Cardiac and Brainstem Neuroinflammatory Pathways Account for Androgenic Incitement of Cardiovascular and Autonomic Manifestations in Endotoxic Male Rats.

ABSTRACT: Inconsistent reports are available on the role of testosterone in end-organ damage caused by endotoxemia. Here, pharmacologic, surgical, and molecular studies were employed to assess the testosterone modulation of cardiovascular, autonomic, and peripheral and central inflammatory derangements caused by endotoxemia. Studies were performed in conscious male rats preinstrumented with femoral indwelling catheters for the measurement of blood pressure and subjected to castration or pharmacologic interventions that interrupt the biosynthetic cascade of testosterone. Compared with the effects of lipopolysaccharide (10 mg/kg intravenously) in sham operated rats, 2-week castration reduced the lipopolysaccharide-evoked (1) falls in blood pressure, (2) decreases in time- and frequency-domain indices of heart rate variability, (3) shifts in spectral measures of cardiac sympathovagal balance toward parasympathetic dominance, and (4) increases in protein expressions of toll-like receptor-4 and monocyte chemoattractant protein-1 in heart and medullary neurons of the nucleus tractus solitarius and rostral ventrolateral medulla. While the ameliorating actions of castration on endotoxic cardiovascular manifestations were maintained after testosterone replacement, the concomitant inflammatory signals were restored to near-sham levels. The favorable influences of castration on inflammatory and cardiovascular abnormalities of endotoxemia were replicated in intact rats pretreated with degarelix (gonadotropin-releasing hormone receptor blocker) or finasteride (5α-reductase inhibitor) but not formestane (aromatase inhibitor). The data signifies the importance of androgens and its biosynthetic enzymes in cardiovascular and autonomic insults induced by the endotoxic inflammatory response. Clinically, the interruption of testosterone biosynthesis could offer a potential strategy for endotoxemia management.

Modulation by Central MAPKs/PI3K/sGc of the TNF-α/iNOS-dependent Hypotension and Compromised Cardiac Autonomic Control in Endotoxic Rats.

Reduced blood pressure (BP) and cardiac autonomic activity are early manifestations of endotoxemia. We investigated whether these effects are modulated by central mitogen-activated protein kinases (MAPKs) and related phosphoinositide-3-kinase (PI3K)/soluble guanylate cyclase (sGC) signaling in conscious rats. The effect of pharmacologic inhibition of these molecular substrates on BP, heart rate (HR), and heart rate variability (HRV) responses evoked by intravascular lipopolysaccharide (LPS) (10 mg/kg) were assessed. LPS (1) lowered BP (2) increased HR, (3) reduced time [SD of beat-to-beat intervals (SDNN), and root mean square of successive differences in R-R intervals (rMSSD)], and frequency domain indices of HRV (total power and spectral bands of low and high-frequency), and (4) elevated serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels. The inhibition of TNF-α (pentoxifylline) or inducible nitric oxide synthase (iNOS, aminoguanidine) abolished hemodynamic, HRV, and inflammatory actions of LPS. Intracisternal (i.c.) injection of ODQ (sGC inhibitor), wortmannin (PI3K inhibitor), and SP600125 (MAPKJNK inhibitor) mitigated the hypotensive and tachycardic actions of LPS but failed to affect associated decreases in HRV. MAPKp38 inhibition by i.c. SB203580 produced exactly opposite effects. None of the LPS effects was altered after i.c. PD98059 (MAPKERK1/2 inhibitor). Overall, central MAPKs/PI3K/sGC pathways variably contribute to the TNF-α/iNOS-dependent reductions in BP and HRV seen during endotoxic shock.

Preeclamptic programming unevenly perturbs inflammatory and renal vasodilatory outcomes of endotoxemia in rat offspring: modulation by losartan and pioglitazone.

Introduction: Preeclampsia (PE) enhances the vulnerability of adult offspring to serious illnesses. The current study investigated whether preeclamptic fetal programming impacts hemodynamic and renal vasodilatory disturbances in endotoxic adult offspring and whether these interactions are influenced by antenatal therapy with pioglitazone and/or losartan. Methods: PE was induced by oral administration of L-NAME (50 mg/kg/day) for the last 7 days of pregnancy. Adult offspring was treated with lipopolysaccharides (LPS, 5 mg/kg) followed 4-h later by hemodynamic and renovascular studies. Results: Tail-cuff measurements showed that LPS decreased systolic blood pressure (SBP) in male, but not female, offspring of PE dams. Moreover, PE or LPS reduced vasodilations elicited by acetylcholine (ACh, 0.01-7.29 nmol) or N-ethylcarboxamidoadenosine (NECA, 1.6-100 nmol) in perfused kidneys of male rats only. The latter effects disappeared in LPS/PE preparations, suggesting a postconditioning action for LPS against renal manifestation of PE. Likewise, elevations caused by LPS in serum creatinine and inflammatory cytokines (TNFα and IL-1ß) as well as in renal protein expression of monocyte chemoattractant protein-1 (MCP-1) and AT1 receptors were attenuated by the dual PE/LPS challenge. Gestational pioglitazone or losartan reversed the attenuated ACh/NECA vasodilations in male rats but failed to modify LPS hypotension or inflammation. The combined gestational pioglitazone/losartan therapy improved ACh/NECA vasodilations and eliminated the rises in serum IL-1ß and renal MCP-1 and AT1 receptor expressions. Conclusion: Preeclamptic fetal programming of endotoxic hemodynamic and renal manifestations in adult offspring depends on animal sex and specific biological activity and are reprogrammed by antenatal pioglitazone/losartan therapy.

The renin-angiotensin system modulates endotoxic postconditioning of exacerbated renal vasoconstriction in preeclamptic offspring.

We recently reported exacerbated endotoxic signs of neuroinflammation and autonomic defects in offspring of preeclamptic (PE) dams. Here, we investigated whether PE programming similarly modifies hemodynamic and renal vasoconstrictor responsiveness to endotoxemia in PE offspring and whether this interaction is modulated by gestational angiotensin 1-7 (Ang1-7). Preeclampsia was induced by gestational treatment with L-NAME. Adult offspring was challenged with lipopolysaccharides (LPS, 5 mg/kg) and systolic blood pressure (SBP) and renal vasoconstrictions were assessed 4 h later. Male, but not female, offspring of PE rats exhibited SBP elevations that were blunted by LPS. Renal vasoconstrictions induced by angiotensin II (Ang II), but not phenylephrine, were intensified in perfused kidneys of either sex. LPS blunted the heightened Ang II responses in male, but not female, kidneys. While renal expressions of AT1-receptors and angiotensin converting enzyme (ACE) were increased in PE offspring of both sexes, ACE2 was upregulated in female offspring only. These molecular effects were diminished by LPS in male offspring. Gestational Ang1-7 caused sex-unrelated attenuation of phenylephrine vasoconstrictions and preferentially downregulated Ang II responses and AT1-receptor and nuclear factor-kB (NFkB) expressions in females. Together, endotoxemia and Ang1-7 offset in sexually-related manners imbalances in renal vasoconstriction and AT1/ACE/ACE2 signaling in PE offspring.

Central α7 and α4ß2 nicotinic acetylcholine receptors offset arterial baroreceptor dysfunction in endotoxic rats.

Cardiac autonomic neuropathy is a prominent feature of endotoxemia. Given the defensive role of the cholinergic pathway in inflammation, we assessed the roles of central homomeric α7 and heteromeric α4ß2 nAChRs in arterial baroreceptor dysfunction caused by endotoxemia in rats. Endotoxemia was induced by i.v. administration of lipopolysaccharides (LPS, 10 mg/kg), and baroreflex activity was measured by the vasoactive method, which assesses reflex chronotropic responses to increments (phenylephrine, PE) or decrements (sodium nitroprusside, SNP) in blood pressure. Shifts caused by LPS in PE/SNP baroreflex curves and associated decreases in baroreflex sensitivity (BRS) were dose-dependently reversed by nicotine (25-100 µg/kg, i.v.). The nicotine effect disappeared after intracisternal administration of methyllycaconitine (MLA) or dihydro-ß-erythroidine (DHßE), selective blockers of α7 and α4ß2 receptors, respectively. The advantageous effect of nicotine on BRSPE was replicated in rats treated with PHA-543613 (α7-nAChR agonist) or 5-iodo-A-85380 (5IA, α4ß2-nAChRs agonist) in dose-dependent fashions. Conversely, the depressed BRSSNP of endotoxic rats was improved after combined, but not individual, treatments with PHA and 5IA. Central α7 and α4ß2 nAChR activation underlies the nicotine counteraction of arterial baroreflex dysfunction induced by endotoxemia. Moreover, the contribution of these receptors depends on the nature of the reflex chronotropic response (bradycardia vs. tachycardia).

Androgenic modulation of arterial baroreceptor dysfunction and neuroinflammation in endotoxic male rats.

Autonomic neuropathy contributes to cardiovascular derangements induced by endotoxemia. In this communication, we tested the hypothesis that androgenic hormones improve arterial baroreflex dysfunction and predisposing neuroinflammatory response caused by endotoxemia in male rats. Baroreflex curves relating changes in heart rate to increases or decreases in blood pressure evoked by phenylephrine (PE) and sodium nitroprusside (SNP), respectively, were constructed in conscious sham-operated, castrated, and testosterone-replaced castrated rats treated with or without lipopolysaccharide (LPS, 10 mg/kg i.v.). Slopes of baroreflex curves were taken as measures of baroreflex sensitivity (BRS). In sham rats, LPS significantly reduced reflex bradycardia (BRSPE) and tachycardia (BRSSNP) and increased immunohistochemical expression of nuclear factor kappa B (NFκB) in heart and brainstem neurons of nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM). The baroreflex depressant effect of LPS was maintained in castrated rats despite the remarkably attenuated inflammatory response. Testosterone replacement of castrated rats counteracted LPS-evoked BRSPE, but not BRSSNP, depression and increased cardiac, but not neuronal, NFκB expression. We also evaluated whether LPS responses could be affected following pharmacologic inhibition of androgenic biosynthetic pathways. Whereas none of LPS effects were altered in rats pretreated with formestane (aromatase inhibitor) or finasteride (5α-reductase inhibitor), the LPS-evoked BRSPE, but not BRSSNP, depression and cardiac and neuronal inflammation disappeared in rats pretreated with degarelix (gonadotropin-releasing hormone receptor blocker). Overall, despite the seemingly provocative role for the hypothalamic-pituitary-gonadal axis in the neuroinflammatory and baroreflex depressant effects of LPS, testosterone appears to distinctly modulate the two LPS effects.

Nicotine uncovers endotoxic-like cardiovascular manifestations in female rats: Estrogen and nitric oxide dependency.

Endotoxic manifestations are diminished in female populations due to immune boosting actions of sex steroids. Considering that tobacco constituents including nicotine inhibit estrogen synthesis, we tested the hypothesis that nicotine exposure unveils cardiovascular anomalies of endotoxemia in female rats. Studies were undertaken in conscious female rats treated with i.v. lipopolysaccharide (LPS, 10 mg/kg) in absence and presence of nicotine. In contrast to no effects for LPS when used alone, dose-related decreases in blood pressure (BP) and serum estrogen were noted in endotoxic rats treated consequently with nicotine (25, 50, or 100 µg/kg i.v.). Signs of cardiac autonomic dysfunction appeared in LPS/nicotine-treated rats such as (i) decreased time-domain indices of heart rate variability (HRV), e.g. standard deviation of R-R intervals (SDNN) and root mean square of successive differences in R-R interval durations (rMSSD), and (ii) reduced total power of the frequency spectrum and shifted cardiac sympathovagal balance toward sympathetic dominance. Nicotine reversed the LPS-evoked modest rises in serum TNFα and IL-1ß while had no effect on associated arterial baroreflex dysfunction, inferring no roles for inflammation or baroreflexes in LPS-nicotine interaction. Estrogen or aminoguanidine (iNOS inhibitor), but not pentoxifylline (TNFα inhibitor), abolished LPS/nicotine hypotension. Together, nicotine acts probably via reducing estrogen availability to uncover nitric oxide-dependent hypotension and autonomic dysregulation in endotoxic female rats.

Brainstem cholinergic pathways diminish cardiovascular and neuroinflammatory actions of endotoxemia in rats: Role of NFκB/α7/α4ß2AChRs signaling.

Nicotine improves endotoxic manifestations of hypotension and cardiac autonomic dysfunction in rats. Here, we test the hypothesis that brainstem antiinflammatory pathways of α7/α4ß2 nicotinic acetylcholine receptors (nAChRs) modulate endotoxic cardiovascular derangements. Pharmacologic and molecular studies were performed to determine the influence of nicotine or selective α7/α4ß2-nAChR ligands on cardiovascular derangements and brainstem neuroinflammation caused by endotoxemia in conscious rats. The i.v. administration of nicotine (50 µg/kg) abolished the lipopolysaccharide (LPS, 10 mg/kg i.v.)-evoked: (i) falls in blood pressure and spectral measure of cardiac sympathovagal balance (ratio of the low-frequency to high-frequency power, LF/HF), (ii) elevations in immunohistochemical protein expressions of NFκB and α4ß2-nAChR in medullary neurons of the nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM), and (iii) decreases in medullary α7-nAChR protein expression. These favorable nicotine influences were replicated in rats treated intracisternally (i.c.) with PHA-543613 (selective α7-nAChR agonist) or 5-iodo-A-85380 (5IA, selective α4ß2-nAChRs agonist). Measurement of arterial baroreflex activity by the vasoactive method revealed that nicotine, PHA, or 5IA reversed the LPS depression of reflex bradycardic, but not tachycardic, activity. Moreover, the counteraction by nicotine of LPS hypotension was mostly inhibited after treatment with i.c. methyllycaconitine (MLA, α7-nAChR antagonist) in contrast to a smaller effect for dihydro-ß-erythroidine (DHßE, α4ß2-nAChR antagonist), whereas the associated increases in LF/HF ratio remained unaltered. The data signifies the importance of brainstem α7, and to a lesser extent α4ß2, receptors in the nicotine counteraction of detrimental cardiovascular and neuroinflammatory consequences of endotoxemia.

Additive counteraction by α7 and α4ß2-nAChRs of the hypotension and cardiac sympathovagal imbalance evoked by endotoxemia in male rats.

The cholinergic antiinflammatory pathway favorably influences end organ damage induced by inflammatory conditions. Here, we hypothesized that α7 and/or α4ß2-nicotinic acetylcholine receptors (nAChRs) protect against cardiovascular and autonomic imbalances induced by endotoxemia in rats. We assessed dose-effect relationships of i.v. nicotine (25, 50, or 100 µg/kg), PHA-543613 (α7-nAChR agonist; 0.2 or 2.0 mg/kg), or 5-iodo-A-85380 (5IA, α4ß2-nAChRs agonist; 0.01 or 0.1 mg/kg) on cardiovascular and inflammatory responses elicited by lipopolysaccharide (LPS, 10 mg/kg i.v.). The two lower doses of nicotine caused dose-dependent attenuation of hypotensive and tachycardic responses of LPS. Nicotine also reversed LPS-evoked reductions in time-domain indices of heart rate variability (HRV) and spectral measure of cardiac sympathovagal balance. Alternatively, hypotensive and tachycardic effects of LPS were (i) partly and dose-dependently reversed after selective activation of α7 (PHA) or α4ß2-nAChRs (5IA), and (ii) completely eliminated after co-treatment with the smaller doses of the two agonists. Further, PHA or 5IA abolished the reducing effect of LPS on time and spectral measures of HRV. Elevations in serum tumor necrosis factor-α (TNF-α) observed in LPS-treated rats were compromised upon co-administration of nicotine, PHA, or 5IA. In conclusion, monomeric α7 or heteromeric α4ß2-nAChRs favorably and additively influence inflammatory and associated cardiovascular anomalies induced by endotoxemia.

Activation of central GABAB receptors offsets the cyclosporine counteraction of endotoxic cardiovascular outcomes in conscious rats.

We have previously shown that cyclosporine (CSA) counteracts cardiovascular manifestations induced by endotoxemia (lipopolysaccharide, LPS) such as hypotension and cardiac autonomic dysfunction in conscious rats. In this study, we investigated whether the facilitation of central γ-amino butyric acid (GABA) neurotransmission blunts these favorable influences of CSA. The LPS-CSA interaction was determined in the absence and presence of drugs that activate GABAA or GABAB receptors or elevate synaptic GABA levels in the central nervous system. The consequent i.v. administration of CSA (10 mg/kg) blunted the LPS-evoked hypotension, tachycardia, and reductions in time- and frequency-domain indices of heart rate variability (measures of cardiac autonomic control) evoked by LPS (10 mg/kg i.v.). The ability of CSA to reverse the LPS effects disappeared in rats treated intracisternally (i.c.) with baclofen (selective GABAB agonist, 2 µg/rat) but not muscimol (selective GABAA agonist, 1 µg/rat), indicating a preferential compromising action for central GABAB receptors on the advantageous effects of CSA. Moreover, the improvement by CSA of LPS-evoked cardiovascular derangements was also eliminated after concurrent i.c. administration of vigabatrin (GABA transaminase inhibitor, 200 µg/rat) or tiagabine (GABA reuptake inhibitor, 100 µg/rat). These results demonstrate that the activation of central GABAB receptors either directly via baclofen or indirectly following interventions that boost GABA levels in central synapses counterbalances the rectifying action of CSA on endotoxemia.

Cyclosporine counteracts endotoxemia-evoked reductions in blood pressure and cardiac autonomic dysfunction via central sGC/MAPKs signaling in rats.

The immunosuppressant drug cyclosporine A (CSA) improves survivability in endotoxemia and offsets associated loss in vascular reactivity and hypotension. We tested the hypothesis that central phosphoinositide-3-kinase (PI3K)/soluble guanylate cyclase (sGC)/mitogen activated protein kinases (MAPKs) cascade modulates the CSA counteraction of endotoxic hypotension and cardiac autonomic dysfunction. The effects of pharmacologic inhibition of these molecular substrates in central pools on CSA interaction with cardiovascular responses evoked by lipopolysaccharide (LPS) were evaluated in conscious rats. CSA (10mg/kg) reversed the LPS-evoked (i) hypotension and tachycardia, (ii) decreases in time and spectral measures of heart rate variability (HRV), and (iii) increases in serum TNFα and IL-6. These CSA effects disappeared after intracisternal (i.c.) administration of ODQ (sGC inhibitor) but not wortmannin (PI3K inhibitor). When used alone, ODQ or wortmannin abolished the LPS-evoked hypotension and tachycardia, but had no effect on the concomitant reductions in HRV. We also report that the reversal by CSA of LPS hypotension disappeared after treatment with i.c SB203580 (MAPKp38 inhibitor) or PD98059 (MAPKERK inhibitor), in contrast to little effect for SP600125 (MAPKJNK inhibitor). Alternatively, the CSA amelioration of LPS-evoked reductions in HRV was abolished in presence of SP600125 or PD98059, but not SB203580. The single exposure to SP600125 reduced the decreases in blood pressure, but not HRV, caused by LPS whereas SB203580 produced the exact opposite effects. Together, while central sGC/MAPKs circuits modulate the CSA counteraction of endotoxic manifestations, the recruitment of individual MAPKs into this interaction depends on the nature of the cardiovascular response.

Central GABAA receptors are involved in inflammatory and cardiovascular consequences of endotoxemia in conscious rats.

γ-Aminobutyric acid (GABA), the principal brain inhibitory neurotransmitter, modulates inflammatory and neurodegenerative disease. Here, we tested the hypothesis that central GABAergic neurotransmission mediates the detrimental inflammatory, hemodynamic, and cardiac autonomic actions of endotoxemia. The effects of drugs that block GABA receptors or interfere with GABA uptake or degradation on blood pressure (BP), heart rate (HR), and HR variability (HRV) responses elicited by i.v. lipopolysaccharide (LPS) were assessed in conscious rats. The hypotensive effect of LPS (10 mg/kg) was blunted after intracisternal (i.c.) administration of bicuculline (GABAA receptor antagonist) or saclofen (GABAB receptor antagonist). By contrast, the concomitant LPS-evoked tachycardia and decreases in time domain and frequency domain indices of HRV (measures of cardiac autonomic control) were abolished upon treatment with bicuculline but not saclofen. Increases in serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) caused by LPS disappeared in the presence of bicuculline or saclofen, whereas LPS-evoked increases in serum nitric oxide metabolites (NOx) were counteracted by bicuculline only. None of the endotoxemia effects was altered in rats treated with i.c. tiagabine (GABA reuptake inhibitor) or vigabatrin (GABA transaminase inhibitor). These data suggest a major role for central GABAA receptors in the inflammatory and cardiovascular effects of endotoxemia.