TABLE I.

Increases in BK_Ca Channel Activity by Different Natural Bile Acids and Synthetic Analogues in Smooth Muscle Cells Freshly Isolated from Rabbit Superior Mesenteric Artery, Recorded in Cell-attached (C-A), Inside-out (I-O), or Outside-out (O-O) Patches

	C-A	I-O	O-O
Unconjugated bile acids
Cholic acid	5/5	7/9	2/2
Deoxycholic acid	2/2	10/10	1/1
Lithocholic acid	5/5	14/14	7/7
Modifications in the lateral chain
Tauroconjugated bile acid:
Taurolithocholic acid	0/5	2/2	3/3
Neutral compounds:
Cholic acid methyl ester	5/6	4/4	n/t
“Cholic alcohol”	5/5	3/3	n/t
Modifications of groups directly attached to the steroid rings
Compounds with polar groups on the side of the molecule opposite to the methyl groups:
“7,12 deoxycholic acid”	4/4	4/4	n/t
lithocholic acid 3-hemisuccinate	5/5	3/3	n/t
ursodeoxycholic acid methyl ester	1/1	2/2	n/t
“epideoxycholic acid”	n/t	4/4	n/t
Compounds without polar groups on the side of the molecule opposite to the methyl groups:
ursocholanic acid	0/3	1/6	4/7
epilithocholic acid	0/2	0/4	n/t
“all β cholic acid”	1/7	2/7	2/2

Results are expressed as the ratio of the number of positive results (i.e., reversible increase in channel NP_o) to the number of patches tested. All compounds were applied at 100-μM concentration in the puffer pipette; n/t: not tested. Lower concentrations also were used for some compounds: lithocholic acid, 3 μM (C-A, 2/2; I-O, 1/2), and 30 μM (I-O, 3/4); deoxycholic acid, 3 μM (I-O, 5/5), 10 μM (I-O, 5/5), and 33 μM (C-A, 1/4; I-O, 12/15); “epideoxycholic acid,” 3 μM (I-O, 6/6), 10 μM (I-O, 6/6), and 33 μM (I-O, 4/4); cholic acid, 33 μM (I-O, 1/1); and cholic acid methyl ester, 33 μM (I-O, 4/5). Compounds were prepared from stock solutions and diluted in bath solution to the final concentration (materials and methods). Solvents used to prepare the stock solutions were equally diluted and applied onto the patches (controls). The DMSO-ethanol dilution mixture did not noticeably modify channel activity (C-A, 0/11; I-O, 0/18; O-O, 0/9). Using O-O patches, we also found that the increase in BK_Ca channel activity by either taurolithocholic acid (1/1) or ursocholanic acid (1/1) when applied to the extracellular surface of the patch in 310 mM NaCl (“hyperosmotic solution”) was similar to that evoked by these compounds in a normosmotic solution, hyperosmotic control solution itself having no noticeable effect on channel activity (n = 2). These recordings were obtained using conditions identical to those described in Fig. 2, except that NaCl was added to the extracellular solution to obtain a high ionic strength solution, which should shield surface charge and, thus, reduce its possible action on channel activity. The number of patches in the outside-out configuration are usually lower than those in the cell-attached or inside-out configurations because of the technical difficulties associated with obtaining outside-out patches in our preparation. The fact that some of the bile acid analogues did not work in some or all of the inside-out patches at the concentration used may be a reflection of the sensitivity of the putative binding site for a particular feature of the structure or the position of the patch in the pipette. For cell-attached patches, the lack of an effect may reflect the additional requirement for the bile acid or bile acid analogue to flip across the bilayer or, less likely, to travel laterally in the bilayer to get to the putative binding site.