Draft - University of Toronto T-Space · 1990; Lessard et al. 1999; Parati et al. 1995; Stauss 2007). Recently, we performed a serial of studies to investigate the causes for CEHP

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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

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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]

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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

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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

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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

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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

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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

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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:

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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).

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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).


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

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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.


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.


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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

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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

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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

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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

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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,

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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

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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

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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

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Defense Medical Center cooperative research project (CH-NDMC-105-4), Taipei,

Taiwan, ROC.

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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####

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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.

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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.

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Documents

Draft - University of Toronto T-Space · 1990; Lessard et al. 1999; Parati et al. 1995; Stauss 2007). Recently, we performed a serial of studies to investigate the causes for CEHP