John N. Buchholz

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.

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.

Regional metabolic changes in the pedunculopontine nucleus of unilateral 6-hydroxydopamine Parkinson's model rats

The pedunculopontine nucleus (PPN) located in the mesopontine tegmentum is innervated by descending projections from nuclei in the basal ganglia. The present study was performed to determine whether nigrostriatal dopaminergic neuron degeneration is associated with changes in PPN metabolic activity. Unilateral nigrostriatal lesioning was performed by injecting 6-hydroxydopamine (6-OHDA) into the substantia nigra pars compacta in 10 rats. Six of these animals exhibited apomorphine-induced rotations contralateral to the lesion and were included in the experimental group for determination of regional cerebral metabolic rate for glucose (rCMRglucose) along with five sham-lesioned and five normal controls. All studies were performed 13-15 days after lesioning using [14]C-2-deoxyglucose autoradiography. Significant hemispheric differences in metabolic activity were observed only in the 6-OHDA lesioned animals. Increased rCMRglucose was found in the globus pallidus (+63%) ipsilateral to the lesion as compared to the contralateral hemisphere, and reduced rCMRglucose in the primary motor, sensory, and auditory cortex (-7%, -12% and -7%, respectively), and in the subthalamic nucleus (-6%). Metabolic activity within the PPN ipsilateral to the lesion was significantly greater than the contralateral hemisphere (P<0.05; lesion 57+/-8, nonlesion 52+/-5), and significantly greater than the sham-lesioned side of the sham rat (P<0.05; sham lesion 47+/-5). No hemispheric differences were observed in the lateral dorsal tegmental nucleus. These observations offer further support for a role of the PPN in Parkinsons and for the utility of the rodent unilateral 6-OHDA model in defining the pathophysiologic significance of the mesopontine tegmental striatal-motor interfaces in basal ganglia disease.

Advancing age alters intracellular calcium buffering in rat adrenergic nerves

There is a marked increase with advancing age of stimulation-evoked neurotransmitter release from vascular adrenergic nerves in the rat, an effect correlated with increased levels of plasma norepinephrine. This increase in norepinephrine release could not be accounted for by an alteration in neuronal and extraneuronal uptake of norepinephrine or a decline in feedback inhibition of release by prejunctional alpha2-adrenergic receptors. Measurement of intracellular calcium in fura-2-labeled superior cervical ganglion cells revealed elevated K+-evoked calcium transients in old compared to young neurons. Blockade of mitochondrial calcium uptake with dinitrophenol resulted in increased calcium transients in old neurons only. Furthermore, following blockade of mitochondrial calcium uptake the rate of return of calcium to resting levels was reduced to a greater degree in old cells as compared to young cells. The effects of dinitrophenol in old cells were attenuated when extracellular calcium was reduced. These findings suggest that older cells are more dependent on mitochondrial calcium buffering, perhaps due to changes in ATP dependent calcium uptake. Increased calcium transients as a result of altered intracellular calcium buffering offer a reasonable explanation for our previous observation of increased stimulation evoked norepinephrine release.

Decreased NR1 Phosphorylation and Decreased NMDAR Function in Hibernating Arctic Ground Squirrels

Heterothermic mammals such as ground squirrels tolerate ischemia and N‐methyl‐D‐aspartate (NMDA) better than homeothermic mammals such as rats both in vivo and in vitro, and this tolerance is enhanced in the hibernating state. However, the cellular mechanisms underlying this tolerance remain unclear. NMDA receptors (NMDAR) play a key role in excitotoxicity. The purpose of the current study was therefore to test the hypothesis that NMDAR are down‐regulated in hibernating Arctic ground squirrels (hAGS; Spermophilus parryii). To address this hypothesis, we used Western blot analysis to investigate NMDAR phosphorylation, an activator of NMDAR function, and internalization in naïve hippocampal tissue from hAGS, interbout euthermic AGS (ibeAGS), and rats. Furthermore, we used fura‐2 calcium imaging to examine NMDAR function in cultured hippocampal slices from hAGS, ibeAGS, and rats. We report that phosphorylation of the NMDAR1 (NR1) subunit is decreased in hippocampal tissue from hAGS and that the NMDAR component of Glu‐induced increase in [Ca2+]i is decreased in hippocampal slices from hAGS. Moreover, the fraction of NR1 in the functional membrane pool in AGS is less than that in rats.

Capsaicin Pre-treatment Provides Neurovascular Protection Against Neonatal Hypoxic-Ischemic Brain Injury in Rats

Capsaicin, a transient receptor potential vanilloid 1 (TRPV1) agonist, has recently been shown to provide neuroprotection against brain injury in experimental adult models of cerebral ischemia. Accordingly, in this study, we investigated the way in which capsaicin-mediated TRPV1 modulation could attenuate damage in an experimental hypoxic-ischemic (HI) neonatal brain injury model. The Rice-Vannucci method was used in 10-day-old rat pups by performing unilateral carotid artery ligation followed by 2 h of hypoxia (8% O2 at 37°C). Capsaicin was administered intraperitoneally (0.2 mg/kg or 2.0 mg/kg) at 3 h pre-HI or 1 h post-HI. Post assessment included measurement of infarction volume at 24 and 72 h in addition to an assessment of the vascular dynamics of the middle cerebral artery (MCA) at 6 h post-HI. The results indicated that pre-treatment with capsaicin reduced infarction volume significantly with either low-dose or high-dose treatment. Pre-treatment also improved myogenic tone and decreased apoptotic changes in the distal MCA. We concluded that capsaicin pre-treatment may provide neurovascular protection against neonatal HI.

Impact of development and chronic hypoxia on NE release from adrenergic nerves in sheep arteries

To examine effects of development and chronic high-altitude hypoxia on sympathetic nerve function in sheep, norepinephrine release was measured in vitro from middle cerebral and facial arteries. Capsaicin was used to test the role of capsaicin-sensitive sensory nerves; norepinephrine release was not altered by capsaicin treatment. Nomega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, decreased stimulation-evoked norepinephrine release in middle cerebral arteries from normoxic sheep with no effect in hypoxic arteries or facial arteries. Thus NO-releasing nerves augmented norepinephrine release. Furthermore, the function of NO-releasing nerves declined after chronic hypoxia. Despite loss of the augmenting effects of NO, stimulation-evoked fractional norepinephrine release was unchanged after chronic hypoxia, suggesting that middle cerebral arteries adapt to hypoxia by increasing stimulation-evoked norepinephrine release. In fetal facial arteries, chronic hypoxia resulted in a decline in stimulation-evoked norepinephrine release, but there was an increase in the adult facial artery. In the adult, adaptation to chronic hypoxia is similar in both cerebral and facial arteries. However, differential adaptation in fetal adrenergic nerves may reflect differences in fetal redistribution of blood flow in the face of chronic hypoxia but could also possibly contribute to increased incidence of fetal morbidity.

Effects of maturation on adrenergic neurotransmission in ovine cerebral arteries

The present studies examine the hypothesis that multiple adrenergic neuroeffector mechanisms are not fully developed in fetal, compared with adult, ovine middle cerebral arteries. In arteries denuded of endothelium and pretreated with 1 microM atropine to block involvement of muscarinic receptors, 10 microM capsaicin to deplete sensory peptidergic neurons, and 10 microM nitro-L-arginine methyl ester (L-NAME) to block possible influences from nitric oxidergic innervation, transmural stimulation at 16 Hz increased contractile tensions to 9.5 +/- 3.7% (n = 6) of the potassium maximum in adult arteries. Corresponding values in fetal arteries, however, were significantly less and averaged only 1.1 +/- 0.6% (n =10). However, postsynaptic sensitivity to norepinephrine (NE) was similar in the two age groups; NE pD(2) values (-log EC(50)) averaged 6.11 +/- 0.12 (n = 6) and 6.33 +/- 0.09 M (n = 9) in fetal and adult arteries, respectively. Similarly, NE content measured via HPLC was also similar in the two age groups and averaged 32.4 +/- 5.0 (n = 17) and 32.5 +/- 3.9 ng/ng wet wt (n = 13) in fetal and adult middle cerebral arteries, respectively. In contrast, stimulation-induced NE release was greater in fetal than in adult arteries, whether calculated as total mass released [883 +/- 184 (n = 17) vs. 416 +/- 106 pg NE/mg wet wt (n = 13)] or as fractional release [51.1 +/- 5.3 (n = 17) vs. 22.8 +/- 3.8 pg/pg NE content per pulse x 10(-6)]. Measured as an index of synaptic density, neuronal cocaine-sensitive NE uptake was similar in fetal and adult arteries [1.55 +/- 0.40 (n = 10) and 1.84 +/- 0.51 pmol/mg wet wt (n = 7), respectively]. Overall, age-related differences in postsynaptic sensitivity to NE, NE release, and NE uptake capacity cannot explain the corresponding age-related differences in response to stimulation. The data thus suggest that total synaptic volume and cleft width, in particular, are probably greater and/or that adrenergic corelease of vasoactive substances other than NE is altered in fetal compared with adult middle cerebral arteries.The present studies examine the hypothesis that multiple adrenergic neuroeffector mechanisms are not fully developed in fetal, compared with adult, ovine middle cerebral arteries. In arteries denuded of endothelium and pretreated with 1 μM atropine to block involvement of muscarinic receptors, 10 μM capsaicin to deplete sensory peptidergic neurons, and 10 μM nitro-l-arginine methyl ester (l-NAME) to block possible influences from nitric oxidergic innervation, transmural stimulation at 16 Hz increased contractile tensions to 9.5 ± 3.7% ( n = 6) of the potassium maximum in adult arteries. Corresponding values in fetal arteries, however, were significantly less and averaged only 1.1 ± 0.6% ( n =10). However, postsynaptic sensitivity to norepinephrine (NE) was similar in the two age groups; NE pD2 values (-log EC50) averaged 6.11 ± 0.12 ( n = 6) and 6.33 ± 0.09 M ( n = 9) in fetal and adult arteries, respectively. Similarly, NE content measured via HPLC was also similar in the two age groups and averaged 32.4 ± 5.0 ( n = 17) and 32.5 ± 3.9 ng/ng wet wt ( n = 13) in fetal and adult middle cerebral arteries, respectively. In contrast, stimulation-induced NE release was greater in fetal than in adult arteries, whether calculated as total mass released [883 ± 184 ( n = 17) vs. 416 ± 106 pg NE/mg wet wt ( n = 13)] or as fractional release [51.1 ± 5.3 ( n = 17) vs. 22.8 ± 3.8 pg/pg NE content per pulse × 10-6]. Measured as an index of synaptic density, neuronal cocaine-sensitive NE uptake was similar in fetal and adult arteries [1.55 ± 0.40 ( n = 10) and 1.84 ± 0.51 pmol/mg wet wt ( n = 7), respectively]. Overall, age-related differences in postsynaptic sensitivity to NE, NE release, and NE uptake capacity cannot explain the corresponding age-related differences in response to stimulation. The data thus suggest that total synaptic volume and cleft width, in particular, are probably greater and/or that adrenergic corelease of vasoactive substances other than NE is altered in fetal compared with adult middle cerebral arteries.

Roles of cytosolic Ca2+ concentration and myofilament Ca2+ sensitization in age-dependent cerebrovascular myogenic tone

In light of evidence that immature arteries contain a higher proportion of noncontractile smooth muscle cells than found in fully differentiated mature arteries, the present study explored the hypothesis that age-related differences in the smooth muscle phenotype contribute to age-related differences in contractility. Because Ca(2+) handling differs markedly between contractile and noncontractile smooth muscle, the present study specifically tested the hypothesis that the relative contributions of Ca(2+) influx and myofilament sensitization to myogenic tone are upregulated, whereas Ca(2+) release is downregulated, in immature [14 days postnatal (P14)] compared with mature (6 mo old) rat middle cerebral arteries (MCAs). Myofilament Ca(2+) sensitivity measured in β-escin-permeabilized arteries increased with pressure in P14 but not adult MCAs. Cyclopiazonic acid (an inhibitor of Ca(2+) release from the sarcoplasmic reticulum) increased diameter and reduced Ca(2+) in adult MCAs but increased diameter with no apparent change in Ca(2+) in P14 MCAs. La(3+) (Ca(2+) influx inhibitor) increased diameter and decreased Ca(2+) in adult MCAs, but in P14 MCAs, La(3+) increased diameter with no apparent change in Ca(2+). After treatment with both La(3+) and CPA, diameters were passive in both adult and P14 MCAs, but Ca(2+) was decreased only in adult MCAs. To quantify the fraction of smooth muscle cells in the fully differentiated contractile phenotype, extents of colocalization between smooth muscle α-actin and SM2 myosin heavy chain were determined and found to be at least twofold greater in adult than pup MCAs. These data suggest that compared with adult MCAs, pup MCAs contain a greater proportion of noncontractile smooth muscle and, as a consequence, rely more on myofilament Ca(2+) sensitization and Ca(2+) influx to maintain myogenic reactivity. The inability of La(3+) to reduce cytosolic Ca(2+) in the pup MCA appears due to La(3+)-insensitive noncontractile smooth muscle cells, which contribute to the spatially averaged measurements of Ca(2+) but not contraction.

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.