draft - university of toronto t-space · 1990; lessard et al. 1999; parati et al. 1995; stauss...
TRANSCRIPT
Draft
Characterization of endogenous nitric oxide role in
myogenic vascular oscillations during cooling-evoked hemodynamic perturbations of rats
Journal: Canadian Journal of Physiology and Pharmacology
Manuscript ID cjpp-2016-0476.R1
Manuscript Type: Article
Date Submitted by the Author: 21-Dec-2016
Complete List of Authors: Lin, Yi-Hsien ; Cheng Hsin General Hospital
Liu, Yia-Ping ; National Defense Medical Center, Physiology Lin, Yu-Chieh ; Cheng Hsin General Hospital, Medical Research and Education Lee, Po-Lei ; National Central University, Electrical Engineering, Tung, Che-Se; Cheng Hsin General Hospital, Medical Research & Education
Keyword: cold stress, hemodynamic perturbations, sympathetic activation, nitric oxide, cardiovascular oscillations
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
1
Characterization of endogenous nitric oxide role in myogenic vascular
oscillations during cooling-evoked hemodynamic perturbations of rats
Yi-Hsien Lin1,4, Yia-Ping Liu2, Yu-Chieh Lin1, Po-Lei Lee3, and Che-Se Tung1
1Division of Medical Research and Education, Cheng Hsin General Hospital, Taipei,
Taiwan
2Department of Physiology, National Defense Medical Center, Ta i p e i , Ta i w a n
3 D e p a r t m e n t o f E l e c t r i c a l E n g i n e e r i n g , National Central University,
Taoyuan, Taiwan
4School of Medicine, National Yang-Ming University, Taipei, Taiwan
Corresponding author Che-Se Tung: Division of Medical Research & Education,
Cheng Hsin General Hospital, Taiwan, ROC.
E-mail address: [email protected]
Page 1 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
2
Abstract: Rapid immersion of a rat’s limbs into 4°C water, a model of cold stress (CS),
can elicit hemodynamic perturbations (CEHP). We have reported that CEHP is highly
relevant to the sympathetic activation and nitric oxide production. This study identifies
the role of nitric oxide in CEHP. Conscious rats were pretreated with the nitric oxide
synthase inhibitor L-NAME alone or following the removal of sympathetic influences
using hexamethonium or guanethidine, and then they were subjected to a 10-min CS
trial. Hemodynamic indices were telemetrically monitored throughout the experiment.
The analyses included measurements of systolic blood pressure; heart rate; dicrotic
notch; short-term cardiovascular oscillations and coherence between blood pressure
variability and heart rate variability at very low- (0.02 to 0.2 Hz), low- (0.2 to 0.6 Hz),
and high-frequency (0.6 to 3.0 Hz) regions. We observed there were different profiles
of hemodynamic reaction between hexamethonium and guanethidine superimposed
on L-NAME, suggesting an essential role for a functional adrenal medulla release
epinephrine under CS. These results indicate that endogenous nitric oxide plays an
important role in the inhibition of the sympathetic activation and cardiovascular
oscillations in CEHP.
Key words: cold stress, hemodynamic perturbations, sympathetic activation, nitric
oxide, cardiovascular oscillations
Page 2 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
3
Introduction
Acute immersion of the limbs of a conscious rat into 4°C water induces pressor
and tachycardia reactions. Cooling-elicited hemodynamic perturbations (CEHP)
represents an ideal model for evaluating of autonomic cardiovascular regulation
(Johnson and Kellogg 2010; Robertson et al. 1979). CEHP is characterized by
hemodynamic instability (irregular blood pressure (BP), heart rate (HR), and
cardiovascular oscillations), an initial vasoconstriction followed by vasodilatation and
a secondary progressive vasoconstriction for blood flow to the cooled areas to avoid
damage, as first described by Lewis (Daanen 2003; Lewis 1926).
Although the underlying mechanisms are still not clear, intact sympathetic and
sensory functions with the compensatory response and humoral factors are known
involving vasoconstrictor responses of CEHP (Daanen 2003). In the periphery, nitric
oxide as a potent vasodilator is crucial in governing vascular resistance and
myocardial contractility (Arnal et al. 1999; Llorens et al. 2002; Rastaldo et al. 2007;
Yamazaki et al. 2006). Emerging evidence indicates that endogenous nitric oxide has
buffering capabilities comparable to baroreflex control in the regulation of blood
pressure (Nafz et al. 1996).
Spectral analysis of BP variability (BPV) and HR variability (HRV) using frequency
domain approaches has been widely applied to investigate the baroreflex function in
homeostasis of cardiovascular oscillations (Akselrod et al. 1985; Japundzic et al.
1990; Lessard et al. 1999; Parati et al. 1995; Stauss 2007). Recently, we performed a
serial of studies to investigate the causes for CEHP. We found that the sympathetic
activation and pressor responses were associated with a significant elevation of the
plasma nitric oxide levels, as well as with marked increases in powers for
low-frequency BPV (LFBPV) and very-low-frequency BPV (VLFBPV). We postulated the
Page 3 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
4
VLFBPV power might reflect the myogenic vascular responsiveness to the stressful
cooling challenge (Liu et al. 2015a; Liu et al. 2015b; Liu et al. 2015c).
The effects of hexamethonium (HEX) and guanethidine (GUA) have been
acknowledged by research (Abercrombie and Davies 1963; Richardson and Wyso
1960; Zimmerman et al. 1960). HEX blocks the transmission across autonomic
ganglia. On the other hand, GUA reaches sites of sympathectomy in the peripheral
neurons through transport by the norepinephrine pump and is familiar with its spared
effects on central adrenergic neurons and adrenal medulla. To clarify the significance
of nitric oxide in the progression of CEHP, we compared by using a constitutive nitric
oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), with the
superimposition of sympathetic removal using HEX or GUA in the present study.
Materials and Methods
Animals
Adult male Sprague-Dawley rats (BioLASCO) weighing between 300 and 350 g
were obtained from the animal center of the National Defense Medical Center
(NDMC), Taiwan, ROC one week before experiments. The experiments were
performed according to a protocol approved by the animal care committee of NDMC.
All efforts were made to keep the number of animals used as low as possible and to
minimize animal suffering during the experiments. All rats were housed in a
temperature and humidity-controlled holding facility with a 12-hour light/dark cycle
(lights on from 07:00 to 19:00), which was maintained by manual light control
switches as required by the experiment. The rats in the same experimental group
were housed together. All rats received food and water ad libitum. The experiments
were performed between 08:30 and 17:30 with individual rats being tested at the
Page 4 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
5
same time every day, when possible.
Experimental protocols and cooling procedure
The timing of the experimental protocols is shown in Fig. 1. The rats were
randomly divided into four experimental groups for interventions with a similar acute
cooling procedure. The control group was given the vehicle (0.9% NaCl solution,
n=12) 0.4 ml via a tail venous bolus injection for baseline comparisons. The other
three groups of rats were given the L-NAME alone (L-NAME, n=12), with the
superimposition of HEX (HEX+L-NAME, n=12) or with GUA (GUA+L-NAME, n=12)
respectively. Experimental procedures for the three group of rats were (a) a tail
venous bolus of L-NAME (30 mg/ml/kg) 15 min before the cold stress (CS) trial, (b) a
jugular venous bolus of HEX (30 mg/ml/kg) followed by continuous infusion (1.5
mg/kg/min) 5 min before the L-NAME intervention throughout the CS trial (around 2
ml), and (c) an intraperitoneal injection of GUA (50 mg/kg/day x 7 days) with a dose
30 min before the CS trial. A separate test to examine the influence of HEX alone or
GUA alone on norepinephrine and epinephrine releases has been conducted at the
end of a 10-min CS trial. Blood was drawn from the tail venous catheter to assay
circulating catecholamines (ELISA Kit, USA).
Following a complete stabilization of BP and HR at room temperature, each rat
was quickly placed in a Plexiglas cage with ice-water (depth=2 cm; temperature=4oC)
to immerse its glabrous palms and soles for a period of 10 min. After this trial, the rat
was removed from the cage, dried with a cloth, and placed in a similar cage for 20 min
to facilitate recovery. The beat-to-beat BP signals were monitored continuously via a
telemetric device (TL11M2-M2-C50-PXT, DSI, USA) at 10-min intervals in the three
experimental conditions, including 10 min before (PreCS), 10 min of a CS trial, and
Page 5 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
6
20-30 min after (PostCS). Successive signals during a period of approximately 5 min
(3 to 8 min) in each condition were taken for spectral analysis because, during this
period, the mean and variance of VLFBPV and systolic blood pressure (SBP) were
stable. The dicrotic notch (Dn) and counts were handled manually.
Surgical intervention
A telemetry transmitter was implanted intra-abdominally into each rat under
anesthesia (sodium pentobarbital, 50 mg/kg). A laparotomy was performed using
aseptic procedures, and the catheter of the transmitter was inserted into the
abdominal aorta, distal to the kidneys, and fixed in place. The experiments were
initiated after the rats had fully recovered from surgery (7 days).
Spectrum signal acquisition and processing
One hour before the experiment in the testing day, the transmitter was
magnetically activated. Pulse signals for calibration were generated as an analog
signal (UA10; DSI, St. Paul, MN) with a range of ±5 V and a 12-bit resolution.
Individual rats in each group were then placed on the top of the receivers (PhysioTel®
RPC-1) for telemetric signal acquisition. Five receivers were connected to a PC
desktop computer via a matrix (Dataquest ART Data Exchange Matrix), and the
received signals were recorded with Dataquest Acquisition software (Dataquest ART
4.33). A series of the successive SBP and the inter-beat interval (IBI) signals
throughout the experiments were then digitized at a 500 Hz sampling rate and
processed off-line using Matlab software (Terasoft Co.).
The beat-by-beat oscillatory SBP and IBI signals were analyzed to quantify their
frequencies and spectral powers regarding BPV and HRV using autoregressive
Page 6 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
7
spectral decomposition. The BPV calculation was based on a software kindly written
for us by Prof. P.L. Lee, National Central University, Taiwan, ROC. Briefly, the
acquired SBP signals were pre-processed by applying a band-pass filter (0.1-18 Hz,
zero-phase 4th-order) to remove the DC components. After identifying all of the SBP
peak maxima between two zero-cross points, the extracted beat-by-beat SBP time
series were detrended, interpolated and resampled at 0.05 s to generate a new time
series of evenly spaced SBP samples, allowing a direct spectral analysis of each
distribution using a Fast Fourier Transform (FFT) algorithm. The HRV calculation was
based on Chart software developed by PowerLab, ADInstruments, USA. In a period
of a 5-min experimental condition, we calculated the powers including total power
(0.00 to 3.0 Hz, TP), very-low-frequency power (0.02 to 0.2 Hz, VLF), low-frequency
power (0.20 to 0.60 Hz, LF), and high-frequency power (0.60 to 3.0 Hz, HF). The
normalized LF and HF were also calculated as nLF (or nHF) = LF (or
HF)/TP-VLF×100%. The modulus of the spectral density for each frequency had units
of BPV: mmHg2 and HRV: ms2. The squared coherence function was computed as
the square of the cross-spectrum normalized by the product of the spectra of the BPV
and HRV signals. When the peak coherence value (K2
IBI/SBP) exceeded 0.58 within a
frequency range, the two signals were considered to covery significantly at that
frequency.
Statistics
The statistical analyses of the present study were conducted with SPSS 18.0 for
Windows (Chicago, IL, USA).The homogeneity of the variance was first confirmed
using the Kolmogorov–Smirnov test. Data were then analyzed by the multiple ways of
analysis of variance (ANOVA) with a within-subject factor, "Trial" (three conditions:
Page 7 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
8
PreCS, CS, and PostCS) and a between-subject factor, "Group" (Four interventions:
Control Vehicle, L-NAME, HEX+L-NAME, and GUA+L-NAME). If necessary, post hoc
comparisons were carried out with Tukey and Student t test. Univariate correlations
were calculated using Pearson’s correlation analysis to provide the associations
between selected frequency bands. The results are expressed as the mean ±
standard error of mean (SE). The statistical significance of probability level was set at
0.05.
Results
Averaged data are shown in Table 1 and Fig. 2-4 as in Table S1 and Table S2
(please see the Data Supplement). Plasma norepinephrine and epinephrine
concentrations in control and infusion of HEX or GUA rats are shown in Table 1.
[To Editor: Please place Figure 1 here]
Responses of SBP, HR, and Dn appearance to various drug interventions
throughout the experimental course
As shown in Table S1 and Fig. 2 (A), inhibition of NO synthesis by L-NAME
significantly increased SBP compared with that of the control vehicle under all
experimental conditions (PreCS, CS, and PostCS) (all p<0.01). The higher SBP
levels in response to L-NAME were attenuated by the superimposition of ganglionic
blockade with HEX (HEX+L-NAME) under CS (p<0.01) and was markedly attenuated
by the superimposition of sympathectomy with GUA (GUA+L-NAME) under all
experimental conditions (all p<0.01). On the other hand, L-NAME caused a significant
decrease in the HR compared with the control vehicle (CS: p<0.01; PostCS: p<0.01).
Page 8 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
9
However, this effect was potentiated (more bradycardia) in the sympathectomized
rats compared to the ganglionic blockade rats under all experimental conditions
(GUA+L-NAME versus HEX+L-NAME: all p<0.01). Nevertheless, all three
interventionsthe administration of L-NAME, HEX+L-NAME,
GUA+L-NAMEcaused tachycardia under CS compared with the respective PreCS
or PostCS (cooling-induced tachycardia, CIT) (L-NAME: 346.40±12.55 versus
277.56±20.49 or 286.09±8.06; HEX+L-NAME: 342.80±12.21 versus 296.11±6.82 or
278.95±9.65; GUA+L-NAME: 284.31±11.92 versus 242.44±4.69 or 244.96±8.100).
[To Editor: Please place Figure 2 here]
As shown in Fig. 2 (B), both L-NAME alone and HEX+L-NAME interventions
generally increased the appearance of the dicrotic notch (with Dn), compared with the
vehicle control under all experimental conditions (p<0.01); however, the increases of
Dn were much more apparent and significant in the GUA+L-NAME intervention.
The effects of L-NAME alone on frequency power and coherence function
As shown in Fig.3 and Table S2, when compared CS with the respective PreCS or
PostCS, the administration of L-NAME had increased the powers for VLFBPV (PreCS
or PostCS versus CS, all p<0.05), LFBPV (PreCS or PostCS versus CS, all p<0.001),
VLFHRV (PostCS versus CS, p<0.05), HFBPV (PreCS or PostCS versus CS, all p<0.05),
and TPBPV (PreCS or PostCS versus CS, all p<0.001), but a non-significant increase
in VLFHRV and a non-significant decrease in LFHRV were observed. When compared
with the control vehicle under PreCS, L-NAME increased the powers for LFBPV
(p<0.05), LFHRV (p<0.05), HFBPV (p<0.01), HFHRV (p<0.01), and TPHRV (p<0.05) and a
Page 9 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
10
non-significantly increasing VLFBPV, VLFHRV, and TPBPV. When compared with the
control vehicle under CS, L-NAME increased the powers for VLFBPV (p<0.05), VLFHRV
(p<0.05), LFHRV (p<0.01), HFHRV (p<0.01), and TPHRV (p<0.01), albeit non-significantly
increasing LFBPV and TPBPV and decreasing for HFBPV and LF/HFHRV. Nevertheless,
the original tendencies for negative correlations of the VLF pair (VLFHRV versus
VLFBPV) (r=-0.32, p=0.39) and the LF pair (LFHRV versus LFBPV) (r=-0.39, p=0.20)
observed for the control vehicle were changed to tendencies for positive correlations
for the VLF pairs (r=0.48, p=0.19) and LF pairs (r=0.61, p<0.05) after the L-NAME
alone intervention.
[To Editor: Please place Figure 3 here]
The linear relationships as assessed by the peak coherence values (K2IBI/SBP)
between BPV and HRV for the three major frequency regions are summarized in Fig.
4. When compared with the control vehicle under all experimental conditions,
L-NAME generally showed large K2IBI/SBP at the LF region (L-NAME versus Control
Vehicle: PreCS: 0.66±0.03 versus 0.55±0.03; CS: 0.62±0.04 versus 0.57±0.03;
PostCS: 0.65±0.02 versus 0.53±0.03) but small K2IBI/SBP at the HF region (L-NAME
versus Control Vehicle: PreCS: 0.63±0.01 versus 0.75±0.03; CS: 0.65±0.03 versus
0.74±0.03; PostCS: 0.62±0.03 versus 0.69±0.03). However, we did not find a
consistent coherence relationship between the BPV and HRV at the VLF region
(K2IBI/SBP<0.58) after the control vehicle or the L-NAME intervention.
[To Editor: Please place Figure 4 here]
Page 10 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
11
Comparisons of the responses of frequency power and coherence function for
HEX versus GUA superimposed on the L-NAME intervention
As shown in Fig. 3 and Table S2, the administration of HEX+L-NAME generally
attenuated the effect of L-NAME on frequency powers throughout the experimental
course. The affected powers included VLFBPV (PreCS: p<0.05; CS: p<0.01; PostCS:
p<0.05), VLFHRV (PreCS: p<0.01; CS: p<0.01), LFBPV (PreCS: p<0.01; CS: p<0.01),
LFHRV (PreCS: p<0.05; CS: p<0.05), HFHRV (PreCS: p<0.01; CS: p<0.01; PostCS:
p<0.05), TPBPV (CS: p<0.01), and TPHRV (PreCS: p<0.05; CS: p<0.01). The
administration of GUA+L-NAME also attenuated the effect of L-NAME on frequency
powers throughout the experimental course. The affected powers included VLFBPV
(CS: p<0.05; PostCS: p<0.05), VLFHRV (PostCS: p<0.01), LFBPV (PreCS: p<0.01; CS:
p<0.01; PostCS: p<0.01), HFBPV (CS: p<0.01), and TPBPV (PrerCS: p<0.01; CS:
p<0.01; PostCS: p<0.01). When compared among groups under CS, the powers
were non-significant larger for LFHRV and HFHRV of GUA+L-NAME than for those of
L-NAME. In addition, when compared with HEX+L-NAME, the effect of
GUA+L-NAME was generally larger for VLFHRV (PreCS: p<0.01; CS: p<0.01), LFHRV
(PreCS: p<0.01; CS: p<0.01), HFHRV (PreCS: p<0.01; CS: p<0.01), and TPHRV
(PreCS: p<0.05; CS: p<0.01) but smaller for VLFHRV (PostCS: p<0.01), LFBPV
(PostCS: p<0.01), and HFBPV (CS: p<0.05). Nevertheless, the positive correlation
tendency for the VLF pair (r=0.48, p=0.19) and the LF pair (r=0.61, p<0.05) observed
for the L-NAME intervention has changed back to a negative correlation tendency
(VLF pairs: r=-0.46, p=0.19; LF pairs: r=-0.48, p<0.05) after the HEX+L-NAME
intervention, that is similar to that observed for the control vehicle intervention.
However, the original negative correlation tendencies for both the VLF pair (r=-0.32,
p=0.39) and LF pair (r=-0.39, p=0.20) observed in the control vehicle were changed
Page 11 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
12
to positive correlation tendencies (VLF pair: r=0.43, p=0.42; LF pair: r=0.76, p<0.01)
after the GUA+L-NAME intervention.
Compared with the respective K2IBI/SBP values for the L-NAME alone intervention
(Fig. 4), there were no consistent coherence relationships between the BPV and HRV
at the LF region after the HEX+L-NAME intervention under any experimental
conditions (PreCS: 0.55±0.03; CS: 0.52±0.03; PostCS: 0.51±0.02). In contrast, in this
region after the GUA+L-NAME intervention, there was still strong coherence linkages
for the PreCS (0.60±0.03), CS (0.59±0.03), and PostCS (0.59±0.03) conditions.
However, compared with the L-NAME alone intervention, the HEX+L-NAME
intervention or the GUA+L-NAME intervention eliminated the coherence relationship
at the HF region under all experimental conditions (K2
IBI/SBP<0.58).
Discussion
In our previous report (Liu et al. 2015b), we pointed out that abolition of adrenergic
influences by HEX or GUA reduced the production of plasma nitric oxide and
decreased SBP with concomitant attenuation of cardiovascular oscillations under
stressful cooling challenge (Table S1 and S2). The results indicated that increasing
VLFBPV power changes are highly relevant to the sympathetic activation and
subsequent nitric oxide production. In the present study, we demonstrated that
abolition of adrenergic influences by HEX drastically reduced the plasma
norepinephrine and also epinephrine throughout the experiment, whereas, by
contrast, GUA increased both norepinephrine and epinephrine (Table 1). Furthermore,
we demonstrated that inhibition of nitric oxide synthase by L-NAME significantly
increased SBP but slightly decreased heart rate with concomitant alterations of
frequency powers and coherence between BPV and HRV. However, the changes
Page 12 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
13
produced by L-NAME alone were differently affected by superimposition of HEX
(HEX+L-NAME) or GUA (GUA+L-NAME), in general, the effects of HEX+L-NAME or
GUA+L-NAME were similar to those achieved with HEX alone or GUA alone in our
previous report. Our present findings clearly offer further support for the sympathetic
activation generated nitric oxide production, which in turn exerted a buffering effect on
the myogenic oscillations in the vasculature to the stressful cooling challenge.
Effects of L-NAME on resting condition
Compared with the vehicle control under resting condition PreCS, L-NAME
significantly increased SBP but decreased HR slightly and intensified the overall
cardiovascular oscillations as increased TPBPV and TPHRV powers, in particular as
increased LFBPV power as sympathetic activation on vasomotor tone (Japundzic et al.
1990; Parati et al. 1995; Stauss 2007). The results indicated the tonic nitric
oxide-dependent vasodilation is exerted irrespective of the presence or absence of
sympathetic influences, as compared with the L-NAME alone still presented a marked
increase of SBP following the superimposition of HEX or GUA under PreCS. The
frequency powers, in general, intensified by L-NAME were attenuated after the
superimposition of HEX or GUA, indicating the involvement of sympathetic activation.
Nevertheless, the L-NAME alone has strengthened the coherence between BPV and
HRV at the LF region (K2
IBI/SBP>0.58), which suggest there is an intact baroreflex
mechanism on sympathetic activation, whereas the superimposition of HEX
weakened but the superimposition of GUA still kept such strengthening effects of
L-NAME (Fig. 4). These findings are in line with the earlier reports showing the tonic
influence of nitric oxide on BP oscillations is exerted independently of the suppression
of the arterial baroreceptor but most likely because of the local dampening effect of
Page 13 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
14
nitric oxide on the vasculature (Nafz et al. 1996; Stauss 2007).
L-NAME appears to affect more than one process to inhibit the effect of nitric
oxide as an inhibitory mediator (Arnal et al. 1999; Llorens et al. 2002; Rastaldo et al.
2007; Yamazaki et al. 2006). The results under PreCS suggest two possible
processes that could be affected by L-NAME. First, from the vasculature point of view,
the increase of SBP is reflective of an enhancement of vascular resistance due to the
inhibition of local BP buffering effect that relies on the endothelial nitric oxide
production (Nafz et al. 1996; Stauss 2007). Second, from the autonomic ganglia point
of view, L-NAME might inhibit the nitric oxide synthase-containing fibers in ganglia to
suppress the nitric oxide-buffering effect on both sympathetic and parasympathetic
discharges and the subsequent cardiovascular oscillations (Ceccatelli et al. 1994;
Elfvin et al. 1997). Indeed, we have demonstrated that L-NAME has increased TPHRV
and all of its associated frequency powers for the effects on the heart, the
superimposition of HEX has abolished almost those effects of L-NAME. The results
are consistent with the finding that the nitric oxide synthase-containing preganglionic
neurons constrain the postganglionic neurons-affected innervating structures
(Morales et al. 1995). Inhibition of nitric oxide production by L-NAME released the
postganglionic sympathetic (LFHRV) and parasympathetic (HFHRV) discharges, leading
to the increases in TPHRV and all its associated frequency powers. Furthermore,
L-NAME released the effects of nitric oxide on negative inotropic and lusitropic
activities (Kojda and Kottenberg 1999), leading to the expression of myocardial
oscillations as an increase in VLFHRV.
Effects of L-NAME on cold stress
In this study, we confirmed our previous findings that compared CS with
Page 14 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
15
respective PreCS, both cooling-induced pressor (CIP) and cooling-induced
tachycardia (CIT) reactions have coexisted in vehicle control treatment. Compared
with the vehicle control under CS, L-NAME increased SBP, decreased heart rate, and
enhanced CEHP by intensified most frequency powers except HFBPV.
We observed that L-NAME has intensified all cooling-elicited frequency powers for
BPV but attenuated most of them for HRV except VLFHRV when compared CS with
respective PreCS. It was evident that the VLFHRV power was considerably intensified
by L-NAME (Fig. 3). The L-NAME-intensified VLFHRV power helped to clarify the
relationship of nitric oxide role and local dampening effect on myocardial oscillations
under CS (Liu et al. 2015a; Liu et al. 2015b). The results also support that
vasoconstrictor tone is essential for the expression of myogenic vascular oscillations
as intensified the VLFBPV power. Endogenous nitric oxide production might provide
background vascular tone against which CIP influences act, thereby generating a
buffering effect on both vasculature and heart under CS.
Nevertheless, we observed a simultaneous increase of VLF pair as of LF pair by
L-NAME under CS. We also observed a CIT tendency with the increase of SBP and
decrease of heart rate throughout the experiment by L-NAME (Fig 2). These findings
suggest that stressful cooling in the presence of L-NAME might intensify the arterial
and cardiac stiffening, a positive correlation tendency for the VLF pair because
eliminated nitric oxide-buffering effect on baroreceptor increased sympathetic
activation, a positive correlation tendency for the LF pair (Edwards et al. 2006). The
L-NAME-induced stiffer arteries and myocardium may also increase arterial
impedance and pulse wave reflection (Hu et al. 1997; Politi et al. 2016) as we
observed an increase of Dn appearance (Fig 2 (B)). To evaluate the possibility that
sympathetic activation and nitric oxide production contribute to the genesis of CEHP,
Page 15 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
16
we performed the following experiments to abolish neural sympathetic influences via
superimposition of HEX or GUA on L-NAME for comparison.
As we found in our previous studies (Liu et al. 2015b), the effects of HEX alone
and GUA alone on SBP and HR showed a marked attenuation of both indices under
the plateau pressor period of CS. However, HEX remained the CIP and CIT reactions,
whereas GUA abolished CIP, attenuated but remained CIT to the stressful cooling
challenge. On the other hand, the effects of HEX alone and GUA alone on myogenic
vascular oscillations showed a marked attenuation of both LFBPV and VLFBPV powers
under this plateau pressor period also. However, HEX and GUA both attenuated but
continued an increasing tendency of the VLFBPV power compared CS with respective
PreCS. The results indicate that sympathetic activation has intensified the VLFBPV
power as an expression of myogenic vascular oscillations in CEHP.
In the present study, compared the effects on SBP and HR between groups (Fig 2
(A)), we observed HEX+L-NAME attenuated the increase in SBP by L-NAME, and
GUA+L-NAME has further attenuated this effect. Whereas the decrease in HR was
equivalent compared HEX+L-NAME with L-NAME, GUA+L-NAME has further
attenuated this effect also. In contrast to a CIP reaction of the vehicle control, both
HEX+L-NAME and GUA+L-NAME have produced the cooling-induced depressor
(CID) reaction. Nevertheless, we observed GUA+L-NAME has further attenuated the
magnitude of CIT attenuated by L-NAME, although L-NAME, HEX+L-NAME, and
GUA+L-NAME all three interventions still exerted a tendency to develop CIT seen in
the vehicle control. The results raised a question about the process of which the
removal of the sympathetic input caused the observed effects of CID and CIT. A
previous report suggested that cooling irritation of the primary afferent C-fibers may
activate the release of calcitonin gene-related peptide (CGRP), subsequently, may
Page 16 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
17
evoke the effects of vasodilation and positive chronotropic and inotropic effects (Kunz
et al. 2007). In this context, the CID and CIT observed in our study could be a
consequence of the CGRP activation produced by stressful cooling presumed a
noxious cold sensation persists the efferent sympathetic influence has been
eliminated.
Compared the effects on the cardiovascular oscillations between groups, we
observed changes produced by L-NAME alone were differently affected by
HEX+L-NAME or GUA+L-NAME. There were different profiles of effects between
HEX+L-NAME and GUA+L-NAME particularly on the aspect of LFHRV and VLFHRV
powers (Fig. 3). In general, the results are consistent with the concept that a complete
vasoconstrictor response to stressful cooling depends on a functional sympathetic
system and the nitric oxide system.
We observed exposure to HEX+L-NAME caused a distinct inhibition of the
L-NAME-induced intensifications of LFBPV, LFHRV, VLFBPV, and VLFHRV powers and
weakened the coherence between the BPV and HRV at the LF region (K2
IBI/SBP<0.58)
under CS. On the basis that HEX interrupts the efferent limb of the baroreflex
feedback process, the results implicated that the elimination of this sympathetically
mediated mechanism responsible for the observed effect. The results also support
our proposition that sympathetic activation initiates the CIP reaction and then
elevates the endothelial nitric oxide production as a secondary BP-buffering system
that serves to modulate CEHP.
On the other hand, we observed exposure to GUA+L-NAME caused a distinct
diminution of the HEX+L-NAME-induced inhibition of LFHRV and VLFHRV powers but
still exerted a significant coherence between BPV and HRV at the LF region. The
results suggest that despite the superimposition of GUA, the sympathetic discharges
Page 17 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
18
were still responsive to the baroreflex feedback under CS. Because of the well-known
sparing effect of GUA on the adrenal medulla (Abercrombie and Davies 1963), the
results could be explained on the basis that stressful cooling causes
sympathoadrenal activation and thus enrich the plasma with epinephrine (Table 1).
The released epinephrine may circulate to the heart to induce the myocardial
oscillations mediated by the β-adrenoreceptors (Liu et al. 2015c).
Finally, we observed L-NAME, HEX+L-NAME, and GUA+L-NAME all three
interventions increased the magnitude of the appearance of Dn under all
experimental conditions (Fig 2 (B)). Overall these data demonstrated that nitric oxide
influences the appearance of Dn in the pressure wave. A higher presence of Dn
suggests the increased vascular resistance by modifying reflected pressure waves in
conduit artery (Politi et al. 2016), and also provides additional information about the
myogenic vascular responses to the hemodynamic perturbations.
In conclusion, the present study provides evidence that nitric oxide production
may contribute to the effects of cold stress on autonomic cardiovascular regulations.
The plasma nitric oxide levels appear to increase during a stressful cooling challenge,
resulting in preventing the pressor response to an increased level of sympathetic
activation and increasing blood flow that prevents tissue damage. Future studies
aimed at identifying the roles of sympathoadrenal activation and essential
adrenoreceptors could be useful to extend our understanding of the CEHP
mechanism.
Acknowledgments
The authors would like to thank Miss Chan-Fan Young for her technical assistance.
This work was supported by grants from the Ministry of Science and Technology
(MOST 102 &103-2320-B-350-001) and the Cheng Hsin General Hospital━National
Page 18 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
19
Defense Medical Center cooperative research project (CH-NDMC-105-4), Taipei,
Taiwan, ROC.
References
Abercrombie, G.F., and Davies, B.N. 1963. The Abercrombie, G.F., and Davies, B.N.
1963. The action of guanethidine with particular reference to the sympathetic
nervous system. Br. J. Pharmacol. Chemother. 20: 171-177.
Akselrod, S., Gordon, D., Madwed, J.B., Snidman, N.C., Shannon, D.C., and Cohen,
R.J. 1985. Hemodynamic regulation: investigation by spectral analysis. Am. J.
Physiol. 249(4 Pt 2): H867-875.
Arnal, J.F., Dinh-Xuan, A.T., Pueyo, M., Darblade, B., and Rami, J. 1999.
Endothelium-derived nitric oxide and vascular physiology and pathology. Cell. Mol.
Life Sci. 55(8-9): 1078-1087.
Ceccatelli, S., Lundberg, J.M., Zhang, X., Aman, K., and Hokfelt, T. 1994.
Immunohistochemical demonstration of nitric oxide synthase in the peripheral
autonomic nervous system. Brain Res. 656(2): 381-395.
Daanen, H.A. 2003. Finger cold-induced vasodilation: a review. Eur. J. Appl. Physiol.
89(5): 411-426. doi: 10.1007/s00421-003-0818-2.
Edwards, D.G., Gauthier, A.L., Hayman, M.A., Lang, J.T., and Kenefick, R.W. 2006.
Acute effects of cold exposure on central aortic wave reflection. J. Appl. Physiol.
100(4): 1210-1214. doi: 10.1152/japplphysiol.01154.2005.
Elfvin, L.G., Holmberg, K., Emson, P., Schemann, M., and Hokfelt, T. 1997. Nitric
oxide synthase, choline acetyltransferase, catecholamine enzymes and
neuropeptides and their colocalization in the anterior pelvic ganglion, the inferior
mesenteric ganglion and the hypogastric nerve of the male guinea pig. J. Chem.
Page 19 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
20
neuroanat. 14(1): 33-49.
Hu, C.T., Chang, K.C., Wu, C.Y., and Chen, H.I. 1997. Acute effects of nitric oxide
blockade with L-NAME on arterial haemodynamics in the rat. Br. J. Pharmacol.
122(6): 1237-1243. doi: 10.1038/sj.bjp.0701496.
Japundzic, N., Grichois, M.L., Zitoun, P., Laude, D., and Elghozi, J.L. 1990. Spectral
analysis of blood pressure and heart rate in conscious rats: effects of autonomic
blockers. J. Auton. Nerv. Syst. 30(2): 91-100.
Johnson, J.M., and Kellogg, D.L., Jr. 2010. Local thermal control of the human
cutaneous circulation. J. Appl. Physiol. 109(4): 1229-1238. doi:
10.1152/japplphysiol.00407.2010.
Kojda, G., and Kottenberg, K. 1999. Regulation of basal myocardial function by NO.
Cardiovasc. Res. 41(3): 514-523.
Kunz, T.H., Scott, M., Ittner, L.M., Fischer, J.A., Born, W., and Vogel, J. 2007.
Calcitonin gene-related peptide-evoked sustained tachycardia in calcitonin
receptor-like receptor transgenic mice is mediated by sympathetic activity. Am. J.
Physiol. 293(4): H2155-2160. doi: 10.1152/ajpheart.00629.2007.
Lessard, A., Salevsky, F.C., Bachelard, H., and Cupples, W.A. 1999. Incommensurate
frequencies of major vascular regulatory mechanisms. Can. J. Physiol. Pharmacol.
77(4): 293-299.
Lewis, T. 1926. The Blood Vessels of the Human Skin. BMJ 2(3418): 61-62.
Liu, Y.P., Lin, Y.H., Chen, Y.C., Lee, P.L., and Tung, C.S. 2015a. Spectral analysis of
cooling induced hemodynamic perturbations indicates involvement of sympathetic
activation and nitric oxide production in rats. Life Sci. 136: 19-27. doi:
10.1016/j.lfs.2015.06.011.
Liu, Y.P., Lin, Y.H., Lin, C.C., Lin, Y.C., Chen, Y.C., Lee, P.L., and Tung, C.S. 2015b.
Page 20 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
21
Role of Efferent Sympathoadrenal Effects in Cooling-Induced Hemodynamic
Perturbations in Rats: An Investigation by Spectrum Analysis. Chinese J. Physiol.
58(5): 312-321. doi: 10.4077/CJP.2015.BAD317.
Liu, Y.P., Lin, Y.H., Chen, Y.C., Lee, P.L., and Tung, C.S. 2015c. Role of
Beta-Adrenoceptors in Cooling-Evoked Hemodynamic Perturbations of Rats:
Investigation by Spectral Analysis, J. Hypertens. (Los Angeles) 4 209
doi:10.4172/2167-1095.1000209
Llorens, S., Jordan, J., and Nava, E. 2002. The nitric oxide pathway in the
cardiovascular system. J. Physiol. Biochem. 58(3): 179-188.
Morales, M.A., Holmberg, K., Xu, Z.Q., Cozzari, C., Hartman, B.K., Emson, P.,
Goldstein, M., Elfvin, L.G., and Hokfelt, T. 1995. Localization of choline
acetyltransferase in rat peripheral sympathetic neurons and its coexistence with
nitric oxide synthase and neuropeptides. Proc. Natl. Acad. Sci. U.S.A. 92(25):
11819-11823.
Nafz, B., Just, A., Stauss, H.M., Wagner, C.D., Ehmke, H., Kirchheim, H.R., and
Persson, P.B. 1996. Blood-pressure variability is buffered by nitric oxide. J. Auton.
Nerv. Syst. 57(3): 181-183.
Parati, G., Saul, J.P., Di Rienzo, M., and Mancia, G. 1995. Spectral analysis of blood
pressure and heart rate variability in evaluating cardiovascular regulation. A critical
appraisal. Hypertension, 25(6): 1276-1286.
Politi, M.T., Ghigo, A., Fernandez, J.M., Khelifa, I., Gaudric, J., Fullana, J.M., and
Lagree, P.Y. 2016. The dicrotic notch analyzed by a numerical model. Comput. Biol.
and Med. 72: 54-64. doi: 10.1016/j.compbiomed.2016.03.005.
Rastaldo, R., Pagliaro, P., Cappello, S., Penna, C., Mancardi, D., Westerhof, N., and
Losano, G. 2007. Nitric oxide and cardiac function. Life Sci. 81(10): 779-793. doi:
Page 21 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
22
10.1016/j.lfs.2007.07.019.
Richardson, D.W., and Wyso, E.M. 1960. Human pharmacology of guanethidine. Ann.
N. Y. Acad. Sci. 88: 944-955.
Robertson, D., Johnson, G.A., Robertson, R.M., Nies, A.S., Shand, D.G., and Oates,
J.A. 1979. Comparative assessment of stimuli that release neuronal and
adrenomedullary catecholamines in man. Circulation, 59(4): 637-643.
Stauss, H.M. 2007. Identification of blood pressure control mechanisms by power
spectral analysisClin. Exp. Pharmacol. Physiol. 34(4): 362-368. doi:
10.1111/j.1440-1681.2007.04588.x.
Yamazaki, F., Sone, R., Zhao, K., Alvarez, G.E., Kosiba, W.A., and Johnson, J.M.
2006. Rate dependency and role of nitric oxide in the vascular response to direct
cooling in human skin. J. Appl. Physiol. 100(1): 42-50. doi:
10.1152/japplphysiol.00139.2005.
Zimmerman, B.G., Brody, M.J., and Beck, L. 1960. Mechanism of the cardiac output
reduction by hexamethonium. Am. J. Physiol. 199: 319-324.
Page 22 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
23
Table 1 and caption
Table 1. Plasma catecholamine concentrations.
[To Editor: Please place Table 1 here]
Note: HEX, hexamethonium; GUA, guanethidine; NE, norepinephrine; EPI,
epinephrine; CS, cold stress (4°C ice-water immersion of the palms and soles);
PreCS, before CS. Data represent means ± SE. #, p<0.05 compared the same
catecholamine of CS to PreCS; a, p<0.05 compared with HEX; b, p<0.05 compared
with GUA.
Control (n=4) Hex (n=4) GUA (n=4)
PreCS NE pg/ml 888800000000.25.25.25.25 ±±±± 2222.30.30.30.30aaaa 53.53.53.53.52525252 ±±±± 0.40.40.40.48888bbbb 960960960960....87878787±±±± 0.10.10.10.12222
Epi pg/ml 315.28315.28315.28315.28 ±±±± 8.878.878.878.87a,ba,ba,ba,b 47.6247.6247.6247.62 ±±±± 1.541.541.541.54bbbb 1010.121010.121010.121010.12 ±±±± 7.177.177.177.17
CS NE pg/ml 888866660000....77779999 ±1.5±1.5±1.5±1.58888aaaa 44442222.90 .90 .90 .90 ±±±± 1.01.01.01.09999bbbb 990990990990.93.93.93.93 ±±±± 0.40.40.40.45555
Epi pg/ml 653.27653.27653.27653.27 ±±±± 9.679.679.679.67#,a,b#,a,b#,a,b#,a,b 58.1258.1258.1258.12 ±±±± 11.611.611.611.6bbbb 1498.261498.261498.261498.26 ±±±± 1.511.511.511.51####
Page 23 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
24
Figure captions
Fig. 1. General protocol for a rat in the experiment: (A) implantation of telemetry
device in rat 14 days before the testing day and (B) the experimental procedures of
the testing day in the following order, PreCS, CS, and PostCS. Three days after the
test, the experimental rats are sacrificed. The experimental groups were 0.9% NaCl
solution (Control Vehicle), the nitric oxide synthase inhibitor (L-NAME) alone,
hexamethonium superimposed on L-NAME (HEX+L-NAME), and guanethidine
superimposed on L-NAME (GUA+L-NAME). CS, cold stress (4 °C ice-water
immersion of the palms and soles); PreCS, 10 min before CS; PostCS, 20-30 min
after CS.
Fig. 2. Effects on (A) systolic blood pressure and heart rate and (B) the appearance
of the dicrotic notch of rats in the four experimental groups throughout the
experimental course. The control group rats were given the vehicle (0.9% NaCl
solution, n=12) 0.4 ml via a tail venous bolus injection for baseline comparisons. The
other three groups of rats were given the L-NAME alone (L-NAME, n=12) or with the
superimposition of hexamethonium (HEX+L-NAME, n=12) or guanethidine
(GUA+L-NAME, n=12). Values represent means ± SE. Note that statistical
significance only shows the differences between experimental groups (**p<0.01).
CS, cold stress (4 °C ice-water immersion of the palms and soles); PreCS, before CS;
PostCS, after CS; SBP, systolic blood pressure ; HR, heart rate; Dn, dicrotic notch.
Page 24 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
25
Fig. 3. Changes in the average spectral powers in the (A) very low-frequency and (B)
low-frequency regions for the blood pressure variability and heart rate variability of
the rats in the four experimental groups throughout the experiments. The module for
blood pressure variability or heart rate variability includes units of mmHg2 or ms2,
respectively. Values represent means ± SE. Note that significance only shows the
differences between experimental groups (*p<0.05, **p<0.01). CS, cold stress (4
°C ice-water immersion of the palms and soles); PreCS, before CS; PostCS, after CS;
VLF, very low frequency; LF, low frequency; BPV, blood pressure variability; HRV,
heart rate variability.
Fig. 4. The relationship between interbeat interval and systolic blood pressure
oscillations as assessed by peak coherence value (K2IBI/SBP) between blood pressure
variability and heart rate variability at the VLF, LF, and HF regions of rats in the four
experimental groups throughout the experiments. Values represent means ± SE. CS,
cold stress (4 °C ice-water immersion of the palms and soles); PreCS, before CS;
PostCS, after CS; K2IBI/SBP, peak coherence value; SBP, systolic blood pressure; IBI,
interbeat interval; VLF, very low frequency; LF, low frequency; HF, high frequency.
Page 25 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
254x190mm (300 x 300 DPI)
Page 26 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
176x243mm (300 x 300 DPI)
Page 27 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology
Draft
209x240mm (300 x 300 DPI)
Page 28 of 29
https://mc06.manuscriptcentral.com/cjpp-pubs
Canadian Journal of Physiology and Pharmacology