Advancing age alters the contribution of calcium release from smooth endoplasmic reticulum stores in superior cervical ganglion cells

Abstract

In superior cervical ganglion (SCG) neurons calcium-induced calcium release (CICR), mediated by ryanodine receptors (RyRs), contributes to stimulation-evoked intracellular calcium ([Ca(2+)](i)) transients.

Hypothesis: The contribution of CICR to electrical field stimulation (EFS)-evoked [Ca(2+)](i) transients in SCG cells declines with senescence and may be partially recovered in the presence of caffeine. We measured EFS-evoked [Ca(2+)](i) transients in isolated fura-2-loaded SCG cells from Fischer-344 rats aged 6, 12, and 24 months with either the RyR antagonist ryanodine to block the contribution of CICR to [Ca(2+)](i) transients or caffeine to sensitize CICR to EFS. EFS-evoked [Ca(2+)](i) transients increased from 6 to 12 months and declined at 24 months and ryanodine decreased [Ca(2+)](i) transients in SCG cells from 6- and 12-month-old animals only. Caffeine significantly increased EFS-evoked [Ca(2+)](i) transients in all age groups. These data suggest that CICR declines with senescence and residual CICR function may be reclaimed in senescent cells with caffeine.

Figure 1.

Model of the superior cervical ganglion (SCG) cell and illustrated protocol used in this study. In SCG cells, calcium transients are modulated by numerous mechanisms including influx via voltage-gated calcium channels, calcium-induced calcium release (CICR), and transport of calcium by smooth endoplasmic reticulum Ca²⁺-ATPases, which both buffers [Ca²⁺]_i transients and refills smooth endoplasmic reticulum (SER) calcium stores to maintain regenerative CICR. CICR is mediated via ryanodine receptors, which can be inhibited or activated using ryanodine or caffeine, respectively (see Methods). In this study, electrical field stimulation was used to evoke calcium influx, which activates CICR from SER calcium stores. Influx and CICR contribute to the magnitude of the measured variable, [Ca²⁺]_i as detected by fura-2 fluorescence.

Figure 2.

Electrical field stimulation (EFS) evokes ryanodine-sensitive calcium responses in rat superior cervical ganglion (SCG) cells. Representative data in a single SCG cell from a (A) 6-month-old and (B) 24-month-old animal. Fifty pulses of EFS (5 Hz) were delivered at varied currents with a 2-minute equilibration separating each stimulation. Following the last stimulation, cells were exposed to Tyrode's buffer containing 100 μM of the ryanodine receptor antagonist ryanodine for 30 minutes, and the stimulation protocol was repeated (see Methods).

Figure 3.

Dependence of electrical field stimulation (EFS)–induced calcium responses on extracellular calcium influx. These data serve as a control to demonstrate that in superior cervical ganglion (SCG) cells, all responses to EFS are lost when calcium influx is blocked with lanthanum (La³⁺). SCG cells were activated with EFS (50 pulses, 5 Hz, 300 mA) in the absence and presence of 100 μM La³⁺. Following two consecutive EFS-evoked [Ca²⁺]_i transients, the cells were incubated with 100 μM La³⁺ for 10 minutes to block all calcium influx. When EFS was reapplied, simulation-evoked [Ca²⁺]_i transients were not observed. Representative data from a group of seven cells from a 6-month-old animal.

Figure 4.

Impact of advancing age on electrical field stimulation–evoked [Ca²⁺]_i transients in the absence and presence of ryanodine in superior cervical ganglion cells from 6- (A), 12- (B), and 24- (C) month-old animals. Cells were activated at various applied currents as per Protocol 1 in the absence and presence of 100 μM ryanodine (see Methods). N = 19–31 cells from eight animals in each age group. *Significantly different from ryanodine treatment, p < .01 by paired t test.

Figure 5.

Impact of advancing age on rate of rise (d[Ca²⁺]_i/dt) of electrical field stimulation–evoked [Ca²⁺]_i transients in the absence and presence of ryanodine in superior cervical ganglion cells from 6- (A), 12- (B), and 24- (C) month-old animals. Cells were activated at various applied currents as per Protocol 1 in the absence (black) and presence (gray) of 100 μM ryanodine (see Methods). N = 19–31 cells from eight animals in each age group. *Significantly different from ryanodine treatment, p < .01 by paired t test.

Figure 6.

Summarized responses of the impact of late maturation and senescence on the magnitude (A) and rate of rise (B) of maximal electrical field stimulation–evoked [Ca²⁺]_i transients. Bars represent the peak values derived from curves in Figures 4 and 5 for control (black) and in the presence of ryanodine (gray). N = 19–31 cells from eight animals in each age group. *Significantly different from ryanodine treatment, p < .01, by paired t test. +Significantly different from 24 months, p < .02, by analysis of variance and Fisher-protected least significant differences test.

Figure 7.

Aging alters electrical field stimulation current (I₅₀) necessary to evoke ½ maximal d[Ca²⁺]_i (A) and rate of rise of [Ca²⁺]_i (B). N = 19–31 cells from eight animals in each age group. *Significantly different from control, p < .05, by paired t test. ++Significantly different from 6 and 12 months, p < .02, by analysis of variance and Fisher-PLSD test.

Figure 8.

Impact of late maturation and senescence, on the magnitude (d[Ca²⁺]_i), of electrical field stimulation–evoked [Ca²⁺]_i transients. Comparisons were made between supermaximal stimulation (5 Hz, 50 pulses) to the stimulation at a lower frequency (3 Hz, 24 pulses) in the absence and presence of caffeine. N = 16–22 cells from five to six animals from each age group. *Significantly different from control, p < .03, by paired t test. **Significantly different from control and supermax, p < .01, by paired t test. +Significantly different from 24 months, p < .05, by analysis of variance (ANOVA) and Fisher-PLSD test. ++Significantly different from 6 and 24 months, p < .03, by ANOVA and Fisher-PLSD test.