Document Detail

Possible regulation of capacitative Ca2+ entry into colonic epithelial cells by NO and cGMP.
MedLine Citation:
PMID:  7545090     Owner:  NLM     Status:  MEDLINE    
A possible role of the nitric oxide (NO)/cGMP pathway in the regulation of Ca2+ entry into HT29/B6 human colonic epithelial cells was investigated using digital image processing of Fura-2 fluorescence and immunoblotting for nitric oxide synthase (NOS). We tested the hypothesis that Ca2+ store depletion causes increased NOS activity and [NO], which is stimulatory to Ca2+ entry by increasing guanylate cyclase (GC) and [cGMP]. Cells were incubated in 95 mM K(+)-containing solutions to depolarize the cell membrane potential and thereby exclude effects of NO and CGMP on K+ or Cl- channels, which might affect Ca2+ entry. Sodium nitroprusside (SNP, 0.5 microM and 30 microM), a NO donor, only slightly raised intracellular [Ca2+] ([Ca2+]i) in resting cells, but in 100 microM carbachol-stimulated cells the sustained, elevated Ca2+ plateau (reflecting Ca2+ entry) as well as Ba2+ entry were increased by 0.5 microM SNP, while 5, 10 or 30 microM SNP either had no effect or were inhibitory. Pretreatment of cells with the NOS inhibitor N-nitro-L-arginine (1 mM) reduced carbachol-stimulated Ca2+ entry, and simultaneous treatment with 0.5 microM (but not 30 microM) SNP restored Ca2+ influx. 8-Br-cGMP (1 mM) had little effect on [Ca2+]i or on rates of Ca2+ or Ba2+ influx into resting cells, but there were large effects on cells in which capacitative Ca2+ entry was activated by carbachol or cyclopiazonic acid (10 microM). The GC inhibitor LY83583 (10 microM) reduced carbachol-stimulated Ca2+ entry, and this entry was restored with 8-Br-cGMP. Western blotting revealed that endothelial-type NOS was present in the particulate fraction of cells. The data are consistent with the notion that Ca2+ entry into HT29/B6 cells is regulated by endothelial NOS/NO and GC/cGMP, but effects are most pronounced in store-depleted cells. Thus, NO and cGMP appear to potentiate the action of messengers released from the store during the emptying process, but NO and cGMP have only small effects of their own to open the Ca2+ channel in the plasma membrane. High [SNP] appeared to be inhibitory while low [SNP] was stimulatory, indicating that a precise range of [NO] may be required for effective stimulation of Ca2+ entry.
G Bischof; J Brenman; D S Bredt; T E Machen
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Cell calcium     Volume:  17     ISSN:  0143-4160     ISO Abbreviation:  Cell Calcium     Publication Date:  1995 Apr 
Date Detail:
Created Date:  1995-10-10     Completed Date:  1995-10-10     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  8006226     Medline TA:  Cell Calcium     Country:  SCOTLAND    
Other Details:
Languages:  eng     Pagination:  250-62     Citation Subset:  IM    
Department of Molecular & Cell Biology, University of California, Berkeley, USA.
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MeSH Terms
Amino Acid Oxidoreductases / antagonists & inhibitors,  metabolism
Aminoquinolines / pharmacology
Arginine / analogs & derivatives,  pharmacology
Barium / metabolism
Biological Transport
Calcium / metabolism*
Carbachol / pharmacology
Colon / metabolism*
Colonic Neoplasms / pathology
Cyclic GMP / analogs & derivatives,  pharmacology,  physiology*
Epithelium / metabolism
Guanylate Cyclase / antagonists & inhibitors,  metabolism
Indoles / pharmacology
Intestinal Mucosa / metabolism
Nitric Oxide / physiology*
Nitric Oxide Synthase
Nitroprusside / pharmacology
Second Messenger Systems*
Tumor Cells, Cultured
Grant Support
Reg. No./Substance:
0/Aminoquinolines; 0/Indoles; 10102-43-9/Nitric Oxide; 15078-28-1/Nitroprusside; 18172-33-3/cyclopiazonic acid; 2149-70-4/Nitroarginine; 31356-94-2/8-bromocyclic GMP; 51-83-2/Carbachol; 74-79-3/Arginine; 7440-39-3/Barium; 7440-70-2/Calcium; 7665-99-8/Cyclic GMP; 91300-60-6/6-anilino-5,8-quinolinedione; EC Oxide Synthase; EC 1.4.-/Amino Acid Oxidoreductases; EC Cyclase

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