INTRODUCTION

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HINDLIMB SUSPENSION OF RATS is currently used to study the effect of simulated weightlessness on cardiovascular activity. Hindlimb suspension has been proposed to elicit many of the hemodynamic alterations that occur after prolonged bed rest with head-down tilt of humans, including transient increases in central venous pressure (24), reduced blood volume (19), tachycardia (12), altered tissue perfusion (19), modified vascular reactivity (7, 22), and decreases in exercise capacity (19). Hindlimb suspension has been also used to simulate the effects of decreased physical activity on vascular reactivity. In the rat aorta, long-term hindlimb suspension (14-15 days) and old age have been shown to result in similar alterations of vascular responsiveness, i.e., a noticeable decrease in maximal contractile force elicited in response to various neuromediators (6). In the rat vena cava, short-term hindlimb suspension (1-2 days) appears to decrease sensitivity to norepinephrine without modification of the maximal contractile response (22). These later effects have been related to a desensitization of ₁-adrenoceptors, depending on increased protein kinase C activity. However, the effects of long-term suspension on vasoconstrictor-induced contractile responses have not been studied in veins.

The present study was undertaken to study the effects of a 14-day hindlimb suspension on increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) evoked by various vasoconstrictors (norepinephrine, angiotensin II, and caffeine) and activation of voltage-dependent Ca²⁺ channels in isolated portal vein myocytes from young adult control and suspended rats. We show a strong reduction in the vasoconstrictor-induced increases in [Ca²⁺]_i in suspended rats, without detectable alterations in the intrinsic properties of ryanodine-sensitive Ca²⁺-release channels, D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]-gated channels, and voltage-dependent Ca²⁺ channels. The reduction of the maximal binding capacity of [³H]ryanodine to vascular membranes suggests that long-term hindlimb suspension decreases the number of functional Ca²⁺ channels in the sarcoplasmic reticulum of rat portal vein myocytes.

	MATERIALS AND METHODS

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Animals and cell preparation. Sixty-two male Wistar rats (160-180 g) were housed in a room with controlled temperature (21 ± 2°C) and a 12:12-h light-dark cycle. Water and rat chow were provided ad libitum. This protocol was approved by the ad hoc committee of the Centre National des Etudes Spatiales and was in accordance with the guidelines on the care and use of animals required by the International Physiological Society. The simulated weightlessness model used was that of Morey (17). Briefly, a plastic disk with a hole in it was attached to the tail with adhesive tape and connected to a pulley by a plastic bar. Rats were able to move freely in the cage with their forelimbs in a 360° arc. The hindlimbs were kept above the cage floor and were nonweight bearing. The head-down tilt position was near 40°. After an acclimatization period in individual cages, the suspended rats were maintained in an antiorthostatic position for 14 days. Control rats and rats submitted to a recovery period were maintained in individual cages. Rats were killed by cervical dislocation. The portal vein was cut into several pieces and incubated for 10 min in low-Ca²⁺ (40 µM) physiological solution, and then 0.8 mg/ml collagenase, 0.25 mg/ml Pronase E, and 1 mg/ml bovine serum albumin (BSA) were added at 37°C for 20 min. After this time, the solution was removed and pieces of portal vein were incubated again in a fresh enzyme solution at 37°C for 20 min. Tissues were then placed in enzyme-free solution and triturated using a fire-polished Pasteur pipette to release cells. Cells were stored on glass coverslips in physiological solution and used on the same day.

Patch-clamp measurements. Voltage-clamp and membrane current recordings were made with a standard patch-clamp technique using a List EPC7 patch-clamp amplifier (Darmstadt, Eberstadt, Germany). The whole cell recording mode was performed with patch pipettes of 2-5 M resistance. Membrane potential and current records were stored and analyzed using an IBM-PC computer (pCLAMP; Axon Instruments, Foster City, CA).

Fluorescence measurements. Cells were loaded by incubation in physiological solution containing 1 µM indo 1-acetoxymethyl ester (indo 1-AM) for 30 min at room temperature. These cells were washed and allowed to cleave the dye to the active indo 1 compound for at least 30 min. Indo 1 repartition was usually homogeneous over the cytoplasm, and no compartmentalization of the dye was observed. Measurement of intracellular Ca²⁺ concentration using this fluorescent dye has previously been published (16). Calibration curves were determined for cells isolated from control and suspended rats, as previously described (16). Indo 1-AM-loaded cells were mounted in a perfusion chamber and placed on the stage of an inverted microscope (Nikon Diaphot, Tokyo, Japan). Some experiments were carried out in the presence of 1 µM oxodipine (a light-stable dihydropyridine derivative) to inhibit voltage-dependent Ca²⁺ channels.

Flash photolysis. Caged Ins(1,4,5)P₃ [D-myo-inositol 1,4,5-trisphosphate, P⁴⁽⁵⁾-1-(2-nitrophenyl)ethyl ester] or caged Ca²⁺ (DM-Nitrophen) were introduced into the cell via the patch pipette, with 3-4 min allowed for equilibration. Photolysis was produced by a 1-ms pulse from a xenon flash lamp (Hi-Tech Scientific, Salisbury, UK) focused to a spot of ~2 mm in diameter around the cell. Light was band-pass filtered with a UG11 glass between 300 and 350 nm. Flash intensity could be adjusted by varying the capacitor-charging voltage between 0 and 385 V, which corresponded to a change in the energy input into the flash lamp from 0 to 240 J. On flash photolysis, Ca²⁺ or Ins(1,4,5)P₃ were released with half-times of 0.03 and 3 ms, respectively. To avoid artifacts in the fluorescence records, flashes were triggered during retrace of the laser scan. A small percentage of conversion of caged compounds (~10%) was useful if repetitive pulses were applied to obtain similar responses. Flash intensities 37 J could be applied repetitively without altering the reserve of caged Ins(1,4,5)P₃ (10 µM) or DM-Nitrophen (1 mM, in the presence of 0.25 mM CaCl₂) and, consequently, the amount of photoreleased compounds.

Crude microsomal preparation. Crude microsomal fractions were prepared from dispersed smooth muscle cells obtained from 3-4 rat portal veins. The portal vein myocytes were stirred at 4°C for 10 min in a medium containing 25 mM sucrose, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 1 mM dithiothreitol (DTT), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) at pH 7.4 and then disrupted with a glass-glass homogenizer. The homogenate was centrifuged at 1,000 g for 5 min, and the resulting supernatant was used for [³H]ryanodine binding experiments. Protein concentration was determined by the method of Bradford (2) with the use of BSA in the standard curve.

[³H]ryanodine binding assay. [³H]ryanodine binding was carried out in a medium containing 1 M KCl, 25 mM HEPES (pH 7.8 at 37°C), 1 mM DTT, 0.1 mM CaCl₂, 1 mg/ml BSA, and 0.1 mM PMSF. [³H]ryanodine was used in the concentration range of 2-30 nM. After a 3-h incubation at 37°C, aliquots were filtered through Whatmann GF/C glass fiber filters and washed three times with 5 ml of ice-cold binding buffer. The filters were placed in scintillation vials filled with 4 ml of liquid scintillation cocktail, shaken for 1 h, and counted in a Packard 1500 Tri-Carb. Nonspecific binding was measured in the presence of 10 µM ruthenium red and was subtracted before calculation. At 20 nM [³H]ryanodine, nonspecific binding was <50% of total binding.

Solutions. The normal physiological solution contained (in mM) 130 NaCl, 5.6 KCl, 1 MgCl₂, 1.7 CaCl₂, 11 glucose, and 10 HEPES, pH 7.4 with NaOH. The basic pipette solution contained (in mM) 130 CsCl and 10 HEPES, pH 7.3 with CsOH. In confocal microscopy experiments, fluo 3 and Fura Red (30 µM) were added to the pipette solution. In flash photolysis experiments with Ins(1,4,5)P₃ (10 µM), fluo-3 (60 µM) was used alone. For the recordings of Ca²⁺-channel current, 5 mM BaCl₂ was substituted for CaCl₂ in the reference solution and the pipette solution contained (in mM) 130 CsCl, 10 HEPES, 10 EGTA, 5 Na₂ATP, and 2 MgCl₂. Substances were applied to the recorded cell by pressure ejection from a glass pipette for the period indicated on the records.

Chemicals and drugs. Collagenase was obtained from Worthington (Freehold, NJ). Pronase E, BSA, norepinephrine, rauwolscine, propranolol, and ruthenium red were from Sigma (St. Louis, MO). Angiotensin II and CGP-42112A were from Neosystem Laboratories (Strasbourg, France). Oxodipine was a gift from Dr. Galiano (Instituto de Investigacion y Desarrollo Quimico Biologico, Madrid, Spain). Caffeine was from Merck (Nogent sur Marne, France). Fluo 3 and Fura Red were from Molecular Probes Europe (Leiden, The Netherlands). Indo 1-AM, DM-Nitrophen, and caged Ins(1,4,5)P₃ were from Calbiochem (Meudon, France). [³H]ryanodine (68.3 Ci/mmol) was from DuPont NEN (Boston, MA).

Data analysis. Data are expressed as means ± SE. Significance was tested by means of Student's t-test. P values <0.05 were considered significant. Concentration-response curves were analyzed using a nonlinear least-squares fitting program according to models involving one or two binding sites.

	RESULTS

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Effects of hindlimb suspension on vasoconstrictor-induced Ca²⁺ response and Ca²⁺-channel current in single myocytes from rat portal vein. In the continuous presence of 1 µM oxodipine to block voltage-dependent Ca²⁺ channels, short (3 s) applications of norepinephrine (in the presence of 10 nM rauwolscine and 1 µM propranolol to block ₂- and -adrenoceptors, respectively) activate transient increases in [Ca²⁺]_i which have been shown to depend essentially on Ca²⁺ release from the intracellular store (10). With time intervals of 3 min between successive short applications of 10 µM norepinephrine, similar increases in [Ca²⁺]_i can be obtained in the same cell, indicating complete refilling of the intracellular Ca²⁺ store within 3 min. In Fig. 1, the concentration-response curve for norepinephrine in control myocytes indicates that maximal Ca²⁺ release was obtained at 100 µM, with a half-maximal concentration (EC₅₀) of 1.05 ± 0.13 µM. The Hill coefficient obtained from Hill plots was close to unity. A concentration-response curve for norepinephrine was also obtained in rats suspended for 14 days. Prolonged suspension resulted in a significant reduction of the maximal norepinephrine-induced increase in [Ca²⁺]_i by ~60% (Fig. 1, A and B; Table 1) without variation of the EC₅₀ value (1.05 ± 0.12 µM). The basal [Ca²⁺]_i level was not significantly affected after a 14-day hindlimb suspension (control: 42 ± 5 nM, n = 25; suspended: 45 ± 4 nM, n = 25; P > 0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effects of hindlimb suspension on norepinephrine (NE)- and caffeine (Caff)-induced increases in cytosolic Ca2+ concentration ([Ca2+]i) in single myocytes from rat portal vein. A: concentration-response curves for NE obtained in control rats (), rats suspended for 14 days (), and rats 4 days after removal of suspension (). Data are means ± SE for 21-73 cells isolated from 8 rats in each group. B: typical increases in [Ca2+]i evoked by 10 µM NE in control rats (a) and 14-day suspended rats (b). NE was ejected by pressure ejection from a glass pipette for period indicated on records. C: typical increases in [Ca2+]i evoked by 10 mM Caff in control rats (a) and 14-day suspended rats (b). Myocytes were loaded with indo 1-acetoxymethyl ester and not patch clamped. External solution contained 1 µM oxodipine to block voltage-dependent Ca2+ channels, 10 nM rauwolscine to inhibit 2-adrenoceptors, and 1 µM propranolol to inhibit -adrenoceptors.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Ca2+ channel currents in control and suspended rats. A: family of Ba2+ currents elicited from a holding potential of 60 mV (a) and current-voltage (I-V) relationship (b) in portal vein myocytes from control rats. B: family of Ba2+ currents (a) and I-V relationship (b) in portal vein myocytes from 14-day suspended rats.

Effects of hindlimb suspension on ryanodine-sensitive Ca²⁺-release channels. In myocytes maintained at a holding potential of 50 mV, spontaneous, spatially localized [Ca²⁺]_i transients, termed Ca²⁺ sparks (Fig. 3), were recorded in about 30% of the cells superfused in 1.7 mM external Ca²⁺ in control and suspended rats (n = 81). Plotting the amplitude of Ca²⁺ sparks as a function of the number of sparks revealed a Gaussian distribution (14) with peak values of 32 and 30 nM in control and suspended rats, respectively (Fig. 3, A and B). In addition, the frequency of Ca²⁺ sparks per line-scan images (control: 0.40 ± 0.20 s¹, n = 12; suspended: 0.35 ± 0.25 s¹, n = 7) and the mean full width at half-maximal amplitude (control: 1.5 ± 0.2 µm, n = 21; suspended: 1.4 ± 0.2 µm, n = 21) were not significantly modified (P > 0.05) by hindlimb suspension. It has been previously demonstrated (4, 5, 14) that the spontaneous Ca²⁺ sparks arise from the opening of a small number of ryanodine-sensitive Ca²⁺-release channels, because they are suppressed after pretreatment with 1 µM ryanodine or 10 mM caffeine.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Spatio-temporal characteristics of spontaneous Ca2+ sparks in voltage-clamped portal vein myocytes from control and suspended rats. Holding potential was 50 mV. A: control rats. a: a line within a cell was repeatedly scanned every 2 ms. Fluorescence ratios of fluo 3 to Fura Red were displayed vertically, and each line was added to right of the preceding line to form line-scan image. Duration of line-scan image was 1,049 ms. A spot of fluorescence was detected in right part of cell. b: fluorescence time course of Ca2+ spark averaged over a region of 2 µm indicated by vertical bar in a. c: frequency of Ca2+ sparks as a function of amplitude. Continuous line represents a Gaussian curve with mean and standard deviation of 32 and 16 nM, respectively. B: suspended rats. a: line-scan image with Ca2+ sparks. b: time course of Ca2+ spark averaged over a region of 2 µm. c: frequency of Ca2+ sparks as a function of amplitude. Mean and standard deviation of Gaussian curve are 30 and 15 nM, respectively. Myocytes were loaded with fluo 3-Fura Red mixture.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Evoked Ca2+ responses in voltage-clamped portal vein myocytes from control and suspended rats. Depolarizing steps were applied from 50 mV to +10 mV. A: traces from control rats show membrane potential (a), membrane current (b), line-scan image (c), and spatial averaged fluorescence (d) from 2-µm region indicated by vertical bar in c. B: traces from suspended rats show membrane potential (a), membrane current (b), line-scan image (c), and spatial averaged fluorescence (d) from 2-µm region indicated by bar in c. Myocytes were loaded with fluo 3-Fura Red mixture and held at 50 mV.

Effects of hindlimb suspension on Ca²⁺ sensitivity of Ca²⁺-release channels. Flash photolysis of DM-Nitrophen (caged Ca²⁺) instantaneously elevated (within 2 ms) the Ca²⁺ concentration and evoked Ca²⁺ transients that appeared to be spatially uniform. The amplitude of Ca²⁺ transients obtained from the entire line-scan image increased as a function of the flash intensity (Fig. 5A), with a maximal value reached at 37 J, and progressively declined within 1 s. Plotting the peak of the Ca²⁺ transients as a function of flash intensity revealed that the Ca²⁺-induced increase in [Ca²⁺]_i in suspended rats was significantly reduced compared with that in control rats (Fig. 5B). The maximal Ca²⁺ transient evoked by flash photolysis of caged Ca²⁺ decreased from 69 ± 10 nM (n = 4) in control rats to 44 ± 9 nM (n = 4) in suspended rats (P < 0.05). In the presence of external and internal applications of 1 µM ryanodine for 5 min to inhibit the Ca²⁺-release channels, the brief and small Ca²⁺ jumps due to flash photolysis of DM-Nitrophen were similar in myocytes from control and suspended rats (n = 8). The Ca²⁺ sensitivity of Ca²⁺-release channels can be examined by plotting the ratio between the peak Ca²⁺ transients and the maximal Ca²⁺ transient at different flash intensities for control and suspended rats (Fig. 5C). The curves are superimposed, indicating no changes in the Ca²⁺ sensitivity of Ca²⁺ release channels after a 14-day hindlimb suspension.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Ca2+ transients induced by flash-photolytic Ca2+ jumps in voltage-clamped portal vein myocytes from control and suspended rats. A: ultraviolet (UV) flashes of increasing energy evoked Ca2+ transients of increasing amplitude. a: line-scan image. b: spatial averaged fluorescence from entire line-scan image. B: increases in [Ca2+]i evoked by flash photolysis of caged Ca2+ in control rats () and in rats suspended for 14 days (). Data are means ± SE for 4 cells. C: normalized curves obtained by plotting [Ca2+]i/maximal [Ca2+]i ([Ca2+]i max) against flash intensity in control () and suspended () rats. Myocytes were loaded with fluo 3-Fura Red mixture and caged Ca2+ DM-Nitrophen and held at 50 mV.

Effects of hindlimb suspension on [³H]ryanodine binding. The [³H]ryanodine binding on rat portal vein microsomal preparations was saturable, reached equilibrium after 90 min of incubation, and remained stable for at least 180 min in control and suspended rats. Saturation isotherms of [³H]ryanodine binding to control and 14-day suspended rats are shown in Fig. 6. The maximal binding capacity decreased by ~50% in suspended rats, whereas the dissociation constants were similar in both control and suspended rats (Table 2). The best fit to the experimental data was obtained with a Hill coefficient close to 2, suggesting a positive allosteric cooperativity between two ryanodine binding proteins, each having one high-affinity binding site (13).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   [3H]ryanodine binding to rat portal vein microsomal preparations. Data points represent averages of at least 3 independent determinations performed on 3 preparations. Solid lines were obtained by fitting Hill equation [(Bmax · CnH)/(CnH + KnHd), where Bmax is maximal binding capacity, C is free ryanodine concentration, and nH is Hill coefficient] to data points. , Control rats; , suspended rats.

Effects of hindlimb suspension on Ins(1,4,5)P₃-gated Ca²⁺ channels. Ins(1,4,5)P₃ was released from a caged precursor by flashes of ultraviolet light. In myocytes from control rats (in the presence of 1 µM ryanodine), application of a single 9-J flash pulse evoked localized elevations of Ca²⁺ throughout the stimulated region, producing a slow and small increase in [Ca²⁺]_i when measurement was made in a 2-µm region of the line-scan image (Fig. 7A). Return to basal [Ca²⁺]_i was obtained within 2-3 s. The Ins(1,4,5)P₃-activated increase in [Ca²⁺]_i was selectively suppressed by intracellular application of 2 mg/ml heparin (data not shown). In myocytes from suspended rats, the increase in [Ca²⁺]_i induced by 9-J flash pulses in a 2-µm region of the line-scan image was not significantly affected (control: 15.5 ± 3.5 nM, n = 14; suspended: 13.5 ± 3.5 nM, n = 15; P > 0.05). Stronger flash pulses (37 J) evoked a large increase in [Ca²⁺]_i which generally started from all the sites of the line-scan image and induced a Ca²⁺ wave (Fig. 7B). The average increase in [Ca²⁺]_i in a 2-µm region of the scanned line appeared significantly reduced after a 14-day hindlimb suspension (control: 62 ± 16 nM, n = 14; suspended: 34 ± 15 nM, n = 16 ; P < 0.05). A similar reduction was obtained from analysis of [Ca²⁺]_i in the entire line-scan image.

View larger version (56K): [in this window] [in a new window]   Fig. 7.   Increases in [Ca2+]i evoked by flash photolysis of caged D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in voltage-clamped portal vein myocytes from control and suspended rats. A: Ca2+ responses evoked by 9-J flash pulses in control (1) and suspended (2) rats, shown as line-scan image (a) and spatial averaged fluorescence (b) from a 2-µm region indicated by vertical bar in a. B: Ca2+ responses evoked by 37-J flash pulses in control (1) and suspended (2) rats, shown as line-scan image (a) and spatial averaged fluorescence (b) from a 2-µm region indicated by vertical bar in a. Myocytes were loaded with fluo 3 and caged Ins(1,4,5)P3 and held at 50 mV.

	DISCUSSION

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The purpose of this paper was to test the effects of a 14-day hindlimb suspension on increases in [Ca²⁺]_i evoked by different stimuli in single myocytes from rat portal vein and to determine whether Ca²⁺ channels of both the plasma membrane and sarcoplasmic reticulum were affected by suspension. The results show that release of Ca²⁺ from the intracellular store elicited by caffeine, norepinephrine, and angiotensin II is reduced by hindlimb suspension and that recovery is obtained 4 days after suspension removal. Ca²⁺ fluxes through voltage-dependent, ryanodine-sensitive, and Ins(1,4,5)P₃-gated Ca²⁺ channels were not modified by long-term suspension. The reduction of [³H]ryanodine binding to vascular membranes by hindlimb suspension suggests that alteration in [Ca²⁺]_i may be due to a reduction in the number of functional ryanodine-sensitive Ca²⁺-release channels in the sarcoplasmic reticulum of rat portal vein myocytes.

Previous studies have provided evidence that vasoconstrictor-induced responses are altered by long-term (14-days) hindlimb suspension of rats. For example, hindlimb suspension has been shown to decrease maximal tension elicited by KCl, norepinephrine, and vasopressin in the rat aorta (6, 7). These results differ from those obtained in the rat vena cava for short-term (1-3 days) suspension which show that the maximal tension elicited by 10-100 µM norepinephrine is not modified but that the sensitivity to norepinephrine is strongly reduced (22). The later observations are also supported by the fact that [³H]prazosin binding to ₁-adrenoceptors indicates an increase in the dissociation constant without variation in maximal binding. However, at submaximal norepinephrine concentrations (1-3 µM), venous contractility appears to be reduced after short-term suspension. The present results demonstrate that long-term suspension reduces maximal increases in [Ca²⁺]_i evoked by various vasoconstrictors in rat portal vein and, consequently, may reduce maximal contractility. In smooth muscle cells, increase in [Ca²⁺]_i is determined mainly by Ca²⁺ influx through voltage-dependent Ca²⁺ channels, Ca²⁺ release from intracellular stores, and Ca²⁺ removal by Ca²⁺-ATPases. Alteration of these mechanisms may account for a reduction in [Ca²⁺]_i in suspended rats. The suspension-induced decrease in [Ca²⁺]_i did not appear to be due to an inhibition of voltage-dependent Ca²⁺ channels, because the Ba²⁺ current density through these channels was identical in control and suspended rats.

Ca²⁺ release from intracellular stores can be promoted by the opening of both ryanodine-sensitive and Ins(1,4,5)P₃-gated Ca²⁺ channels. In portal vein myocytes, the Ins(1,4,5)P₃- and ryanodine-sensitive Ca²⁺ stores appear to represent, at least functionally, a single releasable Ca²⁺ store (9). Using both confocal microscopy and a patch-clamp technique, localized increases in [Ca²⁺]_i have been identified in response to activation of elementary Ca²⁺ units of the sarcoplasmic reticulum in portal vein myocytes. These elementary Ca²⁺ events, termed Ca²⁺ sparks, arise from the gating of a few ryanodine-sensitive Ca²⁺-release channels (1, 14). These Ca²⁺ events have been identified previously in heart and skeletal cells (5, 27) and provide the primary pathway for sarcoplasmic reticulum Ca²⁺ efflux underlying excitation-contraction coupling. In 14-day suspended rats, spontaneous Ca²⁺ sparks and Ca²⁺ sparks activated by Ca²⁺ currents during depolarizing pulses were similar to those obtained in control rats, indicating that suspension affected neither the functioning of ryanodine-sensitive Ca²⁺-release channels nor the local Ca²⁺ content of the sarcoplasmic reticulum. In contrast, propagated Ca²⁺ waves activated by membrane depolarizations were not observed in myocytes from suspended rats. Furthermore, the average [Ca²⁺]_i increase was significantly reduced by long-term suspension in a manner similar to that obtained by measuring the [Ca²⁺]_i with indo 1 loading. Thus the possibility that Ca²⁺ influx was less able to activate sarcoplasmic Ca²⁺-release-channel units and thereby generate Ca²⁺ waves might arise from either a change in the Ca²⁺ dependence of Ca²⁺-release-channel activation or a reduction in the number of functional Ca²⁺-release channels. Our observations that the Ca²⁺ sensitivity of Ca²⁺-release channels is unaffected in suspended rats, whereas the maximal binding capacity of [³H]ryanodine to vascular membrane is strongly reduced, support the conclusion that hindlimb suspension reduces the number of functional Ca²⁺-release channels, leading to diminished increases in [Ca²⁺]_i. Activation of Ins(1,4,5)P₃-gated Ca²⁺ channels has been reported to evoke localized Ca²⁺ transients in various cell types (20, 26). Although elementary Ca²⁺ events were difficult to identify in portal vein myocytes, increases in [Ca²⁺]_i (in the continuous presence of 1 µM ryanodine) in response to photorelease of Ins(1,4,5)P₃ evoked by a reduced light-flash intensity were not significantly modified after a 14-day hindlimb suspension. In contrast, the Ins(1,4,5)P₃-induced Ca²⁺ responses in response to light flashes of maximal intensity were significantly reduced by long-term hindlimb suspension. Although [³H]Ins(1,4,5)P₃-binding experiments have not been performed in this study, it can be postulated that hindlimb suspension probably reduces the number of functional Ins(1,4,5)P₃-gated Ca²⁺ channels in a manner similar to that shown for ryanodine-sensitive channels.

It has been reported that, in skeletal muscle, hindlimb suspension causes a marked increase in protein level of the fast Ca²⁺ pump but does not affect the slow Ca²⁺ pump (23). Therefore, an increased activity of the plasmalemmal Ca²⁺ pump or the Na⁺/Ca²⁺ exchanger by hindlimb suspension may also reduce [Ca²⁺]_i. Although these possibilities cannot be excluded, the possibility of Ca²⁺-pump hyperactivity in smooth muscle after suspension seems unlikely, because the basal [Ca²⁺]_i level is not altered and Ca²⁺ pumps are transcribed from the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2) gene (slow Ca²⁺ pump; Ref. 21). However, measurements of Ca²⁺-pump mRNA levels in smooth muscle of suspended rats are needed to definitively exclude this possibility.

The tail-suspended rat model has been largely used to define hormonal, cardiovascular, and excitation-contraction coupling alterations during simulated weightlessness (3, 8, 15, 18, 25). Interestingly, other authors (6) have used head-down suspension as a model of physical inactivity and demonstrated that both inactivity and old age result in similar reduction of vascular contractility. Therefore, our results may be useful to understand the effects of senescence in vascular reactivity.

In conclusion, long-term hindlimb suspension reduces the release of stored Ca²⁺ in response to vasoconstrictors in rat portal vein myocytes. Because Ca²⁺-channel functioning does not appear to be altered, it is proposed that hindlimb suspension reduces the number of functional ryanodine-sensitive and Ins(1,4,5)P₃-gated Ca²⁺ channels in the sarcoplasmic reticulum, as supported by the reduction of [³H]ryanodine binding to vascular membranes.