A survey of stress, burnout and well-being in UK dentists.

Introduction: It is well established that dentistry is a stressful profession, primarily due to the nature and working conditions in the dental surgery. With dramatic changes taking place in the profession in recent years it is important to establish the impact this has on dentists' well-being. Aims: To determine the levels of stress and burnout in UK dentists and how this relates to well-being and identify the sources of work-related stress dentists report in different fields of practice. Materials and method: An online survey comprising of validated measures examining stress, burnout and well-being in dentists was administered to British Dental Association (BDA) members and non-members. Results: Valid responses were received from 2053 respondents. Dentists working in the UK exhibit high levels of stress and burnout and low well-being. General dental practitioners (GDPs) seem to be particularly affected. Issues relating to regulation and fear of litigation were deemed to be the most stressful aspects of being a dentist. Conclusions: The findings from this study build upon existing research showing that dentistry is a stressful profession. The sources of this stress appear to have shifted over the years, highlighting the changing landscape of dentistry. Interventions should focus on addressing these stressors by making changes to the working conditions of dentists.

Permeability and reflection coefficients of urea and small amides in the human red cell.

Measurement of the transport parameters that govern the passage of urea and amides across the red cell membrane leads to important questions about transport of water. It had initially been thought that small protein channels, permeable to water and small solutes, traversed the membrane (see Solomon, 1987). Recently, however, very strong evidence has been presented that the 28 kDa protein, CHIP28, found in the red cell membrane, is the locus of the water channel (see Agre et al., 1993). CHIP28 transports water very rapidly but does not transport small nonelectrolytes such as urea. The irreversible thermodynamic parameter, sigma i, the reflection coefficient, is a measure of the relationship between the permeability of the solute and that of water. If a solute permeates by dissolution in the membrane, sigma i = 1.0; if it permeates by passage through an aqueous channel, sigma i < 1.0. For urea, Goldstein and Solomon (1960) found that sigma urea = 0.62 +/- 0.03 which meant that urea crosses the red cell membrane in a water-filled channel. This result and many subsequent observations that showed that sigma urea < 1.0 are at variance with the observation that CHIP28 is impermeable to urea. In view of this problem, we have made a new series of measurements of sigma i for urea and other small solutes by a different method, which obviates many of the criticisms Macey and Karan (1993) have made of our earlier method. The new method (Chen et al., 1988), which relies upon fluorescence of the intracellular dye, fluorescein sulfonate, leads to the corrected value, sigma urea,corr = 0.64 +/- 0.03 for ghosts, in good agreement with earlier data for red cells. Thus, the conclusion on irreversible thermodynamic and other grounds that urea and water share a common channel is in disagreement with the view that CHIP28 provides the sole channel for water entrance into the cell.

Inhibition of red cell urea flux by anion exchange inhibitors.

When they studied the chemical properties of red cell anion exchange inhibitors such as DIDS (4,4'-diisothiocyanate-2,2'-stilbene disulfonate), Barzilay et al. (1979) Membr. Biochem. 2, 227-254 also examined the benzene sulfonates. These molecules are structurally similar to half a DIDS molecule and are also specific anion exchange inhibitors with ID50 values measured in mM, rather than microM, as for the stilbene disulfonates. We have studied several inhibitors of the benzene sulfonate (BS) class and found that they also inhibit red cell urea flux by up to 92% and stimulate water flux by up to 58%. The values of Kinhib,app for urea flux inhibition are the same as the ID50 values for anion flux inhibition; covalent DIDS completely suppresses the inhibition. These observations strongly suggest that the effect on urea flux is caused by BS binding at the stilbene site. Comparative studies on the short chain amides exclude lipid solubility and solute molar volume as factors that affect these BS actions. Kstim,app for water flux stimulation is also related to the anion exchange ID50 values; covalent DIDS suppresses the water flux stimulation. These observations on urea and water fluxes are consistent with a common driver, located at the stilbene site, which is responsible for the BS actions on urea, water and anion fluxes. The subsequent steps are independent with separate effectors to modulate each of the individual fluxes. These effectors are presumably located in different regions of the protein or proteins and carry out their separate processes by allosteric means.

Interaction between red cell membrane band 3 and cytosolic carbonic anhydrase.

We have previously proposed that a membrane transport complex, centered on the human red cell anion transport protein, band 3, links the transport of anions, cations and glucose. Since band 3 is specialized for HCO3-/Cl- exchange, we thought there might also be a linkage with carbonic anhydrase (CA) which hydrates CO2 to HCO3-. CA is a cytosolic enzyme which is not present in the red cell membrane. The rate of reaction of CA with the fluorescent inhibitor, dansylsulfonamide (DNSA) can be measured by stopped-flow spectrofluorimetry and used to characterize the normal CA configuration. If a perturbation applied to a membrane protein alters DNSA/CA binding kinetics, we conclude that the perturbation has changed the CA configuration by either direct or allosteric means. Our experiments show that covalent reaction of the specific stilbene anion exchange inhibitor, DIDS, with the red cell membrane, significantly alters DNSA/CA binding kinetics. Another specific anion exchange inhibitor, benzene sulfonate (BSate), which has been shown to bind to the DIDS site causes a larger change in DNSA/CA binding kinetics; DIDS reverses the BSate effect. These experiments show that there is a linkage between band 3 and CA, consistent with CA interaction with the cytosolic pole of band 3.

Transport parameters in the human red cell membrane: solute-membrane interactions of amides and ureas.

We have studied the permeability of a series of hydrophilic amides and ureas through the red cell membrane by determining the three phenomenological coefficients which describe solute-membrane interaction: the hydraulic permeability (Lp), the phenomenological permeability coefficient (omega i) and the reflection coefficient (sigma i). In 55 experiments on nine solutes, we have determined that the reflection coefficient (after a small correction for solute permeation by membrane dissolution) is significantly less than 1.0 (P less than 0.003, t-test), which provides very strong evidence that solute and water fluxes are coupled as they cross the red cell membrane. It is proposed that the aqueous channel is a tripartite assembly, comprising H-bond exchange regions at both faces of the membrane, joined by a narrower sieve-specific region which crosses the lipid. The solutes bind to the H-bond exchange regions to exchange their solvation shell with the H-bonds of the channel; the existence of these regions is confirmed by the finding that the permeation of all the amides and ureas requires binding to well-characterized sites with Km values of 0.1-0.5 M. The sieve-specific regions provide the steric restraints which govern the passage of the solutes according to their size; their existence is shown by the findings that: (1) the reflection coefficient (actually the function [1-corrected sigma i]) is linearly dependent upon the solute molecular diameter; and (2) the permeability coefficient is linearly dependent upon solute molar volume. These several observations, taken together, provide strong arguments which lead to the conclusion that the amides and urea cross the red cell membrane in an aqueous pore.

Transport parameters in the human red cell membrane: solute-membrane interactions of hydrophilic alcohols and their effect on permeation.

A systematic study has been made of the three coefficients that describe the human red cell membrane transport of a series of short straight-chain hydrophilic alcohols: the permeability coefficient, omega i, the reflection coefficient, sigma i, and the hydraulic conductivity, Lp. Ethylene glycol transport is saturable with Km = 220 +/- 50 mM; there is a second, low-affinity, ethylene glycol site which inhibits water transport (K = 570 +/- 140 mM, max. inhib. = 90 +/- 10%). sigma eth gly = 0.71 +/- 0.04 which is significantly less than 1 (n = 6, P less than 0.001), as are sigma i for six other alcohols (n = 23), thus providing strong thermodynamic evidence that water and these alcohols cross the red cell membrane, at least in part, in an aqueous channel. The solute/membrane frictional coefficient, fsm, for all seven alcohols has been determined and found to decrease monotonically as membrane permeability increases. The red cell membrane has been perturbed by treatments with phenylglyoxal and BS3 (bis(succinimidyl suberate]; these treatments are accompanied by correlated modulation of both ethylene glycol and urea permeability. In one set of experiments in control cells, urea permeability is correlated with water permeability; and, in another set, ethylene glycol permeability is correlated with water permeability. All of these observations support the proposition that the urea class of solutes, the ethylene glycol class of solutes and water all cross the membrane through the same aqueous pore. A schematic model of the red cell pore, consistent with the experimental observations, is presented.

Is an intact cytoskeleton required for red cell urea and water transport?

In order to determine the membrane protein(s) responsible for urea and water transport across the human red cell membrane, we planned to reconstitute purified membrane proteins into phosphatidylcholine vesicles. In preparatory experiments, we reconstituted a mixture of all of the red cell integral membrane proteins into phosphatidylcholine vesicles, but found that p-chloromercuribenzenesulfonate (pCMBS), which normally inhibits osmotic water permeability by approximately 90%, has no effect on this preparation. The preparation was also unable to transport urea at the high rates found in red cells, though glucose transport was normal. White ghosts, washed free of hemoglobin and resealed, also did not preserve normal urea and pCMBS-inhibitable water transport. One-step ghosts, prepared in Hepes buffer in a single-step procedure, without washing, retained normal urea and pCMBS-inhibitable water transport. Perturbations of the cytoskeleton in one-step ghosts, by removal of tropomyosin, or by severing the ankyrin link which binds band 3 to spectrin, caused the loss of urea and pCMBS-inhibitable water transport. These experiments suggest that an unperturbed cytoskeleton may be required for normal urea and pCMBS-inhibitable water transport. They also show that the pCMBS inhibition of water transport is dissociable from the water transport process and suggest a linkage between the pCMBS water transport inhibition site and the urea transport protein.

Modulation of water transport in human red cells: effect of urea.

We have studied the effect of urea on water flux in the human red cell and have found that 500 mosmolal urea inhibits osmotic water transport by 39%. The Ki for urea inhibition of water flux is 550 +/- 80 mosmolal, higher than, but comparable with, the Km of urea transport into the red cell of 220-330 mM given by Mayrand and Levitt (J. Gen. Physiol. 55 (1983) 427) and Brahm (J. Gen. Physiol. 82 (1983) 1). Other amides, such as propionamide and valeramide, as well as methyl-substituted ureas, have similar effects, although an indifferent molecule, such as 0.5 M creatinine, has no effect. Urea can be washed off the inhibition site with buffer, and the effects of urea concentrations as high as 1.2 osmolal are entirely reversible. 500 mosmolal urea also significantly increases the reflection coefficient for ethylene glycol, sigma eth gly, from 0.71 +/- 0.03 in control experiments to 0.86 +/- 0.04 (P less than 0.0005, t-test), and propionamide has a similar effect on sigma eth gly. These results show that urea can modulate ethylene glycol transport through the red cell membrane, and are consistent with, but not proof of, the presence of a single class of aqueous channels through which both ethylene glycol and urea enter the red cell. It is suggested that the physiological purpose of these low-affinity urea sites is to modulate water flow out of the red cell during passage through the regions of 0.5-0.6 M urea in the kidney.

Interrelation of ethylene glycol, urea and water transport in the red cell.

The reflection coefficient, sigma j, which measures the coupling between the jth solute and water transport across a semipermeable membrane, varies between 0 and 1.0. Values of sigma j significantly less than 1.0 provide irreversible thermodynamic proof that there is coupling between the transport of solute and solvent and thus that they share a common pathway. We have developed an improved method for measuring sigma and have used it to determine that sigma ethylene glycol = 0.71 +/- 0.03 and sigma urea = 0.65 +/- 0.03, in agreement with many, but not all, previous determinations. Since both of these values are significantly lower than 1.0, they show that there is a common ethylene glycol/water pathway and a common urea/water pathway. Addition of first one and then two methyl groups to urea increases sigma to 0.89 +/- 0.04 for methylurea and 0.98 +/- 0.4 for 1,3-dimethylurea, consistent with passage through an aqueous pore with a sharp cutoff in the 6-7 A region.

Modulation of water and urea transport in human red cells: effects of pH and phloretin.

It has previously been shown by Macey and Farmer (Biochim. Biophys. Acta 211:104-106, 1970) that phloretin inhibits urea transport across the human red cell membrane yet has no effect on water transport. Jennings and Solomon (J. Gen. Physiol. 67:381-397, 1976) have shown that there are separate lipid and protein binding sites for phloretin on the red cell membrane. We have now found that urea transport is inhibited by phloretin binding to the lipids with a KI of 25 +/- 8 microM in reasonable agreement with the KD of 54 +/- 5 microM for lipid binding. These experiments show that lipid/protein interactions can alter the conformational state of the urea transport protein. Phloretin binding to the protein site also modulates red cell urea transport, but the modulation is opposed by the specific stilbene anion transport inhibitor, DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate), suggesting a linkage between the urea transport protein and band 3. Neither the lipid nor the protein phloretin binding site has any significant effect on water transport. Water transport is, however, inhibited by up to 30% in a pH-dependent manner by DIDS binding, which suggests that the DIDS/band 3 complex can modulate water transport.

Control of red cell urea and water permeability by sulfhydryl reagents.

The binding constant for pCMBS (p-chloromercuribenzenesulfonate) inhibition of human red cell water transport has been determined to be 160 +/- 30 microM and that for urea transport inhibition to be 0.09 +/- 0.06 microM, indicating that there are separate sites for the two inhibition processes. The reaction kinetics show that both processes consist of a bimolecular association between pCMBS and the membrane site followed by a conformational change. Both processes are very slow and the on rate constant for the water inhibition process is about 10(5) times slower than usual for inhibitor binding to membrane transport proteins. pCMBS binding to the water transport inhibition site can be reversed by cysteine while that to the urea transport inhibition site can not be reversed. The specific stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamidostilbene-2,2'-disulfonate) causes a significant change in the time-course of pCMBS inhibition of water transport, consistent with a linkage between anion exchange and water transport. Consideration of available sulfhydryl groups on band 3 suggests that the urea transport inhibition site is on band 3, but is not a sulfhydryl group, and that, if the water transport inhibition site is a sulfhydryl group, it is located on another protein complexed to band 3, possibly band 4.5.

Osmotic properties of human red cells.

When an osmotic pressure gradient is applied to human red cells, the volume changes anomalously, as if there were a significant fraction of "nonosmotic water" which could not serve as solvent for the cell solutes, a finding which has been discussed widely in the literature. In 1968, Gary-Bobo and Solomon (J. Gen. Physiol. 52:825) concluded that the anomalies could not be entirely explained by the colligative properties of hemoglobin (Hb) and proposed that there was an additional concentration dependence of the Hb charge (ZHb). A number of investigators, particularly Freedman and Hoffman (1979, J. Gen. Physiol. 74:157) have been unable to confirm Gary-Bobo and Solomon's experimental evidence for this concentration dependence of ZHb and we now report that we are also unable to repeat the earlier experiments. Nonetheless, there still remains a significant anomaly which amounts to 12.5 +/- 0.8% of the total isosmotic cell water (P much less than 0.0005, t test), even after taking account of the concentration dependence of the Hb osmotic coefficient and all the other known physical chemical constraints, ideal and nonideal. It is suggested that the anomalies at high Hb concentration in shrunken cells may arise from the ionic strength dependence of the Hb osmotic coefficient. In swollen red cells at low ionic strength, solute binding to membrane and intracellular proteins is increased and it is suggested that this factor may account, in part, for the anomalous behavior of these cells.

Relation between red cell anion exchange and urea transport.

The new distilbene compound, DCMBT (4,4'-dichloromercuric-2,2,2',2'-bistilbene tetrasulfonic acid) synthesized by Yoon et al. (Biochim. Biophys. Acta 778 (1984) 385-389) was used to study the relation between urea transport and anion exchange in human red cells. DCMBT, which combines properties of both the specific stilbene anion exchange inhibitor, DIDS, and the water and urea transport inhibitor, pCMBS, had previously been shown to inhibit anion transport almost completely and water transport partially. We now report that DCMBT also inhibits urea transport almost completely and that covalent DIDS treatment reverses the inhibition. These observations provide support for the view that a single protein or protein complex modulates the transport of water and urea and the exchange of anions through a common channel.

Binding of DTNB to band 3 in the human red cell membrane.

Inhibition of red cell water transport by the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) has been reported by Naccache and Sha'afi ((1974) J. Cell Physiol. 84, 449-456) but other investigators have not been able to confirm this observation. Brown et al. ((1975) Nature 254, 523-525) have shown that, under appropriate conditions, DTNB binds only to band 3 in the red cell membrane. We have made a detailed investigation of DTNB binding to red cell membranes that had been treated with the sulfhydryl reagent N-ethylmaleimide (NEM), and our results confirm the observation of Brown et al. Since this covalent binding site does not react with either N-ethylmaleimide or the sulfhydryl reagent pCMBS (p-chloromercuribenzenesulfonate), its presence has not previously been reported. This covalent site does not inhibit water transport nor does it affect any transport process we have studied. There is an additional low-affinity (non-covalent) DTNB site that Reithmeier ((1983) Biochim. Biophys. Acta 732, 122-125) has shown to inhibit anion transport. In N-ethylmaleimide-treated red cells, we have found that this binding site inhibits water transport and that the inhibition can be partially reversed by the specific stilbene anion exchange transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), thus linking water transport to anion exchange. DTNB binding to this low-affinity site also inhibits ethylene glycol and methyl urea transport with the same KI as that for water inhibition, thus linking these transport systems to that for water and anions. These results support the view that band 3 is a principal constituent of the red cell aqueous channel, through which urea and ethylene glycol also enter the cell.

Relation between red cell anion exchange and water transport.

A new distilbene compound, 4',4'-dichloromercuric-2,2,2',2'-bistilbene tetrasulfonic acid (DCMBT), has been synthesized for use in studies of anion and water transport in the human red cell. DCMBT combines features of both the specific stilbene anion transport inhibitor, DIDS, and the mercurial water transport inhibitor, pCMBS. This new compound inhibits anion transport almost completely with a Ki of 15 microM. DCMBT also inhibits water transport by about 15-20% with a Ki of about 8 microM. Treatment of red cells with DIDS inhibits the effect of DCMBT on water transport, suggesting that anion transport and water transport are mediated by the same protein.

Effect of thiourea on PCMBS inhibition of osmotic water transport in human red cells.

The organomercurial reagent p-chloromercuribenzene sulfonate (PCMBS) is an inhibitor of osmotic water permeability in the human red cell membrane. We have found that thiourea, when added along with PCMBS to a red cell suspension, interferes with this inhibition and at high enough concentrations prevents the inhibition from developing altogether. For a 2 mM PCMBS concentration Ki = 3 +/- 1 mM. When thiourea is added at a later time, the PCMBS inhibition, which normally takes about 20 min to develop fully, is halted and remains fixed at the value attained by that time. Thiourea also inhibits the reversal of PCMBS inhibition by a 10 mM concentration of cysteine, the half-time for reversal increasing by more than an order of magnitude when [thiourea] = 50 mM. Possible implications for the nature of the water and urea transport pathways across the red cell membrane are discussed.

Site of red cell cation leak induced by mercurial sulfhydryl reagents.

It has been suggested that the human red cell anion transport protein, band 3, is the site not only of the cation leak induced in human red cells by treatment with the sulfhydryl reagent pCMBS (p-chloromercuribenzene sulfonate) but is also the site for the inhibition of water flux induced by the same reagent. Our experiments indicate that N-ethylmaleimide, a sulfhydryl reagent that does not inhibit water transport, also does not induce a cation leak. We have found that the profile of inhibition of water transport by mercurial sulfhydryl reagents is closely mirrored by the effect of these same reagents on the induction of the cation leak. In order to determine whether these effects are caused by band 3 we have reconstituted phosphatidylcholine vesicles containing only purified band 3. Control experiments indicate that these band 3 vesicles do not contain (Na+ + K+)-ATPase as measured by ATP dephosphorylation. pCMBS treatment caused a significant increase in the cation leak in this preparation, consistent with the view that the pCMBS-induced cation leak in whole red cells is mediated by band 3.