Chloride distribution and exchange in rat ventricle

1980 ◽  
Vol 238 (5) ◽  
pp. C169-C176 ◽  
Author(s):  
P. I. Polimeni ◽  
E. Page
Keyword(s):  
Activation Energy ◽  
Equilibrium Potential ◽  
Quasi Steady State ◽  
Cell Water ◽  
Analytical Error ◽  
Rat Ventricle ◽  
Calculated Equilibrium ◽  
Exchange Flux

The cellular Cl content and concentration ([Cl]cell) and the cellular uptake of 36Cl have been measured in the rat left ventricle in vivo. The in vitro efflux of 36Cl from perfused contracting ventricles preequilibrated with 36Cl in vivo was also determined at 22, 30, and 38 degrees C. [Cl]cell was 8.2 +/- 0.5 mmol/kg cell water, corresponding to a calculated equilibrium potential of Cl of -70 to -80 mV. This figure for [Cl]cell is significantly lower than previous estimates in the literature, which were subject to an analytical error leading to overestimation of muscle Cl content obtained coulometrically. At 38 degrees C, Cl exchange under quasi-steady-state conditions was 31.2 mumol . (g dry ventricle . min)-1 or 42.5 pmol . (cm2 plasma membrane . s).-1 Apparent activation energy of the flux was 10.4 kcal/mol. At 22 degrees C, no dependence of the exchange on contraction frequency was detectable over a range of 80-160 contractions/min. The Cl exchange flux is among the fastest, if not the fastest. known for myocardial ion transport.

Dendritic arbor of locus coeruleus neurons contributes to opioid inhibition

1996 ◽  
Vol 75 (5) ◽  
pp. 2029-2035 ◽  
Author(s):  
R. A. Travagli ◽  
M. Wessendorf ◽  
J. T. Williams
Keyword(s):  
Locus Coeruleus ◽  
Horizontal Plane ◽  
Reversal Potential ◽  
Outward Current ◽  
Membrane Properties ◽  
Equilibrium Potential ◽  
Intrinsic Membrane Properties ◽  
In Cells

1. The nucleus locus coeruleus (LC) is made up of noradrenergic cells all of which are hyperpolarized by opioids. Recent work has shown that the reversal potential of the opioid-induced current is more negative than the potassium equilibrium potential. The aim of the present study was to determine whether the extent of the dendritic field could contribute to the very negative opioid reversal potential. 2. Individual LC cells were labeled in the brain slice preparation. The number of dendrites found on cells in slices sectioned in the horizontal plane was greater than cells in coronal slices. However, the dimensions of the cell body slices from each plane were not significantly different. 3. The resting conductance of neurons from slices cut in the horizontal plane was significantly larger than in cells from coronal plane. 4. The amplitude of the outward current induced by [Met5]-enkephalin (ME) was larger in cells from horizontal slices and the reversal potential was more negative than that of cells in coronal slices. 5. The results show that the plane of section influences the membrane properties and opioid actions of LC neurons in vitro and suggest that these differences correlate with the numbers of dendrites. The results suggest that in vivo, in addition to intrinsic membrane properties and synaptic inputs, the structural makeup of the nucleus is an important factor in determining the activity.

Direct Excitation of Mitral Cells Via Activation of α1-Noradrenergic Receptors in Rat Olfactory Bulb Slices

2001 ◽  
Vol 86 (5) ◽  
pp. 2173-2182 ◽  
Author(s):  
Abdallah Hayar ◽  
Phillip M. Heyward ◽  
Thomas Heinbockel ◽  
Michael T. Shipley ◽  
Matthew Ennis
Keyword(s):  
Olfactory Bulb ◽  
Locus Coeruleus ◽  
Equilibrium Potential ◽  
Channel Blockers ◽  
Mitral Cells ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Inward Current

The main olfactory bulb receives a significant modulatory noradrenergic input from the locus coeruleus. Previous in vivo and in vitro studies showed that norepinephrine (NE) inputs increase the sensitivity of mitral cells to weak olfactory inputs. The cellular basis for this action of NE is not understood. The goal of this study was to investigate the effect of NE and noradrenergic agonists on the excitability of mitral cells, the main output cells of the olfactory bulb, using whole cell patch-clamp recording in vitro. The noradrenergic agonists, phenylephrine (PE, 10 μM), isoproterenol (Isop, 10 μM), and clonidine (3 μM), were used to test for the functional presence of α1-, β-, and α2-receptors, respectively, on mitral cells. None of these agonists affected olfactory nerve (ON)–evoked field potentials recorded in the glomerular layer, or ON-evoked postsynaptic currents recorded in mitral cells. In whole cell voltage-clamp recordings, NE (30 μM) induced an inward current (54 ± 7 pA, n= 16) with an EC50 of 4.7 μM. Both PE and Isop also produced inward currents (22 ± 4 pA, n = 19, and 29 ± 9 pA, n = 8, respectively), while clonidine produced no effect ( n = 6). In the presence of TTX (1 μM), and blockers of excitatory and inhibitory fast synaptic transmission [gabazine 5 μM, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) 10 μM, and (±)-2-amino-5-phosphonopentanoic acid (APV) 50 μM], the inward current induced by PE persisted (EC50 = 9 μM), whereas that of Isop was absent. The effect of PE was also observed in the presence of the Ca2+ channel blockers, cadmium (100 μM) and nickel (100 μM). The inward current caused by PE was blocked when the interior of the cell was perfused with the nonhydrolyzable GDP analogue, GDPβS, indicating that the α1 effect is mediated by G-protein coupling. The current-voltage relationship in the absence and presence of PE indicated that the current induced by PE decreased near the equilibrium potential for potassium ions. In current-clamp recordings from bistable mitral cells, PE shifted the membrane potential from the downstate (−52 mV) toward the upstate (−40 mV), and significantly increased spike generation in response to perithreshold ON input. These findings indicate that NE excites mitral cells directly via α1 receptors, an effect that may underlie, at least in part, increased mitral cell responses to weak ON input during locus coeruleus activation in vivo.

scholarly journals Kinetic barriers to SNAREpin assembly in the regulation of membrane docking/priming and fusion

2016 ◽  
Vol 113 (38) ◽  
pp. 10536-10541 ◽  
Author(s):  
Feng Li ◽  
Neeraj Tiwari ◽  
James E. Rothman ◽  
Frederic Pincet
Keyword(s):  
Activation Energy ◽  
Energy Barrier ◽  
Vesicle Docking ◽  
Readily Releasable Pool ◽  
Protein Receptor ◽  
Regulatory Machinery ◽  
Presynaptic Plasma Membrane ◽  
Kinetic Barriers

Neurotransmission is achieved by soluble NSF attachment protein receptor (SNARE)-driven fusion of readily releasable vesicles that are docked and primed at the presynaptic plasma membrane. After neurotransmission, the readily releasable pool of vesicles must be refilled in less than 100 ms for subsequent release. Here we show that the initial association of SNARE complexes, SNAREpins, is far too slow to support this rapid refilling owing to an inherently high activation energy barrier. Our data suggest that acceleration of this process, i.e., lowering of the barrier, is physiologically necessary and can be achieved by molecular factors. Furthermore, under zero force, a low second energy barrier transiently traps SNAREpins in a half-zippered state similar to the partial assembly that engages calcium-sensitive regulatory machinery. This result suggests that the barrier must be actively raised in vivo to generate a sufficient pause in the zippering process for the regulators to set in place. We show that the heights of the activation energy barriers can be selectively changed by molecular factors. Thus, it is possible to modify, both in vitro and in vivo, the lifespan of each metastable state. This controllability provides a simple model in which vesicle docking/priming, an intrinsically slow process, can be substantially accelerated. It also explains how the machinery that regulates vesicle fusion can be set in place while SNAREpins are trapped in a half-zippered state.

scholarly journals Hepatocyte Organization Affects the Translation of Clearance from In Vitro to In Vivo

10.29007/t4kv ◽  
2020 ◽  
Author(s):  
Lopamudra Dutta ◽  
Preethi Krishnan ◽  
Andrew Smith ◽  
Ryan Kennedy ◽  
Glen Ropella ◽  
...  
Keyword(s):  
Hepatic Clearance ◽  
Intrinsic Clearance ◽  
Quasi Steady State ◽  
Exposure Rate ◽  
Virtual Experiments ◽  
Mitigating Factors ◽  
Virtual Mouse ◽  
In Vivo Extrapolation

An improved understanding of in vivo ⇔ in vitro changes is crucial in identifying and mitigating factors contributing to in vitro–in vivo extrapolation (IVIVE) inaccuracies in predicting the hepatic clearance of substances. We argue that a model mechanism-based virtual culture (vCulture) ⇔ virtual mouse (vMouse) (or vRat or vHuman) experiment approach can identify factors contributing to IVIVE disconnects. Doing so depends on having evidence that six Translational Requirements have been achieved. We cite evidence that the first four have been achieved. The fifth Requirement is that differences in measures of vCompound disposition between vCulture and vMouse are attributable solely to the micro-architectural, physiomimetic features, and uncertainties built into the vLiver and vMouse but are absent from the vCulture. The objective of this work is to first improve on a vCulture architecture used previously and then use results of virtual experiments to verify that its use enables the fifth Translational Requirement to be achieved. We employ two different idealized vCompounds, which map to highly permeable real compounds at the extreme ends of the intrinsic clearance spectrum. Virtual intrinsic clearance = Exposure rate per vHPC. At quasi-steady state, results for vCompound-1 are independent of the dosing rate. The average per-vHPC Exposure rates (taken over the whole vLiver in vMouse experiments) are the same (within the variance of the Experiments) as those in vCulture. However, they are location dependent within the vLiver. For vCompound-2, there are dosing rate differences and average per-vHPC Exposure rates within the vLiver are also location dependent. When we account for dosing rate differences, we see again that average per-vHPC Exposure rates averaged over the whole vLiver in vMouse experiments are the same as those in vCulture. Thus, the differences in per vHPC Exposure rate within the vLiver for both vCompounds are attributable solely to the micro-architectural and physiomimetic features built into the vLiver and vMouse but are absent from the vCulture. The results verify that the fifth Translational Requirement has been achieved.

Abstract 475: Beta-adrenoceptor Stimulation Uniquely Regulates Exosome Micro-rna Content Secreted From Cardiomyocytes in vitro and Those Found in vivo Circulating in Blood

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Jin Sook Kwon ◽  
Erhe Gao ◽  
Rajika Roy ◽  
jessica Ibetti ◽  
J Kurt Chuprun ◽  
...  
Keyword(s):  
Microarray Data ◽  
Therapeutic Potential ◽  
Heart Tissue ◽  
Micro Rna ◽  
Neonatal Rat ◽  
Beta Adrenoceptor ◽  
Sympathetic Nervous ◽  
Rat Ventricle

Background and Purpose: The influence of β-adrenoceptor (βAR) signaling on the regulation of exosomes secreted from cardiomyocytes is unknown and since catecholamines are increased in heart failure (HF), there is interest in uncovering whether βARs can induce specific changes in the content of circulating blood exosomes in HF. In this study, we have evaluated whether βAR stimulation by isoproterenol (ISO) on neonatal rat ventricle myocytes (NRVMs) and in vivo in mice can alter the number, size and microRNA (miR) content of secreted exosomes. Methods and Results: ISO treatment of NRVMs did not change exosome number and size of compared than vehicle (PBS). After purifying total RNA from treated myocytes and secreted exosomes, we evaluated the expression level of 37 candidate miR’s, which were selected from previous microarray data. We found that ISO treatment decreased 14 miR’s (miR-222, -106a, -292a, -181b, -210, -489, -214, -1947, -195, -17, -7b, -93, -532 and -19a) in exosomes and in the myocytes themselves, mir-292a and -1947 were up regulated and mir-7b, down regulated. Further, ISO was then used to treat C57 mice via osmotic pump chronically. The number and size of exosomes purified from circulating blood was not changed after 2 and 8 weeks. We evaluated expression of the 14 miRs down-regulated in myocytes as well miR-1 and -21 (important cardiac miRs) both in the blood and hearts of ISO-treated mice. MiR-1 did not change in both blood exosomes and heart tissue and miR-21 was up-regulated in heart tissue but not in blood both at 2 and 8 weeks after ISO treatment. In addition, miRNA-489 and -7b was down-regulated in hearts with miR-214 and -292a up regulated. In blood exosomes, we found only down-regulation of miR-489 and -7b but not miR-214 and 292a. Conclusions: We found that the βAR agonist ISO altered exosomal miR contents but not exosome size and number from myocytes. Importantly, this was found in myocytes in culture and in vivo blood and myocardium that were treated with ISO but several differences were found and changes in blood and myocytes were not homogeneous. However, two miRs, mir-7b and -miR-489, both changes similarly from their origin (NRVMs or mouse hearts) and also in circulating blood exosomes. Therefore, these miRs may represent biomarkers for sympathetic nervous system abnormalities in HF and their therapeutic potential should be evaluated.

scholarly journals Thermal inactivation and reactivation of an enzyme in vivo. Pantothenate hydrolase of Pseudomonas fluorescens

1972 ◽  
Vol 130 (1) ◽  
pp. 111-119 ◽  
Author(s):  
R. Kalervo Airas
Keyword(s):  
Activation Energy ◽  
Pseudomonas Fluorescens ◽  
Rapid Growth ◽  
Thermal Inactivation ◽  
Fast Rate ◽  
Carbon Sources ◽  
Enzyme Inactivation ◽  
Whole Cells

Thermal inactivation and reactivation of pantothenate hydrolase were studied in whole cells of Pseudomonas fluorescens. The enzyme is susceptible to thermal inactivation in whole cells at 37–40°C, and is reactivated when the temperature is lowered again. Chloramphenicol does not prevent reactivation. The activation energy of enzyme inactivation in vivo is about 540kJ/mol. This activation energy is 220kJ/mol in vitro, but it is increased to 550–630kJ/mol by several metabolites, such as succinate, glyoxylate and oxalate. Generally, good carbon sources, causing rapid growth, protect the enzyme from thermal inactivation in vivo, and enable reactivation to occur at a fast rate. The enzyme is also inactivated below 35°C, showing an activation energy of about 35kJ/mol. Good carbon sources prevent this inactivation as well, and cause slight reactivation. Glycine, although not utilized for growth, protects the enzyme well from this inactivation but not from inactivation at 37–40°C, and prevents reactivation totally. From the activation energies of inactivation and the effects of the various carbon sources, it appears possible that changes in the concentrations of intracellular metabolites may be responsible for the changes in inactivation and reactivation.

Oestrogen inhibits salt-dependent hypertension by suppressing GABAergic excitation in magnocellular AVP neurons

2020 ◽  
Author(s):  
Xiangyan Jin ◽  
Woong Bin Kim ◽  
Mi-Na Kim ◽  
Won Woo Jung ◽  
Hyung Kyung Kang ◽  
...  
Keyword(s):  
Antihypertensive Effect ◽  
Protective Role ◽  
Ovariectomized Rats ◽  
Equilibrium Potential ◽  
Salt Treatment ◽  
Abundant Evidence ◽  
Plasma Arginine ◽  
Depressor Effect

Abstract Aims Abundant evidence indicates that oestrogen (E2) plays a protective role against hypertension. Yet, the mechanism underlying the antihypertensive effect of E2 is poorly understood. In this study, we sought to determine the mechanism through which E2 inhibits salt-dependent hypertension. Methods and results To this end, we performed a series of in vivo and in vitro experiments employing a rat model of hypertension that is produced by deoxycorticosterone acetate (DOCA)-salt treatment after uninephrectomy. We found that E2 prevented DOCA-salt treatment from inducing hypertension, raising plasma arginine-vasopressin (AVP) level, enhancing the depressor effect of the V1a receptor antagonist (Phenylac1,D-Tyr(Et)2,Lys6,Arg8,des-Gly9)-vasopressin, and converting GABAergic inhibition to excitation in hypothalamic magnocellular AVP neurons. Moreover, we obtained results indicating that the E2 modulation of the activity and/or expression of NKCC1 (Cl− importer) and KCC2 (Cl− extruder) underpins the effect of E2 on the transition of GABAergic transmission in AVP neurons. Lastly, we discovered that, in DOCA-salt-treated hypertensive ovariectomized rats, CLP290 (prodrug of the KCC2 activator CLP257, intraperitoneal injections) lowered blood pressure, and plasma AVP level and hyperpolarized GABA equilibrium potential to prevent GABAergic excitation from emerging in the AVP neurons of these animals. Conclusion Based on these results, we conclude that E2 inhibits salt-dependent hypertension by suppressing GABAergic excitation to decrease the hormonal output of AVP neurons.

scholarly journals Phosphorylation status of liver by 31P-n.m.r. spectroscopy, and its implications for metabolic control. A comparison of 31P-n.m.r. spectroscopy (in vivo and in vitro) with chemical and enzymic determinations of ATP, ADP and Pi

1985 ◽  
Vol 229 (1) ◽  
pp. 141-151 ◽  
Author(s):  
R A Iles ◽  
A N Stevens ◽  
J R Griffiths ◽  
P G Morris
Keyword(s):  
Metabolic Control ◽  
Rat Liver ◽  
Cell Water ◽  
Organic Phosphates

An investigation into the measurement of Pi and ADP in rat liver in vivo and in freeze-clamped extracts by 31P-n.m.r. spectroscopy was carried out. The concentration of Pi estimated in vivo is less than 25% [1 mM (mumol/ml of cell water)] of the value obtained from freeze-clamped liver (4 mM), whereas ADP in vivo is undetectable (1.4 mM in vitro). At 5 min after infusion of 750 mg of fructose/kg, the Pi content of liver extracts fell to 1.3 mM, whereas Pi is undetectable in vivo under these conditions [Griffiths, Stevens, Gadian, Iles & Porteous (1980) Biochem. Soc. Trans. 8, 641]. The results indicate that the lower Pi and ADP concentrations found in vivo may be due to compartmentation or binding rather than to degradation of labile organic phosphates during extraction. The results are discussed with reference to previous measurements of liver phosphates and investigations of compartmentation in the liver, as are some of the possible consequences for metabolic control in the liver of low ADP and Pi concentrations.

Red cell magnesium in rainbow trout

1988 ◽  
Vol 66 (4) ◽  
pp. 929-933
Author(s):  
A. H. Houston ◽  
J. D. Gray
Keyword(s):  
Magnesium Content ◽  
Magnesium Concentration ◽  
Equilibrium Potential ◽  
Red Cell ◽  
High Potassium ◽  
High Sodium ◽  
Wide Range ◽  
Plasma Magnesium

In vivo erythrocytic magnesium concentration was significantly correlated with red cell potassium level and potassium equilibrium potential, but not with specimen weight, plasma magnesium, red cell sodium and chloride, or chloride equilibrium potential. To examine the relationship between magnesium and potassium, potassium levels were manipulated in vitro with catecholamine, furosemide, ouabain, and valinomycin. Over a wide range of normal and supranormal potassium concentrations, a significant correlation between magnesium and potassium levels was evident. However, depletion of potassium following exposure to ouabain and valinomycin led to a shift from the normal high potassium/low sodium relationship to a high sodium/low potassium state with progressive increases in magnesium content. Correlation of magnesium with potassium was lost, and a significant correlation with red cell sodium, but not with sodium equilibrium potential, was evident. Red cell magnesium concentration appears, therefore, to be influenced by the preponderant univalent cellular cation rather than potassium per se.

The bumetanide-sensitive Na-K-2Cl cotransporter NKCC1 as a potential target of a novel mechanism-based treatment strategy for neonatal seizures

2008 ◽  
Vol 25 (3) ◽  
pp. E22 ◽  
Author(s):  
Kristopher T. Kahle ◽  
Kevin J. Staley
Keyword(s):  
Motor Development ◽  
Specific Inhibitor ◽  
Volume Overload ◽  
Seizure Threshold ◽  
Equilibrium Potential ◽  
Neonatal Seizures ◽  
Mature Adult ◽  
Gabaergic Neurotransmission

Seizures that occur during the neonatal period do so with a greater frequency than at any other age, have profound consequences for cognitive and motor development, and are difficult to treat with the existing series of antiepileptic drugs. During development, γ-aminobutyric acid (GABA)ergic neurotransmission undergoes a switch from excitatory to inhibitory due to a reversal of neuronal chloride (Cl–) gradients. The intracellular level of chloride ([Cl–]i) in immature neonatal neurons, compared with mature adult neurons, is about 20–40 mM higher due to robust activity of the chloride-importing Na-K-2Cl cotransporter NKCC1, such that the binding of GABA to ligand-gated GABAA receptor-associated Cl– channels triggers Cl– efflux and depolarizing excitation. In adults, NKCC1 expression decreases and the expression of the genetically related chloride-extruding K-Cl cotransporter KCC2 increases, lowering [Cl–]i to a level such that activation of GABAA receptors triggers Cl– influx and inhibitory hyperpolarization. The excitatory action of GABA in neonates, while playing an important role in neuronal development and synaptogenesis, accounts for the decreased seizure threshold, increased seizure propensity, and poor efficacy of GABAergic anticonvulsants in this age group. Bumetanide, a furosemide-related diuretic already used to treat volume overload in neonates, is a specific inhibitor of NKCC1 at low doses, can switch the GABA equilibrium potential of immature neurons from depolarizing to hyperpolarizing, and has recently been shown to inhibit epileptic activity in vitro and in vivo in animal models of neonatal seizures. The fundamental role of NKCC1 in establishing excitatory GABAergic neurotransmission in the neonate makes it a tempting target of a novel mechanism-based anticonvulsant strategy that could utilize the well-known pharmacology of bumetanide to help treat neonatal seizures.

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