Subcellular Signal Transduction Research Articles

Atomistic Modeling of Neuro-cardiovascular Coupling Modulation

Cardiac heart rhythm and vascular tone are tightly controlled by sympathetic and parasympathetic autonomic nervous systems and their dysregulation is associated with multiple cardiovascular disorders such as arrythmias and hypertension. At the molecular level such control is established through a complex set of events including synaptic neurotransmitter release, binding to myocyte adrenergic or cholinergic G-protein coupled receptors (GPCRs), subcellular signal transduction through G-proteins, neurotransmitter - GPCR unbinding, reuptake or degradation. In this study, funded by NIH Common Fund program “Stimulating Peripheral Activity to Relieve Conditions” (SPARC), we used atomistic modeling to identify structural, energetic and kinetic determinants of several molecular processes crucial for cardiovascular neuromodulation such as neurotransmitter interactions with GPCR / G-protein complex at neuroeffector junction as well as G-protein interaction with adenylyl cyclase (AC), a key enzyme for downstream subcellular signaling. To this end, we developed atomistic models of human β-adrenergic receptor (βAR) / Gs and muscarinic receptor (MR) / Gi GPCR / G-protein complexes in different conformational states using available structures and Rosetta structural modeling. We tested these models via molecular docking of βAR agonists norepinephrine and isoproterenol and MR agonists acetylcholine and carbachol. Moreover, receptor model structural stabilities were assessed via microsecond-long all-atom molecular dynamics (MD) simulations with and without ligand bound. Enhanced sampling atomistic MD runs were used to estimate receptor - ligand binding affinities and association / dissociation rates. Martini coarse-grained and Brownian dynamics implicit-solvent techniques were utilized to assess G-protein interactions with AC and GPCR as well. This information will be used to inform functional kinetic models of autonomic control of myocyte subcellular signaling, a crucial component of our predictive multi-scale neurocardiovascular simulator.

Time Courses of Subcellular Signal Transduction and Cellular Apoptosis in Remote Viable Myocardium of Rat Left Ventricles Following Acute Myocardial Infarction: Role of Pharmacomodulation

We tested hypothesis that acute myocardial infarction (AMI) induces cellular apoptosis and serial changes of protein kinase C epsilon (PKC-epsilon) and p38 mitogen-activated protein kinase (p38 MAPK), and tested cardio-protective effect of losartan in this condition. The rats were assigned to group A (sacrificed on day 2), group B (sacrificed on day 5), and group C (sacrificed on day 14). Rats in each group were further randomized into the following groups: AMI (ligation of left coronary artery) without losartan (AMI-L0); AMI with losartan 20 mg/ kg/d (AMI-L1); and sham groups (L0 and L1). The PKC-epsilon expression in membrane compartment was increased in AMI-L1 group than in other groups on day 5 and in AMI groups than in sham groups on day 14 (P < .01). Phosphorylated form of cytosolic p38 MAPK level was increased in AMI-L1 than in other groups on day 14 (P < .05). Furthermore, 14-day left ventricular ejection fraction was higher and cellular apoptosis was lower in AMI-L1 group than in AMI-L0 group (P < .0001).

HER-2/neu Expression in Glioblastoma Multiforme

The HER-2/neu oncogene encodes for a transmembrane glycoprotein with intracellular tyrosine kinase activity. The HER-2/neu receptor belongs to the family of epidermal growth factor receptors that are crucial in the activation of subcellular signal transduction pathways controlling epithelial cell growth and differentiation. Overexpression of HER-2/neu is observed in 20% to 40% of breast cancers and other solid tumors. Although information is limited, one study suggested that 15% of glioblastoma multiforme (GBM) express HER-2/neu by immunohistochemistry (IHC); gene amplification by fluorescence in situ hybridization (FISH) was not investigated. Studies in this area are potentially significant owing to the role of recombinant monoclonal anti-HER-2/neu antibody traztuzumab (Herceptin) in the treatment of tumors. A retrospective clinicopathologic review of 49 patients with GBM with HER-2/neu IHC staining and HER-2/neu gene amplification by FISH was performed. The study included 44 patients (17 women, 27 men; age range 20 to 79 y, mean 57.9 y). Initial surgery involved tumor debulking or subtotal resection in 34 patients. Thirty-six patients received adjuvant radiation therapy and 19 patients received adjuvant chemotherapy. At follow-up (range 1.0 to 49.5 mo, mean 10.5 mo), 40 patients died with tumor and 4 patients were lost to follow-up. All tumors were negative for HER-2/neu protein by IHC and for HER-2/neu gene amplification by FISH. No GBM demonstrates HER-2/neu protein by IHC or amplification of the HER-2/neu gene by FISH. The HER-2/neu oncogene does not seem to play a role in the pathogenesis of GBM.

Quantitative Imaging of Subcellular Signal Transduction

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

This month in J Lab Clin Med: Issue highlights for January 2003

This month in J Lab Clin Med: Issue highlights for January 2003

H1-A extracted from Cordyceps sinensis suppresses the proliferation of human mesangial cells and promotes apoptosis, probably by inhibiting the tyrosine phosphorylation of Bcl-2 and Bcl-XL

H1-A, a pure compound used in traditional Chinese medicine, is effective in the treatment of autoimmune disorders of MRL lpr/lpr mice. We have previously reported that after 8 weeks of oral therapy with H1-A, 40 μg/kg/day, MRL lpr/lpr mice demonstrated significantly less proteinuria, lower serum creatinine levels, and less renal mesangial proliferation than mice in an untreated group. To clarify the pharmacologic properties of H1-A, we studied its cellular and subcellular effects in cultured human mesangial cells. Our results show that H1-A inhibits cell proliferation and promotes the apoptosis of interleukin (IL)-1- and platelet-derived growth factor (PDGF)-BB-activated human mesangial cells in vitro. Uptake of tritiated thymidine was nearly totally suppressed by the addition of 12.5 μmol/L H1-A (counts per minute decreased from 3905 ± 70 to 141 ± 5). The population of S-phase cells decreased from 15.5% ± 1.7% to 10.0% ± 0.3%, and G0 + G1 phase cells increased from 68.8% ± 0.07% to 74.6% ± 0.05%. This suppression was not a result of cytotoxicity. Apoptosis of human mesangial cells was detectable after treatment with 12.5 or 25 μmol/L H1-A. Using immunoprecipitation and immunoblotting, we found that H1-A inhibits tyrosine phosphorylation of human mesangial proteins and that Bcl-2 and Bcl-XL were probably among these proteins. These findings suggest that H1-A modulates some subcellular signal-transduction pathways and changes the balance between proliferation and apoptosis of mesangial cells in vitro or in vivo. H1-A may be effective in the management of autoimmune disorders, and the modulation of the signal transduction proteins Bcl-2 and Bcl-XL may represent a target for future pharmacologic interventions. (J Lab Clin Med 2003;141:74-83)

Regional and cellular distribution of protein kinase C in rat cerebellar Purkinje cells.

Protein kinase C (PCK) is a family of isoforms that are implicated in subcellular signal transduction. The authors investigated the distribution of several PKC isoforms (PKC-alpha, PKC-beta, PKC-gamma, PKC-delta, and PKC-epsilon) within major cerebellar cell types as well as cerebellar projection target neurons, including Purkinje neurons, cerebellar nuclear neurons, and secondary vestibular neurons. PKC-alpha, PKC-beta, PKC-gamma, PKC-delta, and PKC-epsilon are found within the cerebellum. Of these isoforms, PKC-gamma and PKC-delta are highly expressed in Purkinje cells. PKC-gamma is expressed in all Purkinje cells, whereas the expression of PKC-delta is restricted to sagittal bands of Purkinje cells in the posterior cerebellar cortex. In the lower folia of the uvula and nodulus, Purkinje cell expression of PKC-delta is uniformly high, and the sagittal banding for PKC-delta expression is absent. Within the cerebellar nuclei, PKC-delta-immunolabeled axons terminate within the medial aspect of the caudal half of the ipsilateral interpositus nucleus. PKC delta-immunolabeled axons also terminated within the caudal medial and descending vestibular nuclei (MVN and DVN, respectively), the parasolitary nucleus (Psol), and the nucleus prepositus hypoglossi (NPH). PKC-gamma-immunolabeled axons terminated in all of the cerebellar nuclei as well as in the lateral and superior vestibular nuclei and the MVN, DVN, Psol, and NPH. The projection patterns of PKC-immunolabeled Purkinje cells were confirmed by lesion-depletion studies in which unilateral uvula-nodular lesions caused depletion of PKC-immunolabeled terminals ipsilateral to the lesion in the vestibular complex. These data identify circuitry that is unique to cerebellar-vestibular interactions.

Prostaglandin E 2 modulates a non-inactivating potassium current in rat neurohypophyseal nerve terminals

A non-inactivating voltage dependent K + channel current was observed in neuro-hypophyseal nerve terminals. This current was sensitive to inhibition by 4-aminopyridine and tetraethyl ammonium chloride, but was not sensitive to inhibition by α- or β-dendrotoxin. Prostaglandin E 2 (PGE 2) modulated the voltage-dependent K + channel, through a receptor-mediated process, as indicated by meclofenamate sensitivity, and this involved the activation of G protein(s), as indicated by sensitivity to guanosine-5′-O-(2-thiodiphosphate) (GDP βS). After short periods of incubation (e.g. 5 min), PGE 2 increased the non-inactivating current. Following longer incubation periods with PGE 2 (e.g. 20 min), the non-inactivating current declined. Forskolin and the cyclic adenosine monophosphate (AMP) analogs 8-bromo- and dibutyryl cyclic AMP, and Sp-cyclic AMPs inhibited the current, but did not mimic the increase in current caused by PGE 2. Also, the cyclic AMP antagonist Rp-cyclic AMPs did not block the increase in current induced by PGE 2. These results indicate that activation of cyclic AMP-dependent protein kinase (PKA) is not involved in mediating the stimulatory actions of PGE 2. These observations provide evidence that PGE 2 may contribute to the regulation of hormone release from the posterior pituitary by modulating K + channels. However, the post-receptor mechanisms of subcellular signal transduction underlying this effect remain unknown

NO and cardiac diastolic function.

Time for primary review 34 days. Since the first report of a role for nitric oxide (NO) in modulating myocardial contractile function in 1991 [1], an enormous number of studies have been published in this field (recently reviewed in Refs. [2–4]). It is now recognised that, under physiological conditions, the major sources of NO that are important for contractile regulation are (1) endothelial-type nitric oxide synthase (eNOS) expressed in cardiac endothelial cells, and (2) eNOS expressed in cardiac myocytes themselves. In a variety of pathological situations, contractile function may also be influenced by inducible NOS (iNOS) expressed in several cell types, including infiltrating inflammatory cells, coronary microvascular and endocardial endothelial cells, coronary vascular smooth muscle, and cardiac myocytes. In contrast to the relatively well-defined, essentially species-independent primary action of NO in the vasculature (i.e., mediation of endothelium-dependent vasodilatation), in the heart multiple and sometimes contradictory actions of NO have been reported (Table 1). Some of these actions may be species-specific while others have not always been reproduced by independent laboratories despite the use of apparently similar experimental preparations and protocols. Apart from species and methodological differences, additional relevant factors that are likely to influence the response to NO include: the cellular source of NO, the amount released (or experimentally studied), prevailing redox balance and antioxidant status, the target tissue (e.g., atrial or ventricular tissue), interactions with neurohumoral and other stimuli, the presence of immune activation or disease, and the activation of distinct cGMP-dependent and -independent subcellular signal transduction pathways. View this table: Table 1 Reported actions of NO on the heart. Due to constraints of space, only a limited number of original references have been cited. Many other original references may be found in the bibliography of Refs. [2–4] Most studies that have investigated the effects of NO on contractile function have focused on … * Corresponding author. Tel.: +32-53-724-433