Elevation of cytosolic [Ca²⁺] due to intracellular Ca²⁺ release retards carbachol stimulation of divalent cation entry in rat parotid gland acinar cells

Summary

This study examines the activation of divalent cation entry into rat parotid gland acinar cells by using Mn²⁺ as a Ca²⁺ surrogate cation. Following muscarinic-cholinergic stimulation of dispersed parotid acini with carbachol (10 μ m), the onset of internal Ca²⁺ release (cytosolic [Ca²⁺], [Ca²⁺] _i , increase) and the stimulation of Mn²⁺ entry (increase in fura2 quenching) are not simultaneously detected. [Ca²⁺] _i elevation, due to intracellular release, is detected almost immediately following carbachol addition and peak [Ca²⁺] _i increase occurs at 6.0 ± 0.8 sec. However, there is an interval (apparent lag) between carbachol addition and the detection of stimulated Mn²⁺ entry. This apparent lag is decreased from 26 ± 3.1 sec to 9.2 ± 1.5 sec when external Mn²⁺ ([Mn²⁺] _o ) is increased from 12.5 to 500 μm. It is not decreased further with increase in [Mn²⁺] _o from 500 μ m to 1 mm(9.8 ± 2.1 sec), although both intracellular free Mn²⁺ and [Mn²⁺-fura2]/[fura2] increase. Thus, at [Mn²⁺] _o <500 μ m, the observed lag time is partially due to a limitation in the magnitude of Mn²⁺ entry. Furthermore, neither peak [Ca²⁺] _i nor the time required to reach peak [Ca²⁺] _i is significantly altered by [Mn²⁺] _o (12.5μ m to 1 mm). At every [Mn²⁺] _o tested (i.e., 12.5 μ m−1 mm), the apparent lag is significantly greater than the time required to reach peak [Ca²⁺] _i . However, when carbachol stimulation of the [Ca²⁺] _i increase is attenuated by loading the acini with the Ca²⁺ chelator, 2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA), there is no detectable lag in carbachol stimulation of Mn²⁺ entry (with 1 mm [Mn²⁺] _o ). Importantly, in BAPTA-loaded acini, carbachol stimulates Mn²⁺ entry via depletion of the internal Ca²⁺ pool and not via direct activation of other divalent cation entry mechanisms. Based on these results, we suggest that the apparent lag in the detection of carbachol stimulation of Mn²⁺ entry into parotid acinar cells is due to a retardation of Mn²⁺ entry by the initial increase in [Ca²⁺] _i , due to internal release, which most likely occurs proximate to the site of divalent cation entry.