Down-regulation of red blood cell beta-adrenoreceptors in response to chronic elevation of plasma catecholamine levels in the rainbow trout.
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Animals↗
Biomedical subjects
S F Perry
Publications and source records attributed to S F Perry.
Antimicrobial susceptibility testing of enterococci: results of a survey conducted by the United Kingdom National External Quality Assessment Scheme for Microbiology.
Six strains of enterococci were distributed to participants in the United Kingdom National External Quality Assessment Scheme for Microbiology with a request that they be tested for susceptibility to ampicillin/penicillin and vancomycin and for high-level resistance to gentamicin. More than 98% of laboratories correctly reported the resistant strains as resistant to penicillin and ampicillin. However, there was a poorer correlation for the susceptible strains, with only 1-6% and 69-83% of laboratories reporting the strains as susceptible to penicillin and ampicillin respectively. Those laboratories which used 5-25 micrograms ampicillin discs or breakpoint methods reported proportionally more results correctly for the susceptible strains than those using 2 micrograms discs. Participants using Escherichia coli or an enterococcus as a control and those not using a control at all were more likely to report ampicillin-susceptible strains correctly than those using Staphylococcus aureus NCTC 6571 (Oxford strain). For vancomycin-susceptible and high-level vancomycin-resistant strains, 93-96% and 96-99% of reports respectively were correct. For the low-level vancomycin-resistant strains, only 50-54% of reports were correct. Participants using a 5 micrograms vancomycin disc reported proportionally more of the low-level resistant strains correctly than those using 10, 20, 25, 30 or 100 micrograms discs. Participants not using controls were proportionally more likely to report the low-level resistant strains as susceptible than those using an enterococcus or S. aureus NCTC 6571 as a control. For the high-level gentamicin-resistant (HLGR) strains, 96-98% of reports were correct. Depending on the strain, between 12% and 41% of participants also reported high-level resistance for the strains which were not HLGR. Those using low-content discs (< or = 30 micrograms) and breakpoint or MIC methods were more likely to report strains which were not HLGR as HLGR than those using high-content discs. Those using an enterococcus as a control were less likely to report strains which were not HLGR as HLGR than those using either S. aureus NCTC 6571 or E. coli as a control and those not using a control.
Ampicillin Resistance↗
Mechanisms of ion and acid-base regulation at the gills of freshwater fish.
This review examines the branchial mechanisms utilized by freshwater fish to regulate internal acid-base status and presents a model to explain the underlying basis of the compensatory processes. Rainbow trout, Oncorhynchus mykiss, and brown bullhead, Ictalurus nebulosus, were examined under a variety of experimental treatments which induced respiratory and metabolic acid-base disturbances. Acid-base regulation was achieved by appropriate adjustments of Na+ and Cl- net fluxes across the gills which, in turn, were accomplished by variable contributions of three different branchial mechanisms: 1) differential changes in Na+ and Cl- diffusive effluxes, 2) changes in internal substrate (H+, HCO3-) availability, and 3) morphological adjustments to the gill epithelium. Differential diffusive efflux of Na+ over Cl- was involved only during periods of metabolic alkalosis. The importance of internal substrate availability was demonstrated using a two-substrate model. According to the model, ionic flux rates (J(in)Cl-, J(in)Na+) are determined not only by the concentration of the external ion (Na+, Cl-) but also by the concentration of the internal counterion (H+, HCO3-). This system provides for an "automatic negative feedback" to aid in the compensation of metabolic acid-base disturbances. Morphological alteration of the gill epithelia and the associated regulation of chloride cell (CC) fractional area is an essential third mechanism which is especially important during respiratory acid-base disturbances. Specifically, fish vary the availability of the CC associated Cl-/HCO3- exchange mechanism by physical covering/uncovering of CCs by adjacent pavement cells.
Acid-Base Equilibrium↗
Control and consequences of adrenergic activation of red blood cell Na+/H+ exchange on blood oxygen and carbon dioxide transport in fish.
The catecholamines, adrenaline and noradrenaline, are released into the circulation of fish during a variety of physical and environmental disturbances that share the common feature of a requirement for enhanced blood oxygen transport. Indeed, the dominant factor controlling the mobilization of catecholamines from chromaffin tissue is a depression of blood oxygen content usually coinciding with a reduction of hemoglobin-O2 (Hb-O2) binding to 50-60% saturation. The elevation of plasma catecholamine levels, under such conditions, activates a beta-adrenergic cyclic AMP-dependent Na+/H+ exchanger on the red blood cell (rbc) membrane. The adrenergic responsiveness AMP-dependent Na+/H+ exchanger on the red blood cell (rbc) membrane. The adrenergic responsiveness of the rbc Na+/H+ exchanger to catecholamines varies both within and between species. Such inter- and intra-specific differences may reflect, in part, the availability of cell surface beta-adrenoceptors that are functionally coupled to adenylate cyclase. The activation of rbc Na+/H+ exchange and the accompanying profound adjustments of intracellular and extracellular acid-base status, nucleoside triphosphate (NTP) levels, and cooperativity of Hb-O2 binding have important consequences on both O2 and CO2 transfer and transport in the blood that vary markedly at the sites of oxygenation (the gill) and deoxygenation (the tissues) thereby enabling simultaneous amelioration of O2 loading and unloading. At the gill, oxygen transfer is enhanced owing to increases in Hb-O2 affinity and capacity while at the tissues, oxygen delivery is facilitated by a reduction of Hb-O2 affinity. This reduction in affinity at the tissues is a consequence of the combined effects of increased cooperativity of Hb-O2 binding and a rise in venous PCO2 (PvCO2) caused by the titration of HCO3- by H+ extruded by the rbc Na+/H+ exchanger. This elevation of PvCO2 may contribute to the rise in arterial PCO2 (PaCO2) observed after adrenergic activation of rbc Na+/H+ exchange that is caused primarily by impairment of rbc CO2 excretion related to modification of the intracellular acid-base status.
Animals↗
Evidence for a morphological component in acid-base regulation during environmental hypercapnia in the brown bullhead (Ictalurus nebulosus).
Exposure of adult brown bullheads Ictalurus nebulosus (120-450 g) to environmental hypercapnia (2% carbon dioxide in air) and subsequent recovery caused transient changes in whole body net sodium flux (JnetNa+) and net chloride flux (JnetCl-) resulting largely from changes in whole body sodium influx (JNa+in) and chloride influx (JinCl-). Scanning electron microscopy (SEM) revealed that the fractional area of chloride cells (CCs) on the interlamellar regions was reduced by 95% during environmental hypercapnia. During post-hypercapnic recovery, gill filament CC fractional area increased. The changes in JinCl- during and after environmental hypercapnia were closely associated with the changes in CC fractional area while the changes in JinNa+ did not correspond to the changes in CC fractional area. Transmission electron microscopy (TEM) supported the SEM observations of CC surface area changes and demonstrated that these changes were caused by covering/uncovering by adjacent pavement cells (PVCs). Lamellar and filament PVC microvilli density increased during hypercapnia while there was a subsequent reduction in the posthypercapnic period. These data suggest that an important mechanism of acid-base regulation during hypercapnic acidosis is modification of the chloride cell-associated Cl-/HCO3- exchange mechanism. We suggest that bullheads vary availability, and thus functional activity, of this transporter via reversible morphological alterations of the gill epithelium. The increase in density of PVC microvilli may be associated with sodium uptake and/or acidic equivalent excretion during acidosis.
Acid-Base Equilibrium↗
Metabolic alkalosis and the response of the trout, Salmo fario, to acute severe hypoxia.
Trout (Salmo fario) were acutely transferred from seawater to freshwater in order to induce blood metabolic alkalosis (cf. Maxime et al., J. Comp. Physiol. 160: 31-39, 1990). After 2 weeks, the fish were exposed to severe environmental hypoxia (final water oxygen partial pressure, PWO2 = 25-45 Torr, reached within 20 min), to assess the impact of the experimentally induced alkalosis, and hence increased haemoglobin-oxygen (Hb-O2) affinity, on various aspects of the hypoxic response. This was accomplished by monitoring oxygen partial pressure and total oxygen content of arterial blood (PaO2 and CaO2), extracellular pH (pHe), red blood cell (RBC) intracellular pH (pHi), and the concentrations of plasma adrenaline, noradrenaline, lactate and haemoglobin (Hb) at 5 min intervals. Blood from normoxic fish exhibited high pHe and RBC pHi values (8.32 +/- 0.02 and 7.53 +/- 0.03, respectively). During hypoxia PaO2 declined to 10 Torr within 25 min; the first 5 min provoked increases of pHe and pHi to 8.43 +/- 0.03 and 7.71 +/- 0.03, respectively; thereafter, pHe decreased whilst pHi remained elevated. The blood lactate concentration increased from 2.30 +/- 0.76 mmol.L-1 in normoxia to 14.94 +/- 5.7 mmol.L-1 at the conclusion (60 min) of the hypoxic exposure and catecholamine levels also increased progressively (from 2.94 +/- 0.51 and 1.90 +/- 0.50 nmol.L-1, in normoxia, to 191.91 +/- 64.25 and 72.00 +/- 25.02 nmol.L-1, at their highest levels, for adrenaline and noradrenaline, respectively). Determination of the PaO2 thresholds for lactate and catecholamine release demonstrated that these substances appeared in the bloodstream when the degree of O2 saturation of the haemoglobin fell below 60%. The results demonstrate that initial blood alkalosis does not prevent the typical physiological responses of trout to hypoxia but simply shifts to lower PaO2 values the threshold at which these responses begin.
Acid-Base Equilibrium↗
Methods used in the United Kingdom for the culture of micro-organisms from blood.
AIMS: To survey blood culture methods in use in the United Kingdom. METHODS: A questionnaire was distributed to the 415 United Kingdom laboratories participating in the clinical bacteriology subscheme of the UK National External Quality Assessment Scheme for Microbiology. RESULTS: Two hundred and eighty seven laboratories completed the questionnaire. The responses indicated wide variations in methods used including considerable differences among laboratories using the same basic methods. The most widely used techniques were the Bactec and conventional broth systems. CONCLUSIONS: Although many published comparisons have failed to show that any basic method is outstandingly successful in the isolation of all organisms, there is evidence that several factors can contribute to good performance. In some laboratories suboptimal conditions were in use. The variations in technique probably reflect differences in practical suitability of a system for any particular laboratory, costs, and personal preferences.
Bacteria↗
Rainbow trout hepatocyte beta-adrenoceptors, catecholamine responsiveness, and effects of cortisol.
Hepatocyte beta-adrenoceptors were characterized in trout with chronically raised plasma cortisol levels (132.1 +/- 14.8 ng/ml, n = 8, 11-12 days) and compared with shams (1.8 +/- 0.9 ng/ml, n = 8) using radioreceptor assay techniques. Hepatocytes prepared from sham trout possessed 1,696 +/- 179 beta-adrenoceptors/cell. These receptors were characterized as beta 2-adrenoceptors based on the potency order of specific inhibition of CGP binding. The number of putative surface beta 2-adrenoceptors significantly increased to 6,005 +/- 1,165 receptors/cell, in hepatocytes from trout exposed to the elevated plasma cortisol concentration. The physiological significance of the increase in hepatocyte surface receptors was assessed by the in vitro responsiveness of hepatocytes to a range of epinephrine concentrations (10-1,000 nmol/l). Both total glucose production and intracellular adenosine 3',5'-cyclic monophosphate content, indicators of epinephrine responsiveness, were enhanced as a result of chronically raised levels of plasma cortisol. We suggest that these cortisol-mediated alterations in the adrenergic responsiveness of trout hepatocytes may ultimately enhance the ability of the liver to supply glucose to the fish during acute stress after extended periods of chronic stress.
Animals↗
Relationship between blood O2 content and catecholamine levels during hypoxia in rainbow trout and American eel.
Plasma catecholamine levels and arterial blood respiratory variables were monitored in rainbow trout (Oncorhynchus mykiss) and American eel (Anguilla rostrata) acutely exposed (30 min) to graded levels of external hypoxia [water PO2 (PWO2) 20-90 Torr]. The experiments were designed to evaluate the factors controlling catecholamine mobilization in hypoxic fish and to elucidate the basis of marked interspecific differences. In trout, plasma catecholamine levels were unchanged when PWO2 remained above 50 Torr but increased markedly when PWO2 was lowered below this value; the predominant catecholamine released into the circulation was epinephrine. In eel, there was no such obvious PWO2 threshold for catecholamine release although plasma levels were consistently elevated above baseline only at PWO2 less than 35 Torr. The magnitude of the catecholamine release in eel was approximately an order of magnitude less than in trout. Unlike in trout, there was no increase in the plasma epinephrine-to-norepinephrine concentration ratio. During hypoxia, the relationship between arterial blood PO2 (PaO2) and PWO2 was similar in both species and thus could not explain the differences in the PWO2 thresholds for catecholamine release. In trout, the calculated PaO2 thresholds for catecholamine release were 25.3 (epinephrine) and 20.5 Torr (norepinephrine) whereas in eel the corresponding values were 12.5 and 11.6 Torr, respectively. These PaO2 thresholds were in good agreement with the in vivo values for PaO2 at half-maximal hemoglobin (Hb)-O2 saturation (P50) for trout and eel blood of 22.9 and 11.1 Torr, respectively. Thus both species displayed essentially equivalent catecholamine release thresholds when expressed in terms of arterial blood O2 content corresponding to approximately 45-60% Hb-O2 saturation.
Animals↗
The relationship between beta-adrenoceptors and adrenergic responsiveness in trout (Oncorhynchus mykiss) and eel (Anguilla rostrata) erythrocytes.
Experiments were performed in vitro specifically to elucidate the underlying mechanism(s) of the attenuated adrenergic responses of eel (Anguilla rostrata) erythrocytes. This was achieved by comparing beta-adrenoceptor numbers and affinities in addition to (i) Na+/H+ exchange activity, (ii) cell swelling and (iii) cyclic AMP formation mediated by catecholamines in eel and trout (Oncorhynchus mykiss) erythrocytes under normoxic and hypoxic conditions. Under normoxic conditions, eel erythrocytes displayed a total absence of Na+/H+ exchange activity (as determined from measurements of extracellular pH) after addition of noradrenaline (50-1000 nmol l-1) in contrast to a pronounced dose-dependent response in trout. Incubation of the blood under hypoxic conditions, to achieve approximately 50% haemoglobin O2-saturation, further increased the extent of Na+/H+ exchange activation in trout and elicited a statistically significant, although physiologically small (10% of the response in trout), activation of H+ extrusion activity in eel. Catecholamine-mediated cell swelling, although obvious in trout, was absent in eel when estimated under hypoxic conditions. Eel erythrocytes possessed approximately 50% fewer surface beta-adrenoceptors than did trout erythrocytes, although the dissociation constants (KD) of these receptors did not differ between eel and trout. The numbers and affinities of the erythrocyte beta-adrenoceptors were not significantly affected by the hypoxic incubation. Both eel and trout erythrocytes displayed a dose-dependent elevation of cyclic AMP concentration in response to noradrenaline that was further increased by hypoxia. Surprisingly, eel erythrocytes produced larger quantities of cyclic AMP despite the lower numbers of surface beta-adrenoceptors. Thus, the absence of adrenergic swelling and the attenuated H+ extrusion response in eel erythrocytes cannot be attributed to insufficient numbers of beta-adrenoceptors or to functional uncoupling of these receptors from adenylate cyclase. Instead, the differences between trout and eel may reflect differing numbers of Na+/H+ exchangers or fundamental differences in the manner by which these exchangers are activated by cyclic AMP.
Anguilla↗
The effects of endogenous or exogenous catecholamines on blood respiratory status during acute hypoxia in rainbow trout (Oncorhynchus mykiss).
An extracorporeal circulation of rainbow trout (Oncorhynchus mykiss) was utilized to continuously monitor the rapid and progressive effects of endogenous or exogenous catecholamines on blood respiratory/acid-base status, and to provide in vivo evidence for adrenergic retention of carbon dioxide (CO2) in fish blood (cf. Wood and Perry 1985). Exposure of fish to severe aquatic hypoxia (final PwO2 = 40-60 torr; reached within 10-20 min) elicited an initial respiratory alkalosis resulting from hypoxia-induced hyperventilation. However, at a critical arterial oxygen tension (PaO2) between 15 and 25 torr, fish became agitated for approximately 5 s and a marked (0.2-0.4 pH unit) but transient arterial blood acidosis ensued. This response is characteristic of abrupt catecholamine mobilization into the circulation and subsequent adrenergic activation of red blood cell (RBC) Na+/H+ exchange (Fievet et al. 1987). Within approximately 1-2 min after the activation of RBC Na+/H+ exchange by endogenous catecholamines, there was a significant rise in arterial PCO2 (PaCO2) whereas arterial PO2 was unaltered; the elevation of PaCO2 could not be explained by changes in gill ventilation. Pre-treatment of fish with the alpha-adrenoceptor antagonist phentolamine did not prevent the apparent catecholamine-mediated increase of PaCO2. Conversely, pre-treatment with the beta-adrenoceptor antagonist sotalol abolished both the activation of the RBC Na+/H+ antiporter and the associated rise in PaCO2, suggesting a causal relationship between the stimulation of RBC Na+/H+ exchange and the elevation of PaCO2.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The effects of catecholamines on ventilation in rainbow trout during hypoxia or hypercapnia.
This study assessed the effects of experimentally elevated plasma catecholamine levels on gill ventilation in rainbow trout (Oncorhyncus mykiss) exposed to various external ventilatory stimulants. Trout were exposed to hypoxia (water PO2 (PwO2) = 90 Torr) or hypercapnia (water PCO2 (PwCO2) = 4.5 Torr) for 30 min. These conditions caused gill ventilation volume (Vw) to increase by 2.3- and 1.5-fold, respectively, but did not stimulate release of catecholamines into the blood. While the stimulus (hypoxia or hypercapnia) was maintained, fish were given a bolus injection (0.3 ml), followed by intra-arterial infusion (0.6 ml.h-1), of a catecholamine mixture (2 x 10(-5) mol.l-1 adrenaline + 5 x 10(-6) mol.l-1 noradrenaline) to mimic the physiological concentrations and ratios of these catecholamines observed under more severe hypoxic or hypercapnic conditions. In hypoxic fish, this treatment caused a significant, but transient (5 min) depression of ventilation while during hypercapnia, the administration of exogenous catecholamines caused a more prolonged hypoventilatory response. These hypoventilatory responses occurred despite a catecholamine-induced blood acidosis (a potential ventilatory stimulant). To assess the importance of initial Vw and/or blood respiratory status on catecholamine-mediated hypoventilation, these experiments were repeated under hyperoxic (PwO2 = 640 Torr) hyperoxic hypercapnic (PwO2 = 510 Torr, PwCO2 = 4.8 Torr) or normoxic (PwO2 = 151 Torr) conditions in which Vw was either depressed (3.9-fold during hyperoxia) or unaffected. Intra-arterial infusion of catecholamines did not affect Vw under either of these experimental conditions. These results demonstrate that during a respiratory challenge, such as hypoxia or hypercapnia, physiologically relevant levels of circulating catecholamines can depress Vw and therefore do not support a stimulatory role for circulating catecholamines in the control of ventilation in fish.
Animals↗
Bioluminescence as a real-time monitor of injury and recovery in Salmonella typhimurium.
Bioluminescence provides a novel tool with which to probe in real time the recovery of microorganisms such as Salmonella typhimurium from sub-lethal injury, a condition of immense importance in the enumeration of microorganisms in food and environmental samples such as from estuarine waters. We describe the application of in vivo bioluminescence to monitor the recovery of S. typhimurium from freeze injury and, in addition, we explore the effect of multiple stress conditions on microbial recovery where we compare bioluminescence with viable count measurement.
Colony Count, Microbial↗
Detection of chloramphenicol resistance in Haemophilus influenzae.
An average of 358 (13%) of United Kingdom laboratories failed to detect resistance to chloramphenicol in strains of Haemophilus influenzae distributed by the United Kingdom National External Quality Assessment Scheme for Microbiology. Clear associations between methods used and results obtained show that error rates are minimised by the use of low content (10 micrograms) disc and a sensitive control strain of H influenzae.
Chloramphenicol Resistance↗
A new in vitro assay for carbon dioxide excretion by trout red blood cells: effects of catecholamines.
A new in vitro assay was developed and critically characterized to measure the rate of CO2 excretion by trout red blood cells (RBCs) from HCO3- in their natural plasma under normal in vivo conditions of acid-base status. The assay is based on the addition of [14C]bicarbonate to the whole blood and collection of the resultant 14CO2 in the overlying gas phase. The assay simulates the exposure of blood passing through the gills, and measured CO2 excretion rates are representative of those occurring in vivo. Rates are linear over the 3 min time course of the assay, related to haematocrit in a non-linear fashion, elevated by the addition of carbonic anhydrase, reduced by blockade with acetazolamide, and sensitive to variations of equilibration PCO2. Large variations in plasma [HCO3-] have only a small effect on CO2 excretion rates when the blood is chronically equilibrated at these levels. Acute elevations in [HCO3-], however, create a non-equilibrium situation, resulting in large increases in CO2 excretion. When the blood is acidified, to duplicate typical post-exercise metabolic acidosis, adrenaline causes a marked inhibition of RBC CO2 excretion. The response is transient, reaching a peak 5-8 min after addition of adrenaline and disappearing by 30-60 min. The magnitude of the adrenergic inhibition is correlated with the magnitude of the RBC pHi regulatory response, expressed as the RBC transmembrane pH difference (pHe-pHi). These results support the 'CO2 retention theory' explaining observed increases in blood PCO2 in vivo after exhaustive exercise and catecholamine infusions in fish.
Animals↗
Adrenergic inhibition of carbon dioxide excretion by trout red blood cells in vitro is mediated by activation of Na+/H+ exchange.
We have used a sensitive new technique to assess the mechanism(s) of adrenergic inhibition of rainbow trout (Oncorhynchus mykiss) red blood cell (RBC) carbon dioxide excretion in vitro. The effect was only apparent using blood acidified to simulate metabolic acidosis. Red blood cell CO2 excretion was inhibited in a dose-dependent manner by physiologically relevant concentrations of noradrenaline (10-1000 nmol l-1) or adrenaline (100-1000 nmol l-1). The beta-adrenoceptor antagonist propranolol abolished the inhibitory effect of adrenaline, whereas the alpha-adrenoceptor antagonist phentolamine was without effect. The action of noradrenaline on RBC CO2 excretion was mimicked by the beta-adrenoceptor agonist isoproterenol, but not by the alpha-adrenoceptor agonist phenylephrine. Therefore, adrenergic inhibition of CO2 excretion is mediated by RBC beta-adrenoceptors, presumably of the beta 1 subtype. The Na+/H+ exchange inhibitor amiloride effectively blocked adrenergic stimulation of Na+/H+ exchange (as indicated from measurements of pHe and RBC pHi) and entirely prevented the inhibition of CO2 excretion. Noradrenaline significantly reduced the rate of CO2 excretion even in the presence of the Cl-/HCO3- exchange inhibitor SITS. Therefore, adrenergic inhibition of CO2 excretion is accomplished via activation of RBC Na+/H+ exchange rather than by a direct inhibition of Cl-/HCO3- exchange. The observed relationship between CO2 excretion rates and the RBC transmembrane pH difference (pHe-pHi) and the occurrence of the inhibition only at low pHe provide further evidence of the linkage with RBC Na+/H+ exchange. We suggest that adrenergic activation of RBC Na+/H+ exchange impedes CO2 excretion by causing a rise in intracellular HCO3- levels concurrent with a reduction of intracellular PCO2. The net result is a reduced gradient for HCO3- entry into the RBC in conjunction with a diminution of the outwardly directed PCO2 gradient. Thus, the rate of formation of CO2 from the dehydration of plasma HCO3- is reduced and, in turn, a portion of this CO2 is not excreted but recycled through the red blood cell.
Adrenergic Antagonists↗
Characterization of beta-adrenoreceptors of rainbow trout (Oncorhynchus mykiss) erythrocytes.
Although many studies have characterized these receptors according to pharmacological criteria, this work represents only the second direct characterization of the rainbow trout beta-adrenergic receptors. Radioligand binding assays using (+/-)-4-(3-t-butylamino-2-hydroxy-propoxy)-[5,7-3H]benzimidazol-2- one ([3H]CGP 12177) and 1-[4,6-propyl 3H]dihydroalprenolol ([3H]DHA) were conducted to determine equilibrium binding times, ligand-receptor dissociation constants (KD) and binding capacities (Bmax). Furthermore, we assessed the influence of erythrocyte handling, suspension medium and endogenous catecholamines on Bmax and KD. Maximal binding was obtained when erythrocytes were handled minimally and maintained suspended in plasma rather than physiological saline. Washing and resuspending the erythrocytes, as well as the transfer of the erythrocytes into saline, significantly impaired apparent radioligand affinity and receptor density. Endogenous catecholamines, at levels considered normal for non-stressed animals, did not interfere with the radioligand binding assays, and thus eliminated the need to wash and resuspend erythrocytes. Based on the binding characteristics after intentional lysis of erythrocytes, it is shown that the total receptor population of trout erythrocytes can be estimated by propranolol-displaceable DHA binding, the density of high-affinity surface receptors can best be determined by isoproterenol-displaceable CGP binding, and the number of receptors located within the erythrocytes can be calculated from the difference between the total receptor density and the number of isoproterenol-displacable DHA binding sites. Each of these components must be considered when performing radioreceptor assays using these radioligands and this has significant implications for the interpretation of erythrocyte beta-adrenorecptor localization and mobilization.
Animals↗
The effects and physiological consequences of raised levels of cortisol on rainbow trout (Oncorhynchus mykiss) erythrocyte beta-adrenoreceptors.
We have investigated the influence of cortisol on the beta-adrenoreceptor population of rainbow trout [Oncorhynchus mykiss (Walbaum)] erythrocytes and determined what impact it has on the adrenergic responsiveness of erythrocytes in vitro to exogenous catecholamines. To do so, the erythrocyte beta-adrenoreceptors were characterized in fish with chronically elevated plasma cortisol levels (118 +/- 5.9 ng ml-1, greater than 10 days) and compared with shams, using radioreceptor assay techniques. The number of 'internalized', low-affinity receptors was increased when cortisol levels were raised, but the number of high-affinity, 'surface' receptors was not altered. The physiological significance of this response was ascertained by assessing the in vitro sensitivity (or responsiveness) of erythrocytes to adrenaline and noradrenaline (10-1000 nmol l-1) under normoxic (PO2 = 16.13 +/- 0.55 kPa, PCO2 = 0.41 +/- 0.01 kPa) or hypoxic (PO2 = 4.13 +/- 0.15 kPa, PCO2 = 0.43 +/- 0.01 kPa) conditions. Erythrocyte sensitivity to catecholamines, as determined by changes in both whole-blood pH (delta pHe) and intracellular cyclic AMP content, was greater in hypoxic than in normoxic blood. Although cortisol further enhanced the responsiveness of erythrocytes to catecholamines, this amplification in sensitivity was observed only during hypoxia. When the radioreceptor assay was conducted using erythrocytes from the catecholamine sensitivity experiments, results were consistent with initial receptor density data. An increase in surface receptor density was associated with hypoxia in vitro. This hypoxia-specific increase in surface beta-adrenoreceptors was significantly enhanced in the cortisol-treated erythrocytes, showing that cortisol had a significant impact on erythrocyte beta-adrenoreceptor dynamics in addition to the direct influence of hypoxia. This study has shown (1) that, by itself, in vitro hypoxia simultaneously initiates the movement of internal receptors to the cell surface, where they become physiologically active, and the replenishment of the internal receptor pool, (2) that cortisol increases receptor availability by increasing the internal pool of low-affinity receptors in the absence of any stimulus for receptor mobilization, and (3) that the sensitivity of erythrocytes to catecholamines is directly proportional to the number of high-affinity receptors present at the erythrocyte surface. Thus, we suggest that, under conditions of chronic stress, cortisol may pre-adapt the erythrocytes to receive additional physiological inputs that can ultimately enhance respiratory performance beyond that which would be possible in the absence of chronically elevated levels of cortisol.
Adrenergic beta-Antagonists↗