Odorant receptors directly activate phospholipase C/inositol-1,4,5-trisphosphate coupled to calcium influx in Odora cells

Mechanisms by which odorants activate signaling pathways in addition to cAMP are hard to evaluate in heterogeneous mixtures of primary olfactory neurons. We used single cell calcium imaging to analyze the response to odorant through odorant receptor (OR) U131 in the olfactory epithelial cell line Odora (Murrell and Hunter 1999), a model system with endogenous olfactory signaling pathways. Because adenylyl cyclase levels are low, agents activating cAMP formation do not elevate calcium, thus unmasking independent signaling mediated by OR via phospholipase C (PLC), inositol-1,4,5-trisphosphate (IP(3)), and its receptor. Unexpectedly, we found that extracellular calcium is required for odor-induced calcium elevation without the release of intracellular calcium, even though the latter pathway is intact and can be stimulated by ATP. Relevant signaling components of the PLC pathway and G protein isoforms are identified by western blot in Odora cells as well as in olfactory sensory neurons (OSNs), where they are localized to the ciliary zone or cell bodies and axons of OSNs by immunohistochemistry. Biotinylation studies establish that IP(3) receptors type 2 and 3 are at the cell surface in Odora cells. Thus, individual ORs are capable of elevating calcium through pathways not directly mediated by cAMP and this may provide another avenue for odorant signaling in the olfactory system.     

Beta-adrenergic receptor-mediated phospholipase C activation independent of cAMP formation in turkey erythrocyte membranes.

The effect of the beta-adrenergic receptor agonist isoproterenol on guanine nucleotide-dependent phospholipase C (PLC) activity was examined in turkey erythrocyte membranes prepared from [3H]inositol-labeled turkey erythrocytes. In the presence of guanosine 5'-(gamma-thiotriphosphate) (GTP[S]) isoproterenol caused a dose-dependent stimulation of [3H]inositol phosphate ([3H]InsP) formation. The activation of PLC by GTP[S] occurred after an initial lag period of 1-2 min and was followed by a sustained rate of [3H]InsP formation which remained linear for 4-5 min. Isoproterenol decreased the lag period for GTP[S]-induced [3H]InsP formation and increased PLC activity at all time points following this lag. Consequently, isoproterenol shifted the dose-response curve for GTP[S] to the left (10-fold) and increased the maximal response. The EC50 value for isoproterenol-induced activation of PLC was 104 +/- 17 nM. Isoproterenol also potentiated GTP-dependent PLC activity but was ineffective in stimulating the enzyme in the presence of AIF4-. The PLC activation by isoproterenol was completely inhibited by propanolol and atenolol but was unaffected by prazosin or yohimbine. Although GTP[S] and isoproterenol could increase cAMP formation in this membrane preparation, the isoproterenol-induced stimulation of PLC occurred in the absence of ATP and was independent of cAMP formation. Furthermore, addition of cAMP, 8-bromo-cAMP, forskolin, or either the regulatory or catalytic subunits of cAMP-dependent protein kinase failed to stimulate [3H]InsP formation and had no effect on the responses elicited by GTP[S] and isoproterenol. Isoproterenol also stimulated [3H]InsP2 and [3H]InsP3 production in intact erythrocytes. Cholera toxin had no effect on [3H]InsP formation in the intact cells under conditions where it stimulated cAMP accumulation. In addition, the activation of PLC by GTP[S] and isoproterenol was unaffected in membranes prepared from cholera toxin-treated erythrocytes. These data demonstrate that stimulation of turkey erythrocyte beta-adrenergic receptors by isoproterenol results in a direct activation of guanine nucleotide-dependent PLC.     

Zinc homeostasis in C6 glioma cells

Zinc homeostasis in mammalian cells is precisely regulated by cellular signal transduction mechanisms. The main result of this study is the finding that modulators of phospholipase C (PLC) activity affect cellular zinc export. Two different PLC inhibitors caused an increase of the total cellular zinc level whereas two different PLC activators caused a decrease. Furthermore, both the inhibition of cyclic nucleotide phosphodiesterases as well as the administration of 8-bromo-cAMP evoked a drop in the intracellular zinc level, indicating the involvement of cAMP in the control of cellular zinc export. It is concluded that the activity of PLC controls cellular zinc transport and that the effect of elevated zinc concentrations on PLC activity might be mediated by cAMP. However, modulation of other major signaling enzymes did not affect the cellular zinc homeostasis. These include activation and inhibition of guanylate cyclase, activation of protein kinase G, activation of protein kinase A, and activation or inhibition of protein kinase C. Furthermore there was no evidence for the existence of a zinc-sensing receptor in C6 glioma cells, which would stimulate PLC activity and evoke a mobilization of intracellular free-calcium levels.     

alpha(2A)-adrenergic receptor stimulation potentiates calcium release in platelets by modulating cAMP levels

alpha(2A)-Adrenergic receptor-mediated Ca(2+) signaling and integrin alpha(IIb)beta(3) exposure were investigated in human platelets under conditions where indirect, thromboxane- or ADP-mediated effects were absent. The alpha(2)-adrenergic receptor agonists, UK14304 and epinephrine (EPI), were unable to raise cytosolic levels of inositol 1,4,5-trisphosphate (InsP(3)) or Ca(2+) but potentiated the [Ca(2+)](i) rises evoked by other agonists that act through stimulation of phospholipase C (thrombin or platelet-activating factor) or stimulation of Ca(2+)-induced Ca(2+) release (CICR) in the absence of InsP(3) generation (thimerosal or thapsigargin). In addition, alpha(2)-adrenergic stimulation resulted in a 20% lowering in the cytosolic cAMP level. In platelets treated with G(salpha)-stimulating prostaglandin E(1), EPI increased the Ca(2+) signal evoked by either phospholipase C- or CICR-stimulating agonists mainly through modulation of the cAMP level. The stimulating effects of UK14304 and EPI on platelet Ca(2+) responses, and also on integrin alpha(IIb)beta(3) exposure and platelet aggregation, were abolished by pharmacological stimulation of cAMP-dependent protein kinase, and these effects were mimicked by inhibition of this activity. In permeabilized platelets, UK14304 and EPI potentiated InsP(3)-induced, CICR-mediated mobilization of Ca(2+) from internal stores in a similar way as did inhibition of cAMP-dependent protein kinase. In summary, a G(ialpha)-mediated decrease in cAMP level appears to play a major role in the platelet-activating effects of alpha(2A)-adrenergic receptor stimulation. Thus, in platelets, unlike other cell types, occupation of the G(ialpha)-coupled alpha(2A)-adrenergic receptors does not result in phospholipase C activation but rather in modulation of the Ca(2+) response by relieving cAMP-mediated suppression of InsP(3)-dependent CICR.     

Membrane signaling and progesterone in female and male osteoblasts. II. Direct involvement of G alpha q/11 coupled to PLC-beta 1 and PLC-beta 3

We have shown that progesterone (10 pM-10 nM) and progesterone covalently bound to bovine serum albumin (P-CMO BSA; 100 pM-1 microM) rapidly increased (within 5 s) the cytosolic free Ca(2+) concentration and inositol 1,4,5 trisphosphate (InsP(3)) formation in confluent female and male rat osteoblasts via a pertussis toxin-insensitive G-protein. The activation of G-proteins coupled to effectors such as phospholipase C (PLC) is an early event in the signal transduction pathway leading to InsP(3) formation. We used antibodies against the various PLC isoforms to show that only PLC-beta1 and PLC-beta 3 were involved in the Ca(2+) mobilization and InsP(3) formation induced by both progestins in female and male osteoblasts, whereas PLC-beta 2, PLC-gamma 1, and PLC-gamma 2 were not. We also used antibodies against the subunits of heterotrimeric G-proteins to show that the activation of PLC-beta 1 and PLC-beta 3 by both progestins involved the G alpha q/11 subunit, which was insensitive to pertussis toxin, whereas G alpha i, G alpha s, and G beta gamma subunits were not. The membrane effects were independent of the concentration of nuclear progesterone receptor, because the concentration of nuclear progesterone receptors was lower in male than in female osteoblasts. These data suggest that progesterone and P-CMO BSA, which does not enter the cell, directly activate G-protein leading to the very rapid formation of second messengers without involving the nuclear receptor.     

Antagonism of rat beta-cell voltage-dependent K+ currents by exendin 4 requires dual activation of the cAMP/protein kinase A and phosphatidylinositol 3-kinase signaling pathways

Antagonism of voltage-dependent K+ (Kv) currents in pancreatic beta-cells may contribute to the ability of glucagon-like peptide-1 (GLP-1) to stimulate insulin secretion. The mechanism and signaling pathway regulating these currents in rat beta-cells were investigated using the GLP-1 receptor agonist exendin 4. Inhibition of Kv currents resulted from a 20-mV leftward shift in the voltage dependence of steady-state inactivation. Blocking cAMP or protein kinase A (PKA) signaling (Rp-cAMP and H-89, respectively) prevented the inhibition of currents by exendin 4. However, direct activation of this pathway alone by intracellular dialysis of cAMP or the PKA catalytic subunit (cPKA) could not inhibit currents, implicating a role for alternative signaling pathways. A number of phosphorylation sites associated with phosphatidylinositol 3 (PI3)-kinase activation were up-regulated in GLP-1-treated MIN6 insulinoma cells, and the PI3 kinase inhibitor wortmannin could prevent antagonism of beta-cell currents by exendin 4. Antagonists of Src family kinases (PP1) and the epidermal growth factor (EGF) receptor (AG1478) also prevented current inhibition by exendin 4, demonstrating a role for Src kinase-mediated trans-activation of the EGF tyrosine kinase receptor. Accordingly, the EGF receptor agonist betacellulin could replicate the effects of exendin 4 in the presence of elevated intracellular cAMP. Downstream, the PKCzeta pseudosubstrate inhibitor could prevent current inhibition by exendin 4. Therefore, antagonism of beta-cell Kv currents by GLP-1 receptor activation requires both cAMP/PKA and PI3 kinase/PKCzeta signaling via trans-activation of the EGF receptor. This represents a novel dual pathway for the control of Kv currents by G protein-coupled receptors.     

Inositol hexakisphosphate increases L-type Ca2+ channel activity by stimulation of adenylyl cyclase.

Inositol hexakisphosphate (InsP6) is a most abundant inositol polyphosphate that changes simultaneously with inositol 1,4,5-trisphosphate in depolarized neurons. However, the role of InsP6 in neuronal signaling is unknown. Mass assay reveals that the basal levels of InsP6 in several brain regions tested are similar. InsP6 mass is significantly elevated in activated brain neurons and lowered by inhibition of neuronal activity. Furthermore, the hippocampus is most sensitive to electrical challenge with regard to percentage accumulation of InsP6. In hippocampal neurons, InsP6 stimulates adenylyl cyclase (AC) without influencing cAMP phosphodiesterases, resulting in activation of protein kinase A (PKA) and thereby selective enhancement of voltage-gated L-type Ca2+ channel activity. This enhancement was abolished by preincubation with PKA and AC inhibitors. These data suggest that InsP6 increases L-type Ca2+ channel activity by facilitating phosphorylation of PKA phosphorylation sites. Thus, in hippocampal neurons, InsP6 serves as an important signal in modulation of voltage-gated L-type Ca2+ channel activity.     

Dopamine receptor-mediated Ca(2+) signaling in striatal medium spiny neurons

Inositol 1,4,5-trisphosphate (InsP(3)) and cAMP are the two second messengers that play an important role in neuronal signaling. Here, we investigated the interactions of InsP(3)- and cAMP-mediated signaling pathways activated by dopamine in striatal medium spiny neurons (MSN). We found that in approximately 40% of the MSN, application of dopamine elicited robust repetitive Ca(2+) transients (oscillations). In pharmacological experiments with specific agonists and antagonists, we found that the observed Ca(2+) oscillations were triggered by activation of D1 class dopamine receptors (DARs). We further demonstrated that activation of phospholipase C was required for induction of dopamine-induced Ca(2+) oscillations and that maintenance of dopamine-evoked Ca(2+) oscillations required both Ca(2+) influx and Ca(2+) mobilization from internal Ca(2+) stores. In "priming" experiments with a type 2 5-hydroxytryptamine receptor agonist, we have shown a likely role for calcyon in coupling D1 class DARs with Ca(2+) oscillations in MSN. In experiments with the DAR-specific agonist SKF83959, we discovered that phospholipase C activation alone could not account for dopamine-induced Ca(2+) oscillations. We further demonstrated that direct activation of protein kinase A by 8-bromo-cAMP or inhibition of protein phosphatase-1 (PP1) or calcineurin (PP2B) resulted in elevation of basal Ca(2+) levels in MSN, but not in Ca(2+) oscillations. In experiments with competitive peptides, we have shown an importance of type 1 InsP(3) receptor association with PP1alpha and with AKAP9.protein kinase A for dopamine-induced Ca(2+) oscillations. In experiments with MSN from DARPP-32 knock-out mice, we demonstrated a regulatory role of DARPP-32 in dopamine-induced Ca(2+) oscillations. Our results indicate that, following D1 class DAR activation, InsP(3) and cAMP signaling pathways converge on the type 1 InsP(3) receptor, resulting in Ca(2+) oscillations in MSN.     

Cyclic adenosine monophosphate signaling in the rat vomeronasal organ: role of an adenylyl cyclase type VI

The present study indicates that male rat urinary components in female rat vomeronasal organ microvillar preparations not only induce a rapid and transient IP(3) signal, but in addition, the level of cAMP decreases with a delayed and sustained time course. This decrease seems to be a consequence of the preceding activation of the phosphoinositol pathway rather than the result of an enhanced phosphodiesterase activity or an inhibition of adenylyl cyclase (AC) via Galpha(i) or Galpha(o). This notion is supported by the finding that activation of the endogenous protein kinase C suppresses basal as well as forskolin-induced cAMP formation. Furthermore, it was observed that elevated levels of calcium inhibit cAMP formation in rat VNO microvillar preparations. These properties of cAMP signaling in the VNO of rats may be mediated by a calcium- and protein kinase C-inhibited AC VI subtype, which is localized in microvillar preparations of the VNO.     

Cross-adaptation between olfactory responses induced by two subgroups of odorant molecules

It has long been believed that vertebrate olfactory signal transduction is mediated by independent multiple pathways (using cAMP and InsP3 as second messengers). However, the dual presence of parallel pathways in the olfactory receptor cell is still controversial, mainly because of the lack of information regarding the single-cell response induced by odorants that have been shown to produce InsP3 exclusively (but not cAMP) in the olfactory cilia. In this study, we recorded activities of transduction channels of single olfactory receptor cells to InsP3-producing odorants. When the membrane potential was held at -54 mV, application of InsP3-producing odorants to the ciliary region caused an inward current. The reversal potential was 0 +/- 7 mV (mean +/- SD, n = 10). Actually, InsP3-producing odorants generated responses in a smaller fraction of cells (lilial, 3.4%; lyral, 1.7%) than the cAMP-producing odorant (cineole, 26%). But, fundamental properties of responses were surprisingly homologous; namely, spatial distribution of the sensitivity, waveforms, I-V relation, and reversal potential, dose dependence, time integration of stimulus period, adaptation, and recovery. By applying both types of odorants alternatively to the same cell, furthermore, we observed cells to exhibit symmetrical cross-adaptation. It seems likely that even with odorants with different modalities adaptation occurs completely depending on the amount of current flow. The data will also provide evidence showing that olfactory response generation and adaptation are regulated by a uniform mechanism for a wide variety of odorants.     

Signaling pathways activated by PACAP in MCF-7 breast cancer cells

PACAP has opposing roles ranging from activation to inhibition of tumor growth and PACAP agonists/antagonists could be used in tumor therapy. In this study, the effect of PACAP stimulation on signaling pathways was investigated in MCF-7 human adenocarcinoma breast cancer cells. Results showed that MCF-7 cells express VPAC1 and VPAC2, but not PAC1, receptors. In addition, PACAP increased the phosphorylation levels of STAT1, Src and Raf within seconds, confirming their involvement in early stages of PACAP signaling whereas maximal phosphorylation of AKT, ERK and p38 was reached 10 to 20 min later. Moreover, selective inhibition of Src or PI3K resulted in a significant decrease in the phosphorylation of ERK and AKT, but not p38, demonstrating that PACAP signaling follows Src/Raf/ERK and PI3K/AKT pathways. On the other hand, selective inhibition of PLC or PKA resulted in a significant decrease in the phosphorylation of p38, but not AKT or ERK, indicating that PACAP signaling also follows the PLC and PKA/cAMP pathways. Furthermore, PACAP induced ROS through H₂O₂ production whereas pretreatment with NAC inhibitor decreased AKT and ERK phosphorylation, but not p38. Selective NOX2 inhibition affected Src/Raf/Erk and PI3K/Akt pathways, without affecting the p38/PLC/PKA pathway whereas other inhibitors (ML171, VAS2870) had no effect on PACAP induced ROS generation. On the other hand, PACAP induced calcium release, which was decreased by pretreatment with PLC inhibitor. Finally, PACAP stimulation promoted apoptosis by increasing Bax and decreasing Bcl2 expression. In conclusion, we demonstrated that PACAP signaling in MCF-7 cells follows the Src/Raf/ERK and PI3K/AKT pathways and is VPAC1 dependent in a ROS dependent manner, whereas it follows PLC and PKA/cAMP pathways and is VPAC2 dependent through p38 MAP kinase activation involving calcium.     

Phosphorylation of inositol 1,4,5-trisphosphate receptors in parotid acinar cells. A mechanism for the synergistic effects of cAMP on Ca2+ signaling.

Acetylcholine-evoked secretion from the parotid gland is substantially potentiated by cAMP-raising agonists. A potential locus for the action of cAMP is the intracellular signaling pathway resulting in elevated cytosolic calcium levels ([Ca(2+)](i)). This hypothesis was tested in mouse parotid acinar cells. Forskolin dramatically potentiated the carbachol-evoked increase in [Ca(2+)](i), converted oscillatory [Ca(2+)](i) changes into a sustained [Ca(2+)](i) increase, and caused subthreshold concentrations of carbachol to increase [Ca(2+)](i) measurably. This potentiation was found to be independent of Ca(2+) entry and inositol 1,4,5-trisphosphate (InsP(3)) production, suggesting that cAMP-mediated effects on Ca(2+) release was the major underlying mechanism. Consistent with this hypothesis, dibutyryl cAMP dramatically potentiated InsP(3)-evoked Ca(2+) release from streptolysin-O-permeabilized cells. Furthermore, type II InsP(3) receptors (InsP(3)R) were shown to be directly phosphorylated by a protein kinase A (PKA)-mediated mechanism after treatment with forskolin. In contrast, no evidence was obtained to support direct PKA-mediated activation of ryanodine receptors (RyRs). However, inhibition of RyRs in intact cells, demonstrated a role for RyRs in propagating Ca(2+) oscillations and amplifying potentiated Ca(2+) release from InsP(3)Rs. These data indicate that potentiation of Ca(2+) release is primarily the result of PKA-mediated phosphorylation of InsP(3)Rs, and may largely explain the synergistic relationship between cAMP-raising agonists and acetylcholine-evoked secretion in the parotid. In addition, this report supports the emerging consensus that phosphorylation at the level of the Ca(2+) release machinery is a broadly important mechanism by which cells can regulate Ca(2+)-mediated processes.     

Phospholipase Cdelta(1) does not mediate Ca(2+) responses in neonatal rat cardiomyocytes

Phospholipase C (PLC) activation in neonatal rat ventricular cardiomyocytes (NRVM) generates inositol(1,4,5)trisphosphate (Ins(1,4,5)P(3)) in response to elevations in Ca(2+) or inositol(1,4)bisphosphate in response to G protein stimulation. Overexpression of PLCdelta(1) increased total [(3)H]inositol phosphate (InsP) content and elevated [(3)H]Ins(1,4,5)P(3), but failed to increase [(3)H]InsP responses to the Ca(2+) ionophore A23187. Antisense PLCdelta(1) expression reduced endogenous PLCdelta(1) content but did not decrease the A23187 response. In permeabilized NRVM, [(3)H]InsP responses to elevated Ca(2+) were not inhibited by Ins(1,4,5)P(3), even at concentrations 1000-fold greater than required for selective inhibition of PLCdelta(1). Taken together these data provide evidence that PLCdelta(1) does not mediate the InsP response to elevated Ca(2+) in NRVM.     

Multiple signaling pathways involved in the effect of endothelin type B receptor in rat median eminence

We assessed the possible link between endothelin receptor mediated phosphoinositide breakdown and NO/cGMP signaling pathways in rat arcuate nucleus-median eminence fragments (AN-ME), brain structures known to contain a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers, together with densely arranged endothelin ETB-receptors-like immunoreactive fibres. Our data show that ET-1, ET-3 and the ETB-receptors agonist, IRL 1620, increased inositol monophosphate (InsP1) accumulation, NOS activity and cGMP formation, in a similar degree. The stimulatory effect of ETs on InsP1 accumulation and cGMP formation was inhibited by the phospholipase C (PLC) inhibitor, neomycin, and the absence of extracellular calcium, suggesting that calcium is involved in endothelin receptor-induced PLC activation. The L-arginine analog, L-NAME, inhibited ET-1 or IRL1620-stimulated cGMP formation. The ETA receptor antagonists BQ 123, did not alter, while the ETB receptor antagonists BQ788 inhibited ETs-induced increase in the PI metabolism, NOS activity and cGMP generation. Our data indicate that in AN-ME, ETB receptor signals through receptor-mediated calcium dependent-stimulation of phosphoinositide breakdown and activation of NOS/cGMP signaling pathway.     

Selective inhibition of β2-adrenergic receptor-mediated cAMP generation by activation of the P2Y2 receptor in mouse pineal gland tumor cells

Rhythmic noradrenergic signaling from the hypothalamic clock in the suprachiasmatic nucleus to the pineal gland causes an increase in intracellular cAMP which regulates the circadian fluctuation of melatonin synthesis. The activation of phospholipase C (PLC)-coupled P2Y(2) receptors upon treatment with ATP and UTP exclusively inhibited the isoproterenol-stimulated cAMP production in mouse pineal gland tumor cells. However, the activation of other PLC-coupled receptors including P2Y(1) and bombesin receptors had little or no effect on the isoproterenol-stimulated cAMP production. Also, ATP did not inhibit cAMP production caused by forskolin, prostaglandin E(2), or the adenosine analog NECA. These results suggest a selective coupling between signalings of P2Y(2) and beta(2)-adrenergic receptors. The binding of [(3)H]CGP12177 to beta(2)-adrenergic receptors was not effected by the presence of ATP or UTP. Ionomycin decreased the isoproterenol-stimulated cAMP production, whereas phorbol 12-myristate 13-acetate slightly potentiated the isoproterenol response. Chelation of intracellular Ca(2+), however, had little effect on the ATP-induced inhibition of cAMP production, while it completely reversed the ionomycin-induced inhibition. Treatment of cells with pertussis toxin almost completely blocked the inhibitory effect of nucleotides. Pertussis toxin also inhibited the nucleotide-induced increase in intracellular Ca(2+) and inositol 1,4,5-trisphosphate production by 30-40%, suggesting that the ATP-mediated inhibition of the cAMP generation and the partial activation of PLC are mediated by pertussis toxin-sensitive G(i)-protein. We conclude that one of the functions of P2Y(2) receptors on the pineal gland is the selective inhibition of beta-adrenergic receptor-mediated signaling pathways via the inhibitory G-proteins.     

Regulation of myometrial phospholipase C system and uterine contraction by beta-adrenergic receptors in midpregnant rat

We investigated whether beta-adrenergic receptors (beta-AR) regulate the phospholipase C (PLC) system in midpregnant rat myometrium. PLCbeta isoforms were characterized, and the effect of isoproterenol (beta-adrenergic agonist) was tested on myometrial inositol phosphate (InsP) production and uterine contraction. Using specific antibodies, we showed that rat myometrium expresses PLCbeta1, PLCbeta3, and PLCbeta4, and to a lesser degree PLCbeta2. Quantitative analysis revealed that PLCbeta isoforms are differentially expressed during pregnancy. Indeed, the amount of PLCbeta4 is increased at midpregnancy, whereas PLCbeta1, PLCbeta2, and PLCbeta3 are up-regulated at term. At midpregnancy, pretreatment of myometrial strips with isoproterenol significantly reduced basal and agonist-stimulated InsP production. Forskolin, a diterpene that increases cAMP accumulation by directly activating adenylyl cyclases, had no effect on InsP production. In contrast, two global potassium (K+) channel inhibitors, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), prevented attenuation of InsP production by isoproterenol. Isoproterenol also significantly decreased spontaneous and agonist-induced contraction of the longitudinal layer of midpregnant rat myometrium. Preincubation of uterine strips with TEA plus 4-AP prior to beta-AR activation blocked only partial uterine relaxation, whereas Forskolin was as potent as isoproterenol. This indicates that beta-AR operate through both K+ channels and cAMP to induce uterine relaxation. In conclusion, we show for the first time that three myometrial PLCbeta isoforms (PLCbeta1, PLCbeta2, and PLCbeta3) are down-regulated at midpregnancy. At this period, beta-AR reduce basal and agonist-stimulated InsP production through activation of K+ channels. Altogether, these mechanisms could act to decrease responsiveness of the longitudinal layer of myometrium to contractant factors.     

Staurosporine potentiates platelet activating factor stimulated phospholipase C activity in rabbit platelets but does not block desensitization by platelet activating factor

The possible involvement of protein kinase C activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets. PAF (100 nM for 5 seconds) stimulated incorporation of 32P into proteins and caused [3H]InsP3 levels to increase about 260% of control. These responses were compared after platelets were pretreated with either PAF, phorbol 12-myristate 13-acetate (PMA) or staurosporine and also after pretreatments with staurosporine followed by PAF or PMA. Pretreating platelets with staurosporine potentiated PAF-stimulated [3H]InsP3 levels by 54% and blocked protein phosphorylation. Pretreatments with PAF and PMA caused PAF-stimulated [3H]InsP3 levels to decrease to 115 and 136%, respectively. Staurosporine pretreatment blocked the decrease caused by the PMA pretreatment but not that by PAF. This study demonstrates that PAF-stimulated PLC activity is negatively affected by protein kinase C (PKC) activation and that inhibition of PKC activity did not prevent desensitization of PLC by PAF.     

Roles of NO Signaling in Long-Term Memory Formation in Visual Learning in an Insect

Many insects exhibit excellent capability of visual learning, but the molecular and neural mechanisms are poorly understood. This is in contrast to accumulation of information on molecular and neural mechanisms of olfactory learning in insects. In olfactory learning in insects, it has been shown that cyclic AMP (cAMP) signaling critically participates in the formation of protein synthesis-dependent long-term memory (LTM) and, in some insects, nitric oxide (NO)-cyclic GMP (cGMP) signaling also plays roles in LTM formation. In this study, we examined the possible contribution of NO-cGMP signaling and cAMP signaling to LTM formation in visual pattern learning in crickets. Crickets that had been subjected to 8-trial conditioning to associate a visual pattern with water reward exhibited memory retention 1 day after conditioning, whereas those subjected to 4-trial conditioning exhibited 30-min memory retention but not 1-day retention. Injection of cycloheximide, a protein synthesis inhibitor, into the hemolymph prior to 8-trial conditioning blocked formation of 1-day memory, whereas it had no effect on 30-min memory formation, indicating that 1-day memory can be characterized as protein synthesis-dependent long-term memory (LTM). Injection of an inhibitor of the enzyme producing an NO or cAMP prior to 8-trial visual conditioning blocked LTM formation, whereas it had no effect on 30-min memory formation. Moreover, injection of an NO donor, cGMP analogue or cAMP analogue prior to 4-trial conditioning induced LTM. Induction of LTM by an NO donor was blocked by DDA, an inhibitor of adenylyl cyclase, an enzyme producing cAMP, but LTM induction by a cAMP analogue was not impaired by L-NAME, an inhibitor of NO synthase. The results indicate that cAMP signaling is downstream of NO signaling for visual LTM formation. We conclude that visual learning and olfactory learning share common biochemical cascades for LTM formation.     

Calcium and fertilization: the beginning of life

The explosive increase in Ca2+ that occurs in the cytosol at fertilization is brought about by the activation of Ca2+-release channels in the intracellular stores. Inositol 1,4,5-trisphosphate (InsP3) is traditionally considered to be the messenger that initiates the increase and spreading of the activating Ca2+ wave. In line with this hypothesis, recent evidence suggests that the penetrating sperm delivers into mammalian eggs a novel isoform of phospholipase C (PLC), which promotes the formation of InsP3. By contrast, data from echinoderms studies indicate that the newly discovered second messenger nicotinic adenine dinucleotide phosphate (NAADP) promotes an initial, localized increase in Ca2+, which is then followed by the InsP3-mediated globalization of the Ca2+ wave. The mechanism by which the interacting sperm triggers the production of NAADP and subsequently that of InsP3 remains obscure.     

Odor suppression of voltage-gated currents contributes to the odor-induced response in olfactory neurons

Olfactorychemotransduction involves a signaling cascade. In addition totriggering transduction, odors suppress ion conductances. Bystimulating with brief odorant pulses, we observed a current associatedwith odor-induced suppression of voltage-gated conductances and studiedits time dependence. We characterized this suppression current inisolated Caudiverbera caudiverberaolfactory neurons. All four voltage-gated currents are suppressed byodor pulses in almost every neuron, and suppression is caused by odorsinducing excitation and by those inducing inhibition, indicating anonselective phenomenon, in contrast to transduction. Suppression has a10-fold shorter latency than transduction. Suppression was morepronounced when odors were applied to the soma than to the cilia,opposite to transduction. Suppression was also present in rat olfactory neurons. Furthermore, we could induce it inDrosophila photoreceptor cells,demonstrating its independence from the chemotransduction cascade. Weshow that odor concentrations causing suppression are similar to thosetriggering chemotransduction and that both suppression and transductioncontribute to the odor response in isolated olfactory neurons.Furthermore, suppression affects spiking, implying a possiblephysiological role in olfaction.