bluuebunny/crossref_metadata_2025 huggingface dataset:
Dependence of L-arginine accumulation on membrane potential in cultured human fibroblasts
Authors: Bussolati O. , Laris P. C. , Nucci F. A. , Dall'Asta V. , Longo N. , Guidotti G. G. , Gazzola G. C.
Published: December 2017

The cell-to-medium distribution ratios at steady state of L-arginine (RArg) and of the lipid-soluble cation tetraphenylphosphonium (RTPP) were studied as a function of the membrane potential (Em) in adult human fibroblasts. The relationship between RArg and Em was qualitatively similar to that of RTPP and Em. Quantitatively, RArg and RTPP differed in that 1) RTPP was much greater than RArg when Em was near zero, indicating a significant binding component in the uptake of TPP+ but not of L-arginine, and 2) after a correction for binding when Em is near zero, RTPP was still greater than RArg so that RT/F . ln RTPP exceeded RT/F . ln RArg by 10-25 mV. The pattern of the redistribution of accumulated TPP+ and arginine after an alteration of Em was identical. In null-point experiments, the external [K+] for which there were no changes in cellular TPP+ or L-arginine in the presence of high valinomycin (the null points) were very similar for the two probes. Em calculated from the null-point measurements (-70(-)-80 mV) was also very similar to RT/F . ln RArg and thus smaller than RT/F.ln RTPP. It was concluded that 1) there was an additional TPP+ binding as cellular [TPP] rose in response to more negative membrane potentials, 2) the transport system for L-arginine in these cells (system y+) operates as a facilitated diffusion system driven by the membrane potential, and 3) in some circumstances, L-arginine could be employed as a probe of Em.
Detection of an oxidizable fraction of cytochrome oxidase in intact rat brain
Authors: LaManna J. C. , Sick T. J. , Pikarsky S. M. , Rosenthal M.
Published: December 2017

Rapid-scanning reflectance spectrophotometry was used to evaluate the reduction-oxidation state of cytochrome oxidase in normoxic rat brain. Reflectance spectra were recorded from intact blood-perfused cerebral cortices after increased oxidative metabolic activity induced by direct cortical stimulation. Reflectance spectra taken from blood-free rat brain and from the rat ear vascular bed were used to identify cytochrome and hemoglobin components of spectra taken from intact brain. Cortical stimulation provoked shifts toward increased oxidation of cytochrome oxidase that were detectable in reflectance spectra. Observations of an oxidizable fraction of cytochrome oxidase demonstrate that a fraction of the cytochrome oxidase pool exists in a reduced state in normoxic brain. The presence of reduced cytochrome oxidase suggests that oxygen delivery to the brain is restricted by microvascular control mechanisms either as a function of brain metabolic physiology or as protection against oxygen toxicity.
Effects of bradykinin and angiotensin II on intracellular Ca2+ dynamics in endothelial cells
Authors: Morgan-Boyd R. , Stewart J. M. , Vavrek R. J. , Hassid A.
Published: December 2017

The purpose of this study was to investigate the effects of angiotensin II and bradykinin on intracellular Ca2+ dynamics in cultured endothelial cells. We used the "second-generation" fluorescent Ca2+ indicator fura-2, in conjunction with dual-wavelength fluorescence spectroscopy, in cultured adherent pulmonary arterial endothelial cells. Angiotensin II (up to 2 microM) had no consistent effect on intracellular Ca2+ levels. In contrast, bradykinin (10 nM) elicited a transient increase of cytosolic free Ca2+, from the resting value of 37 +/- 5 to 647 +/- 123 nM, followed by a decline to a steady-state value of 113 +/- 14 nM, which was significantly higher than the resting Ca2+ levels. Bradykinin's Ca-stimulatory effect was dose dependent, having a half-maximally effective concentration of approximately 1 nM and a maximally effective concentration of 10 nM. A B1-receptor agonist, Des-Arg9-bradykinin, was much less effective than bradykinin as modulator of cytosolic Ca2+. Moreover, a B1-receptor antagonist, Des-Arg9, [Leu8]-bradykinin, did not significantly affect the increase of cytosolic Ca2+ elicited by bradykinin. On the other hand, the bradykinin-elicited increase of Ca2+ was almost completely inhibited by a novel B2-receptor antagonist, D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin. Bradykinin increased cytosolic free Ca2+ levels in cells maintained in Ca2+-deficient extracellular medium, suggesting that the peptide mobilized Ca2+ from intracellular stores. However, the absence of extra-cellular Ca2+ resulted in an 80-90% attenuation of the transient Ca2+ response, whereas the posttransient steady-state response was completely absent. These findings are consistent with the notion that the bradykinin-elicited transient Ca2+ response is dependent on both extra- and intracellular Ca2+ and that the posttransient steady-state response is entirely dependent on extracellular Ca2+. Endothelial cells were responsive to a second dose of bradykinin after a 10-min interim period of incubation in the absence of the peptide hormone. The absence of extracellular Ca2+ during the interim period, or the pretreatment of cells with ionomycin in the absence of extracellular Ca2+, prevented the response of the cells to a second dose of bradykinin. Bradykinin- or ionomycin-desensitized cells could be resensitized by a brief incubation period in Ca2+-replete medium. The results are consistent with the notions that cellular resensitization requires the replenishment of intracellular Ca2+ and that bradykinin, but not angiotensin II, modulates intracellular Ca2+ dynamics in endothelial cells by interacting with a B2-type receptor.
Changes in Na+-H+ exchange regulation upon granulocytic differentiation of HL60 cells
Authors: Restrepo D. , Kozody D. J. , Knauf P. A.
Published: December 2017

The changes in activation of Na+-H+ exchange on granulocytic differentiation of human leukemic promyelocytic HL60 cells have been studied by measuring changes in intracellular pH with the fluorescent pH indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). It was found that the Na+-H+ exchanger is activated by stimulation of protein kinase C in the dimethyl sulfoxide (DMSO)-differentiated (neutrophil-like) HL60 cell, but not in the undifferentiated (promyelocyte-like) cell. In contrast, osmotic shrinkage of the cells triggers the exchanger in the undifferentiated cells, but not in the DMSO-differentiated cells. The data suggest that activation of Na+-H+ exchange by osmotic shock does not occur exclusively through stimulation of kinase C.
Volume-sensitive Cl-dependent K transport in human erythrocytes
Authors: O'Neill W. C.
Published: December 2017

Passive K fluxes, measured with 86Rb, were investigated in osmotically swollen human erythrocytes. K influx and efflux increased progressively with increased hypotonicity up to 167 mosmol/kg. No increase in K flux was seen when NO3 or methylSO4 were substituted for Cl. Substitution of choline or N-methylglucamine for external Na reduced the K flux in swollen cells by only 22%, compared with a 60% reduction in euvolumic cells. However, the magnitude of this Na-dependent component was slightly, but significantly, higher in swollen cells. The presence of Na-dependent K influx in swollen cells was confirmed by measurements of Na influx demonstrating a K-dependent Na influx of similar magnitude in isovolumic and swollen cells. The volume-sensitive K flux was inhibited by bumetanide, but significantly less so than was Cl-dependent flux in isovolumic cells (half-maximal inhibition at 1.0 X 10(-4) vs. 5.8 X 10(-7) M). Kinetic analysis revealed that Cl-dependent K influx had a lower affinity for external K in swollen cells than in euvolumic cells (Km was 29.8 vs. 6.1 mM). The increased K flux in swollen cells was found to be transient, decreasing substantially and reverting back to a predominantly Na-dependent and more bumetanide-sensitive form after 2 h. The results indicate that swelling of human erythrocytes activates a transient Cl-dependent K flux that differs significantly from that in isovolumic cells in that it is less Na dependent, less sensitive to bumetanide, and has a lower affinity for K. Na-K cotransport is either unaffected or slightly increased in swollen cells. The altered flux in swollen cells would thermodynamically favor a volume-regulatory KCl efflux.
Phorbol esters inhibit adenylate cyclase activity in cultured collecting tubular cells
Authors: Dixon B. S. , Breckon R. , Burke C. , Anderson R. J.
Published: December 2017

Activators of protein kinase C, a calcium- and phospholipid-dependent protein kinase, inhibit vasopressin-stimulated water flow in toad bladder. To determine the biochemical mechanisms of this inhibition, we examined the effects of activators of protein kinase C on arginine vasopressin (AVP)-stimulated adenylate cyclase activity in cultured rabbit cortical collecting tubular cells. The phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA), the diacylglycerol, 1-oleyl-2-acetyl glycerol (OAG), and the diacylglycerol kinase inhibitor, R59022, all rapidly activate protein kinase C in collecting tubular cells. Pretreatment with PMA produces a delayed inhibition (greater than or equal to 4 h) of AVP-stimulated adenylate cyclase activity. The 4-h time lag suggests that the effects of protein kinase C are mediated indirectly, possibly as a consequence of stimulating cell proliferation. PMA does not inhibit cholera toxin- or forskolin-stimulated adenylate cyclase activity, suggesting an effect on the vasopressin receptor or coupling of the receptor to the stimulatory guanine nucleotide regulatory protein. Neither prostaglandins nor the inhibitory guanine nucleotide regulatory protein appear to mediate this effect. In contrast, treatment with either OAG or R59022 produces a rapid inhibition of both AVP- and forskolin-stimulated adenylate cyclase activity suggesting a prominent distal site of action, presumably at the catalytic subunit of adenylate cyclase. The results demonstrate that different activators of protein kinase C inhibit AVP-stimulated adenylate cyclase activity by distinctly different mechanisms possibly by altering the substrate specificity or activating multiple forms of the kinase. These results have important implications when using different activators to study the biological effects of protein kinase C.
Specificity of gossypol uncoupling: a comparative study of liver and spermatogenic cells
Authors: Reyes J. , Benos D. J.
Published: December 2017

A comparative study of gossypol uncoupling of rat spermatogenic and liver cells shows that spermatogenic cells metabolizing pyruvate are two to three times more sensitive to gossypol uncoupling than either spermatogenic cells metabolizing glucose or liver cells metabolizing pyruvate or glucose. Direct measurements of in situ rat liver and spermatogenic cell mitochondrial respiration indicate that the pyruvate dependence of the gossypol uncoupling appears to be located in the spermatogenic cell mitochondria. A different type of mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone does not present the substrate-dependent uncoupling effect. This special interaction between spermatogenic cell pyruvate metabolism and gossypol uncoupling confers specificity to a bioenergetic model of gossypol action.
Rectum has abnormal ion transport but normal cAMP-binding proteins in cystic fibrosis
Authors: Goldstein J. L. , Nash N. T. , al-Bazzaz F. , Layden T. J. , Rao M. C.
Published: December 2017

The luminal membranes of involved tissues in cystic fibrosis (CF) are relatively impermeable to Cl and the regulation of Cl transport by adenosine 3',5'-cyclic monophosphate (cAMP)-mediated hormones is abnormal. We investigated the human rectum as a putative model for CF. We compared in vivo transrectal potential difference (PD) in CF and in normal subjects in response to sequential perfusions with various test solutions. The base-line PD was different in normal (-35.5 +/- 4.0 mV; lumen negative; mean +/- SE; n = 9) and CF subjects (-23.4 +/- 3.1 mV; n = 6; P less than 0.025) and was eliminated by amiloride (10(-4) M) perfusion in both groups by 3 min. However, in response to a Cl-free solution with amiloride, all six CF subjects exhibit less of a change in PD (PD, -2.2 +/- 1.2 mV vs. -11.7 +/- 1.5 mV in 6 controls; P less than 0.01). Furthermore, normal subjects (n = 7) respond to a 5 mM theophylline + amiloride perfusion with an increase in lumen-negative PD, whereas, CF subjects (n = 6) show no increase in lumen-negative PD. Rectal biopsy specimens from four normal and four CF subjects exhibit similar (2- to 3-fold) increases in theophylline-induced cAMP content and have similar cAMP-binding proteins (CF, n = 3; control, n = 3). We conclude that the rectum is an involved epithelium in CF in which the aberration may lie at a point beyond the binding of cAMP to its protein kinase.
Different physiological signatures of sweat gland secretory and duct cells in culture
Authors: Jones C. J. , Bell C. L. , Quinton P. M.
Published: December 2017

Human eccrine sweat gland cells grown in culture were found to lose their characteristic shape, becoming flattened and organized into multilayers. The resting membrane potentials of the cultured secretory cells (-35 +/- 2 mV, n = 36) were significantly higher than those measured for cultured duct cells (-22 +/- 1 mV, n = 58, P less than or equal to 0.01). When the cholinergic agonist methacholine (10(-5) or 10(-6) M) was administered, the cultured secretory cells could be distinguished unequivocally by their atropine-sensitive hyperpolarizing response (-20 +/- 2 mV, n = 43), whereas no cultured duct cells responded. When the sodium conductance antagonist amiloride (10(-5) or 10(-6) M) was administered, 44% of cultured secretory cells responded by hyperpolarization (-8 +/- 1 mV, n = 8), whereas 87% of cultured duct cells hyperpolarized (-15 +/- 1 mV, n = 46) and by a significantly greater margin (P less than or equal to 0.01). Substitution of chloride with gluconate in the bathing medium caused membrane potential depolarization in both cultured secretory and duct cell populations, which is consistent with the presence of a chloride conductance in the plasma membrane. The beta-adrenoceptor agonist isoproterenol induced a transient hyperpolarization of 5-10 mV in three out of six cultured secretory cells tested but had no effect on cultured duct cells.
Attachment and maintenance of adult rabbit cardiac myocytes in primary cell culture
Authors: Haddad J. , Decker M. L. , Hsieh L. C. , Lesch M. , Samarel A. M. , Decker R. S.
Published: December 2017

The present observations demonstrate that quiescent calcium-tolerant adult rabbit cardiac myocytes can be isolated by collagenase-hyaluronidase perfusion and maintained in primary culture for at least 2 wk. Culturing large numbers of myocytes requires that the freshly isolated cells be attached to a suitable substratum such as laminin, type IV collagen, or fetal bovine serum. The cultured myocytes retain their rod-like morphology for approximately 7 days before gradually spreading into a flattened conformation by 14 days. During the 1st wk of culture, contaminating interstitial cells rapidly proliferate, making cultures unsuitable for long-term study. Pure myocyte populations can be established and maintained if freshly isolated cells are cultured in the presence of cytosine arabinoside (Ara-C, 10 microM). This antimetabolite does not appear to adversely affect high-energy phosphates, since ATP and creatine phosphate (CrP) content of the myocytes is maintained at levels normally found in biopsy samples of rabbit myocardium. These results illustrate that an energetically stable population of adult cardiac myocytes can be maintained in primary culture in sufficient numbers to make them useful for future investigations of myocyte function.
Effect of hormones on growth and function of cultured canine tracheal epithelial cells
Authors: Van Scott M. R. , Lee N. P. , Yankaskas J. R. , Boucher R. C.
Published: December 2017

Insulin (INS), endothelial cell growth supplement (ECGS), transferrin (TF), cholera toxin (CT), hydrocortisone (HC), triiodothyronine (T3), and epidermal growth factor (EGF) were systematically examined for their effects on proliferation, ion transport activity, and morphological differentiation of canine tracheal epithelial (CTE) cells in culture. INS, ECGS, TF, and CT increase proliferation of CTE cells cultured on plastic Petri dishes but exhibit no acute effect on the bioelectric activity of freshly excised CTE. CT increases amiloride-insensitive transepithelial ion transport across both freshly excised canine trachea and CTE cultures. EGF has no effect on proliferation of CTE cells on plastic but induces hyperplasia of CTE cells on collagen matrices. EGF does not alter basal transepithelial ion transport across cultures but increases cellular responsiveness to beta-adrenergic stimulation. HC and T3 have no effect on proliferation or transepithelial bioelectric properties of CTE cells but improve morphological differentiation yielding cultures of complex epithelia containing cuboidal ciliated and nonciliated cells. These results demonstrate that growth and function of cultured CTE cells are affected by specific growth factors.
Sr2+ activates cross-bridge phosphorylation and latch state in smooth muscle
Authors: Hai C. M. , Murphy R. A.
Published: December 2017

Sr2+ induced myosin phosphorylation and stress development in both skinned and K+-depolarized, Ca2+-depleted, intact swine carotid media. Although higher concentrations of Sr2+ than Ca2+ were required for phosphorylation and stress development, the dependence of stress on phosphorylation was the same in intact tissues. K+ depolarization in the presence of 5 mM Sr2+ produced a transient in phosphorylation (53.2 +/- 5.1% at 1 min, falling to a steady-state value of 21.7 +/- 2.0%) in Ca2+-depleted tissues in which intracellular stores were refilled with Sr2+. Stress developed without a transient (T1/2 = 0.70 min) to a steady state of 89.7 +/- 2.0% of the stress induced by K+ depolarization in the presence of 1.6 mM Ca2+ (K-PSS). Cross-bridge cycling rate as measured by isotonic shortening velocity was proportional to myosin phosphorylation throughout the contraction. When intracellular stores were not refilled with Sr2+, phosphorylation rose to a sustained value of 28.8 +/- 2.7% and stress developed slowly (T1/2 = 2.9 min) to a steady state of 95.9 +/- 1.5% K-PSS-induced stress. Therefore, an initial phosphorylation transient induced by intracellular Sr2+ release only accelerated stress development without significant effects on steady-state stress or phosphorylation (as was true for Ca2+- induced responses). We concluded that Sr2+ substitutes for Ca2+ in phosphorylation and regulation of the latch state in the swine carotid media.
Double-barreled K+-selective microelectrodes based on dibenzo-18-crown-6
Authors: Mooney J. L. , Lyall V. , Acevedo M. , Armstrong W. M.
Published: December 2017

Liquid ion-exchanger microelectrodes based on Corning code 477317 K+ exchanger are known to be much more sensitive to quaternary ammonium ions than to K+. In the presence of such cations, the capability of measuring K+ activities with Corning microelectrodes may be seriously impaired. We have developed a neutral carrier K+-selective microelectrode based on the crown ether dibenzo-18-crown-6. The crown ether cocktail contained (wt/wt) 2.3% dibenzo-18-crown-6, 0.8% Na-tetraphenylborate, 30.1% 2-nitrophenylocylether, and 66.8% O-nitrotoluene. Double-barreled crown ether and Corning microelectrodes were calibrated in KCl solutions with or without choline, acetylcholine, tetramethylammonium, imidazole, Na+, tris(hydroxymethyl)aminomethane (Tris), and N-methyl-D-glucamine. Both kinds of microelectrodes showed similar K+ over Na+, Tris, and N-methyl-D-glucamine selectivities. However, crown ether microelectrodes had immensely greater selectivities of K+ over quaternary ammonium ions and imidazole than Corning microelectrodes. Selectivity factors, defined as log K(ij)K, of crown ether microelectrodes with respect to K+ for tetramethylammonium, choline, acetylcholine, and imidazole were -1.92 +/- 0.13, -2.97 +/- 0.03, -1.75 +/- 0.15, and -1.30 +/- 0.20, respectively. Intracellular K+ activities measured in the same Necturus gallbladders with both kinds of microelectrodes did not differ significantly.
Insulin-like growth factor 1 stimulates renal epithelial Na+ transport
Authors: Blazer-Yost B. L. , Cox M.
Published: December 2017

Insulin-like growth factor 1 (IGF1) stimulates vectorial Na+ transport in a classical model of the mammalian distal nephron, the toad urinary bladder. Net mucosal to serosal Na+ flux is stimulated by concentrations of IGF1 as low as 0.1 nM, and the response is maximal at 10 nM. Na+ transport increases within minutes of the serosal addition of IGF1, reaches a maximum in 2-3 h, and is sustained for at least 5 h. Neither the initial nor the sustained response to IGF1 is dependent on a new protein synthesis. The IGF1 response is inhibited by a concentration of amiloride (10(-5) M) that is known to specifically block the conductive apical Na+ channel but that has little effect on the Na+-H+ antiporter. Further studies will be necessary to establish a role for this growth factor in normal renal epithelial function, but it is possible that the natriferic and growth-stimulatory effects of IGF1 are intimately related.
Effect of leukotriene C4 on electromechanical activity and Ca2+ uptake in taenia coli
Authors: Zschauer A. , Matthews E. K. , Richardson B. P.
Published: December 2017

The actions of leukotriene C4 (LTC4) on electromechanical activity and 45Ca2+ uptake in guinea pig taenia coli were investigated. The contractile action of LTC4 was abolished by the removal of extracellular Ca2+. LTC4 concentrations eliciting a maximal contraction in normal medium produced no response in preparations depolarized with KCl. In single sucrose gap studies, LTC4 increased both the frequency of electrical spiking and tension. These effects were blocked by the dihydropyridine Ca2+-channel antagonist PY 108-068 and by the leukotriene receptor antagonist FPL 55712. In double sucrose gap experiments, LTC4 caused a small depolarization without measurable change in membrane conductivity; increased spontaneous electrical activity was again accompanied by an increase in tension. LTC4 caused a detectable increase in 45Ca2+ uptake only at extracellular Ca2+ concentrations less than 1 mM, and this was again inhibited by PY 108-068 or FPL 55712. It is concluded that the contractile effects of LTC4 in guinea pig taenia coli occur as a consequence of its ability to open voltage-sensitive Ca2+ channels, an effect that may occur independently of membrane depolarization.
Platelet activating factor induces dopamine release in PC-12 cell line
Authors: Bussolino F. , Tessari F. , Turrini F. , Braquet P. , Camussi G. , Prosdocimi M. , Bosia A.
Published: December 2017

The ability of platelet activating factor (PAF) to stimulate dopamine release and modify calcium homeostasis in PC-12 cell line was studied. PAF-induced dopamine release is related to its molecular form, with only the R-form steric configuration [(R)PAF], but not its S-form or its 2-lyso derivative, effective at being active. In addition, PAF acts at very low concentrations in a dose-dependent manner (0.1-30 nM). Preincubation with PAF receptor antagonists (CV-3988 and BN52021) as well as the specific desensitization of PC-12 cells to (R)PAF abolish the (R)PAF-induced dopamine release. Several lines of evidence suggest that dopamine release is dependent on a (R)PAF-induced calcium influx and efflux modulation. Dopamine release by PC-12 cells challenged with (R)PAF is associated with a rapid 45Ca influx and efflux and a rise in cytoplasmic calcium concentrations ([Ca2+]i) evaluated by using the calcium indicators fura-2 and quin2. At 30 nM (R)PAF, the absence of extracellular calcium inhibits the dopamine release but not the rise of [Ca2+]i from the internal stores, suggesting the importance of calcium influx in (R)PAF-induced dopamine release. PAF, which has been reported to be synthesized by stimulated neuronal cells (J. Biol. Chem. 261: 16502-16508, 1986) may thus have a physiological modulatory role on cells with neurosecretory properties.
Fast axonal transport of labeled proteins in motoneurons of exercise-trained rats
Authors: Jasmin B. J. , Lavoie P. A. , Gardiner P. F.
Published: December 2017

In this study, the fast orthograde axonal transport of radiolabeled proteins was measured to determine the effects of endurance-running training on transport velocity and amounts of transported proteins in rat sciatic motoneurons. Female rats were subjected to a progressive running-training program for 10-12 wk. Twenty-four hours after the last training session, rats underwent right L4-L5 dorsal root ganglionectomy. The next day, 20 microCi of [3H]leucine was injected bilaterally in the vicinity of the motoneuronal cell bodies supplying the sciatic nerve, to study axonal transport parameters. Results showed that peak and average transport velocities of labeled proteins were significantly (P less than 0.05) increased by 22 and 29%, respectively, in the deafferented nerves of the runners as compared with controls. Moreover, the amount of total transported protein-bound radioactivity was increased in both left (40%) and right (37%) sciatic nerves of the runners. An exhaustive exercise session reduced (P less than 0.05) peak displacement (8%) and total transported protein-bound radioactivity (36%) in the sciatic nerves of control rats, whereas no changes were noticed in trained animals. The data suggest that chronic endurance running induces significant adaptations in the fast axonal transport of labeled proteins.
Cyclic nucleotide-mediated effects of ANF and VIP on flounder intestinal ion transport
Authors: O'grady S. M.
Published: December 2017

The purpose of this study was to compare the effects of atriopeptin III (AP-III), vasoactive intestinal peptide (VIP), and ionomycin on Na and Cl influx and to correlate changes in transport with effects on intracellular adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 5'-cyclic monophosphate (cGMP) content of the tissue. In addition, the question of whether AP-III inhibits ion transport directly by acting on enterocyte receptors for AP-III or indirectly by stimulation of enteric nerves in the submucosa was also addressed. The results showed that AP-III, ionomycin, and bumetanide all inhibited the initial rate of Na and Cl influx, suggesting that they directly block Na-K-2Cl cotransport activity. VIP had no effect on unidirectional influx of Na and Cl. AP-III caused a fourfold increase in intracellular [cGMP] without any significant effect on [cAMP]. VIP stimulated [cAMP] by fourfold but had no effect on [cAMP]. Ionomycin had no effect on either [cAMP] or [cGMP]. Inhibition of transport by AP-III could not be blocked by tetrodotoxin (TTX), indicating that enteric nerves in the submucosa are not directly involved in mediating the effects of AP-III on epithelial ion transport. The observation that two classes of neuronal depolarizing agents (veratrine and scorpion venom) cause TTX-sensitive inhibition of basal ion transport establishes that NaCl absorption in flounder intestine is subject to regulation by enteric nerves located in the submucosa.
Differing effects of apical and basolateral adenosine on colonic epithelial cell line T84
Authors: Barrett K. E. , Huott P. A. , Shah S. S. , Dharmsathaphorn K. , Wasserman S. I.
Published: December 2017

Adenosine and its agonists, 5'-(N-ethylcarboxamido)adenosine and N6-(D-2-phenylisopropyl)-adenosine, induced a sustained increase in chloride secretion when added to either the apical or basolateral aspect of monolayers of the human colonic epithelial cell line T84. Secretion was induced with identical kinetics by addition to both sides, but apical addition was less potent. The rank order of potency of the agonists on either side was consistent with the presence of an adenosine A2-receptor, but the apical and basolateral receptors differed in both their ability to stimulate increases in adenosine 3',5'-cyclic monophosphate, and their susceptibility to down-modulation by chronic exposure to 5'-(N-ethylcarboxamido) adenosine in culture.
Physiological regulation at the level of mRNA: analysis of steady-state levels of specific mRNAs by DNA-excess solution hybridization
Authors: Hadcock J. R. , Williams D. L. , Malbon C. C.
Published: December 2017

Recent appreciation for how systems may be regulated at the protein and mRNA levels has dictated that physiological questions be addressed at both. The application of novel and sensitive biochemical and molecular biological techniques may be necessary to answer questions fundamental to our understanding of physiological regulation. For measurement of low-abundance proteins, such as receptors for hormones and growth factors, and for measurement of their corresponding mRNAs, the need for highly sensitive assays is critical. The present work focuses both on recent studies of the regulation of steady-state levels of mRNAs and on how mRNAs are quantified by DNA-excess solution hybridization assays using highly specific single-stranded probes. Examples of physiological regulation are provided in which analysis of mRNA levels has been a key facet to our understanding of the mode of regulation. The construction and characterization of probes for solution hybridization assays are described using the hamster beta 2-adrenergic receptor as a model. Studies on the regulation of beta-adrenergic receptors by agonists and by permissive hormones as well as investigations on the regulation of apolipoproteins by estrogen and by cholesterol in vivo are used to illustrate the many advantages of analyzing the steady-state levels of specific mRNAs by DNA-excess solution hybridization.
Transfer of phosphatidic acid from liposomes to cells is collision dependent
Authors: Longmuir K. J. , Malinick L. A.
Published: December 2017

The kinetics of lipid transfer from unilamellar liposomes to cells in monolayer culture were determined for a fluorescent phosphatidic acid, 1-palmitoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] -sn-glycerol 3-phosphate (C6-NBD-PA), and for the analogous phosphatidic acid without the fluorescent NBD group, 1-palmitoyl-2-caproyl-sn-[U-14C] glycerol 3-phosphate (C6-[14C]PA). Initial rates of liposome-to-cell transfer were measured at 2 degrees C under conditions in which the concentration of diffusible monomer in the aqueous medium was constant during the course of an experiment and was independent of total liposome concentration. Rates were similar for C6-NBD-PA and C6-[14C]PA, indicating that the NBD group does not significantly alter the transfer kinetics. It was found that liposome-to-cell transfer was dependent on 1) the mole fraction of diffusible lipid in the liposomes, 2) the liposome concentration, and 3) the cell density. The dependence of rate on the liposome concentration (observed under conditions in which aqueous monomer concentration remained constant) cannot be explained by a liposome-to-cell transfer mechanism involving the free diffusion of monomers through the aqueous medium. Instead, the data are consistent with a collision-dependent mechanism of monomer transfer that occurs when liposome and cell membranes come into contact but do not fuse.
A fluorescent indicator for measuring cytosolic free magnesium
Authors: Raju B. , Murphy E. , Levy L. A. , Hall R. D. , London R. E.
Published: December 2017

The previously developed chelator O-aminophenol-N,N,O-triacetic acid (APTRA) (L. A. Levy, E. Murphy, B. Raju, and R. E. London. Biochemistry 27: 4041-4048, 1988) has been modified to yield a fluorescent analogue which can be utilized as an intracellular probe for ionized Mg2+. The fluorescent analogue, FURAPTRA, with a magnesium dissociation constant of 1.5 mM, is structurally analogous to the calcium chelator fura-2 and exhibits a similar excitation shift on magnesium complexation. Hence, data on the intracellular Mg2+ concentration can be obtained using an analogous ratio method. The acetoxymethyl form of the chelator is readily loaded into cells and has been used to determine a cytosolic free Mg2+ concentration of 0.59 mM for isolated rat hepatocytes. As a consequence of the relatively high levels of cytosolic Mg2+, the problem of ion buffering is much less severe than for the analogous calcium indicators.
A patch-clamp study: secretagogue-induced currents in rat peritoneal mast cells
Authors: Kuno M. , Okada T. , Shibata T.
Published: December 2017

Ca2+ entry through plasma membrane has been considered to play a significant role in elevating cytosolic free Ca2+ concentrations during stimulus-secretion coupling in mast cells, but electrophysiological evidence of the Ca2+ channels is lacking. We examined the properties of secretagogue (compound 48/80)-induced currents in rat peritoneal mast cells, using the patch-clamp technique. In the whole cell recordings, the addition of compound 48/80 induced transient currents that were suppressed by Cd or reduced by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). In Ringer solution containing 2 mM Ca2+, the current-voltage relation was fairly linear from -100 to 50 mV and the reversal potential was 14 +/- 10.1 mV (n = 9). When the external Ca2+ was approximately 1 microM, the compound 48/80-induced currents were marginal, but readmission of Ca2+ or Ba2+ to the bath solution led to an appearance of the currents. In the cell-attached patches, the stimulation enhanced the activity of inward current mediated by Ba2+. The unitary inward Ba2+ current was characterized by the unitary conductance of 10.5 +/- 2.0 pS (n = 10) with isotonic BaCl2 pipette solution, the extrapolated reversal potential of 60.7 +/- 16.0 mV (n = 10) positive to the resting membrane potentials. The percent open time of the inward Ba2+ current channel was not appreciably changed by voltage. In some whole cell recordings, an increase in openings of the cation-selective channel (20-45 pS) was identified in the stimulated cells. When the external Na+ was completely replaced by choline+, the compound 48/80-induced currents had a fairly linear current-voltage relation.(ABSTRACT TRUNCATED AT 250 WORDS)
Autoxidation, dehydration, and adhesivity may be related abnormalities of sickle erythrocytes
Authors: Hebbel R. P. , Ney P. A. , Foker W.
Published: December 2017

The sickle gene has remarkably pleiotropic effects. In an attempt to understand the complex pathobiology of sickle disease, we have searched for potential relationships between various cellular abnormalities. In the present studies, we treated normal erythrocytes (RBCs) with phenazine methosulfate to stimulate intracellular generation of superoxide and thereby stimulate the excessive autoxidation characteristic of sickle RBCs. In response, RBCs show a dose-dependent increase in adherence to endothelium, easily achieving the degree of abnormal adherence typical of unmanipulated sickle RBCs. This is mediated by adverse effects of oxidant on cellular hydration, as evidenced by amelioration of abnormal adhesivity if the drug's dehydrating effect is inhibited. Consistent with potential relevance of this to sickle pathobiology, the abnormal endothelial adherence of sickle RBCs can be worsened or improved by dehydrating or hydrating these cells, respectively. These data suggest an intimate relationship between three abnormalities of sickle RBCs previously thought to be unrelated (autoxidation, dehydration, and adhesivity). Although multiple mechanisms may contribute to abnormal cell-cell interactions in sickle disease, the potentiation of sickle RBC adhesivity by cellular dehydration may help explain why clinical dehydration has been identified as a precipitant of vasocclusive crisis. Insofar as abnormal adhesivity participates in the sickle vasocclusive process, these data provide further rationale for attempting to manipulate RBC hydration as a therapeutic maneuver in this disease.
Skeletal muscle growth is stimulated by intermittent stretch-relaxation in tissue culture
Authors: Vandenburgh H. H. , Hatfaludy S. , Karlisch P. , Shansky J.
Published: December 2017

Avian pectoralis muscle cells differentiated in vitro are mechanically stimulated by repetitive stretch-relaxation of the cell's substratum using a computerized mechanical cell stimulator device. Initiation of mechanical stimulation increases the efflux of creatine kinase from the cells during the first 8-10 h of activity, but the efflux rate returns to control levels after this time period. Decreased total cell protein content accompanies the temporary elevation of creatine kinase efflux. With continued mechanical stimulation for 48-72 h, total cell protein loss recovers and significantly increases in medium supplemented with serum and embryo extract. Myotube diameters increase and cell hyperplasia occurs in the stimulated cultures. In basal medium without supplements, mechanical activity prevents myotube atrophy but does not lead to cell growth. Mechanically induced growth is accompanied by significant increases in protein synthesis rates. The increases in protein synthesis and accumulation induced by mechanical stimulation are not inhibited by tetrodotoxin but are significantly reduced in basal medium without supplements. Mechanically stimulated cell growth is thus dependent on medium growth factors but independent of electrical activity.
Activation of a Cl-dependent K flux by cAMP in pig red cells
Authors: Kim H. D. , Sergeant S. , Forte L. R. , Sohn D. H. , Im J. H.
Published: December 2017

Activation of a Cl-dependent K flux by adenosine 3',5'-cyclic monophosphate (cAMP) was characterized in pig red cells, a cell type that lacks both the Ca-activated K channel and the Na-K-Cl cotransport pathway. As in other red cells, both Cl-dependent K efflux and K influx are stimulated on cell swelling. Although pig red cells fail to respond to beta-adrenergic stimuli, it is possible to raise the intracellular cAMP content by preincubating cells in the presence of 1 mM cAMP. The Cl-dependent K flux was compared in cells having a basal cAMP content of approximately 0.29 nmol/g hemoglobin vs. cAMP-loaded cells having approximately 8.4 nmol cAMP/g hemoglobin. Loading with cAMP stimulated both Cl-dependent K efflux and influx of hypotonically swollen cells. In maximally swollen cells whose volume was increased by approximately 17%, the Cl-dependent Rb influx occurs with a maximum velocity (Vmax) of 17.9 +/- 3.2 mumol.g hemoglobin (Hb)-1.h-1 and Km for Rb of 22.9 +/- 4.1 mM. In cAMP-loaded cells, both Vmax and Km were increased to 59.8 +/- 8.5 mumol.g Hb-1.h-1 and 63.1 +/- 8.8 mM, respectively. The Cl-dependent Rb influx is much larger in young cells than in old cells. However, both cell types respond to cAMP activation. Whereas cAMP and its analogues, 8-bromoadenosine 3',5'-cyclic monophosphate and dibutyryl adenosine 3',5'-cyclic monophosphate are stimulatory, AMP and guanosine 3',5'-cyclic monophosphate (cGMP) are not. These findings suggest that, like other ion transport systems, the Cl-dependent K flux of pig red cells is endowed with the capacity to respond to cAMP.
Interleukin 1 release by human alveolar macrophages and blood monocytes
Authors: Iwamoto G. K. , Monick M. M. , Burmeister L. F. , Hunninghake G. W.
Published: December 2017

These studies utilized a sensitive and specific radioimmunoassay for interleukin 1 beta (IL-1 beta) to compare release of IL-1 by human alveolar macrophages and blood monocytes. The studies demonstrate that alveolar macrophages release amounts of antigenic IL-1 beta that are similar to that of blood monocytes. The amounts of IL-1 released were similar for both cell types at early (4 h) and late (24 h) time points and with differing amounts of stimuli [endotoxin; lipopolysaccharide (LPS)]. In addition, alveolar macrophages actually produced more total IL-1 (intracellular IL-1 plus released IL-1) than did blood monocytes. Alveolar macrophages that were stimulated with LPS released significantly more prostaglandin E2 (PGE2, an inhibitor of IL-1) than did blood monocytes. These studies demonstrate that human alveolar macrophages are not defective in their capacity to release IL-1.
A volume-sensitive chloride conductance in human colonic cell line T84
Authors: Worrell R. T. , Butt A. G. , Cliff W. H. , Frizzell R. A.
Published: December 2017

The chloride-secreting colonic cell line, T84, was studied under whole cell patch clamp with Cl as the permeant ion in pipette and bath solutions. Transmembrane current was initially small (approximately 50 pA at +100 mV) but increased steadily to average values of 1-3 nA within 5-10 min. The development of this current was associated with visible cell swelling, either without a shape change or with membrane blebbing. Basal, preswelling current levels were restored by the addition of 50-75 mM sucrose to the bath or when pipette osmolality was reduced by an equivalent amount. These findings suggest that an isosmotic pipette filling solution behaves as if it is hypertonic by approximately 60 mosmol/kgH2O to the bath. Currents traversing the swelling-induced conductance were outwardly rectified and showed activation at hyperpolarizing voltages and inactivation at depolarizing voltages. They were Cl selective because the reversal potential for current flow approached the Cl equilibrium potential when bath [Cl] was varied. Under nonswelling conditions (bath solution, 300 mosmol/kgH2O; pipette solution, 240 mosmol/kgH2O), single-channel steps (approximately 9 pA at +100 mV) could be resolved. The single-channel characteristics were similar to the macroscopic currents recorded from swollen cells, showing inactivation at positive voltages and an outwardly rectified current-voltage relation. Summation of these single-channel events yielded currents that were similar to those from swollen cells, implying that activation of multiple channels with these properties is the basis of the swelling-induced Cl conductance. This volume-sensitive Cl conductance would contribute to a regulatory volume decrease when T84 cells swell. Its relation to the secretory Cl conductance in these cells is unknown.
Na+-H+ exchange in isolated myocytes from adult rat heart
Authors: Wallert M. A. , Frohlich O.
Published: December 2017

The intracellular pH (pHi) in ventricular myocytes isolated from adult rat heart was measured in suspension using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Steady-state pHi in bicarbonate-free media [extracellular pH (pHo) = 7.4] was 7.16 +/- 0.11 at 37 degrees C. With the use of the ammonium chloride prepulse technique, pHi was acidified, and the rate of return to resting pHi was determined. Initial rate analysis of the recovery was used to characterize the kinetics of proton net efflux via the Na+-H+ exchanger. At pHo = 7.4, proton extrusion was stimulated by extracellular sodium with a K1/2 = 58 +/- 16 mM and a maximal rate of recovery of 55 +/- 7 mmol/(1 cell H2O.min). Amiloride, which inhibited greater than 90% of the observed proton movements, was a competitive inhibitor with respect to Nao, with an inhibition constant of 3.5 microM. Proton net efflux was also inhibited by extracellular protons, with a maximal flux occurring above pHo = 8 and no net efflux occurring below pHo = 6.0. Efflux was stimulated by intracellular protons over a much narrower range (pHi = 6.6-7.1). This steep dependence indicates the involvement of at least one additional proton binding site besides the intracellular transport site, in accordance with the kinetic behavior observed in other cell systems.
Muscarinic suppression of Ca2+-dependent K current in colonic smooth muscle
Authors: Cole W. C. , Carl A. , Sanders K. M.
Published: December 2017

Acetylcholine (ACh) increases the amplitude and duration of colonic electrical slow waves. This suggests that ACh either increases an inward current or suppresses an outward current. The latter hypothesis was tested in whole cell voltage-clamp experiments performed on freshly dispersed smooth muscle cells from canine proximal colon. Addition of ACh (10(-5) M) to solutions bathing cells reduced time-dependent outward currents elicited by depolarizing test pulses in the range of -45 to +30 mV. Analysis of tail currents showed that ACh caused a 10- to 15-mV positive shift in voltage-dependent activation. When cells were pretreated with 10(-6) M nifedipine to abolish the Ca2+-dependent component of the outward current, the reduction of outward current by ACh was blocked. Single-channel experiments were performed to determine whether ACh had a direct effect on Ca2+-activated K channels. ACh, 10(-5) M, added to bath and pipette solutions caused a positive shift in voltage-dependent activation in on-cell experiments. This effect of ACh on Ca2+-activated K channels provides a mechanism for the effects of muscarinic, excitatory stimulation of circular muscle of the colon.
Localization of spermidine uptake in rabbit lung slices
Authors: Saunders N. A. , McGeachie J. K. , Ilett K. F. , Minchin R. F.
Published: December 2017

The lungs have a high polyamine transport capability, and the type II pneumocyte has recently been identified as a major site of putrescine uptake and localization (N. A. Saunders, P. J. Rigby, K. F. Ilett, and R. F. Minchin. Lab. Invest. 59: 380-386, 1988). However, recent evidence suggests that multiple polyamine transport systems exist. In the present study, localization of spermidine uptake in rabbit lung was investigated. Although [14C]spermidine was rapidly accumulated by lung slices, it was not significantly metabolized, and no efflux of the accumulated polyamine was apparent. Autoradiographs prepared after [3H]spermidine transport revealed a localization of uptake activity to cells identified by electron microscopy as type II pneumocytes. Spermidine uptake occurred in all type II cells examined and thus appeared to be a characteristic function of this cell type. In contrast, spermidine uptake was virtually absent in the major airways and blood vessels, whereas moderate uptake was associated with pulmonary alveolar macrophages and alveolar tissue. Subsequent purification and culture of type II pneumocytes showed these cells to have significant polyamine uptake activity. In addition, spermidine uptake activity was positively correlated with the proportion of type II cells present at the various stages of their purification. In other studies, cultured pulmonary alveolar macrophages possessed similar uptake activity to that of cultured type II cells. Combined, these data suggest that both type II cells and pulmonary alveolar macrophages may represent major sites of spermidine uptake in vivo. We also suggest that the transport of polyamines by type II cells may reflect a critical role for polyamines in a characteristic function of this cell type.
Activation kinetics of the acetylcholine-gated potassium current in isolated atrial cells
Authors: Inomata N. , Ishihara T. , Akaike N.
Published: December 2017

Processes involved in activation of the acetylcholine (ACh) receptor-operated K+ current (IK) were examined in atrial cells isolated from guinea pig using a "concentration-clamp" technique. This approach allows for the intracellular perfusion and the rapid change of external solution (time constant 4 ms) with cell-attached condition under a single-electrode voltage-clamp condition. The ACh-induced IK increased in a sigmoidal fashion with increasing concentrations of ACh. The Ka value estimated from the concentration-response curve was 3 X 10(-7) M, and the Hill coefficient was 1.0. The activation phase was accelerated not only by increasing the concentration of ACh but also by elevating the temperature. Before activation of the current, there was a brief latent period after the application of ACh. The latent period was shortened considerably with the increase in ACh concentration and in temperature, i.e., 267 +/- 20 ms at 18 degrees C, 98 +/- 11 ms at 26 degrees C, and 44 +/- 6 ms at 37 degrees C for 10(-6) M ACh. These results suggest that the latent period of ACh response seems to be the time lag needed for the activation of K+ channels using remote sensor.