Functional characterization of voltage-gated K+ channels in mouse pulmonary artery smooth muscle cells

Mice are useful animal models to study pathogenic mechanisms involved in pulmonary vascular disease. Altered expression and function of voltage-gated K⁺ (K_V) channels in pulmonary artery smooth muscle cells (PASMCs) have been implicated in the development of pulmonary arterial hypertension. K_V currents (I_K(V)) in mouse PASMCs have not been comprehensively characterized. The main focus of this study was to determine the biophysical and pharmacological properties of I_K(V) in freshly dissociated mouse PASMCs with the patch-clamp technique. Three distinct whole cell I_K(V) were identified based on the kinetics of activation and inactivation: rapidly activating and noninactivating currents (in 58% of the cells tested), rapidly activating and slowly inactivating currents (23%), and slowly activating and noninactivating currents (17%). Of the cells that demonstrated the rapidly activating noninactivating current, 69% showed I_K(V) inhibition with 4-aminopyridine (4-AP), while 31% were unaffected. Whole cell I_K(V) were very sensitive to tetraethylammonium (TEA), as 1 mM TEA decreased the current amplitude by 32% while it took 10 mM 4-AP to decrease I_K(V) by a similar amount (37%). Contribution of Ca²⁺-activated K⁺ (K_Ca) channels to whole cell I_K(V) was minimal, as neither pharmacological inhibition with charybdotoxin or iberiotoxin nor perfusion with Ca²⁺-free solution had an effect on the whole cell I_K(V). Steady-state activation and inactivation curves revealed a window K⁺ current between –40 and –10 mV with a peak at –31.5 mV. Single-channel recordings revealed large-, intermediate-, and small-amplitude currents, with an averaged slope conductance of 119.4 ± 2.7, 79.8 ± 2.8, 46.0 ± 2.2, and 23.6 ± 0.6 pS, respectively. These studies provide detailed electrophysiological and pharmacological profiles of the native K_V currents in mouse PASMCs. K_V channels     

A Major Role for Transcellular Ca2+ Ion Currents in Cell Elongation in The Unicellular Green Alga Closterium

In semicells of the unicellular green alga Closterium that areundergoing elongation, transcellular ion currents enter theelongating region of the cell and leave via the non-elongatingregion of the cell, as in the case of many other tip-growingorganisms. The density of the inwardly and outwardly directedcurrents was 142.5±63.7 nA cm^–2 (n=42) and 109.3±46.5nA cm^–2 (n=33), respectively, at the respective regionsof the cells. Both currents clearly decreased with decreasesin the external concentration of Ca²⁺ ions, and they were completelyblocked by addition of Ca²⁺-channel blockers, such as 20 µMLaCl₃, to the external medium. Increases in pH up to 10.2 hadno effect on the currents, but a decrease in pH from 7.5 to5.7 or 4.5 resulted in an explosive increase in the currents.Removal of external K⁺ and Cl^– ions induced some increasesin the currents, but removal of external Na⁺ Mg²⁺ plus and ions had little effect on the currents. A major part of thecurrents may be carried by Ca²⁺ ions, while H⁺, K⁺ and Cl^–ions may play a minor role as members of the group of ions thatcarry the currents. Thus, there is a clear relationship betweenCa²⁺ ion currents and elongation in Closterium. (Received April 30, 1992; Accepted July 13, 1992)     

Age-dependent response of the electrocardiogram to K(+) channel blockers in mice

Developmental changes in electrocardiogram (ECG) andresponse to selective K⁺ channelblockers were assessed in conscious, unsedated neonatal (days 1, 7, 14) and adult male mice(>60 days of age). Mean sinus R-R interval decreased from 120 ± 3 ms in day 1 to 110 ± 3 ms inday 7, 97 ± 3 ms inday 14, and 81 ± 1 ms in adultmice (P < 0.001 by ANOVA; all 3 groups different from day 1). Inparallel, the mean P-R interval progressively decreased duringdevelopment. Similarly, the mean Q-T interval decreased from 62 ± 2 ms in day 1 to 50 ± 2 ms inday 7, 47 ± 8 ms inday 14 neonatal mice, and 46 ± 2 ms in adult mice (P < 0.001 byANOVA; all 3 groups are significantly different fromday 1).Q-T_c was calculated asQ- interval.Q-T_c significantly shortened from179 ± 4 ms in day 1 to 149 ± 5 ms in day 7 mice(P < 0.001). In addition, the J junction-S-T segment elevation observed in day1 neonatal mice resolved by day14. Dofetilide (0.5 mg/kg), the selective blocker ofthe rapid component of the delayed rectifier(I_Kr) abolished S-T segment elevation and prolonged Q-T andQ-T_c intervals in day 1 neonates but not in adult mice.In contrast, 4-aminopyridine (4-AP, 2.5 mg/kg) had no effect onday 1 neonates but in adults prolongedQ-T and Q-T_c intervals andspecifically decreased the amplitude of a transiently repolarizingwave, which appears as an r' wave at the end of the apparent QRSin adult mice. In conclusion, ECG intervals and configuration changeduring normal postnatal development in the mouse.K⁺ channel blockers affect themouse ECG differently depending on age. These data are consistent withthe previous findings that the dofetilide-sensitiveI_Kr is dominantin day 1 mice, whereas 4-AP-sensitivecurrents, the transiently repolarizingK⁺ current, and the rapidlyactivating, slowly inactivating K⁺current are the dominant K⁺currents in adult mice. This study provides background information useful for assessing abnormal development in transgenic mice.

     

A new approach to normalize myocardial temperature in the open-chest pig model

To prevent unphysiological temperaturefluctuations in the myocardium in the open-chest model, we constructeda thermocage. Five pigs under pentobarbital sodium anesthesia underwentrepetitive left anterior descending (LAD) coronary arteryocclusions. Myocardial temperature was measured without any thoracictemperature-controlling device and in the presence of either a heatinglamp or the thermocage. Without any thoracic temperature-controllingdevice, the temperature at 5-mm myocardial depth was 1.28 ± 0.33°C below the intra-abdominal temperature(P < 0.05). During a proximal 5-minLAD occlusion, myocardial temperature decreased by 1.86 ± 1.02°C in the ischemic area (P < 0.05). Both the heating lamp and the thermocage abolished thedifference between intra-abdominal and myocardial temperatures andprevented the decrease in myocardial temperature duringischemia. Only the thermocage minimized myocardial temperaturefluctuations due to air currents and prevented epicardial exsiccation.We conclude that either a thermocage or a heating lamp may be used tonormalize myocardial temperature in the open-chest pig model. However,the thermocage is superior to the lamp in minimizing temperaturefluctuations and preventing epicardial exsiccation.

     

Myocardial blood flow heterogeneity in shock and small-volume resuscitation in pigs with coronary stenosis

Kleen, Martin, Martin Welte, Peter Lackermeier, OliverHabler, Gregor Kemming, and Konrad Messmer. Myocardial blood flowheterogeneity in shock and small-volume resuscitation in pigs withcoronary stenosis. J. Appl. Physiol.83(6): 1832-1841, 1997.We analyzed the effects of shock andsmall-volume resuscitation in the presence of coronary stenosis onfractal dimension (D) and spatialcorrelation (SC) of regional myocardial perfusion. Hemorrhagic shockwas induced and maintained for 1 h. Pigs were resuscitated withhypertonic saline-dextran 60 [HSDex, 10% of shed blood volume(SBV)] or normal saline (NS; 80% of SBV). Therapy was continuedafter 30 min with dextran (10% SBV). At baseline, D was 1.39 ± 0.06 (mean ± SE;HSDex group) and 1.34 ± 0.04 (NS group). SC was 0.26 ± 0.07 (HSDex) and 0.26 ± 0.04 (NS). Left anterior descending coronaryartery stenosis changed neither D norSC. Shock significantly reduced D(i.e., homogenized perfusion): 1.26 ± 0.06 (HSDex) and 1.23 ± 0.05 (NS). SC was increased: 0.41 ± 0.1 (HSDex) and 0.48 ± 0.07 (NS). Fluid therapy with HSDex further decreasedD to 1.22 ± 0.05, whereas NS didnot change D. SC was increased by bothHSDex (0.56 ± 0.1) and NS (0.53 ± 0.06). At 1 h afterresuscitation, SC was constant in both groups, andD was reduced only in the NS group(1.18 ± 0.02). We conclude that hemorrhagic shock homogenizedregional myocardial perfusion in coronary stenosis and that fluidtherapy failed to restore this.

     

Modulation of K+ currents in monocytes by VCAM-1 and E-selectin on activated human endothelium

Resting membrane potential (RMP) and whole cell currents wererecorded in human THP-1 monocytes adherent to polystyrene, unstimulated human umbilical vein endothelial cells (HUVECs),lipopolysaccharide (LPS)-treated HUVECs, immobilizedE-selectin, or vascular cell adhesion molecule 1 (VCAM-1)using the patch-clamp technique. RMP after 5 h on polystyrene was24.3 ± 1.7 mV (n = 42) with delayed rectifier K⁺(I_dr) andCl currents(I_Cl) presentin >75% of the cells. Inwardly rectifying K⁺ currents(I_ir) werepresent in only 14% of THP-1 cells. Adherence to unstimulated HUVECsor E-selectin for 5 h had no effect on I_ir orI_Cl but decreasedI_dr. Five hoursafter adherence to LPS-treated HUVECs, outward currents were unchanged,but I_ir waspresent in 81% of THP-1 cells. A twofold increase inI_ir and ahyperpolarization (41.3 ± 3.7 mV,n = 16) were abolished by pretreatmentof THP-1 cells with cycloheximide, a protein synthesis inhibitor, orherbimycin A, a tyrosine kinase inhibitor, or by pretreatment of theLPS-treated HUVECs with anti-VCAM-1. Only a brief (15-min) interactionbetween THP-1 cells and LPS-treated HUVECs was required toinduce I_ir expression 5 h later. THP-1 cells adherent to VCAM-1 exhibited similarconductances to cells adherent to LPS-treated HUVECs. Thus engagementof specific integrins results in selective modulation of differentK⁺ conductances.

     

Electrophysiological characterization of murine HL-5 atrial cardiomyocytes

HL-5 cells are cultured murine atrial cardiomyocytes and have been used in studies to address important cellular and molecular questions. However, electrophysiological features of HL-5 cells have not been characterized. In this study, we examined such properties using whole cell patch-clamp techniques. Membrane capacitance of the HL-5 cells was from 8 to 62 pF. The resting membrane potential was –57.8 ± 1.4 mV (n = 51). Intracellular injection of depolarizing currents evoked action potentials (APs) with variable morphologies in 71% of the patched cells. Interestingly, the incidence of successful, current-induced APs positively correlated with the hyperpolarizing degrees of resting membrane potentials (r = 0.99, P < 0.001). Only a few of the patched cells (4 of 51, 7.8%) exhibited spontaneous APs. The muscarinic agonist carbachol activated the acetylcholine-activated K⁺ current and significantly shortened the duration of APs. Immunostaining confirmed the presence of the muscarinic receptor type 2 in HL-5 cells. The hyperpolarization-activated cation current (I_f) was detected in 39% of the patched cells. The voltage to activate 50% of I_f channels was –73.4 ± 1.2 mV (n = 12). Voltage-gated Na⁺, Ca²⁺, and K⁺ currents were observed in the HL-5 cells with variable incidences. Compared with the adult mouse cardiomyocytes, the HL-5 cells had prolonged APs and small outward K⁺ currents. Our data indicate that HL-5 cells display significant electrophysiological heterogeneity of morphological appearance of APs and expression of functional ion channels. Compared with adult murine cardiomyocytes, HL-5 cells show an immature phenotype of cardiac AP morphology. action potential; ion channel; muscarinic receptor     

Structure, function, and genomic organization of human Na(+)-dependent high-affinity dicarboxylate transporter

We have clonedand functionally characterized the human Na⁺-dependenthigh-affinity dicarboxylate transporter (hNaDC3) from placenta. ThehNaDC3 cDNA codes for a protein of 602 amino acids with 12 transmembrane domains. When expressed in mammalian cells, the clonedtransporter mediates the transport of succinate in the presence ofNa⁺ [concentration of substrate necessary for half-maximaltransport (K_t) for succinate = 20 ± 1 µM]. Dimethylsuccinate also interacts with hNaDC3. TheNa⁺-to-succinate stoichiometry is 3:1 and concentration ofNa⁺ necessary for half-maximal transport(K^Na+_0.5) is 49 ± 1 mM as determined by uptake studies withradiolabeled succinate. When expressed in Xenopuslaevis oocytes, hNaDC3 induces Na⁺-dependent inwardcurrents in the presence of succinate and dimethylsuccinate. At amembrane potential of 50 mV,K^Suc_0.5 is 102 ± 20 µM andK^Na+_0.5 is 22 ± 4 mM as determined by the electrophysiological approach. Simultaneous measurements of succinate-evoked charge transfer andradiolabeled succinate uptake in hNaDC3-expressing oocytes indicate acharge-to-succinate ratio of 1:1 for the transport process, suggestinga Na⁺-to-succinate stoichiometry of 3:1. pH titration ofcitrate-induced currents shows that hNaDC3 accepts preferentially thedivalent anionic form of citrate as a substrate. Li⁺inhibits succinate-induced currents in the presence of Na⁺.Functional analysis of rat-human and human-rat NaDC3 chimeric transporters indicates that the catalytic domain of the transporter lies in the carboxy-terminal half of the protein. The humanNaDC3 gene is located on chromosome20q12-13.1, as evidenced by fluorescent in situ hybridization. Thegene is >80 kbp long and consists of 13 exons and 12 introns.

     

Carbachol induces oscillations in membrane potential and intracellular calcium in a colonic tumor cell line, HT-29

The patch-clamp technique was used to study the effects ofcarbachol (CCh) on HT-29 cells. During CCh exposure, the cells (n = 23) depolarized close to theequilibrium potential forCl(;48 mV) and the membrane potential then started to oscillate(16/23 cells). In voltage-clamp experiments, similar oscillations inwhole cell currents could be demonstrated. The whole cell conductanceincreased from 225 ± 25 pS in control solution to 6,728 ± 1,165 pS (means ± SE, n = 17). Insubstitution experiments (22 mMCl in bath solution, = 0 mV), the reversal potential changed from 41.6 ± 2.2 mV(means ± SE, n = 9) to 3.2 ± 2.0 mV (means ± SE, n = 7).When the cells were loaded with the calcium-sensitive fluorescent dye,fluo 3, and simultaneously patch clamped, CCh caused a synchronousoscillating pattern of fluorescence and membrane potential. Incell-attached patches, the CCh-activated currents reversed at arelative membrane potential of 1.9 ± 3.7 mV (means ± SE,n = 11) with control solution in thepipette and at 46.2 ± 5.3 mV (means ± SE,n = 10) with a 15 mMCl solution in the pipette.High K⁺ (144 mM) did not changethe reversal potential significantly (P  0.05, n = 8). In inside-out patches,calcium-dependent Clchannels could be demonstrated with a conductance of 19 pS(n = 7). It is concluded that CChcauses oscillations in membrane potential that involvecalcium-dependent Clchannels and a K⁺ permeability.

     

{Omega}-3 and {omega}-6 polyunsaturated fatty acids block HERG channels

Dietary polyunsaturated fatty acids (PUFAs) have been reported to exhibit antiarrhythmic properties, which have been attributed to their availability to modulate Na⁺, Ca²⁺, and several K⁺ channels. However, their effects on human ether-a-go-go-related gene (HERG) channels are unknown. In this study we have analyzed the effects of arachidonic acid (AA, -6) and docosahexaenoic acid (DHA, -3) on HERG channels stably expressed in Chinese hamster ovary cells by using the whole cell patch-clamp technique. At 10 µM, AA and DHA blocked HERG channels, at the end of 5-s pulses to –10 mV, to a similar extent (37.7 ± 2.4% vs. 50.2 ± 8.1%, n = 7–10, P > 0.05). 5,6,11,14-Eicosatetrayenoic acid, a nonmetabolizable AA analog, induced effects similar to those of AA on HERG current. Both PUFAs shifted the midpoint of activation curves of HERG channels by –5.1 ± 1.8 mV (n = 10, P < 0.05) and –11.2 ± 1.1 mV (n = 7, P < 0.01). Also, AA and DHA shifted the midpoint of inactivation curves by +12.0 ± 3.9 mV (n = 4; P < 0.05) and +15.8 ± 4.3 mV (n = 4; P < 0.05), respectively. DHA and AA accelerated the deactivation kinetics and slowed the inactivation kinetics at potentials positive to +40 mV. Block induced by DHA, but not that produced by AA, was higher when measured after applying a pulse to –120 mV (IO). Finally, both AA and DHA induced a use-dependent inhibition of HERG channels. In summary, block induced by AA and DHA was time, voltage, and use dependent. The results obtained suggest that both PUFAs bind preferentially to the open state of the channel, although an interaction with inactivated HERG channels cannot be ruled out for AA. K⁺ channel; membrane currents; ion channels; arrhythmia; antiarrhythmics     

cAMP-activated maxi-Cl(-) channels in native bovine pigmented ciliary epithelial cells

The eye’s aqueous humor is secreted by a bilayered ciliary epithelium comprising pigmented (PE) and nonpigmented (NPE) epithelial cell layers. Stromal Cl^– enters the PE cells and crosses gap junctions to the NPE cells for release into the aqueous humor. Maxi-Cl^– channels are expressed in PE cells, but their physiological significance is unclear. To address this question, excised patches and whole native bovine PE cells were patch clamped, and volume was monitored by calcein fluorescence. In symmetrical 130 mM NaCl, cAMP at the cytoplasmic surface of inside-out patches produced concentration-dependent activation of maxi-Cl^– channels with a unitary conductance of 272 ± 2 pS (n = 80). Voltage steps from 0 to ±80 mV, but not to ±40 mV, produced rapid channel inactivation consistent with the typical characteristics of maxi-Cl^– channels. cAMP also activated the maxi-Cl^– channels in outside-out patches. In both cases, maxi-Cl^– channels were reversibly inhibited by SITS and 5-nitro-2-(phenylpropylamino)benzoate (NPPB). Decreasing cytoplasmic Cl^– concentration reduced both open-channel probability and unitary conductance. Similarly, the membrane-permeant 8-bromo-cAMP stimulated outward and inward whole cell currents; the stimulation was larger at higher intracellular Cl^– concentration. As with unitary currents, cAMP-triggered whole cell currents displayed inactivation at ±80 but not at ±40 mV. Moreover, cAMP triggered NPPB-sensitive shrinkage of PE cells. The results suggest that cAMP directly activates maxi-Cl^– channels of native PE cells that contribute to Cl^– release particularly from Cl^–-loaded cells. These cAMP-activated channels provide a potential mechanism for reducing and modulating net aqueous humor secretion by facilitating Cl^– reabsorption into the ciliary stroma. cell volume; chloride secretion; aqueous humor formation     

Changes in agonist-antagonist EMG, muscle CSA, and force during strength training in middle-aged and older people

Effects of 6 mo of heavy-resistance trainingcombined with explosive exercises on neural activation of the agonistand antagonist leg extensors, muscle cross-sectional area (CSA) of thequadriceps femoris, as well as maximal and explosive strength wereexamined in 10 middle-aged men (M40; 42 ± 2 yr), 11 middle-agedwomen (W40; 39 ± 3 yr), 11 elderly men (M70; 72 ± 3 yr) and 10 elderly women (W70; 67 ± 3 yr). Maximal andexplosive strength remained unaltered during a 1-mo control period withno strength training. After the 6 mo of training, maximal isometric anddynamic leg-extension strength increased by 36 ± 4 and 22 ± 2%(P < 0.001) in M40, by 36 ± 3 and 21 ± 3% (P < 0.001) in M70,by 66 ± 9 and 34 ± 4% (P < 0.001) in W40, and by 57 ± 10 and 30 ± 3%(P < 0.001) in W70, respectively.All groups showed large increases (P < 0.05-0.001) in the maximum integrated EMGs (iEMGs) of theagonist vastus lateralis and medialis. Significant(P < 0.05-0.001) increasesoccurred in the maximal rate of isometric force productionand in a squat jump that were accompanied with increased(P < 0.05-0.01) iEMGs of theleg extensors. The iEMG of the antagonist biceps femoris muscle duringthe maximal isometric leg extension decreased in both M70 (from 24 ± 6 to 21 ± 6%; P < 0.05)and in W70 (from 31 ± 9 to 24 ± 4%;P < 0.05) to the same level asrecorded for M40 and W40. The CSA of the quadriceps femoris increasedin M40 by 5% (P < 0.05), in W40 by9% (P < 0.01), in W70 by 6%(P < 0.05), and in M70 by 2% (notsignificant). Great training-induced gains in maximal and explosivestrength in both middle-aged and elderly subjects were accompanied bylarge increases in the voluntary activation of the agonists, withsignificant reductions in the antagonist coactivation in the elderlysubjects. Because the enlargements in the muscle CSAs in bothmiddle-aged and elderly subjects were much smaller in magnitude, neuraladaptations seem to play a greater role in explaining strength andpower gains during the present strength-training protocol.

     

Progressive decrease of intramyocellular accumulation of H+ and Pi in human skeletal muscle during repeated isotonic exercise

The purpose of this study was to evaluatethe hypotheses that accumulation of hydrogen ions and/or inorganicphosphate (Pi) in skeletal muscle increases with repeated bouts ofisotonic exercise. ³¹P-Magnetic resonance spectroscopy wasused to examine the gastrocnemius muscle of seven highly aerobicallytrained females during four bouts of isotonic plantar flexion. Theexercise bouts (EX1-4) of 3 min and 18 swere separated by 3 min and 54 s of complete rest. Muscle ATP did notchange during the four bouts. Phosphocreatine (PCr) degradation duringEX1 (13.3 ± 2.4 mmol/kg wet weight) was higher(P < 0.01) compared with EX3-4(9.7 ± 1.6 and 9.6 ± 1.8 mmol/kg wet weight, respectively).The intramyocellular pH at the end of EX1 (6.87 ± 0.05) was significantly lower (P < 0.001) than thoseof EX2 (6.97 ± 0.02), EX3 (7.02 ± 0.01), and EX4 (7.02 ± 0.02). Total Pi anddiprotonated Pi were significantly higher (P < 0.001)at the end of EX1 (17.3 ± 2.7 and 7.8 ± 1.6 mmol/kg wet weight, respectively) compared with the values at the end of EX3 and EX4. The monoprotonated Pi at the endof EX1 (9.5 ± 1.2 mmol/kg wet weight) was alsosignificantly higher (P < 0.001) than that afterEX4 (7.5 ± 1.1 mmol/kg wet weight). Subjects' ratingof perceived exertion increased (P < 0.001) towardexhaustion as the number of exercises progressed (7.1 ± 0.4, EX1; 8.0 ± 0.3, EX2; 8.5 ± 0.3, EX3; and 9.0 ± 0.4, EX4; scale from 0 to10). The present results indicate that human muscle fatigue during repeated intense isotonic exercise is not due to progressive depletion of high energy phosphates nor to intracellular accumulation of hydrogenions, total, mono-, or diprotonated Pi.

     

Photosynthesis in the Tapinanthus-Diplorhynchus Mistletoe-Host Relationship

Hill reaction activity and gas exchange were studied in thesouthern African mistletoe Tapinanthus vittatus, its host treeDiplorhynchus condylocarpon and uninfected trees of the samespecies. Photosynthetic potential was assessed by measuringHill reaction activities in isolated thylakoids: reaction ratesin the parasite were less than half those of the host or uninfectedtrees. In addition, parasite thylakoids were more sensitiveto heat treatment than those of the trees. Mean maximum CO₂assimilation rates (±s.e.) were significantly lower (4·2±0·24µmol m^-1 s^-1) in the parasite, compared to both uninfectedtrees (7·6±0·22), and those of the infectedhost (5·2±0·27). Chlorophyll contents (±s.e.)were 800±40 mg m^-2 in the parasite, lower in uninfectedtrees (490±80) and lowest in infected trees (310±70).CO₂ assimilation rates calculated on a per unit chlorophyllbasis were similar in parasitized and non-parasitized trees,58±4·3 and 56±2·2 µmol mgchlorophyll^-1 h^-1 respectively, whereas the parasite's maximumCO₂ assimilation rate was considerably lower (20±1·2).Parasite transpiration rates were approximately double thoseof infected trees throughout the daylight period, and somewhathigher than uninfected trees from mid-morning onwards. Overall,parasite were generally less photosynthetically active thanhost plants; additionally, parasite infection was seen to reducehost carbon assimilation rates compared to those of uninfectedtrees.Copyright 1993, 1999 Academic Press Tapinanthus vittatus, Diplorhynchus condylocarpon, mistletoe, photosynthesis, Hill reaction, Zimbabwe     

Influence of K-Cl cotransporter activity on activation of volume-sensitive Cl- channels in human osteoblasts

The whole cell recording mode of the patch-clamp technique was used to study the effect of hypotonic NaCl or isotonic high-KCl solution on membrane currents in a human osteoblast-like cell line, C1. Both hypotonic NaCl or isotonic high-KCl solution activated Cl^– channels expressed in these cells as described previously. The reversal potential of the induced Cl^– current is more negative when activated through hypotonic NaCl solution (–47 ± 5 mV; n = 6) compared with activation through isotonic high-KCl solution (–35 ± 3 mV; n = 8). This difference can be explained by an increase in intracellular [Cl^–] through the activity of a K-Cl cotransporter. Potassium aspartate was unable to activate the current, and furosemide or DIOA suppressed the increase in Cl^– current induced by isotonic high-KCl solution. In addition, we used the polymerase chain reaction to demonstrate the presence of KCC1–KCC4 mRNA in the osteoblast-like cell line. From these results, we conclude that human osteoblasts express functional K-Cl cotransporters in their cell membrane that seem to be able to induce the indirect activation of volume-sensitive Cl^– channels by KCl through an increase in the intracellular ion concentration followed by water influx and cell swelling. potasium-chloride cotransporter; KCC1–KCC4; chloride channels; extracellular potassium concentration buffering     

Characterization of the rat Na+/nucleoside cotransporter 2 and transport of nucleoside-derived drugs using electrophysiological methods

The Na⁺-dependent nucleoside transporter 2 (CNT2) mediates active transport of purine nucleosides and uridine as well as therapeutic nucleoside analogs. We used the two-electrode voltage-clamp technique to investigate rat CNT2 (rCNT2) transport mechanism and study the interaction of nucleoside-derived drugs with the transporter expressed in Xenopus laevis oocytes. The kinetic parameters for sodium, natural nucleosides, and nucleoside derivatives were obtained as a function of membrane potential. For natural substrates, apparent affinity (K_0.5) was in the low micromolar range (12–34) and was voltage independent for hyperpolarizing membrane potentials, whereas maximal current (I_max) was voltage dependent. Uridine and 2'-deoxyuridine analogs modified at the 5-position were substrates of rCNT2. Lack of the 2'-hydroxyl group decreased affinity but increased I_max. Increase in the size and decrease in the electronegativity of the residue at the 5-position affected the interaction with the transporter by decreasing both affinity and I_max. Fludarabine and formycin B were also transported with higher I_max than uridine and moderate affinity (102 ± 10 and 66 ± 6 µM, respectively). Analysis of the pre-steady-state currents revealed a half-maximal activation voltage of about –39 mV and a valence of about –0.8. K_0.5 for Na⁺ was 2.3 mM at –50 mV and decreased at hyperpolarizing membrane potentials. The Hill coefficient was 1 at all voltages. Direct measurements of radiolabeled nucleoside fluxes with the charge associated showed a ratio of two positive inward charges per nucleoside, suggesting a stoichiometry of two Na⁺ per nucleoside. This discrepancy in the number of Na⁺ molecules that bind rCNT2 may indicate a low degree of cooperativity between the Na⁺ binding sites. two-electrode voltage clamp; concentrative nucleoside transport; presteady-state currents     

Changes in regulation of sodium/calcium exchanger of avian ventricular heart cells during embryonic development

It has been suggested that the sodium/calcium exchanger NCX1 may have a more important physiological role in embryonic and neonatal hearts than in adult hearts. However, in chick heart sarcolemmal vesicles, sodium-dependent calcium transport is reported to be small and, moreover, to be 3–12 times smaller in hearts at embryonic day (ED) 4–5 than at ED18, the opposite of what would be expected of a transporter that is more important in early development. To better assess the role of NCX1 in calcium regulation in the chick embryonic heart, we measured the activity of NCX1 in chick embryonic hearts as extracellular calcium-activated exchanger current (I_NCX) under controlled ionic conditions. With intracellular calcium concentration ([Ca²⁺]_i) = 47 nM, I_NCX density increased from 1.34 ± 0.28 pA/pF at ED2 to 3.22 ± 0.55 pA/pF at ED11 (P = 0.006); however, with [Ca²⁺]_i = 481 nM, the increase was small and statistically insignificant, from 4.54 ± 0.77 to 5.88 ± 0.73 pA/pF (P = 0.20, membrane potential = 0 mV, extracellular calcium concentration = 2 mM). Plots of I_NCX density against [Ca²⁺]_i were well fitted by the Michaelis-Menton equation and extrapolated to identical maximal currents for ED2 and ED11 cells (extracellular calcium concentration = 1, 2, or 4 mM). Thus the increase in I_NCX at low [Ca²⁺]_i appeared to reflect a developmental change in allosteric regulation of the exchanger by intracellular calcium rather than an increase in the membrane density of NCX1. Supporting this conclusion, RT-PCR demonstrated little change in the amount of mRNA encoding NCX1 expression from ED2 through ED18. NCX1; chick embryo; allosteric regulation; sodium/calcium exchange current     

Measurement of Yield Threshold and Cell Wal Extensibility of Intact Wheat Roots under Different Ionic, Osmotic and Temperature Treatments

Yield stress threshold (Y) and volumetric extensibility () arethe rheological properties that appear to control root growth.In this study they were measured in wheat roots by means ofparallel measurement of the growth rate (r) of intact wheatroots and of the turgor pressures (P) of individual cells withinthe expansion zone. Growth and turgor pressure were manipulatedby immersion in graded osmoticum (mannitol) solutions. Turgorwas measured with a pressure probe and growth rate by visualobservation. The influence of various growth conditions on Yand was investigated; (a) At 27 °C.In 0.5 mol m^–3 CaCl₂ r, P, Y and were20.7±4.6 µm min^–1, 0.77±0.05 MPa,0.07±0.03 MPa and 26±1.9 µm min^–1MPa^–1 (expressed as increase in length), respectively.Following 24 h growth in 10 mol m^–3 KC1 these parametersbecame 12.3±3.5 µm min^–1, 0.72±0.04MPa, 0.13±0.01 MPa and 21±0.7 µm min^–1MPa^–1. After 24 h osmotic adjustment in 150 mol m^–3mannitol/0.5 mol m^–3 CaCl₂ r= 19.6±4.2 µmmin^–1, P = 0.68±0.05 MPa and Y and were 0.07±0.04MPa and 30±0.2 µm min^–1 MPa^–01, respectively.After 24 h growth in 350 mol m^–3 mannitol/0.5 mol m^–3CaCl₂ r= 13.3±4.1 µm min^–1, P= 0.58±0.07MPa, Y=0.12±0.01 MPa and ø 32±0.2 tim min^–1MPa^–1. During osmotic adjustment in 200 mol m^–3mannitol/0.5 mol m^–3 CaCl₂, with or without KCl, the recoveryof growth rate corresponded to turgor pressure recovery (t1/2approximately 3 h). (b) At 15 °C. Lowered temperature dramatically influencedthe growth parameters which became r= 8.3±2.8 um min^–1,P=0.78 MPa, r=<0.2 MPa and =15±0.1 µm min^–1MPa^–1. Therefore, Y and are influenced by 10 mol m^–3 K⁺ ionsand low temperature. In each case the effective pressure forgrowth (P-Y) was large indicating that small fluctuations ofsoil water potential will not stop root elongation. Key words: Yield threshold, cell wall extensibility, wheat root growth, temperature, turgor pressur     

Recovery of diaphragmatic function in awake sheep after two approaches to thoracic surgery

Imanaka, Hideaki, William R. Kimball, John C. Wain, MasajiNishimura, Kenichi Okubo, Dean Hess, and Robert M. Kacmarek. Recovery of diaphragmatic function in awake sheep after two approaches to thoracic surgery. J. Appl.Physiol. 83(5): 1733-1740, 1997.Video-assistedthoracoscopic surgery (VATS) is replacing thoracotomy, but no study hasaddressed the extent or duration of VATS-induced diaphragmaticalteration. We hypothesized that VATS would impair diaphragmaticfunction less and return diaphragmatic function faster thanthoracotomy. In eight sheep, sonomicrometers were randomly implanted onthe right costal diaphragm via VATS or thoracotomy. Diaphragmaticresting length, shortening fraction, and respiratory function weremeasured weekly during quiet breathing (QB) andCO₂ rebreathing for 4 wk. ForVATS, shortening fraction was smallest onpostoperative days 1 (POD 1) (6.4 ± 3.4 and12.9 ± 8.7% during QB and 10%CO₂ rebreathing, respectively) and7 (6.3 ± 3.4 and 16.9 ± 4.0%during QB and 10% CO₂rebreathing, respectively) and recovered by 3 wk (13.2 ± 1.8 and28.9 ± 8.0% during QB and 10%CO₂ rebreathing, respectively).For thoracotomy, shortening fraction at 10%CO₂ rebreathing was smaller onPODs 1, 7, 14 (15.9 ± 7.1, 13.6 ± 5.4, and 19.0 ± 6.9%) than onPOD 28 (29.9 ± 8.2%), but notduring QB on POD 1 or7 (7.5 ± 3.8 and 3.4 ± 2.6%)compared with POD 28 (10.7 ± 8.7%). Shortening fraction did not differ between surgeries. There wasno group difference in minute ventilation, respiratory rate,transdiaphragmatic pressure, or esophageal and gastric pressures. Inconclusion, although shortening fraction recovered faster for VATS,this translated into insignificant functional differences.

     

Cerebral vasomotor reactivity at high altitude in humans

The purpose of this study was twofold:1) to determine whether at highaltitude cerebral blood flow (CBF) as assessed during CO₂ inhalation and duringhyperventilation in subjects with acute mountain sickness (AMS) wasdifferent from that in subjects without AMS and2) to compare the CBF as assessedunder similar conditions in Sherpas at high altitude and in subjects atsea level. Resting control values of blood flow velocity in themiddle cerebral artery (V_MCA), pulseoxygen saturation (Sa_O2), andtranscutaneous PCO₂ were measured at4,243 m in 43 subjects without AMS, 17 subjects with AMS, 20 Sherpas,and 13 subjects at sea level. Responses ofCO₂ inhalation andhyperventilation onV_MCA,Sa_O2, and transcutaneous PCO₂ were measured, and the cerebralvasomotor reactivity (VMR = V_MCA/PCO₂)was calculated as the fractional change ofV_MCA per Torrchange of PCO₂, yielding ahypercapnic VMR and a hypocapnic VMR. AMS subjects showeda significantly higher resting controlV_MCA than didno-AMS subjects (74 ± 22 and 56 ± 14 cm/s, respectively;P < 0.001), andSa_O2 was significantly lower (80 ± 8 and 88 ± 3%, respectively; P < 0.001). Resting control V_MCA values inthe sea-level group (60 ± 15 cm/s), in the no-AMS group, and inSherpas (59 ± 13 cm/s) were not different. Hypercapnic VMR valuesin AMS subjects were 4.0 ± 4.4, in no-AMS subjects were 5.5 ± 4.3, in Sherpas were 5.6 ± 4.1, and in sea-level subjects were 5.6 ± 2.5 (not significant). Hypocapnic VMR values were significantly higher in AMS subjects (5.9 ± 1.5) compared with no-AMS subjects (4.8 ± 1.4; P < 0.005) but werenot significantly different between Sherpas (3.8 ± 1.1) and thesea-level group (2.8 ± 0.7). We conclude that AMS subjects havegreater cerebral hemodynamic responses to hyperventilation, higherV_MCAresting control values, and lower Sa_O2 compared with no-AMSsubjects. Sherpas showed a cerebral hemodynamic patternsimilar to that of normal subjects at sea level.