Messenger Signaling Cascades Research Articles

Serotonergic dysfunction impairs locomotor coordination in spinal muscular atrophy.

Neuromodulation by serotonin regulates the activity of neuronal networks responsible for a wide variety of essential behaviours. Serotonin (or 5-HT) typically activates metabotropic G protein-coupled receptors, which in turn initiate second messenger signalling cascades and induce short and long-lasting behavioural effects. Serotonin is intricately involved in the production of locomotor activity and gait control for different motor behaviours. Although dysfunction of serotonergic neurotransmission has been associated with mood disorders and spasticity after spinal cord injury, whether and to what extent such dysregulation is implicated in movement disorders has not been firmly established. Here, we investigated whether serotonergic neuromodulation is affected in spinal muscular atrophy (SMA), a neurodegenerative disease caused by ubiquitous deficiency of the SMN protein. The hallmarks of SMA are death of spinal motor neurons, muscle atrophy and impaired motor control, both in human patients and mouse models of disease. We used a severe mouse model of SMA, that closely recapitulates the severe symptoms exhibited by type I SMA patients, the most common and most severe form of the disease. Together, with mouse genetics, optogenetics, physiology, morphology and behavioural analysis, we report severe dysfunction of serotonergic neurotransmission in the spinal cord of SMA mice, both at early and late stages of the disease. This dysfunction is followed by reduction of 5-HT synapses on vulnerable motor neurons. We demonstrate that motor neurons innervating axial and trunk musculature are preferentially affected, suggesting a possible cause for the proximo-distal progression of disease, and raising the possibility that it may underlie scoliosis in SMA patients. We also demonstrate that the 5-HT dysfunction is caused by SMN deficiency in serotonergic neurons in the raphe nuclei of the brainstem. The behavioural significance of the dysfunction in serotonergic neuromodulation is underlined by inter-limb discoordination in SMA mice, which is ameliorated when selective restoration of SMN in 5-HT neurons is achieved by genetic means. Our study uncovers an unexpected dysfunction of serotonergic neuromodulation in SMA and indicates that, if normal function is to be restored under disease conditions, 5-HT neuromodulation should be a key target for therapeutic approaches.

Divalent cation driven liquid‐liquid phase separation of disordered acidic proteins

Divalent cations are essential to cellular processes through interaction with biological macromolecules, involvement in enzyme‐mediated catalysis, and as secondary messengers in signaling cascades. Recently, we have discovered divalent cation driven liquid‐liquid phase separation (LLPS) underlies endoplasmic reticulum Ca2+ stores through disordered acidic calcium binding protein calsequestrin‐1 (CASQ1). CASQ1 interacts with divalent cations to enter a LLPS state via complex coacervation. CASQ1 LLPS propensity is positively regulated by FAM20C‐dependent phosphorylation that induces an order‐to‐disorder transition accompanied by dramatic structural expansion. These events increase intracellular Ca2+ stores and regulate cellular stress response. Proteome wide analysis of disordered acidic proteins suggests divalent cation driven LLPS may be an emerging mechanism extending beyond the ER. Particularly, these proteins are highly enriched in the nucleus and cytosol where they accumulate in a number of biological condensates alongside other polyanions like RNA to regulate gene expression. We hypothesize divalent cation driven LLPS is a widespread mechanism driving both protein‐protein and protein‐nucleic acid interactions.

Bradykinin-Induced Sensitization of Transient Receptor Potential Channel Melastatin 3 Calcium Responses in Mouse Nociceptive Neurons.

TRPM3 is a calcium-permeable cation channel expressed in a range of sensory neurons that can be activated by heat and the endogenous steroid pregnenolone sulfate (PS). During inflammation, the expression and function of TRPM3 are both augmented in somatosensory nociceptors. However, in isolated dorsal root ganglion (DRG) neurons application of inflammatory mediators like prostaglandins and bradykinin (BK) inhibit TRPM3. Therefore, the aim of this study was to examine the effect of preceding activation of cultured 1 day old mouse DRG neurons by the inflammatory mediator BK on TRPM3-mediated calcium responses. Calcium signals were recorded using the intensity-based dye Fluo-8. We found that TRPM3-mediated calcium responses to PS were enhanced by preceding application of BK in cells that responded to BK with a calcium signal, indicating BK receptor (BKR) expression. The majority of cells that co-expressed TRPM3 and BKRs also expressed TRPV1, however, only a small fraction co-expressed TRPA1, identified by calcium responses to capsaicin and supercinnamaldehyde, respectively. Signaling and trafficking pathways responsible for sensitization of TRPM3 following BK were characterized using inhibitors of second messenger signaling cascades and exocytosis. Pharmacological blockade of protein kinase C, calcium–calmodulin-dependent protein kinase II and diacylglycerol (DAG) lipase did not affect BK-induced sensitization, but inhibition of DAG kinase did. In addition, release of calcium from intracellular stores using thapsigargin also resulted in TRPM3 sensitization. Finally, BK did not sensitize TRPM3 in the presence of exocytosis inhibitors. Collectively, we show that preceding activation of DRG neurons by BK sensitized TRPM3-mediated calcium responses to PS. Our results indicate that BKR-mediated activation of intracellular signaling pathways comprising DAG kinase, calcium and exocytosis may contribute to TRPM3 sensitization during inflammation.

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

Immunocytochemical Localization of Olfactory-signaling Molecules in Human and Rat Spermatozoa.

Expression of olfactory receptors (ORs) in non-olfactory tissues has been widely reported over the last 20 years. Olfactory marker protein (OMP) is highly expressed in mature olfactory sensory neurons (mOSNs) of the olfactory epithelium. It is involved in the olfactory signal transduction pathway, which is mediated by well-conserved components, including ORs, olfactory G protein (Golf), and adenylyl cyclase 3 (AC3). OMP is widely expressed in non-olfactory tissues with an apparent preference for motile cells. We hypothesized that OMP is expressed in compartment-specific locations and co-localize with an OR, Golf, and AC3 in rat epididymal and human-ejaculated spermatozoa. We used immunocytochemistry to examine the expression patterns of OMP and OR6B2 (human OR, served as positive olfactory control) in experimentally induced modes of activation and determine whether there are any observable differences in proteins expression during the post-ejaculatory stages of spermatozoal functional maturation. We found that OMP was expressed in compartment-specific locations in human and rat spermatozoa. OMP was co-expressed with Golf and AC3 in rat spermatozoa and with OR6B2 in all three modes of activation (control, activated, and hyperactivated), and the mode of activation changed the co-expression pattern in acrosomal-reacted human spermatozoa. These observations suggest that OMP expression is a reliable indicator of OR-mediated chemoreception, may be used to identify ectopically expressed ORs, and could participate in second messenger signaling cascades that mediate fertility.

G Protein-Coupled Estrogen Receptor: Rapid Effects on Hippocampal-Dependent Spatial Memory and Synaptic Plasticity.

In the hippocampus, estrogen regulates gene transcription linked to neuronal growth, neuroprotection, and the maintenance of memory function (1–3). The mechanism is likely to involve genomic regulation through classic estrogen receptor (ER) signaling cascades that influence transcription. Estrogens binding to classic nuclear ERs, alpha (ERα) and beta (ERβ), and have pleotropic effects on development, behavior, and neurophysiological functions, including synaptic plasticity and memory consolidation (4–7). In addition to ERα and ERβ, estrogen can also initiate activation of classical second messenger signaling cascades to influence the activity of G-proteins and a host of kinases, resulting in rapid changes in physiology (8–14). These rapid effects of estrogen are commonly mediated by membrane receptors. In the late 90s, multiple laboratories cloned cDNA/gene for an orphan G-protein-coupled receptor with very low homology with other G-protein-coupled receptors and named it G-protein-coupled receptor 30 (GPR30) (15–20). Later in 2007, the International Union of Basic and Clinical Pharmacology designated GPR30 as G protein-coupled estrogen receptor (GPER) (21); GPER is a seven-transmembrane G-protein-coupled receptor, predominantly expressed on the cell membrane (22). Interestingly, GPER is reported to mediate many of the rapid responses of estradiol in the adult brain, and is widely distributed in the mammalian brain including the plasma membrane of hippocampal neurons (23–31). GPER modulates second messenger signaling cascades involving Gαs- and Gαi/o-associated increase in cyclic adenosine monophosphate and phosphoinositide 3-kinase or Src protein kinase respectively (32, 33). Activation of GPER is also associated with phospholipase C, and the inositol receptor and ryanodine receptor-mediated increase in intracellular calcium (24, 34). This commentary is concentrated specifically on the possible rapid effects of GPER in hippocampal-dependent spatial memory function and synaptic plasticity.

Interrogating endogenous neuromodulatory GPCR signal processing by real‐time imaging of cAMP dynamics through intact neuronal circuits

The majority of neuromodulators exert their effects by activating G protein coupled receptors (GPCRs) which shape neuronal properties through the regulation of second messengers. The mechanisms of signal decoding at GPCRs amidst diversity of the effects associated with their ctivation remains elusive. Furthermore, there is a paucity of understanding of how GPCRs signal to produce unique responses impacting cellular physiology while converging on a limited set of second messenger signaling cascades. Although significant progress has been made studying GPCRs in reconstituted systems, information decoding processes by GPCRs in an endogenous setting is understood less due to a lack of appropriate tools. Here, we developed a novel in vivo reagent that allows monitoring GPCR signaling in the endogenous setting from genetically defined populations of cells. The approach takes advantage of knock‐in mice engineered to express a genetically encoded FRET‐based cAMP biosensor in a Cre‐dependent fashion. We applied this reagent to study integration of GPCR signals in striatal medium spiny neurons both in culture and in brain slices where intact circuits were activated by optogenetics. Combined with CRISPR/Cas9 genomic editing, this approach enabled us to identify individual receptors and analyze their contributions to downstream signaling in the native environment. We identified key spatial differences in pharmacological responsiveness by comparing dendritic regions with the neuronal cell body as well as plasma membrane and internal cellular signaling compartments. We then conducted comparative analysis of GPCR signaling mediated by two opposing circuits modulated by the neurotransmitter dopamine, revealing the mechanisms of dopaminergic processing as well as major differences in the generation of discrete second messenger signatures. We further demonstrate how cAMP signal processing mechanisms contribute to allostatic adaptations that imbalance tuning between striatal output neurons that may underlie states of reward and aversion.Support or Funding InformationNIH 1R01DK110621, APS STRIDE Undergraduate Summer Research Fellowship 1R25HL115473‐01.This work was supported by NIH grants: DA041207 (to B.S.M.), DA036596 (to K.A.M.), and DA026405 (to K.A.M.).

Expression and immunohistochemical localization of the Gβγ‐activated and calcineurin‐inhibited adenylyl cyclase isoforms in rat articular chondrocytes (922.9)

Aims: Cross‐talk between signal transduction pathways is essential to maintain normal homeostasis. cAMP mediated pathway is archetypical in mammalian cells and adenylyl cyclase (AC) is the effector enzyme that converts ATP into cAMP. We believe that AC/cAMP mediated pathway is involved in homeostasis of the articular cartilage. In this study we report the expression of Gβγ‐activated AC isoforms 2, 4, and 7 and calcineurin‐inhibited AC isoform 9 in rat articular chondrocytes.Main methods: Fresh slices of articular cartilage were taken from various synovial joints of rat and the expression of AC isoforms was determined by RT‐PCR. Immunohistochemistry was performed to localize these isoforms in articular chondrocytes.Key findings: All AC isoforms studied were found differentially expressed in different zones of the rat articular cartilage. Generally, expression of all AC isoforms increased with age.Significance: These data add to the information about signaling cascades possibly involved in articular chondrocytes. Presence of AC isoforms 2, 4, 7, and 9 suggest a role for the cAMP mediated 2nd messenger signaling cascades in articular chondrocytes. Further studies, such as measuring the AC and cAMP levels during development and differentiation of articular chondrocytes would confirm our results and help in understanding the signaling mechanisms in articular chondrocytes.Grant Funding Source: Supported by Seed money grant from the FHS‐Research Committee of the Aga Khan University, Karachi

Lipid imaging by mass spectrometry – a review

Mass spectrometry imaging (MSI) has proven to be extremely useful for applications such as the spatial analysis of peptides and proteins in biological tissue, the performance assessment of drugs in vivo or the measurement of protein or metabolite expression as tissue classifiers or biomarkers from disease versus control tissue comparisons. The most popular MSI technique is MALDI mass spectrometry. First invented by Richard Caprioli in the mid-1990s, it is the highest performing MSI technique in terms of spatial resolution, sensitivity for intact biomolecules and application range today. The unique ability to identify and spatially resolve numerous compounds simultaneously, based on m/z values has inter alia been applied to untargeted and targeted chemical mapping of biological compartments, revealing changes of physiological states, disease pathologies and metabolic faith and distribution of xenobiotics. Many MSI applications focus on lipid species because of the lipids' diverse roles as structural components of cell membranes, their function in the surfactant cycle, and their involvement as second messengers in signalling cascades of tissues and cells. This article gives a comprehensive overview of lipid imaging techniques and applications using established MALDI and SIMS methods but also other promising MSI techniques such as DESI.

Interactions between hormone and redox signalling pathways in the control of growth and cross tolerance to stress

The ability of plants to respond to a wide range of environmental stresses is highly flexible and finely balanced through the interaction of hormonal plant growth regulators and the redox signalling hub, which integrates information from the environment and cellular metabolism/physiology. Plant hormones produce reactive oxygen species (ROS) as second messengers in signalling cascades that convey information concerning changes in hormone concentrations and/or sensitivity to mediate a whole range of adaptive responses. Cellular redox buffering capacity that is determined largely by the abundance of ascorbate has a profound influence on the threshold at which hormone signalling is triggered and on the interactions between different hormones. Other antioxidants such as glutathione, glutaredoxins and thioredoxins are also central redox regulators of hormone signalling pathways. The complex network of cross-communication between oxidants and antioxidants in the redox signalling hub and the different hormone signalling pathways maximises productivity under stress-free situations and regulates plant growth, development, reproduction, programmed cell death and survival upon exposure to stress. This interactive network confers enormous regulatory potential because it allows plants to adapt to changing and often challenging conditions, while preventing boom or bust scenarios with regard to resources, ensuring that energy is produced and utilised in a safe and efficient manner.

Integrating Cardiac PIP 3 and cAMP Signaling through a PKA Anchoring Function of p110γ

Adrenergic stimulation of the heart engages cAMP and phosphoinositide second messenger signaling cascades. Cardiac phosphoinositide 3-kinase p110γ participates in these processes by sustaining β-adrenergic receptor internalization through its catalytic function and by controlling phosphodiesterase 3B (PDE3B) activity via an unknown kinase-independent mechanism. We have discovered that p110γ anchors protein kinase A (PKA) through a site in its N-terminal region. Anchored PKA activates PDE3B to enhance cAMP degradation and phosphorylates p110γ to inhibit PIP(3) production. This provides local feedback control of PIP(3) and cAMP signaling events. In congestive heart failure, p110γ is upregulated and escapes PKA-mediated inhibition, contributing to a reduction in β-adrenergic receptor density. Pharmacological inhibition of p110γ normalizes β-adrenergic receptor density and improves contractility in failing hearts.

Mechanisms Underlying Rapid Aldosterone Effects in the Kidney

The steroid hormone aldosterone is a key regulator of electrolyte transport in the kidney and contributes to both homeostatic whole-body electrolyte balance and the development of renal and cardiovascular pathologies. Aldosterone exerts its action principally through the mineralocorticoid receptor (MR), which acts as a ligand-dependent transcription factor in target tissues. Aldosterone also stimulates the activation of protein kinases and secondary messenger signaling cascades that act independently on specific molecular targets in the cell membrane and also modulate the transcriptional action of aldosterone through MR. This review describes current knowledge regarding the mechanisms and targets of rapid aldosterone action in the nephron and how aldosterone integrates these responses into the regulation of renal physiology.

Melatonin and IP3-induced Ca2+ Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

Ionotropic and metabotropic mechanisms in chemoreception: 'chance or design'?

Chemosensory receptors convert an enormous diversity of chemical signals from the external world into a common language of electrical activity in the brain. Mammals and insects use several families of transmembrane receptor proteins to recognize distinct classes of volatile and non-volatile chemicals that are produced by conspecifics or other environmental sources. A comparison of the signalling mechanisms of mammalian and insect receptors has revealed an unexpected functional distinction: mammals rely almost exclusively on metabotropic ligand-binding receptors, which use second messenger signalling cascades to indirectly activate ion channels, whereas insects use ionotropic receptors, which are gated directly by chemical stimuli, thereby leading to neuronal depolarization. In this review, we consider possible reasons for this dichotomy, taking into account biophysical, cell biological, ecological and evolutionary influences on how information is extracted from chemosensory cues by these animal classes.

Reconstitution of PKA-Dependent Modulation of Cardiac Cav1.2 Channels

L-type calcium currents through Cav1.2 channels initiate contraction in cardiac muscle. Their regulation by neurotransmitters and hormones through second messenger signaling cascades and protein kinase A (PKA) phosphorylation is a key controller of calcium signaling and contractile force. The α1 subunit C-terminus contains binding sites for multiple regulatory proteins including the PKA/A kinase anchoring protein 15 (AKAP15) complex. The C-terminal domain is proteolytically cleaved but reassociates non-covalently with the truncated channel and acts as a potent autoinhibitor of channel activity. Relief of autoinhibition by cellular regulatory signals acting on the C-terminus provides an attractive mechanism for producing the large increases in calcium current that are observed physiologically. In fact, consistent reconstitution of PKA-dependent regulation of Cav1.2 channels in non-muscle has been difficult to achieve. To reconstitute such PKA regulation, we optimized the expression of truncated Cav1.2 channels, the distal C-terminal domain, the α2δ subunit, and the β2b subunit to give a functional autoregulatory complex as assessed by whole-cell voltage clamp recordings of tsA-201 cells. Expression of the truncated Cav1.2 channel with the free distal domain resulted in large decreases in inward barium current and in coupling between voltage-dependent gating and pore opening. We hypothesized that AKAP15 expression would promote PKA association with the distal C-terminal of the channel and increase the likelihood of PKA-dependent phosphorylation. After optimizing AKAP15 expression, currents recorded in 5 μM forskolin were approximately 5-fold larger than those recorded in the presence of kinase inhibitor RO 31-8220 (1 uM). These findings show that the full-range of PKA-dependent modulation of Cav1.2 channels can be reproduced when an autoinhibitory complex is formed in this manner and provide a substrate for further studies of this physiologically important regulatory process.Supported by NIH grant HL007312-31 and AHA fellowship 09POST2080270.

2-Aminoethoxydiphenyl-borate (2-APB) increases excitability in pyramidal neurons

Calcium ions (Ca 2+) released from inositol trisphosphate (IP 3)-sensitive intracellular stores may participate in both the transient and extended regulation of neuronal excitability in neocortical and hippocampal pyramidal neurons. IP 3 receptor (IP 3R) antagonists represent an important tool for dissociating these consequences of IP 3 generation and IP 3R-dependent internal Ca 2+ release from the effects of other, concurrently stimulated second messenger signaling cascades and Ca 2+ sources. In this study, we have described the actions of the IP 3R and store-operated Ca 2+ channel antagonist, 2-aminoethoxydiphenyl-borate (2-APB), on internal Ca 2+ release and plasma membrane excitability in neocortical and hippocampal pyramidal neurons. Specifically, we found that a dose of 2-APB (100 μM) sufficient for attenuating or blocking IP 3-mediated internal Ca 2+ release also raised pyramidal neuron excitability. The 2-APB-dependent increase in excitability reversed upon washout and was characterized by an increase in input resistance, a decrease in the delay to action potential onset, an increase in the width of action potentials, a decrease in the magnitude of afterhyperpolarizations (AHPs), and an increase in the magnitude of post-spike afterdepolarizations (ADPs). From these observations, we conclude that 2-APB potently and reversibly increases neuronal excitability, likely via the inhibition of voltage- and Ca 2+-dependent potassium (K +) conductances.

Multiple roles for sphingolipids in steroid hormone biosynthesis.

Steroid hormones are essential regulators of a vast number of physiological processes. The biosynthesis of these chemical messengers occurs in specialized steroidogenic tissues via a multi-step process that is catalyzed by members of the cytochrome P450 superfamily of monooxygenases and hydroxysteroid dehydrogenases. Though numerous signaling mediators, including cytokines and growth factors control steroidogenesis, trophic peptide hormones are the primary regulators of steroid hormone production. These peptide hormones activate a cAMP/cAMP-dependent kinase (PKA) signaling pathway, however, studies have shown that crosstalk between multiple signal transduction pathways and signaling molecules modulates optimal steroidogenic capacity. Sphingolipids such as ceramide, sphingosine, sphingosine-1-phosphate, sphingomyelin, and gangliosides have been shown to control the steroid hormone biosynthetic pathway at multiple levels, including regulating steroidogenic gene expression and activity as well as acting as second messengers in signaling cascades. In this review, we provide an overview of recent studies that have investigated the role of sphingolipids in adrenal, gonadal, and neural steroidogenesis.

REGULATION OF PANCREATITIS-ASSOCIATED PROTEIN (PAP) INDUCED CYTOKINE EXPRESSION THROUGH SIGNAL TRANSDUCTION PATHWAYS NFKB, JNK, AND ERK

Background: Pancreatitis-associated proteins (PAP) are minimally expressed in normal pancreas but strongly induced in acute pancreatitis. We have previously demonstrated that expression of PAP may modulate the severity of acute pancreatitis, by affecting macrophage activation. Since pancreatitis is associated with immune activation which likely involves second messenger signaling cascades, we investigated the effect of PAP on macrophages and potential intracellular pathways involved in PAP signaling. Methods: Rat macrophages (NR8383 alveolar cell line) were cultured with recombinant PAP (2.5-15 μg/ml) and specific intracellular pathway inhibitors NFκB, JNK, ERK or p38 for 24 hr and evaluated for cytokine (IL-1α, IL-1β, TNFα, IL-6) expression (RT-PCR). Controls consisted of cells cultured with medium, vehicle alone, inhibitors alone or neutralizing PAP antibody. Cells were ≥90% viable in all conditions (trypan blue exclusion dye). Data are reported as fold change or mean percent change + range. Significance between groups was set at p<0.05 (Student's t-test). Results: Macrophages cultured in the presence of PAP resulted in increased expression of IL-1α, IL-1β, TNFα, and IL-6 (69, 40, 750 and 210 fold, respectively at 5ug/ml; p<0.05) when compared with controls, which were reversed (IL-1α, IL-1β) with neutralizing anti-PAP antibody. Treatment of macrophages with NFkB, JNK, or ERK, inhibitors decreased PAP induced inflammatory cytokines (82%±13, 51%±35, 71%±20, respectively; p<0.05), whereas treatment with p38 kinase inhibitor did not. Cells culture with inhibitors alone, in the absence of PAP, did not induce inflammatory cytokine expression. Conclusions: Pancreatitis is an immune phenomenon in which PAP is increased. PAP alone induces macrophage cytokine expression in a dose related fashion. Furthermore, PAP induced cytokine expression is mediated through three well known intracellular pathways involving NFκB, JNK, and ERK. Understanding intracellular pathways involved in PAP induced cytokine expression may provide novel treatments for pancreatitis.

Signal Switching, Crosstalk, and Arrestin Scaffolds

Hormone agonists, including angiotensin II (Ang II), norepinephrine, urotensin II, endothelin-1, vasopressin, and serotonin, mediate a plethora of physiological and pathological cardiovascular events via their cognate 7 membrane-spanning G protein–coupled receptors (GPCRs). On ligand binding, GPCRs undergo conformational changes that enable the activation and dissociation of heterotrimeric guanine nucleotide binding proteins (G proteins) and trigger a range of intracellular second messenger signaling cascades. Because of the immediacy of second messenger generation, GPCRs are able to acutely regulate cardiovascular events such as heart rate, contractile force, and systemic vascular resistance. Compelling evidence for GPCR control in the vasculature comes from both transgenic studies and clinical findings. For example, β-adrenergic receptor blockers and inhibitors of the synthesis and binding of Ang II are proven antihypertensive therapeutics for humans. Furthermore, mice lacking RGS2, a regulatory protein that enhances the speed of GPCR signal termination, display marked hypertension, increased basal vascular tone, and hypersensitivity to vasoconstrictive agonists,1 a phenotype that demonstrates the contribution of immediate GPCR-dependent signals, as well as the effect of GPCR dysregulation on cardiovascular homeostasis. The evidence that signals emanating from GPCRs also contribute to the chronic development of vascular disease is persuasive. Overexpression of the Gαq subunit in cardiomyocytes directly stimulates cardiac hypertrophy and decompensated heart failure,2 whereas transgenic mice expressing an inhibitory fragment of Gαq exhibit reduced hypertrophy in response to pressure overload.3 GPCR agonists like Ang II, endothelin-1, and norepinephrine, act directly on cardiomyocytes to stimulate hypertrophy.4 Meanwhile, Ang II contributes to atherosclerosis via activation of vascular smooth muscle cell (VSMC) migration and hypertrophy; this occurs either directly, via the Ang II type 1 receptor (AT1R), or indirectly by stimulating endothelin-1 expression or activating inflammatory pathways.5 Importantly, these pathological vascular effects require significant modulation of gene transcription, often via activation of …

Retrograde tracing of the subset of afferent connections in mouse barrel cortex provided by zincergic neurons

The barrel cortex of rodents is densely innervated by a prominent subclass of glutamatergic neurons that sequester and release zinc from their synaptic boutons. These neurons may play an important role in barrel cortex function and plasticity, as zinc has been shown to modulate synaptic function by regulating neurotransmitter release, excitatory and inhibitory amino acid receptors, and second messenger signaling cascades. Here, we utilized intracortical infusions of sodium selenite to identify the source of the zincergic innervation to the mouse barrel cortex. Our results demonstrate that the majority of zincergic projections to the barrel cortex arose from ipsilateral and callosal neurons, situated in cortical layers 2/3 and 6. Regionally, these labeled neurons were most abundant within the barrel cortex itself, posterior parietal association cortex, secondary somatosensory cortex, and motor cortex. Labeled neurons were also found in other somatosensory regions corresponding to the trunk, fore- and hindlimb, as well as more distant regions such as the visual, rhinal, dorsal peduncular and insular cortices, the claustrum, and lateral and basolateral amygdaloid nuclei. Further, some mice were injected with the retrograde tracer cholera toxin subunit B to compare retrograde labeling of zincergic neurons with that of the general population of neurons innervating the barrel cortex. Our data indicate that all cortical regions providing inputs to the barrel cortex possess a zincergic component, whereas those from thalamic or brainstem structures do not. These findings demonstrate that zincergic pathways comprise a chemospecific associational network that reciprocally interconnects the barrel cortex with other cortical and limbic structures.