The whole-cell patch-clamp recording technique was used to measure volume-activated currents in K+-free solutions in RINm5F and HIT-T15 insulinoma cells and in dispersed rat islet cells. Cell swelling, induced by intracellular hypertonicity or extracellular hypotonicity, caused activation of an outwardly rectifying conductance which could be subsequently inactivated by hypertonic extracellular solutions. The conductance required adenosine 5'-triphosphate (ATP) in the pipette solution but was Ca2+ independent. Na+ and Cl- substitution studies suggested that the swelling-activated current is Cl- selective with a halide permeability sequence of Br > Cl > I. The conductance was reversibly inhibited by the anion channel inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Further evidence for a volume-activated anion conductance was provided by studies of volume regulation in insulin-secreting cells. When RINm5F cells were exposed to a hypotonic medium, the initial cell swelling was followed by a regulatory volume decrease (RVD). This RVD response was also inhibited by DIDS and by NPPB. These data therefore provide evidence for a volume-activated anion conductance in insulin-secreting cells which could be involved in the RVD following osmotic stress. A possible role for the conductance in hypotonically induced insulin release is also discussed.

Changes in relative cell volume in response to hypotonic solutions and glucose were studied in single isolated rat pancreatic β‐cells using a video‐imaging technique, β‐cell electrical activity was recorded under similar conditions using the perforated patch technique. Exposure of β‐cells to hypotonic solutions (10 and 33% hypotonicity) caused an immediate increase in cell volume to relative values of 1.09 and 1.33, respectively. This was followed by a gradual regulatory volume decrease. Raising the concentration of glucose from 4 to 20 mm or 12 mm (with substitution of mannitol) increased β‐cell volume by 12 and 10%, respectively. This effect of glucose persisted when Co2+ was added to inhibit insulin release. Glucose‐induced volume increases were sustained for the duration of exposure to elevated hexose concentration. The addition of 16 mm 3‐O‐methylglucose, which is transported into the β‐cell but not metabolized, produced only a transient 5% increase in β‐cell volume. Exposure of β‐cells to a 15% hypotonic solution resulted in a transient depolarization and electrical activity. Raising the glucose concentration to 20 or 12 mm caused a sustained depolarization and generation of electrical activity. However, the addition of 16 mm 3‐O‐methylglucose had no effect on β‐cell membrane potential. The glucose‐induced increase in volume and induction of electrical activity, when measured in single β‐cells simultaneously, showed comparable kinetics. The secretion of insulin from intact pancreatic islets was stimulated by exposure to hypotonic solutions (10–33% hypotonicity). A 15% hypotonic solution stimulated insulin release to a peak value comparable to that elicited by raising the glucose concentration from 4 to 20 mm. Whereas hypotonic solutions caused a transient stimulation of insulin release, the effect of glucose was sustained. It is suggested that glucose increases the volume in rat pancreatic β‐cells by a mechanism dependent upon metabolism of the sugar. The extent of cell swelling evoked by raised glucose concentrations is sufficient to depolarize the cells and induce electrical and secretory activity and may involve activation of a volume‐sensitive anion conductance.

No abstract

Aldosterone stimulates sodium transport in the inner medullary collecting duct (IMCD) via the classic genomic pathway, but it is not known whether it also acts via a rapid, non-conventional pathway in this part of the nephron. The IMCD regulates the final sodium content of urine and expresses vasopressin receptors coupled to adenylate cyclase. The recently reported rapid, nongenomic actions of aldosterone have been associated mainly with an increase in intracellular Ca 2+; however, it has also been shown to stimulate cAMP generation. Thus the aim of this study was to determine whether aldosterone stimulates rapid generation of cAMP in isolated IMCD segments. IMCD segments were microdissected from Sprague-Dawley rat kidneys and incubated at 37 C for 4 min with aldosterone (10 12 to 10 6 M), vasopressin (10 12 to 10 6 M), or a combination of hormones in the presence of a phosphodiesterase inhibitor. cAMP was measured by radioimmunoassay. While corticosterone and dexamethasone were ineffective, aldosterone stimulated a dose-dependent increase in cAMP within 4 min (P<0·05). This action of aldosterone was not inhibited by the MR antagonist spironolactone. Co-incubation of aldosterone with vasopressin resulted in a further increase in cAMP generation above that induced by the neurohypophysial hormone alone. Aldosterone-mediated cAMP generation was not inhibited by a vasopressin V 1 or V 2 receptor antagonist. These data support a novel and rapid, non-genomic effect of aldosterone in IMCD. Aldosterone does not apparently interact with the vasopressin receptor to stimulate cAMP generation.

Effective assessment of laboratory practicals is a challenge for large-size classes. To reduce the administrative burden of staff members without compromising the student learning experience, we utilized dedicated computer software for short-answer question assessment for nearly 300 students and compared it with the more traditional, paper-based method of assessment of the same student cohort. Students were generally favorably disposed toward computer-assisted assessment (CAA): 75% of the students responded that for future assignments, they either had no preference for the method of assessment or would prefer CAA. Advantages were perceived to be remote access to the questions and ease of submission. The most common disadvantage cited was lack of internet access. Various advantages of CAA were mentioned by staff members: notably, the reduction in marking time and reduction of paperwork as well as the potential for the software to detect plagiarism and to administer anonymous marking. Disadvantages to CAA were the need to tailor questions to the technology, having to adapt to reading answers and marking onscreen, and the quality of feedback to students. All of the disadvantages could be overcome by training and improved versions of CAA software, currently under development. The use of CAA has proved to be a welcome addition to the tools available to staff members for the assessment of practical classes, and future improved versions of the software will increase the utility of this assessment method.