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Department of Physiology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900
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ABSTRACT |
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 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Autonomic neurons in the hypothalamic paraventricular nucleus (PVN) are innervated by osmotic-sensitive regions of the lamina terminalis, receive input from ANG II-containing cells, and express AT1 ANG II receptors. Therefore, we hypothesized that ANG II actions within the PVN could underlie hyperosmolality-induced increases in renal sympathetic nerve activity (RSNA). In anesthetized baroreceptor-denervated rats, graded concentrations of NaCl (0.30, 0.9, 1.5, and 2.1 osmol/l) were injected (300 µl) centrally via the internal carotid artery (ICA) and produced corresponding increases in mean arterial pressure (MAP) and RSNA. In addition, equivalent hyperosmotic loads (1.5 osmol/l) of NaCl, glucose, and mannitol each significantly (P < 0.05) increased MAP and RSNA. The same stimuli had no effect when administered intravenously. Bilateral PVN microinjections (100 nl) of the AT1-receptor antagonist losartan (80 nmol) before osmotic challenge had no effect on resting RSNA but significantly (P < 0.05) reduced RSNA responses to hyperosmotic NaCl (n = 7), glucose (n = 6), and mannitol (n = 6). Increases in RSNA evoked by hyperosmotic NaCl were significantly (P < 0.05) attenuated ~20 min after losartan injection and recovered within 60-120 min. In contrast, losartan outside the PVN as well as vehicle (saline) within the PVN failed to alter RSNA responses to ICA hyperosmotic NaCl. Results suggest that elevated RSNA after central sodium/osmotic activation is mediated, at least in part, by a synaptic mechanism involving AT1-receptor activation within the PVN.
angiotensin II; osmolality; sodium; sympathetic nerve activity; arterial pressure; paraventricular nucleus
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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CENTRAL HYPEROSMOTIC STIMULATION elicits a variety of responses (13, 19, 20, 23), most notably, a rapid and significant increase in arginine vasopressin (AVP) release (19). It is well established that magnocellular neurosecretory neurons in the hypothalamic paraventricular nucleus (PVN) participate in pituitary release of AVP after hyperosmotic stimulation (3). Additionally, studies indicate that autonomic regions of the PVN are also key targets of forebrain/anterior hypothalamic regions that sense and respond to extracellular fluid hyperosmolality. Indeed, evidence indicates that central hyperosmolality can significantly modulate sympathetic nerve discharge (4, 9, 12, 22, 34, 41). Although autonomic regions of the PVN receive input from a variety of neurotransmitter systems, e.g., adrenergic (15), cholinergic (31), and glutamatergic (10), a major source of excitatory synaptic input arises from cell groups within the lamina terminalis (1, 37) and is conveyed via ANG II-containing nerve fibers (2, 11, 38). Combined, these data suggest that sympathetic nervous system effects of central hyperosmolality may involve actions within the hypothalamic PVN that depend, at least in part, on activation of angiotensinergic input.
In the present study, we tested the hypothesis that central hyperosmotic stimulation increases renal sympathetic nerve activity (RSNA) by a mechanism involving ANG II type 1 (AT1)-receptor activation in the hypothalamic PVN. Given evidence that the AT1 ANG II receptor predominates (5, 14) in the PVN and that responses to PVN-microinjected ANG II can be significantly reduced by local AT1-receptor blockade (5, 18), we examined effects of AT1-receptor antagonism in the PVN on the sympathoexcitatory response to internal carotid artery (ICA) administration of hyperosmotic stimuli. To avoid possible inhibitory influences of arterial baroreceptor activation on the magnitude of evoked sympathetic responses (4, 27, 34), experiments were performed on rats after sinoaortic baroreceptor denervation (SAD). A portion of these results has been previously reported in abstract form (7).
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Experiments were performed using male Sprague-Dawley rats
(350-450 g; n = 39) anesthetized with a mixture of
-chloralose (70 mg/kg) and urethane (700 mg/kg) given
intraperitoneally. An adequate depth of anesthesia was monitored by
observing arterial blood pressure and by the absence of pedal and
corneal reflexes and was maintained by additional anesthesia (10%
initial dose, intravenously) as needed. An arterial catheter was
inserted into the aorta via a femoral artery and was connected to a
pressure transducer to measure arterial blood pressure. A
cardiotachometer was used to obtain heart rate (HR) from the arterial
pressure signal. The left femoral vein was also catheterized and used
to administer drugs. After tracheal cannulation, rats were paralyzed with gallamine triethiodide (25 mg · kg
1 · h1 iv) and
artificially ventilated with oxygen-enriched room air. End-tidal
CO2 was monitored and maintained within normal limits (35-40 mmHg) during experiments by adjusting ventilation rate (80-100 breaths/min) and/or tidal volume (2.0-3.0
ml). Body temperature was maintained at 37 ± 1°C with a
water-circulating pad. All experimental and surgical procedures were
approved by the Institutional Animal Care and Use Committee of The
University of Texas Health Science Center at San Antonio.
SAD
SAD was performed according to standard methods. The superior laryngeal nerves, cervical sympathetic ganglia, and aortic nerves were sectioned bilaterally. Each carotid bifurcation was then stripped of connective tissue and painted with 10% phenol in ethanol, and the ventral cervical incision was closed. A lack of change in HR and sympathetic nerve discharge (see below) during an increase in mean arterial pressure (MAP) induced by the-adrenoceptor agonist
phenylephrine (5-10 µg/kg iv) was used to confirm the completeness of each SAD procedure.
Recording RSNA
Using a flank incision, a left renal sympathetic nerve was isolated from surrounding tissue. Stainless steel wire electrodes (A-M systems, 0.127-mm OD) were placed in contact with the renal nerve bundle and covered with a silicon-based impression material (Coltene, light body) to insulate the nerve-electrode interface from contact with body fluids. Signals were directed to an alternating current amplifier equipped with half-amplitude filters (band pass 100-1,000 Hz) and a 60-Hz notch filter. The processed signal was rectified, integrated (30 ms time constant), and digitized at a frequency of 1,000 Hz and stored on computer disk.Central Hyperosmotic Stimulation
To stimulate osmotically sensitive regions of the brain, aqueous solutions of NaCl, glucose, and mannitol were delivered through a catheter inserted into the ICA. Graded concentrations of NaCl (0.3, 0.9, 1.5, and 2.1 osmol/l) were used to test for effects on MAP and RSNA. Responses to equivalent hyperosmotic loads of glucose and mannitol were compared with those evoked by NaCl (1.5 osmol/l) to test for osmotic vs. sodium effects on MAP and RSNA. Isosmotic (0.3 osmol/l) NaCl was also injected to control for nonspecific effects of the injected volume. Hyperosmotic (1.5 osmol/l) NaCl was injected intravenously to test for possible effects of peripheral osmoreceptor activation. Each solution injected into the ICA or femoral vein was delivered in a volume of 300 µl over a period of 10-15 s.Microinjection of Drugs
Animals were placed in a stereotaxic head frame, and the skull was leveled between bregma and lambda. A section of bone overlying the cortex was removed to allow a glass micropipette to be lowered into the PVN using a piezoelectric microdrive (Burleigh Instruments). All PVN microinjections were made bilaterally using a pneumatic picopump (WPI) at the following stereotaxic coordinates (in mm): 1.0-1.5 caudal to bregma, 0.5-0.7 lateral to midline, and 7.0-7.4 ventral to dura. Each compound injected was dissolved in saline and delivered in a volume of 100 nl/site. Compounds injected were ANG II (Sigma) and the ANG II AT1-receptor antagonist losartan (Merck). To produce a complete and uniform coverage of antagonist treatment, losartan was injected at two sites (1.2 and 1.7 mm caudal to bregma) on each side of the PVN. As an anatomic control, losartan was delivered just outside the PVN, 1.5-1.7 mm lateral to midline.Experimental Protocols
Effects of central hyperosmotic NaCl, glucose, and mannitol.
In preliminary studies it was determined that the magnitude of
increases in MAP and RSNA elicited by a given hyperosmotic dose of NaCl
gradually increased after SAD and reached a maximum after ~5 h, when
MAP and RSNA had returned to their pre-SAD baselines. Based on these
observations, all protocols described below began ~5 h post-SAD.
Effects of ICA injections of NaCl were examined using solutions
containing 0.3, 0.9, 1.5, and 2.1 osmol/l NaCl. Graded doses of NaCl
were injected into the ICA in a random sequence and were separated by
an interval 
15 min to ensure that MAP and RSNA had returned to
baseline before the next injection. Changes in MAP and RSNA were
recorded in response to each injection. In some experiments, the
maximally effective central hyperosmotic stimulus (1.5 osmol/l NaCl)
was injected (300 µl) into the femoral vein to assess the extent to
which evoked responses were influenced by peripheral actions. In three
separate groups of animals, effects on MAP and RSNA of solutions
containing different osmotically active solutes were tested. This was
accomplished by injecting equivalent hyperosmotic loads (1.5 osmol/l in
300 µl) of NaCl, glucose, and mannitol through the ICA as described above.
Effects of AT1-receptor blockade in the PVN on responses to central hyperosmolality. After MAP and RSNA returned to baseline after ICA injection of either hyperosmotic (1.5 osmol/l) NaCl (n = 7), glucose (n = 6), or mannitol (n = 6) (see above), the AT1-receptor antagonist losartan was microinjected bilaterally (40 nmol/side) into the PVN. Responses to ICA hyperosmotic stimuli were tested again 20 and 60 min after PVN losartan. Vehicle (normal saline) was injected into the PVN in four animals to control for nonspecific effects of the injected volume. In addition, experiments (n = 4) were performed to ensure that effects of losartan were due to an action within the PVN by testing effects of losartan microinjected outside the PVN on MAP and RSNA responses to ICA hyperosmotic (1.5 osmol/l) NaCl. Finally, to ensure that the losartan dose produced an effective blockade of AT1 receptors, experiments were carried out in five animals in which MAP and RSNA responses to ANG II (1.0 nmol in 100 nl, 1 site/side) microinjected bilaterally into the PVN were recorded before and after PVN losartan. Effects of losartan on PVN ANG II-evoked responses were tested 20-60 min later.
Histology
At the end of each experiment, Evans blue dye (100 nl) was used to mark the site of each microinjection. Rats were then perfused transcardially with 0.9% saline followed by 4% paraformaldehyde in PBS. Brains were removed and postfixed for 4 h at room temperature in 4% paraformaldehyde. Brains were then transferred to 30% sucrose-PBS for >24 h. The hypothalamic PVN was then cut into 40-µm-thick coronal sections, and microinjection sites were identified under bright-field microscopy.Data Analysis
Baseline values of MAP, HR, and RSNA were measured as the average of each variable taken over a 60-s period immediately before each treatment. RSNA responses to central hyperosmotic stimulation were measured by taking the average of a 5-s period centered on the peak response recorded within 20 s after the start of each ICA injection. Responses of RSNA were measured before, 20 min after, and 60 min after microinjections of vehicle and losartan into or outside the PVN. A 60-s segment of RSNA was averaged to determine the maximum effect of ANG II microinjected into the PVN. Effects of ANG II were determined before, 20 min after, and 60 min after PVN microinjection of losartan.All statistical analyses were performed with a commercially available statistics package (Prism, version 3.0; GraphPad). Effects of ICA hyperosmotic stimuli were determined by comparing values of each recorded variable before and after each microinjected compound using a two-way ANOVA. The same analysis was used to compare responses to graded concentrations of hyperosmotic NaCl and to compare effects of hyperosmotic NaCl to those evoked by glucose and mannitol. For all analyses that revealed a significant interaction, differences among pairwise comparisons were determined by the Tukey post hoc test. All values in the text and in Figs. 2, 4, 5B, and 6 are expressed as means ± SE. Statistical significance was defined as a P value < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of Central Hyperosmotic Stimulation on MAP and RSNA
In arterial baroreceptor-intact animals (n = 3), graded increases in the concentration of ICA-injected NaCl produced small and somewhat variable effects on MAP and RSNA (Fig. 1, top). At the largest hyperosmotic dose of NaCl (2.1 osmol/l), MAP increased an average of 13 ± 4 mmHg from a baseline of 116 ± 2.3 mmHg. At the same dose, RSNA increased an average of 14 ± 3%. In contrast, central hyperosmotic stimulation in SAD rats significantly increased both MAP and RSNA (Fig. 1, bottom, and Fig. 2). In these experiments, graded increases in the concentrations of ICA NaCl (0.3, 0.9, 1.5, and 2.1 osmol/l) produced corresponding and significant (P < 0.005) changes in MAP [
0.5 ± 1.1, 25.3 ± 2.4, 45.1 ± 3.4, and 45.4 ± 1.5 mmHg, respectively (n = 4)] and RSNA [99 ± 5, 165 ± 12, 197 ± 11, and
214 ± 8% of baseline, respectively (n = 4)]. Maximal effects occurred in response to ICA injection of 1.5 osmol/l NaCl. In contrast, injections of isosmotic (0.3 osmol/l) NaCl had no
significant effect on either MAP or RSNA (Fig. 2). No significant change in HR was observed at any concentration of NaCl. Throughout these experiments, resting MAP, RSNA, and HR remained essentially unchanged and averaged 91 ± 1 mmHg, 0.5 ± 0.01 V, and
366 ± 6 beats/min, respectively.
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Effect of Different Osmotically Active Compounds on MAP and RSNA
ICA injection of equivalent hyperosmotic (1.5 osmol/l) loads of NaCl, glucose, and mannitol each produced a similar pattern of sympathoexcitation. There was an initial rapid increase in RSNA that typically lasted ~20 s and reached a peak within 8-10 s of initiating the ICA injection. In some cases the maximum RSNA increase occurred before completion of the ICA injection. This initial response was followed by a second, more sustained elevation that lasted an average of ~60 s (Fig. 3). Although a significant increase in MAP and RSNA was produced by ICA NaCl (n = 7), glucose (n = 6), and mannitol (n = 6), the average RSNA response to ICA NaCl was significantly greater than the response to either hyperosmotic glucose or mannitol (Fig. 4). These differences occurred without a significant difference in either resting MAP (NaCl: 91 ± 5 mmHg; glucose: 92 ± 4 mmHg; mannitol: 105 ± 4 mmHg), RSNA (NaCl: 0.43 ± 0.03 V; glucose: 0.48 ± 0.05 V; mannitol: 0.41 ± 0.04 V), or HR (NaCl: 360 ± 6 beats/min; glucose: 382 ± 13 beats/min; mannitol: 357 ± 8 beats/min) among the three groups. Greater increases in RSNA elicited by ICA NaCl were accompanied by MAP responses that tended to be larger as well, but differences in the magnitude of pressor effects did not reach statistical significance (Fig. 4). The magnitudes of MAP and RSNA responses evoked by ICA glucose and mannitol were not significantly different from each other. Intravenous hyperosmotic (1.5 osmol/l) NaCl stimulation, in contrast, had no significant effect on any recorded variable (Fig. 4).
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Effect of PVN Losartan on MAP and RSNA Responses to Central Hyperosmolality
To test whether increases in MAP and RSNA evoked by central hyperosmotic stimulation involved activation of ANG II AT1 receptors in the PVN, responses to equivalent hyperosmotic (1.5 osmol/l) loads of NaCl, glucose, and mannitol were examined before and after microinjecting the AT1-receptor antagonist losartan bilaterally into the PVN. Microinjection of losartan into the PVN had a small but significant effect, reducing resting MAP without altering either resting HR or RSNA (see Table 1). However, losartan did significantly reduce both the pressor (44.3 ± 3.9 to 18.3 ± 3.7 mmHg) and renal sympathoexcitatory (202 ± 13 to 156 ± 5%) response to hyperosmotic NaCl (Fig. 5). Similar effects of losartan were observed on RSNA responses to ICA hyperosmotic glucose (152 ± 9 to 119 ± 5% of baseline) and mannitol (141 ± 6 to 123 ± 6% of baseline) (Fig. 6). Although MAP responses to hyperosmotic glucose (35.7 ± 2.6 to 28.9 ± 5.7 mmHg) and mannitol (32.5 ± 1.2 to 27.9 ± 2.4 mmHg) tended to be reduced after PVN losartan, these effects did not reach statistical significance. Regardless of the hyperosmotic solute used, recovery from losartan effects could be observed within ~60 min (Figs. 5 and 6). Effects of losartan appeared specific because increases in MAP (38.6 ± 3.8 to 32.4 ± 8.1 mmHg) and RSNA (181 ± 13 to 178 ± 10% of baseline) elicited by ICA hyperosmotic NaCl were unaffected by bilateral PVN microinjection of saline vehicle. Losartan effects appeared to be site specific because microinjections deliberately placed lateral to the PVN had no effect on either resting HR, MAP, or RSNA (Table 1) or the pressor (38.4 ± 6.9 to 43.9 ± 5.5 mmHg) and sympathoexcitatory (186 ± 7 to 204 ± 18% of baseline) response to ICA hyperosmotic NaCl. In preliminary experiments (n = 4), injections made at this lateral position were determined to not significantly impinge on the PVN, because delivery of the GABAA receptor antagonist bicuculline methiodide failed to elicit the well-documented sympathoexcitation that accompanies its direct injection into the PVN (21). Finally, the efficacy of AT1-receptor blockade by losartan was examined by testing its effect on the RSNA response to ANG II bilaterally administered into the PVN. The increase in RSNA produced by PVN ANG II (1 nmol/side) was significantly reduced from an average of 120 ± 2 to 105 ± 1% of baseline (P < 0.01). The PVN ANG II-evoked RSNA response began to recover (116 ± 5% of baseline) ~60 min after microinjection of losartan.
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Histology
Histological analysis of microinjection sites by dye diffusion revealed that injections within the PVN were confined to superior portions of the nucleus near the dorsal cap subnucleus and did not penetrate the ependyma of the third cerebral ventricle. Anatomic control injections located lateral (1.5-1.7 mm) to the PVN were positioned just ventral to the fornix and did not significantly invade lateral portions of the PVN.| |
DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study demonstrated that central hyperosmotic stimulation significantly increased both MAP and RSNA in rats after acute SAD. Hyperosmotically induced increases of RSNA were significantly reduced by microinjection of the AT1 ANG II-receptor antagonist losartan bilaterally into the hypothalamic PVN. Overall, our results indicate that pressor and sympathoexcitatory effects of central hyperosmolality are mediated, in part, by a synaptic mechanism involving AT1-receptor activation in the PVN.
The method used to stimulate neurons in the central nervous system was to deliver compounds through the ICA. The ICA is the major vessel supplying blood flow to limbic regions of brain, including the hypothalamus. As a result, ICA injections are commonly used to deliver stimuli directly to neurons within this region while having little effect in the periphery. In the present study, aqueous solutions of NaCl, glucose, and mannitol were injected through the ICA and elicited significant and highly reproducible increases in MAP and RSNA.
It should be emphasized that the small dose of each hyperosmotic solution used was apparently insufficient to stimulate peripheral osmotic receptors when diluted in the peripheral circulation. This conclusion is supported by our data showing that the same volume of hyperosmotic NaCl injected systemically via the femoral vein failed to produce any significant effect on MAP, RSNA, or HR. This is important because both centrally and peripherally located osmo/sodium sensors appear capable of altering sympathetic nerve discharge (4, 12, 16, 35, 42), but each appears to elicit somewhat different effects. For example, peripherally administered hyperosmotic NaCl has been reported to produce nonuniform effects on sympathetic activity (42), increasing lumbar nerve discharge while concurrently decreasing both renal and splanchnic nerve activity. These data underscore the complex nature of integrated autonomic and cardiovascular effects of hyperosmotic challenge and indicate that sympathetic activity to different target organs may each play a unique role in the overall systemic response when body fluid balance is disturbed (8, 9, 23, 27, 34, 35, 42).
With regard to renal sympathetic activity in particular, peripheral hyperosmotic NaCl has been reported in the majority of studies to elicit inhibition (16, 42), not excitation, of ongoing nerve discharge. A likely mechanism for this effect is activation of gastrointestinal (8) and/or hepatoportal vein (16) sodium/osmoreceptors. By eliciting reductions in renal sympathetic activity, activation of these peripheral sensors has been suggested to promote rapid excretion of sodium when dietary salt intake is elevated (16).
As mentioned previously, however, central hyperosmotic NaCl has also been reported to effectively alter sympathetic nerve activity. This has been reported for a variety of species, including cats (34), sheep (22), dogs (12), and rats (4, 9). Unfortunately, differences in experimental design among these studies complicate assessment of the mechanisms that may determine whether sympathetic activity showed an increase or a decrease. Differences in the species used, the state of anesthesia, the type of anesthetic, and the route of central administration (carotid vs. cerebroventricular) are all factors that complicate interpretation. Irrespective of these differences, however, when central hyperosmotic NaCl challenge has been performed in baroreceptor-denervated animals, increases, not decreases, in sympathetic activity have been consistently reported (6, 34). Indeed, augmented pressor responsiveness to central hypertonic NaCl after SAD led Buñag and Miyajima (4) to propose that increases in sympathetic activity and blood pressure produced by central hyperosmotic NaCl could contribute to salt-induced hypertension, especially when baroreceptor reflex buffering becomes deficient (see also Refs. 27 and 28).
In the present study, RSNA responses to central hyperosmotic stimuli gradually increased after SAD and reached a maximum after ~5 h, when MAP and RSNA had returned to their pre-SAD baselines. At this time, graded concentrations of hyperosmotic NaCl elicited dose-dependent increases in RSNA. In contrast, ICA administration of hyperosmotic NaCl in the baroreceptor-intact animal produced small and variable increases in RSNA. A similar augmentation of the RSNA response to ICA hyperosmolality after SAD has been previously reported in anesthetized cats (34). The mechanisms responsible are not clear at present, but removal of baroreceptor afferents likely prevents reflex suppression of sympathetic responses secondary to the hyperosmotically induced rise in arterial pressure, perhaps in combination with sympathetic regulatory effects of osmotically induced vasopressin release (12, 19, 20). The latter effect may have contributed to the blunting of the sympathoexcitatory response in baroreceptor-intact animals through its reported action to facilitate arterial baroreflex function (24).
Equivalent hyperosmotic loads of NaCl, glucose, and mannitol, in the current study, each evoked a significant increase in MAP and RSNA. Although the cellular mechanisms responsible for hyperosmolality-induced changes in sympathetic nerve discharge are not clear, other studies have demonstrated membrane-depolarizing effects of hyperosmotic solutions among nearby neurosecretory (3, 6, 40) and nonneurosecretory (26) neurons in the hypothalamus. Among magnocellular neurons, this effect appears to depend on an increase in membrane conductance through stretch-inactivated cation channels (6, 40). It is possible that similar mechanisms active among osmosensitive sympathetic regulatory neurons could underlie RSNA and MAP responses observed in the present study. In support of a cationic conductance underlying such responses, a recent study by Nishimura et al. (25) showed that intracerebroventricular administration of the nonselective sodium channel inhibitor benzamil effectively eliminated increases in arterial pressure as well as norepinephrine and vasopressin excretion in response to elevated intracerebroventricular NaCl.
The present study revealed that the increase in RSNA produced by ICA NaCl was significantly greater than that elicited by an equivalent hyperosmotic load of either glucose or mannitol. One explanation for this observation is that hyperosmotic NaCl may have produced a greater net driving force for sodium entry into target neurons compared with either glucose or mannitol. In this regard, ICA NaCl would be expected to increase the concentration of NaCl in the extracellular space surrounding osmosensitive neurons and thus increase the chemical diffusion gradient. In contrast, addition of glucose or mannitol to the interstitial space would be predicted to have opposite effects, namely, a decrease in the normal sodium gradient due to osmotic draw of water to regions adjacent to target neurons. Under these conditions, equivalent increases in extracellular fluid osmolality and thus a similar level of cell shrinkage would be expected to result in a greater increase in ionic conductance through stretch-inactivated cation channels (6, 40). The latter effect would produce a greater membrane depolarization in response to hyperosmotic NaCl treatment compared with either glucose or mannitol. In addition to this mechanism, Voisin et al. (40) recently demonstrated in osmosensitive neurosecretory neurons that for a given level of hyperosmolality, an elevation in extracellular sodium results in a greater increase in membrane permeability. Thus, if similar mechanisms were active in the present study, the enhanced response to hyperosmotic NaCl may have involved both an increase in the sodium driving force as well as an increase in membrane cationic permeability among osmosensitive autonomic regulatory neurons.
A number of studies have demonstrated the presence of osmosensitive neurons in the lamina terminalis (for review see Ref. 3). In vitro recording studies show that even under conditions in which synaptic transmission is blocked, neurons located in the subfornical organ (37), median preoptic nucleus (37, 39), and organum vasculosum laminae terminalis (33) are intrinsically sensitive to changes in fluid osmolality. These same regions of the lamina terminalis are well-recognized circumventricular organs (CVOs) that lack a complete blood-brain barrier. Thus osmotic gradients may build quite rapidly in these regions even when osmotic disturbances are transient, as in the present study. Overall, available evidence indicates that effects of central hyperosmolality on arterial pressure and RSNA involve actions within the lamina terminalis region of the forebrain. Nonetheless, it must also be recognized that central hyperosmolality can open the blood-brain barrier (32), and thus sodium, glucose, or mannitol may be allowed to pass into the interstitium even in regions outside the forebrain CVOs. As a result, hyperosmotic stimuli could act directly within a variety of neuronal cell groups that descend to regions of the brain stem and spinal cord (1, 29, 36) that control sympathetic outflow. Precise determination of the actual neural pathway mediating observed responses will require further electrophysiological studies to identify osmosensitive neurons coupled to autonomic cell groups and to record their gating characteristics during osmotic stimulation.
Although studies have shown that autonomic regions of the PVN are targeted by a number of neurotransmitter systems (5, 31, 38), a major source of synaptic input has been shown to arise from the lamina terminalis region and to be conveyed via angiotensin-containing nerve fibers (for review, see Ref. 11). Combined with data from radioligand-binding studies showing AT1 ANG II-receptor expression in autonomic subdivisions of the PVN (5, 14), we postulated that autonomic PVN neurons might be key targets mediating sympathoexcitatory responses to central hyperosmotic stimulation. In further support of this concept, Chakfe and Bourque (6) recently reported that ANG II activation of AT1 receptors increases gating through effects on stretch-inactivated cation channels present in osmosensitive hypothalamic neurons. Taken together with data from the present study showing that AT1-receptor blockade in the PVN significantly reduced hyperosmolality-evoked pressor and sympathoexcitatory responses, it appears that MAP and RSNA responses could indeed result from release of ANG II as a neurotransmitter within autonomic cell groups of the PVN. Because neither losartan nor vehicle (saline) delivered into the PVN had a significant effect on resting RSNA, it appears unlikely that losartan actions were nonspecific. Data showing that PVN losartan effectively blocked increases in RSNA evoked by local ANG II support this conclusion. Finally, microinjections of losartan outside the PVN failed to significantly alter evoked osmotic responses. Taken together, data from the present study indicate that full manifestation of osmotically induced increases in MAP and RSNA requires activation of ANG II AT1 receptors within autonomic regions of the PVN. It should be emphasized, however, that losartan in the PVN may have reduced neuronal excitability by reducing background angiotensinergic actions. If this is the case, reductions in MAP and RSNA responses to central hyperosmolality by losartan may reflect a permissive rather than a direct role for ANG II in the observed responses.
It should also be emphasized that the sympathoexcitatory response evoked by central hyperosmotic stimulation was not completely blocked by PVN losartan. Although we did not investigate mechanisms for the remaining response, several possibilities exist. First, the dose of losartan used in the present study may not have blocked all AT1 receptors active in the response. This would not appear likely, however, because the dose used was large relative to previous studies (30) and effectively blocked RSNA responses to PVN ANG II. Another possible contribution could have been mediated by interactions of vasopressin with reflex control mechanisms in the brain stem. However, this also seems unlikely because vasopressin release in response to hyperosmotic challenge would likely decrease RSNA by its action to facilitate baroreflex buffering. Moreover, vasopressin augmentation of the arterial baroreceptor reflex has been reported to require baroreceptor afferent input (24). Here, experiments were performed after acute SAD. Another possible mechanism may involve activation of inputs utilizing another neurotransmitter. Glutamatergic, cholinergic, and catecholaminergic inputs, as well as a vast array of other neurotransmitters, have been demonstrated to terminate in the PVN (15, 31, 38). Additional studies remain to be done to investigate the role of other transmitters in cardiovascular responses to hyperosmolality. Finally, PVN autonomic neurons might have intrinsic osmotic sensitivity. Indeed, extracellular recordings have provided evidence of osmotically induced changes in cell discharge among nonneurosecretory neurons in the PVN (26). However, to our knowledge, there is no direct evidence from intracellular recordings showing that autonomic PVN neurons are intrinsically osmosensitive. In the current study the onset of sympathoexcitatory responses was rapid, and the peak response was transient (10-15 s). Whether or not this response pattern could result from increasing osmolality in the interstitium surrounding PVN autonomic neurons is not known. Clearly, additional studies are needed to clarify this important question.
In the present study, PVN losartan produced a slowly developing (60 min) depressor response without an effect on resting RSNA. This result appears in conflict with a previous report that intracerebroventricular administration of losartan (20-40 nmol) did not change baseline blood pressure (17). In the current study, the slowly developing depressor response to centrally administered losartan may indicate involvement of peripheral AT1-receptor blockade. This possibility appears consistent with our observation that the same dose of losartan injected outside the PVN also gradually decreased blood pressure (~10 mmHg), although the decrease did not reach significance. In preliminary experiments, the same dose of the losartan injected intravenously reduced blood pressure 10-15 mmHg in the SAD rat, without changing baseline RSNA. Because SAD has been reported to increase plasma renin concentrations (28), an enhanced contribution of ANG II to the maintenance of arterial pressure in the current study cannot be ruled out. Finally, AT1 receptors are also expressed on vasopressin-synthesizing neurons in the PVN. Given the reduction in baroreceptor-mediated inhibition after SAD, basal AVP release may have been elevated. If so, losartan could have reduced AVP release and thus its pressor actions.
In conclusion, the present study demonstrated that central hyperosmotic stimulation evoked a significant increase in arterial pressure and RSNA in arterial baroreceptor-denervated animals. Observed responses appear to be mediated, at least in part, by a synaptic mechanism involving AT1-receptor activation within the hypothalamic PVN. Further experiments are necessary to define the neuronal cell groups and efferent projection pathways of the PVN activated during central hyperosmotic stimulation and to determine the source(s) of synaptic input that drives osmotically induced increases in sympathetic outflow.
Perspectives
Renal denervation and nerve stimulation studies together suggest that ongoing renal nerve activity promotes sodium reabsorption. This effect appears to increase as nerve activity rises. As the error signal in a negative-feedback control system, increases in extracellular fluid sodium have been reported to reflexly reduce renal nerve activity and thereby promote sodium excretion. Results from the present study appear in conflict with this regulatory scheme because hyperosmotic stimulation increased, not decreased, renal nerve traffic. However, this apparent discrepancy may fit into an alternative and complementary regulatory concept as follows. When sodium is ingested, a rapid reflex sympathoinhibition occurs due to activation of local sensors in the gastrointestinal tract and hepatic portal vein. The reduction in nerve activity promotes sodium loss. If peripherally evoked natriuretic mechanisms are adequate, the absorbed sodium load will be effectively excreted, and extracellular fluid composition of the brain will be protected from major disturbance. However, if sodium rises to a critical level in specific regions of the central nervous system, a renal sympathoexcitatory response will be recruited. The increase in renal nerve discharge is postulated to occur together with sympathoexcitation to other target organs such that arterial and renal perfusion pressures rise. The resulting increase in glomerular filtration evokes a rapid net sodium loss because osmotically induced antidiuretic hormone secretion effectively maintains water reabsorption. If experimentally validated, the central components of this response are envisioned to be active primarily when peripheral reflex mechanisms fail to maintain extracellular fluid composition within an allowable range.| |
ACKNOWLEDGEMENTS |
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We gratefully acknowledge M. Rosas, B. Guler, and M. Cato for excellent technical assistance. We also thank L. LaGrange and Dr. V. S. Bishop for useful discussions of this work.
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FOOTNOTES |
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This project was supported by National Heart, Lung, and Blood Institute Grant HL-56834 and was conducted during the tenure of an American Heart Association Established Investigator Award granted to G. M. Toney.
Address for reprint requests and other correspondence: G. M. Toney, Dept. of Physiology-7756, The Univ. of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900 (E-mail: toney{at}uthscsa.edu).
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. Section 1734 solely to indicate this fact.
Received 8 February 2001; accepted in final form 31 July 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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P < 0.005 vs. 0.9 osmol/l NaCl.
