Background
==========

Tetraethylammonium is known as a pore-occluding blocker of voltage-gated potassium channels \[[@B1]\], but it also blocks other cationic channels such as calcium-dependent K^+^ channels \[[@B2],[@B3]\] and the nicotinic acetylcholine receptor \[[@B4]\]. TEA at 0.1 to 10 mM also inhibits osmotic water flux through human AQP1 channels expressed in *Xenopus* oocytes, decreasing the net swelling rate in hypotonic saline by 30--40% as compared to AQP1-expressing oocytes not treated with TEA \[[@B5]\]. This blocking effect on osmotic water flux was demonstrated to involve AQP1 channels specifically by using site-directed mutagenesis (tyrosine 186 to phenylalanine) to generate a Y186F AQP1 channel that is insensitive to block by TEA, but retains sensitivity to block by mercury. The blocking effect of mercury in AQP1 channels is dependent on a neighboring residue, cysteine 189 \[[@B6]\]. TEA offers attractive advantages over mercury as a reversible and less toxic blocker for AQP1 channels in experimental analyses of water channel function; however, the relevance of TEA as a blocker for AQP1 channels outside the *Xenopus* expression system has not been examined previously.

Differences in properties (including pharmacological sensitivities) have been observed for ion channels expressed in oocytes as compared to those in native tissues. For example, the *Torpedo* acetylcholine receptor and the *Shaker* K^+^ channel protein differ in the fractions of protein glycosylated, the composition of the oligosaccharide chains, and the degree of protein maturation when expressed in *Xenopus* oocytes as compared with channels in native tissue or other expression systems \[[@B7],[@B8]\]. Differences in glycosylation patterns can influence the binding of external blocking agents. The presence of tissue-specific targeting signals not recognized in *Xenopus* oocytes may lead to protein degradation \[[@B9]\]. These discrepancies raised the question of whether AQP1 channels in native tissues are sensitive to TEA, as they are in *Xenopus* oocytes. Data presented here show that TEA is effective as a blocker of AQP1 channels expressed in a mammalian renal cell line and in native renal epithelial membrane.

AQP1 channels are complex solute conductors. They are constitutively permeable to water, and also function as regulated non-selective cation channels \[[@B10]\] when gated by intracellular cyclic GMP \[[@B11],[@B12]\]. Even though only a small proportion of the AQP1 channels that are present in the membrane appear to be available for cGMP-activation of the ionic conductance (1/56,000), model-based calculations nonetheless suggest that this contribution could be meaningful in a physiological setting \[[@B13]\]. Unlike transporters which move substantial amounts of substrate by design, ion channels function to change membrane potential by the net movement of relatively tiny amounts of charge (\~10 picomoles per cm^2^ membrane to generate a change of 100 mV) \[[@B14]\], hence the low proportion of active AQP1 ion channels is consistent with a functional goal of gated ionic signaling that is distinct from that of massive constitutive osmotic water permeability.

The role of the AQP1 channel in mediating both water and ion fluxes raises interesting questions about whether the pathways are shared or structurally distinct. Expression of AQP1 channels in bilayers has shown that the water permeability but not the ionic conductance is blocked by p-chloromethylbenzenesulfonate, indicating that the two fluxes may involve distinct pathways \[[@B12]\]. Work presented here provides an additional line of evidence that the pathways for water and ions in AQP1 can be distinguished pharmacologically, using the blocker TEA as a probe. In combination with data from three-dimensional imaging analyses \[[@B15]\], these results support the suggestion that the AQP1 channel may contain separate parallel pathways for ion and water fluxes. These findings have potential significance in understanding the complex role for AQP1 in processes of salt and water movement across cell membranes.

Results and Discussion
======================

TEA inhibition of water flux in AQP1-expressing MDCK cells
----------------------------------------------------------

TEA inhibited significantly the osmotically-driven net movement of water across monolayers of Madin-Darby Canine Kidney (MDCK) cells expressing human AQP1, as compared to AQP1-infected MDCK cells without TEA treatment (Figure [1](#F1){ref-type="fig"}). MDCK cells were induced to express AQP1 by infection with replication-deficient adenovirus (see Materials and Methods). These cells showed a high rate of water flux, ranging from 4 to 10 μl/cm^2^/hour, and a significant block by 1 mM TEA, ranging from 32 to 45% in 6 sets of replicate experiments. The TEA block of AQP1-mediated water flux for all data combined was 34.2%. In control MDCK cells infected with empty virus, the water flux was significantly lower (approximately 10-fold), ranging from 0.2 to 1.4 μl/cm^2^/hour. In these control cells, the net flux rates with and without treatment were not significantly different. Therefore, the enhanced water permeability and concomitant sensitivity to block by TEA were dependent on the expression of AQP1 channels. MDCK cells transfected with empty virus showed no augmentation of transepithelial water movement, confirming previous work that showed water does not flow across tight junctions in MDCK confluent layers \[[@B16]\]. Cells transfected with empty virus also showed no effect of TEA treatment. The magnitude of block of AQP1-mediated water permeability by TEA is consistent with that reported previously for AQP1 channels expressed in *Xenopus* oocytes \[[@B5]\].

![Block by TEA of AQP1-mediated water permeability in monolayers of adenovirus-infected MDCK cells. (**A**) To account for differences in levels of AQP1 expression between different experimental preparations, data for \"Percent water flux\" were standardized to the mean water flux measured for AQP1-expressing monolayers without TEA on the same experimental day. Cells infected with empty adenovirus were untreated (Control) or treated with TEA (Con TEA). Cells infected with AQP1 adenovirus and expressing AQP1 were untreated (AQP) or treated with TEA (AQP TEA). Box plots show the distribution of data values; the gray box encloses 50% of the data points, the error bars show the full range, and the horizontal bar shows the modal value. Data for mean, standard error and n value are shown below each column. Significant differences were analyzed by T-test and are reported as p \< 0.001 (\*), and not significant (N.S.; p \< 0.50) (**B**). Whole cells lysates were prepared from MDCK cells on filters and 10 μg of protein was loaded onto each lane. Shown is the expression of AQP1 protein in MDCK cells infected with empty (CON) or AQP1 adenovirus. No AQP1 protein was seen in CON lanes even after extended exposures to film (i.e., 30 minutes).](1472-6793-2-4-1){#F1}

TEA inhibition of water flux in renal tubule
--------------------------------------------

Preparations of pure descending thin limbs and pure ascending thin limbs from the inner medulla were segregated by morphological features. Descending thin limbs with their cells with nuclei protruding into the lumen differ markedly in appearance from ascending thin limbs with their large, round, flat nuclei when viewed with DIC optics \[[@B17],[@B18]\]. The differential expression of AQP1 in the two different types of segments (abundant in the descending thin limb and absent in the ascending thin limb), visualized by immunocytochemistry, confirmed the accuracy of the morphological identification (Figure [2](#F2){ref-type="fig"}). AQP1 provides the predominant pathway for water transport in the descending thin limb \[[@B19],[@B20]\]. In preparations of isolated perfused descending thin limbs, net osmotic water absorption rates (J~v~) and water permeability (P~f~) were significantly lower in the presence of 10 mM TEA than in the absence of TEA (Figure [3](#F3){ref-type="fig"}). This sensitivity to TEA correlates with the expression of AQP1 channels in this region. The overall effect of TEA in descending thin limb was to cause a 49% block of J~V~ and a 50% block of P~f~. The pairwise comparison of individual tubule preparations, before and after TEA, showed a significant percent change of (-) 52 ± 6 % (mean ± SEM; n = 10) in descending thin limb. In contrast, preparations of ascending thin limbs that lack AQP1 channels showed essentially no net water flux and no effect of TEA under osmotic gradient conditions. For data from ascending thin limbs, the pairwise comparison of individual tubule preparations before and after TEA showed a non-significant percent change of (+) 17 ± 35 % (n = 9). These data show that the blocking effect of TEA on water permeability originally observed in AQP1-expressing oocytes can be reproduced in a physiologically relevant condition. TEA blocks water permeability in a tissue that has abundant native expression of AQP1 channels, whereas it has no effect in tissue lacking AQP1 expression.

![Immunocytochemical analysis confirms the expression of AQP1 channels in pure descending but not ascending thin limb preparations. Examples are shown for isolated preparations of rat inner medullary descending thin limb(upper panel) and inner medullary ascending thin limb (lower panel) labeled with a rabbit antibody to the AQP1 carboxy tail domain, and visualized with Cy5-labeled antirabbit antibody immunoglobulin-G. The scale bar is 100 μm.](1472-6793-2-4-2){#F2}

![Analysis of water flux in isolated perfused renal ascending and descending thin limbs of Henle\'s loop under osmotic gradient conditions.**A.** Compilation of J~v~ values obtained for descending thin limbs that were untreated (Desc cont) or exposed to 10 mM TEA (Desc TEA), and for ascending thin limbs that were untreated (Asc cont) or exposed to TEA (Asc TEA). **B.** Compilation of calculated osmotic water permeabilities (P~f~) for the same tubules as analyzed in (A). Box plots show the distribution of data values; the gray box encloses 50% of the data points, the error bars show the full range, and the horizontal bar shows the modal value. Data for group means, standard errors and n values are shown below each column. However, since each tubule served as its own control, differences were analyzed by paired T-test and are reported as p \< 0.02 (\*\*), p \< 0.05 (\*), and not significant (N.S.).](1472-6793-2-4-3){#F3}

Lack of TEA block of ion channel conductance
--------------------------------------------

Heterologous expression of human AQP1 in *Xenopus* oocytes, a system that has been well characterized for electrophysiological assays, was used for two-electrode voltage clamp studies to evaluate the effects of TEA on ionic currents. External TEA(10 mM) failed to block the cGMP-dependent ionic current in AQP1-expressing oocytes (Figure [4](#F4){ref-type="fig"}). Current responses were assessed with voltage steps from +40 to -70 mV, from a holding potential of 0 mV (Fig. [4A](#F4){ref-type="fig"}). The initial response shows the current-voltage relationship recorded by two-electrode voltage clamp in an AQP1-expressing oocyte in isotonic 100 mM Na^+^ saline (see Methods). The activated response was measured in the same oocyte at 3--5 minutes after addition of 4 mM sodium nitroprusside (SNP), which elevates endogenous cGMP levels. The effect of 10 mM TEA (with 90 mM NaCl) was assessed by perfusing the recording chamber with TEA saline containing SNP. No appreciable change was observed. For the representative example shown in Figure [4](#F4){ref-type="fig"}, the conductance (measured as the linear slope of the current-voltage relationship between -50 and +30 mV; Fig. [4B](#F4){ref-type="fig"}) for the initial condition was 3.0 nA/mV. After channel activation the conductance was 58 nA/mV in Na^+^ saline and 57 nA/mV in 10 mM TEA saline. The reversal potential of the current (Fig. [4B](#F4){ref-type="fig"}) shifted from approximately -24 mV in the initial state to about -4 mV in the activated state, consistent with the activation of a non-selective cationic conductance permeable to both Na^+^ and K^+^. Similar results were observed for other SNP-activated oocytes (n = 5), as well as for AQP1-expressing oocytes activated by prior incubation in 10 mM 8Br-cGMP (as described in \[[@B11]\]). In AQP1-expressing oocytes, the conductance induced by 8Br-cGMP in control Na^+^ saline did not change significantly \[(+) 11.6 ± 6.2 % (mean ± SD; n = 7)\] after perfusion with 10 mM TEA saline, thus confirming the absence of a blocking effect of TEA on the ion conductance by AQP1 channels. The activated AQP1 ion channel is less permeant to TEA than to Na^+^ or K^+^\[[@B10]\]; however, because of the high extracellular Na^+^ concentration (90 mM) in the TEA bath saline in the present study, the measured reversal potential of the activated current was not appreciably different with and without 10 mM TEA.

![Tetraethylammonium does not block the ionic conductance in cGMP-activated AQP1 channels expressed in Xenopus oocytes.**A.** Recordings of current responses to voltage steps analyzed by two-electrode voltage clamp in an AQP1-expressing oocyte before stimulation (initial), after cGMP-dependent activation in 100 mM Na^+^ saline (activated) and after perfusion with saline containing 10 mM TEA and 90 mM Na^+^ (+TEA). No block of the ionic current was observed. **B.** Plot of the current-voltage relationship for data illustrated in (A). Conductance values calculated from linear fits of the data from -50 to +30 mV were 3.0 μS initial, 57.8 μS after activation in Na^+^ saline, and 57.3 μS with 10 mM TEA.](1472-6793-2-4-4){#F4}

Results presented here show that TEA serves as an effective, though partial, blocker of the osmotic water permeability mediated by human AQP1 channels expressed in a heterologous mammalian cell line and by native AQP1 channels in isolated rat renal descending thin limbs of Henle\'s loops. Before the characterization of TEA, mercurial compounds were among the only known blockers of AQP1, affecting both the constitutive water flux and the regulated ion conductance \[[@B6],[@B10]\]. For AQP1 channels expressed in *Xenopus* oocytes, the block of osmotic water flux is 5% in 0.01 mM TEA, 21% in 1 mM TEA, and 36% in 10 mM TEA, as referenced to the osmotic water flux in control AQP1-expressing oocytes \[[@B5]\]. Data presented here show that the level of block by TEA is comparable or greater for AQP1 channels expressed in mammalian cells. 1 mM TEA produces a 34% block of osmotic water flux in MDCK cells, and 10 mM TEA produces a 50% block in renal descending thin limbs. The effect of TEA on water permeability in cells and tissues that express high levels of AQP1 channels is likely to be significant, given the substantial amount of water that can be moved across AQP1-expressing epithelia (for example, see data on descending thin limbs in Fig. [3](#F3){ref-type="fig"}). The idea that TEA might be a candidate for a lead compound for the development of drugs with possible clinical applications \[[@B5]\] is supported by the present data that show the effectiveness of TEA in two different mammalian models of AQP1-mediated osmotic water permeability. While the usefulness of TEA *in vivo* as a research tool may be limited by toxicity and effects on other channels at higher concentrations, it offers a degree of reversibility and selectivity not seen with mercury, an agent which acts by covalent modification of all exposed cysteine residues \[[@B21],[@B22]\].

The regulated ion channel function of AQP1 is not blocked by external TEA. An intriguing question remains regarding the location of the permeation pathway for cations in AQP1 channels. Analysis of the crystal structure of GlpF suggests that potential ion binding sites may be located in the central pore, at the four-fold axis of symmetry in the tetrameric association of subunits that form the channel \[[@B15]\]. Interestingly, this arrangement of a tetramer of subunits to form a central ion pore is a hallmark of the family of voltage-gated ion channels \[[@B23]\], which rely on a tetrameric organization to create a central pore for ions that is lined by sequences contributed from all four subunits. In contrast, the four individual pores for water or glycerol, located within each subunit of AQP1 or GlpF, appear to be lined with hydrophobic residues expected to preclude ion permeability \[[@B15],[@B24],[@B25]\]. Importantly, TEA distinguishes between the two pathways in AQP1, supporting the idea that the ion and water pores are physically distinct although they co-exist in the same channel complex.

The AQP1-mediated ion conductance may contribute measurably to the function of tissues in which AQP1 is abundantly expressed, such as in the mammalian renal proximal tubule \[[@B13]\]. Further work is needed to assess the additional levels of regulatory control that may constrain the availability of the AQP1 proteins to serve as ion channels gated by cGMP, within a background of constitutive water channel activity. The maintenance of an ionic pore, as well as a conserved cGMP-binding domain in the AQP1 carboxy tail for regulating activation \[[@B11]\], suggests that the ion channel function is likely to be essential for physiological regulatory processes that have yet to be fully appreciated.

Materials and Methods
=====================

MDCK cells
----------

### Cell culture

Madin-Darby Canine Kidney (MDCK) cells were obtained as a gift from Dr. R. Lynch, University of Arizona, and used between passages 13--14. Cells were grown and maintained in Dulbecco\'s Modified Eagle Medium (Life Technologies) that was supplemented with 10% fetal bovine serum (Hyclone), 170 mM glutamine and penicillin/streptomycin (100 μg/ml, Life Technologies) in humidified air containing 5% carbon dioxide at 37°C.

### Adenovirus vector

The adenovirus (AV) backbone for the aquaporin-1 (AQP1) sense and antisense AV constructs was a replication-deficient \"first-generation\" AV with deletions of the E1 and E3 genes \[[@B26]\]. This \"empty\" AV contains the cytomegalovirus (CMV) promoter and bovine growth hormone polyadenylation (bHG) site separated by a polylinker that was used to clone AQP1 DNA as described previously \[[@B27]\]. A recombinant AQP1 virus was constructed using a plasmid containing the coding sequence for AQP1, pCHIPev \[[@B28]\]. pCHIPev was digested and the AQP1 insert was subcloned into the shuttle vector pSKAC, creating pSKAC/AQP1. pSKAC contains map units 0.0--1.3 of the AV which includes the left terminal repeat of AV, a CMV promoter, an AMV translation enhancer and a polylinker region. DNA fragments containing AQP1 DNA were liberated from pSKAC after restriction and ligated into adenovirus as described previously \[[@B27]\]. Human embryonic kidney cells (293 cells) were transfected with ligation mixture and individual viruses were isolated from cell lysates by two consecutive rounds of plaque purification using an agar overlay as described previously \[[@B27]\]. Individual viruses were amplified in 293 cells and purified over cesium step gradient. Individual AV DNA titers were determined by three different methods: 1) plaque titration on 293 cells, 2) immunofluorescence microscopy of AV protein expression (anti-penton group antigen, clone 143, Biodesign, Kennebunk, ME) in 293 cells infected with serial dilutions of AV and 3) absorbance at 260 nm (pfu/ml = A~260~ × dilution × 10^10^).

### Osmotic water flux assay

Water permeability was measured as the net fluid movement driven by an osmotic gradient across intact monolayers of adenovirus-infected MDCK cells, by methods similar to those reported previously \[[@B27],[@B29]\]. Cells were seeded onto Transwell filters (Costar, 1 cm^2^, 0.4 μm pore size) at a density of 1.5 × 10^5^ cells per well. Cells were maintained in humidified air containing 5% CO~2~ for two weeks to allow for cell-cell junctions to mature. Cells were infected at the apical surface with adenovirus containing AQP1 cDNA or with empty adenovirus at a multiplicity of infection (MOI) of 10. Five days post-infection, all medium was removed carefully and completely from both upper (apical) and lower (basolateral) chambers. Exactly one milliliter of fresh prewarmed isotonic medium (\~300 mOsM) was added to the lower chamber and exactly 175 μl of hypertonic medium (\~450 mOsM) was added to the upper chamber at time zero. In experimental groups, TEA chloride was present at a 1 mM concentration in both the upper and lower chambers. After four hours of incubation at 37°C, all of the medium from each of the upper chambers was removed carefully and volume was measured using an analytical balance.

### Immunoblot analysis

Sodium dodecyl sulfate (SDS) solubilized whole cell lysates containing 5% β-mercaptoethanol were electrophoresed into 12% polyacrylamide gels containing 0.1 % SDS. Fractionated proteins were blotted onto nitrocellulose using the Transblot system as per manufacturer instructions (Biorad, Hercules, CA). The blots were preincubated for 30 minutes at 22°C in Tris-buffered saline containing 5% nonfat powdered milk and 0.2% Tween-20 (TBS-T), and were then probed with affinity purified anti-AQP1 IgG (1:5000) for 2 hr at 22°C. The blots were washed (3 × 15 min) in TBS-T and were incubated for 2 hours with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, 1:5000, Pierce). The blots were washed (3 × 15 min) in TBS-T and specific labeling was visualized following enhanced chemiluminescence (Pierce, Rockford, IL) and exposure (10 seconds) to ECL-Hyperfilm (Amersham, Arlington Heights, IL). Immunoblots were digitized using the UVP gel documentation system and densitometry was performed using LabWorks software (Upland, CA).

Isolated perfused rat inner medullary thin limbs
------------------------------------------------

### Animals and dissection of tubules for immunocytochemistry and for in vitro fluid movement measurements

Young male Munich-Wistar rats (Harlan Sprague-Dawley, Indianapolis, In), 90 g average body weight, were maintained on Teklad Mouse/Rat Diet No. 7001 with free access to water. Dissection of inner medullary thin limbs of Henle\'s loop from fresh rat renal tissue for immunocytochemistry and for *in vitro* microperfusion was performed as described in detail previously \[[@B17]\]. We used descending thin limb (DTL) and ascending thin limb (ATL) segments from the top 70% of the inner medulla above the pre-bend level. Descending and ascending thin limbs were identified by cell type using an inverted microscope with Nomarski differential interference contrast (DIC) optics at 400× magnification \[[@B17],[@B18]\].

### Immunocytochemisty

Rabbit antibody recognizing the carboxy tail domain of AQP1 (from W.D. Stamer and J.W. Regan, University of Arizona) was used for immunocytochemical confirmation of protein expression in isolated thin limbs of Henle as described previously \[[@B30]\]. Tubules were positioned onto a glass microscope slide covered with a layer of Cell-Tak^®^ adhesive (Becton Dickinson, Bedford, MA). Tubules were then fixed in 4% paraformaldehyde for 10 min and permeabilized with 100% methanol for 2 min at -20°C. Primary antibody or non-immune rabbit serum (diluted 1:500) was applied overnight at 4°C., followed by incubation with biotinylated goat anti-rabbit antibody (diluted 1:100, 60 min), and incubation with streptavidin conjugated to Cy5 (60 min). Digital fluorescent images were obtained with a Leica-TCS confocal microscope.

### Perfusion and bathing solution for in vitro microperfusions

The Ringer solution used for perfusing and bathing the tubules was that initially used by Chou and Knepper \[[@B18]\] in perfusion of thin limbs from chinchilla kidneys and modified by us. It contained (in mM): 130 NaCl, 20 HEPES, 5 NaHCO~3~, 2.5 K~2~HPO~4~, 2 CaCl~2~, 1.2 MgSO~4~, 5.5 glucose, and 5 urea. The osmolality was about 290 mOsm/kg H~2~O, the pH was adjusted to 7.4 while the solution was gassed with 95 O~2~/5% CO~2~.

### Perfusion of tubules in vitro

We perfused the dissected tubules *in vitro* by a technique essentially the same as that originally described by Burg et al. \[[@B31]\] and modified for use in our laboratory \[[@B17],[@B32],[@B33]\]. The perfusion rate was about 15--20 nl/min. The temperature of the bathing chamber was maintained at 37°C during perfusion and the bath was covered with a layer of light paraffin oil. To check for leaks and to measure net fluid movement across the epithelium with an imposed osmotic gradient, \[^14^C\]dextran (MW \~70,000) was added to the perfusate.

### Determination of net water movement and water permeability of thin limbs perfused in vitro

There is no net transepithelial water movement with solutions of identical osmolalities in the bath and lumen. For this study, however, we determined the occurrence of net water absorption and water permeability when we imposed an osmotic gradient from bath to lumen (see below). Net water absorption, J~v~ (nl min^-1^ mm tubule length^-1^), was determined in each collection period with \[^14^C\]dextran in the perfusate using the following relationship: J~v~= (V~i~ - V~o~)/L. In this equation, V~i~(initial perfusion rate, nl/min) is calculated by dividing the cpm of ^14^C in the collected fluid by the cpm/nl of ^14^C in the initial perfusate and by the time of the collection period; V~o~ (collection rate, nl/min) is measured directly from the collection; and L (mm) is the length of the tubule perfused, measured with an ocular micrometer. For purposes of determining the water permeability of DTL and ATL, we imposed a 100 mOsmol/kg H~2~O osmotic gradient from bath to lumen with sucrose and determined J~v~ as just described. Water permeability (P~f~, μm s^-1^) was then determined from the equation P~f~ = J~v~/(A~s~ V~w~ δC~osmol~) where A~s~ = luminal surface area (π DL), V~w~ = partial molar volume of water (18 ml/M), δC~osmol~ = transepithelial osmolality gradient \[[@B18]\]. When the effect of TEA on water movement was examined, TEA bromide (10 mM) was added to the bathing medium and the NaCl concentration was reduced by 10 mM to maintain the osmolality constant. Collection periods were 5 min in length and each tubule served as its own control for measuring net water flux and water permeability in the presence and absence of TEA. \[^14^C\]dextran (sp act 1.08 mCi/g) was obtained from American Radiolabeled Chemicals, St. Louis, MO., USA. Results are summarized as means ± SE. The *n* value is the number of tubules. Each tubule came from a different animal.

Xenopus oocytes
---------------

### Oocyte preparation

Adult female *Xenopus laevis* were anesthetized with tricaine methane sulfonate (MS-222, Sigma Chemical Co). Stage V-VI oocytes were removed by partial laparotomy and defolliculated by collagenase treatment \[[@B11]\]. Cloned human Aquaporin1 DNA was provided by Dr. P. Agre \[[@B28]\], linearized with XbaI and used as a template for *in vitro* synthesis of cRNA with T3 RNA polymerase. Prepared oocytes were injected with 50 nl of sterile water (control oocytes) or 50 nl of sterile water containing AQP1 cRNA (\~0.2 to 0.5 ng/nl) and were incubated for 2--5 days at 18°C in culture medium (ND96: 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl~2~, 1 mM MgCl~2~, 5 mM Hepes, 2.5 mM pyruvic acid, 100 U/ml penicillin, and 100 μg/ml streptomycin, pH 7.6) to allow protein expression.

### Electrophysiology

Two-electrode voltage clamp recordings were performed at room temperature with electrodes (0.5--3 MΩ) filled with 3 M KCl. Data were recorded with a GeneClamp 500 (Axon Instruments, Foster City, CA), filtered at 2 kHz, digitized at 50 to 2000 μs and analyzed with pClamp software (Axon Instruments). Control recording saline for two-electrode voltage clamp contained (in mM): 100 NaCl, 5 MgCl~2~, and 5 HEPES, pH 7.3. TEA saline was made with (in mM) 10 TEA.Cl, 90 mM NaCl, 5 MgCl~2~, and 5 HEPES, pH 7.3. Activation of the ionic current was induced by preincubation of AQP1-expressing oocytes for 10--30 minutes in 10 mM 8Br-cGMP (Sigma) in high K^+^ saline, containing (in mM): 30 KCl, 80 K gluconate, 5 MgCl~2~, 5 HEPES, pH 7.3, or by addition of 2--4 mM sodium nitroprusside (SNP), a stimulator of guanylate cyclase and cGMP production \[[@B34],[@B35]\]. Control oocytes treated in the same conditions showed no induction of an ionic current. Higher concentrations of SNP (\>10 mM) affected ionic permeability in some batches of control oocytes and were not used. Current voltage relationships were assessed in Na^+^ control saline and in TEA saline to assess potential blocking effects of TEA.

### Chemicals

All chemicals were purchased from standard sources except as specified above and were of the highest purity available.

List of abbreviations
=====================

AQP1 Aquaporin-1

J~v~ Net osmotic water absorption rate

MDCK Madin-Darby Canine Kidney cell

P~f~ Osmotic water permeability

SNP Sodium nitroprusside (nitric oxide donor)

TEA Tetraethylammonium (chloride salt)

Acknowledgements
================

We thank Dr. John Regan for antibodies to AQP1, Amy Marble and Dr. Kathryn Bolles for technical assistance, Dr. Heddwen Brooks for helpful discussions, Neil Atodaria and Eileen Ryan for technical assistance with permeability assays using MDCK cells, and Dr. Ron Lynch for the gift of MDCK cells (University of Arizona). Funding was provided by NIH R01 GM59986 (AJY), American Health Assistance Foundation \#G2001-026 (WDS), and NIH R01 DK16294 (WHD) and Research to Prevent Blindness Foundation.


Background
==========

The extracellular signal regulated kinases ERK1 (p44^mapk^) and ERK2 (p42^mapk^) are activated in response to stimulation of receptor tyrosine kinases (RTKs) as well as heptahelical G protein coupled receptors (GPCR) and transmit signals which regulate cell differentiation and growth \[[@B1]-[@B3]\]. The molecular steps involved in signaling from GPCRs to ERK are incompletely understood. Data obtained in various cell systems have provided evidence in support of several signaling pathways including protein kinase C (PKC) \[[@B4]\], Ca^2+^-mediated mechanisms \[[@B5]-[@B12]\], and transactivation of receptor tyrosine kinases \[[@B13],[@B14]\]. In hepatocytes several hormones, including vasopressin, angiotensin II, norepinephrine, and PGF~2α~, that bind to GPCRs activate ERK \[[@B15]-[@B17]\]. The mechanisms mediating the ERK activation by GPCR agonists are not clarified; there is evidence that protein kinase C is involved \[[@B15],[@B18]\], but a role for Ca^2+^ also appears likely, since all the agents above activate phospholipase C and elevate intracellular Ca^2+^ in hepatocytes \[[@B19],[@B20]\]. Furthermore, agents that elevate intracellular Ca^2+^ through mechanisms bypassing receptors have been found to activate ERK \[[@B15],[@B21]\]. However, agonist-stimulated phospholipase C activity is rapidly down-regulated upon culturing of hepatocytes \[[@B22],[@B23]\], and we recently reported that norepinephrine and PGF~2α~ activate ERK under conditions where the level of inositol 1,4,5-trisphosphate (InsP~3~) was only slightly, and transiently elevated \[[@B17]\]. In the present study we have, therefore, examined more closely the role of Ca^2+^ in ERK activation induced by norepinephrine and PGF~2α~ and mechanisms downstream of elevated \[Ca^2+^\]~i~.

Results
=======

Agents that elevate \[Ca^2+^\]~i~ activate ERK
----------------------------------------------

In agreement with previous observations \[[@B15],[@B21]\] treatment of hepatocytes with thapsigargin, which inhibits Ca^2+^ reuptake to endoplasmatic reticulum \[[@B24]\], and A23187, which induces Ca^2+^ influx, stimulated ERK1/2 activity 2--2.5 fold (Fig. [1A](#F1){ref-type="fig"}). The elevation of intracellular Ca^2+^ resulting from stimulation with thapsigargin is shown in Fig. [1B](#F1){ref-type="fig"}. These observations are compatible with a role for Ca^2+^-elevating mechanisms in the events that trigger ERK1/2 activation in hepatocytes.

![ERK1/2 activation and Ca^2+^ response in hepatocytes. A: At 3 h after the time of seeding hepatocytes were preincubated with timolol (10 μM) for 30 min prior to stimulation with thapsigargin (1 μM), A23187 (10 μM) or norepinephrine (10 μM) for 5 min before they were harvested and ERK 1/2 activity assessed. Results represent mean ± S.E.M. of five different experiments. B: Single cell measurement of \[Ca^2+^\]~i~ as described in Materials and Methods. Results given as ratio (345/385 fluorescence) represent a typical single cell response after stimulation with thapsigargin (10 μM) in a fura-2 AM loaded hepatocyte.](1471-2121-3-5-1){#F1}

Activation of ERK by norepinephrine and PGF~2α~ involves Ca^2+^
---------------------------------------------------------------

We then examined the role of Ca^2+^ in activation of ERK1/2 induced by stimulation of α~1~-adrenoceptors (with norepinephrine in the presence of timolol) and prostaglandin receptors (using PGF~2α~) \[[@B21],[@B25],[@B26]\]. The hepatocytes were pretreated with BAPTA-AM, which is activated intracellularly to bind Ca^2+^, EGTA, which binds extracellular Ca^2+^ and eventually may deplete intracellular Ca^2+^\[[@B27],[@B28]\], or gadolinium, a competitive inhibitor of Ca^2+^ influx \[[@B29]-[@B31]\]. BAPTA-AM completely attenuated the norepinephrine-induced rise of \[Ca^2+^\]~i~ (Fig. [2A](#F2){ref-type="fig"}), while the ERK1/2 activity in response to norepinephrine was partially decreased (Fig. [2B,2C](#F2){ref-type="fig"}). ERK1/2 activity induced by PGF~2α~ and the Ca^2+^ ionophore A23187 was also inhibited by BAPTA-AM, while the TPA response was unaffected (Fig. [2B,2C,2D](#F2){ref-type="fig"}). When the cells were pretreated with EGTA, the initial peak of the Ca^2+^ elevation was only slightly affected, while the prolonged phase of the Ca^2+^-response was abolished (Fig. [3A](#F3){ref-type="fig"}). The activation of ERK1/2 by norepinephrine or PGF~2α~ was partly decreased by EGTA (Fig. [3B,3C,3D](#F3){ref-type="fig"}). EGTA also markedly decreased the ERK1/2 response induced by A23187 and thapsigargin, while the TPA-induced ERK1/2 activation was unaffected (Fig. [3B,3C](#F3){ref-type="fig"}). Pretreatment with gadolinium decreased the adrenergic activation almost to the level obtained by EGTA (Fig. [4A](#F4){ref-type="fig"}). Gadolinium also decreased the A23187-induced activation of ERK1/2 (Fig. [4B](#F4){ref-type="fig"}). Taken together, the results suggest a role for Ca^2+^ in the activation of ERK by norepinephrine and PGF~2α~ and that this involves Ca^2+^ influx as well as release from internal pools.

![Effect of BAPTA-AM on \[Ca^2+^\]~i~ and ERK1/2 activation. A: Measurement of \[Ca^2+^\]~i~. Hepatocytes were preincubated with 0.55 % DMSO or BAPTA-AM (40 μM) during the last 25 minutes of the fura-2 AM loading. After 60 seconds of registration the cells were stimulated with norepinephrine (10 μM) in the presence of timolol (10 μM). Results show a typical single cell response. B-D: ERK1/2 responses. Hepatocytes cultured for 3 h were pretreated for 30 min with BAPTA-AM (40 μM) in the presence of timolol (10 μM) prior to stimulation with norepinephrine (10 μM), A23187 (10 μM), TPA (1 μM) or PGF~2α~ (10 μM) for 5 min before cells were harvested. All cultures contained DMSO at a concentration of 0.5 % during the preincubation and a final concentration of 1 % DMSO during incubation with agonist. B: Activity measurements of ERK1/2 representing mean ± S.E.M. of three experiments. C, D: Immunoblots using antibody against dually phosphorylated ERK1/2.](1471-2121-3-5-2){#F2}

![Effect of EGTA on \[Ca^2+^\]~i~ and ERK 1/2 activation. A: \[Ca^2+^\]~i~ measurements. Hepatocytes were preincubated in Krebs-Ringer-Hepes buffer with or without 5 mM EGTA for 15 min after fura-2 AM loading. After 60 seconds of registration the cells were stimulated with norepinephrine (10 μM) in the presence of timolol (10 μM). Results show a typical single cell response. B-D: ERK1/2 responses. Hepatocytes cultured for 3 h were pretreated with timolol (10 μM) for 30 min and EGTA (5 mM) for 15 min before stimulation with norepinephrine (10 μM), TPA (1 μM), thapsigargin (1 μM), A23187 (10 μM) or PGF~2α~ (10 μM) for 5 min (in the presence of 0.5 % DMSO). B: Activity measurements of ERK1/2 representing the mean ± S.E.M. of three experiments. C, D: Immunoblots using antibody against double phosphorylated, i.e. activated, forms of ERK1/2.](1471-2121-3-5-3){#F3}

![Effect of gadolinium on ERK1/2 responses. A: Hepatocytes cultured for 3 h were pretreated with gadolinium (100 μM) or EGTA (5 mM) for 15 min in the presence of timolol (10 μM) prior to stimulation with norepinephrine (10 μM) for 5 min before cells were harvested. Results are activity measurements of ERK1/2 given as pmol ^32^P incorporated into MBP/mg protein representing mean ± S.E.M. of three experiments. B: Immunoblot showing the effect of pretreatment with gadolinium (100 μM) for 15 min on A23187 (10 μM) induced ERK1/2 response. Antibody against dually phosphorylated ERK1/2 was used.](1471-2121-3-5-4){#F4}

Effect of antagonists of calmodulin and the multifunctional Ca^2+^/calmodulin-dependent protein kinase in ERK activation in hepatocytes
---------------------------------------------------------------------------------------------------------------------------------------

A major mechanism for Ca^2+^-induced signaling is through formation of a complex with calmodulin \[[@B32],[@B33]\]. Calmodulin has been found to stimulate as well as inhibit ERK1/2 activity \[[@B12],[@B34],[@B35]\]. We therefore examined the role of calmodulin in these pathways. Pretreatment of hepatocytes with the calmodulin inhibitors trifluoperazine, J-8, and W-7 markedly inhibited the ERK1/2 activation after stimulation with norepinephrine and PGF~2α~ (Fig. [5](#F5){ref-type="fig"}). The results were confirmed with immunoblots (Fig. [6](#F6){ref-type="fig"}). Activation of ERK1/2 by A23187 was also markedly sensitive to pretreatment with W-7 (Fig. [5](#F5){ref-type="fig"}, [6](#F6){ref-type="fig"}).

![Effect of inhibitors of calmodulin (Trifluoperazine, J-8, W-7) and the multifunctional Ca^2+^/calmodulin dependent protein kinase (KN-93). Hepatocytes were cultured for 3 h before preincubation with timolol (10 μM) for 30 min in the presence or absence of indicated inhibitors prior to stimulation for 5 min with norepinephrine (10 μM), PGF~2α~ (10 μM) or A23817 (10 μM), before cells were harvested and ERK1/2 activation assessed. A: Pretreatment with trifluoperazine (50 μM) in 0.5 % DMSO. The results represent one typical experiment out of three and are expressed as percent of untreated control. B: Pretreatment with J-8 (10, 25, and 50 μM) in 0.5 % DMSO. The results represent mean ± S.E.M of three experiments and are expressed as percent of corresponding control values. C: Pretreatment with W-7 (100 μM) or KN-93 (20 μM) in 0.5 % DMSO, while the final DMSO concentration during incubation with agonist was 1 %. Results represent mean ± S.E.M. of five experiments and are expressed as percent of untreated control.](1471-2121-3-5-5){#F5}

![Immunoblots showing the effect of inhibitors of calmodulin (W-7, J-8, trifluoperazine) and the multifunctional Ca^2+^/calmodulin dependent protein kinase (KN-93) on ERK1/2 activation (A-C). Hepatocytes were cultured for 3 h before preincubation for 30 min with timolol (10 μM) A: in the presence or absence of W-7 (25 μM) before stimulation for 5 min with norepinephrine (10 μM), PGF~2α~ (10 μM) or A23187 (10 μM) or B, C: with or without J-8 (40 μM), trifluoperazine (50 μM) or KN-93 (20 μM) before 5 min of stimulation with norepinephrine (10 μM). Western analyses were based on the use of antibody against dually phosphorylated ERK1/2.](1471-2121-3-5-6){#F6}

Calmodulin may act on several regulatory enzymes \[[@B32],[@B36]-[@B40]\], including the Ca^2+^/calmodulin-dependent protein kinases, which have been implicated in the activation of ERK1/2 \[[@B7]-[@B9]\]. We explored a possible role for the multifunctional Ca^2+^/calmodulin-dependent protein kinase in ERK1/2 activation in hepatocytes stimulated by norepinephrine and PGF~2α~. Pretreatment of the cells with KN-93, an inhibitor of the multifunctional Ca^2+^/calmodulin-dependent protein kinase \[[@B41]\], did not decrease the activation of ERK1/2 either by norepinephrine, PGF~2α~, or A23187 (Fig. [5](#F5){ref-type="fig"}, [6](#F6){ref-type="fig"}). In supplementary experiments we examined the effects of higher concentrations of KN-93 (up to 100 μM) or prolonged exposure times (up to 24 hours) which in none of the cases resulted in a decreased ERK1/2 activation (data not shown). These results suggest that calmodulin might be involved in hormone-induced activation of ERK1/2 in hepatocytes, however the data do not support a role for the multifunctional Ca^2+^/calmodulin-dependent protein kinase in Ca^2+^/calmodulin-mediated activation of ERK1/2 in hepatocytes

Inhibitors of src kinases attenuate ERK activation in hepatocytes
-----------------------------------------------------------------

Src kinases \[[@B42]\] have been implicated in the mechanisms resulting in ERK1/2 activation in response to stimulation of both G~i~- and G~q~- coupled heptahelical receptors \[[@B43]-[@B45]\], and several observations suggest that activation of Src in these pathways involves Ca^2+^\[[@B46],[@B47]\]. Data obtained in this study showed that the Src inhibitors PP1 and PP2, which are reported to primarily inhibit the Lck, Fyn, and Hck subtypes of Src kinases \[[@B48]\], markedly decreased the PGF~2α~-induced ERK1/2 activation and led to partial inhibition of the effect of norepinephrine, while the EGF induced ERK1/2 response was not reduced (Fig. [7](#F7){ref-type="fig"}). Furthermore, ERK1/2 activation induced by A23187 and thapsigargin was also decreased after Src inhibition (Fig. [7](#F7){ref-type="fig"}). The results suggest a role for Src kinases in the mechanisms leading to ERK1/2 activation both by PGF~2α~ and norepinephrine, and that this step at least in part may be located distal to increases in the intracellular level of Ca^2+^.

![Effect of inhibition of Src kinases on ERK1/2 activation. A-D: Hepatocytes were cultured for 3 h prior to preincubation for 30 min with timolol (10 μM) and the Src kinase inhibitors PP1 or PP2 at indicated concentrations in 0.5 % DMSO before stimulation for 5 min with norepinephrine (10 μM), PGF~2α~ (10 μM), EGF (10 nM), thapsigargin (1 μM), or A23187 (10 μM). A: ERK1/2 activity, given as percent of untreated control, induced by hormonal agents expressed as the mean ± S.E.M. of four experiments after treatment with PP1 (10 μM). The inset shows an immunoblot of the effect of PP1 (20 μM) on thapsigargin-induced ERK1/2 response. B: A23187-induced ERK1/2 response after PP1 (10 μM) treatment. Results represent mean ± S.E.M of three experiments. C: Dose-response curve for the effect of PP2 on ERK1/2 activity induced by A23187. Results represent mean ± S.E.M of three experiments. All activity measurements (A-C) are expressed as percent of untreated control. D: Immunoblots showing the effect of PP1 (10 μM) or PP2 (10 μM) on ERK1/2 responses. Antibody against dually phosphorylated ERK1/2 was used.](1471-2121-3-5-7){#F7}

Discussion
==========

The present findings confirm previous reports of a role for Ca^2+^ in ERK1/2 activation in hepatocytes \[[@B15],[@B21]\] and suggest that release of Ca^2+^ from intracellular stores as well as influx of extracellular Ca^2+^ is of importance for the hormone-induced activation of ERK1/2. Furthermore, the results suggest that calmodulin and Src kinases might be involved in the Ca^2+^-dependent activation of ERK1/ERK2.

Evidence from several experimental models suggest that activation of ERK1/2 may occur through Ca^2+^-dependent as well as Ca^2+^-independent mechanisms \[[@B5],[@B28],[@B49]-[@B54]\]. The present data suggest that Ca^2+^ is involved in activation of ERK1/2 in hepatocytes in response to norepinephrine and PGF~2α~. The ERK1/2 response was decreased by chelation of intracellular and extracellular Ca^2+^ with BAPTA-AM and EGTA, respectively, as well as by gadolinium, which competitively inhibits Ca^2+^ influx. It may appear that extracellular and intracellular Ca^2+^ act in a concerted, possibly sequential manner in the mechanisms involved in activation of ERK1/2 by norepinephrine and PGF~2α~. An integration of Ca^2+^ signals from the extracellular and intracellular environment is presumably due to store-operated Ca^2+^ influx \[[@B31],[@B55],[@B56]\]. The mechanisms that initiate Ca^2+^ influx subsequent to depletion of intracellular stores are incompletely understood, but recent studies have suggested that direct interaction between InsP~3~ receptors and calcium channels in the plasma membrane may lead to activation of the calcium channels \[[@B57]\]. A diffusible Ca^2+^ influx factor may also be involved \[[@B58]\]. Previous studies have suggested that hormone-induced Ca^2+^ influx involves heterotrimeric G~i~ proteins in hepatocytes \[[@B59],[@B60]\]. It is notable that norepinephrine and PGF~2α~ activate ERK1/2 in the presence of a barely detectable increase in intracellular InsP~3~\[[@B17]\]. This may suggest either the occurrence of local elevations of InsP~3~ which do not affect global InsP~3~, or that Ca^2+^ pools are regulated by other mechanisms such as generation of sphingosine-1-phosphate \[[@B61]\]. A role for ryanodine-sensitive Ca^2+^ stores in the endoplasmatic reticulum has also been proposed in hepatocytes \[[@B62]\] and EGTA-sensitive pools that are located in plasma membrane micro villar structures have been described \[[@B63]\].

Our data further suggest that calmodulin, which has previously been implicated in growth regulation in liver \[[@B64]\], is involved in activation of ERK1/2. The ERK1/2 responses induced by norepinephrine and PGF~2α~ were markedly decreased after pretreatment with the calmodulin inhibitors trifluoperazine, J-8, or W-7. Besides calmodulin, it is conceivable that the effect of Ca^2+^ is mediated through other Ca^2+^-binding proteins \[[@B39],[@B65]\]. Calmodulin may also act in a Ca^2+^-independent manner \[[@B38],[@B66]\], which might account for the more pronounced inhibition of hormone-stimulated ERK activity by calmodulin antagonists than by agents inhibiting the Ca^2+^ signal. Alternatively, nonspecific effects produced by calmodulin antagonists in higher doses might explain their relatively stronger inhibition. Among the downstream targets of calmodulin, the Ca^2+^/calmodulin-dependent protein kinases have been implicated in ERK1/2 activation in smooth muscle cells \[[@B8],[@B9]\], but not in other cells \[[@B67],[@B68]\]. Furthermore, the multifunctional Ca^2+^/calmodulin-dependent protein kinase was located downstream of ERK1/2 activation by platelet-derived growth factor (PDGF) in vascular smooth muscle cells \[[@B69]\]. Pretreatment of hepatocytes with KN-93 did not decrease ERK1/2 activation induced by hormones or the Ca^2+^ ionophore A23187. Thus, while the multifunctional Ca^2+^ /calmodulin-dependent protein kinase exerts several effects in hepatocytes, including growth inhibition under certain conditions \[[@B70]-[@B72]\], it does not appear to be involved in ERK activation. It is of interest that the α~1~-adrenoceptor-induced c-*fos* expression in fibroblasts was also observed to involve calmodulin, but not the multifunctional Ca^2+^/calmodulin-dependent protein kinase \[[@B73]\].

Increasing evidence suggest a role of Src kinases downstream of Ca^2+^/calmodulin in ERK1/2 signaling \[[@B10],[@B14],[@B46]\]. The present results suggest that Src kinases may be involved in ERK1/2 activation induced by PGF~2α~ and norepinephrine, while the EGF induced ERK1/2 response appears to be independent of these Src kinases. Furthermore, the ERK1/2 activation induced by the Ca^2+^ ionophore A23187 or by thapsigargin was partially decreased by Src inhibition suggesting a role of Src distal to increases in intracellular Ca^2+^. Of the possible downstream targets for Ca^2+^/calmodulin in ERK signaling in hepatocytes our findings thus lend support to a role of Src kinases, although the results do not exclude the possibility that Src kinases and calmodulin act in parallel pathways leading to ERK activation.

While the present results show a role for Ca^2+^ in ERK1/2 activation by norepinephrine and PGF~2α~, it is notable that even complete inhibition of Ca^2+^ signaling only partially inhibited ERK1/2 activity. Taken together with previous observations that inhibition of PKC almost completely inhibited ERK1/2 activation by norepinephrine, vasopressin, and angiotensin II \[[@B18]\], the results suggest that several mechanisms contribute to and may act in concert in the hormonal stimulation of ERK1/2 in hepatocytes.

Conclusion
==========

Our present data indicate that both extracellular and intracellular Ca^2+^ is involved in hormone-induced ERK1/2 activation in cultured hepatocytes, and suggest that calmodulin and Src kinases might play a role in these signaling pathways, while the multifunctional Ca^2+^/calmodulin-dependent protein kinase does not appear to be involved.

Materials and Methods
=====================

Materials
---------

Dulbecco\'s modified Eagle\'s medium, Waymouth\'s medium MAB 87/3, penicillin and streptomycin were from Gibco, Grand Island, NY, U.S.A. Adenosine 5\'-triphosphate, collagen, collagenase, EGTA, phenylmethylsulfonyl fluoride, benzamidine, leupeptin, pepstatin A, myelin basic protein (MBP), norepinephrine, prostaglandin F~2α~, epidermal growth factor, insulin, timolol, gadolinium chloride (hexahydrate), sulfinpyrazone, and 2-mercaptoethanol were from Sigma, St. Louis, MO, USA. A23187, 12-O-tetradecanoyl phorbol-13-acetate, thapsigargin, N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W-7), trifluoperazine dimaleate, 2-\[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)\]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN-93), and l,2-bis(o-aminophenoxy)ethane-N,N,N\',N\'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA-AM), PP1, PP2 were from Calbiochem, La Jolla, CA, USA. (N-8-Aminooctyl)-5-iodo-1-naphtalenesulfonamide (J-8) was from Alexis Biochemicals, Lausen, Switzerland. Fura-2 AM and Pluronic F-127 were from Molecular Probes, Eugene, OR, USA. Sodium(meta)vanadate was from Fluka Chemie AG, Buchs, Switzerland. Phenyl Sepharose CL-4B was from Pharmacia Biotech., Uppsala, Sweden. Dexamethasone was from Norwegian Medicinal Depot, Oslo, Norway, \[γ-^32^P\] Adenosine 5\'-triphosphate (3000 Ci/mol) was from Amersham International, Buckinghamshire, England.

Isolation and culture of hepatocytes
------------------------------------

Male Wistar rats (170--220 g) fed ad libitum were used. Parenchymal liver cells were isolated by in vitro collagenase perfusion and low-speed centrifugation \[[@B74]\] with modifications as previously described \[[@B75]\]. Cell viability was at least 95 %, measured as the ability to exclude trypan blue. The cells were suspended in medium and plated in Costar wells at 20.000 cells/cm^2^, unless otherwise specified. The culture medium (0.2 ml/cm^2^) was a 1:1 mixture of Dulbecco\'s modified Eagle\'s medium and Waymouth\'s medium MAB 87/3 containing 16.8 mM glucose \[[@B76]\], supplemented with penicillin (100 U/ml), streptomycin (0.1 mg/ml), dexamethasone (25 nM) and insulin (100 nM). The cultures were gassed with 95 % air/5 % CO~2~ and kept at 37°C.

Measurement of ERK activity
---------------------------

The measurement of ERK1/2 activity was performed as previously described \[[@B17],[@B77]\]. In brief, the hepatocyte cultures were exposed to agonists for 5 minutes before rinsing and scraping the cells into a 10 % ethylene glycol buffer. The lysate was centrifugated (15,800 × g) for 10 min, and the supernatant was mixed with phenyl-Sepharose which was washed twice in a 10 %, twice in a 35 % ethylene glycol buffer, and finally ERK1/2 was eluted with a 60 % ethylene glycol buffer \[[@B78]\]. The eluate was assayed for ERK1/2 activity with MBP as substrate, thereafter spotted onto P81 paper (Whatman, Maidstone, UK), which was washed, dried and counted in a liquid scintillation counter. Protein content was determined with the BCA Protein Assay (Pierce, Rockford, IL, U.S.A.).

Immunoblotting
--------------

Aliquots with 20 μg cell protein (total cell lysate prepared in Laemmli buffer) were electrophoresed on 10 % polyacrylamide gels ( acrylamide 30:0.8) followed by protein electrotransfer to nitrocellulose membranes and immunoblotting with a polyclonal ERK1/2 antibody against the dually threonine- and tyrosine phosphorylated forms of ERK1 and ERK2 (Promega Corporation, Madison, WI). Assessment of the multifunctional Ca^2+^/calmodulin dependent protein kinase was performed by immunoblotting using an antibody against the phosphorylated from of the enzyme. Immunoreactive bands were visualised with ECL Western blotting detection reagents (Amersham International).

Measurement of cytosolic Ca^2+^ in single hepatocytes
-----------------------------------------------------

The calcium measurements were done as described previously \[[@B20],[@B79]\]. Freshly isolated hepatocytes (50.000/cm^2^) were plated onto glass coverslips coated with collagen and kept in the culture medium for 30 minutes in an atmosphere of 95 % air/5 % CO~2~ at 37°C. The cells were loaded in Krebs-Ringer-Hepes buffer (KRH) with 1 % albumin and 16.8 mM glucose, supplemented with 5 μM fura-2 AM, 0.25 % (v/v) DMSO, 0.025 % Pluronic F-127 and 250 μM sulfinpyrazone for 90 minutes at 37°C. After loading, the cells were washed once and incubated with 400 μl KRH buffer with 16.8 mM glucose. Cells were preincubated with timolol (10 μM) in the presence or absence of EGTA (5 mM) for 15 minutes after loading and the experiments were performed in the same buffer. Preincubation with timolol (10 μM) and DMSO or BAPTA-AM (40 μM) was performed within the last 25 minutes of the loading period. 100 μl norepinephrine was injected after 60 seconds of registration to a final concentration of 10 μM. The experiments were carried out at 37°C. Single cell Ca^2+^ measurements were based on the ratios of the fluorescence with excitation at 345 and 385 nm. Because fura-2 AM is partly compartmentalized in hepatocytes after loading, we did not calculate apparant values for \[Ca^2+^\]~i~ from the ratios. The equipment consisted of a PTI-Δ-scan excitation device, a Nikon inverted microscope, a Hamamatsu CCD video camera and a Sony video recorder.

List of abbreviations
=====================

BAPTA-AM, l,2-bis(o-aminophenoxy)ethane-N,N,N\',N\'-tetraacetic acid tetra (acetoxymethyl)ester; EGF, epidermal growth factor; EGTA, ethyleneglycol-bis(β-aminoethyl)-N,N,N\',N\'-tetraacetic acid; ERK1/2, extracellular signal-regulated kinase 1 and 2; Gd, gadolinium chloride (hexahydrate); GPCR, G protein-coupled receptor; J-8; (N-8-Aminooctyl)-5-iodo-1-naphtalenesulfonamide; InsP~3~, inositol (l,4,5)-trisphosphate; KN-93, 2-\[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)\] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; MBP, myelin basic protein; NE, norepinephrine; PGF~2α~, prostaglandin F~2α~ ; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo \[3,4-d\]pyrimidine; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo \[3,4-d\]pyrimidine; RTK, receptor tyrosine kinase. TFP, trifluoperazine dimaleate; Thaps, thapsigargin; TPA, 12-O-tetradecanoyl phorbol-13-acetate; W-7, N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide.

Acknowledgements
================

This work was supported by The Norwegian Cancer Society, The Research Council of Norway, and the Novo Nordisk Foundation. We thank Eva Østby and Ellen Johanne Johansen for technical help and John-Arne Røttingen for helpful discussions


Background
==========

Non-participation in epidemiological studies has the potential to introduce bias into the results of such studies. Lack of participation can come about through noncontact, refusal or death of the respondent. This problem becomes accentuated in longitudinal studies in which non-random attrition can be expected at each wave. In studies of elderly samples, the problem of attrition is accentuated by the increased rate of deaths. Also, such samples may have reasons for refusal which are specific to older age groups. Therefore, it is important to determine the differences between those who take part in survey research and those who do not.

Often, very little is known about those who do not participate in the initial phase of any study although a number of studies have been able to determine sociodemographic differences and even health differences using hospital records. Romans-Clarkson *et al.*\[[@B1]\] have reviewed the literature on initial non-responders prior to 1988. They found that non-responders are usually older, more often male, of lower socioeconomic status and have less education. They are more likely to smoke, live in urban areas, to have a negative attitude to health surveys and have higher mortality in subsequent years. Their own study, of a random sample of community residing women, found similar results and, by examining hospital records, found no difference in occurrence of physical or psychiatric illness. Similar results for initial refusals have been found in more recent studies \[[@B2],[@B3]\].

In longitudinal studies much more is known about those who do not participate in second and subsequent waves. Although non-response can be due to death of the respondent, no contact or refusal, not all studies examining non-response at follow-up have made this distinction. Lui & Anthony \[[@B4]\] found that non-response in an elderly sample one year after initial interview was associated with lower Mini Mental State Examination (MMSE) scores, being older and having fewer years of education. Schaie *et al*\[[@B5]\] found that non-response in a three wave study over 14 years was associated with lower scores on a number of IQ measures. In a study by Clark *et al.*\[[@B6]\] of depression in people aged 18 and above, non-response was not associated with depression after accounting for demographic variables. However, only one third of non-responders were refusals, most being no contacts.

Looking specifically at refusals, Eaton *et al.*\[[@B7]\], in a follow-up of 18--64 year olds from the Epidemiologic Catchment Area (ECA) Program, found that refusers were older, more likely to be married and had lower educational levels than those interviewed. Psychopathology (assessed by either diagnoses or symptoms) was not significantly associated with refusal. Bucholz *et al*\[[@B8]\] examined refusers amongst an 18--49 year old community sample followed up after 11 years. Refusers were more likely to be male and to have a history of barbiturate abuse or dependence, but were no more likely to be in the problem-drinking, minimal alcoholic group than responders. Studies of elderly refusers have found that they are more likely to be cognitively impaired, to be in poor physical health and to have lower levels of education \[[@B9]-[@B14]\].

Of the possible reasons for non-response, refusal is of particular interest because it is amenable to change. The more we know about those who are alive and have been contacted, but refuse to take part, the more likely we are to be able to improve our approach and hopefully lower our refusal rates. The aim of this paper was to examine the relationship of non-response, particularly refusal, to the physical, psychological and cognitive state of elderly participants at subsequent waves of a longitudinal survey.

Methods
=======

The sample
----------

In 1990--91 a sample of 945 (of 1377 selected) persons drawn from the Electoral Rolls of Canberra and the nearby town of Queanbeyan and representative of the age distribution of this community were interviewed using the Canberra Interview for the Elderly (CIE) \[[@B15]\]. An additional sample of 100 (of 145 selected) residents of Nursing homes and sheltered accommodation were also interviewed. Interviews were sought from both the subject and informant, usually a close relative. The structured interview was administered to the participant by a trained lay interviewer. It was undertaken in the participant\'s home, taking 1.5 to 2 hours to complete. All interviewing and tests were completed in one visit.

Wave 2 of the study was undertaken at a mean of 3.6 (range 3.3--4.2) years later and Wave 3, at a mean of 4.0 (range 3.7--4.4) years after Wave 2. Refusers at Waves 2 and 3 were divided into two types: those who refused all participation and those who were not interviewed themselves but an interview was achieved with a relative or friend. In most cases where only an informant interview was possible, the respondent was approached, initially, to ask if they would take part. However, in some cases, a spouse or child intercepted the interviewer, decided that the respondent was unable to carry out the interview but offered to be interviewed themselves.

For the purposes of this study, those subjects for whom *only* an informant interview was obtained at Wave 1 were excluded from the analysis of refusals at Wave 2, while Informant Only interviews at Wave 2 were excluded from analysis of refusal at Wave 3. No attempt was made at Wave 3, to interview anyone who had refused outright at Wave 2.

Measures
--------

Physical health was assessed using 1) an Activities of Daily Living (ADL) scale \[[@B16]\] which asks the participant to rate their ability to perform a range of everyday activities, with answers ranging from \"no difficulty\" to \"unable to do\", 2) a measure of the number of chronic illnesses suffered by the respondent, and 3) self-reported measures of sight and hearing impairment. The sight impairment scale and the hearing impairment scale were each comprised of 5 questions, including a general question on how they rated their sight or hearing and 4 questions on sight or hearing problems in 4 different situations. Higher scores mean higher impairment. The Goldberg anxiety and depression scales \[[@B17]\] were used as indicators of mental health. This scale contains 18 questions, 9 on depression and 9 on anxiety with answer options being \"yes\" or \"no\". Cognitive impairment was assessed using the MMSE \[[@B18]\], a test covering a range of cognitive abilities including orientation for place and time, memory and spatial ability. A higher score for this test indicates better cognitive function. Premorbid IQ was measured by the National Adult Reading Test (NART) \[[@B19]\]. The NART is a measure of verbal IQ and is commonly used to estimate IQ in elderly subjects because it is highly resistant to the effects of ageing and dementia \[[@B19]\]. It involves the participant reading a list of words of increasing difficulty and being assessed on their pronunciation. Personality was assessed only at Wave 1 by extraversion and neuroticism scales from the short form of the Eysenck Personality Questionnaire-Revised \[[@B20]\], a measure consisting of 24 questions, 12 on extraversion and 12 on neuroticism. DSM-III-R dementia diagnosis was able to be determined from the interview.

At the end of the Wave 1 interview only, participants were asked by the interviewer \"Was the questionnaire at all distressing -- did some of the questions upset you or make you feel anxious?\" with options being \"yes\", \"no\" or \"don\'t know\". Five percent answered \"don\'t know\" or the question had not been asked. This 5% was treated as missing data.

Sociodemographic variables included age, sex, living alone (in community sample only), years of education and previous occupation status (white collar versus blue collar / manual).

Statistical analysis
--------------------

Univariate analyses on continuous variables were performed using one way analysis of variance followed by post hoc modified-Least Significant Difference (LSD) tests to compare the participants and refusers. Categorical variables were analysed using chi-square tests. Multivariate analyses were performed using logistic regression with simultaneous entry of predictor variables.

Results
=======

The response rate at Wave 1 was 69%. The only information available on non-responders was their age and sex. Those who refused participation were not significantly different from participants in age. However, significantly more women than men refused to take part. In the community sample the response rate for males was 76% and for females, 62%. In the nursing home sample, the response rates were 75% and 65% for males and females respectively.

Table [1](#T1){ref-type="table"} gives the breakdown of response status at Wave 2 and Wave 3. Of the 77% of respondents from Wave 1 who were *contacted* at Wave 2, 85% participated, 5% allowed an informant to be interviewed and 10% refused any participation. At Wave 3, of those who were able to be contacted, 86% participated, 5% allowed an informant interview, and 9% refused any participation.

::: {#T1 .table-wrap}
::: {.caption}
######

Response rates at Wave 2 of those respondents interviewed at Wave 1 and response rates at Wave 3 of those interviewed at Wave 2.
:::

                                           **Wave 2**   **Wave 3**
  ---------------------------------------- ------------ ------------ ----- ------
                                           N            \%           N     \%

  Respondent interviews at previous wave   981          100          638   100
  Respondent interviews                    638          65.0         379   59.6
  Informant only interviews                36           3.7          22    3.6
  Refusals                                 78           8.0          39    6.1
  Died since previous wave                 215          21.9         167   26.2
  No contact                               14           1.4          31    4.9
:::

Those who were not contacted were people who either could not be found or had moved too far away to be followed up. This group plus those who had died have been omitted from all further analysis.

Refusal at Wave 2
-----------------

Table [2](#T2){ref-type="table"} compares three groups: those respondents who participated at Wave 2, those with an Informant Only interview and those who refused any participation. Significant differences were found for years of education, occupational status, NART, MMSE and ADL scores, sight impairment and distress at end of Wave 1 interview. Consideration of LSDs for continuous variables and adjusted residuals for categorical variables showed that both groups of refusers, but particularly outright refusers, were less likely to be white collar workers. However, only the outright refusers had significantly fewer years of education. Participants had significantly higher NART and MMSE scores than either those who refused or those with an Informant Only interview. The Informant Only group was significantly more physically impaired in terms of ADL, sight (but not hearing or chronic illness) and had lower MMSE scores. A dementia diagnosis at Wave 1 was significantly more likely in those who allowed an informant interview only at Wave 2, with those refusing being more likely to have had a dementia diagnosis than those who participated at Wave 2. Those who refused any participation were more likely to report distress at the end of the Wave 1 interview than those who participated at Wave 2. There was no difference between the groups on age, sex, anxiety or depression, extraversion or neuroticism.

::: {#T2 .table-wrap}
::: {.caption}
######

Comparison of participants, those respondents who refused but allowed an informant interview, and those who refused all participation at Wave 2: Means (and SDs) or percentages
:::

  Wave 1 variables                       Participants n = 560--638   Refused but informant interview n = 22--36   Refused all Participation N = 60--78   P-value
  -------------------------------------- --------------------------- -------------------------------------------- -------------------------------------- ---------
  Age at Wave 1                          76.5 (4.9)                  77.9 (5.4)                                   75.9 (4.0)                             .123
  Sex (% male)                           48.58                       44.44                                        42.31                                  .532
  Years of education                     11.45 (2.64)                10.82 (1.95)                                 10.56 (2.28)                           .009
  Occupational status (% white collar)   65.78                       48.57                                        42.86                                  .000
  Lives alone (%) ^\*^                   35.78                       31.25                                        49.30                                  .066
  NART                                   113.23 (9.05)               103.23 (11.51)                               105.78 (10.89)                         .000
  MMSE                                   27.69 (2.30)                23.10 (5.49)                                 26.14 (4.48)                           .000
  DSM-III dementia (%)                   4.7                         34.6                                         13.5                                   .000
  ADL                                    1.75 (2.51)                 3.73 (4.56)                                  1.37 (1.56)                            .000
  Chronic illness                        2.94 (1.92)                 2.94 (2.16)                                  2.67 (1.60)                            .498
  Sight problems                         0.88 (1.88)                 2.03 (3.87)                                  0.92 (1.97)                            .005
  Hearing problems                       1.99 (2.45)                 2.87 (3.11)                                  2.14 (2.66)                            .150
  Anxiety                                2.41 (2.23)                 2.18 (2.58)                                  2.84 (2.63)                            .269
  Depression                             1.89 (1.88)                 2.52 (2.38)                                  2.14 (2.15)                            .153
  Extroversion                           5.89 (3.23)                 5.95 (3.14)                                  6.47 (3.11)                            .413
  Neuroticism                            3.04 (2.80)                 4.05 (3.29)                                  3.28 (2.71)                            .223
  Distressed by interview (%)            4.01                        9.68                                         10.96                                  .016

^\*^ Living alone or with others was only examined in those living in the community.
:::

Logistic regression was used to compare 1) participants to refusers and 2) participants to the Informant Only group. Those variables that showed significant differences in Table [2](#T2){ref-type="table"}, were entered simultaneously into the regression. Dementia diagnosis was not included because it was strongly associated with the MMSE score. The results are shown in Table [3](#T3){ref-type="table"}. Premorbid IQ as measured by the NART was the only significant predictor of outright refusal at Wave 2, while both NART and MMSE scores were significant predictors of the Informant Only group at Wave 2. Stepwise entry of variables was also undertaken to further examine the role of predictors in the odds of refusal. The results were essentially the same as for simultaneous entry with the exception that in comparing participants with Informants Only, sight impairment was significantly worse in the latter group. Regression analyses were also undertaken using dichotomised variables: MMSE with a cut point of 23/24, sight impairment versus no impairment, ADL score indicating no need for assistance versus needing assistance. The results were substantively the same as when continuous variables were used.

::: {#T3 .table-wrap}
::: {.caption}
######

Odds ratios (ORs) (and 95% confidence intervals) from logistic regression where the OR of being 1) a refuser or 2) an informant-only respondent at wave 2 is associated with an increase in the predictor.
:::

  Wave 1 Variables                            Refusers compared to Participants n-716   Informant-only groups compared to Participants n-674
  ------------------------------------------- ----------------------------------------- ------------------------------------------------------
  Age                                         0.96 (0.91--1.03)                         1.00 (0.92--1.10)
  Sex (males = 1 females = 2)                 1.25 (0.72--2.18)                         1.21 (0.47--3.08)
  Years of education ^\*^                     1.02 (0.90--1.16)                         1.02 (0.82--1.27)
  Occupational status (white collar)          0.68 (0.38--1.23)                         1.07 (0.42--2.72)
  NART^\*^                                    0.95 (0.92 0.98)                          0.94 (0.89--1.00)
  MMSE^\*^                                    0.91 (0.88--1.02)                         0.75 (0.63--0.89)
  Activities of daily living ^\*^             0.90 (0.76--1.07)                         1.04 (0.89--1.21)
  Sight impairment scale ^\*^                 1.03 (0.88--1.21)                         1.20 (0.98--1.45)
  Distressed by interview (no = 1, yes = 2)   2.30 (0.86--6.15)                         0.94 (0.16--5.37)

^\*^OR associated with an increase of one unit in the scale.
:::

Refusal at Wave 3
-----------------

Refusal at Wave 3 was examined using the same analyses as for refusal at Wave 2. Significant differences between the three groups were found for age, years of education, NART, MMSE and ADL scores. Differences in years of education, NART and ADL scores were essentially the same as for the three Wave 2 groups, while MMSE scores were no different between participants and those refusing any participation but significantly lower in those for whom there was only an informant interview. This latter group was significantly older than the other two groups. Logistic regression comparing participants to outright refusers showed that the only significant predictor was years of education. No individual measure predicted refusal when participants were compared to the Informant Only group, although the block of variables did make a significant contribution.

Change in physical and mental health and cognitive function between Waves 1 and 2 were examined as predictors of refusal at Wave 3. Changes in ADL showed the only significant association, with those providing an informant interview having a greater increase in ADL problems compared to respondents who were interviewed and those who refused any participation.

Discussion
==========

At the third wave of this longitudinal study of an elderly sample, respondent interviews were achieved for 39% of those originally interviewed. Another 39% had died since Wave 1.

At Wave 2, refusal, for both those who allowed an informant to be interviewed and those who refused any participation, was significantly associated with lower occupational status, fewer years of education, lower verbal IQ scores and poorer cognitive function than participants. These results confirm those of other studies which have examined refusal in the elderly \[[@B4],[@B5],[@B7],[@B12],[@B13]\]. A number of these studies also report poorer physical health in refusers. However, in this study, only those with an Informant Only interview showed significantly higher disability (ADL) than participants. Also, the Informant Only group were more likely to have had a diagnosis of dementia at the Wave 1 interview. These findings suggest that including an informant interview in the study has enabled us to gather information on those who were too physically frail or cognitively impaired to participate themselves. These are groups that are important to include in any studies of health in the elderly. Unfortunately, those who refused were also more likely to have had a dementia diagnosis.

It is interesting also to consider those characteristics that were not associated with refusal. It has been suggested that psychological state may contribute to non-participation in community studies \[[@B21]\]. While the data presented here can say nothing about initial refusal, at subsequent waves we found no association between depression and anxiety scores at previous waves and refusal. As the measures of current anxiety and depression were taken 4 years prior to refusal at subsequent waves, it may be reasonable to expect no association. However, the trait measure of neuroticism, which is relatively stable over time and highly correlated to depression and anxiety was also found to have no association. Similarly, Baton *et al*\[[@B7]\], Norris \[[@B22]\] and Clark *et al*\[[@B6]\] did not find any association between non-response and depression symptoms, while Launer \[[@B11]\] found that non-responders had reported more psychiatric symptoms.

Refusers at Wave 2 were no different in age to those who participated. Some other studies \[[@B11]\] have found similar results, while refusers in the study by von Strauss *et al.*\[[@B13]\] were significantly older. This latter study, however, was interviewing people in their mid eighties. At Wave 3 of our study, when the average age of the sample was 84 years, those for whom only an informant interview was achieved were significantly older than those who participated. This present study also found no difference in the proportion of males and females refusing at Waves 2 and 3, although more women refused at the initial approach. Other studies examining refusal at initial approach have found that males are more likely to refuse\[[@B1],[@B8]\], however these tend to be samples covering the adult age range. Studies of older samples \[[@B10],[@B11]\] have found little difference in the gender of initial refusers. It is difficult to know why more women than men refused at the initial approach in this study. Reports of follow-up of older samples \[[@B4],[@B9],[@B13],[@B22]\] have found no differences in gender, as was found here.

Multivariate analysis comparing those who refused any participation to those who participated showed that the verbal IQ score was the only significant predictor of refusal. Comparison of those who either refused or had an Informant Only interview found that both IQ and cognitive impairment contributed. However, the ADL score did not contribute significantly in this analysis. This was somewhat surprising as the ADL score, for those with an informant only interview, was more than double that of those who participated.

Of the 44 people who said they felt some distress during the Wave 1 interview, 34 gave reasons; 17 (50%) gave the cognitive section as the reason for their distress, while a further five felt distressed by their inability to understand or answer the questions. A possible limitation of this measure is that some respondents may have been reluctant to admit to any distress to the interviewer and so the results may not be an adequate indicator of distress or discontent about the interview. Nevertheless, people who refused any participation at Wave 2 were more likely to have experienced distress. Von Strauss *et al.*\[[@B13]\], in a study of attitudes to a longitudinal study, found that those with impaired cognitive functioning and lower education showed the least positive attitude and that the first contact and the cognitive testing were the most stressful part of the study. Similarly, Levin *et al*. \[[@B14]\] examined refusal on follow-up in a sample of Parkinson\'s Disease patients and found that the only significant predictor was cognitive impairment. These authors commented that participants and their spouses were concerned about the development of dementia when contacted at follow-up. As with other studies, this one also found that those who refused had had lower MMSE scores and fewer years of education. One plausible interpretation is that those people who perform poorly on the cognitive tests are aware of this and become distressed by it.

Non-response is a serious issue for survey research, in particular, longitudinal studies in which each wave results in further loss and the possibility of sample bias. In studies of the elderly, loss through death of respondents is inevitable. Kessler *et al*\[[@B23]\] and, more recently, Dunn \[[@B24]\], have examined statistical methods to adjust for non-response. These methods depend on the predictors of missingness and the purpose here has been to identify these predictors. These data can then be used for such methods as multiple imputation or Full Information Maximum Likelihood (FIML) estimation. An examination of the predictors of mortality in this sample \[[@B16]\] found physical ill health, cognitive functioning and being male to be significant predictors. This report has concentrated on those who are alive, have been contacted but refuse to participate.

Conclusions
===========

In conclusion, refusal was predicted by low scores on a verbal IQ test and cognitive impairment, possibly because of distress caused by cognitive testing. It is important that cognitive testing be presented in a non-threatening manner and that participants in the initial wave of such studies be reassured about their performance. However, bias may be reduced by including an informant interview in the study.

Competing interests
===================

None declared

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

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Acknowledgements
================

This work was supported by grant 973302 from the National Health and Medical Research Council and by a grant from the Australian Rotary Health Research Fund. Help in various phases of the study was provided by Suzanne Dee, Ruth Scott, Susan Lindsay, Colleen Doyle, Karen Maxwell, Andrew Mackinnon and Bryan Rodgers.