Skeletal muscle IP3R1 receptors amplify physiological and pathological synaptic calcium signals

Ca(2+) release from internal stores is critical for mediating both normal and pathological intracellular Ca(2+) signaling. Recent studies suggest that the inositol 1,4,5-triphosphate (IP(3)) receptor mediates Ca(2+) release from internal stores upon cholinergic activation of the neuromuscular junction (NMJ) in both physiological and pathological conditions. Here, we report that the type I IP(3) receptor (IP(3)R(1))-mediated Ca(2+) release plays a crucial role in synaptic gene expression, development, and neuromuscular transmission, as well as mediating degeneration during excessive cholinergic activation. We found that IP(3)R(1)-mediated Ca(2+) release plays a key role in early development of the NMJ, homeostatic regulation of neuromuscular transmission, and synaptic gene expression. Reducing IP(3)R(1)-mediated Ca(2+) release via siRNA knockdown or IP(3)R blockers in C2C12 cells decreased calpain activity and prevented agonist-induced acetylcholine receptor (AChR) cluster dispersal. In fully developed NMJ in adult muscle, IP(3)R(1) knockdown or blockade effectively increased synaptic strength at presynaptic and postsynaptic sites by increasing both quantal release and expression of AChR subunits and other NMJ-specific genes in a pattern resembling muscle denervation. Moreover, in two mouse models of cholinergic overactivity and NMJ Ca(2+) overload, anti-cholinesterase toxicity and the slow-channel myasthenic syndrome (SCS), IP(3)R(1) knockdown eliminated NMJ Ca(2+) overload, pathological activation of calpain and caspase proteases, and markers of DNA damage at subsynaptic nuclei, and improved both neuromuscular transmission and clinical measures of motor function. Thus, blockade or genetic silencing of muscle IP(3)R(1) may be an effective and well tolerated therapeutic strategy in SCS and other conditions of excitotoxicity or Ca(2+) overload.     

Inositol trisphosphate (IP3) receptors and epileptic seizure

The effects of anticonvulsants and Ca2+ channel antagonists on the inositol trisphosphate (IP3) binding and IP3-induced Ca2+ release were examined in brain membrane fractions. Anticonvulsant (PHT and valproate) and Ca2+ channel antagonists (verapamil, diltiazem, flunarizine, nicardipine and cinnarizine), examined did not significantly inhibit the IP3-receptor binding. The Kd and Bmax values of the IP3 binding did not change significantly in the various brain regions of the amygdala-kindled rats, killed 10 days after the last seizure, compared to those of controls. On the other hand, PHT, PB and carbamazepine inhibited the Ca2+ releasing activity of IP3 in the cerebellar membrane fractions by approximately 20% at therapeutic concentrations.     

Postsynaptic IP3 receptor-mediated Ca2+ release modulates synaptic transmission in hippocampal neurons

Ca(2+)-dependent mechanisms are important in regulating synaptic transmission. The results herein indicate that whole-cell perfusion of inositol 1,4,5-trisphosphate receptor (IP(3)R) agonists greatly enhanced excitatory postsynaptic current (EPSC) amplitudes in postsynaptic hippocampal CA1 neurons. IP(3)R agonist-mediated increases in synaptic transmission changed during development and paralleled age-dependent increases in hippocampal type-1 IP(3)Rs. IP(3)R agonist-mediated increases in EPSC amplitudes were inhibited by postsynaptic perfusion of inhibitors of Ca(2+)/calmodulin, PKC and Ca(2+)/calmodulin-dependent protein kinase II. Postsynaptic perfusion of inhibitors of smooth endoplasmic reticulum (SER) Ca(2+)-ATPases, which deplete intracellular Ca(2+) stores, also enhanced EPSC amplitudes. Postsynaptic perfusion of the IP(3)R agonist adenophostin (AdA) during subthreshold stimulation appeared to convert silent to active synapses; synaptic transmission at these active synapses was completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Postsynaptic IP(3)R-mediated Ca(2+) release also produced a significant increase in spontaneous EPSC frequency. These results indicate that Ca(2+) release from intracellular stores play a key role in regulating the function of postsynaptic AMPARs.     

IP3 receptor in the hair cells of frog semicircular canal and its possible functional role

The presence and functional role of inositol trisphosphate receptors (IP3R) was investigated by electrophysiology and immunohistochemistry in hair cells from the frog semicircular canal. Intracellular recordings were performed from single fibres of the posterior canal in the isolated, intact frog labyrinth, at rest and during rotation, in the presence of IP3 receptor inhibitors and drugs known to produce Ca2+ release from the internal stores or to increase IP3 production. Hair cell immunolabelling for IP3 receptor was performed by standard procedures. The drug 2-aminoethoxydiphenyl borate (2APB), an IP3 receptor inhibitor, produced a marked decrease of mEPSP and spike frequency at low concentration (0.1 mm), without affecting mEPSP size or time course. At high concentration (1 mm), 2APB is reported to block the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA pump) and increase [Ca2+]i; at the labyrinthine cytoneural junction, it greatly enhanced the resting and mechanically evoked sensory discharge frequency. The selective agonist of group I metabotropic glutamate receptors (RS)-3,5-dihydroxyphenylglycine (DHPG, 0.6 mm), produced a transient increase in resting mEPSP and spike frequency at the cytoneural junction, with no effects on mEPSP shape or amplitude. Pretreatment with cyclopiazonic acid (CPA, 0.1 mm), a SERCA pump inhibitor, prevented the facilitatory effect of both 2APB and DHPG, suggesting a link between Ca2+ release from intracellular stores and quantal emission. Consistently, diffuse immunoreactivity for IP3 receptors was observed in posterior canal hair cells. Our results indicate the presence and a possibly relevant functional role of IP3-sensitive stores in controlling [Ca2+]i and modulating the vestibular discharge.     

Miniature postsynaptic currents depend on Ca2+ released from internal stores via PLC/IP3 pathway.

Miniature postsynaptic currents (mPSCs) were examined on autaptic innervation of single rat retinal ganglion cells in low density cultures. Removal of Ca2+ from bath solution or blocking of Ca2+ channels by Cd2+ had no detectable effect on mPSC frequency or amplitude. Thapsigargin, an agent for mobilization of Ca2+ from internal stores, increased mPSC frequency 3-5-fold in control, Ca2+-free or Cd2+-containing solutions. The inositol 1,4,5-triphosphate (IP3) receptor antago- nist, heparin; the phospholipase C (PLC) inhibitor, U73122; and caffeine abolished mPSC or decreased mPSCs frequency. Calcium imaging showed that cytosolic Ca2+ was increased by thapsigargin and decreased by caffeine. These data demonstrate that internal store-released Ca2+ regulated by the PLC/IP3/IP3-receptor pathway has critical contribution to generation and control of miniature release in retinal ganglion cells.     

Guanosine 5'-0-(3-thiotriphosphate) induced calcium release in human platelets is mediated by inositol 1,4,5-triphosphate.

Guanosine 5'-triphosphate (GTP) and its nonhydrolyzable analogs, such as guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S), induce several responses in platelets including secretion, production of inositol 1,4,5-triphosphate (IP3) and mobilization of Ca2+ from intracellular sites. Because IP3 is well established as a second messenger in mobilizing Ca2+ from intracellular stores it has been generally assumed that Ca2+ release by GTP/GTP gamma S in platelets is mediated by IP3. However, studies in neuronal, hepatic and smooth muscle cells have suggested that IP3 and GTP/GTP gamma S activate Ca2+ release by distinct mechanisms and that IP3-independent mechanisms mediate GTP/GTP gamma S-induced Ca2+ release. In several tissues heparin inhibits binding of IP3 and blocks IP3-stimulated Ca2+ release in a competitive and specific manner. In the present studies, IP3 and GTP gamma S induced Ca2+ release and their relationship was examined in human platelets using heparin as a probe. In saponin permeabilized platelets, IP3 (0.05-5 microM) induced a prompt, dose-dependent release of Ca2+ (EC50 0.5 microM). GTP gamma S (1-50 microM) released Ca2+ in a dose-dependent manner with EC50 of 2 microM but with a time lag of 30-90 seconds. Exposure of platelets to 1 microM IP3 following a submaximal response with GTP gamma S (1 microM) resulted in a further increase in Ca2+ release but no further increase was noted on adding 1 microM IP3 following a maximal response with GTP gamma S (10 microM); similar findings were noted on reversing the order of addition of GTP gamma S and IP3 suggesting that these effectors release Ca2+ from the same source. IP3 (0.5 microM) induced Ca2+ release was blocked by low molecular weight (4000-6000) heparin (IC50 30 micrograms/ml). More importantly, heparin abolished GTP gamma S (2.5 microM) induced Ca2+ release (IC50 10 micrograms/ml). These results indicate that, in contrast to the findings in some other cells, in human platelets GTP gamma S-induced Ca2+ release is mediated largely by a mechanism involving IP3.     

Altered calcium dynamics in cultured cerebellar cells from IP3R1-deficient mice.

Intracellular calcium release in response to bath-applied quisqualate or caffeine was examined in cerebellar primary cultures of type-1-inositol-1,4,5-trisphosphate-receptor (IP3R1)- deficient mice. Under [Ca2+]o-free conditions, calcium release in response to 10 microM quisqualate was significantly reduced in Purkinje cells, but was unaffected in granule cells, suggesting that different subtypes of IP3 receptors contribute to the metabotropic glutamate response in these cells. In addition, calcium release in response to 10 mM caffeine under [Ca2+]o-free conditions was not impaired in cerebellar cells, suggesting that IICR and CICR are independently regulated in cerebellar cells. Moreover, in both wild-type and homozygous mutant mice, CICR in granule cells was approximately 6 times greater than in Purkinje cells.     

Calcium transient activity in cultured murine neural crest cells is regulated at the IP(3) receptor

In a previous study we have shown that cultured neural crest cells exhibit spontaneous calcium transients and that these events are required for neurogenesis. In this study, we examine the mechanism that generates these calcium transients. Extracellular Ca(2+) modulates calcium transient activity. Lanthanum (La(3+)), a general calcium channel antagonist and zero extracellular Ca(2+), reduces the percentage of cells exhibiting calcium transients (26.2 and 40. 5%, respectively) and decreases calcium spiking frequency (4.5 to 1. 0 and 2.5 to 1.0 spikes/30 min, respectively). Intracellular calcium stores also contribute to the generation of calcium transients. Depleting the calcium stores of the endoplasmic reticulum (ER) reduces the percentage of active cells (15.7%) and calcium spiking frequency (2.8 to 1.5 spikes/30 min). Ryanodine (100 microM), which blocks calcium release regulated by the ryanodine receptor (RyR), had no effect on calcium transient activity. Blocking inositol 1,4, 5-triphosphate receptor (IP(3)R)-dependent calcium release, with elevated extracellular Mg(2+) (20 mM), abolished calcium transient activity. Mg(2+) did not block caffeine-sensitive calcium release (RyR-dependent) or voltage dependent calcium channels. Mg(2+) also suppressed thimerosal-induced calcium oscillations (IP(3)R-dependent). Small increases in the intracellular calcium concentration ([Ca(2+)](i)), increases the percentage of active cells and the calcium spiking frequency, while larger increases in [Ca(2+)](i) block the transients. Reducing intracellular IP(3) levels reduces the percentage of active cells and the calcium spiking frequency. We conclude that the mechanism for generating spontaneous calcium transients in cultured neural crest cells fits the model for IP(3)R-dependent calcium excitability of the ER.     

Expression of IP3 receptor isoforms at the nodes of Ranvier in rat sciatic nerve

Inositol 1,4,5-trisphosphate receptors (IP3R) are modulated by the second messenger IP3, which induces intracellular calcium release. Using immunohistochemical techniques, we show that the three isoforms are expressed in sciatic nerve. IP3R1 and IP3R2 are mainly present in the nucleus of Schwann cells. IP3R1 is also expressed in Schmidt-Lanterman incisures. IP3R3 is primarily localized at very high levels in nonmyelinating Schwann cells. Interestingly, the three isoforms are expressed at the nodes of Ranvier. IP3R1 is clustered at the node of Ranvier, in a distribution that is similar to the Nav1.6 sodium channels in the sciatic nerve. IP3R3 is present in the paranodal regions of the nodes. IP3R2 is concentrated in the vicinity of the node, and the outer Schwann cell cytoplasm similar to the Kv1.5 potassium channel.     

Localization of inositol 1,4,5-trisphosphate receptors in mouse retinal ganglion cells

Inositol 1,4,5-trisphosphate receptors (IP(3)R) are ligand-gated intracellular Ca(2+)channels that mediate release of Ca(2+) from intracellular stores into the cytosol on activation by second messenger IP(3.). Similarly, IP(3)R mediated changes in cytosolic Ca(2+) concentrations control neuronal functions ranging from synaptic transmission to differentiation and apoptosis. IP(3)R-generated cytosolic Ca(2+) transients also control intracellular Ca(2+) release and subsequent retinal ganglion cell (RGC) physiology and pathophysiology. The distribution of IP(3)R isotypes in primary adult mouse RGC cultures was determined to identify molecular substrates of IP(3)R mediated signaling in these neurons. Immunocytochemical labeling of IP(3)Rs in retinal sections and cultured RGCs was carried out using isoform specific antibodies and was detected with fluorescence microscopy. RGCs were identified by the use of morphologic criteria and RGC-specific immunocytochemical markers, neurofilament 68 kDa, Thy 1.1, and Thy 1.2. RGC morphology and immunoreactivity to neurofilament 68 kDa and Thy 1.1 or Thy 1.2 were identified in both RGC primary cultures and tissue cryosections. RGCs showed localization on intracellular membranes with a differential distribution of IP(3)R isoforms 1, 2, and 3. IP(3)R Types 1 and 3 were detected intracellularly throughout the cell whereas Type 2 was expressed predominantly in soma. Expression of all three IP(3)Rs by RGCs indicates that all IP(3)R types potentially play a role in Ca(2+) homeostasis and Ca(2+) signaling in these cells. Differential localization of IP(3) receptor subtypes in combination with biophysical properties of IP(3)R types may be an important molecular mechanism by which RGCs organize their cytosolic Ca(2+) signals.     

Decreased olfactory mucus secretion and nasal abnormality in mice lacking type 2 and type 3 IP3 receptors

Although nasal mucus is thought to play important roles in the mammalian olfactory system, the mechanisms of secretion of it and its physiological roles are poorly understood. Here we show that type 2 and type 3 IP3 receptors (IP3R2 and IP3R3) play critical roles in olfactory mucus secretion. Histological studies showed that IP3R2 and IP3R3 are predominantly expressed in two types of nasal glands, the anterior glands of the nasal septum and the lateral nasal glands (LNG), which contain mucosal proteins secreted to the main olfactory epithelium. We therefore examined LNG acinar cells, and found that acetylcholine-mediated calcium responses and fluid- and protein- secretion in the acinar cells were markedly decreased in IP3R2–R3 double-knockout (KO) mice. We also found nasal inflammation and a decrease in olfactory capacity in IP3R2–R3 KO mice. Despite intact signal transduction in the olfactory epithelium, IP3R2–R3 KO mice exhibited elevated threshold sensitivity to odorants on in vivo imaging of olfactory glomerular responses and behavioral tests. Our findings suggest that IP3R2 and IP3R3 mediate nasal mucus secretion, which is important for the maintenance of nasal tissue as well as the perception of odors.     

Diadenosine pentaphosphate increases levels of intracellular calcium in astrocytes by a mechanism involving release from caffeine/ryanodine- and IP3-sensitive stores

Diadenosine polyphosphates (ApnAs, n = 2 to 6 phosphate groups) activate P2-type cell-surface adenine nucleotide purinoreceptors, increase the influx of calcium into neural cells, and modulate the binding of ryanodine to ryanodine receptor-regulated intracellular calcium release channels. In this study, we tested the hypothesis, using single cell fluorescence techniques and cultured human fetal astrocytes, that p1, P5-di(adenosine-5') pentaphosphate (Ap5A)-induced increases in levels of intracellular calcium ([Ca2+]i) resulted from release of calcium from intracellular pools. Basal [Ca2+]i were 141+/-12 nM and Ap5A increased [Ca2+]i to 980+/-150 nM. The effect of Ap5A on [Ca2+]i was mediated in part through activation of purinoceptors and influx of extracellular calcium because the purinoceptor antagonist pyridoxal-phosphate-6-azophenel-2', 4'-disuphonic acid blocked by 52%, and chelation of extracellular calcium with EGTA prevented, almost completely, Ap5A-induced increases in [Ca2+]i. Implicating calcium release from IP3- and ryanodine-regulated pools of intracellular calcium were findings that Ap5A-induced increases in [Ca2+]i were blocked, at least in part, by thapsigargin, ryanodine, caffeine, and xestospongin, and Ap5A increased by 2-fold the production of IP3. Release of calcium from IP3- and ryanodine-regulated intracellular pools may be an important signaling event in neural cells that are exposed to Ap5A.     

Astrocytes in adult rat brain express type 2 inositol 1,4,5-trisphosphate receptors

Astrocytes respond to neuronal activity by propagating Ca(2+) waves elicited through the inositol 1,4,5-trisphosphate pathway. We have previously shown that wave propagation is supported by specialized Ca(2+) release sites, where a number of proteins, including inositol 1,4,5-trisphosphate receptors (IP(3)R), occur together in patches. The specific IP(3)R isoform expressed by astrocytes in situ in rat brain is unknown. In the present report, we use isoform-specific antibodies to localize immunohistochemically the IP(3)R subtype expressed in astrocytes in rat brain sections. Astrocytes were identified using antibodies against the astrocyte-specific markers, S-100 beta, or GFAP. Dual indirect immunohistochemistry showed that astrocytes in all regions of adult rat brain express only IP(3)R2. High-resolution analysis showed that hippocampal astrocytes are endowed with a highly branched network of processes that bear fine hair-like extensions containing punctate patches of IP(3)R2 staining in intimate contact with synapses. Such an organization is reminiscent of signaling microdomains found in cultured glial cells. Similarly, Bergmann glial cell processes in the cerebellum also contained fine hair-like processes containing IP(3)R2 staining. The IP(3)R2-containing fine terminal branches of astrocyte processes in both brain regions were found juxtaposed to presynaptic terminals containing synaptophysin as well as PSD 95-containing postsynaptic densities. Corpus callosum astrocytes had an elongated morphology with IP(3)R2 studded processes extending along fiber tracts. Our data suggest that PLC-mediated Ca(2+) signaling in astrocytes in rat brain occurs predominantly through IP(3)R2 ion channels. Furthermore, the anatomical arrangement of the terminal astrocytic branches containing IP(3)R2 ensheathing synapses is ideal for supporting glial monitoring of neuronal activity.     

Effects of interleukin-1beta on hippocampal glutamate and GABA releases associated with Ca2+-induced Ca2+ releasing systems

Recent clinical and basic studies have demonstrated that hyperactivation of interleukin-1beta (IL-1beta) plays important roles in generation of febrile and epileptic seizures. To clarify this mechanism, the present study determined the effects of IL-1beta on Ca2+-associated releases of glutamate and GABA in mouse hippocampus. Both basal and K+-evoked GABA releases were regulated by Ca2+ influx and Ca2+-induced Ca2+ releasing system (CICR). The K+-evoked glutamate release was also regulated by Ca2+ influx and CICR, whereas basal glutamate release was not affected by them. IL-1beta increased basal releases of glutamate and GABA depending on the activation of Ca2+ influx and ryanodine receptor (RyR)-sensitive CICR, but reduced K+-evoked releases depending on Ca2+ influx, RyR-sensitive and inositol 1,4,5-trisphosphate receptor (IP3R)-sensitive CICRs. During neuronal hyperexcitability, the effect of IL-1beta on GABA release was more predominantly modulated by Ca2+ influx and RyR-sensitive CICR than that on glutamate. These results indicate that hyperactivation of IL-1beta leads to imbalance between glutamatergic and GABAergic transmission via toxic overload response of Ca2+ influx and CICR.     

Calcium-triggered exit of F-actin and IP(3) 3-kinase A from dendritic spines is rapid and reversible

The structure of the actin cytoskeleton in dendritic spines is thought to underlie some forms of synaptic plasticity. We have used fixed and live-cell imaging in rat primary hippocampal cultures to characterize the synaptic dynamics of the F-actin binding protein inositol trisphosphate 3-kinase A (IP3K), which is localized in the spines of pyramidal neurons derived from the CA1 region. IP3K was intensely concentrated as puncta in spine heads when Ca(2+) influx was low, but rapidly and reversibly redistributed to a striated morphology in the main dendrite when Ca(2+) influx was high. Glutamate stimulated the exit of IP3K from spines within 10 s, and re-entry following blockage of Ca(2+) influx commenced within a minute; IP3K appeared to remain associated with F-actin throughout this process. Ca(2+)-triggered F-actin relocalization occurred in about 90% of the cells expressing IP3K endogenously, and was modulated by the synaptic activity of the cultures, suggesting that it is a physiological process. F-actin relocalization was blocked by cytochalasins, jasplakinolide and by the over-expression of actin fused to green fluorescent protein. We also used deconvolution microscopy to visualize the relationship between F-actin and endoplasmic reticulum inside dendritic spines, revealing a delicate microorganization of IP3K near the Ca(2+) stores. We conclude that Ca(2+) influx into the spines of CA1 pyramidal neurons triggers the rapid and reversible retraction of F-actin from the dendritic spine head. This process contributes to changes in spine F-actin shape and content during synaptic activity, and might also regulate spine IP3 signals.     

Astrocytic IP3Rs: Contribution to Ca2+ signalling and hippocampal LTP

Astrocytes regulate hippocampal synaptic plasticity by the Ca²⁺ dependent release of the N‐methyl d ‐aspartate receptor (NMDAR) co‐agonist d ‐serine. Previous evidence indicated that d ‐serine release would be regulated by the intracellular Ca²⁺ release channel IP₃ receptor (IP₃R), however, genetic deletion of IP₃R2, the putative astrocytic IP₃R subtype, had no impact on synaptic plasticity or transmission. Although IP₃R2 is widely believed to be the only functional IP₃R in astrocytes, three IP₃R subtypes (1, 2, and 3) have been identified in vertebrates. Therefore, to better understand gliotransmission, we investigated the functionality of IP₃R and the contribution of the three IP₃R subtypes to Ca²⁺ signalling. As a proxy for gliotransmission, we found that long‐term potentiation (LTP) was impaired by dialyzing astrocytes with the broad IP₃R blocker heparin, and rescued by exogenous d ‐serine, indicating that astrocytic IP₃Rs regulate d ‐serine release. To explore which IP₃R subtypes are functional in astrocytes, we used pharmacology and two‐photon Ca²⁺ imaging of hippocampal slices from transgenic mice (IP₃R2^?/? and IP₃R2^?/?;3^?/?). This approach revealed that underneath IP₃R2‐mediated global Ca²⁺ events are an overlooked class of IP₃R‐mediated local events, occurring in astroglial processes. Notably, multiple IP₃Rs were recruited by high frequency stimulation of the Schaffer collaterals, a classical LTP induction protocol. Together, these findings show the dependence of LTP and gliotransmission on Ca²⁺ release by astrocytic IP₃Rs. GLIA 2017;65:502–513     

Calcium release from the intracellular stores of the soma of the mollusk neuron under the action of inositol triphosphate and its nonhydrolyzable analog GTP

The effects of intracellularly injected inositol trisphosphate (IP3) and nonhydrolyzed GTP analogue (Gpp[NH]p) on intracellular concentration of calcium ions [Ca2+]in was determined by fluorescence signals obtained from Helix pomatia neurons. At the same time these neurons were studied under the current clamp conditions. The IP3 and Gpp[NH]p injection caused a long-lasting [Ca2+]in increase in all neurons. Removal of the Ca2+ ions from external solution did not change the [Ca2+]in values. It is suggested that there is IP3- and GTP-dependent release of Ca2+ from intraneuronal stores in the Helix pomatia neurons.     

Role of presynaptic and postsynaptic IP3‐dependent intracellular calcium release in long‐term potentiation in sympathetic ganglion of the rat

In the rat superior cervical ganglion, a form of long term potentiation (LTP) can be elicited by a brief high frequency stimuli applied to the preganglionic nerve. Cumulative evidence shows that a transient increase in cytoplasmic Ca²⁺ concentration is essential for the generation of the ganglionic LTP. Calcium influx and calcium release from intracellular calcium stores contribute to LTP. However, the differential role of presynaptic and postsynaptic calcium signaling has not been established. Herein, by using heparin, a membrane‐impermeant inositol trisphosphate receptor (IP3R) blocker, we explored the contribution of presynaptic and postsynaptic IP3‐sensitive calcium stores to the ganglionic LTP. The LTP was produced by a conditioning train of 40 Hz for 3 s. We analyzed the effects of heparin on the posttetanic potentiation: PTP magnitude and PTP time constant, and on two parameters that describe the LTP: LTP decay time (elapsed time required by the potentiated response to fall to 20% above the basal value) and LTP extent (the integral of the potentiated response). Heparin (100 and 200 μg/ml) was loaded in the preganglionic, the postganglionic, or in both nerves. We found that in all tested conditions heparin significantly decreased LTP but practically did not affect PTP. The preganglionic and postganglionic inhibitory effects of heparin were not additive. De‐N‐sulfated heparin, an ineffective IP3R blocker, had no effect on LTP, but abolished the heparin blocking effect. Data suggest that presynaptic and postsynaptic IP3‐dependent intracellular calcium release equally contribute to ganglionic LTP, supporting our proposal of a trans‐synaptic mechanism for LTP. Synapse, 2010. © 2010 Wiley‐Liss, Inc.     

Mass contents of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol in human platelets stimulated with a thromboxane analogue and thrombin.

Mass contents of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) were measured in U46619-stimulated human platelets. 1 microM of U46619 induced maximum responses in aggregation, 5-hydroxytryptamine (5HT) secretion and increase in intracellular free Ca2+ concentration ([Ca2+]i). Aggregation was almost comparable to that induced by maximal dose (1 U/ml) of thrombin, while 5HT release was almost half. The initial [Ca2+]i peak in response to U46619 was about half of thrombin stimulation. Production of IP3 and DG was, however, less than one tenth of that seen in thrombin stimulation. The profile (time course and concentration-dependency) of IP3 formation did not correlate with that of [Ca2+]i, suggesting that U46619 stimulates IP3-dependent and -independent Ca2+ mobilization. DG production was small but sustained for more than 5 min. These findings support the recent hypothesis that aggregation is regulated by a delayed accumulation of DG. The low level of 5HT secretion could be explained by the low production of second messengers, IP3 and DG.