The Anx7(+/-) knockout mutation alters electrical and secretory responses to Ca(2+)-mobilizing agents in pancreatic β-cells

Insulin secretion from the pancreatic β-cell is controlled by changes in membrane potential and intracellular Ca(2+). The contribution of intracellular Ca(2+) stores to this process is poorly understood. We have previously shown that β-cells of mice lacking one copy of the Annexin 7 gene (Anx7(+/-)) express reduced levels of IP(3) receptors and defects in IP(3)-dependent Ca(2+) signaling. To further elucidate the effect of the Anx7(+/-) mutation on signaling related to intracellular Ca(2+) stores in the β-cell, we measured the effects of Ca(2+) mobilizing agents on electrical activity, intracellular Ca(2+) and insulin secretion in control and mutant β-cells. We found that the muscarinic agonist carbachol and the ryanodine receptor agonists caffeine and 4-chloro-m-cresol had more potent depolarizing effects on Anx7(+/-) β-cells compared to controls. Accordingly, glucose-induced insulin secretion was augmented to a greater extent by caffeine in mutant islets. Surprisingly, ryanodine receptor-mediated Ca(2+) mobilization was not affected by the Anx7(+/-) mutation, suggesting that the mechanism underlying the observed differences in electrical and secretory responsiveness does not involve intracellular Ca(2+) stores. Our results provide evidence that both IP3 receptors and ryanodine receptors play important roles in regulating β-cell membrane potential and insulin secretion, and that the Anx7(+/-) mutation is associated with alterations in the signaling pathways related to these receptors.     

Role of the inositol 1,4,5-trisphosphate receptor in Ca(2+) feedback inhibition of calcium release-activated calcium current (I(crac)).

We examined the activation and regulation of calcium release-activated calcium current (I(crac)) in RBL-1 cells in response to various Ca(2+) store-depleting agents. With [Ca(2+)](i) strongly buffered to 100 nM, I(crac) was activated by ionomycin, thapsigargin, inositol 1,4,5-trisphosphate (IP(3)), and two metabolically stable IP(3) receptor agonists, adenophostin A and L-alpha-glycerophospho-D-myoinositol-4,5-bisphosphate (GPIP(2)). With minimal [Ca(2+)](i) buffering, with [Ca(2+)](i) free to fluctuate I(crac) was activated by ionomycin, thapsigargin, and by the potent IP(3) receptor agonist, adenophostin A, but not by GPIP(2) or IP(3) itself. Likewise, when [Ca(2+)](i) was strongly buffered to 500 nM, ionomycin, thapsigargin, and adenophostin A did and GPIP(2) and IP(3) did not activate detectable I(crac). However, with minimal [Ca(2+)](i) buffering, or with [Ca(2+)](i) buffered to 500 nM, GPIP(2) was able to fully activate detectable I(crac) if uptake of Ca(2+) intracellular stores was first inhibited. Our findings suggest that when IP(3) activates the IP(3) receptor, the resulting influx of Ca(2+) quickly inactivates the receptor, and Ca(2+) is re-accumulated at sites that regulate I(crac). Adenophostin A, by virtue of its high receptor affinity, is resistant to this inactivation. Comparison of thapsigargin-releasable Ca(2+) pools following activation by different IP(3) receptor agonists indicates that the critical regulatory pool of Ca(2+) may be very small in comparison to the total IP(3)-sensitive component of the endoplasmic reticulum. These findings reveal new and important roles for IP(3) receptors located on discrete IP(3)-sensitive Ca(2+) pools in calcium feedback regulation of I(crac) and capacitative calcium entry.     

Anx7 is required for nutritional control of gene expression in mouse pancreatic islets of Langerhans

BACKGROUND: Gene expression in islets of Langerhans is profoundly sensitive to glucose and other nutrients. Islets of Langerhans in the Anx7(+/-) knockout mouse exhibit a profound reduction in ITPR3 protein expression, defective intracellular calcium signaling, and defective insulin secretion. Additional data presented here also show that mRNA for ITPR3 is virtually undetectable in isolated Anx7(+/-) islets. IP3Receptor type 3 (ITPR3) expression in islets of Langerhans is closely regulated by secretory stimuli, and it has been suggested that the level of the ITPR3 expression controls the ability of the islets to respond to nutritional signals. We report that although control islets respond to glucose in vitro by a transient increment in ITPR3 mRNA, the islets from the Anx7(+/-) mouse remain low. We therefore hypothesized that the Anx7/IP3 Receptor(3)/Ca(2+) signaling pathway plays a role in beta cell responses to glucose, and that in the absence of the Anx7/ITPR3 signaling system, the islets would be unable to discriminate between fed or fasted states in vivo. MATERIALS AND METHODS: To test this hypothesis, we subjected Anx7(+/-) and control mice to either food and water ad libidum or to an overnight fast with access to water only. We then isolated the respective islets and compared nutrient-dependent changes in global gene expression under the four conditions using genome-based microarray technology. RESULTS: Anx7 protein expression in these islets is only about 50% of control levels in normal littermate controls, and IPTR3 message and protein are virtually zero. cDNA microarray analyses show that in control animals gene expression is significantly affected by the fasting state. Many of the affected genes have historical relevance to development and differentiation of islets. These include preproglucagon, APOJ, cadherin2, phosphoglucoisomerase, oncostatin M, PAX6, HGF, and cytokeratin 18. However, there are also many other nutritionally sensitive genes in control islets that are principally associated with cell division and DNA repair. The latter genes have not specifically been associated with islet physiology in the past. By contrast, Anx7(+/-) mouse islets exhibit a greatly reduced ability to discriminate genomically between fed and fasted states for all classes of identified genes. Many of the validated genes are specific to islets in comparison to liver tissue examined. Real-time quantitative RT-PCR analysis of islets from Anx7 heterozygous mice and littermate controls revealed remarkable down-regulation in PTEN, Glut-2, PDX-1, IGF-1, and Neuro D1 expression, but not in liver. CONCLUSIONS: We conclude that reduced gene dosage in the Anx7(+/-) islet, with concomitant loss of ITPR3 expression and consequent defects in Ca(2+) signaling, may substantially contribute to the mechanism of the loss of genomic discrimination, in vivo, between the fed and fasted states. We believe that the requirement for complete Anx7 gene dosage and IPTR3 expression in islets of Langerhans will prove to be of fundamental importance for understanding the mechanism of nutritional sensing in health and disease.     

Calcium pools mobilized by calcium or inositol 1,4,5-trisphosphate are differentially localized in rat heart and brain.

Calcium-induced calcium release (CICR) pools have been demonstrated in brain and heart microsomes biochemically and autoradiographically by the sensitivity of 45Ca2+ accumulation to Mg2+, ATP, ruthenium red, caffeine, and tetracaine. The CICR pool colocalizes with [3H]ryanodine binding sites, supporting the notion that [3H]ryanodine labels CICR pools. Sites of CICR pools in the brain contrast with those of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools with reciprocal localizations between the two Ca2+ pools in several structures. Thus, in the hippocampus CA-1 is enriched in IP3-sensitive Ca2+ pools, whereas CICR pools are highest in CA-3 and the dentate gyrus. The corpus striatum and cerebellum are enriched in IP3 pools, whereas the medial septum and olfactory bulb have high CICR densities. In cardiac tissue, CICR is localized to atrial and ventricular muscle, whereas IP3 pools are concentrated in coronary vessels and cardiac conduction fibers. The reciprocal enrichment of IP3 and CICR Ca2+ pools implies differential regulation of Ca2+ hemostasis in these tissues.     

Modulation of intracellular free Ca2+ concentration by IP3-sensitive and IP3-insensitive nonmitochondrial Ca2+ pools

Intracellular Ca2+ pools play an important role in the adjustment of cytosolic free Ca2+ concentrations. This review summarizes the recent knowledge on receptor-mediated Ca2+ release and Ca2+ uptake mechanisms in Ca2+ stores of exocrine cells taking the exocrine pancreas and the parotid gland as an example. The intracellular mediator for agonist-induced Ca2+ release is inositol 1,4,5-trisphosphate (IP3) which acts by opening Ca2+ channels from the endoplasmic reticulum or a more specialized organelle called 'calciosome'. This Ca2+ release is the major event to increase cytosolic free Ca2+ concentrations of exocrine glands from a resting level of approximately 10(-7) mol/l to approximately 10(-6) mol/l. Subsequently also Ca2+ influx from the extracellular fluid into the cell is increased which involves the action of inositol 1,3,4,5-tetrakisphosphate (IP4). Intracellular nonmitochondrial Ca2+ reuptake occurs into IP3-sensitive (IsCaP) as well as into IP3-insensitive Ca2+ pools Ca2+ pools (IisCaP). While Ca2+ uptake into the IisCaP is mediated by a vanadate-sensitive Ca2+ pump, Ca2+ uptake into the IsCaP is mediated by a Ca2+/H+ exchanger at the expense of an H+ gradient which is established by a vacuolar type H+ pump present in the same Ca2+ pool. During stimulation both Ca2+ pools, IsCaP and IisCaP, are probably connected, the nature of which has not yet been clarified. It is suggested that GTP and/or IP4 control Ca2+ conveyance between intracellular Ca2+ pools by forming Ca2+-carrying junctions between membranes. Other models propose that Ca2+, which is released by IP3, induces Ca2+ release from another Ca2+ pool. Taking into account that H+ transport is present in IP3-sensitive Ca2+ pools the possibility of pH-regulated Ca2+ channels in the IisCaP, located in close neighbourhood to the IsCaP, is also considered.     

H+ uptake increases GTP-induced connection of inositol 1,4,5-trisphosphate- and caffeine-sensitive calcium pools in pancreatic microsomal vesicles.

Evidence suggests that GTP but not GTP gamma S activates Ca2+ movement between myo-inositol 1,4,5-trisphosphate (IP3)-sensitive and -insensitive Ca2+ pools (1). Measuring 45Ca2+ uptake into pancreatic microsomal vesicles we have determined the sizes of three different Ca2+ pools which release Ca2+ in response 1) to IP3, 2) to caffeine, and 3) to both IP3 and caffeine ("common" Ca2+ pool). In the presence of GTP the size of the IP3-sensitive Ca2+ pool is decreased whereas the "common" Ca2+ pool is increased as compared to control Ca2+ pool sizes in the presence of GTP gamma S. This effect of GTP is inhibited by bafilomycin B1, a specific inhibitor of vacuolar type H+ ATPases (2). We conclude that GTP induced connection between IP3- and caffeine-sensitive Ca2+ pools is triggered by intravesicular acidification and involves function of small GTP-binding proteins, known to mediate interorganelle transfer.     

Calcium uptake studies of 1,4-dihydropyridine agonists into rabbit aortic smooth muscle cells in culture

The effects of the three dihydropyridine calcium channel agonists (+/-)BAY K 8644, (+)202-791 and (+/-)CGP 28392 on 45Ca++ uptake were studied in cultures of rabbit aortic smooth muscle cells. At 10(-7) M each agonist enhanced 45Ca++ uptake in 15-50 mM K+ but had no effect on the basal 45Ca++ uptake at 5 mM K+. At the uptake threshold of 15 mM K+ each agonist potentiated 45Ca++ uptake in a dose-dependent manner with half maximal effects at 2.4 nM for (+/-)BAY K 8644, 22 nM for (+)202-791 and 18 nM for (+/-)CGP 28392. The agonists showed no significant antagonistic activity. Responses were antagonized competitively by nifedipine and non-competitively by (+/-)D-600. The 45Ca++ uptake dose-response curves and the half maximal effects of the three agonists were over the same range of concentrations as their inhibition of [3H]nitrendipine binding to rat ventricular receptor membrane preparations. The data suggest that these cells mimic the calcium uptake by the intact aorta better than commercial vascular smooth muscle lines or cardiac cells.     

Distribution of endoplasmic reticulum and calciosome markers in membrane fractions isolated from different regions of the canine brain

Four regions of the canine brain (frontal lobe, parieto-occipital lobe, brainstem, and cerebellum) were each fractionated by differential centrifugation into a crude mitochondrial pellet (P2) and a crude microsomal pellet (P3). Markers of endoplasmic reticulum (glucose-6-phosphate phosphatase and rotenone-insensitive NADPH cytochrome c reductase) and markers of the 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store ([3H]IP3 binding and IP3-induced Ca2+ release) were measured. No correlation was found between the two classes of markers, which suggests that the IP3 receptor does not belong to the endoplasmic reticulum in canine brain. Cerebellum P2 and P3 fractions displayed levels of [3H]IP3 binding 10- to 30-fold higher, and rates of IP3-induced Ca2+ release greater than 15-fold faster than the homologous cerebrum and brainstem fractions. Actively accumulated Ca2+ was only partially released by IP3, both before and after saponin disruption of the plasma membrane compartment. The proportion of the IP3-sensitive Ca2+ store relative to that of the total (IP3-sensitive and IP3-insensitive) Ca2+ store was variable; i.e., it was larger in cerebellum P2 (approximately 90%) than in cerebrum fractions (less than 30%). Cerebellum fractions constitute the best source from which an IP3-sensitive Ca2+ storing organelle can be purified.     

Comparative study of the effect of aliphatic alcohols on energy-dependent accumulation of Ca2+ in intracellular membranes of smooth muscle cells

The effects of ethanol and other aliphatic alcohols on energy-dependent Ca2+ transport in endoplasmic reticulum and mitochondria were studied in digitonin-treated myometrium cells. The Ca2+ uptake in mitochondria increased (on 15-20%) with increasing methanol, ethanol and propanol concentrations in medium, whereas further rise of concentration inhibited this process. Treatments of myometrial cells with short-chain alcohols caused an inhibition of calcium uptake in endoplasmic reticulum. Butanol inhibited both calcium uptake in mitochondria and endoplasmic reticulum. Ca2+ accumulation in intracellular pools is inhibited by aliphatic alcohols in the following order of potency: butanol > propanol > ethanol > methanol. It is concluded that modifying effect of aliphatic alcohols on energy dependent calcium accumulation in intracellular membrane structures is defined as on origin of Ca(2+)-transporting system and (or) properties of these membrane structures so on properties of alcohols.     

Melatonin and IP3-induced Ca2+ release from intracellular stores in the malaria parasite Plasmodium falciparum within infected red blood cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.     

Extracellular calcium-sensing receptor is expressed in rat hepatocytes. coupling to intracellular calcium mobilization and stimulation of bile flow

Liver cells respond to changes in Ca(2+)(o). The hepatic functions affected include bile secretion, metabolic activity, liver regeneration, and the response to xenobiotics. In the present study, we demonstrate the presence, in the liver, of the extracellular calcium-sensing receptor (CASR), described previously in the parathyroid and thyroid glands and kidney. CASR mRNA was specifically expressed in hepatocytes and was absent in nonparenchymal liver cells (stellate, endothelial, and Kupffer cells). Western blot analysis using a specific CASR antibody showed staining in both whole liver and hepatocyte extracts. Immunohistochemistry and in situ hybridization of rat liver sections showed expression of CASR protein and mRNA by a subset of hepatocytes. The known agonists of the CASR, gadolinium (Gd(3+); 0.5-3.0 mm) and spermine (1.25-20 mm), in the absence of Ca(2+)(o), elicited dose-related increases in Ca(2+)(i) in isolated rat hepatocytes loaded with Fura-2/acetoxymethyl ester. There was a greatly attenuated response to a second challenge with either agonist. The response was also abrogated when inositol 1,4,5-trisphosphate (IP(3))-sensitive calcium pools had been depleted by pretreatment with either thapsigargin or phenylephrine, an alpha(1)-adrenergic receptor agonist known to mobilize Ca(2+)(i) from IP(3)-sensitive pools. Addition of the deschloro-phenylalkylamine compound, NPS R-467, but not the S enantiomer, NPS S-467, increased the sensitivity of the Ca(2+)(i) mobilization response to 1.25 mm spermine. Bile flow ceased after Ca(2+)(o) withdrawal, and its recovery was enhanced by spermine in isolated perfused liver preparations. The CASR agonists Ca(2+) and Gd(3+) increased bile flow, and the response to a submaximal Ca(2+) concentration was enhanced by NPS R-467 but not the S compound. Thus, the data indicate that rat hepatocytes harbor a CASR capable of mobilizing Ca(2+)(i) from IP(3)-sensitive stores and that activation of the CASR stimulates bile flow.     

Cyclosporin A inhibits inositol 1,4,5-trisphosphate-dependent Ca2+ signals by enhancing Ca2+ uptake into the endoplasmic reticulum and mitochondria

Cytosolic Ca(2+) ([Ca(2+)](i)) oscillations may be generated by the inositol 1,4,5-trisphosphate receptor (IP(3)R) driven through cycles of activation/inactivation by local Ca(2+) feedback. Consequently, modulation of the local Ca(2+) gradients influences IP(3)R excitability as well as the duration and amplitude of the [Ca(2+)](i) oscillations. In the present work, we demonstrate that the immunosuppressant cyclosporin A (CSA) reduces the frequency of IP(3)-dependent [Ca(2+)](i) oscillations in intact hepatocytes, apparently by altering the local Ca(2+) gradients. Permeabilized cell experiments demonstrated that CSA lowers the apparent IP(3) sensitivity for Ca(2+) release from intracellular stores. These effects on IP(3)-dependent [Ca(2+)](i) signals could not be attributed to changes in calcineurin activity, altered ryanodine receptor function, or impaired Ca(2+) fluxes across the plasma membrane. However, CSA enhanced the removal of cytosolic Ca(2+) by sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), lowering basal and inter-spike [Ca(2+)](i). In addition, CSA stimulated a stable rise in the mitochondrial membrane potential (DeltaPsi(m)), presumably by inhibiting the mitochondrial permeability transition pore, and this was associated with increased Ca(2+) uptake and retention by the mitochondria during a rise in [Ca(2+)](i). We suggest that CSA suppresses local Ca(2+) feedback by enhancing mitochondrial and endoplasmic reticulum Ca(2+) uptake, these actions of CSA underlie the lower IP(3) sensitivity found in permeabilized cells and the impaired IP(3)-dependent [Ca(2+)](i) signals in intact cells. Thus, CSA binding proteins (cyclophilins) appear to fine tune agonist-induced [Ca(2+)](i) signals, which, in turn, may adjust the output of downstream Ca(2+)-sensitive pathways.     

Functional properties of recombinant type I and type III inositol 1, 4,5-trisphosphate receptor isoforms expressed in COS-7 cells

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are ubiquitous intracellular Ca(2+) release channels whose functional characterization by transfection has proved difficult due to the background contribution of endogenous channels. In order to develop a functional assay to measure recombinant channels, we transiently transfected the rat type I IP(3)R into COS-7 cells. Saponin-permeabilized COS cells transfected with type I IP(3)R showed a 50% increase in inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release at saturating [IP(3)] (10 micrometer) but no enhancement at subsaturating [IP(3)] (300 nm). However, cotransfection of the IP(3)R and human sarco/endoplasmic reticulum ATPase (SERCA)-2b ATPase cDNA resulted in 60 and 110% increases in Ca(2+) release at subsaturating and saturating doses of IP(3), respectively. IP(3) or adenophostin A failed to release (45)Ca(2+) from microsomal vesicles prepared from cells expressing either type I IP(3)R or SERCA cDNAs alone. However, microsomal vesicles prepared from cells doubly transfected with IP(3)R and SERCA cDNAs released 33.0 +/- 0.04% of the A23187-sensitive pool within 30 s of 1 micrometer adenophostin A addition. Similarly, the initial rate of (45)Ca(2+) influx into oxalate-loaded microsomal vesicles was inhibited by IP(3) only when the microsomes were prepared from COS cells doubly transfected with SERCA-2b and IP(3)R DNA. The absence of a functional contribution from endogenous IP(3)Rs has enabled the use of this assay to measure the Ca(2+) sensitivities of IP(3)-mediated (45)Ca(2+) fluxes through recombinant neuronal type I (SII(+)), peripheral type I (SII(-)), and type III IP(3)Rs. All three channels displayed a biphasic dependence upon [Ca(2+)](cyt). Introduction of mutations D2550A and D2550N in the putative pore-forming region of the type I IP(3)R inhibited IP(3)-mediated (45)Ca(2+) fluxes, whereas the conservative substitution D2550E was without effect. This assay therefore provides a useful tool for studying the regulatory properties of individual IP(3)R isoforms as well as for screening pore mutations prior to more detailed electrophysiological analyses.     

Inositol 1,4,5-trisphosphate receptors selectively localized to the acrosomes of mammalian sperm

Calcium flux is required for the mammalian sperm acrosome reaction, an exocytotic event triggered by egg binding, which results in a dramatic rise in sperm intracellular calcium. Calcium-dependent membrane fusion results in the release of enzymes that facilitate sperm penetration through the zona pellucida during fertilization. We have characterized inositol 1,4,5-trisphosphate (IP3)-gated calcium channels and upstream components of the phosphoinositide signaling system in mammalian sperm. Peptide antibodies colocalized G alpha q/11 and the beta 1 isoform of phospholipase C (PLC beta 1) to the anterior acrosomal region of mouse sperm. Western blotting using a polyclonal antibody directed against purified brain IP3 receptor (IP3R) identified a specific 260 kD band in 1% Triton X-100 extracts of rat, hamster, mouse and dog sperm. In each species, IP3R immunostaining localized to the acrosome cap. Scatchard analysis of [3H]IP3 binding to rat sperm sonicates revealed a curvilinear plot with high affinity (Kd = 26 nM, Bmax = 30 pmol/mg) and low affinity (Kd = 1.6 microM, Bmax = 550 pmol/mg) binding sites, reflecting among the highest receptor densities in mammalian tissue. Immunoelectron microscopy confirmed the acrosomal localization in rat sperm. The IP3R fractionated with acrosomes by discontinuous sucrose gradient centrifugation and was enriched in the medium of acrosome- reacted sperm. ATP-dependent 45Ca2+ loading of digitonin permeabilized rat sperm was decreased by 45% in the presence of 10 microM IP3. The IP3-mediated release of calcium was blocked by heparin. Thapsigargin, a sequiterpene lactone inhibitor of the microsomal Ca(2+)-ATPase, stimulated the acrosome reaction of mouse sperm to the same extent as the Ca2+ ionophore, A23187. The failure of caffeine and ryanodine to affect calcium accumulation suggested that thapsigargin acted through an IP3-sensitive store. The presence of G alpha q/11, PLC beta 1 and a functional IP3R in the anterior acrosomal region of mammalian sperm, as well as thapsigargin''s induction of the acrosome reaction, implicate IP3-gated calcium release in the mammalian acrosome reaction.     

Characterization of inositol 1,4,5-trisphosphate receptors and calcium mobilization in a hepatic plasma membrane fraction

The distribution of hepatic binding sites for the calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (IP3), was analyzed in subcellular fractions of the rat liver by binding studies with [32P]IP3 and compared with the Ca2+ release elicited by IP3 in each fraction. Three major subcellular fractions enriched in plasma membrane, mitochondria, and endoplasmic reticulum were characterized for their 5'-nucleotidase, glucose-6-phosphatase, succinate reductase, and angiotensin II binding activities. The fraction enriched in plasma membrane showed 7- and 20-fold increases in IP3 binding capacity over those enriched in endoplasmic reticulum and mitochondria, respectively, and contained a single class of high-affinity binding sites with Kd of 1.7 +/- 1.0 nM and concentration of 239 +/- 91 fmol/mg protein. IP3 binding reached equilibrium in 30 min at 0 degrees C, and the half-time of dissociation was about 15 min. The specificity of the IP3 binding sites was indicated by their markedly lower affinities for inositol 1-phosphate, phytic acid, fructose 1,6-bisphosphate, 2,3-bisphosphoglycerate, and inositol 1,3,4,5-tetrakisphosphate. The Ca2+-releasing activity of IP3 in the subcellular fractions was monitored with the fluorescent indicator, Fura-2. All three fractions showed ATP-dependent Ca2+ uptake and rapidly released Ca2+ in response in IP3. The fraction enriched in plasma membrane was the most active in this regard, releasing 174 +/- 67 pmol Ca2+/mg of protein compared to 45 +/- 10 and 48 +/- 7 pmol/mg protein for the fractions enriched in endoplasmic reticulum and mitochondria, respectively. These data suggest that the [32P]IP3 binding sites represent specific intracellular receptors through which IP3 mobilizes Ca2+ from a storage site associated (or co-purifying) with the plasma membrane of the rat liver. It is likely that a specialized vesicular system (to which IP3 can bind and trigger the release of Ca2+) is located in close proximity with the plasma membrane and is thus adjacent to the site at which IP3 is produced during stimulation of the hepatocyte by Ca2+-mobilizing hormones.     

Effects of inositol 1,4,5-trisphosphate on calcium release from the endoplasmic reticulum and Golgi apparatus in mouse mammary epithelial cells: a comparison during pregnancy and lactation

It has been established that inositol 1,4,5-trisphosphate(IP3) is responsible for the mobilization of calcium(Ca2+) from intracellular locations in a wide variety of tissues, and that this response triggers the stimulation of several hormones and neurotransmitters. However, these phenomena have yet to be examined in the mammary epithelium. Ca2+ uptake from the medium into the endoplasmic reticulum(ER) and Golgi apparatus in vitro in both pregnant and lactating mouse mammary epithelial cells was studied and a strong Ca2+ release from these organelles into the medium with the use of IP3 was shown. The Ca2+ uptake and its release due to IP3 was also usually greater during pregnancy than lactation.     

myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes.

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.     

The IP(3) receptor-mitochondria connection in apoptosis and autophagy

The amount of Ca(2+) taken up in the mitochondrial matrix is a crucial determinant of cell fate; it plays a decisive role in the choice of the cell between life and death. The Ca(2+) ions mainly originate from the inositol 1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) stores of the endoplasmic reticulum (ER). The uptake of these Ca(2+) ions in the mitochondria depends on the functional properties and the subcellular localization of the IP(3) receptor (IP(3)R) in discrete domains near the mitochondria. To allow for an efficient transfer of the Ca(2+) ions from the ER to the mitochondria, structural interactions between IP(3)Rs and mitochondria are needed. This review will focus on the key proteins involved in these interactions, how they are regulated, and what are their physiological roles in apoptosis, necrosis and autophagy. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Calcium release in HSY cells conforms to a steady-state mechanism involving regulation of the inositol 1,4,5-trisphosphate receptor Ca2+ channel by luminal [Ca2+]

In many cell types, low concentrations of inositol 1,4,5-trisphosphate (IP3) release only a portion of the intracellular IP3-sensitive Ca2+ store, a phenomenon known as "quantal" Ca2+ release. It has been suggested that this effect is a result of reduced activity of the IP3- dependent Ca2+ channel with decreasing calcium concentration within the IP3-sensitive store ([Ca2+]s). To test this hypothesis, the properties of IP3-dependent Ca2+ release in single saponin-permeabilized HSY cells were studied by monitoring [Ca2+]s using the Ca(2+)-sensitive fluorescent dye mag-fura-2. In permeabilized cells, blockade of the sarco/ER Ca(2+)-ATPase pump in stores partially depleted by IP3 induced further Ca2+ release via an IP3-dependent route, indicating that Ca2+ entry via the sarco/ER Ca(2+)-ATPase pump had been balanced by Ca2+ loss via the IP3-sensitive channel before pump inhibition. IP3- dependent Mn2+ entry, monitored via quenching of luminal mag-fura-2 fluorescence, was readily apparent in filled stores but undetectable in Ca(2+)-depleted stores, indicating markedly reduced IP3-sensitive channel activity in the latter. Also consistent with reduced responsiveness of Ca(2+)-depleted stores to IP3, the initial rate of refilling of these stores was unaffected by the presence of 0.3 microM IP3, a concentration that was clearly effective in eliciting Ca2+ release from filled stores. Analysis of the rate of Ca2+ release at various IP3 concentrations indicated a significant shift of the IP3 dose response toward higher [IP3] with decreasing [Ca2+]s. We conclude that IP3-dependent Ca2+ release in HSY cells is a steady-state process wherein Ca2+ efflux via the IP3 receptor Ca2+ channel is regulated by [Ca2+]s, apparently via changes in the sensitivity of the channel to IP3.     

Effects of methotrexate on calcium flux in rat liver mitochondria, microsomes and plasma membrane vesicles

The metabolic effects of methotrexate in perfused livers are similar to those exerted by hormones acting through Ca(2+)-dependent mechanisms. The aim of the present study was to determine whether the effects of methotrexate are mediated by a direct action on cellular Ca(2+) fluxes. Methotrexate did not affect the ATP-dependent (45)Ca(2+) uptake by mitochondria, microsomes and inside-out plasma membrane vesicles and Ca(2+) efflux from plasma membrane vesicles. However, methotrexate was able to stimulate (45)Ca(2+) release from preloaded microsomes. The amount of Ca(2+) released by methotrexate was similar to that induced by IP(3). Methotrexate could be acting through the capacitative calcium entry mechanism.