Arun R. Wakade

β-amyloid from Alzheimer disease brains inhibits sprouting and survival of sympathetic neurons

The significance of the amyloid plaque core proteins (APCP) in Alzheimers disease (AD) and its consequences for neuronal survival have been controversial. To address this problem we purified the APCP and beta A obtained from brains with AD, and assessed their biological effects in tissue culture. APCP and beta A caused severe toxicity to chick and rat sympathetic and sensory neurons whose survival is dependent upon NGF. This toxicity was dose dependent and reversible at low doses. APCP and beta A prevented sprouting of neurites in freshly plated neurons. In established cultures addition of these molecules caused vacuolation and fragmentation of neurites and disintegration of neuronal soma. We suggest that the deposition of APCP in AD may be partly responsible for the destruction of the neuritic arbor, thereby contributing to the formation of the neuritic plaque and to neuronal death.

Activation and Multiple-Site Phosphorylation of Tyrosine Hydroxylase in Perfused Rat Adrenal Glands

Abstract: Tryptic digestion of tyrosine hydroxylase (TH) isolated from rat adrenal glands labeled with 32Pi produced five phosphopeptides. Based on the correspondence of these phosphopeptides with those identified in TH from rat pheochromocytoma cells, four phosphorylation sites (Ser8, Ser19, Ser31, and Ser40) were inferred. Field stimulation of the splanchnic nerves at either 1 or 10 Hz (300 pulses) increased 32P incorporation into TH. At 10 Hz, the phosphorylation of Ser19 and Ser40 was increased, whereas at 1 Hz, Ser19, Ser31, and Ser40 phosphorylation was increased. Stimulation at either 1 or 10 Hz also increased the catalytic activity of TH, as measured in vitro (pH 7.2) at either 30 or 300 μM tetrahydrobiopterin. Nicotine (3 μM, 3 min) increased Ser19 phosphorylation, vasoactive intestinal polypeptide (10 μM, 3 min) increased Ser40 phosphorylation, and muscarine (100 μM, 3 min) increased TH phosphorylation primarily at Ser19 and Ser31. Vasoactive intestinal polypeptide, but not nicotine or muscarine, mimicked the effects of field stimulation on TH activity. Thus, the regulation of rat adrenal medullary TH phosphorylation by nerve impulses is mediated by multiple first and second messenger systems, as previously shown for catecholamine secretion. However, different sets of second messengers are involved in the two processes. The action of vasoactive intestinal polypeptide as a secretagogue involves the mobilization of intracellular calcium, whereas its effects on TH phosphorylation are mediated by cyclic AMP. This latter effect of vasoactive intestinal polypeptide and the consequent increase in Ser40 phosphorylation appear to be responsible for the rapid activation of TH by splanchnic nerve stimulation.

Stimulated rise in neuronal calcium is faster and greater in the nucleus than the cytosol

Calcium is an important regulator of a variety of neuronal activities including gene expression. However, it is not clear how Ca2+ influx affects intracellular Ca2+ concentration ([Ca2+]i) in the nucleus. We have taken advantage of laser photometry, the Ca2+‐sensitive dye Indo‐1 that allows ratio imaging, and confocal microscopy to eliminate the influences of unequal cell geometry and dye distribution. We show that Ca2+ influx into sympathetic neurons causes a significantly greater and faster increase in [Ca2+]i in the nucleus than in the cytosol. The differential increase in nuclear [Ca2+]i was apparent when Ca2+ entered from the extracellular medium during K+ depolarization, ionomycin or acetylcholine treatment, and brief periods of electrical stimulation. When intracellular Ca2+ was mobilized by caffeine the rise in nuclear [Ca2+]i was again greater than in any other region of the neuron. The increased nuclear Ca2+ levels were uniform throughout the nucleus and not associated with the nuclear envelope. The differential rise in nuclear Ca2+ was eliminated by acridine orange binding to nucleic acids. Nonexcitable cells (astrocytes, oligodendrocytes, and fibroblasts) did not show differential distribution of Ca2+ after ionomycin treatment. These results support the idea that activity‐dependent gene regulation in sympathetic neurons may be mediated by changes in Ca2+ concentration at the level of the chromatin material.—Przywara, D. A.; Bhave, S. V.; Bhave, A.; Wakade, T. D.; Wakade, A. R. Stimulated rise in neuronal calcium is faster and greater in the nucleus than the cytosol. FASEB J. 5: 217–222; 1991.

Differential secretion of catecholamines in response to peptidergic and cholinergic transmitters in rat adrenals.

1. Rat adrenal medulla is stimulated by cholinergic and peptidergic transmitters released from splanchnic nerves. The peptidergic transmitter has been identified as vasoactive intestinal polypeptide (VIP) and its contribution in comparison to that of acetylcholine (ACh) is more prominent at low neuronal activity. The purpose of this study is to determine if ACh and VIP cause differential secretion of adrenaline and noradrenaline and whether the differential secretion also occurs when splanchnic nerves are stimulated at different frequencies. 2. Perfusion of the left adrenal gland with Krebs solution for several hours did not change adrenaline and noradrenaline contents (15.2 micrograms and 3.5 micrograms, respectively) and their ratio (4.4) from those of the unperfused right adrenal medulla (15.2 micrograms, 3.3 micrograms and 4.8, respectively). 3. Perfusion with ACh (10 microM for 4 min) resulted in the secretion of 109 ng of catecholamines and the ratio of adrenaline to noradrenaline was 3.8. Although the secretion increased with increased concentrations of ACh (30 and 100 microM), the ratios remained between 3 and 4. 4. Perfusion with VIP (10 microM for 4 min) resulted in the secretion of 27 ng of catecholamines and the ratio of adrenaline to noradrenaline was 9.7. A higher concentration of VIP (20 microM for 4 min) resulted in the secretion of greater amounts of catecholamines (102 ng) without significantly altering the ratio of adrenaline to noradrenaline (10.9). 5. Perfusion with as low as 0.01 microM pituitary adenylate cyclase‐activating polypeptide (PACAP) increased the secretion of catecholamines to 31 ng and the secretion increased in a dose‐dependent manner up to 0.3 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

The peptide VIP is a neurotransmitter in rat adrenal medulla : physiological role in controlling catecholamine secretion

1. The perfused adrenal gland of the rat was used to establish the identity of a non‐cholinergic substance involved in splanchnic nerve‐mediated secretion of catecholamines. 2. The perfused adrenal medulla was rich in vasoactive intestinal polypeptide (VIP) content (28 pmol g‐1 of wet tissue). VIP‐immunoreactive nerve fibres were present in the adrenal medulla and the adrenal cortex. 3. Field stimulation (10 Hz for 15 min plus 1 Hz for 15 min) caused a large increase in the output of VIP in the perfusate over the spontaneous release of VIP. Secretion of catecholamines was also greatly elevated by field stimulation. Field stimulation‐evoked output of VIP and catecholamines was abolished after chronic denervation of the adrenal glands. 4. Infusion of acetylcholine (ACh) did not increase the output of VIP but caused a robust secretion of catecholamines. 5. The VIP output declined when the stimulation frequency was increased (8.6 x 10(‐3) fmol pulse‐1 at 1 Hz and 4.0 x 10(‐3) fmol pulse‐1 at 10 Hz). 6. In contrast, the output of 3H‐acetylcholine (3H‐ACh, expressed as a fraction of tissue 3H‐ACh content) increased from 7.0 x 10(‐2) pulse‐1 at 1 Hz to 16.3 x 10(‐2) pulse‐1 at 10 Hz. 7. Secretion of catecholamines evoked by low‐frequency stimulation (1 Hz) was reduced by 40% in the presence of cholinergic receptor antagonists (atropine plus hexamethonium). Inclusion of a VIP receptor antagonist ([Ac‐Tyr1, D‐Phe2]‐GRF 1‐29 amide) caused about 75% inhibition. 8. The VIP receptor antagonist inhibited VIP‐evoked secretion of catecholamines without affecting ACh‐evoked secretion. 9. In conclusion, VIP satisfies all the essential criteria to assume the role of a neurotransmitter in the rat adrenal medulla. The contribution of VIP to the secretion of adrenal medullary hormones is more prominent at low rates of neuronal activity whereas ACh is the major contributor at higher activity.

Sites of transmitter release and relation to intracellular Ca2+ in cultured sympathetic neurons.

Fluorescence imaging of indo-1 loaded cells was used to monitor influx and distribution of Ca2+ in cell bodies, neurites and growth cones of sympathetic neurons cultured from embryonic chick. Similar experiments on release of tritiated noradrenaline were performed to assess the relationship between intracellular Ca2+ concentration ([Ca2+]i) and transmitter release. Effects of Ca2+ channel antagonists on electrically stimulated rise in [Ca2+]i were dependent on the neuronal region examined. Cadmium and verapamil blocked Ca2+ entry in cell bodies but were less effective in neurites and growth cones. Nifedipine partially inhibited Ca2+ entry in cell bodies and was less effective in neurites and growth cones. Combination of cadmium and nifedipine blocked [Ca2+]i rise in all neuronal regions. Omega-conotoxin was an effective Ca2+ channel blocker in all regions. Ca2+ channel blockers had effects on [3H]noradrenaline release which paralleled effects on [Ca2+]i in neurites (and growth cones) but not cell bodies. Cadmium, verapamil and nifedipine each caused a partial, reversible block of the evoked release. Combination of cadmium and nifedipine completely blocked evoked [3H]noradrenaline release. Omega-conotoxin caused complete, irreversible block of electrically evoked release. During prolonged depolarization with 125 mM K+ Krebs solution, elevation of [Ca2+]i was maintained in cell bodies but was transient in neurites and growth cones. The amplitude and time course of [3H]noradrenaline release paralleled [Ca2+]i in neurites and growth cones, but not the cell body under the above conditions. A new method is described to study localized uptake and release of [3H]noradrenaline in cell bodies versus neurites of sympathetic neurons. Incubation of these modified cultures with [3H]noradrenaline showed that cell bodies had very low [3H]noradrenaline uptake (0.23 x 10(-6) c.p.m./mg protein), whereas neurites contained approximately 20 times more radioactivity. Depolarization of neurites by excess K+ and field stimulation caused a large increase in the net release of [3H]noradrenaline. The release was unaffected by removal of cell bodies. Neurites remained functionally viable for more than 2 h after separation from their cell bodies. [3H]Noradrenaline release could be evoked repeatedly over this time. [3H]Noradrenaline release from isolated neurites was partially blocked by nifedipine and fully blocked by combination of cadmium and nifedipine or by omega-conotoxin. The uptake and release of [3H]noradrenaline by neurites alone (expressed per mg protein) accounted for the total [3H]noradrenaline in intact cultures with neurites and cell bodies. Therefore, we conclude that neurites (and growth cones) are the prominent sites of uptake, storage and release of sympathetic transmitter.(ABSTRACT TRUNCATED AT 400 WORDS)

Exocytosis from a single rat chromaffin cell by cholinergic and peptidergic neurotransmitters.

Secretion of catecholamines from chromaffin cells is mediated by cholinergic and peptidergic neurotransmitters. The cholinergic transmitter acetylcholine activates both nicotinic and muscarinic receptors to trigger catecholamine secretion in rat adrenal medulla. Vasoactive intestinal polypeptide (VIP) has been identified as the peptidergic transmitter in rat adrenal medulla and may also be the non-cholinergic transmitter in bovine adrenal. Pituitary adenylate cyclase activating polypeptide (PACAP), a VIP-like secretin peptide, is also found in the adrenal, and is a potent secretagogue. Thus, PACAP may be another peptidergic transmitter at the adrenal synapse. A most intriguing property of rat chromaffin cells is that stimulation of nicotinic, muscarinic, VIP or PACAP receptors are each able to produce robust catecholamine secretion on their own. This raises the question of whether a single chromaffin cell can respond to each of the above agonists or whether the secretion is due to subpopulations of chromaffin cells. This issue was addressed by using electrochemical techniques to monitor exocytosis from individual chromaffin cells in culture. We demonstrate that acetylcholine, nicotine, muscarine, VIP and PACAP are each able to evoke catecholamine secretion from a single chromaffin cell. Some cells only responded to acetylcholine. Furthermore, each agonist produced a distinct pattern of exocytosis. Muscarine-evoked secretion exhibited a latency of 0.5-2 s, but exocytosis persisted up to 30 s following 500 ms stimulation. Nicotine produced an immediate response which usually ended within 10 s. The secretory pattern following acetylcholine appeared to be the sum of the nicotinic and muscarinic patterns, showing both rapid onset and longer duration.(ABSTRACT TRUNCATED AT 250 WORDS)

Pituitary adenylate cyclase activating polypeptide-immunoreactive sensory neurons innervate rat adrenal medulla.

Rat adrenal chromaffin cells were invested by a dense network of nerve fibers immunoreactive to pituitary adenylate cyclase activating polypeptide-38 (PACAP-IR). Immunohistochemical studies demonstrated the presence of PACAP-IR in nodose and dorsal root ganglion cells, but not in neurons of the intermediolateral cell column and other autonomic nuclei of the thoracic and upper lumbar spinal cord. Somata of the T7 to T12 paravertebral ganglia were PACAP-negative. A few lightly labeled neurons were occasionally noted in the dorsal motor nucleus of the vagus. Injection of the retrograde tracer Fluorogold into the left adrenal medulla 3 days prior to sacrifice resulted in the labeling of a population of neurons in the ipsilateral spinal cord intermediolateral cell column (T1 to L1), ipsilateral and contralateral nodose ganglia and ipsilateral dorsal root ganglia from T7 to T10 inclusive. A small number of lightly labeled somata was occasionally noted in the dorsal motor nucleus of the vagus. Combined retrograde tracing and PACAP immunohistochemistry showed that a population of Fluorogold-containing nodose and dorsal root ganglion cells were also PACAP-positive. Pre-treatment of the rats with capsaicin caused a marked reduction of the PACAP-IR in the adrenal gland as well as in the superficial layers of the dorsal horn and caudal spinal trigeminal nucleus. These findings, in conjunction with the apparent absence of PACAP-IR in spinal sympathetic preganglionic neurons, sympathetic postganglionic neurons, and dorsal motor nucleus of the vagus, raise the possibility that PACAP-IR fibers observed in the adrenal medulla are primarily sensory in origin. As a corollary, catecholamine secretion from chromaffin cells may be modulated by the peptidergic sensory afferents in addition to the cholinergic sympathetic preganglionic nerve fibers.

Noradrenaline transport and transporter mRNA of rat chromaffin cells are controlled by dexamethasone and nerve growth factor.

1. The biochemical basis for differences in noradrenaline (NA) transporter function between chromaffin cells in the adrenal medulla and those maintained in primary culture was investigated. 2. Intact adrenal medullae of neonatal rats accumulated small amounts of [3H]NA. In contrast, dissociated chromaffin cells placed in culture for 2‐6 days accumulated 100‐1000 times more [3H]NA. 3. Nerve growth factor (NGF) stimulated, whereas glucocorticoids dose dependently and reversibly inhibited, [3H]NA transport in chromaffin cells maintained in culture up to 6 days. During this period, no change in the morphological or biochemical characteristics of either NGF‐treated or ‐untreated chromaffin cells was evident. 4. A rat NA transporter cDNA clone was isolated for use in the quantification of NA transporter mRNA. Intact adrenal medullae contained 40% less NA transporter mRNA than an equivalent number of chromaffin cells in culture. Furthermore, dexamethasone produced nearly 90% loss and NGF elicited approximately 60% increase in NA transporter mRNA levels in cultured cells. 5. In cultured cells, and possibly in vivo, glucocorticoids inhibit NA transporter function of chromaffin cells at least in part through a decrease in NA transporter mRNA.

Activation of K+ channels by lanthanum contributes to the block of transmitter release in chick and rat sympathetic neurons

SummaryWe studied the effects of lanthanum (La3+) on the release of 3H-norepinephrine(3H-NE), intracellular Ca2+ concentration, and voltage clamped Ca2+ and K+ currents in cultured sympathetic neurons. La3+ (0.1 to 10 μm) produced concentration-dependent inhibition of depolarization induced Ca2+ influx and 3H-NE release. La3+ was more potent and more efficacious in blocking 3H-NE release than the Ca2+-channel blockers cadmium and verapamil, which never blocked more than 70% of the release. At 3 μm, La3+ produced a complete block of the electrically stimulated rise in intracellular free Ca2+ ([Ca2+]i) in the cell body and the growth cone. The stimulation-evoked release of 3H-NE was also completely blocked by 3 μm La3+. However, 3 μm La3+ produced only a partial block of voltage clamped Ca2+ current (ICa). Following La3+ (10 μm) treatment 3H-NE release could be evoked by high K+ stimulation of neurons which were refractory to electrical stimulation. La3+ (1 μm) increased the hyperpolarization activated, 4-aminopyridine (4-AP) sensitive, transient K+ current (IA) with little effect on the late outward current elicited from depolarized holding potentials. We conclude that the effective block of electrically stimulated 3H-NE release is a result of the unique ability of La3+ to activate a stabilizing, outward K+ current at the same concentration that it blocks inward Ca2+ current.