Calcium mobilization by inositol phosphates and other intracellular messengers.

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] is now widely recognized as a messenger controlling the release of calcium from intracellular stores. In oocytes, and also probably in excitable cells, another potential calcium-mobilizing messenger is cyclic ADP ribose, although there is as yet little evidence that its levels are regulated by hormones or other extracellular mediators. In addition to signaling intracellular calcium release, [Ins(1,4,5)P(3)] also regulates calcium entry across the plasma membrane, but not in a direct manner. Rather, the depletion of intracellular stores by the calcium-mobilizing action of [Ins (1,4,5)P(3)] initiates a process of retrograde signaling whereby the depleted stores generate or release a diffusible messenger that is believed to act on the plasma membrane. A phosphorylated metabolite of [Ins(1,4,5)P(3)], inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P(4)], has been proposed to modulate this process, but the literature is not consistent on this point. A recently proposed candidate for the retrograde messenger is an activity extracted from Jurkat cells termed CIF (calcium influx factor), which has many properties consistent with such a messenger. There is also evidence that a GTP-dependent process, possibly involving a small G protein, is involved in signaling calcium entry and may be involved in either the formation or action of the diffusible messenger for calcium entry.

cGMP is not required for capacitative Ca2+ entry in Jurkat T-lymphocytes.

In this study, we have addressed the potential role of cGMP in regulating calcium entry in Jurkat T-lymphocytes. These cells display capacitative Ca(2+)-entry in response to the intracellular Ca(2+)-ATPase inhibitor, thapsigargin (TG). In the presence of extracellular Ca2+, TG stimulates a sustained elevation of intracellular cGMP levels. In the absence of extracellular Ca2+, TG induces no apparent increase in the levels of cGMP. However, experiments using Mn2+, as a surrogate for Ca2+, demonstrated that TG increased the rate of divalent cation entry in the absence of extracellular Ca2+. Treatment of Jurkat cells with the guanylyl cyclase inhibitor, LY83583 (20 microM), completely blocked cGMP formation in response to TG. However, LY83583 treated cells still exhibited a sustained, albeit partially reduced, Ca2+ response induced by TG. These data demonstrate that, in Jurkat cells, the sustained formation of cGMP is dependent on elevated intracellular Ca2+, and that elevated levels of cGMP are not necessary for the activation of capacitative Ca2+ entry.

Effect of cytoplasmic Ca2+ on (1,4,5)IP3 formation in vasopressin-activated hepatocytes.

The role of cytoplasmic calcium as a regulator of phospholipase C in vasopressin-activated hepatocytes was examined. According to models in which calcium spiking arises because of a positive feedback by calcium on phospholipase C, Ca2+ is seen as a positive modulator of phospholipase C under conditions of submaximal receptor activation. However, in hepatocytes whose precursor lipids had been labeled by incubation in [3H]-inositol, no increase in [3H]-(1,4,5)IP3 was detected in response to thapsigargin, in either unstimulated cells, or in cells stimulated with 1 nM vasopressin. Addition of a maximal concentration of vasopressin (1 microM) caused a rapid and substantial increase in [3H]-(1,4,5)IP3. These results indicate that changes in cytoplasmic calcium do not influence phospholipase C activity in hepatocytes, even under conditions of submaximal agonist activation. These findings also support models that provide for calcium spiking at constant levels of (1,4,5)IP3 at least in the case of the rat hepatocyte.

Effects of elevated cytoplasmic calcium and protein kinase C on endoplasmic reticulum structure and function in HEK293 cells.

In human embryonic kidney (HEK) cells stably transfected with green fluorescent protein targeted to the endoplasmic reticulum (ER), elevation of intracellular Ca2+ ([Ca2+]i) altered ER morphology, making it appear punctate. Electron microscopy revealed that these punctate structures represented circular and branched rearrangements of the endoplasmic reticulum, but did not involve obvious swelling or pathological fragmentation. Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA), prevented the effects of ionomycin on ER structure without affecting the elevation of [Ca2+]i. These results suggest that protein kinase C activation alters cytoplasmic or ER components underlying the effects of high [Ca2+]i on ER structure. Treatment of HEK cells with PMA also reduced the size of the thapsigargin-sensitive Ca2+ pool and inhibited Ca2+ entry in response to thapsigargin. Thus, protein kinase C activation has multiple actions on the calcium storage and signalling function of the endoplasmic reticulum in HEK cells: (1) reduced intracellular Ca2+ storage capacity, (2) inhibition of capacitative Ca2+ entry, and (3) protection of the endoplasmic reticulum against the effects of high [Ca2+]i.

Mechanisms of activated Ca2+ entry in the rat pancreatoma cell line, AR4-2J.

The characteristics of Ca2+ entry activated by surface receptor agonists and membrane depolarization were studied in the rat pancreatoma cell line, AR4-2J. Ca2+ mobilization activated by substance P, bombesin, or muscarinic receptor stimulation was found to involve both Ca2+ release and entry. In addition, depolarization of the surface membrane of AR4-2J cells with elevated concentrations of K+ activated Ca2+ entry. Ca2+ entry induced by membrane depolarization was inhibited by the L-channel antagonist, nimodipine, while that due to surface receptor agonists was not inhibited by this agent. The microsomal Ca(2+)-ATPase inhibitor, thapsigargin, caused both depletion of the agonist-sensitive intracellular Ca2+ pool and sustained Ca2+ influx indistinguishable from that produced by bombesin or methacholine. These results confirm that, unlike the pancreatic acinar cells from which they are presumably derived, AR4-2J cells express voltage-sensitive, dihydropyridine-inhibitable Ca2+ channels. However, in contrast to previous reports with this cell line, in the AR4-2J cells in use in our laboratory, and under our experimental conditions, surface receptor agonists (including substance P) do not cause Ca2+ influx through voltage-sensitive Ca2+ channels. Instead, we conclude that agonist-activated Ca2+ mobilization is initiated by (1,4,5)IP3-mediated intracellular Ca2+ release and that Ca2+ influx is regulated primarily, if not exclusively, by the state of depletion of the (1,4,5)IP3-sensitive intracellular Ca2+ pool.

A human synaptic vesicle monoamine transporter cDNA predicts posttranslational modifications, reveals chromosome 10 gene localization and identifies TaqI RFLPs.

A human vesicular monoamine transporter cDNA has been identified by screening a human brainstem library using sequences from the rat brain synaptic vesicle monoamine transporter (SVMT) [(1992) Cell 70, 539-551; (1992) Proc. Natl. Acad. Sci. USA 89, 10993-10997]. The hSVMT shares 92% amino acid identity with the rat sequence, but displays one less consensus site for asparagine N-linked glycosylation and one more consensus site for phosphorylation by protein kinase C. The human SVMT gene maps to chromosome 10q25 using Southern blotting analysis of human/rodent hybrid cell lines and fluorescent in situ hybridization approaches. The cDNA, and a subclone, recognize TaqI polymorphisms that may prove useful to assess this gene's involvement in neuropsychiatric disorders involving monoaminergic brain systems.

Human and mouse dopamine transporter genes: conservation of 5'-flanking sequence elements and gene structures.

Synaptic reaccumulation of the neurotransmitter dopamine is mediated by the dopamine transporter (DAT), a member of the family of twelve transmembrane domain, sodium- and chloride-dependent neurotransmitter transporters. Several DAT features, including its exclusive expression in dopaminergic neurons, implication in cocaine action, and prominent role in the mechanisms of Parkinsonism-inducing neurotoxins, make understanding of the DAT gene of interest. Isolation and characterization of the human and mouse DAT genes has allowed elucidation of similarities between each and other members of this transporter gene family. Sequences 5' to transcriptional start sites contain G-C rich, TATA-less, CAAT-less regions with striking conservation between human and mouse gene flanking regions. These studies suggest sequence elements that are candidates to contribute to the dopamine transporter's dopaminergic cell-specific expression.

The inositol phosphate-calcium signalling system in lacrimal gland cells.

From the above discussion, it is clear that the regulation of Ca2+ signalling in exocrine cells is a complex process involving activation of both intracellular Ca2+ release as well as the entry of Ca2+ across the plasma membrane. A poorly understood mechanism links these two phases of Ca2+ signalling thereby providing both rapid as well as sustained signals for the initiation and maintenance of appropriate exocrine responses. Further work is needed to better understand the mechanisms controlling this important and ubiquitous signalling system.

Calcium signalling in exocrine glands.

In exocrine gland cells, stimulation of a variety of surface receptors initiates a Ca2+ signalling system through activation of a polyphosphoinositide-specific phospholipase C. One product of phospholipase C activity, inositol 1,4,5-trisphosphate ((1,4,5)IP3), signals the release of intracellular Ca2+. Release of intracellular Ca2+ is followed by entry of Ca2+ into the cell across the plasma membrane. The mechanism by which Ca2+ entry is regulated is not well understood, although it is clear that (1,4,5)IP3 plays an important role. One hypothesis suggests that Ca2+ entry is triggered by the depletion of intracellular Ca2+ stores by (1,4,5)IP3, a process termed 'capacitative calcium entry'. The purpose of these studies is to gain understanding into the processes controlling capacitative calcium entry in exocrine gland cells.

Inhibition of thapsigargin-induced calcium entry by microinjected guanine nucleotide analogues. Evidence for the involvement of a small G-protein in capacitative calcium entry.

Injection of mouse lacrimal acinar cells with the non-hydrolyzable analogue of GTP, GTP gamma S (guanosine 5'-3-O-(thio)triphosphate), caused a rapid release of intracellular calcium but failed to activate calcium entry. Injection of GTP gamma S together with the inositol 1,4,5-trisphosphate receptor inhibitor, heparin, did not induce calcium release but blocked the activation of capacitative calcium entry by thapsigargin. Injection of GDP beta S (guanosine 5'-O-(thio)diphosphate) produced similar effects. The inhibitor effects of GDP beta S were prevented if an equal concentration of GTP was included in the injection pipette. These findings suggest that one of the steps linking the depletion of intracellular calcium pools to calcium entry across the plasma membrane requires the hydrolysis of GTP and may involve a small G-protein.

Biosynthesis and secretion of insulin.

The mechanisms involved in the biosynthesis, storage and secretion of insulin in the normal pancreatic B-cell have been the subject of intensive investigation during the last twenty years. As a result of this activity a relatively clear picture has emerged about the basic processes involved, and increasing information is available of their regulation and integration. Most of this information is derived from studies with rodent islets, but in general it appears to be applicable to the human B-cell. The article attempts to provide an overview from the vast literature on pancreatic B-cell function; it cannot be entirely comprehensive within the space available.

Effect of inositol 1,3,4,5-tetrakisphosphate on inositol trisphosphate-activated Ca2+ signaling in mouse lacrimal acinar cells.

In mouse lacrimal acinar cells, microinjection of the metabolically stable analog of inositol 1,4,5-trisphosphate, inositol 2,4,5-trisphosphate ((2,4,5)IP3), stimulated both intracellular Ca2+ mobilization and Ca2+ entry. Microinjection of inositol 1,3,4,5-tetrakisphosphate ((1,3,4,5)IP4), the inositol 1,4,5-trisphosphate-3-kinase product, was ineffective at mobilizing intracellular Ca2+ or activating Ca2+ entry. In lacrimal cells previously microinjected with submaximal levels of (2,4,5)IP3, the subsequent microinjection of low to moderate concentrations of (1,3,4,5)IP4 did not result in additional release of intracellular Ca2+, nor did it potentiate the Ca2+ entry phase attributable to (2,4,5)IP3. However, as previously demonstrated (Bird, G. S. J., Rossier, M. F., Hughes, A. R., Shears, S. B., Armstrong, D. L., and Putney, J. W., Jr. (1991) Nature 352, 162-165), additional injections of (2,4,5)IP3 induced further mobilization of intracellular Ca2+ and increased the elevated and sustained Ca2+ entry phase. Introduction of high concentrations of (1,3,4,5)IP4 appeared to inhibit or block the (2,4,5)IP3-induced Ca2+ entry phase. These results were consistent with the observed effect of (1,3,4,5)IP4 in permeabilized lacrimal cells, where (1,3,4,5)IP4 did not release cellular 45Ca2+ but at high concentrations inhibited the ability of submaximal concentrations of (2,4,5)IP3 to release 45Ca2+. Likewise, injection of a high concentration of (1,3,4,5)IP4 prior to injection of (2,4,5)IP3 blocked both release and influx of Ca2+. The inhibitory action of (1,3,4,5)IP4 on Ca2+ signaling observed in intact cells occurred at concentrations that might be obtained in agonist-stimulated cells. However, in permeabilized cells, (1,3,4,5)IP4 inhibited Ca2+ mobilization at concentrations exceeding those likely to occur in agonist-stimulated cells. These results suggest that physiologically relevant levels of (1,3,4,5)IP4 in the cell cytoplasm do not release Ca2+, nor do they potentiate inositol trisphosphate-induced Ca2+ entry across the plasma membrane. Rather, the possibility is raised that (1,3,4,5)IP4 or one of its metabolites could function as a negative feedback on Ca2+ mobilization by inhibiting inositol 1,4,5-trisphosphate-induced Ca2+ release.

Sulfhydryl reagents and cAMP-dependent kinase increase the sensitivity of the inositol 1,4,5-trisphosphate receptor in hepatocytes.

Sulfhydryl reagents such as tert-butyl hydroperoxide (TBHP) have been shown to increase cytosolic Ca2+ concentration ([Ca2+]i) in rat hepatocytes in a way that resembles responses to Ca(2+)-mobilizing hormones (Saikada, I., Thomas, A. P., and Farber, J. L. (1991) J. Biol. Chem. 266, 717-722; Rooney, T. A., Renard, D. C., Sass, E. J., and Thomas, A. P. (1991) J. Biol. Chem. 266, 12272-12282) and to increase the amount of Ca2+ released by inositol 1,4,5-trisphosphate ((1,4,5)IP3) from permeable rat liver cells (Rooney et al., 1991, op. cit.; Missiaen, L., Taylor, C. W., and Berridge, M. J. (1991) Nature 352, 241-244; Renard, D. C., Seitz, M. B., and Thomas, A. P. (1992) Biochem. J. 284, 507-512). The effects of sulfhydryl reagents were studied in fura-2-injected rat and guinea pig hepatocytes and compared with the actions of cAMP (Burgess, G. M., Bird, G. St. J., Obie, J. F., and Putney, J. W., Jr. (1991) J. Biol. Chem. 261, 4772-4781). In rat liver cells, the increases in [Ca2+]i induced by TBHP and thimerosal were prevented by microinjection of the cells with the (1,4,5)IP3 receptor antagonist heparin. In guinea pig hepatocytes, TBHP was not able to increase [Ca2+]i unless the cells were pretreated with angiotensin II to raise endogenous levels of (1,4,5)IP3 or were first injected with a sub-threshold concentration of inositol 2,4,5-trisphosphate ((2,4,5)IP3). The responses to TBHP in (2,4,5)IP3-injected guinea pig cells were also blocked by heparin. In many respects, the actions of TBHP appeared to be similar to those of cAMP, which has previously been shown to increase sensitivity to (1,4,5)IP3 in intact guinea pig hepatocytes (Burgess et al., 1991, op. cit.). TBHP also mimicked the effect of cAMP-dependent kinase (PKA) in permeabilized guinea pig hepatocytes by increasing the amount of Ca2+ released by (1,4,5)IP3. The responses to TBHP and cAMP in (2,4,5)IP3-injected guinea pig hepatocytes differed, however, in that the increase in [Ca2+]i evoked by elevating intracellular cAMP was greatly reduced by Wiptide, an inhibitor of PKA, while Wiptide had no effect on the Ca2+ transients induced by TBHP. This provides evidence that the sensitizing effect of TBHP is not mediated by PKA and is more likely to be a direct effect on the inositol trisphosphate receptor. It is possible, however, that the sulfhydryl reagents and PKA act on a common regulatory site on the receptor protein.

Sustained Ca2+ signaling in mouse lacrimal acinar cells due to photolysis of "caged" glycerophosphoryl-myo-inositol 4,5-bisphosphate.

In saponin-permeabilized mouse lacrimal acinar cells, glycerophosphoryl-myo-inositol 4,5-bisphosphate (GPIP2) activated the release of sequestered Ca2+ to the same extent as inositol 1,4,5-trisphosphate ((1,4,5)IP3) but with a potency about 1/10 that of (1,4,5)IP3. In lacrimal gland homogenates, [3H]GPIP2 was metabolized to two compounds which upon anion exchange high performance liquid chromatography eluted at positions indicating that they were [3H]GPIP and [3H]GPIP3. The rate of metabolism of [3H]GPIP2 was much slower than that of [3H](1,4,5)IP3, and its rate of phosphorylation was less than 1% of that of [3H] (1,4,5)IP3. In intact lacrimal cells, photolysis of a microinjected "caged" derivative of GPIP2, 1-(alpha-glycerophosphoryl)-myo-inositol 4,5-bisphosphate P4(5)-1-(2-nitrophenyl)ethyl ester, resulted in sustained activation of Ca2+ signaling; i.e. intracellular Ca2+ release followed by increased entry of Ca2+ across the plasma membrane. These findings indicate that caged GPIP2 should provide a useful tool for producing photolytically initiated, sustained activation of intracellular (1,4,5)IP3 receptors. They also provide strong support for the idea that sustained Ca2+ signaling can be achieved in lacrimal acinar cells by activation of intracellular receptors for (1,4,5)IP3 in the absence of stimulated production of inositol 1,3,4,5-tetrakisphosphate.

Subcellular distribution of the calcium-storing inositol 1,4,5-trisphosphate-sensitive organelle in rat liver. Possible linkage to the plasma membrane through the actin microfilaments.

The role of Ins(1,4,5)P3 in the mobilization of Ca2+ from intracellular stores of non-muscle cells has been extensively demonstrated; however, the nature of the organelle releasing the Ca2+ is still poorly understood. The distributions of the Ins(1,4,5)P3-binding sites and of the Ins(1,4,5)P3-sensitive Ca2+ pool were investigated in subcellular fractions obtained from rat liver and compared with those of other markers. The Ins(1,4,5)P3-binding vesicles appeared to be completely distinct from the endoplasmic-reticulum-derived microsomes and were enriched in the same fractions which were enriched in alkaline phosphodiesterase I activity. This co-purification of the plasma-membrane marker with the Ins(1,4,5)P3-binding sites was dramatically altered after freezing or after treatment of the homogenate with the microfilament-disruptive drug cytochalasin B, suggesting that the Ins(1,4,5)P3-sensitive organelle may be linked to the plasma membrane through the actin microfilaments. No correlation was observed between the Ins(1,4,5)P3-binding capacity and the portion of the Ca2+ pool that was released by Ins(1,4,5)P3. This may result from the disruption of the native organelle during homogenization, leading to the formation of vesicles containing the Ins(1,4,5)P3 receptor, but lacking the Ca2+ pump. These results are consistent with the idea of a specialized Ins(1,4,5)P3-regulated organelle distinct from the endoplasmic reticulum, and we propose a model of the structural organization of this organelle, in which the anchorage to the cytoskeleton as well as the spatial separation of the Ca2+ pump from the Ins(1,4,5)P3 receptor have important functional significance.

Sinusoidal oscillations in intracellular calcium requiring negative feedback by protein kinase C.

Stimulation of mouse lacrimal acinar cells with submaximal concentrations of the muscarinic agonist, methacholine, resulted in an increase in intracellular calcium ([Ca2+]i), which took the form of sinusoidal oscillations. These oscillations were relatively constant (approximately 4-5/min) regardless of the methacholine concentration, suggesting that the oscillations arise from an oscillating negative feedback in the signal transduction pathway. This negative feedback appears to involve oscillations in protein kinase C activity because the oscillations were prevented by activation, inhibition, or down-regulation of protein C. Activation of protein kinase C with phorbol esters inhibited the methacholine-induced [Ca2+]i signal and formation of the Ca2+ mobilizing messenger, inositol 1,4,5-trisphosphate. [Ca2+]i signals elicited by intracellular introduction of inositol phosphates did not oscillate and were not affected by activators or inhibitors of protein kinase C. Thus, the constant frequency [Ca2+]i oscillations appear to result from a negative feedback loop involving inhibition of inositol trisphosphate production by protein kinase C.

Actions of vasopressin and the Ca(2+)-ATPase inhibitor, thapsigargin, on Ca2+ signaling in hepatocytes.

When hepatocytes were loaded with fura-2 by incubation with the acetoxymethyl ester (fura-2/AM), addition of Mn2+ resulted in a rapid quench of a fraction of cellular fura-2 fluorescence. Addition of vasopressin caused a second, rapid quench of cellular fura-2, whereas the addition of thapsigargin had no effect. When hepatocytes were loaded by microinjection of fura-2 acid, addition of Mn2+ caused a slower, sustained rate of quench, and both vasopressin and thapsigargin increased this rate of quench. When Mn2+ was removed from the medium of fura-2/AM-loaded cells after preincubation with Mn2+, vasopressin still caused quench of cellular fura-2. In contrast, neither vasopressin nor thapsigargin increased fura-2 quench when Mn2+ was removed from fura-2-injected cells. When fura-2/AM-loaded cells were permeabilized with saponin, only a fraction of the cell-associated fura-2 was quenched by addition of Mn2+. A second fraction was then quenched by addition of inositol 1,4,5-trisphosphate. These results indicate that in hepatocytes loaded with the acetoxymethyl ester of fura-2, the increased quench of cellular fura-2 seen with phospholipase C-linked agonists is not due to effects of the agonist on Mn2+ entry across the plasma membrane, but rather is due to agonist activation of Mn2+ penetration into an intracellular organelle, presumably through inositol 1,4,5-trisphosphate-regulated channels. Thus, it appears that compartmentalization of fura-2 accounts for previously reported anomalies in Ca2+ signaling in hepatocytes, such as the apparent failure of Ca(2+)-ATPase inhibition to increase divalent cation entry, as well as the apparent ability of phospholipase C-linked agonists to stimulate efflux of Ca2+.