Agonist-stimulated calcium decreases in ovine ciliated airway epithelial cells: role of mitochondria.

1. In ovine ciliated tracheal epithelial cells, acetylcholine (ACh) activates signal transduction pathways that not only transiently increase cytoplasmic Ca2+ ([Ca2+]i) but also actively lower [Ca2+]i. The pathway for decreasing [Ca2+]i is clearly revealed after depletion of intracellular Ca2+ stores by thapsigargin (Tg), 2,5-di-(tert-butyl)-1,4-benzohydroquinone or NiCl2. Measurements with microinjected fura-2 excluded a [Ca2+] measurement artefact. 2. A four-compartment model to simulate calcium transients in non-excitable cells (consisting of a plasma membrane Ca2+ pump and channel; Ca2+ store with pump and channel; and cytosolic Ca2+ buffer) could not account for the observed [Ca2+]i decrease. We therefore explored, by simulation and experimentation, several different mechanisms that could account for it. 3. The ACh-stimulated [Ca2+]i decrease was not due to an inhibition of Ca2+ influx (Ca2+ channel blockers or absence of extracellular calcium had no effect), activation of a plasma membrane Ca2+-ATPase (two inhibitors, vanadate (30 mM) and lanthanum (10 mM), had no effect) or inhibition of the Na+-Ca2+ exchanger (replacing extracellular Na+ with N-methylglucamine had no effect). 4. The application of mitochondrial uncouplers (5 microM CCCP or 5 microM FCCP), eliminated the ACh-induced [Ca2+]i decrease. Addition of CCCP at the nadir of the decrease restored intracellular calcium levels of Tg-treated cells to baseline faster than controls not exposed to mitochondrial uncouplers. CCCP application to naïve cells did not block the ACh-induced transient increase in [Ca2+]i. 5. These data suggest that ACh-induced [Ca2+]i decreases in ciliated cells are caused by stimulated Ca2+ uptake into mitochondria.

Calcium influx mediates the voltage-dependence of sperm entry into sea urchin eggs.

Sperm entry was monitored in voltage-clamped sea urchin eggs following insemination in a variety of artificial seawaters. In regular seawater, maintaining the membrane potential at increasingly negative values progressively inhibits sperm entry. Reducing [Ca(2+)](o) relieves the inhibition, shifting the sperm entry vs voltage relationship toward more negative potentials. Raising [Ca(2+)](o) shifts the relationship in the other direction. Large changes in [Na(+)](o) or [Mg(2+)](o) do not affect sperm entry although changing [Na(+)](o) dramatically changes the currents following sperm attachment. Applying one of seven different calcium channel blockers or replacing Ca(2+) with Ba(2+) or Sr(2+) or microinjecting calcium chelators into the cytoplasm relieves the block to sperm entry at negative potentials. We conclude that the block to sperm entry at negative potentials is mediated by calcium which crosses the membrane and acts at an intracellular site.

Nicotinic acetylcholine receptors of the neuronal type occur in the plasma membrane of sea urchin eggs.

The addition of acetylcholine (ACh, 100 microliters of 10 microM) to the bath in the vicinity of unfertilised sea urchin eggs (Lytechinus variegatus) suspended in sea water (SW) abruptly depolarises the membrane potential (Vm) of the eggs from the resting value of approximately -70 mV. This results in the firing of the egg's action potential, followed by partial repolarisation. Similar addition of ACh to eggs voltage clamped at -70 mV induces an inward current of abrupt onset with peak amplitude of -1.26 +/- 0.20 nA (SE, n = 81). When the eggs are clamped at a Vm more positive than -70 mV, the peak amplitude of the ACh-induced inward current decreases, becoming 0 at a clamped Vm of approximately -20 mV. Further positive shift of the Vm fails to cause reversal of the current. Oocytes clamped at -70 mV exhibit similar inward current responses following application of ACh. Since ACh stimulates both nicotinic and muscarinic receptors (nACh-R and mACh-R, respectively), the effects of exposing eggs to the agonists and antagonists for each type of receptor were examined. For unfertilised eggs clamped at -70 mV the application of 100 microM (-)-nicotine hydrogen tartrate, an agonist of the nACh-R, induces an inward current response similar to that elicited by 10 microM ACh, but of smaller peak amplitude. In contrast, the application of (+)-muscarine chloride, an agonist of the mACh-R, fails to induce any response. Antagonists of the nACh-R inhibit either the neuronal type of nACh-R or the skeletal muscle type of nACh-R. The effect of the antagonists on the amplitudes of the ACh-induced inward current response was determined by superfusing individual eggs clamped at -70 mV with the desired antagonist dissolved in SW, followed by the addition of 100 microliters of 10 microM ACh in the vicinity of the egg. Mecamylamine chloride, an antagonist of the neuronal nACh-R at a concentration of 1 microM, markedly decreases the response to ACh, while at a concentration of 10 microM the response to ACh is abolished. Hexamethonium chloride, another inhibitor of the nACh-R of the neuronal type, also diminishes the ACh-induced response, but at a concentration of 10 microM the response is not completely abolished. Exposure of eggs to alpha-bungarotoxin, an antagonist of the skeletal muscle nACh-R at concentrations up to 250 nM for periods of 30 min, has no effect on the ACh-induced response. The effects of two antagonists of the mACh-R, atropine sulphate and QNB (R-(-)-3-quinuclidinyl benzilate) were also examined. Exposure of eggs to 1 microM atropine does not affect the ACh-induced response, but at concentrations of 10 microM atropine the amplitude of the ACh-induced inward current is significantly reduced. The exposure of eggs to QNB, a highly specific antagonist of the mACh-R, at concentrations up to 50 nM, has no effect on the ACh-induced response. Consequently, the likely explanation for the inhibitory effect of atropine is that at high concentrations atropine cross-reacts with the nACh-R. These findings reveal the presence in unfertilised sea urchin eggs of an ACh-R resembling the neuronal nACh-R. No evidence could be obtained that these receptors have a role in sperm entry, activation of the egg, or early development.

Muscarinic signaling in ciliated tracheal epithelial cells: dual effects on Ca2+ and ciliary beating.

To examine cholinergic signal transduction pathways that modulate ciliary beat frequency (CBF), cultured ovine tracheal epithelial cells were imaged using a combination of phase-contrast (CBF) and fluorescence (Ca2+) microscopy techniques. In single cells, acetylcholine (ACh) transiently increased CBF and intracellular Ca2+ concentration ([Ca2+]i), mainly by Ca2+ release from internal stores, with a small delayed contribution from Ca2+ influx. Nicotinic agonists did not alter CBF or [Ca2+]i, whereas atropine blocked the ACh-stimulated transients, consistent with the involvement of muscarinic receptors. 4-Diphenylacetoxy-N-methylpiperidine methiodide was approximately 100 times more potent than pirenzepine in inhibiting the ACh-induced [Ca2+]i peaks, suggesting that the receptor is a pharmacologically defined (M3) subtype. Interestingly, after depletion of intracellular Ca2+ stores by thapsigargin, ACh caused a rapid transient decrease in both CBF and [Ca2+]i, again with an antagonist profile of M3 receptors. We conclude that activation of M3 muscarinic receptors initiates specific signaling pathways that act simultaneously to increase and decrease [Ca2+]i and CBF.

Effect on sperm-induced activation current and increase of cytosolic Ca2+ by agents that modify the mobilization of [Ca2+]i. I. Heparin and pentosan polysulfate.

The mechanism of the elevation of intracellular free Ca2+ ([Ca2+]i) induced by a single sperm in eggs of the sea urchin Lytechinus variegatus was investigated. Simultaneous measurements of [Ca2+]i, and of the activation current, were carried out on eggs microinjected with Ca Green-1 or Ca Green dextran, and voltage clamped at -20 mV. The microinjection of 0.5 to 1.0 mg/ml heparin (MW 6000) or pentosan polysulfate (MW 3000), final intracellular concentration, causes a concentration-dependent inhibition in all parameters of the sperm-induced elevation of [Ca2+]i and the phase 2 calcium-activated cation current (Ip). For each: (1) the onset is delayed; (2) the rate of change is slowed; and (3) the peak amplitude attained is diminished. In some experiments at the higher concentrations, the microinjected polysulfates cause the complete suppression of the sperm-induced elevation of [Ca2+]i and Ip. The entry of multiple sperm overcomes the inhibitory effects of the polysulfates. Our data suggest that inositol 1,4,5-trisphosphate is the primary mechanism responsible for the sperm-induced release of Ca2+ from intracellular stores.

Stages leading to and following fusion of sperm and egg plasma membranes.

The site of gamete interaction of electrophysiologically recorded Lytechinus variegatus eggs, fixed with osmium tetroxide (OsO4) and/or glutaraldehyde (GTA) at varying intervals after the onset of the increase in membrane conductance induced by an attached sperm, has been examined by high-voltage and conventional transmission electron microscopy. Although GTA and a GTA-OsO4 mixture induced different electrical responses, specimens prepared with the two fixatives were ultrastructurally similar. In specimens observed within 5 s of the change in conductance, the acrosomal process projected through the vitelline layer and abutted the egg plasma membrane. A conspicuous layer of bindin surrounded the acrosomal process and connected the sperm to the egg's vitelline layer. In a fortuitous specimen fixed within 4 s following the change in conductance, the area of contact between the gamete plasma membranes possessed a trilaminar structure that separated the egg's and sperm's cytoplasms. The morphology of this area of contact was consistent with previously proposed intermediates of membrane fusion. Five to six seconds after the change in conductance, the sperm was connected to the egg via a narrow cytoplasmic bridge that consisted of the former acrosomal process and a projection of the egg cortex. The region of the bridge midway between the fused gametes was encircled by dense material that marked the site of sperm-egg fusion. Gamete interactions in which the activation potential was recorded (unclamped egg) were comparable in time and ultrastructure to events taking place in voltage-clamped eggs except for one major difference. Intact cortical granules (one to three) were observed beneath the tip of the incorporating sperm in unclamped eggs fixed following the onset of the activation potential, whereas all cortical granules dehisced in clamped eggs.

Preparation of individual electrically and video-recorded eggs for integrated temporal and electron microscopic analyses.

A method for correlative studies of early fertilization events that integrates techniques of intracellular electrophysiological recording, video-imaging, and electron microscopy is described. A key feature of the method is its ability to identify the fertilizing sperm and to record the moment of egg excitation. Since the site of gamete interaction is recognizable throughout all stages of preparation, difficulties associated with locating the site of fertilization and determining specimen orientation for microtomy and electron microscopic examination are eliminated. Virtually all samples yield useful information. An example of interacting gametes fixed 4 sec after initiation of the fertilization potential and serial sectioned is described. The method is applicable to systems other than fertilizing eggs when functional, temporal, and spatial relationships of individual cells need to be correlated with changes in ultrastructure.