Role Of Calcium Influx Research Articles

Role of calcium influx and intracellular calcium stores in angiotensin II-mediated calcium hyper-responsiveness in smooth muscle from spontaneously hypertensive rats.

To investigate post-receptor mechanisms that underlie enhanced angiotensin II (Ang II)-stimulated cytosolic free Ca2+ concentration ([Ca2+]i) responses in vascular smooth muscle cells from small arteries of SHR. To determine whether Ca2+ influx is altered in SHR, effects of Ca2+ channel antagonists (nitrendipine and diltiazem) and depletion of extracellular Ca2+ on Ang II-stimulated [Ca2+]i responses in primary cultured unpassaged vascular smooth muscle cells from mesenteric arteries of spontaneously hypertensive rats (SHR), Wistar and Wistar-Kyoto (WKY) rats aged 17 weeks were studied. To assess whether Ca2+ stores contribute to increases in Ang II-stimulated Ca2+ mobilization and [Ca2+]i in SHR, cells were exposed to thapsigargin, a selective reticular Ca2+-ATPase inhibitor. [Ca2+]i was measured by fura-2 methodology. Basal and 1 nmol/l Ang II-stimulated [Ca2+]i were significantly greater in SHR cells (123 +/- 7.1 nmol/l basal; 268 +/- 7.0 nmol/l stimulated) than they were in those from WKY rats (88 +/- 4.8 nmol/l basal; 221 +/- 8.6 nmol/l stimulated) and Wistar rats (85 +/- 3.0 nmol/l basal; 216 +/- 8.3 nmol/l stimulated). In Ca2+-free medium, basal and Ang II-induced [Ca2+]i were reduced in all groups, but Ang II-stimulated [Ca2+]i responses were still significantly enhanced in SHR cells compared with those in Wistar and WKY rat cells (205 +/- 11.2 versus 173 +/- 8.0 and 161 +/- 2.6 nmol/l, respectively). Administrations of 10(-6) mol/l diltiazem and 10(-7) mol/l nitrendipine decreased Ang II-elicited [Ca2+]i responses and normalized basal [Ca2+]i in SHR cells. The inhibition induced by Ca2+ channel antagonists was greater (P < 0.05) in WKY and Wistar rat cells than it was in those from SHR. Administration of thapsigargin, in Ca2+-free buffer, induced a greater (P < 0.05) dose-dependent [Ca2+]i increase in SHR cells than it did in WKY rat cells. Administration of 1 nmol/l Ang II increased [Ca2+]i in thapsigargin-pretreated cells of SHR but not in those of WKY rats. Different mechanisms contribute to increases in basal and Ang II-stimulated [Ca2+]i responses in vascular smooth muscle cells from small arteries of SHR, which contribute to elevated peripheral resistance in hypertension. Increases in basal [Ca2+]i may be partly due to augmentation of Ca2+ influx, whereas Ang II-induced [Ca2+]i hyper-responsiveness might depend primarily on Ca2+ mobilization rather than on influx of extracellular Ca2+.

Modulation of mitochondrial Ca 2+ in ECV304 endothelial cells by agents which elevate CAMP

In the human umbilical vein endothelial cell-derived cell line, ECV304, we have previously shown that the elevation of [Ca 2+] m in response to agonist stimulation is dependent on Ca 2+ influx, i.e. an ATP-induced sustained increase in [Ca 2+] c results in a slow-onset, sustained elevation in [Ca 2+] m [Lawrie A.M., Rizzuto R., Pozzan T., Simpson A.W.M. A role for calcium influx in the regulation of mitochondrial calcium in endothelial cells. J Biol Chem 1996; 271: 10753–10759]. In this study, we have investigated the effect of raising CAMP on ATP-evoked elevations in both [Ca 2+] m and [Ca 2+] c by: (i) activating adenylate cyclase with the forskolin analogue — forskolin 6-[3′-(N,N-dimethylamino-propionyl)]-HCI (1 μM) (FA); (ii) addition of membrane permeable dibutyryl-CAMP (100 μM) (dbcAMP); and (iii) a combination of FA plus inhibition of CAMP phosphodiesterase using RO 20–1724 (17.5 μM) (RO);. We have found that protocols aimed at elevating CAMP significantly reduce the ATP-evoked (1–10 μM) rise in [Ca 2+] m ( n = 14); however, the [Ca 2+] c response to ATP was not affected ( n = 33). This new evidence shows that a second messenger system, other than Ca 2+ itself, may influence [Ca 2+]m changes in response to agonist stimulation.

Ca(2+)-activated K+ channels in rat otic ganglion cells: role of Ca2+ entry via Ca2+ channels and nicotinic receptors.

1. Intracellular recordings were made from neurones in the rat otic ganglion in vitro in order to investigate their morphological, physiological and synaptic properties. We took advantage of the simple structure of these cells to test for a possible role of calcium influx via nicotinic acetylcholine receptors during synaptic transmission. 2. Cells filled with biocytin comprised a homogeneous population with ovoid somata and sparse dendritic trees. Neurones had resting membrane potentials of -53 +/- 0.7 mV (n = 69), input resistances of 112 +/- 7 M omega, and membrane time constants of 14 +/- 0.9 ms (n = 60). Upon depolarization, all cells fired overshooting action potentials which were followed by an apamin-sensitive after-hyperpolarization (AHP). In response to a prolonged current injection, all neurones fired tonically. 3. The repolarization phase of action potentials had a calcium component which was mediated by N-type calcium channels. Application of omega-conotoxin abolished both the repolarizing hump and the after-hyperpolarization suggesting that calcium influx via N-type channels activates SK-type calcium-activated potassium channels which underlie the AHP. 4. The majority (70%) of neurones received innervation from a single preganglionic fibre which generated a suprathreshold excitatory postsynaptic potential mediated by nicotinic acetylcholine receptors. The other 30% of neurones also had one or more subthreshold nicotinic inputs. 5. Calcium influx via synaptic nicotinic receptors contributed to the AHP current, indicating that this calcium has access to the calcium-activated potassium channels and therefore plays a role in regulating cell excitability.

Calcium currents and exocytosis in single isolated pars intermedia cells from tilapia (Oreochromis mossambicus)

The role of calcium influx through voltage-dependent calcium ion channels in the exocytotic response of single isolated pars intermedia cells from the teleost tilapia (Oreochromis mossambicus) was investigated by means of the whole-cell patch-clamp technique and high resolution electrical measurements. Calcium currents differed from barium currents in several ways: the peak ICa was smaller, the current-voltage relationship for calcium attained its maximum at +10 mV instead of 0 mV, and the inward calcium current inactivated more rapidly. Electron micrographs showed that pars intermedia cells possess dense, encored vesicles with an average diameter of 140 nm. Influx of calcium resulted in an increased cell membrane capacitance (Cm) after the depolarizing period, indicating a fast exocytotic response. Comparison with “late” recordings revealed the presence of a transient in Cm, presumably attributable to movement of ion channel gates. The average increase in Cm was 13.4 fF, suggesting the fusion of at least 23 vesicles with the plasma membrane during the depolarizing pulse. In contrast to calcium, barium did not support significant exocytosis. We conclude that calcium entry through voltage-dependent calcium channels rapidly leads to the exocytosis of secretory vesicles from tilapia pars intermedia cells.

A role for calcium influx in setting the threshold for CD4+CD8+ thymocyte negative selection.

We have applied an in vitro system that mimics thymic negative selection to investigate signaling pathways that may be important for the removal of autoreactive cells from the thymus. We sought to more precisely determine the contribution of calcium-dependent pathways to CD4+CD8+ thymocyte deletion that is mediated by either an antigenic peptide or a peptide analogue. We show that the requirement for external calcium influx is dependent upon the strength of the deleting ligand. Furthermore, these results correlate well with a requirement, under certain circumstances, for signaling through the calcium/calmodulin-dependent phosphatase calcineurin. The use of suboptimal stimuli may, therefore, be useful in revealing biochemical pathways important for CD4+CD8+ thymocyte negative selection.

Role of calcium influx during the latent period in sea urchin fertilization.

After ∼7-40 s following gamete fusion, a steadily increasing fraction of a sea urchin's zygotes initiate an activating calcium wave. The fertilization membrane then rises, the cell cycle resumes and development begins. This study focuses on the so-called latent period that occurs between the time that gamete fusion occurs and the initiation of the activating calcium wave. We inhibited calcium influx during this period by adding lanthanum or by reducing external calcium with a buffer at various time points after insemination. Both of these treatments blocked the activation of eggs that had not yet started a wave at the time of treatment. This indicates that an influx of calcium is needed during the latent period to induce egg activation. These results support the sperm conduit model of egg activation in the sea urchin, where calcium flows from the sea through the fused sperms' acrosomal process into a cortical region of the eggs' endoplasmic reticulum.

Metabotropic glutamate receptor modulation of synaptic transmission in corticostriatal co-cultures: Role of calcium influx

Modulation of excitatory glutamatergic transmission at corticostriatal synapses by a metabotropic glutamate receptor (mGluR) was examined using a newly developed cell culture preparation in which small explants of cortical tissue are grown in co-culture with isolated striatal neurons. Electrical stimulation of cortical tissue evoked excitatory postsynaptic currents (eEPSCs) observed during tight-seal, whole-cell recordings from striatal neurons. Transmission was mediated by activation of AMPA/kainate-type glutamate receptors. The mGluR agonists, 1 SR,3 RS-ACPD and DCG-IV, reduced eEPSC amplitude. The effect of 1 SR,3 RS-ACPD increased in a concentration-dependent manner. Application of phorbol diacetate (PDAc) potentiated eEPSC amplitude and reduced the inhibitory effect of mGluR activation. Pretreatment with pertussis toxin (PTX) also reduced inhibition by 1 SR,3 RS-ACPD. Under conditions in which transmission was independent of the function of voltage-gated calcium channels, mGluR activation reduced the frequency of occurrence of miniature EPSCs (mEPSCs), but did not alter mEPSC amplitude. This effect of mGluR activation was reduced by PDAc treatment. mGluR activation modulates glutamatergic transmission via a presynaptic autoreceptor at corticostriatal synapses in this newly-developed corticostriatal co-culture preparation as in striatal slices. Modulation of transmission occurs whether or not transmission involves activation of voltage-gated calcium channels. Furthermore, many of the characteristics of mGluR modulation of eEPSCs are shared by mGluR modulation of mEPSCs. These findings indicate that mechanisms downstream from calcium entry may contribute to modulation of synaptic transmission by mGluR autoreceptors.

The role of calcium influx in cellular proliferation induced by interaction of endogenous ganglioside GM1 with the B subunit of cholera toxin

The B subunit of cholera toxin, which binds specifically to ganglioside GM1, is mitogenic for quiescent Swiss 3T3 fibroblasts. It was previously shown that the B subunit had no effect on cAMP, protein kinase C or phosphoinositide turnover, but did cause an increase in the influx of calcium from extracellular sources (Spiegel, S. and Panagiotopoulos, C. (]988) Exp. Cell Res. [77, 414–427). In contrast to the action of known growth factors, the B subunit induced significant DNA synthesis after only a l–3 h treatment. We utilized this unique property to determine whether the increase in calcium influx plays a role in B subunit-induced mitogenicity. Cells were briefly treated with the B subunit in the presence of calcium channel blockers, followed by removal of the blockers and further incubation in B subunit-free medium for the remaining time required to measure DNA synthesis. When 1 mM cobalt was only present during the first 3 h incubation, DNA synthesis induced by either the B subunit or fetal bovine serum was completely abolished. However, both nickel (1 mM) and the L-type voltage-gated calcium channel inhibitor nicardipin (10 μM) inhibited B subunit-induced cell proliferation without abrogating the response to fetal bovine serum. Using a gel retardation assay, we found that the B subunit markedly stimulated specific DNA-binding activity of the transcription factor, activator protein- 1 (AP-1), which functions as a major convergence point coupling early events induced by a variety of mitogens to long term growth responses. Presence of c-Fos protein in the AP-1 complex was demonstrated as a supershift band in the gel mobility assay using c-Fos polyclonal antibody. Cobalt, which markedly inhibited B subunit-induced DNA synthesis, also completely abolished AP-1 DNA-binding activity stimulated by the B subunit. In sharp contrast, cobalt had no effect on DNA-binding activity of AP-1 induced by the tumor promoter, 12- O-tetradecanoylphorbol 13-acetate. Our results suggest that calcium influx is a key element for both DNA-binding activity of AP-1 and cell proliferation induced by binding of the B subunit of cholera toxin to cell surface ganglioside GM1.

Effect of glutathione depletion and oxidative stress on the in vitro cytotoxicity of velnacrine maleate

Velnacrine maleate (Mentane ®) is an aminoacridine drug developed for the treatment of Alzheimer's disease. Although velnacrine maleate has not been observed to cause prominent cytotoxicity in in vitro hepatocyte cultures, this drug was associated with elevated serum levels of hepatic enzymes in clinical trials. The purpose of the present study was to manipulate cultures of rat hepatocytes in an attempt to elicit a cytotoxic response from this drug and to better understand the in vitro mechanisms of action. Cytotoxicity was evaluated by measuring lactate dehydrogenase (LDH) leakage, neutral red (NR) uptake, and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction. Preliminary studies with fluorescent probes did not indicate a role for calcium influx or the formation of reactive oxygen species in the cytotoxicity of velnacrine maleate. However, depletion of cellular glutathione (GSH) by diamide (DA) pretreatment resulted in a cytotoxic response at concentrations of velnacrine maleate (1 and 10 μg/ml) which were ∼ 25- fold lower than those in the absence of DA. Similarly, pretreatment with velnacrine maleate enhanced the cytotoxicity of DA. Pre-exposure of cells to a mixture of DA and t- butyl hydroperoxide ( t- BHP) at non-toxic concentrations resulted in significant cytotoxicity of the hepatocyte cultures by velnacrine maleate. Results from these studies indicate that oxidative stress and GSH depletion may enhance Alzheimer patients' susceptibility to the hepatotoxic potential of aminoacridine drugs.

Stimulation of prolactin release by dopamine withdrawal: role of calcium influx.

Withdrawal of dopamine (DA), a neurotransmitter that inhibits prolactin (PRL) release from the anterior pituitary, stimulates PRL release with transient (30- to 45-min) secretory rates that exceed those observed before application of DA ("PRL rebound"). Using patch-clamp methods on identified rat lactotropes, we have demonstrated that a period of increased Ca(2+)-spiking activity follows recovery from the DA-induced hyperpolarization. The present experiments used dissociated pituitary cells to identify the relative roles of adenosine 3',5'-cyclic monophosphate (cAMP), inositol phosphates, and the enhanced influx of Ca2+ in the rebound secretion of PRL. Rebound secretion of PRL after DA withdrawal was completely blocked by the Ca2+ channel blocker verapamil (20 microM), which also inhibited spontaneous Ca(2+)-spiking activity. DA-induced changes in cAMP levels could be completely dissociated from the PRL rebound. Production of inositol phosphates rose after DA withdrawal but was secondary to the influx of Ca2+. These data demonstrate that influx of extracellular Ca2+ through verapamil-sensitive channels is a critical step in inducing PRL release after DA withdrawal. This finding supports our theory that DA-induced hyperpolarization recruits previously inactivated Ca2+ channels and upon DA washout the enhanced influx of Ca2+ through these voltage-regulated channels supports the rebound release of PRL.

Role of calcium influx and buffering in the kinetics of Ca(2+)-activated K+ current in rat vagal motoneurons.

1. Intracellular recordings were obtained from neurons of the dorsal motor nucleus of the vagus (DMV) in transverse slices of the rat medulla maintained in vitro. These neurons had a resting potential of -59.8 +/- 8.7 (SD) mV. Single action potentials elicited by brief depolarizing current pulses were followed by a prolonged afterhyperpolarization (AHP). Under voltage clamp, the current underlying the AHP was found to be a calcium-activated potassium current. 2. The outward current (GkCa,1) was voltage insensitive and was not blocked by tetraethylammonium (TEA) (10 mM). Unlike the slower time course calcium-activated potassium current recorded in some other neurons, GkCa,1 was blocked by apamin (25-100 nM), indicating that SK type calcium-activated potassium channels underlie this current. 3. GkCa,1 was maximal within 10 ms of the action potential and its decay was well described by a single exponential. After a single action potential the time constant of decay of GkCa,1 was 155 +/- 66 (+/- SD) ms. 4. Calcium influx was increased by adding TEA to the extracellular solution or by firing more than one action potential. As the calcium load was increased, both the peak amplitude and the time constant of decay of GkCa,1 increased. In cells impaled with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-filled electrodes, the time constant of decay of GkCa,1 after a single action current was 71 +/- 19 ms. 5. A simple diffusion-based model that incorporates two intrinsic calcium buffers is developed that accounts for many of the properties of GkCa,1. It is concluded that the decay of GkCa,1 reflects the time course of removal of calcium that has entered the cell during the action potential.

The Contrasting Role of Calcium Influx in Secretion Induced by Cell Swelling Can Differentiate Normal and Tumor-Derived Rat Pituitary Cells*

We have evaluated whether cell swelling may be a generally useful technique to differentiate normal and neoplastic pituitary cells, making the comparison between normal lactotrophs and thyrotrophs and tumor-derived GH4C1 and MMQ cells. With 1.5 mM medium Ca2+, cell swelling induced by osmotically equivalent stimuli, 27% medium hyposmolarity or 80 mM isotonic urea, caused a prompt increase in both intracellular Ca2+ and hormone secretion by all cell types. Depletion of medium Ca2+ abolished the cell swelling-induced increase in intracellular Ca2+ in all cell types and hormone secretion in the tumor-derived cells. However, it enhanced hormone secretion in normal cells. The critical role of Ca2+ influx in osmotically induced secretion in neoplastic, but not normal, pituitary cells may reflect some fundamental alteration in the intracellular transduction system in tumor cells.

P-592 - Role of calcium influx in the excitation-contraction coupling in cardiac myocytes

P-592 - Role of calcium influx in the excitation-contraction coupling in cardiac myocytes

Calcium-dependent phospholipid catabolism and arachidonic acid mobilization in cerebral minces

Cerebral minces were used to investigate the role of calcium influx on trauma-induced alterations of brain lipid metabolism. Cerebral phospholipids, nonpolar lipids, and free fatty acids were radiolabeled in vivo with [3H]arachidonic acid. Tissue incubation stimulated the time-dependent catabolism of choline and inositol glycerophospholipids, and resulted in the accumulation of [3H]free fatty acids. These effects were attenuated in Ca2(+)-free incubations, and when EGTA or verapamil were present. The inhibition of calcium influx also reduced the labeling of diglycerides, whereas ethanolamine and serine glycerophospholipids were not affected by incubation or treatments. Replacing Ca2+ with other cations also attenuated the incubation-dependent alterations in lipid metabolism. However, only cadmium was able to compete with calcium and reduce the accumulation of [3H]free fatty acids. It appeared that about half of the observed phospholipid catabolism was dependent on Ca2+ influx and that at least 80% of the [3H]free fatty acid accumulation required calcium.

Dexamethasone‐induced killing of neoplastic cells of lymphoid derivation: Lack of early calcium involvement

The role of calcium influx in dexamethasone-induced fragmentation of DNA was studied in the glucocorticoid-sensitive human lymphoid line of T cell derivation (CEM-C7). Reduction of calcium content in the medium or the use of EGTA increased DNA fragmentation and appeared to slightly enhance the effect of dexamethasone. Incubation of isolated nuclei in the presence of high concentrations of calcium did not bring about significant DNA fragmentation. Calmidazolium, an antagonist of calmodulin dependent reactions did not reduce the sensitivity of CEM-C7 cells to dexamethasone nor did it modify the response to dexamethasone of the resistant CEM-C1 line. It appears that in contrast to rodent thymocytes, massive calcium influx is not per se responsible for the initiation of directed cell killing (apoptosis).

Comparison of signals delivered through CD3 and CD2 for T-cell activation: The role of calcium influx and interleukin 1

In the absence of monocytes, resting T lymphocytes extensively purified from human peripheral blood failed to proliferate when stimulated with a mixture of calcium ionophore, which elevates intracellular calcium levels, and TPA, which activated protein kinase C. A third signal, i.e., the triggering via CD3 or CD2 molecules, was necessary in order to observe proliferation. These highly purified T cells required the presence of monocytes in both CD3 and CD2 systems for their proliferation. Exogenous interleukin 1 clearly substituted for monocytes in CD2- but not in CD3- triggered T-cell proliferation. In contrast, the effect of CD2 and CD3 antibodies on Ca ++ influx was apparently not dependent on the presence of monocytes. In the presence or absence of the monocytes, CD3, as well as certain combination of CD2 monoclonal antibodies including the D66 monoclonal antibody, were able to increase the intracellular calcium concentration as measured by Quin 2 fluorescence. EGTA, a Ca + chelator, completely inhibited CD2- and CD3- mediated T-cell proliferation, indicating that calcium uptake is necessary during the T-cell proliferation. The addition of TPA abrogated the inhibitory effect of EGTA and completely restored the response of the T cells stimulated by CD3, but not by CD2, monoclonal antibodies. In the CD2 pathway, EGTA-inhibited proliferation of T cells could be completely restored by addition of exogenous interleukin 2 as well as exogenous recombinant interleukin 1. Our results indicate that EGTA inhibits the production of interleukin 1 but has no direct effect on either interleukin 2 production or on Tac antigen expression. In this system, recombinant interleukin 1 alpha demonstrated a more potent ability for restoring the T-cell response than did recombinant interleukin 1 beta. These results suggest that interleukin 1 could act as a potent costimulatory factor in the non-antigen-specific T-cell activation.

Regulation of sterol carrier protein 2 (SCP 2) levels in the soluble fraction of rat Leydig cells. Kinetics and the possible role of calcium influx

The rate-determining step in steroidogenesis is the conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage enzyme. The transport of substrate for this reaction may be facilitated by sterol carrier protein 2(SCP 2). In rat testis tissue SCP 2 is specifically localized in the Leydig cells and tissue levels of SCP 2 are regulated by luteinizing hormone (LH). The present study concerns short-term regulation of SCP 2 in isolated rat Leydig cells. Levels of SCP 2 in the membrane-free supernatant are increased 2-fold already after 2 min incubation with LH and remain elevated for 24 h. The same response occurs with cells preincubated in the presence of cycloheximide for 4 h. SCP 2 levels are also 2-fold increased after incubation with dibutyryl cAMP or 4β-phorbol 12-myristate 13-acetate (PMA) whereas these compounds stimulate steroid production 5.5- and 2-fold respectively. Luteinizing hormone releasing hormone (LHRH), which can stimulate steroid production more than 3-fold, does not influence SCP 2 levels, neither are SCP 2 levels altered when LH is added in the presence of the Ca 2+-channel blocker diltiazem or in the absence of extracellular Ca 2+. A restoration of the LH effect on SCP 2 levels was already obtained in the presence of 1 μM extracellular Ca 2+. These results suggest that Ca 2+ influx through the plasma membrane may play an important role in the control of SCP 2 levels. In most of the experiments no correlation between steroid production and SCP 2 levels could be observed. The soluble amount of SCP 2 is probably not the rate-limiting factor in the control of cholesterol side-chain cleavage activity but SCP 2 in the membrane-free supernatant may play a permissive role.

Role of calcium in pathogenesis of acute renal failure.

The potential for calcium to play a key role in cell injury has been long suspected. Major sites of calcium action to promote cell injury include the plasma membrane, mitochondria, endoplasmic reticulum, and the cytoskeleton. Major mechanisms of calcium action to promote cell injury include activation of phospholipases, direct and indirect effects on permeability pathways, and effects on contractile and cytoskeletal structure and function. The activation of phospholipases and deterioration of mitochondrial structure and function by calcium appear to be most important in the evolution of cell injury. Tissue calcium levels invariably increase when lethal cell injury develops in a tissue and is due predominantly to mitochondrial accumulation and sequestration. The simultaneous occurrence of cell calcium overload and lethal cell injury, however, only establishes an association between these two events but does not prove causality. Over the past several years, a large amount of data has established that calcium plays a critical modifying role in the pathogenesis of both ischemic and toxic cell injury, but evidence for the thesis that calcium is the "final common pathway" for lethal cell injury is not conclusive. Many studies have emphasized the role of calcium influx from extracellular to intracellular spaces with resulting cellular calcium overload in cell injury. A critical role for intracellular redistribution of calcium pools rather than cellular calcium influx during the important early stages of cell injury may be more important. Modifying alterations in cell calcium redistribution or cellular calcium influx with a variety of agents has been beneficial in ameliorating the degree of cell injury in a number of experimental settings. It is still unclear whether these beneficial effects are due mainly to alterations of calcium-mediated processes that determine the reversibility of injury or are due to alterations in other critical metabolic processes not importantly influenced by calcium.

Ionic control of locomotion and shape of epithelial cells: I. Role of calcium influx.

The role of calcium in the induction of locomotion, control of direction of locomotion, and modulation of shape of epithelial cells derived from Xenopus laevis tadpole epidermis is investigated. Local influx of calcium is achieved by electrophoretic release of small amounts of calcium from a micropipette (tip diameter 0.1-0.5 micron) closely apposed to the cell body or lamella. The cells are made permeable for calcium by calcium ionophore A23187, and they are kept in Ca++-free, Mg++-rich EGTA Ringer. Another method used to induce Ca++ influx is local application of A23187 while cells move in normal culture medium. Influx of Ca++ into the lamella induces a localised increase in thickness and enlargement of the lamella. Stationary cells become active and show movement in the direction of the Ca++ gradient. Fried-egg-shaped cells tend to acquire a semicircular shape and start moving. Moving cells change the direction of their locomotion, following the direction of Ca++ release. Influx of Ca++ in the cell body region induces its contraction concomitant with an increase in lamellar area. These observations suggest the presence of two different Ca++-sensitive components: an actomyosin meshwork in the cell body and an actin gel in the lamella. Influx of Ca++ induces contraction of actomyosin and solation of actin gel. Interaction of these two systems would explain modulation of shape and generation of locomotion in epithelial cells.

Regulation of calcium influx into buccal muscles of Aplysia by acetylcholine and serotonin.

Acetylcholine (ACh) causes contraction of Aplysia buccal muscles E1 and I5, and serotonin (5-hydroxytryptamine, 5-HT) enhances ACh-elicited contractions of these muscles. Possible roles of calcium influx in mediating these responses were examined by studying influx of 45Ca++. 5-HT increased calcium influx into both I5 and E1. Maximal influx occurred at 10(-6) M 5-HT and the increased influx could be sustained in the presence of 5-HT for at least 10 min. ACh also caused calcium influx, and calcium influx increased approximately in proportion to log[ACh] from 10(-5) M to 10(-3) M ACh. 5-HT and ACh probably bring about calcium influx by different mechanisms since the effect of ACh was additive to a maximal 5-HT response, and 10(-4) M hexamethonium bromide inhibited the increased influx caused by ACh but did not affect influx caused by 5-HT. Cyclic AMP analogues and forskolin neither caused an increase in calcium influx nor an increase in the influx caused by ACh. The data support a model in which ACh-elicited contractions of I5 and E1 are due primarily to calcium entry across the extracellular membrane, and 5-HT can "load" an intracellular site by a mechanism different from that activated by ACh. The data do not support a role for cyclic AMP in mediating the calcium influx response to 5-HT.