Extracellular ATP is now recognized as a neurotransmitter and neuromodulator (Bean, 1992) whose effects on bovine adrenal chromaffin cells include both the release of Ca$\sp{2+}$ from intracellular stores and secretion of catecholamines (Kim, 1989). Since chromaffin cells co-secrete 150 mM ATP along with catecholamines and neuropeptides, ATP has considerable opportunity to act as a modulator of secretion via its effects on cytosolic Ca$\sp{2+}$. Nucleotides and their analogues were used to characterize ATP effects, yielding an agonist efficacy order for the release of Ca$\sp{2+}$ from internal stores of ATP = UTP $>$ ADP $>$ 2-MeSATP, $\alpha,\beta$-MeATP, identifying the receptor as a P$\sb{\rm 2U}$ subtype of purinoceptor (O'Connor et al., 1991). In contrast, the efficacy order for the receptor mediating secretion, 2-MeSATP $>$ ATP $> \alpha,\beta$-MeATP, ADP, UTP places it in the P$\sb{\rm 2Y}$ receptor subtype (Gordon, 1986). Thus, two separate receptors are responsible for Ca$\sp{2+}$ release and secretion. Additionally, agonists were found to be more effective at both receptors in the absence of extracellular Mg$\sp{2+}$, suggesting that ATP uncomplexed with divalent cations may bind preferentially to the receptors. The release of Ca$\sp{2+}$ from internal stores was further investigated using ATP and bradykinin (BK), both of which release Ca$\sp{2+}\ via$ inositol (1,4,5)-trisphosphate (Ins(1,4,5)P$\sb3$). In the absence of extracellular Ca$\sp{2+}$, both ATP and BK increased cellular levels of Ins(1,4,5)P$\sb3 \sim 2$-fold within 30 s. Studies of single cells revealed that 93% released Ca$\sp{2+}$ in response to BK whereas ATP triggered release from 40% of the cells. The ATP-responsive cells were a subset of the BK-responsive cells, indicating that spatial separation between ATP and BK-sensitive Ca$\sp{2+}$ stores is intracellular. Furthermore, saturating concentrations of BK and ATP yielded an additive release of Ca$\sp{2+}$. Taken together with previous data from our lab (Kim, 1989), these results indicate that it may be possible for two agonists to activate the release of Ca$\sp{2+}$ from two spatially distinct Ca$\sp{2+}$ stores through a single second messenger, Ins(1,4,5)P$\sb3$. In addition, several parameters of chromaffin cells loaded with fura-2 were examined. The autofluorescence of chromaffin cells illuminated with light from 300-400 nm was found to alter the spectrum of fura-2-loaded cells. Also, fura-2 leakage from the cells over a period of $\sim$1 h was found to reach levels of 40-60% of the initial signal; for comparison of Ca$\sp{2+}$ signals during this time period, a method of normalization of the signal was used. Moreover, in the absence of Mg$\sp{2+}$, the removal of extracellular Ca$\sp{2+}$ from the cells occasioned a large intracellular Ca$\sp{2+}$ transient.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-8903 |
| Date | 01 January 1994 |
| Creators | Reichsman, Frieda |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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