INTRODUCTION

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A CURRENT CONCEPT of importance in the study of thermal homeostasis is that its regulatory parameters are determined by the central and peripheral thermoreceptors. External cooling primarily affects the peripheral skin thermoreceptors. These receptors show static and dynamic activities. The functional significance of the two types of activity is still an open question. It is known that the cold receptors show hardly, if any, dynamic activity during cooling at a rate <0.01-0.02°C/s, whereas, at higher cooling rates, they begin to exhibit dynamic activity that increases with the rate of cooling (1, 12, 13). It is also known that the sympathoadrenal system is involved in the response to cold exposure, which is associated with an elevation of catecholamines in arterial plasma and urine (2, 3).

The aim of this study was to determine the characteristics of the activation of the sympathoadrenal system produced by slow external cooling, when there is no dynamic activity of the skin cold receptors, and by rapid cooling, when this dynamic activity is involved in the cold response.

	MATERIAL AND METHODS

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Animals. Male Wistar rats weighing 180-200 g were used. They were anesthetized with urethan at a dose 1.0-2 g/kg body wt.

Experimental protocol. Rectal and abdominal intracutaneous temperatures were measured with thermocouples. A part of the abdomen, 25 cm², which was depilated, was cooled with a special water thermode and thermostat. Resting temperature of the circulating water was 37.5°C. The cooling rate was either low (0.004-0.006°C/s) or high (0.03-0.05°C/s).

Blood samples (0.5 ml) were drawn from the femoral artery three times from each slowly or rapidly cooled animal: 1) before cooling, 2) at a rectal temperature decreased by 0.5°C, and 3) at a rectal temperature decreased by 3-4°C. Animals were given Ringer solution (0.5 ml iv) after each blood sampling as fluid replacement. Skin tissue samples (30-40 mg) were taken from controls that were not exposed to cold and from animals rapidly or slowly cooled when rectal temperature was decreased by 0.5°C when the dynamic response at rapid cooling should be greatest.

Catecholamine concentration in arterial plasma and in skin tissue was measured by HPLC with electrochemical detection (16).

Chemicals. Tris, sodium octyl sulfonate (SOS), EDTA, NE, Epi, and 3,4-dihydroxybenzylamine (DHBA) were obtained from Sigma (St. Louis, MO). All other reagents of highest purity were purchased from Russian manufacturers (Ministry of Chemical Industry).

Stock solutions of the standards (40 µg/ml) were prepared with the 0.1 M perchloric acid. Working solutions of the standards (10 ng/ml) in 0.1 M perchloric acid were prepared once a week and stored in refrigerator.

Blood probe preparation. Blood samples were collected in 1.5-ml Eppendorf vials containing the mixture of 5% EDTA and sodium methabisulfite solution. After centrifugation in the cold, plasma samples were added to the mixture of 1 M Tris buffer (pH 8.6), DHBA (10 ng/ml), and 10 mg alumina. In parallel standards, mixture (20 µl of each working standard) was prepared in 0.5 ml 0.1 M sodium phosphate buffer (pH 7.1) and processed in the same manner as plasma. After 15 min of shaking and centrifugation, liquid was removed by vacuum aspirator. Alumina was twice washed with 1 ml ice-cold bidistilled water or 0.02 M Tris buffer. Catecholamines were extracted from alumina with 20 µl of 0.1 M perchloric acid. Five microliters of this extract was injected into the column. Five microliters of standards mixture, processed in the same manner, was also injected in an HPLC system for determination of the recovery coefficient.

Chromatographic system. A Glass syringe high-pressure chromatographic pump Milichrom1 and an electrochemical detector with a glassy carbon electrode (both, "Nauchpribor," Orel, Russia) were used for blood catecholamine assay. The system was equipped with a stop-flow injection unit and stainless steel columns of 2 mm ID and 60 mm length, packed with 5 µm spheric sorbent Nucleosil C18 (Macherey-Nagel, Germany).

Mobile phase consisted of aqueous 0.05 M potassium dihydrogen phosphate; 0.05 M citric acid buffer, containing 0.5 g/l SOS; and 60 mg/l EDTA. Twice distilled methanol [10% (vol/vol)] was added to the buffer after titration at pH 4.86 with NaOH. Water was deionized and distilled on glass with the potassium permanganate. The eluent was filtered (0.2 µm, Millipore) and degassed before use. Eluent flow rate was 90 µl/min. The working electrode was operated at potential 0.6 V versus an Ag/AgCl reference electrode. The detector sensitivity range was set at 1 nA/V.

To obtain a homogenate of skin tissue, the skin was frozen in liquid nitrogen and homogenized with a porcelain pestle. Catecholamines were extracted from skin tissue homogenate with a 0.1 M solution of chloric acid. Further treatment of the sample and chromatographic procedure were the same as in the case of plasma measurements.

Evaluation. The data were treated for significance by Student's t-test.

	RESULTS

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The measured rectal temperature varied widely in the range of 35.5-38.5°C from one animal to another (probably due to urethan anesthesia). For this reason, it appeared worthwhile to determine the resting concentration of NE and Epi in arterial plasma. Figure 1 presents plasma concentration of NE (Fig. 1A) and Epi (Fig. 1B) as a function of the resting rectal temperature before cooling. Despite the rather wide individual variations, catecholamine concentration in plasma was not related to the resting rectal temperature.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Resting concentration of norepinephrine (NE; A) and epinephrine (Epi; B) in arterial plasma related to resting rectal temperature [r = 0.16 (NE), P > 0.05; r = 0.22 (Epi), P > 0.05].

The resting mean value for rectal temperature was 36.7 ± 0.3°C. The resting NE and Epi concentrations in plasma and in the skin characterizing urethan-anesthetized animals are given in Table 1.

A decrease in rectal temperature by 0.5°C in response to rapid cooling produced a 2.6-fold increase in plasma NE concentration and a 2.8-fold increase in plasma Epi (Fig. 2). Concomitantly, there was significant decrease by 28% in the skin NE and a greater decrease in skin Epi by 86% (Fig. 3).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Changes in NE and Epi values in arterial plasma produced by rapid (A) and slow (B) cooling of rats. Bars indicate SE. * Significant differences compared with resting values, P < 0.05.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Changes in NE and Epi values in skin produced by rapid (A) and slow (B) cooling of rats. Bars indicate SE. * Significant differences compared with resting values, P < 0.05.

At the same decrease in rectal temperature (by 0.5°C) after slow cooling, plasma catecholamine concentration did not change (Fig. 2), and, at an unaltered skin NE concentration, there occurred a reduction in skin Epi concentration that was, however, less marked compared with the one after rapid cooling (Fig. 3).

At rapid cooling, when rectal temperature was lowered more, by 3-4°C, the increase in plasma catecholamine concentration rose further: plasma NE by 3.7-fold and Epi by 3.5-fold. At the slow cooling rate, a decrease in rectal temperature by 3-4°C was associated with an increase in plasma NE of 3.0-fold and in plasma Epi by 2.2-fold (Fig. 2). It is worthy to note that the increase in plasma NE was virtually the same after deep slow and rapid cooling, whereas the increase in plasma Epi was greater after deep rapid than slow cooling (P < 0.05).

	DISCUSSION

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The results support and extend the idea that the sympathoadrenal system is activated by an external cold stimulus and furthermore reveal that the activation may be different depending on the cooling rate.

We have previously demonstrated the importance of the cooling rate in the formation of the cold defense responses (7, 8, 9). Experimental data we obtained in rats indicated that at rapid cooling, i.e., in the presence of the dynamic activity of the peripheral cold receptors, the metabolic response (an increase in total oxygen consumption) can be initiated with very short latency without any decrease in deep body temperature. This is consistent with the results obtained in humans during sudden cold water immersion (5, 6, 11). Furthermore, we have previously demonstrated that at rapid cooling rates (>0.01-0.02°C/s), the skin temperature threshold for the metabolic response was proportional to the cooling rate: the faster the cooling, the higher the temperature at which the metabolic response was elicited (7, 8). As indicated above, the dynamic activity of the skin cold receptors was initiated at a cooling rate higher than 0.01-0.02°C/s; dynamic activity increased with cooling rate and reached a plateau when the rate exceeded 0.1-0.2°C/s (1, 13). Thus it appeared reasonable to relate changes in the temperature threshold of the metabolic response at different cooling rates to the magnitude of the dynamic activity of the skin cold receptors.

In the discussion of these data we suggested that the changes in the formation of the cold defense responses during cooling at high rates are accompanied by different activation of the sympathetic nervous system. The present results are in agreement with and provide support for the original suggestion. The difference was more apparent at the early steps, when rapid cooling was accompanied by a considerable rise in plasma catecholamines and slow cooling was accompanied by an unaltered catecholamine level in plasma. Deeper cooling (rectal temperature decreased by 3-4°C), irrespective of its rate, produced about the same significant rise in plasma NE, although the rise in plasma Epi remained lower at the slow cooling rate. Here, too, the difference in the activation of the sympathoadrenal system occurring during rapid and slow cooling was retained.

Records of the impulse activity from sympathetic fibers showed that thermal stimuli did not have the same excitatory action on all the peripheral fibers, whereas the activities of the skin and visceral fibers changed in the opposite directions (4, 14, 15, 17). External cold stimuli, as well as local cooling of the hypothalamus and spinal cord, had excitatory effects on sympathetic activity in the skin and produced a reduction in visceral sympathetic effects. Heat elicited reverse actions: cutaneous fibers generally showed inhibitory responses and visceral excitatory effects. One plausible reason why plasma catecholamines rose under the effect of cooling may be their more intense release from the sympathetic nerve endings to blood. This might have caused the decrease in catecholamine concentration in skin that we observed in the present experiments. The skin catecholamines may also be related to constriction of the skin blood vessels, which is an adaptive response to lowered temperatures. It may be assumed that the diminution in the caliber of blood vessels was different in the slowly and rapidly cooled rats. When skin temperature was rapidly and not deeply lowered, the concentration of the two catecholamines in the skin decreased. When it was slowly lowered, the skin Epi level decreased somewhat less and that of NE remained unaltered.

Contrary to the observations made during not-deep rapid cooling, there were no changes in NE concentration in the skin and plasma during slow cooling. This may be because the sympathetic nerves were not activated when cooling was slow. The dynamic activity of the skin cold receptors during rapid cooling presumably provided conditions for an earlier activation of the sympathetic system and, as a consequence, made feasible a more rapid initiation of the metabolic response to a sudden decrease in temperature.

In another series of experiments (unpublished), a 3.5- to 4.0-fold increase in plasma NE and a much greater increase in skin NE was produced by an iontophoretic application of NE (4 mA to a skin surface of 25 cm² for 20 min). The application was not associated with cold exposure, and the rise in plasma and skin NE concentration did not result in an increase in total oxygen consumption. This was taken to mean that the rise in plasma catecholamines, a component of the cold defense response, was not the major determinant in its initiation. Relevant here were the wide variations in plasma catecholamines, showing no noticeable relation to body temperature. The input signals from the thermoreceptors appear to have an important role in triggering the response. One significant component was the dynamic activity of skin thermoreceptors, which could facilitate a more rapid activation of the sympathetic skin fibers and a decrease in the temperature threshold of the metabolic response to the effect of cold.