Activation Of Second Messengers Research Articles

L-365,260 inhibits in vitro acid secretion by interacting with a PKA pathway.

The aim of this study was to analyse the antisecretory mechanism of L-365,260 in vitro in isolated rabbit gastric glands. We showed that compound L-365,260, described as a non-peptide specific competitive CCK-B receptor antagonist, was able to dose-dependently inhibit [14C]-aminopyrine accumulation induced by histamine (10(-4) M), carbachol (5x10(-5) M), 3-isobutyl-1-methyl-xanthine (IBMX) (5x10(-6) M) and forskolin (5x10(-7) M) with similar IC50 values respectively of 1.1+/-0.6x10(-7) M, 1.9+/-1.2x10(-7) M, 4.2+/-2.0x10(-7) M and 4.0+/-2.8x10(-7) M. We showed that L-365,260 acted beyond receptor activation and production of intracellular second messengers and that it had no action on the H+/K+ -ATPase. We found that L-365,260 inhibited cyclic AMP-induced [14C]-aminopyrine accumulation in digitonin-permeabilized rabbit gastric glands, suggesting that this compound acted, at least in part, as an inhibitor of the cyclic AMP-dependent protein kinase (PKA) pathway.

Inflammation-induced up-regulation of protein kinase C γ immunoreactivity in rat spinal cord correlates with enhanced nociceptive processing

Activation of various second messengers contributes to long-term changes in the excitability of dorsal horn neurons and to persistent pain conditions produced by injury. Here, we compared the time-course of decreased mechanical nociceptive thresholds and the density of protein kinase C γ immunoreactivity in the dorsal horn after injections of complete Freund's adjuvant in the plantar surface of the rat hindpaw. Complete Freund's adjuvant significantly increased paw diameter and mechanical sensitivity ipsilateral to the inflammation. The changes peaked one day post-injury, but endured at least two weeks. In these rats, we recorded a 75–100% increase in protein kinase C γ immunoreactivity in the ipsilateral superficial dorsal horn of the L4 and L5 segments at all time-points. Electron microscopy revealed that the up-regulation was associated with a significant translocation of protein kinase C γ immunoreactivity to the plasma membrane. In double-label cytochemical studies, we found that about 20% of the protein kinase C γ-immunoreactive neurons, which are concentrated in inner lamina II, contain glutamate decarboxylase-67 messenger RNA, but none stain for parvalbumin or nitric oxide synthase. These results indicate that persistent changes in protein kinase C γ immunoreactivity parallel the time-course of mechanical allodynia and suggest that protein kinase C γ contributes to the maintenance of the allodynia produced by peripheral inflammation. The minimal expression of protein kinase C γ in presumed inhibitory neurons suggests that protein kinase C γ-mediated regulation of excitatory interneurons underlies the changes in spinal cord activity during persistent nociception.

Regulation of second messengers associated with airway smooth muscle contraction and relaxation.

Agonist-receptor interactions regulate airway smooth muscle tone through activation of guanine nucleotide binding proteins (G proteins), which are coupled to second messenger pathways that mediate changes in the tissue's contractile state. With respect to airway smooth muscle (ASM) contraction, receptor activation elicits phosphatidylinositol turnover that results in the formation of the second messengers, 1,2,-diacylglyserol, which activates protein kinase C (PKC), and inositol 1,4,5,-trisphosphate (Ins[1,4,5]P3), which binds to its intracellular receptor to mobilize intracellular calcium (Ca2+). Both the mobilization of Ca2+ and activation PKC play critical roles in initiating and acutely modulating the intensity and duration of the ASM contraction response. In contrast, bronchodilator agonist-mediated receptor activation is typically coupled to an enhanced accumulation of the second messenger, adenosine 3',5'-cyclic monophosphate (cAMP) which, through activation of cAMP-dependent protein kinase, induces the phosphorylation of specific proteins, leading to ASM relaxation. For activation of both of these functionally distinct signal transduction pathways, the agonist-receptor complexes interact with specific G proteins, which in turn modulate the enzymes regulating the production of their respective second messengers. Perturbations in Ins(1,4,5)P3 accumulation, its metabolism and intracellular binding may underlie changes in ASM contractility. Comparably, changes in ASM relaxation responsiveness, secondary to perturbations in cAMP accumulation, may be due to altered receptor/G protein modulation of adenylate cyclase activity, as well as to altered binding of Ins(1,4,5)P3 to its Ca2+-mobilizing intracellular receptor. This review begins with an overview of the structural and functional characteristics of G protein-linked receptors, followed by descriptions of the role of G proteins, their transmembrane signaling processes, and mechanisms regulating second messenger-coupled ASM contraction and relaxation, and concludes with new information underscoring the important roles of altered receptor/G protein-coupled expression and regulatory interactions between signaling pathways in modulating second-messenger accumulation and action in the "pro-asthmatic" sensitized airway smooth muscle.

SH3 binding domains in the dopamine D4 receptor.

The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit adenylyl cyclase. The dopamine D4 receptor as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the D4 receptor are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. Here we demonstrate that this region of the D4 receptor can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2-SH3 adapter proteins Grb2 and Nck. The repeat sequence itself is not essential in this interaction. The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the dopamine D4 receptor resulted in a receptor that could still bind spiperone and dopamine. Dopamine could not modulate the coupling of these mutant receptors to adenylyl cyclase and MAPK, although dopamine modulated receptor-G protein interaction appeared normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicates that the D4 receptor contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization.

Growth effects of alpha-interferon but not of bombesin or angiotensin II are mediated by activation of STAT proteins.

The recently discovered Jak/STAT signal transduction pathway is associated with cytokine or growth factor receptors; whether members of the G protein-coupled receptor superfamily also activate this pathway is not yet clear. As a first member, the angiotensin (AT)1A receptor has been demonstrated to phosphorylate Jak and STAT proteins. Bombesin, a neurotransmitter and growth factor in many cells and tissues, activates its G protein-coupled receptor and in addition phosphorylates proteins that might be members of the Jak/STAT family. This study investigated whether bombesin- or angiotensin-mediated growth effects are associated with STAT protein activation. Functional receptors were characterized using ligand-binding studies, second-messenger activation and determination of ligand-mediated growth effects. STAT protein activation was analysed by electrophoretic mobility shift assay (EMSA) using labelled DNA response elements recognizing all known STAT proteins. Functional bombesin receptors mediating mitogenic effects were demonstrated on Swiss 3T3 fibroblasts, human melanoma cells (A375-6) and primary human lung fibroblasts; however, bombesin-related STAT protein activation was not observed by EMSA. Interferon-alpha typically activated a STAT1-STAT2-p48 heterotrimer, as well as STAT1-3 hetero- and homodimers in human melanoma cells and significantly inhibited growth of this cell line in vitro. Functional AT1A receptors on primary rat cardiac fibroblasts mediated angiotensin-stimulated growth effects but, in contrast to recently published data, did not activate any known STAT protein. Interferon alpha-stimulated growth inhibition is mediated by activation of the Jak/STAT pathway, whereas bombesin or AT1A receptor-mediated effects on cellular proliferation do not involve phosphorylation of STAT proteins.

Effect of tyrosine kinase inhibition on surfactant protein A gene expression during human lung development

Epidermal growth factor (EGF) stimulates surfactant protein (SP) A synthesis in human fetal lung explants. Ligand binding to the EGF receptor stimulates an intrinsic receptor tyrosine kinase with subsequent activation of second messengers. We hypothesized that inhibition of EGF-receptor tyrosine kinase activity would block SP-A expression in spontaneously differentiating cultured human fetal lung tissue. Midtrimester fetal lung explants were exposed for 4 days to genistein (a broad-range inhibitor of tyrosine kinases) and tyrphostin AG-1478 (a specific inhibitor of EGF-receptor tyrosine kinase). Genistein significantly decreased SP-A and SP-A mRNA levels without affecting either tissue viability or the morphological differentiation of alveolar type II cells. Tyrphostin AG-1478 also decreased SP-A content and SP-A mRNA levels in cultured fetal lung explants. Treatment with EGF could not overcome the inhibitory effects of either genistein or tyrphostin on SP-A; however, only tyrphostin inhibited EGF-receptor tyrosine phosphorylation. We conclude that specific inhibition of EGF-receptor tyrosine kinase with tyrphostin AG-1478 blocks the expression of SP-A during spontaneous differentiation of cultured human fetal lung tissue. Furthermore, exposure to genistein also decreases SP-A expression and blocks the effects of EGF in human fetal lung tissue without inhibiting EGF-receptor tyrosine phosphorylation. These findings support the importance of tyrosine kinase-dependent signal transduction pathways in the regulation of SP-A during human fetal lung development.

A novel interaction between inhibitory melatonin receptors and protein kinase C-dependent signal transduction in ovine pars tuberalis cells.

This study revealed an important and unexpected finding: namely, that inhibitory melatonin receptors can inhibit a phorbol 12,13 myristate acetate (PMA)-induced, protein kinase C (PKC)-dependent increase in c-fos messenger RNA expression in ovine pars tuberalis (PT) cells. PMA induces dose-dependent stimulation of c-fos expression that is attenuated by melatonin in a dose-dependent and pertussis toxin-sensitive manner. The effect of 100 nM PMA is blocked by Ro31-8220 (1 microM), yet is not mimicked by 4alpha-PMA (100 nM). PMA (100 nM) induces PKC activity in PT cells (P < 0.05) within 5 min, but melatonin has no effect on this response. PMA (100 nM) stimulates both phospholipase D and mitogen-activated protein kinase (MAPK) (p42/44) activities in PT cells, but melatonin has no effect on these responses. The results indicate that neither of these second-messenger activities contribute to the melatonin-sensitive pathway of c-fos activation. The MEK (MAPK kinase) inhibitor, PD98059 (50 microM), does not block the induction of c-fos by PMA, although at the same dose it inhibits PMA-mediated activation of p42/44 MAPK by 50-70%, and activation by forskolin or insulin-like growth factor-I by 100%. These data suggest that p42/44 MAPK may not be the primary mediator of PKC-dependent c-fos induction. In contrast to the effect of melatonin on PMA-mediated c-fos induction in PT cells, in L cells stably transfected with the sheep Mel1 alphabeta receptor, melatonin potentiates the c-fos response in a pertussis toxin-sensitive manner. These data indicate the tissue-specific nature of melatonin receptor signaling, and reveal that a pertussis toxin-sensitive pathway can block PKC-mediated c-fos induction in PT cells.

Suppression of phospholipase C beta, gamma, and delta families alters cell growth and phosphatidylinositol 4,5-bisphosphate levels.

Phosphatidylinositol-specific phospholipase C (PLC) activity reflects a summation of the activities of three families, beta, gamma, and delta, each of which is regulated differently. In order to understand the contribution of each family to cell proliferation signaling, expression of each family was suppressed by use of an inducible expression vector for antisense PLC sequences in a single cell line, FTO-2B rat hepatocytes. Activation of second messengers of PLC [diacylglycerol (DAG) and inositol 1,4,5-tris(phosphate) (IP3)] was dramatically reduced, providing a strategy for probing the consequences of PLC deficiency on cell function. Importantly, while one PLC family was suppressed, the other PLCs actively responded to specific stimuli, suggesting parallel and independent signaling pathways for each PLC family in FTO-2B cells. Selective suppression of each PLC family altered cell growth markedly and differentially. The rank order for suppression of cell growth by loss of a PLC family was gamma > delta > beta. Exploration of down-stream growth regulators revealed that loss of beta and gamma, but not delta, families was associated with markedly reduced basal ras and protein kinase C activity. Moreover, suppression of each of the three PLC families caused remarkably reduced basal and stimulated MAP kinase activities. Interestingly, cellular levels of PIP2 were increased and dramatically correlated with growth inhibition rate in the clones with suppressed PLC activity, suggesting that PIP2 itself can serve as a second messenger of cell growth regulation.

Intracellular antagonistic interaction between GnRH and gonadotrophin surge-inhibiting/attenuating factor bioactivity downstream of second messengers involved in the self-priming process.

The antagonistic control of LH concentrations by GnRH and the putative ovarian factor gonadotrophin surge-inhibiting or -attenuating factor (GnSIF/AF) was studied by perifusion of female rat pituitary glands in vitro. LH release and GnRH self-priming were measured in response to five (0.32-10 nmol l-1) or three (10 nmol l-1) 10 min pulses of GnRH. In the latter case, pulses were preceded by five 10 min pulses of either 1 mmol 8-bromo-cAMP l-1 plus 10 mmol theophylline l-1 or 1 mumol phorbol 12-myristate 13-acetate l-1. The stimulations were carried out in the presence or absence of steroid-free bovine follicular fluid, which possesses GnSIF/AF bioactivity, to study the effect of GnSIF/AF on the self-priming process during successive stimulations by GnRH. First, the effect of follicular fluid was studied on GnRH-induced LH release from pituitary glands collected during the ovarian cycle. Only when a clear self-priming effect was evident, as on day 2 of dioestrus and the day of pro-oestrus, follicular fluid antagonized the self-priming effect of GnRH. Second, when glands on day 2 of dioestrus were used, self-priming was displayed by various combinations of GnRH and follicular fluid. The pulses with the lowest concentrations of GnRH together with a high concentration of follicular fluid, however, led to stable low amplitude LH pulses. Finally, priming of the pituitary LH response to GnRH with 8-bromo-cAMP plus theophylline or phorbol 12-myristate 13-acetate was inhibited by follicular fluid. These results confirm the control of LH release by GnRH and GnSIF/AF bioactivity. The effect of GnSIF/AF in follicular fluid is most pronounced on the days before the LH surge, showing evidence of its important role in maintaining low LH concentrations during this period. GnSIF/AF neutralizes downstream actions of second messengers involved in GnRH self-priming.

Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity.

The C-C chemokine receptor 5 (CCR5) plays a crucial role in facilitating the entry of macrophage-tropic strains of the HIV-1 into cells, but the mechanism of this phenomenon is completely unknown. To explore the role of CCR5-derived signal transduction in viral entry, we introduced mutations into two cytoplasmic domains of CCR5 involved in receptor-mediated function. Truncation of the terminal carboxyl-tail to eight amino acids or mutation of the highly conserved aspartate-arginine-tyrosine, or DRY, sequence in the second cytoplasmic loop of CCR5 effectively blocked chemokine-dependent activation of classic second messengers, intracellular calcium fluxes, and the cellular response of chemotaxis. In contrast, none of the mutations altered the ability of CCR5 to act as an HIV-1 coreceptor. We conclude that the initiation of signal transduction, the prototypic function of G protein coupled receptors, is not required for CCR5 to act as a coreceptor for HIV-1 entry into cells.

Development of M1 subtype selective muscarinic agonists for Alzheimer's disease: Translation of in vitro selectivity into in vivo efficacy

Clinical trials in Alzheimer's disease (AD) with first generation muscarinic agonists (e.g., arecoline, oxotremorine, pilocarpine) produced inconsistent results due to poor pharmacokinetic properties and a lack of separation between central and peripheral activities observed with these compounds. Second generation agonists have sought to optimize physicochemical properties, and in most cases, target specific subtypes of muscarinic receptor to overcome these limitations. Based upon receptor distribution in both central and peripheral nervous systems, agonists of the m1/M1 subtype seem to possess the desired profile for AD treatment. For the discovery and characterization of these selective agents, the use of clonal cell lines expressing the five subtypes of muscarinic receptors (m1–m5) has become pivotal. However, their use is not without limitations. Results from functional assays (e.g., activation of second messengers) reliably measures subtype selectivity, whereas receptor binding underestimates selectivity. From a novel series of azabicyclic oximes, PD151832 has been chosen for further development and data obtained with this M1 muscarinic agonist is used to exemplify central and peripheral animal models of cholinergic activity and the ability to translate in vitro subtype selectivity into in vivo efficacy. Drug Dev. Res. 40:133–143, 1997. © 1997 Wiley-Liss, Inc.

Potential cellular signaling mechanisms mediating upregulation of endothelial nitric oxide production by estrogen.

Experimental and clinical studies have provided ample evidence that estrogens exert a significant antiatherosclerotic effect and reduce morbidity and mortality due to cardiovascular diseases. The exact cellular mechanism of this vasculoprotective action of estrogen is not known, but recent work in our and other laboratories suggests that upregulation of endothelial nitric oxide (NO) production may significantly contribute to the mechanism. The vascular endothelium of female animals and humans produces more NO than that of males. Estrogen treatment significantly increases endothelial NO generation in ovariectomized animals and in postmenopausal women. Reduced endothelial NO production in the aorta of estrogen-receptor-deficient mice indicates that the nuclear estrogen receptor mediates the effect of estrogen. The most probable mechanism of estrogen-induced upregulation of endothelial NO production is the transcriptional stimulation of NOS III gene expression. However, the following alternative mechanisms may be involved as well: (1) inhibition of cytokine-induced downregulation of NOS III gene expression, (2) posttranslational modification of NOS III protein, (3) increased cofactor or L-arginine availability, (4) nongenomic activation of second messengers (e.g., Ca2+, cAMP) and tyrosine kinase, and (5) modulation of NO degrading systems (e.g., reactive oxygen radical generation and antioxidants). This paper reviews current data supporting these potential mechanisms.

Calcium-dependent second-messenger regulation of low-density lipoprotein oxidation by human aortic smooth muscle cells

We postulated that the response of vascular smooth muscle cells (SMCs) to oxidized low-density lipoprotein (LDL) may be modulated through calcium and 3', 5' cyclic adenosine monophosphate (Ca+2-cAMP) second-messenger activity. Changes in cytosolic calcium [Ca+2]i in aortic SMCs exposed to native (N-) and oxidized (Ox-) LDL were measured with a Fura 2-AM indicator. The influence of cAMP on this response was determined by incubating the cells with either forskolin or 8-bromo-cAMP (stimulatory) or galanin (inhibitory). The cells were then activated by an initial preincubation with N- or Ox-LDL, and the subsequent cellular oxidation of N-LDL was measured. The effect of cAMP mediators alone or in conjunction with calcium antagonism was studied. Exposure of SMCs to Ox-LDL resulted in a marked elevation of [Ca+2]i(306 +/- 12 nmol/L) compared with that in the control group (192 +/- 15 nmol/Liter; p < 10(-6)). This response was augmented by cAMP stimulation (406 +/- 8 nmol/L; p < 10(-6)) but reduced by cAMP inhibition (247 +/- 8 nmol/L; p < 10(-6)). The activation of intracellular signaling by initial Ox-LDL priming increased the subsequent oxidation of N-LDL (0.40 +/- 0.02 nmol malondialdehyde versus 0.24 +/- 0.02 nmol MDA control; p < 10(-4)). This response was enhanced by cAMP (0.45 +/- 0.03 nmol MDA; p < 10(-4)) and inhibited by galanin (0.26 +/- 0.02; p < 10(-4)). The cAMP effect was reversed by the blockade of calcium mobilization via membrane channels and reticular release. Oxidized LDL-induced Ca+2-cAMP signaling modulates the cellular oxidation of N-LDL. This finding suggests a mechanism through which the scavenger uptake of modified LDL may potentially be regulated.

17β-Estradiol Potentiates Kainate-Induced Currents via Activation of the cAMP Cascade

Evidence for nongenomic actions of steroids is now coming from a variety of fields of steroid research. Mechanisms of steroid action are being studied with regard to the membrane receptors and the activation of second messengers. The present study investigated the mechanism for the rapid effect of estrogen on acutely dissociated hippocampal CA1 neurons by using the whole-cell, voltage-clamp recording. Under the perforated patch configuration, 17 beta-estradiol potentiated kainate-induced currents in 38% of tested neurons. The potentiation was stereospecific, rapid in onset, and reversible after the removal of the steroid. Dose-response curves show that the potentiation by 17 beta-estradiol was evident at a concentration as low as 10 nM and saturated at 10 microM. 17 beta-Estradiol did not affect the kinetics (i.e., affinity and cooperativity) and reversal potential of kainate-induced currents. This suggests that the potentiation did not result from direct interaction with kainate receptors nor the activation of ion channels other than kainate receptor-channels. The potentiation by 17 beta-estradiol was similar to the enhancement of kainate-induced currents evoked by 8-bromo-cAMP, and was modulated by an inhibitor of phosphodiesterase (IBMX). The estrogen potentiation was blocked by a specific blocker of PKA (Rp-cAMPS). Under standard recording configuration, the effect was significantly affected by intracellular perfusing with GDP-beta-S or GTP-gamma-S. The data suggest that the potentiation of kainate-induced currents by 17-beta-estradiol was likely a G-protein(s) coupled, cAMP-dependent phosphorylation event. By involvement of this non-genomic mechanism, estrogen may play a role in the modulation of excitatory synaptic transmission in the hippocampus.

A20 zinc finger protein inhibits TNF and IL-1 signaling.

A20 zinc finger protein is a product of a cytokine-induced primary response gene. In this report, we demonstrate that A20 specifically inhibits signal transduction pathways induced by TNF and IL-1, suggesting that it functions as a negative regulator of the cytokine response. Overexpression of A20 in MCF7 breast carcinoma cells or in WEHI-S fibrosarcoma cells inhibits apoptosis induced by TNF, whereas cytotoxicity induced by anti-Fas (anti-CD95); lymphokine-activated killer (LAK) cells, serum starvation, oxidative stress, or okadaic acid is not inhibited. Overexpression of A20 also inhibits TNF-induced activation of phospholipase A2 in a similar dose-dependent manner as it inhibits TNF-mediated apoptosis, whereas it does not affect the activation of phospholipase A2 by anti-Fas. Interestingly, A20 also blocks TNF-induced signal transduction pathways not directly related to the cytotoxicity, namely activation of NF-kappa B and AP-1 transcription factors. Activation of these transcription factors by a functionally related cytokine, IL-1, is also inhibited by A20, whereas activation induced by hydrogen peroxide or phorbol ester is unaffected. Overexpression of A20 does not affect the binding of TNF to its cell surface receptors. These data suggest that A20 functions as a negative regulator of TNF and IL-1, interfering with signal transduction pathways at an early point following receptor binding but before the activation of various second messengers, leading to the wide variety of effects induced by these cytokines.

Inositol 1,4,5‐trisphosphate receptor localization in the brain of a weakly electric fish (Apteronotus leptorhynchus) with emphasis on the electrosensory system

Inositol 1,4,5-trisphosphate is a widespread intracellular second messenger that mobilizes intracellular Ca2+ stores. The inositol 1,4,5-trisphosphate receptor involved is associated with the endoplasmic reticulum in neurons. In mammalian brain, inositol 1,4,5-trisphosphate receptor-containing neurons are found in many diverse regions, with cerebellar Purkinje cells containing the highest density of these receptors. We used immunohistochemical methods to identify the distribution of inositol 1,4,5-trisphosphate receptor-containing neurons in the brain of the weakly electric fish and Western blotting to confirm that a protein similar to the inositol 1,4,5-trisphosphate receptor of mammalian brain was recognized in the fish brain. In the telencephelon, the dorsal forebrain regions had low amounts of inositol 1,4,5-trisphosphate receptor. In the diencephalon, only the nucleus tuberis posterior was moderately immunoreactive. In the mesencephalon, only the optic tectum contained cells with intense immunoreactivity, similar to our findings for the ryanodine receptor (G.K.H. Zupanc, J.A. Airey, L. Maler, J. Sutko, and M.H. Ellisman, 1992, J. Comp. Neurol. 325:135-151), which also mobilizes intracellular calcium. In the rhombencephalon, a subset of the pyramidal cells of the electrosensory lateral line lobe contained inositol 1,4,5-trisphosphate receptor. These cells have been shown to contain ryanodine receptor (Zupanc et al., 1992). However, unlike the ryanodine receptor, the distribution of inositol 1,4,5-trisphosphate receptor in these cells is constrained to the soma and proximal dendrites. This compartmentalization may indicate the limit of the range of second-messenger action. Other regions containing immunoreactive cells were the nucleus praeminentialis dorsalis (multipolar and boundary cells), nucleus medialis and crista cerebellaris, and the cerebellum, whose Purkinje cells were the most intensely labeled. The functional implications of inositol 1,4,5-trisphosphate receptor localization in the electrosensory lateral line lobe are discussed.

Regulation of cellular calcium through signaling cross-talk involves an intricate interplay between the actions of receptors, G-proteins, and second messengers.

Various external stimuli regulate cellular Ca2+ fluxes. Information from these stimuli is transmitted to the cell interior by signal transducing systems that involve interactions between receptors, G-proteins, and enzymes that generate second messengers. The possible mechanisms by which "cross-talk" within and between these second messenger-generating systems provides an intricate molecular avenue for fine modulation of intracellular Ca2+ are reviewed.

Analysis of the effects of modulatory agents on a modeled bursting neuron: Dynamic interactions between voltage and calcium dependent systems

In a computational model of the bursting neuron R15, we have implemented proposed mechanisms for the modulation of two ionic currents (IR and ISI) that play key roles in regulating its spontaneous electrical activity. The model was sufficient to simulate a wide range of endogenous activity in the presence of various concentrations of serotonin (5-HT) or dopamine (DA). The model was also sufficient to simulate the responses of the neuron to extrinsic current pulses and the ways in which those responses were altered by 5-HT or DA. The results suggest that the actions of modulatory agents and second messengers on this neuron, and presumably other neurons, cannot be understood on the basis of their direct effects alone. It is also necessary to take into account the indirect effects of these agents on other unmodulated ion channels. These indirect effects occur through the dynamic interactions of voltage-dependent and calcium-dependent processes.

Decreased phospholipase C-beta immunoreactivity, phosphoinositide metabolism, and protein kinase C activation in senescent F-344 rat brain.

Phosphoinositide metabolism, phospholipase C immunoreactivity, and protein kinase C translocation were measured in brain slices of 6- and 24-month-old F-344 rats. Basal phosphoinositide labeling and accumulation of [3H]inositol phosphates were reduced in the 24-month-old rats. The cholinergic agonist, carbachol, induced lower net accumulations of inositol phosphates in striatal, hippocampal, and cortical slices of the aged rats. The dose-response curve for carbachol showed a significantly lower maximal response in the striatum of senescent rats, whereas the time course of [3H]inositol incorporation into inositol metabolites and the accumulation of free [3H]inositol in tissues from young and old animals were not different. Quantitative analyses showed marked reductions in endogenous brain levels of the phosphoinositides and in phospholipase C-beta 1 immunoreactivity, but no marked reductions in endogenous brain levels of the phosphoinositides and in phospholipase C-beta 1 immunoreactivity, but no changes in phospholipase C-gamma levels in aged animals. Moreover, basal protein kinase C activity and carbachol-mediated translocation of the enzyme were significantly reduced in the cerebral cortex of the senescent animals. These findings imply that aging is associated with alterations in the brain content, metabolism, and activity of phosphoinositide-derived second messengers in the F-344 rat brain.

Separation of labeled inositol phosphate isomers by high-pressure liquid chromatography (HPLC).

It has now been established that agonist stimulation of a large variety of cell-surface receptors promotes hydrolysis of inositol phospholipids through activation of a phosphoinositide-specific phospholipase C (, ). Phosphodiesteric cleavage of phosphatidylinositol-4,5-bis-phosphate by phospholipase C produces Ins(1,4,5)P3 and sn-1,2-diacylglycerol, which release Ca2+ from intracellular stores and activate protein kinase(s) C, respectively (, , , ). Metabolism of Ins(1,4,5)P3 is rapid and can follow two possible pathways (Fig. 1.; for review, see ref. ). The simple dephosphory lation route proceeds via a 5-phosphatase to Ins(1,4)P2, and Ins(1,3,4)P3/Ins(1,4)P2 1-phosphatase to Ins(4)P1. Alternatively, phosphorylation of Ins(1,4,5)P3 by a 3-kinase generates the putative second messenger Ins(1,3,4,5)P4, which then undergoes sequential dephosphorylation via a 5-phosphatase to Ins(1,3,4)P3 and either a 4-phosphatase, an Ins(1,3,4)P3/Ins(1,4)P2 1-phosphatase, or a 6-kinase route to produce Ins(1,3)P2, Ins(3,4)P2, or Ins(1,3,4,6)P4, respectively. Subsequent dephosphorylation of Ins(1,3)P2 and Ins(3,4)P2 produces Ins(l)P1 and Ins(3)P1, which, along with Ins(4)P1, are metabolized by the inositol monophosphatase to release myo-inositol (). This complex pathway of Ins(1,4,5)P3, serves to terminate second-messenger action and to conserve efficiently the cellular myo-inositol pool for the resynthesis of inositol phospholipids. Fig. 1. The phosphoinositide cycle. Open image in new window Fig. 1. The phosphoinositide cycle.