The effect of water deprivation on signalling molecules that utilise cGMP in the Spinifex Hopping Mouse Notomys alexis

In mammals the natriuretic and guanylin peptides influence renal and intestinal fluid content and electrolyte transport by binding to and activating guanylyl cyclase (GC) receptors that in turn stimulate production of the intracellular second messenger guanosine 3':5'-cyclic monophospate
(cGMP). However, the role of natriuretic and guanylin peptides in desert mammals is not understood. The spinifex hopping-mouse (Notomys alexis), has a suite of behavioural and physiological mechanisms that permits survival for extended periods without access to free water. Because signalling molecules that generate cGMP are known to promote water excretion, it was predicted that natriuretic and guanylin peptide synthesis would be down regulated in water-deprived N. alexis, and thus reduce the amount of water lost in the urine and faeces. However, in the kidney ANP and GC-A mRNA levels were increased in water-deprived mice, but CNP and GC-B mRNA levels were decreased. Water deprivation increased guanylin and uroguanylin mRNA expression in the distal colon, but it remained unchanged in the kidney and proximal colon. The expression of GC-C mRNA increased in the proximal colon but not in the distal colon. This study shows that water deprivation differentially affects the expression of regulatory molecules that stimulate cGMP producti

Nitric oxide control of large veins in the toad Bufo marinus

This study examined the nitric oxide (NO) control of the vascular smooth muscle of the ventral abdominal vein and vena cava of the toad, Bufo marinus, by using anatomical and physiological approaches. Nicotinamide adenine di-nucleotide phosphate-diaphorase histochemistry and immunohistochemistry using endothelial nitric oxide synthase (NOS) and neural NOS antibodies produced no evidence for endothelial NOS in the veins, but, neural NOS-immunoreactive perivascular nerves were present. Acetylcholine (10^–5 M) caused a vasodilation in both veins that was endothelium-independent, and which was blocked by the soluble guanylyl cyclase inhibitor, ODQ (10^–5 M). The NOS inhibitors, L-NNA (10^–4 M) and L-NAME (10^–4 M), did not significantly reduce the vasodilatory effect of acetylcholine in the veins; this suggested that the vasodilation was not due to NO. However, in the presence of phenoxybenzamine (10^–7–10^–8 M), L-NNA significantly reduced the vasodilatory effect of acetylcholine in the veins. This unusual response is due to phenoxybenzamine partially inactivating the muscarinic receptor pool in the veins. In addition, the neural NOS inhibitor, vinyl-L-NIO (10^–5 M), significantly reduced the acetylcholine-mediated vasodilation in the presence of phenoxybenzamine. The results show that in toad veins, nitrergic nerves rather than an endothelial NO system are involved in NO-mediated vasodilation.

The response of the natriuretic peptide system to water deprivation in the desert rodent, Notomys alexis

Natriuretic peptides (NPs) are regulatory molecules that cause cGMP-mediated diuresis and natriuresis in mammals. Accordingly, it is interesting to consider their role in desert-adapted animals in which water is often limited. This study investigated the response of the natriuretic peptide (NP) system to varying periods of water deprivation (WD) in the Australian desert rodent species, Notomys alexis. It was hypothesised that the expression of the NP system will be down-regulated in water-deprived N. alexis compared to water-replete animals. The plasma levels of ANP were significantly reduced after 3 days of WD, but were unaffected by 7, 14 and 28 days of WD. Water deprivation for 3, 7, 14 days had a variable effect on the mRNA expression of ANP, CNP, NPR-A, NPR-B, and NPR-C, and a uniform down-regulation was not observed. However, after 28 days of WD, mRNA expression was similar to water-replete animals, except for NPR-A. Surprisingly, 7 and 14 days of WD caused an up-regulation in the ability of ANP to stimulate cGMP; this also occurred at 14 days for CNP. Taken together, the mRNA expression and peptide mediated guanylyl cyclase activity data after WD were in the opposite direction to what was predicted. Interestingly, after 28 days of WD, most parameters were similar to those of water-replete animals, which indicates that a down-regulation of the NP system is not part of the physiological response to an absence of free water in N. alexis.

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.

Plant natriuretic peptide active site determination and effects on cGMP and cell volume regulation

Natriuretic peptides (NP) were first identified in animals where they play a role in the regulation of salt and water balance. This regulation is partly mediated by intracellular changes in cyclic GMP (cGMP). NP immunoanalogues occur in many plants and have been isolated, with two NP encoding genes characterised in Arabidopsis thaliana L. (AtPNP-A and AtPNP-B). Part of AtPNP-A contains the region with homology to human atrial (A)NP. We report here on the effects of recombinant AtPNP-A and smaller synthetic peptides within the ANP-homologous region with a view to identifying the biologically active domain of the molecule. Furthermore, we investigated interactions between AtPNP-A and the hormone, abscisic acid (ABA). ABA does not significantly affect Arabidopsis mesophyll protoplast volume regulation, whereas AtPNP-A and synthetic peptides promote water uptake into the protoplasts causing swelling. This effect is promoted by the membrane permeable cGMP analogue, 8-Br-cGMP, and inhibited by guanylate cyclase inhibitors indicating that increases in cGMP are an essential component of the plant natriuretic peptides (PNP) signalling cascade. ABA does not induce cGMP transients and does not affect AtPNP-A dependent cGMP increases, hence the two regulators differ in their second messenger signatures. Interestingly, AtPNP-A significantly delays and reduces the extent of ABA stimulated stomatal closure that is also based on cell volume regulation. We conclude that a complex interplay between observed PNP effects (stomatal opening and protoplast swelling) and ABA is likely to be cell type specific.

Mechanisms of vasodilation in the dorsal aorta of the elephant fish, Callorhinchus milii (Chimaeriformes: Holocephali)

This study investigated vasodilator mechanisms in the dorsal aorta of the elephant fish, Callorhinchus milii, using anatomical and physiological approaches. Nitric oxide synthase could only be located in the perivascular nerve fibres and not the endothelium of the dorsal aorta, using NADPH histochemistry and immunohistochemistry. In vitro organ bath experiments demonstrated that a NO/soluble guanylyl cyclase (GC) system appeared to be absent in the vascular smooth muscle, since the NO donors SNP (10⁻⁴ mol l⁻¹) and SIN^-1 (10⁻⁵ mol l⁻¹) were without effect. Nicotine (3 × 10⁻⁴ mol l⁻¹) mediated a vasodilation that was not affected by ODQ (10⁻⁵ mol l⁻¹), _l-NNA (10⁻⁴ mol l⁻¹), indomethacin (10⁻⁵ mol l⁻¹), or removal of the endothelium. In contrast, the voltage-gated sodium channel inhibitor, tetrodotoxin (10⁻⁵ mol l⁻¹), significantly decreased the dilation induced by nicotine, suggesting that it contained a neural component. Pre-incubation of the dorsal aorta with the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP_8–37 (10⁻⁶ mol l^−  1) also caused a significant decrease in the nicotine-induced dilation. We propose that nicotine is mediating a neurally-derived vasodilation in the dorsal aorta that is independent of NO, prostaglandins and the endothelium, and partly mediated by CGRP.

Dual mechanisms for nitric oxide control of large arteries in the estuarine crocodile crocodylus porosus

n reptiles, accumulating evidence suggests that nitric oxide (NO) induces a potent relaxation in the systemic vasculature. However, very few studies have examined the source from which NO is derived. Therefore, the present study used both anatomical and physiological approaches to establish whether NO-mediated vasodilation is via an endothelial or neural NO pathway in the large arteries of the estuarine crocodile Crocodylus porosus. Specific endothelial nitric oxide synthase (NOS) staining was observed in aortic endothelial cells following nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and endothelial NOS immunohistochemistry (IHC), suggesting that an endothelial NO pathway is involved in vascular control. This finding was supported by in vitro organ bath physiology, which demonstrated that the relaxation induced by acetylcholine (10-5 mol l-1) was abolished in the presence of the NOS inhibitor, N-omega-nitro-L-arginine (L-NNA; 10-4 mol l-1), the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10-5 mol l-1), or when the endothelium was removed. Interestingly, evidence for a neural NO pathway was also identified in large arteries of the crocodile. Neural NOS was located in perivascular nerves of the major blood vessels following NADPH-d histochemistry and neural NOS IHC and in isolated aortic rings, L-NNA and ODQ, but not the removal of the endothelium, abolished the relaxation effect of the neural NOS agonist, nicotine (3x10-4 mol l-1). Thus, we conclude that the large arteries of C. porosus are potentially regulated by NO-derived from both endothelial and neural NOS.

Cyclic GMP modulates stomatal opening induced by natriuretic peptides and immunoreactive analogues

In this paper we demonstrate that compounds that promote stomatal opening such as kinetin, atrial natriuretic peptide (ANP) and plant natriuretic peptide immunoanalogues (irPNP) significantly elevate cGMP in guard cell protoplasts. Stomata opened by irPNP are induced to close in the presence of the guanylate cyclase inhibitor, LY 83583. The effect of cGMP on stomatal opening appears to be linked with Ca2+ levels. ANP, irPNP and 8-Br-cGMP all induce stomatal opening and this is inhibited by compounds that lower intracellular Ca2+ levels such as ethylene glycol bis(β-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA), ruthenium red and procaine.

Natriuretic peptide signalling in the gills of freshwater and seawater fishes

The gills are considered major targets for cardiac natriuretic peptides with studies confirming natriuretic peptide receptor presence on vascular and sometimes epithelial tissues. Natriuretic peptide intracellular signalling is via guanylyl cyclase receptors and the cGMP pathway, and via inhibitory G-proteins linked to cyclic AMP pathways. Natriuretic peptides in the gills alter branchial blood flow and may also alter ion transport in various salinities. We present an overview of natriuretic peptide cGMP and cAMP signalling in fishes and consider the implications of the recent discovery of several CNPs and BNP in bony fishes on natriuretic peptide receptor studies.

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.

Neurally-derived nitric oxide regulates vascular tone in pulmonary and cutaneous arteries of the toad, Bufo marinus

In this study, the role of nitric oxide (NO) in regulation of the pulmocutaneous vasculature of the toad, Bufo marinus was investigated. In vitro myography demonstrated the presence of a neural NO signaling mechanism in both arteries. Vasodilation induced by nicotine was inhibited by the soluble guanylyl cyclase (GC) inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, and the NO synthase (NOS) inhibitor, N^ω-nitro-_L-arginine (_L-NNA). Removal of the endothelium had no significant effect on the vasodilation. Furthermore, pretreatment with N⁵-(1-imino-3-butenyl)-_L-ornithine (vinyl-_L-NIO), a more specific inhibitor of neural NOS, caused a significant decrease in the nicotine-induced dilation. In the pulmonary artery only, a combination of _L-NNA and the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP(_8-37), completely blocked the nicotine-induced dilation. In both arteries, the vasodilation was also significantly decreased by glibenclamide, an ATP-sensitive K⁺ (K⁺_ATP) channel inhibitor. Levcromakalim, a K⁺_ATP channel opener, caused a dilation that was blocked by glibenclamide in both arteries. In the pulmonary artery, NO donor-mediated dilation was significantly decreased by pretreatment with glibenclamide. The physiological data were supported by NADPH-diaphorase histochemistry and immunohistochemistry, which demonstrated NOS in perivascular nerve fibers but not the endothelium of the arteries. These results indicate that the pulmonary and cutaneous arteries of B. marinus are regulated by NO from nitrergic nerves rather than NO released from the endothelium. The nitrergic vasodilation in the arteries appears to be caused, in part, via activation of K⁺_ATP channels. Thus, NO could play an important role in determining pulmocutaneous blood flow and the magnitude of cardiac shunting.

Vascular distribution of nitric oxide synthase and vasodilation in the Australian lungfish, Neoceratodus forsteri

The presence of nitric oxide synthase (NOS) and role of nitric oxide (NO) in vascular regulation was investigated in the Australian lungfish, Neoceratodus forsteri. No evidence was found for NOS in the endothelium of large and small blood vessels following processing for NADPH-diaphorase histochemistry. However, both NADPH-diaphorase histochemistry and neural NOS immunohistochemistry demonstrated a sparse network of nitrergic nerves in the dorsal aorta, hepatic artery, and branchial arteries, but there were no nitrergic nerves in small blood vessels in tissues. In contrast, nitrergic nerves were found in non-vascular tissues of the lung, gut and kidney. Dual-wire myography was used to determine if NO signalling occurred in the branchial artery of N. forsteri. Both SNP and SIN-1 had no effect on the pre-constricted branchial artery, but the particulate guanylyl cyclase (GC) activator, C-type natriuretic peptide, always caused vasodilation. Nicotine mediated a dilation that was not inhibited by the soluble GC inhibitor, ODQ, or the NOS inhibitor, L-NNA, but was blocked by the cyclooxygenase inhibitor, indomethacin. These data suggest that NO control of the branchial artery is lacking, but that prostaglandins could be endothelial relaxing factors in the vasculature of lungfish.

Expression of natriuretic peptide receptor mRNA and functional response to atrial natriuretic peptide and C-type natriuretic peptide in rainbow trout (Oncorhynchus mykiss) head kidney leucocytes

The stimulatory effect of vasomodulatory natriuretic peptide hormones on macrophages and peripheral blood leucocytes in mammals is well-established. However, the relationship in lower vertebrates has not been characterised. Expression of atrial natriuretic peptide, ventricular natriuretic peptide and C-type natriuretic peptide-1, and the guanylyl cyclase-linked (GC) natriuretic peptide receptor-A and -B-type receptors (NPR-A and NPR-B, respectively) was determined by PCR from the mRNA of rainbow trout head kidney leucocytes yielding gene fragments with 100% homology to the same respective natriuretic peptide and NPR-A and -B sequences obtained from other rainbow trout tissues. A mixed population of isolated rainbow trout head kidney leucocytes was stimulated in vitro with trout atrial natriuretic peptide (specific NPR-A agonist) and trout C-type natriuretic peptide (NPR-A and -B agonist) as well as the cGMP agonist 8-bromo-cGMP or the GC inhibitor 8-bromo-phenyl-eutheno-cGMP. Respiratory burst was stimulated by trout atrial natriuretic peptide, trout C-type natriuretic peptide-1 and 8-bromo-cGMP in a dose dependant manner with the highest activity as a result of stimulation with trout C-type natriuretic peptide-1 in excess of that achieved by phorbol myristate acetate (PMA). Equimolar concentrations of the inhibitor, inhibited the respiratory burst caused by the natriuretic peptides and 8-bromo-cGMP. The natriuretic peptide receptors on rainbow trout head kidney leucocytes appear to have a stimulatory function with regard to respiratory burst that is activated through a cGMP second messenger pathway and the natriuretic peptides expressed in the head kidney leucocytes may well act in a paracrine/autocrine manner.

GSK3β modulates PACAP-induced neuritogenesis in PC12 cells by acting downstream of Rap1 in a caveolae-dependent manner

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neurotrophic peptide. Here, we show that PACAP recruits Rap1 into caveolin-enriched membrane subdomains in PC12 cells and activates Rap1, nuclear ERK1/2, Elk-1 and CREB in a caveolae-dependent manner. We reveal that GSK3β is a novel modulator in PACAP signalling. PACAP induces phosphorylation of serine 9 in GSK3β, which is inhibited by silencing Rap1. Lithium and valproate promote but wortmannin and LY294002 attenuate PACAP-induced phosphorylation of both GSK3β and ERK1/2, whereas MEK inhibitor PD98059 inhibits nerve growth factor- but not PACAP-induced phosphorylation of GSK3β, suggesting that GSK3β operates downstream of Rapt 1 but upstream of ERK1/2 in PACAP signalling. Inhibition or stimulation of GSK3β results in a 2-fold increase and 6-fold decrease in PACAP-induced neurite outgrowth, respectively. These results reveal an important role of caveolae in the signal transduction of PACAP and that GSK3β is a critical regulator in PACAP-induced neuronal differentiation.

Natriuretic peptides and their receptors in the brain of the amphibian, Bufo marinus

The natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are members of a family of hormones that play an important role in mammalian fluid and electrolyte balance. In the periphery, natriuretic peptides reduce blood volume and subsequently blood pressure by increasing renal natriuresis and diuresis and relaxation of vascular smooth muscle. The actions of natriuretic peptides are mediated via two membrane-linked guanylate cyclase receptors (NPR-GC); natriuretic peptide receptor-A (NPR-A) which has a high affinity for ANP and BNP; and natriuretic peptide receptor-B (NPR-B)which has the greatest affinity for CNP. A third receptor not linked to guanylate cyclase, natriuretic peptide receptor-C (NPR-C) also exists, which binds to ANP, BNP and CNP with a relatively equal affinity, and is involved with clearance of the peptides from the circulation and tissues. The natriuretic peptides are present in the brain and are particularly predominant in cardiovascular and fluid and electrolyte regulating areas such as the anteroventral third ventricle (AV3V) region. This distribution has led to the suggestion natriuretic peptides play a neuromodulatory role in the central control of fluid homeostasis. Natriuretic peptides in the brain have been observed to inhibit the release of other fluid and electrolyte regulating hormones such as arginine vasopressin (AVP) and angiotensin II (AII). Natriuretic peptides have also been identified in the non-mammalian vertebrates although information regarding the distribution of the peptides and their receptors in the non-mammalian brain is limited. In amphibians, immunohistochemical studies have shown that natriuretic peptides are highly concentrated in the preoptic region of the brain, an area believed to be analogous to the A\T3\ region in mammals, which suggests that natriuretic peptides may also be involved in central fluid and electrolyte regulation in amphibians. To date, CNP is the only natriuretic peptide that has been isolated and cloned from the lower vertebrate brain, although studies on the distribution of CNP binding sites in the brain have only been performed in one fish species. Studies on the distribution of ANP binding sites in the lower vertebrate brain are similarly limited and have only been performed in one fish and two amphibian species. Moreover, the nature and distribution of the natriuretic peptide receptors has not been characterised. The current study therefore, used several approaches to investigate the distribution of natriuretic peptides and their receptors in the brain of the amphibian Bufo marinus. The topographical relationship of natriuretic peptides and the fluid and electrolyte regulating hormone arginine vasotocin was also investigated, in order to gain a greater understanding of the role of the natriuretic peptide system in the lower vertebrate brain. Immunohistochemical studies showed natriuretic peptides were distributed throughout the brain and were highly concentrated in the preoptic region and interpeduncular nucleus. No natriuretic peptide-like immunoreactivity (NP-IR) was observed in the pituitary gland. Arginine vasotocin-like immunoreactivity (AvT-IR) was confined to distinct regions, particularly in the preoptic/hypothalamic region and pituitary gland. Double labelling studies of NP-JR and AvT-IR showed the peptides are not colocalised in the same neural pathways. The distribution of natriuretic peptide binding sites using the ligands 125I-rat ANP (125I-rANP) and 125I-porcine CNP (125I-pCNP) showed different distributions in the brain of B. marinus. The specificity of binding was determined by displacement with unlabelled rat ANP, porcine CNP and C-ANF, an NPR-C specific ligand. 125I-rANP binding sites were broadly distributed throughout the brain with the highest concentration in pituitary gland, habenular, medial pallium and olfactory region. Minimal 125I-rANP binding was observed in the preoptic region. Residual 125I-rANP binding in the presence of C-ANF was observed in the olfactory region, habenular and pituitary gland indicating the presence of both NPR-GC and NPR-C in these regions. 125I-pCNP binding was limited to the olfactory region, pallium and posterior pituitary gland. All 125I-pCNP binding was displaced by C-ANF which suggests that CNP in the brain of B. marinus binds only to NPR-C. Affinity cross-linking and SDS-PAGB demonstrated two binding sites at 136 kDa and 65 kDa under reducing conditions. Guanylate cyclase assays showed 0.1 µM ANP increased cGMP levels 50% above basal whilst a 10-fold higher concentration of CNP was required to produce the same result. Molecular cloning studies revealed a 669 base pair fragment showing 91% homology with human and rat NPR-A and 89% homology with human, rat and eel NPR-B. A 432 base pair fragment showing 67% homology to the mammalian NPR-C and 58% homology with eel NPR-D was also obtained. The results show natriuretic peptides and their receptors are distributed throughout the brain of B. marinus which indicates that natriuretic peptides may participate in a range of regulatory functions throughout the brain. The potential for natriuretic peptides to regulate the release of the fluid and electrolyte regulating hormone AVT also exists due to the high number of natriuretic peptide binding sites in the posterior pituitary gland. At least two populations of natriuretic peptide receptors are present in the brain of B. marinus, one linked to guanylate cyclase and one resembling the mammalian clearance receptor. Furthermore, autoradiography and guanylate cyclase studies suggest ANP may be the major ligand in the brain of B. marinus, even though CNP is the only natriuretic peptide that has been isolated from the lower vertebrate brain to date.