The ATP-sensitive K(+) (K(ATP)) channels are composed of the pore-forming K(+) channel Kir6.0 and different sulfonylurea receptors (SURs). SUR1, SUR2A, and SUR2B are sulfonylurea receptors that are characteristic for pancreatic, cardiac, and vascular smooth muscle-type K(ATP) channels, respectively. The structural elements of SURs that are responsible for their different characteristics have not been entirely determined. Here we report that the 42 amino acid segment at the C-terminal tail of SURs plays a critical role in the differential activation of different SUR-K(ATP) channels by ADP and diazoxide. In inside-out patches of human embryonic kidney 293T cells coexpressing distinct SURs and Kir6.2, much higher concentrations of ADP were needed to activate channels that contained SUR2A than SUR1 or SUR2B. In all types of K(ATP) channels, diazoxide increased potency but not efficacy of ADP to evoke channel activation. Replacement of the C-terminal segment of SUR1 with that of SUR2A inhibited ADP-mediated channel activation and reduced diazoxide modulation. Point mutations of the second nucleotide-binding domains (NBD2) of SUR1 and SUR2B, which would prevent ADP binding or ATP hydrolysis, showed similar effects. It is therefore suggested that the C-terminal segment of SUR2A possesses an inhibitory effect on NBD2-mediated ADP-induced channel activation, which underlies the differential effects of ADP and diazoxide on K(ATP) channels containing different SURs.

An ecto-enzyme of NAD glycohydrolase (NADase) induced by retinoic acid in HL-60 cells is attributed to the molecule of CD38 antigen [Kontani, K., Nishina, H., Ohoka, Y., Takahashi, K., and Katada, T. (1993) J. Biol. Chem. 268, 16895-16898]. CD38 antigen has an amino acid sequence homologous to Aplysia ADP-ribosyl cyclase which generates cyclic adenosine diphosphoribose (cADPR) and nicotinamide (NA) from beta-NAD+. On the basis of this sequence homology, we compared enzyme properties between CD38 NADase expressed as a fusion protein in Escherichia coli and ADP-ribosyl cyclase purified from the ovotestis of Aplysia kurodai. 1) beta-NAD+ analogs, nicotinamide 1, N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide, did not serve as good substrates for the ADP-ribosyl cyclase, suggesting that the intact adenine ring of beta-NAD+ was required for the cyclase-catalyzed reaction. On the other hand, CD38 NADase utilized the NAD analogs to form ADP-ribose and NA. 2) Kinetic analyses of the ADP-ribosyl cyclase reaction revealed that NA was first released from the substrate (beta-NAD+)-enzyme complex, followed by the release of another product, cADPR, which was capable of interacting with the free enzyme. 3) The enzyme reaction catalyzed by the ADP-ribosyl cyclase was fully reversible; beta-NAD+ could be formed from cADPR and NA with a velocity similar to that observed in the degradation of beta-NAD+. However, CD38 NADase did not catalyze the reverse reaction to form beta-NAD+ from ADP-ribopase and NA. 4) The CD38 NADase activity was, but the ADP-ribosyl cyclase activity was not, inhibited by dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

The cytotoxic effects of fluoride on odontoblasts are not clear. In this study, we examined whether NaF induces apoptosis in MDPC-23 odontoblast-like cells and the involvement of mitogen-activated protein kinase (MAPK) signaling pathways in NaF-induced apoptosis. MDPC-23 cells incubated with 5 mM NaF for 24 hrs exhibited caspase-3 activation, cleavage of poly(ADP-ribose) polymerase, DNA fragmentation, and an increase in cytoplasmic nucleosomes. Prior to the induction of apoptosis, all MAPKs examined were phosphorylated, but in a different manner. In contrast to the sustained phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and p38, NaF exposure induced a biphasic phosphorylation of extracellular signal-regulated protein kinase (ERK). NaF-induced apoptosis was markedly suppressed by treatment with the JNK inhibitor, SP600125, and mildly suppressed by the MAPK/ERK kinase inhibitor, U0126. Inhibition of p38 activity did not protect cells from apoptosis. Thus, exposure to NaF induces apoptosis in odontoblast-like cells, depending on JNK and, less significantly, ERK pathways.

To reveal the effects of cadmium exposure on the endoplasmic reticulum (ER) stress response, we examined the expression and function of 78-kDa glucose-regulated protein (Grp78), an ER-resident molecular chaperone, in LLC-PK1 cells. In cells treated with 10 μM cadmium chloride, Grp78 protein levels increased after 6 hr and remained elevated at 24 hr. When cells were incubated with 1–20 μM CdCl2 for 6 hr, Grp78 increased in a dose-dependent manner. In addition, Grp78 mRNA levels were elevated in response to CdCl2 exposure. After exposure to 10 μM CdCl2, the levels of activating transcription factor 4 (ATF4) were increased at 2 hr, with a further enhancement after that; this accumulation followed the transient but marked phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) on serine 51. Although ATF4 mRNA levels increased mildly by CdCl2 exposure, treatment with actinomycin D did not suppress CdCl2-induced accumulation of ATF4 protein, suggesting the involvement of posttranscriptional and, in part, transcriptional mechanisms. Compared with other heavy-metal compounds such as manganese chloride, zinc chloride, mercuric chloride, and lead chloride, CdCl2 could increase the levels of Grp78, ATF4, and the phosphorylated form of eIF2α more markedly without definite cellular damage. The silencing of Grp78 expression using short-interference RNA enhanced CdCl2-induced cellular damage. These results show that cadmium induces the expression of Grp78 probably via phosphorylation of eIF2α and resultant translation of ATF4, and this ER stress response plays a role in protection against cadmium cytotoxicity in this renal epithelial cell.

The lymphocyte cell surface antigen, CD38, which has an amino acid sequence similar to Aplysia ADP-ribosyl cyclase, catalyzes not only the hydrolysis of NAD' and l-(S-phospho-P-D-ribosyl)adenosine 5'-phosphate cyclic anhydride (cyclic ADP-ribose) but also the formation of cyclic ADP-ribose from NAD'. To characterize the bifunctional enzyme properties, we produced the recombinant CD38 fused with a maltose-binding protein (MBP-CD38). Zinc ions stimulated the ADP-ribosyl cyclase activity of MBP-CD38, but inversely inhibited its NAD' glycohydrolase activity which was approximately 100-fold dominant to the cyclase activity in the absence of Zn'+. Such dual effects of Zn'+ were also observed in the native membrane-bound CD38 of HL-60 cells which had been caused to differentiate by retinoic acid. Zinc ions inhibited the NAD' glycohydrolase reaction catalyzed by MBP-CD38 in an uncompetitive manner, whereas they enhanced the ADP-ribosyl cyclase reaction without affecting the K,, value for NAD'. There was an increase in the fluorescence intensity of a hydrophobic fluorescent probe, 8-anilino-1 -naphthalenesulfonate, in the presence of MBP-CD38. The fluorescence increase was further enhanced by the addition of Zn2+ with a shift in the maximum emission wavelength from 484nm to 470nm, suggesting that Zn" caused conformational changes of MBP-CD38. These results indicate that Zn2+ directly interacts with CD38 to stimulate its ADP-ribosyl cyclase with inhibition of its NAD' glycohydrolase, probably due to prevention of the access of water molecule to an intermediate of the enzymesubstrate complex.Keywords: CD38 ; cyclic ADP-ribose ; zinc ; NAD' glycohydrolase ; ADP-ribosyl cyclase.The cell-surface antigen, CD38, is a 46-kDa type-I1 singletransmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long C-terminal extracellular domain [I, 21. The expression of CD38 is widely used as a phenotypic marker of the differentiation or activation of T and B lymphocytes [3-51, though its function has not been fully elucidated. We previously reported that an ecto-enzyme activity of NAD' glycohydrolase induced by retinoic acid in HL-60 cells is attributed to the molecule of CD38 [6] and that it has an ability to bind to hyaluronate [7]. Interestingly, CD38 has an amino acid sequence similar to Aplysia ADP-ribosyl cyclase which catalyzes the formation of 1 -(S-phospho-P-D-ribosyl)adenosine 5'-phosphate cyclic anhydride (cyclic ADP-ribose) from NAD' [8 -101 ; there are especially 10 cysteine residues conserved between CD38 and Aplysia ADP-ribosyl cyclase [ll]. Cyclic ADP-ribose has been considered as a new mediator of Ca'+ release from intracellular stores [12, 131.It has been reported that CD38 catalyzes not only the hydrolysis of NAD' but also the formation and hydrolysis of cyclic ADP-ribose [14-161. However, the ADP-ribosyl cyclase activity of CD38 is quite low compared to its NAD' glycohydrolase activity ; the ratio of the two specific activities is approximately 1 : 100 [17]. In this respect, the ADP-ribosyl cyclase acti...