Abstract
Intracellular application of the sulfonylurea tolbutamide during whole- cell patch-clamp recordings stimulated exocytosis >5-fold when applied at a cytoplasmic Ca2+ concentration of 0.17 μM. This effect was not detectable in the complete absence of cytoplasmic Ca2+ and when exocytosis was elicited by guanosine 5'-O-(3-thiotriphosphate) (GTPγS). The stimulatory action could be antagonized by the sulfonamide diazoxide, by the Cl-- channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), by intracellular application of the antibody JSB1 [originally raised against a 170-kDa multidrug resistance (mdr) protein], and by tamoxifen (an inhibitor of the mdr- and volume-regulated Cl- channels). Immunocytochemistry and Western blot analyses revealed that JSB1 recognizes a 65-kDa protein in the secretory granules. This protein exhibited no detectable binding of sulfonylureas and is distinct from the 140-kDa sulfonylurea high-affinity sulfonylurea receptors also present in the granules. We conclude that (i) tolbutamide stimulates Ca2+-dependent exocytosis secondary to its binding to a 140-kDa high-affinity sulfonylurea receptor in the secretory granules; and (ii) a granular 65-kDa mdr-like protein mediates the action. The processes thus initiated culminate in the activation of a granular Cl- conductance. We speculate that the activation of granular Cl- fluxes promotes exocytosis (possibly by providing the energy required for membrane fusion) by inducing water uptake and an increased intragranular hydrostatic pressure.
| Original language | English |
|---|---|
| Pages (from-to) | 5539-5544 |
| Number of pages | 6 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 96 |
| Issue number | 10 |
| DOIs | |
| State | Published - May 11 1999 |
| Externally published | Yes |
Fingerprint
Keywords
- ABC protein
- Insulin
- Sulfonyl urea receptor
ASJC Scopus subject areas
- Genetics
- General
Cite this
The stimulatory action of tolbutamide on Ca2+-dependent exocytosis in pancreatic β cells is mediated by a 65-kDa mdr-like P-glycoprotein. / Barg, S.; Renstrom, E.; Berggren, P. O.; Bertorello, A.; Bokvist, K.; Braun, M.; Eliasson, L.; Holmes, W. E.; Kohler, M.; Rorsman, P.; Thevenod, F.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 96, No. 10, 11.05.1999, p. 5539-5544.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - The stimulatory action of tolbutamide on Ca2+-dependent exocytosis in pancreatic β cells is mediated by a 65-kDa mdr-like P-glycoprotein
AU - Barg, S.
AU - Renstrom, E.
AU - Berggren, P. O.
AU - Bertorello, A.
AU - Bokvist, K.
AU - Braun, M.
AU - Eliasson, L.
AU - Holmes, W. E.
AU - Kohler, M.
AU - Rorsman, P.
AU - Thevenod, F.
PY - 1999/5/11
Y1 - 1999/5/11
N2 - Intracellular application of the sulfonylurea tolbutamide during whole- cell patch-clamp recordings stimulated exocytosis >5-fold when applied at a cytoplasmic Ca2+ concentration of 0.17 μM. This effect was not detectable in the complete absence of cytoplasmic Ca2+ and when exocytosis was elicited by guanosine 5'-O-(3-thiotriphosphate) (GTPγS). The stimulatory action could be antagonized by the sulfonamide diazoxide, by the Cl-- channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), by intracellular application of the antibody JSB1 [originally raised against a 170-kDa multidrug resistance (mdr) protein], and by tamoxifen (an inhibitor of the mdr- and volume-regulated Cl- channels). Immunocytochemistry and Western blot analyses revealed that JSB1 recognizes a 65-kDa protein in the secretory granules. This protein exhibited no detectable binding of sulfonylureas and is distinct from the 140-kDa sulfonylurea high-affinity sulfonylurea receptors also present in the granules. We conclude that (i) tolbutamide stimulates Ca2+-dependent exocytosis secondary to its binding to a 140-kDa high-affinity sulfonylurea receptor in the secretory granules; and (ii) a granular 65-kDa mdr-like protein mediates the action. The processes thus initiated culminate in the activation of a granular Cl- conductance. We speculate that the activation of granular Cl- fluxes promotes exocytosis (possibly by providing the energy required for membrane fusion) by inducing water uptake and an increased intragranular hydrostatic pressure.
AB - Intracellular application of the sulfonylurea tolbutamide during whole- cell patch-clamp recordings stimulated exocytosis >5-fold when applied at a cytoplasmic Ca2+ concentration of 0.17 μM. This effect was not detectable in the complete absence of cytoplasmic Ca2+ and when exocytosis was elicited by guanosine 5'-O-(3-thiotriphosphate) (GTPγS). The stimulatory action could be antagonized by the sulfonamide diazoxide, by the Cl-- channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), by intracellular application of the antibody JSB1 [originally raised against a 170-kDa multidrug resistance (mdr) protein], and by tamoxifen (an inhibitor of the mdr- and volume-regulated Cl- channels). Immunocytochemistry and Western blot analyses revealed that JSB1 recognizes a 65-kDa protein in the secretory granules. This protein exhibited no detectable binding of sulfonylureas and is distinct from the 140-kDa sulfonylurea high-affinity sulfonylurea receptors also present in the granules. We conclude that (i) tolbutamide stimulates Ca2+-dependent exocytosis secondary to its binding to a 140-kDa high-affinity sulfonylurea receptor in the secretory granules; and (ii) a granular 65-kDa mdr-like protein mediates the action. The processes thus initiated culminate in the activation of a granular Cl- conductance. We speculate that the activation of granular Cl- fluxes promotes exocytosis (possibly by providing the energy required for membrane fusion) by inducing water uptake and an increased intragranular hydrostatic pressure.
KW - ABC protein
KW - Insulin
KW - Sulfonyl urea receptor
UR - http://www.scopus.com/inward/record.url?scp=0033545857&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0033545857&partnerID=8YFLogxK
U2 - 10.1073/pnas.96.10.5539
DO - 10.1073/pnas.96.10.5539
M3 - Article
C2 - 10318919
AN - SCOPUS:0033545857
VL - 96
SP - 5539
EP - 5544
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 10
ER -