Using innovative group-work activities to enhance the problem-based learning experience for dental students.

Problem-based learning (PBL) in medical and dental curricula is now well established, as such courses are seen to equip students with valuable transferable skills (e.g. problem-solving or team-working abilities), in addition to knowledge acquisition. However, it is often assumed that students improve in such skills without actually providing direct opportunity for practice, and without giving students feedback on their performance. 'The Manchester Dental Programme' (TMDP) was developed at The University of Manchester, UK as a 5-year, integrated enquiry-led curriculum. The existing PBL course was redesigned to include a unique, additional PBL session ('Session 4') that incorporated an activity for the group to complete, based on the subject material covered during student self-study. A summative mark was awarded for each activity that reflected the teamwork, organisational and overall capabilities of the groups. This paper describes the different types of activities developed for the Session 4 and presents an analysis of the perceptions of the students and staff involved. The student response to the Session 4 activities, obtained via questionnaires, was extremely positive, with the majority finding them fun, yet challenging, and 'worthwhile'. The activities were perceived to enhance subject understanding; develop students' problem-solving skills; allow the application of knowledge to new situations, and helped to identify gaps in knowledge to direct further study. Staff found the activities innovative and exciting learning tools for the students. The Session 4 activities described here are useful educational resources that could be adapted for other PBL courses in a wide variety of subject areas.

Molecular mechanisms of cerebrospinal fluid production.

The epithelial cells of the choroid plexuses secrete cerebrospinal fluid (CSF), by a process which involves the transport of Na(+), Cl(-) and HCO(3)(-) from the blood to the ventricles of the brain. The unidirectional transport of ions is achieved due to the polarity of the epithelium, i.e. the ion transport proteins in the blood-facing (basolateral) membrane are different to those in the ventricular (apical) membrane. The movement of ions creates an osmotic gradient which drives the secretion of H(2)O. A variety of methods (e.g. isotope flux studies, electrophysiological, RT-PCR, in situ hybridization and immunocytochemistry) have been used to determine the expression of ion transporters and channels in the choroid plexus epithelium. Most of these transporters have now been localized to specific membranes. For example, Na(+)-K(+)ATPase, K(+) channels and Na(+)-2Cl(-)-K(+) cotransporters are expressed in the apical membrane. By contrast the basolateral membrane contains Cl(-)- HCO(3) exchangers, a variety of Na(+) coupled HCO(3)(-) transporters and K(+)-Cl(-) cotransporters. Aquaporin 1 mediates water transport at the apical membrane, but the route across the basolateral membrane is unknown. A model of CSF secretion by the mammalian choroid plexus is proposed which accommodates these proteins. The model also explains the mechanisms by which K(+) is transported from the CSF to the blood.

Expression of the Na+K+-2CI- cotransporter in alpha and beta cells isolated from the rat pancreas.

The expression of the Na+-K+-2Cl- cotransporter (NKCC1) in alpha cells and beta cells from the rat pancreas was examined. Isolated alpha cells and beta cells in a mixed islet cell preparation were identified by volume using video-imaging methods, and by the expression of glucagon or insulin. The expression of mRNA for NKCC1 in pancreatic islets was demonstrated by RT-PCR. Immunocytochemical studies showed that the NKCCI protein was expressed in rat beta cells, but not alpha cells. The activity of Na+-K+-2Cl- cotransporter was also examined, by studying cell volume regulation in response to HEPES-buffered, hypertonic solutions. A regulatory volume increase was observed in the beta cells but not the alpha cells. It is concluded that the NKCC1 is expressed in rat pancreatic beta cells but not alpha cells. This is consistent with the hypothesis that Cl- is accumulated above the expected equilibrium distribution in beta cells, but is below equilibrium in alpha cells.

Functional characterisation of the volume-sensitive anion channel in rat pancreatic beta-cells.

The whole-cell and perforated patch configurations of the patch-clamp technique were used to characterise the volume-sensitive anion channel in rat pancreatic beta-cells. The channel showed high permeability (P ) relative to Cl(-) to extracellular monovalent organic anions (P(SCN)/P(Cll) = 1.73, P(acetate)/P(Cll) = 0.39, P(lactate)/P(Cll) = 0.38, P(acetoacetate)/P(Cll) = 0.32, P(glutamate)/P(Cll) = 0.28) but was less permeable to the divalent anion malate (P(malate)/P(Cll) = 0.14). Channel activity was inhibited by a number of putative anion channel inhibitors, including extracellular ATP (10 mM), 1,9-dideoxyforskolin (100 microM) and 4-OH tamoxifen (10 microM). Inclusion of the catalytic subunit of protein kinase A in the pipette solution did not activate the volume-sensitive anion channel in non-swollen cells. Furthermore, addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or forskolin failed to activate the channel in intact cells under perforated patch conditions. Addition of phorbol 12,13-dibutyrate (200 nM), either before or after cell swelling, also failed to affect channel activation. Our findings do not support the suggestion that the volume-sensitive anion channel in pancreatic beta-cells can be activated by protein kinase A. Furthermore, the beta-cell channel does not appear to be subject to regulation via protein kinase C. Experimental Physiology (2001) 86.2, 145-150.

Mechanisms of CSF secretion by the choroid plexus.

The epithelial cells of the choroid plexus secrete cerebrospinal fluid (CSF), by a process that involves the movement of Na(+), Cl(-) and HCO(3)(-) from the blood to the ventricles of the brain. This creates the osmotic gradient, which drives the secretion of H(2)O. The unidirectional movement of the ions is achieved due to the polarity of the epithelium, i.e., the ion transport proteins in the blood-facing (basolateral) are different to those in the ventricular (apical) membranes. Saito and Wright (1983) proposed a model for secretion by the amphibian choroid plexus, in which secretion was dependent on activity of HCO(3)(-) channels in the apical membrane. The patch clamp method has now been used to study the ion channels expressed in rat choroid plexus. Two potassium channels have been observed that have a role in maintaining the membrane potential of the epithelial cell, and in regulating the transport of K(+) across the epithelium. An inward-rectifying anion channel has also been identified, which is closely related to ClC-2 channels, and has a significant HCO(3)(-) permeability. This channel is expressed in the apical membrane of the epithelium where it may play an important role in CSF secretion. A model of CSF secretion by the mammalian choroid plexus is proposed that accommodates these channels and other data on the expression of transport proteins in the choroid plexus.

Modulation of calcium signalling by intracellular pH in exocrine acinar cells.

Cell-permeant weak acids and bases alter the rate of fluid and electrolyte secretion by a range of epithelia, including the exocrine glands. It is widely assumed that weak acids and bases exert these effects by participating in the ion transport mechanism, or by changing intracellular pH (pHi) and hence modulating electrolyte (ion) transporters. An alternative possibility is that these substances act by modifying the intracellular calcium signals which control fluid secretion. In the present study we have examined whether weak acids and bases modify intracellular free calcium ([Ca2+]i) in exocrine acinar cells. Alkalinization with weak bases and acidification with weak acids had quite different effects on [Ca2+]i in resting and agonist-stimulated cells. In unstimulated lacrimal, salivary or pancreatic acinar cells, acidifying the cytosol had no effect on [Ca2+]i, while cytosolic alkalinization caused a modest rise in [Ca2+]i. This alkalinization-induced increase in [Ca2+]i appears to result from Ca2+ release from agonist-sensitive stores, and was probably caused by a small increase in intracellular InsP3 levels. In contrast, [Ca2+]i decreased when intracellular alkalinization was induced during agonist stimulation. Conversely, acidifying the cytosol during agonist stimulation raised [Ca2+]i. This latter effect was particularly dramatic in pancreatic acinar cells, where cytosolic acidification also enhanced agonist-evoked [Ca2+]i oscillations. The effects of pHi on [Ca2+]i in stimulated cells could also be observed in Ca2+-free medium, indicating that pHi altered [Ca2+]i handling by the intracellular stores rather than plasmalemmal Ca2+ transport. The results suggest that modulation of agonist-evoked [Ca2+]i signalling by changes in pHi may constitute a novel mechanism by which weak acids and bases may modulate exocrine fluid and ion transport.

The role of calcium in the volume regulation of rat lacrimal acinar cells.

Earlier studies have suggested a role for Ca2+ in regulatory volume decrease (RVD) in response to hypotonic stress through the activation of Ca2+-dependent ion channels (Kotera & Brown, 1993; Park et al., 1994). The involvement of Ca2+ in regulating cell volume in rat lacrimal acinar cells was therefore examined using a video-imaging technique to measure cell volume. The trivalent cation Gd3+ inhibited RVD, suggesting that Ca2+ entry is important and may be via stretch-activated cation channels. However, Fura-2 loaded cells did not show an increase in [Ca2+]i during exposure to hypotonic solutions. The absence of any changes in [Ca2+]i resulted from the buffering of cytosolic Ca2+ by Fura-2 during hypotonic shock and therefore inhibition of RVD. The intracellular Ca2+ chelator, BAPTA, also inhibited the RVD response to hypotonic shock. An increase in [Ca2+]i induced by either acetylcholine or ionomycin, was found to decrease cell volume under isotonic conditions in lacrimal acinar cells. Cell shrinkage was inhibited by tetraethylammonium ion, an inhibitor of Ca2+-activated K+ channels. On the basis of the presented data, we suggest an involvement of intracellular Ca2+ in controlling cell volume in lacrimal acinar cells.

Modulation of calcium signals by intracellular pH in isolated rat pancreatic acinar cells.

1. We have investigated the interactions between intracellular pH (pH1) and the intracellular free calcium concentration ([Ca2+]i) in isolated rat pancreatic acinar cells. The fluorescent dyes fura-2 and BCECF were used to measure [Ca2+]i and pHi, respectively. 2. Sodium acetate and ammonium chloride (NH4Cl) were used to acidify and alkalinize pHi, respectively. Cytosolic acidification had no effect on [Ca2+]i in resting pancreatic acinar cells, whereas cytosolic alkalinization released Ca2+ from intracellular stores. 3. Cytosolic acidification using either acetate or a CO2-HCO3(-)-buffered medium enhanced Ca2+ signals evoked by acetylcholine (ACh) and cholecystokinin (CCK). In contrast, both NH4Cl and trimethylamine (TMA) inhibited Ca2+ signals during stimulation with either ACh or CCK. This inhibitory effect was also observed in the absence of extracellular Ca2+, and was therefore not due to changes in Ca2+ entry. 4. Calcium oscillations evoked by physiological concentrations of CCK were enhanced by cytosolic acidification and inhibited by cytosolic alkalinization. 5. In order to determine the effects of pHi upon Ca2+ handling by intracellular Ca2+ stores, intraorganellar [Ca2+] was monitored using the low affinity Ca2+ indicator mag-fura-2 in permeabilized cells. Addition of NH4Cl, which is expected to alkalinize intraorganellar pH, did not alter intraorganellar [Ca2+] in permeabilized cells, suggesting that changing intraorganellar pH does not release Ca2+ from intracellular stores. Addition of NH4Cl or acetate also did not affect the rate of Ca2+ release induced by inositol 1,4,5-trisphosphate (InsP3). 6. Modification of extraorganellar ('cytosolic') pH did not affect the rate of ATP-dependent Ca2+ uptake into stores, but did modify the rate of Ca2+ release evoked by submaximal concentrations of InsP3. The rate of Ca2+ release was increased at more alkaline extraorganellar pHs. These results would suggest that manipulation of intraorganellar pH does not affect Ca2+ handling by the intracellular stores. In contrast, extraorganellar ('cytosolic') pH does affect InsP3-induced Ca2+ release from the stores. 7. In conclusion, changes in intracellular pH in pancreatic acinar cells can profoundly alter cytosolic [Ca2+]. This may shed light on earlier observations whereby cell-permeant weak acids and bases can modulate fluid secretion in epithelia.

Intracellular alkalinization mobilizes calcium from agonist-sensitive pools in rat lacrimal acinar cells.

1. We have investigated interactions between intracellular pH (pHi) and the intracellular free calcium concentration ([Ca2+]i) in collagenase-isolated rat lacrimal acinar cells. The fluorescent dyes fura-2 and 2',7'-bis(carboxyethyl)-5-carboxyfluorescein (BCECF) were used to measure [Ca2+]i and pHi, respectively. 2. Application of the weak base NH4Cl alkalinized the cytosol and caused a dose-dependent increase in [Ca2+]i. Trimethylamine (TMA) also alkalinized the cytosol and increased [Ca2+]i. The increase in [Ca2+]i evoked by NH4Cl or TMA was much smaller than that evoked by the secretory agonist acetylcholine (ACh). 3. Application of NH4Cl also increased [Ca2+]i in cells bathed in Ca(2+)-free medium, indicating that NH4Cl released Ca2+ from an intracellular pool. 4. Ammonium chloride had no effect on [Ca2+]i in cells bathed in Ca(2+)-free medium if agonist-sensitive intracellular Ca2+ pools had been depleted with either ACh or the microsomal Ca(2+)-ATPase inhibitor 2,5-di(tert-butyl)hydroquinone. Treatment of cells with NH4Cl in Ca(2+)-free medium reduced the amount of Ca2+ released by ACh. These results suggest that NH4Cl released Ca2+ from the same intracellular pool released by ACh. 5. Calcium release from the agonist-sensitive pool was also triggered when the cytosol was alkalinized by removing the weak acid acetate. 6. Ammonium chloride caused a modest increase in inositol phosphate production, suggesting that NH4Cl may have released stored Ca2+ via an increase in the intracellular inositol 1,4,5-trisphosphate concentration. 7. The increase in [Ca2+]i evoked by NH4Cl was not sustained even in the presence of extracellular Ca2+. In contrast, when a low dose of ACh was used to evoke intracellular Ca2+ release of similar magnitude, sustained Ca2+ entry was observed. 8. Alkalinizing the cytosol appeared to partially inhibit Ca2+ entry triggered by thapsigargin or by ACh. 9. We suggest that alkalinizing the cytoplasm in unstimulated lacrimal acinar cells can release Ca2+ from the intracellular agonist-sensitive Ca2+ pool. However, releasing stored Ca2+ via alkalinization does not appear to trigger significant Ca2+ entry, perhaps because intracellular alkalinization inhibits either the Ca2+ entry pathway or the mechanism which couples the entry pathway to store depletion.