Fluid secretion by interlobular pancreatic ducts was determined by using video microscopy to measure the rate of swelling of isolated duct segments that had sealed following overnight culture. The aim was to compare the HCO 3 − requirement for secretin-evoked secretion in mouse, rat and guinea-pig pancreas. In mouse and rat ducts, fluid secretion could be evoked by 10 nM secretin and 5 µM forskolin in the absence of extracellular HCO 3 − . In guinea-pig ducts, however, fluid secretion was totally dependent on HCO 3 − . Forskolin-stimulated fluid secretion by mouse and rat ducts in the absence of HCO 3 − was dependent on extracellular Cl − and was completely inhibited by bumetanide (30 µM). It was therefore probably mediated by a basolateral Na + -K + -2Cl − cotransporter. In the presence of HCO 3 − , forskolin-stimulated fluid secretion was reduced ∼40% by bumetanide, ∼50% by inhibitors of basolateral HCO 3 − uptake (3 µM EIPA and 500 µM H 2 DIDS), and was totally abolished by simultaneous application of all three inhibitors. We conclude that the driving force for secretin-evoked fluid secretion by mouse and rat ducts is provided by parallel basolateral mechanisms: Na + -H + exchange and Na + -HCO 3 − cotransport mediating HCO 3 − uptake, and Na + -K + -2Cl − cotransport mediating Cl − uptake. The absence or inactivity of the Cl − uptake pathway in the guinea-pig pancreatic ducts may help to account for the much higher concentrations of HCO 3 − secreted in this species.

The pancreatic pathology in cystic fibrosis (CF) is normally attributed to the failure of ductal fluid secretion resulting from the lack of functional CF transmembrane conductance regulator (CFTR). However, murine models of CF show little or no pancreatic pathology. To resolve this dichotomy we analysed the transport mechanisms involved in fluid and electrolyte secretion by pancreatic ducts isolated from CFTR-null mice. Experiments were performed on cultured interlobular duct segments isolated from the pancreas of the Cftr(tm1Cam) strain of CFTR-null mouse. Fluid secretion to the closed luminal space was measured by video microscopy. The secretory response of ducts isolated from CF mice to cAMP-elevating agonists forskolin and secretin was significantly reduced compared with wild type but not abolished. The Cl(-)- and HCO(3) (-) -dependent components of the ductal secretion were affected equally by the absence of CFTR. The secretory response to carbachol stimulation was unaltered in CF ducts. Loading the ductal cells with the Ca2+ chelator BAPTA completely abolished carbachol-evoked secretion, but did not affect forskolin-evoked secretion in CF or wild-type ducts. We conclude that pancreatic duct cells from CF mice can secrete a significant amount of water and electrolytes by a cAMP-stimulated mechanism that is independent of CFTR and cannot be ascribed to the activation of calcium-activated chloride channels.

Increases or decreases in the contractile response of smooth muscle underlie important pathological conditions such as hypertension, incontinence and altered gastrointestinal transit. These disorders are also frequently encountered in the aged population. Oxidative stress and inflammation are key features in the initiation, progression, and clinical manifestations of smooth muscle disorders. Melatonin, the major secretory product of the pineal gland, has free radical scavenging and antioxidative properties and protects against oxidative insult. Recently, widespread interest has grown regarding the apparent protective effects of melatonin on smooth muscle dysfunction. "In vitro" studies have shown that melatonin decreased vascular tone of vascular beds from control, hypertensive or aged animals, through the reduction of adrenergic contraction and the increase in acetylcholine-induced relaxation. "In vivo", melatonin also attenuates sympathetic tone by direct activation of melatonin receptors, scavenging free radicals or increasing NO availability in the central nervous system. In the gastrointestinal tract, melatonin treatment improves age-related impairments in gallbladder contractility and prevents deleterious effects of cholecystitis on smooth muscle and the enteric nervous system through suppression of oxidative stress. In addition, melatonin improves colonic transit time in constipation-predominant IBS patients. Melatonin is also able to restore impaired contractility of the detrusor muscle from old animals through normalization of Ca(2+) dependent and independent contraction, mitochondrial polarity, neuromuscular function and oxidative stress, which would explain the effects of melatonin counteracting cystometric changes in senescent animals. It also reverses bladder damage following ischemia/reperfusion. In conclusion, melatonin may be a promising candidate for future research of agents that modulate smooth muscle motility.

There is increasing evidence that aging is associated with oxidative damage, inflammation, and apoptosis in different cell types. However, there is limited information regarding aging mechanisms in colon smooth muscle. Old male Wistar rats (22 months) were treated for 10 wks with melatonin or growth hormone (GH). Animals were sacrificed at 24 months of age by decapitation. The colon was dissected and the smooth muscle homogenized. H(2)O(2) and malonyl dialdehyde (MDA) content and catalase and glutathione peroxidase (GPX) activities were determined using colorimetric kits. Expression of nuclear factor kappa B (NF-κB), cyclooxygenase 2 (COX-2), caspase-3, and caspase-9 were determined by Western blot. Aging of colon smooth muscle correlated with an increase in H(2)O(2) and MDA levels when compared with young animals in both proximal and distal segments; these changes were associated with a decrease in the catalase activity in the distal colon. Oxidative stress correlated with an increase in COX-2 and NF-κB expression, which were accompanied by an enhanced expression of the pro-apoptotic enzyme caspase-3 and its upstream enzyme, caspase-9. Melatonin treatment normalized the oxidative, inflammatory, and apoptotic patterns, whereas GH replacement, although effective in reducing oxidative stress in distal colon, did not reverse the age-related inflammation or apoptosis. These results suggest that melatonin should be the treatment of choice to most effectively recover physiological functions in aged colonic smooth muscle.

Aging is associated to oxidative damage and alterations in inflammatory and apoptotic pathways. Aging impairs secretion of several hormones, including melatonin and estrogens. However, the mechanisms involved in aging of smooth muscle are poorly known. We have studied the changes induced by aging in the colonic smooth muscle layer of female rats and the protective effect of hormonal therapy. We used young, aged, and ovariectomized aged female rats. Two groups of ovariectomized rats (22 months old) were treated either with melatonin or with estrogen for 10 weeks before sacrifice. Aging induced oxidative imbalance, evidenced by H(2)O(2) accumulation, lipid peroxidation, and decreased catalase activity. The oxidative damage was enhanced by ovariectomy. In addition, aged colonic muscle showed enhanced expression of the pro-inflammatory enzyme cyclooxygenase 2. Expression of the activated forms of caspases 3 and 9 was also enhanced in aged colon. Melatonin and estrogen treatment prevented the oxidative damage and the activation of caspases. In conclusion, aging of colonic smooth muscle induces oxidative imbalance and activation of apoptotic and pro-inflammatory pathways. Hormonal therapy has beneficial effects on the oxidative and apoptotic changes associated to aging in this model.