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First published online September 14, 2007
Journal of Experimental Biology 210, 3451-3460 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.008524
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In vivo red blood cell sickling and mechanism of recovery in whiting, Merlangius merlangus

Pia Koldkjær* and Michael Berenbrink

School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK


Figure 1
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  		Fig. 1. (A) Light microscopy image of whiting sickle cells sampled immediately after capture of fish by hook and line. (B) Sickle cell fraction in rainbow trout (N=8), common carp (N=5) and whiting (N=5) red blood cells (RBCs) immediately after being caught by hook and line, in freshly sampled whiting RBCs after recovery in the holding tank for 24 h or more than one week (N=4 and 8, respectively) and in washed RBCs after 16–24 h storage (N=12). Small light microscopy images show the appearance of representative cells from the column immediately below. In A and B, scale bars correspond to 10 µm, {blacktriangledown} shows examples of cells with granular texture and {blacktriangleup} shows examples of cells with visible bars. In B, * indicates significant difference (P<0.05) from value for whiting immediately after being caught. Values are means ± s.d.

 

Figure 2
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  		Fig. 2. Light microscopy and transmission electron microscopy (TEM) images of representative normal (A,B) and sickle (C–G) cells in vitro at pH 7.97 and 7.03–7.43, respectively. The scale bars correspond to 10 µm (A,C–F) or 200 nm (B,G). In the TEM images, n and cy indicate nucleus and cytosol, respectively. {blacktriangledown} indicates filaments cut in longitudinal direction, and {blacktriangleup} indicates filaments in cross section.

 

Figure 3
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  		Fig. 3. Sickle cell fraction as a function of saline pH for red blood cell (RBC) suspensions incubated under humidified nitrogen (blue; PO2 ~0 kPa; N=4), air (black; PO2 ~20.8 kPa; N=4) or oxygen (red; PO2 ~100 kPa; N=3) for 45 min. The filled symbols represent individual data points. Curves are fitted using the mean pKapp and nH values obtained from individual animals (see Materials and methods and Table 1). The open symbols represent means ± s.d. for pKapp values determined from individual curve fits. In some cases at high and low sickle cell fraction, points are covered by overlaying symbols. See Results for references to in vivo blood pH values as indicated below the x-axis.

 

Figure 4
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  		Fig. 4. Sickle cell fraction as a function of time during in vitro recovery by increasing (A) saline pH in air-equilibrated samples from 7.03 to 7.97 (•; N=3) or (B) PO2 at pH 7.61 from zero to ~100 kPa ({blacksquare}; N=4). Data points on the right-hand side of the dotted line indicate values for cell suspensions kept at pH 7.97 under air (•) or pH 7.61 at a PO2 of ~100 kPa ({blacksquare}) throughout the experiments. Values are means ± s.d. Error bars are in some cases smaller than the symbol.

 

Figure 5
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  		Fig. 5. The ouabain-insensitive Na influx in air-equilibrated red blood cells (RBCs) measured during the initial 5 min at a saline pH of (A) 7.97 or (B) 7.03 in unstimulated RBCs (basal) with or without 10–4 mol l–1 amiloride, or RBCs stimulated by 10–5 mol l–1 isoproterenol alone or together with 10–4 mol l–1 amiloride. Values are means ± s.d. A significant difference (P<0.05) from the respective isoproterenol-stimulated Na influx is indicated by *. N=5.

 

Figure 6
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  		Fig. 6. The sickle cell fraction (A) and ouabain-insensitive Na uptake (B) in cells exposed to a saline pH of 7.97 ({square}); saline pH of 7.03 (•); saline pH of 7.03 with 10–5 mol l–1 isoproterenol alone ({square}) or together with 10–4 mol l–1 amiloride ({blacksquare}). * indicates significant difference (P<0.05) from first sampling time within same treatment, {dagger} indicates cells at pH 7.03 only that are significantly different (P<0.05) from samples at saline pH 7.03 with isoproterenol. Values are means ± s.d. N=8 (A) and 5 (B). Error bars are in some cases smaller than the symbol.

 

Figure 7
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  		Fig. 7. Light microscopy (A) and TEM images (B–E) of globular red blood cells (RBCs). C and E are magnifications of the areas within the squares in B and D, respectively. Scale bars correspond to 10 µm (A), 1 µm (B,D) or 200 nm (C,E). In the TEM images, n and cy indicate nucleus and cytosol, respectively. {blacktriangledown} indicates filaments cut in longitudinal direction, and {blacktriangleup} indicates fragmented filaments.

 




© The Company of Biologists Ltd 2007