Effect of amiloride on electrolyte transport parameters of the main duct of the rabbit mandibular salivary gland

We have studied the response of the rabbit mandibular main duct perfused in vitro to luminally administered amiloride. The half-maximal inhibitory concentrations (K_I) when the duct was bathed in Cl solutions were: for net Na⁺ transport, 3×10^–6 mol l^–1; for transepithelial potential difference, 6×10^–6 mol l^–1; and for transepithelial conductance, 3×10^–7 mol l^–1. Substitution of the impermeant SO ₄ ^2– anion for Cl^– changed the K_I for conductance to 3×10^–6 mol l^–1. Within Cl^–-containing media, the time course of the amiloride effect on potential difference showed an early rapid fall of 10 mV with a half-time 2 s, followed by a slower depolarization of 9 mV, and the conductance change followed the slower component of the potential change. In SO ₄ ^2– -containing media, the potential difference and conductance changes followed time courses similar to one another. Finally, experiments on the effect of serosal applications of ouabain revealed that, although, in general, ouabain reduced resistance, it caused an increase in resistance in those ducts where the initial resistance was low. We conclude that: i) luminal Na⁺ transport occurs via amiloride-sensitive, conductive Na⁺ channels; ii) the Cl^– conductance is the major determinant of transepithelial conductance; iii) the first phase of the potential response is due to blocking of the Na⁺ conductive channels, whilst the slow phase reflects secondary inhibition of an electrogenic Na⁺ pump; and iv) duct resistance changes are secondary to alterations in intracellular Cl^– concentration.     

Effects of acetylcholine and forskolin on the non-electrolyte permeability of the perfused rabbit mandibular gland

Previous studies have suggested that the permeability of exocrine glands to non-electrolytes may change according to the nature and intensity of the stimuli evoking secretion. The purpose of this study was to define the nature of these permeability changes using a method that distinguishes diffusion from solvent drag. Isolated rabbit mandibular salivary glands were perfused with solutions containing¹⁴C-labelled non-electrolytes and stimulated with acetylcholine. Diffusive permeability coefficients (P) and solvent-drag filtration coefficients (1-) were estimated from the relationship between salivary non-electrolyte concentration and salivary flow rate. Filtration coefficients for urea, ethanediol, glycerol, erythritol and sucrose increased with acetylcholine concentration while, with the exception of urea, the diffusive permeabilities remained virtually unchanged. The effect of increasing acetylcholine concentration can best be explained by postulating an increase in the effective channel radius of the water secretion pathway from 0.40 nm to 0.45 nm together with a small increase in the fraction of the total water flow passing through larger non-selective pores. Forskolin had little effect on either of the permeability parameters except for a small increase in the diffusive permeability to ethanediol.     

Kinetics of Na transport in the rat submaxillary main duct perfusedin vitro

Summary The main duct of the rat submaxillary gland has been isolated and perfusedin vitro while incubated in a plasma bath. It reabsorbs Na and secretes K and HCO₃ at rates comparable to those reported for the ductin vivo and develops a transepithelial potential difference with magnitude dependent on the luminal Na concentration.The kinetics of ductal Na transport were studied by varying the Na concentration in the plasma bath step-wise from 149 to 320 mM and then the Na concentration in the luminal perfusate so as to produce zero nett flux of Na and water between lumen and bath. It was found that active Na transport by the duct showed saturation kinetics with a half-saturation constant of about 29 mM. The saturation curve could not be described in terms of a single Michaelis-Menten hyperbola although a more complex expression consisting of two such hyperbolic transport terms could easily be fitted to the data.Utilizing published values for unidirectional Na flux ratios it was calculated that the active Na transport rate when the luminal fluid had a plasma-like Na concentration was 4.37×10^–9 mol·cm^–2·s^–1 and the epithelial Na permeability coefficient was 5.27×10^–6cm·s^–1.Although the kinetics of K and HCO₃ secretion were not studied in detail it was observed incidentally that ductal secretion of these two ions still persisted even when their luminal concentrations were raised, respectively, to 197–225 mM and 80–120 mM.A preliminary report of this work was presented to a meeting of the Australian Physiological and Pharmacological Society and a Regional Meeting of the International Union of Physiological Sciences held at Sydney University in August 1972 [5].     

Acetate stimulates secretion in the rabbit mandibular gland

Abtract In isolated perfused rabbit mandibular glands undergoing stimulation with 0.8M acetylcholine, replacement of HCO ₃ ^– with acetate (25mM) increased fluid secretion by more than 100%. Other short-chain fatty acids, except for propionate, had a similar effect. We focused our further studies on acetate, and in order to find out the cause of its stimulatory effect we investigated whether acetate itself was transported. In the absence of any other transportable anions 25 mM acetate supported secretion at the same rate as 25 mM HCO ₃ ^– or 25 mM Cl^–, i.e. 20% of the control rate. In solutions containing acetate as the only major anion (146 mM), fluid secretion was maintained at about 50% of the control rate. Amiloride (1 mM) inhibited this secretion by about 90%. In glands perfused with acetate/Cl^– solutions, when the stimulatory effect was normally observed, amiloride (1 mM) inhibited secretion by 50–60% and SITS (0.1 mM) had no effect. Probenecid reversibly inhibited 75% of secretion in these glands, but it also inhibited 92% of secretion in glands perfused without any acetate. Interestingly, the acetate effect was abolished in glands stimulated with a higher concentration of acetylcholine (80 M). Results of this study suggest that acetate can be transported by salivary endpieces and that this transport involves an amiloride-sensitive Na⁺–H⁺ antiport. We postulate that acetate may in addition have some regulatory or modifier role in salivary secretion.     

Salivary duct carcinoma: report of a case and review of the literature

Summary Salivary duct carcinoma is a rare primary tumour of the salivary glands arising most frequently in the parotid gland. It has a male preponderance and occurs most often in patients over the age of 50 years. Its distinctive histological features include dilated ducts containing cells arranged in cribriform, papillary or solid patterns often with central necrosis and reminiscent of intraduct carcinoma of the breast. These features are associated with an obvious invasive component. It is an aggressive neoplasm and may metastasize widely, causing death in a high proportion of cases.     

Two independent anion transport systems in rabbit mandibular salivary glands

Cholinergically stimulated Cl and HCO₃ transport in perfused rabbit mandibular glands has been studied with extracellular anion substitution and administration of transport inhibitors. (i) In glands perfused with HCO₃-free solutions, replacement of Cl with other anions supported secretion in the following sequence: Br=>Cl>I=>NO₃>isethionate. Furosemide, 1.0 and 0.1 mmol/l, inhibited Cl-supported secretion by 97–99% and 70–78%, respectively. SITS, 0.1 mmol/l, had no effect and amiloride, 1.0 mmol/l, caused a 55–65% inhibition. Addition of SITS to amiloride-treated glands produced no further effect. (ii) In glands perfused with Cl-free solutions, but containing 25 mM HCO₃, amiloride, 1.0 mmol/l, inhibited secretion by 95% and methazolamide, 0.1 mmol/l, by 55%. (iii) In glands perfused with solutions containing both HCO₃ and Cl, furosemide had smaller effects than in glands perfused with solutions containing only Cl — a dose of 1.0 mmol/l inhibited 60% of the initial fast phase of secretion, and 90% of the later plateau phase, while a dose of 0.1 mmol/l inhibited 30% of the initial phase, but had no effect on the plateau. SITS, 0.1 mmol/l, actually stimulated secretion by about 30%, but when infused in addition to furosemide (0.1 mmol/l), it inhibited by about 20%. Amiloride (1.0 mmol/l) caused no inhibition.The results suggest that there are at least three distinct carriers in the rabbit mandibular gland. One is a furosemidesensitive Na-coupled Cl (probably Na–K–2Cl) symport, responsible for the bulk of normal secretion. The others are an amiloride-sensitive Na–H antiport and a SITS-sensitive Cl–HCO₃ antiport.     

Crystalloids in salivary duct cysts of the human parotid gland

Summary Crystalloids found in salivary duct cysts of the human parotid gland were examined by scanning electron microscopical observations with electron probe X-ray microanalysis. The cystic spaces were filled with numerous crystalloids which had a variety of forms with slight eosinophilic and glassy appearance. Scanning electron microscopically, crystalloids were hexagonal and rhombohedral in shape, and cutting the surface showed a polycyclic structure or regular parallel lamination. By electron probe X-ray microanalysis, sulphur was the only detected element. The present study suggests that crystalloids resulted from deposition from supersaturated saliva containing sulphur containing compounds into the cystic lumen or into epithelial cytoplasm.     

Coupled sodium and chloride transport into plasma membrane vesicles prepared from dogfish rectal gland

A membrane fraction, rich in basal-lateral plasma membranes, was prepared from the rectal gland of the spiny dogfish,Squalus acanthias, and the uptake of²²Na into the plasma membrane vesicles was investigated by a rapid filtration technique. Sodium uptake was greatest in the presence of a chloride gradient directed into the vesicles; it was strikingly reduced when chloride was replaced with nitrate and was even slower with gluconate. If the membrane vesicles were pre-equilibrated with potassium chloride or potassium nitrate plus valinomycin, to minimize any electrical driving forces on sodium movement, the uptake of sodium was still greatest in the presence of chloride and remarkably decreased in the presence of nitrate. Furosemide, 10^–3 and 10^–4 M, decreased sodium uptake into the vesicles in a dose dependent manner only in the presence of chloride. Furthermore, saturation of sodium uptake by increasing sodium chloride concentrations was observed. The above results provide direct evidence for a coupling of sodium and chloride fluxes across the plasma membrane of the rectal gland via a cotransport system sensitive to furosemide. They support the hypothesis that chloride secretion of the rectal gland is a secondary active transport and is driven by the sodium gradient across the basal-lateral membranes of the cell.Abbreviations Dibutyryl cyclic AMP N⁶, O²-dibutyryl adenosine 3: 5-cyclic monophosphoric acid, monosodium salt - ATP adenosine triphosphate, disodium salt - Na K-ATPase, sodiumpotassium stimulated adenosine triphosphatase - Tris tris(hydroxymethyl)aminomethane - HEPES 2-(N-2-hydroxyethylpiperazin-N'yl)ethanesulphonic acid - EDTA ethylenediaminetetraacetate Deceased October 6, 1978     

Pressor effect of angiotensin II in sodium replete and deplete rats

Summary The pressor effect of three doses of angiotensin II (AT) and of single doses of norepinephrine (N) and tyramine (T) was measured in sodium replete and deplete rats before and after ganglionic blockade. In sodium deplete rats, plasma renin concentration (PRC) and hematocrit were significantly higher and plasma sodium was significantly lower than in sodium replete rats. The pressor effect of AT, but not that of N and of T was blunted in sodium deplete rats before and after ganglionic blockade. The relationship between the pressor effect of AT and PRC was significantly negative in the combined material of all groups before ganglionic blockade. The correlation was more pronounced after ganglionic blockade, and it became significant also in the sodium deplete group alone. The pressor effect of AT was not significantly related to plasma sodium concentration and hematocrit, while both exhibited a significant correlation with PRC. Results suggest that the influence of sodium depletion on the pressor action of angiotensin may be mediated by preexisting renin or angiotensin levels and that this relationship is influenced by the autonomous nervous system.Supported by the Deutsche Forschungsgemeinschaft.     

Stimulation of proximal tubular sodium reabsorption by ile5 angiotensin II in the rat kidney

A direct dose-dependent stimulation or inhibition by val⁵-angiotensin II of sodium reabsorption in the rat proximal nephron has been shown using stationary microperfusion combined with perfusion of the peritubular capillaries. Since the circulating form of the hormone in the rat has been suggested to be the ile⁵-analogue these experiments have been repeated using the ile⁵-peptide at peritubular concentrations covering the normal physiological range (10^–10–10^–13M). Comparison of the results with the previously published data shows no significant difference between the actions of these two analogues. At the concentrations tested both caused significant stimulation of sodium reabsorption with a maximum effect at 10^–11M.     

Transcellular sodium transport and basolateral rubidium uptake in the isolated perfused cortical collecting duct

The relation between transcellular Na⁺ absorption, intracellular Na⁺ concentration and Na⁺/K⁺-ATPase activity (the last estimated by the rubidium uptake across the basolateral cell membrane) was examined in the different cell types of the rabbit cortical collecting duct (CCD). Experiments were performed on isolated perfused CCD in which Na⁺ absorption was varied by perfusing the tubule with solutions containing different Na⁺ concentrations (nominally Na⁺-free, 30 mM and 144 mM). Experiments were terminated by shock-freezing the tubules during perfusion. Precisely 30 s before shock-freezing, the K⁺ in the bathing solution was exchanged for Rb⁺. Intracellular element concentrations, including Rb⁺, were determined in freeze-dried cryosections of the tubules using energy-dispersive X-ray analysis. Increasing Na⁺ concentration in the perfusion solution caused significant rises in intracellular Na⁺ concentration and Rb⁺ uptake of principal cells. Principal cell Na⁺ and Rb⁺ concentrations were 7.8±0.9 and 7.0±0.8 mmol/kg wet weight respectively, when the perfusion solution was Na⁺-free, 10.1±0.7 and 11.6±0.6 mmol/kg wet weight with 30 mM Na⁺ in the perfusion solution, and 14.5±1.5 and 14.9 ±0.9 mmol/kg wet weight with 144 mM Na⁺ in the perfusion solution. In contrast, a comparable relationship between lumen Na⁺ concentration, intracellular Na⁺ concentration and basolateral Rb⁺ uptake was not seen in intercalated cells. These results support the notion that principal, but not intercalated, cells are involved in transepithelial Na⁺ absorption. In addition, the data demonstrate that apical Na⁺ entry and basolateral Na⁺/K⁺-AT-Pase activity are closely coupled in principal cells of the rabbit CCD. A rise in lumen Na⁺ concentration leads to increased Na⁺ entry and augmented intracellular Na⁺ concentration, which then secondarily stimulates active basolateral Na⁺/K⁺(Rb⁺) exchange.     

Intracellular pH during secretion in the perfused rabbit mandibular salivary gland measured by31P NMR spectroscopy

Intracellular pH (pH_i) was measured in the isolated, perfused rabbit mandibular salivary gland by³¹P NMR spectroscopy. In the unstimulated gland perfused with HCO ₃ ^– /CO₂-buffered Ringer's solution, pH_i was 7.27±0.01. Continuous stimulation with acetylcholine elicited dose- and time-dependent changes in pH_i. 10^–6 mol/l acetylcholine caused a brief intracellular acidosis (–0.19±0.06 pH units) followed by an increase in pH_i to a more alkaline steady-state value (7.33±0.02). In the absence of perfusate HCO ₃ ^– or in the presence of 10^–4 mol/l DIDS (4,4-diisothiocyanatostilbene-2,2-disulphonic acid), the transient acidosis was abolished and pH_i increased rapidly to give a sustained alkalosis (7.49±0.03 and 7.44±0.03 respectively). In the presence of 10^–3 mol/l amiloride, the response to acetylcholine was a rapid decrease in pH_i to 7.02±0.02. The data suggest that, during perfusion with HCO ₃ ^– /CO₂-buffered solutions, stimulation with acetylcholine results in a transient loss of HCO ₃ ^– from the acinar cells (causing a transient acidosis), and, independently, the activation of Na⁺–H⁺ exchange (causing a sustained alkalosis). In the unstimulated gland, DIDS and the HCO ₃ ^– -free perfusate caused decreases in pH_i to 7.12±0.02 and 7.04±0.01 respectively. In contrast, amiloride had little effect. The relatively high value of pH_i maintained by the unstimulated gland is therefore probably not due to Na⁺–H⁺ exchange.     

人巨细胞病毒对体外培养的人涎腺导管上皮细胞周期的影响及其相关机制

目的 研究人巨细胞病毒（HCMV）对人涎腺导管上皮细胞（HSG）细胞周期的影响及其相关机制.方法 体外培养HSG;用RT-PCR及nest-RT-PCR法检测感染HCMV的HSG中立即早期基因（ie1/ie2）的转录;用流式细胞仪检测HCMV对HSG细胞周期的影响;用蛋白印迹技术检测HCMV感染细胞中周期素D1的表达.结果 感染HCMV的HSG中可检测到HCMV ie1/ie2的转录;HCMV通过影响细胞周期的G1/S关卡,使HSG阻滞于G1期;感染HCMV的HSG周期素D1表达下调.结论 在体外HCMV通过作用于细胞周期的G1/S关卡及下调周期素D1抑制涎腺导管上皮细胞的增殖.     

Direct measurement of Na influx by23Na NMR during secretion with acetylcholine in perfused rat mandibular gland

Intracellular Na content (Na_in) in the perfused rat mandibular gland was measured by using a²³Na NMR spectroscopy at 24°C. An aqueous chemical shift reagent, dysprosium triethylenetetramine-N,N,N,N,NN-hexaacetic acid [Dy(TTHA)] was used in order to discriminate between the intracellular and the extracellular Na signal. The mandibular gland of rat was perfused arterially with a modified Krebs solution containing 10 mM Dy(TTHA). At rest, Na_in was not changed by blocking the Na⁺/K⁺ ATPase with ouabain (1 mM) and atropine (3 M), implying that, in the absence of stimulation, the spontaneous Na influx across the plasma membrane must have been negligibly small. Following onset of stimulation with acetylcholine (1 M), Na_in increased by 9.1±1.5 mmol/l intracellular fluid (mean±SEM,n=13), and remained at this level during stimulation. In the initial phase of secretion (0–5 min), about 50 mmol/min/l intracellular fluid of Na was secreted into the luminal space (estimated from the secretory rate by assuming an isotonic primary secretion) but, in spite of the higher secretion rate, Na_in increased only at an initial rate of 4.1 mmol/min/l intracellular fluid. During the steady phase of secretion (15–30 min) evoked by acetylcholine (1 M), ouabain (1 mM) caused an increment of Na_in of 44±8 mmol/l intracellular fluid (mean±SEM,n=4). From the rate of Na_in increment, the Na influx rate at the steady phase was estimated as 4.5 mmol/min/l intracellular fluid. These results suggest that the influx of Na is caused by stimulation with acetylcholine. The observed Na influx rate was about 50% of the Na secretory rate at the steady phase of secretion, estimated from the secretory rate by assuming an isotonic primary secretion. This is fully compatible with the operation of Na–K-2Cl contransport system for which one would expect a Na influx rate exactly half of the rate of Na and Cl secretion.     

Modification of salivary duct electrolyte transport in rat and rabbit by physalaemin,VIP, GIP and other enterohormones

The effects of various polypeptide enterohormones and the tachykinin secretogogue, physalaemin, on electrolyte transport by the main excretory duct of the mandibular gland of the rabbit were studied in vitro. Vasoactive intestinal peptide (VIP, 2×10^–11 mol l^–1) and gastric inhibitory polypeptide (GIP, 10^–11 mol l^–1) reduced nett Na⁺ movement from lumen to interstitium and VIP also reduced the transepithelial potential difference; the effective concentrations of the two hormones lay within the range of normal plasma concentrations. Gastrin (5×10^–7 mol l^–1) and synthetic secretin (2×10^–7 mol l^–1) had similar effects but only at concentrations well above the normal plasma levels. Caerulein, an analogue of the octapeptide of cholecystokinin, had no effect on duct function even at a concentration of 10^–6 mol l^–1. The potent salivary secretogogue, physalaemin (4×10^–8 mol l^–1), which is an analogue of SubstanceP, a putative mammalian enterohormone and neurotransmitter substance, caused a marked increase in ductal Na transport (in rat as well as rabbit). It is concluded that VIP and GIP would normally play a role in determining salivary electrolyte composition and it is postulated that their action may be antagonized by a tachykinin such as SubstanceP.Supported by the National Health and Medical Research Council of Australia. One of us (A.R.D.) is indebted to the National heart Foundation of Australia, for award of a Summer Vacation Studentship. Preliminary reports of this work have already been presented to the Australian Physiological Society [13] and the Australian Medical Research Society [15]     

Calcium transport across the rabbit thick ascending limb of Henle's loop perfused in vitro

Ca²⁺ transport across the cortical thick ascending limb of Henle's loop (TALH) was studied on the isolated rabbit renal tubule perfused in vitro. Both the efflux (Ke) and influx coefficient (Ki) of Ca²⁺ were determined with⁴⁵Ca at three different levels of the transtubular electrical potential difference (PDt) caused by varying the transtubular Na⁺ concentration gradient. The flux ratios,Ke/Ki, always exceeded those predicted from observed PDt by simple passive diffusion, and the net Ca²⁺ efflux was shown to occur against an electro-chemical potential. An increase in Ca²⁺ concentration in the perfusate was associated with a decrease inKe. Efflux of Ca²⁺, therefore, tended to be saturated as luminal Ca²⁺ concentration was increased.Ke (10⁻⁷ cm²/s) decreased from 4.13±0.56 to 2.02±0.30 (P<0.02) along with a decrease in PDt when 10⁻³ M NaCN was added to the bathing fluid. By contrast, 10⁻³ M iodoacetamide did not affectKe in spite of a significant decrease in PDt. Similarly, neither 10⁻⁵ M ouabain added to the bathing fluid nor 10⁻⁴ M ruthenium red, added either to the bathing fluid or the perfusate, affectedKe despite significant decreases in PDt. Addition of 10⁻⁵ M furosemide in the lumen caused a decrease inKe from 4.31±0.43 to 3.17±0.25 (P<0.01) in association with a decrease in PDt from 6.3±1.04 to 1.7±0.37 mV. These findings suggest; (a) Ca²⁺ transport across the TALH is mainly an active process requiring aerobic metabolism; (b) dissociation of Ca²⁺ and Cl⁻ transport may occur under some experimental conditions.     

Regulation of alveolar fluid clearance and ENaC expression in lung by exogenous angiotensin II

Angiotensin II (Ang II) has been demonstrated as a pro-inflammatory effect in acute lung injury, but studies of the effect of Ang II on the formation of pulmonary edema and alveolar filling remains unclear. Therefore, in this study the regulation of alveolar fluid clearance (AFC) and the expression of epithelial sodium channel (ENaC) by exogenous Ang II was verified. SD rats were anesthetized and were given Ang II with increasing doses (1, 10 and 100 μg/kg per min) via osmotic minipumps, whereas control rats received only saline vehicle. AT1 receptor antagonist ZD7155 (10 mg/kg) and inhibitor of cAMP degeneration rolipram (1 mg/kg) were injected intraperitoneally 30 min before administration of Ang II. The lungs were isolated for measurement of alveolar fluid clearance. The mRNA and protein expression of ENaC were detected by RT-PCR and Western blot. Exposure to higher doses of Ang II reduced AFC in a dose-dependent manner and resulted in a non-coordinate regulation of α-ENaC vs. the regulation of β- and γ-ENaC, however Ang II type 1 (AT1) receptor antagonist ZD7155 prevented the Ang II-induced inhibition of fluid clearance and dysregulation of ENaC expression. In addition, exposure to inhibitor of cAMP degradation rolipram blunted the Ang II-induced inhibition of fluid clearance. These results indicate that through activation of AT(1) receptor, exogenous Ang II promotes pulmonary edema and alveolar filling by inhibition of alveolar fluid clearance via downregulation of cAMP level and dysregulation of ENaC expression.     

Effect of aldosterone and dexamethasone pretreatment on sodium transport in rat distal colon in vitro

Aldosterone and dexamethasone stimulate sodium absorption in the rat colon in vivo. In vitro, increased amiloride inhibitable short-circuit current (I_SC) has been demonstrated following aldosterone or dexamethasone treatment. Since I_SC bears no relationship to sodium flux (J_Na) in the untreated rat colon, we measured J_Na in partially stripped voltage clamped segments of rat distal colon. Our results demonstrate directly that continuous infusion of aldosterone or dexamethasone for 4–7 days stimulated amiloride inhibitable J_Na by stimulating J_NaM-S. The amiloride inhibitable portion of J_NaM-S was highly correlated with and approximately equal to the amiloride inhibitable I_SC. Amiloride had no effect in controls. We conclude that J_NaM-S in the rat distal colon is only sensitive to mucosal amiloride after treatment with aldosterone or dexamethasone. the amiloride sensitive I_SC in these treated tissues was a good measure of the amiloride sensitive J_Na. Small differences between aldosterone and dexamethasone treatment were noted in the effect on transepithelial resistance, potential difference, and the I_SC after amiloride.     

Role of anions in the regulation of cell volume and intracellular pH in perfused rat mandibular salivary gland

 To investigate the role of Cl^– in the regulation of the basolateral transporters of salivary acinar cells, we have measured cell volume and intracellular pH (pH_i) in perfused rat mandibular glands using proton NMR spectroscopy and BCECF fluorometry respectively. When perfusate Cl^– was replaced by glucuronate, isethionate, methylsulphate, nitrate or thiocyanate, cell volume decreased slowly by about 15% over a 10-min period. Replacement with bromide, which substitutes for Cl^– on the Na⁺-K⁺-2Cl^– cotransporter, caused only a small (4%) reduction in cell volume. Replacement of Cl^– by glucuronate, isethionate or methylsulphate evoked a biphasic increase in pH_i consisting of a rapid initial increase followed by a slower secondary rise whose time course was similar to that of cell shrinkage. As judged by the effects of HCO₃ ^– omission, 100 μM 4,4’-diisothiocyanatostilbene-2,2’-disulphonic acid (DIDS) and 1 mM amiloride, the initial rise in pH_i was due to Cl^–/HCO₃ ^– exchange while the secondary rise resulted from activation of Na⁺/H⁺ exchange. Although replacement of Cl^– by nitrate or thiocyanate also caused cell shrinkage, these substituting anions were less effective in activating the exchanger. Therefore, while the upregulation of the exchanger following Cl^– replacement may be due in part to cell shrinkage, there is also evidence for the involvement of an anion-sensitive regulatory mechanism. This would be consistent with the hypothesis that both changes in cell volume and in intracellular Cl^– concentration contribute to the up-regulation of the exchanger following muscarinic stimulation. Received: 12 November 1996 / Received after revision: 11 August 1997 / Accepted: 18 August 1997