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.     

The spleen of the African lungfish Protopterus annectens: freshwater and aestivation

We describe the structure of the spleen of the African lungfish Protopterus annectens in freshwater conditions, and after 6?months of aestivation. The spleen is formed by cortical tissue that surrounds the splenic parenchyma. The cortex is a reticulum that contains two types of granulocytes, developing and mature plasma cells, and melanomacrophage centres (MMCs). The parenchyma is divided into lobules that show a subcapsular sinus and areas of red pulp and white pulp. Red pulp contains vascular sinuses and atypical cords formed by delicate trabeculae. White pulp also contains vascular sinuses and cords. Structural data indicate that red pulp is involved in erythropoiesis, destruction of effete erythrocytes, and plasma cell differentiation. White pulp appears to be involved in the production of immune responses. Macrophages and sinus endothelial cells constitute the reticulo-endothelial system of the spleen. After aestivation, the number of MMCs increases, and spleen tissue is infiltrated by lymphocytes, granulocytes, and monocytes. Also, white pulp is reduced, and sinus endothelial cells undergo vacuolar degeneration. Lungfish spleen shares structural characteristics with secondary lymphoid organs of both ectothermic and endothermic vertebrates, but appears to have evolved in unique ways.     

A local renin-angiotensin-like system in the heart of an African lungfish (Protopterus annectens)

The presence of components of a renin-angiotensin system was investigated in the heart of an African lungfish Protopterus annectens. A renin-like activity is present in heart homogenates. The activity reaches its maximum after 90 min of incubation at 20°C. By a spectrophotometric assay, an angiotensin converting enzyme-like activity was detected. By immunohistochemistry Ile⁵-Angiotensin II immunoreactive cardiocytes were demonstrated in the atrium and bulbus. Our data indicate that probably a local renin-angiotensin-like system is present in the heart of Protopterus annectens.     

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.     

Mitochondria-rich cells in gills and skin of an African lungfish, Protopterus annectens

We used scanning electron microscopy, the vital dye DASPEI and an antibody to the inner mitochondrial membrane to study the presence and localisation of mitochondria-rich cells in the gills and skin (opercular, dorsal and ventral) of the lungfish Protopterus annectens in its free-swimming conditions and at the beginning of aestivation. In the free-swimming period, the gills were short and thick and the pavement cells were extremely large (30-40 microns). The mitochondria-rich cells, which were distributed in the secondary and primary epithelium, occurred as two morphologically different types, i.e. elongated and oval, similar to the alpha and beta chloride cells of fresh water teleosts. In the skin, only one type of mitochondria-rich cells was found, resembling the alpha chloride cells. All the mitochondria-rich cells distributed in the gills and skin were labelled with anti Ca(2+)-ATPase serum indicating the possible uptake of Ca2+ at freshwater chloride cell level. At the start of aestivation, the skin and gills were covered by a thick layer of mucus and the epithelium of the gills was reduced. The mitochondria-rich cells were almost completely covered by the pavement cells.     

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.     

Lympho-granulocytic tissue associated with the wall of the spiral valve in the African lungfish Protopterus annectens

We describe the structure of the lympho-granulocytic tissue associated with the wall of the spiral valve of the African lungfish Protopterus annectens. The study was performed under freshwater conditions and after 6 months of aestivation. The lympho-granulocytic tissue consists of nodes surrounded by reticular tissue. The nodes are formed by an outer and an inner component separated by a thin collagenous layer. The outer component is a reticular-like tissue that contains two types of granulocytes, developing and mature plasma cells and melanomacrophage centres (MMCs). The inner component, the parenchyma, contains a meshwork of trabeculae and vascular sinusoids and shows dark and pale areas. The dark areas contain diffuse lymphoid tissue, with a large number of mitoses and plasma cell clusters. The pale areas contain a small number of macrophages and lymphocytes. Macrophages and sinus endothelial cells are filled with haemosiderin granules and appear to form part of the reticuloendothelial system of the lungfish. The reticular tissue houses granulocytes, plasma cells and MMCs and might serve for the housing and maturation of cells of the white series. After aestivation, the nodes undergo lymphocyte depletion, the suppression of mitosis, granulocyte invasion and the occurrence of cell death. By contrast, few histological changes occur in the reticular tissue. Whereas the nodes appear to be involved in lymphocyte proliferation and plasma cell maturation, the function of the reticular tissue remains obscure.     

Ventricle and outflow tract of the African lungfish Protopterus dolloi

We report a morphologic study of the heart ventricle and outflow tract of the African lungfish Protopterus dolloi. The ventricle is saccular and appears attached to the anterior pericardial wall by a thick tendon. An incomplete septum divides the ventricle into two chambers. Both the free ventricular wall and the incomplete ventricular septum are entirely trabeculated. Only a thin rim of myocardium separates the trabecular system from the subepicardial space. The outflow tract consists of proximal, middle, and distal portions, separated by two flexures, proximal and distal. The proximal outflow tract portion is endowed with a layer of compact, well-vascularized myocardium. This portion is homologous to the conus arteriosus observed in the heart of most vertebrates. The middle and distal outflow tract portions are arterial-like, thus being homologous to the bulbus arteriosus. However, the separation between the muscular and arterial portions of the outflow tract is not complete in the lungfish. A thin layer of myocardium covers the arterial tissue, and a thin layer of elastic tissue underlies the conus myocardium. Two unequal ridges composed of loose connective tissue, the spiral and bulbar folds, run the length of the outflow tract. They form an incomplete division of the outflow tract, but fuse at the distal end. The two folds are covered by endocardium and contain collagen, elastin, and fibroblast-like cells. They appear to be homologous to the dextro-dorsal and sinistro-ventral ridges observed during the development of the avian and mammalian heart. Two to three rows of vestigial arterial-like valves appear in the dorsal and ventral aspects of the conus. These valves are unlikely to have a functional role. The possible functional significance of the "gubernaculum cordis," the thick tendon extending between the anterior ventricular surface and the pericardium, is discussed.     

The gut of the juvenile African lungfish Protopterus annectens: A light and scanning electron microscope study

We describe the microstructure of the alimentary canal of the juvenile lungfish Protopterus annectens. Following the oesophagus, the gut is formed by a long segment that extends down to the pyloric valve. This segment, classically named stomach, is lined by a transitional epithelium but lacks all characteristics of the vertebrate stomach. It has been defined here as the intestinal vestibule. The spiral valve is divided into a first large chamber, which contains mucosal ridges, and a second smooth portion. The entire spiral valve is lined with a pseudostratified columnar epithelium that contains approximately six cell types: enterocytes, goblet cells, ciliated cells, leukocytes, dark pigment cells, and vascular cells. Enterocytes and goblet cells show a high number of cytoplasmic vacuoles. The number and size of the vacuoles, and the number of ciliated cells, decreases from the anterior toward the posterior end, suggesting that most of the digestive processes take place in the anterior part of the spiral valve. The epithelium overlies a lamina propria in the first large chamber and a vascular plexus in the smooth portion. The cloaca has a thick muscular wall covered by a transitional epithelium. An extensive lymphatic system formed by capillaries and lymphatic micropumps is present along the entire wall of the alimentary canal. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Musculoskeletal morphology of the pelvis and pelvic fins in the lungfish Protopterus annectens

The West African lungfish (Protopterus annectens) performs benthic, pelvic fin‐driven locomotion with gaits common to tetrapods, the sister group of the lungfishes. Features of P. annectens movement are similar to those of modern tetrapods and include use of the distal region of the pelvic fin as a “foot,” use of the fin to lift the body above the substrate and rotation of the fin around the joint with the pelvis. In contrast to these similarities in movement, the pelvic fins of P. annectens are long, slender structures that are superficially very different from tetrapod limbs. Here, we describe the musculoskeletal anatomy of the pelvis and pelvic fins of P. annectens with dissection, magnetic resonance imaging, histology and 3D‐reconstruction methods. We found that the pelvis is embedded in the hypaxial muscle by a median rostral and two dorsolateral skeletal projections. The protractor and retractor muscles at the base of the pelvic fin are fan‐shaped muscles that cup the femur. The skeletal elements of the fin are serially repeating cartilage cylinders. Along the length of the fin, repeating truncated cones of muscles, the musculus circumradialis pelvici, are separated by connective tissue sheets that connect the skeletal elements to the skin. The simplicity of the protractor and retractor muscles at the base of the fin is surprising, given the complex rotational movement those muscles generate. In contrast, the series of many repeating segmental muscles along the length of the fin is consistent with the dexterity of bending of the distal limb. P. annectens can provide a window into soft‐tissue anatomy and sarcopterygian fish fin function that complements the fossil data from related taxa. This work, combined with previous behavioral examination of P. annectens, illustrates that fin morphologies that do not appear to be capable of walking can accomplish that function, and may inform the interpretation of fossil anatomical evidence. J. Morphol. 275:431–441, 2014. © 2013 Wiley Periodicals, Inc.     

Proopiomelanocortin (POMC) mRNA and POMC-derived peptides immunolocalization in the skin of Protopterus annectens, an African lungfish

Antisera against adrenocorticotropic hormone (ACTH), alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin were used to localize, by immunohistochemistry, proopiomelanocortin (POMC)-derived peptides in the skin excised from different regions of the African lungfish Protopterus annectens. Immunoreactivity was observed in the epidermis mainly in the germinal layer. Using human POMC cDNA as hybridization probe, POMC-like mRNA was identified in situ in epidermal cells. The demonstration in the same cells of POMC mRNA and POMC-related peptides immunoreactivity indicates a local production of opiate hormones.     

Ionoregulatory physiology of two species of African lungfishes Protopterus dolloi and Protopterus annectens

Basic ionoregulatory physiology was characterized in two species of African lungfish, slender African lungfish Protopterus dolloi and West African lungfish Protopterus annectens , largely under aquatic conditions. There were no substantive differences between the two species. Plasma [Na], [Cl] and [Ca] were only 60–80% of those typical of freshwater teleosts, and plasma Ca activity was particularly low. Unidirectional Na and Cl influx rates from water were also very low, only c . 10% of teleost values, whereas unidirectional Ca influx rates were comparable with teleost rates. Protopterus spp. were fed a 3% ration of bloodworms every 48 h. The bloodworm diet provided similar amounts of Na and Ca as uptake from water, but almost no Cl. Efflux rates of Na and Cl through the urine were greater than via the faeces, whereas the opposite was true for Ca. Net ion flux measurements and ionic balance sheet calculations indicated that (1) both water and dietary uptake routes are important for Na and Ca acquisition; (2) the waterborne route predominates for Cl uptake; (3) unidirectional ion effluxes across the body surface (gills and skin) rather than urine and faeces are the major routes of loss for Na, Cl and Ca. Tissues (muscle, liver, lung, kidney, intestine and heart) and plasma ions were also examined in P. dolloi 'terrestrialized' in air for up to 5 months, during which plasma ion concentrations (Na, Cl, Ca and Mg) did not change and there were only a few alterations in tissue ions, that is, increased [Na] in intestine, decreased [Cl] in kidney and increased [Ca] in liver and kidney.     

Bioassay of insulin activity of pancreatic tissue in active and aestivated African lungfish, Protopterus annectens (Owen)

The insulin activity of the pancreatic tissues of free-swimming and aestivated African lungfish, P. annectens (Owen), was bioassayed against standard insulin in rabbits. The hepatic glycogen content and plasma glucose concentration in free-swimming and aestivated lungfish were also estimated. The liver glycogen content was 64·47 ± 6·32 and 7·19 ± 2·68 mgg⁻¹ net wt and the plasma glucose concentration was 37·62 ± 4·22 and 6·39 ± 1·29% in free-swimming and aestivated lungfish, respectively.
Extracts of pancreatic tissue of the lungfish produced a dose-dependent decrease in the plasma glucose concentration in rabbits. The magnitude of the decrease in plasma glucose concentration produced by extracts from aestivated specimens was significantly smaller (about 26%) compared to that produced by pancreatic extracts from free-swimming active lungfish (about 37%). The insulin activity of the pancreatic tissue was 0·82 ± 0·02 and 1·27 ± 0·25 iu mg⁻¹ dry pancreatic tissue weight in aestivated and free-swimming lungfish, respectively.     

Functional anatomy of the peripheral olfactory system of the African lungfish Protopterus annectens owen: development of the primary olfactory pathway during postembryonic growth

Seven specimens of the African lungfish ranging in size from 4 to 350 gm were used for the study of changes in the olfactory nerve and bulb during postembryonic growth as expressed by the increasing number of lamellae of the olfactory organ. Serial semithin sections were used, and the data were studied mathematically. The number of axons of the olfactory nerve, the surface area of the bulb, and the number of mitral cells increase exponentially; the mitral cell density decreases exponentially; and the calculated average convergence (counted axons/counted mitral cells) of axons onto the cells of the bulbar relay increases exponentially. Convergence figures obtained are smaller than those cited by other authors; two factors might explain this: first the mode of calculation, and second the fact that every year this fish undergoes a 6-month starvation stage marked by degeneration of the primary olfactory structure. A preliminary view of the primary olfactory pathway using serial-section reconstructions is proposed: An orderly projection of the mucosa onto the bulb takes place after a 90 degrees rotation of the system of two perpendicular axes which apply to both the olfactory organ and the olfactory bulb. This study suggests an original attempt at relating the postembryonic change in convergence and projection to the primary processing of the olfactory message.     

Upregulation of intracellular antioxidant enzymes in brain and heart during estivation in the African lungfish Protopterus dolloi

The African slender lungfish, Protopterus dolloi, is highly adapted to withstand periods of drought by secreting a mucous cocoon and estivating for periods of months to years. Estivation is similar to the diapause and hibernation of other animal species in that it is characterized by negligible activity and a profoundly depressed metabolic rate. As is typically observed in quiescent states, estivating P. dolloi are resistant to environmental stresses. We tested the hypothesis that P. dolloi enhances stress resistance during estivation by upregulating intracellular antioxidant defences in brain and heart tissues. We found that most of the major intracellular antioxidant enzymes, including the mitochondrial superoxide dismutase, cytosolic superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, were upregulated in brain tissue of lungfish that had estivated for 60 days. Several of these enzymes were also elevated in heart tissue of estivators. These changes were not due to food deprivation, as they did not occur in a group of fish that were deprived of food but maintained in water for the same period of time. We found little evidence of tissue oxidative damage in estivators. Products of lipid peroxidation (4-hydroxynonenal adducts) and oxidative protein damage (carbonylation) were similar in estivating and control lungfish. However, protein nitrotyrosine levels were elevated in brain tissue of estivators. Taken together, these data indicate that estivating P. dolloi have enhanced oxidative stress resistance in brain and heart due to a significant upregulation of intracellular antioxidant capacity.     

Increased urea synthesis and/or suppressed ammonia production in the African lungfish, Protopterus annectens, during aestivation in air or mud

The objective of this study was to elucidate how the African lungfish, Protopterus annectens, ameliorated ammonia toxicity during 12 or 46 days of aestivation in air or in mud. Twelve days of aestivation in air led to significant increases in contents of urea, but not ammonia, in tissues of P. annectens. The estimated rate of urea synthesis increased 2.7-fold despite the lack of changes in the activities of hepatic ornithine–urea cycle enzymes, but there was only a minor change in the estimated rate of ammonia production. After 46 days of aestivation in air, the ammonia content in the liver decreased significantly and contents of urea in all tissues studied increased significantly, indicating that the fish shifted to a combination of increased urea synthesis (1.4-fold of the day 0 value) and decreased ammonia production (56% of the day 0 value) to defend against ammonia toxicity. By contrast, 12 days of aestivation in mud produced only minor increases in tissue urea contents, with ammonia contents remained unchanged. This was apparently achieved through decreases in urea synthesis and ammonia production (40 and 15%, respectively, of the corresponding day 0 value). Surprisingly, 46 days of aestivation in mud resulted in no changes in tissue urea contents, indicating that profound suppressions of urea synthesis and ammonia production (2.6 and 1.2%, respectively, of the corresponding day 0 value) had occurred. This is the first report on such a phenomenon, and the reduction in ammonia production was so profound that it could be the greatest reduction known among animals. Since fish aestivated in mud had relatively low blood pO₂ and muscle ATP content, they could have been exposed to hypoxia, which induced reductions in metabolic rate and ammonia production. Consequently, fish aestivating in mud had a lower dependency on increased urea synthesis to detoxify ammonia, which is energy intensive, than fish aestivating in air.     

The morphology of the lung of the African lungfish,Protopterus aethiopicus

Summary The lung of the African lungfish (Protopterus aethiopicus) is paired, long and cylindrical. It is situated on the dorsal aspect of the coelomic cavity ventral to the ribs. Much of the gas exchange tissue is found in the proximal aspect of the lung with the caudal part largely taken up by a centrally situated air-duct with a few large peripherally located alveoli. Interalveolar septa, arranged at differing hierarchical levels from the air-duct, subdivide the lung into alveoli, the gas exchange compartments. The alveolar surface is covered by some cells characterized by microvilli on their free surface, while others are devoid of such structures. The general organization of the lung of Protopterus aethiopicus is similar to that of the other genera of Dipnoi, Neoceratodus and Lepidosiren, with the interalveolar septa increasing the surface area for gas exchange through pulmonary compartmentation. The abundant septal smooth muscle fibres and elastic tissue may contribute to the physiomechanical compliance of the lung. The undifferentiated alveolar pneumocytes and the double capillary system, observed in Protopterus, in general appear to characterize the very primitive lungs of the lower air-breathing vertebrates.     

Ultrastructural study on nuclear-cytoplasmic relationships in oocytes of the African lungfish,Protopterus aethiopicus

Summary Growing oocytes of Protopterus, like those of some amphibians and teleosts, show an impressive development of the nucleolar apparatus. Numerous nucleolus-like bodies establish close spatial relationships with the nuclear envelope by extending pedicels and streams of finely dispersed material towards the inner membrane.At such contact points, gaps in the perinuclear cistern are more frequent than elsewhere along the nuclear boundary. Expansion of the outer nuclear membrane gives rise to blebs, with or without visible content, and these become pinched off to form small vesicles in the perinuclear cytoplasm.Small, electron dense aggregates, indistinguishable from nucleolar material occur on both sides of the nuclear envelope opposite to each other, some being connected by a slender portion of the same material within a nuclear pore. Such accumulations are interpreted as detached parts of nucleolar bodies in transit to cytoplasmic sites where they presumably participate in the biogenesis of ribosomes. At the height of nucleolar emission, nucleoplasm and perinuclear cytoplasm are so rich in small electron dense particles that they are almost indistinguishable from each other.At this stage of massive transport, the route provided by the nuclear pores seems to be insufficient and another, more spacious, gateway may be in operation. The latter involves direct passage of material across the nuclear membranes preferentially where these form blebs.This view is supported not only by the overt spatial relationships between nucleolar pedicels and blebs, but by the occurrence within perinuclear lacunae and blebs of particles that seem to be derived from nucleolar bodies. Furthermore, frequent interruptions in the nuclear membranes preferentially located where they expand into outpocketings suggest that at these sites temporary gateways may exist in the living cell that permit easy access of intranuclear components to the cytoplasm.Supported by grants AM-3984, NB-00840, and NB-05219 from the U.S.P.H.S.