Forskolin and PMA pretreatment of HT29 cells alters their chloride conductance induced by cAMP, Ca2+ and hypotonic cell swelling.
HT29 cells were preincubated with forskolin (10(-5) mol/l, FORHT) or phorbol 12-myristate 13-acetate (PMA) (10(-7) mol/l, PMAHT) for 20 h, which has been shown previously and is also shown here, to upregulate and downregulate, respectively, the expression of the cystic fibrosis transmembrane conductance regulator (CFTR). CFPAC-1 cells underwent the same protocols. HT29 cells were examined by slow (SWC) and fast (FWC) whole-cell patch-clamp techniques. The results of SWC and FWC were indistinguishable and were pooled. CFPAC-1 cells were examined with FWC. The membrane voltage (V) of FORHT was -41.8 +/- 1.4 mV (n = 77) and that of PMAHT was -43.6 +/- 2.4 mV (n = 76). The conductance (G) of FORHT (9.4 +/- 0.9 nS, n = 77) was significantly larger than that of PMAHT (3.7 +/- 0.4 nS, n = 76). Acute application of forskolin (10(-5) mol/l, FOR) plus 0.5 mmol/l 8-(4-chlorophenylthio)-cAMP (cAMP) depolarized V by 12 (FORHT) and 8 (PMAHT) mV, respectively. The acute increase of G by FOR plus cAMP was by 7.6 +/- 1.9 nS for FORHT (n = 22) and only 2.2 +/- 1 nS for PMAHT (n = 13). ATP (10(-4) mol/l) depolarized V in both types of cells. It enhanced G by 16.7 +/- 4.1 nS in FORHT (n = 14) and significantly less (by 5.5 +/- 1.2 nS, n = 14) in PMAHT. Also the G increase lasted longer in FORHT. Neurotensin (NT, 10(-8) mol/l) also had a stronger and longer lasting effect in FORHT.(ABSTRACT TRUNCATED AT 250 WORDS)
Kunzelmann
K
K
Physiologisches Institut der Albert-Ludwigs-Universität Freiburg, Germany.
Allert
N
N
Kubitz
R
R
Breuer
W V
WV
Cabantchik
Z I
ZI
Normann
C
C
Schumann
S
S
Leipziger
J
J
Greger
R
R
D000255:Adenosine Triphosphate / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006801:Humans; D007038:Hypotonic Solutions; D015759:Ionomycin / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D009496:Neurotensin / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
C505032:CFTR protein, human; D018118:Chloride Channels; D007038:Hypotonic Solutions; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D009496:Neurotensin; D015759:Ionomycin; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium