Arun R. Wakade

Arun R. Wakade
Wayne State University | WSU · Department of Pharmacology

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128
Publications
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4,165
Citations
Citations since 2016
0 Research Items
174 Citations
201620172018201920202021202205101520253035
201620172018201920202021202205101520253035
201620172018201920202021202205101520253035
201620172018201920202021202205101520253035
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Publications

Publications (128)
Article
Inhibitory effect of adenosine on the isolated heart muscle and vascular system were first described in 1929. Since then, numerous reviews have been published on the diverse actions of this nucleoside on a wide variety of cell types. Essentially all effects of adenosine in neurons and non-neuronal cells are mediated by activation of nucleoside memb...
Article
Full-text available
The novel chromogranin A fragment catestatin (bovine chromogranin A(344-364); RSMRLSFRARGYGFRGPGLQL) is a potent inhibitor of catecholamine release (IC50, approximately 0.2-0.3 microM) by acting as a nicotinic cholinergic antagonist. To define the minimal active region within catestatin, we tested the potencies of synthetic serial three-residue del...
Article
Previous work has shown that nucleosides produce apoptosis in sympathetic ganglion (SG) cells in vitro. The present study examined the effects of nucleosides on the development of the chick embryo in vivo with special attention to the SG and the optic tectum of the central nervous system. In the presence of an adenosine deaminase inhibitor, adenosi...
Article
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...
Article
Our past work on nucleoside toxicity in sympathetic neurons has clearly revealed that adenosine and 2'-deoxyadenosine (dAdo) have different mechanisms of action in inducing apoptotic death. For example, adenosine is toxic to neurons only during early phase of growth whereas dAdo kills even mature neurons. In this study, we hypothesize that dAdo-ind...
Article
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) synthes...
Article
Recently, we have demonstrated that adenosine and 2'-deoxyadenosine are toxic to embryonic sympathetic neurons and proposed that purine and pyrimidine metabolism may play a critical role in the growth and development of sympathetic neurons. To extend this hypothesis further, we examined the effects of these nucleosides on two other neuronal populat...
Article
An increase in the release of norepinephrine (NE) when an identical number of stimulating pulses are delivered at increasing frequencies was first observed in perfused spleen of a cat. A classic agent used to increase neuronal action potential duration is tetraethylammonium (TEA), which selectively inhibits repolarizing K+ currents. The use of cell...
Article
Chronic activation of protein kinase C (PKC) has been implicated in regulation of Ca2+ entry responsible for normal development of transmitter properties in cultured sympathetic neurons. The idea that PKC alters the expression of Ca2+ channels was tested using phorbol 12,13-dibutyrate (PDB) which activates PKC and also supports survival of chick sy...
Article
We show here that 2'-deoxyadenosine (2'-dAdo) but not adenosine was toxic to chromaffin cells of 3-4-week-old rat adrenal glands. More than 75% of the cells plated in culture gradually died over a 3-day period in the presence of 100 microM 2'-dAdo plus 3 microM deoxycoformycin (DCF). Morphological observations together with bisbenzimide staining an...
Article
Increasing evidence suggests that adenosine and ATP not only modulate cell growth and differentiation, but may also act as inducers of cell death. In the session “Purines and cell death,” held during the Purines '96 Symposium and chaired by Claudio Franceschi (Modena, Italy) and Geoffrey Burnstock (London, UK) presentation and discussion of new stu...
Article
We demonstrate that 1-methyl-4-phenylpyridinium (MPP+) is toxic to chick peripheral sympathetic neurons maintained in culture in the presence of nerve growth factor (NGF). When MPP+ was added to the culture medium at the time the neurons were plated, cell loss after 3 days in culture was evident at concentrations as low as 3 nM, and near maximal at...
Article
These experiments characterize the nucleoside transport and quantify the neurotoxicity of adenosine and 2'-deoxyadenosine (dAdo) in chick sympathetic neurons. We show that [3H]adenosine transport was sensitive to low temperature, specific inhibitors of nucleoside transport, and an excess concentration of adenosine. However, many of these treatments...
Article
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 accumu...
Article
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) In...
Article
The hypothesis that multiple trophic inputs are essential for normal development of transmitter release properties in sympathetic neurons was tested using two supportive agents (excess KCl and phorbol 12,13-dibutyrate which produce marked activation of protein kinase C and also support survival of chick sympathetic neurons in culture) in addition t...
Article
Full-text available
Pituitary adenylate cyclase-activating polypeptide (PACAP) is the most potent non-cholinergic neurotransmitter to stimulate catecholamine secretion from rat chromaffin cells; however, the mechanism of action is not clear. We used amperometric detection of exocytosis and indo-1 monitoring of [Ca] to identify PACAP actions in cultured chromaffin cell...
Article
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 nucl...
Article
1. The contribution of target cells in controlling the functional properties of sympathetic neurons was investigated using pure neuronal cultures and co-cultures of neurons with their physiological target cells. 2. Chick embryo sympathetic neurons cultured alone exhibited maximal elevation of cytosolic free Ca2+ ([Ca2+]i) and release of tritiated n...
Article
1. A newly found action of adenosine in neurons, which may have an important physiological function in the growth and development of the sympathetic nervous system, is described. Adenosine (1-100 microM) inhibited neurite outgrowth within the first 24 h and killed about 80% of sympathetic neurons supported by nerve growth factor over the next 2 day...
Article
The stimulated elevation of [Ca2+]i can either promote neuronal survival or lead to Ca(2+)-mediated neurotoxicity. Similarly, growth cone mobility and neurite outgrowth may be promoted or arrested by elevated [Ca2+]i. We examined survival, development and transmitter release properties of chick sympathetic neurons maintained in culture medium conta...
Article
Postmitotic sympathetic neurons are known to undergo a programmed cell death (apoptosis) when they are deprived of nerve growth factor (NGF) or treated with arabinofuranosyl nucleoside antimetabolites. Here we report the existence of a biochemical mechanism for the induction of neuronal death by an endogenous nucleoside in the presence of NGF. In s...
Article
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 det...
Article
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 transmit...
Article
A single stimulus applied to sympathetic neurons co-cultured with cardiac cells produced a very small increase in the release of tritiated norepinephrine and intracellular free Ca2+ concentration ([Ca2+]i), but evoked a typical neuronal action potential. Treatment with 10 mM tetraethylammonium caused a dramatic increase in the responses to a single...
Article
Entry of Ca2+ through voltage-dependent Ca2+ channels is known to be linked to the exocytotic release of transmitter from sympathetic neurons. In this paper we provide evidence that transmitter release can also be stimulated by Ca2+ influx via the Na-Ca exchanger. Furthermore, the release linked to Na-Ca exchange is regulated by cardiac target cell...
Article
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⁺]) and tran...
Article
Full-text available
The concentration of cytosolic free Ca2+ ([Ca2+]i) and the release of tritiated norepinephrine ([3H]NE) were monitored during Ba2+ stimulation of sympathetic neurons cultured from chick embryos. Ba2+ (2.5 mM in Ca(2+)-free medium) caused a rise in [Ca2+]i in all regions (cell bodies, neurites, and growth cones) of sympathetic neurons and evoked [3H...
Article
A study of the effects of dihydropyridine Ca2+ channel modulators on the release of catecholamines from perfused rat adrenal glands, evoked by electrical stimulation of their splanchnic nerves, is presented. Electrically mediated secretory responses were compared to chemically mediated responses (exogenous acetylcholine, nicotine, or high K+). Inte...
Article
We 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 effi...
Article
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 splanchn...
Article
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 adr...
Article
Purification of amyloid plaque core proteins (APCP) from Alzheimer's disease brains to complete homogeneity and in high yield permitted its chemical fractionation and characterization of its components. APCP is mainly made of beta-amyloid (beta A) and an assortment of glycoproteins (accounting for 20%) rich in carbohydrates compatible with N- and O...
Article
1. We studied the relation between cyclic AMP, intracellular Ca2+ concentration and release of [3H]noradrenaline ([3H]NA) in sympathetic neurons cultured from chick embryos. 2. Forskolin (10 microM) and vasoactive intestinal polypeptide (VIP, 3 microM) increased cellular levels of cyclic AMP 8- and 3-fold, respectively, either in the absence or pre...
Article
Full-text available
Several types of extracellular signals affect the function of peripheral neurons. Depolarizing stimuli cause sudden increases in permeability to various ions leading to propagation of nerve impulses and release of transmitter substances. Neurons also receive external signals via neurotransmitter receptors located on the membrane. Different types of...
Article
Effects of the inorganic calcium channel blockers zinc, manganese, cadmium, and nickel on secretion of catecholamines from the perfused adrenal gland of the rat were investigated. Secretion of catecholamines evoked by splanchnic nerve stimulation (1 and 10 Hz) was not affected by nickel (100 microM), partially blocked (50%) by cadmium (100 microM),...
Article
Full-text available
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...
Article
The significance of the amyloid plaque core proteins (APCP) in Alzheimer's 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...
Article
The effects of various neurotransmitters on phosphoinositide hydrolysis, mobilization of Ca2+ and release of [3H]-norepinephrine ([3H]-NE) were studied in cultures of sympathetic neurons of chick embryos. [3H]-inositol-1,4,5-triphosphate ([3H]-IP3) was increased in sympathetic neurons by acetylcholine (ACh), muscarine and serotonin (5-HT). Dopamine...
Article
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...
Article
1. Phorbol esters are known to inhibit phospholipase C-mediated hydrolysis of membrane phosphoinositide. This inhibition is attributed to participation of protein kinase C (PKC) in a negative-feedback control of phosphoinositide metabolism. We have tested this hypothesis by using different types of activators and inhibitors of PKC. 2. Phorbol-12,13...
Article
Full-text available
The effects of neurotransmitters and peptides on phosphoinositide hydrolysis were studied by measuring [3H]inositol monophosphate ([3H]IP) and protein kinase C (PKC) activity in the sympathetic and sensory neuronal cultures of the chick embryo. [3H]IP was increased in sympathetic neurons by acetylcholine (ACh), muscarine, serotonin (5-HT), and vaso...
Article
Forskolin has become an invaluable tool for exploring the involvement of cyclic AMP in a variety of cellular functions. The diterpine directly activates the catalytic subunit of adenylate cyclase, causing a marked increase in cyclic AMP content. Because of this well-characterized action, practically all the observed effects of forskolin are commonl...
Article
Sensory neurons of the chick embryo are supported in culture by several neurotrophic factors, including the phorbol esters. Because phorbol esters are known to activate one of the second messengers, namely, protein kinase C, it was of interest to see if the neurotrophic action of phorbol 12,13-dibutyrate (PDB) was related to the activation of prote...
Article
Veratrine (VT), an alkaloid known to act on the sodium channels and cause depolarization of a cell membrane, was found to support the survival of cultured sympathetic neurons. At 30 microM it was as effective as nerve growth factor (NGF), as determined by the cell counts and [3H]norepinephrine ([3H]NE) uptake. Protein kinase C (PKC) activity of the...
Article
The purpose of the present study was to determine the molecular mechanism of stimulatory actions of ACh and vasoactive intestinal polypeptide (VIP) by determining the role of various second messengers in the neurohumoral secretion. Toward such a goal, we measured cAMP, cGMP, protein kinase (PKC) activity, 3H-inositol triphosphate (3H-IP3), and 45Ca...
Article
Evidence is presented for morphological proof of exocytosis in the rat adrenal medulla in situ. Techniques were modified to allow perfusion of the intact adrenal gland with secretagogues (or electrical stimulation) followed by tannic acid. Unstimulated specimens demonstrated exocytotic (omega-shaped) profiles filled with flocculent material. This f...
Article
Isolated perfused rat adrenal glands were prelabeled with 3H-norepinephrine and catecholamine secretion was evoked by nerve stimulation (10 Hz, supramaximal voltage for 30 seconds) or acetylcholine (ACh)(5.4 micrograms) injection. Nerve stimulation evoked significant increases in tritium (16371 +/- 2109 cpm) and catecholamine (11.5 +/- 1.0 ng norep...
Article
The functional behavior of embryonic chick sympathetic neurons was determined by inducing release of [3H]norepinephrine by electrical stimulation of sympathetic neurons growing in the chick heart and in culture, with and without heart cells. A very close correspondence between the functional behavior of neurons developing with the heart cells, eith...
Article
Experiments were carried out on cultured sympathetic neurons of the chick embryo; first, to demonstrate the presence of adrenergic and dopaminergic receptors, and then to see if these receptors are involved in regulation of transmitter release. We show that alpha 2-agonists, norepinephrine, epinephrine and clonidine, had no effect on neuronal cycli...
Article
The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo we...
Article
Experiments were carried out to determine the cholinergic properties of sensory neurons of the chick embryo by measuring the choline acetyltransferase activity (ChAT) and [3H]choline uptake. The choline acetyltransferase activity in the dorsal root ganglia of an 8-day-old chick embryo was 24.2 +/- 2.52, which increased to 45.4 +/- 9.69 pmol ACh/mg...
Article
The effects of phorbol esters were investigated on the survival of chick sympathetic neurons in a serum-free culture medium. The protein kinase C activator phorbol 12,13-dibutyrate (PDB) supported about 40% of the plated sympathetic neurons. This number was comparable to that supported by nerve growth factor (NGF). A combination of phorbol ester an...
Article
Noradrenergic properties of peripheral sympathetic neurons obtained from 10-12-day-old chick embryos were examined under various culture conditions. Sympathetic neurons supported by nerve growth factor in serum-free or serum-containing medium took up significant and almost equivalent amounts of [3H]norepinephrine. The uptake was markedly enhanced w...
Article
The functional integrity of adrenal chromaffin storage vesicles was studied in the perfused rat adrenal gland subjected to intense exocytosis. Continuous perfusion with 55 mM K+-Krebs solution produced a large and uninterrupted secretion of catecholamines. Total amounts secreted within 45 min were 4.66 μg and represented almost 30% of the total tis...
Article
The effects of continuous exposure of chromaffin cells to nicotine, muscarine and excess potassium on the secretion of catecholamines and uptake of radiolabeled calcium were investigated in the isolated perfused adrenal gland of the rat. Perfusion with 20 microM nicotine-Krebs solution increased catecholamine secretion from a basal value of 5 ng to...
Article
The effect of continuous stimulation of splanchnic nerves at 1, 3, and 10 Hz on the secretion of catecholamines from the isolated rat adrenal gland was examined. Secretion evoked at 10 Hz declined over 60% in 1 h, and by the end of 4 h the secretion was only 10% of the initial value. The secretion evoked at 3 Hz was unchanged in the first hour, but...
Article
We have recently shown that vasoactive intestinal polypeptide (VIP) is as potent as acetylcholine in inducing the secretion of catecholamines from the rat adrenal medulla. In the present study we have investigated the molecular mechanism involved in the exocytotic secretion of catecholamines by VIP and the effects of VIP on Ca45 uptake and phosphoi...
Article
Full-text available
The ability of nerve growth factor to cause rapid activation of the Na+K+ pump of its responsive cells was examined by measuring the uptake of 86Rb+. A significant increase in 86Rb+ uptake in E8 chick dorsal root ganglion sensory neurons after NGF treatment was seen only if the cells had been damaged during the preparation procedure. Such damaged c...
Article
Full-text available
We have recently shown that vasoactive intestinal polypeptide (VIP) is as potent as acetylcholine in inducing the secretion of catecholamines from the rat adrenal medulla. In the present study we have investigated the molecular mechanism involved in the exocytotic secretion of catecholamines by VIP and the effects of VIP on Ca45 uptake and phosphoi...
Article
1. Our previous studies have indicated that splanchnic nerves release a substance(s), other than acetylcholine, that induces the secretion of catecholamines from the rat adrenal medulla. To identify the nature of the non-cholinergic substance, the effects of met-enkephalin and vasoactive intestinal polypeptide (VIP) were investigated in the perfuse...
Article
It had previously been thought that muscarinic cholinergic receptors utilize an influx of extracellular calcium for activation of adrenomedullary catecholamine secretion. However, it has recently been demonstrated that muscarinic receptors on isolated adrenal chromaffin cells can elevate cytosolic free calcium levels in a manner independent of extr...
Article
1. Effects of nicotinic (mecamylamine) and muscarinic (atropine) receptor antagonists were investigated on the secretion of catecholamines evoked by stimulation of splanchnic nerve terminals and acetylcholine in the isolated perfused adrenal gland of the rat to determine whether non-cholinergic substances released from nerve terminals participate i...
Article
Pirenzepine, McN-A-343 and oxotremorine were used to determine the subtypes of muscarinic receptors involved in the secretion of catecholamines from the isolated perfused adrenal gland of the rat. In the presence of 0.1 microM pirenzepine, the concentration-secretion curve for muscarine was shifted in parallel to the right by almost one log unit. W...
Article
Secretion of catecholamines (CA) from the isolated rat adrenal gland during and after perfusion with hypertonic Krebs bicarbonate solution was studied. Perfusion with hypertonic solution made by adding either 156 mM sodium chloride, choline chloride, arginine hydrochloride, sodium thiocyanate, or 312 mM sucrose had no effect on the spontaneous secr...
Article
The uptake and release of catecholamines was investigated in the isolated perfused adrenal gland of the rat after preloading the preparation with [³H]norepinephrine, and the effects of various agents were examined on the stimulation-evoked secretion of catecholamines and total tritium. Large quantities of tritium were found in the adrenal medulla a...
Article
Several investigators have shown that tumour promoter phorbol esters mimic the effects of endogenous diacylglycerol to activate a second messenger, protein kinase C. These phorbol esters have proved to be valuable tools for exploring the role of protein kinase C in many cellular functions. We demonstrate here that secretion of catecholamines evoked...
Article
The question of local regulation of catecholamine secretion by alpha-adrenoceptors of either presynaptic splanchnic nerve terminals or postsynaptic chromaffin cells was investigated in the isolated perfused adrenal gland of the rat. The most selective agonists and antagonists of alpha-adrenoceptors such as clonidine, phenylephrine, yohimbine, etc....
Article
The purpose of the present study was twofold: one, to establish the metabolic pathway responsible for the synthesis of ATP of chick embryonic sympathetic neurons maintained in culture and supplemented with nerve growth factor; two, to establish the relationship between ATP content and the uptake and storage of [3H]norepinephrine. We show that ATP c...
Article
The effect of gallamine on spontaneous and stimulation-evoked overflow of tritium was studied in the submandibular gland of the rat. The gland was perfused retrogradely and labeled with3H-noradrenaline. The stimulation-evoked (1 Hz for 60 s) overflow of tritium was facilitated by increasing concentrations of gallamine (0.3–20 mM). None of the conce...
Article
A culture medium circulated through the rat heart and supplemented with insulin, transferrin and nerve growth factor leads to a massive proliferation of neurite outgrowth from neurons of peripheral sympathetic ganglia of the chick embryo. Addition of 1 mM cyanide or 50 microM dinitrophenol to such medium for 2 days had no adverse effect on the neur...
Article
The effect of phorbol ester, phorbol 12,13-dibutyrate, was investigated on the overflow of tritium from 3H-noradrenaline-loaded sympathetic neurons of the isolated perfused salivary gland of the rat. Stimulation (1 Hz for 60 s)-evoked overflow of tritium was enhanced by phorbol ester. A significant enhancement was seen at 1 nmol/l, which increased...