difference in the maximal response to this drug (Buchholz et al., 1992). Given that fractional stimulation-evoked release increases with age, there would be more to interact with the α2-adrenoceptor and increased competition between higher levels and an antagonist. This chemical competition may possibly reduce the apparent sensitivity of the applied antagonist and account for the decrease in the potency of α2-adrenoceptor antagonists with age (Pottorf et al., 2000b). Overall, in light of others and our studies cited above, it is difficult to explain age-related alterations in release in terms of age-related alterations in the function of uptake mechanisms or α2-adrenoceptor function. Therefore, we looked at other mechanisms that may account for age-related changes in adrenergic nerve activity. These included further studies on stimulation-evoked release with altered extracellular calcium, calcium influx and altered calcium buffering capacity. Effects of altering extracellular calcium, role of calcium influx and altering buffering capacity on function of peripheral adrenergic nerves In light of the studies on the tail artery model cited above, our attention was directed toward the possibility that age-related changes in stimulation-evoked release is possibly due to changes in calcium regulation in the nerve endings. We carried out experiments to examine the effects of increased or lowered extracellular calcium on stimulation-evoked release in tail arteries of young and old rats (Buchholz et al., 1994). Stimulation- evoked fractional release in tail arteries from old animals was found to be more sensitive to an increase and less sensitive to a decrease in extracellular calcium as compared to young animals. The explanation for these results is complex and may be attributable to altered calcium influx, buffering capacity, or sensitivity of the release mechanisms to stimulation-evoked increases in [Ca2+]i. Thus, in another study, we measured release from rat tail arteries via transmural nerve stimulation or KCl. Under both conditions, release was greater in tail arteries from old as compared to young animals (Tsai et al., 1997). These data could be explained in part by an age-related increase in calcium influx with age. However, other reports of the impact of age on calcium influx are mixed. Calcium influx has been reported to increase with age in central neurones (Landfield & Pitler, 1984; Pitler & Landfield, 1990), to decrease in peripheral neurones (Kostyuk et al., 1993), or to remain unchanged in central neurones (Murchinson & Griffith, 1996). Therefore, in another experiment, we bypassed voltage-gated calcium chan- nels using the calcium ionophore, ionomycin, and found that the age-related increase in release persisted (Tsai et al., 1997). While stimulation-evoked calcium influx may possibly increase with age in the tail artery model, from the latter experi- ments we would also argue that other mechanisms such as [Ca2+]i buffering and/or alterations in the sensitivity of the release mechanisms may also change with age. We tested the hypothesis that an age-related alteration in [Ca2+]i buffering may play a partial role in the increase in release Age and Ca2+ homeostasis in peripheral neurones, J. N. Buchholz et al. 289 from tail arteries (Tsai et al., 1997). The addition of the [Ca2+]i chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) decreased stimulation-evoked release to a greater extent in old arteries as compared to young, suggesting that age-related changes in release is in part due to altered calcium-buffering capacity. Overall, the age-related changes in the function of peripheral adrenergic neurones in terms of release appears to be at least due in part to altered [Ca2+]i handling mechanisms. However, altered sensitivity of the release mechanisms has not been ruled out. Alterations in neuronal calcium buffering and extrusion during aging in peripheral neurones The introduction of cell permeant fluorometric calcium indicator dyes such as Fura-2, allowed for the measurement of real-time changes in [Ca2+]i in living cells (Tsien et al., 1985; Roe et al., 1990; Thayer & Miller, 1990; Neher, 1995). Microfluorometry coupled with calcium sensitive dyes allows investigators to measure the impact of advancing age on the mechanisms that modulate stimulation-evoked [Ca2+]i transients and overall calcium homeostasis (Kirischuk et al., 1992; Kirischuk & Verkhratsky, 1996; Neher, 1998; Baylor & Hollingworth, 2000; Pottorf et al., 2002). Each component of the [Ca2+]i buffering and extrusion system participates in controlling the shape and duration of stimulation- evoked [Ca2+]i transients (Fig. 1A). An age-related alteration in any one or a combination of the components of this [Ca2+]i control system may alter the function of neurones and or contribute to neuronal degeneration (Meldrum & Garthwait, 1990; Peterson, 1992; Verkhratsky et al., 1994; Pottorf et al., 2000b, 2002). Given the complex interplay between numerous buffering systems to control [Ca2+]i transients, declining func- tion of one or more systems may be compensated for by increased function of other mechanisms (Pottorf et al., 2000b). Age-related decline in SERCA function in peripheral neurones Active transport of calcium into the SER is mediated by SERCA and an age-related decline in their function has been suggested to contribute to calcium dysregulation in peripheral sympathetic and sensory neurones (Kirischuk et al., 1992; Kirischuk & Verkhratsky, 1996; Pottorf et al., 2002). In addition, a decline in SERCA function with age may have greater consequences. For example, SERCA buffer [Ca2+]i transients and load the SER calcium stores, thus, an age-related decline in their function may possibly cause alterations in CICR from the SER (Murchinson & Griffith, 1999; Usachev & Thayer, 1999a). In our studies in adrenergic nerve endings in tail arteries, the SERCA blocker, thapsigargin, increased stimulation-evoked release in young arteries with no change in arteries from old animals (Tsai et al., 1998). In isolated SCG cells the SERCA blocker cyclopiazonic acid or thapsigargin caused a decline in the rate of recovery of high K+-evoked [Ca2+]i transients only in © 2007 Loma Linda University Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007