Taruna D. Wakade

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.

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)

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.

Exocytosis coupled to mobilization of intracellular calcium by muscarine and caffeine in rat chromaffin cells.

Abstract: We used cultured rat chromaffin cells to test the hypothesis that Ca2+ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca2+ sources: (a) Ca2+ surrounding single cells was transiently displaced by applying agonist with or without Ca2+ from an ejection pipette. (b) Intracellular stores of Ca2+ were depleted by soaking cells in Ca2+‐free plus 1 mM EGTA solution before transient exposure to agonist plus Ca2+. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca2+ concentration ([Ca2+]i) was measured by indo‐1 fluorescence. KCl (35 mM) and nicotine (10 µM) caused an immediate increase in [Ca2+]i and secretion in cells with or without internal Ca2+ stores, but only when applied with Ca2+ in the ejection pipette. Caffeine (10 mM) and muscarine (30 µM) evoked exocytosis whether or not Ca2+ was included in the pipette, but neither produced responses in cells depleted of internal Ca2+ stores. Pretreatment with ryanodine (0.1 µM) inhibited caffeine‐ but not muscarine‐stimulated responses. Elevated [Ca2+]i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 ± 0.38 s) or muscarine (2.2 ± 0.25 s). However, the duration of caffeine‐evoked exocytosis (7.1 ± 0.8 s) was significantly shorter than that evoked by muscarine (33.1 ± 3.5 s). The duration of caffeine‐evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An ∼20‐s refractory period was found between repeated caffeine‐evoked exocytotic bursts even though [Ca2+]i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca2+ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca2+. These results and conclusions are consistent with our original observations in the perfused adrenal gland.

Pituitary adenylyl cyclase-activating polypeptide and nerve growth factor use the proteasome to rescue nerve growth factor-deprived sympathetic neurons cultured from chick embryos.

Abstract: Removal of nerve growth factor (NGF) from sympathetic neurons initiates a neuronal death program and apoptosis. We show that pituitary adenylyl cyclase‐activating polypeptide (PACAP) prevents apoptosis in NGF‐deprived sympathetic neurons. PACAP (100 nM) added to culture medium at the time of plating failed to support neuronal survival. However, in neurons grown for 2 days with NGF and then deprived of NGF, PACAP prevented cell death for the next 24–48 h. Uptake of [3H]norepinephrine ([3H]NE) was used as an index of survival and decreased >50% in NGF‐deprived cultures within 24 h. PACAP (1–100 nM) restored [3H]NE uptake to 92 ± 8% of that of NGF‐supported controls. Depolarization‐induced [3H]NE release in neurons rescued by PACAP was the same as that in NGF‐supported neurons. PACAP rescue was not mimicked by forskolin or 8‐bromo‐cyclic AMP and was not blocked by the protein kinase A inhibitor Rp‐adenosine 3′,5′‐cyclic monophosphothioate. Mobilization of phosphatidylinositol by muscarine failed to support NGF‐deprived neurons. Thus, PACAP may use novel signaling to promote survival of sympathetic neurons. The apoptosis‐associated caspase CPP32 activity increased approximately fourfold during 6 h of NGF withdrawal (145 ± 40 versus 38 ± 17 nmol of substrate cleaved/min/mg of protein) and returned to even below the control level in NGF‐deprived, PACAP‐rescued cultures (14 ± 7 nmol/min/mg of protein). Readdition of NGF or PACAP to NGF‐deprived cultures reversed CPP32 activation, and this was blocked by lactacystin, a potent and specific inhibitor of the 20S proteasome, suggesting that NGF and PACAP target CPP32 for destruction by the proteasome. As PACAP is a preganglionic neurotransmitter in autonomic ganglia, we propose a novel function for this transmitter as an apoptotic rescuer of sympathetic neurons when the supply of NGF is compromised.

Ca2+ Mobilized by Caffeine from the Inositol 1,4,5-Trisphosphate-Insensitive Pool of Ca2+ in Somatic Regions of Sympathetic Neurons Does Not Evoke [3H]Norepinephrine Release

Abstract: The effects of electrical stimulation, muscarinic and serotonergic agonists, and caffeine on [3H]inositol 1,4,5‐trisphosphate ([3H]Ins(1,4,5)P3) content, intracellular free Ca2+ concentration ([Ca2+]i), and release of [3H]norepinephrine ([3H]NE) were studied in cultured sympathetic neurons. Neuronal cell body [Ca2+]i was unaffected by muscarinic or serotonergic receptor stimulation, which significantly increased [3H]Ins(1,4,5)P3 content. Stimulation at 2 Hz and caffeine had no effect on [3H]Ins(1,4,5)P3, but caused greater than two‐fold increase in [Ca2+]i. Only 2‐Hz stimulation released [3H]NE. Caffeine had no effect on the release. When [Ca2+]i was measured in growth cones, only electrical stimulation produced an increase in [Ca2+]i. The other agents had no effect on Ca2+ at the terminal regions of the neurons. We conclude that Ins(1,4,5)P3‐insensitive, but caffeine‐sensitive Ca2+ stores in sympathetic neurons are located only in the cell body and are not coupled to [3H]NE release.

Adenosine induces apoptosis by inhibiting mRNA and protein synthesis in chick embryonic sympathetic neurons

Our previous work has established that adenosine is toxic to chick embryonic sympathetic neurons and kills freshly plated neurons by a process of apoptosis. Although the exact mechanism remains unknown, we found that phosphorylation of adenosine was essential to the toxicity. Using markers for RNA ([3H]uridine) and protein ([35S]methionine) synthesis we demonstrate here that in freshly plated sympathetic neurons adenosine inhibits RNA and protein synthesis by about 50%. The inhibitory effects of adenosine on RNA and protein synthesis, and increased ATP synthesis were blocked by adenosine kinase inhibitor, suggesting that phosphorylated products are responsible for inhibition of RNA and protein synthesis and cell death. Adenosine-induced inhibition of RNA and protein synthesis in neuronal cells provides a new role for adenosine in the regulation of cell function.