scholarly journals Effects of cholecalciferol on the translocation of calcium by non-everted ileum in vitro

1975 ◽  
Vol 152 (2) ◽  
pp. 181-190 ◽  
Author(s):  
Eric S. Holdsworth ◽  
John E. Jordan ◽  
Ellen Keenan
Keyword(s):  
Sodium Dodecyl ◽  
Electrical Potential ◽  
Basement Membranes ◽  
Metabolic Energy ◽  
Temperature Sensitive ◽  
Mucosal Cell ◽  
Electrical Potential Difference ◽  
Mucosal Cells

An apparatus is described that allows perfusion of a non-everted segment of intestine in vitro and the study of the accumulation of substances within the mucosal cells. The translocation of Ca2+ by rachitic-chick ileum and the effect of pretreatment with cholecalciferol was investigated, with the following conclusions. (1) Entry of Ca2+ across the microvilli into mucosal cells is by diffusion; it does not require metabolic energy or the presence of any other inorganic ions. (2) Pretreatment of the chick with cholecalciferol causes increased permeability of the microvillus to Ca2+ in both directions (lumen to cell, cell to lumen). The increased transport brought about by cholecalciferol in vivo can be partially mimicked by sodium dodecyl sulphate added in vitro. (3) The sign and the magnitude of the electrical potential difference prevailing across the ileum does not influence Ca2+ transport. (4) Exit of Ca2+ from the mucosal cell is temperature-sensitive, requires metabolic energy and Na+. (5) Pretreatment with cholecalciferol caused increased movement of Ca2+ out of the cell across the basement membranes. This effect of cholecalciferol given in vivo could be markedly increased by the presence of dicyclohexylcarbodi-imide in the perfusion fluid. These observations suggested that cholecalciferol increased Ca2+ entry (and exit) at the mucosal surface and also caused Ca2+ to be more available to the pump at the serosal surface.

An Investigation of the Role of Possible Neural Mechanisms in Cholera Toxin-Induced Secretion in Rabbit Ileal Mucosa in Vitro

1989 ◽  
Vol 77 (2) ◽  
pp. 161-166 ◽  
Author(s):  
K. J. Moriarty ◽  
N. B. Higgs ◽  
M. Woodford ◽  
L. A. Turnberg
Keyword(s):  
Cholera Toxin ◽  
Fluid Transport ◽  
Short Circuit ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Electrical Potential Difference ◽  
Rabbit Ileum ◽  
Ileal Mucosa

1. Cholera toxin stimulates intestinal secretion in vitro by activation of mucosal adenylate cyclase. However, it has been proposed that cholera toxin promotes secretion in vivo mainly through an indirect mechanism involving enteric neural reflexes. 2. We examined this hypothesis further by studying the influence of neuronal blockade on cholera toxin-induced changes in fluid transport across rabbit ileum in vitro. Mucosa, stripped of muscle layers, was mounted in flux chambers and luminal application of crude cholera toxin (2 μg/ml) caused a delayed but sustained rise in the short-circuit current, electrical potential difference and Cl− secretion. Pretreatment with the nerve-blocking drug, tetrodotoxin (5 × 10−6 mol/l serosal side), failed to influence the secretory response to cholera toxin, and addition of tetrodotoxin at the peak response to cholera toxin also had no effect. 3. That tetrodotoxin could block neurally mediated secretagogues was confirmed by the demonstration that the electrical responses to neurotensin (10−7 mol/l and 10−8 mol/l) were blocked by tetrodotoxin (5 × 10−6 mol/l). Furthermore, the response to cholera toxin of segments of ileum, which included the myenteric, submucosal and mucosal nerve plexuses, was not inhibited by tetrodotoxin. 4. We conclude that cholera toxin-induced secretion in rabbit ileum in vitro is not mediated via a neurological mechanism.

Sodium and chloride transport in the isolated intestine of the earthworm, Lumbricus terrestris (L.)

1982 ◽  
Vol 97 (1) ◽  
pp. 197-216
Author(s):  
J. C. Cornell
Keyword(s):  
Chloride Transport ◽  
Short Circuit ◽  
Electrical Potential ◽  
Lumbricus Terrestris ◽  
Short Circuit Current ◽  
Electrical Potential Difference ◽  
Ionic Basis ◽  
Good Agreement

1. Measurements of electrical potential difference (PD), short-circuit current (SCC) and unidirectional fluxes of sodium and chloride were made across portions of the intestine. Based on the results, the intestine can be divided into at least four physiologically distinct regions. 2. These four physiological regions, designated from anterior to posterior as R I-II, R III A, R III B and R IV, do not completely correspond to the four anatomically distinct regions of the intestine. 3. The PD (serosal side positive) in R I-II, R III A, R III B and R IV is 1.08, 12.4, 5.61 and 31.7 mV, respectively. 4. The SCC in these same regions is 9.9, 50.4, 49.7, and 16.4 micro A cm2, respectively. 5. When short-circuited, net sodium and net chloride fluxes in the above regions are −0.36 and −0.27, 1.46*** and −0.92*, 1.74*** and −0.06 and 1.01*** and 0.07 mumol cm-2 h-1, respectively. Positive fluxes indicate net mucosal to serosal movements and asterisks indicate significant net fluxes (* P less than 0.05, *** P less than 0.001). 6. There is good agreement between the SCC and net sodium transport in R III B. In the other regions of the intestine the ionic basis of the SCC has not been completely explained. 7. The properties of the intestine in vitro appear to make the intestine well suited for the task of conserving sodium, a function which the intestine performs in vivo.

Endometrial transport generates the maternal-fetal electrical potential difference in guinea pigs

1991 ◽  
Vol 261 (2) ◽  
pp. R466-R472 ◽  
Author(s):  
T. G. McNaughton ◽  
L. A. Power ◽  
R. D. Gilbert ◽  
G. G. Power
Keyword(s):  
Guinea Pigs ◽  
Short Circuit ◽  
Electrical Potential ◽  
Uterine Wall ◽  
Late Gestation ◽  
Potential Difference ◽  
Fetal Membranes ◽  
Electrical Potential Difference

These studies examined the transport characteristics of the uterine endometrium with respect to the origin and mechanism of generation of the maternal-fetal electrical potential difference (PD) in pregnant guinea pigs. Late-gestation animals were used in two experimental preparations. In vivo, a sealed uterine pouch that preserved blood flow to the endometrium was prepared by removal of the fetus, placenta, and fetal membranes from the uterus and replacement with Earle's solution, a balanced electrolyte solution. In vitro, sections of uterine wall comprised of myometrium and endometrium without fetal membranes were mounted in Ussing chambers. Transuterine PDs (fetal side negative) were indistinguishable in vivo and in vitro, averaging 29.6 +/- 4.5 and 32.6 +/- 6.1 (95% confidence interval) mV in the respective preparations. Both values are within the range of maternal-fetal PD measured in intact guinea pigs, indicating that the fetoplacental unit is not essential in generating an intrauterine PD. The maternal-fetal PD, therefore, is likely a passive result of the fetus and placenta being immersed in fluids at the intrauterine potential. In vitro, both PD and short-circuit current (Isc) were completely inhibited by ouabain (10(-3) M) at the serosal (maternal) side of the uterine wall but unaffected by the inhibitor from the luminal (fetal) side. Amiloride (10(-5) M) and valinomycin (10(-5) M) caused decreases in the PD when added to the luminal side, both in vivo and in vitro, and were both ineffective from the serosal side in vitro. Isc was reduced 83% from 315 +/- 24 to 53 +/- 6 (SE) microA/cm2 after luminal amiloride (5 x 10(-4) M), indicating that Na+ is the predominant ion actively transported.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of ion transport by bile salts in canine gastric mucosa

1976 ◽  
Vol 231 (5) ◽  
pp. 1433-1437 ◽  
Author(s):  
YJ Kuo ◽  
LL Shanbour
Keyword(s):  
Gastric Mucosa ◽  
Ion Transport ◽  
Bile Salts ◽  
Electrical Potential ◽  
Electrical Potential Difference ◽  
Ph Stat ◽  
Stat Method ◽  
Mucosal Side

Studies were conducted with in vivo and in vitro canine stomach preparations. Instillation of 5, 10, and 20 mM bile salts in TES bufer (pH 7.4) into the nonsecreting stomach in vivo caused a progressive decrease in electrical potential difference (PD) and an increase in electrical resitance (R). The rate of acid secretion, determined by the pH-stat method in the histamine-stimulated stomach, decreased with 5 and 20 mM bile salts. Mucosal adenosine triphosphate (ATP) content of the nonsecreting or secreting stomach was reduced by bile salts. In vitro flux studies demonstrated that within the first hour after 1 mM bile salts were added to the mucosal side of the chamber, PD decreased, R increased, and net sodium transport decreased. In the second hour, unidirectional fluxes of sodium increased, indicating an increase in permeability of the gastric mucosa to sodium. These results demonstrate that the initial action of bile salts is inhibition of ion transport, which is followed by an increase in permeability.

Potassium secretion by nonsensory region of gerbil utricle in vitro

1987 ◽  
Vol 253 (4) ◽  
pp. F613-F621 ◽  
Author(s):  
N. Y. Marcus ◽  
D. C. Marcus
Keyword(s):  
Short Circuit ◽  
Control Level ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Electrical Potential Difference ◽  
Free Solution ◽  
K Secretion

The isolated nonsensory region of the gerbil utricle in vitro produced a lumen-positive transepithelial electrical potential difference (VT) of +5.7 mV and a luminal fluid containing 106 mM K when bathed in mammalian Ringer solution (5 mM K and 150 mM Na). The lumen of this region was perfused in vitro with K-free solution and the luminal [K], VT, and transepithelial resistance (RT) were measured before and following perfusion under control conditions and after addition of bumetanide (0.1 mM) or ouabain (1 mM) to the bath. The perfusate contained a reduced [Ca], since the average value of utricular endolymph in vivo (0.28 +/- 0.03 mM) measured with Ca-selective microelectrodes was 38% of that in perilymph. Under control conditions, the luminal [K] initially increased at a rate of 2.13 mumol X cm-2 X h-1 after perfusion; net secretion continued until the luminal [K] returned to its preperfusion level. This flux rate corresponds to 57 microA/cm2. The “equivalent short-circuit current” (Equiv. Isc; VT/RT) was found to average 61 microA/cm2. Both K secretion and VT were fully inhibited by bumetanide and by ouabain. Luminal application of Ba (5 mM) in K-free solution had no effect on the initial rate of K secretion, but did prevent full recovery of luminal [K] to the control level. These results are the first estimates of K secretion by the nonsensory cells of the utricle and are the first to directly demonstrate inhibition of K secretion in the inner ear by bumetanide and in the nonsensory tissue of the utricle by ouabain.

Mechanism of action of aspirin on canine gastric mucosa

1976 ◽  
Vol 230 (3) ◽  
pp. 762-767 ◽  
Author(s):  
YJ Kuo ◽  
LL Shanbour
Keyword(s):  
Gastric Mucosa ◽  
Electrical Potential ◽  
Electrical Potential Difference ◽  
Atp Content ◽  
Permeability Changes ◽  
In Vitro Exposure ◽  
Initial Action ◽  
Mucosal Side

Using an in vivo chambered canine stomach preparation, exposure of the gastric mucosa to 5, 10, and 20 mM aspirin(pH 3.0) resulted in a decrease in electrical potential difference (PD) and in an increase in resistance (R) within 30 min. In vitro, exposure of the mucosal side of the isolated canine gastric mucosa to 5, 10, and 20 mM aspirin (pH 3.0) for 1 h or of 1 mM aspirin (pH 3.0) for longer than 1 h resulted in marked permeability changes, i.e., increases in the undirectional fluxes of Na+ and Cl-, as well as inhibition of net ion fluxes. These concentrations of nonionized aspirin (pH 3.0) also reduced the R and PD. However, 1 mM aspirin (pH 3.0) or 20 mM ionized aspirin (pH 7.4) depresses the active transport of ion, increases R, but does not increase the ionic permeability. Mucosal adenosine triphosphate (ATP) content is reduced by mucosal instillation of aspirin (pH 3.0). These results demonstrate that the initial action of aspirin is inhibition of ion transport which is followed by an increase in permeability.

OSMOTIC INHIBITION OF THE NATRIFERIC RESPONSE OF THE TOAD URINARY BLADDER TO VASOPRESSIN

1975 ◽  
Vol 67 (1) ◽  
pp. 119-125
Author(s):  
P. J. BENTLEY
Keyword(s):  
Urinary Bladder ◽  
Water Movement ◽  
Short Circuit ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Toad Bladder ◽  
Toad Urinary Bladder ◽  
Electrical Potential Difference ◽  
Osmotic Gradient

SUMMARY The electrical potential difference and short-circuit current (scc, reflecting active transmural sodium transport) across the toad urinary bladder in vitro was unaffected by the presence of hypo-osmotic solutions bathing the mucosal (urinary) surface, providing that the transmural flow of water was small. Vasopressin increased the scc across the toad bladder (the natriferic response), but this stimulation was considerably reduced in the presence of a hypo-osmotic solution on the mucosal side, conditions under which water transfer across the membrane was also increased. This inhibition of the natriferic response did not depend on the direction of the water movement, for if the osmotic gradient was the opposite way to that which normally occurs, the response to vasopressin was still reduced. The natriferic response to cyclic AMP was also inhibited in the presence of an osmotic gradient. Aldosterone increased the scc and Na+ transport across the toad bladder but this response was not changed when an osmotic gradient was present. The physiological implications of these observations and the possible mechanisms involved are discussed.

scholarly journals Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

2021 ◽  
Author(s):  
Li-Nan Wang ◽  
Xiang-Lei Peng ◽  
Min Xu ◽  
Yuan-Bo Zheng ◽  
Yue-Ying Jiao ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Gene Deletion ◽  
Human Respiratory Syncytial Virus ◽  
Temperature Sensitive ◽  
T7 Promoter ◽  
Vaccine Candidates ◽  
Rsv Infection ◽  
Syncytial Virus

AbstractHuman respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed  temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals Effective decellularisation of human saphenous veins for biocompatible arterial tissue engineering applications: Bench optimisation and feasibility in vivo testing

2021 ◽  
Vol 12 ◽  
pp. 204173142098752
Author(s):  
Nadiah S Sulaiman ◽  
Andrew R Bond ◽  
Vito D Bruno ◽  
John Joseph ◽  
Jason L Johnson ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Mechanical Strength ◽  
Vascular Tissue ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Replacement Model ◽  
In Vivo Testing ◽  
Vascular Scaffolds

Human saphenous vein (hSV) and synthetic grafts are commonly used conduits in vascular grafting, despite high failure rates. Decellularising hSVs (D-hSVs) to produce vascular scaffolds might be an effective alternative. We assessed the effectiveness of a detergent-based method using 0% to 1% sodium dodecyl sulphate (SDS) to decellularise hSV. Decellularisation effectiveness was measured in vitro by nuclear counting, DNA content, residual cell viability, extracellular matrix integrity and mechanical strength. Cytotoxicity was assessed on human and porcine cells. The most effective SDS concentration was used to prepare D-hSV grafts that underwent preliminary in vivo testing using a porcine carotid artery replacement model. Effective decellularisation was achieved with 0.01% SDS, and D-hSVs were biocompatible after seeding. In vivo xeno-transplantation confirmed excellent mechanical strength and biocompatibility with recruitment of host cells without mechanical failure, and a 50% patency rate at 4-weeks. We have developed a simple biocompatible methodology to effectively decellularise hSVs. This could enhance vascular tissue engineering toward future clinical applications.

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