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Hydrogen sulfide (H(2)S) is a volatile gas of considerable interest as a physiologically relevant signaling molecule, but this volatility has typically been overlooked in the context of biological experiments. We examined volatility of 10 and 100 μM H(2)S (Na(2)S·9H(2)O) in real time with polarographic electrodes in three commonly employed experimental apparatuses: 24-well tissue culture plates (WP), muscle myograph baths (MB), and the Langendorff perfused heart apparatus (LPH). H(2)S loss from all apparatuses was rapid and exponential, with half-times (t(1/2)) of 5 min (WP), less than 4 min (MB), and less than 0.5 min (LPH). The t(1/2) for H(2)S loss from MB bubbled with 100% oxygen was slightly longer than that for MB bubbled with 100% nitrogen; both were significantly shorter than stirred but unbubbled MB (>9 min). Therefore, even without tissue, H(2)S rapidly disappears from buffer under a variety of experimental conditions, and this is due to volatilization, not oxidation. The inability to maintain H(2)S concentration, even briefly, questions the accuracy of dose-response studies and the relevance of long-term (>10 min) exposure to a single treatment of H(2)S. These results also help to explain the discrepancy between low H(2)S concentrations in blood and tissues versus high concentrations of exogenous H(2)S required to produce physiological responses., (Copyright © 2011 Elsevier Inc. All rights reserved.)

There is considerable controversy surrounding the initial step that transduces a fall in [Formula: see text] into a physiological signal, i.e., the "oxygen sensor" in chemoreceptors. Initial studies on systemic and respiratory vessels suggested that the metabolism of hydrogen sulfide (H(2)S) could serve as the oxygen sensor. This model was subsequently extended to chemoreceptors in fish and tissues of other animals. In this model, constitutive production of biologically active H(2)S is offset by H(2)S oxidation; when oxygen availability falls, production of H(2)S exceeds metabolism, and the resultant increase in intracellular H(2)S initiates the appropriate physiological responses. This model is supported by observations that the effects of hypoxia and H(2)S are similar, if not identical in many tissues: hypoxic responses are inhibited by inhibitors of H(2)S biosynthesis and augmented by sulfur donating molecules, and the tipping point between H(2)S production and oxidation occurs at physiologically relevant [Formula: see text] . Recent studies from other laboratories support this mechanism of O(2) sensing in the carotid body. This review summarizes information that supports the H(2)S metabolic hypothesis in these tissues with emphasis on the carotid chemoreceptors. Evidence suggesting that H(2)S is not involved in oxygen sensing in the carotid body is also critically evaluated., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Hydrogen sulfide (H(2)S) has been shown to affect gastrointestinal (GI) motility and signaling in mammals and O(2)-dependent H(2)S metabolism has been proposed to serve as an O(2) 'sensor' that couples hypoxic stimuli to effector responses in a variety of other O(2)-sensing tissues. The low P(O2) values and high H(2)S concentrations routinely encountered in the GI tract suggest that H(2)S might also be involved in hypoxic responses in these tissues. In the present study we examined the effect of H(2)S on stomach, esophagus, gallbladder and intestinal motility in the rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) and we evaluated the potential for H(2)S in oxygen sensing by examining GI responses to hypoxia in the presence of known inhibitors of H(2)S biosynthesis and by adding the sulfide donor cysteine (Cys). We also measured H(2)S production by intestinal tissue in real time and in the presence and absence of oxygen. In tissues exhibiting spontaneous contractions, H(2)S inhibited contraction magnitude (area under the curve and amplitude) and frequency, and in all tissues it reduced baseline tension in a concentration-dependent relationship. Longitudinal intestinal smooth muscle was significantly more sensitive to H(2)S than other tissues, exhibiting significant inhibitory responses at 1-10 μmol l(-1) H(2)S. The effects of hypoxia were essentially identical to those of H(2)S in longitudinal and circular intestinal smooth muscle; of special note was a unique transient stimulatory effect upon application of both hypoxia and H(2)S. Inhibitors of enzymes implicated in H(2)S biosynthesis (cystathionine β-synthase and cystathionine γ-lyase) partially inhibited the effects of hypoxia whereas the hypoxic effects were augmented by the sulfide donor Cys. Furthermore, tissue production of H(2)S was inversely related to O(2); addition of Cys to intestinal tissue homogenate stimulated H(2)S production when the tissue was gassed with 100% nitrogen (~0% O(2)), whereas addition of oxygen (~10% O(2)) reversed this to net H(2)S consumption. This study shows that the inhibitory effects of H(2)S on the GI tract of a non-mammalian vertebrate are identical to those reported in mammals and they provide further evidence that H(2)S is a key mediator of the hypoxic response in a variety of O(2)-sensitive tissues.

In mammals, natriuretic peptides (NPs) lower blood pressure, reduce blood volume and broadly inhibit cardiovascular remodeling. NPs are often referred to as cardiac hormones, though they also have integral roles in regulating vascular tone, endothelial remodeling and inhibiting vascular smooth muscle cell hypertrophy. Two NPs [atrial (ANP) and C-type (CNP)] have been identified as endogenous constituents in the vasculature of mammals, though such a phenomenon has not previously been described in fishes. Here we describe the endogenous production of B-type NP (BNP) and CNP in multiple blood vessels of the rainbow trout. Western blot analysis showed pro-BNP and pro-CNP production in the efferent branchial artery, celiacomesenteric artery, ventral aorta and anterior cardinal vein. The detection of pro-BNP and pro-CNP was also supported by MALDI-TOF mass spectrometry analysis of NP-enriched tissue extracts. Although vascular pro-peptide levels of BNP and CNP were quantitatively quite comparable to those found in reference tissues (the atrium for BNP and brain for CNP), mRNA levels of these NPs in the vasculature were greatly reduced as determined by quantitative PCR. When the evolutionarily conserved vascular NP (CNP) was infused into un-anesthetized trout, it reduced central venous pressure and mean circulatory filling pressure. CNP also decreased cardiac output via a reduction in preload. The presence of endogenous NP production in the trout vasculature and potent in vivo hypotensive effects further support the numerous functional similarities between teleost and mammalian NP systems.

Hydrogen sulfide (H(2)S) has become the hot new signaling molecule that seemingly affects all organ systems and biological processes in which it has been investigated. It has also been shown to have both proinflammatory and anti-inflammatory actions and proapoptotic and anti-apoptotic effects and has even been reported to induce a hypometabolic state (suspended animation) in a few vertebrates. The exuberance over potential clinical applications of natural and synthetic H(2)S-"donating" compounds is understandable and a number of these function-targeted drugs have been developed and show clinical promise. However, the concentration of H(2)S in tissues and blood, as well as the intrinsic factors that affect these levels, has not been resolved, and it is imperative to address these points to distinguish between the physiological, pharmacological, and toxicological effects of this molecule. This review will provide an overview of H(2)S metabolism, a summary of many of its reported "physiological" actions, and it will discuss the recent development of a number of H(2)S-donating drugs that show clinical potential. It will also examine some of the misconceptions of H(2)S chemistry that have appeared in the literature and attempt to realign the definition of "physiological" H(2)S concentrations upon which much of this exuberance has been established.

This study examines the functional gill morphology of the shortfin mako, Isurus oxyrinchus, to determine the extent to which its gill structure is convergent with that of tunas for specializations required to increase gas exchange and withstand the forceful branchial flow induced by ram ventilation. Mako gill structure is also compared to that of the blue shark, Prionace glauca, an epipelagic species with lower metabolic requirements and a reduced dependence on fast, continuous swimming to ventilate the gills. The gill surface area of the mako is about one-half that of a comparably sized tuna, but more than twice that of the blue shark and other nonlamnid shark species. Mako gills are also distinguished from those of other sharks by shorter diffusion distances and a more fully developed diagonal blood-flow pattern through the gill lamellae, which is similar to that found in tunas. Although the mako lacks the filament and lamellar fusions of tunas and other ram-ventilating teleosts, its gill filaments are stiffened by the elasmobranch interbranchial septum, and the lamellae appear to be stabilized by one to two vascular sacs that protrude from the lamellar surface and abut sacs of adjacent lamellae. Vasoactive agents and changes in vascular pressure potentially influence sac size, consequently effecting lamellar rigidity and both the volume and speed of water through the interlamellar channels. However, vascular sacs also occur in the blue shark, and no other structural elements of the mako gill appear specialized for ram ventilation. Rather, the basic elasmobranch gill design and pattern of branchial circulation are both conserved. Despite specializations that increase mako gill area and efficacy relative to other sharks, the basic features of the elasmobranch gill design appear to have limited selection for a larger gill surface area, and this may ultimately constrain mako aerobic performance in comparison to tunas., (J. Morphol. 271:937-948, 2010. (c) 2010 Wiley-Liss, Inc.)

Vertebrate cardiorespiratory homeostasis is inextricably dependent upon specialized cells that provide feedback on oxygen status in the tissues, blood, and on occasion, environment. These "oxygen sensing" cells include chemoreceptors and oxygen-sensitive chromaffin cells that initiate cardiorespiratory reflexes, vascular smooth muscle cells that adjust perfusion to metabolism or ventilation, and other cells that condition themselves in response to episodic hypoxia. Identification of how these cells sense oxygen and transduce this into the appropriate physiological response has enormous clinical applicability, but despite intense research there is no consensus regarding the initial hypoxia-effector coupling mechanism. This review examines an alternative mechanism of oxygen sensing using oxidation of endogenously produced hydrogen sulfide (H(2)S) as the O(2)-sensitive couple. Support for this hypothesis includes the similarity of effects of hypoxia and H(2)S on a variety of tissues, augmentation of hypoxic responses by precursors of H(2)S production and their inhibition by inhibitors of H(2)S synthesis, and the rapid consumption of H(2)S by O(2) in the range of intracellular/mitochondrial Po(2). These studies also indicate that, under normoxic conditions, it is doubtful that free H(2)S has longer than a transient existence in tissue or extracellular fluid.

Hypoxic pulmonary vasoconstriction (HVC), an intrinsic and assumed ubiquitous response of mammalian pulmonary blood vessels, matches regional ventilation to perfusion via an unknown O(2)-sensing mechanism. Global pulmonary hypoxia experienced by individuals suffering from chronic obstructive pulmonary disease or numerous hypoventilation syndromes, including sleep apnea, often produces maladaptive pulmonary hypertension, but pulmonary hypertension is not observed in diving mammals, where profound hypoxia is routine. Here we examined the response of cow and sea lion pulmonary arteries (PA) to hypoxia and observed the expected HVC in the former and a unique hypoxic vasodilation in resistance vessels in the latter. We then used this disparate response to examine the O(2)-sensing mechanism. In both animals, exogenous H(2)S mimicked the vasoactive effects of hypoxia in isolated PA. H(2)S-synthesizing enzymes, cystathionine beta-synthase, cystathionine gamma-lyase, and 3-mercaptopyruvate sulfur transferase, were identified in lung tissue from both animals by one-dimensional Western blot analysis and immunohistochemistry. The relationship between H(2)S production/consumption and O(2) was examined in real time by use of amperometric H(2)S and O(2) sensors. H(2)S was produced by sea lion and cow lung homogenate in the absence of O(2), but it was rapidly consumed when O(2) was present. Furthermore, consumption of exogenous H(2)S by cow lung homogenate, PA smooth muscle cells, and heart mitochondria was O(2) dependent and exhibited maximal sensitivity at physiologically relevant Po(2) levels. These studies show that HVC is not an intrinsic property of PA and provide further evidence for O(2)-dependent H(2)S metabolism in O(2) sensing.

Natriuretic peptides (NPs) and their receptors (NPRs) comprise an evolutionarily conserved signaling system with profound physiological effects on vertebrate renal and cardiovascular systems. Some NPs (ANP, BNP and VNP) are primarily of cardiac origin whereas CNP is common in the brain. In mammals, non-traditional sites of NPs synthesis, BNP in brain and CNP in atrium, appear to have complementary actions. In the present study, trout were chronically adapted to freshwater (FW) (a volume-loading, salt-depleting environment), saltwater (SW) (a volume-depleting, salt-loading environment), FW and fed a high-salt diet (FW-HSD) (a volume- and salt-loading regime) or acutely volume depleted or expanded by hemorrhage or infusion with dialyzed plasma to perturb volume homeostasis. The responses of brain and atrial BNP and CNP mRNA, pro-peptide, NPR-A and NPR-B were evaluated using quantitative PCR and western analysis. Brain pro-BNP and NPR-A was increased in FW-HSD trout and decreased in SW trout. Brain pro-CNP was largely unaffected whereas NPR-B mRNA was increased in FW-HSD trout. Atrial CNP, although produced at lower levels than other cardiac NPs, was markedly elevated in chronically (FW-HSD) and acutely volume expanded trout (dialyzed-plasma infusion) whereas decreased in hemorrhaged trout. These findings indicate that non-traditional NP synthesis sites in the trout probably complement the broad hypovolemic and hypotensive actions of traditional (cardiac) NP synthesis sites in response to volume expansion but not to plasma osmolarity. This supports the hypothesis that the piscine and mammalian NP systems are fundamentally similar and appear to protect the heart from volume overload.

Hydrogen sulfide (H(2)S) is gaining acceptance as a signaling molecule and has been shown to elicit a variety of biological effects at concentrations between 10 and 1000 micromol/l. Dissolved H(2)S is a weak acid in equilibrium with HS(-) and S(2-) and under physiological conditions these species, collectively referred to as sulfide, exist in the approximate ratio of 20% H(2)S, 80% HS(-) and 0% S(2-). Numerous analyses over the past 8 years have reported plasma or blood sulfide concentrations also in this range, typically between 30 and 300 micromol/l, thus supporting the biological studies. However, there is some question whether or not these concentrations are physiological. First, many of these values have been obtained from indirect methods using relatively harsh chemical conditions. Second, most studies conducted prior to 2000 failed to find blood sulfide in micromolar concentrations while others showed that radiolabeled (35)S-sulfide is rapidly removed from blood and that mammals have a relatively high capacity to metabolize exogenously administered sulfide. Very recent studies using H(2)S gas-sensing electrodes to directly measure sulfide in plasma or blood, or HPLC analysis of head-space gas, have also indicated that sulfide does not circulate at micromolar levels and is rapidly consumed by blood or tissues. Third, micromolar concentrations of sulfide in blood or exhaled air should be, but are not, malodorous. Fourth, estimates of dietary sulfur necessary to sustain micromolar levels of plasma sulfide greatly exceed the daily intake. Collectively, these studies imply that many of the biological effects of sulfide are only achieved at supra-physiological concentrations and they question whether circulating sulfide is a physiologically relevant signaling molecule. This review examines the blood/plasma sulfide measurements that have been reported over the past 30 years from the perspective of the analytical methods used and the potential sources of error.

Natriuretic peptides (NPs) are evolutionarily conserved hormones that affect blood pressure and fluid volume through membrane-bound guanylate cyclase (GC)-linked natriuretic peptide receptors-A and -B (NPR-A and NPR-B, respectively) in a variety of vascular, renal, and other tissues. The principal physiological stimulus for cardiac NPs in fish is somewhat debated between two prominent theories: regulation of salt balance (osmoregulatory hypothesis) or prevention of volume expansion (cardioprotective hypothesis). In the present study, we examined atrial and ventricular expression of trout NPs, atrial (ANP), brain (BNP), and ventricular (VNP) using Northern (mRNA), Western (NP pro-hormone), and qPCR (GC-NPR-A and -B mRNA) blot analysis following independent manipulation of plasma salt and volume levels after chronic exposure to freshwater (FW; volume loaded, salt depleted), saltwater (SW; volume depleted, salt loaded), or freshwater trout fed a high-salt diet (FW-HSD; volume and salt loaded). We also measured NP transcriptional response to acute (2 h) volume expansion with dialyzed plasma (VE; 80% blood vol) or volume depletion by hemorrhage (VD, 20% blood volume every 30 min for 2 h) with real-time PCR. In essentially all instances, increased expression of the NP system was associated with FW-HSD or plasma expansion. There were no differences in NP expression between chronically adapted FW and SW fish, and hemorrhage decreased atrial ANP and VNP mRNA. These results indicate that rainbow trout cardiac NPs and cardiovascular GC-NPRs respond principally to volume, not salt overload, and this suggests that the primary function of trout cardiac NP system is to protect the heart.

Terrestrial, but not aquatic, species of snakes have hepatic portal veins with a corkscrew morphology immediately posterior of the liver. Relatively large volumes of venous blood are associated with this region, and the corkscrew vein has been proposed to function as a bidirectional valve that impedes gravitational shifts of intravascular volume. To better understand the functional significance of the corkscrew anatomy, we investigated the histology and contractile mechanisms in isolated corkscrew segments of the hepatic portal vein of a yellow rat snake (Pantherophis obsoletus). Morphologically, the corkscrew portal vein is here shown to have two distinct layers of smooth muscle--an inner circular layer, and an outer longitudinal layer, separated by a layer of collagen--whereas only a single circular layer of smooth muscle is present in the adjacent posterior caval vein. Low frequency (approximately 0.3 cycles*min(-1)) spontaneous and catecholamine-induced rhythms were observed in 11% and 89% of portal vein segments, respectively, but neither spontaneous nor agonist-induced cycling was observed in adjacent posterior (non-corkscrew) caval veins. Catecholamines, angiotensin II, or stretch increased the amplitude and/or frequency of contractile cycles. Ouabain, verapamil or indomethacin, but not tetrodotoxin, alpha-, or ss-adrenergic receptor antagonists, inhibited cyclical contractions indicating a dependence of these cycles on Na+/K+ ATPase, extracellular Ca2+ and prostanoid(s). These data suggest that the rhythmic contractility of the corkscrew segment of the ophidian portal vein may act in conjunction with its morphological features to improve venous return and to prevent retrograde shifts of blood that might otherwise pool in posterior veins.

Carbon monoxide (CO) is endogenously produced by heme oxygenase (HO) and is involved in vascular, neural, and inflammatory responses in mammals. However, the biological activities of CO in nonmammalian vertebrates is unknown. To this extent, we used smooth muscle myography to investigate the effects of exogenously applied CO (delivered via a water-soluble CO-releasing molecule, CORM-3) on isolated lamprey (Petromyzon marinus) dorsal aortas and examined its mechanisms of action on trout (Oncorhynchus mykiss) efferent branchial (EBA) and celiacomesenteric (CMA) arteries. CORM-3 dose-dependently relaxed all vessels examined. Trout EBA were twofold more sensitive to CORM-3 when precontracted with norepinephrine (NE) than KCl and CORM-3 relaxed five-fold more of the NE- than KCl-induced tension. Glybenclamide (10 microM), an ATP-sensitive potassium channel inhibitor, inhibited NE-induced contraction, but did not affect CORM-3-induced relaxation. NS-2028 (10 microM), a soluble guanylyl cyclase inhibitor, had no effect on a NE-contraction, but inhibited a subsequent CORM-3-induced relaxation. Zinc protopophyrin-IX (ZnPP-IX, 0.3-30 microM), a HO inhibitor, elicited a small, yet dose-dependent and significant, increase in baseline tension but did not have any effect on subsequent NE-induced contractions or a nitric oxide-induced relaxation (via sodium nitroprusside). [ZnPP-IX] greater than 3 microM, however, significantly reduced the predominant vasodilatory response of trout EBA to hydrogen sulfide. These results implicate an active HO/CO pathway in trout vessels having an impact on resting vessel tone and CO-induced vasoactivity that is at least partially mediated by soluble guanylyl cyclase.

The origins and actions of gaseous signaling molecules, nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H(2)S) in the mammalian cardiovascular system have received considerable attention and it is evident that these three "gasotransmitters" perform a variety of homeostatic functions. The origins, actions and disposition of these gasotransmitters in the piscine vasculature are far from resolved. In most fish examined to date, NO or NO donors are generally in vitro and in vivo vasodilators acting via soluble guanylyl cyclase, although there is evidence for NO-mediated vasoconstriction. Injection of sodium nitroprusside into trout causes hypotension that is attributed to a reduction in systemic resistance. Unlike mammals, NO does not appear to have an endothelial origin in fish blood vessels as an endothelial NO synthase has not identified. However, neural NO synthase is prevalent in perivascular nerves and is the most likely source of NO for cardiovascular control in fish. CO is a vasodilator in lamprey and trout vessels, and it, like NO, appears to exert its action, at least in part, via guanylyl cyclase and potassium channel activation. Inhibition of CO production increases resting tone in trout vessels suggestive of tonic CO activity, but little else is known about the origin or control of CO in the fish vasculature. H(2)S is synthesized by fish vessels and its constrictory, dilatory, or even multi-phasic actions, are both species- and vessel-specific. A small component of H(2)S-mediated basal activity may be endothelial in origin, but to a large extent H(2)S affects vascular smooth muscle directly and the mechanisms are unclear. H(2)S injected into the dorsal aorta of unanesthetized trout often produces oscillations in arterial blood pressure suggestive of H(2)S activity in the central nervous system as well as peripheral vasculature. Collectively, these studies hint at significant involvement of the gasotransmitters in piscine cardiovascular function and hopefully provide a variety of avenues for future research.

Although all cells are variously affected by oxygen, a few have the responsibility of monitoring oxygen tensions and initiating key homeostatic responses when P(O2) falls to critical levels. These ;oxygen-sensing' cells include the chemoreceptors in the gills (neuroepithelial cells), airways (neuroepithelial bodies) and vasculature (carotid bodies) that initiate cardiorespiratory reflexes, oxygen sensitive chromaffin cells associated with systemic veins or adrenal glands that regulate the rate of catecholamine secretion, and vascular smooth muscle cells capable of increasing blood flow to systemic tissues, or decreasing it through the lungs. In spite of intense research, and enormous clinical applicability, there is little, if any, consensus regarding the mechanism of how these cells sense oxygen and transduce this into the appropriate physiological response. We have recently proposed that the metabolism of hydrogen sulfide (H2S) may serve as an 'oxygen sensor' in vertebrate vascular smooth muscle and preliminary evidence suggests it has similar activity in gill chemoreceptors. In this proposed mechanism, the cellular concentration of H2S is determined by the simple balance between constitutive H2S production in the cytoplasm and H2S oxidation in the mitochondria; when tissue oxygen levels fall the rate of H2S oxidation decreases and the concentration of biologically active H2S in the tissue increases. This commentary briefly describes the oxygen-sensitive tissues in fish and mammals, delineates the current hypotheses of oxygen sensing by these tissues, and then critically evaluates the evidence for H2S metabolism in oxygen sensing.

We used myography on five different arteries and three veins of the hagfish, Eptatretus cirrhatus, to test the response of vessels to vasoactive drugs. Concentration-response curves were generated for carbachol, endothelin-1, arginine vasotocin and the adrenergic agonists, phenylephrine and isoprenaline. pEC50 values indicated that veins were more sensitive to endothelin-1 than were arteries, but the arteries were more sensitive to the cholinergic agonist, carbachol. Segmental arteries did not react to arginine vasotocin, but all other vessels did, and on a molar basis it was the most potent agonist tested. That ventral and dorsal aortas were more sensitive to arginine vasotocin than smaller vessels might indicate that this neurohypophysial peptide has the potential to exert a profound influence on branchial vascular resistance and cardiac output in hagfishes. The results also demonstrate the potential for a variety of endogenous peptides to contribute to central venous tone.

O2 chemoreceptors elicit cardiorespiratory reflexes in all vertebrates, but consensus on O2-sensing signal transduction mechanism(s) is lacking. We recently proposed that hydrogen sulfide (H2S) metabolism is involved in O2 sensing in vascular smooth muscle. Here, we examined the possibility that H2S is an O2 sensor in trout chemoreceptors where the first pair of gills is a primary site of aquatic O2 sensing and the homolog of the mammalian carotid body. Intrabuccal injection of H2S in unanesthetized trout produced a dose-dependent bradycardia and increased ventilatory frequency and amplitude similar to the hypoxic response. Removal of the first, but not second, pair of gills significantly inhibited H2S-mediated bradycardia, consistent with the loss of aquatic chemoreceptors. mRNA for H2S-synthesizing enzymes, cystathionine beta-synthase and cystathionine gamma-lyase, was present in branchial tissue. Homogenized gills produced H2S enzymatically, and H2S production was inhibited by O2, whereas mitochondrial H2S consumption was O2 dependent. Ambient hypoxia did not affect plasma H2S in unanesthetized trout, but produced a PO2-dependent increase in a sulfide moiety suggestive of increased H2S production. In isolated zebrafish neuroepithelial cells, the putative chemoreceptive cells of fish, both hypoxia and H2S, produced a similar approximately 10-mV depolarization. These studies are consistent with H2S involvement in O2 sensing/signal transduction pathway(s) in chemoreceptive cells, as previously demonstrated in vascular smooth muscle. This novel mechanism, whereby H2S concentration ([H2S]) is governed by the balance between constitutive production and oxidation, tightly couples tissue [H2S] to PO2 and may provide an exquisitely sensitive, yet simple, O2 sensor in a variety of tissues.

Hydrogen sulfide (H(2)S) has been proposed to mediate hypoxic vasoconstriction (HVC), however, other studies suggest the vasoconstrictory effect indirectly results from an oxidation product of H(2)S. Here we examined the relationship between H(2)S and O(2) in isolated hagfish and lamprey vessels that exhibit profound hypoxic vasoconstriction. In myographic studies, H(2)S (Na(2)S) dose-dependently constricted dorsal aortas (DA) and efferent branchial arteries (EBA) but did not affect ventral aortas or afferent branchial arteries; effects similar to those produced by hypoxia. Sensitivity of H(2)S-mediated contraction in hagfish and lamprey DA was enhanced by hypoxia. HVC in hagfish DA was enhanced by the H(2)S precursor cysteine and inhibited by amino-oxyacetate, an inhibitor of the H(2)S-synthesizing enzyme, cystathionine beta-synthase. HVC was unaffected by propargyl glycine, an inhibitor of cystathionine lambda-lyase. Oxygen consumption (M(O(2))) of hagfish DA was constant between 15 and 115 mmHg P(O(2)) (1 mmHg=0.133 kPa), decreased when P(O(2)) <15 mmHg, and increased after P(O(2)) exceeded 115 mmHg. 10 micromol l(-1) H(2)S increased and > or =100 micromol l(-1) H(2)S decreased M(O(2)). Consistent with the effects on HVC, cysteine increased and amino-oxyacetate decreased M(O(2)). These results show that H(2)S is a monophasic vasoconstrictor of specific cyclostome vessels and because hagfish lack vascular NO, and vascular sensitivity to H(2)S was enhanced at low P(O(2)), it is unlikely that H(2)S contractions are mediated by either H(2)S-NO interaction or an oxidation product of H(2)S. These experiments also provide additional support for the hypothesis that the metabolism of H(2)S is involved in oxygen sensing/signal transduction in vertebrate vascular smooth muscle.

Hydrogen sulfide (H(2)S) is rapidly emerging as a biologically significant signaling molecule. Studies published before 2000 report low or undetectable H(2)S (usually as total sulfide) levels in blood or plasma, whereas recent work has reported sulfide concentrations between 10 and 300 microM, suggesting it acts as a circulating signal. In the first series of experiments, we used a recently developed polarographic sensor to measure the baseline level of endogenous H(2)S gas and turnover of exogenous H(2)S gas in real time in blood from numerous animals, including lamprey, trout, mouse, rat, pig, and cow. We found that, contrary to recent reports, H(2)S gas was essentially undetectable (<100 nM total sulfide) in all animals. Furthermore, exogenous sulfide was rapidly removed from blood, plasma, or 5% bovine serum albumin in vitro and from intact trout in vivo. To determine if blood H(2)S could transiently increase, we measured oxygen-dependent H(2)S production by trout hearts in vitro and in vivo. H(2)S has been shown to mediate ischemic preconditioning (IPC) in mammals. IPC is present in trout and, unlike mammals, the trout myocardium obtains its oxygen from relatively hypoxic systemic venous blood. In vitro, myocardial H(2)S production was inversely related to Po(2), whereas we failed to detect H(2)S in ventral aortic blood from either normoxic or hypoxic fish in vivo. These results provide an autocrine or paracrine mechanism for myocardial coupling of hypoxia to H(2)S in IPC, i.e., oxygen sensing, but they fail to provide any evidence that H(2)S signaling is mediated by the circulation.

The natriuretic peptide (NP) family is a seemingly ubiquitous sodium and volume reducing endocrine system of predominantly cardiac origin. Members of the NP system include ANP, BNP, CNP, VNP, their guanylate cyclase (GC)-linked receptors (NPR-A and NPR-B), and clearance receptor (NPR-C). Through the activation of their membrane-bound GC receptors, these small peptides modulate cellular functions that affect both salt and water balance. The elucidation of piscine NP sequences, structure, and functions has steadily advanced over the past 15 years spearheaded by research from Dr. Yoshio Takei's laboratory. The development of these homologous NPs has led to extensive research into both the evolutionary and physiological significance of NPs in fishes. One outcome has been the development of two seemingly disparate hypotheses of NP function; a role in salt excretion, the osmoregulatory hypothesis, versus a role in protecting the heart, the cardioprotective hypotheses. In the osmoregulatory hypothesis NPs are released in response to elevated ambient salinity and inhibit drinking and intestinal uptake of salt, thereby effectively reducing plasma sodium levels. In contrast, the cardioprotective theory depicts NPs acting to prevent debilitating cardiodilation from an excess of either venous or arterial pressure through vasodilation and a reduction of blood volume. These seemingly distinct hypotheses may be elements of a more general regulatory system and certainly require further investigation. Undoubtedly their resolution will not only give us a better perspective of the evolutionary basis of the NP system but will provide us with a greater appreciation of salt and water homeostasis in vertebrates.

Hypoxia contracts mammalian respiratory vessels and increases vascular resistance in respiratory tissues of many vertebrates. In systemic vessels these responses vary, hypoxia relaxes mammalian vessels and contracts systemic arteries from cyclostomes. It has been proposed that hypoxic vasoconstriction in cyclostome systemic arteries is the antecedent to mammalian hypoxic pulmonary vasoconstriction, however, phylogenetic characterization of hypoxic responses is lacking. In this study, we characterized the hypoxic response of isolated systemic and respiratory vessels from a variety of vertebrates using standard myography. Pre-gill/respiratory (ventral aorta, afferent branchial artery, pulmonary artery) and post-gill/systemic (dorsal and thoracic aortas, efferent branchial artery) from lamprey (Petromyzon marinus), sandbar shark (Carcharhinus plumbeus), yellowfin tuna (Thunnus albacares), American bullfrog (Rana catesbeiana), American alligator (Alligator mississippiensis), Pekin duck (Anas platyrhynchos domesticus), chicken (Gallus domesticus) and rat (Rattus norvegicus) were exposed to hypoxia at rest or during pre-stimulation (elevated extracellular potassium, epinephrine or norepinephrine). Hypoxia produced a relaxation or transient contraction followed by relaxation in all pre-gill vessels, except for contraction in lamprey, and vasoconstriction or tri-phasic constriction-dilation-constriction in all pulmonary vessels. Hypoxia contracted systemic vessels from all animals except shark and rat and in pre-contracted rat aortas it produced a transient contraction followed by relaxation. These results show that while the classic "systemic hypoxic vasodilation and pulmonary hypoxic vasoconstriction" may occur in the microcirculation, the hypoxic response of the vertebrate macrocirculation is quite variable. These findings also suggest that hypoxic vasoconstriction is a phylogenetically ancient response., ((c) 2008 Wiley-Liss, Inc.)

Trout are of interest in defining the relationship between fluid and salt balance on cardiovascular function because they thrive in freshwater (FW; volume loading, salt depleting), saltwater (SW; volume depleting, salt loading), and FW while fed a high-salt diet (FW-HS; volume and salt loading). The effects of chronic (>2 wk) adaptation to these three protocols on blood volume (51Cr red cell space), extracellular fluid volume (99mTc-diethylene triaminepenta-acetic acid space), arterial (dorsal aortic; P(DA)) and venous (ductus Cuvier; Pven) blood pressure, mean circulatory filling pressure (zero-flow Pven), and vascular capacitance were examined in the present study on unanesthetized rainbow trout. Blood volume, extracellular fluid volume, P(DA), Pven, and mean circulatory filling pressure progressively increased in the order SW < FW < FW-HS. Vascular capacitance in SW fish appeared to be continuous with the capacitance curve of FW fish and reflect a passive volume-dependent unloading of the venous system of FW fish. Vascular capacitance curves for FW-HS fish were displaced upward and parallel to those of FW fish, indicative of an active increase in unstressed blood volume without any change in vascular compliance. These studies are the first in any vertebrate to measure the relationship between fluid compartments and cardiovascular function during independent manipulation of volume and salt balance, and they show that volume, but not salt, balance is the primary determinant of blood pressure in trout. They also present a new paradigm with which to investigate the relative contributions of water and salt balance in cardiovascular homeostasis.

Experiments were conducted on rainbow trout to determine the impact of dietary salt on arterial blood pressure. After 4-6 wk, fish fed a salt-enriched diet exhibited a 37% elevation of dorsal aortic pressure (from 23.8 +/- 1.2 to 32.6 +/- 1.4 mmHg) and an 18% increase in ventral aortic pressure (from 33.0 +/- 1.5 to 38.9 +/- 1.3 mmHg). The hypertension presumably reflected the increase in cardiac output (from 31.0 +/- 0.8 to 36.4 +/- 2.2 ml.min(-1).kg(-1)) because systemic and branchial resistances were statistically unaltered by salt feeding. The chronic hypertension was associated with a decrease in the pressor responses of the systemic vasculature to catecholamines and hypercapnia in the salt-fed fish. The reduction in alpha-adrenergic responsiveness of the systemic vasculature is consistent with desensitization or loss of functional alpha-adrenoceptors (alpha-ARs). In support of this idea, the salt-fed fish exhibited significantly decreased levels of alpha(1D)-AR mRNA in the dorsal aorta and the afferent (ABA) and efferent branchial arteries (EBA). In contrast, however, the results obtained from norepinephrine dose-response curves for EBA and ABA vascular rings in vitro did not provide evidence for loss of function of branchial artery alpha(1)-ARs in the salt-fed fish. Indeed, the EC(50) for the EBA norepinephrine dose-response curve was significantly reduced (from 3.75 x 10(-7) to 2.12 x 10(-7) M) in the salt-fed fish, indicating an increase in the binding affinity of the alpha(1)-ARs.

How vertebrate blood vessels sense acute hypoxia and respond either by constricting (hypoxic vasoconstriction) or dilating (hypoxic vasodilation) has not been resolved. In the present study we compared the mechanical and electrical responses of select blood vessels to hypoxia and H2S, measured vascular H2S production, and evaluated the effects of inhibitors of H2S synthesis and addition of the H2S precursor, cysteine, on hypoxic vasoconstriction and hypoxic vasodilation. We found that: (1) in all vertebrate vessels examined to date, hypoxia and H2S produce temporally and quantitatively identical responses even though the responses vary from constriction (lamprey dorsal aorta; lDA), to dilation (rat aorta; rA), to multi-phasic (rat and bovine pulmonary arteries; rPA and bPA, respectively). (2) The responses of lDA, rA and bPA to hypoxia and H2S appear competitive; in the presence of one stimulus, the response to the other stimulus is substantially or completely eliminated. (3) Hypoxia and H2S produce the same degree of cell depolarization in bPA. (4) H2S is constitutively synthesized by lDA and bPA vascular smooth muscle. (5) Inhibition of H2S synthesis inhibits the hypoxic response of lDA, rA, rPA and bPA. (6) Addition of the H2S precursor, cysteine, doubles hypoxic contraction in lDA, prolongs contraction in bPA and alters the re-oxygenation response of rA. These studies suggest that H2S may serve as an O2 sensor/transducer in the vascular responses to hypoxia. In this model, the concentration of vasoactive H2S in the vessel is governed by the balance between endogenous H2S production and its oxidation by available O2.

Hydrogen sulfide (H2S) is a recently identified gasotransmitter that may mediate hypoxic responses in vascular smooth muscle. H2S also appears to be a signaling molecule in mammalian non-vascular smooth muscle, but its existence and function in non-mammalian non-vascular smooth muscle have not been examined. In the present study we examined H2S production and its physiological effects in urinary bladder from steelhead and rainbow trout (Oncorhynchus mykiss) and evaluated the relationship between H2S and hypoxia. H2S was produced by trout bladders, and its production was sensitive to inhibitors of cystathionine beta-synthase and cystathionine gamma-lyase. H2S produced a dose-dependent relaxation in unstimulated and carbachol pre-contracted bladders and inhibited spontaneous contractions. Bladders pre-contracted with 80 mmol l(-1) KCl were less sensitive to H2S than bladders contracted with either 80 mmol l(-1) KC2H3O2 (KAc) or carbachol, suggesting that some of the H2S effects are mediated through an ion channel. However, H2S relaxation of bladders was not affected by the potassium channel inhibitors, apamin, charybdotoxin, 4-aminopyridine, and glybenclamide, or by chloride channel/exchange inhibitors 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt, tamoxifen and glybenclamide, or by the presence or absence of extracellular HCO3-. Inhibitors of neuronal mechanisms, tetrodotoxin, strychnine and N-vanillylnonanamide were likewise ineffective. Hypoxia (aeration with N2) also relaxed bladders, was competitive with H2S for relaxation, and it was equally sensitive to KCl, and unaffected by neuronal blockade or the presence of extracellular HCO3-. Inhibitors of H2S synthesis also inhibited hypoxic relaxation. These experiments suggest that H2S is a phylogenetically ancient gasotransmitter in non-mammalian non-vascular smooth muscle and that it serves as an oxygen sensor/transducer, mediating the effects of hypoxia.

pH is recognized as a modulator of vascular smooth muscle (VSM) tone in mammalian vessels, but little is known about its effects on fish VSM. We investigated the effects of extracellular and intracellular pH (pH(o) and pH(i), respectively) on isolated vessels from steelhead and rainbow trout (Oncorhynchus mykiss, Skamania and Kamloops strains, respectively) and of pH(o) on perfused gills from rainbow trout. In otherwise unstimulated (resting) efferent branchial (EBA) and coeliaco-mesenteric arteries (CMA), anterior cardinal veins (ACV) and perfused gills, increasing pH(o) from 6.8 to 8.8-9.0 produced a dose-dependent contraction or increase in gill resistance (R(GILL)) with an estimated half-maximal response of 8.0-8.2. pH(o) interactions with other contractile stimuli were agonist specific; more force was developed at low pH(o) in ligand-mediated (arginine vasotocin) contractions, whereas depolarization-mediated (40-80 mmol l(-1) KCl) contractions were greatest at high pH(o). Increasing pH(i) by application of 40 mmol l(-1) NH4Cl produced sustained contraction in afferent branchial arteries (ABA) suggesting that these vessels could not readily restore pH(i). NH(4)Cl application only transiently contracted EBA and CMA in Hepes buffer, whereas it produced a slight, but prolonged, relaxation of EBA and CMA in Cortland buffer. The buffer effect was due to the presence of Hepes and in this environment EBA and CMA appeared to readily restore pH(i). Increasing pH(i) in KCl-contracted EBA in Hepes produced an additional contraction, whereas ligand-contracted (thromboxane A2 analog, U-46619) EBA relaxed. Reducing pH(i) (NH(4)Cl washout) transiently contracted resting EBA and CMA in both Hepes and Cortland buffer. NH4Cl washout produced an additional, transient contraction of both KCl- and U-46619-contracted EBA in Hepes. EBA contractions produced by increased pH(i) depend primarily on intracellular Ca2+, whereas both intracellular and extracellular Ca2+ contributed to the response to decreased pH(i). Three cycles of perfusate acidification (pH(o) 7.8 to 6.2 and back to 7.8) reproducibly halved, then restored R(GILL) with no adverse effects, indicating that this was not a pathophysiological response. These studies show that the general effects of pH on VSM are phylogenetically conserved from fish to mammals but even within a species there are vessel-specific differences. Furthermore, as fish are exposed to substantial fluctuations in environmental (and therefore plasma) pH, the obligatory response of fish VSM to these changes may have substantial impact on cardiovascular homeostasis.

Hydrogen sulfide (H(2)S) vasoactivity has been observed in isolated vessels from all vertebrate classes, and its effects, which include constriction, dilation, and multiphasic responses, are both species- and vessel-specific. H(2)S is synthesized by mammalian and fish vessels, and because plasma H(2)S titers are also vasoactive in vitro, it is likely that H(2)S is a tonic effector of cardiovascular homeostasis in many vertebrates. Mechanisms of H(2)S vasoactivity in nonmammalian vertebrates have been limited to the trout where the triphasic relaxation-contraction-relaxation includes endothelium-dependent and -independent components, ATP-dependent K(+) channels, and extracellular and intracellular Ca(2+), all independent of cyclic GMP production. The observation that at least some H(2)S constrictory activity has been observed in all vertebrates except sharks suggests that H(2)S may have been an ancestral pressor gasotransmitter. However, the ability of H(2)S to serve as either (or both) an endothelium-independent constrictor or dilator, which is relatively unique among vasoregulatory molecules, is a feature that seems to have been exploited, for unknown reasons, by nearly all vertebrates. Aquatic vertebrates appear particularly vulnerable to H(2)S because of their intrinsically low blood pressure and the potential for increased H(2)S exposure from the environment.

Hydrogen sulfide (H(2)S) is a recently identified endogenous vasodilator in mammals. In steelhead/rainbow trout (Oncorhynchus mykiss, Osteichthyes), H(2)S produces both dose-dependent dilation and a unique dose-dependent constriction. In this study, we examined H(2)S vasoactivity in all vertebrate classes to determine whether H(2)S is universally vasoactive and to identify phylogenetic and/or environmental trends. H(2)S was generated from NaHS and examined in unstimulated and precontracted systemic and, when applicable, pulmonary arteries (PA) from Pacific hagfish (Eptatretus stouti, Agnatha), sea lamprey (Petromyzon marinus, Agnatha), sandbar shark (Carcharhinus milberti, Chondrichthyes), marine toad (Bufo marinus, Amphibia), American alligator (Alligator mississippiensis, Reptilia), Pekin duck (Anas platyrhynchos domesticus, Aves), and white rat (Rattus rattus, Mammalia). In otherwise unstimulated vessels, NaHS produced 1) a dose-dependent relaxation in Pacific hagfish dorsal aorta; 2) a dose-dependent contraction in sea lamprey dorsal aorta, marine toad aorta, alligator aorta and PA, duck aorta, and rat thoracic aorta; 3) a threshold relaxation in shark ventral aorta, dorsal aorta, and afferent branchial artery; and 4) a multiphasic contraction-relaxation-contraction in the marine toad PA, duck PA, and rat PA. Precontraction of these vessels with another agonist did not affect the general pattern of NaHS vasoactivity with the exception of the rat aorta, where relaxation was now dominant. These results show that H(2)S is a phylogenetically ancient and versatile vasoregulatory molecule that appears to have been opportunistically engaged to suit both organ-specific and species-specific homeostatic requirements.

Relations were examined between configurations of Big Five Traits (Extraversion, Agreeableness, Conscientiousness, Neuroticism, Openness to Experience) and 16 fundamental motives (Social Contact, Curiosity, Honor, Power, Order, Idealism, Independence, Status, Vengeance, Romance, Family, Activity, Saving, Acceptance, Eating, Tranquility) in 138 university students (93 women, 45 men; M age= 20.3 yr., SD=4.5). Big Five traits were measured with the NEO-PI-R and motives were measured with the Reiss Profile of Fundamental Goals and Motivation Sensitivities. The traits were significantly related with all the motives (adjusted R2=.06 to .43) except Physical Activity. Four motives were related with only one trait and nine configurations of two or more traits were correlated with the remaining 11 motives. Total motive scores across all participants, an index of the strength of overall motivation, were positively correlated with Extraversion and Neuroticism and negatively with Agreeableness.

Hydrogen sulfide (H(2)S) is an endogenous vasodilator in mammals, but its presence and function in other vertebrates is unknown. We generated H(2)S from NaHS and examined the effects on isolated efferent branchial arteries from steelhead (stEBA) or rainbow (rtEBA) trout. H(2)S concentration was measured colorimetrically (CM) and with ion-selective electrodes (ISE) in rainbow trout plasma. NaHS produced a triphasic response consisting of a relaxation (phase 1), constriction (phase 2), and relaxation (phase 3) in both unstimulated vessels and in stEBA precontracted with carbachol (Carb). Phase 1 and phase 3 in stEBA were decreased and phase 2 increased in unstimulated vessels by K(+)(ATP) channel inhibition (glibenclamide), or a cocktail of inhibitors of cyclooxygenase, lipoxygenase, and cytochrome P-450 (indomethacin, esculetin, and clotrimazole). Inhibition of soluble guanylate cyclase with ODQ o NS-2028 inhibited phase 3 in stEBA, although NaHS decreased cGMP production by tEBA. stEBA phase 2 contractions were partially inhibited by the myosin light chain kinase inhibitor, ML-9, but unaffected by L-type calcium channel inhibition (methoxyverapamil), whereas contraction in tEBA was partially inhibited by nifedipine or removal of extracellular calcium. Phase 3 relaxations were more pronounced in stEBA precontracted with Carb and no epinephrine (NE) than those cont acted by KCl or K(2)SO(4). stEBA phase 2 and phase 3 responses were dose dependent (EC(50) = 1.1 +/- 1.2 x 10(-3) M and 6.7 +/- 0.9 x 10(-5) M, respectively; n = 7). NaHS was also vasoactive in steelhead bulbus arteriosus, celiac mesenteric arteries, and anterior cardinal veins. Rainbow trout plasma sulfide concentration was 4.0 +/- 0.3 x 10(-5) M, n = 4 (CM) and 3.8 +/- 0.4 x 10(-5) M, n = 9 (ISE); similar to phase 3 EC(50). Because NaHS has substantial vasoactive effects at physiological plasma concentrations, we propose that its soluble derivative, H(2)S, is a tonically active endogenous vasoregulator in trout.

Measurement of venous function in vivo is inherently difficult. In this study, we used the Hilbert transform to examine the dynamic relationships between venous pressure and cardiac output (CO) in rainbow trout whose blood volume was continuously increased and decreased by ramp infusion and withdrawal (I/W). The dorsal aorta and ductus Cuvier were cannulated percutaneously and connected to pressure transducers; a flow probe was placed around the ventral aorta. Whole blood from a donor was then I/W via the dorsal aortic cannula at a rate of 10% of the estimated blood volume per minute, and the duration of I/W was varied from 40, 60, 80, 90, 120, 230, 240, 260, 300, and 340 s. Compliance [change in (delta) blood vol/deltavenous pressure] was 2.8 +/- 0.2 ml x mmHg-1x g-1 (N = 25 measurements; 6 fish with closed pericardium) and 2.8 +/- 0.3 ml. mmHg-1x kg-1 (N = 19 measurements, 4 fish with open pericardium). Compliance was positively correlated with the duration of I/W, indicative of cardiovascular reflex responses at longer I/W durations. In trout with closed pericardium, CO followed venous pressure oscillations with an average time lag of 4.2 +/- 1.0 s (N = 9); heart rate (HR) was inversely correlated with CO. These studies show that CO is entrained by modulation of venous pressure, not by HR. Thus, although trout have a rigid pericardium, venous pressure (vis-a-tergo), not cardiac suction (vis-a-fronte), appears to be the primary determinant of CO. Estimation of venous compliance by ramp-modulation of venous pressure is faster and less traumatic than classical capacitance measurements and appears applicable to a variety of vertebrate species, as does the Hilbert transform, which permits analysis of signals with disparate frequencies.

Tunas (family: Scombridae, Tribe: Thunnini) exhibit anatomical, physiological, and biochemical adaptations that dramatically increase the ability of their cardiorespiratory systems to transfer oxygen from the water to the tissues. In the present study the vascular anatomy of the skipjack tuna, Katsuwonus pelamis, gill was examined by light and scanning electron microscopic analysis of methyl methacrylate vascular corrosion replicas prepared under physiological pressure. The gill filament contains three distinct blood pathways, respiratory, interlamellar, and nutrient. The respiratory, or arterio-arterial (AA) pathway, is the site of gas exchange and consists of the afferent and efferent filamental arteries (AFA and EFA) and arterioles (ALA and ELA) and the lamellae. Each ALA in the basal filament supplies ten or more lamellae and they anastomose with their neighbor to form a continuous vascular arcade. Four modifications in the lamellar circulation appear to enhance gas exchange efficiency. 1) The ALA deliver blood directly to the outer margin of the lamellae where unstirred boundary layer effects are predicted to be minimal and water PO2 highest. 2) Pillar cells are closely aligned along the outer boundary of the inlet side and the inner boundary of the outlet side of the lamellae to form multiple distributing and receiving blood channels. 3) Elsewhere in the lamella, pillar cells are aligned to form diagonal channels that direct blood from the outer to the inner lamellar margins, thereby reducing vascular resistance. 4) The lamellar sinusoid is especially widened near the efferent end to augment oxygen saturation of blood flowing through the inner margin. These adaptations, plus the presence of a bow-shaped interlamellar septum, and a thinned filament core appear to decrease gill vascular resistance and maximize gas-exchange efficiency. The interlamellar (IL) and nutrient systems originate from post-lamellar vessels and are arterio-venous (AV) pathways. IL vessels form an extensive ladder-like lattice on both sides of the filamental cartilage and are supplied in part by narrow-bore vessels from the medial wall of the EFA. Their function is unknown. Nutrient vessels are formed from the confluence of a myriad of tortuous, narrow-bore vessels arising from the basal region of the EFA and from efferent branchial arteries. They re-enter the filament and eventually drain into the IL system or filamental veins. As these AV pathways are retained despite considerable reduction in filamental tissue, it is evident that they are integral components of other non-respiratory homeostatic activities of the gill.

Natriuretic peptides (NPs) play important roles in osmoregulatory and cardiovascular systems of vertebrates. For functional studies of NPs, rainbow trout (Oncorhynchus mykiss), a euryhaline fish, is an interesting model. The information on homologous NPs of salmonid fish is, however, still incomplete with respect to C-type NP (CNP). In this study, we isolated cDNAs encoding the precursor of CNP from the brain of trout. Predicted mature CNP (CNP-22) sequence was identical to that of killifish Fundulus heteroclitus, and only one amino acid was different from that of the eel Anguilla japonica, demonstrating a greater conservation among different teleost species than is found with atrial NP (ANP) and ventricular NP (VNP). While the preprosegment of trout CNP retained 57% similarity to the eel sequence, similarities were low to those of sharks and tetrapods. The major site of expression identified by RT-PCR was the brain with minor expression in the atrium. The putative mature CNP-22 was synthesized and its biological activity was compared with other trout NPs (ANP and VNP) using trout ventral aorta, efferent branchial and celiacomesenteric arteries and anterior cardinal vein in vitro. Synthetic trout CNP-22 relaxed all pre-contracted vessels with potencies comparable to trout ANP and VNP.

The kinetics of transvascular fluid transport across fish capillaries and redistribution of fluids between intravascular compartments in intact fish are unknown. Cannulae were placed in the dorsal aorta (DA) and caudal vein (CV) of rainbow trout Oncorhynchus mykiss (mass 0.45-0.85 kg) and the fish spleenectomized. The following day a peristaltic pump was fitted to complete the extracorporeal arterio-venous circulation. Hematocrit (Hct) was monitored in unanesthetized fish either manually, by collecting blood from the extracorporeal loop at 5 min intervals for a period of 1 h (groups 1 and 2), or continuously (instantaneously) with an impedance flow-cell inserted in the aortic cannula (group 3). Fish in group 1 were volume expanded by injecting a volume of saline (0.9 g% NaCl; SI) or trout plasma (PI) equivalent to 40% of the plasma volume. In group 2, 20% or 35% of the blood volume was removed, and in group 3, 35% of the blood volume was removed. Plasma volume (Vp) was calculated from an assumed blood volume of 35 ml kg(-1) and the Hct. Vp declined mono-exponentially after SI with a half-time of 6.8 min and Vp reached a new steady state at 28.1 ml kg(-1); 30% of the injected volume remained in the vasculature. Volume recovery after PI was also mono-exponential, but slower (half-time=15.4 min) than SI, whereas the steady-state Vp (27.3 ml kg(-1)) was similar and 30% of the injected volume remained in the vasculature. Thus the presence of plasma proteins delayed fluid efflux from the vasculature, but did not affect the volume lost. Transvascular fluid filtration coefficients calculated from this data were 5.5 (SI) and 4.5 ml mmHg(-1) kg(-1) min(-1) (PI), and interstitial compliance was 11.8 (SI) and 9.7 ml mmHg(-1) kg(-1) (PI). The rate of volume recovery after 20% or 35% hemorrhage was independent of the hemorrhage volume (half-time=13.3 and 15.1 min, respectively) and similar to the half-time of PI, indicating that protein-rich interstitial fluid is returned to the vasculature. There is a nearly instantaneous change in Hct that occurs during the hemorrhage period; it is dependent on hemorrhage duration and volume and not associated with the subsequent mono-exponential recovery. This initial response is best explained by a rapid fluid shift from a large-volume (approximately 40% of total blood volume), low-hematocrit (less than half of systemic Hct) microcirculation into the higher-hematocrit macrocirculation. These studies are consistent with transcapillary fluid flux across a barrier that is highly permeable to protein, and cannot be explained by fluid shift between primary and secondary circulations, or by transcapilllary flux across a capillary bed that is impermeable to plasma proteins. The results support the hypothesis that whole-body reflection coefficients in trout are very low and that plasma oncotic pressure is not a determinant of transcapillary fluid balance. They also show that both transvascular and intravascular fluid movements are important effectors of central volume homeostasis.

The fish gill is the most physiologically diversified vertebrate organ, and its vasculature the most intricate. Application of vascular corrosion techniques that couple high-fidelity resins, such as methyl methacrylate, with scanning electron microscopy yields three-dimensional replicas of the microcirculation that have fostered a better appreciate gill perfusion pathways. This is the focus of the present review. Three vascular networks can be identified within the gill filament. The arterioarterial (respiratory) pathway consists of the lamellae and afferent and efferent segments of the branchial and filamental arteries and lamellar arterioles. The body of the filament contains two post-lamellar pathways: the interlamellar and nutrient. The interlamellar system is an extensive ladder-like network of thin-walled, highly distensible vessels that traverses the filament between, and parallel to, the lamellae and continues around the afferent and efferent borders of the filament. Interlamellar vessels are supplied by short, narrow-bore feeder vessels from the medial wall of the efferent filamental artery. A myriad of narrow-bore, tortuous arterioles arise from the basal efferent filamental artery and efferent branchial artery and anastomose to form the nutrient circulation of the arch and filament. In the filament body, nutrient capillaries and interlamellar vessels are often closely associated, and the former may ultimately drain into the latter. Many of the anatomical characteristics of interlamellar vessels are strikingly similar to those of mammalian lymphatic capillaries, with the exception that interlamellar vessels are directly fed by arteriovenous-like anastomoses. It is likely that gill interlamellar and mammalian lymphatics are physiologically, if not embryologically, equivalent., (Copyright 2002 Wiley-Liss, Inc.)

The fish gill is the primary regulatory interface between internal and external milieu and a variety of neurocrine, endocrine, paracrine, and autocrine signals coordinate and control gill functions. Many of these messengers also affect gill vascular resistance, and they, in turn, may be inactivated (or activated) by branchial vessels. Few studies have critically addressed how flow is distributed within the gill filament, the physiological consequences thereof, or the impact of gill hormone metabolism on gill and systemic homeostasis. In most fish, the entire cardiac output perfuses the arterioarterial pathway, and this network probably accounts for the majority of passive- and stimulus-induced changes in vascular resistance. The in-series arrangement of the extensive gill microcirculation with systemic vessels is also indicative of a high capacity for metabolism of plasma-borne messengers as well as xenobiotics. Adenosine, arginine vasotocin (AVT), and endothelin (ET) are the most potent gill constrictors identified to date, and all decrease lamellar perfusion. Perhaps not surprising, they are also inactivated by gill vessels. Acetylcholine favors perfusion of the alamellar filamental vasculature, although the physiological relevance of acetylcholine-mediated responses remains unclear. Angiotensin, bradykinin, urotensin, natriuretic peptides, prostaglandins, and nitric oxide are vasoactive to varying degrees, but their effects on intrafilamental blood flow are unknown. If form befits function, then the complex vascular anatomy of the gill suggests a level of regulatory sophistication unparalleled in other vertebrate organs. Resolution of these issues will be technically challenging but unquestionably rewarding., (Copyright 2002 Wiley-Liss, Inc.)

The tegmentum vasculosum of the duckling consists of a highly folded epithelium which extends over the dorsal and lateral walls of the cochlear duct, separating the scala media from the scala vestibuli. This epithelium consists of two distinct cell types, dark cells and light cells, and is well vascularized. The surface of the epithelium is formed by a mosaic of alternating dark and light cells. The goblet-shaped dark cells have an electron-dense, organelle-rich cytoplasm, and are expanded basally by extensive basolateral plasma membrane infoldings, within which are numerous mitochondria. Dark cells are isolated from each other and from the capillaries within the epithelium by intervening light cells. In contrast, columnar light cells exhibit an electron-lucent, organelle-poor cytoplasm and may extend from the underlying capillaries to the endolymphatic surface. Light cells contain abundant, coated endocytic vesicles on their apical surfaces and are bound, apically, to other light cells or to dark cells by tight junctions and desmosomes. Laterally, light cells are linked to each other either by complex, fluid-filled membrane interdigitations or by extensive gap junctions. Plasma membrane interdigitations and obvious, fluid-filled intercellular spaces characterize the lateral borders between light and dark cells. Vascular corrosion casting reveals the three-dimensional anatomy of the cochlear vasculature. A continuous arteriolar loop fed by anterior and posterior cochlear arterioles encircles the cochlear duct. The rich capillary beds of the tegmentum vasculosum are supplied by arching arterioles arising from this loop. These capillaries are the continuous type and are situated primarily within the core of the epithelium or along its border with the scala vestibuli. The structure and blood supply of the tegmentum vasculosum are characteristic of an epithelium involved in active transport.