Jose Ignacio San Roman

Basolateral anion transport mechanisms underlying fluid secretion by mouse, rat and guinea-pig pancreatic ducts.

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 HCO3− 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 HCO3−. In guinea‐pig ducts, however, fluid secretion was totally dependent on HCO3−. Forskolin‐stimulated fluid secretion by mouse and rat ducts in the absence of HCO3− 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 HCO3−, forskolin‐stimulated fluid secretion was reduced ∼40% by bumetanide, ∼50% by inhibitors of basolateral HCO3− uptake (3 μm EIPA and 500 μm H2DIDS), 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+–HCO3− cotransport mediating HCO3− 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 HCO3− secreted in this species.

Cerulein-induced acute pancreatitis in the rat : study of pancreatic secretion and plasma VIP and secretin levels

A study was made with different doses of cerulein (2, 4, 10 and 20 μg/kg) administered subcutaneously to rats by four injections at intervals of 1 hr; the aim of this work was to study exocrine pancreatic secretion of the rat under cerulein-induced acute pancreatitis, analyzing enzyme and hydroelectrolyte secretion of pancreatic juice. A further aim was to study the relationship between the dose of cerulein and the plasma levels of peptides controlling hydroelectrolyte secretion of the pancreas, like secretin and vasoactive intestinal peptide (VIP). At the lowest dose schedule, the amounts of total protein and enzymes (amylase and trypsin) in pancreatic juice decreased significantly, plasma amylase increased, and the pancreas became edematous. Higher doses magnified these effects. By contrast, ductular function (flow and HCO3−) was well preserved in ceruleintreated rats, and this was probably due to the significant increase in plasma levels of immunoreactive secretin whereas VIP levels were unchanged. The secretin released by treatment with cerulein is able to palliate the lack of flow from acinar origin that is affected in the process of acute pancreatitis, being a beneficial response to the cerulein treatment.

Ducts isolated from the pancreas of CFTR-null mice secrete fluid

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 Cftrtm1Cam 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 HCO3−-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.

Heterogeneous decrease of bone mineral density in the vertebral column of ovariectomized rats.

The long-term effect of ovariectomy on the loss of bone mineral density (BMD) was evaluated in rats with and without estrogen treatment; BMD was studied in the lumbar and caudal vertebrae, measured by DXA, to find how the losses of BMD occur in the axial skeleton. Seventy female Wistar rats of 3 months of age were divided into four groups as follows: group 1: control animals; group 2: ovariectomized animals; group 3: ovariectomized animals undergoing treatment with estrogen (0.25 mg/kg per week of 17-beta estradiol); group 4: ovariectomized rats undergoing estrogen treatment only during the last 3 months of the experimental period. No significant differences were found among the groups in regard to the BMD values of the caudal vertebrae at either 3 or 6 months. Likewise, in the lumbar vertebrae there were no significant differences among the groups after 3 months. However, at 6 months, a decrease in the BMDs of the ovariectomized animals with respect to the remaining groups was found: 226 +/- 11 mg/cm2 in the ovariectomized group; 262 +/- 14 mg/cm2 in the controls; 255 +/- 4 mg/cm2 in the rats receiving estrogen treatment for 6 months; and 259 +/- 5 mg/cm2 in the animals receiving estrogen for 3 months. The study also reveals the absence of differences in the bone mineral density between the ovariectomized and control rats when the former received estrogen treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Exocrine pancreatic response to CCK in rats after long-term hydrocortisone administration

The effects of prolonged administration of hydrocortisone (10 mg/kg/day) on exocrine pancreatic secretion were examined, analyzing the quantitative and qualitative variations in secretion at 7, 18 and 30 days of treatment. The weight of the pancreata was found to increase during the period of hydrocortisone treatment. After the 7th day of treatment the hormone significantly decreases basal exocrine pancreatic secretion, maintaining similar values up to the 30th day of treatment. Hydrocortisone enhances the pancreatic response to CCK since the percent increase in acinar secretion under stimulation compared to basal secretion surpassed control levels in all cases. However, the inhibitory effect on secretion shown by hydrocortisone opposes and surpasses the stimulatory effect of the secretagogue, secretion being reduced in the treated animals. Hydrocortisone especially affected the amylase fraction of the juice, to a larger extent inhibiting the isoenzyme A1, with an IEP of 8.5 when the animals were treated over 30 days; thus, a considerable reduction was observed in the amylase secreted both in resting conditions and under stimulation with CCK. There is a possibility that chronic treatment with glucocorticoids may sensitize the acinar cells, alter the composition of the pancreatic juice and inhibit secretion, effects that may involve pancreatic dysfunction.

Effect of secretin on pancreatic juice proteins in caerulein-induced acute pancreatitis in the rat

Nine hours after the start of treatment with caerulein in rats, an increase in the weight of the pancreas and an increase in serum amylase levels were observed. Likewise, a significant increase in endogenous secretin occurred in rats with acute pancreatitis. A dramatic reduction in the secretion of total protein and amylase was also observed. A partial recovery of this latter effect was achieved after an infusion of high doses of secretin. Under our experimental conditions, the volume of secretion did not vary in caerulein-treated rats wtih respect to controls, either in resting conditions or under secretin stimulation, which indicates that the ductular cells were not significantly affected. Isoelectrofocusing (IEF) and crossed-immunoelectrophoresis (CIE) studies revealed important alterations in the proteins of the pancreatic juice of rats with caerulein-induced acute pancreatitis. Trypsinogen appeared to be particularly affected, showing an increase in the T2 acidic form with an IEP of 4.4 and a decrease in the basic form T3 with an IEP of 8.0, which splits in other forms with a clear antigenic community. A hydrolase was also observed with an IEP of 6.2. In this sense, secretin administration may also be said to induce a significant improvement in established acute pancreatitis, since it tended to normalize the structure and proportion of the proteins secreted.

Saline Infusion Through the Pancreatic Duct Leads to Changes in Calcium Homeostasis Similar to Those Observed in Acute Pancreatitis

This work focuses on studying the early events associated with pancreatic damage after retrograde infusion through the pancreatic duct in rats. We have analyzed changes in calcium homeostasis and secretory response in pancreatic acini from rats with taurocholate-induced acute pancreatitis. Moreover, in order to test whether pancreatic duct manipulation can trigger damage inside pancreatic acinar cells, we have studied both parameters in acini from animals infused with saline. Our study demonstrates that taurocholate causes evident damage to acinar cells, impairing both calcium homeostasis and secretory response to CCK. In saline, a significant decrease in calcium cytosolic response to CCK was observed. Calcium disturbances similar to those observed in acute pancreatitis appear before secretion blockade and inflammation processes in saline treated rats. These results could be interesting since pancreatitis is associated to clinical procedures that require duct manipulation such as endoscopic retrograde cholangiopancreatography.

Cardiovascular homeostasis in hypotension associated with initial stages of severe acute pancreatitis.

Objectives: To identify the mechanisms underlying hypotension during the early phase of severe acute pancreatitis (SAP) by analyzing whether an impaired response to vasoactive substances occurs in this pathological process. Methods: Experimental SAP was induced by infusing 5% sodium taurocholate through the main pancreatic duct in rats. Once mean arterial pressure (MAP) in animals with pancreatitis was reduced, different vasoactive substances and inhibitors were administered. Results: Administration of the nonspecific nitric oxide synthase inhibitor N&ohgr;-nitro-l-arginine methyl ester caused a similar increase in MAP in rats with pancreatitis and control rats, whereas inducible nitric oxide synthase inhibition did not cause changes in MAP. Moreover, the hypertensive response to endothelin and angiotensin II was lower in pancreatitis. Inhibition of angiotensin II synthesis by the angiotensin-converting enzyme inhibitor perindopril in animals with pancreatitis caused severe hypotension, causing death in 40% of them. Finally, pressor hyporesponsiveness to angiotensin II in animals with pancreatitis was avoided by previous administration of perindopril and N&ohgr;-nitro-l-arginine methyl ester. Conclusions: The SAP-induced hypotension is associated with a deficient pressor responsiveness to angiotensin II and endothelin-1. The renin-angiotensin system plays an important role in the control of MAP in animals with pancreatitis.

Effect of high fiber intake on pancreatic lysosomal stability in ethanol-fed rats 1

Abstract Chronic ethanol consumption increases the fragility of pancreatic lysosomes, but the effect of a high fiber intake, alone or combined with alcohol abuse, on the lysosomal stability has not been studied. Furthermore, it is not yet known whether these treatments could predispose the exocrine pancreas to a greater damage after cerulein-induced acute pancreatitis. Cytosolic specific activity of three lysosomal enzymes, N-acetyl-β- d -glucosaminidase (NAG), cathepsin B, and β-glucuronidase were measured, as an index of lysosomal stability in pancreas from control rats and rats under chronic alcohol and/or high fiber intake. Cathepsin B is the only enzyme with significantly increased specific activity after chronic ethanol consumption and, moreover, its specific activity undergoes the highest increase after cerulein-induced acute pancreatitis, in all the groups of rats, when compared with the remainder enzymes. When pancreatitis was induced by cerulein, the combination of chronic alcohol and high fiber intake produces a significant decrease in the cytosolic specific activity of N-acetyl-β- d -glucosaminidase and β-glucuronidase when compared with chronic alcohol alone. Our results suggest that fiber partially avoids the damage of ethanol on pancreatic lysosomes, reducing the effects of pancreatitis.