Minor role of a Ca2+-depleted sarcoplasmic reticulum in heterologous desensitization of smooth muscle to K+

Robert L. Wardle and Richard A. Murphy

Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906-0011

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Exposure of porcine carotid artery smooth muscle (PCASM) to histamine was followed by a large reduction in the rate of force generation in response to 40 mM KCl. This was shown to be a manifestation of slow attainment of a steady-state myoplasmic Ca2+ concentration ([Ca2+]i). We hypothesized that if net transsarcolemmal Ca2+ flux into the depolarized PCASM cells is the same before and after a desensitizing histamine treatment, then the transient attenuation of the increase in [Ca2+]i may be due to accelerated uptake of Ca2+ by a partially depleted sarcoplasmic reticulum (SR) acting as a Ca2+ sink or superficial buffer barrier. We tested this hypothesis by eliciting responses of "desensitized PCASM" to 40 mM KCl in the presence of cyclopiazonic acid (CPA), an SR Ca2+-ATPase inhibitor. Contractions of CPA-treated tissues were attenuated less than those of tissues not treated with CPA, but they were not abolished. CPA-insensitive mechanism(s) dominated the desensitization. We conclude that histamine pretreatment reduced net transsarcolemmal Ca2+ flux into PCASM in response to 40 mM KCl.

arterial smooth muscle; latch; excitation-contraction coupling; activation-contraction coupling; calcium transients; attenuation

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

HETEROLOGOUS DESENSITIZATION of smooth muscle can be defined as the reduced contractile response to a stimulus after a limited exposure to a different contractile agonist. Presumably, this capacity to modulate sensitivity to different mediators (many of which induce transsarcolemmal depolarization and L-channel Ca2+ conductance) enables vascular smooth muscle to regulate blood pressure and regional blood flow appropriately in vivo. For example, in the presence of chronic periarteriolar inflammation and local high concentrations of vasoactive mediators (such as histamine released from mast cells), desensitization would prevent excessive vascular smooth muscle contraction that might compromise adequate blood flow to tissues distal to the site of inflammation.

In our experimental model of heterologous desensitization (23), exposure of porcine carotid artery medial rings to histamine was followed by long-lasting reductions in the force elicited by 40 mM KCl (Fig. 1). In contrast to the classic interpretation of desensitization as a reduced activation involving fewer cycling cross bridges, we concluded that the apparent reduction in force-generating capacity was really a manifestation of a slowing in the rates at which the myoplasmic Ca2+ concentration ([Ca2+]i), and consequently myosin regulatory light chain (MRLC) phosphorylation and cross-bridge recruitment (estimated from increases in stiffness), attained steady-state values (23). This retarded stimulus-[Ca2+]i coupling resulted in very slow cross-bridge cycling and rates of force development (latch) (23). The steady-state 40 mM KCl-induced force was not reduced, and there was no detectable change in the Ca2+ sensitivity of MRLC phosphorylation or the dependence of cross-bridge cycling rate on myosin phosphorylation (23).


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Fig. 1.   Desensitization of porcine carotid artery smooth muscle to K+ depolarization. A: relative force induced by 40 mM KCl (arrow) before histamine and 20 min after removal of histamine (10-3 M, 30 min). Note delay of active force development and slower rate of force development after exposure to histamine. B: attenuation of relative force (from A) is difference between time-matched responses to 40 mM KCl before and after histamine (modified from Ref. 23).

The retarded stimulus-[Ca2+]i coupling during K+ depolarization could be due to attenuated Ca2+ influx into the myoplasm or to accelerated Ca2+ extrusion from the myoplasm. We hypothesized that if net transsarcolemmal Ca2+ flux into the depolarized carotid medial cells is the same before and after a desensitizing histamine treatment, then the transient attenuation of the increase in [Ca2+]i and MRLC phosphorylation may be due to accelerated uptake of Ca2+ by a partially depleted sarcoplasmic reticulum (SR) acting as a Ca2+ sink or superficial buffer barrier (Fig. 2) [van Breeman et al. (22)]. Ratz et al. (17) invoked this hypothesis to account for delayed responsiveness of rabbit femoral artery smooth muscle to 23 mM KCl after pretreatment with phenylephrine. We employed two different strategies to test this hypothesis. First, we attempted to create a superficial buffer barrier to increases in [Ca2+]i by exposing tissues to SR Ca2+ depletion protocols. Evidence that Ca2+ depletion desensitized the smooth muscle to K+ depolarization would support the hypothesis. Second, we elicited responses of "desensitized carotid media" to 40 mM KCl in the presence of cyclopiazonic acid (CPA), a selective SR Ca2+-ATPase inhibitor (1, 4, 5, 10, 12, 20, 25). If effective, CPA should prevent the SR from accumulating Ca2+ on K+ depolarization, and the desensitization should be abolished or diminished.


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Fig. 2.   Schematic illustration of sarcoplasmic reticulum (SR) acting as a Ca2+ sink or superficial buffer barrier. We hypothesized that if net transsarcolemmal Ca2+ flux into depolarized carotid media smooth muscle cells is the same before (left) and after (right) a desensitizing histamine treatment, then transient attenuation of increase in myoplasmic Ca2+ concentration may be due to accelerated uptake of Ca2+ by a partially depleted SR acting as a Ca2+ sink or superficial buffer barrier. SL, sarcolemma; L, L-type voltage-dependent channel.

Our objectives were to determine 1) whether SR Ca2+ depletion protocols or histamine pretreatment depleted the SR Ca2+ stores, 2) whether contractile responses to depolarization with 40 mM KCl were attenuated, and 3) whether CPA abolished or diminished any desensitization to K+ depolarization. Contractions induced by phenylephrine (10-4 M in Ca2+-free solution, 5 min) were employed as a rough index of the amount of releasable Ca2+ in the inositol 1,4,5-trisphosphate receptor (IP3R)-mediated SR Ca2+ store. Contractions induced by caffeine (20 mM, 90 s) were employed as a rough index of the amount of releasable Ca2+ in the ryanodine receptor (RyR)-mediated SR Ca2+ store. In contrast to phenylephrine, caffeine bypasses G protein-coupled, plasmalemmal receptor-activated signaling and inositol 1,4,5-trisphosphate generation. Thus brief caffeine-induced contractions should be less affected by any histamine-induced downregulation of signal transduction mechanisms proximal to activation of Ca2+ release from the SR.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Tissue preparation. Porcine carotid arteries obtained from a slaughterhouse were transported to the lab and stored at 4°C in physiological saline solution (PSS) containing (in mM) 140.1 NaCl, 4.7 KCl, 1.2 Na2HPO4, 1.6 CaCl2, 5.6 D-glucose, 1.2 MgCl2, 2.0 MOPS buffer (pH 7.4 at 4 or 37°C), and 0.02 EDTA to chelate trace heavy metal ions. Deendothelialized carotid artery medial rings were prepared as described by Wingard et al. (24).

Drugs and solutions. Histamine dihydrochloride, pyrilamine maleate, cimetidine, phenylephrine hydrochloride, caffeine, CPA, DMSO, and EGTA were purchased from Sigma Chemical (St. Louis, MO). Ca2+-free PSS (also referred to as 0 Ca2+ PSS) contained the Ca2+ chelator EGTA (1.0 mM) and no added CaCl2. Concentrated stock solutions of most drugs were prepared weekly with PSS containing ascorbic acid (0.1% wt/vol), and drugs were diluted in PSS immediately before use. A concentrated stock solution of CPA in DMSO was diluted in PSS immediately before use. KCl was stoichiometrically substituted for NaCl in depolarizing high-K+ PSS (40 or 109 mM KCl).

Force. Deendothelialized carotid artery medial rings (2-3 mm in width) were mounted over two stainless steel posts. One post was connected via a support rod to a micrometer used to adjust the length of the preparation, and the other post was part of a 1-mm-diameter stainless steel wire connected to a Grass FT 03 force transducer. Mounted rings were immersed in water-jacketed baths containing PSS at 37°C and pH 7.4, continuously bubbled with compressed air. Medial rings were adjusted to the length at which the active isometric force generated in response to 109 mM KCl was maximized (Lo). Isometric force was continuously recorded on a polygraph. Stress was calculated as force per tissue cross-sectional area estimated using tissue blotted wet weight, ring length at Lo, and tissue density of 1.055 g/cm3. Contractile responses were normalized to a control 109 mM KCl-induced response (% of K109) elicited before the experimental protocol.

General protocol. Porcine carotid artery medial rings were contracted by 10-4 M phenylephrine in 0 Ca2+ PSS (PhE/0 Ca2+), 20 mM caffeine, or 40 mM KCl both before and after intervening SR Ca2+ depletion protocols (see Fig. 3; protocol modified from Ref. 16) or before and after an intervening treatment with histamine (10-3 M in PSS, 30 or 40 min). The general protocol is illustrated by force tracings in which caffeine-induced contractions were elicited both before and after an intervening treatment with or without histamine (see Fig. 6, top). After the intervening treatment and immediately before the final exposure to PhE/0 Ca2+, caffeine, or KCl (with or without the Ca2+-ATPase inhibitor CPA), all tissues were washed three times over 30 min with PSS (containing 1.6 mM CaCl2) with or without CPA (10-5 M or 3 × 10-5 M) and containing the H1- and H2-receptor antagonists pyrilamine (10-6 M) and cimetidine (10-5 M) to 1) facilitate rapid removal of histamine from the histamine-treated tissues and 2) block response to any histamine that remained after the first two solution changes. Treatment with PSS containing histamine receptor antagonists had no effect on the final 40 mM KCl-induced responses in control experiments.

Data analysis. We previously adopted an alternative form of data presentation, i.e., attenuation analysis, because it has advantages for understanding the mechanisms underlying heterologous desensitization and the complex temporal relationships among the key signaling events regulating smooth muscle contraction (23). Because the hypothesis being tested in the present study was proposed in the discussion section of Ref. 23 and because we desired to readily integrate information gained from the two studies, we performed the same type of data analysis in the present study.

Medial rings that failed to generate 105 N/m2 active stress in response to a 10-min 109 mM KCl exposure were excluded from further analyses. PhE/0 Ca2+-, caffeine-, or KCl-induced active force, basal force, the delay between initiation of 40 mM KCl exposure and onset of active force (see Fig. 1A), and the PhE/0 Ca2+- or 40 mM KCl-induced maximum rate of force development (maximum dF/dt) were determined from chart records. Attenuation of these indexes of medial ring activation was calculated as the difference between values attained before and after intervening treatment. For example, Fig. 1 illustrates how the attenuation of KCl-induced active force was calculated by subtracting time-matched values attained after histamine treatment from the values attained before histamine treatment. The intervening treatment was sometimes associated with increases in basal force. The increase in basal force was calculated as the difference between the values attained immediately before exposures to the contractile agonist. For each contraction, agonist-induced active force was calculated from the basal force immediately before an exposure to the contractile agonist.

Each of the paired rings from three to six different arteries received one intervening treatment between two contractions elicited by one of the contractile agonists (PhE/0 Ca2+, caffeine, or KCl), and the second contraction was elicited in the presence or absence of CPA. The effect of CPA on the intervening treatment-induced attenuation of indexes of medial ring activation was assessed by comparing means or medians of the attenuation for different treatment groups (paired t-test, repeated measures ANOVA, or analogous nonparametric procedures).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of CPA on basal force. Tissues were treated with CPA to selectively inhibit SR Ca2+-ATPase activity after SR Ca2+ depletion protocols or exposure to histamine. Tissues treated with CPA exhibited a greater increase in basal force before the final PhE/0 Ca2+-, caffeine-, or KCl-induced contraction than did tissues that were not treated with CPA (Figs. 3-7, bottom). This difference reflects the contribution of SR Ca2+-ATPase activity to regulation of [Ca2+]i. The CPA-induced increase in basal force (<10% of the control 109 mM KCl-induced active force elicited before the experimental protocol, i.e., <10% of K109) was small relative to PhE/0 Ca2+-, caffeine-, or 40 mM KCl-induced peak active force (~55, 22, and 77% of K109, respectively). Nevertheless, the CPA-induced basal forces were probably large enough to influence estimates of reductions in caffeine-induced peak active force (see Fig. 6, top). Generally a CPA-induced increase in basal force correlated with a subtle change in the attenuation of agonist-induced force development (see Figs. 4-7) but the latter was not always statistically significant at P < 0.05. 


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Fig. 3.   SR Ca2+ depletion-repletion protocols. Isometric force tracings. Medial rings were exposed to 1.0 mM EGTA-buffered Ca2+-free physiological saline solution (0 Ca2+ PSS) changed every 5 min for 40 min total (bottom trace). Some tissues were exposed 3 separate times to 0 Ca2+ PSS containing 0.1 mM histamine (open bars beneath top trace) to ensure depletion of inositol 1,4,5-trisphosphate receptor-releasable SR Ca2+ stores. All tissues were exposed to 20 mM caffeine (solid bars) for 2 min at end of these protocols to ensure depletion of ryanodine receptor-releasable SR Ca2+ stores. All tissues were exposed to 1.6 mM Ca2+ PSS for 30 min to replete SR Ca2+ stores in absence (top trace) or presence (bottom trace) of 0.03 mM cyclopiazonic acid (CPA; hatched bar).


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Fig. 4.   SR Ca2+ depletion protocols depleted phenylephrine-releasable Ca2+ store. Attenuation of peak active force (top) and attenuation of maximum rate of force development (max dF/dt; middle) in response to 10-4 M phenylephrine in 0 Ca2+ PSS (5 min) 30 min after cessation of SR Ca2+ depletion protocols without histamine (Ca2+-free PSS; illustrated in Fig. 3, bottom trace) or SR Ca2+ depletion protocols with histamine (Histamine/0 Ca2+ PSS; illustrated in Fig. 3, top trace); 0 Ca2+ PSS contained the Ca2+ chelator EGTA (1.0 mM) and no added CaCl2. Phenylephrine-induced contractions after depletion protocols were attenuated. Phenylephrine-induced contractions of CPA-treated tissues (shaded bars) were attenuated more than those of tissues not treated with CPA (open bars; * P < 0.05 vs. CPA-treated tissues). Bottom: CPA-treated tissues exhibited a greater increase in basal force before final phenylephrine-induced contraction than tissues that were not treated with CPA (* P < 0.05 vs. CPA-treated tissues). Values are means + SE; paired rings for n = 4-6 arteries in each group. %K109, % of control 109 mM KCl-induced response elicited before experimental protocol.


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Fig. 5.   SR Ca2+ depletion protocols desensitized muscle to K+ depolarization. Top: attenuation of 40 mM KCl-induced active force 30 min after cessation of SR Ca2+ depletion protocols without (-) histamine (Ca2+-free PSS alone; illustrated in Fig. 3, bottom trace) or SR Ca2+ depletion protocols with (+) histamine (Histamine/0 Ca2+ PSS; illustrated in Fig. 3, top trace); 0 Ca2+ PSS (Ca2+-free PSS) contained the Ca2+ chelator EGTA (1.0 mM) and no added CaCl2. * P < 0.05 vs. non-CPA-treated tissues. Bottom: KCl-induced contractions exhibited a delay of active force development (# P < 0.05 only for Ca2+-free PSS alone in absence of CPA group; other groups not different from 0) and an attenuated maximum dF/dt. Note that these KCl-induced contractions were attenuated less than those following exposure to histamine in PSS (cf. top and Fig. 7, top; illustrated together in Fig. 8). KCl-induced contractions of CPA-treated tissues (3 × 10-5 M CPA, filled symbols and shaded bars) after depletion with histamine/0 Ca2+ PSS were attenuated less than KCl-induced contractions of tissues not treated with CPA (open symbols and open bars; * P < 0.05 vs. non-CPA-treated tissues). CPA-treated tissues after depletion with 0 Ca2+ PSS alone exhibited a greater increase in basal force before final KCl-induced contraction than non-CPA-treated tissues after depletion with histamine/0 Ca2+ PSS (dagger  P < 0.05). Values are means ± SE; paired rings for n = 3-5 arteries in each group.


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Fig. 6.   Histamine and CPA depleted caffeine-releasable Ca2+ store. Top: isometric force tracings for 4 carotid medial rings from 1 artery. Tissues were exposed to caffeine (20 mM, 90 s; arrowheads) before and after intervening treatment with PSS alone (trace a), CPA (3 × 10-5 M; trace b), histamine (10-3 M; trace c) or both histamine and CPA (trace d). Force scale (vertical bar) above trace a is applicable for caffeine-induced contractions only (traces a-d). Note that in traces c and d amplification of force signal was halved during histamine activation (early spike) to resolve maximum force on chart record. Amplification of force signal was returned to original sensitivity after response to histamine (apparent sudden increase in basal force) to resolve caffeine-induced force. Bottom: attenuation of caffeine-induced peak active force 30 min after cessation of either PSS alone (a and b) or PSS with histamine (c and d; letters in parentheses correspond to traces in top). Caffeine-induced contractions after histamine were attenuated (dagger  P < 0.05 vs. PSS alone). Caffeine-induced contractions of CPA-treated tissues (shaded bars) were attenuated more than those of tissues not treated with CPA (open bars; * P < 0.05 vs. CPA-treated tissues). CPA-treated tissues exhibited a greater increase in basal force before final caffeine-induced contraction than tissues that were not treated with CPA (* P < 0.05 vs. CPA-treated tissues). Values are means + SE; paired rings for n = 4-5 arteries in each group.


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Fig. 7.   CPA diminished histamine-induced desensitization to K+ depolarization. Top: attenuation of 40 mM KCl-induced active force 30 min after cessation of histamine (10-3 M, 30 min; left) or histamine (10-3 M, 40 min; right). * P < 0.05 vs. non-CPA-treated tissues. Bottom: KCl-induced contractions exhibited a delay of active force development and an attenuated maximum dF/dt as illustrated in Fig. 1. KCl-induced contractions of CPA-treated tissues (10-5 M CPA, left; 3 × 10-5 M CPA, right) were attenuated less than those of tissues not treated with CPA (* P < 0.05 vs. non-CPA-treated tissues; dagger  P < 0.05 vs. non-CPA-, non-DMSO-treated tissues). CPA-treated tissues exhibited a greater increase in basal force before final KCl-induced contraction than tissues that were not treated with CPA (* P < 0.05 vs. non-CPA-treated tissues). Values are means ± SE; paired rings for n = 6 arteries in each group.

Effects of SR Ca2+ depletion protocols and CPA on sensitivity to K+ depolarization. Phenylephrine (PhE/0 Ca2+)-induced contractions before and after intervening treatments were used to assess the amount of releasable Ca2+ in the IP3R-mediated Ca2+ store. PhE/0 Ca2+-induced contractions were attenuated 30 min after cessation of SR Ca2+ depletion protocols (Fig. 4), consistent with partial depletion of the IP3R-mediated Ca2+ store. CPA should inhibit SR Ca2+-ATPases and refilling of SR Ca2+ stores during the 30-min repletion by incubation of tissues in 1.6 mM Ca2+-PSS (illustrated in Fig. 3, bottom). This was manifested by a greater attenuation of SR Ca2+-dependent (PhE/0 Ca2+-induced) contractile responses of CPA-treated tissues than of responses of tissues not treated with CPA (Fig. 4). Although the greater attenuation of PhE/0 Ca2+-induced peak active force in CPA-treated tissues was not statistically significant (P > 0.05; Fig. 4, top), attenuation of the maximum rate of PhE/0 Ca2+-induced force development (maximum dF/dt) of CPA-treated tissues was greater than that exhibited by tissues not treated with CPA (P < 0.05; Fig. 4, middle).

Having indirect evidence that our protocol effectively reduced the amount of PhE/0 Ca2+-releasable Ca2+ in the SR, we exposed another group of tissues to SR Ca2+ depletion protocols to assess their impact on contractile responses to K+ depolarization. These contractions were strictly dependent on extracellular Ca2+ concentration and transsarcolemmal Ca2+ influx. Ca2+-depleted tissues exhibited an attenuated maximum dF/dt in response to 40 mM KCl (Fig. 5, bottom). A modest attenuation of active force relative to the histamine-induced attenuation was observed (cf. Fig. 5, top, and Fig. 7, top; illustrated together in Fig. 8). CPA should inhibit SR Ca2+ uptake during exposure to 40 mM KCl and abolish any attenuation of force due to SR sequestering Ca2+ (see Fig. 2). Indeed, in two of the five tissues that were exposed to histamine during the Ca2+ depletion protocol, CPA abolished attenuation of 40 mM KCl-induced maximum dF/dt and active force. The KCl-induced contractions of the other three CPA-treated tissues were 37-67% less attenuated than KCl-induced contractions of tissues that were not treated with CPA (P < 0.05; Fig. 5, top and bottom).


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Fig. 8.   Comparison of SR Ca2+ depletion-induced and histamine pretreatment-induced desensitization to K+ depolarization. Attenuation of 40 mM KCl-induced active force 30 min after SR Ca2+ depletion protocols with histamine and caffeine in 0 Ca2+ PSS (40 min; from Fig. 5, top) or 30 min after histamine (10-3 M in 1.6 mM Ca2+ PSS, 40 min; from Fig. 7, top right); 0 Ca2+ PSS contained the Ca2+ chelator EGTA (1.0 mM) and no added CaCl2. CPA (3 × 10-5 M) appeared to be as effective in diminishing histamine pretreatment-induced desensitization to 40 mM KCl as it was in abolishing or diminishing SR Ca2+ depletion-induced desensitization to 40 mM KCl. * P < 0.05 vs. non-CPA-treated tissues.

Effects of histamine pretreatment and CPA on SR Ca2+ stores and sensitivity to K+ depolarization. Caffeine-induced contractions were used to assess the amount of releasable Ca2+ in the RyR-mediated Ca2+ store. Caffeine-induced contractions were not attenuated 30 min after cessation of PSS alone but were attenuated 30 min after cessation of histamine (10-3 M, 30 min; Fig. 6). This result is consistent with histamine-induced partial depletion of the RyR-mediated Ca2+ store. CPA should inhibit SR Ca2+ uptake, and CPA alone should deplete the RyR-mediated Ca2+ store. Such was the case: caffeine-induced contractions of CPA-treated tissues were attenuated more than those of tissues not treated with CPA, regardless of whether they had an intervening histamine treatment (Fig. 6). This implies that CPA alone or after histamine was more effective than histamine alone in depleting the RyR-mediated Ca2+ store.

Indirect evidence indicates that histamine pretreatment effectively reduced the amount of caffeine-releasable Ca2+ in the SR. Another group of tissues exposed to histamine exhibited a delay in active force development and an attenuated maximum dF/dt in response to 40 mM KCl (Fig. 7, bottom). This attenuated the active force (Fig. 7, top), as we described previously (23). CPA should prevent the SR from accumulating Ca2+ on K+ depolarization, and the desensitization should be abolished or diminished. The KCl-induced contractions of CPA-treated tissues were attenuated up to 44% less than KCl-induced contractions of tissues that were not treated with CPA (Fig. 7, top), but CPA did not abolish desensitization to K+ depolarization.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The SR is a primary regulator of the cytosolic free Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells and is thereby critically involved in controlling vascular smooth muscle tone. Our current understanding of how the SR contributes to maintenance of homeostasis of [Ca2+]i includes a role as a "superficial buffer barrier" or sink for Ca2+ entering the cell when the SR Ca2+ store is depleted and a role extruding Ca2+ when the SR Ca2+ store is filled [reviewed by Daniel et al. (3) and by van Breeman et al. (22)]. Filling of the SR Ca2+ store is coupled to regulation of transsarcolemmal Ca2+ flux, thereby providing mechanisms whereby depleted stores may be readily refilled ["capacitative Ca2+ entry" reviewed by Putney (13); Daniel et al. (3); "Ca2+ sparks" described by Nelson et al. (9)]. The maintenance of homeostasis of [Ca2+]i in porcine carotid artery smooth muscle subjected to various treatments (18, 19) supports both the buffer barrier hypothesis and the capacitative Ca2+ entry hypothesis (which are not mutually exclusive).

Prior in vitro exposure and withdrawal of contractile agonists such as histamine, phenylephrine, and carbachol may desensitize smooth muscle to depolarizing stimuli for prolonged periods (6, 8, 14, 15, 17, 23). Our findings regarding the putative involvement of a Ca2+-depleted SR in heterologous desensitization of carotid media to depolarizing stimuli can be summarized as follows. Thirty minutes after cessation of SR Ca2+ depletion protocols, 1) the IP3R-releasable Ca2+ store was partially depleted and appeared to be depleted further by CPA; 2) 40 mM KCl-induced contractile responses of tissues not treated with CPA were attenuated, although much less than was observed after histamine treatment; and 3) 40 mM KCl-induced contractile responses of CPA-treated tissues were not attenuated or were attenuated less than those of tissues not treated with CPA. Thirty minutes after cessation of histamine treatment, 1) the RyR-releasable Ca2+ store was partially depleted and was depleted further by CPA; and 2) 40 mM KCl-induced contractile responses of CPA-treated tissues were attenuated less than those of tissues not treated with CPA, but CPA did not abolish desensitization to K+ depolarization. These results are consistent with our hypothesis that accelerated uptake of Ca2+ by a partially depleted SR acting as a Ca2+ sink or superficial buffer barrier contributes to heterologous desensitization to submaximal K+ depolarization. However, CPA-insensitive mechanism(s) appear to be responsible for the majority of the attenuated carotid medial response to 40 mM KCl (see Fig. 8). This suggests that net transsarcolemmal Ca2+ flux into the depolarized smooth muscle cells is not the same before and after a desensitizing histamine treatment.

The contribution of SR Ca2+-ATPase activity to regulation of [Ca2+]i in unstimulated, resting carotid media was revealed by the CPA-induced increase in basal force. These results are consistent with CPA-induced increase of basal force in rat pulmonary artery, aorta, and some superior mesenteric artery smooth muscle (4, 5, 20) and with CPA-induced increase of both basal force and [Ca2+]i in ferret portal vein and rat femoral artery smooth muscle (1, 10). The CPA-induced increase of basal force is likely due to the increase in [Ca2+]i rather than to an increase in the Ca2+ sensitivity of the contractile elements, since CPA did not modify the pCa-tension relationship of beta -escin-skinned rat pulmonary artery smooth muscle (5).

After cessation of SR Ca2+ depletion protocols, the extracellular tissue space of the carotid media should be Ca2+ replete within 10 min of exposure to PSS (16). The IP3R-releasable Ca2+ store of the carotid media was partially depleted (attenuated PhE/0 Ca2+-induced contractions, Fig. 4), coincident with desensitization to K+ depolarization (attenuated 40 mM KCl-induced contractions, Fig. 5) 30 min after cessation of SR Ca2+ depletion protocols. However, this desensitization to K+ depolarization was small compared with that after histamine pretreatment, and it was nearly completely abolished by CPA (cf. Figs. 5 and 7; Fig. 8). The attenuated carotid media contractile responses to K+ depolarization after SR Ca2+ depletion were similar to those of rabbit ear artery and dog mesenteric artery smooth muscle (2, 7). Furthermore, canine mesenteric artery treated with the SR Ca2+-ATPase inhibitor thapsigargin was not desensitized to K+ depolarization after SR Ca2+ depletion (7). These results support at least a minor role for a Ca2+-depleted SR in heterologous desensitization of carotid media to submaximal K+ depolarization.

The attenuation of caffeine-induced contractions of the carotid media 30 min after cessation of histamine (Fig. 6) was consistent with the attenuation of both caffeine-induced stress and [Ca2+]i transients of rabbit femoral artery smooth muscle 10 min after cessation of phenylephrine (17) and the attenuation of caffeine-induced contractions of guinea pig taenia cecum smooth muscle after carbachol exposure (6). These results imply that the RyR-mediated SR Ca2+ store was partially depleted after agonist pretreatment, possibly due to impairment of mechanism(s) required for refilling this store (e.g., reduced transsarcolemmal Ca2+ influx). When we deliberately impaired refilling of the SR Ca2+ store by treating tissues with CPA, caffeine-induced contractions were greatly attenuated regardless of whether tissues were pretreated with histamine (Fig. 6). This was consistent with attenuation of both caffeine-induced tension and [Ca2+]i transients of rat femoral artery smooth muscle after CPA (10). Alternative explanations for the attenuated caffeine-induced contractions of the carotid media are impaired mechanism(s) coupling caffeine to RyR-mediated Ca2+ release from the SR or a decreased Ca2+ sensitivity of the contractile elements. Ratz et al. (15) and Oike et al. (11) reported that agonist pretreatment of vascular smooth muscles modulated the Ca2+ sensitivity of the contractile machinery and thereby impaired contractility to subsequent KCl exposure. However, we did not detect a change in the Ca2+ sensitivity of cross-bridge phosphorylation during 40 mM KCl-induced contractions of histamine-pretreated carotid media (23).

The histamine-induced desensitization of the carotid media to 40 mM KCl was significantly diminished but not abolished by CPA (Fig. 7). We interpret this finding as evidence that some Ca2+ entering the cells through L-channels was diverted from the myofilaments by Ca2+-ATPases of the partially depleted superficial SR, consistent with the superficial buffer barrier hypothesis [reviewed by van Breeman et al. (22)]. Ratz et al. (17) invoked this hypothesis to account for delayed responsiveness of rabbit femoral artery smooth muscle to 23 mM KCl after pretreatment with phenylephrine. Shima and Blaustein (20) suggested the buffer barrier hypothesis could account for enhanced 30 mM K+-evoked contractions of rat mesenteric artery smooth muscle in the presence of CPA. Yoshikawa et al. (25) tested the buffer barrier hypothesis in guinea pig urinary bladder myocytes using voltage-clamp techniques and intracellular Ca2+ microfluorometry. CPA modulated caffeine-induced or depolarization-induced [Ca2+]i transients and the Ca2+-modulated membrane currents, L-channel current and Ca2+-activated K+ current, implying that Ca2+ sequestration by the SR can buffer part of the Ca2+ influx during slow depolarizations (25).

Although CPA nearly abolished the small desensitization of carotid media to K+ depolarization after SR Ca2+ depletion protocols, CPA-insensitive mechanism(s) appear to be responsible for the majority of the attenuated carotid media response to 40 mM KCl after histamine pretreatment (see Fig. 8). The inability of CPA to completely abolish this desensitization to K+ depolarization could be a manifestation of some CPA-insensitive isoform(s) of SR Ca2+-ATPase, or compartmentalized SR Ca2+-ATPases protected from CPA. We found no evidence in the literature to support either of these explanations. The involvement of intracellular compartments other than the SR seems unlikely, given that mitochondria contribute significantly to [Ca2+]i homeostasis only under pathophysiological conditions (21) and that the role of the nucleus and/or nuclear membrane in [Ca2+]i homeostasis has not been established for vascular smooth muscle (12). Finally, we cannot rule out the possibility that histamine pretreatment induces changes in other biochemical signaling pathways that may directly or indirectly affect myofilament activation and force transduction. However, the temporal correlation of the attenuations of aequorin-estimated [Ca2+]i, MRLC phosphorylation, and tissue stiffness (biochemical and mechanical indexes of cross-bridge activation) during desensitization to 40 mM KCl indicate that Ca2+-dependent cross-bridge recruitment per se is not altered significantly (23). Furthermore, as stated previously (23), "the dramatic temporal dissociation between stiffness and stress implies that slow cross-bridge cycling must contribute significantly to reduced mechanical performance and the appearance of desensitization." Consequently we conclude that histamine pretreatment reduced net transsarcolemmal Ca2+ flux into carotid medial smooth muscle cells. This reduced net transsarcolemmal Ca2+ influx impaired both 1) refilling of the SR Ca2+ store upon removal of histamine and 2) the rate of increase of Ca2+ concentration at the myofilaments in response to 40 mM KCl. Putative mechanisms by which this could be accomplished include altered function or number of sarcolemmal proteins involved in Ca2+ fluxes and transient redistribution of ions across the sarcolemma (see Ref. 23).

    ACKNOWLEDGEMENTS

We thank Drs. Paul H. Ratz, Christopher J. Wingard, John D. Strauss, and John S. Walker for thoughtful discussions. Arteries were donated by the Smithfield Company of Smithfield, VA.

    FOOTNOTES

The work was supported by National Heart, Lung, and Blood Institute Grant 5P01-HL-19242 and an American Heart Association (Virginia Affiliate) fellowship to R. L. Wardle.

Address for reprint requests: R. L. Wardle, Molecular Physiology and Biological Physics, University of Virginia, PO Box 10011, Charlottesville, VA 22906-0011.

Received 22 August 1997; accepted in final form 7 July 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Abe, F., H. Karaki, and M. Endoh. Effects of cyclopiazonic acid and ryanodine on cytosolic calcium and contraction in vascular smooth muscle. Br. J. Pharmacol. 118: 1711-1716, 1996[Abstract].

2.   Casteels, R., and G. Droogmans. Exchange characteristics of the noradrenaline-sensitive calcium store in vascular smooth muscle cells of rabbit ear artery. J. Physiol. (Lond.) 317: 263-279, 1981[Abstract].

3.   Daniel, E. E., C. van Breemen, W. P. Schilling, and C.-Y. Kwan. Regulation of vascular tone: cross-talk between sarcoplasmic reticulum and plasmalemma. Can. J. Physiol. Pharmacol. 73: 551-557, 1995[Medline].

4.   Deng, H.-W., and C.-Y. Kwan. Cyclopiazonic acid is a sarcoplasmic reticulum Ca2+-pump inhibitor of rat aortic muscle. Acta Pharm. Sinica 12: 53-58, 1991.

5.   Gonzalez De La Fuente, P., J.-P. Savineau, and R. Marthan. Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum Ca2+ pump blockers: thapsigargin and cyclopiazonic acid. Pflügers Arch. 429: 617-624, 1995[Medline].

6.   Hishinuma, S., and M. K. Uchida. Short-term desensitization of guinea-pig taenia caecum induced by carbachol occurs at intracellular Ca stores and that by histamine at H1-receptors. Br. J. Pharmacol. 94: 882-889, 1988[Abstract].

7.   Low, A. M., V. Gaspar, C.-Y. Kwan, P. J. Darby, J. P. Bourreau, and E. E. Daniel. Thapsigargin inhibits repletion of phenylephrine-sensitive intracellular Ca++ pool in vascular smooth muscles. J. Pharmacol. Exp. Ther. 258: 1105-1113, 1991[Abstract].

8.   Munoz, E., G. Tsujimoto, A. Tsujimoto, S. Azhar, and B. B. Hoffman. Desensitization of alpha 1 adrenoceptor-stimulated glycogen phosphorylase activity in vascular smooth muscle. J. Cardiovasc. Pharmacol. 14: 278-284, 1989[Medline].

9.   Nelson, M. T., H. Cheng, M. Rubart, L. F. Santana, A. D. Bonev, H. J. Knot, and W. J. Lederer. Relaxation of arterial smooth muscle by calcium sparks. Science 270: 633-637, 1995[Abstract].

10.   Nomura, Y., M. Asano, K. Ito, Y. Uyama, Y. Imaizumi, and M. Watanabe. Superficial sarcoplasmic reticulum calcium buffering of resting, voltage-dependent Ca++ influx in rat femoral arterial smooth muscle. J. Pharmacol. Exp. Ther. 279: 830-837, 1996[Abstract].

11.   Oike, M., N. Takahashi, and Y. Ito. Angiotensin II attenuates vascular contractility in the rabbit mesenteric artery. Biochem. Biophys. Res. Commun. 222: 208-214, 1996[Medline].

12.   Orallo, F. Regulation of cytosolic calcium levels in vascular smooth muscle. Pharmacol. Ther. 69: 153-171, 1996[Medline].

13.   Putney, J. W., Jr. Capacitative calcium entry revisited. Cell Calcium 11: 611-624, 1990[Medline].

14.   Ratz, P. H. Receptor activation induces short-term modulation of arterial contractions: memory in vascular smooth muscle. Am. J. Physiol. 269 (Cell Physiol. 38): C417-C423, 1995[Abstract/Free Full Text].

15.   Ratz, P. H., F. A. Lattanzio, Jr., and P. M. Salomonsky. Memory of arterial receptor activation involves reduced [Ca2+]i and desensitization of cross bridges to [Ca2+]i. Am. J. Physiol. 269 (Cell Physiol. 38): C1402-C1407, 1995[Abstract/Free Full Text].

16.   Ratz, P. H., and R. A. Murphy. Contributions of intracellular and extracellular Ca2+ pools to activation of myosin phosphorylation and stress in swine carotid media. Circ. Res. 60: 410-421, 1987[Abstract].

17.   Ratz, P. H., P. M. Salomonsky, and F. A. Lattanzio, Jr. Memory of previous receptor activation induces a delay in Ca2+ mobilization and decreases the [Ca2+]i sensitivity of arterial contractions. J. Vasc. Res. 33: 489-498, 1996[Medline].

18.   Rembold, C. M. Desensitization of swine arterial smooth muscle to transplasmalemmal Ca2+ influx. J. Physiol. (Lond.) 416: 273-290, 1989[Abstract].

19.   Rembold, C. M., D. A. Van Riper, and X.-L. Chen. Focal [Ca2+]i increases detected by aequorin but not by fura-2 in histamine- and caffeine-stimulated swine carotid artery. J. Physiol. (Lond.) 488: 549-564, 1995[Abstract].

20.   Shima, H., and M. P. Blaustein. Modulation of evoked contractions in rat arteries by ryanodine, thapsigargin, and cyclopiazonic acid. Circ. Res. 70: 968-977, 1992[Abstract].

21.   Somlyo, A. P., and B. Himpens. Cell calcium and its regulation in smooth muscle. FASEB J. 3: 2266-2276, 1989[Abstract/Free Full Text].

22.   Van Breemen, C., Q. Chen, and I. Laher. Superficial buffer barrier function of smooth muscle sarcoplasmic reticulum. Trends Pharmacol. Sci. 16: 98-105, 1995[Medline].

23.   Wardle, R. L., J. D. Strauss, C. M. Rembold, and R. A. Murphy. Heterologous desensitization of smooth muscle to K+ depolarization: retarded stimulus-[Ca2+]i coupling. Am. J. Physiol. 272 (Cell Physiol. 41): C1810-C1820, 1997[Abstract/Free Full Text].

24.   Wingard, C. J., A. K. Browne, and R. A. Murphy. Dependence of force on length at constant cross-bridge phosphorylation in the swine carotid media. J. Physiol. (Lond.) 488.3: 729-739, 1995[Abstract].

25.   Yoshikawa, A., C. van Breemen, and G. Isenberg. Buffering of plasmalemmal Ca2+ current by sarcoplasmic reticulum of guinea pig urinary bladder myocytes. Am. J. Physiol. 271 (Cell Physiol. 40): C833-C841, 1996[Abstract/Free Full Text].


Am J Physiol Cell Physiol 275(4):C1095-C1103
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society




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