Maturation and long-term hypoxia alters Ca2+-induced Ca2+ release in sheep cerebrovascular sympathetic neurons.

The contribution of sympathetic nerves arising from the superior cervical ganglia (SCG) toward the growth and function of cerebral blood vessels is pertinent throughout maturation as well as in response to cardiovascular stress imposed by high-altitude long-term hypoxia (LTH). The function of SCG sympathetic neurons is dependent on intracellular Ca2+ concentration ([Ca2+]i) signaling, which is strongly influenced by a process known as Ca(2+)-induced Ca2+ release (CICR) from the smooth endoplasmic reticulum (SER). In this study, we used the sheep SCG neuronal model to test the hypotheses that maturation decreases CICR and high-altitude LTH depresses CICR in fetal SCG neurons but not in those of the adult. We found that the contribution of CICR to electric field stimulation (EFS)-evoked [Ca2+]i transients was greatest in SCG cells from normoxic fetuses and was abolished by LTH. The decline in CICR was associated with a reduction in sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) function in fetal SCG cells during LTH, reducing SER Ca2+ levels below the threshold needed for the coupling of Ca2+ influx and CICR. With respect to the maturation from the fetus to adult, the decrease in CICR may reflect both a reduction in the levels of ryanodine receptor isoforms 2 and 3 and SERCA function. In response to LTH and in contrast to the fetus, CICR function in adult SCG cells is maintained and may reflect alterations in other mechanisms that modulate the CICR process. As CICR is instrumental in the function of sympathetic neurons within the cerebrovasculature, the loss of this signaling mechanism in the fetus may have consequences for the adaptation to LTH in terms of fetal susceptibility to vascular insults.

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

UNLABELLED: 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.

Age-dependent changes in Ca2+ homeostasis in peripheral neurones: implications for changes in function.

Calcium ions represent universal second messengers within neuronal cells integrating multiple cellular functions, such as release of neurotransmitters, gene expression, proliferation, excitability, and regulation of cell death or apoptotic pathways. The magnitude, duration and shape of stimulation-evoked intracellular calcium ([Ca2+]i) transients are determined by a complex interplay of mechanisms that modulate stimulation-evoked rises in [Ca2+]i that occur with normal neuronal function. Disruption of any of these mechanisms may have implications for the function and health of peripheral neurones during the aging process. This review focuses on the impact of advancing age on the overall function of peripheral adrenergic neurones and how these changes in function may be linked to age-related changes in modulation of [Ca2+]i regulation. The data in this review suggest that normal aging in peripheral autonomic neurones is a subtle process and does not always result in dramatic deterioration in their function. We present studies that support the idea that in order to maintain cell viability peripheral neurones are able to compensate for an age-related decline in the function of at least one of the neuronal calcium-buffering systems, smooth endoplasmic reticulum calcium ATPases, by increased function of other calcium-buffering systems, namely, the mitochondria and plasmalemma calcium extrusion. Increased mitochondrial calcium uptake may represent a 'weak point' in cellular compensation as this over time may contribute to cell death. In addition, we present more recent studies on [Ca2+]i regulation in the form of the modulation of release of calcium from smooth endoplasmic reticulum calcium stores. These studies suggest that the contribution of the release of calcium from smooth endoplasmic reticulum calcium stores is altered with age through a combination of altered ryanodine receptor levels and modulation of these receptors by neuronal nitric oxide containing neurones.

Advancing age alters the expression of the ryanodine receptor 3 isoform in adult rat superior cervical ganglia.

Sympathetic nerves arising from the superior cervical ganglion (SCG) protect the cerebrovasculature during periods of acute hypertension and may play a role in homeostasis of target organs. The functions of these nerves depend on calcium release triggered by activation of ryanodine receptor (RyR) channels. The function of RyR channels is in part dependent on genetic expression and regulation by numerous protein modulators such as neuronal nitric oxide synthase (nNOS) neurons also found in the SCG. We have shown that release of calcium in SCG cells is altered during late maturation and advancing age. However, the underlying molecular mechanisms that may in part account for these data are elusive. Therefore we used molecular techniques to test the hypothesis that advancing age alters the pattern of genetic expression and/or protein levels of RyRs and their modulation by nNOS in the SCG in F344 rats aged 6, 12, and 24 mo. Surprisingly, ryr1 expression was undetectable in all age groups and ryr2 and ryr3 are the predominantly transcribed isoforms in the adult rat SCG. mRNA and protein levels for RyR2 isoform did not change with advancing age. However, ryr3 mRNA levels increased from 6 to 12 mo and declined from 12 to 24 mo. Similarly, RyR3 receptor protein levels also increased from 6 to 12 mo and declined from 12 to 24 mo. Because nNOS and the phosphorylation of the RyRs have been shown to modulate the function of RyRs, total phosphorylation and nNOS protein levels were analyzed in all age groups. Phosphorylation levels of the RyRs were similar in all age groups. However, nNOS protein levels increased from 6 to 12 mo followed by decline from 12 to 24 mo. These data suggest that advancing age selectively impacts the genetic expression and protein levels of RyR3 as well as modulatory nNOS protein levels. In addition, these data may part provide some insight into the possible changes in the function of RyRs that may occur with the normal aging process.

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

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.