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The incidence of urinary bladder disturbances increases with age, and free radical accumulation has been proposed as a causal factor. Here we investigated the association between changes in bladder neuromuscular function and oxidative stress in aging and the possible benefits of melatonin treatment. Neuromuscular function was assessed by electrical field stimulation (EFS) of isolated guinea pig detrusor strips from adult and aged female guinea pigs. A group of adult and aged animals were treated with 2.5 mg*[kg.sup.-1]*[day.sup.-1] melatonin for 28 days. Neurotransmitter blockers were used to dissect pharmacologically the EFS-elicited contractile response. EFS induced a neurogenic and frequency-dependent contraction that was impaired by aging. This impairment is in part related to a decrease in detrusor myogenic contractility. Age also decreased the sensitivity of the contraction to pharmacological blockade of purinergic and sensitive fibers but increased the effect of blockade of nitrergic and adrenergic nerves. The density of cholinergic and nitrergic nerves remained unaltered, but aging modified afferent fibers. These changes were associated with an increased level of markers for oxidative stress. Melatonin treatment normalized oxidative levels and counteracted the aging-associated changes in bladder neuromuscular function. In conclusion, these results show that aging modifies neurogenic contraction and the functional profile of the urinary bladder plexus and simultaneously increases the oxidative damage to the organ. Melatonin reduces oxidative stress and improves the age-induced changes in bladder neuromuscular function, which could be of importance in reducing the impact of age-related bladder disorders. detrusor smooth muscle; neuromuscular function; sensory nerves; electrical field stimulation

Transient receptor potential protein family C (TRPC) has been proposed as a candidate for channels involved in capacitative [Ca.sup.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.sup.2+] level ([[[Ca.sup.2+]].sub.i]). Thus lowering the levels of cellular calcium with the chelators EGTA and BAPTA AM results in a downregulation of TRPC 1-TRPC4 gene and protein expression. In contrast, activation of [Ca.sup.2+] influx through L-type [Ca.sup.2+] channels and [Ca.sup.2+] release from intracellular stores induced an increase in TRPC1-TRPC4 mRNA and protein abundance. Activation of [Ca.sup.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.sup.2+] influx. In addition to this mechanism, activation of TRPC gene expression by intracellular [Ca.sup.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.sup.2+] in [Ca.sup.2+] homeostasis at the level of TRPC gene and protein expression. transient receptor protein family C channels; cytosolic calcium levels; gallbladder

The role of Tvbgn3, a [beta]-1,6-glucanase from Trichoderma virens, is examined by comparison of wild-type (WT) and transformant strains in which Tvbgn3 is disrupted (GKO) or constitutively overexpressed (GOE). The results have shown the involvement of [beta]-1,6-glucanase in mycoparasitism and its relevance in the biocontrol activity of Trichoderma virens.

Mitochondria are an important source of reactive oxygen species (ROS) formed as a side product of oxidative phosphorylation. The main sites of oxidant production are complex I and complex III, where electrons flowing from reduced substrates are occasionally transferred to oxygen to form superoxide anion and derived products. These highly reactive compounds have a well-known role in pathological states and in some cellular responses. However, although their link with [Ca.sup.2+] is well studied in cell death, it has been hardly investigated in normal cytosolic calcium concentration ([[[Ca.sup.2+]].sub.i]) signals. Several [Ca.sup.2+] transport systems are modulated by oxidation. Oxidation increases the activity of inositol 1,4,5-trisphosphate and ryanodine receptors, the main channels releasing [Ca.sup.2+] from intracellular stores in response to cellular stimulation. On the other hand, mitochondria are known to control [[[Ca.sup.2+]].sub.i] signals by [Ca.sup.2+] uptake and release during cytosolic calcium mobilization, specially in mitochondria situated close to [Ca.sup.2+] release channels. Mitochondrial inhibitors modify calcium signals in numerous cell types, including oscillations evoked by physiological stimulus. Although these inhibitors reduce mitochondrial [Ca.sup.2+] uptake, they also impair ROS production in several systems. In keeping with this effect, recent reports show that antioxidants or oxidant scavengers also inhibit physiological calcium signals. Furthermore, there is evidence that mitochondria generate ROS in response to cell stimulation, an effect suppressed by mitochondrial inhibitors that simultaneously block [[[Ca.sup.2+]].sub.i] signals. Together, the data reviewed here indicate that [Ca.sup.2+]-mobilizing stimulus generates mitochondrial ROS, which, in turn, facilitate [[[Ca.sup.2+]].sub.i] signals, a new aspect in the biology of mitochondria. Finally, the potential implications for biological modeling are discussed. mitochondria; calcium

Impaired smooth muscle contractility is a hallmark of acute acalculous cholecystitis. Although free cytosolic [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]) is a critical step in smooth muscle contraction, possible alterations in [Ca.sup.2+] homeostasis by cholecystitis have not been elucidated. Our aim was to elucidate changes in the [Ca.sup.2+] signaling pathways induced by this gallbladder dysfunction. [[[Ca.sup.2+]].sub.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. [Ca.sup.2+] responses to the inositol trisphosphate (Ins[P.sub.3]) 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 [Ca.sup.2+] influx through L-type [Ca.sup.2+] channels and capacitative [Ca.sup.2+] entry induced by depletion of intracellular [Ca.sup.2+] pools. In addition, the pharmacological phenotype of these channels was altered in cholecystitic cells. Inflammation impaired contractility further than [Ca.sup.2+] 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 [Ca.sup.2+] release and [Ca.sup.2+] influx in gallbladder smooth muscle, but a loss in the sensitivity of the contractile machinery to [Ca.sup.2+] may also be responsible for the impairment in gallbladder contractility. contractility; calcium stores; calcium channels; F-actin; calcium ion

Coactivation of capacitative calcium entry and L-type calcium channels in guinea pig gallbladder. Am J Physiol Gastrointest Liver Physiol 286: G 1090-G1100, 2004. First published January 22, 2004; 10.1152/ajpgi.00260.2003.--We have evaluated the presence of capacitative [Ca.sup.2+] entry (CCE) in guinea pig gallbladder smooth muscle (GBSM), including a possible relation with activation of L-type [Ca.sup.2+] channels. Changes in cytosolic [Ca.sup.2+] concentration induced by [Ca.sup.2+] entry were assessed by digital microfluorometry in isolated, fura 2-loaded GBSM cells. Application of thapsigargin, a specific inhibitor of the [Ca.sup.2+] store pump, induced a transient C[a.sup.2+] release followed by sustained entry of extracellular [Ca.sup.2+]. Depletion of the stores with thapsigargin, cyclopiazonic acid, ryanodine and caffeine, high levels of the [Ca.sup.2+]-mobilizing hormone cholecystokinin octapeptide, or simple removal of external [Ca.sup.2+] resulted in a sustained increase in [Ca.sup.2+] entry on subsequent reapplication of [Ca.sup.2+]. This entry was attenuated by 2-aminoethoxydiphenylborane, L-type [Ca.sup.2+] channel blockade, pinacidil, and [Gd.sup.3+]. 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 [Ca.sup.2+] channel blockers, and [Gd.sup.3+]. We conclude that, in GBSM, release of [Ca.sup.2+] from internal stores activates a CCE pathway and depolarizes plasma membrane, allowing coactivation of voltage-operated L-type [Ca.sup.2+] channels. This process may play a role in excitation-contraction coupling in GBSM. smooth muscle; contraction; 2-aminoethoxydiphenylborane; nitrendipine; methoxyverapamil

The current study was undertaken to test the existence and possible role of ether-a-go-go-related gene 1 (ERG1) protein [K.sup.+] channels in gallbladder smooth muscle (GBSM). Transcripts encoding ERG1 were detected in human, mouse, and guinea pig GBSM, and ERG1 immunoreactivity was observed in GBSM cells. In intracellular voltage recordings, addition of E-4031 (100 nM-1 [micro]M) or cisapride (100 nM-2 [micro]M) caused concentration-dependent excitation of guinea pig GBSM that was not affected by 500 nM TTX + 5 [micro]M atropine, and E-4031 also depolarized the resting membrane potential. In muscle strip studies, E-4031 either induced phasic contractions or significantly increased the amplitude of phasic contractions in spontaneously active tissues (P = 0.001). E-4031 also potentiated bethanechol-induced contractions. In conclusion, ERG1 channels are expressed in the GBSM, where they play a role in excitation-contraction coupling probably by contributing to repolarization of the plateau phase of the action potential and to the resting membrane potential. biliary motility; rapidly activated delayed rectifier; E-4031; cisapride

[Ca.sup.2+] sparks and BK currents in gallbladder myocytes: role in CCK-induced response. Am J Physiol Gastrointest Liver Physiol 282: G165-G174, 2002. First published October 24, 2001; 10.1152/ajpgi.00326. 2002.--We sought to elucidate the regulation of gallbladder smooth muscle (GBSM) excitability by localized [Ca.sup.2+] release events (sparks) and large-conductance [Ca.sup.2+]-dependent (BK) channels by determining whether sparks exist in GBSM and, if so, whether they activate BK channels. Sparks were identified in isolated GBSM loaded with fluo 4. Each spark was associated with a transient outward current, suggesting communication of ryanodine receptor (RyR) channels with BK channels. This was confirmed by the inhibition of outward currents with iberiotoxin (100 nM), thapsigargin (200 nM), and ryanodine (10 [micro]M). In current clamp mode, the transient BK currents were associated with brief membrane hyperpolarizations (10.9 [+ or -] 1.3 mV). Because transient BK currents could dampen GBSM excitability, we tested whether CCK attenuates these events. CCK (10 nM) reduced the amplitude and frequency of transient BK currents, and subsequent caffeine application restored transient BK current activity. These results support the concept that RyRs and BK channels contribute to the regulation of GBSM excitability and that CCK can act in part by inhibiting this pathway. guinea pig; smooth muscle; sarcoplasmic reticulum; ryanodine receptor; thapsigargin; L-type calcium channels; caffeine

Oligogalacturonides (OGs) are fragments of pectin released from the plant cell wall during insect or pathogen attack. They can be perceived by the plant as damage signals, triggering local and systemic defence responses. Here, we analyse the dynamics of local and systemic responses to OG perception in tomato roots or shoots, exploring their impact across the plant and their relevance in pathogen resistance. Targeted and untargeted metabolomics and gene expression analysis in plants treated with purified OGs revealed that local responses were transient, while distal responses were stronger and more sustained. Remarkably, changes were more conspicuous in roots, even upon foliar application of the OGs. The treatments differentially activated the synthesis of defence‐related hormones and secondary metabolites including flavonoids, alkaloids and lignans, some of them exclusively synthetized in roots. Finally, the biological relevance of the systemic defence responses activated upon OG perception was confirmed, as the treatment induced systemic resistance to Botrytis cinerea. Overall, this study shows the differential regulation of tomato defences upon OGs perception in roots and shoots and reveals the key role of roots in the coordination of the plant responses to damage sensing. Oligogalacturonide perception in tomato roots or shoots triggers differential transient local responses, but also a myriad of systemic responses at the metabolic, transcriptomic and enzymatic level leading to pathogen resistance. The results highlight the key role of roots in the regulation of these systemic responses. [ABSTRACT FROM AUTHOR]