Maria A. Garcia-Espinosa

Branched-chain amino acids and neurotransmitter metabolism: Expression of cytosolic branched-chain aminotransferase (BCATc) in the cerebellum and hippocampus

In the brain, catabolism of the branched‐chain amino acids (BCAAs) provides nitrogen for the synthesis of glutamate and glutamine. Glutamate is formed through transfer of an amino group from BCAA to α‐ketoglutarate in reaction catalyzed by branched‐chain aminotransferases (BCAT). There are two isozymes of BCAT: cytosolic BCATc, which is found in the nervous system, ovary, and placenta, and mitochondrial BCATm, which is found in all organs except rat liver. In cell culture systems, BCATc is found only in neurons and developing oligodendrocytes, whereas BCATm is the isoform in astroglia. In this study, we used immunohistochemistry to examine the distribution of BCATc in the rat brain, focusing on the well‐known neural architecture of the cerebellum and hippocampus. We show that BCATc is expressed only in neurons in the adult rat brain. In glutamatergic neurons such as granule cells of the cerebellar cortex and of the dentate gyrus, BCATc is localized to axons and nerve terminals. In contrast, in GABAergic neurons such as cerebellar Purkinje cells and hippocampal pyramidal basket cells, BCATc is concentrated in cell bodies. A common function for BCATc in these neurotransmitter systems may be to modulate amounts of glutamate available either for release as neurotransmitter or for use as precursor for synthesis of GABA. Particularly striking in our findings is the strong expression of BCATc in the mossy fiber pathway of the hippocampal formation. This result is discussed in light of the effectiveness of the anticonvulsant drug gabapentin, which is a specific inhibitor of BCATc. J. Comp. Neurol. 477:360–370, 2004.

Injections of angiotensin‐converting enzyme 2 inhibitor MLN4760 into nucleus tractus solitarii reduce baroreceptor reflex sensitivity for heart rate control in rats

Injections of the angiotensin(1–7) [Ang(1–7)] antagonist [d‐Ala7]‐Ang(1–7) into the nucleus of the solitary tract (NTS) of Sprague–Dawley rats reduce baroreceptor reflex sensitivity (BRS) for control of heart rate by ∼40%, whereas injections of the angiotensin II (Ang II) type 1 receptor antagonist candesartan increase BRS by 40% when reflex bradycardia is assessed. The enzyme angiotensin‐converting enzyme 2 (ACE2) is known to convert Ang II to Ang(1–7). We report that ACE2 activity, as well as ACE and neprilysin activities, are present in plasma membrane fractions of the dorsomedial medulla of Sprague–Dawley rats. Moreover, we show that BRS for reflex bradycardia is attenuated (1.16 ± 0.29 ms mmHg−1 before versus 0.33 ± 0.11 ms mmHg−1 after; P < 0.05; n= 8) 30–60 min following injection of the selective ACE2 inhibitor MLN4760 (12 pmol in 120 nl) into the NTS. These findings support the concept that within the NTS, local synthesis of Ang(1–7) from Ang II is required for normal sensitivity for the baroreflex control of heart rate in response to increases in arterial pressure.

Angiotensin-(1-7) and baroreflex function in nucleus tractus solitarii of (mRen2)27 transgenic rats.

Endogenous angiotensin (Ang)-(1-7) enhances, while Ang II attenuates, baroreceptor sensitivity (BRS) for reflex control of heart rate (HR) in Sprague-Dawley (SD) rats. In (mRen2)27 renin transgenic rats [(mRen2)], there is overexpression of the mouse Ren2 gene in brain, leading to elevated Ang II and reduced Ang-(1-7) in brain medullary, and associated with hypertension and impaired BRS. Methods: We therefore tested the contribution of endogenous Ang-(1-7) to BRS for control of HR and responses to cardiac vagal chemosensitive afferent fiber activation (CVA) with phenylbiguanide (PBG) in anesthetized SD and (mRen2) 27 rats before and after bilateral nucleus of the solitary tract (nTS) injection of the Ang-(1-7) receptor antagonist (D-Ala7)-Ang-(1-7). Results: (mRen2) 27 rats exhibited a ∼50% impairment in BRS as compared with SD (P < 0.05). (D-Ala7)-Ang-(1-7) attenuated BRS by ∼50% in SD rats, but was without effect in (mRen2) 27 rats. (D-Ala7)-Ang-(1-7) did not alter the responses to CVA by PBG (iv bolus) in either strain. There were no differences in the depressor effects of Ang-(1-7) injected into the nTS, nor were levels of mRNA different for angiotensin-converting enzyme, angiotensin-converting enzyme 2, neprilysin, or the mas receptor in medullary tissue from SD versus (mRen2)27 rats. Conclusion: Endogenous Ang-(1-7) does not provide tonic input in the nTS to modulate BRS for control of HR in (mRen2)27 rats, which may contribute to impairment of BRS in these animals.

Widespread neuronal expression of branched-chain aminotransferase in the CNS: implications for leucine/glutamate metabolism and for signaling by amino acids.

Transamination of the branched‐chain amino acids produces glutamate and branched‐chain α‐ketoacids. The reaction is catalyzed by branched‐chain aminotransferase (BCAT), of which there are cytosolic and mitochondrial isoforms (BCATc and BCATm). BCATc accounts for 70% of brain BCAT activity, and contributes at least 30% of the nitrogen required for glutamate synthesis. In previous work, we showed that BCATc is present in the processes of glutamatergic neurons and in cell bodies of GABAergic neurons in hippocampus and cerebellum. Here we show that this metabolic enzyme is expressed throughout the brain and spinal cord, with distinct differences in regional and intracellular patterns of expression. In the cerebral cortex, BCATc is present in GABAergic interneurons and in pyramidal cell axons and proximal dendrites. Axonal labeling for BCATc continues into the corpus callosum and internal capsule. BCATc is expressed by GABAergic neurons in the basal ganglia and by glutamatergic neurons in the hypothalamus, midbrain, brainstem, and dorsal root ganglia. BCATc is also expressed in hypothalamic peptidergic neurons, brainstem serotoninergic neurons, and spinal cord motor neurons. The results indicate that BCATc accumulates in neuronal cell bodies in some regions, while elsewhere it is exported to axons and nerve terminals. The enzyme is in a position to influence pools of glutamate in a variety of neuronal types. BCATc may also provide neurons with sensitivity to nutrient‐derived BCAAs, which may be important in regions that control feeding behavior, such as the arcuate nucleus of the hypothalamus, where neurons express high levels of BCATc.

Renin-angiotensin System Blockers and Modulation of Radiation-Induced Brain Injury

Radiation-induced brain injury remains a major cause of morbidity in cancer patients with primary or metastatic brain tumors. Approximately 200,000 individuals/year are treated with fractionated partial or whole-brain irradiation, and > half will survive long enough (≤6 months) to develop radiation-induced brain injury, including cognitive impairment. Although short-term treatments have shown efficacy, no long-term treatments or preventive approaches are presently available for modulating radiation-induced brain injury. Based on previous preclinical studies clearly demonstrating that renin-angiotensin system (RAS) blockers can modulate radiation-induced late effects in the kidney and lung, we and others hypothesized that RAS blockade would similarly modulate radiation-induced brain injury. Indeed, studies in the last 5 years have shown that both angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II type 1 receptor antagonists (AT(1)RAs) can prevent/ameliorate radiation-induced brain injury, including cognitive impairment, in the rat. The mechanistic basis for this RAS blocker-mediated effect remains the subject of ongoing investigations. Putative mechanisms include, i] blockade of Ang II/NADPH oxidase-mediated oxidative stress and neuroinflammation, and ii] a change in the balance of angiotensin (Ang) peptides from the pro-inflammatory and pro-oxidative Ang II to the anti-inflammatory and anti-oxidative Ang-1-7). However, given that both ACEIs and AT(1)RAs are 1] well-tolerated drugs routinely prescribed for hypertension, 2] exhibit some antitumor properties, and 3] can prevent/ameliorate radiation-induced brain injury, they appear to be ideal drugs for future clinical trials, offering the promise of improving the quality of life of brain tumor patients receiving brain irradiation.

Chronic immunoneutralization of brain angiotensin-(1-12) lowers blood pressure in transgenic (mRen2)27 hypertensive rats.

Angiotensin-(1-12) [ANG-(1-12)] is a newly identified peptide detected in a variety of rat tissues, including the brain. To determine whether brain ANG-(1-12) participates in blood pressure regulation, we treated male adult (mRen2)27 hypertensive rats (24-28 wk of age) with Anti-ANG-(1-12) IgG or Preimmune IgG via an intracerebroventricular cannula for 14 days. Immunoneutralization of brain ANG-(1-12) lowered systolic blood pressure (-43 +/- 8 mmHg on day 3 and -26 +/- 7 mmHg on day 10 from baseline, P < 0.05). Water intake was lower on intracereroventricular day 6 in the Anti-ANG-(1-12) IgG group, accompanied by higher plasma osmolality on day 13, but there were no differences in urine volume, food intake, or body weight during the 2-wk treatment. In Preimmune IgG-treated animals, there were no significant changes in these variables over the 2-wk period. The antihypertensive effects produced by endogenous neutralization of brain ANG-(1-12) suggest that ANG-(1-12) is functionally active in brain pathways regulating blood pressure.

The brain renin-angiotensin system and cardiovascular responses to stress: insights from transgenic rats with low brain angiotensinogen

The renin-angiotensin system (RAS) has been identified as an attractive target for the treatment of stress-induced cardiovascular disorders. The effects of angiotensin (ANG) peptides during stress responses likely result from an integration of actions by circulating peptides and brain peptides derived from neuronal and glial sources. The present review focuses on the contribution of endogenous brain ANG peptides to pathways involved in cardiovascular responses to stressors. During a variety of forms of stress, neuronal pathways in forebrain areas containing ANG II or ANG-(1-7) are activated to stimulate descending angiotensinergic pathways that increase sympathetic outflow to increase blood pressure. We provide evidence that glia-derived ANG peptides influence brain AT(1) receptors. This appears to result in modulation of the responsiveness of the neuronal pathways activated during stressors that elevate circulating ANG peptides to activate brain pathways involving descending hypothalamic projections. It is well established that increased cardiovascular reactivity to stress is a significant predictor of hypertension and other cardiovascular diseases. This review highlights the importance of understanding the impact of RAS components from the circulation, neurons, and glia on the integration of cardiovascular responses to stressors.

In vivo expression of angiotensin-(1-7) lowers blood pressure and improves baroreflex function in transgenic (mRen2)27 rats.

Abstract: Transgenic (mRen2)27 rats are hypertensive with impaired baroreflex sensitivity for control of heart rate compared with Hannover Sprague–Dawley rats. We assessed blood pressure and baroreflex function in male hemizygous (mRen2)27 rats (30–40 weeks of age) instrumented for arterial pressure recordings and receiving into the cisterna magna either an Ang-(1-7) fusion protein or a control fusion protein (CTL-FP). The maximum reduction in mean arterial pressure achieved was −38 ± 7 mm Hg on day 3, accompanied by a 55% enhancement in baroreflex sensitivity in Ang-(1-7) fusion protein-treated rats. Both the high-frequency alpha index (HF-&agr;) and heart rate variability increased, suggesting increased parasympathetic tone for cardiac control. The mRNA levels of several components of the renin–angiotensin system in the dorsal medulla were markedly reduced including renin (−80%), neprilysin (−40%), and the AT1a receptor (−40%). However, there was a 2-fold to 3-fold increase in the mRNA levels of the phosphatases PTP-1b and dual-specificity phosphatase 1 in the medulla of Ang-(1-7) fusion protein-treated rats. Our finding that replacement of Ang-(1-7) in the brain of (mRen2)27 rats reverses in part the hypertension and baroreflex impairment is consistent with a functional deficit of Ang-(1-7) in this hypertensive strain. We conclude that the increased mRNA expression of phosphatases known to counteract the phosphoinositol 3 kinase and mitogen-activated protein kinases, and the reduction of renin and AT1a receptor mRNA levels may contribute to the reduction in arterial pressure and improvement in baroreflex sensitivity in response to Ang-(1-7).

Proton Magnetic Resonance Spectroscopy Detection of Neurotransmitters in Dorsomedial Medulla Correlate With Spontaneous Baroreceptor Reflex Function

Control of heart rate variability via modulation of sympathovagal balance is a key function of nucleus tractus solitarii and the dorsal motor nucleus of the vagus localized in the dorsomedial medulla oblongata. Normal blood pressure regulation involves precise balance of glutamate (Glu)-glutamine-γ-aminobutyric acid transmitter systems, and angiotensin II modulates these transmitters to produce tonic suppression of reflex function. It is not known, however, whether other brain transmitters/metabolites are indicators of baroreflex function. This study establishes the concept that comprehensive baseline transmitter/metabolite profiles obtained using in vivo 1H magnetic resonance spectroscopy in rats with well-characterized differences in resting blood pressure and baroreflex function can be used as indices of autonomic balance or baroreflex sensitivity. Transgenic rats with over-expression of renin [m(Ren2)27] or under-expression of glial-angiotensinogen (ASrAogen) were compared with Sprague-Dawley rats. Glu concentration in the dorsal medulla is significantly higher in ASrAogen rats compared with either Sprague-Dawley or (mRen2)27 rats. Glu levels and the ratio of Glu:glutamine correlated positively with indices of higher vagal tone consistent with the importance of these neurotransmitters in baroreflex function. Interestingly, the levels of choline-containing metabolites showed a significant positive correlation with spontaneous baroreflex sensitivity and a negative correlation with sympathetic tone. Thus, we demonstrate the concept that noninvasive assessment of neurochemical biomarkers may be used as an index of baroreflex sensitivity.

Regulation of Brain Neprilysin and ACE2 in Transgenic Rats During Aging

Angiotensinogen (Aogen), the precursor to angiotensin (Ang) II and Ang-(1–7) is expressed in astrocytes and neurons. Neprilysin and angiotensin-converting enzyme 2 (ACE2) are potential enzymes for Ang-(1–7) formation from Ang I and Ang II respectively. We assessed their regulation in brain during aging in transgenic rats (ASrAogen) with an Aogen antisense behind a glial-promoter. These exhibit a 90% reduction in brain Aogen levels and low blood pressure that is maintained during aging at 16 and 70 weeks in hypothalamus (HYPO) and medulla (MED). HYPO ACE2 mRNA was 45% higher in ASrAogen than control SD rats and higher in older ASrAogen vs. younger rats. Neprilysin mRNA tended to increase during aging and values were 80% higher in ASrAogen vs. SD. In MED, mRNA of both enzymes during aging tended to decrease with no difference between strains. Immunocytochemistry revealed ACE2-staining in both strains localized to circumventricular organs and plexus choroideus. Thus, the ACE2 distribution suggests the enzyme contributes to Ang-(1–7) in cerebrospinal fluid but not neural pathways. Up regulation of mRNA for both enzymes in HYPO of ASrAogen rats may represent compensation for loss of glial-derived peptides. Lack of differences in MED between strains may reflect distinct sources (neural vs. glial) of peptides responsible for enzyme regulation in the two brain areas.