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Am J Physiol Regul Integr Comp Physiol 277: R47-R55, 1999;
0363-6119/99 $5.00
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Vol. 277, Issue 1, R47-R55, July 1999

Heat acclimation and hypohydration: involvement of central angiotensin II receptors in thermoregulation

Michal Horowitz1, Pavel Kaspler1, Eckhart Simon2, and Ruediger Gerstberger2

1 Department of Physiology, Hadassah Schools of Dental Medicine and Medicine, The Hebrew University, Jerusalem 91120, Israel; and 2 Max-Planck-Institute for Physiological and Clinical Research, W. G. Kerckhoff Institute, D-61231 Bad Nauheim, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This investigation attempted to confirm the involvement of central ANG II-ergic signals in thermoregulation. Experiments were conducted on rats undergoing short (STHA)- and long (LTHA)-term heat acclimation, with and without superimposed hypohydration. Vasodilatation (VTsh) and salivation (STsh) temperature thresholds, tail blood flow, and heat endurance were measured in conscious rats during heat stress (40°C) before and after losartan (Los), an ANG II AT1-selective receptor antagonist, administration either to the lateral ventricle or intravenously. Heat acclimation alone resulted in decreased VTsh. STsh decreased during STHA and resumed the preacclimation value, together with markedly increased heat endurance on LTHA. Hypohydration did not affect this biphasic response, although STsh was elevated in all groups. The enhanced heat endurance attained by LTHA was blunted. Neither Los treatment affected the nonacclimated rats. In the heat-acclimated, euhydrated rats, intracerebroventricular Los resulted in decreased VTsh, whereas intravenous Los resulted in elevated STsh. Both intracerebroventricular and intravenous Los led to markedly enhanced heat endurance of the LTHA hypohydrated rats. It is concluded that the LTHA group showed a loss of the benefits acquired by acclimation on hypohydration, whereas the STHA rats, which show an accelerated autonomic excitability in that phase, gained some benefit. It is suggested that ANG II modulates thermoregulation in conditions of chronic adjustments. Central ANG II signals may lead to VTsh upshift, whereas circumventricular structures, activated via circulating ANG II, decrease STsh. On hypohydration these responses seem to be desensitized.

AT1 receptors; losartan; vasodilatation; salivation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE STATE OF BODY HYDRATION has a marked effect on thermoregulation during heat stress. This is manifested mainly by delayed and attenuated heat dissipation activity, leading to an upward shift of the regulated deep body temperature. To compensate for reduced heat dissipation, basal metabolic rate may be lowered to decrease the internal heat load. Despite these changes, however, ultimately hypohydration interferes with thermal endurance as the need for water conservation overrides thermoregulation. Observations such as elevated temperature threshold (T-Tsh) for activation of the heat-dissipating mechanism (3, 5, 14), reduced sensitivity of the thermoregulatory center in converting temperature signals into heat loss activities (37), and in species that both pant and sweat the transition from sweating to the more economical panting (31, 35) suggests that the rise in body temperature reflects central modulations of the thermoregulatory processes to enhance efficiency of the physical avenues for heat dissipation, rather than their failure (11). Both hyperosmolarity and hypovolemia were identified as putative regulatory signals. These stimuli individually, similarly to hypohydration, elevate T-Tsh for evaporative cooling and the regulated body temperature (4, 13, 15) and lower basal metabolic rate (16). Hyperosmolarity combined with hypovolemia produce an additive effect (13, 15). These findings infer a regulatory role for osmo- and volume receptors, and in turn, osmoregulatory hormones, in the hypohydration-induced altered thermoregulation.

Both arginine vasopressin (AVP) and ANG II are likely osmoregulatory hormone candidates. AVP, as a central neuropeptide, is known to exert antipyretic actions (5). However, modest increases in hypothalamic temperature stimulate AVP release, depending on the state of osmoregulation (19). ANG II, as a peripheral hormone as well as a neuropeptide generated by the renin-angiotensin system (RAS) in the brain (8), was shown to facilitate heat loss in rats by enhancing tail skin vasodilatation (8, 37). Thus in hypohydration both hormones might act in a manner compensating to some extent for the influences of hypovolemia and hyperosmolarity on temperature regulation. For AVP, the widespread preoptic and hypothalamic networks of vasopressinergic neurons (8) and neurons endowed with AVP receptors (19, 35) would form the structural basis for the presumed actions described, with the vasopressinergic innervation of the lateral septal area as currently the most clearly established pathway. Similarly, widespread at the hypothalamic and lamina terminalis level are angiotensinergic neuro-glial elements (21) and ANG II binding sites (1, 28). Their involvement in osmoregulation is well documented (24). However, information as to how ANG II might contribute to the integration of thermal and osmotic stimuli is rather sparse. According to a study on rats receiving losartan (Los) intracerebroventricularly to block the AT1 subtype of ANG II receptors, the full expression of the visceral, sympathoexcitatory, pressor, and AVP responses to heat stress requires ANG II as a brain-intrinsic signal (20). Apart from these direct actions ANG II might stimulate AVP release centrally and thereby modulate temperature regulation. It is not yet clear whether ANG II receptors are restricted to parvocellular neurons of osmoregulatory nuclei or may become expressed also in the magnocellular neurons producing neurohypophysially released AVP, as indicated by our own observation of progressive upregulation of ANG II receptors in the magnocellular portion of the paraventricular nucleus (PVN) coincidentally with progressively increasing thermal (via heat acclimation) and osmotic stress (18). These findings agree with ANG II involvement in central and neurohypophysial AVP release, respectively. Our previous findings that heat acclimation enhances AVP release and alters AVP distribution in hypothalamic vs. extrahypothalamic limbic structures in response to heat or osmotic stress (7) fit with the latter notion as well.

Heat acclimation is a continuum of processes, manifested by two distinct phases varying in the excitability of the thermoregulatory controller (12, 14, 15). The first phase is transient short-term heat acclimation (STHA) and is characterized by augmented autonomic signal/effector output ratio. The second phase is long-term heat acclimation (LTHA), which is stable and is characterized by enhanced physiological efficiency, during which the autonomic signal/effector output ratio decreases compared with the nonacclimated stage. We hypothesized that the acute challenge of hypohydration superimposed on the heat acclimation process might disclose ANG II-mediated modulations in the magnitude of the thermoregulatory responses and in turn provide information about accentuated or diminished functions of the ANG II circuit in the course of heat acclimation.

The purpose of this investigation was twofold: 1) to confirm the involvement of ANG II receptors in heat defense responses and 2) to find out how ANG II receptors and the hypothalamic RAS circuits modulate thermoregulation during hypohydration.

Our results provide evidence for a selective modulatory role of ANG II in heat defense mechanisms in conditions of chronic adjustments, apparently in more than one pathway. Central ANG II signals may lead to vasodilatation temperature threshold (VTsh) upshift, whereas circumventricular structures, activated via circulating ANG II, decrease salivation temperature threshold (STsh). On hypohydration these responses seem to be desensitized.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Male rats (Rattus norvegicus, Sabra strain, albino variety, Harlan, Jerusalem) of initial weight 70-90 g were used. Animals were assigned to normothermic controls (C), and heat-acclimated groups. All animals were kept in a 12:12-h light-dark cycle. Normothermic rats were kept at 24 ± 1°C, whereas heat acclimation was attained by continuous exposure to 34 ± 1°C and 30-40% relative humidity for 2 days (STHA) or 30 days (LTHA) (11, 14). Before the experiments, animals were further divided into euhydrated (E) and hypohydrated (D) groups. Hypohydration was achieved by water deprivation for a period of time, resulting in approximately 10% reduction of body weight. For control-hypohydrated (C-D) rats, this was accomplished by 48 h, and for STHA and LTHA rats by water deprivation for 24 h before the experiments (25). All experiments were performed in accordance with guidelines approved by the Hebrew University Committee for Animal Experimentation.

Acute Heat Load and Heat Defense Response

The heat defense response of the rat is manifested by rapid skin vasodilatation, denoted by an abrupt rise in tail skin temperature (Tt) as an indicator of tail blood flow, together with a rise in colonic temperature (Tc). This early Tc rise is terminated at the onset of salivation, to produce a hyperthermic plateau at which body temperature is regulated. Failure of the evaporative cooling system leads to an explosive rise in Tc and possibly to the development of heat stroke syndrome [Ref. 11 and see Fig. 3, control-euhydrated group (C-E)]. In this investigation, the heat defense response in conscious animals was determined by measuring T-Tshs for the onset of skin vasodilatation and evaporative cooling, Tc of the hyperthermic plateau and heat endurance during subjection to heat stress at 40 ± 1°C. The conscious rats, lightly restrained, were instrumented with colonic and small surface thermistors (YSI 702 and YSI 427, respectively; Yellow Springs Instruments, Yellow Springs, OH), connected to a data acquisition system (Codas, Dataq, OH). The colonic probe was inserted 6 cm beyond the anal sphincter, whereas the small surface probe was attached, isolated from the environment, to the tail surface at a distance of 3-4 cm from its tip, for Tt as an indicator of skin blood flow (16). The rats were then kept at an ambient temperature of 24 ± 1°C for Tc stabilization. This was particularly important for the acclimated rats, which maintain elevated Tc while in the climatic chamber. Immediately afterward the rats were placed in a temperature-controlled chamber at 40°C, with the tip of the tail freely maintained, via a horizontal slit, outside the cage exposed to room temperature. This enables reliable measurement of heat transferred by the vasodilating blood vessels to the tail skin, by the surface thermistor. Tc VTsh, marked by an abrupt rise in Tt, and STsh, marked by the onset of mouth wetness, were recorded. From the Tc records, onset T-Tshs for VTsh and STsh were determined. Heat endurance was determined by the time taken to attain a Tc of 42°C. To determine water loss, body weight was measured (Precisa Electrical Balance, 0.01 g, before and after heat exposure).

Assessment of Role of Central ANG II

For this purpose central AT1-receptor blockade was achieved, using the AT1-receptor selective antagonist Los (a gift from Merck Sharp & Dohme, Petach Tikva, Israel). Two days before the experiments a cannula was inserted into the left lateral brain ventricle, under pentobarbital anesthesia (60 mg/kg body wt). On the experimental day, rats were instrumented as before, and each animal received 50 µg in 5 µl bolus of either Los or saline (preliminary experiments). Animals then underwent heat exposures as previously described. To verify correct cannula placement, Evans blue dye was administered to the ventricle on termination of the experiment. Animals were then killed by pentobarbital sodium overdose; dye distribution in the ventricles and the exact location of the cannulas were validated visually.

Assessment of Role of Peripheral ANG II

In an additional experimental series Los was administered intravenously. One or two days before the experiments, the jugular vein was cannulated (polyethylene PE-50 tubing, Clay Adams). On the experimental day, rats were instrumented as before. Each animal received 1 mg/100 g body weight in 100 µl of either Los or saline. Animals then underwent heat exposures as previously described. Because our main aim was to characterize central pathways of ANG II influence, only thermoregulatory parameters were measured in this series.

Plasma Parameters

To further confirm the effects of hydration level, rats of the experimental groups were instrumented with rectal thermistors as described previously. After withdrawal of a baseline blood sample, rats were placed in the temperature-controlled chamber and were subjected to heat stress at 40°C. Blood samples (70-100 µl each) were taken at every 0.5°C increment in Tc. Hematocrit (Hct) was measured using a Hct centrifuge, and plasma osmolarity with a vapor pressure osmometer (Wescor, Logan, UT). Blood samples were taken from the tip of the tail (via a tiny incision, with microhematocrit capillary). The total amount of blood withdrawn did not exceed 2% of the blood volume, thus not affecting thermoregulation.

Calculations and Data Analysis

All data were presented as a function of Tc or time. For the latter mode of presentation, for within group comparisons, heat endurance of the C-E rats at 42°C was considered 100%. For significance tests, one- and two-way ANOVA were employed using commercially available computer software. Treatments were taken as the fixed effects, and the individual animals were assumed to be random samples from the animal population. Values of P < 0.05 were considered to be statistically significant. For individual comparisons between two groups, Student's unpaired t-test with Bonferroni correction was used. Data are expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Body weight, basal Tc, hypohydration level water loss, and endurance on heat stress of the various groups are presented in Table 1. The average body weights of the different experimental groups determined in the state of normal hydration were similar. Among the groups that were subsequently deprived of water, the hypohydration levels, as indicated by the body weight losses, were similar. Basal Tc at ambient temperature 24°C did not differ significantly, except the higher level of the STHA-E group, which also differed in this respect from the STHA-D group. During heat stress, water (body weight) losses were significantly larger in euhydrated than in hypohydrated groups, and in turn the endurances of the latter were markedly shorter. A significant prolongation of endurance was observed only in the LTHA-E rats.

                              
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  		Table 1.   Colonic temperatures, body weights, and change following water deprivation and/or acute heat stress and heat endurance as influenced by hypohydration and preceding heat acclimation in rats

The parallel measurements of plasma osmolarity and Hct in all groups (Table 2) demonstrated the ability of the animal to adjust their plasma ionic constituents and volume in the attained physiological states. Neither heat acclimation nor water deprivation induced significant osmolarity change, whereas subjection to heat stress resulted in a gradual increase. This increase was highly significant at the termination of heat stress in all groups except for LTHA-E and STHA-D (Table 2). Unlike osmolarity, Hct values of the STHA-E and LTHA-E rats were higher by 17 and 16%, respectively, from the C-E group (P < 0.0005). These data fit with our previous data of absolute plasma volumes of nonacclimated and heat-acclimated rats (25). On hypohydration, Hct changed significantly only in C-D rats. Likewise, under our experimental conditions, on termination of heat stress Hct values remained almost unchanged. These data may suggest that although the thermal dehydration attained was sufficient to induce changes in plasma osmolarity in most groups, greater dehydration levels were required to attain hypovolemia.

                              
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  		Table 2.   Plasma osmolarity and hematocrit before and after water deprivation and/or acute heat stress

Heat Defense Responses in Euhydrated and Hypohydrated Rats

Both acclimation and hypohydration modulated the heat defense response by affecting all or some of the thermoregulatory loops in terms of onset thresholds and intensity.

Figure 1 demonstrates the effects of heat acclimation and hypohydration on the vasomotor response of the tail vasculature to heat stress. As shown by the Tt vs. Tc plots, the initial tail temperatures were sometimes slightly below the average room temperature (24°C), apparently because of an evaporative component in heat transfer from the tail skin to the environment. Furthermore, on the onset of vasodilatation, the rise of Tt was sometimes associated with a slight, transient decrease of Tc, due to blood returning from the initially cool peripheral tissues, before Tt continued to rise further with increasing Tc, although in a more or less decremental fashion. Both STHA and LTHA (Fig. 1A) tended to decrease the T-Tsh for vasodilatation. On hypohydration (Fig. 1B) this effect was partially preserved, but compared with the euhydrated state the rise in Tt per Tc increment was retarded. This was most pronounced in the LTHA rats, suggesting a more gradual vasomotor response in this group.


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  		Fig. 1.   Tail surface temperature, as indicative of tail blood flow plotted vs. colonic temperature (Tc) in euhydrated (A, C, and E) and hypohydrated (B, D, and F) rats during subjection to heat stress at 40 ± 1°C. A-F: tail blood flow (Tt) line obtained for controls (C), short-term heat acclimation (STHA), and long-term heat acclimation (LTHA). Each line represents mean value of 10-14 rats. A and B: euhydrated and hypohydrated untreated rats. C and D: intracerebroventricularly Los-treated rats. E and F: intravenously Los-treated rats. Statistical analysis for vasodilatation temperature thresholds (VTsh) values of each experimental group is given in Fig. 2.

T-Tsh for vasodilatation and salivation are summarized in Fig. 2. The solid bars in Fig. 2, A and B, document the influence of the two investigated phases of heat acclimation on the average VTsh during heat stress. There was a significant tendency for VTsh to decrease in both STHA-E and LTHA-E rats in comparison to the C-E state, with no difference between these groups. In the hypohydrated state (Fig. 2B), VTsh was decreased compared with the euhydrated state (Fig. 2A). However, no effect of heat acclimation was observed. The solid bars in Fig. 2, C and D) document the average threshold temperatures for the onset of salivation (STsh). In the state of euhydration STsh decreased in STHA-E rats and resumed preacclimation levels in LTHA-E. In the state of hypohydration an elevation of STsh in comparison to euhydration could be confirmed for the C-D and STHA-D animals. Moreover, in the latter condition STsh was significantly lower than in the C-D group, whereas no such effect was observed for the LTHA-D group.


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  		Fig. 2.   VTsh and salivation temperature thresholds (STsh) in euhydrated (A and C) and hypohydrated (B and D) rats subjected to heat stress at 40 ± 1°C. UN, untreated, Los-icv, intracerebroventricular lateral ventricle losartan administration, Los-iv, intravenous Los administration. P < 0.025/0.01; n see Fig. 1. Data obtained from same experimental groups as in Fig. 1. * Los intracerebroventricularly vs. untreated, STHA and LTHA vs. controls, circle  hypohydrated vs. euhydrated, + Los intravenously vs. untreated, # Los intravenously vs. Los intracerebroventricularly; *significant difference vs. matched control.

Figure 3 demonstrates the effect of heat stress on thermal endurance and Tc of the acclimating groups in the euhydrated and hypohydrated states. LTHA distinctly prolonged heat endurance (P < 0.01). STHA did not have such an effect. The plateau Tc in this group, however, was significantly lower than that of the C-E and LTHA-E groups because of altered T-Tsh for heat defense responses (Tc plateau for C-E, STHA-E, and LTHA-E, respectively, were 40.14 ± 0.12, 39.43 ± 0.15, and 40.01 ± 0.10°C, P < 0.0003). In the state of hypohydration, Tc of 42°C was generally attained in less than one-half the time elapsed for the euhydrated groups, irrespective of the state of heat adaptation. For the STHA-D animals, a short-term plateau was observed, suggesting a temporary, efficient activation of salivation, which apparently corresponded to the significantly decreased STsh in this group (Fig. 3).


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  		Fig. 3.   Tc of euhydrated and hypohydrated rats during subjection to heat stress at 40 ± 1°C. Each line represents mean value of 10-14 rats. SE range of both time (x-axis) and Tc (y-axis) is denoted by open circle  SE for x and y of C-E group in symbols legend. For further details, as well as significance level among the experimental groups, see data presented in Table 1.

Modulatory Effects of Los on Heat Defense Response

C-E and C-D rats. The effects of Los on Tt and the T-Tsh on VTsh and on STsh is shown in Figs. 1 and 2. An effect of both intracerebroventricularly and intravenously administered Los on VTsh could not be confirmed. T-Tsh for salivation in the corresponding groups increased. This increase was significant, however, only in the intracerebroventricularly Los-administered group. No significant clear effect of Los on T-Tsh of the C-D rats was observed.

Figure 4 summarizes the time courses of the rise in Tc in the untreated and in the two Los-administered control animals in the states of euhydration (left) and hypohydration (right). Whereas the large effect of water deprivation is obvious in each of the treated groups, it is also obvious that neither intracerebroventricular nor intravenous application of Los perceptibly affected the course of Tc during heat stress.


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  		Fig. 4.   Effects of intracerebroventricular and intravenous Los on Tc of euhydrated and hypohydrated rats in course of subjection to heat stress at 40 ± 1°C. Each line represents mean value of 10-14 rats. The following between-group comparisons in heat endurance were found to be significant: STHA-D, UN vs. intravenous Los (P < 0.007); LTHA-E, UN vs. intracerebroventricular Los (P < 0.01); LTHA-D, intracerebroventricular and intravenous Los vs. LTHA-D (P < 0.02 and P < 0.003, respectively). For further details see text; n for various groups 8-10.

STHA-E and STHA-D. Figure 2 summarizes the data from which conclusions can be drawn on the effects of the two Los treatments on T-Tshs after 2 days of heat acclimation without and with superimposed hypohydration. In the STHA-E group, intracerebroventricular Los but not intravenous Los significantly lowered VTsh. For STsh the tendency for an increase by both intracerebroventricular and intravenous Los could be confirmed significantly only for the latter treatment. In the state of hypohydration VTsh was not affected by either Los treatment, but the same significant effect of intravenous Los on STsh as in the euhydrated animals was confirmed for the two modes of Los administration. Interestingly, the rate of rise of tail blood flow vs. Tc rise following intracerebroventricular Los administration was retarded in this group (Fig. 1C).

Figure 4, middle, summarizes the time courses of the rise in Tc with and without the two different Los treatments in the STHA animals in the state of euhydration (left) and of hypohydration (right). The large effect of water deprivation is again obvious, but whereas intracerebroventricular Los treatment had no substantial effects on heat endurance, intravenous Los, together with the significant increase of STsh shown in Fig. 2, significantly reduced the capacity to cope with the heat stress in this condition (P < 0.007).

LTHA-E and LTHA-D groups. Figure 2 summarizes the data from which conclusions can be drawn on the effects of the two Los treatments on the investigated T-Tshs after 30 days of heat acclimation without and with superimposed hypohydration. In the LTHA-E group, intracerebroventricular but not intravenous Los significantly lowered VTsh. STsh was not affected by intracerebroventricular Los, but intravenous Los substantially increased the T-Tsh. In the state of hypohydration, VTsh was reduced by intracerebroventricular but not by intravenous Los, whereas STsh was not affected at all.

Figure 4, bottom, summarizes the time courses of the rise in Tc with and without the two different Los treatments of the LTHA animals in the state of normal hydration (left) and of hypohydration (right). The particularly pronounced endurance of the LTHA-E animals during heat stress was not affected by intracerebroventricular Los, but this adaptive effect was completely abolished by intravenous Los (P < 0.01). This apparently stems from the rise in STsh and the retarded elevation in tail blood flow (Fig. 1) subsequent to the intravenous Los administration. In the LTHA-D animals both intracerebroventricular and intravenous Los moderately but significantly increased the heat-stress endurance in comparison to the untreated LTHA-D animals (P < 0.02 and P < 0.003, respectively). In these groups STsh was not affected (see Fig. 2), but there was a more pronounced rise in Tt compared with the untreated animals, which suggests improved convective heat dissipation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This investigation was concerned first with the consequences of changes in the state of body hydration on the manifestation of heat defense mechanisms in heatacclimating rats and second with the putative roles that ANG II might play under these conditions as an osmoregulatory hormone and as a brain-intrinsic neuropeptide.

Heat Defense Responses in Euhydrated and Hypohydrated Heat-Acclimating Rats

The data presented are in line with our hypothesis that on heat acclimation, thermoregulatory plasticity during heat stress is manifested by biphasic temporal variation of thermoregulatory parameters. As previously reported (10, 12, 14), during heat acclimation there is an early, transient phase (STHA) of augmented autonomic signal/effector output ratio to cope with heat load, followed by a sustained stable phase (LTHA) of efficient thermoregulation characterized by a decreased autonomic signal/effector output ratio. The present study shows that on exposure of euhydrated rats to heat stress this biphasic pattern is expressed in the STHA phase by a more rapid onset of active heat defense documented by reduced T-Tshs for skin vasodilatation and salivation but not by increased endurance. The LTHA condition resulted in considerably prolonged endurance that was associated with a reduced T-Tsh for vasodilatation but apparently not for salivation. A more economical exploitation of the secreted saliva, as previously observed (11), would be a likely explanation for this increased endurance. This is analogous to the phenomenon of "habituation" in sweating (23).

Water deprivation virtually abolished the enhanced efficiency of LTHA-E rats to cope with heat stress. There was, however, some indication that the acutely enhanced stimulus/effector ratio in heat defense activity, typical of STHA, is partly preserved, as indicated by the temporary nonlinearity in the rise of Tc with time and by the reduced T-Tsh for salivation. Nevertheless, it may be stated that on hypohydration the onset and gain of input/output relationships of heat defense effectors are generally impeded in comparison to euhydration. This fits with the notion of Massett et al. (22) that cardiovascular and thermoregulatory adjustments, although capable of compensating for small changes in the hydration state, are severely altered at more pronounced degrees of hypohydration, as indicated in our experiments by body weight reductions exceeding 10%.

In summary, an important conclusion emerging from our data is that when hypohydration is superimposed on heat acclimation, hypohydration does not alter the biphasic sequence of the acclimatory response; however, sensitivity to thermal stress and the mode of coping of the rats in each acclimation phase are reduced compared with the euhydrated state. The LTHA-D group showed a loss of the benefits acquired by acclimation, whereas the STHA-D rats, which rely mostly on accelerated autonomic regulation, gained some benefit from acclimation.

Modulatory Effects of Los on Heat Defense Response

In the present study an attempt was made to differentiate between the still unknown central targets of ANG II that might be involved in the generation of adaptive adjustments to heat per se, as well as in their inhibition following hypohydration. This was attempted by ANG II receptor blockade with the AT1-specific antagonist Los. Whereas intracerebroventricularly administered Los preferentially blocks binding sites on the brain side of the blood-brain barrier (BBB), intravenously administered Los, which is known to cross the BBB (29), binds to both peripheral receptors and to receptors of neuroglial structures on both sides of the BBB.

This study shows that in C-E rats neither treatment perceptively affected VTsh and endurance under heat stress despite elevation of STsh, suggesting that the efficiency of the evaporative heat loss was not impaired. Thus interference by central AT1-receptor blockade of the full expression of the control of the sympathoadrenergic splanchnic vasoconstrictor response pattern (20) does not seem to have been quantitatively relevant in the conditions of these experiments, although the doses used in the present study were higher than those used by Kregel et al. (20). In contrast to the C-E state, in both acclimation phases, intracerebroventricularly administered but not intravenously administered Los brought about a drop in VTsh and attenuated the rate of rise of Tt vs. Tc, suggesting some intracerebroventricular Los effect on vasomotor responses. Current knowledge does not provide any anatomic explanation for these responses; however, the retarded increase in tail blood flow on heat stress may be associated with Los-induced attenuation of splanchnic vasoconstriction in the acclimated groups. The observed differences in Los effects between the acclimated and nonacclimated groups might stem from changes in the responsiveness of AT1 receptors or their signaling pathway in the acclimated vs. the nonacclimated states. Such acclimatory effects (e.g., receptor upregulation, decreased receptor-binding affinity, and altered evoked Ca2+ signal) were previously documented for several peripheral receptors (12, 14).

In contrast to its effect on vasomotor responses, intracerebroventricular Los did not significantly affect STsh or heat endurance of the euhydrated acclimated rats. This may imply that enhanced ANG II binding in some hypothalamic structures, especially in the magnocellular portion of the PVN, as observed, for example, after STHA with and without superimposed hypohydration (18), does not contribute to modulation of the STsh and heat endurance.

Intravenously administered Los only affected STsh, and like intracerebroventricularly administered Los exerted its effect solely on heat-acclimated rats. In the LTHA-E group significant elevation in STsh was accompanied by a marked decrease in heat endurance. Concomitantly, the increment in Tt was similar to that measured following intracerebroventricular Los administration. This suggests a central rather than a peripheral vasomotor effect of intravenous Los, which might have resulted in elevated blood flow due to inhibition of the known vasopressor effect of ANG II.

Hypohydrated acclimated rats displayed different responses to ANG II blockade. The STHA-D rats showed an elevated STsh coincidentally with decreased heat endurance. In the LTHA-D group there was a distinct effect, although directionally different from that seen in the state of euhydration. In the latter, both intravenous and intracerebroventricular Los significantly and equidirectionally increased endurance, with no change in STsh but with a tendency for a reduced VTsh.

Taken together our data enable us to put forward the working hypothesis that adaptive adjustments in the central control of heat-dissipating thermoregulatory effectors are modulated by neuronal systems transducing the ANG II signal, more pronouncedly after heat acclimation. This may suggest that fine-tuning of the autonomic control systems by ANG II occurs in conditions of chronic adjustments. Hence, the impression gained from the present study of mild Los action on heat endurance in the STHA vs. a marked effect in the LTHA rats, depending on the hydration state, might relate to the dose of Los applied and, consequently, the range within which effective concentrations of the drug were reached. This could be either by its diffusion from the site of application when injected into a lateral cerebral ventricle or from the circumventricular structures with their open BBB from neuroglial elements, which could be reached when the drug was injected intravenously.

In this investigation, the modulatory role of ANG II, unraveled by its disruption after intracerebroventricular and intravenous Los administration, is displayed primarily by the fine tuning of VTsh and STsh in two opposing directions: 1) an upward shift of vasodilatation and 2) a downward shift of salivation. In the rat, which relies for its thermoregulation on a hyperthermic plateau and salivation, such fine tuning might be beneficial. On hypohydration, decreased responsiveness of the AT1 receptors might explain the elevated STsh observed in this hydration state. The consistent findings that vasomotor responses were invoked by intracerebroventricular Los, whereas STsh was affected by intravenous Los, permit us to put forward the working hypothesis that angiotensin-associated adaptive adjustments in the central control of the heat-dissipating thermoregulatory effectors are modulated independently by transducing the ANG II signal, either from within BBB structures (vasomotor) or via activation of circumventricular structures which respond to circulating ANG II (STsh). Inasmuch as angiotensinergic-associated changes in our studies include enhanced ANG II binding in the magnocellular component of the PVN (18), this draws attention to the possibility that the vasopressinergic system is involved. The latter assumption fits with the findings of Kregel et al. (20).

Perspectives

The results of the present study together with our previous histochemical data (18, 27) suggest the involvement of brain RAS, as well as of brain structures serving as targets for circulating ANG II, in the linkage that exists between fluid balance and adjustments of autonomic heat defense. In the rat these adjustments depend critically on the responsiveness of the autonomic systems controlling peripheral vasomotor tone and salivary secretion. The network and the ways in which ANG II might act as a linking messenger are not yet elucidated. Our recent study employing c-fos protein immunocytochemistry to identify those neuroglial elements in the preoptic, lamina terminalis, and hypothalamic regions of the basal forebrain that are pertinent for either temperature regulation or for osmoregulation, as well as those integrative structures by which the two control systems are interlinked (27) serve, however, as a guide to eliciting structurally defined connections for which the importance of angiotensinergic components should be elucidated, including their inputs into endocrine and autonomic nervous control systems. With respect to the histochemical approach, studies on ANG II receptor regulation have to be supplemented by studies on signal generation, i.e., on changes of ANG II production and on the transcription of those mRNA signals that ultimately determine the formation of the receptors and of the ANG II precursor angiotensinogen. Data obtained may advance our understanding of the mode of competition between body temperature and osmoregulatory control circuits. To date, the knowledge available on the interaction between these two control systems is confined mostly to phenomenology at the whole body level of organization.


    ACKNOWLEDGEMENTS

The authors appreciate the help of Dr. U. Meiri and J. Shlaier in conducting the study.


    FOOTNOTES

This study was supported by the German Israeli Foundation Research Grant I 0361-108.02/94 to M. Horowitz and R. Gerstberger.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. Horowitz, Dept. of Physiology, Hadassah Medical School, Hebrew Univ., PO Box 12272, Jerusalem 91120, Israel (E-mail: horowitz{at}cc.huji.ac.il).

Received 28 August 1998; accepted in final form 19 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Compar Physiol 277(1):R47-R55
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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