SUMMARY1. We have studied the effects of acetylcholine (ACh) on fluid secretion and intracellular messengers in interlobular ducts isolated from the rat pancreas and maintained in short-term tissue culture.2. Ductal fluid secretion was measured using micropuncture techniques.Intracellular free calcium ([Ca2+]1) and cyclic AMP concentrations were measured in single ducts using fura-2 microspectrofluorimetry and radioimmunoassay techniques respectively. Changes in the levels of these intracellular messengers were correlated with fluid secretion.3. ACh stimulated ductal fluid secretion. The dose required for a half-maximal response was about 0 4 ,/M and maximal secretion was achieved with 10 /LM ACh.These effects of ACh were blocked by atropine and by removal of extracellular Ca2". 4. ACh was about four orders of magnitude less potent as an activator of ductal fluid transport than the hormone secretin; however, the maximal rates of fluid secretion evoked by these two agonists were similar.5. ACh caused a dose-dependent rise in duct cell [Ca2+]i, but had no effect on cyclic AMP. In contrast, secretin increased duct cell cyclic AMP, but had no effect on [Ca2+]1. tTo whom reprint request should be sent.
Newcastle upon Tyne NE2 4HH, UK 1. Interlobular ducts were isolated from the rat pancreas and maintained in short-term tissue culture. Fluid secretion from these isolated ducts was measured using micropuncture techniques, intracellular calcium concentration ([Ca2+]1) by fura-2 microspectrofluorimetry, and cyclic AMP by radioimmunoassay. 2. Applying secretin and ACh simultaneously to ducts caused either a stimulation or an inhibition of fluid secretion depending on the doses employed. 3. The inhibitory effect of secretin and ACh could be relieved by atropine, and by the protein kinase C (PKC) inhibitors staurosporine and 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H-7).4. Activation of PKC by 12-0-tetradecanoylphorbol-13-acetate (TPA) and phorbol 12,13-dibutyrate (PDBu) inhibited secretin-evoked fluid secretion. 5. ACh and TPA also inhibited fluid secretion stimulated by the adenylate cyclase activator, forskolin. 6. Neither secretin nor the PKC activators and inhibitors had any effect on either the increase in [Ca2+]i evoked by ACh or the increase in intracellular cyclic AMP evoked by secretin and forskolin. 7. We conclude that the inhibitory effect of combined doses of secretin and ACh on ductal fluid secretion is probably mediated by PKC at a point in the secretory mechanism distal to the generation of intracellular messengers.
1. We have previously assessed the contributions of diffusion and convection to net potassium reabsorption in the rat proximal convoluted tubule (PCT). The present study was conducted to evaluate the role of active transport in convective potassium reabsorption by measuring the transepithelial potassium reflection coefficient (aK) in the presence and absence of cyanide in anaesthetized rats previously prepared for in vivo microperfusion. 2. Osmotic water permeability (Pf) was measured in double-perfused tubules (lumen and peritubular capillaries) by manipulating the applied transepithelial osmotic gradient between -30 and +40 mosmol (kg H20)-4 using raffinose added to or subtracted from luminal perfusates. Pf was unaffected by the presence of cyanide when the estimated dissipation of osmotic gradients along each tubule were taken into account.3. The proportion of K+ (and Na+) convectively transported with water fluxes (i.e. oJK) was not affected by cyanide. In the absence of active transport and following correction for any diffusive component, 0K was 0-56 + 0-13, indicating substantial solvent drag which probably occurs via the paracellular pathway. 4. However, cyanide caused a reduction in net potassium flux over the entire range of fluid fluxes used in double-perfusion experiments. Subsequent single-perfusion experiments (tubule lumen only) using the specific K+-H+-ATPase inhibitor, SCH28080, failed to reveal any direct evidence for a primary active K+ transporting mechanism involved in K+ reabsorption in the PCT.
1. It has recently been suggested that both formate and oxalate may be involved in the reabsorption of chloride from the proximal convoluted tubule (PCT). Reabsorption is achieved via a mechanism coupling Na+-H+ exchange to C--anion exchange thus facilitating net NaCl transport. This possible mechanism was investigated further by observing the effect of luminal addition of formate and oxalate on proximal tubule transport in anaesthetized rats, previously prepared for in vivo microperfusion. 2. Addition of formate (0-25 mmol I1) to the luminal perfusate resulted in a significant stimulation of fluid reabsorptive rate. 3. Oxalate was found to have a concentration-dependent effect on fluid reabsorption.Luminal oxalate concentrations below 0 05 mmol I1 stimulated fluid reabsorption, while an oxalate concentration of 0'5 mmol 11 inhibited fluid reabsorption.4. The presence of formate (0f25 mmol I' ) and oxalate (0O001 mmol I' ) in the same luminal perfusate stimulated fluid reabsorption from the proximal tubule lumen, but the effects of formate and oxalate were not additive. 5. The removal of bicarbonate from the luminal perfusate and its replacement with phosphate had no significant effect on fluid reabsorptive rate. An alteration in perfusate pH was also ineffective. Addition of oxalate at high and low pH failed to stimulate fluid reabsorption compared with pH-matched control perfusions. 6. The experimental data suggest the involvement of both formate and oxalate in the reabsorption of NaCl from the proximal tubule lumen, though their influence may not be exerted through the same mechanisms.
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