1,25-Dihydroxy-19-nor-vitamin D
2
(19-norD
2
), a new analog of 1,25(OH)
2
D
3
, suppresses parathyroid hormone in renal failure patients and in uremic rats but has less calcemic activity than ...1,25(OH)
2
D
3
. Although 19-norD
2
has high affinity for the vitamin D receptor and similar pharmacokinetics to those of 1,25(OH)
2
D
3
, it has much less bone resorbing activity
in vivo
. The intrinsic activity of 19-norD
2
on osteoclastogenesis and activation of bone resorption in mouse bone marrow cultures was examined to determine the mechanism involved. 19-norD
2
and 1,25(OH)
2
D
3
(10 nM) were equivalent in stimulating the formation and maintenance of large multinucleated, tartrate-resistant acid phosphatase-positive cells. However, the amount of bone resorbed by osteoclasts stimulated by 10 nM 19-norD
2
, as measured by pit-forming assays, was reduced 62% compared with 10 nM 1,25(OH)
2
D
3
-stimulated osteoclasts (
P
< 0.05). This difference could not be attributed to enhanced catabolism or to downregulated vitamin D receptor. The rate of degradation of 19-norD
2
in cultures was approximately 20% greater than 1,25(OH)
2
D
3
, not enough to account for the different effects on bone resorption. The VDR levels were identical in cultures that were treated with 19-norD
2
and 1,25(OH)
2
D
3
. In summary, 19-norD
2
is less effective than 1,25(OH)
2
D
3
in stimulating mouse marrow osteoclasts to resorb bone. The reason for this difference is not clear but seems to involve the late maturation and/or activation of osteoclasts as the number of pits produced by each tartrate-resistant acid phosphatase-positive cell is reduced under stimulation by 19-norD
2
compared with 1,25(OH)
2
D
3
.
Regulation of AE1 anion exchanger and H+-ATPase in rat cortex by acute metabolic acidosis and alkalosis. The cortical collecting duct (CCD) mediates net secretion or reabsorption of protons according ...to systemic acid/base status. Using indirect immunofluorescence, we examined the localization and abundance of the vacuolar H+-ATPase and the AE1 anion exchanger in intercalated cells (IC) of rat kidney connecting segment (CNT) and CCD during acute (6 hr) metabolic (NH4Cl) acidosis and respiratory (NaHCO3) alkalosis. AE1 immunostaining intensity quantified by confocal microscopy was elevated in metabolic acidosis and substantially reduced in metabolic alkalosis. AE1 immunostaining was restricted to Type A IC in all conditions, and the fraction of AE1+ IC was unchanged in CNT and CCD. Metabolic acidosis was accompanied by redistribution of H+-ATPase immunostaining towards the apical surface of IC, and metabolic alkalosis was accompanied by H+-ATPase redistribution towards the basal surface of IC. Therefore, acute metabolic acidosis produced changes consistent with increased activity of Type A IC and decreased activity of Type B IC, whereas acute metabolic alkalosis produced changes corresponding to increased activity of Type B IC and decreased activity of Type A IC. These data demonstrate that acute systemic acidosis and alkalosis modulate the cellular distribution of two key transporters involved in proton secretion in the distal nephron.
Vacuolar H+ ATPases participate in renal hydrogen ion secretion in both the proximal and distal nephron. These plasma membrane forms of the vacuolar H+ ATPase are regulated physiologically to ...maintain the acid-base balance of the organism. Proton transporting renal cells have requirements for constitutive acidification of intracellular compartments for normal endocytic and secretory functions. Recent experiments have begun to reveal how the kidney regulates these proton pumps independently. Vacuolar H+ ATPases are a family of structurally similar enzyme which differ in the composition of specific subunits. Cytosolic regulatory enzymes are present in renal cells which may affect vacuolar H+ ATPases in certain membrane compartments selectively. The vacuolar H+ ATPase in the plasma membrane of intercalated cells resides in a specialized proton-transporting apparatus that translocates the enzyme between an intracellular membrane pool and the plasma membrane in response to physiologic stimuli.
Vacuolar H+-ATPase in Ocular Ciliary Epithelium Wax, Martin B.; Saito, Isao; Tenkova, Tatyana ...
Proceedings of the National Academy of Sciences - PNAS,
06/1997, Letnik:
94, Številka:
13
Journal Article
Recenzirano
Odprti dostop
The mechanisms controlling the production of aqueous humor and the regulation of intraocular pressure are poorly understood. Here, we provide evidence that a vacuolar H+-ATPase (V-ATPase) in the ...ocular ciliary epithelium is a key component of this process. In intracellular pH (pHi) measurements of isolated ciliary epithelium performed with 2′,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF), the selective V-ATPase inhibitor bafilomycin A1 slowed the recovery of pHi in response to acute intracellular acidification, demonstrating the presence of V-ATPase in the plasma membrane. In isolated rabbit ciliary body preparations examined under voltage-clamped conditions, bafilomycin A1 produced a concentration-dependent decrease in short-circuit current, and topical application of bafilomycin A1 reduced intraocular pressure in rabbits, indicating an essential role of the V-ATPase in ciliary epithelial ion transport. Immunocytochemistry utilizing antibodies specific for the B1 isoform of the V-ATPase 56-kDa subunit revealed localization of V-ATPase in both the plasma membrane and cytoplasm of the native ciliary epithelium in both rabbit and rat eye. The regional and subcellular distribution of V-ATPase in specific regions of the ciliary process was altered profoundly by isoproterenol and phorbol esters, suggesting that change in the intracellular distribution of the enzyme is a mechanism by which drugs, hormones, and neurotransmitters modify aqueous humor production.
Most agents that regulate osteoclast bone resorption exert their effects indirectly, through the osteoblast. Nitric oxide, which stimulates soluble guanylyl cyclase, has been reported to inhibit ...osteoclast bone resorption directly, by a cGMP-independent mechanism(1). In this report, we demonstrate that C-type natriuretic peptide (CNP), an activator of membrane-bound guanylyl cyclase, stimulates bone resorption by osteoclast-containing 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated mouse bone marrow cultures. Quantitative reverse transcription polymerase chain reaction assays and anti-CNP immunocytochemistry were used to demonstrate that CNP is expressed in mouse marrow cells cultured in the presence, but not the absence, of 1,25-(OH)2D3. mRNA for guanylyl cyclase type B, the receptor for CNP, was expressed in cultures independent of 1,25-(OH)2D3. CNP (1 and 10 μM) elevated cGMP production in marrow cultures to 350 and 870%, respectively, of control values. 10 μM CNP increased osteoclast bone resorptive activity, measured by the resorption area on whale dentine wafers, or by the NH4Cl-inhibitable release of 3Hproline from radiolabeled bone chips, to 214 and 557% of control, respectively, without affecting osteoclast formation. Bone resorption by the marrow cultures was inhibited by 7F9.1, a monoclonal antibody raised against CNP, but not by control antibodies. These results indicate that CNP is a potent activator of osteoclast activity and may be a novel local regulator of bone remodeling.
To evaluate the efficacy and safety of a selective inhibitor of secretory phospholipase (sPLA2), LY333013, in the treatment of rheumatoid arthritis (RA).
Two hundred and fifty-one patients with ...active RA despite treatment with one or more disease modifying antirheumatic drugs (DMARD) received oral doses of LY333013 (50, 250, and 1000 mg) or placebo once daily for 12 weeks. Concomitant low-dose glucocorticoids (< or = 10 mg/day prednisone equivalent) were allowed. Clinical improvement was assessed using the response criteria of the American College of Rheumatology (ACR20), and safety was evaluated with respect to adverse events and laboratory test abnormalities.
The demographic characteristics of the treatment groups were similar. Dose-response relationships were found for ACR20 responses (p = 0.058) and reductions in C-reactive protein (p = 0.058) at week 1. The proportions of patients with an ACR20 response subsequently increased in all study groups including the placebo group at weeks 4 and 8, and the initial treatment benefit was lost. Adverse events were generally mild in severity and not associated with treatment.
Treatment with LY333013 for 12 weeks was well tolerated but ineffective as an adjunct to DMARD treatment of active RA.
Monocyte-macrophage differentiation was used as a model system for studying gene regulation of the human vacuolar H+-ATPase (V-ATPase). We examined mRNA levels of various V-ATPase subunits during ...differentiation of both native monocytes and the cell line THP-1, and found that transcriptional and post-transcriptional mechanisms could account for increases in cell V-ATPase content. From nuclear runoff experiments, we found that one subunit in particular, the B2 isoform (Mr = 56,000), was amplified primarily by transcriptional means. We have begun to examine the structure of the B2 subunit promoter region. Isolation and sequencing of the first exon and 5′-flanking region of this gene reveal a TATA-less promoter with a high G+C content. Primer extension and ribonuclease protection analyses indicate a single major transcriptional start site. We transfected promoter-luciferase reporter plasmids into THP-1 cells to define sequences that mediate transcriptional control during monocyte differentiation. We found that sequences downstream from the transcriptional start site were sufficient to confer increased expression during THP-1 differentiation. DNase I footprinting and sequence analysis revealed the existence of multiple AP2 and Sp1 binding sites in the 5′-untranslated and proximal coding regions.
Natriuretic peptide receptors A and B have different cellular distributions in rat kidney. The cellular distribution of guanylyl cyclase coupled natriuretic peptide receptors type A (GC-A) and type B ...(GC-B) was examined by immunocytochemistry in normal rat kidney, and compared with the distribution of the vacuolar H+-ATPase. Staining for GC-A was found in glomeruli, thin limbs of Henle's loop, cortical collecting tubule, and inner medullary collecting duct. Staining for GC-B was found in glomeruli and the same nephron sections as GC-A, with the exception of the thin limbs. In the cortical collecting tubule, GC-A was found in both principal and intercalated cells; GC-B was restricted to the apical pole of a intercalated cells. In inner medullary collecting ducts cells, GC-A was located on the basal membrane, whereas GC-B was found in the apical pole. The different pattern of polarization of natriuretic peptide receptors in the inner medulla provides a plausible basis for the different physiologic effects of atrial natriuretic factor and C-type natriuretic peptide. The results also suggest the possibility that GC-B is involved in the regulation of bicarbonate transport in the cortical collecting tubule.
The kidneys of mice (CAR2-null mice) that are genetically devoid of carbonic anhydrase type II (CAII) were screened by immunocytochemistry with antibodies that distinguish intercalated and principal ...cells. Immunofluorescent localization of the anion exchanger AE1 and of the 56-kDa subunit of the vacuolar H(+)-adenosinetriphosphatase (H(+)-ATPase) was used to identify intercalated cells, while the AQP2 water channel was used as a specific marker for principal cells of the collecting duct. The CAII deficiency of the CAR2-null mice was first confirmed by the absence of immunofluorescent staining of kidney sections exposed to an anti-CAII antibody. Cells positive for AE1 and H(+)-ATPase were common in all collecting duct regions in normal mice but were virtually absent from the inner stripe of the outer medulla and the inner medulla of CAR2-null mice. The number of positive cells was also reduced threefold in the cortical collecting duct of CAR2-null animals compared with normal mice. In parallel, the percentage of AQP2-positive cells was correspondingly increased in the collecting tubules of CAII-deficient mice, whereas the total number of cells per tubule remained unchanged. These results suggest that intercalated cells are severely depleted and are replaced by principal cells in CAII-deficient mice. Quantitative analysis and double staining showed that, in the cortex, both type A and type B intercalated cells are equally affected. Elucidation of the mechanism(s) responsible for this phenotype will be of importance in understanding the origin and development of intercalated cells in the kidney.
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