Glycation does not modify bovine serum albumin (BSA)-induced reduction of rat aortic relaxation: The response to glycated and nonglycated BSA is lost in metabolic syndrome

The effects of nonglycated bovine serum albumin (BSA) and advanced glycosylation end products of BSA (AGE-BSA) on vascular responses of control and metabolic syndrome (MS) rats characterized by hypertriglyceridemia, hypertension, hyperinsulinemia, and insulin resistance were studied. Albumin and in vitro prepared AGE-BSA have vascular effects; however, recent studies indicate that some effects of in vitro prepared AGEs are due to the conditions in which they were generated. We produced AGEs by incubating glucose with BSA for 60 days under sterile conditions in darkness and at 37°C. To develop MS rats, male Wistar animals were given 30% sucrose in drinking water since weanling. Six month old animals were used. Blood pressure, insulin, triglycerides, and serum albumin were increased in MS rats. Contraction of aortic rings elicited with norepinephrine was stronger. There were no effects of nonglycated BSA or AGE-BSA on contractions in control or MS rats; however, both groups responded to L-NAME, an inhibitor of nitric oxide synthesis. Arterial relaxation induced using acetylcholine was smaller in MS rats. Nonglycated BSA and AGE-BSA significantly diminished relaxation in a 35% in the control group but the decrease was similar when using nonglycated BSA and AGE-BSA. This decrease was not present in the MS rats and was not due to increased RAGEs or altered biochemical characteristics of BSA. In conclusion, both BSA and AGE-BSA inhibit vascular relaxation in control artic rings. In MS rats the effect is lost possibly due to alterations in endothelial cells that are a consequence of the illness.
Introduction
Reducing sugars including glucose, fructose, trioses, and glyceraldehyde alter proteins by nonenzymatic glycosylation known as glycation or Maillard reaction. This reaction occurs in several steps, the initial ones being relatively fast and reversible, while the latter ones are slower and irreversible originating products known as advanced glycosylation end products (AGEs). AGEs were originally characterized by their yellow-brown fluorescent color and by their ability to cross-link with and between amino groups (Reynolds 1963). However, the term is now used for a broad range of advanced products of the Maillard reaction, including N-carboxymethyllysine (CML) and pyrroline (Reddy et al. 1995; Baynes and Thorpe 1999).
AGEs exert their actions by two different mechanisms: (1) modifying structural intra- and extracellular proteins and (2) binding to their receptors (RAGEs) that belong to the immunoglobulin family and are located in the plasma membranes of monocytes, macrophages, endothelial cells, and vascular smooth muscle cells (Brownlee 1995; Basta et al. 2002). When AGEs bind to their receptors they initiate second messenger cascades. They also generate reactive oxygen species which modulate cellular function and can induce inflammatory processes (Krieglstein and Granger 2001; Basta et al. 2002; Ramasamy et al. 2005).
Albumin is one of the main proteins undergoing glycation reactions due to its abundance in serum and to the fact that it can be glycated at multiple sites (Wautier and Guillausseau 1998). Both bovine serum albumin (BSA) and AGE-BSA have been reported to have vascular effects. BSA reacts with nitric oxide (NO) therefore modulating its biological actions and increasing endothelial permeability (Predescu et al. 2002). Some forms of AGEs also diminish vascular relaxation by decreasing the NO production of endothelial cells (Hogan et al. 1992; Xu et al. 2003). Nevertheless, there have been recent reports that other forms of in vitro prepared AGEs have different effects from the naturally produced AGEs (Valencia et al. 2004).
AGEs have been implicated in aging processes and in the development of chronic complications of diabetes such as renal and vascular damage and could also participate in complications of metabolic syndrome (Brownlee 1995; Wautier and Guillausseau 1998; Valencia et al. 2004). There is a significant association between a high incidence of cardiovascular events and endothelial damage in animal models and diabetic patients and one of the mechanisms underlying this association is the presence of AGEs in plasma (Tan et al. 2002).
A variant of fructose-induced hypertension by giving sucrose to rats has been developed in our institution. The fructose-fed rat becomes hypertensive, hypertriglyceridemic, hyperinsulinemic and has insulin resistance; it exhibits what is known as metabolic syndrome (MS) or “Syndrome X” (Hwang et al. 1987). Some of the characteristics of the model developed in our institution have already been described (Baños et al. 1997) and they coincide with those of fructose fed animals, i.e., our rats also develop hypertension, hypertriglyceridemia, hyperinsulinemia, and insulin resistance. They also show increased oxidative stress (Baños et al. 2005). Some of the characteristics of these animals are accentuated by age and have been associated with renal failure and cardiovascular diseases which are related to alterations in the structure and function of endothelial cells (Rubio et al. 2006). AGEs could play a role in the vascular damage found in MS rats. Therefore, the aim of the present paper was to determine the effect of nonglycated BSA and AGE-BSA on aortic rings from control and MS rats.
Results
Changes in body weight, arterial pressure, triglycerides, glucose, insulin, and serum albumin
Table I shows the values of body weight, arterial pressure, glucose, insulin, homeostasis model assessment (HOMA), triglycerides, and serum albumin for control and MS rats. Experimental animals developed metabolic syndrome characterized by arterial hypertension, hypertriglyceridemia, hyperinsulinemia, and insulin resistance. There was a statistically significant increase in the serum albumin values in MS rats. Although there was no significant change in body weight, the animals with MS clearly developed central adiposity.
Table I
Clinical characteristics and biochemical parameters from control and MS rats
Control
SM
Body weight (g)
558.3 ± 14.9
547.5 ± 38.1
Arterial pressure (mm of Hg)
101.9 ± 1.4
138.9 ± 0.8*
Triglycerides (mg/dL)
55.6 ± 4.7
109.1 ± 12.8*
Glucose (mmol/L)
5.9 ± 0.3
4.8 ± 0.7
Insulin (μU/mL)
6.5 ± 0.9
24.2 ± 5.7*
HOMA
1.02 ± 0.5
5.02 ± 2.1*
Albumin (g/dL)
3.3 ± 0.1
3.6 ± 0.1*
Values are mean ± SEM, n = 8; *P < 0.01.
AGE-BSA preparation
It was possible to produce AGE-BSA under the previously described condition. After incubation with glucose, the albumin solution took a yellow-brown color and showed the characteristic fluorescence spectrum of AGEs. Albumin incubated without glucose did not significantly change its appearance and did not have the characteristic fluorescence spectrum of AGEs (Figure 1).
Fig. 1
Fluorescence spectrum of AGE-BSA and nonglycated BSA. Both solutions were tested at 2 mg/mL of protein. The peak of fluorescence is not present in the nonglycated BSA. The interrupted line represents the saturation level of the fluorescence detection by the equipment employed.
The purification of AGE-BSA on Affi-Gel-Blue is shown in Figure 2; three peaks were observed which corresponded to the highly glycated BSA (first one), the less glycated BSA moderately, and stronger bound to the chromatographic matrix (second and third peaks, respectively). The first peak was employed for subsequent analysis and tested for vasoreactivity.
Fig. 2
Purification of AGE-BSA on Affi-Gel-Blue. Three peaks were observed: the first one corresponds to highly glycated BSA which did not bind to the matrix, the second one corresponds to the less glycated BSA which bound moderately, and the third peak corresponds to the stronger bound BSA. The first peak was the only one to be further analyzed. The arrows indicate the buffer used at each stage.
The AGE-BSA produced had a protein concentration of 53.8 mg/mL and a specific activity, before its purification in Affi-Gel Blue, of 7.9 AU/mg of protein and of 10.5 AU/mg of protein, after purification. Fluorescence was not significantly elevated when protein concentration of nonglycated BSA was increased (Figure 3). The value of specific activity was similar to that obtained by other authors using similar procedures (Verbeke et al. 1997).
Fig. 3
Evaluation of the specific activity (SA) of AGE-BSA. The increase of specific activity obtained by the purification process on Affi-Gel-Blue is observed. Bold circles represent the nonglycated BSA. Bold squares represent the unpurified AGE-BSA and bold triangles represent the purified AGE-BSA. Specific activity increased in approximately 33% by the purification process.
Changes in mass and isoelectric point of BSA and glycated BSA
Table II shows the results of the analysis of the main glycation sites of the BSA structure. The analysis was done using the sequence of bovine serum albumin reported in the GenBank with number CAA76847 (gi:3336842) and the PyMOL v0.99 program (DeLano Scientific LLC, CA). The disposition of the lysine and arginine residues in the three-dimensional structure was evaluated. The analysis shows that a maximum of 52 residues from the 79 present in the molecule can be glycated. The rest of the residues are in the inside of the structure of BSA.
Table II
Distribution of the amino acids lysine and arginine in the three-dimensional structure of BSA
Amino acid position in the three-dimensional structure
Amino acid
Totally exposed
Partially exposed
Nonexposed
Lysine
4,12,20,41,51,64,76, 116,127,132,136,180,187,211,224, 261,273,275,285,312,316,322,350,362,375,377,388,396, 465,474,499,504,520,523,524,535,537,556, 563,573.
159,413,431,533.
93,106,114,131, 204,221,232,239, 242,279,294,439, 544.
Arginine
194,196, 198.
143,185,409,412, 444.
10,81,98,144,208, 217,256,335,336, 347,427,458,483, 484.
Total
43
9
27
The amino acids localized in depression areas are considered as partially exposed.
Figure 4 shows a molecular mass of 66,655.58 Da for nonglycated BSA (Figure 4A). After glycation, the molecular mass increased to 74,461.15 Da (Figure 4B). The net increase of 7,805.57 Da observed corresponds to the addition of 48 glucose molecules, considering that the molecular mass of glucose is 180.2 Da and that during each reaction of Amadori product, a water molecule is liberated (18 Da). The amount of glucose bound to BSA in our preparation was 48, very close to the 52 theoretically possible; therefore, we consider that our AGE-BSA is a highly glycated product.
Fig. 4
Analysis of BSA and AGE-BSA by mass spectrometry. The mass increase of protein after glycation is observed. The mass of nonglycated BSA was 66,655.58 Da (A) and that of AGE-BSA was 74,461.15 Da (B). The net increase of mass was 7,805.57 Da, corresponding to the addition of 48 molecules of glucose to each BSA molecule. Inset: change of isoelectric point of BSA after glycation. Nonglycated BSA had a pI of 4.2 while AGE-BSA had a pI of 6.3. Line 2 is the broad range pI standard kit (pH 3–10).
Nonglycated BSA had an isoelectric point of 4.2. The glycation process changed the isoelectric point to 6.3 (Figure 4, inset).
Vascular responses
Norepinephrine-induced vasoconstriction was higher in MS rats than in control rats (Table III); nevertheless, vasoconstriction was not significantly modified in aortic rings from control or MS rats in the presence of nonglycated BSA or AGE-BSA. L-NAME significantly increased vascular contraction in both groups (154.41 ± 10.35% versus 186.18 ± 7.58%, respectively) since inhibition of NOS induces an imbalance in vasoconstriction and vasodilation which is greater in MS than in control rats (Figure 5).
Fig. 5
Effect of AGE-BSA on vascular contraction in aortic rings from control (solid bars) and MS rats (open bars). The contractions were induced by NE 1 μM and basal tension was normalized to 100% in control and MS rats. Tension values in grams are shown in Table III. Results are the means ± SEM of six independent experiments. *P < 0.05 between MS and control; **P < 0.001 between the Tyrode solution and L-NAME.
Table III
Values of contraction (g) and relaxation (%) of aortic rings in control and experimental rats
Control
MS
Vascular contraction
1.5 ± 0.2
2.2 ± 0.2*
Vascular relaxation
84.4 ± 2.6
64.9 ± 3.3*
The results are expressed as relaxation percentage of the initial precontraction level with NE (1 μM). Final relaxation with Ach (1 × 10−6 M) is reported. Values are mean ± SEM, n = 8; *P < 0.001.
Basal relaxation was diminished in MS rats when compared to control rats (Table III). Figure 6 shows the effect of nonglycated BSA and AGE-BSA upon vascular relaxation in aortic rings from control and MS rats. In control rings nonglycated BSA and AGE-BSA at 40 AU/mL (3.8 mg/mL) significantly inhibited the endothelium-dependent vasorelaxation in a 35%. This effect was not present when a lower dose was used (10 AU/mL; 0.95 mg/mL). Fresh albumin, fatty acid free, diminished vasorelaxation in aortic rings from control rats in the same proportion as pre-incubated control BSA and AGE-BSA at a concentration of 3.8 mg/mL (data not shown).
Fig. 6
Effect of nonglycated BSA and AGE-BSA at 40 AU/mL (3.8 mg/mL) on endothelium-dependent vasorelaxation in aortic rings from control (A) and MS rats (B). Results are expressed as relaxation percentage from the initial precontraction level with NE 1 μM. *P < 0.05 between the Tyrode solution and nonglycated BSA and AGE-BSA; #P < 0.05 between the Tyrode solution in MS and control rats.
In MS aortic rings vasorelaxation was not significantly affected by BSA or AGE-BSA at any of the doses tested.
Immunofluorescence of RAGEs
Results of expression of RAGE are shown in Figure 7. There is an increased RAGE immunoreactivity in aortas from MS rats when compared to control rats. In aortas from C rats fluorescence is observed mainly in the endothelium while in MS aortas both the endothelium and muscular fibers fluoresce. Damage due to the development of the metabolic syndrome can also be appreciated by the presence of buckles and a loss of continuity in elastic fibers.
Fig. 7
Immunohistochemistry for RAGE in aortas of control (Panel A) and MS (Panel B) rats. Negative control (Panel C). Magnification 40×. Asterisk indicates internal elastic lamina. There is an increased RAGE immunoreactivity in MS rats.
Discussion
The aim of the present paper was to determine the effect of nonglycated BSA and AGE-BSA on vascular aortas from control and MS rats in which the condition is induced by a high ingestion of sucrose and where the structure and function of endothelial cells are altered. Both albumin and AGEs have been reported to have vascular effects (Vlassara et al. 1992). However, the vascular effects of AGEs might not be produced by all types of AGEs, since AGEs may have very different structures depending on the reactions generated during the in vitro glycation process of the protein by reducing sugars (Katchalsky and Sharon 1953). Some of the AGEs generated are capable of cross-linking to proteins and others have the capacity to interact with specific receptors (RAGEs) present in some cellular types (Valencia et al. 2004).
There have also been recent reports that some of the effects of in vitro prepared AGEs are due to metal ions which are contaminants commonly found in AGE preparations. Various metal ions have been shown to drive inflammatory responses via generation of oxidative stress (Valencia et al. 2004). The use of a chelator such as EDTA to facilitate the reaction could induce some of the effects reported to be produced by AGEs (Al-Abed et al. 1999).
Our results show that body weight was not significantly modified in MS rats even if the diet of the sucrose-fed rats was hypercaloric (Baños et al. 1997) (Table I). However, sucrose-fed animals showed increased central adiposity. Insulin was increased in MS rats and this increase accompanies an increase in blood pressure. Triglycerides were also elevated. Therefore the MS rats show many of the characteristics of metabolic syndrome. Serum albumin concentration was significantly higher in MS rats when compared to control rats. However, Rumble et al. (1997) reported that diabetic and hypertensive rats had lower serum albumin levels than the normotensive Sprague Dawley rats.
Vascular contraction to norepinephrine increased MS rats in this study. This has been previously described for this model (Baños et al. 1997) and additionally we have previously found that contraction to KCl in MS rats is also increased and that this is due to an elevated response to endothelin (Nava et al. 1999; Rubio et al. 2006). The increase in the circulating levels of albumin in the MS group may contribute to the increased vascular tone observed in this group. Indeed, an increase in circulating albumin levels has been observed in some forms of hypertensive pathology, due to the hemoconcentration induced by edema, which positively correlates with the hypertension levels (Allen and Patterson 1995). AGEs could also participate in the increase in vascular contraction in MS rats since they increase the production of endothelin-1 by endothelial cells (Tan et al. 2002). Our results show that vasoconstriction was not modified by nonglycated BSA or by AGE-BSA (Figure 5). This is in accordance with the results of Xu et al. (2003).
Vasorelaxation in MS rats was significantly diminished in comparison to control rats and could be attributed to an imbalance of constricting and relaxing factors as has been previously reported (Baños et al. 1997). Furthermore, as our model of MS rats is caused by the chronic ingestion of sucrose, vascular damage could be a probable consequence of AGEs increased production. AGEs deposit inside smooth muscle and endothelial cells as well as in extracellular matrix proteins increasing rigidity. Hogan et al. (1992) have reported that AGEs react with NO produced by endothelial cells. They also reported that AGEs reduce the antiproliferative effect of NO on aortic smooth muscle vascular and renal mesangial cells.
Insulin, which is also increased in our model, stimulates collagen synthesis in vascular smooth muscle cells (Ruiz Torres et al. 1998) and could contribute to diminished vasorelaxation in MS rats. Arterial endothelium-dependent relaxation is also diminished hypertensive rats (Ibarra et al. 1995; Küng and Lüscher 1995; Challah et al. 1997; Freitas et al. 2003; Shipley and Muller-Delp 2005). These authors have proposed that the reduced response to Ach could be a consequence of an impairment of either the generation (synthesis or release) of relaxant factors or the cellular response to them.
In our study, both nonglycated BSA and AGE-BSA decreased vasorelaxation in the same proportion in control rats. In arterial rings incubated in the presence of glycated and nonglycated BSA the response to acetylcholine was 35% reduced in comparison to control rings incubated in the absence of BSA (Figure 6A). The reduction in vascular relaxation in the presence of nonglycated BSA is in accordance with the report by Predescu et al. (2002). These authors suggested that BSA reacts with NO therefore modulating its biological actions and increasing endothelial permeability. Although in our experiments we also found a decrease by AGE-BSA, it seems to be due to BSA since there was not a significant difference in the reduction produced by AGE-BSA and by nonglycated BSA.
Previously, Xu et al. (2003) studied the vascular effects of AGEs produced in vitro testing vasoreactivity in rings of thoracic aorta from New Zealand white rabbits and in vitro production of NO in endothelial cells from human umbilical veins. These authors reported that there was not a significant effect of AGEs upon vasoconstriction induced by NE and our results are in accordance with this result. However, they found that AGEs diminished the NO production of isolated endothelial cells and that vasodilation was reduced in thoracic aortic rings while we found that AGEs did not modify vasodilation. Our data suggest that it is BSA that exerts the diminished vasorelaxing effect and not AGEs. In their study the authors produced AGEs incubating BSA with glucose 6-phosphate instead of glucose which is a faster reaction. Glucose 6-phosphate is a reducing sugar of great intracellular importance and can glycate easier than glucose since it is present in a higher proportion in the open-chain form (Hogan et al. 1992). Their reaction was facilitated by the use of EDTA which could induce the effect reported by them (Al-Abed et al. 1999). AGEs derived from a long period of incubation of BSA with glucose, as was done in this paper, conduces mainly to fluorescent cross-linking AGE production. Using specific antibodies, these authors found high levels of carboximetillysine (CML) in their preparation and identified CML as the AGE responsible of the NO synthesis inhibition. Therefore, the kind of AGEs used by them could explain the contradiction between their results and what we observed in the present paper. Furthermore, the difference of the species used for testing vasodilation in both papers (New Zealand rabbits and Wistar rats) might account for the contradicting results found.
The same authors (Xu et al. 2003) did not find the expected previously reported decrease in vascular relaxation when they added nonglycated BSA. The absence of a vascular effect of nonglycated BSA in the above-mentioned study is probably due to the very low doses assayed, in the order of μg/mL. It is known that albumin has a physiologic effect at higher concentration. Indeed, our own results show that the inhibitory effect of BSA or AGE-BSA was only observed when the protein concentration assayed was about of 3.81 mg/mL, but not when it was only of 0.95 mg/mL.
The response to nonglycated BSA and AGEs disappeared in the MS rats (Figure 6B). Alterations in the structure and function of the vascular structures caused by the metabolic syndrome could be the underlying cause of the lack of response in these animals. This physiological adaptation could be considered as a protector effect to counteract the increased basal vasoconstriction observed in metabolic syndrome rats, and must be better studied. We evaluated the presence of AGEs’ receptors (RAGEs) from aortas in MS rats to better explore the mechanism by which AGEs participate in this pathology. Many of the deleterious effects of AGEs are mediated by binding to their receptors which are present in various cellular types. In endothelial cells, the interaction AGE–RAGE modulates the expression of citocynes and adhesion molecules and increases the production of oxygen reactive species (ROS) generating oxidative stress (Ramasamy et al. 2005). Koyama et al. (2005) demonstrated that an alternative form of RAGE, the endogenous secretory RAGE (esRAGE), is associated with the metabolic syndrome or atherosclerosis in the absence of diabetes. Inflammation might be the factor linking the RAGE system with MS. Although a higher number of RAGEs were observed in the aortas from MS rats, AGE-BSA did not have any effect on arterial relaxation in this group. Moreover, the absence of differences in arterial relaxation induced by AGE-BSA or BSA in the control group suggests that the possible effect of AGE-BSA on vascular function in not mediated by RAGEs.
The exposition of the vascular tissue to AGE-BSA during the development of MS could also cause the loss of the relaxation capacity of aortas. The changes in the charge and composition of the BSA molecule produced by glycation increase its aggregation capacity. When the isoelectric point is closer to physiological pH, hydrophobic interactions are favored. These changes could increase the cross-linking activity of AGEs to proteins and the capacity to deposit in the extracellular matrix of the tissues. Correspondingly, we observed an increased aggregation capacity of AGE-BSA during alcohol precipitation or desalting. However, the loss of the relaxing capacity due to biochemical changes of AGE-BSA is not favored by our results since there were no differences in the relaxation in the presence of BSA or AGE-BSA.
We speculate that the cause could be the altered albumin internalization to endothelial cells and therefore a modified neutralization of NO. This should be further evaluated. The molecular structure and charge of albumin facilitate the cotransport of a number of hydrophobic molecules, enzymes, and hormones across the endothelium and the maintenance of vascular integrity and transvascular oncotic pressure gradient (Metha and Malik 2006). Endothelial permeability to plasma proteins and liquid is increased in inflammation, a condition manifested by protein-rich edema (Metha and Malik 2006). Inflammation is present in metabolic syndrome and therefore alterations in albumin transport across endothelial cells are likely to be present. Bevers et al. (2006) reported that in bEnd.3 endothelial cells, which only produce eNOS and have higher levels of NO than other endothelial cells, incubation with low albumin levels increased eNOs activity and the production of NO.
In conclusion both BSA and AGE-BSA inhibit vascular relaxation mediated by NO in control artic rings in the same proportion. This inhibitory effect is likely to be due to the structure of BSA and not to the fluorescent AGEs synthesized by the employed procedure. However, in MS rats this inhibitory vascular relaxation effect is lost, possibly due to alterations in endothelial cells that are a consequence of the illness.
Materials and methods
Animals and arterial pressure determination
Experiments in animals were approved by the Laboratory Animal Care Committee of our institution and were conducted in compliance with our institution's ethical guidelines for animal research.
Weanling male Wistar rats aged 25 days and weighing 50 ± 4 g were separated into two groups—group 1: control rats (C) were given tap water for drinking and group 2: MS rats were given 30% sucrose in drinking water during a 24-week period. All animals were fed Purina 5001 rat chow (Purina Mills Inc., Richmond, IN) ad libitum, which provides 14.63 kJ/g, with 23% protein, 12% fat, and 65% carbohydrate. Animals were kept at controlled temperature and a 12:12-h light-dark cycle.
Systolic arterial blood pressure was measured in conscious animals using the tail cuff method; the cuff was connected to a pneumatic pulse transducer (Narco Bio-Systems Inc., Healthdyne Co., Austin, TX) and a programmed electrosphyngomanometer. The mean of five independent determinations was calculated.
AGE-BSA preparation
AGE-BSA were prepared, purified, and characterized in the reproductive biology laboratory of Instituto Nacional de Ciencias Médicas y Nutrición “Salvador Zubirán” following the technique previously described (Makita et al. 1991, 1992).
AGE-BSA was prepared by incubating 5% of bovine serum albumin (BSA fraction V; Sigma Chemical, St. Louis, MO) in 200 mM of sodium phosphate buffer (pH 7.4) with 500 mM glucose for 60 days under sterile conditions, in darkness and at 37°C. After incubation, the product was reduced with 100 mM sodium borohydride (NaBH4, Sigma Chemical) for 30 min at room temperature to stabilize the Schiff bases formed (Eble et al. 1983; Fluckiger and Gallop 1984; Verbeke et al. 1997). Then, the unbound glucose and unreacted NaBH4 were removed by extensive dialysis against a 500 mM sodium phosphate buffer (pH 7.4). Less glycosylated BSA was removed from AGE-BSA by affinity chromatography on Affi-Gel Blue (Bio-Rad Laboratories, Richmond, CA) (Travis et al. 1976; Verbeke et al. 1997). The Affi-Gel matrix has a blue dye named Cibacron Blue F3GA, with natural affinity for native albumin. A column with 5 mL of matrix with a binding capacity of 170 mg of BSA was employed. 2 mL of AGE-BSA solution with a concentration of 53.8 mg/mL was applied in each purification process, using a flow rate of 1 mL/min. The chromatogram was performed evaluating a dilution 1/10 of each tube.
Protein concentration of AGE-BSA was evaluated by the Lowry method using BSA as standard (Lowry et al. 1951). Finally, AGE-specific fluorescence determination was performed by measuring emission at 440 nm upon excitation at 370 nm using an Aminco fluorescence spectrometer (Aminco-Bowman Series 2 SLM Instruments. Inc., Rochester, NY) (Monnier and Cerami 1981; Makita et al. 1992). Fluorescence value of AGE-BSA was measured at a protein concentration of 1 mg/mL and expressed in arbitrary units (AU). The specific activity (SA) was expressed as arbitrary units of fluorescence per mg of protein (AU/mg).
The control BSA sample (nonglycated BSA) was incubated under identical conditions but without glucose. AGE-BSA and its control (nonglycated BSA) were maintained at −70°C until used.
Mass increase analysis of glycated BSA
The mass change of BSA after glycation was analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF) (Voyager DE-PRO from Applied Biosystems, CA), equipped with a nitrogen laser (337 nm), operating in a positive high-energy linear mode. This analysis was made in the Analysis and Molecular Diagnose Unit of the Insituto Nacional de Salud Pública de México.
Aliquots of 500 μL containing 10 mg/mL of BSA or AGE-BSA were diluted to 1.5 mL with deionized water and centrifuged in Amicon Ultracel-10 K (Millipore Corporation, MA) at 2,000 × g for 30 min to a final volume of 500 μL. Washing was repeated three times. A 10 μL aliquot was taken for MALDI-TOF analysis. The matrix solution employed for BSA analysis contained 10 mg/mL 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) in 30% vol/vol acetonitrile in 0.1% vol/vol aqueous trifluoroacetic acid. For AGE-BSA the matrix solution contained 2,5-dihydroxybenzoic acid (gentisic acid, which is employed for analysis of glycoproteins) to 10 mg/mL in 50% vol/vol acetonitrile. A sample protein solution (1 μL) of a 1:1 vol/vol mixture of 1 mg/mL of total protein and the matrix solution were applied on a sample spot in a steel plate slide and dried in a warm air stream. Bovine's insulin (5,734.59 Da), E. coli's Thioredoxin (11,674.48 Da), and horse's Apomyoglobyn (16,952.56 Da) were used as standard for calibration.
Isoelectric point of glycated BSA
The change in the isoelectric point of BSA after glycation was analyzed by isoelectric focusing in polyacrylamide gels using a FastSystem (Pharmacia, Uppsala, Sweden). The bands were dyed with silver nitrate, and the isoelectric point was evaluated using the analytical unit of FastSystem employing a broad range standard kit (pH 3–10) (Amersham Biosciences, Buckinghamshire, United Kingdom) as a protein calibrator.
Blood sample collection and determination of glucose, insulin, triglycerides, and serum albumin
After overnight fasting (12 h), the animals were killed by decapitation and blood was collected. It was spun and the serum was separated by centrifugation at 600 × g during 15 min at room temperature and stored at −70°C until needed. Serum insulin was determined using a commercial radioimmunoassay (RIA) specific for rat (Linco Research, Inc., Missouri); its sensitivity was 0.1 ng/mL and intra- and interassay coefficients of variation were 5 and 10%, respectively. Glucose concentration was assayed using an enzymatic SERA-PAKR Plus from Bayer Corporation (Sées, France). Homeostasis model assessment (HOMA) was used as an index to measure the degree of insulin resistance and was calculated by the formula [insulin (μU/mL) × glucose (mmol/L)/22.5] (Matthews et al. 1985; Pickavance et al. 1999; Nandhini et al. 2005). Triglycerides (TGs) were determined by commercially available procedures (Randox, Laboratories LTD, Antrim, United Kingdom). Serum albumin was evaluated in both groups of animals using a kit commercialized by Hycel of Mexico which uses bromocresol green (IL-test Instrumentation Laboratory Company, Lexington, MA). Samples were read using an Ilab-600 automatic system.
Sample preparation and tension recording
The animals were killed by decapitation, and aortas were immediately dissected and placed in an oxygenated normal Tyrode solution (mM: 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 5 Hepes, and 5.5 glucose) pH 7.4. Arteries were carefully cleaned from connective and adipose tissue, taking care not to damage the endothelium. Tension measurements were made as previously described (Nava et al. 1999). Briefly, segments of about 2 to 3 mm long were cut and two 250-μm-diameter S-shaped silver wires (Medwire Corp, Mount Vernon, NY) were inserted into the lumen to measure tension developed transversely by rings of the vessel. One of the silver wires was fixed to the bottom of an in vitro chamber, and the other was attached to a tension transducer that was connected to a Grass polygraph model 79D. The chamber was filled with a Tyrode solution, and termorregulated and bubbled with carbogen (95% oxygen, 5% carbon dioxide). A basal passive tension of 2 g was applied after determination in preliminary tests that this was the optimal resting tension under our experimental conditions. Arteries were allowed to rest for 1 h and the solution was changed every 20 min. Contraction was induced twice by the addition of norepinephrine (NE) (1 μM). Arteries were washed by adding a fresh Tyrode solution to the chamber allowing the rings to return to their basal tension (2 g). The mean contraction value was considered as 100% of response. The vasodilator activity was studied by cumulative concentration–response curves to acetylcholine (Ach) (10−9−10−4 M) on precontracted aortic rings in the following experimental groups: (A) incubated only with the Tyrode solution, (B) incubated during 40 min with AGE-BSA at 10 AU/mL, (C) incubated with AGE-BSA at 40 AU/mL, (D) incubated with nonglycated BSA at a protein concentration equivalent to 10 UA/mL of AGE-BSA (0.95 mg/mL), (E) incubated with nonglycated BSA at a protein concentration equivalent to 40 AU/mL of AGE-BSA (3.81 mg/mL), and (F) incubated with L-NAME at 300 μM, in both control and MS rats. The percentage of the response in each experimental group was calculated in relation to the tension developed by the same ring during its basal contraction.
Immunohistochemistry of RAGEs
Aortic rings of both MS and C rats were quickly frozen in Tissue-Tek (Sakura Finetek USA, Inc., Torrance, CA). Sections were fixed with acetone and were blocked with PBS/Azide 0.02%/BSA 1% for 30 min.
Subsequently, sections were incubated during 2 h at room temperature with a rabbit polyclonal antibody against RAGE (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Primary antibodies were detected by using goat anti-rabbit FITC (Jackson ImmunoResearch Laboratories Inc. West Grove, PA), at room temperature for 60 min. Negative controls were prepared by substituting the primary antibody with an irrelevant antibody. Staining was analyzed using fluorescence microscopy.
Statistical analysis
Results are expressed as mean ± standard errors of the mean (SEM) from 6 to 10 different artery preparations. The percentage of contraction in each experiment was calculated, and the mean was then determined. When applicable (comparisons between two values; MS and controls), statistical analysis was done by Student's t-test. Comparisons between groups were done by analysis of variance (ANOVA) or Anova on ranks followed by Student–Newman–Keuls or Dunn's tests, depending on whether the data were normally distributed or not, using the SigmaStat 2.0 program (Jandel Scientific, San Rafael, CA). Differences were considered statistically significant when P < 0.05.

bsa
metabolic syndrome
age-bsa
vascular relaxation


Regulation of intestinal hPepT1 (SLC15A1) activity by phosphodiesterase inhibitors is via inhibition of NHE3 (SLC9A3)

The H+-coupled transporter hPepT1 (SLC15A1) mediates the transport of di/tripeptides and many orally-active drugs across the brush-border membrane of the small intestinal epithelium. Incubation of Caco-2 cell monolayers (15 min) with the dietary phosphodiesterase inhibitors caffeine and theophylline inhibited Gly–Sar uptake across the apical membrane. Pentoxifylline, a phosphodiesterase inhibitor given orally to treat intermittent claudication, also decreased Gly–Sar uptake through a reduction in capacity (Vmax) without any effect on affinity (Km). The reduction in dipeptide transport was dependent upon both extracellular Na+ and apical pH but was not observed in the presence of the selective Na+/H+ exchanger NHE3 (SLC9A3) inhibitor S1611. Measurement of intracellular pH confirmed that caffeine was not directly inhibiting hPepT1 but rather having an indirect effect through inhibition of NHE3 activity. NHE3 maintains the H+-electrochemical gradient which, in turn, acts as the driving force for H+-coupled solute transport. Uptake of β-alanine, a substrate for the H+-coupled amino acid transporter hPAT1 (SLC36A1), was also inhibited by caffeine. The regulation of NHE3 by non-nutrient components of diet or orally-delivered drugs may alter the function of any solute carrier dependent upon the H+-electrochemical gradient and may, therefore, be a site for both nutrient–drug and drug–drug interactions in the small intestine.
1
Introduction
The human intestinal H+-coupled di/tripeptide transporter (hPepT1 or SLC15A1) is responsible for the transport of a significant proportion of dietary protein across the brush-border membrane of the small intestinal epithelium. hPepT1 is also of pharmacological interest as it can transport orally-active drugs such as β-lactam antibiotics and angiotensin converting enzyme inhibitors [1–4]. More recently a number of amino acid modified pro-drugs such as val-acyclovir, l-DOPA-l-Phe and the anti-hypotensive midodrine have been designed specifically to target hPepT1 and thus to enhance oral bioavailability [3–5].
When expressed heterologously, hPepT1 functions as a Na+-independent, pH-dependent, H+-coupled transporter [6,7]. However, until the mid-1980s, it was considered widely that di/tripeptide transport was via a Na+-coupled transport mechanism. Indeed, dipeptides can stimulate Na+ absorption in the human small intestine and Na+ uptake in human intestinal Caco-2 cell monolayers [8–10]. Ganapathy and Leibach proposed a model to account for the interaction of H+-coupled di/tripeptide transport and Na+ absorption [11] whereby the intracellular acidification caused by di/tripeptide associated H+ influx activated an apical Na+/H+ exchanger and thus Na+ absorption. This model also explains the partial Na+-dependence of di/tripeptide uptake observed in mammalian small intestine and Caco-2 cell monolayers [11,12]. Inhibition of Na+/H+ exchange by removal of extracellular Na+, prevents the enterocyte from maintaining intracellular pH (pHi) during H+/solute influx and, therefore, abolishes the driving force (the transmembrane H+ electrochemical gradient) for further transport. The apical Na+/H+ exchanger specifically activated by di/tripeptide uptake is NHE3 (SLC9A3) which plays a significant role in Na+ absorption in the small intestine [7,10,13].
As hPepT1 is involved in both protein assimilation and drug delivery it is important to elucidate how altering hPepT1 function may affect nutritional status and oral bioavailability. Progress has been made in understanding how intestinal di/tripeptide transport is regulated by hormonal and neural signals as well as by disease states and surgical intervention (for review see [14]). However, little is known about how co-administered drugs or dietary factors affect hPepT1 function or the function of other brush-border membrane transport proteins. Alteration of cAMP levels within enterocytes by hormonal, neural and paracrine signals is a key step in the regulation of many brush-border membrane nutrient and electrolyte transporters. NHE3 is inhibited acutely by increasing [cAMP]i
[15]. Therefore, the aim of this study was to identify how dietary and orally-delivered compounds, which act as phosphodiesterase inhibitors (and thus increase [cAMP]i by preventing breakdown), affect hPepT1 function. The dietary phosphodiesterase inhibitors caffeine and theophylline and the orally-delivered drug pentoxifylline all inhibit dipeptide uptake across the brush-border membrane of human intestinal epithelial Caco-2 cell monolayers. This inhibition is not a direct effect on hPepT1 but rather is indirect through inhibition of NHE3.
2
Materials and methods
2.1
Materials
Glycyl[1-14C]sarcosine (specific activity 57 mCi mmol− 1)] was obtained from Cambridge Research Biochemicals (Billingham, UK). [3-3H]β-Alanine (specific activity 50 Ci mmol− 1)] was obtained from American Radiolabeled Chemicals (St. Louis, MO, USA). PACAP (pituitary adenylate cyclase-activating polypeptide) was from Bachem (St. Helens, UK). Biotrak cAMP enzyme immunoassay was from GE Healthcare (Chalfont St. Giles, UK). 2′,7′-Bis(2-carboxyethyl-5(6)-carboxyfluorescein) acetoxymethyl ester (BCECF-AM) was from Invitrogen (Paisley, UK). Transwell polycarbonate filters were from Corning (Schiphol-Rijk, The Netherlands). S1611 was obtained from H.J. Lang (Aventis Pharma Deutschland GmbH, Frankfurt/Main, Germany). Forskolin, phosphodiesterase inhibitors, cell culture media and supplements were from Sigma-Aldrich (Poole, UK). All other chemicals were from Sigma-Aldrich or VWR (Lutterworth, UK) and were of the highest quality available.
2.2
Cell culture
Caco-2 cells (passages 100–119) were cultured as confluent monolayers on permeable polycarbonate filters, as described previously [16]. Monolayers were used at 14–22 days post-seeding and fed approximately 24 h prior to use.
2.3
Measurement of dipeptide uptake
[14C]Gly–Sar (0.5 μCi ml− 1, 0.1 – 10 mM) or [3H]β-alanine (0.5 μCi ml− 1, 0.1 mM) uptake across the apical membrane of Caco-2 cell monolayers was measured, essentially as described previously [16]. Briefly, Caco-2 cells were washed (in 4 × 500 ml) and bathed in modified Krebs' solution (composition (mM): NaCl, 137; KCl, 5.4; MgSO4, 0.99; KH2PO4, 0.34; NaH2PO4, 0.3; CaCl2, 2.8; Glucose, 10) or Na+-free solution (identical except choline chloride replaced NaCl and NaH2PO4 was omitted). Uptake was measured at apical pH 5.5 or pH 6.5 (10 mM MES, adjusted to the correct pH by Tris) with the basolateral solution being pH 7.4 (10 mM HEPES, adjusted to the correct pH by Tris) in all experiments. Uptake was measured over 15 min (37 °C) at the end of which the monolayers were rinsed thoroughly (in 3 × 500 ml, ice-cold, pH 7.4 Krebs' solution). Various compounds were added to the buffers for the duration of uptake (see figure legends for details). Cell monolayer-associated radioactivity was determined by scintillation counting.
2.4
Measurement of pHi
Intracellular pH (pHi) was measured in Caco-2 cell monolayers by microspectrofluorimetry using the pH-sensitive dye BCECF, essentially as described previously [10,17]. Cell monolayers were acidified by superfusion with apical Gly–Sar (20 mM, 5 min) in modified Krebs' solution (Na+-free, apical pH 5.5). The basolateral solution was Na+-free, pH 7.4 throughout all experiments. pHi recovery was then measured in Na+-containing pH 7.4 apical solution until pHi returned to baseline. The monolayers were incubated with caffeine (5 mM, apical and basolateral) for 10 min. The cells were then exposed to Gly–Sar once again but in the continued presence of caffeine (thus total exposure time to the compound before pHi recovery was 15 min). pHi recovery was then measured in the continued presence of caffeine. The initial rate of pHi recovery is expressed as H+ efflux calculated from the gradient of the first 30 s of recovery [18].
2.5
Measurement of intracellular cAMP levels
Caco-2 cell monolayers were washed in 4 × 500 ml Krebs' solution (pH 7.4). Monolayers were incubated for 15 min in the presence or absence of caffeine, pentoxifylline, theophylline (all 5 mM), or forskolin (10 μM) at apical pH 6.5 and basolateral pH 7.4. Monolayers were then washed in 3 × 500 ml, ice-cold Krebs' (pH 7.4) and lysed in 500 μl lysis buffer (from the Biotrak enzyme immunoassay kit) for 10 min. [cAMP]i was measured following the kit protocol.
2.6
Statistical analysis
Data are expressed as mean ± SEM unless stated otherwise. Statistical comparisons of mean values were made using paired two-tailed Student's t-test or one-way analysis of variance (ANOVA) (using the Tukey–Kramer or Bonferroni's multiple comparisons post-test) as appropriate. Significance was assumed if p < 0.05. Curves were fitted using FigP software.
3
Results
3.1
Inhibition of dipeptide uptake by caffeine is via inhibition of NHE3
Uptake of the dipeptide Gly–Sar (glycylsarcosine) across the apical membrane of Caco-2 cell monolayers was measured over 15 min at an apical pH (pH 6.5) representative of that found at the surface of the proximal small intestine. Incubation of Caco-2 cell monolayers with the dietary phosphodiesterase inhibitor caffeine (5 mM) at both the apical and basolateral surfaces (for the duration of the 15 min uptake period) reduced Gly–Sar uptake by around 50% (from 683.6 ± 18.9 to 351.1 ± 10.7 pmol cm− 2 [15 min]− 1, n = 17–18, p < 0.001, Fig. 1A). Caffeine was equally effective at inhibiting Gly–Sar uptake when added solely to either the apical or basolateral chamber (data not shown). Removal of extracellular Na+ reduces Gly–Sar uptake (as described previously [12]) (Fig. 1A). However, in the absence of extracellular Na+, caffeine had no effect on Gly–Sar uptake (uptake being 244.0 ± 9.1 and 242.0 ± 9.5 pmol cm− 2 [15 min]− 1 in the absence and presence of caffeine respectively, n = 17–18, p > 0.05).
Previously we have shown that the Na+-dependence of Gly–Sar uptake can be attributed to a lack of NHE3 activity in the absence of extracellular Na+
[10,12,18]. To test if the lack of effect of caffeine in the absence of extracellular Na+ was due to a requirement for NHE3 activity, dipeptide uptake was measured under other conditions where NHE3 would be inactive (Fig. 1B). At apical pH 6.5, the NHE3-selective inhibitor S1611 (3 μM) significantly reduced Gly–Sar uptake in a manner similar to that caused by the removal of extracellular Na+ (p < 0.001). This concentration of S1611 is sufficient to inhibit NHE3 completely but would not inhibit NHE2 or NHE1 [19]. Previous work has shown that in the absence of Na+, S1611 has no effect on Gly–Sar uptake into Caco-2 cells [12]. In addition, when hPepT1 is expressed in isolation in Xenopus laevis oocytes, dipeptide uptake is not inhibited by S1611 [7]. This observation confirms that the inhibition of dipeptide uptake by S1611 into Caco-2 cells is an indirect effect via inhibition of NHE3. In the presence of S1611, Gly–Sar uptake was no longer inhibited by caffeine (5 mM). The level of inhibition caused by caffeine alone, by S1611 alone and by both compounds together were not significantly different (p > 0.05) suggesting that caffeine and S1611 inhibit hPepT1 activity through a common mechanism (i.e. NHE3 inhibition). Gly–Sar uptake increases with decreasing apical pH, consistent with hPepT1 being a H+/dipeptide symporter. However, NHE3 activity decreases with acidification of the apical solution and is inactive by pH 5.5 due to the unfavourable transmembrane pH gradient [12]. Previous studies have shown that Gly–Sar uptake at apical pH 5.5 is not inhibited by S1611 or by the removal of extracellular Na+
[7,12]. Similarly, Fig. 1B shows that caffeine (5 mM) has no effect on Gly–Sar uptake at apical pH 5.5 (p > 0.05) suggesting again that caffeine is inhibiting Gly–Sar uptake through inhibition of NHE3.
3.2
The inhibition of hPepT1 by caffeine and related compounds is consistent with inhibition of phosphodiesterase activity
Phosphodiesterase inhibitors structurally-related to caffeine and either found in diet (theophylline), or used as orally-delivered therapeutics (pentoxifylline) or used as laboratory tools (IBMX, 3-isobutyl-1-methylxanthine) were tested for inhibition of hPepT1 (Fig. 2). Theophylline (5 mM, Fig. 2A), pentoxifylline (5 mM, Fig. 2B) and IBMX (1 mM, Fig. 2C) reduced dipeptide uptake in the presence of extracellular Na+ (p < 0.001) but not in Na+-free conditions (p > 0.05). Previously, we have shown that the enteric neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) and the related neuropeptide VIP (vasoactive intestinal peptide) reduce dipeptide uptake across the apical membrane of Caco-2 cell monolayers by inhibition of NHE3 [18]. PACAP activates the VPAC1 receptor which is coupled to adenylate cyclase and, therefore, increases [cAMP]i. Fig. 2C shows that when Caco-2 cell monolayers were incubated with PACAP (5 nM) in the basolateral solution for the duration of uptake, Gly–Sar uptake was reduced. Incubation of the cells with both PACAP and IBMX resulted in a reduction in uptake which was not significantly different from the uptake in the presence of either PACAP or IBMX alone suggesting IBMX, like PACAP, is inhibiting the NHE3-dependent component of Gly–Sar uptake.
Phosphodiesterase inhibitors increase the levels of cAMP within cells by preventing cAMP breakdown. However, caffeine can also elicit cell signalling effects such as antagonism of adenosine receptors and activation of calcium release from intracellular stores [20–22]. To confirm that the compounds used in this study inhibit hPepT1 function through increasing cAMP levels, [cAMP]i in Caco-2 cell monolayers was measured. Monolayers were incubated with caffeine (5 mM), theophylline (5 mM), pentoxifylline (5 mM) or the adenylate cyclase activator forskolin (10 μM) which can also inhibit dipeptide uptake [12]. Cells were lysed and [cAMP]i measured by enzyme immunoassay. Caffeine, theophylline and pentoxifylline caused [cAMP]i to more than double from control levels of 950 ± 230 fmol cm− 2 (mean ± SD, n = 3) to 2200 ± 830, 2780 ±300 and 2330 ± 150 fmol cm− 2 (mean ± SD, n = 3), respectively. However, the change in cAMP was modest compared to [cAMP]i detected in the presence of forskolin (92500 ±12990 fmol cm− 2 [mean ± SD, n = 3]).
Inhibition of the Na+-dependent (NHE3-dependent) component of Gly–Sar uptake by phosphodiesterase inhibitors was concentration-dependent (Fig. 3). The IC50 values were 1.0 ±0.2 mM for caffeine, 0.6 ± 0.1 mM for theophylline and 0.5 ±0.1 mM for pentoxifylline. The sensitivity of hPepT1 to these compounds is consistent with the effect being through an inhibition of phosphodiesterase activity [20,21,23–25]. At a concentration of 5 mM all three phosphodiesterase inhibitors almost completely inhibited Na+-dependent Gly–Sar uptake.
3.3
Pentoxifylline decreases the maximal capacity for dipeptide uptake
Gly–Sar uptake across the apical membrane was measured at apical pH 6.5 at various concentrations of Gly–Sar (Fig. 4). In the presence of pentoxifylline (5 mM) Gly–Sar uptake was reduced compared to control at all Gly–Sar concentrations. Analysis of the Michaelis–Menten kinetics confirmed that the maximal capacity (Vmax) for Gly–Sar uptake was significantly reduced from 8246 ± 913 to 5369 ± 117 pmol cm− 2 [15 min]− 1 (p < 0.05, n = 6) by pentoxifylline but the affinity (Km) was not affected (1.0 ± 0.2 mM and 1.6 ± 0.6 mM in the absence and presence of pentoxifylline, respectively, p > 0.05). Reduction in the capacity for dipeptide uptake is consistent with a reduction in the driving force (the H+-electrochemical gradient) for uptake through inhibition of NHE3. Previously, we have shown that S1611 and forskolin similarly decrease the Vmax for dipeptide uptake across the apical membrane of Caco-2 cell monolayers [12].
3.4
Caffeine inhibits pHi recovery (NHE3 function) after dipeptide-induced acidification
Previous work demonstrated that NHE3 (but not NHE1 or NHE2) is selectively activated by the intracellular acidification associated with H+/dipeptide symport in Caco-2 cell monolayers [10,12]. S1611, the removal of extracellular Na+, or factors that increase [cAMP]i, such as forskolin and VIP, all inhibit the ability of the cell to regulate pHi after H+/solute-induced intracellular acidification [10,12,18,26]. pHi was measured using the pH-sensitive dye BCECF (Fig. 5). Caco-2 cell monolayers loaded with BCECF were superfused across the apical membrane with Na+-containing, pH 7.4 modified Krebs' solution. The basolateral solution was maintained as Na+-free, pH 7.4 solution throughout the experiment. Cells were exposed to Gly–Sar (20 mM) under conditions where hPepT1 activity would be maximal but where there would be no concurrent NHE3 activity (pH 5.5, Na+-free). After 5 min, Gly–Sar had induced a large intracellular acidification. When Gly–Sar was removed and the apical solution returned to a Na+-containing, pH 7.4 solution, pHi recovered rapidly back to baseline levels. The same monolayers were then incubated with caffeine (5 mM) in both the apical and basolateral solutions for 10 min. Gly–Sar was then reintroduced for 5 min in the continued presence of caffeine. The degree and rate of acidification caused by Gly–Sar was not altered by caffeine suggesting that hPepT1 is not directly regulated by phosphodiesterase inhibitors. However, the ability of the cell to recover pHi after Gly–Sar-induced acidification was attenuated after the 15 min exposure to caffeine (Fig. 5A). The degree of attenuation was quantified by calculating the H+ efflux rate over the first 30 s of pHi recovery (Fig. 5B). In the presence of caffeine H+ efflux was reduced from 57.6 ± 5.8 to 20.5 ± 3.9 μM s− 1 (n = 4, p < 0.01).
To confirm that phosphodiesterase inhibitors do not have any direct inhibitory effect on dipeptide transport, hPepT1 was expressed in Xenopus laevis oocytes, as described previously [7]. Incubation of hPepT1-expressing oocytes with phosphodiesterase inhibitors such as caffeine (5 mM) had no effect on dipeptide uptake (data not shown).
3.5
Inhibition of NHE3 by caffeine also inhibits the H+/amino acid transporter hPAT1 (SLC36A1)
The indirect nature of the inhibition of hPepT1 by phosphodiesterase inhibitors suggests that any apical solute transporter dependent upon the transmembrane H+-electrochemical gradient will similarly be regulated by these compounds. The H+-coupled amino acid transporter hPAT1 (SLC36A1) has been isolated from Caco-2 cell monolayers [27]. As well as mediating the uptake of a wide variety of amino acids, hPAT1 can also transport orally-active drugs such as the anti-epileptic vigabatrin [28]. Previously we have identified that amino acid uptake into hPAT1-expressing oocytes is Na+-independent but hPAT1-mediated amino acid uptake into Caco-2 cells is partially Na+-dependent [26,29,30]. Intracellular acidification caused by the hPAT1 substrate β-alanine selectively activated Na+/H+ exchange by NHE3 [26]. Like H+-coupled dipeptide uptake, H+-coupled amino acid uptake into Caco-2 cells is inhibited by forskolin, S1611 and VIP in a Na+ and pH-dependent manner via inhibition of NHE3 [26,29,30]. Uptake of the hPAT1 substrate β-alanine [16] was measured across the apical membrane of Caco-2 cell monolayers at apical pH 6.5 for 15 min (Fig. 6). Caffeine (5 mM) reduced β-alanine uptake in the presence (p < 0.001) but not the absence of extracellular Na+ (p > 0.05) suggesting that H+-coupled amino acid uptake via hPAT1 is also modulated indirectly through regulation of NHE3.
4
Discussion
The di/tripeptide transporter hPepT1 acts as a high-capacity route for solutes across the first barrier to oral-bioavailability, the brush-border membrane of the small intestine. Many, orally-active peptidomimetics and amino acid-conjugated pro-drugs have been identified as hPepT1 substrates [3,4]. There is an increasing number of examples of physiological regulation (hormonal, neural, paracrine) of hPepT1 and of regulation of hPepT1 in certain disease states and after surgery (reviewed by [14]). Another, less studied, factor which may affect the degree to which drugs are absorbed across the small intestinal epithelium is interaction with co-administered drugs or components of diet. Exposure of Caco-2 cell monolayers to the hPepT1 substrate Gly–Gln for 4 days resulted in a subsequent increase in capacity for dipeptide uptake and in hPepT1 expression [31]. Another study found that an array of flavonoids, which are found ubiquitously in foods of plant origin, either inhibit, have no effect or increase the hPepT1-mediated uptake of the antibiotic cefixime into Caco-2 cell monolayers [32]. In this study we identify that incubation of human intestinal epithelial cells with either dietary or orally-active therapeutic phosphodiesterase inhibitors reduces Gly–Sar uptake through a reduction in hPepT1 capacity.
The data presented here show that the inhibition of Gly–Sar uptake by phosphodiesterase inhibitors is both Na+- and pH-dependent (Figs. 1 and 2) suggesting that inhibition is not a direct effect on hPepT1 but rather through NHE3. When NHE3 is inhibited (e.g. by the removal of extracellular Na+ or by addition of S1611) the cells are no longer able to maintain pHi during solute-induced acidification and, therefore, the driving force (the transmembrane H+ electrochemical gradient) for further dipeptide uptake is reduced. Previously, we have shown that hPepT1 can be inhibited by other factors which are known to increase cAMP in intestinal epithelial cells such as the enteric neuropeptides VIP and PACAP [18]. Although caffeine, theophylline and pentoxifylline can elicit effects through pathways other than increasing cAMP, a number of factors suggest that they are acting here as phosphodiesterase inhibitors. Firstly, incubating Caco-2 cell monolayers with all three compounds produced an increase in [cAMP]i. The increase is relatively small compared to that produced by forskolin. However, this could be due to the cAMP signal being compartmentalised (as demonstrated in cardiomyocytes [33]) and thus the local change may be much greater than the measured, global change. Secondly, phosphodiesterases can also regulate the levels of cGMP but none of the compounds tested here produced a significant change in [cGMP]i (data not shown). Lastly, the concentrations of caffeine, theophylline and pentoxifylline required to produce inhibition of hPepT1 (IC50 = 1.0, 0.6 and 0.5 mM, respectively, Fig. 3) are much closer to those for inhibition of phosphodiesterases than for other effects such as antagonism of adenosine receptors (IC50≈ 2–80 μM) and release of intracellular calcium (EC50≈ 5–20 mM) [20–22,34–36]. Theophylline (100 μM) has been shown to abolish increased glucose absorption resulting from activation of A2 receptors by luminal adenosine (5 mM) in mouse small intestine in vivo [37]. However, in addition to the significantly higher affinity for theophylline, A2 receptors are positively coupled to adenylate cyclase so antagonism of the receptor should cause a decrease in [cAMP]i and not the increase noted here [22,37].
Caffeine (and to a lesser extent theophylline) is found in high levels in many beverages either naturally or by addition. For example, the average cup of coffee contains 85 mg of caffeine [38]. Pentoxifylline is given orally as 400 mg tablets to treat intermittent claudication as part of peripheral arterial disease [39]. Although it is difficult to predict the actual concentration at the surface of the small intestinal epithelium it is conceivable that caffeine and pentoxifylline may reach concentrations high enough to elicit an effect on hPepT1 similar to that measured here using Caco-2 cell monolayers. There is evidence that caffeine given orally in doses equivalent to diet can elicit changes in small intestinal function. A study using human volunteers found that ingestion or intraluminal jejunal perfusion of caffeine (75–300 mg) caused a rapid reversal of net fluid absorption to net secretion [40]. Although the cell signalling pathway by which caffeine stimulated fluid secretion was not investigated, this study does indicate that the concentrations of caffeine used here in vitro may be sufficient to cause changes in the small intestinal epithelium in vivo.
Since the initial reports of phosphodiesterase activity it has become evident that there are many types of phosphodiesterase. Currently there are 11 different PDE families each of which can contain several isoforms and splice variants [24,41]. Many of these isoforms are tissue or cell-specific and recent studies have shown that they can be expressed in distinct sub-cellular locations where they act to limit site, amplitude and duration of cyclic nucleotide signals [24,33]. Little is known about which PDE isoforms are found in the small intestinal epithelium. Studies reporting tissue distribution of PDE mRNA expression often include a small intestinal sample but generally it is not clear whether the PDE is expressed specifically in enterocytes. A single study of PDE3 isoforms identified that PDE3B but not PDE3A mRNA was highly abundant in the epithelium and underlying smooth muscle layers of developing small intestine of rat [42].
Caffeine, theophylline, IBMX and pentoxifylline generally inhibit all PDE isoforms with roughly equal efficacy [25,43,44]. However, since many PDE isoforms and variants are located in distinctive cell and sub-cellular locations they are now considered to be good targets for specific therapeutic agents. Often the resulting drugs are given orally, for example, the PDE5 inhibitor sildenafil used to treat erectile dysfunction [45]. PDE5 is expressed at the mRNA level in the small intestine [46,47]. Further work is required to identify which PDE isoforms are expressed in small intestinal epithelial cells both along the proximal–distal and crypt–villus axes and which isoforms play a functional role in regulating solute transport at the brush-border membrane.
In conclusion, we have shown that non-nutrient components of diet and orally-delivered therapeutic agents both regulate activity of the nutrient and drug transporter hPepT1. This regulation is an indirect effect through inhibition of apical NHE3 function, which acts as a pH homeostatic mechanism during H+-coupled dipeptide transport. Functional cooperativity between hPepT1 and NHE3 results not only in the absorption of di/tripeptides but also stimulation of Na+ absorption [8–10]. The H+-coupled amino acid transporter hPAT1 (SLC36A1) is regulated by caffeine in a similar, indirect manner to hPepT1 (Fig. 6). Thus, through modulation of a single homeostatic mechanism (NHE3), a luminal compound may influence several different absorptive mechanisms. The results presented here suggest that any regulation of NHE3 activity by luminal contents, co-administered drugs or by neurohormonal signals may have significant impact on the general absorptive state of the cell. Any other nutrient transporter dependent on the maintenance of the H+ electrochemical gradient for function may be regulated in a similar manner.

nhe3
dipeptide transport
na+/h+ exchange
drug transport
pept1
intestinal absorption


Outcome of prenatal depression and risk factors associated with persistence in the first postnatal year: Prospective study from Rawalpindi, Pakistan

Background Rates of prenatal and postnatal depression in developing countries are high. Prolonged depression during the postnatal period is associated with impaired infant growth and development. Little is known about the factors predicting the persistence of prenatal depression beyond the first few postnatal months.
1
Introduction
The rate of postnatal depression in developing countries ranges from 16% to 35% (Ghubash and Abou-Saleh, 1997; Patel et al., 2002; Aydin et al., 2005; Cooper et al., 1999) and is a major contributor to the ‘burden of disease’ in these countries. Depression around childbirth is associated with low birth weight and impaired weight gain in the first year of the infant's life (Rahman et al., 2004). The outcome is worse in infants whose mothers remained persistently depressed from the third trimester throughout the first postnatal year. It is, therefore, important to predict which mothers, depressed prenatally, are likely to remain depressed throughout the next year.
Relatively fewer studies have examined the long-term outcome of postnatal depression. A recent review of studies from developed countries concluded that in about 30% women with postnatal depression, symptoms persist for up to a year after giving birth (Goodman, 2004). There are no studies exploring the long-term outcome of postnatal depression in developing countries. Such longitudinal studies identify the course of depressive disorder and the factors that promote persistence thus helping to formulate effective preventive and treatment strategies (Weich and Araya, 2004). They can also help in targeting limited health resources in developing countries towards those at the greatest risk of poor outcome.
In an earlier paper (Rahman et al., 2003a,b), we assessed 632 women in rural Pakistan and found that 160 (25%) of the sample had ICD-10 Depressive disorder. In this paper, we present the results of the one-year follow-up of those mothers who were depressed in the third trimester of pregnancy.
2
Method
2.1
Study area, subjects and sampling
The study was carried out in a rural sub-district of Rawalpindi, Pakistan. This is a mainly agrarian low-income rural area about 60 km south-east from the city of Rawalpindi. All married women aged 17 to 40 in their third trimester of pregnancy (n = 701) were identified from 10 Union Councils (each consisting of 5–10 villages; total population 150,000) over a period of 4 months (Fig. 1). Subjects were identified by obtaining official lists from 120 government-employed Lady Health Workers (LHWs) working in the area, who routinely collect data on new pregnancies. Six-hundred and seventy out of 701 (95%) agreed to take part. Written informed consent was obtained from all subjects after the procedure had been fully explained. Fourteen (2%) were excluded because of a physical illness or complication of pregnancy, 21 (3%) had anxiety disorder and 3 (0.5%) had learning disability and were excluded. Out of the remaining, 160 were diagnosed with ICD-10 Depressive Episode, giving a prevalence rate of depressive disorder in the prenatal period (T1) of 25%. Out of these 160, four had infants born prematurely and were excluded from this study. Two mothers discontinued due to severity of depression. Ten mothers had stillbirths or neonatal deaths, and 1 newborn had a congenital abnormality and were excluded. Fourteen subjects dropped out of the study. Thus, one hundred and twenty-9 mothers were assessed at 3 months (T2), 6 months (T3) and 12 months (T4) postnatal.
2.2
Data collection
Mental state assessments were carried out at all time points by two trained and experienced clinicians using the Schedules for Clinical Assessment in Neuropsychiatry (SCAN), developed by the World Health Organization as an internationally validated semi-structured interview generating ICD-10 diagnoses of Depressive Disorder (World Health Organization, 1994a). All interviews were carried out after translation, back-translation and cultural adaptation of the interview schedule using an established procedure (Rahman et al., 2003a,b). Interrater reliability was established prior to the study when both interviewers independently assessed 20 women (10 had clinical depression) and agreed on the diagnosis of 19 (κ = 0.90).
Psychological symptoms were assessed at T1 by the same interviewers using the Self-Reporting Questionnaire (SRQ-20) (World Health Organization, 1994b). This consists of twenty items designed to identify psychological symptoms associated with anxiety and depression. Each item has a yes/no answer. The time span refers to the individual's feelings over the past 30 days. Each item is scored 0 or 1. The maximum score is therefore 20.
Disability in mothers was assessed at T1 using the Brief Disability Questionnaire (BDQ) (VonKorff et al., 1996). This is an 8-item questionnaire that rates current problems in carrying out daily activities on a scale of 0 (not at all) to 2 (definitely), with a maximum score of 16. This instrument has been validated in a 15-center cross-national, multilingual study (VonKorff et al., 1996).
Socio-demographic variables (age, education, employment, family structure and composition) were assessed at T1 by the same interviewers using a specially designed Personal Information Questionnaire (PIQ). Education was categorised into no education versus at least four years primary education. Four years of schooling was chosen as a cut-off because many Pakistani female children attend primary school for four years, after which many children from low-income families stop attending. Family structure was categorised into nuclear family (parents and children only) or extended family (three generations, or one or both parents with married sons, their wives and children).
Socioeconomic status was assessed at T1 by inquiring if the household was in debt and by asking Lady Health Workers, who lived in the same locality and had intimate knowledge of the families being studied, to rate the household on a 5-point Likert scale ranging from 1 (richest) to 5 (poorest). A single dichotomous variable of ‘poverty’ was created by combining these 2 measures, i.e., subjects who were both in debt and rated below 3 on the socioeconomic 5-point Likert scale were classified as being poor.
Maternal financial empowerment within the household was measured at T1 by asking the mothers if they were given a lump-sum amount of money for day-to-day household expenses, and whether they could take independent decisions about its use. Mothers who answered ‘yes’ to both questions were classified as financially empowered within the household. Social support was assessed by inquiring if the woman received any support during pregnancy from relatives or friends.
A brief list of life events and difficulties was administered at T1. These were derived from the Life Events and Difficulties Schedule (LEDS) (Brown and Harris, 1989), a semi-structured instrument that measures events and difficulties experienced during the previous year. LEDS has been translated and culturally adapted for use in the study area (Husain et al., 2000). Based on the data from this study, 9 types of events or difficulties that accounted for the majority of the severe events and difficulties reported in that population were used in a modified semi-structured interview. We recorded only those events and difficulties which rated as severe according to the Brown and Harris Rating Scale. In order to do this we discussed the context in which they occurred with the local lady health worker (who lived in the same community and had intimate knowledge of the families being studied).
2.3
Statistical analysis
All analyses were carried out with STATA, version 7 (StataCorp., 2001) Subjects who were depressed at all 4 time points (persistent cases) were compared with the rest (non-persistent cases). T-test was used to compare psychological symptoms, disability and life event scores. Univariate analyses (relative risk, Fisher's two sided exact p) was performed between potential risk factors and chronic depression. Associations were considered significant at the 5% level. The simultaneous effects of the measured risk and protective factors on persistent depression were analyzed using logistic regression analysis, including all the variables studied in the model (listed in Table 1).
The study was approved by the ethics committees of Rawalpindi Medical College, Pakistan and University of Manchester, UK.
3
Results
3.1
Sample characteristics
129 depressed women completed the one-year follow-up. Their average age was 27.5 years (SD = 5.3). All were married, the average age of marriage being 20 years (SD = 2.8). Forty-four percent were uneducated; only 4% were employed outside the home. Eighty-eight percent of the fathers were employed and about 22% of them were absent from home for 6 months or more due to employment in the cities. The average reported monthly family income was 2500 rupees (US$42). Sixty-seven percent of the families were rated below 3 on the 5-point socioeconomic scale by the LHWs and 53% of the households were in debt. Sixty-two (48%) were in debt and rated below 3 by the LHWs and were classified as ‘poor’ in this study.
Nine percent were primigravid, 16% already had one child, 21% two children, 17% three children and the remaining 37% had three or more. Thirty-seven percent lived in nuclear families (parents and children only) while the remaining lived in extended families (three generations, or one or both parents with married sons, their wives and children). Fifty-seven percent delivered at home with a traditional birth attendant, 98% without any reported complication. The gender of newborns was equally distributed.
Of the 129 women, 121 (94%) were depressed at 3 months, 98 (76%) at 6 months and 80 (62%) at 12 months. Eighty out of 129 (62%) mothers depressed during the third trimester of pregnancy were still depressed at 12 months postnatally but 7 of these had not been depressed at 6 months; thus 73 (57%) were depressed at all time points.
A comparison of the 73 women who had persistent depressive disorder with the remainder on scores of the Self-Reporting Questionnaire, Brief Disability Questionnaire and Life Events Checklist is shown in Table 1. Persistent depression was significantly associated with SRQ score and BDQ score, and weakly associated with Life Events score.
Unadjusted relative risks with other factors are shown in Tables 2 and 3. Persistent depressive disorder was associated with having an uneducated husband, family size (5 or more children), poverty and lack of a confiding relationship.
Independent predictors of persistent depression selected by multiple logistic regression were: high score on the SRQ in the third trimester of pregnancy (odds ratio (OR) 1.3, 95% CI 1.1 to 1.6, p < 0.01) and poverty (OR 3.1, 95% CI 1.2–8.4, p > 0.05) (Table 4).
4
Discussion
To our knowledge, this is the first study from the developing world exploring the course of prenatal depression and factors predicting its persistence during the first year of the newborn's life. The study was community based and used standardised and valid instruments to diagnose depression. Depression around childbirth is a serious public health problem in south Asia, affecting about one in four women (Patel et al., 2002; Rahman et al., 2003a,b). Recent studies also provide strong evidence that maternal depression is associated with poor growth in infants living in poor communities in developing countries (Rahman et al., 2004; Patel et al., 2004), and the outcome is worse in infants of mothers with persistent depression. In developed countries, maternal depression is associated with long-term cognitive, emotional and behavioural problems in children, and the impact is worse where the depressive episode is severe or prolonged (Grace et al., 2003; O'Connor et al., 2002). Therefore, prolonged maternal depression has serious consequences not only for the mother but also for infant growth and development.
The main findings of this study are that over half of mothers depressed in the third trimester of pregnancy continued to be depressed one year after giving birth. In similar studies from developed countries, depressive levels have been found to decrease steadily over time. In an early prospective study from USA, O'Hara et al. (1984) followed up 99 women from second trimester of pregnancy to 6 months after giving birth, and found that although almost one half of the subjects had depressive scores that would place them in the mildly depressed range during the second trimester, less than 12% of the subjects were in the mildly depressed range at the 9-week and 6-month follow-ups. In a more recent American study, Campbell and Cohn (1997) followed up 70 women meeting the criteria for clinical depression at 2 months postnatal and found that at 4 months postnatal, 48% continued to be depressed; at 6 months 30% and at 12 months, 24% continued to meet the criteria for depression.
Beeghly et al. (2002) followed up 106 women with high depression scores at 2 months postnatal and found that 35% and 31% continued to have high scores at 6 and 12 months respectively. Rubertsson et al. (2005) followed up a national cohort of Swedish women and found that out of 333 women with high depression scores in early pregnancy, 79 (24%) continued to have high scores at one year postpartum. A follow-up study of an Australian cohort from early pregnancy till five years after birth suggested that in the majority of women who experienced depressed mood after birth, the symptoms were not severe and did not continue beyond a few weeks (Najman et al., 2000). In our study, prevalence of antenatal depression is high and comparatively fewer women recover in the first year after childbirth.
The low rate of recovery could be due to the adverse circumstances experienced by many women in developing countries. Persistent depression was associated with several factors that preceded the birth: poverty, already having 5 or more children, an uneducated husband, and lack of a friend or confidant. Similar factors have been reported in studies from poor communities in developed countries. Horowitz and Goodman (2004) found that women still depressed two years after giving birth were more likely to be poor and have less social support. Bernazzani et al. (1997) found that lower occupational status, lower income, prenatal depression and stressful life events in 12 months prepregnancy were associated with depression persisting at 6 months postnatal. Yonkers et al. (2001) found that persistent postnatal depressive symptoms were linked with the presence of other young children at home. In the current study, multivariate analyses suggested poverty to be the major predictor of persistence of depression after severity of depressive symptoms in the prenatal period was adjusted for.
The gender of the newborn did not predict persistence. In south Asia, giving birth to a female infant was found to be associated with postnatal depression (Patel et al., 2002), especially in mothers who already had more than two girl-children (Rahman et al., 2003a,b). The preference for male children is deeply rooted in South Asia. Women are often blamed for the birth of girls. However, this study suggests that the effects of having a female child on the mother's mood may only be transient. Similarly, lack of social support during pregnancy (in terms of assistance provided in daily activities) was not associated with persistence while lack of a confidant or friend was. It may be that there are qualitative differences in the type of social support that predicts a worse outcome of depression, and studies using better measures of social support may be required.
While only physically healthy mothers were included in the study, we did not include any physical measures to screen mothers for common physical problems such as anaemia or iodine-deficiency. However, chronic malnutrition in the mothers indicated by a low body-mass index was not significantly associated with persistent depression. Other limitations include a relatively small sample size and the fact that all the women came from one sub-district of Rawalpindi.
Cross-sectional epidemiological studies in Pakistan suggest that the prevalence rate of depressive disorder in women is high even in the non-postnatal period (Husain et al., 2000; Mirza and Jenkins, 2004). Other longitudinal studies would be required to try and understand the origins of depression in these women, which may well pre-date the first pregnancy, or even have its onset in adolescence. However, the postnatal period assumes importance because of the impact of maternal depression on the infant's development. Thus, early identification of mothers whose depression is likely to persist and providing extra support could improve outcomes in both mothers and their infants.
The strongest predictor of persistent depression in this study was a high score on the SRQ-20 in the third trimester pregnancy. Recent meta-analyses suggest that psychological symptoms (mainly depression and anxiety) during pregnancy, along with past history of psychiatric illness are primary risk factors for postnatal depression (O'Hara and Swain, 1996; Beck, 2001). The few studies that have explored the long-term outcome of postnatal depression suggest that depressive symptoms during pregnancy (Bernazzani et al., 1997; Verkerk et al., 2003) not only predict early postnatal depression but are also associated with persistence of depression. The utility of the SRQ-20 in predicting persistent depression suggests that this questionnaire, which has been specifically developed for use in primary care in developing countries, could also serve as a useful screening instrument during the antenatal period.

pakistan
postnatal depression
developing countries
depression women
mental health