Benzodiazepines inhibit transport-related oxygen consumption in thick ascending limb

1988 ◽  
Vol 255 (3) ◽  
pp. C385-C392 ◽  
Author(s):  
F. N. Ziyadeh ◽  
Z. S. Agus
Keyword(s):  
Oxygen Consumption ◽  
Amphotericin B ◽  
Binding Sites ◽  
Cell Function ◽  
Chloride Transport ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Thick Ascending Limb ◽  
Total Oxygen ◽  
Sodium Uptake

Specific binding sites for benzodiazepines (BZD) have been identified in several nonneuronal tissues including the kidney where they are localized predominantly to the tubular epithelium of the thick ascending limb of Henle's loop (TALH). The physiological function of these nonneuronal (peripheral) BZD-binding sites is undefined, but it has been suggested that they may represent receptors for putative endogenous ligands that may modulate cell function. In the current study, we examined the in vitro effects of diazepam and Ro5-4864, a specific peripheral BZD-receptor agonist, on the oxygen consumption of medullary TALH tubule suspensions of rabbit kidney. Maximal inhibition of total oxygen consumption was achieved at a dose of 5 X 10(-4) M of either agent. On average, diazepam and Ro5-4864 reduced total oxygen consumption by 41 and 44%, respectively. The predominant inhibition was in the ouabain-sensitive component of oxygen consumption, which suggests that BZDs inhibit active sodium-chloride transport in the TALH. To assess whether this inhibition depends on sodium uptake, TALH tubules were pretreated with amphotericin B (2 X 10(-6) M) to enhance sodium uptake and stimulate basal oxygen consumption; subsequent addition of Ro5-4864 (5 X 10(-4) M) still reduced oxygen consumption to a residual value that was not different from that in TALH tubules treated with Ro5-4864 but without pretreatment with amphotericin B. This suggests that BZD inhibition of transport-related oxygen consumption is not caused by diminution of sodium uptake into cells and thus appears to be distinct from the effect of furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)

Mineralcorticoid receptors along the nephron: [3H]aldosterone binding in rabbit tubules

1981 ◽  
Vol 241 (6) ◽  
pp. F605-F611 ◽  
Author(s):  
A. Doucet ◽  
A. I. Katz
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Scatchard Analysis ◽  
Thick Ascending Limb ◽  
Collecting Tubule ◽  
The Difference ◽  
Collecting Tubules ◽  
Cortical Collecting Tubule

To identify the site of mineralocorticoid action along the nephron, we measured the specific binding of [3H]aldosterone to nephron segments microdissected from aldosterone-deficient rabbits. Specific binding was defined as the difference between binding measured in the absence or in the presence of 2,000-fold excess of unlabeled hormone (in 10(-18) mol X cm tubule length-1 +/- SE). High specific binding capacity was found in the branched collecting tubule (108 +/- 4), the cortical collecting tubule (119 +/- 9), and the outer medullary collecting tubule (115 +/- 16), whereas specific binding was negligible in the proximal convoluted tubule (8 +/- 9), pars recta (2 +/- 6), medullary thick ascending limb (4 +/- 6), cortical thick ascending limb (6 +/- 2), and distal convoluted tubule (6 +/- 6). In cortical collecting tubules, Scatchard analysis of the specific [3H]aldosterone binding indicated a dissociation constant (KD) of 2.2 X 10(-9) M and a maximum number of binding sites of 157 X 10(-18) mol X cm tubule length-1. The steroid specificity was assessed from the competition of various steroids for [3H]aldosterone binding sites. Receptors from the cortical collecting tubule revealed the following sequence of affinities: aldosterone greater than DOCA greater than spironolactone greater than dexamethasone greater than 5 alpha-dihydrotestosterone = progesterone = 17 beta-estradiol, indicating that the binding sites in the collecting tubule are mineralocorticoid receptors. These results demonstrate significant [3H]aldosterone binding to receptors of high affinity and mineralocorticoid specificity only in the collecting tubule and suggest that this nephron segment is the target site of mineralocorticoid action in the rabbit kidney.

Aldosterone binding along the rabbit nephron: an autoradiographic study on isolated tubules

1981 ◽  
Vol 240 (3) ◽  
pp. F172-F179 ◽  
Author(s):  
A. Vandewalle ◽  
N. Farman ◽  
P. Bencsath ◽  
J. P. Bonvalet
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Autoradiographic Study ◽  
Rabbit Kidney ◽  
Thick Ascending Limb ◽  
Collecting Ducts ◽  
Sites Of Action ◽  
Convoluted Tubules ◽  
Silver Grains ◽  
Dry Film

The sites of action of aldosterone (A) along the tubule of rabbit kidney were studied by autoradiographic localization of mineralocorticoid-binding sites on microdissected tubular segments. Kidney pyramids were incubated at 30 degrees C for 1 h in a collagenase solution with [3H]aldosterone at a concentration of 1.5 X 10(-9) M with and without an excess unlabeled A. Tubular segments were then microdissected and transferred onto dry film; fixation and staining were done only after exposure of the film 4 mo later in order to avoid diffusion. Specific nuclear labeling was 19.0 +/- 1.3 silver grains/100 micrometers2 in distal convoluted tubules (n = 28) and 21.0 +/- 1.8 in cortical collecting ducts (n = 18). No difference between these two structures was observed (P greater than 0.1, paired t test, n = 15). No specific binding was found in the proximal tubule (0.5 +/- 0.4, n = 17). In the thick ascending limb of Henle's loop, the labeling was low (3.9 +/- 0.9, n = 16). We conclude that, in the rabbit kidney, nuclear mineralocorticoid-binding sites, presumably receptors, are present in the distal and cortical collecting tubule.

scholarly journals The Plasminogen Activation System and the Regulation of Catecholaminergic Function

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Hongdong Bai ◽  
Samir Nangia ◽  
Robert J. Parmer
Keyword(s):  
Plasminogen Activator ◽  
Binding Sites ◽  
Cell Function ◽  
Specific Binding ◽  
Neurosecretory Cells ◽  
Plasminogen Activation ◽  
Plasmin Activity ◽  
Plasminogen Activation System ◽  
Activation System ◽  
Catecholaminergic Cells

The local environment of neurosecretory cells contains the major components of the plasminogen activation system, including the plasminogen activators, tissue plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), as well as binding sites for t-PA, the receptor for u-PA (uPAR), and also the plasminogen activator inhibitor, PAI-1. Furthermore, these cells express specific binding sites for plasminogen, which is available in the circulation and in interstitial fluid. Colocalization of plasminogen and its activators on cell surfaces provides a mechanism for promoting local plasminogen activation. Plasmin is retained on the cell surface where it is protected from its inhibitor,α2-antiplasmin. In neurosecretory cells, localized plasmin activity provides a mechanism for extracellular processing of secreted hormones. Neurotransmitter release from catecholaminergic cells is negatively regulated by cleavage products formed by plasmin-mediated proteolysis. Recently, we have identified a major plasminogen receptor, Plg-RKT. We have found that Plg-RKTis highly expressed in chromaffin cells of the adrenal medulla as well as in other catecholaminergic cells and tissues. Plg-RKT-dependent plasminogen activation plays a key role in regulating catecholaminergic neurosecretory cell function.

Specific binding sites in the rabbit kidney for prostaglandin a

1973 ◽  
Vol 4 (5) ◽  
pp. 703-709 ◽  
Author(s):  
Ahmad A. Attallah ◽  
James B. Lee
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Specific Binding Sites

Suspension of medullary thick ascending limb tubules from the rabbit kidney

1984 ◽  
Vol 247 (6) ◽  
pp. F955-F964 ◽  
Author(s):  
M. E. Chamberlin ◽  
A. LeFurgey ◽  
L. J. Mandel
Keyword(s):  
Oxygen Consumption ◽  
Cellular Metabolism ◽  
Isolation Procedure ◽  
Rabbit Kidney ◽  
Enzyme Assays ◽  
Thick Ascending Limb ◽  
Marker Enzyme ◽  
Direct Inhibition ◽  
Medullary Thick Ascending Limb ◽  
Carbonyl Cyanide

A procedure for isolating a suspension of tubules derived from the rabbit medullary thick ascending limb is described. The purity of the preparation was assessed by microscopy and enzyme assays and the viability of the preparation was assessed by measuring oxygen consumption. Microscopy revealed that the suspension contains 95% thick ascending limbs and that the isolation procedure preserves the structure of the epithelium except for the loss of the basement membrane. The preparation had a high activity of calcitonin-sensitive adenylate cyclase, a marker enzyme for the medullary thick ascending limb. Control oxygen consumption was considerably higher than that reported for proximal tubules in the literature, and nystatin or carbonyl cyanide p-trifluoromethoxyphenylhydrazone addition produced a more than 100% increase in oxygen consumption. Furosemide inhibited the oxygen consumption by 43% and ouabain inhibited it by 42%. Furosemide inhibited sodium chloride entry without directly affecting the Na-K-ATPase or cellular metabolism. Chloride removal depressed oxygen consumption to the same extent as furosemide, but some of this action was through direct inhibition of cellular metabolism.

Renal adrenergic receptors: localization of [125I]prazosin binding sites along the microdissected rat nephron

1992 ◽  
Vol 70 (7) ◽  
pp. 1016-1020 ◽  
Author(s):  
Herbert T. Cohen ◽  
Fumi Takemoto ◽  
Takeo Satoh ◽  
Adrian I. Katz
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Collecting Duct ◽  
Specific Radioactivity ◽  
Dissociation Rate ◽  
Proximal Convoluted Tubule ◽  
Sodium Reabsorption ◽  
Thick Ascending Limb ◽  
Diluting Segment ◽  
High Specific Radioactivity

Norepinephrine stimulates renal tubular sodium reabsorption, probably through an α1-adrenoceptor-mediated mechanism. Although the distribution of α1-adrenoceptors in the kidney has been studied with autoradiography, the precise location of these receptors in isolated nephron segments is unclear. Using a microassay we determined the specific binding of [125I]iodoarylazidoprazosin ([125I]prazosin), a high specific radioactivity analog of the selective α1-antagonist prazosin, to microdissected glomeruli and tubule segments. Specific binding of [125I]prazosin (3 nM) in the proximal convoluted tubule was time- and concentration-dependent, saturable, and reversible. In this segment the apparent KD by association and dissociation rate constants of [125I]prazosin binding was 0.47 nM, and the maximum receptor density was ~ 0.19 fmol/mm, or 720 fmol/mg protein. Binding specificity was verified in competition studies with excess (3 μM) unlabeled prazosin and probes for α2- (yohimbine), β- (propranolol), dopamine1- (SCH23390), and dopamine2- (S-sulpiride) receptors. [125I]Prazosin binding was inhibited significantly only by unlabeled prazosin. Mapping of prazosin binding along the nephron revealed that the highest density was in the proximal convoluted tubule, followed by the proximal straight tubule. Lesser binding was found in the thick ascending limb and in the distal convoluted tubule, whereas in the cortical and outer medullary collecting duct and in glomeruli, binding was not significantly different from zero. These results demonstrate specific prazosin binding sites in the proximal and early distal nephron where direct innervation by monoaminergic nerves is most abundant, and suggest that portions of the nephron beyond the proximal tubule, specifically the diluting segment, may also be under α1-agonist influence.Key words: α1-adrenoceptor, prazosin, isolated tubule, glomerulus, catecholamine.

Oxygen consumption and active transport of ions by isolated frog gastric mucosa

1963 ◽  
Vol 204 (5) ◽  
pp. 812-816 ◽  
Author(s):  
J. G. Forte ◽  
R. E. Davies
Keyword(s):  
Oxygen Consumption ◽  
Gastric Mucosa ◽  
Chloride Transport ◽  
Short Circuit ◽  
Membrane Conductance ◽  
Short Circuit Current ◽  
Chloride Ions ◽  
Total Oxygen ◽  
Ph Stat ◽  
Stat Method

Isolated bullfrog gastric mucosae were suspended between two glass chambers and bathed with physiological salt solutions equilibrated with 5% CO2 and 95% O2. The transmucosal potential difference, short-circuit current and membrane conductance were measured. Oxygen consumption was measured polarographically and the rate of acid secretion was determined by the pH stat method. Values for the ratio of hydrogen ions secreted to the total oxygen molecules consumed were calculated for 84 experiments and yielded a mean ratio of 1.86 ± 0.51 (sd) with none of the values above 3.5. The ratio of chloride ions transported (calculated as short-circuit current of chloride ions plus hydrochloric acid) to total oxygen consumed for 84 experiments was found to be above 4.0 in 40% of the cases measured (mean 3.80 ± 0.68). A simple redox-pump hypothesis, with molecular oxygen as the electron acceptor, and with one ion moved for each electron transmitted is ruled out as the only operating mechanism to account for active chloride transport by frog gastric mucosa.

Aldosterone binding in isolated tubules I. Biochemical determination in proximal and distal parts of the rabbit nephron

1982 ◽  
Vol 242 (1) ◽  
pp. F63-F68 ◽  
Author(s):  
N. Farman ◽  
A. Vandewalle ◽  
J. P. Bonvalet
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Proximal Tubules ◽  
Rabbit Kidney ◽  
Nuclear Fraction ◽  
Lower Affinity ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Collecting Tubules ◽  
Binding Curve

Microbiochemical methods were applied to proximal tubules (PCT) and a mixture of distal and cortical collecting tubules (D + C) of rabbit kidney in order to define aldosterone binding sites. For each experiment, after incubation of kidney pyramids with [3H]aldosterone ([3H]A), either alone or in the presence of an excess unlabeled A, 100-150 mm of both categories of tubules were microdissected using collagenase. Specific binding was determined on the nuclear fraction of each sample. Aldosterone concentrations ranged from 2 X 10(-9) to 4.5 X 10(-8) M. No specific binding was detectable in PCT. Specific binding in D + C increased rapidly as a function of [3H]A concentration up to 5 X 10(-9) M and then more slowly. No plateau was reached. Both the absence of saturation of the binding curve and the curvilinear aspect of the Scatchard plot suggested the presence of two binding sites, one of high affinity, presumably a mineralocorticoid site, and the other of lower affinity, possibly a glucocorticoid site. These experiments suggest that the distal structures of the nephron, located in the cortex, are the main sites of binding of aldosterone and contain a high number of specific binding sites for this hormone.

scholarly journals Separation of renal medullary cells: isolation of cells from the thick ascending limb of Henle's loop.

1980 ◽  
Vol 87 (3) ◽  
pp. 672-681 ◽  
Author(s):  
J Eveloff ◽  
W Haase ◽  
R Kinne
Keyword(s):  
Oxygen Consumption ◽  
Adenylate Cyclase ◽  
Collecting Duct ◽  
Single Cells ◽  
Rabbit Kidney ◽  
Thick Ascending Limb ◽  
Apical Surface ◽  
Dependent Manner ◽  
Homogeneous Population ◽  
Henle's Loop

A homogeneous population of single cells from the thick ascending limb of Henle's loop (TALH) has been isolated from the rabbit kidney medulla. A total medullary cell suspension was prepared by a series of collagenase, hyaluronidase, and trypsin digestions and separated on a Ficoll gradient (2.6-30.7% wt/wt). Morphologically, the cells isolated from the TALH were homogeneous and showed polarity within their plasma membrane structure, with a few blunt microvilli on their apical surface and deep infoldings of the basal-lateral membrane. Biochemically, the TALH cells were highly enriched in calcitonin-sensitive adenylate cyclase and Na, K-ATPase. Alkaline phosphatase and arginine vasopressin-sensitive adenylate cyclase, highly concentrated in proximal tubule and collecting duct, were present only in low concentrations in the TALH cells. Additionally, furosemide, a diuretic inhibiting sodium chloride transport in the TALH in vivo, inhibited oxygen consumption of the TALH cells in a dose-dependent manner. The TALH cells were viable, as judged by morphological appearance, trypan blue exclusion, the response of oxygen consumption to 2,4-dinitrophenol, succinate and ouabain, and the cellular Na, K and ATP levels.

Autoradiographic determination of dexamethasone binding sites along the rabbit nephron

1983 ◽  
Vol 244 (3) ◽  
pp. F325-F334 ◽  
Author(s):  
N. Farman ◽  
A. Vandewalle ◽  
J. P. Bonvalet
Keyword(s):  
Proximal Tubule ◽  
Binding Sites ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Nuclear Binding ◽  
Specific Binding Sites ◽  
In Vitro Incubation ◽  
Collecting Tubules

Specific binding sites of tritiated dexamethasone ([3H]dex) along the tubule of rabbit kidney were investigated using an autoradiographic method (dry film) on isolated tubular segments. After in vitro incubation of kidney pyramids with [3H]dex (0.15-53 nM) in the presence or absence of an excess (X200) of unlabeled dexamethasone, tubular segments were microdissected and processed for autoradiography. A quantitative analysis of specific labeling over cytoplasm and nuclei was performed. Specific nuclear binding was observed in all tubular segments beyond the pars recta. This binding was dose dependent and reached much higher values than those reported for aldosterone. In the proximal tubule, the specific labeling was also high but remained mostly cytoplasmic. The meaning of these drastically different intracellular localizations is still open to interpretation. Autoradiography was performed after in vivo injection of [3H]dex and [3H]aldosterone. The results were not different from those described here for dexamethasone and from those previously reported for aldosterone after in vitro incubation. We conclude that specific nuclear binding sites for dexamethasone range over the nephron except for proximal tubule, with no great difference among segments, in contrast to specific sites for aldosterone, which are restricted to distal and cortical collecting tubules. The exact significance of the proximal cytoplasmic specific binding of [3H]dex remains to be determined.

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