Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.6.1.1 (adenylate cyclase)
 19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method is described, which allows adenylate cyclase activity measurement in single pieces of various nephron segments. Tubular samples of 0.5 to 2 mm length were isolated by microdissection from collagenase treated slices of rabbit kidney. A photograph of each piece was taken in order to measure its length. After a permeabilisation treatment involving preincubation in a hypoosmotic medium and a freezing step, each sample was incubated for 30 mm at 30 degrees C in a medium containing high specific (alpha-32-P)-ATP 3-10-4 M, final volume 2.5 mu 1. The (32P)-cAMP formed was separated from the other labelled nucleotides by filtering the incubate on a dry aluminium oxide microcolumn, 3H cAMP was added as a tracer for measuring cAMP recovery. The sensitivity of the method was found to be a few fentomoles (10-15 M) cAMP. cAMP generation increased linearly as a function of the incubation time up to more than 30 min, and as a function of the length of the segment used. Control and fluoride (5 mM) stimulated adenvlate cyclase activities were measured in the following segments of the nephron: early proximal convoluted tubule (PCT), pars recta of the proximal tubule (PR), thin descending limb of the loop (TDL), cortical portion of the thick ascending limb (CAL), distal convoluted tubule (dct), first branched portion of the collecting tubule (BCT), further cortical (CCT) and medullary (MCT) portions of the collecting tubule. Mean control adenylate cyclase activity varied from 7 (PR) to 75 (BCT) fmoles/mm/30 min. Flouride addition resulted in a 10 (BCT) to 50 (PR) fold increase in enzyme activity. Series of replicates gave a scatter equal to plus or minus 20% (S.D. as a per cent of the mean). The method described appears to be suitable to determine which nephron segments contain hormone-dependent adenylate cyclase.
 ...
PMID:Adenylate cyclase activity along the rabbit nephron as measured in single isolated segments. 16 67

PTH sensitive adenylate cyclase activity was measured in 9 different segments of the nephron, isolated by microdissection from collagenase-treated rabbit kidney slices. The enzyme of the following segments was stimulated by PTH, 1 U/ml: PCT. (proximal convoluted tubule); PR (pars recta); CAL (cortical portion of the thick ascending limb); DCT (distal convoluted tubule); BCT (first, branched portion of the collecting tubule); the segments which did not respond to PTH were: TDL (thin descending limb): MAL (medullary portion of the thick ascending limb); CCT (cortical portion of the collecting tubule distally adjacent to BCT); MCT (collecting tubule from the outer medulla). PTH sensitive adenylate cyclase per mm tubule in PR was half that measured in PCT. Half maximal stimulation corresponded to 50-100mm U/ml PTH (1-2 times 10-8M) in both PCT and PR, and to about 350 mm U/ml in CAL. PTH (1 U/ml) stimulation factors ranged from 5 to 60 depending on the structures. It is concluded that in addition to PCT and PR, CAL and BCT might be target structures involved in the physiological actions of PTH on the kidney.
 ...
PMID:PTH sensitive adenyl cyclase activity in different segments of the rabbit nephron. 16 68

AVP dependent adenylate cyclase activity was measured in single pieces of 8 different tubular segments isolated from collagenase treated rabbit kidneys. High responses were observed in all the tested portions of the collecting tubule, that is its cortical branched part (BCT), its cortical straight part (CCT) and its outer medullary part (MCT). Dose response curves indicated in CCT: 2 fold threshold stimulation at 10(-11) M AVP, 27 fold stimulation at 10(-6) M AVP, half maximal stimulation at about 10(-9) M AVP. Both the medullary (MAL) and, to a lesser extent, the cortical (CAL) portions of the thick ascending limb were also observed to contain AVP sensitive adenylate cyclase (for MAL: 2 fold threshold stimulation at 10(-9) M AVP, 9 fold stimulation at 10(-7) M AVP, half maximal stimulation at 5 X 10(-9) M AVP). In contrast, nearly no responsiveness to AVP was observed in the proximal convoluted tubule, in the thin descending limb of the loop and in the distal convoluted tubule (DCT). The limited response obtained in DCT (which is a structure generally considered as a target site for AVP) as well as the clearcut effect elicited by AVP in MAL (the functioning of which is not known to be controlled by ADH) were expected observations; their possible physiological implications will be discussed.
 ...
PMID:Vasopressin dependent adenylate cyclase in single segments of rabbit kidney tubule. 17 59

A functional role for the numerically predominant renal alpha2-adrenoceptors, which in other tissues inhibit adenylate cyclase, remains undefined. We therefore examined the effect of alpha2-adrenoceptor stimulation with (-)-epinephrine (E) on cell cAMP content in the isolated proximal convoluted tubule (PCT), medullary and cortical thick ascending limb of Henle, and collecting tubule (MTAL, CTAL, MCT, and CCT, respectively). Parathyroid hormone (1-34 PTH), in PCT or CTAL, or arginine vasopressin (AVP), in MTAL, CTAL, MCT, or CCT, was used to activate adenylate cyclase in intact cells from these microdissected nephron segments in the presence of 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor) and propranolol. Alpha2-Adrenoceptors were activated using varying concentrations of E (37 degrees C, 2 min). Alpha2-Adrenoceptor activation with E (5 X 10(-7) to 5 X 10(-6) M) suppressed cellular cAMP stimulation by PTH by 35% in PCT and stimulation by AVP in CCT by 50%. This suppression by E in PCT and CCT was inhibited by 5 X 10(-6) M yohimbine or 5 X 10(-7) M phentolamine but not by 5 X 10(-6) M prazosin. E also suppressed cAMP stimulated by AVP in MCT, but it did not suppress the PTH-or AVP-stimulated increase in cellular cAMP in CTAL and MTAL. These studies show that there are alpha2-adrenoceptors in the rat nephron. Activation of these alpha 2-adrenoceptors can inhibit cAMP formation stimulated by PTH in PCT and by AVP in the CCT and MCT but not in the CTAL and MTAL. A pathophysiological role of altered regulation of these receptors is yet to be described.
 ...
PMID:Alpha2-adrenoceptors and cellular cAMP levels in single nephron segments from the rat. 299 Feb 39

A calcitonin analogue, MCT-II, having the potential to form an amphiphilic alpha-helix from residue 8 to residue 22 with a continuous surface of aliphatic leucine side chains on the hydrophobic face of the helix has been synthesized, and its physical and biological properties have been characterized. Properties exhibited by this peptide, including self-association in the micromolar concentration range with a concomitant increase in the percentage of alpha-helical structure, formation of stable monolayers at the air-water interface, and adsorption to the surface of egg lecithin single-bilayer vesicles, demonstrate that MCT-II can readily form an amphiphilic alpha-helical structure. Though MCT-II has minimal sequence homology to any particular natural analogue from residue 8 to residue 22, it has biological activity similar to that of salmon calcitonin I for receptor binding in brain and kidney membranes, for activation of adenylate cyclase, and in hypocalcemic potency in vivo. The amphiphilic alpha-helical structure of MCT-II, therefore, is important for binding to calcitonin receptors. It is also apparent that a hydrophilic residue commonly occurring on the hydrophobic face (position 15) in the natural calcitonins is not required for high biological activity.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Design, synthesis, and characterization of a model peptide having potent calcitonin-like biological activity: implications for calcitonin structure/activity. 299 May 46

To further gain insights into the mechanisms underlying impaired urine concentration in hypercalcemia, effects of increasing Ca2+ concentrations in the incubation medium on cAMP production in response to 10(-8) M arginine vasopression (AVP) were examined in thick ascending limbs of Henle (MTAL) and collecting tubules (MCT) dissected from outer medulla of mouse kidney. Increasing Ca2+ in the incubation medium from 1.0 mM to either 2.0 mM or 5.0 mM inhibited AVP-dependent cAMP production in MTAL but not in MCT. This inhibition of AVP-dependent cAMP production by 2.0 mM Ca2+ in MTAL was not reversed by verapamil or diltiazem. Also, Ca2+ ionophore A23187 did not inhibit AVP-dependent cAMP production in MTAL in the presence of 1.0 mM Ca2+. Increasing medium Ca2+ from 1.0 to 5.0 mM inhibited cAMP production in MTAL in response to both glucagon and forskolin by the magnitude comparable to that seen in response to AVP. These results show that high Ca2+ inhibits AVP-dependent cAMP production only in MTAL and not in MCT. In addition, the lack of effects of Ca2+ channel blockers and Ca2+ ionophore suggests that high ambient Ca2+ per se may inhibit AVP-dependent cAMP production in MTAL. The fact that high Ca2+ also suppressed cAMP production in response to glucagon or forskolin suggests that Ca2+ may inhibit AVP-dependent adenylate cyclase at postreceptor site(s), one of which is the catalytic unit of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:High Ca2+ inhibits AVP-dependent cAMP production in thick ascending limbs of Henle. 301 Jul 37

Interactions between AVP and prostaglandins were investigated in MAL and MCT microdissected from the rat outer medulla. Incubation of MCT with 14C-arachidonic acid resulted in the formation of 14C-PGE2 and 14C-PGF2 alpha; however, when MAL was incubated under the same conditions, only traces of prostaglandins were formed. Prostaglandin synthesis in MCT was inhibited (-50%) by the prostaglandin cyclo-oxygenase inhibitor ibuprofen (10(-6)M). Preincubation with ibuprofen enhanced the stimulation of adenylate cyclase by 5 x 10(-9)M AVP in MCT but, in contrast, decreased the stimulation of adenylate cyclase by AVP in MAL. The effects of a second PG cyclo-oxygenase inhibitor naproxen (10(-5)M) were similar to those of ibuprofen. Ibuprofen did not influence cAMP phosphodiesterase activity in MCT or in MAL. Exogenous PGE2 or PGF2 alpha (10(-6)M) had no effect on either basal or AVP-stimulated adenylate cyclase activity in MCT. The present results demonstrated that MCT but not MAL is a site of active synthesis and accumulation of prostaglandin. Although both MAL and MCT have AVP-sensitive adenylate cyclase, incubation with prostaglandin cyclo-oxygenase inhibitors have, in the presence of arachidonic acid, an opposite effect on this enzyme in these two segments, resulting in increased AVP stimulation in MCT and decreased stimulation in MAL. Results also suggest that products of prostaglandin synthesis from arachidonic acid inhibit AVP-sensitive adenylate cyclase activity in MCT but not that located in MAL. Although not totally excluding the primary prostaglandins (PGE2, PGF2 alpha), these observations suggest that they are not responsible for AVP modulation in MCT.
 ...
PMID:Vasopressin-prostaglandin interactions in isolated tubules from rat outer medulla. 624 5

The effects of PDN on VP-sensitive cAMP metabolism were examined in MCT and MAL microdissected from the rat kidney. VP-sensitive adenylate cyclase activity was significantly reduced (delta -46%; p less than 0.05) in MAL of PDN rats but, in sharp contrast, was significantly increased (delta +79%; p less than 0.02) in MCT of PDN rats compared to controls. cAMP phosphodiesterase activity was significantly increased in both MAL (delta +59%; p less than 0.005) and MCT (delta +79%; p less than 0.001) of PDN rats compared to controls. The increase in cAMP accumulation in MAL measured in response to VP in intact tubules did not differ between PDN and controls, whereas cAMP accumulation in response to VP was significantly higher (delta +127%; p less than 0.001) in MCT of PDN rats compared to controls. The present results would indicate that the observed in vivo resistance to the antidiuretic effect of VP that occurs in PDN is not due to an impairment in VP-sensitive cAMP accumulation in MCT, but would rather suggest that a defect exists at a cellular step subsequent to cAMP generation. In addition, our results illustrate that the extent and directionality of in situ accumulation of cAMP measured in intact tubules cannot always be predicted from rhe activities of enzymes controlling its synthesis and degradation (adenylate cyclase and cAMP phosphodiesterase), which are measured in vitro in disrupted tubules.
 ...
PMID:Effect of potassium depletion on the vasopressin-sensitive cyclic AMP system in rat outer medullary tubules. 627 83

The present study was performed to characterize the possible involvement of cAMP synthesis and protein kinase C (PKC) activation in the DNA synthesis-stimulating effect of parathyroid hormone-related protein (PTHrP) in proximal tubule cells. We found that DNA synthesis was stimulated by 10 microM 8BrcAMP, and 1 microM Sp-cDBIMPS, two cAMP analogs, and also by 1 microM phorbol 12-myristate 13-acetate (PMA) and 100 microM 1,2-dioctanoyl-sn-glycerol, two PKC activators, and 10 nM [Cys23] human (h)PTHrP (24-35) amide in rabbit proximal tubule cells (PTC). Both Sp-cDBIMPS and PMA, at 1 microM, also increased DNA synthesis in SV40-immortalized mouse proximal tubule cells MCT. Human PTHrP (7-34) amide [PTHrP (7-34)] dose dependently stimulated DNA synthesis in a similar manner as [34Tyr]PTHrP (1-34) amide [PTHrP (1-34)], in PTC. PMA pre-treatment for 20 h, which downregulates PKC, completely blocked the effect induced by PTHrP (7-34), but not that of PTHrP (1-34), in the latter cells. In contrast, the same PMA pre-treatment abolished the DNA synthesis stimulation by PTHrP (1-34) and PTHrP (7-34) in MCT cells, which appear to have PTH receptors mainly coupled to phospholipase C and not adenylate cyclase. Our results indicate that the stimulatory effect of PTHrP on DNA synthesis in proximal tubule cells is mediated by a cAMP- and PKC-dependent mechanism.
 ...
PMID:Parathyroid hormone-related protein increases DNA synthesis in proximal tubule cells by cyclic AMP- and protein kinase C-dependent pathways. 965 Nov 15