bluuebunny/crossref_metadata_2025 huggingface dataset:
Newly synthesized protein secretion in rat lacrimal gland: post-second messenger synergism
Authors: Mauduit P. , Herman G. , Rossignol B.
Published: December 2017

The vasoactive intestinal peptide (VIP) induces a concentration-dependent secretion of newly synthesized (3H labeled) proteins from lacrimal gland fragments. Maximal secretory response is approximately 20% of total labeled proteins secreted for a 40-min stimulation and half-maximal secretory response is obtained at 3.8 +/- 0.2 nM VIP. The cholinergic (muscarinic) and VIPergic stimulations synergistically interact in eliciting newly synthesized protein secretion. Carbachol (0.3 microM) and the phorbol ester PMA (1 microM) potentiate the secretory response to VIP (10 nM), forskolin (3 microM), and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) (0.5 mM) both in the absence and presence of 2.5 mM extracellular calcium. The calcium ionophore A23187 (1 microM) potentiates the cAMP-dependent responses only in the presence of extracellular calcium. We propose that newly synthesized protein secretion from rat lacrimal glands is controlled by two systems interacting synergistically at a step distal to the production of intracellular second messengers. The potentiating effect of agonists acting through the calcium-dependent pathway on the cAMP-dependent secretory response may involve both calcium and diacylglycerol.
Histamine-induced calcium release and phorbol antagonism in cultured airway smooth muscle cells
Authors: Kotlikoff M. I. , Murray R. K. , Reynolds E. E.
Published: December 2017

Primary cultures of airway smooth muscle cells were exposed to histamine, and intracellular free calcium transients were measured by the calcium-sensitive dye fura-2. Stimulation with 100 microM histamine resulted in a rise in intracellular calcium from an unstimulated level of 178 +/- 25 to 497 +/- 154 nM Ca2+ (SE; n = 14) and a return to base-line free calcium concentration within 1 min of stimulation. Pretreatment of cells with the H1 receptor blocker pyrilamine (2.5 microM) abolished the response; however, the calcium transient was not altered by pretreatment with the H2 blocker cimetidine (50 microM), by chelation of external calcium, or by pretreatment with 2 mM Co2+ or 5 microM nifedipine. Activation of protein kinase c by 200 nM phorbol 12-myristate 13-acetate (PMA) resulted in no detectable rise in cytosolic calcium but completely blocked the release of internal calcium by histamine. We conclude that 1) histamine causes a transient rise of cytosolic calcium in airway smooth muscle, 2) the rise in cytosolic calcium is mediated by H1 receptor coupling that triggers release of internal calcium stores, and 3) activation of protein kinase c blocks the histamine-induced release of intracellular calcium.
Adhesion and differentiation of cultured rat granulosa cells: role of fibronectin
Authors: Morley P. , Armstrong D. T. , Gore-Langton R. E.
Published: December 2017

Because fibronectin is a major secretion product of rat granulosa cells in culture, we have investigated 1) the involvement of endogenous fibronectin in granulosa cell attachment, and 2) the consequences of inhibition of this attachment on follicle-stimulating hormone (FSH)-dependent differentiated responses. Attachment was significantly inhibited for up to 8 h in a concentration-dependent manner by antiserum to rat fibronectin, but not by nonimmune serum. Adsorption of antiserum on fibronectin or addition of exogenous fibronectin eliminated this inhibitory activity. Treatment with antiserum did not significantly alter the FSH-dependent production of adenosine 3',5'-cyclic monophosphate over 2 h or progestins over 48 h, while conversion of testosterone to 17 beta-estradiol over 48 h was suppressed by 60% in the presence of antiserum, regardless of antiserum adsorption on fibronectin. Results indicate that endogenous fibronectin is involved in substratum attachment of rat granulosa cells, but that attachment is not a requisite for FSH responsiveness.
Role of laminin in maintenance of type II pneumocyte morphology and function
Authors: Rannels S. R. , Yarnell J. A. , Fisher C. S. , Fabisiak J. P. , Rannels D. E.
Published: December 2017

Loss of differentiated function by type II pneumocytes plated on plastic surfaces was demonstrated by decreased lamellar body content, increased cellular protein, and rapid cellular flattening, changes that were retarded modestly by plating cells on laminin-coated surfaces. Laminin surfaces also inhibited [3H]thymidine (THM) incorporation into cellular DNA by 40% compared with plastic at 40 h, but did not alter an additional mitogenic effect of rat serum over fetal calf serum. In contrast, cells plated on the laminin-rich basement membrane-like gel formed from an extract of EHS mouse sarcoma, matrix gel (MG), maintained a high content of intracellular lipids in lamellar inclusions and retained a rounded morphology for at least 3 days. MG markedly inhibited THM incorporation and morphological changes when cells were cultured on this surface or when MG was formed over cells initially plated on plastic for various intervals. The importance of the laminin component of MG was demonstrated when these surfaces were pretreated with a highly specific antilaminin serum. Type II cells commenced flattening on the treated MG surface, and THM incorporation increased with the same time course as did control cells on plastic. The data suggest that short-term culture and study of differentiated type II pneumocytes may require a laminin-rich substratum. THM incorporation into type II cell DNA provides an important early and sensitive index of cell-basement membrane interaction and subsequent maintenance of function.
Contractile activation and recovery in skinned frog muscle stimulated by ionic substitution
Authors: Fill M. D. , Best P. M.
Published: December 2017

Contractile activation of skinned (sarcolemma removed) skeletal muscle fibers stimulated by ionic substitution has been studied. Stimulating solutions contained varying amounts of Cl- and K+ with the [K+] x [Cl-] product kept constant at 368 mM2. Activation is a graded function of the ionic content of the stimulating solution. Mechanical threshold is reached when the [Cl-] is changed from 4 to 6.5 mM. Maximal activation occurs at 20 mM Cl-. After stimulation, fibers do not respond to a second stimulus unless allowed to recover. Contractile height reaches 50% of control levels after 30 s in the standard recovery solution (4 mM Cl). Full recovery is reached after approximately 2-4 min. The degree of contractile recovery after a constant interval (30 s) depends on the ionic composition of the recovery solution. Contractile recovery decreases 50% when the [Cl-] of the recovery solution is raised from 4 to 5.5 mM. The activation and recovery phenomena described for skinned fibers stimulated by ionic substitution are similar to those described for intact cells stimulated by elevated [K+]. This finding is consistent with the hypothesis that depolarization of resealed transverse tubules triggers release of Ca2+ from the sarcoplasmic reticulum of skinned fibers.
Asymmetry of Na-K-Cl cotransport in human erythrocytes
Authors: Kracke G. R. , Anatra M. A. , Dunham P. B.
Published: December 2017

The Na-K-Cl cotransport system in human erythrocytes was studied by measuring net influxes and effluxes of Na and K. The influx of K was shown to be stimulated by Na and the influx of Na was stimulated by K, satisfying the fundamental criterion of cotransport. In addition, these mutually stimulating cation influxes had a stoichiometry of 1:1 and were entirely inhibited by furosemide; these results are also consistent with cotransport. Furthermore, the mutually stimulating influxes were entirely dependent on Cl, since they were abolished when nitrate was substituted for Cl. In contrast, cotransport, defined by mutual dependence of fluxes, was not detected in the outward direction over a range of cellular Na and K concentrations from 0 to 50 mmol/l cells. The cotransport pathway did, however, appear to mediate a Na-stimulated K efflux (but no K-stimulated Na efflux), and furosemide-inhibitable effluxes of both Na and K. Nitrate (but not sulfate) appeared to substitute for chloride in promoting Na-stimulated K efflux. Thus the Na-K-Cl cotransport system in human red cells is intrinsically asymmetric, and mediates coupled cation fluxes readily only in the inward direction.
Spatial heterogeneity of metabolism in skeletal muscle in vivo studied by 31P-NMR spectroscopy
Authors: Challiss R. A. , Blackledge M. J. , Radda G. K.
Published: December 2017

Phase-modulated rotating-frame imaging, a localization technique for phosphorus nuclear magnetic resonance spectroscopy, has been applied to obtain information on heterogeneity of phosphorus-containing metabolites in skeletal muscle of the rat in vivo. The distal muscles of the rat hindlimb have been studied at rest and during steady-state isometric twitch contraction; the use of a transmitter surface coil and an electrically isolated, orthogonal receiver Helmholtz coil ensure accurate spatial assignment (1 mm resolution). At rest, intracellular pH was higher and PCr/(PCr + Pi) was lower in deeper muscles compared with superficial muscle of the distal hindlimb. Upon steady-state stimulation, the relatively more alkaline pH of deep muscle was maintained, whereas greater changes in PCr/(PCr + Pi) and Pi/ATP occurred in the superficial muscle layer. This method allows rapid (75 min for each spectral image) acquisition of quantitative information on metabolic heterogeneity in vivo.
Release and reloading of intracellular Ca stores after cholinergic stimulation of the parietal cell
Authors: Negulescu P. A. , Machen T. E.
Published: December 2017

Microspectrofluorimetry was used to measure cytosolic free Ca, Cai, in single parietal cells of intact rabbit gastric glands loaded with the Ca-sensitive fluorescent dye, fura-2. Cells were repeatedly stimulated with the cholinergic agonist carbachol to gain insights into the membrane mechanisms involved in hormonally stimulated Ca metabolism. In either Ca-containing or Ca-free solutions, carbachol (100 microM) caused a rapid (within 30 s) elevation of Cai from a resting level of 100 nM to greater than 600 nM. After the spike, Cai decreased within 3 min to a lower level that was somewhat elevated (greater than 200 nM) over base line. This plateau was dependent on both carbachol and extracellular Ca (Cao) and could be blocked by the addition of atropine (1 microM) or lanthanum ion (La, 50 microM). The spike is due to the release of Ca from internal stores, whereas the plateau is due to Ca entry across the plasma membrane through agonist-controlled, La-inhibitable channels. After a carbachol stimulation of 3 min or longer, reloading of the internal store was absolutely dependent on Cao. Under these conditions, reloading occurred through a La-sensitive (but nifedipine- and verapamil-insensitive) pathway in the plasma membrane. No significant change in Cai was detectable during the reloading. In contrast to the longer treatments, if carbachol stimulation was terminated with atropine while Cai was still elevated, significant reloading occurred from the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)
Comparison of ion transport by cultured secretory and absorptive canine airway epithelia
Authors: Boucher R. C. , Larsen E. H.
Published: December 2017

The use of primary cell culture techniques to predict the function of native respiratory epithelia was tested in studies of dog airway epithelia. Epithelial cells from Cl- secretory (tracheal) and Na+ absorptive (bronchial) airway regions were isolated by enzymatic digestion, plated on collagen matrices, and maintained in serum-free, hormone-supplemented media. Transepithelial and intracellular studies showed that both the tracheal and bronchial culture preparations exhibited bioelectric parameters quantitatively similar to those of intact tissues. Similar to the native tissue, the tracheal preparation exhibited an equivalent short-circuit circuit (Ieq) that was sensitive to inhibitors of Cl- transport (bumetanide, diphenylamine carboxylic acid) but was insensitive to an inhibitor of Na+ transport, amiloride. In contrast, the bronchial preparation, like the native tissue, exhibited an Ieq sensitive to amiloride but insensitive to bumetanide. As compared with the trachea, the bronchial (absorptive) epithelium is characterized by 1) a large amiloride-sensitive cellular conductance and 2) a relatively depolarized basolateral membrane. We conclude that this primary cell culture technique provides epithelial preparations comparable to the native tissue and suitable for quantitative studies of regional differences in ion transport function.
Influx and efflux of 3-O-methyl-D-glucose by cultured human fibroblasts
Authors: Longo N. , Griffin L. D. , Elsas L. J.
Published: December 2017

We measured the initial rates of 3-O-methyl-D-glucose (OMG) fluxes by cultured human fibroblasts. D-Glucose (300 mM) and cytochalasin B (5 microM) inhibited approximately 80% of OMG (1 mM) influx. OMG rapidly entered human fibroblasts, and influx was linear up to 20 s. OMG influx and efflux were about equal. Cytochalasin B inhibited OMG (1 mM) influx and efflux within 20 s of exposure. Cytochalasin B half maximally inhibited OMG influx and efflux at 0.51 and 0.75 microM, respectively. In zero trans conditions, the kinetics of OMG influx and efflux were similar. However, when OMG was present on the trans side of the membrane, OMG influx but not OMG efflux was stimulated. Trans stimulation of OMG influx increased the maximal velocity of this transport process, without affecting its Km. These results suggest that 1) OMG influx and efflux occur through the same transporter, and 2) the glucose transporter of cultured human fibroblasts presents a functional asymmetry when substrate is present on the trans side of the membrane.
Regulation of conductive Cl- transport in human fibroblasts
Authors: Lin P. , Ahluwalia M. , Gruenstein E.
Published: December 2017

Under normal growth conditions, approximately 20% of the efflux of Cl- from human fibroblasts occurs via an electrically conductive pathway or Cl- channel. This basal Cl- conductance is insensitive to the Cl- -anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and to the Cl- -cation cotransport inhibitor bumetanide. Exposure of the cells to dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) for 15 min increases the electrically conductive component of Cl- efflux by approximately 20%. Unlike the basal Cl- conductance, the cAMP-activated channel is DIDS sensitive, indicating that cAMP activates a different Cl- pathway from the one responsible for the basal Cl- conductance. Elevation of intracellular Ca2+ by addition of the ionophore A23187 also stimulates Cl- efflux via a DIDS inhibitable, electrically conductive Cl- pathway. That the cAMP- and Ca2+-stimulated pathways are different is suggested by the observation that simultaneous exposure of cells to optimal levels of dibutyryl cAMP and A23187 results in an increased Cl- efflux equal to the sum of the two factors acting independently. Prostaglandin E1, a known activator of adenylate cyclase, also elevates the levels of intracellular free Ca2+ in these cells and concomitantly activates both the cAMP- and the Ca2+-stimulated Cl- channels. Although regulated, Cl- channels are known to function in the modulation of nerve and muscle excitability, their role in fibroblast function is not clear.
Neutrophil cathepsin G increases permeability of cultured type II pneumocytes
Authors: Rochat T. , Casale J. , Hunninghake G. W. , Peterson M. W.
Published: December 2017

Neutrophils contribute to inflammatory injury in some types of acute and chronic lung disease. Among the injurious agents released by activated neutrophils are cationic proteins such as cathepsin G that may contribute to edema formation. Using cultured rat type II alveolar epithelium, we examined the effect of human neutrophil cathepsin G on permeability to mannitol and electrical resistance across the cultured epithelium. Monolayers maintained in control media alone had constant permeability to mannitol (4.5 +/- 0.3 X 10(-3) cm/h at base line and 4.4 +/- 0.3 X 10(-3) cm/h after 2 h). When monolayers were exposed to cathepsin G at concentrations of 5, 10, and 20 micrograms/ml, there was a progressive increase in mannitol permeability (120 +/- 26% increase at 5 micrograms/ml, 174 +/- 40% increase at 10 micrograms/ml and 301 + 116% increase at 20 micrograms/ml, P less than 0.001). The effect of cathepsin G on mannitol permeability was blocked by the presence of the polyanion-sulfated dextran, and the polycation protamine also increased permeability to mannitol 57 +/- 5% (P less than 0.001), suggesting a role for charge in this effect. Cathepsin G exposure also resulted in a progressive decrease in electrical resistance (67 +/- 2% base line after 15 min, 55 +/- 1% after 30 min, and 43 +/- 2% after 60 min). Cathepsin G at these concentrations was not cytolytic to the cells as measured by lactate dedrogenase release. These functional alterations were accompanied by increase in intercellular gaps in the monolayers seen on scanning electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)
Improved propagation in myometrium associated with gap junctions during parturition
Authors: Miller S. M. , Garfield R. E. , Daniel E. E.
Published: December 2017

The hypothesis that gap junction (GJ) formation between myometrial cells at term improves electrical coupling was tested. We measured the spread of electrical excitation from six extracellular electrodes aligned on uterine strips in either the longitudinal (axial) or transverse (circumferential) direction. Spontaneous bursts propagated over the entire 15-mm recording distance in the axial direction at both preterm and parturition and showed some characteristics of a system of coupled relaxation oscillators. However, individual spikes within the bursts propagated further and with higher velocity at parturition than at preterm. In the circumferential direction, both bursts and individual spikes propagated further at parturition than before. Propagation in this axis at parturition appeared to require an intact circular muscle layer. Spikes evoked by electrical stimulation also propagated further and with higher velocity in both axes at parturition. Electron microscopy showed many GJs between uterine smooth muscle cells during parturition, but few and sometimes no GJs at preterm. Thus improved propagation was associated with increased GJ contact between myometrial cells, consistent with the hypothesis that gap junction formation at term improves electrical coupling.
Effect of amino acid deprivation on initiation of protein synthesis in rat hepatocytes
Authors: Everson W. V. , Flaim K. E. , Susco D. M. , Kimball S. R. , Jefferson L. S.
Published: December 2017

Conditions were defined for maintaining optimal protein synthetic activity in suspensions of freshly isolated rat hepatocytes. Under these conditions, isolated hepatocytes exhibited rates of protein synthesis and levels of polysomal aggregation equivalent to those observed in vivo and in perfused liver. Deprivation of total amino acids or single, essential amino acids resulted in a rapid decrease in the rate of protein synthesis, which was readily reversed by readdition of the deficient amino acid(s). The decrease was accompanied by a disaggregation of polysomes and an inhibition of 43S initiation complex formation, which was indicative of a limitation in the rate of initiation of protein synthesis. Extracts prepared from perfused liver deprived of amino acids were inhibitory to initiation of protein synthesis in reticulocyte lysate. The inhibition in reticulocyte lysate was accompanied by an increase in phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2), suggesting activation of an eIF-2 alpha kinase or inhibition of a phosphatase in amino acid-deprived hepatocytes. This suggestion was confirmed by prelabeling hepatocytes with 32Pi before amino acid deprivation. Incorporation of 32Pi into eIF-2 alpha was two- to threefold higher in lysine-deprived cells than in hepatocytes incubated in fully supplemented medium. Overall, the results indicated that an increase in eIF-2 alpha phosphorylation was responsible for the defect in initiation of protein synthesis caused by amino acid deprivation.
Inhibition of colonic Na+ transport by amiloride analogues
Authors: Bridges R. J. , Cragoe E. J. , Frizzell R. A. , Benos D. J.
Published: December 2017

The potency of several amiloride analogues to inhibit electrogenic Na+ transport in colon from dexamethasone-treated rats was compared. Short-circuit current (Isc) across the colonic mucosa and 22Na+ uptake into membrane vesicles derived from colonic enterocytes was determined in dexamethasone-treated rats. Kinetic analysis of inhibition of Isc and 22Na+ uptake revealed the presence of a high- and low-affinity amiloride pathway. One pathway had a high affinity [(Ki-Isc; Ki uptake] to benzamil (15.5 nM; 5.4 nM), phenamil (19.4 nM; 7.0 nM), 3',4'-dichlorobenzamil (29.0 nM; 25.2 nM), and amiloride (115 nM; 12.4 nM) but a much lower affinity to 5-(N-ethyl-N-isopropyl)amiloride (EIPA) (greater than 100 microM; greater than 9.9 microM) and 5-(N-propyl-N-butyl)-2'-4'-dichlorobenzamil (PBDCB) (greater than microM; greater than 32.8 microM). The high-affinity pathway accounted for 75-83% of the transport of Na+. The second pathway had nearly the same low affinity for each of the analogues (e.g., amiloride Ki-Isc 1 microM; Ki uptake 4 microM) and accounted for only 15-25% of the transport of Na+. The results demonstrate that the structure-inhibitory pattern of these amiloride analogues for the high-affinity pathway is the pattern observed in other electrogenic Na+-transporting epithelia and that this pharmacological profile is preserved in membrane vesicles derived from colonic enterocytes. In addition, the potency of EIPA and benzamil to inhibit electroneutral Na+ transport across the colon from normal rats (i.e., not treated with dexamethasone) was also investigated.(ABSTRACT TRUNCATED AT 250 WORDS)
Inositol lipid turnover and compartmentation in canine trachealis smooth muscle
Authors: Baron C. B. , Pring M. , Coburn R. F.
Published: December 2017

We established conditions for the study of metabolism and compartmentation of inositol phospholipids in canine trachealis muscle. Unstimulated muscle was incubated with myo-[3H]inositol for 30 min at 37 degrees C which resulted in labeling of the tissue free myo-inositol pool, whereas only a small amount of radioactivity was incorporated into inositol phospholipids or inositol phosphates. After addition of 5.5 microM carbachol, phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2), specific radioactivities increased exponentially, reaching apparent constant values in 180-240 min. Initial rates of increases in PI, PIP, and PIP2 specific radioactivities were 39, 32, and 66 times that measured in unstimulated muscle. Metabolic flux rates (nmol.100 nmol total lipid Pi-1.min-1) during development of force averaged 0.42 +/- 0.09 and during force maintenance averaged 0.14 +/- 0.01. Fractions of total PI, PIP, and PIP2 pools that were linked to muscarinic cholinergic activation were estimated to be 0.97, 0.85, and 0.65, respectively. Initial rates of increase in specific radioactivities and specific radioactivities during carbachol activation were similar in PI, PIP, and PIP2 fast active compartments, suggesting metabolic flux from PI to PIP to PIP2 was in near chemical equilibrium. Turnover times for PI, PIP, and PIP2 fast active compartments were estimated to be 21, 1.6, and 4.0 min, respectively.
Angiotensinogen gene expression in 3T3-L1 cells
Authors: Saye J. A. , Cassis L. A. , Sturgill T. W. , Lynch K. R. , Peach M. J.
Published: December 2017

It has previously been established that angiotensinogen mRNA is present in brown and white adipose tissue of the rat. To determine whether angiotensinogen gene expression is present in adipocytes as compared with other cell elements, we have examined angiotensinogen mRNA in 3T3-L1 cells. These cells undergo adipocyte differentiation when the culture reaches confluence. To accelerate the differentiation process, cells were treated with dexamethasone and isobutylmethylxanthine for 3 days. On the 7th day after drug treatment, RNA was extracted from cells and was examined for angiotensinogen mRNA using a full-length rat angiotensinogen cDNA. Angiotensinogen mRNA was readily detected in differentiated 3T3-L1 cells. To determine when the gene is expressed, a 7-day time course from day 0 (before drug treatment) to day 7 was examined for the presence of angiotensinogen mRNA. In addition, C2 cells, a clonal cell line that does not differentiate into adipocytes, were examined. Angiotensinogen mRNA was detected on days 2-7 after drug treatment in 3T3-L1 cells, with no detectable levels in untreated 3T3-C2 cells. When 3T3-C2 cells were subjected to the same drug regimen, angiotensinogen mRNA levels increased in the same time course as 3T3-L1 cells. However, the increase in angiotensinogen message was greater in differentiating 3T3-L1 cells than in the nondifferentiating 3T3-C2 cells. Thus angiotensinogen mRNA is present in both adipocytes and in fibroblast-like cells and appears to be regulated by steroids.
Effect of Li+ substitution for extracellular Na+ on GRF-induced GH secretion from rat pituitary cells
Authors: Kato M. , Suzuki M.
Published: December 2017

Several observations have been made on the mechanism of human growth hormone-releasing factor (hGRF)-induced growth hormone (GH) secretion. 1) hGRF activates adenylate cyclase and the production of adenosine 3',5'-cyclic monophosphate (cAMP). 2) Extracellular Ca2+ is indispensable in both hGRF- and excess K+-induced GH secretion. 3) Extracellular Na+ is also essential in hGRF-induced but not in excess K+-induced GH secretion. 4) Both Ca2+ and Na+ are required in dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-induced GH secretion. Thus hGRF may increase Na+ conductance via cAMP, which in turn depolarizes the somatotrophs and activates voltage-sensitive Ca2+ channels, thereby promoting Ca2+ entry and GH secretion. To further examine this possibility, replacement of Na+ with Li+ (an alkali metal ion permeant to Na+ channel) was studied in perifused dispersed rat anterior pituitary cells. Li+ substitution for extracellular Na+ did not suppress but augmented hGRF-and DBcAMP-induced GH secretion, whereas the rise in cellular cAMP content produced by hGRF was greatly attenuated in Na+-free, Li+ medium. This hGRF-induced GH secretion in Na+-free, Li+ medium was almost completely nullified by removing extracellular Ca2+. Thus Li+ was able to replace Na+, which further suggests the involvement of Na+ channels in hGRF-induced GH secretion. The possible mechanism of the augmented response in Na+-free, Li+ medium is discussed.
Ultrastructure of Amphiuma distal nephron: evidence for cellular heterogeneity
Authors: Biemesderfer D. , Stanton B. , Wade J. B. , Kashgarian M. , Giebisch G.
Published: December 2017

To obtain more information on the ultrastructure of the distal nephron of the salamander, Amphiuma, we conducted freeze-fracture electron microscopy and morphometric experiments. In the early distal tubule, the organization of the tight junction is variable, containing from one to two strands in the proximal region and four strands in distal regions. The length density of the tight junction in this segment varies from greater than 60 m/cm2 of apical membrane surface to less than 10/cm2 of apical membrane surface. These observations agree with a previous study demonstrating that the junction of this segment exhibits considerable axial heterogeneity. The junctions of the late distal tubule and collecting tubule are more complex. In the late distal tubule, the tight junction is composed of 6-8 strands, whereas the tight junction of the collecting tubule is composed of 8-12 strands. The collecting tubule contains principal cells and two types of intercalated cells: alpha and beta. The alpha-cells contain a high density of rod-shaped particles in the apical plasma membrane and in membranes of apical cytoplasmic vesicles. The beta-cells contain rod-shaped particles only in the basolateral membrane. In principal cells, we observed a novel organization of intramembranous particles within the apical plasma membrane. A model describing the relationship of the two types of intramembranous particles within the membrane is presented. This study demonstrates that the amphibian and mammalian distal nephron share many morphological characteristics including cellular and axial heterogeneity.
Intracellular Cl- activity and cellular Cl- pathways in cultured human airway epithelium
Authors: Willumsen N. J. , Davis C. W. , Boucher R. C.
Published: December 2017

Cl- transport was studied in human nasal epithelium, a predominantly Na+-absorbing proximal airway epithelium. Intracellular Cl- activity (aClc) and the electrical potentials across the apical (Va) and basolateral (Vb) membranes were measured with double-barreled, Cl- -selective microelectrodes to characterize the driving forces for Cl- flow across each membrane. Under control conditions (bilateral Krebs-bicarbonate Ringer), Va was -26.1 +/- 1.2 mV, Vb was -36.2 +/- 1.2 mV, and aCL(c) was 42.7 +/- 2.0 mM (n = 34), indicating that Cl- is near electrochemical equilibrium across the apical membrane but significantly above equilibrium across the basolateral membrane. Reduction of luminal [Cl-] from 120 to 3 mM reduced aClc from 42.7 +/- 4.0 to 27.0 +/- 3.5 mM, depolarized Va, and increased fractional apical membrane resistance (fRa) and transepithelial resistance (Rt). Serosal bumetanide reduced aClc by 10 mM without affecting electrical parameters. Reduction of serosal [Cl-] from 120 to 3 mM resulted in a rapid decrease in Vb, a decrease in fRa and an increase in Rt. Also, serosal [Cl-] reduction led to a slow decrease in aClc rom 45.5 +/- 2.5 to 31.1 +/- 4.2 mM) that could be inhibited by bumetanide. The data are consistent with the following conclusions: 1) Cl- is transported across the apical membrane through a conductive pathway; and 2) Cl- is translocated across the basolateral membrane by an electrically silent bumetanide-sensitive cotransport system and by a minor conductive path.
Spontaneous oscillations in cytoplasmic calcium concentration in vascular smooth muscle
Authors: Weissberg P. L. , Little P. J. , Bobik A.
Published: December 2017

Fluorescence measurement of fura-2 and quin2 signals from confluent primary cultures of serum-deprived rat aortic smooth muscle cells have revealed spontaneous oscillations in intracellular calcium concentration ([Ca2+]i). The transients consist of a rapid increase in [Ca2+]i that averages 60 nM and lasts approximately 30 s. They are caused by intracellular calcium release and an influx of extracellular calcium. Exposure of cells to the calcium-channel antagonists verapamil and diltiazem or incubation in nominally calcium-free medium reduced both the duration and amplitude of the transients; in contrast, the calcium-channel agonist (-)BAY K 8644 increased their duration. The transients were abolished by caffeine and 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate, agents that interfere with calcium release from the sarcoplasmic reticulum. These findings demonstrate that the sarcoplasmic reticulum is a primary source for the spontaneous oscillations in cytoplasmic calcium and is closely associated with the influx of extracellular calcium. Although the function of these transients is unclear, they may be involved in the spontaneous contractions observed in some vessels and in the regulation of vascular resistance.
Ion channels in normal human and cystic fibrosis sweat gland cells
Authors: Krouse M. E. , Hagiwara G. , Chen J. , Lewiston N. J. , Wine J. J.
Published: December 2017

Single-channel patch-clamp techniques were used to study the population of apical membrane ion channels in cultured sweat gland secretory cells from normal and cystic fibrosis subjects. Four types of anion channels and two types of cation channels were found. At physiological voltages, anion channels had chord conductances of 10, 18, 24, and greater than 200 pS. All had linear current-voltage relations except the 24 pS channel, which showed outward rectification. Cation channels had chord conductances of 5 and 18 pS, were linear, and were nonselective for a variety of cations. Channel types and proportions were equivalent in control, cystic fibrosis, and cystic fibrosis heterozygote cells. Beyond showing that the distribution of channel types remains unchanged in cystic fibrosis cells, the data provide a basis for comparison with cells cultured under different conditions, with other cell types, and with native tissues.
Energy depletion-repletion and calcium transients in single cardiomyocytes
Authors: Li Q. , Hohl C. M. , Altschuld R. A. , Stokes B. T.
Published: December 2017

Rapid fluctuations of intracellular free calcium in single adult rat heart myocytes were monitored by time-resolved fura-2 fluorescence microscopy. Under controlled aerobic conditions (35 degrees C, pH 7.3), electrical stimulation at 0.5 Hz produced a concave negative staircase of calcium transients. When the myocytes were challenged with 3 mM amobarbital (Amytal) and 2 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP) to deplete ATP, the cells became unresponsive to electrical stimulation within 1 min but responded to 10 mM caffeine with a large increase in free calcium. After the development of rigor contracture, the cellular response to caffeine was blunted. Free calcium increased at a variable rate in individual cells, reaching values of 300-1,000 nM after 15 min. When the inhibitors were removed, calcium declined toward control values, and spontaneous contractile activity and calcium transients were invariably observed. During subsequent electrical stimulation, there was a decrease in the half-widths of the calcium transients and an attenuation of the negative staircase. Parallel experiments with cells in suspension indicated that Amytal and CCCP caused ATP to fall from 27.6 +/- 1.6 to 0.7 +/- 0.2 nmol/mg protein, and the percent rod-shaped cells to fall from 70 to 0% in 5 min. Removal of the inhibitors after 15 min caused a rebound in ATP to 5.3 +/- 1.5 nmol/mg within 2 min and 6.6 +/- 1.3 nmol/mg after 10 min.
G proteins mediate suppression of Ca2+-activated K current by acetylcholine in smooth muscle cells
Authors: Cole W. C. , Sanders K. M.
Published: December 2017

The role of G proteins in cholinergic suppression of Ca2+-activated K current was studied in isolated canine colonic myocytes with the whole cell voltage-clamp technique. Acetylcholine (ACh; 10.0 microM) caused a 64 +/- 2.4% depression in the Ca2+-dependent component of the outward current evoked at potentials between -45 and -15 mV when GTP (0.1 microM) was included in the pipette-filling solution. This effect was reversed within 2-4 min on washout of ACh. Without GTP in the filling solution, ACh caused a 15 +/- 2.5% depression in outward current in 60% of the cells tested. When the non-hydrolyzable GTP analogues, GTP gamma S (0.1 mM) or 5'-guanylylimidodiphosphate (GppNHp; 0.1 mM) were used, the decrease in outward current was greater (85 +/- 4.2 and 78 +/- 6.5%, respectively), and it was not reversed on withdrawal of ACh. Dialysis of the cell interior with pipette solution containing pertussis toxin (1 ng/ml) for 30 min had no effect on the whole cell currents evoked on depolarization, but it abolished the effect of ACh on Ca2+-dependent outward current. These data suggest that coupling of muscarinic receptors to the inhibition of Ca2+-activated K channels is mediated by pertussis toxin-sensitive G proteins in colonic smooth muscle cells. G protein-mediated inhibition is distinctly different from the opening of muscarinic-regulated K channels in other cell types.
Regulation of angiotensin II binding sites in neuronal cultures by catecholamines
Authors: Myers L. M. , Sumners C.
Published: December 2017

Previous studies determined that direct activation of protein kinase C (PKC) with phorbol esters increases the number of angiotensin II (ANG II)-specific binding sites in neuronal cultures prepared from the hypothalamus and brain stem of 1-day-old rats. In the physiological situation, PKC is activated by diacylglycerol, which can be produced by multiple pathways, such as stimulation of inositol phospholipid (IP) hydrolysis, phosphatidylcholine hydrolysis, or by de novo synthesis. In the present study we have examined whether stimulation of IP hydrolysis, and presumably activation of PKC, can mimic the actions of phorbol esters on ANG II-specific binding. We have incubated neuronal cultures with agents that increase IP hydrolysis and have determined the effects on ANG II-specific binding. Incubation of neuronal cultures with norepinephrine (NE) at concentrations (greater than 5 microM) and for times (15-60 min) that cause large increases in IP hydrolysis caused increases in the number of ANG II-specific binding sites, mimicking the actions of phorbol esters. The return of IP hydrolysis to control values was associated with a return of ANG II-specific binding to control levels. The upregulatory action of NE was abolished by prazosin, demonstrating the involvement of alpha 1-adrenergic receptors. In addition, this effect was blunted by the PKC antagonist H 7, suggesting PKC involvement in the response. Thus we have determined a potential physiological mechanism by which stimulation of IP hydrolysis by NE, and possible subsequent activation of PKC, leads to upregulation of ANG II-specific binding sites in neuronal cultures.(ABSTRACT TRUNCATED AT 250 WORDS)
Pacemaker activity recorded in interstitial cells of Cajal of the gastrointestinal tract
Authors: Barajas-Lopez C. , Berezin I. , Daniel E. E. , Huizinga J. D.
Published: December 2017

The hypothesis was tested that interstitial cells of Cajal can generate slow wave activity. Intracellular recordings were performed only in the most superficial cells at the submucosal surface of the canine colonic circular muscle layer. An omnipresent and characteristic slow wave activity was present in all cells with a mean amplitude of 37 +/- 3 mV, a frequency of 4.6 +/- 0.1 counts/min (cpm), and a duration of 5.6 +/- 0.5 s; the average resting membrane potential was -70 +/- 1 mV. To determine the type of cell from which these recordings were obtained, methylene blue was injected by microiontophoresis. The strips were immediately fixed while the microelectrode was kept in the cell. A small segment of the tissue containing this cell was then processed for electron microscopy and serially sectioned. Electron-microscopic evidence showed that the microelectrode tip was positioned in an interstitial cell of Cajal (ICC): 1) several sections were observed with round cytoplasmic lesions of decreasing diameter followed by sections from the same cell without the lesion and 2) electron-dense material was observed in these sections due to the injected methylene blue. These cells were identified as part of the ICC network present at the muscle-submucosa interface of the circular muscle and were positively identified as ICC by the presence of cell processes. This is the first report giving direct evidence for the occurrence of electrical slow waves in ICC. It is essential support for the hypothesis that ICC are the actual pacemaker cells of the gut musculature.
Hyperthyroid adult rat cardiomyocytes. I. Nucleotide content, beta- and alpha-adrenoreceptors, and cAMP production
Authors: Hohl C. M. , Wetzel S. , Fertel R. H. , Wimsatt D. K. , Brierley G. P. , Altschuld R. A.
Published: December 2017

Ventricular myocytes isolated from the hypertrophied hearts of thyrotoxic adult rats have an increase in mean protein content per myocyte (6.3 +/- 0.2 vs. 4.4 +/- 0.2 ng) compared with euthyroid cells. Viability and adenine nucleotide profiles are similar in both populations, but NAD content of the hyperthyroid myocytes is depressed (4.9 +/- 0.2 vs. 5.5 +/- 0.2 nmol/mg for controls) and UTP is higher (1.2 +/- 0.09 vs. 0.9 +/- 0.04 nmol/mg). Binding of (-)-[125I]iodocyanopindolol to intact hyperthyroid myocytes is increased by 42% compared with controls, with no change in the dissociation constant (Kd). This elevation in beta-receptor number is correlated to enhanced beta-agonist-induced adenosine 3',5'-cyclic monophosphate (cAMP) production. The half-maximal effective concentration (EC50) for the euthyroid isoproterenol dose-response curve is 2.14 x 10(-7) M but is decreased to 2.51 x 10(-8) M in hyperthyroid cardiac cells. Basal adenylate cyclase activity is apparently not affected by thyroid hormones, since basal cAMP levels for both groups are identical (5 pmol/mg) and both rise roughly twofold in the presence of a phosphodiesterase inhibitor. Forskolin-induced cAMP production and cAMP-specific phosphodiesterase activity are similar as well. In contrast to beta-adrenergic response, there are no significant differences in alpha 1-antagonist [3H]prazosin binding parameters between hyperthyroid and euthyroid cardiomyocytes.
Regulation of angiotensin II binding sites in neuronal cultures by protein kinase C
Authors: Kalberg C. J. , Sumners C.
Published: December 2017

The radioligand binding of 125I-angiotensin II (ANG II) and calcium phospholipid-dependent protein kinase C (PKC) activity were measured to study the specificity and mechanisms of PKC involvement in the regulation of ANG II-specific binding site expression in neuronal cultures prepared from the brains of 1-day-old rats. Previously, PKC-activating phorbol esters were shown to increase the specific binding of 125I-ANG II in neuronal cultures. However, phorbol esters have many biological effects, which may nonspecifically act to increase 125I-ANG II-specific binding. In the present study, mezerein and teleocidin A, two activators of PKC that are chemically unrelated to phorbol esters, increased the specific binding of 125I-ANG II in a dose- and time-dependent manner with 50% effective dose (ED50) values of 32 and 79 nM, respectively. The PKC antagonist H-7 dose dependently inhibited phorbol 12-myristate 13-acetate (TPA)-stimulated increases in 125I-ANG II binding, whereas downregulation of PKC activity by chronic phorbol ester incubations of 24 and 48 h prevented TPA-stimulated increases in 125I-ANG II-specific binding. TPA (0.8 microM), mezerein (0.76 microM), and teleocidin A (0.5 microM) all caused a rapid translocation of PKC activity from the cytosol to the particulate fraction by 15 min. Temporally, the maximal stimulation of PKC translocation by mezerein, teleocidin A, and TPA preceded their ability to stimulate maximal 125I-ANG II-specific binding. Taken together, these results suggest that PKC is directly involved in the stimulation of ANG II-specific binding site expression and that translocation of PKC is a prerequisite for the increased expression of ANG II binding sites.
Atrial natriuretic factor receptors in cultured renomedullary interstitial cells
Authors: Fontoura B. M. , Nussenzveig D. R. , Pelton K. M. , Maack T.
Published: December 2017

To gain further insight on the cell types that may mediate the effects of atrial natriuretic factor (ANF) in the renal medulla, we determined the distribution and function of biological (B) and clearance (C) receptors of ANF in renomedullary interstitial cells (RMIC). Studies were performed in the 3rd-17th passages of RMIC obtained from a primary culture of the rat renal medulla. Electron microscopy of the cultured cells showed the typical morphological features of RMIC "in vivo," including prominent lipid droplets. RMIC have a very high density of high-affinity specific binding sites of ANF-(1-28) [23,000 sites/cell; dissociation constant (Kd) = 50 pM]. There was only minimal binding of C-ANF-(4-23) (less than 2,500 sites/cell), a specific ligand of C-ANF receptors. ANF-(1-28) markedly increased guanosine 3',5'-cyclic monophosphate (cGMP) from 1.3 +/- 0.3 to 106 +/- 22 pmol cGMP/10(6) cells [50% effective dosage (ED50) = 1.2 nM]. The effect of ANF-(1-28) on cGMP was nearly additive to that of sodium nitroprusside and was not potentiated or antagonized by C-ANF-(4-23). The density of guanylate cyclase-coupled B-ANF receptors and the ANF-induced increase in cGMP in RMIC are higher than those reported to date in other target cells. This suggests that RMIC may mediate some of the known effects of ANF in the renal medulla.
Effects of serosal hypertonicity on water permeability in toad urinary bladder
Authors: Kachadorian W. A. , Spring K. R. , Shinowara N. L. , Muller J. , Palaia T. A. , DiScala V. A.
Published: December 2017

We studied in toad urinary bladder the effects of serosal hypertonicity on tissue water permeability, granular cell luminal membrane water permeability, and granular cell luminal membrane particle aggregates and compared them with effects of antidiuretic hormone (ADH). In tissues challenged by a hypertonic (447 mosmol/kgH2O) serosal bath, luminal membrane aggregates were structurally similar to those caused by ADH. The tissue water permeability increase induced by serosal hypertonicity was much less than that caused by a maximally stimulating concentration of ADH on tissue in isotonic serosal baths with approximately the same transmural gradient. The difference is explained not only by a reduced incidence of luminal membrane aggregates but also by an increased resistance to water movement at a postluminal membrane site. Measurements of luminal membrane water permeability showed a close correlation with luminal membrane aggregate frequency, indicating that the calculated permeability of an individual aggregate was a constant. Thus the relation of luminal membrane aggregates to tissue osmotic permeability is modified by serosal hypertonicity. Morphological examination of these tissues suggested that luminal membrane aggregates may be less stable in the absence of hormone. This was evident by the proportionally greater number of structures interpreted as aggregates captured in the process of disassembly ("patches"). Membrane depressions containing intramembrane particles ("craters") were also observed. They corresponded in terms of frequency and size to coated pits as seen in thin sections.
Neuropeptide Y binding and inhibition of cAMP accumulation in human neuroepithelioma cells
Authors: Lobaugh L. A. , Blackshear P. J.
Published: December 2017

The specific binding of 125I-labeled neuropeptide Y (NPY) and the biological response to NPY receptor activation were measured in cultured human neuroepithelioma (SK-N-MC) cells. A single class of high-affinity binding sites [dissociation constant (KD) = 0.2 nM] was characterized both by equilibrium binding of 125I-NPY concentrations less than 1 nM and kinetically by the initial rates of 125I-NPY association and dissociation. Specific 125I-NPY binding was decreased in a concentration-dependent manner by inclusion of guanine nucleotides in the incubation medium. The existence of multiple affinity states or NPY receptor subtypes was suggested by 1) a Hill coefficient of less than 1.0 obtained when analyzing equilibrium binding with 125I-NPY concentrations greater than 1 nM, 2) biphasic dissociation of 125I-NPY, 3) an increase in the component of rapid dissociation and decrease in the component of slow dissociation when guanine nucleotides were present during dissociation of 125I-NPY, and 4) displacement of 125I-NPY by unlabeled peptide with a slope factor of 0.6. Exposure of intact cells to NPY caused a concentration-dependent pertussis toxin-sensitive inhibition of forskolin-stimulated cellular adenosine 3',5'-cyclic monophosphate (cAMP) accumulation [50% effective concentration (EC50) = 0.4 nM]. In contrast, NPY had no effect on cellular inositol phosphate content or protein kinase C activation. These results demonstrate that NPY binds specifically to a G protein-linked receptor that inhibits adenylate cyclase in SK-N-MC cells.