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P₂ receptor-mediated Ca²⁺ transients in rat cerebral artery smooth muscle cells

Department of Human Anatomy and Cell Biology, University of Liverpool, Liverpool L69 3GE, United Kingdom

Submitted 2 June 2003 ; accepted in final form 16 September 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Significant Ca²⁺ release was previously noted with the activation of L-type Ca²⁺ current in rat superior cerebral artery smooth muscle cells. Here we examined whether the P_2X current that is partly carried by Ca²⁺ also triggers Ca²⁺ release in this preparation. Application of P_2X agonists evoked membrane currents and concomitant Ca²⁺ transients in whole cell voltage-clamped single cells. The expected increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) was calculated from the time-integrated P_2X current by assuming Ca²⁺ is the only charge carrier. The measured increase in [Ca²⁺]_i was plotted as a function of the expected increase in [Ca²⁺]_i, and Ca²⁺-buffering power was obtained as a reciprocal of the linear fit to this relationship. Both ryanodine, a Ca²⁺-induced Ca²⁺-release inhibitor, and cADP ribose, a putative activator of Ca²⁺-induced Ca²⁺ release, had no significant effects on Ca²⁺-buffering power. These results suggest that Ca²⁺ influx through P_2X receptors does not trigger significant Ca²⁺ release. We then examined whether P_2X responses influence the subsequent P_2Y response. P_2Y responses were characterized by measuring the rate of [Ca²⁺]_i increase obtained as the slope of the linear regression to the rising phase of the Ca²⁺ transient. During simultaneous application of the P_2X and P_2Y agonist, the rate of [Ca²⁺]_i increase was facilitated or suppressed depending on the size of the P_2X receptor-mediated [Ca²⁺]_i increase. Membrane depolarization close to the Ca²⁺ equilibrium potential significantly promoted the rate of [Ca²⁺]_i increase. Our results suggest that the [Ca²⁺]_i increase and membrane depolarization caused by the P_2X current may regulate the subsequent P_2Y response.

P_2x receptors; P_2y receptors; voltage clamp; sarcoplasmic reticulum; inositol 1,4,5-trisphosphate

Several pioneering studies characterizing P₂ receptors, although not called so at the time, were carried out using vascular smooth muscle cells (1, 2). In these studies, P₂ receptor stimulation was carried out by application of ATP (1, 2). ATP is an important regulator of vascular tone as it is released not only from sympathetic nerves along with norepinephrine but also from cells that are damaged during vascular injury (24). Furthermore, nucleotides may promote vascular smooth muscle cell proliferation (29), a key event during angiogenesis and atherosclerosis. Thus the understanding of Ca²⁺ homeostasis mediated by nucleotides seems of importance in arteries. Nonetheless, there are still substantial gaps in our knowledge regarding how P₂ receptor activation regulates [Ca²⁺]_i in these cells.

Among the seven P_2X receptor subtypes cloned to date (28), P_2X1 receptor subtypes are thought to be important in smooth muscle cells (41). The defining feature of the P_2X1 receptor is its profound desensitization, a rapid decay of the current during the continuous presence of the drug (28). It follows that the direct responses caused by P_2X1 receptor activation will be short lived. However, there are two possibilities whereby the brief P_2X1 response still imposes substantial impact on the Ca²⁺ homeostasis in vascular smooth muscle cells. First, the P_2X current that is partially carried by Ca²⁺ may trigger further Ca²⁺ release from the sarcoplasmic reticulum, causing amplification of [Ca²⁺]_i increase. P_2X receptor-mediated, Ca²⁺-induced Ca²⁺ release has been suggested in some preparations (27) but not in others (4, 13, 25). Second, P_2X responses may modulate the subsequent activation of the other P₂ receptors, i.e., P_2Y receptors (40).

In the current study, we sought to identify the role of P_2X receptors in Ca²⁺ homeostasis of arterial smooth muscle cells. In particular, we are interested in the contribution of the sarcoplasmic reticulum during P₂ receptor-mediated responses. Experiments were carried out using the combination of electrophysiology and microfluorimetry. This approach has been useful in providing unequivocal evidence for or against the hypothesis that L-type Ca²⁺ currents trigger further Ca²⁺ release (18–20). We used rat superior cerebral artery smooth muscle cells because significant Ca²⁺ release was previously identified in this preparation when the L-type Ca²⁺ current was activated (20). Under our experimental conditions, the P_2X-mediated Ca²⁺ entry did not trigger significant Ca²⁺ release. Rather, our results suggest that activation of the P_2X receptor may modulate the subsequent P_2Y receptor-mediated Ca²⁺ responses.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell dissociation. Male Sprague-Dawley rats (200–300 g) were made unconscious by exposure to a rising concentration of CO₂ and killed by exsanguination in accordance with Schedule 1 of the Animals (Scientific Procedure) Act, 1986. Single smooth muscle cells were dissociated as previously described (20). The brain was removed, and superior cerebral arteries were dissected in a salt solution containing (in mM): 137 NaCl, 0.44 NaH₂PO₄, 0.42 Na₂HPO₄, 4.17 NaHCO₃, 5.6 KCl, 1 MgCl₂, 2 CaCl₂, 10 HEPES, and 10 glucose (pH adjusted to 7.4 with NaOH). Single smooth muscle cells were dissociated by using a two-stage enzyme treatment. The arteries were first digested for 25 min at 35°C with 1.7 mg/ml papain and 0.7 mg/ml dithioerythritol in a low-Ca²⁺ solution containing (in mM): 134 NaCl, 5 KCl, 1 MgCl₂, 0.1 CaCl₂, 10 HEPES, 10 glucose, and 0.2 EDTA (pH adjusted to 7.3 at room temperature using NaOH). The arteries were then further digested for 20 min at 35°C with 1.7 mg/ml collagenase (type F) and 1 mg/ml hyaluronidase (type I-S) in the low-Ca²⁺ solution. The arteries were rinsed with the enzyme-free, low-Ca²⁺ solution, and single smooth muscle cells were obtained by triturating the arteries with a fire-polished Pasteur pipette. The cell suspension was kept in a refrigerator and used the same day.

Current recordings. A conventional whole cell clamp technique (15) was used to record whole cell membrane currents. This configuration also permitted a membrane-impermeant form of the Ca²⁺-sensitive dye fura-2 to be dialyzed into the cell. The composition of the extracellular solution was the following (in mM): 134 NaCl, 14 KCl, 1 MgCl₂, 3 CaCl₂, 10 HEPES, and 10 glucose (pH adjusted to 7.4 with NaOH). For most of the experiments, the pipette solution contained the following (in mM): 140 K aspartate, 15 NaCl, 10 HEPES, and 0.05 fura-2 pentapotassium salt (pH adjusted to 7.2 with KOH). This combination of extracellular and intracellular salt composition makes the equilibrium potentials for K⁺ and Cl^– to be around –60 mV. When Ca²⁺ transients were repeatedly triggered by caffeine application, the composition of the pipette solution was (in mM) 145 KCl, 3 MgCl₂, 3 Na₂ATP, 10 HEPES, and 0.05 fura-2 pentapotassium salt (pH adjusted to 7.2 with KOH). In most cases, the membrane potential was held at –60 mV during the experiments. Stock solutions of ryanodine and cADP-ribose were made using DSMO and water, respectively. The stock solutions were diluted with the intracellular solution by 1,000-fold and applied from the patch pipette. P₂ agonists and caffeine were dissolved in the extracellular solution and applied using a U tube rapid-superfusion system (8). Briefly, the extracellular solution containing the drug was perfused through the U tube with a small hole in its apex. The experimental chamber was continuously perfused with the agonist-free extracellular solution. The U tube was placed downstream of the cell. The outflow of the U tube was fed through a solenoid valve before being finally discarded. The valve was normally kept open. The closure of the valve using pCLAMP software (version 7, Axon Instruments) triggers a rapid ejection of the test solution from the hole, producing a drug concentration jump in the vicinity of the cell. The commencement of valve closure was marked by prior membrane hyperpolarization by 2 mV for 40 ms. The cell is exposed to the drug solution as long as the valve remains closed. The time of the valve closure and hence of the drug application was 1.5 s. When the valve is reopened, the test solution resumes running through the U tube to the waste while the drug is washed out from the chamber. Membrane currents were amplified using an Axopatch 200B (Axon Instruments), filtered at 1 kHz, and sampled at 10 kHz in the case of P_2X experiments and at 2 kHz in the case of P_2Y experiments.

Ca²⁺ measurements. [Ca²⁺]_i was measured using a deltaRAM (Photon Technology International) from voltage-clamped cells as previously described (22). A single cell was dialyzed with a fura-2-containing pipette solution and alternately illuminated for 10 ms with UV light at 340 and 380 nm (bandpass 8 nm). Emission signals were obtained at 510 nm (bandpass 40 nm). The dissociation constant for fura-2 under our experimental conditions was calculated as 180 nM from in vitro calibration. Minimum and maximum ratios (R_min and R_max, respectively) were also determined from in vitro measurements and reduced by 15% to adjust for viscosity (30). Background fluorescence was determined for each cell following formation of a gigaohm seal and subtracted from the measurements obtained during the experiments. Membrane voltage was also recorded by using one of the analog channels to mark valve closure and hence drug application. All experiments were carried out at room temperature.

Statistics. Where appropriate, results were shown as means ± SE of n cells. Significant difference was examined using either Student's unpaired t-test or one-way ANOVA (P < 0.05).

Drugs. Fura-2 pentapotassium salt was obtained from Molecular Probes, and papain was obtained from Worthington Biochemical. Ryanodine and cADP-ribose were purchased from Calbiochem. All other agents were purchased from Sigma or BDH.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Membrane current and Ca²⁺ transient evoked by P₂ agonists. P₂ receptors in arteries are activated by the neurotransmitter ATP that is coreleased from sympathetic neurons along with norepinephrine (24). Therefore, we first examined the effect of ATP on the membrane current and [Ca²⁺]_i in the superior cerebral artery smooth muscle cells. At constant membrane potential of –60 mV, rapid application of 10 µM ATP evoked an inward current (Fig. 1A, bottom) and Ca²⁺ transient (Fig. 1A, top). Figure 1B shows the same result with an expanded time scale. The inward current triggered by the application of ATP decayed during the continuous presence of the drug (Fig. 1B, bottom). The increase in [Ca²⁺]_i consisted of two phases, an initial small increase in [Ca²⁺]_i and a delayed, secondary elevation in [Ca²⁺]_i (Fig. 1B, top). ATP is an agonist for both ligand-gated, ionotropic P_2X receptors and metabotropic, G protein-coupled P_2Y receptors (5). Therefore, the initial [Ca²⁺]_i increase may be caused by the activation of P_2X receptors, whereas the subsequent [Ca²⁺]_i increase may be caused by the activation of P_2Y receptors. However, it is also possible that the secondary increase in [Ca²⁺]_i is caused by the loosely coupled Ca²⁺ release that is triggered by Ca²⁺ influx through P_2X receptors. Hence, we examined the effect of more specific agonists for P₂ receptors. First, we tested the effect of -methylene ATP (-MeATP) on the membrane current and Ca²⁺ transient. The desensitization, the decline of the current while the agonist was still present (see Fig. 1, bottom), strongly suggests the current is caused by the opening of P_2X1 receptors for which -MeATP is a selective agonist (26). Application of 30 µM -MeATP triggered an inward current (Fig. 2A, bottom) and an increase in [Ca²⁺]_i (Fig. 2A, top). Like the inward current evoked by ATP application, the current induced by -MeATP decayed before the termination of drug application (Fig. 2A, bottom). Unlike the Ca²⁺ transient triggered by ATP application, however, there was no secondary increase in [Ca²⁺]_i when -MeATP was the agonist (Fig. 2A, top). Rather, raised [Ca²⁺]_i declined while the drug was still present and more or less returned to the resting level (Fig. 2A, top). The secondary increase in [Ca²⁺]_i was never seen when -MeATP was the agonist, suggesting that the delayed increase in [Ca²⁺]_i seen with the application of ATP is not due to the P_2X current-induced, loosely coupled Ca²⁺ release. We then sought to identify the cause of the secondary [Ca²⁺]_i rise. Application of 100 µM UTP, a specific agonist for some P_2Y receptors, evoked little current (Fig. 2B, bottom) and a Ca²⁺ transient that was slow in onset (Fig. 2B, top). When 30 µM -MeATP and 100 µM UTP were applied together, we detected an inward current (Fig. 2C, bottom) and a Ca²⁺ transient consisting of an initial small [Ca²⁺]_i increase followed by a larger [Ca²⁺]_i increase (Fig. 2C, top). Thus, as reported previously in rat aortic myocytes (29), rat superior cerebral artery smooth muscle cells express both P_2X receptors and P_2Y receptors. The activation of the former presumably corresponds to the first part of the ATP response, whereas the activation of the latter corresponds to the second part of the ATP response.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Membrane current and Ca2+ transient evoked by application of 10 µM ATP. A: application of 10 µM ATP triggered inward current (I, bottom) and Ca2+ transient (top). Membrane potential was clamped at –60 mV (not shown). B: same result as in A, but shown with an expanded time scale. Application of ATP evoked inward current that decayed while the drug was still present (bottom). The inward current coincided with the first phase of the intracellular Ca2+ concentration ([Ca2+]i) increase that was followed by the delayed, larger [Ca2+]i increase (top).

View larger version (20K): [in this window] [in a new window]   Fig. 2. Membrane currents and Ca2+ transients evoked by application of P2 agonists. A: application of 30 µM -methylene ATP (-MeATP) triggered the inward current (I, bottom) and Ca2+ transient (top). Membrane potential was clamped at –60 mV (not shown). Inward current decayed while the drug was still present (bottom) and was accompanied by an increase in [Ca2+]i (top). There was no secondary increase in [Ca2+]i (top). B: application of 100 µM UTP triggered very little current (bottom). There was delay between the beginning of UTP application and the onset of the Ca2+ transient. Also, the initial increase in [Ca2+]i was sluggish, showing a "foot." C: simultaneous application of 30 µM -MeATP and 100 µM UTP triggered inward current (bottom) and Ca2+ transient consisting of initial small [Ca2+]i increase followed by a larger secondary [Ca2+]i increase (top).

Relationship between current and Ca²⁺ transient evoked by P_2X agonist. The observation that application of -MeATP does not produce the secondary increase in [Ca²⁺]_i (Fig. 2A, top) suggests that the P_2X current does not evoke loosely coupled, delayed Ca²⁺ release. However, it is still possible that Ca²⁺ influx through P_2X receptors trigger tightly coupled Ca²⁺ release. Therefore, we tested the hypothesis that the increase in [Ca²⁺]_i noted during activation of the inward current is partially supplied by Ca²⁺ release. To do this, we employed a method previously used to examine whether or not the L-type Ca²⁺ current triggered significant Ca²⁺ release in smooth muscle cells (18–20).

We first made the assumption that the inward current was carried exclusively by Ca²⁺. This assumption is incorrect because a part of the current will be carried by Na⁺. Nonetheless, this assumption allows us to quantitatively test the hypothesis that the activation of P_2X receptors induces significant Ca²⁺ release. By assuming that P_2X current is carried by Ca²⁺, we can calculate the expected increase in [Ca²⁺]_i by using the equation

(1)

where – I dt is the calculated charge entry obtained from time-integrated current, F is the Faraday constant, and V is the estimated cell volume (0.7 pl, 20). Because the holding potential is very close to the equilibrium potentials for K⁺ and Cl^–, we measured very little holding current (Fig. 3, top). Nevertheless, the inward current amplitude was corrected for the holding current measured before application of -MeATP. The result was expressed as a function of time (Fig. 3, bottom, dotted line). The actually measured increase in [Ca²⁺]_i was obtained by subtracting the resting [Ca²⁺]_i from the measured [Ca²⁺]_i and shown also in Fig. 3 (bottom, solid line). The time courses of the measured increase in [Ca²⁺]_i and expected increase in [Ca²⁺]_i were initially similar (0.2 s). However, as the inward current decayed and the expected increase in [Ca²⁺]_i reached a plateau, the measured increase in [Ca²⁺]_i started to decline (>0.2 s). The divergence of the time course of the measured increase in [Ca²⁺]_i from that of the expected increase in [Ca²⁺]_i may reflect changes in Ca²⁺ removal rates (20). We therefore restricted the analysis of the time-integrated inward current and increase in [Ca²⁺]_i to the first 200 ms of the Ca²⁺ transient. Over this period, the measured increase in [Ca²⁺]_i was plotted as a function of the expected increase in [Ca²⁺]_i (summarized in Fig. 4A). The relationship was fitted with a linear regression. As previously described (18), the reciprocal of the slope of this fit yields a Ca²⁺-buffering power, an indicator that describes how many Ca²⁺ have to enter the cell to provide an increase of one free Ca²⁺. The Ca²⁺-buffering power was obtained from each linear fit for each cell and calculated as 4,752 ± 459 for the control experiments (Fig. 5). Next, to examine whether the -MeATP-activated current triggered further Ca²⁺ release from the sarcoplasmic reticulum, similar experiments were performed in the presence of 30 µM ryanodine. Ryanodine is a standard inhibitor of the Ca²⁺-induced Ca²⁺ release. Ca²⁺-induced Ca²⁺ release conventionally refers to Ca²⁺ release that is triggered by L-type Ca²⁺ current and was first described in cardiac myocytes (9). However, its role in smooth muscle cells has been controversial (35). If the Ca²⁺-induced Ca²⁺ release contributes to [Ca²⁺]_i elevation evoked by the application of -MeATP, its inhibition by ryanodine should reduce the Ca²⁺ transient produced by a given I. It follows that the buffering power of the ryanodine-treated cells should be larger than that of the control cells if Ca²⁺-induced Ca²⁺ release is important (20). Figure 4B summarizes the results obtained in the presence of ryanodine. The Ca²⁺-buffering power of the ryanodine-treated cells was calculated as 4,947 ± 845 (Fig. 5). This value was not significantly different from that of control cells when examined using one-way ANOVA. Thus -MeATP-evoked current did not trigger significant Ca²⁺-induced Ca²⁺ release in rat superior cerebral artery smooth muscle cells.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Time course of expected and measured increase in [Ca2+]i evoked by P2X receptor activation. Application of 30 µM -MeATP triggered an inward current (top). The current was converted to the expected increase in [Ca2+]i using Eq. 1 in the text. The expected increase in [Ca2+]i is shown as a function of time (dotted line, bottom). Measured increase in [Ca2+]i was obtained by subtracting resting [Ca2+]i from the measured [Ca2+]i during the experiments (solid line, bottom). Up to 0.2 s, the time course of the measured increase in [Ca2+]i matched that of the expected increase in [Ca2+]i. However, the magnitude of the measured increase in [Ca2+]i (shown in nM, left ordinate, bottom) was 1/4,000th of that of the expected increase in [Ca2+]i (shown in µM, right ordinate, bottom).

View larger version (23K): [in this window] [in a new window]   Fig. 4. Relationship between expected and measured increase in [Ca2+]i in the presence and absence of the Ca2+-induced Ca2+ release antagonist ryanodine. Summarized relationship between the expected and measured increase in [Ca2+]i for control cells (n = 11, A) and 30 µM ryanodine-treated cells (n = 6, B). Data were obtained during the first 200 ms of the current evoked by the application of 30 µM -MeATP. During this period, the relationship was linear, and hence the Ca2+-buffering power was calculated as the reciprocal of the slope of the fit for each experiment.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Ca2+-buffering powers of the control, ryanodine-treated, and cADP-ribose (cADPR)-treated cells were 4,752 ± 459 (open bar, n = 11), 4,947 ± 845 (filled bar, n = 6), and 3,996 ± 303 (hatched bar, n = 5). One-way ANOVA detected no significant difference among these values.

The inability of the P_2X current to induce Ca²⁺ release in rat superior cerebral artery smooth muscle cells was interesting because significant Ca²⁺ release was previously detected in the same cell when the L-type Ca²⁺ current was the trigger (20). This led us to wonder whether or not it is possible to couple P_2X receptors to Ca²⁺-releasing channels by using cADP-ribose. It has been suggested that cADP-ribose is an endogenous activator of Ca²⁺-induced Ca²⁺ release (10, but also see DISCUSSION). It is conceivable that inclusion of cADP-ribose may facilitate Ca²⁺ release that is triggered by the P_2X current. If this is the case, the Ca²⁺-buffering power of the cADP-ribose-treated cells should be significantly smaller than that of the control cells. We chose a concentration of 1 µM that is higher than the maximum cADP-ribose level found in cells (42). The Ca²⁺-buffering power of cADP-ribose-treated cells was 3,996 ± 303, and one-way ANOVA did not detect significant difference (Fig. 5). Therefore, under our experimental conditions, cADP-ribose did not facilitate the Ca²⁺-induced Ca²⁺ release when the P_2X current was the trigger.

Voltage and [Ca²⁺]_i dependency of P_2Y receptor-mediated Ca²⁺ transient. The results described above suggest that the increase in [Ca²⁺]_i caused by P_2X current is not amplified by further Ca²⁺ release. Thus we investigated the other possibility that rapidly desensitizing P_2X current still impacts Ca²⁺ homeostasis by modulating subsequent P_2Y responses. Activation of G protein-coupled P_2Y receptors generates a second messenger, inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]. Ins(1,4,5)P₃, in turn, activates Ca²⁺-releasing channels in the sarco(endo) plasmic reticulum. There are two potential mechanisms by which P_2X current may influence Ins(1,4,5)P₃-mediated Ca²⁺ release. First, the P_2X receptor-mediated initial elevation in [Ca²⁺]_i facilitates the P_2Y responses by priming Ins(1,4,5)P₃ receptors. This mechanism has been suggested by Evans and co-workers in blood cells (40). Second, depolarization caused by the P_2X current may promote the P_2Y receptor-mediated Ca²⁺ transient. The hypothesis that depolarization enhances accumulation of Ins(1,4,5)P₃ has been suggested by Ganitkevich and Isenberg (11, 12) using acetylcholine as the stimulant. We investigated these possibilities by applying UTP either in combination with -MeATP or with/without membrane depolarization.

We first examined more closely the recordings obtained by simultaneously applying UTP and -MeATP. The result shown in Fig. 2C appears to support the hypothesis that the P_2X receptor-mediated elevation in [Ca²⁺]_i facilitates the subsequent P_2Y-induced response by priming Ins(1,4,5)P₃ receptors. However, this may not be always the case. The [Ca²⁺]_i dependency of the Ins(1,4,5)P₃-gated channels is bell shaped, the maximum open probability occurring around a [Ca²⁺]_i of 200 nM with sharp declines either side of this concentration (3). It follows that if the initial P_2X receptor-mediated elevation in [Ca²⁺]_i is sufficiently large, the subsequent P_2Y response may be suppressed, rather than promoted. Figure 6A shows an example where P_2X receptor-mediated [Ca²⁺]_i increase corresponds to the descending limb of Ins(1,4,5)P₃ [Ca²⁺]_i dependency. The secondary [Ca²⁺]_i increase was sluggish in this example, contrasting to the previous example where a robust secondary [Ca²⁺]_i increase was noted following a smaller initial [Ca²⁺]_i rise (Fig. 2C).

View larger version (23K): [in this window] [in a new window]   Fig. 6. Effect of [Ca2+]i increase and depolarization on UTP-induced Ca2+ transients. A: Ca2+ transient was evoked by simultaneous application of 30 µM -MeATP and 100 µM UTP (top). Membrane potential was held at –60 mV (bottom). P2X receptor stimulation by -MeATP evoked an inward current (not shown) and an initial increase in [Ca2+]i reaching 500 nM (top). After this higher [Ca2+]i, the subsequent increase in [Ca2+]i due to P2Y receptor stimulation appears sluggish (compare with Fig. 2C, top). B: effect of depolarization on P2Y receptor-mediated Ca2+ transient was examined. UTP was applied while the membrane potential was depolarized to +100 mV (bottom), evoking a fast [Ca2+]i rise (top).

We next examined the effect of the membrane potential on the P_2Y-mediated Ca²⁺ transient. Under our experimental conditions, the consequence of P_2X receptor activation was a transient increase in [Ca²⁺]_i. However, the P_2X current should also cause membrane depolarization in vivo (36). Membrane depolarization may not only activate L-type Ca²⁺ channels, but also facilitate the accumulation of Ins(1,4,5)P₃ (11). After the 40-ms hyperpolarization to –62 mV that precedes valve closure (see MATERIALS AND METHODS), the membrane potential was depolarized to +100 mV for 7.8 s. Depolarization to +100 mV did not, by itself, cause an increase in [Ca²⁺]_i (data not shown, but see Fig. 6B). Presumably, this was because a membrane potential of +100 mV was virtually the Ca²⁺ equilibrium potential (E_Ca) at which no net Ca²⁺ influx occurs. It was important to hold membrane potential close to E_Ca because the Ca²⁺ influx through the L-type Ca²⁺ channels that occurs negative to E_Ca may facilitate P_2Y-mediated Ca²⁺ responses. When combined with the depolarization to +100 mV, UTP application caused a robust increase in [Ca²⁺]_i (Fig. 6B). This contrasts with the previous example in which UTP was applied while the membrane potential was held at –60 mV (Fig. 2B). These results appear to support the hypothesis that membrane depolarization facilitates the P_2Y receptor-mediated [Ca²⁺]_i increase.

We sought to quantify the observations described above to compare P_2Y receptor-mediated responses under various conditions. One difficulty in examining the Ins(1,4,5)P₃-mediated Ca²⁺ transient is that the response depends on the amount of Ca²⁺ stored in the sarcoplasmic reticulum. The filling state of the sarcoplasmic reticulum greatly varies among cells and is rather difficult to control. We therefore used the rate of [Ca²⁺]_i increase, rather than the maximum amplitude of Ca²⁺ transient, to measure Ins(1,4,5)P₃ responses. The rate of [Ca²⁺]_i increase was obtained as the slope of the linear regression to the fast rising phase of the Ca²⁺ transient (12). Examples are shown in Fig. 7, A and B, the former obtained while the membrane potential was held at –60 mV and the latter at +100 mV. The dotted lines depict the linear fits. The mean rate of [Ca²⁺]_i rise without depolarization was 1,584 ± 369 nM/s (n = 6), whereas that with depolarization was 5,342 ± 954 nM/s (n = 5) (Fig. 7C). In agreement with previous results obtained using acetylcholine as the agonist (12), these values were significantly different (P < 0.05, Student's unpaired t-test). The rate of [Ca²⁺]_i increase was also used to quantify the results obtained by simultaneously applying UTP and -MeATP. In these experiments, membrane potential was held at –60 mV. The examples are presented in Fig. 8, A–C, where the P_2X receptor-mediated [Ca²⁺]_i increase was small, medium, and large, respectively. The linear fit is depicted with the dotted line in each recording. (Note that the initial fast [Ca²⁺]_i increase in Fig. 8C is caused by the P_2X current, not by the P_2Y receptor activation. During the recording that lasted over 40 s, secondary [Ca²⁺]_i increase was virtually absent, suggesting inhibition of P_2Y response by a large increase in [Ca²⁺]_i.) Figure 8D is a summary of 10 such results, with rates of [Ca²⁺]_i increase plotted as a function of highest [Ca²⁺]_i reached during P_2X receptor activation. The [Ca²⁺]_i dependency of the P_2Y receptor-mediated Ca²⁺ transient was bell-shaped, a result in agreement with previous observations (3). Thus our results suggest that both an increase in [Ca²⁺]_i and depolarization evoked by P_2X current may regulate the subsequent P_2Y responses in rat superior cerebral artery smooth muscle cells.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Rate of [Ca2+]i increase induced by application of UTP at a membrane potential of –60 or +100 mV. A: 100 µM UTP was applied while the membrane potential was held at –60 mV (not shown). The rate of [Ca2+]i increase was obtained as the slope of the linear regression, shown with the dotted line, to the fast rising phase of Ca2+ transient. B: 100 µM UTP was applied while the membrane potential was held at +100 mV (not shown). As in A, the rate of [Ca2+]i increase was obtained as the slope of the linear regression, shown with the dotted line. C: the mean rate of [Ca2+]i rise at the holding potential of –60 mV was 1,584 ± 369 nM/s (open bar, n = 6) while that of +100 mV was 5,342 ± 954 nM/s (filled bar, n = 5). These values were significantly different (*P < 0.05, Student's unpaired t-test).

View larger version (22K): [in this window] [in a new window]   Fig. 8. Effect of increase in [Ca2+]i evoked by application of 30 µM -MeATP on 100 µM UTP-induced [Ca2+]i transient. A: P2X current (not shown) induced a small increase in [Ca2+]i. The rate of [Ca2+]i increase was obtained as the slope of the linear regression, shown with the dotted line, to the rising phase of UTP-induced Ca2+ transient. Membrane potential was held at –60 mV (not shown). B: P2X current (not shown) induced a medium increase in [Ca2+]i. As in A, the rate of [Ca2+]i increase was obtained as the slope of the linear fit (dotted line). Membrane potential was held at –60 mV (not shown). C: P2X current (not shown) induced a large increase in [Ca2+]i. As in A, the rate of [Ca2+]i increase was obtained as the slope of the linear fit (dotted line). Membrane potential was held at –60 mV (not shown). Note that the initial fast [Ca2+]i increase is caused by the P2X current, not by the P2Y receptor activation. During the recording that lasted over 40 s, secondary [Ca2+]i increase was virtually absent, suggesting inhibition of P2Y response by a large increase in [Ca2+]i. D: summary of relationship between [Ca2+]i achieved by P2X receptor activation and rate of [Ca2+]i increase evoked by P2Y receptor activation. The relationship was bell shaped with the peak of [Ca2+]i rise occurring around [Ca2+]i of 200 nM. The dotted line is the fourth-order polynomial fit to the data obtained from 10 cells.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
An important objective in this study was to investigate whether or not Ca²⁺ influx through P_2X receptors triggers further Ca²⁺ release from the sarcoplasmic reticulum. We used rat superior cerebral artery smooth muscle cells to address this question, because Ca²⁺ homeostasis including the Ca²⁺-buffering power is well characterized in these cells (20, 21). Previously, significant Ca²⁺ release was noted during the activation of the L-type Ca²⁺ current in these cells (20). However, in the current study, Ca²⁺ influx through P_2X receptors did not trigger further Ca²⁺ release. The Ca²⁺-buffering power was not significantly different between control cells and ryanodine-treated cells (Fig. 5). At a concentration of 30 µM, application of ryanodine may make the sarcoplasmic reticulum leaky by locking ryanodine-sensitive, Ca²⁺-releasing channels into a subconductance state (33). Theoretically, the inhibition of Ca²⁺ release by ryanodine could be cancelled out by the blockade of Ca²⁺ uptake to the sarcoplasmic reticulum. This would provide a false negative result for P_2X current-induced Ca²⁺ release. However, this scenario seems unlikely. Investigation of Ca²⁺ removal profiles in rat cerebral artery smooth muscle cells showed that the activation of Ca²⁺ pumps in the sarcoplasmic reticulum is a time-dependent process that takes more than 10 s to develop (21). This delayed activation of sarcoplasmic reticulum Ca²⁺ uptake may be due to a signal transduction cascade involving Ca²⁺-activated enzymes (18, 21). In the current study, the analysis of Ca²⁺-buffering power was restricted to the first 200 ms of the Ca²⁺ transient during which Ca²⁺ uptake to the sarcoplasmic reticulum would be of little importance. Thus it seems unlikely that the presence of the P_2X current-mediated Ca²⁺ release was overlooked due to a leaky sarcoplasmic reticulum. Our observation is consistent with the finding that the P_2X-mediated mesenteric artery contraction was unaffected by application of cyclopiazonic acid, a functional antagonist of Ca²⁺-induced Ca²⁺ release (13). Similarly, the field stimulation that evoked P_2X receptor-mediated Ca²⁺ transients neither triggers Ca²⁺ waves in rat mesenteric arteries (25) nor releases Ca²⁺ in the mouse vas deferens (4). On the other hand, in rat portal vein myocytes, the P_2X current triggered Ca²⁺ release by activating ryanodine receptors, possibly ryanodine receptor subtype 2 (27). Such discrepancies may be due to differences among preparations.

In a previous study, Ca²⁺ release that was triggered by a given whole cell L-type Ca²⁺ current was larger when the depolarization was smaller (20). As suggested in cardiac cells (43), this observation may be explained with the hypothesis that ryanodine-sensitive, Ca²⁺-releasing channels are colocalized with voltage-dependent Ca²⁺ channels (see Ref. 6) and can sense the size of the unitary Ca²⁺ current. The single Ca²⁺ channel size will be larger when depolarization is smaller (20). The inability of the P_2X receptor current to trigger Ca²⁺ release, therefore, may be because Ca²⁺ influx through P_2X receptors is insufficient for the Ca²⁺-induced Ca²⁺ release. Therefore, we sought to estimate the size of the fractional unitary P_2X current carried by Ca²⁺. When the L-type Ca²⁺ current was used to raise [Ca²⁺]_i, the Ca²⁺-buffering power of ryanodine-treated cells was 250 (20). Voltage-dependent Ca²⁺ channels are highly selective to Ca²⁺. The Ca²⁺-buffering power of the current study where [Ca²⁺]_i was raised by the P_2X current was 4,800. This value was obtained assuming that all P_2X currents were carried exclusively by Ca²⁺. Comparison of Ca²⁺-buffering power for L-type Ca²⁺ channels and P_2X receptors allows us to calculate what proportion of the P_2X current is carried by Ca²⁺. We estimate 5% of the P_2X current is carried by Ca²⁺. This value is very similar to values previously reported (10%, 1; 6%, 2; 6.5%, 32; 67%, 36). Assuming the P_2X unitary current is 0.5 pA at –60 mV (39), we estimate the fractional unitary current carried by Ca²⁺ to be 25 fA. On the other hand, the unitary L-type Ca²⁺ current will be 0.1 pA at 0 mV (with physiological Ca²⁺ as the charge carrier, see Ref. 34). Therefore, it is possible that the fractional unitary P_2X current carried by Ca²⁺ may be too small to trigger Ca²⁺ release from the sarcoplasmic reticulum.

The observation that the P_2X current did not trigger Ca²⁺ release while the L-type Ca²⁺ current did in the same preparation was intriguing. Use of cADP-ribose was an attempt to facilitate the coupling of Ca²⁺ influx through P_2X receptors to the Ca²⁺ release. Since the first demonstration using sea urchin eggs (10), various studies examined the role of cADP-ribose as an endogenous modulator of cardiac ryanodine receptors. To date, evidence for and against the hypothesis that cADP-ribose promotes Ca²⁺-induced Ca²⁺ release in cardiac cells has been provided (14, 16, 37). The discrepancy among these studies may reflect differences in experimental conditions. In the present study, the inclusion of 1 µM cADP-ribose in the intracellular solution had no significant effect on the Ca²⁺-buffering power (Fig. 5). Thus significant Ca²⁺ release was not induced by the activation of P_2X current in the presence of cADP-ribose under our experimental conditions.

We also investigated the hypothesis that the role of P_2X receptors is to prime the subsequent P_2X receptor-mediated Ca²⁺ release (40). Though this hypothesis seems indeed true in some cells (Fig. 2C), higher [Ca²⁺]_i achieved in other cells during P_2X activation was followed by a slow rise in [Ca²⁺]_i (Fig. 6A). P_2Y receptor stimulation presumably activates phospholipase C via Gq proteins (7). This, in turn, would lead to the production of diacylglycerol and Ins(1,4,5)P₃. Of these, Ins(1,4,5)P₃ seems more likely to be responsible for the P_2Y receptor-mediated Ca²⁺ transient because the onset of the Ca²⁺ response seems too quick to be caused by a signal transduction cascade involving diacylglycerol (23). The time delay seen in the response to UTP (e.g., Fig. 2B) presumably reflects the time required by phospholipase C to cleave phosphatidylinositol 4,5-diphosphate to generate Ins(1,4,5)P₃ (12). Like ryanodine-sensitive, Ca²⁺-releasing channels, Ins(1,4,5)P₃-gated channels in the sarco(endo)plasmic reticulum are Ca²⁺ sensitive. [Ca²⁺]_i dependency of the Ins(1,4,5)P₃-gated channels is bell shaped (3), and this may be useful in providing positive feedback as well as negative feedback to the P_2Y receptor-mediated Ca²⁺ release. Indeed, when the rate of [Ca²⁺]_i increase was used to gauge the effect of [Ca²⁺]_i elevation evoked by the P_2X current, the relationship was bell shaped (Fig. 8D). Thus a small to medium-sized [Ca²⁺]_i increase caused by the P_2X current may promote the subsequent P_2Y receptor-mediated Ca²⁺ transient to ensure sufficient increase in [Ca²⁺]_i, whereas a large [Ca²⁺]_i elevation induced by the P_2X receptor activation may suppress the P_2Y response to avoid [Ca²⁺]_i reaching a dangerously high level.

Activation of P_2X receptors not only results in transient elevation in [Ca²⁺]_i but also depolarization (36). Under our experimental condition, depolarization to +100 mV did not cause an increase in [Ca²⁺]_i by itself but seemed to facilitate the P_2Y receptor-mediated Ca²⁺ transient (Fig. 6B). Indeed, the rate of [Ca²⁺]_i increase was significantly different between cells clamped at –60 and +100 mV (Fig. 7). This result suggests that there is a voltage-dependent component in the UTP-induced increase in [Ca²⁺]_i. This effect may be a result of direct coupling of plasma membrane to the sarcoplasmic reticulum, as shown in the skeletal muscle cells (38). Alternatively, the membrane depolarization may indirectly modulate Ca²⁺ release from the sarcoplasmic reticulum (11, 12). To distinguish between these possibilities, we tested the voltage dependence of the caffeine-induced Ca²⁺ transient. Caffeine enhances Ca²⁺ sensitivity of the ryanodine receptors in the sarcoplasmic reticulum so that Ca²⁺ release occurs at the resting [Ca²⁺]_i. The caffeine-induced Ca²⁺ transient does not require generation of second messengers. Therefore, if skeletal muscle-type direct coupling occurs, caffeine-induced Ca²⁺ transients should also be modulated by depolarization. Application of 20 mM caffeine was repeated in the same cell to examine the effect of membrane potential. Figure 9 shows one such example where the first and the third caffeine-induced Ca²⁺ transients were obtained at the holding potential of –60 mV, whereas the second caffeine-induced Ca²⁺ transient was obtained with depolarization to +100 mV (see also Ref. 11). In this cell, Ca²⁺ transients triggered by caffeine application were not altered by depolarization. Similar results were obtained on two other occasions suggesting that the caffeine-induced Ca²⁺ transient is voltage independent. Thus the sarcoplasmic reticulum seems not directly coupled with the cell membrane. Rather, as suggested in guinea pig coronary myocytes (11, 12), the facilitation of UTP-induced Ca²⁺ transient by depolarization may be due to enhanced accumulation of Ins(1,4,5)P₃ (see also Ref. 17). Under our experimental conditions, a 160-mV depolarization (from –60 mV to +100 mV) facilitated the mean rate of [Ca²⁺]_i increase by a factor of 3.4 (see also Ref. 12). Obviously, the magnitude of depolarization used in this study (160 mV) will never occur under physiological conditions. Nevertheless, this potential was chosen to ensure that there is no L-type Ca²⁺ current that may prime the P_2Y receptor-mediated responses. Interestingly, Itoh et al. (17) have suggested that Ins(1,4,5)P₃ production is inhibited by membrane hyperpolarization in the rabbit mesenteric artery. If this is the case in our preparation, the role of membrane depolarization caused by the P_2X current may be to relieve tonic inhibition of Ins(1,4,5)P₃ production.

View larger version (19K): [in this window] [in a new window]   Fig. 9. Depolarization has little effect on caffeine-induced Ca2+ transient. Ca2+ transients were evoked by activating ryanodine-sensitive, Ca2+-releasing channels in the sarcoplasmic reticulum. Caffeine (20 mM) was repeatedly applied. The first and the third caffeine application were conducted while the membrane potential was held at –60 mV, whereas the second caffeine application was carried out with depolarization to +100 mV (bottom). The Ca2+ transients with and without depolarization were virtually indistinguishable (top), suggesting that the sarcoplasmic reticulum Ca2+ release is not directly influenced by the plasma membrane potential.

The present study showed positive responses of rat superior cerebral artery smooth muscle cells to both P_2X and P_2Y agonists. Pharmacology of agonists permits further insight to the receptor subtypes. -MeATP is a selective agonist for receptors containing P_2X1 or P_2X3 subunits (28). During the continued presence of 30 µM -MeATP, membrane current rapidly decayed, showing profound receptor desensitization (Fig. 2, A and C). At this concentration of -MeATP, was determined as 76.1 ± 5.4 ms (n = 20, see also Ref. 26). Moreover, under our experimental conditions, recovery from desensitization was virtually impossible, and therefore a second application of -MeATP triggered only a very small current and Ca²⁺ transient (data not shown). Taken together, these results show that P_2X receptors of rat superior cerebral artery smooth muscle cells seem to bear the characteristics of P_2X1 receptors (28). UTP, on the other hand, is a selective agonist for P_2Y2,P_2Y4, and P_2Y6 receptors. It is unlikely that the delayed increase in [Ca²⁺]_i is caused by the activation of P_2Y6 receptors, to which ATP is ineffective. Therefore, P_2Y receptors present in rat superior cerebral artery smooth muscle cells are likely to be P_2Y2 or P_2Y4 subtypes.

In summary, we have described P₂ responses of rat superior cerebral artery smooth muscle cells. Under our experimental conditions, the increase in [Ca²⁺]_i caused by the activation of P_2X receptors was not amplified by further Ca²⁺ release. Application of cADP-ribose did not facilitate coupling of the P_2X current and Ca²⁺ release. Furthermore, our results suggest that the membrane depolarization and [Ca²⁺]_i increase induced by the P_2X current may regulate subsequent P_2Y responses. Results described here may shed light on the Ca²⁺ homeostasis of vascular smooth muscle cells during P₂ stimulation.

	ACKNOWLEDGMENTS

 
We thank Dr. Richard Evans (Dept. of Cell Physiology & Pharmacology, University of Leicester) for help with the U tube superfusion system.

GRANTS

This work was supported by the British Heart Foundation (FS/2000001) and the Wellcome Trust (055506). T. Kamishima is a British Heart Foundation Intermediate Fellow.

	FOOTNOTES

 

Address for reprint requests and other correspondence: T. Kamishima, Dept. of Human Anatomy and Cell Biology, The Sherrington Bldg. Univ. of Liverpool, Ashton St., Liverpool L69 3GE, UK (E-Mail: kamishi{at}liv.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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