Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple method to determine adenylate cyclase activity in isolated single nephron segments is described. Segments of the proximal convoluted tubule or the cortical collecting tubule were isolated from rabbit kidney slices pretreated with collagenase. After the tubule membranes were made permeable by adding hypotonic medium and freezing-thawing, each sample was incubated at 30 degrees C for 30 min in a medium containing ATP and theophylline. Generated cAMP was succinylated and served for radioimmunoassay. Addition of the incubation medium did not interfere the radioimmunoassay. Recovery of added cAMP was 96%. In the proximal convoluted tubule, either 8 mM NaF or 1 U/ml parathyroid hormone (PTH) markedly stimulated adenylate cyclase activity, but 1 mU/ml arginine vasopressin (AVP) did not. By contrast, in the cortical collecting tubule, either 8 mM NaF or 1 mU/ML AVP markedly stimulated adenylate cyclase activity, but 1 U/ml PTH did not. These data imply that this method is sensitive enough to detect either specific or nonspecific response of adenylate cyclase activity in single nephron segments.
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PMID:A simple method to determine adenylate cyclase activity in isolated single nephron segments by radioimmunoassay for succinyl adenosine 3',5'-cyclic monophosphate. 22 39

Potential regulators of the renal Kallikrein-Kinin System are poorly defined. We have therefore examined the effect of arginine vasopressin and dopamine on the release of kallikrein and kinin from collagenase-dispersed rat and human renal cortical cells.
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PMID:Kallikrein and kinin release using superfused disaggregated cortical cells from rat and human kidney: effects of AVP and dopamine. 146 55

We have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase. Cells were plated on fibronectin-coated culture flasks at a density of 10(4) live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand 600 mOSM for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a marker for endothelial cells) and gamma-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to probe pathogenetic mechanisms of potential nephrotoxins.
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PMID:Characterization of an in vitro system of human renal papillary collecting duct cells. 216 26

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

A mouse monoclonal antibody designated IgG3(rct-30) has been prepared that reacts specifically with an antigen on the surface of all cells comprising the cortical and medullary rabbit renal collecting tubule including the arcades. Plastic culture dishes coated with IgG3(rct-30) were used to isolate collecting tubule cells from collagenase dispersions of rabbit renal cortical cells by immunoadsorption. Typically, 10(6) rabbit cortical collecting tubule (RCCT) cells were obtained from 5 g of renal cortex (2 kidneys). Initial purity was greater than 96% based on immunocytofluorescent staining with three different anti-collecting tubule antibodies. Between 20 and 30% of the RCCT cells were reactive with peanut lectin suggesting that RCCT cells are a mixture of principal and intercalated cells. Approximately 10(7) RCCT cells were obtained after 4 to 5 days in primary culture. Moreover, RCCT cells continued to proliferate after passaging with a doubling time of approximately 32 h. RCCT cells passaged once and then cultured 4-5 days were found 1) to synthesize cAMP in response to arginine vasopressin (AVP), prostaglandin E2 (PGE2), isoproterenol, and parathyroid hormone, but not calcitonin, prostaglandin D2, or prostaglandin I, and 2) to release PGE2 in response to bradykinin but not arginine vasopressin or isoproterenol. Our results indicate that cultured RCCT cells retain many of the hormonal, histochemical, and morphological properties expected for a mixture of principal and intercalated rabbit cortical collecting tubule epithelia. RCCT cells should prove useful both for studying hormonal interactions in the cortical collecting tubule and as a starting population for isolating intercalated collecting tubule epithelia.
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PMID:Immunodissection and culture of rabbit cortical collecting tubule cells. 301 26

Rat renal medullary tubular cells, prepared by collagenase dispersion and hypotonic lysis, were introduced in Teflon chambers for superfusion. Prostaglandin (PG) E2 and cyclic adenosine 5'-monophosphate (cAMP) production was measured in the effluent. Arginine vasopressin (AVP) but not 1-deamino-8-D-arginine vasopressin (dDAVP) (10(-10)-10(-6) M), induced a dose-dependent increase in PGE2 synthesis, whereas AVP and dDAVP produced a similar dose-dependent increase in cAMP synthesis. The Ca2+ ionophore A23187 (10(-6) M) stimulated PGE2 synthesis but not cAMP production. In contrast, forskolin (10(-5) M) stimulated cAMP synthesis without affecting PGE2 generation. The pressor antagonists dEt2AVP and d(CH2)5Tyr(Me)AVP (10(-5) M) completely inhibited the PGE2 response to 10(-8) M AVP, whereas d(CH2)5-D-LeuVAVP (10(-6) M), a mixed pressor-antidiuretic antagonist, inhibited incompletely. dEt2AVP did not significantly affect cAMP synthesis in response to 10(-8) M AVP, whereas d(CH2)5-D-LeuVAVP, and unexpectedly also d(CH2)5Tyr(Me)AVP, were inhibitory. dPTyr(Me)AVP (10(-7) M), a pressor antagonist, had an unexpectedly high cAMP-stimulating capacity. In Ca2+-free media containing EGTA, the PGE2 response to AVP and A23187 was inhibited. Nifedipine (10(-6) M) did not significantly inhibit the AVP-stimulated PGE2 production. Thus AVP-stimulated PGE2 synthesis is linked to a V1-receptor in renal medullary tubular cells and occurs independently to the activation of adenylate cyclase through a V2-receptor.
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PMID:Prostaglandin E2 and cyclic AMP response to vasopressin in renal medullary tubular cells. 301 60

Potassium depletion in rabbits induces a renal concentrating defect in vivo and decreased hydrosmotic response to arginine vasopressin (AVP) in isolated cortical collecting tubules (CCT) perfused in vitro. The molecular basis of the AVP resistance in potassium depletion was investigated by comparing AVP-responsive adenylate cyclase activities in CCT from potassium-depleted and control rabbits. Vasopressin-responsive enzyme activity was impaired in CCT dissected from kidneys of potassium-depleted rabbits but not when kidneys were treated with collagenase to improve microdissection conditions. Potassium depletion also depressed parathyroid hormone (PTH)-stimulated adenylate cyclase activity in proximal straight tubules (PST) dissected from untreated but not collagenase-treated kidneys. Commercially available collagenase, which also contains other proteolytic enzymes, increased AVP-sensitive adenylate cyclase activity in control CCT, and trypsin treatment of CCT dissected without collagenase abolished the decrease in AVP-sensitive activity induced by potassium depletion. Inclusion of trypsin inhibitor during collagenase treatment of kidneys lowered AVP response in CCT from potassium-depleted rabbits. These results demonstrate that potassium depletion impairs hormone-sensitive adenylate cyclase of CCT (and PST) by a protease-sensitive mechanism.
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PMID:Protease effects on adenylate cyclase in potassium-depleted rabbit kidney. 305 38

A superfusion technique was adapted to collagenase-dispersed renal medullary and cortical tubular cells to study prostaglandin (PG) synthesis in response to arginine vasopressin (AVP), angiotensin II (ANG II), bradykinin (BK), Ca2+ ionophore A23187, and to changes in osmolality. Medullary and cortical cells promptly responded to the stimuli by an increase in PGE2 and PGF2 alpha production, whereas 6-keto-PGF1 alpha was not detected. AVP and BK were active on medullary cells, and ANG II was active mainly on cortical cells. A23187 stimulated PG synthesis in both cells but predominantly in the medulla. PG synthesis was dependent on the presence of extracellular Ca2+. The Ca2+ entry blocking agents verapamil and lanthanum did not inhibit the PG response to AVP, BK, and ANG II. Thus peptide hormone-stimulated PG synthesis in renal tubular cells did not depend on Ca2+ influx through channels blocked by these agents. Hyperosmolar NaCl or mannitol stimulated PG synthesis in cortical and, more markedly, in medullary cells. Hyperosmolar urea inhibited PGE2 synthesis stimulated by peptide hormones, NaCl, and A23187 in both cell preparations. In conclusion, the superfusion of isolated tubular cells is a useful method to study the dynamic aspects of renal PG release in response to various sequentially applied stimuli.
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PMID:Dynamic response of PG synthesis to peptide hormones and osmolality in renal tubular cells. 308 19

The effect of vasoactive intestinal peptide (VIP) upon adenylate cyclase (AC) activity has been determined in defined microdissected renal tubules isolated from collagenase-treated rabbit kidneys. In the presence of 10 microM GTP, 1 microM VIP gave marked stimulations of AC over basal values in the bright portion of the distal convoluted tubule (DCTb) (10.1-fold), and in the collecting tubule isolated from the inner stripe of the outer medulla (OMCTi, 7.8-fold). Less pronounced effects of VIP were found in the medullary collecting tubule isolated from the outer stripe (2.5-fold) and in the granular portion of the distal convoluted tubule (2.0-fold). VIP stimulation of AC activity in these segments amounted to 25 to 40% of the effect elicited by other agonists (arginine vasopressin, calcitonin or parathyroid hormone) in their respective target segments. A low response to VIP was observed in the cortical thick ascending limb (1.8-fold) which represented less than 5% of the calcitonin-stimulated AC activity. In the thin descending limb VIP produced a slight and variable stimulation of AC. VIP was without effect upon AC in the convoluted and straight portions of the proximal tubule, the medullary thick ascending limb and the cortical collecting tubule. Half-maximal stimulation of AC by VIP was observed at 26 +/- 10 nM (n = 3) in OMCTi and at 19 nM (n = 2) in DCTb. Related peptides glucagon, secretin and PHI gave lower stimulations of AC compared to VIP in OMCTi. Conversely for rat OMCTi, under identical conditions, glucagon was much more effective than VIP.
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PMID:Distribution of vasoactive intestinal peptide-sensitive adenylate cyclase activity along the rabbit nephron. 317 93

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.
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PMID:Separation of renal medullary cells: isolation of cells from the thick ascending limb of Henle's loop. 625 27


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