Two oxytocin-binding site subtypes in rat kidney: pharmacological characterization, ontogeny and localization by in vitro and in vivo autoradiography

1997 ◽  
Vol 153 (1) ◽  
pp. 49-59 ◽  
Author(s):  
M P Arpin-Bott ◽  
E Waltisperger ◽  
M J Freund-Mercier ◽  
M E Stoeckel
Keyword(s):  
Binding Sites ◽  
Cellular Localization ◽  
Rat Kidney ◽  
Distal Tubule ◽  
Macula Densa ◽  
Pharmacological Characterization ◽  
Henle's Loop ◽  
Semithin Sections

Abstract The localization of oxytocin (OT)-binding sites in the developing rat kidney and their pharmacological characterization were investigated by means of autoradiographic techniques. The cellular localization was studied by application of the histoautoradiographic technique to (1) frozen sections and semithin sections from kidney slices incubated in vitro in the presence of a 125I-labelled OT antagonist and (2) frozen and semithin sections from kidneys after in vivo systemic infusion of the radioligand. Pharmacological characteristics were determined in competition experiments by using quantitative film autoradiography. Specific OT-binding sites were first detected at embryonic day 17 (E17) in the cortex. At early stages up to postnatal days (PN30), the cortical OT-binding sites were highly concentrated on the juxta- and paraglomerular portion of the distal tubule; in the adult they were restricted to the macula densa. In the medulla, OT-binding sites were first detected at E19 when this region is forming; they were localized on the thin limb of Henle's loop. These data obtained by in vitro binding were confirmed by in vivo binding at PN30 which showed, in addition, the presence in one rat of OT-binding sites in the inner stripe of the outer medulla. At all stages examined (PN15 to PN90), cortical OT-binding sites had a higher selectivity for OT versus vasopressin (IC50=0·78 ± 0·04 nm and 8 ± 0·5 nm respectively at PN90) than medullary sites (IC50= 1·9 ± 0·27 nm and 2±1·13 nm respectively at PN90). These data suggest that the OT-binding sites of the macula densa and thin Henle's loop, detected in the rat kidney, represent two subtypes of OT receptors which could mediate distinct effects of OT on kidney function. Journal of Endocrinology (1997) 153, 49–59

Autoradiographic demonstration of oxytocin-binding sites in the macula densa

1989 ◽  
Vol 257 (2) ◽  
pp. F310-F314 ◽  
Author(s):  
M. E. Stoeckel ◽  
M. J. Freund-Mercier
Keyword(s):  
Binding Sites ◽  
Cellular Localization ◽  
Rat Kidney ◽  
Juxtaglomerular Apparatus ◽  
Macula Densa ◽  
Tubuloglomerular Feedback ◽  
Binding Characteristics ◽  
Conventional Film ◽  
High Concentrations ◽  
Autoradiographic Technique

Specific oxytocin (OT)-binding sites were localized in the rat kidney with use of a selective 125I-labeled OT antagonist (125I-OTA). High concentrations of OT binding sites were detected on the juxtaglomerular apparatus with use of the conventional film autoradiographic technique. No labeling occurred on other renal structures. The cellular localization of the OT binding sites within the juxtaglomerular apparatus was studied in light microscope autoradiography, on semithin sections from paraformaldehyde-fixed kidney slices incubated in the presence of 125I-OTA. These preparations revealed selective labeling of the macula densa, mainly concentrated at the basal pole of the cells. Control experiments showed first that 125I-OTA binding characteristics were not noticeably altered by prior paraformaldehyde fixation of the kidneys and second that autoradiographic detection of the binding sites was not impaired by histological treatments following binding procedures. In view of the role of the macula densa in the tubuloglomerular feedback, the putative OT receptors of this structure might mediate the stimulatory effect of OT on glomerular filtration.

EFFECT OF CARBENOXOLONE ON THE CONCENTRATIONS OF ALDOSTERONE IN RAT PLASMA AND KIDNEY

1979 ◽  
Vol 81 (1) ◽  
pp. 143-151 ◽  
Author(s):  
M. J. HUMPHREY ◽  
W. E. LINDUP ◽  
J. CHAKRABORTY ◽  
D. V. PARKE
Keyword(s):  
Binding Sites ◽  
Rat Kidney ◽  
Rat Plasma ◽  
Supernatant Fraction ◽  
Nuclear Fraction ◽  
Toad Skin ◽  
Site Of Action ◽  
Specific Receptors

The renal subcellular distribution of various subcutaneous doses (10−10–10−8 mol) of [3H]aldosterone has been investigated in adrenalectomized rats pretreated with carbenoxolone. Carbenoxolone increased the concentration of plasma [3H]aldosterone, but the uptake of [3H]aldosterone into the kidney nuclear and cytosol fractions was decreased. Pretreatment of rats with carbenoxolone at low doses of aldosterone (0·4 × 10−10 and 1·5 × 10−10 mol) decreased both the amount of [3H]aldosterone bound to the 50% (NH4)2SO4 precipitate, and that present in the supernatant fraction, of a Tris–CaCl2 extract of the kidney nuclear fraction. Carbenoxolone also decreased the rate of biliary excretion of metabolites of aldosterone. When [3H]aldosterone and carbenoxolone were incubated with Tris–CaCl2 extracts of a kidney nuclear fraction in vitro, the carbenoxolone did not decrease the [3H]aldosterone bound to the 50% (NH4)2SO4 precipitate, although the [3H]aldosterone bound to the 50% (NH4)2SO4 supernatant was significantly decreased by carbenoxolone at concentrations known to occur in the kidney in vivo. These results indicate that displacement of aldosterone from binding sites, with a concomitant potential increase in the amount of hormone available to specific receptors may be part of the mechanism by which carbenoxolone potentiates aldosterone action in toad skin in vitro. However, in rat kidney, such a mechanism is unlikely to play a significant role in the manifestation by carbenoxolone of aldosterone-like effects. Data are presented to indicate that carbenoxolone may affect the normal distribution and metabolism of aldosterone, and it is possible that potentiation of aldosterone action may involve receptors in other tissues, such as the gastrointestinal tract which is the major site of action of carbenoxolone.

Cellular distribution of 125I-endothelin-1 binding in rat kidney following in vivo labeling

1994 ◽  
Vol 267 (5) ◽  
pp. F845-F852 ◽  
Author(s):  
R. Dean ◽  
J. Zhuo ◽  
D. Alcorn ◽  
D. Casley ◽  
F. A. Mendelsohn
Keyword(s):  
Cellular Localization ◽  
Rat Kidney ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Endothelin 1 ◽  
Peritubular Capillaries ◽  
Fenestrated Endothelium

Endothelin-1 (ET-1) receptors have previously been demonstrated in the rat kidney by in vitro autoradiography and in cultured renal cell lines by radioreceptor assay, but the precise cellular localization of these receptors under in vivo conditions remains to be determined. We performed electron microscopic autoradiography on rat kidney following intravenous administration of 125I-labeled ET-1. In vivo autoradiographs revealed binding patterns identical to those previously demonstrated following in vitro labeling. Light microscopic autoradiography showed that silver grains occurred exclusively overlaying glomeruli and peritubular capillaries in the cortex, inner stripe of the outer medulla, and the inner medulla. At the electron microscopic level, ET-1 binding was specifically localized to the fenestrated endothelium of glomerular and peritubular capillaries, and to a lesser extent to the vasa recta. No significant grains were seen on mesangial or visceral epithelial cells; nor were any seen on the cells of proximal tubule, the thick and thin limbs of the loop of Henle, the medullary collecting ducts, and renal interstitial cells. These results indicate that the endothelial cells of glomerular and peritubular capillaries are the primary target for the circulating ET-1 in the rat kidney and suggest an autocrine and/or paracrine function of locally synthesized ET-1 in vivo in both physiological and pathophysiological states.

scholarly journals 68Ga-Radiolabeling and Pharmacological Characterization of a Kit-Based Formulation of the Gastrin-Releasing Peptide Receptor (GRP-R) Antagonist RM2 for Convenient Preparation of [68Ga]Ga-RM2

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1160
Author(s):  
Adrien Chastel ◽  
Delphine Vimont ◽  
Stephane Claverol ◽  
Marion Zerna ◽  
Sacha Bodin ◽  
...  
Keyword(s):  
Calcium Mobilization ◽  
Pharmacological Characterization ◽  
Peptide Receptor ◽  
Gastrin Releasing Peptide ◽  
Production Route ◽  
Membrane Bound ◽  
One Year ◽  
Calcium Mobilization Assay

Background: [68Ga]Ga-RM2 is a potent Gastrin-Releasing Peptide-receptor (GRP-R) antagonist for imaging prostate cancer and breast cancer, currently under clinical evaluation in several specialized centers around the world. Targeted radionuclide therapy of GRP-R-expressing tumors is also being investigated. We here report the characteristics of a kit-based formulation of RM2 that should ease the development of GRP-R imaging and make it available to more institutions and patients. Methods: Stability of the investigated kits over one year was determined using LC/MS/MS and UV-HPLC. Direct 68Ga-radiolabeling was optimized with respect to buffer (pH), temperature, reaction time and shaking time. Conventionally prepared [68Ga]Ga-RM2 using an automated synthesizer was used as a comparator. Finally, the [68Ga]Ga-RM2 product was assessed with regards to hydrophilicity, affinity, internalization, membrane bound fraction, calcium mobilization assay and efflux, which is a valuable addition to the in vivo literature. Results: The kit-based formulation, kept between 2 °C and 8 °C, was stable for over one year. Using acetate buffer pH 3.0 in 2.5–5.1 mL total volume, heating at 100 °C during 10 min and cooling down for 5 min, the [68Ga]Ga-RM2 produced by kit complies with the requirements of the European Pharmacopoeia. Compared with the module production route, the [68Ga]Ga-RM2 produced by kit was faster, displayed higher yields, higher volumetric activity and was devoid of ethanol. In in vitro evaluations, the [68Ga]Ga-RM2 displayed sub-nanomolar affinity (Kd = 0.25 ± 0.19 nM), receptor specific and time dependent membrane-bound fraction of 42.0 ± 5.1% at 60 min and GRP-R mediated internalization of 24.4 ± 4.3% at 30 min. The [natGa]Ga-RM2 was ineffective in stimulating intracellular calcium mobilization. Finally, the efflux of the internalized activity was 64.3 ± 6.5% at 5 min. Conclusion: The kit-based formulation of RM2 is suitable to disseminate GRP-R imaging and therapy to distant hospitals without complex radiochemistry equipment.

scholarly journals A Biochemical and Pharmacological Characterization of Phospholipase A2 and Metalloproteinase Fractions from Eastern Russell’s Viper (Daboia siamensis) Venom: Two Major Components Associated with Acute Kidney Injury

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 521
Author(s):  
Janeyuth Chaisakul ◽  
Orawan Khow ◽  
Kulachet Wiwatwarayos ◽  
Muhamad Rusdi Ahmad Rusmili ◽  
Watcharamon Prasert ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Phospholipase A2 ◽  
Protein Identification ◽  
Clinical Manifestations ◽  
Kidney Injury ◽  
Factor X ◽  
Pharmacological Characterization ◽  
Russell’S Viper

Acute kidney injury (AKI) following Eastern Russell’s viper (Daboia siamensis) envenoming is a significant symptom in systemically envenomed victims. A number of venom components have been identified as causing the nephrotoxicity which leads to AKI. However, the precise mechanism of nephrotoxicity caused by these toxins is still unclear. In the present study, we purified two proteins from D. siamensis venom, namely RvPLA2 and RvMP. Protein identification using LCMS/MS confirmed the identity of RvPLA2 to be snake venom phospholipase A2 (SVPLA2) from Thai D. siamensis venom, whereas RvMP exhibited the presence of a factor X activator with two subunits. In vitro and in vivo pharmacological studies demonstrated myotoxicity and histopathological changes of kidney, heart, and spleen. RvPLA2 (3–10 µg/mL) caused inhibition of direct twitches of the chick biventer cervicis muscle preparation. After administration of RvPLA2 or RvMP (300 µg/kg, i.p.) for 24 h, diffuse glomerular congestion and tubular injury with minor loss of brush border were detected in envenomed mice. RvPLA2 and RvMP (300 µg/kg; i.p.) also induced congestion and tissue inflammation of heart muscle as well as diffuse congestion of mouse spleen. This study showed the significant roles of PLA2 and SVMP in snake bite envenoming caused by Thai D. siamensis and their similarities with observed clinical manifestations in envenomed victims. This study also indicated that there is a need to reevaluate the current treatment strategies for Thai D. siamensis envenoming, given the potential for irreversible nephrotoxicity.

scholarly journals Synaptopodin: An Actin-associated Protein in Telencephalic Dendrites and Renal Podocytes

1997 ◽  
Vol 139 (1) ◽  
pp. 193-204 ◽  
Author(s):  
Peter Mundel ◽  
Hans W. Heid ◽  
Thomas M. Mundel ◽  
Meike Krüger ◽  
Jochen Reiser ◽  
...  
Keyword(s):  
Dendritic Spines ◽  
Cultured Cells ◽  
Rat Kidney ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Globular Domain ◽  
Postnatal Maturation ◽  
Human And Mouse

Synaptopodin is an actin-associated protein of differentiated podocytes that also occurs as part of the actin cytoskeleton of postsynaptic densities (PSD) and associated dendritic spines in a subpopulation of exclusively telencephalic synapses. Amino acid sequences determined in purified rat kidney and forebrain synaptopodin and derived from human and mouse brain cDNA clones show no significant homology to any known protein. In particular, synaptopodin does not contain functional domains found in receptor-clustering PSD proteins. The open reading frame of synaptopodin encodes a polypeptide with a calculated Mr of 73.7 kD (human)/74.0 kD (mouse) and an isoelectric point of 9.38 (human)/9.27 (mouse). Synaptopodin contains a high amount of proline (∼20%) equally distributed along the protein, thus virtually excluding the formation of any globular domain. Sequence comparison between human and mouse synaptopodin revealed 84% identity at the protein level. In both brain and kidney, in vivo and in vitro, synaptopodin gene expression is differentiation dependent. During postnatal maturation of rat brain, synaptopodin is first detected by Western blot analysis at day 15 and reaches maximum expression in the adult animal. The exclusive synaptopodin synthesis in the telencephalon has been confirmed by in situ hybridization, where synaptopodin mRNA is only found in perikarya of the olfactory bulb, cerebral cortex, striatum, and hippocampus, i.e., the expression is restricted to areas of high synaptic plasticity. From these results and experiments with cultured cells we conclude that synaptopodin represents a novel kind of proline-rich, actin-associated protein that may play a role in modulating actin-based shape and motility of dendritic spines and podocyte foot processes.

A technique for in vivo and in vitro studies on the preserved and transplanted rat kidney

1980 ◽  
Vol 8 (2) ◽  
pp. 107-112 ◽  
Author(s):  
Bengt Harvig ◽  
Johan Norl�n
Keyword(s):  
Rat Kidney ◽  
In Vitro Studies

Enhanced xenobiotic transporter expression in normal teleost hepatocytes: response to environmental and chemotherapeutic toxins

2001 ◽  
Vol 204 (2) ◽  
pp. 217-227
Author(s):  
J.A. Albertus ◽  
R.O. Laine
Keyword(s):  
Mammalian Cells ◽  
Cellular Localization ◽  
Fundulus Heteroclitus ◽  
Environmental Pollutants ◽  
Human Tumor ◽  
Aquatic Organisms ◽  
In Vivo Studies ◽  
Multixenobiotic Resistance

Many aquatic organisms are resistant to environmental pollutants, probably because their inherent multi-drug-resistant protein extrusion pump (pgp) can be co-opted to handle man-made pollutants. This mechanism of multixenobiotic resistance is similar to the mechanism of multidrug resistance exhibited in chemotherapy-resistant human tumor cells. In the present study, a variety of techniques were used to characterize this toxin defense system in killifish (Fundulus heteroclitus) hepatocytes. The cellular localization and activity of the putative drug efflux system were evaluated. In addition, in vitro and in vivo studies were used to examine the range of expression of this putative drug transporter in the presence of environmental and chemotherapeutic toxins. The broad range of pgp expression generally observed in transformed mammalian cells was found in normal cells of our teleost model. Our findings suggest that the expression of the pgp gene in the killifish could be an excellent indicator of toxin levels or stressors in the environment.

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