Intracellular Free Calcium Ion Concentration Research Articles

Single cell observation of calcium signals in class II specific killer T cells.

A confocal fluorescence microscope with an argon ion laser and a digital imaging fluorescence microscope were used to study calcium signals in class II-specific killer T cells after interaction with target cells. Here, we used two kinds of I-Ak specific allogeneic killer T cell clones, QM11 (CD8+) and QM56 (CD4+), and target cells, B10.A spleen cells (I-Ak) and B cell hybridomas (TP67.21, I-Ak). After interaction with target cells, the intracellular free calcium ion concentration ([Ca2+]i) in QM11 cells (CD8+) increased rapidly with short lag-times (several seconds). The [Ca2+]i in QM56 cells (CD4+), however, increased with much longer lag-times (hundreds of seconds). The results were consistent with previous findings that the cytolytic activity of QM11 cells (CD8+) was much greater than that of QM56 cells (CD4+).

Alteration of the L-type calcium current in guinea-pig single ventricular myocytes by heptaminol hydrochloride.

1. The effects of heptaminol on calcium current amplitude and characteristics were studied in single ventricular myocytes of guinea-pig by use of the whole cell configuration of the patch clamp technique. 2. A concentration-dependent decrease in ICa amplitude was observed. At heptaminol concentration as low as 10(-6) M, this effect was observed in only two cells (n = 6). At 10(-5) M the reduction of ICa was of 30 +/- 15% (n = 11). 3. The current recovery from inactivation at -40 mV holding potential (HP) seemed less sensitive to perfusion with heptaminol (greater than 10(-6) M). However, at -80 mV HP the overshoot of the recovery curve was decreased by heptaminol. 4. Both at -40 mV and -80 mV HP, heptaminol (10(-5) M) significantly increased the steady state inactivation of ICa. 5. As previously proposed by others to explain the effects of membrane active substances, the effects of heptaminol may result from alterations in cell membrane properties and possibly from an increase in intracellular free calcium ion concentration.

Superantigen staphylococcal enterotoxin B-induced T-helper cell activation is independent of CD4 molecules and phosphatidylinositol hydrolysis.

The role of the CD4 molecule in activation of T-helper cells was examined by investigating the effect of an anti-CD4 monoclonal antibody (Leu3a) in conventional peptide antigen-specific cloned T-helper cells that are also reactive to staphylococcal enterotoxin B (SEB). These T-helper cell clones are CD4+/CD45RO+/T-cell antigen receptor beta-chain variable region 12-positive and can respond to nominal peptide antigens and SEB by proliferation in the presence of class II major histocompatibility complex-expressing accessory cells. Although antigen and SEB were comparable in their ability to induce proliferative responses, interleukin 2 (IL-2) production, and IL-2 receptor alpha-chain expression, stimulation with SEB failed to trigger phosphatidylinositol hydrolysis or a rise in the intracellular free calcium ion concentration. Leu3a treatment inhibited antigen-induced proliferative responses of T cells with concomitant suppression of IL-2 production and IL-2 receptor expression. In contrast, SEB-induced responses were unaffected by Leu3a. These findings indicate that the functional consequences of binding (ligation) of conventional antigen and of superantigen with the T-cell receptor are distinct in the context of both signal transduction pathways and participation of CD4 molecules.

A fluorescent molecular rotor probes the kinetic process of degranulation of mast cells.

A confocal fluorescence microscope was used to study the exocytotic secretory processes of mast cells in combination with an fluorescent molecular rotor, 9-(dicyanovinyl)julolidine (DCVJ). DCVJ is known to be an unique fluorescent dye which increases its quantum yield with decreasing intramolecular rotation. Here, DCVJ-loaded peritoneal rat mast cells were stimulated with compound 48/80 and their fluorescence images were compared with fluorescence calcium images of fluo-3-loaded mast cells. Subsequent to transient increases in intracellular free calcium ion concentration, DCVJ fluorescence increased dramatically in the cytoplasm and formed a ring-like structure around the nucleus, suggesting the possibility that the dye bound to the proteins composing the cytoskeletal architecture. Furthermore, the increases of DCVJ fluorescence intensities were mostly blocked in the presence of cytochalasin D (10 microM). However, fluo-3 fluorescence intensities still increased after addition of compound 48/80.

Regulation of programmed death in erythroid progenitor cells by erythropoietin: effects of calcium and of protein and RNA syntheses.

Erythropoietin (EPO) retards DNA breakdown characteristic of programmed cell death (apoptosis) and promotes survival in erythroid progenitor cells. The mechanism by which EPO inhibits programmed death is unknown. In the well-characterized model of glucocorticoid-treated thymocytes, activation of a Ca2+/Mg(2+)-sensitive endonuclease and new protein and RNA syntheses have been found necessary for apoptosis. We examined the effects of EPO on the free intracellular calcium ion concentration ([Ca2+]i), and the roles of Ca2+ and RNA and protein syntheses on DNA cleavage in erythroid progenitor cells. The murine model of erythroid differentiation using Friend leukemia virus-infected proerythroblasts (FVA cells) was used. EPO did not affect the [Ca2+]i in FVA cells. Decreasing [Ca2+]i by extracellular Ca2+ chelation with EGTA facilitated DNA breakdown. Increasing [Ca2+]i with the calcium ionophore 4-bromo-A23187 increased DNA cleavage; however, DNA fragments generated by high [Ca2+]i were much larger than those seen in the absence of EPO or presence of EGTA. Increased [Ca2+]i also inhibited DNA breakdown to small oligonucleosomal fragments characteristic of cells cultured without EPO. However, no concentration of ionophore protected the high molecular weight DNA as did EPO. Cycloheximide inhibited DNA breakdown in a dose dependent manner in cultures lacking EPO, but two other protein synthesis inhibitors, pactamycin and puromycin, did not prevent DNA breakdown. Inhibition of RNA synthesis with actinomycin D did not prevent DNA breakdown. Cells with morphological characteristics similar to those reported in other cells undergoing programmed death accumulated in EPO-derived cultures. These studies demonstrate that although DNA cleavage and morphological changes are common to apoptotic cells, the roles for Ca2+ and protein and RNA syntheses are not universal and suggest that apoptosis can be regulated by different biochemical mechanisms in different cell types.

Activation of protein kinase C reduces L-type calcium channel activity of GH3 pituitary cells.

These studies describe the effect of protein kinase C (PKC) activation on the activity of voltage-sensitive L-type Ca2+ channels of GH3 pituitary cells. The rate of 45Ca2+ uptake was stimulated greater than 25-fold by depolarization in the presence of BAY K 8644; the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) reduced this response by 70% in a concentration-dependent fashion. Phorbol 12,13-dibutyrate (PDBu) inhibited depolarization-induced 45Ca2+ uptake within 1 min and caused a nearly maximal reduction after 1 h; its effects were rapidly reversible. TPA decreased the high K(+)-stimulated increase in intracellular free calcium ion concentration ([Ca2+]i) from 8.5- to 3.2-fold by 5 min and to 2.0-fold after 18 h without altering the peak [Ca2+]i response to the peptide hormone TRH. Ca2+ channel current, measured directly using the whole cell configuration of the patch-clamp technique, declined an average of 6.4% over 5 min for control cells and 28.9% when TPA was added to the bathing medium for 5 min. Treatment with 100 nM TPA for 24 h dramatically reduced peak current without shifting the peak of the current-voltage relationship. The mean peak Ca2+ channel current was reduced from 423 to 128 pA, although a few cells seemed completely resistant. To determine whether the effects of phorbol esters were due to the activation of PKC we tested the potency of several drugs to inhibit L-channel activity and to shift the affinity of the epidermal growth factor (EGF) receptor, an established PKC response.(ABSTRACT TRUNCATED AT 250 WORDS)

Internal calcium release and activation of sea urchin eggs by cGMP are independent of the phosphoinositide signaling pathway.

We show that microinjecting cyclic GMP (cGMP) into unfertilized sea urchin eggs activates them by stimulating a rise in the intracellular free calcium ion concentration ([Ca2+]i). The increase in [Ca2+]i is similar in both magnitude and duration to the transient that activates the egg at fertilization. It is due to mobilization of calcium from intracellular stores but is not prevented by the inositol trisphosphate (InsP3) antagonist heparin. Furthermore, cGMP does not stimulate the eggs Na+/H+ antiport when the [Ca2+]i transient is blocked by the calcium chelator bis-(O-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), suggesting that cGMP does not activate eggs by interacting with the their phosphoinositide signaling pathway. However, the [Ca2+]i increase and activation are prevented in eggs in which the InsP3-sensitive calcium stores have been emptied by the prior microinjection of the InsP3 analogue inositol 1,4,5-trisphosphorothioate. These data indicate that cGMP activates eggs by stimulating the release of calcium from an InsP3-sensitive calcium store via a novel, though unidentified, route independent of the InsP3 receptor.

Responses of the solitary olfactory receptor cell of the guinea pig.

Olfactory receptor cells were isolated from the nasal mucosa of the guinea pig, using enzymatic digestion and mechanical dissociation. Under a phase-contrast microscope, the cells can be readily distinguished from other tissues, because of morphological characteristics. The intracellular free calcium ion concentrations ([Ca2+]i) of these cells were determined using the Ca2+ sensitive dye fura-2 and digital imaging microscopy. In the presence of 1 microM ionomycin, calcium-ionophore, there was an irreversible increase in [Ca2+]i in all the cells. During prolonged stimulation by 10 mM n-Amyl acetate under standard conditions, 16 of the 40 isolated olfactory cells responded with [Ca2+]i. There was no evidence of any increase in [Ca2+]i in the olfactory receptor cells in nominally Ca2+ free solution and stimulated by amyl acetate. When 3 mM CaCl2 was added, the [Ca2+]i increased remarkably. When cells were stimulated with amyl acetate in a CaCl2-depleted extracellular fluid supplemented with 10 microM verapamil (an L-type voltage-dependent calcium channel antagonist), no change occurred in [Ca2+]i. The addition of CaCl2 to this fluid did not elevate [Ca2+]i, in any cell. This would suggest that the [Ca2+]i increase in the presence of amyl acetate may mainly depend on the influx of calcium from the extracellular space. Our finding that the influx of extracellular calcium into cells was suppressed by verapamil suggests that the calcium channel, which is opened by amyl acetate, is a voltage-dependent L-type Ca2+ channel.

Intracellular free calcium concentrations in single taste receptor cells in the guinea pig.

Single, viable taste receptor cells were isolated from the tongue of the guinea pig by enzymatic digestion and mechanical dissociation. The cells could be classified into flask, spindle and intermediate shapes. The intracellular free calcium ion concentrations [(Ca2+)i] of these cells were determined using the Ca2+ sensitive dye fura-2 and digital imaging microscopy. All types of cells produced an irreversible increase in (Ca2+)i upon addition of Ca2+ ionophore ionomycin (1 microM) and denatonium (10 microM). There was no evidence of any increase in (Ca2+)i in the taste receptor cells in nominally Ca2+ free solution, and when stimulated by denatonium (10 microM). When 3 mM CaCl2 was added, the (Ca2+)i remarkably increased. This would suggest that the (Ca2+)i increase in the presence of denatonium mainly depended on calcium influx from the extracellular space. There was no increase in case of high potassium (50 mM and 150 mM) or saccharose (1 mM and 5 mM) stimulation. The hypothesis that the increase in (Ca2+)i controls biochemical mechanisms related to the bitter taste transduction process is worthy of further study.

Free ca2+ gradient in growing pollen tubes of lilium

ABSTRACT Fluorescence ratiometric imaging of Lilium pollen tubes loaded with the Ca2+ indicator Fura-2 dextran has revealed a distinct elevation of free intracellular calcium ion concentration ([Ca2+]i) at the extreme tip of actively growing Lilium pollen tubes that declines to a uniform basal level of ∼170 nM throughout the length of the tube. The calcium gradient occurs within the first 10–20 μM proximal to the tip. Experimental inhibition of tip growth, usually achieved through the injection of the Ca2+ buffer 5,5’-dibromo BAPTA, results in the loss of the [Ca2+]igradient. Occasionally these inhibited cells reinitiate growth, and when they do so ratio imaging reveals that the tip gradient of free [Ca2+Ji re-emerges. The results presented here are very different from those previously published by revealing the presence of the [Ca2+]i gradient that is restricted to the 10–20 μM adjacent to the tube tip. Further, these experiments demonstrate a strict correlation between the presence of a [Ca2+Ji gradient, and tip growth in Lilium pollen tubes.

Dextran sulfate induces changes in the free intracellular calcium ion concentration of a subpopulation of immature thymocytes

Previous studies have shown that certain sulfated polysaccharides, such as dextran sulfate (DxS), can induce a rapid and sustained increase in the free intracellular calcium ion concentration ([Ca2+]i) of thymocytes, whereas they cannot induce a change in the [Ca2+]i of peripheral lymphocytes. This study examined this phenomenon in more detail and clearly demonstrated that only immature thymocytes can respond to DxS. Cortisone-resistant thymocytes failed to exhibit an increase in [Ca2+]i when exposed to DxS. Furthermore, analysis of [Ca2+]i changes at the single cell level using a flow cytometer demonstrated that only a subpopulation of immature thymocytes responded to DxS. Additional flow cytometry studies revealed that DxS and Con-A induce a [Ca2+]i response in different subpopulations of thymocytes, approximately 30% of thymocytes responding to Con-A and 15-20% to DxS. Thus, responsiveness to Con-A and DxS, in terms of increases in [Ca2+]i, may represent a novel marker for two different thymocyte subpopulations. Such metabolic markers could be used to complement the conventional serological methods currently employed to define stages in thymocyte differentiation.

Pharmacological effecs of oral E6123, a novel PAF antagonist, on biological change induced by PAF inhalation in guinea pigs

The effects of a newly synthesized PAF antagonist E6123, (S)-(+)-6-(2-chlorophenyl)-3-cyclopropanecarbonyl-8, 11-dimethyl-2,3,4,5-tetrahydro-8H-pyrido [4′,3′:4,5]thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine, on in vivo inhaled PAF-induced pulmonary changes were investigated. E6123 inihibited PAF inhalation-induced bornchoconstriction in guinea pigs with an ED 50 value (p.o) of 1.3 μg/kg which was lower than those of other PAF-antagonists such as WEB2347 (ED 50 = 26 μg/kg) and Y-24180 (ED 50 = 12 μg/kg). E6123 significantly inhibited PAF inhalation-induec eosinophil infiltraton into the bronchiole and trachea, and bronchial hyperreactivity in guinea pigs after oral administration at 1 and 10 μg/kg, respectively. E6123 inhibited the PAF-induced incrased intracellular free calcium ion concentration ([Ca 2+]i) in guinea pig eosinophils with an IC 5 value of 14 concentration ([Ca 2+]) in guinea pig eosinophils with an IC 50 value of 14 nM. The present results suggest that E6123 may be beneficial for the treatment of asthma, in which PAF is assumed to be involved.

Temperature-dependence of LDL binding and activation of human platelets

The effect of low density lipoprotein (LDL) on intracellular free calcium ion concentration ([Ca 2+] i), taken as an index of the degree of platelet activation, was investigated in normal volunteers. At 37°C LDL, in a dose of 20 μg of protein / ml, increased [Ca 2+] i in all subjects tested (basal 57±11 to 113±19 nM). In contrast, when measurements were performed at 20°C, no effect on [Ca 2+] i was seen following LDL. Thrombin (0.2 U / ml) increased [Ca 2+] i to 455±98 nM. When platelets had been exposed to LDL before thrombin stimulation, this increase was less pronounced (to 301±43 nM). Our finding of a temperature dependence of LDL induced increase in platelet [Ca 2+] i supports the concept of a platelet-LDL receptor mediated mechanism. Furthermore, the lower thrombin response following LDL exposure suggests a LDL-thrombin interaction, possibly at the thrombin receptor level and/or calcium recruitment from the same stores.

Oscillatory cytosolic calcium waves independent of stimulated inositol 1,4,5-trisphosphate formation in hepatocytes

The mechanisms underlying agonist-induced oscillations in intracellular free calcium ion concentration ([Ca2+]i) in hepatocytes were investigated by utilizing tert-butyl hydroperoxide (TBHP) as a tool to perturb hepatocyte Ca2+ homeostasis independent of receptor activation. In permeabilized hepatocytes, TBHP inhibited Ca2+ uptake into the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pool and increased the sensitivity to InsP3 for Ca2+ release. The effects of TBHP could be mimicked by addition of oxidized glutathione (GSSG) and reversed by pretreatment with dithiothreitol. TBHP and GSSG had no effect on the metabolic degradation of [3H]InsP3 in permeabilized cells. The effect of TBHP on [Ca2+]i in intact cells was investigated by digital imaging fluorescence microscopy of Fura-2-loaded primary cultured hepatocytes. TBHP treatment initiated a series of [Ca2+]i oscillations similar to those caused by Ca2(+)-mobilizing hormones. Moreover, in common with the actions of hormones in these cells (Rooney, T.A., Sass, E., and Thomas, A,P. (1990) J. Biol. Chem. 265, 10792-10796), the [Ca2+]i oscillations induced by TBHP propagated through the cell as Ca2+ waves, originating from a discrete subcellular locus identical to that for phenylephrine-induced [Ca2+]i oscillations. The Ca2+ waves induced by TBHP had similar rates of progress (24-27 microns.s-1) to those generated by phenylephrine. Removal of extracellular Ca2+ increased the initial latency of the TBHP responses, but had no effect on the amplitude or rate of propagation of the Ca2+ waves. Addition of TBHP to cells in the presence of phenylephrine converted the oscillatory phenylephrine [Ca2+]i response into a sustained [Ca2+]i increase. The effects of TBHP in intact cells occurred in the absence of any stimulated inositol polyphosphate formation as measured in populations of [3H]inositol-labeled hepatocytes. The data indicate that spatially organized [Ca2+]i oscillations in intact hepatocytes can occur without any requirement for phospholipase C activation. Furthermore, for agents that act by mobilizing Ca2+ from the InsP3-sensitive pool, the kinetics of the Ca2+ release phase of the [Ca2+]i oscillations appears to be independent of the nature of the stimulus.

Calcium homeostasis in bovine somatotrophs: calcium oscillations and calcium regulation by growth hormone-releasing hormone and somatostatin

The free intracellular calcium ion concentration ([Ca 2+] i) was measured in single cells of a population containing 65–80% somatotrophs, using the fluorescent Ca 2+-indicator Fura-2 and digital imaging microscopy. Spontaneous oscillations in [Ca 2+] i ranging in frequency up to 1.5 oscillations per minute were observed in 30% of somatotrophs. These Ca 2+ oscillations were blocked by the Ca 2+ channel blocker CoCl 2 and were thus proposed to be the result of influx of Ca 2+ into the cell, possibly as the result of spontaneous electrical activity [1]. GHRH (10–100 nM) increased [Ca 2+] i in 61% of the cells studied, although the amplitude and dynamics of the response varied from cell to cell. Typically [Ca 2+] i rose from 170 ± 26 nM to 321 ± 44 nM (n = 13) in response to a challenge with 66 nM GHRH. GHRH also increased the frequency of Ca 2+ oscillations in a number of cells, and some previously quiescent cells showed Ca 2+ oscillations following addition of GHRH. Forskolin, which raises cAMP levels in bovine anterior pituitary cells, also stimulated a sustained rise in [Ca 2+] i in 10 out of 14 cells tested. Somatostatin (SS) (10–80 nM) rapidly reduced basal [Ca 2+] i, blocked Ca 2+ oscillations, and blocked the [Ca 2+] i response to GHRH. The Ca 2+ channel blocker CoCl 2 (4 mM) had similar actions on [Ca 2+] i to those of SS. These results suggest that GHRH and SS may regulate GH release by modulating Ca 2+ entry into the cell through the cell membrane. The [Ca 2+] i oscillations seen in a proportion of the somatotrophs were modulated in frequency by GHRH and SS, and are probably generated by influx of Ca 2+ through channels in the cell membrane. Thus GH secretion may be regulated by changes in the mean level of [Ca 2+] i, which in turn, may be influenced by the frequency of [Ca 2+] i oscillations in bovine somatotrophs.

Lysophosphatidylcholine increases cytosolic calcium in ventricular myocytes by direct action on the sarcolemma

Lysophosphatidylcholine (LPC) accumulates in myocardial tissues during ischemia, and has toxic effects which may contribute to the arrhythmias and relaxation abnormalities that occur during acute ischemia. These effects of LPC may be mediated in part by calcium overload. To test this hypothesis, spontaneously contracting cultured embryonic chick ventricular myocytes were superfused with various concentrations of LPC (10, 50 and 100 μ m) while effects on contractile motion (video motion detector) and changes in free intracellular calcium ion concentration ([Ca 2+] i indo-1 fluorescence) were determined. At concentrations greater than or equal to 10 μ m, a dose-related, time-dependent effect occurred after exposure to LPC, consisting of the development of contracture and marked elevation of [Ca 2+] i. LPC also produced a dose-related, time-dependent inhibition of K + uptake, indicating there was inhibition of the Na +K + ATPase Na + pump. However, the LPC-induced increase in [Ca 2+] i was not due to Na + overload caused by inhibition of the Na +K + ATPase Na + pump because superfusion with a zero-Na + solution did not prevent and increase in [Ca 2+] i after LPC exposure; and the increase in [Ca 2+] i after exposure to LPC occurred too rapidly to be accounted for by Na + pump inhibition. Removal of extracellular Ca 2+ prevented the rise in [Ca 2+] i, after exposure to LPC but treatment with verapamil failed to inhibit the increase in [Ca 2+] i induced by LPC. We conclude that LPC produces contracture due to an increase [Ca 2+] i. These effects are seen at concentrations of 10 μ m and greater, are not due to altered Na +K + ATPase Na + pump or calcium channel function, and are probably related to the detergent properties of this amphiphile. There effects may account in part for myocardial dysfunction during ischemia in intact tissue.

Isolation of and Calcium Mobilization in Vestibular Hair Cells of the Guinea Pig (With 1 color plate)

Vestibular hair cells were isolated from the macula utriculi, macula sacculi and crista ampullaris of the guinea pig, using enzymatic and mechanical techniques. The cells could be classified into two types: flask-shaped ones with a neck and rod- or round-shaped ones without a neck. The flask-shaped cells were thought to be type I cells, the rod-shaped cells type II cells, while the round-shaped cells could have been type I or type II cells. The intracellular free calcium ion concentrations [( Ca2+]i) of these cells were determined using the Ca2(+)-sensitive dye Fura-2 and digital imaging miroscopy. In the resting state, [Ca2+]i in the nucleus and cuticular plate was variable in both types of cells. The 150 mM KCl stimulation, which might induce a depolarization, resulted in a temporary increase in [Ca2+]i in both types of cells. In the presence of 1 micron ionomycin, there was an irreversible increase in [Ca2+]i in both types of cells. These observations aid in elucidating vestibular function.

Isolation of and Calcium Kinetics in Cochlear Inner Hair Cells of the Guinea Pig

Single inner hair cells of the guinea pig cochlea were isolated using enzymatic and mechanical techniques. The intracellular free calcium ion concentrations [( Ca2+]i) of the isolated inner hair cells were determined using the Ca2+ sensitive dye fura-2 and digital imaging microscopy. In the presence of 1 micron ionomycin, a Ca2+ ionophore, there was an irreversible increase in [Ca2+]i. The 150 mM KCl stimulation, which induces a depolarization, resulted in a temporary increase in [Ca2+]i. This increase in [Ca2+]i was not observed under conditions of depolarization, in Ca(2+)-free medium. These observations are interpreted to mean that the [Ca2+]i during membrane depolarization mainly originates from an influx of extracellular Ca2+ into the cytoplasm.

Intracellular calcium concentration of acinar cells in feline tracheal submucosal glands

We measured the intracellular free calcium ion concentration [( Ca2+]i) of acinar cells in isolated feline tracheal submucosal glands in response to secretagogues using the Ca2(+)-sensitive fluorescent dye fura-2. The secretagogues included cholinergic, adrenergic agonists, substance P (SP), and vasoactive intestinal polypeptide (VIP) which induce mucus glycoprotein secretion from feline tracheal submucosal glands. Methacholine (MCh) produced a significant increase in [Ca2+]i of up to 9.8 times that of control in a dose-dependent fashion at concentrations of 10(-8) to 10(-3) M. [Ca2+]i increase by MCh reached a peak within 30 s after stimulation and thereafter showed a sustained rise. In Ca2(+)-free medium, MCh produced an initial transient rise, which was less than 30% of that in a Ca2(+)-containing solution, and which lasted for 60 s with no prolonged sustained rise in [Ca2+]i. Atropine abolished MCh-evoked [Ca2+]i increase. Phenylephrine and SP produced a prolonged increase in [Ca2+]i without an initial transient increase. Phenylephrine (up to 10(-4) M) evoked an increase in [Ca2+]i by up to 240% that of control, which was abolished by prazosin. SP (up to 10(-4) M) also evoked an increase in [Ca2+]i by 155% that of control, which was abolished by atropine. By contrast, both isoproterenol (up to 10(-5) M) and VIP (up to 10(-5) M) failed to alter [Ca2+]i. These findings indicate that the mucus glycoprotein secretion evoked by muscarinic cholinergic, alpha-adrenergic agonist or SP can be mediated by intracellular Ca2+, whereas that by beta-adrenergic agonists or VIP cannot.

Free cytosolic calcium during spontaneous contractions in smooth muscle of the guinea-pig mesotubarium

The free intracellular calcium ion concentration ([Ca2+]i) was measured simultaneously with isometric force in strips of guinea-pig mesotubarium using the Fura-2 technique. During the relaxed period (5-15 min) between spontaneous contractions [Ca2+]i continues to decrease after full mechanical relaxation to reach a minimal level of 86 +/- 8 nM (n = 9) just before the start of the next contraction. During the spontaneous contractions (5-15 min) [Ca2+]i reached a maximum of 211 +/- 19 nM and then oscillated between 155 +/- 16 nM and 194 +/- 9 nM. Increased extracellular Ca2+ concentration to 10 mM from the standard concentration of 1.5 mM caused a decreased frequency of spontaneous contractions and an increase in [Ca2+]i both in the relaxed and contracted states. In 10 mM extracellular Ca2+, addition of AlF4-, as 1 mM NaF + 10 microM AlCl3, caused a sustained increase in [Ca2+]i and maintained force. Addition of verapamil (10 microM) in this situation decreased [Ca2+]i to the resting level. The results suggest that the cyclic appearance of trains of action potentials is related to variation in [Ca2+]i, possibly via inactivation of Ca2(+)-dependent K+ channels.