心肌肌浆网膜含有膜质微区结构

细胞膜质微区(microdomain)是细胞膜上特殊的结构域,在细胞信号转导和物质运输过程中起着非常重要的作用.绝大多数膜质微区来源于全细胞膜,即包括质膜和细胞器膜.最新研究表明细胞器膜如高尔基体膜也有膜质微区,因此分离了猪心肌浆网膜的膜质微区.首先获得了没有质膜污染的猪心肌浆网,用去污剂TritonX-100处理该肌浆网,获得了去污剂不溶的质膜微区(SR-DRM),该微区富集胆固醇和鞘磷脂.质膜微区的标记脂和蛋白质:神经节苷脂GM1和Caveolin-3也在该区富集.同时还研究了心肌浆网Ca2 -ATPase(SERCA2a)的分布,结果表明,相当数量的SERCA2a分布在膜质微区,并且有正常的生理功能.上述研究结果表明,在心肌浆网膜上有膜质微区的存在,进一步证明膜质微区不仅存在于细胞质膜,也普遍存在于细胞器膜.     

Chloride-induced Ca2+ Release from the Sarcoplasmic Reticulum of Chemically Skinned Rabbit Psoas Fibers and Isolated Vesicles of Terminal Cisternae

There is increasing evidence that Ca²⁺ release from sarcoplasmic reticulum (SR) of mammalian skeletal muscle is regulated or modified by several factors including ionic composition of the myoplasm. We have studied the effect of Cl⁻ on the release of Ca²⁺ from the SR of rabbit skeletal muscle in both skinned psoas fibers and in isolated terminal cisternae vesicles. Ca²⁺ release from the SR in skinned fibers was inferred from increases in isometric tension and the amount of release was assessed by integrating the area under each tension transient. Ca²⁺ release from isolated SR was measured by rapid filtration of vesicles passively loaded with ⁴⁵Ca²⁺. Ca²⁺ release from SR was stimulated in both preparations by exposure to a solution containing 191 mm choline-Cl, following pre-equilibration in Ca²⁺-loading solution that had propionate as the major anion. Controls using saponin (50 μg/ml), indicated that the release of Ca²⁺ was due to direct action of Cl⁻ on the SR rather than via depolarization of T-tubules. Procaine (10 mm) totally blocked Cl⁻- and caffeine-elicited tension transients recorded using loading and release solutions having ([Na⁺] + [K⁺]) × [Cl⁻] product of 6487.69 mm ² and 12361.52 mm ², respectively, and blocked 60% of Ca²⁺ release in isolated SR vesicles. Surprisingly, procaine had only a minor effect on tension transients elicited by Cl⁻ and caffeine together. The data from both preparations suggests that Cl⁻ induces a relatively small amount of Ca²⁺ release from the SR by activating receptors other than RYR-1. In addition, Cl⁻ may increase the Ca²⁺ sensitivity of RYR-1, which would then allow the small initial release of Ca²⁺ to facilitate further release of Ca²⁺ from the SR by Ca²⁺-induced Ca²⁺ release. Received: 6 February 1996/Revised: 17 July 1996     

Membrane Asymmetry in Isolated Canine Cardiac Sarcoplasmic Reticulum: Comparison with Skeletal Muscle Sarcoplasmic Reticulum

Cardiac sarcoplasmic reticulum (CSR), isolated from dog hearts, was shown to be asymmetric in the distribution of phospholipids across the CSR bilayer. Phosphatidylethanolamine was mostly resident in the outer leaflet, phosphatidylcholine was equally distributed across both monolayers and phosphatidylserine was found primarily in the inner monolayer. This distribution of headgroups is similar to that found in fast skeletal muscle sarcoplasmic reticulum (SSR); however, the asymmetry in CSR is not as striking as that in SSR. Phospholipids retained by the CSR calcium pump protein (CaATPase) after detergent ``stripping' were similar to those intimate to the SSR CaATPase, although the percentages of unsaturated phospholipids and plasmalogenic phospholipids are not as great as in the skeletal system. Lipids associated with the CSR CaATPase following DFDNB cross-linking showed a preference for retention of the aminophospholipids, again similar to the SSR CaATPase. Because the nonrandom distribution of membrane lipids modifies SSR function, it is likely these membrane lipids impact in situ the function of the CSR. Received: 19 December 1997/Revised: 3 April 1998     

Electrokinetic Properties of the Sarcoplasmic Reticulum Membrane Obtained from Reconstitution Studies

Electrophoretic mobility data of SR vesicles reconstituted with uncharged and two mixtures of charged and uncharged lipids (Brethes, D., Dulon, D., Johannin, G., Arrio, B., Gulik-Krzywicki, T., Chevallier, J. 1986. Study of the electrokinetic properties of reconstituted sarcoplasmic reticulum vesicles. Arch. Biochem. Biophys. 246:355–356) were analyzed in terms of four models of the membrane-water interface: (I) a smooth, negatively charged surface; (II) a negatively charged surface of lipid bilayer covered with an electrically neutral surface frictional layer; (III) an electrically neutral lipid bilayer covered with a neutral frictional layer containing a sheet of negative charge at some distance above the surface of the bilayer; (IV) an electrically neutral lipid bilayer covered with a homogeneously charged frictional layer. The electrophoretic mobility was predicted from the numerical integration of Poisson-Boltzmann and Navier-Stokes equations. Experimental results were consistent only with predictions based on Model-III with charged sheet about 4 nm above the bilayer and frictional layer about 10 nm thick. Assuming that the charge of the SR membrane is solely due to that on Ca⁺⁺-ATPase pumps, the dominant SR protein, the mobility data of SR and reconstituted SR vesicles are consistent with 12 electron charges/ATPase. This value compares well to the net charge of the cytoplasmic portion of ATPase estimated from the amino acid sequence (-11e). The position of the charged sheet suggests that the charge on the ATPase is concentrated in the middle of the cytoplasmic portion. The frictional layer of SR can be also assigned to the cytoplasmic portion of Ca⁺⁺-ATPase. The layer has been characterized with hydrodynamic shielding length of 1.1 nm. Its thickness is comparable to the height of the cytoplasmic portion of Ca⁺⁺-ATPase. Received: 15 June 1998/Revised: 8 October 1998     

Effects of ATP-sensitive Potassium Channel Regulators on Chloride Channels in the Sarcoplasmic Reticulum Vesicles from Rabbit Skeletal Muscle

The lipid bilayer technique was used to examine the effects of the ATP-sensitive K⁺ channel inhibitor (glibenclamide) and openers (diazoxide, minoxidil and cromakalim) and Cl⁻ channel activators (GABA and diazepam) on two types of chloride channels in the sarcoplasmic reticulum (SR) from rabbit skeletal muscle. Neither diazepam at 100 μm nor GABA at 150 μm had any significant effect on the conductance and kinetics of the 75 pS small chloride (SCl) channel. Unlike the 150 pS channel, the SCl channel is sensitive to cytoplasmic glibenclamide with K _i ∼ 30 μm. Glibenclamide induced reversible decline in the values of current (maximal current amplitude, I _max and average mean current, I′) and kinetic parameters (frequency of opening F _o , probability of the channel being open P _o and mean open time, T _o , of the SCl channel. Glibenclamide increased mean closed time, T _c , and was a more potent blocker from the cytoplasmic side (cis) than from the luminal side (trans) of the channel. Diazoxide increased I′, P _o , and T _o in the absence of ATP and Mg²⁺ but it had no effect on I _max and also failed to activate or remove the glibenclamide- and ATP-induced inhibition of the SCl channel. Minoxidil induced a transient increase in I′ followed by an inhibition of I _max, whereas cromakalim reduced P _o and I′ by increasing channel transitions to the closed state and reducing T _o without affecting I _max. The presence of diazoxide, minoxidil or cromakalim on the cytoplasmic side of the channel did not prevent [ATP] _cis or [glibenclamide] _cis from blocking the channel. The data suggest that the action(s) of these drugs are not due to their effects on the phosphorylation of the channel protein. The glibenclamide- and cromakalim-induced effects on the SCl channel are mediated via a ``flicker' type block mechanism. Modulation of the SCl channel by [diazoxide] _cis and [glibenclamide] _cis highlights the therapeutic potential of these drugs in regulating the Ca²⁺-counter current through this channel. Received: 2 September 1997/Revised: 20 March 1998     

Clomipramine and Related Structures as Inhibitors of the Skeletal Sarcoplasmic Reticulum Ca2+ Pump

The Ca²⁺-pumping activity of skeletal sarcoplasmic reticulum vesicles is half-maximallyinhibited by 120 M clomipramine, 250 M desipramine, and 500 M imipramine or trimipramine.The inhibition is attributed to the dihydrodibenzazepine moiety, since3-(dimethylamino)propionitrile, reproducing the aliphatic amine chain, has no inhibitory action. The inhibitionis shown as a marked decrease of Ca²⁺ binding at equilibrium in theabsence of ATP and asa reduction of phosphorylation of the Ca²⁺-free conformation byinorganic phosphate. Therefore,the drug effect is consistent with preferential interaction of tricyclic antidepressants withthe Ca²⁺-free conformation of the nonphosphorylated enzyme. An additional decrease in theapparent rate constant of enzyme dephosphorylation, i.e., in the release of phosphate fromATP during enzyme cycling was also noticed.     

大鼠心肌肌浆网的纯化及其性质研究

 用超声波破碎心肌细胞,差速离心法纯化大鼠心肌肌浆网(CSR)。SDS-聚丙烯酰胺凝胶电泳测得Ca~(2+)-ATPase分子量为98kD;电镜观察膜制备为完整的CSR微囊;标志酶哇巴因敏感型Na~(+),K~(+)-ATPase和叠氮化钠敏感型Mg~(2+)-ATPase活性表明膜制备中肌膜含量很低,但仍有线粒体污染。 用~(45)Ca~(2+)示踪微孔滤膜法研究Ca~(2+)跨膜转运,CSRCa~(2+)蓄集最大值为57nmol/mg蛋白。CSR Ca~(2+)-ATPase在4℃—21℃和21℃—49℃两区间反应活化能不同,前者大于后者。酶的最适pH为7.4。以ATP为底物,该酶有两个表观Km值:Km_1为3.7μmol/LKm_2为713μmol/L。     

Characterization of Ryanodine-sensitive Ca2+ Release from Microsomal Vesicles of Rat Parotid Acinar Cells: Regulation by Cyclic ADP-ribose

We have measured ryanodine (caffeine)-sensitive ⁴⁵Ca²⁺ release from isolated microsomal vesicles of endoplasmic reticulum prepared from rat parotid acinar cells. After a steady state of ATP-dependent ⁴⁵Ca²⁺ uptake, the addition of caffeine (40 mm), ryanodine (10∼500 μm) or an NAD⁺ metabolite, cyclic ADP-ribose (cADPR, 4 μm) released about 10% of the ⁴⁵Ca²⁺ that had been taken up. The ⁴⁵Ca²⁺ release was not inhibited by heparin, an antagonist of IP₃ receptor. The effects of caffeine, ryanodine and cADPR on ⁴⁵Ca²⁺ release were also tested in the presence of thapsigargin (TG), an inhibitor of microsomal Ca²⁺-ATPase. When caffeine (10∼40 mm), ryanodine (10 μm) or cADPR (1∼10 μm) was added in the medium with 100 nm TG, a significant ⁴⁵Ca²⁺ release was seen, while higher concentrations of ryanodine (>100 μm) did not cause any ⁴⁵Ca²⁺ release in the presence of TG. The initial rate of caffeine (40 mm)-induced ⁴⁵Ca²⁺ release was increased by a pretreatment with 10 μm ryanodine, whereas the caffeine-induced ⁴⁵Ca²⁺ release was strongly inhibited by the presence of a higher concentration (500 μm) of ryanodine. cADPR-induced ⁴⁵Ca²⁺ release was also inhibited by 500 μm ryanodine. Caffeine (40 mm)- or cADPR (4 μm)-induced ⁴⁵Ca²⁺ release was abolished by a presence of ruthenium red (50∼100 μm). The presence of a low concentration (0.5 μm) of cADPR shifted the dose-response curve of caffeine-induced ⁴⁵Ca²⁺ release to the left. These results indicate the presence of a ryanodine sensitive Ca²⁺ release mechanism in the endoplasmic reticulum of rat parotid acinar cells that is distinct from the IP₃-sensitive Ca²⁺ channel and is activated by caffeine, cADPR and a low concentration (10 μm) of ryanodine, but is inhibited by higher concentrations (>100 μm) of ryanodine and ruthenium red. The properties of the ryanodine-sensitive mechanism are similar to that of the ryanodine receptor as described in muscle cells. Received: 11 June 1996/Revised: 14 November 1996     

pH-Modulation of Chloride Channels from the Sarcoplasmic Reticulum of Skeletal Muscle

The understanding of the role of cytoplasmic pH in modulating sarcoplasmic reticulum (SR) ion channels involved in Ca²⁺ regulation is important for the understanding of the function of normal and adversely affected muscles. The dependency of the SR small chloride (SCl) channel from rabbit skeletal muscle on cytoplasmic pH (pH _cis ) and luminal pH (pH _trans ) was investigated using the lipid bilayer-vesicle fusion technique. Low pH _cis 6.75–4.28 modifies the operational mode of this multiconductance channel (conductance levels between 5 and 75 pS). At pH _cis 7.26–7.37 the channel mode is dominated by the conductance and kinetics of the main conductance state (65–75 pS) whereas at low pH _cis 6.75–4.28 the channel mode is dominated by the conductance and kinetics of subconductance states (5–40 pS). Similarly, low pH _trans 4.07, but not pH _trans 6.28, modified the activity of SCl channels. The effects of low pH _cis are pronounced at 10⁻³ and 10⁻⁴ m [Ca²⁺] _cis but are not apparent at 10⁻⁵ m [Ca²⁺] _cis , where the subconductances of the channel are already prominent. Low pH _cis -induced mode shift in the SCl channel activity is due to modification of the channel proteins that cause the uncoupling of the subconductance states. The results in this study suggest that low pH _cis can modify the functional properties of the skeletal SR ion channels and hence contribute, at least partly, to the malfunction in the contraction-relaxation mechanism in skeletal muscle under low cytoplasmic pH levels. Received: 20 May 1998/Revised: 24 September 1998     

Sarcoplasmic Reticulum Ca2+ Release in Rat Slow- and Fast-Twitch Muscles

The same isoform of ryanodine receptor (RYR1) is expressed in both fast and slow mammalian skeletal muscles. However, differences in contractile activation and calcium release kinetics in intact and skinned fibers have been reported. In this work, intracellular Ca²⁺ transients were measured in soleus and extensor digitorum longus (EDL) single muscle fibers using mag-fura-2 (K _D for Ca²⁺= 49 μm) as Ca²⁺ fluorescent indicator. Fibers were voltage-clamped at V _h =−90 mV and sarcoplasmic reticulum calcium release was measured at the peak (a) and at the end (b) of 200 msec pulses at +10 mV. Values of a-b and b were assumed to correspond to Ca²⁺-gated and voltage-gated Ca²⁺ release, respectively. Ratios (b/a-b) in soleus and EDL fibers were 0.41 ± 0.05 and 1.01 ± 0.13 (n= 12), respectively. This result suggested that the proportion of dihydropyridine receptor (DHPR)-linked and unlinked RYRs is different in soleus and EDL muscle. The number of DHPR and RYR were determined by measuring high-affinity [³H]PN200-110 and [³H]ryanodine binding in soleus and EDL rat muscle homogenates. The B _max values corresponded to a PN200-110/ryanodine binding ratio of 0.34 ± 0.05 and 0.92 ± 0.11 for soleus and EDL muscles (n= 4–8), respectively. These data suggest that soleus muscle has a larger calcium-gated calcium release component and a larger proportion of DHPR-unlinked RYRs. Received: 31 August 1995/Revised: 25 January 1996     

Thermal Stability of the Plasma Membrane Calcium Pump. Quantitative Analysis of Its Dependence on Lipid-Protein Interactions

Thermal stability of plasma membrane Ca²⁺ pump was systematically studied in three micellar systems of different composition, and related with the interactions amphiphile-protein measured by fluorescence resonance energy transfer. Thermal denaturation was characterized as an irreversible process that is well described by a first order kinetic with an activation energy of 222 ± 12 kJ/mol in the range 33–45°C. Upon increasing the mole fraction of phospholipid in the mixed micelles where the Ca²⁺ pump was reconstituted, the kinetic coefficient for the inactivation process diminished until it reached a constant value, different for each phospholipid species. We propose a model in which thermal stability of the pump depends on the composition of the amphiphile monolayer directly in contact with the transmembrane protein surface. Application of this model shows that the maximal pump stability is attained when 80% of this surface is covered by phospholipids. This analysis provides an indirect measure of the relative affinity phospholipid/detergent for the hydrophobic transmembrane surface of the protein (K _LD ) showing that those phospholipids with higher affinity provide greater stability to the Ca²⁺ pump. We developed a method for directly measure K _LD by using fluorescence resonance energy transfer from the membrane protein tryptophan residues to a pyrene-labeled phospholipid. K _LD values obtained by this procedure agree with those obtained from the model, providing a strong evidence to support its validity. Received: 5 August 1999/Revised: 20 October 1999     

Calcium-dependent Enhancement of Depletion-activated Calcium Current in Jurkat T Lymphocytes

We have obtained evidence that the Ca²⁺-selective current activated by Ca²⁺ store depletion (Ca²⁺ release-activated Ca²⁺ current; I _crac) in Jurkat T lymphocytes is augmented in a time-dependent manner by Ca²⁺ itself. Whole cell patch clamp experiments employed high cytosolic Ca²⁺-buffering conditions to passively deplete Ca²⁺ stores. Rapidly switching to nominally Ca²⁺-free extracellular buffer instantaneously reduced I _crac measured at −100 mV to leak current level. Unexpectedly, readmission of 2 mm Ca²⁺ instantaneously restored only 38 ± 5% (mean ±sem; n = 9) of the full I _crac amplitude. The remainder reappeared in a monotonic time-dependent manner over 10 to 20 sec. Rapid vs. slow intracellular Ca²⁺ chelators did not alter this process, and inorganic I _crac blockers did not regenerate it, arguing against an intracellular site of action. The effect was specific to Ca²⁺: introduction of the permeant ions, Ba²⁺ or Sr²⁺, failed to invoke time-dependent I _crac reappearance. Moreover, equimolar substitution of Ba²⁺ for Ca²⁺ initially produced Ba²⁺ current of similar magnitude to the full Ca²⁺ current, but the Ba²⁺ current decayed monotonically to <50% of its initial amplitude in <20 sec. Conversely, return to Ca²⁺ produced a time-dependent increase in I _crac to its larger Ca²⁺ permeation level. Thus Ca²⁺ appears to selectively promote a reversible transition of I _crac that results in larger current flux, and at least partially explains the selectivity of this current for Ca²⁺ over other divalent ions. Received: 30 August 1995/Revised: 7 November 1995     

Modulation of the Calmodulin-induced Inhibition of Sarcoplasmic Reticulum Calcium Release Channel (Ryanodine Receptor) by Sulfhydryl Oxidation in Single Channel Current Recordings and [3H]Ryanodine Binding

The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl–sulfonic acid (4-CMPS) and 4,4′-dithiodipyridine (4,4′-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [³H]ryanodine binding to HSR vesicles. 0.1 μm CaM reduced the open probability (P _o ) of the calcium release channel at maximally activating calcium concentrations (50–100 μm) from 0.502 ± 0.02 to 0.137 ± 0.022 (n= 28), with no effect on unitary conductance. 4-CMPS (10–40 μm) and 4,4′-DTDP (0.1–0.3 mm) induced a concentration dependent increase in P _o (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4′-DTDP to higher concentrations in single channel recordings and [³H]ryanodine binding. 40 μm 4-CMPS induced a near maximal (P _o > 0.9) and 0.3 mm 4,4′-DTDP a submaximal (P _o = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4′-DTDP affected Ca-[¹²⁵I]calmodulin binding to HSR. 1 mm MgCl₂ reduced P _o from 0.53 to 0.075 and 20–40 μm 4-CMPS induced a near maximal channel activation (P _o > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4′-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel. Received: 22 July 1999/Revised: 15 November 1999     

Proton Countertransport and Coupled Gating in the Sarcoplasmic Reticulum Calcium Pump

The calcium pump of the sarcoplasmic reticulum (SERCA) is an ATP-driven active transporter of Ca²⁺ ions that functions via an “alternating-access” cycle mechanism. In each cycle, SERCA transports two Ca²⁺ ions toward the lumen of the sarcoplasmic reticulum and two to three protons to the cytoplasm. How the latter conformational transition is coupled to cytoplasmic release of protons remains poorly understood. The present computational study shows how the mechanism of proton countertransport is coupled to the alternating access gating process in SERCA. Molecular dynamics simulation trajectories are generated starting from a series of configurations taken along the E2 to E1 transition pathway determined by the string method with swarms-of-trajectories. Simulations of different protonation configurations at the binding sites reveal how deprotonation events affect the opening of the cytoplasmic gate. The results show that there is a strong coupling between the chronological order of deprotonation, the entry of water molecules into the TM region, and the opening of the cytoplasmic gate. Deprotonation of E309 and E771 is sequential with E309 being the first to lose the proton. The deprotonation promotes the opening of the cytoplasmic gate but leads to a productive gating transition only if it occurs after the transmembrane domain has reached an intermediate conformation. Deprotonation of E309 and E771 is unproductive when it occurs too early because it causes the re-opening of the luminal gate.     

Inositol 1,4,5-Trisphosphate But Not Ryanodine-Receptor Agonists Induces Calcium Release from Rat Liver Golgi Apparatus Membrane Vesicles

We investigated the direct effect of inositol 1,4,5-trisphosphate (IP₃) and ryanodine receptor agonists on Ca²⁺ release from vesicles of a rat liver Golgi apparatus (GA) enriched fraction, which were actively loaded with ⁴⁵Ca²⁺. Results in GA were compared with those obtained in a rat liver endoplasmic reticulum (ER) enriched fraction. The addition of IP₃ at concentrations ranging from 100 nm to 100 μm, in the presence of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases, promoted a rapid decrease in the Ca²⁺ content of GA vesicles. The amount of Ca²⁺ released from the vesicles was a function of IP₃ concentration, reaching about 60% in both GA and ER fractions at 100 μm IP₃. Calcium release was inhibited by heparin, an antagonist of IP₃ receptors. Calcium exhibited a bell-shaped effect on IP₃-dependent Ca²⁺ released from GA vesicles: it activated Ca²⁺ release at concentrations up to 1 μm, and inhibited it at higher concentrations. In contrast to that found in the endoplasmic reticulum fraction, none of the ryanodine receptor agonists tested (cyclic ADP-ribose, caffeine and ryanodine) significantly induced Ca²⁺ release from GA fraction vesicles in the presence of thapsigargin. Our results indicate the presence of an IP₃-sensitive Ca²⁺ release mechanism in the Golgi apparatus membrane analogous to that of the ER. However, a Ca²⁺ release mechanism sensitive to ryanodine receptor agonists like that of ER is not evident in the GA membrane. Received: 13 March 2000/Revised: 13 July 2000     

Mechanosensitive Calcium Entry and Mobilization in Renal A6 Cells

Using spectrofluorescence imaging of fura-2 loaded renal A6 cells, we have investigated the generation of the cytosolic Ca²⁺ signal in response to osmotic shock and localized membrane stretch. Upon hypotonic exposure, the cells began to swell prior to a transient increase in [Ca²⁺] _i and the cells remained swollen after [Ca²⁺] _i had returned towards basal levels. Exposure to 2/3rd strength Ringer produced a cell volume increase within 3 min, followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also produced a transient increase in [Ca²⁺] after a delay of 22 sec. Both the RVD and [Ca²⁺] _i response to hypotonicity were inhibited in a Ca²⁺-free bathing solution and by gadolinium (10 μm), an inhibitor of stretch-activated channels. Stretching the membrane by application of subatmospheric pressure (-2 kPa) inside a cell-attached patch-pipette induced a similar global increase in [Ca²⁺] _i as occurred after hypotonic shock. A stretch-sensitive [Ca²⁺] _i increase was also observed in a Ca²⁺-free bathing solution, provided the patch-pipette contained Ca²⁺. The mechanosensitive [Ca²⁺] _i response was by gadolinium (10 μm) or Ca²⁺-free pipette solutions, even when Ca²⁺ (2 mm) was present in the bath. Long-term (>10 min) pretreatment of the cells with thapsigargin inhibited the [Ca²⁺] _i response to hypotonicity. These results provide evidence that cell swelling or mechanical stimulation can activate a powerful amplification system linked to intracellular Ca²⁺ release mechanisms. Received: 3 August 1998/Revised: 19 November 1998     

Modulation of the Plasma Membrane Ca2+ Pump

The plasma membrane calcium pump, which ejects Ca²⁺ from the cell, is regulated by calmodulin. In the absence of calmodulin, the pump is relatively inactive; binding of calmodulin to a specific domain stimulates its activity. Phosphorylation of the pump with protein kinase C or A may modify this regulation. Most of the regulatory functions of the enzyme are concentrated in a region at the carboxyl terminus. This region varies substantially between different isoforms of the pump, causing substantial differences in regulatory properties. The pump shares some motifs of the carboxyl terminus with otherwise unrelated proteins: The calmodulin-binding domain is a modified IQ motif (a motif which is present in myosins) and the last 3 residues of isoform 4b are a PDZ target domain. The pump is ubiquitous, with isoforms 1 and 4 of the pump being more widely distributed than 2 and 3. In some kinds of cells isoform 1 or 4 is missing, and is replaced by another isoform. Received: 26 January 1998/Revised: 6 April 1998     

The Role of Calcium in the Volume Regulation of Rat Lacrimal Acinar Cells

Earlier studies have suggested a role for Ca²⁺ in regulatory volume decrease (RVD) in response to hypotonic stress through the activation of Ca²⁺-dependent ion channels (Kotera & Brown, 1993; Park et al., 1994). The involvement of Ca²⁺ in regulating cell volume in rat lacrimal acinar cells was therefore examined using a video-imaging technique to measure cell volume. The trivalent cation Gd³⁺ inhibited RVD, suggesting that Ca²⁺ entry is important and may be via stretch-activated cation channels. However, Fura-2 loaded cells did not show an increase in [Ca²⁺] _i during exposure to hypotonic solutions. The absence of any changes in [Ca²⁺] _i resulted from the buffering of cytosolic Ca²⁺ by Fura-2 during hypotonic shock and therefore inhibition of RVD. The intracellular Ca²⁺ chelator, BAPTA, also inhibited the RVD response to hypotonic shock. An increase in [Ca²⁺] _i induced by either acetylcholine or ionomycin, was found to decrease cell volume under isotonic conditions in lacrimal acinar cells. Cell shrinkage was inhibited by tetraethylammonium ion, an inhibitor of Ca²⁺-activated K⁺ channels. On the basis of the presented data, we suggest an involvement of intracellular Ca²⁺ in controlling cell volume in lacrimal acinar cells. Received: 20 February 1998/Revised: 1 May 1998