significantly higher in the SON of HF rats, while nonselective blockade of glutamate transport activity (100 μM TBOA) evoked an enhanced tonic INMDA activation in HF rats. Steady-state activation of NMDARs by extracellular glutamate levels was diminished during HF. Taken together, these results support a shift in the relative expression and function of two major glial glutamate transporters (from GLT1 to GLAST predominance) during HF. This shift may act as a compensatory mechanism to preserve an adequate basal glutamate uptake level in the face of an enhanced glutamatergic afferent activity in HF rats.

Despite the well-established contribution of neurohumoral activation to morbidity and mortality in heart failure (HF) patients, relatively little is known about the underlying central nervous system mechanisms. In this study, we aimed to determine whether changes in GABAergic inhibitory and glutamatergic excitatory synaptic function contribute to altered hypothalamic magnocellular neurosecretory cell (MNC) activity in HF rats. Patch-clamp recordings were obtained from MNCs in brain slices from sham and HF rats. Glutamate excitatory (EPSCs) and GABAergic inhibitory postsynaptic currents (IPSCs) were simultaneously recorded, and changes in their strengths, as well as their interactions, were evaluated. We found a diminished GABAergic synaptic strength in MNCs of HF rats, reflected as faster decaying IPSCs and diminished mean IPSC charge transfer. Opposite changes were observed in glutamate EPSC synaptic strength, resulting in a shift in the GABA-glutamate balance toward a relatively stronger glutamate influence in HF rats. The prolongation of glutamate EPSCs during HF was mediated, at least in part, by an enhanced contribution of AMPA receptor desensitization to the EPSC decay time course. EPSC prolongation, and consequently increased unitary strength, resulted in a stronger AMPA receptor-mediated excitatory drive to firing discharge in MNCs of HF rats. Blockade of GABAAsynaptic activity diminished the EPSC waveform variability observed among events in sham rats, an effect that was blunted in HF rats. Together, our results suggest that opposing changes in postsynaptic properties of GABAergic and glutamatergic synaptic function contribute to enhanced magnocellular neurosecretory activity in HF rats.

In response to food reward and other pertinent events, midbrain dopaminergic neurons fire short bursts of action potentials causing a phasic release of dopamine in the prefrontal cortex (rapid and transient increases in cortical dopamine concentration). Here we apply short (2s) iontophoretic pulses of glutamate, GABA, dopamine and dopaminergic agonists locally, onto layer 5 pyramidal neurons in brain slices of the rat medial prefrontal cortex (PFC). Unlike glutamate and GABA, brief dopaminergic pulses had negligible effects on the resting membrane potential. However, dopamine altered action potential firing in an extremely rapid (1s) and transient (5 min) manner, as every neuron returned to baseline in less than 5-min post-application. The physiological responses to dopamine differed markedly among individual neurons. Pyramidal neurons with a preponderance of D1-like receptor signaling respond to dopamine with a severe depression in action potential firing rate, while pyramidal neurons dominated by the D2 signaling pathway respond to dopamine with an instantaneous increase in spike production. Increasing levels of dopamine concentrations around the cell body resulted in a dose dependent response, which resembles an "inverted U curve" (Vijayraghavan S, Wang M, Birnbaum SG, Williams GV, Arnsten AF (2007) Inverted-U dopamine D1 receptor actions on prefrontal neurons engaged in working memory. Nat Neurosci 10:376-384), but this effect can easily be caused by an iontophoresis current artifact. Our present data imply that one population of PFC pyramidal neurons receiving direct synaptic contacts from midbrain dopaminergic neurons would stall during the 0.5s of the phasic dopamine burst. The spillover dopamine, on the other hand, would act as a positive stimulator of cortical excitability (30% increase) to all D2-receptor carrying pyramidal cells, for the next 40s.

It is well-known that pH changes can influence a lot of cellular processes. In this work, we have specifically studied the influence of alkalinization, which can be developed in spinal cord neurons during hyperventilation (respiratory alkalosis) and chronic renal failure (metabolic alkalosis) on calcium homeostasis. Application of Tyrode solution with increased pH (pH = 8.8) to secondary sensory neurons isolated from rat spinal dorsal horn induced elevation of intracellular free calcium concentration in the cytosol ([Ca2+]i) if applied after membrane depolarization. Repetitive application of alkaline solution led to disappearance of such elevations. Depletion of endoplasmic reticulum (ER) calcium stores by 30 mM caffeine almost completely blocked the effect of elevated extracellular pH. If caffeine-induced [Ca2+]i transients were evoked during alkalinization, their amplitudes were decreased by 41%. Preapplication of 500 nM ionomycin resulted in disappearance of alkalinization-induced [Ca2+]i transients, whereas prolonged applications (for 20 min) of 200 nM thapsigargin, a blocker of Ca2+ ATPase of the endoplasmic reticulum, resulted in disappearance of the rapid phase of the [Ca2+]i transients induced by alkalinization. Preapplication of the mitochondrial protonophore CCCP (10 microM) also induced changes in the alkalinization-induced calcium response--it lost its peak and was transformed into an irregular wave terminating in several seconds. The data obtained indicate that alkalinization induces an increase of [Ca2+]i level in the investigated neurons via a combined action of both intracellular Ca2+-accumulating structures--the endoplasmic reticulum and mitochondria. This suggestion was supported by morphological data that both structures in these neurons are tightly connected and may interact during release of accumulated calcium ions.

Changes in intracellular Ca2+ induced by extracellular acidification to pH = 6 were studied in isolated rat spinal dorsal horn neurons using indo-1 fluorescent technique. In all neurons such treatment induced a decrease of basal [Ca2+]i level by 20.8%, preceded in some of them by temporary increase. The changes were completely reversible. The depolarization-induced [Ca2+]i transients became strongly and also reversibly depressed. If tested after termination of acidification, they demonstrated substantial prolongation of their decay phase, reaching 310% at 120 sec after the application of depolarization. To analyze the mechanisms of such changes, mitochondrial protonophore CCCP has been applied between the end of acidification and the depolarizing pulse. This completely eliminated the described slowing of the transients' decay. To the contrary, application of caffeine to induce Ca2+ release from the endoplasmic reticulum did not show significant changes in the corresponding [Ca2+]i transients. A conclusion is made that in mammalian neurons extracellular acidification, apart from inhibiting voltage-operated Ca2+ channels, also substantially alters the Ca2+ exchange function of mitochondria responsible for rapid accumulation of ions and their delayed release back into the cytosol.

The role of different Ca2+-regulated mechanisms in the generation of cytosolic Ca2+ transients during neuronal excitation was compared in isolated primary and secondary nociceptive neurons of the rat. Application of carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly increased the peak amplitude of depolarization-induced transients in dorsal root ganglion (DRG) neurons in contrast to what was observed in spinal dorsal horn (DH) neurons. Application of CCCP immediately after termination of depolarization induced in DRG neurons massive Ca2+ release from the mitochondria into the cytosol. Application of CCCP immediately after termination of depolarization elicited a small Ca2+ release in DH neurons, which became more intense when application of the agent was delayed.

The spatiotemporal characteristics of NMDA receptor-mediated changes in intracellular calcium ([Ca2+]i) (NMDAR-[Ca2+]i) is critical in determining the strength of NMDA effects on neuronal excitabil...

An enhanced glutamate excitatory function within the hypothalamic supraoptic and paraventricluar nuclei is known to contribute to increased neurosecretory and presympathetic neuronal activity, and hence, neurohumoral activation, during heart failure (HF). Still, the precise mechanisms underlying enhanced glutamate-driven neuronal activity in HF remain to be elucidated. Here, we performed simultaneous electrophysiology and fast confocal Ca2+imaging to determine whether altered N-methyl-d-aspartate (NMDA) receptor-mediated changes in intracellular Ca2+levels (NMDA-ΔCa2+) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in HF rats. We found that activation of NMDA receptors resulted in a larger ΔCa2+in MNCs from HF when compared with sham rats. The enhanced NMDA-ΔCa2+was neither dependent on the magnitude of the NMDA-mediated current (voltage clamp) nor on the degree of membrane depolarization or firing activity evoked by NMDA (current clamp). Differently from NMDA receptor activation, firing activity evoked by direct membrane depolarization resulted in similar changes in intracellular Ca2+in sham and HF rats. Taken together, our results support a relatively selective alteration of intracellular Ca2+homeostasis and signaling following activation of NMDA receptors in MNCs during HF. The downstream functional consequences of such altered ΔCa2+signaling during HF are discussed.

A dynamic balance between the excitatory and inhibitory neurotransmitters glutamate and GABA is critical for maintaining proper neuronal activity in the brain. This balance is partly achieved via presynaptic interactions between glutamatergic and GABAAergic synapses converging into the same targets. Here, we show that in hypothalamic magnocellular neurosecretory neurons (MNCs), a direct crosstalk between postsynaptic NMDA receptors (NMDARs) and GABAAreceptors (GABAARs) contributes to the excitatory/inhibitory balance in this system. We found that activation of NMDARs by endogenous glutamate levels controlled by astrocyte glutamate transporters, evokes a transient and reversible potentiation of postsynaptic GABAARs. This inter-receptor crosstalk is calcium-dependent and involves a kinase-dependent phosphorylation mechanism, but does not require nitric oxide as an intermediary signal. Finally, we found the NMDAR–GABAAR crosstalk to be blunted in rats with heart failure, a pathological condition in which the hypothalamic glutamate–GABA balance is tipped toward an excitatory predominance. Together, our findings support a novel form of glutamate–GABA interactions in MNCs, which involves crosstalk between NMDA and GABAApostsynaptic receptors, whose strength is controlled by the activity of local astrocytes. We propose this inter-receptor crosstalk to act as a compensatory, counterbalancing mechanism to dampen glutamate-mediated overexcitation. Finally, we propose that an uncoupling between NMDARs and GABAARs may contribute to exacerbated neuronal activity and, consequently, sympathohumoral activation in such disease conditions as heart failure.

Elevations of the cytosolic free Ca2+ concentration ([Ca2+]i) in rat dorsal horn neurons induced by addition of ATP to the medium were compared in spinal cord slices and after isolation of the neurons. In slices, application of ATP results in an increase in the [Ca2+]i by 201 ± 12 nM, on the average; in a Ca2+ -free external solution the respective rise was 156 ± 14 nM (n = 45 of 76 examined cells), which indicate the presence of active purinergic metabotropic receptors in about 59% of the neurons. In freshly isolated neurons with absent dendrites, we found no metabotropic responses. Thus, the results confirm the conclusion on the localization of metabotropic postsynaptic purinoreceptors mostly on the dendritic tree of dorsal horn neurons.

The participation of different calcium-regulated mechanisms in the generation of cytosolic Ca(2+) transients during neuronal excitation has been compared in isolated large and small primary (dorsal root ganglia (DRG)) and secondary (spinal dorsal horn (DH)) rat sensory neurones. As it was shown before in murine primary sensory neurones the application of mitochondrial protonophore CCCP by itself induced only small elevation of [Ca(2+)](i). However, its preceding application substantially increased the peak amplitude of depolarization-induced transients. Application of CCCP immediately after termination of the depolarizing pulse induced in both types of primary neurones a massive release of Ca(2+) from mitochondria into the cytosol. In secondary neurones application of CCCP by itself induced a substantial release of Ca(2+) from the mitochondria, but its preceding application resulted in only an insignificant increase in the peak amplitude of depolarization-triggered calcium transients. Application of CCCP immediately after termination of depolarization elicited a small release of Ca(2+), which became more pronounced when the application was delayed. Preceding application of CCCP increased the amplitude of the transients induced by caffeine-triggered Ca(2+) release from the endoplasmic reticulum in secondary neurones and did not affect those in large primary neurones. These findings may be explained by substantial differences in the density and distribution of mitochondria in the cytosol of primary and secondary sensory neurones. This suggestion was confirmed electronmicroscopically, showing a much lower density of mitochondria near plasmalemma in secondary sensory neurones and predominant clustered location of mitochondria beneath the plasmalemma in the primary cells. The possible functional importance of these differences is discussed.