Advancing age alters rapid and spontaneous refilling of caffeine-sensitive calcium stores in sympathetic superior cervical ganglion cells

Abstract

Intracellular calcium concentration ([Ca2+]i) release from smooth endoplasmic reticulum (SER) stores plays an important role in cell signaling. These stores are rapidly refilled via influx through voltage-gated calcium channels or spontaneously via store-operated calcium channels and subsequent pumping by SER Ca2+-ATPases. We measured [Ca2+]i transients in isolated fura 2-loaded superior cervical ganglion cells from 6-, 12-, 20-, and 24-mo-old Fischer 344 rats. For rapid refilling, [Ca2+]i transients were elicited by a 1) 5-s exposure to K+, 2) caffeine to release Ca2+ from SER stores, 3) K+ to refill SER Ca2+ stores, and 4) caffeine. The percent difference between the peak and rate of rise of the first and second caffeine-evoked [Ca2+]i transient significantly declined over the age range of 12-24 mo. To estimate spontaneous refilling, cells were depolarized for 5 s with 68 mM K+ (control), followed by a 10-s exposure to 10 mM caffeine "conditioning stimulus" to deplete [Ca2+]i stores. Caffeine was then rapidly applied for 5 s at defined intervals from 60 to 300 s. Integrated caffeine-evoked [Ca2+]i transients were measured and plotted as a percentage of the K+ response vs. time. The derivative of the refilling time curves significantly declined over the age range from 12-24 mo. Overall, these data suggest that the ability of superior cervical ganglion cells to sustain release of [Ca2+]i following rapid or spontaneous refilling declines with advancing age. Compromised ability to sustain calcium signaling may possibly alter the overall function of adrenergic neurons innervating the cerebrovasculature.

Fig. 1

Model illustrating overall experimental design of this study. The measured variable is relative cytosolic calcium concentration in response to various protocols used within the study. The overall governing hypothesis is that an age-related decline in SERCA function alters the SER calcium levels and their refilling following depletion. Abbreviations: Smooth endoplasmic reticulum (SER). Smooth endoplasmic reticulum calcium ATP-ase (SERCA). Voltage gated calcium channels (VOCC). Store operated calcium channels (SOCC).

Fig. 2. Protocol 1

(A) Representative data of protocol 1 demonstrating caffeine-evoked release of [Ca²⁺]i from SER stores and fast K⁺-evoked refilling of [Ca²⁺]i stores in a single Fura-2 loaded SCG cell from a 6-month animal. An [Ca²⁺]i transient (S₁) was evoked by 5 sec exposure to 68 mM K⁺ followed by 2 min equilibration. A second [Ca²⁺]i transient (S₂) was evoked by 5 sec exposure to 10 mM caffeine to release calcium from [Ca²⁺]i stores followed by 2 min equilibration. The third [Ca²⁺]i transient (S₃) was evoked by 5 sec exposure to 68 mM K⁺ to refill [Ca²⁺]i stores followed by two min equilibration. A final [Ca²⁺]i transient (S₄) was evoked by 5 sec exposure to 10 mM caffeine followed by 2 min equilibration. (B) Data derived from a single Fura-2 loaded SCG cell from a 6 month animal demonstrating selective caffeine evoked Ca²⁺ release from [Ca²⁺]i stores. An [Ca²⁺]i transient was evoked by 5 sec exposure to buffer containing 68 mM K⁺ and 2 mM extracellular Ca²⁺ followed by 2 min equilibration. A second [Ca²⁺]i transient was evoked by 5 sec exposure to buffer containing 10 mM caffeine and zero extracellular Ca²⁺ (3 mM EGTA) followed by 2 min equilibration. Next the cell was exposed for 5 sec to buffer containing 68 mM K⁺ and zero extracellular Ca²⁺ (3 mM EGTA) followed by two min equilibration. A final [Ca²⁺]i transient was evoked by 5 sec exposure to buffer containing 68 mM K⁺ and 2 mM extracellular Ca²⁺.

Fig. 3. Protocol 2

Example of protocol 2 demonstrating the spontaneous refilling of SER calcium stores following caffeine-evoked emptying in a single SCG cell. Cells were exposed for 5 sec to 68 mM K⁺ and then for 10 sec to 10 mM caffeine representing the control and conditioning response respectively (data not shown). At the time points indicated following the conditioning response, 10 mM caffeine was applied for 5 sec until the maximum response to caffeine was achieved.

Fig. 4

(A, B) Peak [Ca²⁺]i evoked by the first and second exposure to high K⁺ in isolated SCG cells from animals aged 6–24 months. (C, D) Rate of rise of [Ca²⁺]i evoked by the first and second exposure to high K⁺ in isolated SCG cells from animals aged 6–24 months. These data were derived from the protocol shown in figure 2A. Peak [Ca²⁺]i was measured by subtracting basal [Ca²⁺]i from the maximum K+-evoked [Ca²⁺]i transient. Rate of rise of [Ca²⁺]i from baseline to maximum was determined by linear fit using Origin 6.1. Data represent the mean ± S.E. n = 21–42 cells from each age group. ** = significantly different from two other age groups, P<0.05. *** = significantly different from three other age groups, P<0.05.

Fig. 5

(A, B) Peak [Ca²⁺]i evoked by the first and second exposure to 10 mM caffeine in isolated SCG cells from animals aged 6–24 months. (C, D) Rate of rise of [Ca²⁺]i evoked by the first and second exposure to 10 mM caffeine in isolated SCG cells from animals aged 6–24 months. These data were derived from the protocol shown in figure 2A. Peak [Ca²⁺]i was measured by subtracting basal [Ca²⁺]i from the maximum caffeine-evoked [Ca²⁺]i transient. Rate of rise of [Ca²⁺]i from baseline to maximum was determined by linear fit using Origin 6.1. Data represent the mean ± S.E. n = 21–42 cells from each age group. ** = significantly different from two other age groups, P<0.05. *** = significantly different from three other age groups, P<0.05.

Fig. 6

(A, B) Impact of age on the percentage difference between the first and second K⁺-evoked peak and rate of rise of [Ca²⁺]i transients. (C, D) Impact of age on the percentage difference between the first and second caffeine-evoked [Ca²⁺]i transients. The percentage difference was calculated as the difference between the first and second exposure to K⁺ or caffeine divided by the first exposure to K⁺ or caffeine times 100. Data represent the mean ± S.E. n = 21–42 cells from each age group. * = significantly different from one other age group, P<0.05. ** = significantly different from two other age groups, P<0.05. *** = significantly different from three other age groups, P<0.05.

Fig. 7

Validation controls to determine that SOCC channels and SERCA mediate spontaneous refilling of SER calcium stores following caffeine-evoked release. (A) SER calcium stores refill in the presence of calcium channel blockers, nifedipine and ω-contoxin. Cells were exposed for 5 sec to high K⁺ buffer (S₁) followed by a 10 sec exposure to a buffer containing 10 mM caffeine to release [Ca²⁺]i stores (S₂). Next the cells were continually exposed to a buffer containing 10 μM nifedipine and 1 μM ω-conotoxin to block L and N-type calcium channels respectively. As indicated the cells were exposed to again exposed for 5 sec to a buffer containing 10 mM caffeine. Data represent the average for 5 cells from a 6 month-old animal. (B) L and N-type channel antagonists, nifedipine and ω-conotoxin block K⁺-evoked [Ca²⁺]i transients in SCG cells. Cells were exposed for 5 sec to high K⁺ buffer (control). Next cells were exposed for 5 sec to high K⁺ buffer containing 10 μM nifedipine and then for 5 sec to high K⁺ buffer containing 10 μM nifedipine and 1 μM ω-conotoxin. Data represent the mean ± S.E for 6 cells from a 6 month-old animal. (C) Activation of SOCC channels occur following the caffeine-evoked depletion of SER calcium stores and blockade of SERCA with thapsigargin (THAPS). Cells were exposed for 5 sec to high K⁺ (S₁) and then to 10 mM caffeine for 10 sec (S₂). Next cells were continually exposed to a buffer containing the SERCA antagonist THAPS, 1 μM. At the times indicated the cells were exposed for 5 sec to a buffer containing 10 mM caffeine. Data represent the average of 4 cells from a 6 month-old animal. (D) Activation of SOCC channels is blocked with La³⁺ following caffeine-evoked depletion of SER. Cells were exposed for 5 sec to a buffer containing high K⁺ (S₁). Next cells were exposed for 10 sec to a buffer containing 10 mM caffeine (S₂). Following S₂ cells were continually exposed to a buffer containing THAPS (1 μM) and La³⁺ (100 μM). At the times indicated cells were exposed for 5 sec to a buffer containing 10 mM caffeine. Data represent the average for 5 cells from a 6 month-old animal.

Fig. 8

(A) Aging alters the spontaneous refilling of SER calcium stores following caffeine-evoked depletion. Using protocol 2 (Figure 3), data were plotted as integrated caffeine-evoked release of [Ca²⁺]i as a percentage of the K⁺ control vs time after conditioning response. Data represent the mean ± S.E. n = 13–29 cells from 6–24 month-old animals. ** = significantly different from two other age groups, P<0.01. *** significantly different from three other age groups, P<0.01. (B) Aging alters the derivative of caffeine-evoked [Ca²⁺]i transients as a percentage of K⁺ control. Data derived for each individual cell in (A) were plotted and fitted by a Boltzman fit and the derivatives calculated using Origin 6.1. Data represent the mean ± S.E. n = 13–29 cells from 6–24 month-old animals. *** = significantly different from three other age groups, P<0.001. ** = significantly different from two other age groups, P<0.01.