Water balance and renal function in two species of African lungfish Protopterus dolloi and Protopterus annectens

The basic physiology of water balance and kidney function was characterized in two species of African lungfish, Protopterus dolloi and Protopterus annectens. Diffusive water efflux rate constants were low (0.13 h(-1)-0.38 h(-1) in various series) relative to values in freshwater teleost fish. Efflux rate constants increased approximately 3-fold after feeding in both species, and were greatly decreased after 8 months terrestrialization (P. dolloi only tested). Urine flow rates (UFR, 3.9-5.2 mL kg(-1) h(-1)) and glomerular filtration rates (GFR, 6.6-9.3 mL kg(-1) h(-1)) were quite high relative to values in most freshwater teleosts. However urinary ion excretion rates were low, with net re-absorption of >99% Na(+), >98% Cl(-), and >78% Ca(2+) from the primary filtrate, comparable to teleosts. Net water re-absorption was significantly greater in P. dolloi (56%) than in P. annectens (23%). We conclude that renal function in lungfish is similar to that in other primitive freshwater fish, but there is an interesting dichotomy between diffusive and osmotic permeabilities. Aquatic lungfish have low diffusive water permeability, an important pre-adaptation to life on land, and in accord with greatly reduced gill areas and low metabolic rates. However osmotic permeability is high, 4-12 times greater than diffusive permeability. A role for aquaporins in this dichotomy is speculated.     

The African lungfish (Protopterus dolloi): ionoregulation and osmoregulation in a fish out of water

Although urea production and metabolism in lungfish have been thoroughly studied, we have little knowledge of how internal osmotic and electrolyte balance are controlled during estivation or in water. We tested the hypothesis that, compared with the body surface of teleosts, the slender African lungfish (Protopterus dolloi) body surface was relatively impermeable to water, Na(+), and Cl(-) due to its greatly reduced gills. Accordingly, we measured the tritiated water ((3)H-H(2)O) flux in P. dolloi in water and during air exposure. In water, (3)H-H(2)O efflux was comparable with the lowest measurements reported in freshwater teleosts, with a rate constant (K) of 17.6% body water h(-1). Unidirectional ion fluxes, measured using (22)Na(+) and (36)Cl(-), indicated that Na(+) and Cl(-) influx was more than 90% lower than values reported in most freshwater teleosts. During air exposure, a cocoon formed within 1 wk that completely covered the dorsolateral body surface. However, there were no disturbances to blood osmotic or ion (Na(+), Cl(-)) balance, despite seven- to eightfold increases in plasma urea after 20 wk. Up to 13-fold increases in muscle urea (on a dry-weight basis) were the likely explanation for the 56% increase in muscle water content observed after 20 wk of air exposure. The possibility that muscle acted as a "water reservoir" during air exposure was supported by the 20% decline in body mass observed during subsequent reimmersion in water. This decline in body mass was equivalent to 28 mL water in a 100-g animal and was very close to the calculated net water gain (approximately 32 mL) observed during the 20-wk period of air exposure. Tritiated water and unidirectional ion fluxes on air-exposed lungfish revealed that the majority of water and ion exchange was via the ventral body surface at rates that were initially similar to aquatic rates. The (3)H-H(2)O flux declined over time but increased upon reimmersion. We conclude that the slender lungfish body surface, including the gills, has relatively low permeability to water and ions but that the ventral surface is an important site of osmoregulation and ionoregulation. We further propose that an amphibian-like combination of ventral skin water and ion permeability, plus internal urea accumulation during air exposure, allows P. dolloi to extract water from its surroundings and to store water in the muscle when the water supply becomes limited.     

The African lungfish, Protopterus dolloi, detoxifies ammonia to urea during environmental ammonia exposure

The African lungfish, Protopterus dolloi, was able to maintain a low level of blood plasma ammonia during exposure to high concentrations of environmental ammonia. After 6 d of exposure to 30 or 100 mM NH(4)Cl, the total ammonia concentrations in the blood plasma were 0.288 and 0.289 mM, respectively, which were only 1.7-fold greater than the control value of 0.163 mM. In addition, accumulation of ammonia occurred only in the muscle, but not in the liver. This was achieved in part through urea synthesis, as reflected by significant increases in urea contents in the muscle, liver, and plasma of the experimental animals. In contrast with plasma ammonia, the plasma urea concentrations of specimens exposed to 30 or 100 mM NH(4)Cl for 6 d increased 15.4-fold and 18.8-fold, respectively. Taken together, these results suggest that P. dolloi upregulated the rate of urea synthesis to detoxify ammonia during environmental ammonia exposure and that the increased rate of urea synthesis was fast enough to compensate for the rate of endogenous ammonia production plus the net influx of exogenous ammonia in these experimental animals. Simultaneously, there were increases in the rates of urea excretion in the experimental animals between day 2 and day 6 of environmental ammonia exposure. Interestingly, the rates of urea excretion in specimens exposed to 100 mM NH(4)Cl were lower than those exposed to 30 mM NH(4)Cl, despite the presumably greater load of ammonia to be detoxified to urea in the former situation. It would appear that P. dolloi was regulating the rate of urea excretion during ammonia exposure to retain urea, which might have some physiological functions under environmental stresses yet to be determined. There were decreases in the contents of glutamate, glutamine, and total free amino acids in the liver of the experimental animals, which indirectly suggest that a reduction in the rate of proteolysis and/or amino acid catabolism would have occurred that might lead to a decrease in ammonia production. Our results suggest that, unlike marine elasmobranchs and coelacanths, which synthesize and retain urea for osmoregulatory purposes, the ureogenic P. dolloi was adapted to synthesizing and excreting urea for the purpose of ammonia detoxification.     

Effects of intra-peritoneal injection with NH4Cl, urea, or NH4Cl+urea on nitrogen excretion and metabolism in the African lungfish Protopterus dolloi

This study aimed to (1) determine if ammonia (as NH(4)Cl) injected intra-peritoneally into the ureogenic slender African lungfish, Protopterus dolloi, was excreted directly rather than being converted to urea; (2) examine if injected urea was retained in this lungfish, leading to decreases in liver arginine and brain tryptophan levels, as observed during aestivation on land; and (3) elucidate if increase in internal ammonia level would affect urea excretion, when ammonia and urea are injected simultaneously into the fish. Despite being ureogenic, P. dolloi rapidly excreted the excess ammonia as ammonia within the subsequent 12 h after NH(4)Cl was injected into its peritoneal cavity. Injected ammonia was not detoxified into urea through the ornithine-urea cycle, probably because it is energetically intensive to synthesize urea and because food was withheld before and during the experiment. In addition, injected ammonia was likely to stay in extracellular compartments available for direct excretion. At hour 24, only a small amount of ammonia accumulated in the muscle of these fish. In contrast, when urea was injected intra-peritoneally into P. dolloi, only a small percentage (34%) of it was excreted during the subsequent 24-h period. A significant increase in the rate of urea excretion was observed only after 16 h. At hour 24, significant quantities of urea were retained in various tissues of P. dolloi. Injection with urea led to an apparent reduction in endogenous ammonia production, a significant decrease in the hepatic arginine content, and a significantly lower level of brain tryptophan in this lungfish. All three phenomena had been observed previously in aestivating P. dolloi. Hence, it is logical to deduce that urea synthesis and accumulation could be one of the essential factors in initiating and perpetuating aestivation in this lungfish. Through the injection of NH(4)Cl + urea, it was demonstrated that an increase in urea excretion occurred in P. dolloi within the first 12 h post-injection, which was much earlier than that of fish injected with urea alone. These results suggest that urea excretion in P. dolloi is likely to be regulated by the level of internal ammonia in its body.     

Differential NOS expression in freshwater and aestivating Protopterus dolloi (lungfish): heart vs kidney readjustments.

African lungfish Protopterus dolloi is an obligatory air-breather, which aestivates in a cocoon during the dry season. Aestivation associates with functional modifications in many tissues and organs, including heart and kidney. Due to its pleiotropic modulatory effects, nitric oxide (NO), generated by nitric oxide synthases (NOSs), may coordinate organ rearrangement, allowing adaptive adjustments under stressful environmental conditions. By immunofluorescence, Western blotting and NADPH-diaphorase, we examined cardiac and renal localization and activity of NOSs isoforms in both freshwater (FW) and aestivating [6 days (6DA) and 40 days (40DA) of estivation] P. dolloi. In heart and kidney endothelial NOS (eNOS) is the major isoform with respect to inducible and neuronal NOS (iNOS and nNOS, respectively). Cardiac eNOS locates in the epicardium, the trabecular endothelial endocardium, and myocardiocytes of both FW and aestivating fish. Western blotting revealed that cardiac eNOS expression increases in 6DA, but decreases in 40DA fish. In FW fish kidney eNOS is present in vascular endothelial cells and in podocytes of renal corpuscles. In tubular epithelial cells it is restricted to the apical pole. With aestivation, both renal localization and expression of eNOS increase. NADPH-diaphorase revealed an enhancement of cardiac and renal NOS activities during aestivation. Results suggest that in P. dolloi NO contributes, in an autocrine-paracrine fashion, to cardiac and renal readjustments during aestivation. Our findings are of evolutionary interest, since they document for the first time the presence of a NOS system in a ancestral fish, indicative of deep phylogenetic roots of NO bio-synthesis.     

Ornithine-urea cycle and urea synthesis in African lungfishes, Protopterus aethiopicus and Protopterus annectens, exposed to terrestrial conditions for six days

The objectives of this study were (1) to determine the type of carbamoyl phosphate synthetase (CPS) present, and the compartmentalization of arginase, in the livers of the African lungfishes, Protopterus aethiopicus and Protopterus annectens, and (2) to elucidate if these two lungfishes were capable of increasing the rates of urea synthesis and capacities of the ornithine-urea cycle (OUC) during 6 days of aerial exposure without undergoing aestivation. Like another African lungfish, Protopterus dolloi, reported elsewhere, the CPS activities from the livers of P. aethiopicus and P. annectens had properties similar to that of the marine ray (Taeniura lymma), but dissimilar to that of the mouse (Mus musculus). Hence, they possessed CPS III, and not CPS I as reported previously. CPS III was present exclusively in the liver mitochondria of both lungfishes, but the majority of the arginase activities were present in the cytosolic fractions of their livers. Glutamine synthetase (GS) activity was also detected in the hepatic mitochondria of both specimens. Therefore, our results suggest that the evolution of CPS III to CPS I might not have occurred before the evolution of extant lungfishes as suggested previously, prompting an examination of the current view on the evolution of CPS and OUC in vertebrates. Aerial exposure led to significant decreases in rates of ammonia excretion in P. aethiopicus and P. annectens, but there were no accumulations of ammonia in their tissues. However, urea contents in their tissues increased significantly after 6 days of aerial exposure. The estimated rates of urea synthesis in P. aethiopicus and P. annectens increased 1.2- and 1.47-fold, respectively, which were smaller than that in P. dolloi (8.6-fold) reported elsewhere. In addition, unlike P. dolloi, 6 days of aerial exposure had no significant effects on the hepatic CPS III activities of P. aethiopicus and P. annectens. In contrast, aerial exposure induced relatively greater degrees of reductions in ammonia production in P. aethiopicus (34%) and P. annectens (37%) than P. dolloi (28%) as previously reported. Thus, our results suggest that various species of African lungfishes respond to aerial exposure differently with respect to nitrogen metabolism and excretion, and it can be concluded that P. aethiopicus and P. annectens depended more on reductions in ammonia production than on increases in urea synthesis to ameliorate ammonia toxicity when exposed to terrestrial conditions.     

Unpaired Ultimobranchial Glands of the African Lungfish, Protopterus dolloi

The ultimobranchial glands of juvenile African lungfish (Protopterus dolloi) (14 individuals; total body length 25-205 mm) were immunohistochemically examined. In individuals larger than 36 mm, one ultimobranchial gland was close to the left afferent branchial arteries. The topography of the ultimobranchial gland was similar to that of salamanders and sharks, but not to teleosts. With body growth, the ultimobranchial gland was vascularized and the parenchymal cells were gradually immunostained with anti-calcitonin antibody. In all individuals examined, the ultimobranchial gland existed only on the left side of the pharynx. These observations are discussed from a phylogenetic viewpoint.     

Lipid, ketone body and oxidative metabolism in the African lungfish, Protopterus dolloi following 60 days of fasting and aestivation

The potential importance of lipids and ketone bodies as fuels in the African lungfish, Protopterus dolloi, and the role of oxidative metabolism, were examined under control, fasted and aestivated conditions. In aestivating but not fasting lungfish, the activities of citrate synthase (CS) and cytochrome c oxidase (CCO) (enzymes of oxidative metabolism) showed tissue-specific changes. Significant reductions in CS activity occurred in the kidney, heart, gill and muscle, and in CCO in the liver and kidney tissues. Aestivation, but not fasting, also had a tissue-specific effect on mitochondrial state 3 respiration rates (using succinate as a substrate), with a >50% reduction in the liver, yet no change within muscle mitochondria. There is no indication that enzymes involved in lipid catabolism are up-regulated during periods of fasting or aestivation; however, both 3-hydroxyacyl CoA dehydrogenase (HOAD) and carnitine palmitoyl CoA transferase (CPT) activities were sustained in the liver despite the approximately 42% reduction in CCO activity, potentially indicating lipid metabolism is of importance during aestivation. Lungfish are able to utilize both the d- and l-stereoisomers of the ketone body beta-hydroxybutyrate (beta-HB); however, beta-HB does not appear to be an important fuel source during aestivation or fasting as no changes were observed in beta-HB tissue levels. This study demonstrates that an important aspect of metabolic depression during aestivation in lungfish is the tissue-specific down regulation of enzymes of aerobic metabolism while maintaining the activities of enzymes in pathways that supply substrates for aerobic metabolism.     

Carbohydrate and amino acid metabolism in fasting and aestivating African lungfish (Protopterus dolloi)

The potential importance of carbohydrates and amino acids as fuels during periods of fasting and aestivation in the African lungfish, Protopterus dolloi, were examined. No significant decreases in tissue glycogen levels were observed following 60 days of fasting or aestivation, suggesting lungfish may undergo 'glycogen sparing'. Yet glycogenolysis may be important during aestivation based on the differing responses of two flux-generating enzymes of the glycolytic pathway, hexokinase (HK) and pyruvate kinase (PK). PK is required for glycogen breakdown whereas HK is not. HK activity is significantly down-regulated in the heart and gill tissues during aestivation, while PK activity is sustained. The significant negative correlation between the activity of HK and glucose levels in the heart of aestivating lungfish suggests HK may be regulated by glucose concentrations. There was no indication of anaerobic glycolytic flux during aestivation as lactate did not accumulate in any of the tissues examined, and no significant induction of lactate dehydrogenase (LDH)activity was observed. The increase in glutamate dehydrogenase (GDH) and aspartate aminotransferase (Asp-AT) activities in the liver of aestivating P. dolloi suggests some energy may be obtained via increased aminoacid catabolism, leading to the generation of tricarboxylic acid (TCA) cycle intermediates. These findings indicate the importance of both carbohydrate and amino acid fuel stores during aestivation in aphylogenetically ancient, air-breathing fish.     

Heart inflow tract of the African lungfish Protopterus dolloi

We report a morphologic study of the heart inflow tract of the African lungfish Protopterus dolloi. Attention was paid to the atrium, the sinus venosus, the pulmonary vein, and the atrioventricular (AV) plug, and to the relationships between all these structures. The atrium is divided caudally into two lobes, has a common part above the sinus venosus, and appears attached to the dorsal wall of the ventricle and outflow tract through connective tissue covered by the visceral pericardium. The pulmonary vein enters the sinus venosus and runs longitudinally toward the AV plug. Then it fuses with the pulmonalis fold and disappears as an anatomic entity. However, the oxygenated blood is directly conveyed into the left atrium by the formation of a pulmonary channel. This channel is formed cranially by the pulmonalis fold, ventrally by the AV plug, and caudally and dorsally by the atrial wall. The pulmonalis fold appears as a wide membranous fold which arises from the left side of the AV plug and extends dorsally to form the roof of the pulmonary channel. The pulmonalis fold also forms the right side of the pulmonary channel and sequesters the upper left corner of the sinus venosus from the main circulatory return. The AV plug is a large structure, firmly attached to the ventricular septum, which contains a hyaline cartilaginous core surrounded by connective tissue. The atrium is partially divided into two chambers by the presence of numerous pectinate muscles extended between the dorsal wall of the atrium and the roof of the pulmonary channel. Thus, partial atrial division is both internal and external, precluding the more complete division seen in amphibians. The present report, our own unpublished observations on other Protopterus, and a survey of the literature indicate that not only the Protopterus, but also other lungfish share many morphologic traits.     

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.     

Circulating catecholamines and cardiorespiratory responses in hypoxic lungfish (Protopterus dolloi): a comparison of aquatic and aerial hypoxia

Circulating catecholamine levels and a variety of cardiorespiratory variables were monitored in cannulated bimodally breathing African lungfish (Protopterus dolloi) exposed to aquatic or aerial hypoxia. Owing to the purported absence of external branchial chemoreceptors in lungfish and the minor role played by the gill in O2 uptake, it was hypothesized that plasma catecholamine levels would increase only during exposure of fish to aerial hypoxia. The rapid induction of aquatic hypoxia (final PWo2 = 25.9+/-1.6 mmHg) did not affect the levels of adrenaline (A) or noradrenaline (NA) within the plasma. Similarly, none of the measured cardiorespiratory variables--including heart rate (fH), blood pressure, air-breathing frequency (fV), O2 consumption (Mo2), CO2 excretion (Mco2), or blood gases--were influenced by acute aquatic hypoxia. In contrast, however, the rapid induction of aerial hypoxia (inspired Po2=46.6+/-3.3 mmHg) caused a marked increase in the circulating levels of A (from 7.9+/-2.0 to 18.8+/-6.1 nmol L(-1)) and NA (from 7.7+/-2.2 to 19.7+/-6.3 nmol L(-1)) that was accompanied by significant decreases in Mo2, arterial Po2 (Pao2), and arterial O2 concentration (Cao2). Air-breathing frequency was increased (by approximately five breaths per hour) during aerial hypoxia and presumably contributed to the observed doubling of pulmonary Mco2 (from 0.25+/-0.04 to 0.49+/-0.07 mmol kg(-1) h(-1)); fH and blood pressure were unaffected by aerial hypoxia. An in situ perfused heart preparation was used to test the possibility that catecholamine secretion from cardiac chromaffin cells was being activated by a direct localized effect of hypoxia. Catecholamine secretion from the chromaffin cells of the heart, while clearly responsive to a depolarizing concentration of KCl (60 mmol L(-1)), was unaffected by the O2 status of the perfusion fluid. The results of this study demonstrate that P. dolloi is able to mobilize stored catecholamines and increase f(V) during exposure to aerial hypoxia while remaining unresponsive to aquatic hypoxia. Thus, unlike in exclusively water-breathing teleosts, P. dolloi would appear to rely solely on internal/airway O2 chemoreceptors for initiating catecholamine secretion and cardiorespiratory responses.     

Major Central Nervous System Myelin Glycoprotein of the African Lungfish (Protopterus dolloi) Cross-Reacts with Myelin Proteolipid Protein Antibodies, Indicating a Close Phylogenetic Relationship with Amphibians

CNS myelin was isolated from the spinal cord of the African lungfish Protopterus dolloi. Its proteins consisted of (1) two basic proteins (16,000 and 18,500 apparent Mr) that reacted with anti-human CNS myelin basic protein antibodies and (2) a major protein (29,000 apparent Mr) that stained with concanavalin A-horseradish peroxidase and bound to anti-rat CNS myelin proteolipid protein (PLP) antibodies. This dominant 29,000 Mr protein showed no reaction with antibodies against the major bovine PNS myelin glycoprotein P0. Following treatment with endoglycosidase F the 29,000 Mr protein was reduced in size to a 26,000 apparent Mr component that no longer bound concanavalin A but retained the anti-PLP reactivity. These results agree with a concanavalin A-binding oligosaccharide linked through asparagine to a protein backbone of PLP homology. The major 29,000 Mr lungfish CNS myelin protein was therefore termed g-PLP (glycosylated proteolipid protein). This is the first report demonstrating the occurrence of a PLP-cross-reactive protein in CNS myelin of a fish. It attests to the close phylogenetic relationship of lungfishes to amphibians. Amphibians were previously recognized as the oldest class bearing PLP in its CNS myelin.     

Greatly elevated urea excretion after air exposure appears to be carrier mediated in the slender lungfish (Protopterus dolloi)

Under aquatic conditions, Protopterus dolloi is ammoniotelic, excreting only small amounts of urea-N. However, upon return to water after 30 d estivation in air, the lungfish excretes only small amounts of ammonia-N but massive amounts of urea-N. A similar pattern is seen after 21-30 d of terrestrialization, a treatment in which the lungfish is air exposed but kept moist throughout. After both treatments, the time course of urea-N excretion is biphasic with an immediate increase, then a fall, and finally a second larger increase that peaks at about 12 h and may be prolonged for several days thereafter. Urea-N excretion rates during the second peak reach 2,000-6,000 micromol N kg(-1) h(-1), two to three orders of magnitude greater than rates in most fish and comparable only to rates in species known to employ UT-A type facilitated diffusion urea transporters. Divided chamber studies and measurements of the clearance rates of [3H]-PEG-4000 (a glomerular filtration and paracellular diffusion marker) and two structural analogs of urea ([14C]-acetamide and [14C]-thiourea) were performed to characterize the two peaks of urea-N excretion. The smaller first peak was almost equally partitioned between the head (including internal and external gills) and the body compartment (including urinary opening), was accompanied by only a modest increase in [14C]-acetamide clearance equal to that in [14C]-thiourea clearance, and could be accounted for by a large but short-lasting increase in [3H]-PEG-4000 clearance (to about fivefold the terrestrial rate). The delayed, much larger second peak in urea-N excretion represented an elevated efflux into both compartments but occurred mainly (72%) via the body rather than the head region. This second peak was accompanied by a substantial increase in [14C]-acetamide clearance but only a modest further rise in [14C]-thiourea clearance. The acetamide to thiourea permeability ratio was typical of UT-A type transporters in other fish. [3H]-PEG-4000 clearance was stable at this time at about double the terrestrial rate, and excretion rates of urea and its analogs were many fold greater than could be accounted for by [3H]-PEG-4000 clearance. We conclude that the first peak may be explained by elevated urinary excretion and paracellular diffusion across the gills upon resubmergence, while the second peak is attributable to a delayed and prolonged activation of a UT-A type facilitated diffusion mechanism, primarily in the skin and perhaps also in branchial epithelia.     

A glycosylated proteolipid protein is common to CNS myelin of recent lungfish (Ceratodidae, Lepidosirenidae)

1. Myelin proteins from the CNS of recent lungfish (Lepidosiren paradoxa, Protopterus dolloi, Neoceratodus forsteri) were separated and analysed by staining and immunoblotting. 2. All species showed a glycosylated component (g-PLP) that cross-reacted with antibodies against tetrapod proteolipid protein (PLP), indicating phylogenetic relationships with amphibia. 3. Actinopterygian IP or teleostean 36k components were not detectable in lungfish CNS myelin. 4. The identical size of g-PLPs from Lepidosiren and Protopterus (Mr = 29,000) underlines the close relationship of the Lepidosirenidae. The smaller size of g-PLP from the ceratodidan Neoceratodus forsteri (Mr = 27,500) pointed to an earlier diversion.     

Kinetics and Block of Dopamine Uptake in Synaptosomes from Rat Caudate Nucleus

The dopamine (DA) uptake system in mammalian nerve terminals was studied by measuring the unidirectional influx of tritiated DA into synaptosomes prepared from rat caudate nucleus. Two distinct time-dependent components of DA uptake were observed. The principal component was saturable with respect to DA concentration, required both external Na and Cl, and was competitively blocked by micromolar concentrations of the psychotropic agents cocaine, benztropine, nomifensine, amphetamine, and methamphetamine. This principal component of uptake has the properties expected for a carrier-mediated transport system. The second component, which accounted for about 10-30% of the DA uptake at 2 microM DA, was not saturable, and was independent of external Na, Cl, and blockers of the carrier-mediated system. The saturable, Na-dependent component had an apparent Km(DA) of about 0.5 microM. The dependence of DA uptake on external Na was sigmoid [Hill coefficient = 2; Ka(Na) = 45 mM] whereas the dependence on Cl was best described by a rectangular hyperbola [Ka(Cl) = 15 mM]. Depolarizing conditions (elevated external K) reduced the rate of DA influx. The data are consistent with a carrier-mediated DA transport mechanism in which each DA molecule entering the nerve terminal via the carrier is accompanied by two or more Na ions and one Cl ion in a rheogenic process carrying one or more net positive charges into the cell. Net, concentrative accumulation of DA inside nerve terminals may be accomplished by utilizing the Na electrochemical gradient to drive DA against its electrochemical gradient via this carrier system.     

Cerebrovascular Permeability Coefficients to Sodium, Potassium, and Chloride

CSF and regional brain concentrations of 42K, 22Na, 36Cl, and [14C]mannitol were determined 3-45 min after intravenous injection of the tracers in pentobarbital-anesthetized rats. Rapid influx of 36Cl and 22Na into ventricular CSF immediately established concentration gradients from CSF to brain extracellular fluid. The CSF contribution to brain uptake of tracers was greatest in periventricular brain regions, where brain 36Cl concentrations were up to ninefold higher than concentrations in regions distant from ventricular CSF. Acetazolamide (20 mg kg-1 i.p.), an inhibitor of CSF formation, decreased 36Cl uptake into CSF and into periventricular brain regions but not into frontal cortex. 36Cl uptake into brain was unidirectional for 10 min after intravenous injection, and, during that period, diffusion from ventricular CSF did not contribute to uptake in the frontal cortex. Therefore, cerebrovascular permeability coefficients could be calculated from tracer concentrations in frontal cortex at 10 min and equaled, in cm s-1, 13.5 X 10(-7) for 42K, 1.4 X 10(-7) for 22Na, 0.9 X 10(-7) for 36Cl, and 1.5 X 10(-7) for [14C]mannitol. The low cerebrovascular permeabilities to K, Na, and Cl, comparable to those of some cell membranes, and the permselectivity (K much greater than Na greater than Cl) suggest that a significant fraction of ion transport across cerebral capillaries is transcellular, i.e., across the endothelial cell membrane.     

Cardiovascular actions of lungfish bradykinin in the unanaesthetised African lungfish, Protopterus annectens

Bradykinin (BK) isolated from plasma of the African lungfish, Protopterus annectens, contains four amino acid substitutions compared with BK from mammals (Arg(1)-->Tyr, Pro(2)-->Gly, Pro(7)-->Ala, Phe(8)-->Pro). Bolus intra-arterial injections of synthetic lungfish BK (1-1000 pmol/kg body wt.) into unanaesthetised, juvenile lungfish (n=5) produced a dose-dependent increase in arterial blood pressure and pulse pressure. The maximum pressor response occurred 2-3 min after injection and persisted for up to 15 min. The threshold dose producing a significant (P<0.01) rise in pressure was 50 pmol/kg and the maximum increase, following injection of 300 pmol/kg, was 9.3 +/- 2.3 mmHg. Injection of the higher doses of lungfish BK produced a significant (P<0.05) increase in heart rate (2.8 +/- 0.8 beats/min at 100 pmol/kg). In contrast, bolus intra-arterial injections of mammalian BK, in doses up to 1000 pmol/kg, produced no significant cardiovascular effects in the lungfish. The data support the existence of a functioning kallikrein-kinin system in the lungfish and demonstrate that the ligand-binding properties of the receptor(s) mediating the cardiovascular actions of lungfish BK are appreciably different from mammalian B1 and B2 receptors.     

Low unidirectional sodium influx into root cells restricts net sodium accumulation in Thellungiella halophila, a salt-tolerant relative of Arabidopsis thaliana

Thellungiella halophila is a useful model species for research into plant salt tolerance. It is closely related to Arabidopsis thaliana, but shows considerably higher salt tolerance. Comparative analysis of ion homeostasis in the two species allows the identification of ion transport pathways that are critical for salt tolerance and provides the basis for future studies into their molecular features. Previous studies indicated that salt tolerance in T. halophila is accompanied by low accumulation of Na in the leaves. Kinetic analysis of net ion uptake over three days confirmed lower Na uptake and K loss in T. halophila compared with A. thaliana. Differential net Na uptake rates were still apparent after 6 weeks of salt treatment. To assess the contribution of unidirectional Na fluxes to net Na uptake, kinetic studies of (22)Na fluxes were carried out in both species. The results show that unidirectional root Na influx is significantly lower in salt-grown T. halophila plants than in A. thaliana exposed to the same level of salinity (100 mM). Quantitative comparison of unidirectional influx and net Na accumulation suggests that both species operate efficient Na efflux, which partly compensates for Na influx. Kinetic analysis of (22)Na efflux indicated higher root Na efflux in A. thaliana than in T. halophila. Thus A. thaliana appears to spend more energy on Na export while nevertheless accumulating more Na than T. halophila. It is proposed that limitation of Na influx is the main mechanism by which T. halophila secures low net Na accumulation in saline conditions. This strategy provides the basis for a positive balance between growth and net Na uptake rates, which is essential for survival in high salt.