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Query: UNIPROT:P01185 (vasopressin)
 23,126 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Proximal tubules were prepared from rat kidney cortex by collagenase digestion and purified by Percoll gradient centrifugation. Their enrichment was estimated by comparing the specific activities of various cell-specific enzymes in homogenates of renal cortex and of the isolated tubules. The tubules were cultured in a 50:50 mixture of Dulbecco's modified Eagle's and Ham's F12 media supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, and prostaglandin E1. After 2 to 3 d an extensive outgrowth of epithelial cells developed from the attached tubules. After 5 to 7 d near confluent monolayers were obtained. Hormonal responsiveness, marker enzyme activities, and transport properties were determined to further characterize the primary cultures. The cultured cells exhibited increased cyclic AMP production in response to parathyroid hormone but not calcitonin or vasopressin, consistent with the absence of cells derived from distal and collecting tubules. The cells also retained significant levels of 25-hydroxyvitamin D3-1 alpha-hydroxylase, alkaline phosphatase, and gamma-glytamyl-transpeptidase, three enzymes that are primarily associated with the proximal tubule. The cultured epithelial cells also exhibit a Na+-dependent phosphate and glucose transport systems. Therefore, the cells retain many functional properties that are characteristic of proximal tubules. Thus, the primary cultures should be suitable for the study of processes that occur specifically within this segment of the rat nephron.
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PMID:Characterization of primary cell cultures derived from rat renal proximal tubules. 254 89

Prostaglandin (PG) inhibits the hydroosmotic effect of vasopressin. We therefore reexamined the interaction of vasopressin (VP), cAMP, and prostaglandins in toad bladder epithelial cells. Vasopressin slightly, but reproducibly, stimulated PGE2 and thromboxane B2 (TXB2) synthesis in cells prepared by the use of collagenase. When cells were prepared in the presence of a readily reversible cyclooxygenase inhibitor, ibuprofen, subsequent PGE2 synthesis was enhanced sevenfold but that of TXB2 was not. Increasing cAMP by either phosphodiesterase inhibition or 8-bromo-cAMP significantly inhibited both basal and VP-stimulated PGE2 synthesis. This inhibition was overcome by addition of arachidonic acid. Future studies employing these agents will have to consider these effects. VP enhanced 32P labeling of phosphatidylinositol (PI) and phosphatidic acid. This effect was prevented by the phosphodiesterase inhibitor, which also decreased phosphatidylcholine labeling. The results indicate that the phosphodiesterase inhibitor for cAMP may decrease PG formation by interfering with phospholipase activation. Furthermore, VP, similar to its effect in the liver, also increases PI turnover in toad bladder. This may initiate PG synthesis and provide a link among VP, cAMP, and calcium. A double-reciprocal feedback is proposed, whereby VP stimulates PG synthesis in a cAMP-independent manner and also inhibits PG synthesis in a cAMP-dependent manner.
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PMID:Interactions of vasopressin, cAMP, and prostaglandins in toad urinary bladder. 257 84

A series of experiments using isolated rat hepatocytes was carried out to establish rat liver cells in suspension as a physiological model for examining GH responses, and to determine whether acute recombinant bovine GH (rbGH) treatment of rat liver cells increased glucose output and/or suppressed fatty acid synthesis from lactate. Rat liver cells were isolated by collagenase perfusion and incubated in short-term (less than 60 min) suspension. The amount of insulin, glucagon or vasopressin required to elicit a half-maximal response was within the physiological range of the circulating hormone. When hepatocytes from normal rats were acutely (less than 60 min) treated with 0, 0.1, 10, 100 or 1000 nmol rbGH/l, rates of hepatocyte glucose output and fatty acid synthesis were unaltered. In addition, acute rbGH treatment (1000 nmol/l) did not alter hepatocyte responsiveness to insulin or vasopressin. However, acute rbGH treatment of hepatocytes isolated from hypophysectomized rats significantly (P less than 0.05) increased the rate of glucose output twofold and moderately (P less than 0.10) enhanced fatty acid synthesis. The accelerated rate of glucose production was not accompanied by an increase in the amount of glycogen phosphorylase-a. The observations with liver cells from hypophysectomized rats are not consistent with a GH receptor-transducing mechanism which is like that for glucagon (adenylate cyclase-linked) or insulin (tyrosine kinase-linked).
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PMID:Growth hormone acutely increases glucose output by hepatocytes isolated from hypophysectomized rats. 267 Dec 41

Two types of mitochondria-rich (MR) cells have been identified in the rabbit collecting tubule based on differences in immuno- and lectin cytochemistry. We have produced a monoclonal antibody, immunoglobulin (Ig) G1 (mr-mct), that reacts specifically with the MR cells (identified by positive histochemical staining for succinate dehydrogenase) found predominantly in the outer medulla (OM) and cells of the proximal tubule. IgG1 (mr-mct) reacted with 18 +/- 2% of the cells of the outer medullary collecting tubule (OMCT) and did not colocalize with peanut lectin-binding MR cells in the cortex. To isolate MR-OMCT cells, collecting tubule cells from collagenase dispersions of the OM were first adsorbed onto plates treated with a monoclonal antibody reactive against all of the OMCT cells. Of the isolated OMCT cells, 17% reacted with IgG1 (mr-mct). Cells were then detached from the plate and transferred to plates coated with IgG1. Greater than 70% of the adsorbed cells were MR as determined by positive staining with IgG1 (mr-mct). This enrichment of MR-OMCT cells was associated with a severalfold increase in adenosine 3',5' cyclic monophosphate (cAMP) production in response to isoproterenol and an attenuated increase in cAMP production to vasopressin. In summary, we report the isolation of highly enriched populations of MR cells from the OM using two-stage solid-phase immunoadsorption. This approach should provide a useful and convenient method for further investigations of the physiological role of these poorly understood tubular cells.
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PMID:Immunodissection of mitochondria-rich cells from rabbit outer medullary collecting tubule. 283 10

We have previously shown that arginine vasopressin (AVP) directly inhibits testicular steroidogenesis in vitro. In the present study, binding of neurohypophysial peptides to interstitial cells of the rat testis was studied using [3H]AVP as the ligand. Interstitial cells were obtained from adult rat testis after collagenase dispersion and were incubated with [3H]AVP in the presence or absence of unlabeled AVP. Binding equilibrium was reached by 60 min at 4 C, while incubation at higher temperatures (23 and 37 C) resulted in an apparent decrease in binding. Scatchard plot analysis of equilibrium binding data revealed the existence of one class of high affinity, low capacity binding sites (Kd = 1.0 +/- 0.3 nM; maximal binding = 8.5 fmol/10(6) cells). In addition, the rate constants of association and dissociation were calculated to be 0.024 nM-1 min-1 and 0.009 min-1, respectively. Addition of naturally occurring neurohypophysial hormones as well as their synthetic analogs inhibited [3H]AVP binding to testis cells, resulting in parallel displacement curves. The order of potencies for the native peptides was: AVP = lysine vasopressin = arginine vasotocin (IC50, 5 X 10(-10) M) greater than oxytocin = mesotocin (IC50, 4 X 10(-7) M) greater than isotocin = glumitocin (IC50 greater than 10(-6) M). Furthermore, two potent vasopressor antagonists, d(CH2)5Tyr(Me)AVP ([1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine]AVP) and dPTyr(Me)AVP ([1-deaminopenicillamine-2-(O-methyl)tyrosine]AVP) competed for [3H]AVP binding with a higher affinity (IC50, approximately 10(-11) M) than native AVP. In contrast, a selective antidiuretic agonist, dDAVP (1-deamino-8-D-AVP), only competed weakly for receptor binding, while a specific oxytocic agonist, (Thr4,Gly7)oxytocin, did not affect AVP binding. These results suggested that the testis may contain the V1 receptor subtype. Studies on the intratesticular distribution of AVP receptors indicated minimal binding to cells derived from the seminiferous tubule, while most of the AVP-binding sites sediment with enriched fractions of Leydig cells after Metrizamide density gradient centrifugation. AVP-binding sites were also found in rat liver, kidney, and anterior pituitary (10.7, 2.6, and 1.7 fmol/mg protein), whereas adrenal, cerebellum, prostate, and hypothalamus were devoid of AVP-binding sites. Thus, we have demonstrated the presence of high affinity, stereospecific receptors for AVP in the interstitial cell compartment of the rat testis. These V1 receptors may mediate the direct inhibitory action of neurohypophysial hormones on testicular Leydig cell steroidogenesis.
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PMID:Identification and characterization of arginine vasopressin receptors in the rat testis. 298 Oct 73

This paper describes a rapid and simple method for isolation of medullary thick ascending limbs (MTAL) from rat kidney. The technique takes advantage of the fact that MTAL represents a high fraction of the inner stripe (IS) tissue in the outer medulla, and that this nephron segment is more resistant than others to mechanical and enzymatic disruption. Special attention was given in the design of each step of the isolation procedure in order to improve purity and yield of the preparation. Major steps are the following: careful dissection of the IS; cutting IS tissue into small pieces of regular size (approximately equal to 1 mm3); mild and brief enzymatic hydrolysis in a 65 U/ml collagenase solution; separation of long MTAL segments from other tubule fragments and cells, and washing of the collagenase solution, on a nylon sieve (100 microns opening). This technique does not require lengthy centrifugations and provides about 6 mg fresh tissue (= 1 mg protein) from two rat kidneys in 2 h. Light microscopy and transmission electron microscopy show a good purity (at least 95%) and good preservation of TAL ultrastructural morphology. Adenylate cyclase responsiveness to arginine-vasopressin (AVP), glucagon (GLU) and salmon calcitonin (SCT) of the MTAL suspension is similar to that reported for single microdissected rat MTAL. Viability of the MTALs was demonstrated by the ability to accumulate cyclic AMP in presence of AVP, GLU, SCT and forskolin. Normal oxygen consumption was 45.1 +/- 2.4 (SEM) microliter . mg protein-1 . h-1 (n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Quick isolation of rat medullary thick ascending limbs. Enzymatic and metabolic characterization. 301 64

The effects of osmolality on prostaglandin E2 (PGE2) biosynthetic capacity and the interaction between endogenous PGE2 synthesis and vasopressin (AVP)-dependent cyclic AMP generation were examined in papillary collecting ducts (PCD) microdissected from collagenase-digested rat kidneys. Increasing medium osmolality with NaCl:urea (1:2 molar ratio) progressively increased PGE2 synthesis in PCD up to 1,500 mOsm. Addition of NaCl:urea or NaCl alone were equally effective in stimulating PGE2 biosynthetic capacity in PCD. In contrast, addition of urea alone had a much smaller stimulatory effect on PGE2 synthesis. Inhibition of endogenous PGE2 synthesis with naproxen (10(-5)M) suppressed AVP-dependent cAMP formation in PCD when incubated in 300 mOsm medium but had no effect when incubated in 1,500 mOsm medium. Addition of 2.5 X 10(-5) M PGE2 also suppressed AVP-dependent cAMP formation in PCD only when incubated in 300 mOsm medium. The present study suggests that the PCD is a site of active PGE2 synthesis that is modulated by osmolality. Our results do not support the concept that endogenous PGE2 antagonizes vasopressin action via inhibition of AVP-dependent cAMP formation.
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PMID:Prostaglandin E2 synthesis in the inner medullary collecting duct of the rat: implications for vasopressin-dependent cyclic AMP formation. 302 64

Na-K-ATPase activity was measured in individual pieces of nephron microdissected from collagenase-treated kidneys of jerboas, Jaculus orientalis. Na-K-ATPase activity was high in the distal convoluted tubule, intermediate in the thick ascending limb of the loop of Henle and low in the proximal and collecting tubule. When jerboas were adapted for several weeks to a hydrated diet and excreted a more diluted urine, Na-K-ATPase activity was altered in specific segments of the nephron: 1. In the proximal convoluted tubule, Na-K-ATPase activity decreased, especially in the juxtamedullary nephrons, suggesting that internephron heterogeneity was diminished; 2. In the medullary thick ascending limb, but not in the cortical portion, Na-K-ATPase activity decreased by 30%; 3. Na-K-ATPase was also diminished in the cortical collecting tubules (by 20%) but not in the medullary collecting tubule. Morphometric measurements also indicate that changes in Na-K-ATPase activity observed in the thick ascending limb are correlated to a cell atrophy, whereas in the collecting tubule, they occur independently of any visible morphological alteration. These differences in Na-K-ATPase activity are likely to be secondary to the changes in the plasma concentration of vasopressin previously described during such adaptation and to be involved in the control of water and sodium handling.
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PMID:Effect of water intake on Na-K-ATPase in nephron segments of the desert rodent, Jaculus orientalis. 303 78

We modified and improved enzyme digestion and density gradient separation procedures to obtain fractions of proximal and distal renal tubules with high yield and viability. Kidneys from two anesthetized adult Wistar rats were flushed with Krebs-Henseleit buffer (KHB) and then perfused in situ with recirculated KHB containing collagenase and hyaluronidase at 125 mmHg. Cortices were excised, minced, and incubated in KHB containing enzymes for 35 min at 37 degrees C. Dissociated tubules were removed at 10-min intervals, rinsed, and placed in KHB containing 10% calf serum, vitamins, and amino acids at 4 degrees C. Separation was achieved by suspending the tissue in 45% isosmotic Percoll layered over an undiluted Percoll cushion and centrifuging. Proximal tubules sedimented near the cushion. Distal segments were isolated in the uppermost bands of a second 35% Percoll separation. Viability was greater than 95% as measured by lactate dehydrogenase leakage and quantitated by oxygen consumption and ATP content. Basal oxygen consumption was greater than 33 nmol O2 X min-1 X mg protein-1 in all fractions and was stimulated by succinate and inhibited by amiloride and ouabain. Basal ATP content averaged 9.7 nmol/mg ATP. An average 3.3-fold separation for the proximal fraction and 24.5-fold separation for the distal fraction was assessed by the enrichment of six specific enzyme markers, with several of the markers indicating separations up to 32-fold. Isolated tubules also displayed functional responses to parathyroid hormone and vasopressin. Distal, but not proximal, segments demonstrated significantly increased adenosine 3',5'-cyclic monophosphate formation with vasopressin.
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PMID:Improved separation method for rat proximal and distal renal tubules. 303 59

Nerve terminals were isolated from sheep posterior pituitary lobes using a collagenase digestion technique. Freshly dispersed terminals, and terminals cultured for up to 3 days were examined by immunofluorescence microscopy. Immunohistochemical results suggested the presence of immunoreactive-neurophysin, -oxytocin and -vasopressin in membrane-bound structures. Membrane depolarization induced by high concentrations of potassium ions stimulated oxytocin release. This release was attenuated in the absence of calcium ions. The calcium ionophore, A23187, was also an effective stimulator of oxytocin secretion. These data suggest that neurohypophysial nerve terminals prepared by a collagenase dispersion procedure may be suitable for the investigation of posterior pituitary secretion mechanisms.
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PMID:Isolation and properties of sheep neurohypophysial nerve terminals. 309 Apr 71


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