Function of SERCA mediated calcium uptake and expression of SERCA3 in cerebral cortex from young and old rats.

Previous work on peripheral sympathetic neurons indicated that a decline in sarco/endoplasmic reticulum calcium ATPase (SERCA) function occurs with advancing age. Therefore, an age-related decline in mechanisms controlling intracellular calcium homeostasis could contribute to altered neuronal function and/or degeneration. In this study we sought to extend the findings on peripheral neurons and to detect possible age-related declines in SERCA function and expression of SERCA3 in central neurons from cerebral cortex from young (6-month) and old (20-month) rats. Functional studies compared ATP-dependent 45Ca(2+)-uptake into microsomes and plasma membrane vesicles (PMVs). We and found no significant difference in 45Ca(2+)-uptake between microsomes or PMVs between young and old animals. On the other hand expression of SERCA3 mRNA in rat cerebral cortex showed a significant decline with advancing age. However, comparison of SERCA3 protein content did not reveal a corresponding decline; implying that SERCA mRNA turnover rates may be greater in the younger group. Although the present work with rat cerebral cortex does not indicate an age-related decline in SERCA function, previous work from our laboratory on sympathetic nerves and by others on the hippocampus indicate such a decline. In light of our previous and current studies, aging may affect calcium homeostatic mechanisms in central and peripheral autonomic neurons differently.

Adrenergic nerves compensate for a decline in calcium buffering during ageing.

1. The ubiquitous involvement of intracellular calcium ([Ca2+]i) in multiple neuronal pathways has led investigators to suggest that dysfunction of calcium homeostasis may be the primary mediator of age-related neuronal degeneration. Recently, it was shown that sympathetic neurones from superior cervical ganglion (SCG) of aged rats demonstrate decreased sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) function and that aged neurones are more dependent upon mitochondria to control K+-evoked [Ca2+]i transients. 2. Therefore, in the present study we investigated age-related changes in ATP-dependent calcium pumps of plasma membrane Ca2+-ATPase (PMCA) and SERCA in acutely dissociated SCG cells from Fischer-344 rats aged 6 and 20 months. To distinguish between PMCA and SERCA pump activity, we applied the Ca2+-ATPase blocker vanadate and measured rates of recovery of K+-evoked [Ca2+]i transients by fura-2 microfluorometry. 3. Young SCG cells showed a biphasic response to vanadate over the vanadate concentration range (0.01-100 microM); however, old SCG cells showed only a single response over the same concentration range. Additionally, old SCG cells showed a greater sensitivity to Ca2+-ATPase blockade by vanadate. 4. The contribution of mitochondrial calcium uptake to regulate [Ca2+]i was also investigated. To measure the impact of mitochondrial calcium uptake, PMCAs and SERCAs were blocked with vanadate (100 microM) and extracellular sodium was replaced with tetraethylammonium (TEA) to block Na+/Ca2+-exchange. Treated SCG cells showed a decline of 50% in rate of recovery of [Ca2+]i in both 6- and 20-month-old cells; however, this effect did not vary with age. 5. These data suggest that there is an age-related decline in function of SERCAs, with an increased reliance on PMCAs to control high K+-evoked [Ca2+]i transients. In addition, there appears to be no age-related change in the capacity of the mitochondria to restore [Ca2+]i transients to basal levels.

SERCA function declines with age in adrenergic nerves from the superior cervical ganglion.

1. Intracellular calcium is a universal second messenger integrating numerous cellular pathways. An age-related breakdown in the mechanisms controlling [Ca2+]i homeostasis could contribute to neuronal degeneration. One component of neuronal calcium regulation believed to decline with age is the function of sarco/endoplasmic reticulum calcium ATPase (SERCA) pumps. 2. Therefore we investigated the impact of age on the capacity of SERCA pumps to control high (68 mM) [K+]-evoked [Ca2+]i-transients in acutely dissociated superior cervical ganglion (SCG) cells from 6- and 20-month-old Fisher-344 rats. Calcium transients were measured by fura-2 microfluorometry in the presence of vanadate (0.1 microM) to selectively block plasma membrane calcium ATPase (PMCA) pumps, dinitrophenol (100 microM) to block mitochondrial calcium uptake and extracellular sodium replaced with tetraethylammonium to block Na+/Ca2+-exchanger, thus forcing the neuronal cells to rely on SERCA uptake to control [Ca2+]i homeostasis. 3. In the presence of these calcium buffering blockers, the rate of recovery of [Ca2+]i was significantly slower and time to recover to approximately 90% of resting [Ca2+]i was significantly greater in SCG cells from old (20 months) compared with young (6 months) animals. 4. This age-related change in the recovery phase of [K+]-evoked [Ca2+]i-transients could not be explained by differences in the sensitivity of SCG cells to the calcium buffering blockers, as no age-related difference in basal [Ca2+]i was observed. 5. These studies illustrate that when rat SCG cells are forced to rely on SERCAs to buffer [K+]-evoked [Ca2+]i-transients, an age-related decline in SERCA function is revealed. Such age-related declines in calcium regulation coupled with neuronal sensitivity to calcium overload underscore the importance of understanding the components of [Ca2+]i homeostasis and the functional compensation that may occur with advancing age.

Adrenergic nerve smooth endoplasmic reticulum calcium buffering declines with age.

Calcium buffering capacity declines with age in sympathetic nerves of rat tail artery. To test whether smooth endoplasmic reticulum (SER) calcium buffering declines with age, effects of two SER calcium-ATPase inhibitors on norepinephrine release and intracellular calcium were determined. Thapsigargin or cyclopiazonic acid caused a significant increase in stimulation-evoked norepinephrine release from 6 month tail arteries with much less effect in 20 months. In isolated superior cervical ganglion cells, the rate of rise of calcium with K+-depolarization increased only in young cells with either cyclopiazonic acid or thapsigargin, with no effect in the old. In young cells, cyclopiazonic acid significantly influenced time to peak, rate of decline, and time to basal of K+-evoked calcium transients, but had no effect in old cells. Thapsigargin caused a significant increase in rate of decline in young, but not old, cells. These differential effects suggest an age-related decline in function of SER calcium buffering mechanisms in the sympathetic nervous system causing older nerves to become more reliant on mitochondria to buffer calcium.

Sex differences in the effects of 17 beta-estradiol on vascular adrenergic responses.

The in vitro effects of 17 beta-estradiol on vascular responses to adrenergic nerve stimulation were studied in perfused tail arteries from age-matched male and female rats. Nerve stimulation resulted in vasoconstriction that was greater in male arteries. Addition of 17 beta-estradiol (3 x 10(-5) M) reduced the vasoconstrictor responses in both male and female arteries, but the reduction was significantly greater in the females. Gonadectomy of the animals for 1 month prior to the experiment did not alter the in vitro responses to 17 beta-estradiol in either males or females. 17 beta-Estradiol (10(-6) - 3 x 10(-5) M) also relaxed perfused tail arteries precontracted with KCl (50 mM); however the relaxation was not different between males and females, either intact or gonadectomized. Stimulation-evoked release of noradrenaline from adrenergic nerves of perfused tail arteries was measured, but no differences were found between males and females, nor was release modified by in vitro exposure to 17 beta-estradiol (10(-5) M). These results suggest that 17 beta-estradiol acts directly on postjunctional mechanisms to relax tail arteries of either sex. The effect of the hormone on arteries constricted by adrenergic nerve stimulation, however, is greater in females compared to males.

Chronic nicotine administration does not affect peripheral vascular reactivity in the rat.

The effect of chronic nicotine exposure on vascular function and neurotransmitter content was studied in the Fisher 344 rat. Implantation of osmotic mini pumps containing nicotine (0.18-4.7 mg/kg/day) for 14 days resulted in dose-related levels of plasma nicotine (up to 362 +/- 15 ng/ml) and its major metabolite cotinine (1545 +/- 40 ng/ml), as measured by gas chromatography. Rat body weight increase was inhibited significantly by 4.7 mg/kg/day of nicotine exposure. In the isolated perfused mesentery, transmural nerve stimulation of capsaicin-sensitive sensory nerves produced vasodilator responses that were not different between vehicle- and chronic nicotine-treated animals. Endothelium-dependent vasodilation elicited by acetyl-choline also was not influenced by chronic nicotine treatment. Furthermore, chronic nicotine exposure in vivo had no effect on in vitro vasodilator responses caused by nicotine itself, either at low (3 x 10(-5) M) or high (3 x 10(-4) M) concentrations. In tail artery ring segments, vasoconstrictor responses to either transmural adrenergic nerve stimulation or norepinephrine were not different when vehicle and nicotine treatment groups were compared. Prior chronic nicotine exposure also did not alter nicotine's ability to relax ring segments that were precontracted with norepinephrine. Vascular contents of norepinephrine, calcitonin gene-related peptide, neuropeptide Y and substance P were not altered by chronic nicotine treatment. In conclusion, although nicotine has direct vascular actions, chronic nicotine exposure up to 4.7 mg/kg/day does not significantly alter vascular reactivity.