Effect of potassium on ammoniagenesis by renal mitochondria

Ammonia production by rat renal cortical mitochondria was studied with both in vivo and in vitro manipulation of potassium to further elucidate the mechanisms relating potassium homeostasis and renal ammonia production. Mitochondria from potassium-depleted animals demonstrated an increase in ammonia production at all glutamine concentrations studied, which ranged from 0.5 to 10 mM. This increase in ammoniagenesis compared favorably in degree of change with and was of sufficient absolute magnitude to entirely account for the findings observed both in renal cortical slices studied in vitro and in the intact animal. When rotenone is added to the medium, increased ammoniagenesis is still detected, indicating that either glutamine entry into the mitochondria and/or the activity of phosphate-dependent glutaminase are critical rate-controlling steps. In contrast to studies with renal slices, a decrease in ammonia production was not apparent with cortical mitochondria from chronically potassium-loaded animals. In vitro alterations of the potassium homeostasis. Therefore, these experimental manipulations either do not activate an effector mechanism which takes place in vivo, or the alteration in ammoniagenesis requires an adaptation over time that is not achieved with this in vitro approach.

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Influence of potassium on renal ammonia production

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1976.231.4.1178 ◽

1976 ◽

Vol 231 (4) ◽

pp. 1178-1184 ◽

Cited By ~ 38

Author(s):

RL Tannen ◽

J McGill

Keyword(s):

Rat Kidney ◽

Normal Diet ◽

Direct Result ◽

Ammonia Production ◽

Cellular Mechanisms ◽

High Potassium ◽

Potassium Homeostasis ◽

Acute Changes ◽

Cortical Slices

The influence of potassium homeostasis on ammonia production was investigated with both cortical and medullary slices from rat kidney. Renal cortical slices from rats depleted of potassium by dietary restriction produced 31% more NH3 than slices from pair-fed controls. A high-potassium diet for 1 wk diminished ammonia production in cortical slices by 5% in comparison with rats pair fed a normal diet (161 vs. 169 mumol/90 min per g wet wt; P less than 0.05). Pair feeding did not introduce an experimental artifact, since animals ingesting similar K+ diets showed no difference in NH3 production. In contrast to cortex, NH3 production by outer medullary slices from K+-depleted animals was similar to pair-fed controls. Medulla from potassium-loaded rats exhibited an impressive inhibition in NH3 production averaging 36%. These striking differences between cortex and medulla suggest that specific alterations in potassium homeostasis may influence NH3 production selectively at different tubular sites. In vitro manipulation of K+ homeostasis produced by varying bathing media K+ from 0 to 144 mM, with concomitant changes in intracellular K+ from 30 to 130 mM, had no detectable influence on NH3 production by cortical slices. Hence altered cortical ammoniagenesis is not the direct result of acute changes in extracellular or intracellular cortical fluid K+ or in the transcellular gradient for K+. Although the specific cellular mechanisms whereby K+ alters ammoniagenesis remains undefined, the observation that K+ loading diminishes while K+ depletion enhances NH3 production supports the supposition that K+ and NH3 are linked in a physiologic control system.

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The Effect of Phenyl Ethyl Biguanide In Vivo and In Vitro on Gluconeogenesis and Ammonia Production in Rats

Clinical Science ◽

10.1042/cs0400107 ◽

1971 ◽

Vol 40 (2) ◽

pp. 107-115 ◽

Cited By ~ 8

Author(s):

G. A. O. Alleyne ◽

H. S. Besterman ◽

H. Flores

Keyword(s):

Oral Administration ◽

Ammonium Chloride ◽

Specific Site ◽

Ammonia Production ◽

Renal Gluconeogenesis ◽

The Relationship ◽

Cortical Slices

1. The drug phenyl ethyl biguanide (PEBG) was used in vivo and in vitro to study further the relationship between renal gluconeogenesis and ammonia production in the rat. 2. When PEBG was injected intraperitoneally into the rats, it caused a decrease in urinary ammonia in spite of a greater degree of systemic acidosis. PEBG injections also blocked the increase in glucose and ammonia production by renal cortical slices from rats which had been made acidotic by oral administration of ammonium chloride 2 h previously. 3. With increasing concentrations of PEBG in vitro, there was inhibition of gluconeogenesis and ammonia production from glutamine and glutamate as substrates. The two processes were equally inhibited when glutamate was used as substrate but with glutamine, gluconeogenesis was more inhibited than ammonia production. 4. The inhibition of renal gluconeogenesis by PEBG in vitro was similar when succinate, oxaloacetate, fructose, glutamine and glutamate were used as substrates. 5. The results show that PEBG does not inhibit gluconeogenesis by blocking a specific site in the gluconeogenic pathway. In addition, further proof is provided of the physiological interrelationship of renal ammonia production and gluconeogenesis.

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The Development of Homologous (Canine/Anti-Canine) Antibodies in Dogs with Haemophilia A (Factor VIII Deficiency): A Ten-Year Longitudinal Study

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651541 ◽

1993 ◽

Vol 69 (01) ◽

pp. 021-024 ◽

Cited By ~ 20

Author(s):

Shawn Tinlin ◽

Sandra Webster ◽

Alan R Giles

Keyword(s):

Factor Viii ◽

Canine Model ◽

Significant Inhibition ◽

Human Condition ◽

Haemophilia A ◽

Variable Expression ◽

The Family ◽

Over Time

SummaryThe development of inhibitors to factor VIII in patients with haemophilia A remains as a serious complication of replacement therapy. An apparently analogous condition has been described in a canine model of haemophilia A (Giles et al., Blood 1984; 63:451). These animals and their relatives have now been followed for 10 years. The observation that the propensity for inhibitor development was not related to the ancestral factor VIII gene has been confirmed by the demonstration of vertical transmission through three generations of the segment of the family related to a normal (non-carrier) female that was introduced for breeding purposes. Haemophilic animals unrelated to this animal have not developed functionally significant factor VIII inhibitors despite intensive factor VIII replacement. Two animals have shown occasional laboratory evidence of factor VIII inhibition but this has not been translated into clinical significant inhibition in vivo as assessed by clinical response and F.VIII recovery and survival characteristics. Substantial heterogeneity of inhibitor expression both in vitro and in vivo has been observed between animals and in individual animals over time. Spontaneous loss of inhibitors has been observed without any therapies designed to induce tolerance, etc., being instituted. There is also phenotypic evidence of polyclonality of the immune response with variable expression over time in a given animal. These observations may have relevance to the human condition both in determining the pathogenetic factors involved in this condition and in highlighting the heterogeneity of its expression which suggests the need for caution in the interpretation of the outcome of interventions designed to modulate inhibitor activity.

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Abstract 5402: Two Photon Microscopy Measurements Of Sub-epicardial Sarcomere Length In Perfused Rat Hearts

Circulation ◽

10.1161/circ.118.suppl_18.s_543-c ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Patrizia Camelliti ◽

Gil Bub ◽

Daniel J Stuckey ◽

Christian Bollensdorff ◽

Damian J Tyler ◽

...

Keyword(s):

Sarcomere Length ◽

Left Ventricular ◽

Model Systems ◽

Future Research ◽

Fundamental Parameter ◽

Rat Hearts ◽

Perfused Hearts ◽

Over Time

Sarcomere length (SL) is a fundamental parameter underlying the Frank Starling relation in the heart, as it offers an absolute representation of myocardial stretch. Previous studies addressed the Frank Starling relation by measuring SL in isolated myocytes or muscle strips. Here, we report first data obtained using a novel technique to measure sub-epicardial SL in perfused hearts. Rat hearts were Langendorff perfused (normal Tyrode solution) at a constant pressure of 90mmHg, labeled with the fluorescent membrane marker di-4-ANEPPS, and then arrested with high-K + Tyrode for either 2-photon microscopy (n=4) or MRI (n=4). Image analysis software was developed to extract SL at the cell level from >1,400 2-photon images (Fig 1 ) and correct for cell angle. SL increased by 10±2 % between 30 and 80 min of perfusion (1.98±0.04 to 2.17±0.03 μm; p<0.05; Fig 1 ). Measurements of left ventricular myocardial volume (LVMV) were made in vivo and in perfused hearts using 3D MRI. LVMV increased by 24±7% from in vivo to 30 min of perfusion, and by 11±3 % between 30 and 90 min (539±35; 664±44; 737±49 mm 3 , respectively; p<0.05; Fig 1 ). We show that SL can be measured in isolated perfused hearts. The method allowed monitoring of changes in SL over time, and showed that SL and LVMV increase to a similar extent during 30–80 min perfusion with crystalloid solution, probably due to tissue oedema. This result, together with the increase in LVMV during the first 30 min, highlights the pronounced differences between in vivo , in situ , and in vitro model systems for studies of cardiac physiology and mechanics. Future research will compare changes in SL in healthy hearts and disease models involving contractile dysfunction. Figure 1: Left: 2-photon microscopy image of di-4-ANEPPS labeled myocardium. Right: SL and LVMV changes over time.

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A MICROCALORIMETRIC STUDY OF TURTLE CORTICAL SLICES: INSIGHTS INTO BRAIN METABOLIC DEPRESSION

Journal of Experimental Biology ◽

10.1242/jeb.191.1.141 ◽

1994 ◽

Vol 191 (1) ◽

pp. 141-153 ◽

Cited By ~ 1

Author(s):

C Doll ◽

P Hochachka ◽

S Hand

Keyword(s):

Cortical Neurons ◽

Heat Dissipation ◽

Energy Charge ◽

Utilization Rate ◽

Rapid Decline ◽

Metabolic Depression ◽

Slice Preparation ◽

Cortical Slices

In previous papers, we have examined turtle cortical neurons in vitro for mechanisms of anoxic metabolic depression ('channel arrest' and changes in electrical parameters). Negative results prompted the current study with the aim of examining more closely the energy profile and metabolism of turtle cortical slices. Calorimetry is used to measure heat dissipation during normoxia and nitrogen perfusion (120 min) and the results are converted into an ATP utilization rate. These indicate that the control rate of ATP utilization (1.72 µmol ATP g-1 min-1) agrees closely with in vivo whole-brain metabolic measurements. Both nitrogen perfusion and pharmacologically induced anoxic (cyanide+N2) groups depressed heat dissipation considerably compared with the control value (nitrogen 37 %; pharmacological anoxia 49 %). The resulting ATP utilization estimates indicate metabolic depressions of 30 % (nitrogen) and 42 % (pharmacological anoxia). The slice preparation did not exhibit a change in any measured adenylate parameter for up to 120 min of anoxia or pharmacological anoxia. Significant changes did occur in [ADP], ATP/ADP ratio and energy charge after 240 min of exposure to anoxic conditions. These results support the idea that the turtle cortical slice preparation has a profound resistance to anoxia, with both nitrogen perfusion and pharmacological anoxia causing a rapid decline in heat dissipation and metabolism.

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Time-Dependent Reduction of Calcium Oscillations in Adipose-Derived Stem Cells Differentiating towards Adipogenic and Osteogenic Lineage

Biomolecules ◽

10.3390/biom11101400 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1400

Author(s):

Enrico C. Torre ◽

Mesude Bicer ◽

Graeme S. Cottrell ◽

Darius Widera ◽

Francesco Tamagnini

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Adipogenic Differentiation ◽

Specific Treatment ◽

Stem Cell Differentiation ◽

Calcium Oscillations ◽

Multipotent Stem Cells ◽

Over Time

Adipose-derived mesenchymal stromal cells (ASCs) are multipotent stem cells which can differentiate into various cell types, including osteocytes and adipocytes. Due to their ease of harvesting, multipotency, and low tumorigenicity, they are a prime candidate for the development of novel interventional approaches in regenerative medicine. ASCs exhibit slow, spontaneous Ca2+ oscillations and the manipulation of Ca2+ signalling via electrical stimulation was proposed as a potential route for promoting their differentiation in vivo. However, the effects of differentiation-inducing treatments on spontaneous Ca2+ oscillations in ASCs are not yet fully characterised. In this study, we used 2-photon live Ca2+ imaging to assess the fraction of cells showing spontaneous oscillations and the frequency of the oscillation (measured as interpeak interval—IPI) in ASCs undergoing osteogenic or adipogenic differentiation, using undifferentiated ASCs as controls. The measurements were carried out at 7, 14, and 21 days in vitro (DIV) to assess the effect of time in culture on Ca2+ dynamics. We observed that both time and differentiation treatment are important factors associated with a reduced fraction of cells showing Ca2+ oscillations, paralleled by increased IPI times, in comparison with untreated ASCs. Both adipogenic and osteogenic differentiation resulted in a reduction in Ca2+ dynamics, such as the fraction of cells showing intracellular Ca2+ oscillations and their frequency. Adipogenic differentiation was associated with a more pronounced reduction of Ca2+ dynamics compared to cells differentiating towards the osteogenic fate. Changes in Ca2+ associated oscillations with a specific treatment had already occurred at 7 DIV. Finally, we observed a reduction in Ca2+ dynamics over time in untreated ASCs. These data suggest that adipogenic and osteogenic differentiation cell fates are associated with specific changes in spontaneous Ca2+ dynamics over time. While this observation is interesting and provides useful information to understand the functional correlates of stem cell differentiation, further studies are required to clarify the molecular and mechanistic correlates of these changes. This will allow us to better understand the causal relationship between Ca2+ dynamics and differentiation, potentially leading to the development of novel, more effective interventions for both bone regeneration and control of adipose growth.

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Intraperitoneal pressure: Stability over time and validation of Durand’s measurement method

Peritoneal Dialysis International ◽

10.1177/0896860820973120 ◽

2020 ◽

pp. 089686082097312

Author(s):

Alicia Sobrino-Pérez ◽

Alfonso Pérez-Escudero ◽

Lucila Fernández-Arroyo ◽

Ana Dorado-García ◽

Berta Martín-Alcón ◽

...

Keyword(s):

Peritoneal Dialysis ◽

Venous Pressure ◽

Relevant Parameter ◽

Pressure System ◽

Intraperitoneal Pressure ◽

In Vitro Measurements ◽

Long Timescales ◽

Over Time

Intraperitoneal pressure (IPP) is gaining consideration as a relevant parameter of peritoneal dialysis (PD) in adults, although many of its aspects are still pending clarification. We address here its stability over time and the validity of the usual method of clinical measurement, as proposed by Durand in 1992 but never specifically validated. We performed this validation by comparing Durand’s method and direct measurements with a central venous pressure system. We performed a total of 250 measurement pairs in 50 patients with different intraperitoneal volumes plus in-vitro measurements with a simulated peritoneum. Absolute differences between the two systems in vivo were 0.87 ± 0.91 cmH2O (range 0–5 cmH2O); only 6.4% of them were ≥3 cmH2O. In vitro results for both methods were identical. We also compared IPP measurements in the same patient separated by 1–4 h (514 measurement pairs in 136 patients), 1 week (92 pairs in 92 patients), and 2 years (34 pairs in 17 patients). Net differences of measurements separated by hours or 1 week were close to 0 cmH2O, with oscillations of 1.5 cmH2O in hours and 2.3 cmH2O in 1 week. IPP measured 2 years apart presented a net decrease of 2.5 ± 4.9 cmH2O, without correlation with body mass index changes or any other usual parameter of PD. In hours, 7% of IPP differences were >3 cmH2O, 22% in 1 week, and 50% in 2 years. In conclusion, Durand’s method is precise enough to measure IPP in peritoneal dialysis. This parameter is not stable over long timescales, so it is necessary to use recent measurements.

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Presynaptic inhibition of canine renal adrenergic nerves by acetylcholine in vivo

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1979.237.3.h326 ◽

1979 ◽

Vol 237 (3) ◽

pp. H326-H331

Author(s):

N. W. Robie

Keyword(s):

Nerve Stimulation ◽

Neurotransmitter Release ◽

Intact Animal ◽

Inhibitory Effect ◽

Inhibitory Effects ◽

Renal Vasculature ◽

Anesthetized Dogs ◽

Renal Sympathetic

Experiments were performed in anesthetized dogs to determine whether previously reported in vitro inhibition of sympathetic neurotransmitter release by acetylcholine could be demonstrated in the renal vasculature of the intact animal. Vasoconstrictor responses to renal sympathetic nerve stimulation at varying frequencies were compared to intra-arterial injections of norepinephrine before and during intra-arterial infusions of acetylcholine, 2.5--80 micrograms/min. The vasoconstrictor responses to nerve stimulation were inhibited to a greater extent than were responses to norepinephrine during infusions of acetylcholine. The inhibitory effects of acetylcholine on nerve stimulation were dose and frequency dependent. The inhibition was blocked by atropine but not altered by physostigmine. Changes in renal blood flow per se did not contribute to the inhibitory effect of acetylcholine, since another vasodilator agent, sodium acetate, did not affect the nerve stimulation-norepinephrine vasocontriction relationship. Thus, acetylcholine produced inhibition of in vivo renal sympathetic vasoconstrictor responses, and the receptor involved appears to be muscarinic.

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Clathrin structure characterized with monoclonal antibodies. II. Identification of in vivo forms of clathrin.

The Journal of Cell Biology ◽

10.1083/jcb.101.6.2055 ◽

1985 ◽

Vol 101 (6) ◽

pp. 2055-2062 ◽

Cited By ~ 32

Author(s):

F M Brodsky

Keyword(s):

Monoclonal Antibodies ◽

Heavy Chain ◽

Light Chain ◽

Coated Vesicles ◽

Cell Lysates ◽

Over Time

Clathrin was isolated from detergent-solubilized, biosynthetically radiolabeled cells by immunoprecipitation with anti-clathrin monoclonal antibodies. Immunoprecipitates obtained after pulse-chase labeling demonstrated that after biosynthesis the LCa light chain of clathrin could be found either complexed to heavy chain or in a free pool (not associated with heavy chain) which decreased steadily over time. More than half of the free LCa disappeared within the first hour after biosynthesis, but some was still detectable after several hours. Incorporation of clathrin LCa light chain and heavy chain into coated vesicles was coordinate and increased up to 4 h after biosynthesis. Comparison of these kinetics suggested that once incorporated into coated vesicles, LCa and heavy chain did not dissociate, even during depolymerization of the vesicle. There was also little apparent degradation of clathrin found in coated vesicles for up to 22 h after biosynthesis. Immunoprecipitation with anti-clathrin monoclonal antibodies was carried out after fractionation of continuously radiolabeled cell lysates using two different sizing columns. These experiments indicated that the triskelion form of clathrin that has been isolated from coated vesicles in vitro also exists in vivo. They also confirmed the existence of a transient but detectable pool of newly synthesized free LCa light chain.

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