Combined cystourethroplasty and urethral sphincter occluder placement for treatment of urinary incontinence in a cat with genitourinary dysplasia.

A 10-month-old female neutered domestic shorthair cat presented with a history of urinary incontinence and recurrent bacterial cystitis since 2 months of age. Physical examination was unremarkable other than vulval hypoplasia. Genitourinary dysplasia was diagnosed following a physical examination, CT imaging and retrograde positive contrast urethrocystography. Attempted medical management of the urinary incontinence was unsuccessful. Surgical treatment consisted of cystourethroplasty combined with artificial urethral sphincter placement, resulting in marked improvement of urinary continence. Following multiple adjustments of the artificial urethral sphincter near-total continence was achieved, which was maintained at 10 months post-surgery.

Intra-thoracic gossypiboma (textiloma) in a 6-year-old cocker spaniel.

This report documents the first case of gossypiboma (textiloma) identified within the thorax of a dog. CT findings, surgical removal and histopathology are described. Intra-thoracic gossypiboma has not previously been reported in dogs and is rarely reported in the human medical literature, where it is most commonly associated with previous cardiac or pulmonary surgery. This dog had previously had a thoracotomy for attempted surgical correction of a persistent right aortic arch and left ligamentum arteriosum 6 years prior to presentation. A brief review of the previous literature and recommendations for prevention of this condition are provided.

NVP-QBE170: an inhaled blocker of the epithelial sodium channel with a reduced potential to induce hyperkalaemia.

BACKGROUND AND PURPOSE: Inhaled amiloride, a blocker of the epithelial sodium channel (ENaC), enhances mucociliary clearance (MCC) in cystic fibrosis (CF) patients. However, the dose of amiloride is limited by the mechanism-based side effect of hyperkalaemia resulting from renal ENaC blockade. Inhaled ENaC blockers with a reduced potential to induce hyperkalaemia provide a therapeutic strategy to improve mucosal hydration and MCC in the lungs of CF patients. The present study describes the preclinical profile of a novel ENaC blocker, NVP-QBE170, designed for inhaled delivery, with a reduced potential to induce hyperkalaemia. EXPERIMENTAL APPROACH: The in vitro potency and duration of action of NVP-QBE170 were compared with amiloride and a newer ENaC blocker, P552-02, in primary human bronchial epithelial cells (HBECs) by short-circuit current. In vivo efficacy and safety were assessed in guinea pig (tracheal potential difference/hyperkalaemia), rat (hyperkalaemia) and sheep (MCC). KEY RESULTS: In vitro, NVP-QBE170 potently inhibited ENaC function in HBEC and showed a longer duration of action to comparator molecules. In vivo, intratracheal (i.t.) instillation of NVP-QBE170 attenuated ENaC activity in the guinea pig airways with greater potency and duration of action than that of amiloride without inducing hyperkalaemia in either guinea pig or rat. Dry powder inhalation of NVP-QBE170 by conscious sheep increased MCC and was better than inhaled hypertonic saline in terms of efficacy and duration of action. CONCLUSIONS AND IMPLICATIONS: NVP-QBE170 highlights the potential for inhaled ENaC blockers to exhibit efficacy in the airways with a reduced risk of hyperkalaemia, relative to existing compounds.

Hydrogen-bonding in the pyrimidine···NH3 van der Waals complex: experiment and theory.

The pyrimidineNH3 van der Waals complex has been studied using a combination of resonant two-photon ionisation (R2PI) spectroscopy, ab initio molecular orbital calculations and multidimensional Franck-Condon analysis. The R2PI spectrum is assignable to a single stable conformer in which the ammonia molecule binds via two hydrogen bonds within the plane of the ring, in a location which minimises repulsion between the ammonia nitrogen lone pair and that of the second, more remote pyrimidine nitrogen in the 3 position on the opposite side of the ring. Ground state estimated CCSD(T) interaction energies were extrapolated to the complete basis set limit: these calculations found the dissociation energy of the most stable conformer, σ(B), to be 20% larger than that of a second in-plane conformer, σ(A), in which the ammonia forms a similar pseudo five-membered ring, bridging the nitrogen at the 1 position with the carbon at the 2 position. This conformation in turn was found to have a dissociation energy 35% larger than that of a π-complex in which the ammonia binds above the plane of the aromatic ring. The results of multidimensional Franck-Condon simulations based on ab initio ground and excited state CASSCF and RICC2 geometry optimisations and vibrational frequency calculations showed good agreement with experiment. It is postulated that longer-range electrostatic interactions between the ammonia lone pair and the more distant of the two ring nitrogens on the pyrimidine, play a key role in determining which of the two in-plane structures is the more stable and which, therefore, is responsible for all of the spectral features observed in the R2PI spectrum.

Complications of lateral plate fixation compared with tension band wiring and pin or lag screw fixation for calcaneoquartal arthrodesis. Treatment of proximal intertarsal subluxation occurring secondary to non-traumatic plantar tarsal ligament disruption in dogs.

OBJECTIVE: To compare complication rates and the outcomes of these complications after lateral plate fixation with figure-of-eight tension-band-wire and pin or lag screw fixation for arthrodesis of the calcaneoquartal joint, following non-traumatic disruption of the plantar tarsal ligament in dogs. METHODS: Data were collected retrospectively from five UK referral centres. Diplomate specialists and their residents performed all procedures. Referring veterinarians were contacted for long-term follow-up. RESULTS: Seventy-four procedures were undertaken in 61 dogs. There were 58 arthrodeses in the lateral plate group (Plate), nine in the pin and tension-band-wire group (Pin), and seven in the lag screw and tension-band wire-group (Screw). Compared to Plate (17%), further surgical intervention was required more frequently following Pin (56%, OR = 3.2) or Screw (43%, OR = 2.5) fixation. Clinical failure of arthrodesis occurred less frequently with Plate (5%) compared with Screw (43%, OR = 8.6) and Pin fixation (22%, OR = 4.4). Cases managed with external coaptation postoperatively were more likely to suffer from postoperative complications (OR = 2.2). CLINICAL SIGNIFICANCE: Lateral plating was associated with fewer postoperative complications than pin and tension-band-wire fixation for arthrodesis of the calcaneoquartal joint in dogs with non-traumatic disruption of the plantar tarsal ligament.

Camostat attenuates airway epithelial sodium channel function in vivo through the inhibition of a channel-activating protease.

Inhibition of airway epithelial sodium channel (ENaC) function enhances mucociliary clearance (MCC). ENaC is positively regulated by channel-activating proteases (CAPs), and CAP inhibitors are therefore predicted to be beneficial in diseases associated with impaired MCC. The aims of the present study were to 1) identify low-molecular-weight inhibitors of airway CAPs and 2) to establish whether such CAP inhibitors would translate into a negative regulation of ENaC function in vivo, with a consequent enhancement of MCC. To this end, camostat, a trypsin-like protease inhibitor, provided a potent (IC(50) approximately 50 nM) and prolonged attenuation of ENaC function in human airway epithelial cell models that was reversible upon the addition of excess trypsin. In primary human bronchial epithelial cells, a potency order of placental bikunin > camostat > 4-guanidinobenzoic acid 4-carboxymethyl-phenyl ester > aprotinin >> soybean trypsin inhibitor = alpha1-antitrypsin, was largely consistent with that observed for inhibition of prostasin, a molecular candidate for the airway CAP. In vivo, topical airway administration of camostat induced a potent and prolonged attenuation of ENaC activity in the guinea pig trachea (ED(50) = 3 microg/kg). When administered by aerosol inhalation in conscious sheep, camostat enhanced MCC out to at least 5 h after inhaled dosing. In summary, camostat attenuates ENaC function and enhances MCC, providing an opportunity for this approach toward the negative regulation of ENaC function to be tested therapeutically.

The guinea-pig tracheal potential difference as an in vivo model for the study of epithelial sodium channel function in the airways.

BACKGROUND AND PURPOSE: The epithelial sodium channel (ENaC) is a key regulator of airway mucosal hydration and mucus clearance. Negative regulation of airway ENaC function is predicted to be of clinical benefit in the cystic fibrosis lung. The aim of this study was to develop a small animal model to enable the direct assessment of airway ENaC function in vivo. EXPERIMENTAL APPROACH: Tracheal potential difference (TPD) was utilized as a measure of airway epithelial ion transport in the guinea-pig. ENaC activity in the trachea was established with a dose-response assessment to a panel of well-characterized direct and indirect pharmacological modulators of ENaC function, delivered by intra-tracheal (i.t.) instillation. KEY RESULTS: The TPD in anaesthetized guinea-pigs was attenuated by the direct ENaC blockers: amiloride, benzamil and CF552 with ED(50) values of 16, 14 and 0.2 microg kg(-1) (i.t.), respectively. 5-(N-Ethyl-N-isopropyl) amiloride, a structurally related compound but devoid of activity on ENaC, was without effect on the TPD. Intra-tracheal dosing of the Kunitz-type serine protease inhibitors aprotinin and placental bikunin, which have previously been demonstrated to inhibit proteolytic activation of ENaC, likewise potently attenuated TPD in guinea-pigs, whereas alpha(1)-antitrypsin and soya bean trypsin inhibitor were without effect. CONCLUSIONS AND IMPLICATIONS: The pharmacological sensitivity of the TPD to amiloride analogues and also to serine protease inhibitors are both consistent with that of ENaC activity in the guinea-pig trachea. The guinea-pig TPD therefore represents a suitable in vivo model of human airway epithelial ion transport.

Identification and mapping of human saphenous vein medial smooth muscle proteins by two-dimensional polyacrylamide gel electrophoresis.

Changing smooth muscle phenotype and abnormal cell proliferation are important features of vascular pathology, including the failure of saphenous vein bypass grafts. We have characterised and mapped protein expression in human saphenous vein medial smooth muscle, using two-dimensional (2-D) polyacrylamide gel electrophoresis. The 2-D system comprised a nonlinear immobilised pH 3-10 gradient in the first dimension (separating proteins with isoelectric point values between pH 3-10), and 12%T total gel concentration sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension (separating proteins in the range 14,000-200,000 Daltons). Using a combination of peptide mass fingerprinting by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and partial amino acid sequencing by nanospray tandem mass spectrometry, a subset of 149 protein spots was analysed, with 129 protein spots being identified and mapped. The data presented here are an important addition to the limited knowledge of venous medial smooth muscle protein expression in vivo. Our protein map will facilitate the identification of proteins differentially expressed in human saphenous vein bypass grafts. In turn, this may lead to the elucidation of molecular events involved in saphenous vein bypass graft failure. The map should also provide a basis for comparative studies of protein expression in vascular smooth muscle of varying origins.

Arterial flow induces changes in saphenous vein endothelium proteins transduced by cation channels.

OBJECTIVES: expression of leukocyte adhesins and proteins controlling thrombosis is likely to be an important determinant of graft patency early following vein bypass. We have previously demonstrated rapid increase in endothelial ICAM-1 and nitric oxide synthase (eNOS) concentrations in human saphenous vein exposed to arterial flow. The aim of this study was to investigate whether ion-channel-blocking drugs could alter these flow-induced changes. METHODS: human saphenous vein segments, freshly excised from patients, were placed in a validated in vitro circuit using flow conditions shown to simulate arterial or venous circulations for 90 min, in the presence or absence of ion-channel blockers. The concentrations of ICAM-1, VCAM-1, eNOS and tissue factor (TF) were assessed by quantitative immunohistochemistry in vein exposed to flow and compared with that in freshly excised vein from the same patient. The endothelial protein concentration was calculated as the mean area of staining as percentage of that for the control protein CD31, using computer-aided image analysis. RESULTS: after arterial flow conditions the area ratio of ICAM-1 increased from 21.4+/-1.4 to 44.6+/-2.0%, of eNOS increased from 50.0+/-5.6 to 70.1+/-5.0%, of VCAM-1 decreased from 16.6+/-3.4 to 3.6+/-1.0%, whereas TF staining area ratio was unchanged. Inclusion of the non-selective K(+)channel blocker, tetraethylammonium, in the arterial perfusion solution abolished all these arterial flow-induced changes. Inclusion of the K(+)ATP channel blocker, glibenclamide, selectively abolished the arterial flow-induced changes in ICAM-1 and VCAM-1. Inclusion of the calcium channel blocker, nifedipine, abolished the arterial flow-induced changes in eNOS and VCAM-1 but increased the TF staining area ratio from 3.0+/-0.4 to 8.5+/-0.7%, p=0.01. Inclusion of the stretch-activated cation-channel blocker, gadolinium, enhanced the arterial flow-induced increase in eNOS, but prevented the arterial flow-induced increase in ICAM-1. CONCLUSIONS: perfusion of veins under arterial flow conditions with gadolinium was associated with low endothelial concentrations of ICAM-1, VCAM-1 and TF, but high levels of eNOS. Such a concentration of endothelial proteins may be advantageous in newly implanted vein grafts. In contrast, nifedipine could have adverse effects by promoting increase in TF concentration.

Rapid changes in the coagulant proteins on saphenous vein endothelium in response to arterial flow.

Healthy endothelium provides a nonthrombogenic surface. In this study the authors investigated the effect of arterial flow on the saphenous vein endothelial expression of proteins controlling thrombosis. Human saphenous vein segments, freshly excised from patients, were placed in a validated in vitro circuit with flow conditions shown to simulate arterial or venous circulations. In separate experiments, placement of an external polytetrafluoroethylene (PTFE) stent was used to differentiate the effects of pulsatile wall deformation and shear stress, while addition of drugs to the vein perfusate allowed study of the role of ion channels in transducing the response of the vein to arterial flow. Endothelial concentrations of thrombomodulin, nitric oxide synthase, tissue factor, and tissue plasminogen activator were assessed by quantitative immunohistochemistry and Western blotting of endothelial cell lysates, in paired vein samples, in comparison to control proteins. Arterial flow conditions caused a rapid and significant reduction in the endothelial concentration of thrombomodulin: The immunostaining area decreased from 80.1 +/- 7.0 to 48.3 +/- 5.0 and 32.9 +/- 3.0% at 45 and 90 minutes respectively, p = 0.01. These findings were confirmed by Western blotting. The reduction in thrombomodulin concentration was unaffected by eliminating vein wall deformation by placement of an external PTFE stent or by including the K+ channel blocker tetraethylammonium (TEA) in the vein perfusate. In contrast, thrombomodulin concentrations remained high when blockers of stretch-activated cation and calcium channels were included in the vein perfusate. The endothelial concentration of nitric oxide synthase increased after 90 minutes of arterial flow and this change was abolished when TEA was included in the vein perfusate. Arterial flow induced rapid changes in saphenous vein antithrombotic proteins. Different cation channels mediated the flow-induced changes in thrombomodulin and nitric oxide synthase.

Arterial flow conditions downregulate thrombomodulin on saphenous vein endothelium.

BACKGROUND: The antithrombogenic properties of venous endothelium may be attenuated when vein is implanted in the arterial circulation. Such changes may facilitate thrombosis, which is the final common pathway for saphenous vein arterial bypass graft occlusion. METHODS AND RESULTS: Using human saphenous vein in a validated ex vivo flow circuit, we investigated (1) the possibility that arterial flow conditions (mean pressure, 100 mm Hg, 90 cpm, approximately 200 mL/min) alter the concentration of proteins involved in regulating thrombosis at the vessel wall and (2) the influence of ion channel blockade on such effects. Concentrations of thrombomodulin and tissue factor were quantified by Western blotting (ratio of von Willebrand factor staining) and immunohistochemistry (as a percentage of CD31-staining area). Thrombomodulin concentrations after 90 minutes of venous and arterial flow conditions were quantified by immunostaining (68.9+/-4.8% and 41.0+/-3.0% CD31, respectively; P<0.01) and by Western blotting (1.35+/-0.20 and 0. 15+/-0.03 ratio of von Willebrand factor, respectively; P<0.01). The ability of endothelial cells to generate activated protein C also decreased from 62+/-14 to 19+/-10 ng. min-1. 1000 cells-1 (P=0.01). The significant reduction in thrombomodulin was attenuated if calcium was removed from the perfusate but not by external vein stenting. Inclusion in the vein perfusate of drugs that reduce calcium entry (including Gd3+, to block stretch-activated ion channels, and nifedipine) abolished the reduction in thrombomodulin concentration observed after arterial flow conditions. In freshly excised vein, negligible concentrations of tissue factor were detected on the endothelium and concentrations did not increase after 90 minutes of arterial flow conditions, although the inclusion of nifedipine caused the immunostaining to increase from 3.0+/-0.4% to 8.5+/-0.7% CD31 (P<0.02). CONCLUSIONS: In saphenous vein endothelium exposed to arterial flow conditions, there is rapid downregulation of thrombomodulin, sufficient to limit protein C activation, by a calcium-dependent mechanism.

Molecular and cellular changes in vein grafts: influence of pulsatile stretch.

Adaptation of saphenous vein, with its intrinsic myogenic tone, from the low-pressure, minimally pulsatile flow of the venous system to the pulsatile flow of the arterial circulation is a minor miracle. Changes in gene expression caused by the pulsatile circumferential deformation (cyclic strain) initiate changes in gene expression, which lead to both vein graft adaptation and pathology. Removal of circumferential deformation by external stenting attenuates the early changes in gene expression and the later development of intimal hyperplasia. Pathways for the transduction of cyclic strain into cellular events have been elucidated in cultured vascular cells. The important second messengers include calcium and reactive oxygen species.

Human saphenous vein endothelial cells express a tetrodotoxin-resistant, voltage-gated sodium current.

Whole-cell patch-clamp electrophysiological investigation of endothelial cells cultured from human saphenous vein (HSVECs) has identified a voltage-gated Na+ current with a mean peak magnitude of -595 +/- 49 pA (n = 75). This current was inhibited by tetrodotoxin (TTX) in a concentration-dependent manner, with an IC50 value of 4.7 microM, suggesting that it was of the TTX-resistant subtype. An antibody directed against the highly conserved intracellular linker region between domains III and IV of known Na+ channel alpha-subunits was able to retard current inactivation when applied intracellularly. This antibody identified a 245-kDa protein from membrane lysates on Western blotting and positively immunolabeled both cultured HSVECs and intact venous endothelium. HSVECs were also shown by reverse transcription-polymerase chain reaction to contain transcripts of the hH1 sodium channel gene. The expression of Na+ channels by HSVECs was shown using electrophysiology and cell-based enzyme-linked immunosorbent assay to be dependent on the concentration and source of human serum. Together, these results suggest that TTX-resistant Na+ channels of the hH1 isoform are expressed in human saphenous vein endothelium and that the presence of these channels is controlled by a serum factor.

The effects of potassium channel openers on saphenous vein exposed to arterial flow.

OBJECTIVES: To assess the sensitivity of saphenous vein to potassium channel opening drugs (KCOs). METHODS: Saphenous vein, harvested at bypass surgery or high ligation for correction of varicose veins, was exposed to an in vitro flow circuit and vasomotor responses assessed by organ bath pharmacology. OUTCOME MEASURES: Effective drug concentrations for 50% reduction in vein ring tension (IC50). RESULTS: Vein rings pre-contracted with phenylephrine showed a concentration-dependent relaxation to all the KCOs tested with a potency ranking of HOE 234 > cromakalim > pinacidil > diazoxide. The relaxation to cromakalim was endothelium-independent and was inhibited by glibenclamide (an ATP-sensitive K+ channel blocker). The sensitivity of vein rings to cromakalim increased after exposure to arterial flow conditions for 90 minutes (IC50 before 1.7 +/- 0.25 microM and after 0.25 +/- 0.08 microM, p > 0.001). This effect was not evident after 90 min of venous flow conditions, 2.19 +/- 0.49 microM. When the workload on vein, exposed to arterial flow conditions, was reduced mechanically by external stenting with PTFE the increased sensitivity to cromakalim was abolished. CONCLUSIONS: Saphenous vein has ATP-sensitive K+ channels responsive to KCOs. The increased sensitivity to cromakalim, induced by arterial flow conditions, may represent an endogenous protective mechanism limiting ischaemic damage resulting from the higher workload imposed on grafted vein.

Direct inhibitory effects of an antisense oligodeoxynucleotide upon the volume-sensitive chloride current in rat osteoblast-like (ROS 17/2.8) cells.

The effects of a 15-mer antisense c-myc phosphorothioate modified oligodeoxynucleotide (OdN) upon the volume-sensitive Cl- current in ROS 17/2.8 cells were investigated using the whole-cell configuration of the patch clamp technique. At 5 microM, the OdN reversibly inhibited the current in a voltage- and time-dependent fashion. This was evident from the reduction in the peak current as assessed at the termination of each voltage pulse and an acceleration of the time-dependent inactivation present at strongly depolarised potentials. The kinetic modifications induced by the OdN suggest it may act by blocking the pore of open channels when the cell membrane potential is depolarised.

Effects of arachidonic acid upon the volume-sensitive chloride current in rat osteoblast-like (ROS 17/2.8) cells.

1. The effects of arachidonic acid upon the volume-sensitive Cl- current present in cultured osteoblastic cells (ROS 17/2.8) was studied using the whole-cell patch-clamp technique. 2. Arachidonate produced two distinct phases of inhibition, a rapid phase occurring within 10-15 s of application, preceding a slower phase that occurred 2 min after onset of arachidonate superfusion. Accompanying the slower inhibitory phase was an acceleration of the time-dependent inactivation exhibited by the current at strongly depolarized potentials (> + 50 mV). The half-maximal inhibitory concentrations (IC50) were 177 +/- 31 and 10 +/- 4 microM for the two phases, respectively. 3. Arachidonate was still effective in the presence of inhibitors of cyclo-oxygenase (indomethacin, 10 microM), lipoxygenase (nordihydroguaretic acid, 10-100 microM) and cytochrome P450 (SKF525A, 100 microM; ethoxyresorufin, 10 microM; metyrapone, 500 microM; piperonyl butoxide, 500 microM; cimetidine, 1 mM). The effects of arachidonate could not be produced by another cis unsaturated fatty acid, oleic acid. 4. Measurements of cell volume showed that arachidonate effectively inhibited the regulatory volume decrease elicited by ROS 17/2.8 cells in response to a reduction in extracellular osmolarity. 5. It is concluded that the volume-sensitive Cl- conductance in ROS 17/2.8 cells is directly modulated by arachidonate and may represent a physiological mechanism by which volume regulation can be controlled in these cells.

Activation of calcium-sensitive potassium channels in L6 skeletal myocytes by arginine vasopressin requires extracellular calcium.

The effects of extracellular application of arginine vasopressin (AVP) upon membrane currents in L6 skeletal myocytes was investigated using the whole-cell configuration of the patch-clamp technique. At 0 mV AVP produced large amplitude, transient outward currents that reversed when the clamping potential was changed to -100 mV (negative to EK). The effects of alterations in the extracellular K+ concentration upon the current reversal potential suggested that the current elicited by AVP was carried mainly by K+ ions. Intracellular dialysis with 10 microM inositol 1,4,5-trisphosphate (InsP3) elicited similar currents but only in 6/14 cells. Inclusion of 5 mg ml-1 heparin in the intracellular solutions was ineffective at inhibiting the current responses to AVP. The AVP-induced current was totally abolished when the intracellular EGTA concentration was increased from 0.05 mM to 10 mM or Ca2+ was removed from the extracellular perfusing solution. These results suggest that AVP produces activation of a Ca(2+)-sensitive K+ conductance in L6 skeletal myocytes by a process dependent upon extracellular Ca2+ and not intracellular Ca2+ release.

Characterization of a volume-sensitive chloride current in rat osteoblast-like (ROS 17/2.8) cells.

1. During osmotic swelling, cultured osteoblastic cells (ROS 17/2.8) exhibited activation of large amplitude Cl- currents in the whole-cell configuration of the patch-clamp technique. Effects of hypotonic shock on cell volume and membrane conductance were rapidly reversed on return to isotonic conditions. 2. Voltage command pulses in the range -80 to +50 mV produce instantaneous activation of Cl- currents. At potentials more positive than +50 mV the current exhibited time-dependent inactivation. The instantaneous current-voltage relationship was outwardly rectifying. 3. The anion permeability sequence of the induced current was SCN- (2.2) > i- (1.9) > Br- (1.5) > Cl- (1.0) > F- (0.8) > gluconate- (0.2). This corresponds to Eisenman's sequence I. 4. The volume-sensitive Cl- current was effectively inhibited by the Cl- channel blockers 4,4'- diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Outward currents were more effectively suppressed by DIDS than inward currents. The concentrations for 50% inhibition (IC50) of outward and inward currents were 81 and 298 microM, respectively. NPPB was equally effective at inhibiting outward and inward currents (IC50 of 64 microM). The current was relatively insensitive to diphenylamine-2-carboxylate (DPC), 500 microM producing only 22.5 +/- 4.0% inhibition. 5. Inhibitors of protein kinase A (H-89, 1 microM) and tyrosine kinase (tyrphostin A25, 200 microM) were without effect upon activation of Cl- currents in response to hypotonic shock. Under isotonic conditions, elevation of intracellular Ca2+ by ionomycin (1 microM) or activation of protein kinase C by 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.1 microM) failed to evoke increases in basal Cl- conductance levels. 6. It is concluded that an outwardly rectifying Cl- conductance is activated upon osmotic swelling and may be involved in cell volume regulation of ROS 17/2.8 cells.