Natriuretic peptides in fish physiology.

Natriuretic peptides exist in the fishes as a family of structurally-related isohormones including atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) and ventricular natriuretic peptide (VNP); to date, brain natriuretic peptide (or B-type natriuretic peptide, BNP) has not been definitively identified in the fishes. Based on nucleotide and amino acid sequence similarity, the natriuretic peptide family of isohormones may have evolved from a neuromodulatory, CNP-like brain peptide. The primary sites of synthesis for the circulating hormones are the heart and brain; additional extracardiac and extracranial sites, including the intestine, synthesize and release natriuretic peptides locally for paracrine regulation of various physiological functions. Membrane-bound, guanylyl cyclase-coupled natriuretic peptide receptors (A- and B-types) are generally implicated in mediating natriuretic peptide effects via the production of cyclic GMP as the intracellular messenger. C- and D-type natriuretic peptide receptors lacking the guanylyl cyclase domain may influence target cell function through G(i) protein-coupled inhibition of membrane adenylyl cyclase activity, and they likely also act as clearance receptors for circulating hormone. In the few systems examined using homologous or piscine reagents, differential receptor binding and tissue responsiveness to specific natriuretic peptide isohormones is demonstrated. Similar to their acute physiological effects in mammals, natriuretic peptides are vasorelaxant in all fishes examined. In contrast to mammals, where natriuretic peptides act through natriuresis and diuresis to bring about long-term reductions in blood volume and blood pressure, in fishes the primary action appears to be the extrusion of excess salt at the gills and rectal gland, and the limiting of drinking-coupled salt uptake by the alimentary system. In teleosts, both hypernatremia and hypervolemia are effective stimuli for cardiac secretion of natriuretic peptides; in the elasmobranchs, hypervolemia is the predominant physiological stimulus for secretion. Natriuretic peptides may be seawater-adapting hormones with appropriate target organs including the gills, rectal gland, kidney, and intestine, with each regulated via, predominantly, either A- or B-type (or C- or D-type?) natriuretic peptide receptors. Natriuretic peptides act both directly on ion-transporting cells of osmoregulatory tissues, and indirectly through increased vascular flow to osmoregulatory tissues, through inhibition of drinking, and through effects on other endocrine systems.

Snapshot of a large dynamic replicon in a halophilic archaeon: megaplasmid or minichromosome?

Extremely halophilic archaea, which flourish in hypersaline environments, are known to contain a variety of large dynamic replicons. Previously, the analysis of one such replicon, pNRC100, in Halobacterium sp. strain NRC-1, showed that it undergoes high-frequency insertion sequence (IS) element-mediated insertions and deletions, as well as inversions via recombination between 39-kb-long inverted repeats (IRs). Now, the complete sequencing of pNRC100, a 191,346-bp circle, has shown the presence of 27 IS elements representing eight families. A total of 176 ORFs or likely genes of 850-bp average size were found, 39 of which were repeated within the large IRs. More than one-half of the ORFs are likely to represent novel genes that have no known homologs in the databases. Among ORFs with previously characterized homologs, three different copies of putative plasmid replication and four copies of partitioning genes were found, suggesting that pNRC100 evolved from IS element-mediated fusions of several smaller plasmids. Consistent with this idea, putative genes typically found on plasmids, including those encoding a restriction-modification system and arsenic resistance, as well as buoyant gas-filled vesicles and a two-component regulatory system, were found on pNRC100. However, additional putative genes not expected on an extrachromosomal element, such as those encoding an electron transport chain cytochrome d oxidase, DNA nucleotide synthesis enzymes thioredoxin and thioredoxin reductase, and eukaryotic-like TATA-binding protein transcription factors and a chromosomal replication initiator protein were also found. A multi-step IS element-mediated process is proposed to account for the acquisition of these chromosomal genes. The finding of essential genes on pNRC100 and its property of resistance to curing suggest that this replicon may be evolving into a new chromosome.

Local synthesis of natriuretic peptides in the eel intestine.

The intestine is a major osmoregulatory organ in euryhaline fishes which allows them to survive in the sea, and natriuretic peptides have been implicated in regulation of transmural transport. Atrial (ANP) and ventricular natriuretic peptide (VNP) were identified in eel intestine. Elution profiles of ANP and VNP from high-performance liquid chromatography (HPLC) were determined by radioimmunoassay using highly specific antisera. Elution times of immunoreactive ANP and VNP in HPLC were identical to those of authentic peptide standards and were consistent with the relative molecular masses of these peptides. Tissue localization of ANP and VNP was accomplished by fluorescence immunohistochemistry. Immunoreactive cells were observed in the epithelium of anterior, middle, and posterior regions of intestine. Reverse transcription of mRNA isolated from intestine and subsequent polymerase chain reaction amplification yielded appropriate-size products consistent with ANP and VNP expression. Together, these data show that natriuretic peptides are synthesized locally in eel intestine, rather than trapped from the circulation.

Inhibition of goby posterior intestinal NaCl absorption by natriuretic peptides and by cardiac extracts.

Natriuretic peptides abolish active Na+ and Cl- absorption across the posterior intestine of the euryhaline goby Gillichthys mirabilis. Inhibition by eel and human natriuretic peptides is dose-dependent with the following sequence of potencies based on experimentally determined ID50 values for inhibition of short-circuit current: eel ventricular natriuretic peptide (78 nmol.l-1), eel atrial natriuretic peptide (156 nmol.l-1), human brain natriuretic peptide (326 nmol.l-1), human alpha atrial natriuretic peptide (1.05 mumol.l-1), and eel C-type natriuretic peptide (75 mumol.l-1). Natriuretic peptides also significantly increase transcellular conductance. The observed sequence of natriuretic peptide potencies is suggestive of cellular mediation by GC-A-type NP-R1 receptors in this tissue; as expected for guanylyl-cyclase-coupled NP-R1 receptors, cyclic GMP mimics the action of natriuretic peptides on the goby intestine. Crude aqueous extracts of goby atrium and ventricle inhibited short circuit current and increased tissue conductance in a dose-dependent manner. Ventricular extract was more potent than atrial extract on both a per organ and per milligram basis.

DPC blockade of transepithelial chloride absorption and single anion channels in teleost urinary bladder.

The columnar cell epithelium of the euryhaline goby (Gillichthys mirabilis) urinary bladder actively absorbs NaCl from the lumen, thereby driving water transport and reducing water loss to the hypertonic external environment. Transcellular transport of Cl- involves apical membrane entry via Na(+)-coupled cotransport driven by the Na+ electrochemical gradient and subsequent basolateral membrane exit. An anion channel in the basolateral cell membrane of columnar epithelial cells was identified using patch-clamp technique. This channel may be one avenue for basolateral Cl- exit from the urinary bladder columnar cell. Single-channel conductance (Gc) of channels in excised, inside-out membrane patches was approximately 75 pS in symmetrical solutions containing 140 mM Cl-. The channel was selective to Cl- over other anions [Cl- > 2-(N-morpholino)ethanesulfonic acid (MES) > F- approximately Br- approximately I- > NO3- approximately SO4(2-)). Channel activity, expressed as the open probability (Po), was voltage dependent in the physiological range of membrane potential, with membrane depolarization increasing Po. Decreasing the pH of the solution bathing the cytoplasmic face of the membrane patch over the range 8.4-6.0 reduced Po. There was no effect of pH on either Gc or ionic selectivity. Radiochloride flux technique was also applied to intact columnar epithelial cell sheets to relate anion channel activity to macroscopic transcellular transport. Serosal exposure to the anion channel blocker diphenylamine-2-carboxylic acid (DPC, 30 microM to 3 mM) reduced and abolished transcellular radiochloride fluxes and net Cl- absorption across short-circuited tissues in a dose-dependent fashion. DPC addition (10 microM to 1 mM) to the solution bathing the cytoplasmic face of excised, inside-out membrane patches reduced Po in a dose-dependent manner and had no effect on Gc. These parallel findings of DPC blockade on intact epithelia and on single anion channels support the notion that this anion channel is a basolateral membrane component of the pathway for Cl- movement in transcellular Cl- absorption.

Identification of a stretch-activated monovalent cation channel from teleost urinary bladder cells.

The urinary bladder of euryhaline teleost is an important osmoregulatory organ which absorbs Na+, Cl-, and water from urine. Using patch clamp technique, single stretch-activated channels, which were permeable to K+ and Na+ (PNa/PK approximately 0.75) and had conductances of 55 and 116 pS, were studied. In excised, inside-out patches which were voltage-clamped in the physiological range of membrane potential, the single-channel open probability (Po) was low (approximately 0.02), and increased to a maximum of 0.9 with applied pipette suction. Single-channel conductance also increased with suction. The channels showed adaptation to applied suction and relaxed to a steady-state activity about 20 seconds after application of suction. The Po increased up to 0.9 with strong membrane depolarization (Vm = 0 to +80 mV); however, there was little dependence of Po on membrane potential in the physiological range. The kinetic data suggest that there is one conducting state and at least two non-conducting states of the channel. The open-time constant increased with suction but remained unchanged with membrane potential (Vm = -70 to +60 mV). The mean closed-time of the channel decreased with suction and membrane depolarization. These results demonstrate the presence of a non-selective monovalent cation channel which may be involved in cell volume regulation in the goby urinary bladder. Additionally, this channel may function as an enhancer of Na+ influx and K+ efflux across the bladder cell as part of transepithelial ion transport if it is located in apical membrane.

Recognition by goby intestine of a somatostatin analog, SMS 201-995.

The caudal neurosecretory peptide urotensin II and its partial structural analog the hypothalamic and gastroenteropancreatic peptide somatostatin can be distinguished by the goby posterior intestine; urotensin II stimulates Na+ and Cl- absorption whereas somatostatin is without effect. Sandostatin (Sandoz compound SMS 201-995) is a partial structural analog of somatostatin, possessing the active core common to both urotensin II and somatostatin but lacking the C- and N-terminal tails which apparently confer recognition in species specificity. SMS 201-995 mimicked the effects of urotensin II, namely, increased net Cl- absorption and decreased radiochloride backflux consistent with the observed increase in transepithelial resistance and the cellular depolarization typical of transport stimulation in this tissue. Speculation on the role of the tails in species-specific recognition is supported by the nature of amino acid residue substitutions in urotensin II, somatostatin, and SMS 201-995 and suggests the existence of multiple receptor types for this group of peptides.

Functional characterization of a voltage-gated anion channel from teleost fish intestinal epithelium.

An anion channel was isolated, using patch-clamp technique, from the basolateral membrane of goby intestinal epithelial cells. Single-channel conductance varied over a range from 20 to 90 pS. The channel was voltage-gated over the physiological range of cell membrane potential with depolarization increasing the proportion of time in the open state. There was no Ca2+ sensitivity. The selectivity sequence was SO4(2-) greater than Cl- greater than Mes-. The channel may function in vivo as one of several avenues of basolateral membrane Cl- exit with the voltage-gating property serving to match basolateral Cl- exit to apical entry.

Regulation of goby intestinal ion absorption by the calcium messenger system.

The role of the calcium messenger system in the regulation of ion absorption across the teleost intestine was studied using pharmacological intervention. Radiochloride transport was independent of external Ca2+ over the range 10 microM to 2.5 mM. Treatment with the Ca2+ ionophore A23187 (to hyperpolarization of the apical membrane potential of intestinal epithelial cells. The Ca2+-calmodulin antagonists trifluoperazine (TFP) and calmidazolium (R24571) produced opposite effects, i.e., stimulation of Cl- absorption and cellular depolarization. Treatment with TFP or R24571 will block or override the inhibitory action of A23187. These data suggest a regulatory role for Ca2+ in the control of intestinal NaCl absorption and mediation via calmodulin.

Differential effects of two bile salts on ion transport characteristics of teleost intestine.

The effects of a dihydroxy and a trihydroxy bile salt on the Na+- and Cl(-)-absorbing, goby posterior intestine are quite different. Taurochenodeoxycholate, a dihydroxy bile salt, increases tissue permeability to Cl-, primarily by opening the paracellular shunt pathway. The trihydroxy bile salt taurocholate lacks these effects and may, in fact, decrease tissue permeability. In light of the general structural similarity of these two molecules, a detergent action is considered unlikely and, instead, a more specific (perhaps receptor-mediated) mechanism is suggested.

Urotensin II lowers cytoplasmic free calcium concentration in goby enterocytes: measurements using quin2.

A technique is described for the measurement of cytoplasmic free calcium concentration ([Ca2+]c) in a suspension of goby intestinal epithelial cells using the fluorescent probe quin2. For 18 cell suspensions, [Ca2+]c was determined to be 142 +/- 14 nM. The caudal neurosecretory peptide urotensin II at 10(-7) to 10(-6) M concentration reduced [Ca2+]c by 20-50%. Together with previous findings linking the calcium-calmodulin system with the stimulation of Na+ and Cl- absorption in this tissue, it appears that alterations in cytoplasmic Ca2+ may mediate urotensin II-induced stimulation of ion transport.

Luminal alkalinization in the intestine of the goby.

The rate of luminal alkalinization in vitro by Gillichthys mirabilis posterior intestine as measured by a manual pH stat technique was 0.70 +/- 0.05 mu Equiv/cm2 h; acidification of the mucosal medium was never observed. The rate of HCO-3 secretion (JHCO3) was reduced by ouabain, serosally-applied DIDS, removal of serosal HCO3- and replacement of media Cl- with gluconate. HCO3- secretion was enhanced by replacement of Cl- with isethionate and unaffected by mucosal DIDS, furosemide or acetazolamide. JHCO3 was reduced at mucosal pH above or below 7.5. These results support active HCO3- secretion via a Cl-/HCO3- exchange mechanism on the basolateral membrane and a conductive exit pathway for HCO-3, H+ or OH- on the apical membrane.

Ion transport in goby intestine: cellular mechanism of urotensin II stimulation.

The Na- and Cl-absorbing goby posterior intestinal epithelium is composed predominantly of mitochondria-rich, tall columnar cells. Glass intracellular microelectrode recording technique was applied to absorptive cells of this relatively leaky epithelium to measure apical cell membrane potential difference (psi mc) and apical membrane fractional resistance. As determined by ion-substitution studies, absorptive cells are characterized by a large, Ba2+-inhibitable apical K conductance, which is a major factor determining psi mc and smaller Cl and Na conductances. Inhibition of the apical Na-Cl-coupled influx directly by furosemide or indirectly by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine produced hyperpolarization of psi mc, consistent with the greater apical membrane conductance to Cl than Na. The urophysial neurosecretory peptide urotensin II, which stimulates Na-Cl-coupled absorption, markedly depolarized psi mc in posterior intestinal tissues from 5% seawater-adapted gobies. This response is consistent with a stimulatory effect of urotensin II at the apical membrane carrier rather than at the basolateral Na-K-ATPase. Urotensin II is without effect on psi mc in tissues from seawater-adapted fish and somatostatin, a natural analogue of urotensin II, is without effect on tissues from fish adapted to either salinity. This specificity parallels that determined using radiotracer fluxes.

Specificity of response of intestinal ion transport systems to a pair of natural peptide hormone analogs: somatostatin and urotensin II.

Specificity of two intestinal ion transport systems toward the natural peptide hormone analogs somatostatin and urotensin II has been demonstrated by electrophysiological and radiotracer studies in vitro. Somatostatin inhibits active C1 secretion across the theophylline-treated rat colon but urotensin II, a dodecapeptide somatostatin analog from the teleost caudal neurosecretory system, is without effect. Conversely, urotensin II stimulates active Na and C1 absorption across the posterior intestine of the 5% seawater-adapted goby, Gillichthys mirabilis, but somatostatin is ineffective. From these and others' studies, it appears that in both systems, increased net absorption results from increased mucosal-to-serosal unidirectional ion fluxes. Based on structure-activity relationships in these and other systems, it is speculated that the difference in amino acid residues at position 4 (somatostatin-Lys, urotensin II-Ala) may contribute to the observed specificity.

Control of ion transport by Gillichthys mirabilis urinary bladder.

The columnar epithelial cell region of the goby Gillichthys mirabilis urinary bladder is the region responsible for active Na and Cl reabsorption from bladder urine. In 5% seawater-adapted fish, reabsorption occurs via an electrically silent coupled NaCl transport mechanism. Bladder reabsorption is increased in seawater-adapted fish above that observed in 5% seawater-adapted fish; incremental reabsorption results from an electrogenic Na transport in addition to the neutral component. Hypophysectomy of seawater-adapted fish reduces the electrogenic Na transport (measured as short-circuit current, ISC) and increases the transepithelial resistance (R) to values near those of 5% seawater-adapted fish. Cortisol restores the ISC and R to normal seawater-adapted values and will initiate electrogenic Na transport in 5% seawater-adapted fish. Ovine prolactin will also restore the ISC and R of columnar cell regions of hypophysectomized seawater-adapted fish to normal seawater-adapted levels; this effect appears to be due to contamination or to inherent stimulatory activity of the ovine prolactin preparation, since endogenous prolactin is ineffective in the restoration of ISC or R.

Ion transport by the urinary bladder of the gobiid teleost, Gillichthys mirabilis.

Transport characteristics of the two epithelial cell types lining the urinary bladder of the goby can be studied in Ussing-type chambers. Cuboidal cell regions from seawater- and 5% seawater-acclimated fish exhibit little mucosal-to-serosal Na or Cl transport. In contrast, columnar cell regions from both seawater- and 5% seawater-acclimated fish transport substantial amounts of Na and Cl. Whereas all electrolyte transport in 5% seawater-acclimated fish is electrically silent (neutral), columnar cell regions from seawater-acclimated fish possess an electrogenic Na transport component in addition to the neutral transport. Columnar cell regions also possess a Cl/Cl exchange diffusion mechanism. Neutral Na and Cl transport are not altered in response to external salinity; electrogenic transport varies both with acclimation to salinity and season. Reabsorption of Na and Cl from urine reduces excretory losses of these ions from 5% seawater-acclimated fish and drives urinary bladder water reabsorption in seawater-acclimated fish.