Intracarotid hypertonic sodium chloride differentially modulates sympathetic nerve activity to the heart and kidney

Abstract

Hypertonic NaCl infused into the carotid arteries increases mean arterial pressure (MAP) and changes sympathetic nerve activity (SNA) via cerebral mechanisms. We hypothesized that elevated sodium levels in the blood supply to the brain would induce differential responses in renal and cardiac SNA via sensors located outside the blood-brain barrier. To investigate this hypothesis, we measured renal and cardiac SNA simultaneously in conscious sheep during intracarotid infusions of NaCl (1.2 M), sorbitol (2.4 M), or urea (2.4 M) at 1 ml/min for 4 min into each carotid. Intracarotid NaCl significantly increased MAP (91 ± 2 to 97 ± 3 mmHg, P < 0.05) without changing heart rate (HR). Intracarotid NaCl was associated with no change in cardiac SNA (11 ± 5.0%), but a significant inhibition of renal SNA (−32.5 ± 6.4%, P < 0.05). Neither intracarotid sorbitol nor urea changed MAP, HR, central venous pressure, cardiac SNA, and renal SNA. The changes in MAP and renal SNA were completely abolished by microinjection of the GABA agonist muscimol (5 mM, 500 nl each side) into the paraventricular nucleus of the hypothalamus (PVN). Infusion of intracarotid NaCl for 20 min stimulated a larger increase in water intake (1,100 ± 75 ml) than intracarotid sorbitol (683 ± 125 ml) or intracarotid urea (0 ml). These results demonstrate that acute increases in blood sodium levels cause a decrease in renal SNA, but no change in cardiac SNA in conscious sheep. These effects are mediated by cerebral sensors located outside the blood-brain barrier that are more responsive to changes in sodium concentration than osmolality. The renal sympathoinhibitory effects of sodium are mediated via a pathway that synapses in the PVN.

increases in the concentration of extracellular fluid sodium [Na⁺] initiate a coordinated series of homeostatic responses to return plasma [Na⁺] to normal levels. The responses are mediated, in part, by the brain in response to the accompanying increase in [Na⁺] within the brain. Understanding the effects of an increase in brain [Na⁺] is of interest because it may play a role in the hypertension due to mineralocorticoid excess, and in the natriuretic and antidiuretic responses to dehydration (27, 38). Furthermore, intravenous hypertonic saline is used as a treatment for hemorrhagic shock, and we have shown that this beneficial action occurs partly because the increase in brain [Na⁺] stimulates cardiac sympathetic nerve activity (CSNA) and thus cardiac output (12).

Numerous studies have shown that intracerebroventricular infusion of hypertonic saline increases arterial blood pressure in several mammalian species (1, 21, 25, 58). The reported effects on renal sympathetic nerve activity (RSNA) are less consistent. Many previous studies have found that acute central or peripheral administration of hypertonic saline decreases RSNA. This has been observed with intravenous hypertonic saline in conscious rabbits (3) and anesthetized rats (37, 54), with intracarotid NaCl in anesthetized cats (50) and with intracerebroventricular NaCl in anesthetized dogs (20). In contrast, an increase in RSNA during intracerebroventricular administration of hypertonic saline was reported in conscious rats (17) and after intracarotid infusion of hypertonic saline in anesthetized rats (7, 44), but the reasons for these differences are unclear. In contrast to RSNA, there is a differential increase in CSNA with intracerebroventricular infusion of hypertonic NaCl in conscious sheep (53). It is not known whether intracarotid infusion of hypertonic saline, which may cause a more physiological change in sodium concentration, results in differential responses in CSNA and RSNA. The first aim of our study was to test the effects on CSNA and RSNA of intracarotid infusion of hypertonic NaCl. In addition, we also tested the effects of two osmotic substitutes, urea, which readily enters cells but does not cross the blood-brain barrier, and sorbitol, which, assuming it is similar to mannitol, neither enters cells nor crosses the blood-brain barrier (30, 34, 57). Furthermore, to ascertain that hypertonic sorbitol but not urea-stimulated central osmoreceptors, we determined in a separate group of animals whether these physiological changes in osmolarity or sodium concentration led to changes in water intake.

The brain regions driving any putative changes in CSNA or RSNA in response to a raised circulating concentration of sodium are not completely clear. A brain area crucial for the regulation of body fluid homeostasis is the paraventricular nucleus of the hypothalamus (PVN). Located lateral to the third ventricle, it is anatomically and functionally connected to neurons residing in forebrain sensory circumventricular organs, parts of the brain lacking a functional blood-brain barrier and known for their ability to sense and respond to small changes in sodium concentration/osmolality (48). The PVN includes vasopressin-producing magnocellular neurons and parvocellular neurons, some of which project to either spinal preganglionic sympathetic neurons or to premotor sympathetic neurons in the brain stem, or to both (43). Thus, the PVN is a plausible site for the coordination of neurogenic and hormonal responses of the cardiovascular system and the kidney to changes in body fluid sodium concentration/osmolality. Indeed, in anesthetized rats, blockade of ANG II type 1 receptors in the PVN attenuates the acute renal sympathoexcitation that occurs after intracarotid infusion of hypertonic NaCl (7). However, the influence of the PVN on homeostatic responses caused by elevated intracarotid [Na⁺] has not been investigated in conscious nonstressed animals. Our aim was to test the hypothesis that mean arterial pressure (MAP) and RSNA responses to intracarotid infusion of hypertonic saline are mediated by the PVN.

METHODS

Adult merino ewes (35–45 kg body wt) were housed in individual metabolism cages in association with other sheep. Experiments were started when sheep were accustomed to laboratory conditions and human contact. Sheep were fed a diet of oaten chaff (800 g/day) and water ad libitum. All experiments were approved by the Animal Experimentation Ethics Committee of the Howard Florey Institute under guidelines laid down by the National Health and Medical Research Council of Australia.

Surgical procedures.

Prior to the studies, sheep underwent two aseptic surgical procedures, separated by a recovery period of at least 2 wk. Anesthesia was induced with intravenous sodium thiopental (15 mg/kg) and following tracheal intubation, anesthesia was maintained with 1.5–2.0% isoflurane/O₂. The first operation involved construction of a carotid arterial loop and consisted of exteriorizing both carotid arteries in a skin fold to facilitate arterial cannulation to measure arterial pressure, as well as infuse drugs. After at least a 2-wk recovery, surgery was conducted to implant electrodes in the cardiothoracic and left renal sympathetic nerves, as described previously (39, 52). Briefly, an incision was made above the fourth rib, the periosteum was opened, and the rib was removed. The thoracic cardiac nerves were identified, and the fascia over the nerves was removed. Up to five electrodes were inserted obliquely through the nerve sheath, ensuring that the tip was positioned in the center of the nerve. Electrodes were fixed in place with cyanoacrylate glue, the implantation site was covered with a layer of Kwik-Sil (World Precision Instruments, Glen Waverly, Vic, Australia), and the wires were exteriorized next to the sutured wound. A stainless-steel ring implanted subcutaneously was used as a ground electrode. The renal artery was exposed via a paracostal retroperitoneal approach. The renal nerves were identified, and up to five electrodes were implanted, as described for the cardiac sympathetic nerves.

In a separate group of animals (n = 6), an additional surgery was performed before the nerve surgery to allow microinjections in the PVN. Animals were placed in a stereotaxic frame, and two stainless-steel guide tubes were inserted, so the tips were 5 mm above the lateral cerebral ventricles. Contrast media (0.3 ml, Omnipaque; GE Healthcare Dassel, Germany) were injected into the lateral ventricles, and two microinjection guide tubes were placed bilaterally 5 mm above the PVN using the ventricular anatomy on overhead and lateral radiographs to guide the placement (11). All cannulas were fixed in place with dental acrylic and sealed with obturators. Because of the low success rates of obtaining combined cardiac SNA recording and successful PVN probe placement, only the MAP and RSNA responses were studied in animals with PVN microinjection probes.

Antibiotic (900 mg procaine penicillin, Troy Laboratories, NSW, Australia) was administered prophylactically at the start of surgery and for 2 days postsurgery. Postsurgical analgesia was maintained with intramuscular injection of flunixin meglumine (1 mg/kg; Mavlab, Qld, Australia) at the start of surgery, and after 24 h recovery.

A day prior to electrode placement surgery, using aseptic techniques, cannulas were inserted 20 cm into a jugular vein for measurement of central venous pressure and for intravenous infusions. One day prior to experiments, a cannula was inserted 15 cm proximally into the carotid artery exteriorized in a loop for measurement of arterial pressure. Experiments were started at least 4 days after implantation of electrodes. On the day of the experiments, both carotid arteries were cannulated by insertion of a 23-gauge needle attached to a polyethylene cannula to enable bilateral intracarotid infusions.

Nerve recording.

Sympathetic nerve activity was recorded differentially between pairs of electrodes, and the pair with the best signal-to-noise ratio was selected (41, 52). The signal was amplified (×100,000) and filtered (bandpass 400 to 1,000 Hz), displayed on an oscilloscope and passed through an audio amplifier and loudspeaker. Sympathetic nerve activity and blood pressure were recorded on computer using a CED micro 1401 interface and Spike 2 software (Cambridge Electronic Design, Cambridge, UK).

Effect of intracarotid infusions of hypertonic saline, sorbitol, and urea on SNA.

These experiments were carried out on separate days in conscious sheep. After a 20-min baseline recording, NaCl (1.2 M) dissolved in water, sorbitol (2.4 M), or urea (2.4 M) dissolved in isotonic saline, were infused at 1 ml/min for 4 min into each carotid artery. Venous blood samples were taken before and at the end of the intracarotid infusion to determine plasma sodium and protein concentrations.

Effects of intravenous infusions of phenylephrine on MAP and SNA.

After a 20-min baseline recording, the responses to intravenous infusion of phenylephrine (33 mg·ml⁻¹·min⁻¹ for 5 min), which caused a similar increase in MAP to intracarotid infusion of NaCl, was examined. The changes in MAP, HR, CSNA, and RSNA were recorded.

Effect of intracarotid infusions of hypertonic saline, sorbitol, and urea on drinking responses.

The effects of bilateral intracarotid infusion of NaCl (1.2 M), sorbitol (2.4 M), or urea (2.4 M) at 1 ml/min for 20 min into each carotid artery on water intake determined in a separate group of sheep (n = 4). The infusions of the different solutions were carried out on separate days. Access to water was removed for 30 min prior to beginning the experiment, and the amount of water drunk over the 20 min of infusion was calculated.

Role of the PVN in mediating the SNA responses to intracarotid hypertonic saline.

After a 20-min baseline recording, muscimol (5 mM, 500 nl each side) or artificial cerebrospinal fluid (CSF) (500 nl) was bilaterally microinjected into the PVN in conscious sheep (n = 6). This was immediately followed by intracarotid infusion of NaCl (1.2 M at 1 ml/min for 5 min into each carotid artery). Sheep were treated with muscimol or vehicle in random order on separate days.

Data analysis.

Data were analyzed on a beat-to-beat basis using custom-written routines in the Spike 2 program, as previously described (41, 52). For each heartbeat, the program determined diastolic, systolic, and MAP, heart period, and the number of discriminated spikes over threshold between the following diastolic pressures (a measure of burst size). The threshold was set just above background so that spikes from small bursts could be counted. As a measure of CSNA or RSNA, the spikes over threshold were calculated for each heartbeat. The accuracy of burst determination was checked by eye over the entire recording file. Post hoc analysis of data was conducted by using custom-written scripts for Spike 2, and data were exported to a spreadsheet for further analysis.

Verification of PVN cannula placement.

All sheep were killed with an overdose of pentobarbital sodium (100 mg/kg). Blue dye (100 nl) was microinjected into the PVN to verify the position of the injection. In all animals, brains were instantly perfused via the carotid arteries with isotonic saline followed by 4% paraformaldehyde and were later transferred to 20% sucrose. Two days later, brain sections (60 μm) were cut on a freezing microtome in a coronal plane. Sections were mounted on glass slides and stained with cresyl violet. The injection sites were verified using a light microscope by following the tracks of the microinjection cannula. Only animals in which the center of the blue dye was within the boundaries of the PVN were included in this study.

Statistics.

Results are expressed as means ± SE. Paired t-tests were used to compare changes in plasma sodium and plasma protein before and 4 min postinfusion of solutes. One-way ANOVA was used to test for differences between baseline and the different time points for hypertonic sodium chloride, urea, and sorbitol infusions. A significant result was considered to be P < 0.05.

RESULTS

There were no differences in the levels of MAP, CSNA, or RSNA in the baseline periods before each intracarotid infusion (Table 1). The baseline levels of CSNA were significantly lower than that of RSNA (P < 0.05) (Table 1). Bilateral intracarotid infusion of hypertonic saline (1.2 M) significantly increased MAP (Figs. 1 and 2). There was no change in the levels of CSNA (11 ± 5.0%), and there was a significant decrease in the levels of RSNA (−32.5 ± 6.4%; P < 0.05) within the 4 min of the infusion (Fig. 3). Intracarotid infusion of either sorbitol (2.4 M) or urea (2.4 M) had no effect on the baseline levels of MAP, CSNA, or RSNA (Figs. 2 and 3). In addition, intravenous infusion of NaCl (1.2 M) was also without effect. There was no change in plasma sodium or plasma protein levels after any of the infusions (Table 2).

Table 1.

Baseline levels of mean arterial pressure, heart rate, central venous pressure, cardiac, and renal sympathetic nerve activity just before the different infusions were administered

	1.2 M NaCl ic	n	2.4 M Sorbitol ic	n	2.4 M Urea ic	n	1.2 M NaCl iv	n
Mean arterial pressure, mmHg	91 ± 3	7	92 ± 2	7	91 ± 2	4	90 ± 3	4
Heart rate, beats/min	84 ± 5	7	86 ± 5	7	96 ± 5	4	87 ± 7	4
Central venous pressure, mmHg	2 ± 1	7	1.0 ± 0.5	7	0.8 ± 0.2	4	1.2 ± 0.6	4
CSNA, spikes/s	8.5 ± 3.2	6	10.2 ± 5.7	6	7.0 ± 1.0	4	7.8 ± 1.0	3
RSNA, spikes/s	15.5 ± 1.4	7	15.1 ± 1.9	7	16.1 ± 2.3	4	17.6 ± 1.9	4

Values are expressed as means ± SE.

ic, intracarotid; iv, intravenous; CSNA, cardiac sympathetic nerve activity; RSNA, renal sympathetic nerve activity.

Fig. 1.

Representative recordings of arterial pressure, cardiac sympathetic nerve activity (CSNA), and renal sympathetic nerve activity (RSNA) before and after bilateral intracarotid infusion (dashed line) of NaCl (1.2 M at 1ml/min ic bilaterally).

Fig. 2.

Effects of intracarotid infusions of hypertonic NaCl (1.2 M), sorbitol (2.4 M), urea (2.4 M), and intravenous NaCl (1.2 M) on mean arterial pressure (MAP), heart rate (HR), and central venous pressure (CVP). *Significant effect of the infusion, P < 0.05.

Fig. 3.

Effects of intracarotid infusions of hypertonic NaCl (1.2 M), sorbitol (2.4 M), urea (2.4 M), and intravenous NaCl (1.2 M) on cardiac sympathetic nerve activity (CSNA) and renal sympathetic nerve activity (RSNA). *Significant effect of the infusion, P < 0.05.

Table 2.

Changes in plasma sodium and plasma protein levels after intracarotid infusion of NaCl (1.2 M), sorbitol (2.4 M), or urea (2.4 M) and intravenous infusion of NaCl (1.2 M)

Group	1.2 M NaCl ic	n	2.4 M Sorbitol ic	n	2.4 M Urea ic	n	1.2 M NaCl iv	n
Plasma sodium, mmol/l
Baseline	142.8 ± 0.5	7	145.0 ± 1.1	7	143.5 ± 0.4	4	142.5 ± 0.4	4
4 min	143.4 ± 1.0	7	143.8 ± 1.2	7	143.5 ± 0.4	4	144.5 ± 1.2	4
Plasma-Protein, g/l
Baseline	74 ± 2.0	7	77.6 ± 0.6	7	77.0 ± 0.8	4	71.0 ± 2.4	4
4 min	74.2 ± 1.4	7	76.8 ± 0.4	7	77.5 ± 0.4	4	72.0 ± 0.8	4

Values are expressed as means ± SE.

Phenylephrine was infused intravenously at doses titrated to increase MAP (5.5 ± 0.8 mmHg) to a similar extent as intracarotid infusion of 1.2 M NaCl (5.2 ± 0.7 mmHg) (Fig. 4). Phenylephrine caused a significant bradycardia and inhibition of both CSNA and RSNA. In contrast, hypertonic saline had no effect on heart rate or CSNA, but it caused a greater inhibition of RSNA than phenylephrine (all P < 0.05 vs. phenylephrine) (Fig. 4).

Fig. 4.

Comparison of the effects of intracarotid infusion of hypertonic NaCl (●) and intravenous phenylephrine (○) on heart rate, cardiac sympathetic nerve acticity (CSNA), and RSNA. #Significant difference between the two infusions.

Thirst responses to intracarotid infusion of saline.

After the end of the intracarotid infusion of hypertonic saline when the water was returned to the animals, 1,100 ± 75 ml of water was drunk (n = 4; Fig. 5). Intracarotid infusion of hypertonic sorbitol led to an increase in water intake (683 ± 125 ml of water). In contrast, no water was drunk at the end of the intracarotid infusion of urea.

Fig. 5.

Amount of water drunk by sheep during 20 min of intracarotid infusion of hypertonic NaCl (1.2 M), sorbitol (2.4 M), and urea (2.4 M). There was a significant difference in the amount of water drunk after the different infusions. #Significant difference from hypertonic NaCl, P < 0.05.

Role of the PVN in mediating the SNA responses to intracarotid hypertonic saline.

Intracarotid infusion of hypertonic saline in sheep given a microinjection of artificial CSF into the PVN was associated with a similar increase in MAP and decrease in RSNA to that observed in the previous group of animals. Inhibition of neurons in the PVN using muscimol (5 mM) did not significantly change MAP or RSNA, but it significantly attenuated the increase in MAP and prevented the inhibition of RSNA caused by subsequent intracarotid infusion of hypertonic NaCl (Fig. 6).

Fig. 6.

Effects of intracarotid infusions of hypertonic NaCl (1.2 M) on mean arterial pressure and RSNA in animals with prior microinjection of artificial cerebrospinal fluid (aCSF) (500 nl each side; ●) or muscimol (5 mM, 500 nl each side; ○) into the PVN. #Significant effect between the aCSF and mucimol. B: distribution of all injection sites in a schematic drawing adapted from coronal sections from one animal. The dots show injection sites as visualized by the distribution of blue dye. OC, optic chiasm; PVN, paraventricular nucleus.

DISCUSSION

Our study is the first to examine the differential responses in cardiac and renal SNA during intracarotid infusion of hypertonic saline in conscious sheep. Our main findings are 1) increased sodium levels in the blood supply to the brain result in an increase in arterial pressure but a significant decrease in RSNA and no change in CSNA; 2) the MAP and SNA responses are mediated by cerebral sensors located outside the blood-brain barrier that are more responsive to changes in NaCl concentration per se compared with osmolality or tonicity, and 3) the changes in MAP and renal SNA in response to increased blood sodium levels are mediated by a neural pathway though the PVN.

Changes in SNA with hypertonic saline.

We have previously shown that infusion of hypertonic saline into the cerebral ventricles increased MAP, stimulated CSNA, and inhibited RSNA (21, 53). In the present study, we observed similar responses in MAP and RSNA when hypertonic saline was given into the carotid arteries, a more physiological route. The role that baroreceptor-mediated sympathoinhibition played in determining the responses to hypertonic saline was evaluated by studying the responses to an equivalent pressor dose of phenylephrine. A similar increase in MAP induced by phenylephrine caused less renal sympathoinhibition, indicating that hypertonic saline has a direct action to inhibit RSNA, as well as inducing a baroreceptor-mediated inhibition. In contrast to the decreases in heart rate and CSNA with phenylephrine, there were no changes with hypertonic saline, indicating that the baroreceptor-mediated inhibition of CSNA in response to the increase on MAP may have been countered by sodium-induced selective cardiac sympathoexcitation.

There are extensive data in the literature from both our laboratory and others that intracarotid or systemic infusion of hypertonic NaCl or hyperosmolar urea elevates the CSF sodium concentration and tonicity (9, 24, 42, 51) and almost certainly brain or interstitial sodium concentration and tonicity. Therefore, it is unlikely that a central osmoreceptor or sodium sensor within the blood-brain barrier mediates the changes in MAP and RSNA caused by intracarotid hypertonic NaCl. While sorbitol, unlike urea, should cause cellular dehydration outside the blood-brain barrier in the circumventricular organs (30, 34), it is unlikely that the changes in SNA in response to intracarotid hypertonic NaCl are mediated by osmoreceptors either inside or outside the blood-brain barrier because both intracarotid sorbitol and urea were ineffective in changing MAP or RSNA. Hence, we concluded that the changes were due to actions of NaCl on sodium receptors outside the blood-brain barrier. In this regard, one of the important areas in the brain sensing changes in plasma and CSF sodium is the lamina terminalis in the anterior wall of the third ventricle (29). Previous studies have shown that lamina terminalis lesions abolished the renal blood flow responses to intravenous hypertonic NaCl (36). In addition, we have shown that the inhibition of RSNA and reduction in plasma renin activity in response to intracerebroventricular hypertonic NaCl is abolished following lamina terminalis lesions (22), suggesting that the lamina terminalis plays an important role in mediating the inhibition of RSNA. Chemical inhibition or electrolytic lesion of the organum vasculosum of the lamina terminalis (OVLT) also attenuated changes in RSNA after injections of hypertonic NaCl into the carotid artery of anesthetized rats (44), consistent with an important role of the lamina terminalis in sensing sodium.

The pathway from the lamina terminalis that leads to renal sympathoinhibition and cardiac sympathoexcitation is unclear. There is extensive evidence of anatomical and functional links between the lamina terminalis and the PVN. Neurons in the OVLT innervate both the magnocellular and the parvocellular neurons of the PVN (26), and studies using c-Fos as a marker of activation have shown that OVLT neurons projecting to the PVN are activated by systemically infused hypertonic saline (29). A previous study in rats has indicated an important role of the PVN in mediating the pressor response to intracerebroventricular hypertonic saline (13, 14). In addition, studies in conscious rabbits have also indicated that the PVN plays an important role in mediating the changes in RSNA to intravenous hypertonic saline (3). In the present study, the pressor and sympathetic actions of intracarotid hypertonic saline were blocked by inhibition of neurons in the PVN with muscimol (Fig. 6), indicating that these responses are mediated via the PVN. Similarly, we have shown that intracerebroventricular hypertonic saline-induced renal sympathoinhibition is mediated by neurons in the PVN, since this response was blocked by glycine microinjection into the PVN (11). In addition to changes induced by hypertonic saline, our previous findings also indicate a role for the PVN in mediating the inhibition in renal SNA after volume expansion in conscious animals (40), indicating that this nucleus is essential for both sodium and volume homeostasis. It is important to point out that while previous studies have explored the role of the PVN in control of RSNA, the majority of these studies have been conducted in anesthetized animals. Given that a previous study has shown that anesthesia can significantly modify the cardiovascular responses to stimulation of the PVN (19), it was important to conduct these studies in conscious animals.

Our study does not allow us to ascertain the specific neurotransmitters in the PVN that mediate the responses to hypertonic saline, although previous studies have indicated a role for both angiotensin (21) and glutamate (2). In addition, a recent study has suggested that the increase in renal SNA observed during intracarotid infusion of hypertonic saline in the anesthetized rat is dependent on release of vasopressin within the PVN (46).

Inhibition of neurons in the PVN also abolished the increase in MAP caused by hypertonic saline. It is likely that the increase in MAP is driven by a combination of neurally mediated vasoconstriction (16) and vasopressin release (4). While we measured a small nonsignificant increase in cardiac SNA, it is unlikely that this would be sufficient to drive the increase in MAP. Previous studies have shown that hypertonicity increases lumbar sympathetic nerve activity in anesthetized rats (54), as well as humans (10), suggesting that this contributes to the pressor response. Whether the PVN mediates the increase in muscle SNA during increases in brain sodium levels needs to be directly tested. In addition, studies in rats have shown an important role for vasopressin in mediating the increase in MAP after acute hyperosmolarity (4), indicating both vasopressin and SNA contribute to the increase in MAP. It appears that in sheep, the increase in SNA is the most important mechanism to increase MAP, since infusion of vasopressin at high doses does not increase MAP (33).

It should also be noted that hypertonic saline may act on neural afferent pathways to modulate SNA. A possible action on the carotid bodies is suggested by the finding that sinoaortic denervation attenuated the recovery of MAP in response to hypertonic saline (8). In addition, recent studies have also suggested a role for A2 noradrenergic neurons since the decrease in RSNA in anesthetized rats following hypertonic saline infusion is abolished with lesion of these neurons (35).

Regarding the physiological relevance of our dose, previous studies have indicated that intracarotid infusion of hypertonic saline in conscious sheep raises CSF sodium by 2 mM, which is well within the physiological range. Indeed, the increase in CSF sodium during 48-h water dehydration is close to 5 mM (42). It is difficult to directly estimate the osmolality of the fluid surrounding the OVLT neurons. We know there are parallel changes in the CSF osmolarity during the hypertonic saline infusion, although this change in osmolarity does not mediate the changes in SNA, given our results with intracarotid sorbitol and urea. In contrast to our study, the increases in plasma osmolality produced by intracarotid injections of hypertonic saline in the anesthetized rat (7, 44) are estimated to exceed levels observed under physiological or pathophysiological conditions, as acknowledged by the authors of the rat studies (44). More importantly, the changes in osmolarity induced by the hypertonic saline in those studies (7, 44) also have independent effects on MAP and RSNA (7), suggesting the stimulus for the increase in RSNA is hyperosmolarity, as opposed to increased sodium levels. In addition, the species, as well as the state of the animal, may play an important role. In this context, our findings in conscious animals are important since previous studies have clearly shown that anesthesia can both alter baseline levels of SNA and dampen reflex responses to SNA (45).

Water drinking responses to hypertonic NaCl.

Our data indicating increased water intake in response to hypertonic saline is in agreement with similar findings in a previous study (28). These data showing increased water intake with both intracarotid hypertonic sorbitol, as well as hypertonic saline, but not urea, confirms that sorbitol is an effective stimulus to the central osmoreceptors, although somewhat less than hypertonic NaCl. Because access to the water was delayed in this experiment, we cannot rule out the possibility that osmoreceptors outside the forebrain, including in the periphery, may have been activated by the hypertonic saline. In addition, it is possible that the lesser drinking response to intracarotid sorbitol infusion may have been influenced by the systemic hyponatremia that results from osmotic movement of intracellular water to the blood. However, this is not the case for intracarotid infusion of hypertonic sucrose, which causes similar water intake as hypertonic NaCl; the majority of evidence favoring an osmoreceptor for thirst outside the blood-brain barrier (24, 49). Similar to the role of the OVLT in mediating the SNA responses, the OVLT also plays an important role in mediating the increases in water intake during intracarotid hypertonic saline infusion (26). Studies using c-Fos expression have shown increased neuronal excitability in the OVLT in response to hypertonicity (31), and lesions of the OVLT combined with the MnPO reduce the amount of water drunk in conscious sheep (28).

Perspectives and Significance

These findings have important implications for understanding sympathetic control under physiological conditions characterized by increased sodium levels in the blood. One such situation is water deprivation, which results in a significant increase in the daily output of [Na⁺] in the urine (23). The present study in conscious, normovolemic sheep has demonstrated a significant role for PVN neurons in mediating the decrease in renal SNA evoked by intracarotid hypertonic sodium chloride challenge. This decrease in RSNA aids in acutely removing excess sodium via the urine. This effect appears to be mediated by sensors located outside the blood-brain barrier that are more responsive to [Na⁺] per se, as opposed to osmolarity or tonicity. This result clearly implicates the PVN as part of the neural circuitry stimulated by increased sodium concentration. In addition, osmotic regulation of SNA is also important for maintenance of MAP during water deprivation (6, 47). The present study also demonstrates that an increase in circulating [Na⁺] stimulates the PVN, leading to an increase in blood pressure. This is presumably due to peripheral vasoconstriction of both arteries and veins. This activation of the sympathetic nervous system in response to increased sodium concentrations results in increased peripheral vasoconstriction (5) and has been proposed to cause hypertension. Consistent with our findings of differential regulation of sympathetic outflow by hypertonic saline, a previous study in conscious rats indicated a chronic decrease in directly recorded renal SNA, but no change in lumbar SNA during hypertension induced by high salt and ANG II (56). In addition, denervation of the renal nerves does not attenuate the hypertension mediated by DOCA salt (18) or by a high-salt diet in the Dahl-sensitive rat (32, 55), suggesting the pressor response is not mediated by an increase in renal SNA. In accordance with studies performed in rats (2, 7, 15), these results suggest that the PVN may be part of the neural circuitry stimulated by increased brain sodium concentration that is responsible for slowly developing sodium-sensitive hypertension.

GRANTS

This work was supported by National Health and Medical Research Council of Australia Grant 628573 and the Victorian Government's Operational Infrastructure Support Program. R. Frithiof was supported by funds from the Swedish Research Council (Grant 521-2011-2843). R. Ramchandra was the recipient of National Health and Medical Research Council/National Heart Foundation Postdoctoral Fellowship 07M 3293, and C. N. May was supported by a National Health and Medical Research Council Research Fellowship 566819.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: R.F. and R.R. conception and design of research; R.F., T.X., and R.R. performed experiments; R.F., T.X., and R.R. analyzed data; R.F., T.X., M.J.M., C.N.M., and R.R. interpreted results of experiments; R.F., T.X., and R.R. prepared figures; R.F., M.J.M., C.N.M., and R.R. edited and revised manuscript; R.F., T.X., M.J.M., and R.R. approved final version of manuscript; R.R. drafted manuscript.