Estudio de los datos

In addition to intracellular Ca2+ _{mobilisation by IP}

3, there is a substantial body

of evidence supporting the role of two pyridine nucleotides in Ca2+ _{mobilisation,}

cyclic adenosine diphosphate-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP).

1.4.4.1 Ca2+ mobilisation by cADPR

In 1987 Lee and colleagues discovered that, in addition to IP3, 2 pyridine

nucleotides were capable of mediating Ca2+ release in sea urchin egg homogenate. These nucleotides were identified as β-nicotinamide adenine dinucleotide (β-NAD+)

and β-nicotinamide adenine dinucleotide phosphate (β-NADP+) (Clapper et al.,

1987). Prior desensitisation of the homogenates with any of the 3 messengers did not inhibit further Ca2+ release by subsequent application of a different molecule. This illustrated that the 2 pyridine nucleotides not only mobilised Ca2+ by a mechanism different to that of IP3 but also via mechanisms independent of each other (Clapper et al., 1987). However, the authors also observed that β-NAD+ mobilised Ca2+ after a

notable delay of approximately 1-2 minutes which was absent with both IP3 and β-

NADP+ mediated Ca2+ release (Clapper et al., 1987). This delay was later demonstrated to be due to the enzymatic conversion of β-NAD+ into an active, Ca2+

mobilising metabolite identified as cADPR (Lee et al., 1989). Following this discovery, enzymes capable of synthesising cADPR were identified in sea urchin eggs and a number of mammalian tissues, suggesting that cADPR is a ubiquitous intracellular Ca2+ mobilising messenger (Koshiyama et al., 1991; Lee et al., 1991; Rusinko et al., 1989; Walseth et al., 1991).

1.4.4.2 Synthesis of cADPR

The synthesis of cADPR may be stimulated by the activation of a number of GPCR signalling pathways (Higashida, 1997). cADPR is synthesised from β-NAD+

and this reaction is thought to be mediated by ADP-ribosyl cyclases (ARCs) which are a family of multifunctional enzymes, that exhibit a number of activities such as base-exchange, glycohydrolase and cyclase activity (Lee, 1999; Lee, 2000). The first ARC to be isolated was from the ovetestis of Aplysia californica (Hellmich et al., 1991) and two mammalian homologues of the Aplysia ARC have also been identified: the type II membrane glycoprotein CD38 (Howard et al., 1993; States et al., 1992) and a GPI-anchored protein, CD157 (Hirata et al., 1994). However investigations into the topology of the CD38 enzyme revealed that that the catalytic site is located on the extracellular face of the plasma membrane. This poses a ‘topological paradox’

whereby cADPR is potentially synthesised extracellularly, only to then mobilise Ca2+ via interaction with its intracellularly located receptor (De Flora et al., 2000). A solution to this paradox was proposed by De Flora and colleagues, basing their suggestion on the finding that β-NAD+ can be transported outside the cell via

connexin 43 hemichannels where it is then converted to cADPR, and transported back into the cell (Bruzzone et al., 2001). However this process would appear to be energy inefficient and more suited to an autocrine / paracrine signalling mechanism rather than a rapid intracellular signalling system. A more probable solution is that an as yet unidentified CD38 homologue could be found on the surface of intracellular organelles. Consistent with this view, 3 ARC isoforms from the sea urchin egg, ARCα, ARCβ and ARCγ, have been cloned and characterised by Galione and co-

workers (Davis et al., 2008). Though not closely related to CD38 the distribution of these isoforms revealed that ARCα was ectocellular, while ARCβ and ARCγ were

located within the lumen of acidic organelles and in a pattern reminiscent of the distribution of cortical granules (Davis et al., 2008). It is therefore clear, that in sea urchin eggs there exists a closer coupling between the site of cADPR synthesis and its target receptor, than would be indicative of an extracellularly orientated ARC. Furthermore, in mammalian cells subpopulations of CD38 appear to be localised on the ER and nuclear membranes (Sun et al., 2002; Yalcintepe et al., 2005). Taken together these data suggest that future investigations may identify a mammalian equivalent of the intracellular ARCβ and ARCγ, yet with limited homology to the sea

urchin isoforms. Such an enzyme that is distinct from CD38 and CD157 would therefore be capable of cADPR synthesis intracellularly and thus resolve the paradox associated with CD38 and CD157.

Some but not all ARCs are multifunctional, and it has been shown that metabolism of cADPR may be mediated by ARCs as, in addition to being able to catalyse the generation of cADPR from β-NAD+, both CD38 and CD157 have also

been shown to catalyse the hydrolysis of cADPR to ADP-ribose (Hirata et al., 1994; Howard et al., 1993).

1.4.4.3 cADPR-mediated Ca2+ mobilisation via ryanodine receptors

Ca2+ release by cADPR involves the regulation of RyRs present on the ER/SR. This was first demonstrated in sea urchin egg homogenates whereby caffeine and ryanodine desensitised sea urchin egg homogenate to Ca2+ release by cADPR but not IP3 (Galione et al., 1991; Lee et al., 1993). Also, the RyR antagonist ruthenium red

inhibited cADPR-mediated Ca2+ release (Galione et al., 1991). Furthermore, it was later shown that lower, subthreshold concentrations of caffeine potentiated Ca2+ release by cADPR (Lee, 1993). This suggested that cADPR not only released Ca2+ from the same store as caffeine but also acted on the same Ca2+ release channels, RyRs (Lee, 1993). The role of RyRs in cADPR-mediated Ca2+ release has since been shown in a broad range of cell types from protozoa to mammals. In the protozoan parasite, T.gondii ruthenium red inhibited cADPR-mediated Ca2+ release but not IP3-

mediated Ca2+ release (Chini et al., 2005). Consistent with cADPR-mediated Ca2+ release via RyRs in the sea urchin egg, inhibitory concentrations of ryanodine and ruthenium red block cADPR-mediated Ca2+ _{release in a number of mammalian}

preparations including rat brain microsomes and mesangial cells (Meszaros et al., 1993; White et al., 1993; Yusufi et al., 2001). Furthermore, ryanodine also inhibits cADPR-mediated hyperpolarisation and dilation in rat pulmonary arterial smooth muscle cells (Boittin et al., 2003), and reduced expression of the RyR subtype 3 in Jurkat T-cells results in a significant reduction in Ca2+ release in response to cADPR without affecting Ca2+ release by IP3 (Schwarzmann et al., 2002).

RyRs are large (~2000 kDa) homotetramers that regulate Ca2+ release from the S / ER. There are currently 3 known mammalian subtypes of RyR (RyR1, RyR2 and RyR3) which have approximately 65% amino acid homology (Hamilton, 2005). RyRs may be activated by Ca2+ directly or Ca2+ may facilitate further Ca2+ release via RyRs in a process known as Ca2+-induced Ca2+ release (CICR) (Endo et al., 1970). This process of CICR allows the amplification of small, localised Ca2+ signals into global Ca2+ waves via the progressive recruitment of subpopulations of RyRs. Ca2+ at millimolar concentrations may also inhibit RyRs and thus RyRs exhibit a bell-shaped concentration-response to cytoplasmic Ca2+ (Fill et al., 2002). As previously mentioned, in addition to Ca2+ modulation of RyRs, cADPR is also an endogenous regulator that can either activate RyRs directly or facilitate CICR via RyRs by sensitising RyRs to Ca2+ (Galione et al., 1991; Meszaros et al., 1993). Further

complexity is provided by the fact that Ca2+ can sensitise RyRs to activation by cADPR (Panfoli et al., 1999). It is therefore important to consider these combinatorial effects of cADPR and Ca2+ on the regulation of RyRs when investigating Ca2+ signals involving RyR and / or CICR (Evans et al., 2005).

Despite the fact that cADPR has been shown to regulate Ca2+ signalling via RyRs, investigations on the mechanism of binding / regulation of RyRs by cADPR have proved inconclusive. Initial experiments regarding cADPR binding used [32P]N3-cADPR as a photoaffinity probe for cADPR binding sites. Photoaffinity

labelling identified two putative cADPR binding proteins (Walseth et al., 1993). These proteins of approximately 100 and 140 kDa in size are smaller than RyRs (~2000 kDa) (Hamilton, 2005) and were therefore considered to be either RyR fragments or distinct cADPR binding/accessory proteins (Walseth et al., 1993). However, the former suggestion was deemed ‘unlikely’ by the authors as no larger labelled proteins, indicative of less fragmented sections, were detected. Furthermore, binding of cADPR to the two detected proteins was inhibited by caffeine to a differing extent, suggesting that they are separate proteins rather than resultant fragments from proteolysis of a larger protein (Walseth, et al., 1993). In recent years evidence has also been presented to suggest that FK506-binding protein 12.6 (FKBP12.6) is involved in cADPR-mediated Ca2+ release via RyRs. Based on studies involving microsomes from rat pancreatic islets it was proposed that FKBP12.6 normally binds to RyRs and has an inhibitory effect. Upon cADPR binding to FKBP12.6 it has been proposed that the FKBP12.6-cADPR complex dissociates from the RyR resulting in an increased open probability (Noguchi et al., 1997). Similar results have also been demonstrated in other cell types including coronary arterial smooth muscle and tracheal smooth muscle (Tang et al., 2002; Wang et al., 2004) thus adding further support for FKBP12.6 acting as a cADPR binding protein. However, contrary to this proposal, it was observed that cADPR did not affect the binding of recombinant FKBPs to RyR3 in HEK293 cells (Bultynck et al., 2001). Furthermore, it has also been demonstrated that in the sea urchin egg, antagonists of FKBP, rapamycin and FK-506, failed to inhibit [32P]cADPR binding (Thomas et al., 2001). Taken together, these data would suggest that either the cADPR binding site is an as yet unidentified homologue of the FKBPs or that cADPR binding to FKBP12.6 could be RyR subtype-specific.

Fig. 1.2. Summary schematic representation of the mechanisms involved in Ca2+ _{extrusion/influx}

across the plasma membrane and sequestering/release of Ca2+ _{by intracellular Ca}2+ _stores

Depicted are some of the mechanisms by which Ca2+ _{is able cross the plasma membrane and the}

membranes of some of the intracellular Ca2+ stores. Cav, voltage gated Ca2+ channel; ROC, receptor

operated channel; PMCA, plasma membrane Ca2+ ATPase; NCX, Na+/Ca2+ exchanger; KCa, Ca2+-

activated K+ _{channel; Cl}

Ca, Ca2+-activated Cl- channel; STIM, stromal interaction molecule; SERCA,

Sarco/endoplasmic reticulum Ca2+ _{ATPase; RyR, Ryanodine receptor; IP}

3R, IP3 receptor; SPCA,

secretory pathway ATPase; MCU, mitochondrial Ca2+ _{uniporter; RaM, rapid mode of mitochondrial}

Ca2+ _uptake.