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.     

Presence in the heart of Protopterus annectens (Fishes, Dipneuste) of nodal and conducting cells histologically distinct from common myocardial cells]

Contrary to Francis and Davies's assumption according to which the impulse-conducting system of the Homoiothermal Vertebrates is a neomorphic structure, we have been able to identify in the heart of a Poikilothermal (Protopterus) impulse-conducting cells which possess distinctive histological characters.     

Histochemical studies of the adrenocortical homologue in the kidney of the Australian lungfish,Neoceratodus forsteri

A histochemical study has been made of groups of cells lying adjacent to the blood vessels of the kidney of Neoceratodus forsteri. The cells contain lipid droplets of varying sizes, stain positively for cholesterol and its esters and contain 3betaol-steroid dehydrogenase activity. Similar cells have previously been described in another dipnoan fish, Protopterus, and in a urodele, Pleurodeles waltlii and are considered to be the adrenocortical tissue of these species. Because of the histochemical characteristics of the cells together with their location, we conclude that they represent the adrenocortical homologue of N. forsteri. As is the case in Protopterus, no chromaffin tissue was detected in the kidney of N. forsteri.     

Histochemical study on the innervation of the heart of Pteropus giganteus.

Histochemical study of the innervation of the heart of the Indian flying fox Pteropus giganteus has been made using Coupland and Holmes' technique for cholinesterase. Distribution of nerve plexuses in the heart wall has been described. Nerve cells, nerve endings and various types of synapses have been reported. Specialized muscle fibres are found to contain less nerve fibres and conduction of the impulse of contraction is reported to take place through muscle fibres.     

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.     

Capsaicin-sensitive nerves influence the release of atrial natriuretic factor by atrial stretch in the rat.

Although many factors may modulate the release of atrial natriuretic factor (ANF), the primary mechanism has been demonstrated to be atrial stretch. Recent studies have led to the suggestion that the peptidergic innervation of the heart, through the release of peptides, may be involved in the control of ANF secretion. We have examined the influence of chronic capsaicin treatment on three models of atrial stretch that release ANF. This treatment inhibited ANF released through in vivo blood volume expansion and through balloon inflation in the right atrium of in vitro isolated perfused hearts. Immunohistochemical and electron microscopical analysis confirmed the absence of innervation of the heart by calcitonin gene related peptide and substance P immunoreactive nerve fibres and apparent lack of effect on atrial granules in capsaicin treated rats. We conclude that capsaicin-sensitive cardiac innervation is a component modulating the release of ANF, stimulated by atrial stretch in the rat.     

Cardiac responses in relation to heart size

There is a correlation between heart size and the propensity to develop and maintain fibrillation. Atrial and ventricular fibrillation is more easily induced and sustained in large than in small hearts. But other factors are at work as well, such as the ability to hibernate. The hibernator's heart has an adrenergic innervation which is different in distribution than that in nonhibernators with adrenergic nerves in the ventricles exclusively accompanying the blood vessels, thus leaving the myocardium proper without adrenergic innervation. This indicates that the risk of inhomogeneity of the electrophysiological parameters of the myocardial cells at a high sympathetic tone is less in the heart of a hibernator than in that of a nonhibernator.     

The distribution of acetylcholine and choline in guinea pig heart

The regional distributions of acetylcholine (ACh) and choline (Ch) in the guinea pig heart were investigated with a pyrolysis-mass fragmentography technique. Using ACh as a marker for cholinergic neurons, we have described a pattern of parasympathetic innervation in the guinea pig heart. This distribution is very similar to that suggested by studies using several different cholinergic indicators in various species. Atrial areas receive richer parasympathetic innervation than ventricular areas, with the right portions receiving more than the left. The nodal areas were the most abundantly innervated regions examined. Ch content is not a good indicator for cholinergic innervation as the regional distribution of ACh and Ch throughout the guinea pig heart are not strongly associated.     

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.     

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.     

Adrenergic innervation of the large arteries and veins of the semiarboreal rat snake Elaphe obsoleta

Fluorescence histochemistry was used to study the adrenergic innervation of the large arteries and veins at six points along the body of the semiarboreal rat snake Elaphe obsoleta. Apart from the vessels adjacent to the heart, there was a marked contrast in the density of adrenergic innervation of anterior and posterior systemic arteries and veins. The anterior arteries and veins have little adrenergic innervation in contrast to the extremely dense innervation of the arteries and veins posterior to the heart. The innervation pattern is consistent with known physiological adjustments to gravity and suggests a mechanism for regulating dependent blood flow via sympathetic nerves. In comparison to the posterior systemic arteries, parallel segments of pulmonary artery taken from the same body position of Elaphe contained a much sparser innervation by adrenergic nerves. The sparser innervation can be correlated with less gravitational disturbance in the pulmonary artery, which is relatively short in this and in other arboreal snakes.     

The cardiac innervation of a marsupial heterotherm,the fat-tailed dunnart (Sminthopsis crassicaudata)

This study investigated the pattern of autonomic innervation of the heart of the fat-tailed dunnart (Sminthopsis crassicaudata) using isolated cardiac preparations. While the pattern of autonomic innervation of the atria was consistent with that found in other mammals, the ventricles displayed an unusual pattern of mammalian cardiac innervation. Transmural stimulation of the intramural nerves of isolated right ventricular preparations caused a decrease in the force of contraction of 46.8+/-3.2% followed by a rebound increase in the force of contraction beyond basal levels of 40.9+/-6.9%. These responses could be blocked independently by the application of the muscarinic receptor antagonist hyoscine and beta-adrenoreceptor antagonist propranolol respectively and could also be mimicked by the application of the agonists acetylcholine (Ach) and noradrenaline (NA). These findings indicated the presence of a functional cholinergic innervation of the ventricles that was capable of reducing the force of contraction below basal levels. This pattern of innervation has only been found previously in one other mammal, the bent-winged bat (Miniopterus schreibersii). Given that both of these species are heterotherms, it is possible that such a pattern of innervation may relate to the control of cardiac output during torpor. These findings are the first that demonstrate the homogeneity of a physiological control mechanism in a so-called 'shallow, daily torpidator' (S. crassicaudata) and a 'deep hibernator' (M. schreibersii) that is absent in mammalian homeotherms. These findings are consistent with recent work suggesting that there may be little difference between these types of heterothermy.     

Demonstration of choline acetyltransferase of a peripheral type in the rat heart.

Cholinergic innervation of the heart has been analyzed using cholinergic markers including acetylcholinesterase, choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT). In the present study we demonstrate putative cholinergic nerves in the rat heart using an antibody to ChAT of a peripheral type (pChAT), which is the product of a splice variant of ChAT mRNA and preferentially localized to peripheral cholinergic nerves. Expression of mRNAs for pChAT and the conventional form of ChAT (cChAT) were verified in the rat atrium by RT-PCR. Localization of both protein products in the atrium was confirmed by Western blotting. Virtually all neurons and small intensely fluorescent cells in the intrinsic cardiac ganglia were stained immunohistochemically for pChAT. The density of pChAT-positive fibers was very high in the conducting system, high in both atria, the right atrium in particular, and low in the ventricular walls. pChAT and VAChT immunoreactivities were closely associated in some fibers and fiber bundles in the ventricular walls. These results indicate that intrinsic cardiac neurons homogeneously express both pChAT and cChAT. Furthermore, innervation of the ventricular walls by pChAT- and VAChT-positive fibers provides morphological evidence for a significant role of cholinergic mechanisms in ventricular functions.     

Intraclass diversification of immunoglobulin heavy chain genes in the African lungfish

Lungfish (Dipnoi) are the closest living relatives to tetrapods, and they represent the transition from water to land during vertebrate evolution. Lungfish are armed with immunoglobulins (Igs), one of the hallmarks of the adaptive immune system of jawed vertebrates, but only three Ig forms have been characterized in Dipnoi to date. We report here a new diversity of Ig molecules in two African lungfish species (Protopterus dolloi and Protopterus annectens). The African lungfish Igs consist of three IgMs, two IgWs, three IgNs, and an IgQ, where both IgN and IgQ originated evidently from the IgW lineage. Our data also suggest that the IgH genes in the lungfish are organized in a transiting form from clusters (IgH loci in cartilaginous fish) to a translocon configuration (IgH locus in tetrapods). We propose that the intraclass diversification of the two primordial gnathostome Ig classes (IgM and IgW) as well as acquisition of new isotypes (IgN and IgQ) has allowed lungfish to acquire a complex and functionally diverse Ig repertoire to fight a variety of microorganisms. Furthermore, our results support the idea that “tetrapod-specific” Ig classes did not evolve until the vertebrate adaptation to land was completed ~360 million years ago.     

Distribution of vasoactive intestinal polypeptide-like immunoreactivity in the mammalian heart

Summary The distribution of vasoactive intestinal polypeptide (VIP) immunoreactivity has been studied in the mammalian heart and compared with that of neurotensin and substance P by use of light-microscopic peroxidase-antiperoxidase immunohistochemistry. VIP-immunoreactive cell bodies are present in intracardiac ganglia in various locations. VIP-immunoreactive nerve fibers predominate in the atria and the conduction system but are rare in the ventricles and occur in cardiac ganglia, endocardium, and epicardium. VIP-ergic nerves supply the coronary vasculature having a preference for the microvasculature and the nodal cells of the sinuatrial node. The large vessels of the heart and periarterial cardiac glomera also receive a VIP-immunoreactive nerve supply. There is partial co-distribution with neurotensin- and substance P-immunoreactive nerve fibers but no co-location in identical nerve fibers is detectable. The VIP-ergic cardiac innervation, which is probably predominantly intrinsic, may stem from postganglionic parasympathetic neurons and is less substantial than the more homogeneous neurotensin-ergic and substance P-ergic nervous supply which is probably extrinsic. The occurrence of an extrinsic VIP-ergic cardiac innervation cannot be excluded however. The differential histotopography of the multitarget cardiac nerves containing the cardiovascular active peptides VIP, neurotensin and substance P may suggest multiple and complex peptide-peptide and peptide-classical transmitter interactions. These may contribute to the regulation of various cardiac functions.     

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.     

Targeting neuronal dysfunction and receptor imaging

The sympathetic nervous system has great influence on cardiovascular physiology, and the importance of cardiac innervation abnormalities in the physiopathology of various cardiac diseases has been emphasized. Cardiac neurotransmission imaging with single-photon emission computed tomography (SPECT) allows in vivo assessment of the myocardial nervous system. At present, the most commonly used SPECT tracer to assess cardiac neurotransmission is metaiodobenzylguanidine labelled with iodine-123 ((123)I-MIBG). In patients with heart transplantation, ischemic heart disease, dysautonomias and drug-induced cardiotoxicity, assessment of neuronal function can help characterise the disease and improve the prognostic stratification. Cardiac (123)I-MIBG scintigraphy allows autonomic neuropathy to be detected in the early stages of diabetes mellitus. In patients with heart failure, the assessment of cardiac sympathetic activity has important prognostic implications. Future directions in cardiac sympathetic neurotransmission include the development of new tracers, targeting of second-messenger molecules and early assessment of cardiac neurotransmission in genetically predisposed subjects for prevention of heart failure.     

Monoamine storage in relation to cardiac regulation in the port jackson shark heterodontus portusjacksoni

Summary A study has been made of catecholamine stores that may be involved in cardiac regulation in the shark Heterodontus portusjacksoni. The anatomy of the anterior chromaffin bodies associated with the sympathetic chain is described. A fluoresent histochemical study shows that the chromaffin cells contain a monoamine, probably noradrenaline. The chromaffin cells have a fine structure comparable to that of chromaffin cells in other vertebrates. The heart is devoid of histochemically-demonstrable chromaffin cells or adrenergic nerve fibres, with the exception of a very sparse adrenergic innervation of the sinus venosus. It is argued that adrenergic control of the heart in Heterodontus might occur via amines released from the anterior chromaffin masses into the blood in the posterior cardinal sinus, which is then aspirated directly into the heart.     

Evolution of tissue-specific keratins as deduced from novel cDNA sequences of the lungfish Protopterus aethiopicus

Lungfishes are possibly the closest extant relatives of the land vertebrates (tetrapods). We report here the cDNA and predicted amino acid sequences of 13 different keratins (ten type I and three type II) of the lungfish Protopterus aethiopicus. These keratins include the orthologs of human K8 and K18. The lungfish keratins were also identified in tissue extracts using two-dimensional polyacrylamide gel electrophoresis, keratin blot binding assays and immunoblotting. The identified keratin spots were analyzed by peptide mass fingerprinting which assigned seven sequences (inclusively Protopterus K8 and K18) to their respective protein spot. The peptide mass fingerprints also revealed the fact that the major epidermal type I and type II keratins of this lungfish have not yet been sequenced. Nevertheless, phylogenetic trees constructed from multiple sequence alignments of keratins from lungfish and distantly related vertebrates such as lamprey, shark, trout, frog, and human reveal new insights into the evolution of K8 and K18, and unravel a variety of independent keratin radiation events.