Validation of a rapid , non-radioactive method to quantify internalisation of G-protein coupled receptors Agonist exposure can cause internalisation of G-protein coupled receptors -LRB- GPCRs -RRB- , which may be a part of desensitisation but also of cellular signaling . Previous methods to study internalisation have been tedious or only poorly quantitative . Therefore , we have developed and validated a quantitative method using a sphingosine-1-phosphate -LRB- S1P -RRB- receptor as a model . Because of a lack of suitable binding studies , it has been difficult to study S1P receptor internalisation . Using a N-terminal HisG-tag , S1P1 receptors on the cell membrane can be visualised via immunocytochemistry with a specific anti-HisG antibody . S1P-induced internalisation was concentration dependent and was quantified using a microplate reader , detecting either absorbance , a fluorescent or luminescent signal , depending on the antibodies used . Among those , the fluorescence detection method was the most convenient to use . The relative ease of this method makes it suitable to measure a large number of data points , e.g. to compare the potency and efficacy of receptor ligands . Introduction When chronically exposed to an agonist , G-protein coupled receptors -LRB- GPCRs -RRB- can undergo desensitisation , i.e. the response to a given level of agonist exposure declines , and/or more agonist is required to maintain the same response . Various mechanisms can be involved in desensitisation , some of which directly affect receptor responsiveness . One of these mechanisms involves phosphorylation of activated receptors by GPCR kinases -LRB- GRKs -RRB- . This enables binding of arrestins to the receptor , leading to uncoupling from the G-protein and hence impaired signaling . Another , not necessarily mutually exclusive mechanism of desensitisation is internalisation of the receptor , which can be either homologous -LRB- because of activation of the receptor -RRB- or heterologous -LRB- because of activation of other receptors or to other stimuli -RRB- . Receptor internalisation can be arrestin-dependent or independent -LRB- Bünemann and Hosey 1999 -RRB- and involves translocation of the receptor from the cell membrane to the inside of the cell . There , it is entrapped inside clathrin-coated pits and can undergo either degradation or resensitisation . Several approaches have been used in the past to detect and quantify GPCR internalisation . One classical method involves homogenisation of tissues or cells , differential centrifugation to yield a plasma membrane and a microsomal fraction followed by electrophoresis and autoradiographical analyses of the receptor of interest in both fractions -LRB- Simpson et al. 1984 -RRB- . Another classical method involves labeling of all receptors in intact cells with a lipophilic radioligand and competition by a hydrophilic ligand , which will replace the radioligand from sites at the cell surface but not from those entrapped intracellularly -LRB- Staehelin et al. 1983 -RRB- . More recently , genetic approaches have been used . An example is the use of a C-terminal green fluorescent protein -LRB- GFP ; Barak et al. 1997 ; Kallal et al. 1998 -RRB- to monitor receptor localisation after ligand stimulation sometimes combined with an algorithm to discriminate between internalised and membrane-bound receptors -LRB- Conway et al. 1999 ; Fukunaga et al. 2006 ; Schlag et al. 2004 ; Hirasawa et al. 2005 -RRB- . This latter method uses a cell-based fluorescence imaging system ArrayScanII , where the amount of internalised receptor/GFP spots are counted and normalised against the number of nuclei stained with Hoechst 33342 . However , a GFP , or other fluorescent tag , is relatively large -LRB- 200 -- 300 amino acids -RRB- and can influence receptor function -LRB- McLean and Milligan 2000 -RRB- . Other methods use a combination of techniques , like quantitative flow cytometry analysis of the cell surface receptors and immunocytochemical confocal microscopy analysis to visualise the sub-cellular localisation of the receptor of interest -LRB- Hirasawa et al. 1998 -RRB- . Detailed quantitative studies of internalisation are of interest as this process can be the mechanism of underlying physiological and pharmacological effects . For example , receptor internalisation is thought to underlie the modulation of immune function by sphingosine-1-phosphate -LRB- S1P ; Matloubian et al. 2004 -RRB- . This also appears to be the mechanism of action of the immunosuppressive drug FTY720 -LRB- fingolimod ; Chiba et al. 2006 ; LaMontagne et al. 2006 -RRB- , which is undergoing clinical trials for the treatment of multiple sclerosis and previously also for renal graft rejection -LRB- Budde et al. 2002 ; Kappos et al. 2006 -RRB- . In our search to quantify internalisation of membrane receptors in a fast and easy way , we have setup a method , which uses an N-terminal HisG -LRB- 6xHis + Gly -RRB- - tagged S1P1 receptor . Because the HisG-tag is N-terminal , it is extracellular . Therefore , when immunocytochemistry is performed without permeabilisation , only membrane-bound , tagged receptors will be detected . This way , internalisation is quantified as a difference in membrane-fluorescence between unstimulated and agonist-stimulated cells . The use of small , N-terminal tags has been described before -LRB- Daunt et al. 1997 ; Vicentic et al. 2002 ; Wozniak and Limbird 1996 ; Von Zastrow et al. 1993 ; Miller 2004 -RRB- . Of these , Daunt et al. 1997 described a method where cells were plated onto 24 well tissue culture dishes . After stimulations with an agonist , incubations with first and second antibodies followed . Measurements were done after adding alkaline phosphatase substrate , resulting in a colorimetric reading . In this paper , we now describe the validation of a similar method by testing various antibody combinations and detection methods to quantify internalisation of N-terminal HisG-tagged receptors . This resulted in immunocytochemical method using a specific anti-HisG first antibody combined with a fluorescent second antibody -LRB- AlexaFluor ® 488 -RRB- as our preferred choice . The signal can either be visualised under the fluorescence microscope or measured in a fluorescence microplate reader . The latter results in quantitative measurement of the decrease in signal caused by receptor internalisation , in a direct , sensitive and fast way . Materials and methods Materials pcDNA3 .1 containing the entire coding region of the human S1P1 receptor was purchased from UMR cDNA Resource Center -LRB- Rolla , MO -RRB- . Cell culture media , hygromycine B , Lipofectamine ™ 2000 , pcDNA3 .1 / His C , pOG44 , CHO-FlpIn cells , Alexa Fluor ® 488 goat anti-mouse -LRB- IgG -RRB- , anti-HisG -LRB- mouse monoclonal IgG2a -RRB- , anti-HisG-horse radish peroxidase -LRB- HRP ; mouse monoclonal IgG2a -RRB- , pcDNA5/FRT/TO , NuPAGE ® System , NuPAGE ® Novex 4 -- 12 % Bis -- Tris gel , MES running buffer , NuPAGE ® transfer buffer and Invitrolon ™ PVDF blotting membranes were obtained from Invitrogen -LRB- Breda , The Netherlands -RRB- . BCA ™ Protein Assay Kit was obtained from Pierce -LRB- Etten-Leur , The Netherlands -RRB- . Formaldehyde solution was obtained from Sigma -LRB- Zwijndrecht , The Netherlands -RRB- . Restriction enzymes -LRB- BamHI , XhoI , EcoRI and HindIII -RRB- were obtained from Fermentas Life Sciences -LRB- St. Leon-Rot , Germany -RRB- . Black , clear bottom 96 well plates were obtained from Greiner Bio One -LRB- Alphen aan den Rijn , The Netherlands -RRB- . S1P was obtained from Avanti-Polar Lipids -LRB- via Instruchemie B.V. , Delfzijl , The Netherlands -RRB- . Chamber slides were obtained from Nunc -LRB- Amsterdam , The Netherlands -RRB- . BM Chemiluminescence Blotting Substrate -LSB- POD -RSB- and ABTS -LSB- 2,2 ′ - azino-bis -LRB- 3-ethylbenzthiazoline-6-sulphonic acid -RRB- -RSB- were obtained from Roche -LRB- Mannheim , Germany -RRB- . Hyperfilm ECL was obtained from Amersham Biosciences -LRB- Diegem , Belgium -RRB- . SEW2871 -LRB- 5 - -LRB- 4-Phenyl-5-trifluoromethylthiophen-2-yl -RRB- -3 - -LRB- 3-trifluoromethylphenyl -RRB- -1,2,4-oxadiazole -RRB- was obtained from Calbiochem -LRB- via VWR , Amsterdam , The Netherlands -RRB- . Goat anti-mouse-HRP was obtained from Jackson ImmunoResearch Europe -LRB- via Sanbio B.V. , Uden , The Netherlands -RRB- . RIPA buffer was obtained from Boston bio products -LRB- Worcester , UK -RRB- . Halt protease inhibitor cocktail was obtained from Pierce -LRB- via Perbio Science Nederland B.V. Etten-Leur , The Netherlands -RRB- . Molecular cloning and transfection An N-terminal HisG-tag was added to the S1P1 receptor via cloning into pcDNA3 .1 / HisC using BamHI and XhoI . A second cloning step was done using HindIII and XhoI to clone the HisG-tagged S1P1 receptor into the expression vector pcDNA5/FRT/TO . Transfection of plasmids into CHO-FlpIn cells and cell culture was done as described previously -LRB- Jongsma et al. 2006 -RRB- . Western blot Cells were washed and scraped in PBS , spun down and lysed in RIPA buffer with protease inhibitor . Protein concentration was determined using the BCA kit according to the manufacturer 's protocol . Ten micrograms of protein were loaded onto a 4 -- 12 % NuPAGE ® Bis -- Tris Gel . Electrophoresis was carried out at 200 V for 40 min in NuPAGE ® MES running buffer . Protein was transferred to Invitrolon ™ PVDF blotting membranes at 30 V for 1 h . The membrane was blocked for 1 h at room temperature in 50 mg/ml non-fat dry milk in PBS followed by overnight incubation at 4 °C with a dilution of 1:5,000 for mouse anti-HisG . After washing with PBS-Tween -LRB- 0.1 % -RRB- , the membrane was incubated with a 1:20,000 dilution of goat anti-mouse-HRP IgG at room temperature . Detection was done using BM Chemiluminescence Blotting Substrate and Hyperfilm ECL . Immunocytochemistry Cells -LRB- 40,000 cells/well -RRB- were plated on day 1 in 96 wells black , clear bottom plates or eight well chamber slides . The next day , the medium was changed to serum-free medium . After incubation overnight , cells were stimulated with the indicated ligand in serum-free medium for 30 min at 37 °C , unless otherwise indicated . The stimulation solutions were removed , and cells were fixed by applying 4 % v/v formaldehyde in PBS for 15 min at room temperature . After washing three times with PBS , cells were incubated with the first antibody for 1 h at room temperature . Cells were washed again three times with PBS and , if necessary , incubated with the second antibody for 1 h at room temperature . Cells were washed three times with PBS followed by measurements on a Victor2 -LRB- Wallac , Perkin Elmer -RRB- . The following antibody combinations were used : mouse anti-HisG 1:200 with AlexaFluor ® 488 goat anti-mouse 1:500 , mouse anti-HisG-HRP 1:200 and mouse anti-HisG 1:200 with goat anti-mouse-HRP 1:500 . In case an HRP-bound antibody was used , either ABTS solution -LRB- 50 μg/ml -RRB- or BM Chemiluminescence Blotting Substrate was added to generate an absorbance or luminescence signal , respectively . For the absorbance signal , a 405-nm filter was used ; for the fluorescent signal , a 490-nm excitation and a 535-nm emission filter were used . Data analysis Concentration response curves were analysed by fitting sigmoidal functions to the experimental data using Prism 4 -LRB- Graphpad Software , San Diego , CA -RRB- . Data are expressed as means ± SEM . Differences between groups were compared using a one way analysis of variance with a Dunnett 's correction or a Student 's t test where appropriate . P < 0.05 was considered significant . Results Characterisation of the HisG-tagged S1P1 receptor The presence of HisG-S1P1 receptor in stably transfected CHO-FlpIn cells was confirmed in Western blot analysis as a band of approximately 50 kDa -LRB- Fig. 1 -RRB- . The HisG-S1P1 receptor was also visualised in the cell membrane by fluorescence microscopy using AlexaFluor ® 488 goat anti-mouse as a secondary antibody -LRB- Fig. 2 -RRB- . CHO-FlpIn cells stably expressing the untagged S1P1 receptor were used as a negative control for both detection methods -LRB- Figs. 1 and 2 -RRB- . Addition of the N-terminal HisG-tag to the S1P1 receptor did not influence signaling via this receptor as it did not significantly alter the potency of S1P to inhibit forskolin-stimulated cAMP accumulation -LRB- pEC50 S1P1 9.0 ± 0.1 vs HisG-S1P1 8.9 ± 0.1 ; n = 6 -RRB- , measured as described previously -LRB- Jongsma et al. 2006 -RRB- . Fig. 1Western blot of CHO-FlpIn cells stably expressing the S1P1 receptor -LRB- lane 1 -RRB- and HisG-S1P1 receptor -LRB- lane 2 -RRB- using a mouse anti-HisG first antibody and a goat anti-mouse HRP second antibody . HisG-S1P1 is detected at approximately 50 kDa as indicated by the arrow . The band at 80 kDa is nonspecific . The blot shown is from a typical experiment out of fourFig . 2Fluorescence microscope pictures of CHO-FlpIn cells stably expressing the HisG-S1P1 receptor -LRB- a -RRB- and the S1P1 receptor -LRB- b -RRB- using a mouse anti-HisG first antibody and AlexaFluor ® 488 goat anti-mouse second antibody . Images shown are from one typical experiment out of nine Internalisation of the S1P1 receptor A 30-min incubation with 1 μM of S1P or 10 μM of the selective S1P1 agonist SEW2871 -LRB- Sanna et al. 2004 -RRB- at 37 °C decreased fluorescence -LRB- Fig. 3a , c , e -RRB- . No such decrease in fluorescence was seen when cells were incubated with either agonist at 4 °C -LRB- Fig. 3b , d , f -RRB- . S1P concentration dependently internalised the HisG-S1P1 receptor as visualised with a fluorescence microscope -LRB- Fig. 4 -RRB- . Such decrease in fluorescence was also measured using a fluorescence microplate reader . The fluorescent signal of cells stably expressing the HisG-S1P1 receptor was significantly higher than for cells stably expressing the untagged S1P1 receptor . Stimulation with S1P -LRB- 1 μM -RRB- for 30 min significantly decreased this signal -LRB- Fig. 5 -RRB- . Fig. 3Fluorescence microscope pictures of CHO-FlpIn cells stably expressing the HisG-S1P1 using a mouse anti-HisG first antibody and AlexaFluor ® 488 goat anti-mouse second antibody . Cells were stimulated for 30 min with 1 μM S1P or 10 μM SEW2871 at either 37 °C -LRB- left panel -RRB- or 4 °C -LRB- right panel -RRB- . Images shown are from one typical experiment out of fourFig . 4Fluorescence microscope pictures of CHO-FlpIn cells stably expressing the HisG-S1P1 receptor using a mouse anti-HisG first antibody and AlexaFluor ® 488 goat anti-mouse second antibody . Cells were stimulated with S1P at the indicated concentrations at 37 °C for 30 min . Images shown are from one typical experiment out of four experimentsFig . 5Quantitative measurements of cells stably expressing HisG-S1P1 using a mouse anti-HisG first antibody and AlexaFluor ® 488 goat anti-mouse second antibody . Cells were stimulated at 37 °C for 30 min at the indicated S1P concentrations . The first bar represents cells stably expressing the S1P1 receptor without a HisG-tag . The second bar represents cells stably expressing HisG-S1P1 incubated without the first antibody . Statistical significance was shown between ` vehicle ' and ` 1 μM S1P ' as well as between ` no HisG ' and ` vehicle ' . Data are shown as relative fluorescent units -LRB- RFU -RRB- ± S.E.M. -LRB- n = 3 -RRB- Choice of detection method Instead of the antibody combination mouse anti-HisG and AlexaFluor ® 488 goat anti-mouse -LRB- Fig. 6a -RRB- , other combinations were tested to optimise the method . The use of a first antibody mouse anti-HisG-HRP -LRB- Fig. 6b -RRB- or a first antibody mouse anti-HisG with a second antibody goat anti-mouse-HRP -LRB- Fig. 6c -RRB- combined with the BM Chemiluminescence Blotting Substrate -LRB- POD -RRB- resulted in a luminescence signal , which proved to be very sensitive . However , the signal was very unstable over time , which required the addition of the substrate by the microplate reader . The combination of a first antibody mouse anti-HisG-HRP -LRB- Fig. 6d -RRB- or a first antibody mouse anti-HisG with a second antibody goat anti-mouse-HRP -LRB- Fig. 6e -RRB- combined with ABTS resulted in an absorbance signal . This signal however , had less sensitivity compared to the other two detection methods . The use of a first antibody mouse anti-HisG-HRP resulted in a loss of sensitivity compared to the combination of a first antibody mouse anti-HisG with a second antibody goat anti-mouse-HRP . When the combination of a first antibody mouse anti-HisG and a second antibody AlexaFluor ® 488 goat anti-mouse was used , we measured a concentration-dependent internalisation of HisG-S1P1 after stimulation with either S1P or SEW2871 -LRB- pEC50 S1P = 7.4 ± 0.1 -LSB- n = 8 -RSB- and pEC50 SEW2871 = 5.7 ± 0.2 -LSB- n = 3 -RSB- , respectively ; Fig. 7 -RRB- . Fig. 6Quantitative measurements of HisG-S1P1 . Ligand stimulations are carried out at 37 °C for 30 min at the indicated concentrations -LRB- n = 3 -- 4 -RRB- . Antibody and detection combinations used are mouse anti-HisG first antibody and AlexaFluor ® 488 goat anti-mouse second antibody -LRB- a -RRB- ; mouse anti-HisG-HRP first antibody + POD -LRB- b -RRB- ; mouse anti-HisG first antibody and goat anti-mouse-HRP second antibody + POD -LRB- c -RRB- ; mouse anti-HisG-HRP first antibody + ABTS -LRB- d -RRB- ; mouse anti-HisG first antibody and goat anti-mouse-HRP second antibody + ABTS -LRB- e -RRB- . Data are shown as percentage of unstimulated ± S.E.M. . An asterisk indicates significant difference compared to unstimulated cells ; a pound sign indicates significant difference compared to S1P -LRB- 10 nM -RRB- stimulated cellsFig . 7Effect of increasing concentrations of S1P -LRB- closed squares -RRB- or SEW2871 -LRB- open squares -RRB- on CHO-FlpIn cells stably expressing the HisG-S1P1 receptor , using a mouse anti-HisG first antibody and AlexaFluor ® 488 goat anti-mouse second antibody . Stimulations were carried out at 37 °C for 30 min . The data shown represent the mean ± S.E.M. of six independent experiments performed in triplicate Discussion Previously reported methods to study the internalisation of GPCRs were very labor intensive , required specific equipment , added bulky tags and/or were poorly quantitative . We have developed and validated a method that uses stably transfected cells expressing a receptor with a minor modification only , i.e. an N-terminal HisG-tag , which did not influence receptor signaling . Several antibody combinations and detection methods were tested . The luminescent approach was very sensitive but highly dependent on strict adherence to a specific time interval between substrate addition and actual signal measurement . The absorbent signal obtained using another detection substrate resulted in a loss of sensitivity . The antibody combination of a first HisG specific antibody and a second AlexaFluor ® 488 fluorescent antibody proved the most convenient , resulting in a sensitive and stable signal . Compared to previously published methods using a similar approach -LRB- Daunt et al. 1997 ; Vicentic et al. 2002 ; Wozniak and Limbird 1996 ; Von Zastrow et al. 1993 -RRB- , this immunocytochemical detection of the N-terminal HisG-tag is suitable to assess a large number of samples , e.g. to construct detailed concentration response curves and/or compare multiple ligands . In this regard , we showed that S1P was approximately 50 times more potent than SEW2871 in internalising the HisG-S1P1 receptor . This is in good agreement with the previously reported potency difference between the two agonists for activating the receptor -LRB- Sanna et al. 2004 -RRB- . As comparable methods have been described for adrenergic receptors as mentioned above -LRB- Daunt et al. 1997 ; Vicentic et al. 2002 ; Wozniak and Limbird 1996 ; Von Zastrow et al. 1993 -RRB- , it is likely that the method we described here is also suitable for receptors other than our model receptor , S1P1 . While our technique does not allow following internalisation in real time , the ability to process a large number of samples compensates for that by studying multiple time points . The only limitation to our method is the inability to measure internalisation of endogenous receptors . However , this would be possible when specific receptor antibodies are available , which is not yet the case for our model receptor . In conclusion , we have validated a method to quantitatively measure receptor internalisation in which we use an N-terminal HisG-tag combined with immunocytochemistry . It proves to be sensitive enough to discriminate between different receptor ligands in a fast and non-radioactive way .

O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

sphingosine-1-phosphate receptors
hisg-tag
quantification
cellular trafficking
g-protein coupled receptor


Preconception Healthcare : What Women Know and Believe Objectives : The objectives of this study were to determine if women realize the importance of optimizing their health prior to a pregnancy , whether the pregnancy is planned or not ; and to evaluate their knowledge level and beliefs about preconception healthcare . Additionally , we sought to understand how and when women wanted to receive information on preconception health . Methods : A survey study was performed using consecutive patients presenting to primary care practices for an annual well-woman exam . Patients were recruited based on appointment type and willingness to complete the survey at the time of their appointment , but prior to being seen by the physician . Results : A total of 499 women completed the survey . Nearly all women -LRB- 98.6 % -RRB- realized the importance of optimizing their health prior to a pregnancy , and realized the best time to receive information about preconception health is before conception . The vast majority of patients surveyed -LRB- 95.3 % -RRB- preferred to receive information about preconception health from their primary care physician . Only 39 % of women could recall their physician ever discussing this topic . The population studied revealed some significant knowledge deficiencies about factors that may threaten the health of mother or fetus . Conclusions : A majority of women do understand the importance of optimizing their health prior to conception , and look to their Primary care physician as their preferred source for such information . Study participants demonstrated deficiencies in their knowledge of risk factors that impact maternal and fetal health suggesting that physicians are not addressing preconception healthcare during routine care . Introduction Preconception care is defined as the promotion of the health and well being of a woman and her partner before pregnancy -LSB- 1 -RSB- . The goal of the preconception visit is to identify medical and social conditions that may put the mother or fetus at risk . The concepts of preconception care have been articulated for over a decade -- but unfortunately have not become part of the routine practice . Although many studies -LSB- 2 -- 9 -RSB- document the effectiveness of interventions targeted to increase awareness of preconception folic acid supplementation , however , little evidence links comprehensive preconception health promotion to improved pregnancy outcomes . Only one study using data gathered more than 10 years ago demonstrated a greater likelihood of pregnancy intendedness in a low-income cohort of woman exposed to information on preconception health during routine family planning visits at a community health department -LSB- 10 -RSB- . Studies in the United Kingdom -LSB- 11 , 12 -RSB- of knowledge and attitudes toward preconception care among primary health care teams show widespread consensus among the healthcare workers of the importance of the topic . One of these studies included the attitudes of women of childbearing age and noted that the view of the importance of preconception care was less strongly held by the female population studied -LSB- 11 -RSB- . Other studies have focused on more specific topics , such as rubella immunity , folic acid supplementation and glycemic control in diabetics -LSB- 13 -- 15 -RSB- . Approximately 2 % to 3 % of all pregnancies result in a neonate with a serious genetic disease or a birth defect that can cause disabilities , mental retardation , and in some cases early death -LSB- 16 -RSB- . We are unaware of additional studies assessing the general knowledge and beliefs that women possess about optimizing their health prior to conception , or their preferences for obtaining such information . Methods A survey study of consecutive patients presenting to primary care practices for an annual well-woman exam was performed in accord with prevailing ethical principles . The selected primary care practices , occupying the same building at the Mayo Clinic Arizona , represented a women 's health general internal medicine practice -LRB- 5 physicians -RRB- and a family medicine practice -LRB- 9 faculty physicians and 18 family medicine residents -RRB- . Women were recruited for the study by nursing staff working within the practices , with permission and informed consent completed prior to distributing the survey tool . The Mayo Clinic IRB approved the study . Patients were recruited based on appointment type -LRB- e.g. , well woman exam , annual Pap exam , and annual preventive medicine exam -RRB- and consenting to complete the survey questionnaire at the time of their appointment , but prior to being seen by the physician . The enrollment period was between August 2004 and July 2005 . Women were considered eligible for the study if they were between the ages of 18 and 45 years , understood English , and gave their permission . The survey instrument was a four-page questionnaire and required approximately 10 minutes to complete . The survey included questions about demographics , pregnancy intendedness , knowledge and attitudes about preconception care , and personal preferences about sources of health information about preconception care . Data from each survey was entered into a database at the Research Survey Center , Mayo Clinic , Rochester , Minnesota , and the aggregate data made available for analysis to the research team . Results A total of 570 women were invited to participate in the study ; 58 declined and 13 did not meet eligibility criteria , leaving 499 women who completed surveys for data analysis . The demographic profile of the study population is shown in Table 1 . In this study population , the majority -LRB- 70.6 % -RRB- of women were not currently attempting to conceive as noted in Table 2 . Approximately 5 % were actively trying to conceive , with 13.5 % and 8.4 % considering a pregnancy in either the next 1 -- 2 or 3 -- 5 years respectively . Interestingly , of the women who had previously been pregnant , pregnancies had actually been planned in only 47.2 % of instances . Nearly all women in the study -LRB- 98.6 % -RRB- realized the importance of optimizing their health prior to a pregnancy , again as noted in Table 2 . However , only 39 % could ever recall their physician discussing preconception health . The majority of the women in this study population who were interested in preconception health education preferred the information prior to a pregnancy -LRB- 74.8 % -RRB- or at the time of their annual medical exam -LRB- 11.9 % -RRB- , as displayed in Fig. 1 . Table 1Study population demographic -LRB- n = 499 -RRB- Age Range : 18 to 45 years 18 to 25 years24 % 26 to 35 years30 % 36 to 45 years46 % Mean : 33 yearsEthnicity White84 .8 % Asian3 .6 % African-American1.3 % Native American1 .4 % Other9 .0 % Education -LRB- highest level -RRB- 11th grade or less0 .4 % Graduated High School7 .5 % Some college or technical school33 .0 % Graduated college38 .5 % Some graduate work6 .7 % Graduate degree14 .0 % Household income Less than $ 25,00010.5 % $ 26,000 to $ 50,00019.7 % $ 51,000 to $ 75,00018.6 % $ 76,000 to $ 99,00013.0 % $ 100,000 to $ 125,00013.2 % $ 126,000 to $ 150,0006.8 % $ 151,000 to $ 200,0006.4 % Greater than $ 200,00011.8 % Table 2Conception history and planningPlans about getting pregnant No plans at present time70 .6 % Currently trying4 .6 % Considering in next 1 to 2 years13 .5 % Considering in next 3 to 5 years8 .4 % Have tried , unable to get pregnant2 .8 % Ever been pregnant ? Yes50 .7 % No49 .3 % If ever pregnant , where previous pregnancies planned ? Yes47 .2 % No52 .8 % Affect of optimizing health of mother and pregnancy ? Has a good effect on the pregnancy98 .6 % Has no effect on the pregnancy0 .8 % Has a bad effect on the pregnancy0 .6 % Doctor ever spoken to you about preconception health ? Yes39 .0 % No61 .0 % Are you interested in receiving preconception health education ? Very interested34 .8 % Somewhat interested21 .6 % Unsure10 .1 % Not at all interested33 .5 % If interested or unsure about education , when would you prefer ? ` At the time I become pregnant7 .6 % Before I try to get pregnant74 .8 % During pregnancy0 .7 % Every time I get an annual medical exam11 .9 % Unsure5 .0 % Fig. 1Are you interested in preconception health education ? The women who were interested in preconception health education , or unsure of such interest , were asked their preferences for sources for such information -LRB- Table 3 and Fig. 1 -RRB- . The vast majority preferred their physician , either a primary care physician -LRB- 51.3 % -RRB- or Obstetrician/gynecologist -LRB- 44.0 % -RRB- . Only a fraction would primarily seek their information from sources other than their physician . The survey included questions about patient awareness of key preconception risk factors which may influence the outcome of a future pregnancy . The study population demonstrated high awareness of certain risk factors , such as tobacco , alcohol , drug use , and domestic abuse -LRB- Table 4 -RRB- . There were , however , opportunities for improvement in basic understanding of the risk to maternal/fetal health as it relates to fish consumption , exposure to cat litter , and the impact of family and/or genetic history . Discussion Preconception care is the primary prevention of maternal and perinatal morbidity and mortality . It is an important issue in women 's health that is easily overlooked by physicians and patients . The results of this study demonstrate that the vast majority of study participants understood the importance of preconception healthcare and realized that it should be obtained prior to conception . Interestingly , many of the women who expressed an interest in receiving information about preconception healthcare perceived the annual exam as the appropriate venue for exploring this topic . The study participants showed a strong preference for obtaining information about preconception healthcare from their personal physician and the majority eschewed the use of technology such as the Internet as a source of such information . The study revealed that a large percentage of women expected their primary care physician or OB/GYN physician to address preconception healthcare with them.Table 3Patient preferences for sources of preconception information -LRB- Percentage ranking the choice as first preference -RRB- Primary care physician51 .3 % Obstetrician/gynecologist44 .0 % Family , friends0 .3 % Magazine , newspaper0 .3 % Internet/world wide web3 .1 % Other0 .7 % These data demonstrate that the study population exhibited gaps in knowledge about specific preconception health topics , but confirmed previous findings that women do have an increased awareness of the importance of folic acid supplementation -LSB- 2 -- 9 -RSB- . Additionally , the study population showed a high awareness of the risks associated with tobacco , alcohol and drug use , but were much less aware of the risks to fetal health associated with fish consumption and exposure to cat litter . Such findings point out the need to continue efforts at increasing public awareness of other modifiable risks to fetal health . Unfortunately , fewer than 40 % of the women surveyed recalled discussing preconception healthcare with their physician at the time of their annual exam . Clearly , there is a gap in patient expectation and delivery of healthcare services in this population of educated middle class women . This study only surveyed women who had chosen to obtain their primary care in Internal Medicine and Family Medicine practices of a private health care facility , suggesting that there may be obstacles to delivering preconception care even in an academic outpatient setting . Potential explanations for this gap in the delivery of preconception healthcare may include : age of patients studied , time constraints in the outpatient setting or insufficient training and/or content knowledge in preconception healthcare of the physicians . This study demonstrates that an opportunity to better understand the gap in care delivery and to evaluate possible solutions in private middle class settings . More exposure on the part of residents to a curriculum on preconception healthcare may provide an opportunity for physicians in these specialty fields to improve their skills and the delivery of this much-needed care to women.Table 4Preconception health knowledge and opinions -LRB- Percentage awarea of risk factor potentially affecting a pregnancy -RRB- Consumption of certain fish54 .2 % Exposure to cat liter64 .9 % Folic acid use79 .6 % Impact of family and/or genetic history84 .1 % Infectious diseases -LRB- need to screen for -RRB- 89.3 % Immunizations -LRB- up to date -RRB- 91.2 % Alcohol use95 .8 % Abuse -LRB- verbal , sexual and/or physical -RRB- 97.2 % Medication use -LRB- prescription and nonprescription -RRB- 97.4 % Tobacco use98 .2 % Illicit drug use98 .8 % aAgreed or strongly agreed on a five point Likert scale One of the limitations of our study was the homogeneity of our patient population . The majority of our study participants were middle class , Caucasian and had at least some college education . Our findings may not be broadly applicable to women of other socioeconomic backgrounds , ethnicity and educational levels . The lack of diversity of our population reflects the demographics of this community and further study in other populations should be pursued . As all women of reproductive age and potential presenting for continuing care in the primary care setting are candidates for preconception care , the essential and critical role of primary care physicians and providers in the provision of preconception care is apparent . The Centers for Disease Control -LRB- CDC -RRB- has published their `` Recommendations for Improving Preconception Health and Health Care '' -LSB- 17 -RSB- . These recommendations were developed through a collaborative effort during which the CDC successfully aligned the missions of a number of its external partners and internal programs to ultimately draft these national recommendations for preconception care . These national recommendations can now serve as a roadmap for both graduate medical education and continuing medical education curricula to improve the knowledge and skill of the physician workforce in the delivery of comprehensive preconception health care .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O

preconception care
preconception health
primary care