Expression of yeast INM1 encoding inositol monophosphatase is regulated by inositol, carbon source and growth stage and is decreased by lithium and valproate

Inositol monophosphatase plays a vital role in the de novo biosynthesis of inositol and in the phosphoinositide second messenger signalling pathway. We cloned the Saccharomyces cerevisiae open reading frame (ORF) YHR046c (termed INM1), which encodes inositol monophosphatase, characterized the protein Inm1p and analysed expression of the INM1 gene. INM1 was expressed in bacteria under the control of the lacZ promoter. The purified protein has inositol monophosphatase activity that is inhibited by the antibipolar drug lithium, but not valproate. In the inm1Delta:URA3 null mutant, inositol monophosphatase activity was reduced but not eliminated. The disruption had little effect on growth in the presence of lithium or valproate and no effect on growth in the absence of inositol. To characterize the regulation of INM1, we examined the effects of inositol, carbon source, growth phase, and the antibipolar drugs lithium and valproate on INM1 expression using an INM1-lacZ reporter gene. Unlike all other phospholipid biosynthetic enzyme-encoding genes studied, which contain the UASINO regulatory element, INM1 expression is increased in the presence of inositol. In addition, INM1 expression was repressed during growth in glycerol and derepressed as glucose-grown cells entered stationary. Both lithium and valproate, which cause a decrease in intracellular inositol, effect a decrease in INM1 expression. A model is presented to account for regulation of INM1 expression.     

Altered Na+ and Li+ Homeostasis in Saccharomyces cerevisiae Cells Expressing the Bacterial Cation Antiporter NhaA

The bacterial Na⁺(Li⁺)/H⁺ antiporter NhaA has been expressed in the yeast Saccharomyces cerevisiae. NhaA was present in both the plasma membrane and internal membranes, and it conferred lithium but not sodium tolerance. In cells containing the yeast Ena1-4 (Na⁺, Li⁺) extrusion ATPase, the extra lithium tolerance conferred by NhaA was dependent on a functional vacuolar H⁺ ATPase and correlated with an increase of lithium in an intracellular pool which exhibited slow efflux of cations. In yeast mutants without (Na⁺, Li⁺) ATPase, lithium tolerance conferred by NhaA was not dependent on a functional vacuolar H⁺ ATPase and correlated with a decrease of intracellular lithium. NhaA was able to confer sodium tolerance and to decrease intracellular sodium accumulation in a double mutant devoid of both plasma membrane (Na⁺, Li⁺) ATPase and vacuolar H⁺ ATPase. These results indicate that the bacterial antiporter NhaA expressed in yeast is functional at both the plasma membrane and the vacuolar membrane. The phenotypes conferred by its expression depend on the functionality of plasma membrane (Na⁺, Li⁺) ATPase and vacuolar H⁺ ATPase.     

Lithium and valproate decrease inositol mass and increase expression of the yeast INO1 and INO2 genes for inositol biosynthesis

Bipolar affective disorder (manic-depressive illness) is a chronic, severe, debilitating illness affecting 1-2% of the population. The Food and Drug Administration-approved drugs lithium and valproate are not completely effective in the treatment of this disorder, and the mechanisms underlying their therapeutic effects have not been established. We are employing genetic and molecular approaches to identify common targets of lithium and valproate in the yeast Saccharomyces cerevisiae. We show that both drugs affect molecular targets in the inositol metabolic pathway. Lithium and valproate cause a decrease in intracellular myo-inositol mass and an increase in expression of both a structural (INO1) and a regulatory (INO2) gene required for inositol biosynthesis. The opi1 mutant, which exhibits constitutive expression of INO1, is more resistant to inhibition of growth by lithium but not by valproate, suggesting that valproate may inhibit the Ino1p-catalyzed synthesis of inositol 1-phosphate. Consistent with this possibility, growth in valproate leads to decreased synthesis of inositol monophosphate. Thus, both lithium and valproate perturb regulation of the inositol biosynthetic pathway, albeit via different mechanisms. This is the first demonstration of increased expression of genes in the inositol biosynthetic pathway by both lithium and valproate. Because inositol is a key regulator of many cellular processes, the effects of lithium and valproate on inositol synthesis have far-reaching implications for predicting genetic determinants of responsiveness and resistance to these agents.     

The effect of acetylcholine on inositol lipid metabolism and intracellular calcium concentrations in bovine anterior pituitary cells

Acetylcholine (ACh) increased the intracellular calcium concentration in bovine anterior pituitary cells. In the presence of the calcium channel antagonists verapamil (20 microM) or nitrendepine (1 microM) the increase in calcium was partially inhibited but showed both transient and sustained components. In the presence of EGTA (2.5 mM) only the transient component was observed. ACh also decreased inositol radioactivity in phosphatidylinositides and increased it in inositol phosphates. It is concluded that the increase in calcium caused by acetylcholine requires both the entry of external calcium and mobilisation of internal calcium. Replacement of external sodium by N-methyl-D-glucamine inhibited the rises in calcium and inositol phosphate labelling in response to ACh. Tetrodotoxin (3 microM) or ouabain (50 microM) did not affect either response to ACh. Verapamil did not affect the calcium rise induced by ACh in the absence of external sodium. The phorbol ester PMA (10 nM) caused a transient rise in calcium and inhibited the calcium rise caused by acetylcholine: it did not modify the effect of acetylcholine on inositol phosphates. The dependence of the stimulation of external calcium entry and inositol phosphate production on external sodium ions and protein kinase C is discussed.     

A direct interaction between IP(3) receptors and myosin II regulates IP(3) signaling in C. elegans

Molecular and physiological studies of cells implicate interactions between the cytoskeleton and the intracellular calcium signalling machinery as an important mechanism for the regulation of calcium signalling. However, little is known about the functions of such mechanisms in animals. A key component of the calcium signalling network is the intracellular release of calcium in response to the production of the second messenger inositol 1,4,5-trisphosphate (IP(3)), mediated by the IP(3) receptor (IP(3)R). We show that C. elegans IP(3)Rs, encoded by the gene itr-1, interact directly with myosin II. The interactions between two myosin proteins, UNC-54 and MYO-1, and ITR-1 were identified in a yeast two-hybrid screen and subsequently confirmed in vivo and in vitro. We defined the interaction sites on both the IP(3)R and MYO-1. To test the effect of disrupting the interaction in vivo we overexpressed interacting fragments of both proteins in C. elegans. This decreased the animal's ability to upregulate pharyngeal pumping in response to food. This is a known IP(3)-mediated process [15]. Other IP(3)-mediated processes, e.g., defecation, were unaffected. Thus it appears that interactions between IP(3)Rs and myosin are required for maintaining the specificity of IP(3) signalling in C. elegans and probably more generally.     

Regulation of inositol monophosphatase in Saccharomyces cerevisiae

Inositol monophosphatase is a key enzyme in the de novo biosynthesis of inositol and in the phosphoinositide second-messenger signalling pathway. Inhibition of this enzyme is a proposed mechanism for lithium's pharmacological action in bipolar illness (manic depression). Very little is known about how expression of this enzyme is regulated. Because the yeast Saccharomyces cerevisiae has been shown to be an excellent model system in which to understand the regulation of inositol metabolism, we characterized inositol monophosphatase in this yeast. Lithium inhibited monophosphatase activity in vitro . Growth in the presence of inositol resulted in increased expression of the enzyme in vivo , although inositol had no effect on enzyme activity in vitro . The inositol effect was apparent when cells were grown in glucose but not in glycerol/ethanol. Monophosphatase activity was derepressed as cells entered stationary phase. This effect was apparent only during growth in glucose plus inositol. The results demonstrate that S. cerevisiae monophosphatase is inhibited by lithium and regulated by factors affecting phospholipid biosynthesis.     

Formation of second messengers in response to activation of ion channels in excitable cells

1. Depolarization of excitable cells of the central nervous system results in the formation of the second messengers cyclic AMP, cyclic GMP, inositol phosphates, and diacylglycerides. 2. Depolarization-evoked accumulation of cyclic AMP in brain preparations can be accounted for mainly by the release of adenosine, which subsequently interacts with stimulatory adenosine receptor linked to adenylate cyclase. 3. Depolarization-evoked formation of cyclic GMP in brain preparations is linked to activation of voltage-dependent calcium channels, presumably leading to activation of guanylate cyclase by calcium ions. 4. In brain slices depolarization-evoked stimulation of phosphoinositide breakdown and subsequent formation of inositol phosphates and diacylglycerides are linked to activation of voltage-dependent calcium channels, which are sensitive to dihydropyridines, presumably leading to activation of phospholipase(s) C by calcium ions. 5. In the synaptoneurosome preparation depolarization-evoked stimulation of phosphoinositide breakdown does not involve activation of dihydropyridine-sensitive calcium channels and, instead, appears to be regulated primarily by the intracellular concentration of sodium ions. Thus, agents that induce increases in intracellular sodium--such as toxins that open or delay inactivation of voltage-dependent sodium channels; ouabain, an inhibitor of Na+/K+ ATPase that transports sodium outward and a sodium ionophore--all stimulate phosphoinositide breakdown. Mechanistically, increases in intracellular sodium either might directly affect phospholipase(s) C or might lead to influx of calcium ions through Na+/Ca2+ transporters. 6. Depolarization-evoked stimulation of cyclic AMP formation and phosphoinositide breakdown can exhibit potentiative interactions with responses to receptor agonists, thereby providing mechanisms for modulation of receptor responses by neuronal activity. 7. Since all these second messengers can induce phosphorylation of ion channels through the activation of specific kinases, it is proposed that depolarization-evoked formation of second messengers represents a putative feedback mechanism to regulate ion fluxes in excitable cells.     

Interaction of sodium with the sarcolemmal calcium system.

Sarcolemma isolated from guinea pig heart binds calcium in an ATP-dependent manner. Sodium ions decrease the total amount of calcium bound by the membranes. ATP-dependent calcium binding is more sensitive to sodium than the non-ATP-dependent calcium binding. The ATPase active during calcium binding is affected by sodium ions to the same extent as the ATP-dependent calcium binding process. The inhibition of the calcium binding process and of ATPase activity by sodium was more pronounced when the membranes were preincubated with sodium. The effect of sodium on calcium binding is dependent on both the time of contact between sodium and the membranes and the concentration of sodium. It is suggested that the effect of sodium on the calcium binding system in the sarcolemma may be a link between the inhibition of Na+K+-ATPase (EC 3.6.1.3) by cardiac glycosides and the subsequent increase in intracellular calcium.     

Spatial and temporal aspects of cell signalling

As new techniques are developed to measure intracellular messengers it becomes increasingly apparent that there is a remarkable spatial and temporal organization of cell signalling. Cells possess a small discrete hormone-sensitive pool of inositol lipid. In some cells such as Xenopus oocytes and Limulus photoreceptors this phosphoinositide signalling system is highly concentrated in one region of the cell, so establishing localized calcium gradients. Another example is the hydrolysis of inositol lipids in eggs at the point of sperm entry resulting in a localized increase in Ins(1,4,5)P3 and calcium which spreads like a wave throughout the egg. In hamster eggs this burst of calcium at fertilization recurs at 1-3 min intervals for over 100 min, a particularly dramatic example of spontaneous activity. Spontaneous oscillations in intracellular calcium exist in many different cell types and are often induced by agonists that hydrolyse inositol lipids. We have made a distinction between oscillations that are approximately sinusoidal and occur at a higher frequency where free calcium is probably continuously involved in the oscillatory cycle and those where calcium falls to resting levels for many seconds between transients. In the former case, the oscillations are thought to be induced through a cytoplasmic oscillator based on the phenomenon of calcium-induced calcium release. Such oscillations can be induced in Xenopus oocytes after injection with Ins(1,4,5)P3. A receptor-controlled oscillator based on the periodic formation of Ins(1,4,5)P3 is probably responsible for the generation of the widely spaced calcium transients. The function of such calcium oscillations is currently unknown. They may be a reflection of the feedback interactions that operate to control intracellular calcium. Another possibility emerged from observations that in some cells the frequency of calcium oscillations varied with agonist concentration, suggesting that cells might employ these oscillations as a way of encoding information. One advantage of using such a frequency-dependent mechanism may lie in an increase in fidelity, especially at low agonist concentrations. Whatever these functions might be, it is clear that uncovering the mechanisms responsible for such oscillatory activity will greatly enhance our understanding of the relation between the phosphoinositides and calcium signalling.     

Induction of a sodium-dependent depolarization by external calcium removal in human sperm

Removal of external calcium with EGTA (from 2.5 mm to nanomolar levels) caused a remarkable depolarization in human sperm. This depolarization was initially fast. It was followed by a slow phase that brought the Vm to values of over 0 mV in 1-2 min. The slow and sustained phase correlated with a sustained decrease in intracellular calcium. However, calcium removal still induced depolarization in sperm with enhanced intracellular calcium (induced by progesterone), indicating that the sustained depolarization was not caused by a sustained intracellular calcium decrease. The depolarization was reduced as the external sodium content was substituted with choline, indicating that it was due to a sodium current, and was observed in lithium but not in tetramethylammonium-containing medium. In low sodium medium, the addition of sodium after calcium removal induced depolarization to the extent of which slightly increased in 2 min. The depolarization was completely inhibited by external magnesium (Ki = 1.16 mm). The addition of calcium or magnesium to calcium removal-induced depolarized sperm induced hyperpolarization that was inhibited by ouabain and was also prevented in medium without potassium, suggesting that the activity of the electrogenic Na+,K+-ATPase was involved. The conductance activated by calcium removal might unveil the presence of a calcium channel that in the absence of external calcium allows sodium permeation and that in normal conditions might contribute to the resting intracellular calcium concentration.     

X-ray structure of yeast Hal2p, a major target of lithium and sodium toxicity, and identification of framework interactions determining cation sensitivity

The product of the yeast HAL2 gene (Hal2p) is an in vivo target of sodium and lithium toxicity and its overexpression improves salt tolerance in yeast and plants. Hal2p is a metabolic phosphatase which catalyses the hydrolysis of 3'-phosphoadenosine-5'-phosphate (PAP) to AMP. It is, the prototype of an evolutionarily conserved family of PAP phosphatases and the engineering of sodium insensitive enzymes of this group may contribute to the generation of salt-tolerant crops. We have solved the crystal structure of Hal2p in complex with magnesium, lithium and the two products of PAP hydrolysis, AMP and Pi, at 1.6 A resolution. A functional screening of random mutations of the HAL2 gene in growing yeast generated forms of the enzyme with reduced cation sensitivity. Analysis of these mutants defined a salt bridge (Glu238 ellipsis Arg152) and a hydrophobic bond (Va170 ellipsis Trp293) as important framework interactions determining cation sensitivity. Hal2p belongs to a larger superfamily of lithium-sensitive phosphatases which includes inositol monophosphatase. The hydrophobic interaction mutated in Hal2p is conserved in this superfamily and its disruption in human inositol monophosphatase also resulted in reduced cation sensitivity.     

Involvement of phosphoinositides,calmodulin and glyoxalase-I in cell proliferation in callus cultures of Amaranthus paniculatus

The effect of lithium and trifluoperazine (TFP) was studied on cell proliferation in callus cultures of Amaranthus paniculatus. TFP (20 μM) and lithium (40 mM) inhibited the callus growth by 50% and 80%, respectively. The inhibition by lithium was reversed by the addition of myoinositol (2.5 mM). Equimolar concentration of NaCl, as that of LiCl, had no significant effect on callus growth. The activity of calmodulin was inhibited by TFP as tested both by in vivo and in vitro experiments. The level of phosphatidylinositol (PI) in calli grown on lithium was lower than the calli grown on the medium containing inositol alone. The activity of the enzyme glyoxalase-I was inhibited by lithium and TFP. The inhibition of the enzyme activity by lithium was reversed by the addition of inositol. Possible involvement of phosphoinositide cycle, calcium and calmodulin in cell proliferation in in vitro cultures is suggested.     

Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation.

Cold shock elicits an immediate rise in cytosolic free calcium concentration ([Ca2+]cyt) in both chilling-resistant Arabidopsis and chilling-sensitive tobacco (Nicotiana plumbaginifolia). In Arabidopsis, lanthanum or EGTA caused a partial inhibition of both cold shock [Ca2+]cyt elevation and cold-dependent kin1 gene expression. This suggested that calcium influx plays a major role in the cold shock [Ca2+]cyt response and that an intracellular calcium source also might be involved. To investigate whether the vacuole (the major intracellular calcium store in plants) is involved, we targeted the calcium-dependent photoprotein aequorin to the cytosolic face of the vacuolar membrane. Cold shock calcium kinetics in this microdomain were consistent with a cold-induced vacuolar release of calcium. Treatment with neomycin or lithium, which interferes with phosphoinositide cycling, resulted in cold shock [Ca2+]cyt kinetics consistent with the involvement of inositol trisphosphate and inositide phosphate signaling in this response. We also investigated the effects of repeated and prolonged low temperature on cold shock [Ca2+]cyt. Differences were observed between the responses of Arabidopsis and N. plum-baginifolia to repeated cold stimulation. Acclimation of Arabidopsis by pretreatment with cold or hydrogen peroxide caused a modified calcium signature to subsequent cold shock. This suggests that acclimation involves modification of plant calcium signaling to provide a "cold memory."     

Neural function: metabolism and actions of inositol metabolites in mammalian brain

In the nervous system, a variety of cell types respond to external stimuli through the inositol lipid signalling pathways. The stimulus-coupled sequence of intracellular events has been investigated in a homogeneous model system, the cloned mammalian neural cell line NG115-401L. The neural peptide bradykinin stimulates a rapid production of identified inositol phosphate isomers and an intracellular Ca2+ discharge followed by a persistent plasma membrane influx. The temporal sequence suggests that Ins(1,4,5)P3 or Ins(1,3,4,5)P4 or both may coordinate these events in a neuronal cell, as has been suggested in other cell types. Thapsigargin, an irritant and tumour-promoting plant product, produces calcium transients in the absence of inositol phosphate production, and may provide a new tool for investigating the interactions between inositol phosphates and changes in cellular calcium homeostasis. In the 401L line, high levels of radiolabelled InsP5 and InsP6 have been detected, which has led to the evaluation of their possible occurrence and actions in normal brain. Both InsP5 and InsP6 are produced from a radiolabelled myo-inositol precursor in intact mature brain in a region-specific manner. This suggests that both inositol polyphosphates may be end products of regionally regulated biosynthetic pathways. When microinjected into a nucleus of the brainstem, or iontophoretically applied to the dorsal horn of the spinal cord, both InsP5 and InsP6, but not Ins(1,3,4,5)P4 isomers, appear to be potent neural stimulants. These results suggest that the inositol lipid signalling pathways may generate both intracellular and extracellular signals in brain.     

Thapsigargin defines the roles of cellular calcium in secretagogue-stimulated enzyme secretion from pancreatic acini.

In the present study we used thapsigargin (TG), an inhibitor of microsomal calcium ATPase, to evaluate the roles of free cytoplasmic calcium and intracellular stored calcium in secretagogue-stimulated enzyme secretion from rat pancreatic acini. Using microspectrofluorimetry of fura-2-loaded pancreatic acini, we found that TG caused a sustained increase in free cytoplasmic calcium by mobilizing calcium from inositol 1,4,5-trisphosphate-sensitive intracellular stores and by increasing influx of extracellular calcium. TG also caused a small increase in basal amylase secretion, inhibited the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate, and potentiated the stimulation of amylase secretion caused by 12-O-tetradecanoylphorbol-13-acetate or secretagogues that increase cyclic adenosine 3',5'-monophosphate. Bombesin, which like TG increased free cytoplasmic calcium, also potentiated the stimulation of amylase secretion caused by secretagogues that increase cyclic adenosine 3',5'-monophosphate, but did not inhibit the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate. Finally, TG inhibited the sustained phase of cholecystokinin-stimulated amylase secretion and potentiated the time course of vasoactive intestinal peptide-stimulated amylase secretion. The present findings indicate that stimulation of amylase secretion by secretagogues that increase inositol 1,4,5-trisphosphate does not depend on increased free cytoplasmic calcium per se. In contrast, TG-induced potentiation of the stimulation of secretagogues that increase cellular cyclic adenosine 3',5'-monophosphate appears to result from increased free cytoplasmic calcium per se.     

Interaction of intracellular ion buffering with transmembrane-coupled ion transport

The role of the Na/Ca exchanger in the control of cellular excitability and tension development is a subject of current interest in cardiac physiology. It has been suggested that this coupled transporter is responsible for rapid changes in intracellular calcium activity during single beats, generation of plateau currents, which control action potential duration, and control of intracellular sodium during Na/K pump suppression, which may occur during terminal states of ischemia. The actual behavior of this exchanger is likely to be complex for several reasons. First, the exchanger transports two ionic species and thus its instantaneous flux rate depends on both intracellular sodium and calcium activity. Secondly, the alteration in intracellular calcium activity, which is caused by a given transmembrane calcium flux, and which controls the subsequent exchanger rate, is a complex function of available intracellular calcium buffering. The buffers convert the ongoing transmembrane calcium fluxes into changes in activity that are a small and variable fraction of the change in total calcium concentration. Using a number of simple assumptions, we model changes in intracellular calcium and sodium concentration under the influence of Na/Ca exchange, Na/K ATPase and Ca-ATPase pumps, and passive sodium and calcium currents during periods of suppression and reactivation of the Na/K ATPase pump. The goal is to see whether and to what extent general notions of the role of the Na/Ca exchanger used in planning and interpreting experimental studies are consistent with its function as derived from current mechanistic assumptions about the exchanger. We find, for example, that based on even very high estimates of intracellular calcium buffering, it is unlikely that Na/Ca exchange alone can control intracellular sodium during prolonged Na/K pump blockade. It is also shown that Na/Ca exchange can contaminate measurements of Na/K pump currents under a variety of experimental conditions. The way in which these and other functions are affected by the dissociation constants and total capacity of the intracellular calcium buffers are also explored in detail.     

Pressure injection of calcium both excites and adapts Limulus ventral photoreceptors

Single pressure injections of 1-2 mM calcium aspartate into the light-sensitive region of Limulus ventral photoreceptors resulted in a rapid, 20-40-mV depolarization lasting approximately 2 s. The depolarization closely followed the rise in intracellular free calcium caused by the injection, as indicated by aequorin luminescence. The depolarization was followed by reversible desensitization (adaptation) of responses to both light and inositol 1,4,5 trisphosphate. Similar single injections of calcium into the light-insensitive region of the receptor were essentially without effect, even though aequorin luminescence indicated a large, rapid rise in intracellular free calcium. The depolarization caused by injection of calcium arose from the activation of an inward current with rectification characteristics and a reversal potential between +10 and +20 mV that were similar to those of the light-activated conductance, which suggests that the same channels were activated by light and by calcium. The reversal potentials of the light- and calcium-activated currents shifted similarly when three-fourths of the extracellular sodium was replaced by sucrose, but were not affected by a similar replacement of sodium by lithium. The current activated by calcium was abolished by prior injection of a calcium buffer solution containing EGTA. The responses of the same cells to brief light flashes were slowed and diminished in amplitude, but were not abolished after the injection of calcium buffer. Light adaptation and prior injection of calcium diminished the calcium-activated current much less than they diminished the light-activated current.