Rise In Intracellular Free Calcium Research Articles

Effects of metalloendoproteinase inhibitors on secretion and intracellular free calcium in bovine adrenal chromaffin cells

The possible role of metalloendoproteinase in stimulus-secretion coupling in adrenal chromaffin cells was examined using the metalloendoproteinase inhibitors 1,10-phenanthroline and carbobenzoxy-Gly-Phe-NH 2. Catecholamine release elicited by nicotine or by depolarisation with 55 mM K + was almost completely abolished by 0.5 mM 1,10-phenanthroline. Carbobenzoxy-Gly-Phe-NH 2 (2.5 mM) inhibited catecholamine release in response to nicotine but enhanced that due to 55 mM K +. The rise in intracellular free calcium, [Ca 2+] i, in response to either nicotine or 55 mM was inhibited by about 50% by both inhibitors. One site of action of metalloendoproteinase inhibitors may, therefore, be at the level of the regulation of [Ca 2+] i. Catecholamine release and the rise in [Ca 2+] i elicited by the calcium ionophore ionomycin were not reduced by the inhibitors. These results show that metalloendoproteinase inhibitors have complex effects on chromaffin cells including effects on the regulation of [Ca 2+] i but do not inhibit calcium-activated exocytosis itself.

Pressure injection of calcium both excites and adapts Limulus ventral photoreceptors.

Single pressure injections of 1-2 mM calcium aspartate into the light-sensitive region of Limulus ventral photoreceptors resulted in a rapid, 20-40-mV depolarization lasting approximately 2 s. The depolarization closely followed the rise in intracellular free calcium caused by the injection, as indicated by aequorin luminescence. The depolarization was followed by reversible desensitization (adaptation) of responses to both light and inositol 1,4,5 trisphosphate. Similar single injections of calcium into the light-insensitive region of the receptor were essentially without effect, even though aequorin luminescence indicated a large, rapid rise in intracellular free calcium. The depolarization caused by injection of calcium arose from the activation of an inward current with rectification characteristics and a reversal potential between +10 and +20 mV that were similar to those of the light-activated conductance, which suggests that the same channels were activated by light and by calcium. The reversal potentials of the light- and calcium-activated currents shifted similarly when three-fourths of the extracellular sodium was replaced by sucrose, but were not affected by a similar replacement of sodium by lithium. The current activated by calcium was abolished by prior injection of a calcium buffer solution containing EGTA. The responses of the same cells to brief light flashes were slowed and diminished in amplitude, but were not abolished after the injection of calcium buffer. Light adaptation and prior injection of calcium diminished the calcium-activated current much less than they diminished the light-activated current.

Excitation and adaptation of Limulus ventral photoreceptors by inositol 1,4,5 triphosphate result from a rise in intracellular calcium.

Single pressure injections of 1-10 pl of inositol 1,4,5 triphosphate (IP3) or inositol 4,5 bisphosphate [I(4,5)P2] excite Limulus ventral photoreceptors by inducing rapid bursts of inward current. After excitation by IP3, responses to subsequent injections of IP3 or light flashes are often reversibly diminished (adapted). Single injections of IP3 and I(4,5)P2 are effective at concentrations in the injecting pipette of 20 microM to 1 mM. Single injections of inositol 1,4 bisphosphate are ineffective at concentrations of 100-500 microM. Excitation by IP3 or I(4,5)P2 is accompanied by a rise in intracellular free calcium, as indicated by aequorin luminescence. Prior injection of calcium buffer solutions containing 100 mM EGTA greatly diminishes the total charge transferred across the plasma membrane during excitation by IP3 or I(4,5)P2, which suggests that a rise in Cai is necessary for excitation by the inositol polyphosphates. Adaptation of the response to light by IP3 is also abolished by prior injection of EGTA. In the same cells, the response to brief light flashes is slowed and diminished in amplitude by the injection of calcium buffer, but the charge transferred during the response is not significantly diminished. This suggests that light has access to a pathway of excitation in the presence of EGTA that is not accessible to intracellularly injected IP3.

Sequential control of meiosis reinitiation by pH and Ca 2+ in oocytes of the prosobranch mollusk Patella vulgata

The dependence of meiosis reinitiation upon pH and calcium has been investigated in oocytes of a prosobranch mollusk, the limpet Patella vulgata. In this species, 10 to 80% of the population of prophase-blocked oocytes with an intact germinal vesicle (GV) eventually reach the metaphase 1 stage (spontaneous maturation) and they do so even in the absence of external calcium and sodium. Release of the metaphase block was obtained by fertilization or treatment with ionophore A 23187 or excess KCl. Ionophore application is effective in the absence of calcium. These treatments are ineffective when applied to GV prophase-blocked oocytes. In contrast, such oocytes resumed maturation up to metaphase 1 when incubated in alkaline complete or calcium and sodium-free seawater in the presence of millimolar concentrations of weak bases (ammonia, procaine, or nicotine) or after microinjection with alkaline Hepes-KOH buffers. Furthermore, when insemination, ionophore or KCl treatments preceded the application of weak bases, full activation was obtained. These results suggest that activation of P. vulgata oocytes normally proceeds in two steps involving, respectively, a change in intracellular pH followed by a rise in intracellular free calcium.

Regulation of cortical vesicle exocytosis in sea urchin eggs by inositol 1,4,5-trisphosphate and GTP-binding protein.

To investigate the roles of inositol 1,4,5-trisphosphate (InsP3) and guanyl nucleotide binding proteins (G-proteins) in the transduction mechanism coupling fertilization and exocytosis of cortical vesicles in sea urchin eggs, we microinjected InsP3 and guanyl nucleotide analogs into eggs of Lytechinus variegatus. Injection of 28 nM InsP3 caused exocytosis. However, if the egg was first injected with EGTA ([Cai] less than or equal to 0.1 microM; EGTA = 1.6 mM), InsP3 injection did not cause exocytosis, supporting the hypothesis that InsP3 acts by causing a rise in intracellular free calcium. Injection of 28 microM guanosine-5'-0-(3-thiotriphosphate) (GTP-gamma-S), a hydrolysis-resistant analog of GTP, caused exocytosis, but exocytosis did not occur if the egg was pre-injected with EGTA. Injection of 3 mM guanosine-5'-0-(2-thiodiphosphate) (GDP-beta-S), a metabolically stable analog of GDP, prevented sperm from stimulating exocytosis. However, injection of GDP-beta-S did not prevent the stimulation of exocytosis by InsP3. These results suggested the following sequence of events. The sperm activates a G-protein, which stimulates production of InsP3. InsP3 elevates intracellular free calcium, which causes exocytosis.

Post-synaptic calcium influx at the giant synapse of the squid during activation by glutamate.

Changes in free calcium were monitored in the post-synaptic axon of the giant synapse of the squid, using the calcium indicators aequorin and Arsenazo III. The peak size of the calcium-dependent optical signals recorded from aequorin and Arsenazo III both showed a linear relation with the amount of calcium injected ionophoretically into the axon, but the Arsenazo signal had a slower time course than the aequorin. Ionophoretic application of glutamate to the post-synaptic axon depolarized the axon and caused a rise in intracellular free calcium. Aequorin signals were detected in natural sea water, and their size increased when the calcium concentration in the sea water was raised. Arsenazo signals could be detected only in high-calcium (55 mM) sea water. Intracellular calcium signals were detected also during bath application of several glutamate analogues, including kainate, ibotenate, and aspartate. The peak amplitude of the intracellular calcium signal, monitored with both indicators, increased with increasing ionophoretic glutamate dose, and varied linearly with the integral of the glutamate-induced membrane depolarization. No calcium signals were detected when depolarizations, similar to those produced by glutamate, were induced by current injection in the absence of glutamate. We conclude that glutamate increases the calcium permeability of the post-synaptic membrane, independently of the glutamate-induced depolarization. The glutamate-induced depolarization and the rise in intracellular free calcium increased roughly linearly as the membrane potential was made more negative. Extrapolation of these data indicated that the glutamate depolarization would reduce to zero at about -30 mV, while the calcium signals would be suppressed at about +50 mV.

Functional substance P receptors on a rat pancreatic acinar cell line

A pancreatic acinar cell line, AR4-2J, that expresses a high density of substance P (SP)-binding sites has been identified. SP-binding sites on intact AR4-2J cells were detected with 125I-Bolton-Hunter SP (125I-BHSP). 125I-BHSP binding to AR4-2J cells has an apparent Kd of 40 pm with slow rates of association and dissociation. The number of high affinity binding sites was about 10(4)/cell. Binding of 125I-BHSP was inhibited by SP and by structurally related peptides. Physalaemin was a more potent inhibitor of binding than SP, whereas kassinin, eledoisin, and neurokinin A (substance K, neuromedin alpha, or neurokinin L) were much less potent. SP-free acid and SP (7-11) were 3 to 4 orders of magnitude less potent than SP itself. The membrane, intracellular, and secretory events elicited by exposure of AR4-2J cells to SP have also been examined. Intracellular recording from AR4-2J cells revealed resting membrane potentials of -40 to -65 mV. Pressure application of SP (100 pM to 100 nM) evoked depolarizations of 20 to 40 mV which were maintained for prolonged periods. The intracellular free calcium concentration in AR4-2J cells, measured with (2-[2-amino-5-methylphenoxy)-methyl)-6-methoxy-8-aminoquinolone tetra-acetoxy methyl ester), was between 100 and 500 nM. Addition of SP (100 pM to 10 nM) or physalaemin (1 nM) induced a transient rise in intracellular free calcium. AR4-2J cells synthesize amylase, and exposure of cells to SP resulted in a dose-dependent increase in amylase secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

A transient rise in intracellular free calcium is not a sufficient stimulus for respiratory burst activation in human polymorphonuclear leukocytes

Binding of murine monoclonal antibody PMN7C3 to human neutrophils results in a large rapid, dose dependent transient increase in intracellular free calcium as measured by QUIN-2 fluorescence. Unlike other calcium mobilizing agents PMN7C3 does not induce any increase in respiratory burst activity over basal level. The PMN7C3 effect requires multivalent binding. Chelation of extracellular calcium does not significantly decrease the fluorescence transient generated by exposure to PMN7C3. Lowering of basal levels of intracellular free calcium concentration by maintaining QUIN-2-loaded PMN in calcium free medium eliminates the fluorescence transient. The observations demonstrate that a cell surface receptor mediated intracellular free calcium transient may be generated without any associated respiratory burst activation.

Effect of activation of muscarinic receptors on intracellular free calcium and secretion in bovine adrenal chromaffin cells

Stimulation of the nicotinic receptor of bovine chromaffin cells results in a rise in intracellular free calcium ([Ca 2+] i) and subsequent release of catecholamine. This response is totally dependent on the presence of external Ca 2+. Monitoring [Ca 2+] i using quin-2 demonstrated a rise in [Ca 2+] i in response to muscarinic agonists which was approximately 4-times less than that obtained in response to nicotinic stimulation. This atropine-sensitive [Ca 2+] i rise occurred after a 10-s lag and was found to be independent of the external Ca 2+, implying the existence of an intracellular Ca 2+ source. Despite producing this [Ca 2+] i rise low concentrations of the muscarinic agonist, methacholine (under 1·10 −3 M), failed to trigger secretion itself and did not effect the secretory response elicited by nicotine. Challenging the cells with higher methacholine concentrations (over 1·10 −3 M) resulted in the same [Ca 2+] i rise, no secretion, but an inhibition of secretion due to nicotine. This latter response, however, was found to be atropine-insensitive and therefore non-muscarinic. The [Ca 2+] i rise and secretion due to depolarization by 55 mM K + were largely unaffected by prior addition 1·10 −2 M methacholine, inferring that high concentrations of methacholine inhibit nicotine-induced secretion by interacting with the nicotinic receptor. These results provide evidence consistent with the existence of an intracellular Ca 2+ store mobilized by muscarinic receptor activation in bovine chromaffin cells and show that, despite causing a rise in [Ca 2+] i, bovine chromaffin cell muscarinic stimulation does not effect secretion itself or secretion induced by either nicotine or high K +.

Measurement of calcium transients in frog muscle by the use of arsenazo III.

Intracellular injection of the calcium indicator dye arsenazo III was used to measure the calcium transients occurring during depolarization of frog skeletal muscle fibres. A quantitative estimate of the rise in intracellular free calcium during a twitch was made, and the relation between membrane potential and calcium release was examined. The results also indicate that calcium in the bathing solution plays no important part in the generation of calcium transients during single twitches.