Subcellular distribution of nucleotide cyclases in rat intestinal epithelium.

1978 ◽  
Vol 235 (5) ◽  
pp. E539 ◽  
Author(s):  
M W Walling ◽  
A K Mircheff ◽  
C H Van Os ◽  
E M Wright
Keyword(s):  
Adenylate Cyclase ◽  
Guanylate Cyclase ◽  
Brush Border ◽  
Linear Regression Analysis ◽  
Basolateral Membrane ◽  
Multiple Linear Regression Analysis ◽  
Isolated Cells ◽  
Basolateral Membranes ◽  
Rat Duodenum ◽  
Triton X

The subcellular distributions of adenylate cyclase and guanylate cyclase were determined for the mature enterocyte from the rat duodenum. Brush-border and basolateral membranes were prepared from isolated cells by an analytical isolation procedure, and multiple linear regression analysis was used to obtain a quantitative estimate of the distribution of recovered cyclase activities between the brush borders and basolateral membranes. Adenylate cyclase was largely confined to the basolateral surface of the epithelium, whereas guanylate cyclase was found on the brush-border and basolateral membrane fractions in the ratio 2.4:1. There was no evidence for the presence of nucleotide cyclases in the cytosol. Guanylate cyclase in both the brush-border and basolateral membranes was stimulated by epinephrine, insulin, and Triton X-100, but not by carbachol. Adenylate cyclase was not influenced by epinephrine, but was markedly stimulated by NaF and vasoactive intestinal peptide. These results are discussed in relation to the effects of hormones on transport across the small intestine.

scholarly journals The metabolism of cyclic nucleotides in the guinea-pig pancreas. Adenylate cyclase and guanylate cyclase

1980 ◽  
Vol 186 (2) ◽  
pp. 499-505 ◽  
Author(s):  
M Lemon ◽  
P Methven ◽  
K Bhoola
Keyword(s):  
Adenylate Cyclase ◽  
Guinea Pig ◽  
Guanylate Cyclase ◽  
Cyclic Nucleotides ◽  
Cyclase Activity ◽  
Dependent Manner ◽  
Guanylate Cyclase Activity ◽  
Triton X ◽  
Dose Dependent ◽  
Significant Activation

Adenylate cyclase from the guinea-pig pancreas was activated in a dose-dependent manner by both secretin and cholecystokinin-pancreozymin, but in contrast with results in other species the hormones were approximately equipotent. All other hormones and transmitter substances tested were without any effect on adenylate cyclase activity. Guanylate cyclase activity was shown to have both particulate and supernatant components in the guinea-pig pancreas. The particulate enzyme, but not the supernatant enzyme, was markedly activated by Triton X-100, and most of the induced activity was released into the supernatant. The supernatant enzyme was specifically Mn2+-dependent, but, even though Mn2+ was maximally effective at a concentration of 3 mM, activity could be raised further by increasing Ca2+ concentration. The particulate enzyme, by contrast, was relatively Mn2+-independent. Activity of the particulate guanylate cyclase was enhanced by phosphatidylserine. The supernatant enzyme displayed classical Michaelis-Menten kinetics, but the particulate enzyme deviated markedly from such kinetics. Under none of the conditions used was any significant activation of guanylate cyclase observed with any of the secretogen hormones or transmitter substances.

scholarly journals Adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterases of guinea-pig cardiac sarcolemma

1976 ◽  
Vol 158 (3) ◽  
pp. 535-541 ◽  
Author(s):  
P J St Louis ◽  
P V Sulakhe
Keyword(s):  
Adenylate Cyclase ◽  
Guinea Pig ◽  
Guanylate Cyclase ◽  
Cyclic Gmp ◽  
Maximal Activity ◽  
Cardiac Sarcolemma ◽  
Guinea Pig Heart ◽  
Membrane Bound ◽  
Triton X ◽  
Assay Conditions

1. The activities of the enzymes involved in the metabolism of cyclic nucleotides were studied in sarcolemma prepared front guinea-pig heart ventricle; the enzyme activities reported here were linear under the assay conditions. 2. Adenylate cyclase was maximally activated by 3mM-NaF; NaF increased the Km for ATP (from 0.042 to 0.19 mM) but decreased the Ka for Mg2+ (from 2.33 to 0.9 mM). In the presence of saturating Mg2+ (15 mM), Mn2+ enhanced adenylate cyclase, whereas Co2+ was inhibitory. beta-Adrenergic amines (10-50 muM) stimulated adenylate cyclase (38+/-2%). When added to the assay mixture, guanyl nucleotides (GTP and its analogue, guanylyl imidophosphate) stimulated basal enzyme activity and enhanced the stimulation by isoproterenol. By contrast, preincubation of sarcolemma with guanylyl imidodiphosphate stimulated the formation of an ‘activated’ form of the enzyme, which did not reveal increased hormonal sensitivity. 3. The guanylate cyclase present in the membranes as well as in the Triton X-100-solubilized extract of membranes exhibited a Ka for Mn 2+ of 0.3 mM; Mn2+ in excess of GTP was required for maximal activity. Solubilized guanylate cyclase was activated by Mg2+ only in the presence of low Mn2+ concentrations; Ca2+ was inhibitory both in the absence and presence of low Mn2+. Acetylcholine as well as carbamolycholine stimulated membrane-bound guanylate cyclase. 4. Cylic nucleotide phosphodiesterase activities of sarcolemma exhibited both high-and low-Km forms with cyclic AMP and with cyclic GMP as substrate. Ca2+ ions increased the Vmax. of the cyclic GMP-dependent enzyme.

Inorganic anion transport in kidney and intestinal brush border and basolateral membranes

1980 ◽  
Vol 238 (6) ◽  
pp. F452-F460 ◽  
Author(s):  
S. Grinstein ◽  
R. J. Turner ◽  
M. Silverman ◽  
A. Rothstein
Keyword(s):  
Brush Border ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Anion Transport ◽  
Significant Inhibition ◽  
Kidney Cortex ◽  
Basolateral Membrane Vesicles ◽  
Inorganic Anion ◽  
Transport Pathways ◽  
Basolateral Membranes

The efflux of inorganic anions from purified brush border and basolateral membrane vesicles from dog kidney cortex was measured under equilibrium exchange conditions. Marked differences in temperature sensitivity and effects of inhibitors were found between the Cl and SO4 transport pathways and between the two types of membranes. SO4 transport in both brush border and basolateral membranes was markedly reduced by cooling, but significant inhibition by 4,4'–diisothiocyano-2,2'–disulfonic stilbene (DIDS) was only observed in basolateral vesicles. In contrast, Cl efflux from both types of vesicles was neither substantially inhibited by DIDS nor by lowering the temperature to 0 degrees C. Phosphate efflux from basolateral membrane vesicles was found to be only partially sensitive to DIDS. Attempts to label the stilbene-sensitive SO4 pathway in basolateral vesicles using [3H2]DIDS as a marker were unsuccessful due to the nonspecific labeling of many membrane components. The asymmetry in inorganic anion transport behavior exhibited by brush border and basolateral membrane vesicles from dog renal proximal tubule was also observed in equivalent vesicles prepared from rat small intestine.

[3H]Angiotensin II binding to basolateral membranes from rat proximal renal tubule: effect of sodium intake and captopril

1989 ◽  
Vol 122 (2) ◽  
pp. 499-NP ◽  
Author(s):  
N. P. Lewis ◽  
D. R. Ferguson
Keyword(s):  
Angiotensin Ii ◽  
Adenylate Cyclase ◽  
Binding Site ◽  
Renal Tubule ◽  
Sodium Intake ◽  
Basolateral Membrane ◽  
Membrane Preparation ◽  
Basolateral Membranes ◽  
Proximal Renal Tubule ◽  
Site Density

ABSTRACT Basolateral membranes were prepared from rat renal cortex by density gradient centrifugation. Their purity was confirmed by electron microscopy and by marker enzyme enrichment. The basolateral membrane preparation was shown to be derived predominantly from the proximal renal tubule by measurement of hormone-stimulated adenylate cyclase; marked stimulation of adenylate cyclase was found with parathyroid hormone, but not with isoprenaline, antidiuretic hormone or calcitonin. A single class of specific high-affinity [3H]angiotensin II-binding site was identified in the basolateral membrane preparation which, after correction of results for tracer degradation, showed equilibrium dissociation constant of 0·23 nmol/l and binding site concentration of 485·8 fmol/mg protein. Binding sites for [3H]angiotensin II were measured in basolateral membranes prepared from rats fed diets with a low, normal or high sodium content. A trend of increased binding site density with reduced sodium intake was found which did not reach statistical significance. No effect on affinity was demonstrated. Treatment of rats on a low-sodium diet with captopril (500 mg/l drinking water) caused a significant reduction in binding site density; no effect on affinity was demonstrated. These findings suggest that the density of angiotensin II receptors at this site is altered by the activity of the renin-angiotensin system. Journal of Endocrinology (1989) 122, 499–507

Sensitivity of rat renal luminal and contraluminal sulfate transport systems to DIDS

1986 ◽  
Vol 250 (2) ◽  
pp. F226-F234 ◽  
Author(s):  
C. Bastlein ◽  
G. Burckhardt
Keyword(s):  
Transport System ◽  
Brush Border ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Transport Systems ◽  
Basolateral Membrane Vesicles ◽  
Sulfate Transport ◽  
Sulfate Uptake ◽  
Basolateral Membranes ◽  
Irreversible Inhibition

4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was tested as an inhibitor of the sulfate transport systems in rat renal brush border and basolateral membrane vesicles. Na+-driven sulfate uptake into brush border membrane vesicles was half-maximally inhibited at 350 microM DIDS. Proton gradient-driven sulfate uptake into basolateral membrane vesicles was competitively inhibited by DIDS with a Ki of 2.4 microM. The Km for delta pH-driven sulfate uptake was 5.4 microM. The different affinities of the sulfate transport systems for DIDS correlated with different substrate specificities. The luminal transport system accepted a smaller range of anions than the contraluminal system and did not operate as a Na+-independent anion exchanger. After treatment of basolateral membrane vesicles with 50 microM DIDS at pH 8.4 for 30 min, an irreversible inhibition of sulfate uptake was observed. With brush border membranes, only a small irreversible inhibition was obtained. Lack of inhibition after treatment of basolateral membranes with DIDS at pH 6.4 indicated that DIDS reacted with deprotonated amino groups of the transport protein. Sulfate was protected from the irreversible inhibition by DIDS. Sodium-driven uptake of L-glutamate and methylsuccinate into basolateral membrane vesicles was not irreversibly inhibited by DIDS, indicating a specific action of DIDS on the contraluminal sulfate transport system. Irreversible and substrate-protectable inhibition of sulfate transport render DIDS suitable for future affinity labeling studies on the sulfate transport system in basolateral membranes.

Na+ gradient-dependent Pi uptake in basolateral membrane vesicles from dog kidney

1984 ◽  
Vol 246 (5) ◽  
pp. F663-F669 ◽  
Author(s):  
S. J. Schwab ◽  
S. Klahr ◽  
M. R. Hammerman
Keyword(s):  
Brush Border ◽  
Renal Cortex ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Basolateral Membrane Vesicles ◽  
Basolateral Membranes ◽  
Solute Uptake ◽  
Dog Kidney ◽  
Proximal Tubular

To ascertain whether Na+ gradient-stimulated 32Pi uptake was demonstrable in renal basolateral membrane vesicles, we measured 32Pi uptake in basolateral membrane suspensions isolated from canine renal cortex and compared solute uptake in basolateral suspensions with that measured in brush border suspensions. Measurements revealed Na+ gradient-dependent 32Pi transport in basolateral preparations. D-[3H] Glucose uptakes in basolateral suspensions were not stimulated by the Na+ gradient in contrast to findings in brush border suspensions. Na+ gradient-dependent 32Pi transport in basolateral suspensions was electrogenic in contrast to that measured in brush border preparations. Unlike 32Pi uptake in brush border preparations, Na+ gradient-dependent 32Pi uptake in basolateral suspensions did not increase as extravesicular pH was increased from 6.5 to 7.5. Na+ gradient-dependent 32Pi uptake in basolateral membranes showed saturation over the range of [Pi] from 5 to 100 microM (apparent Km, 14 +/- 2 microM; apparent Vmax, 34 +/- 2 pmol Pi X mg protein-1 X 30s-1). Our findings are compatible with the presence of an electrogenic Na+-Pi cotransporter in the canine proximal tubular basolateral membrane.

Fluidity of brush border and basolateral membranes from human kidney cortex

1983 ◽  
Vol 245 (2) ◽  
pp. F227-F231 ◽  
Author(s):  
C. Le Grimellec ◽  
S. Carriere ◽  
J. Cardinal ◽  
M. C. Giocondi
Keyword(s):  
Brush Border ◽  
Liquid Crystalline ◽  
Plasma Membranes ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Human Kidney ◽  
Kidney Cortex ◽  
Lipid Phase ◽  
Basolateral Membranes ◽  
Normal Human Kidney

The physical state of lipids in brush border and basolateral membrane vesicles prepared from normal human kidney cortex was investigated by fluorescence polarization and electron spin resonance. At physiologic temperature, lipids were significantly less ordered, i.e., more fluid, in basolateral than in brush border membranes. This difference was also observed using corresponding liposomes made from total lipid extracts. For both brush border and basolateral membranes, temperature-dependent experiments revealed the existence of a broad thermotropic transition extending approximately from 20 to 42 degrees C. These data are interpreted to indicate that plasma membranes from human kidney cortex function physiologically at the upper critical temperature of a transition that probably corresponds to a liquid crystalline-to-gel lipid phase separation.

Fluidity and composition of brush border and basolateral membranes from rat kidney

1984 ◽  
Vol 247 (3) ◽  
pp. F434-F439 ◽  
Author(s):  
M. K. Hise ◽  
W. W. Mantulin ◽  
E. J. Weinman
Keyword(s):  
Brush Border ◽  
Fluorescence Anisotropy ◽  
Brush Border Membrane ◽  
Membrane Lipids ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Fatty Acyl ◽  
Protein Ratio ◽  
Basolateral Membranes ◽  
Relative Contribution

The physical state of rat renal brush border and basolateral membranes was examined using the technique of electron spin resonance. As estimated from the order parameters of both 5- and 12-doxyl spin-labeled phosphatidylcholine, the brush border membrane was significantly more ordered than the basolateral membrane. To confirm these findings, fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene was studied. Fluorescence anisotropy at 25 and 37 degrees C was significantly greater in the brush border membrane when compared with the basolateral membrane. The relative contribution of the lipid component to this difference was examined using multilamellar liposomes prepared from lipid extracts of these membranes. Fluorescence anisotropy in the brush border membrane lipids was significantly greater than that in the basolateral membrane lipids at both 25 and 37 degrees C. Compositional determinants of fluidity were examined in both membranes. Factors that may contribute to the more fluid state of the basolateral membrane include 1) a greater lipid-to-protein ratio; 2) a greater ratio of phosphatidylcholine to sphingomyelin; and 3) a tendency toward shorter-length fatty acyl chains.

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