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First published online June 15, 2006
Journal of Experimental Biology 209, 2586-2594 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02290
Effect of pH on trout blood vessels and gill vascular resistance
Indiana University School of Medicine-South Bend Center, 1234 Notre Dame Avenue, South Bend, IN 46617, USA and Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA
* Author for correspondence (e-mail: olson.1{at}nd.edu)
Accepted 20 April 2006
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Summary |
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Key words: pH, fish, rainbow trout, Oncorhynchus mykiss, vessels, gills, vascular tone
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;
Aalkjær and Peng, 1997;
Smith et al., 1998;
Austin and Wray, 2000;
Wray and Smith, 2004). Because
it is somewhat difficult to manipulate pHo and pHi
independently, apparent pHo-dependent vascular responses may be due
to concomitant changes in pHi
(Roos and Boron, 1981;
Smith et al., 1998;
Wray and Smith, 2004).
However, pHo may be more physiologically relevant when considering
the effects of acid–base status on vascular resistance, because most (if
not all) acid–base disturbances arise from non-vascular tissue.
pH-induced vasoactivity is most often correlated with changes in intracellular
calcium ([Ca2+i]), which is then followed by a
corresponding change in force
(Aalkjær and Poston,
1996; Aalkjær and Peng,
1997; Smith et al.,
1998; Austin and Wray,
2000; Wray and Smith,
2004). Much of this Ca2+ may be from an extracellular
source as L-type Ca2+ channels have been shown to be closed by
acidosis and opened by alkalosis. Other pH-sensitive targets that have been
identified include potassium channels, intracellular Ca2+ stores
(sarcoplasmic reticulum; SR), and Ca2+-ATPases involved in
translocating Ca2+ into the SR (SERCA pumps). Upon these
generalizations there is a continuum of variables that include differences
between vessels, differences between resting or pre-contracted vessels, the
nature of the stimulant used for precontraction, and the mechanism with which
the change in pH is achieved.
These vagaries notwithstanding, acidotic vasodilation and alkalotic
vasoconstriction are assumed to be physiologically significant. In systemic
vessels, where blood flow is coupled to metabolism, it is easy to envision how
acidosis attendant with hypoxia would contribute to the well-known hypoxic
vasodilation. Conversely, hypoxia contracts pulmonary arteries
(Madden et al., 1992), and
although both acidosis and alkalosis have been reported to produce
vasoconstriction (Krampetz and Rhoades,
1991), Madden et al. (Madden
et al., 2001) clearly showed in canine pulmonary arteries that the
effects of hypoxia on vessel tension and pHi depend on the size of
the vessel; hypoxia relaxes large vessels and decreases pHi,
whereas it contracts small arteries and increases pHi. Thus,
although the responses of systemic and large pulmonary arteries to hypoxia are
different from those of small pulmonary arteries, all responses appear to be
consistent with a corresponding change in pHi. It should be noted,
however, that a mechanistic link between hypoxia and pHi has not
been clearly demonstrated (Taggart and
Wray, 1998) and it is unclear if this connection is
coincidental.
Acid–base status in mammals is largely regulated from within by the
interplay between metabolism, ventilation and renal function. Other than diet,
there is little, if any, environmental load of either acid or base
equivalents. Conversely, acid–base status in fish is strongly linked to
the environment. Ambient water is a large sink for respiratory CO2.
This keeps blood PCO2 low (
1–2 mmHg)
(Janssen and Randall, 1975),
but increases sensitivity of blood pH to ambient
PCO2. Many aquatic environments experience
rapid and large variations in ambient PO2,
PCO2 and pH, singularly and in various
combinations, and these have a substantial impact on blood pH
(Dejours, 1972;
Janssen and Randall, 1975;
Thomas and Le Ruz, 1982).
Although the effect of acid–base disturbances on the fish heart has been
examined in some detail (Farrell et al.,
1983), there is scant information regarding the effects of pH on
fish blood vessels (Canty and Farrell,
1985).
The purpose of the present experiments was to examine the effect of
pHo and pHi on fish VSM in vitro. Afferent and efferent
branchial (ABA and EBA) and celiaco-mesenteric (CMA) arteries, ventral aorta
(VA) and anterior cardinal veins (ACV) were mounted in myograph chambers and
the vasoactive effects of manipulating pHo, and in some instances
(EBA) pHi, were examined in unstimulated vessels and in vessels
pre-contracted with ligand- and voltage-mediated agonists. We also examined
the effects of pHo on vascular resistance of the isolated perfused
gill whose sensitivity to hypoxia (Smith
et al., 2001), multifunctionality
(Olson, 2002) and close
apposition to the environment make it especially relevant.
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Materials and methods |
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Isolated vessels
Steelhead were anesthetized in (25 mg l–1) tricaine
methanesulfonate (MS-222) and efferent branchial arteries (EBA) from the third
and fourth gill arches, afferent branchial arteries (ABA) from the first arch,
ventral aorta (VA), anterior cardinal vein (ACV), and coeliaco-mesenteric
arteries (CMA) were removed, gently cleaned of extra-adventitial tissue, and
placed in 4°C Hepes-buffered saline. Vessels were cut transaxially into 2-
to 3-mm-long segments, individually mounted on 280 µm diameter stainless
steel hooks and suspended in 5 or 20 ml water-jacketed (12–14°C)
smooth muscle chambers. The baths were aerated with room air (Hepes buffer) or
21% O2 1% CO2 78% N2 (Cortland buffer).
Tension was measured by a Grass FT-03 force-displacement transducer and
recorded on a Grass model 7 polygraph calibrated to detect tension changes as
low as 5 mg. Analog data was digitally converted by computer interface and
written directly to disk by Labtech Notebook (Laboratory Technologies,
Andover, MA, USA) and displayed with Sigmaplot software (Jandel Scientific,
San Rafael, CA, USA). Resting tension (equilibrium tension) of 500–1000
mg was applied to all arteries and 300 mg was applied to the ACV. The rings
were then allowed to stabilize for at least 30 min, contracted with 80 mmol
l–1 KCl, washed with buffer, and allowed to equilibrate an
additional 30 min before experimentation. Resting tension was continuously
adjusted to the desired level during this period. The response of EBAs to
contractile agonists and pH was generally more pronounced than those of other
vessels and these vessels were selected for additional study.
pHo was adjusted by replacing the entire bath with pretitrated
14°C buffers. The ammonium-pulse technique was used to change
pHi. With this technique, 40 mmol l–1
NH4Cl is added to the bath and the dissociate NH3
rapidly diffuses into the cell and produces a transient intracellular
alkalosis as the NH3 buffers intracellular H+
(Roos and Boron, 1981). After
the cells have presumably re-established pHi (30 min), the
bath is replaced with ammonium-free buffer creating an outward diffusion of
NH3 and a transient intracellular acidosis. Other
agonists/antagonists present during the pHo perturbation were added
to the new buffers prior to addition to the cells. The L-type calcium channel
blocker methoxyverapamil (D600, 0.1 mmol–1) was added 30 min
prior to experimentation.
Perfused gills
Rainbow trout were killed by a blow to the head and vessels removed as
above, or the gill arches isolated and cannulated as described elsewhere
(Olson et al., 1986). Briefly,
after the fish was stunned, the heart was exposed through a midventral
incision and 1 ml of heparinized (15 mg ml–1) saline (0.9 g%
NaCl) was injected into the ventricle and allowed to circulate for 2 min.
The head was then severed and the second pair of gill arches were isolated.
The afferent branchial artery was cannulated with an 18-gauge beveled
(45°) needle connected to polyethylene tubing (PE-90). A `T' was inserted
8–10 cm proximal to the cannula through which perfusion pressure could
be monitored via a Gould-Statham (Detroit, MI, USA) pressure
transducer and Grass polygraph (Astromed, Providence, RI, USA). The gill was
suspended in aerated tapwater (<1 mOsm) and continuously perfused with
filtered (0.2 µm) phosphate-buffered saline (PBS) at 12°C via
a peristaltic pump. Pump speed was adjusted (nominally 0.6 ml
min–1) to produce stable input pressures of 40 mmHg (1
mmHg=133 Pa). This was estimated to be equivalent to 35 mmHg at the gill
arch. A four-way stopcock on the aspiration end of the pump was used to switch
between perfusates without interrupting flow. All treatments were perfused for
a sufficient time (10 min or more) to achieve steady-state input
pressures.
Chemicals
The composition of Hepes
(N-2-hydroxyethyliperazine-N'-2-ethanesulfonic acid)
buffer was (in mmol l–1): NaCl, 145; KCl, 3; MgSO4
7H2O, 0.57; CaCl2 2H2O, 2; Hepes acid, 3;
Hepes Na+ salt, 7; glucose, 5. The composition of Cortland saline
was (in mmol l–1): NaCl, 124; KCl, 3; CaCl2
2H2O, 2; MgSO4 7H2O, 1.1;
NaH2PO4, 0.09; Na2HPO4, 1.8;
NaHCO3; glucose, 5.5. The composition of PBS was as follows (in
mmol l–1): NaCl, 126; KCl, 4.16; CaCl2
2H2O, 0.68; MgSO4 7H2O, 0.57;
KH2PO4, 3.38; Na2HPO4, 14.23;
glucose, 5. Buffers were set to pH 7.8 (control) or adjusted to the desired pH
with 1 mol l–1 NaOH or 1 mol l–1 HCl.
Changes in bath or perfusate pH were made by complete exchange of appropriate
buffer at constant temperature. If pH was changed during an agonist
contraction, the agonist was added to the new buffer prior to exchange.
Choice of contractile agonist and dose (50–80% of maximal
contraction; EC50 to EC80) was based on previous
experience with these vessels. Stock solutions were prepared as follows:
arginine vasotocin (AVT; 1 µmol l–1), acetylcholine (ACh;
10 mmol l–1), the thromboxane A2 mimetics U-44069
or U-46619; 10 mmol l–1) and epinephrine (EPI; 10 mmol
l–1). Propanolol (final concentration 0.1 mmol
l–1) was added to the baths 15 min prior to EPI to block
ß-receptor-mediated relaxation in CMAs
(Olson and Meisheri, 1989).
All compounds except U-46619 were purchased from Sigma (Chemical Co., St
Louis, MO, USA) and dissolved in distilled H2O. U-44069 was a
generous gift from Dr K. Meisheri of the Upjohn Company (Kalamazoo MI, USA).
Both U-44069 and U-46619 were dissolved in 95% ethanol. Ethanol was not
vasoactive at the concentrations used in these studies.
Data analysis
Values are expressed as mean ± s.e.m., unless indicated otherwise.
Vessel tension is presented in mg. Gill resistance (RGILL)
was calculated from input pressure (in mmHg) divided by pump flow (ml
min–1) and normalized for gill wet mass after blotting.
Venous pressure was assumed to be zero because the efferent branchial artery
was not cannulated.
Comparisons were made by Students' t-test or paired t-test where appropriate. One-way ANOVA followed with Student-Newman-Keul's test was used for multiple comparisons of means. Significance was assumed when P<0.05.
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Effects of pHi on isolated vessels
Application of NH4Cl increases pHi in cells and its
removal from the extracellular compartment decreases pHi
(Roos and Boron, 1981). The
increased pHi following addition of NH4Cl, contracted
ABA in either Hepes or Cortland buffer and tension was sustained until
pHi was lowered by removing NH4Cl
(Fig. 4). 41 out of 48 EBA and
all (eight) CMA in Hepes buffer transiently contracted when pHi was
increased and then returned toward near baseline within 10 min
(Fig. 4). The ensuing decrease
in pHi during NH4Cl washout produced an even stronger,
but also transient, contraction. The response of the other seven EBA in Hepes
was similar to the ABA and these vessels were not examined further. Increasing
pHi in Cortland buffer produced a slight, but prolonged relaxation
of EBA and CMA, whereas the fall in pHi during NH4Cl
washout again produced a strong, transient, contraction similar to that in
Hepes buffer (Fig. 4). EBA
responses to increased or decreased pHi in Hepes buffer were not
affected by addition of 10 mmol l–1 NaHCO3
(Fig. 5), 1 mmol
l–1 NaH2PO4 (N=8; not shown),
or 10 mmol l–1 NaHCO3 plus 1 mmol
l–1 NaH2PO4 (N=8; not shown).
In Cortland buffer, both the alkalotic relaxation and enhanced acidotic
contraction were significantly different from the respective responses in
Hepes and in Hepes buffer with bicarbonate
(Fig. 5), Hepes buffer with
phosphate, and Hepes buffer with bicarbonate plus phosphate.
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The effect of increasing pHi on pre-contracted EBA in Hepes buffer was dependent upon the nature of the pre-contraction stimulus; KCl-contracted vessels contracted further following NH4Cl application, while vessels contracted with the thromboxane A2 agonist, U-46619, relaxed (Figs 6, 7, 8). Both responses lasted 20 min or longer (Fig. 6). NH4Cl washout transiently contracted both KCl and U-46619 pre-contracted vessels (Figs 6, 7, 8). Responses of pre-contracted vessels in Cortland buffer were qualitatively similar to those in Hepes although the acidotic contraction of KCl-contracted vessels in Cortland buffer was significantly weaker than the corresponding contraction in Hepes buffer (Fig. 7) and the alkalotic relaxation in U-46619-contracted vessels in Cortland buffer was significantly greater than the corresponding relaxation in Hepes buffer (Fig. 8).
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Relationship between pHi and [Ca2+]o in isolated vessels
The contribution of extracellular calcium ([Ca2+]o)
to contractions accompanying intracellular alkalosis and acidosis in otherwise
un-stimulated EBA in Hepes buffer is shown in
Fig. 9. The magnitude of
contraction during the initial increase in pHi following
NH4Cl application was unaffected by either D600 or zero
extracellular calcium (0[Ca2+]o). The second
contraction, associated with the decrease in pHi, was significantly
lower in 0[Ca2+]o and appeared to be reduced by D600,
although this was not statistically significant.
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In many EBA, U-46619 produced an initial peak contraction that was followed by a sustained plateau at lower tension. This peak contraction was not affected by either D600 or 0[Ca2+]o, whereas the plateau was partially inhibited in 0[Ca2+]o and appeared to be inhibited by D600, although this was not significant (Fig. 11). The relaxation accompanying NH4Cl addition to U-46619-contracted vessels was unaffected by D600 or 0[Ca2+]o, whereas the contraction accompanying NH4Cl washout was partially inhibited by both treatments (Fig. 11).
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Effects of pHo on vascular resistance of the perfused gill
Vascular resistance of the perfused gill significantly increased when
perfusate pH was increased from 7.8 to 8.8 and decreased when perfusate pH was
lowered from 7.8 to 6.6 or below (Fig.
1). Even the lowest pH was well tolerated by the gill as the
acidotic vasodilation and ensuing recovery was reproducible when perfusate pH
was cycled between 7.8 and 6.2 (Fig.
12).
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pHo and pHi
Essentially all mammalian vessels dilate when pHo falls, and
constrict when pHo increases
(Aalkjær and Poston,
1996; Aalkjær and Peng,
1997; Smith et al.,
1998; Austin and Wray,
2000; Wray and Smith,
2004). Much, but not all, of this response in both systemic and
pulmonary vessels appears to be due to concomitant and parallel changes in
pHi when pHo is manipulated
(Madden et al., 2001;
Wray and Smith, 2004). As
these responses are also observed in trout arteries, veins, perfused gills
(Fig. 1), and the perfused
trunk of the ocean pout (Canty and Farrell,
1985) it is likely that they are indicative of fundamental
properties of vertebrate vascular smooth muscle. The ammonium pulse technique,
which increases pHi when ammonium is applied due to rapid non-ionic
diffusive entry of NH3 and subsequent absorption of intracellular
H+, and which lowers pHi when the process is reversed,
has been used extensively in mammalian vessels to alter pHi
independent of pHo (Roos and
Boron, 1981). In our experiments, the mechanical response of trout
vessels to alterations in pHi did not always correlate with those
produced by a change in pHo.
Afferent branchial arteries (ABA) contract when ammonium is added to the bath and they remain contracted for the duration of the ammonium exposure, independent of the incubation buffer (Fig. 4). This implies that intracellular alkalosis produces contraction and it suggests that these vessels are unable to restore pHi, with or without extracellular bicarbonate. ABA are pre-gill vessels and their unique response to pH may be related to their association with systemic venous blood.
Efferent branchial (EBA) and celiacomesenteric (CMA) arteries are post-gill, systemic, vessels and their response to ammonium was similar, but unlike that of the ABA. Both EBA and CMA in Hepes buffer contract after ammonium addition (Fig. 4), but the contraction wanes within 10–20 min. This suggests that these vessels are able to restore pHi. Conversely, in Cortland buffer, both EBA and CMA slightly relax after ammonium addition and recovery is slow (Fig. 4). Hepes buffers have been shown to inhibit contraction in mammalian vessels (see below), however, this is clearly different from our findings in that trout vessels contract in the presence of Hepes, Hepes plus bicarbonate, Hepes plus phosphate, and Hepes plus bicarbonate and phosphate, yet they relax in Cortland. This suggests that the effect is due to Hepes, but the mechanism is different from that in mammalian vessels.
A number of studies have shown that Hepes buffers inhibit mammalian
vascular smooth muscle (Altura et al.,
1980a; Altura et al.,
1980b; Kane et al.,
1997) whereas others show little effect
(Sigurdsson, 1983;
Douglas et al., 1993). In many
of the earlier studies, Hepes buffer was substituted for bicarbonate-based
buffers and the authors did not examine whether the effect was in fact due to
the presence of Hepes or the absence of bicarbonate. In studies where Hepes
solutions contained bicarbonate, the inhibition has been variously attributed
to Hepes [i.e. canine basilar arteries
(Kane et al., 1997)], or
bicarbonate [i.e. rat aortas (Lamb and
Barna, 1998)]. How Hepes acts is unclear. In canine basilar
arteries, Hepes inhibits KCl, serotonin and prostaglandin F2
(PGF2) contractions by an unknown mechanism that does not
include chloride channels, generation of H2O2, or
release of vasodilators from the endothelium
(Kane et al., 1997).
Regardless, the inhibitory effects of Hepes or bicarbonate are not observed in
trout EBA as these vessels contract during ammonium alkalinization in the
presence of Hepes, with or without bicarbonate, but relax in a bicarbonate
buffer. Thus in trout EBA, Hepes appears to promote alkaline-initiated
contraction and its absence prevents it. The nature of the vascular response
becomes even more problematic in prestimulated EBA where the type of
pre-stimulation (voltage or ligand) determines whether ammonium alkalinization
contracts or relaxes vessels in Hepes buffer. Furthermore, Hepes effects
appear to be vessel specific as they were observed in EBA and CMA, but not in
ABA. Yamamoto and Suzuki proposed
(Yamamoto and Suzuki, 1987)
that Hepes enters Drosophila neurons and blocks Cl–
channels on the cytoplasmic side of the membrane. If Hepes affects
transmembrane Cl– flux in EBA it would either have to
decrease Cl– entry or increase Cl– efflux,
neither of which have been demonstrated.
Calcium signaling and alkalosis
Whereas both extracellular and intracellular alkalosis contract EBA, the
magnitude of the response in pre-contracted vessels varies with the
pre-stimulus, i.e. alkalosis enhances voltage-mediated (KCl) contractions and
decreases ligand-mediated (AVT or U-46619) contractions. As described below,
the interaction between alkalosis and voltage-mediated (KCl) contractions
appears relatively straightforward, how alkalosis affects ligand-mediated
contractions is less obvious.
EBA responses to alkalosis and KCl appear to be independent and additive.
The contraction produced by an increase in pHi in otherwise
un-stimulated EBA in Hepes buffer does not utilize extracellular calcium
[Ca2+]o: it is not significantly affected by the L-type
calcium channel inhibitor, D600, or by removal of
[Ca2+]o (Fig.
9). This is different from the responses of mammalian vessels
where L-type channels are generally involved in alkalotic contractions
(Wray and Smith, 2004).
Alkalotic contraction of trout must therefore depend either on release of
intracellular calcium (Ca2+i) or Ca2+
sensitization of the contractile apparatus; this was not addressed in our
experiments. Lack of involvement of Ca2+o in an alkaline
contraction of EBA is even more evident in KCl-contracted vessels where the
KCl response was greatly decreased by D600 and completely inhibited in zero
[Ca2+]o, whereas the response to ammonium application
was not significantly affected by either treatment
(Fig. 10). As is evident from
our study (Fig. 10), and
others on mammalian vessels (Nobe and
Paul, 2001), KCl-mediated contraction of vascular smooth muscle is
essentially due to influx of Ca2+o. Our study shows that
this is probably the result of a potassium-xmediated cellular depolarization,
because identical results were observed when acetate was substituted for
chloride (Fig. 2). Thus the
total contraction in KCl-alkalinized (either by increased pHi or
pHo) vessels probably represents the sum of two independent events;
(1) KCl-mediated depolarization and resultant influx of
Ca2+o, and (2) alkalinity-mediated release of
Ca2+ from an intracellular store. This also explains why increasing
either pHi or pHo augments KCl contractions.
It is not clear how alkalosis relaxes ligand (AVT and U-46619)-contracted
vessels (Figs 2,
3,
6). Ligand-mediated contraction
of mammalian vessels is usually a two-step process, involving an initial
increase in intracellular Ca2+ (Ca2+i)
firstly due to Ca2+ release from intracellular stores, and secondly
due to entry of Ca2+o. Entry of
Ca2+o is brought about by the initial increase in
[Ca2+i] which opens calcium-activated chloride channels
(ClC) thereby increasing Cl– efflux. This depolarizes the
cell membrane and opens the L-type (voltage-gated) calcium channels
(Lamb and Barna, 1998).
U-46619 appears to contract EBAs through a similar two-step process in that
removal of Ca2+o does not affect the initial contraction
but reduces the plateau (Fig.
11). However, an alkalosis-mediated inhibition of either ClC or
L-type Ca2+ channels does not appear to be involved in the
alkalotic relaxation of U-46619-contracted vessels because the relaxation is
independent of Ca2+o
(Fig. 11). Thus the increase
in pHi either decreases total Ca2+ release from
intracellular stores, or desensitizes the contractile proteins to
Ca2+.
Calcium signaling and acidosis
All trout vessels relax when pHo is decreased
(Fig. 1), whereas, with the
exception of ABA, they transiently contract when pHi is increased,
irrespective of pre-stimulation, the nature of the pre-stimulus or the
presence or absence of bicarbonate in the buffer (Figs
4,
5,
6,
7,
8). It is generally accepted
that the relaxation accompanying extracellular acidosis in mammalian vessels
is due to decreased [Ca2+]i
(Austin and Wray, 2000). A
number of mechanisms have been shown to contribute to this effect in mammalian
vessels, including inhibition of L-type channels, inhibition of
receptor-operated channels, opening of ATP-dependent (KATP),
voltage-dependent (KV) and Ca2+-activated
(KCa) potassium channels, inhibition of passive and capacitative
Ca2+ entry, and possibly systems that remove
Ca2+i or affect myofilament Ca2+ sensitivity
(Austin and Wray, 2000). These
mechanisms may be operative in trout as well, although trout do not appear to
have KATP channels (Smith and Olson, unpublished observation).
Brief, but substantial contractions are also frequently observed in
mammalian vessels when pHi is transiently decreased
(Aalkjær and Poston,
1996). This is similar to our findings in EBA, CMA and ACV
(Fig. 4). In mammalian vessels,
this has been attributed to a rise in [Ca2+]i from both
extracellular and intracellular sources
(Aalkjær and Poston,
1996). It is unclear how trout vessels regulate
Ca2+o during ammonium washout. In both un-stimulated and
U-46619 pre-contracted vessels, 0[Ca2+]o and D600 only
partially inhibited the acidotic contraction (Figs
9,
11) suggesting that both
Ca2+o and Ca2+i are involved.
However, in KCl-contracted vessels the acidotic contraction was completely
inhibited by 0[Ca2+]o
(Fig. 10). In all vessels D600
was less effective than 0[Ca2+]o in inhibiting the
transient acidotic contraction. This could be due to the presence of
non-voltage gated Ca2+ channels as in mammalian vessels
(Austin and Wray, 2000), or
poor specificity of D600 for trout Ca2+ channels. The latter seems
more likely as D600 only inhibited around 70% of the KCl response
(Fig. 10).
pHo effects in the gill
The effect of pHo on vascular resistance of the perfused gill
was consistent with its effect on isolated conductance arteries (EBA) and
veins (ACV), even to the degree of pH sensitivity
(Fig. 1). Tissue hypoxia
resulting from a decrease in the ratio of O2 delivery (perfusion)
to O2 consumption (metabolism), such as that encountered by fish
during exercise, typically results in tissue and circulatory acidosis
(Milligan, 1996). In mammalian
vessels, both acidosis (Aalkjær and
Poston, 1996; Wray and Smith,
2004) and hypoxia (Thorne et
al., 2004) dilate systemic vessels thereby producing a concerted
increase in blood flow. Even in large mammalian pulmonary arteries a hypoxic
vasodilation is accompanied by a fall in pHi
(Madden et al., 2001).
However, the response of small mammalian pulmonary arteries is different as
hypoxia produces vasoconstriction and an increase in pHi
(Madden et al., 2001). We did
not measure pHi in our study, but it is probable that resistance
vessels in the gill are similar to small pulmonary arteries; they are relaxed
by acidosis (Fig. 1) and
constricted by hypoxia (Smith et al.,
2001). Conversely, hypoxic vasoconstriction in conductance
arteries, such as EBA, is uncommon (Smith
et al., 2001).
Acidotic dilation (Figs 1,
12) and hypoxic
vasoconstriction (Smith et al.,
2001) of gill resistance vessels is undoubtedly of homeostatic
benefit. Acidotic dilation may enhance gas exchange and it certainly would
decrease cardiac afterload at a time, such as that accompanying exhaustive
exercise in trout (Milligan,
1996), when myocardial contractility would be most vulnerable
(Farrell et al., 1986).
Hypoxic vasoconstriction in fish would have similar beneficial effects as it
does in the mammalian lung by preventing over perfusion of under ventilated
lamellae thereby maintaining O2 saturation of systemic arterial
blood. In fact, it is likely that this unique response originally developed in
the gill microcirculation and was retained during evolution to become an
integral component of the mammalian pulmonary circulation.
Homeostatic and environmental implications
Pollution aside, fish often encounter rapid and long-term variations in
ambient pH, PO2 and
PCO2 that directly affect blood acid–base
status (Dejours, 1972;
Janssen and Randall, 1975;
Thomas and Le Ruz, 1982;
Moyle and Cech, Jr, 1996). The
consequences of these perturbations on homeostatic mechanisms regulating
systemic and branchial perfusion, as well as blood pressure are unknown, but
clearly deserve further investigation.
pH or pOH?
Although the difficulty in separating pH from pOH effects is well known
(Roos and Boron, 1981),
virtually all recent reviews and primary articles on acid–base effects
in mammalian vascular smooth muscle discuss the relative contribution of
pHo and pHi (i.e. [H+]) to vascular smooth
muscle tension, but do not consider OH–
(Aalkjær and Poston,
1996; Aalkjær and Peng,
1997; Smith et al.,
1998; Austin and Wray,
2000; Wray and Smith,
2004). We feel that a case can be made for OH– as
the vasoactive moiety. First, an increase in tension is directly correlated
with an increase in [OH–], but inversely related to
[H+]. It seems intuitively easier to envision how a contraction
would dose-dependently increase with increasing agonist concentrations, rather
than decreasing concentrations. This is especially evident in otherwise
un-stimulated vessels (Fig. 1)
where there does not appear to be large resting tone. When these (resting)
vessels are acidified from physiological pH (7.8) to 6.8 there is little
further change in tonus, even though [H+] has now increased 10-fold
from 0.016 to 0.16 µmol l–1. However, increasing
[OH–] from physiological pH of 7.8 to 8.8 increases
[OH–] from 0.63 µmol l–1 to 6.3 µmol
l–1 and more than doubles the tension. This response is also
obvious in pre-contracted vessels. Second, at the upper range of pH effects
(e.g. pH 9; Fig. 1), the
concentration of H+ is 1 nmol l–1, whereas the
OH– concentration is 10 µmol l–1. Not
only is this a 10 000-fold difference, but it seems more realistic that a 10
µmol l–1 increase in OH– (pH 8 to 9)
would produce a half-maximal contraction, than would a 10 nmol
l–1 decrease in H+. Granted, H+ can
have substantial effects on amphoteric molecules and buffers, however, when
one considers the alkalinity at which most vasoactivity is observed we feel
that it is more likely an OH– effect. Perhaps this is through
the variety of anion channels and transporters present in smooth muscle.
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different from
AVT at higher pH.
