It has been suggested that Ca2+ sensitization mechanisms might contribute to myogenic tone, however, specific mechanisms have not yet been fully identified. Therefore, we investigated the role of protein kinase C (PKC)- or RhoA-induced Ca2+ sensitization in myogenic tone of the rabbit basilar vessel. Myogenic tone was developed by stretch of rabbit basilar artery. Fura-2 Ca2+ signals, contractile responses, PKC immunoblots, translocation of PKC and RhoA, and phosphorylation of myosin light chains were measured. Stretch of the resting vessel evoked a myogenic contraction and an increase in the intracellular Ca2+ concentration ([Ca2+]i) only in the presence of extracellular Ca2+. Stretch evoked greater contraction than high K+ at a given [Ca2+]i. The stretch-induced increase in [Ca2+]i and contractile force were inhibited by treatment of the tissue with nifedipine, a blocker of voltage-dependent Ca2+ channel, but not with gadolinium, a blocker of stretch-activated cation channels. The PKC inhibitors, H-7 and calphostin C, and a RhoA-activated protein kinase (ROK) inhibitor, Y-27632, inhibited the stretch-induced myogenic tone without changing [Ca2+]i. Immunoblotting using isoform-specific antibodies showed the presence of PKCalpha and PKCepsilon in the rabbit basilar artery. PKCalpha, but not PKCepsilon, and RhoA were translocated from the cytosol to the cell membrane by stretch. Phosphorylation of the myosin light chains was increased by stretch and the increased phosphorylation was blocked by treatment of the tissue with H-7 and Y-27632, respectively. Our results are consistent with important roles for PKC and RhoA in the generation of myogenic tone. Furthermore, enhanced phosphorylation of the myosin light chains by activation of PKCalpha and/or RhoA may be key mechanisms for the Ca2+ sensitization associated with myogenic tone in basilar vessels.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; Antibodies ; Basilar Artery ; Calcium ; Cell Membrane ; Cytosol ; Fura-2 ; Gadolinium ; Immunoblotting ; Myosin Light Chains ; Nifedipine ; Phosphorylation ; Protein Kinase C ; Protein Kinases

gamma-Aminobutyric Acid (GABA) is contained in pancreatic islet beta-cells although its physiological role in pancreatic exocrine function is completely unknown at the present time. Recently, we have reported that exogenous GABA enhances secretagogue-evoked exocrine secretion in the isolated, perfused rat pancreas. This study was aimed to investigate an effect of exogenous GABA on pancreatic exocrine secretion in vivo evoked by intestinal stimulation. Rats were anesthetized with urethane (1.4 g/kg) after 24-h fast with free access to water. GABA (10, 30 and 100micromol/kg/h), given intravenously, did not change spontaneous pancreatic amylase secretion but dose-dependently elevated the amylase secretion evoked by intraduodenal sodium oleate (0.05 mmol/h). GABA (30micromol/kg/h) also further increased the amylase secretion stimulated by CCK+(30 pmol/kg/h) plus secretin (20 pmol/kg/h) but failed to modify the amylase secretion induced by secretin alone. GABA (10, 30 and 100micromol/kg/h) also dose-dependently elevated pancreatic amylase secretion evoked by CCK+alone. Bicuculline (100micromol/kg/h), a GABAA-receptor antagonist, markedly reduced the GABA-enhanced pancreatic responses to sodium oleate, CCK+plus secretin or CCK+alone. The results indicate that GABA enhances the sodium oleate-evoked pancreatic amylase secretion via GABAA-receptors in anesthetized rats, which may account for elevating the action of CCK+released by sodium oleate.
Amylases* ; Animals ; Bicuculline ; Cholecystokinin ; gamma-Aminobutyric Acid* ; Islets of Langerhans ; Oleic Acid* ; Pancreas ; Rats* ; Secretin ; Sodium* ; Urethane ; Water

Effects of cadmium (Cd) intoxication on renal endosomal accumulation of organic cations (OC ) were studied in rats using 14C-tetraethylammnium (TEA) as a substrate. Cd intoxication was induced by s.c. injections of 2 mg Cd/kg/day for 2-3 weeks. Renal cortical endosomes were isolated and the endosomal acidification (acridine orange fluorescence change) and TEA uptake (Millipore filtration technique) were assessed. The TEA uptake was an uphill transport mediated by H /OC antiporter driven by the pH gradient established by H -ATPase. In endosomes of Cd-intoxicated rats, the ATP-dependent TEA uptake was markedly attenuated due to inhibition of endosomal acidification as well as H /TEA antiport. In kinetic analysis of H /TEA antiport, Vmax was reduced and Km was increased in the Cd group. Inhibition of H /TEA antiport was also observed in normal endosomes directly exposed to free Cd (but not Cd-metallothionein complex, CdMt) in vitro. These data suggest that during chronic Cd exposure, free Cd ions liberated by lysosomal degradation of CdMt in proximal tubule cells may impair the endosomal accumulation of OC by directly inhibiting the H /OC antiporter activity and indirectly by reducing the intravesicular acidification, the driving force for H /OC exchange.
Animals ; Biological Transport, Active ; Cadmium ; Cations ; Citrus sinensis ; Endosomes* ; Filtration ; Fluorescence ; Ion Transport ; Ions ; Kidney ; Proton-Motive Force ; Rats* ; Tea ; Tetraethylammonium*

The role of vascular NAD(P)H oxidase in subarachnoid hemorrhage (SAH)-induced vasospasm in the basilar artery was examined in a rat model. Arterial vasospasm characterized by increased wall thickness and decreased lumen size was observed at 5 to 7 days after 2nd injection of blood into cisterna magna, and these changes were significantly ameliorated by pretreatment of diphenyleneiodonium (DPI, 25microl of 100microM), an inhibitor of NAD(P)H oxidase. To determine the time course of changes in the vascular NAD(P)H oxidase activity, cerebral vasculature was isolated at different time intervals from 12 hrs to 14 days after injection of autologous blood. At 24 hrs after the second injection of blood, the NAD(P)H oxidase activity was markedly increased with an enhanced membrane translocation of p47phox, but by 48 hours both the enzyme activity and p47phox translocation regained normal values, and were remained unchanged up to 14 days after SAH. However, no significant changes in the expression of p22phox mRNA was observed throughout the experiments. These findings suggest that the activation of NAD(P)H oxidase by which assembly of the oxidase components enhanced and subsequent production of superoxide in the early stages of SAH might contribute to the delayed cerebral vasospasm in SAH rats.
Animals ; Basilar Artery ; Cisterna Magna ; Membranes ; Models, Animal ; NADPH Oxidase ; Oxidoreductases ; Rats* ; Reference Values ; RNA, Messenger ; Subarachnoid Hemorrhage* ; Superoxides* ; Vasospasm, Intracranial*

In the present study, we used the microdialysis technique combined with high performance liquid chromatography (HPLC) and electrochemical detection to measure the extracellular levels of norepinephrine (NE) in the posterior hypothalamus in vivo, and to examine the effects of various drugs, affecting central noradrenergic transmission, on the extracellular concentration of NE in the posterior hypothalamus. Microdialysis probes were implanted stereotaxically into the posterior hypothalamus (coordinates: posterior 4.3 mm, lateral 0.5 mm, ventral 8 mm, relative to bregma and the brain surface, respectively) of rats, and dialysate collection began 2 hr after the implantation. The baseline level of monoamines in the dialysates were determined to be: NE 0.17 +/- 0.01, 3,4-dihydroxyphenylacetic acid (DOPAC) 0.94 +/- 0.07, homovanillic acid (HVA) 0.57 +/- 0.05 pmol/sample (n=8). When the posterior hypothalamus was perfused with 90 mM potassium, maximum 555% increase of NE output was observed. Concomitantly, this treatment significantly decreased the output of DOPAC and HVA by 35% and 28%, respectively. Local application of imipramine (50microM) enhanced the level of NE in the posterior hypothalamus (maximum 200%) compared to preperfusion control values. But, DOPAC and HVA outputs remained unchanged. Pargyline, an irreversible monoamine oxidase inhibitor, i.p. administered at a dose of 75 mg/kg, increased NE output (maximum 165%), while decreased DOPAC and HVA outputs (maximum 13 and 12%, respectively). These results indicate that NE in dialysate from the rat posterior hypothalamus were neuronal origin, and that manipulations which profoundly affected the levels of extracellular neurotransmitter had also effects on metabolite levels.
3,4-Dihydroxyphenylacetic Acid ; Animals ; Brain* ; Chromatography, Liquid ; Dialysis Solutions ; Homovanillic Acid ; Hypothalamus ; Hypothalamus, Posterior* ; Imipramine ; Microdialysis* ; Monoamine Oxidase Inhibitors ; Neurons ; Neurotransmitter Agents ; Norepinephrine* ; Pargyline ; Potassium ; Rats*

Congenital Long QT syndrome (LQTs) is a relatively rare pathologic disorder but results frequently in sudden cardiac death. Of the six LQTs that have been clinically described, five have been worked out for their genetic and biophysical profile. Most are generated by mutations which cause a loss of function in two delayed K+currents, iKs and iKr. One syndrome is generated by mutations in the Na channel which causes essentially a gain of function in the channel. Clinically the syndromes are characterized by slowed repolarization of the cardiac ventricular action potential and the occurrence of typical arrhythmias with undulating peaks in the electrocardiogram, called Torsade de Pointes. Arrhythmias are initiated by early or delayed afterdepolarizations and continue as reentry. Triggers for cardiac events are exercise (swimming; LQT1), emotion (arousal; LQT2) and rest/sleep (LQT3). beta-blockers have a high efficacy in the treatment of LQT1 and LQT2. In LQT3 their use is questionable. The study of congenital LQTsyndromes is a remarkable example of how basic and clinical science converge and take profit of each other's contribution.
Action Potentials ; Arrhythmias, Cardiac ; Death, Sudden, Cardiac ; Electrocardiography ; Long QT Syndrome ; Torsades de Pointes*

Diclofenac, a phenylacetic acid derivative, is a widely used non-steroidal anti-inflammatory drug (NSAID) to provide effective relief of inflammation and pain. Nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been implicated as a mediator of inflammation. We examined the inhibitory effects of diclofenac on the induction of iNOS in RAW 264.7 macrophages which were activated with lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). Treatment of RAW 264.7 cells with diclofenac and other NSAIDs (aspirin and indomethacin) significantly inhibited NO production and iNOS protein expression induced by LPS plus IFN-gamma. Also, diclofenac but not aspirin and indomethacin, inhibited iNOS mRNA expression and nuclear factor-kappa B (NF-kappaB) binding activity concentration-dependently. Furthermore, transfection of RAW 264.7 cells with iNOS promoter linked to a CAT reporter gene revealed that only diclofenac inhibited the iNOS promoter activity induced by LPS plus IFN-gamma through the NF-kappaB sites of iNOS promoter. Taken together, these suggest that diclofenac may exert its anti-inflammatory effect by inhibiting iNOS gene expression at the transcriptional level through suppression of NF-kappaB activation.
Animals ; Anti-Inflammatory Agents, Non-Steroidal ; Aspirin ; Cats ; Diclofenac* ; Gene Expression* ; Genes, Reporter ; Indomethacin ; Inflammation ; Interferon-gamma ; Macrophages* ; NF-kappa B* ; Nitric Oxide ; Nitric Oxide Synthase Type II ; RNA, Messenger ; Transfection

Nimopidine, one of dihydropyridine derivatives, has been widely used to pharmacologically identify L-type Ca currents. In this study, it was tested if nimodipine is a selective blocker for L-type Ca currents in sensory neurons and heterologous system. In mouse dorsal root ganglion neurons (DRG), low concentrations of nimodipine (<10 muM), mainly targeting L-type Ca currents, blocked high-voltage-activated calcium channel currents by apprx38%. Interestingly, high concentrations of nimodipine (>10 muM) further reduced the "residual" currents in DRG neurons from alpha1E knock-out mice, after blocking L-, N- and P/Q-type Ca currents with 10 muM nimodipine, 1 muM omega-conotoxin GVIA and 200 nM omega-agatoxin IVA, indicating inhibitory effects of nimodipine on R-type Ca currents. Nimodipine (>10 muM) also produced the inhibition of both low-voltage-activated calcium channel currents in DRG neurons and alpha1B and alpha1E subunit based Ca channel currents in heterologous system. These results suggest that higher nimodipine (>10 muM) is not necessarily selective for L-type Ca currents. While care should be taken in using nimodipine for pharmacologically defining L-type Ca currents from native macroscopic Ca currents, nimodipine (>10 muM) could be a useful pharmacological tool for characterizing R-type Ca currents when combined with toxins blocking other types of Ca channels.
Animals ; Calcium Channels ; Calcium* ; Diagnosis-Related Groups ; Ganglia, Spinal ; Mice ; Mice, Knockout ; Neurons ; Nimodipine* ; omega-Agatoxin IVA ; omega-Conotoxin GVIA ; Sensory Receptor Cells

Long-term treatment of cultured bovine adrenal medullary chromaffin (BAMC) cells with arachidonic acid (100 muM), angiotesnin II (100 nM), prostaglandin E2 (PGE2; 10 muM), veratridine (2 muM) or KCl (55 mM) for 24 hrs increased both norepinephrine and epinephrine levels in the supernatant. Pretreatment with staurosporine (10 nM), a protein kinase C (PKC) inhibitor, completely blocked increases of norepinephrine and epinephrine secretion induced by arachidonic acid, angiotensin II, PGE2, veratridine or KCl. In addition, K252a, another PKC inhibitor whose structure is similar to that of staurosporine, effectively attenuated both norepinephrine and epinephrine secretion induced by arachidonic acid. However, K252a did not affect the catecholamine secretion induced by angiotensin II, PGE2, veratridine or KCl. Our results suggest that staurosporine may inhibit long-term catecholamine secretion induced by various secretagogues in a mechanism other than inhibiting PKC signaling. Furthermore, long-term secretion of catecholamines induced by arachidonic acid may be dependent on PKC pathway.
Angiotensin II ; Arachidonic Acid ; Catecholamines* ; Chromaffin Cells* ; Dinoprostone ; Epinephrine ; Norepinephrine ; Protein Kinase C ; Staurosporine* ; Veratridine

We have cloned the mouse neurotensin/neuromedin N (NT/N) gene from the murine mast cell line C1.MC/C57.1 for the first time. The murine NT/N cDNA clone consisted of 765 nucleotides and coded for 169 peptide residues with an N-terminal signal peptide, and the C-terminal region contained of one copy of neurotensin (NT) and one copy of neuromedin N (NN). Total of four Lys-Arg dibasic motifs were present; one each at the middle of the open reading frame, at the N-terminal of NN, at the C-terminal of NT, and between NN and NT. Amino acid sequence analysis of the mouse NT/N revealed 90% homology to that of the rat NT/N gene. NT/N is expressed in routine mast cell lines (C1.MC/C57.1 and P815), but not in murine bone marrow-derived mast cells (BMMCs), murine macrophage cell line (RAW 264.7), nor in murine T cell line (EL-4). NT/N mRNA in C1.MC/C57.1 is highly inducible by IgE cross-linking, phorbol myristate acetate, neurotensin, and substance P. Following the treatment of demethylating agent, 5-azacytidine (5-azaC), the NT/N gene was induced in BMMCs in response to IgE cross-linking. 5-azaC-treated BMMCs did not express the NT/N gene without additional stimuli. These findings suggested that the regulation of NT/N gene expression was dependent on the effects of not only gene methylation but also enhancer and/or repressor proteins acting on the NT/N promoter.
Animals ; Azacitidine ; Cell Line ; Clone Cells ; DNA, Complementary ; Gene Expression ; Immunoglobulin E ; Macrophages ; Mast Cells* ; Methylation ; Mice ; Neurotensin ; Nucleotides ; Open Reading Frames ; Protein Sorting Signals ; Rats ; Repressor Proteins ; RNA, Messenger ; Sequence Analysis, Protein ; Substance P ; Tetradecanoylphorbol Acetate