Hyperosmotic activation of CNS sympathetic drive: implications for cardiovascular disease

Transcription

1 J Physiol (2010) pp SYMPOSIUM REVIEW Hyperosmotic activation of CNS sympathetic drive: implications for cardiovascular disease Glenn M. Toney 1 and Sean D. Stocker 2 1 Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX , USA 2 Department of Cellular and Molecular Physiology, Pennsylvania State College of Medicine, Hershey, PA 17033, USA Evidence now indicates that exaggerated sympathetic nerve activity (SNA) significantly contributes to salt-sensitive cardiovascular diseases. Although CNS mechanisms that support the elevation of SNA in various cardiovascular disease models have been intensively studied, many mechanistic details remain unknown. In recent years, studies have shown that SNA can rise as a result of both acute and chronic increases of body fluid osmolality. These findings have raised the possibility that salt-sensitive cardiovascular diseases could result, at least in part, from direct osmosensory activation of CNS sympathetic drive. In this brief review we emphasize recent findings from several laboratories, including our own, which demonstrate that neurons of the forebrain organum vasculosum laminae terminalis (OVLT) play a pivotal role in triggering hyperosmotic activation of SNA by recruiting neurons in specific regions of the hypothalamus, brainstem and spinal cord. Although OVLT neurons are intrinsically osmosensitive and shrink when exposed to extracellular hypertonicity, it is not yet clear if these processes are functionally linked. Whereas acute hypertonic activation of OVLT neurons critically depends on TRPV1 channels, studies in TRPV1 / mice suggest that acute and long-term osmoregulatory responses remain largely intact. Therefore, acute and chronic osmosensory transduction by OVLT neurons may be mediated by distinct mechanisms. We speculate that organic osmolytes such as taurine and possibly novel processes such as extracellular acidification could contribute to long-term osmosensory transduction by OVLT neurons and might therefore participate in the elevation of SNA in salt-sensitive cardiovascular diseases. (Received 21 April 2010; accepted after revision 27 June 2010; first published online 5 July 2010) Corresponding author G. M. Toney: Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX , USA. toney@uthscsa.edu Abbreviations ICV, intracerebroventricular; IML, intermediolateral cell column; MNC, magnocellular neurosecretory cell; MnPO, median preoptic nucleus; OVLT, organum vasculosum laminae terminalis; PVN, paraventricular nucleus; RVI, regulatory volume increase; RVLM, rostral ventrolateral medulla; SFO, subfornical organ; SIC, stretch-inhibited cation (channel); SNA, sympathetic nerve activity; TRPV, transient receptor potential vanilloid (channel). Glenn Toney obtained his bachelor s degree from Weber State University (Ogden, Utah) and his PhD in physiology from the University of Louisville College of Medicine. His first faculty position was at the University of Texas Health Science Center at San Antonio where he remains, now as Professor. He has spent most of his career studying neural circuits in the mammalian brain that regulate autonomic function in relation to cardiovascular and metabolic diseases. He is currently on the editorial boards of the American Journal of Physiology: Regulatory, Integrative, and Comparative Physiology and The Journal of Physiology. Sean Stocker received his PhD in neurocience from the University of Pittsburgh in 2002 followed by postdoctoral training in physiology at UT Health Science Center. He is currently Associate Professor in the Department of Cellular & Molecular Physiology at Penn State College of Medicine. Dr Stocker s laboratory is funded by the NIH and American Heart Association. His research focuses on the neural pathways and cellular mechanisms that contribute to elevated sympathetic outflow in obesity and salt-sensitive hypertension. He is currently a member of the editorial board of the journal Hypertension. This review was presented at The Journal of Physiology Symposium on Regulation of neuronal cell volume: From activation to inhibition to degeneration, which took place at Experimental Biology 2010, Anaheim, CA, USA, 26 April It was commissioned by the Editorial Board and reflects the views of the authors. DOI: /jphysiol

2 3376 G. M. Toney and S. D. Stocker J Physiol Throughout evolution, dehydration has been a persistent challenge to body fluid osmotic homeostasis. Among an array of known osmoregulatory responses to dehydration, pituitary release of the antidiuretic hormone vasopressin and thirst are perhaps the best characterized and extensive reviews have been published on these subjects (McKinley et al. 1992; Johnson et al. 1996; Bourque, 2008). Vasopressin preserves fluid volume by reducing urinary water loss and thirst stimulates drinking to restore fluid volume and tonicity. These responses are stimulated, in large part, by CNS osmosensory neurons that respond to body fluid hyperosmolality and activate appropriate downstream circuitry (Johnson et al. 1996; McKinley et al. 2003; Bourque, 2008). Recent work has established that acute (Chen & Toney, 2001; Shi et al. 2007, 2008) and chronic (Stocker et al. 2006; Freeman & Brooks, 2007) hyperosmotic challenges also cause prompt and persistent increases of peripheral sympathetic nerve activity (SNA). These findings have also been the subject of several recent reviews (Toney et al. 2003; Stocker et al. 2008, 2010). Although the role that elevated SNA might play in restoring body fluid homeostasis is not known (Toney et al. 2003; Brooks et al. 2005a; Stocker et al. 2008), elevated SNA to the kidneys during dehydration could help to preserve blood volume by mitigating Na + excretion that would normally occur as a result of pituitary release of oxytocin (Johnson et al. 1996; McKinley et al. 2003). More generalized sympathetic activation could help to maintain cardiovascular function and preserve the ability to forage for water (Toney et al. 2003; Stocker et al. 2008). In modern times humans have begun to experience a different form of osmotic challenge: that imposed by ingesting foods high in salt (NaCl). Coincident with the trend to increase salt consumption there has been a dramatic increase in the prevalence of salt-sensitive cardiovascular diseases such as hypertension (Weinberger, 1996) and heart failure (DiBona & Sawin, 1991; Adams, 2004). A large and growing body of literature now links sodium-retaining cardiovascular diseases with elevated SNA (DiBona & Sawin, 1991; Weinberger, 1996; Adams, 2004; Brooks et al. 2005a; O Donaughy et al. 2006). Given emerging evidence that body fluid hyperosmolality can stimulate SNA (Chen & Toney, 2001; Stocker et al. 2006; Freeman & Brooks, 2007; Shi et al. 2007, 2008), an important question naturally follows. Does body fluid hyperosmolality stimulate CNS sympathetic drive and might this contribute to salt-sensitive cardiovascular diseases? It should be emphasized that the answer to the above question is not known. Indeed, the concept that body fluid hyperosmolality is capable of stimulating regional sympathetic outflow is not without controversy as a number of studies report an opposite effect (i.e. a decrease of renal SNA). For more detailed information on osmoregulatory control of body fluid homeostasis and cardiovascular function the reader should consult comprehensive reviews that have appeared in the literature (Johnson et al. 1996; McKinley et al. 2003; Toney et al. 2003; Bourque, 2008; Stocker et al. 2008, 2010). Here, our first goal is to present a concise summary of recent information on CNS circuitry that drives increases of SNA in response to acute and chronic hyperosmotic challenges. Much of this work originated from the laboratories of the authors, with significant contributions from our esteemed colleagues. A major conclusion is that osmosensory neurons in the ventral forebrain/organum vasculosum laminae terminalis (OVLT) are critical for translating body fluid hyperosmolality into increases of SNA. Given the functional importance of OVLT neurons, it is apparent that their osmosensory transduction mechanisms must be elucidated if we hope to fully grasp the role of body fluid hyperosmolality in driving sympathoexcitation and whether this plays a role in salt-sensitive cardiovascular diseases. Toward this end, the second part of this report underscores how little is presently known about OVLT neuronal osmo-transduction by contrasting this with more detailed information on osmo-transduction by hypothalamic magnocellular neurosecretory cells (MNCs). To stimulate dialogue and encourage additional work in the area, we conclude this report by speculating on possible OVLT neuronal osmo-transduction mechanisms and their potential involvement in salt-sensitive cardiovascular disease. CNS circuitry mediating sympathetic activation by body fluid hyperosmolality There is now compelling evidence from studies in humans and rodents that acute increases in plasma osmolality or Na + concentration can result in a prompt increase of SNA (Stocker et al. 2008). The CNS circuitry that has been shown to mediate this effect is shown in Fig. 1. It is important to note that whereas both intravenous and intracarotid infusions of hypertonic NaCl raise SNA and arterial blood pressure (Weiss et al. 1996; Chen & Toney, 2001; Shi et al. 2007), intracarotid hypotonic saline has been shown to do the opposite, i.e. reduce ongoing SNA and arterial blood pressure, in water-deprived (Brooks et al. 2005b) and deoxycorticosterone acetate (DOCA)+ salt-treated hypertensive rats (O Donaughy et al. 2006) (Fig. 2). The principle osmoreceptors that regulate antidiuretic hormone secretion, thirst and SNA, are located in the forebrain lamina terminalis (Johnson et al. 1996; McKinley et al. 2003; Toney et al. 2003; Bourque, 2008; Stocker et al. 2008), which is a collection of interconnected structures located along the rostral wall of the third cerebral ventricle. It consists of the organum vasculosum laminae terminalis

3 J Physiol Hyperosmotic activation of SNA in cardiovascular disease 3377 (OVLT), the median preoptic nucleus (MnPO) and the subfornical organ (SFO). The OVLT and SFO each lack a complete blood brain barrier, and evidence indicates that neurons within these regions are activated by hypernatraemia (Toney et al. 2003; Stocker et al. 2008) and are intrinsically osmosensitive (Anderson et al. 2000; Ciura & Bourque, 2006). Stimulation of OVLT or SFO neurons raises SNA (Toney et al. 2003; Stocker et al. 2008), and inhibition of the OVLT (Shi et al. 2007) or knife cuts placed immediately caudal to the lamina terminalis attenuate the sympathoexcitatory response to acute hypernatraemia (Antunes et al. 2006). The major downstream target of neurons in the lamina terminalis is the hypothalamic paraventricular nucleus (PVN). PVN neurons modulate SNA through mono- and polysynaptic pathways to several sympathetic-regulatory regions including the rostral ventrolateral medulla (RVLM) and spinal intermediolateral cell column (IML) (Toney et al. 2003; Brooks et al. 2005a; Guyenet, 2006; Stocker et al. 2008). The discharge of PVN neurons is altered by a number of sensory inputs including those responsive to changes in plasma osmolality or Na + concentration. The contribution of PVN neurons to osmotic-induced changes in SNA is synaptically driven rather than involving an intrinsic osmosensitivity as direct injection of hypertonic NaCl into the PVN does not affect SNA (Antunes et al. 2006), but blockade of local ionotropic glutamate (Antunes et al. 2006) or angiotensin II type 1 (Chen & Toney, 2001) receptors significantly attenuates acute hyperosmolality-induced increases of SNA. PVN neurons may increase SNA during hyperosmolality through direct projections to the IML or indirectly via the RVLM. In regard to the former, blockade of spinal vasopressin receptors eliminates the sympathoexcitatory response to increased systemic osmolality (Antuneset al. 2006). Inhibition of the RVLM, on the other hand, prevents SNA responses to intracarotid injection of hypertonic NaCl (Stocker et al. 2008), and blockade of ionotropic glutamate receptors in the RVLM lowers arterial blood pressure in rats infused with hypertonicnaclordeprivedofwaterfor48h(brookset al. 2004a,b). While most humans only rarely experience chronic dehydration due to prolonged water deprivation, modern diets are typically high in salt (NaCl), and evidence suggests that high dietary salt intake raises plasma sodium concentration in both humans and rodents (Habecker et al. 2003; He et al. 2005; Schmidlin et al. 2007; Adams et al. 2009). Although technical limitations of multi-fibre sympathetic nerve recordings do not permit unequivocal assessment of the impact of high dietary salt on the level of ongoing SNA (Stocker et al. 2010), evidence indicates that elevated salt intake exaggerates a number of sympathetic reflexes and enhances the excitability of sympathetic neurons in the RVLM (Adams et al. 2007, 2008, 2009; Stocker et al. 2010). The RVLM contains tonically active bulbospinal neurons and has been implicated in elevated SNA of many cardiovascular diseases including salt-sensitive hypertension (Guyenet, 2006). Increased dietary salt intake enhances SNA and blood pressure responses to RVLM injection of various receptor agonists including L-glutamate, angiotensin II, and GABA (Adams et al. 2007, 2008). In addition, increased dietary salt enhances several sympathetic reflexes that depend upon Figure 1. Schematic representation of osmosensitive sympathetic-regulatory CNS neural pathways See text for details.

4 3378 G. M. Toney and S. D. Stocker J Physiol RVLM neurotransmission (Stocker et al. 2010). To date, it is not clear how dietary salt causes changes to these neurons; however, as noted above, several laboratories have reported that increased dietary salt intake can elevate plasma sodium concentration (Habecker et al. 2003; Adams et al. 2009) within less than 48 h. Thus it seems conceivable that hypernatraemia/plasma hyperosmolality might act directly to stimulate forebrain osmosensory neurons. Support for this comes from evidence that lesion of the ventral lamina terminalis or OVLT (i) prevents dietary salt-induced functional changes in RVLM (Adams et al. 2009), (ii) prevents exaggeration of sympathetic reflexes (S. D. Stocker, unpublished observations), and (iii) attenuates or prevents the development of hypertension in several experimental models (Brody, 1988). In specific regard to functional sensitization of the RVLM Figure 2 Antihypertensive effect of acute hypotonicity Effect of intracarotid (IC) hypotonic fluid infusion on mean arterial pressure (MAP) and heart rate (HR) in deoxycorticosterone acetate (DOCA)-salt rats either untreated (Intact) or pretreated with a V 1 vasopressin antagonist (V 1 blocked) or with hexamethonium and the V 1 antagonist (ganglionic and V 1 blocked). P < 0.05, within group. #P < 0.05, between groups separated by # (DOCA-salt IC and other 2 groups; ganglionic and V 1 blocked and other 2 groups). (Reproduced with permission from O Donaughy et al ) by high dietary salt, it is noteworthy that the underlying mechanisms are not known. However, it seems likely that sensitization involves neuronal adaptive responses (i.e. plasticity) that develop and resolve over time such that onset and recovery are delayed relative to the occurrence of elevated plasma Na + (Stocker et al. 2010). Mechanisms of neuronal osmosensory transduction From the foregoing discussion is seems that hyperosmotic activation of SNA is largely dependent on neuronal osmosensory mechanisms in the ventral lamina terminalis/ovlt. This is underscored by evidence that acute inhibition of OVLT neuronal activity largely prevents increases of SNA evoked by internal carotid artery (ICA) injectionofgradedconcentrationsofnacl(shiet al. 2007) (Fig. 3). Although in vitro patch clamp recordings of enzymatically dissociated OVLT neurons have shown that many are responsive to brief hypertonic stimulation (Nissen et al. 1993; Ciura & Bourque, 2006), precisely which neurons in the intact OVLT are intrinsically responsive to hyperosmolality is unresolved. It may even be possible that distinct populations of neurons transduce acute versus chronic increases in osmolality. In this regard, the OVLT is known to consist of two sub-regions, a dorsal cap and lateral margins, which surround a highly vascular core (McKinley et al. 2003; Shi et al. 2008). Neurons of the dorsal cap are generally credited with playing a dominant role in the activation of osmoregulatory responses (McKinley et al. 2003). Arecent study indicated that a similar number of neurons in the dorsal cap and lateral margins express c-fos in response to graded hypertonic NaCl challenges achieved by bolus intracarotid injections (Shi et al. 2008). Collectively it would appear that if different subsets of OVLT neurons mediate responses to acute and chronic hyperosmotic disturbances, then most likely they are interspersed among one another and not segregated into different OVLT sub-regions. Another recent observation is that a nearly equal number of neurons in each OVLT sub-region with axonal projections to the PVN were activated by acute hypertonic NaCl challenge (Shi et al. 2008). Whether c-fos expression among OVLT PVN output neurons reflects their intrinsic osmosensitivity or their synaptic activation by other intrinsically osmosensitive OVLT interneurons is not presently known. Neuronal osmo-transduction. Because osmolality rises progressively with dehydration and can remain elevated for a prolonged period during chronic salt loading, effective osmosensory mechanisms should not be rapidly adapting. It is therefore important to evaluate the temporal response characteristics of intrinsically osmosensitive neurons and establish whether the same or different osmosensory mechanisms mediate rapid and long-term

5 J Physiol Hyperosmotic activation of SNA in cardiovascular disease 3379 osmoregulatory responses. In the following sections we discuss cell volume defence mechanisms as these might contribute to acute versus sustained intrinsic osmo-transduction by OVLT neurons. As recently reviewed by Bourque (2008), most of the evidence related to cell volume defence mechanisms in neuronal osmo-transduction derives from studies of hypothalamic MNCs. The extent to which these mechanisms contribute to OVLT neuronal osmo-transduction is largely a matter of speculation at the present time. Acute hyperosmotic challenges. Role of cell shrinkage. Most cells exposed to extracellular hyperosmolality rapidly shrink due to osmotic loss of water (Yancey et al. 1982; Burg et al. 2007). Therefore, a possible mechanism of rapid neuronal osmosensory transduction could rely on shrinkage itself to trigger increased excitability and action potential discharge (Law, 1994). This possibility has been explored in OVLT neurons (Ciura & Bourque, 2006) and vasopressin-releasing MNCs of the supraoptic nucleus (Sharif Naeini et al. 2006), both of which are intrinsically osmosensitive and lose volume (i.e. shrink) when exposed to acute increases in extracellular osmolality (Ciura & Bourque, 2006; Bourque, 2008). In MNCs, stretch-inactivated cation (SIC) channels are expressed that are partially inactivated by membrane stretch at normal (isotonic) cell volume (Oliet & Bourque, 1993). In voltage-clamp studies, SIC channels exhibit cell volume-dependent gating, consistent with their ability to encode rapid changes in osmolality-associated cell shrinkage (Sharif Naeini et al. 2006). Interestingly, gating of SIC channels is also modulated by the concentration of external Na +.Asaresult,SICchannelopenprobability (P o ) increases under fixed anisotonic conditions (above 275 mosmol kg 1 )asextracellularna + concentrations rise (Bourque, 2008). The latter feature could allow responses to be prolonged during chronic salt loading. The molecular identity of SIC channels in MNCs remains to be unequivocally demonstrated, though an N-terminal variant of transient receptor potential vanilloid type-1 (TRPV1) channels is expressed that has features closely resembling those of SIC channels (Sharif Naeini et al. 2006). In OVLT neurons, ionic conductances resembling those mediated by SIC/TRPV1 channels have been described (Ciura & Bourque, 2006). Interestingly, OVLT neurons isolated from TRPV1 / mice have been reported to be unresponsive to acute hypertonicity (Fig. 4), even though they undergo the same degree of shrinkage as neurons from wild-type mice (Ciura & Bourque, 2006). Interestingly, the lack of acute electrophysiological responses to hyperosmolality does not correspond to loss of acute osmoregulatory drinking. This is based on evidence that TRPV1 / mice drank nearly two-thirds (Ciura & Bourque, 2006) or equivalent (Taylor et al., 2008) amounts ofwateraswild-typemicewhengivenanacutenacl load. Thus, although SIC/TRPV1 channels are required for increasing discharge of intrinsically osmosensitive neurons, precisely what role these channels play in mediating in vivo osmoregulatory responses remains to be fully determined. Role of regulatory volume increase (RVI). Very soon after shrinking in response to hypertonicity, most CNS neurons studied in vitro promptly undergo RVI (Law, 1994), which Figure 3. Effects on arterial blood pressure (ABP), integrated renal sympathetic nerve activity (RSNA) and integrated lumbar sympathetic nerve activity (LSNA) of internal carotid artery (ICA) injections of graded concentrations of NaCl before and after acute chemical inhibition of the OVLT A, microinjection of vehicle into the OVLT had no effect on responses to ICA hypertonic NaCl (top, left vs. right). In contrast, responses were blunted following microinjection of the GABA A receptor agonist muscimol into the OVLT (bottom, left vs. right). (Reproduced with permission from Shi et al )

6 3380 G. M. Toney and S. D. Stocker J Physiol is characterized by an initial influx of inorganic ions (Na +,Cl,K + ) and osmotic draw of water back into the intracellular compartment, thus restoring cell volume toward normal (Arieff & Guisado, 1976). With time, ions are replaced by organic osmolytes such as amino acids, choline, creatine, inositol and especially taurine (Burg et al. 2007), allowing normal cell and brain volume to be maintained in spite of prevailing extracellular hyperosmolality (Arieff & Guisado, 1976). It is tempting to Figure 4. Current-clamp analysis of osmotically stimulated OVLT neurons A, representative examples of current-clamp recordings from osmotically stimulated (bar) OVLT neurons acutely isolated from wild-type (WT, left column) and TRPV1 / (right column) mice. Note that hypertonic stimuli consistently depolarize and excite WT but not TRPV1 / neurons. B, bar graphs show the mean ± S.E.M. membrane potential (V m ) observed under control and osmotically stimulated conditions in OVLT neurons from WT and TRPV1 / mice. Note that no significant depolarization was observed in neurons from TRPV1 / mice. C, bar graphs show the mean ± S.E.M. changes in firing rate observed under control and osmotically stimulated conditions in OVLT neurons from WT and TRPV1 / mice. Note the absence of osmotically induced excitation in neurons from TRPV1 / mice. P < (Reproduced with permission from Ciura & Bourque, 2006.) speculate that mechanisms leading to RVI might not only be used for defence of cell volume, but also for neuronal osmo-transduction. Does cation influx during the initial phase of RVI increase neuronal excitability and action potential discharge? The answer to this question is unknown, though SIC/TRPV1 channels in MNCs apparently do not participate in the initial phase of RVI. This is the case because significant SIC channel current is recorded in MNCs under hypertonic conditions even though these neurons do not undergo RVI and do not acutely defend their volume (Zhang & Bourque, 2003). It should be recognized, however, that the apparent lack of RVI among very large diameter MNCs might reflect mechanisms that restore volume too slowly to be readily detected during acute in vitro imaging studies. If shrinkage followed by RVI occurs among even a sub-set of intrinsically osmosensitive CNS neurons, the transient nature of the associated cation influx would probably encode only abrupt increases of osmolality. A recent study by Taylor et al. reported that acute salt loading, 24 h water deprivation and 48 h salt loading each evoked nearly identical drinking responses in wild-type and TRPV1 / mice (Taylor et al. 2008). In addition to similar drinking responses, expression of c-fos as a marker of neuronal activation was similar in the OVLT and other body fluid regulatory brain regions of wild-type and TRPV1 null mice. Collectively, these data suggest that if RVI is required for a full manifestation of acute or chronic osmosensory responses, then it is capable of occurring without involvement of TRPV1 channels. Does evidence of normal osmoregulatory drinking and circuit activation in TRPV1 null mice imply that cationic influx (through SIC/TRPV1) in response to shrinkage/rvi is of limited physiological relevance for long-term regulation of body fluid osmolality? We believe that such a conclusion is premature when one considers that functional channels may form from co-assembly of TRPV1, TRPV2 and TRPV4 channel subunits (Liedtke & Friedman, 2003; Bourque, 2008). Thus, elimination of TRPV1 alone may not be sufficient to prevent osmosensory transduction. In regard to involvement of TRPV4, knock-out mice have been shown to exhibit persistent deficits in both basal and hypertonicity-stimulated drinking (Liedtke & Friedman, 2003). Thus, TPRV4 may be able to substitute for TRPV1 in subserving neuronal osmo-transduction. Persistent osmotic deficits in TRPV4 / mice, however, suggest that theconversemaynotbetrue. Possible mechanisms of chronic neuronal osmotransduction. The following section is intended to emphasize that more information is needed before a comprehensive understanding of OVLT neuronal

7 J Physiol Hyperosmotic activation of SNA in cardiovascular disease 3381 osmo-transduction will emerge. We speculate on the possibility that a cell volume defence mechanism might be at work and we discuss two possibilities based on evidence from other osmosensitive cell systems. First we speculate on the possible neuromodulatory role of organic osmolytes, which are continuously transported in and out of various cell types (including neurons and astrocytes) during regulatory volume increases that often occur after hypertonic cell shrinkage. Next we take our lead from studies of vascular smooth muscle and renal cells, which document a significant efflux of protons (H + ) during hypertonic exposure by activating sodium proton (Na + H + ) exchange. We speculate that if such a process were to occur in OVLT, then it may be possible for neurons to increase discharge during hypertonic challenge because of activation of various acid-sensing ion channels. Role of organic osmolytes. As noted above, the initial phase of RVI in the CNS involves the influx of mostly inorganic cations (Arieff & Guisado, 1976; Law, 1994; Burg et al. 2007) followed, usually within hours, by accumulation of organic osmolytes (Arieff & Guisado, 1976). Whether newly synthesized or transported from the extracellular space, organic osmolytes replace the inorganic ions that entered during the initial phase of RVI. Whereas inorganic ions perturb neuronal function by disrupting normal ionic gradients and altering the structure/interaction of intracellular proteins, organic osmolytes are considered to be compatible with normal cellular function (Yancey et al. 1982). In neurons, perhaps the most important intracellular organic osmolyte that accumulates during long-term hyperosmolality is taurine (Law, 1994; Burg et al. 2007). In MNCs of the supraoptic nucleus, taurine has been reported to contribute to acute hypertonicity-evoked activity. The mechanism involves local astrocytes that extrude taurine under isotonic conditions by efflux through an osmolyte-sensitive anion channel (Deleuze et al. 1998; Bourque, 2008). During hypertonic challenge, taurine efflux is reduced. In this regard it could perhaps be said that astrocytes possess a form of intrinsic osmosensitivity. With the reduction of taurine efflux, its tonic agonist effect on glycine receptors is also reduced (Hussy et al. 1997). The resulting decrease in Cl permeability of MNCs leads to depolarization and increased discharge, thereby encoding the increase of extracellular osmolality (Bourque, 2008). The role played by taurine in long-term neuronal osmo-transduction has not yet been fully tested. Given that intracellular taurine levels most often rise during sustained exposure to hypertonicity, it may be that extracellular taurine concentration falls because of increased influx during the late phase of RVI. Alternatively or in combination, reduced efflux through astrocytic anion channels could account for a reduction of extracellular taurine during sustained hyperosmolality. In the specific case of OVLT neurons, no study to date has directly tested involvement of taurine or any other organic osmolyte in osmosensory transduction. Role of extracellular acidification. Exposure of vascular smooth muscle cells (Soleimani et al. 1994) and renal mesangial cells (Miyata et al. 2000) to hyperosmolality has been reported to increase activity/expression of Na + /H + exchange (NHE). This can result in a prompt and persistent acidification of extracellular fluid (Miyata et al. 2000). It is therefore tempting to speculate that such a mechanism operating in the OVLT could underlie osmosensory transduction. Of course, this would requirethatthedegreeofextracellularacidificationbe sufficient to increase neuronal activity. Although no study to date has tested this possibility, if this does occur then neuronal activation could potentially result from cation influx through a variety of ph-sensitive ion channels, including TRPV1 (Schumacher, 2010; Neelands et al. 2010) as well as acid-sensing ion channels (ASIC) (Neelands et al. 2010). As indicated above, TRPV1 channels are expressed in OVLT (Ciura & Bourque, 2006) and preliminary RT-QPCR studies performed in our laboratory indicate that mrna encoding NHE1 Figure 5. Effect of ICV infusion of benzamil HCl on the development of angiotensin II (Ang II) salt hypertension Male Sprague Dawley rats consuming a high salt (2% NaCl) diet received Ang II (150 ng kg 1 day 1 )via osmotic minipump starting on day four (4). Rats treated with an ICV infusion of vehicle (saline, n = 2) developed a large increase of mean arterial pressure (MAP), whereas rats that received an ICV infusion of benzamil (8 nmol day 1 ; n = 2) remained normotensive throughout the Ang II infusion period.

8 3382 G. M. Toney and S. D. Stocker J Physiol and all four ASIC channel members (1a, 2ab, 3 and 4) is expressed in the ventral lamina terminalis/ovlt (G. M. Toney, unpublished observations). As discussed above, if TRPV1 were the main channel activated by acidification, then data fromtrpv1 null mice suggest that acidification would not account for long-term activation of osmoregulatory responses. A thorough search of the literature indicates that NHE/ASIC activation has not yet been studied as a mechanism for either acute or chronic neuronal osmo-transduction. If such a mechanism were involved, treatment with amiloride would be predicted to restore normal extracellular ph and interrupt osmosensory transduction as both NHE (Soleimani et al. 1994; Miyata et al. 2000) and ASIC (Neelands et al. 2010) are blockable by amiloride analogues. Although no study to date has specifically tested this possibility, intracerebroventricular (ICV) administration of the amiloride analogue benzamil effectively interferes with deoxycorticosterone acetate (mineralocorticoid)-salt hypertension (Nishimura et al. 1998) and preliminary data indicate that ICV benzamil also interrupts the neurogenic component of angiotensin-dependent salt-sensitive hypertension (Hirsch et al. 2010) (Fig. 5). Interestingly, sustained hypertension in the angiotensin salt model appears to depend on activation of splanchnic SNA (Toney et al. 2010). Summary and conclusions A large and growing body of evidence indicates that exaggerated SNA contributes significantly to the pathogenesis of chronic salt-sensitive cardiovascular diseases. Studies in recent years now clearly demonstrate that acute and chronic increases in body fluid osmolality can profoundly modulate the activity of sympatheticregulatory circuits in the CNS. Whereas the osmosensory transduction process has been reported to largely depend on OVLT/lamina terminalis neurons, the cellular mechanisms remain to be fully explored. Although OVLT neurons are intrinsically osmosensitive and shrink when exposed to extracellular hypertonicity, it is not yet clear if osmosensitive discharge depends on cell shrinkage. Activation of TRPV1 channels is critical for acute hypertonic activation of OVLT neurons invitro,but TRPV1 null mice have essentially normal long-term osmoregulatory drinking responses and c-fos expression patterns following dehydration and salt loading. We speculate that long-term osmosensory transduction by OVLT neurons could involve modulation by local organic osmolytes such as taurine and possibly novel processes such as extracellular acidification. Whatever the mechanism(s) might be, we speculate that persistent osmosensory activation of SNA could potentially contribute to the aetiology of chronic cardiovascular diseases. References Adams JM, Bardgett ME & Stocker SD (2009). Ventral lamina terminalis mediates enhanced cardiovascular responses of rostral ventrolateral medulla neurons during increased dietary salt. Hypertension 54, Adams JM, McCarthy JJ & Stocker SD (2008). Excess dietary salt alters angiotensinergic regulation of neurons in the rostral ventrolateral medulla. Hypertension 52, Adams JM, Madden CJ, Sved AF & Stocker SD (2007). Increased dietary salt enhances sympathoexcitatory and sympathoinhibitory responses from the rostral ventrolateral medulla. Hypertension 50, Adams KF Jr (2004). Pathophysiologic role of the renin-angiotensin-aldosterone and sympathetic nervous systems in heart failure. Am J Health Syst Pharm 61 (Suppl. 2), S4 13. Anderson JW, Washburn DL & Ferguson AV (2000). Intrinsic osmosensitivity of subfornical organ neurons. Neuroscience 100, AntunesVR,YaoST,PickeringAE,MurphyD&PatonJF (2006). A spinal vasopressinergic mechanism mediates hyperosmolality-induced sympathoexcitation. JPhysiol576, Arieff AI & Guisado R (1976). Effects on the central nervous system of hypernatremic and hyponatremic states. Kidney Int 10, Bourque CW (2008). Central mechanisms of osmosensation and systemic osmoregulation. Nat Rev Neurosci 9, Brody MJ (1988). Central nervous system and mechanisms of hypertension. Clin Physiol Biochem 6, Brooks VL, Freeman KL & Clow KA (2004a). Excitatory amino acids in rostral ventrolateral medulla support blood pressure during water deprivation in rats. Am J Physiol Heart Circ Physiol 286, H1642 H1648. Brooks VL, Freeman KL & O DonaughyTL (2004b). Acute and chronic increases in osmolality increase excitatory amino acid drive of the rostral ventrolateral medulla in rats. Am J Physiol Regul Integr Comp Physiol 287, R1359 R1368. Brooks VL, Haywood JR & Johnson AK (2005a). Translation of salt retention to central activation of the sympathetic nervous system in hypertension. Clin Exp Pharmacol Physiol 32, Brooks VL, Qi Y & O Donaughy TL (2005b). Increased osmolality of conscious water-deprived rats supports arterial pressure and sympathetic activity via a brain action. Am J Physiol Regul Integr Comp Physiol 288, R1248 R1255. Burg MB, Ferraris JD & Dmitrieva NI (2007). Cellular response to hyperosmotic stresses. Physiol Rev 87, Chen QH & Toney GM (2001). AT 1 -receptor blockade in the hypothalamic PVN reduces central hyperosmolality-induced renal sympathoexcitation. Am J Physiol Regul Integr Comp Physiol 281, R1844 R1853. Ciura S & Bourque CW (2006). Transient receptor potential vanilloid 1 is required for intrinsic osmoreception in organum vasculosum lamina terminalis neurons and for normal thirst responses to systemic hyperosmolality. JNeurosci26,

9 J Physiol Hyperosmotic activation of SNA in cardiovascular disease 3383 Deleuze C, Duvoid A & Hussy N (1998). Properties and glial origin of osmotic-dependent release of taurine from the rat supraoptic nucleus. JPhysiol507, DiBona GF & Sawin LL (1991). Role of renal nerves in sodium retention of cirrhosis and congestive heart failure. Am J Physiol Regul Integr Comp Physiol 260, R298 R305. Freeman KL & Brooks VL (2007). AT 1 and glutamatergic receptors in paraventricular nucleus support blood pressure during water deprivation. Am J Physiol Regul Integr Comp Physiol 292, R1675 R1682. Guyenet PG (2006). The sympathetic control of blood pressure. Nat Rev Neurosci 7, HabeckerBA,GrygielkoET,HuhtalaTA,FooteB&BrooksVL (2003). Ganglionic tyrosine hydroxylase and norepinephrine transporter are decreased by increased sodium chloride in vivo and in vitro. Auton Neurosci 107, He FJ, Markandu ND, Sagnella GA, de Wardener HE & MacGregor GA (2005). Plasma sodium: ignored and underestimated. Hypertension 45, Hirsch DM, Guzman PA, Rodriguez M, Toney G & Osborn JW (2010). Effect of intracerebroventricular and subcutaneous benzamil administration on AngII-salt hypertension. FASEB J 22, Hussy N, Deleuze C, Pantaloni A, Desarmenien MG & Moos F (1997). Agonist action of taurine on glycine receptors in rat supraoptic magnocellular neurones: possible role in osmoregulation. JPhysiol502, Johnson AK, Cunningham JT & Thunhorst RL (1996). Integrative role of the lamina terminalis in the regulation of cardiovascular and body fluid homeostasis. Clin Exp Pharmacol Physiol 23, Law RO (1994). Regulation of mammalian brain cell volume. JExpZool268, Liedtke W & Friedman JM (2003). Abnormal osmotic regulation in trpv4 / mice. Proc Natl Acad Sci U S A 100, McKinley MJ, Bicknell RJ, Hards D, McAllen RM, Vivas L, Weisinger RS & Oldfield BJ (1992). Efferent neural pathways of the lamina terminalis subserving osmoregulation. Prog Brain Res 91, McKinley MJ, McAllen RM, Davern P, Giles ME, Penschow J, Sunn N, Uschakov A & Oldfield BJ (2003). The sensory circumventricular organs of the mammalian brain. Adv Anat EmbryolCellBiol172, III XII, 1 122, back cover. Miyata Y, Muto S, Yanagiba S & Asano Y (2000). Extracellular Cl modulates shrinkage-induced activation of Na + /H + exchanger in rat mesangial cells. Am J Physiol Cell Physiol 278, C1218 C1229. Neelands TR, Zhang XF, McDonald H & Puttfarcken P (2010). Differential effects of temperature on acid-activated currents mediated by TRPV1 and ASIC channels in rat dorsal root ganglion neurons. Brain Res 1329, Nishimura M, Ohtsuka K, Nanbu A, Takahashi H & Yoshimura M (1998). Benzamil blockade of brain Na + channels averts Na + -induced hypertension in rats. Am J Physiol Regul Integr Comp Physiol 274, R635 R644. Nissen R, Bourque CW & Renaud LP (1993). Membrane properties of organum vasculosum lamina terminalis neurons recorded in vitro. Am J Physiol Regul Integr Comp Physiol 264, R811 R815. O Donaughy TL, Qi Y & Brooks VL (2006). Central action of increased osmolality to support blood pressure in deoxycorticosterone acetate-salt rats. Hypertension 48, Oliet SH & Bourque CW (1993). Mechanosensitive channels transduce osmosensitivity in supraoptic neurons. Nature 364, Schmidlin O, Forman A, Sebastian A & Morris RC Jr (2007). Sodium-selective salt sensitivity: its occurrence in blacks. Hypertension 50, Schumacher MA(2010). Transient receptor potential channels in pain and inflammation: therapeutic opportunities. Pain Pract 10, Sharif Naeini R, Witty MF, Seguela P & Bourque CW (2006). An N-terminal variant of Trpv1 channel is required for osmosensory transduction. Nat Neurosci 9, Shi P, Martinez MA, Calderon AS, Chen Q, Cunningham JT & Toney GM (2008). Intra-carotid hyperosmotic stimulation increases Fos staining in forebrain organum vasculosum laminae terminalis neurones that project to the hypothalamic paraventricular nucleus. JPhysiol586, Shi P, Stocker SD & Toney GM (2007). Organum vasculosum laminae terminalis contributes to increased sympathetic nerve activity induced by central hyperosmolality. Am J Physiol Regul Integr Comp Physiol 293, R2279 R2289. Soleimani M, Bookstein C, McAteer JA, Hattabaugh YJ, Bizal GL, Musch MW, Villereal M, Rao MC, Howard RL & Chang EB (1994). Effect of high osmolality on Na + /H + exchange in renal proximal tubule cells. JBiolChem269, Stocker SD, Madden CJ & Sved AF (2010). Excess dietary salt intake alters the excitability of central sympathetic networks. Physiol Behav 100, Stocker SD, Osborn JL & Carmichael SP (2008). Forebrain osmotic regulation of the sympathetic nervous system. Clin Exp Pharmacol Physiol 35, Stocker SD, Simmons JR, Stornetta RL, Toney GM & Guyenet PG (2006). Water deprivation activates a glutamatergic projection from the hypothalamic paraventricular nucleus to the rostral ventrolateral medulla. JCompNeurol494, Taylor AC, McCarthy JJ & Stocker SD (2008). Mice lacking the transient receptor vanilloid potential 1 channel display normal thirst responses and central Fos activation to hypernatremia. Am J Physiol Regul Integr Comp Physiol 294, R1285 R1293. Toney GM, Chen QH, Cato MJ & Stocker SD (2003). Central osmotic regulation of sympathetic nerve activity. Acta Physiol Scand 177, Toney GM, Pedrino GR, Fink GD & Osborn JW (2010). Does enhanced respiratory-sympathetic coupling contribute to peripheral neural mechanisms of AngII-salt hypertension? Exp Physiol 95, Weinberger MH (1996). Salt sensitivity of blood pressure in humans. Hypertension 27, Weiss ML, Claassen DE, Hirai T & Kenney MJ (1996). Nonuniform sympathetic nerve responses to intravenous hypertonic saline infusion. JAutonNervSyst57,

10 3384 G. M. Toney and S. D. Stocker J Physiol Yancey PH, Clark ME, Hand SC, Bowlus RD & Somero GN (1982). Living with water stress: evolution of osmolyte systems. Science 217, Zhang Z & Bourque CW (2003). Osmometry in osmosensory neurons. Nat Neurosci 6, Author contributions G.M.T. and S.D.S. contributed equally to the conception and design of the article, to drafting the article and revising it for intellectual content. Both authors approved the final version to be published. Acknowledgements The authors gratefully acknowledge funding from the NIH (HL and HL076312, G.M.T.; HL090826, S.D.S.). We thank Dr John W. Osborn for preliminary data on the effect of ICV benzamil in angiotensin II-salt hypertension.