Shun-Guang Wei

Does Aldosterone Upregulate the Brain Renin-Angiotensin System in Rats With Heart Failure?

The brain renin-angiotensin system (RAS) contributes to increased sympathetic drive in heart failure (HF). The factors upregulating the brain RAS in HF remain unknown. We hypothesized that aldosterone (ALDO), a downstream product of the systemic RAS that crosses the blood-brain barrier, signals the brain to increase RAS activity in HF. We examined the relationship between circulating and brain ALDO in normal intact rats, in adrenalectomized rats receiving subcutaneous infusions of ALDO, and in rats with ischemia-induced HF and sham-operated controls. Brain ALDO levels were proportional to plasma ALDO levels across the spectrum of rats studied. Compared with sham-operated controls rats, HF rats had higher plasma and hypothalamic tissue levels of ALDO. HF rats also had higher expression of mRNA and protein for angiotensin-converting enzyme and angiotensin type 1 receptors in the hypothalamus, increased reduced nicotinamide-adenine dinucleotide phosphate oxidase activity and superoxide generation in the paraventricular nucleus of the hypothalamus, increased excitation of paraventricular nucleus neurons, and increased plasma norepinephrine. HF rats treated for 4 weeks with intracerebroventricular RU28318 (1 &mgr;g/h), a selective mineralocorticoid receptor antagonist, had less hypothalamic angiotensin-converting enzyme and angiotensin type 1 receptor mRNA and protein, less reduced nicotinamide-adenine dinucleotide phosphate–induced superoxide in the paraventricular nucleus, fewer excited paraventricular nucleus neurons, and lower plasma norepinephrine. RU28318 had no effect on plasma ALDO or on angiotensin-converting enzyme or angiotensin type 1 receptor expression in brain cortex. The data demonstrate that ALDO of adrenal origin enters the hypothalamus in direct proportion to plasma levels and suggest that ALDO contributes to the upregulation of hypothalamic RAS activity and sympathetic drive in heart failure.

Angiotensin II upregulates hypothalamic AT1 receptor expression in rats via the mitogen-activated protein kinase pathway.

ANG II type 1 receptors (AT(1)R) mediate most of the central effects of ANG II on cardiovascular function, fluid homeostasis, and sympathetic drive. The mechanisms regulating AT(1)R expression in the brain are unknown. In some tissues, the AT(1)R can be upregulated by prolonged exposure to ANG II. We examined the hypothesis that ANG II upregulates the AT(1)R in the brain by stimulating the intracellular mitogen-activated protein kinase (MAPK) signaling pathway. Using molecular and immunochemical approaches, we examined expression of the AT(1)R and phosphorylated MAPK in the paraventricular nucleus of the hypothalamus (PVN) and the subfornical organ (SFO) of rats receiving a chronic (4-wk) subcutaneous infusion of ANG II (0.6 microg/h) or saline (vehicle control), with or without concomitant (4-wk) intracerebroventricular (ICV) infusions of MAPK inhibitors or the AT(1)R blocker losartan. Subcutaneous infusion of ANG II markedly increased phosphorylation of MAPK and expression of AT(1)R mRNA and protein and AT(1)R-like immunoreactivity in the PVN and SFO. ANG II-induced AT(1)R expression was blocked by ICV infusion of the p44/42 MAPK inhibitor PD-98059 (0.025 microg/h) and the JNK inhibitor SP-600125 (0.125 microg/h), but not by the p38 MAPK inhibitor SB-203580 (0.125 microg/h). Upregulation of the AT(1)R in the PVN and SFO by peripheral ANG II was abolished by ICV losartan (10 microg/h). The data indicate that blood-borne ANG II upregulates brain AT(1)R by activating intracellular p44/42 MAPK and JNK signaling pathways.

11β-Hydroxysteroid Dehydrogenase Type 2 Activity in Hypothalamic Paraventricular Nucleus Modulates Sympathetic Excitation

Aldosterone stimulates the sympathetic nervous system by binding to a select population of brain mineralocorticoid receptors (MR). These MR have an equal affinity for corticosterone that is present in substantially higher concentrations, but are held in reserve for aldosterone by activity of the enzyme 11&bgr;-hydroxysteroid dehydrogenase type 2 (11&bgr;-HSD-2), which converts corticosterone to an inactive metabolite. Thus, colocalization of MR and 11&bgr;-HSD-2 activity may help identify brain regions that mediate the effects of aldosterone. The present studies tested the hypothesis that 11&bgr;-HSD-2 activity regulates MR-mediated responses in the paraventricular nucleus (PVN) of the hypothalamus, a forebrain region implicated in sympathetic regulation. Real-time–polymerase chain reaction revealed the presence of 11&bgr;-HSD-2 mRNA in PVN. In anesthetized adult male Sprague-Dawley rats, microinjection of the 11&bgr;-HSD-2 inhibitor carbenoxolone (CBX) into PVN increased mean arterial pressure, heart rate, and renal sympathetic nerve activity. Intracerebroventricular injections of CBX excited PVN neurons and increased mean arterial pressure, heart rate, and renal sympathetic nerve activity. The ability of CBX to increase sympathetic activity by inhibiting 11&bgr;-HSD-2, thereby permitting corticosterone to activate MR, was confirmed by the following: Intracerebroventricular glycyrrhizic acid, another 11&bgr;-HSD-2 inhibitor, mimicked the sympathoexcitatory effects of CBX; the sympathoexcitatory effects of CBX were blocked by spironolactone, a MR antagonist. Neither CBX nor glycyrrhizic acid elicited a response in adrenalectomized rats. These findings suggest that MR in PVN contribute to sympathetic regulation and may be activated by aldosterone or corticosterone (or cortisol in humans) depending on the state of 11&bgr;-HSD-2 activity.

Brain Perivascular Macrophages and the Sympathetic Response to Inflammation in Rats After Myocardial Infarction

Inflammation is associated with increased sympathetic drive in cardiovascular diseases. Blood-borne proinflammatory cytokines, markers of inflammation, induce cyclooxygenase 2 (COX-2) activity in perivascular macrophages of the blood-brain barrier. COX-2 generates prostaglandin E2, which may enter the brain and increase sympathetic nerve activity. We examined the contribution of this mechanism to augmented sympathetic drive in rats after myocardial infarction (MI). Approximately 24 hours after acute MI, rats received an intracerebroventricular injection (1 &mgr;L/min over 40 minutes) of clodronate liposomes (MI+CLOD) to eliminate brain perivascular macrophages, liposomes alone, or artificial cerebrospinal fluid. A week later, COX-2 immunoreactivity in perivascular macrophages and COX-2 mRNA and protein had increased in hypothalamic paraventricular nucleus of MI rats treated with artificial cerebrospinal fluid or liposomes alone compared with sham-operated rats. In MI+CLOD rats, neither perivascular macrophages nor COX-2 immunoreactivity was seen in the paraventricular nucleus, and COX-2 mRNA and protein levels were similar to those in sham-operated rats. Prostaglandin E2 in cerebrospinal fluid, paraventricular nucleus neuronal excitation, and plasma norepinephrine were less in MI+CLOD rats than in MI rats treated with artificial cerebrospinal fluid or liposomes alone but more than in sham-operated rats. Intracerebroventricular CLOD had no effect on interleukin 1β and tumor necrosis factor-α mRNA and protein in the paraventricular nucleus or plasma interleukin-1β and tumor necrosis factor-α, which were increased in MI compared with sham-operated rats. In normal rats, pretreatment with intracerebroventricular CLOD reduced (P<0.05) the renal sympathetic, blood pressure, and heart rate responses to intracarotid artery injection of tumor necrosis factor-α (0.5 &mgr;g/kg); intracerebroventricular liposomes had no effect. The results suggest that proinflammatory cytokines stimulate sympathetic excitation after MI by inducing COX-2 activity and prostaglandin E2 production in perivascular macrophages of the blood-brain barrier.

Central Gene Transfer of Interleukin-10 Reduces Hypothalamic Inflammation and Evidence of Heart Failure in Rats After Myocardial Infarction

The expression of proinflammatory cytokines increases in hypothalamus of rats with myocardial infarction (MI) and heart failure. We used central gene transfer of human interleukin (IL)-10, a potent antiinflammatory cytokine, to counter the effects of brain proinflammatory cytokines and examine their functional significance. Sprague–Dawley rats underwent coronary ligation to induce MI or sham surgery (SHAM). One week later, adenoviral vectors encoding human IL-10 (AdIL-10) or &bgr;-galactosidase (&bgr;Gal) were injected (30 &mgr;L over 30 minutes) into lateral ventricle. One week after injection, there was abundant expression of human IL-10 in the brain of MI+AdIL-10 and SHAM+AdIL-10 rats. Compared with SHAM+&bgr;Gal, MI+&bgr;Gal had increased (P<0.05) IL-1&bgr; and cyclooxygenase-2 mRNA and protein and nuclear factor &kgr;B activity in the hypothalamus, cyclooxygenase-2 fluorescence in perivascular cells of the paraventricular nucleus of hypothalamus, prostaglandin E2 in cerebrospinal fluid, and Fra-like activity (indicating neuronal excitation) in paraventricular nucleus. Plasma norepinephrine levels, lung/body weight, right ventricle/body weight, and left ventricular end-diastolic pressure were increased and maximal left ventricular dP/dt was decreased. All of these findings were ameliorated in MI rats treated with AdIL-10. Hypothalamic tumor necrosis factor-&agr; and circulating tumor necrosis factor-&agr; and IL-1&bgr; levels, also increased in MI+&bgr;Gal, were not affected by AdIL-10 treatment. Rat native IL-10 was not affected by MI or AdIL-10. AdIL-10 had no effects on SHAM rats. The results demonstrate that cardiovascular and autonomic mechanisms leading to heart failure after MI can be modulated by manipulating the balance between proinflammatory and antiinflammatory cytokines in the brain.

Angiotensin II–Triggered p44/42 Mitogen-Activated Protein Kinase Mediates Sympathetic Excitation in Heart Failure Rats

Angiotensin II (Ang II), acting via angiotensin type 1 receptors in the brain, activates the sympathetic nervous system in heart failure (HF). We reported recently that Ang II stimulates mitogen-activated protein kinase (MAPK) to upregulate brain angiotensin type 1 receptors in HF rats. In this study we tested the hypothesis that Ang II-activated MAPK signaling pathways contribute to sympathetic excitation in HF. Intracerebroventricular administration of PD98059 and UO126, 2 selective p44/42 MAPK inhibitors, induced significant decreases in mean arterial pressure, heart rate, and renal sympathetic nerve activity in HF rats, but had no effect on these variables in sham-operated rats. Pretreatment with losartan attenuated the effects of PD98059. Intracerebroventricular administration of the p38 MAPK inhibitor SB203580 and the c-Jun N-terminal kinase inhibitor SP600125 had no effect on mean arterial pressure, heart rate, or renal sympathetic nerve activity in HF. The phosphatidylinositol 3-kinase inhibitor LY294002 induced a small decrease in mean arterial pressure and heart rate but no change in renal sympathetic nerve activity. Immunofluorescent staining demonstrated increased p44/42 MAPK activity in neurons of the paraventricular nucleus of the hypothalamus of HF rats, colocalized with Fra-like activity (indicating chronic neuronal excitation). Intracerebroventricular PD98059 and UO126 reduced Fra-like activity in the paraventricular nucleus of the hypothalamus neurons in HF rats. In confirmatory acute studies, intracerebroventricular Ang II increased mean arterial pressure, heart rate, and renal sympathetic nerve activity in baroreceptor-denervated rats and Fra-like immunoreactivity in the paraventricular nucleus of the hypothalamus of neurally intact rats. Central administration of PD98059 markedly reduced these responses. These data demonstrate that intracellular p44/42 MAPK activity contributes to Ang II-induced neuronal excitation in the paraventricular nucleus of the hypothalamus and augmented sympathetic nerve activity in rats with HF.

Mitogen-Activated Protein Kinases Mediate Upregulation of Hypothalamic Angiotensin II Type 1 Receptors in Heart Failure Rats

In heart failure (HF), angiotensin II type 1 receptor (AT1-R) expression is upregulated in brain regions regulating sympathetic drive, blood pressure, and body fluid homeostasis. However, the mechanism by which brain AT1-R are upregulated in HF remains unknown. The present study examined the hypothesis that the angiotensin II (Ang II)–triggered mitogen-activated protein kinases (MAPKs) p44/42, p38, and c-Jun N-terminal kinase contribute to upregulation of the AT1-R in the hypothalamus of rats with HF. AT1-R protein, AT1-R mRNA, and AT1-R immunoreactivity increased in the paraventricular nucleus of hypothalamus and the subfornical organ of rats with ischemia-induced HF compared with sham-operated controls. Phosphorylated p44/42 MAPK, c-Jun N-terminal kinase, and p38 MAPK also increased in paraventricular nucleus and subfornical organ. A 4-week ICV infusion of the AT1-R antagonist losartan decreased AT1-R protein and phosphorylation of p44/42 MAPK, c-Jun N-terminal kinase, and p38 MAPK in the HF rats. A 4-week ICV infusion of the p44/42 MAPK inhibitor PD98059 or the c-Jun N-terminal kinase inhibitor SP600125 significantly decreased AT1-R protein and AT1-R immunoreactivity in the paraventricular nucleus and subfornical organ, but the p38 MAPK inhibitor SB203580 did not. Treatment with ICV losartan, PD98059, and SP600125 had no effect on AT1-R expression by Western blot in sham-operated rats. In untreated HF rats 4 weeks after coronary ligation, a 3-hour ICV infusion of PD98059, SP600125, or losartan reduced AT1-R mRNA in paraventricular nucleus and subfornical organ. These data indicate that MAPK plays an important role in the upregulation of AT1-R in the rat forebrain in HF and suggest that Ang II upregulates its own receptor by this mechanism.

Centrally administered lipopolysaccharide elicits sympathetic excitation via NAD(P)H oxidase-dependent mitogen-activated protein kinase signaling

Objective The mechanisms by which inflammation activates sympathetic drive in heart failure and hypertension remain ill-defined. In this study, an intracerebroventricular injection of lipopolysaccharide (LPS) was used to induce the expression of cytokines and other inflammatory mediators in the brain, in the absence of other excitatory mediators, and the downstream signaling pathways leading to sympathetic activation were examined using intracerebroventricular injections of blocking or inhibiting agents. Methods and results In anesthetized rats, intracerebroventricular injection of LPS (5 μg) increased (P < 0.05) renal sympathetic nerve activity, blood pressure and heart rate. LPS increased (P < 0.05) hypothalamic mRNA for NAD(P)H oxidase subunits p47phox and gp91phox, NAD(P)H oxidase-dependent superoxide generation, hypothalamic mRNA for tumor necrosis factor-α, cyclooxygenase-2 and cerebrospinal fluid levels of tumor necrosis factor-α and prostaglandin E2. In the paraventricular nucleus of hypothalamus, dihydroethidium staining for superoxide expression and c-Fos activity (indicating neuronal excitation) increased. The superoxide scavenger tempol significantly (P < 0.05) diminished the expression of inflammatory mediators, as well as superoxide expression and neuronal excitation in paraventricular nucleus. SB203580 (p38 mitogen-activated protein kinase inhibitor) also reduced the expression of inflammatory mediators in hypothalamus and cerebrospinal fluid. Tempol, apocynin [NAD(P)H oxidase inhibitor], SB203580 and NS398 (cyclooxygenase-2 inhibitor) all reduced cerebrospinal fluid prostaglandin E2 and the sympathoexcitatory response to LPS. LPS also increased angiotensin II type 1 receptor mRNA, a response blocked by apocynin and tempol but not by SB203580. Conclusion These findings suggest that central inflammation in pathophysiological conditions activates the sympathetic nervous system via NAD(P)H oxidase and p38 mitogen-activated protein kinase-dependent synthesis of prostaglandin E2.

Proinflammatory Cytokines Upregulate Sympathoexcitatory Mechanisms in the Subfornical Organ of the Rat

Our previous work indicated that the subfornical organ (SFO) is an important brain sensor of blood-borne proinflammatory cytokines, mediating their central effects on autonomic and cardiovascular function. However, the mechanisms by which SFO mediates the central effects of circulating proinflammatory cytokines remain unclear. We hypothesized that proinflammatory cytokines act within the SFO to upregulate the expression of excitatory and inflammatory mediators that drive sympathetic nerve activity. In urethane-anesthetized Sprague–Dawley rats, direct microinjection of tumor necrosis factor (TNF)-&agr; (25 ng) or interleukin (IL)-1&bgr; (25 ng) into SFO increased mean blood pressure, heart rate, and renal sympathetic nerve activity within 15 to 20 minutes, mimicking the response to systemically administered proinflammatory cytokines. Pretreatment of SFO with microinjections of the angiotensin II type-1 receptor blocker losartan (1 &mgr;g), angiotensin-converting enzyme inhibitor captopril (1 &mgr;g) or cyclooxygenase-2 inhibitor NS-398 (2 &mgr;g) attenuated those responses. Four hours after the SFO microinjection of TNF-&agr; (25 ng) or IL-1&bgr; (25 ng), mRNA for angiotensin-converting enzyme, angiotensin II type-1 receptor, TNF-&agr; and the p55 TNF-&agr; receptor, IL-1&bgr; and the IL-1R receptor, and cyclooxygenase-2 had increased in SFO, and mRNA for angiotensin-converting enzyme, angiotensin II type-1 receptor, and cyclooxygenase-2 had increased downstream in the hypothalamic paraventricular nucleus. Confocal immunofluorescent images revealed that immunoreactivity for the p55 TNF-&agr; receptor and the IL-1 receptor accessory protein, a subunit of the IL-1 receptor, colocalized with angiotensin-converting enzyme, angiotensin II type-1 receptor–like, cyclooxygenase-2, and prostaglandin E2 EP3 receptor immunoreactivity in SFO neurons. These data suggest that proinflammatory cytokines act within the SFO to upregulate the expression of inflammatory and excitatory mediators that drive sympathetic excitation.

Subfornical Organ Mediates Sympathetic and Hemodynamic Responses to Blood-Borne Proinflammatory Cytokines

Proinflammatory cytokines play an important role in regulating autonomic and cardiovascular function in hypertension and heart failure. Peripherally administered proinflammatory cytokines, such as tumor necrosis factor-&agr; (TNF-&agr;) and interleukin-1&bgr; (IL-1&bgr;), act on the brain to increase blood pressure, heart rate, and sympathetic nerve activity. These molecules are too large to penetrate the blood–brain barrier, and so the mechanisms by which they elicit these responses remain unknown. We tested the hypothesis that the subfornical organ (SFO), a forebrain circumventricular organ that lacks a blood–brain barrier, plays a major role in mediating the sympathetic and hemodynamic responses to circulating proinflammatory cytokines. Intracarotid artery injection of TNF-&agr; (200 ng) or IL-1&bgr; (200 ng) dramatically increased mean blood pressure, heart rate, and renal sympathetic nerve activity in rats with sham lesions of the SFO (SFO-s). These excitatory responses to intracarotid artery TNF-&agr; and IL-1&bgr; were significantly attenuated in SFO-lesioned (SFO-x) rats. Similarly, the increases in mean blood pressure, heart rate, and renal sympathetic nerve activity in response to intravenous injections of TNF-&agr; (500 ng) or IL-1&bgr; (500 ng) in SFO-s rats were significantly reduced in the SFO-x rats. Immunofluorescent staining revealed a dense distribution of the p55 TNF-&agr; receptor and the IL-1 receptor accessory protein, a subunit of the IL-1 receptor, in the SFO. These data suggest that SFO is a predominant site in the brain at which circulating proinflammatory cytokines act to elicit cardiovascular and sympathetic responses.