Rapid saline infusion and/or drinking enhance skin sympathetic nerve activity components reduced by hypovolaemia and hyperosmolality in hyperthermia

Abstract

Key points

• In hyperthermia, plasma hyperosmolality suppresses both cutaneous vasodilatation and sweating responses and this suppression is removed by oropharyngeal stimulation such as drinking. Hypovolaemia suppresses only cutaneous vasodilatation, which is enhanced by rapid infusion in hyperthermia.

• Our recent studies suggested that skin sympathetic nerve activity (SSNA) involves components synchronized and non‐synchronized with the cardiac cycle, which are associated with an active vasodilator and a sudomotor, respectively.

• In the present study, plasma hyperosmolality suppressed both components; drinking removed the hyperosmolality‐induced suppressions, simultaneously with increases in cutaneous vasodilatation and sweating, while not altering plasma volume and osmolality.

• Furthermore, a rapid saline infusion increased the synchronized component and cutaneous vasodilatation in hypovolaemic and hyperthermic humans.

• The results support our idea that SSNA involves an active cutaneous vasodilator and a sudomotor, and that a site where osmolality signals are projected to control thermoregulation is located more superior than the medulla where signals from baroreceptors are projected.

Abstract

We reported that skin sympathetic nerve activity (SSNA) involved components synchronized and non‐synchronized with the cardiac cycle; both components increased in hyperthermia and our results suggested that the components are associated with an active vasodilator and a sudomotor, respectively. In the present study, we examined whether the increases in the components in hyperthermia would be suppressed by plasma hyperosmolality simultaneously with suppression of cutaneous vasodilatation and sweating and whether this suppression was released by oropharyngeal stimulation (drinking). Also, effects of a rapid saline infusion on both components and responses of cutaneous vasodilatation and sweating were tested in hypovolaemic and hyperthermic subjects. We found that (1) plasma hyperosmolality suppressed both components in hyperthermia, (2) the suppression was released by drinking 200 mL of water simultaneously with enhanced cutaneous vasodilatation and sweating responses, and (3) a rapid infusion at 1.0 and 0.2 ml min⁻¹ kg⁻¹ for the first 10 min and the following 20 min, respectively, increased the synchronized component and cutaneous vasodilatation in diuretic‐induced hypovolaemia greater than those in a time control; at 0.1 ml min⁻¹ kg⁻¹ for 30 min no greater increases in the non‐synchronized component and sweating responses were observed during rapid infusion than in the time control. The results support the idea that SSNA involves components synchronized and non‐synchronized with the cardiac cycle, associated with the active cutaneous vasodilator and sudomotor, and a site of osmolality‐induced modulation for thermoregulation is located superior to the medulla where signals from baroreceptors are projected.

Key points

• In hyperthermia, plasma hyperosmolality suppresses both cutaneous vasodilatation and sweating responses and this suppression is removed by oropharyngeal stimulation such as drinking. Hypovolaemia suppresses only cutaneous vasodilatation, which is enhanced by rapid infusion in hyperthermia.

• Our recent studies suggested that skin sympathetic nerve activity (SSNA) involves components synchronized and non‐synchronized with the cardiac cycle, which are associated with an active vasodilator and a sudomotor, respectively.

• In the present study, plasma hyperosmolality suppressed both components; drinking removed the hyperosmolality‐induced suppressions, simultaneously with increases in cutaneous vasodilatation and sweating, while not altering plasma volume and osmolality.

• Furthermore, a rapid saline infusion increased the synchronized component and cutaneous vasodilatation in hypovolaemic and hyperthermic humans.

• The results support our idea that SSNA involves an active cutaneous vasodilator and a sudomotor, and that a site where osmolality signals are projected to control thermoregulation is located more superior than the medulla where signals from baroreceptors are projected.

Introduction

A human is unique in having a greater thermoregulatory capacity than other animal species, mediated by an increase in cutaneous blood flow and large amounts of perspiration in hyperthermia. Cutaneous vasodilatation decreases total peripheral resistance and pools blood in the peripheral veins in an upright position, and a loss of hypotonic sweat reduces plasma volume and elevates plasma osmolality. Both blood pooling in the peripheral veins and hypovolaemia decrease venous return to the heart and then threaten the maintenance of arterial blood pressure, resulting in syncope (Rowell 1986; Nose & Takamata 1997). To prevent this, cutaneous vasodilatation and sweating are modulated by non‐thermal factors, such as baroreflexes and/or osmoregulation.

Plasma hyperosmolality suppresses both cutaneous vasodilatation and sweating responses in hyperthermia (Fortney et al. 1984; Takamata et al. 1997, 1995; Ichinose et al. 2005; Kamijo et al. 2005 b; Shibasaki et al. 2009). On the other hand, hypovolaemia is not likely to suppress the sweating response but suppresses cutaneous vasodilatation in hyperthermic humans (Kamijo et al. 2005 b, 2011; Ikegawa et al. 2011; Ogawa et al. 2011), although there is a contradictory result (Dodt et al. 1995). The previous studies from our laboratory suggest that skin sympathetic nerve activity (SSNA) involved components synchronized and non‐synchronized with the cardiac cycle; the components increased with an increase in body core temperature, and are associated with an active vasodilator and a sudomotor, respectively (Kamijo et al. 2011; Ogawa et al. 2011). However, it remained unknown whether hyperosmolality would suppress both components in hyperthermia. Therefore, neural pathways for cutaneous vasodilatation and sweating modulated by plasma osmolality were not fully explained.

Although the microneurography technique to assess sympathetic nervous activity in humans was established in the 1970s (Hagbarth et al. 1972), there is a concern over comparison of the electrical signals between subjects and its reproducibility (Young et al. 2009). Thus, if hyperosmolality‐ and hypovolaemia‐induced suppressions could be released acutely while the position of an electrode was held constant, the technique may be able to assess changes in SSNA signals in response to the removal and to evaluate influences from hypovolaemia and/or hyperosmolality. It is well known that in hyperosmotic and hyperthermic subjects, drinking a glass of water immediately reduces plasma concentration of arginine vasopressin and the thirst sensation (Geelen et al. 1979; Takamata et al. 1995; Figaro & Mack 1997), the so‐called ‘oropharyngeal reflex’, which is a mechanism to prevent humans or animals from overhydration. Hyperosmotic suppression of sweating is also released by oropharyngeal stimulation, because it may attenuate an inhibitory input from the osmoregulatory centre to the thermoregulatory centre in the central nervous system (Takamata et al. 1995). On the other hand, saline infusion rapidly enhances an increase in blood flow to the skin during exercise (Nose et al. 1990) or passive heating (Crandall et al. 1999).

In the present study, we assessed thermoregulatory responses and the SSNA components in plasma hyperosmolality during passive warming and changes in those responses after drinking. Furthermore, the effects were tested of an acute restoration of plasma volume by rapid saline infusion on the synchronized component and cutaneous vasodilatation response in hypovolaemic and passively warmed men. Our hypotheses were that: (1) hyperosmolality would suppress both synchronized and non‐synchronized components in SSNA compared with isosmolality in hyperthermia, (2) oropharyngeal stimulation by drinking would release the hyperosmotic suppressions, simultaneously with enhancing cutaneous vasodilatation and sweating, and (3) the synchronized component and cutaneous vasodilatation response in hypovolaemia would be enhanced by an acute plasma volume restoration by rapid saline infusion in passively warmed men but the non‐synchronized component and sweating response would not.

Methods

Subjects

The procedures in this study conformed to the guidelines in the Declaration of Helsinki and were approved by the Review Board on Human Experiments, Shinshu University School of Medicine. After the experimental protocols had been fully explained, non‐smoking young males gave their written informed consent before participating in the study. Female subjects were excluded from the study because it was difficult for all female subjects to schedule an experiment in the same period of the menstrual cycle. In the first experiment (Exp. 1), 14 subjects were divided into isosmolality (Iso, n = 7) and hyperosmolality (Hos, n = 7) groups; there were no significant differences in any physical characteristics between the Iso and Hos groups: 25 ± 4 and 24 ± 4 years of age, 172 ± 6 and 176 ± 8 cm in height, and 65.8 ± 6.1 and 66.8 ± 7.3 kg body weight, respectively (all P > 0.353). In the second experiment (Exp. 2), 18 subjects were divided into rapid infusion (RI, n = 9) and time control (TC, n = 9) groups; there were no significant differences in any physical characteristics between the two groups: 25 ± 2 and 25 ± 2 years of age, 173 ± 2 and 173 ± 2 cm in height, 62.77 ± 2.20 and 67.12 ± 1.79 kg body weight, and 52.2 ± 2.9 and 52.9 ± 1.9 mL kg⁻¹ min⁻¹ peak oxygen consumption, respectively (all P > 0.144). To confirm the effects of hypovolaemia on thermoregulatory responses, values between Iso in Exp. 1 and RI in Exp. 2 were compared. There were no significant differences in characteristics between Iso and RI (all P > 0.367).

Experimental protocol

Subjects in both groups reported to the laboratory at 06.30 h with no breakfast. After they had emptied their bladders, they sat in a test room at ∼28°C ambient temperature and ∼50% relative humidity for insertion of a Teflon catheter (18 gauge) into the left antecubital vein. After the first blood sample was taken at 07.00 h as baseline, subjects ate a light meal (200 kcal; Calorie Mate, Otsuka Pharmaceutical Co., Ltd, Tokyo, Japan).

In the Hos group of Exp. 1, subjects were then given 0.5 mg kg⁻¹ of furosemide. After the first 1.5 h of the waiting period, hypertonic NaCl infusion (3%) was started at a rate of 0.16 ± 0.06 mL kg⁻¹ min⁻¹ for 70.8 ± 7.7 min (means ± SD); drinking was restricted until the time of drinking during the experiment and subjects did not consume any foods until the end of the experiment. In the Iso group, subjects rested in the room without taking any medicine or infusion for the 3.5 h waiting period; they could drink tap water for the first 2.5 h of the period and were able to void their bladder when necessary. The total urine volume in each person during the waiting period was 1384 ± 225 mL, significantly higher than Iso (577 ± 148 mL; P = 0.011). Fluid balance (= total water intake − total urine volume) during this period was −224 ± 200 and −461 ± 178 mL in the Iso and Hos groups, respectively, which was not significantly different (P = 0.393). In Exp. 2, all subjects were also given 0.5 mg kg⁻¹ of furosemide and then rested in the room for 3.5 h while voiding their bladder when necessary; they could drink water ad libitum for the first 2.5 h to prevent hyperosmolality, and after that, drinking and consuming food were restricted until the end of the experiment. Fluid balance during this period was −1236 ± 123 and −1521 ± 174 mL in the RI and TC groups, respectively, which was not significantly different (P = 0.201).

After the waiting period, subjects wore a tube‐lined perfusion suit covering the entire skin surface except the face, hands, feet and a leg where SSNA was measured. Subjects entered an experimental chamber at 28–29°C ambient temperature and ∼20% relative humidity and sat in a semi‐recumbent position. All measurement devices were applied, while water at 34°C was perfused through the suit to provide thermoneutrality. First, a thermoneutral measurement was performed for 10 min and a second blood sample was taken at the fifth minute of this condition. Then the temperature of perfused water was changed to 47°C to warm the whole body and data were collected continuously during the experimental trial.

In Exp. 1, the whole body was warmed for at least 45 min or until oesophageal temperature (T _oes) increased by 0.5°C. Then subjects drank 200 mL of tap water warmed to 37°C within a minute. Two subjects in the Hos group, whose increases in T _oes was over 0.5°C at 40 min after the onset of passive warming, drank tap water at 40 min of passive warming. Data were collected during the thermoneutral condition and passive warming until the 10th minute after drinking. In Exp. 2, after 45 min passive warming, subjects were infused with intravenous saline warmed with 37°C water at 1.0 mL kg⁻¹ min⁻¹ for the first 10 min and 0.2 mL kg⁻¹ min⁻¹ for the following 20 min in the RI group (the average rate for 30 min was 0.7 mL kg⁻¹ min⁻¹); in the TC group, the infusion rate was 0.1 mL kg⁻¹ min⁻¹ for 30 min.

The third, fourth and fifth blood samples were taken just before drinking and at the fifth and 10th minutes following drinking in Exp. 1 and just before the start of infusion and at 10 and 30 min following infusion in Exp. 2. After each trial, we determined maximal skin blood flow (SkBF), heating the skin site to ∼44°C for 30 min using an incandescent lamp, as reported previously (Kamijo et al. 2005 a, 2011).

Measurements

Microneurography

A tungsten microelectrode with an impedance of 4 MΩ at 1 kHz, 35 mm length, <5 μm tip and 200 μm shank diameter was inserted percutaneously into cutaneous nerve fascicles in the superficial peroneal nerve at the posterior aspect of the head of the fibula to record multiple‐unit postganglionic SSNA. To set a reference, an Ag–AgCl electrode (Vitrode Bs; Nihon Kohden Corp., Tokyo, Japan) was attached on the surface of the skin ∼5 cm from the recording electrode. The nerve signal was pre‐amplified 10000‐fold (DAM80; WPI Inc., Sarasota, FL, USA), transferred to a digital tape recorder and passed through a band‐pass filter of 700–2000 Hz, and then the signal was sent to a loud speaker and in parallel to a resistance–capacitance circuit to rectify and filter as time constant 0.1 s. We confirmed that the signals after rectifying and filtering met the following traditional criteria for SSNA: (1) subjects felt paraesthesia in the dorsal foot without any numbness, (2) a loud sound was evoked through the speaker by gentle touch within the innervated area, arousal stimulation and a deep breath, but (3) was not evoked by a Valsalva's manoeuvre as previously shown (Hagbarth et al. 1972; Kamijo et al. 2005 a, 2011).

SkBF and sweat rate

SkBF was measured by laser‐Doppler velocimetry (time constant = 0.1 s; moor VMS‐LDF2, Moor instruments, Axminster, UK) at the site of the ipsilateral dorsal foot, innervated by the peroneal nerve, but devoid of any superficial cutaneous veins. Sweat rate (SR) was measured by a capacitance hygrometer (Hygro Flex1, Rotronic Inst., Huntington, NY, USA). A small capsule was placed on the dorsal foot (0.79 cm² area) beside the laser‐Doppler probe to detect sweating. Dry air was suppiled through a small capsule placed on the foot at 60 ml min⁻¹. Relative humidity and temperature in the sweat capsule were monitored and SR was calculated from them. SkBF and SR were shown as averaged values every minute. Cutaneous vascular conductance (CVC) was calculated as SkBF/mean arterial pressure and is presented as a percentage of the maximal SkBF (% max).

Body temperatures

T _oes was measured by thermocouples in a polyethylene tube (PE90; Nippon Becton Dickinson Co., Tokyo, Japan). The tip of the tube was advanced at a distance of one‐fourth of the subject's standing height through the external nares. Skin temperatures were also measured by thermocouples at three sites and mean skin temperature (T _sk) was determined by the same method as in our previous study (Roberts et al. 1977).

Cardiovascular and respiratory responses

ECG was recorded by using an amplifier (Bioelectric Amplifier 1253A; San‐ei, Tokyo, Japan). Heart rate was calculated by averaged R–R interval of ECG every minute. Systolic and diastolic blood pressures were measured from the right upper arm placed at the heart level by inflation of the cuff with a sonometric pickup of Korotkoff's sound (STBP‐780, Colin, Komaki, Japan). Pulse pressure (=systolic blood pressure–diastolic blood pressure) and mean arterial pressure (=pulse pressure/3 + diastolic blood pressure) were calculated. Respiratory movement was monitored with a piezoelectric belt transducer (MLT1132; ADInstruments, Colorado Springs, CO, USA). Respiratory rate was calculated from voluntary respiratory movement.

Blood composition

A 1 mL aliquot of each 4‐mL blood sample was used to determine the haematocrit (as a percentage) and haemoglobin concentration (in g dL⁻¹). Haematocrit and haemoglobin concentration ([Hb]) were determined by the microcentrifuge and sodium lauryl sulfate haemoglobin methods, respectively. These values were used to calculate the percentage change (%Δ) in plasma volume (Greenleaf et al. 1979). Three millilitres of the aliquot was immediately centrifuged at room temperature and used to determine plasma osmolality (mosmol (kg H₂O)⁻¹) by freezing point depression method (one‐ten osmometer; FISK, Norwood, MA, USA).

Data acquisition

The rectified and filtered SSNA, ECG, SkBF and voluntary respiratory movement were recorded at a sampling rate of 200 Hz through an A/D converter using a computerized data‐acquisition system (AD16‐16U (PCI) EH, Contec, Tokyo). Skin temperatures and T _oes were recorded every 30 s with another A/D converter (34970A; Agilent Technologies, Santa Clara, CA, USA) and averaged every minute. The original SSNA signal was recorded off‐line from a digital tape recorder (RD‐180T, TEAC, Tama, Japan) at 20 kHz after passing through a band‐pass filter of 700–2000 Hz as in previous studies (Kamijo et al. 2005 a, 2011).

Analyses

Total SSNA (tSSNA) was determined by previous methods as follows. Peaks and leading and trailing edges of each SSNA burst were identified from a trace of the rectified and filtered SSNA signal using a program developed in our laboratory (MATLAB 7.1; The MathWorks, Natick, MA, USA). The amplitude of a burst was obtained by subtracting either the leading or the trailing edge value, whichever was lower, from the peak value (amplitude = peak value − lower edge value). If the amplitude was less than the level of twofold the baseline fluctuation during a 5–30 s silent period with no bursts in the thermoneutral condition, it was excluded from the following analyses. The area of a burst was determined by integrating the voltage every 1/200 s from the time at the leading edge to that at the trailing edge of each burst as given in the following formula:

$${\displaystyle \mathrm{area}=\frac{1}{200}\times {\int }_{t_{\mathrm{lead}}}^{t_{\mathrm{trail}}}A(t)\mathrm{d}t·[\mathrm{volt}·\mathrm{s}]}$$

where A(t) is the rectified and filtered signal of SSNA, and t _lead and t _trail are the times at leading and trailing edges, respectively. Then, the area was expressed as a percentage of the maximal values determined every minute under thermoneutral conditions (% max). SSNA was expressed as follows:

$${\displaystyle \mathrm{tSSNA}[n]=\sum ^{\mathrm{burst}\mathrm{number}\mathrm{per}\mathrm{the}n-min}\mathrm{area}(k)[\%max{min}^{-1}]}$$

The original SSNA spikes were also analysed with another program that was prepared in our laboratory by using the same software as above (Kamijo et al. 2011; Ogawa et al. 2011). As action potentials of SSNA were recorded extracellularly, the spikes were supposed to be negative. Thus, a negative threshold for detecting spikes was calculated as follows: (1) ∼0.5 s of the raw data before rectification and filtering with no incidence of spikes appearing during TN was selected, (2) we picked up all valley values from the selected data, (3) we then calculated the mean −3SD, (4) which was defined as the threshold. We chose SSNA spikes meeting the criterion of their valleys being below the threshold. Further, we excluded extremely high spikes of apparent artefacts from the following analysis. As we did not perform other noise cancelations, spikes detected by the present method might include some degree of artefacts with no relation to real nerve activities.

The SSNA signals synchronized and non‐synchronized with the cardiac cycles were determined as follows. First, a 5 s dataset of the original signals of SSNA triggered by a given R‐wave was selected (Fig. 1 B). The datasets were divided into 100 bins, of which each duration was 0.05 s. Spikes picked up by the above method were counted every 0.05 s bin and presented as a histogram (Kamijo et al. 2011). The same numbers of the 5 s incidence histograms as heart rate in each corresponding time were averaged every minute from the fifth minute of the thermoneutral condition to the 45th min of passive warming (50 min) and from 9 min before to 30 min after the onset of infusion (Fig. 1 C). As we found more than three peaks in the averaged spike incidence, we determined peak to peak intervals for the averaged histogram of SSNA (T _S) and that for R‐wave incidence (T _R) where the maximal value of the auto‐correlation function was observed. Similarly, we determined the latency of peak firing for SSNA after a given R‐wave (T _RS) by cross‐correlation analysis. We confirmed that the maximal values of auto‐correlation and cross‐correlation functions were significant (P < 0.05). If maximal correlation coefficients were not significant in some periods, we interpolated the values from the values at the closest times. Furthermore, the area above a valley of a periodic cycle in the histogram (the upper area; UA) was calculated as the synchronized component, and that below the valley (the lower area; LA) as the non‐synchronized component. UA and LA per minute (UA_min and LA_min, respectively) were calculated by multiplying UA and LA for 5 s with 60 s/5 s (Fig. 1 C).

Figure 1. The procedure of SSNA analyses.

A, ECG, raw, rectified and filtered (integrated) spikes of the skin sympathetic nerve activity (SSNA; left panels) and muscle sympathetic nerve activity (MSNA; right panels). B, ECG (upper) and original recording of SSNA (lower). The width of the dashed square represents 5 s. C, R‐wave and spike incidence histograms from the time of a given R‐wave of ECG in the 5‐s window with 0.05‐bin. The histograms were averaged for every minute (right panels). T _R and T _S, the peak‐to‐peak intervals of the R‐wave and the SSNA spike incidences, respectively, determined from the averaged histograms of each minute by auto‐correlation analysis. T _RS, the time between a given peak of R‐wave incidence and the following first peak of SSNA spike incidence by cross‐correlation analysis. The upper (UA) and the lower (LA) areas are defined as the components synchronized and non‐synchronized with the cardiac cycle, respectively. Data shown here are from one of each subject from the previous studies (A, Ogawa et al. 2017; B and C, Kamijo et al. 2011) with permission from the authors.

In Exp. 1, cardiovascular and respiratory responses were averaged during the thermoneutral condition, 40–45 min of passive warming (shown as 45 min of warming for convenience), and the period from 9 to 5 min before drinking and 0–5 and 6–10 min after drinking, shown as before drinking, 5 min and 10 min after drinking as a matter of convenience (Table 2). To determine sensitivities of increases in tSSNA, UA_min, LA_min, CVC and SR at a given increase in T _oes, we pooled whole data from the onset of warming to just before drinking and calculated regression equations between T _oes and them by using the standard least‐square method in each subject. The slope of the equation was defined as sensitivity (Fig. 3). T _oes, T _sk, tSSNA, UA_min, LA_min, CVC and SR were averaged during the thermoneutral condition, 38–40 min or 43–45 min of warming (shown as 45 min of warming in Results) and the period from 9 to 5 min before drinking (Table 3). To evaluate effects of drinking on tSSNA, UA_min, LA_min, CVC and SR, changes (Δ) in values at 5 min and 10 min after drinking from averaged values during 5 min before drinking were calculated (Fig. 4).

Table 2.

Cardiovascular and respiratory responses

Group		Passive warming				P
Exp. 1	TN	PW	Before	5AD	10AD	Group	Time	G × T
Heart rate (beats min−1)
Iso	64 ± 5	81 ± 7†	81 ± 7†	86 ± 6† , §	85 ± 6† , §	0.968	<0.0001	0.612
Hos	59 ± 3	81 ± 2†	79 ± 2†	86 ± 1† , §	84 ± 2† , §
Pulse pressure (mmHg)
Iso	50 ± 3	54 ± 3	55 ± 3	58 ± 2†	56 ± 3†	0.216	<0.0001	0.155
Hos	49 ± 3	60 ± 4†	58 ± 3†	58 ± 3†	60 ± 5†
Mean artrial pressure (mmHg)
Iso	89 ± 3	90 ± 2	89 ± 2	89 ± 2	89 ± 3	0.351	0.477	0.304
Hos	81 ± 3	85 ± 2	84 ± 3	84 ± 2	84 ± 2
Respiratory rate (breaths min−1)
Iso	17 ± 1	19 ± 1†	19 ± 1†	19 ± 1†	20 ± 1†	0.369	<0.0001	0.098
Hos	18 ± 1	20 ± 1†	20 ± 1†	20 ± 1†	20 ± 1†

Exp. 2	TN	PW	Before	Inf10	Inf30	Group	Time	G × T
Heart rate (beats min−1)
RI	66 ± 2	85 ± 5†	90 ± 6†	88 ± 5†	89 ± 5†	0.440	<0.0001	0.616
TC	69 ± 3	89 ± 3†	92 ± 4†	93 ± 4†	97 ± 5†
Pulse pressure (mmHg)
RI	45 ± 4	46 ± 5	44 ± 5	57 ± 4† , §	60 ± 6† , §	0.763	<0.0001	0.023
TC	43 ± 4	47 ± 5	48 ± 5	47 ± 6	53 ± 6†
Mean artrial pressure (mmHg)
RI	87 ± 2	87 ± 2	85 ± 2	87 ± 3	88 ± 2	0.793	0.038	0.276
TC	89 ± 4	87 ± 4	87 ± 4	84 ± 3	90 ± 5
Respiratory rate (breaths min−1)
RI	18 ± 1	20 ± 1	21 ± 1†	20 ± 1†	21 ± 1†	0.374	<0.0001	0.994
TC	17 ± 1	18 ± 1	19 ± 1†	20 ± 1†	19 ± 1†

Data are means ± SEM for 7 subjects of isosmolality (Iso) and hyperosmolality (Hos) groups in Exp. 1 and 9 subjects of rapid infusion (RI) and time control (TC) groups in Exp. 2. †Significant difference from TN; §difference from Before at the level of P < 0.05. P‐values are shown as main effects of group and time and an interactive effect of group and time (G × T). 5AD and 10AD, 5 and 10 min after drinking in Exp. 1; Inf10 and Inf30, 10 and 30 min after the onset of infusion, respectively, in Exp. 2; TN, thermoneutral condition; PW, ∼45 min after the onset of passive warming; Before, just before the onset of infusion.

Figure 3. Changes (Δ) in thermoregulatory responses at a given increase in oesophageal temperature (T _oes).

Values are means ± SEM for 7 subjects of each group. ^*Significant difference between groups, P < 0.05. All abbreviations are the same as Fig. 2.

Table 3.

Thermoregulatory responses during passive warming before drinking and infusion in the two experiments

				P
Group	TN	PW	Before	Group	Time	G × T
Exp. 1
T sk (°C)
Iso	34.9 ± 0.2	36.9 ± 0.1†	36.9 ± 0.1†	0.053	<0.0001	0.788
Hos	35.4 ± 0.1	37.2 ± 0.1†	37.2 ± 0.1†
T oes (°C)
Iso	36.6 ± 0.1	37.0 ± 0.1†	37.0 ± 0.1†	0.148	<0.0001	0.015
Hos	36.7 ± 0.1	37.2 ± 0.1* , †	37.3 ± 0.1†
tSSNA (% thermoneutral)
Iso	567 ± 66	1254 ± 262†	1372 ± 215†	0.028	<0.0001	0.061
Hos	396 ± 43	709 ± 124* , †	702 ± 98* , †
UAmin (counts min−1)
Iso	18.1 ± 2.7	28.1 ± 4.0†	28.1 ± 3.9†	0.105	0.0008	0.0056
Hos	17.4 ± 1.3	19.2 ± 2.9	17.7 ± 1.8*
LAmin (counts min−1)
Iso	45.7 ± 2.6	95.9 ± 13.0†	114.6 ± 8.6†	0.002	<0.0001	0.117
Hos	35.1 ± 3.1	59.9 ± 13.7* , †	66.6 ± 7.8* , †
CVC (% max)
Iso	6.4 ± 1.9	21.7 ± 2.4†	22.9 ± 2.4†	0.582	<0.0001	0.163
Hos	11.8 ± 2.9	24.5 ± 5.1†	22.1 ± 4.0†
SR (mg cm−2 min−1)
Iso	0.00 ± 0.01	0.33 ± 0.08†	0.34 ± 0.09†	0.522	<0.0001	0.566
Hos	0.00 ± 0.01	0.28 ± 0.08†	0.24 ± 0.06†
Exp. 2
T sk (°C)
RI	34.8 ± 0.1	36.8 ± 0.2†	37.0 ± 0.2†	0.369	<0.0001	0.201
TC	35.2 ± 0.1	37.0 ± 0.1†	37.0 ± 0.2†
T oes (°C)
RI	36.6 ± 0.1	37.1 ± 0.1†	37.2 ± 0.1†	0.580	<0.0001	0.512
TC	36.7 ± 0.1	37.2 ± 0.1†	37.2 ± 0.1†
tSSNA (% thermoneutral)
RI	506 ± 102	991 ± 356†	1110 ± 410†	0.870	0.0004	0.868
TC	623 ± 88	1054 ± 151†	1088 ± 151†
UAmin (counts min−1)
RI	16.4 ± 2.5	21.2 ± 3.8	19.8 ± 3.3†	0.448	0.015	0.587
TC	14.0 ± 1.7	17.2 ± 1.3	18.9 ± 1.7†
LAmin (counts min−1)
RI	39.5 ± 5.8	136.0 ± 65.7†	104.7 ± 24.4†	0.472	0.021	0.592
TC	39.0 ± 7.9	84.4 ± 10.5	91.1 ± 12.9
CVC (% max)
RI	9.2 ± 1.1	28.5 ± 7.3†	30.6 ± 6.8†	0.260	<0.0001	0.757
TC	14.9 ± 2.8	38.8 ± 7.4†	41.4 ± 8.2†
SR (mg cm−2 min−1)
RI	0.03 ± 0.02	0.34 ± 0.04†	0.39 ± 0.04†	0.714	<0.0001	0.752
TC	0.03 ± 0.01	0.33 ± 0.05†	0.35 ± 0.05†

Data are means ± SEM for 7 subjects of isosmolality (Iso) and hyperosmolality (Hos) groups in Exp. 1 and 9 subjects of rapid infusion (RI) and time control (TC) groups in Exp. 2. ^*Significant differences between the groups; ^†significant differences from TN at the level of P < 0.05. P‐values are shown as main effects of group and time and an interactive effect group and time (G x T). T _sk, mean skin temperature; T _oes, oesophageal temperature; tSSNA, total skin sympathetic nerve activity; UA_min, upper area per min; LA_min, lower area per min; CVC, cutaneous vascular conductance; SR, sweat rate; TN, thermoneutral condition; PW, ∼45 min after the onset of passive warming; Before, just before drinking and the onset of infusion.

Figure 4. Changes (Δ) in skin sympathetic nerve activity (SSNA) and effector‐organ responses after drinking.

Values are means ± SEM for 7 subjects of each group, isosmolality (Iso) and hyperosmolality (Hos). 5AD and 10AD, averaged values for 0–5 min and 6–10 min after drinking. ^*Significant difference between groups; #significant difference vs. just before drinking; P < 0.05. All abbreviations are the same as Fig. 2.

In Exp. 2, cardiovascular and respiratory responses (Table 2), T _oes, T _sk, tSSNA, UA_min, LA_min, CVC and SR were averaged during the thermoneutral condition, 43–45 min of warming (shown as 45 min of warming in Results) and the period from 10 to 6 min before infusion (Table 3), and during 9–11 min, 19–21 min and the last 3 min after the onset of infusion, shown as 10, 20 and 30 min after drinking, respectively, in Results for convenience. Changes (Δ) in these infusion variables were calculated from the values averaged during the 5‐min period before infusion (Fig. 5).

Figure 5. Changes (Δ) in skin sympathetic nerve activity (SSNA) and effector‐organ responses after the onset of infusion.

Values are means ± SEM for 9 subjects in both the rapid infusion (RI) and time control groups (TC) of Exp. 2. All abbreviations are the same as Fig. 4. ^*Significant difference between groups; †significant difference vs. baseline before infusion; P < 0.05.

Statistics

Two‐way (1 between (trial) and 1 within (time)) ANOVA for repeated measures was used for comparison of the variables between the groups and change from the baseline, thermoneutral condition, or just before drinking in Exp. 1 and the onset of infusion in Exp. 2 (Tables 1, 2, 3; Figs. 4 and 5). Assuming that main effects of d (= |μ₁–μ₂|/SD) for the variables in Figs. 4 and 5 within subjects were 1.5 and statistical power (1 – β) was >0.85 for ΔtSSNA, ΔUA, ΔLA,  ΔCVC and ΔSR after drinking at α of 0.05, we could determine that sample size was 7. Subsequent post hoc tests to determine significant differences in the various pairwise comparisons were performed using the Tukey–Kramer method. A sensitivity of increasing each response at a given increase in T _oes between the groups was analysed by Student's unpaired t test (Fig. 3). The standard least‐square method was used to determine regression equations between changes in UA_min and CVC and between changes in LA_min and SR (Fig. 6). All values are means ± SEM except where noted. The null hypothesis was rejected at P < 0.05.

Table 1.

Changes in plasma volume and osmolality in the two experiments

Group			Passive warming			P
Exp. 1	BL	TN	Before	5AD	10AD	Group	Time	G × T
ΔPV (% control)
Iso	0.0 ± 0.0	−0.7 ± 1.6	−4.1 ± 1.6‡	−5.0 ± 1.6† , ‡	−4.8 ± 1.8† , ‡	0.266	<0.0001	0.231
Hos	0.0 ± 0.0	3.2 ± 2.0	−0.6 ± 2.1†	−1.8 ± 2.2†	−3.2 ± 1.9† , ‡
Posmol (mosmol (kg H2O)−1)
Iso	294.9 ± 2.8	291.0 ± 1.2	292.6 ± 1.5	293.1 ± 0.8	294.6 ± 1.6	0.004	0.0027	<0.0001
Hos	292.2 ± 1.1	299.4 ± 0.7* , ‡	299.9 ± 1.4* , ‡	302.1 ± 1.6* , ‡	300.8 ± 1.5* , ‡

Exp. 2	BL	TN	Before	Inf10	Inf30
ΔPV (% control)
RI	0.0 ± 0.0	−14.1 ± 1.5‡	−17.4 ± 2.4‡	−0.4 ± 3.8† , §	−3.3 ± 2.8† , §	0.021	<0.0001	<0.0001
TC	0.0 ± 0.0	−13.1 ± 1.5‡	−17.7 ± 1.7† , ‡	−16.7 ± 2.1‡ , *	−18.3 ± 1.9† , ‡ , *
Posmol (mosmol (kg H2O)−1)
RI	290.6 ± 0.9	289.4 ± 1.0	291.8 ± 1.1‡	291.5 ± 0.9	291.3 ± 1.1	0.571	<0.0001	0.0047
TC	289.6 ± 1.4	288.7 ± 1.4	292.6 ± 1.4† , ‡	293.5 ± 1.6† , ‡ , *	294.9 ± 1.7† , ‡ , *

Data are means ± SEM for 7 subjects of isosmolality (Iso) and hyperosmolality (Hos) groups in Exp. 1 and 9 subjects of rapid infusion (RI) and time control (TC) groups in Exp. 2. ^*Significant differences between groups in each experiment; ^†differences from TN; ‡differences from BL; ^§differences from Before at the level of P < 0.05. P‐values are shown as main effects of group and time and an interactive effect of group and time (G × T). BL, baseline before the waiting period; TN, thermoneutral condition; Before, just before drinking and the onset of infusion in Exp. 1 and Exp. 2, respectively; 5AD and 10AD, 5 and 10 min after drinking in Exp. 1; Inf10 and Inf30, 10 and 30 min after the onset of infusion, respectively, in Exp. 2; P_osmol, plasma osmolality.

Figure 6. Relationships between the SSNA components and effector‐organ responses.

Values are means ± SEM for 7 subjects of isosmolality (Iso; open circle) and hyperosmolality (Hos; filled circle) groups in Exp. 1, and 9 subjects of rapid infusion (RI; filled circle) and time control (TC; open circle) groups in Exp. 2. A, relationship between changes (Δ) of upper area per min (UA_min) and cutaneous vascular conductance (CVC) from value before drinking in Exp. 1 (r = 0.956; P = 0.0013). B, relationship between changes (Δ) of lower area per min (LA_min) and sweat rate (SR) from value before drinking in Exp. 1 (r = 0.961; P = 0.0001). C, relationship between ΔUA_min and CVC from before the onset of infusion in Exp. 2 (r = 0.949; P = 0.00015). D, relationship between ΔLA_min and SR from before the onset of infusion in Exp. 2 (r = 0.924; P = 0.00059).

Results

Comparisons between RI and Iso (effects of hypovolaemia)

To confirm the effects of hypovolaemia, we compared increases in tSSNA, UA_min, LA_min, CVC and SR at an increase in T _oes in the RI (hypovolaemia) and Iso (normovolaemia) groups, even though the two were in different experiments. Before the treatment in the morning, [Hb] was ∼15.5 g dL⁻¹ in both RI and Iso groups (P = 0.618). After the treatment (taking diuretics), plasma volume was 13% lower in the RI than in the Iso group during the thermoneutral condition and passive warming just before drinking (P < 0.0001). Plasma osmolality was similar between the two groups (P = 0.124). tSSNA, UA_min and LA_min increased with an increase in T _oes in the Iso and RI groups with increases in CVC and SR. Slopes of tSSNA, LA_min and SR in response to an increase in T _oes were similar between the two groups (P = 0.145, 0.706 and 0.336, respectively). A slope of UA_min was significantly lower in the RI than in the Iso group (P = 0.038). A slope of CVC to T _oes was also significantly lower in the RI than in the Iso group (P = 0.04) after excluding one outlier value by the Smirnoff–Grubbs test of rejection.

Comparisons between Iso and Hos (effects of hyperosmolality; Exp. 1)

There were no significant changes in Δplasma volume before and after drinking in the Iso and Hos groups and no significant differences between the two groups (Table 1; P = 0.266). Plasma osmolality was 6–9 mosmol (kg H₂O)⁻¹ higher in the Hos than in the Iso group (P = 0.004) during passive warming, before and after drinking, from the thermoneutral condition with a significant interactive effect of Group × Time (P < 0.0001). Increases in heart rate during passive warming were similar (Group × Time; P = 0.612) and mean arterial pressure remained unchanged through the experiment in both groups (Table 2; P = 0.477). T _sk was ∼35°C during the thermoneutral condition and increased by ∼2°C during passive warming for 40 or 45 min, with no significant differences between the groups (P = 0.053). T _oes increased by 0.4 and 0.6°C during passive warming before drinking with significant interaction of Group × Time (Table 3; P = 0.015). tSSNA, UA_min, LA_min, CVC and SR increased during passive warming (Fig. 2; Table 3; P = 0.006), but the increases in these at a given increase in T _oes were significantly suppressed in the Hos compared with the Iso group except for SR (Figs. 2 and 3; P = 0.017, 0.006, 0.008, 0.022, and 0.090, respectively).

Figure 2. Thermoregulatory responses to increased oesophageal temperature (T _oes) during passive warming in the isosmolality (Iso; open circles) and the hyperosmolality (Hos; grey circles) groups of Exp. 1.

Values are means ± SEM for 7 subjects of each group. CVC, cutaneous vascular conductance; SR, sweat rate; tSSNA, total skin sympathetic nerve activity; UA_min and LA_min, upper and lower areas of spike firing curve per minute, respectively.

ΔPlasma volume was similar between groups and plasma osmolality was 7 mosmol (kg H₂O)⁻¹ higher in the Hos group but both values remained unchanged before and after drinking (Table 1). ΔtSSNA, ΔUA_min and ΔLA_min were significantly higher at 5 and 10 min after drinking than the values before drinking in the Hos group (all P < 0.025), while no significant increases were observed in the Iso group (Fig. 4). ΔCVC in the Hos group was significantly increased at 5 and 10 min after drinking (P = 0.003), while that in the Iso group remained unchanged (P = 0.161). Furthermore, ΔSR was significantly increased at 5 and 10 min after drinking in both groups (P < 0.0001), but the increase was higher in the Hos than in the Iso group (P = 0.003). ΔCVC and ΔSR were positively correlated with ΔUA_min (r = 0.956; P = 0.0013) and ΔLA_min (r = 0.961; P = 0.0001), respectively (see Fig. 6).

Heart rate during the thermoneutral condition was 64 beats min⁻¹ in the Iso group, similar to that in the Hos group, and increased to ∼85 beats min⁻¹ just before drinking (P < 0.0001), with no significant differences between the groups (P = 0.968). After drinking, heart rate increased by ∼6 beats min⁻¹ in both groups with no significant interaction of Group × Time (Table 2; P = 0.612). Mean arterial pressure remained unchanged before and after drinking in both groups (Table 2; P = 0.477).

Comparisons between RI and TC (effects of a rapid infusion; Exp. 2)

ΔPlasma volume was −17.4% and −17.7% before infusion in the RI and TC groups, respectively, with no significant difference between the groups (Table 1). Plasma volume returned to baseline level 10 min after infusion in the RI group but not in the TC group; it was significantly higher in the RI than in the TC group at 10 and 30 min after drinking (Table 1; both P < 0.002). Plasma volume remained unchanged before and during infusion. There were no significant differences in T _sk and T _oes before and during infusion in the two groups.

ΔUA_min and ΔCVC were increased at 30 min and at 20 and 30 min after drinking, respectively, in the RI group, while those in the TC group remained unchanged, with significant interactions of Group × Time (Fig. 5; P = 0.027 and 0.0001, respectively). ΔtSSNA, ΔLA_min and ΔSR increased during infusion in both groups but there were no significant differences between the groups. As shown in Fig. 6, we also found significant correlations between ΔUA_min and ΔCVC (r = 0.936; P = 0.0003) and between ΔLA_min and ΔSR (r = 0.938; P = 0. 0003) when the averaged values in the RI and TC groups were pooled. On the other hand, there were no significant correlations between ΔLA_min and ΔCVC (r = 0.302; P = 0.463) or between ΔUA_min and ΔSR (r = 0.532; P = 0.168) (not shown).

Heart rate during the thermoneutral condition was 66 beats min⁻¹ in the RI group, similar to that in the TC group, and increased to ∼85 beats min⁻¹ just before infusion (P < 0.0001), with no significant differences between the groups (P = 0.440). ΔHeart rate was increased at 20 and 30 min after drinking from the values before infusion in the TC group (all P < 0.0003) and was significantly higher than in the RI group at 10, 20 and 30 min after drinking (all P < 0.0008) with significant interactive effects of Group × Time (P = 0.0003). ΔMean arterial pressure remained unchanged in the RI group during the infusion period (all P > 0.29) but was significantly increased at 30 min after drinking from the values before infusion in the TC group (P = 0.0007). Pulse pressure increased during infusion in both groups (all P < 0.03) and the increases were higher in the RI than in the TC group at 10, 20 and 30 min after drinking (all P < 0.0009) with significant interactive effects of Group × Time (P < 0.0001).

Discussion

In the present study, we found in Exp. 1 that (1) increases in UA_min and LA_min were suppressed in the Hos compared to those in the Iso group simultaneously with suppressed CVC and a tendency for suppressed SR, (2) both the components CVC and SR were enhanced by drinking only in the Hos group, and in Exp. 2 that (3) UA_min and CVC were enhanced in the RI group, while they remained unchanged in the TC group, but (4) the tSSNA, LA_min and SR responses were not enhanced by the rapid infusion. The results support our concept that cutaneous vasodilatation and sweating responses are associated with synhcronized and non‐synchronized components of SSNA with the cardiac cycle, respectively (Kamijo et al. 2011; Ogawa et al. 2017).

SSNA and CVC between normovolaemia (Iso) and hypovolaemia (RI)

We have confirmed that an increase in UA_min was suppressed in the RI group (i.e. the hypovolaemic condition) compared with that in the Iso group (i.e. euhydration) with similar plasma hyperosmolality and that an increase in CVC at a given increase in T _oes also tended to be lower than that in the Iso group but increases in LA_min and SR were similar between the groups (see Results). The results suggest that SkBF control is modulated by unloading baroreceptors, but the sweating response is not.

As reported previously, cutaneous vasodilatation was suppressed by lower body negative pressure less than −20 mmHg, while arterial pressure remained unchanged (Johnson et al. 1974); was enhanced with negative‐pressure breathing, even though arterial blood pressure decreased (Nagashima et al. 1998); and was not altered by unloading of carotid baroreceptors with neck compression (Crandall et al. 1996). The results suggest that control of SkBF is likely to be modulated by unloading or loading of cardiopulmonary baroreceptors.

Effects of plasma hyperosmolality on SSNA components and thermoregulatory responses

Plasma hyperosmolality suppresses both cutaneous vasodilatation and sweating in hyperthermia (Fortney et al. 1984; Takamata et al. 1997, 1995; Ichinose et al. 2005; Kamijo et al. 2005 b; Shibasaki et al. 2009). However, there has been no direct evidence that SSNA components synchronized and non‐synchronized with cardiac cycles would be involved in these mechanisms. Based on the previous findings, we believed that UA_min and LA_min are associated with cutaneous vasodilatation and sweating responses in hyperthermia, respectively. Both increases in UA_min and LA_min were attenuated by hyperosmolality with suppressed increases in CVC and SR. Although response patterns of UA_min and CVC and those of LA_min and SR after drinking were not exactly match as shown in Fig. 4, the high correlations between changes in UA_min and CVC and between LA_min and SR suggest that UA_min and LA_min are associated with CVC and SR, respectively.

Even slopes of increases in CVC and SR at a given increase in T _oes were suppressed by hyperosmolality in the present study; the results did not seem to be consistent with several previous studies (Takamata et al. 1997; Ichinose et al. 2005; Shibasaki et al. 2009), which showed that plasma hyperosmolality delayed the onset of cutaneous vasodilatation and sweating but did not suppress the slopes of both cutaneous vasodilatation and sweating at a given increase in T _oes. In the present study, we pooled whole data from the onset of warming to just before drinking and calculated a slope of regression equation in each subject; because the range of increase in T _oes was smaller than that in the previous studies, technically it was hard to distinguish a threshold. Another reason for the discrepancy is explained by regional difference at the measurement sites. There could be regional difference in CVC and SR responses in hyperthermia, e.g. peak forearm blood flow during one‐legged exercise was about 3 times that in the calf in the non‐active leg (Nishiyasu et al. 1992). We measured these responses on the dorsal foot. This was different from the previous studies where measurements were taken at sites such as on the chest and/or forearm (Takamata et al. 1997; Ichinose et al. 2005; Shibasaki et al. 2009). Differences in analysis procedure and measurement sites would account for the discrepancy.

The level of osmolality modulation for cutaneous vasodilatation and sweating in the central nervous system may be superior to that for baroreflexes. Nakashima et al (1985) assessed a firing rate of warm‐sensitive neurons in a slice of hypothalamus in rats that was soaked in reflux fluid with isosmolality or hyperosmolality (+15 mosmol (kg H₂O)⁻¹), while fluid temperature was changed from 34 to 40°C. They reported that a threshold for enhanced firing rate at a given increase in fluid temperature was shifted toward higher temperature in the hyperosmolality condition. The results suggested that hyperosmolality is able to suppress firing rate of warm sensitive neurons in the thermoregulatory centre, i.e. the hypothalamus, including the pre‐optic area.

Effects of drinking on SSNA components in plasma hyperosmolality

A previous study suggested that oropharyngeal stimulation by drinking released hyperosmotic suppression of sweating simultaneously with reduced thirst sensation and plasma concentration of arginine vasopressin reflexively (Takamata et al. 1995). Because a level of plasma volume and plasma osmolality were not altered by drinking, the mechanism of the increments would involve a central nervous pathway. However, there was no direct evidence for it. We asked subjects to drink 200 mL of warmed water as in the previous studies (Takamata et al. 1995; Kamijo et al. 2005 b) and the level of plasma volume and plasma osmolality remained unchanged before and after drinking in the present study (Table 1). Although we did not assess plasma concentration of arginine vasopressin and/or the thirst sensation before and after drinking in the present study, the enhancements of UA_min and LA_min after drinking were likely to be caused by reflex effects of oropharyngeal stimulation.

The present results partially conflicted with the previous study (Takamata et al. 1995), which showed no increase in SkBF after drinking, while arterial blood pressure remained unchanged. This discrepancy may be explained by the level of SkBF before drinking. The previous study showed that SkBF had a similar level in hyperosmolality to that in euhydration. On the other hand, the sweating response was attenuated in hyperosmolality by ∼60% compared with euhydration before drinking even T _oes was 0.6°C higher in hyperosmolality. There seemed to be not enough reserve to increase CVC like SR in the previous study.

Effects of a rapid infusion on synchronized component of SSNA and CVC in hypovolaemia

Several previous studies have evaluated the effects of venous saline infusion in hyperthermic subjects. Crandall et al. (1999) reported that saline infusion at the rate of 1.5–2.1 mL kg⁻¹ min⁻¹, which is 50–100% higher than in the present study, increased cutaneous vasodilatation responses by 30% during passive heating, which elevated T _oes by 0.7–0.8°C, while central venous pressure increased by 4 mmHg. Even a decrease in right atrial pressure by ∼2 mmHg during exercise due to reduced venous return to the heart suppresses further increase in forearm SkBF (Nose et al. 1994), and the attenuation was removed by venous saline infusion at ∼0.30 ml kg⁻¹ min⁻¹ (Nose et al. 1990). As venous compliance during exercise is at least one‐fifth of that at rest, the rate of infusion would correspond to over 1.0 ml kg⁻¹ min⁻¹. The rate of the rapid infusion would be enough to restore cardiac filling pressure by an increase in venous return to the heart.

In the present study, UA_min and CVC in the RI group increased after the onset of infusion (Fig. 5), while UA_min and CVC in the TC group remained unchanged throughout the infusion period, even though T _oes significantly increased in the TC group. Moreover, the increase in CVC during infusion correlated with that in UA_min (Fig. 6), tendencies that matched the results in our previous studies (Kamijo et al. 2011; Ogawa et al. 2017). In addition, heart rate and mean arterial pressure remained unchanged in the RI group during the infusion period, and even pulse pressure increased by ∼15 mmHg during infusion in the RI group. Therefore, the restoration of plasma volume possibly enhanced UA_min through removing unloading of baroreceptors, such as the cardiopulmonary baroreceptor, and then increased CVC, although it remains controversial whether arterial‐baroreceptor distention would contribute to this mechanism.

Despite the correlation between ΔUA_min and ΔCVC, the increase in UA_min was delayed compared with that in CVC during rapid infusion. The reasons are not clear, but two explanations are adapted. One is that the initial increase in CVC would not be completely related to sympathetic nervous activity, but partially related to local effects, such as a shear stress as suggested by an increase in pulse pressure. Mechanical signals including shear stress produces nitric oxide through an activation of endotherial nitric oxide synthase (eNOS), inducing smooth muscle cells via cGMP (Hellsten et al. 2012). Smooth muscle cells in cutaneous vasculature have eNOS and cutaneous vasodilatation induced by local heating is mediated by eNOS (Kellogg et al. 2009). Therefore, an increase in shear stress caused by rapid infusion would partially initiate cutaneous vasodilatation with local mechanisms.

The other explanation is that UA_min is possibly underestimated as an index of cutaneous vasodilatation; in other words, LA_min partially contributes to the initial increase in CVC. The possibility cannot be completely ruled out that LA_min contains a component related to cutaneous vasodilatation. However, because we also have found no correlation between LA_min and CVC during the infusion period, the contribution would be low.

Effects of a rapid infusion on non‐synchronized component of SSNA and SR in hypovolaemia

The present study confirmed that LA_min and SR increased after the onset of infusion in both groups, but there were no significant interactions of Group × Time (Fig. 5). A significant correlation between the increases in LA_min and SR was also observed (Fig. 6). In our previous study (Kamijo et al. 2011), LA_min increased by ∼60 counts min⁻¹ in the normovolaemic and hypovolaemic groups with no significant difference, while T _oes increased by ∼0.6°C at the end of 45 min of passive warming. This was accompanied by ∼0.35 mg cm⁻² min⁻¹ increase in SR in both groups, corresponding to the present results.

Central pathways for cutaneous vasodilatation and sweating

Collectively, the present results and our previous findings suggest that central pathways for cutaneous vasodilatation and sweating would be different. All thermal information, such as increases in core and skin temperatures, integrates in the thermoregulatory centre located in the pre‐optic area in the hypothalamus (Boulant, 1996). A pathway for cutaneous vasodilatation travels via the cardiovascular centre in the medulla to the spinal cord, and then a signal is conducted to skin sympathetic nerves, inducing cutaneous vasodilatation. Thus, unloading baroreceptors due to hypovolaemia could modulate the cutaneous vasodilatation response at the level of the medulla, where signals from baroreceptors are projected. However, that for sweating may not go through the sites in the medulla, because the previous studies reported that the sweating response was not altered by unloading baroreceptors (Vissing et al. 1994; Wilson et al. 2001, 2005; Kamijo et al. 2005 b, 2011; Ikegawa et al. 2011; Ogawa et al. 2017). Moreover, as the sweating response is suppressed by hyperosmolality simultaneously with suppressed cutaneous vasodilatation in hyperthermia, a site where the sweating response is modulated by osmolality is located at a higher level than the medulla.

Possible sex differences

We did not recruit females in the present study, even though sex differences in SSNA and cutaneous vascular and sweating responses were expected. Previous studies reported that thermoregulatory responses in females were different from those in males; e.g. cholinergic cutaneous vasodilatation was modulated by a lower concentration of ATP in females than males while modulation by ATP of the sweating response was similar (Fujii et al. 2015); the sweating response to increase in mean body temperature was attenuated in females compared with in males (Inoue et al. 2005; Gagnon et al. 2013) even though increased cutaneous vasodilatation was similar between both groups (Gagnon et al. 2013); and a SSNA response during exposure to emotional stimulus was different between females and males, while sweating and cutaneous vasoconstriction were not always consistent with the increased SSNA (Brown & Macefield 2014). There were no sex differences in NO‐dependent cutaneous vasodilatation during local heating (Stanhewicz et al. 2014) and cutaneous adrenergic vasoconstriction to exogenous noradrenaline (Greaney et al. 2014). Because of the non‐uniform results from these previous studies, it is difficult to interpret whether sex differences in SSNA and the end‐organ responses are present.

Limitations

As shown in Table 1, plasma osmolality was 2.0–3.6 mosmol (kg H₂O)⁻¹ higher in the TC than in the RI group after 10 min of infusion. As the T _oes thresholds for cutaneous vasodilatation and sweating increased by ∼0.2°C when plasma osmolality was increased by ∼4 mosmol (kg H₂O)⁻¹ (Takamata et al. 1997), the increase in CVC in the present study was suppressed by ∼7%. However, ΔCVC was ∼15% higher in the RI than in the TC group at the end of infusion (Fig. 5), equivalent to ∼40% of the peak CVC observed during ∼45 min of passive warming before infusion in both groups. Additionally, the lower ΔSR in the RI compared with the TC group after infusion was likely explained by the attenuated increase in ΔT _oes. Thus, the increase in plasma osmolality observed in the TC group is unlikely to have contributed to the suppression of cutaneous vasodilatation and SR.

The increase in T _sk (Table 3) was significantly lower in the RI than in the TC group during infusion while the body was passively warmed. This may be due to the use of insufficiently warmed water for infusion; however, the difference in T _sk between the RI and TC groups was only ∼0.7°C and changes in T _sk during infusion in both groups were less than 0.3°C. This was too small to influence thermoregulatory responses (Nadel et al. 1971; Wenger et al. 1975).

In conclusion, the results support our concept that the SSNA components synchronized and non‐synchronized with the cardiac cycle are an active cutaneous vasodilator and a sudomotor, respectively, and that the osmoregulatory site is located at a higher level of the medulla where signals from baroreceptors are projected.

Additional information

Competing interests

The authors disclose any conflict of interests.

Author contributions

Y‐i.K. and H.N. designed the study; Y‐i.K., K.O., S.I. and Y.O. performed the experiments; Y‐i.K., Y.O., S I. and H.N. analysed the data; Y‐i.K. wrote the paper; all authors contributed to revise the paper. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

This work was supported in part by grants from The Ministry of Education, Culture, Sports, Science, and Technology of Japan (18700529 to Y‐i.K. and 24240089 to H.N.) and a fellowship from The Uehara Memorial Foundation (200810009 to Y‐i.K.).

Biography

Yoshi‐ichiro Kamijo is an Associate Professor at the Department of Rehabilitaion Medicine, Wakayama Medical University. He graduated from Shinshu University, School of Medicine in 1997, and obtained a PhD in the Department of Sports Medical Science under the guidance of H.N. He worked in the John B. Pierce Lab., Yale University from 2001 to 2003 (PI: Dr Gary W. Mack), and then returned to the same department in Shinshu University until 2015, while carrying out research into body fluid regulation and thermoregulation in humans.