Calcium Regulation—Section 17.2

Intracellular Ca²⁺ levels modulate a multitude of vital cellular processes—including gene expression, cell viability, cell proliferation, cell motility, cell shape and volume regulation—thereby playing a key role in regulating cell responses to external signals. These dynamic changes in Ca²⁺ levels are regulated by ligand-gated and G-protein–coupled ion channels in the plasma membrane and by mobilization of Ca²⁺ from intracellular stores. The generation of cytosolic Ca²⁺ spikes and oscillations typically involves the coordinated release and uptake of Ca²⁺ from these stores, mediated by intracellular Ca²⁺ channels and their response to several second messengers such as Ca²⁺ itself, cyclic ADP ribose and inositol triphosphate.

This section includes several Molecular Probes reagents for studying Ca²⁺ regulation in live cells. Fluorescent nucleotides, including analogs of ATP, ADP, AMP, GTP, GDP and GMP, are described in Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3. Our GTP analogs may be particularly useful in the assay of G-protein–coupled receptors. Probes for Lipid Metabolism and Signaling—Section 17.4 discusses several selective phosopholipase substrates, as well as labeled ceramide and sphingomyelin probes.

D-myo-1,4,5-Inositol Triphosphate and Caged D-myo-1,4,5-Inositol Triphosphate

We offer the potassium salt of D-myo-inositol 1,4,5-triphosphate (Ins 1,4,5-P₃, I3716) for researchers investigating inositol triphosphate–dependent Ca²⁺ mobilization and signal transduction mechanisms. Cytoplasmic Ins 1,4,5-P₃ is a potent intracellular second messenger that induces Ca²⁺ release from membrane-bound stores in many tissues. Our Ins 1,4,5-P₃ is at least 99% pure, as determined by paper chromatography and by ¹H NMR and ³¹P NMR.

NPE-caged Ins 1,4,5-P₃ can be used to generate rapid and precisely controlled release of Ins 1,4,5-P₃ in intact cells and is widely employed in studies of Ins 1,4,5-P₃–mediated second messenger pathways. Our NPE-caged Ins 1,4,5-P₃ (I23580) is a mixture of the physiologically inert, singly esterified P⁴ and P⁵ esters () and does not contain the somewhat physiologically active P¹ ester. NPE-caged Ins 1,4,5-P₃ exhibits essentially no biological activity prior to photolytic release of the biologically active Ins 1,4,5-P₃.

Fluorescent Heparin

Fluorescein-labeled heparin (H7482) should be a useful tool for studying binding of this mucopolysaccharide in cells and tissues. In addition to its well-known anticoagulant activity, heparin binds to the Ins 1,4,5-P₃ receptor and inhibits the biological cascade of events mediated by Ins 1,4,5-P₃. Heparin exhibits a number of other biological properties, including modulation of the structure, function and metabolism of many proteins and enzymes. This mucopolysaccharide binds to thrombin, low-density lipoproteins, lipoprotein lipase, circulatory serine proteases and proteinase inhibitors, as well as to blood vessel–associated proteins such as fibronectin and laminin. Heparin also interacts with heparin-binding growth factors (HBGFs) and, for HBGF-1, amplifies its mutagenic and neurotrophic activity. Fluorescein-labeled heparin can be prepared by different methods, which may influence its applications. Therefore, fluorescein heparin from Molecular Probes may not be identical to those used in all of the reported applications.

Cyclic ADP ribose (cADP-ribose) is a potent Ca²⁺-mobilizing agent that functions as a second messenger in an Ins 1,4,5-P₃–independent pathway and is involved in Ca²⁺-induced Ca²⁺ release in both mammalian cells and plants. cADP-ribose is a putative physiological regulator of certain isoforms of ryanodine-activated Ca²⁺ channels that may act through a calmodulin-mediated mechanism. cADP-ribose is reported to mobilize Ca²⁺ in dorsal root ganglion cells, pituitary cells and sea urchin eggs, where it can also act synergistically with ryanodine to release Ca²⁺ from intracellular Ca²⁺ stores. This Ca²⁺-mobilizing agent is also produced in pancreatic islets by glucose stimulation and has been shown to mediate glucose-induced insulin release in pancreatic cells and to induce release of Ca²⁺ from stores in and around the nuclear envelope. Researchers have investigated the role of cADP-ribose in agonist-evoked Ca²⁺ oscillations using pancreatic acinar cells. In this study, cADP-ribose–induced Ca²⁺ spikes could be blocked with either ryanodine or heparin, implicating both ryanodine and Ins 1,4,5-P₃ receptors in the Ca²⁺ spike generation.

Caged Cyclic ADP Ribose

The application of cADP-ribose in cells and tissues can be controlled spatially and temporally by flash photolysis of NPE-caged cADP-ribose (C7074, ). Photorelease of cADP-ribose from NPE-caged cADP-ribose results in initiation of Ca²⁺ release and the cortical reaction in sea urchin eggs.

Cyclic ADP Ribose Antagonist

Inhibition of cADP-ribose–induced Ca²⁺ mobilization can be achieved specifically and reversibly with the cADP-ribose antagonist 8-amino-cADP-ribose. 8-Amino-cADP-ribose (A7621, ) has been shown both to block binding of radiolabeled cADP-ribose to sea urchin egg microsomes and to inhibit cADP-ribose–mediated release of Ca²⁺ from egg homogenates, but it does not block caffeine- or ryanodine-induced Ca²⁺ release.

Caged ions and caged chelators can be used to influence the ionic composition of both solutions and cells, particularly for ions such as Ca²⁺ that are present at low concentrations. The properties and uses of caged probes are described in Photoactivatable Reagents, Including Photoreactive Crosslinkers and Caged Probes—Section 5.3.

NP-EGTA: A Caged Ca²⁺ Reagent

Developed by Ellis-Davies and Kaplan, the photolabile chelator o-nitrophenyl EGTA (NP-EGTA) exhibits a high selectivity for Ca²⁺, a dramatic 12,500-fold decrease in affinity for Ca²⁺ upon UV illumination (its K_d increases from 80 nM to >1 mM) and a high photochemical quantum yield (~0.2). Furthermore, with a K_d for Mg²⁺ of 9 mM, NP-caged EGTA does not perturb physiological levels of Mg²⁺. We offer both the potassium salt (N6802) and the acetoxymethyl (AM) ester (N6803) of NP-EGTA. The NP-EGTA salt can be complexed with Ca²⁺ to generate a caged calcium complex that will rapidly deliver Ca²⁺ upon photolysis (Figure 17.4). We have loaded similar chelators into cells with the Influx pinocytic cell-loading reagent (I14402, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8). The cell-permeant AM ester of NP-EGTA does not bind Ca²⁺ unless the AM esters are removed. It can potentially serve as a photolabile buffer in cells because, once converted to NP-EGTA by intracellular esterases, it will bind Ca²⁺ with high affinity until photolyzed with UV light. NP-EGTA has been used to measure the calcium buffering capacity of cells.

DMNP-EDTA: A Caged Ca²⁺ Reagent

The first caged Ca²⁺ reagent described by Ellis-Davies and Kaplan was 1-(4,5-dimethoxy-2-nitrophenyl) EDTA (DMNP-EDTA, D6814), which they named DM-Nitrophen (now a trademark of Calbiochem-Novabiochem Corp.). Because its structure better resembles that of EDTA than EGTA, we named it as a caged EDTA derivative (Figure 17.5). Upon illumination, DMNP-EDTA's K_d for Ca²⁺ increases from 5 nM to 3 mM. Thus, photolysis of DMNP-EDTA complexed with Ca²⁺ results in a pulse of free Ca²⁺. Furthermore, DMNP-EDTA has significantly higher affinity for Mg²⁺ (K_d = 2.5 µM) than does NP-EGTA (K_d = 9 mM). The photolysis product's K_d for Mg²⁺ is ~3 mM, making DMNP-EDTA an effective caged Mg²⁺ source, in addition to its applications for photolytic Ca²⁺ release. Photorelease of Ca²⁺ has been shown to occur in <180 microseconds, with even faster photorelease of Mg²⁺. A paper by Neher and Zucker discusses the uses and limitations of DMNP-EDTA.

Diazo-2: A Photoactivatable Ca²⁺ Scavenger

In contrast to NP-EGTA and DMNP-EDTA, diazo-2 is a photoactivatable Ca²⁺ scavenger. Diazo-2 (), which was introduced by Adams, Kao and Tsien, is a relatively weak chelator (K_d for Ca²⁺ = 2.2 µM). Following flash photolysis at ~360 nm, however, cytosolic free Ca²⁺ rapidly binds to the diazo-2 photolysis product, which has a high affinity for Ca²⁺ (K_d = 73 nM). Photolysis of diazo-2 has been used to decrease cytosolic Ca²⁺ in less than two milliseconds in tensed frog muscle cells and rabbit arterial smooth muscle. Microinjecting a relatively low concentration of fluo-3, fluo-4, or one of the Calcium Green or Oregon Green 488 BAPTA indicators (Fluorescent Ca{2+} Indicators Excited with Visible Light—Section 19.3), along with a known quantity of diazo-2, permits measurement of the extent of depletion of cytosolic Ca²⁺ following photolysis. Diazo-2 can be microinjected into cells as its potassium salt (D3034). Diazo-2 also has been loaded into cells permeabilized with Staphylococcus aureus α-toxin and can potentially be loaded into cells using our Influx pinocytic cell-loading reagent (I14402, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8).

Thapsigargin and Fluorescent Thapsigargin

Thapsigargin is a naturally occurring sesquiterpene lactone isolated from the umbelliferous plant Thapsia garganica. This tumor promoter releases Ca²⁺ from intracellular stores by specifically inhibiting the endoplasmic reticulum Ca²⁺-ATPase; it does not directly affect plasma membrane Ca²⁺-ATPases, Ins 1,4,5-P₃ production or protein kinase C activity. Reports have described the effects of thapsigargin-induced Ca²⁺ signals on the transient suppression of c-myb mRNA levels, as well as the regulation of such Ca²⁺ signals by sphingomyelinase and sphingosine.

Thapsigargin is available in 1 mg units (T7458) and specially packaged in 20 vials containing 50 µg each (T7459). We have also prepared the green-fluorescent BODIPY FL thapsigargin (B7487, ) and red-fluorescent BODIPY TR-X thapsigargin (B13800, ). These membrane-permeant probes have spectral properties similar to those of fluorescein and the Texas Red dye, respectively, and may be useful for localization of thapsigargin binding sites in live cells.

Luminescent Calcium Analog

The trivalent lanthanide terbium (III), which is supplied as its chloride salt (T1247), is a luminescent analog of Ca²⁺ that can be used to study structure–function relationships in Ca²⁺-binding proteins such as calmodulin, oncomodulin, lactalbumin and ATPases. The long-lived luminescence of Tb³⁺ has also been use to probe Ca²⁺-binding sites of alkaline phosphatase, glutamine synthetase, integrins, protein kinase C and ryanodine-sensitive Ca²⁺ channels. Tb³⁺ reportedly binds most strongly to the I and II sites of calmodulin.