bluuebunny/crossref_metadata_2025 huggingface dataset:
Safety and tolerability of DepoFoam® encapsulated bupivacaine for epidural nerve block in rats and dogs
Authors: Richard Brigitte M. , Ott Laura R. , Haan Dean , Brubaker Abram N. , Cole Phaedra I. , Nelson Keith G. , Ross Paul E. , Rebelatto Marlon C. , Newton Paul E.
Published: June 2021

Pacira Pharmaceuticals, Inc. is developing DepoBupivacaineTM (DB; bupivacaine extended‐release liposome injection) using multivesicular DepoFoam® technology for prolonged postoperative analgesia. The safety and tolerability of DB by epidural (ED) and "accidental" intrathecal (IT) routes were investigated. A sensitive silver stain method was used to evaluate the spinal cord. Gait abnormalities were recorded for up to 22 days, after dosing on day 1 in dogs. In rats, lethality was observed with bupivacaine solution (Bsol, 0.75 mg; 14/38) related to an exaggerated response to bupivacaine (BUP). No effects were seen with DB alone (1.5 mg). In dogs, limb impairment with Bsol (4/6, 15 mg) or DB (5/6, 45 mg) with 1.5% lidocaine/1:200,000 epinephrine was limited to day 1; no effects were seen with DB alone (45 mg; ED). With Bsol (15 mg), limb impairment was more frequent and longer lasting compared with DB (45 mg; IT). There was no histological evidence of damage to the spinal cord and adjacent tissues at the injection site. Depot effect was evidenced by 18% higher peak plasma concentration of BUP, despite threefold higher dose of DB vs. Bsol (45 vs. 15 mg). Data reported here are the first demonstration of the safety of DB after epidural administration in relevant toxicology species. Based on preclinical data, DB poses no risk beyond that of Bsol.

Research support was provided by Pacira Pharmaceuticals, Inc.
Identification of two potent and selective histamine H4 receptor antagonists with antipruritic activity
Authors: Milicic Ivan , Witte David G. , Miller Thomas L. , Vortherms Timothy A. , Strakhova Marina I. , Manelli Arlene M. , Carr Tracy L. , Coward Marlon D. , Altenbach Robert J. , Liu Huaqing , Brioni Jorge D. , Esbenshade Timothy A.
Published: June 2021

It is widely recognized that histamine is a mediator of itch and recent reports implicate the histamine H4 receptor (H4R) in this response. We have synthesized two novel H4R antagonists, A‐943931 [4‐ ((R) ‐3‐amino‐ pyrrolidin‐1‐yl) ‐6,7‐ dihydro ‐ 5H‐benzo [6,7] cyclohepta [1,2‐d] pyrimidin‐2‐ylamine] and A‐987306 [4‐piperazin‐1‐yl‐5,6,7a,8,9,10,11,11a‐octahydro‐7‐oxa‐1,3‐diaza‐benzo[c] fluoren‐2ylamine] as potent and selective H4R antagonists with high affinity at both human (Kis = 4.6 and 5.7 nM) and rat (Kis = 3.8 and 3.4 nM) receptors, respectively. A‐943931 and A‐987306 competitively and potently antagonize rat H4R agonist‐mediated responses in [35S]GTPγS binding assays (Kbs = 28 and 6 nM) and intracellular calcium mobilization at rat and human receptors (Kbs from 5‐10 nM). When tested in vivo in a mouse model of clobenpropit (10 nM, i.d.) induced scratching, A‐943931 inhibited the scratching response (IC50 = 26 μmoles/kg, i.p.) with significant inhibition (69%) at the highest tested dose (30 μmoles/kg, i.p.). A‐987306 also inhibited the scratching response (IC50 = 0.4 μmoles/kg, i.p.) with (85%) inhibition seen at 30 μmoles/kg, i.p. Our results show that A‐943931 and A‐987306 are potent and selective H4R antagonists with robust antipruritic activity and are useful tools for further exploration of the role of H4R receptors in vivo. Research supported by Abbott Laboratories.
Ccl2 and Ccl3 mediate neutrophil recruitment through induction of protein synthesis and secondary generation of lipid mediators
Authors: Reichel Christoph Andreas , Rehberg Markus , Lerchenberger Max , Berberich Nina , Bihari Peter , Zahler Stefan , Krombach Fritz
Published: June 2021

CC chemokines Ccl2 and Ccl3 have recently been implicated in neutrophil migration. Here, we analyzed the role of endogenously generated lipid mediators for neutrophil recruitment in Ccl2‐ and Ccl3‐elicited inflammation in the mouse cremaster muscle using in vivo microscopy. Stimulation with Ccl2 and Ccl3 induced a significant increase in numbers of firmly adherent and transmigrated leukocytes (> 80% Gr‐1+ neutrophils) as compared to controls, respectively. This increase was significantly attenuated in mice receiving inhibitors of RNA synthesis (actinomycin D) as well as antagonists of PAF (BN52021) and LTB4 (MK‐886). In contrast, leukocyte responses elicited by PAF and LTB4 themselves were not affected by actinomycin D, BN52021, or MK‐886. Conversely, PAF and LTB4, but not Ccl2 and Ccl3, induced shedding of CD62L and increased expression of CD11b on neutrophils as assessed by flow cytometry. Moreover, Ccl2‐ and Ccl3‐elicited leakage of FITC dextran as well as collagen IV remodeling within the venular basement membrane (BM) were completely absent in neutropenic mice. These data demonstrate that Ccl2‐ and Ccl3‐elicited firm adherence and (subsequent) transmigration of neutrophils require protein synthesis and secondary generation of lipid mediators such as PAF and LTB4 which in turn directly activate neutrophils. Thereby, neutrophils facilitate BM remodeling and promote the microvascular leakage.
Outside‐in signaling via LFA‐1 in acute inflammation
Authors: Dixit Neha , Tsuzuki Yuki , Simon Scott I.
Published: June 2021

Circulating neutrophils bind to ICAM‐1 upregulated on inflamed endothelium via high affinity clustered LFA‐1. This results in an outside‐in signal that recruits Src family kinases, Rap‐1 and cytoskeletal elements like Talin and F‐actin to stabilize the high affinity integrin bond with ICAM‐1. We hypothesize that dimerization of the LFA‐1 bond to ICAM‐1 provides a critical submicron configuration to elicit outside‐in signaling critical for rearrangement of cytoskeletal components leading to directed polarization and migration of the neutrophil. Real time imaging of neutrophils rolling and arresting on recombinant ICAM‐1 and E‐selectin elicited colocalization of Rap‐1, Talin and high affinity LFA‐1 upon arrest only on dimeric and not on monomeric ICAM‐1. Calcium flux mediated by ligand induced arrest was markedly higher in neutrophils bound to dimeric ICAM‐1 as compared to the monomer. We conclude that integrin mediated outside‐in signal is dependent on the valency of the LFA‐1/ICAM‐1 bond thereby providing a key gate keeping mechanism necessary for subsequent and efficient cell polarization and transmigration. This work was supported by AI47294 to S.I.S.
Heme Oxygenase‐1 Upregulation Following Prolyl Hydroxylase Inhibition Attenuates Hypoxia‐Induced Microvascular Inflammation.
Authors: Wood John G. , Font Ruben , Moncure Michael , Thomas James H. , Holloway Naomi B. , Blanco V. Gustavo , Copple Bryan L. , Casillan Alfred J. , West Cameron E. , Gonzalez Norberto C.
Published: June 2021

Acute systemic hypoxia (Hx) promotes leukocyte adherence in post‐capillary venules of rats. When animals are chronically hypoxic, the initial microvascular inflammation resolves in part due to upregulation of heme oxygenase‐1 (HO‐1). Inhibition of prolyl hydroxylase (PHD) has been shown to increase hypoxia‐inducible factor‐1 (HIF‐1) levels, which can increase HO‐1 expression. Increased tissue levels of HO‐1 have been shown to attenuate microvascular injury after hemorrhagic shock.

Objective

This study evaluated whether inhibition of PHD attenuated Hx‐induced microvascular inflammation, and if upregulation of HO‐1 was involved.

Methods

Intravital microscopy was used to measure leukocyte adherence in mesenteric venules of anesthetized rats. The PHD inhibitor EDHB was administered in carboxymethyl cellulose (vehicle) s.c. to rats 24 hrs prior to experiments. HO protein levels in mesentery were measured by western blotting.

Results

Hx caused a rapid increase in leukocyte adherence in vehicle‐ but not in EDHB‐treated rats. Superfusion of the mesentery with the HO inhibitor zinc protoporphyrin caused a significant increase in adherent leukocytes during Hx in EDHB‐ but not in vehicle‐treated rats. HO protein levels were significantly elevated in mesenteries of EDHB‐treated rats compared to vehicle‐treated animals.

Conclusion

These results show that inhibition of PHD reduces Hx‐induced microvascular inflammation in part through HO upregulation, and are consistent with a HIF‐1‐dependent mechanism.
The effect of annexin 1 on inflammatory stress in the microcirculation
Authors: gavins felicity n.e. , hughes ellie , buckingham julia
Published: June 2021

The bodies' response mechanisms against inflammation can be influenced by gender and sex hormone status, and can become deregulated and detrimental, leading to tissue damage. This study has investigated the anti‐inflammatory protein annexin 1 and its role in the sexual dimorphism of inflammatory disease.

Male and proestrous female WT (C57BL/6) and male AnxA1 null (ANXA1‐/‐) mice were challenged with lipopolysaccharide (LPS). The mesentery was exteriorised and leukocyte‐endothelial cell interactions were recorded using intravital microscopy.

In both male and female WT mice, LPS caused a significant increase in leukocyte adherence and emigration in venules vs. saline group, but with no difference between sexes. LPS administration had a modest effect in AnxA1‐/‐ mice, with only venular leukocyte rolling being decreased, which may be indicative of the initial phase of inflammation. Further studies in which the time course of the response to LPS is examined are now required. However, our findings fit with previous data concerning the survival rate of AnxA1‐/‐ mice, which is significantly decreased at 24hr, but not 2hr (1).

To conclude, these data suggest that there is no sexual dimorphism in the inflammatory response following 2hr LPS challenge. Furthermore, this inflammatory response is not observed in AnxA1‐/‐ mice.

1. Damazo AS et al., Am J Pathol. 2005;166:1607‐17.
Neutrophil‐derived heparin binding protein (HBP) is an endogenous activator of the kallikrein‐kinin system
Authors: Kenne Ellinor , Soehnlein Oliver , Herwald Heiko , Lindbom Lennart
Published: June 2021

Adhesion of polymorphonuclear leukocytes (PMN) to the endothelial cell (EC) lining leads to release of heparin binding protein (HBP) causing increased vascular permeability. We have previously reported data suggesting that the kallikrein‐kinin system (KKS) is involved in PMN‐evoked plasma extravasation. Plasma prekallikrein (PK), factor XII (FXII) and high molecular weight kininogen (HK) are principal components of the KKS. Activated PK cleaves HK, liberating bradykinin (BK) which triggers EC contraction. Endogenous activators of this system are poorly defined. Here, we investigated the role of HBP in the activation of the KKS. Stimulation of EC monolayers with HBP caused an increase in paracellular permeability that was abolished with BK B1 and B2 receptor antagonists or a peptide displacing HK from the EC surface. Western blot analysis further revealed that HBP is able to induce cleavage of HK on EC. Thioglycollate‐induced pleurisy in the mouse was used to study PMN‐evoked plasma changes in vascular permeability in vivo. Plasma extravasation was markedly increased 4 h after thioglycollate injection. Treatment with BK receptor antagonists or an antibody to HBP significantly reduced the plasma exudation. These results demonstrate a critical role of the KKS in PMN‐evoked increase in vascular permeability. The data suggest that PMN‐derived HBP may constitute an endogenous activator of this system.
Ischemic Preconditioning Protects Mitochondrial Respiratory Function In Complex I With Decreased Free Radical Production
Authors: Lee Daniel Sungkeun , Steinbaugh Gregory , Pfeiffer Douglas , Zweier Jay Louis , Crestanello Juan
Published: June 2021

Mitochondrial respiratory complexes are damaged by ischemia reperfusion injury. Oxygen free radicals (OFR) may be involved in the cardioprotective mechanism of ischemic preconditioning (IPC). The purpose of this study was to determine the effects of IPC on mitochondrial respiratory complex I activity and OFR production.

Rat hearts (n=6/group) were subjected to either A) 30 minutes (min) of equilibration (EQ), 30 min of ischemia (I), and 30 min of reperfusion (R) (CONTROL) or B) 10 min of EQ, three 5 min episodes of IPC, 30 min I, and 30 min of R (IPC group). A third set of non‐ischemic control hearts were also studied (NIC). Mitochondria were isolated at end reperfusion. Respiratory complex I activity was assessed by polarography using specific substrates and inhibitors for complex I in the presence of ADP. Complex I OFR production was measured by Electron Paramagnetic Resonance (EPR) spectrometry. Complex I substrates were glutamate and malate (4.7 mM) and the inhibitor was rotenone (188 nM). Data is expressed as mean±SEM.

Ischemia reperfusion impairs mitochondrial complex I activity and increases complex I OFR production. IPC preserves mitochondrial respiratory complex I activity and decreases mitochondrial free radical generation at end reperfusion.
Activation of AMP‐activated protein kinase protects endothelial barrier against reperfusion‐induced failure
Authors: Assad Muhammad , Arshad Muhammad , Haertel Frauke V. , Aslam Muhammad , Fleming Ingrid , Piper H Michael , Noll Thomas
Published: June 2021

Ischemia‐reperfusion causes capillary leakage leading to edema formation and organ failure. AMPK has been shown to stabilize barrier function of endothelial cells (EC). we hypothesized that targeted activation of AMPK protects EC barrier against reperfusion‐induced failure. Methods and Results: Downregulation of AMPK by siRNA (~80%) leads to disintegration of VE‐cadherin‐mediated cell adhesion structures, alterations in actin cytoskeleton (confocal microscopy) and gap formation (GF: video‐imaging technique) between EC. Exposure of EC to ischemia (40 min, Po2<5 mmHg; pH 6.4) caused an increase in GF and a 3‐fold increase in AMPK activity (AMPK~P: AMPK phosphorylation; Western blot) after 40 min. During reperfusion (40 min, Po2=140 mm Hg; pH 7.4) GF was further increased by 307+ 9 % (P<0.05, n=5) within 40 min. In contrast, AMPK~P declined to basal level within first 10 min. of reperfusion. Addition of AICAR (AMPK activator; 5‐aminoimidizole‐4‐carboxamide riboside) at the onset of reperfusion induced AMPK~P above the endischemic level, VE‐cadherin translocation to cell borders and abolished reperfusion‐induced GF.

Conclusion

AMPK is involved in maintenance of endothelial barrier. Activation of AMPK at the onset of reperfusion abolishes reperfusion‐induced hyperpermeability. Hence, AMPK activation may provide a new therapeutic option to prevent reperfusion‐induced endothelial barrier failure.
Vascular thromboxane generation restrains arteriolar hypoxic dilation in skeletal muscle of obese zucker rats
Authors: Goodwill Adam G. , Hollander John M. , Brock Robert W. , James Milinda E. , Frisbee Jefferson C.
Published: June 2021

We determined if altered vascular PGI2 or TxA2 production with reduced PO2 contributes to impaired hypoxic dilation of skeletal muscle arterioles of obese Zucker rats (OZR) vs. lean Zucker rats (LZR). Mechanical responses were assessed in isolated arterioles following reduced PO2 under control conditions and with pharmacological interventions inhibiting arachidonic acid metabolism, NO synthase, and alleviating vascular oxidant stress. Hypoxic dilation, endothelium‐dependent in both, was attenuated in OZR; NOS inhibition had no impact on dilation in either strain. COX inhibition attenuated hypoxic dilation in LZR and abolished responses in OZR. Treating arterioles from OZR with PEG‐SOD improved hypoxic dilation; the improvement was entirely COX‐dependent. Vascular PGI2 production with hypoxia was similar between strains, although TxA2 production was increased in OZR; a difference that was attenuated by treatment with PEG‐SOD. Both blockade of PGH2/TxA2 receptors and inhibition of TxA2 synthase increased hypoxic dilation in OZR arterioles. These results suggest that a contributor to impaired hypoxic dilation of arterioles of OZR may be increased TxA2 production, which opposes the dilator influences of PGI2. These results also suggest that elevated vascular oxidant stress may contribute to the increased TxA2 production and may blunt vascular sensitivity to PGI2. (NIH R01 DK64668, AHA EIA 0740129N)
Length adaptation of the active length‐tension curve in rabbit detrusor smooth muscle
Authors: Speich John E. , Almasri Atheer M. , Bhatia Hersch , Ratz Paul H.
Published: June 2021

Until the 1990s, the active and passive length‐tension (L‐T) curves for smooth muscle were believed to be static, with single passive tension (Tp) and maximum active tension (Ta) values for each length. However, recent studies have shown that the L‐Ta curves in airway and vascular smooth muscles can adapt to length changes. Our prior studies have shown that the L‐Tp curve in detrusor smooth muscle (DSM) is not static and can shift along the length axis as a function of strain and activation history, a trait we labeled adjustable passive stiffness (APS). In the present study, calcium‐free Tp and peak total tension (Tt) during a maximal KCl‐induced contraction were measured in DSM strips, and Ta was found by subtracting Tp from Tt. Strips were contracted at their passive slack length (Ls, ~3 mm) and then passively stretched to and contracted 5 times at 3‐fold Ls. These contractions produced increasingly greater Ta that approached a steady value. Next, strips were contracted once at 4‐fold Ls, returned to 3‐fold Ls and contracted 5 times. Ta at 3‐fold Ls was reduced after the contraction at 4‐fold Ls, and the 4 subsequent contractions at 3‐fold Ls produced increasingly greater Ta that returned to the initial steady Ta. These results provide evidence that the L‐Ta curve in DSM can adapt to changes in length. Support was provided by the Edwin Beer Research Program in Urology and Urology‐Related Fields of The New York Academy of Medicine.
Neuroanatomical characterization of the phrenic nucleus in adult mice
Authors: Qiu Kai , Michael Lane A , Reier Paul J , Fuller David D
Published: June 2021

Targeted gene delivery to phrenic motoneurons (PhMNs) may be important in transgenic mouse models of respiratory insufficiency including glycogen storage disease and muscular dystrophy. The present study was conducted to determine how the distribution of PhMNs and pre‐phrenic interneurons in the mouse compared with that seen in the rat. PhMNs were retrogradely labeled in adult C57BL/6 mice with cholera toxin (β‐subunit) and a transneuronal tracer (pseudo‐rabies virus; PRV). Both tracers revealed that PhMN distribution was similar to what has been reported in the rat (HG Goshgarian and JA Rafols, J. Comp. Neurol., 1981), with minor differences. Retrograde labeling of PhMNs was most robust in the C3‐C4 segments and 191±9 cholera‐toxin labeled cells were observed on each side. PRV studies also indicated the presence of pre‐phrenic cervical interneurons as reported in rat (MA Lane et al., J. Comp. Neurol., 2008). No dendrites were observed to cross the spinal midline. Anterograde tracing experiments further demonstrated that some projections from the region of the ventral respiratory column in the medulla cross the spinal midline at the level of PhMNs. These data provide an anatomical basis for targeted gene delivery to phrenic motoneurons. Funding: NIH 1R01HD052682‐01A1 (DDF) and NIH RO1 NS054025 (PJR) grants.
Chronic blockade of 5‐HT and NE reuptake alters the timing and patterning of phrenic nerve discharge during eupnea, but not gasping, in arterially‐perfused adult mouse
Authors: Warren Kelly Ann , Solomon Irene C.
Published: June 2021

Previous in vivo and in vitro studies have demonstrated that a long‐term increase in either 5‐HT or NE exerts complex modulatory effects on central respiratory neural control, resulting in a depression of respiratory motor output. The influence of excess endogenous 5‐HT and NE chronically on respiratory motor output, however, requires clarification. Therefore, in an arterially‐perfused adult mouse preparation, we examined phrenic nerve discharge following intraperitoneal (ip) administration for 28‐d of the 5‐HT and NE reuptake inhibitor venlafaxine hydrochloride (VHCL). The results from these experiments were compared with those obtained from a separate group of mice that received ip injections of 0.9% NaCl (vehicle). We found that in mice receiving VHCL, basal phrenic burst frequency was slower by ~45% (P<0.05), with the effects on TI and TE being somewhat variable. Further, post‐inspiratory activity and spurious expiratory bursts were noted. In response to hypoxia/ischemia, we found that both groups exhibited similar acute hypoxic ventilatory responses and ischemia‐induced gasping responses, with gasps exhibiting similar timing and patterning characteristics. These results demonstrate that chronic excess endogenous 5‐HT and NE depress basal respiratory motor output, but exert minimal effects on hypoxic responses. Supported by NS045321
Acid sensing ion and epithelial sodium channels contribute to the metaboreceptor component of the exercise pressor reflex
Authors: McCord Jennifer Lockwood , Tsuchimochi Hirotsugu , Kaufman Marc P
Published: June 2021

The exercise pressor reflex is due to activation of thin fiber afferents within contracting muscle. Acid sensing ion channels (ASICs), which are blocked by amiloride, play a role in stimulating the endings of these afferents during contraction. Recently, amiloride had no effect on the mechanoreceptor component of the reflex, prompting us to determine if ASICs contributed to the metaboreceptor component of the exercise pressor reflex. The metaboreceptor component can be assessed by measuring mean arterial pressure during post contraction ischemia when only metabolic afferents are stimulated. We examined the effect of amiloride (0.5 µg/kg) injected into the popliteal artery on the pressor response to and after ischemic static contraction of the triceps surae muscles in 11 decerebrated cats. Amiloride attenuated the pressor response both to ischemic contraction (before amiloride Δ 32 ± 4 mmHg; after amiloride Δ 19 ± 3 mmHg; P<0.05) and to post contraction ischemia (before amiloride Δ 19 ± 3 mmHg; after amiloride Δ 9 ± 3 mmHg; P<0.05). Saline, however, injected as a control, had no effect on the pressor response to ischemic contraction (before saline Δ 24 ± 2 mmHg; after saline Δ 25 ± 4 mmHg; P=0.99) or to post contraction ischemia (before saline Δ 12 ± 2 mmHg; after saline Δ 10 ± 2 mmHg; P=0.97) in 6 cats. Our findings lead us to conclude that ASICs contribute to the metaboreceptor component of the exercise pressor reflex.µ
Hemodynamics during whole body heating in individuals with type 2 diabetes mellitus
Authors: Sawyer Jessica Rose , Strom Nicholas A. , Kingsley‐Berg Shirley M. , Roberts Shelly K. , Charkoudian Nisha
Published: June 2021

Thermoregulatory increases in skin blood flow in humans are brought about by a combination of cutaneous vasodilation and increased cardiac output. As such, whole body heating (WBH) in humans is associated with increased heart rate and decreased blood pressure compared to normothermic baseline conditions. We hypothesized that the tachycardia and hypotension associated with WBH in resting humans would be diminished in individuals with type 2 diabetes mellitus (T2DM) compared to controls (C) of similar age due to altered autonomic control of cardiac function in T2DM. We measured heart rate (HR; ECG) and beat‐to‐beat blood pressure (Finometer) in 6 individuals with T2DM and 6 control subjects at baseline and during WBH with a water‐perfused suit. Resting HR and blood pressure were similar in C vs. T2DM (HR: C: 59 ± 2; T2DM: 62 ± 4, P > 0.05; MAP: C: 93 ± 7; T2DM: 95 ± 7, P > 0.05). During body heating, heart rate increased significantly in both groups; at peak of WBH, HR was 91 ± 4 in C and 92 ± 1 in T2DM. This represented a change of 32 ± 3 in C and 27 ± 3 in T2DM (P > 0.05).

Our preliminary data suggest a similar increase in HR and decrease in BP during body heating in T2DM when compared with C. This may have implications for clinical applications in the treatment and management of T2DM, particularly with regard to heat exposure and/or exercise.
Mechanisms related to endothelial nitric oxide synthase (eNOS) uncoupling in myocardial ischemia/reperfusion (MI/R)
Authors: Perkins Kerry‐Anne Andreen , Pershad Sailesh , Chen Qian , McGraw Sloane , Adams Jovan , Young Lindon H
Published: June 2021

MI/R results in endothelial and cardiac contractile dysfunction in the presence of polymorphonuclear leukocytes (PMNs). The role of eNOS in reperfusion injury was studied in a rat PMN‐induced MI/R injury model. Isolated hearts subjected to ischemia (20 min) and reperfused (45 min) with activated PMNs were studied. Cell permeable protein kinase C (PKC) epsilon activator (ε+) or inhibitor (ε‐) peptides were used to increase or decrease eNOS activity respectively. Tetrahydrobiopterin (BH4) or dihydrobiopterin (BH2) are cofactors of eNOS that regulate nitric oxide or superoxide (SO) release respectively. The combination of PKC ε+ (10µM)/BH4 (5µM) (n=7) given during reperfusion significantly restored post‐reperfusion cardiac function compared to I/R PMN hearts (n=11, P <0.01) or PKC ε+ (n=6) or BH4 (n=6) by itself. By contrast, the combination of PKC ε+/BH2 (100µM) (n=6) compromised post‐reperfusion cardiac function suggesting that increased eNOS activity compromises cardiac function when eNOS becomes uncoupled by BH2 or can be cardioprotective when eNOS is coupled to BH4. Moreover, the combination of PKC ε‐ (5µM)/BH2 (n=6) is cardioprotective compared to I/R PMN hearts (P <0.01) or BH2 (n=7) by itself suggesting that decreased eNOS activity during reperfusion attenuates SO release from eNOS. This study was supported by NHLBI Grant 2R15HL‐076235‐02A1 and the Center for the Chronic Disorders of Aging at PCOM.
Characterization of Cardiac Stem Cells Derived from Right Atrial Myocardium in Young Patients
Authors: Kaushal Sunjay , Vijayan Kalpana , Patel Shivani , Backer Carl Lewis , Harrington Daniel
Published: June 2021

The adult human heart contains cardiac stem cells (CSCs) that can repair myocardial damage. We examined CSCs in young patients (age 13 days‐9 years) with congenital heart defects (CHD pts) to determine if CHD pts have similar CSCs with respect to CSC markers or regenerative potential. Right atrial appendage biopsy samples were obtained, with parental consent, at routine CHD surgery procedures. Tissues were characterized by immunohistochemistry for expression of CSC markers, minced, and serially plated to obtain an efficient and reproducible CSC isolation method. All heart tissue samples, mean weight 61.3 mgs, generated CSCs despite different patient ages and CHD. We generated 65419.7±502.6 cells/mg of tissue maintained up to passage 4. Flow cytometry analysis demonstrated CSCs were hematopoietic lineage (Lin1), and were positive for c‐kit+ (48±25.5%), representing higher c‐kit+ percentage than reported for adult patients (~20%). No age‐related difference was observed when comparing c‐kit+ cell percentage between patients <1 versus >1 year of age. Immunocytochemical staining showed CSCs expressed alpha‐sarcomeric actin, sarcoplasmic reticulum Ca2+ ATPase‐2 and connexin 43. Despite small tissue samples, CSCs are reproducibly attainable from CHD pts, regardless of age or diagnosis. In future these cells may be used to treat CHD pts to improve myocardial function or engineer cardiovascular structures.
Survival of human red cells (RCS) determined using multiple densities of biotin label
Authors: Matthews Nell I , Mock Donald M , Strauss Ronald G , Burmeister Leon F , Schmidt Robert , Widness John A
Published: June 2021

Anemia is a serious problem in the neonate. To investigate the pathophysiology of neonatal anemia, to assess responses to blood transfusions and therapy, and to comparatively evaluate various sources of RBC and methods for RBC storage, direct measurement of RCS would be useful.

Methods

To develop a method for simultaneous measurements of RCS, the density of biotin label on RBCs (BioRBC) was incrementally increased to produce 4 distinct BioRBC populations. Following reinfusion, blood was sampled over the next 4 mo, and the decline in BioRBCs was determined by flow cytometric enumeration.

Results

BioRBC at reagent concentrations < 81 μg/mL RBC produced superimposable linear disappearance curves (Fig) and normal survival (mean potential lifespan=117±5 d). BioRBC at 243 µg/mL disappeared rapidly and were not suitable for RCS.

Conclusion

RCS of distinct populations of infused RBCs in humans can be measured accurately using enumeration of BioRBC. RCS of different blood sources (e.g., placenta, donor, and autologous) can be simultaneously tracked. Supported by NIH P01 HL046925; Thrasher Research Foundation 02825‐3.
Expression of porcine jejunal excitatory amino acid carrier 1 (EAAC1) and glutamate transporter associated protein 3‐18 (GTRAP3‐18) during postnatal development
Authors: Yang Chengbo , Swanson Kendall , Fan Ming Z.
Published: June 2021

The excitatory amino acid carrier 1 (EAAC1) is expressed in both the small bowel and the central nervous system and is responsible for the homeostasis of the neurotransmitter L‐glutamate. Glutamate transporter associated protein 3‐18 (GTRAP3‐18) is an inhibitory regulator of EAAC1 by affecting its trafficking and through protein‐protein interactions. We examined jejunal EAAC1 protein and mRNA abundances and GTRAP3‐18 protein abundance during the postnatal development in pigs at the ages of d 1, 4, 6, 12, 20, 28 (1‐wk post‐weaning), and 70 (mature gut), respectively. There were linear decreases (P<0.05) in the jejunal EAAC1 and GTRAP3‐18 protein abundances in homogenate, cytosol and the apical membrane, whereas a cubic pattern was observed (P<0.05) in the jejunal EAAC1 mRNA abundance during postnatal growth. Furthermore, EAAC1 protein abundances were correlated with GTRAP 3‐18 protein abundances in homogenate (r=0.92, P<0.01), cytosol (r=0.91, P<0.01) and the apical membrane (r=0.81, P<0.01), respectively. However, the EAAC1 protein abundances in jejunal homogenate, cytosol and the apical membrane were poorly correlated (P>0.05) with jejunal EAAC1 mRNA abundance. These results suggest that there is a postnatal decrease in the jejunal EAAC1 expression likely regulated at the post‐transcriptional level. A parallel decline in GTRAP3‐18 protein abundance may further reduce EAAC1 function in the jejunum during postnatal growth. Supported by NSERC Discovery Program of Canada.
Low dose ouabain regulation of Na‐K ATPase requires NHE‐1 scaffolding properties.
Authors: Khundmiri Syed Jalal , Holthouser Kristine , Mandal Amritlal , Merchant Michael L , Schelling Jeffery , Delamere Nicholas A , Lederer Eleanor D
Published: June 2021

Plasma cardioglycosides (ouabain) levels increase in rat models of hypertension and in humans with heart and kidney failure. Ouabain activates Src, ERK, and Akt, resulting in increased 86Rb uptake and cell proliferation. NHE1 is also activated by Akt, suggesting coordinate regulation of NHE1 and Na‐K ATPase (Na‐K). We hypothesize that ouabain regulates Na‐K through an NHE1 dependent mechanism. In the present study we determined the role of ouabain in NHE1‐mediated pH recovery and regulation of NHE1 and Na‐K association and phosphorylation. Pretreatment of OK cells with 10nM ouabain, 5uM EIPA (NHE1inhibitor), or both inhibited intracellular pH recovery in a bicarbonate‐free buffer. Ouabain (10nM) stimulated Akt phosphorylation, 86Rb uptake, and phosphorylation and association of Na‐K with NHE1. These effects of ouabain were prevented by EIPA. In HKC‐2 cells that lack NHE‐1 expression, 10pM ouabain had no effect on 86Rb uptake and Akt phosphorylation. Transfection with wild type NHE1 but not with a mutant NHE1 that prevents NHE‐1 association with cytoskeleton, into HKC‐2 cells rescued ouabain‐mediated Akt phosphorylation. These results suggest that NHE1 plays a critical role in ouabain‐mediated increase in Na‐K activity and Akt phosphorylation through its scaffolding function. The role of NHE1 in regulation of proximal renal tubule intracellular pH is not clear.

Support from VAMR and AHA
Antibiotic inhibitors of protein synthesis on the ribosome
Authors: Douthwaite Stephen R.
Published: June 2021

Many types of chemically diverse antibiotics inhibit bacterial growth by binding to the ribosome and blocking protein synthesis. The large majority of these antibiotics have a distinct target site on either the 30S or the 50S ribosomal subunit. Thus, these antibiotics are generally specific inhibitors of steps in protein synthesis that are associated with either the 30S subunit (such as mRNA decoding) or the 50S subunit (such as peptide bond formation or controlled GTP hydrolysis). An important exception to this rule is the cyclic peptide drug, capreomycin, which is currently used to treat strains of Mycobacterium tuberculosis that have become resistant to first‐line drug therapy. Capreomycin and related drugs function by binding across the ribosomal subunit interface to simultaneously interact with both the 30S and the 50S, where they block the translocation of tRNA along the messenger RNA. Resistance to many ribosome‐targeting drugs is often conferred by addition of one or more methyl groups to specific nucleotides in the rRNA, and thereby sterically hindering the binding of the antibiotic to its target. Resistance to capreomycin has been analyzed in clinical strains of M. tuberculosis, and the mechanism is exceptional in that it is conferred by loss of natural methylations on the rRNA, rather than by gaining extra methylations.
Physiology Pathway diagrams: new interactive online tools that provide efficient access to genomic and phenomic information through biological pathway analysis.
Authors: Munzenmaier Diane H. , Dwinell Melinda , Petri Victoria , Shimoyama Mary , Jacob Howard J.
Published: June 2021

Aim

The Rat Genome Database (RGD) has been developing tools and resources to aid investigators in the integration of genotype and phenotype data derived from rat research for the past 10 years. The new Physiological Pathway diagrams consist of navigational hubs that allow physiologists to easily link phenotype and genotype data through intuitive mechanistic pathway flowcharts.

Methods

RGD has always contained valuable information for physiologists, however the focus has been primarily on genomic data, making it somewhat challenging for physiologists to navigate. Physiological Pathway diagrams provide a familiar map for the user, including links to relevant genotype and phenotype data through flowcharts depicting related physiological and regulatory mechanisms and pathways. Additionally, pharmacological actions and drug‐gene interactions that influence these pathways are depicted within the diagrams and also link to related phenotype and genotype data. Examples of the types of pathways included in this portal are regulatory and physiological systems such as blood pressure regulation, hematopoiesis and angiogenesis.

Conclusion

The Physiological Pathway diagrams at RGD promise to be effective and user‐friendly ways to access and retrieve physiological and genomic data for researchers who use the rat as a model to study physiological processes and pharmacological modulation of these processes.
Low luminal pH aggravates fatty acid bound albumin induced O2·− production in renal proximal tubular cell
Authors: Souma Tomokazu , Abe Michiaki , Akiyama Yasutoshi , Toyohara Takafumi , Suzuki Takehiro , Shiwaku Hiromi O. , Tanemoto Masayuki , Abe Takaaki , Ito Sadayoshi
Published: June 2021

It is known that fatty acid bound albumin induces oxidative stress in proximal tubular cells. Although luminal pH of proximal tubule decreases according to the reabsorption of bicarbonate, it is not examined well how luminal pH affects the production of superoxide, O2·−. Therefore, we examined the role of luminal pH in the O2·− production.

We applied oleic acid bound albumin(OA‐alb, 15g/L) to the HK‐2 cells, human proximal tubule cell line, and measured the production with dihydroethidium. The production was evaluated as the increasing rate of ethidium intensity under real time fluorescent microscopy. When buffer pH is above 6.6, the production was not significant(O2·− production; 0.15 ± 0.06 unit/sec at pH 6.9 and 0.34 ± 0.15 unit/sec at pH6.6, p=0.297). However, by decreasing buffer pH to 6.4, OA‐alb increased the production to 1.45 ± 0.28 unit/sec (p=0.026 versus pH 6.6). A Na/H exchanger inhibitor, dimethylamiloride blunted the production by 62%.

In summary, the luminal acidic pH, which activates Na/H exchanger through the reduction of intracellular pH, aggravates OA‐alb induced O2·− production in proximal tubular cells.
Cross‐talk between NAD(P)H oxidase and AT1 receptors in the proximal tubules of obese Zucker rats
Authors: Sabuhi Rifat , Siddiqui Athar , Hussain Tahir
Published: June 2021

Abnormal regulation of renin‐angiotensin system and increased level of oxidative stress have been implicated in renal/cardiovascular diseases associated with obesity. Earlier, we have shown that Angiotensin II AT1 receptor activity causes greater antinatriuresis via enhanced stimulation of tubular Na,K‐ATPase (NKA) in obese Zucker rats. The present study investigated the role of NAD(P)H oxidase in the enhanced AT1 receptor function in obese Zucker rats. Lean and obese Zucker rats were given NAD(P)H oxidase inhibitor, apocynin (3.5 μg/kg/min; i.v infusion) and/or AT1 receptor antagonist, candesartan (100 μg/kg; i.v bolus). Pretreatment with apocynin completely abolished candesartan‐induced diuresis/natriuresis in obese Zucker rats. In lean Zucker rats, apocynin alone increased diuresis/natriuresis and had an additive effect on diuresis/natriuresis when given along with candesartan. The blood pressure and GFR remained unchanged during drug treatment indicating a tubular effect. Apocynin treatment abolished and brought the AngII‐stimulated NKA activity below the basal activity in the isolated proximal tubules of obese Zucker rats. The data suggest NAD(P)H oxidase as an antinatriuretic molecule that switches to AT1 receptor signaling in the proximal tubules of obese Zucker rats and thereby contributes to the enhanced antinatriuresis. (NIH‐R01‐DK‐61578; GEAR‐UH)
NAADP contributes to norepinephrine and endothelin‐1‐mediated Ca2+ stimulation in rat renal afferent arterioles
Authors: Thai Tiffany L , Churchill Grant C , Arendshorst William J
Published: June 2021

Nicotinic acid adenine dinucleotide phosphate (NAADP) may be produced by ADPR cyclase residing in acidic vesicles in response to G protein‐coupled receptor (GPCR) stimulation in vascular smooth muscle cells. We tested the hypothesis that NAADP receptors mediate increases in cytosolic Ca2+ ([Ca2+]i ) induced by norepinephrine (NE) and endothelin‐1 (ET‐1) in isolated renal afferent arterioles via a lysosome‐dependent pathway. To test this, we measured [Ca2+]i responses to NE and ET‐1 in individual arterioles of Sprague‐Dawley rats. ET‐1 increased peak [Ca2+]i by 100‐130 nM. This response was attenuated by lysosome disrupters concanamycin A1 (CCA) and bafilomycin A (Baf) to 58 ± 8 and 64 ± 10 nM, respectively (P < 0.05). The NAADP receptor inhibitor NAADP EON discovered molecule 14 (Ned‐14) reduced ET‐1 stimulation to 34 ± 11 nM (P < 0.005). Control NE increased immediate [Ca2+]i 70‐80 nM. CCA and Baf decreased the response to 37 ± 3 and 47 ± 7 nM, respectively (P < 0.05). Ned‐14 decreased NE responses to 25 ± 8 nM (P < 0.005). We conclude that NAADP receptors and lysosomal vesicles contribute to Ca2+ signaling pathways evoked acutely by ET‐1 and NE in renal afferent arterioles.
Tumor necrosis factor‐alpha (TNF‐α) induced natriuresis is prevented by blockade of distal tubular sodium transport in mice.
Authors: Castillo Alexander , Shahid Mohd , Islam Mohammed Toriqul , Majid Dewan Syed Abdul
Published: June 2021

We demonstrated earlier that acute TNF‐α administration in mice resulted in marked natriuresis despite its induction of renal vasoconstriction and hypofiltration. To examine whether this natriuresis is caused by a direct inhibitory action of TNF‐α on epithelial sodium transport, we assessed the renal responses to TNF‐α infusion (0.3 ng/min/g; i.v.) in anesthetized mice (n=6) pretreated with combined administration of amiloride (AM; 17 μg/kg/min; i.v.) and bendroflumethiazide (BZ; 2.8 μg/kg/min, i.v.) that would block distal tubular sodium transport. Renal blood flow (RBF) and glomerular filtration rate (GFR) were determined by PAH and inulin clearances. TNF‐α infusion in AM+BZ pretreated mice caused reductions of 35+8% in RBF (10.2±1.7 to 6.5±1.2 mL/min/g) and 43±6% in GFR (1.02±0.1 to 0.60±0.8 mL/min/g) with a slight decrease in mean arterial pressure (87±4 to 79+3 mmHg) which were similar to those observed in non‐treated mice reported earlier. However, contrary to the natriuretic response observed in non‐treated mice in our earlier study, TNF‐α in these AM+BZ pretreated mice resulted in a marked reduction of 64+9% in sodium excretion (4.4±0.3 to 1.5±0.3 μmol/min/g) with no significant change in fractional excretion of sodium (3.1±0.2 to 2.2±0.7%). These data suggest that TNF‐α induced natriuretic response results from its direct inhibitory action on distal tubular sodium transport.
Membrane and Nuclear Progestin Receptors in Oocyte Maturation of Zebrafish
Authors: Hanna Richard Ned , Daly Sean CJ , Zhu Yong
Published: June 2021

Progestins induce final oocyte maturation in fish and amphibian species, but the identity of the receptor(s) mediating these progestin actions has been a subject of heated debate. We have examined the functions and signaling of zebrafish progestin receptors in both mammalian cell lines and zebrafish germ cells, in order to establish models for studying the physiological actions and molecular mechanisms of nuclear progestin (nPR) and membrane progestin receptors (mPRα and mPRα). Rapid, progestin mediated signaling via inhibition of cAMP and activation of MAPK was observed in mPRα and mPRα transfected mammalian cell lines deficient for nuclear progesterone receptors, while overexpression of nPR increased progestin induced transcriptional activities as measured by a progesterone responsive reporter assay. Immunohistochemical analysis with progestin receptor specific antibodies revealed both similar and distinct localization of mPRs and nPR in the major reproductive tissues of the ovary, testis and pituitary. In the ovary, mPRα and mPRα expression is found at or near the oocyte membrane and in the follicle layer surrounding oocytes, while nPR expression is constricted to the follicle layer. Microinjection of either mPRα or nPR into late stage oocytes significantly accelerated the rate of oocyte maturation, implying a potential role of both receptors in induction of final oocyte maturation. However, the increase in oocyte maturation induced by overexpression of nPR was blocked by the transcriptional inhibitor Actinomycin D, while the mPRα mediated increase was unaffected. Our findings suggest that actions of mPRs and nPR in zebafish oocytes are distinct and mediated by two separate signaling pathways (genomic for nPR and rapid non‐genomic signaling for mPRs).
Matrix metalloproteinase‐2 co‐localizes with titin in cardiac myocytes and contributes to its proteolysis in ischemia‐reperfusion injury
Authors: Ali Mohammad Abdel‐Motaal , Cho Woo Jung , Granzier Henk , Schulz Richard
Published: June 2021

Titin, the largest known protein in mammalian cells, plays a central role as a molecular spring of the cardiomyocyte and thus is an important determinant of cardiac function. Although loss of titin in ischemic hearts has been reported, the mechanism of this is unclear. Matrix metalloproteinase‐2 (MMP‐2) is a protease ubiquitously expressed in cardiomyocytes. We have shown that MMP‐2 is localized to the cardiac sarcomere and proteolyzes specific myofilament proteins in ischemia‐reperfusion (I‐R) injury.

Hypothesis

Titin is an intracellular substrate for MMP‐2 and its degradation during I‐R contributes to cardiac dysfunction.

Results

Immunohistochemistry showed the clear co‐localization of MMP‐2 and titin using an antibody to the Z disc region. In vitro incubation of purified titin with MMP‐2 led to titin degradation which was prevented by inhibiting MMP‐2. Isolated working rat hearts were subjected to I‐R injury (25 min global, no‐flow ischemia followed by 60 min aerobic reperfusion) with or without ONO4817 (MMP‐2/9 inhibitor). Titin in heart samples was analyzed by gel electrophoresis (1% agarose‐SDS) and immunohistochemistry. I‐R caused a loss of titin in the heart. ONO4817 prevented I‐R‐induced titin degradation and improved the recovery of mechanical function.

Conclusion

MMP‐2 localizes to titin at the Z disc region of the cardiac sarcomere and contributes to titin degradation in I/R injury.
Prognostic value of resting pulmonary function in heart failure
Authors: Denzer Dustin L , Sinnett William L , Wilson Ted , Olson Thomas P , Johnson Bruce D
Published: June 2021

Pulmonary function (PF) may be influenced by a deterioration of cardiac function. As such, resting measures of PF may be prognostically useful in heart failure patients (HF). This study examined the prognostic value of resting PF in a cohort of HF. This included 113 patients (enrolled 1999‐2005, EF=28±11%; age=55±12yr; 62% male) followed for 64±34mo with death confirmed via the SSI & Mayo Clinic registry. PF included, forced vital capacity, FVC; diffusing capacity, DLCO; forced expiratory volume, FEV1; alveolar volume, VA. Patients were divided in tertiles according to PF with survival analysis via Log‐rank Mantel‐Cox Test with Chi squared analysis (Chi‐sq). Groups for FVC: 1)=80, 2)81‐97, 3)=98, %pre (Chi‐sq 21.5, p<0.01). Bonferonni correction (BC) suggested differences (diff) between groups 1 & 2 and 1 & 3 (p=0.01) with no diff (ND) between 2 & 3 (p=0.62). Groups for FEV1: 1)=77, 2)78‐95, 3)=96, %pre (Chi‐sq 16.3, p<0.01). BC suggested diff between groups 1 & 2 (p=0.02) and 1 & 3 (p<0.01). Groups for DLCO: 1)=75, 2)76‐90, 3)=91, %pre (Chi‐sq=8.2, p=0.02). BC suggested diff between groups 1 & 3 (p=0.003) with ND between 1 & 2 or 2 & 3 (p>0.05). Groups for VA: 1)=87, 2)88‐97, 3)=98, %pre (Chi‐sq=8.9, p=0.01). BC suggested diff between 1 & 3 (p<0.01) with ND between 1 & 2 or 2 & 3 (p>0.05). In a well defined cohort of HF patients, resting measures of PF appear to be predictive of all cause mortality. NIH HL71478
Cardioprotective effect of bicyclol in isolated rat ventricular myocytes subjected to anoxia and reoxygenation
Authors: Cui Jie , Xue Meng , Xia Qiang
Published: June 2021

Bicyclol is a novel antihepatitis drug with cytoprotective effects. The present study was to determine whether bicyclol played a protective role in myocardial ischemia and reperfusion. Myocytes isolated from adult male Sprague‐Dawley rats by enzymatic dissociation were subjected to 60 min anoxia and reoxygenation for 30 min. Trypan blue was used as the index of the viability of the isolated cardiomyocytes. Reactive oxygen species (ROS) generation and mitochondrial membrane potential were measured spectrophotofluorometrically with the fluorescent dye 2',7′‐dichlorfluorescin‐diacetate (DCFH‐DA) and tetramethyl‐rhodamine ethyl ester (TMRE) respectively. Absorbance at 520 nm was measured in isolated cardiac mitochondria exposed to 200 μM CaCl2 to show the mitochondrial permeability transition pore opening. In the isolated ventricular myocytes, pretreatment with bicyclol (1 μM) for 5 min increased the cell viability, and inhibited the increase intensity of DCFH‐DA and TMRE. The decrease of absorbance at 520 nm evoked by 200 μM CaCl2 was greatly inhibited by bicyclol. But the inhibition effect was attenuated by atractyloside (20 μM), inhibitor of mitochondrial permeability transition pore. The results indicate that bicyclol prevented cell death induced by simulated ischemia and reperfusion and the release of ROS. Bicyclol also inhibited the depolarization of the mitochondrial membrane induced by anoxia and reoxygenation. Bicyclol reduced Ca2+ induced mitochondrial swelling via inhibiting MPTP opening. We get that bicyclol is cardioprotective against ischemia and reperfusion, probably via inhibiting ROS generation and mitochondrial permeability transition pore opening.