Phospholipase C-protein Kinase Research Articles

Baicalin inhibited PANX-1/P2Y6 signaling pathway activation in porcine aortic vascular endothelial cells infected by Glaesserella parasuis

Glaesserella parasuis can induce endothelial barrier damage in piglets, although the mechanism by which this pathogen triggers inflammatory damage remains unclear. Baicalin possesses anti-inflammatory and anti-oxidant activities. However, whether baicalin can relieve endothelial barrier damage caused by Glaesserella parasuis infection has not yet been studied. Hence, we evaluated the ability of baicalin to counteract the changes induced by Glaesserella parasuis in porcine aortic vascular endothelial cells. The results showed that Glaesserella parasuis could upregulate the expression of pannexin 1 channel protein and promote the release of adenosine triphosphate, adenosine diphosphate, adenosine 3′-monophosphate, uridine triphosphate, uridine diphosphate, and uridine monophosphate in porcine aortic vascular endothelial cells. The expression level of purinergic receptor P2Y6 was upregulated in porcine aortic vascular endothelial cells triggered by Glaesserella parasuis. In addition, Glaesserella parasuis could activate phospholipase C-protein kinase C and myosin light chain kinase-myosin light chain signaling pathways in porcine aortic vascular endothelial cells. Baicalin could inhibit pannexin 1 channel protein expression, reduce adenosine triphosphate, adenosine diphosphate, adenosine 3′-monophosphate, uridine triphosphate, uridine diphosphate, and uridine monophosphate release, and attenuate the expression level of P2Y6 in porcine aortic vascular endothelial cells induced by Glaesserella parasuis. Baicalin could also reduce the activation of phospholipase C-protein kinase C and myosin light chain kinase-myosin light chain signaling pathways in porcine aortic vascular endothelial cells triggered by Glaesserella parasuis. Our study report that Glaesserella parasuis could promote pannexin 1 channel protein expression, induce nucleosides substance release, and P2Y6 expression in porcine aortic vascular endothelial cells and baicalin could inhibit the expression levels of pannexin 1, nucleosides substance, and P2Y6 in the porcine aortic vascular endothelial cells induced by Glaesserella parasuis, which might be served as some targets for treatment of inflammation disease caused by Glaesserella parasuis.

A new therapeutic target for patent ductus arteriosus

The ductus arteriosus (DA) maintains the fetal circulation by connecting the aorta and pulmonary arteries. Patent ductus arteriosus (PDA) occurs in >70% extremely-low-birth-weight infants. Patients with PDA exhibit circulatory failure, which is caused by left-to-right shunt. The DA immediately contracts after birth in response to the elevation of blood oxygen tension and to the decline in circulating prostaglandin E2 (PGE2). Cyclooxygenase inhibitors targeting smooth muscle cell (SMC) contraction represent only pharmacological treatment for PDA. However, it is important for DA anatomical closure that intimal thickening (IT) is appropriately formed between SMC layer and endothelial cells (EC). IT begins to form before the second-trimester and becomes prominent toward the end of third-trimester as an increase in placenta-derived PGE2. Immature DAs frequently fail to be close due to poorly formed IT. IT consists of extracellular matrices (ECM) and migrated DA-SMCs from the tunica media. A glycoprotein fibulin-1 is expressed in developing cardiovascular system and binds to multiple ECMs. We found that PGE2 increased fibulin-1 via EP4 in DA-SMCs, and Fbln1-deficient mice exhibited PDA with poor IT formation. Although EP4 is a Gs-coupled GPCR, fibulin-1 was secreted from DA-SMCs through the phospholipase C-protein kinase C-non-canonical NFκB signaling pathway. Fibulin-1 bound to DA-EC-derived versican which is a binding partner of hyaluronan, which promoted directional DA-SMC migration toward ECs and contributed to IT formation in the DA. Fibulin-1 upregulation by the activation of specific downstream pathway of EP4 may serve a new pharmacological strategy for PDA.

Gq Protein-Coupled Membrane-Initiated Estrogen Signaling Rapidly Excites Corticotropin-Releasing Hormone Neurons in the Hypothalamic Paraventricular Nucleus in Female Mice.

CRH neurons in the hypothalamic paraventricular nucleus (PVN) play a central role in regulating the hypothalamus-pituitary-adrenal (HPA) axis and are directly influenced by 17β-estradiol (E2). Although compelling evidence has suggested the existence of membrane-associated estrogen receptors (mERs) in hypothalamic and other central nervous system neurons, it remains unknown whether E2 impacts CRH neuronal excitability through this mechanism. The purpose of the current study is to examine the existence and function of mER signaling in PVN CRH neurons. Whole-cell recordings were made from CRH neurons identified by Alexa Fluor 594 labeling and post hoc immunostaining in ovariectomized female mice. E2 (100nM) rapidly suppressed the M-current (a voltage-dependent K(+) current) and potentiated glutamatergic excitatory postsynaptic currents. The putative Gq-coupled mER (Gq-mER) characterized in hypothalamic proopiomelanocortin neurons initiates a phospholipase C-protein kinase C-protein kinase A pathway; therefore, we examined the involvement of this pathway using selective inhibitors. Indeed, the ER antagonist ICI 182780 and inhibitors of Gq-phospholipase C-protein kinase C-protein kinase A blocked E2's actions, suggesting dependence on the Gq-mER. Furthermore, STX, a selective ligand for the Gq-mER, mimicked E2's actions. Finally, to examine the in vivo effect of Gq-mER activation, E2 or STX injection increased c-fos expression in CRH neurons in the PVN, suggesting CRH neuronal activation. This corresponded to an increase in plasma corticosterone. We conclude that the Gq-mER plays a critical role in the rapid regulation of CRH neuronal activity and the HPA axis. Our findings provide a potential underlying mechanism for E2's involvement in the pathophysiology of HPA-associated mood disorders.

Crosstalk between dopamine receptors and the Na+/K+-ATPase (Review)

Dopamine (DA) receptors, which belong to the G protein-coupled receptor family, are the target of ~50% of all modern medicinal drugs and constitute a large and diverse class of proteins whose primary function is to transduce extracellular stimuli into intracellular signals. Na+/K+-ATPase (NKA) is ubiquitous and crucial for the maintenance of intracellular ion homeostasis and excitability. Furthermore, it plays a critical role in diverse effects, including clinical cardiotonic and cardioprotective effects, ischemic preconditioning in the brain, natriuresis, lung edema clearance and other processes. NKA regulation is of physiological and pharmacological importance and has species- and tissue-specific variations. The activation of DA receptors regulates NKA expression/activity and trafficking in various tissues and cells, for example in the kidney, lung, intestine, brain, non-pigmented ciliary epithelium and the vascular bed. DA receptor-mediated regulation of NKA mediates a diverse range of cellular responses and includes endocytosis/exocytosis, phosphorylation/dephosphorylation of the α subunit of NKA and multiple signaling pathways, including phosphatidylinositol (PI)-phospholipase C/protein kinase (PK) C, cAMP/PKA, PI3K, adaptor protein 2, tyrosine phosphatase and mitogen-activated protein kinase/extracellular signal-regulated protein kinase. Furthermore, in brain and HEK293T cells, D1 and D2 receptors exist in a complex with NKA. Among D1 and D2 receptors and NKA, regulations are reciprocal, which leads to crosstalk between DA receptors and NKA. In the present study, the current understanding of signaling mechanisms responsible for the crosstalk between DA receptors and NKA, as well as with specific consequent functions, is reviewed.

Protein kinase Cβ deficiency attenuates obesity syndrome of ob/ob mice by promoting white adipose tissue remodeling

To explore the role of leptin in PKCβ action and to determine the protective potential of PKCβ deficiency on profound obesity, double knockout (DBKO) mice lacking PKCβ and ob genes were created, and key parameters of metabolism and body composition were studied. DBKO mice had similar caloric intake as ob/ob mice but showed significantly reduced body fat content, improved glucose metabolism, and elevated body temperature. DBKO mice were resistant to high-fat diet-induced obesity. Moreover, PKCβ deficiency increased β-adrenergic signaling by inducing expression of β1- and β3-adrenergic receptors (β-ARs) in white adipose tissue (WAT) of ob/ob mice. Accordingly, p38(MAPK) activation and expression of PGC-1α and UCP-1 were increased in WAT of DBKO mice. Consistent with results of in vivo studies, inhibition of PKCβ in WAT explants from ob/ob mice also increased expression of above β-ARs. In contrast, induction of PGC-1α and UCP-1 expression in brown adipose tissue of DBKO mice was not accompanied by changes in the expression of these β-ARs. Collectively, these findings suggest that PKCβ deficiency may prevent genetic obesity, in part, by remodeling the catabolic function of adipose tissues through β-ARs dependent and independent mechanisms.

Atrial Natriuretic Factor Stimulates Efflux of cAMP in Rat Exocrine Pancreas via Multidrug Resistance–Associated Proteins

Atrial natriuretic factor (ANF) prevents increases in intracellular levels of cAMP that are induced by secretin in the exocrine pancreas. We investigated the contribution of cyclic adenosine monophosphate (cAMP) efflux to ANF inhibition of secretin signaling. Intracellular and extracellular cAMP were measured by radio-binding assays in isolated pancreatic acini exposed to secretin and other secretagogues, alone or with ANF. Levels of messenger RNA for multidrug resistance-associated protein (MRP)4, MRP5, and MRP8 were measured by real-time polymerase chain reaction. MRP4 was knocked down in AR42J cells by small interfering RNA. In vivo studies were performed in rats. Pancreatic secretagogues increased levels of intracellular cAMP, but only secretin and vasoactive intestinal peptide promoted cAMP efflux; efflux was increased by ANF, through signaling via natriuretic peptide receptor-C and phospholipase C-protein kinase C. In time-course studies with active phosphodiesterases, levels of intracellular and extracellular cAMP increased earlier after the addition of secretin and ANF (1 min) than after the addition of secretin alone (3 min). Similar kinetic patterns occurred with a phosphodiesterase inhibitor. A probenecid-sensitive transporter mediated cAMP egression. The main cAMP transporter, MRP4, was expressed in AR42J cells and pancreas. cAMP egression occurred in AR42J cells exposed to secretin, but this response was reduced in cells that expressed MRP4 small interfering RNA. In rats, levels of cAMP in plasma and pancreatic juice increased after infusion with secretin alone or secretin plus ANF. ANF signals via natriuretic peptide receptor-C coupled to the phospholipase C-protein kinase C pathway to increase secretin-induced efflux of cAMP, probably through MPR-4. Cyclic AMP extrusion might be a mechanism, in addition to phosphodiesterase action, to regulate intracellular cAMP levels in pancreatic acinar cells.

Mediation of Endogenous β‐Endorphin in the Plasma Glucose‐Lowering Action of Herbal Products Observed in Type 1‐Like Diabetic Rats

Recently, there have been advances in the development of new substances effective in managing diabetic disorders. Opioid receptors couple multiple systems to result in various biological effects, although opioids are best known for analgesia. In the present review, we used our recent data to describe the advance in plasma glucose-lowering action of herbal products, especially the mediation of β-endorphin in glucose homeostasis of insulin-deficient diabetes. In type 1-like streptozotocin-induced diabetic rats, we identified many products purified from herbs that show a dose-dependent plasma glucose-lowering action. Increase in β-endorphin secretion from the adrenal gland may activate peripheral opioid μ-receptors (MOR) to enhance the expression of muscle glucose transporters and/or to reduce hepatic gluconeogenesis at the gene level, thereby leading to improved glucose utilization in peripheral tissues for amelioration of severe hyperglycemia. It has also been observed that stimulation of α 1-adrenoceptors (α 1-ARs) in the adrenal gland by some herbal products is responsible for the increase in β-endorphin secretion via a phospholipase C-protein kinase dependent pathway. However, an increase in β-endorphin secretion from the adrenal gland by herbal products can function via another receptor. New insights into the mediation of endogenous β-endorphin activation of peripheral MOR by herbal products for regulation of glucose homeostasis without the presence of insulin have been established. Therefore, an increase in β-endorphin secretion and/or direct stimulation of peripheral MOR via an insulin-independent action might serve as the potential target for development of a therapeutic agent or promising adjuvant in intensive plasma glucose control.

Urotensin II Activates Sarcolemmal Na+/H+ Exchanger in Adult Rat Ventricular Myocytes

Our aims in the present study were (1) to determine the effects of urotensin II (UT-II) on the sarcolemmal Na/H exchanger (NHE1) activity, and (2) to investigate possible kinase pathways for UT-II-mediated NHE1 stimulation. In single rat ventricular myocytes (n = 5-10/group) loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1, acid efflux rates (JH) were determined as an index of NHE1 activity by rate of recovery of intracellular pH (pHi) from NH4Cl-induced acidosis and the intrinsic buffering power. Phosphorylation of extracellular signal-regulated kinase (ERK), a key kinase of NHE1 activation, was determined by Western blot analysis. JH increased by 31%-71% relative to control in the presence of 100 nmol/L UT-II at pHi range of 6.6-7.0. Stimulation of NHE1 activity by UT-II was abolished by inhibitors of phospholipase C, protein kinase C, and ERK kinase; 2-nitro-4-carboxyphenil-N,N-diphenilcarbamate at 100 micromol/L, GF109203X at 300 nmol/L, and PD98059 at 50 micromol/L, respectively. Moreover, UT-II at 100 nmol/L produced a significant increase in cellular ERK1/2 phosphorylation, which was also inhibited by those inhibitors. Our study was the first to demonstrate that UT-II activates the cardiac sarcolemmal NHE1 and that the phenomenon may involve, at least in part, the phospholipase C-protein kinase C-ERK pathway.

Undiscovered role of endogenous thromboxane A2 in activation of cardiac sympathetic afferents during ischaemia

Myocardial ischaemia activates blood platelets, which in turn stimulate cardiac sympathetic afferents, leading to chest pain and sympathoexcitatory reflex cardiovascular responses. Previous studies have shown that activated platelets stimulate ischaemically sensitive cardiac sympathetic afferents, and that thromboxane A(2) (TxA(2)) is one of the mediators released from activated platelets during myocardial ischaemia. The present study tested the hypothesis that endogenous TxA(2) stimulates cardiac afferents during ischaemia through direct activation of TxA(2) (TP) receptors coupled with the phospholipase C-protein kinase C (PLC-PKC) cellular pathway. Nerve activity of single unit cardiac sympathetic afferents was recorded from the left sympathetic chain or rami communicantes (T(2)-T(5)) in anaesthetized cats. Single fields of 39 afferents (conduction velocity = 0.27-3.65 m s(-1)) were identified in the left or right ventricle initially with mechanical stimulation and confirmed with a stimulating electrode. Five minutes of myocardial ischaemia stimulated all 39 cardiac afferents (8 Adelta-, 31 C-fibres) and the responses of these 39 afferents to chemical stimuli were further studied in the following four protocols. In the first protocol, 2.5, 5 and 10 microg of the TxA(2) mimetic, U46619, injected into the left atrium (LA), stimulated seven ischaemically sensitive cardiac afferents in a dose-dependent manner. Second, BM13,177, a selective TxA(2) receptor antagonist, abolished the responses of six afferents to 5 microg of U46619 injected into the left atrium and attenuated the ischaemia-related increase in activity of seven other afferents by 44%. In contrast, cardiac afferents, in the absence of TP receptor blockade responded consistently to repeated administration of U46619 (n = 6) and to recurrent myocardial ischaemia (n = 7). In the fourth protocol, administration of PKC-(19-36), a selective PKC inhibitor, attenuated the responses of six other cardiac afferents to U46619 by 38%. Finally, using an immunohistochemical staining approach, we observed that TP receptors were expressed in cardiac sensory neurons in thoracic dorsal root ganglia. Taken together, these data indicate that endogenous TxA(2) contributes to the activation of cardiac afferents during myocardial ischaemia through direct stimulation of TP receptors probably located in the cardiac sensory nervous system and that the stimulating effect of TxA(2) on cardiac afferents is dependent, at least in part, upon the PLC-PKC cellular pathway.

Expression of α-AR Subtypes in T Lymphocytes and Role of the α-ARs in Mediating Modulation of T Cell Function

Objectives: Previous work in our laboratory has shown that α-adrenoreceptors (α-ARs) and β-ARs exist on lymphocytes from functional profile, and that the receptors mediate the regulation of lymphocyte function by catecholamines. In the present study, we directly examined the expression of α-AR subtypes, α₁-AR and α₂-AR mRNAs, in T lymphocytes and explored the roles of the α-AR subtypes and intracellular signal transduction mechanisms linked to the receptors in mediating the modulation of T lymphocyte function. Methods: T lymphocytes from mesenteric lymph nodes of rats were purified by using a nylon wool column. Reverse transcription polymerase chain reaction was used to detect the expression of α₁-AR and α₂-AR mRNAs in the freshly isolated T cells and the mitogen concanavalin A (Con A)-activated lymphocytes. Colorimetric methylthiazoletetrazolium assay was employed to measure lymphocyte proliferation induced by Con A. Interferon-γ (IFN-γ) and interleukin-4 (IL-4) levels in the Con A-stimulated lymphocyte culture supernatants were examined by enzyme-linked immunosorbent assay. Results: T cells expressed both α₁-AR and α₂-AR mRNAs. The expression of both α₁-AR and α₂-AR mRNAs was significantly higher in the Con A-activated lymphocytes than in the resting lymphocytes. Phenylephrine, a selective α₁-AR agonist, had no evident effect on lymphocyte proliferation nor on IFN-γ and IL-4 production induced by Con A. However, the selective α₂-AR agonist clonidine attenuated Con A-induced lymphocyte proliferation as well as IFN-γ and IL-4 production. The inhibited lymphocyte proliferation and IFN-γ and IL-4 production by clonidine were blocked by yohimbine, an α₂-AR antagonist. Either phospholipase C inhibitor U-73122 or protein kinase C inhibitor chelerythrine partially prevented the suppressive effect of clonidine on Con A-stimulated lymphocyte proliferation and IL-4 production. Conclusions: T lymphocytes express both α₁-ARs and α₂-ARs, but only the α₂-ARs participate in the suppressive modulation of lymphocyte proliferation and cytokine production in vitro. The inhibitory effect of α₂-AR stimulation on lymphocyte function is partially mediated via the phospholipase C-protein kinase C pathway.

Diadenosine tetraphosphate stimulates atrial ANP release via A 1 receptor: Involvement of K ATP channel and PKC

Diadenosine polyphosphates (APnAs) are endogenous compounds and exert diverse cardiovascular functions. However, the effects of APnAs on atrial ANP release and contractility have not been studied. In this study, the effects of diadenosine tetraphosphate (AP4A) on atrial ANP release and contractility, and their mechanisms were studied using isolated perfused rat atria. Treatment of atria with AP4A resulted in decreases in atrial contractility and extracellular fluid (ECF) translocation whereas ANP secretion and cAMP levels in perfusate were increased in a dose-dependent manner. These effects of AP4A were attenuated by A 1 receptor antagonist but not by A 2A or A 3 receptor antagonist. Other purinoceptor antagonists also did not show any effects on AP4A-induced ANF release and contractility. The increment of ANP release and negative inotropy induced by AP4A was similar to those induced by AP3A, AP5A, and AP6A. Protein kinase A inhibitors accentuated AP4A-induced ANP secretion. In contrast, an inhibitor of phospholipase C, protein kinase C or sarcolemma K ATP channel completely blocked AP4A-induced ANP secretion. However, an inhibitor of adenylyl cyclase or mitochondria K ATP channel had no significant modification of AP4A effects. These results suggest that AP4A regulates atrial inotropy and ANP release mainly through A 1 receptor signaling involving phospholipase C-protein kinase C and sarcolemmal K ATP channel and that protein kinase A negatively modulates the effects of AP4A.

Possible roles of neuropeptide Y Y3-receptor subtype in rat aortic endothelial cell proliferation under hypoxia, and its specific signal transduction

The present study was undertaken to determine whether neuropeptide Y (NPY) induces proliferation of rat aortic endothelial cells (RAECs). Since NPY increased the permeability of RAEC monolayers to large molecules via the NPY Y(3) receptor, RAEC proliferation has been evaluated in terms of NPY-receptor subtypes and also intracellular mechanisms. RAECs were incubated with gases containing 20, 15, or 10% O(2) and a certain amount of N(2), depending on the O(2) content in 5% CO(2) incubators. NPY (10(-9)-10(-6) M) increased the RAEC numbers under hypoxic conditions, such as 15 or 10% O(2). Peptide YY elicited no proliferative effect on RAEC, and NPY-(18-36) inhibited the NPY-induced increase in cell number, suggesting that NPY increases the RAEC count through the NPY Y(3) receptor. Pertussis toxin, U-73122, GF-109203X, myristorylated autocamtide-2-related inhibitory peptide, and wortmannin inhibited the NPY-induced proliferation of RAEC concentration dependently. DY9760e little affected the proliferation caused by NPY. ML-9 and imatinib actually enhanced the NPY-induced proliferation of cells. These results indicated that the NPY Y(3) receptor is coupled with G(i) protein, and that NPY-induced increases in RAEC proliferation are mediated by phospholipase C-protein kinase C and/or phosphatidylinositol 3-kinase pathways. In intracellular Ca(2+)-calmodulin-dependent pathways, calmodulin-dependent protein kinase II partly participates in the NPY-induced cell proliferation. Regarding the previously reported effect of NPY on the permeability of RAEC monolayers to large molecules, it is probable that protein kinase C and phosphatidylinositol 3-kinase pathways are activated for both permeability and cell proliferation induced by NPY under hypoxia, relevant to new insights into the roles of NPY in ischemia-hypoxia.

Vascular Endothelial Growth Factor (VEGF)-Receptor2: Its Biological Functions, Major Signaling Pathway, and Specific Ligand VEGF-E

Two high-affinity receptors for vascular endothelial growth factor (VEGF)-A, VEGFR1 and VEGFR2, cooperate for physiological vasculogenesis and angiogenesis in embryogenesis. VEGFR2 transduces the major signals for angiogenesis via its strong tyrosine kinase activity. However, unlike other representative tyrosine kinase receptors, VEGFR2 does not use the Ras pathway as a major downstream signaling but rather uses the phospholipase C-protein kinase C pathway to signal mitogen-activated protein (MAP)-kinase activation and DNA synthesis. Cell migration signals from VEGFR2 were recently shown to use, at least partly, a pathway dependent on the adaptor molecule TSAd from the kinase-insert region of VEGFR2. VEGFR2 is a direct and major signal transducer for pathological angiogenesis, including cancer and diabetic retinopathy, in cooperation with many other signaling partners; thus, VEGFR2 and its downstream signaling appear to be critical targets for the suppression of these diseases. More than 10 antagonists of VEGFR2, including kinase inhibitors and neutralizing antibodies, are now under clinical trials. Recently, the VEGFR2-specific ligand VEGF-E (also known as Orf-VEGF) family was extensively characterized. Interestingly, activation of VEGFR2 via VEGF-E in vivo results in a strong angiogenic response in mice, with minor effects on inflammation and hypervascular permeability compared with VEGF-A, suggesting that VEGF-E is a useful tool for proangiogenic therapy in ischemic diseases.

Mechanism of VEGF-Induced Uterine Venous Hyperpermeability

Background: Transfer of molecular signals from veins to adjacent arteries is an established mechanism of vascular communication in the uterine circulation, which ultimately depends on venous permeability. This study tests the hypotheses that: (1) uterine veins are permeable to intravenous solutes, using a 3-kDa dextran tracer and (2) this permeability is enhanced in response to vascular endothelial growth factor (VEGF). Additionally, the involvement of nitric oxide (NO), calcium, and the phospholipase C-protein kinase C (PLC-PKC) cascade in VEGF-enhanced permeability were investigated, and the impact of pregnancy-induced uterine vascular remodeling on permeability was evaluated. Methods: Studies utilized fluorimetry to quantitate solute flux in isolated segments of rat uterine vein as a function of endothelial surface area and time under basal and VEGF-stimulated conditions. VEGF signaling was probed using NO synthase inhibition (L-NNA), calcium channel blockade (lanthanum chloride) and withdrawal (calcium-free solution), and inhibitors of the PLC-PKC cascade (U-73122 and chelerythrine chloride, respectively). Gestational effects were assessed using vessels from late pregnant (day 20) rats. Results: Basal flux (control) in nonpregnant animals was 26 ± 2.5 molecules/µm²/min × 1,000 and was increased significantly by VEGF in a concentration-dependent manner (1 nM ≈ 3.3-fold, 10 nM ≈ 4.6-fold). Inhibition of PLC, PKC, and calcium signaling, but not NO, attenuated the response to VEGF. Gestation significantly increased flux (78 ± 9.3 molecules/µm²/min × 1,000), and maintained responsiveness to VEGF. Conclusions: These results demonstrate uterine venous permeability to intermediate-sized solutes through a VEGF-sensitive pathway involving calcium and PLC-PKC, but not NO, and further substantiate a role for veno-arterial communication in uterine blood flow regulation during pregnancy.

Effects of anesthetic agents on focal adhesion kinase (pp125FAK) tyrosine phosphorylation in rat hippocampal slices.

Tyrosine protein kinase proteins exert a prominent control on signaling pathways and may couple rapid events, such as action potential and neurotransmitter release, to long-lasting changes in synaptic strength and survival. Whether anesthetics modulate tyrosine kinase activity remains unknown. The aim of the current study was therefore to examine the effects of intravenous and volatile anesthetics on the phosphorylation of focal adhesion kinase (ppFAK), a functionally important nonreceptor tyrosine kinase, in the rat hippocampus. Phosphorylation of ppFAK was examined in hippocampal slices by immunoblotting with both antiphosphotyrosine and specific anti-ppFAK antibodies. Experiments were performed in the absence (control) or presence of various concentrations of pharmacologic or anesthetic agents or both. Clinically relevant concentrations of thiopental, propofol, etomidate, isoflurane, sevoflurane, and desflurane induced a concentration-related increase in tyrosine phosphorylation. In contrast, ketamine (up to 100 microm) and the nonimmobilizer F6 (1,2-dichlorohexafluorocyclobutane, 25 microm) did not significantly affect ppFAK phosphorylation. The anesthetic-induced increase in ppFAK phosphorylation was blocked by GF 109203X, RO 318220, and chelerythrin (100 microm), three structurally distinct inhibitors of protein kinase C and U 73122 (50 microm), an inhibitor of phospholipase C. The propofol- and isoflurane-induced increase in ppFAK phosphorylation was reversible and showed nonadditivity of effects with phorbol 12-myristate 13-acetate (an activator of protein kinase C, 0.1 microm). In contrast, ketamine (up to 100 microm), MK801 (10 microm, an N-methyl-d-aspartate receptor antagonist), bicuculline (10 microm, a gamma-aminobutyric acid type A receptor antagonist), and dantrolene (30 microm, an inhibitor of the ryanodine receptor) were ineffective in blocking anesthetic-induced activation of tyrosine phosphorylation. Except for ketamine, anesthetic agents markedly increase tyrosine phosphorylation of ppFAK in the rat hippocampus, most likely via the phospholipase C-protein kinase C pathway, whereas the nonimmobilizer F6 does not. These results suggest that ppFAK represents a target for anesthetic action in the brain.

Histamine Enhances the Production of Nerve Growth Factor in Human Keratinocytes

Nerve growth factor induces innervation and epidermal hyperplasia in inflammatory skin diseases like psoriasis. Nerve growth factor production by keratinocytes is increased in the inflammatory lesions. Nerve growth factor induces histamine release from mast cells. We examined the in vitro effects of histamine on nerve growth factor production in human keratinocytes. Histamine enhanced nerve growth factor secretion, mRNA expression, and promoter activity in keratinocytes. Two TPA-response elements on the nerve growth factor promoter were responsible for the activation by histamine. Histamine enhanced transcriptional activity and DNA binding of activator protein 1 at the two TPA-response elements. It shifted the TPA-response-element-binding activator protein 1 composition from c-Jun homodimers to c-Fos/c-Jun heterodimers. Histamine transiently induced c-Fos mRNA expression, which was not detectable in unstimulated keratinocytes, whereas c-Jun mRNA expression was constitutive and was not altered by histamine. Histamine-induced enhancement of nerve growth factor secretion, promoter activity, activator protein 1 transcriptional activity, and c-Fos expression was suppressed by H1 antagonist pyrilamine, protein kinase C inhibitor calphostin C, and PD98059, an inhibitor of mitogen-activated protein kinase kinase 1. Histamine induced the translocation of protein kinase C activity from cytosol to membrane, which was suppressed by phospholipase C inhibitor U73122. It stimulated the phosphorylation of extracellular signal-regulated kinase, which was blocked by pyrilamine, calphostin C, and PD98059. These results suggest that histamine may enhance nerve growth factor production by inducing c-Fos expression in keratinocytes. These effects may be mediated by the H1-receptor-induced signaling cascade of phospholipase C-protein kinase C-mitogen-activated protein kinase kinase 1-extracellular signal-regulated kinase.

Phospholipase C-protein kinase C mediated phospholipase D activation pathway is involved in tamoxifen induced apoptosis.

Tamoxifen (TAM) is the endocrine therapeutic agent the most widely used in the treatment of breast cancer, and it operates primarily through the induction of apoptosis. In this study, we attempted to elucidate the non-ER mediated mechanism behind TAM treatment, involving the phospholipase C-protein kinase C (PLC-PKC) mediated phospholipase D (PLD) activation pathway, using multimodality methods. In TAM treated MCF7 cells, the PLC and PLD protein and mRNA levels increased. Phosphatidylethanol (PEt) and diacylglycerol (DAG) generation also increased, showing increased activity of PLD and PLCgamma1. Translocation of PKCalpha, from cytosol to membrane, was observed in TAM treated cells. By showing that both PKC and PLC inhibitors could reduce the effects of TAM-induced PLD activation, we confirmed the role of PKC and PLC as upstream regulators of PLD. Finally, we demonstrated that TAM treatment reduced the viability of MCF7 cells and brought about rapid cell death. From these results, we confirmed the hypothesis that TAM induces apoptosis in breast cancer cells, and that the signal transduction pathway, involving PLD, PLC, and PKC, constitutes one of the possible mechanisms underlying the non-ER mediated effects associated with TAM.

The vascular response to the K + channel inhibitor 4-aminopyridine in hypertensive rats

The K + channel inhibitor 4-aminopyridine induced an immediate increase in blood pressure and tension in spontaneously hypertensive rats (SHR). Further analysis strongly suggested this to be due to closure of vascular smooth muscle K + channels, as previously concluded for normotensive rats (WKY). The tension response was greater in SHR than WKY, suggesting an increased channel activity in order to compensate for the high total peripheral vascular resistance in SHR. The response was enhanced after nitric oxide (NO) synthase inhibitor in both strains, probably reflecting increased channel activity after elimination of the NO–cGMP pathway. The response in SHR but not WKY was increased after α 1-adrenoceptor inhibition and adrenalectomy but not sympathetic nerve transmitter depletion. It increased also after angiotensin AT 1 and endothelin ET A receptor antagonists and protein kinase C inhibitor. These results indicated an increased adrenal catecholamine, angiotensin AT 1 and endothelin ET A activation of the phospholipase C-protein kinase C pathway in SHR, inhibiting the 4-aminopyridine-sensitive K + channels.

Leptin Constrains Phospholipase C-Protein Kinase C-Induced Insulin Secretion via a Phosphatidylinositol 3-Kinase-Dependent Pathway

Leptin-deficient Lep(ob)/Lep(ob)mice hypersecrete insulin in response to acetylcholine stimulation of the phospholipase C-protein kinase C (PLC-PKC) pathway, and leptin constrains this hypersecretion. Leptin has been reported to activate phosphatidylinositol 3-kinase (PI 3-K) and subsequently phosphodiesterase (PDE) to impair protein kinase A (PKA)-induced insulin secretion from cultured islets of neonatal rats. We determined if PKA-induced insulin secretion was also hyperresponsive in islets from Lep(ob)/Lep(ob)mice, and if leptin impaired this pathway in islets from these mice. Additionally, the possible role for PI 3-K and PDE in leptin-induced control of acetylcholine-induced insulin secretion was examined. Stimulation of insulin secretion with GLP-1, forskolin (an activator of adenylyl cyclase), or IBMX (an inhibitor of PDE) did not cause hypersecretion of insulin from islets of young Lep(ob)/Lep(ob)mice, and leptin did not inhibit GLP-1-induced insulin secretion from islets of these mice. Inhibition of PDE with IBMX also did not block leptin-induced inhibition of acetylcholine-mediated insulin secretion from islets of Lep(ob)/Lep(ob)mice. But, preincubation of islets with wortmannin, an inhibitor of PI 3-K activity, blocked the ability of leptin to constrain acetylcholine-induced insulin secretion from islets of Lep(ob)/Lep(ob)mice. We conclude that the capacity of the PKA pathway to stimulate insulin secretion is not increased in islets from young Lep(ob)/Lep(ob)mice, and that leptin does not regulate this pathway in islets from mice. Leptin may stimulate PI 3-K to constrain PLC-PKC-induced insulin secretion from islets of Lep(ob)/Lep(ob)mice.

Signal transduction-associated and cell activation-linked antigens expressed in human mast cells.

Mast cells (MCs) are multifunctional hematopoietic effector cells that produce and release an array of biologically active mediator substances. Growth and functions of MCs are regulated by cytokines, other extracellular factors, surface and cytoplasmic receptors, oncogene products, and a complex network of signal transduction cascades. Key regulators of differentiation of MCs appear to be stem cell factor (SCF) and its tyrosine kinase receptor KIT (c-kit proto-oncogene product=CD117), downstream-acting elements, and the mi transcription factor (MITF). Signaling through KIT is negatively regulated by the signal regulatory protein (SIRP)-alpha (CD172a)-SHP-1-pathway that is disrupted in neoplastic MCs in MC proliferative disorders. Both KIT and FcepsilonRI are involved in MC activation and mediator release. Activation of MCs through FcepsilonRI is associated with increased expression of activation-linked membrane antigens as well as with signaling events involving Lyn and Syk kinases, the phosphatidylinositol-3-kinase-pathway, Ras pathway, and the phospholipase C-protein kinase C pathway. A similar network of signaling is found in SCF-activated MCs. The current article gives an overview on signal transduction-associated and activation-linked antigens expressed in human MCs. Wherever possible the functional implication of signaling pathways and antigen expression are discussed.