Analysis of Ca2+ and Sr2+ activation characteristics in skinned muscle fibre preparations with different proportions of myofibrillar isoforms

Summary To understand how the coexistence of fast and slow contractile and regulatory systems within single skeletal muscle fibres might affect contractile behaviour, fibre segments from the fast-twitch extensor digitorum longus and predominantly slow-twitch soleus muscle of the adult rat were tied together, either in parallel or in series, and then activated in Ca²⁺-and Sr²⁺-buffered solutions. Experimental force-pCa and force-pSr relations were compared with theoretical force-pCa and force_pSr curves predicted by a model for composite fibres, which accounted for the coexistence of fast and slow myosin within the contractile unit and enabled an estimate to be made of the relative contribution of fast- and slow-twitch elements within the tied-fibre combinations. The contractile behaviour of a fast-twitch and a slow-twitch muscle fibre tied either in series or in parallel, were compared with the force-pCa and force-pSr data predicted from the composite fibre model. Interestingly, the resultant force-pCa(-pSr) curves of the parallel-tied fibre combinations were well fitted with those predicted by the composite model. However, the experimental force-pCa(-pSr) curves of the series-tied fibres were not well fitted by a composite curve based on the known proportion of fast- and slow-twitch fibre components. A total force-length diagram was devised to take into account changes in the length of the fibre segments tied in series during activation, as well as possible differences in fibre diameter. Using this diagram it was possible to explain accurately the Ca²⁺ and Sr²⁺ activation curves of known fast- and slow-twitch segments tied in series. The results from this study are important for the interpretation of contractile date obtained from single muscle fibres exhibiting mixed fast- and slow-twitch contractile characteristics. Such muscle fibres have previously been identified in animals affected by muscular diseases (e.g. dystrophy), in mammalian extraocular muscles and in animals subjected to long-term exercise training.     

Stretch-induced increase in the Ca2+ sensitivity of myofibrillar ATPase activity in skinned fibres from pig ventricles

The increase in force development in the heart with increase in end-diastolic pressure (Frank-Starling mechanism) has been ascribed to an increase in contractile responsiveness of the myofibrils to calcium. We now show that this calcium sensitization is also associated with an increase in calcium responsiveness of the myofibrillar ATPase. Thus, at submaximal Ca activation (pCa 6.0), the ATPase activity of skinned fibres from pig right ventricles is increased from 57.9±4.4% to 70.6±4.4% of the maximal Ca²⁺ activation of ATPase by stretching (by 15%l _o). At maximal Ca²⁺ activation, ATPase was barely altered by stretching. The relationship between ATPase activity of skinned trabecula of pig right ventricle and ATPase-Ca²⁺ concentrations is shifted (by 0.1 pCa unit) to higher pCa values after a stretch-induced increase of the sarcomere length from 2.1 μm to 2.4 μm. The relationship between force and pCa was affected in a similar way by extension. This increased calcium sensitivity is, however, not associated with an alteration in the relationship between ATPase activity and force development (tension cost). In accordance with Brenner's hypothesis, we propose therefore that stretch activation of ATPase is associated with an increase in the apparent rate constant of crossbridge attachment rather than with a decrease in the apparent rate constant of crossbridge detachment.     

Associated changes in Ca2+ and Sr2+ activation properties and fiber proteins in cross-reinnervated rabbit soleus

Changes in the Ca²⁺ and Sr²⁺ activation properties of functionally skinned slow-twitch soleus fibers were measured and compared with those of normal fast-twitch extensor digitorum longus (EDL) following cross-reinnervation of soleus with the nerve to EDL. Most of the fibers showed either complete transformation of activation properties (66%) or remained unchanged (34%). The change in sensitivity to divalent cations was correlated with changes in the proteins present in fibers pooled on the basis of their activation properties. The banding patterns of the 35,000- and 37,500-dalton proteins (tropomyosin and troponin T) in cross-reinnervated soleus were correspondingly transformed to those of normal EDL. Slow and fast myosin light chains were present in the pooled cross-reinnervated fibers. Fiber distributions based on activation properties were confirmed by histochemical features. For the first time it has been demonstrated that cross-reinnervation produced changes in the activation properties of soleus fibers and associated changes in the regulatory proteins measured.     

Probing a Ca2+-activated K+ channel with quaternary ammonium ions

A series of quaternary amonium (QA) ions were used to probe the gross architecture of the ion conduction pathway in a Ca²⁺-activated K⁺ channel from rat muscle membrane. The channels were inserted into planar phospholipid membranes and the single channel currents were measured in the presence of the different QA ions. Internally applied monovalent QA ions (e.g. tetramethylammonium and analogues) induced a voltage-dependent blockade with a unique effective valence of the block equal to 0.30, and blocking potency increases as the compound is made more hydrophobic. Blockade is relieved by increasing the K⁺ concentration of the internal or external side of the channel. The effective valence of block is independent of K⁺ concentration. These results suggest that, from the internal side, all monovalent QA ions interact with a site located in the channel conduction system. Divalent QA ions of the type n-alkylbis-,-trimethylammonium (bisQn) applied internally also block the channel in a voltage dependent fashion. For short chains (bisQ2-bisQ5), the effective valence decreases with chain length from 0.41 to 0.27, it remains constant for bisQ5 to bisQ6 and increases up to 0.54 for bisQ10. This dependence of block with chain length implies that 27% of the voltage drop within the channel occurs over a distance of 1 nm. Externally applied monovalent QA ions also block the channel. The site is specific for tetraethylammonium; increasing or decreasing the side chains in one methylene group decrease potency by about 400-fold. It is concluded that the Ca²⁺-activated K⁺ channel has wide mouths located at each end and that they are different in molecular nature.     

兔膈肌肌浆网Ca2+-ATPase活性及钙离子的转运

本文分别用定磷法测定兔膈肌SR Ca 2 -APTase 活性、Fura-2 荧光法测定SR Ca 2 释放、摄取动力学和[ 3 H] -Ryanodine 与RyR 结合实验测定SR RyR 的量,分析其功能特性。 结果显示兔隔肌、心肌和骨骼肌SR Ca 2 -APTase 活性分别为70.13 ±8.25、 41.25 ±6.25 和120.17± 17.03 m mol/L pi/mg 蛋白/h1 。膈肌的SRCa 2 -APTase 活性显著高于心肌P<0.01 。但明显低于骨骼肌P<0.01; 膈肌SR Ca 2 释放量和摄取速度显著快于心肌（P<0.01）,但明显低于骨骼肌（P<0.01）;膈肌SR RyR 同[ 3 H] Ryanodine 的最大结合值（Bmax）是0.78 ±0.05pmol/mg 蛋白,其解离常数（KD）是6.93 1.13nmol/L,分别位于心肌和骨骼肌范围内。本文认为膈肌Ca 2 释放单位、SR Ca 2 -APTase 和SR Ca 2 释放摄取动力学分别具有心肌和骨骼肌的一些特征,其Ca 2 释放可能具有变构偶联和CICR 偶联两种形式,心肌型DHPR 亚型,RyR 3 和SERCA 2 a 的存在可能是膈肌ECC 依赖于细胞外Ca 2 的主要原因。     

Glutamate receptor activation in cultured cerebellar granule cells increases cytosolic free Ca2+ by mobilization of cellular Ca2+ and activation of Ca2+ influx

Summary The Ca²⁺ sensitive fluorescent probe, fura-2 has been used to monitor cytosolic free calcium levels in mature primary cultures of cerebellar granule cells during exposure to L-glutamate and other excitatory amino acids: quisqualate (QA), kainate (KA) and N-methyl-D-aspartate (NMDA). Glutamate at micromolar concentrations produced a prompt and dose-related increase in the intracellular concentration of free Ca²⁺, ([Ca²⁺]_i), whereas QA, KA and NMDA had no effect. This increase was also seen in the absence of extracellular Ca²⁺, suggesting that L-glutamate promotes mobilization of Ca²⁺ from intracellular stores. In the presence of extracellular calcium, the elevation of [Ca²⁺]_i was, in part, mediated by an increase in the plasma membrane permeability to Ca²⁺. This Ca²⁺ influx was not affected by the Ca²⁺-channel antagonist 1-Verapamil. However, L-Verapamil did block the increase in [Ca²⁺]_i seen after depolarization of the cells with potassium. The Ca²⁺ response elicited by glutamate was partially blocked by the excitatory amino acid antagonist glutamate diethyl ester (GDEE). Furthermore, glutamate stimulated the formation of inositol mono-, bis-, tris and tetrakisphosphates (IP1, IP2, IP3, and IP4) suggesting a role for these compounds for the increase in [Ca²⁺]_i.     

Effect of diadenosine polyphosphates on Ca2+ ATPase activity

 Diadenosine tri-, tetra-, penta-, and hexaphosphate (Ap₃A, Ap₄A, Ap₅A and Ap₆A) have been described as having various effects on vascular tone depending on the number of phosphate groups. This study examined the effect of diadenosine polyphosphates on Ca²⁺ ATPase activity. The activity of the enzyme was measured spectrophotometrically as the difference in hydrolysis of ATP in the presence and absence of Ca²⁺ with various concentrations of ATP and diadenosine polyphosphates. The diadenosine polyphosphates increased the activity of the Ca²⁺ ATPase. The effect tended to be stronger with Ap₅A and Ap₆A than with Ap₃A and Ap₄A in the order of potency: Ap₃A≈AP₄A<Ap₅A≈AP₆A. The stimulatory effect of diadenosine polyphosphates was not competitive with that of ATP, suggesting an allosteric activation of Ca²⁺ ATPase by diadenosine polyphosphates. This effect may be physiologically relevant for limiting the increase in cytosolic free Ca²⁺ concentration elicited by diadenosine polyphosphates by receptor activation and modulating Ca²⁺ ATPase function under resting conditions. Received: 24 January 1997 / Accepted: 9 May 1997     

Ryanodine- and thapsigargin-insensitive Ca2+-induced Ca2+ release is primed by lowering external Ca2+ in rabbit autonomic neurons

Rises in cytosolic Ca2+ induced by a high K+ concentration (30 or 60 mM) (K+-induced Ca2+ transient) were recorded by fluorimetry of Ca2+ indicators in cultured rabbit otic ganglion cells. When external Ca2+ ([Ca2+]o) was reduced to a micromolar (10-40 microM) or nanomolar (<10 nM) level prior to high-K+ treatment, K+-induced Ca2+ transients of considerable amplitude (50% of control) were generated in most cells, although those initiated at normal [Ca2+]o were reduced markedly or abolished by reducing [Ca2+]o during exposure to a high K+ concentration. Lowering [Ca2+]o alone occasionally caused a transient rise in cytosolic Ca2+. K+-induced Ca2+ transients at micromolar [Ca2+]o were repeatedly generated and propagated inwardly at a speed slower than that at normal [Ca2+]o, while those at nanomolar [Ca2+]o occurred only once. K+-induced Ca2+ transients at micromolar [Ca2+]o were not blocked by ryanodine (10 microM), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP, 5 microM: at 20-22 degrees C but blocked at 31-34 degrees C) or thapsigargin (1-2 microM), but were blocked by Ni2+ (1 mM) or nicardipine (10 microM). Thus, there is a ryanodine-insensitive Ca2+-release mechanism in FCCP- and thapsigargin-insensitive Ca2+ stores in rabbit otic ganglion cells, which is primed by lowering [Ca2+]o and then activated by depolarization-induced Ca2+ entry. This Ca2+-induced Ca2+ release may operate when [Ca2+]o is decreased by intense neuronal activity.     

Estimation of myofibrillar responsiveness to Ca2+ in isolated rat ventricular myocytes

 To estimate myofibrillar responsiveness to Ca²⁺, we used the relation between cell length and intracellular [Ca²⁺] ([Ca²⁺]_i) during tetanic contractions of isolated ventricular myocytes. Enzymatically isolated rat ventricular myocytes were loaded with fura-2 AM (4 μM for 10 min) and excited alternately at 340 nm and 380 nm. The ratio (R) of fura-2 fluorescence at these wavelengths [F(340)/F(380), an index of [Ca²⁺]_i] and cell length (L) were measured simultaneously. Following treatment with thapsigargin (0.2 μM), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction every min and an instantaneous plot of R vs L (R-L trajectory) was constructed. The R-L trajectory followed the same path during cell shortening and re-lengthening, suggesting that dynamic equilibrium between R and L was achieved during tetanus. Increasing the extracellular [Ca²⁺] from 1 to 8 mM extended the R-L trajectory without a substantial shift of the relation. The Ca²⁺-sensitizing thiadiazinone derivative, EMD57033 (1 μM), shifted the R-L trajectory to the left (sensitization of the myofibrils to Ca²⁺), whereas the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxantine (200 μM), shifted the R-L trajectory to the right (desensitization of the myofibrils to Ca²⁺), in agreement with previous results obtained using skinned preparations. We conclude that the R-L trajectory is useful for estimating the myofibrillar responsiveness to Ca²⁺ in isolated myocytes and may be beneficial for the evaluation of inotropic agents. Received: 13 March 1998 / Received after revision: 4 May 1998 / Accepted: 2 June 1998     

Myofibrillar ATPase activity and mechanical performance of skinned fibres from rabbit psoas muscle.

1. The relationship between energy turnover and mechanical performance was investigated in chemically skinned single fibres from rabbit psoas muscle at 15 degrees C, pH = 7.1, with MgATP, 5 mM; free Mg2+, 1 mM; ionic strength, 200 mM and sarcomere length, 2.4 microns by measuring force production and myofibrillar ATP turnover during isometric contractions as well as during repetitive changes in length. ATP hydrolysis was stoichiometrically coupled to the breakdown of NADH, which was measured photometrically via the absorption of near UV light at 340 nm. 2. Force and ATPase activity were measured during square-wave length changes of different amplitudes (1-10% of the fibre length, Lo) and different frequencies (2.5-167 Hz). The average force during the length changes was less than the isometric value and decreased with increasing amplitude and frequency. At full activation (pCa 4.5), the isometric ATP turnover rate (+/- S.E.M.) was 2.30 +/- 0.05 s-1 per myosin head. ATP turnover increased monotonically with increasing amplitude as well as with increasing frequency until saturation was reached. The greatest increase observed was 2.4 times the isometric value. 3. Force and ATPase activity were also determined for ramp shortenings followed by fast restretches. The average force decreased with increasing shortening velocity in a hyperbolic fashion. The ATP turnover increased with ramp velocity up to 0.5 L0 s-1 and stayed almost constant (at 2.2 times the isometric value) for larger velocities. 4. Isometric force and ATPase activity both decreased as the calcium concentration was decreased. They did not vary in proportion at low Ca2+ concentrations, but this could largely be accounted for by the presence of a residual, Ca(2+)-dependent, membrane-bound ATPase. At high calcium concentrations ATPase activity during square-wave length changes was higher than the isometric value, but at low calcium concentrations (pCa > 6.1), the ATPase activity during the length changes decreased below the isometric value and reached a minimum of 40% of the isometric level. 5. ATPase activity and average force obtained during changes in length show a high, movement protocol-independent correlation. During the length changes the rate of ATP turnover divided by the average force level (tension cost) was larger than the isometric tension cost. The largest value found, for 10% length changes at 23 Hz, was 17 times the tension cost under isometric conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of calcium ions in the activation of rabbit psoas muscle

Summary This study tested the effects of free Ca⁺⁺ on both the small-amplitude mechanical behaviour (dynamic stiffness and phase between 1 and 500 Hz) and the large-scale filament-sliding behaviour (V _max) of single fibres of chemically-activated glycerol-extracted rabbit psoas muscle. Small-amplitude vibrations (0.1% peak-to-peak of initial lengthL _o) were used to elicitf _w, the frequency for maximum oscillatory work-production per cycle. The unloaded contraction velocityV _max was measured during the same contractions, using the slack-test method with shortening length steps of up to 10%L _o. These produced nonlinear length-time plots demonstrating that the unloaded contraction velocity was not constant as contraction progressed, but fell with time. This behaviour was approximated by two velocities,V ₁ the velocity observed for about the first 15 ms andV ₂ the velocity after this time.V ₁ andV ₂ were found to have different sensitivities to Ca⁺⁺. The value ofV ₂ fell as the level of [Ca⁺⁺] was reduced, and was linearly proportional to the active tension over the range 0.2P _max toP _max (whereP _max is the isometric tension for saturating amounts of Ca⁺⁺). In contrast to thisV ₁ remained insensitive to changes in [Ca⁺⁺] for levels of activation corresponding to active tensions ranging fromP _max to 0.6P _max, and then fell as the level of activation was further reduced. It was found that the level of [Ca⁺⁺] did not affect the magnitude off _w over the range of concentrations yielding active tensions from 0.2P _max toP _max. These results are discussed in terms of the kinetic processes underlying transient readjustments to perturbations from isometric equilibrium.     

The effects of caffeine and Ca2+ on rigor tension in triton-treated rat ventricular trabeculae

Ventricular trabeculae from rat heart were chemically skinned with Triton-X100, which disrupts all cellular membranes including the sarcoplasmic reticulum. Trabeculae developed a maintained rigor contracture when adenosine triphosphate was withdrawn from the bathing medium. In all preparations, the final level of rigor force developed in the presence of caffeine (10–40 mM) was greater than under control conditions. However, caffeine failed to increase rigor tension when applied after contracture had fully developed. The effect of caffeine on rigor was maximal at about 15 mM; concentrations greater or less than 15 mM were less effective. On average, caffeine decreased the time required to develop half-maximum rigor force. The caffeine-induced potentiation of rigor force occurred in the effective absence of Ca²⁺ (10^–9 M), in solutions strongly Ca²⁺-buffered with [ethylenebis(oxonitrilo)]tetraaceticacid (10–50 mM). In all preparations, rigor force was found to be independent of [Ca²⁺] over the range 10^–10 M to about 10^–7 M. These results suggest that caffeine affects rigor force by a direct effect on the myofilaments via a mechanism that is independent of Ca²⁺.     

Effect of free sarcoplasmic Ca2+ concentration on maximum unloaded shortening velocity: measurements on single glycerinated rabbit psoas muscle fibres

Summary Isotonic shortening velocity in tension-controlled quick releases was studied at different sarcomere lengths, loads and Ca²⁺ concentrations using single glycerinated rabbit psoas muscle fibres. During a single quick release shortening velocity decreases with increasing shortening. Length-velocity relations, that is, momentary shortening velocity as a function of the corresponding sarcomere length, of a single quick release yield straight lines when graphed on a logarithmic scale. This indicates an exponential decrease of the isotonic shortening velocity during a quick release. A given shortening affects the subsequent part of the length-velocity relation in a manner which can be simulated by loading the fibre. The decrease in shortening velocity during a quick release could therefore be interpreted as the result of a shortening-induced increase in contractile system load. Thetotal amount of this additional load is proportional to the amount of shortening but is independent of the Ca²⁺ concentration. It follows that the change in loadper crossbridge due to a given shortening l, is inversely proportional to the degree of activation of the contractile system, provided that the number of cycling crossbridges during isotonic shortening is dependent on sarcoplasmic Ca²⁺ concentration as is known for the isometric state. These shortening-induced changes in crossbridge load should be considered in interpreting Ca²⁺ effects on shortening velocity. One approach may be extrapolation of the length-velocity relation to the starting length of the respective quick release, that is, the point at which all length-velocity relations of different Ca²⁺ concentrations converge. Thus, when an increase in crossbridge load due to shortening is taken into account, an effect of the free Ca²⁺ concentration on maximum unloaded shortening velocity cannot be proved.     

The contribution of the sarcoplasmic reticulum Ca2+-transport ATPase to caffeine-induced Ca2+ transients of murine skinned skeletal muscle fibres

The present study was carried out to investigate the contribution of the Ca²⁺-transport ATPase of the sarcoplasmic reticulum (SR) to caffeine-induced Ca²⁺ release in skinned skeletal muscle fibres. Chemically skinned fibres of balb-C-mouse EDL (extensor digitorum longus) were exposed for 1 min to a free Ca²⁺ concentration of 0.36 μM to load the SR with Ca²⁺. Release of Ca²⁺ from the SR was induced by 30 mM caffeine and recorded as an isometric force transient. For every preparation a pCa/force relationship was constructed, where pCa = −log₁₀ [Ca²⁺]. In a new experimental approach, we used the pCa/force relationship to transform each force transient directly into a Ca²⁺ transient. The calculated Ca²⁺ transients were fitted by a double exponential function: Y ₀ + A ₁⋅exp (−t/t ₁) + A ₂⋅exp(t/t ₂), with A ₁ < 0 < A ₂, t ₁ < t ₂ and Y ₀, A ₁, A ₂ in micromolar. Ca²⁺ transients in the presence of the SR Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) were compared to those obtained in the absence of the drug. We found that inhibition of the SR Ca²⁺-ATPase during caffeine-induced Ca²⁺ release causes an increase in the peak Ca²⁺ concentration in comparison to the control transients. Increasing CPA concentrations prolonged the time-to-peak in a dose-dependent manner, following a Hill curve with a half-maximal value of 6.5 ± 3 μM CPA and a Hill slope of 1.1 ± 0.2, saturating at 100 μM. The effects of CPA could be simulated by an extended three-compartment model representing the SR, the myofilament space and the external bathing solution. In terms of this model, the SR Ca²⁺-ATPase influences the Ca²⁺ gradient across the SR membrane in particular during the early stages of the Ca²⁺ transient, whereas the subsequent relaxation is governed by diffusional loss of Ca²⁺ into the bathing solution. Received: 2 February 1996/Accepted: 1 April 1996     

Vasopressin-stimulated Ca2+ reabsorption in rabbit cortical collecting system: effects on cAMP and cytosolic Ca2+

 The effect of arginine vasopressin (AVP) on transepithelial Ca²⁺ transport in primary cultures of rabbit cortical collecting system cells was examined. Addition of AVP to the basolateral side of the monolayer dose-dependently (EC₅₀ = 0.7 nM) increased active Ca²⁺ reabsorption from a basal value of 85 ± 2 nmol·h^–1·cm^–2 to a maximum value of 124 ± 3 nmol·h^–1·cm^–2. This was paralleled by a dose-dependent (EC₅₀ = 1.1 nM) increase in cellular adenosine 3′,5′-cyclic monophosphate (cAMP) content. Both effects of AVP were mimicked by the V₂ agonist deamino-Cys,D-Arg⁸-vasopressin (dDAVP) and forskolin. Addition of either AVP or dDAVP to the basolateral side evoked a sustained increase in cytosolic free Ca²⁺ concentration, which resulted from both Ca²⁺ entry and release from internal stores. Only the effect on Ca²⁺ entry was mimicked by forskolin, demonstrating that cAMP acts by activating a Ca²⁺ influx pathway. The present findings demonstrate that AVP stimulates transcellular Ca²⁺ transport in the cortical collecting system through activation of basolateral V₂ receptors coupled to adenylyl cyclase to increase the cellular cAMP content. Received: 4 July 1996 / Received after revision and accepted: 3 September 1996     

Relationship between global cytosolic Ca2+ concentration and Ca2+-activated K+ current in rabbit cerebral arterial myocyte.

In smooth muscle cells, the sarcoplasmic reticulum (SR) has been identified as the primary storage site for intracellular Ca2+. The peripheral SR is in close proximity with plasma membrane to make a narrow subsarcolemmal space. In this study, we investigated the regulation of subsarcolemmal [Ca2+] ([Ca2+]sl) and global cytosolic [Ca2+] ([Ca2+]c) of rabbit arterial smooth muscle using whole cell patch clamp technique and microspectrofluorimetry. The Ca2+-activated K+ current (IK(Ca)) and the ratio of fura-2 fluorescence (R340/380) were considered to reflect the [Ca2+]sl and [Ca2+]c, respectively. At a holding potential of 0 mV, extracellular application of 10 mM caffeine, a well known Ca2+-releasing agent, induced transient increase of IK(Ca) and R340/380 (IK(Ca)-transient and R340/380-transient, respectively). The increase and decay of IK(Ca) transient was faster than R340/380-transient. By repetitive application of caffeine, when the refilling state of SR was supposed to be lower than the control condition, IK(Ca)-transient and R340/380 transient were suppressed to different levels; e.g. the second application 20 sec after the first could induce smaller IK(Ca) transient than R340/380-transient. Dissociation of IK(Ca)-transient and R340/380-transient was removed by sufficient (>3 min) washout of caffeine. Recovery from the dissociation was also dependent upon the membrane potential; faster recovery was observed at negative (-40 mV) holding potential than at depolarized (0 mV) condition. Dissociation of IK(Ca) from [Ca2+]c was also partially prevented by perfusion with Na+-free (replaced by NMDG+) extracellular solution. These results suggest that, 1) there is prominent spatial inhomogeneity of [Ca2+] in cerebral arterial myocyte, 2) [Ca2+]Sl is preferentially affected by the interference from nearby plasmalemmal Ca2+ regulation mechanism which is partly dependent upon extracellular Na+.