Báo cáo khoa học: Calcium and polyamine regulated calcium-sensing receptors in cardiac tissues

doi:10.1046/j.1432-1033.2003.03645.x

Eur. J. Biochem. 270, 2680–2688 (2003) (cid:1) FEBS 2003

Calcium and polyamine regulated calcium-sensing receptors in cardiac tissues

Rui Wang1, Changqing Xu2, Weimin Zhao1, Jing Zhang1, Kun Cao1, Baofeng Yang2 and Lingyun Wu3 1Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada; 2Department of Pathophysiology, Harbin Medical University, Harbin, P.R. China; 3Department of Pharmacology, University of Saskatchewan, Saskatoon, SK, Canada

isolated ventricular myocytes from adult rats. Spermine (1–10 mM) also increased [Ca2+]i. Pre-treatment of cardiac myocytes with thapsigargin or U73122 abolished the extra- cellular calcium, gadolinium or spermine-induced increase in [Ca2+]i. The blockade of Na+/Ca2+ exchanger or voltage- dependent calcium channels did not alter the extracellular calcium-induced increase in [Ca2+]i. Finally, extracellular calcium, gadolinium and spermine all increased intracellular inositol 1,4,5-triphosphate (IP3) levels. Our results demon- strated that Ca-SR was expressed in cardiac tissue and car- diomyocytes and its function was regulated by extracellular calcium and spermine.

Keywords: calcium-sensing receptor; heart; IP3; RT-PCR; spermine.

Activation of a calcium-sensing receptor (Ca-SR) leads to increased intracellular calcium concentration and altered cellular activities. The expression of Ca-SR has been iden- tified in both nonexcitable and excitable cells, including neurons and smooth muscle cells. Whether Ca-SR was expressed and functioning in cardiac myocytes remained unclear. In the present study, the transcripts of Ca-SR were identified in rat heart tissues using RT-PCR that was further confirmed by sequence analysis. Ca-SR proteins were detected in rat ventricular and atrial tissues as well as in isolated cardiac myocytes. Anti-(Ca-SR) Ig did not detect any specific bands after preadsorption with standard Ca-SR antigens. An immunohistochemistry study revealed the presence of Ca-SR in rat cardiac as well as other tissues. An increase in extracellular calcium or gadolinium induced a concentration-dependent sustained increase in [Ca2+]i in

This observation led to the conclusion that Ca-SR was actually expressed in perivascular nerves with the corres- ponding mRNA residing in the neuronal soma away from the isolated blood vessel wall. Interestingly, a recent study claimed that Ca-SR was present in smooth muscle cells of spiral modiolar artery of gerbils [8]. The functions of Ca-SR in these smooth muscle cells were not studied.

Calcium ions were the first identified endogenous substance to function as both a first and second messenger via the stimulation of an extracellular calcium sensing receptor (Ca- SR). The binding of extracellular calcium (first messenger) to Ca-SR in plasma membrane activates Gq proteins, stimulates phospholipase C (PLC)-b activity, and increases intracellular IP3 levels, leading to intracellular calcium release (second messenger) [1,2]. The expression of Ca-SR has been identified in parathyroid [2], thyroid [3], kidney [4], bone [5] and GI tract [6], the organs involved in systemic calcium home- ostasis. Defective Ca-SRs are involved in genetic diseases linked to calcium homeostasis. Ca-SR and its isoforms or homologous receptors may represent novel clinical targets for treatment of these diseases and others like osteoporosis. Calcium handling is essential for the homeostatic control of cardiovascular functions, which may not couple directly to systemic calcium homeostasis. Whether Ca-SR has a functional role to play in the cardiovascular system is unclear. Ca-SR proteins were detected, but not the corres- ponding transcripts, in mesenteric resistant artery tissues [7].

Cardiac tissue is very sensitive to calcium homeostasis. An increased intracellular calcium concentration, either due to the increased extracellular calcium entry through voltage- gated calcium channels or the increased intracellular calcium release, would trigger the contraction of myocytes. Overloading of cellular calcium, on the other hand, leads to cell death and heart injury. To date, the expression of Ca-SR in cardiomyocytes had not been reported, less alone the function of these receptors. Several lines of evidence are presented in this communication that demonstrate the existence of Ca-SR in rat heart by identifying the mRNA and proteins of Ca-SR in cardiac tissues and by delineating the functional regulation of Ca-SR in cardiac myocytes. Ca-SR may present itself as a novel target by which the cardiac functions can be modulated.

Materials and methods

Correspondence to: R. Wang, Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E5. Fax: + 1 306 966 6532, Tel.: + 1 306 966 6592, E-mail: wangrui@duke.usask.ca Abbreviations: Ca-SR, calcium-sensing receptor; IP3, inositol 1,4,5- triphosphate; TG, Thapsigargin. (Received 9 March 2003, revised 10 April 2003, accepted 30 April 2003)

RT-PCR analysis of the Ca-SR

Male Sprague–Dawley rats (10–12 weeks old) were used with an approved protocol (University Committee on Animal Care and Supply of University of Saskatchewan).

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International) was used at a dilution of 1 : 400 to detect the expression level of a-actin in the isolated tissues as the house-keeping internal control.

Immunohistochemistry study

sodium pentobarbital

Total RNA was extracted from isolated tissue with an RNeasy Total RNA Kit (Qiagen) and treated with RNase- free DNase I (Ambion). First-strand cDNA was made by reverse transcribing 2 lg of DNase I-treated total RNA with MMuLV reverse transcriptase (Perkin-Elmer) using random hexamers in a total volume of 20 lL. The RT reaction was carried out at room temperature for 15 min followed by incubation at 42 (cid:2)C for 1 h. Five microliters of RT reaction mixture were used for PCR amplification in a volume of 50 lL using Advanced PCR II mixer (Clontech) with gene specific primers designed on reported sequences of rat Ca-SR (GenBank accession number U20289). Portions of the Ca-SR cDNA were amplified using the primer pairs that were Ca-SR (forward), 5¢-ttcggcatcagctttgtg-3¢; and Ca-SR (reverse), 5¢-tgaagatgatttcgtcttcc-3¢. PCR amplifica- tion consisted of 35 cycles of denaturation at 94 (cid:2)C for 20 s, annealing at 60 (cid:2)C for 20 s, and polymerization at 68 (cid:2)C for 30 s. Aliquots (5 lL) of PCR reactions were electropho- resed through ethidium bromide-stained 1.2% agarose gels.

Nucleotide sequence analysis

Gel purified PCR-amplified Ca-SR products were cloned into the pCR2.1 TA cloning vector (Invitrogen). The automated sequence analysis was performed on three independent clones using an ABI-373A (Applied Biosys- tems Inc.) sequencer.

Western blot analysis

Sprague–Dawley rats were anasthetized by intraperitoneal (60 mgÆkg)1 body injection of weight). The rats were perfused through the left ventricle with ice cold NaCl/Pi (pH 7.4) for 1 min and ice cold 4% paraformaldehyde in NaCl/Pi for 2 min. The tissues were removed and fixed in 4% paraformaldehyde in NaCl/Pi at 4 (cid:2)C overnight. Specimens were dehydrated with 20% sucrose in NaCl/Pi for 24 h. Cryostat sections (5 lm) were cut on a Micron cryostat at )20 (cid:2)C and thaw-mounted onto ethanol-cleaned slides coated with 1% gelatin. Sections were postfixed in 4% paraformaldehyde for 20 min, followed by 15 min incubation in 5 lgÆmL)1 proteinase K (Ambion) for antigen retrieval at 37 (cid:2)C. After washing with NaCl/Pi, the sections were blocked with 5% normal horse serum in NaCl/Pi for 1 h at room temperature and then incubated with 1 : 500 polyclonal Ig against Ca-SR (Alpha Diagnostic International) in NaCl/Pi containing 2.5% normal horse serum and 0.1% Triton X-100 overnight at 4 (cid:2)C. After rinsing with NaCl/Pi, staining was performed with the Vectastain Universal Elite ABC Kit (Vector Laboratories, Burlington) according to manufacturer’s instructions. Briefly, after washing three times in NaCl/Pi, sections were incubated for 30 min with diluted biotinylated universal secondary IgG. After washing with NaCl/Pi, the sections were exposed to Vector ABC reagent (avidin coupled to biotinylated horseradish peroxidase) for 30 min. Sections were washed again in NaCl/Pi and visualized by incubating with horseradish peroxidase substrate containing 0.02% diaminobenzidine, 0.3% nickel ammonium sulfate and 0.002% hydrogen peroxide (Vector Laboratories). The appearance of reaction product was monitored and photo- graphed under bright-field illumination. As a control, some sections were not incubated with primary antibody.

Adult rat myocyte isolation

Membrane proteins were prepared as described previously [9]. Briefly, tissues were homogenized with a Polytron homogenizer in 1 mL Tris-buffered saline (10 mM Tris, 0.3 M sucrose and 1 mM EDTA) containing protease inhibitor mixture [9]. The homogenate was centrifuged at 6000 g for 15 min at 4 (cid:2)C to remove nuclei and undisrupted cells. The supernatant was further centrifuged at 40 000 g for 1 h at 4 (cid:2)C. Resulting pellets were then washed and resuspended with the same Tris-buffered saline without sucrose. Protein concentration was determined using a Bio-Rad protein assay solution with BSA as standard. Membrane proteins (20 lg) were electrophoresed through standard 10% SDS-PAGE in Tris-glycine electrophoresis buffer [125 mM Tris, 959 mM glycine (pH 8.3), and 0.5% SDS] and blotted onto nitrocellulose membrane in transfer- ring buffer [39 mM glycine, 48 mM Tris (pH 8.3) and 20% methanol] at 80 mA for 1.5 h in a water-cooled transfer apparatus. The membrane was blocked in a blocking buffer NaCl/Pi containing 3% skimmed milk at room temperature for 2 h. The membrane was then incubated overnight at 4 (cid:2)C with 1 : 500 diluted affinity-purified polyclonal antibody against Ca-SR in blocking buffer. Unless otherwise specified, anti-(Ca-SR) Igs were from Alpha Diagnostic International Inc. (San Antonio, TX, USA).

After the membrane was washed five times in NaCl/Pi, it was incubated with goat anti-(rabbit IgG) Ig conjugated with horseradish peroxidase diluted to 1 : 5000 in the blocking buffer for 2 h at room temperature. Antibody– antigen complexes were detected by chemiluminescence using chemiluminescent substrate kit (NEN Life Sciences). As a control, immunoblotting was carried out as described above without anti-(Ca-SR) Ig. Anti-actin Ig (Chemicon Adult (6–8 weeks old) male Sprague–Dawley rats were anesthetized with pentobarbital sodium (50 mgÆkg)1, i.p.). The heart was removed and firstly perfused via the aorta at 37 (cid:2)C with standard Tyrode’s solution for about 5 min until the effluent was clear. Standard Tyrode’s solution was composed of (in mM): NaCl, 136; KCl, 5.4; NaH2PO4, 0.33; MgCl2, 1.0; CaCl2, 2.0; dextrose, 10 and Hepes, 10 (pH adjusted to 7.4 with NaOH), and was maintained at room temperature and equilibrated with 95% O2 and 5% CO2. Then the heart was perfused with Ca2+-free Tyrode’s solution for 5 min and Ca2+-free Tyrode’s solution con- taining 120 UÆmL)1 collagenase for (cid:1) 70 min. Ventricular tissues (2–3 mm in diameter) were excised and placed in a high [K+] solution composed of (in mM): glutamic acid, 70; taurin, 15; KCl, 30; KH2PO4, 10; Hepes, 10; MgCl2, 0.5; EDTA, 0.5 and glucose, 10 (pH adjusted to 7.3–7.4 with KOH). Myocytes were isolated by trituration with a Pasture pipette and collected by centrifuging at 600 r.p.m. for 1 min at room temperature. Cells were re-suspended in the high [K+] solution and kept at room temperature [10].

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indicating the tested RNA samples were free of ted, genomic DNA contamination. This 234 bp PCR fragment was gel-purified, subcloned into plasmid vectors, and sequenced. The derived sequences from three independent clones were identical to the Ca-SR cDNAs from rat parathyroid, kidney [4] and brain [11]. The expression level of Ca-SR mRNA in thyroid appears to be much greater than that in cardiac tissues. However, these results were derived from RT-PCR, which is a qualitative rather than quantitative mRNA assay. Therefore, it would be inappropriate to predict the protein levels based on RT-PCR results shown in Fig. 1A.

Protein expression of Ca-SR in rat cardiac tissues

Fura-2 measurements of [Ca2+]i Single ventricular myocytes attached to the glass bottom of Petri dishes coated with laminin (10 lgÆmL)1, 500 lL per dish, with blow-drying). Cells were loaded with 2 lM fura 2-AM (Sigma) for 60 min at room temperature in a Hepes buffer composed of (in mM): NaCl, 125; KCl, 3.0; MgSO4, 1.2; Na2PO4, 2.0; CaCl2, 1.8; dextrose, 10.5; Hepes, 32 and 0.1% BSA (pH 7.4). Thereafter, myocytes were rinsed with normal Hepes buffer twice to remove the remaining dye and then equilibrated for 30 min at room temperature. The tested compounds were added directly to petri dishes to reach the desired final concentrations. The fura-2 loaded myocytes were alternatively excited at 340 and 380 nm from a monochromator (SpectraMASTER, Olympus America, Melville, NY, USA). Fluorescent images of ventricular myocytes were observed through an inverted phase-contrast microscope (Olympus IX70, Tokyo) and video images of fluorescence at 510 nm emission were collected at 2 Hz using an intensified CCD camera system (AstroCam, Olympus Life Science resources, Cambridge, UK) with the output digitized at 768 · 512 pixels. The ratio of the fluorescence intensities at 340 : 380 nm excitations was monitored and processed with computer software (ULTRA- VIEW, PerkinElmer Life Sciences Inc., Boston, MA, USA).

Measurement of IP3 formation

The expression of Ca-SR protein was examined using Western blotting on whole-tissue extract. Ca-SR proteins with a relative molecular mass between 120 and 140 kDa were detected in rat atrium and ventricle (Fig. 1B) or in whole heart tissues (Fig. 1C). The same 120–140 kDa band was also detected in thyroid, liver, parathyroid and kidney tissues, which serve as positive control. While the band of PCR product for atrium was faint (Fig. 1A), the expression levels of Ca-SR proteins were similar between atrium and ventricular tissues (Fig. 1B), which may indicate a relative instability of Ca-SR mRNA in rat atrium. In the absence of antibody, no positive band was identified (Fig. 1B). Fur- thermore, preadsorption of anti-Ca-SR antibody with standard Ca-SR antigen eliminated the 140 kDa band (Fig. 1C). Together, these results indicate the specificity of the anti-(Ca-SR) Ig.

Immunohistochemistry study on the expression of Ca-SR protein in different tissues

Isolated rat ventricular myocytes were incubated for 4 h in serum-free and inositol-free DMEM, to which 5 lCiÆmL)1 myo-2-[3H]inositol (Du Pont Canada Inc.) were added. The cells were subjected to different stimuli for 60 mins, and the reaction was terminated by adding 0.9 mL methanol/ inositol- chloroform/HCl (40 : 20 : 1, v/v/v). The initial phosphate (IP) pool of the aqueous phase composed of inositol 4-phosphate, inositol 4,5-biphosphate and IP3 was eluted consecutively by ion-exchange chromatography (AG1-X8 resin, Bio-Rad Laboratories). The lipid phase was counted to measure the phosphatidylinositol phosphate (PIP) lipid pool. IP3 was expressed as a relative value of (IP3/PIP) · 103 (arbitrary units) to correct for the variation in the labeling of the lipid pool. Strong immunostaining was observed in liver cells (Fig. 2A and B) as reported by Canaff et al. [12]. In heart, deep brown immunostaining was present throughout all cardio- myocytes (Fig. 2D,E), indicating the expression of Ca-SR at protein level in rat heart. Lack of specific staining was demonstrated in control sections in the absence of anti- Ca-SR antibody (Fig. 2C,F).

Chemicals and data analysis Protein expression of Ca-SR in isolated rat cardiac myocytes

Thapsigargin (TG) was purchased from Calbiochem. U73122, U73343, spermine, nifedipine, CdCl2 and other chemicals were from Sigma. Data were expressed as means ± SEM. Differences between treatments in the same cells were evaluated by paired Student’s t-test or in conjunction with Newman–Keuls test. A significant level of difference was determined when P < 0.05.

Results

Transcriptional expression of Ca-SRin rat cardiac tissues

Expression of Ca-SR mRNA was examined using RT- PCR. A cDNA fragment of 234 bp corresponding to the selected Ca-SR mRNA sequence was detected in both rat atrium and ventricle (Fig. 1A). In the absence of reverse transcriptase, no PCR-amplified fragment could be detec- To confirm that Ca-SR was expressed in cardiac myocytes, rather than neuronal or other types of cell in heart tissue, ventricular and atrial myocytes were isolated separately and the expression of Ca-SR proteins in these cells was examined. Similar to the observations on whole heart tissue, Ca-SR proteins were identified in the isolated myocytes (Fig. 3). Compared to ventricular and atrial membrane preparations, membrane preparation from liver cells had a low protein content as evidenced by low actin level (Figs 1 and 3). Preadsorption of the anti-Ca-SR Igs with standard Ca-SR antigen completely eliminated the 140 kDa band (Fig. 3B). In these experiments, the anti- (Ca-SR) Ig was from Affinity BioRegents, Inc. (Golden, CO, USA) at dilution of 1 : 400. In all other Western blot and immnunostaining studies, anti-(Ca-SR) Ig from Alpha Diagnostic International were used. The same results using

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Fig. 1. Expression of Ca-SR in rat cardiac tissues. (A) Detection of Ca-SR mRNA by RT-PCR in rat heart in the presence or absence of reverse transcriptase (RT). M, DNA marker; bp, base pairs. Similar results were obtained in four other experiments. (B) Detection of Ca-SR proteins by Western blot in various rat tissues using anti-(Ca-SR) Ig (left) or in the absence of anti-(Ca-SR) Ig (right). (C) Detection of Ca-SR proteins by Western blot in various rat tissues using anti- (Ca-SR) Ig without preadsorption (left) or after incubation with excess Ca-SR antigens overnight at 4 (cid:2)C (right).

the anti-(Ca-SR) Igs from different suppliers further validate the specificity of Ca-SR proteins detected in rat cardio- myocytes.

application, all cells in the observation field contracted and quickly (cid:2)exploded(cid:3) (Fig. 5A and B). This (cid:2)calcium burst(cid:3), however, was not observed after calcium was removed completely from the bath solution. As shown in Fig. 5C,D, spermine still increased intracellular calcium but in a less dramatic way and all cells survived from this spermine treatment.

Extracellular calcium, gadolinium and spermine induced changes in intracellular calcium concentration Elevating [Ca2+]o from 0 mM to > 1.5 mM evoked an increase in intracellular calcium concentration in more than 90% of isolated ventricular myocytes in a given observation field (Fig. 4A). The maximal increase in intracellular calcium concentrations was obtained with 5–10 mM extra- cellular calcium (Fig. 4B). After changing extracellular calcium concentration back to 0 mM, the increased intra- cellular calcium concentration declined gradually (Fig. 4C). Consecutive exposure of freshly isolated rat ventricular myocytes to extracellular gadolinium also induced a concentration-dependent increase in intracellular calcium (Fig. 4D).

The role of intracellular calcium release and the phospholipase C (PLC) pathway in the extracellular calcium-induced increase in [Ca2+]i Isolated myocytes were pretreated for 10 min with 10 lM TG that inhibits the refilling of the IP3-sensitive calcium release pools [12,13]. Subsequently, extracellular calcium was changed from 0–1.5 mM, which failed to elicit any increase in [Ca2+]i. This effect was observed in a total of 25 cells from six Petri dishes (n ¼ 6, P < 0.05) (Fig. 6A). Preincubation of myocytes with TG also abolished 0.3 mM Gd3+-induced (n ¼ 8) or 5 mM spermine-induced (n ¼ 6) increase in the [Ca2+]i level (not shown). U73122 is a phosphatidylinositol-specific PLC blocker [3,14]. Pretreat- ment with U73122 for 10 min eliminated the effect of extracellular calcium-induced intracellular calcium release With 1 mM Ca2+ in the bath solution, spermine from 1–10 mM induced a time- and concentration-dependent increase in intracellular calcium (Fig. 5). At 10 mM, sper- mine produced a (cid:2)calcium burst(cid:3) in a total of 27 cells from five dishes (P < 0.05). In less than 1 min after spermine

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Fig. 2. Immunohistochemical detection of Ca-SR in rat cardiac tissues. Tissue sections of rat liver (A–C) and rat heart (D–F) were processed in the presence (A,B,D,E) or absence of anti-Ca-SR Ig (C and F). Magnification was · 95 (A,D,F); · 190 (B,C); · 380 (E). Representative results were shown from three different experiments.

another series of experiments, myocytes were pretreated with 200 lM CdCl2 for 10 min. CdCl2 treatment alone did not alter [Ca2+]i. With CdCl2 pretreatment, an increase in [Ca2+]i induced by extracellular calcium was again observed (Fig. 7A). Furthermore, increasing [Ca2+]o from 0–1.5 mM still significantly increased intracellular calcium in 30 cells from five Petri dishes in the presence of nifedipine (10 lM) (not shown).

Changes in intracellular IP3 levels in response to different Ca-SR stimuli

(n ¼ 4) (Fig. 6B). This treatment also abolished 0.3 mM Gd3+-induced (n ¼ 4) or 5 mM spermine-induced (n ¼ 4) increase in intracellular calcium (not shown). Under the same condition but without TG or U73122 pretreatment, extracellular calcium induced significant increase in [Ca2+]i (Fig. 4C). On the other hand, pretreatment of cells with U73334 at 10 lM, an inactivated analogue of U73122 [8], for 10 min did not prevent the increase in [Ca2+]i induced by extracellular calcium (n ¼ 5, P < 0.05) (Fig. 6C). These results suggest that activation of Ca-SR resulted in stimu- lation of PLC pathway, and the subsequent production of IP3 stimulated the TG-sensitive IP3 receptors, leading to intracellular calcium increase.

An increased IP3 formation in rat ventricular myocytes was observed after incubation with 3 mM calcium, 0.3 mM gadolinium, or 1 mM spermine (Fig. 7B). The largest IP3 response was induced by extracellular calcium when com- pared with the effects of gadolinium and spermine.

Discussion

Involvement of extracellular calcium entry in the extracellular calcium-induced increase in [Ca2+]i To examine whether the increased [Ca2+]i was related to the activity of Na+/Ca2+ exchanger, NiCl2 (10 mM) was applied to the isolated myocytes [15]. Under this condition, increasing [Ca2+]o from 0–1.5 mM significantly increased [Ca2+]i (data not shown). Thus, the activity of Na+/Ca2+ exchanger in plasma membrane could not explain the increase in [Ca2+]i upon the stimulation of Ca-SR. In Expression of Ca-SR in cells with functions unrelated to systemic calcium homeostasis has been demonstrated in many cases. For instance, expression of Ca-SR in neurons suggests the coupling of [Ca2+]o to neuronal activities [11].

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Fig. 3. Detection of Ca-SR receptor in isolated rat atrial and ventricular myocytes using the anti-(Ca-SR) Igs (Affinity BioRegents, Inc.). (A) Ca- SR proteins were detected in ventricular and atrial myocytes as well as in liver. (B) Anti-(Ca-SR) Igs were incubated with excess Ca-SR antigens overnight at 4 (cid:2)C before being used in Western blot experi- ments.

Fig. 4. Extracellular calcium-induced intracellular calcium increase in freshly isolated rat ventricular myocytes. (A) The same groups of ven- tricular myocytes were exposed consecutively to different [Ca2+]o. Changes in the density of pseudo-greyscale indicate different levels of intracellular calcium concentrations with the black representing lower [Ca2+]. (B) Concentration dependent effects of extracellular calcium on [Ca2+]i in ventricular myocytes. Changes in 4–6 cells in each culture dish were analysed and a total of four culture dishes were used at each calcium concentration. *P < 0.05 vs. data obtained at 0 mM of extracellular calcium. (C) Reversibility of the extracellular calcium- induced [Ca2+]o change. (D) The same groups of ventricular myocytes were exposed consecutively to different [Gd3+]o.

or a Na+/Ca2+ exchanger. Release of intracellular calcium from thapsigargin-sensitive calcium pools after activation of PLC pathway was responsible for the extracellular calcium- induced [Ca2+]i ; (e) [Ca2+]i increase in isolated ventricular myocytes was induced by spermine at concentrations between 1–10 mM, which was the concentration range used in many other studies to elucidate the presence of Ca-SR in different preparations [6,12,16].

Ca-SR in cardiac cells senses the changes in extracellular calcium concentrations An increase from 0–1.5 mM in [Ca2+]o triggered an increase in intracellular calcium and this effect was maximal at Identification of Ca-SR in spiral modiolar artery, located between the eighth cranial nerve and the bond of the cochlear modiolus, also indicates that the changes in [Ca2+]o may somehow affect smooth muscle functions. The involvement of Ca-SR in diverse cellular functions implies broad physiological functions beyond the regulation of systemic calcium homeostasis. Our present study for the first time demonstrated the existence of Ca-SR in cardiac myocytes. This conclusion is based on several lines of evidence: (a) transcripts of Ca-SR were clearly detected in cardiac tissue and the sequences of these transcripts were confirmed as identical to the known sequence of Ca-SR; (b) Ca-SR proteins were identified in cardiac tissue as well as in isolated atrial and ventricular myocytes; (c) Immunohisto- logical staining clearly located Ca-SR proteins in cardiac tissues; (d) increase in [Ca2+]o increased intracellular free calcium levels, which was not mediated by extracellular calcium entry through either voltage-gated Ca2+ channels

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Fig. 6. Signal transduction pathways involved in the extracellular cal- cium-induced increase in [Ca2+]i in isolated rat ventricular myocytes. (A) Thapsigargin blocked the effect of extracellular calcium-induced increase in [Ca2+]i. (B) Pretreatment of cells with 10 lM U73122 eliminated the effect of extracellular calcium-induced intracellular calcium release. (C) Pretreatment of cells with 10 lM U73343 did not prevent the increase in [Ca2+]i. induced by extracellular calcium.

Fig. 5. Extracellular spermine-induced [Ca2+]i in freshly isolated rat ventricular myocytes. Changes in the density of pseudo-greyscale indicate different levels of [Ca2+]i with black representing lower cal- cium levels. (A) A sudden exposure of ventricular myocytes to 10 mM spermine triggered an intracellular calcium burst and cell death with 1 mM calcium in the bath solution. (B) Time course of the increase in [Ca2+]i induced by a sudden exposure to 10 mM spermine with 1 mM calcium in the bath solution. (C) Spermine induced a gradual increase in [Ca2+]i with 0 mM calcium in the bath solution. All cells survived under this gradual spermine exposure condition. (D) Time course of the increase in [Ca2+]i induced by various concentrations of spermine with 0 mM calcium in the bath solution (total 10 cells from three dif- ferent Petri dishes).

[Ca2+]o would reduce the activity of Ca-SR in cardio- myocytes, lowering [Ca2+]i and protecting cardiac muscles from sustained contraction. Upon repolarization, [Ca2+]o can be restored to a physiological level around 1.5 mM. The consequent re-activation of Ca-SR would then restore normal contractility of cardiac muscles by normalizing [Ca2+]i. Can [Ca2+]o be further elevated from 1.5–5 mM in cardiac muscle? Similar to our results in cardiac myocytes, Ca-SR in human antral gastrin cells has been reported to be sensitive to extracellular calcium concentrations ranged from 1.8–5.4 mM [6]. Under certain in vivo conditions, the luminal surface of the gastrin cells can be exposed to 15 mM extracellular calcium [20]. As high as 40 mM of extracellular calcium in the direct vicinity of bone-osteoclasts has been observed [8]. There are several scenarios for which [Ca2+]o in the vicinity of cardiac myocytes, especially in T-tubule system, may temporarily increase, such as the extrusion of intracellular calcium from the excited myocytes and the (cid:1) 5 mM extracellular calcium. The physiological relevance of this narrow range of [Ca2+]o in cardiac myocytes should be commented on. Under what circumstances would the extracellular calcium be in the range of 0–1.5 mM in heart? Intracellular calcium changes have been observed in para- thyroid hormone-releasing and calcitonin cells in response to [Ca2+]o changes from 0.75– 3 mM [17]. Brown et al. described a steep dose–response relationship of the activa- tion of Ca-SR by extracellular calcium in parathyroid cells [2]. The plasma levels of ionized Ca2+ are between 1.0– 1.3 mM [6]. The [Ca2+]o can be significantly lowered within the interstitial fluid of the beating heart [18], especially within the T-tubular system of heart. This system is a restricted plasma membrane invagination and the calcium content therein is limited. The sustained membrane depo- larization of heart membrane has been reported to lead to calcium depletion in T-tubular system [19]. The lowering of

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ion channels the activation of different plasmalemmal [26–28] as well as the stimulation of Ca-SR [29]. In our study, spermine elicited an extracellular calcium-dependent intracellular calcium response in isolated cardiomyocytes. In the absence of extracellular calcium, the spermine-induced [Ca2+]i increase was less dramatic than that in the presence of 1 mM extracellular calcium and no (cid:2)calcium burst(cid:3) and cellular destruction were observed. Similar extracellular calcium dependency of the effects of spermine on Ca-SR has been noticed in other previous studies [6,29].

Fig. 7. Changes in intracellular calcium and IP3 levels. (A) Effect of CdCl2 on the extracellular calcium-induced increase in [Ca2+]i. in isolated rat ventricular myocytes (total of 32 cells from five Petri dishes). [Ca2+]i. was determined when the changes reached the maxi- mum levels. (B) IP3 formation in isolated rat ventricular myocytes. n ¼ 5 for each group. *P < 0.05 compared with control group.

tissues was

release of calcium from necrotic myocytes. The healthy myocyte in the neighborhood of necrotic myocytes would face relatively high [Ca2+]o and increase their Ca-SR activity. Thus, the contractility of these healthy myocytes would be increased to maintain the pump function by increasing their intracellular calcium levels. Contrary to the conventional thought of a static extracellular calcium level, [Ca2+]o in heart tissues may undergo fluctuations depending on the activity of the heart. The presence of Ca-SR in cardiac myocytes may co-ordinate cellular activities with the dynamic changes in [Ca2+]o in the vicinity of cardiomyocytes [1].

The physiological concentration of plasma spermine is in the low micromolar range [29,30]. In the study by Quinn et al. [29], spermine was used at concentrations from 0.1–1 mM to test the Ca-SR-mediated intracellular calcium response in Ca-SR-expressing HEK cells. Ray et al. [6] reported the effect of spermine on Ca-SR at concentrations between 0.1–1 mM. spermine-induced In hepatocytes, [Ca2+]i increase was manifested at spermine concentrations from 1.25–10 mM [12]. Similarly, in our study, a spermine response was observed at concentrations between 1–10 mM. No effect was observed when spermine concentration was lower than 1 mM. Nevertheless, the physiological signifi- cance of this spermine effect at these concentrations can still be appreciated. Polyamine secretion from some neurons has been indicated [31], presenting the possibility that local concentration of spermine can be much higher than the circulating concentration. Moreover, the tissue spermine content of ventricular increased from 68 pmolÆmg)1 in normotensive Wistar–Kyoto rats to 376 pmolÆmg)1 in spontaneously hypertensive rats [24]. This observation may also shed light on the pathophysio- logical significance of the effect of spermine at relatively high concentrations on Ca-SR in hearts. The spermin- induced increase in [Ca2+]i alone may not suffice to demonstrate conclusively the involvement of Ca-SR but when taken in conjunction with the effects of extracellular calcium and gadolinium on [Ca2+]i, as well as the detection of Ca-SR at mRNA and protein levels, does provide a line of evidence for the presence and function of Ca-SR in cardiac myocytes. The physiological importance of Ca-SR in cardiomyocytes can be better understood by comparing the structure and function of hearts with or without Ca-SR deficiency. Ca-SR knock-out mice provide an avenue for this kind of study. However, cardiovascular functions of Ca-SR knock-out mice have not been reported to our knowledge. Loss of Ca-SR in parathyroid gland in knock- out mice results in hyperparathyroidism, hypercalcemia, and growth retardation [32]. These alterations may also significantly and indirectly affect cardiac function, mingled with any potential direct cardiac outcome due to the lack of cardiac Ca-SR. Therefore, organ-selective or heart-selective inactivation or activation of Ca-SR in living animals should be engineered, which may help to better determine the direct cardiac outcome of cardiac Ca-SR deficiency.

In summary, Ca-SR may play an important physiological role in the modulation of cardiac functions under both physiological and pathophysiological conditions. Increased local calcium concentration is sensed by myocytes via Ca-SR and lead to increased cardiac activity. Increased extracellular polyamine concentration in heart, on the other hand, may stimulate Ca-SR on cardiomyocytes to promote cardiac hypertrophy. Due to the limited access to specific The naturally occurring polyamines, including spermine, spermidine and putrescine, are involved in the synthesis of nucleic acids and proteins in eukaryotic and prokaryotic cells. They play an important role in the regulation of cellular proliferation and differentiation [21]. For the regulation of cardiac function, polyamines are also import- ant. Previous studies have provided evidence that polyam- ines promote cardiac hypertrophy [22,23]. In spontaneously hypertensive rats, an increased left ventricular mass [24] or cardiac hypertrophy [25] was associated with increased spermine and spermidine contents. The molecular mecha- nisms underlying the cellular actions of polyamines include

2688 R. Wang et al. (Eur. J. Biochem. 270)

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of phospholipase C-beta and protein kinase C in MC3T3–E1 osteoblasts. Bone 30, 559–566.

15. Janiak, R., Lewartowski, B. & Langer, G.A. (1996) Functional coupling between sarcoplasmic reticulum and Na/Ca exchange in single myocytes of guinea-pig and rat heart. J. Mol. Cell. Cardiol. 28, 253–264.

blockers of Ca-SR, whether polyamine-induced cardiac hypertrophy is mediated by Ca-SR cannot be readily tested at the moment. Nevertheless, delineation of the interaction among extracellular calcium levels, polyamine concentra- tions, functional status of Ca-SR, and myocyte apoptosis and proliferation would help better understand the mech- anisms of cardiac hypertrophy as well as its management.

Acknowledgements

16. Sanders, J.L., Chattopadhyay, N., Kifor, O., Yamaguchi, T., Butters, R.R. & Brown, E.M. (2000) Extracellular calcium-sensing receptor expression and its potential role in regulating parathyroid hormone-related peptide secretion in human breast cancer cell lines. Endocrinology 141, 4357–4364.

This study was supported by an operating grant from Canadian Institutes of Health Research (CIHR). R. Wang is an Investigator of CIHR. L. Wu is a New Investigator of CIHR.

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