![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 281, Issue 2, 634-642, 1997
Department of Physiology and Pharmacology, The City University of New York Medical School, The Sophie Davis School of Biomedical Education, New York, New York
 |
Abstract |
|---|
 Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Calcium antagonists have routinely been assumed to inhibit the
contractions of arterial smooth muscle through block of membrane channels. The effects of nifedipine and diltiazem on contractions were
examined in in vitro preparations of cattle coronary
artery, one of the key therapeutic targets of calcium antagonists, to determine if alternate mechanisms of action are involved. Contractions elicited in calcium-free Krebs, in the presence of U 46619, to potassium channel inhibitors (4-aminopyridine and tetraethylammonium) and to a Na+-K+-ATPase inhibitor (ouabain),
were antagonized by low concentrations of nifedipine (3 × 10
9 - 3 × 108
M) and by diltiazem (3 × 108 and 1 × 107 M). Contractions produced in
calcium-free Krebs to KCl (50 mM) were antagonized similarly by calcium
antagonists. Contractions to depolarizing agents in calcium-free Krebs
were antagonized at lower concentrations of nifedipine than comparably
elicited responses in Krebs containing 2.3 mM calcium. In addition,
higher concentrations of nifedipine were required to antagonize
contractions to extracellular calcium, produced in preparations
maintained in calcium-free Krebs in the presence of KCl (50 mM), than
were needed to block contractions to KCl or to potassium channel
inhibitors elicited in calcium-free Krebs. Pretreatment of preparations
in calcium-free Krebs with ryanodine (30 µM) did not reduce the
nifedipine-sensitive contractions elicited in calcium-free Krebs. It is
concluded that at least some of the therapeutic effects of calcium
antagonists on arterial tone may be the consequence of antagonism at
vascular smooth muscle cell site(s) at which calcium is released or
interacts, rather than of block of calcium entry through membrane L
channels.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
The therapeutic efficacy of
calcium antagonists on blood vessels is routinely attributed to block
of calcium influx through specific membrane channels (e.g.,
Willerson et al., 1995
; Robertson and Robertson, 1996). This
mechanism of action was initially put forward by Kalsner and associates
(1970). They provided the first description of the antagonism by an
organic compound of arterial contractions to potassium-induced
depolarization and to increases in extracellular calcium, as well as to
certain agonists, that could be reasonably assigned to block of calcium
influx. This model for the action of calcium antagonists, investigated
in depth by Fleckenstein and co-workers (see Fleckenstein, 1988) has
been expanded by others and is currently widely accepted.
In the intervening years, an occasional investigator has postulated
that calcium antagonists, particularly those that are highly active on
blood vessels, might also act intracellularly (e.g., Johnson
et al., 1982; Ratz and Flaim, 1985; Kanaide et al., 1988). Invariably, however, evidence for intracellular sites of action could be obtained only when calcium antagonists were used in
concentrations well above those associated with blockade of membrane L
channels (e.g., Saida and van Breeman, 1983; Kanaide et al., 1988; Hagiwara et al., 1993). For
example, Saida and van Breemen (1983) reported that diltiazem, in
concentrations of more than 105 M, inhibited
calcium release from an intracellular store in skinned rabbit
mesenteric artery preparations. Kanaide and associates (1988) noted
that norepinephrine-induced calcium transients, observed in cultured
vascular smooth muscle cells maintained in a calcium-free medium, were
inhibited partially by 106 to
104 M concentrations of verapamil and
diltiazem. Another group (Salag et al., 1993) reported that
in the absence of extracellular calcium, nifedipine and verapamil when
used in very high concentrations, more than
105 M, significantly inhibited
norepinephrine-induced contractions.
In our study, the effects of calcium antagonists on vascular contractions were examined in in vitro preparations of cattle coronary artery tissue, immersed in calcium-free Krebs-Henseleit solution under conditions that yield contractile responses. These experiments were done to determine if the calcium antagonists have inhibitory effects on contractions that cannot be attributed to blockade of the influx of extracellular calcium. Responses were also examined in Krebs solution containing 2.3 mM calcium. It was found that certain classes of contractions obtained in calcium-free solution are highly sensitive to surprisingly low concentrations of calcium antagonists. These antagonist concentrations are well within the range used therapeutically, and appear to be below those needed to block responses associated with calcium influx. The role of ryanodine resistant and ryanodine sensitive pools of calcium in these contractions was explored.
| |
Methods |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Cattle hearts were removed immediately after slaughter, immersed
in chilled Krebs-Henseleit solution, transported promptly to the
laboratory, and prepared as described previously (Kalsner, 1993). After
removal of visible fat and connective tissue the left descending,
circumflex and right coronary arteries and their branches were cut into
4-mm rings which were then opened transversely (flaps). Preparations
were routinely denuded of endothelium by rubbing the intimal surface
with a glass rod six times. That this procedure successfully denudes
the vessel segments was confirmed previously by histological
examination (Kalsner, 1985a). Rabbit aorta segments were trimmed of
adherent tissue, denuded of endothelium, and prepared similarly to
coronary artery segments.
Vascular segments were immersed in 15 ml muscle baths maintained at 37°C and under 4 g of tension (g/t) for isometric recording with a Grass polygraph. Preparations were allowed to equilibrate a minimum of 60 min before drug testing. The composition of the standard Krebs solution (with a pH of 7.4) was (mM): NaCl, 114.3; KCl, 4.6; CaCl2, 2.3; MgSO4, 1.1; NAHCO3, 22.1; KH2PO4, 1.1; disodium EDTA, .03; and glucose, 7.8. For calcium-free Krebs the CaCl2 was omitted from the recipe and the disodium EDTA increased to 0.06 mM. KCl (30 or 50 mM) was added to either calcium-free or regular Krebs solution, as appropriate, with a compensating reduction in NaCl to maintain osmolarity.
Drugs were prepared immediately before use and dissolved in 0.9% NaCl
(containing .01 N HCl) except that nifedipine was first dissolved in
ethanol at 103 M. Ryanodine was dissolved in
10% DMSO with the balance ethanol. Vehicle controls indicated no
effects of the solvent, in the volumes utilized, on any aspect of
coronary vascular performance. The drugs used and their sources were: U
46619 (The Upjohn Company, Kalamazoo, MI and Cayman Chemicals Company,
Ann Arbor, MI), nifedipine and ouabain (Sigma Chemical Co., St. Louis,
MO), ryanodine (Research Biochemicals International, Natick, MA),
tetraethylammonium chloride (Eastman Kodak, Rochester, NY), diltiazem
(Marion Labs, Kansas City, MO) and 4-aminopyridine (Aldrich, Milwaukee,
WI).
Mean data are presented with their S.E. and Student's t test was used for comparisons between two groups: a P value of 0.05 or less between groups was considered to represent a significant difference. Multiple comparisons of dependent samples were done using one-way analysis of variance and Bonferroni's inequality.
| |
Results |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Coronary artery responses.
Coronary artery segments from
cattle heart immersed in Krebs-Henseleit solution containing 2.3 mM
calcium respond with contractions to several distinct classes of
agonists such as histamine and thromboxane analogues, as well as to
increases in the extracellular potassium concentration
(e.g., Kalsner, 1993). The dihydropyridine calcium
antagonist nifedipine, used in the concentration range that is
typically employed in vascular studies, approximately 5 × 107 to 3 × 106 M, almost completely antagonizes
contractions to potassium chloride (5-100 mM), but only slightly
attenuates contractions to the thromboxane analogue U 46619 (Kalsner,
1993).
).
Contractions that were slightly above threshold to the thromboxane
analogue U 46619 were obtained after a minimum of 30 min in
calcium-free Krebs. After responses had reached plateau values, individual inhibitors of potassium channels were added to the muscle
baths. Contractions to U 46619 (usually 6 × 109 M), which reached a mean of 0.8 ± 0.1 g/t, were increased in amplitude by addition to the muscle chambers
of 4-AP (3 mM), an inhibitor of the delayed rectifier class of
potassium channels (Hille, 1992), to 279.0 ± 60.9% of their
initial value (n = 9). Whereas tone increments induced
by 4-AP in Krebs containing calcium (2.3 mM) are generally attributed
to influx of calcium through membrane L-channels opened by membrane
depolarization, increases seen in calcium-free Krebs likely signify
other events, such as those associated with the mobilization and
utilization of bound calcium. In other experiments it was shown that
TEA (1-3 mM), a known blocker of the moderate to large conductance
calcium-activated class of potassium channels, also increased
contractions to U 46619 in some preparations in calcium-free Krebs.
Contractions to U 46619, a mean of 0.9 ± 0.1 g/t were increased
to 244.3 ± 52.1% of their initial value by TEA 3 mM
(n = 3).
Potassium channel antagonists and nifedipine.
Contractions
elicited by potassium channel antagonists in the presence of a
threshold concentration of U 46619 in calcium-free Krebs were clearly
antagonized by the dihydropyridine calcium antagonist nifedipine.
Nifedipine, added to the muscle chambers after the contractile
increments induced by the potassium channel antagonists reached plateau
levels, inhibited the contractile effects of 4-AP and also those of
TEA, as well as those of the combination of potassium channel
inhibitors. Typical polygraph traces are shown in Figure 1a and b.
Nifedipine was effective in a surprisingly low concentration, with
3 × 108 M producing almost complete
inhibition of contractions to 4-AP (3 mM), a reduction in tone of
97.2 ± 1.7% (n = 5). In other tissues, it was
determined that a 10-fold lower concentration of nifedipine reduced the
tone increments to 4-AP by 25.4 ± 12.1% (n = 4),
and it was calculated that a concentration of 6.4 × 109 M, produced 50% inhibition. The tone
increment produced by TEA (3 mM) on small contractions to U 46619, to
244.3 ± 52.1% of initial values, was reduced 81.9 ± 34.0%
(n = 3) by nifedipine (3 × 108 M).
|
9 M in
calcium-free Krebs (n = 3). Contractions to U 46619 alone were not detectably diminished by these concentrations of
nifedipine in calcium-free Krebs. In the presence of TEA and 4-AP,
inhibition of Na+-K+-ATPase with ouabain
(5 × 107 M) usually elicited a
significant tone increment, a mean of 1.8 ± 0.6 g/t. The
ouabain-induced tone increments, along with those to the potassium
channel antagonists were almost completely and rapidly reversed by
nifedipine (9 × 109-3 × 108 M), a mean reduction of 86.6 ± 1.7% (n = 4) (e.g., fig.
1C).
Potassium contractions in calcium-free Krebs.
Contractions
were elicited after 30 to 60 min in calcium-free Krebs by replacing the
bathing solution with iso-osmotic calcium-free Krebs containing 50 mM
KCl. The contractions to potassium, which reached 7.5 ± 0.8 g/t
(n = 15) were reduced by concentrations of nifedipine
as low as 3 × 109 M. Maximal inhibition
was seen with nifedipine 9 × 109 M, a
tone reduction of 77.6 ± 1.3%, and the IC 50 was 6.1 × 109 M. Contractions to KCl (50 mM) in
calcium-free Krebs, in the absence of nifedipine, generally maintain a
stable plateau, over a comparable time period. Typical polygraph traces
are shown in figure 2 and the data are summarized in
figure 3.
|
|
Calcium-free Krebs.
Evidence was obtained that contractions to
KCl in calcium-free Krebs involves a cellular pool of calcium that
slowly depletes over time. Contractions to a low concentration of KCl
(30 mM) were repeated at 30, 75, 120 and 160 min of exposure to
calcium-free Krebs. Initial contractions to KCl in calcium-free Krebs
(30 min), which were 2.6 = 0.7 g/t (n = 4),
declined progressively, at the indicated time intervals, to 0.9 ± 0.2, 0.6 ± 0.2 and 0.2 ± 0.1 g/t. These values are
significantly different from each other. The re-addition of
CaCl2 (50 µM) to the muscle chambers, after 172 min in
calcium-free Krebs, partially restored responses to the subsequent
readdition of KCl (30 mM) in calcium-free Krebs, which reached a mean
of 1.5 ± 0.3 g/t or 56.8 ± 3.5% of their initial values
(n = 4). A typical experiment is shown in figure 4.
|
|
Contractions to calcium repletion.
To assess the degree of
functional blockade of the membrane L channels at the low
concentrations of nifedipine that effectively antagonize contractions
to potassium elicited in calcium-free Krebs, experiments were done in
which calcium was incrementally readded to the muscle chambers
containing KCl (50 mM) in calcium-free Krebs. This was done, in the
presence and in the absence of nifedipine, on matched coronary segments
taken from the same vessels. Concentrations of calcium as low as 100 µM produced distinct contractions in the absence of nifedipine (fig.
5). In contrast to the effects of the calcium antagonist on
contractions to KCl (fig. 3), nifedipine (3 × 109 M) did not antagonize significantly
contractions to CaCl2, and at 9 × 109 M of nifedipine the response to the
readdition of 2.3 mM calcium was significantly depressed only at the
lowest test concentration (100 µM). Nifedipine at 3 × 108 M was required to inhibit the maximal
contraction to calcium by 50% whereas full antagonism of contractions
to calcium readdition required a substantially higher concentration of
nifedipine (9 × 107 M) (fig.
6). A typical polygraph trace of one of these
experiments is shown in figure 7.
|
|
Diltiazem.
The benzothiazepine antagonist diltiazem was also
effective in antagonizing contractions in calcium-free Krebs.
Preparations were contracted with low concentrations of U 46619 (1.5-30 × 109 g/ml) and then exposed to
4-AP (3 mM), as was done in experiments with nifedipine. Diltiazem
3 × 108 M relaxed the 4-AP-induced tone
increments by 23.7% ± 13.2 (n = 2) and 1 × 107 M did so by 87.4 ± 11.8%
(n = 5), with an IC 50 of 5.0 × 108 M. Similarly, tone increments due to the
combination of 4-AP, TEA and ouabain, which reached 250.2 ± 15.7% of initial U 46619-induced amplitudes, were relaxed 88.8 ± 8.5% by diltiazem (1 × 107 M)
(n = 9) (e.g., fig. 1D).
Ryanodine treatment.
Preparations maintained in calcium-free
Krebs for a minimum of 30 min were exposed to ryanodine (30 µM)
(n = 6) or vehicle (n = 6), and 30 min
later all tissues were exposed to U 46619 and to 4-AP (1 mM).
Contractions to 4-AP were not significantly different in the
ryanodine-treated tissues compared with vehicle treated segments from
the same experiments; with mean increments of 1.0 ± 0.1 and
0.9 ± 0.2 g/t, respectively. Additionally, other experiments done
in calcium-free Krebs revealed that responses to 50 mM KCl were similar
regardless of the presence or absence of ryanodine; a mean of 4.9 ± .8 and 4.1 ± .8 g/t, respectively (n = 7 in
each group). In addition, nifedipine 9 × 109 M relaxed similarly matched ryanodine-
and vehicle-treated preparations contracted with KCl, with means of
67.6% ± 6.1 and 71.4% ± 8.0, respectively.
Nifedipine in standard Krebs.
To assess the potency of
nifedipine as a calcium channel antagonist in a standard Krebs medium,
contractions to potassium chloride (50 mM) were obtained in 2.3 mM
calcium Krebs and once stable response levels were reached, a mean of
12.7 ± 1.9 g/t in 10 preparations, nifedipine (3 × 109 M and higher) was added to the muscle
chambers. As shown in figure 8, nifedipine antagonized
depolarization-induced contractions to potassium, but at concentrations
that were significantly higher than those used to antagonize
contractions elicited by potassium channel inhibitors in calcium-free
Krebs. For example, 9 × 109 M of
nifedipine produced a greater inhibition of potassium-induced contractions in calcium-free Krebs than did a 10-fold higher
concentration in Krebs with 2.3 mM calcium. Also, in other experiments,
contractions to low concentrations of potassium chloride (5-15 mM),
which reached a mean amplitude of 6.9 ± 1.4 g/t were decreased
only 3.6 ± 1.9, 37.7 ± 11.1, 49.1 ± 13.6 and
42.2 ± 13.2% by nifedipine 3 and 9 × 108 and 3 and 9 × 107 M, respectively, which was much less than
the effect of nifedipine on contractions to KCl (50 mM) in calcium-free
Krebs (fig. 3).
|
9 M
clearly antagonized such contractions in calcium-free Krebs a 10-fold
higher concentration was required for equivalent blockade in Krebs
containing 2.3 mM calcium, in the presence or absence of U 46619 (fig.
9).
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
The vascular relaxant actions of calcium antagonists are generally
attributed to combination with a discrete site on membrane calcium
channels preventing the conformational changes associated with calcium
entry. This view was initially proposed to account for what was then
the unique capacity of SKF 525A
(diethylaminoethyl-diphenylpropylacetate) to antagonize vascular
contractions to KCl, also to the readdition of extracellular calcium,
and to a varying extent the contractions to a range of other agonists
in rabbit aorta (Kalsner et al., 1970). It is generally
accepted that the action of calcium antagonists to inhibit membrane L
channels is relatively specific, and that inordinately high
concentrations of the antagonists are needed for meaningful action at
other loci. Consequently, the therapeutic effects of this broad and
diverse class of drugs on vascular smooth muscle routinely are assigned
to interactions with calcium L channels (e.g., Robertson and
Robertson, 1996). Surprisingly, the available evidence does not
conclusively support this view, and this provides the basis for our
study, which focussed on a coronary artery preparation as an
appropriate model system.
The large (epicardial) coronary arteries of the heart are considered a
major therapeutic target of calcium antagonists (e.g., Habib
and Roberts, 1994; Willerson et al., 1995), and considerable background information on this widely used vessel system is available (see Kalsner, 1985b, 1995). Of the three prototype calcium antagonists, verapamil, diltiazem and nifedipine, the dihydropyridine nifedipine is
the most potent on vascular tissue, both in vivo and
in vitro (e.g., Robertson and Robertson, 1996),
and consequently nifedipine was used as the primary drug in our study.
Work from my laboratory on cattle coronary arteries indicates that
depolarizing stimuli elicit contractions both by promoting the entry of
extracellular calcium, presumably via membrane L channels, and by
altering calcium availability from voltage sensitive stores on/in
vascular muscle cells (Kalsner, 1994). Earlier work with other vascular
tissue had already pointed to this possibility (e.g.,
Kobayashi et al., 1985). Our study suggests that calcium antagonists have at least two distinct loci of action in antagonizing vascular contractile responses, and that the locus most sensitive to
blockade is not the membrane channels that permit calcium influx but a
site(s) linked to contractions elicited in the absence of extracellular
calcium. Contractions in a calcium-free medium were elicited by using a
variety of depolarizing stimuli ranging from inhibition of potassium
channels and consequent block of potassium efflux to inhibition of
Na+-K+-ATPase. Elevated extracellular potassium
was also used to depolarize vascular muscle cells.
A class of potassium channels, known as delayed rectifier channels, has
been identified by patch clamp analyses in arterial cells in culture
and it appears to play a role in maintaining the membrane potential of
some vascular tissue (Hille, 1992; Schochan et al., 1994).
These channels have been described as being particularly sensitive to
inhibition by 4-AP, and this compound has been used for their selective
blockade (e.g., Schochan et al., 1994). In our
study, it was found that contractions to U 46619 were increased in
calcium-free Krebs by 4-AP (1 and 3 mM). These 4-AP-induced increments
were almost totally antagonized by low concentrations of nifedipine,
with an IC50 of 6.4 × 109 M
in coronary artery segments. Nifedipine, used in this concentration, had no detectable effect on contractions to U 46619 alone in
calcium-free Krebs. TEA, an antagonist of the moderate to large
conductance calcium activated potassium channels (KCa)
(Hille, 1992), also increased the tone in calcium-free Krebs in the
presence of U 46619, but did so less reliably than did 4-AP. These
increments were again antagonized by low concentrations of nifedipine,
as was the contractile effects of the combination of TEA and 4-AP. The
use of a cardiac glycoside, ouabain, to block
Na+-K+- ATPase, in combination with the
potassium channel blockers, also increased tone to U-46619, and these
increments were again antagonized by low concentrations of nifedipine
(1 × 108-3 × 108 M). Experiments with diltiazem, a
benzothiazepine, confirmed that antagonism of tone generated by 4-AP,
TEA and ouabain in calcium-free Krebs was not limited to
dihydropyridine compounds.
It is well known that contractions of vascular smooth muscle may
involve influx of extracellular calcium and contributions from membrane
and intracellular sources (e.g., Rasmussen et
al., 1990; Endo et al., 1990; Greenberg et
al., 1991). The calcium sources seem to vary dependent on agonist,
species and vessel. The previous work of Ratz and Flaim (1984, 1985)
with cattle coronary artery segments showed that contractions to some
agonists, i.e., acetylcholine and 5-hydroxytryptamine, are
partially preserved in calcium-free Krebs. These workers further
reported that in isolated cattle coronary artery segments a high
concentration of diltiazem (105 M) inhibited
contractions to 5-hydroxytryptamine more than did exposure to
calcium-free Krebs (Ratz and Flaim, 1985). An earlier report (Flaim and
DiPette, 1979) showed that the cardiac glycoside digoxin potentiates
responses to norepinephrine in intact segments of perfused rabbit
carotid artery in calcium free Krebs. Noting that digoxin did not
potentiate the response to norepinephrine in the presence of a high
concentration (5 × 105 M) of verapamil
in standard (CaCl2) Krebs solution, they concluded that
participation of a cellular calcium storage site is likely. Others have
reported that the dihydropyridine calcium antagonist felodipine, in
near mM concentrations, interact with calcium binding proteins such as
calmodulin, based on changes in the Cd-NMR spectrum (Boström
et al., 1981). The implications of this latter work for
intact vascular smooth muscle cells, and with more moderate levels of
calcium antagonists are not clear. From another perspective, some
investigators have been unable to detect a decrease in 45Ca uptake
after treatment with nifedipine or diltiazem in rat portal vein, when
the antagonist is used in concentrations that clearly inhibit tone
(Church and Zsoter, 1980; Boström et al., 1981).
Evidence that contractions to elevated extracellular potassium in
smooth muscle may involve mobilization of calcium from cellular stores,
as well as the influx of extracellular calcium, has been specifically
provided by others (e.g., Khoyi et al., 1989; Low et al., 1992). In one study, the release of calcium from
intracellular stores by high extracellular potassium was demonstrated
in rat aorta maintained in calcium-free Krebs using a fluorescent dye (Kobayashi et al., 1985). Similarly, an elevation of
intracellular calcium by high extracellular KCl, attributable to
mobilization from internal storage sites was described in cell
suspensions of rat parotid gland cells maintained in vitro
in calcium-free medium (Takemura and Ohshika, 1988). In our study,
contractions to potassium chloride (50 mM) were readily elicited in
calcium-free Krebs and these contractions were antagonized by low
concentrations of nifedipine, with a concentration of 9 × 109 M producing 50% inhibition of
potassium-induced contractions. This is in contrast to the
significantly higher concentration of nifedipine needed for an
equivalent degree of blockade in Krebs containing 2.3 mM calcium. The
contractions to potassium (50 mM) in 2.3 mM calcium Krebs reached a
mean of 13.4 ± 2.4 g/t vs. a mean of 7.5 ± .8 g/t in calcium-free Krebs, suggesting that both extracellular and
stored calcium likely contributed to the larger contractions seen in
calcium containing Krebs.
Regarding the composition of the calcium-free Krebs used in our
experiments. The Krebs contained a 60 µM concentration of chelator
(disodium EDTA), as used in previous studies (e.g., Kalsner, 1994). Work by Daniel and his group (see Guan et al., 1988)
has shown that over time high concentrations of chelator "remove
superficially bound Ca++ and subsequently reduce the
intracellular Ca++ pool via extraction of the intracellular
Ca++ at the cell membrane surfaces" (Guan et
al., 1988; Low et al., 1994), making it necessary that
low concentrations be used. Evidence was provided that makes it highly
unlikely that responses seen in calcium-free Krebs in our experiments
represent the meaningful participation of residual levels of
extracellular calcium in the bathing medium. Repeated exposure to KCl
(30 mM) in calcium-free Krebs demonstrated progressively diminishing
responses over the course of 160 min, providing evidence for the
depletion of a tissue store of calcium that was utilized in the
responses. Also, readdition of a small concentration of
CaCl2 (50 uM) restored contractions to KCl in calcium-free
Krebs in these tissues.
Other evidence that nifedipine-sensitive responses seen in calcium-free Krebs were not likely attributable to the presence of extracellular calcium sufficient to maintain contractions to agonists dependent on calcium influx, was the finding that spontaneously generated contractions, seen in Krebs containing 2.3 mM calcium, were virtually entirely eliminated by a 15-min exposure to calcium free Krebs. Also, in contrast to data with coronary arteries, brief exposure of rabbit aorta to calcium-free Krebs almost entirely antagonized contractions to KCl. The aorta is a highly elastic conducting artery, showing characteristics of multiunit vascular tissue, very different from epicardial coronary arteries and probably from many peripheral resistance vessels.
The concentration of nifedipine that successfully antagonizes contractions to potassium depends on the source of calcium. It appears that nifedipine blocks calcium influx through membrane channels at higher concentrations than are needed to block the alternate site(s) susceptible to nifedipine in calcium-free Krebs. When the absolute reduction in grams of tension generated by elevated extracellular potassium is considered, it is possible that approximately similar concentrations of nifedipine cause equivalent inhibition of a vulnerable component of contraction present in the two media, but that an additional component present only in 2.3 mM calcium requires higher concentrations of nifedipine. This is supported by the observation that contractions to very low concentrations of added potassium (5-15 mM), that seem to rely predominantly on extracellular calcium, because they are absent in calcium-free Krebs, were not readily antagonized by nifedipine although comparable magnitude contractions in calcium-free Krebs were highly susceptible to nifedipine.
The likelihood that multiple sources of calcium are involved in the responses studied here was supported by observations on the antagonism by nifedipine of tone increments produced by 4-AP in 2.3 mM calcium Krebs. Contractions to 4-AP occurred at lower concentrations in Krebs containing 2.3 mM calcium than in calcium-free Krebs, probably reflecting participation of a calcium source not available in calcium-free Krebs. These contractions were inhibited by nifedipine in Krebs with 2.3 mM calcium, but higher concentrations of the antagonist were required than in calcium-free Krebs.
Experiments were done in which calcium was readded in a calcium free
medium in the presence of 50 mM KCl. Contractions elicited under these
conditions are ordinarily attributed by investigators to calcium entry
via membrane channels (e.g., Kalsner et al., 1970) and they were blocked in our experiments by a sufficiently high
concentration of nifedipine. However, it was clear that block of
calcium-induced contractions in calcium-free Krebs required a
significantly higher concentration of nifedipine than was needed to
antagonize contractions to potassium in calcium-free Krebs. This
provided strong evidence for an alternate site of action for
nifedipine, and one more sensitive to the antagonist than are the
membrane channels.
Ryanodine is an antagonist of one class of calcium storage sites in
vascular smooth muscle presumably by emptying them of calcium
(e.g., Wagner-Mann et al., 1992). Previous work
indicated that both ryanodine-sensitive and ryanodine-insensitive
stores of calcium likely are present in cattle coronary artery segments (Kalsner, 1994). Responses to endothelin in calcium-free Krebs were
potentiated by ryanodine pretreatment but those to U 46619 were
slightly but significantly depressed, at the uppermost portion of the
concentration-response curve. Presumably, enhanced contractions to
endothelin were attributable to loss of a ryanodine-sensitive "sink" for calcium whereas partially compromised responses to the
thromboxane analogue signified contributions from both types of calcium
pool (Kalsner, 1994). In my experiments with ryanodine (30 uM),
contractions to potassium in calcium-free Krebs were not inhibited nor
did the alkaloid alter the inhibitory effects of nifedipine. Further,
tone increments to 4-AP in calcium-free Krebs were not significantly
altered by ryanodine pretreatment. These experiments indicate that the
relevant target of nifedipine action is not the ryanodine sensitive
storage depots for calcium.
The vascular efficacy of calcium antagonists, as anti-hypertensive agents and as antianginal drugs is presumed to rest on their block of membrane calcium L channels, and a therapeutically beneficial decrease in peripheral vascular resistance, or increase in coronary blood flow. Our findings suggest that at least some of the effects of calcium antagonists may be the consequence of antagonism at other vascular cell site(s) with which calcium interacts. Although our study, examining the contractile response of isolated vascular segments, needed to be done to demonstrate the capacity of calcium antagonists in calcium-free Krebs to alter contractile tension, future studies using electrophysiological and biochemical techniques would be worthwhile to provide insight into the identity of the mechanisms involved.
| |
Acknowledgments |
|---|
The author thanks Mr. Amir S. Abdali provided expert technical assistance and Ms. Maria Velazquez for skillful formatting of the manuscript.
| |
Footnotes |
|---|
Accepted for publication January 17, 1997.
Received for publication September 9, 1996.
1 This research was supported, in part, by a grant from PSC/CUNY.
Send reprint requests to: Dr. Stanley Kalsner, Professor and Chair, Department of Physiology and Pharmacology, The City University of New York Medical School, The Sophie Davis School of Biomedical Education, 138th Street and Convent Avenue, New York, NY 10031.
| |
Abbreviations |
|---|
TEA, tetraethylammonium chloride; 4-AP, 4-aminopyridine; SKF 525A, diethylaminoethyl-diphenylpropylacetate; g/t, grams of tension.
| |
References |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  A. del Valle-Rodriguez, E. Calderon, M. Ruiz, A. Ordonez, J. Lopez-Barneo, and J. Urena Metabotropic Ca2+ channel-induced Ca2+ release and ATP-dependent facilitation of arterial myocyte contraction. PNAS, March 14, 2006; 103(11): 4316 - 4321. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. C. Ng, S. M Wilson, and J. R Hume Mobilization of sarcoplasmic reticulum stores by hypoxia leads to consequent activation of capacitative Ca2+ entry in isolated canine pulmonary arterial smooth muscle cells J. Physiol., March 1, 2005; 563(2): 409 - 419. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.-L. Li, W.-X. Tang, H. H. Valdivia, A.-P. Zou, and W. B. Campbell cADP-ribose activates reconstituted ryanodine receptors from coronary arterial smooth muscle Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H208 - H215. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
