Biomedical subjects

S F Perry

Publications and source records attributed to S F Perry.

At least 19 recordsLinked to original sources

PubMed2006

Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias).

Physiological and biochemical studies have provided indirect evidence for a membrane-associated carbonic anhydrase (CA) isoform, similar to mammalian type IV CA, in the gills of dogfish (Squalus acanthias). This CA isoform is linked to the plasma membrane of gill epithelial cells by a glycosylphosphatidylinositol anchor and oriented toward the plasma, such that it can catalyze the dehydration of plasma HCO(3)(-) ions. The present study directly tested the hypothesis that CA IV is present in dogfish gills in a location amenable to catalyzing plasma HCO(3)(-) dehydration. Homology cloning techniques were used to assemble a 1,127 base pair cDNA that coded for a deduced protein of 306 amino acids. Phylogenetic analysis suggested that this protein was a type IV CA. For purposes of comparison, a second cDNA (1,107 base pairs) was cloned from dogfish blood; it encoded a deduced protein of 260 amino acids that was identified as a cytosolic CA through phylogenetic analysis. Using real-time PCR and in situ hybridization, mRNA expression for the dogfish type IV CA was detected in gill tissue and specifically localized to pillar cells and branchial epithelial cells that flanked the pillar cells. Immunohistochemistry using a polyclonal antibody raised against rainbow trout type IV CA revealed a similar pattern of CA IV immunoreactivity and demonstrated a limited degree of colocalization with Na(+)-K(+)-ATPase immunoreactivity. The presence and localization of a type IV CA isoform in the gills of dogfish is consistent with the hypothesis that branchial membrane-bound CA with an extracellular orientation contributes to CO(2) excretion in dogfish by catalyzing the dehydration of plasma HCO(3)(-) ions.

Amino Acid Sequence↗

PubMed2006

Acid-base balance and CO2 excretion in fish: unanswered questions and emerging models.

Carbon dioxide (CO(2)) excretion and acid-base regulation in fish are linked, as in other animals, though the reversible reactions of CO(2) and the acid-base equivalents H(+) and HCO(3)(-): CO(2)+H(2)O<-->H(+)+HCO(3)(-). These relationships offer two potential routes through which acid-base disturbances may be regulated. Respiratory compensation involves manipulation of ventilation so as to retain CO(2) or enhance CO(2) loss, with the concomitant readjustment of the CO(2) reaction equilibrium and the resultant changes in H(+) levels. In metabolic compensation, rates of direct H(+) and HCO(3)(-) exchange with the environment are manipulated to achieve the required regulation of pH; in this case, hydration of CO(2) yields the necessary H(+) and HCO(3)(-) for exchange. Because ventilation in fish is keyed primarily to the demands of extracting O(2) from a medium of low O(2) content, the capacity to utilize respiratory compensation of acid-base disturbances is limited and metabolic compensation across the gill is the primary mechanism for re-establishing pH balance. The contribution of branchial acid-base exchanges to pH compensation is widely recognized, but the molecular mechanisms underlying these exchanges remain unclear. The relatively recent application of molecular approaches to this question is generating data, sometimes conflicting, from which models of branchial acid-base exchange are gradually emerging. The critical importance of the gill in acid-base compensation in fish, however, has made it easy to overlook other potential contributors. Recently, attention has been focused on the role of the kidney and particularly the molecular mechanisms responsible for HCO(3)(-) reabsorption. It is becoming apparent that, at least in freshwater fish, the responses of the kidney are both flexible and essential to complement the role of the gill in metabolic compensation. Finally, while respiratory compensation in fish is usually discounted, the few studies that have thoroughly characterized ventilatory responses during acid-base disturbances in fish suggest that breathing may, in fact, be adjusted in response to pH imbalances. How this is accomplished and the role it plays in re-establishing acid-base balance are questions that remain to be answered.

Acid-Base Equilibrium↗

PubMed2006

Roles of cytosolic and membrane-bound carbonic anhydrase in renal control of acid-base balance in rainbow trout, Oncorhynchus mykiss.

We tested the hypothesis that cytosolic and membrane-associated carbonic anhydrase (CA IV) are involved in renal urinary acidification and bicarbonate reabsorption in rainbow trout. With the use of homological cloning techniques, a 1,137-bp cDNA was assembled that included an open reading frame encoding for a deduced protein of 297 amino acids. Phylogenetic analysis revealed that this protein was likely a CA IV isoform. With the use of this sequence and a previously described trout cytosolic isoform [tCAc (13)], tools were developed to quantify and localize mRNA and protein for the two CA isoforms. Unlike tCAc, which displayed a broad tissue distribution, trout CA IV mRNA (and to a lesser extent protein) was highly and preferentially expressed in the posterior kidney. The results of in situ hybridization, immunocytochemistry, and standard histological procedures demonstrated that CA IV was likely confined to epithelial cells of the proximal tubule with the protein being expressed on both apical and basolateral membranes. The CA IV-containing tubule cells were enriched with Na(+)-K(+)-ATPase. Similar results were obtained for tCAc except that it appeared to be present in both proximal and distal tubules. The levels of mRNA and protein for tCAc increased significantly during respiratory acidosis (hypercapnia). Although tCA IV mRNA was elevated after 24 h of hypercapnia, tCA IV protein levels were unaltered. By using F3500, a membrane-impermeant (yet filtered) inhibitor of CA, in concert with blood and urine analyses, we demonstrated that CA IV (and possibly other membrane-associated CA isoforms) plays a role in urinary acidification and renal bicarbonate reabsorption.

Absorption↗

PubMed2006

Does bradycardia or hypertension enhance gas transfer in rainbow trout (Oncorhynchus mykiss)?

Experiments were conducted to test the hypothesis that branchial gas transfer is enhanced in rainbow trout during hypoxia or hypercarbia by bradycardia and systemic vasoconstriction. Gas transfer was indirectly assessed by continuous monitoring of arterial blood gases, PaO2 and PaCO2. Cardiac frequency was maximally decreased by 34.9+/-4.3 and 8.6+/-3.2 bpm in hypoxic and hypercarbic fish, respectively. Pre-treating fish with atropine (1micromol kg(-1)) attenuated or abolished the bradycardia during hypoxia and hypercarbia, respectively. However, there were no significant differences in the arterial blood gases between the control and atropinized fish. Dorsal aortic blood pressure was increased maximally by 11.3+/-2.8 and 17.7+/-2.0mm Hg in the hypoxic and hypercarbic fish. Pre-treatment of fish with prazosin (2.4micromol kg(-1)) prevented these increases in blood pressure. Blood gases were unaltered by prazosin treatment in the hypercarbic fish. However, in the hypoxic fish, gas transfer appeared to be impaired by prazosin on the basis of lowered PaO2 (by approximately 35 mm Hg compared to control fish) and increased PaCO2 (by approximately 0.3mm Hg). Because the normal hyperventilatory response to hypoxia was prevented by prazosin, it is possible that the impairment of gas transfer was related to inadequate ventilation rather than to any differences in the pressor response. The present results provide no evidence that gas transfer in rainbow trout is enhanced by bradycardia nor do they reveal any obvious benefit associated with the increases in blood pressure that accompany hypoxia and hypercarbia.

Adrenergic alpha-Antagonists↗

PubMed2006

Developmental plasticity of ventilatory control in zebrafish, Danio rerio.

To determine whether development of ventilatory control in zebrafish (Danio rerio) exhibits plasticity, embryos were exposed to hypoxia, hyperoxia or hypercapnia for the first 7 days post-fertilization. Their acute reflex breathing responses to ventilatory stimuli (hypoxia, hypercapnia and external cyanide) were assessed when they had reached maturity (3 months or older). Zebrafish reared under hyperoxic conditions exhibited significantly higher breathing frequencies at rest (283+/-27min(-1) versus 212+/-16min(-1) in control fish); breathing frequency was unaffected in adult fish subjected to hyperoxia for 7 days. The respiratory responses of fish reared in hyperoxic water to acute hypoxia, hypercapnia or external cyanide were blunted (hypoxia, cyanide) or eliminated (hypercapnia). Adult fish exposed for 7 days to hyperoxia showed no change in acute responses to these stimuli. The respiratory responses to acute hypoxia, hypercapnia or external cyanide of fish reared under hypoxic or hypercapnic conditions were similar to those in fish reared under normal conditions. A subset of all fish examined exhibited episodic breathing; an analysis of breathing patterns demonstrated that fish reared under hypercapnic conditions had an increased tendency to display episodic breathing. The results of this study reveal that there is flexibility in the design and functioning of the embryonic or larval respiratory system in zebrafish.

Aging↗

PubMed2006

The role of branchial carbonic anhydrase in acid-base regulation in rainbow trout (Oncorhynchus mykiss).

The objective of the present study was to examine the branchial distribution of the recently identified rainbow trout cytoplasmic carbonic anhydrase isoform (tCAc) and to investigate its role in the regulation of acid-base disturbances in rainbow trout (Oncorhynchus mykiss). In situ hybridization using an oligonucleotide probe specific to tCAc revealed tCAc mRNA expression in both pavement cells and mitochondria-rich cells (chloride cells). Similarly, using a homologous polyclonal antibody, tCAc immunoreactivity was localized to pavement cells and mitochondria-rich cells in the interlamellar region and along the lamellae of the gills. Exposure of rainbow trout to hypercarbia (approximately 0.8% CO2) for 24 h resulted in significant increases in tCAc mRNA expression (approximately 20-fold; quantified by real-time PCR) and protein levels (approximately 1.3-fold; quantified by western analysis) but not enzyme activity (assessed on crude gill homogenates using the delta-pH CA assay). Inhibition of branchial CA activity in vivo using acetazolamide reduced branchial net acid excretion significantly by 20%. This effect was enhanced to a 36% reduction in branchial net acid excretion by subjecting the trout to hypercarbia (approximately 0.8% CO2) for 10 h prior to acetazolamide injection, an exposure that significantly increased branchial net acid excretion. The results of the present study support the widely held premise that branchial intracellular CA activity (tCAc) plays a key role in regulating acid-base balance in freshwater teleost fish.

Acid-Base Equilibrium↗

PubMed2006

Chemoreceptor plasticity and respiratory acclimation in the zebrafish Danio rerio.

The goals of this study were to assess the respiratory consequences of exposing adult zebrafish Danio rerio to chronic changes in water gas composition (hypoxia, hyperoxia or hypercapnia) and to determine if any ensuing effects could be related to morphological changes in branchial chemoreceptors. To accomplish these goals, we first modified and validated an established non-invasive technique for continuous monitoring of breathing frequency and relative breathing amplitude in adult fish. Under normal conditions 20% of zebrafish exhibited an episodic breathing pattern that was composed of breathing and non-breathing (pausing/apneic) periods. The pausing frequency was reduced by acute hypoxia (Pw(O)2<130 mmHg) and increased by acute hyperoxia (Pw(O)2>300 mmHg), but was unaltered by acute hypercapnia. Fish were exposed for 28 days to hyperoxia (Pw(O)2>350 mmHg), or hypoxia (Pw(O)2=30 mmHg) or hypercapnia (Pw(CO)2=9 mmHg). Their responses to acute hypoxia or hypercapnia were then compared to the response of control fish kept for 28 days in normoxic and normocapnic water. In control fish, the ventilatory response to acute hypoxia consisted of an increase in breathing frequency while the response to acute hypercapnia was an increase in relative breathing amplitude. The stimulus promoting the hyperventilation during hypercapnia was increased Pw(CO)2 rather than decreased pH. Exposure to prolonged hyperoxia decreased the capacity of fish to increase breathing frequency during hypoxia and prevented the usual increase in breathing amplitude during acute hypercapnia. In fish previously exposed to hyperoxia, episodic breathing continued during acute hypoxia until Pw(O)2 had fallen below 70 mmHg. In fish chronically exposed to hypoxia, resting breathing frequency was significantly reduced (from 191+/-12 to 165+/-16 min(-1)); however, the ventilatory responses to hypoxia and hypercapnia were unaffected. Long-term exposure of fish to hypercapnic water did not markedly modify the breathing response to acute hypoxia and modestly blunted the response to hypercapnia. To determine whether branchial chemoreceptors were being influenced by long-term acclimation, all four groups of fish were acutely exposed to increasing doses of the O(2) chemoreceptor stimulant, sodium cyanide, dissolved in inspired water. Consistent with the blunting of the ventilatory response to hypoxia, the fish pre-exposed to hyperoxia also exhibited a blunted response to NaCN. Pre-exposure to hypoxia was without effect whereas prior exposure to hypercapnia increased the ventilatory responses to cyanide. To assess the impact of acclimation to varying gas levels on branchial O(2) chemoreceptors, the numbers of neuroepithelial cells (NECs) of the gill filament were quantified using confocal immunofluorescence microscopy. Consistent with the blunting of reflex ventilatory responses, fish exposed to chronic hyperoxia exhibited a significant decrease in the density of NECs from 36.8+/-2.8 to 22.7+/-2.3 filament(-1).

Adaptation, Physiological↗

PubMed2006

Characterization of a branchial epithelial calcium channel (ECaC) in freshwater rainbow trout (Oncorhynchus mykiss).

The entry of calcium (Ca2+) through an apical membrane epithelial calcium channel (ECaC) is thought to a key step in piscine branchial Ca2+ uptake. In mammals, ECaC is a member of the transient receptor potential (TRP) gene family of which two sub-families have been identified, TRPV5 and TPRV6. In the present study we have identified a single rainbow trout (Oncorhynchus mykiss) ECaC (rtECaC) that is similar to the mammalian TRPV5 and TRPV6. Phylogenetic analysis of the protein sequence suggests that an ancestral form of the mammalian genes diverged from those in the lower vertebrates prior to the gene duplication event that gave rise to TRPV5 and TRPV6. The putative model for Ca2+ uptake in fish proposes that the mitochondria-rich cell (also termed ionocyte or chloride cell) is the predominant or exclusive site of transcellular Ca2+ movements owing to preferential localisation of ECaC to the apical membrane of these cells. However, the results of real-time PCR performed on enriched gill cell populations as well as immunocytochemistry and in situ hybridisation analysis of enriched cells, cell cultures and whole gill sections strongly suggest that ECaC is not exclusive to mitochondria-rich cells but that it is also found in pavement cells. Not only was ECaC protein localized to areas of the gill normally having few mitochondria-rich cells, but there was also no consistent co-localization of ECaC- and Na+/K+-ATPase-positive (a marker of mitochondria rich cells) cells. Taken together, the results of the present study suggest that although ECaC (mRNA and protein) does exist in trout gill, its cellular distribution is more extensive than previously thought, thus suggesting that Ca2+ uptake may not be restricted to mitochondria-rich cells as was proposed in previous models.

Amino Acid Sequence↗

PubMed2005

Pulmonary oxygen diffusing capacity of the South American lungfish Lepidosiren paradoxa: physiological values by the Bohr method.

Lungfish (Dipnoi) may represent the sister group to all land vertebrates and are therefore important for reconstructing the conquest of land by tetrapods. We determined venous and arterial blood gases, pulmonary O(2) uptake, and the form of the hemoglobin-O(2) dissociation curves in the South American lungfish Lepidosiren paradoxa. Measurements were performed at 25 degrees and 35 degrees C. Based on this information, we calculated its pulmonary O(2) diffusing capacity (D(L)O(2)), using the Bohr integration procedure. D(L)O(2) increased with temperature to reach about 0.04 mL stpd kg(-1) min(-1) mmHg(-1) at 35 degrees C. This value represents about 40% of the morphometric diffusing capacity and is similar to physiological values in some amphibians and reptiles.

Analysis of Variance↗

PubMed2005

Circulating catecholamines and cardiorespiratory responses in hypoxic lungfish (Protopterus dolloi): a comparison of aquatic and aerial hypoxia.

Circulating catecholamine levels and a variety of cardiorespiratory variables were monitored in cannulated bimodally breathing African lungfish (Protopterus dolloi) exposed to aquatic or aerial hypoxia. Owing to the purported absence of external branchial chemoreceptors in lungfish and the minor role played by the gill in O2 uptake, it was hypothesized that plasma catecholamine levels would increase only during exposure of fish to aerial hypoxia. The rapid induction of aquatic hypoxia (final PWo2 = 25.9+/-1.6 mmHg) did not affect the levels of adrenaline (A) or noradrenaline (NA) within the plasma. Similarly, none of the measured cardiorespiratory variables--including heart rate (fH), blood pressure, air-breathing frequency (fV), O2 consumption (Mo2), CO2 excretion (Mco2), or blood gases--were influenced by acute aquatic hypoxia. In contrast, however, the rapid induction of aerial hypoxia (inspired Po2=46.6+/-3.3 mmHg) caused a marked increase in the circulating levels of A (from 7.9+/-2.0 to 18.8+/-6.1 nmol L(-1)) and NA (from 7.7+/-2.2 to 19.7+/-6.3 nmol L(-1)) that was accompanied by significant decreases in Mo2, arterial Po2 (Pao2), and arterial O2 concentration (Cao2). Air-breathing frequency was increased (by approximately five breaths per hour) during aerial hypoxia and presumably contributed to the observed doubling of pulmonary Mco2 (from 0.25+/-0.04 to 0.49+/-0.07 mmol kg(-1) h(-1)); fH and blood pressure were unaffected by aerial hypoxia. An in situ perfused heart preparation was used to test the possibility that catecholamine secretion from cardiac chromaffin cells was being activated by a direct localized effect of hypoxia. Catecholamine secretion from the chromaffin cells of the heart, while clearly responsive to a depolarizing concentration of KCl (60 mmol L(-1)), was unaffected by the O2 status of the perfusion fluid. The results of this study demonstrate that P. dolloi is able to mobilize stored catecholamines and increase f(V) during exposure to aerial hypoxia while remaining unresponsive to aquatic hypoxia. Thus, unlike in exclusively water-breathing teleosts, P. dolloi would appear to rely solely on internal/airway O2 chemoreceptors for initiating catecholamine secretion and cardiorespiratory responses.

Analysis of Variance↗

PubMed2005

Cardiorespiratory responses to hypercarbia in tambaqui Colossoma macropomum: chemoreceptor orientation and specificity.

Experiments were carried out to test the hypothesis that ventilatory and cardiovascular responses to hypercarbia (elevated water P(CO2)) in the tambaqui Colossoma macropomum are stimulated by externally oriented receptors that are sensitive to water CO(2) tension as opposed to water pH. Cardiorespiratory responses to acute hypercarbia were evaluated in both the absence and presence of internal hypercarbia (elevated blood P(CO2)), achieved by treating fish with the carbonic anhydrase inhibitor acetazolamide. Exposure to acute hypercarbia (15 min at each level, final water CO(2) tensions of 7.2, 15.5 and 26.3 mmHg) elicited significant increases in ventilation frequency (at 26.3 mmHg, a 42% increase over the normocarbic value) and amplitude (128%), together with a fall in heart rate (35%) and an increase in cardiac stroke volume (62%). Rapid washout of CO(2) from the water reversed these effects, and the timing of the changes in cardiorespiratory variables corresponded more closely to the fall in water P(CO2) (Pw(CO2)) than to that in blood P(CO2) (Pa(CO2)). Similar responses to acute hypercarbia (15 min, final Pw(CO2) of 13.6 mmHg) were observed in acetazolamide-treated (30 mg kg(-1)) tambaqui. Acetazolamide treatment itself, however, increased Pa(CO2) (from 4.81+/-0.58 to 13.83+/-0.91 mmHg, mean +/-S.E.M.; N=8) in the absence of significant change in ventilation, heart rate or cardiac stroke volume. The lack of response to changes in blood P(CO2) and/or pH were confirmed by comparing responses to the bolus injection of hypercarbic saline (5% or 10% CO(2); 2 ml kg(-1)) into the caudal vein with those to the injection of CO(2)-enriched water (1%, 3%, 5% or 10% CO(2); 50 ml kg(-1)) into the buccal cavity. Whereas injections of hypercarbic saline were ineffective in eliciting cardiorespiratory responses, changes in ventilation and cardiovascular parameters accompanied injection of CO(2)-laden water into the mouth. Similar injections of CO(2)-free water acidified to the corresponding pH of the hypercarbic water (pH 6.3, 5.6, 5.3 or 4.9, respectively) generally did not stimulate cardiorespiratory responses. These results are in agreement with the hypothesis that in tambaqui, externally oriented chemoreceptors that are predominantly activated by increases in water P(CO2), rather than by accompanying decreases in water pH, are linked to the initiation of cardiorespiratory responses to hypercarbia.

Acetazolamide↗

PubMed2005

Activity of three muscles associated with the uncinate processes of the giant Canada goose Branta canadensis maximus.

The activity of three muscles associated with the uncinate processes, (i) external intercostal, (ii) appendicocostalis and (iii) external oblique, were examined using patch and sew-through electrodes during sitting, standing and moderate speed treadmill running in the giant Canada goose. The external intercostal muscles demonstrated no respiratory activity, being active only during running, suggesting they play some role in trunk stabilisation. The appendicocostalis and external oblique muscles are respiratory muscles, being active during inspiration and expiration, respectively. The activity of the appendicocostalis muscles increased during sitting, suggesting that the uncinate processes in birds play an important role in facilitating lateral flaring of the rib cage when sternal rocking is prevented. We suggest that the uncinate processes in birds facilitate movements of the ribs and sternum during breathing and therefore are integral to the breathing mechanics of birds.

Animals↗

PubMed2005

Cytoplasmic carbonic anhydrase isozymes in rainbow trout Oncorhynchus mykiss: comparative physiology and molecular evolution.

It is well established that the gills of teleost fish contain substantial levels of cytoplasmic carbonic anhydrase (CA), but it is unclear which CA isozyme(s) might be responsible for this activity. The objective of the current study was to determine if branchial CA activity in rainbow trout was the result of a general cytoplasmic CA isozyme, with kinetic properties, tissue distribution and physiological functions distinct from those of the red blood cell (rbc)-specific CA isozyme. Isolation and sequencing of a second trout cytoplasmic CA yielded a 780 bp coding region that was 76% identical with the trout rbc CA (TCAb), although the active sites differed by only 1 amino acid. Interestingly, phylogenetic analyses did not group these two isozymes closely together, suggesting that more fish species may have multiple cytoplasmic CA isozymes. In contrast to TCAb, the second cytoplasmic CA isozyme had a wide tissue distribution with high expression in the gills and brain, and lower expression in many tissues, including the red blood cells. Thus, unlike TCAb, the second isozyme lacks tissue specificity and may be expressed in the cytoplasm of all cells. For this reason, it is referred to hereafter as TCAc (trout cytoplasmic CA). The inhibitor properties of both cytoplasmic isozymes were similar (Ki acetazolamide 1.21+/-0.18 nmol l(-1) and 1.34+/-0.10 nmol l(-1) for TCAc and TCAb, respectively). However, the turnover of TCAb was over three times greater than that of TCAc (30.3+/-5.83 vs 8.90+/-1.95 e4 s(-1), respectively), indicating that the rbc-specific CA isoform was significantly faster than the general cytoplasmic isoform. Induction of anaemia revealed differential expression of the two isozymes in the red blood cell; whereas TCAc mRNA expression was unaffected, TCAb mRNA expression was significantly increased by 30- to 60-fold in anaemic trout.

Amino Acid Sequence↗

PubMed2005

Nitric oxide and the control of catecholamine secretion in rainbow trout Oncorhynchus mykiss.

An in situ saline-perfused posterior cardinal vein preparation was used to assess the role of nitric oxide (NO) in the regulation of basal and stimulus-evoked catecholamine secretion from rainbow trout Oncorhynchus mykiss chromaffin cells. Addition of the NO donor, sodium nitroprusside (SNP) to the inflowing perfusate abolished catecholamine secretion during electrical field stimulation, thereby establishing the potential for NO to act as a potent inhibitor of catecholamine release. A possible role for endogenously produced NO was established by demonstrating that stimulus-evoked (depolarizing levels of KCl or electrical field stimulation) catecholamine secretion was markedly stimulated in the presence of the nitric oxide synthase (NOS) inhibitors l-NAME and 7-NI. Although in vitro experiments demonstrated that catecholamine degradation was enhanced by NO in a dose-dependent manner, the dominant factor contributing to the reduction in catecholamine appearance in the perfusate was specific inhibition of catecholamine secretion. Subsequent experiments were performed to identify the NOS isoform(s) contributing to the inhibition of stimulus-evoked catecholamine secretion. Inducible NOS (iNOS; an enzyme that can be activated in the absence of Ca2+), although present in the vicinity of the chromaffin cells (based on mRNA measurements), does not appear to play a role because stimulus-evoked NO production was eliminated during perfusion with Ca2+-free saline. The potential involvement of endothelial NOS (eNOS) was revealed by showing that hypoxic perfusate evoked NO production and corresponded with an inhibition of stimulus-evoked catecholamine secretion; chemical removal of the endothelium (using saponin) prevented the production of NO during hypoxia. However, because removal of the endothelium did not affect NO production during electrical field stimulation, it would appear that the neuronal form of NOS (nNOS) is the key isoform modulating catecholamine secretion from trout chromaffin cells.

Analysis of Variance↗

PubMed2005

An investigation of the role of carbonic anhydrase in aquatic and aerial gas transfer in the African lungfish Protopterus dolloi.

Experiments were performed on bimodally breathing African lungfish Protopterus dolloi to examine the effects of inhibition of extracellular vs total (extracellular and intracellular) carbonic anhydrase (CA) activity on pulmonary and branchial/cutaneous gas transfer. In contrast to previous studies on Protopterus, which showed that the vast majority of CO(2) is excreted into the water through the gill and/or skin whereas O(2) uptake largely occurs via the lung, P. dolloi appeared to use the lung for the bulk of both O(2) uptake (91.0+/-2.9%) and CO(2) excretion (76.0+/-6.6%). In support of the lung as the more important site of CO(2) transfer, aerial hypercapnia (P(CO(2))=40 mmHg) caused a significant rise in partial pressure of arterial blood CO(2) (Pa(CO(2))) whereas a similar degree of aquatic hypercapnia was without effect on Pa(CO(2)). Intravascular injection of low levels (1.2 mg kg(-1)) of the slowly permanent CA inhibitor, benzolamide, was without effect on red blood cell CA activity after 30 min, thus confirming its suitability as a short-term selective inhibitor of extracellular CA. Benzolamide treatment did not affect CO(2) excretion, blood acid-base status or any other measured variable within the 30 min measurement period. Injection of the permeant CA inhibitor acetazolamide (30 mg kg(-1)) resulted in the complete inhibition of red cell CA activity within 10 min. However, CO(2) excretion (measured for 2 h after injection) and arterial blood acid-base status (assessed for 24 h after injection) were unaffected by acetazolamide treatment. Intra-arterial injection of bovine CA (2 mg kg(-1)) caused a significant increase in overall CO(2) excretion (from 0.41+/-0.03 to 0.58+/-0.03 mmol kg(-1) h(-1)) and an increase in air breathing frequency (from 19.0+/-1.3 to 24.7+/-1.8 breaths min(-1)) that was accompanied by a slight, but significant, reduction in Pa(CO(2)) (from 21.6+/-1.6 to 19.6+/-1.8 mmHg). The findings of this study are significant because they (i) demonstrate that, unlike in other species of African lungfish that have been examined, the gill/skin is not the major route of CO(2) excretion in P. dolloi, and (ii) suggest that CO(2) excretion in Protopterus may be less reliant on carbonic anhydrase than in most other fish species.

Acetazolamide↗

PubMed2004

Buffering limits plasma HCO3- dehydration when red blood cell anion exchange is inhibited.

Theory suggests that HCO3- dehydration in the plasma of rainbow trout is limited by both the absence of carbonic anhydrase (CA) activity and the low non-bicarbonate buffer capacity of the plasma (betaplasma). The potential for betaplasma to limit plasma HCO3- dehydration was assessed in rainbow trout in which HCO3- dehydration via the red blood cell (RBC) was inhibited using the anion exchange blocker 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). DIDS administration reduced the rate of RBC HCO3- dehydration by 68-80% for at least 6h, resulting in the elevation of arterial CO2 tension (PaCO2) by 3.07 +/- 0.45 Torr (N = 6). Addition of bovine CA to the circulation of DIDS-treated trout caused PaCO2 to decrease significantly. This effect was increased significantly in rainbow trout in which betaplasma was elevated experimentally by intravascular injection of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), supporting the hypothesis that CA-catalysed HCO3- dehydration in the plasma of rainbow trout is limited by proton availability.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2004

A comparison of adrenergic stress responses in three tropical teleosts exposed to acute hypoxia.

Experiments were performed to assess the afferent and efferent limbs of the hypoxia-mediated humoral adrenergic stress response in selected hypoxia-tolerant tropical fishes that routinely experience environmental O(2) depletion. Plasma catecholamine (Cat) levels and blood respiratory status were measured during acute aquatic hypoxia [water Po(2) (Pw(O(2))) = 10-60 mmHg] in three teleost species, the obligate water breathers Hoplias malabaricus (traira) and Piaractus mesopotamicus (pacu) and the facultative air breather Hoplerythrinus unitaeniatus (jeju). Traira displayed a significant increase in plasma Cat levels (from 1.3 +/- 0.4 to 23.3 +/- 15.1 nmol/l) at Pw(O(2)) levels below 20 mmHg, whereas circulating Cat levels were unaltered in pacu at all levels of hypoxia. In jeju denied access to air, plasma Cat levels were increased markedly to a maximum mean value of 53.6 +/- 19.1 nmol/l as Pw(O(2)) was lowered below 40 mmHg. In traira and jeju, Cat release into the circulation occurred at abrupt thresholds corresponding to arterial Po(2) (Pa(O(2))) values of approximately 8.5-12.5 mmHg. A comparison of in vivo blood O(2) equilibration curves revealed low and similar P(50) values (i.e., Pa(O(2)) at 50% Hb-O(2) saturation) among the three species (7.7-11.3 mmHg). Thus Cat release in traira and jeju occurred as blood O(2) concentration was reduced to approximately 50-60% of the normoxic value. Intravascular injections of nicotine (600 nmol/kg) elicited pronounced increases in plasma Cat levels in traira and jeju but not in pacu. Thus the lack of Cat release during hypoxia in pacu may reflect an inoperative or absent humoral adrenergic stress response in this species. When allowed access to air, jeju did not release Cats into the circulation at any level of aquatic hypoxia. The likeliest explanation for the absence of Cat release in these fish was that air breathing, initiated by aquatic hypoxia, prevented Pa(O(2)) values from falling to the critical threshold required for Cat secretion. The ventilatory responses to hypoxia in each species were similar, consisting generally of increases in both frequency and amplitude. These responses were not synchronized with or influenced by plasma Cat levels. Thus the acute humoral adrenergic stress response does not appear to stimulate ventilation during acute hypoxia in these tropical species.

Animals↗

PubMed2004

Branchial membrane-associated carbonic anhydrase activity maintains CO2 excretion in severely anemic dogfish.

Plasma CO(2) reactions in Pacific spiny dogfish (Squalus acanthias) have access to plasma and gill membrane-associated carbonic anhydrase (CA). Acute severe experimental anemia and selective CA inhibitors were used to investigate the role of extracellular CA in CO(2) excretion. Anemia was induced by blood withdrawal coupled to volume replacement with saline. Lowering hematocrit from 14.2 +/- 0.4% (mean +/- SE; N = 31) to 5.2 +/- 0.1% (N = 31) had no significant impact on arterial or venous CO(2) tensions (Pa(CO(2)) and Pv(CO(2)), respectively) over the subsequent 2 h. PCO(2) was maintained despite the reduction in red cell number and a significant 32% increase in cardiac output (V(b)), both of which have been found to cause Pa(CO(2)) increases in teleost fish. By contrast, treatment of anemic dogfish with the CA inhibitors benzolamide (1.3 mg/kg) or F3500 (50 mg/kg), to selectively inhibit extracellular CA, elicited rapid and significant increases in Pa(CO(2)) of 0.68 +/- 0.17 Torr (N = 6) and 0.53 +/- 0.11 Torr (N = 7), respectively, by 30 min after treatment. These findings provide a functional context in which extracellular CA in dogfish contributes substantially to CO(2) excretion. Additionally, the apparent lack of effect of V(b) changes on PCO(2) suggests that, in contrast to teleost fish, CO(2) excretion in dogfish does not behave as a diffusion-limited system.

Acid-Base Equilibrium↗