bluuebunny/crossref_metadata_2025 huggingface dataset:
Structurally Diverse Positive Allosteric Modulators of the D1 Dopamine Receptor Potentiate G‐Protein and β‐Arrestin‐Mediated Signaling
Authors: Luderman Kathryn D. , Conroy Jennie L. , Free R Benjamin , Southall Noel T. , Ferrer Marc , Aubé Jeffrey , Frankowski Kevin J. , Sibley David R.
Published: November 2023

The D1 dopamine receptor (D1R) is linked to a variety of neuropsychiatric disorders and represents an attractive drug target for the enhancement of cognition. Agents that enhance D1R signaling in the prefrontal cortex may be useful in the treatment of cognitive decline in schizophrenia, Alzheimer's disease and other disorders. Unfortunately, orthosteric D1R agonists have proved problematic as they frequently induce hypotension and exhibit a narrow therapeutic window. An alternative approach is to potentiate D1R activity using allosteric modulation. Hypothetically, a positive allosteric modulator of the D1R may exhibit highly selective actions and exhibit a larger therapeutic window. In order to identify allosteric ligands of the D1R, we implemented a high throughput screen of the NIH Molecular Libraries Program 400,000+ small molecule library. A D1R‐G15 calcium mobilization assay was used to identify library compounds that potentiated an EC20 concentration of dopamine. Initial hit compounds were characterized using endogenous G‐protein (cAMP stimulation) and ®‐arrestin (recruitment) signaling pathways, and parental cell lines were used as negative controls. A pool of compounds that exhibited positive allosteric modulation (PAM) of the D1R were identified and further counter‐screened against other dopamine receptor subtypes to establish subtype selectivity. Two structurally diverse compounds, MLS6585 and MLS1082, with PAM activity at the D1R were selected as lead compounds. These compounds potentiate both G‐protein and ®‐arrestin‐mediated D1R signaling, increasing the potency of dopamine by 5–10 fold for stimulating cAMP accumulation and 4–8 fold and for β‐arrestin recruitment. Both compounds also increased the maximum dopamine‐stimulated responses by 25–30%. Neither compound displayed any intrinsic agonist activity in either assay. Further, the two compounds displayed minimal to no ability to inhibit radioligand binding to the orthosteric‐binding site of the D1R. Both compounds potentiated the agonist activity of dopamine, as well as other full and partial agonists, in a dose‐dependent manner with EC50 values in the micromolar range. Importantly, they showed no ability to potentiate forskolin‐stimulated cAMP accumulation, or β‐arrestin recruitment by other receptor subtypes. MLS1082 and MLS6585 may serve as important scaffolds for the future development of optimized D1R PAMs for in vivo use and the discovery of therapeutic lead compounds.

Support or Funding Information

NINDS Intramural Research Program
Small Molecule Inhibition of MEKK2 Signal Transduction in Cancer Cells
Authors: Noll Jessica , Scott John E. , Cuevas Bruce
Published: November 2023

Most cancer patient deaths are due to secondary tumors called metastases that are frequently resistant to therapy. Effective treatment options for metastatic cancer are very limited; therefore the development of new drugs to treat metastases is necessary to improve cancer patient survival. An enzyme critical to metastases can be utilized as a potential therapeutic target if a corresponding small‐molecule inhibitor can be identified and optimized. Our group has demonstrated that the protein kinase, MEKK2, is required for xenograft breast tumor growth and metastasis. We screened small molecule libraries for potential MEKK2 inhibitors and identified several lead compounds that inhibit MEKK2 in vitro that could be modified as future cancer therapeutics. In this work, we examined how these lead compounds affect MEKK2‐dependent functions of tumor cells in culture and if that effect could be extended as a potential therapeutic. Four analogues of one hit were further developed as potential lead compounds (referred to as #1, #2, 82A, and 527), and then we determined whether exposure to the compounds inhibited MEKK2‐dependent tumor cell functions of ERK5 activation, cell migration, and gene expression. We investigated ERK5 pathway activation by ERK5 electrophoretic mobility shift, and MEKK2‐dependent gene expression was analyzed via luciferase‐based reporter assays of NFκB and AP‐1 activation. A modified wound assay was developed to measure velocity in correspondence to cell migration. We discovered that 10μM 82A significantly reduced MEKK2‐dependent NFκB and AP‐1 reporter activity, blocked MEKK2‐dependent ERK5 phosphorylation, and inhibited breast tumor cell migration. Taken together, our data strongly suggest that the 82A lead compound significantly inhibits multiple known MEKK2‐dependent cellular functions. We conclude that the 82A small‐molecule is a suitable lead molecule that can be developed into novel therapeutic MEKK2 inhibitors for treatment of cancer metastases and other MEKK2‐dependent diseases.

Support or Funding Information

This work was supported by a grant from the National Institutes of Health (NIH) and the American Society for Pharmacology and Experimental Therapeutics (ASPET).
Resveratrol can enhance as well as relax contractions of the rat tail artery. Possible role of endothelial potassium channels
Authors: Peuler Jacob D. , Stom Sayra M. , Phelps Laura E.
Published: November 2023

Last year we reported that resveratrol can directly relax adrenergically‐precontracted rings prepared from the ventral tail artery of the rat (FASEB J. 29, supplement 1, 783.6, 2015). In addition, this effect was no different in proximal (conductance) vs. distal (resistance) portions of the same vessel. Our present aim was to determine if that relaxation could be blocked by agents known to block different subpopulations of potassium (K) channels in arterial smooth muscle. We found that it could not. However, we uncovered a novel unanticipated action which has not been reported by others. In over half of our arterial ring preparations (proximal and distal alike), resveratrol enhanced the magnitude of adrenergically‐induced precontractions beginning well before its more delayed and sustained relaxant effect became apparent. Although transient in duration, this enhancing action lasted nearly two hours at the lower more therapeutically‐relevant test concentrations that we employed. This action provides the first reasonable explanation for previously unexplained increases in arterial pressures observed during acute intravenous administration of resveratrol to animal models of severe ischemic tissue injury, in which marked hypotension is often present and in need of correction. Also unanticipated, this same resveratrol‐induced enhancement of adrenergic contraction was notably inhibited by some of the same K channel blockers (particularly tetraethylammonium and glibenclamide) that failed to influence its relaxant effect. As smooth muscle is not a likely site for such a paradoxical finding, we suspect resveratrol is acting on K‐selective mechano‐sensitive ion channels located in the endothelium where they are thought to participate in release of contracting factors.

Support or Funding Information

Midwestern University Master's Program
Renal Oxygenation and Microcirculation in Relation to Fluid Therapy During Kidney Transplantation in a Porcine Model
Authors: Moeslund Niels , Nielsen Lise Have , Eriksen Jonathan Kunisch
Published: November 2023

The Study is ongoing hence no conclusive results can be described

The objective of the study is to investigate the renal oxygenation and microcirculation in a high volume fluid therapy against an individualized goal directed fluid therapy (IGDT) during renal transplantation in a porcine model.

Background

A significant number of kidney recipients experience complications after transplantation ranging from delayed graft function to obstipation and poor wound healing. Iatrogenic over hydration peri‐ and postoperatively is a suspected culprit. Complications are more common in kidneys from brain dead donors. A pilot study showed that the kidney increase in weight and volume after reperfusion due to edema; it is not known if tissue oxygenation is affected by this.

Methods

The study is a prospective randomized animal study, with four groups: high volume fluid therapy, individualized goal directed fluid therapy each group is subdivided so half receives a low dose norepinephrine.

In order to mimic the clinical situation a brain dead donor model is used. Brain death is achieved by raising the intracranial pressure until it exceeds the systolic BP, this is done by inflating a 60 cc urinary catheter epidural. After four hours the kidneys are perfused with “Bridge to life®” cold storage solution and kept on cold ischemia for 18 hours. Two recipient swine have their native kidneys removed and each get one kidney from the donor. After the transplantation the recipients are monitored under anesthesia for ten hours.

Fluid responsiveness in the individualized goal directed fluid therapy group is determined by change in stroke volume after fluid challenges.

The renal oxygenation and microcirculation are measured using the NF‐BF/OFT/E combined oxygen and the microcirculation probe with the OxyLite™ and OxyFlo™ system from Oxford Optronix. The probes are inserted in the medulla, as it is prone to ischemia if the microcirculatory blood flow is compromised.

Results

The preliminary results indicate that the renal oxygenation is correlated to urine production, with high oxygen tension in kidneys with high urine production. The fluid therapy does not seem to influence on the oxygenation other than by urine production. The edema seems to equal in the high volume and the individualized fluid therapy. The amount of fluid administered in the individualized group is significantly less than the high volume therapy

Preliminary conclusions

Invasive measurements of oxygen and microcirculation is a indicator for the metabolic activity in the kidney, from the preliminary results we can se the metabolic oxygen demand is increased in a low volume fluid therapy due to increased reabsorption.

Support or Funding Information

Novo Nordisk Fondennnfond@novo.dk+45 35276600
Systemic and Microcirculatory Effects of Severe Hemorrhage and Resuscitation Using a PEGylated Carboxyhemoglobin (PEG‐COHb)
Authors: Song Bjorn K , Nugent William H
Published: November 2023

This study investigated the systemic (heart rate, mean arterial pressure) and microcirculatory (vasoactivity, interstitial fluid oxygenation [ISF PO2]) impact of two resuscitation fluids: a novel carbon monoxide/oxygen delivery agent (PEG‐COHb [Sanguinate™] Prolong Pharmaceuticals™, South Plainfield, NJ), and a non‐oxygen carrying colloid volume control (Hextend™; Hospira, Inc, Lake Forest, IL). Male Sprague‐Dawley rats underwent a controlled 45% blood volume hemorrhage at 3.5 ml × min−1 × kg−1 through a cannulated carotid artery and were maintained in this hypovolemic, hemorrhagic shock phase for 30 minutes. Resuscitation fluids were infused at a rate of 3.5 ml × min−1 × kg−1 to a volume equal to 20% of the total estimated blood volume through a cannulated jugular vein. Systemic measurements were recorded via a cannulated femoral artery that was connected to a pressure transducer (MP150; Biopac Systems, Inc, Goleta, CA) while microcirculatory parameters were collected through phosphorescence quenching and intravital microscopic examination of the exteriorized spinotrapezius muscle. Compared to baseline, the 45% hemorrhage produced a significant reduction in heart rate, blood pressure, arterial diameter and ISF PO2 in all animals. Resuscitation with either PEG‐COHb or Hextend™ animals improved the systemic parameters towards baseline, but only PEG‐COHb‐treated animals showed an improvement in ISF PO2. The impact of improved systemic variables was evident in mortality with untreated animals (sham) expiring an hour after hemorrhage, while Hextend resuscitated animals expired after 4 hours. PEG‐COHb animals survived for the entire 8 hour observation period. In addition to extended survival times, PEG‐COHb animals showed steady systemic and microcirculatory parameters. Hextend™ resuscitated animals, however, developed rigor mortis in their limbs at approximately 3 hours post‐hemorrhage, which was concurrent with a rapid decline in systemic variables. These results demonstrate that while reestablishing blood pressure is acutely important for post‐hemorrhage survival, restoring oxygen delivery to peripheral tissues is critical for improving long‐term outcomes.

Support or Funding Information

Support: Prolong Pharmaceuticals™
Evaluation of Stem Cell Fate in Cultured Microvascular Networks
Authors: Azimi Mohammad Sadegh , Strong Amy , Dutreil Maria , Fishel Ryan , Bunnell Bruce , Murfee Walter Lee
Published: November 2023

In vitro models of angiogenesis are valuable tools for understanding the underlying mechanisms and the pre‐clinical evaluation of therapies. Our laboratory recently introduced the rat mesentery culture model as a new tool for investigating the mechanistic cell–cell interactions at specific locations across intact blood and lymphatic microvascular networks ex vivo. One potential application of the model is its use to screen the fate of exogenously delivered stem cells. The objective of this study was to examine the effect of aging on exogenously delivered human stem cell differentiation into pericytes along capillaries using angiogenesis in cultured microvascular networks. DiI‐labeled stem cells were seeded onto the harvested adult Wistar rat mesenteric tissues and cultured in alpha‐MEM+1% serum for up to five days according to four experimental groups: 1) adult human adipose‐derived stem cells (hASCs), 2) aged hASCs, 3) adult human bone marrow‐derived stem cells (hBMSCs), and 4) aged hBMSCs. Lectin labeling of endothelial cells on different time points enabled simultaneous visualization of blood vessels. Angiogenesis per experimental group was supported by observation of increased vessel density and capillary sprouting. For each tissue per experimental group, a subset of cells was observed in typical pericyte location wrapped along blood capillaries (Adult hASC=4.96±1.58; Aged hASC=4.51±1.07; Aged hBMSC=20.63±5.46; Adult hBMSC=7.13±1.73). Stem cell differentiation into pericytes was supported by the adoption of typical elongated pericyte morphology along endothelial cells and positive NG2 labeling. The percentage of cells in pericyte location was significantly increased for the Aged hBMSC group compared to the Adult hBMSC and hASC groups. Our results highlight the potential effect of aging on stem cell type specific differentiation during angiogenesis and showcase the application of rat mesentery culture model for evaluating stem cell fate.
Transient exposure to Serotonin + L‐NAME activates early processes mediating inward remodeling of cremasteric arterioles, in vivo
Authors: Foote Christopher
Published: November 2023

Hypertension and other cardiovascular pathologies are associated with inward remodeling of the resistance vasculature. It has been demonstrated ex vivo that prolonged (4 hr) exposure to vasoconstrictor agonists, is sufficient to induce remodeling via mechanisms that include both the polymerization of actin and the activation of transglutaminases. Here we report that in vivo, a brief, 10 min exposure to serotonin (5‐Hydroxytryptamine, 5‐HT) + Nω‐nitro‐L‐arginine methyl ester hydrochloride (L‐NAME) induces inward remodeling of rat cremasteric feed arterioles (100–200 μm lumen diameter), absent changes to systemic blood pressure. This process was blocked by the addition of the transglutaminase inhibitor, cystamine. Furthermore, we demonstrate ex vivo, that 5‐HT + L‐NAME activates transglutaminases and increases the phosphorylation state of cofilin, a negative regulator of actin polymerization. In addition, we demonstrate a new approach to examine actin polymerization dynamics in live vessels. We show that in isolated functional arterioles, the ratio of filamentous actin (labeled with silicon rhodamine jasplakanolide) to globular actin (labeled with DNAse1‐488) increases in individual smooth muscle cells exposed to 5‐HT + L‐NAME. We also report that, in isolated arterioles, 5‐HT + L‐NAME induced remodeling that is blocked by inhibiting Lim Kinase, an enzyme that phosphorylates cofilin and positively regulates actin polymerization. These results are the first to examine the remodeling process as it unfolds in vivo in real time, and further bolster the hypothesis that both actin polymerization and the activation of transglutaminases mediate early stages of the transition from a structurally normal vessel to an inwardly remodeled one.

Support or Funding Information

NIH HL‐088105 to LAM, 1PO1HL095486 to GAM
Metaboreflex Contribution to Peak Pressor Response Achieved During Fatiguing Intermittent Static Exercise Is Attenuated in Postmenopausal Women
Authors: Barrett Megan A , Kim Jin‐Kwang , Garvin Nathan M , De Souza Mary Jane , Pawelczyk James A , Proctor David N
Published: November 2023

Postmenopausal women have been shown to experience greater increases in blood pressure during static exercise than their premenopausal counterparts. However, the mechanisms by which this augmented pressor response is achieved remains incompletely understood. As a result, our study sought to determine the relative contribution of the muscle metaboreflex to the peak blood pressure response during fatiguing, intermittent static exercise with graded occlusion in pre and postmenopausal women. In this study, we hypothesized that the muscle metaboreflex would provide a larger contribution to the peak pressor response in postmenopausal women than in premenopausal women. Eighteen healthy women participated in this study (8 postmenopausal women, between 57–64 years; 10 premenopausal women, between 20–28 years). On the experimental day, after quiet rest, subjects were asked to squeeze a handgrip device (Stoelting Co.) at a rate of 30 contractions/minute at 10% of their maximum voluntary contraction (MVC). Following a 4‐minute free flow exercise period, a blood pressure cuff on the subject's upper arm was progressively inflated at a rate of 20 mmHg/minute. Subjects continued to exercise until volitional fatigue. Blood pressure was recorded continuously on the contralateral hand (Finometer). As expected, postmenopausal women had a significantly higher systolic blood pressure at fatigue compared to premenopausal women (DSBP from rest of 40 mmHg and 24 mmHg, respectively) (p = 0.005). Consistent with our working hypothesis, postmenopausal women exhibited a metaboreflex mediated blood pressure response that tended to be greater than that in premenopausal women (DSBP from rest of 27 mmHg and 20 mmHg, respectively)(p = 0.11). However, this response contributed significantly less to the peak blood pressure response in postmenopausal women than it did in premenopausal women (SBPpeak – SBPPECA of 13 mmHg and 4 mmHg, respectively) (p = 0.037). These findings suggest that while postmenopausal women exhibit a greater metaboreflex mediated blood pressure response compared to premenopausal women, the response contributes less to the peak blood pressure response in postmenopausal women. This suggests that postmenopausal women may be less reliant on the metaboreflex to generate the peak blood pressure response during fatiguing small muscle mass exercise than their premenopausal counterparts.

Support or Funding Information

Penn State Ambulatory Research Fund (Jaffe Endowment)
The early postmenopausal phase is associated with a decline in vascular function: is the prostanoid system to blame?
Authors: Nyberg Michael , Egelund Jon , Nielsen Mads Bernhard , Mogensen Alexander , Hellsten Ylva
Published: November 2023

Menopause is associated with a decline in vascular function; however, whether this is an effect of age and/or menopause and the mechanisms underlying this decline remains unclear. We examined the vascular response to femoral arterial infusion of vasoactive compounds in 20 pre‐and 17 recent postmenopausal women of similar age (49±2 vs. 53±2 years). The postmenopausal women had not experienced a menstrual cycle during the previous 12 months but were less than 5 years past their final menstrual period (2.9±0.6 years). The change in leg vascular conductance (LVC) in response to infusion of the endothelium‐dependent vasodilator acetylcholine (ACh) was lower (~25%, P<0.05) in the postmenopausal compared to the premenopausal women. Co‐infusion of ketorolac to inhibit prostanoid formation reduced the vasodilator response to ACh to a similar extent in the two groups of women. Despite the similar effect of ketorolac, the change in LVC with infusion of the prostacyclin analog epoprostenol was lower (~40%, P<0.05) in the postmenopausal compared to the premenopausal women. These findings suggest that vascular function is adversely altered already within the first years following menopause. The early onset of this altered function indicates that this change is more related to menopausal status than aging per se. The reduced endothelial function does not appear to be related to an altered function of the prostanoid system despite a reduced vascular sensitivity to prostacyclin.

Support or Funding Information

Financial support for the study was provided by the faculty of SCIENCE at University of Copenhagen.
Quorum Sensing And Its Control
Authors: Bassler Bonnie L.
Published: November 2023

Bacteria communicate with one another via the production and detection of secreted signal molecules called autoinducers. This cell‐to‐cell communication process, called “Quorum Sensing”, allows bacteria to synchronize behavior on a population‐wide scale. Behaviors controlled by quorum sensing are usually ones that are unproductive when undertaken by an individual bacterium acting alone but become effective when undertaken in unison by the group. For example, quorum sensing controls virulence factor production, biofilm formation, and the excretion of public goods such as enzymes that solubilize solid food sources making them accessible for consumption. We developed small molecule quorum‐sensing agonists and antagonists to discover the principles underlying the exquisite selectivity quorum‐sensing receptors have for their cognate ligands. Our results suggest mechanisms bacteria use in the wild to ensure the proper ligand has interacted with its partner receptor prior to eliciting signal transduction. We suggest that, in their native environments, bacteria encounter mixtures of autoinducers produced by other species occupying the same niche. Precise autoinducer discrimination enables a particular species of bacteria to respond exclusively to its own signal even in the face of fierce competition. This ability prevents the leakage of benefits of quorum‐sensing‐controlled public goods to non‐kin. Beyond learning about fundamental principles underlying quorum sensing, another use for our synthetic molecules is to control quorum sensing on demand. Indeed, our most potent quorum sensing antagonist protects animals from quorum‐sensing‐mediated killing by pathogenic bacteria and prevents biofilm formation in model microfluidics chambers that mimic medical devices. These results validate the notion that targeting quorum sensing has potential for antimicrobial drug development.
AMELIORATIVE EFFECT OF GALLIC ACID ON DOXORUBICIN INDUCED CARDIOTOXICITY IN ADULT WISTAR RATS
Authors: Adejumobi Olumuyiwa A , Orotusin Oyetola , Oyagbemi Ademola , Adedapo Adeolu , Olaogun Charles , Omóbòwálé Temidayo Olutayo
Published: November 2023

Doxorubicin is a potent antineoplastic anthracycline drug however, its use is greatly limited by its toxic side effects which occurs by formation of reactive oxygen species that disruption of cell function. This present study was conducted to assess the possible cardioprotective effect of gallic acid against doxorubicin‐ induced cardiotoxicity in rats.

In this study, sixty (60) male Wistar rats were divided into six groups. Group A was the control. Group B rats was intoxicated with Doxorubicin (Dox) at 15 mg/kg body weight i.p. Group C pre‐treated with 60 mg/kg Gallic Acid (GA) orally for 7 days prior to administration of Dox 15 mg/kg, Group D pre‐treated with 120 mg/kg Gallic Acid (GA) orally for 7 days prior to administration of Dox 15 mg/kg, Group E received 60 mg/kg Gallic Acid (GA) orally alone for 7 days and Group F received 120 mg/kg Gallic Acid (GA) orally alone for 7 days. The parameters of study were cardiac antioxidant status, serum biomarker of cardiac damage and electrocardiography measurements.

Exposure of rats to doxorubicin led to a significant (p < 0.05) decrease in cardiac glutathione peroxadise (GPx), glutathione‐S‐transferase (GST), reduced glutathione (GSH) content, superoxide dismutase (SOD) and catalase (CAT) activity. Similarly, a significant (p < 0.05) increase in protein thiol was recorded in doxorubicin treated rats while it was reduced in those pre‐treated with GA. Dox also caused elevation of Creatinine kinase‐muscle brain (CK‐MB) and Lactate Dehydrogenase (LDH). Histopathology of rats exposed to Dox showed focal areas of infiltration by inflammatory cells and degeneration of myofibres. Electrocardiography results showed a significant decrease in heart rate; increase in P wave duration, R‐amplitude, PR interval, QRS and QT‐segment prolongation.

Pre‐treatment with GA significantly alleviated Dox associated ECG abnormalities, restored the antioxidant enzyme and decreased serum Creatinine kinase‐muscle brain CK‐MB and LDH. However, administration of GA alone in a set of groups led to a significant prolongation of QT interval which could predispose to the potentially fatal ventricular tachycardia, torsades de pointes. All these findings confirm the efficacy of GA against Dox induced cardiotoxicity but caution must be taken in its use.

Support or Funding Information

None
Renal Salt and Water Homeostasis: a New Regulatory Pathway Involving the Ser/Thr Kinase PIM‐3
Authors: Spirli Alessia , Ronzaud Caroline , Debonneville Anne , Staub Olivier
Published: November 2023

Since many years now, scientists believe that a great credit for the blood pressure regulation goes to the kidneys, as their inability in maintaining salt balance and water homeostasis can result in the development of hypertension, the most common disease in the human population. Aldosterone is the key hormone in the control of sodium balance, blood volume and blood pressure, acting in the aldosterone‐sensitive distal nephron (ASDN) and stimulating a complex transcriptional, translational and cellular program.

We have carried out a gene expression profiling in a mouse cortical collecting duct cell model (mpkCCD), stimulated or not by aldosterone and identified the PIM‐3 Ser/Thr kinase as a novel aldosterone‐induced protein. PIM kinases (PIM‐1, ‐2 and ‐3), and in particular PIM‐3, are overexpressed in different tumors and are known to be regulators of apoptosis, cell proliferation and cell metabolism. PIM‐3, the PIM kinase family member most expressed in the kidney, has similar substrate specificities to other Ser/Thr kinases (e.g. SGK1, PKB), and despite its circadian expression in several organs including the kidney, its renal role is largely unknown.

Here we studied a possible new role of PIM‐3 in the regulation of renal salt and water handling, both in vitro (mCCDcl1 cells) and in vivo (PIM‐3 KO mice). In mCCD cells, we confirmed that PIM‐3 expression is stimulated by aldosterone in a dose dependent manner and observed that shRNA‐based suppression of PIM‐3 reduces the aldosterone‐mediated sodium currents as well as the activation of the aldosterone‐mineralocorticoid receptor pathway. In PIM‐3 KO mice, we found decreased body weight, increased circulating renin activity and elevated plasma aldosterone levels compared to control littermates. Moreover, challenging WT and KO mice with standard, low Na+ or high‐Na+ diet, we observed a tendency of impaired expression of the most important electrolytes transporters in case of PIM‐3 deficiency, leading to a different sodium and water handling between WT and KO mice.

In conclusion, our data suggest a possible novel function of the PIM‐3 kinase and its potentially important role in the control of sodium and water homeostasis to finely regulate blood pressure.
Dose‐dependent effects of Angiotensin‐(1–7) and Angiontensin II on the NHE3 exchanger in in vivo proximal tubule of spontaneously hypertensive rats
Authors: Branco Regiane Cardoso Castelo , Leite‐Dellova Deise Carla Almeida , Malnic Gerhard , Mello‐Aires Margarids
Published: November 2023

The action of Angiotensin‐(1‐7) [Ang‐(1‐7) on the NHE3 exchanger contributes to the understanding of the etiology of hypertension in spontaneously hypertensive rats (SHR) and their controls, Wistar‐Kyoto rats (WKY). This action was evaluated by bicarbonate reabsorption (JHCO3−) of in vivo proximal renal tubules, using microperfusion with H ion‐sensitive microelectrodes. In the WKY rats, the mean control value of JHCO3− was 2,41 ± 0,12 nmol.cm−2.s−1 (N=14). Losartan [10−7 M; an Ang II receptor AT1 antagonist] alone decreased the JHCO3−to 1,64 ± 0,09 nmol.cm−2.s−1 (11); A779 [10−6 M; an Ang‐(1‐7) receptor Mas antagonist] alone decreased the JHCO3− to 1,75 ± 0,08 nmol.cm−2.s−1 (09). These data indicated that in the basal situation intratubular Ang II and Ang‐(1‐7) stimulates the JHCO3−. Ang‐(1‐7) (10−9 M) injected into luminally perfused tubules causes a significant decrease [to 1,73 ± 0,09 nmol.cm−2.s−1 (12) but Ang‐(1‐7) (10−6 M) increases it to 3,00 ± 0,11 nmol.cm−2.s−1 (18). However, A779 and losartan did not affect the inhibitory effect but prevents the stimulatory effects of Ang‐(1‐7) in JHCO3−. S3226 (10−6 M; an inhibitor of the NHE3 isoform of Na+/H+) alone decreases JHCO3− to 1,19 ± 0,05 nmol.cm−2.s−1 (14), does not affect the inhibitory effect of Ang‐(1‐7) and changes its stimulatory effect to an inhibitory effect [1,30 ± 0,07 nmol.cm−2.s−1 (19)]. In the SHR rats, JHCO3 was 2,00 ± 0,13 nmol.cm−2.s−1 (09). Losartan alone decreased the JHCO3 to 1,11 ± 0,07 nmol.cm−2.s−1 (15); A779 alone decreased the JHCO3− to 1,22 ± 0,09 nmol.cm−2.s−1 (11). These data indicated that in the basal situation endogenous intratubular Ang II and Ang‐(1‐7) stimulate JHCO3−. Ang‐(1‐7) (10−9 M) in luminally perfused tubules causes a significant increase [to 2,50 ± 0,12 nmol.cm−2.s−1 (24) but Ang‐(1‐7) (10−6 M) decreased it to 1,40 ± 0,07 nmol.cm−2.s−1 (08). However, losartan did not affect the inhibitory effect but prevented the stimulatory effect of Ang‐(1‐7) on JHCO3−. A779 prevents the inhibitory and the stimulatory effects of Ang‐(1‐7) on JHCO3−. S3226 alone decreases the JHCO3− to 1,03 ± 0,05 nmol.cm−2.s−1 (11), does not affect the inhibitory effect of Ang‐(1‐7) and changes its stimulatory effect to an inhibitory effect [0,99 ± 0,09 nmol.cm−2.s−1 (19)]. So, our data are showing that in WKY and SHR rats, the inhibitory dose‐dependent effect of Ang‐(1‐7) on JHCO3− is partially mediated by the AT1 receptor, demonstrating a possible inhibitory beneficial effect of Ang‐(1‐7) in hypertensive rats opposite to the deleterious stimulatory effects of Ang II. It is therefore reasonable to assume that, in the intact animal, the interaction of the opposite dose‐dependent effects of Ang‐(1‐7) and Ang II on the Na+/H+ may represent an important physiological regulation in terms of intra‐ and extracellular volume and/or pH changes.

Support or Funding Information

FAPESP, CNPq, CAPES
Remodeling of the Purinergic Receptors Profile in the ARPKD Cystic Epithelia
Authors: Ilatovskaya Daria , Palygin Oleg , Levchenko Vladislav , Pavlov Tengis , Staruschenko Alexander
Published: November 2023

Polycystic kidney diseases (PKD) are a group of inherited nephropathies marked with the formation of fluid‐filled cysts along the nephron. Autosomal Recessive PKD (ARPKD) has an incidence of approximately 1 in 20,000 live births; infants with ARPKD that survive beyond the perinatal period develop chronic renal failure by adolescence. Growing evidence suggests that autocrine and paracrine effects of purinergic signaling via P2 receptors could detrimentally contribute to cyst expansion. Studies have shown that extracellularly PKD cysts can accumulate up to 10 μM of ATP. We have proposed that ATP through associated intracellular signaling can contribute to a pathological increase of fluid secretion into the cysts by modulation of calcium influx and/or G‐protein coupled signaling. PCK/CrljCrl‐Pkhd1pck/CRL (PCK) rat, an established model of ARPKD, was used here to test this hypothesis. PCK rats were initially derived from Sprague Dawley rats (SD), and this strain was used as an additional control. First, we have implemented enzymatic biosensors to assess the levels of ATP in the cortical tissue of the isolated SD and PCK rat kidneys, and collected PCK cystic fluid. These levels were found to be 0.2, 1.1 and 2.1 μM, respectively. Therefore, cortical tissues and especially cystic fluid of PCK rats exhibited significantly higher levels of ATP compared to SD rat kidney interstitium. We have further employed immunohistochemistry and Western blotting to determine specific P2 receptors involved in the transduction of the ATP signal in the cystic epithelia. IHC demonstrated profound expression of the P2Y2 and P2X7 receptors in the healthy and dilated cortical tubules of the PCK rats. Western blotting suggested a significant increase in the P2X7 expression in the PCK rat kidney cortex with age (12 vs 20 week old animals). Life confocal calcium imaging of the freshly isolated cystic monolayers revealed a dose‐dependent response to ATP in a range of 10 to 100 μM. The use of PPADS and 5‐BDBD (potent antagonists of P2X1/P2X2/P2X3/P2X5 and P2X4 receptors, respectively), and experiments conducted in calcium‐free solutions suggested a significant involvement of the P2X7 signaling axis in the regulation of cytosolic calcium level in the cystic epithelia. In conclusion, our study provides evidence that the profile of P2 receptors is altered in the ARPKD cystic epithelia; pharmacological manipulation of purinergic signaling and/or suppression of the cystic ATP levels may be used as a novel target to affect cystogenesis in ARPKD. Supported by NIH K99 DK105160, R01 HL108880, and R01 HL122662.

Support or Funding Information

Supported by NIH K99 DK105160, R01 HL108880, and R01 HL122662.
Enhancement of neutrophil antimicrobial activity by the breast cancer drug tamoxifen
Authors: Flores Roxana , Insel Paul A , Nizet Victor , Corriden Ross
Published: November 2023

Neutrophils, the primary phagocytic leukocytes in the blood, play a central role in the innate immune response to pathogens such as methicillin‐resistant Staphylococcus aureus (MRSA). Given the rapid emergence of antibiotic resistance and slow development of antibiotics, new approaches to treat infections are urgently needed. We found that the breast cancer drug tamoxifen enhances several pro‐inflammatory pathways in human neutrophils, including chemotaxis, phagocytosis and neutrophil extracellular trap (NET) formation. Microscopy and mass spectrometry revealed that tamoxifen‐induced NET production occurs via an accumulation of intracellular ceramide (specifically C16 and C24 ceramide species) and subsequent activation of PKCζ. In contrast, enhancement of chemotaxis and phagocytosis occur via tamoxifen's agonist activity at the G protein‐coupled estrogen receptor GPER; indeed, G‐36, a selective antagonist of GPER, is a potent inhibitor of these processes. Pretreatment of human neutrophils with tamoxifen boosts neutrophil bactericidal capacity against MRSA, Pseudomonas aeruginosa and Escherichia coli in vitro and enhances survival and bacterial clearance (with 2.4–4.2 log reductions in bacterial counts in peritoneal lavage, blood, kidney, liver and spleen tissue samples) in an in vivo model of MRSA infection. Our results suggest that tamoxifen, and the lipid/GPCR signaling pathways it modulates, appear to be previously unrecognized ways to boost host innate immune function (in particular of neutrophils) and through such actions, to be a novel means to aid in the treatment of infections, especially of antibiotic‐resistant bacteria.

Support or Funding Information

Research was supported by NIH grants HD071600, AI057153 and AI052453.
Deletion of Hepatic AMPK Leads to Metabolic Inflexibility in Low‐ and High‐Protein Diets but Not in Normo‐Protein Diet
Authors: Chalvon‐Demersay Tristan , Even Patrick Christian , Chaumontet Catherine , Piedcoq Julien , Viollet Benoit , Gaudichon Claire , Tomé Daniel , Foretz Marc , Azzout‐Marniche Dalila
Published: November 2023

The AMP‐activated protein kinase (AMPK) is involved in energy signalling and in the control of the protein synthesis in the liver. The present study attempted to define the role of this ubiquitous kinase in the adaptation of energy metabolism in response to changes in the protein content of the diet.

52 wild‐type mice (WT) and 46 KO‐AMPK liver specific male mice (KOLS) were separated in 3 groups fed during 3 weeks with a low (LP‐5%), normal (NP‐14%) or high (HP‐55%) protein diet. Food intake and changes in body composition were measured throughout the experiment. At the end of the first week, changes in the rates of glucose and fatty acid oxidations (indirect calorimetry) were followed during 6 hours after ingestion of a 1g‐calibrated meal of their test‐diets given after an overnight fast. At the end of the experiment, the same test‐meal procedure was repeated and mice were killed either two or four hours after meal onset for measurements of hepatic gene expression and triglyceride content.

Two hours after ingestion of the LP or HP test‐meals, KOLS mice showed smaller increase in glucose oxidation and decrease in fatty acid oxidation than WT mice. Food intake, plasmatic concentration of FGF21 (Fibroblast growth factor‐21), hepatic triglyceride content and expression of genes involved in fatty acid oxidation were not altered. In contrast, in NP‐fed mice, glucose and fatty acid oxidation profiles did not differ between the WT and KOLS mice. Intriguingly, the results showed an increase in cumulative food intake and plasma FGF21 levels in NP‐fed KOLS mice, these mice also displayed an increase in hepatic triglyceride content associated with higher expressions of genes involved in liver fatty acid oxidation and ketogenesis.

Taken together, these results show that the consequences of AMPK deletion on energy metabolism depend on the protein content of the diet. In NP‐fed mice, deletion of AMPK in the liver led to an adaptation of liver metabolism resulting in an increased secretion of FGF21 and changes in gene expression encoding proteins involved in liver fatty acid oxidation and ketogenesis. These changes possibly compensated for the absence of hepatic AMPK as these mice exhibited normal post‐prandial changes in glucose and lipid oxidation. In contrast, in LP‐and HP‐fed mice, the lack of adjustment in liver metabolism in KOLS mice resulted in a metabolic inflexibility leading to reduced amplitude of meal‐induced changes in glucose and fatty acid oxidation.
Mouse Slc4a11 Mediates Extracellular‐pH‐Sensitive H+‐Conduction in Xenopus oocytes
Authors: Myers Evan J , Marshall Aniko , Parker Mark D
Published: November 2023

The SLC4 gene family includes ten members, eight of which encode bicarbonate transporters involved in intracellular pH (pHi) regulation. SLC4A11, a gene mutated in corneal dystrophy, has been proposed to encode a H+‐permeable pHi regulator although its roster of substrates and their mode of transport remain controversial. In this study we set out to determine the molecular action of mouse Slc4a11 (mSlc4a11) expressed in isolation in Xenopus laevis oocytes. Using two‐electrode voltage‐clamp circuitry, we assessed the membrane conductance (Gm) and membrane potential (Vm) of oocytes as they were exposed to nominally HCO3−‐free solutions of pH 6.5, 7.5, and 8.5. In pH 6.5 and 7.5 solutions, Gm of mSlc4a11‐expressing oocytes was not significantly different from Gm of H2O‐injected oocytes although Vm of these cells was slightly more depolarized than Vm of H2O‐injected cells at pH 6.5 and 7.5. For mSlc4a11‐expressing oocytes, but not for H2O‐injected oocytes, a switch from pH 7.5 to 8.5 solution elicited a significant increase in Gm (P<0.01, n=6) and a hyperpolarization of Vm to −73 ± 1 mV (P<0.01, n=6), a value consistent with the estimated reversal potential (Erev) for H+ (EH). The conductance persisted with unaltered Erev even in the absence of extracellular Na+, K+, and Cl− (n=3). During prolonged (25 min) exposure to pH 8.5 solution, Gm continued to rise without any further change in Vm, but both Gm and Vm were immediately reset when pH 7.5 solution was restored (n=4). Notably, oocytes expressing the mutant mSlc4a11‐Arg99His, a mimic of the R125H mutation linked to human corneal dystrophy, behaved like H2O‐injected oocytes upon exposure to pH 8.5 solution (n=3) despite apparently normal accumulation of the mutant in the oocyte plasma membrane, as determined by biotinylation. Using a H+‐selective microelectrode, we monitored pHi (with extracellular pH at 8.5) as we clamped oocyte Vm to values more positive or more negative than predicted EH. The pHi of H2O‐injected oocytes was not substantially influenced by these maneuvers. On the other hand, the pHi of oocytes expressing mSlc4a11 increased when Vm was >EH and decreased when Vm was <EH (n=3 or each). We find that the response of Vm to changes in the transmembrane H+‐gradient is close to Nernstian (52 ± 12 mV/pH unit, n=3) in mSlc4a11‐expressing cells, demonstrating H+‐selectivity. Together these data are consistent with the hypothesis that mSlc4a11 mediates an uncoupled, extracellular‐pH‐sensitive H+‐conduction that is related to its physiological role.

Support or Funding Information

Supported by Start‐up Funds from UB:SUNY (to MDP).
Influence of Blood Flow Velocity on Arterial Distensibility of Carotid Artery in Healthy Men
Authors: Sugawara Jun , Tomoto Tsubasa , Maeda Seiji
Published: November 2023

Decreased distensibility of carotid artery is independently associated with incident cardiovascular events and all‐cause mortality. Thus, the elucidation of physiological determinants responsible for carotid arterial distensibility might give us distinct target for prevention of future cardiovascular disease. We hypothesized that local arterial blood flow velocity (BFV), a major determinant of wall shear stress, influences vascular tone via endothelium‐derived vasoactive substances and regulates regional arterial distensibility. To test our hypothesis, using cross‐sectional study design we determine influence of carotid arterial BFV on its arterial distensibility. A total of 73 apparent healthy men (18–64 years) were studied. Carotid arterial properties, including measures of carotid arterial distensibility and BFV at rest, were evaluated via B‐mode and Doppler ultrasound imaging and applanation tonometry system. Distensibility coefficient of carotid artery correlated with peak (i.e., systolic) BFV (r=0.305, P<0.01) and more strongly with pulsatile (i.e., systolic minus diastolic) BFV (r=0.406, P<0.0001) and normalized pulsatile BFV by time‐averaged velocity (r=0.591, P<0.0001). Multi‐regression analysis revealed that age (β=−0.57, P<0.0001) was the primary independent determinant for distensibility coefficient. In addition, carotid lumen diameter (β= −0.202, P<0.01) and normalized pulsatile BFV (β=0.237, P<0.05) were also significant independent determinants of distensibility coefficient. Qualitatively similar results (although inverse in direction) were obtained by use of β‐stiffness index. Taken together, distensibility of carotid artery retards with aging but is independently and favorably associated with smaller lumen diameter and greater gradient of BFV during a cardiac cycle, suggesting the important contribution of baseline shear stress on distensibility of carotid artery.

Support or Funding Information

This study was supported in part by a Grant‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, (No. 25702045 and 26670116) (JS).
Pretreatment with Ampakine CX717 Enhances Long‐Term Facilitation (LTF) of Inspiratory Hypoglossal (XII) Bursting, But Only When Initial Burst Output is Low
Authors: Turner Sara , Elmallah Mai , Hoyt Aaron , Greer John , Fuller David
Published: November 2023

Glutamatergic synaptic transmission is a fundamental component of the neural drive to breathe, and AMPA‐mediated synaptic currents contribute to respiratory rhythmogenesis and respiratory motoneuron depolarization during inspiration. Ampakines are positive allosteric modulators of AMPA receptors, and have been shown to potentiate neuroplasticity in models of long‐term potentiation. The aim of this study was to explore the impact of ampakine CX717 on intermittent‐hypoxia (IH) induced respiratory LTF in urethane anesthetized, vagotomized, and mechanically ventilated adult mice. Ampakine was administered at 15 mg/kg (intraperitoneal injection) 10‐min prior to 3, 1‐min bouts of hypoxia (10% O2). Initial control experiments indicated that a single 15 mg/kg ampakine injection had no persistent impact on XII motor output in this preparation. However, pairing ampakine with IH resulted in enhanced LTF. Thus, over a 90‐min period following IH, the amplitude of the XII inspiratory burst was significantly higher in mice pretreated with ampakine (485 ± 96% of baseline) vs. sham‐injected mice (198 ± 32% of baseline; 2 way repeated measure ANOVA, P<0.05). In addition, the absolute value of the initial pre‐IH baseline XII inspiratory burst amplitude (i.e., raw voltage or “arbitrary units”) was robustly correlated (P<0.001) with the magnitude of LTF (%baseline). Thus, the probability of the ampakine injection evoking a greater LTF was much greater when baseline outputs were low. A similar relationship was not detectable in the non‐ampakine treated mice (P=0.852). We conclude that IH‐induced respiratory LTF can be enhanced by pre‐treatment with ampakine, but the drug is not effective when initial respiratory output is high. These data suggest that ampakines have potential to enhance the impact IH as a neurorehabilitation modality for neurologic conditions associated with reduced respiratory motor output.

Support or Funding Information

1 R01 NS080180‐01A1 (DDF), State of Florida Brain and Spinal Cord Injury Research Program (DDF), Canadian Institutes for Health Research (JJG), Craig H. Neilsen Foundation 313369 (SMFT)