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J. Biol. Chem., Vol. 278, Issue 34, 32058-32067, August 22, 2003
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From the Department of Medicine and Center for Molecular Genetics, University of California, and Veterans Affairs San Diego Healthcare System, San Diego, California 92161, and the Department of Medicine, University of Massachusetts, Worcester, Massachusetts 01655
Received for publication, May 27, 2003 , and in revised form, June 5, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chromogranin A, the major soluble protein in the core of amine and peptide hormone and neurotransmitter secretory vesicles (2, 3), plays both intracellular and extracellular roles. Within the catecholamine storage vesicle, chromogranin A plays a necessary role in vesiculogenesis and the ability to conduct regulated catecholamine secretion (4). Its extracellular roles derive from its biologically active proteolytic cleavage fragments (3): the catecholamine release-inhibitory fragment catestatin (bovine chromogranin A344–364) (1), the vasodilator vasostatin (bovine chromogranin A1–76) (5), and the dysglycemic peptide pancreastatin (porcine chromogranin A240–288) (6).
When secretory stimuli (such as acetylcholine interacting with nicotinic cholinergic receptors) trigger transmitter release from chromaffin cells or sympathetic axons, is the resynthesis of just released transmitters also initiated by the secretory stimulus? We have characterized this process (sometimes called "stimulus-secretion-synthesis coupling" or "stimulus-transcription coupling") in chromaffin cells in vitro (7–9) and established transcriptional activation of chromogranin A by nicotinic cholinergic (physiologic pathway) stimulation. The process occurs at the level of transcript initiation (7), requires particular elements in cis in the proximal promoter (7), and has well defined signal transduction pathways in trans (8, 9). However, whether this nicotinic cholinergic stimulation of chromogranin A occurs in vivo is uncertain.
We therefore set out to explore whether chromaffin cell stimulus-transcription coupling (specifically nicotinic cholinergic transcriptional stimulation of chromogranin A) occurs in vivo. To test this possibility, we employed a transgenic strain in which a mouse chromogranin A 4.8-kbp proximal promoter drives the expression of firefly luciferase, an extraordinarily sensitive reporter of gene expression (10–12). In the in vivo experiments reported here, we used nicotine (mimicking the autonomic ganglionic transmitter acetylcholine) and reserpine (indirect stimulation by vesicular depletion) to evoke catecholamine release and expression of the transgene in the adrenal gland, sympathetic nerve, and brain.
At the same time, we wished to test whether the catecholamine release-inhibitory fragment catestatin (1) functions in vivo. Our previous characterization of catestatin in chromaffin cells in vitro (1, 13–16) documented its mechanism as a nicotinic cholinergic antagonist (17), thereby acting as an autocrine, negative feedback inhibitor of catecholamine release (1). However, it is not yet clear whether catestatin inhibits catecholamine release in vivo (i.e. in an intact organism).
Our in vivo findings confirm in vitro studies of nicotinic signaling to both catecholamine secretion and gene transcription, documenting the role of catestatin in both processes. The studies also provide a novel in vivo photoprobe to investigate stimulus-transcription coupling in experimental cardiovascular disease states.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation of Transgenic Mice—The mouse chromogranin A promoter/firefly luciferase reporter transgenic mouse was created by first digesting the above pXP1 mouse chromogranin A 4.8-kbp promoter/firefly luciferase reporter construct with PvuI and BamHI, to linearize and excise the promoter/reporter transgene from the vector, and then purifying the linear transgene for microinjection using Sepharose gel separation, CsCl density gradient centrifugation, and dialysis against microinjection buffer (20). The transgene also contains an SV40 polyadenylation signal and SV40 3'-untranslated sequence, containing an SV40 small T intron (splice donor and acceptor sites) (Fig. 1A). The male pronucleus of fertilized eggs from the FVB inbred strain (Taconic Farms Inc., Germantown, NY) was used for microinjection of the transgene. The presence of the transgene was evaluated by either Southern blot or polymerase chain reaction. The transgenic animals were then inbred to homozygosity.
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mRNA Abundance by RT1-PCR—Total RNA was prepared from freshly dissected mouse tissues with the RNeasy minikit (Qiagen, Valencia, CA), followed by RNase-free DNase I (Qiagen) treatment (to eliminate any residual genomic DNA). Integrity of the RNA was confirmed by the appearance of 28 s and 18 S rRNA bands on ethidium bromide-stained gels. RT-PCR was performed using PTC-200 DNA Engine thermal cyclers (MJ Research, Watertown, MA), employing a Qiagen one-step RT-PCR kit and the following primer pairs (NCBI accession numbers): mouse chromogranin A mRNA (NM_007693 [GenBank] ), forward chromogranin A 256–278 (5'-AAGTGCGTCCTGGAAGTCATCTC-3') and reverse chromogranin A 859–840 (5'-GCTTGGCTTTTCTGGCTTGC-3'); firefly luciferase mRNA (M15077 [GenBank] ), forward firefly 603–626 (5'-TACTGGGTTACCTAAGGGTGTGGC-3') and reverse firefly 1002–982 (5'-TGGAAGATGGAAGCGTTTTGC-3'); mouse cyclophilin mRNA (X52803 [GenBank] ), forward cyclophilin 232–255 (5'-GTGGTGACTTTACACGCCATAATG-3') and reverse cyclophilin 488–467 (5'-ATTCCTGGACCCAAAACGCTCC-3').
First strand cDNA was prepared from 500 ng of total RNA template by reverse transcription (using OmniscriptTM and SensiscriptTM reverse transcriptases) at 54 °C for 30 min, followed by PCR. The PCR protocol began with a 95 °C/15-min step (for simultaneous inactivation of the reverse transcriptases and activation of the HotstarTaqTM DNA polymerase), followed by a three-step amplification profile (94 °C denaturing step for 30 s, 55 °C annealing step for 30 s, and 72 °C extension step for 1 min) for 25 cycles, 72 °C for 10 min, and finally holding at 4 °C. The reaction mixture was composed of 10 µl of 5x RT-PCR buffer, 2 µl of dNTP mix (each at 10 mM), 2 µl each of forward and reverse primers (each at 10 µM), 2 µl of RT-PCR enzyme mix, 0.2 µl of RNasin® ribonuclease inhibitor (at 40 units/µl; Promega), and 5–15 µl (depending on the concentration) of the template RNA and RNase-free water to achieve the final desired total volume (50 µl/amplification).
After PCR, the products were visualized/photographed on 1.5% agarose gels by ethidium bromide staining/310-nm UV fluorescence. PCR products were then purified with the QIAquick PCR purification kit (Qiagen, Valencia, CA), and amplification of the correct targets was verified by DNA sequencing.
As a negative control, when RNA was pretreated with RNase A (Qiagen), no product in the RT-PCR assay was detected after gel electrophoresis. As a second negative control, no PCR product was obtained when water was included instead of RNA samples in the reaction mixture.
Drug Treatments and Tissue Harvesting—The drugs used were
nicotine (2.5 mg/kg intraperitoneally; Sigma), the vesicular monoamine
transporter inhibitor reserpine (2.5 mg/kg intraperitoneally; Sigma), the
neuronal nicotinic cholinergic antagonist chlorisondamine (5 mg/kg
intraperitoneally; Tocris-Cookson), the novel nicotinic antagonist catestatin
(20 nmol/25-g mouse intraperitoneally), or vehicle. Catestatin (bovine
chromogranin A344–358; RSMRLSFRARGYGFR) was synthesized and
then purified to >95% homogeneity by RP-HPLC; the identity and purity of
the final product were verified by re-HPLC and matrix-assisted laser
desorption ionization mass spectrometry. The catestatin dose (20 nmol/25-g
mouse) was calculated so as to achieve a concentration of 
4
µM in the extracellular space (estimated as 20% of body
weight).
In some studies, stimulation of sympathetic outflow was undertaken (directly by nicotine; indirectly by reserpine or vehicle). In dose- and time-dependent studies, reserpine at several doses (1, 5, and 10 mg/kg intraperitoneally) versus mock was injected for 4 and 18 h. In other studies, animals were first pretreated with either a nicotinic cholinergic antagonist (chlorisondamine (5 mg/kg intraperitoneally) or catestatin (20 nmol/25 g intraperitoneally)) or vehicle. 30 min later, the sympathetic stimulant was administered: nicotine (2.5 mg/kg intraperitoneally), reserpine (2.5 mg/kg intraperitoneally), or vehicle.
For studies of the effects of the stimulants on plasma catecholamines, mice were treated with drugs (versus vehicle) as noted above, and 30 min later they were anesthetized intraperitoneally with a rodent anesthesia mixture (ketamine (60 mg/kg of body weight); xylazine (6.4 mg/kg of body weight); acepromazine (1.2 mg/kg of body weight)). After anesthesia was achieved (1–2 min), blood was then collected from the left ventricle into Eppendorf tubes containing acid/citrate/dextrose as anticoagulant. Blood was kept on ice and centrifuged, and the plasma stored at –70 °C until the assay. Plasma catecholamines were assayed by a sensitive radioenzymatic method (21).
In studies of transcriptional activation of the chromogranin A/luciferase transgene, tissues were obtained from control or drug-treated mice after sacrifice by cervical dislocation, typically 16 h after administration of the drug (nicotine, reserpine, or vehicle) perturbing chromogranin A biosynthesis.
Quantitative Assay of the Chromogranin A/Luciferase Transgene Enzymatic Activity—Freshly dissected tissue samples were collected in 500 µl of ice-cold lysis buffer (0.1 M potassium phosphate buffer, pH 7.8, 1 mM dithiothreitol, 0.1% Triton X-100) and briefly (15–30 s, depending on the volume of the tissue) homogenized by a Tissuemizer homogenizer with a TR-10 power control device (Tekmar, Cincinnati, OH) set at 50% power output. Typical buffer homogenization volumes were 0.25 ml/adrenal gland, and 1.0 ml/brain. The homogenate was centrifuged twice at 14,000 rpm for 30 min at 4 °C, collecting the supernatant in a fresh microcentrifuge tube after the first spin.
100 µl of the clear lysates were then placed in clear plastic polystyrene 12 x 47-mm tubes in an ultrasensitive, low noise luminometer with a 12-watt Peltier-cooled (8 °C) photomultiplier tube, a dark count rate of <100 counts/s (even at 20 °C), spectral sensitivity of 390–620 nm, 20-ns resolution, and quantum efficiency of 24% (AutoLumat LB 953, EG&G Berthold, Bad Wildbad, Germany) with AutoLumat-PC-Control software in a personal computer running DOS on an Intel Celeron RAM chip. 100 µl of the luciferase assay buffer (final concentrations: 100 mM Tris acetate, pH 7.8, 10 mM magnesium acetate, 1 mM EDTA, 0.1 mM luciferin substrate, 3 mM ATP cofactor) were injected to each tube, and flash luminescence was recorded for 10 s and saved in a Kaleidagraph spreadsheet in a personal computer. 3–10 µl (depending on the tissue type) of the tissue lysates were used for total protein measurement (22), by a Coomassie Brilliant Blue dye binding assay reagent (Bio-Rad). Luciferase activities in the various tissues were normalized to protein concentration and expressed as mean relative light units (RLU) ± S.E. One RLU represents 10 pulses released by light quanta from the photon counter. Assay blanks were obtained by measuring luciferase activity in buffer alone (without tissue homogenate).
Data Analysis and Statistics—Data are reported as the mean value ± one S.E. When only two conditions (e.g. control and experimental) were compared, the data were evaluated by unpaired t tests. When multiple conditions were compared, we used one-way analysis of variance, followed by the Dunnett multiple comparison post hoc test, if appropriate. Statistical significance was concluded at p < 0.05. Statistics were computed with the programs InStat (GraphPad Software, San Diego, CA), SPSS (Statistical Package for the Social Sciences, Chicago, IL), or Kaleidagraph (Synergy/Abelbeck Software, Reading, PA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Assay of chromogranin A/luciferase in the adrenal gland of transgenic mice
showed luciferase expression more than 1000 times background
(nonspecific) luminescence in control adrenal glands
(Fig. 2A).
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In agreement with the typical cell type-specific pattern of expression of the endogenous chromogranin A gene (23), we found that the chromogranin A-luciferase transgene was expressed in endocrine (adrenal » pituitary > testis > ovary > pancreas) and neuronal tissues (brain stem, hippocampus, frontal cortex, thalamus, olfactory bulb > striatum, hypothalamus, cerebellum) but not in control tissues (liver, spleen) (Fig. 2B).
Tissue Distribution of Transgene and Endogenous Chromogranin A
Transcripts: RT-PCR
To directly compare expression of the two forms of the chromogranin A
promoter (endogenous gene versus transgenic), we employed RT-PCR in
the same tissues (Fig. 3).
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Consistent with the luminescence data (Fig. 2B), both the endogenous chromogranin A gene (chromogranin A mRNA) and the chromogranin A/luciferase transgene (luciferase mRNA) were expressed in endocrine (adrenal > gut > pituitary > testis, ovary) and neuronal tissues (brain > vas deferens). The endogenous and transgenic mRNAs displayed similar rank orders of expression in these neuroendocrine sites. There was no detectable expression of either endogenous or transgenic mRNAs in control tissues (skeletal muscle, liver, spleen, heart). Comparability of mRNA load per lane was confirmed by RT-PCR of a "housekeeping" transcript, cyclophilin.
Expression of the Chromogranin A-Luciferase Transgene during
Ontogenetic Development: Brain and Adrenal Gland
Previous in situ hybridization studies on the ontogeny (day E16 to
P9) of chromogranin A mRNA in rat brain revealed expression in the
intermediate cortical layer and dentate gyrus by day E16
(24). In the present study, we
detected expression of the chromogranin A/luciferase transgene as early as day
E6 (Fig. 4A). A steady
increase in expression of the transgene in brain was seen up to day E18, with
a subsequent abrupt decline after birth (day P1) and then a further slow
decline up to day P30, after which expression stabilized up to day P360
(Fig. 4A). In
contrast, expression of the transgene in the adrenal gland displayed a
substantial increment from day E18 to P1 but was followed thereafter by only
20% fluctuations up to day P360 (Fig.
4B).
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Direct Nicotinic Cholinergic Stimulation of the Sympathoadrenal
System: Acute Transmitter Release, Subsequent Adrenal Stimulus-Transcription
Coupling, and Role of Catestatin in Vivo
Acute Transmitter Release—Direct activation of nicotinic
cholinergic receptors by nicotine caused acute (30 min) 2.7-fold release
of catecholamines (norepinephrine and epinephrine) from storage vesicles into
the bloodstream (Fig. 5). When
animals were pretreated with the classical neuronal nicotinic antagonist
chlorisondamine, the catecholamine increment was blunted by >80%,
confirming specific mediation of the response by nicotinic receptors.
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Stimulus-Transcription Coupling—Chromogranin A
(25) and its catestatin
fragment (14) are co-released
by exocytosis with catecholamines. In cultured chromaffin cells in
vitro
(7–9),
exocytotic stimuli also program the resynthesis of just released catecholamine
storage vesicle contents, a process known as
"stimulus-secretion-synthesis coupling" or
"stimulus-transcription coupling." Does this phenomenon occur
in vivo? Treatment with nicotine resulted in a 2-fold increment
in expression of the chromogranin A/luciferase transgene, confirming the
phenomenon in vivo, and the increment was >80% inhibited by the
classical nicotinic cholinergic antagonist chlorisondamine
(Fig. 6A).
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Catestatin in Vivo—The chromogranin A fragment catestatin (1), a novel nicotinic cholinergic antagonist stored and released with catecholamines by exocytosis (14), also blocked the catecholamine secretory response to nicotine by >80% (Fig. 5), indicating that this peptide functions as a nicotinic antagonist upon secretion in vivo, extending its role as an autocrine, negative feedback inhibitor of catecholamine release (1). Furthermore, catestatin also blocked the transcriptional response to nicotine by >90% (Fig. 6A), establishing an entirely new role for the peptide on gene expression in vivo.
Stimulus-Transcription Coupling in Brain after Direct Nicotinic
Cholinergic Stimulation
Besides stimulation of neurotransmitter release, nicotine triggers
expression of genes encoding enzymes involved in neurotransmitter synthesis
such as tyrosine hydroxylase
(26), neurotransmitter
transporters such as the vesicular acetylcholine transporter
(27), neuropeptides such as
neuropeptide Y (28), and
transcription factors such as c-Fos and the cAMP-response element-binding
protein (29).
Nicotinic stimulation elevated brain expression of the chromogranin A
transgene by 26% (Fig.
6B). This increment was blocked 70% by the classical
nicotinic antagonist chlorisondamine and 80% by the novel nicotinic
antagonist catestatin.
Indirect Stimulation of the Sympathoadrenal System by Vesicular
Depletion: Initial Transmitter Release, Subsequent Adrenal
Stimulus-Transcription Coupling, and Role of Catestatin in Vivo
Initial Transmitter Release and Catestatin—Vesicular
monoamine transporter (VMAT) inhibition by reserpine causes an initial release
of stored catecholamines in vitro
(30) and in vivo
(31), followed by a prolonged
inhibition of catecholamine uptake into storage granules in the adrenal
medulla (32) and noradrenergic
nerves (33,
34). Treatment with reserpine
(Fig. 7) caused acute
catecholamine secretion, to plasma levels 30% greater than basal.
Pretreatment with the classical nicotinic cholinergic antagonist
chlorisondamine completely blocked the acute secretory response
(Fig. 7), suggesting that the
acute secretion might be, at least in part, mediated by reflex increments in
efferent preganglionic sympathetic (splanchnic) nerve traffic
(35,
36).
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Stimulus-transcription coupling—We also tested whether
initial depletion of stored catecholamines by reserpine, followed by reflex
stimulation of efferent sympathetic/splanchnic outflow
(35,
36), might also cause
stimulation of chromogranin A/luciferase transgene expression. Reserpine
caused time- and dose-dependent increments in transgene expression in the
adrenal gland (Fig.
8A; 4-fold stimulation at 10 mg/kg). The
reserpine-stimulated adrenal transcriptional response was blocked >70% by
the nicotinic antagonist chlorisondamine
(Fig. 8B). Thus,
prolonged catecholamine depletion seems to reflexively trigger increments in
efferent preganglionic sympathetic (splanchnic) nerve traffic
(35,
36).
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Catestatin in Vivo—Catestatin not only completely blocked depletion-induced increments of plasma catecholamines (Fig. 7) but also diminished by >60% the chromogranin A transgene activation after reserpine (Fig. 8B). Thus, catestatin in vivo seems to exert nicotinic cholinergic antagonist activity on both secretory and transcriptional processes in the sympathoadrenal system.
Stimulus-Transcription Coupling in Brain after Indirect Stimulation
by Vesicular Depletion
Depletion of neurotransmitter storage by reserpine also increases gene
expression of enzymes involved in neurotransmitter synthesis, such as tyrosine
hydroxylase (37), as well as
neurotransmitter transporters, such as the serotonin transporter
(38), and neuropeptides
including preproenkephalin
(39), preprotachykinin
(39), galanin, vasopressin
(40), neuropeptide Y
(41), chromogranin B, and
secretogranin II (40).
In the present study, transmitter depletion caused time- and dose-dependent
increments of transgene expression in the brain, with 3.3-fold
stimulation at 5 mg of reserpine/kg (Fig.
8C). The increment was blocked 30% by the classical
nicotinic antagonist chlorisondamine and 35% by the novel nicotinic
antagonist catestatin (Fig.
8D).
Stimulus-Transcription Coupling in Peripheral Postganglionic
Sympathetic Axons (vas Deferens)
The male vas deferens is a rich source of postganglionic sympathetic nerve
terminals (25,
31). Contraction of smooth
muscle in the vas deferens is elicited by sympathetic nerves co-releasing the
neurotransmitters ATP and norepinephrine from large dense core vesicles
(42,
43). Reserpine depletes
catecholamines from these vesicles
(44). Since nicotinic
cholinergic receptors are expressed on vas deferens nerve terminals
(45), nicotinic cholinergic
stimulation should evoke catecholamine and chromogranin A co-release from
these same vesicles.
In these studies of the vas deferens
(Fig. 9), vesicular depletion
by reserpine increased chromogranin A transgene expression by 1.75-fold,
whereas direct nicotinic cholinergic stimulation activated the transgene by
1.5-fold.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The 4.8-kbp mouse proximal chromogranin A promoter directs correct neuroendocrine expression of the gene in transfected neuroendocrine cells in vitro (19, 47). Does this promoter region also direct appropriate expression in vivo? The rank orders of endogenous chromogranin A mRNA and transgenic luciferase mRNA expression were across cell types (Fig. 3), indicating that the 4.8-kbp mouse chromogranin A proximal promoter fragment contains information sufficient to direct correct neuroendocrine specific expression in vivo.
Detection of the chromogranin A/luciferase transgene in testis, vas deferens, and ovary supports earlier immunohistological findings of endogenous chromogranin A expression in testis (48, 49), vas deferens (50, 51), and ovary (52, 53).
Neuroendocrine Ontogeny of Transgene Expression—Ontogenetic expression of chromogranin A has been explored previously in birds and mammals. In the chick, chromogranin A was detected in the carotid body (day E9) (54) and gizzard (day E12) (55). Chromogranin A mRNA was detected in rat brain at day E16 (24) and in rat enterochromaffin-like cells at day E18 (56). In the fetal pig, chromogranin A immunoreactivity was detected at days F24–F27 in sympathetic ganglia, days F37–F42 in chromaffinoblasts, and days F54–F56 in adrenal medullary cells, declining after day F76 and postnatally (57). In the human adrenal gland, chromogranin A was detected in chromaffin cell precursors by 9 weeks of gestation (58), whereas in the fetal lung chromogranin A was detected as early as 12–14 weeks (59). In the present study, detection of chromogranin A/luciferase transgene expression at day E6 may indicate an especially early role in embryonic development.
Stimulus-Transcription Coupling in Vivo and the Role of Nicotinic
Cholinergic Receptors—Nicotine is a powerful stimulant of the
sympathoadrenal system, causing release of catecholamines from postganglionic
sympathetic neurons and adrenal medulla
(60). Nicotine also acts
centrally to stimulate dopamine release from nigrostriatal and
mesocorticolimbic neurons and norepinephrine release from hippocampus,
cerebellum, and locus coeruleus neurons
(61–63).
In agreement with these findings as well as our previous in vitro
studies (1,
7), here we documented acute
nicotinic stimulation of catecholamine release as reflected by 2.7-fold
increments in plasma catecholamine levels
(Fig. 5).
Such nicotinic stimulation in cultured chromaffin cells in vitro (7–9) triggers the resynthesis of chromogranin A at the transcriptional level. This process of "stimulus-secretion-synthesis coupling" or "stimulus-transcription coupling" may serve to replete storage vesicles of just released transmitters (7–9).
Enkephalins (64) and
catecholamine biosynthetic enzymes (tyrosine hydroxylase, dopamine
-hydroxylase, and phenylethanolamine-N-methyltransferase) are
up-regulated in response to nicotinic cholinergic stimulation
(7,
29,
66,
67). Nicotine can also
function centrally to activate tyrosine hydroxylase in the adrenal medulla,
both transcriptionally and post-translationally
(67). Consistent with our
in vitro studies of the transfected chromogranin A promoter in
chromaffin cells
(7–9),
we documented 2-fold direct nicotinic stimulation of chromogranin
A/luciferase transgene expression in the adrenal gland
(Fig. 6A), and the
increment was blocked >80% by chlorisondamine. Three conclusions can be
drawn: 1) the stimulus-transcription mechanism for repletion of vesicular
contents postexocytosis also functions in vivo; 2) the
stimulus-transcription process is specifically triggered by the nicotinic
cholinergic receptor; 3) a 4.8-kbp proximal chromogranin A promoter is
sufficient to confer the nicotinic transcriptional response in
vivo.
Reserpine was also used to explore the stimulus-transcription process (Figs. 7, 8, 9). The mechanism of action of reserpine involves catecholamine store depletion from sympathetic nerve endings and chromaffin cells (33, 34) through reversible competitive inhibition of VMATs (69). Previous reports revealed that splanchnic denervation diminished reserpine-induced adrenal medullary catecholamine secretion and depletion (70–72), indicating efferent sympathetic neural mediation of reserpine action on chromaffin cell catecholamine release. In agreement with these reports, we found complete blockade of reserpine-stimulated catecholamine secretion by the classical nicotinic cholinergic antagonist chlorisondamine (Fig. 7). >4-fold transcriptional activation of the chromogranin A/luciferase transgene was also inhibited >70% by chlorisondamine, documenting the participation of splanchnic nerve traffic in a reflex response (Fig. 8, A and B).
Thus, both nicotine and reserpine activate not only acute catecholamine release (Figs. 5 and 7) but also chronic stimulus-transcription coupling (Figs. 6 and 8), albeit by rather different initial mechanisms. Nonetheless, each process involves an obligate role for nicotinic cholinergic receptors: in the case of nicotine itself, a direct role (Figs. 5 and 6), and an indirect, reflex role in splanchnic neurotransmission in the case of vesicular depletion by reserpine (Figs. 7 and 8).
Stimulus-Transcription Coupling in Neurons and Brain— Both nicotine (Figs. 6 and 9) and reserpine (Figs. 8 and 9) provoked increments in chromogranin A/luciferase transgene expression, in both peripheral postganglionic sympathetic axons (Fig. 9) and brain (Figs. 6B and 8, C and D). Reserpine was more powerful than nicotine in stimulating the transgene, whether in the adrenal gland (Fig. 8, A and B), peripheral noradrenergic nerve terminals (Fig. 9), or brain (Fig. 8, C and D).
In neurons, the relative degree of transgene activation by either nicotine
(1.26- to 1.5-fold) (Figs.
6 and
9) or reserpine (1.75- to
3.3-fold) (Figs. 8, C and
D, and 9) was
quantitatively similar in brain (Figs.
6B and
8, C and D)
and peripheral sympathetic nerves (Fig.
9). However, the degree of luciferase stimulation in neurons was
substantially less than the corresponding increment in the adrenal gland after
either nicotine (2-fold) (Fig.
6A) or reserpine (4-fold)
(Fig. 8, A and
B). Impaired passage of nicotine or reserpine across the
blood-brain barrier (73)
cannot easily explain the adrenal versus brain/neuron disparity in
transgene activation, since vas deferens nerve termini are certainly outside
the brain, and both nicotine and reserpine display at least some brain
penetration (73), especially
over a 16–18-h time course. Reasons for these adrenal versus
neuronal discrepancies in transgene response are not immediately apparent but
might include the well known differences in the subunit composition of
nicotinic receptors in the two sites
(17) or the marked
quantitative differences in catecholamine storage vesicle size, composition,
and abundance in chromaffin cells versus noradrenergic nerves
(25,
31,
74).
The ability of the classical nicotinic cholinergic antagonist
chlorisondamine to block stimulus-transcription coupling
(Fig. 6) also differed somewhat
by site (adrenal versus brain) and by stimulus (nicotine
versus reserpine). In the adrenal gland, both stimuli to
transcription (nicotine (Fig.
6A) and reserpine
(Fig. 8B) were blocked
>70% by chlorisondamine. By contrast, in brain only nicotine-stimulated
transcription (Fig.
6B) was substantially (>70%) blocked by
chlorisondamine, whereas reserpine-stimulated transcription
(Fig. 8D) was reduced
only 30% by chlorisondamine. Here we must consider the potential role of
the blood-brain barrier in hindering transport of chlorisondamine into the
brain; chlorisondamine's two obligate positively charged quaternary amines
(i.e. bis-quaternary amine structure), coupled with the relatively
brief (30-min) period between chlorisondamine pretreatment and subsequent
stimulus (nicotine or reserpine) administration, would diminish the
probability of effective and timely brain penetration
(73) for central nicotinic
cholinergic blockade. Indeed, other investigators
(68,
75) have found that effective
central nervous system nicotinic cholinergic blockade by parenteral
chlorisondamine may require larger doses or longer periods of time (as long as
12–21 days of pretreatment).
Role of Catestatin in Vivo—The chromogranin A biologically active fragment catestatin (e.g. bovine chromogranin A344–364) is proteolytically cleaved from chromogranin A in catecholamine storage vesicles in vivo (14), whereupon it can be co-released by exocytosis along with catecholamines (14). It then acts as a nicotinic cholinergic antagonist (17) to block catecholamine release in response to the physiological trigger, acetylcholine (1). Whereas catestatin's nicotinic cholinergic blocking actions have been extensively characterized in cultured chromaffin cells in vitro (1, 13–16), its potential actions in vivo have not been extensively explored.
Here we found that catestatin in vivo effectively blocked not only
the catecholamine secretory responses (nicotine
(Fig. 5) and reserpine
(Fig. 7)) but also the adrenal
transcriptional responses (nicotine (Fig.
6) and reserpine (Fig.
8)) to nicotinic cholinergic-mediated stimuli. In each case
(secretion or transcription), blockade by catestatin was comparable in
magnitude with blockade achieved by the classical nicotinic cholinergic
antagonist chlorisondamine. In one setting (reserpineinduced brain
transcription) (Fig.
8D), catestatin and chlorisondamine were only partially
(30–35%) effective in blocking stimulus-induced transcription. In
this setting, it is noteworthy that both chlorisondamine and catestatin are
predicted to exhibit poor penetration of the blood-brain barrier
(73) (chlorisondamine because
of its two obligate quaternary amine moieties (i.e. bis-quaternary
amine structure) and catestatin (as used here: bovine chromogranin
A344–358; RSMRLSFRARGYGFR) because of its highly cationic
nature (calculated pI of 12.7, with 33% Arg content) and relatively high
molecular weight (1861 g/mol) for a drug). Finally, catestatin is a somewhat
less potent inhibitor of catecholamine release from neurites than chromaffin
cells (1).
Conclusion and Perspectives—Thus, chromogranin A (and its catestatin fragment) seem to be at the virtual nexus of nicotinic cholinergic signaling to both secretion and transcription in vivo. The catecholamine release-inhibitory peptide catestatin seems clearly to function in vivo as a nicotinic cholinergic antagonist, blocking agonist-induced increments in both secretion (Figs. 5 and 7) and transcription (Figs. 6 and 8, B and D) in sympathochromaffin cells. These actions of exogenous catestatin clarify and put into functional perspective recent observations in humans on the inferred actions of endogenous catestatin in vivo (65), in which we observed that a diminution of plasma catestatin was associated with augmented risk of developing hypertension and increased pressor responses to environmental stressors.
These in vivo findings extend the significance of our previous in vitro studies of nicotinic signaling to catecholamine secretion and gene transcription in chromaffin cells (1, 13–16), establish a fundamental role for chromogranin A and its catestatin fragment at the nexus of nicotinic cholinergic signaling (Fig. 10), and provide a sensitive, novel in vivo photoprobe to investigate stimulus-transcription coupling in experimental cardiovascular disease states.
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Fig. 10 presents a diagram summarizing our principal findings and conclusions. Although simply a model, it does present a framework for integrating the results and formulating new hypotheses for future testing.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Medicine and Center for
Molecular Genetics, University of California at San Diego, and VASDHS (9111H),
3350 La Jolla Village Dr., San Diego, CA 92161. Tel.: 858-552-8585 (ext.
7373); Fax: 858-642-6331; E-mail:
doconnor{at}ucsd.edu.
1 The abbreviations used are: RT, reverse transcription; VMAT, vesicular
monoamine transporter; HPLC, high pressure liquid chromatography; RLU,
relative light unit(s); En, embryonic day n; Pn,
postnatal day n; Fn, fetal day n.
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