Magnitude of the Root effect in red blood cells and haemoglobin solutions of fishes: a tribute to August Krogh.

AIM: The ability of high carbon dioxide tensions or low pH to reduce blood oxygen binding even at high oxygen tensions, first observed by August Krogh and Isabella Leitch in 1919 and now known as the Root effect, was studied in red blood cells and haemoglobin solutions of several fish species. METHODS: Red blood cells in physiological saline were acidified at atmospheric oxygen tension by increasing carbon dioxide tensions and the percentage decrease in oxygen content was used to quantify the Root effect. Haemoglobin was incubated in air-equilibrated citrate buffers between pH 5 and 7 and the percentage decrease in oxygen saturation relative to pH 8 determined by spectral deconvolution. RESULTS: The maximal magnitude of the Root effect in citrate-buffered haemoglobin solutions closely matched the value in blood or red blood cells of 11 vertebrates over a Root effect range between 3 and 80%. Contrary to previous reports, there was no evidence for a significant Root effect in red blood cells or haemoglobin solutions of the wels catfish, but a significant Root effect under both conditions in the Siberian sturgeon. CONCLUSIONS: Under the conditions employed in this study, the maximal Root effect of citrate-buffered haemoglobin solutions closely resembles the maximal Root effect in red blood cells. This strengthens previous studies on the evolution of the Root effect and its role in oxygen concentration at the retina and swimbladder of a large number of fishes that were based on Root effect measurements in haemoglobin solutions.

Seasonality of the red blood cell stress response in rainbow trout (Oncorhynchus mykiss).

The beta-adrenergic stress response in red blood cells (RBCs) of rainbow trout shows seasonal changes in expression. We have explored the mechanisms underpinning this response by following, over a period of 27 months, changes in beta-adrenergic receptor (beta-AR) binding characteristics, beta-adrenergically stimulated RBC Na(+)/H(+) exchanger (betaNHE) activity, together with beta-AR and betaNHE mRNA levels and plasma steroid hormone and lactate levels. These parameters were measured at approximately monthly intervals in a single population of fish held under semi-natural conditions. Membrane-bound, high-affinity beta-ARs were present in RBCs at all sampling times, varying from 668+/-112 receptors cell(-1) to 2654+/-882 receptors cell(-1) (mean +/- S.E.M.; N=8). betaNHE activity, however, was reduced by 57% and 34% in December 1999 and February 2001, respectively, compared with an otherwise sustained influx that averaged 110.4+/-2.3 mmol l(-1) RBCs h(-1) (N=119). Only one reduction coincided with a spawning period but both were preceded by transient increases in circulating testosterone. betaNHE activity measured under standard conditions was not correlated with the number or affinity of beta-ARs nor with water temperature, but both beta-AR numbers and betaNHE activity were positively related to their respective mRNA levels (P=0.005 and 0.038, respectively). Pharmaceutical intervention in the transduction cascade linking the beta-AR and betaNHE failed to indicate any failure of the transduction elements in RBCs displaying low betaNHE activity. Similarly, we failed to demonstrate any link between seasonal cortisol fluctuations and seasonally reduced betaNHE activity. However, the betaNHE activity of age-separated RBC fractions showed that younger RBCs had a significantly higher betaNHE response than older RBCs, consistent with the seasonal reductions in betaNHE being linked to turnover of RBCs and erythropoiesis. Testosterone is known to induce erythropoiesis and we conclude that seasonal reductions in betaNHE are not caused by changes in beta-AR numbers but may be linked to testosterone-induced erythropoiesis.

Adrenergic receptors, Na+/H+ exchange and volume regulation in lungfish erythrocytes.

Aestivation in African and South American lungfish (Protopterus and Lepidosiren, respectively) is associated with elevations of extracellular osmolarity. Osmotic shrinkage of Protopterus red blood cells (RBCs) caused a small but significant stimulation of the Na influx that was amiloride-sensitive. suggesting involvement of the Na+/H+ exchanger (NHE). The associated in vitro regulatory volume increase was insignificant within a time frame of 120 min, but the shrinkage-activated Na+ influx may be sufficient for slow regulatory volume increase during aestivation in vivo. Osmotic swelling of the RBCs induced an incomplete regulatory volume decrease that was statistically significant after 180 min. The RBCs of Protopterus were very large (mean cellular volume of 6939 +/- 294 microm3) and possessed 23,066 +/- 7,326 beta-adrenoceptors cell(-1) with a Kd value of 6.1 +/- 3.2 nM. The number of receptors per unit surface area of lungfish RBCs was calculated to be twice that of trout RBCs and 70% that of cod RBCs. There was, however, no adrenergic stimulation of the NHE in either Protopterus or Lepidosiren. Acidification of the extracellular medium also failed to activate the NHE.

The differential cardio-respiratory responses to ambient hypoxia and systemic hypoxaemia in the South American lungfish, Lepidosiren paradoxa.

Lungfishes (Dipnoi) occupy an evolutionary transition between water and air breathing and possess well-developed lungs and reduced gills. The South American species, Lepidosiren paradoxa, is an obligate air-breather and has the lowest aquatic respiration of the three extant genera. To study the relative importance, location and modality of reflexogenic sites sensitive to oxygen in the generation of cardio-respiratory responses, we measured ventilatory responses to changes in ambient oxygen and to reductions in blood oxygen content. Animals were exposed to aquatic and aerial hypoxia, both separately and in combination. While aerial hypoxia elicited brisk ventilatory responses, aquatic hypoxia had no effect, indicating a primary role for internal rather than branchial receptors. Reducing haematocrit and blood oxygen content by approximately 50% did not affect ventilation during normoxia, showing that the specific modality of the internal oxygen sensitive chemoreceptors is blood PO(2) per se and not oxygen concentration. In light of previous studies, it appears that the heart rate responses and the changes in pulmonary ventilation during oxygen shortage are similar in lungfish and tetrapods. Furthermore, the modality of the oxygen receptors controlling these responses is similar to tetrapods. Because the cardio-respiratory responses and the modality of the oxygen receptors differ from typical water-breathing teleosts, it appears that many of the changes in the mechanisms exerting reflex control over cardio-respiratory functions occurred at an early stage in vertebrate evolution.

Lactate sequestration in the carapace of the crayfish Austropotamobius pallipes during exposure in air.

When held in air for up to 24 h, crayfish accumulated Ca(2+) and Mg(2+) in their haemolymph in direct proportion to raised levels of lactate. K(+) levels were highly variable, with elevated levels associated with morbidity. Lactate accumulation in the haemolymph was reflected in proportional increases in lactate levels in the carapace and muscle. Pieces of carapace incubated in saline containing elevated levels of lactate accumulated lactate to up to half the dissolved concentration. Measured levels in the carapace, relative to its water content, implied that lactate accumulated in the carapace in a combined form, possibly complexed to calcium. The exoskeleton seems to provide a reserve of buffering capacity and a sink for lactate during anaerobic metabolism. A similar mechanism has been identified in pond turtles.

Anion uptake and acid-base and ionic effects during isolated and combined exposure to hypercapnia and nitrite in the freshwater crayfish, Astacus astacus.

Nitrite influx into crayfish showed saturation kinetics, supporting a carrier-mediated uptake. Addition of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS: at 10(-5), 10(-4) and 10(-3) M) and bumetanide (at 10(-5) M and 10(-4) M) to the ambient water did not significantly affect nitrite influx. Rather than suggesting that neither Cl-/HCO3- exchange nor K+/Na+/2Cl- cotransport were involved in the transport, this may reflect that the gill cuticle has a low permeability to the pharmacological agents, or that the sensitivity of the transport mechanism to the inhibitors is low. Nitrite accumulation in the haemolymph was significantly decreased during hypercapnic conditions compared to normocapnic conditions. This supports the idea that an acid-base regulatory decrease in Cl-(influx)/HCO3- (efflux) induced by hypercapnia should decrease NO2- uptake if NO2- and Cl- share this uptake route. The respiratory acidosis caused by exposure to hypercapnia alone was partially compensated by HCO3- accumulation in the haemolymph. Combined exposure to hypercapnia and nitrite improved pH recovery, mainly by augmenting the [HCO3-] increase, but also by decreasing haemolymph PCO2. Exposure to nitrite in normocapnic water induced an initial increase in haemolymph [HCO3-] and later also a decrease in PCO2. Thus, the improved acid-base compensation during combined hypercapnia and nitrite exposure was an amplification of this nitrite-induced response. Haemolymph base excess rose much more than haemolymph [Ca], suggesting that transfer of acid-base equivalents between animal and water was more important than H+ buffering by exoskeletal CaCO3 in mediating the increase in haemolymph [HCO3-].

Red blood cells from the South American rattlesnake (Crotalus durissus terrificus) regulate volume incompletely following osmotic shrinkage and swelling in vitro.

The ability of rattlesnake (Crotalus durissus terrificus) red blood cells to volume regulate in vitro has been investigated. Blood was drawn through a catheter inserted in the dorsal aorta and equilibrated to gas mixtures of different composition. Cells shrunken osmotically by increasing the extracellular osmolarity from approximately 291 mosm l(-1) (n=3) to approximately 632 mosm l(-1) (calculated) only partially regulated their volume back towards the original volume either at pH 7.51+/-0.05 (mean+/-S.D., n=5) or pH 7. 20+/-0.06 (mean+/-S.D., n=3). There was no improvement of the regulatory volume increase at low haemoglobin oxygen saturation. The limited volume restoration was inhibited by separate additions of amiloride (10(-4) M) or DIDS (10(-4) M) suggesting involvement of the Na(+)/H(+) and Cl(-)/HCO(3)(-) exchangers. Cells that were swollen osmotically by an approximately 30% dilution of the extracellular medium also exhibited a limited ability to recover their volume. Therefore, these cells show little ability to volume regulate when exposed to in vitro conditions that shrink or swell the cell.