Extracellular synthesis of cADP-ribose from nicotinamide-adenine dinucleotide by rat cortical astrocytes in culture

Abstract

cADPR is an endogenous calcium-mobilizing agent that in vertebrates is synthesized from nicotinamide-adenine dinucleotide (NAD) by bifunctional enzymes with ADP-ribosyl cyclase and cADPR hydrolase activity. ADP-ribosyl cyclase and cADPR hydrolase activity have been reported in the brain, but the cellular localization of these activities has not been determined previously. In the present study, selective culturing techniques were employed to localize ADP-ribosyl cyclase activity and cADPR hydrolase activity to astrocytes or neurons in cultures derived from rat embryonic cerebral cortex. ADP-ribosyl cyclase activity was determined by incubating cultures with 1 mM NAD in the extracellular medium for 60 min at 37 degrees C and measuring formation of cADPR by bioassay and by HPLC. Astrocyte cultures and mixed cultures of astrocytes and neurons had mean specific activities of 0.84 +/- 0.06 and 0.9 +/- 0.18 nmol cADPR produced/mg protein/hr, respectively. No detectable ADP-ribosyl cyclase activity was found in neuron-enriched/ astrocyte-poor cultures. cADPR hydrolase activity was detectable by incubating cultures with 300 microM cADPR for 60 min at 37 degrees C and assaying loss of cADPR or accumulation of ADPR. The demonstration of extracellular ADP-ribosyl cyclase and cADPR hydrolase activities associated with astrocytes may have important implications for the role of extracellular cADPR in signal transduction and in intercellular communication in the nervous system.

Fig. 1.

Calcium release by cADPR and IP₃ in sea urchin egg homogenates. A, Representative traces of Fluo 3 fluorescence increase attributable to calcium release in egg homogenate injected with authentic cADPR standards (0–100 nm final concentrations). Injections were performed at 50 sec. The horizontal axes show time,tick = 500 sec, except for the far left trace where tick = 200 sec. Vertical axis = 600 fluorescence units. B, Standard curve of the initial rate of fluorescence increase (measured at 20 sec) in egg homogenate injected with authentic cADPR standards.C, Representative trace of Fluo 3 fluorescence increase attributable to calcium release in egg homogenate injected with IP₃ (final concentration 1 μm). Time axistick = 500 sec; vertical axistick = 600 fluorescence units. D, Representative trace of Fluo 3 fluorescence after injection of NAD (10 μm final concentration).

Fig. 2.

Production of calcium-releasing activity in media of AR cultures incubated with NAD. AR well cultures were incubated at room temperature with calcium-free HBSS buffer containing NAD (100 μm). Samples were assayed for calcium-releasing activity in sea urchin egg homogenates with Fluo 3 fluorescence detection. Horizontal axis: tick = 500 sec. Vertical axistick = 400 fluorescence units. Trace 1, HBSS + NAD control (no exposure to culture); trace 2, 15 min incubation of culture with HBSS + NAD; trace 3, 30 min incubation of culture with HBSS + NAD.

Fig. 3.

Positive identification of cADPR in media of astrocyte cultures incubated with 1 mm NAD. Astrocyte (A) dish cultures were incubated 60 min at 37°C with calcium-free HBSS containing NAD (1 mm). Samples were assayed for calcium-releasing activity in egg homogenates desensitized to nicotinamide and to IP₃ or cADPR. Time axistick = 500 sec. A, Heparin (250 μg/ml), IP₃ (1 μm), and nicotinamide (2 × 100 μm) did not desensitize egg homogenate to calcium-releasing activity in the astrocyte incubation medium (A under arrow). B, Nicotinamide (2 × 100 μm) and cADPR (2 × 100 nm) almost completely desensitized egg homogenate to calcium-releasing activity in the astrocyte incubation medium. C, The astrocyte incubation medium desensitized egg homogenate to repeated injections of media and to cADPR (100 nm) but not to IP₃ (1 μm).

Fig. 4.

Positive identification of cADPR in media of AR cultures incubated with 1 mm NAD. Experimental protocol as in Figure 3. Time axis tick = 500 sec.A, Heparin (250 μg/ml), IP₃ (1 μm), and nicotinamide (2 × 100 μm) did not desensitize egg homogenate to calcium-releasing activity in the AR incubation medium. B, Nicotinamide (2 × 100 μm) and cADPR (2 × 100 nm) almost completely desensitized egg homogenate to calcium-releasing activity in the AR incubation medium. C, The calcium-releasing activity in the AR incubation medium desensitized egg homogenate to itself and to cADPR (100 nm) but not to IP₃.

Fig. 5.

Representative time course of cADPR production in astrocyte cultures incubated with or without calcium. Astrocyte dish cultures were incubated at 37°C with complete (filled circles) or calcium-free (open circles) HBSS buffer containing NAD (1 mm). cADPR production was quantified by measurement of the initial rate of Fluo 3 fluorescence increase attributable to calcium mobilization in egg homogenate previously desensitized to nicotinamide. Calcium in HBSS was removed by treatment with Chelex before assay.

Fig. 6.

Comparison of ADP-ribosyl cyclase activity in astrocyte, AR cultures, and AP cultures by assaying conversion of NGD to the fluorescent compound cGDP-ribose. Cultures grown in 24-well plates were incubated with NGD (100 μm) in EBSS at 37°C for selected intervals of time, after which medium was removed and assayed for the appearance of fluorescence emission at 410 nm with excitation at 300 nm. Both astrocyte and AR cultures caused a time-dependent increase in fluorescence intensity. AP cultures caused an increase in fluorescence that was significantly greater than control, but at 60 min was only ∼5% of the intensity seen with astrocyte and AR cultures.

Fig. 7.

Confirmation by HPLC of cADPR production by astrocyte cultures. Astrocyte dish cultures were incubated with complete HBSS with NAD (1 mm) at 37°C for 120 min. Media samples (50 μl) were run on a two-column HPLC system (PL-SAX column followed by a C₁₈ reverse-phase column) (see Materials and Methods) and assayed by absorbance detection. The selective degradation of cADPR by boiling for 30 min was used as an additional test for cADPR. The solid upward arrows indicate the time of injection of sample. A, Degradation by boiling of 6.2 min peak in astrocyte incubation medium sample by boiling. Upper trace, Astrocyte medium sample (120 min incubation) before boiling. A peak is present at 6.2 min, indicated by downward open arrow, which is the retention time of cADPR. Lower trace, Astrocyte medium sample (120 min incubation) after boiling for 30 min. Boiling has removed the peak at 6.2 min, indicated by downward open arrow. B, Boiling for 30 min degrades authentic cADPR to ADPR but does not affect ADPR.Upper trace, ADPR (5 μm) standard after 30 min boiling. The retention time is 10.8 min, the value previously determined for unboiled ADPR. Middle trace, cADPR (2 μm) standard after boiling for 30 min. The cADPR peak at 6.2 min is no longer visible; instead, a peak has appeared at 10.8 min, which is the ADPR retention time. Bottom trace, cADPR (2 μm) standard before boiling for 30 min. cADPR elutes as a sharp peak at 6.2 min.