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Alterations of Calcium Uptake in Renovascular Hypertensive Rat Aorta
ISSN 0306-3623/98 $19.00 1 .00 PII S0306-3623(97)00445-X All rights reserved
Gen. Pharmac. Vol. 31, No. 2, pp. 265–270, 1998 Copyright 1998 Elsevier Science Inc. Printed in the USA.
Alterations of Calcium Uptake in Renovascular Hypertensive Rat Aorta: Functional Assessment with Thapsigargin P. I. B. Ceron and L. M. Bendhack* Laboratory of Pharmacology, College of Pharmaceutical Sciences, University of Sa˜o Paulo, Ribeira˜o Preto, SP-14040-903, Brazil [Tel: 55-16-6024165; Fax: 55-16-6331092; E-mail: [email protected]] ABSTRACT. 1. The aim of this study was to test the hypothesis that impaired calcium (Ca21) recycling by sarcoplasmic reticulum (SR) Ca21-ATPase takes place in aortae from 1 kidney–1 clip (1K-1C) hypertensive rats. 2. The contractile response elicited when Ca21 is released from the SR with phenylephrine and caffeine in Ca21-free Krebs solution was greater in 1K-1C than in 1K rat aorta. In the arteries submitted to intracellular Ca21 store depletion and reloading, this response was not different between 1K-1C and 1K rat aortae. Thapsigargin decreased the phasic contractile responses to phenylephrine in 1K and 1K1C rat aortae and increased the tone that developed during the refilling period in 1K-1C rat aortae. 3. Our data support the hypothesis that the 1K-1C rat aorta has defective intracellular Ca21 regulation that may be implicated in an inadequate SR buffering ability. gen pharmac 31;2:265–270, 1998. 1998 Elsevier Science Inc. KEY WORDS. Renal hypertension, thapsigargin, 1 kidney–1 clip, sarcoplasmic reticulum INTRODUCTION 21
Calcium (Ca ) is a basic component of vascular smooth muscle contraction (Bohr, 1963; Defeo and Morgan, 1985; Rembold and Murphy, 1988). Several lines of evidence have suggested that a defect in the regulation of intracellular Ca21 concentration may play a role in the augmented vascular reactivity that is characteristic of experimental models of hypertension (Bohr and Webb, 1984; Kwan, 1985). Mulvany and Nyborg (1980) demonstrated that Ca21 influx induced by noradrenaline is abnormally high in vessels from spontaneously hypertensive rats (SHRs). Furthermore, other authors demonstrated that intracellular Ca21 levels are elevated in the walls of mesenteric arterioles from renovascular hypertensive rats (Tobian and Chesley, 1966), in aortic smooth muscle cells from SHRs (Sugiyama et al., 1990) and in vessels of rats submitted to aorta coarctation (Papageorgiou and Morgan, 1991). The levels of free intracellular Ca21 within the smooth muscle cells are precisely regulated by sequestration and extrusion mechanisms (Morgan, 1987; van Breemen et al., 1986). The sarcoplasmic reticulum (SR) acts as a buffer for intracellular Ca21, and it is the primary source of the intracellular Ca21 taking part in the contractile process (van Breemen, 1977). Several studies on the ability of the SR to sequester Ca21 in vessels from hypertensive animals have shown variable results. Webb and Bhalla (1976) demonstrated that the microsomal fraction of vascular smooth muscle of the SHR has reduced Ca21 sequestering ability compared with normotensive Wistar rats. Kwan et al., (1994) suggested that an impaired SR Ca21-ATPase pump may be an important factor in the pathogenesis of hypertension, whereas Shibata et al. (1975), Aqel et al. (1987) and Hermsmeyer and Erne (1989) reported a significant increase in the capacity of SR for Ca21 storage. The Ca21-ATPase pump is a major protein of the SR membrane that regulates intracellular Ca21 transport from the cytoplasm to the SR lumen (Sumida et al., 1984). * To whom correspondence should be addressed. Received 20 September 1997.
The selective inhibitor thapsigargin has been used to study the SR Ca21-ATPase. Thapsigargin is a tumor-promoting sesquiterpene lactone that binds stoichiometrically to the SR Ca21-ATPase and causes an essentially irreversible inhibition of its activity by blocking the enzyme in the Ca21-free E2 state (Lytton et al., 1991; Thastrup et al 1990; Wictome et al., 1992). Therefore, thapsigargin is a pharmacological tool that can be a source of insight into the ability of the SR to sequester Ca21. The hypothesis tested in this study is that ATP-dependent Ca21 uptake is depressed in the SR in vessels from 1 kidney–1 clip (1K-1C) hypertensive rats. We performed contractile experiments with the use of caffeine, an agonist that activates the ryanodine-sensitive Ca21 channel in the SR, and phenylephrine, which induces Ca21 release through inositoltrisphosphate (IP3)-sensitive Ca21 channels. The effect of thapsigargin on phenylephrine-stimulated Ca21 release was evaluated. METHODS
Rats Male Wistar rats (170–190 g) were used in this study. Renal hypertension was produced by placing a silver clip (0.2 mm ID) on the left renal artery, and the contralateral kidney was removed (1 kidney–1 clip) under ether anesthesia. In the control rats (1K), only the right kidney was removed under ether anesthesia. The rats were maintained on standard rat chow, and 7 days after the surgery, under ether anesthesia, the femoral artery was cannulated for arterial pressure recording. Twenty-four hours later, when the rats had completely recovered from anesthesia, they were connected to the recording system, and the arterial pressure recording was initiated after adaptation of conscious freely moving rats to the environment of a quiet laboratory, with temperature control. After the initial period of adaptation, arterial pressure was recorded for 30 min by using a Narco pressure transducer RP 1500, connected to a four channel Narco polygraph (Narco Biosystems, Inc., Houston, TX). The rats were considered hypertensive when mean arterial pressure was higher than 130 mm Hg.
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Preparation of aortic rings After the arterial pressure recordings, the rats were sacrificed, and the thoracic aortae were isolated. Aorta rings 3 mm in length were cut and mounted between two steel hooks to measure the isometric tension. The arterial rings were placed in bath chambers for isolated organs containing Krebs solution of the following composition (in mM): NaCl 118.0, CaCl2 2.5, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.0, dextrose 11.2, at 378C, and the pH 7.4 was adjusted with 95% O2 and 5% CO2. The system was connected to an F-60 force-displacement transducer, and the contractile responses were recorded on a polygraph (Narco Biosystems, Inc., Houston, TX). After 60-min equilibration under a resting tension of 1 g, the aorta rings were continuously stimulated with 30 mM phenylephrine until reproducible contractile responses were obtained. After the period of equilibration, specific protocols were performed.
Contractile response to phenylephrine and caffeine Phenylephrine (10 mM)- and caffeine (20 mM)-induced contractile responses in Ca21-free Krebs solution were evaluated. Briefly, aortic rings of 1K-1C hypertensive and 1K control rats were induced by 10 mM phenylephrine in Krebs solution containing 2.5 mM Ca21. Then, the rings were washed with Krebs solution until the preparations were totally relaxed. The bathing medium was then replaced with Ca21-free Krebs solution and, after 1 min, contractile responses to phenylephrine (10 mM) or caffeine (20 mM) were stimulated. The results were expressed as the percentage of contractile response induced by 10 mM phenylephrine or 20 mM caffeine in Ca21-free Krebs solution in relation to the initial contraction induced by 10 mM phenylephrine in 2.5 mM Ca21 Krebs solution.
Effects of thapsigargin on phenylephrine-induced contractile responses Aortic rings of 1K-1C hypertensive and 1K control rats were induced by 10 mM phenylephrine to produce a maximal contractile response. When the contraction reached a plateau, vessels were rinsed in Ca21-free 1 mM EGTA Krebs solution for 10 min to deplete intracellular Ca21 stores. Depletion was demonstrated by no contraction in response to phenylephrine or caffeine. After Ca21 depletion, intracellular Ca21 stores were loaded by placing the rings in Krebs solution containing 2.5 mM Ca21 for 20 min (loading period). The bathing medium was then replaced with Ca21-free 50 mM EGTA Krebs solution, and, after 1 min, contractile responses to phenylephrine (10 mM) were induced. The protocol was performed in the presence of thapsigargin (1 mM) or vehicle (dimethylsulfoxide, DMSO). To investigate the role of endothelium in the response induced by thapsigargin, endothelium was removed by gently rubbing the lumen side of the aorta rings. The functional state of the endothelium was assessed by the lack of relaxation in response to acetylcholine (1 mM) after contraction with phenylephrine (100 nM).
Drugs All drugs were kept on ice during the experiments. Phenylephrine, caffeine, and thapsigargin were purchased from Sigma Chemical Co. (St Louis, MO, USA). Thapsigargin was dissolved in DMSO.
Statistical analysis Values are expressed as mean6SEM of contractile response in relation to 10 mM phenylephrine-induced contractile response (100%) in Krebs solution. Student’s t-test was used to compare observations
FIGURE 1. Caffeine-induced contractile response in aorta rings from 1K-1C and 1K rats in Ca21-free Krebs solution. Values are mean6SEM. The results are expressed as a percentage of the contraction induced by 20 mM caffeine in Ca21-free Krebs solution in relation to the contraction induced by 10 mM phenylephrine in Krebs solution. *Statistical difference (P,0.05) between 1K-1C (h, n513) and 1K (j, n59) rat aortae. between 1K-1C hypertensive and 1 K rat aortae and to compare differences related to the treatment. RESULTS Mean arterial pressure values at 7 days after surgery were significantly higher in 1K-1C rats (14965 mm Hg, n58) compared with 1K control rats (10063 mm Hg, n57).
Contractile responses to caffeine and phenylephrine After stimulation with phenylephrine in 2.5 mM Ca21-Krebs solution, 20 mM caffeine in Ca21-free Krebs solution produced contractile responses that were compared with initial phenylephrineinduced contractions. Caffeine-stimulated contractile responses were higher in 1K-1C rat aortae (36.464.9%, n513) than in 1K rat aortae (21.063.3%, n59), as shown in Figure 1. Compared with the contractile responses induced by phenylephrine in 2.5 mM Ca21Krebs solution, caffeine-stimulated contractions were smaller than the contractile responses induced by 10 mM phenylephrine both in 1K-1C rat aortae and in 1K rat aortae. Similarly, the contractile responses induced by 10 mM phenylephrine (Fig. 2) in Ca21-free Krebs solution were greater in 1K-1C hypertensive rat aortae (74.166.8%, n55) than in 1K rat aortae (56.562.1%, n57).
Effects of thapsigargin on phenylephrine-induced contractile responses Figure 3 illustrates the protocol employed in this study. The resulting phasic contractile response induced by phenylephrine in Ca21-free Krebs solution was taken as an indication of Ca21 released from intracellular stores. In this protocol, phasic contractile responses induced by 10 mM phenylephrine in Ca21-free Krebs solution after intracellular Ca21 store depletion and reloading were not different between aortae from 1K-1C rats, as shown in Figure 3B (37.568.5%, n57) and aortae from 1K rats (Fig. 3A) (46.864.6%,
Ca21 Uptake in 1K-1C Rat Aorta
FIGURE 2. Phenylephrine-induced contraction of aorta rings from 1K-1C and 1K rats in Ca21-free Krebs solution. Values are mean6SEM. The results are expressed as a percentage of the contraction induced by phenylephrine in Ca21-free Krebs solution in relation to the contraction induced by 10 mM phenylephrine in Krebs solution. *Statistical difference (P,0.05) between 1K-1C (h, n55) and 1K (j, n57) rat aortae.
FIGURE 3. Aorta rings from 1K1C and 1K rats were stimulated with phenylephrine (10 mM) and allowed to reach a plateau level of contraction. Ca21-free Krebs solution containing thapsigargin (TG, 1 mM) or vehicle (DMSO) and EGTA (1 mM) was then introduced into the muscle bath, and rings were allowed to equilibrate for 10 min. After this depletion period, 2.5 mM Ca21 Krebs solution containing vehicle (A, B) or TG (C, D) was placed in the muscle bath and rings were allowed to equilibrate for an additional 20 min. After the loading period, Ca21-free EGTA (50 mM) Krebs solution was introduced into the bath and allowed to equilibrate for 1 min before the addition of 10 mM phenylephrine. All responses are expressed as mean6SEM of contractile response in relation to the initial response to phenylephrine in 2.5 mM Ca21 Krebs solution, and data were compared by Student’s t-test with P,0.05. Tracings are representative of at least six experiments.
267 n56). This result contradicts the data shown in Figure 2, where the contractile response induced by phenylephrine in Ca21-free Krebs solution before the protocol that produces depletion and reloading of intracellular Ca21 stores was greater in aortae from 1K-1C hypertensive rats than in aortae from 1K control rats. The effect of thapsigargin, or inhibition of SR Ca21-ATPase, on the refilling of intracellular Ca21 stores also was evaluated. Thapsigargin (1 mM) significantly and equally reduced phenylephrineinduced phasic contraction in aortae from 1K-1C rats (Fig. 3D) and in aortae from 1K rats (Fig. 3C). The effects of thapsigargin on phenylephrine-induced phasic contraction were not different between 1K-1C hypertensive (17.264.6%, n57) and 1K control rat aortae (22.865.9%, n56). Aortae from both control and hypertensive rats also increased the tone that developed during the Ca21 loading period (Fig. 3A, 44.3621.7%, n58; Fig. 3B, 54.8617.7%, n59). Thapsigargin (1 mM) caused a significant increase in the tone that developed during the Ca21 loading period in arteries from 1K-1C rats (Fig. 3D, 106.0616.3%, n59) but left unchanged the state of contraction during the loading period in control arteries (Fig. 3C, 53.6628.8%, n58). Removal of vascular endothelium did not alter contractile responses in this protocol (data not shown).
DISCUSSION Calcium plays a vital role in the control of contractile function of all types of muscles including vascular smooth muscles, and a dysfunction of its regulation under pathological conditions, such as hypertension, could conceivably lead to an altered contractile state of
268 smooth muscle (Daniel and Kwan, 1981). Evidence suggests that intracellular Ca21 levels are elevated in aortic smooth muscle cells from hypertensive animals (Papageorgiou and Morgan, 1991; Sugiyama et al., 1990; Tobian and Chesley, 1966). The aorta is a conduit artery that does not contribute to vascular resistance. However, in hypertension, this artery undergoes several functional changes that also take place in resistance arteries. It has been proposed that alterations in the storage or Ca21 release from intracellular sites may be an important cellular change that contributes to increased vascular reactivity in hypertension (Sharma and Bhalla, 1988). A decrease in the ability of the SR to sequester Ca21 could contribute to augmented intracellular Ca21 levels and vascular hyperreactivity. The present study was performed to determine wheter there is a decrease in the SR buffering capacity in aortae from 1K-1C hypertensive rats. Thapsigargin, a specific and irreversible inhibitor of the SR Ca21ATPase, was used. Thapsigargin is a tumor-promoting sesquiterpene lactone from the plant Thapsia garganica, which binds stoichiometrically to the SR Ca21-ATPase and causes an essentially irreversible inhibition of its activity by blocking the enzymes in the Ca21-free E2 state (Lytton et al., 1991; Thastrup et al., 1990; Wictome et al., 1992). One of the specific protocols used in these experiments (shown in Fig. 3) has been used in other studies to analyze the intracellular Ca21 stores in several models of hypertensive rats (Kanagy et al., 1994; Karaki et al., 1979; Perry and Webb, 1991; Tostes et al., 1995; Turla and Webb, 1990; Watts and Webb, 1994). Caffeine- and phenylephrine-induced contractions in Ca21-free Krebs solution were used as functional measures of intracellular Ca21 released by ryanodine and IP3-sensitive Ca21 channels in the SR (Ehrlich et al., 1994). Caffeine releases Ca21 from the SR independently of second messengers (Hisayama and Takayanagi, 1988; Leijten and van Breemen, 1984) and phenylephrine releases Ca21 from IP3-sensitive intracellular stores (Berridge, 1984). Our results demonstrate that phasic contractile responses to phenylephrine and caffeine in Ca21free Krebs solution are enhanced in aortae from 1K-1C rats (Figs. 1 and 2), suggesting an increase in intracellular Ca21 stores in aortae from 1K-1C hypertensive rats. Sugiyama et al. (1990) also observed a greater increase in [Ca21] in response to stimulation with caffeine and angiotensin II in SHRs than in Wistar-Kyoto (WKY) vascular smooth muscle cells. Turla and Webb (1990) demonstrated that concentration–effect curves for serotonin were shifted to the left in arteries from SHR-stroke prone (SHRSP) compared with WKY. Phasic contractile responses to serotonin in Ca21-free conditions in aortic strips from SHRSP were significantly higher than phasic responses in WKY. The authors suggest that (1) the intracellular Ca21 store that could be released by serotonin may be larger than that in WKY or (2) the action of second messengers such as IP3 is greater in SHRSP than in WKY or (3) that receptor activation by serotonin may cause greater second-messenger release in arteries from SHRSP than in arteries from WKY or all three. Perry and Webb (1991) demonstrated in mesenteric arteries from DOCA hypertensive rats, that phasic contractile responses to noradrenaline, serotonin and angiotensin II, in Ca21-free buffer, were higher compared with arteries from normotensive rats. The authors demonstrated that phasic contractions in response to caffeine in hypertensive rat arteries were not different from those of normotensive rat arteries, suggesting that an alteration in phosphoinositide signaling, instead of an increased intracellular Ca21 store, may be altered in hypertensive rat arteries. On the other hand, Kanagy et al. (1994) demonstrated that phasic contractile responses to caffeine (20 mM) in Ca21-free buffer were higher in aortic strips from SHRSP than in strips from WKY rats, suggesting that the Ca21 stored in the SR is
P. I. B. Ceron and L. M. Bendhack larger in arteries from SHRSP than in arteries from WKY. Similarly, Tostes et al.(1995) showed that phasic contractile responses to serotonin and caffeine in Ca21-free buffer are enhanced in aortae from DOCA rats, suggesting an increase in the intracellular Ca21 store in vessels from DOCA hypertensive rats. The differences between these results may be related to the vessels studied or to the hypertension models used. When we performed the protocol that produces depletion and reloading of the intracellular Ca21 stores, we observed that phasic contractile responses to phenylephrine in Ca21-free Krebs solution were not different in 1K-1C rat aortae and 1K rat aortae (Fig. 3). This result differs from the data shown in Figure 2, where the aortae from 1K-1C hypertensive rats showed greater contractile responses induced by phenylephrine than did the aortae from 1K control rats. However, in this protocol, the aortae were submitted to depletion and reloading of intracellular Ca21 stores. The Ca21 reloading depends on Ca21-ATPase activity; and, if SR Ca21-ATPase activity is reduced, there may be a reduction in reloading in aortae from 1K1C rats. Thus, we suggest that Ca21-ATPase activity was reduced in aortae from these rats. SR Ca21-ATPase inhibition with thapsigargin decreased the phasic contractile response to 10 mM phenylephrine in 1K and 1K-1C aortae (Fig. 3), consistent with an inhibitory action of thapsigargin on the refilling of intracellular Ca21 stores. No differences were observed on the effect of thapsigargin in aortae from 1K-1C rats and from 1K rats. These results indicate that there are no differences in the inhibitory properties of thapsigargin. Kanagy et al. (1994) described similar findings in aortic strips from genetically hypertensive rats (SHRSP). These authors observed that treatment with thapsigargin similarly inhibited caffeine-induced contractile responses in SHRSP and WKY. During the refilling period, an increased basal tone was observed in aortae from both 1K-1C and 1K rats. Thapsigargin increased the tone that developed during the refilling period in aortae from 1K1C rats, but the state of contraction was unchanged during the loading period in 1K rat arteries. These results may indicate a decreased buffering capacity of SR in 1K-1C rat aortae. Another explanation is that Ca21 influx is increased in 1K-1C rat aortae. Similar results were described by Kanagy et al. (1994), with thapsigargin causing a greater force development in aortic strips from SHRSP than in aortic strips from WKY rats. The authors suggest that the effect of thapsigargin is caused by enhanced Ca21 influx across the sarcolemma. Studies with cyclopiazonic acid (CPA), another inhibitor of SR Ca21-ATPase, also support this hypothesis. CPA caused greater contractile responses in aortic segments from SHRs than in those from WKY rats, and these contractions were absent when the arteries were incubated in Ca21-free solution (Low et al., 1993). Enhanced CPA-induced contractions have also been observed in aortae from DOCA hypertensive rats during the refilling period, which strongly supports the idea of defective Ca21 regulation and may suggest a decreased capacity of the SR in buffering intracellular Ca21. However, CPA increased the tone that developed during the refilling period, and aortae from DOCA rats were more sensitive to CPA in this protocol. These data suggest that the SR of the DOCA rat aorta can properly buffer the intracellular Ca21 (Tostes et al., 1995). The authors suggest an increased extracellular Ca21 influx, and not a decreased buffering ability of the SR, contributing to the enhanced vascular reactivity observed in DOCA hypertension. In summary, the present study demonstrates that: 1. The contractile response elicited when Ca21 is released from the SR with phenylephrine and caffeine in Ca21-free Krebs solution
Ca21 Uptake in 1K-1C Rat Aorta is greater in 1K-1C hypertensive rat arteries than in arteries from 1K control rats, suggesting enlarged intracellular Ca21 stores in 1K-1C hypertension. 2. With the protocol that produces depletion and reloading of intracellular Ca21 stores, the contractile responses to phenylephrine in Ca21 -free Krebs solution were not different in aortae from 1K1C and aortae from 1K rats. This Ca21 reloading depends on Ca21-ATPase activity. If Ca21 reloading is reduced in aortae from 1K-1C rats, we could suggest that the Ca21-ATPase activity is reduced in aortae from 1K-1C rats. 3. Thapsigargin decreased phasic contractile responses to phenylephrine in 1K and 1K-1C aortae, consistent with an inhibitory action of thapsigargin on the refilling of intracellular Ca21 stores. 4. Thapsigargin increased the tone that developed during the refilling period in aortae from 1K-1C rats, suggesting a decreased buffering capacity of SR or increased Ca21 influx in 1K-1C aortae. The data presented here support the hypothesis that vascular smooth muscle from 1K-1C hypertensive rats has defective intracellular Ca21 regulation that may contribute to an inadequate SR buffering ability. SUMMARY The aim of this study was to test the hypothesis that impaired Ca21 recycling by SR Ca21-ATPase occurs in the aortae from 1K-1C hypertensive rats. The contractile force in aortae from 1K-1C and 1K control rats was measured. Force development in response to caffeine (20 mM) and phenylephrine (10 mM) in Ca21-free Krebs solution was compared in aortae from 1K-1C and 1K rats. In addition, the aortae were submitted to depletion and reloading of the intracellular stores, and the contractile responses to phenylephrine in Ca21-free Krebs solution were compared in 1K-1C and 1K rat aortae. The effects of thapsigargin were used to evaluate Ca21 buffering. The contractile response elicited when Ca21 is released from the SR with phenylephrine and caffeine in Ca21-free Krebs solution was greater in 1K-1C arteries than in 1K rat aortae. In the arteries submitted to intracellular Ca21 store depletion and reloading, the contractile responses induced by phenylephrine in Ca21-free EGTA Krebs solution were not different in 1K-1C and 1K rat aortae. Because this reloading depends on Ca21-ATPase activity and Ca21 reloading was reduced in 1K-1C rat aortae, we suggest that Ca21ATPase activity is reduced in 1K-1C rat aortae. Thapsigargin decreased the phasic contractile responses to phenylephrine in 1K and 1K-1C rat aortae. Thapsigargin increased the tone that developed during the refilling period in 1K-1C rat aortae, which suggests decreased buffering capacity of the SR or increased Ca21 influx in 1K1C aortae. Our data support the hypothesis that 1K-1C rat aortae have defective intracellular Ca21 regulation that may contribute to an inadequate SR buffering ability. The authors wish to thank Mr. Carlos A. A. da Silva and Mrs. Miriam C. C. de Melo for technical assistance. This study was supported by FAPESP 95/4685-8 and PRONEX 357/96.
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Gen. Pharmac. Vol. 31, No. 2, pp. 265–270, 1998 Copyright 1998 Elsevier Science Inc. Printed in the USA.
Alterations of Calcium Uptake in Renovascular Hypertensive Rat Aorta: Functional Assessment with Thapsigargin P. I. B. Ceron and L. M. Bendhack* Laboratory of Pharmacology, College of Pharmaceutical Sciences, University of Sa˜o Paulo, Ribeira˜o Preto, SP-14040-903, Brazil [Tel: 55-16-6024165; Fax: 55-16-6331092; E-mail: [email protected]] ABSTRACT. 1. The aim of this study was to test the hypothesis that impaired calcium (Ca21) recycling by sarcoplasmic reticulum (SR) Ca21-ATPase takes place in aortae from 1 kidney–1 clip (1K-1C) hypertensive rats. 2. The contractile response elicited when Ca21 is released from the SR with phenylephrine and caffeine in Ca21-free Krebs solution was greater in 1K-1C than in 1K rat aorta. In the arteries submitted to intracellular Ca21 store depletion and reloading, this response was not different between 1K-1C and 1K rat aortae. Thapsigargin decreased the phasic contractile responses to phenylephrine in 1K and 1K1C rat aortae and increased the tone that developed during the refilling period in 1K-1C rat aortae. 3. Our data support the hypothesis that the 1K-1C rat aorta has defective intracellular Ca21 regulation that may be implicated in an inadequate SR buffering ability. gen pharmac 31;2:265–270, 1998. 1998 Elsevier Science Inc. KEY WORDS. Renal hypertension, thapsigargin, 1 kidney–1 clip, sarcoplasmic reticulum INTRODUCTION 21
Calcium (Ca ) is a basic component of vascular smooth muscle contraction (Bohr, 1963; Defeo and Morgan, 1985; Rembold and Murphy, 1988). Several lines of evidence have suggested that a defect in the regulation of intracellular Ca21 concentration may play a role in the augmented vascular reactivity that is characteristic of experimental models of hypertension (Bohr and Webb, 1984; Kwan, 1985). Mulvany and Nyborg (1980) demonstrated that Ca21 influx induced by noradrenaline is abnormally high in vessels from spontaneously hypertensive rats (SHRs). Furthermore, other authors demonstrated that intracellular Ca21 levels are elevated in the walls of mesenteric arterioles from renovascular hypertensive rats (Tobian and Chesley, 1966), in aortic smooth muscle cells from SHRs (Sugiyama et al., 1990) and in vessels of rats submitted to aorta coarctation (Papageorgiou and Morgan, 1991). The levels of free intracellular Ca21 within the smooth muscle cells are precisely regulated by sequestration and extrusion mechanisms (Morgan, 1987; van Breemen et al., 1986). The sarcoplasmic reticulum (SR) acts as a buffer for intracellular Ca21, and it is the primary source of the intracellular Ca21 taking part in the contractile process (van Breemen, 1977). Several studies on the ability of the SR to sequester Ca21 in vessels from hypertensive animals have shown variable results. Webb and Bhalla (1976) demonstrated that the microsomal fraction of vascular smooth muscle of the SHR has reduced Ca21 sequestering ability compared with normotensive Wistar rats. Kwan et al., (1994) suggested that an impaired SR Ca21-ATPase pump may be an important factor in the pathogenesis of hypertension, whereas Shibata et al. (1975), Aqel et al. (1987) and Hermsmeyer and Erne (1989) reported a significant increase in the capacity of SR for Ca21 storage. The Ca21-ATPase pump is a major protein of the SR membrane that regulates intracellular Ca21 transport from the cytoplasm to the SR lumen (Sumida et al., 1984). * To whom correspondence should be addressed. Received 20 September 1997.
The selective inhibitor thapsigargin has been used to study the SR Ca21-ATPase. Thapsigargin is a tumor-promoting sesquiterpene lactone that binds stoichiometrically to the SR Ca21-ATPase and causes an essentially irreversible inhibition of its activity by blocking the enzyme in the Ca21-free E2 state (Lytton et al., 1991; Thastrup et al 1990; Wictome et al., 1992). Therefore, thapsigargin is a pharmacological tool that can be a source of insight into the ability of the SR to sequester Ca21. The hypothesis tested in this study is that ATP-dependent Ca21 uptake is depressed in the SR in vessels from 1 kidney–1 clip (1K-1C) hypertensive rats. We performed contractile experiments with the use of caffeine, an agonist that activates the ryanodine-sensitive Ca21 channel in the SR, and phenylephrine, which induces Ca21 release through inositoltrisphosphate (IP3)-sensitive Ca21 channels. The effect of thapsigargin on phenylephrine-stimulated Ca21 release was evaluated. METHODS
Rats Male Wistar rats (170–190 g) were used in this study. Renal hypertension was produced by placing a silver clip (0.2 mm ID) on the left renal artery, and the contralateral kidney was removed (1 kidney–1 clip) under ether anesthesia. In the control rats (1K), only the right kidney was removed under ether anesthesia. The rats were maintained on standard rat chow, and 7 days after the surgery, under ether anesthesia, the femoral artery was cannulated for arterial pressure recording. Twenty-four hours later, when the rats had completely recovered from anesthesia, they were connected to the recording system, and the arterial pressure recording was initiated after adaptation of conscious freely moving rats to the environment of a quiet laboratory, with temperature control. After the initial period of adaptation, arterial pressure was recorded for 30 min by using a Narco pressure transducer RP 1500, connected to a four channel Narco polygraph (Narco Biosystems, Inc., Houston, TX). The rats were considered hypertensive when mean arterial pressure was higher than 130 mm Hg.
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Preparation of aortic rings After the arterial pressure recordings, the rats were sacrificed, and the thoracic aortae were isolated. Aorta rings 3 mm in length were cut and mounted between two steel hooks to measure the isometric tension. The arterial rings were placed in bath chambers for isolated organs containing Krebs solution of the following composition (in mM): NaCl 118.0, CaCl2 2.5, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.0, dextrose 11.2, at 378C, and the pH 7.4 was adjusted with 95% O2 and 5% CO2. The system was connected to an F-60 force-displacement transducer, and the contractile responses were recorded on a polygraph (Narco Biosystems, Inc., Houston, TX). After 60-min equilibration under a resting tension of 1 g, the aorta rings were continuously stimulated with 30 mM phenylephrine until reproducible contractile responses were obtained. After the period of equilibration, specific protocols were performed.
Contractile response to phenylephrine and caffeine Phenylephrine (10 mM)- and caffeine (20 mM)-induced contractile responses in Ca21-free Krebs solution were evaluated. Briefly, aortic rings of 1K-1C hypertensive and 1K control rats were induced by 10 mM phenylephrine in Krebs solution containing 2.5 mM Ca21. Then, the rings were washed with Krebs solution until the preparations were totally relaxed. The bathing medium was then replaced with Ca21-free Krebs solution and, after 1 min, contractile responses to phenylephrine (10 mM) or caffeine (20 mM) were stimulated. The results were expressed as the percentage of contractile response induced by 10 mM phenylephrine or 20 mM caffeine in Ca21-free Krebs solution in relation to the initial contraction induced by 10 mM phenylephrine in 2.5 mM Ca21 Krebs solution.
Effects of thapsigargin on phenylephrine-induced contractile responses Aortic rings of 1K-1C hypertensive and 1K control rats were induced by 10 mM phenylephrine to produce a maximal contractile response. When the contraction reached a plateau, vessels were rinsed in Ca21-free 1 mM EGTA Krebs solution for 10 min to deplete intracellular Ca21 stores. Depletion was demonstrated by no contraction in response to phenylephrine or caffeine. After Ca21 depletion, intracellular Ca21 stores were loaded by placing the rings in Krebs solution containing 2.5 mM Ca21 for 20 min (loading period). The bathing medium was then replaced with Ca21-free 50 mM EGTA Krebs solution, and, after 1 min, contractile responses to phenylephrine (10 mM) were induced. The protocol was performed in the presence of thapsigargin (1 mM) or vehicle (dimethylsulfoxide, DMSO). To investigate the role of endothelium in the response induced by thapsigargin, endothelium was removed by gently rubbing the lumen side of the aorta rings. The functional state of the endothelium was assessed by the lack of relaxation in response to acetylcholine (1 mM) after contraction with phenylephrine (100 nM).
Drugs All drugs were kept on ice during the experiments. Phenylephrine, caffeine, and thapsigargin were purchased from Sigma Chemical Co. (St Louis, MO, USA). Thapsigargin was dissolved in DMSO.
Statistical analysis Values are expressed as mean6SEM of contractile response in relation to 10 mM phenylephrine-induced contractile response (100%) in Krebs solution. Student’s t-test was used to compare observations
FIGURE 1. Caffeine-induced contractile response in aorta rings from 1K-1C and 1K rats in Ca21-free Krebs solution. Values are mean6SEM. The results are expressed as a percentage of the contraction induced by 20 mM caffeine in Ca21-free Krebs solution in relation to the contraction induced by 10 mM phenylephrine in Krebs solution. *Statistical difference (P,0.05) between 1K-1C (h, n513) and 1K (j, n59) rat aortae. between 1K-1C hypertensive and 1 K rat aortae and to compare differences related to the treatment. RESULTS Mean arterial pressure values at 7 days after surgery were significantly higher in 1K-1C rats (14965 mm Hg, n58) compared with 1K control rats (10063 mm Hg, n57).
Contractile responses to caffeine and phenylephrine After stimulation with phenylephrine in 2.5 mM Ca21-Krebs solution, 20 mM caffeine in Ca21-free Krebs solution produced contractile responses that were compared with initial phenylephrineinduced contractions. Caffeine-stimulated contractile responses were higher in 1K-1C rat aortae (36.464.9%, n513) than in 1K rat aortae (21.063.3%, n59), as shown in Figure 1. Compared with the contractile responses induced by phenylephrine in 2.5 mM Ca21Krebs solution, caffeine-stimulated contractions were smaller than the contractile responses induced by 10 mM phenylephrine both in 1K-1C rat aortae and in 1K rat aortae. Similarly, the contractile responses induced by 10 mM phenylephrine (Fig. 2) in Ca21-free Krebs solution were greater in 1K-1C hypertensive rat aortae (74.166.8%, n55) than in 1K rat aortae (56.562.1%, n57).
Effects of thapsigargin on phenylephrine-induced contractile responses Figure 3 illustrates the protocol employed in this study. The resulting phasic contractile response induced by phenylephrine in Ca21-free Krebs solution was taken as an indication of Ca21 released from intracellular stores. In this protocol, phasic contractile responses induced by 10 mM phenylephrine in Ca21-free Krebs solution after intracellular Ca21 store depletion and reloading were not different between aortae from 1K-1C rats, as shown in Figure 3B (37.568.5%, n57) and aortae from 1K rats (Fig. 3A) (46.864.6%,
Ca21 Uptake in 1K-1C Rat Aorta
FIGURE 2. Phenylephrine-induced contraction of aorta rings from 1K-1C and 1K rats in Ca21-free Krebs solution. Values are mean6SEM. The results are expressed as a percentage of the contraction induced by phenylephrine in Ca21-free Krebs solution in relation to the contraction induced by 10 mM phenylephrine in Krebs solution. *Statistical difference (P,0.05) between 1K-1C (h, n55) and 1K (j, n57) rat aortae.
FIGURE 3. Aorta rings from 1K1C and 1K rats were stimulated with phenylephrine (10 mM) and allowed to reach a plateau level of contraction. Ca21-free Krebs solution containing thapsigargin (TG, 1 mM) or vehicle (DMSO) and EGTA (1 mM) was then introduced into the muscle bath, and rings were allowed to equilibrate for 10 min. After this depletion period, 2.5 mM Ca21 Krebs solution containing vehicle (A, B) or TG (C, D) was placed in the muscle bath and rings were allowed to equilibrate for an additional 20 min. After the loading period, Ca21-free EGTA (50 mM) Krebs solution was introduced into the bath and allowed to equilibrate for 1 min before the addition of 10 mM phenylephrine. All responses are expressed as mean6SEM of contractile response in relation to the initial response to phenylephrine in 2.5 mM Ca21 Krebs solution, and data were compared by Student’s t-test with P,0.05. Tracings are representative of at least six experiments.
267 n56). This result contradicts the data shown in Figure 2, where the contractile response induced by phenylephrine in Ca21-free Krebs solution before the protocol that produces depletion and reloading of intracellular Ca21 stores was greater in aortae from 1K-1C hypertensive rats than in aortae from 1K control rats. The effect of thapsigargin, or inhibition of SR Ca21-ATPase, on the refilling of intracellular Ca21 stores also was evaluated. Thapsigargin (1 mM) significantly and equally reduced phenylephrineinduced phasic contraction in aortae from 1K-1C rats (Fig. 3D) and in aortae from 1K rats (Fig. 3C). The effects of thapsigargin on phenylephrine-induced phasic contraction were not different between 1K-1C hypertensive (17.264.6%, n57) and 1K control rat aortae (22.865.9%, n56). Aortae from both control and hypertensive rats also increased the tone that developed during the Ca21 loading period (Fig. 3A, 44.3621.7%, n58; Fig. 3B, 54.8617.7%, n59). Thapsigargin (1 mM) caused a significant increase in the tone that developed during the Ca21 loading period in arteries from 1K-1C rats (Fig. 3D, 106.0616.3%, n59) but left unchanged the state of contraction during the loading period in control arteries (Fig. 3C, 53.6628.8%, n58). Removal of vascular endothelium did not alter contractile responses in this protocol (data not shown).
DISCUSSION Calcium plays a vital role in the control of contractile function of all types of muscles including vascular smooth muscles, and a dysfunction of its regulation under pathological conditions, such as hypertension, could conceivably lead to an altered contractile state of
268 smooth muscle (Daniel and Kwan, 1981). Evidence suggests that intracellular Ca21 levels are elevated in aortic smooth muscle cells from hypertensive animals (Papageorgiou and Morgan, 1991; Sugiyama et al., 1990; Tobian and Chesley, 1966). The aorta is a conduit artery that does not contribute to vascular resistance. However, in hypertension, this artery undergoes several functional changes that also take place in resistance arteries. It has been proposed that alterations in the storage or Ca21 release from intracellular sites may be an important cellular change that contributes to increased vascular reactivity in hypertension (Sharma and Bhalla, 1988). A decrease in the ability of the SR to sequester Ca21 could contribute to augmented intracellular Ca21 levels and vascular hyperreactivity. The present study was performed to determine wheter there is a decrease in the SR buffering capacity in aortae from 1K-1C hypertensive rats. Thapsigargin, a specific and irreversible inhibitor of the SR Ca21ATPase, was used. Thapsigargin is a tumor-promoting sesquiterpene lactone from the plant Thapsia garganica, which binds stoichiometrically to the SR Ca21-ATPase and causes an essentially irreversible inhibition of its activity by blocking the enzymes in the Ca21-free E2 state (Lytton et al., 1991; Thastrup et al., 1990; Wictome et al., 1992). One of the specific protocols used in these experiments (shown in Fig. 3) has been used in other studies to analyze the intracellular Ca21 stores in several models of hypertensive rats (Kanagy et al., 1994; Karaki et al., 1979; Perry and Webb, 1991; Tostes et al., 1995; Turla and Webb, 1990; Watts and Webb, 1994). Caffeine- and phenylephrine-induced contractions in Ca21-free Krebs solution were used as functional measures of intracellular Ca21 released by ryanodine and IP3-sensitive Ca21 channels in the SR (Ehrlich et al., 1994). Caffeine releases Ca21 from the SR independently of second messengers (Hisayama and Takayanagi, 1988; Leijten and van Breemen, 1984) and phenylephrine releases Ca21 from IP3-sensitive intracellular stores (Berridge, 1984). Our results demonstrate that phasic contractile responses to phenylephrine and caffeine in Ca21free Krebs solution are enhanced in aortae from 1K-1C rats (Figs. 1 and 2), suggesting an increase in intracellular Ca21 stores in aortae from 1K-1C hypertensive rats. Sugiyama et al. (1990) also observed a greater increase in [Ca21] in response to stimulation with caffeine and angiotensin II in SHRs than in Wistar-Kyoto (WKY) vascular smooth muscle cells. Turla and Webb (1990) demonstrated that concentration–effect curves for serotonin were shifted to the left in arteries from SHR-stroke prone (SHRSP) compared with WKY. Phasic contractile responses to serotonin in Ca21-free conditions in aortic strips from SHRSP were significantly higher than phasic responses in WKY. The authors suggest that (1) the intracellular Ca21 store that could be released by serotonin may be larger than that in WKY or (2) the action of second messengers such as IP3 is greater in SHRSP than in WKY or (3) that receptor activation by serotonin may cause greater second-messenger release in arteries from SHRSP than in arteries from WKY or all three. Perry and Webb (1991) demonstrated in mesenteric arteries from DOCA hypertensive rats, that phasic contractile responses to noradrenaline, serotonin and angiotensin II, in Ca21-free buffer, were higher compared with arteries from normotensive rats. The authors demonstrated that phasic contractions in response to caffeine in hypertensive rat arteries were not different from those of normotensive rat arteries, suggesting that an alteration in phosphoinositide signaling, instead of an increased intracellular Ca21 store, may be altered in hypertensive rat arteries. On the other hand, Kanagy et al. (1994) demonstrated that phasic contractile responses to caffeine (20 mM) in Ca21-free buffer were higher in aortic strips from SHRSP than in strips from WKY rats, suggesting that the Ca21 stored in the SR is
P. I. B. Ceron and L. M. Bendhack larger in arteries from SHRSP than in arteries from WKY. Similarly, Tostes et al.(1995) showed that phasic contractile responses to serotonin and caffeine in Ca21-free buffer are enhanced in aortae from DOCA rats, suggesting an increase in the intracellular Ca21 store in vessels from DOCA hypertensive rats. The differences between these results may be related to the vessels studied or to the hypertension models used. When we performed the protocol that produces depletion and reloading of the intracellular Ca21 stores, we observed that phasic contractile responses to phenylephrine in Ca21-free Krebs solution were not different in 1K-1C rat aortae and 1K rat aortae (Fig. 3). This result differs from the data shown in Figure 2, where the aortae from 1K-1C hypertensive rats showed greater contractile responses induced by phenylephrine than did the aortae from 1K control rats. However, in this protocol, the aortae were submitted to depletion and reloading of intracellular Ca21 stores. The Ca21 reloading depends on Ca21-ATPase activity; and, if SR Ca21-ATPase activity is reduced, there may be a reduction in reloading in aortae from 1K1C rats. Thus, we suggest that Ca21-ATPase activity was reduced in aortae from these rats. SR Ca21-ATPase inhibition with thapsigargin decreased the phasic contractile response to 10 mM phenylephrine in 1K and 1K-1C aortae (Fig. 3), consistent with an inhibitory action of thapsigargin on the refilling of intracellular Ca21 stores. No differences were observed on the effect of thapsigargin in aortae from 1K-1C rats and from 1K rats. These results indicate that there are no differences in the inhibitory properties of thapsigargin. Kanagy et al. (1994) described similar findings in aortic strips from genetically hypertensive rats (SHRSP). These authors observed that treatment with thapsigargin similarly inhibited caffeine-induced contractile responses in SHRSP and WKY. During the refilling period, an increased basal tone was observed in aortae from both 1K-1C and 1K rats. Thapsigargin increased the tone that developed during the refilling period in aortae from 1K1C rats, but the state of contraction was unchanged during the loading period in 1K rat arteries. These results may indicate a decreased buffering capacity of SR in 1K-1C rat aortae. Another explanation is that Ca21 influx is increased in 1K-1C rat aortae. Similar results were described by Kanagy et al. (1994), with thapsigargin causing a greater force development in aortic strips from SHRSP than in aortic strips from WKY rats. The authors suggest that the effect of thapsigargin is caused by enhanced Ca21 influx across the sarcolemma. Studies with cyclopiazonic acid (CPA), another inhibitor of SR Ca21-ATPase, also support this hypothesis. CPA caused greater contractile responses in aortic segments from SHRs than in those from WKY rats, and these contractions were absent when the arteries were incubated in Ca21-free solution (Low et al., 1993). Enhanced CPA-induced contractions have also been observed in aortae from DOCA hypertensive rats during the refilling period, which strongly supports the idea of defective Ca21 regulation and may suggest a decreased capacity of the SR in buffering intracellular Ca21. However, CPA increased the tone that developed during the refilling period, and aortae from DOCA rats were more sensitive to CPA in this protocol. These data suggest that the SR of the DOCA rat aorta can properly buffer the intracellular Ca21 (Tostes et al., 1995). The authors suggest an increased extracellular Ca21 influx, and not a decreased buffering ability of the SR, contributing to the enhanced vascular reactivity observed in DOCA hypertension. In summary, the present study demonstrates that: 1. The contractile response elicited when Ca21 is released from the SR with phenylephrine and caffeine in Ca21-free Krebs solution
Ca21 Uptake in 1K-1C Rat Aorta is greater in 1K-1C hypertensive rat arteries than in arteries from 1K control rats, suggesting enlarged intracellular Ca21 stores in 1K-1C hypertension. 2. With the protocol that produces depletion and reloading of intracellular Ca21 stores, the contractile responses to phenylephrine in Ca21 -free Krebs solution were not different in aortae from 1K1C and aortae from 1K rats. This Ca21 reloading depends on Ca21-ATPase activity. If Ca21 reloading is reduced in aortae from 1K-1C rats, we could suggest that the Ca21-ATPase activity is reduced in aortae from 1K-1C rats. 3. Thapsigargin decreased phasic contractile responses to phenylephrine in 1K and 1K-1C aortae, consistent with an inhibitory action of thapsigargin on the refilling of intracellular Ca21 stores. 4. Thapsigargin increased the tone that developed during the refilling period in aortae from 1K-1C rats, suggesting a decreased buffering capacity of SR or increased Ca21 influx in 1K-1C aortae. The data presented here support the hypothesis that vascular smooth muscle from 1K-1C hypertensive rats has defective intracellular Ca21 regulation that may contribute to an inadequate SR buffering ability. SUMMARY The aim of this study was to test the hypothesis that impaired Ca21 recycling by SR Ca21-ATPase occurs in the aortae from 1K-1C hypertensive rats. The contractile force in aortae from 1K-1C and 1K control rats was measured. Force development in response to caffeine (20 mM) and phenylephrine (10 mM) in Ca21-free Krebs solution was compared in aortae from 1K-1C and 1K rats. In addition, the aortae were submitted to depletion and reloading of the intracellular stores, and the contractile responses to phenylephrine in Ca21-free Krebs solution were compared in 1K-1C and 1K rat aortae. The effects of thapsigargin were used to evaluate Ca21 buffering. The contractile response elicited when Ca21 is released from the SR with phenylephrine and caffeine in Ca21-free Krebs solution was greater in 1K-1C arteries than in 1K rat aortae. In the arteries submitted to intracellular Ca21 store depletion and reloading, the contractile responses induced by phenylephrine in Ca21-free EGTA Krebs solution were not different in 1K-1C and 1K rat aortae. Because this reloading depends on Ca21-ATPase activity and Ca21 reloading was reduced in 1K-1C rat aortae, we suggest that Ca21ATPase activity is reduced in 1K-1C rat aortae. Thapsigargin decreased the phasic contractile responses to phenylephrine in 1K and 1K-1C rat aortae. Thapsigargin increased the tone that developed during the refilling period in 1K-1C rat aortae, which suggests decreased buffering capacity of the SR or increased Ca21 influx in 1K1C aortae. Our data support the hypothesis that 1K-1C rat aortae have defective intracellular Ca21 regulation that may contribute to an inadequate SR buffering ability. The authors wish to thank Mr. Carlos A. A. da Silva and Mrs. Miriam C. C. de Melo for technical assistance. This study was supported by FAPESP 95/4685-8 and PRONEX 357/96.
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