bluuebunny/crossref_metadata_2025 huggingface dataset:
Altered Ca2+ dynamics in single cardiac myocytes from renovascular hypertensive rats
Authors: Moore R. L. , Yelamarty R. V. , Misawa H. , Scaduto R. C. , Pawlush D. G. , Elensky M. , Cheung J. Y.
Published: December 2017

Several functional and biochemical characteristics of hypertrophied hearts isolated from rats with renovascular hypertension provide indirect evidence that cellular Ca2+ dynamics during myocardial contraction-relaxation are altered. In this study, intracellular Ca2+ concentration ([Ca2+]i) dynamics were examined in paced left ventricular (LV) myocytes isolated from rats with hypertension (HYP) induced by partial occlusion of the left renal artery and from normotensive rats (Sham). Characteristic myocardial changes produced by renovascular hypertension included a 40% increase in LV weight and a 3.6-fold increase in the fractional expression of the beta-heavy chain of myosin in isolated LV myocytes. In periods of mechanical quiescence between contractions, basal [Ca2+]i values were similar in Sham and HYP LV myocytes. During a contraction-relaxation cycle in HYP myocytes, peak [Ca2+]i, +d[Ca2+]i/dt, and -d[Ca2+]i/dt were reduced, whereas the time required for [Ca2+]i to rise from a basal value to a peak value (time-to-peak [Ca2+]i) was unaffected. In both Sham and HYP myocytes, the fall in [Ca2+]i from peak to basal values could be approximated by a monoexponential rate constant, kf. Values for kf were significantly smaller in HYP than in Sham myocytes. After treatment with 4 microM isoproterenol, peak [Ca2+]i, +[Ca2+]i/dt, -d[Ca2+]i/dt, and kf increased in both Sham and HYP myocytes. In contrast, basal [Ca2+]i and time-to-peak [Ca2+]i did not change. Thus, despite recent reports of inefficiencies of beta-adrenergic receptor coupling, there was no evidence of blunted beta-adrenergic responsiveness in HYP myocytes with respect to [Ca2+]i dynamics during contraction-relaxation. Finally, no Sham vs. HYP differences in the number of specific [3H]-PN200-110 binding sites per cell in quiescent, rod-shaped myocytes were detected, but a significant reduction in [3H]-PN200-110 binding sites in an enriched sarcolemmal membrane fraction isolated from HYP animals was observed. These observations are suggestive of a reduction in slow, Ca2+ channel surface density in HYP myocytes. The results of this study clearly indicate that [Ca2+]i dynamics during contraction-relaxation in single left ventricular myocytes are affected by residence in a chronic setting of renovascular hypertension. In addition, the prolonged [Ca2+]i removal phase observed in HYP myocytes can be restored toward normal by beta-adrenergic agonists.
Ca2+ channel blockers distinguish between G protein-coupled pharmacomechanical Ca2+ release and Ca2+ sensitization
Authors: Kobayashi S. , Gong M. C. , Somlyo A. V. , Somlyo A. P.
Published: December 2017

The effects of Ca2+ channel blockers on two modes of G protein-mediated pharmacomechanical coupling, Ca2+ release and modulation of Ca2+ sensitivity of the contractile apparatus, were investigated. Smooth muscles were permeabilized with Staphylococcal alpha-toxin or with beta-escin to avoid effects due to block of sarcolemmal Ca2+ channels, while retaining receptor/G protein coupling. In permeabilized portal vein smooth muscle, verapamil and nifedipine inhibited Ca2+ release induced by an alpha 1-adrenergic agonist (phenylephrine) and by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), but not that induced by inositol 1,4,5-trisphosphate (InsP3). These Ca2+ channel blockers also did not block the phenylephrine- or GTP gamma S-induced force development at constant cytoplasmic Ca2+ ("Ca2+ sensitization"). An alpha 1-blocker (prazosin) inhibited both the Ca2(+)-releasing and Ca2(+)-sensitizing effects of phenylephrine, but not those of GTP gamma S, nor did it block InsP3-induced Ca2+ release. We conclude that Ca2+ channel blockers selectively uncouple the Ca2(+)-releasing, but not the Ca2(+)-sensitizing, component of pharmacomechanical coupling. These findings raise the possibility that pharmacomechanical Ca2+ release may be modulated by dihydropyridine binding proteins at the level of G proteins/phospholipase C and also indicate a divergence of the Ca2(+)-releasing and Ca2(+)-sensitizing effects at some step prior to phospholipase C.
Differential expression of amino acid transport systems A and ASC during erythroleukemia cell differentiation
Authors: Vadgama J. V. , Chan M. N. , Wu J. M.
Published: December 2017

The human erythroleukemic cell K-562 serves as an in vitro model to study changes in cell surface antigens and mechanisms regulating globin gene expression associated with in vivo erythropoiesis. In this report we have examined the regulation of amino acid transport systems, in particular, systems A and ASC, during differentiation of erythroleukemic cells. For additional comparison we examined the uptake of leucine, 3-aminoendobicyclo-(3,2,1)-octane-3-carboxylic acid (BCO), arginine, and glutamate. Hexamethylene-bis-acetamide (HMBA), dimethyl sulfoxide, and butyrate induce cell differentiation with a block in G1-G0 phase of the cell cycle. These agents caused a significant downregulation of 2-(methylamino)isobutyric acid uptake by system A. In contrast, the Na(+)-dependent threonine uptake by system ASC remained unaltered. The uptake of leucine, BCO, arginine, and glutamate by as yet unidentified systems was, however, stimulated after HMBA treatment. Hemin, a potent inducer of hemoglobin synthesis in K-562 cells, does not block cell cycle events and, interestingly, had no significant effect on both systems A and ASC. These differences in inducer actions suggest that system A activity may be related to specific stages of cell differentiation and perhaps to other cellular signals.
Characterization of ANF receptors in cultured renal cortical vascular smooth muscle cells
Authors: Bea M. L. , Dussaule J. C. , Bens M. , Ardaillou R.
Published: December 2017

Because atrial natriuretic factor (ANF) has been demonstrated to decrease resistances in cortical renal vessels in vivo, we studied 125I-ANF binding and ANF-dependent guanosine 3',5'-cyclic monophosphate (cGMP) production in subcultured vascular smooth muscle cells (VSMC) prepared from the rabbit renal cortex. 125I-ANF specific binding at 4 degrees C represented 70% of total binding and reached a plateau at 30-60 min. Equilibrium saturation binding curves suggested one group of high-affinity receptor sites (KD = 78 +/- 16 pM, Bmax = 45 +/- 11 fmol/mg) but were compatible with several groups exhibiting close binding parameters. ANF, [Ala7,Ala23]ANF (a linear analogue), and C-ANF-(4-23) (a ligand of C receptors) inhibited 125I-ANF binding at 37 degrees C with nearly similar potencies. In contrast, at 4 degrees C, complete or nearly complete inhibition of binding was obtained with ANF and linear ANF, the latter exhibiting the weakest potency, whereas C-ANF-(4-23) displaced only 35% of the tracer. ANF markedly stimulated cGMP accumulation, with a threshold concentration of 10 pM and a stimulation of 115 times basal value at 0.1 microM. Linear ANF was also stimulatory with a much weaker potency. Around 25% of 125I-ANF bound to cell surface was internalized at 37 degrees C. Phenylarsine oxide partially inhibited internalization as well as the inhibitory potency of C-ANF-(4-23) on 125I-ANF binding. As shown by high-performance liquid chromatography extracellular 125I-ANF was rapidly degraded at 37 degrees C into its 125I-COOH-terminal tripeptide and 125I-Tyr.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of endothelin-mediated calcium mobilization in vascular smooth muscle cells by isoproterenol
Authors: Xuan Y. T. , Watkins W. D. , Whorton A. R.
Published: December 2017

We have investigated the effect of isoproterenol on endothelin-induced Ca2+ mobilization in A10 vascular smooth muscle cells. Endothelin (ET) stimulates a rapid and sustained elevation of intracellular Ca2+ mediated by production of inositol phosphates, release of intracellular Ca2+, and activation of a plasmalemmal Ca2+ influx pathway. This influx pathway appears to be a L-type channel because it is inhibited by nicardipine and activated by BAY K 8644. Depolarization of the cells, by elevating extracellular K+, activated a pharmacologically similar channel and produced a similar change in intracellular Ca2+ concentration. Preincubation of cells with isoproterenol reduced the peak Ca2+ response to endothelin and blocked the sustained elevation. However, isoproterenol did not alter K(+)-induced Ca2+ entry. Thus it appears that ET-induced entry is mediated by intracellular signals and not by depolarization. With the use of cells incubated in Ca2(+)-free medium containing 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, isoproterenol was shown to inhibit Ca2+ release from intracellular pools by 36 +/- 3%. Furthermore, isoproterenol pretreatment or addition of adenosine 3',5'-cyclic monophosphate (cAMP) to saponin-permeabilized cells inhibited inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-induced Ca2+ release from intracellular sites. Similar effects were seen with forskolin. Propranolol reversed the inhibitory effects of isoproterenol. Isoproterenol pretreatment also inhibited the rapid formation of Ins(1,4,5)P3 and [2-3H]inositol 1,3,4,5-tetrakisphosphate stimulated by endothelin and reduced the sustained formation of these compounds. Finally, isoproterenol and forskolin led to a greater than 10-fold increase in intracellular cAMP levels. This stimulation of adenylate cyclase by isoproterenol was completely blocked by propranolol. It appears then that the beta-agonist isoproterenol interacts with a beta-adrenergic receptor, elevates cAMP, and thereby alters endothelin-induced Ca2+ mobilization. Inhibition of Ins(1,4,5)P3 formation, reduction in the responsiveness of the Ins(1,4,5)P3 intracellular receptor, and perhaps inhibition of ET-induced Ca2+ entry appear to be involved.
Long-term regulation of Na(+)-H+ exchange in vascular smooth muscle cells: role of protein kinase C
Authors: Mitsuka M. , Berk B. C.
Published: December 2017

Regulation of intracellular pH (pHi) plays an important role in vascular smooth muscle cell (VSMC) contractile tone and growth. We have shown that pHi in proliferating VSMC is more alkaline (7.25) than in growth-arrested cells (7.10). To study the Na(+)-H+ exchanger in the growth-dependent regulation of VSMC pHi, ethylisopropylamiloride (EIPA)-sensitive Na+ influx was measured. Exposure of growth-arrested VSMC to 10% serum initially increased Na+ influx (145% of baseline at 30 min), which then decreased (52% of baseline at 24 h). Serum-induced alterations in the kinetic properties of the Na(+)-H+ exchanger were studied by analysis of its external Na+ binding site properties. Exposure of growth-arrested VSMC to 10% serum for 24 h increased the Km for external Na+ from 54 to 380 mM, with a change in the Vmax from 155 to 199 nmol Na+.mg protein-1.min-1. The change in Km was due to activation of protein kinase C (PKC). Phorbol 12,13-dibutyrate caused a 48% decrease in EIPA-sensitive influx, the inactive 4 alpha-phorbol 12,13-didecanoate had no effect, and the PKC inhibitor sphingosine reversed the effect. Therefore, the Na(+)-H+ exchanger in VSMC is regulated in a growth-dependent manner via PKC.
Differential sorting of two glucose transporters expressed in insulin-sensitive cells
Authors: Piper R. C. , Hess L. J. , James D. E.
Published: December 2017

The insulin-regulatable glucose transporter (IRGT) is specifically expressed in muscle and fat cells and undergoes translocation from an intracellular compartment to the cell surface following acute insulin treatment. This study examined sorting differences between the IRGT and the homologous HepG2/erythrocyte/brain glucose transporter (HepG2 GT) when expressed together in insulin-responsive 3T3-L1 adipocytes. The ratio of the amount of transporter per unit protein in the plasma membrane fraction vs. the intracellular membrane fraction was 1:2 for the HepG2 GT and 1:30 for the IRGT. Insulin treatment increased the plasma membrane concentration of the IRGT by 10-fold and the HepG2 GT by 3.5-fold. This distribution was confirmed by confocal immunofluorescence microscopy. Differential sorting within intracellular organelles was evident by sucrose gradient analysis and immunoisolation of transporter vesicles and by double immunofluorescence labeling. We propose that differential sorting at an intracellular locus preferably withdraws the IRGT from a pathway which is in close communication with the plasma membrane, thus allowing the IRGT to regulate glucose entry into fat and muscle cells in a highly insulin-regulated fashion.
Skeletal muscle bioenergetics during frequency-dependent fatigue
Authors: Bridges C. R. , Clark B. J. , Hammond R. L. , Stephenson L. W.
Published: December 2017

The bioenergetic correlates of skeletal muscle fatigue were assessed in vivo with phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy. After surgical construction of latissimus dorsi muscle ventricles, seven beagles underwent 31P-NMR spectroscopy during 12-min exercise protocols at 25- and 85-Hz stimulation frequencies and during both isovolumetric and dynamic contractions. Exercise at 85 Hz was associated with significantly greater fatigue than exercise at 25 Hz. At both frequencies, the onset of exercise was associated with a marked increase in inorganic phosphate (Pi) and a decrease in phosphocreatine (PCr). As the muscle fatigued at 85 Hz but not at 25 Hz, the phosphorus spectra returned to near baseline with a decrease in Pi and increase in PCr. For a given amount of force generated, the Pi-to-PCr ratio was higher for dynamic contractions than for isovolumetric contractions. This study indicates that high-frequency fatigue is unlikely to result from the direct effects of high-energy phosphate metabolism and that contractions producing external work consume more metabolic energy than equally forceful isometric contractions.
Direct measurement of intracellular pressure
Authors: Kelly S. M. , Macklem P. T.
Published: December 2017

The feasibility of using the servo-null technique for direct measurement of intracellular pressure (Pin) was investigated. A large cell, the Xenopus laevis oocyte, was chosen for study, and it was established that Pin obtained with this method was both stable and accurate in these cells. Median resting Pin in oocytes was found to be 0.27 cmH2O, range 0.14-0.61 cmH2O. During osmotic swelling Pin increased, in a non-linear fashion, to a value of 4.11 cmH2O, range 2.61-8.91 cmH2O, with increases in cell volume (Vc) of 24 +/- 3% (SE). This technique may be of use in the study of cellular mechanics.
Gestational change in Na+ and Ca2+ channel current densities in rat myometrial smooth muscle cells
Authors: Inoue Y. , Sperelakis N.
Published: December 2017

The change of Na+ and Ca2+ channel currents during gestation was investigated using the whole cell voltage-clamp method on single smooth muscle cells freshly isolated from the longitudinal layer of pregnant rat uterus. The current-voltage relationships for both the Na+ and Ca2+ currents did not change during gestation. The threshold voltage, the voltage at the peak inward current, and the reversal potential (extrapolated) were virtually identical. The averaged current densities of Ca2+ channel were almost unchanged between days 9 and 21; this value at day 5 was somewhat lower. In contrast, the averaged current density of fast Na+ channels increased markedly in the myometrium during gestation: from 0 at day 5 to 0.19 +/- 0.16 at day 9, to 0.56 +/- 0.13 at day 14, to 0.90 +/- 0.13 at day 18, and to 0.86 +/- 0.14 pA/pF at day 21. This almost linear increase in the averaged density of fast Na+ channels during gestation occurs because of an increase in the fraction of cells which possessed fast Na+ channels. These results suggest that the role of fast Na+ channels in myometrial activity becomes more and more important as term approaches. We suggest that the fast Na+ current may be involved in spread of excitation.
External ATP-induced changes in [Ca2+]i and membrane currents in mammalian atrial myocytes
Authors: Hirano Y. , Abe S. , Sawanobori T. , Hiraoka M.
Published: December 2017

Fura-2 fluorescent digital-imaging microscopy and whole cell patch-clamp recordings were used to study the effects of externally applied ATP on atrial myocytes isolated from rabbit and guinea pig hearts. Application of 100 microM ATP elicited a transient increase in intracellular calcium concentration ([Ca2+]i), which was not suppressed by theophylline, whereas adenosine and ADP failed to evoke the response. The Ca2+ transients were suppressed by the application of Co2+, Ni2+, or verapamil and by the removal of extracellular Ca2+, indicating that the inflow of external Ca2+ is necessary to evoke the response. The Ca2+ transient was suppressed also by ryanodine, suggesting that the mobilization of intracellular Ca2+ is another important factor. In the whole cell recordings, ATP induced a transient depolarization of the membrane potential due to the activation of a rapidly desensitizing inward current which persisted in the presence of Co2+, Ni2+, verapamil, or ryanodine. These results indicate that in mammalian atrial myocytes, ATP evoked transient increase in [Ca2+]i via P2-receptor, through the release of internally stored Ca2+ associated with the inflow of external Ca2+. This response seemed to be triggered mainly by the influx of Ca2+ through L-type Ca2+ channel activated by membrane depolarization, which was caused by the ATP-induced inward current.
Oxygen partial pressure and free intracellular adenosine of isolated cardiomyocytes
Authors: Smolenski R. T. , Schrader J. , de Groot H. , Deussen A.
Published: December 2017

Adenosine formation by the heart is known to critically depend on the ratio of oxygen supply to oxygen demand, but the sensitivity of cardiomyocytes to defined changes in PO2 is not known. Isolated metabolically stable rat cardiomyocytes were incubated up to 45 min at constant PO2 values ranging from 0.1 to 100 mmHg using a feedback-controlled incubation system (oxystat system). Changes of the free intracellular adenosine concentration were measured after trapping of adenosine by cytosolic S-adenosylhomocysteine (SAH) hydrolase in the presence of 200 microM L-homocysteine thiolactone. Rate of SAH formation was constant at a PO2 between 3 and 100 mmHg and gradually increased at PO2 less than 3 mmHg. Cellular ATP decreased only at PO2 less than 1 mmHg, and this was accompanied by a decline of oxygen consumption. Treatment of cells with 5.5 mM deoxyglucose and 4 micrograms/ml oligomycin increased SAH formation 60-fold and was associated with elevated intra- and to a lesser extent extracellular adenosine levels. Inhibition of nucleoside transport with 20 microM S-(p-nitrobenzyl)-6-thioinosine steepened the transmembrane adenosine gradient. Our findings suggest that the cardiomyocyte responds to metabolic poisoning and oxygen deprivation with an enhanced formation of adenosine. This adenosine is mainly formed intracellularly and reaches the extracellular space by diffusion. Threshold for adenosine formation is as low as 3 mmHg.
gamma-Aminobutyric acid-induced response in acutely isolated nucleus solitarii neurons of the rat
Authors: Nakagawa T. , Wakamori M. , Shirasaki T. , Nakaye T. , Akaike N.
Published: December 2017

The gamma-aminobutyric acid (GABA)-induced macroscopic Cl- current (ICl) was investigated in acutely isolated nucleus tractus solitarii (NTS) neurons by a conventional patch-clamp technique combined with a rapid drug application method. The GABA- and muscimol-induced ICl increased in a concentration-dependent manner. The reversal potentials were close to the Cl- equilibrium potential. Pentobarbital sodium (PB) itself elicited a current. Bicuculline (BIC), strychnine (STR), picrotoxin, benzylpenicillin (PCG), Cd2+, and Zn2+ suppressed the GABA response in a concentration-dependent manner. Both BIC and STR shifted the concentration-response curve for GABA response to the right, whereas PCG suppressed the maximum response without affecting the threshold, indicating that BIC and STR antagonized competitively and PCG noncompetitively. The inhibitory action of PCG on GABA response was in a highly voltage-dependent manner. PB shifted the concentration-response curve for GABA response to the left. The augmentatory effect of PB was voltage dependent.
Effect of external Ca2+ concentration on stretch-augmented natriuretic peptide secretion by rat atria
Authors: Page E. , Upshaw-Earley J. , Goings G. E. , Hanck D. A.
Published: December 2017

An in vitro noncontracting rat atrial preparation stretched at 37 degrees C by a distending pressure of 5.1 mmHg was used to examine effects of external Ca2+ concentration ([Ca2+]out, 0.05-3.0 mM) on secretion of immunoreactive atrial natriuretic peptide (ANP) in presence of saxitoxin (STX) and in presence or absence of ryanodine. Under these conditions, the time course of the amount (y) of ANP secreted per milligram dry atrium during 44 min could be approximated by a rate coefficient (k) according to the relation y = s[1 - e(-kt)], where s is the maximal amount secreted after a long time (t). Although k, the rate coefficient for stretch-augmented secretion, increased significantly as [Ca2+]out was raised, secretion inactivated progressively in a time- and [Ca2+]out-dependent manner. This time-dependent decrease was not prevented by ryanodine. We conclude that a component of ANP secreted by quiescent atria in vitro is positively modulated by [Ca2+]out and does not require ryanodine-sensitive Ca2+ release from sarcoplasmic reticulum. The [Ca2+]out-sensitive processes underlying time-dependent inactivation of secretion remain undetermined.
Primary action of endothelin on Ca release in bovine coronary artery smooth muscle cells
Authors: Wagner-Mann C. , Bowman L. , Sturek M.
Published: December 2017

Intracellular free Ca concentrations (Cai) were determined by fura-2 microfluorometry in single freshly dispersed cells to differentiate endothelin (ET)-induced Ca release from Ca influx through voltage-gated Ca channels (VGCC). In physiological solution ET (10(-8) M) significantly (P less than 0.05) increased Cai 23 +/- 3% (+/- SE) above baseline; this increase was not significantly attenuated by 2 x 10(-4) M lanthanum, a blocker of VGCC, or Ca-free solution. When the sarcoplasmic reticulum was depleted of Ca by prolonged treatment with 5 x 10(-3) M caffeine, depolarization with 80 mM K (80K; or 30K) plus ET did not increase Cai above that induced by 80K (or 30K) in caffeine alone. In contrast, 10(-6) M BAY K 8644, instead of ET in the protocol, significantly (P less than 0.05) increased Cai above that induced by 80K (or 30K). ET released Ca from the caffeine-sensitive internal store but was not rapid and transient like caffeine-induced release, which elicited a peak Cai increase in less than 1 min; instead, release was more gradual and prolonged with Cai peaking in greater than 2 min, thus resembling the response to 10(-5) M ryanodine. With two ET exposures, either a transient nonrepeatable increase in Cai or a delayed, but sustained, increase in Cai resulted, similar to the response to ryanodine. These data indicate that in freshly dispersed bovine cells the predominant mechanism by which ET increases Cai is release of Ca from the sarcoplasmic reticulum; if any increase in L-type voltage-gated Ca influx occurred, it was minimal and matched by efflux.
Endothelin mediates Ca influx and release in porcine coronary smooth muscle cells
Authors: Wagner-Mann C. , Sturek M.
Published: December 2017

Endothelin (ET)-induced changes in intracellular free Ca (Cai) in freshly dispersed coronary artery smooth muscle cells were determined using fura-2 microfluorometry to differentiate the action of ET on Ca influx vs. release from internal stores. Comparison was made with caffeine (CAF)-induced Ca release from the sarcoplasmic reticulum (SR) to determine whether ET acts on the same Ca store. In physiological external solution, ET (5 x 10(-8) M) induced a rapid (within 90 s), transient (less than 2.5-min duration) 70% increase in Cai above baseline (n = 20). Pretreatment with diltiazem (10(-4) M; n = 10) did not change the amplitude or shape of the ET-induced Cai transient. In Ca-free solution, ET elicited a Cai response similar in duration but smaller (P less than 0.05) in peak magnitude (31% increase; n = 7). CAF (5 x 10(-3) M) also elicited a rapid (less than 60 s), transient 82% increase in Cai (n = 7). In the continued presence of CAF, ET caused no change in Cai. In contrast, ET elicited a transient 69% increase in Cai (n = 8), and in the continued presence of ET, CAF caused a 24% increase in Cai. Ryanodine (5 x 10(-5) M) suppressed the subsequent ET-induced Cai transient. These data on porcine cells suggest ET induces a rapid release of Ca from a CAF- and ryanodine-sensitive store and causes rapid influx of Ca, which is different from bovine smooth muscle cells. The return of Cai to baseline values in the continued presence of ET suggests the ET-sensitive store is depleted and increased Ca efflux matches Ca influx.
Effects of cGMP on [Ca2+]i, myosin light chain phosphorylation, and contraction in human myometrium
Authors: Word R. A. , Casey M. L. , Kamm K. E. , Stull J. T.
Published: December 2017

Adenosine 3',5'-cyclic monophosphate (cAMP) is believed to be an important mediator of myometrial relaxation, and there is evidence to suggest that guanosine 3',5'-cyclic monophosphate (cGMP) is a mediator of smooth muscle relaxation in vascular and probably nonvascular tissues. To investigate the biochemical mechanisms involved in regulation of human myometrial contractility, we studied the effects of analogues of cAMP and cGMP, as well as activators of adenylate and guanylate cyclases, on uterine smooth muscle contractile activity. We found that myometrial smooth muscle cells in culture respond to analogues of cGMP and cAMP, as well as activators of guanylate cyclase, with a significant decrease in the resting and endothelin-induced increase in [Ca2+]i. Treatment of human uterine smooth muscle strips with sodium nitroprusside or isoproterenol results in diminished force and frequency of contraction as well as a decrease in the rate and extent of myosin light chain phosphorylation in spontaneous contractions of human myometrium. cGMP did not effect relaxation of endothelin-stimulated contractions of human myometrium, but the relaxation effects of cGMP were dramatic in precontracted bovine tracheal and human fetal aortic smooth muscles. Whereas cGMP and cAMP act to promote a decrease in the force and frequency of spontaneous contractions in human myometrium, this tissue is not as responsive to the actions of cyclic nucleotides as are other types of smooth muscle.
Length vs. active force relationship in single isolated smooth muscle cells
Authors: Harris D. E. , Warshaw D. M.
Published: December 2017

The length vs. active force relationship (L-F) may provide information about changes in smooth muscle contractile protein interactions as muscle length changes. To characterize the L-F in single toad stomach smooth muscle cells, cells were attached to a force measurement system, electrically stimulated, and isometric force and elastic modulus (an estimate of the number of attached cross bridges) determined at different cell lengths. Cells generated maximum stress (Pmax = 152.5 mN/mm2) and elastic modulus (Eact = 0.68 x 10(4) mN/mm2) at their rest length (Lcell = 78.0 microns; distance between cell attachments). At shorter lengths, active force and elastic modulus declined proportionally with active force eliminated at 0.4 Lcell. Stretching the relaxed cells up to 1.4 Lcell shifted the subsequent L-F along the length axis by the amount of the stretch but did not change Pmax or the shape of the L-F. In activated cells, force was a function of cell length rather than of shortening history. We interpret these findings as evidence that 1) Lcell is close to the optimum length for force generation, 2) the decline in force at lengths less than Lcell results from a reduced number of attached cross bridges, and 3) stretching relaxed smooth muscle cells may not move the contractile units to new positions on their L-F.
Citrate decreases contraction and Ca current in cardiac muscle independent of its buffering action
Authors: Bers D. M. , Hryshko L. V. , Harrison S. M. , Dawson D. D.
Published: December 2017

Extracellular Ca (Cao) depletions that occur during cardiac muscle contractions are indicative of net Ca entry. Buffering Cao concentration ([Ca]o) with citrate can limit the magnitude of these Cao depletions [e.g., Shattock and Bers. Am. J. Physiol. 256 (Cell Physiol. 25): C813-C822, 1989] which theoretically would allow more Ca entry and consequently greater force at the same free [Ca]o. However, Shimoni and Ginsburg [Am. J. Physiol. 252 (Cell Physiol. 21): C248-C252, 1987] have shown that citrate inhibits cardiac contractions and suggested that this was due to its Ca-buffering action (i.e., dissipating a local elevation of [Ca] at the outer sarcolemmal surface and thereby decreasing Ca influx). To examine the effects of Ca buffering per se, we compared the effects of four low-affinity Ca buffers [citrate, nitrilotriacetic acid (NTA), dipicolinic acid (DPA), and acetamidoiminodiacetic acid (ADA)] on several cardiac preparations. In Mg-free medium with 2 mM free Ca (measured using murexide), citrate, DPA, and ADA (10 mM) decreased the force of twitch contractions in rabbit ventricle to 76 +/- 2, 60 +/- 2, and 85 +/- 2%, respectively, but 10 mM NTA increased force slightly to 105 +/- 2%. No simple correlation was observed between the Ca affinity of the buffer and its effect on tension. These effects were not due to changes in sarcoplasmic reticulum (SR) Ca loading because rapid cooling contractures were not affected and similar results were observed in the presence of caffeine or ryanodine. The depressant effects of citrate and ADA on tension were greater at pH 5.5-6 and ADA had no effect at pH 8.5. Thus the depressant effect is stronger with more protonated forms of citrate and ADA, which are also poorer Ca buffers. Citrate (but not NTA) decreased Ca current in whole cell voltage clamp and shifted the current-voltage relationship and reversal potential to more negative potentials. Citrate decreased Ca current more effectively at higher citrate and lower Ca concentrations. We conclude that citrate (and some other weak Ca buffers) may directly decrease Ca current and contraction in a manner independent of Ca buffering ability.
Simultaneous measurement of membrane potential, cytosolic Ca2+, and tension in intact smooth muscles
Authors: Ozaki H. , Stevens R. J. , Blondfield D. P. , Publicover N. G. , Sanders K. M.
Published: December 2017

Microelectrode techniques and the fluorescent Ca2+ indicator indo-1 were used to measure membrane potential, cytosolic Ca2+ ([Ca2+]cyt), and muscle tension simultaneously in canine antral smooth muscles. Responses of muscles from the myenteric and submucosal regions were compared, since electrical activity and excitation-contraction coupling in these regions differ. The upstroke phase of electrical slow waves in both regions induced an increase in [Ca2+]cyt. In myenteric muscles the plateau phase of slow waves often caused either a further rise in [Ca2+]cyt or maintenance of the level reached during the upstroke event. In submucosal muscles, the plateau phase was significantly smaller and did not induce a second phase in the Ca2+ transient. Contractions were related to the amplitudes of Ca2+ transients. Acetylcholine (ACh; 3 x 10(-8)-10(-6) M) increased the amplitude and duration of the plateau phase of slow waves in a concentration-dependent manner. ACh also increased the second phase of Ca2+ transients and contractile responses associated with the plateau potential. In submucosal muscles ACh induced a significant increase in the plateau phase of the slow wave and increased the corresponding phase of Ca2+ transient. Nicardipine (10(-6) M) inhibited plateau phase of slow waves and the associated increases in [Ca2+]cyt and muscle tension. BAY K 8644 (10(-7) M) augmented the plateau potential and increased [Ca2+]cyt and muscle tension. These results suggest that dihydropyridine-sensitive Ca2+ currents participate in the plateau potential. Cholinergic stimulation modulates [Ca2+]cyt and therefore force by regulating the amount of Ca2+ entering cells through these channels.
Hypokalemia decreases Na(+)-K(+)-ATPase alpha 2- but not alpha 1-isoform abundance in heart, muscle, and brain
Authors: Azuma K. K. , Hensley C. B. , Putnam D. S. , McDonough A. A.
Published: December 2017

K+ deficiency has been linked to a loss of K+ from muscle associated with a decrease in ouabain binding and K(+)-dependent phosphatase activity. This study aimed to quantitate the Na(+)-K(+)-ATPase alpha- and beta-isoform-specific responses to hypokalemia in vivo in heart, skeletal muscle, and brain at pre- and posttranslational levels. Two-week dietary K+ restriction resulted in decreases in alpha 2-mRNA in heart and skeletal muscle to 0.60 and 0.65, and in alpha 2-protein abundance to 0.38 and 0.18 of control, respectively. The decrease in alpha 2-protein was greater than the decrease in mRNA in both tissues, suggesting translational and/or posttranslational mechanism(s) of regulation as well as pretranslational regulation in response to hypokalemia. K(+)-dependent p-nitrophenyl phosphatase (pNPPase) activity decreased in heart and skeletal muscle to 0.67 and 0.58, respectively. There were no changes in alpha 1-. or beta-mRNA or protein levels in skeletal muscle or heart. In brain, there was a similar pattern of regulation. While brain alpha 2-mRNA did not change in hypokalemia, protein levels decreased to 0.72 of control. In conclusion, hypokalemia is associated with a large decrease in expression of the alpha 2-isoform of Na(+)-K(+)-ATPase. These results support the hypothesis that in skeletal and heart muscle hypokalemia induces a decrease in Na(+)-K(+)-ATPase activity (measured as K(+)-dependent pNPPase activity) by specifically decreasing the expression of the alpha 2-isoform of Na(+)-K(+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of apical vs. basolateral palytoxin on LLC-PK1 renal epithelia
Authors: Mullin J. M. , Snock K. V. , McGinn M. T.
Published: December 2017

Research on palytoxin focuses on its action as a tumor promoter and its ionophoretic action in cell membranes. The first property is unusual because palytoxin is not a protein kinase C activator. The second property is remarkable in that it may require interaction with the Na(+)-K(+)-adenosinetriphosphatase (ATPase). Our studies here with palytoxin exposure to the LLC-PK1 renal epithelial cells have yielded the following results: 1) unlike protein kinase C-activating tumor promoters (tetradecanoylphorbol 12,13-acetate or teleocidin), palytoxin does not produce a specific effect on the tight junctions between epithelia; 2) palytoxin instead produces an irreversible cytotoxic effect characterized by a pronounced cell swelling associated with sharply elevated levels of intracellular Na+ and decreased levels of intracellular K+; 3) these fluctuations in intracellular Na+ and K+ levels are explained by marked elevations in the membrane flux of 22Na+ and 86Rb+; 4) the electrophysiological reflection of these altered ion fluxes is a pronounced depolarization of the cell sheet if palytoxin is presented to the basolateral cell surface and a pronounced hyperpolarization (due to sharply elevated apical Na+ flux and transepithelial short-circuit current) if palytoxin is administered apically; 5) the apical effect of palytoxin can be blocked by apical ouabain; and 6) this apical effect of palytoxin decreases as a function of the age of the cell sheet. This first report of palytoxin action in a polar epithelial cell system provides additional evidence for palytoxin effects being mediated by contact with the Na(+)-K(+)-ATPase. It also adds to a growing literature suggesting the existence of Na(+)-K(+)-ATPase in the apical cell surface of epithelia under certain conditions.
Regulation of epithelial chloride channels by protein phosphatase
Authors: La B. Q. , Carosi S. L. , Valentich J. , Shenolikar S. , Sansom S. C.
Published: December 2017

A combination of planar bilayer and patch-clamp techniques was used to determine whether apical membrane Cl- channels of shark (Squalus acanthias) rectal gland (SRG) were regulated by a phosphorylating and dephosphorylating cycle. In channel reconstitution studies, apical membrane vesicles of SRG were purified, incubated in ATP-Mg2+ and the presence or absence (control) of catalytic subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (cAMP-PK) and incorporated into planar lipid bilayers. In the presence of cAMP-PK, two distinct Cl- channels were found when imposing either 450/50 or 300/50 mM KCl (cis/trans) gradients. The most frequently observed channels (G beta 1) were open greater than 80% at all potentials between -60 and +20 mV (trans ground) and were inactivated by alkaline phosphatase added to the cis chamber. The single-channel conductance of G beta 1 was 42 pS between -60 and +20 mV with a 300/50 mM KCl gradient. The second channel (G beta 2) was always observed in pairs of 62-pS subchannels and was not affected by alkaline phosphatase, but the open probability increased with depolarizing potentials. G beta 2 was observed once, but G beta 1 was never observed in the absence of cAMP-PK. In parallel patch-clamp studies of the apical membrane of cultured SRG, a 50-pS channel similar to G beta 1 was noted after incubating cells with either forskolin, an activator of adenylate cyclase, or okadaic acid, an inhibitor of protein phosphatases 1 and 2A. It is concluded that G beta 1 of SRG can be studied in both patch-clamp and bilayer preparations and that G beta 1 is regulated by reversible phosphorylation by cAMP-PK and dephosphorylation by a protein phosphatase.
Inner medullary collecting duct Na(+)-H+ exchanger
Authors: Hering-Smith K. S. , Cragoe E. J. , Weiner D. , Hamm L. L.
Published: December 2017

Cells from the inner medullary collecting duct (IMCD) exhibit Na(+)-H+ exchange. The present studies were performed to address certain important characteristics of this process in cultured IMCD cells. First, Na(+)-H+ exchange was found to be present both at 37 degrees C and at 25 degrees C, in contrast to Na(+)-independent H+ extrusion, which was only observed in some cultures and only at 37 degrees C. Second, with the use of image analysis techniques, virtually all cells in IMCD cultures were demonstrated to possess Na(+)-H+ exchange, whether or not the cells exhibited Na(+)-independent intracellular pH recovery from acid loads. Also, Na(+)-H+ exchange was found to be expressed on the basolateral aspect of these cells, but not on the apical membrane. These properties of IMCD Na(+)-H+ exchange are consistent with a function to regulate intracellular pH rather than mediate transepithelial acid-base transport. Na(+)-H+ exchange in IMCD cells was also compared with that in cultured renal proximal tubule cells. Despite physiologically distinct roles in vivo, Na(+)-H+ exchange in these two cell types in culture was found to be similar with respect to the Km for Na+ and the Ki for 5-(N-ethyl-N-isopropyl)amiloride. These data are consistent with functionally similar (if not identical) processes mediating Na(+)-H+ exchange in these two cell types, but with opposite polarity
Passive chloride permeability charge coupled to H(+)-ATPase of avian osteoclast ruffled membrane
Authors: Blair H. C. , Teitelbaum S. L. , Tan H. L. , Koziol C. M. , Schlesinger P. H.
Published: December 2017

We prepared proton-transporting membrane vesicles from the avian osteoclast's ruffled membrane, a specialized region of the cell surface that acidifies the bone resorption space. We demonstrated a unique conductive Cl- permeability that is charge coupled to the vesicle H(+)-ATPase and is required for acidification. Ion replacement indicated an anion selectivity of Br- approximately Cl- greater than SO4(2-) greater than NO3- approximately SCN- in supporting acidification. The anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (10 microM) was a competitive inhibitor of acidification and raised the Michaelis constant for ATP of the proton pump approximately 11-fold in 120 mM KCl. Inhibition was reversed by valinomycin, which provides an alternate path for charge neutralization. The Cl- dependence of acidification was nonlinear and yielded a Hill coefficient of 3-4, showing that it is distinct from a linear Cl- dependence reported for acidification of renal cortical endosomes. The K+ ionophore valinomycin augmented H+ transport in K2SO4, and not in KCl. Dependence of Cl- transport on membrane potential was confirmed by direct measurement of 36Cl- transport. We uncoupled charge transport from proton transport with a large excess of ammonia, which had no effect on 36Cl- accumulation in vesicles, and by measuring 36Cl- accumulation in response to a membrane diffusion potential, produced with a [K+] gradient and valinomycin in the absence of ATP. These experiments demonstrate that the electrogenic proton pump of the osteoclast ruffled membrane is charge coupled to a passive Cl- permeability in the same membrane.
Inwardly rectifying potassium current in mammalian lens epithelial cells
Authors: Cooper K. , Rae J. L. , Dewey J.
Published: December 2017

Lens potassium conductance is essential for the maintenance of lens volume and transparency. Recent work has identified three major potassium currents in lens: 1) an outwardly rectifying current, 2) an inwardly rectifying current, and 3) a calcium-activated current. This paper presents a study of the lens inward rectifier using whole cell and single-channel patch-clamp techniques. Inwardly rectifying potassium current is present in isolated human, rabbit, rat, and mouse lens epithelia. The voltage about which rectification occurs depends on the external potassium concentration. Internal magnesium is not necessary for rectification. In physiological saline, a time-dependent decrease in current during sustained hyperpolarization is seen. This “droop” is due to voltage-dependent block by external sodium. The inward rectifier is also effectively blocked by external cesium or barium but not by tetraethylammonium or 4-aminopyridine. The mouse lens inward rectifier has a single-channel conductance of 32 pS (measured on-cell with 150 mM potassium in the pipette). The single-channel current-voltage relationship is linear in the inward direction. In contrast to the macroscopic case, no outward current was measurable. The inward rectifier in lens has the necessary properties to be involved in setting resting voltage.
Prolonged incubation of skeletal muscle in vitro: prevention of increases in glucose transport
Authors: Gulve E. A. , Cartee G. D. , Holloszy J. O.
Published: December 2017

During experiments involving prolonged incubation of skeletal muscle, we observed large increases in glucose transport activity. The basal rate of 3-O-methylglucose (3-MG) transport increased two- to fourfold in rat epitrochlearis muscles incubated for 9 h without insulin in Krebs-Henseleit buffer supplemented with 8 mM glucose. The stimulatory effect of a low concentration of insulin (30 microU/ml, added during the final 30 or 60 min of incubation) on glucose transport activity was enhanced 2.5-fold after 6 h and approximately 5-fold after 9 h of incubation. Exposure of muscles to 100 microU/ml of insulin for the first 8 h inhibited slightly but significantly the increase in insulin-stimulated 3-MG transport over a 9-h incubation period. Incubation of muscles in minimal essential medium (MEM) for 9 h inhibited the time-dependent rise in basal and insulin-stimulated transport by approximately 45%. The effect of MEM was reproduced with MEM essential, but not nonessential, amino acids. Incubation of muscles with MEM plus 100 microU/ml of insulin for the first 8 h prevented the increases in 3-MG transport activity measured after a 9-h incubation period. Muscles incubated for 9 h maintained ATP and phosphocreatine concentrations, and changes in glycogen concentrations were small. Thus we have defined conditions for long-term incubation of skeletal muscle under which a progressive increase in glucose transport is prevented.
Disruption of microtubules alters polarity of basement membrane proteoglycan secretion in epithelial cells
Authors: De Almeida J. B. , Stow J. L.
Published: December 2017

Basement membrane proteins such as the heparan sulfate proteoglycan (HSPG) are secreted in a polarized fashion from the basolateral membrane of epithelial cells. We have used the microtubule-disrupting drug colchicine to study the role of the microtubule network in directing constitutive secretion to the basolateral membrane of LLC-PK1 renal epithelial cells. Microtubule depolymerization induced by colchicine resulted in fragmentation and redistribution of fluorescently labeled trans-Golgi membranes. Increased immunofluorescent staining of HSPG was associated with these dispersed Golgi cisternae. The biosynthetic processing of HSPG was not significantly altered by the loss of microtubules or by the dispersal of the Golgi elements. The most striking effect of microtubule disruption was the loss of polarity of HSPG secretion. Immunoprecipitation studies showed that HSPG was secreted from both apical and basolateral surfaces of LLC-PK1 cells treated with colchicine, and a similar result was found for the delivery of laminin, another basement membrane protein. In contrast, there was no change in the distribution of an integral basolateral membrane protein, Na(+)-K(+)-ATPase, following colchicine treatment. Our results provide the first demonstration that microtubules are involved in the constitutive trafficking of basolateral secretory proteins. These data also suggest that there may be an inherent difference in the targeting or delivery of membrane and secretory proteins to the basolateral cell surface.
Blockade by lithium ions of potassium channels in rat anterior pituitary cells
Authors: Kato M. , Lledo P. M. , Vincent J. D.
Published: December 2017

Extracellular Li+ has been known to facilitate the basal secretion of growth hormone from anterior pituitary cells and of catecholamine from chromaffin cells. In both cases, the intracellular accumulation of Li+ seems to be the prerequisite, and the presence of extracellular Ca2+ is indispensable. In this series of experiments, we examined whether Li+ blocked K+ currents by using primary cultured anterior pituitary cells from male rats. K+ currents were measured in the whole cell configuration of the patch-clamp technique. Extracellular Li+ (140 mM) suppressed both the delayed rectifier K+ current (IK) and the transient outward K+ current to 71 and 69% of control, respectively, in a reversible manner. IK elicited by a voltage step to +70 mV from holding potential of -70 mV was suppressed by 32.5 mM internal Li+ to 28% of control. Half-maximal suppression of K+ conductance by internal Li+ was 16 mM. Furthermore, Ca(2+)-channel blocker methoxyverapamil potently suppressed Li(+)-induced growth hormone secretion. From these results we propose that the blockade by Li+ of K+ channels could depolarize the cells and activate Ca2+ channels, thereby promoting the influx of Ca2+ and hormone secretion as a mechanism of Li(+)-induced hormone secretion.