bluuebunny/crossref_metadata_2025 huggingface dataset:
Second messenger-specific protein kinases in a salt-absorbing intestinal epithelium
Authors: Toskulkao C. , Nash N. T. , Leach K. , Rao M. C.
Published: December 2017

Calcium, adenosine 3',5'-cyclic monophosphate (cAMP), and guanosine 3',5'-cyclic monophosphate (cGMP) can regulate the same or different ion transport processes within an epithelium, presumably via independent protein phosphorylation mechanisms. Because there have been few detailed studies characterizing these processes in epithelia, we examined the distribution of Ca-, cAMP-, and cGMP-specific protein kinases and substrates in vitro in a homogenous salt-absorbing epithelium, the winter flounder intestine. In this tissue cGMP and Ca inhibit Na-K-2Cl cotransport, cAMP increases anion permeability, and phorbol esters do not affect ion transport. The Ca-specific kinases are calmodulin (CaM) dependent. The tissue possesses type III Ca-CaM protein kinase and its specific substrate elongation factor 2 and type II but not type I Ca-CaM kinase. Addition of phosphatidylserine (PS) and Ca to crude or DEAE-cellulose-purified cytosol neither increased the phosphorylation of exogenous histone H1 substrate nor that of any endogenous substrates. Although these suggest the absence of Ca-phospholipid-dependent kinase (PKC), the cytosol has a 78-kDa protein recognizable by a highly specific polyclonal sheep antibody to rat brain PKC. Both the particulate and cytosolic fractions possess cAMP-specific binding proteins and cAMP-specific phosphoprotein substrates. The particulate fraction cAMP-binding proteins are of molecular mass 50 kDa (pI 5.2) and 48 kDa with multiple isoforms (pI 5.6-6.2); these proteins generate different peptide maps. The cytosol chiefly contains a 50-kDa (pI 5.2) cAMP binding protein that is similar to the particulate 50-kDa protein on peptide mapping. The flounder cAMP binding proteins have the same pI but lower molecular mass and different peptide profiles than the rat brain RII (54/52 kDa) and RI (50 kDa) cAMP regulatory proteins. The cGMP-specific protein kinase was less prominent, very low levels of cGMP-specific binding proteins being detected either by equilibrium binding or by photoaffinity labeling. A prominent kinase substrate in homogenates is a 50-kDa protein, the phosphorylation of which is increased by Ca and cGMP but decreased by cAMP. When intact tissue was prelabeled with 32Pi and then exposed to cGMP, the phosphorylation of a number of substrates including that of a 50-kDa protein was increased. In summary, the flounder intestine possesses the necessary protein phosphorylation mechanisms to account for the regulation of its ion transport processes by second messengers.
Identification of a 50-kDa Ca-, cAMP-, and cGMP-dependent epithelial phosphoprotein as a cAMP regulatory protein
Authors: Toskulkao C. , Rao M. C.
Published: December 2017

Multiple second messengers, presumably acting via protein phosphorylation mechanisms, regulate flounder intestinal ion transport. We recently reported [C. Toskulkao, N. T. Nash, K. Leach, and M. C. Rao. Am. J. Physiol. 258 (Cell Physiol. 27): C879-C888, 1990] that this tissue possesses adenosine 3',5'-cyclic monophosphate (cAMP)- and guanosine 3',5'-cyclic monophosphate (cGMP)-specific protein kinases, types II and III Ca-calmodulin kinases, and very low levels of protein kinase C. These results correlate with ion transport studies in which cGMP and Ca were shown to inhibit salt absorption, cAMP to increase anion permeability, and phorbol esters to have no effect. In the present study we characterized in detail a 50-kDa protein the phosphorylation of which is regulated by more than one second messenger. The 50-kDa (pI 5.2) phosphoprotein is present in both the cytosol (50 kDa-C) and particulate (50 kDa-P) fractions and appears to be regulated by Ca, cAMP, and cGMP. Although the pI and Mr of the regulated proteins are identical, there are differences in the regulation of 50 kDa-P and 50 kDa-C. The phosphorylation of 50 kDa-P is high in the basal state, and Ca and cGMP enhance this. cAMP has a biphasic effect, increasing it at low and decreasing it at high protein concentrations. The isoquinoline derivatives H-8 [50% effective dose (ED50) approximately 2.3 microM] and H-7 (ED50 approximately 45 microM) inhibit basal 50 kDa-P phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
Immune-related intestinal chloride secretion. II. Effect of adenosine on T84 cell line
Authors: Barrett K. E. , Cohn J. A. , Huott P. A. , Wasserman S. I. , Dharmsathaphorn K.
Published: December 2017

The inflammatory mediator adenosine caused sustained Cl- secretion across monolayers of T84 cells. The effect was promptly reversed by the adenosine receptor antagonist 8-phenyltheophylline and appeared to be mediated through an adenosine A2-receptor [rank order of potency: 5'-(N-ethyl)-carboxamido-adenosine (NECA) greater than adenosine greater than (-)-N6-(phenylisopropyl)adenosine (PIA) greater than or equal to (+)-PIA]. High doses of adenosine and its analogues increased cellular adenosine 3',5'-cyclic monophosphate (cAMP) but not guanosine 3',5'-cyclic monophosphate (cGMP) or free cytosolic Ca2+. However, lower concentrations of adenosine had maximal effects on Cl- secretion with little or no effect on cAMP. In other respects, Cl- secretion resembled that induced by cAMP-mediated secretagogues such as vasoactive intestinal peptide (VIP). Addition of both low and high doses of NECA activated basolateral K+ and apical Cl- channels, exhibited synergism with Ca2(+)-mediated secretagogues, did not produce additive effects with VIP or Escherichia coli heat-stable enterotoxin, and was associated with cAMP-dependent protein kinase-mediated protein phosphorylation. The results suggest that either adenosine mobilizes an intracellular pool of cAMP that is extremely efficiently coupled to the cAMP-dependent protein kinase and is thereafter rapidly destroyed or that second messenger(s) other than cAMP, cGMP, or Ca2+ are able to activate Cl- secretion in the T84 cell line. In the latter case, such messenger(s), as yet unidentified, might represent a final common pathway for cyclic nucleotide-activated Cl- secretion.
Metabolism and force in hypertrophic smooth muscle from rat urinary bladder
Authors: Arner A. , Malmqvist U. , Uvelius B.
Published: December 2017

Ten days of urinary outlet obstruction in the rat induced a threefold increase in bladder weight. Active force of control and hypertrophic bladder muscle strips was measured at varying PO2 levels after high-K+, carbachol, or electrical field stimulation. Highest force output was obtained with carbachol. Force per muscle area was lower in the hypertrophic muscles. The basal rates of oxygen consumption and lactate formation were similar in the two groups. The metabolic tension cost (ATP turnover/active force) was similar in the two groups for activation with high K+ and carbachol. In anoxia the active force decreased, but this was less pronounced in the hypertrophied muscle. Hypertrophied muscle could, in contrast to the controls, maintain a sustained K+ contracture in anoxia. Basal metabolic rates and tension cost were markedly reduced in anoxia for both groups. The lower force per area with unaltered tension cost, in hypertrophic muscles under all experimental conditions, may reflect unaltered intrinsic properties of the contractile system, although the amount of contractile material has decreased relative to cell volume. The increased resistance to anoxia may reflect a metabolic adaptation to impaired oxygen supply to the hypertrophied tissue.
Binding of bumetanide to microsomes from optic ganglia of the squid, Loligo pealei
Authors: Altamirano A. A. , Watts B. A. , Russell J. M.
Published: December 2017

Saturable high-affinity binding of [3H] bumetanide [dissociation constant (KD) = 80 nM] was measured in microsomal membranes prepared from squid optic ganglia. Under control conditions, the maximal specific binding of labeled bumetanide (Bmax) was approximately 6-7 pmol/mg protein. Binding had a higher relative affinity for bumetanide than for furosemide and depended on the presence of Cl- and K+, but not Na+, in the incubation media. In the case of K+, [3H]bumetanide binding was half-saturated at [K+] = 100 mM. The Cl- effect was biphasic. At [Cl-] between 0 and 150 mM, [3H]bumetanide binding increased with increasing [Cl-]. However, when [Cl-] was increased above 150 mM, [3H]bumetanide binding was progressively reduced. ATP acted as a nonessential activator [mean affinity constant (K0.5) approximately 1 microM] of the ion-dependent [3H]bumetanide binding by increasing the apparent binding capacity. The activation by ATP did not require Mg2+. Other adenosine analogues also stimulated the binding of bumetanide.
Effects of MeCh, thapsigargin, and La3+ on plasmalemmal and intracellular Ca2+ transport in lacrimal acinar cells
Authors: Kwan C. Y. , Takemura H. , Obie J. F. , Thastrup O. , Putney J. W.
Published: December 2017

The Ca2(+)-mobilizing actions of the muscarinic receptor agonist, methacholine (MeCh), and the microsomal Ca2+ pump inhibitor, thapsigargin, were investigated in lacrimal acinar cells. As previously shown for parotid cells (J. Biol. Chem. 264: 12266-12271, 1989), thapsigargin activates both internal Ca2+ release and Ca2+ entry from the extracellular space without increasing cellular inositol phosphates. The inorganic Ca2+ antagonist La3+ inhibited MeCh- or thapsigargin-activated Ca2+ entry. However, when added before MeCh or thapsigargin, La3+ inhibited the extrusion of Ca2+ at the plasma membrane. This phenomenon was exploited in protocols designed to investigate the pathways for filling agonist-sensitive Ca2+ stores in lacrimal cells. The results show that, in contrast to previous suggestions that external Ca2+ is required to replenish agonist-regulated Ca2+ stores, the inhibition of Ca2+ extrusion permits recycling of Ca2+ released by MeCh back into an MeCh- and thapsigargin-sensitive pool. Thus, although extracellular Ca2+ is the major source for refilling the intracellular Ca2+ stores under physiological conditions, the pathway by which this Ca2+ enters the pool need not be a direct one. These results are consistent with the recently revised capacitative model for the refilling of intracellular Ca2+ stores through Ca2+ influx subsequent to Ca2+ depletion, according to which refilling of intracellular Ca2+ stores occurs via a cytoplasmic route rather than a direct channel between intracellular Ca2+ stores and the extracellular space.
Nickel inhibits endothelin-induced contractions of vascular smooth muscle
Authors: Blackburn K. , Highsmith R. F.
Published: December 2017

Endothelin (ET)-induced contractions of vascular smooth muscle (VSM) are dependent on extracellular Ca2+ yet display only partial sensitivity to L-type Ca2+ antagonists. The purpose of this study was to evaluate the effect of nickel (Ni2+), a Ca2+ channel antagonist with clearly documented differential potency toward L- vs. T-type Ca2+ currents on ET-mediated contractions in VSM. Treatment of rings of left anterior descending porcine coronary artery (LAD) with Ni2+ produced a profound dose-dependent inhibition of isometric force development in response to porcine ET (ET-1). At a concentration of 360 microM, Ni2+ exerted a significant inhibitory effect on contracture in response to doses of ET-1 ranging from 3 to 100 nM. In contrast, the same concentration of Ni2+ failed to significantly affect peak force development in response to KCl depolarization (5-77 mM) or to phenylephrine (0.3-30 mM). In addition, 360 microM Ni2+ significantly inhibited the contractile response of rat aorta to 10 nM ET-1. We conclude that ET-1 activates a Ni2(+)-sensitive process in VSM which may signal an additional Ca2+ influx pathway that appears to be functionally distinct from the L-type Ca2+ channel.
Olfactory neurons exhibit heterogeneity in depolarization-induced calcium changes
Authors: Restrepo D. , Teeter J. H.
Published: December 2017

Olfactory neurons from the channel catfish, Ictalurus punctatus, were isolated by a brief (15 min) treatment with papain. After incubation with fura-2 acetoxymethyl ester (fura-2/AM) for 1 h, the isolated olfactory receptor cells are found to hydrolyze fura-2/AM to fura-2 free acid without detectable traces of intermediate products of hydrolysis. Intracellular calcium measured with fura-2 in single cells covers a wide range (from less than 2 to 100 nM with a median of 17.6 nM, n = 140 cells). Twenty-one percent of the cells respond to potassium-induced depolarization with an increase in intracellular calcium mediated by influx of extracellular calcium. The L-type calcium channel antagonist nimodipine inhibits the increase in intracellular calcium triggered by membrane depolarization and blocks small unitary barium currents displaying the characteristics of L-type calcium currents (unitary conductance of 29 +/- 5 pS in 55 mM BaCl2 and high selectivity for Ba2+ over Na+ and K+) recorded from azolectin bilayers at the tip of patch pipettes into which isolated olfactory cilia membrane vesicles had been incorporated. Olfactory neurons are found to be functionally heterogeneous in their response to membrane depolarization and can be separated into three groups: one in which the increase in intracellular calcium is rapid and transient, another in which calcium increases slowly, and a third group of cells in which depolarization causes no change in intracellular calcium.
Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release. II. Effect of cAMP-dependent protein kinase
Authors: Volpe P. , Alderson-Lang B. H.
Published: December 2017

The effect of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) on Ca2+ loading, inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release, and [3H]IP3 binding of canine cerebellar membrane fractions was investigated. PKA in the presence of cAMP and the catalytic subunit of PKA did not change Ca2+ loading yet increased the extent of IP3-induced Ca2+ release by approximately 35%. Hill plot analysis indicated that the catalytic subunit of PKA increased the apparent Michaelis constant of IP3-induced Ca2+ release twofold, from 0.3 to 0.7 microM IP3. The protein kinase inhibitor reversed these changes. cAMP affected neither Ca2+ loading nor IP3-induced Ca2+ release. The catalytic subunit of PKA did not appreciably affect the maximum binding and dissociation constant of [3H]IP3 binding, as judged by Scatchard analysis. Thus the catalytic subunit of PKA influences the opening of Ca2+ channels by IP3 without interfering with the binding of IP3 to its receptor sites.
Activation of human platelets by C5a-stimulated neutrophils: a role for cathepsin G
Authors: Ferrer-Lopez P. , Renesto P. , Schattner M. , Bassot S. , Laurent P. , Chignard M.
Published: December 2017

Human platelets can be stimulated by recombinant human fifth component of complement (rhC5a) in the presence of human neutrophils. After challenge with N-formyl-Met-Leu-Phe or rhC5a, concentrated neutrophils release cathepsin G into the supernatant. The concentrations of cathepsin G recovered by titration of the enzymatic activity correlate with the capability of these supernatants to induce platelet stimulation as measured by serotonin release. Cathepsin G purified from neutrophil granules triggered platelet aggregation and serotonin release independent of arachidonic acid metabolites and platelet-activating factor formation. A concentration of 100 nM of cathepsin G, which was reached in the surrounding space of activated neutrophils, induced a 50% platelet stimulation. Three distinct antiproteinases were tested against cathepsin G-induced platelet activation. Z-Gly-Leu-Phe-CH2Cl, a specific inhibitor of cathepsin G enzymatic activity, proved to be nonspecific in our biological system. By contrast, alpha 1-antichymotrypsin and alpha 1-antitrypsin displayed specific activities. The physiological specific inhibitor of cathepsin G, alpha 1-antichymotrypsin, was the most potent and was used in the rhC5a-induced neutrophils-mediated platelet activation. A complete inhibition was achieved, showing that release of cathepsin G from neutrophils accounts for platelet activation. Such a chain of events involving C5a, neutrophils, cathepsin G, and platelets may be of relevance in certain inflammatory states, particularly the adult respiratory distress syndrome.
Role of Na(+)-H+ and Cl(-)-HCO3- antiports in the regulation of cytosolic pH near neutrality
Authors: Tonnessen T. I. , Sandvig K. , Olsnes S.
Published: December 2017

In Vero cells, Na(+)-H+ antiport as well as Na(+)-coupled and Na(+)-independent Cl(-)-HCO3- antiport are involved in regulation of cytosolic pH (pHi) after large (unphysiological) deviations from neutrality. In this paper we have studied to which extent each of the three antiports is involved in regulation of pHi after small deviations from neutrality expected to occur under physiological conditions. At physiological extracellular pH (pHo), inhibition of Na(+)-H+ exchange by amiloride did not alter pHi. At neutral and alkaline pHo, pHi was found to be lower in the presence of HCO3- than in its absence, whereas at acidic pHo, pHi was higher in the presence of HCO3- than in its nominal absence. Above pHi 6.5, the activity of the Na(+)-coupled Cl(-)-HCO3- antiport was higher than the Na(+)-H+ antiport. After a small reduction of pHi, the recovery of steady-state pHi was entirely dependent on Na(+)-coupled Cl(-)-HCO3- antiport, whereas after more pronounced acidification, also Na(+)-H+ exchange contributed to the acid extrusion. The Na(+)-independent Cl(-)-HCO3- antiport, which acts as an acidifying mechanism, was strongly activated at pHi greater than 7.1. The results indicate that at physiological pHo the steady-state pHi is largely determined by the activity of the two Cl(-)-HCO3- antiports, and they suggest that Na(+)-H+ exchange does not influence the resting pHi under these conditions.
Polyaspartic acid inhibits gentamicin-induced perturbations of phospholipid metabolism
Authors: Ramsammy L. , Josepovitz C. , Lane B. , Kaloyanides G. J.
Published: December 2017

We investigated whether polyaspartic acid (PAA) can inhibit aminoglycoside-induced perturbations of phospholipid metabolism in cultured renal cells of opossum and rabbit and examined the mechanism involved. Cells incubated in medium containing gentamicin (10(-3) M) manifested a time-dependent increase in total phospholipid in association with the appearance of lysosomal myeloid bodies, impaired degradation of phospholipid, and disruption of the phosphatidylinositol (PI) cascade in response to bradykinin stimulation. These alterations of phospholipid metabolism were either completely or almost completely prevented in cells grown in medium containing gentamicin (10(-3) M) and PAA (3 x 10(-4) M, mol wt 11,000) even though PAA did not inhibit the cellular accumulation of gentamicin (40 +/- 1 vs. 42 +/- 1 micrograms/mg protein). In other in vitro studies, we demonstrated that gentamicin depressed the permeability of phosphatidylcholine (PC)/PI liposomes to glycerol and promoted liposomal aggregation. Both effects were blocked by prior addition of PAA. Methylene blue, a cationic dye, was shown to form an electrostatic complex with PAA; gentamicin competitively displaced methylene blue bound to PAA. Our results support the conclusion that the protective effect of PAA is related to its ability to serve as an anionic substrate that electrostatically binds aminoglycoside antibiotics and, thereby, prevents these polycationic drugs from interacting electrostatically with anionic phospholipid of cell membranes.
Furosemide blocks basolateral membrane Cl- permeability in gallbladder epithelium
Authors: Stoddard J. S. , Altenberg G. A. , Ferguson M. L. , Reuss L.
Published: December 2017

In Necturus gallbladders bathed in a NaCl Ringer solution buffered with 10 mM HCO3(-)-1% CO2, furosemide (added to the serosal solution) caused a concentration-dependent hyperpolarization of both cell membranes that was slow and reversible. At 10(-3) M furosemide, the basolateral membrane voltage (Vcs) increased significantly from -71 +/- 3 to -85 +/- 3 mV, the depolarization of Vcs elicited by a 10-fold rise in serosal [K+] increased from 34 +/- 4 to 50 +/- 1 mV, the depolarization elicited by lowering serosal [Cl-] from 98 to 8.1 mM was reduced from 15 +/- 1 to 1 +/- 1 mV, and the depolarization in response to lowering serosal [HCO3-] from 10 to 1 mM was reduced from 13 +/- 1 to 5 +/- 0.4 mV. Furosemide could in principle decrease the basolateral membrane Cl- conductance (Gcl), increase the basolateral membrane K+ conductance, or have a combined effect. To distinguish among these possibilities, we estimated the resistance of the basolateral membrane (Rb) by means of two-point intraepithelial cable analysis experiments. Furosemide increased Rb by 22%, which indicates that furosemide reduces basolateral membrane Gcl. The effect cannot be attributed to inhibition of apical membrane anion exchange by serosal addition of furosemide, because base secretion from cells to lumen is unchanged. We conclude that furosemide blocks reversibly basolateral membrane electrodiffusive Cl- permeability. A concomitant stimulation of basolateral membrane electrodiffusive K+ permeability is also possible.
A nonenzymatic preparation of epithelial basolateral membrane for patch clamp
Authors: Wehner F. , Garretson L. , Dawson K. , Segal Y. , Reuss L.
Published: December 2017

A preparation has been developed that permits patch clamping of the basolateral membrane of Necturus gallbladder epithelial cells with a high success rate. The epithelium is separated from the underlying tissues mechanically, without enzymatic treatment. Its apical surface is attached to a plastic cover slip, and the basolateral surface, facing up, is cleaned with a suction pipette under microscopic observation. With this cleaning procedure, the success rate in obtaining gigaohm seals increases from less than 1% to approximately 10% of the attempts. The cells appear to retain their structural and functional integrity, as evidenced by electron-microscopic appearance and magnitude of cell membrane voltages. Major advantages of the preparation are that the basolateral membrane domain is preserved and that enzymatic treatment, which could potentially alter membrane proteins, is not necessary.
A simple model of aerobic metabolism: applications to work transitions in muscle
Authors: Funk C. I. , Clark A. , Connett R. J.
Published: December 2017

Adding kinetics to the model of the phosphate energy system [Connett. Am. J. Physiol. 254 (Regulatory Integrative Comp. Physiol. 23): R949-R959, 1988], we provide a framework for analyzing metabolic transients in muscle tissue. We modify the formalism of the earlier model and introduce a buffering factor, which measures buffering of adenine nucleotides by phosphocreatine. The time course of the phosphate energy state can be calculated given the following: 1) adenosinetriphosphatase (ATPase) rate, 2) pH, and 3) a mitochondrial driving function, i.e., ATP production in terms of the phosphate energy state. We use mitochondrial driving functions derived from steady-state measurements to predict the time courses for rest-work transitions. Predictions for transitions in the rat gastrocnemius muscle agree with published values. The model is used to test different existing hypotheses of oxygen consumption (VO2) regulation. Each hypothesis generates a specific mitochondrial driving function, which in turn generates a specific time course of phosphate energy state during transitions. A mitochondrial driving function based on enzyme kinetics with ADP as a substrate leads to time courses not matching the data. Mitochondrial driving functions that are linear with phosphocreatine, Pi, phosphorylation potential, or the pool of high-energy phosphate bonds (phosphate potential energy) gave good agreement with the data.
Liver glucose transporter: a basolateral protein in hepatocytes and intestine and kidney cells
Authors: Thorens B. , Cheng Z. -Q. , Brown D. , Lodish H. F.
Published: December 2017

Pages C279–C294: B. Thorens, Z.-Q. Cheng, D. Brown, and H. F. Lodish. “Liver glucose transporter: a basolateral protein in hepatocytes and intestine and kidney cells.” B. Thorens, H. F. Lodish, and D. Brown. “Differential localization of two glucose transporter isoforms in rat kidney.” Because of the disappointing reproduction of the halftones, these articles are reprinted at the end of the December issue.
Morphological and functional characterization of the endosarcomeric elastic filament
Authors: Salviati G. , Betto R. , Ceoldo S. , Pierobon-Bormioli S.
Published: December 2017

The elastic filament was studied in chemically skinned fibers from rabbit psoas muscle by electron microscopy and resting tension measurements. Extraction of skinned fibers with 40 mM sodium pyrophosphate caused a selective removal of about two-thirds of the thick filaments and formed a gap between the remaining portion of the A band and the I band. Very thin filaments were seen in the gap and were decorated by anti-titin antibody. The resting tension of these fibers was comparable to that of unextracted control fibers. When the M band was completely extracted by a solution containing 0.6 M NaCl, the resting tension completely disappeared at sarcomere lengths from 2.8 to approximately 3.4 microns. These results suggest that the elastic force of short sarcomeres is endowed in the titin filaments and that these filaments are anchored to some structures of the Z and M lines. Other filaments were found in the gap between the two I bands of NaCl-extracted sarcomeres. These filaments differed from titin filaments by a larger diameter and the anchoring points. They may represent the sarcomeric structures responsible for the resting tension of extracted fibers stretched at sarcomere lengths longer than 3.4 microns.
Acidification and intracellular sodium ion activity during stimulated myocardial ischemia
Authors: Vanheel B. , de Hemptinne A. , Leusen I.
Published: December 2017

With the use of microelectrodes, intracellular pH (pHi), surface pH (pHs), and intracellular Na+ activity (aiNa) were measured in isolated guinea pig papillary muscles during normal superfusion and during a reversible condition of simulated ischemia. Acid loading by NH+4 prepulse or by CO2-HCO3- addition during superfusion with pH 7.4 solutions caused internal acidification followed by a recovery of pHi, which could be inhibited by amiloride. pHi recovery was associated with an amiloride-sensitive peak rise of aiNa and membrane hyperpolarization, indicative of Na(+)-H+ exchange. Peak increase of aiNa was absent if the pH of the superfusion solution was concomitantly lowered. Imposed ischemia after control superfusion caused membrane depolarization and acidification of pHi and pHs. The change of pHs consistently was larger than that of pHi. aiNa decreased from 5.5 to 4.6 mM after 10-min ischemia. Enlarging the pHi (and pHs) decrease in ischemia by prior reduction of the tissue buffer capacity (CO2-HCO3(-)-free superfusion) was unable to induce a rise of aiNa during the subsequent ischemic period. Amiloride had no significant effect on aiNa during ischemia. It is concluded that the important acidification of pHs reduces the rate of pHi regulatory Na(+)-H+ exchange and thereby contributes to a longer maintenance of the Na+ electrochemical gradient in ischemic cardiac muscle.
Failure of glutamate dehydrogenase system to predict oxygenation state of human skeletal muscle
Authors: Katz A. , Spencer M. K. , Sahlin K.
Published: December 2017

In a recent study, the total tissue contents of glutamate (Glu), ammonium (NH+4), and 2-oxoglutarate (2-OG) were used to estimate changes in the mitochondrial redox state ([NAD+]/[NADH]) of contracting skeletal muscle with intact circulation [Am. J. Physiol. 253 (Cell Physiol. 22): C263-C268, 1987]. These metabolites participate in the glutamate dehydrogenase (GDH) reaction, which, based on a number of assumptions, theoretically enables calculation of the mitochondrial redox state as follows (brackets indicate concentrations): [NAD+]/[NADH] = ([NH+4] [2-OG])/[( Glu]Kapp), where Kapp is the apparent equilibrium constant for GDH. The purpose of this study was to determine whether changes in the total tissue contents of Glu, NH+4, and 2-OG could be used to predict a reduction of the mitochondrial redox state in anoxic skeletal muscle. Anoxia was induced in the quadriceps femoris muscle by 10 min of circulatory occlusion (low metabolic rate) and isometric contraction to fatigue (high metabolic rate). The mean (+/- SE) value for the metabolite ratio ([NH+4][2-OG]/[Glu]) at rest was 6 +/- 3 mmol/kg dry wt (x 10(-4]. No significant change occurred after circulatory occlusion (4 +/- 2 x 10(-4); P greater than 0.05), whereas an almost 60-fold increase was observed after isometric contraction (P less than 0.05). Because the muscle was anoxic under both conditions, a significant decrease in the metabolite ratio should have occurred. These data demonstrate that changes in total tissue contents of Glu, NH+4, and 2-OG cannot be used to estimate changes in the redox and oxygenation state of mitochondria in intact human skeletal muscle.
Na(+)-K(4)-Cl- cotransport in cultured cells derived from human retinal pigment epithelium
Authors: Kennedy B. G.
Published: December 2017

Unidirectional fluxes of Rb+ and Cl- were measured in cultured human retinal pigment epithelium (HRPE) to characterize the Na(+)-K(+)-Cl-cotransport system in this epithelial tissue. In HRPE, the ouabain-sensitive fraction of Rb+ influx comprises 65% of the total Rb+ influx. Bumetanide [inhibition constant (KI) = 160 nM] inhibited the residual ouabain-insensitive fraction of Rb+ influx by 70%. The bumetanide-sensitive fraction of Rb+ influx was dependent on the presence of both extracellular Na+ and Cl-. Cl- influx was similarly inhibited by bumetanide and also dependent on extracellular Na+ and Rb+. The stoichiometry of bumetanide-sensitive Cl- influx to Rb+ influx was 2:1. Elevation of extracellular osmolarity (by 30%) caused a 2.5-fold increase in Na(+)-K(+)-Cl- cotransport activity in cultured HRPE. The present study provides direct evidence for the occurrence of Na(+)-K(+)-Cl- cotransport-mediated cation and anion transport in HRPE.
H+ and HCO3- flux across apical surface of rat distal colon
Authors: Feldman G. M. , Stephenson R. L.
Published: December 2017

Colonic ion transport is postulated to occur via simultaneous operation of Na(+)-H+ exchange and Cl(-)-HCO3- exchange. Accordingly H+ and HCO3- should be transported simultaneously by the colon. To assess simultaneous H+ and HCO3- transport, net acid-base flux was measured in isolated segments of rat distal colon. When both tissue surfaces were bathed in symmetrical solutions containing Cl-, net base was secreted (-1.0 +/- 0.1 mu eq.cm-2.h-1). Cl- substitution with gluconate in the mucosal medium caused net base flux to switch from secretion to absorption (2.0 +/- 0.2 mu eq.cm-2.h-1). To evaluate whether base absorption was dependent on H+ secretion via Na(+)-H+ exchange, mucosal Na+ was substituted with N-methylglucamine, and amiloride, an inhibitor of Na(+)-H+ exchange, was applied. Na+ substitution and 1 mM amiloride inhibited base absorption by 37 and 38%, respectively, suggesting operation of Na(+)-H+ exchange. Because base absorption persisted, an additional mechanism was considered, HCO3- absorption via Cl(-)-HCO3- exchange. This was evaluated with an inhibitor of Cl(-)-HCO3- exchange 4-acetamido-4'-isothiostilbene-2,2'-disulfonic acid (SITS). SITS (1 mM) inhibited HCO3- absorption by 40%. The effects of amiloride and SITS were additive, suggesting that the Na(+)-H+ and Cl(-)-HCO3- exchangers operate simultaneously. Amiloride also inhibited H+ secretion when net HCO3- was secreted, suggesting that the direction of HCO3- movement does not influence Na(+)-H+ exchange activity. These data suggest that the colon transports both H+ and HCO3- across the apical surface via Na(+)-H+ exchange and Cl(-)-HCO3- exchange; H+ is secreted via Na(+)-H+ exchange, whereas HCO3- can be secreted or absorbed via Cl(-)-HCO3- exchange.
Beta-adrenoceptor metabolism in wild-type, Gs, and protein kinase A-variant S49 cells
Authors: Jasper J. R. , Motulsky H. J. , Mahan L. C. , Insel P. A.
Published: December 2017

To determine the role of the stimulatory guanine nucleotide-binding protein, Gs, and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase in the basal metabolism of beta-adrenergic receptors in S49 lymphoma cells, we measured the return of receptor number and function after irreversible blockade of receptors. After inactivation of receptors with the irreversible ligand N8-(bromoacetyl)-N'-[3-(4-indolyoxy)-2-hydroxypropyl]-(2)-1,8-diam ino-p- methane (BIM), beta-adrenergic receptors (defined as [125I]iodocyanopindolol binding sites) reappeared in a biphasic manner, the faster phase having a half-time (t 1/2) of 3-8 h (approximately 50% of the sites) and the slower phase greater than 40 h. Although the slow phase is not readily explained, recovery of binding sites during the first 10 h matched recovery of receptor function after BIM treatment (as measured by stimulation of cAMP accumulation) and recovery of receptor sites after downregulation induced by the agonist isoproterenol. Thus quantifying receptor recovery during the first 10 h after BIM treatment appears to be a reasonable method for examining basal receptor metabolism in S49 cells. Measured in this way, metabolism of beta-adrenergic receptors is very similar in wild-type S49 and the following variant clones: cyc- (absent Gs alpha), UNC and H21a (defective Gs alpha), and kin- (lacking cAMP-dependent protein kinase activity). Although previous data have demonstrated that agonist-promoted downregulation of beta-adrenergic receptors requires functional receptor-Gs coupling, the current data suggest that neither Gs nor cAMP-dependent protein kinase activity plays an important role in the regulation of basal metabolism of beta-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
Anionic amino acid transport systems in isolated basal plasma membrane of human placenta
Authors: Hoeltzli S. D. , Kelley L. K. , Moe A. J. , Smith C. H.
Published: December 2017

The placenta absorbs anionic amino acids from the maternal and fetal circulations but does not significantly transfer these amino acids from mother to fetus. Uptake of L-aspartate and L-glutamate by basal (fetal-facing) plasma membrane vesicles from placental syncytiotrophoblast was stimulated by an inward sodium and an outward potassium gradient. Measurable saturable uptake was entirely sodium dependent and electrogenic. Studies of concentration dependence resolved a high-affinity (microM) system that has characteristics of the X-AG system found in other tissues including the placental microvillous plasma membrane. Uptake of 0.2 microM L-glutamate was inhibited by 2 mM L-glutamate, L-aspartate, D-aspartate, L-cysteate, and L-cysteinesulfinic acid and was uninhibited by 2 mM D-glutamate, L-glutamine, L-alanine, L-serine, L-asparagine, and taurine or by 1 mM methylaminoisobutyric acid. The X-AG system in the two membranes of the placental syncytiotrophoblast may mediate the concentrative uptake of anionic amino acids from the maternal and fetal circulations into the placenta.
A23187 increases permeability of MDCK monolayers independent of phospholipase activation
Authors: Peterson M. W. , Gruenhaupt D.
Published: December 2017

Changes in intracellular calcium influence epithelial barrier integrity, but the mechanism of action is unknown. One possibility is that calcium may work by increasing phospholipase A2 (PLA2) and/or phospholipase C (PLG) activity. Measuring the mannitol permeability (Pmann) of cultured monolayers of Madin-Darby canine kidney (MDCK) epithelium cells as a measure of barrier integrity, we found that exposure of the monolayers to 5 and 10 microM A23187 produced an increase in Pmann whereas 1 microM A23187 did not. Exposure of MDCK cells labeled with [3H]arachidonate to A23187 resulted in an increase in both PLA2 activity, as measured by an increase in free fatty acids, and in PLC activity, as measured by an increase in diacylglycerol (DAG). The increase in DAG was due to an increase in phosphatidylcholine-specific PLC activity. The relationship of phospholipolysis to Pmann was evaluated further by the use of mepacrine and dexamethasone. Mepacrine (10 microM) decreased PLA2 activity by 60% but had no effect on increased Pmann after exposure to A23187. Preexposure of the monolayers to dexamethasone (10 microM) blocked both PLA2 activity and PLC activity and also prevented the increase in Pmann after exposure to A23187. To evaluate whether this protective effect of dexamethasone was due to PLC blockade, we incubated the cells with the protein kinase C blocker H-7. Incubation with H-7 offered no protection from increased Pmann after A23187. These results demonstrate that increased intracellular calcium decreases the barrier integrity of epithelium and increases both PLA2 and phosphatidylcholine-specific PLC activity. The increase in Pmann, however, appears to occur through mechanisms other than phospholipase activation.
Calcium and the heat-shock response in the human monocytic line U-937
Authors: Kantengwa S. , Capponi A. M. , Bonventre J. V. , Polla B. S.
Published: December 2017

In the human monocytic line U-937, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] increases cytosolic free calcium concentration ([Ca2+]i). 1,25-(OH)2D3 also enhances the synthesis of heat-shock proteins (HSPs) when U-937 cells are exposed to elevated temperatures. To establish whether these two effects of 1,25-(OH)2D3 are related, we examined the effects of calcium on the heat-shock (HS) response, as well as the influence of 1,25-(OH)2D3 on this system. The equilibrium dissociation constant (Kd) of the fluorescent probe used to measure [Ca2+]i, fura-2, at 37 and 45 degrees C was found to be 191 and 234 nM, respectively. Exposure of U-937 cells to 45 degrees C did not increase [Ca2+]i under conditions in which active efflux of the dye was prevented by the organic anion transport inhibitor probenecid (1 mM). In cells preincubated in calcium-free medium, with subsequent addition of 4 mM EGTA before HS, or exposed to the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), the increase in HSPs synthesis was not affected. Cell viability, assessed by [3H]thymidine uptake, was not different between cells exposed to HS in calcium-containing or calcium-free media. Moreover, the effects of 1,25-(OH)2D3 on the HS response were also observed in a calcium-depleted medium, indicating that the effects of 1,25-(OH)2D3 on HSP synthesis were not mediated by [Ca2+]i.
Uptake and metabolism of putrescine in confluent LLC-PK1 cells
Authors: Hauser M. R. , Cook J. S.
Published: December 2017

Putrescine is taken up by confluent pig kidney (LLC-PK1) cells at roughly equal rates over both Na(+)-dependent and Na(+)-independent pathways. The former is sensitive to 1 mM amiloride, but the latter is not. Uptake rates are similar at both the apical and basolateral surfaces. The principal fate of the putrescine is oxidative deamination, yielding a product that appears to be either gamma-aminobutyraldehyde or delta 1-pyrroline. Most of the remainder is converted to products tentatively identified as spermidine, spermine, or another unidentified product; these products as well as putrescine itself are lost from the cell at either surface. Changing the extracellular pH in the range of 6.8-8.0 has no affect on putrescine uptake. Cells acidified to intracellular pH 6.8 show a reduced capacity to incorporate radioactivity, an effect that may be due to inhibition of diamine oxidase. Depletion of ATP stores by treating cells with 2-deoxy-D-glucose and NaN3 does not reduce putrescine uptake, suggesting that the mechanism is not a primary active transporter. The Na(+)-dependent component of uptake is inhibited by 5-50 microM Hg2+ in a dose-dependent manner. p-Chloromercuribenzene sulfonic acid (p-CMBS) at high concentrations (500-1,000 microM) does not affect Na(+)-independent uptake but in the presence of Na+ depresses total uptake more than Na+ depletion alone, suggesting that Na+ enhances the binding of p-CMBS to both transporters. Spermidine and spermine compete with putrescine for uptake, but a variety of other organic bases and amino acids do not, indicating that polyamines are transported by mechanisms distinct from the transporters for those other compounds.
Distribution of F-actin in bipolar and horizontal cells of bass retinas
Authors: Vaughan D. K. , Lasater E. M.
Published: December 2017

F-actin distribution was studied in bipolar and horizontal interneurons of white bass retina. Cryosections of intact retina and isolated cells in culture were labeled with the F-actin specific probe fluorescent phalloidin. In intact retina, labeling was heaviest in the photoreceptor-pigment epithelium region, outer limiting membrane, horizontal cell somata, and plexiform layers. In isolated bipolar and horizontal cells, labeling patterns specific to each cell type were obtained, as were patterns in distinct categories of neurites extending from cells maintained in culture for several days. Label was especially heavy where contacting horizontal cells apposed and were coupled via extensive gap junctions both in the intact retina and in culture. This observation led to the hypothesis that this F-actin domain might modulate junctional conductance. However, cytochalasin D-induced disruption of the F-actin cytoskeletons of coupled, cultured horizontal cells had no effect on normal coupling or on dopamine-induced uncoupling. The F-actin domain may instead mediate gap junction turnover.
Forskolin-induced HCO3- current across apical membrane of the frog corneal epithelium
Authors: Candia O. A.
Published: December 2017

Forskolin (and other Cl- secretagogues) does not affect the very small Na(+)-originated short-circuit current (Isc) across frog corneal epithelium bathed in Cl- free solutions. However, forskolin in combination with increased PCO2 bubbling of the solutions (5-20% CO2) stimulated Isc proportionally to PCO2 to a maximum of approximately 8 microA/cm2. This current could be eliminated and reinstated by sequentially changing the gas composition of the bubbling to 100% air and 20% CO2-80% air. The same effects were observed when PCO2 changes were limited to the apical-side solution. Stroma-to-tear HCO3- movement was deemed unlikely, since the increase in Isc was observed with a HCO3(-)-free solution on the stromal side and CO2 gassing limited to the tear side. From the effects of ouabain and tryptamine, at least 80% of the Isc across the basolateral membrane can be accounted for by the Na+ pump current plus K+ movement from cell to bath. Methazolamide also inhibited Isc. Current across the apical membrane cannot be attributed to an electronegative Na(+)-HCO3- symport given the insensitivity of Isc to a disulfonic stilbene and the fact that stroma-to-tear Na+ fluxes did not increase on stimulation of Isc. The tear-to-stroma Na+ flux also remained unaltered, negating an increased apical bath-to-cell Na+ flow. The forskolin-20% CO2 manipulation produced a depolarization of the intracellular potential, a reduction in the apical-to-basolateral resistance ratio, and a decrease in transepithelial resistance.(ABSTRACT TRUNCATED AT 250 WORDS)
Stretch-induced prostaglandins and protein turnover in cultured skeletal muscle
Authors: Vandenburgh H. H. , Hatfaludy S. , Sohar I. , Shansky J.
Published: December 2017

Intermittent repetitive mechanical stimulation of differentiated avian skeletal muscle cells in vitro for 48 h stimulates skeletal muscle growth [Am. J. Physiol. 256 (Cell Physiol. 25): C674-C682, 1989]. During the first 2-3 h of stimulation, temporary muscle damage occurs based on increases in creatine kinase efflux, total protein degradation rates, and several proteinase activites. With continued mechanical stimulation for several days in serum-containing medium, the proteinase activities return to control levels, and total protein degradation rates decrease to levels less than static controls. Decreased protein degradation thus contributes to stretch-induced cell growth. The efflux of prostaglandins (PG) E2 and F2 alpha but not 6-keto-PGF1 alpha increase with mechanical stimulation. During the first 5 h of stimulation, PGE2 and PGF2 alpha efflux rates increase 101 and 41%, respectively. PGE2 efflux returns to control levels by 24 h of mechanical stimulation, whereas PGF2 alpha efflux is continuously elevated (41-116%) for at least 48 h. The long-term stretch-induced elevation of PGF2 alpha efflux correlates with a 52-98% long-term increase in total protein synthesis rates. The prostaglandin synthesis inhibitor indomethacin partially blocks early stretch-induced cell damage and long-term stretch-induced cell growth. The results indicate that both of these processes are partially dependent on stretch-induced increases in prostaglandin synthesis.
Substance P contracts bovine tracheal smooth muscle via activation of myosin light chain kinase
Authors: Corson M. A. , Sellers J. R. , Adelstein R. S. , Schoenberg M.
Published: December 2017

At near-threshold substance P concentrations, the isometric tension response of bovine tracheal strips is almost completely abolished by atropine, indicating mediation of contraction via substance P-stimulated release of acetylcholine from prejunctional nerve terminals. At near-maximal concentrations, the atropine-inhibited component of the tension response is less than 25%, indicating mainly direct activation. Under conditions in which activation by substance P is direct, peak tension is reached in approximately 11 min. Immunoblot analysis of the time course of phosphorylation of the 20-kDa myosin light chain (LC20) reveals incorporation of approximately 0.5 mol phosphate/mol light chain at 10 min. Two-dimensional tryptic phosphopeptide analysis of phosphorylated light chain reveals a single major phosphopeptide. The peptide migrates identically with that produced by myosin light chain kinase phosphorylation of purified tracheal myosin in vitro. Contraction stimulated by acetylcholine is more rapid, with attainment of peak tension in 2.5 min and a peak LC20 phosphorylation of 0.65 mol/mol. These results indicate that 1) substance P mediates contraction of bovine trachea both directly and indirectly, and 2) under conditions in which activation is direct, the tension and phosphorylation responses qualitatively resemble those observed with acetylcholine.
Peptide modulators of myosin light chain kinase affect smooth muscle cell contraction
Authors: Kargacin G. J. , Ikebe M. , Fay F. S.
Published: December 2017

To examine the importance of myosin light chain kinase (MLCK) in the initiation of contraction in smooth muscle, we used a constitutively active form of MLCK (IMLCK) and two specific peptide inhibitors of MLCK to study the activation of skinned single smooth muscle cells. Although unregulated by Ca-calmodulin, IMLCK, in vitro, was found to have biochemical properties like those of MLCK. Upon photolysis of caged ATP, IMLCK caused Ca-free shortening of skinned cells similar in time course and extent to that induced by Ca2+. Two peptide probes, RS-20 and SM-1, patterned after the Ca-calmodulin binding site and a pseudosubstrate inhibitory site, respectively, of the native MLCK molecule, were shown to specifically inhibit MLCK in in vitro experiments. Both peptides dose dependently inhibited Ca-induced shortening of skinned single cells. These results indicate that MLCK plays an essential role in the activation process in the smooth muscle cell in that activation of this enzyme is both necessary and sufficient for the initiation of contraction.