A role for the sodium pump in H2O2-induced vasorelaxation in porcine isolated coronary arteries.

Hydrogen peroxide (H2O2) has been proposed to act as a factor for endothelium-derived hyperpolarization (EDH) and EDH may act as a 'back up' system to compensate the loss of the NO pathway. Here, the mechanism of action of H2O2 in porcine isolated coronary arteries (PCAs) was investigated. Distal PCAs were mounted in a wire myograph and pre-contracted with U46619 (1nM-50µM), a thromboxane A2-mimetic or KCl (60mM). Concentration-response curves to H2O2(1µM-1mM), bradykinin (0.01nM-1µM), sodium nitroprusside (SNP) (10nM-10µM), verapamil (1nM-10µM), KCl (0-20mM) or Ca(2+)-reintroduction (1µM-10mM) were constructed in the presence of various inhibitors. Activity of the Na(+)/K(+)-pump was measured through rubidium-uptake using atomic absorption spectrophotometry. H2O2 caused concentration-dependent vasorelaxations with a maximum relaxation (Rmax) of 100±16% (mean±SEM), pEC50=4.18±0.20 (n=4) which were significantly inhibited by PEG-catalase at 0.1-1.0mM H2O2 (P<0.05). 10mM TEA significantly inhibited the relaxation up to 100µM H2O2 (P<0.05). 60mM K(+) and 500nM ouabain significantly inhibited H2O2-induced vasorelaxation producing a relaxation of 40.8±8.5% (n=5) and 47.5±8.6% (n=6) respectively at 1mM H2O2 (P<0.0001). H2O2-induced vasorelaxation was unaffected by the removal of endothelium, inhibition of NO, cyclo-oxygenase, gap junctions, SKCa, IKCa, BKCa Kir, KV, KATP or cGMP. 100µM H2O2 had no effects on the KCl-induced vasorelaxation or Ca(2+)-reintroduction contraction. 1mM H2O2 inhibited both KCl-induced vasorelaxation and rubidium-uptake consistent with inhibition of the Na(+)/K(+)-pump activity. We have shown that the vascular actions of H2O2 are sensitive to ouabain and high concentrations of H2O2 are able to modulate the Na(+)/K(+)-pump. This may contribute towards its vascular actions.

Bradykinin protects nucleus pulposus cells from tert-butyl hydroperoxide-induced damage and delays intervertebral disc degeneration.

Intervertebral disc degeneration (IVDD) is a leading cause of degenerative spinal disorders, involving complex biological processes. This study investigates the role of the kallikrein-kinin system (KKS) in IVDD, focusing on the protective effects of bradykinin (BK) on nucleus pulposus cells (NPCs) under oxidative stress. Clinical specimens were collected, and experiments were conducted using human and rat primary NPCs to elucidate BK's impact on tert-butyl hydroperoxide (TBHP)-induced oxidative stress and damage. The results demonstrate that BK significantly inhibits TBHP-induced NPC apoptosis and restores mitochondrial function. Further analysis reveals that this protective effect is mediated through the BK receptor 2 (B2R) and its downstream PI3K/AKT pathway. Additionally, BK/PLGA sustained-release microspheres were developed and validated in a rat model, highlighting their potential therapeutic efficacy for IVDD. Overall, this study sheds light on the crucial role of the KKS in IVDD pathogenesis and suggests targeting the B2R as a promising therapeutic strategy to delay IVDD progression and promote disc regeneration.

Exercise training-enhanced, endothelium-dependent dilation mediated by altered regulation of BK(Ca) channels in collateral-dependent porcine coronary arterioles.

OBJECTIVE: Test the hypothesis that exercise training increases the contribution of BK(Ca) channels to endothelium-mediated dilation in coronary arterioles from collateral-dependent myocardial regions of chronically occluded pig hearts and may function downstream of H2O2. METHODS: An ameroid constrictor was placed around the proximal left circumflex coronary artery to induce gradual occlusion in Yucatan miniature swine. Eight weeks postoperatively, pigs were randomly assigned to sedentary or exercise training (treadmill; 14 week) regimens. RESULTS: Exercise training significantly enhanced bradykinin-mediated dilation in collateral-dependent arterioles (~125 µm diameter) compared with sedentary pigs. The BK(Ca) -channel blocker, iberiotoxin alone or in combination with the H2O2 scavenger, polyethylene glycol catalase, reversed exercise training-enhanced dilation in collateral-dependent arterioles. Iberiotoxin-sensitive whole-cell K+ currents (i.e., BK(Ca)-channel currents) were not different between smooth muscle cells of nonoccluded and collateral-dependent arterioles of sedentary and exercise trained groups. CONCLUSIONS: These data provide evidence that BK(Ca)-channel activity contributes to exercise training-enhanced endothelium-dependent dilation in collateral-dependent coronary arterioles despite no change in smooth muscle BK(Ca)-channel current. Taken together, our findings suggest that a component of the bradykinin signaling pathway, which stimulates BK(Ca) channels, is enhanced by exercise training in collateral-dependent arterioles and suggest a potential role for H2O2 as the mediator.

In vivo up-regulation of kinin B1 receptors after treatment with Porphyromonas gingivalis lipopolysaccharide in rat paw.

It has been demonstrated that kinin B(1) receptors are highly up-regulated under several stressful stimuli, such as infection. However, there is no evidence indicating whether Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) might lead to B(1) receptor up-regulation. In this study, we demonstrate that Pg-LPS injection into the rat paw resulted in a marked functional up-regulation of B(1) receptors (as measured by an increase of B(1) receptor-induced edema), which was preceded by a rapid rise in B(1) receptor mRNA expression. The local administration of Pg-LPS also resulted in a prominent production of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha), followed by an increase of neutrophil influx; both events were observed at periods before B(1) receptor induction. The functional and molecular Pg-LPS-elicited B(1) receptor up-regulation was significantly reduced by the glucocorticoid dexamethasone (0.5 mg/kg s.c.), and to a lesser extent by the chimeric anti-TNF-alpha antibody infliximab (1 mg/kg s.c.). Of high relevance, we show for the first time that a single administration of the proresolution lipid mediator (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid (resolvin E1; 300 ng/rat i.p.) was able to markedly down-regulate Pg-LPS-driven B(1) receptor expression, probably by inhibiting TNF-alpha production and neutrophil migration. Collectively, the present findings clearly suggest that Pg-LPS is able to induce the up-regulation of B(1) receptors through mechanisms involving TNF-alpha release and neutrophil influx, which are largely sensitive to resolvin E1. It is tempting to suggest that kinin B(1) receptors might well represent a pivotal pathway for the inflammatory responses evoked by P. gingivalis and its virulence factors.

Effects of interleukin-1alpha on the production and release of basic fibroblast growth factor in cultured nifedipine-reactive gingival fibroblasts.

The effect of interleukin-1alpha (IL-1alpha) on the production of basic fibroblast growth factor (bFGF) in human gingival fibroblasts originated from a nifedipine-reactive patient was investigated. Ca(2+)-mobilizing agents, thapsigargin and bradykinin, were also tested to determine whether they affected the production and release of bFGF. The release of bFGF from IL-1alpha-pretreated cells in relation to the transient increase in intracellular Ca(2+)([Ca(2+)]i) and extracellular Ca(2+)levels was also investigated. IL-1alpha and thapsigargin yielded significantly higher bFGF production, and also enhanced bFGF mRNA expression. IL-1alpha-pretreated cells showed significantly greater release of bFGF under the present experimental conditions. Levels of released bFGF were significantly higher in cells pretreated with IL-1alpha, followed by bradykinin and thapsigargin in the presence of extracellular Ca(2+). The transient mobilization of intracellular Ca(2+) accelerated the release of bFGF in IL-1alpha-pretreated cells, but not in untreated cells. Thus, IL-1alpha increases bFGF production in nifedipine-reactive gingival fibroblasts and also influences the release of bFGF in the IL-1alpha-pretreated cells.

Endothelial nitric oxide synthase activation leads to dilatory H2O2 production in mouse cerebral arteries.

OBJECTIVE: Hydrogen peroxide (H2O2) produced by the vascular endothelium is a signaling molecule regulating vascular tone. We hypothesized that H2O2 derived from eNOS activity could play a physiological role in endothelium-dependent dilation of mouse cerebral arteries. METHODS: Simultaneous endothelium-dependent dilation and fluorescence-associated free radical (DCF-DA) or NO (DAF-2) production were recorded in isolated and pressurized (60 mm Hg) cerebral artery of C57Bl/6 male mice. RESULTS: Without synergism, N-nitro-L-arginine (L-NNA) or the H2O2 scavengers catalase, PEG-catalase and pyruvate reduced (P < 0.05) by 50% the endothelium-dependent dilation induced by acetylcholine (ACh). Simultaneously with the dilation, H2O2--but not NO--production, sensitive to either L-NNA or catalase, was detected. In cerebral arteries from C57Bl/6.eNOS-/- mice, catalase had no effect on ACh-induced dilation and no H2O2-associated fluorescence was observed. In C57Bl/6 mice, silver diethyldithiocarbamate (DETC), a superoxide dismutase (SOD) inhibitor, but not the specific NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl3-oxide (PTIO), prevented ACh-induced dilation and H2O2 production suggesting that eNOS-derived superoxide is an intermediate in the production of H2O2. The catalase-sensitive ACh-induced dilation was restored by the eNOS cofactor tetrahydrobiopterin (BH4). This reversal was associated with a NO-associated fluorescence sensitive to PTIO but not to catalase. Soluble guanylate cyclase inhibition with 1H-[1,2,4]-oxadiazole-4,3-aquinoxalin-1-one (ODQ) prevented the dilation induced by ACh and by exogenous H2O2. Lastly, L-NNA, PTIO and ODQ--but not DETC, catalase or pyruvate--increased the pressure-dependent myogenic tone, suggesting that eNOS produces NO at rest, but leads to H2O2 during muscarinic stimulation. CONCLUSION: H2O2-dependent dilation in mouse cerebral arteries appears to be a physiological eNOS-derived mechanism.

Antimicrobial peptide encapsulation and sustained release from polymer network particles prepared in supercritical carbon dioxide.

Antimicrobial peptide loaded poly(2-hydroxyethyl methacrylate) particles were synthesized in supercritical carbon dioxide via one-pot free-radical dispersion polymerisation of 2-hydroxyethyl methacrylate and a cross-linker. Discrete particles with a well-defined spherical morphology and a diameter as low as 450 nm have been obtained in mild conditions. The encapsulation and release of the peptide were confirmed by antimicrobial tests that demonstrated for the first time a sustained release of the peptide from poly(2-hydroxyethyl methacrylate) microgels prepared by one-pot dispersion polymerization in supercritical carbon dioxide and then dispersed in water.

The effect of serotonin (5-hydroxytryptamine) and derivatives on guanosine 3',5'-monophosphate in human monocytes.

The effects of several putative mediators of inflammatory responses on the cyclic nucleotide content of mononuclear leukocytes from human peripheral blood were investigated. Incubation of mononuclear cells with 100 muM serotonin (a maximally effective concentration) for 5 min caused a five- to eightfold increase in their content of guanosine 3',5'-monophosphate (cAMP)...

Descriptive and functional neuroanatomy of locus coeruleus-noradrenaline-containing neurons involvement in bradykinin-induced antinociception on principal sensory trigeminal nucleus.

The present study was carried out in Wistar rats, using the jaw-opening reflex and dental pulp stimulation, to investigate noradrenaline- and serotonin-mediated antinociceptive circuits. The effects of microinjections of bradykinin into the principal sensory trigeminal nucleus (PSTN) before and after neurochemical lesions of the locus coeruleus noradrenergic neurons were studied. Neuroanatomical experiments showed evidence for reciprocal neuronal pathways connecting the locus coeruleus (LC) to trigeminal sensory nuclei and linking monoaminergic nuclei of the pain inhibitory system to spinal trigeminal nucleus (STN). Fast blue (FB) injections in the locus coeruleus/subcoeruleus region retrogradely labeled neurons in the contralateral PSTN and LC. Microinjections of FB into the STN showed neurons labeled in both ipsilateral and contralateral LC, as well as in the ipsilateral Barrington's nucleus and subcoeruleus area. Retrograde tract-tracing with FB also showed that the mesencephalic trigeminal nucleus sends neural pathways towards the ipsilateral PSTN, with outputs from cranial and caudal aspects of the brainstem. In addition, neurons from the lateral and dorsolateral columns of periaqueductal gray matter also send outputs to the ipsilateral PSTN. Microinjections of FB in the interpolar and caudal divisions of the STN labeled neurons in the caudal subdivision of STN. Microinjections in the STN interpolar and caudal divisions also retrogradely labeled serotonin- and noradrenaline-containing nucleus of the brainstem pain inhibitory system. Finally, the gigantocellularis complex (nucleus reticularis gigantocellularis/paragigantocellularis), nucleus raphe magnus and nucleus raphe pallidus also projected to the caudal divisions of the STN. Microinjections of bradykinin in the PSTN caused a statistically significant long-lasting antinociception, antagonized by the damage of locus coeruleus-noradrenergic neuronal fibres with (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) (DSP4), a neurotoxin that specifically depleted noradrenaline from locus coeruleus terminal fields. These data suggest that serotonin- and noradrenaline-containing nuclei of the endogenous pain inhibitory system exert a key-role in the antinociceptive mechanisms of bradykinin and the locus coeruleus is crucially involved in this effect.

Depletion of intracellular Ca2+ stores sensitizes the flow-induced Ca2+ influx in rat endothelial cells.

Hemodynamic shear stress elicits a rise in endothelial [Ca2+]i, which may serve as a key second messenger to regulate many flow-associated physiological and biochemical processes. In the present study, we used Mn2+ quenching of fluorescent dye Fluo3 as an assay to investigate the Ca2+ influx of rat aortic endothelial cells in response to flow. We found that the Ca2+ signaling in response to flow could be greatly influenced by the status of intracellular Ca2+ stores. Depletion of intracellular Ca2+ stores by thapsigargin (4 micromol/L) or cyclopiazonic acid (10 micromol/L) drastically sensitized the Ca2+ influx in response to flow. Ca2+-mobilizing agonist bradykinin (100 nmol/L) or ATP (100 micromol/L) had similar sensitizing effect. The effect of bradykinin or ATP was blocked by Xestospongin C and U73122, suggesting that the sensitization was related to the IP3-mediated store depletion. On the other hand, the Mn2+ quenching in response to flow was greatly reduced by ochratoxin A (100 nmol/L), an agent that could increase the filling state of intracellular Ca2+ stores. In addition, we found that depletion-sensitized Ca2+ influx in response to flow was mediated by a PKG-inhibitable cation channel and that the influx was affected by membrane potential and K+ channel activity. In conclusion, the present study argues for a critical role of intracellular Ca2+ status in determining the Ca2+ signaling in response to flow and it provides a general mechanistic explanation for the stimulatory role of blood-borne agonists on flow-induced Ca2+ influx.

Bradykinin mediates phosphorylation of eNOS in odontoblasts.

While the activation of eNOS by Akt/PKB-dependent phosphorylation, leading to NO release, and the inhibition of enzyme activity by bradykinin (BK)-mediated phosphorylation of eNOS in endothelial cells are established, the phosphorylation of eNOS in odontoblasts is unknown. To clarify the regulation of eNOS in odontoblasts by BK, we examined the phosphorylation of eNOS, Akt/PKB, and ERK1/2 in odontoblasts of rat molars. BK (10(-7) M) transiently induced the phosphorylation of eNOS at Ser1177, Akt/PKB in odontoblasts, while it induced the phosphorylation of eNOS at Thr495 throughout the entire period of BK treatment. BK receptor 2 antagonist HOE 140 (10(-6) M) significantly reduced signal intensities of phosphorylated-eNOS at Ser1177, Thr495, and phosphorylated-Akt/PKB. These results suggest that BK has dual effects on the activation of eNOS in odontoblasts, the Akt/PKB-dependent up-regulation of eNOS by the transient phosphorylation at Ser1177, and the ERK1/2-independent down-regulation of eNOS by the phosphorylation at Thr495.

Effects of acrylic resin monomers on porcine coronary artery reactivity.

The purpose of the present investigation was to assess the reactivity of porcine coronary arteries under in vitro conditions following their exposure to methyl methacrylate (MMA) and hydroxyethyl methacrylate (HEMA) monomers. Confirming previous studies using rat aortas, both MMA and HEMA induced acute/direct relaxation of coronary ring preparations, which was partly dependent on the endothelium. With prolonged tissue exposure, both monomers caused time- and concentration-dependent inhibition of receptor-mediated contraction of the vascular smooth muscle caused by prostaglandin F2∝ (PGF2∝), with HEMA causing more inhibition than MMA. Hydroxyethyl methacrylate, but not MMA, also produced impairment of non-receptor-mediated contraction of the coronary smooth muscle induced by KCl. On the other hand, neither HEMA nor MMA altered relaxation of the smooth muscle produced by the direct-acting pharmacological agent, sodium nitroprusside (SNP). While exposure to HEMA impaired endothelium-dependent vasorelaxation caused by bradykinin (BK), MMA markedly enhanced this endothelial-mediated response of the arteries. The enhanced endothelial response produced by MMA was linked to nitric oxide (NO) release. In conclusion, with prolonged tissue exposure, MMA causes less pronounced effects/adverse consequences on coronary smooth muscle function relative to the effect of HEMA, while enhancing vasorelaxation associated with release of NO from the endothelium. Accordingly, MMA-containing resin materials appear to be safer for human applications than materials containing HEMA.

Bradykinin upregulates IL-8 production in human gingival fibroblasts stimulated by interleukin-1beta and tumor necrosis factor alpha.

The proinflammatory mediator bradykinin (BK) is suggested to play an important role in the pathogenesis of various inflammatory diseases including periodontitis. In this study, BK per se stimulated interleukin-8 (IL-8) production in human gingival fibroblasts in vitro. Furthermore, BK upregulated the stimulatory effect of the cytokines IL-1beta and TNFalpha on the production of IL-8. The stimulatory effect of BK on the IL-1beta- or TNFalpha-stimulated IL-8 production was reduced in the presence of BK B2 receptor antagonist HOE 140, whereas the B1 receptor antagonist Lys-(des-arg9, Leu8)-BK had no effect. Similar to BK, the calcium ionophore A23187 also upregulated the stimulatory effect of IL-1beta and TNFalpha on IL-8 production. The protein kinase C (PKC) inhibitor bisindolylmaleimide, BIS, significantly reduced the stimulatory effect of BK on IL-1beta and TNFalpha increased IL-8 production but did not affect the production of IL-8 stimulated by cytokines alone. The specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 reduced IL-8 production stimulated by the combination of BK and IL-1beta as well as the IL-1beta-stimulated IL-8 production. In conclusion, this study shows that BK upregulates IL-1beta- and TNFalpha-stimulated IL-8 production via BK B2 receptor and that PKC signal pathway seems to be involved in the upregulation of the cytokine-induced IL-8 production in gingival fibroblasts. This stimulatory effect of BK on IL-8 production may contribute to the maintenance of the gingival inflammation and enhanced risk for destruction of gingival connective tissue.

Electron spin resonance detection of hydrogen peroxide as an endothelium-derived hyperpolarizing factor in porcine coronary microvessels.

OBJECTIVE: Endothelium-derived hyperpolarizing factor (EDHF) plays an important role in modulating vascular tone, especially in microvessels, although its nature has yet to be elucidated. This study was designed to examine whether hydrogen peroxide (H2O2) is an EDHF in porcine coronary microvessels with use of an electron spin resonance (ESR) method to directly detect H2O2 production from the endothelium. METHODS AND RESULTS: Isometric tension and membrane-potential recordings demonstrated that bradykinin and substance P caused EDHF-mediated relaxations and hyperpolarizations of porcine coronary microvessels in the presence of indomethacin and Nomega-nitro-L-arginine. The contribution of H2O2 to the EDHF-mediated responses was demonstrated by the inhibitory effect of catalase and by the relaxing and hyperpolarizing effects of exogenous H2O2. Endothelial production of H2O2 was quantified in bradykinin- or substance P-stimulated intact blood vessels by ESR spectroscopy. Tiron, a superoxide scavenger that facilitates H2O2 formation, enhanced bradykinin-induced production of H2O2, as well as the EDHF-mediated relaxations and hyperpolarizations. By contrast, cytochrome P-450 inhibitors (sulfaphenazole or 17-octadecynoic acid) or a gap junction inhibitor (18alpha-glycyrrhetinic acid) failed to inhibit the EDHF-mediated relaxations. Involvement of endothelium-derived K+ was not evident in experiments with ouabain plus Ba2+ or exogenous K+. CONCLUSIONS: These results provide ESR evidence that H2O2 is an EDHF in porcine coronary microvessels.

Perfusion-induced changes in cardiac contractility and oxygen consumption are not endothelium-dependent.

OBJECTIVE: Are substances released from rat coronary endothelial cells responsible for the increase in contractility and oxygen consumption (Gregg phenomenon) seen with an increase in cardiac perfusion? METHODS: In an isovolumically contracting, Langendorff, crystalloid perfused rat heart (n = 6) at 27 degrees C, coronary flow was changed (from 4.4 to 15.4 ml.min-1.gww(-1)) before and after the endothelium was made dysfunctional by Triton X-100. Vascular endothelium and smooth muscle function were tested with bradykinin (BK, 1 microM, an endothelium-dependent dilator) and papaverine (PAP, 1 microM, an endothelium-independent dilator) in a preconstricted vascular bed (vasopressin, VP, 3 nM). RESULTS: Before Triton X-100, coronary resistance (at constant flow) decreased significantly in response to BK and to PAP. After Triton X-100 treatment the dilatory response to BK was abolished while the PAP response was still present, suggesting endothelial dysfunction with intact smooth muscle function. Due to Triton X-100 treatment, coronary resistance increased significantly. Therefore coronary flow changes were also applied during a similar increase in coronary resistance induced by VP infusion (3 nM) before Triton X-100 treatment. During control, developed left ventricular pressure (dev Plv) increased with 68 +/- 21% and oxygen consumption (VO2) increased with 122 +/- 25% in response to the maximal increase in coronary flow. During increased coronary resistance with and without functional endothelium, dev Plv increased by 57 +/- 16 and 64 +/- 22%, respectively, and VO2 increased by 126 +/- 21 and 103 +/- 20%, respectively, in response to the maximal increase in flow. These changes were not significantly different from control. CONCLUSION: The results suggest that the arterial endothelium is not involved in the Gregg phenomenon.

Bradykinin induces a rapid cyclooxygenase-2 mRNA expression via Ca2+ mobilization in human gingival fibroblasts primed with interleukin-1 beta.

We have previously demonstrated that bradykinin potentiates prostaglandin E(2)release in human gingival fibroblasts pretreated with interleukin-1 beta (priming). In this study, we demonstrate a potentiating effect of bradykinin on cyclooxygenase-2 mRNA expression in the interleukin-1 beta-primed fibroblasts. Interleukin-1 beta (200 pg/ml) induced cyclooxygenase-2 mRNA expression, but not bradykinin (1 microM). However, bradykinin rapidly and markedly increased the cyclooxygenase-2 mRNA expression in the fibroblasts primed with interleukin-1 beta. In the primed fibroblasts, ionomycin and thapsigargin mimicked the potentiating effect of bradykinin on the cyclooxygenase-2 mRNA expression. Dexamethasone and actinomycin D completely suppressed not only the interleukin-1 beta-induced cyclooxygenase-2 mRNA expression, but also the bradykinin-induced cyclooxygenase-2 mRNA expression in the interleukin-1 beta-primed fibroblasts, although cycloheximide did not inhibit the effects of interleukin-1 beta and bradykinin. These results suggest that bradykinin-induced prostaglandin E2 synthesis is regulated at the level of the transcription of cyclooxygenase-2 mRNA via Ca2+ mobilization in the interleukin-1 beta-primed human gingival fibroblasts.

Inositol phosphate formation in the human squamous cell carcinoma line SCC-12 F: studies with bradykinin, the calcium ionophore A23187, and sodium fluoride.

The phospholipase C (PLC)-mediated hydrolysis of membrane phosphoinositides is an important signal transduction pathway coupled to the cell-surface receptors for several hormones and growth factors. In addition, PLC activity can be modulated by changes in intracellular calcium and activation of GTP binding proteins. In this report, differential activation of PLC in the human keratinocyte cell line SCC-12F was studied as judged by specific patterns of inositol phosphate formation. Several hormones and growth factors previously shown to stimulate PLC in a variety of cell types were screened for activity in SCC-12F cells. Only bradykinin was active, stimulating the PLC-dependent generation of inositol (1,4,5) triphosphate (Ins(1,4,5)P3). Ins(1,4,5)P3 was rapidly metabolized to inositol(1,4)biphosphate (Ins(1,4)P2) and inositol(1,3,4,5)tetrakisphosphate (Ins(1,3,4,5)P4), and subsequently degraded to inositol monophosphates. The response elicited by bradykinin was concentration dependent (EC50 value of 50 nM), suggesting involvement of a specific bradykinin receptor. Treatment of these cells with the calcium ionophore A23187 appeared to result in the direct formation of Ins(1,4)P2 without Ins(1,4,5)P3 as precursor. Treatment of the cells with AIF4-, a putative activator of GTP binding proteins, resulted in the generation of inositol monophosphates as the major metabolites in the absence of detectable Ins(1,4,5)P3 formation. Taken together, these observations suggest that the PLC complex present in SCC-12F cells can be differentially activated to yield either Ins(1,4,5)P3, Ins(1,4)P2, or InsP. The observed effects may be due to a direct PLC-dependent hydrolysis of the appropriate membrane phosphoinositide.

Attenuation of pressor responses to norepinephrine and pitressin and potentiation of pressor response to angiotensin II by captopril in human subjects.

The present study was conducted to investigate the influence of captopril on cardiochemodynamic responses in 38 normal volunteers (20- to 35-year-old men) to exogenously administered vasopressor substances. Norepinephrine (NE), 0.05, 0.1, and 0.2 micrograms/kg min-1; angiotensin II (AII), 5, 10, and 20 ng/kg min-1; and pitressin (2 mU/kg min-1) were infused for 10 minutes. Each infusion was reported twice, and the responses were reproducible. Captopril (50 mg by mouth) significantly attenuated the pressor responses to NE and pitressin, but the decrease in heart rate in response to NE and pitressin was almost the same before and after captopril treatment, suggesting that captopril potentiates reflex slowing of the heart. Captopril significantly potentiated the pressor response to AII. Attenuation of pressor response and potentiation of reflex slowing of the heart, in response to NE and pitressin, disappeared when a subdepressor dose of AII (1 ng/kg min-1) was infused in addition to captopril. Infusion of a subdepressor dose of bradykinin (BK), 0.1 microgram/kg min-1, had no influence on the pressor response to NE. In the subjects treated with indomethacin (225 mg/54 hrs), captopril still attenuated the pressor response to NE. These results suggest that captopril attenuates the pressor responses to NE and pitressin primarily by depletion of endogenous AII; decreased AII may desensitize the contraction of arterial smooth muscle and may potentiate the compensatory reflex mechanism.

Role of the cyclic adenosine 3',5'-monophosphate and the phosphatidylinositol-Ca2+ cascades in mediating the effects of thyrotropin and iodide on hormone synthesis and secretion in human thyroid slices.

There are two major known regulatory pathways in human thyrocytes: the phosphatidylinositol-Ca2+ cascade (PiP2 cascade) and the cAMP cascade. We study here the regulation of the PiP2 cascade by TSH, ATP, NaF, and bradykinin. Our data show that protein iodination and, thus, the synthesis of thyroid hormones in human thyroid is under the control of both the PiP2 cascade and the cAMP cascade. Activation of the PiP2 cascade by TSH (10 mU/mL), NaF, bradykinin, ionomycin, and 12-O-tetradecanoylphorbol-13-acetate stimulates iodide organification. Conversely, activation of the cAMP cascade by forskolin, TSH (0.3 mU/mL), and dibutyryl cAMP inhibits iodide organification. These metabolic effects are correlated to activations and inhibitions of the H2O2-generating system, showing that H2O2 is a limiting factor for protein iodination in these cells. The cascades also regulate in parallel the activity of the pentose phosphate pathway. The effects of various concentrations of TSH on H2O2 generation and [1-14C]glucose oxidation were tested, showing a dual effect with an inhibition of these metabolisms for low concentrations of TSH (that stimulate the cAMP cascade) and an activation for high concentrations of TSH (that stimulate the PiP2 cascade). The control of thyroid secretion differs from that of protein iodination, in that the cAMP cascade greatly enhances secretion, whereas the PiP2 cascade has no effect on basal secretion and even an inhibitory effect on TSH-stimulated secretion (1 mU/mL). We also demonstrate here the presence of an inhibitory effect of iodide on its own organification in human thyroid (Wolff-Chaikoff effect). This effect is probably mediated through an inhibition of the inositol trisphosphate response to TSH and of the H2O2 response to Ca2+.

Jaw electromyographic activity induced by the application of algesic chemicals to the rat tooth pulp.

The aim of this study was to determine if the application of mustard oil (MO), a small-fiber excitant and inflammatory irritant, or other algesic chemicals (capsaicin, CAP, and bradykinin, BK) to the rat maxillary molar tooth pulp induces electromyographic (EMG) activity of the masseter and digastric muscles, and also to determine if endogenous opioid mechanisms may be involved in any documented EMG changes. Application of MO to the tooth pulp induced a significant increase in EMG activity of the ipsilateral masseter up to 30 min. The application of mineral oil to the pulp or MO application to the pulp-extirpated tooth did not induce any significant EMG increases. The application of CAP or BK to the pulp in contrast had much weaker effects on EMG activity of the jaw muscles. CAP produced a small but prolonged increase in masseter EMG activity, and BK induced a short-lasting increase in digastric EMG activity. The systemic administration of the opiate antagonist naloxone significantly reactivated (i.e. rekindled) the EMG response evoked by MO application to the pulp. Naloxone did not produce any such significant rekindling effect on EMG activity following CAP, BK or mineral oil application to the pulp or following MO application to the pulp-extirpated tooth. The MO, BK and especially CAP groups showed histological evidence of vasodilatation and polymorphonuclear leukocyte infiltration in the pulp tissue and a significant increase in plasma extravasation of Evans Blue dye, whereas mineral oil did not induce these changes. These findings suggest that pulp afferent inputs to the central nervous system evoked by BK. CAP and especially MO may induce enhanced jaw muscle activity. In addition, the naloxone data suggest that an opioid suppresive mechanism may be induced by the pulpal afferent inputs evoked by MO, and may serve to limit the jaw muscle activity elicited by these inputs.