bluuebunny/crossref_metadata_2025 huggingface dataset:
Advances in structure based drug design and virtual screening
Authors: Abagyan Ruben
Published: June 2021

The rapid growth of the number of structures in the Protein Data Bank and the emergence of the chemogenomics databases combined with the advances in computational methods created a unique opportunity for a wider application of rational drug design and structure based virtual screening. Another factor is the determination of the three dimensional structure of beta 2 adrenergic receptor. The main challenge, however, remains to be the understanding and treatment of the receptor induced fit effects.

We built the largest structural chemogenomic database and presented a docking algorithm that reliably docks ligands to their pockets despite the induced fit effect. The new GPCR structure and its implications for rational design and screening of potent and specific GPCR agonists and antagonists are reviewed.

The success of the ligand guided models is demonstrated on several cases studies including drug repurposing.

This work was supported by NIH grants 5R01‐GM074832‐02 and 5R01‐GM071872‐02.
[beta]1 adrenergic receptor ([beta]1AR)‐epidermal growth factor receptor (EGFR) interaction regulates ERK cellular activity
Authors: Tilley Douglas G. , Rockman Howard A.
Published: June 2021

Recently, we showed that β1AR stimulation mediates transactivation of the EGFR and induces ERK activation in a βarrestin‐dependent manner to confer cardioprotection. However, ERK activation is associated with increased cardiac growth and hypertrophy, frequently a detrimental process in the failing heart. Therefore, we hypothesized that the β1AR and EGFR form a βarrestin‐dependent signaling complex in which ERK may be differentially targeted within the cell. Here, via (A) confocal microscopy, (B) immunoprecipitation and (C) FRET‐based assays, we show the physical association between β1AR and EGFR in a HEK 293 cell‐based system that is regulated by agonist stimulation. The interaction between β1AR and EGFR and the stimulation‐induced recruitment of βarrestin to the receptor complex is dependent on G protein receptor‐coupled kinase (GRK) phosphorylation sites in the C‐terminal tail of β1AR. While both β1AR stimulation and EGF ligand induce EGFR activation, EGF ligand causes translocation of activated ERK to the nucleus, whereas β1AR‐activated ERK is restricted to the cytoplasm. These data reveal a new signaling paradigm in which a β1AR‐EGFR‐βarrestin complex acts to retain activated ERK in the cytoplasm, resulting in differential intracellular targeting of ERK signaling. Supported by the Heart and Stroke Foundation of Canada and the National Institutes of Health.
Functional characterization of the alpha1D‐AR/syntrophin signaling complex
Authors: Lyssand John Sverre , DeFino Mia C , Hertz Angie L , Feller David B , Tang Xiaobo , Adams Marvin E , Wacker Jennifer L , Hague Chris
Published: June 2021

Previously, we reported the identification of syntrophins as novel accessory proteins for α1D‐adrenergic receptors (ARs). To characterize the functional importance of this interaction, we examined the role of syntrophins on α1D‐AR function in vitro and in vivo. Syntrophins increase α1D‐AR binding site density and functional coupling in HEK293 cells, with β2>β1>β1. To examine the importance of each domain in syntrophin to α1D‐AR function, an α1D‐AR/α1‐syntrophin fusion construct was created with α1‐syntrophin tethered to the α1D‐AR C‐terminus. Using this technique, we determined the syntrophin unique domain is critical for α1D‐AR function. Mass spectrometry was next used to identify potential accessory proteins recruited by α1‐syntrophin to the α1D‐AR signaling complex. In vivo, syntrophin KO mice have a 10% decrease in resting MAP and i.p. phenylephrine (PE) causes minor increases in MAP. Interestingly, PE effects are unaffected by the α1D‐AR antagonist BMY 7378, suggesting α1D‐AR stimulated increases in MAP are abrogated. From these studies, we conclude syntrophins are essential regulators of α1D‐AR function in vivo through regulation of the α1D‐AR protein signaling network.
Agonist and antagonist activation of signalling pathways in CHO‐K1 cells stably expressing the human β3‐adrenoceptor
Authors: Summers Roger J , Hutchinson Dana S , Evans Bronwyn A , Sato Masaaki
Published: June 2021

The human β3‐adrenoceptor (hβ3‐AR) couples to Gs to activate adenylyl cyclase, leading to lipolysis or thermogenesis in adipose tissue. hβ3‐AR also couple to Gi, to modulate adenylyl cyclase and activate mitogen‐activated protein kinase (MAPK) pathways. Here, we have examined the action of the β2‐AR agonist zinterol, the selective hβ3‐AR agonist L755507 and the selective hβ3‐AR antagonist L748337 on hβ3‐AR stably expressed in CHO‐K1 cells. Zinterol and L755507 caused dose‐dependent cAMP accumulation (pEC50′ 8.47±0.11 and 12.28±0.03) while L748337 had little effect in this assay. In contrast, in the cytosensor microphysiometer, the maximum extracellular acidification rate (ECAR) responses elicited by L748337 (pEC50 7.14±0.03) were almost identical to those obtained with zinterol and L755507 (pEC50 8.13±0.03 and 8.63±0.10 respectively). This suggested that L748337 can selectively activate pathways other than cAMP. Western blots of p38 MAPK and Erk1/2 phosphorylation showed strong activation in response to both L748337 and to L755507. Activation in response to L748337 was almost completely abolished by PTX pre‐treatment that only slightly affected responses to L755507. We conclude that L755507 and L748337 utilize discrete pathways to activate p38 MAPK and Erk1/2; L755507 by signalling through Gs and L748337 by signalling through Gi.

This research was supported by National Health and Medical Research Council Project Grants 236884 and 491190.
S1P receptor localization confers selectivity for Gi mediated signaling pathways
Authors: Means Chris , Miyamoto Shigeki , Chun Jerold , Brown Joan Heller
Published: June 2021

Adult mouse ventricular myocytes (AMVMs) express S1P1, S1P2, and S1P3 receptors. S1P activates Akt and ERK in AMVMs through a pertussis toxin (PTX)‐sensitive (Gi‐mediated) pathway. S1P‐mediated activation of ERK and Akt is partially reduced in both S1P2 and S1P3 receptor null AMVMs and abolished in S1P2,3 receptor null AMVMs. In addition S1P treatment leads to PTX‐sensitive inhibition of isoproterenol (iso)‐stimulated cAMP accumulation in WT myocytes, and this response in unchanged in S1P2,3 null myocytes, demonstrating that the S1P1 receptor also couples to Gi. Similarly an S1P1 receptor selective agonist, SEW2871, also decreased iso‐stimulated cAMP accumulation but failed to activate either ERK or Akt. To determine whether localization of the S1P1 receptor and its effectors is important in mediating this signaling specificity, the caveolae disrupting agent methyl‐β‐cyclodextrin (MβCD) was used. The S1P1 receptor was concentrated in caveolar fractions and this localization was disrupted by MβCD treatment. MβCD did not prevent S1P‐mediated activation of ERK or Akt, but fully abolished the inhibition of cAMP accumulation and negative inotropic effects of both S1P and SEW2871. Together, the data indicate that localization of S1P1 receptors to caveolae is required for this receptor to inhibit adenylyl cyclase and decrease contractility, yet compromises the coupling of this receptor to Akt and ERK pathways.

This work was supported by National Institutes of Health Grants HL28143 and HL46345 (to J.H. Brown), NS048478 and DA019674 (to J. Chun), and by an American Heart Association predoctoral fellowship (to C.K. Means)
The pleiotropic physiological effects of relaxin are determined by the signalling pathway profile activated in target cells
Authors: Halls Michelle L , Bathgate Ross A , Summers Roger J
Published: June 2021

Relaxin is a two‐chain hormone, related to insulin, that mediates pleiotropic effects in physiological systems. The relaxin receptor RXFP1, is a G‐protein coupled receptor that increases cAMP accumulation via a biphasic pathway when expressed in HEK293T cells: initially RXFP1 couples to Gαs and GαoB, to increase and modulate cAMP respectively, but with time recruits coupling to Gαi3 and activates a Gβγ‐PI3K‐PKCζ pathway to further increase cAMP. However, the signalling transduction mechanisms activated by this hormone in physiological target cells and tissues are unclear. This study examines the signalling pathways activated by relaxin in cells that endogenously express RXFP1 using an α‐screen cAMP accumulation assay. RXFP1 expression was identified in three cell lines and two primary cell cultures: THP‐1, T‐47D, Colo16, rat cardiac fibroblasts and rat renal fibroblasts from obstructed kidneys. Only in THP‐1 cells and rat cardiac fibroblasts was there activation of the delayed Gαi3 pathway. In all other cells there was activation of a combination of the initial pathways. T‐47D cells could only activate Gαs, whereas Colo16 and rat renal fibroblasts could activate both Gαs and GαoB. Thus the signalling pathways activated by relaxin are highly dependent upon the cell under investigation, and may help explain the varied physiological responses exerted by relaxin in different target tissues.

This research was supported by National Health and Medical Research Council Project Grants 436713 and 454375.
PACAP regulates transcription of the Ier3 gene during PC12 cell differentiation via calcium‐dependent activation of a non‐canonical (PKA‐independent) cAMP signaling pathway that activates ERK and Elk
Authors: Gerdin Matthew J. , Eiden Lee E.
Published: June 2021

PC12 cells are a model to study PACAP‐mediated differentiation. PACAP, forskolin or KCl increase neuritogenesis in PC12 cells suggesting a role for cAMP and calcium. Neuritogenesis is ERK dependent and PACAP, forskolin or KCl increase ERK phosphorylation identifying ERK as a critical signaling node in PC12 cell neuritogenesis. Interestingly, PACAP activation of ERK is independent of PKA as it is not blocked following pharmacological inhibition of PKA or in a PC12 cell deficient in PKA, while forskolin and KCl appear PKA‐dependent. Combinatorial activation by cAMP and calcium, however, exhibits a synergistic activation of an Elk reporter whose inhibition in the presence of a PKA inhibitor is equivalent to PACAP suggesting that this combinatorial regulation unmasks a PKA‐independent signaling cassette not seen with either agent alone. Microarray analysis identified cAMP and ERK dependent genes potentially involved in PC12 cell neuritogenesis. One gene, Ier3, exhibited a similar combinatorial regulation by cAMP and calcium as seen with the Elk reporter and PACAP‐induction of Ier3 was significantly blunted by pharmacological inhibitors targeting calcium signaling, but independent of PKA. These data suggest that forskolin and KCl alone activate a PKA‐dependent signaling pathway but that co‐stimulation with both forskolin and KCl unmasks a PKA‐independent pathway, which is used by PACAP via the PAC1 receptor.
Poly(ADP‐ribose) polymerase‐1 mediated glycolytic inhibition as a mechanism of targeted necrosis following renal ischemia
Authors: Devalaraja‐Narashimha Kishor , Padanilam Babu J
Published: June 2021

Following ischemic renal injury (IRI), selective damage occurs in the S3 segment of the proximal tubule (PST) due to glycolytic inhibition. However, the exact mechanism by which glycolysis is inhibited is not elucidated. We reported that inhibition of PARP‐1 activity offers protection against necrotic cell death in PSTs following IRI by preserving ATP levels. We hypothesize that discriminating PARP‐1 activation in PSTs following IRI causes poly(ADP‐ribosyl)ation and inhibition of the key glycolytic enzyme GAPDH, leading to inhibition of glycolysis and anaerobic ATP synthesis. We demonstrate using in vitro and in vivo models that GAPDH is poly(ADP‐ribosyl)ated and as a consequence, its activity is inhibited under hypoxic conditions. Inhibition of PARP activity restored GAPDH activity and ATP levels. Supplementation of NAD+ in simulated hypoxia had only a minimal effect on ATP synthesis indicating that glycolytic inhibition is not due to depletion of NAD+ as previously proposed. Inhibition of GAPDH using iodoacetamide, however, exacerbated ATP depletion and necrotic cell death under hypoxic conditions when compared to controls, while inhibition of PARP activity protected the cells. In conclusion, our data indicate that GAPDH‐poly(ADP‐ribosyl)ation and subsequent inhibition of anaerobic respiration exacerbate ATP depletion in PST to induce selective PST cell injury following IRI. AHA and NKF‐Nebraska.
CaMKII δ Deletion Protects Against Apoptosis In Myocardial Ischemia/Reperfusion Injury In Vivo
Authors: Ling Haiyun , Zhang Tong , Brown Joan Heller
Published: June 2021

Background: The δ subunit of Ca2+/Calmodulin‐dependent protein kinase II (CaMKII) predominates in the heart. There is controversy regarding the role of CaMKII in myocardial ischemia/reperfusion, based largely on in vitro experiments using a pharmacological CaMKII inhibitor (KN‐93). The best evidence for in vivo function of CaMKII would come from the use of mice in which the gene is deleted. A mouse with CaMKII δ deletion has been recently developed in our lab. Methods and Results: CaMKII δ knockout mice and littermate controls were subjected to 1 h myocardial ischemia followed by 6 h reperfusion. Infarct size was measured by Evans blue/TTC staining. Apoptosis was assayed by DNA laddering. The expression of apoptosis related proteins (caspase‐3 and Bax) was also examed by Western blotting. Compared to WT controls, mice in which CaMKII δ was deleted showed a significant reduction in infarct size and DNA laddering, accompanied by attenuated Bax and caspase‐3 in the ischemic myocardium. Conclusion: CaMKII δ deletion protects against apoptosis in myocardial ischemia/reperfusion injury in vivo.

This work was supported by HL80101.
Imaging of regional ischemia and altered conduction patterns in excised rat hearts
Authors: Swift Luther M , Bhatt Jay , Arutunyan Ara , Sarvazyan Narine , Kay Matthew W
Published: June 2021

We describe a new approach that combines several techniques to visualize abnormal electrical activity caused by regional hypoperfusion of myocardial tissue. A flexible microcannula was inserted into the left anterior descending artery of Langendorff perfused rat hearts, an air‐tight seal between the coronary artery and the cannula was created, and an HPLC pump was used to deliver a specified flowrate through the microcannula. The size and geometry of the affected coronary bed and the degree of ischemia was monitored by the acquiring the epicardial fluorescence of nicotinamide adenine dinucleotide (NADH). High resolution optical mapping of NADH and transmembrane potential was then conducted using a dual CCD camera system. The ECG was acquired using surface electrodes. The described method provides a powerful new tool to assess how coronary flow rate affects the degree of local ischemia and to associated it with abnormal patterns of electrical activity. Financial support by the American Heart Association, MidAtlantic Affiliate and the NIH (HL076722) is gratefully acknowledged.
Expression of Smac/DIABLO and Caspase‐3 Proteins in Rat Myocardium Suffered from Hypothermic Preservation
Authors: Zheng Ming‐zhi , Jiang Jian‐ping , Chen Ying‐ying , Shen Yue‐liang , Zheng Liang‐rong
Published: June 2021

Objective: To investigate the effect of different duration of hypothermic preservation on the expression of Smac/DIABLO and caspase‐3 proteins in rat hearts.

Methods: The Langendorff model of isolated rat heart was used. The hearts were stored in 4°C Celsior solution for different time (0, 3, 6, 9 or 12 h) followed by 60 min of reperfusion. The recovery of rate‐pressure product (RPP) in heart mitochondrial was measured at the end of reperfusion. Apoptotic cardiomyocytes were detected by TUNEL technique. The expression of Smac/DIABLO and Caspase‐3 proteins was also analyzed by Western blotting.

Results: Prolonged the hypothermic preservation, the recovery of RPP decreased and the percentage of apoptotic cells enhanced in rat hearts. Long‐term preservation time‐dependently increased Smac/DIABLO and caspase‐3 proteins expression, the peak protein levels appeared at 9 h after preservation. Then levels of two proteins fell after 12 h of preservation.

Conclusion: The results shown that prolonged the hypothermic preservation might lead to the expression of Smac/DIABLO protein and induce cardiomyocytes apoptosis, which may in turn result in malfunction of cardiomyocytes.

This work was supported by the Science Foundation of Zhejiang Medical College (No.2006XZ04) and the National Natural Science Foundation of China (No.30470635).
Enhanced Matrix Metalloproteinase Activity in Microvascular Endothelium of the Spontaneously Hypertensive Rat
Authors: Tran Edward D , Schmid‐Schonbein Geert W
Published: June 2021

Elevated arterial pressure is associated with enhanced microvascular complications and organ injury, but the mechanisms for complications are not well defined. The spontaneously hypertensive rat (SHR) has a significantly elevated level of proteolytic activity in microvascular cells compared to its normotensive controls (WKY and Wistar rats). Our objective was to identify the type of matrix metalloproteinases (MMP) involved. Enzymatic activities of MMP‐1, ‐2, ‐3, ‐7, and ‐8, and lipase in rat mesenteric microvessels were measured with in‐vivo micro‐zymography. Fluorogenic peptide substrates were used to visualize enzyme activity on freshly exposed mesentery for 60 min with digital fluorescent microscopy. Significantly enhanced levels of MMP‐1, ‐7, and ‐8, and lipase activities were observed in microvascular endothelium, but not in the interstitium of the SHR. There was a readily detectable pattern of MMP‐1, ‐7, and ‐8, and lipase activities in the SHR with enhanced level in venules compared to arterioles and capillaries. Enhanced levels of MMP‐2 and lipase activities were observed in microvascular endothelium subject to spontaneous occlusion irrespective of rat strain. These results suggest that elevated microvascular MMP‐1, ‐7, and ‐8, and lipase activities in the SHR. Supported by HL 10881.
Effects of HET0016 and estrogen on postischemic pial artery dilatory response
Authors: Miyasaki Chikao , Koehler Raymond C. , Littleton‐Kearney Marguerite T.
Published: June 2021

We determined if inhibiting hydroxyeicosatetraenoic acid (20‐HETE) synthesis by HET0016 improves postischemic pial dilatory function and if estrogen status and the timing of estrogen treatment alters these effects. Pial artery dilation to acetylcholine (ACh) (10 μM) was measured through closed cranial windows before and after 15‐min forebrain ischemia (4‐vessel occlusion) in male (M), intact female (F), ovariectomized female (OVX), OVX + immediate 17 β‐estradiol (E) treated (E1) and OVX + delayed E treated (E10) rats (n=6/group). The E1 rats were implanted with E slow release pellets after ovariectomy. The E10 rats were treated 10 weeks after ovariectomy. Compared to preischemia, postischemic dilation to ACh was depressed in M (21.9 ± 1.8 vs 3.0 ± 0.6 %), OVX (26.9 ± 14.5 vs 8.0 ± 0.7 %) and E10 (23.7 ± 2.2 vs 4.0 ± 0.5 %) but was improved in F(28.3 ± 2.6 vs 20 ± 1.5%), and E1 (26.4 ± 3.2 vs 19.7 ± 1.6 %). HET0016 (1 μM), superfused after 1 h of reperfusion, increased baseline diameter in all groups but did not improve postischemic dilation to ACh in any group. We conclude that timing of E treatment is important to obtain the benefits of E on postischemic pial dilatory response. Although 20‐HETE contributes to postischemic vasomotor tone independent of sex hormone status, 20‐HETE does not appear to be responsible for impaired endothelial‐dependent dilation in pial arteries. (Supported by NR005339 and HL59996)
Inhibition of Mannose‐Binding Lectin Decreases Infarction Size After Ischemic Stroke and Reperfusion
Authors: Morrison Helena , McDonagh Paul , Stahl Gregory , Ritter Leslie
Published: June 2021

Components of the alternative and classical complement pathways have been implicated in contributing to brain injury after ischemic stroke and reperfusion (ISR). Inhibition of components in these two pathways are neuroprotective in animal models of ISR. The role of mannose binding lectin (MBL) in the lectin complement pathway has not been examined in the setting of ISR. The purpose of this study was to determine if treatment with anti‐MBL monoclonal antibody (mAb) is effective in reducing cerebral infarction size after ISR. Male, Sprague‐Dawley rats (275–325g) underwent middle cerebral artery occlusion (MCAO) and reperfusion via the filament method for four hours. Thirty minutes before reperfusion, animals were treated with either anti‐MBL mAb (10mg/kg, i.p.) (N=3) or with normal saline (0.3ml, i.p.) (N=3). After 24 hours of reperfusion, brains were excised, sliced into seven 2mm sections, and stained with 2% TTC, and cerebral infarction volume was calculated (% of the contralateral hemisphere). Treatment with anti‐MBL mAb reduced cerebral infarction size by 50%. These findings indicate that in a rodent model, the MBL‐dependent portion of the lectin complement pathway may contribute to brain injury after ISR. MBL inhibition may represent a novel neuroprotective strategy worthy of further investigation. Supported by NIH NR 05028 and NIH HL 58859
Rho signaling is involved in HIV‐1 Tat‐induced dysfunction of brain endothelial cells and alterations of claudin‐5 expression
Authors: Zhong Yu , Couraud Pierre‐Olivier , Hennig Bernhard , Toborek Michal
Published: June 2021

The blood‐brain barrier (BBB) is the critical structure preventing HIV trafficking into the brain. Specific HIV proteins, such as Tat, can contribute to the dysfunction of brain microvascular endothelial cells (BMEC) and facilitate HIV entry into the brain. The Rho cascades of small GTPases (RhoA, Rac1 and Cdc42) have gained considerable recognition as powerful regulators of actin cytoskeletal organization. In addition, the Rho pathway is critical for the integrity of the BBB. Therefore, the present study was focused on the effects of Tat on the Rho signaling. Treatment with Tat markedly increased membrane RhoA levels in human BMEC, while the total level of RhoA was not changed. Tat also elevated GTP‐RhoA levels as assessed by the pull‐down assay. Moreover, we found that Tat increased ROCK phosphorylation. Because integrity of the brain endothelium is regulated by tight junctions, these structural elements of the BBB were also evaluated in the present study. Most interestingly, Tat induced a decrease in claudin‐5 expression by a Rho‐dependent mechanism. Indeed, inhibition of the Rho signaling by the C3 exoenzyme blocked Tat‐induced disruption of claudin‐5 expression in human BMEC. The present data indicate the importance of the Rho signaling in disruption of the integrity of the BBB in response to Tat exposure. Supported by NS39254, MH63022, and MH072567.
BBB Na/H exchange: plasma membrane distribution of NHE1 and NHE2 isoforms and stimulation by arginine vasopressin
Authors: Lam Tina I. , O’Donnell Martha E.
Published: June 2021

Cerebral edema formation in ischemic stroke involves increased activity of blood‐brain barrier (BBB) Na transporters through mechanisms not well understood. Na/H exchange inhibitors can reduce brain edema in rat models of ischemic stroke. We have found previously that cerebral microvascular endothelial cells (CMEC) express both NHE1 and NHE2 isoforms of the Na/H exchanger and that CMEC exchanger activity is stimulated by the ischemic factors hypoxia, aglycemia and arginine vasopressin (AVP). In the present study we evaluated BBB in situ for the presence of NHE isoforms and further, investigated the mechanism by which AVP stimulates the BBB Na/H exchanger. Western blot analysis and immunoelectron microscopy were used to evaluate NHE isoforms in freshly isolated rat cerebral microvessels and perfusion‐fixed rat brain, respectively. In addition, Na/H exchange activity of CMEC was evaluated by microspectrofluorometry, using the pH‐sensitive dye BCECF. NHE1 and NHE2 isoforms were found in the microvessels and in BBB in situ, with both isoforms predominantly in the luminal BBB membrane (70% for NHE1, 75% for NHE2). CMEC Na/H exchange activity was increased 2.3‐fold by AVP and 2.5‐fold by the V1 agonist Orn VP (both 100 nM, 5 min) but not by a V2 agonist (DDAVP). Our findings suggest that AVP stimulates luminal BBB NHE1 and/or NHE2 activity via a V1 AVP receptor pathway.

Supported by NINDS and AHA Western States Affiliate
Effects of Aging and Renin‐Angiotensin System (RAS) Blockade on the Intra‐renal RAS in Older Fischer 344 X Brown Norway Rats
Authors: Kasper Sherry O , Gilliam‐Davis Shea , Groban Leanne , Carter Christy S , Sonntag William E , Chappell Mark C , Diz Debra I
Published: June 2021

Activation of the intrarenal RAS occurs during aging in Wistar, Sprague‐Dawley and Fischer 344 (F344) rats as indicated by increased urinary angiotensin (Ang) II and proteinuria, with or without systolic blood pressure elevations. Long‐term RAS blockade when started early (2–4 months of age) prevents intra‐renal RAS activation. The present study assessed the effects of aging and RAS blockade on kidney function in F344 X Brown Norway rats, a model of healthy aging in terms of overall longevity. Rats (n = 6 – 9/group) received daily subcutaneous injections of the ACE inhibitor enalapril (MED, 40 mg/kg; HIGH, 80 mg/kg) or saline (CON) for 6 months starting at 24 months of age; a young group (YO;9 months) was also studied. At 30 months of age, urine volume and body weight were similar among older groups and higher than YO rats. Urinary Ang II was significantly lower in older CON rats relative to YO rats, but did not differ among other groups (HIGH = 6 ± 1; MED = 7 ± 1; CON = 6 ± 1; YO = 11 ± 2 pmol/kg/day). Protein excretion did not increase in the older CON rats relative to YO animals and was not altered by treatment. Therefore, in animals with healthy aging there is a lack of activation of the intra‐renal RAS and no development of proteinuria. Moreover, in the absence of apparent age‐related indices of renal injury, there is no effect of long‐term RAS blockade on these variables. Support: HL51952, AHA‐MA021515U, AG026764, AG10484, AG11370
Greater Renal Sensitivity to Angiotensin II in Rats with a Lower Nephron Number
Authors: Reverte Virginia , Salazar Francisco , Saez Fara , Loria Analia S. , Llinas Maria T , Salazar F. Javier
Published: June 2021

Our group has reported that the blockade of AngII effects during nephrogenic period in rats reduces nephron number by 37%, modifies renal function and elicits the development of hypertension. Our aim was to evaluate whether the renal hemodynamics effects of AngII are enhanced in these rats, and to examine whether these effects are sex‐dependent. Experiments were performed in 3 months old anesthetized rats treated with vehicle or an AT1 receptor antagonist (ARA) (7 mg/kg) during the first 14 postnatal days. The renal hemodynamics response to an AngII infusion (30 ng/kg/min, iv.) were examined in male and female rats. AngII infusion in control males (n=9) leads to a fall in GFR (1,3±0,2 to 1,0±0,1 ml/min/gr kw; P<0.05) and RPF (4.7±0,3 to 3,4±0,2 ml/min/gr kw; P<0.05). Basal GFR and ERPF levels were lower (P<0.05) in ARA‐treated than in control male rats. The renal hemodynamics response to AngII was enhanced in ARA‐treated males (n=9) (P<0.05), since AngII infusion induced a fall in GFR (0.9±0,1 to 0,5±0,1 ml/min/gr kw) and RPF (3,1±0,2 to 1,7±0,2 ml/min/gr kw). The enhancement of the renal hemodynamics response to AngII was similar in male and female ARA‐treated rats. Our results show that the renal sensitivity to AngII is similarly elevated in male and female rats with a reduced nephron number. This greater sensitivity can contribute to the important structural and functional renal changes previously found in these rats.
Acute exercise alters circulating and local tissue renin‐angiotensin systems
Authors: Chung Justin C.S. , Rush James W.E.
Published: June 2021

Although the renin‐angiotensin system (RAS) plays an intricate role in coordinating vascular adaptations to exercise, there is a paucity of information regarding changes to circulating and local RAS associated with acute exercise. Thus, we investigated the modifications to circulating, aorta and kidney RAS in exercised male Sprague Dawley rats. Rats were exercised for 1h and euthanized immediately (IPEx) or 24h post exercise (24PEx, n=10 per group). Compared to non exercised control (CON), plasma ACE activity decreased IPEx, while 24PEx activity decreased further (85.61 ± 3.53 U/L; 75.28 ± 2.87 U/L; 62.03 ± 3.33 U/L, p<0.05 (U = μmol/min)). Plasma angiotensin II levels were increased IPEx (77.04 ± 16.71 pg/mL vs. 151.22 ± 25.42 pg/mL, p<0.05), but 24PEx levels were comparable to CON (81.19 ± 13.62 pg/mL, p>0.05). Compared to CON, aorta ACE activity decreased IPEx and remained blunted 24PEx (0.798 ± 0.03 U/g; 0.661 ± 0.05 U/g; 0.572 ± 0.03 U/g, p<0.05). Aorta ACE protein decreased ∼42% IPEx compared to CON and 24PEx (p<0.05). Kidney ACE activity increased IPEx (CON: 0.526 ± 0.03 U/g; IPEx: 0.648 ± 0.04 U/g; 24PEx: 0.489 ± 0.03 U/g, p<0.05). Upregulation of kidney ACE protein was observed IPEx (+42%) and 24PEx (+77%) (p<0.05). This study highlights changes in systemic and organ‐specific local RAS in response to acute exercise, which may be essential to the beneficial changes observed in chronic exercise.

Funding: NSERC / HSFO
Cardiovascular Effects of L‐glu Microinjection into the Paraventricular Nucleus (PVN) of Unanesthetized Rats
Authors: Busnardo Cristiane , Tavares Rodrigo Fiacadori , Corrêa Fernando Morgan de Aguiar
Published: June 2021

Objective: Our objective was to investigate the cardiovascular effects of the chemical stimulation of the PVN with L‐glutamate (L‐glu) as well as the central and peripheral mechanisms involved. Methods: Male Wistar rats were used. Guide cannulas were implanted in the PVN. A catheter was introduced into the right femoral artery for blood pressure and heart rate recording. Results: Microinjection of L‐glu in 100 nL into the PVN caused dose‐related pressor and tachycardiac responses in unanesthetized rats, with an ED50 of approximately 10nmol. The cardiovascular response to L‐glu into the PVN was blocked by intravenous pretreatment with the ganglion blocker pentolinium, suggesting that this response is mediated by an increase in sympathetic activity. The response to L‐glu was not affected by local microinjection of the selective non‐NMDA receptor antagonist NBQX (2 nmol). The pressor response was not affected, but the tachycardia was converted into bradycardia by local pretreatment with the selective NMDA receptor antagonist LY235959 (2 nmol), indicating the involvement of NMDA receptors in these responses. Conclusion: The results suggest that injection of L‐glu into the PVN activates the sympathetic output and that the tachycardiac response to the injection of L‐glu into the PVN is mediated by NMDA receptors. Financial Support: FAPESP (06/52446‐9).
Purinergic transmission into the NTS mediates cardiovascular adjustments during hypothalamic electrical stimulation
Authors: Korim Willian Seiji , Ferreira‐Neto Marcos Luis , Cravo Sergio Luiz
Published: June 2021

This study sought to determine the role of NTS purinergic receptors in cardiovascular adjustments to hypothalamic defense area electrical stimulation (HDAS). Male Wistar rats were anesthetized and prepared for mean arterial pressure (MAP), heart rate (HR), hindquarter blood flow (HQBF) and vascular conductance (HQVC) recordings. HDAS (150 μA; 0.6 ms; 100 Hz; N=6) evoked hypertension (21±5 mmHg), tachycardia (38±7 bpm) and hindquarter vasodilation indicated by the maximum peak (161±13% of baseline) and the area under the HQVC curve (AUC: 5958±778%). After bilateral microinjections of suramin (10 mM) into the NTS, pressor and HR responses to HDAS were unchanged (35±11 mmHg; 29±6 bpm respectively), whereas hindlimb vasodilation was extensively reduced (peak:71±7%, AUC: 782±180%). In another group (N=5) bilateral microinjections of PPADS (5mM) into the same NTS sites markedly reduced hypertension (16±4 mmHg vs 7±1 mmHg), tachycardia (7±2 bpm vs 2±1 bpm) and hindlimb vasodilation (max: 114±23% vs 28±9%, AUC: 3107±824% vs 145±49%) induced by HS. Basal levels of MAP, HR and HQVC remained unchanged after suramin or PPADS microinjections into the NTS. Our results indicated the participation of NTS different purinergic receptors subtypes in the hindquarter vasodilation during defense reactions.
Acute volume expansion preserves orthostatic tolerance during whole‐body heat stress
Authors: Keller David M , Low David A. , Wingo Jonathan E. , Brothers R. Matthew , Davis Scott L. , Crandall Craig G.
Published: June 2021

Whole‐body heat stress reduces orthostatic tolerance. The mechanisms responsible remain incompletely understood. The purpose of this study was to test the hypothesis that acute volume expansion improves orthostatic tolerance during passive heat stress (Δ core temperature ∼1.5 °C). Seven subjects participated in the following experimental protocol. Orthostatic tolerance was determined on 3 successive days using graded lower‐body negative pressure during normothermia (NT), whole‐body heating (WBH) and whole‐body heating with infusion of a volume expander (Dextran; WBH‐DEX) to restore central venous pressure (measured via a central venous catheter) to pre‐heat stress values. NT and WBH were randomized between Days 1 and 2, while WBH‐DEX was always performed on Day 3. As expected, orthostatic tolerance, expressed as cumulative stress index (CSI, mm Hg • min), was markedly reduced during WBH (182 ± 57 CSI) compared to NT (938 ± 152 CSI, P < 0.0001). Interestingly, WBH‐DEX (1109 ± 152 CSI) not only improved orthostatic tolerance relative to WBH (P < 0.0001), it also preserved tolerance compared to NT (P = 0.47). Therefore, despite marked reductions in orthostatic tolerance due to heat stress alone, acute volume expansion restored orthostatic tolerance similar to that observed during normothermic conditions.

Supported by NIH Grant NIH‐HL61388 & HL84072
Evaluation of 24h blood pressure by telemetry in Yucatan miniature swine: Absence of dipping
Authors: Myrie Semone B , McKnight Leslie L , Van Vliet Bruce N , Bertolo Robert F
Published: June 2021

A “non‐dipping” blood pressure (BP) profile is regarded as an independent risk factor for various cardiovascular events and target organ damage. In the present study, we investigated the dipping phenomenon in Yucatan miniature swine. Piglets (∼1000g, N=6) were weaned at 31 days old and then fed standard chow ad libitum. At 8 mo, BP telemeters (DSI model D70) were implanted via the femoral artery to measure hemodynamics and locomotor activity in unrestrained pigs. Following 8 days of recovery, heart rate (HR) and indices of locomotor activity showed pronounced diurnal variation, both being significantly (P<0.05) greater in the daytime than nighttime (HR: 84.4 vs 75.7 bpm; Activity index: 2.6 vs. 0.5). In contrast, BP indices showed little diurnal variation, the values being unimodally distributed over the 24 h period. The phenomenon of BP dipping was absent, with nighttime BP actually being slightly higher than the daytime BP (mean: 110 vs. 113 mmHg; systolic: 130 vs. 134 mmHg; diastolic: 91 vs. 94 mmHg). During a 7‐day salt challenge (from 0.5 % to 5.0 % salt diet), BP rose from 110 mmHg to 119 mmHg indicating that the pigs were moderately sodium sensitive. However, BP dipping was absent on either diet. Our results demonstrate that in this swine model, BP dipping is absent and unaffected by locomotor activity and/or dietary sodium. (Supported by CIHR).
Role of phosphoinositide‐3‐kinase (PI3K) in the nucleus of the solitary tract (NTS) in the modulation of baroreceptor reflex function in the hypertensive rat
Authors: Zubcevic Jasenka , Polson Jaimie W , Kasparov Sergey , Diez‐Freire Carlos C , Raizada Mohan K , Paton Julian F.R.
Published: June 2021

Previous in vitro studies indicate an angiotensin II (AngII)‐dependent increase in PI3K signaling in the brainstem neurones of the spontaneously hypertensive (SHRs) but not Wistar Kyoto rats (WKYs; Yang & Raizada, 1999; Veerasingham et al. 2005). Here, we chronically blocked PI3K activity in the NTS of SHR by lentiviral overexpression of a dominant negative form of the p85α regulatory subunit of PI3K (DNp85α). Perfusion pressure, heart rate, and activities of the phrenic nerve and lower thoracic sympathetic chain were measured in an in situ arterially perfused rat preparation. Baroreceptors were stimulated by increasing perfusion pressure by 30–50 mmHg before and after NTS microinjection of AngII. Transgene expression and sites of AngII injections were confirmed post hoc. PI3K blockade alone reduced both the cardiac (from −3.1±0.1 to −1.4±0.5 bpm/mmHg) and sympathetic (from −5.7±3.4 to −1.0±0.6 %baseline/mmHg) baroreflex components when compared to the control rats overexpressing GFP in the NTS. AngII (500 fmol) in the NTS reduced the gain of the cardiac baroreflex in both groups to a similar extent. In contrast, the AngII‐mediated depression of the sympathetic component of baroreflex seen in controls (by 65±17%) was abolished in the DNp85α group (15±57%). We conclude that in SHR, AngII via the PI3K signalling modulates the sympathetic but not the cardiac component of the baroreflex. NIH grant #HL33610.
Aldosterone acutely increases the production of reactive oxygen species (ROS) and intracellular calcium concentration [Ca2+]i in neurons of the paraventricular nucleus (PVN): An in vitro study
Authors: Badaue‐Passos Daniel , Xue Baojian , Johnson Ralph Fred , Beltz Terry G , Johnson Alan Kim , Hay Meredith
Published: June 2021

Besides the well known genomic or transcriptional effects of aldosterone (ALDO), non‐transcription‐like (rapidly acting) actions have been recently demonstrated. We investigated the effects of ALDO on changes in [Ca2+]i and ROS production in PVN cells in vitro. Neurons in the PVN were visualized by confocal microscopy. Changes in [Ca2+]i were measured as changes in the fluorescence of Fluo‐4 AM, a [Ca2+]i marker dye. To estimate ROS, we measured the changes in the ethidium fluorescence which is directly proportional to the oxidation of dihydroethidium (DHE) by ROS. Male rats were decapitated and the brains were promptly removed, cooled in an artificial cerebrospinal fluid (aCSF) containing 7.5% sucrose and cut on a vibratome. The cells were loaded with Fluo‐4 (18 μM) or DHE (20 μM) added to oxygenated aCSF (34±0.5°C) for 1h. The slices were then placed in a chamber for microscopic visualization of fluorescence. The chamber was perfused with heated and oxygenated aCSF and ALDO (1 μM) or KCl (60 mM). Three‐dimensional images were obtained every minute for 1h. The perfusion of ALDO increased both [Ca2+]i (Δ=69.9±10.6%) and ROS (Δ=26.0±2.5%) within 15–30 min in the PVN neurons (i.e., KCl responsive cells). The increase in [Ca2+]i suggests that ALDO can acutely activate neurons in the PVN, and the enhanced ROS production implies that ROS may mediate a non‐transcriptional effect of ALDO. (Support: NIH HL‐59676 & HL‐62261)
Effects of fluoxetine on cardiac remodeling and autonomic control of blood pressure and heart rate after myocardial infarction in mice
Authors: Janssen Ben , Debets Jacques , Smits Jos
Published: June 2021

Selective serotonin reuptake inhibitors (SSRIs) are used to treat mental depression after myocardial infarction (MI). It is unknown if SSRIs influence cardiac remodeling after MI. SSRIs may modulate cell proliferation and modify autonomic tone by inhibiting the reuptake of catecholamines. We examined the effects of 2 weeks fluoxetine (FLU) treatment (6 mg/kg.day) on cardiac remodeling in male Swiss mice subjected to coronary artery ligation. 11 days after MI, catheters were implanted and 2 days later blood pressure variability (BPV) and heart rate variability (BPV) were measured (by spectral analysis) in the conscious state as an index of autonomic tone. Compared to non‐treated MI mice, FLU treatment did not alter infarct size, cardiac hypertrophy and cardiac hyperplasia (assessed by BrdU labeling). Basal BP and HR were not altered by FLU treatment. In the absence of treatment low‐frequency BPV and HRV (0.1–1 Hz) were lower in MI than in sham‐MI mice. FLU treatment reduced low‐frequency BPV and HRV in sham and MI mice, reaching values comparable to those observed in non‐treated MI mice. All between group differences in BPV and HRV were eliminated after acute pharmacological blockade with atropine and metoprolol. These data indicate that, in mice, FLU treatment does not aversely modify cardiac remodeling after MI and is associated with a sympatholytic effect.
Gestational administration of glucocorticoid alters baroreceptor and chemoreceptor reflexes in rat
Authors: Polson Jaimie William , Paton Julian FR , Wolf Andrew R
Published: June 2021

In the rat, administration of high levels of glucocorticoid during pregnancy produces late onset hypertension in the offspring. The causes of hypertension are not known, but may involve alterations in cardiovascular autonomic control. We examined baro‐ and chemoreflex changes in heart rate (HR) and sympathetic nerve activity (SNA) in the rat following gestational dexamethasone administration (DEX), using the in situ working heart brainstem preparation.

Pregnant dams were administered DEX (200 μg/kg s.c.) at E15–16. At 3–5 weeks, perfusion pressure, HR, and activity of the phrenic nerve and thoracic sympathetic chain were measured in the in situ preparation of DEX offspring and naive controls. HR and SNA responses to baroreceptor (increase in pressure of 20–30 mmHg) and chemoreceptor (injection of 20–100 μL 0.03% NaCN) stimulation were measured. Baroreflex changes in HR gain (control, 1.3+0.2 v 0.8+0.2 bpm/mmHg) and SNA (16+4 v 6+1 %baseline/mmHg) were lower in the DEX rat than control (P<0.05, n=4), whereas the chemoreflex responses were augmented (bradycardia of 161+30 v 217+15 bpm and sympathoexcitation of 60+14 v 106+13 %baseline, P<0.05, n=4). The results indicate that cardiovascular autonomic reflexes are altered in the DEX hypertension in such a way as to facilitate an increase in sympathetic drive. This may contribute to the ontogeny of hypertension in this model.

Support: British Heart Foundation
A2 noradrenergic neurons inhibit osmoreceptor‐induced pressor responses.
Authors: Freiria‐Oliveira Andre Henrique , Blanch Graziela Torres , Pedrino Gustavo R. , Cravo Sergio L. , Colombari Eduardo , Menani Jose Vanderlei , Colombari Debora S. A.
Published: June 2021

A previous study showed that intragastric (ig) 2 M NaCl load increases mean arterial pressure (MAP) in rats with chronic electrolytic lesions of the commissural nucleus of the solitary tract (commNTS), but not in sham rats, suggesting that commNTS mechanisms inhibit pressor responses to osmoreceptor activation. In the present study we investigated the effects of ig 2 M NaCl load on MAP and heart rate (HR) in rats with lesions of the A2 noradrenergic neurons located in the NTS. Male Wistar rats (280–320 g) were submitted to lesions of DβH‐containing neurons in the commNTS achieved by injections of anti‐DβH‐saporin (12.6 ng/60 nl, n=11). Sham rats received injections of immunoglobulin‐G‐saporin (12.6 ng/60 nl, n = 5). Thirty days after saporin injections, MAP and HR were recorded for 60 min before and 60 min after ig 2 M NaCl load (2 ml). Intragastric 2 M NaCl increased MAP in A2 lesioned rats (14 ± 2 mmHg), but not in sham rats (3 ± 2 mmHg), without changing HR in both groups. A2 lesions produced no change on baseline MAP (112 ± 2 vs. sham: 118 ± 4 mmHg) and HR (341 ± 12 vs. sham: 382 ± 26 bpm) and also on baro and chemoreflex tested previously to ig 2 M NaCl. The results show that lesions of A2 noradrenergic neurons in the NTS facilitate pressor responses produced by increases in plasma osmolality, suggesting that A2 neurons are part of the mechanisms that inhibits osmoreceptor‐induced pressor responses.

Supported by CNPq, FAPESP.
A novel implantable blood pressure telemetry device; Comparison between Data Sciences and Telemetry Research systems.
Authors: Malpas Simon C , Lim Matthew , McCormick Daniel , Kirton Robert , Van Vliet Bruce , Easteal Alan , Barrett Carolyn , Guild Sarah , Budgett David
Published: June 2021

The pending expiry (May 2008) of a Data Sciences (DSI) patent in the area of blood pressure telemetry permits the development of alternative technologies. A key aspect in providing new telemetry systems is a comparison to existing technology. Important aspects include stability of the calibration over time and the ability to capture the pulsitile blood pressure waveform. In a group of 6 rats and 5 rabbits DSI blood pressure transmitters (C40 or D70 models) were implanted in conjunction with Telemetry Research (TR) transmitters. Both systems incorporate a fluid filled catheter of similar dimensions with a biocompatible gel in the tip. The blood pressure waveform was collected via telemetry for up to 2 months after implantation. The signal was sampled at 500 Hz and digitally transmitted to a receiver up to 5 m away The battery of TR transmitter was recharged within the rat using inductive power transfer technology. The pulsitile waveform associated with each heart beat was reflected similarly in all cases although the frequency response of DSI telemeters was limited to ~40 Hz (−3 dB rolloff point). The calibrated offset level between the two transmitters was not more than 5 mmHg at all times over a 2 month period. We conclude that the Telemetry Research blood pressure transmitters offer comparable performance to existing technology but with extra design advantages (rechargeable, co‐housing of animals, greater range).
Chronic Intracerebroventricular Immunoneutralization of the Novel Angiotensin Peptide, Angiotensin‐(1–12), Lowers Blood Pressure in Transgenic (mRen2)27 Hypertensive Rats
Authors: Isa Katsunori , García‐Espinosa Maria Antonia , Arnold Amy C. , Pirro Nancy T. , Tommasi Ellen N. , Chappell Mark C. , Ferrario Carlos M. , Diz Debra I.
Published: June 2021

Angiotensin (Ang)‐(1–12) is a novel peptide that is present in a variety of rat tissues including brain. To identify the role of Ang‐(1–12) in the brain, we treated hypertensive rats with an antibody to Ang‐(1–12). Anti‐Ang‐(1–12)‐IgG or pre‐immune IgG was administrated via intracerebroventricular (icv) cannula connected to an osmotic mini‐pump for 14 days (2.7 microgram/hr, each group) in male adult (mRen2)27 hypertensive rats. The transgenic (mRen2)27 rat over‐expresses the murine Ren2 gene exhibiting elevated brain tissue Ang peptides and hypertension. Immunoneutralization of brain Ang‐(1–12) lowered systolic blood pressure (SBP; 143 ± 7 vs. 170 ± 6 mm Hg, baseline; p < 0.05) with no significant change in heart rate (HR) on days 9–11 of icv administration. In pre‐immune IgG treated animals, there were no significant differences in SBP on days 9–11, but HR was higher than baseline on day 11 (314 ± 15 vs. 268 ± 17 bpm baseline; p < 0.05). Water intake was lower on day 6 in Ang‐(1–12)‐IgG group and there were no differences in urine volume, food intake or body weight during the 2 week‐treatment. These results suggest that endogenous brain Ang‐(1–12) exerts a centrally‐mediated pressor effect. Since Ang‐(1–12) was processed in the serum via ACE to Ang I and then to Ang II (Chappell et al., 2007), we propose that Ang‐(1–12) serves as a precursor to Ang II in brain pathways regulating sympathetic outflow. HL51952, HL56973