Different Types Of Ca2 Research Articles

Three types of Ca2+ channel trigger secretion with different efficacies in chromaffin cells.

To determine whether the different types of Ca2+ channels present in the same secretory cell contribute equally to secretion, we used chromaffin cells to analyse the coupling between three distinct types of Ca2+ channel and exocytosis. These are omega-conotoxin-GVIA-sensitive N-type channels, omega-agatoxin-IVA-sensitive P-type Ca2+ channels and dihydropyridine-sensitive facilitation Ca2+ channels, which are normally quiescent but are activated by depolarizing pre-pulses, repetitive depolarizations to physiological potentials, or agents that raise cyclic AMP. We have simultaneously monitored changes in capacitance as an assay of catecholamine secretion, and Ca2+ currents. Although all three types of Ca2+ channel trigger secretion individually, facilitation channels produce much greater secretion for a given size of Ca2+ current, indicating that they are coupled more efficiently to exocytosis. These results indicate that facilitation Ca2+ channels may be physically nearer vesicle release sites. They also show that low efficiency P- and N-type channels could trigger mild release and that high-efficiency facilitation channels may underlie the massive catecholamine release that occurs during the 'fight or flight' response.

Chapter 6 Application of Patch Clamp Methods to the Study of Calcium Currents and Calcium Channels

Chapter 6 Application of Patch Clamp Methods to the Study of Calcium Currents and Calcium Channels

Functional identification and quantitation of three intracellular calcium pools in GH4C1 cells: evidence that the caffeine-responsive pool is coupled to a thapsigargin-resistant, ATP-dependent process.

We have recently reported that basal oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) in intact GH4C1 cells are dependent on a Ca(2+)-induced Ca2+ release (CICR) mechanism. The purpose of the present study was to characterize the uptake and release pathways for intracellular Ca2+ in GH4C1 cells. We have used both permeabilized cells and microsome preparations, and we have monitored the change in ambient [Ca2+] using the dye, fluo 3. We find that there are three functionally distinct nonmitochondrial, ATP-dependent Ca2+ pools in these cells: Pool 1 is an inositol 1,4,5-trisphosphate (InsP3) responsive pool which is filled by a thapsigargin (Tg) sensitive Ca(2+)-ATPase; pool 2 is a second Tg-sensitive pool which is InsP3-unresponsive; and pool 3 is a Tg-resistant pool, at least a part of which has the characteristics of a CICR mechanism. These pools were established as follows. Tg caused additional Ca2+ release after maximum release was induced by prior addition of InsP3. In contrast, the InsP3 response was abolished in a time-dependent manner after pretreatment with Tg. Ambient Ca2+, added after maximum blockade by Tg, was still able to be sequestered. Ionomycin released Ca2+ even after maximum depletion by Tg. The ionomycin-releasable pool remaining after Tg treatment was also ATP-dependent, because this pool was completely discharged by ATP-depletion. Two additional inhibitors of intracellular Ca(2+)-ATPases, 2,4-di(tert-butyl)hydroquinone and cyclopiazonic acid, which are structurally unrelated to Tg, acted on the same targets as Tg. To estimate accurately the distribution of Ca2+ among compartments, we developed a new approach based on the analysis of two equilibrium states of Ca2+ distribution. Using this method, the size of the Tg-sensitive pools (pools 1 + 2) was estimated to be 63 +/- 2.5% of total non-mitochondrial Ca2+ in our preparation. Caffeine induced Ca2+ release, and this action was observed even after complete depletion of the Tg-sensitive pool, indicating that pool 3 had the characteristics of a CICR compartment. Because caffeine pretreatment caused an increase in the size of pools 1 + 2, the CICR-like mechanism operated primarily on pool 3. These new results strengthen our model, in which a distinct CICR-like pool is responsible for Ca2+ oscillations in GH4C1 cells, and also support the concept that different types of Ca2+ efflux pathways occur in Ca(2+)-storing nonmitochondrial organelles containing different types of Ca(2+)-ATPases.

Bell-shaped activation of inositol-1,4,5-trisphosphate-induced Ca2+ release by thimerosal in permeabilized A7r5 smooth-muscle cells.

There is no consensus about the different types of Ca2+ transport processes in the endoplasmic reticulum that are targeted by the sulphydryl reagent thimerosal. We have therefore investigated how thimerosal affects the various Ca2+ transport processes in permeabilized A7r5 smooth-muscle cells, using an unidirectional 45Ca2+ flux technique. Thimerosal up to a concentration of 32 microM did not have an effect on the passive 45Ca2+ leak from the stores, while higher concentrations increased this aspecific leak. Thimerosal inhibited the endoplasmic reticulum Ca2+ pump with an EC50 of 9 microM. Thimerosal exerted a biphasic effect on the Ca2+ release induced by inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] with a stimulation of the release at thimerosal concentrations below 10 microM, and an inhibitory effect at higher concentrations. Thimerosal (2.5-250 microM) did not exert an effect on the specific binding of [3H]Ins(1,4,5)P3 to its receptor, indicating that it probably did not act at the level of the binding site. This finding contrasts with the effect of the closely related sulphydryl reagent parachloromercuriphenylsulphonate, which, at high concentrations, inhibited [3H]Ins(1,4,5)P3 binding. The effects of thimerosal were largely prevented by the sulphydryl reducing agent dithiothreitol (3 mM). We conclude that thimerosal concentrations ranging from 0.32 to 1 microM can stimulate the Ins(1,4,5)P3-induced Ca2+ release without inhibiting the Ca2+ pumps or without increasing the passive Ca2+ permeability of the endoplasmic reticulum.

Inhibition of Ca2+ currents by a mu-opioid in a defined subset of rat sensory neurons

Activation of the endogenous opioid system can suppress pain without affecting other sensations, but the cellular mechanism of this selectivity is unclear. The analgesia might be due to inhibitory synapses arranged only on neurons whose activity leads to pain sensations. Alternatively, opioids might be released broadly, with neurons involved in pain sensation being especially sensitive. Therefore, we asked whether different subsets of rat dorsal root ganglion (DRG) sensory neurons vary in their sensitivity to opioids. Dissociated neurons were subdivided according to the spinal laminae to which they likely had projected, and whether they had innervated muscle. Using the patch-clamp method, we measured the inhibition of Ca2+ current by DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol), a peptide that selectively activates the mu (morphine) receptor. We also investigated the presence of different types of Ca2+ channels. In DRG neurons chosen at random, Ca2+ currents were inhibited by DAGO to widely varying degrees, with an average inhibition of 38%. Ca2+ currents in neurons in a subset that projects to laminae I and II had a lower average inhibition, and unlike the randomly selected cells, the responses were predictable and tightly distributed about the mean. This indicates that the variability of opioid sensitivity among DRG neurons reflects the presence of different subsets of cells. Since neurons projecting to laminae I and II, the projection site of nociceptive neurons, did not show high opioid sensitivity, there is no evidence that nociceptive neurons have stronger responses to opioids. But a firm conclusion is impossible because projection site does not strictly define sensory modality.

Role of different types of Ca 2+ channels and a reticulum-like Ca 2+ pump in neurotransmitter release

The factors controlling the Ca 2+ concentration directly responsible for triggering acetylcholine (ACh) release were investigated at an identified neuro-neuronal synapse of the Aplysia buccal ganglion. The types of presynaptic voltage-gated Ca 2+ channels associated with transmitter release were determined by using selective blockers such as nifedipine, ω-conotoxin and a partially purified extract from the venom of a funnel web spider (FTx). L-type, N-type and P-type Ca 2+ channels are present in the presynaptic neuron. The influx of Ca 2+ through both N- and P-types induces the release of ACh whereas Ca 2+ flowing through L-type channels modulates the duration of the presynaptic action potential by controlling the Ca 2+-dependent K + current. tBuBHQ, a blocker of the reticulum Ca 2+ pump, induces a potentiation of evoked release without modifying the presynaptic Ca 2+ influx. This seems to indicate that a part of the Ca 2+ entering the presynaptic terminal through N- and P-type Ca 2+ channels is sequestered in a presynaptic reticulum-like Ca 2+ buffer preventing these ions from contributing to ACh release. To exert its control, this Ca 2+ buffer must be located close to both the presynaptic Ca 2+ channels and the transmitter release mechanism.

Differential expression of voltage-gated Ca 2+-currents in cultivated aortic myocytes

The expression of different types of Ca 2+-channels was studied using the whole-cell patch-clamp technique in cultured rat aortic smooth-muscle myocytes. Ca 2+-currents were identified as either low- or high voltage-activated ( I Ca,LVA or I Ca,HVA, respectively) based on their distinct voltage-dependences of activation and inactivation, decay kinetics using Ba 2+ as the charge carrier and sensitivity to dihydropyridines. The heterogeneity in the functional expression of the two types of Ca 2+-channels in the cultured myocytes delineated four distinct phenotypes; (i), cells exhibiting only LVA currents; (ii), cells exhibiting only HVA currents; (iii), cells exhibiting both LVA and HVA currents and (iv), cells exhibiting no current. The myocytes exclusively expressed HVA currents both during the first five days in primary culture and after the cells had reached confluence (> 15 days). In contrast, LVA currents were expressed transiently between 5 and 15 days, during which time the cells were proliferating and had transient loss of contractility. Thus both LVA and HVA Ca 2+-current types contribute to Ca 2+-signalling in cultured rat aortic myocytes. However, the differential expression of the two Ca 2+ current types associated with differences in contractile and proliferative phenotypes suggest that they serve distinct cellular functions. Our results are consistent with the idea that LVA current expression is important for cell proliferation.

Intracellular Ca2+ dynamics in response to Ca2+ influx and Ca2+ release in autonomic neurones.

Spatial and temporal changes in the intracellular free Ca2+ concentration in response to Ca2+ influx at the cell membrane and to Ca2+ release from intracellular organelles were studied by recording fluorescence of Ca(2+)-sensitive probes, fura 2 or indo 1, with conventional epifluorescence or confocal laser-scanning microscopy combined with recordings of Ca(2+)-dependent membrane responses in bullfrog sympathetic ganglion cells. It was found that an increase in the intracellular Ca2+ induced by (an) action potential(s) in freshly ganglion cells bathed in Ringer's solution was solely a result of Ca2+ influx, while a rise in the intracellular Ca2+ by Ca2+ current in voltage-clamped cultured neurones was caused by not only Ca2+ influx but also Ca2+ release. This Ca2+ release was suggested to occur by a voltage-dependent (and graded) mode of activation of a Ca(2+)-induced Ca2+ release mechanism, explaining the lack of Ca2+ release by action potentials (because of their short-lasting depolarization) in freshly isolated neurons. In both cases, there was an inward spread of an increase in intracellular Ca2+. On the other hand, all or nothing activation of Ca(2+)-induced Ca2+ release occurred in the presence of caffeine, leading to the oscillation of Ca2+ in the cells. Characteristics of this mode of Ca2+ release and unique properties of drugs to block Ca2+ release were described. Finally, the physiological significance of different types of Ca2+ release was discussed.

Two types of high-threshold calcium currents inhibited by omega-conotoxin in nerve terminals of rat neurohypophysis.

1. The neurohypophysis comprises the nerve terminals of hypothalamic neurosecretory cells, which contain arginine vasopressin (AVP) and oxytocin. The secretory terminals of rat neurohypophyses were acutely dissociated. The macroscopic calcium currents (ICa) of these isolated peptidergic terminals were studied using 'whole-cell' patch-clamp recording techniques. 2. There are two types ('Nt' (where the subscript 't' denotes terminal) and 'L') of high-threshold voltage-activated ICa in the terminals, which can be distinguished by holding at different potentials i.e. -90 and -50 mV. Replacement of Ca2+ in the bathing solution by Ba2+ increased the amplitude of ICa, primarily due to an increase in the L-type component. Both inward currents were eliminated by adding 50 microM-Cd2+ or when in a Ca(2+)-free bathing solution. 3. omega-Conotoxin GVIA (omega-CgTx) has been widely used as a Ca2+ channel blocker. However, whether this toxin can discriminate between different types of Ca2+ channels is still a subject of controversy. We applied omega-CgTx over a wide range of concentrations (0.01-2 microM) to examine its effects on both Nt- and L-type ICa in these terminals. At a concentration of 30 nM, omega-CgTx selectively reduced, by 48%, the amplitude of Nt-type ICa. In contrast, a higher concentration (300 nM) of omega-CgTx was necessary to inhibit the L-type ICa. 4. omega-CgTx inhibited both Nt- and L-type ICa in a dose-dependent manner, and the half-maximum inhibition (IC50) of the ICa by the toxin was 50 and 513 nM, respectively, which was approximately a tenfold difference. The reduction in both types of currents did not result from any shift in their current-voltage or steady-state inactivation relationships. 5. In contrast, omega-CgTx, at a concentration of 300 nM, had no effect on the tetrodotoxin-sensitive sodium current (INa) of the isolated peptidergic nerve terminals. Furthermore, omega-CgTx did not reduce the long-lasting, non-inactivating ICa in the isolated non-neuronal secretory cells of the pars intermedia (PI) (intermediate lobe of the pituitary). 6. Our studies suggest that omega-CgTx might exert specific blocking effects on both Nt- and L-type Ca2+ channels, but that in the isolated peptidergic nerve terminals, the Nt-type component is more susceptible to this toxin.

Adrenergic and 5-HT 2 receptors on the same astroglial cell. A microspectrofluorimetric study on cytosolic Ca 2+ responses in single cells in primary culture

Noradrenaline (NA) or 5-hydroxytryptamine (5-HT) evoked cytosolic Ca 2+ mobilization in single type 1 astrocytes in primary culture from the cerebral cortex of newborn rat. The Ca 2+ indicator dye fura-2/AM was used in a microspectrofluorimetric system to visualize fluctuations in the intracellular Ca 2+ concentration. Activation of the adrenergic receptors α 2, α 2 and β, or activation of the 5-HT 2 receptors elicited different responses of Ca 2+ mobilization with different types of Ca 2+ spikes or oscillations. Principally, 4 different types of Ca 2+ responses could be obtained: a sharp spike, which declined back to baseline; an initial sharp spike, which declined to a smaller but sustained Ca 2+ elevation; an initial sharp spike which declined and showed low amplitude oscillations; and a sharp spike which declined back to baseline with baseline oscillations. Applications of the a 2 adrenoceptor agonist clonidine to individual astroglial cells evoked Ca 2+ transients mostly in young cultures (cultivated for 7–10 days), while applications of the α 1 adrenoceptor agonist phenylephrine evoked Ca 2+ transients mostly in older cultures (17–21 days of cultivation). Applications of the β adrenoceptor agonist isoproterenol evoked Ca 2+ transients in both young and older cultures, however, more frequent in older cultures. The a 2 and β receptor responses were dependent on external Ca 2+ levels. The NA-evoked Ca 2+ responses were seen in cultivated cells at all ages, but were more frequent in older cultures. Approximately 50% of the astroglial cells in 8 day old cultures responded to 5-HT with a cytosolic Ca 2+ mobilization and 80% of the cells in 21 day old cultures responded. Calcium transients evoked by different receptor agonists on the same astrocyte were obtained. Most cells which responded to 5-HT 2 receptor stimulation also responded to NA. The responses were blocked by specific receptor antagonists whereafter the receptors could be restimulated. Single type 1 astrocytes responded to α 1, β and 5-HT 2 receptor stimulation. Other cells responded to α 1, β and 5-HT 2 receptor agonists. Some cells only responded to 5-HT 2 receptor stimulation while others only responded to α 2 or NA, per se. Three conclusions can be made. (1) Adrenergic and 5-HT 2 receptors, the activation of which elicited cytosolic Ca 2+ mobilization, are expressed on single type 1 astrocytes. (2) Adrenergic and 5-HT 2 receptors are expressed on the same type 1 astrocyte in primary culture. (3) There seems to be a prominent heterogeneity among cells concerning membrane receptor sets.

Voltage sensor of excitation-contraction coupling in skeletal muscle.

Voltage sensor of excitation-contraction coupling in skeletal muscle.

Multiple conductance levels of the dihydropyridine-sensitive calcium channel in GH3 cells

Calcium channels in GH3 cells exhibit at least five conductance levels when examined in cell-attached or outside-out patches. These channels resemble the high threshold Ca2+ current in their range of activation and inactivation, and in their sensitivity to dihydropyridines (DHP). Mean open times for the five levels were brief (less than 1 msec) in control solutions but increased in the presence of BAY K 8644. In 100 mM Ba2+ and BAY K 8644, the five predominant slope conductances were 8-9, 12-13, 16-18, 23-24, and 28 pS. The present study is the first report of multiple levels of the DHP-sensitive Ca2+ channel occurring with high frequency in native membranes. The range of conductance levels that we observed encompasses the range of conductances found for two other different types of Ca2+ channels and indicates that unit conductance should be used with caution as a distinguishing characteristic for identification of different channel types.

Ca 2+ channels and intracellular Ca 2+ stores in neuronal and neuroendocrine cells

Changes in [Ca 2+] i are essential in modulating a variety of cellular functions. In no other cell type does the regulation of [Ca 2+] i reach the level of sophistication observed in cells of neuronal origin. Because of its physicochemical characteristics, the fluorescent Ca 2+ indicator Fura-2 has become extremely popular among neuroscientists. The use of this probe, however, has generated a number of problems, in particular, extracytosolic trapping and leakage from intact cells. In the first part of this contributin we briefly discuss the practical application of Fura-2 to the study of [Ca 2+] i in primary cultures of neurons and astrocytes. In the second part, we review some recent data (mainly from our laboratories) obtained in neurons and neuroendocrine cells, concerning the regulation of different types of Ca 2+ channels and the role and mechanism of intracellular Ca 2+ mobilization. The experimental evidence supporting the existence of a previously unrecognised organelle, the calciosome, that we hypothesize represents the functional equivalent in non-muscle cells of sarcoplasmic reticulum, will also briefly be discussed.

ω-Conotoxin GVIA and dihydropyridines discriminate two types of Ca 2+ channels involved in GABA release from striatal neurons in culture

We previously reported that the opening of the L-type Ca 2+ channel was only partly involved in the K +-evoked Ca 2+-dependent γ-aminobutyric acid (GABA) release from striatal neurons, suggesting that probably different types of voltage-sensitive Ca 2+ channels were implicated in this physiological process. Here we demonstrate that ω-contoxin GVIA, which has been reported to block L- and N-type neuronal Ca 2+ channels, also partly inhibits the Ca 2+-dependent GABA release. The maximal effects of ω-conotoxin GVIA and nifedipine, a highly specific antagonist of the L-type channels, were additive, a total inhibition of the Ca 2+-dependent GABA release being obtained in the presence of both drugs. We therefore propose that ω-conotoxin GVIA and nifedipine block two different types of Ca 2+ channels, both involved in the GABA release process.

Separate Binding and Functional Sites for ω co‐Conotoxin and Nitrendipine Suggest Two Types of Calcium Channels in Bovine Chromaffin Cells

Purified adrenomedullary plasma membranes contain two high-affinity binding sites for 125I-omega-conotoxin, with KD values of 7.4 and 364 pM and Bmax values of 237 and 1,222 fmol/mg of protein, respectively. Dissociation kinetics showed a biphasic component and a high stability of the toxin-receptor complex, with a t1/2 of 81.6 h for the slow dissociation component. Unlabeled omega-conotoxin inhibited the binding of the radioiodinated toxin, adjusting to a two-site model with Ki1 of 6.8 and Ki2 of 653 pM. Specific binding was not affected by Ca2+ channel blockers or activators, cholinoceptor antagonists, adrenoceptor blockers, Na+ channel activators, dopaminoceptor blockers, or Na+/H+ antiport blockers, but divalent cations (Ca2+, Sr2+, and Ba2+) inhibited the toxin binding in a concentration-dependent manner. The binding of the dihydropyridine [3H]nitrendipine defined a single specific binding site with a KD of 490 pM and a Bmax of 129 fmol/mg of protein. At 0.25 microM, omega-conotoxin was not able to block depolarization-evoked Ca2+ uptake into cultured bovine adrenal chromaffin cells depolarized with 59 mM K+ for 30 s, whereas under the same conditions, 1 microM nitrendipine inhibited uptake by approximately 60%. When cells were hyperpolarized with 1.2 mM K+ for 5 min and then Ca2+ uptake was subsequently measured during additions of 59 mM K+. Omega-conotoxin partially inhibited Ca2+ uptake in a concentration-dependent manner. These results suggest that two different types of Ca2+ channels might be present in chromaffin cells. However, the molecular identity of omega-conotoxin binding sites remains to be determined.

Diversity and novel pharmacological properties of Ca2+ channels in Drosophila brain membranes.

Binding studies as well as affinity labelling and immunoblot techniques were used to identify and characterize the receptors for Ca2+ channel blockers in Drosophila brain membranes. Despite structural analogies with mammalian receptors, Drosophila binding sites for phenylalkylamines and 1,4-dihydropyridines, unlike those described in skeletal and cardiac muscle, were found to be located on separate Ca2+ channels. Single-channel bilayer recordings from reconstituted membranes revealed the presence of eight distinct cobalt-sensitive Ba2+-conducting channels in Drosophila brain membrane preparations. In good agreement with binding studies, the most frequently observed Ca2+ channel type (Ba2+ conductance of 13 pS) was extremely sensitive to phenylalkylamines but not affected by micromolar concentrations of 1,4-dihydropyridines. Distinct 1,4-dihydropyridine-sensitive and phenylalkylamine-insensitive channels were also identified. They had unitary Ba2+ conductances of 21 and 31 pS. A detailed analysis of drug action showed that both 1,4-dihydropyridines and phenylalkylamines first increased channel open state probability before fully blocking channel activity. Other types of channels have been identified with unitary Ba2+ conductances of 9, 41, 53, 64 and 81 pS. They were insensitive to the previously described organic Ca2+ channel blockers. The Drosophila system seems to be a unique model to analyse the properties of several different types of Ca2+ channels and particularly those of channel types that are uniquely blocked by phenylalkylamines or uniquely blocked by 1,4-dihydropyridines.

Voltage‐gated sodium and calcium currents in rat osteoblasts.

1. The whole-cell voltage-clamp mode of the patch-clamp technique was used to investigate the presence of voltage-gated inward currents in osteoblasts from newborn rat calvaria. 2. In K+-free solutions, three kinds of inward currents could be activated by depolarization: a voltage-gated Na+ current and two different types of Ca2+ currents. 3. The Na+ current was activated by depolarization above -40 mV in all the cells. It was reduced by half by 10 nM-TTX (tetrodotoxin). 4. In an isotonic Ba2+ external solution containing TTX, and with a Cs-EGTA internal solution buffered at pCa 8, depolarizing jumps induced both a transient Ba2+ current and a sustained Ba2+ current. The relative proportions of these two currents varied greatly among cells. 5. The transient and sustained Ba2+ currents differ with respect to their time course and their voltage dependence. 6. The depolarization-activated inward currents were also observed under more physiological conditions, in the presence of only 2 mM-external Ca2+ and with a K+ internal solution buffered at pCa 7. 7. A few records obtained in current clamp showed that it is possible to induce action potentials in osteoblasts.

Calcium conductance and firing properties of spinal motoneurones in the turtle.

1. The contribution of Ca2+ conductance to the firing properties of motoneurones was investigated in transverse slices of the turtle spinal cord. 2. In the presence of tetrodotoxin (TTX), tetraethylamonium (TEA) in low extracellular concentration (less than 5 mM) promoted Ca2+ spikes. In higher concentrations of TEA, a suprathreshold depolarizing current pulse was followed by an after-discharge of Ca2+ spikes riding on a Ca2+ plateau potential. 3. The Ca2+-dependent plateau was also promoted by Cs+, 4-aminopyridine (4-AP) and apamin. However, Ca2+ spikes during plateaux were an order of magnitude faster when promoted by Cs+ or 4-AP rather than TEA, and apamin did not promote Ca2+ spikes at all. 4. Ca2+ plateaux but not Ca2+ spikes were blocked by nifedipine. 5. In normal medium all effects of the transient Ca2+ influx during action potentials were attributable to its influence on the slow after-hyperpolarization. The nifedipine-sensitive, sustained Ca2+ influx was expressed exclusively as plateau potentials and only under conditions of reduced K+ current. 6. It is concluded that the transient and the sustained Ca2+ fluxes in spinal motoneurones are curtailed by different K+ conductances. The two Ca2+ responses are suggested as being mediated by two different types of Ca2+ channels.

Calcium channels in hepatocytes

In living cells, the concentration of cytosolic Ca :+ ([Ca2+]i) is maintained at a level ranging from 50 to 200 nM. The cytosolic Ca 2+ is actively extruded from the cell by a Ca2+-pump present in the plasma membrane and/or by Ca2+/Na + exchange. In resting cells, Ca 2+ is accumulated essentially in the endoplasmic ' reticulum and, to a lesser extent, in mitochondria. The [Ca2+]i is about four orders of magnitude lower than extracellular ([Ca2+]o) and intrareticular [Ca-'+]. In these conditions, small variations in Ca 2+ permeabilities of the plasma membrane or the membrane of the intracellular store can induce fast and large variations of the [Ca2+]i. Ca 2+ can then be used as an intracellular signal for the transduction of extracellular signals carried by hormones or neuromediators_ The resulting increase in [Ca2+]i modulates a number of cellular functions such as contraction, secretion and activation of the enzymes of cell metabolism. It is well known that the initial phase of the [Ca2+]i rise initiated by hormones results essentially from the release of Ca ,-+ accumulated in the intracellular store. The sustained phase is due to an influx of extracellular Ca :+ [1,2]. In a stimulated cell, the two Ca 2+ fluxes coming from an intracellular store and from the extracellular medium converge in the cytosol to maintain an elevated [Ca2+]i. The mechanism of the diffusional transport of Ca 2+ through the biological membranes has been the subject of a number of studies. In many cases, it has been shown to result from the activation of specific channels. Different types of Ca 2+ channels have been found in eucaryotes. Those gated by the membrane potential are called voltage-operated channels (VOCs). Others, opened by the binding of ligands (hormones or neurotransmitters) to specific receptors are called receptor-operated channels (ROCs). The VOCs studied by electrophysiological techniques have been differentiated into: T-(transient), N-(neuronal) and L-(long-lasting) channels_ N-channels are present only in neuronal cells, whereas Tand L-channels have been identified in most excitable cells. The L-type channel can be modulated by hormones through different cellular effectors such as cAMP-dependent or cGMP-dependent protein kinases, protein kinase C and G-proteins (see Ref. 3). These L-channels are inhibited by specific inhibitors,

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.