A perspective on the role of natriuretic peptides in amphibian osmoregulation

The natriuretic peptide (NP) system is a complex family of peptides and receptors that is primarily linked to the maintenance of osmotic and cardiovascular homeostasis. In amphibians, the potential role(s) of NPs is complicated by the range of osmoregulatory strategies found in amphibians, and the different tissues that participate in osmoregulation. Atrial NP, brain NP, and C-type NP have been isolated or cloned from a number of species, which has enabled physiological studies to be performed with homologous peptides. In addition, three types of NP receptors have been cloned and partially characterised. Natriuretic peptides are always potent vasodilators in amphibian blood vessels, and ANP has been shown to increase the permeability of the microcirculation. In the perfused kidney, ANP causes vasodilation, diuresis and natriuresis that are caused by an increased GFR rather than effects in the renal tubules. These data are supported by the presence of ANP receptors only on the glomeruli and renal blood vessels. In the bladder and skin, the function of NPs is enigmatic because physiological analysis of the effects of ANP on bladder and skin function has yielded conflicting data with no clear role for NPs being revealed. Overall, NPs often have no direct effect, but in some studies they have been shown to inhibit the function of AVT. In addition, there is evidence that ANP can inhibit salt retention in amphibians since it can inhibit the ability of adrenocorticotrophic hormone or angiotensin II to stimulate corticosteroid secretion. It is proposed that an important role for cardiac NPs could be in the control of hypervolaemia during periods of rapid rehydration, which occurs in terrestrial amphibians.

Osmoregulation in elephant fish Callorhinchus Milii (Holocephali), with special reference to the rectal gland

Osmoregulatory mechanisms in holocephalan fishes are poorly understood except that these fish are known to conduct urea-based osmoregulation as in elasmobranchs. We, therefore, examined changes in plasma parameters of elephant fish Callorhinchus milii, after gradual transfer to concentrated (120%) or diluted (80%) seawater (SW). In control fish, plasma Na and urea concentrations were about 300 mmol l^–1 and 450 mmol l^–1, respectively. These values were equivalent to those of sharks and rays, but the plasma urea concentration of elephant fish was considerably higher than that reported for chimaeras, another holocephalan. After transfer to 120% SW, plasma osmolality, urea and ion concentrations were increased, whereas transfer to 80% SW resulted in a fall in these parameters. The rises in ion concentrations were notable after transfer to 120% SW, whereas urea concentration decreased predominantly following transfer to 80% SW. In elephant fish, we could not find a discrete rectal gland. Instead, approximately 10 tubular structures were located in the wall of post-valvular intestine. Each tubular structure was composed of a putative salt-secreting component consisting of a single-layered columnar epithelium, which was stained with an anti-Na⁺,K⁺-ATPase serum. Furthermore, Na⁺,K⁺-ATPase activity in the tubular structures was significantly increased after acute transfer of fish to concentrated SW (115%). These results suggest that the tubular structures are a rectal gland equivalent, functioning as a salt-secreting organ. Since the rectal gland of elephant fish is well developed compared to that of Southern chimaera, the salt-secreting ability may be higher in elephant fish than chimaeras, which may account for the lower plasma NaCl concentration in elephant fish compared to other chimaeras. Since elephant fish have also attracted attention from a viewpoint of genome science, the availability of fish for physiological studies will make this species an excellent model in holocephalan fish group.

Osmoregulation in elephant fish, CALLORHYNCHUS MILLII (HOLOCEPHALI)

Osmoregulatory mechanisms in holocephalan fishes are unknown except that they conduct urea-based osmoregulation as in elasmobranchs. We, therefore, examined changes in plasma parameters of elephant fish, Callorhynchus millii, after gradual transfer to concentrated (120%) or diluted (80%) seawater (SW). In control fish, plasma Na and urea concentrations were about 300mM and 450mM, respectively. These values were equivalent to those of sharks and rays, but the plasma urea concentration of elephant fish was considerably higher than that reported for chimaeras, another holocephalan. After transfer to 120% SW, the plasma Na concentration markedly increased, while a conspicuous decrease in plasmaurea concentration was observed following transfer to 80% SW. In elephant fish, we could not find a discrete rectal gland. Instead, approximately 10 tubular structures were located in the wall of post-valvular intestine. Each tubular structure was composed of a putative salt-secreting component consisting of a single-layered columnar epithelium, which was stained with anti-Na+,K±ATPase serum. It is most likely that the tubular structures in the posterior intestine represent a primitive form of the rectal gland in elephant fish. In addition, we have identified two C-type natriuretic peptides (CNPs) from the heart and brain of elephant fish, which may contribute to the control of NaCl excretion from the rectal gland of elephant fish as it does in elasmobranchs.

Urea-based osmoregulation in the developing embryo of oviparous cartilaginous fish (Callorhinchus milii) : contribution of the extraembryonic yolk sac during the early developmental period

Marine cartilaginous fish retain a high concentration of urea to maintain the plasma slightly hyperosmotic to the surrounding seawater. In adult fish, urea is produced by hepatic and extrahepatic ornithine urea cycles (OUCs). However, little is known about the urea retention mechanism in developing cartilaginous fish embryos. In order to address the question as to the mechanism of urea-based osmoregulation in developing embryos, the present study examined the gene expression profiles of OUC enzymes in oviparous holocephalan elephant fish (Callorhinchus milii) embryos. We found that the yolk sac membrane (YSM) makes an important contribution to the ureosmotic strategy of the early embryonic period. The expression of OUC enzyme genes was detectable in the embryonic body from at least stage 28, and increased markedly during development to hatching, which is most probably due to growth of the liver. During the early developmental period, however, the expression of OUC enzyme genes was not prominent in the embryonic body. Meanwhile, we found that the mRNA expression of OUC enzymes was detected in the extra-embryonic YSM; the mRNA expression of cmcpsIII in the YSM was much higher than that in the embryonic body during stages 28-31. Significant levels of enzyme activity and the existence of mitochondrial-type cmgs1 transcripts in the YSM supported the mRNA findings. We also found that the cmcpsIII transcript is localized in the vascularized inner layer of the YSM. Taken together, our findings demonstrate for the first time that the YSM is involved in urea-based osmoregulation during the early to mid phase of development in oviparous cartilaginous fish.

Microfluidic platforms for the investigation of intercellular signalling mechanisms

Intercellular signalling has been identified as a highly complex process, responsible for orchestrating many physiological functions. While conventional methods of investigation have been useful, their limitations are impeding further development. Microfluidics offers an opportunity to overcome some of these limitations. Most notably, microfluidic systems can emulate the in-vivo environments. Further, they enable exceptionally precise control of the microenvironment, allowing complex mechanisms to be selectively isolated and studied in detail. There has thus been a growing adoption of microfluidic platforms for investigation of cell signalling mechanisms. This review provides an overview of the different signalling mechanisms and discusses the methods used to study them, with a focus on the microfluidic devices developed for this purpose.

Functional analysis of natriuretic peptide receptors in the bladder of the toad, bufo marinus

This study aimed to localize and characterize natriuretic peptide binding sites in the urinary bladder of Bufo marinus and to then examine the effect of natriuretic peptides on the bladder vascular tone and water reabsorption in isolated perfused bladder preparations. Specific ¹²⁵I-rat atrial natriuretic peptide (¹²⁵I-rANP) binding sites were present on blood vessels, muscle, and epithelium. In tissue sections and/or isolated membranes, the binding was completely displaced by frog ANP, rat ANP, and porcine C-type natriuretic peptide (CNP; membranes only). However, a reduction in binding was observed after incubation with ¹²⁵I-rANP and 1 μM of the natriuretic peptide receptor-C (NPR-C) ligand C-ANF, but residual binding remained suggesting the presence of two distinct binding sites. Electrophoresis of bladder membranes cross-linked to ¹²⁵I-rANP identified two bands at approximately 70 and 140 kDa that correspond to the monomeric mass of NPR-C and the guanylate cyclase receptors, respectively. Furthermore, the presence of natriuretic peptide receptor-A and NPR-C mRNA in the bladder was demonstrated with reverse transcription–polymerase chain reaction. In addition, rat ANP, frog ANP, and porcine CNP stimulated a significant increase in cGMP generation in bladder membrane preparations, which indicated the presence of guanylate cyclase-linked receptors. In perfused bladder preparations, arginine vasotocin increased perfusion pressure and water permeability. The infusion of frog ANP or porcine CNP failed to alter perfusion pressure or water reabsorption in the presence or absence of arginine vasotocin. This study identified a well-developed natriuretic peptide receptor system in the urinary bladder of B. marinus but the function of the receptors remains unclear.

Ecotypic variation in the response of Arabidopsis thaliana to Phytophthora cinnamomi

A variety of reactions to inoculation with Phytophthora cinnamomi ranging from high susceptibility to moderate resistance were found in 20 ecotypes of Arabidopsis thaliana. P. cinnamomi zoospores successfully colonised both root and leaf tissue of Arabidopsis and sporulation in the form of chlamydospores and sporangia occurred in leaves and roots of each ecotype but the number varied considerably between ecotypes. In the more susceptible ecotypes, colonisation was characterised by rapid intercellular growth and sporulation of the pathogen from 48 h post inoculation. In less susceptible ecotypes, P. cinnamomi was limited to a defined region within tissues. In response to P. cinnamomi infection, several ecotypes expressed active defence responses in both root and leaf tissue. Callose formation was closely associated with lesion restriction as was the production of the reactive oxygen species, hydrogen peroxide. The oxidative burst was not limited to the site of pathogen ingress but also occurred in distant, uninfected tissues. We have characterised an Arabidopsis–P. cinnamomi system that will be useful for further studies of active resistance mechanisms.

Comparative aspects of natriuretic peptide physiology in non-mammalian vertebrates: a review

The natriuretic peptide system is a complex family of peptides and receptors that is primarily linked to the maintenance of osmotic and cardiovascular homeostasis. A natriuretic peptide system is present in each vertebrate class but there are varying degrees of complexity in the system. In agnathans and chondrichthyians, only one natriuretic peptide has been identified, while new data has revealed that multiple types of natriuretic peptides are present in bony fish. However, it seems in tetrapods that there has been a reduction in the number of natriuretic peptide genes, such that only three natriuretic peptides are present in mammals. The peptides act via a family of guanylyl cyclase receptors to generate the second messenger cGMP, which  mediates a range of physiological effects at key targets such as the gills, kidney and the cardiovascular system. This review summarises the current knowledge of the natriuretic peptide system in non-mammalian vertebrates and discusses the physiological actions of the peptides.

Natriuretic peptide guanylyl cyclase receptors in the kidney of the Japanese eel, Anguilla japonica

Natriuretic peptides are linked to osmoregulation, cardiovascular and volume regulation in fishes. The peptides bind to two guanylyl-cyclase-linked receptors, natriuretic peptide receptor-A (NPR-A) and NPR-B, to elicit their effects. Atrial natriuretic peptide (ANP) binds principally to NPR-A, whereas C-type natriuretic peptide (CNP) binds to NPR-B. The teleost kidney has an important role in the maintenance of fluid and electrolyte balance; therefore, the location of NPR-A and NPR-B in the kidney could provide insights into the functions of natriuretic peptides. This study used homologous, affinity purified, polyclonal antibodies to NPR-A and NPR-B to determine their location in the kidney of the Japanese eel, Anguilla japonica. Kidneys from freshwater and seawater acclimated animals were fixed overnight in 4% paraformaldehyde before being paraffin-embedded and immunostained. NPR-A immunoreactivity was found on the apical membrane of proximal tubule 1 and the vascular endothelium including the glomerular capillaries. In contrast, NPR-B immunoreactivity was located on the smooth muscle of blood vessels including the glomerular afferent and efferent arterioles, and on smooth muscle tissue surrounding the collecting ducts. No difference in the distribution of NPR-A and NPR-B was observed between freshwater and seawater kidneys. Immunoreactivity was not observed in any tissue in which the antibodies had been preabsorbed. In addition, there was no difference in NPR-A and NPR-B mRNA expression between freshwater-acclimated and seawater-acclimated eels. These results suggest that, although utilizing the same second messenger system, ANP and CNP act on different targets within the kidney and presumably elicit different effects.

Impaired expression of notch signaling genes in aged human skeletal muscle

Notch signaling is essential for myogenesis and the regenerative potential of skeletal muscle: however, its regulation in human muscle is yet to be fully characterized. Increased expression of Notch3, Jagged1. Hes1, and Hes6 gene transcripts were observed during differentiation of cultured human skeletal muscle cells. Furthermore, significantly lower expressions of Notch1, Jagged1, Numb, and Delta-like 1 were evident in muscle biopsies from older men (60-75 years old) compared to muscle from younger men (18-25 years old). Importantly, with supervised resistance exercise training, expression of Notch1 and Hes6 genes were increased and Delta-like 1 and Numb expression were decreased. The differences in Notch expression between the age groups were no longer evident following training. These results provide further evidence to support the role of Notch in the impaired regulation of muscle mass with age and suggest that some of the benefits provided by resistance training may be mediated through the Notch signaling pathway.

Identification and expression of natriuretic peptide receptor Type-A and-B mRNA in freshwater and seawater rainbow trout

Natriuretic peptide receptors mediate the physiological response of  natriuretic peptide hormones. One of the natriuretic peptide receptor types is the particulate guanylyl cyclase receptors, of which there are two identified: NPR-A and NPR-B. In fishes, these have been sequenced and characterized in eels, medaka, and dogfish shark (NPR-B only). The euryhaline rainbow trout provides an opportunity to further pursue examination of the system in teleosts. In this study, partial rainbow trout NPR-A-like and NPR-B-like mRNA sequences were identified via PCR and cloning. The sequence information was used in real-time PCR to examine mRNA expression in a variety of tissues of freshwater rainbow trout and rainbow trout acclimated to 35 parts per thousand seawater for a period of 10 days. In the excretory kidney and posterior intestine, real-time PCR analysis showed greater expression of NPR-B in freshwater fish than in those adapted to seawater; otherwise, there was no difference in the expression of the individual receptors in fresh water or seawater. In general, the expression of the NPR-A and NPR-B type receptors was quite low. These findings indicate that NPR-A and NPR-B mRNA expression is minimally altered under the experimental regime used in this study.

Immunolocalization of Na+/K+-ATPase, carbonic anhydrase II, and vacuolar H+-ATPase in the gills of freshwater adult lampreys, Geotria australis

As adults, anadromous lampreys migrate from seawater into freshwater rivers, where they require branchial ion (NaCl) absorption for osmoregulation. In teleosts and elasmobranchs, pharmological, immunohistochemical, and molecular data support roles for Na⁺/K⁺-ATPase (NPPase), carbonic anhydrase II (CAII), and vacuolar H⁺-ATPase (V-ATPase) in two different models of branchial ion absorption. To our knowledge, these transport-related proteins have not been studied in adult freshwater lampreys, and therefore it is not known if they are expressed, or have similar functions, in lampreys. The purpose of this study was to localize NPPase, CAII, and V-ATPase in the gills of adult freshwater lampreys and determine if any of these transport-related proteins are expressed in the same cells. Heterologous antibodies were used to localize the three proteins in gill tissue from pouched lamprey (Geotria australis). Immunoreactivity (IR) for all three proteins occurred between, and at the base of, lamellae in cells that match previous descriptions of mitochondrion-rich-cells (MRCs). NPPase-IR was always on the basolateral side of cells that did not stain for CAII or V-ATPase. In contrast, CAII-IR was always on the apical side of cells that also contained diffuse V-ATPase-IR. Therefore, we have identified two types of MRC in adult freshwater lamprey gills based on immunohistochemical staining for three transport proteins. A model of ion transport, based on our results, is proposed for adult freshwater lampreys. 

Potassium phosphonate alters the defence response of Xanthorrhoea australis following infection by Phytophthora cinnamomi

Potassium phosphonate (phosphite) is widely used in the management of Phytophthora diseases in agriculture, horticulture and natural environments. The Austral grass tree, Xanthorrhoea australis, a keystone species in the dry sclerophyll forests of southern Australia, is susceptible to Phytophthora cinnamomi, but is protected by applications of phosphite. We examined the effect of phosphite application on the infection of X. australis seedlings and cell suspension cultures by zoospores of P. cinnamomi. Phosphite induced more intense cellular responses to pathogen challenge and suppressed pathogen ingress in both seedlings and cell cultures. In untreated X. australis seedlings, hyphal growth was initially intercellular, became intracellular 24 h after inoculation, and by 48 h had progressed into the vascular tissue. In phosphite-treated seedlings, growth of P. cinnamomi remained intercellular and was limited to the cortex, even at 72 h after inoculation. The cell membrane retracted from the cell wall and phenolic compounds and electron dense substances were deposited around the wall of infected and neighbouring cells. Suspension cells were infected within 6 h of inoculation. Within 24 h of inoculation, untreated cells were fully colonised, had collapsed cytoplasm and died. The protoplast of phosphite-treated suspension cells collapsed within 12 h of inoculation, and phenolic material accumulated in adjacent, uninfected cells. No anatomical response to phosphite treatment was observed before infection of plant tissues, suggesting that the phosphite-associated host defence response is induced following pathogen challenge. Anatomical changes provide evidence that phosphite stimulates the host defence system to respond more effectively to pathogen invasion.

Amoebic gill disease and natriuretic peptide receptors in the gills of Atlantic salmon

Amoebic gill disease (AGD) is a problem in the farming of Atlantic salmon, and may compromise osmoregulatory, cardiovascular and respiratory functions. We examined the effects of AGD on atrial and C-type natriuretic peptide (ANP and CNP) stimulated branchial cyclic GMP formation, since natriuretic peptides (NPs) are involved in cardiovascular function and osmoregulation. NPs act via guanylyl cyclase receptors (NPR), which stimulate cGMP formation. NPR activity was measured by ANP and CNP stimulation of branchial cGMP formation, and compared between diseased and healthy salmon over an 11 day AGD infection. We also measured plasma osmolality. Osmolality increased in AGD infected salmon from an initial 355 mmol.kg-1 to 411 mmol.kg-1 at 11 days. There was no evidence that branchial cGMP formation changed in response to AGD. In all groups, CNP stimulation of guanylyl cyclase was 190% of basal rate, whereas ANP was 150% of basal. After 11 days, all groups were given a 4 h freshwater bath, the usual treatment for AGD. Another group was given a seawater to seawater transfer, to control for handling. In this group, plasma osmolality at 11 days was the same as in AGD fish. This elevation may be due to these fish experiencing disturbance for the first time in 11 days. ANP and CNP branchial NPR activity at the conclusion of the 4 h transfers was elevated in all groups compared to that at 11 days. The increased cGMP formation in the handling control suggests a NPR response to the transfer/handling stress. AGD fish demonstrated the greatest elevation in ANP and CNP guanylyl cyclase activity immediately following the bath; these values were greater than in the control groups. The AGD infected salmon, therefore, responded more emphatically to the freshwater treatment, suggesting that the NP system is involved in some aspects of AGD.

Osmoregulatory balance in Murray cod, Maccullochella peelii peelii (Mitchell), affected with chronic ulcerative dermatopathy

This study examined the osmoregulatory capability of Murray cod, Maccullochella peelii peelii, affected by chronic ulcerative dermatopathy (CUD) in intensive aquaculture. This condition appears to arise only in facilities utilizing groundwater, with the causative agent suggested to be a water-borne factor. Healthy Murray cod (~ 700 g) were transferred to a CUD-affected farm to monitor the progression of the syndrome and began to show signs of CUD after approximately five months. In order to evaluate possible effects of CUD on osmoregulation; plasma electrolyte concentrations, osmolality, and Na⁺,K⁺-ATPase activities were measured, and gill histology and immunohistochemistry were analyzed. Plasma electrolyte concentrations and osmolality of CUD-affected Murray cod were consistent with reference values determined for non CUD-affected fish. A greater number of gill mucous cells were observed in Murray cod cultured at the CUD-affected farm compared to non CUD-affected fish. We also found an un-identified cell type that was present solely in the gills of CUD-affected Murray cod. Gill Na⁺,K⁺-ATPase activity was significantly higher in severely CUD-affected Murray cod compared to individuals transferred to the CUD-affected farm. While there appeared to be some minor changes in the gills of CUD-affected fish, this study demonstrated that Murray cod were able to effectively osmoregulate, although, perhaps at an energetic cost.