Injection Of InsP3 Research Articles

Calcium signalling during mammalian fertilization.

The fertilized mammalian egg is a nice model system for analysing spatiotemporal Ca2+ signalling in the intact cell. Hamster eggs show repetitive Ca2+ transients, associated in the initial response with Ca2+ waves which begin from the site of sperm attachment and are propagated across the deep cytoplasm to the opposite pole. In unfertilized eggs, a regenerative Ca2+ wave is induced by injection of either inositol 1,4,5-trisphosphate (InsP3) or Ca2+, and Ca2+ oscillations are produced by continuous injection of InsP3. These Ca2+ waves and oscillations in both fertilized and unfertilized eggs are inhibited in a dose-dependent manner by a monoclonal antibody to the type 1 InsP3 receptor. Ryanodine receptors (both skeletal and cardiac types) are not detected by physiological or immunoblot analyses. Positive and negative feedback between cytosolic Ca2+ and Ca2+ release from InsP3-sensitive pools accounts for the spatiotemporal Ca2+ signalling. In addition to intracellular Ca2+ release, Ca2+ entry from outside the egg is necessary to refill the Ca2+ pools and maintain Ca2+ oscillations. Evidence suggests that inositol 1,3,4,5-tetrakisphosphate activates the Ca2+ influx. The signal transduction process leading to the production of InsP3 and the mechanism of egg activation following the Ca2+ response still remain to be elucidated.

The excitation cascade of Limulus ventral photoreceptors: guanylate cyclase as the link between InsP3-mediated Ca2+ release and the opening of cGMP-gated channels

BackgroundEarly stages in the excitation cascade of Limulus photoreceptors are mediated by activation of Gq by rhodopsin, generation of inositol-1,4,5-trisphosphate by phospholipase-C and the release of Ca2+. At the end of the cascade, cGMP-gated channels open and generate the depolarizing receptor potential. A major unresolved issue is the intermediate process by which Ca2+ elevation leads to channel opening.ResultsTo explore the role of guanylate cyclase (GC) as a potential intermediate, we used the GC inhibitor guanosine 5'-tetraphosphate (GtetP). Its specificity in vivo was supported by its ability to reduce the depolarization produced by the phosphodiesterase inhibitor IBMX. To determine if GC acts subsequent to InsP3 production in the cascade, we examined the effect of intracellular injection of GtetP on the excitation caused by InsP3 injection. This form of excitation and the response to light were both greatly reduced by GtetP, and they recovered in parallel. Similarly, GtetP reduced the excitation caused by intracellular injection of Ca2+. In contrast, this GC inhibitor did not affect the excitation produced by injection of a cGMP analog.ConclusionWe conclude that GC is downstream of InsP3-induced Ca2+ release and is the final enzymatic step of the excitation cascade. This is the first invertebrate rhabdomeric photoreceptor for which transduction can be traced from rhodopsin photoisomerization to ion channel opening.

NAADP+initiates the Ca2+response during fertilization of starfish oocytes

We have explored the role of the recently discovered second messenger nicotinic acid adenine nucleotide phosphate (NAADP+) in Ca2+ swings that accompany the fertilization process in starfish oocytes. The injection of NAADP+ deep into the cytoplasm of oocytes matured by the hormone 1-methyladenine (1-MA), mobilized Ca2+ exclusively in the cortical layer, showing that the NAADP+-sensitive Ca2+ pool is restricted to the subplasma membrane region of the cell. At variance with this, InsP3 initiated the liberation of Ca2+ next to the point of injection in the center of the cell. The initial cortical Ca2+ liberation induced by NAADP+ was followed by a spreading of the Ca2+ wave to the remainder of the cell and by a massive cortical granule exocytosis similar to that routinely observed on injection of InsP3. A striking difference in the responses to NAADP+ and InsP3 was revealed by the removal of the nucleus from immature oocytes, i.e., from oocytes not treated with 1-MA. Whereas the Ca2+ response and the cortical granule exocytosis induced by NAADP+ were unaffected by the removal of the nucleus, the Ca2+ response promoted by InsP3 was significantly slowed. In addition, the cortical granule exocytosis was completely abolished. When enucleated oocytes were fertilized, the spermatozoon still promoted the Ca2+ wave and normal cortical exocytosis, strongly suggesting that the Ca2+ response was mediated by NAADP+ and not by InsP3. InsP3-sensitive Ca2+ stores may mediate the propagation of the wave initiated by NAADP+ since its spreading was strongly affected by removal of the nucleus.

Cellular Mechanisms of Acrolein-Induced Alteration in Calcium Signaling in Airway Smooth Muscle

Acrolein, an unsaturated aliphatic aldehyde, is a potent respiratory irritant. We have previously observed that acrolein administered ex vivo to isolated airways alters subsequent airway responsiveness to muscarinic agonists in terms of both mechanical activity of rings and calcium signaling in isolated cells. In the present study, we have examined the mechanisms by which acrolein alters Ca2+ signaling. In freshly isolated rat tracheal smooth muscle cells, preexposure to acrolein increased the [Ca2+]i oscillation frequency in response to endothelin 1 (ET-1, 0.1 μM), a contractile agonist that acts via the activation of a receptor different from the muscarinic cholinoceptor. We then studied acrolein-induced alteration in cell signaling with special attention to the steps downstream of membrane receptor activation i.e., the inositol 1,4,5-trisphosphate (InsP3) signaling pathway. Pretreatment of cells with LiCl (20 mM), a modulator of InsP3 concentration, mimicked the effect of acrolein exposure on agonist-induced [Ca2+]i response, i.e., increased the amplitude of the first Ca2+ rise and the oscillation frequency in response to 0.1 and 10 μM acetylcholine (ACh), respectively. Moreover, in tracheal smooth muscle, preexposure to acrolein significantly increased carbachol-induced [3H]inositol-phosphates accumulation, up to 34 ± 11% above unexposed tissue values. Finally, in β-escin permeabilized cells, injection of InsP3 (0.1–10 μM) induced a concentration-dependent [Ca2+]i rise followed, for high InsP3 concentration, by [Ca2+]i oscillations, a calcium response whose pattern was similar to that induced by ACh. Exposure to acrolein did not alter the InsP3-induced [Ca2+]i response. These results indicate that the effect of acrolein exposure on Ca2+ responses in airway smooth muscle is not restricted to activation of the muscarinic cholinoceptor and is due to an enhancement in agonist-induced InsP3 production. Since acrolein does not modify InsP3 receptor channel sensitivity, we conclude that acrolein-induced alteration in calcium signaling can be ascribed to its sole effect on InsP3 production.

Protein Kinase C Activators Inhibit the Visual Cascade inLimulusVentral Photoreceptors at an Early Stage

The phosphoinositide cascade mediates visual transduction in invertebrate photoreceptors. Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol bisphosphate, producing inositol trisphosphate (InsP(3)) and diacylglycerol (DAG). Protein kinase C (PKC) is a major target of DAG in many cell types. We have used PKC activators to investigate the function of the kinase in the phototransduction cascade in Limulus polyphemus ventral photoreceptors. Extracellular application of (-)-indolactam V (0. 03-30 microM) or phorbol-12,13-dibutyrate (10 microM) reversibly reduced the sensitivity of the electrical response of the photoreceptors to light by up to 1000-fold. The inert stereoisomer (+)-indolactam V and 4alpha-phorbol had no effect. The effect of (-)-indolactam V was antagonized by the PKC inhibitors bisindolylmaleimide I and Gö 6976. Coapplication of bisindolylmaleimide V, used as a negative control compound for PKC inhibition, did not reduce the effectiveness of (-)-indolactam V. These findings are consistent with (-)-indolactam V activating PKC and desensitizing the light response. Furthermore, our pharmacological results indicate that PKC activation does not appear to play a role in light adaptation. We localized the position of the target of PKC in the visual cascade. We chemically excited the cascade at various stages to determine the kinase's target. PKC activation by (-)-indolactam V decreased the light-induced elevation of intracellular calcium but had no effect on the photoreceptor's excitatory response to intracellular injection of InsP(3). However, the PKC activator greatly reduced the excitation caused by GTP-gamma-S injection. We propose that PKC inhibits the visual transduction cascade at the G-protein and/or PLC stage.

Intracellular calcium responses in bovine oocytes induced by spermatozoa and by reagents

The objectives of the present study were to clarify and compare the characteristics of the transient rises in intracellular calcium concentrations ([Ca 2+] i) induced either by spermatozoa or by stimulation with artificial activators in bovine oocytes. These transient rises in [Ca 2+] i in oocytes matured in vitro were recorded with Ca 2+ imaging using the Ca 2+ indicator fura-2. During fertilization, a series of transient rises in [Ca 2+] i was observed. The first Ca 2+ response peaked at a concentration of 521±39 nM (n=20) and lasted for 4 min, while the subsequent Ca 2+ responses were significantly smaller and shorter, with a peak of 368±13 nM (n=23) and a duration of 2 min. Injection of inositol 1,4,5- triphosphate (InsP3) into unfertilized oocytes caused a transient rise in [Ca 2+] i in a dose-dependent manner. The maximum response was induced by 20 nA × 1 sec injection of InsP3. Thimerosal, a sulfhydryl reagent, induced the repetitive transient rises in [Ca 2+] i. The peak and the duration of the rises in [Ca 2+] i induced by InsP3 or thimerosal were smaller and shorter, respectively, than those of the first rise induced by spermatozoa. Ethanol and Ca 2+ ionophore IA23187, which are general parthenogenetic activators of unfertilized oocytes, each induced a single transient rise in [Ca 2+] i. The duration of the rise in [Ca 2+] i by ethanol or Ca 2+ ionophore was significantly longer than that by spermatozoa at fertilization, although the peaks were smaller. These results clarified the characteristics of the rises in [Ca 2+] i induced by spermatozoa and by several artificial reagents, and showed that the first rise in [Ca 2+] i induced by spermatozoa had a higher peak [Ca 2+] i and a longer duration compared with each the subsequent rises in [Ca 2+] i and the rises in [Ca 2+] i induced by artificial reagents. These indicate that a mode like as the first rise in [Ca 2+] i induced by spermatozoa is an effective trigger for artificial activation of oocytes.

Mechanism of Calcium Oscillations in Fertilized Rabbit Eggs

Changes in intracellular calcium concentrations ([Ca2+]i) occur at regular intervals following fertilization in eggs of all mammalian species studied to date. To investigate the mechanisms of their generation, rabbit eggs were injected with the fluorescent Ca2+ indicator fura-2 dextran. [Ca2+]i oscillations were associated with fertilization (n = 10) and inositol 1,4,5-trisphosphate (InsP3; IICR) appears to participate in their generation because injection of heparin (100 mg/ml in the pipette), a competitive InsP3 receptor antagonist, blocked or considerably delayed the fertilization [Ca2+]i rises in all eggs (8/8). Injection of guanosine 5′-0-(2-thiodiphosphate) (GDPβ[S]), a G-protein antagonist, which possibly reduced the production of InsP3, also resulted in inhibition of [Ca2+]i oscillations (n = 7). Ca2+ injection-induced Ca2+ release in fertilized eggs was observed by injection of CaCl2, which evoked intracellular Ca2+ release in all oscillating eggs (n = 14), but only in a few late fertilization stage nonoscillating eggs (7/19 eggs) and in none of the unfertilized eggs (n = 11). Injection of InsP3 (5 μM) between fertilization [Ca2+]i rises also elicited Ca2+ responses that were similar in peak [Ca2+]i to the fertilization [Ca2+]i rises (n = 5). In unfertilized eggs, injection of guanosine 5′-0-(3-thiotriphosphate) (GTP[S]; 5-20 mM), a stimulator of G-proteins, induced [Ca2+]i oscillations. CaCl2 injections, delivered between GTP[S]-induced [Ca2+]i rises, resulted in increased Ca2+ responses in 5/7 eggs. The results of this study indicate that IICR participates in the generation of fertilization-associated [Ca2+]i rises and that Ca2+ injection-induced Ca2+ release appears to be stimulated by a product of the phosphoinositide pathway. Furthermore, the time to reach threshold levels of InsP3 may dictate the periodicity of fertilization [Ca2+]i rises in rabbit eggs.

Regenerative and non-regenerative calcium transients in hamster eggs triggered by inositol 1,4,5-trisphosphate.

1. Inositol 1,4,5-trisphosphate (InsP3) injected into unfertilized golden hamster eggs elicits a hyperpolarizing response (HR) that is due to stimulation of calcium-activated potassium channels in the egg plasma membrane. 2. A single injection of InsP3 gave a single HR above a threshold value of 0.3 nM. At 5 nM and above, InsP3 induced HRs with no detectable latency. At concentrations between these two values a latency was observed. The amplitude of the HR was independent of InsP3 concentration. 3. A second HR could be elicited by injection of InsP3, but five times more InsP3 was required to trigger a second HR, and 10-100 times more to give an HR of similar magnitude to the first, and there was no latency. 4. The increase in [Ca2+]i in response to an initial injection of 1 nM InsP3 could be resolved into two distinct components: a slow, early rise immediately after InsP3 injection (phase I) followed by a larger and more rapid increase (phase II). The initiation of an HR coincided with the second component of the [Ca2+]i increase. 5. Further injection of InsP3 resulted only in slow, smaller increases in [Ca2+]i that resembled phase I and often did not cause an HR. Phase II appeared to be absent. However, 100-fold greater InsP3 concentrations gave slow, larger Ca2+ transients (and HRs) with no detectable latency. 6. If large amounts of InsP3 were allowed to leak into the eggs constantly from a pipette, repetitive calcium transients were seen. Unlike the sustained repetitive responses seen at fertilization, they were often smaller than the initial transient and less well sustained. However, a subsequent transient could still be elicited on injection of very large concentrations of InsP3. 7. InsP3 can induce regenerative, all-or-none [Ca2+]i increase (CICR) in hamster eggs, often with a long latency, as well as non-regenerative increases. InsP3 injections desensitize CICR and cannot mimic all the features of Ca2+ signalling at fertilization in the hamster egg, in particular, the sensitization of CICR caused by the sperm.

Ca2+-dependent K+ channel activity in rat glioma cells induced by bradykinin stimulation and by inositol 1,4,5-trisphosphate injection

1. A glial cell line derived from C6 rat glioma cells has been shown previously to respond to extracellular pulses of bradykinin or intracellular injection of inositol 1,4,5-trisphosphate (Ins-P3) with a slow hyperpolarizing response due to activation of a K+ current (G. Reiser et al., Brain Res. 506, 205-214; 1990). 2. We determined the ensuing single-channel activity, which is most likely caused by Ca2+ released from internal stores after bradykinin stimulation. Bradykinin-activated channels were selectively permeable to K+, but not to Na+ or to Cl-, and exhibited conductances of mainly 40 and 50 pS. In glioma cells the same type of channel was activated by intracellular injection of Ins-P3 and by extracellular bradykinin pulses.

Depletion of InsP3 stores activates a Ca2+ and K+ current by means of a phosphatase and a diffusible messenger.

In non-excitable cells, release of Ca2+ from the inositol 1,4,5-trisphosphate (InsP3)-sensitive store can activate Ca2+ entry. Very little is known about the signal mechanism relating store emptying to plasma membrane Ca2+ influx. It has been suggested that the signal may be either a diffusible messenger like an inositol phosphate, or the InsP3 receptor itself, which, by physically coupling to some component of Ca2+ entry in the plasma membrane, may link store release to Ca2+ entry. The nature of the Ca2+ entry pathway is also unclear. Only in mast cells has a very selective Ca2+ current been observed after store emptying. Activation of exogenous 5-hydroxytryptamine (5-HT) receptors expressed in Xenopus oocytes or direct injection of InsP3 evokes Ca2+ entry activated by InsP3 pool depletion. Here we investigate the nature of this influx pathway and find a current activated by pool depletion. This has an unusual selectivity in that it is more permeable to Ca2+ ions than to other divalent cations (Ba2+, Sr2+ or Mn2+). Moreover, a K+ permeability is also stimulated after pool depletion. The activation of this store depletion current involves both a phosphatase and an unidentified diffusible messenger. Both the Ca2+ entry pathway and the activating factors found here may be relevant to pool-depleted Ca2+ entry in a variety of non-excitable cells.

Inositol 1,4,5-trisphosphate mediates adrenaline activation of K+ conductance in mouse peritoneal macrophages

In mouse peritoneal macrophages, alpha 1-adrenoceptor stimulation evokes a Ca(2+)-dependent K+ current [Io(Adr)][Hara et al. (1991) Pflügers Arch 419:371-379]. The roles of D-myo-inositol 1,4,5-trisphosphate (InsP3) and a GTP-binding protein (G protein) in Io(Adr) were investigated with tight-seal whole-cell recordings and fura-2 fluorescence measurements. Intracellular injection of InsP3 (5-50 microM) evoked transient outward currents [Io(InsP3)] with or without damped oscillations in membrane currents at -40 mV. Dialysis with 0.2 mM guanosine 5'-[3-thio]triphosphate (GTP[gamma S], a poorly hydrolysable GTP analogue) at -40 mV activated oscillatory outward currents or a slowly developing steady current on which such oscillations were superimposed after a delay of 10-90 s. Io(InsP3) and the GTP[gamma S]-induced current [Io(GTP[gamma S])] were accompanied by an increase in conductance. Reversal potentials of both responses closely depended on the extracellular K+ concentration. Fura-2 measurements revealed that Io(InsP3) and Io(GTP[gamma S]) result from a rise in intracellular free Ca2+ concentration ([Ca2+]i). Removal of extracellular Ca2+ did not abolish Io(InsP3) and Io(GTP[gamma S]). Both were blocked by bath-applied charybdotoxin. Intracellular D-myo-inositol 1,3,4,5-tetrakisphosphate (InsP4, 50 microM) did not evoke any responses, whereas D-myo-inositol 2,4,5-trisphosphate [InsP3(2,4,5), 20 microM] elicited an outward current at -40 mV. Io(InsP3) was completely blocked by prior dialysis with the InsP3 receptor antagonist heparin (5 mg/ml). Inclusion of guanosine 5'-[2-thiol] diphosphate (GDP[beta S], 2 mM) or heparin (5 mg/ml) together with GTP[gamma S] in the patch pipette solution completely blocked Io(GTP[gamma S]). These results indicate that intracellular injection of InsP3 or GTP[gamma S] mimic Io(Adr).(ABSTRACT TRUNCATED AT 250 WORDS)

A lingering elevation of Cai accompanies inhibition of inositol 1,4,5 trisphosphate-induced Ca release in Limulus ventral photoreceptors.

Injection of inositol 1,4,5 trisphosphate (InsP3) into Limulus ventral photoreceptors causes an elevation of intracellular free Ca concentration (Cai) and depolarizes the photoreceptors. When measured with the photoprotein aequorin, the InsP3-induced Cai increase follows the time course of depolarization and declines within 1-2 s. However, sensitivity to further injections of InsP3 remains suppressed for several tens of seconds. The possibility that the suppression of Ca release (feedback inhibition) is due to a small lingering elevation of Cai, below the existing detection limit of aequorin, was investigated by measuring Cai with Ca-sensitive electrodes. Double-barreled, Ca-selective microelectrodes were used to pressure inject InsP3 and measure Cai at the same point. Light or InsP3 injections into the light-sensitive compartment depolarized the photoreceptors and induced an elevation of Cai that persisted for tens of seconds. Injections of InsP3 during the decay of Cai showed that sensitivity to InsP3 recovered as resting Cai approached the prestimulus level. The relationship between elevated Cai and feedback inhibition was very steep. An elevation of Cai of 1 microM or more was associated with inhibitions of 79 +/- 12.4% (SEM; n = 7) for the InsP3-induced Cai increase and of 76 +/- 8% for depolarizations. With a residual Cai elevation of 0.01 microM or less, the mean inhibition was 10 +/- 7.4% for InsP3-induced Cai increase and 6.6 +/- 4% for InsP3-induced depolarization. Injections of InsP3 into a light-insensitive compartment within the cell induced elevations of Cai with no associated depolarizations or feedback inhibition. To verify that a sustained elevation of Cai is necessary for inhibition of InsP3-induced Cai increase and depolarization, we injected ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) between two injections of InsP3. Injection of 1 mM EGTA or the related Ca chelator BAPTA, delivered 750 ms after the first injection of InsP3, restored the peak depolarization caused by the second injection of InsP3 to > 80 +/- 3% of control, compared with 13 +/- 8% without an intervening injection of EGTA. Measurement of Cai with aequorin showed that an intervening injection of EGTA partially restored the InsP3-induced Cai increase. The results suggest that feedback inhibition of InsP3-induced Cai increase and depolarization is mediated by a lingering elevation of Cai and not by depletion of intracellular Ca stores.

Effect of intracellular injection of inositol trisphosphate on cytosolic calcium and membrane currents in Aplysia neurons

Pacemaker cells of Aplysia californica display a regular bursting that results from a complex interplay of Ca(2+)-mediated conductances and a continuous influx and extrusion of Ca2+. The effect of the second messenger 1,4,5-inositol trisphosphate (InsP3) on intracellular free Ca2+ concentration (Cai) regulation and electrical properties was investigated in identified neurons of the abdominal ganglion (R15, L2-L4, L6). Double-barreled Ca-selective microelectrodes were used to pressure inject InsP3 and measure Cai at the same point. Brief injection of InsP3 resulted in an average increase of Cai of 9.2 +/- 10.0 microM (+/- SE; n = 14) that decayed in about 1 min. The InsP3-induced elevation of Cai increased in a dose-dependent manner and saturated when large amounts of InsP3 were injected. The InsP3-induced Cai increase was the result of mobilization from intracellular stores; Cai could be repeatedly mobilized by InsP3 in cells superfused with 0 Ca artificial seawater for more than 60 min. Following multiple injections of InsP3, there was no evidence of immediate inhibition or facilitation. the spatial nature of the InsP3-induced Cai increase was investigated by moving the double-barreled Ca-selective microelectrode tip in a stepwise manner relative to the membrane surface. The largest InsP3-induced Cai increases were measured in an area 0-80 microns from the membrane surface; some cells had their largest InsP3-induced Cai increase some 120-160 microns away from the membrane. Injection of InsP3 in a bursting neuron induced an immediate train of action potentials followed by membrane hyperpolarization and a decrease in the burst frequency. Injection of InsP3 in voltage-clamped cells induced a biphasic response: a rapid inward current followed by a more prolonged outward current; the temporal overlap of the currents was depth dependent. Injection of InsP3 or Ca2+ from a double-barreled injecting electrode induced currents that were different in waveform and time course, indicating that part of the conductance change induced by InsP3 is direct and not mediated by the mobilized Ca2+. In BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'tetra-acetic acid]-loaded cells, the InsP3-induced inward current was mostly unaffected while the Ca-induced outward current was largely attenuated. The results suggest that InsP3 mobilizes Ca2+ from discrete intracellular compartments and induces distinct changes in membrane currents that seem to be independent of the Cai increase.

Spontaneous cytosolic calcium oscillations driven by inositol trisphosphate occur during in vitro maturation of mouse oocytes.

Immature mouse oocytes undergo spontaneous meiotic maturation when released from antral follicles into culture media. The first sign of meiotic resumption is germinal vesicle breakdown (GVB). Cytosolic free Ca2+ was measured in mouse oocytes during spontaneous maturation by monitoring fluorescence of indo-1 or fluo-3. The majority of oocytes showed a series of Ca2+ oscillations that continued for 1-3 h. Repetitive Ca2+ increases occurred every 1-3 min and lasted for 10-60 s. The Ca2+ oscillations appeared to be caused by an increase in inositol 1,4,5-trisphosphate (InsP3) because once they ceased, similar oscillations were triggered by injection of exogenous InsP3. Also, injection of the InsP3 receptor antagonist heparin (final concentration, 100 micrograms/ml) blocked the spontaneous Ca2+ oscillations. In contrast, Ca2+ oscillations induced by thimerosal were not inhibited by heparin. Treating oocytes with media containing 20 microM BAPTA/AM abolished Ca2+ oscillations in oocytes but did not affect the rate of GVB. The data show that cytosolic Ca2+ oscillations apparently caused by polyphosphoinositide turnover occur during mammalian oocyte maturation. However, the spontaneous oscillations do not appear to trigger GVB. Also, the data indicate that there are two separate Ca2+ release mechanisms in mouse oocytes, one sensitive to InsP3, the other to thimerosal.

Effects of alcohols on responses evoked by inositol trisphosphate in Xenopus oocytes.

1. The effects of ethanol and other alcohols on inositol 1,4,5-trisphosphate (InsP3) signalling were studied in Xenopus oocytes by the use of flash photolysis of caged InsP3. Calcium liberation induced by InsP3 was monitored by voltage-clamp recording of Ca(2+)-activated membrane currents, and by fluorescence of the Ca2+ indicator Fluo-3. 2. Membrane current and fluorescence Ca2+ signals evoked by light flashes giving small responses were initially potentiated by bath application of ethanol (80-400 mM). However, the responses subsequently declined while ethanol was present and were strongly reduced or suppressed when it was removed. 3. These effects did not arise artifactually from changes in photolysis of caged InsP3, as similar results were seen with responses evoked by intracellular injections of InsP3. Also, the effects on the membrane current did not arise primarily through actions on the Ca(2+)-dependent Cl- channels, since currents evoked by intracellular injections of Ca2+ were little changed by ethanol. 4. Ethanol reduced the threshold level of InsP3 required to cause Ca2+ liberation. Thus, potentiation was most prominent with small responses evoked by brief light flashes, whereas the predominant effect on larger responses was inhibitory. 5. The facilitatory and inhibitory actions of ethanol persisted after removing extracellular Ca2+. 6. Intracellular injections of ethanol produced an initial inhibition of InsP3 responses, followed, in some oocytes, by a potentiation. 7. Methanol had little effect on InsP3 responses, whereas butanol and other long-chain alcohols produced strong inhibition, but little or no potentiation. 8. We conclude that extracellular application of ethanol produces a rapid potentiation of InsP3-mediated Ca2+ liberation, and a more slowly developing inhibition. The potentiation may arise through stimulation of InsP3 formation at the plasma membrane, whereas the inhibition occurs more deeply in the cell. Both actions were evident at relatively low concentrations (a few tens of millimoles per litre), and might thus be important in the behavioural effects of ethanol intoxication.

Muscarinic enhancement of the voltage‐dependent calcium current in an identified snail neuron.

1. In the F1 neuron of the snail Helix aspersa bathed in a Ba2+ and 4-aminopyridine-containing saline, carbamylcholine (CCh) enhanced the inward current carried by Ba2+ through the voltage-dependent Ca2+ channels. 2. This effect of CCh on the F1 neuron was not affected by the nicotinic antagonists (+)-tubocurarine and hexamethonium, but it was mimicked by oxotremorine and blocked by both atropine and pirenzepine. 3. The intracellular injection of GTP gamma S (guanosine 5'-O-(3- thiotriphosphate] into the F1 neuron caused both a decrease in Ca2+ current and a blockade of the CCh-induced enhancement of the Ca2+ current. 4. Neither cyclic AMP, cyclic GMP nor arachidonic acid mimicked the effect of CCh on the Ca2+ current in the F1 neuron. In contrast, the intracellular injection of EGTA blocked the CCh-induced enhancement of the Ca2+ current thus suggesting that cytosolic Ca2+ is involved in the CCh-induced response. 5. We then investigated the possible role of inositol 1,4,5-trisphosphate (InsP3) and Ca(2+)-dependent protein kinases in the CCh-induced enhancement of the Ca2+ current. The intracellular injection of InsP3 in the F1 neuron elicited no consistent change in the Ca2+ current. Diacylglycerol analogues (OAG and DOG) decreased the Ca2+ current amplitude, i.e. an effect opposite to that produced by CCh. This effect of the diacylglycerol analogues resulted from the activation of protein kinase C (PKC) since it was blocked by staurosporine. In addition, staurosporine did not affect the CCh-induced increase in Ca2+ current. 6. The intracellular injection of either Ca(2+)-calmodulin-dependent protein kinase II (Ca(2+)-CaM-PK) or a peptide inhibitor of this enzyme into the F1 neuron affected neither the Ca2+ current nor its enhancement by CCh. 7. We conclude that the CCh-induced enhancement of the Ca2+ current in the snail F1 neuron involves the activation via muscarinic receptors of an intracellular transduction mechanism in which cytosolic Ca2+ plays a key role. However, InsP3, protein kinase C and Ca(2+)-CaM-PK do not appear to be directly involved in this CCh-induced response.

Bradykinin-evoked acetylcholine release via inositol trisphosphate-dependent elevation in free calcium in neuroblastoma x glioma hybrid NG108-15 cells.

The mechanism underlying the bradykinin (BK)-induced increase of acetylcholine (ACh) release was studied in neuroblastoma x glioma hybrid NG108-15 cells and their synapses formed onto mouse muscle cells. External application of BK or iontophoretic injection of extrinsic inositol 1,4,5-trisphosphate (InsP3) into the cytoplasm of NG108-15 cells produced membrane hyperpolarization in the hybrid cells and an increase in the frequency of miniature end-plate potentials (MEPPs) in paired myotubes. Ba2+ blocked the hyperpolarization in response to BK, but facilitation of MEPPs was still observed. InsP3-dependent facilitation of MEPPs was also observed in cells where the InsP3 injections produced no detectable hyperpolarization or even depolarization. Real-time quantitative monitoring of intracellular free Ca2+ concentration [( Ca2+]i) with fura-2 in single NG108-15 cells showed that BK application or InsP3 injection induced an elevation of [Ca2+]i which coincided in time with membrane hyperpolarization recorded from the same cell. The [Ca2+]i rise produced by InsP3 injection started from the single site of injection and that produced by BK began from a deep compartment of the cytoplasm of the NG108-15 cells. The BK- and InsP3-evoked facilitation of MEPPs and the [Ca2+]i rise were relatively independent of extracellular Ca2+. These findings suggest that the BK-induced ACh release results not from membrane potential changes but from a transient InsP3-dependent elevation of [Ca2+]i.

Bradykinin induces inositol 1,4,5‐trisphosphate‐dependent hyperpolarization in K+ M‐current‐deficient hybrid NL308 cells: Comparison with NG108–15 neuroblastoma × glioma hybrid cells

External application of bradykinin (BK) to mouse neuroblastoma X mouse fibroblast hybrid NL308 cells and mouse neuroblastoma X rat glioma hybrid NG108-15 cells produced a transient outward (hyperpolarizing) current. In NG108-15 cells, BK also induced an inward (depolarizing) current associated with a decrease in input membrane conductance, which results from the inhibition of a voltage-sensitive potassium current, the M-current. However, in NL308 cells, either no depolarization was elicited by BK or, even if the BK-induced depolarization was evoked, it was associated with an increased conductance. To explain the above difference, the intracellular second messenger system of NL308 cells was examined in detail. BK induced the rapid accumulation (three- to fivefold higher than the control level) of inositol 1,4,5-trisphosphate (InsP3) in NL308 cells. The cytosolic Ca2+ concentration was also elevated to 540 nM from 180 nM at a basal level. This seems to be enough to activate a voltage-independent and Ca2(+)-sensitive K+ current, resulting in the hyperpolarization. Intracellular injection of InsP3 replicated the hyperpolarization. NL308 cells possess protein kinase C (C-kinase), with specific activities of C-kinase in cytosolic and membrane fractions being 233 and 24 pmol/min/mg protein, respectively. The activity associated with particulates became higher after phorbol dibutyrate (PDBu) treatment. But NL308 cells did not show the characteristic inward relaxation by step hyperpolarizations and the outward rectification in the current-voltage relationship, indicating that the M current is deficient in NL308 cells. Therefore, application of PDBu failed to mimic the inward current. The results suggest the role of InsP3 and C-kinase in controlling two K+ currents.

Hepatocyte gap junctions are permeable to the second messenger, inositol 1,4,5-trisphosphate, and to calcium ions.

Hepatocytes are well coupled by gap junctions, which allow the diffusion of small molecules between cells. Although gap junctions in many tissues are permeable to molecules larger than cAMP and in several preparations gap junctions pass cAMP itself, little direct evidence supports permeation by other second-messenger species. Ca2+, perhaps the smallest second messenger, would be expected to cross gap junctions, but the issue is complicated because gap-junction channels are closed when intracellular free Ca2+ concentration, [Ca2+]i, is elevated to micromolar levels or above. Inositol 1,4,5-trisphosphate (InsP3), a second messenger that can evoke Ca2+ release, might also reduce junctional permeability by this mechanism. We report here evidence for transjunctional flux of Ca2+ and InsP3 in freshly isolated pairs or small clusters of rat hepatocytes. The Ca2+ indicator fura-2 was used to monitor transjunctional diffusion of Ca2+ directly or to detect passage of InsP3 by localized Ca2+ release. Fura-2 injected as the free acid passed between cells. Injection of InsP3 or CaCl2 immediately increased [Ca2+]i in the injected cell (peak values less than 1 microM), and [Ca2+]i increased rapidly in contacting cells (within seconds). The initial rise in [Ca2+]i induced by InsP3 was greater at discrete regions in the cytoplasm of both injected and uninjected cells and was inconsistent with simple diffusion of Ca2+. In the coupled cells the regions of greatest increase were not necessarily near the contact zone. In contrast, the rise induced in [Ca2+]i by CaCl2 injection when cells were bathed in normal Ca2+ was always more diffuse than with InsP3 injection, and in cells coupled to a cell injected with CaCl2 the earliest and maximal increases occurred at the region of cell contact. This difference in distribution indicates that injected InsP3 (or an active metabolite, but not Ca2+) diffused between cells to cause localized release of Ca2+ from intracellular stores. Ca2+ injection induced a rise in [Ca2+]i in coupled cells even when cells were maintained in Ca2+-free saline, suggesting that changes in [Ca2+]i seen in adjacent cells were due to transjunctional diffusion from the injected cell and not to uptake from the extracellular solution. However, in Ca2+-free saline, [Ca2+]i distribution was nonuniform, indicating that Ca2+-releasing mechanisms contribute to the observed changes. No increase in [Ca2+]i was seen in adjacent cells when Ca2+ was injected after treatment with the uncoupling agent octanol (500 microM), which itself did not change [Ca2+]i. These data provide evidence that the second messengers Ca2+ and InsP3 can be transmitted from cell to cell through gap junctions, a process that may have an important role in tissue function.

Protein kinase C activators reduce the inositol trisphosphate‐Induced outward current and the CA2+‐activated outward current in identified neurons of aplysia

Effects of intracellularly injected activators of protein kinase C on the InsP3-induced K+ current and the Ca2+-activated K+ current recorded from identified neurons (R9-R12) of Aplysia kurodai were investigated with conventional voltage-clamp and pressure-injection techniques. Intracellular injection of InsP3 into identified neurons produced a 4-aminopiridine (4-AP)-resistant, tetraethylammonium (TEA)-sensitive, and quinidine-sensitive K+ current similar to the Ca2+ activated K+ current elicited by direct injection of Ca2+ ions into the same neurons. The diacylglycerol analogue 1,2-oleoylacetylglycerol (OAG) at an intracellular concentration of 65 nM produced irreversible decreases in both the InsP3-induced K+ current and the Ca2+-activated K+ current. The phorbol 12,13-dibutyrate (PDBu) at an intracellular concentration of 150 nM also decreased irreversibly both the InsP3-induced K+ current and the Ca2+-activated K+ current. These results suggest that protein kinase C activators reduce both the InsP3-induced K+ current and the Ca2+-activated K+ current recorded from certain identified neurons of Aplysia and that protein kinase C reduces the ability of Ca2+ to open K+ channels rather than affecting the ability of InsP3 to release Ca2+ from intracellular stores.