A radiographic study of dental health in adult patients with dental anxiety

We used Northern blot hybridization to determine whether 9-amino-1,2,3,4-tetrahydroacridine (THA), a potential antidementia drug, selectively altered the levels of muscarinic acetylcholine receptor (mAChR) mRNA in differentiating cerebellar granule cells. Granule cells were cultured for 8 days in media containing 15 mM K+, 25 mM K+ or 15 mM K+ plus 30 microM THA. High K+ markedly increased the levels of m2- and m3-mAChR mRNA in the surviving cells. In contrast, THA increased the levels of m3-mAChR mRNA, but had little or no effect on m2-mAChR mRNA levels. These results suggest that THA selectively up-regulates the synthesis of m3-mAChR mRNA.     

Calcium-regulated protein tyrosine phosphorylation is required for endothelin-1 to induce prostaglandin endoperoxide synthase-2 mRNA expression and protein synthesis in mesangial cells

The role of endothelin (ET)-1-mediated cytosolic calcium ([Ca2+]i) elevation in regulating ET-1-induced prostaglandin endoperoxide synthase, prostaglandin G/H synthase (PGHS)-2 mRNA expression and protein synthesis was investigated in mesangial cells (MC). Ionomycin, a calcium ionophore, and thapsigargin, an inhibitor of calcium ATPase, mimicked the ET-1-stimulated PGHS-2 mRNA and protein induction. Inhibition of [Ca2+]i increases with (2-?C2-bis-(carboxymethyl)-amino-5 methylphenoxy]methyl?-6-methoxy-8-bis-(carboxymethyl)-aminoquinoline tetra-(acetoxymethyl)ester (Quin/AM), a calcium chelator, or with the combined presence of [8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, HCl] (TMB), an inhibitor of intracellular calcium stores release, and ethyleneglycol-bis-(beta-aminoethyl)- N,N,N',N'-tetra-acetic acid (EGTA) suppressed ET-1, as well as ionomycin and thapsigargin-mediated PGHS-2 mRNA and protein formation. Also, the ET-1-, ionomycin-, and thapsigargin-induced PGHS-2 mRNA expression and protein formation was inhibited in MC pretreated with inhibitors of calcium calmodulin kinase. In contrast, these conditions did not inhibit interleukin (IL)-1-induced PGHS-2 mRNA expression and protein synthesis. Pretreatment with tyrosine kinase inhibitors abolished the ET-1-, ionomycin-, thapsigargin-, and IL-1-mediated PGHS-2 mRNA and protein induction. ET-1-, ionomycin-, and thapsigargin- induced protein tyrosine phosphorylation, but not IL-1-induced protein tyrosine phosphorylation, was suppressed by inhibiting either [Ca2+]i elevation or calcium calmodulin kinase activation. It was concluded that elevation of [Ca2+]i and activation of calcium calmodulin kinases are upstream mediators of ET-1-induced PGHS-2 gene expression through activation of non-receptor-linked protein tyrosine kinase in MC.     

Research on the mechanism of endothelin inflammatory effects on human mesangial cells

OBJECTIVE: To investigate the mechanism of endothelin (ET) inflammatory effects on human mesangial cells (HMC). METHODS: The following experiments were performed on cultured HMC after ET-1 stimulation: (1) the expression of tumor necrosis factor-alpha (TNF alpha), interleukin-1 beta (IL-1 beta), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelin-1 (ET-1) itself messenger ribonucleic acid (mRNA) was determined by Northern Blot analysis; (2) the TNF alpha concentration was tested with radioimmunoassay; the IL-1 activity was assayed by the enhancement of thymocyte proliferation in response to mitogen; the surface expression of ICAM-1 and VCAM-1 was measured with cell enzyme linked immunoadsorbent assay (ELISA) analysis. RESULTS: ET-1 (10(-7) mol/L) induced the following changes on HMC: (1) up-regulation of the expression of TNF alpha mRNA and protein; (2) up-regulation of the expression of ICAM-1 and VCAM-1 mRNA and protein; (3) up-regulation of the expression of ET-1 itself mRNA. However, the expression of IL-1 mRNA and protein was not changed. CONCLUSIONS: ET-1 can stimulate HMC to produce TNF alpha, ICAM-1 and VCAM-1, and thereby induce inflammatory effects. ET-1 can also stimulate HMC to up-regulate the expression of ET-1 itself, so as to amplify inflammatory effects. So, ET-1 is actually an inflammatory mediator and may play an important role in the pathogenesis of glomerulonephritis.     

Endothelin-1 as a putative modulator of gene expression and cellular physiology in cultured human corporal smooth muscle cells

PURPOSE: Increases in cytosolic calcium levels trigger smooth muscle contraction while nuclear calcium increases are thought to regulate gene expression. Endothelin-1 (ET-1) affects both. The goal of these studies was to further investigate the importance of ET-1 to corporal physiology by examining its actions on proliferation and immediate early gene (IEG) expression in cultured human corporal smooth muscle cells. MATERIALS & METHODS: Early passage (1-3) smooth muscle cells were grown in culture and exposed to either phenylephrine (PE) or ET-1 in the absence and presence of serum, the ET(A) or ET(B) selective antagonist BQ123 or IRL1038, or the L-type Ca2+ channel blocker, verapamil. Cell proliferation was assessed with a hemocytometer. The effects of ET-1 on c-myc and c-fos were evaluated using Northern blot analysis. Parametric or nonparametric statistics were used as appropriate. RESULTS: Addition of ET-1 (100 nM) to serum-starved cultured corporal smooth muscle cells was associated with a nearly 2-fold increase in cell number, as well as 2 to 6-fold increases in c-myc and c-fos levels. Cellular proliferation was inhibited by ET(A)- or ET(B)-receptor subtype blockade with BQ123 (1 microM) or IRL1038 (1 microM), respectively, or blockade of Ca2+ channels with verapamil (10 microM). PE (3 microM) had no detectable effect on smooth muscle proliferation. CONCLUSIONS: Cell proliferation was mediated by activation of the ET(A) and ET(B) receptor subtypes, dependent on transmembrane Ca2+ flux, and correlated with significant increases in c-myc and c-fos mRNA levels. These studies extend previous observations to indicate the potential pleotropic actions of this peptide in the regulation of human corporal smooth muscle physiology in vivo.     

Comparison of the stability of technetium-labeled peptides to challenge with cysteine

-Tacrolimus (FK 506) is a powerful, widely used immunosuppressant. The clinical utility of FK 506 is complicated by substantial hypertension and nephrotoxicity. To clarify the mechanisms of FK 506-induced hypertension, we studied the chronic effects of FK 506 on the synthesis of endothelin-1 (ET-1), the expression of mRNA of ET-1 and endothelin-converting enzyme-1 (ECE-1), the endothelial nitric oxide synthase (eNOS) activity, and the expression of mRNA of eNOS and C-type natriuretic peptide (CNP) in rat blood vessels. In addition, the effect of the specific endothelin type A receptor antagonist FR 139317 on FK 506-induced hypertension in rats was studied. FK 506, 5 mg. kg-1. d-1 given for 4 weeks, elevated blood pressure from 102+/-13 to 152+/-15 mm Hg and increased the synthesis of ET-1 and the levels of ET-1 mRNA in the mesenteric artery (240% and 230%, respectively). Little change was observed in the expression of ECE-1 mRNA and CNP mRNA. FK 506 decreased eNOS activity and the levels of eNOS mRNA in the aorta (48% and 55%, respectively). The administration of FR 139317 (10 mg. kg-1. d-1) prevented FK 506-induced hypertension in rats. These results indicate that FK 506 may increase blood pressure not only by increasing ET-1 production but also by decreasing NO synthesis in the vasculature.     

The effect of cycloheximide on the regulation of proenkephalin and prodynorphin gene expressions induced by kainic acid in rat hippocampus

The effect of cycloheximide (CHX), a protein synthesis inhibitor, on the regulation of proenkephalin (proENK) and prodynorphin (proDYN) mRNA levels, proto-oncogenes, such as c-fos, 35-kDa fra and c-jun mRNA, and the levels of their products induced by kainic acid (KA) in rat hippocampus was studied. The proENK and proDYN mRNA levels were markedly increased 4 and 8 h after KA (10 mg/kg i.p.) administration. However, the intracellular proENK protein level was not affected by KA. The elevations of both proENK and proDYN mRNA levels induced by KA were inhibited by pre-administration of CHX (15 mg/kg i.p.). The increases of proENK and proDYN mRNA levels induced by KA were well-correlated with the increases of c-Fos, 35-kDa Fra and c-Jun protein levels. KA administration increased the hippocampal levels of c-Fos, 35-kDa Fra and c-Jun proteins with the time. The increases of c-Fos, 35-kDa Fra and c-Jun protein levels induced by KA administration were also inhibited by CHX pre-administration. KA administration markedly increased both c-fos and c-jun mRNA levels during 1 and 4 h and the increased levels of these proto-oncogene mRNA were further prolonged by the treatment with CHX. In addition, CHX alone increased both c-fos and c-jun mRNA levels although the onset times of induction were different. In electrophoretic mobility shift-assay, both AP-1 and ENKCRE-2 DNA-binding activities were increased by KA. KA-induced increases of AP-1 and ENKCRE-2 DNA-binding activities were also attenuated by CHX. In addition, KA-induced AP-1 and ENKCRE-2 DNA-binding activities were diminished by the antibodies against Fos and Jun family proteins. Furthermore, the cross-competition studies revealed that AP-1 proteins actively participated in ENKCRE-2 DNA domain. The results suggest that KA-induced proENK and proDYN mRNA expressions may require on-going synthesis of proteins, such as c-Fos, c-Jun and 35-kDa Fra, which may have a possible role in the up-regulation of proENK and proDYN gene expression through the binding with AP-1 and ENKCRE-2 DNA-binding motifs.     

Coronary pressure as a determinant of B-type natriuretic peptide gene expression in isolated perfused adult rat heart

The role of coronary flow in the regulation of ventricular B-type natriuretic peptide (BNP) gene expression was studied in isolated perfused rat heart preparation. The increase of coronary flow from 5 ml/min to 20 ml/min for 2 h resulted in a 132+/-6 mm Hg increase in aortic perfusion pressure. The changes in BNP mRNA and immunoreactive BNP (IR-BNP) levels in response to hemodynamic stress were compared to those of c-fos and adrenomedullin (ADM) gene expression. The increase of coronary flow resulted in 1.5-fold increases in the left ventricular BNP mRNA (P < 0.001) and IR-BNP (P < 0.05) levels in 2-month old rats. There was also a 1.5-fold (P < 0.05) increase in ventricular c-fos mRNA levels, whereas ADM mRNA levels decreased by 74% (P < 0.001) in the left ventricle. In 18-month old rats, the increase in coronary flow decreased left and right ventricular BNP mRNA levels by 18% (P < 0.05) and 39% (P < 0.001), respectively. There were no changes in IR-BNP peptide and c-fos mRNA levels, whereas ADM mRNA levels decreased by 46% (P < 0.001) in the left ventricles. The results show that increased aortic perfusion pressure results in differential expression of cardiac genes including up-regulation of ventricular BNP and c-fos gene expression and down-regulation of ADM gene expression. Furthermore, aging seems to elevate the threshold at which hemodynamic stress of the heart results in a response at BNP gene level.     

Calcium influx via L-type voltage-gated channels mediates the delayed, elevated increases in steady-state c-fos mRNA levels in cerebellar granule cells exposed to excitotoxic levels of glutamate

The altered kinetics of steady-state c-fos mRNA production in cultured cerebellar granule cells under excitotoxic conditions was investigated in neurons subjected to depolarising stimuli, namely, high KCl and L-glutamate (Glu), in which Ca2+ influx occurs by differing routes. Increases in intracellular-free calcium levels ([Ca2+]i) stimulated by nontoxic or toxic levels of Glu were blocked by selective N-methyl-D-aspartate (NMDA) receptor antagonism; were blocked only partially by the L-type channel blocker, nifedipine; and were unaffected by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptor antagonists. Glu-induced cell death was prevented only by NMDA receptor blockade. Exposure of cells to nontoxic levels of Glu resulted in a transient increase in c-fos mRNA levels, whereas an excitotoxic dose produced a delay in the appearance of c-fos mRNA but a subsequent, progressive, and sustained (>4 hr) increase. An excitotoxic dose of Glu in combination with either nifedipine or selective NMDA receptor antagonists resulted in the normal, transient increase of c-fos mRNA levels. Chronic exposure to 55 mM KCl caused no cytotoxicity, although it resulted in a delayed, elevated increase in c-fos mRNA levels that was unaffected by NMDA receptor blockade but reverted to the normal, transient profile of c-fos mRNA formation when it was coadministered with nifedipine. The KCl-induced increase in [Ca2+]i levels was inhibited dramatically by nifedipine but was unaffected by any of the ionotropic Glu receptor antagonists. The results support the notion that the appearance of a delayed but elevated increase in steady-state c-fos mRNA levels following exposure to excitotoxic doses of Glu is mediated specifically by calcium influx via L-type voltage-gated channels.     

Cortical induction of c-fos by intrastriatal endothelin-1 is mediated via NMDA receptors

Endothelin (ET) is a potent vasoconstrictor which has also been proposed to act as a neuromodulator. We have investigated the action of ET-1 on neurones in vivo, using c-fos as a marker of neuronal activation. Intrastriatal injection of ET-1 caused seizures and barrel rolling which were prevented by pretreatment with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 and attenuated by the nitric-oxide synthase inhibitor N omega-nitro-L-arginine (L-NNA). In association with these behaviours, a dramatic increase in c-fos mRNA expression was seen in the cerebral cortex. This increase was blocked by both MK-801 and L-NNA. We suggest that ET-1 modulates the activity of cortical afferents to the striatum, and causes seizures via an NMDA receptor-dependent mechanism.     

Up-regulation of angiotensin type 2 receptor mRNA by angiotensin II in rat cortical cells

The present experiment demonstrates that the exposure of angiotensin II (AII) produced an up-regulation of the AT2 receptor mRNA level in rat cortical cells. AII (10(-9)-10(-5) M) exerted a marked increase of AT2 receptor mRNA in a dose-dependent manner. The maximum increase was observed at 3 hr of AII stimulation and lasted 3 hr. The up-regulation of AT2 receptor mRNA was antagonized by PD123319, an AT2 receptor antagonist, but not by SC-52458, an AT1 receptor antagonist, thus suggesting that the increase in AT2 receptor mRNA is mediated via AT2 receptor. This increase is blocked by serine/threonine phosphatase inhibitor okadaic acid, but not by the phosphotyrosine phosphatase inhibitor sodium vanadate, thus suggesting the involvement of serine/threonine phosphatase in this process. Protein kinase C inhibitor, H-7 and calphostin C, did not inhibit the AII-induced up-regulation significantly. In addition, calcium ionophore, A23187 had no effect. These findings suggest that the AT2 receptor mRNA expression by AII is regulated by the activity of serine/threonine phosphatase in the cortical neurons. This observation is also the first example concerning the regulation of AT2 receptor within the brain.     

Systemic interleukin-1 induces early and late patterns of c-fos mRNA expression in brain

This study was designed to examine the mechanisms by which systemic interleukin-1 affects neuroendocrine systems in the brain. Intraperitoneal injections of interleukin-1 beta (1.25 micrograms/rat) were administered to rats. One or three hours after injection, the expression levels of the immediate-early gene c-fos and of genes for several neuropeptides, receptors, and enzymes were examined by in situ hybridization histochemistry. In the brainstem at 1 hr, c-fos mRNA was elevated in the area postrema and nucleus of the solitary tract, but not in the locus coeruleus. At 3 hr, the c-fos mRNA levels had increased further in the nucleus of the solitary tract. Rostrally, elevations in c-fos mRNA levels were found in the hypothalamic and thalamic paraventricular nuclei, central nucleus of amygdala, bed nucleus of the stria terminalis, and medial preoptic area, peaking at 1 hr and diminishing at 3 hr. In addition, at 3 hr a new pattern of c-fos activity emerged--the arcuate nucleus and cells at the external margins throughout the brain now expressed c-fos mRNA. Corticotropin-releasing hormone mRNA levels were doubled in the paraventricular nucleus at 1 and 3 hr, concomitant with elevations in plasma adrenocorticotrophic hormone (ACTH) and corticosterone. Tyrosine hydroxylase mRNA levels in the brainstem did not change. The c-fos mRNA induction patterns reveal a temporally dynamic response to interleukin-1 administration. We propose that the early set of structures responding to interleukin-1 initiates the neuroendocrine response to cytokines. Coactivation of the area postrema and nucleus of the solitary tract may reflect entry into the brain and neural transduction of the peripheral signal. The late set--including the nucleus of the solitary tract, arcuate nucleus, and the brain's edge--may reflect cellular activation along the diffusion routes traveled by interleukin-1 or a bioactive transduction product, because the pattern of edge labeling is similar to the autoradiographic pattern of flow lf radiolabeled tracer substances in the cerebrospinal fluid. The late c-fos mRNA response to interleukin-1, therefore, may represent a demonstration of information transfer in the parasynaptic mode, also known as volume transmission.     

Atrial natriuretic peptide inhibits endothelin-1-induced activation of JNK in glomerular mesangial cells

Atrial natriuretic peptide (ANP) has been shown to counteract various actions of endothelin-1 (ET-1) in mesangial cells. We have reported that both extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) are activated by ET-1 and ET-1-induced activation of ERK is inhibited by ANP. To further clarify the action of ANP, we examined the effect of ANP on ET-1-induced activation of JNK. ANP inhibited ET-1-induced activation of JNK in a dose-dependent manner. This inhibitory effect of ANP was reversed by HS-142-1, an antagonist for biological receptors of ANP, while C-ANP, an analog specific to clearance receptors of ANP, failed to inhibit ET-1-induced activation of JNK. 8-Bromo-cGMP and sodium nitroprusside were also able to inhibit ET-1-induced activation of JNK, suggesting cGMP-dependent action of ANP. In contrast, ANP failed to inhibit interleukin-1 beta (IL-1 beta)-induced activation of JNK. Since an increase in intracellular calcium ([Ca2+]i) was shown to be necessary for ET-1-induced activation of JNK in mesangial cells, we measured [Ca2+]i using fura-2. ANP attenuated the ET-1-induced increase in [Ca2+]i in concentrations enough to inhibit ET-1-induced activation of JNK. Finally, ANP was able to inhibit ET-1-, but not IL-1 beta-induced increase in DNA-binding activity of AP-1 by gel shift assay. These results indicate that ANP is able to inhibit ET-1-induced activation of AP-1 by inhibiting both ERK and JNK, suggesting that ANP might be able to counteract the expression of AP-1-dependent genes induced by ET-1.