Tasa interna de retorno (TIR)

Abstract.

Immunohistochemistry and western blotting were carried out with fresh frozen sections and crude extracts from adult rat thyroids. The histochemical and immunoblotting studies were carried out with P2X-receptor antibodies from two different sources. P2X immunopositive cells were identified by fluorescence double labelling and confocal microscopy.

Results o f the western blotting experiments showed double bands o f approximately 70 kDa and 140 kDa for all seven P2X receptor subtypes with both sets o f antibodies. Histochemical stains with antibodies from both sources also gave essentially identical results. P2X i, P2X2 and P2X^ receptors were detected exclusively in vascular smooth

muscle; while, P2X$ and P2Xy receptors were also present on vascular smooth muscle. Endothelial cells stained for P2Xg, P2X^ and P2Xy receptors. Thyroid follicular cells displayed immunoreactivity for P2X], P2X4 and P2X$ receptors. No immunostaining

for P2X-receptors was observed on C-cells. Possible roles for the broad expression o f P2X-receptor subtypes in the rat thyroid are discussed.

Specific materials and methods.

Anim al handling.

Thyroids were taken from 250g, male Sprague-Dawley rats (n=12).

Immunohistochemistry. Tissue handling.

Tissue was collected as described in General M aterials and Methods. C ryostat- sections o f 7 |i,m were cut for immunohistochemical staining and conventional fluorescence microscopy. Sections o f 14p,m were cut for immunofluorescence detection by confocal microscopy. The sections were placed on gelatine coated slides.

Comparing immunostaining fo r P2X-receptors with antibodies from Roche Bioscience and Alomone Labs.

One set o f P2X antibodies (against P2X receptor subtypes 1 to 7) was obtained from Roche Bioscience (Palo Alto, CA) as described in General Materials and M ethods. A second set o f antibodies (against P2X receptor subtypes 1,2,4 and 7) was purchased from Alomone Labs, Israel. The P2X receptor antibodies from Alomone Labs were also raised in rabbit against an antigenic sequence at the carboxy-terminus o f the receptors. All Alomone Labs antibodies were purchased as affinity purified antibodies.

The Alomone Labs antibodies were made monospecific, by elution against their cognate peptides. Thus, this set o f antibodies contains mainly immunoglobulins with high avidity fo r P 2 X receptors. The antibodies from Roche Bioscience consist ofpurified immunoglobulin, but were not eluted against their cognate peptides. Comparing the data

give clues whether the polyclonal Roche antibodies consist ofhigh-avidity

immunoglobulins, specific fo r P 2X receptors. Staining and preabsorption o f the Alomone P2X] receptor antibody is shown (Fig. 4.1).

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Fig 4.1. Im m u n oh istoch em ical stain in g for P 2 X -recep tors with A lom on e antibody. (A) Alomone P2Xi antibody labels the vascular media; (B ) Labelling with Alomone P2Xi is completely preabsorbable.

Comparing strategies fo r immunohistochemical and immunofluorescence labelling.

Immunohistochemistry using ‘high salt ’ medium.

Primary antibodies were diluted in phosphate buffer/10%NHS containing 1.5% (w/v) NaCl (high-salt medium). In experiments using high-salt medium the washing step after incubation with primary antibody was carried out in phosphate buffer containing 1.5% (w/v) NaCl.

It was previously shown that variations in the salt concentration o f the medium used fo r antibody dilution can have profound effects on antibody-binding (Labrousse et a l

1994). Comparing both sets o f antibodies after incubation in isotonic and ‘high s a lt’ medium can provide information on their vulnerability to changes in electrostatic interactions with their antigen.

Comparing conventional immunofluorescence with tyramide amplified

immunofluorescence.

Roche P2X receptor antibodies for conventional immunofluorescence were used at 2.5 to 6p,g/ml (protein) in 10% NHS (in detail: P2Xi, 2.5p,g/ml; P2X2, 2.5pig/ml; P2Xg,

5(a,g/ml; P2X4, 6 p-g/ml; P2X$, 2.5pig/ml; P2Xg, 3 pig/ml; P2Xy, 4pig/ml). P2X receptor

antibodies (Roche) for tyramide amplification (Renaissance, TSA indirect, NEN, USA) were used at 0.4 to 0.625 pig/ml in blocking solution according to the manufacturers instructions. Primary antibodies were detected as described in General Materials and Methods.

o f the staining, but may give relatively high background. Immunofluorescent labelling after tyramide amplification can yield high sensitivity and low background, as reported previously by Shindler and Roth (1996).

Immunofluorescence and double-labelling.

Three different immunofluorescence double labelling techniques for P2X-receptor detection and cell identification were used.

First, P2X receptor antibodies (from Roche) were applied as for conventional histochemical staining (as described above). The primary antibody was detected by biotinylated donkey anti-rabbit antibody (Jackson, UK), applied together with mouse monoclonal antibodies directed against a-sm ooth muscle actin (Sigma, UK) to identify smooth muscle cells (Shalli et al. 1988) or against cytokeratins 1, 4, 5, 6, 8, 10, 13, 18, and 19 (pan cytokeratin antibody; Sigma, UK), to identify epithelial cells (Dockhom- Dwomiczak et al. 1987). Slides were then (simultaneously) incubated with streptavidin- coupled Texas-Red (TR) (Amersham, UK) and goat anti-mouse immunoglobulin G (IgG; Sigma, UK). Finally, the anti-mouse IgG was detected with fluorescein-isothiocyanate (FITC) coupled to chicken anti-goat antibody (ICN, CA). For co-labelling with endothelial cells the P2X antibodies were used as above, but no other primary antibody was applied. Endothelial cells were identified w ith FITC-coupled lectin from Bandeira

simplicifolia (Sigma, UK), as previously described by Bankston et al. (1991).

In a second immunofluorescence double-labelling experiment primary antibody against P2X receptors (from Roche) was enhanced with tyramide signal amplification (as described above). P2X receptors were then again visualised with strepavidin- coupled TR. Labelling o f vascular smooth muscle, epithelial cells and endothelial cell was

performed as above.

In a third series o f experiments, co-localisation studies for C-cells and P2X- receptors were performed, using two primary antibodies raised in the same species. The experiments were carried out as described by Shindler and Roth (1996). Briefly, P2X receptors were detected by tyramide amplification using P2X antibody concentrations below the detection limit o f a fluorophore-coupled secondary antibody (goat anti-rabbit, FITC-coupled; Nordic, NE). C-cells were then identified by incubation with rabbit anti- CGRP antibody (Affinity, UK) and by FITC-coupled goat anti-rabbit antibody (Arias et al. 1989).

Western blots.

Thyroids were taken from three 250g, male Sprague/Dawley rats and immunoblotting was performed as described in General Materials and Methods using a homogenisation buffer composed o f either (50 mM Tris-HCL, pH 7.4, ISOmM NaCl, 1% SDS,I% NP40, lOpig/ml PMSF, 30pl/ml aprotinin, Im M sodium orthovanadate) or o f (50 mM Tris-HCL, pH 7.4, 6M urea; 150mM NaCl, 1% SDS,1% NP40, lOpg/ml

PMSF, 30pl/ml aprotinin, Im M sodium orthovanadate).

RESULTS.

Western Blotting,

Both sets o f antibodies for P2X-receptors and both homogenisation buffers gave identical results in western blotting. Antibodies from Roche Bioscience against P2Xi through P2X7 receptors and antibodies from Alomone Labs against P2Xi, P2X2, P2X4

140 kDa (Fig. 4.2). As the antibodies from Alomone Labs are supplied with sufficient antigenic peptide, preabsoptions o f the immunoblots were carried out with this set o f antibodies. Immunostaining o f both bands o f P2X-receptors was completely preabsorbable.

Fig. 4.2. W estern blots o f crude extracts from rat thyroid. Gels were run under reducing conditions, proteins were transferred onto nitrocellulose, incubated w ith antibodies for P2X-receptors and were visualised using chemiluminescence. In panel (A) P2X-receptor antibodies from Roche Bioscience were tested (subtypes 1 to 7), in panel (B) antibodies for P2X-receptors from Alomone Labs were used (subtypes 1, 2, 4, and 7). Antibodies from Alomone Labs were also preabsorbed in immunoblotting, lane a showing the immunodetection, lane b giving results from the preabsorption experiments. Running o f molecular weight markers is indicated by triangles and the respective molecular weights are given in kDa. The number on each lane indicates for the respective P2X-receptor subtypes; (1) P2Xi, (2) P2Xz, (3) P2Xg, (4) P2X4, (5) P2Xg, (6) P2Xg and (7) P2Xy.

kDa 205 116 97.4 58.1 39.8 > 29.0 >

B

Comparing immunohistochemical staining fo r P2X-receptors with Alomone Labs and Roche antibodies.

Results o f the immunohistochemical experiments were principally identical. However Alomone Labs P2X2 antibody produced broad nuclear staining and Alomone Labs P2X7 antibody was not completely preabsorbable.

Comparing strategies fo r immunohistochemical and immunofluorescence labelling.

Immunohistochemistry using ‘high sa lt’ medium.

The labelling appeared now slightly weaker, but no qualitative differences in immunostaining for P2X-receptors were observed.

Conventional immunofluorescence experiments.

Background staining obtained after ‘conventional immunofluorescence’ experiments was relatively high. However, incubation with cyanoborohydride-coupling buffer significantly reduced thyroid autofluorescence.

Tyramide amplified immunofluorescence.

The background obtained with ‘conventional immunofluorescence’ was greatly reduced after using tyramide amplification.

P2X-receptors on blood vessels.

The media o f blood-vessels was found to be immunopositive for P2Xi receptors and the staining was completely preabsorbable (Fig. 4.3). Results o f preabsorption and immunostaining experiments were generally shown for corresponding locations o f the tissue. The media o f blood-vessels were immunopositive for P2X2 receptors and the

immunoreactivity for P2X$ and receptors in the median layer, which was

completely abolished after preabsorption. Staining for P2X7 receptors was also regularly

found in the media o f smaller and bigger vessels o f the thyroid.

Immunolabelling for P2Xg and P2X4 receptors was found in the intima o f thyroid vessels and in some capillaries (Fig. 4.4). Antibodies against P2X^ receptors consistently gave immunostaining not only o f the media, but also o f the o f the intima o f thyroid blood vessels and appeared to label the endothelium o f inter-follicular capillaries. All intimai staining for P2X-receptors was preabsorbable.

Immunostaining for P2X-receptors was also observed in follicle-like structures. P2X], P2X4 and P2X$ receptors were observed on the thyroid follicular epithelium,

Fig. 4.3. Light microscopical detection o f im m unohistochem ical labelling with antibodies against P2X-receptors. Staining was performed with antibodies against P2X- receptor subtypes 1-7. Preabsorption experiments were carried out for each P2X- antibody on sections that were consecutive to those used for immunostaining. (A) Vascular smooth muscle stained with P2X% antibody ; (B) preabsorption o f P2Xi; (C) immunostaining for P2X2 receptors on vascular smooth muscle; (D) preabsorption o f P2X2 antibody; (E) Vascular smooth muscle immunopositive for P2X$ receptors ; (F) preabsorbed P2X$ antibody; (G) P2Xg antibody on vascular smooth muscle; (H) staining with P2Xé antibody was preabsorbable.

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Fig. 4.4. L igh t m icroscop ical detection o f im m u n o h isto ch em ica l lab ellin g w ith an tib od ies again st P 2X -recep tors. (A ) staining for P2X ] receptors on endothelial cells

(arrowheads) o f a vessel, that is adjacent to a (also immunopositive) follicle (arrows),

(B) staining for P2X] receptors is preabsorbable (endothelial cells are still present,

arrow), (C) immunoreactivity for P2X4 receptors on endothelial cells; staining for P2X4

is preabsorbed in (D); (E) P2X? antibody staining an endothelial cell; (F) P2X? antibody preabsorbed.

A ll scale bars: 10pm .

Immunofluorescence double labelling.

Thyroid vascular smooth muscle was shown to express P2X i, P2X2, P2X$, P2Xg

and P2X7 receptors (Fig. 4.5). Colocalisation experiments o f P2X-receptor antibodies with a marker for endothelial cells showed, that thyroid endothelial cells express P2Xg, P2X4 and P2Xy receptors (Fig. 4.6). A colocalisation study with antibodies for P2X-

receptors and a marker for cytokeratins revealed, that thyroid follicular epithelial cell express P2X], P2X4 and P2X5 receptors (Fig. 4.7).

Co-localisation experiments fo r P2X-receptors and C-cells.

Fig. 4.5.Colocalization study, identifying P2X-receptors on vascular smooth muscle by confocal microscopy. Sections from rat thyroid were incubated with antibodies for P2X-receptors and were visualised with Texas-Red (red fluorescence). Parallel staining with a marker for vascular smooth muscle was performed, the markers were detected with FITC (green fluorescence). Areas o f colocalisation (red and green fluorescence overlap) appear yellow. (A) P2Xi receptors are expressed on vascular smooth muscle cells, but not all smooth muscle cells express P2X% (arrow)', (B) P2X2

receptors colocalize with vascular smooth muscle cells; (C) vascular smooth muscle cells express P2X$ receptors; (D) P2Xg receptors could be detected in vascular smooth muscle; (E) immunoreactivity for P2X? receptors was seen in vascular smooth muscle;

Fig. 4.6.Colocalization study, identifying P2X-receptors on endothelial cells by confocal microscopy. Sections from rat thyroid were incubated with antibodies for P2X- receptors and were visualised with Texas-Red (red fluorescence). Parallel staining with a marker for rat endothelial cells was performed, the markers were detected with FITC (green fluorescence). Areas of colocalisation (red and green fluorescence overlap) appear yellow. (A) endothelial cells stain for P2X] receptors (vascular lumen is indicated by asterisk); (B) P2X4 receptors were detected on endothelial cells (vascular lumen is indicate by asterisks); (C) immunostaining for P2X? receptors was observed in endothelial cells (arrow) and in vascular smooth muscle (arrowheads, lumina of the vessels are indicated by asterisks).

Fig 4.7. Identification of P2X-receptor immunopositive epithelial cells by confocal microscopy. Sections were incubated with antibodies for P2X-receptors and visualised by red fluorescence. Epithelial cells were identified by a pan-cytokeratin marker, which was coupled to green fluorescence. Areas of colocalisation appear yellow. Figure (A) shows widespread immunoreactivity for P2Xs receptors on thyroid follicular cells (single

arrows) and in some nearby endothelial cells (double arrows); (B) higher magnification

figure showing immunoreactivity for P2Xg receptors in thyroid follicular cells; (C) thyroid follicular cells stain for P2X4 receptors; (D) P2X5 receptors were detected on thyroid follicular cells.

Cell types

P2X receptors