Articles in PresS. Am J Physiol Heart Circ Physiol (August 4, 2017). doi:10.1152/ajpheart.00702.2016
1 2 3
Modulation of Mesenteric Collecting Lymphatic Contractions by Sigma-1 Receptor Activation and Nitric Oxide Production
4 5 6 7
Andrea N. Trujillo, Christopher Katnik, Javier Cuevas, Byeong Jake Cha, Thomas E. Taylor-Clark, Jerome W. Breslin
8 9 10
Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida
11 12 13
Running Head: Sigma-1 Receptor and Lymphatic Contractions
14 15 16 17
This project was supported by the University of South Florida and NIH (L40 HL097863 & R01 HL098215).
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Address Correspondence to: Jerome W. Breslin, PhD Department of Molecular Pharmacology and Physiology, MDC8 University of South Florida 12901 Bruce B. Downs Blvd. Tampa, FL 33612 USA Phone: +1 (813) 974-1554 FAX: +1 (813) 974-3079 Email: [email protected]
33
Copyright © 2017 by the American Physiological Society.
34
ABSTRACT
35
Recently it was reported that a sigma receptor antagonist could reduce inflammation-
36
induced edema. Lymphatic vessels play an essential role in removing excess interstitial
37
fluid. We tested the hypothesis that activation of sigma receptors would reduce or
38
weaken collecting lymphatic contractions. We utilized isolated, cannulated rat
39
mesenteric collecting lymphatic vessels for study of contractions in response to the
40
sigma receptor agonist afobazole in the absence or presence of different sigma receptor
41
antagonists. We also investigated whether these vessels express the sigma-1 receptor
42
using RT-PCR and Western blotting, and localization of the sigma-1 receptor in the
43
collecting lymphatic wall by immunofluorescence confocal microscopy. We tested the
44
role of nitric oxide (NO) signaling using L-NAME pretreatment prior to afobazole in
45
isolated lymphatics. Lastly, we tested whether afobazole increases NO release in
46
cultured lymphatic endothelial cells using DAF-FM fluorescence as an indicator. Our
47
results show that the afobazole (50-150 µM) elevated end-systolic diameter and
48
generally reduced pump efficiency, and this response could be partially blocked with the
49
sigma-1 receptor antagonists BD 1047 and BD 1063 but not the sigma-2 receptor
50
antagonist SM-21. Sigma-1 receptor mRNA and protein were detected in lysates from
51
isolated rat mesenteric collecting lymphatics. Confocal images with anti-sigma-1
52
receptor antibody labeling suggested localization in the lymphatic endothelium.
53
Blockade of NO synthases with L-NAME inhibited the effects of afobazole. Lastly,
54
afobazole elicited increases in NO production from cultured lymphatic endothelial cells.
55
Our findings suggest that the sigma-1 receptor limits collecting lymphatic pumping
56
through a NO-dependent mechanism.
57
New and Noteworthy: Relatively little is known about the mechanisms that govern
58
contractions of lymphatic vessels. Sigma-1 receptor activation was shown to reduce the
59
fractional pump flow of isolated rat mesenteric collecting lymphatics. The sigma-1
60
receptor was localized mainly in the endothelium and blockade of nitric oxide synthase
61
inhibited the effects of afobazole.
62 63
Key
64
Endothelium, Nitric Oxide
65
Words:
Collecting
Lymphatic,
Afobazole,
Sigma-1
Receptor,
Lymphatic
66
INTRODUCTION
67
Afobazole is an anxiolytic drug that acts upon Sigma (σ) receptors, a family of
68
proteins that modulate a variety of signals within cells (11, 12, 40). The σ receptor family
69
consists of two known subtypes, σ1 and σ2. While σ1 and σ2 are highly expressed in
70
the central nervous system, they have also been found in the liver, kidney, heart, eye
71
and endocrine organs (9). The σ1 and σ2 subtypes have also been shown to
72
differentially regulate acid-sensing ion channels and voltage-gated calcium channel
73
activity (23, 55). Within cells, σ receptors localize to either the plasma membrane or to
74
intracellular membranes, and have the ability to translocate within different subcellular
75
compartments when treated with agonistic compounds (9). In the mitochondria-
76
associated membrane of the endoplasmic reticulum, σ1 receptors appear to act as
77
chaperones stabilizing the IP3 receptor, and are involved in Ca2+ transfer from the
78
endoplasmic reticulum to mitochondria (22). Many drugs used to treat Alzheimer
79
Disease or drug/alcohol dependence have moderate to high affinity for σ1 receptors (9).
80
Endogenous sphingolipids, N,N-dimethyltryptamine, and neuroactive steroids such as
81
dehydroepiandosterone can bind to σ1 receptors, but their potential roles as
82
physiological ligands remain to be determined (15, 28, 33, 42).
83
Drugs that affect intracellular free Ca2+ ([Ca2+]i) have previously been shown to
84
have mild to profound effects on the pump function of collecting lymphatics (5, 7, 14, 26,
85
38, 46). Unlike blood vessels, which have the heart as a central pump to drive flow,
86
collecting lymphatics are organized into a series of segments that contract phasically
87
and drive lymph flow (6). These segments, called lymphangions, are separated by one-
88
way luminal valves to prevent backflow of lymph, and have a smooth muscle layer that
89
expresses a unique combination of contractile proteins found in both cardiac and
90
smooth muscle (13, 35). Some researchers have adopted the term “lymphatic muscle”
91
to describe this smooth muscle layer because of these unique properties (35). Like
92
other smooth muscle cell types, contraction of lymphatic smooth muscle can be driven
93
by increases in [Ca2+]i (4, 37, 49).The phasic contractions are driven by oscillating action
94
potentials that elicit transient increases in [Ca2+]i within lymphatic smooth muscle cells
95
(24). In addition, these vessels display some degree of tone between phasic
96
contractions, which is also a Ca2+-dependent process (3, 26, 46). L- and T-type voltage
97
gated Ca2+ channels appear to have an important role in the mechanism (27), although
98
there is some controversy about this (51). Release and uptake of Ca2+ from internal
99
stores also plays an important role (46, 53). In addition, other signaling pathways, such
100
as those driven by PKC and Rho/Rho kinase, also affect the contractile mechanisms in
101
lymphatic vessels, probably by affecting the sensitivity to Ca2+ (26, 35, 47). Lastly, the
102
lymphatic endothelium can also affect lymphatic pumping. Endothelial cells may
103
respond to agonists or physical forces like shear stress, which can cause activation of
104
endothelial nitric oxide (NOS) and release of NO, which can then elicit relaxation of
105
lymphatic smooth muscle through a cGMP-dependent mechanism (16, 18-20, 25).
106
Collectively, many important details about the lymphatic contractile mechanism are
107
known, however much remain unknown, and the potential role of σ receptors in the
108
control of lymphatic pumping has not been previously explored.
109
In the current study, we investigated how afobazole affects the contractions of
110
isolated rat mesenteric collecting lymphatic vessels. We tested the hypothesis that the σ
111
receptor agonist afobazole would depress lymphatic contractions, and investigated the
112
expression and localization of the σ1 receptor in rat mesenteric collecting lymphatics.
113
The experimental model consisted of isolated, perfused collecting lymphatics from the
114
rat mesentery. This model was chosen because it allowed us to study the direct effect of
115
afobazole on lymphatic contractions without confounding influences from interstitial fluid
116
flow, and with tight control of the inflow and outflow pressures (6).
117
MATERIALS AND METHODS
118
Materials
119
Afobazole was generously provided by IBC Genarium (Moscow, Russia). BD
120
1047 dihydrobromide (cat. no. 0956), BD 1063 dihydrochloride (cat. no. 0883), SM-21
121
maleate (cat. no. 0751), and L-NAME hydrochloride (cat. no. 0665) were obtained from
122
Tocris Bioscience/R&D Systems (Minneapolis, MN). DAF-FM (4-Amino-5-Methylamino-
123
2’, 7’- Difluorofluorescein Diacetate, cat. no. D23844) was purchased from Life
124
Technologies (Carlsbad, CA). Acetylcholine Chloride (cat. no. A9101-10VL) and
125
Bradykinin (cat. no. B3259) were obtained from (Sigma-Aldrich, St. Louis, MO).
126
Crystallized bovine albumin (purified BSA; cat. no. 10856) used for preparing
127
physiological salt solutions was purchased from Affymetrix (Santa Clara, CA). For
128
blocking solutions, donkey serum (cat. no. D9663) was purchased from Sigma-Aldrich.
129
Rabbit anti-Sigma-1 Receptor (cat. no. 42-3300) was purchased from Invitrogen
130
Corporation (Camarillo, CA). Mouse anti-α/γ-smooth muscle actin (cat. no. Mab1522)
131
was purchased from Millipore (Billerica, MA). Goat anti-VE-Cadherin (cat. no. sc-6458)
132
was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Donkey anti-rabbit IgG-
133
HRP (cat. no. ab97064) was obtained from Abcam (Cambridge, MA). Alexa-488-donkey
134
anti-rabbit IgG (cat. no. A21206), Alexa-488-donkey anti-mouse IgG (A21202), Alexa-
135
555-donkey anti-goat IgG (cat. no. A21432), Alexa-647-donkey anti-mouse IgG (cat. no.
136
A31571) and Alexa Fluor® 633 hydrazide were obtained from Life Technologies (Grand
137
Island,
138
Rn01775763_g1) primers (TaqMan® Gene Expression Assays) for PCR were
139
purchased from Applied Biosystems/ThermoFisher Scientific (Waltham, MA). All other
NY).
SIGMAR1
(cat.
no.
Rn00578590_m1)
and
GAPDH
(cat.
no.
140
chemicals and reagents, unless otherwise specified, were purchased from Sigma-
141
Aldrich.
142
Animal Care and Use
143
All procedures were approved by the Institutional Animal Care and Use
144
Committee (IACUC) at the University of South Florida under protocol number
145
IS00002179. These studies were performed in accordance with the National Institute of
146
Health Guide for the Care and Use of Laboratory Animals (8th Edition, 2011) and are
147
reported here in accordance with the ARRIVE Guidelines. A total number of 42 male
148
Sprague-Dawley rats (5-9 weeks of age) were purchased from Charles River
149
(Wilmington, MA), housed in a controlled temperature (22 °C) and controlled illumination
150
(12:12 h light dark cycle) environment. After arrival, the rats were submitted to a one-
151
week acclimation period and were provided standard rat chow (2018 Teklad Global 18%
152
Protein Rodent Diet, Harlan, Indianapolis, IN) and water ad libitum. When possible,
153
multiple lymphatics or tissue samples were obtained from a single rat for different
154
experiments, in order to minimize the total number of rats utilized. All possible measures
155
were taken to minimize pain or suffering, including the administration of general
156
anesthesia prior to performing experiments (details provided in next sub-section). All
157
rats were euthanized by extending the laparotomy into the chest cavity and injecting
158
Euthasol/Somnasol (0.1 ml/450 g body weight) directly into the cardiac ventricle, in
159
accordance with American Veterinary Medical Association guidelines for the euthanasia
160
of animals.
161
Mesenteric Collecting Lymphatic Isolation
162
Collecting lymphatics were isolated as previously described (25). Briefly, rats
163
were anesthetized (i.p. ketamine and xylazine at 90 and 9 mg/kg, respectively), and
164
depth of anesthesia was checked using inter-digital pinch and palpebral blink reflex. A
165
midline laparotomy was performed, and the small intestine and mesentery were
166
exteriorized, excised, and placed in ice-cold albumin physiological salt solution (APSS:
167
NaCl, 120 mM; KCl, 4.7 mM; CaCl2·2H2O, 2 mM; MgSO4·7H2O, 1.2 mM; NaH2PO4, 1.2
168
mM; Na pyruvate, 2 mM; glucose, 5 mM; EDTA, 0.02 mM; MOPS, 3 mM and 1% BSA).
169
Rats were then immediately euthanized as indicated above. In each experiment, a
170
section of mesentery that included the terminal ileum was pinned in a dissection
171
chamber containing ice-cold APSS, and with the aid of a stereomicroscope, a collecting
172
lymphatic vessel (60–150 µm internal diameter and 0.5 cm of length) was carefully
173
dissected from surrounding adipose and connective tissue. The isolated lymphatic was
174
transferred to an isolated vessel chamber (Living Systems Instrumentation, Burlington,
175
VT) and was mounted onto two resistance-matched glass micropipettes and secured
176
with nylon thread. The chamber was transferred to an Accu-Scope® 3032 inverted
177
microscope, equipped with a halogen lamp, 10X objective and CCD camera for video
178
image acquisition (Living Systems). Intraluminal pressure was imposed using APSS-
179
filled gravity manometers that fed to each micropipette. All experiments were performed
180
with the vessel bathed in 37 °C APSS with the imposed pressure from both
181
micropipettes set at 2 cm H2O, and with a 30-45 min stabilization period to let the vessel
182
equilibrate for baseline measurements. Only lymphatic vessels that displayed phasic
183
contractions that reduced internal diameter during systole by at least 25% of the
184
diastolic diameter were considered sufficiently viable and used for these studies.
185
186
Experimental Protocols
187
After cannulation, the vessels were allowed to equilibrate for 30-45 minutes in order to
188
establish baseline contractions. Upon achieving a steady baseline pumping, the impact
189
of the σ receptor agonist afobazole on lymphatic contractions was evaluated by adding
190
it to achieve final concentrations of 50, 100 and 150 µM in the bath. These
191
concentrations were chosen based on previous work in which this range was effective
192
for inhibiting ATP-induced migration of glial cells and also mitigating acidosis-induced
193
calcium overload in neurons (11, 12). To investigate the roles of σ1 versus σ2 receptor
194
activation by afobazole, lymphatics were pretreated for 20 min with either the selective
195
σ1 receptor antagonists BD 1047 (200 nM) or BD 1063 (200 nM), or the selective σ2
196
receptor antagonist SM-21 (2 µM), prior to the addition of afobazole (50, 100, and 150
197
µM). These concentrations were also chosen based on previous work (11, 12) . The role
198
of NO signaling was investigated using the nitric oxide synthase inhibitor L-NAME (200
199
µM), which we have previously used to block NOS (8, 25). Vessel diameters were
200
tracked throughout each experiment and the bath solution was changed to Ca2+-free
201
APSS at the end of each experiment to determine the maximal passive diameter
202
(MaxD) at the same luminal pressure, 2 cm H2O. Parameters used to characterize
203
lymphatic pump function were calculated from the data (outlined below under Data
204
Analyses).
205 206
Total RNA Isolation and PCR
207
Mesenteric collecting lymphatic vessels (10 vessels per rat) were freshly isolated,
208
placed on ice, sonicated, snap frozen in 300 µl QIAzol® Lysis Reagent (Qiagen Inc.,
209
Valencia, CA) and stored at -80 °C. Total RNA was extracted using chloroform and
210
isopropanol and the samples were treated with DNase to remove genomic DNA. The
211
RNA was then purified using the RNeasy® MinElute® Cleanup Kit (Qiagen Inc.,
212
Valencia, CA) according to the manufacturer’s specifications, and the RNA pellet was
213
eluted in 14 µl of RNase-free water. RNA concentration and purity were determined
214
using the NanoVue Plus™ (GE Healthcare, Piscataway, NJ). Total RNA (50 ng) was
215
reverse-transcribed into cDNA using the ProtoScript® First Strand cDNA Synthesis Kit
216
(New England Biolabs Inc., Ipswich, MA). The cDNA was combined with TaqMan® Fast
217
Universal PCR Master Mix (Applied Biosystems/ThermoFisher Scientific) and primers
218
specific for either rat SIGMAR1 or rat GAPDH. PCR reactions were performed using a
219
10-min hold at 95 °C, 95 °C for 15 s and 60 °C for 1 min (40 cycles) using a CFX
220
Connect Real-Time System (Bio-Rad, Hercules, CA). Primer sequences (NCBI
221
Reference Sequence) used were: NM_030996.1(SIGMAR1) and NM_017008.4
222
(GAPDH). PCR reaction products were run on a 2 % agarose gel and bands were made
223
detectable with SYBR Green I nucleic acid gel stain (Life Technologies/ThermoFisher
224
Scientific, Waltham, MA) and visualized under UV, using the BioRad Chemi Doc XRS+
225
System with Quantity 1-D Analysis Software (Bio-Rad, Hercules, CA).
226
Immunoblotting Protocol
227
Approximately 10 collecting lymphatic vessel segments per rat, freshly isolated
228
from the mesentery, were placed in 200 µl 1X RIPA (4 °C) containing HALT Protease
229
and Phosphatase Inhibitor Cocktail (Pierce, Rockford, IL). The mixture was sonicated
230
twice at 4 °C for 5 s using a Fisher Sonic Dismembranator, model FB-120 (Fisher
231
Scientific, Asheville, NC) and frozen at -80 °C. Protein concentrations were determined
232
using the BCA protein assay (Pierce/Thermo Scientific, Waltham, MA) to equilibrate
233
samples. Lysate was mixed with 4X NuPage LDS sample buffer containing reducing
234
agent (Invitrogen, Grand Island, NY). Samples containing 30 µg of protein were loaded
235
into 4-20% Novex Bis-Tris gels (Invitrogen) for SDS-PAGE to separate proteins. The
236
NexusPointer prestained protein ladder (BioNexus, Oakland, CA) was used to
237
determine molecular weight vs. mobility. The proteins were transferred from the gels to
238
Immobilon-P PVDF membranes (Millipore) by wet transfer. Membranes were blocked
239
with 5% BSA in Tris-buffered saline with Tween® 20 (TBST). Primary antibody was
240
1:1000 rabbit anti-sigma 1 receptor (Invitrogen 42-3300). The secondary antibody was
241
1:10,000 donkey anti-rabbit IgG-HRP (ab97064). Bands were visualized using WestPico
242
Supersignal reagent (Pierce/Thermo Scientific) and a BioRad Chemi Doc XRS+ System
243
with Quantity 1-D Analysis Software (5 min exposure), (Bio-Rad, Hercules, CA).
244
Immunofluorescence Labeling and Confocal Microscopy
245
Rat mesenteric lymphatics were isolated and excised, and for each isolated vessel,
246
one end was mounted onto a glass micropipette filled with APSS in a custom chamber
247
for fixation and labeling as previously described (25). The other end of the lymphatic
248
was left free to float in an APSS bath, to allow for APSS to be pulled into or pushed out
249
of the vessel lumen by gently applying negative or positive pressure, respectively, on
250
the micropipette via an attached 1 ml syringe. Each vessel was fixed with 4%
251
paraformaldehyde for 10-15 min at room temperature with positive pressure in the
252
vessel lumen, followed by two 5-min washes with 100 mM glycine buffer and one 5-min
253
wash with Ca2+/Mg2+-free Dulbecco’s PBS (CMF-DPBS). Ice-cold acetone (-10 to -20
254
°C) was applied for 5 min to permeabilize the cell membranes, followed by three 5-min
255
washes with CMF-DPBS. A blocking solution consisting of 5% normal donkey serum in
256
CMF-DPBS was applied for 30 min at room temperature, followed by overnight
257
incubation with combinations of primary antibodies in antibody dilution buffer (151 mM
258
NaCl, 17 mM trisodium citrate, 2% donkey serum, 1% BSA, 0.05% Triton X-100, 0.02%
259
NaN3) at 4 °C: 1:3000 mouse anti-α/γ-smooth muscle actin (Mab1522); 1:50 goat anti-
260
VE-cadherin (sc-6458); and 1:60 rabbit anti-sigma 1 receptor. Labeling controls
261
received antibody dilution buffer containing no primary antibody. After overnight
262
incubation, three 10-min rinses with antibody wash solution (151 mM NaCl, 17 mM
263
trisodium citrate, 0.05% Triton X-100) were performed. The vessels were incubated for
264
30-60 min at room temperature with antibody dilution buffer containing secondary
265
antibodies: 1:400 Alexa-488-donkey anti-rabbit IgG (A21206), 1:400 Alexa-488-donkey
266
anti-mouse IgG (A21202), 1:400 Alexa-555-donkey-anti-goat IgG (A21432) and 1:400
267
Alexa-647-donkey anti-mouse IgG (A31571). Alexa Fluor® 633 hydrazide (1.0 μM) was
268
also added during this time period to label extracellular matrix (ECM) elastin fibers (10,
269
41) in some of the whole mounts. Three 10-min rinses with antibody wash solution were
270
performed, and each vessel was removed from its cannula, placed on a glass slide with
271
a Secure-Seal™ imaging spacer in 20 µl of Prolong Gold Anti-Fade reagent containing
272
DAPI (Life Technologies) or VECTASHIELD® with DAPI (Vector Laboratories, Inc.,
273
Burlingame, CA) and covered with a #1 glass coverslip. Care was taken to keep the
274
lymphatic vessel lumen patent for better view of vessel structure. Confocal z-stack
275
images (step size = 0.5-0.75 μm) of the vessels were obtained with an Olympus FV1200
276
spectral inverted laser scanning confocal microscope, and a 60X (PLAPON60XOil,
277
1.42NA or 1.30NA) objective with 1.3-1.5X zoom at the Lisa Muma Weitz Advanced
278
Microscopy and Cell Imaging Core at the University of South Florida. Imaris software
279
(Bitplane, Concord, MA) was used to produce 3D models of z-stacks for interpretation.
280
FIJI/ImageJ open source imaging software (http://fiji.sc) was also used to view and
281
process confocal image stacks into the figures.
282
Cell Culture
283
Primary human dermal lymphatic endothelial cells (HDLEC-juvenile) (PromoCell,
284
Heidelberg, Germany) were seeded onto gelatin-coated, (0.75%) 100-mm culture
285
dishes. Cells were routinely maintained in Endothelial Cell Growth Medium2-MV
286
(EGM2-MV) (Lonza, Walkersville, MD) with medium changed every 48 h. For all
287
experiments, passage 2-3 endothelial cells were used. For NO imaging studies,
288
confluent endothelial monolayers were washed with 1X DPBS, incubated in trypsin-
289
EDTA, 0.25%, neutralized with EGM2-MV and pelleted at 2000 rpm/min. Resuspended
290
endothelial cells were seeded on 18mm round microscope cover glasses coated with
291
gelatin, (0.75%) at a density of 0.1 x 106 cells and grown to 70-80% confluency.
292 293
Nitric Oxide Imaging Measurements
294
Changes in intracellular NO concentrations were measured in cultured primary
295
endothelial cells using fluorescent imaging techniques and the NO sensitive dye DAF-
296
FM. Cells were loaded using membrane permeable DAF-FM. Cells seeded on
297
coverslips (as above) were incubated for 1 hour at 37˚ in HBSS (NaCl, 135 mM; KCl, 5
298
mM; CaCl2, 1.5 mM; MgCl2, 1 mM; glucose, 10 mM; HEPES, 10 mM pH 7.4) with 8 μM
299
DAF-FM and 0.8 % dimethyl sulfoxide (DMSO). The coverslips were washed in DAF-
300
FM-free HBSS prior to experiments being performed. Cells were illuminated with 495
301
nm light for 400 msec at 0.3 Hz (Lambda DG-4, Sutter Instruments, Novato CA) and
302
fluorescent emissions at 535 nm were collected using a Sensicam digital CCD camera
303
(Cooke Corp., Auburn Hills, MI). Imaged cells were perfused with control solution and
304
solutions containing 150 μM afobazole, 10 μM Acetylcholine or 1 μM bradykinin using a
305
perfusion system consisting of 250 μm diameter glass tubes positioned 500 μm away
306
with flow rates of 300 μl/min.
307
Data Analyses
308
For the lymphatic pumping studies, datasets of lymphatic diameter over time,
309
before and after various interventions such as pressure steps or addition of drugs, were
310
used for analysis. Parameters used to characterize lymphatic pump function were
311
determined from the lymphatic intraluminal diameter measurements in the same
312
manner as previously described (16, 26). These include contraction frequency (CF), end
313
diastolic diameter (EDD), end systolic diameter (ESD), amplitude of contraction (AMP =
314
EDD - ESD), ejection fraction (EF) = (EDD2-ESD2)/(EDD2), and fractional pump flow
315
(FPF; FPF = CF X EF). EDD, ESD, and AMP were normalized to MaxD to account for
316
variability in the resting diameter of different lymphatics. Baseline data for all drug
317
experiments were the averages for the 2-min period prior to drug administration. For
318
each concentration of afobazole, the 2-min increment starting 4 min post addition was
319
used to calculate the mean data (i.e. means represent 4-6 min after addition of each
320
concentration and each concentration was held for 10 min). These time points after
321
afobazole administration represented the peak responses. When σ receptor antagonists
322
or L-NAME were added, we utilized the last 2-min period just prior to the addition of
323
afobazole to calculate the mean parameters due to any effects of the antagonist by
324
itself.
325
Summarized data are presented as mean ± SE. For all experiments, the
326
hypothesis tested was that the various experimental interventions would produce a
327
difference in the baseline pumping parameters just before afobazole was added, which
328
served as controls. GraphPad Prism 6 software was used for statistical analyses
329
(GraphPad Software, LaJolla, CA) with the threshold for significance set at P
1 2 3
Modulation of Mesenteric Collecting Lymphatic Contractions by Sigma-1 Receptor Activation and Nitric Oxide Production
4 5 6 7
Andrea N. Trujillo, Christopher Katnik, Javier Cuevas, Byeong Jake Cha, Thomas E. Taylor-Clark, Jerome W. Breslin
8 9 10
Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida
11 12 13
Running Head: Sigma-1 Receptor and Lymphatic Contractions
14 15 16 17
This project was supported by the University of South Florida and NIH (L40 HL097863 & R01 HL098215).
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Address Correspondence to: Jerome W. Breslin, PhD Department of Molecular Pharmacology and Physiology, MDC8 University of South Florida 12901 Bruce B. Downs Blvd. Tampa, FL 33612 USA Phone: +1 (813) 974-1554 FAX: +1 (813) 974-3079 Email: [email protected]
33
Copyright © 2017 by the American Physiological Society.
34
ABSTRACT
35
Recently it was reported that a sigma receptor antagonist could reduce inflammation-
36
induced edema. Lymphatic vessels play an essential role in removing excess interstitial
37
fluid. We tested the hypothesis that activation of sigma receptors would reduce or
38
weaken collecting lymphatic contractions. We utilized isolated, cannulated rat
39
mesenteric collecting lymphatic vessels for study of contractions in response to the
40
sigma receptor agonist afobazole in the absence or presence of different sigma receptor
41
antagonists. We also investigated whether these vessels express the sigma-1 receptor
42
using RT-PCR and Western blotting, and localization of the sigma-1 receptor in the
43
collecting lymphatic wall by immunofluorescence confocal microscopy. We tested the
44
role of nitric oxide (NO) signaling using L-NAME pretreatment prior to afobazole in
45
isolated lymphatics. Lastly, we tested whether afobazole increases NO release in
46
cultured lymphatic endothelial cells using DAF-FM fluorescence as an indicator. Our
47
results show that the afobazole (50-150 µM) elevated end-systolic diameter and
48
generally reduced pump efficiency, and this response could be partially blocked with the
49
sigma-1 receptor antagonists BD 1047 and BD 1063 but not the sigma-2 receptor
50
antagonist SM-21. Sigma-1 receptor mRNA and protein were detected in lysates from
51
isolated rat mesenteric collecting lymphatics. Confocal images with anti-sigma-1
52
receptor antibody labeling suggested localization in the lymphatic endothelium.
53
Blockade of NO synthases with L-NAME inhibited the effects of afobazole. Lastly,
54
afobazole elicited increases in NO production from cultured lymphatic endothelial cells.
55
Our findings suggest that the sigma-1 receptor limits collecting lymphatic pumping
56
through a NO-dependent mechanism.
57
New and Noteworthy: Relatively little is known about the mechanisms that govern
58
contractions of lymphatic vessels. Sigma-1 receptor activation was shown to reduce the
59
fractional pump flow of isolated rat mesenteric collecting lymphatics. The sigma-1
60
receptor was localized mainly in the endothelium and blockade of nitric oxide synthase
61
inhibited the effects of afobazole.
62 63
Key
64
Endothelium, Nitric Oxide
65
Words:
Collecting
Lymphatic,
Afobazole,
Sigma-1
Receptor,
Lymphatic
66
INTRODUCTION
67
Afobazole is an anxiolytic drug that acts upon Sigma (σ) receptors, a family of
68
proteins that modulate a variety of signals within cells (11, 12, 40). The σ receptor family
69
consists of two known subtypes, σ1 and σ2. While σ1 and σ2 are highly expressed in
70
the central nervous system, they have also been found in the liver, kidney, heart, eye
71
and endocrine organs (9). The σ1 and σ2 subtypes have also been shown to
72
differentially regulate acid-sensing ion channels and voltage-gated calcium channel
73
activity (23, 55). Within cells, σ receptors localize to either the plasma membrane or to
74
intracellular membranes, and have the ability to translocate within different subcellular
75
compartments when treated with agonistic compounds (9). In the mitochondria-
76
associated membrane of the endoplasmic reticulum, σ1 receptors appear to act as
77
chaperones stabilizing the IP3 receptor, and are involved in Ca2+ transfer from the
78
endoplasmic reticulum to mitochondria (22). Many drugs used to treat Alzheimer
79
Disease or drug/alcohol dependence have moderate to high affinity for σ1 receptors (9).
80
Endogenous sphingolipids, N,N-dimethyltryptamine, and neuroactive steroids such as
81
dehydroepiandosterone can bind to σ1 receptors, but their potential roles as
82
physiological ligands remain to be determined (15, 28, 33, 42).
83
Drugs that affect intracellular free Ca2+ ([Ca2+]i) have previously been shown to
84
have mild to profound effects on the pump function of collecting lymphatics (5, 7, 14, 26,
85
38, 46). Unlike blood vessels, which have the heart as a central pump to drive flow,
86
collecting lymphatics are organized into a series of segments that contract phasically
87
and drive lymph flow (6). These segments, called lymphangions, are separated by one-
88
way luminal valves to prevent backflow of lymph, and have a smooth muscle layer that
89
expresses a unique combination of contractile proteins found in both cardiac and
90
smooth muscle (13, 35). Some researchers have adopted the term “lymphatic muscle”
91
to describe this smooth muscle layer because of these unique properties (35). Like
92
other smooth muscle cell types, contraction of lymphatic smooth muscle can be driven
93
by increases in [Ca2+]i (4, 37, 49).The phasic contractions are driven by oscillating action
94
potentials that elicit transient increases in [Ca2+]i within lymphatic smooth muscle cells
95
(24). In addition, these vessels display some degree of tone between phasic
96
contractions, which is also a Ca2+-dependent process (3, 26, 46). L- and T-type voltage
97
gated Ca2+ channels appear to have an important role in the mechanism (27), although
98
there is some controversy about this (51). Release and uptake of Ca2+ from internal
99
stores also plays an important role (46, 53). In addition, other signaling pathways, such
100
as those driven by PKC and Rho/Rho kinase, also affect the contractile mechanisms in
101
lymphatic vessels, probably by affecting the sensitivity to Ca2+ (26, 35, 47). Lastly, the
102
lymphatic endothelium can also affect lymphatic pumping. Endothelial cells may
103
respond to agonists or physical forces like shear stress, which can cause activation of
104
endothelial nitric oxide (NOS) and release of NO, which can then elicit relaxation of
105
lymphatic smooth muscle through a cGMP-dependent mechanism (16, 18-20, 25).
106
Collectively, many important details about the lymphatic contractile mechanism are
107
known, however much remain unknown, and the potential role of σ receptors in the
108
control of lymphatic pumping has not been previously explored.
109
In the current study, we investigated how afobazole affects the contractions of
110
isolated rat mesenteric collecting lymphatic vessels. We tested the hypothesis that the σ
111
receptor agonist afobazole would depress lymphatic contractions, and investigated the
112
expression and localization of the σ1 receptor in rat mesenteric collecting lymphatics.
113
The experimental model consisted of isolated, perfused collecting lymphatics from the
114
rat mesentery. This model was chosen because it allowed us to study the direct effect of
115
afobazole on lymphatic contractions without confounding influences from interstitial fluid
116
flow, and with tight control of the inflow and outflow pressures (6).
117
MATERIALS AND METHODS
118
Materials
119
Afobazole was generously provided by IBC Genarium (Moscow, Russia). BD
120
1047 dihydrobromide (cat. no. 0956), BD 1063 dihydrochloride (cat. no. 0883), SM-21
121
maleate (cat. no. 0751), and L-NAME hydrochloride (cat. no. 0665) were obtained from
122
Tocris Bioscience/R&D Systems (Minneapolis, MN). DAF-FM (4-Amino-5-Methylamino-
123
2’, 7’- Difluorofluorescein Diacetate, cat. no. D23844) was purchased from Life
124
Technologies (Carlsbad, CA). Acetylcholine Chloride (cat. no. A9101-10VL) and
125
Bradykinin (cat. no. B3259) were obtained from (Sigma-Aldrich, St. Louis, MO).
126
Crystallized bovine albumin (purified BSA; cat. no. 10856) used for preparing
127
physiological salt solutions was purchased from Affymetrix (Santa Clara, CA). For
128
blocking solutions, donkey serum (cat. no. D9663) was purchased from Sigma-Aldrich.
129
Rabbit anti-Sigma-1 Receptor (cat. no. 42-3300) was purchased from Invitrogen
130
Corporation (Camarillo, CA). Mouse anti-α/γ-smooth muscle actin (cat. no. Mab1522)
131
was purchased from Millipore (Billerica, MA). Goat anti-VE-Cadherin (cat. no. sc-6458)
132
was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Donkey anti-rabbit IgG-
133
HRP (cat. no. ab97064) was obtained from Abcam (Cambridge, MA). Alexa-488-donkey
134
anti-rabbit IgG (cat. no. A21206), Alexa-488-donkey anti-mouse IgG (A21202), Alexa-
135
555-donkey anti-goat IgG (cat. no. A21432), Alexa-647-donkey anti-mouse IgG (cat. no.
136
A31571) and Alexa Fluor® 633 hydrazide were obtained from Life Technologies (Grand
137
Island,
138
Rn01775763_g1) primers (TaqMan® Gene Expression Assays) for PCR were
139
purchased from Applied Biosystems/ThermoFisher Scientific (Waltham, MA). All other
NY).
SIGMAR1
(cat.
no.
Rn00578590_m1)
and
GAPDH
(cat.
no.
140
chemicals and reagents, unless otherwise specified, were purchased from Sigma-
141
Aldrich.
142
Animal Care and Use
143
All procedures were approved by the Institutional Animal Care and Use
144
Committee (IACUC) at the University of South Florida under protocol number
145
IS00002179. These studies were performed in accordance with the National Institute of
146
Health Guide for the Care and Use of Laboratory Animals (8th Edition, 2011) and are
147
reported here in accordance with the ARRIVE Guidelines. A total number of 42 male
148
Sprague-Dawley rats (5-9 weeks of age) were purchased from Charles River
149
(Wilmington, MA), housed in a controlled temperature (22 °C) and controlled illumination
150
(12:12 h light dark cycle) environment. After arrival, the rats were submitted to a one-
151
week acclimation period and were provided standard rat chow (2018 Teklad Global 18%
152
Protein Rodent Diet, Harlan, Indianapolis, IN) and water ad libitum. When possible,
153
multiple lymphatics or tissue samples were obtained from a single rat for different
154
experiments, in order to minimize the total number of rats utilized. All possible measures
155
were taken to minimize pain or suffering, including the administration of general
156
anesthesia prior to performing experiments (details provided in next sub-section). All
157
rats were euthanized by extending the laparotomy into the chest cavity and injecting
158
Euthasol/Somnasol (0.1 ml/450 g body weight) directly into the cardiac ventricle, in
159
accordance with American Veterinary Medical Association guidelines for the euthanasia
160
of animals.
161
Mesenteric Collecting Lymphatic Isolation
162
Collecting lymphatics were isolated as previously described (25). Briefly, rats
163
were anesthetized (i.p. ketamine and xylazine at 90 and 9 mg/kg, respectively), and
164
depth of anesthesia was checked using inter-digital pinch and palpebral blink reflex. A
165
midline laparotomy was performed, and the small intestine and mesentery were
166
exteriorized, excised, and placed in ice-cold albumin physiological salt solution (APSS:
167
NaCl, 120 mM; KCl, 4.7 mM; CaCl2·2H2O, 2 mM; MgSO4·7H2O, 1.2 mM; NaH2PO4, 1.2
168
mM; Na pyruvate, 2 mM; glucose, 5 mM; EDTA, 0.02 mM; MOPS, 3 mM and 1% BSA).
169
Rats were then immediately euthanized as indicated above. In each experiment, a
170
section of mesentery that included the terminal ileum was pinned in a dissection
171
chamber containing ice-cold APSS, and with the aid of a stereomicroscope, a collecting
172
lymphatic vessel (60–150 µm internal diameter and 0.5 cm of length) was carefully
173
dissected from surrounding adipose and connective tissue. The isolated lymphatic was
174
transferred to an isolated vessel chamber (Living Systems Instrumentation, Burlington,
175
VT) and was mounted onto two resistance-matched glass micropipettes and secured
176
with nylon thread. The chamber was transferred to an Accu-Scope® 3032 inverted
177
microscope, equipped with a halogen lamp, 10X objective and CCD camera for video
178
image acquisition (Living Systems). Intraluminal pressure was imposed using APSS-
179
filled gravity manometers that fed to each micropipette. All experiments were performed
180
with the vessel bathed in 37 °C APSS with the imposed pressure from both
181
micropipettes set at 2 cm H2O, and with a 30-45 min stabilization period to let the vessel
182
equilibrate for baseline measurements. Only lymphatic vessels that displayed phasic
183
contractions that reduced internal diameter during systole by at least 25% of the
184
diastolic diameter were considered sufficiently viable and used for these studies.
185
186
Experimental Protocols
187
After cannulation, the vessels were allowed to equilibrate for 30-45 minutes in order to
188
establish baseline contractions. Upon achieving a steady baseline pumping, the impact
189
of the σ receptor agonist afobazole on lymphatic contractions was evaluated by adding
190
it to achieve final concentrations of 50, 100 and 150 µM in the bath. These
191
concentrations were chosen based on previous work in which this range was effective
192
for inhibiting ATP-induced migration of glial cells and also mitigating acidosis-induced
193
calcium overload in neurons (11, 12). To investigate the roles of σ1 versus σ2 receptor
194
activation by afobazole, lymphatics were pretreated for 20 min with either the selective
195
σ1 receptor antagonists BD 1047 (200 nM) or BD 1063 (200 nM), or the selective σ2
196
receptor antagonist SM-21 (2 µM), prior to the addition of afobazole (50, 100, and 150
197
µM). These concentrations were also chosen based on previous work (11, 12) . The role
198
of NO signaling was investigated using the nitric oxide synthase inhibitor L-NAME (200
199
µM), which we have previously used to block NOS (8, 25). Vessel diameters were
200
tracked throughout each experiment and the bath solution was changed to Ca2+-free
201
APSS at the end of each experiment to determine the maximal passive diameter
202
(MaxD) at the same luminal pressure, 2 cm H2O. Parameters used to characterize
203
lymphatic pump function were calculated from the data (outlined below under Data
204
Analyses).
205 206
Total RNA Isolation and PCR
207
Mesenteric collecting lymphatic vessels (10 vessels per rat) were freshly isolated,
208
placed on ice, sonicated, snap frozen in 300 µl QIAzol® Lysis Reagent (Qiagen Inc.,
209
Valencia, CA) and stored at -80 °C. Total RNA was extracted using chloroform and
210
isopropanol and the samples were treated with DNase to remove genomic DNA. The
211
RNA was then purified using the RNeasy® MinElute® Cleanup Kit (Qiagen Inc.,
212
Valencia, CA) according to the manufacturer’s specifications, and the RNA pellet was
213
eluted in 14 µl of RNase-free water. RNA concentration and purity were determined
214
using the NanoVue Plus™ (GE Healthcare, Piscataway, NJ). Total RNA (50 ng) was
215
reverse-transcribed into cDNA using the ProtoScript® First Strand cDNA Synthesis Kit
216
(New England Biolabs Inc., Ipswich, MA). The cDNA was combined with TaqMan® Fast
217
Universal PCR Master Mix (Applied Biosystems/ThermoFisher Scientific) and primers
218
specific for either rat SIGMAR1 or rat GAPDH. PCR reactions were performed using a
219
10-min hold at 95 °C, 95 °C for 15 s and 60 °C for 1 min (40 cycles) using a CFX
220
Connect Real-Time System (Bio-Rad, Hercules, CA). Primer sequences (NCBI
221
Reference Sequence) used were: NM_030996.1(SIGMAR1) and NM_017008.4
222
(GAPDH). PCR reaction products were run on a 2 % agarose gel and bands were made
223
detectable with SYBR Green I nucleic acid gel stain (Life Technologies/ThermoFisher
224
Scientific, Waltham, MA) and visualized under UV, using the BioRad Chemi Doc XRS+
225
System with Quantity 1-D Analysis Software (Bio-Rad, Hercules, CA).
226
Immunoblotting Protocol
227
Approximately 10 collecting lymphatic vessel segments per rat, freshly isolated
228
from the mesentery, were placed in 200 µl 1X RIPA (4 °C) containing HALT Protease
229
and Phosphatase Inhibitor Cocktail (Pierce, Rockford, IL). The mixture was sonicated
230
twice at 4 °C for 5 s using a Fisher Sonic Dismembranator, model FB-120 (Fisher
231
Scientific, Asheville, NC) and frozen at -80 °C. Protein concentrations were determined
232
using the BCA protein assay (Pierce/Thermo Scientific, Waltham, MA) to equilibrate
233
samples. Lysate was mixed with 4X NuPage LDS sample buffer containing reducing
234
agent (Invitrogen, Grand Island, NY). Samples containing 30 µg of protein were loaded
235
into 4-20% Novex Bis-Tris gels (Invitrogen) for SDS-PAGE to separate proteins. The
236
NexusPointer prestained protein ladder (BioNexus, Oakland, CA) was used to
237
determine molecular weight vs. mobility. The proteins were transferred from the gels to
238
Immobilon-P PVDF membranes (Millipore) by wet transfer. Membranes were blocked
239
with 5% BSA in Tris-buffered saline with Tween® 20 (TBST). Primary antibody was
240
1:1000 rabbit anti-sigma 1 receptor (Invitrogen 42-3300). The secondary antibody was
241
1:10,000 donkey anti-rabbit IgG-HRP (ab97064). Bands were visualized using WestPico
242
Supersignal reagent (Pierce/Thermo Scientific) and a BioRad Chemi Doc XRS+ System
243
with Quantity 1-D Analysis Software (5 min exposure), (Bio-Rad, Hercules, CA).
244
Immunofluorescence Labeling and Confocal Microscopy
245
Rat mesenteric lymphatics were isolated and excised, and for each isolated vessel,
246
one end was mounted onto a glass micropipette filled with APSS in a custom chamber
247
for fixation and labeling as previously described (25). The other end of the lymphatic
248
was left free to float in an APSS bath, to allow for APSS to be pulled into or pushed out
249
of the vessel lumen by gently applying negative or positive pressure, respectively, on
250
the micropipette via an attached 1 ml syringe. Each vessel was fixed with 4%
251
paraformaldehyde for 10-15 min at room temperature with positive pressure in the
252
vessel lumen, followed by two 5-min washes with 100 mM glycine buffer and one 5-min
253
wash with Ca2+/Mg2+-free Dulbecco’s PBS (CMF-DPBS). Ice-cold acetone (-10 to -20
254
°C) was applied for 5 min to permeabilize the cell membranes, followed by three 5-min
255
washes with CMF-DPBS. A blocking solution consisting of 5% normal donkey serum in
256
CMF-DPBS was applied for 30 min at room temperature, followed by overnight
257
incubation with combinations of primary antibodies in antibody dilution buffer (151 mM
258
NaCl, 17 mM trisodium citrate, 2% donkey serum, 1% BSA, 0.05% Triton X-100, 0.02%
259
NaN3) at 4 °C: 1:3000 mouse anti-α/γ-smooth muscle actin (Mab1522); 1:50 goat anti-
260
VE-cadherin (sc-6458); and 1:60 rabbit anti-sigma 1 receptor. Labeling controls
261
received antibody dilution buffer containing no primary antibody. After overnight
262
incubation, three 10-min rinses with antibody wash solution (151 mM NaCl, 17 mM
263
trisodium citrate, 0.05% Triton X-100) were performed. The vessels were incubated for
264
30-60 min at room temperature with antibody dilution buffer containing secondary
265
antibodies: 1:400 Alexa-488-donkey anti-rabbit IgG (A21206), 1:400 Alexa-488-donkey
266
anti-mouse IgG (A21202), 1:400 Alexa-555-donkey-anti-goat IgG (A21432) and 1:400
267
Alexa-647-donkey anti-mouse IgG (A31571). Alexa Fluor® 633 hydrazide (1.0 μM) was
268
also added during this time period to label extracellular matrix (ECM) elastin fibers (10,
269
41) in some of the whole mounts. Three 10-min rinses with antibody wash solution were
270
performed, and each vessel was removed from its cannula, placed on a glass slide with
271
a Secure-Seal™ imaging spacer in 20 µl of Prolong Gold Anti-Fade reagent containing
272
DAPI (Life Technologies) or VECTASHIELD® with DAPI (Vector Laboratories, Inc.,
273
Burlingame, CA) and covered with a #1 glass coverslip. Care was taken to keep the
274
lymphatic vessel lumen patent for better view of vessel structure. Confocal z-stack
275
images (step size = 0.5-0.75 μm) of the vessels were obtained with an Olympus FV1200
276
spectral inverted laser scanning confocal microscope, and a 60X (PLAPON60XOil,
277
1.42NA or 1.30NA) objective with 1.3-1.5X zoom at the Lisa Muma Weitz Advanced
278
Microscopy and Cell Imaging Core at the University of South Florida. Imaris software
279
(Bitplane, Concord, MA) was used to produce 3D models of z-stacks for interpretation.
280
FIJI/ImageJ open source imaging software (http://fiji.sc) was also used to view and
281
process confocal image stacks into the figures.
282
Cell Culture
283
Primary human dermal lymphatic endothelial cells (HDLEC-juvenile) (PromoCell,
284
Heidelberg, Germany) were seeded onto gelatin-coated, (0.75%) 100-mm culture
285
dishes. Cells were routinely maintained in Endothelial Cell Growth Medium2-MV
286
(EGM2-MV) (Lonza, Walkersville, MD) with medium changed every 48 h. For all
287
experiments, passage 2-3 endothelial cells were used. For NO imaging studies,
288
confluent endothelial monolayers were washed with 1X DPBS, incubated in trypsin-
289
EDTA, 0.25%, neutralized with EGM2-MV and pelleted at 2000 rpm/min. Resuspended
290
endothelial cells were seeded on 18mm round microscope cover glasses coated with
291
gelatin, (0.75%) at a density of 0.1 x 106 cells and grown to 70-80% confluency.
292 293
Nitric Oxide Imaging Measurements
294
Changes in intracellular NO concentrations were measured in cultured primary
295
endothelial cells using fluorescent imaging techniques and the NO sensitive dye DAF-
296
FM. Cells were loaded using membrane permeable DAF-FM. Cells seeded on
297
coverslips (as above) were incubated for 1 hour at 37˚ in HBSS (NaCl, 135 mM; KCl, 5
298
mM; CaCl2, 1.5 mM; MgCl2, 1 mM; glucose, 10 mM; HEPES, 10 mM pH 7.4) with 8 μM
299
DAF-FM and 0.8 % dimethyl sulfoxide (DMSO). The coverslips were washed in DAF-
300
FM-free HBSS prior to experiments being performed. Cells were illuminated with 495
301
nm light for 400 msec at 0.3 Hz (Lambda DG-4, Sutter Instruments, Novato CA) and
302
fluorescent emissions at 535 nm were collected using a Sensicam digital CCD camera
303
(Cooke Corp., Auburn Hills, MI). Imaged cells were perfused with control solution and
304
solutions containing 150 μM afobazole, 10 μM Acetylcholine or 1 μM bradykinin using a
305
perfusion system consisting of 250 μm diameter glass tubes positioned 500 μm away
306
with flow rates of 300 μl/min.
307
Data Analyses
308
For the lymphatic pumping studies, datasets of lymphatic diameter over time,
309
before and after various interventions such as pressure steps or addition of drugs, were
310
used for analysis. Parameters used to characterize lymphatic pump function were
311
determined from the lymphatic intraluminal diameter measurements in the same
312
manner as previously described (16, 26). These include contraction frequency (CF), end
313
diastolic diameter (EDD), end systolic diameter (ESD), amplitude of contraction (AMP =
314
EDD - ESD), ejection fraction (EF) = (EDD2-ESD2)/(EDD2), and fractional pump flow
315
(FPF; FPF = CF X EF). EDD, ESD, and AMP were normalized to MaxD to account for
316
variability in the resting diameter of different lymphatics. Baseline data for all drug
317
experiments were the averages for the 2-min period prior to drug administration. For
318
each concentration of afobazole, the 2-min increment starting 4 min post addition was
319
used to calculate the mean data (i.e. means represent 4-6 min after addition of each
320
concentration and each concentration was held for 10 min). These time points after
321
afobazole administration represented the peak responses. When σ receptor antagonists
322
or L-NAME were added, we utilized the last 2-min period just prior to the addition of
323
afobazole to calculate the mean parameters due to any effects of the antagonist by
324
itself.
325
Summarized data are presented as mean ± SE. For all experiments, the
326
hypothesis tested was that the various experimental interventions would produce a
327
difference in the baseline pumping parameters just before afobazole was added, which
328
served as controls. GraphPad Prism 6 software was used for statistical analyses
329
(GraphPad Software, LaJolla, CA) with the threshold for significance set at P
