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.

Transient receptor potential protein family C (TRPC) has been proposed as a candidate for channels involved in capacitative Ca(2+) entry (CCE) mechanisms, but the modulation of their gene expression remains unexplored. In this study we show that guinea pig gallbladder smooth muscle contains mRNA encoding TRPC1, TRPC2, TRPC3, and TRPC4 proteins whose abundance depends on cytosolic Ca(2+) level ([Ca(2+)](i)). Thus lowering the levels of cellular calcium with the chelators EGTA and BAPTA AM results in a downregulation of TRPC1-TRPC4 gene and protein expression. In contrast, activation of Ca(2+) influx through L-type Ca(2+) channels and Ca(2+) release from intracellular stores induced an increase in TRPC1-TRPC4 mRNA and protein abundance. Activation of Ca(2+)/calmodulin-dependent kinases (CaMK) and phosphorylation of cAMP-response element binding protein accounts for the increase in TRPC mRNA transcription in response to L-type channel-mediated Ca(2+) influx . In addition to this mechanism, activation of TRPC gene expression by intracellular Ca(2+) release also involves calcineurin pathway. According to the proposed role for these channels, activation of CCE induced an increase in TRPC1 and TRPC3 mRNA abundance, which depends on the integrity of the calcineurin and CaMK pathways. These findings show for the first time an essential autoregulatory role of Ca(2+) in Ca(2+) homeostasis at the level of TRPC gene and protein expression.

Diabetic nephropathy (DN), a major cause of morbidity and mortality in diabetes, will develop within a subset of type 1 diabetes mellitus (T1DM) patients, and oxidative stress has been implicated in its pathogenesis. To investigate the relationship between indicators of early DN stages (hyperfiltration estimated by creatinine clearance > or =150 ml/min per 1.73 m(2), microalbuminuria) and oxidative stress, a prospective study was conducted in 29 T1DM patients (age 13.89 +/- 4.61 years) and 18 control subjects (age 13.23 +/- 3.99 years). Blood samples were collected to assay for biomarkers of oxidative stress (malondialdehyde and carbonyl groups) and antioxidants (glutathione peroxidase, reduced glutathione, alpha-tocopherol, and beta-carotene). With respect to control subjects, in T1DM patients, an increase was found in biomarkers of oxidative stress (p < 0.05), mainly due to the group of subjects with hyperfiltration, and a decrease in the ratio alpha-tocopherol/lipids (p < 0.05). In multiple regression analyses, age at disease onset, glycated hemoglobin, microalbuminuria, and oxidative stress biomarkers remained as explicative variables of hyperfiltration (R (2) adjusted = 0.731, p = 0.000). These findings support the importance of the oxidative damage to lipids and proteins, which is linked to hyperfiltration and which could contribute to the development of DN in patients with T1DM.

The existence of functionally distinct intracellular Ca(2+) stores has been proposed in some types of smooth muscle. In this study, we sought to examine Ca(2+) stores in the gallbladder by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura 2-loaded isolated myocytes, membrane potential in intact smooth muscle, and isometric contractions in whole mount preparations. Exposure of isolated myocytes to 10 nM CCK caused a transient elevation in [Ca(2+)](i) that persisted in Ca(2+)-free medium and was inhibited by 2-aminoethoxydiphenylborane (2-APB). Application of caffeine induced a rapid spike-like elevation in [Ca(2+)](i) that was insensitive to 2-APB but was abolished by pretreatment with 10 muM ryanodine. These data support the idea that both inositol trisphosphate (IP(3)) receptors (IP(3)R) and ryanodine receptors (RyR) are present in this tissue. When caffeine was applied in Ca(2+)-free solution, the [Ca(2+)](i) transients decreased as the interval between Ca(2+) removal and caffeine application was increased, indicating a possible leakage of Ca(2+) in these stores. The refilling of caffeine-sensitive stores involved sarcoendoplasmic reticulum Ca(2+)-ATPase activation, similar to IP(3)-sensitive stores. The moderate Ca(2+) elevation caused by CCK was associated with a gallbladder contraction, but caffeine or ryanodine failed to induce gallbladder contraction. Nevertheless, caffeine caused a concentration-dependent relaxation in gallbladder strips either under resting tone conditions or precontracted with 1 muM CCK. Taken together, these results suggest that, in gallbladder smooth muscle, multiple pharmacologically distinct Ca(2+) pools do not exist, but IP(3)R and RyR must be spatially separated because Ca(2+) release via these pathways leads to opposite responses.

Impaired smooth muscle contractility is a hallmark of acute acalculous cholecystitis. Although free cytosolic Ca2+ ([Ca2+]i) is a critical step in smooth muscle contraction, possible alterations in Ca2+ homeostasis by cholecystitis have not been elucidated. Our aim was to elucidate changes in the Ca2+ signaling pathways induced by this gallbladder dysfunction. [Ca2+]i was determined by epifluorescence microscopy in fura 2-loaded isolated gallbladder smooth muscle cells, and isometric tension was recorded from gallbladder muscle strips. F-actin content was quantified by confocal microscopy. Ca2+ responses to the inositol trisphosphate (InsP3) mobilizing agonist CCK and to caffeine, an activator of the ryanodine receptors, were impaired in cholecystitic cells. This impairment was not the result of a decrease in the size of the releasable pool. Inflammation also inhibited Ca2+ influx through L-type Ca2+ channels and capacitative Ca2+ entry induced by depletion of intracellular Ca2+ pools. In addition, the pharmacological phenotype of these channels was altered in cholecystitic cells. Inflammation impaired contractility further than Ca2+ signal attenuation, which could be related to the decrease in F-actin that was detected in cholecystitic smooth muscle cells. These findings indicate that cholecystitis decreases both Ca2+ release and Ca2+ influx in gallbladder smooth muscle, but a loss in the sensitivity of the contractile machinery to Ca2+ may also be responsible for the impairment in gallbladder contractility.