Evidence for two types of internal Ca2+ stores in canine mesenteric artery with different refilling
mechanisms
A. M. LOW, C. Y. KWAN, AND E. E. DANIEL Smooth Muscle Research Program, Pharmacology and Physiology Division, McMaster University Health Science Centre, Hamilton, Ontario L8N 325, Canada
Low, A. M., C. Y. Kwan, and E. E. Daniel. Evidence for two types of internal Ca2+ stores in canine mesenteric artery with different refilling mechanisms. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H31-H37, 1992.-Novel transient biphasic responses of the dog mesenteric artery to phenylephrine hydrochloride (PE, 10 PM) in Ca2+-free medium containing 50 PM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) have been analyzed. The initial component was significantly inhibited by ryanodine (30-100 PM), an agonist enhancing Ca2+ release from the sarcoplasmic reticulum, whereas the second was significantly inhibited by nifedipine (1 PM), an L-type Ca2’ channel antagonist, or EGTA, to chelate Ca2+, and was potentiated by BAY K 8644 (1 PM), an L-type Ca2’ channel agonist. After repletion of Ca2+ stores in normal Krebs solution or in high KC1 (60 mM) Krebs, the first component was inhibited by cyclopiazonic acid (CPA, 30 PM), a putative, reversible, and selective microsomal Ca2+ pump adenosinetriphosphatase inhibitor. BAY K 8644 potentiated the second component in the presence of CPA. The inhibition of the first component by CPA suggests that the refilling ultimately requires the CPA-sensitive Ca2’ pump for Ca2’ resequestration. However, the second component may refill by a CPA-independent route opened by BAY K 8644. These results, taken as a whole, indicate that the biphasic PE response in Ca2+-free medium may reflect compartmentalization of Ca2’ storage related to the different routes of refilling. ryanodine; cyclopiazonic acid; vascular smooth muscle; calcium mobilization; calcium pump; endoplasmic reticulum
for a physiological response in many systems usually requires an elevation of intracellular Ca2+ concentration. In vascular smooth muscles, contractility can be modulated by the release of Ca2+ from intracellular storage and influx of Ca2+ from the extracellular space (1, 2, 5). The two sources of Ca2+ correspond to the biphasic components of an agonist-induced contraction in physiological saline solution (PSS). The initial phase is a consequence of Ca2+ release from the internal Ca2+ store, presumably located in the endoplasmic reticulum (ER), whereas the tonic phase requires Ca2+ influx from the exterior of the cell via Ca2+ channels of the plasma membrane (1, 2, 5, 7, 24). Apart from the observation of a biphasic agonistinduced contraction in normal PSS, Heaslip and Rahwan (12) have reported that norepinephrine induced a biphasic contraction in Ca2’ -free medium containing ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), a Ca2+ chelating agent, in the rat aorta. The first component was transient, and the second was sustained and repeatedly reproducible for as long as 5 h in Ca2+-free Krebs containing EGTA with concentrations ranging between 0 and 10 mM. According to the accumulating evidence for the role of diacylglycerol in protein kinase C activation and in the phosphorylation THE TRIGGER
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$2.00 Copyright
of the myosin light chain kinase, the sustained contraction in Ca2+-free medium as described by Heaslip and Rahwan (12) could be accounted for by the activation of the diacylglycerol branch of the phospholipase C (13, 21). Unlike the dual component of the norepinephrineinduced contraction in Ca2+-free medium containing a Ca2+ chelator observed by Heaslip and Rahwan (12), the present study describes a novel short-lived biphasic phenylephrine (PE)-induced contraction in Ca2+-free medium containing 50 PM EGTA in the dog mesenteric artery (DMA). We attempt to explain the mechanisms of this response by using selective modulators of ER function such as ryanodine (RYA), an agonist for the ER Ca2+-release channel and cyclopiazonic acid (CPA), a novel inhibitor of the ER Ca2+ pump adenosinetriphosphatase (ATPase) (6, 20). MATERIALS Animal
AND
METHODS
and Tissue Handling
Mongrel dogs (-20 kg) of either sex were used. The dogs were given an overdose of pentobarbitone sodium (100 mg/kg). The DMA was isolated from freshly killed animals and placed in Krebs solution at pH 7.4 containing (in mM) 119 NaCl, 5 KCl, 2.5 CaC12, 2 MgCl,, 25 NaHC03, 1 NaH2P04, and 11 glucose. Fat and connective tissues were removed under a dissecting microscope, and 3- to 4-mm rings whose endothelial cells were removed by rubbing against the teeth of a pair of forceps were mounted on a 15-ml organ bath connected to a force transducer (Grass FT03C) and a pen recorder (Gould 2800). The organ baths and Krebs solution were bubbled continuously with 95% 02-5% CO2 and warmed to 37’C. The rings were equilibrated for 20 min before stretching the arteries to their optimal resting tension of -5 g. Stimulation of the arteries with 60 mM KC1 was repeated every 15-20 min until a reproducible contractile response was obtained. For Ca2+-free Krebs, Ca2+ was omitted and 50 PM EGTA was added. This concentration of EGTA was used because it was found to be sufficient to chelate the contaminating level of Ca2+ occurring in Ca2’free medium without an additional membrane-destabilizing effect (11). Contractility
Studies
The experimental setup and protocol have been described previously (16). After a period of equilibration with KC1 stimulations, the artery was stimulated with PE (10 PM) in normal Krebs solution, a concentration that produced maximum contraction in the DMA. All subsequent responses were expressed as a percentage of this maximum contraction. RYA (30-100 PM), CPA (lo-30 PM), or vehicle controls [dimethyl sulfoxide (DMSO) for CPA or absolute ethanol for RYA] were added, and the arterial rings were incubated for 30-60 min. The effects of these compounds are optimal at these concentrations (16). Because both CPA and RYA effects were found to be reversible
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on washing, these were replaced as needed. After the incubation of the tissuewith CPA, RYA, or vehicle and after the development of basal active tension, the arterial rings werewashedand incubated in Ca2+-freeKrebs for -5 min to remove extracellular Ca2+.PE was added to the bath for a final concentration of 10 PM, a just submaximalconcentration. Exposure to PE was maintained until the contraction had returned to its original baseline or within 10% thereof. For convenience in subsequentdescription and discussion, this transient responseto PE in Ca2+-freemedium is hereafter referred to as a “Ca2+-release”response.A subsequentapplication of PE (10 PM) wasineffective in producing a response, although parallel controls in normal Krebs solution continued to respondmaximally to PE. At this point the tissue was said to be depletedof its PE-sensitive Ca2’ pool. The tissueswere allowed to replete their storesin the Ca2’containing normal Krebs solution for ~30-40 min. Subsequent PE (10 PM) stimulation wasusedto assess the effectivenessof the refilling processand thus will be referred to as “repletion” (16). Drugs Unlessotherwise stated, all drugs were prepared in doubledistilled water. CPA (Sigma, St. Louis, MO) was dissolvedin DMSO to makea stock solution of 10 mM, and RYA (Research Biochemical, Natick, MA) waspreparedin absoluteethanol for a stock solution of 10 mM. Nifedipine (Sigma), BAY K 8644 (Miles Laboratories), and U-46619 (Sigma) were prepared in ethanol to a stock solution of 10 mM and stored in darkness. PE was purchasedfrom Sigma. All other chemicals were of laboratory standard from various commercialsources.
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Fig. 1. After phenylephrine hydrochloride (PE)-sensitive stores were depleted in dog mesenteric artery as described in MATERIALS AND METHODS, tissues were allowed to replete their store for 30 min in normal Krebs solution containing 2.5 mM Ca2+. All arterial rings had approximately similar-sized PE reference contraction in normal Krebs at start of experiment (not shown). A: subsequent PE (10 PM) contraction in Ca2+-free medium was found biphasic. C: when repletion was performed in presence of nifedipine (1 PM), second component was abolished. B and D: ryanodine (RYA) (100 PM) caused basal tension and was found to inhibit selectively first component of biphasic response.
A
Statistical Analysis All the responses(maximum amplitude of contraction attained) are expressedas a percentageof responseto 10 pM PE in normal Krebs solution unlessotherwise stated. Where applicable, data are expressedas meanst SE. The significant difference wascalculatedby usingthe Student’s unpaired t test (two tailed) or one-wayanalysisof variance where appropriate. When the F ratio is significant, differing pairs were determined by Bonferroni’s method. The minimal P value accepted for statistical significancewas0.05. RESULTS
Qualitative Characterization of Biphasic Response in Ca2+-Free Medium
We recently reported that repletion of the PE-sensitive store after depletion with repetitive PE (10 PM) stimulation in Ca2+-free medium was enhanced in the presence of 60 mM KC1 (16). Refilling of the PE store was slow when the tissue, initially depleted of its PE-sensitive store, was incubated in normal Krebs solution for l-2 h. Reducing the repletion period resulted in a transient biphasic response to PE in Ca2+-free medium, and this pattern was reproducible as long as repletion was performed in normal Krebs solution for Cl h. Figures lA, ZA, 3A, and 4A show typical PE (10 PM)-induced biphasic responses in Ca2’ -free medium after a repletion period of -30-40 min in normal Krebs solution containing 2.5 mm Ca2+. This biphasic response was not a unique feature of the agonist PE; U-46619 produced a similar biphasic response (not shown). In contrast, when repletion was performed in the presence of 60 mM potassium,
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Fig. 2. Left: reference PE contraction in normal Krebs before exposure with BAY K 8644 (1 PM). Right: PE-induced contraction in Ca2+-free medium plus 50 PM EGTA after depletion of PE-sensitive store and subsequent repletion in normal Krebs in absence (A) and presence (B) of BAY K 8644. In presence of BAY K 8644, second component was enhanced selectively compared with controls.
a uniphasic response probably representing an unresolved biphasic response was observed as illustrated in Fig. 5A. Repletion in high KC1 also resulted in this uniphasic response to other constrictor agonists such as prostaglandin F2a and U-46619 (not shown). Effects of RYA and nifedipine. The initial phase of the contraction could be abolished by pretreatment of tissue with RYA (30-100 PM) as illustrated in Fig. 1B. RYA pretreament caused a small increase in basal tension during repletion. In the presence of nifedipine (1 pM) the second component was abolished, leaving only the initial component as seen in Figs. 1C and 3C, compared with corresponding controls (Figs. 1A and 3A). Figure 1D shows that incubation of the tissue with both RYA
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repletion period of -30 min, the biphasic response to PE in Ca2+-free medium was restored (Fig. 4B, right) and was comparable with controls (Fig. 4A). When repletion was performed in the presence of both BAY K 8644 (1 PM) and CPA (30 PM) in normal Krebs solution, it was found that the first component was suppressed but the second was relatively unaffected (Fig. 40, left). After CPA was removed (Fig. 40, right), the first component was restored (Fig. 40) and was comparable with controls (Fig. 4C). It appears that, in the presence of BAY K 8644, the refilling of one of the compartments is independent of the ER Ca2+ pump.
c
D PE Ca-free+EGTA
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59
Fig. 3. Effect of cyclopiazonic acid (CPA; 10 PM) on repletion of PEsensitive Ca2’ store in normal Krebs for 30 min was investigated in dog mesenteric artery. All arterial rings had approximately similarsized PE reference contraction in normal Krebs at start of experiment. A: subsequent PE-induced contraction in control demonstrated biphasic response. B: in presence of CPA, both components of PEinduced contraction in Ca2+-free medium containing 50 PM EGTA were greatly reduced, although reduction of first component was statistically significant (see Fig. 7). C: in presence of nifedipine (1 PM), only first component of response was observed. D: CPA was found to reduce this first component to the same extent as it did in B.
and nifedipine inhibited nearly all of the PE response in Ca2+-free medium. These results suggest that such a biphasic contraction in Ca2+ -free medium may represent dual compartmentalization of the intracellular Ca2+ store. Effects of BA Y K 8644. Because repletion in the presence of nifedipine, a Ca2+ channel antagonist, abolished the second component (Fig. lC), refilling such a compartment required Ca2+ influx from the extracellular space via the control of voltage-operated Ca2+ channels. It was therefore hypothesized that a Ca2+ channel agonist such as BAY K 8644 could potentiate it by promoting refilling of this compartment. Figure 2 is a recording that clearly shows that when repletion was performed in the presence of BAY K 8644 (1 PM, Fig. 2B), the second component of the PE-induced biphasic response was potentiated compared with control (Fig. 2A). In some experiments, the potentiation of the second component by BAY K 8644 resulted in a uniphasic response as seen in Fig. 4C compared with controls (Fig. 4A ). Effects of CPA and nifedipine. The putative Ca2+ pump ATPase inhibitor CPA (30-100 PM), after repletion in normal Krebs, suppressed the first component with a tendency to reduce the second (Figs. 3B and 4B), although only the reduction of the first component was statistically significant (Fig. 8). CPA pretreatment reduced the first component usually present after repletion in the presence of nifedipine, as illustrated in Fig. 30 (cf. with Fig. 3C). Therefore, CPA was effective in reducing the PE-induced contraction in Ca2+-free medium, and its effect, like that of RYA, was selective for the first component of the response. This suggests that the uptake of Ca2+ by the ER Ca2+ pump from the cytosol is also in operation. The reversible effect of CPA is demonstrated in Fig. 4. Repletion in the presence of CPA suppressed the biphasic response (Fig. 4B, left). After washout and a
Effects of BAY K
8644 and CPA on repletion with 60
mA4 KCI. Repletion performed in the presence of 60 mM KC1 was followed by an apparent uniphasic contraction to PE in Ca2+-free Krebs (Fig. 5A). In the presence of CPA (30 PM), repletion was nearly abolished, as seen in Fig. 5B. Statistically, CPA appeared to inhibit only the first component of the contraction (Fig. 7). In the presence of BAY K 8644 (1 PM), repletion as denoted by the PE-induced contraction in Ca2+-free medium was potentiated (Fig. 5C) compared with controls (Fig. 5A). BAY K 8644, added together with CPA, partially prevented its inhibitory effect (cf. Fig. 5B with 0). Thus the effect wash
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Figures 6 and 8 summarize quantitatively the effects of various agents on the repletion of the PE-sensitive store in normal Krebs solution (Fig. 8) and the concentration effect of EGTA on the components of the biphasic response to PE in Ca2+ -free medium in the presence and absence of BAY K 8644 (Fig. 6). The fact that BAY K 8644 promoted refilling of the compartment (represented by the second component of the biphasic contraction to PE), which was also EGTA-sensitive, further supports our contention that this compartment of the Ca2+ stores may be communicating directly with the extracellular space. DISCUSSION
D
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Fig. 5. Repletion of PE-sensitive store was performed in high potassium-containing Krebs. In presence of CPA (30 PM, B), PE-induced contraction in Ca2+-free medium was inhibited markedly compared with controls. In presence of BAY K 8644 (1 PM, C), repletion was enhanced compared with controls. CPA inhibited some subsequent PEinduced contraction in Ca2+-free medium, with first component of response more affected than second as denoted by slowly rising contraction in Ca2+-free medium containing 50 PM EGTA.
As stated earlier, it is well recognized that in vascular smooth muscle acl-adrenoceptor agonist mobilizes Ca2+ from intracellular stores to cause a transient contraction in Ca2+-free medium (8). Refilling of these Ca2’ stores after depletion under physiological conditions requires entry of extracellular Ca2+. However, as in nonexcitable cells (14), reuptake of the previously released Ca2+ by the ER Ca2+ pump in vascular smooth muscle is also possible under special, nonphysiological experimental conditions (3). Whereas the role of ER in agonist-induced Ca2+ release involving the generation of inositol l&S-trisA
*Y I
of BAY K 8644 appears to be quite different from that of 60 mM KC1 on the refilling mechanism in the presence of CPA. Effect of EGTA on components of biphasic response.
After a repletion period of -30 min, the tissues were washed with Ca2+-free Krebs (containing 50 PM EGTA) and exposed to different concentrations of EGTA for Z3 min before PE stimulation. EGTA reduced the second component in a concentration-dependent manner, but the first component remained relatively intact. To amplify the effect of EGTA (showing clearly that it was selective for the second component), an additional series of experiments was performed in the presence of BAY K 8644 because repletion in the presence of BAY K 8644 (1 PM) enhanced the second component (see Fig. 8 for data). Figure 7 demonstrates the selective and concentration-dependent effect of EGTA on the second component. In addition, both components of the PE contraction in Ca2+-free medium were reduced simply by incubating the tissues in Ca2+ -free medium containing an addition of 100 PM EGTA over lo-30 min. Both components were virtually abolished by 20-30 min after incubation (data not shown). These results suggest that a portion of the PE-sensitive Ca2+ stores may be located very close and perhaps connected to the plasma membrane.
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Fig. 6. Summary of effect of EGTA on components of PE contraction in Ca’+-free medium when repletion was performed in normal Krebs (A) or in presence of BAY K 8644 (1 PM; B). CO, controls; TC, time controls. Open bars, 1st component; filled bars, 2nd component. Nos. are concentrations of EGTA used in PM. No. in parentheses, no. of dog mesenteric artery preparations in separate experiments. Minimal P value accepted for statistical significance was 0.05, where *P C 0.05; ***p < 0.01; ****p < 0.001.
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with the contention that Ca2+ store repletion limits the contraction during the initial developing phase of high KCl-induced contraction. It is therefore likely that Ca2+ uptake into the empty stores limits the increase in intracellular Ca2+ near contractile proteins and that Ca2+ entry during high KC1 contraction partly contributes to refilling of the internal Ca2+ store. Although the subsequent PE-induced contraction in Ca2+-free medium was enhanced markedly by repletion in the presence of 60 mM KCl, the PE contraction was inhibited markedly in tissues pretreated with the ER Ca2+ pump inhibitors thapsigargin or CPA (6, 16). Such observations suggest that such a thapsigargin- or CPA-sensitive route of refilling by Ca2+ entered via voltage-operated Ca2+ channels must involve Ca2+ passing through the cytosolic space before entering the store. Furthermore, these data suggest that the Ca2’ influx from the extracellular space instigated by high KC1 is through the voltage-sensitive Ca2+ channels and that these channels have no direct connection with the intracellular store. However, further experimentation with the use of BAY K 8644 and CPA revealed that this statement is incomplete.
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Fig. 7. Recordings show selective and concentration-dependent effect of EGTA on second component of PE contraction in Ca2+-free medium in presence of BAY K 8644 (1 PM).
phosphate (IPa) is reasonably well established, the refilling process of the ER remains obscure. The route(s) of entry of Ca2+ into the agonist-sensitive internal Ca2+ store remains an interesting dispute (see reviews in Refs. 18, 19). The present study-provides a novel observation of a biphasic contractile response of DMA to PE in Ca2+free medium. The routes of Ca2+ entry into the agonistsensitive Ca2+ stores during the refilling process proved to be manipulated selectively by various pharmacological tools. Our results, as discussed below, are consistent with the model in which the agonist-sensitive Ca2’ stores, presumably the ER, could be refilled from the cytosol. This route is dependent on the sequestration of Ca2+ by the ER Ca2’ pump. In addition, we have obtained evidence for a route in which another agonist-sensitive Ca2+ store appears to be connected directly to the extracellular space. Both routes ultimately require extracellular Ca2+. Evidence for Contribution of ER Ca2+Pump to Refilling of Ca2+Store
We have observed previously that, when repletion was performed in the presence of a depolarizing concentration of KCl, the subsequent PE-induced contraction in Ca2+-free medium was enhanced markedly compared with when repletion was performed in normal Krebs solution (16). In addition, the slowly developing KC1 contracture immediately after PE-induced depletion of its store, which was replaced by an abrupt KC1 contracture in the presence of thapsigargin (another putative microsomal Ca2+ pump inhibitor) (16, 23), is consistent
Evidence for Ca2+Entry Into Agonist-Sensitive Store That Bypasses ER Ca2+Pump
When repletion was performed in the presence of BAY K 8644, as with high KCl, the subsequent PE-induced contraction was enhanced compared with controls, and it was not differentiated into two components (Fig. 5, A and C). In the presence of CPA alone, both the components of the PE-induced contraction were inhibited markedly (Fig. 50). However, in the presence of both BAY K 8644 and CPA, the resultant PE tension increase in Ca2+-free medium was slow in onset, revealing only the second component. The second component was not inhibited by CPA in the presence of BAY K 8644 (Fig. 4, C and 0). This observation is consistent with the idea that BAY K 8644 may selectively open a route for refilling the internal Ca2+ store independent of CPA inhibition. We suggest that BAY K 8644 may open a direct passage connecting the extracellular space to the internal Ca2+ store. The hypothesis for a direct route of entry into the agonist-sensitive Ca2+ store is also supported by the fact that the second component was very sensitive to EGTA in the presence as well as in the absence of BAY K 8644 (Fig. 6). A Model Proposed for Ca2+Entry Into Internal Store for Refilling
In view of the above findings from the DMA, we propose a model for Ca2+ entry in the internal Ca2+ store of vascular muscle as shown in Fig. 9. In this model, the agonist-sensitive internal Ca2+ store consists of two internal Ca2+ storage sites, each representing one functional component of the biphasic response to PE in Ca2+free medium. Compartment A represents the source of Ca2+ for the initial component of the biphasic response to the agonist. This component is sensitive to depletion of Ca2+ by RYA, which opens the ER Ca2+-release channels (lo), or by CPA, which inhibits the ER Ca2+ pump
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(6, 20). Based on the observations that the first component can be inhibited by selective modulators of the ER, we suggest that the first component is a consequence of Ca2+ release from the ER proper. Therefore, it possibly contains IP3 receptors that cause Ca2+ release and subsequent contraction on agonist activation. Ca2+ refilling occurs from the cytosol, which in turn depends on Ca2+
conme
Fig. 8. Summary of effect of various compounds on biphasic contraction to PE in Ca2+-free medium after repletion in normal Krebs. Con, control; BAYK, BAY K 8644; NIF, nifedipine; KCP, high KC1 + CPA. Open bars, 1st component; filled bars, 2nd component. No. in parentheses, no. of experiments. For statistical analysis, responses were compared with corresponding controls in Con (A), BAYK (B), NIF (C), RYA (D), and CPA (E). Only statistically significant differences are indicated. *‘P < 0.05; **P < 0.02; ***P < 0.01; ****p < 0.001.
CPCI-
Fig. 9. Schematic model proposed to explain biphasic contraction in dog mesenteric artery. Agonist such as PE activates formation of inositol 1,4,&trisphosphate (IP,) from phospholipase C (PLC). IP3 stimulates release of Ca2’ from ER proper (A: site of Ca2’ for 1st component of biphasic contraction in Ca2+-free medium). RYA stimulates (RYA+) Ca2’ release from, whereas CPA inhibits (CPA-) Ca2’ uptake into ER. Released Ca2’ activates contractile proteins nearby to cause contraction. Directly connected to plasma membrane is superficial ER (B: site of Ca2’ for 2nd component). It is gated by nifedipine (inhibitory, NIF-) or BAY K 8644 (stimulatory, BAYK+). Release of Ca2’ from superficial ER can be induced by increased cytosolic concentration of Ca2’ ([Cali) such as via Ca2’-induced Ca2+-release mechanism. This can therefore account for slower-onset second component contraction. Other nifedipine-sensitive Ca2’ channels (VOC, BAYK+) or receptor-operated Ca2’ channels (ROC) on plasma membrane can also admit Ca2’ from extracellular space, but this Ca2’ influx must be taken up into ER proper via CPA-sensitive Ca2’ pump.
CPAfBayK (21)
entry from the extracellular space. The entry can be gated by various Ca2’ channels present in the plasma membrane. Slower in onset, the second component may be a consequence of Ca2+ release distant from the contractile filaments. Alternatively, it may be slow because it represents the Ca2+-induced Ca2+-release mechanism, and intracellular concentration of Ca2+ must rise to a certain level, as a result of Ca2+ entry or Ca2+ release from the first component, to initiate it. The second, slower component of the biphasic response to the agonist may be represented by compartment B. This Ca2+ storage compartment resides close to, and possibly is directly connected to, the plasma membrane. Although the idea of a pool of a superficially located ER has been postulated previously (25) and supported by ultrastructural evidence (9), the observation that the second component of the PE contraction in Ca2+ -free medium was susceptible to modulation by L-type Ca2+ channel agents and EGTA and unaffected by the inhibitory action of CPA on the ER Ca2+ pump has led us to extend the postulation to include the presence of a direct passage for Ca2’ entry in vascular smooth muscle for the refilling of the internal Ca2+ pool. The gate at this direct passage may be voltage operated, although the voltage sensor as well as the voltage sensed at the direct channel site between these compartments and that at the plasma membrane proper may be quite different entities. Comparison With Other Models
The idea of a close integration between the ER and the plasma membrane originated as a result of studies on vascular smooth muscle (4, 24, 25) and evolved into a
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“capacitative Ca2+-entry” model in nonexcitable cells (18). In this hypothetical model, agonist-induced depletion of Ca2+ store signals the entry of Ca2’ to refill the empty store through a direct channel that links the store to the extracellular space. However, during the past few years, more sophisticated experimental protocols (e.g., the use of fluorescent Ca2+-indicators) and more selective pharmacological tools (e.g., the use of selective ER Ca2’ pump inhibitors such as thapsigargin) have provided a wide array of evidence that led to a substantially modified version of the capacitative model. This revised model encompasses a cytosolic route of Ca2+ entry for internal pool repletion that depends ultimately on the ER Ca2+ pump (as reviewed in Ref. 19). In our model, the routes of Ca2’ entry as proposed in both versions of Putney’s capacitative model seem likely in vascular smooth muscle. The lack of BAY K 8644sensitive Ca2+ entry in many nonexcitable cells may limit the routes of Ca2+ entry and make the direct route of Ca2’ entry to the internal stores of these cells less likely (14, l&22). In vascular smooth muscle, recycling of Ca2+ by ER as reflected by repeated contractions to norepinephrine in Ca2’ -free medium has been reported in portal vein smooth muscle under the experimental condition when the Ca2’ entry and Ca2’ extrusion processes were blocked by 10 mM La3+ (3). On the other hand, the direct route of Ca2+ entry into the store as an alternative mechanism for the agonist-induced Ca2’ entry also has received experimental support from the recent study of Missiaen et al. (17) on rat aortic smooth muscle cell line A7r5. They have demonstrated substantial accumulation of Mn2+, a poor substrate for ER Ca2+ pump, in the previously depleted internal Ca2’ store when Mn2+ was introduced extracellularly. Unlike our previous findings in rat aortic ring preparations (16) and the present findings in the DMA, RYA and KC1 depolarization did not modify the Ca2’ uptake in A7r5 ceils. This raises the caution put forth by Putney (19) that “it seems doubtful that capacitative Ca2+ entry is the only mechanism by which agonists increase Ca2+ entry in cells. It seems more likely that it is but one of several mechanisms by which this might be accomplished, depending on the cell and stimulus involved.” In sum, we have presented evidence suggesting that both direct and cytosolic pathways of Ca2+ entry into the internal store are operative in DMA.
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17.
18. 19.
This work was supported by grants-in-aid from the Medical Research Council of Canada (MRC) and the Heart and Stroke Foundation of Ontario (HSFO). C. Y. Kwan is a recipient of an HSFO Career Investigator Award, and A. M. Low is a recipient of an MRC postdoctoral fellowship award. Address for reprint requests: C. Y. Kwan, .HSC-4N50, Faculty of Health Sciences, McMaster Univ., 1200 Main St. W., Hamilton, Ontario L8N 325, Canada. Received
15 April
1991; accepted
in final
form
8 August
20.
21.
22.
1991.
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