Coactivation of capacitative calcium entry and L-type calcium channels in guinea pig gallbladder

Abstract

We have evaluated the presence of capacitative Ca2+ entry (CCE) in guinea pig gallbladder smooth muscle (GBSM), including a possible relation with activation of L-type Ca2+ channels. Changes in cytosolic Ca2+ concentration induced by Ca2+ entry were assessed by digital microfluorometry in isolated, fura 2-loaded GBSM cells. Application of thapsigargin, a specific inhibitor of the Ca2+ store pump, induced a transient Ca2+ release followed by sustained entry of extracellular Ca2+. Depletion of the stores with thapsigargin, cyclopiazonic acid, ryanodine and caffeine, high levels of the Ca2+-mobilizing hormone cholecystokinin octapeptide, or simple removal of external Ca2+ resulted in a sustained increase in Ca2+ entry on subsequent reapplication of Ca2+. This entry was attenuated by 2-aminoethoxydiphenylborane, L-type Ca2+ channel blockade, pinacidil, and Gd3+. Accumulation of the voltage-sensitive dye 3,3′-dipentylcarbocyanine and direct intracellular recordings showed that depletion of the stores is sufficient for depolarization of the plasma membrane. Contractility studies in intact gallbladder muscle strips showed that CCE induced contractions. The CCE-evoked contraction was sensitive to 2-aminoethoxydiphenylborane, L-type Ca2+ channel blockers, and Gd3+. We conclude that, in GBSM, release of Ca2+ from internal stores activates a CCE pathway and depolarizes plasma membrane, allowing coactivation of voltage-operated L-type Ca2+ channels. This process may play a role in excitation-contraction coupling in GBSM.

Role of mitochondria in spontaneous rhythmic activity and intracellular calcium waves in the guinea pig gallbladder smooth muscle

“…Furthermore, we have previously demonstrated that VDCC and nonselective cation channels in the plasma membrane (27,30,33,54) and the SR (2,3,27,34) are also essential for rhythmic activity in the GBSM. Collectively, these findings indicate that the mitochondrion, SR, and plasma membrane channels constitute key components of the GBSM pacemaker.…”

Melatonin Treatment Reverts Age-Related Changes in Guinea Pig Gallbladder Neuromuscular Transmission and Contractility

“…The cell length was similar in the three age groups (young adult 44.68 Ϯ 1.62, aged 45.49 Ϯ 1.83, and melatonin 43.07 Ϯ 1.36 m; n ϭ 98, 61, and 96 cells, respectively) Cell Loading and [Ca 2؉ ] i Determination. [Ca 2ϩ ] i was determined by epifluorescence microscopy using the fluorescent ratiometric Ca 2ϩ indicator fura 2 as described previously (Morales et al, 2004). Isolated cells were loaded with 4 M fura 2/AM at room temperature for 25 min.…”

“…at ASPET Journals on May 11, 2018 jpet.aspetjournals.org capacitative calcium entry (CCE) is another influx route activating contraction in gallbladder smooth muscle (Morales et al, 2004). As previously reported, after depletion of the stores by incubation of control gallbladder strips with 1 M TPS in Ca 2ϩ -free medium for 30 min, reintroduction of extracellular Ca 2ϩ induced a sustained contraction (3.69 Ϯ 0.53 mN/mg; n ϭ 12) that was significantly smaller in aged strips (2.68 Ϯ 0.29 mN/mg; n ϭ 9, P Ͻ 0.01) but similar in melatonin-treated animals (4.41 Ϯ 0.39 mN/mg; n ϭ 8) (Fig.…”

Cyclic AMP‐mediated inhibition of gallbladder contractility: role of K+ channel activation and Ca2+ signaling

“…Another Ca 2 þ influx pathway that contributes to the CCKinduced gallbladder contraction is the activation of CCE mechanisms (Morales et al, 2004). To test whether cAMP could affect this pathway, CCE was activated by depletion of the stores with thapsigargin in a free Ca 2 þ medium and then Ca 2 þ was restored in the extracellular medium in the presence of 1 mM nitrendipine to block non-CCE.…”

“…Recently, CCE, which has been shown to be essential in nonexcitable cells (Putney, 1986), is gaining attention in the area of smooth muscle physiology (Gibson et al, 1998) where it participates in contraction (Morales et al, 2004). However, the possible role of cAMP controlling this process remains unknown.…”