bluuebunny/crossref_metadata_2025 huggingface dataset:
Stromatoxin‐1‐sensitive voltage‐gated K+ channels regulate rat urinary bladder smooth muscle contractility
Authors: Chen Muyan , Kellett Whitney F. , Petkov Georgi V.
Published: June 2021

Recent studies report that stromatoxin‐1 (ScTx1), a 34‐amino acid peptide, selectively inhibits the voltage‐gated K+ (Kv) channel members, Kv2.1, Kv2.2, Kv4.2, and Kv2.1/9.3 heteromultimers with high affinity. The objective of this study was to identify if any voltage‐gated Kv channels sensitive to ScTx1 participate in the regulation of rat urinary bladder smooth muscle (UBSM) contractility. Freshly isolated rat UBSM strips and single UBSM cells were studied using molecular, immunological, and physiological techniques. RT‐PCR experiments on isolated UBSM cells show mRNA expression of Kv2.1, Kv2.2, and Kv9.3 but not Kv4.2 subunits (n=3). Protein expression of Kv2.1 and Kv2.2 was further confirmed in single UBSM cells by applying immunological methods with specific antibodies (n=3). Isometric UBSM tension recordings of isolated UBSM strips showed that ScTx1 (100 nM) increased spontaneous phasic contraction amplitude and muscle force by about 20% without any effect on contraction frequency or muscle tone (n=5). The ScTx1 (100 nM) effect on UBSM contraction amplitude was observed even after the UBSM strips were depolarized with 20 mM K+ (n=3). Our studies indicate that Kv2.1 and Kv2.2 members of the Kv family play a key role in shaping rat UBSM contractility. New drugs targeting specific Kv channel subunits in the UBSM may prove useful for overactive bladder.

Supported by NIH DK‐070909 to G.V. Petkov.
NF‐κB inhibition and ischemic preconditioning ameliorate ischemia‐reperfusion injury in dystrophin‐deficient muscles of mdx mice
Authors: Ye Jianfeng , Thomas Gail D
Published: June 2021

Mutations of the dystrophin gene cause Duchenne muscular dystrophy, but the pathogenetic mechanisms are poorly understood. We previously reported that ischemia and reperfusion (I/R) cause excessive injury to dystrophin‐deficient muscles, which is ameliorated by antioxidant treatment. We therefore hypothesized that the enhanced susceptibility of the dystrophin‐deficient muscles of mdx mice to I/R‐induced injury is mediated by activation of the redox‐sensitive transcription factor NF‐κB. Because brief exposure to ischemia induces tolerance to subsequent I/R and is thought to be mediated in part by reactive oxygen species, we also hypothesized that I/R‐induced injury would be reduced by ischemic preconditioning (IPC). In anesthetized mdx and C57BL10 mice, I/R (1 h/2 h) induced greater sarcolemmal damage as reflected by Evans blue dye (EBD) uptake (EBD+ area: 29±3% vs 6±2%; P<0.05) and greater activation of NF‐κB (+115±32% vs +36±16%; P<0.05). I/R‐induced injury in mdx mice was attenuated by pharmacological inhibition of NF‐κB (EBD+ area: 17±3%; P<0.05 vs I/R alone) and by 3 cycles of IPC consisting of 5 min ischemia and 10 min reperfusion (EBD+ area: 11±4%; P<0.05 vs I/R alone). These data suggest that activation of NF‐κB plays a role in the excessive I/R‐induced injury in dystrophin‐deficient muscle and provide support for a protective effect of IPC to ameliorate this muscle damage. Supported by NIH AR051034.
Chronic sustained hypoxia (CSH) alters the function of adenosine A2A receptor in the nucleus of solitary tract (NTS)
Authors: Zhang Weirong , Mifflin Steve
Published: June 2021

Adenosine modulates cardio‐respiratory activity in the NTS via both A1 and A2A receptors. Normal ambient extracellular ADO primarily activates A1 receptors, which have a higher affinity than A2A receptors. However, A2A receptors may be important when extracellular ADO levels increase during hypoxia. To examine how CSH exposure modulates adenosine A2A receptor function in the NTS, whole‐cell recordings of NTS second‐order neurons identified following DiA labeling of the carotid bodies were obtained in a brainstem slice. We tested the effect of A2A receptor agonist CGS 21680 on spontaneous release of glutamate in NTS slices collected from normoxic rats (NORM) and rats exposed to CSH (10% FIO2 for 7 days) by measuring miniature EPSCs (mEPSCs). CGS 21680 (10 nM) increased the frequency of mEPSCs in both NORM and CSH neurons, but the increase was significantly greater in CSH than in NORM (48±3%, n=5 vs 25±6%, n=6, p<0.01). In addition, 4/5 CSH neurons showed significant increase in the amplitude of mEPSCs (12±4%) while only 1/6 NORM neuron showed increase (8%). The results indicate that activation of adenosine A2A receptors increases pre‐synaptic glutamate release in the NTS. Exposure to CSH enhances A2A receptor‐induced pre‐synaptic glutamate release and induces a de novo, post‐synaptic response. This work is supported by AHA BGIA 0865269F and NIH HL41894.
Sympathetic neural responses to lower body negative pressure during early follicular and midluteal phases of the menstrual cycle
Authors: Carter Jason R , Klein Jenna C , Lawrence Johnathan E , Cross Jessica L
Published: June 2021

Sympathetic neural responses to an orthostatic challenge have not been compared during early follicular (EF) and midluteal (ML) phases of the menstrual cycle. We hypothesize an augmented sympathetic neural response to lower body negative pressure (LBNP) during the ML phase of the menstrual cycle. Muscle sympathetic nerve activity (MSNA), mean arterial pressure (MAP), and heart rate (HR) were recorded during progressive LBNP (‐5, ‐10, ‐15, ‐20, ‐30, and ‐40 mmHg; 3 min per stage) in 12 healthy, eumenorrheic women (age 21 ± 1 years). Plasma estradiol (42 ± 6 vs. 112 ± 12 pg/ml; p < 0.001) and progesterone (2 ± 0 vs. 10 ± 2 ng/ml; p < 0.001) were elevated during the ML phase. Resting MSNA (8 ± 1 vs. 11 ± 1 bursts/min), MAP (79 ± 2 vs. 78 ± 2 mmHg), and HR (58 ± 2 vs. 60 ± 2 beats/min) were not different during EF and ML phases. MSNA and HR increased during progressive LBNP (p < 0.001), and the increases in MSNA burst frequency (bursts/min) and HR were similar during both phases. In contrast, increases in total MSNA (arbitrary units) during progressive LBNP were augmented during the ML phase (p < 0.05). Progressive LBNP did not change MAP during either phase. Our results demonstrate an augmentation of the MSNA response to progressive LBNP during the ML phase of the menstrual cycle. These findings suggest that hormonal fluctuations of eumenorrheic women may influence sympathoexcitation during an orthostatic challenge. Supported by NIH (HL‐088689).
The effect of large fluctuations in arterial blood pressure on cerebral autoregulation during heat stress
Authors: Brothers R. Matthew , Zhang Rong , Wingo Jonathan E. , Hubing Kimberly A. , Crandall Craig G.
Published: June 2021

This study tested the hypothesis that passive heat stress attenuates cerebral autoregulation during large swings in blood pressure (MAP). MAP and middle cerebral artery blood velocity (MCAVmean) were continuously recorded over ~6 min during normothermia (NT; body temperature (Tc) = 37.0±0.13 °C) and heat stress (HS; Tc = 38.1±0.14 °C, P<0.001) conditions in 9 healthy individuals. Swings in MAP were induced by oscillatory lower body negative pressure (LBNP) of 70 mmHg (NT) and 20 mmHg (HS) at a frequency of ~0.03 Hz (i.e., 15 s on 15 s off) and ~0.1 Hz (i.e., 5 s on 5 s off). Transfer function gain was calculated as power spectral density of MCAVmean / power spectral density of MAP. At 0.03 Hz LBNP, swings in MAP resulted in smaller swings in MCAVmean during HS relative to NT resulting in a reduced transfer function gain (NT: 0.38±0.4, HS: 0.14±0.06 cm•s‐1•mmHg‐1; P=0.01), which is reflective of improved cerebrovascular autoregulation. At 0.1 Hz LBNP, swings in MAP resulted in similar swings in MCAVmean between NT and HS resulting in similar transfer function gains (NT: 1.5±1.6, HS: 1.0±0.4 cm▸s‐1▸mmHg‐1; P=0.65). Contrary to our hypothesis these data indicate that cerebral autoregulation during large swings in MAP at a low frequency (0.03 Hz) is improved during HS; however, autoregulation is similar between thermal conditions when these swings occur at a higher frequency (0.1 Hz).

Supported by NIH Grants HL61388 & HL84072
HCN channel blockers increase parasympathetic ganglionic transmission in guinea pig airways
Authors: Mazzone Stuart B , McGovern Alice E
Published: June 2021

Hyperpolarization activated cyclic nucleotide gated (HCN) channels regulate neuronal membrane potential. We assessed whether HCN channel blockers (ZD7288 or Cs+) alter vagal control of airway smooth muscle. In anaesthetized, ventilated guinea pigs bilateral electrical stimulation (32Hz, 10 sec) of the vagi produced voltage dependent (1‐12V), atropine sensitive airway contractions. The voltage producing 50% maximum contraction was reduced from 6.0±0.7 in vehicle (n=6) to 3.3±0.7 in ZD7288 (2mg/kg, iv, n=7) treated animals, P<0.05. Vagus nerve‐evoked contractions in vitro were potentiated by 5mM Cs+ (n=8). At stimulus intensities of 10.3±1.3 volts, tracheal contractions averaged 25.4±4.5 and 62.7±12.5 percent maximum before and after Cs+. Cs+ also evoked spontaneous rhythmical airway contractions (onset=17.0±4.9 mins, peak magnitude=29.6±8.2 percent maximal tracheal contraction). Spontaneous contractions were (a) abolished by 1μM atropine (n=3) and 1μM tetrodotoxin (n=3), (b) reversed by the ganglionic blocker hexamethonium (100mM, n=4) and (c) almost absent in tissues following 48 hours in organotypic culture. Preliminary immunohistochemical experiments show that preganglionic neurons in the nucleus ambiguus may express HCN1 and HCN4 channel subtypes. These data suggest that inhibition of HCN channels on vagal preganglionic neurons may increase ganglionic transmission in the airways.
Pregnancy decreases baroreflex gain: role of GABA in the paraventricular nucleus
Authors: Brooks Virginia L. , Page Mollie
Published: June 2021

Pregnancy (P) decreases baroreflex gain (BRG) via a mechanism that includes reduced actions of insulin in the hypothalamus. However, specific brain sites and mechanisms are unclear. The paraventricular nucleus (PVN) influences BRG and contains numerous insulin receptors (IR). Because insulin is inhibitory, we hypothesized that PVN insulin increases BRG by suppression of inhibitory GABA inputs and that loss of this action during P leads to enhanced GABAergic inhibition of BRG. To test this hypothesis, we determined if PVN microinjection of bicuculline (BIC, 60 pmol), to block GABA receptors, increases BRG more in late P than in virgin (V) urethane‐anesthetized rats. P decreased (P<0.05) BRG (in bpm/mmHg: 1.9±0.2, V, n=12; 0.9±0.2, P, n=8) and AP (in mmHg: 98±4, V; 81±5, P). In both V and P rats, PVN BIC increased (P<0.05) AP (in mmHg: by 13±2, V; by 11±3, P) and BRG (in bpm/mmHg: by 2.4±0.9, V; by 0.6±0.1, P); however, the increment in BRG was less in P rats (P<0.05). Use of immunocytochemistry, to further test the hypothesis that insulin acts via inhibition of GABAergic inputs, revealed that IR are in both magnocellular and parvocellular neuronal soma in PVN regions that also contain GABAergic (GAD67) terminals. However, no evidence of colocalization of IR in GABAergic terminals was obtained. Together, these results do not support the hypothesis that P impairs BRG by reducing insulin's action to inhibit PVN GABAergic inputs.
Activation of peripheral delta‐2 opioid receptors increases tolerance to ischemia/reperfusion injury
Authors: Oeltgen Peter R. , Maslov Leonid N. , Brown Stephen A.
Published: June 2021

We investigated the role of peripheral δ2 opioid receptors (OR) in cardiac tolerance to ischemia/reperfusion injury and examined the contribution of Protein Kinase C (PKC), tyrosine kinase (TK), potassium ATP (KATP) channels and the autonomic nervous system in δ2 cardioprotection. Deltorphin II and inhibitors were administered prior to coronary artery occlusion and reperfusion in a rat model. Rats were monitored for the development of arrhythmias and infarct development. Pretreatment with δ2 specific OR antagonist naltriben completely abolished the cardioprotective effects of Deltorphin II. In contrast the selective δ1 OR antagonist 7‐Benzylidenenaltrexone had no effect. The PKC inhibitor chelerythrine and the nitric oxide synthase (NOS) inhibitor L‐NAME (N‐nitro‐L‐arginine methyl ester) also reversed both Deltorphin II effects. The nonselective KATP channel inhibitor glibenclamide and the selective mitochondrial KATP channel inhibitor 5‐hydroxydecanoic acid abolished the infarct‐sparing effect of Deltorphin II. Inhibition of TK with genistein, the ganglion blocker hexamethonium and the depletion of endogenous catecholamine storage with guanethidine reversed the antiarrhythmic action of deltorphin II, but did not change its infarct sparing action. The cardioprotective mechanism of Deltorphin II is mediated via stimulation of peripheral δ2 opioid receptors.
Role of BH4 in resistance to myocardial ischemia in the BN/Mcw vs. SS/Mcw rats
Authors: An Jianzhong , Du Jianhai , Wei Na , Xu Hao , Pritchard kirkwood A , Shi Yang
Published: June 2021

BH4, an essential cofactor for NOS was shown to contribute to ischemia/reperfusion (I/R) injury. Provision of Sepiapterin (Sep), an intermediate in the salvage pathway of BH4, improved cardioprotection. In contrast, inhibiting GTP cyclohydrolase I (GCH1), the rate‐limiting enzyme for BH4 synthesis by 2, 4‐Diamino‐6‐hydroxypyrimidine (DAHP) increased I/R injury. We previously showed SS/Mcw rats were more susceptible to myocardial I/R than BN/Mcw rats due to reduced •NO production. This study is testing if decreased BH4 levels are responsible for the increased susceptibility to I/R injury in SS/Mcw rats, and if Sep supplementation in SS/Mcw hearts or GCH1 inhibition by DAHP in BN/Mcw hearts could increase/decrease resistance to I/R injury, respectively. Hearts were perfused with Sep or DAHP for 40 min before I/R. Cardiac BH4 levels were much lower in SS/Mcw hearts than in BN/Mcw hearts (27.1±2.2 vs. 37.3±5.5 pmols/mg protein). After I/R, BH4 levels were decreased and this decrease was even more in SS/Mcw hearts. Real‐time PCR revealed that GCH‐1 mRNA was transcribed less in SS/Mcw hearts than BN/Mcw hearts. Sep increased recovery of left ventricular developed pressure (RVDP) following I/R in SS/Mcw hearts (from 37.1±4.3% to 54.3±6.4%). DAHP decreased RVDP in BN/Mcw hearts compared to controls (38.2±4.4% vs. 59.6±4.1%). These results indicate that BH4 plays a crucial role in •NO‐mediated resistance to I/R in rat.
Phorbol Esters Stimulate Ubiquitination of NCC By Activating ERK1/2
Authors: Ko Benjamin S. , Cooke Leslie L , Hoover Robert S
Published: June 2021

The sodium chloride co‐transporter (NCC) is the principal salt‐absorptive pathway in the distal convoluted tubule (DCT). We have described a pathway of NCC regulation in which phorbol esters (PE) stimulate Ras guanyl‐releasing protein 1 (RasGRP1), activating ERK1/2 MAPK and decreasing NCC activity. We have also shown that PEs trigger the ubiquitination and internalization of NCC. To test whether these findings are related, we examined the ability of the PE TPA to increase NCC ubiquitination while inhibiting the RasGRP1/ERK1/2 pathway. mDCT cells were treated with 100nM TPA and the MEK1/2 inhibitor U0126 for 15 minutes. Lysis, immunoprecipitation for NCC, and immunoblotting for ubiquitin showed that U0126 prevented any PE‐induced increase in ubiquitination (no change compared to a 493±4% increase in ubiquitination with TPA alone). To examine RasGRP1's role, its expression was silenced using siRNA. These cells showed no increase in ubiquitination compared to RasGRP1 knockdown alone. These data indicate that TPA's ability to enhance NCC ubiquitination is dependent upon activation of RasGRP1 and ERK1/2.
Dual role of the Na+/H+ exchanger isoform 3 for PEPT1‐mediated H+/dipeptide cotransport in native murine intestine
Authors: Chen Mingmin , Singh Anurag , Dringenberg Ulrike , Yeruva Sunil , Wang Jian , Engelhardt Regina , Riederer Brigitte , Manns Michael , Rubio‐Aliaga Isabel , Näßl Anna‐Maria , Soleimani Manoocher , Shull Gary , Daniel Hannelore , Seidler Ursula
Published: June 2021

This study investigates the molecular function of NHE3 in H+‐dipeptide transport regulation in the native intestine. Luminal application of the dipeptide Gly‐Sar resulted in a strong increase in murine small intestinal fluid absorption in vivo, Isc response in chambered jejunal mucosa, and a decrease in pHi in BCECF‐loaded enterocytes in isolated jejunal villi. Genetic ablation of PEPT1 abolished Gly‐Sar‐induced fluid absorption, Isc response, and enterocyte acidification. Genetic ablation, pharmacological or second‐messenger‐mediated inhibition of NHE3 also reduced Gly‐Sar‐induced fluid absorption in vivo and Isc response in vitro, but enhanced enterocyte acidification. Genetic ablation of Slc26a6 also enhanced enterocyte acidification, but did not alter either Gly‐Sar induced fluid absorption or Isc response. The transmembrane proton gradient was irrelevant for sustained H+/dipeptide transport rates, but the luminal Na+ concentration, enterocyte Na+/K+ ATPase activity, and luminal membrane potential were essential determinants. PEPT1 is the only H+/dipeptide uptake pathway, and NHE3 function is essential for sustained H+/dipeptide uptake rates in the native intestine. We speculate that NHE3 is the major Na+‐uptake pathway into enterocytes during dipeptide‐induced volume increase, and therefore essential to the maintenance of membrane potential negativity.
Translocating cell‐impermeable molecules through the plasma membrane of cancer cells
Authors: THEVENIN Damien , An Ming , Andreev Oleg , Reshetnyak Yana K. , Engelman Donald M.
Published: June 2021

We have discovered a novel approach to specifically target tumors based on their acidity. Our method is based on pHLIP (pH Low Insertion Peptide) ‐ a peptide that is soluble at physiological pH and inserts across lipid bilayers as a transmembrane helix at acidic pH. Unlike other strategies, pHLIP can simultaneously target tumors, carry a cargo linked to its C‐terminus and translocate the payload across the plasma membrane. In drug design, a constraint has been to create molecules that are sufficiently hydrophobic and small to traverse membranes (Log P from ‐0.4 to +5.6 and MW of 160 to 480 g.mol‐1). As a result, a broad range of cell‐impermeable molecules is excluded from drug design and discovery. Our goal is to define the properties of cell‐impermeable cargo molecules linked to pHLIP to guide the use of this novel delivery system. In this study, peptides linked to the C‐terminus of pHLIP served as model cargos. These peptides follow a host‐guest model, where single amino acids can be modified to allow the fine adjustment of cargo properties and were designed to be membrane‐impermeable. We show that pHLIP can successfully translocate such membrane‐impermeable cargos (Log P < ‐2.5; MW ≈ 800) across lipid bilayers and cancer cell membranes. Thus, pHLIP can broaden the useful range of polarity and size of potential therapeutic agents as well as targeting them to tumors.

Support: NIH CA133890 and GM073857
Resistance to Theophylline Therapy
Authors: Henkin Robert I. , Schmidt Loren , Velicu Irina
Published: June 2021

In a recent clinical trial theophylline was given to 312 patients with smell loss (hyposmia) and over 50% improved. While all patients exhibited lower levels of parotid saliva cAMP and cGMP prior to treatment lack of improvement among all patients raised questions about drug efficacy. To investigate reasons for response lack analysis of changes in smell function and in parotid saliva cAMP and cGMP resulted in separation of patients into two groups ‐ those who improved in smell function (responders) and those who did not (non‐responders). Analysis of saliva cAMP and cGMP levels in responders demonstrated significant improvement in smell function and significantly higher levels of both cyclic nucleotides above baseline whereas among non‐responders there was little change from baseline in either smell function or saliva cyclic nucleotides. While plasma theophylline increased in both groups plasma theophylline among responders was consistently higher than among non‐responders and significantly higher as theophylline dose increased. These results indicate that some patients with smell loss treated with theophylline are resistant to the drug and may explain some aspects of decreased response to its use in improving smell function. Mechanism(s) of this resistance is unclear but could relate to individual genetic differences, differences in drug metabolism or apoptotic changes related to drug responsiveness.
The N‐terminal tail of the Na‐K‐2Cl cotransporter serves as a regulatory scaffold
Authors: Gagnon Kenneth B.E. , Delpire Eric
Published: June 2021

The Na‐K‐2Cl cotransporter (NKCC1) participates in cell volume maintenance and regulation by mediating the movement of ions and water across the plasma membrane. Our laboratory identified two Ste20p‐like serine/threonine kinases (SPAK/OSR1) which mediate the phosphorylation and activation of NKCC1. In addition to two putative kinase binding motifs, the N‐terminal tail of NKCC1 contains a putative binding motif for protein phosphatase 1 (PP1). Here we show that heterologous expression of PP1 and NKCC1 in Xenopus laevis oocytes reduces hyperosmotically‐stimulated cotransporter activity to the level of water‐injected oocytes. Immunofluorescence microscopy of oocytes injected with PP1 and EGFP‐tagged NKCC1 demonstrate that the phosphatase does not alter membrane expression of the cotransporter. Mutation of key residues in the PP1 binding motif partially ameliorates the inhibition of hyperosmotically‐stimulated NKCC1 in Xenopus laevis oocytes. In vitro phosphorylation experiments demonstrate that both NKCC1 and SPAK are de‐phosphorylated by PP1, as de‐phosphorylation of SPAK is significantly greater when the N‐terminal tail of NKCC1 is present in the in vitro reaction to scaffold the phosphatase and kinase in proximity to one another. Taken together, our data are consistent with the N‐terminal tail of NKCC1 scaffolding the regulatory phosphatase, PP1, in proximity to the activating kinase, SPAK.
Using Fresh Tissue to Teach Anatomy
Authors: Acland Robert D
Published: June 2021

This presentation summarizes the author's experience over 25 years as Director of an active Fresh Tissue Dissection Laboratory in a large academic center. During that time the Laboratory has become an important part of the center's infrastructure. Three of its many roles will be highlighted: 1) it is a center for surgeons to re‐learn anatomy and practice new skills; 2) it enables first year students to understand anatomy as seen in the living body; and 3) it has made possible the creation of a comprehensive anatomy video series.

The presentation will also describe one center's experience with light embalming as a practical alternative to "fresh" tissue; and will explore the several different meanings of the latter term.
Circular permutants of the ribosomes in the cell
Authors: Suzuki Tsutomu , Kitahara Kei
Published: June 2021

Ribosome biogenesis is a highly complex, energy‐comsuming and rapid event in the cell. The transcription of the ribosomal (r)RNAs is coupled to associate with ribosomal proteins, which enables an ordered and hierarchical assembly of the ribosomes. To investigate whether the ordered transcription of rRNA domains is required for the efficient biogenesis of the ribosome, we constructed four 'circular permutants (CPs)' of E. coli rRNAs, in which the original 5' and 3' termini of 16S or 23S rRNAs were genetically connected, and new termini were created in the helix 33 (cp33) of 16S rRNA, or in the helices 45 (CP45), 63 (CP63) or 78 (CP78) of 23S rRNA. We could unexpectedly isolate surviving clones for all CPs that enabled rescue of the E. coli strain (Δrrn) in which all chromosomal rRNA operons were depleted. The result demonstrated that hierarchical assembly from the 5′‐terminal domain of both 16S and 23S rRNAs is not essential for ribosomal formation in the cell. CP78 showed a severe growth phenotype resulted from the assembly defect of the 50S subunit, suggesting that the ordered transcription from the domains IV to V is critical for efficient assembly of the 50S subunit. Growth defect of all CPs was found in the absence of the DEAD‐box RNA helicase DeaD, indicating that DeaD assists efficient assembly of CPs in the cell. The CPs of rRNAs probe a hierarchical and coordinated assembly of the ribosomes in the cell.
Blood pressure, blood flow and oxygenation in the clipped kidney of long‐term Goldblatt hypertensive rats
Authors: Palm Fredrik , Onozato Maristela , Welch William J , Wilcox Christopher S
Published: June 2021

Angiotensin II (Ang II) maintains blood flow (RBF) and oxygenation (pO2) in the clipped kidney of early two‐kidney, one clip Goldblatt hypertensive rats (2K,1C). However, most cases of human renovascular disease are chronic. The involvement of Ang II declines whereas oxidative stress increases during progression of 2K,1C hypertension. We hypothesized that acute administration of drugs to inhibit reactive oxygen species (ROS; tempol), angiotensin converting enzyme (ACE; enalaprilat), Ang II type 1 or 2 receptors (AT1 or AT2; candesartan or PD‐123,319) lower mean arterial pressure (MAP), RBF and pO2 in the clipped kidney of chronic 2K,1C. Twelve months after left renal artery clipping, MAP, RBF and pO2 were measured after acute administration these drugs. MAP of 2K,1C was unchanged by PD123,319, reduced by ACE inhibition and AT1 blockade, and especially by tempol. Only tempol increased RBF and pO2 in clipped kidneys.

In conclusion, only tempol effectively reduces MAP and increases RBF and pO2 in the clipped kidneys in the chronic model of Goldblatt hypertension. These beneficial renal effects of tempol can not be achieved by conventional interventions directed to the renin‐angiotensin system.
Gender Differences of Creatinine Excretion in Mice
Authors: Eisner Christoph Benedikt , Faulhaber‐Walter Robert , Wang Yaohui , Levine Mark , Briggs Josephine , Schnermann Jurgen
Published: June 2021

Previous experiments from our laboratory have shown that about 50% of urinary creatinine excretion is derived from tubular secretion. In the present study we determined gender differences in creatinine excretion and examined the effect of cimetidine on creatinine excretion. Under anesthesia C57 mice were infused with 0.5 % FITC‐inulin at 0.25 μl/min g BW; after 30 min the first of three blood samples (0, 30 and 60 min) was taken while urine was collected in 10 minute periods. FITC‐inulin fluorescence was determined by spectrometer and the creatinine concentration was measured by HPLC in both plasma and urine samples. The difference between the clearances of inulin (Cin) and creatinine (Ccr), the relative secretory component (RSC), was significantly higher in male mice (Cin: 222 ± 30 μl/min, Ccr: 456 ± 69 μl/min, RSC 50 ± 3 %; n = 7) than female mice (Cin: 329 ± 21 μl/min, Ccr: 525 ± 49 μl/min, RSC 35 ± 5 %; n = 8; p < 0.05). Cimetidine (iv bolus of 30 mg/kg BW and infusion at 10 mg/kg BW/h) reduced creatinine secretion in male mice (Cin: 259 ± 42 μl/min, Ccr: 361 ± 65 μl/min, RSC 27 ± 10 %; n = 4; p < 0.05 compared to control), and less so in female mice (Cin: 295 ± 13 μl/min, Ccr: 364 ± 50 μl/min, RSC 20 ± 7 %; n = 6). In both genders plasma creatinine increased when creatinine secretion was blocked. Thus, secretion of creatinine in male mice is twice that in female mice, suggesting a major difference in the expression of the responsible organic transporters.
Norepinephrine Treatment Enhances the Constriction of the Afferent Arterioles to Angiotensin II by Increasing the Calcium Sensitivity
Authors: Lai En Yin , Fähling Michael , Ma Zufu , Källskog Örjan , Persson Pontus B. , Patzak Andreas , Persson A. Erik G. , Hultström Michael
Published: June 2021

Afferent arterioles (Af) are the main site for regulation of renal blood flow. The aim of the present study was to investigate the effect of transient treatment with 10−4 mol/L norepinephrine (NE) for 2 min with 10 min washout on the Ang II concentration response curve, and to analyze the intracellular pathways of this interaction. NE treatment increased the constriction to low concentrations of Ang II (‐4.4 ± 4.2 % vs ‐46.3 ± 15.8 % at 10−11 mol/L Ang II). The increased constriction was not accompanied by a significant increase in calcium signalling (14.4 ± 2.3 nM non‐treated vs 24.6 ± 8.8 nM in NE treated at 10−11 mol/L Ang II). The effect of NE treatment on Ang II constriction was inhibited by both the alpha‐1 blocker prazosin and the alpha‐2 blocker yohimbine. Western blot analysis for phosphorylation of MYPT1 at thr696 and of MLC(20) at ser19 showed an increase of the 10−11 mol/L Ang II induced phosphorylation after NE treatment (MYPT1(thr696): 0.85 ± 0.14 to 1.4 ± 0.5, P<0.05, and MLC(20): 1.3 ± 0.57 to 1.81 ± 0.18, P<0.05). Combination of NE and Ang II 10−11 mol/L did not influence phosphorylation of p38‐MAPK. In conclusion, transient application of NE increases the sensitivity of the Af to Ang II. This is a receptor dependent reaction, and the constriction thus elicited is dependent on calcium sensitization mediated by MYPT1 induced inhibition of MLCP.
Voltage‐dependent calcium channels in human renal arteries
Authors: Hansen Pernille B , Walter Steen , Marcussen Niels , Jensen Boye L , Skott Ole
Published: June 2021

Several types of voltage‐dependent calcium channels (VDCC) Cav1.2 (L‐type), Cav2.1 (P/Q‐ type) and Cav3.1/ Cav3.2 (T‐ type) are expressed in vascular smooth muscle cells from rodents and are important for renal resistance. We examined whether human renal arteries express several types of VDCC and assessed their functional importance. The expression of Cav 1.2, mRNA was demonstrated in all human renal and interlobular arteries tested whereas Cav 2.1, Cav 3.1 and Cav 3.2 only were observed in some patients. Immunolabeling revealed expression of Cav1.2 and Cav2.1 in human arteries. Using a myograph set‐up, the functional importance of different types of VDCC was investigated in freshly isolated human interlobular arteries. High K+ led to a dose‐dependent constriction of the arteries. Nifedipine blocked the K+ (70 mM) induced constriction with an EC50 of 1.2 × 10−8 M. Blockade of T‐type channels by mibefradil inhibited the constriction with an EC50 of 6.8 × 10−7 M. The response was very variable between individual patients. Blocking P/Q‐type channels using ω‐agatoxin IVA (10−8 nmol/L) had no significant effect (14.8 ± 5.0 % decrease) on the constriction.

We conclude that all human renal arteries express functionally significant L‐type, VDCC while there is a more variable presence and significance of P/Q‐ and T‐type VDCC.

Research Support: Lundbeck Foundation, Danish Medical Research Council, Danish Heart Foundation
Myogenic Responses of Mouse Isolated Perfused Renal Afferent Arterioles: Effect of Salt Intake and Reduced Renal Mass
Authors: Lai En Yin , Welch William J. , Wilcox Christopher S.
Published: June 2021

Renal autoregulation is mediated by myogenic vasoconstriction and tubuloglomerular feedback. Since defects may contribute to renal barotrauma in salt‐sensitive hypertension and chronic kidney disease, we tested the hypothesis that the myogenic response is diminished by dietary salt or the reduced renal mass (RRM). Afferent arterioles (Af) were dissected from 1 2/3 nephrectomized RRM mice and sham‐operated controls and perfused during step changes in pressure from 0 to 140 mmHg. The active wall tension (AWT) was the differences with physiologic perfusate, compared to zero calcium + EGTA. The MAP of conscious mice by telemetry (85 ± 4 mmHg) was unaffected by salt intake or RRM. Af developed a linear increase in AWT above a perfusion pressure of 60 ± 6 mmHg without plateau. The slope of AWT vs perfusion pressure defined the myogenic response. This response was similar in mice fed normal or high salt for 3 months (3.5±0.3 vs 3.9 ± 0.5 dynes μcm−1/mmHg, p>0.05). It was unaffected after 3 days or 3 weeks of RRM but was reduced by 28±3% and 60±7% (p<0.05) in the normal and high salt RRM groups at 3 months (2.5 ± 0.3 and 1.6 ± 0.3 dynes·cm−1/mmHg). We conclude that the mouse renal Af develops a linear increase in myogenic tone above ambient perfusion pressure that could safeguard against renal barotrauma. The normal myogenic response is not dependent on salt intake, but is impaired substantially in RRM, especially during high salt intake.
Tumor necrosis factor and Angiotensin II interaction modulates class I HDAC expression in neuronal cell culture
Authors: Agarwal Deepmala , Haque Masudul , Francis Joseph
Published: June 2021

Pro‐inflammatory cytokine (PIC) induced gene expression is an extremely complex process and poorly understood. PIC‐signals have been shown to alter the interactions of Histone deacetylases (HDACs) with specific transcription factors such as NF‐κB, which in turn induces several inflammatory and anti‐apoptotic genes. HDACs play dual roles as repressors and activators of gene expression. In present study, attempt was made to unravel the TNFα‐mediated HDAC regulation in association with the Renin‐Angiotensin system (RAS). Neuronal cell line (NG108‐15) was treated with either rat TNFα or Angiotensin II. In a few groups, cells were pretreated with AT1 receptor blocker Losartan or TNF blocker Etanercept. HDACs mRNA levels were measured by real time RT‐PCR and protein by immunoflourescence. TNFα caused a reduction in mRNA expression of class I HDACs (1,2,3 and 8). Effect of TNFα on all above mentioned HDACs was partially but significantly restored by Losartan. Similarly, Ang II caused depletion of class I HDACs. ETN partially but significantly restored the Ang II induced‐reduced expression of HDACs. However, Losartan or ETN by itself did not cause any changes in HDAC expression. These observations suggest that the interaction between TNF and Angiotensin plays an important role in modulating the expression of class I HDAC's. Grant support: National Heart, Lung and Blood Institute grant HL‐80544 for Dr. Joseph Francis.
Soluble fms‐like tyrosine‐1 (sFlt‐1) is enhanced in response to chronic tumor necrosis factor‐ alpha excess during pregnancy
Authors: Murphy Sydney R. , LaMarca Babbette , Speed Joshua , Keiser Sharon , Cockrell Kathy , Granger Joey
Published: June 2021

Recent studies have shown that an imbalance of pro‐angiogenic factors and the anti‐angiogenic factor, sFlt‐1, exists in preeclampsia. While increases in sFlt‐1 are thought to play an important role in the pathogenesis of hypertension during preeclampsia, the mechanisms that lead to increased production are unclear. Although preeclampsia in women and placental ischemia induced hypertension in pregnant rats is associated with enhanced production of inflammatory cytokines such as tumor necrosis factor‐alpha (TNF‐ a), the role of TNF‐a in the regulation of sFlt‐1 production is unknown. Therefore, the purpose of this study was to determine the effect of chronic TNF‐a excess on plasma sFlt‐1 levels in pregnant rats. Chronic infusion of TNF‐a into normal pregnant (NP) rats significantly increased arterial pressure from 101±2 to 125±1 mmHg. Associated with the hypertension were significant increases in circulating sFlt‐1 (180±5 NP vs 2907±412 pg/ml TNF‐a treated). Moreover, sFlt‐1 production was elevated in cultured placental explants of TNF‐a induced hypertensive pregnant rats (2544±1132 pg/ml) vs NP rats (2189±586 pg/ml). In summary, we report circulating and placental sFlt‐1 to be elevated in response to chronic TNF‐α excess during pregnancy. These data support a potential role for inflammatory cytokines, such as TNF‐α, as an important stimulus for sFlt‐l production during preeclampsia. NIH grant HL51971.
Renal infiltration of T‐lymphocytes is associated with elevated intrarenal angiotensin II (AngII) and the development of hypertension and kidney damage in Dahl salt‐sensitive (SS) rats
Authors: De Miguel Carmen , Das Satarupa , Lund Hayley , Mattson David L
Published: June 2021

This study examined mechanisms by which immune cells participate in hypertension and renal disease development in Dahl SS rats. Increasing dietary salt from low (LS, 0.4% NaCl) to high salt (4.0% NaCl, HS, n=7) significantly increased renal infiltration of T‐lymphocytes (from 8.8 ± 1.2 x 105 to 14.4 ± 2.0 x 105 cells/2 kidneys), increased arterial blood pressure (from 131±2 to165±6 mmHg), increased albumin excretion rate (from 17±3 to 129±20 mg/day), and resulted in renal glomerular and tubular damage. Furthermore, renal tissue AngII failed to suppress in SS rats on HS (LS: 133.7 ± 26.3 pg/g; HS: 134.1 ± 42.3 pg/g). Administration of the immunosuppressive agent mycophenolate mofetil (MMF; 20mg/kg/day) prevented the infiltration of T‐lymphocytes (HS/MMF: 5.4 ± 1.0 x 105 cells/2 kidneys vs. HS/Vehicle: 9.3 ± 1.5 x 105 cells/2 kidneys) and attenuated Dahl SS hypertension and renal disease. Of importance, rats treated with MMF demonstrated a suppression of renal tissue AngII (from 163.4 ± 25.7 to 87.7 ± 9.3 pg/g of tissue; n=6/each) when fed HS. Finally, infiltrating T‐cells contain renin activity (0.9 ± 0.8 ng AngI/ml/hr/106 cells, n=3). These data indicate that infiltrating T‐cells can participate in AngII production and are associated with increased intrarenal AngII, hypertension, and renal disease; suppression of T cell infiltration decreased AngII and prevented Dahl SS hypertension and kidney damage. Supported by HL‐29587.
Effect of testosterone on obesity‐related cardiac hypertrophy and fibrosis
Authors: Wilson Ana K , Winters Beatrice A , Mallet Robert T , Caffrey James L , Carroll Joan F
Published: June 2021

Testosterone and obesity each increase renin‐angiotensin system (RAS) activity. Thus, testosterone may worsen obesity‐related RAS activation, and contribute to cardiovascular dysfunction in obese men. This study determined whether testosterone potentiated effects of obesity on left ventricular (LV) hypertrophy and cardiac fibrosis. Male New Zealand White rabbits were fed a lean or 10% added fat diet. After 12 wks, obese rabbits exhibited increased resting heart rate, systolic blood pressure, plasma renin activity (PRA) and plasma aldosterone. Obese rabbits also exhibited increased LV mass (6.05 ± 0.16 vs. 4.75 ± 0.10 g, respectively, p=0.05) and cardiomyocyte cross‐sectional area (372 ± 19 vs. 305 ± 13 μm2;, respectively; p=0.01) compared to lean rabbits. These hypertrophic effects were attenuated by both castration (5.31 ± 0.26 g; 328 ± 21 μm2) and treatment with the angiotensin type 1 receptor blocker, losartan (5.52 ± 0.23 g; 288 ± 35 μm2). However, castration in obese rabbits did not attenuate obesity‐related increases in PRA or aldosterone. Both castration and losartan in obese rabbits increased matrix metalloproteinase‐2 activity but did not alter tissue inhibitor of matrix metalloproteinase‐1 expression. These results suggest that testosterone contributes to obesity‐related LV hypertrophy and collagen degradation, but does so independently of PRA. Supported by AHA GIA 0655019Y.
Overexpression of Mst1 induces apoptosis but does not impair contraction of surviving myocytes
Authors: Cook Jeffrey R , Sadoshima Junichi , Crystal George J , Kim Song‐Jung
Published: June 2021

Mammalian sterile 20‐like kinase 1 (Mst1) is activated by proapoptotic stimuli, such as ischemia/reperfusion. Our previous study found increased apoptosis and dilated cardiomyopathy without compensatory hypertrophy in mice with cardiac‐specific overexpression of Mst1. The current study evaluated whether defective contractile function of surviving myocytes contributed to the reduced LV function in vivo in Mst1 mice. Findings from 10‐16 wk old Mst1 (n=4) and wild‐type (WT) mice (n = 4) were compared. LV function (ejection fraction; EF) in vivo was assessed with echocardiography. Myocytes were isolated and paced at 1 Hz (37̈¬ C). Intrinsic myocyte function (% contraction) was measured using video edge detection at baseline and during inotropic stimulation with Ca+2 (2, 3, 4 mM) and isoproterenol (10‐8, 10‐7, 10‐6 M). EF was reduced in Mst1 compared to WT (53¡3/43 % vs. 68¡3/42 %, p<0.05). % contraction of isolated myocytes was similar at baseline for Mst1 and WT (3.5¡3/40.2 % vs. 4.1¡¾ 0.3 %, NS). There was also no difference in % contraction of inotropically‐stimulated myocytes from Mst1 and WT. Our findings suggest that impaired contraction of surviving myocytes did not contribute to LV dysfunction in Mst1 overexpressed mice in vivo. Potential causes for this dysfunction include apoptotic loss of myocardium, increased fibrosis, altered loading conditions, and reduced neurohormonal stimulation.
Cardiovascular function and plasma lipid profiles in rats exposed to petroleum products and water contaminated with hydrocarbons
Authors: Anigbogu Chikodi Nnanyelu , Ojo Olumide Alaba
Published: June 2021

Diffuse and chronic exposure to petroleum products and contamination is increasing both in the rural areas and urban areas especially with increased use of petroleum products for power generation in domestic and industrial environment. Acute exposure also occurs following spillages, blowouts, and pipeline vandalisation and tanker accidents. This study therefore investigated the effects of diesel, kerosene, petrol and their water soluble fractions on cardiovascular function and lipid profile and organ weights in experimental animals.

Contaminated water and petroleum products were administered via the oral route to various groups of Sprague Dawley rats for three weeks. The rats were anaesthetised and cannulated for recording blood pressure and heart rate while samples of blood were taken from the venous catheter for plasma lipid profiling.

The results showed that while most petroleum products had no significant effect on systolic blood pressure, the diesel‐water fraction (WSFD) lowered systolic blood pressure (80 ±10.6mmHg vs. 120±4.9mmHg), increased pulse pressure and heart rate (p<0.05). Diastolic pressure was reduced by most fractions (p<0.05). It was also observed that total cholesterol, HDL‐ cholesterol, and LDL cholesterol where lowered while plasma triglycerides increased. The organ weight index was increased. These results suggest that acute exposure to these petroleum products and their water soluble components may affect aspects of cardiovascular function and other organs of the body.

This work was supported by University of Lagos, Postgraduate intramural fund.
TARGETED MODIFICATION OF THE RAT GENOME USING ZINC FINGER NUCLEASES
Authors: Geurts Aron M , Choi Vivian , DeKelver Russell C , Miller Jeffrey C , Zeitler Bryan , Rebar Edward J , Wood Adam , Schilling Rebecca , Kalloway Shawn , Davis Greg , Urnov Fyodor D , Gregory Phillip D , Jacob Howard
Published: June 2021

Targeted modification of the genome has been a holy grail of genetic research involving the laboratory rat. While breakthroughs in stem cell research may lead to methods for homologous recombination‐mediated approaches, we have pursued an approach for generating heritable mutations in rats which does not involve the use of stem cells. Engineered Zinc Finger Nucleases (ZFNs) combine the DNA binding specificity of the C2H2 zinc finger protein motif with a restriction endonuclease to bind DNA with high sequence specificity and introduce a site‐specific double‐strand break (DSB) into the targeted chromosome. Host cellular mechanisms which repair the ZFN‐induced DSB via the non‐homologous end joining (NHEJ) pathway frequently add or subtract sequence information, leading to the introduction of mutations in the investigator‐specified gene. ZFNs have been used to generate mono‐ and bi‐allelic sequence alterations in plants, cultured human and mouse cells, and can be used to generate heritable mutations in zebrafish embryos. Using engineered ZFNs designed to bind to and cleave sequences within a green fluorescent protein (GFP) and endogenous rat genes, we demonstrate specific disruption of target genes in living rat cells in vitro. This technology represents a breakthrough in targeted modification of the rat genome and can pave the way for creating novel genetic disease models with specific genome alterations.
Effects of hydrogen peroxide on neuronal firing in cultured rat celiac neurons
Authors: Wang Xiaohong , Kreulen David L
Published: June 2021

Elevated reactive oxygen species (ROS) and altered patterns of firing associated with reduced potassium currents were found in celiac ganglion neurons from hypertensive rats. In this study, we investigated whether these changes could be mimicked by application of H2O2. Using whole‐cell patch clamp, dissociated celiac neurons from 3‐week old SD rats were divided into three groups based on their action potential (AP) firing patterns in response to a suprathreshold depolarizing current step (100pA; 5sec). Phasic neurons fired several APs and adapted quickly; tonic‐phasic neurons fired several APs and adapted slowly; tonic neurons fired continuously. After a 1 min exposure to 3mM H2O2, 20% of tonic neurons were converted to phasic neurons; 80% of tonic neurons continued to fire tonically but at a reduced frequency. These effects were reversible in 77% of neurons. Tonic‐phasic and phasic neurons showed decreased firing frequency. The amplitude of the sustained potassium current measured at 50mV from a holding potential of ‐70 mV was significantly increased by 21% in these neurons. When catalase (100 kU/ml) was applied intracellularly in the patch electrode, subsequent H2O2 application had no effect. These findings demonstrate that H2O2 acts intracellularly in celiac neurons to reduce neuronal firing frequency and alter firing patterns. These changes in firing properties are associated with increased amplitude of steady‐state potassium currents.
Scapular Anastomoses: the Nature of Vascular Blood Flow Around the Scapula
Authors: Obourn Chelsea Ann , Qureshi Naema , Márquez Samuel
Published: June 2021

Documenting normal variation of neurovascular structures continues to be of great value to the surgeon as they formulate and implement surgical strategies during pre‐operative preparation. Scapular anastomoses (SA) are a network of streams that generate variable arterial patterns of blood supply whose origins are from multiple sources (i.e., dorsal scapular, suprascapular and circumflex scapular arteries). These ensure adequate delivery of nutrients to the 17 scapular muscles. This study examined the arterial supply pattern of 19 scapulas (cadaveric material n=10 from College of Medicine at SUNY Downstate). The pattern of SA saw the majority of our sample (86.7%) exhibiting the typical pattern of branching as described in anatomy texts. An anastomosis of the dorsal scapular artery with an intercostal artery was observed in four specimens. One arterial pattern saw the suprascapular and dorsal scapular branching from the transverse scapular artery. Another variant saw two branches of the transverse cervical artery that looped around the clavicle joining together before bifurcating, with one targeting the trapezius muscle and the other forming the dorsal scapular. This study showed SA to be highly intricate arterial patterns connecting each subclavian and the corresponding axillary artery and allowing blood to flow past the joint regardless of the position of the arm.

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