A Self-restricted CD38-connexin 43 Cross-talk Affects NAD+ and Cyclic ADP-ribose Metabolism and Regulates Intracellular Calcium in 3T3 Fibroblasts

Abstract

CD38, a type II transmembrane glycoprotein widely expressed in mammalian cells, is both a receptor/co-receptor initiating signal-transducing processes and also a multifunctional enzyme (1-3). Its known enzyme activities are synthesis from NAD ϩ of nicotinamide and of the potent calcium mobilizer cyclic ADP-ribose (cADPR) 1 (ADP-ribosyl cyclase) and degradation of cADPR to ADP-ribose (cADPR hydrolase); in addition, CD38 has been demonstrated to catalyze a number of baseexchange reactions that include conversion of NADP ϩ , in specific conditions and in the presence of nicotinic acid, to an additional calcium mobilizer, i.e. NAADP ϩ (4, 5). Since the discovery of its role in cADPR metabolism, it soon became clear that CD38 is an ectoenzyme (6 -11). This property raised a number of obvious questions on: (i) how the cyclase substrate NAD ϩ can become accessible to the catalytic site of CD38 at the outer surface of many cells, and (ii) even assuming ectocellular cADPR generation, how the CD38 product cADPR can reach the intracellular ryanodine-sensitive channels from which it releases calcium into the cytosol (12-15). The latter question was also supported by failure to identify any functional effect of cADPR other than its intracellular calcium releasing activity.Similar topological questions arose for that fraction of CD38 which is localized to intracellular membrane vesicles. These mediate the export of "de novo" synthesized CD38 to the plasma membrane (16) and also the opposite process of CD38 endocytosis that is observed upon incubating several cells types with specific ligands, e.g. 18). In both cases, the active site of CD38 is intravesicular and therefore unavailable to cytosolic NAD ϩ . Moreover, any intravesicularly generated cADPR would be sequestered inside the vesicles and therefore be unable to target the ryanodine-sensitive calcium stores.This "topological paradox" of the CD38/cADPR system appeared to be challenging, in view of the powerful calcium releasing activity of cADPR and of the remarkably increased cytosolic calcium ([Ca 2ϩ ] i ) levels (and consequent triggering of calcium-stimulated cell functions) that are observed during both de novo expression of CD38 (16) and its ligand-induced internalization (18). Both findings clearly indicated, in the absence of obvious underlying mechanisms, that intracellularly localized CD38 (e.g. vesicle-bound) can in fact convert cytosolic NAD ϩ to cADPR and that the cyclic nucleotide is functionally active as it can have accessibility to the calcium stores from which it up-modulates calcium release (19).Recently, three different transporters for NAD ϩ and cADPR have been identified, which have elucidated the topological

New roles for astrocytes: Gap junction hemichannels have something to communicate

“…The precursor NAD + is released from the cytoplasm into the medium through Cx43 hemichannels, as indicated by: (i) dependence on level of Cx43 expression in transfected or antisensetreated cells; (ii) sensitivity to gap-junction blockers; and (iii) influx into and efflux from liposomes incorporating isolated Cx43. cADPR is also synthesized in intracellular vesicles transporting CD38 to or from the surface; NAD + enters the vesicles through Cx43 hemichannels and cADPR moves to the cytoplasm by action of the same nucleotide transporters that operate in the surface membrane [33,55]. This pathway of synthesis would prevent loss of NAD + and cADPR by diffusion away from an ectoenzyme, but also prevent paracrine action.…”

“…Surprisingly, it is synthesized by the ectoenzyme CD38, an integral membrane protein with an extracellular active site that cyclizes NAD + to form cADPR ( Figure 2). cADPR, which does not permeate Cx43 hemichannels [31,33], reaches its site of action on ryanodine receptors by both active and passive transport across the surface membrane [55]. It can also have a paracrine action, which might be why an ectoenzyme is used.…”

“…Although Cx43 is largely dephosphorylated by the time metabolic inhibition is inducing hemichannel opening, cell-cell coupling is not abolished, and dephosphorylation might not play a role in hemichannel opening. Protein kinase C (PKC)-mediated phosphorylation reduces hemichannel permeability to NAD + [33]. Cx43 hemichannels are opened by alendronate, a bisphosphonate that inhibits dexamethasoneinduced apoptosis and is used to promote survival of osteoblasts in the prevention of osteoporosis [12].…”

Connexin channel permeability to cytoplasmic molecules

“…43h (Kam et al, 1998) 3f 43h (Romanello and D'Andrea P, 2001) 3a 3c 3e 43j (Venance et al, 1997) 1a 1b 2 5 43j (Paemeleire et al, 2000) 1b 2 43j (Romanello and D'Andrea P, 2001) 1a 1b 2 32j > 43j > 26j 1b 2 (normalized by Mn spread, not junctional conductance) mobile pH buffers 43j (Swietach and Vaughan-Jones, 2004) 1a 57 NAD+ 43h (Bruzzone et al, 2001a;Bruzzone et al, 2001a) 3a 3b 3c 3f peptides ≤ 10mers 43j (Neijssen et al, 2005) 1a prostaglandin E2 43h (Jiang and Cherian, 2003;Cherian et al, 2005) 3a 3b 3c 3e RNA 12mer φ 26/32j (Valiunas et al, 2005) 1a 43j (Valiunas et al, 2005) 1a RNA 16mer 43j (Valiunas et al, 2005) 1a RNA 24mer 43j (Valiunas et al, 2005) 1a dsRNA 12mer φ 43j (Valiunas et al, 2005) 1a siRNA 22mer φ 26/32j (Valiunas et al, 2005) 1a 43j (Valiunas et al, 2005) 1a 5…”

“…φ ATP during paracellular propagation of Ca waves cAMP (Kam et al, 1998) 3f IP3 (Kam et al, 1998) 3f IP3 (Romanello and D'Andrea P, 2001) 3a 3c 3e NAD+ (Bruzzone et al, 2001a;Bruzzone et al, 2001a) 3a 3b 3c 3f prostaglandin E2 (Jiang and Cherian, 2003;Cherian et al, 2005) 3a 3b 3c 3e 43j ADP/ATP (Goldberg et al, 1998) 1a φ adenosine (Goldberg et al, 1999) 1a cAMP (Bedner et al, 2003;Bedner et al, 2006) 1a cAMP (Ponsioen et al, 2007) 1a IP3 (Venance et al, 1997) 1a 1b 2 5 IP3 1b 2…”