Robert C. Speth

Pancreatic angiotensin-converting enzyme 2 improves glycemia in angiotensin II-infused mice

An overactive renin-angiotensin system (RAS) is known to contribute to type 2 diabetes mellitus (T2DM). Although ACE2 overexpression has been shown to be protective against the overactive RAS, a role for pancreatic ACE2, particularly in the islets of Langerhans, in regulating glycemia in response to elevated angiotensin II (Ang II) levels remains to be elucidated. This study examined the role of endogenous pancreatic ACE2 and the impact of elevated Ang II levels on the enzymes ability to alleviate hyperglycemia in an Ang II infusion mouse model. Male C57bl/6J mice were infused with Ang II or saline for a period of 14 days. On the 7th day of infusion, either an adenovirus encoding human ACE2 (Ad-hACE2) or a control adenovirus (Ad-eGFP) was injected into the mouse pancreas. After an additional 7-8 days, glycemia and plasma insulin levels as well as RAS components expression and oxidative stress were assessed. Ang II-infused mice exhibited hyperglycemia, hyperinsulinemia, and impaired glucose-stimulated insulin secretion from pancreatic islets compared with control mice. This phenotype was associated with decreased ACE2 expression and activity, increased Ang II type 1 receptor (AT1R) expression, and increased oxidative stress in the mouse pancreas. Ad-hACE2 treatment restored pancreatic ACE2 expression and compensatory activity against Ang II-mediated impaired glycemia, thus improving β-cell function. Our data suggest that decreased pancreatic ACE2 is a link between overactive RAS and impaired glycemia in T2DM. Moreover, maintenance of a normal endogenous ACE2 compensatory activity in the pancreas appears critical to avoid β-cell dysfunction, supporting a therapeutic potential for ACE2 in controlling diabetes resulting from an overactive RAS.

Immunohistochemical Localization of AT1a, AT1b, and AT2 Angiotensin II Receptor Subtypes in the Rat Adrenal, Pituitary, and Brain with a Perspective Commentary.

Angiotensin II increases blood pressure and stimulates thirst and sodium appetite in the brain. It also stimulates secretion of aldosterone from the adrenal zona glomerulosa and epinephrine from the adrenal medulla. The rat has 3 subtypes of angiotensin II receptors: AT1a, AT1b, and AT2. mRNAs for all three subtypes occur in the adrenal and brain. To immunohistochemically differentiate these receptor subtypes, rabbits were immunized with C-terminal fragments of these subtypes to generate receptor subtype-specific antibodies. Immunofluorescence revealed AT1a and AT2 receptors in adrenal zona glomerulosa and medulla. AT1b immunofluorescence was present in the zona glomerulosa, but not the medulla. Ultrastructural immunogold labeling for the AT1a receptor in glomerulosa and medullary cells localized it to plasma membrane, endocytic vesicles, multivesicular bodies, and the nucleus. AT1b and AT2, but not AT1a, immunofluorescence was observed in the anterior pituitary. Stellate cells were AT1b positive while ovoid cells were AT2 positive. In the brain, neurons were AT1a, AT1b, and AT2 positive, but glia was only AT1b positive. Highest levels of AT1a, AT1b, and AT2 receptor immunofluorescence were in the subfornical organ, median eminence, area postrema, paraventricular nucleus, and solitary tract nucleus. These studies complement those employing different techniques to characterize Ang II receptors.

Angiotensin‐converting enzyme 2: a new target for neurogenic hypertension

Overactivity of the renin–angiotensin system (RAS) is involved in the pathogenesis of hypertension, and an overactive brain RAS has been highlighted in several genetic and experimental models. Until now, angiotensin II (Ang II) was thought to be the main effector of this system, and the angiotensin‐converting enzyme (ACE)–Ang II–Ang II type 1 receptor axis was the main target for antihypertensive therapies. A new member of the RAS, ACE2 (angiotensin‐converting enzyme type 2), has been identified in organs and tissues related to cardiovascular function (e.g. heart, kidney and blood vessels) and appears to be part of a counter‐regulatory pathway to buffer the excess of Ang II. We recently identified the ACE2 protein in brain regions involved in the central regulation of blood pressure and showed that it regulates, and is regulated by, other components of the RAS. Here, we present evidence for the involvement of brain ACE2 in the central regulation of blood pressure, autonomic and cardiac function. We show that lack of ACE2 is deleterious for the central regulation of blood pressure and that brain ACE2 gene therapy can restore baroreflex and autonomic functions and prevent the development of hypertension. Additionally, and independently of a reduction in Ang II levels, we will highlight some of the mechanisms responsible for the beneficial effects of central ACE2 in cardiovascular function.

A consensus definitive classification of scavenger receptors and their roles in health and disease

Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a diverse variety of ligands including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the United States National Institute of Allergy and Infectious Diseases, National Institutes of Health, to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of nonself or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. This classification was discussed at three national meetings and input from participants at these meetings was requested. The following manuscript is a consensus statement that combines the recommendations of the initial workshop and incorporates the input received from the participants at the three national meetings.

Atypical signaling and functional desensitization response of MAS receptor to peptide ligands

MAS is a G protein-coupled receptor (GPCR) implicated in multiple physiological processes. Several physiological peptide ligands such as angiotensin-(1–7), angiotensin fragments and neuropeptide FF (NPFF) are reported to act on MAS. Studies of conventional G protein signaling and receptor desensitization upon stimulation of MAS with the peptide ligands are limited so far. Therefore, we systematically analyzed G protein signals activated by the peptide ligands. MAS-selective non-peptide ligands that were previously shown to activate G proteins were used as controls for comparison on a common cell based assay platform. Activation of MAS by the non-peptide agonist (1) increased intracellular calcium and D-myo-inositol-1-phosphate (IP1) levels which are indicative of the activation of classical Gαq-phospholipase C signaling pathways, (2) decreased Gαi mediated cAMP levels and (3) stimulated Gα12-dependent expression of luciferase reporter. In all these assays, MAS exhibited strong constitutive activity that was inhibited by the non-peptide inverse agonist. Further, in the calcium response assay, MAS was resistant to stimulation by a second dose of the non-peptide agonist after the first activation has waned suggesting functional desensitization. In contrast, activation of MAS by the peptide ligand NPFF initiated a rapid rise in intracellular calcium with very weak IP1 accumulation which is unlike classical Gαq-phospholipase C signaling pathway. NPFF only weakly stimulated MAS-mediated activation of Gα12 and Gαi signaling pathways. Furthermore, unlike non-peptide agonist-activated MAS, NPFF-activated MAS could be readily re-stimulated the second time by the agonists. Functional assays with key ligand binding MAS mutants suggest that NPFF and non-peptide ligands bind to overlapping regions. Angiotensin-(1–7) and other angiotensin fragments weakly potentiated an NPFF-like calcium response at non-physiological concentrations (≥100 µM). Overall, our data suggest that peptide ligands induce atypical signaling and functional desensitization of MAS.

Angiotensin type 1a receptors on corticotropin-releasing factor neurons contribute to the expression of conditioned fear.

Although generally associated with cardiovascular regulation, angiotensin II receptor type 1a (AT1aR) blockade in mouse models and humans has also been associated with enhanced fear extinction and decreased post‐traumatic stress disorder (PTSD) symptom severity, respectively. The mechanisms mediating these effects remain unknown, but may involve alterations in the activities of corticotropin‐releasing factor (CRF)‐expressing cells, which are known to be involved in fear regulation. To test the hypothesis that AT1aR signaling in CRFergic neurons is involved in conditioned fear expression, we generated and characterized a conditional knockout mouse strain with a deletion of the AT1aR gene from its CRF‐releasing cells (CRF‐AT1aR(−/−)). These mice exhibit normal baseline heart rate, blood pressure, anxiety and locomotion, and freeze at normal levels during acquisition of auditory fear conditioning. However, CRF‐AT1aR(−/−) mice exhibit less freezing than wild‐type mice during tests of conditioned fear expression—an effect that may be caused by a decrease in the consolidation of fear memory. These results suggest that central AT1aR activity in CRF‐expressing cells plays a role in the expression of conditioned fear, and identify CRFergic cells as a population on which AT1R antagonists may act to modulate fear extinction.

AT1 angiotensin II receptor and novel non-AT1, non-AT2 angiotensin II/III binding site in brainstem cardiovascular regulatory centers of the spontaneously hypertensive rat

Spontaneously hypertensive rats (SHR) have an activated brain angiotensin system that contributes to the elevation of blood pressure in this animal model. Physiological and pharmacological studies suggest that hyperactivation of brain AT₁ angiotensin receptors is a major pathophysiological factor. Consistent with these observations, radioligand binding studies indicate widespread up-regulation of brain angiotensin receptors in SHR. One key brainstem site in which AT₁ receptor stimulation appears to contribute to the elevated blood pressure in SHR is the rostral ventrolateral medulla (RVLM). However, no quantitative comparison of AT₁ receptor binding in the RVLM has been made in SHR versus normotensive rats. A novel, non-AT₁, non-AT₂ binding site, specific for angiotensins II and III, has recently been discovered in the brain. To determine if radioligand binding to either AT₁ receptors or this novel angiotensin binding site is altered in the RVLM and other caudal brainstem regions of SHR, a quantitative densitometric autoradiographic comparison of radioligand binding in SHR versus normotensive Wistar-Kyoto rats was made. In both the RVLM and caudal ventrolateral medulla (CVLM) as well as dorsomedial medulla (DMM), there was increased expression of AT₁ receptor binding in SHR (13%, 9%, and 23%, respectively). Conversely, expression of the novel, non-AT₁, non-AT₂, angiotensin II and III binding site was decreased in the RVLM and DMM of SHR (37% and 13%, respectively). This increased AT₁ receptor binding in the RVLM may contribute to the hypertension of SHR. Reduced radioligand binding to the novel, non-AT₁, non-AT₂, angiotensin binding site in the RVLM of SHR may indicate a role for this binding site to reduce blood pressure via its interactions with angiotensins II and III.

Neuronal differentiation of NG108-15 cells has impact on nitric oxide- and membrane (natriuretic peptide receptor-A) cyclic GMP-generating proteins.

Cyclic GMP (cGMP), produced in response to either nitric oxide (NO) or certain peptides, controls important neuronal functions. NG108-15 cells were used to characterize the expression of NO- and cGMP-generating proteins and to identify potential alterations associated with neuronal differentiation (neurite outgrowth). We find that these cells contain exclusively neuronal NO synthase (nNOS) isoforms as well as both NO- (soluble guanylyl cyclase, sGC) and natriuretic peptide- (natriuretic peptide receptor-A, NPR-A) responsive cGMP-producing enzymes. The sGC beta(1) subunit (unlike protein phosphatase 2A subunits) is highly membrane-associated. Membrane concentrations of NPR-A and nNOS, but not sGC beta(1) protein are up-regulated with neuronal differentiation. Intriguingly, the rate of hormone-induced cGMP production by NPR-A is significantly diminished in differentiated cells. These findings support roles for NPR-A, the common receptor of atrial (ANP) and B-type (BNP) natriuretic peptide in mature neurons and provide evidence for pronounced changes in neuronal submembrane cGMP signalling during neuronal differentiation.

Regulation of cardiac miR‐208a, an inducer of obesity, by rapamycin and nebivolol

Resistance to obesity is observed in rodents and humans treated with rapamycin (Rap) or nebivolol (Neb). Because cardiac miR‐208a promotes obesity, this study tested whether the modes of actions of Rap and Neb involve inhibition of miR‐208a.

Water deprivation increases angiotensin-converting enzyme but not AT1 receptor expression in brainstem and paraventricular nucleus of the hypothalamus of the rat

The rostral ventrolateral medulla (RVLM) is critical to the maintenance of blood pressure. It has been proposed that blood-borne Ang II can influence the RVLM via a neural connection between the circumventricular organs and paraventricular nucleus of the hypothalamus (PVH) and that a component of this pathway is angiotensinergic. A period of water deprivation leads to increased ability of angiotensin type 1 (AT(1)) receptor antagonists to reduce blood pressure when administered into the RVLM and PVH. We studied the differences in AT(1) receptor and angiotensin-converting enzyme (ACE) expression in these and other brain regions involved in blood pressure regulation and water intake following dehydration. AT(1) receptor and ACE expression in brains of rats deprived of water for 48 h were compared to that of water-replete rats by quantitative receptor autoradiography. AT(1) receptor expression was increased in the subfornical organ and periventricular nucleus of the hypothalamus, but not in other brain regions measured. ACE expression was increased in the RVLM, PVH, choroid plexus, median preoptic nucleus, and organosum vasculosum of the lamina terminalis. These findings suggest that increased Ang II production but not increased receptor expression in the PVH and RVLM is the mechanism by which Ang II in the brain helps to sustain systemic blood pressure during periods of water deprivation.