bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneity of expression of apical membrane determinants in A6 epithelial cells
Authors: Moberly J. B. , Fanestil D. D.
Published: December 2017

Expression of apical membrane antigens by A6 cells is heterogeneous. The A6 cell line is a model system for sodium transport and hormone action in “tight” epithelia. To create probes for the study of sodium transport and aldosterone action, we generated monoclonal antibodies (MoAbs) by immunization of BALB/c mice with intact A6 cells. MoAbs binding to apical membrane determinants were selected by an enzyme-linked immunoadsorbance assay (ELISA) against the apical surface of confluent A6 monolayers. Examination of ELISA-positive MoAbs by indirect immunofluorescence on confluent cells grown on glass cover slips revealed heterogeneity of apical expression: individual cells comprising the monolayer were fluorescently labeled to variable degrees and some cells were unlabeled. Expression of one antigen, recognized by MoAb I4C1, was heterogeneous in cells at low density as well as for confluent cells. Another antibody, 2B8, exhibited heterogeneity of labeling in confluent A6 monolayers, but no labeling of any cells at low density. This heterogeneity of antigen expression suggested that A6 cells are not a single cell type. However, cloned A6 cells established by limiting dilution were also labeled heterogeneously by both antibodies. Thus the heterogeneity observed is not due to genetic inhomogeneity between cells. Expression of the antigens on apical membranes of cells grown on Millipore filters was heterogeneous for the antigen recognized by 2B8, but essentially homogeneous for that recognized by I4C1, suggesting that the degree of differentiation of the cells plays a role in the heterogeneous expression of apical antigens. This heterogeneity of expression of apical antigens adds new complexity to the study of A6 cells and the differentiation of epithelial cells in culture.
[Na]i modulates isoproterenol's effect on Ca permeability in cultured heart cells
Authors: Kim D. , Smith T. W.
Published: December 2017

Isoproterenol (ISO) augments the slow inward Ca current in cardiac muscle cells. We examined the role of intracellular Na (Nai) on ISO-mediated alterations in Ca uptake in cultured chick heart cells. In 140 mM Na medium, 1 microM ISO did not measurably alter 45Ca uptake. When cells were first preincubated in Na-free medium for 5 min and then incubated in control medium with 45Ca, ISO increased 45Ca uptake by 30%. Nifedipine (10 microM), verapamil (1 microM), or dl-propranolol (1 microM) abolished the effect of ISO on 45Ca uptake. CGP 28392 (1 microM), a Ca channel agonist, increased Ca influx in a manner that was augmented by decreased Nai, similar to the ISO response. Neither ISO nor CGP 28392 altered 45Ca uptake when cells preincubated in Na-free medium were further incubated in Na-free medium containing 45Ca. Exposure of cells to Na-free medium or 25 mM K+ medium caused depolarization of the resting membrane potential to approximately -40 mV. In the absence of ISO, the 45Ca uptake in cells preincubated in Na-free or 25 mM extracellular K (Ko) medium was significantly greater than in cells preincubated in control medium. This appeared to be due partly to increased 45Ca uptake via nifedipine-sensitive pathways. These findings support the hypothesis that reduction in Nai concentration ([Na]i) enhances the ISO-induced augmentation of Ca uptake via nifedipine-sensitive pathways (presumably via slow Ca channels), probably by a direct effect on the channels.
Three distinct cell populations in rat kidney collecting duct
Authors: Holthofer H. , Schulte B. A. , Pasternack G. , Siegel G. J. , Spicer S. S.
Published: December 2017

The morphologically heterogeneous cell populations in the collecting ducts of the rat kidney were studied using immunocytochemical detection of Na+-K+-ATPase and the anion channel (band 3) glycoprotein. Both enzymes were localized to the basal aspect of separate and morphologically distinct subpopulations of cells in various segments of the collecting duct. Na+-K+-ATPase appeared to be present exclusively in principal cells as identified by their morphology, whereas band 3 antibodies reacted only with intercalated cells. However, 5-20% of cells with the morphological characteristics of intercalated cells failed to react with either antisera in various segments of collecting ducts. As band 3 glycoprotein serves in exchanging intracellular bicarbonate for chloride, it is highly likely that the cells positive for this antigen secrete protons. The method introduced here appears thus useful for distinguishing between principal and intercalated cells by differences in their enzyme content and further for revealing two subpopulations of intercalated cells. This method promises to provide a useful approach for studying the principal and intercalated cell populations in various metabolic states.
Ryanodine and the calcium content of cardiac SR assessed by caffeine and rapid cooling contractures
Authors: Bers D. M.
Published: December 2017

The ability of rabbit ventricular muscle sarcoplasmic reticulum (SR) to accumulate and retain Ca during Na-free perfusion was assessed using caffeine contractures and rapid cooling contractures (RCC). Muscles were exposed to a Na-free medium for 15 min, during which time a transient contracture developed and relaxed back to the resting tension level. Muscles were then either exposed to 20 mM caffeine or rapidly cooled to less than 5 degrees C, both of which produced large contractures. The magnitudes of those contractures are believed to be a relative index of SR Ca content. Reduction of extracellular [Ca] from 2 to 0.2 mM did not significantly alter the magnitude of either Na-free caffeine contractures or RCC. These are not the maximum contractures that can be obtained. The possibility that low extracellular [Ca] ([Ca]o) may increase passive Ca permeability is suggested in explanation of this effect. After equilibration with 100 nM ryanodine, both Na-free caffeine contractures and RCC are virtually eliminated. This suggests that even if the SR could accumulate Ca during the initial Na-free exposure in the presence of ryanodine, it could not retain that Ca in Na-free medium. It is proposed that the sarcolemmal Ca pump can extrude Ca from the cells at a rate sufficient to deplete the ryanodine-treated SR. When removal of Na was accompanied by increase of [K]o (to 12, 20, or 30 mM), the Na-free RCC were enhanced. Increasing [K]o during Na-free superfusion in the presence of ryanodine resulted in demonstrable RCC that were graded with [K] and completely suppressed by nifedipine.(ABSTRACT TRUNCATED AT 250 WORDS)
ATP depletion in slow-twitch red muscle of rat
Authors: Whitlock D. M. , Terjung R. L.
Published: December 2017

Rat slow-twitch muscle, in contrast to fast-twitch muscle, maintains its ATP content near normal during intense stimulation conditions that produce rapid fatigue. An extensive depletion of adenine nucleotide content by the deamination of AMP to IMP + NH3, typical of fast-twitch muscle, does not occur. We evaluated whether this response of slow-twitch muscle could be simply due to failure of synaptic transmission or related to cellular conditions influencing enzyme activity. Stimulation of soleus muscles in situ via the nerve or directly in the presence of curare at 120 tetani/min for 3 min resulted in extensive fatigue but normal ATP contents. Thus the lack of ATP depletion must be related to cellular events distal to neuromuscular transmission. Even nerve and direct muscle stimulation (with curare) during ischemia did not cause a large depletion of ATP or a large elevation of lactate content (12.0 +/- 0.7 mumol/g), even though the decline in tension was essentially complete. However, if the same tension decline during ischemia was prolonged by stimulating for 10 min at 12 tetani/min a large decrease in ATP (2.24 +/- 0.09 mumol/g) and increase in IMP (2.47 +/- 0.16 mumol/g) and lactate (30.4 +/- 2.0 mumol/g) content occurred. Thus adenine nucleotide deamination to IMP can occur in slow-twitch muscle during specific contraction conditions. The cellular events leading to the activation of AMP deaminase require an intense contraction condition and may be related to acidosis caused by a high lactate content.
A distal nephron glycoprotein that has different cell surface distributions on MDCK cell sublines
Authors: Ojakian G. K. , Romain R. E. , Herz R. E.
Published: December 2017

Monoclonal antibodies that recognize three distinct epitopes of a 23-kDa glycoprotein (gp23) on the plasma membrane of MDCK cells were used to study cell-surface polarity. Immunofluorescence microscopy of MDCK cells obtained from the American Type Culture Collection demonstrated that gp23 was nonpolarized in approximately 50% of the cells (on both apical and basolateral membranes), whereas, in the remaining cells, gp23 had a polarized distribution (basolateral only). This heterogeneous gp23 cell-surface staining was not observed in a variety of other MDCK sublines. Instead, gp23 was found to have a nonpolarized distribution on MDCK cells that produced monolayers with low transepithelial electrical resistances (less than 220 omega X cm2) and was localized only to the basolateral membrane of MDCK cell lines capable of generating considerably higher transepithelial electrical resistances (770-2,220 omega X cm2). Immunofluorescence and immunoelectron microscopy of dog, rat, and rabbit kidney demonstrated that gp23 is a nephron segment-specific glycoprotein localized only to the distal and collecting tubules. These observations provide further evidence for the heterogeneity of the MDCK cell line. They also support a proposal that the origin of MDCK cells is the renal distal nephron.
Diamide stimulates calcium-sodium exchange in dog red blood cells
Authors: Parker J. C.
Published: December 2017

Calcium influx can be stimulated in dog red blood cells by preexposure to diamide under certain conditions. Diamide-activated calcium influx resembles swelling-induced Ca2+-Na+ exchange in several respects. These include saturation of calcium influx at external calcium levels greater than 0.5 mM, suppression of calcium influx by external sodium, and inhibition by quinidine. The ability of diamide to stimulate this transport pathway depends critically on the ionic composition of the medium in which the cells are bathed at the time of diamide exposure. The effect is greatest if the diamide preincubation is conducted in a hypotonic lithium chloride medium containing at least 1 microM calcium. Stimulation of Ca2+-Na+ exchange is seen at diamide concentrations (0.10-0.33 mM) that are lower than those reported to cause major spectrin cross-linking, glutathione depletion, Ca2+-ATPase inhibition, or ion channel formation. The results suggest that dog red cells have a large latent capacity for Ca2+-Na+ exchange.
Mechanics of tonus fibers of frog muscle
Authors: Bozler E.
Published: December 2017

Contractions with two phases of relaxation are induced by brief strong stimulation in some frog muscles. The first phase with rapid relaxation is produced by the twitch fibers; the second phase, which is very slow and is only present after strong stimulation, represents the relaxation of the tonus fibers. At moderate loads, half time of isotonic relaxation of these fibers is as long as 30 min at 2 degrees C, but the rate varies with the load and depends on the condition of the frogs. With regard to the rate of relaxation, the tonus fibers resemble molluscan catch muscles. In tonus fibers, rapid isotonic and isometric relaxation can be induced by a small extension; shortening opposes this effect. These responses are like the length responses previously found in various types of striated muscle. They go in the same direction as the well-known metabolic effects of length changes (Fenn effect). After a large extension by an increase in load there is no active shortening when the load is returned to the previous value. This and other observations show that the slowness of relaxation is not due to sustained activity, but is determined by the strength of the contractile bonds formed during contraction. Because activity during relaxation is very low, it is unlikely that length responses are caused by a modification of the cross-bridge cycle. It is suggested that length changes act through a mechanism that is separate from that initiating contraction, but alters the speed of relaxation by making the cross bridges weaker or stronger.
Interactions of NIP-taurine, NAP-taurine, and Cl- with the human erythrocyte anion exchange system
Authors: Knauf P. A. , Mann N. A. , Kalwas J. E. , Spinelli L. J. , Ramjeesingh M.
Published: December 2017

N-(4-isothiocyano-2-nitrophenyl)-2-aminoethanesulfonate (NIP-taurine), a newly synthesized isothiocyano derivative of N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonate (NAP-taurine), is a potent inhibitor of human erythrocyte chloride exchange. At 0 degrees C, the inhibition is reversible, but at 37 degrees C, NIP-taurine inhibits irreversibly, indicating that it may be a useful label for its binding site. When present at the outside of the cell, NIP-taurine binds with low affinity to a site that seems to be the Cl- transport site (on the basis of its affinity for Cl-) and with much higher affinity to a different site, MN, which has a much lower affinity for Cl-. In this respect, NIP-taurine resembles NAP-taurine, and an analysis of interactions between these two probes is consistent with the idea that they bind to the same two sites. The affinity of NIP-taurine for the high-affinity MN site is enhanced by about fourfold when the transport protein, band 3, is in the conformation with the transport site facing outward (Eo), as compared with the conformation with the transport site facing inward (Ei). External Cl-, but not cytoplasmic Cl-, competes with NIP-taurine for binding to the external, high affinity site. Thus NIP-taurine provides a label for an external site, at which Cl- and perhaps other anions bind, which is separate from both the transport site and the cytoplasmic modifier site at which high Cl- concentrations inhibit Cl- exchange.
Effect of secretagogues on cytoplasmic free calcium in alveolar type II epithelial cells
Authors: Sano K. , Voelker D. R. , Mason R. J.
Published: December 2017

Pulmonary surfactant is synthesized and secreted by alveolar type II epithelial cells. Although intracellular calcium and other second messengers have been implicated in secretion by type II cells, this is the first report on measurement of cytoplasmic free calcium concentration ([Ca2+]i). Known secretagogues, 12-O-tetradecanoylphorbol-13-acetate (TPA) and terbutaline, were tested to see if they caused rapid increases in cytoplasmic calcium. Ionomycin, a calcium ionophore, was used to increase cytoplasmic free calcium concentration, to determine if a rapid increase in cytoplasmic free calcium would stimulate secretion, and to measure interactions with other secretagogues. Ionomycin increased both [Ca2+]i and pulmonary surfactant secretion from alveolar type II cells. A low concentration of ionomycin (100 nM) greatly enhanced secretion stimulated by terbutaline or by 8-bromo-cAMP but only had an additive effect on secretion stimulated by TPA. Terbutaline transiently increased [Ca2+]i by 24% over control basal condition, and the increase in [Ca2+]i produced by terbutaline occurred in the absence of extracellular calcium. TPA itself did not change [Ca2+]i. However, TPA completely inhibited the terbutaline-induced increase of [Ca2+]i but not the increase due to ionomycin. When alveolar type II cells were loaded with 2-(2-bis-[carboxymethyl]-amino-5-methyl-phenoxy)-methyl-6-methoxy-8-bis carboxymethylaminoquinoline (quin2) in calcium-free buffer, [Ca2+]i decreased from 143 +/- 10 to 31 +/- 8 nM. Lowering [Ca2+]i inhibited TPA- or terbutaline-induced secretion by 22 and 40%, respectively. Although the precise role of cytoplasmic free calcium on surfactant secretion cannot be established on the basis of current data, our results indicate that an increase in cytoplasmic free calcium produced by ionomycin stimulates secretion and that an increase in [Ca2+]i affects cAMP-induced secretion more than protein kinase C-mediated secretion in alveolar type II cells.
Regulation of ovarian ornithine decarboxylase by human chorionic gonadotrophin
Authors: Sertich G. J. , Persson L. , Pegg A. E.
Published: December 2017

Treatment of 29-day-old female Sprague-Dawley rats with human chorionic gonadotrophin (hCG) produced a large and rapid increase in the activity of ornithine decarboxylase. Measurements that use a specific radioimmunoassay showed that the increased activity could be accounted for by a parallel change in the amount of ornithine decarboxylase protein. The increased protein content was caused by an increased rate of synthesis, since the half-life of ornithine decarboxylase was not changed by the hormone treatment. The content of mRNA for ornithine decarboxylase was determined by hybridization with a cDNA probe, and it was found that the increased amount of protein was correlated with a change in the amount of mRNA. These results indicate that treatment with hCG induces ornithine decarboxylase in the rat ovary by increasing the production or the stability of the mRNA for this enzyme. The increased amount of ornithine decarboxylase led to an increase in putrescine in the ovary but did not increase the content of the polyamines spermidine and spermine. These findings show that, despite its rapid and large scale induction, ornithine decarboxylase is not the rate-limiting step that determines the content of these polyamines in this tissue. They also suggest that putrescine itself may play an important role in the ovary.
SITS-sensitive Cl- conductance pathway in chick intestinal cells
Authors: Montrose M. , Randles J. , Kimmich G. A.
Published: December 2017

The unidirectional influx of 36Cl- into isolated chick epithelial cells is 30% inhibited by 300 microM SITS. Characteristics of the SITS-sensitive flux pathway were examined in terms of sensitivity to changes in membrane potential and intracellular pH. Potential dependence was evaluated using unidirectional influx of [14C]tetraphenylphosphonium ([14C]-TPP+) as a qualitative sensor of diffusion potentials created by experimentally imposed gradients of Cl-. Steady-state distribution of [14C]methylamine ([14C]MA) was used to examine for Cl(-)-dependent changes in intracellular pH. Imposed Na+ gradients, but not Cl- gradients, induce changes in [14C]MA distribution. SITS does not alter the [14C]MA distribution observed in cells with imposed gradients of Na+ and Cl-. Both results suggest that inhibition of Cl(-)-OH- exchange system is not the basis for the SITS effect on Cl- influx. However, if relative permeabilities for ion pairs via conductance pathways are compared, it can be shown that SITS causes a marked reduction of Pcl relative to either PNa or PK. SITS also inhibits electrically induced influx of [14C]TPP+ or [14C] alpha-methylglucoside driven by imposed Cl- gradients. Conversely, electrically driven Cl- influx can be blocked by SITS. These observations are all consistent with a SITS-sensitive Cl- conductance pathway associated with the plasma membrane of chick intestinal cells. No Cl(-)-OH- exchange capability can be detected for chick intestinal cells.
Na-K pump site density and ouabain binding affinity in cultured chick heart cells
Authors: Lobaugh L. A. , Lieberman M.
Published: December 2017

The possible existence of multiple [3H]ouabain binding sites and the relationship between ouabain binding and Na-K pump inhibition in cardiac muscle were studied using cultured embryonic chick heart cells. [3H]ouabain bound to a single class of sites in 0.5 mM K (0.5 Ko) with an association rate constant (k+1) of 3.4 X 10(4) M-1.s-1 and a dissociation rate constant (k-1) of 0.0095 s-1 [corrected]. Maximal specific [3H]ouabain binding RT to myocyte-enriched cultures is 11.7 pmol/mg protein and Kd is 0.43 microM in 0.5 Ko, whereas Kd,apparent is 6.6 microM in 5.4 Ko. The number of binding sites per myocyte was calculated by correcting for the contribution of fibroblasts in myocyte-enriched cultures using data from homogeneous fibroblast cultures (RT = 3.3 pmol/mg protein; Kd = 0.19 microM in 0.5 Ko). Equivalence of [3H]ouabain binding sites and Na-K pumps was implied by agreement between maximal specific binding of [3H]ouabain and 125I-labeled monoclonal antibody directed against Na+-K+-ATPase (approximately 2 X 10(6) sites/cell). However, [3H]ouabain binding occurred at lower concentrations than inhibition of ouabain-sensitive 42K uptake in 0.5 Ko. Further studies in both 0.5 K and 5.4 Ko showed that ouabain caused cell Na content Nai to increase over the same range of concentrations that binding occurred, implying that increased Nai may stimulate unbound Na-K pumps and prevent a proportional decrease in 42K uptake rate. The results show that Na-K pump inhibition occurs as a functional consequence of specific ouabain binding and indicate that the Na-K pump is the cardiac glycoside receptor in cultured heart cells.
Fura-2 fluorescence is localized to mitochondria in endothelial cells
Authors: Steinberg S. F. , Bilezikian J. P. , Al-Awqati Q.
Published: December 2017

The new, highly fluorescent, calcium-sensitive dye, fura-2, can be loaded nondisruptively into intact cells by means of its permeant ester and used to measure the free calcium ion concentration in individual cells. For fura-2 to signal cytosolic calcium, it must be distributed homogeneously and exclusively throughout the cytoplasmic space. However, microscopic examination of bovine aortic endothelial cells loaded with fura-2 by exposure to its permeant ester reveals fluorescence associated with discrete intracellular structures rather than the homogeneous distribution expected for a cytosolic stain. Simultaneous labeling of bovine aortic endothelial cells with fura-2 and rhodamine 123 (a mitochondrial fluorescent vital stain) identifies these structures as mitochondria. Subcellular dye localizations are not observed when the cells are loaded with other putative cytosolic stains that gain access to the cytosol by means of a membrane permeant ester. Both carboxyfluorescein and indo-1 (another member of the family of second generation calcium indicators) stain the cytoplasm diffusely. It is suggested that fura-2 fluorescence accumulates in certain cells in association with mitochondria. It is important to assess the intracellular distribution of fura-2 when this indicator is used to measure the free cytosolic calcium ion concentration.
Na+-Ca2+ exchange in human neutrophils
Authors: Simchowitz L. , Cragoe E. J.
Published: December 2017

The pathway for inward Ca2+ movements in isolated human neutrophils was investigated. One-way 45Ca2+ influx into resting, steady-state cells amounted to approximately 6 mumol.l cell water-1.min-1. This uptake can be entirely accounted for on the basis of a carrier-mediated exchange of external Ca2+ for internal Na+. The counter transport exhibited trans stimulation of 45Ca2+ influx by internal Na+ (Km approximately equal to 26 mM) with a Hill coefficient of approximately equal to 2.6. There was substrate saturation by external Ca2+ (Km approximately equal to 0.15 mM) and by external Na+ (Km approximately equal to 30 mM); the two ions competed for the same binding sites. The exchange was noncompetitively inhibited by benzamil (Ki approximately 75 microM) and by some other amiloride analogues bearing a substituent on the terminal nitrogen atom of the guanidino group. Membrane depolarization enhanced the rate of 45Ca2+ entry, suggesting an electrogenic process; this voltage dependence was consistent with a coupling ratio of approximately 3 Na+:1 Ca2+. Hence, the Na+-Ca2+ exchange mechanism of neutrophils displays many features in common with those of other cell types. Studies in cells exposed to the tripeptide N-formylmethionyl-leucyl-phenylalanine (FMLP) indicate activation of the counter-transport system by the chemotactic factor. Thus, Na+-Ca2+ exchange may be at least partly responsible for the increase in 45Ca2+ influx and transient rise in intracellular free Ca2+ that are seen following stimulation.
Effect of seeding density and time in culture on vascular smooth muscle cell proteins
Authors: Seidel C. L. , White V. , Wallace C. , Amann J. , Dennison D. , Schildmeyer L. A. , Vu B. , Allen J. C. , Navarro L. , Eskin S.
Published: December 2017

The purpose of this work was to determine the effect of seeding density and time in culture on quantitative and qualitative characteristics of myosin in primary cultures of vascular smooth muscle cells (VSMCs). Enzymatically dispersed VSMCs from femoral arteries and saphenous veins of adult dogs were seeded at a density of 10(3)-10(5) cells/cm2 and assayed after 7 days or at 10(5) cells/cm2 and assayed between 1 and 10 days. Myosin, actin, and total protein contents as well as electrophoretic and immunoreactive characteristics of myosin heavy chains (MHCs) were determined. Total and contractile protein contents were independent of seeding density and increased with time in culture. Freshly dispersed cells exhibited two MHCs (MHC-1 and MHC-2) but, within 24 h after culturing, only cells attached to the dish expressed a third protein band (MHC-3), which had electrophoretic mobility and immunoreactivity similar to purified platelet MHC. MHC-3 appeared before onset of cell division and, by 4 days in culture when cells were proliferating, became the dominant MHC form. Loss of MHC-1 and MHC-2 could be prevented by growing cells in a serum-free, defined media that prevented proliferation. These data indicate that seeding density does not affect myosin content, but that with time in culture expression of a MHC with characteristics similar to nonmuscle myosin occurs. Expression of MHC-3 is associated with cell attachment, whereas loss of MHC-1 and MHC-2 requires proliferation.
Volume response of quiescent and interleukin 2-stimulated T-lymphocytes to hypotonicity
Authors: Lee S. C. , Price M. , Prystowsky M. B. , Deutsch C.
Published: December 2017

Regulatory volume decreased (RVD) in lymphocytes in response to hyptonically induced swelling is dependent on the membrane permeabilities of K+, Cl-, and H2O. We used electronic cell sizing, cell water determination, and the whole cell patch-clamp method to study these membrane permeabilities in the cloned mouse T-lymphocyte, L2. Quiescent L2 cells express low levels of a voltage-gated K+ channel and show no RVD at 25 degrees C. In contrast, L2 cells stimulated to proliferate with the growth factor interleukin 2 have increased K+ conductance and show RVD in response to hypotonicity. RVD in stimulated cells is blocked by quinine and verapamil at levels that also completely block the voltage-gated K+ conductance. Swollen, unstimulated L2 cells can be induced to shrink by addition of the monovalent cation ionophore gramicidin in the presence of impermeant extracellular organic cations; gramicidin also enhances the rate of RVD in stimulated cells. Additionally, the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) blocks this gramicidin-facilitated RVD. These results suggest that a minimum requisite Cl- permeability is present even in the unstimulated L2 cells, that a necessary and limiting K+ permeability determines the rate of RVD, and that this K+ permeability increases after growth-factor stimulation as predicted from the direct measurement of voltage-gated K+ conductance. The hydraulic permeability is approximately 70% greater in proliferating L2 cells than in quiescent cells. At 37 degrees C, some RVD occurs in unstimulated L2 cells, and stimulated cells show faster and more complete shrinkage. These results are discussed with respect to the underlying membrane permeabilities and their relation to stimulated cell proliferation.
Complex response of epithelial cells to inhibition of Na+ transport by amiloride
Authors: Fisher R. S. , Lockard J. W.
Published: December 2017

When toad urinary bladder or frog skin epithelia are treated with amiloride, short-circuit current (Isc), which represents the net active transepithelial Na+ transport rate from the apical to basolateral surface, decreases rapidly (2-5 s) to approximately 15-20% of control values and then slowly, over several minutes, continues falling toward zero. The contribution of this second phase of the decline is dependent on the transporting condition of the tissue before administration of amiloride. Attenuation of the second phase was observed if tissues were subjected to a period of transport inhibition. Tissues preincubated in 0 Na+ Ringer solution on the apical surface were returned to control Na+ Ringer, which caused an approximately 25% increase of Isc above control values. Immediate reapplication of amiloride caused Isc to decrease more rapidly than the previous exposure to values near zero, substantially reducing or eliminating the secondary slow decline. After long-term reincubation of tissues in control, 100 mM Na+ solution, another treatment with amiloride indicated that the magnitude of the secondary decline increased in frog skin but not in urinary bladder epithelia. We conclude that the effect of amiloride is complex and may cause additional effects besides simply blocking entry of Na+ into the apical membrane channel, and we suggest that regulatory mechanisms may be invoked in response to transport inhibition.
Tumor-promoting phorbol esters induce angiogenesis in vivo
Authors: Morris P. B. , Hida T. , Blackshear P. J. , Klintworth G. K. , Swain J. L.
Published: December 2017

It has been hypothesized that tumor growth is dependent on the concomitant growth of its vascular supply, and thus agents that stimulate angiogenesis may help support tumor growth. Phorbol esters are potent tumor promoters that induce a variety of biochemical effects in cells, including activation of protein kinase C. The specific mechanisms responsible for tumor promotion by phorbol esters are unknown. The objective of this study was to determine whether the tumor-promoting phorbol esters can induce vascular growth. Phorbol esters were tested for their ability to stimulate angiogenesis in vivo using the chick chorioallantoic membrane and rabbit cornea assays. The active tumor promoters 12-O-tetradecanoyl phorbol-13-acetate and phorbol 12,13-didecanoate, which activate protein kinase C, were found to stimulate angiogenesis in a dose-dependent manner. In contrast, 4 alpha-phorbol 12,13-didecanoate, which is inactive as a tumor promoter and does not activate protein kinase C, did not stimulate angiogenesis. Phorbol esters may be indirect angiogenic factors, since no mitogenic effect on bovine capillary endothelial cells in culture could be detected. The results demonstrate that the tumor-promoting activity of phorbol esters may, in part, be secondary to stimulation of neovascularization to support tumor growth and suggest a role for the activation of protein kinase C in this process.
(Na + K + 2Cl) cotransport in cultured embryonic chick heart cells
Authors: Liu Shi , Jacob Ron , Piwnica-Worms David , Lieberman Melvyn
Published: December 2017

Pages C72–C730: Shi Liu, Ron Jacob, David Piwnica-Worms, and Melvyn Lieberman. “(Na + K + 2Cl) cotransport in cultured embryonic chick heart cells.” Page C721: first and second sentences, second paragraph, second column, should read “In this study, coupled ion movements are investigated using Na-, K-, and Cl-selective microelectrodes, in contrast to previous work (30), in which we determined the contents of Na, K, and Cl by atomic absorption spectrophotometry (Nai and Ki) or coulometric titration (Cli). We find that Cl-HCO3 exchange does not appear to contribute to the maintenance of a high agrCli in the resting steady state.” C722: Eq. 1 should read

(See PDF)
Role of sodium pump in membrane potential gradient of canine proximal colon
Authors: Burke E. P. , Reed J. B. , Sanders K. M.
Published: December 2017

A large gradient in membrane potential exists through the thickness of the circular layer in canine colonic muscles. This study tested the effects of several experimental manipulations known to block electrogenic sodium pumping on the resting potentials of colonic muscles. Membrane potentials were recorded with microelectrodes from cells through the circular muscle layer. In cells adjacent to the submucosal surface of the circular layer, application of ouabain (10(-6) to 10(-5) M) caused an average membrane depolarization of 36 mV. Removal of the external K+ resulted in depolarizations similar to the effect of ouabain. Readmission of K+ (5.9 mM) produced repolarization and an additional hyperpolarization that averaged 13 mV beyond the resting potential. When exposed to 15 mM K+, cells hyperpolarized well beyond the estimated potassium equilibrium potential (EK). Ouabain blocked the repolarization in response to reintroduction of external K+. Lowering the bath temperature to 20 degrees C rapidly depolarized membrane potential; rewarming repolarized cells. Ouabain and K+-free solutions blocked the repolarization response to rewarming. Cells also depolarized when exposed to solutions in which the NaCl was replaced with LiCl. Membrane potentials of cells within the bulk of the circular layer decreased as a function of distance from the submucosal border. Cells at the myenteric border of the circular muscle were not significantly affected by ouabain and K+-free solution, but these treatments abolished the gradient in membrane potential across the circular layer.(ABSTRACT TRUNCATED AT 250 WORDS)
Caffeine suppresses length dependency of Ca2+ sensitivity of skinned striated muscle
Authors: de Beer E. L. , Grundeman R. L. , Wilhelm A. J. , Caljouw C. J. , Klepper D. , Schiereck P.
Published: December 2017

Freeze-dried skinned cardiac and skeletal muscle preparations of the rabbit were immersed in Ca2+-containing solutions with different concentrations of caffeine. The relation between the negative logarithm of the Ca2+ concentration (pCa) and normalized developed force was studied. The exact position of these Ca2+-sensitivity functions proved to be dependent on both the sarcomere length (monitored by means of laser diffraction) and caffeine concentration. High concentrations of caffeine induce a reversible fall in tension, particularly at low binding site saturation (low pCa) and long sarcomere lengths. At a concentration of 10 mM caffeine, the sarcomere length dependency of the Ca2+-sensitivity curves is markedly reduced for the rising part of the curve. Only the depressive effect of caffeine at high pCa remains. A possible mechanism of caffeine action is discussed.
Effect of fasting on myocardial substrates in male and female rats
Authors: Arnall D. A. , Palmer W. K. , Miller W. C. , Oscai L. B.
Published: December 2017

Adult male and female rats were fasted for 1, 2, or 3 days to determine its effect on circulating and endogenous fuels available to the heart. Liver glycogen was depleted within the first 24 h of food restriction. Plasma glucose decreased approximately 2.5 mM in both sexes during the 3 days. Fasting significantly increased plasma beta-hydroxybutyrate to approximately the same level in female and male rats. Plasma free fatty acid (FFA) increased approximately 0.2 mM in both groups during the first 24 h without food and remained elevated over the next 2 days. FFA concentrations were higher in fed female than in fed male rats and remained significantly higher in female rats throughout the experimental period. Myocardial glycogen increased 64% during the first 2 days of fasting in the male rats and stayed elevated on the third day of fasting. In contrast, heart glycogen of female rats remained unchanged from an initial value of 3.13 mg/g throughout the 3-day fasting period. Endogenous triglyceride (TG) of male rats decreased from 2.14 +/- 0.09 to 1.41 +/- 0.21 mumol/g during the first 24 h without food and remained at that level during the second and third days. Heart TG in female rats fell progressively from 2.36 +/- 0.19 to 1.02 +/- 0.12 mumol/g during the fasting period. Cardiac FFA were higher in female than in male animals throughout the entire experiment. These data indicate that quantitative and qualitative metabolic differences exist between male and female rats stressed by fasting.(ABSTRACT TRUNCATED AT 250 WORDS)
Kinetics of high-affinity Ca2+ sequestration in permeabilized rat pancreatic acini
Authors: Hurley T. W.
Published: December 2017

Energy-dependent subcellular Ca2+ sequestration was studied in the presence of ruthenium red using rat pancreatic acini, which had been permeabilized by exposure to medium nominally free of Ca2+. The initial rate of Ca2+ uptake (approximately 2,800 pmol.min-1.mg acinar protein-1) quickly slowed, and a mean steady-state Ca2+ content of approximately 3,000 pmol/mg was reached after 5-10 min of incubation at 37 degrees C. Ca2+ uptake was stimulated by submicromolar Ca2+ concentrations (K0.5 = 156 nM); required Mg2+-ATP (K0.5 = 0.78 mM) was greatest at a pH of 7.0 and was abolished by the Ca2+ ionophore A23187. Other nucleotide phosphates as well as p-nitrophenylphosphate were relatively poor substrates, supporting Ca2+ uptake at initial rates that were 6-14% of those measured in the presence of ATP. These results show that pancreatic acini permeabilized without detergents possess a nonmitochondrial Ca2+ transporting system not located in the plasma membrane but with the properties expected of a major regulator of acinar cytosolic Ca2+ concentration.
Na+-H+ and Na+-Ca2+ exchange in glomerulosa cells: possible role in control of aldosterone production
Authors: Hunyady L. , Kayser S. , Cragoe E. J. , Balla I. , Balla T. , Spat A.
Published: December 2017

Sodium uptake by rat adrenal glomerulosa cells was stimulated by intracellular acidosis evoked by Na+-propionate. This process was inhibited by 5-(N,N-hexamethylene) amiloride (HMA), a known inhibitor of the Na+-H+ exchange. These experiments demonstrate the existence of the Na+-H+ exchange in glomerulosa cells. Although amiloride inhibited the angiotensin II- and adrenocorticotropic hormone (ACTH)-induced aldosterone response, HMA, a more specific inhibitor of Na+-H+ exchange, failed to do that. 45Ca2+ influx and efflux were dependent on intra- and extracellular Na+ concentrations. Amiloride analogues, known to inhibit Na+-Ca2+ exchange, reduced basal 45Ca influx. Although we could not reveal the activation of Na+-Ca2+ exchange by angiotensin II, inhibitors of Na+-Ca2+ exchange also inhibited the angiotensin- and ACTH-induced aldosterone response of glomerulosa cells. Our results suggest that Na+-Ca2+ exchange supports the maintenance of basal Ca2+ level in the cytoplasma of glomerulosa cells, and amiloride derivatives inhibit aldosterone production by reducing Ca2+ level below resting values.
Characterization of cAMP-induced activation of epithelial sodium channels
Authors: Lester D. S. , Asher C. , Garty H.
Published: December 2017

Incubating toad bladder with 10 mU/ml vasopressin increases the amiloride-blockable Na+ flux in membrane vesicles derived from the epithelial cells by about twofold. This stimulation is further enhanced by 3-isobutyl-1-methylxanthine and can be mimicked by 8-bromoadenosine 3', 5'-cyclic monophosphate. Thus the natriferic action of cAMP involves a sustained change of the apical membrane preserved by the isolated vesicles. The possibility that transport is modulated by direct phosphorylation/dephosphorylation of the Na+ channel was tested. Trapping purified cAMP-dependent protein kinase, cAMP, and ATP in apical vesicles failed to alter Na+ transport even though the enzyme proved active and could phosphorylate intravesicular proteins. Trapping several phosphatases partially purified from toad bladder in vesicles was ineffective as well. These data suggest that the cAMP-induced increase in Na+ conductance involves processes other than phosphorylation of the channel protein or direct channel-cAMP interaction.
In search of a second messenger for insulin
Authors: Saltiel A. R. , Cuatrecasas P.
Published: December 2017

Despite significant advances in the past few years on the chemistry and biology of insulin and its receptor, the molecular events that couple the insulin-receptor interaction to the regulation of cellular metabolism remain uncertain. Progress in this area has been complicated by the pleiotropic nature of insulin's actions. These most likely involve a complex network of pathways resulting in the coordination of mechanistically distinct cellular effects. Because the well-recognized mechanisms of signal transduction (i.e., cyclic nucleotides, ion channels) appear not to be central to insulin action, investigators have searched for a novel second messenger system. A low-molecular-weight substance has been identified that mimics certain actions of insulin on metabolic enzymes. This substance has an inositol glycan structure and is produced by the insulin-sensitive hydrolysis of a glycosyl-phosphatidylinositol in the plasma membrane. This hydrolysis reaction, which is catalyzed by a specific phospholipase C, also results in the production of a structurally distinct diacylglycerol that may selectively regulate one or more of the protein kinases C. The glycosyl-phosphatidyl-inositol precursor for the inositol glycan enzyme modulator is structurally analogous to the recently described glycosyl-phosphatidylinositol membrane protein anchor. Preliminary studies suggest that a subset of proteins anchored in this fashion might be released from cells by a similar insulin-sensitive, phospholipase-catalyzed reaction. Efforts are underway to determine the precise role of the metabolism of glycosyl-phosphatidylinositols in insulin action.
Pathways of Cl- transport in human fibroblasts
Authors: Lin P. , Gruenstein E.
Published: December 2017

Three pathways of Cl- efflux were identified in normal human fibroblasts. Twenty percent of the total Cl- efflux is via an electrically conductive pathway with an efflux constant of 0.016 min-1. This pathway is insensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and bumetanide but is partially inhibited by anthracene-9-carboxylic acid. Twenty-five percent of the Cl- efflux occurs via Cl- with cation cotransport having an efflux constant of 0.020 min-1. This pathway is inhibited by bumetanide and is dependent on the simultaneous presence of Na+, K+, and Cl-. Under basal conditions, the energetics of this pathway indicate that it is operating close to equilibrium. Fifty percent of the Cl- efflux occurs via an anion exchange pathway having an efflux constant of 0.040 min-1 that is inhibited by DIDS or by removal of Cl- from the extracellular medium. Together these pathways account for 95% of the total Cl- efflux.
Modulation of Na+-Pi cotransport in opossum kidney cells by extracellular phosphate
Authors: Biber J. , Forgo J. , Murer H.
Published: December 2017

The effect of the extracellular concentration of Pi on the Na+-dependent phosphate transport activity of OK cells was investigated. When incubated with extracellular Pi at concentrations of 200 microM or less, Na+-Pi cotransport increased approximately twofold in OK cells compared with control cells (kept in 0.85 mM Pi), whereas other Na+-dependent transport activities were not affected. After Pi deprivation, Na+-Pi cotransport could be inhibited to a similar extent (80%) by parathyroid hormone (PTH) as in control cells, suggesting that the PTH-sensitive Na+-Pi cotransport activity is also regulated by extracellular Pi. The increase of Na+-Pi cotransport was maximally expressed after 6 h and could be prevented by cycloheximide (70 microM) but not by actinomycin D (0.5-5 g/ml). However, the adaptive response was completely blocked by 3'-deoxyadenosine (cordycepin) at 100 microM. From these data, it is concluded that the upregulation of Na+-Pi cotransport in OK cells due to low extracellular Pi is controlled at a posttranscriptional level.
Myocyte deenergization and intracellular free calcium dynamics
Authors: Li Q. A. , Altschuld R. A. , Stokes B. T.
Published: December 2017

Intracellular free calcium in adult rat heart ventricular myocytes was monitored by single cell fura-2 fluorescence microscopy. The average resting free calcium in rod-shaped quiescent cells was 125 nM (range 70-200 nM). When cells were deenergized with an inhibitor (amytal) and an uncoupler (carbonyl-cyanide m-chlorophenylhydrazone) of oxidative phosphorylation, there was a small but significant increase (125-380 nM) in intracellular free calcium during the transition to a highly contracted (square) rigor form. After the onset of contracture, which occurred 5-15 min after addition of the above compounds, the increase in free calcium was slow for the first 20 min, reaching a value of only 750 nM. Thereafter, the rate of increase accelerated and 50 min after contracture, free calcium was approximately 3 microM. The increase in free calcium was absolutely dependent on extracellular calcium but was not inhibited by high concentrations of verapamil (2-7 microM), suggesting influx via the Na+-Ca2+ exchange transporter as the cause of calcium increase. However, in calcium repletion protocols the rate of increase in sodium-loaded myocytes was greatly accelerated if cells were not depleted of ATP, confirming suggestions that ATP loss partially inhibits Na+-Ca2+ exchange.
Effects of carbon monoxide on cardiac muscle cells in culture
Authors: Nag A. C. , Chen K. C. , Cheng M.
Published: December 2017

Embryonic rat cardiac muscle cells grown in the presence of various tensions of CO (5-95%) without the presence of O2 survived and exhibited reduced cell growth, which was concentration dependent. When cardiac muscle cells were grown in the presence of a mixture of CO (10-20%) and O2 (10-20%), the growth rate of these cells was comparable to that of the control cells. Cardiac myocytes continued to beat when exposed to varying tensions of CO, except in the case of 95% CO. The cells exposed to different concentrations of CO contained fewer myofibrils of different stages of differentiation compared with the control and the culture exposed to a mixture of 20% O2 and 20% CO, with cells that contained abundant, highly differentiated myofibrils. There was no significant difference in the structural organization of mitochondria between the control and the surviving experimental cells. It is evident from the present studies that O2 is required for the optimum in vitro cellular growth of cardiac muscle. Furthermore, CO in combination with O2 at a concentration of 10 or 20% can produce optimal growth of cardiac muscle cells in culture.
Effect of superoxide radical on Ca2+ pumps of coronary artery
Authors: Grover A. K. , Samson S. E.
Published: December 2017

The effect of superoxide radical on the azide-insensitive ATP-dependent Ca2+-transport by a plasma membrane (PM)-enriched fraction (F2) and an endoplasmic reticulum (ER)-enriched fraction (F3) isolated from pig coronary artery was examined using xanthine oxidase plus xanthine to generate superoxide ions. A preincubation with xanthine oxidase plus xanthine at 37 degrees C preferentially inactivated the oxalate-stimulated Ca2+ uptake by the F3 fraction rather than the phosphate-stimulated uptake by the F2 fraction, indicating that the Ca2+ pump in the ER was more susceptible to this free radical. The inactivation of the Ca2+ uptake depended on the concentrations of xanthine oxidase and xanthine in the preincubation mixture as well as on the preincubation time. Furthermore, the inclusion of superoxide dismutase in the preincubation mixture prevented the inactivation. Thus the inactivation was caused by superoxide radical. Preincubation with xanthine oxidase plus xanthine, however, altered the half-life of efflux of Ca2+ from these vesicles only marginally. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the F3 fraction showed formation of a Ca2+-dependent acid stable phosphoenzyme at 0 degree C predominantly at a protein band corresponding to 100 kDa. The level of the 100-kDa acylphosphate intermediate was inhibited in parallel with the inhibition of the Ca2+ uptake by preincubation with xanthine oxidase plus xanthine. We conclude that superoxide radical inactivates the ER Ca2+ transport by lowering the level of the phosphoenzyme.